CA2216057A1 - Polymorphic cag repeat-containing gene, diagnosis of psychiatric diseases and therapeutic uses thereof - Google Patents
Polymorphic cag repeat-containing gene, diagnosis of psychiatric diseases and therapeutic uses thereof Download PDFInfo
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Abstract
The present invention relates to hGTl gene, a polymorphic CAG repeat-containing gene and its uses thereof for the diagnosis, prognosis and treatment of psychiatric diseases, such as schizophrenia.
Description
CA 022160~7 1997-09-19 POLYMORPHIC CAG REPEAT-CONTAINING GENE, DIAGNOSIS OF
PSYCHIATRIC DT~~.S AND THERAPEUTIC USES T~R~OF
R~CK~OUND OF THE INV~NTION
(a) Field of the Invention The invention relates to hGTl gene, a polymor-phic CAG repeat-containing gene and its uses thereof for the diagnosis, prognosis and treatment of psychiat-ric diseases, such as schizophrenia.
(b) DescriPtion of Prior Art Schizophrenia is a chronic brain disorder char-acterized by a behavioral syndrome combining in various degrees hallucinations, delusions, social withdrawal, affective flattening, disorganized behavior and formal thought disorders. It affects up to 1% of the general population and results in a lower level of social and occupational functioning. Many recent studies indicate that schizophrenia may originate from neural cell dis-turbances occurring in the developing/maturing brain.
Genetic factors are known to play a major role in the etiology of this disorder as demonstrated by extensive family, twin and adoption studies. However, the quest for genes conferring susceptibility to schizophrenia has been difficult and has not yielded consistent find-ings using both association and linkage studies. It isthought that these difficulties are in part due to het-erogeneity in etiology, both of genetic and non-genetic origins, resulting in a highly variable phenotype with respect to age at onset, symptom profile, course of illness, response to medication, long term outcome and performance on neuropsychological tests.
One promising avenue to guide research in this search for genes increasing susceptibility to schizo-phrenia may be to distinguish patients on the basis of therapeutic response to neuroleptics. Indeed, while most schizophrenic patients are improved by neuroleptic CA 022160~7 1997-09-19 medication, a substantial number of subjects (15 to 25%) remains severely symptomatic despite multiple and adequate neuroleptic therapeutic attempts. In contrast to this between subject variability, within subject (from one episode to the other) consistency of neu-roleptic response have been reported. Clinical pre-treatment characteristics that correlate with good neu-roleptic response include spontaneous high blink-rate and blink-rate decrease under Haloperidol challenge, absence of spontaneous movement disorders, and absence of dysphoric reaction within 24-48 hours of neuroleptic initiation. On a long term basis, it has been demon-strated that good response to neuroleptics (but not the severity of the symptoms prior to neuroleptic medica-tion) in the early stages of the disease, predicts abetter outcome. Neurophysiological characteristics that correlate with good neuroleptic response include high-frequency waves and few alpha and slow waves in computerized EEG prior to the treatment with neurolep-tics, a specific profile of changes in quantified EEGspectrum under neuroleptics and high degree of elec-trodermal activity prior to neuroleptic treatment. An important number of studies indicate that dopamine neu-rotransmission is disturbed predominantly in the responsive schizophrenic patients. High pre-treatment plasma levels of HVA have been shown to predict good response to neuroleptics in most of the studies. Pre-liminary genetic epidemiological data indicate that poor or delayed response to neuroleptic treatment is associated with an increased prevalence of schizophre-nia spectrum disorders in relatives of schizophrenic probands. These convergent lines of evidence suggest that long term response to neuroleptic medication may be considered as a bioclinical dimension with an etiologically relevant significance; the two extremes -CA 022160~7 1997-09-19 of this dimension being occupied by two groups of schizophrenic patients, at least partially, distinct with respect to the pathogeny of their illness.
It would be highly desirable to be provided with a tool for the diagnosis, prognosis and treatment of psychiatric diseases, such as schizophrenia.
SUMMARY OF THE INVENTION
One aim of the present invention is to provide a tool for the diagnosis, prognosis and treatment of psychiatric diseases, such as schizophrenia.
Another aim of the present invention is to detect association between allelic variants of CAG
repeat-containing genes and schizophrenia or its pheno-typic variability with respect to long term response toneuroleptic medication.
In accordance with the present invention, we compared the allelic frequencies of various polymorphic candidate genes between two groups of schizophrenic patients carefully screened on the basis of their long term response to typical neuroleptics (excellent responders, Rs; non-responders, NRs) and controls.
This report summarizes our finding while considering CAG containing genes as candidates for schizophrenia.
This family of candidate genes was deemed attractive for the following reasons: (1) CAG repeat instability was associated with several neurodegenerative brain diseases that display genetic anticipation, a feature believed to be present in schizophrenia, (Z) some iso-lated, though promising, reports indicate that expandedCAG repeats are more prevalent in schizophrenic patients compared to normal controls, (3) CAG repeats are often very polymorphic and have been found to be over represented in coding sequences of the human genome particularly those coding for DNA-binding pro-teins/transcription factors. These factors are impor-CA 022160~7 1997-09-19 tant actors in the regulation of the genetic program and neurodevelopmental processes and have been impli-cated in several human neurodevelopmental diseases including one that may present with schizophrenia-like symptoms, and; (4) CAG repeats (or the polyglutamine stretches for which they encode) might modulate the function of the genes (or protein) they are part of suggesting that they might be functional polymorphisms and not silent ones.
In accordance with the present invention there is provided a hGTl gene containing transcribed polymor-phic CAG repeat, which comprises a sequence as set forth in Fig. 3.
The allelic variants of CAG repeat of hGTl gene may be associated with schizophrenia, affective dis-eases such as manic depression, neurodevelopmental brain diseases or with phenotypic variability with respect to long term response to neuroleptic medica-tion.
More precisely, there are 5 allelic variants of CAG repeat which are identified as follows:
Size of PCR amplifiedPredicted No. of CAG repeats SIIOll~al to longest r~ay~"~lS (bp) In accordance with the present invention there is provided a method for the prognosis of severity of schizophrenia of a patient, which comprises the steps of:
a) obtaining a nucleic acid sample of the patient;
and CA 022160~7 1997-09-19 b) determining allelic variants of CAG repeat of the hGTl gene, and wherein long allelic vari-ants are indicative of severe schizophrenia.
The preferred nucleic acid sample used in accordance with the present invention is DNA. For RNA
sample, an additional step is carried out, which con-sists in using a reverse transcriptase to transcribe the RNA into DNA.
More precisely, the allelic variants identified as short or as having between about 171 and 177 bp (referred to as -3, -2 and -1) are associated with mild schizophrenia and long or as having between about 180 and 183 bp (referred to as 0 and 1) are associated with severe schizophrenia.
In accordance with the present invention there is provided a method for the identification of patient responding to neuroleptic medication, which comprises the steps of:
a) obtaining a nucleic acid sample of the patient;
and b) determining allelic variants of CAG repeat of the hGTl gene, and wherein short allelic vari-ants are indicative of neuroleptic response.
More precisely, the allelic variants identified as short or as having between about 171 and 177 bp (referred to as -3, -2 and -1) are associated with patient capable of neuroleptic response and long or as having between about 180 and 183 bp (referred to as 0 and 1) are associated with non-response to neuroleptic medication.
In accordance with the present invention there is provided a non-human mammal model for the hGTl gene, whose germ cells and somatic cells are modified to express at least one allelic variant of the hGTl gene and wherein the allelic variant of the hGTl being CA 022160~7 1997-09-19 introduced into the mammal, or an ancestor of the mam-mal, at an embryonic stage.
In accordance with the present invention there is provided a method for the identification of patient responding to neuroleptic medication, which comprises the steps of:
a) obtaining a nucleic acid sample of the patient;
and b) determining allelic variants of CAG repeat of the hGTl gene, and wherein short allelic vari-ants (from about 171 to about 177 bp) are indicative of neuroleptic response.
In accordance with the present invention there is provided a method for the screening of therapeutic agents for the prevention and/or treatment of schizo-phrenia, which comprises the steps of:
a) administering said therapeutic agents to the non-human mammal of the present invention or schizophrenia patients; and b) evaluating the prevention and/or treatment of development of schizophrenia in said mammal or said patients.
In accordance with the present invention there is provided a method to identify genes part of or interacting with a biochemical pathway affected by hGTl gene, which comprises the steps of:
a) designing probes and/or primers using the hGTl gene of the present invention and screening psychiatric patients samples with said probes and/or primers; and b) evaluating the identified gene role in psychi-atric patients.
In accordance with the present invention there is provided a method of stratifying psychiatric CA 022160~7 1997-09-19 patients based on the allelic variants of the hGTl gene for clinical trials purposes, which comprises:
a) obtaining a nucleic acid sample of the patients; and b) determining allelic variants of CAG repeat of the hGTl gene, wherein patients are stratified with respect to their allelic variants and wherein short allelic variants are indicative of neuroleptic response.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 illustrates the average allelic lengths of the GCTlOD04 EST CAG repeat in controls, responsive (R) and non-responsive (NR) patients, showing the shorter (S) allele only, longer (L) allele only and the sum (L+S) of the two alleles in the three groups of subjects;
Fig. 2 illustrates the correlation between the average length of the (CAG)n polymer of the short (a), the long (b) alleles and the sum of 2 alleles (C) and seventy of schizophrenia in the different classes of severity of the disease; and Fig. 3 illustrates the sequence homology between the human GCTlOD04 sequence and the mouse GTl gene.
