U.S. patent application number 14/691498 was filed with the patent office on 2015-11-19 for autism-associated biomarkers and uses thereof.
The applicant listed for this patent is THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK. Invention is credited to Mady HORNIG, W. Ian LIPKIN, Brent L. WILLIAMS.
Application Number | 20150329909 14/691498 |
Document ID | / |
Family ID | 46637038 |
Filed Date | 2015-11-19 |
United States Patent
Application |
20150329909 |
Kind Code |
A1 |
LIPKIN; W. Ian ; et
al. |
November 19, 2015 |
AUTISM-ASSOCIATED BIOMARKERS AND USES THEREOF
Abstract
The invention discloses biomarkers for human autism. The
invention provides methods for treating, preventing, and diagnosing
human autism and autism-related disorders.
Inventors: |
LIPKIN; W. Ian; (New York,
NY) ; HORNIG; Mady; (New York, NY) ; WILLIAMS;
Brent L.; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW
YORK |
New York |
NY |
US |
|
|
Family ID: |
46637038 |
Appl. No.: |
14/691498 |
Filed: |
April 20, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13328982 |
Dec 16, 2011 |
9050276 |
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14691498 |
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PCT/US2010/034254 |
May 10, 2010 |
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13328982 |
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61527313 |
Aug 25, 2011 |
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61187606 |
Jun 16, 2009 |
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Current U.S.
Class: |
506/4 ; 435/6.11;
435/6.12; 435/6.15; 435/7.4; 435/7.92; 506/9 |
Current CPC
Class: |
C12Q 1/689 20130101;
G01N 2800/28 20130101; C12Q 2600/158 20130101; C12Q 1/6883
20130101; A61P 29/00 20180101; C12Q 2600/106 20130101; A61K 38/47
20130101; A61K 2300/00 20130101; G01N 2333/62 20130101; G01N
2333/924 20130101; A61K 38/1709 20130101; C12Q 2600/112 20130101;
A61K 2300/00 20130101; G01N 33/56911 20130101; A61K 38/47 20130101;
A61K 38/1709 20130101; A61P 1/00 20180101; C12Q 2600/156 20130101;
A61K 31/00 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/569 20060101 G01N033/569 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The work described herein was supported in whole, or in
part, by National Institute of Health Grant No. U01 NS047537 and
Grant No. AI57158. Thus, the United States Government has certain
rights to the invention.
Claims
1. A method for detecting a predisposition to autism or an autism
spectrum disorder (ASD) in a human subject, the method comprising:
(a) obtaining a biological sample from a human subject; and (b)
detecting whether or not there is an alteration in the expression
of a carbohydrate metabolic enzyme protein in the subject as
compared to a non-autistic subject.
2. (canceled)
3. The method of claim 1, wherein the subject is a child of a human
subject.
4. The method of claim 1, wherein the carbohydrate metabolic enzyme
comprises sucrase isomaltase, maltase glucoamylase, lactase, or a
combination thereof.
5. (canceled)
6. The method of claim 1 further comprising detecting a decrease in
Bacteriodetes, an increase in the Firmicute/Bacteroidete ratios, an
increase in cumulative levels of Firmicutes and Proteobacteria, an
increase in Beta-proteobacteria, or an increase in Sutterella sp.
in the small intestine or large intestine of the subject.
7. The method of claim 1 further comprising detecting an increase
in Sutterella sp. in the small intestine or large intestine of the
subject.
8. The method of claim 1, wherein the detecting comprises detecting
whether there is an alteration in a gene locus that encodes the
carbohydrate metabolic enzyme.
9. (canceled)
10. The method of claim 1, wherein the detecting comprises
detecting whether mRNA expression of the protein is reduced.
11. The method of claim 1, wherein the subject is a human embryo, a
human fetus, or an unborn human child.
12. The method of claim 1, wherein the sample comprises blood,
serum, sputum, lacrimal secretions, semen, vaginal secretions,
fetal tissue, small intestine tissue, large intestine tissue, liver
tissue, amniotic fluid, or a combination thereof.
13-48. (canceled)
Description
[0001] This application is a continuation-in-part of International
Application Number PCT/US2010/034254, filed on May 10, 2010, which
claims priority to Provisional Application 61/187,606, filed on
Jun. 16, 2009, the contents of each which are hereby incorporated
by reference in their entireties. This application also claims
priority to Provisional Application No. 61/527,313 filed on Aug.
25, 2011, the contents of which are hereby incorporated by
reference in its entirety.
[0003] This patent disclosure contains material that is subject to
copyright protection. The copyright owner has no objection to the
facsimile reproduction by anyone of the patent document or the
patent disclosure as it appears in the U.S. Patent and Trademark
Office patent file or records, but otherwise reserves any and all
copyright rights.
[0004] This patent disclosure contains material that is subject to
copyright protection. The copyright owner has no objection to the
facsimile reproduction by anyone of the patent document or the
patent disclosure as it appears in the U.S. Patent and Trademark
Office patent file or records, but otherwise reserves any and all
copyright rights.
BACKGROUND OF THE INVENTION
[0005] Autistic disorder is one of five pervasive developmental
disorders defined in the Diagnostic and Statistical Manual of
Mental Disorders, Fourth Edition, Text Revision DSM-IV-TR (2000).
Autistic disorder is a developmental disorder of the human brain
that manifests during infancy or childhood and is characterized by
behavioral and social abnormalities that appear to be
developmentally based (for example, impairments in social
interaction and communication). In addition, autism interferes with
imagination and the ability to reason. Autism is frequently
associated with other disorders such as attention
deficit/hyperactivity disorder (AD/HD) and can be associated with
psychiatric symptoms such as anxiety and depression. In the last
decade, autism diagnoses have increased by 300% to 500% in the
United States and many other countries. A means of prevention and
treatment is needed for this health crisis that addresses the
underlying mechanisms leading to the development of autism versus
those that merely address the symptoms.
[0006] Pervasive developmental disorders (PDDs) are also part of
the Autism Spectrum Disorders (ASDs). PDD is used to categorize
children who do not meet the strict criteria for Autistic Disorder
but who come close, either by manifesting atypical autism or by
nearly meeting the diagnostic criteria in two or three of the key
areas. Some of these children meet criteria for the ASD known as
Asperger's Disorder (ASP), wherein language capacities are
relatively spared compared to children with Autistic Disorder.
Others meet criteria for the PDDs known as Childhood Disintegrative
Disorder, which begins at a slightly later age than the other ASDs,
or Rett's Disorder, which is related to a mutation in a DNA
methylation binding protein gene called MeCP2 and usually occurs in
girls.
[0007] Many children with autism have gastrointestinal (GI)
disturbances that affect their quality of life. Although some of
these children have been investigated through GI immunopathology,
molecular studies are lacking that characterize host gene
expression or survey microflora using pyrosequencing methods.
SUMMARY OF THE INVENTION
[0008] The invention is based, at least in part, on the finding
that decreased levels in sucrase isomaltase, maltase glucoamylase,
lactase, GLUT2, and SGLT1 can serve as markers for human Autism
Spectrum Disorders. Accordingly, in one aspect, the invention
provides a method for detecting the presence of or a predisposition
to autism or an autism spectrum disorder (ASD) in a human subject
or a child of a human subject. The method comprises: (1) obtaining
a biological sample from a human subject; and (2) detecting whether
or not there is an alteration in the expression of a carbohydrate
metabolic enzyme protein or a carbohydrate transporter protein in
the subject as compared to a non-autistic subject. In one
embodiment, the carbohydrate metabolic enzyme comprises sucrase
isomaltase, maltase glucoamylase, lactase, or a combination
thereof. In another embodiment, the carbohydrate transporter
comprises GLUT2, SGLT1, or a combination thereof. In some
embodiments, the method further comprises detecting a decrease in
Bacteriodetes, an increase in the Firmicute/Bacteroidete ratios, an
increase in cumulative levels of Firmicutes and Proteobacteria, an
increase in Beta-proteobacteria, and an increase in Sutterella sp.
in the small or large intestine of the subject. In one embodiment,
the detecting comprises detecting whether there is an alteration in
the gene locus that encodes the carbohydrate metabolic enzyme
protein or the carbohydrate transporter protein. In a further
embodiment, the detecting comprises detecting whether expression of
the carbohydrate metabolic enzyme protein or the carbohydrate
transporter protein is reduced. In some embodiments, the detecting
comprises detecting in the sample whether there is a reduction in
the mRNA expression of the carbohydrate metabolic enzyme protein or
the carbohydrate transporter protein. In some embodiments of the
invention, the subject is a human embryo, a human fetus, or an
unborn human child. In other embodiments, the sample comprises
blood, serum, sputum, lacrimal secretions, semen, vaginal
secretions, fetal tissue, skin tissue, small intestine tissue
(e.g., the ileum), large intestine tissue (e.g., the cecum), muscle
tissue, amniotic fluid, or a combination thereof.
[0009] An aspect of the invention provides a method for treating or
preventing autism or an autism spectrum disorder in a subject in
need thereof. The method comprises administering to the subject a
therapeutic amount of a pharmaceutical composition comprising a
functional carbohydrate metabolic enzyme molecule or a carbohydrate
transporter molecule, thereby treating or preventing autism or an
autism spectrum disorder. In a further embodiment, the
administering comprises a subcutaneous, intra-muscular,
intra-peritoneal, or intravenous injection; an infusion; oral,
nasal, or topical delivery; or a combination of the delivery modes
described. In some embodiments, the administering comprises
delivery of a carbohydrate metabolic enzyme molecule or a
carbohydrate transporter molecule to the alimentary canal or
intestine of the subject. In other embodiments, the administering
comprises feeding the human subject or child thereof a
therapeutically effective amount of the carbohydrate metabolic
enzyme molecule or a carbohydrate transporter molecule. In further
embodiments, the administering occurs daily, weekly, twice weekly,
monthly, twice monthly, or yearly.
[0010] In other aspects, the invention provides for a
pharmaceutical composition comprising: a carbohydrate metabolic
enzyme molecule or a carbohydrate transporter molecule; and a
pharmaceutically acceptable carrier.
[0011] An aspect of the invention provides for an isolated nucleic
acid composition. In one embodiment, the composition comprises a
nucleic acid molecule having at least about 80% identity to SEQ ID
NO: 11, 12, 13, or 14. In one embodiment, the composition comprises
a nucleic acid molecule having at least about 85% identity to SEQ
ID NO: 11, 12, 13, or 14. In one embodiment, the composition
comprises a nucleic acid molecule having at least about 90%
identity to SEQ ID NO: 11, 12, 13, or 14. In one embodiment, the
composition comprises a nucleic acid molecule having at least about
95% identity to SEQ ID NO: 11, 12, 13, or 14. In one embodiment,
the composition comprises a nucleic acid molecule having at least
about 98% identity to SEQ ID NO: 11, 12, 13, or 14. In one
embodiment, the composition comprises a nucleic acid molecule
having at least about 99% identity to SEQ ID NO: 11, 12, 13, or 14.
In one embodiment, the composition is SEQ ID NO: 11, 12, 13, or
14.
[0012] An aspect of the invention provides for a diagnostic kit for
detecting the presence of Sutterella sp. in a sample. In one
embodiment, the kit comprises a nucleic acid molecule that
specifically hybridizes to or a primer combination that amplifies a
Sutterella sp. 16S nucleic acid sequence. In one embodiment, the
nucleic acid molecule comprises a nucleic acid primer or nucleic
acid probe. In another embodiment, the 16S nucleic acid sequence
comprises at least about 80% of SEQ ID NO: 59 or SEQ ID NO: 60. In
some embodiments, the 16S nucleic acid sequence comprises at least
about 85% of SEQ ID NO: 59 or SEQ ID NO: 60. In further
embodiments, the 16S nucleic acid sequence comprises at least about
90% of SEQ ID NO: 59 or SEQ ID NO: 60. In other embodiments, the
16S nucleic acid sequence comprises at least about 95% of SEQ ID
NO: 59 or SEQ ID NO: 60. In another embodiment, the 16S nucleic
acid sequence comprises at least about 98% of SEQ ID NO: 59 or SEQ
ID NO: 60. In some embodiments, the 16S nucleic acid sequence
comprises at least about 99% of SEQ ID NO: 59 or SEQ ID NO: 60. In
further embodiments, the 16S nucleic acid sequence is SEQ ID NO: 59
or SEQ ID NO: 60. In one embodiment, the probe comprises a
nucleotide sequence having SEQ ID NOS: 13 or 14 in Table 1, or the
italicized nucleotide of sequence SEQ ID NO: 19. In a further
embodiment, the probe comprises at least 10 consecutive nucleotide
bases comprising SEQ ID NO: 19, wherein S is a G nucleotide and/or
a C nucleotide, wherein Y is a C nucleotide and/or T nucleotide,
wherein R is an A nucleotide and/or G nucleotide, wherein W is an A
nucleotide and/or T nucleotide, and wherein H is an A nucleotide
and/or T nucleotide and/or C nucleotide. In some embodiments, the
probe comprises a reverse complement of SEQ ID NOS: 11, 12, 15, 16,
17, 18, or 19, wherein S is a G nucleotide and/or a C nucleotide,
wherein Y is a C nucleotide and/or T nucleotide, wherein R is an A
nucleotide and/or G nucleotide, wherein W is an A nucleotide and/or
T nucleotide, and wherein H is an A nucleotide and/or T nucleotide
and/or C nucleotide. In other embodiments, the primer comprises a
nucleotide sequence selected from the group consisting of SEQ ID
NOS: 11, 12, 15, 16, 17, or 18, wherein, wherein S is a G
nucleotide and/or a C nucleotide, wherein Y is a C nucleotide
and/or T nucleotide, wherein R is an A nucleotide and/or G
nucleotide, wherein W is an A nucleotide and/or T nucleotide, and
wherein H is an A nucleotide and/or T nucleotide and/or C
nucleotide. In one embodiment, the sample is from a human or
non-human animal. In other embodiments, the sample comprises
intestinal tissue (e.g., the small intestine or large intestine),
feces, blood, skin, or a combination of the mentioned tissues.
[0013] An aspect of the invention provides for a diagnostic kit for
determining whether a sample from a subject exhibits a presence of
or a predisposition to autism or an autism spectrum disorder (ASD).
In one embodiment, the kit comprising a nucleic acid primer that
specifically hybridizes to an autism biomarker, wherein the primer
will prime a polymerase reaction only when an autism biomarker is
present. In another embodiment, the primer comprises a nucleotide
sequence selected from the group consisting of SEQ ID NOS: 11, 12,
15, 16, 17, or 18, wherein, wherein S is a G nucleotide and/or a C
nucleotide, wherein Y is a C nucleotide and/or T nucleotide,
wherein R is an A nucleotide and/or G nucleotide, wherein W is an A
nucleotide and/or T nucleotide, and wherein H is an A nucleotide
and/or T nucleotide and/or C nucleotide. In some embodiments, the
autism biomarker is a carbohydrate trasporter molecule, a
carbohydrate metabolic enzyme molecule, or a gastrointestinal
Sutterella sp. bacterium. In a further embodiment, the carbohydrate
trasporter molecule is GLUT2 or SGLT1. In other embodiments, the
carbohydrate metabolic enzyme molecule is SI, MGAM, or LCT. In one
embodiment, the sample is from a human or non-human animal. In
other embodiments, the sample comprises intestinal tissue (e.g.,
the small intestine or large intestine), feces, blood, skin, or a
combination of the mentioned tissues.
[0014] An aspect of the invention provides for a method of treating
or preventing a disease associated with elevated levels of
Beta-proteobacteria. The method of the invention comprises
administering to a subject in need thereof a therapeutic amount of
an antimicrobial composition effective against Beta-proteobacteria
for treating the disease. In one embodiment, the antimicrobial
composition is an antibiotic, a probiotic agent, or a combination
thereof. In another embodiment, the disease is ASD, autism, or a
gastrointestinal disease. In a further embodiment, the
gastrointestinal disease is diarrhea, inflammatory bowel disease,
antimicrobial-associated colitis, or irritable bowel syndrome. In
some embodiments, the diarrhea or inflammatory bowel diseases is
ulcerative colitis or Crohn's disease. In one embodiment, the
antibiotic comprises lincosamides, chloramphenicols, tetracyclines,
aminoglycosides, beta-lactams, vancomycins, bacitracins,
macrolides, amphotericins, sulfonamides, methenamin,
nitrofurantoin, phenazopyridine, trimethoprim; rifampicins,
metronidazoles, cefazolins, lincomycin, spectinomycin, mupirocins,
quinolones, novobiocins, polymixins, gramicidins, antipseudomonals,
or a combination of the stated antibiotics. In another embodiment
of the invention, the probiotic agent comprises Bacteroides,
Prevotella, Porphyromonas, Fusobacterium, Sutterella, Bilophila,
Campylobacter, Wolinella, Butyrovibrio, Megamonas, Desulfomonas,
Desulfovibrio, Bifidobacterium, Lactobacillus, Eubacterium,
Actinomyces, Eggerthella, Coriobacterium, Propionibacterium, other
genera of non-sporeforming anaerobic gram-positive bacilli,
Bacillus, Peptostreptococcus, newly created genera originally
classified as Peptostreptococcus, Peptococcus, Acidaminococcus,
Ruminococcus, Megasphaera, Gaffkya, Coprococcus, Veillonella,
Sarcina, Clostridium, Aerococcus, Streptococcus, Enterococcus,
Pediococcus, Micrococcus, Staphylococcus, Corynebacterium, species
of the genera comprising the Enterobacteriaceae and
Pseudomonadaceae, or a combination of the listed probiotic
agents.
[0015] An aspect of the invention provides for a method of
detecting a Sutterella sp. in a sample. The method comprises: (a)
selecting a Sutterlla sp.-specific primer pair, wherein the primer
pair mediates amplification of a polynucleotide amplicon of a
selected, known length from a nucleic acid of a Sutterlla sp.;
contacting a nucleic acid from the sample with the Sutterlla
sp.-specific primer pair in a reaction mixture under conditions
that promote amplification of a polynucleotide amplicon, wherein
the primer pair will prime a polymerase reaction only when the
nucleic acid of a Sutterlla sp. is present; and detecting the
amplicons, wherein the detection of an amplicon of a selected,
known length is indicative of the sample containing the nucleic
acid of a Sutterlla sp. In one embodiment, the sample comprises
intestinal tissue (e.g., the small intestine or large intestine),
feces, blood, skin, or a combination of the listed tissues. In one
embodiment, the primer pair comprises a forward primer and a
reverse primer. In some embodiments, the forward primer comprises
SEQ ID NO: 11 or 17, wherein S is a G nucleotide and/or a C
nucleotide, wherein Y is a C nucleotide and/or T nucleotide,
wherein R is an A nucleotide and/or G nucleotide, wherein W is an A
nucleotide and/or T nucleotide, and wherein H is an A nucleotide
and/or T nucleotide and/or C nucleotide. In other embodiments, the
reverse primer comprises SEQ ID NO: 12 or 18, wherein S is a G
nucleotide and/or a C nucleotide, wherein Y is a C nucleotide
and/or T nucleotide, wherein R is an A nucleotide and/or G
nucleotide, wherein W is an A nucleotide and/or T nucleotide, and
wherein H is an A nucleotide and/or T nucleotide and/or C
nucleotide. In further embodiments, the forward primer comprises at
least 10 consecutive nucleotide bases comprising SEQ ID NO: 17 or
19, wherein S is a G nucleotide and/or a C nucleotide, wherein Y is
a C nucleotide and/or T nucleotide, wherein R is an A nucleotide
and/or G nucleotide, wherein W is an A nucleotide and/or T
nucleotide, and wherein H is an A nucleotide and/or T nucleotide
and/or C. In some embodiments, the reverse primer comprises at
least 10 consecutive nucleotide bases comprising SEQ ID NO: 18 or
19, wherein S is a G nucleotide and/or a C nucleotide, wherein Y is
a C nucleotide and/or T nucleotide, wherein R is an A nucleotide
and/or G nucleotide, wherein W is an A nucleotide and/or T
nucleotide, and wherein H is an A nucleotide and/or T nucleotide
and/or C nucleotide, wherein B is a T nucleotide, C nucleotide, or
G nucleotide, wherein V is an A nucleotide, G nucleotide, or C
nucleotide; wherein D is an A nucleotide, G nucleotide, or T
nucleotide; and wherein K is a G nucleotide or T nucleotide.
BRIEF DESCRIPTION OF THE FIGURES
[0016] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0017] FIG. 1 is a schematic depicting carbohydrate metabolizing
enzymes (e.g., sucrase isomaltase, maltase glucoamylase, and
lactase) and carbohydrate transporter proteins (e.g., GLUT2 and
SGLT1) involved in carbohydrate metabolism, uptake, and absorption
in the enterocytes of the ileum.
[0018] FIG. 2 shows bar graphs depicting that carbohydrate
metabolizing enzyme mRNAs are reduced in the ileum of ASD subjects.
Graphs are shown for sucrase isomaltase (left), maltase
glucoamylase (center), and lactase (right).
[0019] FIG. 3 shows bar graphs depicting that carbohydrate
transporter mRNAs are reduced in the ileum of ASD subjects. Graphs
are shown for SGLT1 (Top) and GLUT2 (Bottom).
[0020] FIG. 4 shows graphs depicting that mRNA for ileal
inflammatory markers are increased in the ileum of ASD subjects.
Graphs are shown for C1QA (Top Left), Resistin (Top Right), and
IL17F (Bottom Left and Right).
[0021] FIG. 5 shows bar graphs depicting the differences in
bacteria phylum found in the ileum of ASD subjects. Changes at the
phylum level were observed. Bar graphs show a decrease in
Bacteroidetes (left) and increase in Firmicute/Bacteroidete ratios
in ileum of AUT-GI children.
[0022] FIG. 6 is a bar graph depicting the copy number of
bacteroidetes found in the ileum of ASD subjects. Real-time PCR
confirmed a decrease in Bacteroidete. Bacteroidete 16S rDNA copies
(Normalized to Total Bacterial 16S rDNA).
[0023] FIG. 7 is a schematic summarizing the interplay between
expression levels of carbohydrate metabolic enzymes (e.g., sucrase
isomaltase, maltase glucoamylase, and lactase), carbohydrate
transporters (e.g., GLUT2 and SGLT1) and the population of bacteria
in the ileum of ASD subjects.
[0024] FIGS. 8A-B show the Presence of Sutterella sequences in a
subset of AUT-GI patients: Detection by pyrosequencing of the
V2-region of the 16S rRNA gene. FIGS. 8A-B are bar graphs showing
the abundance of Sutterella sp. in the ileum (FIG. 8A) and cecum
(FIG. 8B) of autism and control patients. Distribution of
Sutterella sequences as a percentage of total bacterial 16S rRNA
gene reads from ileal (FIG. 8A; Mann-Whitney, tied p=0.022) and
cecal (FIG. 8B; Mann-Whitney, tied p=0.037) biopsies from AUT-GI
and Control-GI patients.
[0025] FIGS. 8C-D show the presence of Sutterella sequences in a
subset of AUT-GI patients: Detection by pyrosequencing of the
V2-region of the 16S rRNA gene. FIGS. 8C-D are bar graphs showing
the abundance of Sutterella sp. sequences in the ileum (FIG. 8C)
and cecum (FIG. 8D) of autism and control patients. FIGS. 8C-D
shows the distribution of Sutterella sequences by individual
patient as a percentage of total bacteria 16S rRNA reads from ileal
(FIG. 8C) and cecal (FIG. 8D) biopsies from AUT-GI (patients #1-15)
and Control-GI (patients #16-22) patients. *, p<0.05.
[0026] FIGS. 8E-F show the presence of Sutterella sequences in a
subset of AUT-GI patients: Detection by pyrosequencing of the
V2-region of the 16S rRNA gene. FIGS. 8E-F are bar graphs showing
the abundance of Sutterella sp. sequences comprising the
Beta-proteobacteria sequences in the ileum (FIG. 8E) and cecum
(FIG. 8F) of autism and control patients. FIGS. 8E-F show the
distribution of Sutterella sequences by individual patient as a
percentage of total Betaproteobacteria 16S rRNA reads from ileal
(FIG. 8E) and cecal (FIG. 8F) biopsies from AUT-GI (patients #1-15)
and Control-GI (patients #16-22) patients. *, p<0.05.
[0027] FIG. 9 is a photograph of an agarose gel showing the results
of classical PCR experiments for the detection of Sutterella.
Sutterella-specific 16S rRNA gene (V6-V8) PCR amplification of
10-fold dilutions of Sutterella plasmid DNA standards spiked into
ileal DNA from a Sutterella-negative Control-GI patient. Note
linear amplification down to 5.times.10.sup.2 copies and an
endpoint detection limit of 5.times.10' copies
[0028] FIG. 10A is an amplification plot of Sutterella sp. through
cycles of Real-time PCR experiments. The figure depicts Real-time
PCR amplification plot of 10-fold serial dilutions of Sutterella
plasmid DNA standards.
[0029] FIG. 10B is a standard curve graph showing the copy number
of Sutterella sp. from Real-time PCR experiments.
[0030] FIG. 11 is a photograph of an agarose gel showing the
results of Sutterella detection in the ileum and cecum of patients
using the V6-V8 Sutterella sp.-specific PCR.
[0031] FIGS. 12A-B are bar graphs showing the copy number of
Sutterella sp. in the ileum (FIG. 12A) and cecum (FIG. 12B) of
autism and control patients using the V6-V8 Sutterella sp.-specific
PCR.
[0032] FIGS. 12C-D are bar graphs showing the copy number of
Sutterella sp. in the ileum (FIG. 12C) and cecum (FIG. 12D) of
autism and control patients using the V6-V8 Sutterella sp.-specific
PCR.
[0033] FIG. 13 is a sequence alignment for the V6-V8 region of
Sutterella sp. obtained from biological samples of Autism patients
1, 3, 10, 11, and 12 (SEQ ID NO: 59), and Autism patients 5 and 7
(SEQ ID NO: 60).
[0034] FIG. 14 depicts Sutterella sp. sequence clustering from the
Operational Taxonomic Unit (OTU) analysis of V2 pyrosequencing
reads.
[0035] FIG. 15A is a schematic depicting Sutterella sp. treeing
analysis of the V6-V8 sequences.
[0036] FIG. 15B is a schematic depicting Sutterella sp. treeing
analysis of the V2 sequence.
[0037] FIGS. 16A-G show graphs of quantitative real-time PCR
analysis of disaccharidases, hexose transporters, villin and CDX2
transcripts. Box-and-whisker plots displaying (FIG. 16A) SI
(Mann-Whitney; p=0.001), (FIG. 16B) MGAM (Mann-Whitney; p=0.003),
(FIG. 16C) LCT (Mann-Whitney; p=0.032), (FIG. 16D) SGLT1
(Mann-Whitney; p=0.008), (FIG. 16E) GLUT2 (Mann-Whitney; p=0.010),
(FIG. 16F) Villin (Mann-Whitney; p=0.307), and (FIG. 16G) CDX2
(Mann-Whitney; p=0.192) mRNA expression normalized to GAPDH mRNA in
ileal biopsies from AUT-GI (AUT) and Control-GI (Control) patients.
Box-and-whisker plots show the median and the interquartile
(midspread) range (boxes containing 50% of all values), the
whiskers (representing the 25.sup.th and 75.sup.th percentiles) and
the extreme data points (open circles). *p<0.05; **, p<0.01;
n.s., not significant.
[0038] FIGS. 17A-H show graphs depicting pyrosequencing analysis of
intestinal microbiota in AUT-GI children. (FIGS. 17A-B)
Phylum-level comparison of the average relative abundance of
bacterial taxa in ileal (FIG. 17A) and cecal (FIG. 17B) biopsies
from AUT-GI and Control-GI patients. (FIGS. 17C-D) Box-and-whisker
plot displaying the distribution of Bacteroidetes as a percentage
of total bacterial 16S rRNA V2 pyroseqeuncing reads from ileal (C;
Mann-Whitney, p=0.012) and cecal (FIG. 17D; Mann-Whitney, p=0.008)
biopsies from AUT-GI and Control-GI patients. (FIGS. 17E-F)
Bacteroidete-specific quantitative real-time PCR analysis of ileal
(FIG. 17E; Mann-Whitney, p=0.003) and cecal (FIG. 17F;
Mann-Whitney, p=0.022) biopsies from AUT-GI and Control-GI
patients. (FIGS. 17G-H) Heatmaps displaying abundance distributions
(% of total sequence reads per patient) of Bacteroidetes classified
at the family level in ileal (FIG. 17G) and cecal (FIG. 17H)
biopsies from AUT-GI and Control-GI children (Bottom row displays
cumulative levels of all family members by patient). copy number
values are normalized relative to total bacteria copy numbers; *,
p<0.05, **p<0.01.
[0039] FIGS. 18A-J show graphs of Firmicute abundance in AUT-GI and
Control-GI children. (FIGS. 18A-18B) Box-and-whisker plots
displaying the Firmicute/Bacteroidete ratio from pyrosequencing
reads obtained from ileal (FIG. 18A; Mann-Whitney, p=0.026) and
cecal (FIG. 18B; Mann-Whitney, p=0.032) biopsies of AUT-GI and
Control-GI patients. (FIGS. 18C-18D) Box-and-whisker plots
displaying the cumulative levels of members of the families
Lachnospiraceae and Ruminococcaceae in ileal (FIG. 18C;
Mann-Whitney; p=0.062) and cecal (FIG. 18D; Mann-Whitney; p=0.098)
biopsies from AUT-GI and Control-GI children. (FIGS. 18E-18F)
Heatmaps displaying abundance distribution (% of total sequence
reads per patient) of family members in the class Clostridia in
ileum (FIG. 18E) and cecum (FIG. 18F) of AUT-GI and Control-Gi
children (Bottom row displays cumulative levels of all family
members by patient). (FIGS. 18G-18H) Box-and-whisker plots
displaying the cumulative abundance of Firmicutes and
Proteobacteria from ileal (FIG. 18G; Mann-Whitney, p=0.015) and
cecal (FIG. 18H; Mann-Whitney, p=0.007) biopsies from AUT-GI and
Control-GI patients. (FIGS. 18I-18J) Heatmaps displaying the
abundance distribution (% of total sequence reads per patient) of
Firmicutes and Proteobacteria by patient in ilea (FIG. 18I) and
ceca (FIG. 18J) of AUT-GI and Control-GI children (Bottom row
displays cumulative levels of Firmicutes and Proteobacteria by
patient). *, p<0.05, **, p<0.01, t, p<0.1 (trend).
[0040] FIGS. 19A-F graphs of the abundance of Proteobacteria in
AUT-GI and Control-GI children. (FIGS. 19A-19B) Box-and-whisker
plots displaying the phyla level abundance of Proteobacteria
members in ilea (FIG. 19A; Mann-Whitney, p=0.549) and ceca (FIG.
19B; Mann-Whitney, p=0.072) of AUT-GI and Control-GI children
biopsies obtained by pyrosequencing. (FIGS. 19C-19D)
Box-and-whisker plots displaying the class level abundance of
Betaproteobacteria members in ilea (FIG. 19C; Mann-Whitney,
p=0.072) and ceca (FIG. 19D; p=0.038) of AUT-GI and Control-GI
children. (FIGS. 19E-19F) Heatmaps displaying the abundance
distribution (% of total sequence reads per patient) of family
members within the classes Alpha-, Beta-, and Gammaproteobacteria
in the ilea (FIG. 19E) and ceca (FIG. 19F) of AUT-GI and Control-GI
children (Bottom row of each heatmap displays the cumulative levels
of family members in each class by patient). *, p<0.05,
.dagger., p<0.1 (trend); n.s., not significant.
[0041] FIGS. 20A-C show schematics depicting factors that mediate
GI disease in AUT-GI children. (FIG. 20A) Schematic representation
of enterocyte-mediated digestion of disaccharides and
absorption/transport of monosaccharides in the small intestine.
Disaccharidase enzymes (SI, MGAM, and LCT) in the enterocyte brush
border break down disaccharides into their component
monosaccharides. The monosaccharides, glucose and galactose, are
transported from the small intestinal lumen into the enterocyte by
the sodium-dependent transporter SGLT1. On the basolateral
enterocyte membrane, the facilitative transporter, GLUT2,
transports glucose, galactose and fructose out of the enterocyte
and into the circulation, thus regulating postprandial blood
glucose levels. GLUT2 can also be transiently inserted into the
apical enterocyte membrane, contributing a diffusive component to
monosaccharide absorption in certain circumstances (Kellet et al.,
2008). The expression levels of disaccharidases and hexose
transporters can be controlled by the transcription factor CDX2.
(FIG. 20B) In the normal small intestine, where expression of
disaccharidases and hexose transporters are high, the majority, if
not all, of disaccharides are efficiently digested and
monosaccharides are absorbed from the lumen. Thus, only complex
polysaccharides reach the large intestine and serve as growth
substrates for colonic bacteria. Those bacteria best suited for
growth on polysaccharides (i.e., Bacteroidetes) outcompete other
bacteria and dominate the colonic space. In the normal intestine,
colonic (i.e., cecal) microbial community structure can be kept
within a normal homeostatic range by the level of expression of
disaccharidases and hexose transporters upstream in the small
intestine. The constraint on bacterial structure regulated by ileal
gene expression would constrain bacterial byproducts of
fermentation such as SCFAs, and limit the growth of potential
pathogens. (FIG. 20C) In the AUT-GI intestine, where expression of
disaccharidases and hexose transporters are deficient, mono- and
disaccharides accumulate in the lumen of the distal small intestine
(ileum) and proximal colon (cecum), and can exert extraintestinal
effects by reducing postprandial blood glucose. The presence of
additional carbohydrate substrates in the lumen abrogates the
growth advantage of bacteria best suited for growth on
polysaccharides (i.e., Bacteroidetes) and promotes the growth of
other bacteria. In ASD-GI this specifically manifests as an
increase in Firmicute/Bacteroidete ratios, cumulative levels of
Firmicutes and Proteobacteria, and in levels of Betaproteobacteria
in both the ileum and cecum. The level of dysbiosis in the ileum
and cecum can thus be controlled by the degree and type of
deficiency of carbohydrate metabolism and transport in the small
intestine. Within the intestine, malabsorbed monosaccharides can
lead to osmotic diarrhea; non-absorbed sugars can also serve as
substrates for intestinal microflora, that produce fatty acids and
gases (methane, hydrogen, and carbon dioxide) and promote
additional GI symptoms of bloating and flatulence. Additional
effects of dysbiosis can manifest in changes in SCFAs that can
reduce colonic pH, further inhibiting the growth of Bacteroidetes.
Disruption of symbiotic relationships between the host and the
intestinal microbial ecosystem as a result of dysbiosis can also
play a fundamental role in development, distribution, activation
and differentiation of immune cells within the intestine (Abt and
Artis, 2009; Mazmanian et al., 2008), thus providing a framework
for understanding previous reports of inflammatory indices in the
AUT-GI intestine.
[0042] FIGS. 21A-E depict lactase genotyping. (FIG. 21A)
Representative agarose gel banding patterns observed for LCT-13910
and LCT-22018 polymorphisms. (FIG. 21B) Distribution of genotypes
for 13910 and 22018 polymorphisms between AUT-GI (n=15) and
Control-GI (n=7) patients (chi-squared test, p=0.896). (FIG. 21C)
Box-and-whisker plot displaying the distribution of LCT mRNA
expression in all individuals (AUT-GI and Control-GI) with the
homozygous adult-type hypolactasia genotype (13910-C/C; 22018-G/G)
compared to all individuals (AUT-GI and Control-GI) possessing at
least one copy of the normal allele (heterozygous: 13910-C/T;
22018-G/A and homozygous: 13910-T/T; 22018-A/A); Mann-Whitney,
p=0.033. (FIG. 21D) Distribution of LCT mRNA expression levels
split by genotype and group (AUT-GI and Control-GI);
Kruskal-Wallis, p=0.097. (FIG. 21E) Distribution of LCT mRNA
expression for all patients possessing at least one copy of the
normal (lactase persistence) allele for AUT-GI (n=12) and
Control-GI (n=6); Mann-Whitney, p=0.0246. Adult-type hypolactasia
genotype is highlighted in red. *, p<0.05.
[0043] FIGS. 22A-E show graphs depicting Villin normalization and
CDX2 expression stratified by total disaccharidase and transporter
deficiencies. Disaccharidase or transporter mRNA/villin mRNA ratio
for SI (FIG. 22A; Mann-Whitney, p=0.001), MGAM (FIG. 22B;
Mann-Whitney, p=0.001), LCT (FIG. 22C; Mann-Whitney, p=0.005),
SGLT1 (FIG. 22D; Mann-Whitney, p=0.0008), and GLUT2 (FIG. 22E;
Mann-Whitney, p=0.002). *, p<0.05, **, p <0.01,***,
p<0.001; .dagger., p<0.1 (trend).
[0044] FIGS. 23A-D show graphs of the diversity of AUT-GI and
Control-GI phylotypes. (FIGS. 23A-23B) Rarefaction curves assessing
the completeness of sampling from pyrosequencing data obtained for
individual AUT-GI (red) and Control-GI (blue) subjects' ileal (FIG.
23A) and cecal (FIG. 23B) biopsies. The y-axis indicates the number
of OTUs detected (defined at 97% threshold for sequence
similarity), the x-axis indicates the number of sequences sampled.
(FIGS. 23C-23D) Rarefaction curves to estimate phylotype diversity,
using the Shannon Diversity Index, from pyrosequencing data
obtained for individual AUT-GI (red) and Control-GI (blue)
subjects' ileal (FIG. 23C) and cecal (FIG. 23D) biopsies.
[0045] FIGS. 24A-D show graphs depicting the distribution of
pyrosequencing reads by patient. (FIGS. 24A-24B) Phylum level
distribution of bacteria by patient obtained from 16S rRNA gene
barcoded pyrosequencing for ilea (FIG. 24A) and ceca (FIG. 24B).
(FIGS. 24C-D) Distribution of low abundance bacterial phyla
obtained by barcoded pyroseqeuncing. By-patient distribution of low
abundance bacterial phyla in ilea (FIG. 24C) and ceca (FIG. 24D)
from AUT-GI (patients 1-15) and Control-GI (patients 16-22).
[0046] FIGS. 25A-E show the OTU analysis of Bacteroidete
phylotypes. (FIGS. 25A-25B) Heatmaps displaying abundance
distributions (% of total sequence reads per patient) of the 12
most abundant Bacteroidete OTUs (defined at 97% threshold) in ileal
(FIG. 25A) and cecal (FIG. 25B) biopsies from AUT-GI and Control-GI
children (Bottom row displays cumulative levels of all 12 OTUs by
patient). (FIGS. 25C-25D) Box-and-whisker plots displaying the
cumulative abundance of the 12 OTUs in ilea (FIG. 25C;
Mann-Whitney, p=0.008) and ceca (FIG. 25D; Mann-Whitney, p=0.008)
of AUT-GI and Control-GI children. (FIG. 25E) Greengenes- or
microbial blast(*)-derived classification of representative
sequences obtained from each Bacteroidete OTU. Color code denotes
the family-level, Ribosomal Database-derived taxonomic
classification of each representative OTU sequence. **,
p<0.01
[0047] FIGS. 26A-D show graphs depicting order-level analysis of
Firmicute/Bacteroidete ratio and confirmation by real-time PCR.
(FIGS. 26A-26B) Box-and-whisker plot displaying the order-level
distribution of the Clostridiales/Bacteroidales ratio from
pyrosequencing reads obtained from ileal (FIG. 26A; Mann-Whitney,
p=0.012) and cecal (FIG. 26B; Mann-Whitney, p=0.032) biopsies from
AUT-GI and Control-GI patients. (FIGS. 26C-26D) Box-and-whisker
plot displaying the Firmicute/Bacteroidete ratios obtained by
real-time PCR for ilea (FIG. 26C; Mann-Whitney, p=0.0006) and ceca
(FIG. 26D; Mann-Whitney, p=0.022) of AUT-GI and Control-GI
children. *, p<0.05, ***, p<0.001.
[0048] FIGS. 27A-F show graphs of the abundance of Firmicutes
assayed by pyrosequencing and real-time PCR. (FIGS. 27A-27B)
Box-and-whisker plots displaying the phyla level abundance of
Firmicutes in the ilea (FIG. 27A; Mann-Whitney, p=0.098) and ceca
(FIG. 27B; Mann-Whitney, p=0.148) of AUT-GI and Control-GI children
obtained by pyrosequencing. (FIGS. 27C-27D) Box-and-whisker plots
displaying the phyla level abundance of Firmicutes in the ilea
(FIG. 27C; Mann-Whitney, p=0.245) and ceca (FIG. 27D; Mann-Whitney,
p=0.053) of AUT-GI and Control-GI children obtained by real-time
PCR. Copy number values for Firmicutes are normalized relative to
total bacteria copy numbers. (FIGS. 27E-27F) Box-and-whisker plots
displaying the abundance of Clostridiales from ileal (FIG. 27E;
Mann-Whitney, p=0.072) and cecal (FIG. 27F; Mann-Whitney, p=0.098)
biopsies from AUT-GI and Control-GI patients obtained by
pyrosequencing. *, p<0.05; .dagger., p<0.1 (trend); n.s., not
significant.
[0049] FIGS. 28A-B show genus-level distribution of members of the
families Ruminococcaceae and Lachnospiraceae. (FIGS. 28A-28B)
Heatmap representation of the individual patient distributions (by
patient) of Ruminococcaceae and Lachnospiraceae genus members in
ileal (FIG. 28A) and cecal (FIG. 28B) biopsies from AUT-GI
(Patients 1-15) and Control-GI (Patients 16-22) patients. *, genus
members contributing to the trend toward increased Firmicutes in
AUT-GI children.
[0050] FIGS. 29A-F show graphs depicting increases in inflammatory
markers, such as C1Q, Resistin, CD163, Tweak, IL17F, and nNOS.
These inflammatory markers can also serve as biomarkers for
diagnosis of human Autism Spectrum Disorders, as well as for
detecting the presence of or a predisposition to autism or an
autism spectrum disorder.
[0051] FIGS. 30A-B depict graphs of Firmicute/Bacteroidete ratios
obtained by real-time PCR for ilea (FIG. 30A; Mann-Whitney,
p=0.0006) and ceca (FIG. 30B; Mann-Whitney, p=0.022).
[0052] FIGS. 31A-D show levels of Clostridiales members in AUT-GI
patients stratified by timing of GI onset. FIGS. 31A-B show the
abundance of Clostridiales from ileal (FIG. 31A) and cecal (FIG.
31B) biopsies from AUT-GI and Control-GI patients (n=7), with
AUT-GI stratified by whether the onset of GI symptoms occurred
after (n=5) the onset of autism symptoms (GI-After) or before and
at the same time (n=10) as autism symptoms (GI-Before/Same). [FIG.
31A: AUT (GI-After) vs. AUT (GI-Before/Same), Mann-Whitney,
p=0.028; AUT (GI-Before/Same) vs. Control-GI, Mann-Whitney,
p=0.015; AUT (GI-After) vs. Control-GI, Mann-Whitney, p=0.935]
[FIG. 31B: AUT (GI-After) vs. AUT (GI-Before/Same), Mann-Whitney,
p=0.037; AUT (GI-Before/Same) vs. Control-GI, Mann-Whitney,
p=0.019; AUT (GI-After) vs. Control-GI, Mann-Whitney, p=0.935].
FIGS. 31C-D show the cumulative abundance of Lachnospiraceae and
Ruminococcaceae from ileal (FIG. 31C) and cecal (FIG. 31D) biopsies
from AUT-GI and Control-GI patients (n=7), with AUT-GI stratified
by whether the onset of GI symptoms occurred after (n=5) the onset
of autism symptoms or before and at the same time (n=10) as autism
symptoms [FIG. 31C: AUT (GI-After) vs. AUT (GI-Before/Same),
Mann-Whitney, p=0.028; AUT (GI-Before/Same) vs. Control-GI,
Mann-Whitney, p=0.015; AUT (GI-After) vs. Control-GI, Mann-Whitney,
p=0.808] [FIG. 31D: AUT (GI-After) vs. AUT (GI-Before/Same),
Mann-Whitney, p=0.020; AUT (GI-Before/Same) vs. Control-GI,
Mann-Whitney, p=0.011; AUT (GI-After) vs. Control-GI, Mann-Whitney,
p=0.685]. *, p<0.05; **, p <0.01; n.s., not significant.
[0053] FIG. 31E shows the age at GI onset (in months) for AUT-GI
and Control-GI patients, with AUT-GI stratified by whether GI onset
occurred after (n=5) the onset of autism symptoms or before and at
the same time (n=10) as autism symptoms [FIG. 31E: AUT (GI-After)
vs. AUT (GI-Before/Same), Mann-Whitney, tied p=0.007; AUT
(GI-Before/Same) vs. Control-GI, Mann-Whitney, tied p=0.757; AUT
(GI-After) vs. Control-GI, Mann-Whitney, tied p=0.027]. *,
p<0.05; **, p<0.01; n.s., not significant.
[0054] FIG. 32 is a schematic representation of enterocyte-mediated
digestion of disaccharides and transport of monosaccharides in the
small intestine. Disaccharidases (SI, MGAM, and LCT) in the
enterocyte brush border break down disaccharides into their
component monosaccharides. The monosaccharides, glucose and
galactose, are transported from the small intestinal lumen into
enterocytes by the sodium-dependent transporter SGLT1. On the
basolateral enterocyte membrane, GLUT2, transports glucose,
galactose and fructose out of enterocytes and into the circulation.
The expression levels of disaccharidases and hexose transporters
can be controlled, in part, by the transcription factor CDX2.
[0055] FIG. 33 is a bar graph showing CDX2 mRNA expression in
AUT-GI children stratified by number of total disaccharidase and
transporter deficiencies [All 5 deficient (n=10) or fewer than 5
deficient (n=5)] compared to all Control-GI children (n=7). AUT
(All 5) vs AUT (<5); Mann-Whitney, p=0.037. AUT (All 5) vs
Control; Mann-Whitney, p=0.064. *, p<0.05; **, p<0.01;***,
p<0.001; .dagger., p<0.1 (trend).
[0056] FIGS. 34A-B show the percent difference in abundance of
Bacteroidetes, Firmicutes, and Proteobacteria in individual AUT-GI
patients. (FIGS. 34A-B show bar graphs indicating the percent
difference in phylotype abundance for Bacteroidetes, Firmicutes and
Proteobacteria in AUT-GI patients (#1-15) relative to the
Control-GI mean abundance for each of the three phylotypes obtained
by pyrosequencing of ieal (FIG. 34A) and cecal (FIG. 34B)
biopsies.
[0057] FIGS. 35A-D show graphs of increased Betaproteobacteria in
AUT-GI children is associated with total deficiencies in
disaccharidases and hexose transporters and CDX2 mRNA expression.
FIGS. 35A-B show the abundance of Betaproteobacteria in AUT-GI
children with deficiency in all 5 disaccharidases and transporters
(All 5; n=10), AUT-GI children with deficiency in fewer than 5
disaccharidases and transporters (<5; n=5), and Control-GI
children (n=7) in ileum (FIG. 35A) and cecum (FIG. 35B). (FIG. 35A)
Ileum: AUT-GI (All 5) vs. AUT-GI (<5), Mann-Whitney, p=0.028;
AUT-GI (All 5) vs. Control-GI, Mann-Whitney, p=0.015; AUT-GI
(<5) vs. Control-GI, Mann-Whitney, p=0.935. (FIG. 35B) Cecum:
AUT-GI (All 5) vs. AUT-GI (<5), Mann-Whitney, p=0.014; AUT-GI
(All 5) vs. Control-GI, Mann-Whitney, p=0.006; AUT-GI (<5) vs.
Control-GI, Mann-Whitney, p=0.808. FIGS. 35C-D show Ileal CDX2 mRNA
expression in AUT-GI children with Betaproteobacteria above the
75.sup.th percentile of Control-GI children [AUT (+)
.beta.-proteo], AUT-GI children with Betaproteobacteria levels
below the 75.sup.th percentile of Control-GI children [AUT (-)
.beta.-proteo], and Control-GI children in ileum (FIG. 35C) and
cecum (FIG. 35D). (FIG. 35C) Ileum: AUT (+) .beta.-proteo. (n=8)
vs. AUT (-) .beta.-proteo. (n=7), Mann-Whitney, p=0.037; AUT (+)
.beta.-proteo. vs. Control-GI (n=7), Mann-Whitney, p=0.064; AUT (-)
.beta.-proteo. vs. Control-GI, Mann-Whitney, p=0.749. (FIG. 35D)
Cecum: AUT (+) .beta.-proteo. (n=10) vs. AUT (-) .beta.-proteo.
(n=5), Mann-Whitney, p=0.028; AUT (+) .beta.-proteo. vs. Control-GI
(n=7), Mann-Whitney, p=0.097; AUT (-) .beta.-proteo. vs.
Control-GI, Mann-Whitney, p=0.808. *, p<0.05; **, p<0.01;
.dagger., p<0.1 (trend); n.s., not significant.
[0058] FIG. 36 shows the distribution of Sutterella sequences by
individual patient as a percentage of total bacteria 16S rRNA reads
from cecal biopsies from AUT-GI (patients #1-15) and Control-GI
(patients #16-22) patients. *, p<0.05.
[0059] FIG. 37 is a pie chart indicating the percentage of
Sutterella sequences in the dominant OTU (either OTU 1 or OTU 2)
relative to sequences from subdominant Sutterella OTUs in ileum and
cecum of the seven Sutterella-positive patients. The percentage of
the dominant OTU is shown per individual patient.
[0060] FIG. 38 is a schematic representation showing the location
of PCR primers and products evaluated in Sutterella-specific PCR
assays.
[0061] FIGS. 39A-B are photographic images of gels showing
PCR-based detection of Sutterella 16S rRNA gene sequences (V6-V8
region and C4-V8 region) in biopsies from AUT-GI and Control-GI
patients. FIG. 39A shows agarose gel detection of 260 bp Sutterella
products in ileal (4 biopsies/patient) and cecal (4
biopsies/patient) biopsy DNA using SuttFor and SuttRev primers
(V6-V8 region) in conventional PCR assays. FIG. 39B shows agarose
gel detection of 715 bp Sutterella products in ileal and cecal
biopsy DNA using pan-bacterial primer 515For and SuttRev primer
(C4-V8) in conventional PCR assays. Negative control is PCR
reagents with water substituted for DNA. Positive control is DNA
isolated from cultured S. wadsworthensis (ATCC, #51579).
[0062] FIG. 40 is a graph that shows quantitation of Sutterella
sequences in ileal and cecal biopsies from AUT-GI and Control-GI
patients using a novel Sutterella-specific real-time PCR assay.
Bars in graph show mean copy number in 4 biopsies from ileum (blue)
and 4 biopsies from cecum (red)+the standard error mean for each
individual patient.
[0063] FIGS. 41A-L show pie charts of the distribution of
Sutterella species in ileal and cecal biopsies of AUT-GI patients
based on C4-V8 products. The closest sequence match to known
Sutterella isolates was determined using the RDP seqmatch tool. The
frequency of Sutterella species matches in ileal and cecal clone
libraries are shown as pie charts for patient #1 (FIG. 41A),
patient #3 (FIG. 41B), patient #5 (FIG. 41C), patient #7 (FIG.
41D), patient #10 (FIG. 41E), patient #11 (FIG. 41F), patient #12
(FIG. 41G), patient #24a (FIG. 41H), patient #25a (FIG. 41I),
patient #27a (FIG. 41J), patient #28a (FIG. 41K), patient #29a
(FIG. 41L). *, Note: Sutterella 16S sequences obtained from patient
#28a were less than 97% similar to the 16S sequence of all known
isolates of Sutterella species.
[0064] FIG. 42 is a schematic of a phylogenetic tree based on
predominant 16S rRNA gene sequences obtained by C4-V8 Sutterella
PCR from AUT-GI patients, Sutterella species isolates, and related
species. The tree was constructed using the Neighbor joining
method. Bootstrap values (>60%) based on 1000 replicates are
shown next to the branches. There were a total of 653 positions in
the final dataset. The evolutionary distances were computed using
the Jukes-Cantor method and are in the units of the number of base
substitutions per site. The optimal tree with sum of branch
length=0.66371685 is shown. The tree is rooted to the outgroup
Escherichia coli. Accession numbers are shown in parentheses.
AUT-GI patient sequences are boxed in red.
[0065] FIGS. 43A-B show western immunoblot analysis of AUT-GI and
Control-GI patients' plasma antibody immunoreactivity against S.
wadsworthensis antigens. FIG. 43A depicts patients' plasma IgG
antibody immunoreactivity against S. wadsworthensis antigens. FIG.
43B depicts patients' IgM antibody immunoreactivity against S.
wadsworthensis antigens. 2.degree.=Secondary antibody control.
[0066] FIGS. 44A-D show graphs of the abundance distribution of all
genus level classifications of sequences from pyrosequencing for
patients #1, 3, 5 and 7. Bar graph showing all ileal genera, in
order of highest abundance (top) to lowest abundance (bottom), from
(FIG. 44A) patient #1 (32 total genera), (FIG. 44B) patient #3 (35
total genera), (FIG. 44C) patient #5 (39 total genera), and (FIG.
44D) patient #7 (39 total genera). The abundances of Sutterella
sequences are indicated in red. Note unclassified family members
can represent more than one genus (i.e. Unclassified
Lachnospiraceae).
[0067] FIGS. 45A-C show graphs of the abundance distribution of all
genus level classifications of sequences from pyrosequencing for
patients #10, 11, and 12. Bar graph showing all ileal genera, in
order of highest abundance (top) to lowest abundance (bottom) from
(FIG. 45A) patient #10 (32 total genera), (FIG. 45B) patient #11
(39 total genera), and (FIG. 45C) patient #12 (44 total genera).
The abundances of Sutterella sequences are indicated in red. Note
unclassified family members can represent more than one genus (i.e.
Unclassified Lachnospiraceae).
[0068] FIGS. 46A-B depict Sutterella OTU analysis. Heatmap
generated from OTU analysis of all Sutterella sequences by patient.
Note patients #1, 3, 10, 11, and 12 cluster together and the
majority of Sutterella sequences are present in OTU 2. Patients #5
and 7 cluster together and the majority of Sutterella sequences are
present in OTU 1. Heatmap scale represents OTU abundance (expressed
as % of total bacterial pyrosequencing reads per patient).
[0069] FIG. 47 is a schematic of a phylogenetic tree based on the
representative 16S rRNA gene sequences obtained by V2 region
pyrosequencing (OTU 1 and OTU 2) from AUT-GI patients, Sutterella
species isolates, and related species. The tree was constructed
using the Neighbor joining method. Bootstrap values based on 1000
replicates are shown next to the branches (% bootstrap support).
There were a total of 218 positions in the final dataset. The
evolutionary distances were computed using the Jukes-Cantor method
and are in the units of the number of base substitutions per site.
The optimal tree with sum of branch length=1.01142743 is shown. The
tree is rooted to the outgroup Escherichia coli. Accession numbers
are shown in parentheses. The location of AUT-GI patients'
representative OTU 1 and OTU 2 sequences are boxed in red.
[0070] FIG. 48 is a schematic of a phylogenetic tree based on
predominant 16S rRNA gene sequences obtained by V6-V8 Sutterella
PCR from AUT-GI patients, Sutterella species isolates, and related
species. The tree was constructed using the Neighbor joining
method. Bootstrap values based on 1000 replicates are shown next to
the branches (% bootstrap support). There were a total of 215
positions in the final dataset. The evolutionary distances were
computed using the Jukes-Cantor method and are in the units of the
number of base substitutions per site. The optimal tree with sum of
branch length=0.67013793 is shown. The tree is rooted to the
outgroup Escherichia coli. Accession numbers are shown in
parentheses. The location of AUT-GI patients' sequences are boxed
in red.
[0071] FIG. 49 shows a Sutterella sequence alignment. Clustal W
alignment of the most abundant Sutterella 16S rRNA gene (C4-V8
region) sequences in the 12 Sutterella-positive patients. Sequences
have had the 515For and SuttRev primer sequences removed. The
positions of the beginning (nucleotide position 501) and end
(nucleotide position 1176) of the sequences are relative to the 16S
rRNA gene of S. wadsworthensis (Accession L37785). Patients 1, 24a
(SEQ ID NO: 61); Patients 3, 10, 11, 12, 27a, 29a (SEQ ID NO: 62);
Patients 5, 7, 25a (SEQ ID NO: 63); Patient 28a (SEQ ID NO:
64).
ABBREVIATIONS USED HEREIN
[0072] ASD, autism spectrum disorders; GI, gastrointestinal;
AUT-GI, children with autistic disorder and GI disease; Control-GI,
normally developing children with GI disease; FA, food allergy; MA,
milk-related allergy; WA, wheat-related allergy; AD, atopic
disease; SI, sucrase isomaltase; MGAM, maltase glucoamylase; LCT,
lactase; SGLT1, sodium-dependent glucose cotransporter; GLUT2,
glucose transporter 2; CDX2, caudal type homeobox 2; OTU,
operational taxonomic unit.
DETAILED DESCRIPTION OF THE INVENTION
[0073] Autism, one of the ASDs, is mostly diagnosed clinically
using behavioral criteria because few specific biological markers
are known for diagnosing the disease. Autism is a neuropsychiatric
developmental disorder characterized by impaired verbal
communication, non-verbal communication, and reciprocal social
interaction. It is also characterized by restricted and stereotyped
patterns of interests and activities, as well as the presence of
developmental abnormalities by 3 years of age (Bailey et al.,
(1996) J Child Psychol Psychiatry 37(1):89-126). Autism-associated
disorders, diseases or pathologies can comprise any metabolic,
immune or systemic disorders; gastrointestinal disorders; epilepsy;
congenital malformations or genetic syndromes; anxiety, depression,
or AD/HD; or speech delay and motor in-coordination.
[0074] Autism spectrum disorders (ASD) are defined by impairments
in verbal and non-verbal communication, social interactions, and
repetitive and stereotyped behaviors (DSM-IV-TR criteria, American
Psychiatric Association, 2000). In addition to these core deficits,
previous reports indicate that the prevalence of gastrointestinal
symptoms ranges widely in individuals with ASD, from 9 to 91% (Buie
et al., 2010). Macroscopic and histological observations in ASD
include findings of ileo-colonic lymphoid nodular hyperplasia
(LNH), enterocolitis, gastritis and esophagitis (Wakefield et al.,
2000; Wakefield et al., 2005; Furlano et al., 2001; Torrente et
al., 2002; Horvath et al., 1999). Associated changes in intestinal
inflammatory parameters include higher densities of lymphocyte
populations, aberrant cytokine profiles, and deposition of
immunoglobulin (IgG) and complement C1q on the basolateral
enterocyte membrane (Furlano et al., 2001; Ashwood and Wakefield,
2006). Functional disturbances include increased intestinal
permeability (D'Eufemia et al., 1996), compromised
sulphoconjugation of phenolic compounds (O'Reilly and Waring, 1993;
Alberti et al., 1999), deficient enzymatic activity of
disaccharidases (Horvath et al., 1999), increased secretin-induced
pancreatico-biliary secretion (Horvath et al., 1999), and abnormal
Clostridia taxa (Finegold et al., 2002; Song et al., 2004; Parracho
et al., 2005). Some children placed on exclusion diets or treated
with the antibiotic vancomycin are reported to improve in cognitive
and social function (Knivsberg et al., 2002; Sandler et al.,
2000).
[0075] The gastrointestinal tract is exposed to an onslaught of
foreign material in the form of food, xenobiotics, and microbes.
The intestinal muco-epithelial layer must maximize nutritional
uptake of dietary components while maintaining a barrier to toxins
and infectious agents. Although some aspects of these functions are
host-encoded, others are acquired through symbiotic relationships
with microbial flora. Dietary carbohydrates enter the intestine as
monosaccharides (glucose, fructose, and galactose), disaccharides
(lactose, sucrose, maltose), or complex polysaccharides. Following
digestion with salivary and pancreatic amylases, carbohydrates are
further digested by disaccharidases expressed by absorptive
enterocytes in the brush border of the small intestine and
transported as monosaccharides across the intestinal epithelium.
However, humans lack the glycoside hydrolases and polysaccharide
lyases necessary for cleavage of glycosidic linkages present in
plant cell wall polysaccharides, oligosaccharides, storage
polysaccharides, and resistant starches. Intestinal bacteria
encoding these enzymes expand the capacity to extract energy from
dietary polysaccharides (Sonnenburg et al., 2008; Flint et al.,
2008). As an end product of polysaccharide fermentation, bacteria
produce short-chain fatty acids (butyrate, acetate, and propionate)
that serve as energy substrates for colonocytes, modulate colonic
pH, regulate colonic cell proliferation and differentiation, and
contribute to hepatic gluconeogenesis and cholesterol synthesis
(Wong et al., 2006; Jacobs et al., 2009). Indigenous microflora
also mediate postnatal development of the muco-epithelial layer,
provide resistance to potential pathogens, regulate development of
intraepithelial lymphocytes and Peyer's patches, influence cytokine
production and serum immunoglobulin levels, and promote systemic
lymphoid organogenesis (O'Hara and Shanahan, 2006; Macpherson and
Harris, 2004).
[0076] The prevalence of autism in the US is about 1 in 91 births
and, largely due to changes in diagnostic practices, services, and
public awareness. Autism is growing at the fastest pace of any
developmental disability (10-17%) (Fombonne, E. (2003). The
prevalence of autism. JAMA 289(1): 87-9). Care and treatment of
autism costs the U.S. healthcare system $90B annually. Early
detection and intervention can result in reducing life-long costs.
In the last 5 years, federal funding for autism research rose by
16.1%. The Autism Society is currently lobbying Congress for $37
million for autism monitoring and studies, another $16.5 million
for autism screening and academic research. At present, few tools
outside psychiatric evaluation are available for diagnosing autism.
While a causative link between GI abnormalities and pathology of
autism has yet to be established, a correlation between the two
disorders is relatively well established. Thus, technologies
facilitating detection and treatment of abnormal gut flora in
autistic patients has great potential utility for diagnosis and
treatment.
[0077] The present invention provides the discovery and the
identification of GLUT2 as well SGLT1 as biomarkers for human
Autism Spectrum Disorders. The present invention provides for
methods to use genes encoding carbohydrate metabolic enzyme
molecules (such as sucrase isomaltase, maltase glucoamylase, and
lactase) or carbohydrate transporter molecules, or a combination of
the two, and corresponding expression products for the diagnosis,
prevention and treatment of autism and autism spectrum
disorders.
[0078] The methods of the invention are useful in various subjects,
such as humans, including adults, children, and developing human
fetuses at the prenatal stage.
[0079] The GLUT2 gene locus can comprise all GLUT2 sequences or
products in a cell or organism, including GLUT2 coding sequences,
GLUT2 non-coding sequences (e.g., introns), GLUT2 regulatory
sequences controlling transcription and/or translation (e.g.,
promoter, enhancer, terminator).
[0080] A GLUT2 gene, also known as SLC2A2, encodes the glucose
transporter 2 isoform. It is an integral plasma membrane
glycoprotein of the liver, pancreatic islet beta cells, intestine,
and kidney epithelium. GLUT2 mediates the bidirectional transport
of glucose. In the context of the invention, the GLUT2 gene also
encompasses its variants, analogs and fragments thereof, including
alleles thereof (e.g., germline mutations) which are related to
susceptibility to autism and/or autism spectrum disorders.
[0081] The SGLT1 gene locus can comprise all SGLT1 sequences or
products in a cell or organism, including SGLT1 coding sequences,
SGLT1 non-coding sequences (e.g., introns), SGLT1 regulatory
sequences controlling transcription and/or translation (e.g.,
promoter, enhancer, terminator).
[0082] A SGLT1 gene, also known as SLC5A1, encodes the
sodium/glucose co-transporter 1. The sodium dependent glucose
transporter is an integral plasma membrane glycoprotein of the
intestine. SGLT1 mediates glucose and galactose uptake from the
intestinal lumen. Mutations in this gene have been associated with
glucose-galactose malabsorption. In the context of the invention,
the SGLT1 gene also encompasses its variants, analogs and fragments
thereof, including alleles thereof (e.g., germline mutations) which
are related to susceptibility to autism and/or autism spectrum
disorders.
[0083] As used herein, "carbohydrate transport activity" means the
ability of a polypeptide to bind a carbohydrate, such as glucose,
to a transporter protein, and subsequently facilitate uptake of the
carbohydrate from the serum or extracellular millieu into a cell
(e.g., a liver cell, or pancreatic .beta.-cell). Glucose transport
activity can be measured as described by Hissin et al., 1982, J.
Clin. Invest. 70(4): 780-90. In one embodiment, the carbohydrate
transport activity is glucose transport activity, and the activity
can be measured by determining glucose transport activity as
described in Hissin as well as the ability to decrease
extracellular or serum glucose levels. Non-limiting examples of a
carbohydrate transporter include GLUT1, GLUT2, GLUT3, GLUT4, GLUT5,
GLUT6, GLUT7, GLUT5, GLUT9, GLUT10, GLUT11, GLUT12, and HMIT (see
Scheepers et al., JPEN J Parenter Enteral Nutr. 2004
September-October; 28(5):364-71).
[0084] A sucrase isomaltase (SI) gene encodes a sucrase-isomaltase
protein, which is a glucosidase enzyme, that is expressed in the
intestinal brush border. The encoded protein is synthesized as a
precursor protein that is cleaved by pancreatic proteases into two
enzymatic subunits, sucrase and isomaltase. The two subunits
heterodimerize to form the sucrose-isomaltase complex, which is
essential for the digestion of dietary carbohydrates including
starch, sucrose and isomaltose. Mutations in this gene are the
cause of congenital sucrase-isomaltase deficiency. In the context
of the invention, the SI gene also encompasses its variants,
analogs and fragments thereof, including alleles thereof (e.g.,
germline mutations) which are related to susceptibility to autism
and/or autism spectrum disorders.
[0085] A maltase glucoamylase (MGAM) gene encodes a
maltase-glucoamylase enzyme. It is localized to the brush border
membrane and plays a role in the final steps of digestion of
starch. The protein has two catalytic sites identical to those of
sucrase-isomaltase, but the proteins are only 59% homologous. Both
are members of glycosyl hydrolase family 31, which has a variety of
substrate specificities. In the context of the invention, the MGAM
gene also encompasses its variants, analogs and fragments thereof,
including alleles thereof (e.g., germline mutations) which are
related to susceptibility to autism and/or autism spectrum
disorders.
[0086] A lactase (LCT) gene encodes a glycosyl hydrolase of family
1. The protein is integral to plasma membrane and has both
phlorizin hydrolase activity and lactase activity.
[0087] As used herein, "carbohydrate metabolic enzyme activity"
includes "sucrase isomaltase activity", "maltase glucoamylase
activity", "lactase activity", "sucrase activity", "maltase
activity", "trehalase activity", "amylase activity", "cellulase
activity", "glucosidase activity", "pullulanase activity",
"galactosidase activity", "alpha-Mannosidase activity",
"glucuronidase activity", "hyaluronidase activity", "glycosylase
activity", "fucosidase activity", "hexosaminidase activity",
"iduronidase activity", or "maltase-glucoamylase activity".
"Sucrase isomaltase activity" means the ability of a polypeptide to
catalyze the hydrolysis of sucrose to fructose and glucose and to
enzymatically digest polysaccharides at the alpha 1-6 linkages.
Sucrase and isomaltase activities can be measured as described by
Dahlqvist, A. (1964) Anal. Biochem. 7, 18-25 and the enzyme assays
described by Goda et al., Biochem J. 1988 Feb. 15; 250(1): 41-46.
"Maltase glucoamylase activity" means the ability of a polypeptide
to enzymatically digest starch, releasing malstose and free
glucose, as well as to catalyze the hydrolysis of the disaccharide
maltose. Maltase and glucoamylase activities can be measured as
described by Dahlqvist A. Specificity of the human intestinal
disaccharidases and implications for hereditary disaccharide
intolerance. J Clin Invest. 1962; 41:463-9; Dahlqvist A. Assay of
intestinal disaccharidases. Scand J Clin Lab Invest. 1984;
44:169-72; and Quezada-Calvillo et al., J. Nutr. 137:1725-1733,
July 2007. "Lactase activity" means the ability of a polypeptide to
hydrolyze lactose to galactose and glucose. Lactase activity can be
measured as described by Dahlqvist A. Specificity of the human
intestinal disaccharidases and implications for hereditary
disaccharide intolerance. J Clin Invest. 1962; 41:463-9; Dahlqvist
A. Assay of intestinal disaccharidases. Scand J Clin Lab Invest.
1984; 44:169-72; and Quezada-Calvillo et al., J. Nutr.
137:1725-1733, July 2007. "Trehalase activity" means the ability of
a polypeptide to catalyze the conversion of the dissacharide
trehalose (.alpha.-D-glucopyranosyl-1,1-.alpha.-D-glucopyranoside)
to glucose.
