U.S. patent application number 13/011362 was filed with the patent office on 2011-05-19 for microsatellite markers of schizophrenia.
This patent application is currently assigned to UNIVERSITY OF LOUISVILLE RESEARCH FOUNDATION, INC.. Invention is credited to Mark David Brennan, Jodi Ann Condra, Amy Tabb Massey, Holly Neibergs, Wei Wei.
Application Number | 20110117215 13/011362 |
Document ID | / |
Family ID | 36615576 |
Filed Date | 2011-05-19 |
United States Patent
Application |
20110117215 |
Kind Code |
A1 |
Brennan; Mark David ; et
al. |
May 19, 2011 |
Microsatellite Markers of Schizophrenia
Abstract
The invention includes method of determining if a subject is at
risk for developing schizophrenia (SZ), schizotypal personality
disorder (SPD), or schizoaffective disorder (SD).
Inventors: |
Brennan; Mark David;
(Jeffersonville, IN) ; Condra; Jodi Ann;
(Louisville, KY) ; Massey; Amy Tabb; (Louisville,
KY) ; Wei; Wei; (Galveston, TX) ; Neibergs;
Holly; (Palouse, WA) |
Assignee: |
UNIVERSITY OF LOUISVILLE RESEARCH
FOUNDATION, INC.
Louisville
KY
|
Family ID: |
36615576 |
Appl. No.: |
13/011362 |
Filed: |
January 21, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11323061 |
Dec 30, 2005 |
7892735 |
|
|
13011362 |
|
|
|
|
60640707 |
Dec 30, 2004 |
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Current U.S.
Class: |
424/722 ;
514/221; 514/253.04; 514/651 |
Current CPC
Class: |
C12Q 1/6883 20130101;
C12Q 2600/156 20130101; C12Q 2600/172 20130101; A61P 25/18
20180101 |
Class at
Publication: |
424/722 ; 435/6;
514/253.04; 514/651; 514/221 |
International
Class: |
A61K 33/00 20060101
A61K033/00; C12Q 1/68 20060101 C12Q001/68; A61K 31/497 20060101
A61K031/497; A61K 31/137 20060101 A61K031/137; A61K 31/5513
20060101 A61K031/5513; A61P 25/18 20060101 A61P025/18 |
Claims
1. A method of determining a human subject's risk of developing
schizophrenia (SZ), the method comprising: obtaining a sample
comprising DNA from the subject; and determining the size of both
alleles of microsatellite marker D22s256 in a sample from the
subject, wherein the presence of apparently homozygous alleles
indicates that the subject has an increased risk of developing the
disorder.
2. The method of claim 1, wherein the size of microsatellite marker
D22s256 is determined using PCR.
3. The method of claim 2, wherein the PCR is performed with a first
primer comprising SEQ ID NO:3, and a second primer comprising SEQ
ID NO:4.
4. The method of claim 1, wherein the sample is obtained from the
subject by a health care provider.
5. The method of claim 1, wherein the sample is provided by the
subject without the assistance of a health care provider.
6. The method of claim 1, further comprising determining the
presence or absence of one or more additional markers associated
with schizophrenia.
7. The method of claim 1, wherein the subject is a patient having,
or at risk of, schizophrenia.
8. The method of claim 1, wherein the subject is suffering from
early, intermediate or aggressive schizophrenia.
9. The method of claim 1, wherein the subject has one or more risk
factors associated with SZ.
10. The method of claim 9, wherein the risk factors associated with
SZ include one or more of: a relative afflicted with schizophrenia,
a genetically based phenotypic trait associated with risk for SZ;
deficits in working memory; and mixed-handedness, particularly in
females.
11. The method of claim 10, wherein the subject has one or more of
a grandparent, parent, uncle or aunt, sibling, or child who has or
had SZ.
12. The method of claim 10, wherein the genetically based
phenotypic is eye tracking dysfunction.
13. The method of claim 1, wherein the subject is a child, fetus,
or embryo, and one of the relatives of the subject has SZ.
14. The method of claim 1, further comprising administering a
treatment to a subject identified as being at increased risk for
developing SZ.
15. The method of claim 14, wherein the treatment is a
pharmacological or psychosocial treatment for SZ.
16. The method of claim 1, further comprising using the information
to select a subject population for a clinical trial.
17. The method of claim 1, further comprising using the information
to stratify a subject population in a clinical trial.
18. The method of claim 1, further comprising using the information
to stratify subjects that respond to a treatment from those who do
not respond to a treatment, or subjects that have negative side
effects from those who do not have negative side effects.
19. A method of selecting a human subject for inclusion or
exclusion in a clinical trial, the method comprising: obtaining a
sample comprising DNA from the subject; and determining the size of
both alleles of microsatellite marker D22s256 in a sample from the
subject, wherein the presence of homozygous alleles indicates that
the subject has an increased risk of developing the disorder; and
including or excluding the subject based on presence of homozygous
alleles.
20. The method of claim 19, wherein the clinical trial is of a
treatment for SZ.
Description
CLAIM OF PRIORITY
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/323,061, filed on Dec. 30, 2005, which claims the
benefit of U.S. Provisional Patent Application Ser. No. 60/640,707,
filed on Dec. 30, 2004. The entire contents of the foregoing are
hereby incorporated by reference.
STATE SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made in part with an award from the
Kentucky Science and Technology Corporation under Contract No.
144-401-06.
TECHNICAL FIELD
[0003] This invention relates to genetic markers of schizophrenia,
and methods of use thereof.
BACKGROUND
[0004] Numerous linkage and association studies have implicated
chromosome 22q in the etiology of schizophrenia (Vallada et al.,
Psychiatr. Genet. 5:127-30 (1995); Gill et al., Am. J. Med. Genet.
16:40-5 (1996); Myles-Worsley et al., Am. J. Med. Genet. 88:544-50
(1999); Jorgensen et al., Am. J. Med. Genet. 114:245-52 (2002);
DeLisi et al., Am. J. Psychiatry 159:803-12 (2002); Lewis et al.,
Am. J. Hum. Genet. 73:34-48 (2003); Takahashi et al., Am. J. Med.
Genet. 120B:11-7 (2003)). Nonetheless, the precise location of the
genes involved has yet to be resolved.
[0005] Possibly owing to genetic heterogeneity, analyses of
positional candidates on this chromosome have resulted in
conflicting results. The 22q11 region has received much attention,
as its deletion in velo-cardio-facial syndrome correlates with
increased propensity to develop schizophrenia (Ivanov et al., Br J
Psychiatry. 183:409-13 (2003); van Amelsvoort et al., Genetic Curr.
Psychiatry. Rep. 6:176-82 (2004); Williams and Owen, Curr
Psychiatry Rep. 6(3):176-82 (2004)). Candidates identified in this
region include the catechol-O-methyltransferase (COMT) gene, an
attractive candidate whose role has recently been challenged, and
proline dehydrogenase, a gene whose role may be limited to Chinese
lineages (Shifman et al., Am. J. Hum. Genet. 71:1296-302 (2002);
Williams and Owen, (2004), supra; McGuffin et al., Curr.
Psychiatry. Rep. 5:121-7 (2003); Williams et al., Am. J. Med.
Genet. 120B:42-6 (2003); Handoko et al., Mol Psychiatry. 10:589-597
(2005) [Epub ahead of print Oct. 26, 2004]; Shirts and Nimgaonkar,
Curr. Psychiatry. Rep. 6:303-12 (2004)). Other studies suggest a
more distal location for a susceptibility gene in 22q12 or 22q13
(DeLisi et al., 2002, supra; Takahashi et al., Am. J. Med. Genet.
120B:11-7 (2003) et al., 2003). Here again, however, family-based
transmission studies and evaluation of specific candidate genes
have provided somewhat modest or, at times, contradictory, results
(Vallada et al., Psychiatr. Genet. 5:127-30 (1995); Stober et al.,
Am. J. Med. Genet. 96:392-7 (2000); Meyer et al., Mol. Psychiatry
6:302-6 (2001); Takahashi et al., Am. J. Med. Genet. 120B:11-7
(2003); Georgieva et al., Psychiatr. Genet. 13:103-6 (2003);
Kaganovich et al., Am. J. Med. Genet. 125B:31-7 (2004)).
[0006] Due to the severity of the disorder, the negative impact of
a psychotic episode on a patient, and the diminishing recovery
after each psychotic episode, there is a need to more conclusively
identify individuals who have or are at risk of developing
schizophrenia (SZ), schizotypal personality disorder (SPD) or
schizoaffective disorder (SD), for example, to confirm clinical
diagnoses, to allow for prophylactic therapies, to determine
appropriate therapies based on their genotypic subtype, and to
provide genetic counseling for prospective parents with a history
of the disorder.
SUMMARY
[0007] In previous work the present inventors developed a high
quality linkage genetic map of chromosome 22 that included two
extreme distal markers (Brennan et al., Genomics 63:430-432 (2000);
Matise et al., Am. J. Hum. Genet. 70:1398-410 (2002)). These and
other highly informative microsatellite markers (including a new
microsatellite marker targeting the promoter region of the Sult4a1
gene) were used to evaluated 27 families from the NIMH
Schizophrenia Genetics Initiative. Based on the linkage and
family-based association patterns that were observed, a multi-locus
model involving at least the Sult4A1 region and a more distal
region near marker D22s256 in 22q13 is described herein. Thus, the
invention includes methods of determining risk of developing
schizophrenia (SZ), schizotypal personality disorder (SPD) or
schizoaffective disorder (SD) as described herein.
[0008] In one aspect, the invention includes methods for obtaining
information regarding a subject's risk for developing SZ, SD or
SPD. The methods include obtaining a test haplotype associated with
schizophrenia as described herein. The methods can also include
obtaining a sample comprising genomic DNA (gDNA) from the subject,
and determining the identity, absence or presence of a test
haplotype associated with SZ, SD or SPD as described herein. In
some embodiments, the methods include obtaining a test haplotype
for the subject comprising at least one test marker that is within
1 linkage disequilibrium unit (1 LDU) of a marker listed in Table
4, 6, 7, 8 or 9, wherein the haplotype provides information
regarding the subject's risk of developing SZ, SPD, or SD. In some
embodiments, the test marker is a marker listed in one or more of
tables 4, 6, 7, 8, or 9, or a marker within 1 linkage
disequilibrium unit (1 LDU) or >0.5 D' of a polymorphism
described herein, e.g., markers in a region of chromosome 22, e.g.,
in 22q13, e.g., in 22q13.3, that is between and including SNPs
rs738596, rs738598, or rs135221 on the proximal end, and rs13884 or
rs137853 on the distal end, e.g., between rs738596 and
rs137853.
[0009] In some embodiments, the test marker is within 1 LDU of a
marker listed in Table 6, 7, 8, or 9, and is in a region of 22q13
that is between and including SNPs rs738596, rs738598, or rs135221
on the proximal end, and rs137853 or rs13884 on the distal end.
[0010] In some embodiments, the test haplotype includes at least
one marker listed in Table 4, 6, 7, 8 or 9.
[0011] In some embodiments, the test haplotype includes one or more
of: microsatellite marker D22S526, and/or a polymorphism of
Sulfotransferase 4A1 (Sult4a1), e.g., rs138060, rs138097, rs138110,
and/or D22s1749e. In some embodiments, the polymorphism is an
allele of Sult4a1 at microsatellite marker D22s1749e comprising
more than 207 nucleotides, and indicates that the subject has an
increased risk of developing SZ, SPD, or SD.
[0012] In some embodiments, the test haplotype includes at least
two markers, one of which is microsatellite marker D22S526.
[0013] In some embodiments, the test haplotype includes at least
one marker listed in Table 4 or 9, or in bold in table 8, and
provides information regarding a subject's risk of developing SZ,
under a narrower (DSM III) disease definition.
[0014] The methods described herein can include obtaining a
haplotype that includes two or more, e.g., two, three, four, five,
or six markers.
[0015] Additionally, the methods can include determining the
presence or absence of other markers known to be associated with
SZ, SD or SPD, e.g., outside of a region identified herein. A
number of other such markers are known in the art, e.g., as
described herein.
[0016] The subject can be a mammal, e.g., a primate, preferably a
higher primate, e.g., a human (e.g., a patient having, or at risk
of, SZ, SD or SPD). In one embodiment, the subject is a patient
having SZ, SD or SPD (e.g., a patient suffering from early,
intermediate or aggressive SZ, SD or SPD). In some embodiments, the
methods described herein are used to obtain information regarding a
subject's risk of developing SZ, SD or SPD, wherein the disorder is
other than catatonic schizophrenia. In some embodiments, the
subject is of African American (AA) or European American (EA)
descent, i.e., has one or more ancestors who are AA or EA.
[0017] In one embodiment, a subject to be evaluated by a method
described herein is a subject having one or more risk factors
associated with SZ, SPD or SD. For example, the subject may have a
relative afflicted with SZ, e.g., one or more of a grandparent,
parent, uncle or aunt, sibling, or child who has or had SZ, SPD or
SD; the subject may have a genetically based phenotypic trait
associated with risk for SZ, SPD or SD (e.g., eye tracking
dysfunction); deficits in working (short-term) memory; and/or
mixed-handedness (the use of different hands for different tasks),
particularly in females.
[0018] In some embodiments, the subject is a child, fetus, or
embryo, and one of the subject's relatives, e.g., a parent or
sibling, of the child, fetus, or embryo has SZ, SPD or SD. In this
case, the presence in the child, fetus, or embryo of a haplotype
described herein that is shared with the affected parent, but not
with the non-affected parent, indicates that the child, fetus, or
embryo has an increased risk of developing SPD, SD, or SZ. In some
embodiments, the subject has no overt or clinical signs of SZ, SPD,
or SD.
[0019] In some embodiments, obtaining a test haplotype includes
obtaining a sample comprising DNA from the subject; and determining
the identity, presence or absence of at least one test marker that
is within 1 LDU of a marker listed in Table 4, 6, 7, 8 or 9 in the
DNA. The sample can be obtained, e.g., from the subject by a health
care provider, or provided by the subject without the assistance of
a health care provider.
[0020] In some embodiments, obtaining a test haplotype includes
reviewing a subject's medical history, wherein the medical history
includes information regarding the presence or absence of at least
one test marker that is within 1 LDU of a marker listed in Table 4,
6, 7, 8 or 9 in the subject.
[0021] In some embodiments, the methods described herein include
obtaining a reference haplotype including a reference marker that
corresponds to a test marker, and comparing the test haplotype to
the reference haplotype. A reference marker that "corresponds to" a
test marker is the same marker. For example, if the test haplotype
includes D22S526, then the reference haplotype should also include
D22S526 for comparison purposes. The sharing of a haplotype (e.g.,
of some or all of the markers) between the test haplotype and a
reference haplotype is indicative of whether there is an increased
likelihood that the subject will develop SZ, SPD, or SD.
[0022] In some embodiments, the methods include administering a
treatment to a subject identified as being at increased risk for
developing SZ, SPD, or SD, e.g., a pharmacological or psychosocial
treatment as described herein. In some embodiments, the subject has
no overt or clinical signs of SZ, SPD, or SD, and the treatment is
administrated before any such signs appear.
[0023] Information obtained using a method described herein can be
used, e.g., to select a subject population for a clinical trial, to
stratify a subject population in a clinical trial, and/or to
stratify subjects that respond to a treatment from those who do not
respond to a treatment, or subjects that have negative side effects
from those who do not.
[0024] In another aspect, the invention provides methods for
selecting a subject for inclusion in a clinical trial, e.g., a
trial of a treatment for SZ, SPD, or SD. The methods include
obtaining a haplotype for the subject including at least one marker
that is within 1 linkage disequilibrium unit (1 LDU) of a marker
listed in Tables 4, 6, 7, 8 or 9; determining whether the haplotype
is associated with an increased risk of developing schizophrenia
(SZ), schizotypal personality disorder (SPD), or schizoaffective
disorder (SD); and including the subject in the trial if the
haplotype indicates that the subject has an increased risk of
developing SZ, SPD, or SD.
[0025] In another aspect, the invention provides methods for
selecting a subject for administration of a treatment for
schizophrenia (SZ), schizotypal personality disorder (SPD), or
schizoaffective disorder (SD). The methods include obtaining a
haplotype for the subject, wherein the haplotype comprises at least
one marker that is within 1 linkage disequilibrium unit (1 LDU) of
a marker listed in Tables 4, 6, 7, 8 or 9; determining whether the
haplotype is associated with an increased risk of developing SZ,
SPD, or SD; and administering the treatment to the subject if the
haplotype indicates that the subject has an increased risk of
developing SZ, SPD, or SD.
[0026] In another aspect, the invention provides methods for
selecting a treatment for administration to a subject. The methods
include obtaining a haplotype for the subject, wherein the
haplotype comprises at least one marker that is within 1 linkage
disequilibrium unit (1 LDU) of a marker listed in Tables 4, 6, 7, 8
or 9; determining whether the haplotype is associated with an
increased risk of developing schizophrenia (SZ), schizotypal
personality disorder (SPD), or schizoaffective disorder (SD); and
administering the treatment for SZ, SPD, or SD to the subject if
the haplotype indicates that the subject has an increased risk of
developing SZ, SPD, or SD.
[0027] In another aspect, the invention provides methods for
evaluating the effect of a haplotype on the outcome of a treatment
for schizophrenia (SZ), schizotypal personality disorder (SPD), or
schizoaffective disorder (SD). The methods include obtaining
information regarding outcome of the treatment, wherein the
information comprises a parameter relating to the treatment of each
subject in a population of subjects; obtaining haplotypes for each
subject in the population, wherein the haplotype comprises at least
one marker that is within 1 linkage disequilibrium unit (1 LDU) of
a marker listed in Tables 4, 6, 7, 8 or 9; and correlating the
information regarding outcome with the haplotypes; thereby
evaluating the effect of the haplotype on the outcome of the
treatment.
[0028] In some embodiments, the method includes selecting a
treatment for administration to a subject who has a selected
haplotype, based on the effect of the haplotype on the outcome of
the treatment.
[0029] In some embodiments, the information regarding outcome of
the treatment is from a completed clinical trial, and the analysis
is retrospective.