DET~TT~n DESCRIPTION OF THE INVENTION
In accordance with the present invention, the main objective was to detect allelic variants of CAG
repeat containing genes associated with schizophrenia or its phenotypic variability with respect to the pres-ence or absence of schizophrenia and long term response to neuroleptic medication.
Accordingly, CAG repeat allelic variants were compared between three groups of subjects: two groups CA 022160~7 1997-09-19 of schizophrenic patients, one neuroleptic-responsive (Rs; n=43) and one -non-responsive (NRs; n=63), and; a group of controls screened out for DMS-IV axis I psy-chiatric disorders (Ci n=87). Assessment of response to conventional neuroleptics was based on a comprehen-sive review of medical files according to a priori defined criteria and blind to genotyping. Genes con-taining polymorphic CAG repeats were identified by means of genetic sequences data base searches.
The results in accordance with the present invention shows that short CAG repeat allelic variants of the hGTl gene were associated with schizophrenia irrespective of neuroleptic response (% short alleles SCZ=45%; C=31%, p=0.005). This association was highly significant in Rs (52%, p=0.0009) and marginal in NRs (40%, p=0.12) groups. A statistically significant cor-relation (Gamma=0.37, p=0.0024) between the CAG repeat length and the overall pattern of severity of schizo-phrenia was also observed.
Surprisingly and in accordance with the present invention, CAG repeat allelic variants of the hGTl gene show strong association with neuroleptic responsive schizophrenia and length correlation with the overall pattern of severity of the disease.
Clinical The study was conducted between 1994 and summer 1997. Patients have been recruited in the Douglas hos-pital (n=82), the Clinique Jeunes Adultes of L.H.
Lafontaine Hospital (n=15) and the Schizophrenia Clinic of the Royal Ottawa Hospital (n=9). 333 schizophrenic patients were identified as potential subjects for this study. 123 patients did not meet the criteria for schizophrenia or Rs/NRs (undifferentiated response) diagnoses. 125 and 85 patients met respectively the criteria for NRs-schizophrenia and Rs-schizophrenia. 62 CA 022160~7 1997-09-19 NRs and 42 Rs subjects were not included in the study because refusal or other exclusion criteria.
NRs schizophrenic patients were recruited according to the following criteria: (1) they all met axis I diagnosis of schizophrenia, according to the Diagnostic and Statistical Manual of Mental Disorders, version IV (American Psychiatric association, Diagnos-tic and Statistical Manual of Mental Disorders, APA;
1994) (DSM-IV), (2) they did not experience remission of psychotic symptoms within the past 2 years, (3) in the preceding 5 years, all patients underwent at least 3 periods of treatment with typical neuroleptics, from at least two distinct families of drugs, at therapeutic dosage (equal to or greater than 750 mg Chlorpromazine equivalent in monotherapy or 1000 mg chlorpromazine equivalent, when a combination of neuroleptics is used), for a continuous period of at least 6 weeks at a time, with no significant relief of symptoms, and; (4) Unable to function without supervision in all or nearly all domains of social and vocational activities with a Global Assessment Score (GAS) < 40 within the last 12 months.
Criteria for the selection of neuroleptic Rs patients were as follows: (1) all patients met the cri-teria for schizophrenia according to DSM-IV, (2) all were admitted at least once to a psychiatric care facility because of acute psychotic episode, (3) during all hospitalizations, patients experienced full or par-tial remission in response to treatment with typical neuroleptics, at recommended dosage, within six-eight weeks of continuous treatment; remission being defined as a rapid reduction of schizophrenic symptoms with limited residual symptoms, (4) all patients were able to function with only occasional supervision in all or nearly all domains of social and vocational activities CA 022160~7 1997-09-19 with a GAS score > 60 within the last 12 months, (5) no patients had to be admitted to hospitals because of psychotic exacerbation, if and when compliant to treat-ment and treated continuously with typical neurolep-tics, and; (6) at least one psychotic relapse when neu-roleptic medication is reduced or interrupted. Exclu-sion criteria for schizophrenic patients were brain trauma, any neurological condition, drug or alcohol abuse in the last two years.
All schizophrenic patients were directly inter-viewed by the PI, a research psychiatrist trained in the use of the Diagnostic Interview for Genetic Stud-ies (DIGS) (Nurnberger JI et al., A~chives of General Psychiatry. 1994jSl:849-59) and their medical records were comprehensively reviewed. Complementary informa-tion from the treating physician and nurses in charge of the patients and their close relatives was obtained, whenever possible. A best estimate diagnosis was established on the basis of all the available data.
Responsiveness to typical neuroleptic medication was evaluated according to a 7 point's scale. The severity of symptoms and overall psychosocial functioning were assessed using the following instruments: (1) Brief Psychiatric Rating Scale (BPRS) (Woerner MG et al., Psychopharmacology Bulletin. 1988j24:112-117), (2) the Scale for the Assessment of Negative Symptoms (SANS), the (3) Scale for the Assessment of Positive Symptoms, (4) the GAS, (5) the Pattern of Severity Scale, a 5 point's scale assessing overall course and outcome of the disease (American Psychiatric association, Diagnos-tic and Statistical Manual of Mental Disorders, Fourth Edition, American Psychiatric association, Washington D.C.; 1994), and; (7) the Pattern of Symptoms subtypes, a categorical classification of patients according to the combination and changes over the course of the dis-CA 022160~7 1997-09-19 ease of positive and negative symptoms (Nurnberger JI
et al., Archives of General Psychiatry. 1994;51:849-59). All these evaluation tools, except the BPRS are part of the DIGS.
The control group (C) was made by healthy vol-unteers recruited through advertisement in local papers (n=49) and married-in individuals from a linkage study (n=38). All subjects in this group underwent a struc-tured psychiatric interview in order to exclude those who meet DSM-IV axis I disorders. Subjects recruited through advertisement have also been screened for schizophrenia spectrum disorders and have been tightly matched for ethnic background (mother and father eth-nicity) with schizophrenic patients. All, (except one responsive~, patients and controls were Caucasians.
All of them gave informed and written consent. The research protocol has been approved by the three hospi-tals ethic committee where the research took place.
Genetic methods To identify sequences potentially encoding polymorphic polyglutamine tracts, we conducted a number of Basic Local Alignment Search Tool (BLAST) (Altschul SF et al., Journal of Molecular Biology. 1990;215:403-410) searches using the following sequences: (1) (CAG)30 or (CAA)30 (BLASTn, unfiltered against the non-redundant nucleic acid and the expressed sequence Tag (dbEST) databases) and, (2) Q30 (BLASTp, unfiltered, against the non-redundant protein database or tBLASTn against dbEST). Sequences containing homopolymer tracts of >7 CAG or CAA repeats or potentially encoding a tract of >12 glutamine residues were used to design PCR primers able to amplify the CAG or CAA repeats.
PCR primers were designed using DNASTAR Inc. (Madison, Wisconsin) software.
CA 022160~7 1997-09-19 Genomic DNA was isolated from peripheral lym-phocytes using standard methods. CAG repeat-containing fragments were amplified by PCR using specific primers for each repeat. PCR was performed in a total volume of 13 ~1 containing 30 ng of human genomic DNA, lOmM
Tris-HCl (pH 8.8), 1.5 mM MgC12, 50mM KCl, 1% Dimethyl-sulfoxide, 250 mM each of dCTP, dGTP, and dTTP, 25 mM
dATP, 1.5 uCi alpha 35S-dATP, 100 ng of each primer, and 3 units of Taq polymerase (Perkin-Elmer). DNA was denatured at 94~C for 5 min., then subjected to 30 cycles of a 1 min. denaturation at 94~C, a 1 min.
annealing at the optimized annealing temperature for each primer pair and a 1 min. elongation at 72~C. This was followed by a final extension at 72~C for 5 min.
PCR products were electrophoresed on denaturing 6% Polyacrylamide gels and visualized by autoradiogra-phy. Absolute allele sizes were estimated according to an M13 sequence ladder. Since differences in absolute allele sizes were in all cases multiples of 3 base pairs, we assumed that variations in allele sizes were due to differences in the number of trinucleotide repeat units in the amplified sequences. By conven-tion, we designated the most common allele as 0, with less common alleles as positive or negative integers according to their number of trinucleotide repeats (e.g. if allele 0 had 20 repeats, allele +2 and -2 would have respectively 22 and 18 repeats).
Analysis:
Each subject was assigned two numeric values which represent respectively the lengths of his short (S) and long (L) alleles. Under the assumption of a quantitative effect of the CAG tract length, data were initially analyzed using a non-parametric analysis of variance (Kruskal-Wallis median statistic) where the independent variable is the diagnostic status (Rs, NRs CA 022160~7 1997-09-19 and C) and the dependent variable is the length of the CAG repeat of S, L or the sum of the two alleles. In the case of a significant overall group effect in the ANOVA, pair-wise contrasts between the different groups were performed using the Mann and Whitney U-statistic.
This approach allows to control for the inflation of type I error secondary to multiple testing.
We also analyzed data by contrasting allelic frequencies in different pairs of groups using the %Z
statistic. Alleles were grouped in different classes in accordance to the pattern of results found in the analysis of variance. Since both patient and control groups include an important number of subjects with a French Canadian ethnic origin, we reanalyzed any asso-ciation finding after stratifying subjects according tothe ethnic origin of their parents (both parents from French Canadian origin vs. at least one parent with non-French Canadian origin). This analysis allows to control for associations resulting from ethnically based differences in allelic frequencies (population stratification) as opposed to those attributable to the pathological condition under study (true association).
When a particular EST showed allelic or size association with schizophrenia and/or responsiveness to medication, further analyses were performed to investi-gate the putative relation linking various clinical dimensions (age at onset, pattern of severity) to the length of the CAG repeat. For this purpose, we used the Gamma correlation statistic, a non parametric statistic recommended when there are many ties in the data set.