TABLE-US-00001 SEQ ID NO: 1 is the human wild type amino acid
sequence corresponding to the GLUT2 enzyme (residues 1-524) having
GenBank Accession No. NP_000331: 1 MTEDKVTGTL VFTVITAVLG SFQFGYDIGV
INAPQQVIIS HYRHVLGVPL DDRKAINNYV 61 INSTDELPTI SYSMNPKPTP
WAEEETVAAA QLITMLWSLS VSSFAVGGMT ASFFGGWLGD 121 TLGRIKAMLV
ANILSLVGAL LMGFSKLGPS HILIIAGRSI SGLYCGLISG LVPMYIGEIA 181
PTALRGALGT FHQLAIVTGI LISQIIGLEF ILGNYDLWHI LLGLSGVRAI LQSLLLFFCP
241 ESPRYLYIKL DEEVKAKQSL KRLRGYDDVT KDINEMRKER EEASSEQKVS
IIQLFTNSSY 301 RQPILVALML HVAQQFSGIN GIFYYSTSIF QTAGISKPVY
ATIGVGAVNM VFTAVSVFLV 361 EKAGRRSLFL IGMSGMFVCA IFMSVGLVLL
NKFSWMSYVS MIAIFLFVSF FEIGPGPIPW 421 FMVAEFFSQG PRPAALAIAA
FSNWTCNFIV ALCFQYIADF CGPYVFFLFA GVLLAFTLFT 481 FFKVPETKGK
SFEEIAAEFQ KKSGSAHRPK AAVEMKFLGA TETV SEQ ID NO: 2 is the human
wild type nucleic acid sequence corresponding to the GLUT2 enzyme
(bps 1-3439) having GenBank Accession No. NM_000340: 1 tctggtttgt
aacttatgcc taagggacct gctcccattt tctttcctag tggaacaaag 61
gtattgaagc cacaggttgc tgaggcaaag cacttattga ttagattccc atcaatattc
121 agctgccgct gagaagatta gacttggact ctcaggtctg ggtagcccaa
ctcctccctc 181 tccttgctcc tcctcctgca atgcataact aggcctaggc
agagctgcga ataaacaggc 241 aggagctagt caggtgcatg tgccacactc
acacaagacc tggaattgac aggactccca 301 actagtacaa tgacagaaga
taaggtcact gggaccctgg ttttcactgt catcactgct 361 gtgctgggtt
ccttccagtt tggatatgac attggtgtga tcaatgcacc tcaacaggta 421
ataatatctc actatagaca tgttttgggt gttccactgg atgaccgaaa agctatcaac
481 aactatgtta tcaacagtac agatgaactg cccacaatct catactcaat
gaacccaaaa 541 ccaacccctt gggctgagga agagactgtg gcagctgctc
aactaatcac catgctctgg 601 tccctgtctg tatccagctt tgcagttggt
ggaatgactg catcattctt tggtgggtgg 661 cttggggaca cacttggaag
aatcaaagcc atgttagtag caaacattct gtcattagtt 721 ggagctctct
tgatggggtt ttcaaaattg ggaccatctc atatacttat aattgctgga 781
agaagcatat caggactata ttgtgggcta atttcaggcc tggttcctat gtatatcggt
841 gaaattgctc caaccgctct caggggagca cttggcactt ttcatcagct
ggccatcgtc 901 acgggcattc ttattagtca gattattggt cttgaattta
tcttgggcaa ttatgatctg 961 tggcacatcc tgcttggcct gtctggtgtg
cgagccatcc ttcagtctct gctactcttt 1021 ttctgtccag aaagccccag
atacctttac atcaagttag atgaggaagt caaagcaaaa 1081 caaagcttga
aaagactcag aggatatgat gatgtcacca aagatattaa tgaaatgaga 1141
aaagaaagag aagaagcatc gagtgagcag aaagtctcta taattcagct cttcaccaat
1201 tccagctacc gacagcctat tctagtggca ctgatgctgc atgtggctca
gcaattttcc 1261 ggaatcaatg gcatttttta ctactcaacc agcatttttc
agacggctgg tatcagcaaa 1321 cctgtttatg caaccattgg agttggcgct
gtaaacatgg ttttcactgc tgtctctgta 1381 ttccttgtgg agaaggcagg
gcgacgttct ctctttctaa ttggaatgag tgggatgttt 1441 gtttgtgcca
tcttcatgtc agtgggactt gtgctgctga ataagttctc ttggatgagt 1501
tatgtgagca tgatagccat cttcctcttt gtcagcttct ttgaaattgg gccaggcccg
1561 atcccctggt tcatggtggc tgagtttttc agtcaaggac cacgtcctgc
tgctttagca 1621 atagctgcat tcagcaattg gacctgcaat ttcattgtag
ctctgtgttt ccagtacatt 1681 gcggacttct gtggacctta tgtgtttttc
ctctttgctg gagtgctcct ggcctttacc 1741 ctgttcacat tttttaaagt
tccagaaacc aaaggaaagt cttttgagga aattgctgca 1801 gaattccaaa
agaagagtgg ctcagcccac aggccaaaag ctgctgtaga aatgaaattc 1861
ctaggagcta cagagactgt gtaaaaaaaa aaccctgctt tttgacatga acagaaacaa
1921 taagggaacc gtctgttttt aaatgatgat tccttgagca ttttatatcc
acatctttaa 1981 gtattgtttt atttttatgt gctctcatca gaaatgtcat
caaatattac caaaaaagta 2041 tttttttaag ttagagaata tatttttgat
ggtaagactg taattaagta aaccaaaaag 2101 gctagtttat tttgttacac
taaagggcag gtggttctaa tatttttagc tctgttcttt 2161 ataacaaggt
tcttctaaaa ttgaagagat ttcaacatat cattttttta acacataact 2221
agaaacctga ggatgcaaca aatatttata tatttgaata tcattaaatt ggaattttct
2281 tacccatata tcttatgtta aaggagatat ggctagtggc aataagttcc
atgttaaaat 2341 agacaactct tccatttatt gcactcagct tttttcttga
gtactagaat ttgtattttg 2401 cttaaaattt tacttttgtt ctgtattttc
atgtggaatg gattatagag tatactaaaa 2461 aatgtctata gagaaaaact
ttcatttttg gtaggcttat caaaatcttt cagcactcag 2521 aaaagaaaac
cattttagtt cctttattta atggccaaat ggtttttgca agatttaaca 2581
ctaaaaaggt ttcacctgat catatagcgt gggttatcag ttaacattaa catctattat
2641 aaaaccatgt tgattccctt ctggtacaat cctttgagtt atagtttgct
ttgcttttta 2701 attgaggaca gcctggtttt cacatacact caaacaatca
tgagtcagac atttggtata 2761 ttacctcaaa ttcctaataa gtttgatcaa
atctaatgta agaaaatttg aagtaaagga 2821 ttgatcactt tgttaaaaat
attttctgaa ttattatgtc tcaaaataag ttgaaaaggt 2881 agggtttgag
gattcctgag tgtgggcttc tgaaacttca taaatgttca gcttcagact 2941
tttatcaaaa tccctattta attttcctgg aaagactgat tgttttatgg tgtgttccta
3001 acataaaata atcgtctcct ttgacatttc cttctttgtc ttagctgtat
acagattcta 3061 gccaaactat tctatggcca ttactaacac gcattgtaca
ctatctatct gcctttacct 3121 acataggcaa attggaaata cacagatgat
taaacagact ttagcttaca gtcaatttta 3181 caattatgga aatatagttc
tgatgggtcc caaaagctta gcagggtgct aacgtatctc 3241 taggctgttt
tctccaccaa ctggagcact gatcaatcct tcttatgttt gctttaatgt 3301
gtattgaaga aaagcacttt ttaaaaagta ctctttaaga gtgaaataat taaaaaccac
3361 tgaacatttg ctttgttttc taaagttgtt cacatatatg taatttagca
gtccaaagaa 3421 caagaaattg tttcttttc SEQ ID NO: 3 is human wild
type amino acid sequence corresponding to the SGLT1 enzyme
(residues 1-664) having GenBank Accession No. NP_000334: 1
MDSSTWSPKT TAVTRPVETH ELIRNAADIS IIVIYFVVVM AVGLWAMFST NRGTVGGFFL
61 AGRSMVWWPI GASLFASNIG SGHFVGLAGT GAASGIAIGG FEWNALVLVV
VLGWLFVPIY 121 IKAGVVTMPE YLRKRFGGQR IQVYLSLLSL LLYIFTKISA
DIFSGAIFIN LALGLNLYLA 181 IFLLLAITAL YTITGGLAAV IYTDTLQTVI
MLVGSLILTG FAFHEVGGYD AFMEKYMKAI 241 PTIVSDGNTT FQEKCYTPRA
DSFHIFRDPL TGDLPWPGFI FGMSILTLWY WCTDQVIVQR 301 CLSAKNMSHV
KGGCILCGYL KLMPMFIMVM PGMISRILYT EKIACVVPSE CEKYCGTKVG 361
CTNIAYPTLV VELMPNGLRG LMLSVMLASL MSSLTSIFNS ASTLFTMDIY AKVRKRASEK
421 ELMIAGRLFI LVLIGISIAW VPIVQSAQSG QLFDYIQSIT SYLGPPIAAV
FLLAIFWKRV 481 NEPGAFWGLI LGLLIGISRM ITEFAYGTGS CMEPSNCPTI
ICGVHYLYFA IILFAISFIT 541 IVVISLLTKP IPDVHLYRLC WSLRNSKEER
IDLDAEEENI QEGPKETIEI ETQVPEKKKG 601 IFRRAYDLFC GLEQHGAPKM
TEEEEKAMKM KMTDTSEKPL WRTVLNVNGI ILVTVAVFCH 661 AYFA SEQ ID NO: 4
is the human wild type nucleic acid sequence corresponding to the
SGLT1 enzyme(bps 1-5061) having GenBank Accession No. NM_000343: 1
ccccattcgc aggacagctc ttacctgccg ggccgccgcc ccagccaaca gctcagccgg
61 gtgctccttc ctgggctcca cgcccggagc tgcttcctga cggtgcagcc
gcaaggcatc 121 gcaggggccc cgcgctactg ccctgctccc tcaaagtccc
aggtcccctc ccctggtgct 181 gatcattaac caggaggccg tataaggagc
tagcggccct ggcgagaggg aaggacgcaa 241 cgctgccacc atggacagta
gcacctggag ccccaagacc accgcggtca cccggcctgt 301 tgagacccac
gagctcattc gcaatgcagc cgatatctcc atcatcgtta tctacttcgt 361
ggtagtgatg gccgtcggac tgtgggctat gttttccacc aatcgtggga ctgttggagg
421 cttcttcctg gcaggccgaa gtatggtgtg gtggccgatt ggagcctccc
tctttgctag 481 taacattgga agtggccact ttgtggggct ggccgggact
ggggcagctt caggcatcgc 541 cattggaggc tttgaatgga atgccctggt
tttggtggtt gtgctgggct ggctgtttgt 601 ccccatctat attaaggctg
gggtggtgac aatgccagag tacctgagga agcggtttgg 661 aggccagcgg
atccaggtct acctttccct tctgtccctg ctgctctaca ttttcaccaa 721
gatctcggca gacatcttct cgggggccat attcatcaat ctggccttag gcctgaatct
781 gtatttagcc atctttctct tattggcaat cactgccctt tacacaatta
cagggggcct 841 ggcggcggtg atttacacgg acaccttgca gacggtgatc
atgctggtgg ggtctttaat 901 cctgactggg tttgcttttc acgaagtggg
aggctatgac gccttcatgg aaaagtacat 961 gaaagccatt ccaaccatag
tgtctgatgg caacaccacc tttcaggaaa aatgctacac 1021 tccaagggcc
gactccttcc acatcttccg agatcccctc acgggagacc tcccatggcc 1081
tgggttcatc tttgggatgt ccatccttac cttgtggtac tggtgcacag atcaggtcat
1141 tgtgcagcgc tgcctctcag ccaagaatat gtctcacgtg aagggtggct
gcatcctgtg 1201 tgggtatcta aagctgatgc ccatgttcat catggtgatg
ccaggaatga tcagccgcat 1261 tctgtacaca gaaaaaattg cctgtgtcgt
cccttcagaa tgtgagaaat attgcggtac 1321 caaggttggc tgtaccaaca
tcgcctatcc aaccttagtg gtggagctca tgcccaatgg 1381 actgcgaggc
ctgatgctat cagtcatgct ggcctccctc atgagctccc tgacctccat 1441
cttcaacagc gccagcaccc tcttcaccat ggacatctac gccaaggtcc gcaagagagc
1501 atctgagaaa gagctcatga ttgccggaag gttgtttatc ctggtgctga
ttggcatcag 1561 catcgcctgg gtgcccattg tgcagtcagc acaaagtggg
caactcttcg attacatcca 1621 gtccatcacc agttacttgg gaccacccat
tgcggctgtc ttcctgcttg ctattttctg 1681 gaagagagtc aatgagccag
gagccttttg gggactgatc ctaggacttc tgattgggat 1741 ttcacgtatg
attactgagt ttgcttatgg aaccgggagc tgcatggagc ccagcaactg 1801
tcccacgatt atctgtgggg tgcactactt gtactttgcc attatcctct tcgccatttc
1861 tttcatcacc atcgtggtca tctccctcct caccaaaccc attccggatg
tgcatctcta 1921 ccgtctgtgt tggagcctgc gcaacagcaa agaggagcgt
attgacctgg atgcggaaga 1981 ggagaacatc caagaaggcc ctaaggagac
cattgaaata gaaacacaag ttcctgagaa 2041 gaaaaaagga atcttcagga
gagcctatga cctattttgt gggctagagc agcacggtgc 2101 acccaagatg
actgaggaag aggagaaagc catgaagatg aagatgacgg acacctctga 2161
gaagcctttg tggaggacag tgttgaacgt caatggcatc atcctggtga ccgtggctgt
2221 cttttgccat gcatattttg cctgagtcct accttttgct gtagatttac
catggctgga 2281 ctcttactca ccttccttta gtctcgtcct gtggtgttga
agggaaatca gccagttgta 2341 aattttgccc aggtggataa atgtgtacat
gtgtaattat aggctagctg gaagaaaacc 2401 attagtttgc tgttaattta
tgcatttgaa gccagtgtga tacagccatc tgtacctact 2461 ggagctgcag
aagggaagtc cactcagtca catccagaaa aaggcagact aagaatcaga
2521 agccatgtga ttgatgtctg acgtgagtct gtctcaggta gattccgggt
gtcagtgtgg 2581 tttataatcc ttgaatattg ttttagaaac tttggtctcc
ctggttcctg ccacttttcc 2641 tgtccgtcct cctccccatt ttttttttaa
aagaaagctg ttttcccctc atcatatccc 2701 tcttgagttt tgcctggact
ttccctctca agtgtgtcaa tcaggtaaac tgaggaatgc 2761 atggaagctg
aggatggagc ttgatgggct ccctgtcctg ggtgtttgct ctctgaagtg 2821
gaggcctgag gaaggtagta cttccacaaa agggagggac ccgggcccca gcctcaagct
2881 agtgggggag gcagatagcc tgaatccagg ggattttctg ggcttcttaa
aatgtccatt 2941 gtgagttccc cgtgtttggg attccactca ttttggcatt
cacagtgcct ggaatgtctt 3001 agattttcag caatgcgtgt tgaataaatg
aatgacatag gcatttattt ttaaatcttt 3061 gcttgctttt tacatgagcc
tggcccttag ttaacctttt cttgtggcta cacaaagtat 3121 gctcactggt
tactaatgac ttgggatgca tttgtcaaac tgattatatt agttttctag 3181
ggatgccata acaaagtagc acagaccaga tggctcaagc agcagacatt tattttctca
3241 cagttctaga ggctagaagt tggaggccaa gatgtcagca gggttggttt
cttctgaggc 3301 ctctctcctt ggttgcagat ggtcatatct cactctgtct
tccgtggcct tccttttgtc 3361 tgtgtcctaa atctactctt ctgataagga
catcagtcat attggaatag gacccaccct 3421 aatgtcttca ttttaatcac
ctctttaaag cccctacctc caaatacagt cacactgtga 3481 gaaactgagg
gttaggaagt cagcaagtga gtcttgaaga gatactaaac aaacccacaa 3541
cacagataaa gtatgcattt tggagatttc caagccagag tctcccgtga aaaaggtaaa
3601 cggaagcagt tattgtgcag caaaaggaaa aagaattaca aactgaacgt
atgtaggtga 3661 ggcaaggcag ggtagggcag ggcctttggg taggctgatc
agagggtttt tcaacaataa 3721 atcaatggga atgcatttgt tgctcccagg
accctggcac cttgactctg gtactatagc 3781 atgtcagcaa atacaagcaa
agcccaacac tctgatttgc atttatgcca atctaaacta 3841 tccggtgttt
agtttgattt tttgagtgca ggttcattca aggaccaggt tcccttgtgc 3901
tcagggtgaa gtagaaccag aaaacatcgt tatccattcc cagaagtttt ggaagagcct
3961 tggtagaaaa gcagaagctg ctttgaccgt gaaaatattt gactcctatc
agtttttggt 4021 caggagaaga tatccaccta gaccaacctg aggagaaggc
tcagagtaca gatatacccc 4081 gagcaacgtg atcaatgtcc ttgaaccttc
atttttcatc tgaaaacaga gacataaatg 4141 cctggctcac agatttaaat
gttatacatt gacagcattt atcagtataa catttattta 4201 aataagtagg
tgctcaatag gtgttggtct tctaacttgt ctacatccca tccccattcc 4261
agggtcttca gaattgaagg agagatgttg tatcactgtt agaaggctgc tttgggacat
4321 tctgcagcag ggaggaggga ctgtcaaccc ctacaccatg accaccaagt
tcctcacctt 4381 ggctgagtcc ctaaaactct ctgaacctca ggttcctcca
agcataatgc agacttcaca 4441 gagctgttgt aaagattagg tgaggtcaat
tgatactgct taaaaggccc ggtccgtaga 4501 aaatgcccaa taaacattac
tgctttcccc ctcaccctac tgcctgaaaa aatattacac 4561 ctgtgagact
gactttgaga accagtgtgg gtggggagtt gtgcatataa actatttaat 4621
gagtaccaaa cacaaaagtc aagcttgtaa aatatcaggc cttgccccag aaagacaaat
4681 accacatgat ctcactgata tgtagaatct taaaaagtca aactcagaag
cagagagtag 4741 aatgatggtt atcaagggct gggggaggga gggactgggg
agatgttggt caaatgatac 4801 aaaggtttag ttaggtggaa taagttcaga
aaatcaattg tacaatgtat caattatagt 4861 taatagcaat ataacatata
cttgaaaatt gctgagagta gtgtgagtgt tctaccacaa 4921 aaaaatatgt
gcagtaatag atgttaatta ccttaattta gtcatttcac aatatgtaca 4981
tatataaaaa tatgttgtat gccatgagta tatataatta ttatttgtga atttaaaaaa
5041 taaaaataat ttccaaaaaa a SEQ ID NO: 5 is the human wild type
amino acid sequence corresponding to the sucrase isomaltase (SI)
enzyme (residues 1-1827) having GenBank Accession No. NP_001032: 1
MARKKFSGLE ISLIVLFVIV TIIAIALIVV LATKTPAVDE ISDSTSTPAT TRVTTNPSDS
61 GKCPNVLNDP VNVRINCIPE QFPTEGICAQ RGCCWRPWND SLIPWCFFVD
NHGYNVQDMT 121 TTSIGVEAKL NRIPSPTLFG NDINSVLFTT QNQTPNRFRF
KITDPNNRRY EVPHQYVKEF 181 TGPTVSDTLY DVKVAQNPFS IQVIRKSNGK
TLFDTSIGPL VYSDQYLQIS TRLPSDYIYG 241 IGEQVHKRFR HDLSWKTWPI
FTRDQLPGDN NNNLYGHQTF FMCIEDTSGK SFGVFLMNSN 301 AMEIFIQPTP
IVTYRVTGGI LDFYILLGDT PEQVVQQYQQ LVGLPAMPAY WNLGFQLSRW 361
NYKSLDVVKE VVRRNREAGI PFDTQVTDID YMEDKKDFTY DQVAFNGLPQ FVQDLHDHGQ
421 KYVIILDPAI SIGRRANGTT YATYERGNTQ HVWINESDGS TPIIGEVWPG
LTVYPDFTNP 481 NCIDWWANEC SIFHQEVQYD GLWIDMNEVS SFIQGSTKGC
NVNKLNYPPF TPDILDKLMY 541 SKTICMDAVQ NWGKQYDVHS LYGYSMAIAT
EQAVQKVFPN KRSFILTRST FAGSGRHAAH 601 WLGDNTASWE QMEWSITGML
EFSLFGIPLV GADICGFVAE TTEELCRRWM QLGAFYPFSR 661 NHNSDGYEHQ
DPAFFGQNSL LVKSSRQYLT IRYTLLPFLY TLFYKAHVFG ETVARPVLHE 721
FYEDTNSWIE DTEFLWGPAL LITPVLKQGA DTVSAYIPDA IWYDYESGAK RPWRKQRVDM
781 YLPADKIGLH LRGGYIIPIQ EPDVTTTASR KNPLGLIVAL GENNTAKGDF
FWDDGETKDT 841 IQNGNYILYT FSVSNNTLDI VCTHSSYQEG TTLAFQTVKI
LGLTDSVTEV RVAENNQPMN 901 AHSNFTYDAS NQVLLIADLK LNLGRNFSVQ
WNQIFSENER FNCYPDADLA TEQKCTQRGC 961 VWRTGSSLSK APECYFPRQD
NSYSVNSARY SSMGITADLQ LNTANARIKL PSDPISTLRV 1021 EVKYHKNDML
QFKIYDPQKK RYEVPVPLNI PTTPISTYED RLYDVEIKEN PFGIQIRRRS 1081
SGRVIWDSWL PGFAFNDQFI QISTRLPSEY IYGFGEVEHT AFKRDLNWNT WGMFTRDQPP
1141 GYKLNSYGFH PYYMALEEEG NAHGVFLLNS NAMDVTFQPT PALTYRTVGG
ILDFYMFLGP 1201 TPEVATKQYH EVIGHPVMPA YWALGFQLCR YGYANTSEVR
ELYDAMVAAN IPYDVQYTDI 1261 DYMERQLDFT IGEAFQDLPQ FVDKIRGEGM
RYIIILDPAI SGNETKTYPA FERGQQNDVF 1321 VKWPNTNDIC WAKVWPDLPN
ITIDKTLTED EAVNASRAHV AFPDFFRTST AEWWAREIVD 1381 FYNEKMKFDG
LWIDMNEPSS FVNGTTTNQC RNDELNYPPY FPELTKRTDG LHFRTICMEA 1441
EQILSDGTSV LHYDVHNLYG WSQMKPTHDA LQKTTGKRGI VISRSTYPTS GRWGGHWLGD
1501 NYARWDNMDK SIIGMMEFSL FGMSYTGADI CGFFNNSEYH LCTRWMQLGA
FYPYSRNHNI 1561 ANTRRQDPAS WNETFAEMSR NILNIRYTLL PYFYTQMHEI
HANGGTVIRP LLHEFFDEKP 1621 TWDIFKQFLW GPAFMVTPVL EPYVQTVNAY
VPNARWFDYH TGKDIGVRGQ FQTFNASYDT 1681 INLHVRGGHI LPCQEPAQNT
FYSRQKHMKL IVAADDNQMA QGSLFWDDGE SIDTYERDLY 1741 LSVQFNLNQT
TLTSTILKRG YINKSETRLG SLHVWGKGTT PVNAVTLTYN GNKNSLPFNE 1801
DTTNMILRID LTTHNVTLEE PIEINWS SEQ ID NO: 6 is the human wild type
nucleic acid sequence corresponding to the sucrase isomaltase (SI)
enzyme (bps 1-6023) having GenBank Accession No. NM_001041: 1
ttattttggc agccttatcc aagtctggta caacatagca aagagaacag gctatgaaat
61 aagatggcaa gaaagaaatt tagtggattg gaaatctctc tgattgtcct
ttttgtcata 121 gttactataa tagctattgc cttaattgtt gttttagcaa
ctaagacacc tgctgttgat 181 gaaattagtg attctacttc aactccagct
actactcgtg tgactacaaa tccttctgat 241 tcaggaaaat gtccaaatgt
gttaaatgat cctgtcaatg tgagaataaa ctgcattcca 301 gaacaattcc
caacagaggg aatttgtgca cagagaggct gctgctggag gccgtggaat 361
gactctctta ttccttggtg cttcttcgtt gataatcatg gttataacgt tcaagacatg
421 acaacaacaa gtattggagt tgaagccaaa ttaaacagga taccttcacc
tacactattt 481 ggaaatgaca tcaacagtgt tctcttcaca actcaaaatc
agacacccaa tcgtttccgg 541 ttcaagatta ctgatccaaa taatagaaga
tatgaagttc ctcatcagta tgtaaaagag 601 tttactggac ccacagtttc
tgatacgttg tatgatgtga aggttgccca aaacccattt 661 agcatccaag
ttattaggaa aagcaacggt aaaactttgt ttgacaccag cattggtccc 721
ttagtgtact ctgaccagta cttacagatc tcaacccgtc ttccaagtga ttatatttat
781 ggtattggag aacaagttca taagagattt cgtcatgatt tatcctggaa
aacatggcca 841 atttttactc gagaccaact tcctggtgat aataataata
atttatacgg ccatcaaaca 901 ttctttatgt gtattgaaga tacatctgga
aagtcattcg gtgttttttt aatgaatagc 961 aatgcaatgg agatttttat
ccagcctact ccaatagtaa catatagagt taccggtggc 1021 attctggatt
tttacatcct tctaggagat acaccagaac aagtagttca acagtatcaa 1081
cagcttgttg gactaccagc aatgccagca tattggaatc ttggattcca actaagtcgc
1141 tggaattata agtcactaga tgtagtgaaa gaagtggtaa ggagaaaccg
ggaagctggc 1201 ataccatttg atacacaggt cactgatatt gactacatgg
aagacaagaa agactttact 1261 tatgatcaag ttgcgtttaa cggactccct
caatttgtgc aagatttgca tgaccatgga 1321 cagaaatatg tcatcatctt
ggaccctgca atttccatag gtcgacgtgc caatggaaca 1381 acatatgcaa
cctatgagag gggaaacaca caacatgtgt ggataaatga gtcagatgga 1441
agtacaccaa ttattggaga ggtatggcca ggattaacag tataccctga tttcactaac
1501 ccaaactgca ttgattggtg ggcaaatgaa tgcagtattt tccatcaaga
agtgcaatat 1561 gatggacttt ggattgacat gaatgaagtt tccagcttta
ttcaaggttc aacaaaagga 1621 tgtaatgtaa acaaattgaa ttatccaccg
tttactcctg atattcttga caaactcatg 1681 tattccaaaa caatttgcat
ggatgctgtg cagaactggg gtaaacagta tgatgttcat 1741 agcctctatg
gatacagcat ggctatagcc acagagcaag ctgtacaaaa agtttttcct 1801
aataagagaa gcttcattct tacccgctca acatttgctg gatctggaag acatgctgcg
1861 cattggttag gagacaatac tgcttcatgg gaacaaatgg aatggtctat
aactggaatg 1921 ctggagttca gtttgtttgg aatacctttg gttggagcag
acatctgtgg atttgtggct 1981 gaaaccacag aagaactttg cagaagatgg
atgcaacttg gggcatttta tccattttcc 2041 agaaaccata attctgacgg
atatgaacat caggatcctg cattttttgg gcagaattca 2101 cttttggtta
aatcatcaag gcagtattta actattcgct acaccttatt acccttcctc 2161
tacactctgt tttataaagc ccatgtgttt ggagaaacag tagcaagacc agttcttcat
2221 gagttttatg aggatacgaa cagctggatt gaggacactg agtttttgtg
gggccctgca 2281 ttacttatta ctcctgttct aaaacaggga gcagatactg
tgagtgccta catccctgat 2341 gctatttggt atgattatga atctggtgca
aaaaggccat ggaggaaaca acgggttgat 2401 atgtatcttc cagcagacaa
aataggatta catcttagag gaggttatat catccccatt 2461 caagaaccag
atgtaacaac aacagcaagc cgtaagaatc ctctaggact tatagtcgca 2521
ttaggtgaaa acaacacagc caaaggagac tttttctggg atgatggaga aactaaagat
2581 acaatacaaa atggcaacta catattatat acattttcag tttctaataa
cacattagat 2641 attgtgtgca cacattcatc atatcaggaa ggaactacct
tagcatttca gactgtaaaa 2701 atccttgggt tgacagacag tgttacagaa
gttagagtgg cggaaaataa tcaaccaatg 2761 aacgctcatt ccaatttcac
ttatgatgct tctaaccagg ttctcctaat tgcagatctc 2821 aaacttaatc
ttggaagaaa ctttagtgtt caatggaatc aaattttctc agaaaatgaa
2881 agatttaatt gttatccaga tgcagatttg gcaactgaac aaaagtgcac
acaacgtggc 2941 tgtgtatgga gaacgggttc ttctctatcc aaagcacctg
agtgttactt tcccagacaa 3001 gataactctt attcagtcaa ctcagctcgc
tattcatcca tgggtataac agctgacctc 3061 caactaaata ctgcaaatgc
cagaataaag ttaccttctg accccatctc aactcttcgt 3121 gtggaggtga
aatatcacaa aaatgatatg ttgcagttta agatttatga tccccaaaag 3181
aagagatatg aagtaccagt accgttaaac attccaacca ccccaataag tacttatgaa
3241 gacagacttt atgatgtgga aatcaaggaa aatccttttg gcatccagat
tcgacggaga 3301 agcagtggaa gagtcatttg ggattcttgg ctgcctggat
ttgcttttaa tgaccagttc 3361 attcaaatat cgactcgcct gccatcagaa
tatatatatg gttttgggga agtggaacat 3421 acagcattta agcgagatct
gaactggaat acttggggaa tgttcacaag agaccaaccc 3481 cctggttaca
aacttaattc ctatggattt catccctatt acatggctct ggaagaggag 3541
ggcaatgctc atggtgtttt cttactcaac agcaatgcaa tggatgttac attccagcca
3601 actcctgctc taacttaccg tacagttgga gggatcttgg atttttatat
gtttttgggc 3661 ccaactccag aagttgcaac aaagcaatac catgaagtaa
ttggccatcc agtcatgcca 3721 gcttattggg ctttgggatt ccaattatgt
cgttatggat atgcaaatac ttcagaggtt 3781 cgggaattat atgacgctat
ggtggctgct aacatcccct atgatgttca gtacacagac 3841 attgactaca
tggaaaggca gctagacttt acaattggtg aagcattcca ggaccttcct 3901
cagtttgttg acaaaataag aggagaagga atgagataca ttattatcct ggatccagca
3961 atttcaggaa atgaaacaaa gacttaccct gcatttgaaa gaggacagca
gaatgatgtc 4021 tttgtcaaat ggccaaacac caatgacatt tgttgggcaa
aggtttggcc agatttgccc 4081 aacataacaa tagataaaac tctaacggaa
gatgaagctg ttaatgcttc cagagctcat 4141 gtagctttcc cagatttctt
caggacttcc acagcagagt ggtgggccag agaaattgtg 4201 gacttttaca
atgaaaagat gaagtttgat ggtttgtgga ttgatatgaa tgagccatca 4261
agttttgtaa atggaacaac tactaatcaa tgcagaaatg acgaactaaa ttatccacct
4321 tatttcccag aactcacaaa aagaactgat ggattacatt tcagaacaat
ttgcatggaa 4381 gctgagcaga ttcttagtga tggaacatca gttttgcatt
acgatgttca caatctctat 4441 ggatggtcac agatgaaacc tactcatgat
gcattgcaga agacaactgg aaaaagaggg 4501 attgtaattt ctcgttccac
gtatcctact agtggacgat ggggaggaca ctggcttgga 4561 gacaactatg
cacgatggga caacatggac aaatcaatca ttggtatgat ggaatttagt 4621
ctgtttggaa tgtcatatac tggagcagac atctgtggtt ttttcaacaa ctcagaatat
4681 catctctgta cccgctggat gcaacttgga gcattttatc catactcaag
gaatcacaac 4741 attgcaaata ctagaagaca agatcccgct tcctggaatg
aaacttttgc tgaaatgtca 4801 aggaatattc taaatattag atacacctta
ttgccctatt tttacacaca aatgcatgaa 4861 attcatgcta atggtggcac
tgttatccga ccccttttgc atgagttctt tgatgaaaaa 4921 ccaacctggg
atatattcaa gcagttctta tggggtccag catttatggt taccccagta 4981
ctggaacctt atgttcaaac tgtaaatgcc tacgtcccca atgctcggtg gtttgactac
5041 catacaggca aagatattgg cgtcagagga caatttcaaa catttaatgc
ttcttatgac 5101 acaataaacc tacatgtccg tggtggtcac atcctaccat
gtcaagagcc agctcaaaac 5161 acattttaca gtcgacaaaa acacatgaag
ctcattgttg ctgcagatga taatcagatg 5221 gcacagggtt ctctgttttg
ggatgatgga gagagtatag acacctatga aagagaccta 5281 tatttatctg
tacaatttaa tttaaaccag accaccttaa caagcactat attgaagaga 5341
ggttacataa ataaaagtga aacgaggctt ggatcccttc atgtatgggg gaaaggaact
5401 actcctgtca atgcagttac tctaacgtat aacggaaata aaaattcgct
tccttttaat 5461 gaagacacta ccaacatgat attacgtatt gatctgacca
cacacaatgt tactctagaa 5521 gaaccaatag aaatcaactg gtcatgaaga
tcaccatcaa ttttagttgt caatgggaaa 5581 aaacaccagg atttaagttt
cacagcactt acaattttcc ctcttcactt ggttcttgta 5641 ctctacaaaa
tatagctttc ataacatcga aaagttattt tgtagcgtac atcaatgata 5701
atgctaattt tattatagta atgtgacttg gattcaattt taaggcatat ttaacaaaat
5761 ttgaatagcc ctatttatcc ttgttaagta tcagctacaa ttgtaaacta
gttactaaac 5821 atgtatgtaa atagctaaga tataatttaa acgtgatttt
taaattaaat aaaattttta 5881 tgtaattata tatactatat ttttctcaat
gtttagcaga tttaagatat gtaacaacaa 5941 ttatttgaag atttaattac
ttcttagtat gtgcatttaa ttagaaaaag agaataaaaa 6001 atgtaagtgt
aaaaaaaaaa aaa SEQ ID NO: 7 is the human wild type amino acid
sequence corresponding to the maltase glucoamylase (MGAM) enzyme
(residues 1-1857) having GenBank Accession No. NP_004659: 1
MARKKLKKFT TLEIVLSVLL LVLFIISIVL IVLLAKESLK STAPDPGTTG TPDPGTTGTP
61 DPGTTGTTHA RTTGPPDPGT TGTTPVSAEC PVVNELERIN CIPDQPPTKA
TCDQRGCCWN 121 PQGAVSVPWC YYSKNHSYHV EGNLVNTNAG FTARLKNLPS
SPVFGSNVDN VLLTAEYQTS 181 NRFHFKLTDQ TNNRFEVPHE HVQSFSGNAA
ASLTYQVEIS RQPFSIKVTR RSNNRVLFDS 241 SIGPLLFADQ FLQLSTRLPS
TNVYGLGEHV HQQYRHDMNW KTWPIFNRDT TPNGNGTNLY 301 GAQTFFLCLE
DASGLSFGVF LMNSNAMEVV LQPAPAITYR TIGGILDFYV FLGNTPEQVV 361
QEYLELIGRP ALPSYWALGF HLSRYEYGTL DNMREVVERN RAAQLPYDVQ HADIDYMDER
421 RDFTYDSVDF KGFPEFVNEL HNNGQKLVII VDPAISNNSS SSKPYGPYDR
GSDMKIWVNS 481 SDGVTPLIGE VWPGQTVFPD YTNPNCAVWW TKEFELFHNQ
VEFDGIWIDM NEVSNFVDGS 541 VSGCSTNNLN NPPFTPRILD GYLFCKTLCM
DAVQHWGKQY DIHNLYGYSM AVATAEAAKT 601 VFPNKRSFIL TRSTFAGSGK
FAAHWLGDNT ATWDDLRWSI PGVLEFNLFG IPMVGPDICG 661 FALDTPEELC
RRWMQLGAFY PFSRNHNGQG YKDQDPASFG ADSLLLNSSR HYLNIRYTLL 721
PYLYTLFFRA HSRGDTVARP LLHEFYEDNS TWDVHQQFLW GPGLLITPVL DEGAEKVMAY
781 VPDAVWYDYE TGSQVRWRKQ KVEMELPGDK IGLHLRGGYI FPTQQPNTTT
LASRKNPLGL 841 IIALDENKEA KGELFWDNGE TKDTVANKVY LLCEFSVTQN
RLEVNISQST YKDPNNLAFN 901 EIKILGTEEP SNVTVKHNGV PSQTSPTVTY
DSNLKVAIIT DIDLLLGEAY TVEWSIKIRD 961 EEKIDCYPDE NGASAENCTA
RGCIWEASNS SGVPFCYFVN DLYSVSDVQY NSHGATADIS 1021 LKSSVYANAF
PSTPVNPLRL DVTYHKNEML QFKIYDPNKN RYEVPVPLNI PSMPSSTPEG 1081
QLYDVLIKKN PFGIEIRRKS TGTIIWDSQL LGFTFSDMFI RISTRLPSKY LYGFGETEHR
1141 SYRRDLEWHT WGMFSRDQPP GYKKNSYGVH PYYMGLEEDG SAHGVLLLNS
NAMDVTFQPL 1201 PALTYRTTGG VLDFYVFLGP TPELVTQQYT ELIGRPVMVP
YWSLGFQLCR YGYQNDSEIA 1261 SLYDEMVAAQ IPYDVQYSDI DYMERQLDFT
LSPKFAGFPA LINRMKADGM RVILILDPAI 1321 SGNETQPYPA FTRGVEDDVF
IKYPNDGDIV WGKVWPDFPD VVVNGSLDWD SQVELYRAYV 1381 AFPDFFRNST
AKWWKREIEE LYNNPQNPER SLKFDGMWID MNEPSSFVNG AVSPGCRDAS 1441
LNHPPYMPHL ESRDRGLSSK TLCMESQQIL PDGSLVQHYN VHNLYGWSQT RPTYEAVQEV
1501 TGQRGVVITR STFPSSGRWA GHWLGDNTAA WDQLKKSIIG MMEFSLFGIS
YTGADICGFF 1561 QDAEYEMCVR WMQLGAFYPF SRNHNTIGTR RQDPVSWDVA
FVNISRTVLQ TRYTLLPYLY 1621 TLMHKAHTEG VTVVRPLLHE FVSDQVTWDI
DSQFLLGPAF LVSPVLERNA RNVTAYFPRA 1681 RWYDYYTGVD INARGEWKTL
PAPLDHINLH VRGGYILPWQ EPALNTHLSR QKFMGFKIAL 1741 DDEGTAGGWL
FWDDGQSIDT YGKGLYYLAS FSASQNTMQS HIIFNNYITG TNPLKLGYIE 1801
IWGVGSVPVT SVSISVSGMV ITPSFNNDPT TQVLSIDVTD RNISLHNFTS LTWISTL SEQ
ID NO: 8 is the human wild type nucleic acid sequence corresponding
to the maltase glucoamylase (MGAM) enzyme (bps 1-6513) having
GenBank Accession No. NM_004668: 1 attgctaagc catccttcag acagagaggg
agcggctgca agaggtaatg agagatggca 61 agaaagaagc tgaaaaaatt
tactactttg gagattgtgc tcagtgttct tctgcttgtg 121 ttgtttatca
tcagtattgt tctaattgtg cttttagcca aagagtcact gaaatcaaca 181
gccccagatc ctgggacaac tggtacccca gatcctggga caactggtac cccagatcct
241 ggaacaactg gtaccacaca tgctaggaca acgggtcccc cagatcctgg
aacaactggt 301 accactcctg tttctgctga atgtccagtg gtaaatgaat
tggaacgaat taattgcatc 361 cctgaccagc cgccaacaaa ggccacatgt
gaccaacgtg gctgttgctg gaatccccag 421 ggagctgtaa gtgttccctg
gtgctactat tccaagaatc atagctacca tgtagagggc 481 aaccttgtca
acacaaatgc aggattcaca gcccggttga aaaatctgcc ttcttcacca 541
gtgtttggaa gcaatgttga caatgttctt ctcacagcag aatatcagac atctaatcgt
601 ttccacttta agttgactga ccaaaccaat aacaggtttg aagtgcccca
cgaacacgtg 661 cagtccttca gtggaaatgc tgctgcttct ttgacctacc
aagttgaaat ctccagacag 721 ccatttagca tcaaagtgac cagaagaagc
aacaatcgtg ttttgtttga ctcgagcatt 781 gggcccctac tgtttgctga
ccagttcttg cagctctcca ctcgactgcc tagcactaac 841 gtgtatggcc
tgggagagca tgtgcaccag cagtatcggc atgatatgaa ttggaagacc 901
tggcccatat ttaacagaga cacaactccc aatggaaacg gaactaattt gtatggtgcg
961 cagacattct tcttgtgcct tgaagatgct agtggattgt cctttggggt
gtttctgatg 1021 aacagcaatg ccatggaggt tgtccttcag cctgcgccag
ccatcactta ccgcaccatt 1081 gggggcattc tcgacttcta tgtgttcttg
ggaaacactc cagagcaagt tgttcaagaa 1141 tatctagagc tcattgggcg
gccagccctt ccctcctact gggcgcttgg atttcacctc 1201 agtcgttacg
aatatggaac cttagacaac atgagggaag tcgtggagag aaatcgcgca 1261
gcacagctcc cttatgatgt tcagcatgct gatattgatt atatggatga gagaagggac
1321 ttcacttatg attcagtgga ttttaaaggc ttccctgaat ttgtcaacga
gttacacaat 1381 aatggacaga agcttgtcat cattgtggat ccagccatct
ccaacaactc ttcctcaagt 1441 aaaccctatg gcccatatga caggggttca
gatatgaaga tatgggtgaa tagttcagat 1501 ggagtgactc cactcattgg
ggaggtctgg cctggacaaa ctgtgtttcc tgattatacc 1561 aatcccaact
gtgctgtttg gtggacaaag gaatttgagc tttttcacaa tcaagtagag 1621
tttgatggaa tctggattga tatgaatgaa gtctccaact ttgttgatgg ttcggtctca
1681 ggatgttcca caaacaacct aaataatccc ccattcactc ccagaatcct
ggatgggtac 1741 ctgttctgca agactctctg tatggatgca gtgcagcact
ggggcaagca gtatgacatt 1801 cacaatctgt atggctactc catggcggtc
gccacagcag aagctgccaa gactgtgttc 1861 cctaataaga gaagcttcat
tctgacccgt tctacctttg cgggctctgg caagtttgca 1921 gcacattggt
taggagacaa cactgccacc tgggatgacc tgagatggtc catccctggc 1981
gtgcttgagt tcaacctttt tggcatccca atggtgggtc ctgacatatg tggctttgct
2041 ttggacaccc ctgaggagct ctgtaggcgg tggatgcagt tgggtgcatt
ttatccgttt 2101 tctagaaatc acaatggcca aggctacaag gaccaggatc
ctgcctcctt tggagctgac 2161 tccctgctgt tgaattcctc caggcactac
cttaacatcc gctatactct attgccctac 2221 ctatacaccc tcttcttccg
tgctcacagc cgaggggaca cggtggccag gccccttttg 2281 catgagttct
acgaggacaa cagcacttgg gatgtgcacc aacagttctt atgggggccc
2341 ggcctcctca tcactccagt tctggatgaa ggtgcagaga aagtgatggc
atatgtgcct 2401 gatgctgtct ggtatgacta cgagactggg agccaagtga
gatggaggaa gcaaaaagtc 2461 gagatggaac ttcctggaga caaaattgga
cttcaccttc gaggaggcta catcttcccc 2521 acacagcagc caaatacaac
cactctggcc agtcgaaaga accctcttgg tcttatcatt 2581 gccctagatg
agaacaaaga agcaaaagga gaacttttct gggataatgg ggaaacgaag 2641
gatactgtgg ccaataaagt gtatctttta tgtgagtttt ctgtcactca aaaccgcttg
2701 gaggtgaata tttcacaatc aacctacaag gaccccaata atttagcatt
taatgagatt 2761 aaaattcttg ggacggagga acctagcaat gttacagtga
aacacaatgg tgtcccaagt 2821 cagacttctc ctacagtcac ttatgattct
aacctgaagg ttgccattat cacagatatt 2881 gatcttctcc tgggagaagc
atacacagtg gaatggagca taaagataag ggatgaagaa 2941 aaaatagact
gttaccctga tgagaatggt gcttctgccg aaaactgcac tgcccgtggc 3001
tgtatctggg aggcatccaa ttcttctgga gtcccttttt gctattttgt caacgaccta
3061 tactctgtca gtgatgttca gtataattcc catggggcca cagctgacat
ctccttaaag 3121 tcttccgttt atgccaatgc cttcccctcc acacccgtga
acccccttcg cctggatgtc 3181 acttaccata agaatgaaat gctgcagttc
aagatttatg atcccaacaa gaatcggtat 3241 gaagttccag tccctctgaa
catacccagc atgccatcca gcacccctga gggtcaactc 3301 tatgatgtgc
tcattaagaa gaatccattt gggattgaaa ttcgccggaa gagtacaggc 3361
actataattt gggactctca gctccttggc tttaccttca gtgacatgtt tatccgcatc
3421 tccacccgcc ttccctccaa gtacctctat ggctttgggg aaactgagca
caggtcctat 3481 aggagagact tggagtggca cacttggggg atgttctccc
gagaccagcc cccagggtac 3541 aagaagaatt cctatggtgt ccacccctac
tacatggggc tggaggagga cggcagtgcc 3601 catggagtgc tcctgctgaa
cagcaatgcc atggatgtga cgttccagcc cctgcctgcc 3661 ttgacatacc
gcaccacagg gggagttctg gacttttatg tgttcttggg gccgactcca 3721
gagcttgtca cccagcagta cactgagttg attggccggc ctgtgatggt accttactgg
3781 tctttggggt tccagctgtg tcgctatggc taccagaatg actctgagat
cgccagcttg 3841 tatgatgaga tggtggctgc ccagatccct tatgatgtgc
agtactcaga catcgactac 3901 atggagcggc agctggactt caccctcagc
cccaagtttg ctgggtttcc agctctgatc 3961 aatcgcatga aggctgatgg
gatgcgggtc atcctcattc tggatccagc catttctggc 4021 aatgagacac
agccttatcc tgccttcact cggggcgtgg aggatgacgt cttcatcaaa 4081
tacccaaatg atggagacat tgtctgggga aaggtctggc ctgattttcc tgatgttgtt
4141 gtgaatgggt ctctagactg ggacagccaa gtggagctat atcgagctta
tgtggccttc 4201 ccagactttt tccgtaattc aactgccaag tggtggaaga
gggaaataga agaactatac 4261 aacaatccac agaatccaga gaggagcttg
aagtttgatg gcatgtggat tgatatgaat 4321 gaaccatcaa gcttcgtgaa
tggggcagtt tctccaggct gcagggacgc ctctctgaac 4381 caccctccct
acatgccaca tttggagtcc agggacaggg gcctgagcag caagaccctt 4441
tgtatggaga gtcagcagat cctcccagac ggctccctgg tgcagcacta caacgtgcac
4501 aacctgtatg ggtggtccca gaccagaccc acatacgaag ccgtgcagga
ggtgacggga 4561 cagcgagggg tcgtcatcac ccgctccaca tttccctctt
ctggccgctg ggcaggacat 4621 tggctgggag acaacacggc cgcatgggat
cagctgaaga agtctatcat tggcatgatg 4681 gagttcagcc tcttcggcat
atcctatacg ggagcagata tctgtgggtt ctttcaagat 4741 gctgaatatg
agatgtgtgt tcgctggatg cagctggggg ccttttaccc cttctcaaga 4801
aaccacaaca ccattgggac caggagacaa gaccctgtgt cctgggatgt tgcttttgtg
4861 aatatttcca gaactgtcct gcagaccaga tacaccctgt tgccatatct
gtataccttg 4921 atgcataagg cccacacgga gggcgtcact gttgtgcggc
ctctgctcca tgagtttgtg 4981 tcagaccagg tgacatggga catagacagt
cagttcctgc tgggcccagc cttcctggtc 5041 agccctgtcc tggagcgtaa
tgccagaaat gtcactgcat atttccctag agcccgctgg 5101 tatgattact
acacgggtgt ggatattaat gcaagaggag agtggaagac cttgccagcc 5161
cctcttgacc acattaatct tcatgtccgt gggggctaca tcctgccctg gcaagagcct
5221 gcactgaaca cccacttaag ccgccagaaa ttcatgggct tcaaaattgc
cttggatgat 5281 gaaggaactg ctgggggctg gctcttctgg gatgatgggc
aaagcattga tacctatggg 5341 aaaggactct attacttggc cagcttttct
gccagccaga atacgatgca aagccatata 5401 attttcaaca attacatcac
tggtacaaat cctttgaaac tgggctacat tgaaatctgg 5461 ggagtgggca
gtgtccccgt taccagtgtc agcatctctg tgagtggcat ggtcataaca 5521
ccctccttca acaatgaccc cacgacacag gtattaagca tcgatgtgac tgacagaaac
5581 atcagcctac ataattttac ttcattgacg tggataagca ctctgtgaat
ttttacagca 5641 agattctaac taactatgaa tgactttgaa actacttata
cttcatactc ataaaaatta 5701 ttgtgtgttg ctaatttgtt catacccact
attggtgaaa tatttctgtt aattttgtta 5761 tatgtttttt gtgtgaaccc
taaaggttaa accttagccc tgtgggatag gcagttaggg 5821 aggtgtggaa
aatctatgca ttaccttaat gtctctgtgt ggttagtatg gtagtgactg 5881
ttcatcatat gacatttact gaagatgaac tgggtccatg atgaagtgtg tgtatgtcca
5941 cgtttgtaat catagaatgg accccattct tttgttaaat acacaagaga
aagctttctg 6001 tgacagttcc aggtcttgaa gctaatcagc atctcaagaa
agtatccaga aagaacatct 6061 gctagttggt tataggcggt gggaggaata
atatacctaa ttggttatag gtggggggag 6121 catgataagc aaagaaaagg
caaacacaag gaaagatcag atgaaacaga agatgatagt 6181 aaaagtgatc
ctaagtaaga acataatgta aaattgtcag cagcctcatg gggaggaaaa 6241
aggaagagtc aactcacttg aagaagaggg tcttgagaaa tccttagcat aaagggctac
6301 tggtgagatt gagatctgag caggcaaagc tcaaaagaga gtttggaggt
taaaaataat 6361 ttatttttgc agtagtgtgc tttgaaatgt gtaaatctta
tttctaatgt atacaaccac 6421 atttcacata aaaatatgca atttatatgc
cagataaaaa taaaacaagt gaatttgcaa 6481 gtgaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaa SEQ ID NO: 9 is the human wild type amino acid
sequence corresponding to the lactase (LCT) enzyme (residues
1-1927) having GenBank Accession No. NP_002290: 1 MELSWHVVFI
ALLSFSCWGS DWESDRNFIS TAGPLTNDLL HNLSGLLGDQ SSNFVAGDKD 61
MYVCHQPLPT FLPEYFSSLH ASQITHYKVF LSWAQLLPAG STQNPDEKTV QCYRRLLKAL
121 KTARLQPMVI LHHQTLPAST LRRTEAFADL FADYATFAFH SFGDLVGIWF
TFSDLEEVIK 181 ELPHQESRAS QLQTLSDAHR KAYEIYHESY AFQGGKLSVV
LRAEDIPELL LEPPISALAQ 241 DTVDFLSLDL SYECQNEASL RQKLSKLQTI
EPKVKVFIFN LKLPDCPSTM KNPASLLFSL 301 FEAINKDQVL TIGFDINEFL
SCSSSSKKSM SCSLTGSLAL QPDQQQDHET TDSSPASAYQ 361 RIWEAFANQS
RAERDAFLQD TFPEGFLWGA STGAFNVEGG WAEGGRGVSI WDPRRPLNTT 421
EGQATLEVAS DSYHKVASDV ALLCGLRAQV YKFSISWSRI FPMGHGSSPS LPGVAYYNKL
481 IDRLQDAGIE PMATLFHWDL PQALQDHGGW QNESVVDAFL DYAAFCFSTF
GDRVKLWVTF 541 HEPWVMSYAG YGTGQHPPGI SDPGVASFKV AHLVLKAHAR
TWHHYNSHHR PQQQGHVGIV 601 LNSDWAEPLS PERPEDLRAS ERFLHFMLGW
FAHPVFVDGD YPATLRTQIQ QMNRQCSHPV 661 AQLPEFTEAE KQLLKGSADF
LGLSHYTSRL ISNAPQNTCI PSYDTIGGFS QHVNHVWPQT 721 SSSWIRVVPW
GIRRLLQFVS LEYTRGKVPI YLAGNGMPIG ESENLFDDSL RVDYFNQYIN 781
EVLKAIKEDS VDVRSYIARS LIDGFEGPSG YSQRFGLHHV NFSDSSKSRT PRKSAYFFTS
841 IIEKNGFLTK GAKRLLPPNT VNLPSKVRAF TFPSEVPSKA KVVWEKFSSQ
PKFERDLFYH 901 GTFRDDFLWG VSSSAYQIEG AWDADGKGPS IWDNFTHTPG
SNVKDNATGD IACDSYHQLD 961 ADLNMLRALK VKAYRFSISW SRIFPTGRNS
SINSHGVDYY NRLINGLVAS NIFPMVTLFH 1021 WDLPQALQDI GGWENPALID
LFDSYADFCF QTFGDRVKFW MTFNEPMYLA WLGYGSGEFP 1081 PGVKDPGWAP
YRIAHAVIKA HARVYHTYDE KYRQEQKGVI SLSLSTHWAE PKSPGVPRDV 1141
EAADRMLQFS LGWFAHPIFR NGDYPDTMKW KVGNRSELQH LATSRLPSFT EEEKRFIRAT
1201 ADVFCLNTYY SRIVQHKTPR LNPPSYEDDQ EMAEEEDPSW PSTAMNRAAP
WGTRRLLNWI 1261 KEEYGDIPIY ITENGVGLTN PNTEDTDRIF YHKTYINEAL
KAYRLDGIDL RGYVAWSLMD 1321 NFEWLNGYTV KFGLYHVDFN NTNRPRTARA
SARYYTEVIT NNGMPLARED EFLYGRFPEG 1381 FIWSAASAAY QIEGAWRADG
KGLSIWDTFS HTPLRVENDA IGDVACDSYH KIAEDLVTLQ 1441 NLGVSHYRFS
ISWSRILPDG TTRYINEAGL NYYVRLIDTL LAASIQPQVT IYHWDLPQTL 1501
QDVGGWENET IVQRFKEYAD VLFQRLGDKV KFWITLNEPF VIAYQGYGYG TAAPGVSNRP
1561 GTAPYIVGHN LIKAHAEAWH LYNDVYRASQ GGVISITISS DWAEPRDPSN
QEDVEAARRY 1621 VQFMGGWFAH PIFKNGDYNE VMKTRIRDRS LAAGLNKSRL
PEFTESEKRR INGTYDFFGF 1681 NHYTTVLAYN LNYATAISSF DADRGVASIA
DRSWPDSGSF WLKMTPFGFR RILNWLKEEY 1741 NDPPIYVTEN GVSQREETDL
NDTARIYYLR TYINEALKAV QDKVDLRGYT VWSAMDNFEW 1801 ATGFSERFGL
HFVNYSDPSL PRIPKASAKF YASVVRCNGF PDPATGPHAC LHQPDAGPTI 1861
SPVRQEEVQF LGLMLGTTEA QTALYVLFSL VLLGVCGLAF LSYKYCKRSK QGKTQRSQQE
1921 LSPVSSF SEQ ID NO: 10 is the human wild type nucleic acid
sequence corresponding to the lactase (LCT) enzyme (bps 1-6274)
having GenBank Accession No. NM_002299: 1 gttcctagaa aatggagctg
tcttggcatg tagtctttat tgccctgcta agtttttcat 61 gctgggggtc
agactgggag tctgatagaa atttcatttc caccgctggt cctctaacca 121
atgacttgct gcacaacctg agtggtctcc tgggagacca gagttctaac tttgtagcag
181 gggacaaaga catgtatgtt tgtcaccagc cactgcccac tttcctgcca
gaatacttca 241 gcagtctcca tgccagtcag atcacccatt ataaggtatt
tctgtcatgg gcacagctcc 301 tcccagcagg aagcacccag aatccagacg
agaaaacagt gcagtgctac cggcgactcc 361 tcaaggccct caagactgca
cggcttcagc ccatggtcat cctgcaccac cagaccctcc 421 ctgccagcac
cctccggaga accgaagcct ttgctgacct cttcgccgac tatgccacat 481
tcgccttcca ctccttcggg gacctagttg ggatctggtt caccttcagt gacttggagg
541 aagtgatcaa ggagcttccc caccaggaat caagagcgtc acaactccag
accctcagtg 601 atgcccacag aaaagcctat gagatttacc acgaaagcta
tgcttttcag ggcggaaaac 661 tctctgttgt cctgcgagct gaagatatcc
cggagctcct gctagaacca cccatatctg 721 cgcttgccca ggacacggtc
gatttcctct ctcttgattt gtcttatgaa tgccaaaatg 781 aggcaagtct
gcggcagaag ctgagtaaat tgcagaccat tgagccaaaa gtgaaagttt 841
tcatcttcaa cctaaaactc ccagactgcc cctccaccat gaagaaccca gccagtctgc
901 tcttcagcct ttttgaagcc ataaataaag accaagtgct caccattggg
tttgatatta 961 atgagtttct gagttgttca tcaagttcca agaaaagcat
gtcttgttct ctgactggca 1021 gcctggccct tcagcctgac cagcagcagg
accacgagac cacggactcc tctcctgcct 1081 ctgcctatca gagaatctgg
gaagcatttg ccaatcagtc cagggcggaa agggatgcct
1141 tcctgcagga tactttccct gaaggcttcc tctggggtgc ctccacagga
gcctttaacg 1201 tggaaggagg ctgggccgag ggtgggagag gggtgagcat
ctgggatcca cgcaggcccc 1261 tgaacaccac tgagggccaa gcgacgctgg
aggtggccag cgacagttac cacaaggtag 1321 cctctgacgt cgccctgctt
tgcggcctcc gggctcaggt gtacaagttc tccatctcct 1381 ggtcccggat
cttccccatg gggcacggga gcagccccag cctcccaggc gttgcctact 1441
acaacaagct gattgacagg ctacaggatg cgggcatcga gcccatggcc acgctgttcc
1501 actgggacct gcctcaggcc ctgcaggatc atggtggatg gcagaatgag
agcgtggtgg 1561 atgccttcct ggactatgcg gccttctgct tctccacatt
tggggaccgt gtgaagctgt 1621 gggtgacctt ccatgagccg tgggtgatga
gctacgcagg ctatggcacc ggccagcacc 1681 ctcccggcat ctctgaccca
ggagtggcct cttttaaggt ggctcacttg gtcctcaagg 1741 ctcatgccag
aacttggcac cactacaaca gccatcatcg cccacagcag caggggcacg 1801
tgggcattgt gctgaactca gactgggcag aacccctgtc tccagagagg cctgaggacc
1861 tgagagcctc tgagcgcttc ttgcacttca tgctgggctg gtttgcacac
cccgtctttg 1921 tggatggaga ctacccagcc accctgagga cccagatcca
acagatgaac agacagtgct 1981 cccatcctgt ggctcaactc cccgagttca
cagaggcaga gaagcagctc ctgaaaggct 2041 ctgctgattt tctgggtctg
tcgcattaca cctcccgcct catcagcaac gccccacaaa 2101 acacctgcat
ccctagctat gataccattg gaggcttctc ccaacacgtg aaccatgtgt 2161
ggccccagac ctcatcctct tggattcgtg tggtgccctg ggggataagg aggctgttgc
2221 agtttgtatc cctggaatac acaagaggaa aagttccaat ataccttgcc
gggaatggca 2281 tgcccatagg ggaaagtgaa aatctctttg atgattcctt
aagagtagac tacttcaatc 2341 aatatatcaa tgaggtgctc aaggctatca
aggaagactc tgtggatgtt cgttcctaca 2401 ttgctcgttc cctcattgat
ggcttcgaag gcccttctgg ttacagccag cggtttggcc 2461 tgcaccacgt
caacttcagc gacagcagca agtcaaggac tcccaggaaa tctgcctact 2521
ttttcactag catcatagaa aagaacggtt tcctcaccaa gggggcaaaa agactgctac
2581 cacctaatac agtaaacctc ccctccaaag tcagagcctt cacttttcca
tctgaggtgc 2641 cctccaaggc taaagtcgtt tgggaaaagt tctccagcca
acccaagttc gaaagagatt 2701 tgttctacca cgggacgttt cgggatgact
ttctgtgggg cgtgtcctct tccgcttatc 2761 agattgaagg cgcgtgggat
gccgatggca aaggccccag catctgggat aactttaccc 2821 acacaccagg
gagcaatgtg aaagacaatg ccactggaga catcgcctgt gacagctatc 2881
accagctgga tgccgatctg aatatgctcc gagctttgaa ggtgaaggcc taccgcttct
2941 ctatctcctg gtctcggatt ttcccaactg ggagaaacag ctctatcaac
agtcatgggg 3001 ttgattatta caacaggctg atcaatggct tggtggcaag
caacatcttt cccatggtga 3061 cattgttcca ttgggacctg ccccaggccc
tccaggatat cggaggctggg agaatcctg 3121 ccttgattga cttgtttgac
agctacgcag acttttgttt ccagacctttg gtgatagag 3181 tcaagttttg
gatgactttt aatgagccca tgtacctggc atggctaggtt atggctcag 3241
gggaatttcc cccaggggtg aaggacccag gctgggcacc atataggatag cccacgccg
3301 tcatcaaagc ccatgccaga gtctatcaca cgtacgatga gaaatacaggc
aggagcaga 3361 agggggtcat ctcgctgagc ctcagtacac actgggcaga
gcccaagtcac caggggtcc 3421 ccagagatgt ggaagccgct gaccgaatgc
tgcagttctc cctgggctggt ttgctcacc 3481 ccatttttag aaacggagac
tatcctgaca ccatgaagtg gaaagtgggga acaggagtg 3541 aactgcagca
cttagccacc tcccgcctgc caagcttcac tgaggaagaga agaggttca 3601
tcagggcgac ggccgacgtc ttctgcctca acacgtacta ctccagaatcg tgcagcaca
3661 aaacacccag gctaaaccca ccctcctacg aagacgacca ggagatggctg
aggaggagg 3721 acccttcgtg gccttccacg gcaatgaaca gagctgcgcc
ctgggggacgc gaaggctgc 3781 tgaactggat caaggaagag tatggtgaca
tccccattta catcaccgaaa acggagtgg 3841 ggctgaccaa tccgaacacg
gaggatactg ataggatatt ttaccacaaaa cctacatca 3901 atgaggcttt
gaaagcctac aggctcgatg gtatagacct tcgagggtatg tcgcctggt 3961
ctctgatgga caactttgag tggctaaatg gctacacggt caagtttggac tgtaccatg
4021 ttgatttcaa caacacgaac aggcctcgca cagcaagagc ctccgccaggt
actacacag 4081 aggtcattac caacaacggc atgccactgg ccagggagga
tgagtttctgt acggacggt 4141 ttcctgaggg cttcatctgg agtgcagctt
ctgctgcata tcagattgaag gtgcgtgga 4201 gagcagatgg caaaggactc
agcatttggg acacgttttc tcacacaccac tgagggttg 4261 agaacgatgc
cattggagac gtggcctgtg acagttatca caagattgctg aggatctgg 4321
tcaccctgca gaacctgggc gtgtcccact accgtttttc catctcctggt ctcgcatcc
4381 tccctgatgg aaccaccagg tacatcaatg aagcgggcct gaactactacg
tgaggctca 4441 tcgatacact gctggccgcc agcatccagc cccaggtgac
catttaccact gggacctac 4501 cacagacgct ccaagatgta ggaggctggg
agaatgagac catcgtgcagc ggtttaagg 4561 agtatgcaga tgtgctcttc
cagaggctgg gagacaaggt gaagttttgga tcacgctga 4621 atgagccctt
tgtcattgct taccagggct atggctacgg aacagcagctc caggagtct 4681
ccaataggcc tggcactgcc ccctacattg ttggccacaa tctaataaagg ctcatgctg
4741 aggcctggca tctgtacaac gatgtgtacc gcgccagtca aggtggcgtga
tttccatca 4801 ccatcagcag tgactgggct gaacccagag atccctctaa
ccaggaggatg tggaggcag 4861 ccaggagata tgttcagttc atgggaggct
ggtttgcaca tcctattttca agaatggag 4921 attacaatga ggtgatgaag
acgcggatcc gtgacaggag cttggctgcag gcctcaaca 4981 agtctcggct
gccagaattt acagagagtg agaagaggag gatcaacggca cctatgact 5041
tttttgggtt caatcactac accactgtcc tcgcctacaa cctcaactatg ccactgcca
5101 tctcttcttt tgatgcagac agaggagttg cttccatcgc agatcgctcgt
ggccagact 5161 ctggctcctt ctggctgaag atgacgcctt ttggcttcag
gaggatcctga actggttaa 5221 aggaggaata caatgaccct ccaatttatg
tcacagagaa tggagtgtccc agcgggaag 5281 aaacagacct caatgacact
gcaaggatct actaccttcg gacttacatca atgaggccc 5341 tcaaagctgt
gcaggacaag gtggaccttc gaggatacac agtttggagtg cgatggaca 5401
attttgagtg ggccacaggc ttttcagaga gatttggtct gcattttgtga actacagtg
5461 acccttctct gccaaggatc cccaaagcat cagcgaagtt ctacgcctctg
tggtccgat 5521 gcaatggctt ccctgacccc gctacagggc ctcacgcttg
tctccaccagc cagatgctg 5581 gacccaccat cagccccgtg agacaggagg
aggtgcagtt cctggggctaa tgctcggca 5641 ccacagaagc acagacagct
ttgtacgttc tcttttctct tgtgcttcttg gagtctgtg 5701 gcttggcatt
tctgtcatac aagtactgca agcgctctaa gcaagggaaaa cacaacgaa 5761
gccaacagga attgagcccg gtgtcttcat tctgatgagt taccacctcaa gttctatga
5821 agcaggccta gtttcttcat ctatgtttac cggccaccaa acaccttaggg
tcttagact 5881 ctgctgatac tggacttctc cataaagtcc tgctgcaccg
ttagagatgac tttaatctt 5941 gaatgatttc gacttgctga gtaaaatgga
aatatctcca tcttgctccag tatcagagt 6001 tcatttgggc atttgagaag
caagtagctc ttgcggaaac gtgtagatact ggtctagtg 6061 ggtctgtgaa
ccacttaatt gaacttaaca gggctgtttt aagtttcagag ttgttaagg 6121
gttgttaagg gagcaaaaac cgtaaaaatc cttcctataa gaagaaatcaa ctccattgc
6181 atagactgca atatcatctc ctgcccttct gcaagctctc cctagcttcac
atcttgtgt 6241 tttccagaaa ataaaaacag cagactgtcc tttc
[0088] As used herein, a "carbohydrate transporter molecule" means
a nucleic acid which encodes a polypeptide that exhibits
carbohydrate transporter activity, or a polypeptide or
peptidomimetic that exhibits carbohydrate transporter activity. For
example, a carbohydrate transporter molecule can include the human
GLUT2 protein (e.g., having the amino acid sequence shown in SEQ ID
NO: 1), or a variant thereof, such as a fragment thereof, that
exhibits carbohydrate transporter activity. For example, a
carbohydrate transporter molecule can include the human SGLT1
protein (e.g., having the amino acid sequence shown in SEQ ID NO:
3), or a variant thereof, such as a fragment thereof, that exhibits
carbohydrate transporter activity. The nucleic acid can be any type
of nucleic acid, including genomic DNA, complementary DNA (cDNA),
synthetic or semi-synthetic DNA, as well as any form of
corresponding RNA. For example, a carbohydrate transporter molecule
can comprise a recombinant nucleic acid encoding human GLUT2
protein or human SGLT1 protein. In one embodiment, a carbohydrate
transporter molecule can comprise a non-naturally occurring nucleic
acid created artificially (such as by assembling, cutting, ligating
or amplifying sequences). A carbohydrate transporter molecule can
be double-stranded. A carbohydrate transporter molecule can be
single-stranded. The carbohydrate transporter molecules of the
invention can be obtained from various sources and can be produced
according to various techniques known in the art. For example, a
nucleic acid that is a carbohydrate transporter molecule can be
obtained by screening DNA libraries, or by amplification from a
natural source. The carbohydrate transporter molecules of the
invention can be produced via recombinant DNA technology and such
recombinant nucleic acids can be prepared by conventional
techniques, including chemical synthesis, genetic engineering,
enzymatic techniques, or a combination thereof. Non-limiting
examples of a carbohydrate transporter molecule, that is a nucleic
acid, is the nucleic acid having the nucleotide sequence shown in
SEQ ID NO: 2 or SEQ ID NO: 4. Another example of a carbohydrate
transporter molecule is a fragment of a nucleic acid having the
sequence shown in SEQ ID NO: 2 or SEQ ID NO:4, wherein the fragment
is exhibits carbohydrate transporter activity.
[0089] As used herein, a "carbohydrate metabolic enzyme molecule"
means a nucleic acid which encodes a polypeptide that exhibits
carbohydrate metabolic enzyme activity, or a polypeptide or
peptidomimetic that exhibits carbohydrate metabolic enzyme
activity. For example, a carbohydrate metabolic enzyme molecule can
include the human sucrase-isomaltase (SI) protein (e.g., having the
amino acid sequence shown in SEQ ID NO: 5), or a variant thereof,
such as a fragment thereof, that exhibits carbohydrate metabolic
enzyme activity. For example, a carbohydrate metabolic enzyme
molecule can include the human maltase-glucoamylase protein (e.g.,
having the amino acid sequence shown in SEQ ID NO: 7), or a variant
thereof, such as a fragment thereof, that exhibits carbohydrate
metabolic enzyme activity. For example, a carbohydrate metabolic
enzyme molecule can include the human lactase protein (e.g., having
the amino acid sequence shown in SEQ ID NO: 9), or a variant
thereof, such as a fragment thereof, that exhibits carbohydrate
metabolic enzyme activity. The nucleic acid can be any type of
nucleic acid, including genomic DNA, complementary DNA (cDNA),
synthetic or semi-synthetic DNA, as well as any form of
corresponding RNA. For example, a carbohydrate metabolic enzyme
molecule can comprise a recombinant nucleic acid encoding human
sucrase-isomaltase (SI) protein, human maltase-glucoamylase
protein, or human lactase protein. In one embodiment, a
carbohydrate metabolic enzyme molecule can comprise a non-naturally
occurring nucleic acid created artificially (such as by assembling,
cutting, ligating or amplifying sequences). A carbohydrate
metabolic enzyme molecule can be double-stranded. A carbohydrate
metabolic enzyme molecule can be single-stranded. The carbohydrate
metabolic enzyme molecules of the invention can be obtained from
various sources and can be produced according to various techniques
known in the art. For example, a nucleic acid that is a
carbohydrate metabolic enzyme molecule can be obtained by screening
DNA libraries, or by amplification from a natural source. The
carbohydrate metabolic enzyme molecules of the invention can be
produced via recombinant DNA technology and such recombinant
nucleic acids can be prepared by conventional techniques, including
chemical synthesis, genetic engineering, enzymatic techniques, or a
combination thereof. A non-limiting example of a carbohydrate
metabolic enzyme, that is a nucleic acid, is the nucleic acid
having the nucleotide sequence shown in SEQ ID NO: 6, 8, or 10.
Another example of a carbohydrate metabolic enzyme molecule is a
fragment of a nucleic acid having the sequence shown in SEQ ID NO:
6, 8, or 10, wherein the fragment is exhibits carbohydrate
metabolic enzyme activity.
[0090] According to this invention, a carbohydrate transporter
molecule encompasses orthologs of human GLUT2 and SGLT1. According
to this invention, a carbohydrate metabolic enzyme molecule
encompass orthologs of human sucrase-isomaltase (SI), human
maltase-glucoamylase, and human lactase. For example, a
carbohydrate transporter molecule or a carbohydrate metabolic
enzyme molecule encompass the orthologs in mouse, rat, non-human
primates, canines, goat, rabbit, porcine, feline, and horses. In
other words, a carbohydrate transporter molecule or a carbohydrate
metabolic enzyme molecule can comprise a nucleic acid sequence
homologous to the human nucleic acid that encodes a human GLUT2 and
SGLT1 protein, or human sucrase-isomaltase (SI), human
maltase-glucoamylase, and human lactase protein, respectively,
wherein the nucleic acid is found in a different species and
wherein that homolog encodes a protein with a glucose transporter
function similar to a carbohydrate transporter molecule or an
enzymatic function similar to a carbohydrate metabolic enzyme
molecule.
[0091] A carbohydrate transporter molecule of this invention also
encompasses variants of the human nucleic acid encoding the GLUT2
or SGLT1 proteins that exhibit carbohydrate transporter activity,
or variants of the human GLUT2 or SGLT1 proteins that exhibit
carbohydrate transporter activity. A carbohydrate transporter
molecule of this invention also includes a fragment of the human
GLUT2 or SGLT1 nucleic acid which encodes a polypeptide that
exhibits carbohydrate transporter activity. A carbohydrate
transporter molecule of this invention encompasses a fragment of
the human GLUT2 or SGLT1 protein that exhibits carbohydrate
transporter activity.
