Germ cell development and maintenance:
Approximately two percent of human males are infertile due to severe defects in sperm production. In 50-60 % of individuals the etiology is unknown. A number of studies have suggested that sperm counts in certain European countries may be declining, the reasons for this decline are unclear but are associated with an increase in other urogenital anomalies such as hypospadias and cryptorchidism. In Paris sperm counts have been declining by two percent per year since the mid 1970's. The genetics of male infertility is complex. Infertile couples have fewer sibs compared with fertile control groups and male infertility shows a distinct pattern of familial aggregation, suggesting that non-Mendelian multifactorial inheritance could play a significant role. Approximately 15% of men with unexplained azoospermia or severe oligozoospermia have microdeletions of the Y chromosome that remove genes that are involved in male germ cell development and maintenance. Despite the recent interest in Y chromosome microdeletions and infertility, there remains a large group of men with unexplained infertility. Premature ovarian failure (POF) has an incidence of approximately 1 in 100. Although immunological disorders are frequently associated with POF, the etiology is unknown in approximately 60 % of cases. Remarkably this relatively common phenotype has received very little genetic investigation. Recent studies suggest that between 30-50% of all POF cases may be familial suggesting an important genetic contribution to the phenotype.
(I) Identify factors involved in human sex determination and differentiation
Active recruitment of familial cases of pathologies associated with human sex determination (XY gonadal dysgenesis, XX ovarian dysgenesis and XX males) is underway. Already this project has resulted in the recruitment of >90% of all published familial cases. Additional efforts to recruit families, particularly in the Indian subcontinent are underway (in collaboration with several pediatric centres, both nationally and internationally). Linkage analysis has been performed in some cases and novel loci implicated in human sex determination have been identified.
-Identify novel sex determining genes by the detailed analysis of chromosomal breakpoints associated with sex reversal (eg 9p)
-Identify novel sex determining/differentiation genes by sequence homology and database mining.
-Identify protein partners of SRY by Far-Western blotting techniques. Several candidates have been identified and are under characterisation
-Comparative Genome Hybridisation has identified a number of novel loci associated with anomalies of gonadal development. These are currently being characterised.
(II) Identify factors necessary for human germ cell development and maintenance
A large scale effort has been made to identify familial cases of male and female infertility.
- Genes involved in mouse premature ovarian failure are being identified in the human and their contribution to ovarian dysgenesis and POF evaluated.
- Specific cases of infertility are under study for mutations in meiotic genes. This study has already lead to a sequence variant identified in cyclin A1 associated with oligozoospermia.
(III) Identify genetic risk factors associated with infertility and gonadal cancers in defined populations/ethnic groups.
This has already been performed on an infertile Danish population and is being extended to include a larger sample size and to include testicular cancer (TC) patients. Epidemiological evidence indicates that the genetics of unexplained infertility and unexplained ambiguous genitalia are non-Mendelian. We have begun to identify some of these factors in one European population.. The aim of this project is to recruit familial cases in France of unexplained ambiguous genitalia. Active recruitment will be for three years, following which, linkage or association studies will be performed depending on the population structure.
Role of sexual dimorphism on brain development and psychiatric diseases
Identification and characterisation of new candidate genes for schizophrenia and autism
Based on whole genome scan results, several regions of the human genome were studied on chromosome 6, X and Y.
On chromosome 6p23, five independent studies have localised a locus for predisposition to schizophrenia between markers D6S274 and D6S285. Within this 2 cM genetic interval, we isolated and characterised two novel genes Autosomal Highly Conserved Protein (AHCP) and KIF13A that we are currently testing for association in patients with schizophrenia.
Autosomal Highly Conserved Protein (AHCP) : At the beginning of this study (1998), AHCP was the first gene identified between D6S274 et D6S285. This gene code for a putative membrane protein, specific to metazoans. We also identified the conserved orthologous genes in C. elegans et D. melanogaster. AHCP does not contain any known motif and its function is still not elucidated. AHCP is ubiquitously expressed in the adult with a testis specific transcript. During mouse development, the expression is restricted to the central nervous system (CNS). There are five unexpressed pseudogenes of AHCP (AHCPp) in the human genome ) each one inserted within a human endogenous retrovirus (HERV). The capture of the AHCP cellular mRNA by ERV occurred during primate evolution and resemble to the transduction of cellular oncogene. After the capture, this retroelement (ERV + AHCP) was transposed in several place of the genome on human chromosome 2, 6p21, 6q16, 11 et Y. Before AHCP, this transduction mechanism has never been observed in the human. The coding regions of the functional gene were tested by SSCP in 73 schizophrenic patients and no association was observed.
KIF13A : We identified this gene within the same genetic interval than AHCP. KIF13A is a new member of the unc-104/KIF1A kinesin family involved in the anterograde transport (from the cell body to the synapses) of cellular organites and synaptic vesicules (cargos). Mutations in the unc-104 from C. elegans results in abnormal behaviour (unc = uncoordinated) and a decrease of synaptic vesicules. During early mouse development, KIF13A expression is restricted to the CNS. Several single nucleotide polymorphisms (SNPs) within KIF13A will be tested in schizophrenic patients.
A genome scan was previously performed and pointed chromosome 6q21 as a candidate region for autism. This region contains the glutamate receptor 6 (GluR6 or GRIK2) gene, a functional candidate for the syndrome. Glutamate is the principal excitatory neurotransmitter in the brain and is directly involved in cognitive functions such as memory and learning. We used two different approaches, the affected sib-pair (ASP) method and the transmission disequilibrium test (TDT), to investigate the linkage and association between GluR6 and autism. The ASP method, conducted on 59 families, showed a significant excess of allele sharing, generating an elevated multipoint maximum LOD score (GENEHUNTER-PLUS NPL= 3.28; P = 0.0005). Using 107 additional families with a single affected child, a significant maternal transmission disequilibrium was observed (TDT linkage P = 0.0004). Furthermore, TDT (with only one affected proband per family) and Haplotype Relative Risk (HRR) analyses showed significant association between GluR6 and autism (TDT association P = 0.008; HRR P = 0.01). In contrast to maternal transmission, paternal transmission of GluR6 alleles was as expected in the absence of linkage, suggesting a maternal effect such as imprinting. Mutation screening was performed in 33 affected individuals, revealing several nucleotide polymorphisms (SNPs), including one amino acid change (M867I) found in 8% of the autistic subjects. This change takes place in a highly conserved domain of the protein and seems to be more maternally transmitted than expected to autistic males (P = 0.007). Presently, we are testing for the imprinting status of GluR6 and in collaboration with C. Mulle at the University of Bordeaux, we are testing the functional consequence of the amino acid change (M867I) on the receptor properties.
Chromosomes X et Y
Due to the gender difference in autism (male to female sex ratio 4:1), sexual chromosomes are also investigated. Several regions of the X and the Y chromosome were associated with autism on the bases of cytogenetic rearrangements. Chromosome Xp22.3 closed to the pseudo autosomal region (PAR) was found deleted in three girls with autism. This region is also the most significant region identified by the whole genome scan of the PARIS study. Several candidate genes are localised within this interval and are currently tested. In order to determine a Y chromosome effect in the predisposition of the male to the syndrome, we analysed the Y chromosome haplogroup frequencies in 107 autistic subjects from France, Sweden and Norway. No significant difference in Y-haplotype distribution between the affected and control groups was observed. Although this study cannot exclude the presence of a Y susceptibility gene, our results are not suggestive of a Y chromosome effect in autism.