U.S. patent application number 13/323413 was filed with the patent office on 2012-05-10 for system and method for diagnosis and treatment of neuropsychiatric disorders.
This patent application is currently assigned to THE RESEARCH FOUNDATION FOR MENTAL HYGIENE. Invention is credited to Catherine L. Clelland, James D. Clelland.
Application Number | 20120114767 13/323413 |
Document ID | / |
Family ID | 39492916 |
Filed Date | 2012-05-10 |
United States Patent
Application |
20120114767 |
Kind Code |
A1 |
Clelland; James D. ; et
al. |
May 10, 2012 |
System and Method for Diagnosis and Treatment of Neuropsychiatric
Disorders
Abstract
An assay for a GCH1 allele and associated genotype for the
screening, prediction, diagnosis, prognosis, treatment and
treatment response of psychiatric, neuropsychiatric, and
neurological disorders, such as schizophrenia, schizoaffective
disorder and bipolar disorder, and for defining treatments of such
disorders. The presence of a variant in the GCH1 gene, alone or in
conjunction with a measurement of low or altered biopterin, or
altered BH4 system measures, is used to screen or diagnose subjects
at risk for developing a psychiatric, neuropsychiatric, or
neurological disorder. The genetic assay, with or without a
biopterin or BH4 system assay, may also be used to determine
treatment regimens. For subjects with an impaired BH4 system,
treatments to increase or normalize biopterin, BH4, or the BH4
system can also be used, such as BH4 supplementation, lithium
treatment, phenylalanine treatment, or other treatments and
therapies.
Inventors: |
Clelland; James D.; (New
York, NY) ; Clelland; Catherine L.; (New York,
NY) |
Assignee: |
THE RESEARCH FOUNDATION FOR MENTAL
HYGIENE
Menands
NY
|
Family ID: |
39492916 |
Appl. No.: |
13/323413 |
Filed: |
December 12, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11873971 |
Oct 17, 2007 |
8076075 |
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13323413 |
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60829856 |
Oct 17, 2006 |
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Current U.S.
Class: |
424/677 ;
435/6.11; 514/225.8; 514/226.2; 514/250; 514/255.02; 514/259.41;
514/327; 514/567 |
Current CPC
Class: |
A61K 31/5415 20130101;
A61K 31/519 20130101; C12Q 1/68 20130101; A61K 31/7052 20130101;
C12Q 2600/118 20130101; A61K 45/06 20130101; C12Q 2600/158
20130101; G01N 2800/50 20130101; A61K 33/14 20130101; C12Q 1/6883
20130101; C12Q 2600/156 20130101; A61P 25/18 20180101; A61K 33/00
20130101; A61P 25/00 20180101; G01N 2800/302 20130101; G01N 33/50
20130101; A61K 31/7052 20130101; A61K 2300/00 20130101; A61K 33/00
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/677 ;
435/6.11; 514/250; 514/567; 514/255.02; 514/327; 514/259.41;
514/226.2; 514/225.8 |
International
Class: |
A61K 33/14 20060101
A61K033/14; A61K 31/519 20060101 A61K031/519; A61P 25/18 20060101
A61P025/18; A61K 31/495 20060101 A61K031/495; A61K 31/451 20060101
A61K031/451; A61K 31/5415 20060101 A61K031/5415; C12Q 1/68 20060101
C12Q001/68; A61K 31/198 20060101 A61K031/198 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Work on this invention was supported, in part, by U.S.
Government Funds under Grant Nos. NIMH R21MH066883, NIMH
R01MH44153, and 1R21MH070601-01A2 of the National Institutes of
Health (NIH). The government has certain rights in the invention.
Claims
1. A method of evaluating a subject for schizophrenia or
schizoaffective disorder, comprising the step of determining a GTP
cyclohydrase I (GCH1) genotype of the patient.
2. The method of claim 1, wherein the GCH1 genotype of the subject
comprises the GCH1-959 nt G/A GCH1 genotype.
3. The method of claim 1, further comprising the step of
determining the level of at least one of total biopterins, BH4,
7,8-dihydrobiopterin, and fully oxidized biopterin of the
subject.
4. The method of claim 2, further comprising the step of diagnosing
the subject as having schizophrenia or a schizoaffective disorder
based on the GCH1 genotype and said level of at least one total
biopterins, BH4, 7,8-dihydrobiopterin, and fully oxidized biopterin
of the subject.
5. The method of claim 4, wherein the GCH1 genotype of the subject
comprises the GCH1-959 nt G/A GCH1 genotype.
6. The method of claim 5, wherein the step of diagnosing the
subject as having schizophrenia or a schizoaffective disorder
comprises the step of determining the presence and allele dose of
the "A" allele of the GCH1-959 nt G/A GCH1 genotype.
7. The method of claim 6, wherein the step of diagnosing the
subject as having schizophrenia or a schizoaffective disorder
further comprises determining that the subject has a decreased
levels of at least one of total biopterins, BH4,
7,8-dihydrobiopterin, and fully oxidized biopterin
8. A method of treating a subject for the risk or presence of
schizophrenia or schizoaffective disorder, comprising the steps of:
determining a GTP cyclohydrase I (GCH1) genotype of the subject;
determining the levels of at least one of total biopterins, BH4,
7,8-dihydrobiopterin, and fully oxidized biopterin of the subject;
and treating the subject based on the genotype or biopterin
level.
9. The method of claim 8, wherein the step of treating the subject
comprises the administration of a therapeutic amount of natural or
synthetic tetrahydrobiopterin (BH4).
10. The method of claim 8, wherein the step of treating the subject
comprises modulating the level of at least one of, total
biopterins, BH4, 7,8-dihydrobiopterin, and fully oxidized biopterin
in the subject.
11. The method of claim 10, wherein modulating the level of at
least one of, total biopterins, BH4, 7,8-dihydrobiopterin, and
fully oxidized biopterin in the subject comprises increasing the
level of at least one of, total biopterins, BH4,
7,8-dihydrobiopterin, and fully oxidized biopterin in the subject
if the at least one of total biopterins, BH4, 7,8-dihydrobiopterin,
and fully oxidized biopterin levels are below a predetermined
threshold.
12. The method of claim 8, wherein the step of treating the subject
comprises providing the subject with an effective amount of at
least one compound selected from the group consisting of pterin,
biopterin, dihydrobiopterin, neopterin,
6-pyruvoyl-tetrahydrobiopterin, tetrahydrobiopterin, phenylalanine,
aspartame, sepiapterin, and sapropterin dihydrochloride.
13. The method of claim 8, wherein the step of treating the subject
comprises providing the subject with an effective amount of at
least one compound selected from the group consisting of
haloperidol, risperidone, Chlorpromazine, Fluphenazine,
Perphenazine, Prochlorperazine, Thioridazine, Trifluoperazine,
Mesoridazine, Promazine, Triflupromazine, Levomepromazine,
Chlorprothixene, Flupenthixol, Thiothixene, Zuclopenthixol,
Droperidol, Pimozide, Melperone, Clozapine, Olanzapine,
Risperidone, Quetiapine, Ziprasidone, Amisulpride, fluoxetine,
Paliperidone, Aripiprazole, LY2140023, Lithium compounds, Lithium
chloride, Lithium carbonate, Valproic acid, divalproate, divalproex
sodium, sodium valproate, valproate, Lamotrigine, Carbamazepine,
Gabapentin, Oxcarbazepine, and Topiramate
14. The method of claim 8, further comprising the step of
determining the level of at least one of total biopterins, BH4,
7,8-dihydrobiopterin, and fully oxidized biopterin of the subject
after the step of treating the subject.
15. A method of treating a subject for the risk or presence of
schizophrenia or schizoaffective disorder, comprising the step of
administering of a therapeutic amount of one or more of natural or
synthetic pterin, biopterin, dihydrobiopterin, neopterin,
sepiapterin, 6-pyruvoyl-tetrahydrobiopterin, tetrahydrobiopterin,
sapropterin.
16. The method of claim 15, further comprising the step of
determining the levels of at least one of total biopterins, BH4,
7,8-dihydrobiopterin, and fully oxidized biopterin of the subject
prior to and after administering said one or more of natural or
synthetic pterin, biopterin, dihydrobiopterin, neopterin,
sepiapterin, 6-pyruvoyl-tetrahydrobiopterin, tetrahydrobiopterin,
sapropterin, or synthetic forms of the above.
17. The method of claim 16, further comprising the step of
determining a GTP cyclohydrase I (GCH1) genotype of the subject.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 11/873,971, filed on Oct. 17, 2007, which
claimed priority to U.S. Provisional Application Ser. No.
60/829,856, filed Oct. 17, 2006.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to psychiatric and
neuropsychiatric disorders, such as schizophrenia, schizoaffective
disorder, bipolar disorder, and Alzheimer's disease and, more
particularly, to the role of GTP cyclohydrolase I gene, GTP
cyclohydrolase (GTPCH), biopterins, and tetrahydrobiopterin in the
detection, diagnosis, prognosis and treatment of such
disorders.
[0005] 2. Description of the Related Art
[0006] Schizophrenia (SZ) and schizoaffective disorder (SaD) are
among the most common forms of mental illness, and have large
genetic and heritable components, indicated by studies showing
increased risk among first degree relatives, and concordance
between mono- and dizygotic twins. The genetic components of SZ and
SaD appear to involve multiple genes. Individuals with these
psychiatric disorders can display an overlapping range of symptoms
and there appears to be increased prevalence of SZ in the families
of SaD sufferers, and vice versa. There have also been reports of
shared genetic susceptibility loci for these disorders.