[0092] A carbohydrate metabolic enzyme molecule of this invention
also encompasses variants of the human nucleic acid encoding the
sucrase-isomaltase (SI), human maltase-glucoamylase, and human
lactase proteins that exhibit carbohydrate metabolic enzyme
activity, or variants of the human sucrase-isomaltase (SI), human
maltase-glucoamylase, and human lactase proteins that exhibit
carbohydrate metabolic enzyme activity. A carbohydrate metabolic
enzyme molecule of this invention also includes a fragment of the
human sucrase-isomaltase (SI), human maltase-glucoamylase, and
human lactase nucleic acid which encodes a polypeptide that
exhibits carbohydrate metabolic enzyme activity. A carbohydrate
metabolic enzyme molecule of this invention encompasses a fragment
of the human sucrase-isomaltase (SI), human maltase-glucoamylase,
and human lactase protein that exhibits carbohydrate metabolic
enzyme activity.
[0093] The variants can comprise, for instance, naturally-occurring
variants due to allelic variations between individuals (e.g.,
polymorphisms), mutated alleles related to autism, or alternative
splicing forms. In one embodiment, a carbohydrate transporter
molecule is a nucleic acid variant of the nucleic acid having the
sequence shown in SEQ ID NO: 2, wherein the variant has a
nucleotide sequence identity to SEQ ID NO: 2 of at least about 65%,
at least about 75%, at least about 85%, at least about 90%, at
least about 91%, at least about 92%, at least about 93%, at least
about 94%, at least about 95%, at least about 96%, at least about
97%, at least about 98%, or at least about 99% with SEQ ID NO: 2.
In another embodiment, a carbohydrate transporter molecule is a
nucleic acid variant of the nucleic acid having the sequence shown
in SEQ ID NO: 4, wherein the variant has a nucleotide sequence
identity to SEQ ID NO: 4 of at least about 65%, at least about 75%,
at least about 85%, at least about 90%, at least about 91%, at
least about 92%, at least about 93%, at least about 94%, at least
about 95%, at least about 96%, at least about 97%, at least about
98%, or at least about 99% with SEQ ID NO: 4. In one embodiment, a
carbohydrate metabolic enzyme molecule is a nucleic acid variant of
the nucleic acid having the sequence shown in SEQ ID NO: 6, wherein
the variant has a nucleotide sequence identity to SEQ ID NO: 6 of
at least about 65%, at least about 75%, at least about 85%, at
least about 90%, at least about 91%, at least about 92%, at least
about 93%, at least about 94%, at least about 95%, at least about
96%, at least about 97%, at least about 98%, or at least about 99%
with SEQ ID NO: 6. In another embodiment, a carbohydrate metabolic
enzyme molecule is a nucleic acid variant of the nucleic acid
having the sequence shown in SEQ ID NO: 8, wherein the variant has
a nucleotide sequence identity to SEQ ID NO: 8 of at least about
65%, at least about 75%, at least about 85%, at least about 90%, at
least about 91%, at least about 92%, at least about 93%, at least
about 94%, at least about 95%, at least about 96%, at least about
97%, at least about 98%, or at least about 99% with SEQ ID NO: 8.
In a further embodiment, a carbohydrate metabolic enzyme molecule
is a nucleic acid variant of the nucleic acid having the sequence
shown in SEQ ID NO: 10, wherein the variant has a nucleotide
sequence identity to SEQ ID NO: 10 of at least about 65%, at least
about 75%, at least about 85%, at least about 90%, at least about
91%, at least about 92%, at least about 93%, at least about 94%, at
least about 95%, at least about 96%, at least about 97%, at least
about 98%, or at least about 99% with SEQ ID NO: 10.
[0094] In one embodiment, a carbohydrate transporter molecule
encompasses any portion of at least about 8 consecutive nucleotides
of SEQ ID NO: 2 or 4. In one embodiment, the fragment can comprise
at least about 15 nucleotides, at least about 20 nucleotides, or at
least about 30 nucleotides of SEQ ID NO: 2 or 4. Fragments include
all possible nucleotide lengths between about 8 and 100
nucleotides, for example, lengths between about 15 and 100, or
between about 20 and 100. In one embodiment, a carbohydrate
metabolic enzyme molecule encompasses any portion of at least about
8 consecutive nucleotides of SEQ ID NO: 6, 8, or 10. In one
embodiment, the fragment can comprise at least about 15
nucleotides, at least about 20 nucleotides, or at least about 30
nucleotides of SEQ ID NO: 6, 8, or 10. Fragments include all
possible nucleotide lengths between about 8 and 100 nucleotides,
for example, lengths between about 15 and 100, or between about 20
and 100.
[0095] The invention further provides for nucleic acids that are
complementary to a nucleic acid encoding GLUT2, SGLT1,
sucrase-isomaltase (SI), human maltase-glucoamylase, or human
lactase proteins. Such complementary nucleic acids can comprise
nucleic acid sequences, which hybridize to a nucleic acid sequence
encoding a GLUT2, SGLT1, sucrase-isomaltase (SI),
maltase-glucoamylase, or lactase protein under stringent
hybridization conditions. Non-limiting examples of stringent
hybridization conditions include temperatures above 30.degree. C.,
above 35.degree. C., in excess of 42.degree. C., and/or salinity of
less than about 500 mM, or less than 200 mM. Hybridization
conditions can be adjusted by the skilled artisan via modifying the
temperature, salinity and/or the concentration of other reagents
such as SDS or SSC.
[0096] In one embodiment, a carbohydrate transporter molecule
comprises a protein or polypeptide encoded by a carbohydrate
transporter nucleic acid sequence, such as the sequence shown in
SEQ ID NO: 2 or 4. In another embodiment, the polypeptide can be
modified, such as by glycosylations and/or acetylations and/or
chemical reaction or coupling, and can contain one or several
non-natural or synthetic amino acids. An example of a carbohydrate
transporter molecule is the polypeptide having the amino acid
sequence shown in SEQ ID NO: 1 or 3. In one embodiment, a
carbohydrate metabolic enzyme molecule comprises a protein or
polypeptide encoded by a carbohydrate metabolic enzyme nucleic acid
sequence, such as the sequence shown in SEQ ID NO: 6, 8, or 10. In
another embodiment, the polypeptide can be modified, such as by
glycosylations and/or acetylations and/or chemical reaction or
coupling, and can contain one or several non-natural or synthetic
amino acids. An example of a carbohydrate transporter molecule is
the polypeptide having the amino acid sequence shown in SEQ ID NO:
5, 7, or 9.
[0097] In another embodiment, a carbohydrate transporter molecule
can be a fragment of a carbohydrate transporter protein, such as
GLUT2 or SGLT1. For example, the carbohydrate transporter molecule
can encompass any portion of at least about 8 consecutive amino
acids of SEQ ID NO: 1 or 3. The fragment can comprise at least
about 10 amino acids, a least about 20 amino acids, at least about
30 amino acids, at least about 40 amino acids, a least about 50
amino acids, at least about 60 amino acids, or at least about 75
amino acids of SEQ ID NO: 1 or 3. In another embodiment, a
carbohydrate metabolic enzyme molecule can be a fragment of a
carbohydrate metabolic enzyme protein, such as sucrase-isomaltase
(SI), maltase-glucoamylase, or lactase. For example, the
carbohydrate metabolic enzyme molecule can encompass any portion of
at least about 8 consecutive amino acids of SEQ ID NO: 5, 7, or 9.
The fragment can comprise at least about 10 amino acids, a least
about 20 amino acids, at least about 30 amino acids, at least about
40 amino acids, a least about 50 amino acids, at least about 60
amino acids, or at least about 75 amino acids of SEQ ID NO: 5, 7,
or 9. Fragments include all possible amino acid lengths between
about 8 and 100 about amino acids, for example, lengths between
about 10 and 100 amino acids, between about 15 and 100 amino acids,
between about 20 and 100 amino acids, between about 35 and 100
amino acids, between about 40 and 100 amino acids, between about 50
and 100 amino acids, between about 70 and 100 amino acids, between
about 75 and 100 amino acids, or between about 80 and 100 amino
acids.
[0098] In certain embodiments, the carbohydrate transporter
molecule of the invention includes variants of the human GLUT2 or
SGLT1 protein (having the amino acid sequence shown in SEQ ID NO: 1
and 3, respectively). Such variants can include those having at
least from about 46% to about 50% identity to SEQ ID NO: 1 or 3, or
having at least from about 50.1% to about 55% identity to SEQ ID
NO: 1 or 3, or having at least from about 55.1% to about 60%
identity to SEQ ID NO: 1 or 3, or having from at least about 60.1%
to about 65% identity to SEQ ID NO: 1 or 3, or having from about
65.1% to about 70% identity to SEQ ID NO: 1 or 3, or having at
least from about 70.1% to about 75% identity to SEQ ID NO: 1 or 3,
or having at least from about 75.1% to about 80% identity to SEQ ID
NO: 1 or 3, or having at least from about 80.1% to about 85%
identity to SEQ ID NO: 1 or 3, or having at least from about 85.1%
to about 90% identity to SEQ ID NO: 1 or 3, or having at least from
about 90.1% to about 95% identity to SEQ ID NO: 1 or 3, or having
at least from about 95.1% to about 97% identity to SEQ ID NO: 1 or
3, or having at least from about 97.1% to about 99% identity to SEQ
ID NO: 1 or 3.
[0099] In certain embodiments, the carbohydrate metabolic enzyme
molecule of the invention includes variants of the human
sucrase-isomaltase (SI), maltase-glucoamylase, or lactase protein
(having the amino acid sequence shown in SEQ ID NO: 5, 7, and 9,
respectively). Such variants can include those having at least from
about 46% to about 50% identity to SEQ ID NO: 5, 7, or 9, or having
at least from about 50.1% to about 55% identity to SEQ ID NO: 5, 7,
or 9, or having at least from about 55.1% to about 60% identity to
SEQ ID NO: 5, 7, or 9, or having from at least about 60.1% to about
65% identity to SEQ ID NO: 5, 7, or 9, or having from about 65.1%
to about 70% identity to SEQ ID NO: 5, 7, or 9, or having at least
from about 70.1% to about 75% identity to SEQ ID NO: 5, 7, or 9, or
having at least from about 75.1% to about 80% identity to SEQ ID
NO: 5, 7, or 9, or having at least from about 80.1% to about 85%
identity to SEQ ID NO: 5, 7, or 9, or having at least from about
85.1% to about 90% identity to SEQ ID NO: 5, 7, or 9, or having at
least from about 90.1% to about 95% identity to SEQ ID NO: 5, 7, or
9, or having at least from about 95.1% to about 97% identity to SEQ
ID NO: 5, 7, or 9, or having at least from about 97.1% to about 99%
identity to SEQ ID NO: 5, 7, or 9.
[0100] In another embodiment, the carbohydrate transporter molecule
of the invention encompasses a peptidomimetic which exhibits
carbohydrate transporter activity. In another embodiment, the
carbohydrate transporter molecule of the invention encompasses a
peptidomimetic which exhibits carbohydrate transporter activity. In
another embodiment, the carbohydrate metabolic enzyme molecule of
the invention encompasses a peptidomimetic which exhibits
carbohydrate metabolic enzyme activity. In another embodiment, the
carbohydrate metabolic enzyme molecule of the invention encompasses
a peptidomimetic which exhibits carbohydrate metabolic enzyme
activity. A peptidomimetic is a small protein-like chain designed
to mimic a peptide that can arise from modification of an existing
peptide in order to protect that molecule from enzyme degradation
and increase its stability, and/or alter the molecule's properties
(for example modifications that change the molecule's stability or
biological activity). These modifications involve changes to the
peptide that can not occur naturally (such as altered backbones and
the incorporation of non-natural amino acids). Drug-like compounds
can be able to be developed from existing peptides. A
peptidomimetic can be a peptide, partial peptide or non-peptide
molecule that mimics the tertiary binding structure or activity of
a selected native peptide or protein functional domain (e.g.,
binding motif or active site). These peptide mimetics include
recombinantly or chemically modified peptides.
[0101] In one embodiment, a carbohydrate transporter molecule
comprising SEQ ID NO: 1, SEQ ID NO: 3, variants of each, or
fragments thereof, can be modified to produce peptide mimetics by
replacement of one or more naturally occurring side chains of the
20 genetically encoded amino acids (or D amino acids) with other
side chains. In one embodiment, a carbohydrate metabolic enzyme
molecule comprising SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,
variants of, or fragments thereof, can be modified to produce
peptide mimetics by replacement of one or more naturally occurring
side chains of the 20 genetically encoded amino acids (or D amino
acids) with other side chains. This can occur, for instance, with
groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered
alkyl, amide, amide lower alkyl, amide di(lower alkyl), lower
alkoxy, hydroxy, carboxy and the lower ester derivatives thereof,
and with 4, 5-, 6-, to 7-membered heterocyclics. For example,
proline analogs can be made in which the ring size of the proline
residue is changed from 5 members to 4, 6, or 7 members. Cyclic
groups can be saturated or unsaturated, and if unsaturated, can be
aromatic or non-aromatic. Heterocyclic groups can contain one or
more nitrogen, oxygen, and/or sulphur heteroatoms. Examples of such
groups include the furazanyl, ifuryl, imidazolidinyl imidazolyl,
imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g.
morpholino), oxazolyl, piperazinyl (e.g. 1-piperazinyl), piperidyl
(e.g. 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl,
pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl,
pyrrolidinyl (e.g. 1-pyrrolidinyl), pyrrolinyl, pyrrolyl,
thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g.
thiomorpholino), and triazolyl. These heterocyclic groups can be
substituted or unsubstituted. Where a group is substituted, the
substituent can be alkyl, alkoxy, halogen, oxygen, or substituted
or unsubstituted phenyl. Peptidomimetics can also have amino acid
residues that have been chemically modified by phosphorylation,
sulfonation, biotinylation, or the addition or removal of other
moieties. For example, peptidomimetics can be designed and directed
to amino acid sequences encoded by a carbohydrate transporter
molecule comprising SEQ ID NO: 1 or 3. For example, peptidomimetics
can be designed and directed to amino acid sequences encoded by a
carbohydrate metabolic enzyme molecule comprising SEQ ID NO: 5, 7,
or 9.
[0102] A variety of techniques are available for constructing
peptide mimetics with the same or similar desired biological
activity as the corresponding native but with more favorable
activity than the peptide with respect to solubility, stability,
and/or susceptibility to hydrolysis or proteolysis (see, e.g.,
Morgan & Gainor, Ann. Rep. Med. Chem. 24,243-252, 1989).
Certain peptidomimetic compounds are based upon the amino acid
sequence of the peptides of the invention. Peptidomimetic compounds
can be synthetic compounds having a three-dimensional structure
(i.e. a peptide motif) based upon the three-dimensional structure
of a selected peptide. The peptide motif provides the
peptidomimetic compound with the desired biological activity,
wherein the binding activity of the mimetic compound is not
substantially reduced, and is often the same as or greater than the
activity of the native peptide on which the mimetic is modeled.
Peptidomimetic compounds can have additional characteristics that
enhance their therapeutic application, such as increased cell
permeability, greater affinity and/or avidity and prolonged
biological half-life. Peptidomimetic design strategies are readily
available in the art (see, e.g., Ripka & Rich, Curr. Op. Chem.
Biol. 2, 441452, 1998; Hruby et al., Curr. Op. Chem. Biol. 1,
114119, 1997; Hruby & Balse, Curr. Med. Chem. 9,
945-970,-2000).
[0103] Diagnosis
[0104] The invention provides diagnosis methods based on monitoring
a gene encoding a carbohydrate metabolic enzyme molecule (such as
sucrase isomaltase, maltase glucoamylase, or lactase) or a
carbohydrate transporter molecule (such as GLUT2 or SGLT1). As used
herein, the term "diagnosis" includes the detection, typing,
monitoring, dosing, comparison, at various stages, including early,
pre-symptomatic stages, and late stages, in adults, children, and
unborn human children. Diagnosis can include the assessment of a
predisposition or risk of development, the prognosis, or the
characterization of a subject to define most appropriate treatment
(pharmacogenetics).
[0105] The invention provides diagnostic methods to determine
whether an individual is at risk of developing autism or an autism
spectrum disorder (ASD), or suffers from autism or an ASD, wherein
the disease reflects an alteration in the expression of a gene
encoding a carbohydrate metabolic enzyme molecule (such as sucrase
isomaltase, maltase glucoamylase, or lactase) or a carbohydrate
transporter molecule (such as GLUT2 or SGLT1). Subjects diagnosed
with autism, as well as ASD, can display some core symptoms in the
areas of a) social interactions and relationships, b) verbal and
non-verbal communication, and c) physical activity, play, physical
behavior. For example, symptoms related to social interactions and
relationships can include but are not limited to the inability to
establish friendships with children the same age, lack of empathy,
and the inability to develop nonverbal communicative skills (for
example, eye-to-eye gazing, facial expressions, and body posture).
For example, symptoms related to verbal and nonverbal communication
comprises delay in learning to talk, inability to learn to talk,
failure to initiate or maintain a conversation, failure to
interpret or understand implied meaning of words, and repetitive
use of language. For example, symptoms related to physical
activity, play, physical behavior include, but are not limited to
unusual focus on pieces or parts of an object, such as a toy, a
preoccupation with certain topics, a need for routines and rituals,
and stereotyped behaviors (for example, body rocking and hand
flapping).
[0106] In one embodiment, a method of detecting the presence of or
a predisposition to autism or an autism spectrum disorder in a
subject is provided. The subject can be a human or a child thereof.
The subject can also be a human embryo, a human fetus, or an unborn
human child. The method can comprise detecting in a sample from the
subject the presence of an alteration in the expression of a gene
of a carbohydrate metabolic enzyme molecule (such as sucrase
isomaltase, maltase glucoamylase, or lactase) or a carbohydrate
transporter molecule (such as GLUT2 or SGLT1). In one embodiment,
the detecting comprises detecting whether there is an alteration in
the gene locus encoding a carbohydrate metabolic enzyme molecule
(such as sucrase isomaltase, maltase glucoamylase, or lactase) or a
carbohydrate transporter molecule (such as GLUT2 or SGLT1). In a
further embodiment, the detecting comprises detecting whether
expression of a carbohydrate metabolic enzyme molecule (such as
sucrase isomaltase, maltase glucoamylase, or lactase) or a
carbohydrate transporter molecule (such as GLUT2 or SGLT1) is
reduced. In some embodiments, the detecting comprises detecting in
the sample whether there is a reduction in an mRNA encoding a
carbohydrate metabolic enzyme molecule or a carbohydrate
transporter molecule, or a reduction in either the carbohydrate
metabolic enzyme protein or a carbohydrate transporter protein, or
a combination thereof. The presence of such an alteration is
indicative of the presence or predisposition to autism or an autism
spectrum disorder. The presence of an alteration in a gene encoding
a carbohydrate metabolic enzyme molecule or a carbohydrate
transporter molecule in the sample is detected through the
genotyping of a sample, for example via gene sequencing, selective
hybridization, amplification, gene expression analysis, or a
combination thereof. In one embodiment, the sample can comprise
blood, serum, sputum, lacrimal secretions, semen, vaginal
secretions, fetal tissue, skin tissue, ileum tissue, cecum tissue,
muscle tissue, amniotic fluid, or a combination thereof
[0107] The invention also provides a method for treating or
preventing autism or an autism spectrum disorder in a subject. In
one embodiment, the method comprises (1) detecting the presence of
an alteration in a carbohydrate transporter gene or a carbohydrate
metabolic enzyme in a sample from the subject, where the presence
of the alteration is indicative of autism or an ASD, or the
predisposition to autism or ASD, and, (2) administering to the
subject in need a therapeutic treatment against autism or an autism
spectrum disorder. In one embodiment, the carbohydrate transporter
gene can be a GLUT2 gene or a SGLT1 gene. In another embodiment,
the carbohydrate metabolic enzyme gene can be a sucrase isomaltase
gene, a maltase glucoamylase gene, or a lactase gene. The
therapeutic treatment can be a drug administration (for example, a
pharmaceutical composition comprising a functional carbohydrate
transporter molecule or a functional carbohydrate metabolic enzyme
molecule). In one embodiment, the molecule comprises a carbohydrate
transporter polypeptide comprising at least about 75%, at least
about 80%, at least about 85%, at least about 90%, at least about
93%, at least about 95%, at least about 97%, at least about 98%, at
least about 99%, or 100% of the amino acid sequence of SEQ ID NO: 1
or SEQ ID NO: 3, and exhibits the function of restoring functional
carbohydrate transporter expression in deficient individuals, thus
restoring the capacity for carbohydrate transport. In another
embodiment, the molecule comprises a carbohydrate metabolic enzyme
polypeptide comprising at least about 75%, at least about 80%, at
least about 85%, at least about 90%, at least about 93%, at least
about 95%, at least about 97%, at least about 98%, at least about
99%, or 100% of the amino acid sequence of SEQ ID NO: 5, 7, or 9,
and exhibits the function of restoring functional carbohydrate
metabolic enzyme expression in deficient individuals, thus
restoring the capacity for carbohydrate metabolism.
[0108] In some embodiments, the molecule comprises a nucleic acid
encoding a carbohydrate transporter polypeptide comprising at least
about 75%, at least about 80%, at least about 85%, at least about
90%, at least about 93%, at least about 95%, at least about 97%, at
least about 98%, at least about 99%, or 100% of the nucleic acid
sequence of SEQ ID NO: 2 or 4 and encodes a polypeptide with the
function of restoring functional carbohydrate transporter
expression in deficient individuals, thus restoring the capacity
for carbohydrate transport. In further embodiments, the molecule
comprises a nucleic acid encoding a carbohydrate metabolic enzyme
polypeptide comprising at least about 75%, at least about 80%, at
least about 85%, at least about 90%, at least about 93%, at least
about 95%, at least about 97%, at least about 98%, at least about
99%, or 100% of the nucleic acid sequence of SEQ ID NO: 6, 8, or
10, and encodes a polypeptide with the function of restoring
functional carbohydrate metabolic enzyme expression in deficient
individuals, thus restoring the capacity for carbohydrate
metabolism.
[0109] The alteration can be determined at the DNA, RNA or
polypeptide level of the carbohydrate transporter or carbohydrate
metabolic enzyme. The detection can also be determined by
performing an oligonucleotide ligation assay, a confirmation based
assay, a hybridization assay, a sequencing assay, an
allele-specific amplification assay, a microsequencing assay, a
melting curve analysis, a denaturing high performance liquid
chromatography (DHPLC) assay (for example, see Jones et al, (2000)
Hum Genet., 106(6):663-8), or a combination thereof. In some
embodiments, the detection is performed by sequencing all or part
of a carbohydrate transporter or carbohydrate metabolic enzyme gene
or by selective hybridization or amplification of all or part of a
carbohydrate transporter or carbohydrate metabolic enzyme gene. A
carbohydrate transporter or carbohydrate metabolic enzyme gene
specific amplification can be carried out before the alteration
identification step.
[0110] An alteration in a carbohydrate transporter gene locus
(e.g., where GLUT2 or SGLT1 are located) or a carbohydrate
metabolic enzyme gene locus (e.g., where SI, MGAM, or LCT are
located) can be any form of mutation(s), deletion(s),
rearrangement(s) and/or insertions in the coding and/or non-coding
region of the locus, alone or in various combination(s). Mutations
can include point mutations. Insertions can encompass the addition
of one or several residues in a coding or non-coding portion of the
gene locus. Insertions can comprise an addition of between 1 and 50
base pairs in the gene locus. Deletions can encompass any region of
one, two or more residues in a coding or non-coding portion of the
gene locus, such as from two residues up to the entire gene or
locus. Deletions can affect smaller regions, such as domains
(introns) or repeated sequences or fragments of less than about 50
consecutive base pairs, although larger deletions can occur as
well. Rearrangement includes inversion of sequences. The
carbohydrate transporter gene locus alteration or carbohydrate
metabolic enzyme gene locus alteration can result in amino acid
substitutions, RNA splicing or processing, product instability, the
creation of stop codons, frame-shift mutations, and/or truncated
polypeptide production. The alteration can result in the production
of a carbohydrate transporter polypeptide or a carbohydrate
metabolic enzyme with altered function, stability, targeting or
structure. The alteration can also cause a reduction in protein
expression. In one embodiment, the alteration in a carbohydrate
transporter gene locus can comprise a point mutation, a deletion,
or an insertion in the carbohydrate transporter gene or
corresponding expression product. In another embodiment, the
alteration in a carbohydrate metabolic enzyme gene locus can
comprise a point mutation, a deletion, or an insertion in the
carbohydrate metabolic enzyme gene or corresponding expression
product. In one embodiment, the alteration can be a deletion or
partial deletion of a carbohydrate transporter gene or a
carbohydrate metabolic enzyme gene. The alteration can be
determined at the level of the DNA, RNA, or polypeptide of a
carbohydrate transporter or a carbohydrate metabolic enzyme.
[0111] In another embodiment, the method can comprise detecting the
presence of an altered RNA expression of a carbohydrate transporter
or a carbohydrate metabolic enzyme. Altered RNA expression includes
the presence of an altered RNA sequence, the presence of an altered
RNA splicing or processing, or the presence of an altered quantity
of RNA. These can be detected by various techniques known in the
art, including by sequencing all or part of the RNA of a
carbohydrate transporter or a carbohydrate metabolic enzyme, or by
selective hybridization or selective amplification of all or part
of the RNA. In a further embodiment, the method can comprise
detecting the presence of an altered polypeptide expression of a
carbohydrate transporter or a carbohydrate metabolic enzyme.
Altered polypeptide expression includes the presence of an altered
polypeptide sequence, the presence of an altered quantity of
carbohydrate transporter polypeptide or carbohydrate metabolic
enzyme polypeptide, or the presence of an altered tissue
distribution. These can be detected by various techniques known in
the art, including by sequencing and/or binding to specific ligands
(such as antibodies).
[0112] Various techniques known in the art can be used to detect or
quantify altered gene expression, RNA expression, or sequence,
which include, but are not limited to, hybridization, sequencing,
amplification, and/or binding to specific ligands (such as
antibodies). Other suitable methods include allele-specific
oligonucleotide (ASO), oligonucleotide ligation, allele-specific
amplification, Southern blot (for DNAs), Northern blot (for RNAs),
single-stranded conformation analysis (SSCA), PFGE, fluorescent in
situ hybridization (FISH), gel migration, clamped denaturing gel
electrophoresis, denaturing HLPC, melting curve analysis,
heteroduplex analysis, RNase protection, chemical or enzymatic
mismatch cleavage, ELISA, radio-immunoassays (RIA) and
immuno-enzymatic assays (IEMA). Some of these approaches (such as
SSCA and CGGE) are based on a change in electrophoretic mobility of
the nucleic acids, as a result of the presence of an altered
sequence. According to these techniques, the altered sequence is
visualized by a shift in mobility on gels. The fragments can then
be sequenced to confirm the alteration. Some other approaches are
based on specific hybridization between nucleic acids from the
subject and a probe specific for wild type or altered gene or RNA.
The probe can be in suspension or immobilized on a substrate. The
probe can be labeled to facilitate detection of hybrids. Some of
these approaches are suited for assessing a polypeptide sequence or
expression level, such as Northern blot, ELISA and RIA. These
latter require the use of a ligand specific for the polypeptide,
for example, the use of a specific antibody.
[0113] Sequencing.
[0114] Sequencing can be carried out using techniques well known in
the art, using automatic sequencers. The sequencing can be
performed on the complete gene or on specific domains thereof, such
as those known or suspected to carry deleterious mutations or other
alterations.
[0115] Amplification.
[0116] Amplification is based on the formation of specific hybrids
between complementary nucleic acid sequences that serve to initiate
nucleic acid reproduction. Amplification can be performed according
to various techniques known in the art, such as by polymerase chain
reaction (PCR), ligase chain reaction (LCR), strand displacement
amplification (SDA) and nucleic acid sequence based amplification
(NASBA). These techniques can be performed using commercially
available reagents and protocols. Useful techniques in the art
encompass real-time PCR, allele-specific PCR, or PCR-SSCP.
Amplification usually requires the use of specific nucleic acid
primers, to initiate the reaction. For example, nucleic acid
primers useful for amplifying sequences from the gene or locus of a
carbohydrate transporter (such as GLUT2 or SGLT1) or a carbohydrate
metabolic enzyme (such as SI, MGAM, or LCT) are able to
specifically hybridize with a portion of the gene locus that flanks
a target region of the locus, wherein the target region is altered
in certain subjects having autism or an autism spectrum disorder.
In one embodiment, amplification comprises using forward and
reverse RT-PCR primers comprising nucleotide sequences of SEQ ID
NOS: 26, 27, 29, 30, 32, 33, 35, 36, 38, or 39.
[0117] The invention provides for a nucleic acid primer, wherein
the primer can be complementary to and hybridize specifically to a
portion of a coding sequence (e.g., gene or RNA) of a carbohydrate
transporter (such as GLUT2 or SGLT1) or a carbohydrate metabolic
enzyme (such as SI, MGAM, or LCT) that is altered in certain
subjects having autism or an autism spectrum disorder. Primers of
the invention can thus be specific for altered sequences in a gene
or RNA of a carbohydrate transporter or a carbohydrate metabolic
enzyme. By using such primers, the detection of an amplification
product indicates the presence of an alteration in the gene or the
absence of such gene. Examples of primers of this invention can be
single-stranded nucleic acid molecules of about 5 to 60 nucleotides
in length, or about 8 to about 25 nucleotides in length. The
sequence can be derived directly from the sequence of the
carbohydrate transporter or the carbohydrate metabolic enzyme gene
(e.g., GLUT2 or SGLT1, and SI, MGAM, or LCT, respectively). Perfect
complementarity is useful, to ensure high specificity. However,
certain mismatch can be tolerated. In one embodiment, the primer
can be an isolated nucleic acid comprising a nucleotide sequence of
SEQ ID NOS: 26, 27, 29, 30, 32, 33, 35, 36, 38, or 39. For example,
a nucleic acid primer or a pair of nucleic acid primers as
described above can be used in a method for detecting the presence
of or a predisposition to autism or an autism spectrum disorder in
a subject.
[0118] Amplification methods include, e.g., polymerase chain
reaction, PCR (PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS,
ed. Innis, Academic Press, N.Y., 1990 and PCR STRATEGIES, 1995, ed.
Innis, Academic Press, Inc., N.Y., ligase chain reaction (LCR)
(see, e.g., Wu, Genomics 4:560, 1989; Landegren, Science 241:1077,
1988; Barringer, Gene 89:117, 1990); transcription amplification
(see, e.g., Kwoh, Proc. Natl. Acad. Sci. USA 86:1173, 1989); and,
self-sustained sequence replication (see, e.g., Guatelli, Proc.
Natl. Acad. Sci. USA 87:1874, 1990); Q Beta replicase amplification
(see, e.g., Smith, J. Clin. Microbiol. 35:1477-1491, 1997),
automated Q-beta replicase amplification assay (see, e.g., Burg,
Mol. Cell. Probes 10:257-271, 1996) and other RNA polymerase
mediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario);
see also Berger, Methods Enzymol. 152:307-316, 1987; Sambrook;
Ausubel; U.S. Pat. Nos. 4,683,195 and 4,683,202; Sooknanan,
Biotechnology 13:563-564, 1995. All the references stated above are
incorporated by reference in their entireties.
[0119] Selective Hybridization.
[0120] Hybridization detection methods are based on the formation
of specific hybrids between complementary nucleic acid sequences
that serve to detect nucleic acid sequence alteration(s). A
detection technique involves the use of a nucleic acid probe
specific for wild type or altered gene or RNA, followed by the
detection of the presence of a hybrid. The probe can be in
suspension or immobilized on a substrate or support (for example,
as in nucleic acid array or chips technologies). The probe can be
labeled to facilitate detection of hybrids. In one embodiment, the
probe according to the invention can comprise a nucleic acid having
SEQ ID NOS: 28, 31, 34, 37, or 40. For example, a sample from the
subject can be contacted with a nucleic acid probe specific for a
wild type carbohydrate transporter or carbohydrate metabolic enzyme
gene or an altered carbohydrate transporter or carbohydrate
metabolic enzyme gene, and the formation of a hybrid can be
subsequently assessed. In one embodiment, the method comprises
contacting simultaneously the sample with a set of probes that are
specific, respectively, for the wild type carbohydrate transporter
or carbohydrate metabolic enzyme gene and for various altered forms
thereof. Thus, it is possible to detect directly the presence of
various forms of alterations in the carbohydrate transporter gene
(e.g., GLUT2 or SGLT1) or carbohydrate metabolic enzyme gene (e.g.,
SI, MGAM, or LCT) in the sample. Also, various samples from various
subjects can be treated in parallel.
[0121] According to the invention, a probe can be a polynucleotide
sequence which is complementary to and specifically hybridizes with
a, or a target portion of a, carbohydrate transporter or
carbohydrate metabolic enzyme gene or RNA, and that is suitable for
detecting polynucleotide polymorphisms associated with alleles of
such, which predispose to or are associated with autism or an
autism spectrum disorder. Useful probes are those that are
complementary to the carbohydrate transporter or carbohydrate
metabolic enzyme gene, RNA, or target portion thereof. Probes can
comprise single-stranded nucleic acids of between 8 to 1000
nucleotides in length, for instance between 10 and 800, between 15
and 700, or between 20 and 500. Longer probes can be used as well.
A useful probe of the invention is a single stranded nucleic acid
molecule of between 8 to 500 nucleotides in length, which can
specifically hybridize to a region of a gene or RNA that carries an
alteration.
[0122] The sequence of the probes can be derived from the sequences
of the carbohydrate transporter or carbohydrate metabolic enzyme
genes provided herein. Nucleotide substitutions can be performed,
as well as chemical modifications of the probe. Such chemical
modifications can be accomplished to increase the stability of
hybrids (e.g., intercalating groups) or to label the probe. Some
examples of labels include, without limitation, radioactivity,
fluorescence, luminescence, and enzymatic labeling.
[0123] A guide to the hybridization of nucleic acids is found in
e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL
(2.sup.ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, 1989;
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley
& Sons, Inc., New York, 1997; LABORATORY TECHNIQUES IN
BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID
PROBES, PART I. Theory and Nucleic Acid Preparation, Tijssen, ed.
Elsevier, N.Y., 1993.
[0124] Specific Ligand Binding.
[0125] As indicated herein, alteration in a carbohydrate
transporter or carbohydrate metabolic enzyme gene locus or in
carbohydrate transporter or carbohydrate metabolic enzyme
expression can also be detected by screening for alteration(s) in
corresponding polypeptide sequence or expression levels. Different
types of ligands can be used, such as specific antibodies. In one
embodiment, the sample is contacted with an antibody specific for a
carbohydrate transporter or carbohydrate metabolic enzyme
polypeptide and the formation of an immune complex is subsequently
determined. Various methods for detecting an immune complex can be
used, such as ELISA, radioimmunoassays (RIA) and immuno-enzymatic
assays (IEMA).
[0126] For example, an antibody can be a polyclonal antibody, a
monoclonal antibody, as well as fragments or derivatives thereof
having substantially the same antigen specificity. Fragments
include Fab, Fab'2, or CDR regions. Derivatives include
single-chain antibodies, humanized antibodies, or poly-functional
antibodies. An antibody specific for a carbohydrate transporter or
a carbohydrate metabolic enzyme polypeptide can be an antibody that
selectively binds a carbohydrate transporter or carbohydrate
metabolic enzyme polypeptide, respectively, namely, an antibody
raised against a carbohydrate transporter or carbohydrate metabolic
enzyme polypeptide or an epitope-containing fragment thereof.
Although non-specific binding towards other antigens can occur,
binding to the target polypeptide occurs with a higher affinity and
can be reliably discriminated from non-specific binding. In one
embodiment, the method comprises contacting a sample from the
subject with an antibody specific for a wild type or an altered
form of a carbohydrate transporter or carbohydrate metabolic enzyme
polypeptide, and determining the presence of an immune complex.
Optionally, the sample can be contacted to a support coated with
antibody specific for the wild type or altered form of a
carbohydrate transporter or carbohydrate metabolic enzyme
polypeptide. In one embodiment, the sample can be contacted
simultaneously, or in parallel, or sequentially, with various
antibodies specific for different forms of a carbohydrate
transporter or carbohydrate metabolic enzyme polypeptide, such as a
wild type and various altered forms thereof.
[0127] The invention also provides for a diagnostic kit comprising
products and reagents for detecting in a sample from a subject the
presence of an alteration in a carbohydrate transporter gene (e.g.,
GLUT2 or SGLT1) or a carbohydrate metabolic enzyme gene (e.g., SI,
MGAM, or LCT), or a carbohydrate transporter polypeptide or
carbohydrate metabolic enzyme polypeptide; alteration in the
expression of a carbohydrate transporter gene (e.g., GLUT2 or
SGLT1) or carbohydrate metabolic enzyme gene (e.g., SI, MGAM, or
LCT), or a carbohydrate transporter or carbohydrate metabolic
enzyme polypeptide; and/or an alteration in carbohydrate
transporter or carbohydrate metabolic enzyme activity. The kit can
be useful for determining whether a sample from a subject exhibits
reduced carbohydrate transporter or carbohydrate metabolic enzyme
expression or exhibits a gene deletion of a carbohydrate
transporter (e.g., GLUT2 or SGLT1) or carbohydrate metabolic enzyme
(e.g., SI, MGAM, or LCT). For example, the diagnostic kit according
to the present invention comprises any primer, any pair of primers,
any nucleic acid probe and/or any ligand, (for example, an antibody
directed to a carbohydrate transporter or carbohydrate metabolic
enzyme). The diagnostic kit according to the present invention can
further comprise reagents and/or protocols for performing a
hybridization, amplification or antigen-antibody immune reaction.
In one embodiment, the kit can comprise nucleic acid primers that
specifically hybridize to and can prime a polymerase reaction from
a carbohydrate transporter (e.g., GLUT2 or SGLT1) or carbohydrate
metabolic enzyme (e.g., SI, MGAM, or LCT). In another embodiment,
the primer can comprise a nucleotide sequence of SEQ ID NOS: 26,
27, 29, 30, 32, 33, 35, 36, 38, or 39.
[0128] The diagnosis methods can be performed in vitro, ex vivo, or
in vivo. These methods utilize a sample from the subject in order
to assess the status of a carbohydrate transporter gene locus or a
carbohydrate metabolic enzyme gene locus. The sample can be any
biological sample derived from a subject, which contains nucleic
acids or polypeptides. Examples of such samples include, but are
not limited to, fluids, tissues, cell samples, organs, or tissue
biopsies. Non-limiting examples of samples include blood, plasma,
saliva, urine, or seminal fluid. Pre-natal diagnosis can also be
performed by testing fetal cells or placental cells, for instance.
Screening of parental samples can also be used to determine
risk/likelihood of offspring possessing the germline mutation. The
sample can be collected according to conventional techniques and
used directly for diagnosis or stored. The sample can be treated
prior to performing the method, in order to render or improve
availability of nucleic acids or polypeptides for testing.
Treatments include, for instance, lysis (e.g., mechanical,
physical, or chemical), centrifugation. Also, the nucleic acids
and/or polypeptides can be pre-purified or enriched by conventional
techniques, and/or reduced in complexity. Nucleic acids and
polypeptides can also be treated with enzymes or other chemical or
physical treatments to produce fragments thereof. In one
embodiment, the sample is contacted with reagents, such as probes,
primers, or ligands, in order to assess the presence of an altered
carbohydrate transporter gene locus or carbohydrate metabolic
enzyme gene locus. Contacting can be performed in any suitable
device, such as a plate, tube, well, or glass. In specific
embodiments, the contacting is performed on a substrate coated with
the reagent, such as a nucleic acid array or a specific ligand
array. The substrate can be a solid or semi-solid substrate such as
any support comprising glass, plastic, nylon, paper, metal, or
polymers. The substrate can be of various forms and sizes, such as
a slide, a membrane, a bead, a column, or a gel. The contacting can
be made under any condition suitable for a complex to be formed
between the reagent and the nucleic acids or polypeptides of the
sample.
[0129] Identifying an altered polypeptide, RNA or DNA of a
carbohydrate transporter (e.g., GLUT2 or SGLT1) or a carbohydrate
metabolic enzyme (e.g., SI, MGAM, or LCT) in the sample is
indicative of the presence of an altered carbohydrate transporter
or carbohydrate metabolic enzyme gene in the subject, which can be
correlated to the presence, predisposition or stage of progression
of autism or an autism spectrum disorder. For example, an
individual having a germ line mutation in a carbohydrate
transporter gene (e.g., GLUT 2 or SGLT1) or a carbohydrate
metabolic enzyme gene (e.g., SI, MGAM, or LCT) has an increased
risk of developing autism or an autism spectrum disorder. The
determination of the presence of an altered gene locus in a subject
also allows the design of appropriate therapeutic intervention,
which is more effective and customized. Also, this determination at
the pre-symptomatic level allows a preventive regimen to be
applied.
[0130] GI Bacterial Colonization in ASD Subjects
[0131] An aspect of the invention provides for a new PCR strategy
for the identification, quantitation, and taxonomic classification
of Sutterella bacterial colonization from biological samples. As
shown in Example 2 herein, intestinal biopsies of children with
autism accompanied by gastrointestinal (GI) complaints showed
highly significant elevation of intestinal levels of Sutterella
bacteria. These findings can provide insights into pathogenesis of
autism associated with GI disorder, enabling new strategies for
therapeutic intervention.
[0132] Bacterial members of the genus Sutterella, a class of
Beta-proteobacteria in the order Burkholderiales and the family
Alcaligenaceae have been associated with human infections below the
diaphragm (A1). Furthermore, Sutterella sp. sequences have been
identified in intestinal biopsies and fecal samples from
individuals with Crohn's disease and ulcerative colitis (A2, A3).
Sutterella sp. have also been found in canine faeces and the cecal
microbiota of domestic and wild turkeys (A4, A5). However, little
is known about the pathogenic potential of Sutterella sp. According
to the Sutterella sp.-specific PCR methods described herein,
Sutterella detection can be achieved in a mammal, such as a dog, a
cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, a
turkey, or a human.
[0133] Sutterella bacterial infections have been associated with
ASD in addition to Crohn's disease and ulcerative colitis.
Bacterial infections are also associated with various intestinal
diseases, such as irritable bowel syndrome (IBS). Over 40 million
people in the U.S. suffer from irritable bowel syndrome (IBS), a
type of inflammatory bowel disease. IBS, though not fatal, has a
huge impact on quality-of-life. After the common cold, IBS is the
second most common reason for missed work and is estimated to
generate $30B in healthcare costs. Few simple molecular diagnostic
tests for IBS/IBD are presently available. Diagnosis usually relies
upon symptom analysis and/or invasive colonoscopy procedures. The
IBD/IBS diagnostics market has significant growth potential.
[0134] Little is known of the epidemiology and pathogenesis of
Sutterella infection and their role in Crohn's disease, ASD, and
ulcerative colitis. Current methods for Sutterella biopsies are
costly, laborious and non-specific. There are no known rapid,
specific, or cost-effective technologies to identify Sutterella sp.
in biological samples.
[0135] An aspect of the invention provides for a PCR assay that
allows for rapid identification, quantification, classification,
and diagnosis of Sutterella sp. in biological or industrial
samples. This would allow for specific therapies to be implemented
in subjects in need (e.g., ASD patients, IB patients, intestinal
disease patients, etc.) following identification of Sutterella in
infections. Directed administration of antimicrobial agents (e.g.,
antibiotics) that limit the growth of Sutterella could be
fascilitated rapidly following identification of Sutterella
species. An antibiotic refers to any compound known to one of
ordinary skill in the art that will inhibit the growth of, or kill,
bacteria. Useful, non-limiting examples of an antibiotic include
lincosamides (clindomycin); chloramphenicols; tetracyclines (such
as Tetracycline, Chlortetracycline, Demeclocycline, Methacycline,
Doxycycline, Minocycline); aminoglycosides (such as Gentamicin,
Tobramycin, Netilmicin, Amikacin, Kanamycin, Streptomycin,
Neomycin); beta-lactams (such as penicillins, cephalosporins,
Imipenem, Aztreonam); vancomycins; bacitracins; macrolides
(erythromycins), amphotericins; sulfonamides (such as
Sulfanilamide, Sulfamethoxazole, Sulfacetamide, Sulfadiazine,
Sulfisoxazole, Sulfacytine, Sulfadoxine, Mafenide, p-Aminobenzoic
Acid, Trimethoprim-Sulfamethoxazole); Methenamin; Nitrofurantoin;
Phenazopyridine; trimethoprim; rifampicins; metronidazoles;
cefazolins; Lincomycin; Spectinomycin; mupirocins; quinolones (such
as Nalidixic Acid, Cinoxacin, Norfloxacin, Ciprofloxacin,
Perfloxacin, Ofloxacin, Enoxacin, Fleroxacin, Levofloxacin);
novobiocins; polymixins; gramicidins; and antipseudomonals (such as
Carbenicillin, Carbenicillin Indanyl, Ticarcillin, Azlocillin,
Mezlocillin, Piperacillin) or any salts or variants thereof. Such
antibiotics can be obtained commercially, e.g., from Daiichi
Sankyo, Inc. (Parsipanny, N.J.), Merck (Whitehouse Station, N.J.),
Pfizer (New York, N.Y.), Glaxo Smith Kline (Research Triangle Park,
N.C.), Johnson & Johnson (New Brunswick, N.J.), AstraZeneca
(Wilmington, Del.), Novartis (East Hanover, N.J.), and
Sanofi-Aventis (Bridgewater, N.J.). The antibiotic used will depend
on the type of bacterial infection.
[0136] In one embodiment, the invention provides for a method of
detecting Sutterella sp. DNA from biological or industrial sources,
e.g. intestinal tissue, feces, blood, or skin. In another
embodiment, the invention provides for Sutterella diagnostics to
detect Sutterella sp. in samples from children with autism as well
as patients with intestinal disease, e.g. irritable bowel syndrome
(IBS). In some embodiments, the invention provides for PCR-based
methods of assessing a subject's response to exposure to
therapeutic treatments directed at bacterial infections, for
example, Sutterella sp. infections, or exposure to other
pathogens.
[0137] For example, primers having SEQ ID NOS: 11, 12, 15, or 16
can be used for detecting Sutterella sp. DNA. SEQ ID NOS: 17 and 18
can also be used for detecting Sutterella sp. DNA.
TABLE-US-00002 Sutt For Primer (SEQ ID NO: 17)- ##STR00001## Sutt
Rev Primer (SEQ ID NO: 18)- 5'-CCCTCTGTTCCGACCATTGTATGACGTGTGA GCCC
AGC C TAAGGGCCATGAGGACTT-3' Sutt Probe 3 (SEQ ID NO: 19)-
##STR00002##
[0138] In addition to the primers having SEQ ID NOS: 11, 12, and
15-18, additional primers containing any part of SEQ ID NOS: 17,
18, or 19 and containing any portion of the italicized DNA sequence
regions can be used to assess the presence or absence of Sutterella
species. Further, inclusion of degenerate bases (bolded and
underlined) can be used to increase coverage of Sutterella species
(for example, where S can be a G nucleotide and/or a C nucleotide;
where Y can be a C nucleotide and/or T nucleotide; where R can be
an A nucleotide and/or G nucleotide; where W can be an A nucleotide
and/or T nucleotide; where H can be an A nucleotide and/or T
nucleotide and/or C nucleotide; where B can be a T nucleotide, C
nucleotide, or G nucleotide; where V can be an A nucleotide, G
nucleotide, or C nucleotide; where D can be an A nucleotide, G
nucleotide, or T nucleotide; where K can be a G nucleotide or T
nucleotide).
[0139] In addition to the highlighted probe sequence of SEQ ID
NO:19 as well as SEQ ID NOS: 13 and 14, any portion of SEQ ID NO:
19 shown above can be used for Sutterella species detection and
quantitation. The reverse complement of SEQ ID NOS: 11, 12, or
15-19 can also be used to detect the opposite DNA strand of
Sutterella species 16S rRNA genes.
[0140] The invention can be used to detect Sutterella sp. 16S rRNA
sequences in small amounts of DNA from any biological or industrial
source. These sources include, but are not limited to human or
animal intestinal tissue, feces, blood, or skin (swabs or tissue).
Based on these findings, the invention can be used to detect,
quantitate, and classify Sutterella sp. in biological samples from
children with Autism. In one embodiment, the invention can be used
to detect Sutterella sp. in biological samples from individuals
with various forms of intestinal disease. Intestinal diseases
include, but are not limited to, Crohn's disease and Ulcerative
colitis. In one embodiment, detection of Sutterella sp. can occur
in biological samples from any undiagnosed infection below or above
the diaphragm. The invention will allow for large cohort
investigations of Sutterella sp. in the aforementioned, and as yet
to be determined, diseases in order to establish an association
between Sutterella sp. and disease manifestation. In one
embodiment, the presence and quantity of Sutterella sp. in
intestinal tissues can be investigated following any number of
experimental manipulations. Experimental manipulations include, but
are not limited to, responses to chemicals (i.e. antibiotics),
changes in diet, pathogen exposure (i.e. pathogenic viruses,
bacteria, fungi), or probiotic usage. The rapid identification of
Sutterella sp. in human and animal samples facilitated by this
invention can lead to rapid diagnosis and directed antimicrobial
treatment of infections caused by Sutterella sp.
[0141] Gene, Vectors, Recombinant Cells, and Polypeptides
[0142] The invention encompasses an altered or mutated genes of a
carbohydrate transporter or carbohydrate metabolic enzyme, or a
fragment thereof. The invention also encompasses nucleic acid
molecules encoding an altered or mutated polypeptide of s
carbohydrate transporter or carbohydrate metabolic enzyme, or a
fragment thereof. The alteration or mutation of the nucleotide or
amino acid sequence modifies the carbohydrate transporter or
carbohydrate metabolic enzyme activity, respectively. The invention
provides for a vector that comprises a nucleic acid encoding a
carbohydrate transporter or carbohydrate metabolic enzyme
polypeptide (for example, a nucleic acid comprising SEQ ID NO: 2 or
4, and a nucleic acid comprising SEQ ID NO: 6, 8, or 10,
respectively) or mutant thereof. The vector can be a cloning vector
or an expression vector, i.e., a vector comprising regulatory
sequences causing resulting in the expression of carbohydrate
transporter or carbohydrate metabolic enzyme polypeptides from the
vector in a competent host cell. These vectors can be used to
express polypeptides, or mutants thereof, of carbohydrate
transporters or carbohydrate metabolic enzymes in vitro, ex vivo,
or in vivo, to create transgenic or Knock-Out non-human animals, to
amplify the nucleic acids, or to express antisense RNAs.
[0143] The nucleic acids used to practice the invention, whether
RNA, RNAi, antisense nucleic acid, cDNA, genomic DNA, vectors,
viruses or hybrids thereof, can be produced or isolated from a
variety of sources, genetically engineered, amplified, and/or
expressed/generated recombinantly. Recombinant polypeptides
generated from these nucleic acids can be individually isolated or
cloned and tested for a desired activity. Any recombinant
expression system can be used, including bacterial, mammalian,
yeast, insect or plant cell expression systems. Alternatively,
these nucleic acids can be synthesized in vitro by well-known
chemical synthesis techniques, as described in, e.g., Adams, J. Am.
Chem. Soc. 105:661, 1983; Belousov, Nucleic Acids Res.
25:3440-3444, 1997; Frenkel, Free Radic. Biol. Med. 19:373-380,
1995; Blommers, Biochemistry 33:7886-7896, 1994; Narang, Meth.
Enzymol. 68:90, 1979; Brown Meth. Enzymol. 68:109, 1979; Beaucage,
Tetra. Lett. 22:1859, 1981; U.S. Pat. No. 4,458,066, all of which
are incorporated by reference in their entireties.
[0144] The invention provides oligonucleotides comprising sequences
of the invention, e.g., subsequences of the exemplary sequences of
the invention. Oligonucleotides can include, e.g., single stranded
poly-deoxynucleotides or two complementary polydeoxynucleotide
strands which can be chemically synthesized.
[0145] Techniques for the manipulation of nucleic acids, such as,
subcloning, labeling probes (for example, random-primer labeling
using Klenow polymerase, nick translation, amplification),
sequencing, and hybridization are well described in the scientific
and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING:
A LABORATORY MANUAL (2.sup.ND ED.), Vols. 1-3, Cold Spring Harbor
Laboratory, 1989; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel,
ed. John Wiley & Sons, Inc., New York, 1997; LABORATORY
TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION
WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid
Preparation, Tijssen, ed. Elsevier, N.Y., 1993.
[0146] Nucleic acids, vectors, or polypeptides can be analyzed and
quantified by any of a number of general means well known to those
of skill in the art. These include, for example, analytical
biochemical methods such as radiography, electrophoresis, NMR,
spectrophotometry, capillary electrophoresis, thin layer
chromatography (TLC), high performance liquid chromatography
(HPLC), and hyperdiffusion chromatography; various immunological
methods, such as immuno-electrophoresis, Southern analysis,
Northern analysis, dot-blot analysis, fluid or gel precipitation
reactions, immunodiffusion, quadrature radioimmunoassay (RIAs),
enzyme-linked immunosorbent assays (ELISAs), immuno-fluorescent
assays, gel electrophoresis (e.g., SDS-PAGE), nucleic acid or
target or signal amplification methods, radiolabeling,
scintillation counting, and affinity chromatography.
[0147] Obtaining and manipulating nucleic acids used to practice
the methods of the invention can be done by cloning from genomic
samples, and, if desired, screening and re-cloning inserts isolated
or amplified from, e.g., genomic clones or cDNA clones. Sources of
nucleic acid used in the methods of the invention include genomic
or cDNA libraries contained in, e.g., mammalian artificial
chromosomes (MACs), see, e.g., U.S. Pat. Nos. 5,721,118; 6,025,155;
human artificial chromosomes, see, e.g., Rosenfeld, Nat. Genet.
15:333-335, 1997; yeast artificial chromosomes (YAC); bacterial
artificial chromosomes (BAC); P1 artificial chromosomes, see, e.g.,
Woon, Genomics 50:306-316, 1998; P1-derived vectors (PACs), see,
e.g., Kern, Biotechniques 23:120-124, 1997; cosmids, recombinant
viruses, phages or plasmids
[0148] The vectors of this invention can comprise a coding sequence
for a carbohydrate transporter molecule or a carbohydrate metabolic
enzyme molecule that is operably linked to regulatory sequences,
e.g., a promoter, or a polyA tail. Operably linked indicates that
the coding and regulatory sequences are functionally associated so
that the regulatory sequences cause expression (e.g.,
transcription) of the coding sequences. The vectors can further
comprise one or several origins of replication and/or selectable
markers. The promoter region can be homologous or heterologous with
respect to the coding sequence, and can provide for ubiquitous,
constitutive, regulated and/or tissue specific expression, in any
appropriate host cell, including for in vivo use. Examples of
promoters include bacterial promoters (T7, pTAC, Trp promoter),
viral promoters (LTR, TK, CMV-IE), mammalian gene promoters
(albumin, PGK), etc.
[0149] The vector can be a plasmid, a virus, a cosmid, a phage, a
BAC, a YAC. Plasmid vectors can be prepared from commercially
available vectors such as pBluescript, pUC, or pBR. Viral vectors
can be produced from baculoviruses, retroviruses, adenoviruses, or
AAVs, according to recombinant DNA techniques known in the art. In
one embodiment, a recombinant virus can encode a polypeptide of a
carbohydrate transporter or carbohydrate metabolic enzyme of the
invention. The recombinant virus is useful if
replication-defective, for example, if selected from E1- and/or
E4-defective adenoviruses, Gag-, pol- and/or env-defective
retroviruses and Rep- and/or Cap-defective AAVs. Such recombinant
viruses can be produced by techniques known in the art, such as by
transfecting packaging cells or by transient transfection with
helper plasmids or viruses. Examples of virus packaging cells
include PA317 cells, PsiCRIP cells, GPenv+ cells, or 293 cells.
Detailed protocols for producing such replication-defective
recombinant viruses can be found for instance in WO95/14785,
WO96/22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S.
Pat. No. 4,861,719, U.S. Pat. No. 5,278,056 and WO94/19478, which
are all hereby incorporated by reference.
[0150] In another embodiment, the invention provides for a
recombinant host cell comprising a recombinant carbohydrate
transporter gene (e.g., GLUT2 or SGLT1) or a carbohydrate metabolic
enzyme gene (e.g., SI, MGAM, or LCT), or a recombinant vector as
described herein. Suitable host cells include, without limitation,
prokaryotic cells (such as bacteria) and eukaryotic cells (such as
yeast cells, mammalian cells, insect cells, or plant cells).
Specific examples include E. coli, the yeasts Kluyveromyces or
Saccharomyces, mammalian cell lines (e.g., Vero cells, CHO cells,
3T3 cells, or COS cells) as well as primary or established
mammalian cell cultures (e.g., produced from fibroblasts, embryonic
cells, epithelial cells, nervous cells, or adipocytes). In a
further embodiment, the invention provides a method for producing a
recombinant host cell expressing a polypeptide of a carbohydrate
transporter or carbohydrate metabolic enzyme. The method can entail
(a) introducing in vitro or ex vivo into a competent host cell a
recombinant nucleic acid or a vector as described herein, (b)
culturing in vitro or ex vivo the recombinant host cells obtained,
and (c) optionally, selecting the cells which express the
polypeptide of a carbohydrate transporter or carbohydrate metabolic
enzyme. Such recombinant host cells can be used for the production
of carbohydrate transporter or carbohydrate metabolic enzyme
polypeptides, as well as for screening of active molecules, as
described below. Such cells can also be used as a model system to
study autism. These cells can be maintained in suitable culture
media, such as HAM, DMEM, or RPMI, in any appropriate culture
device (plate, flask, dish, tube, or pouch).
[0151] The practice of aspects of the present invention can employ,
unless otherwise indicated, conventional techniques of cell
biology, cell culture, molecular biology, transgenic biology,
microbiology, recombinant DNA, and immunology, which are within the
skill of the art. Such techniques are explained fully in the
literature. See, for example, Molecular Cloning A Laboratory
Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring
Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.
N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed.,
1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid
Hybridization (B. D. Hames & S. J. Higgins eds. 1984);
Transcription And Translation (B. D. Hames & S. J. Higgins eds.
1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc.,
1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal,
A Practical Guide To Molecular Cloning (1984); the treatise,
Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer
Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,
1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols.
154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And
Molecular Biology (Caner and Walker, eds., Academic Press, London,
1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.
Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse
Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1986). All patents, patent applications and references cited
herein are incorporated in their entirety by reference.
[0152] Administration and Dosing
[0153] A carbohydrate transporter molecule (e.g., GLUT2 or SGLT1)
or carbohydrate metabolic enzyme molecule (e.g., SI, MGAM, or LCT)
can be administered to the subject once (e.g., as a single
injection or deposition). Alternatively, a carbohydrate transporter
or carbohydrate metabolic enzyme molecule of the invention can be
administered once or twice daily to a subject in need thereof for a
period of from about two to about twenty-eight days, or from about
seven to about ten days. It can also be administered once or twice
daily to a subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12 times per year, or a combination thereof. Furthermore, the
carbohydrate transporter or carbohydrate metabolic enzyme molecule
of the invention can be co-administrated with another therapeutic,
such as an anti-depressant, an anti-psychotic, a benzodiazepine
drug, or a combination thereof. Where a dosage regimen comprises
multiple administrations, the effective amount of the carbohydrate
transporter or carbohydrate metabolic enzyme molecule administered
to the subject can comprise the total amount of gene product
administered over the entire dosage regimen.
[0154] The carbohydrate transporter or carbohydrate metabolic
enzyme molecules of the invention can be administered to a subject
by any means suitable for delivering the carbohydrate transporter
or carbohydrate metabolic enzyme molecules to cells of the subject,
such as ileum cell or cecum cells. For example, carbohydrate
transporter or carbohydrate metabolic enzyme molecules can be
administered by methods suitable to transfect cells. Transfection
methods for eukaryotic cells are well known in the art, and include
direct injection of the nucleic acid into the nucleus or pronucleus
of a cell; electroporation; liposome transfer or transfer mediated
by lipophilic materials; receptor mediated nucleic acid delivery,
bioballistic or particle acceleration; calcium phosphate
precipitation, and transfection mediated by viral vectors.
[0155] The compositions of this invention can be formulated and
administered to reduce the symptoms associated with autism or an
ASD by any means that produces contact of the active ingredient
with the agent's site of action in the body of an animal. They can
be administered by any conventional means available for use in
conjunction with pharmaceuticals, either as individual therapeutic
active ingredients or in a combination of therapeutic active
ingredients. They can be administered alone, but are generally
administered with a pharmaceutical carrier selected on the basis of
the chosen route of administration and standard pharmaceutical
practice.
[0156] Pharmaceutical compositions for use in accordance with the
invention can be formulated in conventional manner using one or
more physiologically acceptable carriers or excipients. The
therapeutic compositions of the invention can be formulated for a
variety of routes of administration, including systemic and topical
or localized administration. Techniques and formulations generally
can be found in Remmington's Pharmaceutical Sciences, Meade
Publishing Co., Easton, Pa. (1985), the entire disclosure of which
is herein incorporated by reference. For systemic administration,
an injection is useful, including intramuscular, intravenous,
intraperitoneal, and subcutaneous. For injection, the therapeutic
compositions of the invention can be formulated in liquid
solutions, for example in physiologically compatible buffers such
as Hank's solution or Ringer's solution. In addition, the
therapeutic compositions can be formulated in solid form and
redissolved or suspended immediately prior to use. Lyophilized
forms are also included. Pharmaceutical compositions of the present
invention are characterized as being at least sterile and
pyrogen-free. These pharmaceutical formulations include
formulations for human and veterinary use.
[0157] Pharmaceutical formulations of the invention can comprise a
carbohydrate transporter or carbohydrate metabolic enzyme molecule
(e.g., 0.1 to 90% by weight), or a physiologically acceptable salt
thereof, mixed with a pharmaceutically-acceptable carrier. The
pharmaceutical formulations of the invention can also comprise the
carbohydrate transporter or carbohydrate metabolic enzyme molecules
of the invention which are encapsulated by liposomes and a
pharmaceutically-acceptable carrier. Useful
pharmaceutically-acceptable carriers are water, buffered water,
normal saline, 0.4% saline, 0.3% glycine, or hyaluronic acid.
[0158] Pharmaceutical compositions of the invention can also
comprise conventional pharmaceutical excipients and/or additives.
Suitable pharmaceutical excipients include stabilizers,
antioxidants, osmolality adjusting agents, buffers, and pH
adjusting agents. Suitable additives include physiologically
biocompatible buffers (e.g., tromethamine hydrochloride), additions
of chelants (such as, for example, DTPA or DTPA-bisamide) or
calcium chelate complexes (as for example calcium DTPA,
CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium
salts (for example, calcium chloride, calcium ascorbate, calcium
gluconate or calcium lactate). Pharmaceutical compositions of the
invention can be packaged for use in liquid form, or can be
lyophilized.
[0159] For solid pharmaceutical compositions of the invention,
conventional nontoxic solid pharmaceutically-acceptable carriers
can be used; for example, pharmaceutical grades of mannitol,
lactose, starch, magnesium stearate, sodium saccharin, talcum,
cellulose, glucose, sucrose, or magnesium carbonate.
[0160] Solid formulations can be used for enteral (oral)
administration. They can be formulated as, e.g., pills, tablets,
powders or capsules. For solid compositions, conventional nontoxic
solid carriers can be used which include, e.g., pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharin, talcum, cellulose, glucose, sucrose, or magnesium
carbonate. For oral administration, a pharmaceutically acceptable
nontoxic composition is formed by incorporating any of the normally
employed excipients, such as those carriers previously listed, and
generally 10% to 95% of active ingredient (e.g., peptide). A
non-solid formulation can also be used for enteral administration.
The carrier can be selected from various oils including those of
petroleum, animal, vegetable or synthetic origin, e.g., peanut oil,
soybean oil, mineral oil, or sesame oil. Suitable pharmaceutical
excipients include e.g., starch, cellulose, talc, glucose, lactose,
sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium
stearate, sodium stearate, glycerol monostearate, sodium chloride,
dried skim milk, glycerol, propylene glycol, water, ethanol.
[0161] Nucleic acids, peptides, or polypeptides of the invention,
when administered orally, can be protected from digestion. This can
be accomplished either by complexing the nucleic acid, peptide or
polypeptide with a composition to render it resistant to acidic and
enzymatic hydrolysis or by packaging the nucleic acid, peptide or
polypeptide in an appropriately resistant carrier such as a
liposome. Means of protecting compounds from digestion are well
known in the art, see, e.g., Fix, Pharm Res. 13: 1760-1764, 1996;
Samanen, J. Pharm. Pharmacol. 48: 119-135, 1996; U.S. Pat. No.
5,391,377, describing lipid compositions for oral delivery of
therapeutic agents (for example, liposomal delivery). In one
embodiment, the carbohydrate transporter molecule (e.g., GLUT2 or
SGLT1) or carbohydrate metabolic enzyme molecule (e.g., SI, MGAM,
or LCT) can be delivered to the alimentary canal or intestine of
the subject via oral administration that is can withstand digestion
and degradation.
[0162] For oral administration, the therapeutic compositions can
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets can be
coated by methods well known in the art. Liquid preparations for
oral administration can take the form of, for example, solutions,
syrups or suspensions, or they can be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations can be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., ationd oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations can
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate.
[0163] Preparations for oral administration can be suitably
formulated to give controlled release of the active agent. For
buccal administration the therapeutic compositions can take the
form of tablets or lozenges formulated in a conventional manner.
For administration by inhalation, the compositions for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebuliser, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit can be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g., gelatin for use in an inhaler or insufflate or
can be formulated containing a powder mix of the therapeutic agents
and a suitable powder base such as lactose or starch.
[0164] The therapeutic compositions can be formulated for
parenteral administration by injection, e.g., by bolus injection or
continuous infusion. Formulations for injection can be presented in
unit dosage form, e.g., in ampoules or in multi-dose containers,
with an added preservative. The compositions can take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and can contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient can
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0165] Suitable enteral administration routes for the present
methods include oral, rectal, or intranasal delivery. Suitable
parenteral administration routes include intravascular
administration (e.g. intravenous bolus injection, intravenous
infusion, intra-arterial bolus injection, intra-arterial infusion
and catheter instillation into the vasculature); peri- and
intra-tissue injection (e.g., peri-tumoral and intra-tumoral
injection, intra-retinal injection, or subretinal injection);
subcutaneous injection or deposition including subcutaneous
infusion (such as by osmotic pumps); direct application to the
tissue of interest, for example by a catheter or other placement
device (e.g., a retinal pellet or a suppository or an implant
comprising a porous, non-porous, or gelatinous material); and
inhalation. For example, the carbohydrate transporter or
carbohydrate metabolic enzyme molecules of the invention can be
administered by injection, infusion, or oral delivery.
[0166] In addition to the formulations described previously, the
therapeutic compositions can also be formulated as a depot
preparation. Such long acting formulations can be administered by
implantation (for example subcutaneously or intramuscularly) or by
intramuscular injection. For example, the therapeutic compositions
can be formulated with suitable polymeric or hydrophobic materials
(for example as an emulsion in an acceptable oil) or ion exchange
resins, or as sparingly soluble derivatives, for example, as a
sparingly soluble salt.
[0167] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration bile
salts and fusidic acid derivatives. In addition, detergents can be
used to facilitate permeation. Transmucosal administration can be
through nasal sprays or using suppositories. For topical
administration, the compositions of the invention are formulated
into ointments, salves, gels, or creams as generally known in the
art. A wash solution can be used locally to treat an injury or
inflammation to accelerate healing. For oral administration, the
therapeutic compositions are formulated into conventional oral
administration forms such as capsules, tablets, and tonics.
[0168] A composition of the present invention can also be
formulated as a sustained and/or timed release formulation. Such
sustained and/or timed release formulations can be made by
sustained release means or delivery devices that are well known to
those of ordinary skill in the art, such as those described in U.S.
Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719;
4,710,384; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543;
5,639,476; 5,354,556; and 5,733,566, the disclosures of which are
each incorporated herein by reference. The pharmaceutical
compositions of the present invention can be used to provide slow
or sustained release of one or more of the active ingredients
using, for example, hydropropylmethyl cellulose, other polymer
matrices, gels, permeable membranes, osmotic systems, multilayer
coatings, microparticles, liposomes, microspheres, or the like, or
a combination thereof to provide the desired release profile in
varying proportions. Suitable sustained release formulations known
to those of ordinary skill in the art, including those described
herein, can be readily selected for use with the pharmaceutical
compositions of the invention. Single unit dosage forms suitable
for oral administration, such as, but not limited to, tablets,
capsules, gel-caps, caplets, or powders, that are adapted for
sustained release are encompassed by the present invention.
[0169] In the present methods, the carbohydrate transporter or
carbohydrate metabolic enzyme molecules can be administered to the
subject either as RNA, in conjunction with a delivery reagent, or
as a nucleic acid (e.g., a recombinant plasmid or viral vector)
comprising sequences which expresses the gene product. Suitable
delivery reagents for administration of the carbohydrate
transporter or carbohydrate metabolic enzyme molecules include the
Minis Transit TKO lipophilic reagent; lipofectin; lipofectamine;
cellfectin; or polycations (e.g., polylysine), or liposomes.
[0170] The dosage administered can be a therapeutically effective
amount of the composition sufficient to result in amelioration of
symptoms of autism or an autism spectrum disorder in a subject, and
can vary depending upon known factors such as the pharmacodynamic
characteristics of the active ingredient and its mode and route of
administration; age, sex, health and weight of the recipient;
nature and extent of symptoms; kind of concurrent treatment,
frequency of treatment and the effect desired. For example, an
effective enzyme unit of amount of SI, MGAM, and/or LCT can be
administered to a subject in need thereof. The enzyme unit (U) is a
unit for the amount of a particular enzyme. One U is defined as the
amount of the enzyme that catalyzes the conversion of 1 micro mole
of substrate per minute. In one embodiment, the therapeutically
effective amount of the administered carbohydrate enzyme (e.g., SI,
MGAM, or LCT) is at least about 1 U, at least about 10 U, at least
about 20 U, at least about 25 U, at least about 50 U, at least
about 100 U, at least about 150 U, at least about 200 U, at least
about 250 U, at least about 300 U, at least about 350 U, at least
about 400 U, at least about 450 U, at least about 500 U, at least
about 550 U, at least about 600 U, at least about 650 U, at least
about 700 U, at least about 750 U, at least about 800 U, at least
about 850 U, at least about 900 U, at least about 950 U, at least
about 1000 U, at least about 1250 U, at least about 1500 U, at
least about 1750 U, at least about 2000 U, at least about 2250 U,
at least about 2500 U, at least about 2750 U, at least about 3000
U, at least about 3250 U, at least about 3500 U, at least about
4000 U, at least about 4500 U, at least about 5000 U, at least
about 5500 U, at least about 6000 U, at least about 6500 U, at
least about 7000 U, at least about 7500 U, at least about 8000 U,
at least about 8500 U, at least about 9000 U, at least about 9250
U, at least about 9500 U, or at least about 10,000 U.
