Identification et caractérisation de gènes impliqués dans l'infertilité masculine

Près de 15% des couples sont confrontés à des problèmes d'infertilité. Dans près de la moitié des cas, une composante masculine est retrouvée, avec souvent une anomalie des paramètres du spermogramme montrant une diminution de la qualité du sperme. L'étiologie de la grande majorité des infertilités masculines reste inconnue et une origine génétique est probablement responsable d'une proportion importante des troubles de la spermatogénèse. Ce travail comporte deux parties: dans la 1ère partie, l'analyse d'une large cohorte de patients (n=87), nous a permis d'identifier deux nouvelles mutations du gène AURKC. La mutation [c.36-2A>G] a été identifiée uniquement à l'état hétérozygote chez deux frères et le 2ème variant identifié [p.Y248*]: est une mutation récurrente retrouvée chez 11 patients non apparenté d'origine maghrébine et européenne. La 2ème partie de notre étude a été réalisée sur 20 patients infertiles présentant un phénotype homogène d'anomalies flagellaire de type flagelles courts, absents et de calibre irrégulier associé a une asthénozoospermie. Nous avons appliqué la stratégie d'homozygotie par filiation qui a permis de mettre en évidence deux régions d'homozygoties communes: la 1ère région, située sur le chromosome 3, est commune à 9/20 patients et la 2ème sur le chromosome 20 commune à 13/20 patients. Trois gènes candidats présents dans ces régions ont été sélectionnés : les gènes KIF9, SPAG4 et DNAH1. Le séquençage du gène DNAH1 a permis de mettre en évidence des mutations de type faux-sens [c.3877G>A], run-on [c.12796 T>C] et d'épissage [c.5094+1G>A] [c.11958-1G>A]. L'absence de la protéine DNAH1 a pu être mise en évidence par immunomarquage sur les spermatozoïdes d'un patients porteur de la mutation [c.11958-1G>A] et confirme la dégradation du transcrit muté par NMD également observé. Les analyses par microscopie électronique sur les spermatozoïdes d'un patient de la cohorte ont permis de mettre en évidence des anomalies de la structure de l'axonème. Cette étude précise le diagnostic d'infertilité masculine et élargit les connaissances sur les gènes impliquées dans la spermatogenèse.About 15% of couples are confronted with infertility problems. In half of the cases, a male factor component is found, often with abnormal semen parameters. The etiology of the large majority of male infertility remains unknown and genetic origin is probably responsible of a significant proportion of spermatogenesis disorders. This work comprises two parts: in the first part, the analysis of a large cohort of patients (n = 87), allowed us to identify two new mutations in AURKC gene. A splice site mutation [c.36-2A> G] was identified in only two brothers and the second variant identified [p.Y248*] is a recurrent mutation found in 11 unrelated patients. The second part of our study was carried out on 20 infertile patients with flagellar abnormalities associated with asthenozoospermia. We have applied the strategy of homozygosity by descent who has bring out two regions of homozygosity: the first region, located on chromosome 3, is common for 9/20 patients and the second one, located on chromosome 20, is common for 13/20 patients. Three candidate genes present in these regions were selected: KIF9, SPAG4 and DNAH1. Sequencing of DNAH1 gene has bring out three type of mutations: missense mutation [c.3877G> A], run-on mutation [c.12796 T> C] and splice site mutation [c.5094 +1 G> A] [c.11958-1G> A]. The absence of dnah1 protein has been shown by immunostaining of spermatozoa of a patient carrier the mutation [c.11958-1G> A] and confirms the degradation of the mutated transcript by NMD. An electron microscopic analysis of spermatozoa of one patient of the cohort reveals axoneme abnormalities. This study clarifies the diagnosis of male infertility and broadens the knowledge of the genes involved in spermatogenesis.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Chromosomal abnormalities in 163 Tunisian couples with recurrent miscarriages

Recurrent miscarriage (RM) is defined as three or more consecutive pregnancy losses before 24 weeks of gestation. Parental chromosomal abnormalities represent an important etiology of RM. The aim of the present study was to identify the distribution of chromosome abnormalities among Tunisian couples with RM referred to the Department of Cytogenetic at the Pasteur Institute of Tunis (Tunisia) during the last five years. Standard cytogenetic analysis was carried out in a total of 163 couples presenting with two or more spontaneous abortions. Karyotypes were analyzed by R-banding. We identified 14 chromosomal abnormalities including autosomal reciprocal translocation, Robertsonian translocation, inversion, mosaic aneuploidy and heteromorphysm. The overall prevalence of chromosomal abnormalities was 8.5% in our cohort. This finding underlies the importance of cytogenetic investigations in the routine management of RM

