A novel mutation in CFAP47 causes male infertility due to multiple morphological abnormalities of the sperm flagella.

A previous study suggested that loss of CFAP47 function is involved in multiple morphological abnormalities of the sperm flagella (MMAF) in humans and mice. However, the comprehensive role of CFAP47 in spermatogenesis is largely unknown. Whole-exome sequencing (WES) was conducted to identify pathogenic variant in two patients with MMAF. The functional effect of the identified mutations was investigated by immunofluorescence staining and western blotting. Intracytoplasmic sperm injection (ICSI) was used to assist fertilization for the patient with MMAF. In this study, we identified a novel missense mutation (c.1414G>A; p.V472M) in CFAP47 in two unrelated patients with oligoasthenoteratozoospermia. Intriguingly, in addition to the MMAF phenotype very analogous to the previous report, the two patients notably presented abnormal morphology of sperm heads, the sperm mitochondrial sheath was obviously disorganized, and the sperm annulus were almost defective. Further functional experiments confirmed that the expression of CFAP47 was markedly reduced in the spermatozoa of the patients. Mechanism analysis suggested that CFAP47 might regulate the expression of CFAP65, CFAP69 and SEPTIN4 through their physical interactions and thus modulating sperm morphogenesis. we revealed a novel mutation in CFAP47 and further expanded the phenotype and mutation spectrum of CFAP47, as well as the potential mechanism of CFAP47 manipulating spermatogenesis, finally providing important guidance for genetic counseling and targeted treatment for CFAP47 mutation-related male infertility.

Liu M ，Dai S ，Zhang J ，Yang Y ，Shen Y ，Liu H ，Yang Y ，Jiang C ，Tian E ... -  《Frontiers in Endocrinology》

Deleterious variants in X-linked CFAP47 induce asthenoteratozoospermia and primary male infertility.

Asthenoteratozoospermia characterized by multiple morphological abnormalities of the flagella (MMAF) has been identified as a sub-type of male infertility. Recent progress has identified several MMAF-associated genes with an autosomal recessive inheritance in human affected individuals, but the etiology in approximately 40% of affected individuals remains unknown. Here, we conducted whole-exome sequencing (WES) and identified hemizygous missense variants in the X-linked CFAP47 in three unrelated Chinese individuals with MMAF. These three CFAP47 variants were absent in human control population genome databases and were predicted to be deleterious by multiple bioinformatic tools. CFAP47 encodes a cilia- and flagella-associated protein that is highly expressed in testis. Immunoblotting and immunofluorescence assays revealed obviously reduced levels of CFAP47 in spermatozoa from all three men harboring deleterious missense variants of CFAP47. Furthermore, WES data from an additional cohort of severe asthenoteratozoospermic men originating from Australia permitted the identification of a hemizygous Xp21.1 deletion removing the entire CFAP47 gene. All men harboring hemizygous CFAP47 variants displayed typical MMAF phenotypes. We also generated a Cfap47-mutated mouse model, the adult males of which were sterile and presented with reduced sperm motility and abnormal flagellar morphology and movement. However, fertility could be rescued by the use of intra-cytoplasmic sperm injections (ICSIs). Altogether, our experimental observations in humans and mice demonstrate that hemizygous mutations in CFAP47 can induce X-linked MMAF and asthenoteratozoospermia, for which good ICSI prognosis is suggested. These findings will provide important guidance for genetic counseling and assisted reproduction treatments.

Liu C ，Tu C ，Wang L ，Wu H ，Houston BJ ，Mastrorosa FK ，Zhang W ，Shen Y ，Wang J ，Tian S ，Meng L ，Cong J ，Yang S ，Jiang Y ，Tang S ，Zeng Y ，Lv M ，Lin G ，Li J ，Saiyin H ，He X ，Jin L ，Touré A ，Ray PF ，Veltman JA ，Shi Q ，O'Bryan MK ，Cao Y ，Tan YQ ，Zhang F ... -  《-》

A novel CCDC39 mutation causes multiple morphological abnormalities of the flagella in a primary ciliary dyskinesia patient.

Male infertility is a widespread symptom in patients with primary ciliary dyskinesia (PCD). PCD-related male infertility is often caused by asthenozoospermia, with barely normal sperm morphology. Multiple morphological abnormalities of the sperm flagella (MMAF) are a major cause of asthenozoospermia, characterized by various malformed morphologies of sperm flagella. To date, a limited number of genes have been suggested to be involved in the pathogenesis of both PCD and MMAF. What other genes associated with both PCD and MMAF are waiting to be discovered? Whole-exome sequencing (WES) was performed to identify the pathogenic mutation associated with MMAF in a PCD patient. Peripheral venous blood and semen samples were collected from the PCD patient. Transmission electron microscopy (TEM), immunofluorescence staining and western blotting were conducted to confirm the pathogenicity of the identified mutation. A novel homozygous mutation in CCDC39, c.983 T>C (p. Leu328Pro), was identified in two PCD-affected siblings of a consanguineous family showing a typical PCD phenotype, while the proband was infertile, which is associated with characterized MMAF. Furthermore, TEM revealed the abnormal ultrastructure of the patient's sperm flagella. Moreover, immunofluorescence staining revealed that CCDC39 was almost undetectable in the spermatozoa, which was further confirmed by western blotting. The outcome of intracytoplasmic sperm injection (ICSI) in the patient with the CCDC39 mutation was also favourable. This study demonstrates that a novel loss-of-function mutation of CCDC39 is involved in the pathogenesis of PCD and MMAF and initially reported that ICSI treatment has a good outcome. Therefore, the novel variant of CCDC39 contributes to the genetic diagnosis, counselling and treatment of male infertility in PCD patients with MMAF phenotype.