[0030] In another aspect, the invention features methods of
predicting a subject's risk of developing SZ, SPD, or SD. The
methods include obtaining a reference haplotype. In some
embodiments, the reference haplotype is from at least one of the
following relatives of the subject: (i) a parent who has SZ, SPD,
or SD; (ii) a sibling who has SZ, SPD, or SD, and an unaffected
parent; or (iii) a second degree relative (e.g., aunt, uncle, or
grandparent) who has SZ, SPD, or SD, and an unaffected parent;
obtaining a test haplotype from the subject in the same region; and
comparing the test haplotype to a reference haplotype. The sharing
of a haplotype in this region between the test haplotype and a
reference haplotype from a relative having the disorder is an
indication of an increased likelihood that the subject will develop
SZ, SPD, or SD. In some embodiments, the reference haplotype is
from an unaffected individual, and sharing of a haplotype indicates
that there is no increased likelihood that the subject will develop
SZ, SD, or SD.
[0031] In a further aspect, the invention features methods for
detecting the presence of a haplotype associated with
susceptibility to SZ, SPD, or SD in a subject, by analyzing a
sample of DNA from the subject.
[0032] Additionally, the invention features methods of predicting a
test subject's risk of developing SZ, SPD, or SD. The methods
include obtaining a reference haplotype of a reference subject,
wherein the reference subject has SZ, SPD, or SD; determining a
test haplotype of the test subject in the same region; and
comparing the test haplotype to the reference haplotype, wherein
the sharing of a haplotype in this region between the test subject
and the reference subject is an indication of an increased
likelihood that the test subject will develop SZ, SPD, or SD. In
some embodiments, the method further includes comparing the
subject's haplotype to a reference subject who does not have SZ,
SPD, or SD.
[0033] Further, the invention features methods for predicting a
test subject's risk of developing SZ. The methods include obtaining
a reference haplotype of a reference subject in a region described
herein, wherein the reference subject has SZ; obtaining a test
haplotype of the test subject in the same region; and comparing the
test haplotype to the reference haplotype. The sharing of a
haplotype in this region between the test subject and the reference
subject is an indication of an increased likelihood that the test
subject will develop SZ. In some embodiments, the method also
includes comparing the test subject's haplotype to a reference
subject who does not have SZ.
[0034] In another aspect, the invention features methods for
predicting a subject's risk of developing SZ, SPD, or SD. The
methods include obtaining genomic DNA (gDNA) from the subject; and
determining the absence or presence of a haplotype associated with
SZ at human chromosome 22q13 as described herein. The presence of a
haplotype associated with SZ, SPD, or SD indicates that the subject
has an increased risk of developing SZ, SD or SPD.
[0035] The invention further features nucleic acid probes having a
nucleotide sequence that hybridizes with a nucleotide sequence
within human chromosome 22q13 and allows detection of a
microsatellite marker at D22s1749E, e.g., under hybridization
conditions of a 50% formamide, 2.times.SSC wash for 10 minutes at
45.degree. C. followed by a 2.times.SSC wash for 10 minutes at
37.degree. C. In some embodiments, the probes are at least 20
nucleotides long and include all or part of
5'-CAGCCGCACGCCATGGAACTCGAAG-3'(SEQ ID NO:1) or
5'-GGCGCCATGACGTCACGCCTGC-3'(SEQ ID NO:2). In some embodiments, the
probes are no longer than 30, 50, 100, 200, or 500 nucleotides
long.
[0036] Also provided herein are kits for use in detection of
haplotypes associated with SZ, SD or SPD, including at least one
nucleic acid probe that hybridizes to a sequence that includes a
polymorphism described herein, or can be used to amplify a sequence
that includes a polymorphism described herein.
[0037] Also provided are arrays that include a substrate having a
plurality of addressable areas, wherein one or more of the
addressable areas includes one or more probes that can be used to
detect a polymorphism described herein.
[0038] In another aspect, the invention provides methods for
providing information regarding a subject's risk of developing
schizophrenia (SZ), schizotypal personality disorder (SPD), or
schizoaffective disorder (SD). The methods include obtaining a
sample from the subject at a first site; transferring the sample to
a second site for analysis, wherein the analysis provides data
regarding the identity, presence or absence of at least one test
marker that is within 1 LDU of a marker listed in Tables 4, 6, 7, 8
or 9; and transferring the data to one or more of a health care
provider, the subject, or a healthcare payer. In some embodiments,
the first site is a health care provider's place of business, or is
not a health care provider's place of business, e.g., the subject's
home.
[0039] In some embodiments, the data is transferred to a healthcare
payer and used to decide whether to reimburse a health care
provider.
Definitions
[0040] As used herein, a "haplotype" is a set of signature genetic
changes (polymorphisms) that are normally grouped closely together
on the DNA strand, and are usually inherited as a group; the
polymorphisms are also referred to herein as "markers." A
"haplotype" as used herein is information regarding the presence or
absence of one or more genetic markers in a subject. A haplotype
can consist of a variety of genetic markers, including indels
(insertions or deletions of the DNA at particular locations on the
chromosome); single nucleotide polymorphisms (SNPs) in which a
particular nucleotide is changed; microsatellites; and
minisatellites.
[0041] Microsatellites (sometimes referred to as a variable number
of tandem repeats or VNTRs) are short segments of DNA that have a
repeated sequence, usually about 2 to 5 nucleotides long (e.g.,
CACACA), that tend to occur in non-coding DNA. Changes in the
microsatellites sometimes occur during the genetic recombination of
sexual reproduction, increasing or decreasing the number of repeats
found at an allele, changing the length of the allele.
Microsatellite markers are stable, polymorphic, easily analyzed and
occur regularly throughout the genome, making them especially
suitable for genetic analysis.
[0042] "Linkage disequilibrium" refers to when the observed
frequencies of haplotypes in a population does not agree with
haplotype frequencies predicted by multiplying together the
frequency of individual genetic markers in each haplotype.
[0043] The term "chromosome" as used herein refers to a gene
carrier of a cell that is derived from chromatin and comprises DNA
and protein components (e.g., histones). The conventional
internationally recognized individual human genome chromosome
numbering identification system is employed herein. The size of an
individual chromosome can vary from one type to another with a
given multi-chromosomal genome and from one genome to another. In
the case of the human genome, the entire DNA mass of a given
chromosome is usually greater than about 100,000,000 base pairs.
For example, the size of the entire human genome is about
3.times.10.sup.9 base pairs. Chromosome 22 contains about
5.3.times.10.sup.7 base pairs (see, e.g., Yunis, Science
191:1268-1270 (1976), and Kavenoff et al., Cold Spring Harbor
Symposia on Quantitative Biology 38:1-8 (1973)).
[0044] The term "gene" refers to a DNA sequence in a chromosome
that codes for a product (either RNA or its translation product, a
polypeptide). A gene contains a coding region and includes regions
preceding and following the coding region (termed respectively
"leader" and "trailer"). The coding region is comprised of a
plurality of coding segments ("exons") and intervening sequences
("introns") between individual coding segments.
[0045] The term "probe" refers to an oligonucleotide. A probe can
be single stranded at the time of hybridization to a target. As
used herein, probes include primers, i.e., oligonucleotides that
can be used to prime a reaction, e.g., a PCR reaction.
[0046] The term "label" or "label containing moiety" refers in a
moiety capable of detection, such as a radioactive isotope or group
containing same, and nonisotopic labels, such as enzymes, biotin,
avidin, streptavidin, digoxygenin, luminescent agents, dyes,
haptens, and the like. Luminescent agents, depending upon the
source of exciting energy, can be classified as radioluminescent,
chemiluminescent, bioluminescent, and photoluminescent (including
fluorescent and phosphorescent). A probe described herein can be
bound, e.g., chemically bound to label-containing moieties or can
be suitable to be so bound. The probe can be directly or indirectly
labeled.
[0047] The term "direct label probe" (or "directly labeled probe")
refers to a nucleic acid probe whose label after hybrid formation
with a target is detectable without further reactive processing of
hybrid. The term "indirect label probe" (or "indirectly labeled
probe") refers to a nucleic acid probe whose label after hybrid
formation with a target is further reacted in subsequent processing
with one or more reagents to associate therewith one or more
moieties that finally result in a detectable entity.
[0048] The terms "target," "DNA target," or "DNA target region"
refers to a nucleotide sequence that occurs at a specific
chromosomal location. Each such sequence or portion is preferably
at least partially, single stranded (e.g., denatured) at the time
of hybridization. When the target nucleotide sequences are located
only in a single region or fraction of a given chromosome, the term
"target region" is sometimes used. Targets for hybridization can be
derived from specimens which include, but are not limited to,
chromosomes or regions of chromosomes in normal, diseased or
malignant human cells, either interphase or at any state of meiosis
or mitosis, and either extracted or derived from living or
postmortem tissues, organs or fluids; germinal cells including
sperm and egg cells, or cells from zygotes, fetuses, or embryos, or
chorionic or amniotic cells, or cells from any other germinating
body; cells grown in vitro, from either long-term or short-term
culture, and either normal, immortalized or transformed; inter- or
intraspecific hybrids of different types of cells or
differentiation states of these cells; individual chromosomes or
portions of chromosomes, or translocated, deleted or other damaged
chromosomes, isolated by any of a number of means known to those
with skill in the art, including libraries of such chromosomes
cloned and propagated in prokaryotic or other cloning vectors, or
amplified in vitro by means well known to those with skill; or any
forensic material, including but not limited to blood, or other
samples.
[0049] The term "hybrid" refers to the product of a hybridization
procedure between a probe and a target.
[0050] The term "hybridizing conditions" has general reference to
the combinations of conditions that are employable in a given
hybridization procedure to produce hybrids, such conditions
typically involving controlled temperature, liquid phase, and
contact between a probe (or probe composition) and a target.
Conveniently and preferably, at least one denaturation step
precedes a step wherein a probe or probe composition is contacted
with a target. Guidance for performing hybridization reactions can
be found in Ausubel et al., Current Protocols in Molecular Biology,
John Wiley & Sons, N.Y. (2003), 6.3.1-6.3.6. Aqueous and
nonaqueous methods are described in that reference and either can
be used. Hybridization conditions referred to herein are a 50%
formamide, 2.times.SSC wash for 10 minutes at 45.degree. C.
followed by a 2.times.SSC wash for 10 minutes at 37.degree. C.
[0051] Calculations of "identity" between two sequences can be
performed as follows. The sequences are aligned for optimal
comparison purposes (e.g., gaps can be introduced in one or both of
a first and a second nucleic acid sequence for optimal alignment
and non-identical sequences can be disregarded for comparison
purposes). The length of a sequence aligned for comparison purposes
is at least 30%, e.g., at least 40%, 50%, 60%, 70%, 80%, 90% or
100%, of the length of the reference sequence. The nucleotides at
corresponding nucleotide positions are then compared. When a
position in the first sequence is occupied by the same nucleotide
as the corresponding position in the second sequence, then the
molecules are identical at that position. The percent identity
between the two sequences is a function of the number of identical
positions shared by the sequences, taking into account the number
of gaps, and the length of each gap, which need to be introduced
for optimal alignment of the two sequences.
[0052] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In some embodiments, the percent identity
between two nucleotide sequences is determined using the GAP
program in the GCG software package, using a Blossum 62 scoring
matrix with a gap penalty of 12, a gap extend penalty of 4, and a
frameshift gap penalty of 5.
[0053] As used herein, the term "substantially identical" is used
to refer to a first nucleotide sequence that contains a sufficient
number of identical nucleotides to a second nucleotide sequence
such that the first and second nucleotide sequences have similar
activities. Nucleotide sequences that are substantially identical
are at least 80%, e.g., 85%, 90%, 95%, 97% or more, identical.
[0054] The term "nonspecific binding DNA" refers to DNA which is
complementary to DNA segments of a probe, which DNA occurs in at
least one other position in a genome, outside of a selected
chromosomal target region within that genome. An example of
nonspecific binding DNA comprises a class of DNA repeated segments
whose members commonly occur in more than one chromosome or
chromosome region. Such common repetitive segments tend to
hybridize to a greater extent than other DNA segments that are
present in probe composition.
[0055] As used herein, the term "stratification" refers to the
creation of a distinction between subjects on the basis of a
characteristic or characteristics of the subjects. Generally, in
the context of clinical trials, the distinction is used to
distinguish responses or effects in different sets of patients
distinguished according to the stratification parameters. In some
embodiments, stratification includes distinction of subject groups
based on the presence or absence of particular markers or
haplotypes described herein. The stratification can be performed,
e.g., in the course of analysis, or can be used in creation of
distinct groups or in other ways.
[0056] 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. Methods
and materials are described herein for use in the present
invention; other, suitable methods and materials known in the art
can also be used. The materials, methods, and examples are
illustrative only and not intended to be limiting. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control.
[0057] Other features and advantages of the invention will be
apparent from the following detailed description and figures, and
from the claims.
DESCRIPTION OF DRAWINGS
[0058] FIG. 1 is a line graph illustrating LOD scores for markers
at the indicated locations on the long arm of chromosome 22. The
locations of markers D22s683, D22s270, and sJCW16, which are
associated with the highest LOD scores, are shown.
[0059] FIG. 2 is a line graph illustrating LOD scores for markers
at the indicated locations on the long arm of chromosome 22,
including the new marker D22S1749E, the location of which is
indicated.
[0060] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
[0061] The methods described herein are based, at least in part, on
the discovery of haplotypes and markers in 22q13 that are
associated with increased risk of developing schizophrenia (SZ),
schizotypal personality disorder (SPD) or schizoaffective disorder
(SD). As described herein, TDT analysis provided suggestive
evidence of role of Sult4a1 in this set of families (P=0.002 for
narrowly-defined SZ, 0.04 for SZ+SPD), with a tendency for one of
the longer alleles of D22S1749E to be preferentially transferred to
affected children (See Examples, below). Additionally, TDT analysis
for the other microsatellite markers suggests that a region near
marker D22S526 plays a role in SZ (P=0.003, for narrowly-defined
SZ; P=0.00009 for SZ+SD+SPD). Thus, segments of chromosome 22 near
Sult4a1 and D22S526 contain sequences that are linked to a
predisposition to SPD, SD and SZ.
Methods of Diagnoses and Evaluation of Risk
[0062] Described herein are a variety of methods for the diagnosis
of susceptibility to SZ, SPD or SD. "Susceptibility" does not
necessarily mean that the subject will develop SZ, SPD or SD, but
rather that the subject is, in a statistical sense, more likely to
develop SZ than an average member of the population, i.e., has an
increased risk of developing SZ, SPD, or SD. As used herein,
susceptibility to SZ exists if the subject has a haplotype
associated with an increased risk of SZ, SPD, or SD as described
herein. Ascertaining whether the subject has such a haplotype is
included in the concept of diagnosing susceptibility to SZ, SPD or
SD as used herein. Such determination is useful, for example, for
purposes of diagnosis, treatment selection, and genetic counseling.
Thus, the methods described herein can include obtaining a
haplotype associated with an increased risk of SZ, SPD, or SD as
described herein for the subject.
[0063] As used herein, "obtaining a haplotype" includes obtaining
information regarding the identity, presence or absence of one or
more genetic markers in a subject. Obtaining a haplotype can, but
need not, include obtaining a sample comprising DNA from a subject,
and/or assessing the identity, presence or absence of one or more
genetic markers in the sample. The individual or organization who
obtains the haplotype need not actually carry out the physical
analysis of a sample from a subject; the haplotype can include
information obtained by analysis of the sample by a third party.
Thus the methods can include steps that occur at more than one
site. For example, a sample can be obtained from a subject at a
first site, such as at a health care provider, or at the subject's
home in the case of a self-testing kit. The sample can be analyzed
at the same or a second site, e.g., at a laboratory or other
testing facility.
[0064] Obtaining a haplotype can also include or consist of
reviewing a subject's medical history, where the medical history
includes information regarding the identity, presence or absence of
one or more genetic markers in the subject, e.g., results of a
genetic test.
[0065] In some embodiments, to detect the presence of a haplotype
described herein, a biological sample that includes nucleated cells
(such as blood, a cheek swab or mouthwash) is prepared and analyzed
for the presence or absence of preselected markers. Such diagnoses
may be performed by diagnostic laboratories, or, alternatively,
diagnostic kits can be manufactured and sold to health care
providers or to private individuals for self-diagnosis. Diagnostic
or prognostic tests can be performed as described herein or using
well known techniques, such as described in U.S. Pat. No.
5,800,998.
[0066] Results of these tests, and optionally interpretive
information, can be returned to the subject, the health care
provider or to a third party payor. The results can be used in a
number of ways. The information can be, e.g., communicated to the
tested subject, e.g., with a prognosis and optionally interpretive
materials that help the subject understand the test results and
prognosis. The information can be used, e.g., by a health care
provider, to determine whether to administer a specific drug, or
whether a subject should be assigned to a specific category, e.g.,
a category associated with a specific disease endophenotype, or
with drug response or non-response. The information can be used,
e.g., by a third party payor such as a healthcare payer (e.g.,
insurance company or HMO) or other agency, to determine whether or
not to reimburse a health care provider for services to the
subject, or whether to approve the provision of services to the
subject. For example, the healthcare payer may decide to reimburse
a health care provider for treatments for SZ, SPD or SD if the
subject has an increased risk of developing SZ, SPD or SD. As
another example, a drug or treatment may be indicated for
individuals with a certain haplotype, and the insurance company
would only reimburse the health care provider (or the insured
individual) for prescription or purchase of the drug if the insured
individual has that haplotype. The presence or absence of the
haplotype in a patient may be ascertained by using any of the
methods described herein.
[0067] Information gleaned from the methods described herein can
also be used to select or stratify subjects for a clinical trial.
For example, the presence of a selected haplotype described herein
can be used to select a subject for a trial. The information can
optionally be correlated with clinical information about the
subject, e.g., diagnostic or endophenotypic information.
