Genotype-phenotype correlation of morphological abnormalities of the sperm flagella (MMAF) in infertile men by Whole exome sequencing

Background: Multiple morphological abnormalities of the flagella (MMAF), is one of the most severe form of asthenozoospermia and is characterized by the simultaneous presence of five morphological defects of the sperm flagella (absent, short, bent, coiled flagella and flagella of irregular width). Many cases of infertile males are categorized to be idiopathic, indicating that the cause is unknown and the mechanisms responsible for their condition are to be found. To date, a short list of genes was identified which is in sharp contrast with the fact that several hundreds of genes are estimated to be involved in spermatogenesis and male reproduction. However, gene identification is the key to improving knowledge of the pathophysiology of MMAF and opens new perspectives for diagnosis and treatment of infertile patients.Objective: Further genetic studies are therefore warranted to identify other genes involved in MMAF to better characterize the genetic etiology of the MMAF phenotype and to improve the management of patients diagnosed with flagellar defects.Materials and Methods: In our study, we analyzed 78 MMAF patients using WES and showed that mutations in DNAH1, CFAP43, CFAP44, CFAP69, WDR66, and FSIP2 are responsible for MMAF syndrome. After Sanger Sequencing verification of all candidate variants including, Relative mRNA expression levels for the selected candidate genes were assessed by qRT-PCR. To characterize the structural and ultrastructural anomalies present in patients sperm, immunofluorescence was performed on sperm samples from the subjects with a mutation and one control and transmission electron microscopy (TEM) analyses was performed on sperm samples. Most importantly, we investigated the role of some of these novel genes by performing gene invalidation and silencing in two evolutionary distant models sharing an extremely conserved flagellar structure, Trypanosoma and mouse.Results: Overall, DNAH1, CFAP43, CFAP44, CFAP69, WDR66, and FSIP2 mutations were identified in 45% of the analyzed subjects (35 out of 78 patients) originating from the Middle East, North Africa, and Europe. None of these mutations were reported in control sequence databases. TEM analyses showed a complete disorganization of the fibrous sheet associated with axonemal defects. Immunofluorescence analyses confirmed that the central-pair microtubules and the inner and outer dynein arms of the axoneme were abnormal in the patients carrying these mutations. Conclusion: Altogether, our results underline the global importance of these 6 genes in the MMAF syndrome and will improve the genetic diagnosis efficiency of infertile MMAF patients. In our study, WES revealed that the aforementioned genes are the main genes involved in MMAF phenotypes. Our work illustrates the efficiency of the combination of WES with original workflow for the validation of the candidate genes that are identified in male infertility due to a MMAF phenotype and exploit the WES data to the benefit of the patients.

Keywords:

Male infertility , Genetic diagnosis , Exome sequencing , Teratozoospermia , MMAF