Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-12-22
2001-04-03
Sisson, Bradley L. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S270000, C204S456000, C436S094000
Reexamination Certificate
active
06210879
ABSTRACT:
The present invention relates to a method for diagnosing schizophrenia. It relates, more particularly, to a process for detecting variations in a repeated DNA sequence which is present in the TH gene, certain forms of which appear specifically in schizophrenic patients. The invention also relates to primers which can be used for detecting specific alleles of this repeated sequence, which alleles are associated with schizophrenia.
The presence of mutations in all classes of repeated DNA sequences is a known phenomenon. However, the functional significance of these mutations is unknown. Thus, microsatellites represent an abundant class of repeated DNA sequences which exhibit a high degree of polymorphism linked to variations in the number of repeated motifs (ref. 1) and/or in the sequence of these motifs. For this reason, these microsatellites have been used as genetic markers for constructing genetic maps and for identifying loci which are involved in pathologies (ref. 2). The size of the different alleles of a microsatellite depends on variations in the number of repeated motifs. Thus, sequencing experiments carried out on repeated dimers have demonstrated a variation in the number of repeated motifs and in their sequence. These variations can correspond, in particular, to “perfect” repeats, that is to say without interruption in the base sequence, or to “imperfect” repeats, which contain one or more interruptions in the sequences of the motifs, which interruption(s) can be (a) deletion(s) or (an) insertion(s) (ref. 3). In the same way, variations in length and/or sequence have also been observed in the repeated trimeric or tetrameric motifs. Thus, variations of this type have been observed in the HUMHPRTB (ref. 4) and HUMTH01 (ref. 5) microsatellites.
The Applicant has been interested, more particularly, in searching for genetic alterations which are linked to schizophrenia. To this end, the Applicant has carried out a study of the association between the gene for tyrosine hydroxylase (TH) and schizophrenia, which study has been orientated more particularly towards the HUMTH01 microsatellite. TH is the limiting enzyme in the pathway for biosynthesizing catecholamines. Various genetic studies of psychiatric and neurological pathologies have been carried out using markers which are located in the TH gene (refs 6 and 7). However, there has so far been no demonstration in the literature of a genetic association with schizophrenia.
The HUMTH01 microsatellite is located in the first intron of the tyrosine hydroxylase gene. This microsatellite consists of repeated tetrameric TCAT motifs. It exhibits a certain degree of polymorphism, with different alleles possessing variable numbers of repeated motifs having been described. The allele which is most frequently encountered comprises 10 repeated motifs and a deletion of one base pair in the fifth repeated motif, which motif has the sequence CAT.
The Applicant has consequently examined the involvement of the TH gene in schizophrenia by looking for the presence of sequence and length variations in the HUMTH01 microsatellite. The results which were obtained showed that the perfect allele was very rare and was only present in schizophrenic patients. These results were replicated in patient populations of different ethnic origin. Thus, 6 different alleles, designated A, B, C, D, Ei and Ep, were detected in a French population of 239 subjects, 94 of which were suffering from schizophrenia. These different alleles differ from each other by 4 base pairs (1 complete motif), with the exception of the two longest alleles, which differ by one single base pair. Sequencing these different alleles demonstrated that the repeated motif of the HUMTH01 microsatellite, having the sequence TCAT, is perfectly repeated in the A, B, C and D alleles, which respectively contain 6, 7, 8 and 9 repeated motifs. On the other hand, the E allele, which comprises 10 repeat motifs, exhibits the same deletion of one thymidine in the fifth repeated motif in the majority of cases. This deletion results in an imperfect allele, which has the sequence (TCAT)4(CAT)(TCAT)5 and is designated Ei (for imperfect). The Ei allele is the allele which is most frequently found in the Caucasian population (ref. 5). By contrast, the perfect E allele, having the sequence (TCAT)10 (designated Ep), is very rare. Thus, of the entire schizophrenic population which was tested, only 5 patients possessed the perfect allele. The results which were obtained unexpectedly demonstrate that all the subjects harbouring the Ep allele are schizophrenic patients, whereas this allele is not present in 145 healthy control subjects (cf. Table 1). These results demonstrate a highly significant association between the presence of the Ep allele and schizophrenia. Furthermore, the 5 schizophrenic patients harbouring the Ep allele are sporadic cases of schizophrenia whose clinical subtype is a paranoid schizophrenia in one case, an undifferentiated schizophrenia in 3 cases and a disorganized schizophrenia in 1 case.
The Applicant then extended this study to another population of different ethnic origin. Thus, a similar association study was undertaken on a population of 88 Tunisians. The results obtained demonstrate that the frequency of the different A-E alleles encountered in the Tunisian population tested is significantly different from that observed in the French population (cf. Table 1). Nevertheless, only 4 individuals proved to harbour the perfect Ep allele, with all these individuals being schizophrenic patients (1 paranoid schizophrenia and 3 undifferentiated schizophrenias); the schizophrenic forms were sporadic forms in this instance as well.
These results constitute the first demonstration of an association between TH and schizophrenia. They clearly demonstrate that the perfect Ep allele of the HUMTH01 microsatellite, having the sequence (TCAT)10, can be significantly associated with schizophrenia and for this reason represents a genetic tool for screening for this type of pathology.
The specific nature of the association of this allele with schizophrenia has furthermore been confirmed by a study on a population of 100 patients suffering from sporadic manic-depressive psychosis. The Ep allele was not found to be present in any of these patients.
The demonstration of the association between this allele and schizophrenia offers a large number of applications in the diagnostic and therapeutic fields. Thus, the present invention now offers, for the first time, the possibility of diagnosing schizophrenia by means of a biological test and no longer exclusively by means of clinical reasoning. One part of the subject-matter of the present invention is, more particularly, a method for diagnosing schizophrenia, which method consists in detecting the presence of the Ep allele of the HUMTH01 microsatellite in the TH gene. This method can also be applied to diagnosing pathologies of the schizophrenia spectrum, such as, in particular, schizotypy, schizoid individuals, etc. The invention thus makes it possible to refine the criteria for diagnosing these pathologies, which criteria are currently solely of a clinical nature. Furthermore, the invention also makes it possible to demonstrate predispositions to this type of psychiatric disorder by means of identifying a genetic vulnerability in the families of patients which harbour this Ep allele. From a therapeutic point of view, the invention advantageously makes it possible to define medical treatments which are more appropriate in terms of the type of schizophrenia. By confirming the hypothesis of an involvement of the catecholamine pathway, the invention makes it possible to use therapies which are more targeted. Furthermore, even if the functional implications of the presence of the Ep allele have not been definitely established, it is to be noted that the HUMTH01 microsatellite is located in the first intron of the TH gene, that is the region in which alternative splicing, leading to 4 isoforms of TH, has been demonstrated (ref. 8). It is therefore possible that
Laurent Claudine
Mallet Jacques
Meloni Rolando
Rhone-Poulenc Rorer S.A.
Sisson Bradley L.
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