The compound heterozygous mutations of CFAP65 cause multiple morphological abnormalities of sperm flagella in infertile men

Cilia and flagella-associated protein 65 (CFAP65), located on the acrosome and flagellar midpiece of the spermatozoon, is essential for spermatogenesis and male fertility. Here, we report novel CFAP65 stop-gain bi-allelic variants in a Chinese infertile patient presenting with multiple morphological abnormalities of sperm flagella. The proband was a 48-year-old Han Chinese man with a 15-year history of infertility. Semen analysis (Supplementary Table S1) showed the patient had normal levels of semen volume, pH, color and viscosity and a mildly reduced sperm concentration (12.08 × 106 spermatozoa/ml; reference range, ≥15 spermatozoa/ml). However, no motile sperm was found (Supplementary Table S1). Transmission electron microscopy demonstrated striking defects in the flagellar, including axonemal and mitochondrial sheath malformations, as well as a variety of sperm-head shape abnormalities (Figure 1A). Overall, the semen characteristics of the patient displayed obvious MMAF phenotypes. Furthermore, the proband exhibited classic primary ciliary dyskinesia (PCD)-like clinical features, with nasosinusitis and tympanitis. The proband had a normal karyotype (46, XY) without Y chromosome microdeletions. His younger brother was fertile and had a child through natural conception (Figure 1B). Identification of the compound heterozygous variants of CFAP65 in an MMAF-affected Chinese man. (A) TEM analysis of the spermatozoa from the patient. Misshapen heads (asterisk) and morphologically defective acrosomes (blue arrow) were observed. Sperm tails revealed poorly assembled mitochondria (green arrow) and typical axonemal anomalies. The axoneme microtubule structure, including DMT (purple arrow and loop) and CP (red arrow and loop), was visible. Scale bars: (a and b) 2.0 μm; (c) 500 nm; and (d) 200 nm. (B) Family tree of the patient. The proband was indicated by the black arrow. (C) Sanger sequencing validated the compound heterozygous variants of CFAP65 in the patient, his parents and his young brother. The red arrow and pane point to the variant sites. Ac, acrosome; CP, central pair of microtubules; Mi, mitochondria; DMTs, peripheral doublet microtubules; MMAF, multiple morphological abnormalities of the sperm flagella; TEM, transmission electron microscopy. Whole exome sequencing was performed to investigate the genetic cause of infertility. After bioinformatic analysis and variant filtering, 28 rare and likely deleterious mutations in 20 genes, including 11 homozygous variants, 16 heterozygous variants and 2 hemizygous variants, remained for consideration (Supplementary Table S2). Among them, we noted bi-allelic protein-truncating variants (NM_194302:c.G5341T:p.E1781X; NM_001278296:c.1580_1581del:p.P527Rfs*73) in the CFAP65 gene, one of which was previously linked to male infertility and abnormal sperm morphology.1 Co-segregation analysis via Sanger sequencing confirmed the p.E1781X and p.P527Rfs*73 variants were maternally and paternally inherited, respectively (Figure 1C). The patient’s fertile brother only carried the heterozygous p.E1781X variant. Therefore, the proband was compound heterozygous for the bi-allelic CFAP65 variants. The nonsense c.G5341T:p.E1781X variant was previously described by Wang et al.1 The c.1580_1581del:p.P527Rfs*73 variant, introducing a premature stop codon due to a 2-bp frameshift deletion, was novel. Both CFAP65 variants were absent from the GnomAD database (Supplementary Table S3). The p.E1781X variant was predicted to be deleterious by Combined Annotation Dependent Depletion (score of 39) and Mutation Taster (Disease_causing_automatic) analysis (Supplementary Table S3). These findings indicated that the compound heterozygous variants of CFAP65 (NM_194302:c.G5341T:p.E1781X; NM_001278296:c.1580_1581del:p.P527Rfs*73) were pathogenic according to The American College of Medical Genetics and Genomics guidelines and likely the genetic cause of infertility in this patient. CFAP65, also named CCDC108 (coiled-coil domain containing 108), participates in acrosome biogenesis and flagella assembly during spermatogenesis and is associated with MMAF and ciliopathy in humans.2 To date, several studies have reported a series of genetic variants of CFAP65 leading to MMAF.1,3–6 Wang et al. reported a novel homozygous CFAP65 nonsense mutation (p.E1781X) in an infertile man manifesting typical MMAF phenotypes, as well as typical PCD-like symptoms, including a history of chronic wet cough, nasal obstruction and pneumonia in early life, and the gradual development of recurrent respiratory tract infection, bronchitis and rhinosinusitis confirmed by computer tomography.1 The patient in our study also exhibited MMAF phenotypes and had a positive history of nasosinusitis and tympanitis. However, the patient did not undergo further examination, including computer tomography, magnetic resonance imaging, ciliary biopsy or nasal nitric oxide measurement, for definitive diagnosis of PCD. In summary, our study identified bi-allelic loss-of-function variants of CFAP65, one of which was novel, in a Chinese infertile man with MMAF, further broadening the variant spectrum of the CFAP65 gene. Supplementary material is available at QJMED online. Our study received ethical approval from the institutional ethics committee of the National Research Institute for Family Planning. Participants who were included in the study provided informed consent. The final version of the article for submission had been read and approved by all co-authors. All authors agreed to its submission for publication and signed informed consent about the publication of data and images. We would like to acknowledge the patient and his family members for participating in our research. This work was supported by the Beijing Municipal Natural Science Foundation (7232112). H.L. and B.W. were involved in the conception and design of the study. Y.L., L.R., S.L., J.W. and T.L. performed experiments and data analysis. All authors read and contributed to previous versions of the manuscript. All authors approved the final article for submission. None declared. In our study, all data and materials are available from the corresponding author on reasonable request.