Mutations in TTC29, Encoding an Evolutionarily Conserved Axonemal Protein, Result in Asthenozoospermia and Male Infertility.

In humans, structural or functional defects of the sperm flagellum induce asthenozoospermia, which accounts for the main sperm defect encountered in infertile men. Herein we focused on morphological abnormalities of the sperm flagellum (MMAF), a phenotype also termed "short tails," which constitutes one of the most severe sperm morphological defects resulting in asthenozoospermia. In previous work based on whole-exome sequencing of a cohort of 167 MMAF-affected individuals, we identified bi-allelic loss-of-function mutations in more than 30% of the tested subjects. In this study, we further analyzed this cohort and identified five individuals with homozygous truncating variants in TTC29, a gene preferentially and highly expressed in the testis, and encoding a tetratricopeptide repeat-containing protein related to the intraflagellar transport (IFT). One individual carried a frameshift variant, another one carried a homozygous stop-gain variant, and three carried the same splicing variant affecting a consensus donor site. The deleterious effect of this last variant was confirmed on the corresponding transcript and protein product. In addition, we produced and analyzed TTC29 loss-of-function models in the flagellated protist T. brucei and in M. musculus. Both models confirmed the importance of TTC29 for flagellar beating. We showed that in T. brucei the TPR structural motifs, highly conserved between the studied orthologs, are critical for TTC29 axonemal localization and flagellar beating. Overall our work demonstrates that TTC29 is a conserved axonemal protein required for flagellar structure and beating and that TTC29 mutations are a cause of male sterility due to MMAF.

Genetic causes of male infertility: snapshot on morphological abnormalities of the sperm flagellum.

Male infertility due to Multiple Morphological Abnormalities of the sperm Flagella (MMAF), is characterized by nearly total asthenozoospermia due to the presence of a mosaic of sperm flagellar anomalies, which corresponds to short, angulated, absent flagella and flagella of irregular calibre. In the last four years, 7 novel genes whose mutations account for 45% of a cohort of 78 MMAF individuals were identified: DNAH1, CFAP43, CFAP44, CFAP69, FSIP2, WDR66 (CFAP251), AK7. This successful outcome results from the efficient combination of high-throughput sequencing technologies together with robust and complementary approaches for functional validation, in vitro, and in vivo using the mouse and unicellular model organisms such as the flagellated parasite T. brucei. Importantly, these genes are distinct from genes responsible for Primary Ciliary Dyskinesia (PCD), an autosomal recessive disease associated with both respiratory cilia and sperm flagellum defects, and their mutations therefore exclusively lead to male infertility. In the future, these genetic findings will definitely improve the diagnosis efficiency of male infertility and might provide genotype-phenotype correlations, which could be helpful for the prognosis of intracytoplasmic sperm injection (ICSI) performed with sperm from MMAF patients. In addition, functional study of these novel genes should improve our knowledge about the protein networks and molecular mechanisms involved in mammalian sperm flagellum structure and beating.

Les infertilités masculines dues au phénotype de « flagelles courts ¼ ou « Multiple Morphological Abnormalities of the sperm Flagella ¼ (MMAF), sont caractérisées par une asthénozoospermie quasi totale associée à la présence d'une mosaïque d'anomalies flagellaires correspondant à des flagelles courts, angulés, absent ou de calibre irrégulier. Durant les quatre dernières années, une approche génétique par séquençage d'exome de 78 patients MMAF a permis l'identification de mutations causales dans 7 gènes: DNAH1, CFAP43, CFAP44, CFAP69, FSIP2, WDR66 (CFAP251), AK7, permettant ainsi un diagnostic pour près de 45% des sujets de la cohorte. Ce succès remarquable résulte de la combinaison efficace de technologies de séquençage à haut débit et d'approches complémentaires de validation fonctionnelle des mutations, in vitro et in vivo, dans le modèle murin et les modèles unicellulaires tels que le parasite flagellé T. brucei.De manière importante, les gènes identifiés sont distincts des gènes responsables de Dyskinésie Ciliaire Primitive (DCP), une maladie autosomale récessive associée à des défauts des cils et du flagelle, et leurs mutations induisent par conséquent une infertilité masculine isolée. Dans le futur, ces résultats génétiques vont permettre d'améliorer le diagnostic des infertilités masculines humaines et potentiellement de fournir des corrélations génotype-phénotype, utiles pour le pronostic de la fécondation in vitro par injection intra-cytoplasmique des spermatozoïdes de sujets MMAF. Par ailleurs, les études fonctionnelles de ces nouveaux gènes identifiés, permettront de mieux définir les mécanismes moléculaires et les complexes protéiques impliqués dans l'assemblage et le battement du flagelle.