Clinical dimension that were used as criteria to define the two groups of patients (GAS, severity of current symptoms, neuroleptic responsiveness scores) were not included in this analysis. Relations between categori-cal variables (schizophrenia subtypes of illness, pat-CA 022160~7 1997-09-19 tern of symptoms) and the CAG alleles were explored by a % statistic with the appropriate degree of freedom.
Logistic regression was used to determine the attribut-able risk conferred by any EST allelic variants which showed a positive association with schizophrenia or neuroleptic responsiveness. All analyses were made using the Statistica software (Statsoft).
Table 1 Demo~raphic and clinical characteristics of patients and controls Non-respongive(62) Responsive (43) Controls (C) (87) Mean age in y~ars +SD (n~ 38J 7 (62) 40i lo (43) 44* 13 (87) Education inyears+ SD (n) 11+ 2 0 (59) 11+ 2 ~ (43) 14+ 3 3 (49) SES of HH+SD (n)- 54+ 24 (53) 51+ 24(41) 59+ 20(49) Sex, % M 74% 67% 45%
Ethnic origin FCIOB 27/35 26117 40147 Subtype, UIPIDIC 271301411 61361110 Mean age at C, in years +SD (n) 181 3 9(55) 24:t 4.8 (43) Illness duration in years +SD (n) 20+ 7 0 (55) 16i 8 8 (43) ~/O time as in-patient~(n) 62% (61)-- 8 2% (43) BPRS total score i SD (n) 49+ 8 9 (53) ~- 241 3 9 (53) NLP response score 1 83 + 0 74 (58) ~ 6 3 ~ 0 67 (43) Patternofseverity 40+00(55)~- 19+05(43) SES of HH indicates socioeconomic status head of house hold; FC/OB, French Canadian/other ethnic background; U, undifferentiated; P, paranoid; D, disorganized; C, catatonic SrLi ~j L .,..ia; C~, first consultation; BPRS. Brief Psychiatric Rating Scale; and, NLP neuroleptic. ~-, p<0 001 Table 1 shows the demographic and clinical characteristics of the three groups of subjects Rs, NRs, and C. The two groups of patients were comparable with respect to age, level of education and socio-economic status of the head of household. As expected,they differed significantly according to the severity of psychosis ~BPRS scores, F=280, p<0.000), the percent of time spent as inpatient since their first contact with the psychiatric institution (F=81, p<0.000) and the age at first contact with psychiatric care facili-ties (F=47, p<O.OOO).
Table 2 shows the sixteen different candidate expressed CAG repeats identified and analyzed and includes mapping, homology, and polymorphism informa-tion.
Table 2~ it of diffelent sLufied ESTs Seq~elD PCR RT-PCR PNQ 1~ ,potentialf~lion PolynK~phic M~pdata T08930 + na 15 1 ',~ ." ab~RNA!' ' _, ' - -CUG~BPmNabS0 and xc~us E~R-I gene R98242 - + 27 1 ' ._,~." acAMP~pQlsivei '.'' ~ ,! geneexE~ssion L37868 + na 21 N-Oct-3TF,P~Ud~mainTF H.
U23868 + na 26 No known l~mology. + HCHI
U23862OS + na 7 pc6sible l ' ~, I . ' - -p~tein(S. cercisiae) N55395 + na 15 b.~n _~ _ ~ ' TF +
L10379 + na 28 noknownlKmology +++
Z78314 + na 20 nol X85326 - notdale 11 noknownb.~logy na T90581 - + 10 nokn~wnbunology L10375 - + 16 no l I ',, +
X82209 + na 27 27Q, l~nnnl gene disnQted byabalanced ++
1~26155 + na 23 SW12/SN~2,awidelangci '.' factor, ++
idaacls wi~ ERand RAreceptors GCT5EII + na 22 noknownb.~nology +++ HCH3 TATABF + na 39 TFllDTATAbo~bir~lingp~teir~gen~
factor GCIIOD04 + na 14 b,amology to a rn~3se retinoic acid ind~ceble gene +++ HCH17 1~ ~ ' PDGFTF, ER, . ~ R~ ~c~n~ic acid ra~r.
Seven of the candidate sequences showed homol-ogy or identity with DNA binding domains or transcrip-tion factors. Most of the candidates (12/16) gave a PCR product with the predicted size. Candidates that amplified a larger than expected fragment or no prod-ucts at all were further analyzed by RT-PCR to control for possible intronic interruptions in the genomic DNA.
Three candidates gave an RT-PCR product of the pre-CA 022160~7 1997-09-19 dicted size; only one was polymorphic using a small sample of chromosomes. Overall, 10/16 candidate sequences contained a polymorphic CAG repeat. Allelic frequencies of these polymorphic CAG repeats were com-pared in the four groups of subjects.
Only allelic variants of the GCTlOD04 locus (primers; SCZ15:GGGGCAGCGGGTCCAGAATCTTC, SCZ16: TGGCCTTGCTGCCCGTAGTGCT; annealing temperature 62~C) showed an overall significant group effect for the L allele (Kruskal-Wallis H (2, N = 194) = 12.18, p = .002)i the CAG repeat average length being the shortest in the neuroleptic-responders (Rs), intermediate in the non-responders (NRs) and longest in the control group (C) (Fig. 1).
The reference point to measure the CAG repeat length is the most common allele (180 bp fragment or 14 predicted repeats), which is taken as 0. Alleles with n repeats above or below the 0 allele are scored +n or -n. C indicates the control groupi Rs, neuroleptic responsive schizophrenic patients group; and NRs, neu-roleptic non-responsive schizophrenic patients group.
A similar trend was observed for the S allele (Kruskal-Wallis H (2, N= 194) = 5.32, p =0.06). Post-hoc analysis using the U-statistic showed that this global effect was mainly due to the difference between neuroleptic-responders and normal controls (C) (L
allele: adjusted-Z=-3.52, p=0.0004; S allele: adjusted-Z= -2.28, p=0.02). Resistant schizophrenic patients showed also a trend toward smaller CAG repeat average size of the L allele compared to controls (C) (adjusted-Z=-1,68, p=0.09). When we analyzed the sum of the two alleles, the three groups were statistically different (p=0.01) and the difference between controls and Rs was significant at the level of p=0.004 (adjusted-z=-2.8). Further analysis were carried out, testing the hypothesis that short alleles of the GCTlOD04 were more frequent in schizophrenic patients.
For that purpose, two distinct classes of alleles, long (O,l) and short (-3, -2, -1), were defined and allelic frequencies between the four groups were reexamined (Table 3).
Table 3: frequencies of the CAG Allele short variants of hGT 1 gene p~ ~ ~ patientS
Controls SCZ Rs NRs Number(2n) 174 212 86 126 % of short alleles 31% 45% 52% 40%
%2 p=0.005 p=0.0009 1'=0.12 Both parents are French C' Nurnber (2n) 80 106 52 54 % of short alleles 35% 47% 54% 41%
%2 p=O O9 P=0.03 P=0.5 at least one par0t is non French C - ) Number (2n) 94 106 34 72 % of short alleles 28% 42% 50% 39%
%2 1'=0.03 P=0.018 1~0.12 Allelic r~ ;;C;, are given as percent of alleles shorter than 0 (<0) . Fre~pl~.n~ i~s are analyzed according to different diagnosis groups and ethnic l~c~uulld of parents. All rl~u~ were c~ ast~Al with the frequencies of the alleles shorter than 0 in the control group. SCZ indicates ~l~h~ c patients; Rs, neuroleptic~ ol~ive schizophrenic patients; NRs, n~ ~ non-l~ol~iv~ GO~JIII~ patients, and; x2, Chi 2 statistic with I degree of freedom Schizophrenic patients, irrespective of their neuroleptic response status were more likely to carry one of the short alleles compared to controls (%2=7.6, df=l, p=0.005). This difference was mainly due to Rs schizophrenic patients who were significantly more CA 022160~7 1997-09-19 likely to have small alleles compared to controls (% =11.0, df=l; p=0.0009) and to NRs patients (% =3.30, df=l, p=0.07). Neuroleptic-non responders were margin-ally different from controls (X =2.41, df=l, p=0.12).
When subjects with both parents of French Canadian ori-gin or those with at least one parent from non French Canadian origin were analyzed separately, the same pat-tern emerged (Rs vs. C: %2=4.6, df=l, p=0.03i schizo-phrenics vs. C: % =2.7, df=l, p=0.09).
Finally, a correlation analysis indicated that the size of the CAG repeat tract is linearly related to the pattern of severity of schizophrenia (measured blindly to genotype, using a 1-5 scoring system defined as follows: (1, episodic shift) episodes of illness interspersed between periods of health or near normal-ity, (2, mild deterioration) periods of illness occur, but there are periods of return to near normality, with some ability to work at a job and near normal or normal social functioning, (3, moderate deterioration), the subject may occasionally experience some resolution of symptoms, but overall the course is downhill culminat-ing in a relatively sever degree of social and occupa-tional incapacitation, (4, severe deterioration), the subject illness has become chronic resulting in inabil-ity to maintain employment (outside of a shelteredworkshop) and social impairment, and; (5, relatively stable), the subject illness has not changed signifi-cantly (since it started at a severe level of impair-ment); the longer the size, the worse and poorer is the outcome (Gamma statistic for S, L and L+S alleles respectively: 0.25, p=0.01; 0.37, p= 0.002; 0.29, p=0.002) (Fig. 2).