[0171] In some embodiments, the effective amount of the
administered carboydrate transporter molecule (e.g., GLUT2 or
SGLT1) is at least about 0.01 .mu.g/kg body weight, at least about
0.025 .mu.g/kg body weight, at least about 0.05 .mu.g/kg body
weight, at least about 0.075 .mu.g/kg body weight, at least about
0.1 .mu.g/kg body weight, at least about 0.25 .mu.g/kg body weight,
at least about 0.5 .mu.g/kg body weight, at least about 0.75
.mu.g/kg body weight, at least about 1 .mu.g/kg body weight, at
least about 5 .mu.g/kg body weight, at least about 10 .mu.g/kg body
weight, at least about 25 .mu.g/kg body weight, at least about 50
.mu.g/kg body weight, at least about 75 .mu.g/kg body weight, at
least about 100 .mu.g/kg body weight, at least about 150 .mu.g/kg
body weight, at least about 200 .mu.g/kg body weight, at least
about 250 .mu.g/kg body weight, at least about 300 .mu.g/kg body
weight, at least about 350 .mu.g/kg body weight, at least about 400
.mu.g/kg body weight, at least about 450 .mu.g/kg body weight, at
least about 500 .mu.g/kg body weight, at least about 550 .mu.g/kg
body weight, at least about 600 .mu.g/kg body weight, at least
about 650 .mu.g/kg body weight, at least about 700 .mu.g/kg body
weight, at least about 750 .mu.g/kg body weight, at least about 800
.mu.g/kg body weight, at least about 850 .mu.g/kg body weight, at
least about 900 .mu.g/kg body weight, at least about 950 .mu.g/kg
body weight, or at least about 1000 .mu.g/kg body weight.
[0172] Toxicity and therapeutic efficacy of therapeutic
compositions of the present invention can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., for determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio LD.sub.50/ED.sub.50. Therapeutic agents that
exhibit large therapeutic indices are useful. Therapeutic
compositions that exhibit some toxic side effects can be used.
[0173] A therapeutically effective dose of carbohydrate transporter
or carbohydrate metabolic enzyme molecules can depend upon a number
of factors known to those or ordinary skill in the art. The dose(s)
of the carbohydrate transporter or carbohydrate metabolic enzyme
molecules can vary, for example, depending upon the identity, size,
and condition of the subject or sample being treated, further
depending upon the route by which the composition is to be
administered, if applicable, and the effect which the practitioner
desires the carbohydrate transporter or carbohydrate metabolic
enzyme molecules to have upon the nucleic acid or polypeptide of
the invention. These amounts can be readily determined by a skilled
artisan.
[0174] Pharmaceutical Composition and Therapy
[0175] The invention provides methods for treating or preventing
autism or an autism spectrum disorder in a subject. In one
embodiment, the method can comprise administering to the subject a
functional (e.g., wild-type) carbohydrate transporter molecule
(e.g., GLUT2 or SGLT1) or carbohydrate metabolic enzyme molecule
(e.g., SI, MGAM, or LCT), which can be a polypeptide or a nucleic
acid.
[0176] Various approaches can be carried out to restore the
carbohydrate transporter or carbohydrate metabolic enzyme activity
or function in a subject, such as those carrying an altered gene
locus comprising a carbohydrate transporter gene (e.g., GLUT2 or
SGLT1) or a carbohydrate metabolic enzyme gene (e.g., SI, MGAM, or
LCT). Supplying wild-type function of the carbohydrate transporter
or carbohydrate metabolic enzyme to such subjects can suppress
phenotypic expression of autism or an autism spectrum disorders in
a pathological cell or organism. Increasing carbohydrate
transporter or carbohydrate metabolic enzyme activity can be
accomplished through gene or protein therapy as discussed later
herein.
[0177] A nucleic acid encoding a carbohydrate transporter or
carbohydrate metabolic enzyme or a functional part thereof can be
introduced into the cells of a subject in one embodiment of the
invention. The wild-type carbohydrate transporter gene or
carbohydrate metabolic enzyme gene (or a functional part thereof)
can also be introduced into the cells of the subject in need
thereof using a vector as described herein. The vector can be a
viral vector or a plasmid. The gene can also be introduced as naked
DNA. The gene can be provided so as to integrate into the genome of
the recipient host cells, or to remain extra-chromosomal.
Integration can occur randomly or at precisely defined sites, such
as through homologous recombination. For example, a functional copy
of the carbohydrate transporter gene or a carbohydrate metabolic
enzyme gene can be inserted in replacement of an altered version in
a cell, through homologous recombination. Further techniques
include gene gun, liposome-mediated transfection, or cationic
lipid-mediated transfection. Gene therapy can be accomplished by
direct gene injection, or by administering ex vivo prepared
genetically modified cells expressing a functional polypeptide.
[0178] Gene Therapy and Protein Replacement Methods
[0179] Delivery of nucleic acids into viable cells can be effected
ex vivo, in situ, or in vivo by use of vectors, and more
particularly viral vectors (e.g., lentivirus, adenovirus,
adeno-associated virus, or a retrovirus), or ex vivo by use of
physical DNA transfer methods (e.g., liposomes or chemical
treatments). Non-limiting techniques suitable for the transfer of
nucleic acid into mammalian cells in vitro include the use of
liposomes, electroporation, microinjection, cell fusion,
DEAE-dextran, and the calcium phosphate precipitation method (see,
for example, Anderson, Nature, supplement to vol. 392, no. 6679,
pp. 25-20 (1998)). Introduction of a nucleic acid or a gene
encoding a polypeptide of the invention can also be accomplished
with extrachromosomal substrates (transient expression) or
artificial chromosomes (stable expression). Cells can also be
cultured ex vivo in the presence of therapeutic compositions of the
present invention in order to proliferate or to produce a desired
effect on or activity in such cells. Treated cells can then be
introduced in vivo for therapeutic purposes.
[0180] Nucleic acids can be inserted into vectors and used as gene
therapy vectors. A number of viruses have been used as gene
transfer vectors, including papovaviruses, e.g., SV40 (Madzak et
al., 1992), adenovirus (Berkner, 1992; Berkner et al., 1988;
Gorziglia and Kapikian, 1992; Quantin et al., 1992; Rosenfeld et
al., 1992; Wilkinson et al., 1992; Stratford-Perricaudet et al.,
1990), vaccinia virus (Moss, 1992), adeno-associated virus
(Muzyczka, 1992; Ohi et al., 1990), herpesviruses including HSV and
EBV (Margolskee, 1992; Johnson et al., 1992; Fink et al., 1992;
Breakfield and Geller, 1987; Freese et al., 1990), and retroviruses
of avian (Biandyopadhyay and Temin, 1984; Petropoulos et al.,
1992), murine (Miller, 1992; Miller et al., 1985; Sorge et al.,
1984; Mann and Baltimore, 1985; Miller et al., 1988), and human
origin (Shimada et al., 1991; Helseth et al., 1990; Page et al.,
1990; Buchschacher and Panganiban, 1992). Non-limiting examples of
in vivo gene transfer techniques include transfection with viral
(typically retroviral) vectors (see U.S. Pat. No. 5,252,479, which
is incorporated by reference in its entirety) and viral coat
protein-liposome mediated transfection (Dzau et al., Trends in
Biotechnology 11:205-210 (1993), incorporated entirely by
reference). For example, naked DNA vaccines are generally known in
the art; see Brower, Nature Biotechnology, 16:1304-1305 (1998),
which is incorporated by reference in its entirety. Gene therapy
vectors can be delivered to a subject by, for example, intravenous
injection, local administration (see, e.g., U.S. Pat. No.
5,328,470) or by stereotactic injection (see, e.g., Chen, et al.,
1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical
preparation of the gene therapy vector can include the gene therapy
vector in an acceptable diluent, or can comprise a slow release
matrix in which the gene delivery vehicle is imbedded.
Alternatively, where the complete gene delivery vector can be
produced intact from recombinant cells, e.g., retroviral vectors,
the pharmaceutical preparation can include one or more cells that
produce the gene delivery system.
[0181] For reviews of gene therapy protocols and methods see
Anderson et al., Science 256:808-813 (1992); U.S. Pat. Nos.
5,252,479, 5,747,469, 6,017,524, 6,143,290, 6,410,010 6,511,847;
and U.S. Application Publication Nos. 2002/0077313 and 2002/00069,
which are all hereby incorporated by reference in their entireties.
For additional reviews of gene therapy technology, see Friedmann,
Science, 244:1275-1281 (1989); Verma, Scientific American: 68-84
(1990); Miller, Nature, 357: 455-460 (1992); Kikuchi et al., J
Dermatol Sci. 2008 May; 50(2):87-98; Isaka et al., Expert Opin Drug
Deliv. 2007 September; 4(5):561-71; Jager et al., Curr Gene Ther.
2007 August; 7(4):272-83; Waehler et al., Nat Rev Genet. 2007
August; 8(8):573-87; Jensen et al., Ann Med. 2007; 39(2):108-15;
Herweijer et al., Gene Ther. 2007 January; 14(2):99-107; Eliyahu et
al., Molecules, 2005 Jan. 31; 10(1):34-64; and Altaras et al., Adv
Biochem Eng Biotechnol. 2005; 99:193-260, all of which are hereby
incorporated by reference in their entireties.
[0182] Protein replacement therapy can increase the amount of
protein by exogenously introducing wild-type or biologically
functional protein by way of infusion. A replacement polypeptide
can be synthesized according to known chemical techniques or can be
produced and purified via known molecular biological techniques.
Protein replacement therapy has been developed for various
disorders. For example, a wild-type protein can be purified from a
recombinant cellular expression system (e.g., mammalian cells or
insect cells-see U.S. Pat. No. 5,580,757 to Desnick et al.; U.S.
Pat. Nos. 6,395,884 and 6,458,574 to Selden et al.; U.S. Pat. No.
6,461,609 to Calhoun et al.; U.S. Pat. No. 6,210,666 to Miyamura et
al.; U.S. Pat. No. 6,083,725 to Selden et al.; U.S. Pat. No.
6,451,600 to Rasmussen et al.; U.S. Pat. No. 5,236,838 to Rasmussen
et al. and U.S. Pat. No. 5,879,680 to Ginns et al.), human
placenta, or animal milk (see U.S. Pat. No. 6,188,045 to Reuser et
al.), or other sources known in the art. After the infusion, the
exogenous protein can be taken up by tissues through non-specific
or receptor-mediated mechanism.
[0183] A polypeptide encoded by a carbohydrate transporter gene
(e.g., GLUT2 or SGLT1) or a carbohydrate metabolic enzyme gene (for
example, SI, MGAM, or LCT) can also be delivered in a controlled
release system. For example, the polypeptide can be administered
using intravenous infusion, an implantable osmotic pump, a
transdermal patch, liposomes, or other modes of administration. In
one embodiment, a pump can be used (see is Langer, supra; Sefton,
CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery
88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In
another embodiment, polymeric materials can be used (see Medical
Applications of Controlled Release, Langer and Wise (eds.), CRC
Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,
Drug Product Design and Performance, Smolen and Ball (eds.), Wiley,
New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev.
Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190
(1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al.,
J. Neurosurg. 71:105 (1989)). In yet another embodiment, a
controlled release system can be placed in proximity of the
therapeutic target thus requiring only a fraction of the systemic
dose (see, e.g., Goodson, in Medical Applications of Controlled
Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled
release systems are discussed in the review by Langer (Science
249:1527-1533 (1990)).
[0184] These methods described herein are by no means
all-inclusive, and further methods to suit the specific application
is understood by the ordinary skilled artisan. Moreover, the
effective amount of the compositions can be further approximated
through analogy to compounds known to exert the desired effect.
[0185] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Exemplary methods and materials are described below, although
methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention.
[0186] All publications and other references mentioned herein are
incorporated by reference in their entirety, as if each individual
publication or reference were specifically and individually
indicated to be incorporated by reference. Publications and
references cited herein are not admitted to be prior art.
EXAMPLES
[0187] Examples are provided below to facilitate a more complete
understanding of the invention. The following examples illustrate
the exemplary modes of making and practicing the invention.
However, the scope of the invention is not limited to specific
embodiments disclosed in these Examples, which are for purposes of
illustration only, since alternative methods can be utilized to
obtain similar results.
Example 1
Identification of Carbohydrate Transporters and Carbohydrate
Metabolic Enzymes as Biomarkers in a Subset of Autism Spectrum
Disorders (ASD)
[0188] Gastrointestinal disturbances complicate clinical management
in some children with autism. Reports of ileo-colonic lymphoid
nodular hyperplasia and deficiencies in disaccharidase enzymatic
activity led to the survey of intestinal gene expression and
microflora in children with autism and gastrointestinal disease
(AUT-GI) or gastrointestinal disease alone (Control-GI). In AUT-GI
subjects, ileal transcripts for the disaccharidases sucrase
isomaltase, maltase glucoamylase, and lactase, and the
monosaccharide transporters, sodium-dependent glucose
co-transporter, and glucose transporter 2 were significantly
decreased. Alterations in intestinal carbohydrates as a result of
these deficiencies would have a distinct impact on the composition
of AUT-GI intestinal microbiota. Bacterial 16S rRNA gene
pyrosequencing analysis of biopsy material from ileum and cecum
revealed decreased Bacteroidetes, increased Firmicute/Bacteroidete
ratios, higher cumulative levels of Firmicutes and Proteobacteria,
and increased Betaproteobacteria in AUT-GI as compared with
Control-GI biopsies. These results indicate a complex dependence
between intestinal gene expression and bacterial community
structure that contributes to gastrointestinal dysfunction in
AUT-GI children.
[0189] Deficiencies in intestinal disaccharidase and/or
glucoamylase activity are reported in over half of autistic
children with gastrointestinal disturbances (AUT-GI) (Horvath et
al., 1999). To determine whether functional deficits reflect
decreased levels of mRNA encoding these enzymes transcript levels
were examined for three primary brush border disaccharidases
(sucrase isomaltase [SI], maltase glucoamylase [MGAM], and lactase
[LCT]) in ileal biopsies of AUT-GI and Control-GI children by real
time PCR. Levels of mRNA for all three enzymes were decreased in
AUT-GI: SI (FIG. 16A: Mann-Whitney, p=0.001), MGAM (FIG. 16B:
Mann-Whitney, p=0.003) and LCT (FIG. 16C: Mann-Whitney, p=0.032).
Deficiencies in LCT mRNA in AUT-GI children were not attributable
to disproportionate adult-type hypolactasia genotypes in the AUT-GI
group relative to the Control-GI group (FIGS. 21A-21E and Methods).
Within the ASD-GI group, 86.7% (SI), 80% (MGAM), and 80% (LCT) of
children had transcript levels below the 25.sup.th percentile of
Control-GI children (Table 5A). Nearly all (14/15, or 93.3%) AUT-GI
children had deficiencies in at least one disaccharidase enzyme;
80% had deficiencies in 2 or more enzymes; and 73.3% had
deficiencies in all three enzymes (Table 5A). Tables 5A-C are
summary tables for gene expression and bacterial assays. Increases
or decreases in AUT-GI children in both gene expression and
bacterial parameters were determined for each individual based on
the levels of each parameter in the Control-GI group. The values
for a given parameter in the AUT-GI children that exceeded the
75.sup.th (arrow pointing up) percentile or were below the
25.sup.th percentile (arrow pointing down) for the corresponding
parameter in the Control-GI children were scored as an increase or
decrease, respectively. Values that were also above the 90.sup.th
or below the 10.sup.th percentiles of Control-GI children are
indicated by double arrows.
TABLE-US-00003 TABLE 5A Summary tables for gene expression and
bacterial assays. ASD Patient # SI MGAM LCT SGLT1 GLUT2 CDX2 Villin
# Disaccharidases # Transporters Total 1 n.c. 3/3 2/2 5/5 2 3/3 2/2
5/5 3 3/3 2/2 5/5 4 n.c. 3/3 2/2 5/5 5 3/3 2/2 5/5 6 n.c. n.c. n.c.
n.c. n.c. n.c. 1/3 0/2 1/5 7 n.c. n.c. n.c. n.c. 0/3 0/2 0/5 8 n.c.
3/3 2/2 5/5 9 n.c. n.c. 3/3 1/2 4/5 10 n.c. 3/3 2/2 5/5 11 n.c. 3/3
2/2 5/5 12 n.c. n.c. 3/3 2/2 5/5 13 3/3 2/2 5/5 14 n.c. n.c. n.c.
2/3 1/2 3/5 15 n.c. n.c. n.c. n.c. n.c. n.c. 1/3 0/2 1/5 % below
86.7% 80.0% 80.0% 73.3% 73.3% 33.3% 26.7% Summary Summary Summary
controls All 3 = 73.3% Both = 66.7% All 5 = 66.7% At least 2 = 80%
At least 1 = 80% At least 4 = 73.3% At least 1 = 93.3% At least 3 =
80% At least 1 = 93.3%
TABLE-US-00004 TABLE 5B Summary tables for gene expression and
bacterial assays. ##STR00003##
TABLE-US-00005 TABLE 5C Summary tables for gene expression and
bacterial assays Firm./ Firm./ Firm./ Firm./ Clostridiales/
Clostridiales/ Firm. + Firm. + Bacteroid. Bacteroid. Bacteroid.
Bacteroid. Bacteroidales Bacteroidales Proteobac. Proteobac. ASD
Ratio-RT Ratio-RT Ratio-454 Ratio-454 Ratio-454 Ratio-454 Ratio-454
Ratio-454 Patient # Ileum Cecum Ileum Cecum Ileum Cecum Ileum Ileum
1 2 3 4 5 n.c. n.c. n.c. n.c. 6 n.c. n.c. n.c. n.c. 7 8 9 n.c. 10
n.c. 11 12 n.c. n.c. n.c. n.c. n.c. n.c. n.c. 13 14 n.c. n.c. n.c.
n.c. 15 % below 100% 60% 73.3% 66.7% 80.0% 66.7% 80% 73.3% or above
controls
[0190] Two hexose transporters, SGLT1 and GLUT2, mediate transport
of monosaccharides in the intestine. SGLT1, located on the luminal
membrane of enterocytes, is responsible for the active transport of
glucose and galactose from the intestinal lumen into enterocytes.
GLUT2 transports glucose, galactose and fructose across the
basolateral membrane into the circulation and can also translocate
to the apical membrane (Kellett et al., 2008). Real-time PCR
revealed a decrease in SGLT1 mRNA (FIG. 16D: Mann-Whitney, p=0.008)
and GLUT2 mRNA (FIG. 16E: Mann-Whitney, p=0.010) in AUT-GI
children. For SGLT1, 73.3% of AUT-GI children had transcript levels
below the 25th percentile of Control-GI children; 73.3% of AUT-GI
children had GLUT2 transcript levels below the 25.sup.th percentile
of Control-GI children (Table 5A). Deficiencies were found in at
least one hexose transporter in 80% of AUT-GI children; 66.7% had
deficiencies in both transporters. In total, 66.7% of AUT-GI
children had mRNA deficiencies in all 5 molecules involved in
carbohydrate digestion and transport (Table 5 .ANG.). Expression
levels were correlated (Bonferroni-adjusted Spearman rank order
correlations) in the AUT-GI group for all gene combinations except
LCT and GLUT2, for which only a trend was observed. In the
Control-GI group, significance was limited to correlations of
SI-MGAM, MGAM-SGLT1, and LCT-SGLT1 (Table 2).
TABLE-US-00006 TABLE 2 Spearman correlations between ileal gene
expression and bacterial abundance variables. Spearman correlations
are shown for the AUT-GI group alone (AUT) and the Control-GI group
alone (Control). Group SI MGAM LCT SGLT1 GLUT2 Villin CDX2 SI AUT 1
0.89*** 0.59* 0.88** 0.76** 0.24 0.59* Control 1 0.93* 0.54
0.68.dagger. 0.75.dagger. 0.57 0.68.dagger. MGAM AUT -- 1 0.56*
0.86** 0.75** 0.31 0.63* Control -- 1 0.75.dagger. 0.82* 0.64
0.71.dagger. 0.82* LCT AUT -- -- 1 0.62* 0.52.dagger. 0.58* 0.65*
Control -- -- 1 0.86* 0.57 0.82* 0.86* SGLT1 AUT -- -- -- 1 0.71**
0.34 0.54* Control -- -- -- 1 0.64 0.96* 1.00* GLUT2 AUT -- -- --
-- 1 0.51.dagger. 0.69** Control -- -- -- -- 1 0.54 0.64 Villin AUT
-- -- -- -- -- 1 0.60* Control -- -- -- -- -- 1 0.96* CDX2 AUT --
-- -- -- -- -- 1 Control -- -- -- -- -- -- 1 Bacteroidetes AUT 0.33
0.10 0.31 0.52.dagger..sup.a 0.07 0.02 -0.01 Ileum Control -0.29
-0.29 -0.32 -0.18 -0.75.dagger..sup.a 0.00 -0.18 Bacteroidetes AUT
0.18 0.06 0.23 0.33 0.05 0.12 0.10 Cecum Control -0.93* -1.00*
-0.75.dagger. -0.82* -0.64 -0.71.dagger. -0.82* Firmicutes AUT
-0.61*.sup.a -0.55*.sup.a -0.00 0.12 0.23 0.64* 0.48.dagger..sup.a
Ileum Control 0.43 0.36 0.18 0.32 0.61 0.14 0.32 Firmicutes AUT
-0.06 0.05 -0.05 0.04 0.15 0.58* 0.14 Cecum Control 0.86* 0.86*
0.68.dagger. 0.89.sup.* 0.86* 0.79.dagger. 0.89* Firm./Bacteroid.
AUT -0.72**.sup.a -0.65*.sup.a -0.61*.sup.a -0.65*.sup.a
-0.55*.sup.a 0.36 -0.58*.sup.a Ileum Control 0.43 0.36 0.18 0.32
0.61 0.14 0.32 Firm./Bacteroid. AUT -0.51.dagger..sup.a -0.08 -0.11
-0.23 0.00 0.42 0.06 Cecum Control 0.86* 0.86* 0.68.dagger. 0.89*
0.86* 0.79.dagger. 089* Betaproteo. AUT -0.63* -0.60* -0.56*
-0.44.dagger. -0.60* -0.45.dagger. -0.70** Ileum Control
-0.75.dagger. -0.82* -0.54 -0.61 -0.57 -0.39 -0.61 Betaproteo. AUT
-0.56* -0.59* -0.64* -0.51.dagger. -0.61* -0.61* -0.85** Cecum
Control -0.43 -0.43 0.14 -0.00 0.14 0.14 -0.00 *= p < 0.05, **=
p < 0.01, ***= p < 0.001, ****= p < 0.0001, .dagger.= p
< 0.1 (trend) .sup.a= values obtained from bacteria-specific
real-time PCR
[0191] To determine whether reductions in disaccharidase and
transporter transcript levels reflected loss of or damage to
intestinal epithelial cells, mRNA levels associated with a
tissue-specific marker restricted to these cells, villin (Khurana
and George, 2008) were measured. Ileal villin mRNA levels were not
decreased in AUT-GI children (Mann-Whitney, p=0.307) (FIG. 16F).
Normalization of SI, MGAM, LCT, SGLT1 and GLUT2 to villin mRNA did
not correct AUT-GI deficits in gene expression for these
transcripts (FIGS. 22A-22E).
[0192] CDX2, a member of the caudal-related homeobox transcription
factor family, regulates expression of SI, LCT, GLUT2, SGLT1 and
villin (Suh and Traber, 1996; Troelsen et al., 1997; Uesaka et al.,
2004; Balakrishnan et al., 2008; and Yamamichi et al., 2009).
Real-time PCR experiments demonstrated lower levels of CDX2 mRNA in
some AUT-GI subjects as compared with controls, but group
differences were not significant (FIG. 16G: Mann-Whitney, p=0.192).
Only 33.3% of AUT-GI patients had CDX2 mRNA levels below the
25.sup.th percentile of the Control-GI group (FIG. 23A). However,
86.7% of AUT-GI children had CDX2 levels below the 50.sup.th
percentile of Control-GI children. Only one AUT-GI child (patient
7) had CDX2 levels above the 75.sup.th percentile of Control-GI
children. This child was the only subject who did not show signs of
deficiencies in any disaccharidases or transporters (Table 5A). In
the AUT-GI group, expression of CDX2 was correlated with that of
SI, MGAM, LCT, SGLT1, GLUT2, and villin (Bonferroni-adjusted
Spearman rank order correlations; Table 2). Among Control-GI
subjects, the expression of CDX2 was correlated only with that of
MGAM, LCT, SGLT1, and villin (Table 2).
[0193] To determine whether deficient carbohydrate digestion and
absorption influenced the composition of intestinal microflora,
ileal and cecal biopsies from AUT-GI and Control-GI children were
analyzed by bacterial 16S rRNA gene pyrosquencing (See also Methods
and FIGS. 23A-23D). Bacteroidetes and Firmicutes were the most
prevalent taxa present in the ileal and cecal tissues of AUT-GI
children, with the exception of the ileal samples of patients 2,
15, and 19 and cecal samples of patient 15, wherein levels of
Proteobacteria exceeded those of Firmicutes and/or Bacteroidetes
(FIGS. 17A-B and FIGS. 24A-B). Other phyla identified at lower
levels included Verrucomicrobia, Actinobacteria, Fusobacteria,
Lentisphaerae, TM7, and Cyanobacteria, as well as unclassified
bacterial sequences (FIGS. 17A-B and FIGS. 24A-24D). The abundance
of Bacteroidetes was lower in AUT-GI ileal (FIG. 17C: Mann-Whitney,
p=0.012) and cecal samples (FIG. 17D: Mann-Whitney, p=0.008) as
compared with the abundance of Bacteroidetes in Control-GI samples.
Real-time PCR using Bacteroidete-specific primers confirmed
decreases in Bacteroidetes in AUT-GI ilea (FIG. 17E: Mann-Whitney,
p=0.003) and ceca (FIG. 17F: Mann-Whitney, p=0.022), with levels
below the 25.sup.th percentile of Control-GI children in 100% of
AUT-GI ilea and 86.7% of AUT-GI ceca (Table 5B). Family-level
analysis of Bacteroidete diversity from pyrosequencing reads
indicateed that losses among members of the family Bacteroidaceae
in AUT-GI patient samples contributed substantially to overall
decreases in Bacteroidete levels in ilea (FIG. 17G) and ceca (FIG.
17H). OTU (Operational Taxonomic Unit) analysis of Bacteroidete
sequences indicateed that deficiencies in Bacteroidete sequences in
AUT-GI subjects were attributable to cumulative losses of 12
predominant phylotypes of Bacteroidetes, rather than loss of any
one specific phylotype (FIGS. 25A-25E and Methods).
[0194] Analysis of pyrosequencing reads revealed an increase in
Firmicute/Bacteroidete ratios in AUT-GI ilea (FIG. 18A:
Mann-Whitney, p=0.026) and ceca (FIG. 18B: Mann-Whitney, p=0.032).
An increase was also observed at the order level for
Clostridiales/Bacteroidales ratios in ilea (FIG. 26A: Mann-Whitney,
p=0.012) and ceca (FIG. 26B: Mann-Whitney, p=0.032). Real-time PCR
using Firmicute- and Bacteroidete-specific primers confirmed these
increases in Firmicute/Bacteroidete ratios in AUT-GI ilea (FIG.
26C: Mann-Whitney, p=0.0006) and ceca (FIG. 26D: Mann-Whitney,
p=0.022). Firmicute/Bacteroidete ratios were above the 75.sup.th
percentile of Control-GI values in 100% of AUT-GI ilea and 60% of
AUT-GI ceca (Table 5C). Order-level analysis of pyrosequencing
reads indicated trends toward increased Clostridiales in AUT-GI
ilea (FIG. 27E: Mann-Whitney, p=0.072) and ceca (FIG. 27F:
Mann-Whitney, p=0.098). Family-level analysis revealed that
increased Clostridiales levels in AUT-GI patient samples were
largely attributable to increases in members of the families
Lachnospiraceae and Ruminococcaceae (FIGS. 18C-18F). Cumulative
levels of Lachnospiraceae and Ruminococcaceae above the 75.sup.th
percentile of the corresponding levels in Control-GI samples were
found in 60% of AUT-GI ileal and 53.3% of AUT-GI cecal samples
(FIGS. 18E-18F and Table 5B). Genus-level analysis indicated that
members of the genus Faecalibacterium within the family
Ruminococcaceae contributed to the overall trend toward increased
Clostridia levels (FIGS. 28A-B). Within Lachnospiraceae, members of
the genus Lachnopsiraceae Incertae Sedis, Unclassified
Lachnospiraceae, and to a lesser extent Bryantella (cecum only)
contributed to the overall trend toward increased Clostridia in
ASD-GI patients (FIGS. 28A-B).
[0195] The cumulative level of Firmicutes and Proteobacteria was
higher in AUT-GI group in both ileal (FIG. 18G: Mann-Whitney,
p=0.015) and cecal samples (FIG. 18H: Mann-Whitney, p=0.007) (FIGS.
18I-J); however, neither Firmicute nor Proteobacteria levels showed
significant differences on their own (FIGS. 19A-19B and FIGS.
27A-27D). Levels of Betaproteobacteria tended to be higher in the
ilea of AUT-GI patients (FIG. 19C: Mann-Whitney, p=0.072);
significantly higher levels of Betaproteobacteria were found in
AUT-GI ceca (FIG. 19D: Mann-Whitney, p=0.038). Levels of
Betaproteobacteria were above the 75.sup.th percentile of
Control-GI children in 53.3% of AUT-GI ilea and 66.7% of AUT-GI
ceca (Table 5B). Family-level analysis revealed that members of the
families Alcaligenaceae and Incertae Sedis 5 (patient 2 only)
contributed substantively to the observed increases in
Beta-Proteobacteria in ilea (FIG. 19E) and ceca (FIG. 19F).
Alcaligenaceae sequences were detected in 46.7% of AUT-GI children
and none of the Control-GI children. Overtly elevated levels of
Proteobacteria in AUT-GI ilea and ceca reflected increased Alpha-
(families Methylo-bacteriaceae and Unclassified Rhizobiales) and
Betaproteobacteria (family Incertae Sedis 5) for patient #2 and
increased Gammaproteobacteria (family Enterobacteriaceae) for
patients #8 and #15 (FIGS. 19E-19F). Levels of Alpha-, Delta-,
Gamma-, and Epsilonproteobacteria were not significantly different
between AUT-GI and Control-GI samples.
[0196] The relationships between ileal and cecal microflora and
levels of disaccharidases, transporters, villin, and CDX2 were
assessed (Table 2). In the AUT-GI group, significant inverse
Spearman correlations were found for ileal Firmicutes vs. SI and
MGAM; the ileal Firmicute/Bacteroidete ratio vs. SI, MGAM, LCT,
SGLT1, GLUT2, and CDX2; and ileal and cecal Betaproteobacteria vs.
SI, MGAM, LCT, GLUT2, and CDX2. In the Control-GI group significant
inverse Spearman correlations were found for cecal Bacteroidetes
vs. SI, MGAM, SGLT1, and CDX2; as well as ileal Betaproteobacteria
vs. MGAM. Positive Spearman correlations were also found in the
Control-GI group: cecal Firmicutes vs. SI, MGAM, SGLT1, GLUT2, and
CDX2; and cecal Firmicute/Bacteroidete ratio vs. SI, MGAM, SGLT1,
GLUT2, and CDX2 (Table 2). These results indicate a complex
dependence between carbohydrate metabolizing and transporting genes
and the composition of the intestinal microbiome (See FIG.
20A-20C).
[0197] Discussion
[0198] ASD are brain disorders defined using behavioral criteria;
however, many affected individuals also have substantial GI
morbidity. A previous report on GI disturbances in ASD found low
activities of at least one disaccharidase or glucoamylase in
duodenum in 58% of children examined (21 of 36) (Horvath et al.,
1999). As described herein, 93.3% of AUT-GI children had decreased
mRNA levels for at least one of the three disaccharidases (SI,
MGAM, or LCT). In addition, decreased levels of mRNA were found for
two important hexose transporters, SGLT1 and GLUT2. Transcripts for
the enterocyte marker, villin, were not deficient in AUT-GI ilea;
thus these deficiencies are unlikely to be due to a general loss of
enterocytes. However, defects in enterocyte maturational or
migration along the crypt-villus axis can compromise ranscriptional
regulation of ileal enzymes and transporters (Hodin et al., 1995).
The expression of CDX2, a master transcriptional regulator in the
intestine, was correlated with expression of disaccharidases and
transporters in AUT-GI children. Therefore, CDX2 could play a role
in the observed expression deficits for these genes. Whatever the
mechanism, reduced capacity for digestion and transport of
carbohydrates can have profound effects. Within the intestine
malabsorbed monosaccharides can lead to osmotic diarrhea;
non-absorbed sugars can also serve as substrates for intestinal
microflora that produce fatty acids and gases (methane, hydrogen,
and carbon dioxide), promoting additional GI symptoms such as
bloating and flatulence. The deficiency of even a single gene in
this important pathway can result in severe GI disease, as occurs
with Glucose-galactose malabsorption syndrome caused by SGLT1
deficiency, Fanconi-Bickel syndrome resulting from GLUT2 mutations,
sucrase-isomaltase deficiency, and congenital lactase deficiency.
Without being bound by theory, a potential link between
neurological dysfunction and malabsorption in childhood autism has
been indicated (Goodwin et al., 1971). Extra-intestinal
manifestations of GI disease, including neurologic presentation,
are described in patients with inflammatory bowel disease and
celiac disease (Bushara 2005; Lossos et al., 1995; Gupta et al.,
2005). An association between language regression and GI symptoms
has been reported in ASD, supporting a link between GI disease and
behavioral outcomes (Valicenti-McDermott et al., 2008). Outside the
intestine, the major role of dietary carbohydrates is to serve as
the primary source of cellular energy throughout the body.
Following digestion, nearly all ingested carbohydrates are
converted to glucose, which serves a central role in metabolism and
cellular homeostasis. The brain, of all organs, is quantitatively
the most energy-demanding, accounting for 50% of total body glucose
utilization (Owen et al., 1967). Abnormalities in glucose
metabolism and homeostasis have been documented in ASD: recovery of
blood glucose levels was delayed in ASD children following
insulin-induced hypoglycemia (Maher et al., 1975). Brain glucose
metabolism is decreased in ASD by positron emission tomography
(Toal et al., 2005; Haznedar et al., 2000; Haznedar et al., 2006).
Without being bound by theory, a reduced capacity to digest
carbohydrates and absorb glucose due to deficient expression of
disaccharidases and hexose transporters explains these previous
observations in ASD.
[0199] Changes in diet can influence composition of intestinal
microflora; thus, without being bound by theory carbohydrate
malabsorption can have similar effects in AUT-GI subjects. 16S rRNA
pyrosequencing revealed multicomponent dysbiosis in AUT-GI children
including decreased levels of Bacteroidetes, an increase in the
Firmicute/Bacteroidete ratio, increased cumulative levels of
Firmicutes and Proteobacteria, and an increase in the class
Betaproteobacteria. Bacteroidetes are implicated in mediating
maturational and functional processes in the intestine as well as
immune modulation. Monocolonization of mice with the prototypic gut
symbiont, Bacteroides thetaiotaomicron, reverses the maturational
defect in ileal epithelial glycan fucosylation that occurs in
germ-free mice and regulates the expression of host genes,
including SGLT-1 and LCT, that participate in key intestinal
functions (i.e., nutrient absorption, metabolism, epithelial
barrier function, and intestinal maturation) (Hooper et al.,
2001).
[0200] A direct role for Bacteroidetes in carbohydrate metabolism
is also evident. B. thetaiotaomicron encodes in its genome an
expansive number of genes dedicated to polysaccharide acquisition
and processing, including 236 glycoside hydrolases and 15
polysaccharide lyases (Flint et al., 2008). Thus, deficient
digestion and absorption of di- and monosaccharides in the small
intestine can alter the milieu of growth substrates in the ileum
and cecum. As such, the growth advantages that Bacteroidetes enjoy
in the healthy intestine as a result of their expansive capacity to
thrive on polysaccharides can be compromised in AUT-GI children as
bacterial species better suited for growth on undigested and
unabsorbed carbohydrates flourish. Furthermore, polysaccharide A
(PSA), a single molecule from another Bacteroidete member,
Bacteroides fragilis, protects germ-free mice from Helicobacter
hepaticus- and chemically-induced colitis by correcting defects in
T-cell development, suppressing production of IL-17 and TNF-alpha,
and inducing IL-10 (Mazmanian et al., 2008). These reports
highlight the multiple roles Bacteroidete members play in the
maintenance of intestinal homeostasis, including maturation of
epithelium; regulation of intestinal gene expression, including
carbohydrate metabolizing genes and transporters; metabolism of
polysaccharides in the colon; and development of a competent immune
system. Thus, deficient levels of Bacteroidetes in the
muco-epithelium of AUT-GI children can directly compromise
carbohydrate metabolism and trigger inflammatory pathways.
[0201] Mice that are genetically obese (ob/ob) have 50% fewer
Bacteroidetes. A lower abundance of Bacteroidetes is reported in
stool samples from obese individuals (Ley et al., 2005; Ley et al.,
2006). Using Bacteroidete-specific real-time PCR, dramatic
decreases were found in the ilea (.about.50% lower abundance) as
well as significantly lower levels in the ceca (.about.25% lower
abundance) of AUT-GI compared to Control-GI children. In ob/ob
mice, diet-induced obese mice, and in obese humans, the decrease in
Bacteroidetes is accompanied by an increase in Firmicutes
(Turnbaugh et al., 2008; Ley et al., 2005; Ley et al., 2006). The
increased Firmicute/Bacteroidete ratio in obesity increases the
capacity to harvest energy from the diet (Turnbaugh et al., 2006).
As discussed herein, the trend toward increased Firmicutes and the
significant decrease in Bacteriodetes led to a significant increase
in the Firmicute/Bacteroidete ratio in ilea and ceca of AUT-GI
compared to Control-GI children. The trend toward increased
Firmicutes was largely attributable to Clostridia members; based on
pyrosequencing result, members of Ruminococcaceae and
Lachnospiraceae were the major contributors.
[0202] Several members of Ruminococcaceae and Lachnospiraceae are
known butyrate producers and can thus influence short-chain fatty
acid (SCFA) levels (Louis et al., 2010). SCFA influence colonic pH
and Bacteroides sp. are relatively sensitive to acidic pH (Duncan
et al., 2009). Three reports indicated differences in Clostridia
species in stool samples from ASD-GI as compared to control
children, including greater abundance of Clostridium clusters I,
II, XI and C. bolteae (Finegold et al, 2002; Song et al., 2004;
Parracho et al., 2005). Although only a trend was observed for
increased Firmicutes in AUT-GI children, the cumulative levels of
Firmicutes and Proteobacteria were significantly higher. Three
AUT-GI patients had extremely high levels of Alpha- and Beta-, or
Gammaproteobacteria. In addition, the AUT-GI group had elevated
levels of Betaproteobacteria compared to the Control-GI group,
reflecting the presence of Alcaligenaceae members in the ilea and
ceca of 46.7% of AUT-GI children. Alcaligenaceae sequences were not
detected in tissues from Control-GI children.
[0203] Conclusions:
[0204] Metabolic interactions between intestinal symbionts and the
human host are only beginning to be understood. Increasing evidence
shows that gastrointestinal disease and dysbiosis exert system-wide
effects on normal host physiology. As discussed herein, GI disease
in autism has a molecular profile distinct from GI disease in
normally-developing children. AUT-GI children have deficiencies in
disaccharidase and hexose transporter gene expression that likely
promote malabsorption and multicomponent, compositional dysbiosis.
Although the extra-intestinal effects these changes can elicit
remain speculative, the identification of specific molecular and
microbial signatures that define gastrointestinal pathophysiology
in AUT-GI children sets the stage for further research aimed at
defining the epidemiology, diagnosis and informed treatment of GI
symptoms in autism.
[0205] Materials and Methods:
[0206] Patient Samples.
[0207] Patient biopsies were collected as part of a study to assess
the frequency of measles virus transcripts in ilea and ceca of
children with autistic disorder and gastrointestinal complaints
(AUT-GI, n=15) and children with gastrointestinal complaints
without brain disorder (Control-GI, n=7). This cohort has been
previously described in detail (Hornig et al., 2008). The present
study restricted to male, Caucasian children from the original
cohort between 3 and 5 years of age to control for confounding
effects of gender, race and age on intestinal gene expression and
bacterial microbiota. The age at biopsy was similar for AUT-GI and
Control-GI subjects (median, in years [interquartile range, IQR]:
AUT-GI, 4.5 (1.2); Control-GI, 3.98 (0.9); Mann-Whitney, p=0.504]
(See Table 3).
TABLE-US-00007 TABLE 3 Patient information Table. Age LCT Patient #
Group (yrs.) (13910:22018) 215 1 ASD 4.35 C/T:G/A 478 2 ASD 5.94
T/T:A/A 513 3 ASD 4.66 T/T:A/A 530 4 ASD 5.46 C/T:G/A 554 5 ASD
4.01 T/T:A/A 562 6 ASD 3.80 C/T:G/A 566 7 ASD 3.49 T/T:A/A 581 8
ASD 4.29 T/T:A/A 589 9 ASD 5.62 C/C:G/G* 648 10 ASD 4.71 C/T:G/A
678 11 ASD 5.28 T/T:A/A 686 12 ASD 5.03 C/T:G/A 688 13 ASD 4.00
C/C:G/G* 733 14 ASD 4.53 T/T:A/A 800 15 ASD 3.51 C/C:G/G* 667 16
Control 3.98 T/T:A/A 755 17 Control 5.06 T/T:A/A 760 18 Control
3.89 C/T:G/A 796 19 Control 5.48 C/T:G/A 797 20 Control 3.98
C/T:G/A 814 21 Control 3.95 C/C:G/G* 842 22 Control 4.12
T/T:A/A
[0208] RNA and DNA Extraction.
[0209] RNA and DNA were extracted sequentially from individual
ileal and cecal biopsies (total of 176 biopsies: 88 ileal and 88
cecal biopsies; 4 biopsies per patient per region; 15 AUT-GI
patients and 7 Control-GI patients) in TRIzol using standard
protocols. RNA and DNA concentrations and integrity were determined
using a Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies,
Wilmington, Del.) and Bioanalyzer (Agilent Technologies, Foster
City, Calif.) and stored at -80.degree. C.
[0210] Quantitative Real-Time PCR of Human mRNA.
[0211] Intron/exon spanning, gene-specific PCR primers and probes
for sucrase isomaltase, maltase glucoamylase, lactase, SGLTI,
GLUT2, Villin, and CDX2, with GAPDH and Beta-actin as dual
housekeeping gene controls were designed for real-time PCR using
Primer Express 1.0 software (Applied Biosystems, Foster City,
Calif.). Taqman probes were labeled with the reporter FAM
(6-carboxyfluorescein) and the quencher BBQ (Blackberry) (TIB
MolBiol). PCR standards for determining copy numbers of target
transcripts were generated from amplicons cloned into the vector
pGEM-T easy (Promega Corporation, Madison, Wis.). Linearized
plasmids were quantitated by UV spectroscopy and 10-fold serial
dilutions (ranging from 5.times.10.sup.5 to 5.times.10.degree.
copies) were created in water containing yeast tRNA (1 ng/.mu.l).
Unpooled RNA from individual ileal biopsies were used for real time
PCR assays; each individual biopsy was assayed in duplicate. cDNA
was synthesized using Taqman reverse transcription reagents
(Applied Biosystems) from 2 .mu.g unpooled RNA per 100 .mu.l
reaction. Each 25-.mu.l amplification reaction contained 10 .mu.l
template cDNA, 12.5 .mu.l Taqman Universal PCR Master Mix (Applied
Biosystems), 300 nM gene-specific primers and 200 nM gene-specific
probe (Table 2). The thermal cycling profile using a ABI
StepOnePlus Real-time PCR System (Applied Biosystems) consisted of:
Stage 1, one cycle at 50.degree. C. for 2 min; Stage 2, 1 cycle at
95.degree. C. for 10 min; Stage 3, 45 cycles at 95.degree. C. for
15 s and 60.degree. C. for 1 min (1 min 30 s for LCT). GAPDH and
B-actin mRNA were amplified in duplicate reactions by real-time PCR
from the same reverse transcription reaction as was performed for
the gene of interest. The mean concentration of GAPDH or Beta-actin
in each sample was used to control for integrity of input RNA and
to normalize values of target gene expression to those of the
housekeeping gene expression. The final results shown were
expressed as the mean copy number from replicate biopsies per
patient, relative to values obtained for GAPDH mRNA. Beta-actin
normalization gave similar results to GAPDH normalization for all
assays. Due to insufficient or poor quality RNA, only 3 of the 4
biopsies were included for 3 patients (Patient #s 4, 7, 10) and
only 2 of the 4 biopsies were included for 1 patient (Patient #2).
Thus, 83 of the original 88 ileal biopsies were used in real-time
PCR experiments.
[0212] Lactase Genotyping.
[0213] Genomic DNA from AUT-GI (n=15) and Control-GI (n=7) patients
was subjected to previously-described PCR-restriction fragment
length polymorphism (PCR-RFLP) analysis for the C/T-13910 and
G/A-22018 polymorphisms associated with Adult-type Hypolactasia
with minor modifications (Buning et al., 2003). Genotyping primers
for C/T-13910 and G/A-22018 polymorphisms are as follows:
C/T-13910For (5'-GGATGCACTGC TGTGATGAG-3'[SEQ ID NO: 20]),
C/T-13910Rev (5'-CCCACTGACCTATCCTCGTG-3' [SEQ ID NO: 21]),
G/A-22018For (5'-AACAGGCACGTGGAGGAGTT-3' [SEQ ID NO: 22]), and
G/A-22018Rev (5'-CCCACCTCAGCCTCTTGAGT-3'[SEQ ID NO: 23]). Each
50-.mu.l amplification reaction contained 500 ng genomic DNA, 400
nM forward and reverse primers, and 25 .mu.l High Fidelity PCR
master mix. Thermal cycling consisted of 1 cycle at 94.degree. C.
for 4 min followed by 40 cycles at 94.degree. C. for 1 min,
60.degree. C. for 1 min, and 72.degree. C. for 1 min. PCR reactions
for C/T-13910 were directly digested with the restriction enzyme
BsmFI at 65.degree. C. for 5 hrs. PCR reactions for G/A-22018 were
resolved on 1% agarose gels followed by gel extraction of the
prominent 448 bp amplicon. Gel extracted G/A-22018 amplicons were
then digested with the restriction enzyme HhaI at 37.degree. C. for
5 hrs. Restriction digests of C/T-13910 and G/A-22018 were resolved
on 1.5% ethidium-stained agarose gels for genotyping analysis.
BsmFI digestion of the C/T-13910 amplicons generates two fragments
(351 bp and 97 bp) for the hypolactasia genotype (C/C), four
fragments (35 lbp, 253 bp, 98 bp, and 97 bp) for the heterozygous
genotype (C/T), and three fragments (253 bp, 98 bp, and 97 bp) for
the normal homozygous allele (T/T). HhaI digestion of the G/A-22018
amplicons generates two fragments (284 bp and 184 bp) for the
hypolactasia genotype (G/G), three fragments (448 bp, 284 bp, and
184 bp) for the heterozygous genotype (G/A), and a single fragment
(448 bp) for the normal homozygous allele (A/A).
[0214] PCR Amplification of Bacterial 16S rRNA Gene and Barcoded
454 Pyrosequencing of Intestinal Microbiota.
[0215] For DNA samples from 88 ileal biopsies (4 biopsies per
patient; 15 AUT-GI patients, 7 Control-GI patients) and 88 cecal
biopsies from the same patients, PCR was carried out using
bacterial 16S rRNA gene-specific (V2-region), barcoded primers as
previously described (Hamady et al., 2008). Composite primers were
as follows:
TABLE-US-00008 (For) [SEQ ID NO: 24]
5'-GCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG-3', (Rev) [SEQ ID NO:
25] 5'-GCCTCCCTCGCGCCATCAGNNNNNNNNCATGCTGCCTCCCGTAGGAG T-3'.
Underlined sequences in the Forward and Reverse primers represent
the 454 Life Sciences@ primer B and primer A, respectively. Bold
sequences in the forward and reverse primers represent the
broadly-conserved bacterial primer 27F and 338R, respectively. NNN
represents the eight-base barcode, which was unique for each
patient. PCR reactions consisted of 8 .mu.l 2.5.times.5 PRIME
HotMaster Mix (5 PRIME Inc., Gaithersburg, Md.), 6 .mu.l of 4 .mu.M
forward and reverse primer mix, and 200 ng DNA in a 20-.mu.l
reaction volume. Thermal cycling consisted of one cycle at
95.degree. C. for 2 min; and 30 cycles at 95.degree. C. for 20
seconds, 52.degree. C. for 20 seconds, and 65.degree. C. for 1 min.
Each of 4 biopsies per patient was amplified in triplicate, with a
single, distinct barcode applied per patient. Ileal and cecal
biopsies were assayed separately. Triplicate reactions of
individual biopsies were combined, and PCR products were purified
using Ampure magnetic purification beads (Beckman Coulter Genomics,
Danvers, Mass.) and quantified with the Quanti-iT PicoGreen dsDNA
Assay Kit (Invitrogen, Carlsbad, Calif.) and Nanodrop ND-1000
Spectrophotometer (Nanodrop Technologies, Wilmington, Del.).
Equimolar ratios were combined to create two master DNA pools, one
for ileum and one for cecum, with a final concentration of 25
ng/.mu.l. Master pools were sent for unidirectional pyrosequencing
with primer A at 454 Life Sciences (Branford, Conn.) on a GS FLX
sequencer.
[0216] Real-Time PCR of Bacteroidete and Firmicute 16S rRNA
Genes.
[0217] Primer sequences used for real-time PCR are listed in Table
4.
TABLE-US-00009 TABLE 4 Real-time PCR primers and probes used for
gene expression and bacterial quantitative analysis. SEQ ID Name
NO. Primers and Probe Amplicon size (bp) SI 26 For
5'-TCTTCATGAGTTTTATGAGGATACGAAC-3' 150 27 Rev:
5'-TTTGCACCAGATTCATAATCATACC-3' 28 Probe:
5'-CAGATACTGTGAGTGCCTACATCCCTGATGCTATT-3' MGAM 29 For:
5'-TACCTTGATGCATAAGGCCCA-3' 150 30 Rev: 5'-GGCATTACGCTCCAGGACA-3'
31 Probe: 5'-CGTCACTGTTGTGCGGCCTCTGC-3' LCT 32 For:
5'-CAGGAATCAAGAGCGTCACAACT-3' 180 33 Rev: 5'-AAATCGACCGTGTCCTGGG-3'
34 Probe: 5'TCCTGCTAGAACCACCCATATCTGCGCT-3' SGLT1 35 For:
5'-GCTCATGCCCAATGGACTG-3' 125 36 Rev: 5'-CGGACCTTGGCGTAGATGTC-3' 37
Probe: 5'-ACAGCGCCAGCACCCTCTTCACC-3' Glut2 38 For:
5'-AGTTAGATGAGGAAGTCAAAGCAA-3' 164 39 Rev: 5'-TAGGCTGTCGGTAGCTGG-3'
40 Probe: 5'-ACAAAGCTTGAAAAGACTCAGAGGATATGATGATGTC-3' Villin 41
For: 5'-CATGCGCTGAACTTCATCAAA-3' 120 42 Rev:
5'-GGTTGGACGCTGTCCACTTC-3' 43 Probe: 5'-CGGCCGTCTTTCAGCAGCTCTTCC-3'
CDX2 44 For 5'-GGCAGCCAAGTGAAAACCAG-3' 112 45 Rev:
5'-TCCGGATGGTGATGTAGCG-3' 46 Probe: 5'-ACCACCAGCGGCTGGAGCTGG-3'
.beta.-Actin 47 For: 5'-AGCCTCGCCTTTGCCGA-3' 175 48 Rev:
5'-CTGGTGCCTGGGGCG-3' 49 Probe: 5'-CCGCCGCCCGTCCACACCCGCC GAPDH 50
For: 5'-CCTGTTCGACAGTCAGCCG-3' 100 51 Rev:
5'-CGACCAAATCCGTTGACTCC-3' 52 Probe: 5'-CGTCGCCAGCCGAGCCACA-3'
Bacteroidetes 53 For: 5'-AACGCTAGCTACAGGCTT-3' ~293 54 Rev:
5'-CCAATGTGGGGGACCTTC-3' (Frank et al.) Firmicutes 55 For:
5'-GGAGYATGTGGTTTAATTCGAAGCA-3' ~126 56 Rev:
5'-AGCTGACGACAACCATGCAC-3' (Guo et al.) Total Bacteria 57 For:
5'-GTGCCAGCMGCCGCGGTAA-3' ~295 58 Rev: 5'-GACTACCAGGGTATCTAAT-3'
(Frank et al.)
[0218] PCR standards for determining copy numbers of bacterial 16S
rDNA were prepared from representative amplicons of the partial 16S
rRNA genes of Bacteroidetes and Firmicutes and total Bacteria
cloned into the vector PGEM-T easy (Promega). A representative
amplicon with high homology to Bacteroides Vulgatus (Accession #:
NC 009614) was used with Bacteroidete-specific primers. A
representative amplicon with high homology to Faecalibacterium
prausnitzii (Accession #: NZ.sub.-- ABED02000023) was used with
Firmicute-specific primers. A representative amplicon with high
homology to Bacteroides intestinalis (Accession #: NZ_ABM02000007)
16S rRNA gene was used with total Bacteria primers. Cloned
sequences were classified using the RDP Seqmatch tool and confirmed
by the Microbes BLAST database. Plasmids were linearized with the
SphI restriction enzyme and ten-fold serial dilutions of plasmid
standards were created ranging from 5.times.10.sup.7 to
5.times.10.degree. copies for Bacteroidetes, Firmicutes and total
Bacteria. Amplification and detection of DNA by real-time PCR were
performed with the ABI StepOnePlus Real-time PCR System (Applied
Biosystems). Cycling parameters for Bacteroidetes and total
Bacteria were as previously described (Frank et al., 2007), as were
cycling parameters for Firmicutes (Guo et al., 2008). Each 25-.mu.l
amplification reaction mixture contained 50 ng DNA, 12.5 .mu.l SYBR
Green Master Mix (Applied Biosystems), and 300 nM bacteria-specific
(Bacteroidete, Firmicute or total Bacteria) primers. DNA from each
of 88 ileal biopsies (4 biopsies per patient) and 88 cecal biopsies
(4 biopsies per patient) was assayed in duplicate. The final
results were expressed as the mean number of Bacteroidete or
Firmicute 16S rRNA gene copies normalized to 16S rRNA gene copies
obtained using total Bacterial primers. Eight water/reagent
controls were included for all amplifications. The average copy
number for water/reagent controls (background) was subtracted from
each ileal and cecal amplification prior to normalization. For the
Bacteroidete assay all water controls contained undetectable levels
of amplification. For the Firmicute assay average amplification
signal from water samples were minimal, 12.03+/-15.0 copies.
[0219] Bioinformatic Analysis of Pyrosequencing Reads.
[0220] Pyrosequencing reads ranging from 235 to 300 base pairs in
length (encompassing all sequences within the major peak obtained
from pyrosequencing) were filtered for analysis. Low-quality
sequences--i.e., those with average quality scores below 25--were
removed based on previously described criteria (Huse et al., 2007;
Hamady et al., 2008). Additionally, reads with any ambiguous
characters were omitted from analysis. Sequences were then binned
according to barcode, followed by removal of primer and barcode
sequences. Taxonomic classifications of bacterial 16S rRNA
sequences were obtained using the RDP Classifier with a minimum 80%
bootstrap confidence estimate. To normalize data for differences in
total sequences obtained per patient, phylotype abundance was
expressed as a percentage of total bacterial sequence reads per
patient at all taxonomic levels.
[0221] Statistical Analysis.
[0222] Data were not normally distributed, based on
Kolmogorov-Smirnov test and evaluation of skewness and kurtosis;
thus, the non-parametric Mann-Whitney U test was performed using
StatView (Windows version 5.0.1; SAS Institute, Cary, N.C.). The
comparative results of gene expression and bacteria levels were
visualized as box-and-whisker plots showing: the median and the
interquartile (midspread) range (boxes containing 50% of all
values), the whiskers (representing the 25.sup.th and 75.sup.th
percentiles) and the extreme data points (open circles).
Associations between different variables were assessed by Spearman
rank correlation test. Chi-squared test was used to evaluate
between-group genotypes for adult-type hypolactasia. Kruskal-Wallis
one-way analysis of variance was employed to assess significance of
LCT mRNA expression levels split by genotype and group.
Significance was accepted at p<0.05.
[0223] Genetically Determined Lactase Non-Persistence is not
Responsible for Deficient Lactase mRNA in AUT-GI.
[0224] Although it is beyond the scope of this study to evaluate
all possible mutations in carbohydrate genes that can affect
expression, deficient LCT mRNA is not a result of the common
adult-type hypolactasia genotype. LCT mRNA levels can be affected
by two single nucleotide polymorphisms that determine adult-type
hypolactasia; therefore, we genotyped these children using PCR-RFLP
analysis (FIG. 21A). The homozygous, hypolactasia variant alleles
were found in 20% (3 out of 15) of AUT-GI children and 14.3% (1 out
of 7) of Control-GI children (chi-squared test, p=0.896) (FIG.
21B). LCT mRNA expression was significantly lower in individuals
with the homozygous hypolactasia genotype compared to all other
genotypes (FIG. 21C: Mann-Whitney, p=0.033). Comparison of LCT mRNA
expression across genotype and group failed to reach significance
(FIG. 21D: Kruskal-Wallis, p=0.097). Comparison of mRNA expression
in subjects carrying at least one copy of the normal allele
confirmed a significant decrease in LCT mRNA in AUT-GI relative to
Control-GI subjects, independent of the individuals with the
homozygous hypolactasia genotype (FIG. 21E: Mann-Whitney, p=0.025).
In summary, although the data support the notion that LCT genotype
affects gene expression, deficient LCT mRNA in AUT-GI was not
attributable to disproportionate hypolactasia genotypes between the
AUT-GI and Control-GI groups.
[0225] Barcoded 16S rRNA Gene Pyrosequencing.
[0226] A total of 525,519 sequencing reads (representing 85% of the
initial number of sequencing reads) remained after filtering based
on read length, removing low-quality sequences and combining
duplicate pyrosequencing runs (271,043 reads for ilea; 254,476
reads for ceca). Binning of sequences by barcode revealed similar
numbers of 16S rRNA gene sequence reads per patient (average #
sequences per patient+/-STD for ilea=12,320+/-1220; average #
sequences per patient+/-STD for ceca=11,567+/-1589). There was not
a significant difference between the AUT-GI and Control-GI groups
in terms of the number of reads per patient. In order to assess
whether sufficient sampling was achieved in the total
pyrosequencing data set for all AUT-GI and Control-GI subjects,
OTUs (Operational Taxonomic Units) were defined at a threshold of
97% identity, split by data for ileum and cecum, and rarefaction
analysis was carried out (FIGS. 23A-23B). Rarefaction curves showed
a tendency toward reaching plateau for all subjects; however
failure to reach plateau means that additional sampling would be
required to achieve complete coverage of all OTUs present in ileal
and cecal biopsies. Investigation of diversity in AUT-GI and
Control-GI patients was carried out using the Shannon Diversity
Index calculated from OTU data for each subject. Rarefaction
analysis revealed that all Shannon Diversity estimates had reached
stable values (FIGS. 23C-23D). While Shannon Diversity estimates
varied widely between individuals, there was not an apparent
overall difference (loss or gain of diversity) between the AUT-GI
and Control-GI groups in ileal (FIG. 23C) or cecal (FIG. 23D)
biopsies.
[0227] OTU Analysis of Bacteroidetes.
[0228] In order to determine whether the decreased abundance of
Bacteroidete members was attributable to the loss of specific
Bacteroidete phylotypes, the distribution of Bacteroidete OTUs
(defined using a threshold of 97% identity or greater, 3% distance)
was investigated. The number of Bacteroidete OTUs per patient
ranged from 23 to 102 for ileal samples and 10 to 130 for cecal
samples. Interestingly, no single OTU was significantly over or
underrepresented between AUT-GI and Control-GI children and many
OTUs contained single sequences. Thus, it was determined whether,
the decrease in OTUs could be attributed to overall losses of the
most prevalent Bacteroidete phylotypes. In both ileal and cecal
samples, 12 OTUs accounted for the majority of Bacteroidete
sequences (FIGS. 25A-25B). The cumulative levels of these 12 OTUs
were significantly lower in AUT-GI compared to Control-GI children
in both the ileum (FIG. 25C: Mann-Whitney, p=0.008) and cecum (FIG.
25D: Mann-Whitney, p=0.008). Representative sequences from each of
these 12 OTUs were classified using Green Genes Blast
(greengenes.lbl.gov) and microbial blast alignment (NCBI) (FIG.
25E). The majority of sequences were members of the family
Bacteroidaceae (OTUs 3, 5, 6, 7, and 19), except in the case of
patient 20, where Prevotellaceae were the dominant phylotype. These
results indicate that the loss of Bacteroidetes in AUT-GI children
is primarily attributable to overall decreases in the dominant
phylotypes of Bacteroidetes.
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Example 2
Intestinal Inflammation, Impaired Carbohydrate Metabolism and
Transport, and Microbial Dysbiosis in Autism
[0334] The objective of this study was to survey host gene
expression and microflora in intestinal biopsies from children with
autistic disorder and gastrointestinal complaints (AUT-GI) vs
children with gastrointestinal complaints alone (Control-GI).
[0335] This example herein describes a rapid and specific PCR-based
assay for diagnostic detection of Sutterella species in biological
samples. It is a PCR-based detection scheme utilizing new genomic
16S rRNA sequences to allow rapid, sensitive, and specific species
identification from gut samples.
[0336] Overview
[0337] Methods.
[0338] Transcription profiling was pursued by cDNA microarray using
RNA extracted from ileal biopsies (4 per patient) of 15 male AUT-GI
and 7 age-matched, male Control-GI patients. Pathway analysis was
performed using Ingenuity Pathway Analysis and GO Ontology. Changes
in gene expression were confirmed by quantitative real-time PCR.
Intestinal microbiota were investigated in ileal and cecal biopsies
from AUT-GI and Control-GI children using amplicon-based, bar-coded
pyrosequencing of the V2 region of bacterial 16S rDNA. Taxonomic
classification of 525,519 bacterial sequences was accomplished
using the Ribosomal Database Project classifier tool. Differences
in microbiota between the two groups were further evaluated and
confirmed using Bacteroidete-, Firmicute-, and Sutterella-specific
real-time PCR.
[0339] Results.
[0340] Microarray and pathway analysis revealed significant changes
in genes involved in carbohydrate metabolism and transport and
inflammation in ileal biopsies from AUT-GI as compared to
Control-GI subjects. Real-time PCR confirmed significant decreases
in the AUT-GI group in the primary brush border disaccharidases,
sucrase isomaltase (p=0.0013), maltase glucoamylase (p=0.0027), and
lactase (p=0.0316) as well as in two enterocyte hexose
transporters, sodium glucose co-transporter 1 (p=0.0082) and
glucose transporter 2 (p=0.0101). In contrast, increases were
confirmed for inflammation-related genes in AUT-GI subjects:
complement component 1, q subcomponent, A chain (p=0.0022),
resistin (p=0.0316), CD163 (p=0.0150), tumor necrosis factor-like
weak inducer of apoptosis (p=0.015), and interleukin 17F
(p=0.0220). No significant group differences were observed for the
enterocyte-specific marker, villin. In conjunction with changes in
intestinal gene expression, bacterial content differed between the
AUT-GI and Control-GI groups: pyrosequencing and real-time PCR
revealed lower levels of Bacteroidetes (ileum: 50% reduction,
p=0.0027; cecum: 25% reduction, p=0.0220, and higher
Firmicute/Bacteroidete ratios in AUT-GI children (ileum: p=0.0006;
cecum: p=0.0220). High levels of Sutterella species were found in
47% of AUT-GI biopsies (7/15), whereas Sutterella was not detected
in any Control-GI biopsies (0/7; ileum: p=0.0220; cecum:
p=0.0368).
[0341] Conclusions.
[0342] A syndrome in autistic children is described wherein
gastrointestinal dysfunction is associated with altered gene
expression reflecting intestinal inflammation, impaired
carbohydrate metabolism and transport, and dysbiosis. These
findings provide insights into pathogenesis and allow for new
strategies for therapeutic intervention.
[0343] In this study, high levels of Sutterella sp. were found in
ileal and cecal biopsies from children with autism spectrum
disorders (ASD) and gastrointestinal disease, while Sutterella sp.
were undetectable in control children with gastrointestinal
disease. Little is known about the epidemiology and pathogenesis of
Sutterella sp. and their role in infectious diseases of humans and
animals. Current methods for detecting Sutterella sp. are costly,
labor intensive, and non-specific requiring isolation and anaerobic
culture of the bacteria or generation, screening, sequencing, and
sequence analysis of hundreds to thousands of bacterial 16S rRNA
gene sequences from bacterial libraries or pyrosequencing analysis
of hundreds of thousands of sequences. These methods can be costly,
lack specificity, ease of execution, and are not strictly
quantitative.
[0344] A rapid and specific PCR-based assay is described for the
diagnostic identification, quantification, and phylogenetic
analysis of Sutterella sp. in biological samples based on the
variable sequence (V6-V8 region) of the 16S rRNA gene of Sutterella
sp.
[0345] Study Background
[0346] An association between autistic spectrum disorder (ASD) and
gastrointestinal (GI) immunopathology is supported by reports of a
higher incidence of GI complaints, ileo-colonic lymphoid nodular
hyperplasia, and enterocolitis in children with autism. In this
study, intestinal bacteria were assessed in ileal (4 biopsies per
patient) and cecal (4 biopsies per patient) biopsies from male ASD
children (aged 3-5 years) with gastrointestinal symptoms (ASD-GI;
n=15) and normally developing age-matched, male controls with
gastrointestinal symptoms (Control-GI; n=7) by 454 pyrosequencing
of the V2 region of the bacterial 16S rRNA gene. Taxonomic
classification of 525,519 bacterial sequences was performed using
the Ribosomal Database Project classifier tool. Genus-level
analysis of pyrosequencing reads revealed a significant increase in
Sutterella sp. The average confidence estimate of all genus-level
Sutterella sequences identified using the RDP Classifier was high
(99.1%) with the majority of sequences at 100% confidence.
[0347] Comparison of ASD-GI and Control-GI patients revealed
significant increases in Sutterella sp. In the ileum (FIG. 8A:
Mann-Whitney U, p=0.022) and cecum (FIG. 8B: Mann-Whitney U,
p=0.0368). Sutterella sp. sequences were completely absent from all
Control-GI samples (% of total bacteria=0). Individual analysis of
ASD-GI patients revealed that 7 out of 15 ASD-GI patients (46.7%)
had high levels of Sutterella sp. sequences in both the ileum and
cecum (FIG. 8C and FIG. 8D). By patient, ileal Sutterella sp.
sequence abundance ranged from 1.7 to 6.7% of total bacterial reads
(FIG. 8C). Similarly, in the Cecum Sutterella sp. sequence
abundance ranged from 1.9 to 7.0% of total bacterial reads for the
same patients (FIG. 8D). Sutterella sp. Sequences represented the
majority of sequences present in the class Beta-proteobacteria in
these select ASD-GI patients. In the Ileum of these ASD-GI
patients, Sutterella sp. sequences accounted for 75.6% to 97.8% of
all Beta-proteobacteria sequences (FIG. 8E). In the cecum,
Sutterella sp. sequences accounted for 92.7% to 98.2% of all
Beta-proteobacteria sequences (FIG. 8F). The results of this
costly, time consuming, non-specific pyrosequencing analysis
prompted the design of a Sutterella sp.-specific PCR assay to
confirm, quantitate, and determine taxonomy of Sutterella sp. in
the same samples analyzed by pyrosequencing.
[0348] Methods
[0349] Primer and Probe Design:
[0350] Sutterella sp.-specific 16S rRNA gene PCR primers and probe
were designed against the 16S sequence for Sutterella
wadsworthensis (Genbank Accession # L37785) and Sutterella clone
LW53 (Genbank Accession # AY976224) using Primer Express 1.0
software (Applied Biosystems, Foster City, Calif.). Genus
specificity of candidate primers was evaluated using the RDP Probe
Match tool. While several potential primer pairs were identified,
only one pair showed high specificity for Sutterella sp. In PCR
assays. These primers are designated here as SuttFor and SuttRev
(Sequences of primers and probe are shown in Table 1).
TABLE-US-00010 TABLE 1 Sutterella sp.-specific primers and probes
for classical and real- time PCR assays and pan-bacterial primers
used for normalization. SEQ ID NO: Primers and Probe Amplicon size
(bp) 11 SuttFor: 5'-CGCGAAAAACCTTACCTAGCC-3' ~260 12 SuttRev:
5'-GACGTGTGAGGCCCTAGCC-3' 13
SuttProbe1:5'-CACAGGTGCTGCATGGCTGTCGT-3' 14 SuttProbe2: 5'-CCG
CAAGGGAATCTGGACACAGGT-3' 15 515For: 5'-GTGCCAGCMGCCGCGGTAA-3' ~295
(Frank et al.) 16 805Rev: 5'-GACTACCAGGGTATCTAAT-3'
[0351] Evaluation of good quality sequences that were >1200
bases in the RDP database revealed a total of 248 Sutterella
sequences at the time of analysis. SuttFor and SuttRev_primers
showed high exclusivity for the genus Sutterella. Approximately 90%
of RDP matches for SuttFor were in the genus Sutterella and 100% of
matches for the reverse primer were Sutterella sequences. The
SuttFor primer sequence matched exactly with approximately 91%
(225/248 Sutterella sequences) of all Sutterella sequences, while
the SuttRev primer matched exactly with approximately 81% (200/248
Sutterella sequences) of all Sutterella sequences. The SuttProbe1
(SEQ ID NO: 13) used for real-time PCR had low exclusivity but high
coverage of Sutterella sequences (100%). An additional probe (SEQ
ID NO: 143) with high exclusivity, but low coverage of Sutterella
sequences (58.8%) was also designed and can be used when sequence
information is available for Sutterella sp. in biological
samples.
[0352] Classical PCR. The SuttFor and SuttRev primers amplify a 260
bp region between variable regions 6, 7 and 8 (V6-V8) of the 16S
rRNA of Sutterella. Classical PCR for detection of Sutterella was
carried out in 25 ul reactions consisting of 25 ng genomic DNA, 300
nm each SuttFor and SuttRev primers, 2 ul dNTP mix (10 mM; Applied
Biosystems), 2.5 ul of 10.times.PCR Buffer (Qiagen), 5U of
HotStarTaq DNA polymerase (Qiagen), and 5 ul Q-solution (Qiagen).
Cycling parameters consisted of an initial denaturation step at 950
C for 15 min, followed by 30 cycles of 940 C for 1 min, 600 C for 1
min, and 720 C for 1 min and a final extension at 720 C for 5 min.
Amplified products were run on a 1.5% agarose gel, extracted from
the gel and either sent for direct PCR product sequencing using
SuttFor and SuttRev primers or cloned into PGEM-T easy cloning
vector for construction of bacterial libraries followed by
sequencing using vector primers. Specificity of the assay was
confirmed through direct sequence analysis of PCR products and
clone sequences using the RDP Seqmatch and Classifier tools. All
PCR products and clones were classified as Sutterella by RDP. In
order to test linearity and sensitivity of the assay, the
Sutterella clone used for real-time PCR standards was tested by
classical PCR using the same conditions as all intestinal DNA. Ten
fold dilutions of the Sutterella clone ranging from 5.times.105 to
5.times.100 were amplified by classical PCR alone as well as spiked
into ileal DNA from a Sutterella negative patient. Both in the
presence and absence of background ileal DNA, the Classical PCR was
linear in the range of 5.times.105 to 5.times.102 copies and had an
end-point detection limit of 5.times.101 copies (FIG. 9).