[0007] Tetrahydrobiopterin (BH4) is a vital cofactor maintaining
availability of the amine neurotransmitters, dopamine (DA),
noradrenaline (NA), and serotonin (5-HT). BH4 is also involved in
regulating the synthesis of nitric oxide (NO) by nitric oxide
synthases (NOS), and stimulating and modulating the glutamatergic
system. In the central nervous system (CNS), BH4 has also been
shown to stimulate the release of DA, 5-HT and glutamate, as well
as regulating the expression of tyrosine hydroxylase at nerve
terminals. Plasma total
[0008] biopterins level (biopterin) is a measure of BH4
(approximately 80-90% in the form of BH4) and are correlated with
CNS biopterin levels.
[0009] Conventional methods do not rely on a genetically based
method for assessing the presence or risk of schizophrenia or
schizoaffective disorder using the GTP cyclohydrolase I (GCH1) gene
alone or in conjunction with a biochemical assay. These method also
fail to disclose a method of treating psychiatric and
neuropsychiatric diseases, such as schizophrenia, by addressing
genetic deficiencies in the GCH1 gene and/or in the BH4 system. For
example, there is currently no useful genetic test for determining
subjects that are at-risk for developing schizophrenia and, as a
result, treatment approaches have limited success.
BRIEF SUMMARY OF THE INVENTION
[0010] It is therefore a principal object and advantage of the
present invention to provide a system and method for assessing the
presence or risk or severity or progression or prognosis of certain
psychiatric and neuropsychiatric diseases, and neurological
disorders.
[0011] It is an additional object and advantage of the present
invention to provide a system and method for testing for the
presence or risk of psychiatric, neuropsychiatric, and neurological
disorders.
[0012] It is a further object and advantage of the present
invention to provide a system and method for the determination of
treatment of psychiatric and neuropsychiatric diseases, and
neurological disorders.
[0013] It is a further object and advantage of the present
invention to provide a treatment for psychiatric and
neuropsychiatric diseases, and neurological disorders.
[0014] In accordance with the foregoing objects and advantages, the
present invention provides for genetic testing of GCH1, either as a
stand-alone test, or in conjunction with assay of biopterin, BH4,
and/or BH4 system measures for the assessment of psychiatric and
neuropsychiatric diseases, such as schizophrenia, either alone or
in conjunction with assays of other genes. Symptom scales,
behavioral measures, physiological testing, biological or molecular
or genetic testing, and imaging analyses, will also be used where
appropriate, in conjunction with genetic testing, assay of
biopterin, BH4, or BH4 system measures. In particular, a GTP
cyclohydrolase I (GCH1) gene variant (nucleotide variant -959 nt
G/A: rs10137071, NCBI dbSNP database) "A" allele, is present in a
much larger than expected proportion of psychiatric patients
(schizophrenics (SZ) and schizoaffective disorder (SaD)), than in
healthy people. For example, the odds ratio of having the GCH1
variant genotype was over five fold higher in SZ and SaD patients
when compared to healthy control subjects. The risk of having a
psychiatric disorder is therefore multiplied by five times for
people who carry the "A/A" variant.
[0015] The present invention is based on an assay of GCH1 genotype,
separately and/or in conjunction with assay of biopterin or other
pterins in blood, plasma, serum, CSF, or other fluids or tissues,
and/or assays of other BH4 system measures, and is useful in the
prediction, diagnosis and prognosis of psychiatric disorders, and
for defining treatments. In addition, treatments such as BH4,
biopterin, other pterin species, phenylalanine, lithium, or other
treatments designed or known to increase biopterin or BH4 (or
normalize the BH4 system) in persons with a variant GCH1 genotype
(and thus to alleviate biopterin deficit or to prevent a deficit in
persons at-risk for the disorders), may be administered to provide
a therapeutic or preventative response or treatment in patients
with the disorders or at-risk for developing the disorders. The
assay of GCH1 genotype, with or without biopterin or BH4 assay, may
also be used to determine antipsychotic or mood stabilizer
medication, as well as other treatment requirements. For subjects
with an impaired BH4 system, treatments to increase BH4 or
normalize BH4 can be used, such as supplementation with BH4 (or
other pterin molecule species), lithium treatment, phenylalanine
treatment, or other useful treatments, such as ECT.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The present invention will be more fully understood and
appreciated by reading the following Detailed Description in
conjunction with the accompanying drawings, in which:
[0017] FIGS. 1A and B are schematics of biosynthesis pathways of
BH4 and its roles in the hydroxylation of the aromatic amino acids
to the amine neurotransmitters, nitric oxide (also as a cofactor)
and its roles in stimulation and modulation of neurotransmitter
synthesis and release.
[0018] FIG. 2 is a chart illustrating the change in biopterin
levels with lithium as a covariate.
[0019] FIG. 3 is a graph of the ranked plasma biopterin levels and
GCH1 genotype within test diagnostic groups.
[0020] FIG. 4 is a graph of the plasma biopterin values and GCH1
genotypes for the test subject data.
[0021] FIG. 5 illustrates the sequence of the "g" allele of the
GHC1 variant of the present invention (SEQ ID NO:1), the sequence
of the "a" allele of the GHC1 variant of the present invention (SEQ
ID NO:2), the sequence of the forward primer according to the
present invention (SEQ ID NO:3), the sequence of the reverse primer
according to the present invention (SEQ ID NO:4).
[0022] FIG. 6 is a graph illustrating leukocyte GCH1 expression in
the SZ A allele group when compared to SZ and BpD G/G subjects.
[0023] FIG. 7 is a graph illustrating leukocyte gene expression for
BPD subjects.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The present invention provides a screening (including
population screening), risk assessment, prognostic and diagnostic
test for SZ and SaD, and, if genetic and biochemical testing is
combined, a screening, risk assessment, diagnostic and prognostic
test for bipolar disorder (BpD). In addition, the present invention
provides for treatments such as biopterin, BH4 or other pterin
species (which have been successfully used to treat autism and
other disorders in children, and depression and other disorders in
adults, and so safe therapeutic doses have previously been
determined), other treatments that increase BH4 (such as lithium)
or biopterin, treatments which alleviate the biopterin deficit or
the potential biopterin deficit in those at risk, or treatments
which may be used to normalize BH4. The present invention is
therefore be used to allow determinations of treatment use, and to
provide treatments for alleviating psychiatric, neuropsychiatric,
or neurological symptoms. The present invention will also provide
for early detection and prophyphylactic or preventative treatment
for those at risk for developing the disorders, such as children
and adolescents, or others at risk.
[0025] The present invention further allows for assessment of GCH1
genotype alone, or in conjunction with assessment of one or more of
biopterin, BH4, neopterin or other pterins, GCH1 RNA, epigenetic
modifications including DNA methylation, GTPCH protein levels,
GTPCH enzyme activity and/or the BH4 system, that can be used to
determine treatment needs. The present invention may also be used
to diagnose and/or prognose and/or determine treatment, for other
disorders including but not limited to those described herein.
[0026] The term "BH4 system" includes biopterin (total biopterins
or individual biopterin species), BH4, neopterin (total neopterins
or individual neopterin, such as dihydroneopterin trophosphate),
other pterin species, and/or the genes, RNA, proteins, and enzymes
that form the pathways of BH4 biosynthesis and metabolism
(including the de novo synthesis, regeneration, and salvage
pathways, and cofactor reaction pathways). The term also includes
genes, RNA, proteins, enzymes and metabolites that can directly
and/or indirectly influence the biosynthesis of BH4 and/or the
pathways of BH4 biosynthesis and metabolism, including the de novo
synthesis, regeneration, and salvage pathways, and cofactor
reaction pathways and associated genes, including the genes GCH1
GCHFR, PTS, SPR, PCBD, QDPR, NOS(I,II,III), PAH, TH, TPH, DHFR,
MTHFR, AKR1B1, AKR1C3, AKR1C1, CBR1, NR4A2, CTF1, IL6, MTPN, LIF,
CNTF, PRKG2, and the genes, proteins, enzymes, and/or metabolites
relating to GSK3 alpha and/or beta, NURR1 and/or Nur77, Nurr1
and/or NOR1, IMPase and/or inositol phosphate-1-phosphotase (IPP),
v-akt murine thymoma viral oncogene homolog 1 (AKT1), AKT/PKB,
AKT2, AKT3, etc. The term BH4 system also includes the promoters,
enhancers, supressors, and other regulatory regions, regulatory
RNAs (such as microRNA) of "BH4 system" genes. The term BH4 system
also includes epigenetic regulation such as methylation of BH4
system genes, and acetylation and/or ubiquitylation of
chromosome-associated proteins such as histones.
