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Genetics and Pathophysiology of Morphological Abnormalities of the Sperm Flagellum associated with human male infertility. Morphological Abnormalities of Sperm Flagella: MAS Flagella – MAS Flagella

Genetics and Pathophysiology of Morphological Abnormalities of the Sperm Flagellum associated with human male infertility.

Despite a strong genetic basis, only a handful of genes have so far been formally correlated with male infertility. We intend to sequence the exome of 150 infertile men presenting with asthenozoospermia due to multiple morphological abnormalities of the flagella (MMAF phenotype). We are confident that this project will enable us to identify many additional genes. We believe it will improve our understanding of the molecular mechanisms underlying the biogenesis and the structure of the flagellum.

Identification and characterization of new genetic defects leading to male infertility.

En dépit d’une forte base génétique, seuls quelques gènes ont pu être formellement corrélés à l'infertilité masculine. Nous proposons de réaliser le séquençage exomique de 50 patients présentant une asthénozoospermie sévère causée par des anomalies morphologiques multiples des flagelles. La validation fonctionnelle des gènes identifiés sera réalisées dans trois modèles complémentaires: Trypanosoma brucei, Chlamydomonas reinhardtii et les cellules humaines RPE1. Une étude préliminaire réalisée sur une petite cohorte nous a permis d’identifier des mutations tronquantes dans 5 nouveaux gènes, validant ainsi la pertinence de notre recrutement et de notre stratégie expérimentale. Ce projet permettra d’identifier plusieurs nouveaux gènes et d’améliorer nos connaissances et notre compréhension de la structure et de la biosynthèse des flagelles et par extension des cils mobiles. Il permettra également d’augmenter le nombre de diagnostics génétiques disponibles pour l’homme infertile.

We will use the 5 methods and technics described in the following 5 work packages to identify and characterize new genes involved in the MMAF phenotype.

• WP 1: Exome sequencing of 150 patients with severe MMAF. Patients have been recruited by four reproductive biology centers (Cochin-Paris, Tunis, Constantine, Teheran).
• WP2: Identification of candidate genes. The exome-seq data will be analyzed using an in house bioinformatics pipeline.
• WP 3: Phenotypic characterization and classification of patients with identified mutations. This will be accomplished by immunodetection of structural components of the axoneme and by electron microscopy on patient’s sperm sample. This will permit to establish a genotype-phenotype correlation for all the identified genes.
• WP 4: Validation and characterisation of the function of the best candidate genes in Trypanosoma brucei (T. brucei) which is a parasitic protist that causes African trypanosomiasis (or sleeping sickness) in humans and is commonly used for flagellar investigations and validation as it harbours a flagellum with the conserved 9+2 axonemal structure. Chlamydomonas, a green algae, comprises two cilia with a conserved structure.
• WP 5: Validation and characterisation of the function of the best candidate genes in the mouse. We will take advantage of public consortiums such as the NIH Knock Out Mouse Project (KOMP). When the KOs of interest are not available, we intend to create our own KOs using the CRISPR/Cas9 technology. We aim to obtain KO lines for at least 5 of our candidate genes. This will allow us to study the physiopathology of the studied genes and to gain insight into the molecular mechanisms underlying cilia/flagella assembly and activity.

We are convinced that the genetics will allow to identify many genes (at least 15 new genes). We hope that the functional work will 1) confirm the implication of these genes in the investigated phenotype, 2) elucidate the function of the corresponding proteins in the physiological context, and 3) significantly contribute to our understanding of the physiology of the flagella and by extension of the motile cilia biogenesis/structure.

We are confident that by the end of our study a genetic diagnostic will be obtained for more than >60% of MMAF patients. Most of the patients analysed in this study have recourse to the assisted reproductive technologies (ART), in general in vitro fertilisation (IVF) followed by Intra Cytoplasmic Sperm injection (ICSI). At the end of this study we will try to establish a “phenotype- genotype” correlation: the genotype being the affected gene, the phenotype being the ICSI results. We therefore believe that this work and the genetic diagnoses that it will allow to carry out in the future will permit to provide the patients with a better prognostic for ICSI success thus allowing to rapidly orientate them towards the best course of treatment.