To evaluate the proportion of variance attrib-utable to the CAG polymorphism in the phenotype respon-sive schizophrenia (as contrasted to the phenotype nor-CA 022160~7 1997-09-19 mal controls), we performed a logistic regression where the S and L alleles were the independent variables.
This analysis indicate that the length of the two alleles contribute 10 % to the variance of this pheno-type.
A sequence homology search was performed using the GCTlOD04 nucleic acid sequence (GenBank acc. no.
G09710) against the non-redundant nucleic acid database (BLASTn, GenBank). The GCTlOD04 sequence was 84%
homologous to a mouse gene (GTl, GenBank D29801, see figure 3) from which is transcribed a 7.2 kb cDNA
encoding a 196 kDa protein of unknown function, sug-gesting that GCTlOD04 represents a portion of the human homologue, which we term hGTl. The murine GTl gene is inducible with retinoic acid in the mouse embryonic carcinoma cell line Pl9 and is expressed at highest levels in neurons but not in glial cells. A sequence homology search using the mGTl protein sequence identi-fied several conserved domains in another mouse gene (stromelysin PDGF responsive element binding protein transcription factor, GenBank U20282) and in its human homologue (ARl, GenBank U19345), suggesting that the hGTl protein may also function as a transcription fac-tor.
Common allelic variants, rather than rare muta-tions, may be responsible for the familial aggregation observed in complex diseases such as schizophrenia.
Allelic variants that are neither necessary nor suffi-cient to cause a disease may not be identified by link-age analysis, particularly when the attributable risk is less than 10%. In contrast, association studies are sensitive to detect such variants.
To identify genes that may confer susceptibil-ity to schizophrenia and/or its phenotypic variability with respect to neuroleptic responsiveness, we CA 022160~7 1997-09-19 recruited patient according to their long term respon-siveness to neuroleptic medication, a strategy that might reduce the putative genetic heterogeneity of schizophrenia. Control and patient groups were strati-fied according to the ethnic background of parents;thus reducing the risk of population stratification bias.
In accordance with the present invention, neu-roleptic-responsive-schizophrenic patients were sig-nificantly more likely to have hGTl gene alleles withshort CAG repeats as compared to patients who are char-acterize by long term poor response to neuroleptics and outcome. Furthermore, a significant correlation between the size of the hGTl CAG repeat and the pattern of severity of the disease (the longer is the CAG
repeat the more severe is the outcome) was identified in the group of schizophrenic patients regardless of the quality of their response to neuroleptic medica-tion.
One major limitation of association studies with a relatively small number of subjects and a poten-tially high number of genes to be tested is an increased risk of false positive findings (type error). In this study, we focused on candidate genes containing expressed and polymorphic CAG repeats, thus markedly reducing the number of genes to be tested;
the number of CAG repeats is thought to be around 700 in the total human genome. Polymorphic CAG repeats containing transcripts might be much less represented.
Based on these numbers, the Bonferroni corrected p-value for our tested hypothesis ought to be between 2X
10 4 and 7x10 5. In our study, and in spite of the small sample sizes, short alleles were likely to be more frequent in responsive schizophrenia compared to controls at a p-value of 9x10 , which is suggestive of CA 022160~7 1997-09-19 a true association in the case of a complex disease such as schizophrenia. Moreover, the fact that the association is detected in an ethnically very homoge-nous subgroup (both parents French Canadians) as well as in a mixed subgroup (at least one parent is non French Canadian~, suggests that this allelic associa-tion is very likely not to be due to stratified popula-tion bias. Furthermore, the fact that hGTl gene has a high homology with a mouse gene involved in neural cell differentiation induced by retinoic acid is consistent with both the neurodevelopmental and retinoic acid hypotheses of schizophrenia.
Patients who presented with episodic shifts and good between-episode recovery were more likely to have shorter CAG repeats in both of their hGTl alleles. This finding could be interpreted in various different ways:
(1) it could indicate that hGTl short alleles have a causative effect in the disease of patients with favor-able outcome (good between episodes recovery, slow pro-gression of functional deficits) whereas resistantpatients with sever pattern of severity (continuos psy-chosis, no psychotic free episodes, rapid decline of psychosocial functioning) have other genetic or envi-ronmental factors involved in their disease. Patients falling between these two levels of severity may be a more mixed group difficult to relate to either one of the two extremes using clinical criteria (heterogeneity hypothesis), (2) it could indicate that the hGTl poly-morphism modulates the pattern of severity of the schizophrenia phenotype but not the susceptibility to schizophrenia per se (modifier gene hypothesis), and (3); hGTl gene could influence susceptibility to schizophrenia irrespective of the pattern of severity and responsiveness to neuroleptics; the weak associa-tion in the group of resistant schizophrenic patients CA 022160~7 1997-09-19 being the result of a selection bias. Indeed, should another gene with a higher attributable risk than the hGTl be acting in the resistant form, the enrichment of hGTl short alleles in non-responsive patients with severe pattern of the disease would be relaxed and the association would be more difficult to identify in this group. In accordance with this hypothesis, family studies have suggested that neuroleptic-delayed response and marked deterioration in the psychosocial functioning are associated with a higher degree of familial aggregation of the disease; suggesting the presence of gene(s) with relatively high penetrance.
Transcription factors are major actors in all neurodevelopmental phases, and might be very important when developmental activity is intensive such as brain fetal development or synaptic pruning occurring in the adolescence phase of human development. They have been implicated in animal complex behavioral traits and have also a major role in the transduction pathways involved in the biological adaptation of the central nervous system to environmental changes (ranging from physical conditions such as viral infections to psychological conditions such as nurturing behavior in mice). It is also of interest to note that all antipsychotic drugs 2 5 modulate DNA transcription in specific areas of the brain and ultimately results in modifications of neu-ronal interconnectivity. Variable number of tandem repeats, including trinucleotide repeats, have been found to be over represented in genes coding for DNA-binding proteins/transcription factors. Such repeatsmay be the basis of a fine modulation of gene activity.
We speculate that one or multiple transcription factors might be involved in the etiology of schizophrenia or its phenotypic variability (including the quality of the response to different drugs). It is therefore of CA 022160~7 1997-09-19 interest to consider transcription factors containing polymorphic CAG repeats as a putative candidate "family of genes" for schizophrenia and other psychiatric disorders thought to be of a neurodevelopmental origin.
The present invention will be more readily un-derstood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
While the invention has been described in con-nection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any varia-tions, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.
PSYCHIATRIC DT~~.S AND THERAPEUTIC USES T~R~OF
R~CK~OUND OF THE INV~NTION
(a) Field of the Invention The invention relates to hGTl gene, a polymor-phic CAG repeat-containing gene and its uses thereof for the diagnosis, prognosis and treatment of psychiat-ric diseases, such as schizophrenia.
(b) DescriPtion of Prior Art Schizophrenia is a chronic brain disorder char-acterized by a behavioral syndrome combining in various degrees hallucinations, delusions, social withdrawal, affective flattening, disorganized behavior and formal thought disorders. It affects up to 1% of the general population and results in a lower level of social and occupational functioning. Many recent studies indicate that schizophrenia may originate from neural cell dis-turbances occurring in the developing/maturing brain.
Genetic factors are known to play a major role in the etiology of this disorder as demonstrated by extensive family, twin and adoption studies. However, the quest for genes conferring susceptibility to schizophrenia has been difficult and has not yielded consistent find-ings using both association and linkage studies. It isthought that these difficulties are in part due to het-erogeneity in etiology, both of genetic and non-genetic origins, resulting in a highly variable phenotype with respect to age at onset, symptom profile, course of illness, response to medication, long term outcome and performance on neuropsychological tests.
One promising avenue to guide research in this search for genes increasing susceptibility to schizo-phrenia may be to distinguish patients on the basis of therapeutic response to neuroleptics. Indeed, while most schizophrenic patients are improved by neuroleptic CA 022160~7 1997-09-19 medication, a substantial number of subjects (15 to 25%) remains severely symptomatic despite multiple and adequate neuroleptic therapeutic attempts. In contrast to this between subject variability, within subject (from one episode to the other) consistency of neu-roleptic response have been reported. Clinical pre-treatment characteristics that correlate with good neu-roleptic response include spontaneous high blink-rate and blink-rate decrease under Haloperidol challenge, absence of spontaneous movement disorders, and absence of dysphoric reaction within 24-48 hours of neuroleptic initiation. On a long term basis, it has been demon-strated that good response to neuroleptics (but not the severity of the symptoms prior to neuroleptic medica-tion) in the early stages of the disease, predicts abetter outcome. Neurophysiological characteristics that correlate with good neuroleptic response include high-frequency waves and few alpha and slow waves in computerized EEG prior to the treatment with neurolep-tics, a specific profile of changes in quantified EEGspectrum under neuroleptics and high degree of elec-trodermal activity prior to neuroleptic treatment. An important number of studies indicate that dopamine neu-rotransmission is disturbed predominantly in the responsive schizophrenic patients. High pre-treatment plasma levels of HVA have been shown to predict good response to neuroleptics in most of the studies. Pre-liminary genetic epidemiological data indicate that poor or delayed response to neuroleptic treatment is associated with an increased prevalence of schizophre-nia spectrum disorders in relatives of schizophrenic probands. These convergent lines of evidence suggest that long term response to neuroleptic medication may be considered as a bioclinical dimension with an etiologically relevant significance; the two extremes -CA 022160~7 1997-09-19 of this dimension being occupied by two groups of schizophrenic patients, at least partially, distinct with respect to the pathogeny of their illness.
It would be highly desirable to be provided with a tool for the diagnosis, prognosis and treatment of psychiatric diseases, such as schizophrenia.