[0027] The term "assay of BH4 system measures" includes assays
and/or measurements of biopterin or BH4 or BH4 system DNA, RNA,
genes, miRNA, methylation, acetylation, ubiquitylation, proteins,
enzymes, and/or metabolites, other pterin species, GCH1 RNA, GTPCH
protein, and/or GTPCH enzyme activity (and the RNA, protein and
enzyme activities of the other BH4 system genes, including GCH1
GCHFR, PTS, SPR, PCBD, QDPR, NOS(I,II,III), PAH, TH, TPH, DHFR,
MTHFR, AKR1B1, AKR1C3, AKR1C1, CBR1, NR4A2, CTF1, IL6, MTPN, LIF,
CNTF, PRKG2), whether in plasma, serum, blood, CSF, urine, or
saliva, as well as other tissues, fluids, cells, organs or
substances. Assays of BH4 system measures, e.g., assays of
biopterins, BH4, neopterins, and pterin species, includes the use
of HPLC methods. Assay of BH4 system measures also includes use of
mass spectrometry methods, immunoassay, radioimmunoassay,
immunohistochemistry, MRS, spectroscopy methods, radio-tracer assay
and/or imaging methods, electrophoresis methods, molecular biology
methods, including PCR and real time PCR, genetic sequencing
methods, SNP assay methods, biochemical measurement methods, enzyme
activity measurement methods, protein measurement methods, DNA,
RNA, miRNA or protein microarray assay methods, other DNA, RNA and
protein multiplex assay methods, and other assay and measurement
methods known to those skilled in the art.
[0028] The term "psychiatric disorders" includes but is not limited
to: SZ, SaD, BpD, mood disorders and personality disorders,
unipolar depressive disorder, psychotic disorders, major depressive
and other depressive disorders, other affective disorders,
attention deficit disorder, delusional disorder, anxiety disorders,
obsessive compulsive disorder, paranoid schizotypal or schizoid
personality.
[0029] The term "neuropsychiatric disorders" includes, but is not
limited to, neurological, neuropsychiatric and neurodegenerative
disorders, including Alzheimer's disease, Pick's disease,
Parkinson's disease, Huntington's disease, multiple sclerosis,
Wilson's disease, Creutzfeldt-Jakob disease and other disorders of
the central nervous system and the peripheral nervous system
including abnormal or heightened pain sensitivity, as well as
movement disorders including dyskinesias, dystonias and akathisias,
autism, Asperger's syndrome and spectrum disorders, and autism
spectrum disorders, and also dementias, cognitive status and
decline, intellectual status and decline, learning or memory status
decline, and other intellectual disabilities and disorders. The
term "patient" includes human subjects, including fetuses, as well
as includes organ, tissue, cell, fluid, DNA, RNA, protein, chemical
and/or material samples from patients and other organisms (e.g.,
animals). The term model includes animal models of diseases and
states, and chimeric models.
[0030] The term "assay of GCH1 genotype" includes, but is not
limited to, determination of the -959 nt G/A: rs10137071 GCH1
genotype and/or one or more other genetic change affecting GCH1, as
well as genetic or epigenetic difference such as a nucleotide
variant affecting the GCH1 gene or a copy number polymorphism or
duplication, deletion or other mutation or change, in GCH1 or its
promoters, enhancers, suppressors and other regulatory regions, and
any other RNA species, e.g., regulatory RNAs such as miRNAs that
regulate GHC1 gene expression. The term "assay of GCH1 genotype"
also includes determination of epigenetic regulation such as DNA
methylation, and/or acetylation and/or ubiquitylation of
chromosome-associated proteins such as histones, affecting the GCH1
gene.
[0031] The term "normalize their BH4 system levels" includes
treatment to normalize biopterin(s), other pterin species levels,
BH4 levels, GCH1 RNA, GTPCH protein, tyrosine hydroxylase,
tryptophan hydroxylase levels, the activities of the BH4 system
enzymes, and/or the function of the BH4 system. The term "normalize
their BH4 system levels" also includes treatment to normalize or
treat or alter or supplement, the sequences and/or function of BH4
system genes and/or their regulatory regions or epigenetic
regulators, including by gene therapy methods. For example,
treatment may occur by introduction of nucleic acids to provide
adequate and/or functional and/or normal gene functioning, to those
in need of treatment, including those with, or at risk of
developing a psychiatric or neuropsychiatric or neurological
disorder. One or more nucleic acids can be provided or introduced
to patient's, bodies, cells, organs, tissues or fluids. Nucleic
acids to be provided can include for example one, or more than one
different, or several different, GCH1 nucleic acids, such as an
oligonucleotide incorporating the gene variant (nucleotide variant
-959 nt G/A: rs10137071, NCBI dbSNP database) "G" nucleotide, using
methods known to those skilled in the art.
[0032] The term "a GCH1 variant" includes the -959 nt
guanine/adenine (G/A): rs10137071 GCH1, "A" allele and/or "G/A" or
"A/A" genotype and/or other genetic change(s) affecting GCH1.
[0033] The term "useful treatments" includes treatment with
pterin(s) and/or other molecules, including BH4, biopterin,
dihydrobiopterin, sepiapterin, sapropterin, dihydroneopterin
triphosphate or other neopterins, Lithium (Li) or Li-based
medications, phenylalanine, aspartame, sapropterin dihydrochloride,
peptide molecules containing residues such as phenylalanine,
tyrosine, and/or tryptophan, inhibitors of glycogen synthase kinase
3 (beta and/or alpha) expression, enzyme activity, or catalysis,
inositol phosphate-1-phosphotase (IPP) expression, enzyme activity,
or catalysis, Inosotiol monophosphate (IMPase) expression, enzyme
activity, or catalysis, promoters or inhibitors of AKT expression,
enzyme activity, or catalysis, promoters of glycogen synthase
kinase 3 (beta and/or alpha) phosphorylation, administration of
electrical stimulation and/or electroconvulsive therapy (ECT),
transcranial magnetic stimulation (TMS), electrical brain
stimulation, deep brain stimulation and/or other electrical,
magnetic or radiowave general or focally targeted brain
stimulation, and/or inositol depletion treatments. Useful
treatments may also include neuroleptic(s) and/or other
antipsychotic and/or mood stabilizer(s) and/or other
psychotropic(s) and/or other medication(s)
[0034] The term "low biopterin" includes, but is not limited to,
biopterin levels (or other BH4 system measures) that are either
lower than the mean, median, mode, "normal" level, or reference
range level for control or healthy subject groups (groups can be
defined by factors including but not limited to, ethnicity, race,
age, gender, BMI, weight, nutritional status, health status). The
term "low biopterin" also includes levels that are outside of
levels needed for optimal or normal brain, CNS, biological,
biochemical, and/or physiological function. Subject biopterin level
can also be adjusted to reflect the influence of modifiers of
biopterin level, BH4 level, and/or Phe levels, such as that due to
nicotine, or to treatments such as lithium, ECT, TMS, deep brain
stimulation, or other treatments or therapies. Low biopterin can
also be defined as a range or level compared to different
population(s) and/or group norms and/or arbitrary cutoffs. Abnormal
biopterin levels can also include elevated or "high" BH4 system
measures.
[0035] The definitions, such as those described above, can also be
utilized to define "low" biopterin, BH4 or other measures, such as
other pterin species, GCH1 RNA, GTPCH protein, and/or GTPCH enzyme
activity, or other BH4 system measures. Definitions can also be
based upon comparisons (e.g., with reference ranges) and/or
statistical descriptions including those such as percentiles,
standard deviations, confidence intervals, standard errors etc.
[0036] The present invention is based on the fact that a GTP
cyclohydrolase I (GCH1) homozygous gene variant (nucleotide variant
-959 nt G/A: rs10137071, NCBI dbSNP database) "A" allele, is
present in a much larger than expected proportion of psychiatric
patients (schizophrenics (SZ) and schizoaffective disorder (SaD)),
than in healthy people. For example, the odds ratio of having the
GCH1 variant genotype was nearly five fold higher in SZ and SaD
patients when compared to healthy control subjects. The risk of
having a psychiatric disorder is therefore multiplied by five times
for people who carry the "A/A" variant.
[0037] Patients with the GCH1 "A" allele and, in particular, the
"A/A" genotype, have decreased fasting blood plasma levels of a
total biopterins, known as "biopterin," that is from, and is a
measure of tetrahydrobiopterin (BH4). BH4 is a vital cofactor that
is required for the maintenance of neurotransmitters in the brain
and periphery, and these neurotransmitters have been implicated in
psychiatric disorders. The GTP cyclohydrolase I (GCH1) gene encodes
GTP-cyclohydrolase (GTPCH), the first enzyme in BH4 biosynthesis.
Our finding directly links the GCH1 gene "A" variant to the
increased risk for these psychiatric disorders.
[0038] As the GCH1 "A" variant is a risk allele for SZ, SaD, and
BpD, having the GCH1 variant genotype has biological consequences
that likely results in decreased GCH1 gene expression or altered
GCH1 splicing, and thus the decreased biopterin level observed in
patients with the variant "A" allele. As GCH1 encodes an enzyme
that is required for BH4 biosynthesis (called GTPCH), lower or
altered GCH1 expression likely results in the plasma biopterin
deficit in SZ and SaD populations. SZ patients have lower GCH1 gene
expression than healthy control subjects, and SZ subjects with the
GCH1 "A" allele have lower GCH1 expression than SZ without the "A"
allele, and thus are likely to exhibit a similar brain biopterin
deficit (or BH4 system deficit) that will result in the
dysregulation of neurotransmitters, such as dopamine,
noradrenaline, serotonin and the glutamatergic system, nitric
oxide, and with result in an SZ, SaD, or other psychiatric or
neuropsychiatric disorder or neurological disorder phenotype.
Similarly, GCH1 expression is lower in "A" allele BpD patients
compared to BPD without the A allele, and these BpD patients are
also likely to exhibit a similar brain biopterin deficit (or BH4
system deficit) that will result in the dysregulation of
neurotransmitters, such as dopamine, noradrenaline, serotonin and
the glutamatergic system, and nitric oxide.