[0353] Quantitative Real-Time PCR.
[0354] PCR standards for determining copy numbers of bacterial 16S
rDNA were prepared from representative clones of the partial 16S
rDNA of Sutterella obtained using the Classical PCR assay. Cloned
sequences were classified using the RDP Seqmatch tool and confirmed
by the Microbes BLAST database. Plasmids were linearized with the
SphI restriction enzyme and ten fold serial dilutions of plasmid
standards were created ranging from 500,000 to 5 copies for
Sutterella (FIG. 10A and FIG. 10B). Amplification and detection of
DNA by real-time PCR were performed with the ABI StepOnePlus
Real-time PCR System (Applied Biosystems). For Sutterella
sp.-specific real-time PCR, each 25 ul reaction contained 50 ng
DNA, 12.5 ul Taqman universal master mix (ABI), 300 nm each of
SuttFor and SuttRev primers, and 200 nm SuttProbe1 (Reporter=FAM,
Quencher=BBQ). The standard curve had sensitivity down to 5 copies
of plasmid, with a slope of -3.08, y-intercept of 41.787, and with
an R2 value of 0.996 (FIG. 10A and FIG. 10B). DNA from each of 88
ileal biopsies and 88 cecal biopsies was assayed in duplicate. The
final results were expressed as the mean number of copies
normalized to 16S rRNA copies obtained using Pan-bacterial primers
(Table 1: primers 515For and 805Rev) in a SYBR Green Real-time PCR
assay (see Ref. 6 for more information). While normalization to
total bacteria is not necessary, we have implemented its use in
this study to control for variation in input DNA. Eight
water/reagent controls were included for all amplifications. The
average copy number for water controls (background) was subtracted
from each ileal and cecal amplification prior to normalization.
Where background copy number values exceeded amplification values
in ileal and cecal samples, copy number was set to a value of 0.
Average amplification signal from water samples with the Sutterella
assay were very low (125.8+/-40 copies) compared to amplification
in Sutterella positive samples (all ranging between 50,000 and
1,000,000 copies). Average copy numbers for all ileum and cecum
Sutterella-negative amplifications was 26.6+/-21.0 copies (all were
lower than the background controls).
[0355] Taxonomic Classification of Sutterella sp.
[0356] Sequence alignments using sequences obtained by direct
sequencing of Sutterella sp. from the classical PCR assay and
phylogenetic analyses were conducted using MEGA4 software. Primer
sequences were trimmed from the sequences obtained by direct
sequencing of amplicons. Classification was confirmed using the RDP
classifier and seqmatch tools. Sutterella sequences obtained from
ileal and cecal biopsies were aligned with sequences from the 11
known isolates of Sutterella sp. found in the RDP database.
Sequences from known Sutterella sp. Isolates were trimmed to the
length of the sequences obtained from ileal and cecal biopsies.
Phylogenetic trees were constructed according to the neighbour
joining method, rooted to the outgroup Burkholderia pseudomallei,
and the stability of the groupings was estimated by bootstrap
analysis (1000 replications) using MEGA4.
[0357] Results
[0358] Implementation of Sutterella sp.-Specific Classical PCR for
Detection.
[0359] Classical PCR analysis of Sutterella sp. using DNA from all
88 ileal and 88 cecal biopsies showed that the same individuals
identified as having high levels of Sutterella by V2 pyrosequencing
were also positive by the V6-V8 Sutterella sp.-specific PCR.
Additionally, all 4 biopsies per region in all 7
Sutterella-positive patients showed Sutterella amplicons, while no
amplicons were observed in any Control-GI patients or ASD-GI
patients that lacked Sutterella sequences in V2 pyrosequencing
experiments (FIG. 11). All patients amplicons were confirmed to
represent Sutterella by direct sequencing of PCR products and
cloning of individual amplicons to create bacterial libraries
followed by sequencing of 50 individual clones.
[0360] Implementation of Sutterella sp.-Specific Real-Time PCR for
Quantification.
[0361] Real-time PCR analysis using the same V6-V8 primers and a
high coverage Taqman probe (SuttProbe1), revealed significant
increases in Sutterella in ASD-GI compared to Control-GI patients
for both the ileum (FIG. 12A:Mann-Whitney U, p=0.0368) and cecum
(FIG. 12B:Mann-Whitney U, p=0.0368). Sutterella copy numbers were
quite high in both the ileum and cecum (in the range of 10.sup.4 to
10.sup.5 copies) of Sutterella-positive patients (FIG. 12C and FIG.
12D). The distribution of Sutterella abundance by patient and the
copy number revealed by V2 pyrosequencing and V6-V8 real-time PCR,
respectively, were in striking concordance (Compare ileum FIG. 8C
with FIG. 12C and compare cecum FIG. 8D with FIG. 12D). There was
100% congruence between V2 region 454 pyrosequencing and both
classical and real-time PCR using the V6-V8 region Sutterella
sp.-specific primers.
[0362] Implementation of Sutterella sp.-Specific Classical PCR for
Taxonomic Classification.
[0363] Sequences obtained from direct cloning and clone libraries
of the V6-V8 regions of each patient were aligned following removal
of primer sequences. This analysis revealed that the consensus
sequence obtained in ileal biopsies matched exactly with sequences
in cecal biopsies from the same patient. Furthermore, alignment of
sequences revealed that patients 1, 3, 10, 11, and 12 had the exact
same sequence for the V6-V8 region, while patients 5 and 7 had a
distinct, but identical sequence (FIG. 13). These findings are in
agreement with OTU analysis of V2 pyrosequencing reads in which
patients 1, 3, 10, 11, and 12 clustered together with OTU 11
containing the majority of Sutterella sequences and patient 5 and 7
clustered together with OTU 38 containing the majority Sutterella
sequences (FIG. 14). Treeing analysis of the V6-V8 sequences
revealed that Sutterella sp. found in patients 1, 3, 10, 11, and 12
were phylogenetically most closely associated with the isolates
Sutterella stercoricanis (supported by a bootstrap resampling value
of 70%) and Parasutterella sp. (supported by a bootstrap resampling
value of 68%). In contrast, treeing analysis revealed that
Sutterella sp. sequences found in patients 5 and 7 were most
closely associated with the isolate Sutterella wadsworthensis
(supported by a bootstrap resampling value of 94%) (FIG. 15A).
These findings were consistent with treeing analysis obtained from
V2 sequences obtained from pyrosequencing analysis in which V2
Sutterella sequences from patients 1, 3, 10, 11, and 12 were most
closely associated with the isolates Sutterella stercoricanis and
Sutterella sanguinus (supported by a bootstrap resampling value of
67%) while the V2 Sutterella sequences from patients 5 and 7 were
most closely associated with the isolates of Sutterella
wadsworthensis (supported by a bootstrap resampling value of 100%)
(FIG. 15B). Thus, sequences from patients 5 and 7 clustered with
Sutterella wadsworthensis isolates using both the V2 pyrosequencing
reads and the V6-V8 sequences obtained from this assay. In
contrast, sequences from patients 1, 3, 10, 11, and 12 clustered
with Sutterella stercoricanis using both the V2 pyrosequencing
reads and the V6-V8 sequence obtained from this assay. However,
there was some divergence between the V2 and V6-V8 regions in
determining relationships to other isolates (i.e. relatedness to
Sutterella sanguinus from the V2 sequences and relatedness to
Parasutterella sp. from the V6-V8 sequences).
REFERENCES
[0364] A1.) Wexler H M, Reeves D, Summanen P H, Molitoris E,
McTeague M, Duncan J, Wilson K H, Finegold S M. 1996. Sutterella
wadsworthensis gen. nov., sp. nov., bile-resistant microaerophilic
Campylobacter gracilis-like clinical isolates. Int J Syst
Bacteriol, 46(1): 252-258. [0365] A2.) Mangin I, Bonnet R, Seksik
P, Rigottier-Gois L, Sutren M, Bouhnik Y, Neut C, Collins M D,
Colombel J F, Marteau P, Dore J. 2004. Molecular inventory of
faecal microflora in patients with Crohn's disease. FEMS Microbiol
Ecol, 50(1): 25-36. [0366] A3.) Gophna U, Sommerfeld K, Gophna S,
Doolittle W F, Veldhuyzen van Zanten S J. 2006. Differences between
tissue-associated intestinal microfloras of patients with Crohn's
disease and ulcerative colitis. J Clin Microbiol, 44(11):
4136-4141. [0367] A4.) Greetham H L, Collins M D, Gibson G R,
Giffard C, Falsen E, Lawson P A. 2004. Sutterella stercoricanis sp.
nov., isolated from canine faeces. Int J Syst Evol Microbiol. 54:
1581-1584. [0368] A5.) J Scupham A, Patton T G, Bent E, Bayles D O.
2008. Comparison of the cecal microbiota of domestic and wild
turkeys. Microb Ecol. 56: 322-331. [0369] A6.) Frank D N, St Amand
A L, Feldman R A, Boedeker E C, Harpaz N, Pace N R. 2007.
Molecular-phylogenetic characterization of microbial community
imbalances in human inflammatory bowel diseases. Proc Natl Acad Sci
USA. 104: 13780-13785. [0370] King A, Downes J, Nord C E, Phillips
I; European Study Group. 1999. Antimicrobial susceptibility of
non-Bacteroides fragilis group anaerobic Gram-negative bacilli in
Europe. Clin Microbiol Infect. 5: 404-416. [0371] Goldstein E J,
Citron D M. 2009. Activity of a novel carbapenem, doripenem,
against anaerobic pathogens. Diagn Microbiol Infect Dis. 63:
447-454. [0372] Wexler H M, Molitoris D, St John S, Vu A, Read E K,
Finegold S M. 2002. In vitro activities of faropenem against 579
strains of anaerobic bacteria. Antimicrob Agents Chemother. 46:
3669-3675. [0373] Wexler H M, Molitoris D, Finegold S M. 2000. In
vitro activities of M K-826 (L-749,345) against 363 strains of
anaerobic bacteria. Antimicrob Agents Chemother. 44: 2222-2224.
[0374] Molitoris E, Wexler H M, Finegold S M. 1997. Sources and
antimicrobial susceptibilities of Campylobacter gracilis and
Sutterella wadsworthensis. Clin Infect Dis. Suppl 2: S264-265.
[0375] Wexler H M, Molitoris E, Molitoris D, Finegold S M. 1996. In
vitro activities of trovafloxacin against 557 strains of anaerobic
bacteria. Antimicrob Agents Chemother. 40: 2232-2235.
Example 3
Impaired Carbohydrate Digestion and Transport and Mucosal
Dysbiosis
[0376] Gastrointestinal disturbances are commonly reported in
children with autism, complicate clinical management, and can
contribute to behavioral impairment. Reports of deficiencies in
disaccharidase enzymatic activity and of beneficial responses to
probiotic and dietary therapies led to the survey gene expression
and the mucoepithelial microbiota in intestinal biopsies from
children with autism and gastrointestinal disease and children with
gastrointestinal disease alone. Ileal transcripts encoding
disaccharidases and hexose transporters were deficient in children
with autism, indicating impairment of the primary pathway for
carbohydrate digestion and transport in enterocytes. Deficient
expression of these enzymes and transporters was associated with
expression of the intestinal transcription factor, CDX2.
Metagenomic analysis of intestinal bacteria revealed compositional
dysbiosis manifest as decreases in Bacteroidetes, increases in the
ratio of Firmicutes to Bacteroidetes, and increases in
Betaproteobacteria. Expression levels of disaccharidases and
transporters were associated with the abundance of affected
bacterial phylotypes. These results indicate a relationship between
human intestinal gene expression and bacterial community structure
and provide insights into the pathophysiology of gastrointestinal
disturbances in children with autism.
[0377] Autism spectrum disorders (ASD) are defined by impairments
in verbal and non-verbal communication, social interactions, and
repetitive and stereotyped behaviors. In addition to these core
deficits, the prevalence of gastrointestinal (GI) symptoms ranges
widely in individuals with ASD, from 9 to 91% in different study
populations [1]. Macroscopic and histological observations in ASD
include findings of ileo-colonic lymphoid nodular hyperplasia,
enterocolitis, gastritis and esophagitis [2, 3, 4, 5, 6, 7].
Associated changes in intestinal inflammatory parameters include
higher densities of lymphocyte populations, aberrant cytokine
profiles, and deposition of immunoglobulin (IgG) and complement C1q
on the basolateral enterocyte membrane [5, 8, 9, 10, 11, 12].
Reported functional disturbances include increased intestinal
permeability [13], deficient enzymatic activity of disaccharidases
[7], increased secretin-induced pancreatico-biliary secretion [7],
and abnormal Clostridia taxa [14, 15, 16]. Some children placed on
exclusion diets or treated with the antibiotic vancomycin are
reported to improve in cognitive and social function [17, 18].
Furthermore, a strong correlation between GI symptoms and autism
severity was found [19].
[0378] The intestinal mucoepithelial layer must maximize
nutritional uptake of dietary components while maintaining a
barrier to toxins and infectious agents. Although some aspects of
these functions are host-encoded, others are acquired through
symbiotic relationships with microbial flora. Dietary carbohydrates
enter the intestine as monosaccharides (glucose, fructose, and
galactose), disaccharides (lactose, sucrose, maltose), or complex
polysaccharides. Following digestion with salivary and pancreatic
amylases, carbohydrates are further digested by disaccharidases
expressed by absorptive enterocytes in the brush border of the
small intestine and transported as monosaccharides across the
intestinal epithelium. Although humans lack the glycoside
hydrolases and polysaccharide lyases necessary for cleavage of
glycosidic linkages present in plant cell wall polysaccharides,
oligosaccharides, storage polysaccharides, and resistant starches,
intestinal bacteria encoding these enzymes expand the capacity to
extract energy from dietary polysaccharides [20,21]. As an end
product of polysaccharide fermentation, bacteria produce
short-chain fatty acids (butyrate, acetate, and propionate) that
serve as energy substrates for colonocytes, modulate colonic pH,
regulate colonic cell proliferation and differentiation, and
contribute to hepatic gluconeogenesis and cholesterol synthesis
[22,23]. Intestinal microbes also mediate postnatal development of
the gut mucoepithelial layer, provide resistance to potential
pathogens, regulate development of intraepithelial lymphocytes and
Peyer's patches, influence cytokine production and serum
immunoglobulin levels, promote systemic lymphoid organogenesis, and
influence brain development and behavior [24, 25, 26].
[0379] Although bacteria have been examined in fecal material from
children with autism, no study to date has reported analyses of
microbiota adherent to their intestinal mucoepithelium.
Furthermore, there are no reports wherein intestinal gene
expression in children with autism has been correlated with
alterations in intestinal microbiota. GI dysfunction is commonly
reported in children with autism; however, it remains unclear how
or whether GI dysfunction in children with autism differs from GI
dysfunction found in typically developing children. Expression of
human genes involved in carbohydrate digestion and transport was
investigated along with bacterial community composition in
intestinal biopsies from children with autistic disorder and GI
disease (AUT-GI) compared to children with GI disease alone
(Control-GI). Results from gene expression assays and metagenomic
analysis of over half a million bacterial 16S rRNA gene sequences
revealed decreased mRNA expression for human disaccharidases and
hexose transporters and compositional dysbiosis in children in the
AUT-GI group compared to those in the Control-GI group. Results
described herein show the complex relationship between human
intestinal gene expression and bacterial community structure, and
provide insights into the molecular mechanisms underlying the
pathophysiology of gastrointestinal disturbances in children with
autism.
Results
[0380] Patient Characteristics
[0381] All AUT-GI and Control-GI children evaluated were male
(Table 6A). Mean onset age for autism in AUT-GI was 13.4+/-5.4
months. Median age at biopsy was similar for AUT-GI and Control-GI
children [median age in years (interquartile range, IQR), AUT-GI,
4.5 (1.3); and Control-GI, 4.0 (1.1)]. Median number of medications
used and the IQR for number of medications used per subject were
identical in AUT-GI and Control-GI children. Food allergies (FA)
were commonly reported in both AUT-GI (67%) and Control-GI (71%)
subjects. The majority of children with FA had reported
milk-related allergy (90% for AUT-GI and 100% for Control-GI)
and/or wheat-related allergy (80% for AUT-GI and 80% for
Control-GI). Beneficial effects of dietary intervention on GI
disturbances were reported for all AUT-GI and Control-GI subjects
with FA. Comorbid conditions were reported in 67% of AUT-GI
children and 100% of Control-GI children. The most commonly
reported comorbid conditions were atopic manifestations (asthma,
atopic dermatitis, and allergic rhinitis). Atopic manifestations
were more common in Control-GI children (100%) than AUT-GI children
(53%) (Table 6A). The frequency of individual atopic manifestations
was higher in Control-GI children. The largest difference in
frequency was for asthma, which was only reported in 20% of AUT-GI
children compared to 71% of Control-GI children (Table 6A).
Established intestinal disorders were only reported in a few
subjects: two AUT-GI subjects (13%: 1 with IBD, 1 with Celiac
disease) and one Control-GI subject (14%: IBD). For detailed
information related to medication use, food allergy and comorbid
conditions in individual AUT-GI and Control-GI children see Table
7. The prevalence of specific GI symptoms was similar in AUT-GI and
Control-GI children (Table 6B). The most frequently reported GI
symptoms in both groups were diarrhea (AUT-GI, 80%; Conrol-GI, 71%)
and changes in stool frequency (AUT-GI, 87%; Control-GI, 71%) and
consistency (AUT-GI, 80%; Control-GI, 86%). Mucus in stool was more
frequent in Control-GI (86%) compared to AUT-GI (40%) children;
bloating was more frequent in AUT-GI (60%) compared to Control-GI
(29%) children. Regression (loss of language and/or other skills
following acquisition) is reported in 20% to 40% of individuals
with autism, and some studies indicate higher rates of GI symptoms
in ASD subjects with regression than those without regression [27].
87% of the AUT-GI subjects had behavioral regression (Table 8).
TABLE-US-00011 TABLE 6A, B Summary of patient characteristics.
AUT-GI Control-GI Subject Characteristic Subcategory (n = 15) (n =
7) Autism onset age in months, AUT-GI subjects 13.4 .+-. 5.4 --
mean .+-. SD Gender All subjects All male All male Ethnicity, n (%)
Caucasian 14 (93) 6 (86) Hispanic 1 (7) 0 (0) African-American 0
(0) 1 (14) Age at biopsy in years, All subjects 4.5 (1.3) [3.5-5.9]
4.0 (1.1) [3.9-5.5] median (IQR) [range] Medications-number per All
subjects 5 (7) [1-21] 5 (7) [0-8] subject.sup.a, median (IQR)
[range] Food allergies, n (% of All subjects 10 (67) 5 (71)
subjects) Milk-related allergy.sup.b, n (% Subjects reporting 9
(90) 5 (100) of subjects with food allergy) any food allergy
Wheat-related allergy.sup.c, n (% Subjects reporting 8 (80) 4 (80)
of subjects with food allergy) any food allergy Diet improvement of
GI Subjects reporting 10 (100) 5 (100) problems, n (% of subjects
any food allergy with food allergy) Current comorbid All subjects 1
(1.75) [0-5] 2 (2.75) [1-6] conditions-number per subject, median
(IQR) [range] Comorbid atopic disease All subjects 8 (53) 7 (100)
manifestations.sup.d, n (% of subjects) Asthma, n (% of subjects)
All subjects 3 (20) 5 (71) Atopic dermatitis, n (% of All subjects
4 (27) 4 (57) subjects) Allergic rhinitis, n (% of All subjects 4
(27) 3 (43) subjects) .sup.aNumber of prescription drugs and
alternative agents taken regularly, per subject .sup.bAllergy to
milk, casein, lactose or dairy .sup.cAllergy to wheat or gluten
.sup.dAsthma, Allergic rhinitis, or Atopic dermatitis
TABLE-US-00012 TABLE 6B Summary of patients' GI symptoms. GI
Symptoms AUT-GI, n (%) Control-GI, n (%) Diarrhea 12 (80) 5 (71)
Diarrhea w/ Vomiting 2 (13) 2 (29) Vomiting 2 (13) 1 (14) Bloating
9 (60) 2 (29) .DELTA. Stool Frequency 13 (87) 5 (71) .DELTA. Stool
Consistency 12 (80) 6 (86) Mucus in Stool 6 (40) 6 (86) Blood in
Stool 2 (13) 1 (14) Pain 8 (53) 5 (71) Weight Loss 3 (20) 0 (0)
Fever 1 (7) 0 (0)
TABLE-US-00013 TABLE 7 Reported comorbid conditions, food
allergies, and medication use by patient Current Comorbid Food
Allergy Patient # Group Conditions Reported Medications 1 AUT-GI
asthma, atopic milk, gluten, Vitamin B1, B2, B3, B6, B9,
dermatitis, celiac eggs, B12, C, E; Ca, Zn, Fish oil, disease,
movement peanuts, tree Omega-3-fatty acids, disorder, myopathy
nuts, soy, Probiotic, Ibuprofen, corn, peas Lanzoprazole,
Montelukast sodium, Levalbuterol inhaler, Albuterol inhaler 2
AUT-GI allergic rhinitis milk, gluten, Vitamin C; MVM, Ca/Mg eggs
supplement, Omeprazole 3 AUT-GI IBD Milk, gluten, Vitamin B12, C;
MVM, dyes Ca/Mg supplement, Zn, flaxseed oil, antifungal herbal
agent, digestive enzymes 4 AUT-GI allergic rhinitis, casein, gluten
Vitamin A, C, Methyl-B12, asthma, atopic Folinic acid; MVM, Ca/Mg
dermatitis, migraine supplement, Zn, Mb, Fish oil, Omega-3-fatty
acids, SAMe, Inositol, Selenomethionine, Trimethylglycine, 5-
methyl-tetrahydrofolate, Transdermal glutathione, MgSO4 cream, Zn
soy cream, DMAE, DMPS, Alpha lipoic acid, Montelukast sodium 5
AUT-GI atopic dermatitis lactose MVM 6 AUT-GI allergic rhinitis,
gluten, corn, Vitamin D; Ca, Zn, Mg, P, frequent URI, soy Flaxseed
oil, Probiotic, epilepsy Artichoke extract, Sarsaparilla extract,
Wasabi powder, Lipase, Amylase, Protease 7 AUT-GI allergic
rhinitis, milk, gluten, Folinic acid; MVM, Ca/Mg frequent otitis
sweet supplement, media potatoes, Trimethylglycine, Lipase,
oranges, Amylase, Protease, berries Cellulase, Lactase 8 AUT-GI
none none reported Vitamin B complex, L- carnitine, Lipase,
Amylase, Protease, Diphenhydramine, Acetaminophen, Ibuprofen,
Melatonin, Sertraline, Valproic acid 9 AUT-GI none none reported
MVM, Ca 10 AUT-GI none none reported Omeprazole 11 AUT-GI atopic
dermatitis cow's milk, Flaxseed oil, Coenzyme goat's milk, Q10,
Cell signal barley, enhancers (CSE-14, 15), carrots, Probiotic,
Lipase bananas, cantelope, coffee, cranberry, lamb, lettuce 12
AUT-GI Epstein-Barr virus dairy, wheat, Methyl-B12, DMSA, infection
salicylates, Amphoterecin B Phenols 13 AUT-GI asthma dairy, wheat,
Vitamin B12; Ca/Mg yeast supplement, Zn, Probiotic, Clonidine,
Secretin 14 AUT-GI none none reported MVM, F 15 AUT-GI none none
reported Lipase, Amylase, Protease, Milk of magnesia, Lansoprazole
16 Control- allergic rhinitis, none reported MVM, Montelukast
sodium, GI asthma, atopic Fluticasone propionate, dermatitis,
frequent Lansoprazole, Amoxicillin sinusitis 17 Control- atopic
dermatitis none reported Ca citrate, Mg/amino acid GI complex,
Hydroxyzine, Budesonide, Prednisolone, Montelukast sodium,
Levalbuterol inhaler, Tacrolimus 18 Control- asthma dairy, peanuts
Ibuprofen GI 19 Control- asthma, atopic milk, wheat, Lipase,
Amylase, Protease, GI dermatitis, IBD, eggs, oats, Diphenhydramine,
Cetirizine dysphagia, salmon, soy, hydrochloride, Omeprazole,
microcytic anemia, peanut, tree Budesonide, Montelukast pancreatic
nut, chicken, sodium, Levalbuterol inhaler insufficiency turkey,
beef, broccoli, cabbage, lentils, legumes 20 Control- allergic
rhinitis, dairy, gluten, Vitamin B12, Fish oil, Milk GI asthma,
atopic eggs, soy, thistle, DMSA, Allithiamine dermatitis citrus 21
Control- asthma dairy, wheat, Probiotic GI eggs, fruit 22 Control-
allergic rhinitis, dairy, wheat, none reported GI vitiligo eggs,
peanuts, beef IBD--Inflammatory Bowel Disease; URI--Upper
respiratory tract infection; MVM--multivitamin with minerals;
SAMe--S-adenosylmethionine; DMAE--dimethylaminoethanol;
DMPS--2,3-Dimercapto-1-propanesulfonic acid;
DMSA--Dimercaptosuccinic acid
TABLE-US-00014 TABLE 8 Reported behavioral regression in AUT-GI
children. AUT/GI cases (n = 15) PHENOTYPIC CHARACTERISTICS n (%)
ANY REPORTED LOSS (ADI-R or CDI) 13 (87) ADI-R LOSS Language loss
11 (73) ITEMS Other skill loss 12 (80) (with or without language
loss) Other skill loss 2 (13) without language loss CPEA Word loss
regression 12 (80) REGRESSION Non-word loss regression 1 (7)
CATEGORY No regression 2 (13) Legend: ADI-R, Autism Diagnostic
Interview-Revised; CDI, MacArthur Communicative Development
Inventory; CPEA, Collaborative Program for Excellence in
Autism.
[0382] Deficient Ileal mRNA Expression of Disaccharidases and
Hexose Transporters in AUT-GI Children
[0383] Transcript levels were examined for three primary brush
border disaccharidases (sucrase isomaltase [SI], maltase
glucoamylase [MGAM], and lactase [LCT]) in ileal biopsies of AUT-GI
and Control-GI children by real time PCR. Levels of mRNA for all
three enzymes were decreased in AUT-GI children: SI (FIG. 16A:
Mann-Whitney, p=0.001), MGAM (FIG. 16B: Mann-Whitney, p=0.003) and
LCT (FIG. 16C: Mann-Whitney, p=0.032). Within the AUT-GI group,
86.7%, 80%, and 80% of children had deficient transcript levels
(defined as below the 25.sup.th percentile of values obtained for
Control-GI children and at least two-fold below Control-GI mean
values) for SI, MGAM, and LCT, respectively (Table 9A and Table
10). Nearly all (14/15, or 93.3%) AUT-GI children had deficiencies
in at least one disaccharidase enzyme; 80% had deficiencies in 2 or
more enzymes; 73.3% had deficiencies in all three enzymes (Table
9A). Deficiencies in LCT mRNA in AUT-GI children were not
attributable to disproportionate adult-type hypolactasia genotypes
in the AUT-GI group relative to the Control-GI group (FIG.
36A-D).
TABLE-US-00015 TABLE 9 Patient summary tables for gene expression
and bacterial assays. (A-C) Legend: Increases or decreases in
AUT-GI children in both gene expression (A) and bacterial
parameters (B and C) were determined for each individual based on
the levels of each parameter in the Control-GI group. (A) The gene
expression levels in the AUT-GI children that exceeded the
75.sup.th percentile of Control-GI values and were at least 2-fold
increased relative to the Control-GI mean (arrow pointing up) or
below the 25.sup.th percentile of Control-GI values and at least
2-fold decreased relative to the Control-GI mean (arrow pointing
down) were scored as an increase or decrease, respectively. (B and
C) Bacterial parameters in AUT-GI children that exceeded the
75.sup.th percentile of Control-GI values (arrows pointing up) or
were below the 25.sup.th percentile of Control-GI values (arrows
pointing down) were scored as an increase or decrease,
respectively. Values above the 90.sup.th or below the 10.sup.th
percentiles of Control-GI children are indicated by double arrows.
Results arc shown for data obtained by real-time PCR (RT), where
performed, and pyroscquencing (454). (n.c. - no change relative to
defined cut-off values for Control-GI children). A ##STR00004## B
##STR00005## C Firm./ Firm./ Firm./ Firm./ Clostridiales/
Clostridiales/ Firm. + Firm. + Bacteroid. Bacteroid. Bacteroid.
Bacteroid. Bacteroidates Bacteroidates Proteobac. Proteobac. AUT-GI
Ratio- Ratio- Ratio- Ratio- Ratio- Ratio- Ratio- Ratio- Patien # RT
Ileum RT Cecum 454 Ileum 454 Cecum 454 Ileum 454 Cecum 454 Ileum
454 Ileum 1 2 3 4 5 n.c. n.c. n.c. n.c. 6 n.c. n.c. n.c. n.c. 7 8 9
n.c. 10 n.c. 11 12 n.c. n.c. n.c. n.c. n.c. n.c. n.c. 13 14 n.c.
n.c. n.c. n.c. 15 % above 100% 60% 73.3% 66.7% 80.0% 66.7% 80%
73.3% controls
TABLE-US-00016 TABLE 10 Fold-change in gene expression in AUT-GI
children. Legend: Fold-change values were calculated relative to
the mean expression level obtained for all Control-GI children for
each gene. Expression levels for individual patients that were at
least 2-fold increased (>2) or decreased (<0.5) relative to
the Control-GI mean (grey*) are highlighted in gray, and dark gray,
respectively. ##STR00006##
[0384] Two hexose transporters, sodium-dependent glucose
cotransporter (SGLT1) and glucose transporter 2 (GLUT2), mediate
transport of monosaccharides in the intestine. SGLT1, located on
the luminal membrane of enterocytes, is responsible for the active
transport of glucose and galactose from the intestinal lumen into
enterocytes. GLUT2 transports glucose, galactose and fructose
across the basolateral membrane into the circulation and can also
translocate to the apical membrane [28]. Real-time PCR revealed a
decrease in ileal SGLT1 mRNA (FIG. 16D: Mann-Whitney, p=0.008) and
GLUT2 mRNA (FIG. 16E: Mann-Whitney, p=0.010) in AUT-GI children.
For SGLT1, 73.3% of AUT-GI children had deficient transcript
levels; 73.3% of AUT-GI children had deficient GLUT2 transcript
levels relative to Control-GI children (Table 9A). Deficiencies
were found in at least one hexose transporter in 80% of AUT-GI
children; 66.7% had deficiencies in both transporters.
[0385] In total, 93.3% (14/15) of AUT-GI children had mRNA
deficiencies in at least one of the 5 genes involved in
carbohydrate digestion or transport; 66.7% (10/15) had mRNA
deficiencies in all 5 genes (Table 9A).
[0386] To determine whether reductions in disaccharidase and
transporter transcript levels reflected loss of or damage to
intestinal epithelial cells, mRNA levels associated with a
tissue-specific marker restricted to these cells, villin [29,30]
was measured. Ileal villin mRNA levels were not decreased in AUT-GI
children (Mann-Whitney, p=0.307) (FIG. 16F). Normalization of SI,
MGAM, LCT, SGLT1 and GLUT2 to villin mRNA levels did not correct
deficits (FIG. 22A-E).
[0387] The transcription factor, caudal type homeobox 2 (CDX2),
regulates expression of SI, LCT, GLUT2, and SGLT1 [31, 32, 33, 34].
Real-time PCR experiments demonstrated lower levels of CDX2 mRNA in
some AUT-GI subjects versus controls; however, group differences
were not significant (FIG. 16G: Mann-Whitney, p=0.192). Although
only 33.3% of AUT-GI patients had deficient CDX2 mRNA levels (Table
9A), 86.7% of AUT-GI children had CDX2 levels below the 50.sup.th
percentile of Control-GI children and 46.7% of AUT-GI children had
at least a two-fold decrease in CDX2 expression relative to the
Control-GI mean. Only one AUT-GI child (patient #7) had CDX2 levels
above the 75.sup.th percentile of Control-GI children and a near
2-fold (1.95-fold) increase in CDX2 expression (Table 9A and Table
10). This child was the only AUT-GI subject who did not show signs
of deficiencies in disaccharidases or transporters.
[0388] AUT-GI children with deficiencies in all five
disaccharidases and tranporters had significantly lower levels of
CDX2 mRNA compared to AUT-GI children with fewer than five
deficiencies (FIG. 33: Mann-Whitney, p=0.037). However, only a
trend toward decreased CDX2 levels was found when comparing AUT-GI
children with deficiencies in all five disaccharidases and
transporters and Control-GI children (FIG. 33: Mann-Whitney,
p=0.064).
[0389] Multiple linear regression analysis was conducted to
determine whether diagnostic status (AUT-GI or Control-GI), CDX2
mRNA expression, or villin mRNA expression (predictor variables)
was associated with mRNA expression levels of individual
disaccharidases (SI, MGAM, LCT) or transporters (SGLT1, GLUT2)
(Table 11). In each of the five models, where the expression of SI,
MGAM, LCT, SGLT1, or GLUT2 served as outcome variables, CDX2
contributed significantly to the model. As the level of CDX2
increased by one unit of standard deviation, there was a
concomitant approximate one unit increase in log-transformed
disaccharidase and transporter transcript levels (ranging from 0.78
for SGLT1 to 1.30 for LCT). None of the interaction terms between
CDX2 and status were significant, indicating that the magnitude of
the effect of CDX2 on log-transformed enzyme and transporter levels
was the same for AUT-GI and Control-GI children. For SGLT1 and
GLUT2 expression, CDX2 was the sole significant predictor variable
in the model. Status and CDX2 were significant predictors of SI,
MGAM, and LCT expression, indicateing that additional factors
associated with status must also contribute to expression levels
for these enzymes. Villin was not a significant predictor of the
expression levels of any of the five genes after adjusting for
CDX2.
TABLE-US-00017 TABLE 11 Multiple linear regression analysis
examining CDX2 and villin as predictors of disaccharidase and
transporter mRNA expression among AUT-GI and Control-GI children.
Ad- Predictor Variables: Outcome F.sub.3,18 justed Coefficient
Estimate Variable (p-value) R.sup.2 Status CDX2.sup.STDev
Villin.sup.STDev SI 10.35 0.57 -1.83* 0.93* -0.19 (0.0003)*** MGAM
8.78 0.53 -2.10* 1.15* -0.20 (0.0008)*** LCT 10.87 0.59 -2.25*
1.30* 0.65 (0.0003)*** SGLT1 6.88 0.46 -1.36.dagger. 0.78* 0.12
(0.0030)** GLUT2 6.06 0.42 -1.90.dagger. 1.06* 0.03 (0.0050)**
.sup.STDevChange in log-transformed outcome variable levels per
unit standard deviation increase in predictor variable *p <
0.05; **p < 0.01; ***p < 0.001; .dagger.p < 0.1
(trend)
[0390] Mucosal Dysbiosis in AUT-GI Children
[0391] To determine whether deficient carbohydrate digestion and
absorption influenced the composition of intestinal microflora,
ileal and cecal biopsies from AUT-GI and Control-GI children were
analyzed by bacterial 16S rRNA gene pyrosquencing. The use of
biopsies rather than fecal material allowed us to assess the
mucoepithelia-associated microbiota, as these likely establish more
intimate interactions with the human intestinal epithelium and
immune cells [35]. A total of 525,519 bacterial sequences were
subjected to OTU (Operational Taxonomic Unit; defined at 97%
identity) analysis and classified with RDP (Ribosomal Database
Project). Rarefaction analysis of OTUs did not indicate a loss or
gain of overall diversity based on Shannon Diversity estimates in
AUT-GI compared to Control-GI children (See FIG. 23A-D).
[0392] Classification of pyrosequencing reads revealed that
Bacteroidetes and Firmicutes were the most prevalent taxa in ileal
and cecal tissues of AUT-GI and Control-GI children, followed by
Proteobacteria (FIG. 17A, B). Other phyla identified at lower
levels included Verrucomicrobia, Actinobacteria, Fusobacteria,
Lentisphaerae, and TM7, as well as "unclassified bacteria"
(sequences that could not be assigned at the phylum-level) (FIG.
17A, B). The abundance of Bacteroidetes was lower in AUT-GI ileal
(FIG. 17C: Mann-Whitney, p=0.012) and cecal biopsies (FIG. 17D:
Mann-Whitney, p=0.008) as compared with the abundance of
Bacteroidetes in Control-GI biopsies. Real-time PCR using
Bacteroidete-specific primers confirmed decreases in Bacteroidetes
in AUT-GI ilea (FIG. 17E: Mann-Whitney, p=0.003; Table 12: 50%
average reduction in Bacteroidete 16S rDNA copies; range, 24.36% to
76.28% decrease) and ceca (FIG. 17F: Mann-Whitney, p=0.022; Table
12: 29% average reduction in 13 of 15 patients with reduced
Bacteroidetes; range, 7.22% to 56.54% decrease), with levels below
the 25.sup.th percentile of Control-GI children in 100% of AUT-GI
ilea and 86.7% of AUT-GI ceca (Table 9B). OTU analysis of
Bacteroidete sequences indicateed that deficiencies in Bacteroidete
sequences in AUT-GI subjects were attributable to cumulative losses
of 12 predominant phylotypes of Bacteroidetes, rather than loss of
any one specific phylotype (FIG. 25A-E).
TABLE-US-00018 TABLE 12 Percent change in bacterial levels in
AUT-GI children. Bacteroidetes Bacteroidetes Bacteroidetes
Bacteroidetes Firmicutes Firmicutes Firmicutes Firmicutes RT-Ileum
RT-Cecum 454-Ileum 454-Cecum RT-Ileum RT-Cecum 454-Ileum 454-Cecum
Patient # % Change % Change % Difference % Difference % Change %
Change % Difference % Difference 1 -38.45 -45.21 -12.97 -8.04 22.53
39.63 12.22 8.24 2 -76.28 -32.49 -41.41 -18.39 57.67 96.65 -6.97
1.43 3 -54.81 -27.47 -9.17 -13.61 26.31 88.51 6.54 7.84 4 -61.97
-16.71 -18.16 -11.57 132.28 77.63 17.25 13.88 5 -48.68 -22.27 5.65
3.02 5.58 -3.18 -5.16 -5.10 6 -38.60 0.94 0.80 4.77 19.05 131.95
5.23 -0.66 7 -38.60 -14.12 -4.85 -12.24 -2.18 48.42 10.23 14.63 8
-53.67 -50.41 -20.58 -21.64 -13.25 2.35 3.26 3.41 9 -41.25 -17.14
-12.58 -10.11 24.21 22.05 16.30 8.73 10 -40.14 -9.41 -10.88 -12.04
-13.93 18.59 13.89 12.11 11 -70.52 -56.54 -13.25 -16.26 45.33 83.50
3.06 3.85 12 -35.81 -7.22 -0.12 -4.52 17.67 30.06 2.06 3.13 13
-47.99 -40.26 -6.34 -20.95 14.14 49.50 8.90 10.92 14 -24.36 13.00
-7.63 -3.02 8.88 15.44 12.74 5.19 15 -75.62 -34.67 -29.30 -14.41
-60.76 -62.03 -14.79 -16.49 Clostridia Clostridia Lach. + Rumino.
Lach. + Rumino. Proteobacteria Proteobacteria Beta-Proteoabact.
Beta-Proteoabact. 454-Ileum 454-Cecum 454-Ileum 454-Cecum RT-Ileum
RT-Cecum 454-Ileum 454-Cecum Patient # % Difference % Difference %
Difference % Difference % Difference % Difference % Difference %
Difference 1 13.33 8.84 14.51 9.92 1.52 0.82 4.36 3.00 2 -6.26 1.56
-6.50 1.87 47.77 16.23 24.82 7.16 3 7.34 8.11 5.45 6.39 -0.57 1.95
3.10 3.12 4 17.72 14.20 16.72 15.29 1.90 -1.27 1.55 0.02 5 -4.24
-4.49 -3.18 -3.61 -0.02 2.72 4.46 3.89 6 6.14 0.06 5.81 0.35 -5.00
-2.99 -0.56 -0.78 7 10.91 15.17 12.31 16.49 -4.34 -1.26 0.17 0.89 8
3.04 2.34 3.97 3.24 18.40 19.27 -1.35 -0.42 9 17.34 9.46 17.21 9.35
-3.27 1.85 0.54 2.69 10 14.97 12.76 15.52 13.44 -1.98 0.73 2.43
2.79 11 3.91 4.30 3.85 4.18 8.05 10.52 5.55 6.16 12 2.79 3.56 2.05
2.15 -1.82 1.25 2.34 2.90 13 9.86 11.17 10.47 11.74 -3.60 8.33 0.06
4.70 14 13.66 0.03 15.50 7.61 -4.09 -1.09 -0.56 -0.15 15 -15.60
-16.02 -14.38 -14.15 44.83 31.90 -0.43 -0.79 Firm./Bac- Firm./Bac-
Firm./Bac- Firm./Bac- Clostrid./Bac- Clostrid./Bac- Firm. + Firm. +
teroid. Ratio teroid. Ratio teroid. Ratio teroid. Ratio teroid.
Ratio teroid. Ratio Proteobact. Proteobact. RT-Ileum RT-Cecum
454-Ileum 454-Cecum 454-Ileum 454-Cecum 454-Ileum 454-Cecum Patient
# % Change % Change % Change % Change % Change % Change % Change %
Change 1 85.88 130.48 80.63 48.14 89.92 52.85 13.73 9.06 2 520.47
163.47 81.04 39.53 83.47 39.47 40.79 17.86 3 160.98 132.59 43.00
60.37 47.41 62.46 5.97 9.79 4 470.19 92.89 126.70 84.43 133.22
67.82 19.15 12.62 5 92.09 12.65 -28.53 -26.23 -26.69 -25.13 -5.18
-2.36 6 61.61 107.84 17.06 -10.99 21.09 -6.72 0.24 -3.67 7 49.24
56.32 48.85 60.21 53.38 94.97 5.89 13.37 8 74.81 86.66 59.00 60.28
57.63 53.77 21.65 22.88 9 97.40 33.24 99.17 55.50 107.51 59.77
13.04 10.59 10 34.24 18.40 81.88 77.16 90.50 82.97 11.91 12.84 11
360.23 281.88 38.65 46.99 40.77 49.43 11.11 14.37 12 71.17 28.80
6.13 16.04 8.99 20.18 0.24 4.39 13 104.90 126.51 46.68 101.59 55.16
107.58 5.30 19.25 14 34.37 -7.59 66.89 24.23 74.34 28.28 8.65 4.09
15 50.31 -47.27 -30.42 -62.02 -43.09 -63.48 30.04 15.41 Legend:
Percent change values were calculated for real-time PCR and ratio
data relative to the mean levels obtained for all Control-GI
children for each bacterial variable. Percent difference values
were calculated for pyrosequencing data by subtracting the mean
percent abundance of Control-GI children from the percent abundance
of each AUT-GI patient for each variable.
[0393] Analysis of pyrosequencing reads revealed a significant
increase in Firmicute/Bacteroidete ratios in AUT-GI ilea (FIG. 18A:
Mann-Whitney, p=0.026) and ceca (FIG. 18B: Mann-Whitney, p=0.032).
An increase was also observed at the order level for
Clostridiales/Bacteroidales ratios in ilea (FIG. 26A: Mann-Whitney,
p=0.012) and ceca (FIG. 26B: Mann-Whitney, p=0.032). Real-time PCR
using Firmicute- and Bacteroidete-specific primers confirmed
increases in Firmicute/Bacteroidete ratios in AUT-GI ilea (FIG.
30C: Mann-Whitney, p=0.0006) and ceca (FIG. 30D: Mann-Whitney,
p=0.022). Based on real-time PCR results, Firmicute/Bacteroidete
ratios were above the 75.sup.th percentile of Control-GI values in
100% of AUT-GI ilea and 60% of AUT-GI ceca (Table 9C).
[0394] The cumulative level of Firmicutes and Proteobacteria was
significantly higher in the AUT-GI group in both ileal (FIG. 18G:
Mann-Whitney, p=0.015) and cecal (FIG. 18H: Mann-Whitney, p=0.007)
biopsies; however, neither Firmicute nor Proteobacteria levels
showed significant differences on their own (FIG. 27A-D and FIG.
19A, B). These results indicate that the observed decrease in
Bacteroidetes in AUT-GI children is accompanied by an increase in
Firmicutes (Ileal biopsies--Patients 1, 3, 4, 6, 7, 9, 10, 13, and
14; Cecal biopsies-Patients 1, 3, 4, 7, 9, 10, and 13), or
Proteobacteria (Ileal biopsies--Patients 2, 8, 11 and 15; Cecal
biopsies--Patients 2, 5, 8, 11, 13, and 15), or both (Cecal
biopsies--Patient 13) (Table 9B and FIG. 34A-B).
[0395] Within the Firmicute phyla, order-level analysis of
pyrosequencing reads indicated trends toward increases in
Clostridiales in AUT-GI ilea (FIG. 27E: Mann-Whitney, p=0.072) and
ceca (FIG. 27F: Mann-Whitney, p=0.098). Family-level analysis
revealed that increased Clostridiales levels in AUT-GI patient
samples were largely attributable to increases in Lachnospiraceae
and Ruminococcaceae (FIG. 18C-F) Cumulative levels of
Lachnospiraceae and Ruminococcaceae above the 75.sup.th percentile
of the corresponding levels in Control-GI samples were found in 60%
of AUT-GI ileal and 53.3% of AUT-GI cecal samples (Table 9B).
Genus-level analysis indicated that members of the genus
Faecalibacterium within the family Ruminococcaceae contributed to
the overall trend toward increased Clostridia levels (FIG. 28A-B).
Within Lachnospiraceae, members of the genus Lachnopsiraceae
Incertae Sedis, Unclassified Lachnospiraceae, and to a lesser
extent Bryantella (cecum only), contributed to the overall trend
toward increased Clostridia (FIG. 28A-B).
[0396] Within the Proteobacteria phyla, levels of
Betaproteobacteria tended to be higher in the ilea of AUT-GI
patients (FIG. 19C: Mann-Whitney, p=0.072); significantly higher
levels of Betaproteobacteria were found in AUT-GI ceca (FIG. 19D:
Mann-Whitney, p=0.038). Levels of Betaproteobacteria were above the
75.sup.th percentile of Control-GI children in 53.3% of AUT-GI ilea
and 66.7% of AUT-GI ceca (Table 9B). Family-level analysis revealed
that members of the families Alcaligenaceae (patients #1, 3, 5, 7,
10, 11, and 12) and Incertae Sedis 5 (patient #2 only) contributed
to the increases in Betaproteobacteria in ilea (FIG. 19E) and ceca
(FIG. 19F). Alcaligenaceae sequences were detected in 46.7% of
AUT-GI children and none of the Control-GI children. Elevated
levels of Proteobacteria in AUT-GI ilea and ceca reflected
increased Alpha- (families Methylobacteriaceae and Unclassified
Rhizobiales) and Betaproteobacteria (family Incertae Sedis 5) for
patient #2 and increased Gammaproteobacteria (family
Enterobacteriaceae) for patients #8 and #15 (FIG. 19E-F). Levels of
Alpha-, Delta-, Gamma-, and Epsilonproteobacteria were not
significantly different between AUT-GI and Control-GI samples.
[0397] The use of probiotics, proton-pump inhibitors, or
antibiotics has been shown to impact the intestinal microbiome [36,
37, 38]. Analysis of the potential effects of these agents in this
cohort revealed only one potential confounding effect: a
correlation between the ratio of Firmicutes to Bacteroidetes in the
cecum obtained by real-time PCR in AUT-GI children who had taken
probiotics (Table 13A). No effect of proton-pump inhibitors was
observed for any of the significant variables assessed in this
study (Table 13B). Only one patient, a control (Control-GI patient
#16), had taken an antibiotic (amoxicillin) in the three months
prior to biopsy (See Table 13C).
TABLE-US-00019 TABLE 13A Evaluation of confounding effects
attributed to the use of probiotics (Pb). AUT(-Pb) vs. AUT(-Pb) vs.
Control(-Pb).sup.a, AUT(+Pb).sup.b, p-value.sup.MW, p-value.sup.MW,
Variable [effect in AUT(-Pb)] [effect in AUT(+Pb)] SI 0.007**,
[decreased] 0.602, [no change] MGAM 0.007**, [decreased] 0.240, [no
change] LCT 0.012*, [decreased] 0.695, [no change] SGLT1 0.021*,
[decreased] 0.433, [no change] GLUT2 0.021*, [decreased] 0.794, [no
change] Bacteroidetes 0.009**, [decreased] 0.602, [no change]
IL(RT) Bacteroidetes CEC(RT) 0.056.dagger., [decreased] 0.192, [no
change] Bacteroidetes IL(454) 0.035*, [decreased] 0.602, [no
change] Bacteroidetes CEC(454) 0.009**, [decreased] 0.999, [no
change] Firm./Bacteroid. Ratio 0.004**, [increased] 0.361, [no
change] IL(RT) Firm./Bacteroid. Ratio 0.159, [no change] 0.037*,
[increased] CEC(RT) Firm./Bacteroid. 0.070.dagger., [increased]
0.514, [no change] Ratio IL(454) Firm./Bacteroid. Ratio
0.056.dagger., [increased] 0.896, [no change] CEC(454)
Clostridiales/ 0.044*, [increased] 0.695, [no change] Bacteroidales
IL(454) Clostridiales/ 0.070.dagger., [increased] 0.896, [no
change] Bacteroidales CEC(454) Beta-proteobacteria 0.108, [not
significant] 0.361, [no change] CEC(454) .sup.aAUT(-Pb), n = 11;
Control (-Pb), n = 6 .sup.bAUT(-Pb), n = 11; AUT(+Pb), n = 4
.sup.MWMann-Whitney test
TABLE-US-00020 TABLE 13B Evaluation of confounding effects
attributed to use of proton-pump inhibitors (PPI). AUT(-PPI) vs.
AUT(-PPI) vs. Control(-PPI).sup.a, AUT(+PPI).sup.b, p-value.sup.MW,
p-value.sup.MW, Variable [effect in AUT(-PPI)] [effect in
AUT(+PPI)] SI 0.003**, [decreased] 0.794, [no change] MGAM 0.006**,
[decreased] 0.695, [no change] LCT 0.234, [no change] 0.192, [no
change] SGLT1 0.036*, [decreased] 0.896, [no change] GLUT2 0.036*,
[decreased] 0.602, [no change] Bacteroidetes IL(RT) 0.002**,
[decreased] 0.433, [no change] Bacteroidetes CEC(RT) 0.011*,
[decreased] 0.433, [no change] Bacteroidetes IL(454) 0.036*,
[decreased] 0.050.dagger., [decreased] Bacteroidetes CEC(454)
0.036*, [decreased] 0.514, [no change] Firm./Bacteroid. Ratio
0.004**, [increased] 0.602, [no change] IL(RT) Firm./Bacteroid.
Ratio 0.011*, [increased] 0.896, [no change] CEC(RT)
Firm./Bacteroid. Ratio 0.027*, [increased] 0.514, [no change]
IL(454) Firm./Bacteroid. Ratio 0.036*, [increased] 0.514, [no
change] CEC(454) Clostridiales/ 0.015*, [increased] 0.514, [no
change] Bacteroidales IL(454) Clostridiales/ 0.036*, [increased]
0.514, [no change] Bacteroidales CEC(454) Beta-proteobacteria
0.047*, [increased] 0.794, [no change] CEC(454) .sup.aAUT(-PPI), n
= 11; Control(-PPI), n = 5 .sup.bAUT(-PPI), n = 11; AUT(+PPI), n =
4 .sup.MWMann-Whitney test
TABLE-US-00021 TABLE 13C Evaluation of confounding effects
attributed to the use of antibiotics. Including Antibiotic
Excluding Antibiotic User (Ab) User (Ab) AUT (-Ab) vs. Control (+Ab
AUT (-Ab) vs. Control and -Ab).sup.a, p-value.sup.MW, (-Ab).sup.b,
p-value.sup.MW, Variable [effect in AUT(-Ab)] [effect in AUT(-Ab)]
SI 0.001**, [decreased] 0.003**, [decreased] MGAM 0.003**,
[decreased] 0.010**, [decreased] LCT 0.032*, [decreased]
0.062.dagger., [decreased] SGLT1 0.008**, [decreased] 0.020*,
[decreased] GLUT2 0.010*, [decreased] 0.024*, [decreased]
Bacteroidetes IL (RT) 0.003**, [decreased] 0.0005***, [decreased]
Bacteroidetes CEC (RT) 0.022*, [decreased] 0.002**, [decreased]
Bacteroidetes IL (454) 0.012*, [decreased] 0.005**, [decreased]
Bacteroidetes CEC (454) 0.008**, [decreased] 0.008**, [decreased]
Firm./Bacteroid. Ratio IL (RT) 0.0006***, [increased] 0.001**,
[increased] Firm./Bacteroid. Ratio CEC (RT) 0.022*, [increased]
0.008**, [increased] Firm./Bacteroid. Ratio IL (454) 0.026*,
[increased] 0.013*, [increased] Firm./Bacteroid. Ratio CEC (454)
0.032*, [increased] 0.029*, [increased] Clostridiales/Bacteroidales
IL 0.012*, [increased] 0.008**, [increased] (454)
Clostridiales/Bacteroidales CEC 0.032*, [increased] 0.024*,
[increased] (454) Beta-proteobacteria CEC (454) 0.038*, [increased]
0.120, [no change] .sup.aAUT(-Ab), n = 15; Control(+Ab and -Ab), n
= 7 .sup.bAUT(-Ab), n = 15; Control(-Ab), n = 6 .sup.MWMann-Whitney
test
[0398] Disaccharidase and Transporter mRNA Levels as Predictors of
Bacterial Abundance
[0399] Multiple linear regression analysis was conducted to
determine whether diagnostic status (AUT-GI or Control-GI) and mRNA
expression of disaccharidases (SI, MGAM and LCT) and transporters
(SGLT1 and GLUT2) (predictor variables) were associated with
bacterial levels as outcome variables (Table 14). For
Bacteroidetes, SGLT1 (ileum and cecum) and SI (cecum only) were
significant predictors. In both the ileum and cecum, Bacteroidete
levels increased as SGLT1 transcript levels increased. In the
cecum, Bacteroidete levels significantly decreased as the levels of
SI increased (a similar marginal effect was observed in ileum).
Bacteroidete levels were lower among AUT-GI children compared to
Control-GI children even after adjusting for the expression of all
disaccharidases and transporters.
TABLE-US-00022 TABLE 14 Multiple linear regression analysis
examining disaccharidases and transporters as predictors of
bacterial levels among AUT-GI and Control-GI children. Interaction
F- Terms with Outcome statistic Adjusted Main Effects: Coefficient
Estimate Status (Co- Variable (p-value) R.sup.2 Status SI.sup.STDev
MGAM.sup.STDev LCT.sup.STDev SGLT1.sup.STDev GLUT2.sup.STDev
efficient.sup.STDev) Bacteroidetes, 5.52.sup.a 0.56 -0.86***
-0.54.dagger. 0.05 -0.02 0.35* 0.05 none Ileum-RT (0.003)**
Bacteroidetes, 2.61.sup.a 0.31 -0.36* -0.60* 0.27 -0.08 0.29* 0.08
none Cecum-RT (0.062).dagger. Firmicutes, 2.50.sup.b 0.33 0.40
-0.57.dagger. 0.44 -0.01 0.10 0.10 MGAM (-0.52)* Ileum-RT
(0.068).dagger. Firmicutes, 6.98.sup.c 0.69 1.29*** -0.99** 0.86**
0.18.dagger. 0.06 0.40* MGAM (-0.50)*, Cecum-RT (0.001)** GLUT2
(-0.46)* Firm./Bac., 3.43.sup.b 0.45 1.43** -0.19 0.19 0.04 -0.27
0.48.dagger. GLUT2 (-0.61)* Ileum-RT (0.024)* Firm./Bac.,
5.13.sup.b 0.58 1.47*** 0.27 0.21 0.19 -0.22 -0.02 SI (-0.93)**
Cecum-RT (0.005)** Proteobacteria, 2.47.sup.b 0.33 -1.05 2.76**
-2.31* 0.01 -0.79.dagger. -0.59.dagger. MGAM (1.21).dagger.
Ileum-454 (0.071).dagger. Proteobacteria, 5.41.sup.b 0.59 -1.21
3.34*** -3.56*** -0.03 -0.68.dagger. -0.38 MGAM (1.59)** Cecum-454
(0.004)** BetaProteobact- 1.14.sup.a 0.04 -0.14 0.61 -0.87 0.05
-0.26 -0.16 none eria, Ileum-454 (0.385) BetaProteobact- 5.64.sup.a
0.57 -0.16 1.43* -2.07** 0.27 -0.44 0.08 none eria, Cecum-
(0.003)** 454 .sup.aon 6 and 15 degrees of freedom .sup.bon 7 and
14 degrees of freedom .sup.con 8 and 13 degrees of freedom
.sup.STDevChange in log-transformed outcome variable levels per
unit standard deviation increase in predictor variable (main effect
variables or interaction terms) *p < 0.05; **p < 0.01; ***p
< 0.001; .dagger.p < 0.1 (trend)
[0400] Firmicute levels significantly decreased as SI levels
increased in cecum. Cecal Firmicute levels were increased as the
levels of MGAM and GLUT2 increased. The levels of Firmicutes in the
cecum were higher in AUT-GI compared to Control-GI children after
adjusting for the expression of disaccharidases and transporters.
Significant interaction was found between status and MGAM and GLUT2
levels in the Firmicute models. Whereas higher levels of MGAM and
GLUT2 were associated with higher levels of Firmicutes among
Control-GI children, the effects of MGAM and GLUT2 on Firmicutes
was not present in AUT-GI children.
[0401] Disaccharidases and transporter levels were not significant
predictors of the ratios of Firmicutes to Bacteroidetes in ileum or
cecum. However, the interaction terms with GLUT2 in the ileum and
SI in the cecum were significant.
[0402] Proteobacteria abundance significantly increased as the
levels of SI increased, but decreased as MGAM increased for both
ileum and cecum. However, the interaction terms with MGAM in both
ileum and cecum were significant, indicating that the magnitude of
decline is significantly smaller among AUT-GI children.
Betaproteobacteria abundance was positively associated with SI and
inversely associated with MGAM only in cecum; none of the
interactions were significant. In addition, Proteobacteria and
Betaproteobacteria abundance were not significantly different
between AUT-GI and Control-GI children after adjusting for the
expression of all disaccharidases and transporters. Overall, these
results indicate that expression levels of disaccharidases and
transporters are associated with the abundance of Bacteroidetes,
Firmicutes, and Betaproteobacteria in the mucoepithelium.
[0403] The levels of Betaproteobacteria in the ileum and cecum were
higher in AUT-GI children with deficiencies in all 5
disaccharidases and transporters versus AUT-GI children with fewer
than 5 disaccharidase and transporter deficiencies (FIG. 35A-B).
Levels of CDX2 were lower in AUT-GI children with levels of
Betaproteobacteria above the 75.sup.th percentile of Control-GI
children compared to AUT-GI children with levels of
Betaproteobacteria below the 75.sup.th percentile of Control-GI
children (FIG. 35C-D). These results indicate a potential link
between increased levels of Betaproteobacteria, reduced levels of
CDX2 expression, and overall deficiencies in disaccharidases and
transporters.
[0404] Timing of GI Disturbances Relative to Onset of Autism is
Associated with Changes in Clostridiales Members
[0405] In this cohort, the onset of GI symptoms was reported to
occur before or at the same time as the development of autism in
67% of AUT-GI children. As a sub-analysis, it was determined
whether the timing of GI onset relative to autism onset was
associated with gene expression and bacterial variables.
[0406] Patients were stratified based on whether the first episode
of GI symptoms occurred before or at the same time (within the same
month) as the onset of autism (AUT-GI-Before or Same group) or
whether the first episode of GI symptoms occurred after the onset
of autism (AUT-GI-After group). The timing of GI onset was not
associated with levels of disaccharidase, hexose transporter or
CDX2 transcripts, Bacteroidetes, Proteobacteria or
Beta-proteobacteria (data not shown). However, a significant effect
was observed for the levels of Clostridiales and cumulative levels
of Lachnospiraceae and Ruminococcaceae in both the ileum and cecum
(FIG. 31A-D). Whereas only a trend toward increased Clostridiales
and cumulative levels of Lachnospiraceae and Ruminococcaceae were
observed when comparing all AUT-GI and Control-GI children (FIG.
27E-F and FIG. 18C-D), stratification based on timing of GI onset
revealed a significant increase in these variables in both the
ileum and cecum of the AUT-GI-Before or Same group relative to all
Control-GI children (FIG. 31A: Clostridiales-ileum, Mann-Whitney,
p=0.015; FIG. 31B: Clostridiales-cecum, Mann-Whitney, p=0.019; FIG.
31C: Lach.+Rum.-ileum, Mann-Whitney, p=0.015; FIG. 31D:
Lach.+Rum.-cecum, Mann-Whitney, p=0.011). Furthermore, the levels
of Clostridiales and cumulative levels of Lachnospiraceae and
Ruminococcaceae were significantly higher in the AUT-GI-Before or
Same group relative to the AUT-GI-After group (FIG. 31A:
Clostridiales-ileum, Mann-Whitney, p=0.028; FIG. 31B:
Clostridiales-cecum, Mann-Whitney, p=0.037; FIG. 31C:
Lach.+Rum.-ileum, Mann-Whitney, p=0.028; FIG. 31D:
Lach.+Rum.-cecum, Mann-Whitney, p=0.020); the AUT-GI-After group
was not significantly different from the Control-GI group. As
expected, the AUT-GI-After group had a significantly older age at
first onset of GI symptoms [median age in months, (interquartile
range, IQR)=36, (22.5)] compared to the AUT-GI-Before or Same group
[median age in months, (interquartile range, IQR)=1, (12)] (FIG.
31E: Mann-Whitney, p=0.007), and was also higher than the
Control-GI group [median age in months, (interquartile range,
IQR)=1, (14)] (FIG. 31E: Mann-Whitney, p=0.027). The age at first
GI onset was not significantly different between the AUT-GI-Before
or Same group and the Control-GI group (FIG. 31E: Mann-Whitney,
p=0.757). Thus, the increased levels of Clostridiales in the
AUT-GI-Before or Same group as compared to the Control-GI group
were not influenced by differences in age of onset of GI symptoms
between these two groups. These results indicate that the timing of
onset of GI symptoms relative to onset of autism or the age at
first GI onset can be associated with increases in
Clostridiales.
[0407] Associations Between Gene Expression, Bacterial Abundance,
and Food Allergies and Other Comorbid Atopic Manifestations
[0408] A National Survey of Children's Health performed under the
auspices of the Centers for Disease Control reported that parents
of autistic children reported more allergy symptoms than control
children, and FA were the most prevalent complaint [39]. Parental
reports of FA in the cohort were reported with similar frequency in
AUT-GI (67%) and Control-GI (71%) children. Milk-related (MA) and
wheat-related (WA) allergies were the most commonly reported
allergies in both groups (Table 6 and Table 7). To determine
whether FA was associated with gene expression or bacterial levels,
patients in the AUT-GI group and Control-GI group were stratified
based on reports of any FA (Table 15A), MA (Table 15B), or WA
(Table 15C).
[0409] Stratification by any FA revealed a significant effect for
levels of GLUT2, ileal and cecal Firmicutes, ileal and cecal ratios
of Firmicutes to Bacteroidetes, and cecal Betaproteobacteria (Table
15A). No effect was observed for the levels of Bacteroidetes, which
were significantly reduced in AUT-GI children independent of FA
status.
[0410] Stratification by MA status revealed even more significant
effects (Table 15B). Significant effects were observed for MGAM,
GLUT2, and CDX2 expression, as well as ileal and cecal ratios of
Firmicutes to Bacteroidetes, and ileal and cecal
Beta-proteobacteria. Additional trends were observed for SI
expression and ileal and cecal Firmicutes. No effect was observed
for the levels of Bacteroidetes, which were significantly reduced
in AUT-GI children independent of MA status.
[0411] Stratification by WA status was associated with a
significant effect only for cecal levels of Firmicutes, though this
effect was highly significant [AUT (+WA) vs. AUT (-WA):
Mann-Whitney, p-value=0.008], and the cecal ratio of Firmicutes to
Bacteroidetes (Table 15C).
[0412] These results indicate that changes in the expression of
some disaccharidases and transporters and CDX2, as well as changes
in the abundance of some bacterial phylotypes, are significantly
associated with reported FA, especially MA. Whereas the levels of
Firmicutes, the ratio of Firmicutes to Bacteroidetes, and levels of
Betaproteobacteria were increased in AUT-GI children with FA, the
levels of Bacteroidetes were not significantly different. This
indicates that the levels of Bacteroidetes were significantly
decreased in AUT-GI children, independent of FA status.
[0413] Atopic disease manifestations (AD: asthma, allergic
rhinitis, or atopic dermatitis) were the most commonly reported
comorbid conditions in both AUT-GI and Control-GI children. The
frequency of AD tended to be higher in the Control-GI group (100%)
than in the AUT-GI group (53%) (Table 6: Fisher's Exact Test,
2-sided p=0.051). In the combined group (all AUT-GI and Control-GI
patients), 86.7% of children with reported FA had at least one
reported AD; only 28.6% of children without reported food allergy
had one or more AD (Fisher's Exact Test, 2-sided p=0.014). As AD
was associated with reported FA, it was determined whether AD
manifestation was also associated with changes in disaccharidases
and transporters or bacterial parameters. Stratification of
subjects by AD status revealed that cecal Firmicutes and the cecal
ratio of Firmicutes to Bacteroidetes were higher in AUT-GI children
with AD compared to Control-GI children with AD [Table 15D:
AUT(+AD) vs. Control(+AD); Firmicutes CECRT, Mann-Whitney, p=0.015;
Firm./Bacteroid. Ratio CECRT, Mann-Whitney, p=0.002] and AUT-GI
children without AD [Table 15D: AUT(-AD) vs. AUT(+AD); Firmicutes
CEC(RT), Mann-Whitney, p=0.049; Firm./Bacteroid. Ratio CEC(RT),
Mann-Whitney, p=0.049].
TABLE-US-00023 TABLE 15A Association of food allergies (FA) with
host gene expression and bacterial phylotypes in AUT-GI children.
AUT(+FA) vs. AUT(-FA) vs. Control(+FA).sup.a, AUT(+FA).sup.b,
p-value.sup.MW, p-value.sup.MW, Variable [effect in AUT(+FA)]
[effect in AUT(+FA)] GLUT2 0.014*, [decreased] 0.037*, [decreased]
Bacteroidetes IL(RT) 0.002**, [decreased] 0.806, [no change]
Bacteroidetes CEC(RT) 0.005**, [decreased] 0.713, [no change]
Bacteroidetes IL(454) 0.037*, [decreased] 0.221, [no change]
Bacteroidetes CEC(454) 0.050*, [decreased] 0.713, [no change]
Firmicutes IL(RT) 0.221, [no change] 0.037*, [increased] Firmicutes
CEC(RT) 0.037*, [increased] 0.010*, [increased] Firm./Bacteroid.
Ratio 0.003**, [increased] 0.037*, [increased] IL(RT)
Firm./Bacteroid. Ratio 0.005**, [increased] 0.020*, [increased]
CEC(RT) Beta-proteobacteria 0.050.dagger., [increased]
0.066.dagger., [increased] IL(454) Beta-proteobacteria 0.028*,
[increased] 0.037*, [increased] CEC(454) .sup.aAUT(+FA), n = 10;
Control(+FA), n = 5 .sup.bAUT(-FA), n = 5; AUT(+FA), n = 10
.sup.MWMann-Whitney test
TABLE-US-00024 TABLE 15B Association of milk allergies (MA) with
host gene expression and bacterial phylotypes in AUT-GI children.
AUT(+MA) vs. AUT(-MA) vs. Control(+MA).sup.a, AUT(+MA).sup.b,
p-value.sup.MW, p-value.sup.MW, Variable [effect in AUT(+MA)]
[effect in AUT(+MA)] SI 0.006**, [decreased] 0.099.dagger.,
[decreased] MGAM 0.006**, [decreased] 0.045*, [decreased] GLUT2
0.009**, [decreased] 0.013*, [decreased] CDX2 0.072.dagger.,
[decreased] 0.034*, [decreased] Bacteroidetes IL(RT) 0.003**,
[decreased] 0.480, [no change] Bacteroidetes CEC(RT) 0.003**,
[decreased] 0.289, [no change] Bacteroidetes IL(454) 0.028*,
[decreased] 0.637, [no change] Bacteroidetes CEC(454) 0.020*,
[decreased] 0.637, [no change] Firmicutes IL(RT) 0.205, [no change]
0.059.dagger., [increased] Firmicutes CEC(RT) 0.053.dagger.,
[increased] 0.099.dagger., [increased] Firm./Bacteroid. Ratio
0.004**, [increased] 0.034*, [increased] IL(RT) Firm./Bacteroid.
Ratio 0.006**, [increased] 0.045*, [increased] CEC(RT)
Beta-proteobacteria 0.020*, [increased] 0.013*, [increased] IL(454)
Beta-proteobacteria 0.009**, [increased] 0.007**, [increased]
CEC(454) .sup.aAUT(+MA), n = 9; Control(+MA), n = 5 .sup.bAUT(-MA),
n = 6; AUT(+MA), n = 9 .sup.MWMann-Whitney test
TABLE-US-00025 TABLE 15C Association of wheat allergies (WA) with
host gene expression and bacterial phylotypes in AUT-GI children.