SUMMARY OF THE INVENTION
One aim of the present invention is to provide a tool for the diagnosis, prognosis and treatment of psychiatric diseases, such as schizophrenia.
Another aim of the present invention is to detect association between allelic variants of CAG
repeat-containing genes and schizophrenia or its pheno-typic variability with respect to long term response toneuroleptic medication.
In accordance with the present invention, we compared the allelic frequencies of various polymorphic candidate genes between two groups of schizophrenic patients carefully screened on the basis of their long term response to typical neuroleptics (excellent responders, Rs; non-responders, NRs) and controls.
This report summarizes our finding while considering CAG containing genes as candidates for schizophrenia.
This family of candidate genes was deemed attractive for the following reasons: (1) CAG repeat instability was associated with several neurodegenerative brain diseases that display genetic anticipation, a feature believed to be present in schizophrenia, (Z) some iso-lated, though promising, reports indicate that expandedCAG repeats are more prevalent in schizophrenic patients compared to normal controls, (3) CAG repeats are often very polymorphic and have been found to be over represented in coding sequences of the human genome particularly those coding for DNA-binding pro-teins/transcription factors. These factors are impor-CA 022160~7 1997-09-19 tant actors in the regulation of the genetic program and neurodevelopmental processes and have been impli-cated in several human neurodevelopmental diseases including one that may present with schizophrenia-like symptoms, and; (4) CAG repeats (or the polyglutamine stretches for which they encode) might modulate the function of the genes (or protein) they are part of suggesting that they might be functional polymorphisms and not silent ones.
In accordance with the present invention there is provided a hGTl gene containing transcribed polymor-phic CAG repeat, which comprises a sequence as set forth in Fig. 3.
The allelic variants of CAG repeat of hGTl gene may be associated with schizophrenia, affective dis-eases such as manic depression, neurodevelopmental brain diseases or with phenotypic variability with respect to long term response to neuroleptic medica-tion.
More precisely, there are 5 allelic variants of CAG repeat which are identified as follows:
Size of PCR amplifiedPredicted No. of CAG repeats SIIOll~al to longest r~ay~"~lS (bp) In accordance with the present invention there is provided a method for the prognosis of severity of schizophrenia of a patient, which comprises the steps of:
a) obtaining a nucleic acid sample of the patient;
and CA 022160~7 1997-09-19 b) determining allelic variants of CAG repeat of the hGTl gene, and wherein long allelic vari-ants are indicative of severe schizophrenia.
The preferred nucleic acid sample used in accordance with the present invention is DNA. For RNA
sample, an additional step is carried out, which con-sists in using a reverse transcriptase to transcribe the RNA into DNA.
More precisely, the allelic variants identified as short or as having between about 171 and 177 bp (referred to as -3, -2 and -1) are associated with mild schizophrenia and long or as having between about 180 and 183 bp (referred to as 0 and 1) are associated with severe schizophrenia.
In accordance with the present invention there is provided a method for the identification of patient responding to neuroleptic medication, which comprises the steps of:
a) obtaining a nucleic acid sample of the patient;
and b) determining allelic variants of CAG repeat of the hGTl gene, and wherein short allelic vari-ants are indicative of neuroleptic response.
More precisely, the allelic variants identified as short or as having between about 171 and 177 bp (referred to as -3, -2 and -1) are associated with patient capable of neuroleptic response and long or as having between about 180 and 183 bp (referred to as 0 and 1) are associated with non-response to neuroleptic medication.
In accordance with the present invention there is provided a non-human mammal model for the hGTl gene, whose germ cells and somatic cells are modified to express at least one allelic variant of the hGTl gene and wherein the allelic variant of the hGTl being CA 022160~7 1997-09-19 introduced into the mammal, or an ancestor of the mam-mal, at an embryonic stage.
In accordance with the present invention there is provided a method for the identification of patient responding to neuroleptic medication, which comprises the steps of:
a) obtaining a nucleic acid sample of the patient;
and b) determining allelic variants of CAG repeat of the hGTl gene, and wherein short allelic vari-ants (from about 171 to about 177 bp) are indicative of neuroleptic response.
In accordance with the present invention there is provided a method for the screening of therapeutic agents for the prevention and/or treatment of schizo-phrenia, which comprises the steps of:
a) administering said therapeutic agents to the non-human mammal of the present invention or schizophrenia patients; and b) evaluating the prevention and/or treatment of development of schizophrenia in said mammal or said patients.
In accordance with the present invention there is provided a method to identify genes part of or interacting with a biochemical pathway affected by hGTl gene, which comprises the steps of:
a) designing probes and/or primers using the hGTl gene of the present invention and screening psychiatric patients samples with said probes and/or primers; and b) evaluating the identified gene role in psychi-atric patients.
In accordance with the present invention there is provided a method of stratifying psychiatric CA 022160~7 1997-09-19 patients based on the allelic variants of the hGTl gene for clinical trials purposes, which comprises:
a) obtaining a nucleic acid sample of the patients; and b) determining allelic variants of CAG repeat of the hGTl gene, wherein patients are stratified with respect to their allelic variants and wherein short allelic variants are indicative of neuroleptic response.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 illustrates the average allelic lengths of the GCTlOD04 EST CAG repeat in controls, responsive (R) and non-responsive (NR) patients, showing the shorter (S) allele only, longer (L) allele only and the sum (L+S) of the two alleles in the three groups of subjects;
Fig. 2 illustrates the correlation between the average length of the (CAG)n polymer of the short (a), the long (b) alleles and the sum of 2 alleles (C) and seventy of schizophrenia in the different classes of severity of the disease; and Fig. 3 illustrates the sequence homology between the human GCTlOD04 sequence and the mouse GTl gene.
DET~TT~n DESCRIPTION OF THE INVENTION
In accordance with the present invention, the main objective was to detect allelic variants of CAG
repeat containing genes associated with schizophrenia or its phenotypic variability with respect to the pres-ence or absence of schizophrenia and long term response to neuroleptic medication.
Accordingly, CAG repeat allelic variants were compared between three groups of subjects: two groups CA 022160~7 1997-09-19 of schizophrenic patients, one neuroleptic-responsive (Rs; n=43) and one -non-responsive (NRs; n=63), and; a group of controls screened out for DMS-IV axis I psy-chiatric disorders (Ci n=87). Assessment of response to conventional neuroleptics was based on a comprehen-sive review of medical files according to a priori defined criteria and blind to genotyping. Genes con-taining polymorphic CAG repeats were identified by means of genetic sequences data base searches.
The results in accordance with the present invention shows that short CAG repeat allelic variants of the hGTl gene were associated with schizophrenia irrespective of neuroleptic response (% short alleles SCZ=45%; C=31%, p=0.005). This association was highly significant in Rs (52%, p=0.0009) and marginal in NRs (40%, p=0.12) groups. A statistically significant cor-relation (Gamma=0.37, p=0.0024) between the CAG repeat length and the overall pattern of severity of schizo-phrenia was also observed.
Surprisingly and in accordance with the present invention, CAG repeat allelic variants of the hGTl gene show strong association with neuroleptic responsive schizophrenia and length correlation with the overall pattern of severity of the disease.
Clinical The study was conducted between 1994 and summer 1997. Patients have been recruited in the Douglas hos-pital (n=82), the Clinique Jeunes Adultes of L.H.
Lafontaine Hospital (n=15) and the Schizophrenia Clinic of the Royal Ottawa Hospital (n=9). 333 schizophrenic patients were identified as potential subjects for this study. 123 patients did not meet the criteria for schizophrenia or Rs/NRs (undifferentiated response) diagnoses. 125 and 85 patients met respectively the criteria for NRs-schizophrenia and Rs-schizophrenia. 62 CA 022160~7 1997-09-19 NRs and 42 Rs subjects were not included in the study because refusal or other exclusion criteria.
NRs schizophrenic patients were recruited according to the following criteria: (1) they all met axis I diagnosis of schizophrenia, according to the Diagnostic and Statistical Manual of Mental Disorders, version IV (American Psychiatric association, Diagnos-tic and Statistical Manual of Mental Disorders, APA;
1994) (DSM-IV), (2) they did not experience remission of psychotic symptoms within the past 2 years, (3) in the preceding 5 years, all patients underwent at least 3 periods of treatment with typical neuroleptics, from at least two distinct families of drugs, at therapeutic dosage (equal to or greater than 750 mg Chlorpromazine equivalent in monotherapy or 1000 mg chlorpromazine equivalent, when a combination of neuroleptics is used), for a continuous period of at least 6 weeks at a time, with no significant relief of symptoms, and; (4) Unable to function without supervision in all or nearly all domains of social and vocational activities with a Global Assessment Score (GAS) < 40 within the last 12 months.
Criteria for the selection of neuroleptic Rs patients were as follows: (1) all patients met the cri-teria for schizophrenia according to DSM-IV, (2) all were admitted at least once to a psychiatric care facility because of acute psychotic episode, (3) during all hospitalizations, patients experienced full or par-tial remission in response to treatment with typical neuroleptics, at recommended dosage, within six-eight weeks of continuous treatment; remission being defined as a rapid reduction of schizophrenic symptoms with limited residual symptoms, (4) all patients were able to function with only occasional supervision in all or nearly all domains of social and vocational activities CA 022160~7 1997-09-19 with a GAS score > 60 within the last 12 months, (5) no patients had to be admitted to hospitals because of psychotic exacerbation, if and when compliant to treat-ment and treated continuously with typical neurolep-tics, and; (6) at least one psychotic relapse when neu-roleptic medication is reduced or interrupted. Exclu-sion criteria for schizophrenic patients were brain trauma, any neurological condition, drug or alcohol abuse in the last two years.