[0039] BpD patients treated with Li, have higher levels mRNA levels
of GCH1, compared to patients not treated with Li. Also, BpD
subjects treated with Li and who have the GCH1 "A" allele had lower
GCH1 expression than BpD subjects who were also treated with Li,
but did not have the "A"allele. It is likely that Li upregulation
of GCH1 is modulated by the GCH1 gene sequence, and Li treatment
may be less effective, or have different efficacy, in patients with
the GCH1 "A" allele.
[0040] The present invention therefore involves the assay of GCH1
genotype, separately and/or in conjunction with assay of biopterin,
BH4, other pterins in plasma, other BH4 system measures, CSF,
serum, urine, or other cells, tissues, organs, and fluid, and is
useful in the screening, prediction, diagnosis and prognosis of
psychiatric disorders, and for defining treatments. In addition,
treatments such as BH4, biopterin, other pterin species, Phe,
lithium, or other treatments designed or known or considered to
increase biopterin or BH4 in persons with a variant GCH1 genotype
(and thus to alleviate biopterin or BH4 system deficit, or prevent
manifestation of a biopterin deficit in those at risk), will be
administered to provide a therapeutic or preventative response or
treatment in patients with the disorders or at-risk for developing
the disorders. The present invention will be useful for
determination of risk, screening, early detection, diagnosis, and
treating psychiatric disorders including SZ, SaD, bipolar disorder
(BpD), mood disorders and personality disorders, unipolar
depressive disorder, psychotic disorders, major depressive and
other depressive disorders, other affective disorders, attention
deficit disorder, delusional disorder, anxiety disorders, obsessive
compulsive disorder, paranoid schizotypal or schizoid
personality.
[0041] Patients with Alzheimer's disease (AD) also have decreased
biopterin and/or BH4 levels. The present invention may thus be
useful for determination of risk, screening, early detection,
diagnosis, and treating AD and other neurological and
neuropsychiatric and neurodegenerative disorders, including Pick's
disease, Parkinson's disease, Huntington's disease, multiple
sclerosis, Wilson's disease, Creutzfeldt-Jakob disease and other
disorders of the central nervous system and the peripheral nervous
system, and movement disorders including dyskinesias, dystonias and
akathisias, and dementias, as well as in intellectual or cognitive
status impairment or decline. The same diagnostic and/or prognostic
and/or treatment approaches would apply for these disorders, as
with psychiatric disorders such as those outlined below for SZ
and/or SaD and/or BpD.
[0042] The present invention also encompasses the screening,
detection, prediction, diagnosis, early detection, prognosis,
and/or treatment of disorders where a genetic or epigenetic (e.g.,
methylation and/or acetylation and/or ubiquitylation) difference
affecting the GCH1 gene and/or its promoters, enhancers,
suppressors and other regulatory regions and/or RNA species (e.g.,
regulatory RNAs, such as miRNAs or non-coding RNA), is present,
such as a nucleotide variant, deletion, duplication, mutation, or
change. The present invention further includes the generation of
genetic therapy treatments designed to provide GCH1 DNA, RNA and/or
GTPCH protein to subjects in need of treatment, and that do or do
not carry deleterious variants. The present invention further
includes the generation of genetic therapy treatments designed to
provide BH4 system DNA, RNA, and/or protein(s) to subjects in need
of treatment. Subjects determined to be at-risk for development of
a psychiatric, neuropsychiatric or neurological disorder, can
include infants, children, adults, gametes, embryos, and/or
fetuses, and assessment of risk status can be made from the
individual, parental, and/or prenatal testing.
[0043] The present invention also encompasses kits for the
screening, detection, prediction, diagnosis, early detection,
prognosis, and/or treatment of disorders where a genetic or
epigenetic (e.g., methylation and/or acetylation and/or
ubiquitylation) difference affecting the GCH1 gene and/or its
promoters, enhancers, suppressors and other regulatory regions
and/or RNA species (e.g., regulatory RNAs, such as miRNAs or
non-coding RNA), is present, such as a nucleotide variant,
deletion, duplication, mutation, or change. As described herein,
the present invention includes primers that may be used to identify
the presence of the GCH1 alleles in humans.
[0044] Assessment of risk or screening is performed by genotyping
subjects, along with, or without, a biopterin, BH4, BH4 system,
other pterin system measurement (e.g., fasting plasma biopterin,
GCH1 RNA, GTPCH protein and/or neopterin measurement. Subjects with
the rs10137071 GCH1 "A/A" variant genotype alone or in conjunction
with a "low" or "altered" BH4 system measure (e.g., biopterin), or
the "A/G" or "A/A" genotype (or other DNA variants of the GCH1
gene) in conjunction with a measurement of "low" or "altered"
biopterin (BH4, marker of low BH4, GCH1 RNA, and/or GTPCH protein),
and/or other BH4 system measures, would be considered at an
increased risk for developing a psychiatric disorder.
[0045] In addition, subjects with the variant genotype, low or
altered biopterin, BH4, or BH4 system, altered GCH1 RNA, and/or
GTPCH protein levels would be medicated with one or more of,
biopterin or BH4 or other pterin species, or Phe or other treatment
that increases BH4 (such as lithium) or inositol depletion, to
alleviate the deficit, or treatment to increase or supplement BH4
or biopterins, such as electrical brain stimulation or
electroconvulsive therapy (ECT) or transcranial magnetic
stimulation (TMS).
[0046] Assessment of biopterin and/or BH4 and/or other pterin
levels, along with GCH1 genotyping will also be used for screening
or assessing those at-risk for developing a psychiatric,
neuropsychiatric, or neurological disorder, allowing for heightened
monitoring, early detection, and early, prodromal or prophylactic
initiation of treatments. For those at-risk or with the disorders,
treatment decisions can be made based on genotyping and/or
biopterin or BH4 level assay, and/or GCH1 RNA, GTPCH protein
level(s), and/or BH4 system measures.
[0047] The assay of GCH1 genotype with or without biopterin or BH4
assay, can be used to determine antipsychotic and/or mood
stabilizer medication, and/or other treatment requirements. The
efficacy of different medications and/or dosages will vary with
genotype and biopterin and/or BH4 and/or GCH1 RNA and/or GTPCH
protein level(s). For subjects with an impaired BH4 system
(indicated by low biopterin, for example), treatments to increase
BH4 can be used, such as BH4 supplementation, lithium treatment
(patients treated with lithium have increased biopterin levels),
phenylalanine treatment, or other treatments, such as ECT, TMS,
etc.
[0048] FIGS. 1A and B show the biosynthesis pathways of BH4 and its
central role in the hydroxylation of the aromatic amino acids to
the amine neurotransmitters, and its role as a cofactor in nitric
oxide synthesis, and in the synthesis and release of
neurotransmitters. In FIGS. 1A and B, GTPCH refers to GTP
cyclohydrolase I, encoded by the GCH1 gene, PTPS refers to
6-pyruvoyl-tetrahydropterin synthase, encoded by the PTS gene, SR
refers to sepiapterin reductase, encoded by the SPR gene, DA refers
to dopamine, NA refers to noradrenaline, 5-HT refers to
5-Hydroxytryptamine, serotonin, NO refers to nitric oxide, and Glu
refers to Glutamate.
[0049] As the amine neurotransmitter and glutamatergic systems and
NO activity have been implicated in the etiology of schizophrenia
and affective disorders, a study of fasting plasma total biopterin
(a measure of BH4) was performed. Study subjects included patients
with BpD (n=27), SZ (n=154), SaD (n=59), and control subjects
(n=37). For each patient, a lifetime psychiatric diagnosis (using
DSM-III-R criteria) was determined based on (a) clinical data
collected from current and previous admissions, and (b) diagnostic
interviews. For control subjects, an interview and internal scale
were completed to determine the presence of personal and/or family
history for psychiatric, neurological, and medical conditions.
Demographics were collected for each of the subject groups, that
included age, gender, ethnicity, years on neuroleptics (NL), and
chlorpromazine equivalent dose (CPZE) (for the 21 days prior to
blood draw), and the 4 groups (controls, BPD, SZ, SaD) were not
significantly different for the variables of age, gender, ethnicity
distribution and body mass index. There was a significant
difference in CPZ equivalence among patient groups, but no
correlation between CPZ equivalence (i.e., neuroleptic use) and
biopterin levels was observed.
[0050] To test for differences in biopterin levels between the
diagnostic groups an initial GLM was used, with total biopterin as
the primary outcome variable. This model, with the study group, the
covariates of gender, age, ethnicity, years of NL group, and CPZE
group, 24 hour dietary Phe/ protein ratio and plasma Phe, showed
significant or near significant main effects, or trends (on primary
outcome variable of fasting total biopterin), only for the
variables of study group, ethnicity, and plasma Phe. A final model
revealed only study group and plasma Phe as significant predictors
of variation in total biopterin with ethnicity approaching
significance. Analysis of the main effects revealed that study
group explained more than four times the amount of variation in log
biopterin (Partial Eta Squared=0.112) than the variance explained
by log Phe (Partial Eta squared=0.024). Pairwise comparisons of log
biopterin estimated marginal means (controlling for log Phe and
ethnicity) revealed significantly lower biopterin in SaD patients
(p<0.001) and SZ patients (p<0.0001) than controls;
specifically a plasma biopterin deficit of 34 percent in SZ
patients and of 25 percent in SaD patients, when compared to the
healthy control subjects, after partialling out the effects of
potential confounds including gender, age, ethnicity, neuroleptic
use history and dose of current use, 24-hour dietary
phenylalanine/protein ratio (relevant to BH4 synthesis) and plasma
phenylalanine (Phe) which stimulates BH4 synthesis. Of interest,
BPD patients had significantly higher biopterin than SZ patients
(mean difference.+-.standard error=0.200.+-.0.071, p<0.031), and
were not significantly different from controls. SaD patients showed
no significant difference from SZ or BPD patients.