Chen D ，Liang Y ，Li J ，Zhang X ，Zheng R ，Wang X ，Zhang H ，Shen Y ... -  《-》

WDR87 interacts with CFAP47 protein in the middle piece of spermatozoa flagella to participate in sperm tail assembly.

Spermatogenesis is a complex process that includes spermatogonia self-renewal, spermatocyte meiosis and spermatozoa assembly. Recent studies have revealed that WD40-repeat domain-containing (WDR) proteins play important roles in spermatocyte division, spermatozoa flagella assembly and head shaping. In this study, we investigated the expression pattern of WDR87 and found that it was highly expressed in the testis of both humans and mice. Immunofluorescence staining revealed that mouse WDR87 was distributed in the perinuclear cytoplasm of primary spermatocytes, secondary spermatocytes and round spermatids. In the spermiogenesis stage, with extension of the nucleus, WDR87 migrated to the manchette and finally localized to the middle piece of the spermatozoa tail. Furthermore, we identified a cilia- and flagella-associated protein, CFAP47, which interacted with WDR87 in the flagellar midpiece of the spermatozoa, suggesting that WDR87 may be associated with multiple morphological abnormalities of the flagella (MMAF). Subsequently, we screened gene mutations in seven MMAF individuals and found two novel mutations in CFAP47 (c.706G>A, Val236Met; c.1337C>T, Thr446Met) in one case. Immunoblotting and immunofluorescence revealed that CFAP47 was dramatically reduced in spermatozoa from the CFAP47-mutated man. Meanwhile, the expression of WDR87 was also significantly decreased, and weak signals were detected adjacent to the spermatozoa nuclei, indicating that CFAP47 was necessary for WDR87 transportation during spermatozoa flagella biogenesis. These data indicate that WDR87 is located in the middle piece of the sperm tail and interacts with CFAP47 to form a complex which is involved in spermatozoa tail assembly.

Liao HQ ，Guo ZY ，Huang LH ，Liu G ，Lu JF ，Zhang YF ，Xing XW ... -  《-》

Absence of CFAP69 Causes Male Infertility due to Multiple Morphological Abnormalities of the Flagella in Human and Mouse.

The multiple morphological abnormalities of the flagella (MMAF) phenotype is among the most severe forms of sperm defects responsible for male infertility. The phenotype is characterized by the presence in the ejaculate of immotile spermatozoa with severe flagellar abnormalities including flagella being short, coiled, absent, and of irregular caliber. Recent studies have demonstrated that MMAF is genetically heterogeneous, and genes thus far associated with MMAF account for only one-third of cases. Here we report the identification of homozygous truncating mutations (one stop-gain and one splicing variant) in CFAP69 of two unrelated individuals by whole-exome sequencing of a cohort of 78 infertile men with MMAF. CFAP69 encodes an evolutionarily conserved protein found at high levels in the testis. Immunostaining experiments in sperm from fertile control individuals showed that CFAP69 localized to the midpiece of the flagellum, and the absence of CFAP69 was confirmed in both individuals carrying CFPA69 mutations. Additionally, we found that sperm from a Cfap69 knockout mouse model recapitulated the MMAF phenotype. Ultrastructural analysis of testicular sperm from the knockout mice showed severe disruption of flagellum structure, but histological analysis of testes from these mice revealed the presence of all stages of the seminiferous epithelium, indicating that the overall progression of spermatogenesis is preserved and that the sperm defects likely arise during spermiogenesis. Together, our data indicate that CFAP69 is necessary for flagellum assembly/stability and that in both humans and mice, biallelic truncating mutations in CFAP69 cause autosomal-recessive MMAF and primary male infertility.

Dong FN ，Amiri-Yekta A ，Martinez G ，Saut A ，Tek J ，Stouvenel L ，Lorès P ，Karaouzène T ，Thierry-Mieg N ，Satre V ，Brouillet S ，Daneshipour A ，Hosseini SH ，Bonhivers M ，Gourabi H ，Dulioust E ，Arnoult C ，Touré A ，Ray PF ，Zhao H ，Coutton C ... -  《-》