1 INTRODUCTION
The etiology of male infertility is highly heterogeneous, and genetic factors account for at least 15% of cases (Jiao et al., 2021). The integrity of the flagella is crucial for sperm motility, and any structural and functional defects of sperm flagella could impair sperm function, which is indispensable for spontaneous fertilization (Inaba et al., 2011). Multiple morphological abnormalities of the sperm flagella (MMAF) is a rare asthenoteratozoospermia with severely impaired sperm motility (Ben et al., 2014). This conception was initially reported in 2014 and has attracted increasing attention (Ben et al., 2014). Recent research based on animal models and genetic analysis has uncovered certain disease-causing or disease-promoting genes. To date, 23 MMAF-associated genes have been identified, including AK7 (MIM: 615364), ARMC2 (MIM:618424), CEP135 (MIM: 611423),CFAP43 (MIM: 617558), CFAP44 (MIM: 617559), CFAP47(MIM: 301057), CFAP58 (MIM: 619129), CFAP65 (MIM: 614270),CFAP69 (MIM: 617949), CFAP70 (MIM: 618661), CFAP91(MIM: 609910), CFAP251 (MIM: 618146), DNAH1 (MIM: 603332),DNAH2 (MIM: 603333), DNAH6 (MIM: 603336), DNAH8(MIM: 603337), DNAH17 (MIM: 610063), DZIP1 (MIM: 608671),FSIP2 (MIM: 618153), QRICH2 (MIM: 618304), SPEF2(MIM: 610172), TTC21A (MIM: 611430), and TTC29 (MIM: 618735) (Jiao et al., 2021; Toure´ et al., 2020). However, mutations in each gene are possibly responsible for only a small fraction of pathogenic factors, and a mass of cases could not be explained. Therefore, a more comprehensive investigation of the pathology and molecular mechanisms of MMAF is needed to further boost diagnosis efficacy.
The cilia and flagella-associated protein (CFAP) protein family is associated with the development and function of sperm flagellum (Tang et al., 2017). This family plays an important role in the generation, assembly, and maintenance of motor functions of cilia and flagella. To date, multiple CFAP gene deficiencies have been reported to be associated with MMAF, such as several mutations in CFAP43, CFAP44, CFAP58, CFAP65, CFAP69, CFAP70, and CFAP251 (Tang et al., 2017; Coutton et al., 2018; Li et al., 2020; He et al., 2020; He et al., 2019; Beurois et al., 2019; Auguste et al., 2018). To date, only one report revealed that cilia and flagella-associated protein 47 (CFAP47,NG_016381.2) leads to MMAF in an X-chromosome-linked inheritance pattern (Liu et al., 2021). Males with hemizygous CFAP47 variants exhibit a typical MMAF phenotype, and the ultrastructure shows an abnormal axoneme, including disorganized outer dense fibers (ODF), and peripheral microtubule doublets (DMTs), as well as central pair of microtubules (CPs), are missing (Liu et al., 2021).CFAP47 -mutated male mice are sterile, with decreased sperm motility and abnormal flagella morphology (Liu et al., 2021). Hence, future studies should evaluate CFAP47 in larger cohorts to corroborate the relationship between genotype and phenotype.
Herein, we report two MMAF patients who carried a novel hemizygous mutation c.1414G>A [p.V472M] in CFAP47 . Bioinformatics analysis and functional studies in vitro validated the pathogenicity of the mutation. Furthermore, we first revealed thatCFAP47 mutations are also related to malformed sperm heads and annulus, as well as suggested the potential mechanism by whichCFAP47 regulates spermatogenesis. Our work provides strong evidence to confirm the causative relationship between CFAP47variants and MMAF.