Haplotypes Associated with SZ, SPD and SD
[0068] As described herein, haplotypes associated with SZ, SPD or
SD include markers in the distal region of the long arm of
chromosome 22 (i.e., in 22q13.3) as exemplified by the transmission
disequilibrium results shown in tables 4, 6, 7, 8 or 9.
[0069] As one example, haplotypes associated with a broader
disorder definition including SZ, SPD and SD include one or more
markers on chromosome 22 that are within 1 linkage disequilibrium
unit (1 LDU) of a marker listed in Tables 4, 6, 7, 8 or 9. In some
embodiments, the haplotype includes one or more of the markers
listed in tables 4, 6, 7, 8 or 9. In some embodiments, the markers
are in a region of 22q13 that is between and includes SNPs
rs738596, rs738598, or rs135221 on the proximal end, and rs13884 or
rs137853 on the distal end. In some embodiments, the markers are in
a region of 22q13 that is between and includes SNPs rs738598 and
rs13884. In some embodiments, the markers are in a region of 22q13
that is between and includes SNPs rs135221 and rs13884.
[0070] Haplotypes associated with a narrower disorder definition of
SZ can include one or more markers that are within 1 LDU of a
marker listed in Table 4 or 9, or in bold in table 8. In some
embodiments, the haplotype includes one or more of the markers
listed in Tables 4 or 9, or in bold in table 8. In some
embodiments, the markers are in a region of 22q13 that is between
and includes rs135221 on the proximal end, and rs13884 on the
distal end.
[0071] In some embodiments, the methods include determining the
presence of a haplotype that includes one or more polymorphisms
near D22S526 and/or the polymorphisms in the Sult4a1 gene listed in
Table 4, and/or polymorphisms within 1 LDU of these markers.
[0072] In some embodiments, the methods described herein do not
include detecting polymorphisms within the MLC1 gene.
[0073] Sulfotransferase-4A1 (Sult4a1)
[0074] Using samples obtained from the National Institutes of
Mental Health Schizophrenia Genetics Initiative, 27 nuclear
families having multiple siblings with schizophrenia and
schizophrenia-spectrum disorders were evaluated for linkage to
chromosome 22 markers. Analysis with 14 highly informative
microsatellite markers provided evidence for linkage near marker
D22s270. Assuming heterogeneity, a maximum LOD score of 2.90 was
obtained using DSM IV criteria, and a maximum LOD score of 3.96 was
obtained for a broader disease definition that included schizotypal
personality disorder (SPD). Nonparametric linkage analysis provided
suggestive evidence for linkage at the same location (LOD scores of
2.6 and 2.8 for the narrow and broad definitions,
respectively).
[0075] This segment of chromosome 22 contains the
sulfotransferase-4A1 (Sult4a1) gene, which encodes a brain-specific
sulfotransferase believed to be involved in metabolism of
neurotransmitters (Falany et al., Biochem J. 346:857-64 (2000);
Sakakibara et al., Gene 285:39-47 (2002); Liyou et al., J.
Histochem. Cytochem. 51:655-64 (2003)). This positional candidate
was evaluated by family-based TDT analysis of 27 families from the
NIMH Schizophrenia Genetics Initiative. To evaluate this candidate
gene, a microsatellite marker (D22S1749E) targeting a promoter
polymorphism in the gene was developed, and transmission
disequilibrium (TDT) analysis of this marker and three single
nucleotide polymorphisms spanning a 37 kb region containing the
gene was performed.
[0076] As described herein, TDT analysis provided suggestive
evidence of role of Sult4a1 in this set of families (P=0.002 for
narrowly-defined SZ, 0.04 for SZ+SPD), with a tendency for one of
the longer alleles (213nt) of D22S1749E to be preferentially
transferred to affected children (See Examples 1-4, below).
[0077] The sample was expanded by the addition of 17 further
families to the original 27 families. Using the D22S1749E marker in
linkage analysis for the pooled sample (using a dominant model
assuming genetic heterogeneity, a penetrance of 50% for a
heterozyote and a 1% allele frequency) a single point heterogeneous
LOD score of 4.78 was obtained for the combined sample of 44
families (.alpha.=0.7). Consistent with the initial findings, for
the pooled sample, D22S1749E shows significant deviation from
expectation for transmission to affected offspring using TRANSMIT
(P=0.015 for SZ, and P=0.006 for the broader definition including
SPD).
[0078] Thus, the methods described herein can include detecting the
identity, presence or absence of one or more polymorphisms of the
Sult4a1 gene, e.g., polymorphisms described herein. For example,
the methods described herein can include determining the presence
of a polymorphism at D22S1749E, e.g., determining the length of the
alleles at D22S1749E. In some embodiments the methods also include
detecting the presence of a SNP in the Sult4a1 gene, e.g., one or
more of rs138060, rs138097, and rs138110 (see, e.g., Example 3 and
Table 4).
[0079] D22S526 and the Distal Region of 22q13
[0080] Numerous two and three SNP haplotypes spanning the distal
region of 22q13 show highly significant distortions in transmission
ratios for DSM-IIIR diagnosed SZ and broader disease definitions
(see the Examples, below; P<10.sup.-5). Some of these remain
significant even after the most parsimonious corrections for
multiple comparisons (Risch and Merikangas, Science
273(5281):1516-7 (1996); Sabatti et al., Genetics 164(2):829-33
(2003)). One SNP by itself, rs1573726, shows significant TDT values
by this method (X.sup.2=15.6, ldf, P=7.8.times.10.sup.-5).
[0081] Thus, the methods described herein include identifying
subjects on the basis of having a haplotype that includes
polymorphisms that are in the region of chromosome 22 that is
defined by the SNPs rs738596 (on the proximal end) and rs137853 (on
the distal end). In some embodiments, the methods include
identifying haplotypes that include polymorphisms between SNPs
rs738598 (proximal) and rs137853 or rs138844 (distal), or between
rs135221 (proximal) and rs137853 or rs138844 (distal). Proximal
refers to a location that is nearer the centromere, distal is
further away. In some embodiments, the methods do not include the
evaluation of polymorphisms at microsatellite D22s1169.
[0082] A close evaluation of the haplotypes revealed an interesting
pattern. Indeed, there are particular SNP haplotypes preferentially
transmitted, and these differ somewhat in EA (European American)
and AA (African American) families. Some are not rare, but fairly
common haplotypes having 25 to 40% expected frequencies based on
information now available through the haplotyping consortium (on
the world wide web at hapmap.org). However, in about half of the
NIMH families, these SNP haplotypes occur as part of a larger
haplotype involving a small subset (two to four per population) of
the 23 alleles of the highly polymorphic marker D22s526. TDT
analysis for the other microsatellite markers suggests that a
region near marker D22S526 plays a role in SZ (P=0.003, for
narrowly-defined SZ; P=0.00009 for SZ+SPD+SD), possibly due to
microdeletions of the region immediately surrounding and including
this highly polymorphic marker (see Examples 4 and 7, below). Thus,
in some embodiments, the methods described herein include the
evaluation of polymorphisms of D22S526, to detect microdeletions
e.g., microdeletions that include D22S526, e.g., microdeletions of
at least 50, 100, 200, 300, 400, 500 or more Kb. In some
embodiments, the microdeletions appear as apparent homozygosity,
and the presence of homozygosity at D22S526 is indicative of an
increased risk of developing SZ, SD, or SPD.
[0083] Linkage Disequilibrium Analysis
[0084] Linkage disequilibrium (LD) is a measure of the degree of
association between alleles in a population. One of skill in the
art will appreciate that haplotypes involving markers within 1
Linkage Disequilibrium Unit (LDU) of the polymorphisms described
herein can also be used in a similar manner to those described
herein. LDUs share an inverse relationship with LD so that regions
with high LD (such as haplotype blocks) have few LDUs and low
recombination, whilst regions with many LDUs have low LD and high
recombination. Methods of calculating LDUs are known in the art
(see, e.g., Morton et al., Proc Natl Acad Sci USA 98(9):5217-21
(2001); Tapper et al., Proc Natl Acad Sci USA 102(33):11835-11839
(2005); Maniatis et al., Proc Natl Acad Sci USA 99:2228-2233
(2002)).
[0085] Thus, in some embodiments, the methods include analysis of
polymorphisms that are within 1 LDU of a polymorphism described
herein. Methods are known in the art for identifying such
polymorphisms; for example, the International HapMap Project
provides a public database that can be used, see hapmap.org, as
well as The International HapMap Consortium, Nature 426:789-796
(2003), and The International HapMap Consortium, Nature
437:1299-1320 (2005). Generally, it will be desirable to use a
HapMap constructed using data from individuals who share ethnicity
with the subject, e.g., a HapMap for African Americans would
ideally be used to identify markers within 1 LDU of a marker
described herein for use in genotyping a subject of African
American descent.
[0086] Exemplary polymorphisms that are within 1 LDU of some of the
markers described herein are included in the Examples.
[0087] Alternatively, methods described herein can include analysis
of polymorphisms that are within a value defined by Lewontin's D'
(linkage disequilibrium parameter, see Lewontin, Genetics 49:49-67
(1964)) of a polymorphism described herein. Results can be
obtained, e.g., from on line public resources such as HapMap.org.
The simple linkage disequilibrium parameter (D) reflects the degree
to which alleles at two loci (for example two SNPs) occur together
more often (positive values) or less often (negative values) than
expected in a population as determined by the products of their
respective allele frequencies. For any two loci, D can vary in
value from -0.25 to +0.25. However, the magnitude of D (Dmax)
varies as function of allele frequencies. To control for this,
Lewontin introduced the D' parameter, which is D/Dmax and varies in
value from -1 (alleles never observed together) to +1 (alleles
always observed together). Typically, the absolute value of D'
(i.e., |D'|) is reported in online databases, because it follows
mathematically that positive association for one set of alleles at
two loci corresponds to a negative association of equal magnitude
for the reciprocal set. This disequilibrium parameter varies from 0
(no association of alleles at the two loci) to 1 (maximal possible
association of alleles at the two loci).
[0088] Thus, in some embodiments, the methods include analysis of
polymorphisms that are within D'>0.5, D'>0.75, or D'=1, for
pairwise comparisons, of a polymorphism described herein.
[0089] Identification of Additional Markers for Use in the Methods
Described Herein
[0090] In general, genetic markers can be identified using any of a
number of methods well known in the art. For example, numerous
polymorphisms in the regions described herein are known to exist
and are available in public databases, which can be searched using
methods and algorithms known in the art. Alternately, polymorphisms
can be identified by sequencing either genomic DNA or cDNA in the
region in which it is desired to find a polymorphism. According to
one approach, primers are designed to amplify such a region, and
DNA from a subject is obtained and amplified. The DNA is sequenced,
and the sequence (referred to as a "subject sequence" or "test
sequence") is compared with a reference sequence, which can
represent the "normal" or "wild type" sequence, or the "affected"
sequence. In some embodiments, a reference sequence can be from,
for example, the human draft genome sequence, publicly available in
various databases, or a sequence deposited in a database such as
GenBank. In some embodiments, the reference sequence is a composite
of ethnically diverse individuals.
[0091] In general, if sequencing reveals a difference between the
sequenced region and the reference sequence, a polymorphism has
been identified. The fact that a difference in nucleotide sequence
is identified at a particular site that determines that a
polymorphism exists at that site. In most instances, particularly
in the case of SNPs, only two polymorphic variants will exist at
any location. However, in the case of SNPs, up to four variants may
exist since there are four naturally occurring nucleotides in DNA.
Other polymorphisms, such as insertions and deletions, may have
more than four alleles.
[0092] Other Genetic Markers of Schizophrenia
[0093] The methods described herein can also include determining
the presence or absence of other markers known or suspected to be
associated with SZ, or with SZ, SD or SPD, e.g., markers outside of
a region identified herein, see, e.g., Harrison and Owen, Lancet,
361(9355):417-419 (2003), including, for example, markers on
chromosome 22 and other chromosomes, e.g., in the region of 22q12.3
(e.g., near D22S283), 22q11.2, 22q11.2, 22q11-q13, 1q42.1, 1q42.1,
4p, 18p, 15q15, 14q32.3, 13q34, 13q32, 12q24, 11q14-q21, 1q21-q22,
10p15-p13 (e.g., near D105189), 10q22.3, 8p12-21, 6q13-q26, 6p22.3,
6p23, 5q11.2-q13.3, and/or 3p25. In some embodiments, the methods
include determining the presence or absence of one or more other
markers that are or may be associated with SZ, or with SZ, SD or
SPD, e.g., in one or more genes, e.g., ADRA1A (Clark et al., Biol
Psychiatry. 58(6):435-9 (2005)); AKT1 (Emamian et al., Nature
Genet. 36:131-137 (2004)); ALDH3B1 (Sun et al. Sci. China C. Life.
Sci. 48(3):263-9 (2005)); ARSA (Marcao et al., Mol Genet Metab.
79(4):305-7 (2003); ARVCF (Chen et al., Schizophr Res.
72(2-3):275-7 (2005)); BDNF (Neves-Pereira et al., Molec. Psychiat.
10:208-212 (2005)); BZRP (Kurumaji et al., J Neural Transm.
107(4):491-500 (2000)); DAO (Owen et al., Trends Genet.
21(9):518-25 (2005)); DAOA (Owen et al., 2005, supra); CAPON
(Brzustowicz et al., Am J Hum Genet. 74(5):1057-63 (2004)); CHRNA2
(Blaveri et al., Europ. J. Hum. Genet. 9: 469-472 (2001)); COMT
(Shifman et al., Am. J. Hum. Genet. 71:1296-1302 (2002)); CPLX2
(Lee et al., Behav Brain Funct. 1:15 (2005)); DGCR8 (Jacquet et
al., Hum Mol Genet. 11(19):2243-9 (2002)); DISC1 (Owen et al.,
2005, supra; see, e.g., the D1S2709 marker (Ekelend et al., Hum.
Molec. Genet. 10:1611-1617 (2001), HEP3 haplotype, Hennah et al.,
Hum. Molec. Genet. 12: 3151-3159 (2003), and Leu607Pro, Hodgkinson
et al., Am. J. Hum. Genet. 75:862-872 (2004), Erratum: Am. J. Hum.
Genet. 76:196 (2005)); DISC2 (Millar et al., Ann Med. 36(5):367-78
(2004)); DPYSL2 (Hong et al., Am J Med Genet B Neuropsychiatr
Genet. 136(1):8-11 (2005)); DRD1 (Coon et al., Am. J. Hum. Genet.
52: 327-334 (1993)); DRD2 (Glatt et al., Am. J. Psychiat.
160:469-476 (2003)); DRD3 (Rybakowski et al., Molec. Psychiat.
6:718-724 (2001)); DTNBP1 (Owen et al., 2005, supra); EPSIN4 (Am J
Hum Genet. 76(5):902-7 (2005)); ErbB; EGF (Futamura et al., Am. J.
Hum. Genet. 52: 327-334 (2002)); GABRA1, GABRA2, GABRA6, GABRP
(Petryshen et al., Mol Psychiatry. 10(12):1057 (2005)); GFRA1
(Semba et al., Brain Res Mol Brain Res. 124(1):88-95 (2004)); GNB3
(Kunugi et al., J. Neural Transm. 109(2):213-8 (2002)); GRIK1
(Shibata et al., Psychiatr Genet. 11(3):139-44 (2001)); GRIK2
(Shibata et al., Psychiatry Res. 113(1-2):59-67 (2002)); GRIN1 (Qin
et al., Eur J Hum Genet. 13(7):807-14 (2005)); GRIN2A, GRIN2B
(Abdolmaleky et al., Am J Pharmacogenomics. 5(3):149-60 (2005));
GRIN2D (Makino et al., Psychiatr Genet. 15(3):215-21 (2005)); GRM3
(Egan et al., Proc Natl Acad Sci U S A. 101(34):12604-9 (2004));
GRM4 (Ohtsuki et al., Psychiatr Genet. 11(2):79-83 (2001)); G30/G72
(Schulze et al., Am J Psychiatry. 162(11):2101-8 (2005)); HTR2A
(Baritaki et al., Eur J Hum Genet. 12(7):535-41 (2004)); HLA-DRB1
(Schwab et al., Am J Med Genet. 114(3):315-20 (2002)); HLA-BRB3 (Yu
et al., Zhonghua Liu Xing Bing Xue Za Zhi. 24(9):815-8 (2003));
IL2RB (Schwab et al., Am J Med Genet. 60(5):436-43 (1995)); KCNN3
(Ujike et al., Psychiatry Res. 101(3):203-7 (2001)); KIF13A (Jamain
et al., Genomics. 74(1):36-44 (2001)); KPNA3 (Wei and Hemmings,
Neurosci Res. 52(4):342-6 (2005)); LGI1 (Fallin et al. A J Hum
Genet. 77:918-36 (2005)); MAG (Wan et al., Neurosci Lett.
388(3):126-31 (2005)); MLC1 (Verma et al., Biol Psychiatry.
58(1):16-22 (2005)); MTHFR (Lewis et al., Am. J. Med. Genet.
(Neuropsychiat. Genet.) 135B:2-4 (2005)); NOS1 (Liou et al.,
Schizophr Res. 65(1):57-9 (2003)); NOTCH4 (Wei and Hemmings,
(Letter) Nature Genet. 25:376-377 (2000)); NRG1 (Owen et al., 2005,
supra); NRG3 (Fallin et al. A J Hum Genet. 77:918-36 (2005)); PCQAP
(Sandhu et al., Psychiatr Genet. 14(3):169-72 (2004)); PIK4CA
(Saito et al., Am J Med Genet B Neuropsychiatr Genet. 116(1):77-83
(2003)); PLA2G4A, PLA2G4C (Yu et al., Prostaglandins Leukot Essent
Fatty Acids. 73(5):351-4 (2005)); PPP3CC (Gerber et al., Proc Natl
Acad Sci U S A. 100(15):8993-8 (2003)); PNOC (Blaveri et al.,
2001); PRODH (Chakravarti, Proc. Nat. Acad. Sci. 99:4755-4756
(2002)); QKI (Aberg et al., Am J Med Genet B Neuropsychiatr Genet.