AUT(+WA) vs. AUT(-WA) vs. Control(+WA).sup.a, AUT(+WA).sup.b,
p-value.sup.MW, p-value.sup.MW, Variable [effect in AUT(+WA)]
[effect in AUT(+WA)] Bacteroidetes IL(RT) 0.007**, [decreased]
0.643, [no change] Bacteroidetes CEC(RT) 0.017*, [decreased] 0.643,
[no change] Bacteroidetes IL(454) 0.017*, [decreased] 0.488, [no
change] Bacteroidetes CEC(454) 0.089.dagger., [decreased] 0.908,
[no change] Firmicutes IL(RT) 0.174, [no change] 0.083.dagger.,
[increased] Firmicutes CEC(RT) 0.089.dagger., [increased] 0.008*,
[increased] Firm./Bacteroid. Ratio 0.011*, [increased] 0.203, [no
change] IL(RT) Firm./Bacteroid. Ratio 0.011*, [increased] 0.049*,
[increased] CEC(RT) Beta-proteobacteria 0.089.dagger., [increased]
0.643, [no change] IL(454) Beta-proteobacteria 0.042*, [increased]
0.418, [no change] CEC(454) .sup.aAUT(+WA), n = 8; Control(+WA), n
= 4 .sup.bAUT(-WA), n = 7; AUT(+WA), n = 8 .sup.MWMann-Whitney
test
TABLE-US-00026 TABLE 15D Association of atopic disease (AD) status
with host gene expression and bacterial phylotypes in AUT-GI
children. AUT(+AD) vs. AUT(-AD) vs. Control(+AD).sup.a,
AUT(+AD).sup.b, p-value.sup.MW, p-value.sup.MW, Variable [effect in
AUT(+AD)] [effect in AUT(+AD)] Bacteroidetes IL(RT) 0.008**,
[decreased] 0.563, [no change] Bacteroidetes CEC(RT) 0.028*,
[decreased] 0.418, [no change] Bacteroidetes IL(454) 0.049*,
[decreased] 0.643, [no change] Bacteroidetes CEC(454)
0.064.dagger., [decreased] 0.908, [no change] Firmicutes IL(RT)
0.064.dagger., [increased] 0.133, [no change] Firmicutes CEC(RT)
0.015*, [increased] 0.049*, [increased] Firm./Bacteroid. Ratio
0.002**, [increased] 0.064.dagger., [increased] IL(RT)
Firm./Bacteroid. Ratio 0.006**, [increased] 0.049*, [increased]
CEC(RT) Beta-proteobacteria 0.049*, [increased] 0.203, [no change]
IL(454) Beta-proteobacteria 0.028*, [increased] 0.133, [no change]
CEC(454) .sup.aAUT(+AD), n = 8; Control(+AD), n = 7 .sup.bAUT(-AD),
n = 7; AUT(+AD), n = 8 .sup.MWMann-Whitney test
[0414] Discussion
[0415] Although the major deficits in ASD are social and cognitive,
many affected individuals with ASD also have substantial GI
morbidity. Findings in this study that can shed light on GI
morbidity in ASD include the observations that: (1) levels of
transcripts for disaccharidases and hexose transporters are reduced
in AUT-GI children; (2) AUT-GI children have microbial dysbiosis in
the mucoepithelium; and (3) dysbiosis is associated with
deficiencies in host disacharidase and hexose transporter mRNA
expression. Without being bound by theory, deficiencies in
disaccharidases and hexose transporters alter the milieu of
carbohydrates in the distal small intestine (ileum) and proximal
large intestine (cecum), resulting in the supply of additional
growth substrates for bacteria. These changes manifest in
significant and specific compositional changes in the microbiota of
AUT-GI children (see FIG. 32, FIG. 20).
[0416] A previous report on GI disturbances in ASD found low
activities of at least one disaccharidase or glucoamylase in
duodenum in 58% of children [7]. In this study, 93.3% of AUT-GI
children had decreased mRNA levels for at least one of the three
disaccharidases (SI, MGAM, or LCT). In addition, decreased levels
of mRNA were found for two important hexose transporters, SGLT1 and
GLUT2. Congenital defects in these enzymes and transporters are
extremely rare [40,41], and even the common variant for adult-type
hypolactasia was not responsible for reduced LCT expression in
AUT-GI children in this cohort. It is unlikely, therefore, that the
combined deficiency of disaccharidases (maldigestion) and
transporters (malabsorption) are indicative of a primary
malabsorption resulting from multiple congenital or acquired
defects in each of these genes. Transcripts for the enterocyte
marker, villin, were not reduced in AUT-GI ilea and did not predict
the expression levels of any of the disaccharidases or transporters
in multiple regression models. This indicates that a general loss
of enterocytes is unlikely. Without being bound by theory, defects
in the maturational status of enterocytes or enterocyte migration
along crypt-villus axis can contribute to deficits in
disaccharidase and transporter expression [42].
[0417] The ileal expression of CDX2, a master transcriptional
regulator in the intestine, was a significant predictor of mRNA
expression of all five disaccharidases and transporters in AUT-GI
and Control-GI children based on linear regression models. However,
as ASD status remained a significant predictor of disaccharidase
mRNA expression even after adjusting for CDX2 and villin,
additional factors must also contribute. One factor is diet.
Dietary intake of carbohydrates can regulate the mRNA expression of
disaccharidases and hexose transporters in mice and rats [43, 44,
45]. ASD children exhibit feeding selectivity and aberrant nutrient
consumption [46, 47, 48, 49, 50, 51, 52]. However, of the four
studies reporting carbohydrate intake, none found differences in
total carbohydrate intake in ASD children [47, 48, 49, 50].
Furthermore, one study found no association between dietary intake
of macronutrients (i.e., carbohydrates, proteins, or fats) and GI
symptoms [47]. Unfortunately, dietary diaries for the period
immediately preceding biopsy were not available for the children
evaluated in this study; hence, the extent to which dietary intake
affected intestinal gene expression could not be determined.
[0418] Hormonal and growth factor regulation of some
disaccharidases and hexose transporters have been reported in in
vitro studies and in animals [53,54]. Inflammatory cytokines can
regulate SI gene expression in human intestinal epithelial cells in
vitro [55]. Thus, immunological or hormonal imbalances reported in
ASD children [5, 8, 9, 10, 11, 12, 56, 57, 58] can also contribute
to expression deficits. Additionally, intestinal microbes can
influence the expression of disaccharidases and transporters [59]
through the influence of pathogen-associated molecular patterns
(PAMPs) and butyrate (a byproduct of bacterial fermentation) on
CDX2 expression and activity [60, 61, 62, 63]. In this regard, the
observation that CDX2 was decreased in AUT-GI children with
increased levels of Betaproteobacteria can be important.
[0419] Whatever the underlying mechanisms, reduced capacity for
digestion and transport of carbohydrates can have profound effects.
Within the intestine, malabsorbed carbohydrates can lead to osmotic
diarrhea [64]; non-absorbed sugars can also serve as substrates for
intestinal microflora that produce fatty acids and gases (methane,
hydrogen, and carbon dioxide), promoting additional GI symptoms
such as bloating and flatulence [65]. The deficiency of even a
single gene in this important pathway can result in severe GI
disease, as occurs with glucose-galactose malabsorption syndrome
caused by SGLT1 deficiency, Fanconi-Bickel syndrome resulting from
GLUT2 mutations, sucrase-isomaltase deficiency, and congenital
lactase deficiency [40,41].
[0420] Changes in the type and quantity of dietary carbohydrates
can influence composition and function of intestinal microflora
[66, 67, 68]; thus, we reasoned that carbohydrate maldigestion and
malabsorption, resulting from deficient expression of
disaccharidases and hexose transporters, might have similar effects
in AUT-GI subjects. Pyrosequencing analysis of mucoepithelial
bacteria revealed significant multicomponent dysbiosis in AUT-GI
children, including decreased levels of Bacteroidetes, an increase
in the Firmicute/Bacteroidete ratio, increased cumulative levels of
Firmicutes and Proteobacteria, and an increase in levels of
bacteria in the class Betaproteobacteria.
[0421] A recent pyrosequencing study reported an increase in
Bacteroidetes in fecal samples of ASD subjects [69]. Although these
findings can appear to be incongruent with those reported here, the
data were obtained using biopsies rather than free fecal material.
Others have reported differences in the composition of fecal versus
mucosal microflora [35, 70, 71, 72]. Only about 50% of cells in
feces are viable, with dead and injured cells making up the
remaining fractions [73]. The loss of Bacteroidetes from the
mucoepithelium as a result of death, injury, or competition for
binding in the mucosal space can result in increased wash out of
Bacteroidete cells into the fecal stream. Thus, higher levels of
Bacteroidetes in feces could be indicative of an inability to
thrive in the mucosal microbiome rather than an indication that
Bacteroidetes are found at higher levels in the microbiome.
[0422] The trend toward increased Firmicutes was largely
attributable to Clostridia with Ruminococcaceae and Lachnospiraceae
as major contributors. Several Ruminococcaceae and Lachnospiraceae
are known butyrate producers and can thus influence short-chain
fatty acid (SCFA) levels [74]. SCFA influence colonic pH, and some
Bacteroides sp. are sensitive to acidic pH [75]. Three previous
reports indicated differences in Clostridia species in children
with ASD, including greater abundance of Clostridium clusters I,
II, XI and C. bolteae [14, 15, 16]. Stratification of AUT-GI
children based on the timing of GI symptom development relative to
autism onset revealed that the levels of Clostridiales and
cumulative levels of Lachnospiraceae and Ruminococcaceae were
significantly higher in AUT-GI children for whom GI symptoms
developed before or at the same time as the onset of autism
symptoms compared to AUT-GI children for whom GI symptoms developed
after the onset of autism and compared to Control-GI children.
However, we cannot discern whether changes in Clonstridiales
members occurred before the onset of autism in this subgroup. We
can only conclude that increased levels of Clostridiales members in
biopsies taken after the development of both GI symptoms and autism
are associated with the timing of GI onset relative to autism onset
in this cohort. Although the reason for this association remains
unclear, this finding can indicate that the timing of GI onset
relative to autism is an important variable to consider in the
design of future prospective studies investigating the microbiota
of children with autism.
[0423] Although we found only a trend for increased Firmicutes in
AUT-GI children, the cumulative levels of Firmicutes and
Proteobacteria were significantly higher. These results indicate
that in some patients the decrease in Bacteroidetes is associated
with an increase in Firmicutes, whereas in others increases in
Proteobacteria are associated with a reduced abundance of
Bacteroidetes. Three AUT-GI patients had high levels of Alpha-,
Beta-, or Gammaproteobacteria. In addition, the AUT-GI group had
elevated levels of Betaproteobacteria compared to the Control-GI
group, primarily reflecting the presence of Alcaligenaceae.
Alcaligenaceae sequences were not detected in any tissues from
Control-GI children.
[0424] Deficient digestion and absorption of di- and
monosaccharides in the small intestine can alter the balance of
growth substrates, eliminating the growth advantages that
Bacteroidetes enjoy in the healthy intestine and enabling
competitive growth of bacterial phylotypes better suited for growth
on undigested and unabsorbed carbohydrates. In support of this
hypothesis, multiple linear regression models demonstrated that
levels of ileal SGLT1 and SI mRNA were associated with levels of
Bacteroidetes in ileum and cecum, or cecum alone, respectively.
Levels of ileal SI, MGAM and GLUT2 mRNA were associated with levels
of cecal Firmicutes, although the magnitude of the effects of MGAM
and GLUT2 differed between AUT-GI and Control-GI children.
Significant associations were also observed between levels of SI
and MGAM mRNA and of Proteobacteria in ileum and cecum, and of
Betaproteobacteria in cecum. Although deficiencies in
disaccharidase and transporter expression appear to at least
partially contribute to these alterations in the AUT-GI microbiota,
diagnostic status remained a significant predictor of Bacteroidete
and cecal Firmicute abundance even after adjusting for gene
expression.
[0425] Metabolic interactions between intestinal microflora and
their hosts are only beginning to be understood. Nonetheless, there
is already abundant evidence that microflora can have system-wide
effects [76, 77, 78, 79, 80, 81, 82, 83] and influence immune
responses, brain development and behavior [24, 25, 26, 84, 85]. We
acknowledge that this is a small study comprising 22 subjects. The
small sample size evaluated in this study is a limitation arising
from the difficulty in obtaining biopsies from young children
undergoing invasive endoscopic examination. Without being bound by
theory, the data show that at least some children with autism have
a distinct intestinal profile that is linked to deficient
expression of disaccharidases and hexose transporters, potentially
promoting maldigestion, malabsorption and multicomponent,
compositional dysbiosis. Although the underlying cause of these
changes and the extra-intestinal effects these changes can elicit
remain speculative, the identification of specific molecular and
microbial signatures that define GI pathophysiology in AUT-GI
children sets the stage for further research aimed at defining the
epidemiology, diagnosis and informed treatment of GI symptoms in
autism.
[0426] Materials and Methods
[0427] All samples were analyzed anonymously. Samples assessed in
this example were restricted to those derived from male children
from the original cohort between 3 and 5 years of age to control
for confounding effects of gender and age on intestinal gene
expression and the microbiota. This subset comprised 15 AUT-GI
(Patient #1-15) and 7 Control-GI (Patient #16-22) patients.
[0428] Clinical Procedures:
[0429] Specific clinical procedures for defining neuropsychiatric
and regression status in this cohort have been previously described
[86]. Briefly, neuropsychiatric status was established for all
subjects using Diagnostic and Statistical Manual-Fourth Edition,
Text Revision (DSM-IV-TR) diagnostic criteria. Only cases meeting
full DSM-IV-TR criteria for Autistic Disorder (AUT) were included
for further analysis. DSM-IV-TR diagnosis of AUT was confirmed by
certified raters using the Autism Diagnostic Interview-Revised
(ADI-R). Regression status was determined based on ADI-R and
Shortened CPEA Regression Interview. Control-GI children were
evaluated in the same manner as cases to exclude subjects with any
developmental disturbances, including ASD. Age of AUT onset was
determined by an ADI-R certified interviewer. Questions posed to
parents in standardized data collection forms regarding GI symptoms
were based on previous work [27]. Symptoms were only reported if
the child had experienced the specific GI symptoms, including food
allergies, for 3 consecutive months. History of medication use,
presence of comorbid conditions, age at first GI episode, and
presence and type of food allergies were also acquired through
parental questionnaires.
[0430] RNA and DNA Extraction:
[0431] All biopsies were snap frozen at collection and stored at
-80.degree. C. until extraction. RNA and DNA were extracted
sequentially from individual ileal and cecal biopsies [total of 176
biopsies from 15 AUT-GI patients and 7 Control-GI patients: 8
biopsies per patient (4 each from ileum and cecum), yielding 88
ileal and 88 cecal biopsies] in TRIzol (Invitrogen) using standard
protocols. RNA from half of the biopsies (2 ileal and 2 cecal
biopsies per AUT-GI or Control-GI patient) was derived from
residual extracts from the original study completed in 2008 [86].
RNA from the other half of the biopsies (the remaining 2 ileal and
2 cecal biopsies per AUT-GI or Control-GI patient) was newly
extracted from stored biopsies (stored undisturbed at -80.degree.
C.) at the inception of the current study in 2008. The interphase
and organic phase fractions were stored at -80.degree. C.,
following RNA extraction, for subsequent DNA extraction. All
extractions were stored in aliquots at -80.degree. C. to avoid
repeated freeze thawing of samples. RNA and DNA concentrations,
purity and integrity were determined for all residual extracts and
newly extracted biopsies just prior to cDNA synthesis for mRNA
expression studies and just prior to PCR of newly extracted DNA
using a Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies)
and Bioanalyzer (Agilent Technologies).
[0432] Quantitative Real-Time PCR of Human mRNA:
[0433] Intron/exon spanning, gene-specific PCR primers and probes
(Table 16) for SI, MGAM, LCT, SGLTI, GLUT2, villin, and CDX2, with
GAPDH and .beta.-actin as dual housekeeping gene controls were
designed for real-time PCR using Primer Express 1.0 software
(Applied Biosystems). Taqman probes were labeled with the reporter
FAM (6-carboxyfluorescein) and the quencher BBQ (Blackberry) (TIB
MolBiol). Assays were designed and implemented as previously
described [87, 88, 89]. Levels of mRNA expression for each gene and
in each AUT-GI individual were considered significantly increased
or decreased if they were above the 75.sup.th percentile or below
the 25.sup.th percentile, respectively, of gene expression obtained
for all Control-GI children and were at least 2-fold increased or
decreased from the Control-GI mean (Table 9 and Table 10).
TABLE-US-00027 TABLE 16 Real-time PCR primers and probes used for
gene expression and bacterial quantitative analysis. SEQ ID
Amplicon Name NO. Primers and Probe size (bp) SI 26 For
5'-TCTTCATGAGTTTTATGAGGATACGAAC-3' 150 27 Rev:
5'-TTTGCACCAGATTCATAATCATACC-3' 28 Probe:
5'-CAGATACTGTGAGTGCCTACATCCCTGATGCTATT-3' MGAM 29 For:
5'-TACCTTGATGCATAAGGCCCA-3' 150 30 Rev: 5'-GGCATTACGCTCCAGGACA-3'
31 Probe: 5'-CGTCACTGTTGTGCGGCCTCTGC-3' LCT 32 For:
5'-CAGGAATCAAGAGCGTCACAACT-3' 180 33 Rev: 5'-AAATCGACCGTGTCCTGGG-3'
34 Probe: 5'-TCCTGCTAGAACCACCCATATCTGCGCT-3' SGLT1 35 For:
5'-GCTCATGCCCAATGGACTG-3' 125 36 Rev: 5'-CGGACCTTGGCGTAGATGTC-3' 37
Probe: 5'-ACAGCGCCAGCACCCTCTTCACC-3' Glut2 38 For:
5'-AGTTAGATGAGGAAGTCAAAGCAA-3' 164 39 Rev: 5'-TAGGCTGTCGGTAGCTGG-3'
40 Probe: 5'-ACAAAGCTTGAAAAGACTCAGAGGATATGATGATGTC-3' Villin 41
For: 5'-CATGCGCTGAACTTCATCAAA-3' 120 42 Rev:
5'-GGTTGGACGCTGTCCACTTC-3' 43 Probe: 5'-CGGCCGTCTTTCAGCAGCTCTTCC-3'
CDX2 44 For 5'-GGCAGCCAAGTGAAAACCAG-3' 112 45 Rev:
5'-TCCGGATGGTGATGTAGCG-3' 46 Probe: 5'-ACCACCAGCGGCTGGAGCTGG-3'
.beta.-Actin 47 For: 5'-AGCCTCGCCTTTGCCGA-3' 175 48 Rev:
5'-CTGGTGCCTGGGGCG-3' 49 Probe: 5'-CCGCCGCCCGTCCACACCCGCC GAPDH 50
For: 5'-CCTGTTCGACAGTCAGCCG-3' 100 51 Rev:
5'-CGACCAAATCCGTTGACTCC-3' 52 Probe: 5'-CGTCGCCAGCCGAGCCACA-3'
Bacteroidetes 53 For: 5'-AACGCTAGCTACAGGCTT-3' ~293 54 Rev:
5'-CCAATGTGGGGGACCTTC-3' Firmicutes 55 For:
5'-GGAGYATGTGGTTTAATTCGAAGCA-3' ~126 56 Rev:
5'-AGCTGACGACAACCATGCAC-3' Total Bacteria 57 For:
5'-GTGCCAGCMGCCGCGGTAA-3' ~295 58 Rev:
5'-GACTACCAGGGTATCTAAT-3'
[0434] Lactase Genotyping:
[0435] Genomic DNA from AUT-GI and Control-GI patients was
subjected to previously-described, PCR-restriction fragment length
polymorphism (PCR-RFLP) analysis for the C/T-13910 and G/A-22018
polymorphisms associated with adult-type hypolactasia with minor
modifications [90]. For details, see FIG. 21B-E.
[0436] Barcoded Pyrosequencing of Intestinal Microbiota:
[0437] PCR was carried out using bacterial 16S rRNA gene-specific
(V2-region), barcoded primers as previously described [91].
Barcoded 16S rRNA genes were amplified from DNA samples from the 88
ileal biopsies and 88 cecal biopsies. Amplicons were sequenced at
454 Life Sciences on a GS FLX sequencer.
[0438] Quantitative Real-Time PCR of Bacteroidete and Firmicute 16S
rRNA Genes:
[0439] Primer sequences and PCR conditions used for bacterial
real-time PCR assays to quantify Bacteroidetes, Firmicutes, and
total Bacterial 16S rRNA genes have been previously described
[92,93]; primer sequences are listed in Table 16. Copy numbers of
Bacteroidetes, Firmicutes, or Firmicute to Bacteroidete ratios that
were above the 75.sup.th percentile or below the 25.sup.th
percentile of Control-GI children were scored as an increase or
decrease, respectively (Table 9). Percent changes in bacterial
parameters for individuals in the AUT-GI group were determined
based on the mean levels in Control-GI children (Table 12).
[0440] Bioinformatic Analysis of Pyrosequencing Reads:
[0441] Pyrosequencing reads ranging from 235 to 300 base pairs in
length (encompassing all sequences within the major peak obtained
from pyrosequencing) were filtered for analysis. Low-quality
sequences--i.e., those with average quality scores below 25--were
removed based on previously described criteria [91,94].
Additionally, reads with any ambiguous characters were omitted from
analysis. Sequences were then binned according to barcode, followed
by removal of primer and barcode sequences. Taxonomic
classifications of bacterial 16S rRNA sequences were obtained using
the RDP classifier tool (http://rdp.cme.msu.edu/) with a minimum
80% bootstrap confidence estimate. To normalize data for
differences in total sequences obtained per patient, phylotype
abundance was expressed as a percentage of total bacterial sequence
reads per patient at all taxonomic levels. Taxonomy note: the RDP
classifier binned all of the limited number of sequences obtained
for the phylum Cyanobacteria into the chloroplast-derived genus
Streptophyta. Heatmaps were constructed with MeV (Version 4.5.0),
using abundance data from pyrosequencing reads. Heatmap scales were
made linear where possible, with the upper limit reflecting the
highest abundance recorded for any taxa in a given heatmap (red),
the lower limit reflecting sequences above 0% abundance (green),
and the midpoint limit (white) set to the true midpoint between 0%
and the upper limit. In some instances, the midpoint limit was
adjusted to highlight salient differences between the AUT-GI and
Control-GI groups. Gray cells in all heatmaps reflect the complete
absence of sequences detected for a given taxa in a given
patient.
[0442] OTU-based analysis was carried out in MOTHUR (version 1.8.0)
[95]. Filtered sequences generated from 454 pyrosequencing were
aligned to the greengenes reference alignment (greengenes.lbl.gov),
using the Needleman-Wunsch algorithm with the "align.seqs" function
(ksize=9). Pairwise genetic distances among the aligned sequences
were calculated using the "dist.seqs" function (calc=onegap,
countends=T). Sequences were assigned to OTUs (97% identity) using
nearest neighbor clustering. Rarefaction curves to assess coverage
and diversity (Shannon Diversity Index) were constructed in MOTHUR.
For OTU analysis of Bacteroidete sequences, phylum level
classification in RDP was used to subselect all Bacteroidete
sequences, followed by OTU assignment at 97% identity.
Representative sequences (defined as the sequence with the minimum
distance to all other sequences in the OTU) from each OTU were
obtained using the get.oturep command in MOTHUR. Representative
sequences were classified using the nearest species match from
Greengenes Blast (greengenes.lbl.gov) and NCBI BLAST alignment. OTU
abundance by patient was expressed as percent relative abundance,
determined by dividing the number of reads for an OTU in a given
patient sample by the total number of bacterial reads obtained
through pyrosequencing for that sample.
[0443] Statistical Analysis:
[0444] Most of the data were not normally distributed, based on
Kolmogorov-Smirnov test and evaluation of skewness and kurtosis;
thus, the non-parametric Mann-Whitney U test was performed to
evaluate differences between groups using StatView (Windows version
5.0.1; SAS Institute). The comparative results of gene expression
and bacteria 16S rRNA gene levels were visualized as
box-and-whisker plots showing: the median and the interquartile
(midspread) range (boxes containing 50% of all values), the
whiskers (representing the 25.sup.th and 75.sup.th percentiles) and
the extreme data points (open circles). Chi-squared test was used
to evaluate between-group genotypes for adult-type hypolactasia as
well as differences in the frequency of atopic disease between
groups. Kruskal-Wallis one-way analysis of variance was employed to
assess significance of LCT mRNA expression levels split by genotype
and group. To evaluate the effects of CDX2 and/or villin on enzyme
and transporter levels and the effects of levels of enzymes and
transporters on bacterial levels, multiple linear regression
analyses were conducted. For details on multiple linear regression
analyses see Table 11, and Table 14. Significance was accepted for
all analyses at p<0.05.
[0445] Supporting Results
[0446] Genetically Determined Lactase Non-Persistence is not
Responsible for Deficient Lactase mRNA in AUT-GI Children (FIG.
21):
[0447] Although it is beyond the scope of this study to evaluate
all possible mutations in carbohydrate genes that can affect
expression, it was confirmed that deficient LCT mRNA in AUT-GI
children is not a result of the common adult-type hypolactasia
genotype. LCT mRNA levels can be affected by two single nucleotide
polymorphisms that determine adult-type hypolactasia; therefore,
these children were genotyped using PCR-RFLP analysis. The
homozygous, hypolactasia variant alleles were found in 20% (3 out
of 15) of AUT-GI children and 14.3% (1 out of 7) of Control-GI
children. Genotype proportions were not significantly different
between the two groups (chi-squared test, p=0.896) (FIG. 21B). LCT
mRNA expression was significantly lower in individuals with the
homozygous hypolactasia genotype compared to all other genotypes
(FIG. 21C: Mann-Whitney, p=0.033). Comparison of LCT mRNA
expression across genotype and group failed to reach significance
(FIG. 21D: Kruskal-Wallis, p=0.097). Comparison of mRNA expression
in subjects carrying at least one copy of the normal allele
confirmed a significant decrease in LCT mRNA in AUT-GI relative to
Control-GI subjects, independent of the individuals with the
homozygous hypolactasia genotype (FIG. 21E: Mann-Whitney, p=0.025).
In summary, although the data support the notion that LCT genotype
affects gene expression, deficient LCT mRNA in AUT-GI was not
attributable to disproportionate hypolactasia genotypes between the
AUT-GI and Control-GI groups.
[0448] Barcoded 16S rRNA Gene Pyrosequencing (FIG. 23):
[0449] A total of 525,519 sequencing reads (representing 85% of the
initial number of sequencing reads) remained after filtering based
on read length, removing low-quality sequences and combining
duplicate pyrosequencing runs (271,043 reads for ilea; 254,476
reads for ceca). Binning of sequences by barcode revealed similar
numbers of 16S rRNA gene sequence reads per patient (average #
sequences per patient+/-STD for ilea=12,320+/-1220; average #
sequences per patient+/-STD for ceca=11,567+/-1589). There was not
a significant difference between the AUT-GI and Control-GI groups
in terms of the number of reads per patient. In order to assess
whether sufficient sampling was achieved in the total
pyrosequencing data set for all AUT-GI and Control-GI subjects,
OTUs (Operational Taxonomic Units) were defined at a threshold of
97% identity, split by data for ileum and cecum, and rarefaction
analysis was carried out (FIG. 23A-B). Rarefaction curves showed a
tendency toward reaching plateau for all subjects; however failure
to reach plateau indicates that additional sampling would be
required to achieve complete coverage of all OTUs present in ileal
and cecal biopsies. Investigation of diversity in AUT-GI and
Control-GI patients was carried out using the Shannon Diversity
Index calculated from OTU data for each subject. Rarefaction
analysis revealed that all Shannon Diversity estimates had reached
stable values (FIG. 23C-D). While Shannon Diversity estimates
varied widely between individuals, there was not an apparent
overall difference (loss or gain of diversity) between the AUT-GI
and Control-GI groups in ileal (FIG. 23C) or cecal (FIG. 23D)
biopsies.
[0450] OTU Analysis of Bacteroidetes (FIG. 25):
[0451] In order to determine whether the decreased abundance of
Bacteroidete members was attributable to the loss of specific
Bacteroidete phylotypes, the distribution of Bacteroidete OTUs
(defined using a threshold of 97% identity or greater; 3% distance)
were investigated. The number of Bacteroidete OTUs per patient
ranged from 23 to 102 for ileal samples and 10 to 130 for cecal
samples. Interestingly, no single OTU was significantly over or
underrepresented between AUT-GI and Control-GI children and many
OTUs contained single sequences. Furthermore, high inter-subject
variability in the distribution and abundance of individual
Bacteroidete phylotypes was observed, as has been previously
described [B1]. Thus, it was determined whether the decrease in
Bacteroidete abundance in AUT-GI children could be attributed to
overall losses of the most prevalent Bacteroidete phylotypes. In
both ileal and cecal samples, 12 OTUs accounted for the majority of
Bacteroidete sequences (FIG. 25A-B). The cumulative levels of these
12 OTUs were significantly lower in AUT-GI compared to Control-GI
children in both the ileum (FIG. 25C: Mann-Whitney, p=0.008) and
cecum (FIG. 25D: Mann-Whitney, p=0.008). Representative sequences
from each of these 12 OTUs were classified using Greengenes Blast
and microbial blast alignment (NCBI) (FIG. 25E). The majority of
sequences were members of the family Bacteroidaceae (OTUs 1, 3, 5,
6, 7, and 19), except in the case of patient 20, where
Prevotellaceae were the dominant phylotype (OTU #21). These results
indicate that the loss of Bacteroidetes in AUT-GI children is
primarily attributable to overall decreases in the dominant
phylotypes of Bacteroidetes in individual patients
[0452] Evaluation of Confounding Effects of Probiotic, Proton-Pump
Inhibitor and Antibiotic Use:
[0453] The use of probiotics (Pb), proton-pump inhibitors (PPI),
and antibiotics are reported to exert effects on the composition of
the intestinal microbiota [B2, B3]. As some patients in both the
AUT-GI and Control-GI groups had taken these medications, we sought
to determine whether potential confounding effects of these
medications on the findings could be excluded. Probiotics had been
used by both AUT-GI (n=4; 27%) and Control-GI (n=1; 14%) children.
If probiotics use determined the outcome of gene expression and
bacterial variables, then the significant effect for a given
variable should not be present when comparing individuals that had
not taken probiotics in the AUT-GI and Control-GI groups [Table
13A: AUT(-Pb) vs. Control(-Pb)]. For each of the 16 variables,
except the ratio of Firmicutes to Bacteroidetes in the cecum (RT)
and Betaproteobacteria in the cecum (454), either a significant
result or trend was observed between the AUT(-Pb) and Control(-Pb)
groups. If the cecal ratio of Firmicutes to Bacteroidetes and
Betaproteobacteria are affected by probiotic use, then a difference
in the levels of these bacterial parameters should be evident when
comparing AUT-GI probiotic non-users vs. AUT-GI probiotic users
[Table S5 .ANG.: AUT(-Pb) vs. AUT(+Pb)]. There was not a
significant difference in Betaproteobacteria levels between these
groups; however, the ratio of Firmicutes to Bacteroidetes in the
cecum, determined by real-time PCR, was significantly higher in the
AUT(+Pb) group compared to the AUT(-Pb) group (Table 13A:
Mann-Whitney, p=0.037). Thus an effect mediated by probiotics on
this variable cannot be excluded. This effect, however, was not
apparent in the corresponding ratio of Firmicutes to Bacteroidetes
in the cecum, determined by pyrosequencing.
[0454] The use of proton-pump inhibitors (PPI: Lanzoprazole or
Omeprazole) was similarly examined. PPI had been used by both
AUT-GI (n=4; 27%) and Control-GI (n=2; 29%) children. A significant
difference was found for all variables, except LCT, when comparing
AUT(-PPI) children with Control(-PPI) children [Table S5B:
AUT(-PPI) vs. Control(-PPI)]. Thus a potential effect of PPI use
should only be considered for LCT. As LCT levels were not
significantly different between AUT(-PPI) and AUT(+PPI) children,
it is unlikely that PPIs exerted any major effect on LCT
expression. A trend toward an effect in the levels of Bacteroidetes
in the ileum, determined by pyrosequencing, was evident between
AUT(-PPI) and AUT(+PPI) children; however, a significant effect was
observed between AUT(-PPI) and Control(-PPI) children. This
indicates that this potential effect was not a major determinant of
the difference in ileal Bacteroidetes between AUT-GI and Control-GI
children. Only one patient (AUT-GI patient #1) had used both
probiotics and proton-pump inhibitors, thus an additive effect was
not evaluated. Grouping of patients based on whether they had taken
either probiotics or proton-pump inhibitors did not reveal any
significant effects in the 16 variables.
[0455] Only one individual had taken an antibiotic (amoxicillin) in
this cohort (Control-GI patient #16). This patient had high levels
of mRNA expression for all disaccharidases and transporters, within
the range of other Control-GI children and at least above the
90.sup.th percentile of all AUT-GI children. Thus, exclusion of
this patient from the analysis had a negative effect on
significance values obtained for gene expression assays (Table
13C). These results indicate that antibiotic use had no effect on
disaccharidase and hexose transporter levels in this patient. In
contrast, Control-GI patient #16 consistently had the lowest levels
of Bacteroidetes (representing the low-range outlier) compared to
all other Control-GI children in pyrosequencing and real-time PCR
assays. Thus, exclusion of this patient from analysis of bacterial
phylotypes generally improved the significance of results obtained
for Bacteroidetes, ratios of Firmicutes to Bacteroidetes, and
ratios of Clostridiales to Bacteroidales. Levels of
Beta-proteobacteria in the cecum for this patient were near the
median value of all other Control-GI children. Thus, it is likely
that antibiotic use in this patient had some effect on Bacteroidete
levels, but no effect on Betaproteobacteria or gene expression for
disaccharidases and transporters. As the effect of antibiotic use
in this patient did not affect all variables and exclusion of this
patient did not affect the interpretation of results, this patient
was not excluded from the overall analysis.
REFERENCES FOR SUPPORTING RESULTS
[0456] B1. Eckburg P B, Bik E M, Bernstein C N, Purdom E,
Dethlefsen L, et al. (2005) Diversity of the human intestinal
microbial flora. Science 308: 1635-1638. [0457] B2. Reid G, Younes
J A, Van der Mei H C, Gloor G B, Knight R, et al. (2011) Microbiota
restoration: natural and supplemented recovery of human microbial
communities. Nat Rev Microbiol 9: 27-38. [0458] B3. Lombardo L,
Foti M, Ruggia 0, Chiecchio A (2010) Increased incidence of small
intestinal bacterial overgrowth during proton pump inhibitor
therapy. Clin Gastroenterol Hepatol 8: 504-508.
[0459] Supporting Methods
[0460] Quantitative Real-Time PCR of Human mRNA:
[0461] PCR standards for determining copy numbers of target
transcripts were generated from amplicons of SI, MGAM, LCT, SGLT1,
GLUT2, Villin, CDX2, GAPDH, and Beta-actin cloned into the vector
pGEM-T easy (Promega Corporation). Linearized plasmids were
quantitated using a Nanodrop ND-1000 Spectrophotometer, and 10-fold
serial dilutions (ranging from 5.times.10.sup.5 to
5.times.10.degree. copies) were created in water containing yeast
tRNA (1 ng/.mu.l). Unpooled RNA from individual ileal biopsies were
used for real time PCR assays; each individual biopsy was assayed
in duplicate. cDNA was synthesized using Taqman reverse
transcription reagents (Applied Biosystems) from 2 .mu.g unpooled
RNA per 100 .mu.l reaction. Each 25-.mu.l amplification reaction
contained 10 .mu.l template cDNA, 12.5 .mu.l Taqman Universal PCR
Master Mix (Applied Biosystems), 300 nM gene-specific primers and
200 nM gene-specific probe (Table 16). The thermal cycling profile
using a ABI StepOnePlus Real-time PCR System (Applied Biosystems)
consisted of: Stage 1, one cycle at 50.degree. C. for 2 min; Stage
2, 1 cycle at 95.degree. C. for 10 min; Stage 3, 45 cycles at
95.degree. C. for 15 s and 60.degree. C. for 1 min (1 min 30 s for
LCT). GAPDH and B-actin mRNA were amplified in duplicate reactions
by real-time PCR from the same reverse transcription reactions as
were used for the genes of interest. The mean concentration of
GAPDH or Beta-actin in each sample was used to control for
integrity of input RNA and to normalize values of target gene
expression to those of the housekeeping gene expression. GAPDH and
Beta-actin have been shown to be the most stable reference genes in
small bowel and colonic biopsies from healthy patients and
pediatric patients with inflammatory bowel disease [C1]. The final
results shown were expressed as the mean copy number from replicate
biopsies per patient, relative to values obtained for GAPDH mRNA.
Beta-actin normalization gave similar results to GAPDH
normalization for all assays (data not shown). Due to insufficient
or poor quality RNA (OD 260/280 ratio<1.7, or RNA integrity
number<7.0), only 3 of the 4 biopsies were included for 3
patients (Patient #s 4, 7, 10) and only 2 of the 4 biopsies were
included for 1 patient (Patient #2). Thus, 83 of the original 88
ileal biopsies were used in real-time PCR experiments.
[0462] Lactase Genotyping:
[0463] Genotyping primers for the LCT C/T-13910 and G/A-22018
polymorphisms are as follows: C/T-13910For
(5'-GGATGCACTGCTGTGATGAG-3' [SEQ ID NO: 20]), C/T-13910Rev
(5'-CCCACTGACCTATCCTCGTG-3' [SEQ ID NO: 21]), G/A-22018For
(5'-AACAGGCACGTGGAGGAGTT-3' [SEQ ID NO: 22]), and G/A-22018Rev
(5'-CCCACCTCAGCCTCTTGAGT-3' [SEQ ID NO: 23]). Each 50-.mu.l
amplification reaction contained 500 ng genomic DNA, 400 nM forward
and reverse primers, and 25 .mu.l High Fidelity PCR master mix
(Roche). Thermal cycling consisted of 1 cycle at 94.degree. C. for
4 min followed by 40 cycles at 94.degree. C. for 1 min, 60.degree.
C. for 1 min, and 72.degree. C. for 1 min. PCR reactions for
C/T-13910 were directly digested with the restriction enzyme BsmFI
at 65.degree. C. for 5 hrs. PCR reactions for G/A-22018 were
resolved on 1% agarose gels followed by gel extraction of the
prominent 448 bp amplicon. Gel extracted G/A-22018 amplicons were
then digested with the restriction enzyme HhaI at 37.degree. C. for
5 hrs. Restriction digests of C/T-13910 and G/A-22018 were resolved
on 1.5% ethidium-stained agarose gels for genotyping analysis.
BsmFI digestion of the C/T-13910 amplicons generates two fragments
(35 lbp and 97 bp) for the hypolactasia genotype (C/C), four
fragments (35 lbp, 253 bp, 98 bp, and 97 bp) for the heterozygous
genotype (C/T), and three fragments (253 bp, 98 bp, and 97 bp) for
the normal homozygous allele (T/T). HhaI digestion of the G/A-22018
amplicons generates two fragments (284 bp and 184 bp) for the
hypolactasia genotype (G/G), three fragments (448 bp, 284 bp, and
184 bp) for the heterozygous genotype (G/A), and a single fragment
(448 bp) for the normal homozygous allele (A/A).
[0464] Barcoded Pyrosequencing of Intestinal Microbiota:
[0465] Composite primers used for pyrosequencing analysis were as
follows
TABLE-US-00028 (For) [SEQ ID NO: 24]
5'-GCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG-3', (Rev) [SEQ ID NO:
25] 5'-GCCTCCCTCGCGCCATCAGNNNNNNNNCATGCTGCCTCCCGTAGGAG T-3'.
Underlined sequences in the Forward and Reverse primers represent
the 454 Life Sciences@ primer B and primer A, respectively. Bold
sequences in the forward and reverse primers represent the
broadly-conserved bacterial primer 27F and 338R, respectively.
NNNNNNNN represents the eight-base barcode, which was unique for
each patient. PCR reactions consisted of 8 .mu.l 2.5.times.5 PRIME
HotMaster Mix (5 PRIME Inc), 6 .mu.l of 4 .mu.M forward and reverse
primer mix, and 200 ng DNA in a 20-.mu.l reaction volume. Thermal
cycling consisted of one cycle at 95.degree. C. for 2 min; and 30
cycles at 95.degree. C. for 20 seconds, 52.degree. C. for 20
seconds, and 65.degree. C. for 1 min. Each of 4 biopsies per
patient was amplified in triplicate, with a single, distinct
barcode applied per patient. Ileal and cecal biopsies were assayed
separately. Reagent controls were included (negative controls) to
control for any background contamination. Triplicate reactions of
individual biopsies and reagent controls were combined, and PCR
products were purified using Ampure magnetic purification beads
(Beckman Coulter Genomics) and quantified with the Quanti-iT
PicoGreen dsDNA Assay Kit (Invitrogen) and Nanodrop ND-1000
Spectrophotometer (Nanodrop Technologies). Equimolar ratios were
combined to create two master DNA pools, one for ileum and one for
cecum, with a final concentration of 25 ng/.mu.l. Master pools were
sent for unidirectional pyrosequencing with primer A at 454 Life
Sciences on a GS FLX sequencer. Each master pool was sequenced in
duplicate on different days to control for variability in the
sequencing reactions. Sequences obtained from duplicate runs were
combined for the final analysis. No sequences were obtained from
reagent controls, indicating that no background contamination was
present.
[0466] Quantitative Real-Time PCR of Bacteroidete and Firmicute 16S
rRNA Genes:
[0467] PCR standards for determining copy numbers of bacterial 16S
rDNA were prepared from representative amplicons of the partial 16S
rRNA genes of Bacteroidetes, Firmicutes and total Bacteria cloned
into the vector PGEM-T easy (Promega). A representative amplicon
with high sequence similarity to Bacteroides Vulgatus (Accession #:
NC 009614) was used with Bacteroidete-specific primers. A
representative amplicon with high sequence similarity to
Faecalibacterium prausnitzii (Accession #: NZ.sub.-- ABED02000023)
was used with Firmicute-specific primers. A representative amplicon
with high sequence similarity to Bacteroides intestinalis
(Accession #: NZ_ABM02000007) 16S rRNA gene was used with total
Bacteria primers (primers 515F and 805R). Cloned sequences were
classified using the Ribosomal Database Project (RDP, release 10)
Seqmatch tool and confirmed by the Microbes BLAST database.
Plasmids were linearized with the SphI restriction enzyme,
quantitated, and ten-fold serial dilutions of plasmid standards
were created ranging from 5.times.10.sup.7 to 5.times.10.degree.
copies for Bacteroidetes, Firmicutes and total Bacteria.
Amplification and detection of DNA by real-time PCR were performed
with the ABI StepOnePlus Real-time PCR System (Applied Biosystems).
Cycling parameters for Bacteroidetes and total Bacteria were as
previously described [C2], as were cycling parameters for
Firmicutes [C3]. Each 25-.mu.l amplification reaction mixture
contained 50 ng DNA, 12.5 .mu.l SYBR Green Master Mix (Applied
Biosystems), and 300 nM bacteria-specific (Bacteroidete, Firmicute
or total Bacteria) primers. DNA from each of 88 ileal biopsies (4
biopsies per patient) and 88 cecal biopsies (4 biopsies per
patient) was assayed in duplicate. The final results were expressed
as the mean number of Bacteroidete or Firmicute 16S rRNA gene
copies normalized to 16S rRNA gene copies obtained using total
Bacterial primers. Eight water/reagent controls were included for
all amplifications. The average copy number for water/reagent
controls (background) was subtracted from each ileal and cecal
amplification prior to normalization. For the Bacteroidete assay
all water controls contained undetectable levels of amplification.
For the Firmicute assay average amplification signal from water
samples were minimal, 12.03+/-15.0 copies.
[0468] Statistical Analysis:
[0469] To evaluate the effects of CDX2 and/or villin on enzyme and
transporter levels and the effects of levels of enzymes and
transporters on bacterial levels, multiple linear regression
analyses were conducted. For assessing the affects of CDX2 and
villin on disaccharidase and transporter expression levels,
disaccharidase and transporter levels were log-transformed to
stabilize the variance. Using each log-transformed disaccharidase
and transporter mRNA expression level as an outcome, three models
were fitted: first with CDX2 only as independent variable; second
with CDX2 and status (dummy coded; AUT-GI=1 vs. Control-GI=0); and
third with CDX2, status, and the interaction term between CDX2 and
status. The interaction term allowed us to evaluate whether the
effect of CDX2 on disaccharidases and transporters was similar for
AUT-GI and Control-GI children. The same models were fitted after
adding villin and the interaction term between villin and status.
The coefficient estimates in Table 11 represent change in
log-transformed disaccharidase or transporter mRNA levels per unit
standard deviation increase in CDX2 and villin mRNA levels.
[0470] To delineate the effects of disaccharidases and transporters
on bacterial levels in ileal and cecal biopsies, bacterial 16S rRNA
gene quantities (obtained from real-time PCR for Bacteroidetes and
Firmicutes) or abundance (obtained from 454 pyrosequencing data for
Proteobacteria and Betaproteobacteria) were log-transformed to
stabilize variance. For each of the log-transformed bacterial
levels, enzyme levels were first fitted simultaneously as the main
effects (SI, MGAM, LCT, SGLT1, and GLUT2) to evaluate the effects
of enzymes on a given bacterial taxa. Status was added to the model
to determine whether there was a residual difference in bacterial
levels between AUT-GI and Control-GI children after adjusting for
the levels of disaccharidases and transporters. It was further
examined whether the effect of disaccharidases or transporters on
bacterial levels was the same depending on the status by examining
two-way interaction terms between status and each disaccharidase
and transporter. The final model was derived by including all the
main effect terms and selectively including two-way interaction
terms using the backward elimination method starting from all
possible two-way interaction terms with status and the individual
disaccharidases and transporters. The coefficient estimates in
Table 14 represent change in log-transformed bacterial levels per
unit standard deviation increase in disaccharidase or transporter
levels. The statistical package R (version 2.7.0) was used for
regression analysis.
SUPPORTING METHODS REFERENCES
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method for Firmicutes and Bacteroidetes in faeces and its
application to quantify intestinal population of obese and lean
pigs. Lett Appl Microbiol 47: 367-373.
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Example 4
Application of Sutterella-Specific PCR-Based Methods for Detection,
Quantitation, and Phylogenetic Characterization of Sutterella
Species
[0570] Abstract.
[0571] Gastrointestinal disturbances are commonly reported in
children with autism and can be associated with compositional
changes in intestinal bacteria. In a previous report we surveyed
intestinal microbiota in ileal and cecal biopsies from children
with autism and gastrointestinal dysfunction (AUT-GI) and children
with only gastrointestinal dysfunction (Control-GI). The results
demonstrated the presence of members of the family Alcaligenaceae
in some AUT-GI children, while no Control-GI children had
Alcaligenaceae sequences. Here we demonstrate that increased levels
of Alcaligenaceae in intestinal biopsies from AUT-GI children
result from the presence of high levels of members of the genus
Sutterella. We also report the first Sutterella-specific polymerase
chain reaction assays for detecting, quantitating, and genotyping
Sutterella species in biological and environmental samples.
Sutterella 16S rRNA gene sequences were found in 12 of 23 AUT-GI
children but in none of 9 Control-GI children. Phylogenetic
analysis revealed a predominance of either the species Sutterella
wadsworthensis or Sutterella stercoricanis in 11 of the individual
Sutterella-positive AUT-GI patients; in one AUT-GI patient,
Sutterella sequences were obtained that could not be given a
species level classification based on the 16S rRNA gene sequences
of known Sutterella isolates. Western immunoblots revealed plasma
IgG or IgM antibody reactivity to Sutterella wadsworthensis
antigens in 11 AUT-GI patients, 8 of whom were also PCR-positive,
indicating the presence of an immune response to Sutterella in some
children.
[0572] Autism spectrum disorders affect approximately 1% of the
population. Many children with autism have gastrointestinal (GI)
disturbances that can complicate clinical management and contribute
to behavioral problems. Understanding the molecular and microbial
underpinnings of these GI issues is of paramount importance for
elucidating pathogenesis, rendering diagnosis, and administering
informed treatment. An association between high levels of
intestinal, mucoepithelial-associated Sutterella species and GI
disturbances in children with autism is described. These findings
elevate this little-recognized bacterium to the forefront by
demonstrating that Sutterella is a major component of the
microbiota in over half of AUT-GI children and is absent in
Control-GI children evaluated in this study. Furthermore, these
findings bring into question the role Sutterella plays in the human
microbiota in health and disease. With the Sutterella-specific
molecular assays described herein, some of these questions are
addressed.
[0573] Introduction
[0574] Autism spectrum disorders (ASD) are pervasive developmental
disorders that depend on triadic presentation of social
abnormalities, communication impairments, and stereotyped and
repetitive behaviors for diagnosis (DSM-IV-TR criteria, American
Psychiatric Association, 2000). Gastrointestinal (GI) symptoms are
commonly reported in children with autism and can correlate with
autism severity (D1, D2). Intestinal disturbances in autism have
been associated with macroscopic and histological abnormalities,
altered inflammatory parameters, and various functional
disturbances (D3-9).
[0575] In a previous study, we showed that a complex interplay
exists between human intestinal gene expression for disaccharidases
and hexose transporters and compositional differences in the
mucoepithelial microbiota of children with autism and
gastrointestinal disease (AUT-GI children) compared to children
with GI disease but typical neurological status (Control-GI
children). Significant compositional changes in Bacteroidetes,
Firmicute/Bacteroidete ratios, and Betaproteobacteria in AUT-GI
intestinal biopsies were reported (D10). Although others have
demonstrated changes in fecal bacteria of children with autism (D2,
D11-15), the study differed from these by investigating
mucoepithelial microbiota (D10). The GI microbiota plays an
essential role in physiological homeostasis in the intestine and
periphery, including maintaining resistance to infection,
stimulating immunological development, and perhaps even influencing
brain development and behavior (D16-19). Thus, disruption of the
balanced communication between the microbiota and the human host
could have profound effects on human health.
[0576] In the previous metagenomic study, sequences were found to
correspond to members of the family Alcaligenaceae in the class
Betaproteobacteria that were present in ileal and cecal biopsies
from 46.7% (7/15) of AUT-GI children. Alcaligenaceae sequences were
completely absent from biopsies of Control-GI children (D10).
Members of the family Alcaligenaceae inhabit diverse habitats,
ranging from humans and animals to soil (D20). Several members of
Alcaligenaceae cause clinically relevant infections or are
suspected opportunistic pathogens in humans and animals, including
members of the genus Bordetella (the human respiratory pathogens,
B. pertussis and B. parapertussis; the mammalian respiratory
pathogen B. bronchiseptica; and the poultry respiratory pathogen,
B. avium); a member of the genus Alcaligenes (the human
opportunistic pathogen A. faecalis); members of the genus
Achromobacter (the human opportunistic pathogens A. xylosoxidans
and A. piechaudii), members of the genus Oligella (the potential
opportunistic genitourinary species O. urethralis and O.
ureolytica); a member of the genus Taylorella (the equine
urogenital pathogen, T. equigenitalis); and a member of the genus
Pelistega (the pigeon respiratory pathogen, P. europaea) (D20).
[0577] In some cases the pathogenic potential of Alcaligenaceae
members is unclear. The genus Sutterella represents one such
Alcaligenaceae member. Members of the genus Sutterella are
anaerobic, bile-resistant, asaccharolytic, Gram-negative, short
rods (D21). Members of the genus Sutterella have been isolated from
human infections below the diaphragm (D22, D23). Sutterella 16S
rRNA gene sequences have also been identified in intestinal
biopsies and fecal samples from individuals with Crohn's disease
and ulcerative colitis (D24, D25). Whether the presence of
Sutterella species at sites of human infection and inflammation
represents cause or consequence, or whether Sutterella is a normal
part of the microbiota in some individuals, remains unclear. The
dearth of knowledge concerning the epidemiology and pathogenic
potential of Sutterella derives in part from the lack of specific
assays to detect and characterize members of this genus.
[0578] Alcaligenaceae sequences identified were further
characterized in AUT-GI children and describe PCR assays for
detection, quantitation, and genotyping of Sutterella as well as
serological assays for detection of immunological responses to
Sutterella.
[0579] Results
[0580] High Levels of Sutterella in a Subset of AUT-GI Patients
Identified by Pyrosequencing:
[0581] Previous pyrosequencing results (D10) demonstrated a high
abundance of sequences from the family Alcaligenaceae in nearly
half of AUT-GI children (Patients #1-15) and the absence of
corresponding sequences in Control-GI children (Patients #16-22),
and prompted a more detailed investigation of these taxa of
bacteria. Genus level analysis of pyrosequencing reads revealed
that all sequences of Alcaligenaceae found in AUT-GI patients'
biopsies were classified as members of the genus Sutterella. The
average confidence estimate of all genus level, RDP (Ribosomal
Database Project)-classified Sutterella sequences was high (99.1%),
with the majority of sequences classified at 100% confidence.
[0582] Comparison of Sutterella abundance from pyrosequencing reads
revealed significant increases in Sutterella in the ilea (FIG. 8A:
Mann-Whitney, tied p-value=0.022) and ceca (FIG. 8B: Mann-Whitney,
tied p-value=0.037) of AUT-GI children compared to Control-GI
children. Individual analysis of AUT-GI patients revealed that
46.7% (7/15) of AUT-GI patients (Patients #1, 3, 5, 7, 10, 11, 12)
had high levels of Sutterella 16S rRNA gene sequences in both the
ileum (FIG. 8C and Table 19) and cecum (FIGS. 8D and 36 and Table
19). Sutterella sequences were absent from all Control-GI samples
(Patients #16-22). In those seven AUT-GI patients with Sutterella
sequences, ileal Sutterella sequence abundance ranged from 1.7 to
6.7% of total bacterial reads (FIG. 8C and Table 19). For the same
patients, cecal Sutterella sequence abundance ranged from 2.0 to
7.0% of total bacterial reads (FIGS. 8D and 36 and Table 19).
TABLE-US-00029 TABLE 19 Summary of total bacteria,
Betaproteobacteria, and Sutterella sequences obtained by 16S rRNA
gene (V2-region) pyrosequencing from ileal and cecal biopsies of
AUT-GI and Control-GI children. # of Total Bacteria # of Total
Bacteria # of Betaproteobacteria Patient # AUT/Control Reads-Ileum
Reads-Cecum Reads-Ileum 1 AUT-GI 11,881 13,032 706 2 AUT-GI 13,734
7,647 3627 3 AUT-GI 11,434 10,147 536 4 AUT-GI 12,756 11,779 400 5
AUT-GI 10,708 10,502 647 6 AUT-GI 14,739 11,075 137 7 AUT-GI 11,941
11,246 209 8 AUT-GI 11,348 11,754 27 9 AUT-GI 12,320 10,661 262 10
AUT-GI 12,483 12,295 501 11 AUT-GI 11,211 12,436 800 12 AUT-GI
11,055 11,103 434 13 AUT-GI 10,420 10,670 171 14 AUT-GI 12,217
11,012 123 15 AUT-GI 12,002 12,561 138 16 Control-GI 13,758 13,630
129 17 Control-GI 12,246 14,956 147 18 Control-GI 11,888 14,330 315
19 Control-GI 11,290 10,136 377 20 Control-GI 14,844 11,794 134 21
Control-GI 13,308 11,567 145 22 Control-GI 13,460 10,143 131 # of
Betaproteobacteria # of Sutterella Sutterella-Ileum (% of
Sutterella-Ileum (% of Patient # Reads-Cecum Reads-Ileum Total
Bacteria) Betaproteobacteria) 1 535 534 4.5 75.6 2 632 0 0 0 3 428
503 4.4 93.8 4 132 0 0 0 5 535 581 5.4 89.8 6 36 0 0 0 7 224 201
1.7 96.2 8 80 0 0 0 9 404 0 0 0 10 478 490 3.9 97.8 11 903 747 6.7
93.4 12 444 408 3.7 94.0 13 619 0 0 0 14 105 0 0 0 15 39 0 0 0 16
136 0 0 0 17 116 0 0 0 18 404 0 0 0 19 151 0 0 0 20 58 0 0 0 21 34
0 0 0 22 85 0 0 0 # of Sutterella Sutterella-Cecum (% of
Sutterella-Cecum (% of Patient # Reads-Cecum Total Bacteria)
Betaproteobacteria) 1 520 4.0 97.2 2 0 0 0 3 403 4.0 94.2 4 0 0 0 5
498 4.7 93.1 6 0 0 0 7 220 2.0 98.2 8 0 0 0 9 0 0 0 10 459 3.7 96.0
11 870 7.0 96.3 12 409 3.7 92.1 13 0 0 0 14 0 0 0 15 0 0 0 16 0 0 0
17 0 0 0 18 0 0 0 19 0 0 0 20 0 0 0 21 0 0 0 22 0 0 0
[0583] To put the levels of Sutterella in these patients into
perspective, the abundance of all ileal and cecal genus level
classifications were ranked from pyrosequencing results. In the
ileum, Sutterella sequences represented the 4.sup.th most abundant
genera for patient #1, the 6.sup.th most abundant genera for
patient #3, the 5.sup.th most abundant genera for patient #5, the
5.sup.th most abundant genera for patient #7, the 3.sup.rd most
abundant genera for patient #10, the 8.sup.th most abundant genera
for patient #11, and the 5.sup.th most abundant genera for patient
#12 (FIG. 44 and FIG. 45). Similar rankings were obtained in the
cecum of these patients.
[0584] Sutterella sequences represented the majority of sequences
present in the class Betaproteobacteria in these seven AUT-GI
patients. In ileal biopsies from the seven AUT-GI patients with
Sutterella sequences, Sutterella sequences accounted for 75.6% to
97.8% of all Betaproteobacteria sequences (FIG. 8E and Table 19).
In cecal biopsies, Sutterella sequences accounted for 92.1% to
98.2% of all Betaproteobacteria sequences (FIG. 8F and Table
19).
[0585] OTU and Sequence Analysis of Sutterella Sequences in AUT-GI
Children:
[0586] OTU (Operational Taxonomic Unit) analysis of V2
pyrosequencing reads in ileum (FIG. 46A) and cecum (FIG. 46B)
revealed that sequences from patients #1, 3, 10, 11, and 12
clustered together with OTU 2 containing the majority of Sutterella
sequences, and patients #5 and 7 clustered together with OTU 1
containing the majority of Sutterella sequences. OTU 2 accounted
for 87% and 84% for patient #1, 85% and 87% for patient #3, 66% and
66% for patient #10, 87% and 85% for patient #11, and 81% and 81%
for patient #12 of all Sutterella sequences obtained by
pyrosequencing of the 16S rRNA gene in ileum and cecum,
respectively (FIG. 37). OTU 1 accounted for 88% and 86% for patient
#5 and 88% and 83% for patient #7 of all Sutterella sequences
obtained by pyrosequencing of the V2 region of the 16S rRNA gene in
ileum and cecum, respectively (FIG. 37). Subdominant OTUs can
represent true phylotypes, but could also arise from PCR or
sequencing artifacts. The analysis was focused on those OTUs
containing the majority of Sutterella sequences, namely OTU 1 and
OTU 2.
[0587] The representative sequences from OTU 1 and OTU 2 were
aligned and used for phylogenetic analysis (FIG. 47. The
representative sequence from OTU 1 was phylogenetically most
closely related to the species S. wadsworthensis; the
representative sequence from OTU 2 was most closely related to S.
stercoricanis. Although some branches in the tree are clearly
differentiated by high bootstrap values, others are differentiated
poorly by low bootstrap values. Furthermore, members of the genus
Comamonas and Burkholderia were grouped with members of the genus
Sutterella. This indicates that sequences from the V2 region alone
can be insufficient for accurate species level phylogenetic
analysis of Sutterella sequences.
[0588] Confirmation and Quantitation of Sutterella Sequences Using
New PCR Assays:
[0589] To independently verify V2 pyrosequencing results for
Sutterella, Sutterella-specific PCR assays were designed that could
be used in both conventional and real-time PCR, using primers that
amplify a 260 by region spanning the V6 to V8 regions of the 16S
rRNA gene (SuttFor and SuttRev primers) (FIG. 38, FIG. 9, FIG.
10A-B). Conventional PCR analysis using DNA from each of 4 ileal
and 4 cecal biopsies per patient showed that the same individuals
identified as having high levels of Sutterella by V2 pyrosequencing
(Patients #1, 3, 5, 7, 10, 11, 12) were also positive by the novel
V6-V8 Sutterella-specific PCR (FIG. 39A). All 4 biopsies from ileum
and cecum, in all seven Sutterella-positive patients, showed
Sutterella products. A single 260 by product was observed in
positive amplifications, and non-specific products were never
observed. No products were observed in any Control-GI patients that
were evaluated by pyrosequencing (Patients #16-22), the AUT-GI
patients that were negative for Sutterella sequences by V2
pyrosequencing (Patients #2, 4, 6, 8, 9, 13, 14, 15), or
water/reagent controls (FIG. 39A). Furthermore, the positive
control (DNA from a cultured S. wadsworthensis isolate) was
positive by PCR. In addition to those patients evaluated by
pyrosequencing, ileal and cecal biopsies were assessed from eight
additional male AUT-GI (Patients #23a-30a) and two additional male
Control-GI (Patients #31a and 32a) children using the V6-V8
Sutterella PCR. Of these additional samples, 5 of the 8 AUT-GI
patients were positive for Sutterella in ileal and cecal biopsies
(Patients #24a, 25a, 27a, 28a, and 29a). All biopsies from the two
additional Control-GI patients were PCR-negative (Patients #31a and
32a). In summary, whereas 12 of 23 (52%) AUT-GI children were
PCR-positive for Sutterella, 0 of the 9 Control-GI children were
PCR-positive for Sutterella.
[0590] In addition, the broadly conserved, pan-bacterial primer
515For was used in combination with the SuttRev primer in
conventional PCR assays (FIG. 39B). These primers amplify a 715 bp
region of the 16S rRNA gene from conserved region 4 (C4) to
variable region 8 (V8) (see FIG. 38A). Results of the C4-V8
amplification were identical to the V6-V8 assay. All products were
confirmed to represent Sutterella by sequencing of V6-V8 and C4-V8
products. These results indicate that the SuttRev primer is
sufficient to confer specificity for Sutterella amplification.
[0591] In addition, Sutterella 16S rRNA gene sequences were
quantified in biopsies from AUT-GI and Control-GI patients using
real-time PCR (FIG. 10A-B). Real-time PCR analysis using the
SuttFor and SuttRev (V6-V8) primers and a high coverage Taqman
probe revealed similar results to conventional PCR assays. By
real-time PCR, Sutterella was detected in patients #1, 3, 5, 7, 10,
11, 12, 24a, 25a, 27a, 28a, and 29a (FIG. 40), consistent with both
pyrosequencing and conventional PCR results. Sutterella was
undetectable in all Control-GI and Sutterella-negative AUT-GI
patients' samples. Mean Sutterella copy numbers were high in both
the ileum and cecum [in the range of 10.sup.3 to 10.sup.5 copies]
of Sutterella-positive patients.
[0592] Phylogenetic Analysis of Sutterella Sequences Obtained by
Novel PCR Assays:
[0593] The predominant Sutterella sequence from the ileum and cecum
of each patient was determined following alignment of all V6-V8
sequences obtained by library cloning of products. This analysis
revealed that the predominant sequences obtained in ileal biopsies
were identical to the predominant sequences in cecal biopsies from
each individual patient. Thus, a single predominant sequence was
further assessed for each patient.
[0594] Phylogenetic analysis of the predominant V6-V8 sequences
obtained by PCR revealed that the dominant Sutterella species found
in patients #1, 3, 10, 11, 12, 24a, 27a, and 29a were most closely
associated with the isolates S. stercoricanis and Parasutterella
secunda; the dominant V6-V8 Sutterella sequences found in patients
#5, 7, and 25a were most closely associated with isolates of S.
wadsworthensis (FIG. 48). Sequences from patient #28a were most
closely associated with Sutterella sp. YIT 12072. Thus, sequences
from patients #5 and 7 grouped with S. wadsworthensis isolates
using both the V2 pyrosequencing reads and the V6-V8 sequences
obtained by PCR, while sequences from patients #1, 3, 10, 11, and
12 grouped with S. stercoricanis using both the V2 pyrosequencing
reads and the V6-V8 sequences obtained by PCR. However, as was the
case from phylogenetic analysis of V2 pyrosequencing, bootstrap
values were low at many branches, indicateing that neither the V2
nor V6-V8 regions provide sufficient information for accurate
species level differentiation. Furthermore, members of the genus
Sutterella did not all group together based on the V6-V8 region
sequences, with Parasutterella secunda, S. stercoricanis, S.
sanguinus, and S. morbirenis being more closely associated with
other Alcaligenaceae and Burkholderiales members.
[0595] 480 sequences (40 sequences per patient; 20 ileal sequences
and 20 cecal sequences) obtained from clone libraries of C4-V8
products were analyzed from the 12 Sutterella-positive patients
(FIG. 41). No sequences were obtained from any genus other than
Sutterella from any cloned PCR products. The majority or all of the
C4-V8 sequences from patients #1, 3, 10, 11, 12, 24a, 27a, and 29a
were most closely matched with S. stercoricanis, the majority or
all of the C4-V8 sequences obtained from patients #5, 7, and 25a
matched most closely with S. wadsworthensis, and all sequences
obtained from patient #28a matched most closely with Sutterella sp.
YIT 12072. It was evident from this analysis that although one
species predominated in each patient, mixed populations were
detected in many patients. Most individuals with mixed populations
harbored sequences of S. wadsworthensis and S. stercorcanis.
Patient #24a had species matches for S. stercoricanis, S.
wadsworthensis, and S. parvirubra.
[0596] To determine the accuracy of the C4-V8 region for
confirmation of species level classification, the predominant C4-V8
16S rRNA gene sequences obtained from the ileum and cecum of each
patient were analyzed. Similar to the results obtained with the
V6-V8 region, this analysis revealed that the predominant
Sutterella 16S rRNA gene sequences identified in ileal biopsies
were identical to the predominant Sutterella sequences in cecal
biopsies for each of the individual patients. Thus, a single
predominant sequence was further assessed for each patient.
[0597] Alignment of the predominant C4-V8 sequence from each
patient revealed that patients #1 and 24a had identical predominant
sequences, but that these were distinct from all other patients;
patients #3, 10, 11, 12, 27a, and 29a had identical sequences,
distinct from all other patients; patients #5, 7, and 25a had
identical sequences that were distinct from all other patients; and
patient #28a had a unique sequence (FIG. 49).
[0598] Comparison of percent sequence similarity between these
groups (Table 17) revealed 99.9% similarity between sequences of
patients #1 and 24a and those of patients #3, 10, 11, 12, 27a, and
29a. This value is above the cut-off value of 97% similarity,
commonly applied for bacterial species definition (D26),
indicateing that the predominant sequences from these two groups
are likely the same species.
TABLE-US-00030 TABLE 17 Sequence similarity between 16S rRNA gene
(C4-V8 region) of Sutterella from AUT-GI children and Sutterella
isolates. Highest sequence similarities are shown in bold. Patients
3, Sutterella Sutterella Sutterella Sutterella Sutterella sp.
Patients 10, 11, 12, Patients Patient stercorcanis wadsworthensis
parvirubra sanguinus YIT 12072 % Similarity 1, 24a 27a, 29a 5, 7,
25a 26a (AJ566848) (GU585669) (AB300989) (AJ748647) (AB491210)
Patients 1, 24a -- 99.9% 94.8% 93.8% 98.5% 94.8% 95.4% 96.3% 93.2%
Patients 3, 10, -- 94.7% 93.6% 98.4% 94.7% 95.4% 96.4% 93.3% 11,
12, 27a, 29a Patients 5, 7, 25a -- 92.8% 94.7% 100% 96.6% 93.6%
92.9% Patient 26a -- 93.0% 92.8% 93.6% 92.0% 95.3% Sutterella --
94.7% 94.7% 96.6% 93.2% stercorcanis (AJ566849) Sutterella -- 96.6%
93.6% 92.9% wadsworthensis (GU585669) Sutterella -- 95.0% 92.6%
parvirubra (AB300989) Sutterella -- 92.2% sanguinus (AJ749647)
Sutterella sp. -- YIT 12072 (AB491210)
[0599] The predominant sequences from patients #1 and 24a and
patients #3, 10, 11, 12, 27a, and 29a had the highest percent
similarity to the isolate S. stercorcanis (98.5% similarity and
98.4% similarity, respectively) (Table 17). The percent similarity
of sequences from patients #1, 24a, 3, 10, 11, 12, 27a, and 29a
were below 97% compared to the other Sutterella isolates,
indicateing that the predominant species in these patients is
likely S. stercorcanis. In addition, the 16S rRNA gene sequence
from patients #1 and 24a shared 100% similarity with 16S rRNA gene
sequences from uncultured bacteria in genbank, such as those
derived from intestinal biopsies from an ulcerative colitis patient
(i.e., Accession FJ512128) (D27) and mucosal biopsies from the
intestinal pouch of a familial adenomatous polyposis patient (i.e.,
Accession GQ159316). Similarly, the sequences from patients #3, 10,
11, 12, 27a, and 29a shared 100% similarity with 16S rRNA gene
sequences from uncultured bacteria in genbank, including sequences
derived from intestinal biopsies from a patient with ulcerative
colitis (i.e., Accession 512152) (D27) and fecal samples from
bovines (i.e., Accession FJ682648) (D28).
[0600] Sequences from patients #5, 7, and 25a had 100% sequence
similarity to S. wadsworthensis and below 97% sequence similarity
to all other Sutterella isolates (Table 17). Thus, the predominant
sequences from patients #5, 7, and 25a are likely S.
wadsworthensis. The sequence from patients #5, 7, and 25a also
shared 100% sequence similarity to 16S rRNA sequences in genbank,
such as those derived from intestinal biopsies from an ulcerative
colitis patient (i.e., Accession FJ509042) (D27).
[0601] The unique sequence found in patient #28a matched most
closely with the isolate, Sutterella sp. YIT 12072; however, the
percent similarity was only 95.3% (Table 17). Thus, based on
sequence analysis alone, Sutterella sequences from patient #28a
cannot be classified as Sutterella sp. YIT 12072 or any of the
other known isolates. Despite the closest association of sequences
from patient #28a with the sequence of the isolate Sutterella sp.
YIT 12072, the 16S rRNA gene sequence from patient #28a shared 100%
similarity with 16S rRNA gene sequences from uncultured bacteria in
genbank that were derived from intestinal biopsies from a Crohn's
disease patient (i.e., Accession FJ503635) (D27), human skin
popliteal fossa swab (i.e., Accession HM305996), and feces from a
95-year old woman (i.e., Accession EF401376) (D29). Thus, the 16S
rRNA gene sequences from patient #28a and identical genbank
sequences likely represent an uncharacterized species of
Sutterella.
[0602] Phylogenetic analysis of the predominant sequences obtained
from patient biopsies using the C4-V8 PCR assay revealed high
bootstrap values at most branches and good grouping of members of
the genus Sutterella from other Alcaligenaceae family members and
other Burkholderiales order members (FIG. 42). Thus, sequences
obtained by C4-V8 PCR can be used for accurate species level
classification of Sutterella sequences. This tree demonstrates that
sequences from patients #1, 24a, 3, 10, 11, 12, 27a, and 29a
grouped most closely with S. stercoricanis (supported by a
bootstrap resampling value of 92%); sequences from patients #5, 7,
and 25a grouped most closely with S. wadsworthensis (supported by a
bootstrap resampling value of 99%); and sequences from patient #28a
grouped most closely with the isolate Sutterella sp. YIT 12072
(supported by a bootstrap resampling value of 97%) but formed a
distinct phylogenetic lineage.
[0603] AUT-GI Plasma Antibodies Bind to S. wadsworthensis
Proteins:
[0604] It was also determined whether systemic antibody responses
to Sutterella were present in this cohort. The antigens used for
western blot analysis were whole protein lysates from cultured S.
wadsworthensis containing a wide range of proteins, as observed on
Coommassie-stained SDS-polyacrylamide gels. Individual patient's
plasma was assessed for IgG (FIG. 43A) and IgM (FIG. 43B) antibody
immunoreactivity against the bacterial antigens. Immunoreactive
bands were visible for 11 out of 23 (48%) AUT-GI patients. In ten
AUT-GI children the immunoreactive antibodies were IgG (FIG. 43A);
one (patient #26a) had IgM antibodies (FIG. 43B). In contrast, only
1 of the 9 (11%) Control-GI patients (patient #21) had weak
immunoreactivity to 84-kDa and 41-kDa Sutterella proteins. A total
of 11 distinct immunoreactive protein bands were identified, based
on size (104-, 89-, 84-, 62-, 56-, 50-, 48-, 44-, 41-, 30-, and
27-kDa). AUT-GI patients #1 and #5 (both positive by PCR) had the
most immunoreactive protein bands with four protein bands in common
(89-, 62-, 56-, and 41-kDa). The 89-kDa band was detected by IgG or
IgM antibodies in seven AUT-GI patients. The 56-, 41-, and 30-kDa
bands were detected by IgG antibodies in each of three patients.