[0051] A previous study of urine biopterin excretion showed no
elevation in SZ subjects when compared to controls, suggesting that
the plasma biopterin deficit results from a BH4 synthesis defect
rather than increased urine excretion.
[0052] In a further effort to explore the differences in plasma
biopterin deficits between the psychiatric patient groups a second
analysis was performed employing Lithium (Li) and mood stabilizer
use as covariates on the model of plasma biopterin. In this new
model, Li was found to have a significant main effect on plasma
biopterin. Post-hoc analysis adjusted for multiple testing showed
that, in this model, all patient groups had plasma biopterin levels
significantly lower than control subjects. FIG. 2 illustrates the
change in biopterin levels with Li as a covariate. This data
suggests that Li treatment increases plasma biopterin levels. The
percent change in adjusted least squares means is shown for each
patient group in FIG. 2 when lithium (yes/no) was added to the
final model (Li, Phe and ethnicity as covariates, on biopterin as
the outcome variable). Post-hoc Tukey test (adjusted) demonstrated
that SZ (p<0.0001), SaD (p<0.0001), and BPD (p=0.0179) differ
from controls. No significant differences were found between
patient groups. 89 percent of the BPD patients were treated with
Li, approximately 50 percent of the SaD patients, and 2 percent of
the SZ patients. The final model demonstrated a plasma biopterin
deficit of 32 percent, 27 percent, and 21.5 percent for SZ, SaD and
BPD subjects respectively, when compared to controls.
[0053] DNA variants in a BH4 biosynthesis pathway gene thus appear
to have a central role in the etiology of SZ, via dysregulation of
BH4 Synthesis, which would manifest as a biopterin deficit. The
rate-limiting and initial step in the de novo BH4 synthesis pathway
is catalyzed by the enzyme GTP cyclohydrolase I (GTPCH (EC
3.5.4.16)) encoded by the GCH1 gene, which maps to chromosome
14q22. The present invention thus considered the genotype data for
a biallelic nucleotide variant (-959 nt G to A: rs10137071, NCBI
dbSNP database) in the GCH1 gene promoter sequence, previously
described as having association with bipolar disorder. The -959 nt
G/A variant lies in the 5' upstream promoter of GCH1, and has a
reported heterozygosity of 0.46 (23). Testing for association of
this common variant with SZ and SaD in a mixed US sample of 174
subjects (86 SZ subjects, 42 SaD subjects and 46 control subjects)
and testing for an association of GCH1 with the biopterin deficit
in subject groups demonstrate that a GCH1 gene variant increases
risk of major psychiatric disorders, and is associated with low
biopterin levels in psychiatric patients. The results support a
genetic basis for BH4 deficiencies in psychiatric disorders.
[0054] For each patient subject, a lifetime psychiatric diagnosis
(using DSM-III-R criteria) was determined based on (a) clinical
data collected from current and previous admissions, and (b)
diagnostic interviews conducted by the research team. For control
subjects a questionnaire was completed to determine the presence of
personal and/or family history for psychiatric illness. Control
subjects had no history of psychiatric disorders, although 1
control subject had previously experienced mild, age-related
depression, but had not sought treatment. After complete
description of the study to the subjects, written informed consent
was obtained.
[0055] Genomic DNA was extracted from whole blood using standard
methods (Gentra Systems, Inc). Primers were utilized to amplify a
351 bp genomic region spanning the G/A variant in a 35 cycle PCR
reaction. The restriction enzyme BsrD1 recognizes and cleaves the A
allele resulting in the formation of a 191/160 bp doublet. Digested
products were visualized following electrophoreses. Subjects
homozygous for the G allele were genotyped by the presence of a
single 351 bp band, heterozygous subjects have two visible bands,
the 351 bp G allele and the 191/160 bp A allele, while A/A subjects
have only the 191/160 bp doublet.
[0056] Sample preparation and assay for plasma total biopterin has
previously been reported and measurement of plasma Phe (assayed due
to its known role in the regulation of BH.sub.4 synthesis) has also
been reported. Tests for Hardy Weinberg equilibrium, ethnic
distribution of study groups, and initial model of association
between GCH1 genotypes (A/A, G/A, G/G) and diagnostic group (SZ,
SaD, C) were performed using exact tests. Under a recessive model
(testing the difference between the minor allele (A) homozygotes
versus G/A heterozygotes and major allele (G) homozygotes),
logistic regression was employed to test for association with
psychiatric group status. Wald chi-squares and odds ratios (OR)
with Wald 95% Confidence Intervals (Wald CI) were reported with and
without ethnicity as a covariate.
[0057] Functional analyses of the GCH1 genotype using logistic
regression and general linear models were conducted on a subset of
subjects with plasma biopterin data available. For logistic models,
dependent variables were psychiatric status and independent
variables were GCH1 genotype, biopterin level and the interaction
of GCH1 genotype with biopterin level. Ethnicity was used as a
covariate in the initial model.
[0058] A general linear model was used to test the effect of GCH1
genotype, psychiatric disorder status and their interaction on
biopterin level. As the plasma concentration of Phe in fasting
subjects has a significant effect on plasma total biopterin levels,
and Phe was employed as a covariate to measure the effect of GCH1
genotype on biopterin level. Post-hoc significance testing was
reported using a Tukey-Kramer adjustment for multiple comparisons
in all logistic and linear models. Statistical analyses were
conducted using SAS 9.1.2 (FREQ, LOGISTIC and GLM Procedures, SAS
Institute Inc, 2004) and exact tests for Hardy Weinberg equilibrium
were calculated using R with the genetics library (R: A language
and environment for statistical computing. R Foundation for
Statistical Computing, Vienna, Austria, 2006, version 1.2.0,
2005).
[0059] Exact tests for Hardy-Weinberg equilibrium demonstrated
equilibrium for the patient (n=128, p=0.1534), and control groups
(n=46, p=0.1299). Ethnic distributions of SZ, SaD, and control
samples were not significantly different (Fisher exact test, n=174,
p=0.4234). Overall frequencies: Patient group-42.2% Caucasian,
39.8% African-American, 18.0% Hispanic; Control group-56.5%
Caucasian, 30.4% African-American, 13.1% Hispanic. Subject group
demographics are presented in Table 1 below. Exact counts (and %)
of GCH1 genotypes are shown for each of the study and ethnic
groups. The A/A genotype was significantly associated with both SZ
and SaD. Significance was observed in the African-American group
and a trend toward significance in the Hispanic group. After
adjusting for ethnicity, the associations detected in the recessive
model remained significant in all groups tested.
TABLE-US-00001 TABLE 1 Diagnostic and Ethnic Group Comparison of
GCH1 Genotype. Wald Chi-Square (p =) GCH1 Genotype Recessive Model
Subjects (n = 174) A/A G/A G/G Model (adjusted) SZ (n = 86) 30
(34.9) 34 (39.5) 22 (25.6) 0.0024 0.0057 Controls (n = 46) 4 (8.7)
27 (58.7) 15 (32.6) SaD (n = 42) 12 (29) 21 (50) 9 (21) 0.0216
0.0288 Controls (n = 46) 4 (8.7) 27 (58.7) 15 (32.6) Patients (n =
128) 42 (32.8) 55 (43) 31 (24.2) 0.0033 0.0066 Controls (n = 46) 4
(8.7) 27 (58.7) 15 (32.6) African-American (n = 65) Patients (n =
51) 25 (49) 21 (41.2) 5 (9.8) 0.0312 -- Controls (n = 14) 2 (14.3)
12 (85.7) 0 (0) Caucasian (n = 80) Patients (n = 54) 7 (13) 28
(51.8) 19 (35.2) n/s -- Controls (n = 26) 2 (7.7) 12 (46.15) 12
(46.15) Hispanic (n = 29)* Patients (n = 23) 10 (43.5) 6 (26.1) 7
(30.4) 0.0676 -- Controls (n = 6) 0 (0) 3 (50) 3 (50) *In the
Hispanic group (n = 29), a valid maximum likelihood estimate could
not be calculated and a Fisher Exact Test was performed. n/s, not
significant
[0060] In an initial analysis, an association between GCH1 genotype
(A/A, A/G and G/G) and SZ (Fisher exact test, p=0.0027), and SaD
(Fisher exact test, p=0.05) was revealed. When patient groups were
combined, a significant association remained (n=174, Fisher exact
test, p=0.0038). Most notably, under a recessive model we found a
highly significant association between the A/A genotype and SZ
(Wald chi-square=9.2, df=1, p=0.0024) and significant association
with SaD (Wald chi-square=5.3, df=1, p=0.0216). When the patients
were combined (patients n=128 and controls n=46), a highly
significant association remained (Wald chi-square=8.6, df=1,
p=0.0033). The odds ratios (OR) of having a SZ or SaD diagnosis
amongst the homozygous A/A population were 5.6, and 4.2
respectively. Combining the patient groups yielded an OR of
5.1.