2005 Dec. 9; [Epub ahead of print]); RGS4 (Chowdari et al., Hum.
Molec. Genet. 11:1373-1380 (2002), Erratum: Hum. Molec. Genet.
12:1781 (2003)); RELN (Costa et al., Mol Interv. 2(1):47-57
(2002)); SCA1 (Culkjovic et al., Am J Med Genet. 96(6):884-7
(2000)); SLC15A1 (Maheshwari et al., BMC Genomics. 3(1):30 (2002));
SLC18A1 (Bly, Schizophr Res. 78(2-3):337-8 (2005)); SNAP29 (Saito
et al., Mol Psychiatry 6(2):193-201 (2001); Erratum in: Mol
Psychiatry 6(5):605 (2001); SYNGR1 (Verma et al., Biol Psychiatry.
55(2):196-9 (2004)); SYN2 (Chen et al., Bio. Psychiat. 56:177-181
(2004)); SYN3 (Porton et al. Biol Psychiatry. 55(2):118-25 (2004));
TBP/SCA17 (Chen et al., Schizophr Res. 78(2-3):131-6 (2005)); TPP2
(Fallin et al. A J Hum Genet. 77:918-36 (2005)); TRAR4 (Am J Hum
Genet. 75(4):624-38 (2004)); TRAX (Thomson et al., Mol Psychiatry.
10(7):657-68, 616 (2005)); UFD1L (De Luca et al., Am J Med Genet.
105(6):529-33 (2001)); YWHAH (Toyooka et al., Am J Med Genet.
88(2):164-7 (1999)); ZDHHC8 (Mukai et al., Nature Genet. 36:725-731
(2004)); or ZNF74 (Takase et al., Schizophr Res. 52(3):161-5
(2001)). See also, e.g., OMIM entry no. 181500 (SCZD).
Methods of Determining the Presence or Absence of a Haplotype
Associated with SZ, SPD or SD
[0094] The methods described herein include determining the
presence or absence of haplotypes associated with SZ, SPD or SD. In
some embodiments, an association with SZ is determined by the
presence of a shared haplotype between the subject and an affected
reference individual, e.g., a first or second-degree relation of
the subject, and the absence of the haplotype in an unaffected
reference individual. Thus the methods can include obtaining and
analyzing a sample from a suitable reference individual.
[0095] Samples that are suitable for use in the methods described
herein contain genetic material, e.g., genomic DNA (gDNA).
Non-limiting examples of sources of samples include urine, blood,
and tissue. The sample itself will typically consist of nucleated
cells (e.g., blood or buccal cells), tissue, etc., removed from the
subject. The subject can be an adult, child, fetus, or embryo. In
some embodiments, the sample is obtained prenatally, either from a
fetus or embryo or from the mother (e.g., from fetal or embryonic
cells in the maternal circulation). Methods and reagents are known
in the art for obtaining, processing, and analyzing samples. In
some embodiments, the sample is obtained with the assistance of a
health care provider, e.g., to draw blood. In some embodiments, the
sample is obtained without the assistance of a health care
provider, e.g., where the sample is obtained non-invasively, such
as a sample comprising buccal cells that is obtained using a buccal
swab or brush, or a mouthwash sample.
[0096] The sample may be further processed before the detecting
step. For example, DNA in a cell or tissue sample can be separated
from other components of the sample. The sample can be concentrated
and/or purified to isolate DNA. Cells can be harvested from a
biological sample using standard techniques known in the art. For
example, cells can be harvested by centrifuging a cell sample and
resuspending the pelleted cells. The cells can be resuspended in a
buffered solution such as phosphate-buffered saline (PBS). After
centrifuging the cell suspension to obtain a cell pellet, the cells
can be lysed to extract DNA, e.g., gDNA. See, e.g., Ausubel et al.,
2003, supra. All samples obtained from a subject, including those
subjected to any sort of further processing, are considered to be
obtained from the subject.
[0097] The absence or presence of a haplotype associated with SZ,
SPD or SD as described herein can be determined using methods known
in the art, e.g., gel electrophoresis, capillary electrophoresis,
size exclusion chromatography, sequencing, and/or arrays to detect
the presence or absence of the marker(s) of the haplotype.
Amplification of nucleic acids, where desirable, can be
accomplished using methods known in the art, e.g., PCR.
[0098] Methods of nucleic acid analysis to detect polymorphisms
and/or polymorphic variants include, e.g., microarray analysis.
Hybridization methods, such as Southern analysis, Northern
analysis, or in situ hybridizations, can also be used (see Current
Protocols in Molecular Biology, Ausubel, F. et al., eds., John
Wiley & Sons 2003). To detect microdeletions, fluorescence in
situ hybridization (FISH) using DNA probes that are directed to a
putatively deleted region in a chromosome can be used. For example,
probes that detect all or a part of microsatellite marker D22s526
can be used to detect microdeletions in the region that contains
that marker.
[0099] Other methods include direct manual sequencing (Church and
Gilbert, Proc. Natl. Acad. Sci. USA 81:1991-1995 (1988); Sanger et
al., Proc. Natl. Acad. Sci. 74:5463-5467 (1977); Beavis et al. U.S.
Pat. No. 5,288,644); automated fluorescent sequencing;
single-stranded conformation polymorphism assays (SSCP); clamped
denaturing gel electrophoresis (CDGE); two-dimensional gel
electrophoresis (2DGE or TDGE); conformational sensitive gel
electrophoresis (CSGE); denaturing gradient gel electrophoresis
(DGGE) (Sheffield et al., Proc. Natl. Acad. Sci. USA 86:232-236
(1989)), mobility shift analysis (Orita et al., Proc. Natl. Acad.
Sci. USA 86:2766-2770 (1989)), restriction enzyme analysis (Flavell
et al., Cell 15:25 (1978); Geever et al., Proc. Natl. Acad. Sci.
USA 78:5081 (1981)); quantitative real-time PCR (Raca et al., Genet
Test 8(4):387-94 (2004)); heteroduplex analysis; chemical mismatch
cleavage (CMC) (Cotton et al., Proc. Natl. Acad. Sci. USA
85:4397-4401 (1985)); RNase protection assays (Myers et al.,
Science 230:1242 (1985)); use of polypeptides that recognize
nucleotide mismatches, e.g., E. coli mutS protein; allele-specific
PCR, for example. See, e.g., U.S. Patent Publication No.
2004/0014095, to Gerber et al., which is incorporated herein by
reference in its entirety. In some embodiments, the methods
described herein include determining the sequence of the entire
region of 22q13 described herein as being of interest, e.g.,
between and including SNPs rs738596, rs738598, or rs135221 on the
proximal end, and rs13884 or rs137853 on the distal end. In some
embodiments, the sequence is determined on both strands of DNA.
[0100] In order to detect polymorphisms and/or polymorphic
variants, it will frequently be desirable to amplify a portion of
genomic DNA (gDNA) encompassing the polymorphic site. Such regions
can be amplified and isolated by PCR using oligonucleotide primers
designed based on genomic and/or cDNA sequences that flank the
site. See e.g., PCR Primer: A Laboratory Manual, Dieffenbach and
Dveksler, (Eds.); McPherson et al., PCR Basics: From Background to
Bench (Springer Verlag, 2000); Mattila et al., Nucleic Acids Res.,
19:4967 (1991); Eckert et al., PCR Methods and Applications, 1:17
(1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S.
Pat. No. 4,683,202. Other amplification methods that may be
employed include the ligase chain reaction (LCR) (Wu and Wallace,
Genomics, 4:560 (1989), Landegren et al., Science, 241:1077 (1988),
transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci.
USA, 86:1173 (1989)), self-sustained sequence replication (Guatelli
et al., Proc. Nat. Acad. Sci. USA, 87:1874 (1990)), and nucleic
acid based sequence amplification (NASBA). Guidelines for selecting
primers for PCR amplification are well known in the art. See, e.g.,
McPherson et al., PCR Basics: From Background to Bench,
Springer-Verlag, 2000. A variety of computer programs for designing
primers are available, e.g., `Oligo` (National Biosciences, Inc,
Plymouth Minn.), MacVector (Kodak/IBI), and the GCG suite of
sequence analysis programs (Genetics Computer Group, Madison, Wis.
53711).
[0101] In one example, a sample (e.g., a sample comprising genomic
DNA), is obtained from a subject. The DNA in the sample is then
examined to determine a haplotype as described herein. The
haplotype can be determined by any method described herein, e.g.,
by sequencing or by hybridization of the gene in the genomic DNA,
RNA, or cDNA to a nucleic acid probe, e.g., a DNA probe (which
includes cDNA and oligonucleotide probes) or an RNA probe. The
nucleic acid probe can be designed to specifically or
preferentially hybridize with a particular polymorphic variant.
[0102] In some embodiments, a peptide nucleic acid (PNA) probe can
be used instead of a nucleic acid probe in the hybridization
methods described above. PNA is a DNA mimetic with a peptide-like,
inorganic backbone, e.g., N-(2-aminoethyl)glycine units, with an
organic base (A, G, C, T or U) attached to the glycine nitrogen via
a methylene carbonyl linker (see, e.g., Nielsen et al.,
Bioconjugate Chemistry, The American Chemical Society, 5:1 (1994)).
The PNA probe can be designed to specifically hybridize to a
nucleic acid comprising a polymorphic variant conferring
susceptibility to or indicative of the presence of SZ.
[0103] In some embodiments, restriction digest analysis can be used
to detect the existence of a polymorphic variant of a polymorphism,
if alternate polymorphic variants of the polymorphism result in the
creation or elimination of a restriction site. A sample containing
genomic DNA is obtained from the individual. Polymerase chain
reaction (PCR) can be used to amplify a region comprising the
polymorphic site, and restriction fragment length polymorphism
analysis is conducted (see Ausubel et al., Current Protocols in
Molecular Biology, supra). The digestion pattern of the relevant
DNA fragment indicates the presence or absence of a particular
polymorphic variant of the polymorphism and is therefore indicative
of the presence or absence of susceptibility to SZ.
[0104] Sequence analysis can also be used to detect specific
polymorphic variants. A sample comprising DNA or RNA is obtained
from the subject. PCR or other appropriate methods can be used to
amplify a portion encompassing the polymorphic site, if desired.
The sequence is then ascertained, using any standard method, and
the presence of a polymorphic variant is determined.
[0105] Allele-specific oligonucleotides can also be used to detect
the presence of a polymorphic variant, e.g., through the use of
dot-blot hybridization of amplified oligonucleotides with
allele-specific oligonucleotide (ASO) probes (see, for example,
Saiki et al., Nature (London) 324:163-166 (1986)). An
"allele-specific oligonucleotide" (also referred to herein as an
"allele-specific oligonucleotide probe") is typically an
oligonucleotide of approximately 10-50 base pairs, preferably
approximately 15-30 base pairs, that specifically hybridizes to a
nucleic acid region that contains a polymorphism. An
allele-specific oligonucleotide probe that is specific for
particular a polymorphism can be prepared using standard methods
(see Ausubel et al., Current Protocols in Molecular Biology,
supra).
[0106] Generally, to determine which of multiple polymorphic
variants is present in a subject, a sample comprising DNA is
obtained from the individual. PCR can be used to amplify a portion
encompassing the polymorphic site. DNA containing the amplified
portion may be dot-blotted, using standard methods (see Ausubel et
al., Current Protocols in Molecular Biology, supra), and the blot
contacted with the oligonucleotide probe. The presence of specific
hybridization of the probe to the DNA is then detected. Specific
hybridization of an allele-specific oligonucleotide probe (specific
for a polymorphic variant indicative of susceptibility to SZ) to
DNA from the subject is indicative of susceptibility to SZ.
[0107] In some embodiments, fluorescence polarization
template-directed dye-terminator incorporation (FP-TDI) is used to
determine which of multiple polymorphic variants of a polymorphism
is present in a subject (Chen et al., (1999) Genome Research,
9(5):492-498). Rather than involving use of allele-specific probes
or primers, this method employs primers that terminate adjacent to
a polymorphic site, so that extension of the primer by a single
nucleotide results in incorporation of a nucleotide complementary
to the polymorphic variant at the polymorphic site.
[0108] Real-time pyrophosphate DNA sequencing is yet another
approach to detection of polymorphisms and polymorphic variants
(Alderborn et al., (2000) Genome Research, 10(8):1249-1258).
Additional methods include, for example, PCR amplification in
combination with denaturing high performance liquid chromatography
(dHPLC) (Underhill, P. A., et al., Genome Research, Vol. 7, No. 10,
pp. 996-1005, 1997).
[0109] The methods can include determining the genotype of a
subject with respect to both copies of the polymorphic site present
in the genome. For example, the complete genotype may be
characterized as -/-, as -/+, or as +/+, where a minus sign
indicates the presence of the reference or wild type sequence at
the polymorphic site, and the plus sign indicates the presence of a
polymorphic variant other than the reference sequence. If multiple
polymorphic variants exist at a site, this can be appropriately
indicated by specifying which ones are present in the subject. Any
of the detection means described herein can be used to determine
the genotype of a subject with respect to one or both copies of the
polymorphism present in the subject's genome.
[0110] In some embodiments, it is desirable to employ methods that
can detect the presence of multiple polymorphisms (e.g.,
polymorphic variants at a plurality of polymorphic sites) in
parallel or substantially simultaneously. Oligonucleotide arrays
represent one suitable means for doing so. Other methods, including
methods in which reactions (e.g., amplification, hybridization) are
performed in individual vessels, e.g., within individual wells of a
multi-well plate or other vessel may also be performed so as to
detect the presence of multiple polymorphic variants (e.g.,
polymorphic variants at a plurality of polymorphic sites) in
parallel or substantially simultaneously according to certain
embodiments of the invention.
Probes
[0111] Nucleic acid probes can be used to detect and/or quantify
the presence of a particular target nucleic acid sequence within a
sample of nucleic acid sequences, e.g., as hybridization probes, or
to amplify a particular target sequence within a sample, e.g., as a
primer. Probes have a complimentary nucleic acid sequence that
selectively hybridizes to the target nucleic acid sequence. In
order for a probe to hybridize to a target sequence, the
hybridization probe must have sufficient identity with the target
sequence, i.e., at least 70%, e.g., 80%, 90%, 95%, 98% or more
identity to the target sequence. The probe sequence must also be
sufficiently long so that the probe exhibits selectivity for the
target sequence over non-target sequences. For example, the probe
will be at least 20, e.g., 25, 30, 35, 50, 100, 200, 300, 400, 500,
600, 700, 800, 900 or more, nucleotides in length. In some
embodiments, the probes are not more than 30, 50, 100, 200, 300,
500, 750, or 1000 nucleotides in length. Probes are typically about
20 to about 1.times.10.sup.6 nucleotides in length. Probes include
primers, which generally refers to a single-stranded
oligonucleotide probe that can act as a point of initiation of
template-directed DNA synthesis using methods such as PCR
(polymerase chain reaction), LCR (ligase chain reaction), etc., for
amplification of a target sequence.
[0112] In some embodiments, the probe is a test probe, e.g., a
probe that can be used to detect polymorphisms in a region
described herein, e.g., polymorphisms as described herein,
including D22S526, D22S1749E, and/or other polymorphisms of the
Sult4a1 gene lying between SNP markers rs138060 and rs138110. In
some embodiments, the probe can hybridize to a target sequence
within a region delimited by SNP rs738596 and SNP rs743615
(described on the internet at
ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=738596 and
ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=743615, respectively).
[0113] In some embodiments, the probe can bind to another marker
sequence associated with SZ, SPD or SD, as described herein.
[0114] Control probes can also be used. For example, a probe that
binds a less variable sequence, e.g., repetitive DNA associated
with a centromere of a chromosome, can be used as a control. Probes
that hybridize with various centromeric DNA and locus-specific DNA
are available commercially, for example, from Vysis, Inc. (Downers
Grove, Ill.), Molecular Probes, Inc. (Eugene, Oreg.), or from
Cytocell (Oxfordshire, UK). Probe sets are available commercially,
e.g., from Applied Biosystems, e.g., the Assays-on-Demand SNP kits.
Alternatively, probes can be synthesized, e.g., chemically or in
vitro, or made from chromosomal or genomic DNA through standard
techniques. For example, sources of DNA that can be used include
genomic DNA, cloned DNA sequences, somatic cell hybrids that
contain one, or a part of one, human chromosome along with the
normal chromosome complement of the host, and chromosomes purified
by flow cytometry or microdissection. The region of interest can be
isolated through cloning, or by site-specific amplification via the
polymerase chain reaction (PCR). See, for example, Nath and
Johnson, Biotechnic. Histochem., 1998, 73(1):6-22, Wheeless et al.,
Cytometry 1994, 17:319-326, and U.S. Pat. No. 5,491,224.
[0115] In some embodiments, the probes are labeled, e.g., by direct
labeling, with a fluorophore, an organic molecule that fluoresces
after absorbing light of lower wavelength/higher energy. A directly
labeled fluorophore allows the probe to be visualized without a
secondary detection molecule. After covalently attaching a
fluorophore to a nucleotide, the nucleotide can be directly
incorporated into the probe with standard techniques such as nick
translation, random priming, and PCR labeling. Alternatively,
deoxycytidine nucleotides within the probe can be transaminated
with a linker. The fluorophore then is covalently attached to the
transaminated deoxycytidine nucleotides. See, e.g., U.S. Pat. No.
5,491,224.
[0116] Fluorophores of different colors can be chosen such that
each probe in a set can be distinctly visualized. For example, a
combination of the following fluorophores can be used:
7-amino-4-methylcoumarin-3-acetic acid (AMCA), Texas Red.TM.
(Molecular Probes, Inc., Eugene, Oreg.),
5-(and-6)-carboxy-X-rhodamine, lissamine rhodamine B, 5- (and-
6)-carboxyfluorescein, fluorescein-5-isothiocyanate (FITC),
7-diethylaminocoumarin-3-carboxylic acid,
tetramethylrhodamine-5-(and -6)-isothiocyanate, 5-(and
-6)-carboxytetramethylrhodamine, 7-hydroxycoumarin-3-carboxylic
acid, 6-[fluorescein 5-(and -6)-carboxamido]hexanoic acid,
N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a diaza-3-indacenepropionic
acid, eosin-5-isothiocyanate, erythrosin-5-isothiocyanate, and
Cascade.TM. blue acetylazide (Molecular Probes, Inc., Eugene,
Oreg.). Fluorescently labeled probes can be viewed with a
fluorescence microscope and an appropriate filter for each
fluorophore, or by using dual or triple band-pass filter sets to
observe multiple fluorophores. See, for example, U.S. Pat. No.