We believe that our work will permit the publication of at least 3 major manuscripts in high impacts journals (IF>10) and at least 5 more in good journals ( IF between 3 and 8).

Male infertility affects more than 20 million men worldwide and represents a major health concern. Although multifactorial, male infertility has a strong genetic basis, which has so far not been extensively studied. We have previously demonstrated that macrozoospermia and globozoospermia, two rare forms of teratozoospermia, were caused in more than 70% of cases by mutations in AURKC and DPY19L2 respectively, thus confirming that transmittable mutations are often the cause of male infertility.

Asthenozoospermia, defined by the decrease or the absence of sperm motility, likely accounts for up to 50% of male infertility and subfertility. It is often caused by morphological and ultrastructural abnormalities of the sperm flagellum. Sperm flagellum and motile cilia share a common axonemal structure composed of a central pair of microtubules surrounded by nine doublets of microtubules (9+2). Hence flagellar abnormalities have also been reported in men with Primary Ciliary Dyskinesia (PCD), a group of mainly autosomal recessive disorders caused by dysfunctions of motile cilia leading primarily to respiratory infections and often to situs invertus. We previously analyzed 20 subjects presenting with impaired sperm motility due to a mosaic of multiple morphological abnormalities of the flagella (MMAF) characterized by the combination of morphological defects of the sperm flagella including absent, short, bent or coiled and flagella of irregular width. Interestingly, these men did not present any of the additional symptoms associated with PCD although we could not exclude subclinical or late onset PCD symptoms. Homozygosity mapping allowed us to identify a candidate region and we could demonstrate that 7 men (35%) carried a homozygous mutation in DNAH1, a gene encoding for an inner arm dynein heavy chain, which we showed to have a stabilizing effect on the axoneme. We subsequently sequenced the exome of the 13 remaining patients with no DNAH1 mutations. Six patients carried unreported homozygous truncating mutations in 5 new genes, which we considered, due to their expression profile and their presumed functions, to be very good candidates. This work confirms that like PCD, MMAF is genetically heterogeneous and we can expect many other genes to be involved in this newly described syndrome. We can expect a continuum of clinical manifestations ranging from infertile PCD patients (which corresponds to the most investigated phenotype so far) to MMAF patients with no or subtle PCD manifestations.

We now would like to analyse the coding sequences (exome) of another 50 subjects presenting with MMAF. To facilitate the identification of causal mutations we focused our recruitment on familial cases (n=10) and on subjects born from related parents (consanguinity). We are confident that the analysis of this already constituted cohort will permit to confirm the importance of the 5 newly identified genes while allowing identifying a minimum of 10 additional candidates. Targeted sequencing of 15-25 candidate genes will then be carried out on a larger cohort of 250 MMAF patients. A phenotypic characterization and classification of patients will be carried out for all patients. The best candidate genes will be characterized functionally on at least two of four models available through the partners of this project: study of the orthologs in Trypanosoma brucei and/or Chlamydomonas reinhardtii, inactivation of the gene in RPE1-cells and study of KO mice. Such functional analysis should provide a better understanding of the molecular mechanisms underlying the assembly and the activity of cilia and flagella.

We are confident that this work will allow the identification of at least 15 new genes involved in male infertility and cilium-related diseases and that a genetic diagnosis will be achieved in more than 50% of MMAF patients. Patients will benefit by having a better counsel and prognosis.

Project coordination

Pierre RAY (CHU de Grenoble)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


UMR 5234 CNRS - UMR 5234 Microbiologie Fondamentale et Pathogénicité - MFP Group Cytoskeleton Biogenesis in Trypanosomes Université Bordeaux 2
INSERM U1016 Intitut National de la Santé et Recherche Medicale, Unité 1016
CHUG CHU de Grenoble
TIMC-IMAG Laboratoire Techniques de l’Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble

Help of the ANR 150,336 euros
Beginning and duration of the scientific project: September 2014 - 48 Months

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