[0061] Although subject ethnicities were not different between our
patient and control groups, assuming a degree of stratification in
our mixed US population we added ethnicity as a covariate in our
model. The resulting OR's adjusted for ethnicity were 5.0
(p=0.0057, 95% Wald CI: 1.601-15.886), for SZ and 4.1 for SaD
(p=0.0288, 95% Wald CI: 1.157-14.389), 4.7 combined patient group
(p=0.0066, 95% Wald CI: 1.54-14.48). Thus, the A/A genotype confers
a highly significant increased risk of having a psychiatric
disorder.
[0062] The association of the A/A genotype with psychiatric
disorders differed between ethnic groups within the study sample
(see Table 1). In patient and control subject groups, the highest
prevalence of the A/A genotype was found in the African-American
subjects (patients 49.0%, controls 14.3%). Further association
testing performed within the specific ethnic groups illustrated a
significant association of the A/A genotype with diagnostic group
in the African-American subset of patients, which was a trend in
the Hispanic subjects. The A/A genotype was 69% more frequent in
patients than controls in the Caucasian group.
[0063] The biological relevance of the GCH1 genotype was initially
investigated through analysis of plasma biopterin and genotype
interaction. The plasma biopterin level is significantly lower in
SZ when compared to control subjects. There is seen in FIG. 3, an
individual subject's ranked plasma biopterin levels and GCH1
genotype within each diagnostic group displayed by Study Group,
Plasma Biopterin level and GCH1 Genotype. Subjects are ranked by
biopterin level (increasing left-right) within their study group,
illustrating both the decreased biopterin levels in the SZ (n=52)
and SaD (n=32) subject groups compared to the control group (C,
n=32), and the relative preponderance of A/A genotype among
patients with the lowest biopterin levels. Plasma biopterin levels
of the SZ and SaD subjects were not significantly different from
each other, however both were significantly different from control
subjects (p<0.01 for both SZ and SaD groups). BpD patients also
had significantly lower biopterin than controls when biopterin
levels were adjusted for the effects of Lithium treatment.
[0064] Table 2 below shows descriptive statistics for biopterin
level (and standard deviations (SD)) for each subject group,
separated by GCH1 genotype. Biopterin Values and GCH1 Genotypes for
subject groups (Patient and Control). The mean biopterin levels
(nM) and standard deviation (SD) within each subject group is shown
for each GCH1 genotype. Final biopterin values were adjusted by
fasting Phe measurements.
TABLE-US-00002 TABLE 2 Mean Biopterin Adjusted Mean Subjects n (nM)
SD Biopterin (nM) Patients 84 AA 29 10.7 3.9 10.7 AG 35 10.8 3.4
10.8 GG 20 13.6 5.5 13.9 A/A A/G 64 10.7 3.6 10.8 G 20 13.6 5.5
13.9 Controls 32 AA 3 17.5 6.5 17.2 AG 22 15.1 3.7 15.0 GG 7 15.1
5.2 14.7 AA/AG 25 15.4 4.0 15.2 G 7 15.1 5.2 14.7
[0065] The plasma biopterin values and GCH1 Genotypes for the
subject group data is plotted in the FIG. 4 for patients (Panel A)
and controls (Panel B). Plasma biopterin values (nM) (unadjusted
for fasting phenylalanine levels) were plotted for patient subject
genotype (Panel A), A/A (n=29), G/A (n=35) and G/G (n=20) and for
control subject genotypes (Panel B), A/A (n=3), G/A (n=22) and G/G
(n=7). No significant difference in biopterin level was observed
between A/A or G/A genotypes in the patient group, however both the
A/A and G/A patient groups were significantly different from the
G/G patients (Tukey adjusted p-values for multiple comparisons:
p=0.0165 and p=0.0168, respectively). Patient subject A allele
carriers were combined for further analysis (patient A allele
carriers-blue box-plots). No significant difference in biopterin
level was observed among the control genotypes (control A allele
carriers-pink box-plots). Panel C shows patient A allele carriers
(n=64) have decreased biopterin levels compared to homozygous G/G
patients (n=20), and to all control subject groups (lines indicate
Tukey adjusted p-values for multiple comparisons: ** p<0.05, ***
p<0.0001). Box plots display the median (horizontal line in
box), first (Q1) and third (Q3) quartiles (ends of the boxes). Bars
outside the boxes represent the extreme values within 1.5 times the
inter-quartile range (IQR) from the upper or lower quartile. Points
at a greater distance from the median than 1.5 times the IQR are
plotted individually as small circles.
[0066] A logistic regression analysis was performed using all
subjects with both plasma biopterin level and GCH1 genotype
(patient group n=84; control group n=32) and the interaction
between plasma biopterin and GCH1 genotype was tested for the
outcome variable "study group," controlling for ethnicity. Although
the interaction between A/A genotype and biopterin on study group
(Patient, Control) was not significant, and post-hoc comparisons
did not show a significant difference between biopterin levels of
patient subjects with the A/A and G/A genotypes, the mean biopterin
levels of both A/A and G/A patient groups were significantly
different to mean biopterin for G/G patients (A/A p=0.0165, A/G
p=0.0168) and so the A/A and G/A patient groups were combined to
form an A allele carrier group.
[0067] The interaction of GCH1 A allele and low biopterin level,
was found to be a significant predictor of diagnostic group
(patient or control) (Wald chi-square=4.8, df=1, p=0.0286).
Ethnicity was not a significant predictor of study group (Wald
chi-square=0.1, df=1, p=0.8962) and was thus removed from the final
model (adjusted p-value of final model interaction, p=0.0276, model
concordance at 79.8%). Table 3 below shows parameters from the
logistic models. Logistic regression was employed to test the
interaction between plasma biopterin level and GCH1 genotype, as a
significant predictor of study group (patient or control, n=116),
using ethnicity as a covariate in the initial model.
TABLE-US-00003 TABLE 3 GCH1 A allele Status and Biopterin Level as
a Predictor of Study Group. Standard Wald Pr > Parameter DF
Estimate Error Chi-Square ChiSq Initial Model.sup.a Intercept 1
3.2294 0.8042 16.1268 <.0001 GCH1 A allele 1 1.4525 0.8122
3.1982 0.0737 Biopterin (n/M) 1 -0.1693 0.0535 10.0163 0.0016
Biopterin * GCH1 1 -0.1179 0.0537 4.8213 0.0281 A allele Caucasian
vs 1 -0.0178 0.3400 0.0028 0.9582 African American Hispanic vs
African 1 0.1531 0.4509 0.1153 0.7342 American Final Model.sup.b
Intercept 1 3.2055 0.7970 16.1774 <.0001 GCH1 A allele 1 1.4206
0.7970 3.1772 0.0747 Biopterin (n/M) 1 -0.1693 0.0536 9.9888 0.0016
Biopterin * GCH1 1 -0.1180 0.0536 4.8531 0.0276 A allele
.sup.aOverall model: Likelihood Ratio chi-square = 22.9, df = 5, p
= 0.0004, 80.1% Concordance rate .sup.bOverall model: Likelihood
Ratio chi-square = 22.7, df = 3, p < .0001, 79.8% Concordance
rate
[0068] Further supporting this detected interaction, tests showed
that GCH1 allele status is itself a significant predictor of
biopterin level. As fasting phenylalanine (Phe) levels are known to
be predictors of plasma biopterin levels, adjustments were made for
Phe. While the GCH1 allele did not have a significant main effect
on plasma biopterin, levels within the patient group were
significantly lower in patients with the A allele compared to those
without (Tukey adjusted p-values for multiple comparisons,
p=0.0208). In control subjects no significant difference in
biopterin levels for GCH1 allele status was detected (Tukey
adjusted p=0.9924) (see FIG. 3, which shows for unadjusted
biopterin levels and significance testing between subject
groups).
[0069] The sequence of the GCH1 promoter in which the GCH1 variant
of the present invention lies is seen in FIG. 5. Also seen in FIG.
5 are the sequences for the forward and reverse primers that may be
used to identify the GHC1 alleles of the present invention. The
nucleotide sequence of the 351 bp DNA product that is amplified
using the primer pair (green) is shown. The published SNP
rs10137071 (the SNP rs10137071 is published as a C/T variant as the
GCH1 gene is transcribed in the reverse DNA strand), also referred
to as the -959 nt G/A variant, is underlined in the sequence (red)
and the two possible alleles of the present invention are shown.
Human subjects can be G/G, A/G or A/A at the relevant nucleotide
position.
[0070] GCH1 mRNA transcripts are lower in peripheral tissues from A
allele patient carriers. Whole blood leukocytes collected from
subjects with SZ (both medicated and non-medicated SZ subjects),
and BPD, and also ethnicity and gender matched control subjects,
and global leukocyte gene expression were measured using Affymetrix
microarrays. For each subject recruited, a 15 ml blood sample was
collected. Immediately after blood collection, leukocytes were
isolated by lysis of red cells, centrifugation and washing
(Qiagen). Purified leukocytes were stored at -70.degree. C. prior
to RNA extraction (and are stable for periods of >1 year). Total
RNA was extracted using RNEasy columns (Qiagen), and quantified by
UV spectrometry using RNA standards for normalization or using an
Agilent Bioanalyzer. Subjects were also genotyped for the GCH1
promoter variant, as described herein.