5,776,688. Alternatively, techniques such as flow cytometry can be
used to examine the hybridization pattern of the probes.
Fluorescence-based arrays are also known in the art.
[0117] In other embodiments, the probes can be indirectly labeled
with, e.g., biotin or digoxygenin, or labeled with radioactive
isotopes such as .sup.32P and .sup.3H. For example, a probe
indirectly labeled with biotin can be detected by avidin conjugated
to a detectable marker. For example, avidin can be conjugated to an
enzymatic marker such as alkaline phosphatase or horseradish
peroxidase. Enzymatic markers can be detected in standard
colorimetric reactions using a substrate and/or a catalyst for the
enzyme. Catalysts for alkaline phosphatase include
5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium.
Diaminobenzoate can be used as a catalyst for horseradish
peroxidase.
[0118] Oligonucleotide probes that exhibit differential or
selective binding to polymorphic sites may readily be designed by
one of ordinary skill in the art. For example, an oligonucleotide
that is perfectly complementary to a sequence that encompasses a
polymorphic site (i.e., a sequence that includes the polymorphic
site, within it or at one end) will generally hybridize
preferentially to a nucleic acid comprising that sequence, as
opposed to a nucleic acid comprising an alternate polymorphic
variant.
Arrays and Uses Thereof
[0119] In another aspect, the invention features arrays that
include a substrate having a plurality of addressable areas, and
methods of using them. At least one area of the plurality includes
a nucleic acid probe that binds specifically to a sequence in
chromosome 22q13, and can be used to detect the absence or presence
of a polymorphism, e.g., one or more SNPs, microsatellites,
minisatellites, or indels, as described herein, to determine a
haplotype in this region. For example, the array can include one or
more nucleic acid probes that can be used to detect a polymorphism
listed in table 4, 6, 7, 8, or 9. In some embodiments, the array
further includes at least one area that includes a nucleic acid
probe that can be used to specifically detect another marker
associated with SZ, SPD or SD, as described herein. The substrate
can be, e.g., a two-dimensional substrate known in the art such as
a glass slide, a wafer (e.g., silica or plastic), a mass
spectroscopy plate, or a three-dimensional substrate such as a gel
pad. In some embodiments, the probes are nucleic acid capture
probes.
[0120] Methods for generating arrays are known in the art and
include, e.g., photolithographic methods (see, e.g., U.S. Pat. Nos.
5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g.,
directed-flow methods as described in U.S. Pat. No. 5,384,261),
pin-based methods (e.g., as described in U.S. Pat. No. 5,288,514),
and bead-based techniques (e.g., as described in PCT US/93/04145).
The array typically includes oligonucleotide probes capable of
specifically hybridizing to different polymorphic variants.
According to the method, a nucleic acid of interest, e.g., a
nucleic acid encompassing a polymorphic site, (which is typically
amplified) is hybridized with the array and scanned. Hybridization
and scanning are generally carried out according to standard
methods. See, e.g., Published PCT Application Nos. WO 92/10092 and
WO 95/11995, and U.S. Pat. No. 5,424,186. After hybridization and
washing, the array is scanned to determine the position on the
array to which the nucleic acid hybridizes. The hybridization data
obtained from the scan is typically in the form of fluorescence
intensities as a function of location on the array.
[0121] Arrays can include multiple detection blocks (i.e., multiple
groups of probes designed for detection of particular
polymorphisms). Such arrays can be used to analyze multiple
different polymorphisms. Detection blocks may be grouped within a
single array or in multiple, separate arrays so that varying
conditions (e.g., conditions optimized for particular
polymorphisms) may be used during the hybridization. For example,
it may be desirable to provide for the detection of those
polymorphisms that fall within G-C rich stretches of a genomic
sequence, separately from those falling in A-T rich segments.
Additional description of use of oligonucleotide arrays for
detection of polymorphisms can be found, for example, in U.S. Pat.
Nos. 5,858,659 and 5,837,832. In addition to oligonucleotide
arrays, cDNA arrays may be used similarly in certain embodiments of
the invention.
[0122] The methods described herein can include providing an array
as described herein; contacting the array with a sample, e.g., a
portion of genomic DNA that includes at least a portion of human
chromosome 22, e.g., a region between SNP rs738596 and SNP
rs743615, and, optionally, a different portion of genomic DNA,
e.g., a portion that includes a different portion of human
chromosome 22 or another chromosome, e.g., including another region
associated with SZ, SPD or SD., and detecting binding of a nucleic
acid from the sample to the array. Optionally, the method includes
amplifying nucleic acid from the sample, e.g., genomic DNA that
includes a portion of human chromosome 22q13 described herein, and,
optionally, a region that includes another region associated with
SZ, SPD, or SD, prior to or during contact with the array.
[0123] In some aspects, the methods described herein can include
using an array that can ascertain differential expression patterns
or copy numbers of one or more genes in samples from normal and
affected individuals. For example, arrays of probes to a marker
described herein can be used to measure polymorphisms between DNA
from a subject having SZ, SPD, or SD, and control DNA, e.g., DNA
obtained from an individual that does not have SZ, SPD, or SD, and
has no risk factors for SZ, SPD, or SD. Since the clones on the
array contain sequence tags, their positions on the array are
accurately known relative to the genomic sequence. Different
hybridization patterns between DNA from an individual afflicted
with SZ and DNA from a normal individual at areas in the array
corresponding to markers in human chromosome 22q13 described
herein, and, optionally, one or more other regions associated with
SZ, SPD, or SD, are indicative of a risk of SZ. Methods for array
production, hybridization, and analysis are described, e.g., in
Snijders et al., (2001) Nat. Genetics 29:263-264; Klein et al.,
(1999) Proc. Natl Acad. Sci. U.S.A. 96:4494-4499; Albertson et al.,
(2003) Breast Cancer Research and Treatment 78:289-298; and
Snijders et al. "BAC microarray based comparative genomic
hybridization." In: Zhao et al. (eds), Bacterial Artificial
Chromosomes: Methods and Protocols, Methods in Molecular Biology,
Humana Press, 2002.
[0124] In another aspect, the invention features methods of
determining the absence or presence of a haplotype associated with
SZ as described herein, using an array described above. The methods
include providing a two dimensional array having a plurality of
addresses, each address of the plurality being positionally
distinguishable from each other address of the plurality having a
unique nucleic acid capture probe, contacting the array with a
first sample from a test subject who is suspected of having or
being at risk for SZ, and comparing the binding of the first sample
with one or more references, e.g., binding of a sample from a
subject who is known to have SZ, SPD, or SD, and/or binding of a
sample from a subject who is unaffected, e.g., a control sample
from a subject who neither has, nor has any risk factors for SZ,
SPD, or SD. In some embodiments, the methods include contacting the
array with a second sample from a subject who has SZ, SPD or SD;
and comparing the binding of the first sample with the binding of
the second sample. In some embodiments, the methods include
contacting the array with a third sample from a cell or subject
that does not have SZ and is not at risk for SZ; and comparing the
binding of the first sample with the binding of the third sample.
In some embodiments, the second and third samples are from first or
second-degree relatives of the test subject. Binding, e.g., in the
case of a nucleic acid hybridization, with a capture probe at an
address of the plurality, can be detected by any method known in
the art, e.g., by detection of a signal generated from a label
attached to the nucleic acid.
Schizophrenia, Schizotypal Personality Disorder, and
Schizoaffective Disorder
[0125] The methods described herein can be used to determine an
individual's risk of developing schizophrenia (SZ), schizotypal
personality disorder (SPD), and/or a schizoaffective disorder
(SD).
[0126] Schizophrenia (SZ)
[0127] SZ is considered a clinical syndrome, and is probably a
constellation of several pathologies. Substantial heterogeneity is
seen between cases, which is thought to reflect multiple
overlapping etiologic factors, including both genetic and
environmental contributions. A diagnosis of SZ is typically
indicated by chronic psychotic symptoms, e.g., hallucinations and
delusions. Disorganization of thought and behavior are common and
are considered distinguishing factors in the diagnosis of SZ.
Patients typically have some subtle impairments in cognition.
Reduced emotional experience and expression, low drive, and
impaired speech are observed in a subgroup of patients. Cognitive,
emotional and social impairments often appear early in life, while
the psychotic symptoms typically manifest in late adolescence or
early adulthood in men, a little later in women.
[0128] A diagnosis of SZ can be made according to the criteria
reported in the Diagnostic and Statistical Manual of Mental
Disorders, Fourth Edition, Text Revision, American Psychiatric
Association, 2000, (referred to herein as DSM-IV) as follows:
[0129] Diagnostic Criteria for SZ
[0130] All six criteria must be met for a diagnosis of SZ.
[0131] A. Characteristic symptoms: Two (or more) of the following,
each present for a significant portion of time during a one month
period (or less if successfully treated):
[0132] (1) delusions
[0133] (2) hallucinations
[0134] (3) disorganized speech (e.g., frequent derailment or
incoherence)
[0135] (4) grossly disorganized or catatonic behavior
[0136] (5) negative symptoms, e.g., affective flattening, alogia,
or avolition
[0137] Only one criterion A symptom is required if delusions are
bizarre or hallucinations consist of a voice keeping up a running
commentary on the person's behavior or thoughts, or two or more
voices conversing with each other.
[0138] B. Social/occupational dysfunction: For a significant
portion of the time since the onset of the disturbance, one or more
major areas of functioning such as work, interpersonal relations,
or self-care are markedly below the level achieved prior to the
onset (or when the onset is in childhood or adolescence, failure to
achieve expected level of interpersonal, academic, or occupational
achievement).
[0139] C. Duration: Continuous signs of the disturbance persist for
at least 6 months. This 6-month period must include at least 1
month of symptoms (or less if successfully treated) that meet
Criterion A (i.e., active-phase symptoms) and may include periods
of prodromal or residual symptoms. During these prodromal or
residual periods, the signs of the disturbance may be manifested by
only negative symptoms or two or more symptoms listed in Criterion
A present in an attenuated form (e.g., odd beliefs, unusual
perceptual experiences).
[0140] D. Schizoaffective and Mood Disorder Exclusion:
Schizoaffective Disorder and Mood Disorder With Psychotic Features
have been ruled out because either (1) no major depressive, manic,
or mixed episodes have occurred concurrently with the active-phase
symptoms; or (2) if mood episodes have occurred during active-phase
symptoms, their total duration has been brief relative to the
duration of the active and residual periods.
[0141] E. Substance/General Medical Condition Exclusion: The
disturbance is not due to the direct physiological effects of a
substance (e.g., a drug of abuse, a medication) or a general
medical condition.
[0142] F. Relationship to a Pervasive Developmental Disorder: If
the patient has a history of Autistic Disorder or another Pervasive
Developmental Disorder, the additional diagnosis of SZ is made only
if prominent delusions or hallucinations are also present for at
least a month (or less if successfully treated).
[0143] Schizoaffective Disorder (SD)
[0144] SD is characterized by the presence of affective (depressive
or manic) symptoms and schizophrenic symptoms within the same,
uninterrupted episode of illness.
[0145] Diagnostic Criteria for Schizoaffective Disorder
[0146] The DSM-IV Criteria for a diagnosis of schizoaffective
disorder is as follows:
[0147] An uninterrupted period of illness during which, at some
time, there is either (1) a Major Depressive Episode (which must
include depressed mood), (2) a Manic Episode, or (3) a Mixed
Episode, concurrent with symptoms that meet (4) Criterion A for SZ,
above.
[0148] A. Criteria for Major Depressive Episode
[0149] At least five of the following symptoms must be present
during the same 2-week period and represent a change from previous
functioning; at least one of the symptoms is either (1) depressed
mood or (2) loss of interest or pleasure.
[0150] (1) depressed mood most of the day, nearly every day, as
indicated by either subjective report (e.g., feels sad or empty) or
observation made by others (e.g., appears tearful). In children and
adolescents, this can be an irritable mood.
[0151] (2) markedly diminished interest or pleasure in all, or
almost all, activities most of the day, nearly every day (as
indicated by either subjective account or observation made by
others)
[0152] (3) significant weight loss when not dieting or weight gain
(e.g., a change of more than 5% of body weight in a month), or
decrease or increase in appetite nearly every day. (In children,
failure to make expected weight gains is considered).
[0153] (4) insomnia or hypersomnia nearly every day
[0154] (5) psychomotor agitation or retardation nearly every day
(observable by others, not merely subjective feelings of
restlessness or being slowed down)
[0155] (6) fatigue or loss of energy nearly every day
[0156] (7) feelings of worthlessness or excessive or inappropriate
guilt (which may be delusional) nearly every day (not merely
self-reproach or guilt about being sick)
[0157] (8) diminished ability to think or concentrate, or
indecisiveness, nearly every day (either by subjective account or
as observed by others)
[0158] (9) recurrent thoughts of death (not just fear of dying),
recurrent suicidal ideation without a specific plan, or a suicide
attempt or a specific plan for committing suicide
[0159] In addition, the symptoms do not meet criteria for a Mixed
Episode. The symptoms cause clinically significant distress or
impairment in social, occupational, or other important areas of
functioning. The symptoms are not due to the direct physiological
effects of a substance (e.g., a drug of abuse, a medication) or a
general medical condition (e.g., hypothyroidism).
[0160] The symptoms are not better accounted for by Bereavement,
i.e., after the loss of a loved one, the symptoms persist for
longer than 2 months, or are characterized by marked functional
impairment, morbid preoccupation with worthlessness, suicidal
ideation, psychotic symptoms, or psychomotor retardation.
[0161] B. Criteria for Manic Episode
[0162] A manic episode is a distinct period of abnormally and
persistently elevated, expansive, or irritable mood, lasting at
least one week (or any duration, if hospitalization is
necessary).
[0163] During the period of mood disturbance, three (or more) of
the following symptoms have persisted (four if the mood is only
irritable) and have been present to a significant degree:
[0164] (1) inflated self-esteem or grandiosity
[0165] (2) decreased need for sleep (e.g., feels rested after only
3 hours of sleep)
[0166] (3) more talkative than usual or pressure to keep
talking
[0167] (4) flight of ideas or subjective experience that thoughts
are racing
[0168] (5) distractibility (i.e., attention too easily drawn to
unimportant or irrelevant external stimuli)
[0169] (6) increase in goal-directed activity (either socially, at
work or school, or sexually) or psychomotor agitation
[0170] (7) excessive involvement in pleasurable activities that
have a high potential for painful consequences (e.g., engaging in
unrestrained buying sprees, sexual indiscretions, or foolish
business investments)
[0171] The symptoms do not meet criteria for a Mixed Episode. The
mood disturbance is sufficiently severe to cause marked impairment
in occupational functioning or in usual social activities or
relationships with others, or to necessitate hospitalization to
prevent harm to self or others, or there are psychotic features.
The symptoms are not due to the direct physiological effects of a
substance (e.g., a drug of abuse, a medication, or other treatment)
or a general medical condition (e.g., hyperthyroidism).
[0172] C. Criteria for Mixed Episode
[0173] A mixed episode occurs when the criteria are met both for a
Manic Episode and for a Major Depressive Episode (except for
duration) nearly every day during at least a 1-week period. The
mood disturbance is sufficiently severe to cause marked impairment
in occupational functioning or in usual social activities or
relationships with others, or to necessitate hospitalization to
prevent harm to self or others, or there are psychotic
features.
[0174] The symptoms are not due to the direct physiological effects
of a substance (e.g., a drug of abuse, a medication, or other
treatment) or a general medical condition (e.g.,
hyperthyroidism).
[0175] D. Criterion A of SZ
[0176] See above.
[0177] E. Types of SD
[0178] The type of SD may be may be specifiable, as either Bipolar
Type, if the disturbance includes a Manic or a Mixed Episode (or a
Manic or a Mixed Episode and Major Depressive Episodes), or
Depressive Type, if the disturbance only includes Major Depressive
Episodes.
[0179] F. Associated Features
[0180] Features associated with SD include Learning Problems,
Hypoactivity, Psychotic, Euphoric Mood, Depressed Mood,
Somatic/Sexual Dysfunction, Hyperactivity,
[0181] Guilt/Obsession, Odd/Eccentric/Suspicious Personality,
Anxious/Fearful/Dependent Personality, and
Dramatic/Erratic/Antisocial Personality.
[0182] Schizotypal Personality Disorder (SPD)
[0183] Diagnostic Criteria for SPD
[0184] A diagnosis of SPD under the criteria of the DSM-IV is
generally based on a pervasive pattern of social and interpersonal
deficits marked by acute discomfort with, and reduced capacity for,
close relationships as well as by cognitive or perceptual
distortions and eccentricities of behavior, beginning by early
adulthood and present in a variety of contexts, as indicated by
five (or more) of the following:
[0185] (1) ideas of reference (excluding delusions of
reference)
[0186] (2) odd beliefs or magical thinking that influences behavior
and is
[0187] (3) inconsistent with subcultural norms (e.g.,
superstitiousness, belief in clairvoyance, telepathy, or "sixth
sense;" in children and adolescents, bizarre fantasies or
preoccupations)
[0188] (4) unusual perceptual experiences, including bodily
illusions
[0189] (5) odd thinking and speech (e.g., vague, circumstantial,
metaphorical, overelaborate, or stereotyped)
[0190] (6) suspiciousness or paranoid ideation
[0191] (7) inappropriate or constricted affect
[0192] (8) behavior or appearance that is odd, eccentric, or
peculiar
[0193] (9) lack of close friends or confidants other than
first-degree relatives
[0194] (10) excessive social anxiety that does not diminish with
familiarity and tends to be associated with paranoid fears rather
than negative judgments about self
[0195] SPD is diagnosed if the symptoms do not occur exclusively
during the course of SZ, a Mood Disorder With Psychotic Features,
another Psychotic Disorder, or a Pervasive Developmental Disorder,
and the disturbance is not due to the direct physiological effects
of a substance (e.g., a drug of abuse, a medication) or a general
medical condition.