All schizophrenic patients were directly inter-viewed by the PI, a research psychiatrist trained in the use of the Diagnostic Interview for Genetic Stud-ies (DIGS) (Nurnberger JI et al., A~chives of General Psychiatry. 1994jSl:849-59) and their medical records were comprehensively reviewed. Complementary informa-tion from the treating physician and nurses in charge of the patients and their close relatives was obtained, whenever possible. A best estimate diagnosis was established on the basis of all the available data.
Responsiveness to typical neuroleptic medication was evaluated according to a 7 point's scale. The severity of symptoms and overall psychosocial functioning were assessed using the following instruments: (1) Brief Psychiatric Rating Scale (BPRS) (Woerner MG et al., Psychopharmacology Bulletin. 1988j24:112-117), (2) the Scale for the Assessment of Negative Symptoms (SANS), the (3) Scale for the Assessment of Positive Symptoms, (4) the GAS, (5) the Pattern of Severity Scale, a 5 point's scale assessing overall course and outcome of the disease (American Psychiatric association, Diagnos-tic and Statistical Manual of Mental Disorders, Fourth Edition, American Psychiatric association, Washington D.C.; 1994), and; (7) the Pattern of Symptoms subtypes, a categorical classification of patients according to the combination and changes over the course of the dis-CA 022160~7 1997-09-19 ease of positive and negative symptoms (Nurnberger JI
et al., Archives of General Psychiatry. 1994;51:849-59). All these evaluation tools, except the BPRS are part of the DIGS.
The control group (C) was made by healthy vol-unteers recruited through advertisement in local papers (n=49) and married-in individuals from a linkage study (n=38). All subjects in this group underwent a struc-tured psychiatric interview in order to exclude those who meet DSM-IV axis I disorders. Subjects recruited through advertisement have also been screened for schizophrenia spectrum disorders and have been tightly matched for ethnic background (mother and father eth-nicity) with schizophrenic patients. All, (except one responsive~, patients and controls were Caucasians.
All of them gave informed and written consent. The research protocol has been approved by the three hospi-tals ethic committee where the research took place.
Genetic methods To identify sequences potentially encoding polymorphic polyglutamine tracts, we conducted a number of Basic Local Alignment Search Tool (BLAST) (Altschul SF et al., Journal of Molecular Biology. 1990;215:403-410) searches using the following sequences: (1) (CAG)30 or (CAA)30 (BLASTn, unfiltered against the non-redundant nucleic acid and the expressed sequence Tag (dbEST) databases) and, (2) Q30 (BLASTp, unfiltered, against the non-redundant protein database or tBLASTn against dbEST). Sequences containing homopolymer tracts of >7 CAG or CAA repeats or potentially encoding a tract of >12 glutamine residues were used to design PCR primers able to amplify the CAG or CAA repeats.
PCR primers were designed using DNASTAR Inc. (Madison, Wisconsin) software.
CA 022160~7 1997-09-19 Genomic DNA was isolated from peripheral lym-phocytes using standard methods. CAG repeat-containing fragments were amplified by PCR using specific primers for each repeat. PCR was performed in a total volume of 13 ~1 containing 30 ng of human genomic DNA, lOmM
Tris-HCl (pH 8.8), 1.5 mM MgC12, 50mM KCl, 1% Dimethyl-sulfoxide, 250 mM each of dCTP, dGTP, and dTTP, 25 mM
dATP, 1.5 uCi alpha 35S-dATP, 100 ng of each primer, and 3 units of Taq polymerase (Perkin-Elmer). DNA was denatured at 94~C for 5 min., then subjected to 30 cycles of a 1 min. denaturation at 94~C, a 1 min.
annealing at the optimized annealing temperature for each primer pair and a 1 min. elongation at 72~C. This was followed by a final extension at 72~C for 5 min.
PCR products were electrophoresed on denaturing 6% Polyacrylamide gels and visualized by autoradiogra-phy. Absolute allele sizes were estimated according to an M13 sequence ladder. Since differences in absolute allele sizes were in all cases multiples of 3 base pairs, we assumed that variations in allele sizes were due to differences in the number of trinucleotide repeat units in the amplified sequences. By conven-tion, we designated the most common allele as 0, with less common alleles as positive or negative integers according to their number of trinucleotide repeats (e.g. if allele 0 had 20 repeats, allele +2 and -2 would have respectively 22 and 18 repeats).
Analysis:
Each subject was assigned two numeric values which represent respectively the lengths of his short (S) and long (L) alleles. Under the assumption of a quantitative effect of the CAG tract length, data were initially analyzed using a non-parametric analysis of variance (Kruskal-Wallis median statistic) where the independent variable is the diagnostic status (Rs, NRs CA 022160~7 1997-09-19 and C) and the dependent variable is the length of the CAG repeat of S, L or the sum of the two alleles. In the case of a significant overall group effect in the ANOVA, pair-wise contrasts between the different groups were performed using the Mann and Whitney U-statistic.
This approach allows to control for the inflation of type I error secondary to multiple testing.
We also analyzed data by contrasting allelic frequencies in different pairs of groups using the %Z
statistic. Alleles were grouped in different classes in accordance to the pattern of results found in the analysis of variance. Since both patient and control groups include an important number of subjects with a French Canadian ethnic origin, we reanalyzed any asso-ciation finding after stratifying subjects according tothe ethnic origin of their parents (both parents from French Canadian origin vs. at least one parent with non-French Canadian origin). This analysis allows to control for associations resulting from ethnically based differences in allelic frequencies (population stratification) as opposed to those attributable to the pathological condition under study (true association).
When a particular EST showed allelic or size association with schizophrenia and/or responsiveness to medication, further analyses were performed to investi-gate the putative relation linking various clinical dimensions (age at onset, pattern of severity) to the length of the CAG repeat. For this purpose, we used the Gamma correlation statistic, a non parametric statistic recommended when there are many ties in the data set.
Clinical dimension that were used as criteria to define the two groups of patients (GAS, severity of current symptoms, neuroleptic responsiveness scores) were not included in this analysis. Relations between categori-cal variables (schizophrenia subtypes of illness, pat-CA 022160~7 1997-09-19 tern of symptoms) and the CAG alleles were explored by a % statistic with the appropriate degree of freedom.
Logistic regression was used to determine the attribut-able risk conferred by any EST allelic variants which showed a positive association with schizophrenia or neuroleptic responsiveness. All analyses were made using the Statistica software (Statsoft).
Table 1 Demo~raphic and clinical characteristics of patients and controls Non-respongive(62) Responsive (43) Controls (C) (87) Mean age in y~ars +SD (n~ 38J 7 (62) 40i lo (43) 44* 13 (87) Education inyears+ SD (n) 11+ 2 0 (59) 11+ 2 ~ (43) 14+ 3 3 (49) SES of HH+SD (n)- 54+ 24 (53) 51+ 24(41) 59+ 20(49) Sex, % M 74% 67% 45%
Ethnic origin FCIOB 27/35 26117 40147 Subtype, UIPIDIC 271301411 61361110 Mean age at C, in years +SD (n) 181 3 9(55) 24:t 4.8 (43) Illness duration in years +SD (n) 20+ 7 0 (55) 16i 8 8 (43) ~/O time as in-patient~(n) 62% (61)-- 8 2% (43) BPRS total score i SD (n) 49+ 8 9 (53) ~- 241 3 9 (53) NLP response score 1 83 + 0 74 (58) ~ 6 3 ~ 0 67 (43) Patternofseverity 40+00(55)~- 19+05(43) SES of HH indicates socioeconomic status head of house hold; FC/OB, French Canadian/other ethnic background; U, undifferentiated; P, paranoid; D, disorganized; C, catatonic SrLi ~j L .,..ia; C~, first consultation; BPRS. Brief Psychiatric Rating Scale; and, NLP neuroleptic. ~-, p<0 001 Table 1 shows the demographic and clinical characteristics of the three groups of subjects Rs, NRs, and C. The two groups of patients were comparable with respect to age, level of education and socio-economic status of the head of household. As expected,they differed significantly according to the severity of psychosis ~BPRS scores, F=280, p<0.000), the percent of time spent as inpatient since their first contact with the psychiatric institution (F=81, p<0.000) and the age at first contact with psychiatric care facili-ties (F=47, p<O.OOO).
Table 2 shows the sixteen different candidate expressed CAG repeats identified and analyzed and includes mapping, homology, and polymorphism informa-tion.
Table 2~ it of diffelent sLufied ESTs Seq~elD PCR RT-PCR PNQ 1~ ,potentialf~lion PolynK~phic M~pdata T08930 + na 15 1 ',~ ." ab~RNA!' ' _, ' - -CUG~BPmNabS0 and xc~us E~R-I gene R98242 - + 27 1 ' ._,~." acAMP~pQlsivei '.'' ~ ,! geneexE~ssion L37868 + na 21 N-Oct-3TF,P~Ud~mainTF H.
U23868 + na 26 No known l~mology. + HCHI
U23862OS + na 7 pc6sible l ' ~, I . ' - -p~tein(S. cercisiae) N55395 + na 15 b.~n _~ _ ~ ' TF +
L10379 + na 28 noknownlKmology +++
Z78314 + na 20 nol X85326 - notdale 11 noknownb.~logy na T90581 - + 10 nokn~wnbunology L10375 - + 16 no l I ',, +
X82209 + na 27 27Q, l~nnnl gene disnQted byabalanced ++
1~26155 + na 23 SW12/SN~2,awidelangci '.' factor, ++
idaacls wi~ ERand RAreceptors GCT5EII + na 22 noknownb.~nology +++ HCH3 TATABF + na 39 TFllDTATAbo~bir~lingp~teir~gen~
factor GCIIOD04 + na 14 b,amology to a rn~3se retinoic acid ind~ceble gene +++ HCH17 1~ ~ ' PDGFTF, ER, . ~ R~ ~c~n~ic acid ra~r.