[0071] Only RNA samples with good quality ribosomal RNA, and
satisfactory O.D. 260/280 ratios were processed to completion. 8
.mu.g of total RNA was employed as a cDNA synthesis template, using
an oligo-dT primer and Reverse Transcriptase (RT) enzyme, according
to standard Affymetrix protocols. Purified cDNA, was then used as a
template to generate biotin labeled cRNA (Enzo). cRNA samples were
quantified and stored at -70.degree. C. prior to fragmentation. 20
ng of each fragmented cRNA product was hybridized to an Affymetrix
TEST3 array to check sample quality and then hybridized to an HU133
plus 2.0 array (containing over 40,000 transcripts).
[0072] Initial comparison of GCH1 leukocyte mRNA levels between SZ,
BPD, and control subjects showed that GCH1 was downregulated in
patient groups, which is consistent to that found in the CNS. SZ
patients with the A allele has significantly lower plasma biopterin
levels when compared to controls, while G/G SZ patients had similar
levels to controls (and there were no differences between any of
the control subject genotypes). As a result, differences in GCH1
transcript levels in A allele patients were tested as compared to
G/G patients. Referring to FIG. 6, significantly lower leukocyte
GCH1 expression was found in the SZ A allele group (n=12) when
compared to SZ G/G subjects (n=7), which is consistent with plasma
biopterin data obtained from this subject group. Data in FIG. 6 are
natural log (ln) normalized via RMA, where the first panel
illustrates GCH1 expression is significantly lower in peripheral
leukocytes from A allele SZ patients compared to SZ G/G patients
(p=0.024). The second panel of FIG. 6 illustrates that GCH1
expression is lower in A allele BPD patients compared to G/G BPD
patients (p=0.2). The box plots display the mean (+), median
(horizontal line in box), first (Q1) and third (Q3) quartiles (ends
of the boxes). Analysis of 17 BPD subjects also demonstrated lower
levels of GCH1 in A allele carriers (n=9), when compared to G/G BPD
patients (n=8), although this result was not significant.
[0073] Ten of the BPD subjects employed for this analysis had been
treated with lithium, seven had not (16 of the 17 BPD subjects were
also receiving neuroleptics). Subjects were therefore separated for
both genotype and Li treatment groups, with the means calculated
and plotted in FIG. 7, which illustrates mean log expression for
BPD subjects without Li treatment (solid line) with the AA (n=2),
A/G (n=4) and G/G (n=4) genotypes. Log mean expression for BPD
subjects with Li treatment (dashed line) with the A/A (n=0), A/G
(n=3) and G/G (n=4) genotypes. Although the results were not
significant (likely due to small number in each group), the data
was striking and consistent with all previous results, i.e., levels
of GCH1 in the BPD Li untreated group (lowest in the A/A subjects
and highest in the G/G BPD subjects) had a similar expression
profile to the plasma biopterin levels in SZ subjects (see FIG. 4).
In addition, subjects treated with Li had higher expression levels
than subjects without Li treatment. Plasma biopterin levels show
that Li-treatment likely increases plasma biopterin levels, which
is likely accomplished via increased GCH1 expression.
[0074] The data thus suggests that subjects with the GCH1
BPD-associated genotype will have downregulated GCH1 mRNA (in both
the periphery and CNS) that alters BH4 biosynthesis. The resultant
BH4 deficit will thus contribute to BPD susceptibility, likely via
altered CNS neurotransmission. The data also suggests that for Li
treated patients, the biopterin deficit can be alleviated via
increased GCH1 expression, which is supported by a similar GHC1
mRNA rise observed in the rat brain following Li-induced inositol
depletion (via Li administration). Li has a major effect on plasma
biopterin levels, thus suggesting that the increased biopterin
levels in BPD subjects is due, in part, to Li upregulating GCH1
transcription and subsequently BH4 biosynthesis. Li treatment may
thus be less effective in subjects with the BPD associated allele,
and the data supports targeting of treatment for BPD based on GCH1
genotype.
[0075] With respect to the prediction of risk and/or screening, the
present invention provides for the assay of GCH1 genotype for the
presence of a GCH1 variant as a method for prediction of risk for
SZ or SaD or a psychiatric disorder, neuropsychiatric disorder, or
neurological disorder. The present invention also provides for the
assay of GCH1 genotype for the presence of a GCH1 variant, in
conjunction with the assay of BH4 system measures (e.g., plasma
biopterin assay), as a method for prediction of risk and/or
screening for SZ, SaD, or BpD, or other psychiatric disorders,
neuropsychiatric disorders, or neurological disorders.
[0076] With respect to diagnosis, the present invention provide for
the assay of GCH1 genotype for the presence of a GCH1 variant, in
conjunction with the assay of BH4 system measures (e.g., plasma
biopterin assay), as a method for diagnosis of SZ or SaD, or BpD or
a psychiatric disorder, neuropsychiatric disorder, or neurological
disorder. The present invention also provides for the assay of GCH1
genotype for the presence of a GCH1 variant, as a method for
diagnosis of SZ or SaD, or other psychiatric disorders,
neuropsychiatric disorders, or neurological disorders.
[0077] With respect to diagnosis incorporating symptoms, the
present invention provides for the assay of GCH1 genotype for the
presence of a GCH1 variant, in conjunction with assessment of
specific symptoms and symptoms types, and in conjunction with the
assay of BH4 system measures (e.g., plasma biopterin assay), as a
method for diagnosis of SZ or SaD, or BpD or a psychiatric disorder
or neuropsychiatric disorder, and/or the analysis of symptoms for
those disorders. The present invention also provides for the assay
of GCH1 genotype for the presence of a GCH1 variant, in conjunction
with assessment of specific symptoms and symptoms types, as a
method for diagnosis of SZ or SaD, or other psychiatric disorders,
neuropsychiatric disorders, or neurological disorders.
[0078] With respect to prediction of prognosis, the present
invention provides for the assay of GCH1 genotype for the presence
of a GCH1 variant, in conjunction with the assay of BH4 system
measures (e.g., plasma biopterin assay), as a method for prediction
of prognosis of SZ or SaD, or BpD or a psychiatric disorder or
neuropsychiatric disorder. The present invention also provides for
the assay of GCH1 genotype for the presence of a GCH1 variant as a
method for prediction of prognosis of SZ or SaD, or other
psychiatric disorders, neuropsychiatric disorders, or neurological
disorders.
[0079] With respect to the assessment of patient treatment benefit,
the present invention provides for the assay of GCH1 genotype for
the presence of a GCH1 variant, in conjunction with the assay of
BH4 system measures (e.g., plasma biopterin assay), as a method for
defining those patients with a psychiatric disorder,
neuropsychiatric disorder, or neurological disorder (e.g., SZ, SaD,
BpD) who would benefit from treatment designed to normalize their
BH4 system levels, or prevent a potential biopterin or BH4 system
deficit in those at-risk. The present invention also provides for
the assay of GCH1 genotype for the presence of a GCH1 variant, as a
method for defining those patients with a psychiatric disorder,
neuropsychiatric disorder, or neurological disorder (e.g., SZ, SaD,
BpD) who would benefit from treatment designed to normalize their
BH4 system levels and/or improve their symptoms.
[0080] With respect to defining at-risk subjects, the present
invention provides for the assay of GCH1 genotype for the presence
of a GCH1 variant as a method for defining those at risk for SZ or
SaD or other psychiatric, neuropsychiatric, or neurological
disorder. The present invention also provides for the assay of GCH1
genotype for the presence of a GCH1 variant, in conjunction with
the assay of BH4 system measures (e.g., plasma biopterin assay), as
a method for defining those at risk for SZ, SaD, or BpD, or other
psychiatric disorders, neuropsychiatric disorders, or neurological
disorders.
[0081] With respect to defining at-risk subjects who would benefit
from prophylactic treatment, the present invention provides for the
assay of GCH1 genotype for the presence of a GCH1 variant, in
conjunction with the assay of BH4 system measures, as a method for
defining those at risk for development of SZ or SaD or BpD or a
psychiatric disorder, neuropsychiatric disorder, or neurological
disorder who would benefit from treatment designed to normalize
their BH4 system levels and/or improve their symptoms. The present
invention also provides for the assay of GCH1 genotype for the
presence of a GCH1 variant as a method for defining those at risk
for development of a psychiatric, neuropsychiatric or neulogical
disorders, including SZ or SaD, and those who would benefit from
useful treatments designed to normalize or improve their BH4 system
levels, improve their symptoms, and/or benefit from Li,
neuroleptic(s), and/or other antipsychotic or mood stabilizers,
and/or treatments (e.g., administration of electrical stimulation
and/or electroconvulsive therapy (ECT), transcranial magnetic
stimulation (TMS), electrical brain stimulation, deep brain
stimulation, and/or inositol depletion treatments).