[0196] Associated features of SPD include Depressed Mood and
Odd/Eccentric/Suspicious Personality.
Endophenotypes in SZ
[0197] A number of endophenotypes, i.e., intermediate phenotypes,
that may more closely reflect biological mechanisms behind SZ, have
been suggested, such as prepulse inhibition, structural
abnormalities evident in MRI scans, specific domains of cognition
(e.g., executive function), fine motor performance, working memory,
etc.
[0198] Endophenotypes can also include clinical manifestations such
as hallucinations, paranoia, mania, depression,
obsessive-compulsive symptoms, etc., as well as response or lack of
response to drugs and comorbidity for substance and alcohol
abuse.
[0199] See, e.g., Kendler et al., Am J Psychiatry 152(5):749-54
(1995); Gottesman and Gould, Am J Psychiatry 160(4):636-45 (2003);
Cadenhead, Psychiatric Clinics of North America. 25(4):837-53
(2002); Gottesman and Gould, American Journal of Psychiatry.
160(4):636-45 (2003); Heinrichs, Neuroscience & Biobehavioral
Reviews. 28(4):379-94 (2004); and Zobel and Maier, Nervenarzt.
75(3):205-14 (2004).
[0200] There is now evidence that some candidate genes that were
identified using DSM-IV type categorical definitions for "affected"
individuals may influence specific endophenotypes, see, e.g., Baker
et al., Biol Psychiatry 58(1):23-31 (2005); Cannon et al., Arch Gen
Psychiatry 62(11):1205-13 (2005); Gothelf et al., Nat Neurosci
8(11):1500-2 (2005); Hallmayer et al., Am J Hum Genet 77(3):468-76
(2005); Callicott et al., Proc Natl Acad Sci USA 102(24):8627-32
(2005); Gornick et al., J Autism Dev Disord 1-8 (2005). Thus, the
methods described herein can be used to associate haplotypes of
22q13 with specific endophenotypes.
Current Treatment of SZ, SD, or SPD
[0201] Subjects with SZ typically require acute treatment for
psychotic exacerbations, and long-term treatment including
maintenance and prophylactic strategies to sustain symptom
improvement and prevent recurrence of psychosis. Subjects with
schizoaffective disorder experience the symptoms of both SZ and
affective disorder (manic and/or depressive), thus require the
specific treatments for each disorder. Subjects with SPD sometimes
require medication for acute psychotic episodes but are often
treated using psychosocial methods. The methods described herein
can include the administration of one or more accepted or
experimental treatment modalities to a person identified as at risk
of developing SZ, SPD, or a SD, based on the presence of a
haplotype associated with SZ, SPD, or SD. Currently accepted
treatments presently include both pharmacologic and psychosocial
management, and occasionally electroconvulsive therapy (ECT).
[0202] Standard pharmacologic therapies for SZ and SD include the
administration of one or more antipsychotic medications, which are
typically antagonists acting at postsynaptic D.sub.2 dopamine
receptors in the brain. Antipsychotic medications include
conventional, or first generation, antipsychotic agents, which are
sometimes referred to as neuroleptics because of their neurologic
side effects, and second generation antipsychotic agents, which are
less likely to exhibit neuroleptic effects and have been termed
atypical antipsychotics.
[0203] In some embodiments, the methods described herein include
the administration of one or more antipsychotic medications to a
person identified by a method described herein as being at risk of
developing SZ, SPD, or SD. Antipsychotic medications substantially
reduce the risk of relapse in the stable phase of illness. In some
embodiments, the methods include the administration of a first
generation antipsychotic medication at a dose that is around the
"extrapyramidal symptom (EPS) threshold" (i.e., the dose that will
induce extrapyramidal side effects, e.g., bradykinesia, rigidity,
or dyskinesia, with minimal rigidity detectable on physical
examination, and/or a second-generation antipsychotics at a dose
that is therapeutic, yet below the EPS threshold.
[0204] Standard pharmacologic therapies for SD also include the
administration of a combination of antidepressant, and anti-anxiety
medication. Suitable antidepressants include serotonergic
antidepressants, e.g., fluoxetine or trazodone. Suitable
anxiolytics include benzodiazepines, e.g., lorazepam, clonazepam.
Lithium can also be administered. Thus, in some embodiments, the
methods can include the administration of one or more
antidepressant and/or anti-anxiety medications to a person
identified as at risk of developing SZ, SPD, or SD.
[0205] The methods can also include psychosocial and rehabilitation
interventions, e.g., interventions that are generally accepted as
therapeutically beneficial, e.g., cognitive-behavioral therapy for
treatment-resistant positive psychotic symptoms; supportive,
problem-solving, educationally oriented psychotherapy; family
therapy and education programs aimed at helping patients and their
families understand the patient's illness, reduce stress, and
enhance coping capabilities; social and living skills training;
supported employment programs; and/or the provision of supervised
residential living arrangements.
[0206] Currently accepted treatments for SZ are described in
greater detail in the Practice Guideline for the Treatment of
Patients With Schizophrenia American Psychiatric Association,
Second Edition, American Psychiatric Association, 2004, which is
incorporated herein by reference in its entirety.
Methods of Determining Treatment Regimens and Methods of Treating
SZ, SPD or SD
[0207] As described herein, the presence of haplotypes described
herein at chromosome 22q13 has been correlated with poor patient
prognosis. Thus, the new methods can also include selecting a
treatment regimen for a subject determined to be at risk for
developing SZ, SPD or SD, based upon the absence or presence of a
haplotype associated with SZ as described herein. The determination
of a treatment regimen can also be based upon the absence or
presence of other risk factors associated with SZ, e.g., as
described herein. Therefore, the methods of the invention can
include selecting a treatment regimen for a subject having one or
more risk factors for SZ, and having a haplotype described herein
at chromosome 22q13. The methods can also include administering a
treatment regimen to a subject having, or at risk for developing,
SZ to thereby treat, prevent or delay further progression of the
disease. A treatment regimen can include the administration of
antipsychotic medications to a subject identified as at risk of
developing SZ before the onset of any psychotic episodes.
[0208] As used herein, the term "treat" or "treatment" is defined
as the application or administration of a treatment regimen, e.g.,
a therapeutic agent or modality, to a subject, e.g., a patient. The
subject can be a patient having SZ, a symptom of SZ or at risk of
developing (i.e., a predisposition toward) SZ. The treatment can be
to cure, heal, alleviate, relieve, alter, remedy, ameliorate,
palliate, improve or affect SZ, the symptoms of SZ or the
predisposition toward SZ.
[0209] The methods of the invention, e.g., methods of determining a
treatment regimen and methods of treatment or prevention of SZ, can
further include the step of monitoring the subject, e.g., for a
change (e.g., an increase or decrease) in one or more of the
diagnostic criteria for SZ listed herein, or any other parameter
related to clinical outcome. The subject can be monitored in one or
more of the following periods: prior to beginning of treatment;
during the treatment; or after one or more elements of the
treatment have been administered. Monitoring can be used to
evaluate the need for further treatment with the same or a
different therapeutic agent or modality. Generally, a decrease in
one or more of the parameters described above is indicative of the
improved condition of the subject, although with red blood cell and
platelet levels, an increase can be associated with the improved
condition of the subject.
[0210] The methods can be used, e.g., to evaluate the suitability
of, or to choose between alternative treatments, e.g., a particular
dosage, mode of delivery, time of delivery, inclusion of adjunctive
therapy, e.g., administration in combination with a second agent,
or generally to determine the subject's probable drug response
genotype. In a preferred embodiment, a treatment for SZ can be
evaluated by administering the same treatment or combinations or
treatments to a subject having SZ, SPD or SD and a haplotype as
described herein at human chromosome 22q13 and to a subject that
has SZ but does not have a haplotype as described herein at human
chromosome 22q13. The effects of the treatment or combination of
treatments on each of these subjects can be used to determine if a
treatment or combination of treatments is particularly effective on
a sub-group of subjects having SZ, SPD or SD. In other embodiments,
various treatments or combinations of treatments can be evaluated
by administering two different treatments or combinations of
treatments to at least two different subjects having SZ, SPD or SD
and a haplotype as described herein in human chromosome 22q13. Such
methods can be used to determine if a particular treatment or
combination of treatments is more effective than others in treating
this subset of SZ, SPD and/or SD patients.
[0211] Various treatment regimens are known for treating SZ, e.g.,
as described herein.
Pharmacogenomics
[0212] With regards to both prophylactic and therapeutic methods of
treatment of SZ, such treatments may be specifically tailored or
modified, based on knowledge obtained from the field of
pharmacogenomics. "Pharmacogenomics," as used herein, refers to the
application of genomics technologies such as structural chromosomal
analysis, to drugs in clinical development and on the market. See,
for example, Eichelbaum et al., Clin. Exp. Pharmacol. Physiol.
23:983-985 (1996) and Linder et al., Clin. Chem. 43:254-266 (1997).
Specifically, as used herein, the term refers the study of how a
patient's genes determine his or her response to a drug (e.g., a
patient's "drug response phenotype," or "drug response genotype").
Thus, another aspect of the invention provides methods for
tailoring an individual's prophylactic or therapeutic treatment
according to that individual's drug response genotype.
[0213] Information generated from pharmacogenomic research using a
method described herein can be used to determine appropriate dosage
and treatment regimens for prophylactic or therapeutic treatment of
an individual. This knowledge, when applied to dosing or drug
selection, can avoid adverse reactions or therapeutic failure and
thus enhance therapeutic or prophylactic efficiency when
administering a therapeutic composition, e.g., a cytotoxic agent or
combination of cytotoxic agents, to a patient, as a means of
treating or preventing SZ.
[0214] In one embodiment, a physician or clinician may consider
applying knowledge obtained in relevant pharmacogenomics studies,
e.g., using a method described herein, when determining whether to
administer a pharmaceutical composition, e.g., an antipsychotic
agent or a combination of antipsychotic agents, to a subject. In
another embodiment, a physician or clinician may consider applying
such knowledge when determining the dosage, e.g., amount per
treatment or frequency of treatments, of a treatment, e.g., a
antipsychotic agent or combination of antipsychotic agents,
administered to a patient.
[0215] As one example, a physician or clinician may determine (or
have determined, e.g., by a laboratory) the haplotype of a subject
at chromosome 22q13, and optionally one or more other markers
associated with SZ, SPD, or SD, of one or a group of subjects who
may be participating in a clinical trial, wherein the subjects have
SZ, SPD, or SD, and the clinical trial is designed to test the
efficacy of a pharmaceutical composition, e.g., an antipsychotic or
combination of antipsychotic agents, and wherein the physician or
clinician attempts to correlate the genotypes of the subjects with
their response to the pharmaceutical composition.
[0216] As another example, information regarding a haplotype
associated with an increased risk of SZ, SPD or SD, as described
herein, can be used to stratify or select a subject population for
a clinical trial. The information can, in some embodiments, be used
to stratify individuals that may exhibit a toxic response to a
treatment from those that will not. In other cases, the information
can be used to separate those that will be non-responders from
those who will be responders. The haplotypes described herein can
be used in pharmacogenomics-based design and manage the conduct of
a clinical trial, e.g., as described in U.S. Pat. Pub. No.
2003/0108938.
Theranostics
[0217] Also included herein are compositions and methods for the
identification and treatment of subjects who have an increased risk
of SZ, SPD or SD, such that a theranostic approach can be taken to
test such individuals to determine the effectiveness of a
particular therapeutic intervention (e.g., a pharmaceutical or
non-pharmaceutical intervention as described herein) and to alter
the intervention to 1) reduce the risk of developing adverse
outcomes and 2) enhance the effectiveness of the intervention.
Thus, in addition to diagnosing or confirming the predisposition to
SZ, SPD or SD, the methods and compositions described herein also
provide a means of optimizing the treatment of a subject having
such a disorder. Provided herein is a theranostic approach to
treating and preventing SZ, SPD or SD, by integrating diagnostics
and therapeutics to improve the real-time treatment of a subject.
Practically, this means creating tests that can identify which
patients are most suited to a particular therapy, and providing
feedback on how well a drug is working to optimize treatment
regimens.
[0218] Within the clinical trial setting, a theranostic method or
composition of the invention can provide key information to
optimize trial design, monitor efficacy, and enhance drug safety.
For instance, "trial design" theranostics can be used for patient
stratification, determination of patient eligibility
(inclusion/exclusion), creation of homogeneous treatment groups,
and selection of patient samples that are representative of the
general population. Such theranostic tests can therefore provide
the means for patient efficacy enrichment, thereby minimizing the
number of individuals needed for trial recruitment. "Efficacy"
theranostics are useful for monitoring therapy and assessing
efficacy criteria. Finally, "safety" theranostics can be used to
prevent adverse drug reactions or avoid medication error.
[0219] The methods described herein can include retrospective
analysis of clinical trial data as well, both at the subject level
and for the entire trial, to detect correlations between a
haplotype as described herein and any measurable or quantifiable
parameter relating to the outcome of the treatment, e.g., efficacy
(the results of which may be binary (i.e., yes and no) as well as
along a continuum), side-effect profile (e.g., weight gain,
metabolic dysfunction, lipid dysfunction, movement disorders, or
extrapyramidal symptoms), treatment maintenance and discontinuation
rates, return to work status, hospitalizations, suicidality, total
healthcare cost, social functioning scales, response to
non-pharmacological treatments, and/or dose response curves. The
results of these correlations can then be used to influence
decision-making, e.g., regarding treatment or therapeutic
strategies, provision of services, and/or payment. For example, a
correlation between a positive outcome parameter (e.g., high
efficacy, low side effect profile, high treatment maintenance/low
discontinuation rates, good return to work status, low
hospitalizations, low suicidality, low total healthcare cost, high
social function scale, favorable response to non-pharmacological
treatments, and/or acceptable dose response curves) and a selected
haplotype can influence treatment such that the treatment is
recommended or selected for a subject having the selected
haplotype.
Kits
[0220] Also within the scope of the invention are kits comprising a
probe that hybridizes with a region of human chromosome at 22q13
and can be used to detect a polymorphism described herein. The kit
can include one or more other elements including: instructions for
use; and other reagents, e.g., a label, or an agent useful for
attaching a label to the probe. Instructions for use can include
instructions for diagnostic applications of the probe for assessing
risk of SZ in a method described herein. Other instructions can
include instructions for attaching a label to the probe,
instructions for performing in situ analysis with the probe, and/or
instructions for obtaining a sample to be analyzed from a subject.
As discussed above, the kit can include a label, e.g., any of the
labels described herein. In some embodiments, the kit includes a
labeled probe that hybridizes to a region of human chromosome at
22q13, e.g., a labeled probe as described herein.
[0221] The kit can also include one or more additional probes that
hybridize to the same chromosome, e.g., chromosome 22, or another
chromosome or portion thereof that can have an abnormality
associated with risk for SZ. For example, the additional probe or
probes can be: a probe that hybridizes to human chromosome 22q11-12
or a portion thereof, (e.g., a probe that detects a sequence
associated with SZ in this region of chromosome 22), or probes that
hybridize to all or a portion of 22q12.3 (e.g., near D22S283),
22q11.2, 22q11.2, 22q11-q13, 1q42.1, 1q42.1, 18p, 15q15, 14q32.3,
13q34, 13q32, 12q24, 11q14-q21, 1q21-q22, 10p15-p13 (e.g., near
D10S189), 10.sub.822.3, 8p21, 6q13-q26, 6p22.3, 6p23, 5q11.2-q13.3,
and/or 3p25. A kit that includes additional probes can further
include labels, e.g., one or more of the same or different labels
for the probes. In other embodiments, the additional probe or
probes provided with the kit can be a labeled probe or probes. When
the kit further includes one or more additional probe or probes,
the kit can further provide instructions for the use of the
additional probe or probes.
[0222] Kits for use in self-testing can also be provided. For
example, such test kits can include devices and instructions that a
subject can use to obtain a sample, e.g., of buccal cells or blood,
without the aid of a health care provider. For example, buccal
cells can be obtained using a buccal swab or brush, or using
mouthwash.
[0223] Kits as provided herein can also include a mailer, e.g., a
postage paid envelope or mailing pack, that can be used to return
the sample for analysis, e.g., to a laboratory. The kit can include
one or more containers for the sample, or the sample can be in a
standard blood collection vial. The kit can also include one or
more of an informed consent form, a test requisition form, and
instructions on how to use the kit in a method described herein.
Methods for using such kits are also included herein. One or more
of the forms, e.g., the test requisition form, and the container
holding the sample, can be coded, e.g., with a bar code, for
identifying the subject who provided the sample.
[0224] Databases
[0225] Also provided herein are databases that include a list of
polymorphisms as described herein, and wherein the list is largely
or entirely limited to polymorphisms identified as useful in
performing genetic diagnosis of or determination of susceptibility
to SZ, SPD or SD as described herein. The list is stored, e.g., on
a flat file or computer-readable medium. The databases can further
include information regarding one or more subjects, e.g., whether a
subject is affected or unaffected, clinical information such as
endophenotype, age of onset of symptoms, any treatments
administered and outcomes (e.g., data relevant to pharmacogenomics,
diagnostics or theranostics), and other details, e.g., about the
disorder in the subject, or environmental or other genetic factors.
The databases can be used to detect correlations between a
particular haplotype and the information regarding the subject,
e.g., to detect correlations between a haplotype and a particular
endophenotype, or treatment response.
[0226] Transgenic Animals and Cells
[0227] Also provided herein are non-human transgenic animals and
cells that harbor one or more polymorphism described herein, e.g.,
one or more polymorphisms that constitute a haplotype associated
with SZ, SPD, or SD. Such animals and cells are useful for studying
the effect of a polymorphism on physiological function, and for
identifying and/or evaluating potential therapeutic agents for the
treatment of SZ, SPD, or SD, e.g., anti-psychotics.