The other bands (104-, 84-, 62-, 50-, 48-, and 44-kDa) were less
frequent.
[0605] Of the 12 AUT-GI patients that were PCR-positive for
Sutterella, 8 (66.7%) had plasma IgG antibodies against S.
wadsworthensis proteins (patients #1, 3, 5, 7, 10, 11, 24a, and
25a). Three AUT-GI patients (patients #4, 23a, and 26a) had IgG or
IgM antibodies against S. wadsworthensis proteins, but were
PCR-negative. In total, 15 out of 23 (65.2%) AUT-GI children had
evidence of Sutterella either by PCR or serology (Table 18).
TABLE-US-00031 TABLE 18 Summary of PCR assays and western
immunoblot analysis. MW of Any Ig Any Patient AUT/Control PCR IgG
MW of Bands IgM Bands Positive Positive 1 AUT-GI + ++ 89, 62, 56,
41 - - Yes Yes 2 AUT-GI - - - - - No No 3 AUT-GI + + 30 - - Yes Yes
4 AUT-GI - ++ 89 - - Yes Yes 5 AUT-GI + ++ 89, 62, 56, 48, 44, 41 -
- Yes Yes 6 AUT-GI - - - - - No No 7 AUT-GI + ++ 50, 44 - - Yes Yes
8 AUT-GI - - - - - No No 9 AUT-GI - - - - - No No 10 AUT-GI + ++ 30
- - Yes Yes 11 AUT-GI + + 89, 48 - - Yes Yes 12 AUT-GI + - - - - No
Yes 13 AUT-GI - - - - - No No 14 AUT-GI - - - - - No No 15 AUT-GI -
- - - - No No 23a AUT-GI - ++ 104, 30, 27 - - Yes Yes 24a AUT-GI +
+ 89 - - Yes Yes 25a AUT-GI + ++ 89, 56 - - Yes Yes 26a AUT-GI - -
- ++ 89 Yes Yes 27a AUT-GI + - - - - No Yes 28a AUT-GI + - - - - No
Yes 29a AUT-GI + - - - - No Yes 30a AUT-GI - - - - - No No 16
Control-GI - - - - - No No 17 Control-GI - - - - - No No 18
Control-GI - - - - - No No 19 Control-GI - - - - - No No 20
Control-GI - - - - - No No 21 Control-GI - + 84, 41 - - Yes Yes 22
Control-GI - - - - - No No 31a Control-GI - - - - - No No 32a
Control-GI - - - - - No No % AUT-GI + 52% 43% - 4% - 48% 65% %
Control-GI + 0% 11% - 0% - 11% 11%
[0606] Discussion
[0607] Detection by pyrosequencing of Alcaligenaceae sequences in
AUT-GI children (10) was previously reported. More focused analysis
revealed that this finding reflects the presence of Sutterella
species. Whereas 12 of 23 AUT-GI patients (52%) were PCR-positive
both in ileum and cecum, 0 of 9 Control-GI children were
PCR-positive for Sutterella. Sutterella abundance in the seven
Sutterella-positive AUT-GI patients, assessed by pyrosequencing,
ranged from 1 to 7% of total bacterial sequences. Novel real-time
PCR assays confirmed high copy numbers of Sutterella species in DNA
from ileal and cecal biopsies of Sutterella-positive patients, with
averages ranging from 10.sup.3 to 10.sup.5 Sutterella 16S rRNA gene
copies amplified from only 25 ng of total genomic biopsy DNA.
[0608] OTU analysis of V2-region pyrosequencing reads indicated
that only two OTUs accounted for the majority of Sutterella
sequences in the seven AUT-GI patients that were
Sutterella-positive by pyrosequencing. Sequencing of PCR products
from V6-V8 and C4-V8 Sutterella-specific PCR assays corroborated
this finding. The analysis also indicates that C4-V8 Sutterella
products can be accurately classified at the species level.
Classification with RDP and phylogenetic analysis of Sutterella
sequences obtained from C4-V8 Sutterella-specific PCR indicated
that the predominant sequences obtained from patients #1, 3, 10,
11, 12, 24a, 27a, and 29a were most closely related to the isolate
S. stercoricanis, supported by a sequence similarity of over 98%.
The predominant C4-V8 sequences obtained from patients #5, 7, and
25a were most closely related to the isolate S. wadsworthensis,
supported by a sequence similarity of 100%. The results indicate
that these two species of Sutterella are the dominant phylotypes
present at high levels in the intestines of AUT-GI children in this
cohort. Of the known isolates, the predominant C4-V8 sequence
obtained from patient #28a was most closely related to Sutterella
sp. YIT 12072. However, the low sequence similarity (95.3%) between
sequences from patient #28a and Sutterella sp. YIT 12072 indicates
that these are not likely to be the same species. Sequences from
patient #28a did have 100% sequence similarity with uncultured
Sutterella sequences in genbank, indicating that this undefined
species has been detected previously in human samples using
non-specific techniques.
[0609] Sutterella species have been isolated from human and animal
feces (D30-D32) and have also been isolated from human infections
below the diaphragm; most often from patients with appendicitis,
peritonitis or rectal or perirectal abscesses (D22, D23).
Sutterella sequences have been identified in fecal samples and
intestinal biopsies from individuals with Crohn's disease and
ulcerative colitis but also from apparently healthy adults (D24,
D25, D27, D33). Without being bound by theory, Sutterella species
can contribute to inflammation and infection or are simply normal
inhabitants of the human microbiota in some individuals. Even if
the latter is the case, the results demonstrate that Sutterella is
a major component of the mucoepithelial microbiota in some
children, accounting for up to 7% of all bacteria. Relative to all
other bacterial genera identified in biopsies, Sutterella ranged
from the 3.sup.rd to 8.sup.th most abundant genera in the patients
assessed by pyrosequencing. Only the most abundant Bacteroidete and
Firmicute genera outnumbered Sutterella sequences. This result is
remarkable given that Sutterella is not reported as a major
component of the microbiota (D34).
[0610] Loss of commensals in the intestine can affect immune
responses and disrupt colonization resistance to potentially
pathogenic bacteria (D17, D19). A significant loss of commensals,
namely members of the Bacteroidete phyla, were found in AUT-GI
biopsies (D10). Thus, the loss of Bacteroidetes in AUT-GI children
could facilitate the growth of opportunistic pathogens. Whether
Sutterella is pathogenic in AUT-GI children cannot be determined
from current data. However, the observation that some AUT-GI
children have antibodies that react with S. wadsworthensis proteins
is generally consistent with infection. We detected either IgG or
IgM antibodies against S. wadsworthensis proteins in approximately
48% (11/23) of AUT-GI children. Only one Control-GI child had very
weak IgG immunoreactivity against S. wadsworthensis proteins. Of
the 12 patients that were positive for Sutterella by PCR, 8 (66.7%)
demonstrated plasma IgG antibodies against S. wadsworthensis
proteins. In total, 65.2% (15 out of 23) of AUT-GI children were
either positive by PCR assays or had immunoglobulin reactivity to
S. wadsworthensis proteins. Three AUT-GI patients were negative by
PCR but had IgG or IgM antibodies against S. wadsworthensis
proteins. Without being bound by theory, Sutterella species can
also be present in other regions of the small or large intestine or
elsewhere in the body of these three patients, explaining the
presence of Sutterella-specific antibodies without detection of the
agent by PCR. Alternatively, IgG antibodies can persist long after
antigenic exposure; thus, the presence of IgG antibodies can
indicate past exposure in some children. The IgM immunoreactivity
of patient #26a indicates recent or current exposure to Sutterella
antigen in this patient. It is well recognized that the use of
different strains and species as antigen leads to variations in the
immunoreactive profile of immunogenic proteins (D35). Several
Sutterella-positive patients in this study had S. stercoricanis as
the dominant Sutterella species.
[0611] The nature of intestinal damage in autism has not been fully
defined. Abnormalities in intestinal permeability in children with
autism have been reported in two studies (D8, D9). In Crohn's
disease, a condition associated with increased intestinal
permeability, a generalized enhancement of antimicrobial IgG to
many members of the intestinal microbiota is reported (D36). A
defective epithelial barrier could lead to enhanced contact between
many members of the microbiota and antigen-presenting cells in the
lamina propria. If this turns out to be the case in autism, then
antibodies against Sutterella proteins can reflect
inter-individual, compositional variation in the microbiota, rather
than an indication of Sutterella infection.
[0612] In conclusion, Sutterella 16S rRNA gene sequences were
identified in mucoepithelial biopsies from AUT-GI children using
non-specific, pan-microbial pyrosequencing. New Sutterella-specific
PCR assays were designed and applied that confirmed high levels of
Sutterella species in over half of AUT-GI children and the complete
absence of Sutterella in Control-GI children tested in this study.
The Sutterella-specific molecular assays reported in this study
will enable more directed studies to detect, quantify, and classify
this poorly understood bacterium in biological and environmental
samples. With such specific techniques, the following can be
understood: the epidemiology of this bacterium and its associations
with human infections and inflammatory diseases; the role
Sutterella plays in the microbiota, and the extent to which
Sutterella can contribute to the pathogenesis of GI disturbances in
children with autism.
[0613] Materials and Methods
[0614] Clinical Samples:
[0615] Clinical procedures for this study population are previously
described (D10, D37). The Institutional Review Board (IRB) at
Columbia University Medical Center reviewed and approved the use of
de-identified residual ileal and cecal samples, obtained as
described in an earlier publication (D37), and waived the need for
patient consent for these analyses, as all samples were analyzed
anonymously. Patients assessed by pyrosequencing were restricted to
male children between 3 to 5 years of age to control for
confounding effects of gender and age on the microbiota (D10). This
subset comprised 15 AUT-GI (patients #1-15) and 7 Control-GI
(patients #16-22) children. For assessment of Sutterella sequences
in ileal and cecal biopsies, we also included 8 additional male
AUT-GI children (patients #23a-30a: 6 children between 6 and 7
years of age, and 2 children between 8 and 10 years of age) and 2
additional male Control-GI children (patients #31 a and 32a: 1
child between 6 and 7 years of age and 1 child between 8 and 10
years of age) from the initial cohort (D37).
[0616] Bacterial Culture:
[0617] S. wadsworthensis was obtained from American Type Culture
Collection (ATCC, #51579). The isolate was grown in chopped meat
broth in Hungate capped tubes (Anaerobe Systems, Morgan Hill,
Calif.), supplemented with sodium formate and fumaric acid at a
final concentration of 0.3% each. Inoculated cultures were
incubated at 37.degree. C. and growth was monitored at 0, 6, 12,
24, and 48 hours using a Sutterella-specific real-time PCR assay
(see below).
[0618] DNA Extraction:
[0619] DNA was extracted from individual ileal and cecal biopsies
(total of 256 biopsies: 128 ileal biopsies and 128 cecal biopsies;
8 biopsies per patient [4 from ileum and 4 from cecum]; 23 AUT-GI
patients and 9 Control-GI patients) and bacterial cultures of S.
wadsworthensis in TRIzol (Invitrogen, Carlsbad, Calif.) using
standard protocols. DNA concentrations and integrity were
determined using a Nanodrop ND-1000 Spectrophotometer (Nanodrop
Technologies, Wilmington, Del.) and Bioanalyzer (Agilent
Technologies, Foster City, Calif.) and stored at -80.degree. C.
[0620] Pyrosequencing:
[0621] Barcoded pyrosequencing of the bacterial V2 region of the
16S rRNA gene and analyses are previously described for ileal and
cecal biopsies from AUT-GI patients #1-15 and Control-GI patients
#16-22 (D10). The pan-bacterial barcoded V2 primers, designated
V2For and V2Rev, amplify a region of the 16S rRNA gene from
nucleotide position 27 to 338 (D38) (FIG. 38).
[0622] Sutterella-Specific PCR Assay Design:
[0623] Sutterella-specific 16S rRNA PCR primers were designed
against the 16S rRNA gene sequence for S. wadsworthensis (Accession
L37785) using Primer Express 1.0 software (Applied Biosystems,
Foster City, Calif.). Genus specificity of candidate primers was
evaluated using the RDP (Ribosomal Database Project) probe match
tool. Several potential primer pairs were identified but only one
pair showed high specificity for Sutterella. These primers are
designated here as SuttFor (nucleotide position 936-956 of S.
wadsworthensis: Accession L37785) and SuttRev (nucleotide position
1177-1195 of S. wadsworthensis: Accession L37785) [Table 20].
SuttFor and SuttRev primers amplify a 260 base pair (bp) region
between variable regions 6, 7 and 8 (V6-V8) of the 16S rRNA gene of
Sutterella (FIG. 38).
TABLE-US-00032 TABLE 20 Primers and probes used for conventional
PCR or real-time PCR amplification and quantitation of Sutterella
species. Nucleotide Amplicon SEQ Name Primers and Probe (5'-3'
Position* size (bp) ID NO: Sutterella (V6-V8) SuttFor:
CGCGAAAAACCTTACCTAGCC 936-956 ~260 11 SuttRev: GACGTGTGAGGCCCTAGCC
1177-1195 12 SuttProbe: FAM-CACAGGTGCTGCATGGCTGTCGT-NFQ 1011-1033
13 Sutterella (C4-V8) 515For: GTGCCAGCMGCCGCGGTAA 482-500 ~715 66
SuttRev: GACGTGTGAGGCCCTAGCC 1177-1195 66 Total Bacteria 515For:
GTGCCAGCMGCCGCGGTAA 482-500 ~292 15 805Rev: GACTACCAGGGTATCTAATT
754-772 16 *Nucleotide position relative to the 16S rRNA gene of
Suftterellia wadworthensis (Accession #L37785)
[0624] Conventional PCR Assays:
[0625] Conventional PCR for detection of Sutterella was carried out
in 25 .mu.l reactions consisting of 25 ng of biopsy DNA or 25 .mu.g
of genomic DNA from cultured S. wadsworthensis (ATCC, #51579:
positive control), 300 nm each SuttFor and SuttRev primers (for
V6-V8 amplification) or 515For and SuttRev (for C4-V8
amplification), 2 .mu.l dNTP Mix (10 mM; Applied Biosystems, Foster
City, Calif.), 2.5 .mu.l of 10.times.PCR Buffer (Qiagen, Valencia,
Calif.), 5U of HotStarTaq DNA polymerase (Qiagen), and 5 .mu.l
Q-solution (Qiagen). Cycling parameters consisted of an initial
denaturation step at 95.degree. C. for 15 min, followed by 30
cycles of 94.degree. C. for 1 min, 60.degree. C. for 1 min,
72.degree. C. for 1 min, and a final extension at 72.degree. C. for
5 min. The amplified product was detected by electrophoresis on a
1.5% agarose gel stained with ethidium bromide. To confirm
specificity of PCR amplification, V6-V8 products were gel-extracted
and sent for direct sequencing with SuttFor and SuttRev primers.
Additionally, V6-V8 and C4-V8 products were subcloned into the
vector PGEM-T easy (Promega, Madison, Wis.) and bacterial libraries
were created. One hundred and twenty V6-V8 plasmid clones were
sequenced. A total of 480 C4-V8 colonies were sequenced and
analyzed (40 sequences from each of the 12 PCR-positive patients;
20 sequences from ileal and 20 sequences from cecal biopsies). All
V6-V8 and C4-V8 plasmid clones were found to contain Sutterella
sequences using the RDP classifier tool with a minimum 80%
bootstrap confidence estimate. The closest sequence match to
Sutterella isolates was determined using the RDP seqmatch tool.
Sequences from each individual patient were aligned using
MacVector, and a consensus sequence was determined from the
predominant Sutterella species in each patient.
[0626] Quantitative Real-Time PCR Assay:
[0627] PCR standards for determining copy numbers of bacterial 16S
rRNA genes were prepared from products of the partial 16S rRNA gene
(V6-V8 region) of S. wadsworthensis (Accession GU585669). A
representative product with high sequence similarity to Bacteroides
intestinalis (Accession NZ_ABM02000007) 16S rRNA gene was used with
broadly conserved total bacteria primers (D10, D39). Products were
cloned into the vector PGEM-T easy (Promega) and ten-fold serial
dilutions of linearized plasmid standards were created ranging from
5.times.10.sup.5 to 5.times.10.degree. copies. Amplification and
detection of DNA by real-time PCR were performed with the ABI
StepOne Plus Real-time PCR system (Applied Biosystems). Linearity
and sensitivity of plasmid standards were tested with SuttFor and
SuttRev primers and the SuttProbe. Amplification plots of plasmid
standards indicated sensitivity of detection down to 5 copies of
plasmid (FIG. 10A), and standard curves generated from plasmid
dilutions had correlation coefficients of 0.996 (FIG. 10B).
[0628] Bioinformatics Analysis:
[0629] Operational taxonomic unit (OTU)-based analysis of
pyrosequencing data was carried out in MOTHUR (version 1.8.0) and
as previously described (D10, D40).
[0630] Phylogenetic Analysis of Sutterella Sequences:
[0631] Phylogenetic analyses were conducted in MEGA4 (D41).
Sequence alignments were based on representative sequences from OTU
1 and OTU 2, obtained from pyrosequencing analysis of the V2 region
of the 16S rRNA gene, as well as sequences of Sutterella from the
V6-V8 (SuttFor and Sutt Rev amplifications) conventional PCR assay,
and the predominant sequences obtained from clone libraries of the
C4-V8 (515For and SuttRev amplifications) conventional PCR assay.
Primer sequences were trimmed from the sequences. Classification
was confirmed using the RDP classifier and seqmatch tools.
Sutterella sequences obtained from ileal and cecal biopsies were
aligned with sequences from 8 isolates of Sutterella found in the
RDP database and sequences from 14 additional related species
(members of the family Alcaligenaceae and order Burkholderiales).
Sequences from Sutterella isolates and related species were trimmed
to the length of the sequences obtained from ileal and cecal
biopsies of AUT-GI patients. Phylogenetic trees were constructed
according to the neighbour joining method with evolutionary
distances determined using the Jukes-Cantor method (D42, D43).
Trees were rooted to the outgroup Escherichia coli (Accession
X80725). The stability of the groupings was estimated by bootstrap
analysis (1000 replications) using MEGA4. The percentages of 16S
rRNA gene sequence similarity were determined for Sutterella C4-V8
products and Sutterella isolates using the EzTaxon server 2.1
(www.eztaxon.org/) (D44).
[0632] Western Immunoblots:
[0633] Soluble proteins of cultured S. wadsworthensis (ATCC,
#51579) were extracted and used as antigen in immunoblot assays. S.
wadsworthensis antigens were separated by SDS-PAGE and transferred
to nitrocellulose membranes. Membranes were blocked, incubated with
each patent's plasma (diluted 1:100 in blocking solution), probed
with secondary antibodies [either peroxidase-conjugated goat
anti-human IgG (Fc.sub..gamma. fragment-specific; Jackson
ImmunoResearch, West Grove, Pa.) or peroxidase-conjugated goat
anti-human IgM (Fc.sub.5.mu. fragment-specific; Jackson
ImmunoResearch)], and developed with ECL Plus Western blot
detection system (Amersham Biosciences, Arlington Heights,
Ill.).
[0634] Supplemental Materials and Methods
[0635] Pyrosequencing:
[0636] 16S rRNA genes were amplified using V2-region specific,
barcoded primers (E1) and products were sequenced at 454 Life
Sciences on a GS FLX sequencer as previously described (E2). A
total of 525,519 16S rRNA gene (V2 region) sequencing reads
remained after filtering based on read length, removing low-quality
sequences and sequences with ambiguous characters, and combining
duplicate pyrosequencing runs (271,043 reads for ilea; 254,476
reads for ceca). Binning of sequences by barcode revealed similar
numbers of 16S rRNA gene sequence reads per patient (average #
sequences per patient+/-standard deviation [SD], ilea:
12,320+/-1220; ceca: 11,567+/-1589) (see Table 19). Taxonomic
classifications of bacterial 16S rRNA gene sequences were obtained
using the Ribosomal Database Project (RDP), Release 10, classifier
tool (http://rdp.cme.msu.edu/) with a minimum 80% bootstrap
confidence estimate. To normalize data for differences in the total
number of sequences obtained per patient, the abundance of
sequences corresponding to members of the genus Sutterella and all
other genera were expressed as a percentage of total bacterial
sequence reads. The abundance of Sutterella was also expressed as a
percentage of total class Betaproteobacteria sequence reads per
patient (see Table 19).
[0637] Operational Taxonomic Unit (OTU) Analysis:
[0638] For OTU analysis of Sutterella sequences, genus level
classification from RDP was used to subselect all Sutterella
sequences. Sutterella sequences generated from 454 pyrosequencing
were aligned to the greengenes reference alignment using the
Needleman-Wunsch algorithm with the "align.seqs" function
(ksize=9). Pairwise genetic distances among the aligned sequences
were calculated using the "dist.seqs" function (calc=onegap,
countends=T). Sequences were assigned to OTUs (defined at 97%
sequence similarity) using average neighbor clustering.
Representative sequences (the sequence which is the minimum
distance to all other sequences in an OTU) from OTU 1 and OTU 2
were obtained using the get.oturep command in MOTHUR. OTU abundance
by patient was expressed as percent relative abundance, determined
by dividing the number of reads for an OTU in a given patient by
the total number of bacterial reads obtained by pyrosequencing for
that patient. Heatmaps were constructed using MeV (Version 4.5.0)
using OTU abundance data from pyrosequencing reads. Heatmaps were
drawn using Pearson's correlation as the similarity metric and
complete linkage clustering. The upper limit approximately reflects
the highest abundance recorded for any taxa in the heatmap (6%;
red), and the lower limit reflects sequences above 0% abundance
(0%; green); the midpoint limit (1%; white) is adjusted to
highlight salient differences between the AUT-GI and Control-GI
groups. Gray cells in the heatmaps represent instances wherein no
sequences were detected for a given taxa in a given patient.
[0639] Sutterella-Specific PCR Primers and Probe
Bioinformatics:
[0640] Evaluation of good quality sequences greater than or equal
to 1200 nucleotides in length revealed a total of 724 Sutterella
sequences in the RDP database at the time of most recent analysis
(RDP Release 10, Update 27: Aug. 9, 2011). SuttFor and SuttRev
primers showed high exclusivity for the genus Sutterella.
Approximately 90% (692/768 bacterial 16S sequence matches) of all
SuttFor matches and 98% (674/688 bacterial 16S sequence matches) of
all SuttRev matches were specific to the genus Sutterella. The
SuttFor primer sequence matched exactly with approximately 96%
(692/724 Sutterella 16S sequences) of all Sutterella sequences,
while the SuttRev primer matched exactly with approximately 93%
(674/724 Sutterella 16S sequences) of all Sutterella sequences in
the RDP database. The SuttProbe (nucleotide position 1011-1033 of
S. wadsworthensis: Accession L37785) (Table 20) used for real-time
PCR had low exclusivity but high coverage of Sutterella sequences
(99%). The SuttProbe was labeled with the reporter FAM
(6-carboxyfluorescein) and the nonfluorescent quencher BBQ
(Blackberry) (TIB MolBiol, Berlin, Germany).
[0641] Sutterella V6-V8 PCR Sensitivity, Linearity, and End-Point
Detection:
[0642] To determine V6-V8 assay sensitivity, Sutterella plasmid
standards (see quantitative real-time PCR methods) were tested by
conventional PCR using the same conditions as for the biopsy DNA.
Ten-fold dilutions of the Sutterella clone ranging from
5.times.10.sup.5 to 5.times.10.degree. were spiked into ileal DNA
(25 ng) from a Sutterella-negative patient. We previously
demonstrated that the ileal DNA from this Sutterella-negative
patient contains 16S rRNA genes from a broad range of bacterial
phylotypes dominated by Bacteroidetes, Firmicutes and
Proteobacteria, but does not contain any Sutterella 16S rRNA
sequences (2). The conventional V6-V8 PCR was linear in the range
of 5.times.10.sup.5 to 5.times.10.sup.2 copies and had an end-point
detection limit of 5.times.10.sup.1 copies in the presence of
background ileal DNA (FIG. 9).
[0643] Quantitative Real-time PCR Assay Details: For
Sutterella-specific real-time PCR on biopsy material, each 25 .mu.l
reaction contained 25 ng biopsy DNA, 12.5 .mu.l Taqman universal
master mix (ABI), 300 nm each of SuttFor and SuttRev primers, and
200 nm SuttProbe. The cycling protocol for Sutterella amplification
consisted of denaturation at 95.degree. C. (10 min) followed by 45
cycles of 95.degree. C. (15 sec) and 60.degree. C. (1 min). For
total bacteria real-time PCR, each 25 .mu.l of amplification
reaction mixture contained 25 ng DNA, 12.5 .mu.l SYBR Green Master
Mix (Applied Biosystems), and 300 nM each of the pan-bacterial
primers (515For and 805Rev: Table 20). The cycling protocol for
total bacteria consisted of denaturation at 95.degree. C. (10 min)
followed by 45 cycles of 95.degree. C. (15 sec), 56.degree. C. (15
sec), and 60.degree. C. (1 min). DNA from each of 128 ileal (4
biopsies per patient) and 128 cecal biopsies (4 biopsies per
patient) was assayed in duplicate. The final results were expressed
as the mean number of Sutterella 16S rRNA gene copies normalized to
the average 16S rRNA gene copies obtained using total bacterial
primers. Eight water/reagent controls were included for all
amplifications and the average copy number for water/reagent
controls (background) was subtracted from each ileal and cecal
amplification prior to normalization.
[0644] Western Immunoblots (Detailed Protocol):
[0645] Anaerobic cultures of S. wadsworthensis (ATCC, #51579) were
pelleted by centrifugation at 5000.times.g for 10 minutes and
stored at -80.degree. C. Protein lysates were prepared from S.
wadsworthensis bacterial pellets using B-PER Solution (Thermo
Scientific, Rockford, Ill.) supplemented with DNase I (2 .mu.l/ml
B-PER), lysozyme (2 .mu.l/ml B-PER), and protease inhibitor
cocktail and incubated for 10 minutes at room temperature. The
lysate was centrifuged at 15,000.times.g for 5 minutes to remove
insoluble proteins. The protein concentration of the soluble
fraction was determined using the BCA protein assay kit (Pierce
Biotechnology; Rockford, Ill.). Protein lysates (200 .mu.g) in
sample buffer (10 mM Tris-Hcl, pH 7.5; 10 mM EDTA, 20% v/v
glycerol; 1% w/v SDS; 0.005% w/v bromophenol blue; 100 mM
dithiothreitol; 1% v/v beta-mercaptoethanol) were boiled for 5 min
and size-fractionated by 10% SDS-PAGE using a single large well on
each gel to achieve uniform separation of proteins. Proteins were
transferred to nitrocellulose membranes using the iBlot Gel
Transfer System (Invitrogen). Membranes were blocked in 5% nonfat
milk powder in TTBS (20 mM Tris-Hcl, pH 7.6; 137 mM NaCl; 0.3%
Tween 20) for 1 hour at room temperature. Blocked membranes were
transferred to a Mini-Protean II MultiScreen apparatus (BioRad,
Hercules, Calif.). Plasma from each individual patient was diluted
1:100 in blocking solution (650 .mu.l) and loaded onto the membrane
in the individual chambers of the Mini-Protean II MultiScreen
apparatus and incubated overnight at 4.degree. C. Membranes were
then removed from the apparatus and washed three times with TTBS
for 10 minutes each wash. Secondary antibodies, either
peroxidase-conjugated goat anti-human IgG (Fc.sub..gamma.
fragment-specific; Jackson ImmunoResearch, West Grove, Pa.) or
peroxidase-conjugated goat anti-human IgM (Fc.sub.5.mu.
fragment-specific; Jackson ImmunoResearch) were diluted 1:50,000 in
blocking solution and incubated with the membranes for one hour at
room temperature, followed by three washes with TTBS for 10 minutes
each wash. Membranes were developed using ECL Plus Western blot
detection system (Amersham Biosciences, Arlington Heights, Ill.)
and scanned for chemiluminescence using a Typhoon Trio imager (GE
Healthcare Life Sciences, Piscataway, N.J.). Western blots were
performed three times to confirm reproducibility of results.
Secondary antibody alone controls were included for all immunoblots
to control for nonspecific binding. Background adjustments using
ImageQuant (Molecular Dynamics) were applied equally to all
immunoblots.
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Sequence CWU 1
1
661524PRTHomo sapiens 1Met Thr Glu Asp Lys Val Thr Gly Thr Leu Val
Phe Thr Val Ile Thr 1 5 10 15 Ala Val Leu Gly Ser Phe Gln Phe Gly
Tyr Asp Ile Gly Val Ile Asn 20 25 30 Ala Pro Gln Gln Val Ile Ile
Ser His Tyr Arg His Val Leu Gly Val 35 40 45 Pro Leu Asp Asp Arg
Lys Ala Ile Asn Asn Tyr Val Ile Asn Ser Thr 50 55 60 Asp Glu Leu
Pro Thr Ile Ser Tyr Ser Met Asn Pro Lys Pro Thr Pro 65 70 75 80 Trp
Ala Glu Glu Glu Thr Val Ala Ala Ala Gln Leu Ile Thr Met Leu 85 90
95 Trp Ser Leu Ser Val Ser Ser Phe Ala Val Gly Gly Met Thr Ala Ser
100 105 110 Phe Phe Gly Gly Trp Leu Gly Asp Thr Leu Gly Arg Ile Lys
Ala Met 115 120 125 Leu Val Ala Asn Ile Leu Ser Leu Val Gly Ala Leu
Leu Met Gly Phe 130 135 140 Ser Lys Leu Gly Pro Ser His Ile Leu Ile
Ile Ala Gly Arg Ser Ile 145 150 155 160 Ser Gly Leu Tyr Cys Gly Leu
Ile Ser Gly Leu Val Pro Met Tyr Ile 165 170 175 Gly Glu Ile Ala Pro
Thr Ala Leu Arg Gly Ala Leu Gly Thr Phe His 180 185 190 Gln Leu Ala
Ile Val Thr Gly Ile Leu Ile Ser Gln Ile Ile Gly Leu 195 200 205 Glu
Phe Ile Leu Gly Asn Tyr Asp Leu Trp His Ile Leu Leu Gly Leu 210 215
220 Ser Gly Val Arg Ala Ile Leu Gln Ser Leu Leu Leu Phe Phe Cys Pro
225 230 235 240 Glu Ser Pro Arg Tyr Leu Tyr Ile Lys Leu Asp Glu Glu
Val Lys Ala 245 250 255 Lys Gln Ser Leu Lys Arg Leu Arg Gly Tyr Asp
Asp Val Thr Lys Asp 260 265 270 Ile Asn Glu Met Arg Lys Glu Arg Glu
Glu Ala Ser Ser Glu Gln Lys 275 280 285 Val Ser Ile Ile Gln Leu Phe
Thr Asn Ser Ser Tyr Arg Gln Pro Ile 290 295 300 Leu Val Ala Leu Met
Leu His Val Ala Gln Gln Phe Ser Gly Ile Asn 305 310 315 320 Gly Ile
Phe Tyr Tyr Ser Thr Ser Ile Phe Gln Thr Ala Gly Ile Ser 325 330 335
Lys Pro Val Tyr Ala Thr Ile Gly Val Gly Ala Val Asn Met Val Phe 340
345 350 Thr Ala Val Ser Val Phe Leu Val Glu Lys Ala Gly Arg Arg Ser
Leu 355 360 365 Phe Leu Ile Gly Met Ser Gly Met Phe Val Cys Ala Ile
Phe Met Ser 370 375 380 Val Gly Leu Val Leu Leu Asn Lys Phe Ser Trp
Met Ser Tyr Val Ser 385 390 395 400 Met Ile Ala Ile Phe Leu Phe Val
Ser Phe Phe Glu Ile Gly Pro Gly 405 410 415 Pro Ile Pro Trp Phe Met
Val Ala Glu Phe Phe Ser Gln Gly Pro Arg 420 425 430 Pro Ala Ala Leu
Ala Ile Ala Ala Phe Ser Asn Trp Thr Cys Asn Phe 435 440 445 Ile Val
Ala Leu Cys Phe Gln Tyr Ile Ala Asp Phe Cys Gly Pro Tyr 450 455 460
Val Phe Phe Leu Phe Ala Gly Val Leu Leu Ala Phe Thr Leu Phe Thr 465
470 475 480 Phe Phe Lys Val Pro Glu Thr Lys Gly Lys Ser Phe Glu Glu
Ile Ala 485 490 495 Ala Glu Phe Gln Lys Lys Ser Gly Ser Ala His Arg
Pro Lys Ala Ala 500 505 510 Val Glu Met Lys Phe Leu Gly Ala Thr Glu
Thr Val 515 520 2 3439DNAHomo sapiens 2tctggtttgt aacttatgcc
taagggacct gctcccattt tctttcctag tggaacaaag 60gtattgaagc cacaggttgc
tgaggcaaag cacttattga ttagattccc atcaatattc 120agctgccgct
gagaagatta gacttggact ctcaggtctg ggtagcccaa ctcctccctc
180tccttgctcc tcctcctgca atgcataact aggcctaggc agagctgcga
ataaacaggc 240aggagctagt caggtgcatg tgccacactc acacaagacc
tggaattgac aggactccca 300actagtacaa tgacagaaga taaggtcact
gggaccctgg ttttcactgt catcactgct 360gtgctgggtt ccttccagtt
tggatatgac attggtgtga tcaatgcacc tcaacaggta 420ataatatctc
actatagaca tgttttgggt gttccactgg atgaccgaaa agctatcaac
480aactatgtta tcaacagtac agatgaactg cccacaatct catactcaat
gaacccaaaa 540ccaacccctt gggctgagga agagactgtg gcagctgctc
aactaatcac catgctctgg 600tccctgtctg tatccagctt tgcagttggt
ggaatgactg catcattctt tggtgggtgg 660cttggggaca cacttggaag
aatcaaagcc atgttagtag caaacattct gtcattagtt 720ggagctctct
tgatggggtt ttcaaaattg ggaccatctc atatacttat aattgctgga
780agaagcatat caggactata ttgtgggcta atttcaggcc tggttcctat
gtatatcggt 840gaaattgctc caaccgctct caggggagca cttggcactt
ttcatcagct ggccatcgtc 900acgggcattc ttattagtca gattattggt
cttgaattta tcttgggcaa ttatgatctg 960tggcacatcc tgcttggcct
gtctggtgtg cgagccatcc ttcagtctct gctactcttt 1020ttctgtccag
aaagccccag atacctttac atcaagttag atgaggaagt caaagcaaaa
1080caaagcttga aaagactcag aggatatgat gatgtcacca aagatattaa
tgaaatgaga 1140aaagaaagag aagaagcatc gagtgagcag aaagtctcta
taattcagct cttcaccaat 1200tccagctacc gacagcctat tctagtggca
ctgatgctgc atgtggctca gcaattttcc 1260ggaatcaatg gcatttttta
ctactcaacc agcatttttc agacggctgg tatcagcaaa 1320cctgtttatg
caaccattgg agttggcgct gtaaacatgg ttttcactgc tgtctctgta
1380ttccttgtgg agaaggcagg gcgacgttct ctctttctaa ttggaatgag
tgggatgttt 1440gtttgtgcca tcttcatgtc agtgggactt gtgctgctga
ataagttctc ttggatgagt 1500tatgtgagca tgatagccat cttcctcttt
gtcagcttct ttgaaattgg gccaggcccg 1560atcccctggt tcatggtggc
tgagtttttc agtcaaggac cacgtcctgc tgctttagca 1620atagctgcat
tcagcaattg gacctgcaat ttcattgtag ctctgtgttt ccagtacatt
1680gcggacttct gtggacctta tgtgtttttc ctctttgctg gagtgctcct
ggcctttacc 1740ctgttcacat tttttaaagt tccagaaacc aaaggaaagt
cttttgagga aattgctgca 1800gaattccaaa agaagagtgg ctcagcccac
aggccaaaag ctgctgtaga aatgaaattc 1860ctaggagcta cagagactgt
gtaaaaaaaa aaccctgctt tttgacatga acagaaacaa 1920taagggaacc
gtctgttttt aaatgatgat tccttgagca ttttatatcc acatctttaa
1980gtattgtttt atttttatgt gctctcatca gaaatgtcat caaatattac
caaaaaagta 2040tttttttaag ttagagaata tatttttgat ggtaagactg
taattaagta aaccaaaaag 2100gctagtttat tttgttacac taaagggcag
gtggttctaa tatttttagc tctgttcttt 2160ataacaaggt tcttctaaaa
ttgaagagat ttcaacatat cattttttta acacataact 2220agaaacctga
ggatgcaaca aatatttata tatttgaata tcattaaatt ggaattttct
2280tacccatata tcttatgtta aaggagatat ggctagtggc aataagttcc
atgttaaaat 2340agacaactct tccatttatt gcactcagct tttttcttga
gtactagaat ttgtattttg 2400cttaaaattt tacttttgtt ctgtattttc
atgtggaatg gattatagag tatactaaaa 2460aatgtctata gagaaaaact
ttcatttttg gtaggcttat caaaatcttt cagcactcag 2520aaaagaaaac
cattttagtt cctttattta atggccaaat ggtttttgca agatttaaca
2580ctaaaaaggt ttcacctgat catatagcgt gggttatcag ttaacattaa
catctattat 2640aaaaccatgt tgattccctt ctggtacaat cctttgagtt
atagtttgct ttgcttttta 2700attgaggaca gcctggtttt cacatacact
caaacaatca tgagtcagac atttggtata 2760ttacctcaaa ttcctaataa
gtttgatcaa atctaatgta agaaaatttg aagtaaagga 2820ttgatcactt
tgttaaaaat attttctgaa ttattatgtc tcaaaataag ttgaaaaggt
2880agggtttgag gattcctgag tgtgggcttc tgaaacttca taaatgttca
gcttcagact 2940tttatcaaaa tccctattta attttcctgg aaagactgat
tgttttatgg tgtgttccta 3000acataaaata atcgtctcct ttgacatttc
cttctttgtc ttagctgtat acagattcta 3060gccaaactat tctatggcca
ttactaacac gcattgtaca ctatctatct gcctttacct 3120acataggcaa
attggaaata cacagatgat taaacagact ttagcttaca gtcaatttta
3180caattatgga aatatagttc tgatgggtcc caaaagctta gcagggtgct
aacgtatctc 3240taggctgttt tctccaccaa ctggagcact gatcaatcct
tcttatgttt gctttaatgt 3300gtattgaaga aaagcacttt ttaaaaagta
ctctttaaga gtgaaataat taaaaaccac 3360tgaacatttg ctttgttttc
taaagttgtt cacatatatg taatttagca gtccaaagaa 3420caagaaattg
tttcttttc 34393664PRTHomo sapiens 3Met Asp Ser Ser Thr Trp Ser Pro
Lys Thr Thr Ala Val Thr Arg Pro 1 5 10 15 Val Glu Thr His Glu Leu
Ile Arg Asn Ala Ala Asp Ile Ser Ile Ile 20 25 30 Val Ile Tyr Phe
Val Val Val Met Ala Val Gly Leu Trp Ala Met Phe 35 40 45 Ser Thr
Asn Arg Gly Thr Val Gly Gly Phe Phe Leu Ala Gly Arg Ser 50 55 60
Met Val Trp Trp Pro Ile Gly Ala Ser Leu Phe Ala Ser Asn Ile Gly 65
70 75 80 Ser Gly His Phe Val Gly Leu Ala Gly Thr Gly Ala Ala Ser
Gly Ile 85 90 95 Ala Ile Gly Gly Phe Glu Trp Asn Ala Leu Val Leu
Val Val Val Leu 100 105 110 Gly Trp Leu Phe Val Pro Ile Tyr Ile Lys
Ala Gly Val Val Thr Met 115 120 125 Pro Glu Tyr Leu Arg Lys Arg Phe
Gly Gly Gln Arg Ile Gln Val Tyr 130 135 140 Leu Ser Leu Leu Ser Leu
Leu Leu Tyr Ile Phe Thr Lys Ile Ser Ala 145 150 155 160 Asp Ile Phe
Ser Gly Ala Ile Phe Ile Asn Leu Ala Leu Gly Leu Asn 165 170 175 Leu
Tyr Leu Ala Ile Phe Leu Leu Leu Ala Ile Thr Ala Leu Tyr Thr 180 185
190 Ile Thr Gly Gly Leu Ala Ala Val Ile Tyr Thr Asp Thr Leu Gln Thr
195 200 205 Val Ile Met Leu Val Gly Ser Leu Ile Leu Thr Gly Phe Ala
Phe His 210 215 220 Glu Val Gly Gly Tyr Asp Ala Phe Met Glu Lys Tyr
Met Lys Ala Ile 225 230 235 240 Pro Thr Ile Val Ser Asp Gly Asn Thr
Thr Phe Gln Glu Lys Cys Tyr 245 250 255 Thr Pro Arg Ala Asp Ser Phe
His Ile Phe Arg Asp Pro Leu Thr Gly 260 265 270 Asp Leu Pro Trp Pro
Gly Phe Ile Phe Gly Met Ser Ile Leu Thr Leu 275 280 285 Trp Tyr Trp
Cys Thr Asp Gln Val Ile Val Gln Arg Cys Leu Ser Ala 290 295 300 Lys
Asn Met Ser His Val Lys Gly Gly Cys Ile Leu Cys Gly Tyr Leu 305 310
315 320 Lys Leu Met Pro Met Phe Ile Met Val Met Pro Gly Met Ile Ser
Arg 325 330 335 Ile Leu Tyr Thr Glu Lys Ile Ala Cys Val Val Pro Ser
Glu Cys Glu 340 345 350 Lys Tyr Cys Gly Thr Lys Val Gly Cys Thr Asn
Ile Ala Tyr Pro Thr 355 360 365 Leu Val Val Glu Leu Met Pro Asn Gly
Leu Arg Gly Leu Met Leu Ser 370 375 380 Val Met Leu Ala Ser Leu Met
Ser Ser Leu Thr Ser Ile Phe Asn Ser 385 390 395 400 Ala Ser Thr Leu
Phe Thr Met Asp Ile Tyr Ala Lys Val Arg Lys Arg 405 410 415 Ala Ser
Glu Lys Glu Leu Met Ile Ala Gly Arg Leu Phe Ile Leu Val 420 425 430
Leu Ile Gly Ile Ser Ile Ala Trp Val Pro Ile Val Gln Ser Ala Gln 435
440 445 Ser Gly Gln Leu Phe Asp Tyr Ile Gln Ser Ile Thr Ser Tyr Leu
Gly 450 455 460 Pro Pro Ile Ala Ala Val Phe Leu Leu Ala Ile Phe Trp
Lys Arg Val 465 470 475 480 Asn Glu Pro Gly Ala Phe Trp Gly Leu Ile
Leu Gly Leu Leu Ile Gly 485 490 495 Ile Ser Arg Met Ile Thr Glu Phe
Ala Tyr Gly Thr Gly Ser Cys Met 500 505 510 Glu Pro Ser Asn Cys Pro
Thr Ile Ile Cys Gly Val His Tyr Leu Tyr 515 520 525 Phe Ala Ile Ile
Leu Phe Ala Ile Ser Phe Ile Thr Ile Val Val Ile 530 535 540 Ser Leu
Leu Thr Lys Pro Ile Pro Asp Val His Leu Tyr Arg Leu Cys 545 550 555
560 Trp Ser Leu Arg Asn Ser Lys Glu Glu Arg Ile Asp Leu Asp Ala Glu
565 570 575 Glu Glu Asn Ile Gln Glu Gly Pro Lys Glu Thr Ile Glu Ile
Glu Thr 580 585 590 Gln Val Pro Glu Lys Lys Lys Gly Ile Phe Arg Arg
Ala Tyr Asp Leu 595 600 605 Phe Cys Gly Leu Glu Gln His Gly Ala Pro
Lys Met Thr Glu Glu Glu 610 615 620 Glu Lys Ala Met Lys Met Lys Met
Thr Asp Thr Ser Glu Lys Pro Leu 625 630 635 640 Trp Arg Thr Val Leu
Asn Val Asn Gly Ile Ile Leu Val Thr Val Ala 645 650 655 Val Phe Cys
His Ala Tyr Phe Ala 660 4 5061DNAHomo sapiens 4ccccattcgc
aggacagctc ttacctgccg ggccgccgcc ccagccaaca gctcagccgg 60gtgctccttc
ctgggctcca cgcccggagc tgcttcctga cggtgcagcc gcaaggcatc
120gcaggggccc cgcgctactg ccctgctccc tcaaagtccc aggtcccctc
ccctggtgct 180gatcattaac caggaggccg tataaggagc tagcggccct
ggcgagaggg aaggacgcaa 240cgctgccacc atggacagta gcacctggag
ccccaagacc accgcggtca cccggcctgt 300tgagacccac gagctcattc
gcaatgcagc cgatatctcc atcatcgtta tctacttcgt 360ggtagtgatg
gccgtcggac tgtgggctat gttttccacc aatcgtggga ctgttggagg
420cttcttcctg gcaggccgaa gtatggtgtg gtggccgatt ggagcctccc
tctttgctag 480taacattgga agtggccact ttgtggggct ggccgggact
ggggcagctt caggcatcgc 540cattggaggc tttgaatgga atgccctggt
tttggtggtt gtgctgggct ggctgtttgt 600ccccatctat attaaggctg
gggtggtgac aatgccagag tacctgagga agcggtttgg 660aggccagcgg
atccaggtct acctttccct tctgtccctg ctgctctaca ttttcaccaa
720gatctcggca gacatcttct cgggggccat attcatcaat ctggccttag
gcctgaatct 780gtatttagcc atctttctct tattggcaat cactgccctt
tacacaatta cagggggcct 840ggcggcggtg atttacacgg acaccttgca
gacggtgatc atgctggtgg ggtctttaat 900cctgactggg tttgcttttc
acgaagtggg aggctatgac gccttcatgg aaaagtacat 960gaaagccatt
ccaaccatag tgtctgatgg caacaccacc tttcaggaaa aatgctacac
1020tccaagggcc gactccttcc acatcttccg agatcccctc acgggagacc
tcccatggcc 1080tgggttcatc tttgggatgt ccatccttac cttgtggtac
tggtgcacag atcaggtcat 1140tgtgcagcgc tgcctctcag ccaagaatat
gtctcacgtg aagggtggct gcatcctgtg 1200tgggtatcta aagctgatgc
ccatgttcat catggtgatg ccaggaatga tcagccgcat 1260tctgtacaca
gaaaaaattg cctgtgtcgt cccttcagaa tgtgagaaat attgcggtac
1320caaggttggc tgtaccaaca tcgcctatcc aaccttagtg gtggagctca
tgcccaatgg 1380actgcgaggc ctgatgctat cagtcatgct ggcctccctc
atgagctccc tgacctccat 1440cttcaacagc gccagcaccc tcttcaccat
ggacatctac gccaaggtcc gcaagagagc 1500atctgagaaa gagctcatga
ttgccggaag gttgtttatc ctggtgctga ttggcatcag 1560catcgcctgg
gtgcccattg tgcagtcagc acaaagtggg caactcttcg attacatcca
1620gtccatcacc agttacttgg gaccacccat tgcggctgtc ttcctgcttg
ctattttctg 1680gaagagagtc aatgagccag gagccttttg gggactgatc
ctaggacttc tgattgggat 1740ttcacgtatg attactgagt ttgcttatgg
aaccgggagc tgcatggagc ccagcaactg 1800tcccacgatt atctgtgggg
tgcactactt gtactttgcc attatcctct tcgccatttc 1860tttcatcacc
atcgtggtca tctccctcct caccaaaccc attccggatg tgcatctcta
1920ccgtctgtgt tggagcctgc gcaacagcaa agaggagcgt attgacctgg
atgcggaaga 1980ggagaacatc caagaaggcc ctaaggagac cattgaaata
gaaacacaag ttcctgagaa 2040gaaaaaagga atcttcagga gagcctatga
cctattttgt gggctagagc agcacggtgc 2100acccaagatg actgaggaag
aggagaaagc catgaagatg aagatgacgg acacctctga 2160gaagcctttg
tggaggacag tgttgaacgt caatggcatc atcctggtga ccgtggctgt
2220cttttgccat gcatattttg cctgagtcct accttttgct gtagatttac
catggctgga 2280ctcttactca ccttccttta gtctcgtcct gtggtgttga
agggaaatca gccagttgta 2340aattttgccc aggtggataa atgtgtacat
gtgtaattat aggctagctg gaagaaaacc 2400attagtttgc tgttaattta
tgcatttgaa gccagtgtga tacagccatc tgtacctact 2460ggagctgcag
aagggaagtc cactcagtca catccagaaa aaggcagact aagaatcaga
2520agccatgtga ttgatgtctg acgtgagtct gtctcaggta gattccgggt
gtcagtgtgg 2580tttataatcc ttgaatattg ttttagaaac tttggtctcc
ctggttcctg ccacttttcc 2640tgtccgtcct cctccccatt ttttttttaa
aagaaagctg ttttcccctc atcatatccc 2700tcttgagttt tgcctggact
ttccctctca agtgtgtcaa tcaggtaaac tgaggaatgc 2760atggaagctg
aggatggagc ttgatgggct ccctgtcctg ggtgtttgct ctctgaagtg
2820gaggcctgag gaaggtagta cttccacaaa agggagggac ccgggcccca
gcctcaagct 2880agtgggggag gcagatagcc tgaatccagg ggattttctg
ggcttcttaa aatgtccatt 2940gtgagttccc cgtgtttggg attccactca
ttttggcatt cacagtgcct ggaatgtctt 3000agattttcag caatgcgtgt
tgaataaatg aatgacatag gcatttattt ttaaatcttt 3060gcttgctttt
tacatgagcc tggcccttag ttaacctttt cttgtggcta cacaaagtat
3120gctcactggt tactaatgac ttgggatgca tttgtcaaac tgattatatt
agttttctag 3180ggatgccata acaaagtagc acagaccaga tggctcaagc
agcagacatt tattttctca 3240cagttctaga ggctagaagt tggaggccaa
gatgtcagca gggttggttt cttctgaggc 3300ctctctcctt ggttgcagat
ggtcatatct cactctgtct tccgtggcct tccttttgtc 3360tgtgtcctaa
atctactctt ctgataagga catcagtcat attggaatag gacccaccct
3420aatgtcttca ttttaatcac ctctttaaag cccctacctc caaatacagt
cacactgtga 3480gaaactgagg gttaggaagt cagcaagtga gtcttgaaga
gatactaaac aaacccacaa 3540cacagataaa gtatgcattt tggagatttc
caagccagag tctcccgtga aaaaggtaaa 3600cggaagcagt tattgtgcag
caaaaggaaa aagaattaca aactgaacgt atgtaggtga 3660ggcaaggcag
ggtagggcag ggcctttggg taggctgatc agagggtttt tcaacaataa
3720atcaatggga atgcatttgt tgctcccagg
accctggcac cttgactctg gtactatagc 3780atgtcagcaa atacaagcaa
agcccaacac tctgatttgc atttatgcca atctaaacta 3840tccggtgttt
agtttgattt tttgagtgca ggttcattca aggaccaggt tcccttgtgc
3900tcagggtgaa gtagaaccag aaaacatcgt tatccattcc cagaagtttt
ggaagagcct 3960tggtagaaaa gcagaagctg ctttgaccgt gaaaatattt
gactcctatc agtttttggt 4020caggagaaga tatccaccta gaccaacctg
aggagaaggc tcagagtaca gatatacccc 4080gagcaacgtg atcaatgtcc
ttgaaccttc atttttcatc tgaaaacaga gacataaatg 4140cctggctcac
agatttaaat gttatacatt gacagcattt atcagtataa catttattta
4200aataagtagg tgctcaatag gtgttggtct tctaacttgt ctacatccca
tccccattcc 4260agggtcttca gaattgaagg agagatgttg tatcactgtt
agaaggctgc tttgggacat 4320tctgcagcag ggaggaggga ctgtcaaccc
ctacaccatg accaccaagt tcctcacctt 4380ggctgagtcc ctaaaactct
ctgaacctca ggttcctcca agcataatgc agacttcaca 4440gagctgttgt
aaagattagg tgaggtcaat tgatactgct taaaaggccc ggtccgtaga
4500aaatgcccaa taaacattac tgctttcccc ctcaccctac tgcctgaaaa
aatattacac 4560ctgtgagact gactttgaga accagtgtgg gtggggagtt
gtgcatataa actatttaat 4620gagtaccaaa cacaaaagtc aagcttgtaa
aatatcaggc cttgccccag aaagacaaat 4680accacatgat ctcactgata
tgtagaatct taaaaagtca aactcagaag cagagagtag 4740aatgatggtt
atcaagggct gggggaggga gggactgggg agatgttggt caaatgatac
4800aaaggtttag ttaggtggaa taagttcaga aaatcaattg tacaatgtat
caattatagt 4860taatagcaat ataacatata cttgaaaatt gctgagagta
gtgtgagtgt tctaccacaa 4920aaaaatatgt gcagtaatag atgttaatta
ccttaattta gtcatttcac aatatgtaca 4980tatataaaaa tatgttgtat
gccatgagta tatataatta ttatttgtga atttaaaaaa 5040taaaaataat
ttccaaaaaa a 506151827PRTHomo sapiens 5Met Ala Arg Lys Lys Phe Ser
Gly Leu Glu Ile Ser Leu Ile Val Leu 1 5 10 15 Phe Val Ile Val Thr
Ile Ile Ala Ile Ala Leu Ile Val Val Leu Ala 20 25 30 Thr Lys Thr
Pro Ala Val Asp Glu Ile Ser Asp Ser Thr Ser Thr Pro 35 40 45 Ala
Thr Thr Arg Val Thr Thr Asn Pro Ser Asp Ser Gly Lys Cys Pro 50 55
60 Asn Val Leu Asn Asp Pro Val Asn Val Arg Ile Asn Cys Ile Pro Glu
65 70 75 80 Gln Phe Pro Thr Glu Gly Ile Cys Ala Gln Arg Gly Cys Cys
Trp Arg 85 90 95 Pro Trp Asn Asp Ser Leu Ile Pro Trp Cys Phe Phe
Val Asp Asn His 100 105 110 Gly Tyr Asn Val Gln Asp Met Thr Thr Thr
Ser Ile Gly Val Glu Ala 115 120 125 Lys Leu Asn Arg Ile Pro Ser Pro
Thr Leu Phe Gly Asn Asp Ile Asn 130 135 140 Ser Val Leu Phe Thr Thr
Gln Asn Gln Thr Pro Asn Arg Phe Arg Phe 145 150 155 160 Lys Ile Thr
Asp Pro Asn Asn Arg Arg Tyr Glu Val Pro His Gln Tyr 165 170 175 Val
Lys Glu Phe Thr Gly Pro Thr Val Ser Asp Thr Leu Tyr Asp Val 180 185
190 Lys Val Ala Gln Asn Pro Phe Ser Ile Gln Val Ile Arg Lys Ser Asn
195 200 205 Gly Lys Thr Leu Phe Asp Thr Ser Ile Gly Pro Leu Val Tyr
Ser Asp 210 215 220 Gln Tyr Leu Gln Ile Ser Thr Arg Leu Pro Ser Asp
Tyr Ile Tyr Gly 225 230 235 240 Ile Gly Glu Gln Val His Lys Arg Phe
Arg His Asp Leu Ser Trp Lys 245 250 255 Thr Trp Pro Ile Phe Thr Arg
Asp Gln Leu Pro Gly Asp Asn Asn Asn 260 265 270 Asn Leu Tyr Gly His
Gln Thr Phe Phe Met Cys Ile Glu Asp Thr Ser 275 280 285 Gly Lys Ser
Phe Gly Val Phe Leu Met Asn Ser Asn Ala Met Glu Ile 290 295 300 Phe
Ile Gln Pro Thr Pro Ile Val Thr Tyr Arg Val Thr Gly Gly Ile 305 310
315 320 Leu Asp Phe Tyr Ile Leu Leu Gly Asp Thr Pro Glu Gln Val Val
Gln 325 330 335 Gln Tyr Gln Gln Leu Val Gly Leu Pro Ala Met Pro Ala
Tyr Trp Asn 340 345 350 Leu Gly Phe Gln Leu Ser Arg Trp Asn Tyr Lys
Ser Leu Asp Val Val 355 360 365 Lys Glu Val Val Arg Arg Asn Arg Glu
Ala Gly Ile Pro Phe Asp Thr 370 375 380 Gln Val Thr Asp Ile Asp Tyr
Met Glu Asp Lys Lys Asp Phe Thr Tyr 385 390 395 400 Asp Gln Val Ala
Phe Asn Gly Leu Pro Gln Phe Val Gln Asp Leu His 405 410 415 Asp His
Gly Gln Lys Tyr Val Ile Ile Leu Asp Pro Ala Ile Ser Ile 420 425 430
Gly Arg Arg Ala Asn Gly Thr Thr Tyr Ala Thr Tyr Glu Arg Gly Asn 435
440 445 Thr Gln His Val Trp Ile Asn Glu Ser Asp Gly Ser Thr Pro Ile
Ile 450 455 460 Gly Glu Val Trp Pro Gly Leu Thr Val Tyr Pro Asp Phe
Thr Asn Pro 465 470 475 480 Asn Cys Ile Asp Trp Trp Ala Asn Glu Cys
Ser Ile Phe His Gln Glu 485 490 495 Val Gln Tyr Asp Gly Leu Trp Ile
Asp Met Asn Glu Val Ser Ser Phe 500 505 510 Ile Gln Gly Ser Thr Lys
Gly Cys Asn Val Asn Lys Leu Asn Tyr Pro 515 520 525 Pro Phe Thr Pro
Asp Ile Leu Asp Lys Leu Met Tyr Ser Lys Thr Ile 530 535 540 Cys Met
Asp Ala Val Gln Asn Trp Gly Lys Gln Tyr Asp Val His Ser 545 550 555
560 Leu Tyr Gly Tyr Ser Met Ala Ile Ala Thr Glu Gln Ala Val Gln Lys
565 570 575 Val Phe Pro Asn Lys Arg Ser Phe Ile Leu Thr Arg Ser Thr
Phe Ala 580 585 590 Gly Ser Gly Arg His Ala Ala His Trp Leu Gly Asp
Asn Thr Ala Ser 595 600 605 Trp Glu Gln Met Glu Trp Ser Ile Thr Gly
Met Leu Glu Phe Ser Leu 610 615 620 Phe Gly Ile Pro Leu Val Gly Ala
Asp Ile Cys Gly Phe Val Ala Glu 625 630 635 640 Thr Thr Glu Glu Leu
Cys Arg Arg Trp Met Gln Leu Gly Ala Phe Tyr 645 650 655 Pro Phe Ser
Arg Asn His Asn Ser Asp Gly Tyr Glu His Gln Asp Pro 660 665 670 Ala
Phe Phe Gly Gln Asn Ser Leu Leu Val Lys Ser Ser Arg Gln Tyr 675 680
685 Leu Thr Ile Arg Tyr Thr Leu Leu Pro Phe Leu Tyr Thr Leu Phe Tyr
690 695 700 Lys Ala His Val Phe Gly Glu Thr Val Ala Arg Pro Val Leu
His Glu 705 710 715 720 Phe Tyr Glu Asp Thr Asn Ser Trp Ile Glu Asp
Thr Glu Phe Leu Trp 725 730 735 Gly Pro Ala Leu Leu Ile Thr Pro Val
Leu Lys Gln Gly Ala Asp Thr 740 745 750 Val Ser Ala Tyr Ile Pro Asp
Ala Ile Trp Tyr Asp Tyr Glu Ser Gly 755 760 765 Ala Lys Arg Pro Trp
Arg Lys Gln Arg Val Asp Met Tyr Leu Pro Ala 770 775 780 Asp Lys Ile
Gly Leu His Leu Arg Gly Gly Tyr Ile Ile Pro Ile Gln 785 790 795 800
Glu Pro Asp Val Thr Thr Thr Ala Ser Arg Lys Asn Pro Leu Gly Leu 805
810 815 Ile Val Ala Leu Gly Glu Asn Asn Thr Ala Lys Gly Asp Phe Phe
Trp 820 825 830 Asp Asp Gly Glu Thr Lys Asp Thr Ile Gln Asn Gly Asn
Tyr Ile Leu 835 840 845 Tyr Thr Phe Ser Val Ser Asn Asn Thr Leu Asp
Ile Val Cys Thr His 850 855 860 Ser Ser Tyr Gln Glu Gly Thr Thr Leu
Ala Phe Gln Thr Val Lys Ile 865 870 875 880 Leu Gly Leu Thr Asp Ser
Val Thr Glu Val Arg Val Ala Glu Asn Asn 885 890 895 Gln Pro Met Asn
Ala His Ser Asn Phe Thr Tyr Asp Ala Ser Asn Gln 900 905 910 Val Leu
Leu Ile Ala Asp Leu Lys Leu Asn Leu Gly Arg Asn Phe Ser 915 920 925
Val Gln Trp Asn Gln Ile Phe Ser Glu Asn Glu Arg Phe Asn Cys Tyr 930
935 940 Pro Asp Ala Asp Leu Ala Thr Glu Gln Lys Cys Thr Gln Arg Gly
Cys 945 950 955 960 Val Trp Arg Thr Gly Ser Ser Leu Ser Lys Ala Pro
Glu Cys Tyr Phe 965 970 975 Pro Arg Gln Asp Asn Ser Tyr Ser Val Asn
Ser Ala Arg Tyr Ser Ser 980 985 990 Met Gly Ile Thr Ala Asp Leu Gln
Leu Asn Thr Ala Asn Ala Arg Ile 995 1000 1005 Lys Leu Pro Ser Asp
Pro Ile Ser Thr Leu Arg Val Glu Val Lys 1010 1015 1020 Tyr His Lys
Asn Asp Met Leu Gln Phe Lys Ile Tyr Asp Pro Gln 1025 1030 1035 Lys
Lys Arg Tyr Glu Val Pro Val Pro Leu Asn Ile Pro Thr Thr 1040 1045
1050 Pro Ile Ser Thr Tyr Glu Asp Arg Leu Tyr Asp Val Glu Ile Lys
1055 1060 1065 Glu Asn Pro Phe Gly Ile Gln Ile Arg Arg Arg Ser Ser
Gly Arg 1070 1075 1080 Val Ile Trp Asp Ser Trp Leu Pro Gly Phe Ala
Phe Asn Asp Gln 1085 1090 1095 Phe Ile Gln Ile Ser Thr Arg Leu Pro
Ser Glu Tyr Ile Tyr Gly 1100 1105 1110 Phe Gly Glu Val Glu His Thr
Ala Phe Lys Arg Asp Leu Asn Trp 1115 1120 1125 Asn Thr Trp Gly Met
Phe Thr Arg Asp Gln Pro Pro Gly Tyr Lys 1130 1135 1140 Leu Asn Ser
Tyr Gly Phe His Pro Tyr Tyr Met Ala Leu Glu Glu 1145 1150 1155 Glu
Gly Asn Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met 1160 1165
1170 Asp Val Thr Phe Gln Pro Thr Pro Ala Leu Thr Tyr Arg Thr Val
1175 1180 1185 Gly Gly Ile Leu Asp Phe Tyr Met Phe Leu Gly Pro Thr
Pro Glu 1190 1195 1200 Val Ala Thr Lys Gln Tyr His Glu Val Ile Gly
His Pro Val Met 1205 1210 1215 Pro Ala Tyr Trp Ala Leu Gly Phe Gln
Leu Cys Arg Tyr Gly Tyr 1220 1225 1230 Ala Asn Thr Ser Glu Val Arg
Glu Leu Tyr Asp Ala Met Val Ala 1235 1240 1245 Ala Asn Ile Pro Tyr
Asp Val Gln Tyr Thr Asp Ile Asp Tyr Met 1250 1255 1260 Glu Arg Gln
Leu Asp Phe Thr Ile Gly Glu Ala Phe Gln Asp Leu 1265 1270 1275 Pro
Gln Phe Val Asp Lys Ile Arg Gly Glu Gly Met Arg Tyr Ile 1280 1285
1290 Ile Ile Leu Asp Pro Ala Ile Ser Gly Asn Glu Thr Lys Thr Tyr
1295 1300 1305 Pro Ala Phe Glu Arg Gly Gln Gln Asn Asp Val Phe Val
Lys Trp 1310 1315 1320 Pro Asn Thr Asn Asp Ile Cys Trp Ala Lys Val
Trp Pro Asp Leu 1325 1330 1335 Pro Asn Ile Thr Ile Asp Lys Thr Leu
Thr Glu Asp Glu Ala Val 1340 1345 1350 Asn Ala Ser Arg Ala His Val
Ala Phe Pro Asp Phe Phe Arg Thr 1355 1360 1365 Ser Thr Ala Glu Trp
Trp Ala Arg Glu Ile Val Asp Phe Tyr Asn 1370 1375 1380 Glu Lys Met
Lys Phe Asp Gly Leu Trp Ile Asp Met Asn Glu Pro 1385 1390 1395 Ser
Ser Phe Val Asn Gly Thr Thr Thr Asn Gln Cys Arg Asn Asp 1400 1405
1410 Glu Leu Asn Tyr Pro Pro Tyr Phe Pro Glu Leu Thr Lys Arg Thr
1415 1420 1425 Asp Gly Leu His Phe Arg Thr Ile Cys Met Glu Ala Glu
Gln Ile 1430 1435 1440 Leu Ser Asp Gly Thr Ser Val Leu His Tyr Asp
Val His Asn Leu 1445 1450 1455 Tyr Gly Trp Ser Gln Met Lys Pro Thr
His Asp Ala Leu Gln Lys 1460 1465 1470 Thr Thr Gly Lys Arg Gly Ile