[0082] With respect to the treatment and prevention of disorders,
the present invention provides for the treatment of subjects with,
or who are at risk of developing, SZ or SaD and/or a psychiatric
disorder, neuropsychiatric or neurological disorder, who either
carry a GCH1 variant and/or a BH4 system gene variant and/or a
variant in another gene that can lead to BH4 system deficit or
alteration, or who have (or are at risk for developing) a fasting
or non-fasting biopterin level (e.g. plasma biopterins) and/or BH4
and/or pterin levels, and/or GCH1 RNA and/or GTPCH protein that lie
in the range defined as "low" or "altered" or is different from the
mean (or median, mode, normal range, expected value, etc.) compared
to controls, by treatment with useful treatments. Treatments will
be used as a way to boost and/or alter BH4 levels, biopterin
levels, and/or affected BH4 system component levels in those in
need of BH4 system supplementation and/or normalization, with
and/or at-risk of developing a disorder. Such treatment(s) can be
used either alone or in conjunction with other "useful treatments"
which include, but are not limited to: treatment with pterin(s)
and/or other molecules, including BH4 and/or biopterin and/or
dihydrobiopterin and/or sepiapterin and/or sapropterin
dihydrochloride. Other medications that may improve BH4 system
status or that can be used in conjunction with such treatments may
include neuroleptic or other psychotropic medications (e.g.,
Phenothiazines, Chlorpromazine, Fluphenazine, Perphenazine,
Prochlorperazine, Thioridazine, Trifluoperazine, Butyrophenones,
Haloperidol, Droperidol, Pimozide, Clozapine, Olanzapine,
Risperidone, Quetiapine, Ziprasidone, Aripiprazole, Bifeprunox;
norclozapine (ACP-104), Symbyax, Tetrabenazine, and lithium), ECT,
TMS, and other like medications. Such treatment(s) may be provided
to those with SZ, SaD, BpD, or BH4-responsive PKU,
hyperphenylalanenemia (Phe and/or aspartame and/or Phe-containing
polypeptides will not be used for hyperphenylalaninemia), and/or
dystonias. Such treatments will also be used for other psychiatric
and/or neuropsychiatric disorders and/or neurological
disorders.
[0083] With respect to the treatment of patients with useful
treatments designed or used to normalize BH4 and/or BH4 system
levels, the present invention also provides for the treatment of
SZ, SaD, or BpD who carry a GCH1 variant (or a BH4 system gene
variant), in conjunction with a fasting or non-fasting biopterins
(e.g., plasma biopterins), BH4, BH4 system measures, pterin levels,
GCH1 RNA and/or GTPCH protein, that lie in the range defined as
"low" or "altered" compared to controls, with therapeutic amounts
of useful treatments. The present invention also provides for the
treatment of SZ, SaD, or BpD (or other psychiatric disorders,
neuropsychiatric disorders, or neurological disorders in need of
treatment), in subjects who carry a GCH1 gene (or a BH4 system
gene) variant, by treatment with therapeutic amounts of useful
treatments. It should also be recognized by those of skill in the
art that the present invention may be used to determine the
presence of a GCH1 G/G genotype, and any BH4 system surplus, such
as high BH4 and/or bopterin, that may factor into diseases
symptoms, disease risk, and/or the applicable treatment
response.
[0084] With respect to the treatment of at-risk subjects with
useful treatments designed or used to normalize BH4, the present
invention provides for the treatment of those at risk of developing
SZ, SaD, or BpD, or other psychiatric disorders or neuropsychiatric
disorders or neurological disorders (or other disorders in need of
treatment) who carry a GCH1 variant (or a BH4 system gene variant)
in conjunction a "low" or "altered" BH4 system and/or with a
fasting or non-fasting biopterin levels (e.g., plasma biopterins),
BH4 levels, and/or pterin levels, as well as GCH1 RNA and/or GTPCH
proteins that lie in the range defined as "low" or "altered" or is
different from the mean (or median, mode, normal range, expected
value(s) etc.) compared to controls and/or other patient groups, by
treatment with useful treatments. The present invention also
provides for the treatment of those at risk of developing SZ or SaD
or BpD (or other disorders in need of treatment) who carry a GCH1
variant (or a BH4 system gene variant), by treatment with useful
treatments. Subjects determined to be at-risk for development of a
psychiatric, neuropsychiatric or neurological disorders can include
gametes, embryos, and/or fetuses, and assessment of risk status can
be made from parental and/or prenatal testing.
[0085] With respect to prevention and/or treatment using gene or
protein therapies, the present invention provides for the treatment
of those with, or at risk of developing, SZ or SaD or a
psychiatric, neuropsychiatric, or neurological disorder (or other
disorders in need of treatment) who carry a GCH1 variant (or a BH4
system gene variant), either alone or in conjunction with a fasting
or non-fasting biopterins (e.g. plasma biopterins) and/or BH4
and/or pterin levels, and/or GCH1 RNA and/or GTPCH protein that lie
in the range defined as "low" or "altered" or different from the
mean (or median, mode, normal range, expected value, etc.) compared
to controls, by treatment with therapeutic DNA and/or RNA molecules
to cells, tissues, or organs, or locally or systemically (including
injection, intravenously, orally, suppository, intranasally,
intracerebrallly, intrathecal, parenterally, or by infusion), or by
administration to isolated cells, tissues, organs, fluids, etc.
Therapeutic DNA and/or RNA molecules include, but are not limited
to, sequences related to those of GCH1 (such as for example
sequence containing the -959 nt G to A: rs10137071, "G" allele),
other BH4 system genes, and/or related genes, and can be introduced
by using vectors, viral particles, capsules, liposomes, and other
containers or delivery methods, or by binding to other molecules or
particles, surfaces, or materials. Therapeutic DNA and/or RNA
molecules can also be introduced to sperm, eggs, embryos, fetuses,
stem cells, or other cells. Additionally, in conjunction with these
procedures, one or more other useful treatments may also be
used.
[0086] With respect to imaging correlates in the screening,
detection, diagnosis, prognosis, definition of at-risk, and
assessment of treatment response, the present invention provides
for the assay of GCH1 genotype (or assay of a BH4 system gene
variant), altered biopterin level, and/or BH4 system measures
(e.g., biopterin), for those with or at risk of developing a
psychiatric, neuropsychiatric, or neurological disorder or other
disorder, and the determination use of MRI, MRS, fMRI, PET, SPECT,
CT, or other imaging technologies and methods to assess CNS,
peripheral, structural, biochemical activity, physiological, and/or
other differences or changes in subjects.
[0087] With respect to gene or protein expression in the screening,
detection, diagnosis, prognosis, definition of at-risk, and
assessment of treatment response, the present invention provides
for the assay of GCH1 genotype (or of a detected BH4 system gene
variant) and/or assay of the BH4 system or BH4 system measures, for
those with or at risk of developing a psychiatric,
neuropsychiatric, or neurological disorder, and the use of any RNA
(including miRNA) and/or protein and/or peptide expression
measurement, such as microarray analysis, mass spectrometry
analysis, protein microarray analysis or electrophoresis analysis,
and can be used for disease and/or symptom detection, diagnosis,
prognosis, definition of at-risk, determination of treatment,
assessment of treatment response. Expression analysis in any
tissue, and/or organs and/or cells, from a subject or animal model
could be employed. Expression measurements, profiling, and
detection methods are included to assess CNS, peripheral,
structural, biochemical activity, physiological, and/or other
differences or changes in subjects.
[0088] With respect to the detection, diagnosis and medication of
subjects having BH4 system gene mutations and variants and
alterations, the present invention provides for the screening,
determination of at-risk, detection, diagnosis, prognosis,
determination of treatment and treatment of disorders, including
whether there is a genetic or epigenetic (e.g., methylation,
acetylation, and/or ubiquitylation) difference affecting the
gene(s), as well as their promoters, enhancers, suppressors and
other regulatory regions, regulatory RNAs (such as miRNAs), such as
whether there is a nucleotide variant (e.g., a G/A nucleotide
substitution or a nucleotide deletion), or copy number polymorphism
and/or duplication, deletion or other mutation or change, whether
newly detected or previously known, such as for GCH1, NURR1, GSK
(including GSK 3 beta and alpha), IMPase, AKT1, AKT/PKB, AKT2, AKT3
or IPP mutations and variants, and for other BH4 system genes.
[0089] With respect to the generation of, and/or breeding with,
and/or use of a GCH1 transgenic animal model and/or animal model
with a targeted disruption of the GCH1 gene, the present invention
provides for research into the etiology and pathogenesis of, as
well as the development of mediations and treatments, for
neuropsychiatric, and neurological disorders, including SZ, SaD and
BpD.
Sequence CWU 1
1
41351DNAHomo sapiens 1tcaagtgagg aaaaaggtcc atttattaat ctcaaagaaa
acagttacag cagatgtcac 60tggttaagag ttcagttggt gaatagcatt tcacaatttg
taccaacatc tggggaaaga 120cgctttgcat ggaactgtaa aacaattgag
caccaaatct gcacaactgc gtttatagaa 180aatgcgatgg gttttataga
gatgaggtct tgctatgttt tccaggctgg tctcgaactc 240ttggcctcaa
gcgatcctcc cgcctcggtc tccccaagcg ccgggagtac aggcgtgagc
300caccgacgga aatggatttt aagtgaaagt cctatcttcg tttgcaaatc a
3512351DNAHomo sapiens 2tcaagtgagg aaaaaggtcc atttattaat ctcaaagaaa
acagttacag cagatgtcac 60tggttaagag ttcagttggt gaatagcatt tcacaatttg
taccaacatc tggggaaaga 120cgctttgcat ggaactgtaa aacaattgag
caccaaatct gcacaactgc gtttatagaa 180aatgcaatgg gttttataga
gatgaggtct tgctatgttt tccaggctgg tctcgaactc 240ttggcctcaa
gcgatcctcc cgcctcggtc tccccaagcg ccgggagtac aggcgtgagc
300caccgacgga aatggatttt aagtgaaagt cctatcttcg tttgcaaatc a
351321DNAArtificial SequenceForward primer for identifying allele
3tcaagtgagg aaaaaggtcc a 21422DNAArtificial SequenceReverse primer
for identifying allele 4tgatttgcaa acgaagatag ga 22
* * * * *