[0228] As used herein, a "transgenic animal" is a non-human animal,
preferably a mammal in which one or more of the cells of the animal
includes a transgene. Examples of transgenic animals include
rodents (e.g., rats or mice), non-human primates, rabbits, sheep,
dogs, cows, goats, chickens, amphibians, and the like. A transgene
as used herein replicates a polymorphism described herein and is
integrated into or occurs in the genome of the cells, e.g., the
cultured cells or the cells of a transgenic animal. As one example,
included herein are cells in which one of the various alleles of
the Sult4a1 polymorphism has be re-created, e.g., an allele of
D22S1749E. Thus, a transgenic animal or cell can be one in which an
endogenous Sult4a1 gene has been altered to include an allele of
D22S1749E, e.g., an allele that is associated with an increased
risk of SZ, SD, or SPD. Methods are known in the art for generating
such animals and cells. e.g., by homologous recombination between
the endogenous gene and an exogenous DNA molecule introduced into a
cell, e.g., a cell of an animal, e.g., an embryonic cell of an
animal, prior to development of the animal.
[0229] A transgenic founder animal can be identified based upon the
presence of a transgene in its genome. A transgenic founder animal
can then be used to breed additional animals carrying the
transgene. Moreover, transgenic animals carrying one transgene
protein can further be bred to other transgenic animals carrying
other transgenes. The invention also includes populations of cells
from a transgenic animal as described herein.
[0230] Also provided are cells, preferably mammalian cells, e.g.,
neuronal type cells, in which an endogenous gene has been altered
to include a polymorphism as described herein. Techniques such as
targeted homologous recombinations, can be used to insert the
heterologous DNA as described in, e.g., Chappel, U.S. Pat. No.
5,272,071; WO 91/06667, published in May 16, 1991.
[0231] The invention is further illustrated by the following
examples, which should not be construed as further limiting. The
contents of all references, pending patent applications and
published patents, cited throughout this application, are hereby
expressly incorporated by reference.
EXAMPLES
Example 1
Analysis of Microsatellite Markers in Chromosome 22
[0232] Twenty-seven nuclear families, comprising 212 individuals,
each having multiple affected siblings were provided by the
Institutes of Mental Health (NIMH) Schizophrenia Genetics
Initiative. Self-description of heritage was a follows:
African-American, 12 families; European/Mediterranean, 11 families;
Hispanic, 2 families; other 2 families. DSM-III criteria were
compiled for all subjects by researchers at Columbia University,
Harvard University and Washington University. Detailed information
on ascertainment, diagnosis and informed consent has been
previously provided by these groups (Colinger et al., 1998; Faranoe
et al., 1998; Kaufmann et al., Am. J. Genet. 81:282-289 (1998)).
Using the DSM-III criteria for SZ, the sample contained 55 affected
sibling pairs, and using a broader disease definition that include
schizotypal personality disorder and schizoaffective disorder, the
sample contained 100 affected sibling pairs.
[0233] Initially, linkage was analyzed using a set of 14
microsatellite markers, which are listed in Table 1. As an example,
PCR was performed using primers for the microsatellite marker
D22s1749e. The upstream primer sequence was
5'-CAGCCGCACGCCATGGAACTCGAAG-3' (SEQ ID NO:1) and the downstream
primer sequence was 5'-GGCGCCATGACGTCACGCCTGC-3' (SEQ ID NO:2).
Each 10 .mu.l reaction contained a final concentration of 5 ng of
genomic DNA, 10.times. buffer (Roche), 0.16 U of AmpliTaq Gold, 2.0
mM MgCl.sub.2, 1 mM of each dNTP, 0.33 .mu.M of forward and reverse
primers, and 10% DMSO. PCR conditions consisted of an initial
enzyme activation at 95.degree. C. for 5 min, followed by 35 cycles
of 93.degree. C. for 2 min, 92.degree. C. for 1 min, 71.degree. C.
for 30 s, and 72.degree. C. for 2 min, and a final incubation at
72.degree. C. for 5 min. PCR products were analyzed and fragment
size was determined using the Biomek CEQ 8000 Analysis System.
TABLE-US-00001 TABLE 1 Markers on Human Chromosome 22q Marker.sup.a
Kosambi cM.sup.b Distance Mb.sup.b,c D22s311 D22s446 2.6000
20.3437-20.3439 D22s315 11.5000 24.3404-24.3406 D22s275 22.8000
D22s683 30.2000 34.8384-34.8389 D22s270 41.5000 41.3780-41.3782
rs138060 44.4000 42.5477 rs138097 44.4678 42.5755 D22s1749e 44.4863
42.5831-42.5833 rs138110 44.4897 42.5847 D22s274 47.0187
43.5897-43.5899 D22s1149 51.3187 44.9934-44.9935 D22s1170 56.7187
46.6712-46.6714 rs738596 59.4417 47.6932 rs738598 59.4857 47.7102
D22s1169 59.5187 47.7230-47.7231 rs2073224 59.6587 47.7490 rs738615
59.6617 47.7495 rs135221 60.1217 47.8325 rs767219 60.8517 47.9633
sJCW16 60.8917 47.9709 rs848768 61.7811 48.0389 rs848728 62.0582
48.0616 rs2269523 62.3402 48.0898 rs737734 62.4936 48.1051 rs136770
62.6528 48.1211 D22s526 63.0417 48.1599-48.1602 rs134474 63.7602
48.2321 rs134472 63.7671 48.2328 rs134454 63.8946 48.2455 rs135819
64.2029 48.2763 rs763126 64.2360 48.3094 rs916363 64.2650 48.3384
rs1573726 64.2803 48.3537 rs138817 64.3833 48.4839 rs138844 64.4047
48.5053 rs137853 64.6264 48.7459 rs1053744 65.0191 49.1759 D22s1744
65.1608 49.3178 D22s1743 65.3608 49.3418 .sup.aMarkers are listed
from 22cen to 22qter .sup.bBrennan et al. Genomics 63, 430-423
(2000) .sup.cEnsembl
[0234] Simple parametric models did not give significant evidence
for linkage, regardless of the mode of inheritance or the degree of
penetrance assumed. However, a model assuming genetic heterogeneity
resulted in maximum LOD score of 2.6 at marker D22s270 (.theta.=0)
for SZ and a value of 3.6 for a broader definition of disease that
included schizotypal personality disorder (SPD; FIG. 1). In
agreement with the findings of others, some evidence for linkage
near marker D22s683 was seen using the narrow definition (Vallada
et al., Psychiatr. Genet. 5:127-30 (1995); DeLisi et al., Am. J.
Psychiatry 159:803-12 (2002); Takahashi et al., Am. J. Med. Genet.
120B:11-7 (2003)). Similar peaks at D22s270 resulted from
nonparametric linkage analysis giving LOD scores of 2.5 and 2.7,
for the narrow and broad disease definitions, respectively (FIG.
1). Note that the broader disease definition results both in higher
LOD scores for D22s270 and an increase in the smaller, more distal
peak centered at sJCW16.
[0235] Initial mapping of D22s1749e was performed using the
MultiMap program (version 2.40) as described previously (Cox Matise
et al., Nature Genetics 6:384-390 (1994); Cox-Matise et al.,
Multimap, Automated genetic linkage mapping, version 2.4. (1996);
Brennan et al., Genomics 63:430-432 (2000)). TDT analysis was
performed using TRANSMIT (version 2.5.2) (Clayton, Am. J. Hum.
Genet. 65(4):1170-7 (1999)), with rare haplotypes aggregated so as
to prevent elevation of X.sup.2 values that can arise due to
expectations for rare haplotypes. The resulting global P values for
the X.sup.2 analyses estimate the significance of the transmission
distribution for all haplotypes combined, with rare haplotypes
being treated as a single haplotype. Similarly, X.sup.2 values for
transmission of individual genotypes and haplotypes, with one
degree of freedom, are determined by TRANSMIT.
Example 2
Identification of Sult4a1 as a Candidate Gene
[0236] A search for candidate genes near marker D22s270, performed
using public database resources, identified the
sulfotransferase-4A1 gene (Sult4a1), which is located within 1.2 Mb
of this microsatellite marker, and encodes a brain-specific
sulfotransferase believed to be involved in dopamine catabolism
(Falany et al., Biochem J. 346:857-64 (2000); Sakakibara et al.,
Gene 285:39-47 (2002); Liyou et al., J. Histochem. Cytochem.
51:655-64 (2003)).
[0237] Alignment of the genomic sequences with several
corresponding cDNA sequences (Z97055, AF176342, AF188698, AF251263,
AK091700, A1832543) indicated, in all likelihood, that the DNA
encoding the 5' non-translated leader region of the Sult4a1 mRNA
was polymorphic, having a varying number of imperfect GCC repeats
(primary accession numbers Z97055, AF176342, AF188698, AF251263,
AK091700, A1832543). To evaluate this possibility, a PCR procedure
was developed to amplify the genomic region at the 5' end of the
gene.
[0238] Briefly, SNPs were analyzed using Applied Biosystems
Assays-on-Demand SNP kits. Each 5 .mu.l reaction contained 2.5
.mu.l of Taq Man polymerase, 0.25 .mu.l of 20.times.SNP assay and
2.25 .mu.l of 10 ng genomic DNA. PCR conditions consisted of an
initial enzyme activation at 95.degree. C. for 10 min, followed by
40 cycles of 95.degree. C. for 15 sec, and 60.degree. C. for 1 min.
PCR products were analyzed using the ABI Prism 7900HT Sequence
Detection System. The region is very G-C rich and refractory to
amplification. Nonetheless, reproducible amplification of the
region was obtained for all families, confirming Mendelian
inheritance in all cases.
[0239] In this sample of families, seven alleles, with one two five
nucleotides separating adjacent alleles in the series, were
observed. The MultiMap program was used to confirm that D22s1749e
mapped approximately 10 cM distal to D22s683. Table 2 lists the
location, in mb, of this new microsatellite marker and the three
nearby SNPs that were used for TDT analysis.
TABLE-US-00002 TABLE 2 Markers Used Marker Location on Chromosome
22 (Mb.sup.a) rs138060 42.5477 rs138097 42.5755 D22s1749e 42.5831
rs138110 42.5847 .sup.aNCBI: www.ncbi.nlm.nih.gov/SNP/
[0240] In this sample of families, seven alleles of marker
D22s1749e ranging in size from 198 to 216 nucleotides were
observed. (Table 3).
TABLE-US-00003 TABLE 3 Observed Alleles of D22sl749e Size (nt)
Observed frequency.sup.a 198 0.0022 202 0.0088 207 0.385 209 0.033
212 0.286 213 0.165 216 0.022 .sup.aFrequency for the NIMH sample
using only those parental genotypes that were directly observed or
that could be unambiguously inferred.
[0241] Including this new marker in the linkage analysis did not
alter the location of the maximum LOD scores, which were still
observed at marker D22S270. Owing to the increased information
content, the maximum LOD values increased somewhat. Assuming
heterogeneity, a maximum LOD score of 2.90 was obtained using DSM
IIIR criteria, and a maximum LOD score of 3.96 was obtained for a
broader disease definition that included schizotypal personality
disorder (FIG. 2). Again, nonparametric linkage analysis provided
suggestive evidence for linkage at the same location, with LOD
scores of 2.6 and 2.8 for the narrow broad definitions,
respectively (FIG. 1, solid and broken black lines).
[0242] The sample was further expanded by adding 17 more families
to the original 27 families. Using the D22s1749e marker in linkage
analysis for the pooled sample (using a dominant model assuming
genetic heterogeneity, a penetrance of 50% for a heterozygote and a
1% allele) a single point heterogeneous LOD score of 4.78 was
obtained for the combined sample of 44 families (.alpha.=0.7).
[0243] Consistent with the initial findings, for the pooled sample,
D22S1749E showed significant deviation from expectation for
transmission to affected offspring using TRANSMIT (Clayton Am J Hum
Genet. 65(4):1170-7 (1999) (P=0.015 for SZ, and P=0.006 for the
broader definition including SPD).
Example 3
Identification of Haplotypes Including Markers in Sult4a1
[0244] The Sult4a1 candidate gene was further evaluated by
Transmission/Disequilibrium Test (TDT) analysis employing the new
microsatellite marker, along with three SNPs in the gene. Table 4
summarizes the results of the TDT analysis for these polymorphisms
and haplotypes involving them. Significant results were seen for
D22s1749e and various haplotypes involving D22s1749e and the three
SNPs in Sult4a1. In most cases, the results were more significant
for a narrow definition of schizophrenia (SZ), than for broader
definitions that included schizotypal personality disorder (SPD) or
schizoaffective disorder (SD).
TABLE-US-00004 TABLE 4 TDT Analysis of Sult4a1 Markers P Value for
Disease Definition .sup.a SZ + SZ + SPD + Marker(s) df SZ SPD SD
D22s1749e 4 .sup.b 0.04 0.05 0.04 rs138060-rs138097 3 .sup.b 0.04
0.12 0.12 rs138060- D22s1749e 7 .sup.b 0.008 0.004 0.17
rs138060-rs138097-D22s1749e 9 .sup.c 0.0014 0.0006 0.0055 rs138060-
D22s1749e- 10 .sup.c 0.0095 0.0017 0.018 rs138110 rs138097-
D22s1749e- 7 .sup.c 0.04 0.03 0.05 rs138110 rs138060-rs138097- 11
.sup.c 0.014 0.0064 0.04 D22s1749e-rs138110 .sup.a SZ =
schizophrenia, SPD = schizophrenia + schizotypal personality
disorder, SD = schizoaffective disorder. .sup.b Global chi square
values as determined by Transmit, with haplotypes having a
frequencies of 3% or less aggregated. .sup.c Global chi square
values as determined by Transmit with haplotypes having a
frequencies of 14% or less aggregated.
Example 4
Identification of Microsatellite Markers in 22q13 Showing TD
[0245] All of the microsatellite polymorphisms listed in Table 1
were tested for evidence of transmission disequilibrium. Other than
D22s1749e, only D22s256 showed significant results (Table 5).
[0246] D22s256 was evaluated using PCR with the following
conditions: 95.degree. C., 12 min, 1 cycle; 94.degree. C., 15 sec,
60.degree. C., 15 sec, 72.degree. C., 30 sec, 10 cycles; 89.degree.
C., 15 sec, 60.degree. C., 15 sec, 72.degree. C., 30 sec, 25
cycles; 72.degree. C., 30 min, 1 cycle. The primers were: Left:
5'-AGAGCAAGACTCTGTCTCAACA-3' (SEQ ID NO:3); Right,
5'-TTCTCCTTCACTTTCTGCCATG-3' (SEQ ID NO:4s). The left primer has a
HEX florescent label at the 5' end. PCR products were analyzed
using an ABI PRISM 377 DNA Sequencer with GeneScan and Genotyper
software packages. The expected product size was 250 to 308 nt.
[0247] In this sample of families, 16 of the 23 alleles of D22s256,
ranging in size from 258 to 305 nt, were observed. Using the narrow
DSM-III criteria for SZ provided significant results for this
marker (P=0.003). Broader disease definitions including SPD or both
SPD and schizoaffective disorder (SD) provided even more striking
results (P=0.002 and P=0.00009, respectively).
TABLE-US-00005 TABLE 5 TDT Analysis of Marker D22s526 Disease
definition Chi Square .sup.a P .sup.b SZ 24.97 0.003 SZ + SPD 31.93
0.0002 SZ + SPD + SD 33.95 0.00009 .sup.a Global chi square values
as determined by Transmit, with haplotypes having a frequencies of
3% or less aggregated. .sup.b Probability with 9 df.
Example 5
Identification of Haplotypes Associated with SZ, SPD, and SD-2 and
3 Marker Haplotypes
[0248] Tables 6 and 7 list two and three marker haplotypes,
respectively, that showed highly significant deviations from
expected transmission frequencies for affected offspring under the
broadest disease definition, including SZ, schizoaffective
disorder, and schizotypal personality disorder. The distances are
taken from the NCBI database (SNPdb build 125; Genome Build 35.1,
September 2005).
TABLE-US-00006 TABLE 6 TDT: Two marker haplotype p .ltoreq. 0.001
SZ + SD + SPD Marker p value Distances Mb rs2073224 - D22s526
1.5217E-05 47.7490-48.1602 rs738615- D22s526 1.5217E-05
47.7495-48.1602 rs767219- D22s526 0.0010 47.9633-48.1602 rs767219 -
rs1573726 0.0002 47.9633-48.3537 D22s526 - rs134472 0.0003
48.1602-48.2328 D22s526 - rs763126 3.787E-07 48.1602-48.3094
D22s526 - rs1573726 0.0004 48.1602-48.3537 D22s526 - rs138817
8.741E-09 48.1602-48.4839 D22s526 - rs138844 7.976E-10
48.1602-48.5053
TABLE-US-00007 TABLE 7 TDT: Three marker haplotype p .ltoreq. 0.001
SZ + SD + SPD Markers p value Distances Mb rs2073224-rs135221-
rs767219 7.4504E-06 47.7490-47.9633 rs2073224- rs135221- rs138817
4.0105E-09 47.7490-48.4839 rs2073224- rs767219- rs737734 8.3037E-29
47.7490-48.1051 rs2073224- rs767219-rs1573726 4.26677E-05
47.7490-48.3537 rs2073224-rs2269523-rs134472 1.9771E-09
47.7490-48.2328 rs135221- rs767219- rs916363 9.7080E-15
47.8325-48.3384 rs135221- rs767219- rs1573726 0.0002
47.8325-48.3537 rs135221- rs848768- rs1573726 4.7207E-05
47.8325-48.3537 rs135221- rs848768- rs138817 6.6501E-08
47.8325-48.4839 rs135221- rs848768-rs1053744 1.1167E-08
47.8325-49.1759 rs135221-rs737734-rs134472 9.8432E-06
47.8325-48.2328 rs135221-rs134474- rs1053744 1.6512E-12
47.8325-49.1759 rs135221-rs916363- rs138817 8.9512E-242
47.8325-48.4839 rs135221- rs916363- rs1053744 2.2041E-05
47.8325-49.1759 rs767219- rs848768- rs2269523 0.0001
47.9633-48.0898 rs767219- rs848768- rs737734 9.9720E-06
47.9633-48.1051 rs767219- rs848768- rs1573726 5.1963E-05
47.9633-48.3537 rs767219- rs848768- rs138817 2.5709E-08
47.9633-48.4839 rs767219-rs848728- rs737734 0.0009 47.9633-48.1051
rs767219-rs848728-rs136770 1.4604E-10 47.9633-48.1211
rs767219-rs848728-rs134472 9.8306E-23 47.9633-48.2328
rs767219-rs848728- rs1573726 0.0006 47.9633-48.3537 rs767219-
rs2269523-rs737734 4.0063E-16 47.9633-48.1051 rs767219- rs2269523-
rs1573726 0.0004 47.9633-48.3537 rs767219- rs737734-rs916363
3.8423E-06 47.9633-48.3384 rs848768- rs2269523-rs138817 3.3795E-08
48.0389-48.4839 rs136770-rs134474-rs763126 3.4059E-05
48.1211-48.3094
[0249] Additional haplotypes within this region were also
evaluated, and the results are presented in Table 8. Haplotypes
listed in bold show highly significant results for the narrowest
disease definition of SZ.