Seven of the candidate sequences showed homol-ogy or identity with DNA binding domains or transcrip-tion factors. Most of the candidates (12/16) gave a PCR product with the predicted size. Candidates that amplified a larger than expected fragment or no prod-ucts at all were further analyzed by RT-PCR to control for possible intronic interruptions in the genomic DNA.
Three candidates gave an RT-PCR product of the pre-CA 022160~7 1997-09-19 dicted size; only one was polymorphic using a small sample of chromosomes. Overall, 10/16 candidate sequences contained a polymorphic CAG repeat. Allelic frequencies of these polymorphic CAG repeats were com-pared in the four groups of subjects.
Only allelic variants of the GCTlOD04 locus (primers; SCZ15:GGGGCAGCGGGTCCAGAATCTTC, SCZ16: TGGCCTTGCTGCCCGTAGTGCT; annealing temperature 62~C) showed an overall significant group effect for the L allele (Kruskal-Wallis H (2, N = 194) = 12.18, p = .002)i the CAG repeat average length being the shortest in the neuroleptic-responders (Rs), intermediate in the non-responders (NRs) and longest in the control group (C) (Fig. 1).
The reference point to measure the CAG repeat length is the most common allele (180 bp fragment or 14 predicted repeats), which is taken as 0. Alleles with n repeats above or below the 0 allele are scored +n or -n. C indicates the control groupi Rs, neuroleptic responsive schizophrenic patients group; and NRs, neu-roleptic non-responsive schizophrenic patients group.
A similar trend was observed for the S allele (Kruskal-Wallis H (2, N= 194) = 5.32, p =0.06). Post-hoc analysis using the U-statistic showed that this global effect was mainly due to the difference between neuroleptic-responders and normal controls (C) (L
allele: adjusted-Z=-3.52, p=0.0004; S allele: adjusted-Z= -2.28, p=0.02). Resistant schizophrenic patients showed also a trend toward smaller CAG repeat average size of the L allele compared to controls (C) (adjusted-Z=-1,68, p=0.09). When we analyzed the sum of the two alleles, the three groups were statistically different (p=0.01) and the difference between controls and Rs was significant at the level of p=0.004 (adjusted-z=-2.8). Further analysis were carried out, testing the hypothesis that short alleles of the GCTlOD04 were more frequent in schizophrenic patients.
For that purpose, two distinct classes of alleles, long (O,l) and short (-3, -2, -1), were defined and allelic frequencies between the four groups were reexamined (Table 3).
Table 3: frequencies of the CAG Allele short variants of hGT 1 gene p~ ~ ~ patientS
Controls SCZ Rs NRs Number(2n) 174 212 86 126 % of short alleles 31% 45% 52% 40%
%2 p=0.005 p=0.0009 1'=0.12 Both parents are French C' Nurnber (2n) 80 106 52 54 % of short alleles 35% 47% 54% 41%
%2 p=O O9 P=0.03 P=0.5 at least one par0t is non French C - ) Number (2n) 94 106 34 72 % of short alleles 28% 42% 50% 39%
%2 1'=0.03 P=0.018 1~0.12 Allelic r~ ;;C;, are given as percent of alleles shorter than 0 (<0) . Fre~pl~.n~ i~s are analyzed according to different diagnosis groups and ethnic l~c~uulld of parents. All rl~u~ were c~ ast~Al with the frequencies of the alleles shorter than 0 in the control group. SCZ indicates ~l~h~ c patients; Rs, neuroleptic~ ol~ive schizophrenic patients; NRs, n~ ~ non-l~ol~iv~ GO~JIII~ patients, and; x2, Chi 2 statistic with I degree of freedom Schizophrenic patients, irrespective of their neuroleptic response status were more likely to carry one of the short alleles compared to controls (%2=7.6, df=l, p=0.005). This difference was mainly due to Rs schizophrenic patients who were significantly more CA 022160~7 1997-09-19 likely to have small alleles compared to controls (% =11.0, df=l; p=0.0009) and to NRs patients (% =3.30, df=l, p=0.07). Neuroleptic-non responders were margin-ally different from controls (X =2.41, df=l, p=0.12).
When subjects with both parents of French Canadian ori-gin or those with at least one parent from non French Canadian origin were analyzed separately, the same pat-tern emerged (Rs vs. C: %2=4.6, df=l, p=0.03i schizo-phrenics vs. C: % =2.7, df=l, p=0.09).
Finally, a correlation analysis indicated that the size of the CAG repeat tract is linearly related to the pattern of severity of schizophrenia (measured blindly to genotype, using a 1-5 scoring system defined as follows: (1, episodic shift) episodes of illness interspersed between periods of health or near normal-ity, (2, mild deterioration) periods of illness occur, but there are periods of return to near normality, with some ability to work at a job and near normal or normal social functioning, (3, moderate deterioration), the subject may occasionally experience some resolution of symptoms, but overall the course is downhill culminat-ing in a relatively sever degree of social and occupa-tional incapacitation, (4, severe deterioration), the subject illness has become chronic resulting in inabil-ity to maintain employment (outside of a shelteredworkshop) and social impairment, and; (5, relatively stable), the subject illness has not changed signifi-cantly (since it started at a severe level of impair-ment); the longer the size, the worse and poorer is the outcome (Gamma statistic for S, L and L+S alleles respectively: 0.25, p=0.01; 0.37, p= 0.002; 0.29, p=0.002) (Fig. 2).
To evaluate the proportion of variance attrib-utable to the CAG polymorphism in the phenotype respon-sive schizophrenia (as contrasted to the phenotype nor-CA 022160~7 1997-09-19 mal controls), we performed a logistic regression where the S and L alleles were the independent variables.
This analysis indicate that the length of the two alleles contribute 10 % to the variance of this pheno-type.
A sequence homology search was performed using the GCTlOD04 nucleic acid sequence (GenBank acc. no.
G09710) against the non-redundant nucleic acid database (BLASTn, GenBank). The GCTlOD04 sequence was 84%
homologous to a mouse gene (GTl, GenBank D29801, see figure 3) from which is transcribed a 7.2 kb cDNA
encoding a 196 kDa protein of unknown function, sug-gesting that GCTlOD04 represents a portion of the human homologue, which we term hGTl. The murine GTl gene is inducible with retinoic acid in the mouse embryonic carcinoma cell line Pl9 and is expressed at highest levels in neurons but not in glial cells. A sequence homology search using the mGTl protein sequence identi-fied several conserved domains in another mouse gene (stromelysin PDGF responsive element binding protein transcription factor, GenBank U20282) and in its human homologue (ARl, GenBank U19345), suggesting that the hGTl protein may also function as a transcription fac-tor.
Common allelic variants, rather than rare muta-tions, may be responsible for the familial aggregation observed in complex diseases such as schizophrenia.
Allelic variants that are neither necessary nor suffi-cient to cause a disease may not be identified by link-age analysis, particularly when the attributable risk is less than 10%. In contrast, association studies are sensitive to detect such variants.
To identify genes that may confer susceptibil-ity to schizophrenia and/or its phenotypic variability with respect to neuroleptic responsiveness, we CA 022160~7 1997-09-19 recruited patient according to their long term respon-siveness to neuroleptic medication, a strategy that might reduce the putative genetic heterogeneity of schizophrenia. Control and patient groups were strati-fied according to the ethnic background of parents;thus reducing the risk of population stratification bias.
In accordance with the present invention, neu-roleptic-responsive-schizophrenic patients were sig-nificantly more likely to have hGTl gene alleles withshort CAG repeats as compared to patients who are char-acterize by long term poor response to neuroleptics and outcome. Furthermore, a significant correlation between the size of the hGTl CAG repeat and the pattern of severity of the disease (the longer is the CAG
repeat the more severe is the outcome) was identified in the group of schizophrenic patients regardless of the quality of their response to neuroleptic medica-tion.
One major limitation of association studies with a relatively small number of subjects and a poten-tially high number of genes to be tested is an increased risk of false positive findings (type error). In this study, we focused on candidate genes containing expressed and polymorphic CAG repeats, thus markedly reducing the number of genes to be tested;
the number of CAG repeats is thought to be around 700 in the total human genome. Polymorphic CAG repeats containing transcripts might be much less represented.
Based on these numbers, the Bonferroni corrected p-value for our tested hypothesis ought to be between 2X
10 4 and 7x10 5. In our study, and in spite of the small sample sizes, short alleles were likely to be more frequent in responsive schizophrenia compared to controls at a p-value of 9x10 , which is suggestive of CA 022160~7 1997-09-19 a true association in the case of a complex disease such as schizophrenia. Moreover, the fact that the association is detected in an ethnically very homoge-nous subgroup (both parents French Canadians) as well as in a mixed subgroup (at least one parent is non French Canadian~, suggests that this allelic associa-tion is very likely not to be due to stratified popula-tion bias. Furthermore, the fact that hGTl gene has a high homology with a mouse gene involved in neural cell differentiation induced by retinoic acid is consistent with both the neurodevelopmental and retinoic acid hypotheses of schizophrenia.