FANCA Gene Mutations in North African Fanconi Anemia Patients

Populations in North Africa (NA) are characterized by a high rate of consanguinity. Consequently, the proportion of founder mutations might be higher than expected and could be a major cause for the high prevalence of recessive genetic disorders like Fanconi anemia (FA). We report clinical, cytogenetic, and molecular characterization of FANCA in 29 North African FA patients from Tunisia, Libya, and Algeria. Cytogenetic tests revealed high rates of spontaneous chromosome breakages for all patients except two of them. FANCA molecular analysis was performed using three different molecular approaches which allowed us to identify causal mutations as homozygous or compound heterozygous forms. It included a nonsense mutation (c.2749C > T; p.Arg917Ter), one reported missense mutation (c.1304G > A; p.Arg435His), a novel missense variant (c.1258G > A; p.Asp409Glu), and the FANCA most common reported mutation (c.3788_3790delTCT; p.Phe1263del). Furthermore, three founder mutations were identified in 86.7% of the 22 Tunisian patients: (1) a deletion of exon 15, in 36.4% patients (8/22); (2), a deletion of exons 4 and 5 in 23% (5/22) and (3) an intronic mutation c.2222 + 166G > A, in 27.3% (6/22). Despite the relatively small number of patients studied, our results depict the mutational landscape of FA among NA populations and it should be taken into consideration for appropriate genetic counseling

Identification of a Novel Mutation in FOXL2 Gene That Leads to Blepharophimosis Ptosis Epicanthus Inversus and Telecanthus Syndrome in a Tunisian Consanguineous Family

International audienceMutations in FOXL2 gene are responsible for blepharophimosis ptosis epicanthus inversus and telecanthus syndrome (BPES). The BPES syndrome is a rare autosomal dominant genetic disease characterized by eyelid malformations associated with premature ovarian failure (BPES type I) or not (BPES type II). The human FOXL2 protein (376 aa) contains a 100 amino-acid DNA-binding forkhead domain (residues 52-152) and a polyalanine tract (residues 221-234). In the present study, we report the molecular investigation of four affected members with BPES syndrome in a Tunisian consanguineous family. To identify the causative mutation, we performed a direct sequencing of the FOXL2 gene. The sequence analysis of the coding exon revealed a novel frameshift mutation g.1113 dup C, c.876 dup C, p.P292 Fs. The mutation is located downstream of the polyalanine tract and causes the protein extension to 532 aa. This study reports for the first time a novel frameshift mutation in two-generation consanguineous Tunisian family with BPES. Our results expand the spectrum of FOXL2 mutations

Fluorescent In Situ Hybridization, Psychological, and Psychiatric Studies in Children With Supravalvular Aortic Stenosis

International audienceObjective. To estimate the frequency and investigate the clinical features of 7q11.23 microdeletion in unselected patients with supravalvular aortic stenosis, a total of 7 patients originating from the south of Tunisia were evaluated prospectively by molecular cytogenetic studies. Methods. The clinical analysis was performed according to a specific clinical protocol for the diagnosis of congenital cardiovascular malformations. Cytogenetic analysis with RHG banding was used to detect chromosome rearrangements. Cytogenetic molecular analysis was undertaken using one probe: LSI Williams-Beuren Syndrome (WBS) region probe D7S486/D7S522. For the 3 patients carrying a 7q11.2 microdeletion, psychological and psychiatric tests were performed. Results.-All patients had normal karyotype 46,XX or 46,XY. Three patients were found to have a 7q11.2 deletion, whereas all of them had clinically typical WBS features. Conclusions: The clinical observation noted in this study emphasizes the need for more detailed phenotypic studies in patients and their families, We have seen a wide range of phenotypes associated with a deletion at the elastin locus in this series

Downregulation of miR-451 in Tunisian chronic myeloid leukemia patients: potential implication in imatinib resistance

International audienceObjectives: Resistance to imatinib has been recognized as a major challenge for the treatment of chronic myeloid leukemia (CML). Aberrant expression of miR-451 has been reported to participate in anticancer drug resistance. However, the role of miR-451 in imatinib resistance has not been investigated. The present study was undertaken to determine the expression of miR-451 in order to find a possible association between the expression of this miRNA and imatinib resistance in Tunisian CML patients.Methods: First, real-time RT-PCR was performed to identify the expression of miR-451 in peripheral leukocytes of 59 CML patients treated with imatinib. Then, bioinformatics analysis was carried out to understand the regulatory roles of miR-451 in imatinib-resistant process.Results: Downregulated miR-451 was observed in imatinib-resistant CML cases. In silico analysis identified MYC as a potential target of miR-451. We further revealed the existence of an MYC-binding site in MiR-451 promoter region. On the other hand, increased level of MYC was detected in imatinib-resistant CML cases which may explain the causative role of MYC in CML cases and the downregulation of miR-451.Conclusion and discussion: Taken together, our findings suggest that miR-451 and MYC form together a regulatory loop which may act as a potential therapeutic target, and disruption of suggested regulatory loop could help to improve CML therapy