TABLE-US-00008 TABLE 8 Examples of additional haplotypes p .ltoreq.
0.001 for various disease definitions.sup.a Disease definition
Single Nucleotide Haplotypes SZ.sup.b SZ + SD.sup.c SZ + SPD.sup.d
SZ + SD + SPD.sup.e rs1355221-rs1053744 0.1309 0.0648 0.0003
3.96E-05 rs738596-rs763126 0.082 0.1049 0.4214 1.60E-16
rs135221-rs48768-rs1053744 0.0717 0.6809 0.0018 3.26E-27
rs135221-rs738615-rs138817 0.0677 0.8333 0.3203 5.47E-11
rs136770-rs134474-rs763126 0.0635 0.0792 2.28E-05 3.4E-05
rs135221-rs738598-rs2269523 0.0476 0.0021 0.0233 0.0011
rs135221-rs916363-rs138817 0.0320 0.0002 0.0214 2.2E-05
rs135221-rs763126-rs1573726 0.0154 0.0005 0.0162 0.0017
rs848768-rs738598-rs1573726 0.0065 0.0031 0.0109 1.48E-26
rs738598-rs2269523-rs1573726 0.0059 0.0015 0.0109 7.51E-17
rs738598-rs738615-rs1573726 0.0041 0.0010 0.5106 0.6582
rs737734-rs136770-rs763126 0.0010 0.0002 0.0241 0.0126
rs738598-rs1573726-rs138844 0.0008 0.0002 0.0003 7.18E-05
rs738596-rs738598-rs1573726 0.0007 0.0003 0.0031 0.0023
rs738596-rs1573726 0.0004 0.0007 0.0024 0.0040 rs738598-rs1573726
0.0004 0.0002 0.0109 0.0058 rs135819-rs1573726 0.0004 0.0008 0.0448
0.0467 rs738598-rs138844-rs1053744 0.0002 2.39E-05 0.0043 0.0012
rs1573726 8.06E-05 0.0001 0.0013 0.0015 rs153221-rs763126 4.28E-05
0.0073 0.0021 0.0310 rs135221-rs2269523-rs737734 9.76E-06 0.1564
0.0001 0.0560 rs737734-rs134474-rs134454 5.76E-07 3.85E-05 0.7276
0.5819 rs135221-rs848768-rs763126 3.80E-11 0.0273 0.0951 0.1246
rs2073224-rs763126-rs138844 4.10E-53 0.0023 0.0061 0.0071
rs738615-rs763126-rs138844 4.10E-53 0.0023 0.0061 0.0071 .sup.aAs
determined by TRANSMIT (rare haplotypes pooled) .sup.bSZ =
schizophrenia .sup.cSZ + SD = schizophrenia + schizoaffective
disorder .sup.dSZ + SPD = schizophrenia + schizotypal personality
disorder .sup.eSZ + SD + SPD = schizophrenia + schizoaffective
disorder + schizotypal personality disorder
Example 6
Identification of Haplotypes Associated with SZ--Alleles of
Sult4A
[0250] Table 9 summarizes X.sup.2 tests for specific haplotypes
that were determined to be transferred more frequently or less
frequently than expected to affected offspring using the narrow
DSM-III definition of SZ. The 213 nt allele for D22s1749e was
transmitted more often than expected, and the 207 nt allele less
often than expected to affected offspring. None of the SNPs, when
used alone, showed X.sup.2 values for transmission disequilibrium
that were significant at the P<0.01 level. However, several
haplotypes involving these SNPs in combination with D22s1749e
showed significant transmission distortion (Table 9).
TABLE-US-00009 TABLE 9 TDT Analysis for Specific Haplotypes (P <
0.01) Transmission to Affected Offspring (DSM-III schizophrenia)
Higher/Lower Marker(s) .sup.a Haplotype .sup.b than expected
.chi..sup.2 (1 df) P D22s1749e 213 higher 7.23 0.0071 rs138060-
D22s1749e A-213 higher 7.89 0.0049 rs138060- D22s1749e C-207 lower
7.58 0.0059 rs138060-rs138097-D22s1749e C-T-207 lower 6.73 0.0094
rs138060-rs138097-D22s1749e A-T-213 higher 8.02 0.0046 rs138060-
D22s1749e-rs138110 A-213-G higher 7.66 0.0056 rs138097-
D22s1749e-rs138110 T-213-G higher 8.01 0.0046 rs138060-rs138097-
D22s1749e- A-T-213-G higher 7.83 0.0051 rs138110 .sup.a
Polymorphisms are listed in proximal to distal order on the
chromosome. .sup.b Genotypes give the length (nt) for D22s1749e and
specific nucleotide descriptions for each SNP, listed in the same
order as the marker names.
[0251] The 213 nt allele of Sult4a1 appears to be transmitted more
often than expected to affected children. The 216 nt allele
occurred too rarely in this small sample for the TDT analysis to be
statistically valid, but tentatively, it too appears to be
preferentially transmitted to affected offspring. These alleles are
predicted to encode an mRNA with a longer 5' nontranslated leader
sequence than the shorter alleles. As one theory, not meant to be
binding, the longer 5' leader sequences might lower translatability
of the mRNAs and result in lower final levels of the Sult4a1
enzyme. At present, the major physiological substrate(s) of the
Sult4a1 isozyme is unknown, but in vitro, it functions on a variety
of phenolic compounds structurally resembling the catecholamines
(Sakakibara et al., Gene 285:39-47 (2002)).
[0252] These findings add to a body of results pointing to a role
for chromosome 22q in the etiology of SZ. In agreement with the
findings of others (Vallada et al., Psychiatr. Genet. 5:127-30
(1995); DeLisi et al., Am J Psychiatry 159:803-12 (2002); Takahashi
et al., Am. J. Med. Genet. 120B:11-7 (2003)), evidence for linkage
near marker D22s683 is seen at about 30 cM on the linkage map, but
the highest LOD score was obtained at 41.5 cM corresponding to
marker D22s270. The smaller peak at D22s683 was most prominent with
the narrow disease definition, while a broader disease definition
results in an additional distal linkage peak centered at
sJCW16.
[0253] Based on TDT analysis, both the Sult4a1 candidate gene and
the more distal region of 22q appear to contribute to the genetic
predisposition to SZ. In this sample of families, TDT provided
suggestive evidence for a role of the Sult4a1 candidate gene
located near marker D22s270, representing the major LOD score peak
observed in linkage analysis. In contrast, no evidence of
transmission disequilibrium was seen for most microsatellite
markers, including D22s683 and D22s270. However, highly significant
results, particularly for broader disease definitions, were seen
for marker D22s526, which is located within 200 kb of marker
sJCW16, corresponding to the more distal peak we see in linkage
analysis.
[0254] Taken together, these results support a two locus model,
involving a proximal locus, perhaps most significant for a narrowly
defined SZ and a more distal locus near D22s526, most likely
contributing additionally to SPD, SD and other SZ-spectrum
disorders. It now seems clear that sequences within these
chromosomal segments contribute to the genetic predisposition to
these disorders.
Example 7
Identification of Haplotypes Associated with SZ--Microdeletions at
D22s526
[0255] As described above, numerous two and three SNP haplotypes
spanning the distal region show highly significant distortions in
transmission ratios for DSM-IIIR diagnosed SZ and broader disease
definitions (P<10.sup.-5). A close evaluation of the haplotypes
revealed particular SNP haplotypes that are preferentially
transmitted. In about half of the NIMH families, these SNP
haplotypes occur as part of a larger haplotype involving a small
subset (two to four per population) of the 23 alleles of a highly
polymorphic marker (D22s526).
[0256] The D22s526 microsatellite marker was evaluated in 561
unrelated individuals from the Louisville Twin Family Study
(comprising approximately 70% EA, 25% AA and 5% other). As
described in Brennan et al., Genomics 63(3):430-2 (2000), a total
of 23 alleles of D22s526 were observed, ranging in size from 254 nt
to 308 nt inclusive. These alleles are numbered from 1 to 23
(smallest to largest) in Table 10.
TABLE-US-00010 TABLE 10 Analysis of D22s526 in Control Sample and
NIMH Schizophrenia Families Observed Observed Occurrences of
frequency of frequency of allele in NIMH apparent apparent
sample.sup.c homozygotes Allele Expected homozygotes (N=52) in NIMH
frequency in frequency of in controls [observed sample.sup.d Allele
controls.sup.a homozygotes.sup.b (N = 561) frequency] (N = 26) 1
0.007 <0.1% 0 0 0 2 0.005 <0.1% 0 0 0 3 0.065 0.4% 0 2
[0.038] 0 4 0.102 .sup. 1% 0.4% 3 [0.058] 0 5 0.041 0.2% 0 0 0 6
0.112 1.3% 0.4% 1 [0.019] 0 7 0.041 0.2% 0 2 [0.038] 0 8 0.107 1.1%
0 6 [0.115] 0 9 0.033 0.1% 0.2% 2 [0.038] 3.8% 10 0.149 2.2% 0.7% 9
[0.173] 7.7% 11 0.033 0.1% 0.2% 1 [0.019] 0 12 0.102 1.0% 0.2% 9
[0.173] 7.7% 13 0.080 0.6% 0.7% 1 [0.019] 0 14 0.051 0.2% 0 6
[0.115] 3.8% 15 0.074 0.5% 0.5% 0 0 16 0.020 <0.1% 0.2% 0 0 17
0.063 0.4% 0.2% 6 [0.115] 11.5% 18 0.003 <0.1% 0 1 [0.019] 0 19
0.038 0.1% 0.2% 1 [0.019] 0 20 0.002 <0.1% 0 0 0 21 0.018
<0.1% 0 1 [0.019] 0 22 0.009 <0.1% 0 1 [0.019] 0 23 0.054
0.3% 0.2% 0 0 .sup.aFrequency observed in 561 unrelated individuals
representing a cross section of the population in the Louisville
metropolitan area. The values do not add to 1.00 due to rounding.
.sup.bExpected frequency of homozygous individuals for an
unselected sample given Hardy-Weinberg assumptions.
.sup.cOccurrences and empirical frequencies of the alleles in 26
probands from NIHM Schizophrenia Genetics Initiative.
.sup.dObserved frequency of apparently homozygous (or hemizygous)
individuals in a sample of 26 probands from NIHM Schizophrenia
Genetics Initiative.
[0257] An apparent heterozygosity of 97.5% was found in the
unselected sample of 561 unrelated individuals. In other words, one
expects only about 2.5% of randomly sampled individuals to be
homozygous for this marker. By contrast, 9 of 27 (33%) of the NIMH
probands (i.e., the individuals first identified as affected for
each particular family) are apparent homozygotes (5 of 13 EA; 2 of
12 AA; 1 of 2 "other"; Fisher Exact Test P=2 X 10.sup.-5; OR 15.3;
Log odds=2.7).
[0258] At least a portion of the apparent homozygosity for this
region appears to be due to microdeletions segregating in some
families. Mendelian inheritance patterns for the control sample
showed that 4 of the 15 apparently homozygous individuals could be
hemizygous, because they fail to transmit the expected allele to
one or more children. Thus, perhaps about 0.5% of individuals from
the unselected population are hemizygous for D22s526.
[0259] A closer look at the NIMH families indicates that
microdeletions are likely. Six of the eight probands with apparent
homozygosity for the microsatellite polymorphism also have an
adjacent region of presumptive hemizygosity extending over
approximately 200 to 300 kb in one or both directions. Furthermore,
in AA families in particular, there are five additional probands
who appear to carry deletions that do not uncover the
microsatellite but do uncover nearby extended regions of at least
100 to 500 kb, as judged by apparent homozygosity for certain (and
various) infrequent haplotypes.
[0260] DNA from one or both parents and multiple siblings can be
used to rule out most trivial explanations for these results. Loss
of homozygosity during immortalization and propagation of cell
lines is unlikely, as the same presumptive deletion is carried by
multiple family members. Consanguinity and resulting extended
regions of homozygosity cannot explain the results either, because
other polymorphic markers, even on the same chromosome, do show
extensive homozygosity.
[0261] These novel microdeletions may confer significant risk of
developing schizophrenia spectrum disorders (SZ, SD, and/or SPD).
As one theory, not meant to be limiting, using the 22q11 deletion
syndrome as a prototype, is that the microdeletions either uncover
one or more specific "risk" alleles, or that haploinsufficiency per
se confers increased risk.
Example 8
Exemplary Markers within 1 Linkage Disequilibrium Unit (1 LDU)
[0262] On-line public resources (HapMap.org) were used to identify
exemplary SNPs that are in linkage disequilibrium with some of the
SNPs described herein, as follows:
[0263] rs738596
[0264] SNPs within 1 LDU of marker rs738596 in African American
populations include: rs5770635, rs17000207; in European American
Populations include: rs4823940, rs13053183, rs4824067, rs9628096;
in Chinese populations include: rs5770579, rs8136613, rs4824067,
rs17824774, rs5770632, rs9628096, rs5770634, rs5770635, rs9628100;
and in Japanese populations include: rs2024698, rs9616622,
rs5770581, rs4824067.
[0265] rs2073224
[0266] SNPs within 1 LDU of marker rs2073224 in African American
populations include: rs9616222, rs5769820, rs5769821, rs17178537,
rs4823974, rs2064542; in European American populations include:
rs9616222, rs5769820, rs5769821, rs17178537, rs6009133, rs6009134,
rs4823974, rs2064542.
[0267] rs738615
[0268] SNPs within 1 LDU of marker rs738615 in Chinese populations
include: rs4823908, rs2073225, rs761666, rs2064542; and in Japanese
populations include: rs9616409, rs4823908, rs2073225, rs761666,
rs2064542.
[0269] rs848768
[0270] SNPs within 1 LDU of marker rs848768 in African American
populations include: rs12165304, rs9627698, rs9616561, rs9627966,
rs12169496, rs12628115, rs11090946, rs848751, rs848750; in European
American Populations include: rs2319174, rs739049, rs7287432,
rs9616561, rs848764, rs848750; in Chinese populations include:
rs739049, rs848764, rs12628115, rs11090946, rs848751, rs848750; and
in Japanese populations include: rs8135938, rs848751, rs848752.
[0271] rs737734
[0272] SNPs within 1 LDU of marker rs737734 in European American
populations include: rs5769607, rs5770363, rs714007, rs713997,
rs5770369, rs7285315, rs2873922, rs2097363, rs7510746; and in
Chinese populations include: rs5769607, rs8140231, rs713919,
rs2097363, rs4824032, rs2187751.
[0273] rs134474
[0274] SNPs within 1 LDU of marker rs134474 in African American
populations include: rs6520121, rs17001084, rs17001087, rs3810643;
in European American Populations include: rs9616663, rs2873932,
rs470019, rs470018, rs470017, rs134459, rs134458, rs134456.
[0275] rs763126
[0276] SNPs within 1 LDU of marker rs763126 in African American
populations include: rs135786, rs135787, rs135788, rs135789,
rs135791, rs763124, rs135800, rs135804, rs8140984, rs135821,
rs135827, rs135832, rs2319345, rs135833, rs135846, rs6009767,
rs5769691, rs2071894, rs5770562, rs2071893, rs135854, rs135855,
rs17001439, rs5770567, rs2007024, rs17182154, rs17001168,
rs2187891, rs9616687, rs17001172, rs739247, rs2071890; in European
American populations include: rs9616685, rs5769691, rs2071894,
rs135853, rs5770562, rs2071893, rs135854, rs135855, rs5770567,
rs2007024, rs739247, rs135861, rs2071890, rs12628438, rs10854876,
rs6009782, rs135875, rs135876, rs135877; in Chinese populations
include: rs763124, rs135845, rs135846, rs135853, rs135827,
rs135854, rs135855; rs2319345, rs9616685, rs5769691, rs2071894,
rs5770562, rs135854, rs135855, rs5770567; and in Japanese
populations include: rs135819, rs135827, rs135831, rs470058,
rs2319345, rs135846, rs1008320, rs5769691, rs2071894, rs135853,
rs2071893, rs2071892, rs135854, rs135855, rs135856, rs10854874.
[0277] rs138844
[0278] SNPs within 1 LDU of marker rs138844 in African American
populations include: rs138841, rs139818, rs10483250; in European
American populations include: rs139818, rs138840, rs138841; in
Chinese populations include: rs6009860, rs10483250, rs138841; and
in Japanese populations include: rs10483250, rs6009870, rs5770689,
rs138816, rs138820, rs138821, rs138823, rs138827, rs6009874,
rs138840, rs138841, rs138843.
Other Embodiments
[0279] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
4125DNAArtificial SequencePrimer 1cagccgcacg ccatggaact cgaag
25222DNAArtificial SequencePrimer 2ggcgccatga cgtcacgcct gc
22322DNAArtificial SequencePrimer 3agagcaagac tctgtctcaa ca
22422DNAArtificial SequencePrimer 4ttctccttca ctttctgcca tg 22
* * * * *
References