Patients who presented with episodic shifts and good between-episode recovery were more likely to have shorter CAG repeats in both of their hGTl alleles. This finding could be interpreted in various different ways:
(1) it could indicate that hGTl short alleles have a causative effect in the disease of patients with favor-able outcome (good between episodes recovery, slow pro-gression of functional deficits) whereas resistantpatients with sever pattern of severity (continuos psy-chosis, no psychotic free episodes, rapid decline of psychosocial functioning) have other genetic or envi-ronmental factors involved in their disease. Patients falling between these two levels of severity may be a more mixed group difficult to relate to either one of the two extremes using clinical criteria (heterogeneity hypothesis), (2) it could indicate that the hGTl poly-morphism modulates the pattern of severity of the schizophrenia phenotype but not the susceptibility to schizophrenia per se (modifier gene hypothesis), and (3); hGTl gene could influence susceptibility to schizophrenia irrespective of the pattern of severity and responsiveness to neuroleptics; the weak associa-tion in the group of resistant schizophrenic patients CA 022160~7 1997-09-19 being the result of a selection bias. Indeed, should another gene with a higher attributable risk than the hGTl be acting in the resistant form, the enrichment of hGTl short alleles in non-responsive patients with severe pattern of the disease would be relaxed and the association would be more difficult to identify in this group. In accordance with this hypothesis, family studies have suggested that neuroleptic-delayed response and marked deterioration in the psychosocial functioning are associated with a higher degree of familial aggregation of the disease; suggesting the presence of gene(s) with relatively high penetrance.
Transcription factors are major actors in all neurodevelopmental phases, and might be very important when developmental activity is intensive such as brain fetal development or synaptic pruning occurring in the adolescence phase of human development. They have been implicated in animal complex behavioral traits and have also a major role in the transduction pathways involved in the biological adaptation of the central nervous system to environmental changes (ranging from physical conditions such as viral infections to psychological conditions such as nurturing behavior in mice). It is also of interest to note that all antipsychotic drugs 2 5 modulate DNA transcription in specific areas of the brain and ultimately results in modifications of neu-ronal interconnectivity. Variable number of tandem repeats, including trinucleotide repeats, have been found to be over represented in genes coding for DNA-binding proteins/transcription factors. Such repeatsmay be the basis of a fine modulation of gene activity.
We speculate that one or multiple transcription factors might be involved in the etiology of schizophrenia or its phenotypic variability (including the quality of the response to different drugs). It is therefore of CA 022160~7 1997-09-19 interest to consider transcription factors containing polymorphic CAG repeats as a putative candidate "family of genes" for schizophrenia and other psychiatric disorders thought to be of a neurodevelopmental origin.
The present invention will be more readily un-derstood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
While the invention has been described in con-nection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any varia-tions, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.
Claims (10)
1. A hGTl gene containing transcribed polymorphic CAG repeat, which comprises a sequence as set forth in Fig. 3.
2. The gene of claim 1, wherein allelic variants of CAG repeat are associated with schizophrenia, affective disorders, neurodevelopmental brain diseases or with phenotypic variability with respect to long term response to neuroleptic medication.
3. The gene of claim 2, wherein said affective disorder is manic depression.
4. A method for the prognosis of severity of schizophrenia of a patient, which comprises the steps of:
a) obtaining a nucleic acid sample of said patient; and b) determining allelic variants of CAG repeat of the gene of claim 1, and wherein short allelic variants are indicative of non-severe schizophrenia.
a) obtaining a nucleic acid sample of said patient; and b) determining allelic variants of CAG repeat of the gene of claim 1, and wherein short allelic variants are indicative of non-severe schizophrenia.
5. A method for the identification of patient responding to neuroleptic medication, which comprises the steps of:
a) obtaining a nucleic acid sample of said patient; and b) determining allelic variants of CAG repeat of the gene of claim 1, and wherein short allelic variants are indicative of neuroleptic response
a) obtaining a nucleic acid sample of said patient; and b) determining allelic variants of CAG repeat of the gene of claim 1, and wherein short allelic variants are indicative of neuroleptic response
6. The method of claim 5, wherein said short allelic variants have from about 171 to about 177 bp in length.
7. A non-human mammal model for the hGTl gene of claim 1, whose germ cells and somatic cells are modified to express at least one allelic variant of the hGTl gene and wherein said allelic variant of the hGTl being introduced into the mammal, or an ancestor of the mammal, at an embryonic stage.
8. A method for the screening of therapeutic agents for the prevention and/or treatment of schizophrenia, which comprises the steps of:
a) administering said therapeutic agents to the non-human mammal of claim 7 or schizophrenia patients; and b) evaluating the prevention and/or treatment of development of schizophrenia in said mammal or said patients.
a) administering said therapeutic agents to the non-human mammal of claim 7 or schizophrenia patients; and b) evaluating the prevention and/or treatment of development of schizophrenia in said mammal or said patients.
9. A method to identify genes part of or interacting with a biochemical pathway affected by hGTl gene, which comprises the steps of:
a) designing probes and/or primers using the hGTl gene of claim 1 and screening psychiatric patients samples with said probes and/or primers; and b) evaluating the identified gene role in psychiatric patients.
a) designing probes and/or primers using the hGTl gene of claim 1 and screening psychiatric patients samples with said probes and/or primers; and b) evaluating the identified gene role in psychiatric patients.
10. A method of stratifying psychiatric patients based on the allelic variants of the hGTl gene of claim 1 for clinical trials purposes, which comprises:
a) obtaining a nucleic acid sample of said patients; and b) determining allelic variants of CAG repeat of the gene of claim 1, wherein patients are stratified with respect to their allelic variants and wherein short allelic variants are indicative of neuroleptic response.
a) obtaining a nucleic acid sample of said patients; and b) determining allelic variants of CAG repeat of the gene of claim 1, wherein patients are stratified with respect to their allelic variants and wherein short allelic variants are indicative of neuroleptic response.
Priority Applications (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2216057 CA2216057A1 (en) | 1997-09-19 | 1997-09-19 | Polymorphic cag repeat-containing gene, diagnosis of psychiatric diseases and therapeutic uses thereof |
| AU91495/98A AU757556B2 (en) | 1997-09-19 | 1998-09-18 | Polymorphic CAG repeat-containing gene and uses thereof |
| CA002303106A CA2303106A1 (en) | 1997-09-19 | 1998-09-18 | Polymorphic cag repeat-containing gene and uses thereof |
| EP98943607A EP1015574A1 (en) | 1997-09-19 | 1998-09-18 | Polymorphic cag repeat-containing gene and uses thereof |
| PCT/CA1998/000884 WO1999015639A1 (en) | 1997-09-19 | 1998-09-18 | Polymorphic cag repeat-containing gene and uses thereof |
| BR9812254-1A BR9812254A (en) | 1997-09-19 | 1998-09-18 | Polymorphic cag gene of contained repetition and its uses |
| JP2000512932A JP2001517432A (en) | 1997-09-19 | 1998-09-18 | Genes containing polymorphic CAG repeats and uses thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2216057 CA2216057A1 (en) | 1997-09-19 | 1997-09-19 | Polymorphic cag repeat-containing gene, diagnosis of psychiatric diseases and therapeutic uses thereof |
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| CA2216057A1 true CA2216057A1 (en) | 1999-03-19 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2216057 Abandoned CA2216057A1 (en) | 1997-09-19 | 1997-09-19 | Polymorphic cag repeat-containing gene, diagnosis of psychiatric diseases and therapeutic uses thereof |
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| Country | Link |
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| EP (1) | EP1015574A1 (en) |
| JP (1) | JP2001517432A (en) |
| AU (1) | AU757556B2 (en) |
| BR (1) | BR9812254A (en) |
| CA (1) | CA2216057A1 (en) |
| WO (1) | WO1999015639A1 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19835910C1 (en) * | 1998-08-07 | 2000-05-04 | Deutsches Krebsforsch | Gene isolated on the short arm of human chromosome 17 |
| WO2002020850A2 (en) | 2000-09-08 | 2002-03-14 | Iowa State Universtiy Research Foundation, Inc. | Novel prkag3 alleles and use of the same as genetic markers for reproductive and meat quality traits |
| GB0228079D0 (en) * | 2002-12-02 | 2003-01-08 | Laxdale Ltd | Huntington's Disease |
| WO2008032056A2 (en) * | 2006-09-16 | 2008-03-20 | Genophrenix Limited | Dopamine receptor interacting proteins as markers for disease |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5834183A (en) * | 1993-06-29 | 1998-11-10 | Regents Of The University Of Minnesota | Gene sequence for spinocerebellar ataxia type 1 and method for diagnosis |
| WO1997018825A1 (en) * | 1995-11-17 | 1997-05-29 | The University Of British Columbia | PROTEIN WHICH INTERACTS WITH THE HUNTINGTON'S DISEASE GENE PRODUCT, cDNA CODING THEREFOR, AND ANTIBODIES THERETO |
-
1997
- 1997-09-19 CA CA 2216057 patent/CA2216057A1/en not_active Abandoned
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1998
- 1998-09-18 BR BR9812254-1A patent/BR9812254A/en not_active Application Discontinuation
- 1998-09-18 JP JP2000512932A patent/JP2001517432A/en active Pending
- 1998-09-18 AU AU91495/98A patent/AU757556B2/en not_active Ceased
- 1998-09-18 EP EP98943607A patent/EP1015574A1/en not_active Withdrawn
- 1998-09-18 WO PCT/CA1998/000884 patent/WO1999015639A1/en active IP Right Grant
Also Published As
| Publication number | Publication date |
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| BR9812254A (en) | 2002-07-09 |
| JP2001517432A (en) | 2001-10-09 |
| WO1999015639A1 (en) | 1999-04-01 |
| EP1015574A1 (en) | 2000-07-05 |
| AU757556B2 (en) | 2003-02-27 |
| AU9149598A (en) | 1999-04-12 |
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