Val Ile Ser Arg Ser Thr Tyr Pro 1475 1480 1485 Thr Ser Gly Arg Trp
Gly Gly His Trp Leu Gly Asp Asn Tyr Ala 1490 1495 1500 Arg Trp Asp
Asn Met Asp Lys Ser Ile Ile Gly Met Met Glu Phe 1505 1510 1515 Ser
Leu Phe Gly Met Ser Tyr Thr Gly Ala Asp Ile Cys Gly Phe 1520 1525
1530 Phe Asn Asn Ser Glu Tyr His Leu Cys Thr Arg Trp Met Gln Leu
1535 1540 1545 Gly Ala Phe Tyr Pro Tyr Ser Arg Asn His Asn Ile Ala
Asn Thr 1550 1555 1560 Arg Arg Gln Asp Pro Ala Ser Trp Asn Glu Thr
Phe Ala Glu Met 1565 1570 1575 Ser Arg Asn Ile Leu Asn Ile Arg Tyr
Thr Leu Leu Pro Tyr Phe 1580 1585 1590 Tyr Thr Gln Met His Glu Ile
His Ala Asn Gly Gly Thr Val Ile 1595 1600 1605 Arg Pro Leu Leu His
Glu Phe Phe Asp Glu Lys Pro Thr Trp Asp 1610 1615 1620 Ile Phe Lys
Gln Phe Leu Trp Gly Pro Ala Phe Met Val Thr Pro 1625 1630 1635 Val
Leu Glu Pro Tyr Val Gln Thr Val Asn Ala Tyr Val Pro Asn 1640 1645
1650 Ala Arg Trp Phe Asp Tyr His Thr Gly Lys Asp Ile Gly Val Arg
1655 1660 1665 Gly Gln Phe Gln Thr Phe Asn Ala Ser Tyr Asp Thr Ile
Asn Leu 1670 1675 1680 His Val Arg Gly Gly His Ile Leu Pro Cys Gln
Glu Pro Ala Gln 1685 1690 1695 Asn Thr Phe Tyr Ser Arg Gln Lys His
Met Lys Leu Ile Val Ala 1700 1705 1710 Ala Asp Asp Asn Gln Met Ala
Gln Gly Ser Leu Phe Trp Asp Asp 1715 1720 1725 Gly Glu Ser Ile Asp
Thr Tyr Glu Arg Asp Leu Tyr Leu Ser Val 1730 1735 1740 Gln Phe Asn
Leu Asn Gln Thr Thr Leu Thr Ser Thr Ile Leu Lys 1745 1750 1755 Arg
Gly Tyr Ile Asn Lys Ser Glu Thr Arg Leu Gly Ser Leu His 1760 1765
1770 Val Trp Gly Lys Gly Thr Thr Pro Val Asn Ala Val Thr Leu Thr
1775 1780 1785 Tyr Asn Gly Asn Lys Asn Ser Leu Pro Phe Asn Glu Asp
Thr Thr 1790 1795 1800 Asn Met Ile Leu Arg Ile Asp Leu Thr Thr His
Asn Val Thr Leu 1805 1810 1815 Glu Glu Pro Ile Glu Ile Asn Trp Ser
1820 1825 6 6023DNAHomo sapiens 6ttattttggc agccttatcc aagtctggta
caacatagca aagagaacag gctatgaaat 60aagatggcaa gaaagaaatt tagtggattg
gaaatctctc tgattgtcct ttttgtcata 120gttactataa tagctattgc
cttaattgtt gttttagcaa ctaagacacc tgctgttgat 180gaaattagtg
attctacttc aactccagct actactcgtg tgactacaaa tccttctgat
240tcaggaaaat gtccaaatgt gttaaatgat cctgtcaatg tgagaataaa
ctgcattcca 300gaacaattcc caacagaggg aatttgtgca cagagaggct
gctgctggag gccgtggaat 360gactctctta ttccttggtg cttcttcgtt
gataatcatg gttataacgt tcaagacatg 420acaacaacaa gtattggagt
tgaagccaaa ttaaacagga taccttcacc tacactattt 480ggaaatgaca
tcaacagtgt tctcttcaca actcaaaatc agacacccaa tcgtttccgg
540ttcaagatta ctgatccaaa taatagaaga tatgaagttc ctcatcagta
tgtaaaagag 600tttactggac ccacagtttc tgatacgttg tatgatgtga
aggttgccca aaacccattt 660agcatccaag ttattaggaa aagcaacggt
aaaactttgt ttgacaccag cattggtccc 720ttagtgtact ctgaccagta
cttacagatc tcaacccgtc ttccaagtga ttatatttat 780ggtattggag
aacaagttca taagagattt cgtcatgatt tatcctggaa aacatggcca
840atttttactc gagaccaact tcctggtgat aataataata atttatacgg
ccatcaaaca 900ttctttatgt gtattgaaga tacatctgga aagtcattcg
gtgttttttt aatgaatagc 960aatgcaatgg agatttttat ccagcctact
ccaatagtaa catatagagt taccggtggc 1020attctggatt tttacatcct
tctaggagat acaccagaac aagtagttca acagtatcaa 1080cagcttgttg
gactaccagc aatgccagca tattggaatc ttggattcca actaagtcgc
1140tggaattata agtcactaga tgtagtgaaa gaagtggtaa ggagaaaccg
ggaagctggc 1200ataccatttg atacacaggt cactgatatt gactacatgg
aagacaagaa agactttact 1260tatgatcaag ttgcgtttaa cggactccct
caatttgtgc aagatttgca tgaccatgga 1320cagaaatatg tcatcatctt
ggaccctgca atttccatag gtcgacgtgc caatggaaca 1380acatatgcaa
cctatgagag gggaaacaca caacatgtgt ggataaatga gtcagatgga
1440agtacaccaa ttattggaga
ggtatggcca ggattaacag tataccctga tttcactaac 1500ccaaactgca
ttgattggtg ggcaaatgaa tgcagtattt tccatcaaga agtgcaatat
1560gatggacttt ggattgacat gaatgaagtt tccagcttta ttcaaggttc
aacaaaagga 1620tgtaatgtaa acaaattgaa ttatccaccg tttactcctg
atattcttga caaactcatg 1680tattccaaaa caatttgcat ggatgctgtg
cagaactggg gtaaacagta tgatgttcat 1740agcctctatg gatacagcat
ggctatagcc acagagcaag ctgtacaaaa agtttttcct 1800aataagagaa
gcttcattct tacccgctca acatttgctg gatctggaag acatgctgcg
1860cattggttag gagacaatac tgcttcatgg gaacaaatgg aatggtctat
aactggaatg 1920ctggagttca gtttgtttgg aatacctttg gttggagcag
acatctgtgg atttgtggct 1980gaaaccacag aagaactttg cagaagatgg
atgcaacttg gggcatttta tccattttcc 2040agaaaccata attctgacgg
atatgaacat caggatcctg cattttttgg gcagaattca 2100cttttggtta
aatcatcaag gcagtattta actattcgct acaccttatt acccttcctc
2160tacactctgt tttataaagc ccatgtgttt ggagaaacag tagcaagacc
agttcttcat 2220gagttttatg aggatacgaa cagctggatt gaggacactg
agtttttgtg gggccctgca 2280ttacttatta ctcctgttct aaaacaggga
gcagatactg tgagtgccta catccctgat 2340gctatttggt atgattatga
atctggtgca aaaaggccat ggaggaaaca acgggttgat 2400atgtatcttc
cagcagacaa aataggatta catcttagag gaggttatat catccccatt
2460caagaaccag atgtaacaac aacagcaagc cgtaagaatc ctctaggact
tatagtcgca 2520ttaggtgaaa acaacacagc caaaggagac tttttctggg
atgatggaga aactaaagat 2580acaatacaaa atggcaacta catattatat
acattttcag tttctaataa cacattagat 2640attgtgtgca cacattcatc
atatcaggaa ggaactacct tagcatttca gactgtaaaa 2700atccttgggt
tgacagacag tgttacagaa gttagagtgg cggaaaataa tcaaccaatg
2760aacgctcatt ccaatttcac ttatgatgct tctaaccagg ttctcctaat
tgcagatctc 2820aaacttaatc ttggaagaaa ctttagtgtt caatggaatc
aaattttctc agaaaatgaa 2880agatttaatt gttatccaga tgcagatttg
gcaactgaac aaaagtgcac acaacgtggc 2940tgtgtatgga gaacgggttc
ttctctatcc aaagcacctg agtgttactt tcccagacaa 3000gataactctt
attcagtcaa ctcagctcgc tattcatcca tgggtataac agctgacctc
3060caactaaata ctgcaaatgc cagaataaag ttaccttctg accccatctc
aactcttcgt 3120gtggaggtga aatatcacaa aaatgatatg ttgcagttta
agatttatga tccccaaaag 3180aagagatatg aagtaccagt accgttaaac
attccaacca ccccaataag tacttatgaa 3240gacagacttt atgatgtgga
aatcaaggaa aatccttttg gcatccagat tcgacggaga 3300agcagtggaa
gagtcatttg ggattcttgg ctgcctggat ttgcttttaa tgaccagttc
3360attcaaatat cgactcgcct gccatcagaa tatatatatg gttttgggga
agtggaacat 3420acagcattta agcgagatct gaactggaat acttggggaa
tgttcacaag agaccaaccc 3480cctggttaca aacttaattc ctatggattt
catccctatt acatggctct ggaagaggag 3540ggcaatgctc atggtgtttt
cttactcaac agcaatgcaa tggatgttac attccagcca 3600actcctgctc
taacttaccg tacagttgga gggatcttgg atttttatat gtttttgggc
3660ccaactccag aagttgcaac aaagcaatac catgaagtaa ttggccatcc
agtcatgcca 3720gcttattggg ctttgggatt ccaattatgt cgttatggat
atgcaaatac ttcagaggtt 3780cgggaattat atgacgctat ggtggctgct
aacatcccct atgatgttca gtacacagac 3840attgactaca tggaaaggca
gctagacttt acaattggtg aagcattcca ggaccttcct 3900cagtttgttg
acaaaataag aggagaagga atgagataca ttattatcct ggatccagca
3960atttcaggaa atgaaacaaa gacttaccct gcatttgaaa gaggacagca
gaatgatgtc 4020tttgtcaaat ggccaaacac caatgacatt tgttgggcaa
aggtttggcc agatttgccc 4080aacataacaa tagataaaac tctaacggaa
gatgaagctg ttaatgcttc cagagctcat 4140gtagctttcc cagatttctt
caggacttcc acagcagagt ggtgggccag agaaattgtg 4200gacttttaca
atgaaaagat gaagtttgat ggtttgtgga ttgatatgaa tgagccatca
4260agttttgtaa atggaacaac tactaatcaa tgcagaaatg acgaactaaa
ttatccacct 4320tatttcccag aactcacaaa aagaactgat ggattacatt
tcagaacaat ttgcatggaa 4380gctgagcaga ttcttagtga tggaacatca
gttttgcatt acgatgttca caatctctat 4440ggatggtcac agatgaaacc
tactcatgat gcattgcaga agacaactgg aaaaagaggg 4500attgtaattt
ctcgttccac gtatcctact agtggacgat ggggaggaca ctggcttgga
4560gacaactatg cacgatggga caacatggac aaatcaatca ttggtatgat
ggaatttagt 4620ctgtttggaa tgtcatatac tggagcagac atctgtggtt
ttttcaacaa ctcagaatat 4680catctctgta cccgctggat gcaacttgga
gcattttatc catactcaag gaatcacaac 4740attgcaaata ctagaagaca
agatcccgct tcctggaatg aaacttttgc tgaaatgtca 4800aggaatattc
taaatattag atacacctta ttgccctatt tttacacaca aatgcatgaa
4860attcatgcta atggtggcac tgttatccga ccccttttgc atgagttctt
tgatgaaaaa 4920ccaacctggg atatattcaa gcagttctta tggggtccag
catttatggt taccccagta 4980ctggaacctt atgttcaaac tgtaaatgcc
tacgtcccca atgctcggtg gtttgactac 5040catacaggca aagatattgg
cgtcagagga caatttcaaa catttaatgc ttcttatgac 5100acaataaacc
tacatgtccg tggtggtcac atcctaccat gtcaagagcc agctcaaaac
5160acattttaca gtcgacaaaa acacatgaag ctcattgttg ctgcagatga
taatcagatg 5220gcacagggtt ctctgttttg ggatgatgga gagagtatag
acacctatga aagagaccta 5280tatttatctg tacaatttaa tttaaaccag
accaccttaa caagcactat attgaagaga 5340ggttacataa ataaaagtga
aacgaggctt ggatcccttc atgtatgggg gaaaggaact 5400actcctgtca
atgcagttac tctaacgtat aacggaaata aaaattcgct tccttttaat
5460gaagacacta ccaacatgat attacgtatt gatctgacca cacacaatgt
tactctagaa 5520gaaccaatag aaatcaactg gtcatgaaga tcaccatcaa
ttttagttgt caatgggaaa 5580aaacaccagg atttaagttt cacagcactt
acaattttcc ctcttcactt ggttcttgta 5640ctctacaaaa tatagctttc
ataacatcga aaagttattt tgtagcgtac atcaatgata 5700atgctaattt
tattatagta atgtgacttg gattcaattt taaggcatat ttaacaaaat
5760ttgaatagcc ctatttatcc ttgttaagta tcagctacaa ttgtaaacta
gttactaaac 5820atgtatgtaa atagctaaga tataatttaa acgtgatttt
taaattaaat aaaattttta 5880tgtaattata tatactatat ttttctcaat
gtttagcaga tttaagatat gtaacaacaa 5940ttatttgaag atttaattac
ttcttagtat gtgcatttaa ttagaaaaag agaataaaaa 6000atgtaagtgt
aaaaaaaaaa aaa 602371857PRTHomo sapiens 7Met Ala Arg Lys Lys Leu
Lys Lys Phe Thr Thr Leu Glu Ile Val Leu 1 5 10 15 Ser Val Leu Leu
Leu Val Leu Phe Ile Ile Ser Ile Val Leu Ile Val 20 25 30 Leu Leu
Ala Lys Glu Ser Leu Lys Ser Thr Ala Pro Asp Pro Gly Thr 35 40 45
Thr Gly Thr Pro Asp Pro Gly Thr Thr Gly Thr Pro Asp Pro Gly Thr 50
55 60 Thr Gly Thr Thr His Ala Arg Thr Thr Gly Pro Pro Asp Pro Gly
Thr 65 70 75 80 Thr Gly Thr Thr Pro Val Ser Ala Glu Cys Pro Val Val
Asn Glu Leu 85 90 95 Glu Arg Ile Asn Cys Ile Pro Asp Gln Pro Pro
Thr Lys Ala Thr Cys 100 105 110 Asp Gln Arg Gly Cys Cys Trp Asn Pro
Gln Gly Ala Val Ser Val Pro 115 120 125 Trp Cys Tyr Tyr Ser Lys Asn
His Ser Tyr His Val Glu Gly Asn Leu 130 135 140 Val Asn Thr Asn Ala
Gly Phe Thr Ala Arg Leu Lys Asn Leu Pro Ser 145 150 155 160 Ser Pro
Val Phe Gly Ser Asn Val Asp Asn Val Leu Leu Thr Ala Glu 165 170 175
Tyr Gln Thr Ser Asn Arg Phe His Phe Lys Leu Thr Asp Gln Thr Asn 180
185 190 Asn Arg Phe Glu Val Pro His Glu His Val Gln Ser Phe Ser Gly
Asn 195 200 205 Ala Ala Ala Ser Leu Thr Tyr Gln Val Glu Ile Ser Arg
Gln Pro Phe 210 215 220 Ser Ile Lys Val Thr Arg Arg Ser Asn Asn Arg
Val Leu Phe Asp Ser 225 230 235 240 Ser Ile Gly Pro Leu Leu Phe Ala
Asp Gln Phe Leu Gln Leu Ser Thr 245 250 255 Arg Leu Pro Ser Thr Asn
Val Tyr Gly Leu Gly Glu His Val His Gln 260 265 270 Gln Tyr Arg His
Asp Met Asn Trp Lys Thr Trp Pro Ile Phe Asn Arg 275 280 285 Asp Thr
Thr Pro Asn Gly Asn Gly Thr Asn Leu Tyr Gly Ala Gln Thr 290 295 300
Phe Phe Leu Cys Leu Glu Asp Ala Ser Gly Leu Ser Phe Gly Val Phe 305
310 315 320 Leu Met Asn Ser Asn Ala Met Glu Val Val Leu Gln Pro Ala
Pro Ala 325 330 335 Ile Thr Tyr Arg Thr Ile Gly Gly Ile Leu Asp Phe
Tyr Val Phe Leu 340 345 350 Gly Asn Thr Pro Glu Gln Val Val Gln Glu
Tyr Leu Glu Leu Ile Gly 355 360 365 Arg Pro Ala Leu Pro Ser Tyr Trp
Ala Leu Gly Phe His Leu Ser Arg 370 375 380 Tyr Glu Tyr Gly Thr Leu
Asp Asn Met Arg Glu Val Val Glu Arg Asn 385 390 395 400 Arg Ala Ala
Gln Leu Pro Tyr Asp Val Gln His Ala Asp Ile Asp Tyr 405 410 415 Met
Asp Glu Arg Arg Asp Phe Thr Tyr Asp Ser Val Asp Phe Lys Gly 420 425
430 Phe Pro Glu Phe Val Asn Glu Leu His Asn Asn Gly Gln Lys Leu Val
435 440 445 Ile Ile Val Asp Pro Ala Ile Ser Asn Asn Ser Ser Ser Ser
Lys Pro 450 455 460 Tyr Gly Pro Tyr Asp Arg Gly Ser Asp Met Lys Ile
Trp Val Asn Ser 465 470 475 480 Ser Asp Gly Val Thr Pro Leu Ile Gly
Glu Val Trp Pro Gly Gln Thr 485 490 495 Val Phe Pro Asp Tyr Thr Asn
Pro Asn Cys Ala Val Trp Trp Thr Lys 500 505 510 Glu Phe Glu Leu Phe
His Asn Gln Val Glu Phe Asp Gly Ile Trp Ile 515 520 525 Asp Met Asn
Glu Val Ser Asn Phe Val Asp Gly Ser Val Ser Gly Cys 530 535 540 Ser
Thr Asn Asn Leu Asn Asn Pro Pro Phe Thr Pro Arg Ile Leu Asp 545 550
555 560 Gly Tyr Leu Phe Cys Lys Thr Leu Cys Met Asp Ala Val Gln His
Trp 565 570 575 Gly Lys Gln Tyr Asp Ile His Asn Leu Tyr Gly Tyr Ser
Met Ala Val 580 585 590 Ala Thr Ala Glu Ala Ala Lys Thr Val Phe Pro
Asn Lys Arg Ser Phe 595 600 605 Ile Leu Thr Arg Ser Thr Phe Ala Gly
Ser Gly Lys Phe Ala Ala His 610 615 620 Trp Leu Gly Asp Asn Thr Ala
Thr Trp Asp Asp Leu Arg Trp Ser Ile 625 630 635 640 Pro Gly Val Leu
Glu Phe Asn Leu Phe Gly Ile Pro Met Val Gly Pro 645 650 655 Asp Ile
Cys Gly Phe Ala Leu Asp Thr Pro Glu Glu Leu Cys Arg Arg 660 665 670
Trp Met Gln Leu Gly Ala Phe Tyr Pro Phe Ser Arg Asn His Asn Gly 675
680 685 Gln Gly Tyr Lys Asp Gln Asp Pro Ala Ser Phe Gly Ala Asp Ser
Leu 690 695 700 Leu Leu Asn Ser Ser Arg His Tyr Leu Asn Ile Arg Tyr
Thr Leu Leu 705 710 715 720 Pro Tyr Leu Tyr Thr Leu Phe Phe Arg Ala
His Ser Arg Gly Asp Thr 725 730 735 Val Ala Arg Pro Leu Leu His Glu
Phe Tyr Glu Asp Asn Ser Thr Trp 740 745 750 Asp Val His Gln Gln Phe
Leu Trp Gly Pro Gly Leu Leu Ile Thr Pro 755 760 765 Val Leu Asp Glu
Gly Ala Glu Lys Val Met Ala Tyr Val Pro Asp Ala 770 775 780 Val Trp
Tyr Asp Tyr Glu Thr Gly Ser Gln Val Arg Trp Arg Lys Gln 785 790 795
800 Lys Val Glu Met Glu Leu Pro Gly Asp Lys Ile Gly Leu His Leu Arg
805 810 815 Gly Gly Tyr Ile Phe Pro Thr Gln Gln Pro Asn Thr Thr Thr
Leu Ala 820 825 830 Ser Arg Lys Asn Pro Leu Gly Leu Ile Ile Ala Leu
Asp Glu Asn Lys 835 840 845 Glu Ala Lys Gly Glu Leu Phe Trp Asp Asn
Gly Glu Thr Lys Asp Thr 850 855 860 Val Ala Asn Lys Val Tyr Leu Leu
Cys Glu Phe Ser Val Thr Gln Asn 865 870 875 880 Arg Leu Glu Val Asn
Ile Ser Gln Ser Thr Tyr Lys Asp Pro Asn Asn 885 890 895 Leu Ala Phe
Asn Glu Ile Lys Ile Leu Gly Thr Glu Glu Pro Ser Asn 900 905 910 Val
Thr Val Lys His Asn Gly Val Pro Ser Gln Thr Ser Pro Thr Val 915 920
925 Thr Tyr Asp Ser Asn Leu Lys Val Ala Ile Ile Thr Asp Ile Asp Leu
930 935 940 Leu Leu Gly Glu Ala Tyr Thr Val Glu Trp Ser Ile Lys Ile
Arg Asp 945 950 955 960 Glu Glu Lys Ile Asp Cys Tyr Pro Asp Glu Asn
Gly Ala Ser Ala Glu 965 970 975 Asn Cys Thr Ala Arg Gly Cys Ile Trp
Glu Ala Ser Asn Ser Ser Gly 980 985 990 Val Pro Phe Cys Tyr Phe Val
Asn Asp Leu Tyr Ser Val Ser Asp Val 995 1000 1005 Gln Tyr Asn Ser
His Gly Ala Thr Ala Asp Ile Ser Leu Lys Ser 1010 1015 1020 Ser Val
Tyr Ala Asn Ala Phe Pro Ser Thr Pro Val Asn Pro Leu 1025 1030 1035
Arg Leu Asp Val Thr Tyr His Lys Asn Glu Met Leu Gln Phe Lys 1040
1045 1050 Ile Tyr Asp Pro Asn Lys Asn Arg Tyr Glu Val Pro Val Pro
Leu 1055 1060 1065 Asn Ile Pro Ser Met Pro Ser Ser Thr Pro Glu Gly
Gln Leu Tyr 1070 1075 1080 Asp Val Leu Ile Lys Lys Asn Pro Phe Gly
Ile Glu Ile Arg Arg 1085 1090 1095 Lys Ser Thr Gly Thr Ile Ile Trp
Asp Ser Gln Leu Leu Gly Phe 1100 1105 1110 Thr Phe Ser Asp Met Phe
Ile Arg Ile Ser Thr Arg Leu Pro Ser 1115 1120 1125 Lys Tyr Leu Tyr
Gly Phe Gly Glu Thr Glu His Arg Ser Tyr Arg 1130 1135 1140 Arg Asp
Leu Glu Trp His Thr Trp Gly Met Phe Ser Arg Asp Gln 1145 1150 1155
Pro Pro Gly Tyr Lys Lys Asn Ser Tyr Gly Val His Pro Tyr Tyr 1160
1165 1170 Met Gly Leu Glu Glu Asp Gly Ser Ala His Gly Val Leu Leu
Leu 1175 1180 1185 Asn Ser Asn Ala Met Asp Val Thr Phe Gln Pro Leu
Pro Ala Leu 1190 1195 1200 Thr Tyr Arg Thr Thr Gly Gly Val Leu Asp
Phe Tyr Val Phe Leu 1205 1210 1215 Gly Pro Thr Pro Glu Leu Val Thr
Gln Gln Tyr Thr Glu Leu Ile 1220 1225 1230 Gly Arg Pro Val Met Val
Pro Tyr Trp Ser Leu Gly Phe Gln Leu 1235 1240 1245 Cys Arg Tyr Gly
Tyr Gln Asn Asp Ser Glu Ile Ala Ser Leu Tyr 1250 1255 1260 Asp Glu
Met Val Ala Ala Gln Ile Pro Tyr Asp Val Gln Tyr Ser 1265 1270 1275
Asp Ile Asp Tyr Met Glu Arg Gln Leu Asp Phe Thr Leu Ser Pro 1280
1285 1290 Lys Phe Ala Gly Phe Pro Ala Leu Ile Asn Arg Met Lys Ala
Asp 1295 1300 1305 Gly Met Arg Val Ile Leu Ile Leu Asp Pro Ala Ile
Ser Gly Asn 1310 1315 1320 Glu Thr Gln Pro Tyr Pro Ala Phe Thr Arg
Gly Val Glu Asp Asp 1325 1330 1335 Val Phe Ile Lys Tyr Pro Asn Asp
Gly Asp Ile Val Trp Gly Lys 1340 1345 1350 Val Trp Pro Asp Phe Pro
Asp Val Val Val Asn Gly Ser Leu Asp 1355 1360 1365 Trp Asp Ser Gln
Val Glu Leu Tyr Arg Ala Tyr Val Ala Phe Pro 1370 1375 1380 Asp Phe
Phe Arg Asn Ser Thr Ala Lys Trp Trp Lys Arg Glu Ile 1385 1390 1395
Glu Glu Leu Tyr Asn Asn Pro Gln Asn Pro Glu Arg Ser Leu Lys 1400
1405 1410 Phe Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Ser Phe
Val 1415 1420 1425 Asn Gly Ala Val Ser Pro Gly Cys Arg Asp Ala Ser
Leu Asn His 1430 1435 1440 Pro Pro Tyr Met Pro His Leu Glu Ser Arg
Asp Arg Gly Leu Ser 1445 1450 1455 Ser Lys Thr Leu Cys Met Glu Ser
Gln Gln Ile Leu Pro Asp Gly 1460 1465 1470 Ser Leu Val Gln His Tyr
Asn Val His Asn Leu Tyr Gly Trp Ser 1475 1480 1485 Gln Thr Arg Pro
Thr Tyr Glu Ala Val Gln Glu Val Thr Gly Gln 1490 1495 1500 Arg Gly
Val Val Ile Thr Arg Ser Thr Phe Pro Ser Ser Gly Arg 1505 1510 1515
Trp Ala Gly His Trp Leu Gly Asp Asn Thr Ala Ala Trp Asp Gln 1520
1525 1530 Leu Lys Lys Ser Ile Ile Gly Met Met Glu Phe Ser Leu Phe
Gly 1535 1540 1545 Ile Ser Tyr Thr Gly Ala Asp Ile Cys Gly Phe Phe
Gln Asp Ala 1550 1555 1560 Glu Tyr Glu Met Cys Val Arg Trp Met Gln
Leu Gly Ala Phe Tyr
1565 1570 1575 Pro Phe Ser Arg Asn His Asn Thr Ile Gly Thr Arg Arg
Gln Asp 1580 1585 1590 Pro Val Ser Trp Asp Val Ala Phe Val Asn Ile
Ser Arg Thr Val 1595 1600 1605 Leu Gln Thr Arg Tyr Thr Leu Leu Pro
Tyr Leu Tyr Thr Leu Met 1610 1615 1620 His Lys Ala His Thr Glu Gly
Val Thr Val Val Arg Pro Leu Leu 1625 1630 1635 His Glu Phe Val Ser
Asp Gln Val Thr Trp Asp Ile Asp Ser Gln 1640 1645 1650 Phe Leu Leu
Gly Pro Ala Phe Leu Val Ser Pro Val Leu Glu Arg 1655 1660 1665 Asn
Ala Arg Asn Val Thr Ala Tyr Phe Pro Arg Ala Arg Trp Tyr 1670 1675
1680 Asp Tyr Tyr Thr Gly Val Asp Ile Asn Ala Arg Gly Glu Trp Lys
1685 1690 1695 Thr Leu Pro Ala Pro Leu Asp His Ile Asn Leu His Val
Arg Gly 1700 1705 1710 Gly Tyr Ile Leu Pro Trp Gln Glu Pro Ala Leu
Asn Thr His Leu 1715 1720 1725 Ser Arg Gln Lys Phe Met Gly Phe Lys
Ile Ala Leu Asp Asp Glu 1730 1735 1740 Gly Thr Ala Gly Gly Trp Leu
Phe Trp Asp Asp Gly Gln Ser Ile 1745 1750 1755 Asp Thr Tyr Gly Lys
Gly Leu Tyr Tyr Leu Ala Ser Phe Ser Ala 1760 1765 1770 Ser Gln Asn
Thr Met Gln Ser His Ile Ile Phe Asn Asn Tyr Ile 1775 1780 1785 Thr
Gly Thr Asn Pro Leu Lys Leu Gly Tyr Ile Glu Ile Trp Gly 1790 1795
1800 Val Gly Ser Val Pro Val Thr Ser Val Ser Ile Ser Val Ser Gly
1805 1810 1815 Met Val Ile Thr Pro Ser Phe Asn Asn Asp Pro Thr Thr
Gln Val 1820 1825 1830 Leu Ser Ile Asp Val Thr Asp Arg Asn Ile Ser
Leu His Asn Phe 1835 1840 1845 Thr Ser Leu Thr Trp Ile Ser Thr Leu
1850 1855 8 6513DNAHomo sapiens 8attgctaagc catccttcag acagagaggg
agcggctgca agaggtaatg agagatggca 60agaaagaagc tgaaaaaatt tactactttg
gagattgtgc tcagtgttct tctgcttgtg 120ttgtttatca tcagtattgt
tctaattgtg cttttagcca aagagtcact gaaatcaaca 180gccccagatc
ctgggacaac tggtacccca gatcctggga caactggtac cccagatcct
240ggaacaactg gtaccacaca tgctaggaca acgggtcccc cagatcctgg
aacaactggt 300accactcctg tttctgctga atgtccagtg gtaaatgaat
tggaacgaat taattgcatc 360cctgaccagc cgccaacaaa ggccacatgt
gaccaacgtg gctgttgctg gaatccccag 420ggagctgtaa gtgttccctg
gtgctactat tccaagaatc atagctacca tgtagagggc 480aaccttgtca
acacaaatgc aggattcaca gcccggttga aaaatctgcc ttcttcacca
540gtgtttggaa gcaatgttga caatgttctt ctcacagcag aatatcagac
atctaatcgt 600ttccacttta agttgactga ccaaaccaat aacaggtttg
aagtgcccca cgaacacgtg 660cagtccttca gtggaaatgc tgctgcttct
ttgacctacc aagttgaaat ctccagacag 720ccatttagca tcaaagtgac
cagaagaagc aacaatcgtg ttttgtttga ctcgagcatt 780gggcccctac
tgtttgctga ccagttcttg cagctctcca ctcgactgcc tagcactaac
840gtgtatggcc tgggagagca tgtgcaccag cagtatcggc atgatatgaa
ttggaagacc 900tggcccatat ttaacagaga cacaactccc aatggaaacg
gaactaattt gtatggtgcg 960cagacattct tcttgtgcct tgaagatgct
agtggattgt cctttggggt gtttctgatg 1020aacagcaatg ccatggaggt
tgtccttcag cctgcgccag ccatcactta ccgcaccatt 1080gggggcattc
tcgacttcta tgtgttcttg ggaaacactc cagagcaagt tgttcaagaa
1140tatctagagc tcattgggcg gccagccctt ccctcctact gggcgcttgg
atttcacctc 1200agtcgttacg aatatggaac cttagacaac atgagggaag
tcgtggagag aaatcgcgca 1260gcacagctcc cttatgatgt tcagcatgct
gatattgatt atatggatga gagaagggac 1320ttcacttatg attcagtgga
ttttaaaggc ttccctgaat ttgtcaacga gttacacaat 1380aatggacaga
agcttgtcat cattgtggat ccagccatct ccaacaactc ttcctcaagt
1440aaaccctatg gcccatatga caggggttca gatatgaaga tatgggtgaa
tagttcagat 1500ggagtgactc cactcattgg ggaggtctgg cctggacaaa
ctgtgtttcc tgattatacc 1560aatcccaact gtgctgtttg gtggacaaag
gaatttgagc tttttcacaa tcaagtagag 1620tttgatggaa tctggattga
tatgaatgaa gtctccaact ttgttgatgg ttcggtctca 1680ggatgttcca
caaacaacct aaataatccc ccattcactc ccagaatcct ggatgggtac
1740ctgttctgca agactctctg tatggatgca gtgcagcact ggggcaagca
gtatgacatt 1800cacaatctgt atggctactc catggcggtc gccacagcag
aagctgccaa gactgtgttc 1860cctaataaga gaagcttcat tctgacccgt
tctacctttg cgggctctgg caagtttgca 1920gcacattggt taggagacaa
cactgccacc tgggatgacc tgagatggtc catccctggc 1980gtgcttgagt
tcaacctttt tggcatccca atggtgggtc ctgacatatg tggctttgct
2040ttggacaccc ctgaggagct ctgtaggcgg tggatgcagt tgggtgcatt
ttatccgttt 2100tctagaaatc acaatggcca aggctacaag gaccaggatc
ctgcctcctt tggagctgac 2160tccctgctgt tgaattcctc caggcactac
cttaacatcc gctatactct attgccctac 2220ctatacaccc tcttcttccg
tgctcacagc cgaggggaca cggtggccag gccccttttg 2280catgagttct
acgaggacaa cagcacttgg gatgtgcacc aacagttctt atgggggccc
2340ggcctcctca tcactccagt tctggatgaa ggtgcagaga aagtgatggc
atatgtgcct 2400gatgctgtct ggtatgacta cgagactggg agccaagtga
gatggaggaa gcaaaaagtc 2460gagatggaac ttcctggaga caaaattgga
cttcaccttc gaggaggcta catcttcccc 2520acacagcagc caaatacaac
cactctggcc agtcgaaaga accctcttgg tcttatcatt 2580gccctagatg
agaacaaaga agcaaaagga gaacttttct gggataatgg ggaaacgaag
2640gatactgtgg ccaataaagt gtatctttta tgtgagtttt ctgtcactca
aaaccgcttg 2700gaggtgaata tttcacaatc aacctacaag gaccccaata
atttagcatt taatgagatt 2760aaaattcttg ggacggagga acctagcaat
gttacagtga aacacaatgg tgtcccaagt 2820cagacttctc ctacagtcac
ttatgattct aacctgaagg ttgccattat cacagatatt 2880gatcttctcc
tgggagaagc atacacagtg gaatggagca taaagataag ggatgaagaa
2940aaaatagact gttaccctga tgagaatggt gcttctgccg aaaactgcac
tgcccgtggc 3000tgtatctggg aggcatccaa ttcttctgga gtcccttttt
gctattttgt caacgaccta 3060tactctgtca gtgatgttca gtataattcc
catggggcca cagctgacat ctccttaaag 3120tcttccgttt atgccaatgc
cttcccctcc acacccgtga acccccttcg cctggatgtc 3180acttaccata
agaatgaaat gctgcagttc aagatttatg atcccaacaa gaatcggtat
3240gaagttccag tccctctgaa catacccagc atgccatcca gcacccctga
gggtcaactc 3300tatgatgtgc tcattaagaa gaatccattt gggattgaaa
ttcgccggaa gagtacaggc 3360actataattt gggactctca gctccttggc
tttaccttca gtgacatgtt tatccgcatc 3420tccacccgcc ttccctccaa
gtacctctat ggctttgggg aaactgagca caggtcctat 3480aggagagact
tggagtggca cacttggggg atgttctccc gagaccagcc cccagggtac
3540aagaagaatt cctatggtgt ccacccctac tacatggggc tggaggagga
cggcagtgcc 3600catggagtgc tcctgctgaa cagcaatgcc atggatgtga
cgttccagcc cctgcctgcc 3660ttgacatacc gcaccacagg gggagttctg
gacttttatg tgttcttggg gccgactcca 3720gagcttgtca cccagcagta
cactgagttg attggccggc ctgtgatggt accttactgg 3780tctttggggt
tccagctgtg tcgctatggc taccagaatg actctgagat cgccagcttg
3840tatgatgaga tggtggctgc ccagatccct tatgatgtgc agtactcaga
catcgactac 3900atggagcggc agctggactt caccctcagc cccaagtttg
ctgggtttcc agctctgatc 3960aatcgcatga aggctgatgg gatgcgggtc
atcctcattc tggatccagc catttctggc 4020aatgagacac agccttatcc
tgccttcact cggggcgtgg aggatgacgt cttcatcaaa 4080tacccaaatg
atggagacat tgtctgggga aaggtctggc ctgattttcc tgatgttgtt
4140gtgaatgggt ctctagactg ggacagccaa gtggagctat atcgagctta
tgtggccttc 4200ccagactttt tccgtaattc aactgccaag tggtggaaga
gggaaataga agaactatac 4260aacaatccac agaatccaga gaggagcttg
aagtttgatg gcatgtggat tgatatgaat 4320gaaccatcaa gcttcgtgaa
tggggcagtt tctccaggct gcagggacgc ctctctgaac 4380caccctccct
acatgccaca tttggagtcc agggacaggg gcctgagcag caagaccctt
4440tgtatggaga gtcagcagat cctcccagac ggctccctgg tgcagcacta
caacgtgcac 4500aacctgtatg ggtggtccca gaccagaccc acatacgaag
ccgtgcagga ggtgacggga 4560cagcgagggg tcgtcatcac ccgctccaca
tttccctctt ctggccgctg ggcaggacat 4620tggctgggag acaacacggc
cgcatgggat cagctgaaga agtctatcat tggcatgatg 4680gagttcagcc
tcttcggcat atcctatacg ggagcagata tctgtgggtt ctttcaagat
4740gctgaatatg agatgtgtgt tcgctggatg cagctggggg ccttttaccc
cttctcaaga 4800aaccacaaca ccattgggac caggagacaa gaccctgtgt
cctgggatgt tgcttttgtg 4860aatatttcca gaactgtcct gcagaccaga
tacaccctgt tgccatatct gtataccttg 4920atgcataagg cccacacgga
gggcgtcact gttgtgcggc ctctgctcca tgagtttgtg 4980tcagaccagg
tgacatggga catagacagt cagttcctgc tgggcccagc cttcctggtc
5040agccctgtcc tggagcgtaa tgccagaaat gtcactgcat atttccctag
agcccgctgg 5100tatgattact acacgggtgt ggatattaat gcaagaggag
agtggaagac cttgccagcc 5160cctcttgacc acattaatct tcatgtccgt
gggggctaca tcctgccctg gcaagagcct 5220gcactgaaca cccacttaag
ccgccagaaa ttcatgggct tcaaaattgc cttggatgat 5280gaaggaactg
ctgggggctg gctcttctgg gatgatgggc aaagcattga tacctatggg
5340aaaggactct attacttggc cagcttttct gccagccaga atacgatgca
aagccatata 5400attttcaaca attacatcac tggtacaaat cctttgaaac
tgggctacat tgaaatctgg 5460ggagtgggca gtgtccccgt taccagtgtc
agcatctctg tgagtggcat ggtcataaca 5520ccctccttca acaatgaccc
cacgacacag gtattaagca tcgatgtgac tgacagaaac 5580atcagcctac
ataattttac ttcattgacg tggataagca ctctgtgaat ttttacagca
5640agattctaac taactatgaa tgactttgaa actacttata cttcatactc
ataaaaatta 5700ttgtgtgttg ctaatttgtt catacccact attggtgaaa
tatttctgtt aattttgtta 5760tatgtttttt gtgtgaaccc taaaggttaa
accttagccc tgtgggatag gcagttaggg 5820aggtgtggaa aatctatgca
ttaccttaat gtctctgtgt ggttagtatg gtagtgactg 5880ttcatcatat
gacatttact gaagatgaac tgggtccatg atgaagtgtg tgtatgtcca
5940cgtttgtaat catagaatgg accccattct tttgttaaat acacaagaga
aagctttctg 6000tgacagttcc aggtcttgaa gctaatcagc atctcaagaa
agtatccaga aagaacatct 6060gctagttggt tataggcggt gggaggaata
atatacctaa ttggttatag gtggggggag 6120catgataagc aaagaaaagg
caaacacaag gaaagatcag atgaaacaga agatgatagt 6180aaaagtgatc
ctaagtaaga acataatgta aaattgtcag cagcctcatg gggaggaaaa
6240aggaagagtc aactcacttg aagaagaggg tcttgagaaa tccttagcat
aaagggctac 6300tggtgagatt gagatctgag caggcaaagc tcaaaagaga
gtttggaggt taaaaataat 6360ttatttttgc agtagtgtgc tttgaaatgt
gtaaatctta tttctaatgt atacaaccac 6420atttcacata aaaatatgca
atttatatgc cagataaaaa taaaacaagt gaatttgcaa 6480gtgaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaa 651391927PRTHomo sapiens 9Met Glu Leu Ser
Trp His Val Val Phe Ile Ala Leu Leu Ser Phe Ser 1 5 10 15 Cys Trp
Gly Ser Asp Trp Glu Ser Asp Arg Asn Phe Ile Ser Thr Ala 20 25 30
Gly Pro Leu Thr Asn Asp Leu Leu His Asn Leu Ser Gly Leu Leu Gly 35
40 45 Asp Gln Ser Ser Asn Phe Val Ala Gly Asp Lys Asp Met Tyr Val
Cys 50 55 60 His Gln Pro Leu Pro Thr Phe Leu Pro Glu Tyr Phe Ser
Ser Leu His 65 70 75 80 Ala Ser Gln Ile Thr His Tyr Lys Val Phe Leu
Ser Trp Ala Gln Leu 85 90 95 Leu Pro Ala Gly Ser Thr Gln Asn Pro
Asp Glu Lys Thr Val Gln Cys 100 105 110 Tyr Arg Arg Leu Leu Lys Ala
Leu Lys Thr Ala Arg Leu Gln Pro Met 115 120 125 Val Ile Leu His His
Gln Thr Leu Pro Ala Ser Thr Leu Arg Arg Thr 130 135 140 Glu Ala Phe
Ala Asp Leu Phe Ala Asp Tyr Ala Thr Phe Ala Phe His 145 150 155 160
Ser Phe Gly Asp Leu Val Gly Ile Trp Phe Thr Phe Ser Asp Leu Glu 165
170 175 Glu Val Ile Lys Glu Leu Pro His Gln Glu Ser Arg Ala Ser Gln
Leu 180 185 190 Gln Thr Leu Ser Asp Ala His Arg Lys Ala Tyr Glu Ile
Tyr His Glu 195 200 205 Ser Tyr Ala Phe Gln Gly Gly Lys Leu Ser Val
Val Leu Arg Ala Glu 210 215 220 Asp Ile Pro Glu Leu Leu Leu Glu Pro
Pro Ile Ser Ala Leu Ala Gln 225 230 235 240 Asp Thr Val Asp Phe Leu
Ser Leu Asp Leu Ser Tyr Glu Cys Gln Asn 245 250 255 Glu Ala Ser Leu
Arg Gln Lys Leu Ser Lys Leu Gln Thr Ile Glu Pro 260 265 270 Lys Val
Lys Val Phe Ile Phe Asn Leu Lys Leu Pro Asp Cys Pro Ser 275 280 285
Thr Met Lys Asn Pro Ala Ser Leu Leu Phe Ser Leu Phe Glu Ala Ile 290
295 300 Asn Lys Asp Gln Val Leu Thr Ile Gly Phe Asp Ile Asn Glu Phe
Leu 305 310 315 320 Ser Cys Ser Ser Ser Ser Lys Lys Ser Met Ser Cys
Ser Leu Thr Gly 325 330 335 Ser Leu Ala Leu Gln Pro Asp Gln Gln Gln
Asp His Glu Thr Thr Asp 340 345 350 Ser Ser Pro Ala Ser Ala Tyr Gln
Arg Ile Trp Glu Ala Phe Ala Asn 355 360 365 Gln Ser Arg Ala Glu Arg
Asp Ala Phe Leu Gln Asp Thr Phe Pro Glu 370 375 380 Gly Phe Leu Trp
Gly Ala Ser Thr Gly Ala Phe Asn Val Glu Gly Gly 385 390 395 400 Trp
Ala Glu Gly Gly Arg Gly Val Ser Ile Trp Asp Pro Arg Arg Pro 405 410
415 Leu Asn Thr Thr Glu Gly Gln Ala Thr Leu Glu Val Ala Ser Asp Ser
420 425 430 Tyr His Lys Val Ala Ser Asp Val Ala Leu Leu Cys Gly Leu
Arg Ala 435 440 445 Gln Val Tyr Lys Phe Ser Ile Ser Trp Ser Arg Ile
Phe Pro Met Gly 450 455 460 His Gly Ser Ser Pro Ser Leu Pro Gly Val
Ala Tyr Tyr Asn Lys Leu 465 470 475 480 Ile Asp Arg Leu Gln Asp Ala
Gly Ile Glu Pro Met Ala Thr Leu Phe 485 490 495 His Trp Asp Leu Pro
Gln Ala Leu Gln Asp His Gly Gly Trp Gln Asn 500 505 510 Glu Ser Val
Val Asp Ala Phe Leu Asp Tyr Ala Ala Phe Cys Phe Ser 515 520 525 Thr
Phe Gly Asp Arg Val Lys Leu Trp Val Thr Phe His Glu Pro Trp 530 535
540 Val Met Ser Tyr Ala Gly Tyr Gly Thr Gly Gln His Pro Pro Gly Ile
545 550 555 560 Ser Asp Pro Gly Val Ala Ser Phe Lys Val Ala His Leu
Val Leu Lys 565 570 575 Ala His Ala Arg Thr Trp His His Tyr Asn Ser
His His Arg Pro Gln 580 585 590 Gln Gln Gly His Val Gly Ile Val Leu
Asn Ser Asp Trp Ala Glu Pro 595 600 605 Leu Ser Pro Glu Arg Pro Glu
Asp Leu Arg Ala Ser Glu Arg Phe Leu 610 615 620 His Phe Met Leu Gly
Trp Phe Ala His Pro Val Phe Val Asp Gly Asp 625 630 635 640 Tyr Pro
Ala Thr Leu Arg Thr Gln Ile Gln Gln Met Asn Arg Gln Cys 645 650 655
Ser His Pro Val Ala Gln Leu Pro Glu Phe Thr Glu Ala Glu Lys Gln 660
665 670 Leu Leu Lys Gly Ser Ala Asp Phe Leu Gly Leu Ser His Tyr Thr
Ser 675 680 685 Arg Leu Ile Ser Asn Ala Pro Gln Asn Thr Cys Ile Pro
Ser Tyr Asp 690 695 700 Thr Ile Gly Gly Phe Ser Gln His Val Asn His
Val Trp Pro Gln Thr 705 710 715 720 Ser Ser Ser Trp Ile Arg Val Val
Pro Trp Gly Ile Arg Arg Leu Leu 725 730 735 Gln Phe Val Ser Leu Glu
Tyr Thr Arg Gly Lys Val Pro Ile Tyr Leu 740 745 750 Ala Gly Asn Gly
Met Pro Ile Gly Glu Ser Glu Asn Leu Phe Asp Asp 755 760 765 Ser Leu
Arg Val Asp Tyr Phe Asn Gln Tyr Ile Asn Glu Val Leu Lys 770 775 780
Ala Ile Lys Glu Asp Ser Val Asp Val Arg Ser Tyr Ile Ala Arg Ser 785
790 795 800 Leu Ile Asp Gly Phe Glu Gly Pro Ser Gly Tyr Ser Gln Arg
Phe Gly 805 810 815 Leu His His Val Asn Phe Ser Asp Ser Ser Lys Ser
Arg Thr Pro Arg 820 825 830 Lys Ser Ala Tyr Phe Phe Thr Ser Ile Ile
Glu Lys Asn Gly Phe Leu 835 840 845 Thr Lys Gly Ala Lys Arg Leu Leu
Pro Pro Asn Thr Val Asn Leu Pro 850 855 860 Ser Lys Val Arg Ala Phe
Thr Phe Pro Ser Glu Val Pro Ser Lys Ala 865 870 875 880 Lys Val Val
Trp Glu Lys Phe Ser Ser Gln Pro Lys Phe Glu Arg Asp 885 890 895 Leu
Phe Tyr His Gly Thr Phe Arg Asp Asp Phe Leu Trp Gly Val Ser 900 905
910 Ser Ser Ala Tyr Gln Ile Glu Gly Ala Trp Asp Ala Asp Gly Lys Gly
915 920 925 Pro Ser Ile Trp Asp Asn Phe Thr His Thr Pro Gly Ser Asn
Val Lys 930 935 940 Asp Asn Ala Thr Gly Asp Ile Ala Cys Asp Ser Tyr
His Gln Leu Asp 945 950 955 960 Ala Asp Leu Asn Met Leu Arg Ala Leu
Lys Val Lys Ala Tyr Arg Phe 965 970 975 Ser Ile Ser Trp Ser Arg Ile
Phe Pro Thr Gly Arg Asn Ser Ser Ile 980 985 990 Asn Ser His Gly Val
Asp Tyr Tyr
Asn Arg Leu Ile Asn Gly Leu Val 995 1000 1005 Ala Ser Asn Ile Phe
Pro Met Val Thr Leu Phe His Trp Asp Leu 1010 1015 1020 Pro Gln Ala
Leu Gln Asp Ile Gly Gly Trp Glu Asn Pro Ala Leu 1025 1030 1035 Ile
Asp Leu Phe Asp Ser Tyr Ala Asp Phe Cys Phe Gln Thr Phe 1040 1045
1050 Gly Asp Arg Val Lys Phe Trp Met Thr Phe Asn Glu Pro Met Tyr
1055 1060 1065 Leu Ala Trp Leu Gly Tyr Gly Ser Gly Glu Phe Pro Pro
Gly Val 1070 1075 1080 Lys Asp Pro Gly Trp Ala Pro Tyr Arg Ile Ala
His Ala Val Ile 1085 1090 1095 Lys Ala His Ala Arg Val Tyr His Thr
Tyr Asp Glu Lys Tyr Arg 1100 1105 1110 Gln Glu Gln Lys Gly Val Ile
Ser Leu Ser Leu Ser Thr His Trp 1115 1120 1125 Ala Glu Pro Lys Ser
Pro Gly Val Pro Arg Asp Val Glu Ala Ala 1130 1135 1140 Asp Arg Met
Leu Gln Phe Ser Leu Gly Trp Phe Ala His Pro Ile 1145 1150 1155 Phe
Arg Asn Gly Asp Tyr Pro Asp Thr Met Lys Trp Lys Val Gly 1160 1165
1170 Asn Arg Ser Glu Leu Gln His Leu Ala Thr Ser Arg Leu Pro Ser
1175 1180 1185 Phe Thr Glu Glu Glu Lys Arg Phe Ile Arg Ala Thr Ala
Asp Val 1190 1195 1200 Phe Cys Leu Asn Thr Tyr Tyr Ser Arg Ile Val
Gln His Lys Thr 1205 1210 1215 Pro Arg Leu Asn Pro Pro Ser Tyr Glu
Asp Asp Gln Glu Met Ala 1220 1225 1230 Glu Glu Glu Asp Pro Ser Trp
Pro Ser Thr Ala Met Asn Arg Ala 1235 1240 1245 Ala Pro Trp Gly Thr
Arg Arg Leu Leu Asn Trp Ile Lys Glu Glu 1250 1255 1260 Tyr Gly Asp
Ile Pro Ile Tyr Ile Thr Glu Asn Gly Val Gly Leu 1265 1270 1275 Thr
Asn Pro Asn Thr Glu Asp Thr Asp Arg Ile Phe Tyr His Lys 1280 1285
1290 Thr Tyr Ile Asn Glu Ala Leu Lys Ala Tyr Arg Leu Asp Gly Ile
1295 1300 1305 Asp Leu Arg Gly Tyr Val Ala Trp Ser Leu Met Asp Asn
Phe Glu 1310 1315 1320 Trp Leu Asn Gly Tyr Thr Val Lys Phe Gly Leu
Tyr His Val Asp 1325 1330 1335 Phe Asn Asn Thr Asn Arg Pro Arg Thr
Ala Arg Ala Ser Ala Arg 1340 1345 1350 Tyr Tyr Thr Glu Val Ile Thr
Asn Asn Gly Met Pro Leu Ala Arg 1355 1360 1365 Glu Asp Glu Phe Leu
Tyr Gly Arg Phe Pro Glu Gly Phe Ile Trp 1370 1375 1380 Ser Ala Ala
Ser Ala Ala Tyr Gln Ile Glu Gly Ala Trp Arg Ala 1385 1390 1395 Asp
Gly Lys Gly Leu Ser Ile Trp Asp Thr Phe Ser His Thr Pro 1400 1405
1410 Leu Arg Val Glu Asn Asp Ala Ile Gly Asp Val Ala Cys Asp Ser
1415 1420 1425 Tyr His Lys Ile Ala Glu Asp Leu Val Thr Leu Gln Asn
Leu Gly 1430 1435 1440 Val Ser His Tyr Arg Phe Ser Ile Ser Trp Ser
Arg Ile Leu Pro 1445 1450 1455 Asp Gly Thr Thr Arg Tyr Ile Asn Glu
Ala Gly Leu Asn Tyr Tyr 1460 1465 1470 Val Arg Leu Ile Asp Thr Leu
Leu Ala Ala Ser Ile Gln Pro Gln 1475 1480 1485 Val Thr Ile Tyr His
Trp Asp Leu Pro Gln Thr Leu Gln Asp Val 1490 1495 1500 Gly Gly Trp
Glu Asn Glu Thr Ile Val Gln Arg Phe Lys Glu Tyr 1505 1510 1515 Ala
Asp Val Leu Phe Gln Arg Leu Gly Asp Lys Val Lys Phe Trp 1520 1525
1530 Ile Thr Leu Asn Glu Pro Phe Val Ile Ala Tyr Gln Gly Tyr Gly
1535 1540 1545 Tyr Gly Thr Ala Ala Pro Gly Val Ser Asn Arg Pro Gly
Thr Ala 1550 1555 1560 Pro Tyr Ile Val Gly His Asn Leu Ile Lys Ala
His Ala Glu Ala 1565 1570 1575 Trp His Leu Tyr Asn Asp Val Tyr Arg
Ala Ser Gln Gly Gly Val 1580 1585 1590 Ile Ser Ile Thr Ile Ser Ser
Asp Trp Ala Glu Pro Arg Asp Pro 1595 1600 1605 Ser Asn Gln Glu Asp
Val Glu Ala Ala Arg Arg Tyr Val Gln Phe 1610 1615 1620 Met Gly Gly
Trp Phe Ala His Pro Ile Phe Lys Asn Gly Asp Tyr 1625 1630 1635 Asn
Glu Val Met Lys Thr Arg Ile Arg Asp Arg Ser Leu Ala Ala 1640 1645
1650 Gly Leu Asn Lys Ser Arg Leu Pro Glu Phe Thr Glu Ser Glu Lys
1655 1660 1665 Arg Arg Ile Asn Gly Thr Tyr Asp Phe Phe Gly Phe Asn
His Tyr 1670 1675 1680 Thr Thr Val Leu Ala Tyr Asn Leu Asn Tyr Ala
Thr Ala Ile Ser 1685 1690 1695 Ser Phe Asp Ala Asp Arg Gly Val Ala
Ser Ile Ala Asp Arg Ser 1700 1705 1710 Trp Pro Asp Ser Gly Ser Phe
Trp Leu Lys Met Thr Pro Phe Gly 1715 1720 1725 Phe Arg Arg Ile Leu
Asn Trp Leu Lys Glu Glu Tyr Asn Asp Pro 1730 1735 1740 Pro Ile Tyr
Val Thr Glu Asn Gly Val Ser Gln Arg Glu Glu Thr 1745 1750 1755 Asp
Leu Asn Asp Thr Ala Arg Ile Tyr Tyr Leu Arg Thr Tyr Ile 1760 1765
1770 Asn Glu Ala Leu Lys Ala Val Gln Asp Lys Val Asp Leu Arg Gly
1775 1780 1785 Tyr Thr Val Trp Ser Ala Met Asp Asn Phe Glu Trp Ala
Thr Gly 1790 1795 1800 Phe Ser Glu Arg Phe Gly Leu His Phe Val Asn
Tyr Ser Asp Pro 1805 1810 1815 Ser Leu Pro Arg Ile Pro Lys Ala Ser
Ala Lys Phe Tyr Ala Ser 1820 1825 1830 Val Val Arg Cys Asn Gly Phe
Pro Asp Pro Ala Thr Gly Pro His 1835 1840 1845 Ala Cys Leu His Gln
Pro Asp Ala Gly Pro Thr Ile Ser Pro Val 1850 1855 1860 Arg Gln Glu
Glu Val Gln Phe Leu Gly Leu Met Leu Gly Thr Thr 1865 1870 1875 Glu
Ala Gln Thr Ala Leu Tyr Val Leu Phe Ser Leu Val Leu Leu 1880 1885
1890 Gly Val Cys Gly Leu Ala Phe Leu Ser Tyr Lys Tyr Cys Lys Arg
1895 1900 1905 Ser Lys Gln Gly Lys Thr Gln Arg Ser Gln Gln Glu Leu
Ser Pro 1910 1915 1920 Val Ser Ser Phe 1925 10 6274DNAHomo sapiens
10gttcctagaa aatggagctg tcttggcatg tagtctttat tgccctgcta agtttttcat
60gctgggggtc agactgggag tctgatagaa atttcatttc caccgctggt cctctaacca
120atgacttgct gcacaacctg agtggtctcc tgggagacca gagttctaac
tttgtagcag 180gggacaaaga catgtatgtt tgtcaccagc cactgcccac
tttcctgcca gaatacttca 240gcagtctcca tgccagtcag atcacccatt
ataaggtatt tctgtcatgg gcacagctcc 300tcccagcagg aagcacccag
aatccagacg agaaaacagt gcagtgctac cggcgactcc 360tcaaggccct
caagactgca cggcttcagc ccatggtcat cctgcaccac cagaccctcc
420ctgccagcac cctccggaga accgaagcct ttgctgacct cttcgccgac
tatgccacat 480tcgccttcca ctccttcggg gacctagttg ggatctggtt
caccttcagt gacttggagg 540aagtgatcaa ggagcttccc caccaggaat
caagagcgtc acaactccag accctcagtg 600atgcccacag aaaagcctat
gagatttacc acgaaagcta tgcttttcag ggcggaaaac 660tctctgttgt
cctgcgagct gaagatatcc cggagctcct gctagaacca cccatatctg
720cgcttgccca ggacacggtc gatttcctct ctcttgattt gtcttatgaa
tgccaaaatg 780aggcaagtct gcggcagaag ctgagtaaat tgcagaccat
tgagccaaaa gtgaaagttt 840tcatcttcaa cctaaaactc ccagactgcc
cctccaccat gaagaaccca gccagtctgc 900tcttcagcct ttttgaagcc
ataaataaag accaagtgct caccattggg tttgatatta 960atgagtttct
gagttgttca tcaagttcca agaaaagcat gtcttgttct ctgactggca
1020gcctggccct tcagcctgac cagcagcagg accacgagac cacggactcc
tctcctgcct 1080ctgcctatca gagaatctgg gaagcatttg ccaatcagtc
cagggcggaa agggatgcct 1140tcctgcagga tactttccct gaaggcttcc
tctggggtgc ctccacagga gcctttaacg 1200tggaaggagg ctgggccgag
ggtgggagag gggtgagcat ctgggatcca cgcaggcccc 1260tgaacaccac
tgagggccaa gcgacgctgg aggtggccag cgacagttac cacaaggtag
1320cctctgacgt cgccctgctt tgcggcctcc gggctcaggt gtacaagttc
tccatctcct 1380ggtcccggat cttccccatg gggcacggga gcagccccag
cctcccaggc gttgcctact 1440acaacaagct gattgacagg ctacaggatg
cgggcatcga gcccatggcc acgctgttcc 1500actgggacct gcctcaggcc
ctgcaggatc atggtggatg gcagaatgag agcgtggtgg 1560atgccttcct
ggactatgcg gccttctgct tctccacatt tggggaccgt gtgaagctgt
1620gggtgacctt ccatgagccg tgggtgatga gctacgcagg ctatggcacc
ggccagcacc 1680ctcccggcat ctctgaccca ggagtggcct cttttaaggt
ggctcacttg gtcctcaagg 1740ctcatgccag aacttggcac cactacaaca
gccatcatcg cccacagcag caggggcacg 1800tgggcattgt gctgaactca
gactgggcag aacccctgtc tccagagagg cctgaggacc 1860tgagagcctc
tgagcgcttc ttgcacttca tgctgggctg gtttgcacac cccgtctttg
1920tggatggaga ctacccagcc accctgagga cccagatcca acagatgaac
agacagtgct 1980cccatcctgt ggctcaactc cccgagttca cagaggcaga
gaagcagctc ctgaaaggct 2040ctgctgattt tctgggtctg tcgcattaca
cctcccgcct catcagcaac gccccacaaa 2100acacctgcat ccctagctat
gataccattg gaggcttctc ccaacacgtg aaccatgtgt 2160ggccccagac
ctcatcctct tggattcgtg tggtgccctg ggggataagg aggctgttgc
2220agtttgtatc cctggaatac acaagaggaa aagttccaat ataccttgcc
gggaatggca 2280tgcccatagg ggaaagtgaa aatctctttg atgattcctt
aagagtagac tacttcaatc 2340aatatatcaa tgaggtgctc aaggctatca
aggaagactc tgtggatgtt cgttcctaca 2400ttgctcgttc cctcattgat
ggcttcgaag gcccttctgg ttacagccag cggtttggcc 2460tgcaccacgt
caacttcagc gacagcagca agtcaaggac tcccaggaaa tctgcctact
2520ttttcactag catcatagaa aagaacggtt tcctcaccaa gggggcaaaa
agactgctac 2580cacctaatac agtaaacctc ccctccaaag tcagagcctt
cacttttcca tctgaggtgc 2640cctccaaggc taaagtcgtt tgggaaaagt
tctccagcca acccaagttc gaaagagatt 2700tgttctacca cgggacgttt
cgggatgact ttctgtgggg cgtgtcctct tccgcttatc 2760agattgaagg
cgcgtgggat gccgatggca aaggccccag catctgggat aactttaccc
2820acacaccagg gagcaatgtg aaagacaatg ccactggaga catcgcctgt
gacagctatc 2880accagctgga tgccgatctg aatatgctcc gagctttgaa
ggtgaaggcc taccgcttct 2940ctatctcctg gtctcggatt ttcccaactg
ggagaaacag ctctatcaac agtcatgggg 3000ttgattatta caacaggctg
atcaatggct tggtggcaag caacatcttt cccatggtga 3060cattgttcca
ttgggacctg ccccaggccc tccaggatat cggaggctgg gagaatcctg
3120ccttgattga cttgtttgac agctacgcag acttttgttt ccagaccttt
ggtgatagag 3180tcaagttttg gatgactttt aatgagccca tgtacctggc
atggctaggt tatggctcag 3240gggaatttcc cccaggggtg aaggacccag
gctgggcacc atataggata gcccacgccg 3300tcatcaaagc ccatgccaga
gtctatcaca cgtacgatga gaaatacagg caggagcaga 3360agggggtcat
ctcgctgagc ctcagtacac actgggcaga gcccaagtca ccaggggtcc
3420ccagagatgt ggaagccgct gaccgaatgc tgcagttctc cctgggctgg
tttgctcacc 3480ccatttttag aaacggagac tatcctgaca ccatgaagtg
gaaagtgggg aacaggagtg 3540aactgcagca cttagccacc tcccgcctgc
caagcttcac tgaggaagag aagaggttca 3600tcagggcgac ggccgacgtc
ttctgcctca acacgtacta ctccagaatc gtgcagcaca 3660aaacacccag
gctaaaccca ccctcctacg aagacgacca ggagatggct gaggaggagg
3720acccttcgtg gccttccacg gcaatgaaca gagctgcgcc ctgggggacg
cgaaggctgc 3780tgaactggat caaggaagag tatggtgaca tccccattta
catcaccgaa aacggagtgg 3840ggctgaccaa tccgaacacg gaggatactg
ataggatatt ttaccacaaa acctacatca 3900atgaggcttt gaaagcctac
aggctcgatg gtatagacct tcgagggtat gtcgcctggt 3960ctctgatgga
caactttgag tggctaaatg gctacacggt caagtttgga ctgtaccatg
4020ttgatttcaa caacacgaac aggcctcgca cagcaagagc ctccgccagg
tactacacag 4080aggtcattac caacaacggc atgccactgg ccagggagga
tgagtttctg tacggacggt 4140ttcctgaggg cttcatctgg agtgcagctt
ctgctgcata tcagattgaa ggtgcgtgga 4200gagcagatgg caaaggactc
agcatttggg acacgttttc tcacacacca ctgagggttg 4260agaacgatgc
cattggagac gtggcctgtg acagttatca caagattgct gaggatctgg
4320tcaccctgca gaacctgggc gtgtcccact accgtttttc catctcctgg
tctcgcatcc 4380tccctgatgg aaccaccagg tacatcaatg aagcgggcct
gaactactac gtgaggctca 4440tcgatacact gctggccgcc agcatccagc
cccaggtgac catttaccac tgggacctac 4500cacagacgct ccaagatgta
ggaggctggg agaatgagac catcgtgcag cggtttaagg 4560agtatgcaga
tgtgctcttc cagaggctgg gagacaaggt gaagttttgg atcacgctga
4620atgagccctt tgtcattgct taccagggct atggctacgg aacagcagct
ccaggagtct 4680ccaataggcc tggcactgcc ccctacattg ttggccacaa
tctaataaag gctcatgctg 4740aggcctggca tctgtacaac gatgtgtacc
gcgccagtca aggtggcgtg atttccatca 4800ccatcagcag tgactgggct
gaacccagag atccctctaa ccaggaggat gtggaggcag 4860ccaggagata
tgttcagttc atgggaggct ggtttgcaca tcctattttc aagaatggag
4920attacaatga ggtgatgaag acgcggatcc gtgacaggag cttggctgca
ggcctcaaca 4980agtctcggct gccagaattt acagagagtg agaagaggag
gatcaacggc acctatgact 5040tttttgggtt caatcactac accactgtcc
tcgcctacaa cctcaactat gccactgcca 5100tctcttcttt tgatgcagac
agaggagttg cttccatcgc agatcgctcg tggccagact 5160ctggctcctt
ctggctgaag atgacgcctt ttggcttcag gaggatcctg aactggttaa
5220aggaggaata caatgaccct ccaatttatg tcacagagaa tggagtgtcc
cagcgggaag 5280aaacagacct caatgacact gcaaggatct actaccttcg
gacttacatc aatgaggccc 5340tcaaagctgt gcaggacaag gtggaccttc
gaggatacac agtttggagt gcgatggaca 5400attttgagtg ggccacaggc
ttttcagaga gatttggtct gcattttgtg aactacagtg 5460acccttctct
gccaaggatc cccaaagcat cagcgaagtt ctacgcctct gtggtccgat
5520gcaatggctt ccctgacccc gctacagggc ctcacgcttg tctccaccag
ccagatgctg 5580gacccaccat cagccccgtg agacaggagg aggtgcagtt
cctggggcta atgctcggca 5640ccacagaagc acagacagct ttgtacgttc
tcttttctct tgtgcttctt ggagtctgtg 5700gcttggcatt tctgtcatac
aagtactgca agcgctctaa gcaagggaaa acacaacgaa 5760gccaacagga
attgagcccg gtgtcttcat tctgatgagt taccacctca agttctatga
5820agcaggccta gtttcttcat ctatgtttac cggccaccaa acaccttagg
gtcttagact 5880ctgctgatac tggacttctc cataaagtcc tgctgcaccg
ttagagatga ctttaatctt 5940gaatgatttc gacttgctga gtaaaatgga
aatatctcca tcttgctcca gtatcagagt 6000tcatttgggc atttgagaag
caagtagctc ttgcggaaac gtgtagatac tggtctagtg 6060ggtctgtgaa
ccacttaatt gaacttaaca gggctgtttt aagtttcaga gttgttaagg
6120gttgttaagg gagcaaaaac cgtaaaaatc cttcctataa gaagaaatca
actccattgc 6180atagactgca atatcatctc ctgcccttct gcaagctctc
cctagcttca catcttgtgt 6240tttccagaaa ataaaaacag cagactgtcc tttc
62741121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 11cgcgaaaaac cttacctagc c 211219DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
12gacgtgtgag gccctagcc 191323DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 13cacaggtgct gcatggctgt cgt
231424DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 14ccgcaaggga atctggacac aggt 241519DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
15gtgccagcmg ccgcggtaa 191619DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 16gactaccagg gtatctaat
191776DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 17cggtggatga tgtggattaa ttcgaygcaa cgcgaaaaac
cttacsyagc cttgacatgy 60crrgaabbyb bvdkrr 761860DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
18ccctctgttc cgaccattgt atgacgtgtg argcccyagc crtaagggcc atgaggactt
6019100DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 19gtrcccghaa grgaryyygr rcacaggtgc tgcatggctg
tcgtcagctc gtgtcgtgrg 60atrtyrrgtt argtcccgca rcragcgcaa ccctygwcay
1002020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 20ggatgcactg ctgtgatgag 202120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
21cccactgacc tatcctcgtg 202220DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 22aacaggcacg tggaggagtt
202320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 23cccacctcag cctcttgagt 202441DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
24gccttgccag cccgctcagt cagagtttga tcctggctca g 412548DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
25gcctccctcg cgccatcagn nnnnnnncat gctgcctccc gtaggagt
482628DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 26tcttcatgag ttttatgagg atacgaac
282725DNAArtificial SequenceDescription of Artificial Sequence
Synthetic
primer 27tttgcaccag attcataatc atacc 252835DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
28cagatactgt gagtgcctac atccctgatg ctatt 352921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
29taccttgatg cataaggccc a 213019DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 30ggcattacgc tccaggaca
193123DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 31cgtcactgtt gtgcggcctc tgc 233223DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
32caggaatcaa gagcgtcaca act 233319DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 33aaatcgaccg tgtcctggg
193428DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 34tcctgctaga accacccata tctgcgct
283519DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 35gctcatgccc aatggactg 193620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
36cggaccttgg cgtagatgtc 203723DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 37acagcgccag caccctcttc acc
233824DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 38agttagatga ggaagtcaaa gcaa 243918DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
39taggctgtcg gtagctgg 184037DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 40acaaagcttg aaaagactca
gaggatatga tgatgtc 374121DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 41catgcgctga acttcatcaa a
214220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 42ggttggacgc tgtccacttc 204324DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
43cggccgtctt tcagcagctc ttcc 244420DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
44ggcagccaag tgaaaaccag 204519DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 45tccggatggt gatgtagcg
194621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 46accaccagcg gctggagctg g 214717DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
47agcctcgcct ttgccga 174815DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 48ctggtgcctg gggcg
154922DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 49ccgccgcccg tccacacccg cc 225019DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
50cctgttcgac agtcagccg 195120DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 51cgaccaaatc cgttgactcc
205219DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 52cgtcgccagc cgagccaca 195318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
53aacgctagct acaggctt 185418DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 54ccaatgtggg ggaccttc
185525DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 55ggagyatgtg gtttaattcg aagca 255620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
56agctgacgac aaccatgcac 205719DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 57gtgccagcmg ccgcggtaa
195819DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 58gactaccagg gtatctaat 1959220DNASutterella sp.
59ttgacatgcc aggaatcctg aagagattcg ggagtgcccg caagggaatc tggacacagg
60tgctgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg
120caacccttgt cactagttgc tacgcaagag cactctagtg agactgccgg
tgacaaaccg 180gaggaaggtg gggatgacgt caagtcctca tggcccttat
22060220DNASutterella sp. 60ttgacatgcc aggaaggcct gagagatcag
gccgtgcccg caagggaatc tggacacagg 60tgctgcatgg ctgtcgtcag ctcgtgtcgt
gagatgttgg gttaagtccc gcaacgagcg 120caacccttgt cattagttgc
tacgaaaggg cactctaatg agactgccgg tgacaaaccg 180gaggaaggtg
gggatgacgt caagtcctca tggcccttat 22061676DNASutterlla sp.
61tacgtagggt gcaagcgtta atcggaatta ctgggcgtaa agcgtgcgca ggcggttctg
60taagacagat gtgaaatccc cgggctcaac ctgggaattg catttgtgac tgcaggacta
120gagttcatca gaggggggtg gaattccaag tgtagcagtg aaatgcgtag
atatttggaa 180gaacaccaat ggcgaaggca gccccctggg atgcgactga
cgctcatgca cgaaagcgtg 240gggagcaaac aggattagat accctggtag
tccacgccct aaacgatgtc tactggttgt 300tggggtttat taaccttggt
aacgaagcta acgcgtgaag tagaccgcct ggggagtacg 360gtcgcaagat
taaaactcaa aggaattgac ggggacccgc acaagcggtg gatgatgtgg
420attaattcga tgcaacgcga aaaaccttac ctagccttga catgccagga
atcctgaaga 480gattcgggag tgcccgcaag ggaatctgga cacaggtgct
gcatggctgt cgtcagctcg 540tgtcgtgaga tgttgggtta agtcccgcaa
cgagcgcaac ccttgtcact agttgctacg 600caagagcact ctagtgagac
tgccggtgac aaaccggagg aaggtgggga tgacgtcaag 660tcctcatggc ccttat
67662676DNASutterlla sp. 62tacgtagggt gcaagcgtta atcggaatta
ctgggcgtaa agcgtgcgca ggcggttctg 60taagacagat gtgaaatccc cgggctcaac
ctgggaattg catttgtgac tgcaggacta 120gagttcatca gaggggggtg
gaattccaag tgtagcagtg aaatgcgtag atatttggaa 180gaacaccaat
ggcgaaggca gccccctggg atgcgactga cgctcatgca cgaaagcgtg
240gggagcaaac aggattagat accctggtag tccacgccct aaacgatgtc
tactggttgt 300tggggttttt taaccttggt aacgaagcta acgcgtgaag
tagaccgcct ggggagtacg 360gtcgcaagat taaaactcaa aggaattgac
ggggacccgc acaagcggtg gatgatgtgg 420attaattcga tgcaacgcga
aaaaccttac ctagccttga catgccagga atcctgaaga 480gattcgggag
tgcccgcaag ggaatctgga cacaggtgct gcatggctgt cgtcagctcg
540tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgtcact
agttgctacg 600caagagcact ctagtgagac tgccggtgac aaaccggagg
aaggtgggga tgacgtcaag 660tcctcatggc ccttat 67663676DNASutterlla sp.
63tacgtagggt gcaagcgtta atcggaatta ctgggcgtaa agcgtgcgca ggcggttctg
60taagatagat gtgaaatccc cgggctcaac ctgggaattg catatatgac tgcaggactt
120gagtttgtca gaggagggtg gaattccacg tgtagcagtg aaatgcgtag
atatgtggaa 180gaacaccgat ggcgaaggca gccctctggg acatgactga
cgctcatgca cgaaagcgtg 240gggagcaaac aggattagat accctggtag
tccacgccct aaacgatgtc tactagttgt 300tggggacgat agtccttggt
aacgcagcta acgcgtgaag tagaccgcct ggggagtacg 360gtcgcaagat
taaaactcaa aggaattgac ggggacccgc acaagcggtg gatgatgtgg
420attaattcga tgcaacgcga aaaaccttac ctagccttga catgccagga
aggcctgaga 480gatcaggccg tgcccgcaag ggaatctgga cacaggtgct
gcatggctgt cgtcagctcg 540tgtcgtgaga tgttgggtta agtcccgcaa
cgagcgcaac ccttgtcatt agttgctacg 600aaagggcact ctaatgagac
tgccggtgac aaaccggagg aaggtgggga tgacgtcaag 660tcctcatggc ccttat
67664678DNASutterlla sp. 64tacgtagggt gcgagcgtta atcggaatta
ctgggcgtaa agcgtgcgca ggcggttggg 60taagacagat gtgaaatccc cgggcttaac
ctgggaactg catttgtgac tgtccgactg 120gagtatgtca gaggggggtg
gaattccaag tgtagcagtg aaatgcgtag atatttggaa 180gaacaccgat
ggcgaaggca gccccctggg gcaaaactga cgctcatgca cgaaagcgtg
240gggagcaaac aggattagat accctggtag tccacgccct aaacgatgtc
tactggttgt 300tggagggtaa aaccttcagt aacgaagcta acgcgtgaag
tagaccgcct ggggagtacg 360gtcgcaagat taaaactcaa aggaattgac
ggggacccgc acaagcggtg gatgatgtgg 420attaattcga tgcaacgcga
aaaaccttac ctagccttga catgtcagga acgctccgga 480gatggggcgg
tgcccgcaag ggaacctgag cacaggtgct gcatggctgt cgtcagctcg
540tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgtcact
agttgctacg 600taacagagca ctctagtgag actgccggtg acaaaccgga
ggaaggtggg gatgatgtca 660agtcctcatg gcccttat 6786519DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
65gtgccagcmg ccgcggtaa 196619DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 66gacgtgtgag gccctagcc 19
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References