Lack of CCDC146, a ubiquitous centriole and microtubule-associated protein, leads to non-syndromic male infertility in human and mouse

Abstract

Genetic mutations are a recurrent cause of male infertility. Multiple morphological abnormalities of the flagellum (MMAF) syndrome is a heterogeneous genetic disease, with which more than 50 genes have been linked. Nevertheless, for 50% of patients with this condition, no genetic cause is identified. From a study of a cohort of 167 MMAF patients, pathogenic bi-allelic mutations were identified in theCCDC146gene in two patients. This gene encodes a poorly characterized centrosomal protein which we studied in detail here. First, protein localization was studied in two cell lines. We confirmed the centrosomal localization in somatic cells and showed that the protein also presents multiple microtubule-related localizations during mitotic division, suggesting that it is a microtubule-associated protein (MAP). To better understand the function of the protein at the sperm level, and the molecular pathogenesis of infertility associated withCCDC146mutations, two genetically modified mouse models were created: aCcdc146knock-out (KO) and a knock-in (KI) expressing a HA-tagged CCDC146 protein. KO male mice were completely infertile, and sperm exhibited a phenotype identical to our two MMAF patient’s phenotype withCCDC146mutations. No other pathology was observed, and the animals were viable. CCDC146 expression starts during late spermiogenesis, at the time of flagellum biogenesis. In the spermatozoon, the protein is conserved but is not localized to centrioles, unlike in somatic cells, rather it is present in the axoneme at the level of microtubule doublets. Expansion microscopy associated with the use of the detergent sarkosyl to solubilize microtubule doublets, provided evidence that the protein could be a microtubule inner protein (MIP). At the subcellular level, the absence of CCDC146 affected the formation, localization and morphology of all microtubule-based organelles such as the manchette, the head–tail coupling apparatus (HTCA), and the axoneme. Through this study, we have characterized a new genetic cause of infertility, identified a new factor in the formation and/or structure of the sperm axoneme, and demonstrated that the CCDC146 protein plays several cellular roles, depending on the cell type and the stages in the cell cycle.

Deep learning modeling of rare noncoding genetic variants in human motor neurons definesCCDC146as a therapeutic target for ALS

“…Flagella are functionally mirrored by cilia and it is notable that primary cilia dyskinesia has been associated with ALS 66,67 . We note that frank primary ciliary dyskinesia has not been observed in CCDC146 knockout mice 17 , but we speculate a more subtle phenotype may be present and should be the subject of further research. The variants we have identified are all rare in the population; we have previously speculated that variants that modify ALS pathogenesis might be rare, despite the fact that ALS onset is typically later than reproductive age, because they have a pleiotropic effect on gamete function 5 .…”

“…The predicted structure mainly consists of coiled coils linked by flexible regions, without ligand binding sites detected on its surface 74 ( Methods ), suggesting that it would be challenging to design small-molecule drugs for this target. As we already discussed, in the cellular environment the CCDC146 protein is likely to be located in centriole and microtubule 17 , which may further limit its accessibility for ligand binding. Taken together, CCDC146 could be difficult-to-drug via traditional small-molecule drug discovery strategies, underscoring the importance of alternative targeting approaches such as ASO.…”

“…It is notable that, aside from the fertility defect, loss of CCDC146 is well tolerated, suggesting our ASO approach may be viable without significant toxicity. Indeed, loss-of-function (LOF) variants within CCDC146 cause male infertility but are otherwise well tolerated in both mice and humans 17 . Additionally, this gene is not well conserved 68 (probability of being loss-of-function intolerant (pLI) = 0).…”

“…CCDC146 is a microtubule associated protein (MAP) and localized to sperm flagella. Biallelic variants in CCDC146 have been associated with reduced sperm viability and male infertility in both mice and humans 17 . Dysfunction of MAP is a recognized upstream driver of ALS pathogenesis 65 .…”

“…4e,f ). Indeed, LOF of CCDC146 has been shown to be well tolerated in humans 17 . Of note, none of the experimentally-tested patient-derived 3D-iN lines exhibited elevated CCDC146 expression at baseline ( Extended Data Fig.…”