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. 2017 Jun 1;100(6):854-864.
doi: 10.1016/j.ajhg.2017.04.012. Epub 2017 May 25.

Biallelic Mutations in CFAP43 and CFAP44 Cause Male Infertility With Multiple Morphological Abnormalities of the Sperm Flagella

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Free PMC article

Biallelic Mutations in CFAP43 and CFAP44 Cause Male Infertility With Multiple Morphological Abnormalities of the Sperm Flagella

Shuyan Tang et al. Am J Hum Genet. .
Free PMC article

Abstract

Sperm motility is vital to human reproduction. Malformations of sperm flagella can cause male infertility. Men with multiple morphological abnormalities of the flagella (MMAF) have abnormal spermatozoa with absent, short, coiled, bent, and/or irregular-caliber flagella, which impair sperm motility. The known human MMAF-associated genes, such as DNAH1, only account for fewer than 45% of affected individuals. Pathogenic mechanisms in the genetically unexplained MMAF remain to be elucidated. Here, we conducted genetic analyses by using whole-exome sequencing and genome-wide comparative genomic hybridization microarrays in a multi-center cohort of 30 Han Chinese men affected by MMAF. Among them, 12 subjects could not be genetically explained by any known MMAF-associated genes. Intriguingly, we identified compound-heterozygous mutations in CFAP43 in three subjects and a homozygous frameshift mutation in CFAP44 in one subject. All of these recessive mutations were parentally inherited from heterozygous carriers but were absent in 984 individuals from three Han Chinese control populations. CFAP43 and CFAP44, encoding two cilia- and flagella-associated proteins (CFAPs), are specifically or preferentially expressed in the testis. Using CRISPR/Cas9 technology, we generated two knockout models each deficient in mouse ortholog Cfap43 or Cfap44. Notably, both Cfap43- and Cfap44-deficient male mice presented with MMAF phenotypes, whereas the corresponding female mice were fertile. Our experimental observations on human subjects and animal models strongly suggest that biallelic mutations in either CFAP43 or CFAP44 can cause sperm flagellar abnormalities and impair sperm motility. Further investigations on other CFAP-encoding genes in more genetically unexplained MMAF-affected individuals could uncover novel mechanisms underlying sperm flagellar formation.

Keywords: CFAP43; CFAP44; CFAP65; CGH; cilia; flagella; male infertility; motility; sequencing; sperm.

Figures

Figure 1
Figure 1
Biallelic Mutations in CFAP43 and CFAP44 Identified in Subjects with MMAF (A) Biallelic CFAP43 mutations (M1–M5) were identified in three subjects with MMAF (P003, P028, and P029). M1 was recurrent in unrelated individuals P003 and P029. The parental origins of all CFAP43 mutations are shown. (B) The positions of four point mutations in CFAP43 (M1–M3 and M5) are shown. The affected amino acid residues are conserved across species. (C) The homozygous frameshift mutation in CFAP44 (M6) was identified in one subject with MMAF (P002). Both of his parents carried the heterozygous mutation. All CFAP43 and CFAP44 mutations were verified by Sanger sequencing. Red arrows indicate the positions of point mutations, and red rectangles indicate the shifted sequences after deletion start points. Abbreviation: WT, wide-type.
Figure 2
Figure 2
Sperm Morphology in the Subjects Carrying Biallelic Mutations in CFAP43 or CFAP44 (A) Light microscopy shows a spermatozoon with normal morphology from a healthy man. (B–D) Most spermatozoa of the subjects with biallelic CFAP43 mutations (B, P003; C, P028; D, P029) presented with morphological abnormalities of the sperm flagella, such as short and coiled flagella, flagella of irregular caliber, and other malformations diagnosed as MMAF. (E) In individual P002, who carries homozygous frameshift mutations in CFAP44, most spermatozoa had short or absent flagella. (F) SEM shows a spermatozoon with a normal flagellum. (G–I) SEM shows that most spermatozoa of the subjects with biallelic CFAP43 mutations (G, P003; H, P028; I, P029) presented with absent, short, or coiled flagella and other MMAF phenotypes. (J) The spermatozoa in CFAP44-deficient subject P002 were abnormal. One of the major abnormalities was short flagella. The results of SEM were consistent with those of light microscopy.
Figure 3
Figure 3
Sperm Ultrastructures in MMAF-Affected Individuals with Mutations in CFAP43 and CFAP44 (A) The ultrastructure of a cross section in a normal spermatozoon from a healthy man. The typical “9+2” microtubule structure (nine peripheral microtubule doublets paired with nine outer dense fibers and the central pair of microtubules) is shown. (B and C) In CFAP43-deficient (B) and CFAP44-deficient (C) subjects, most spermatozoa lacked the central pair of microtubules and had hyperplasia of fibrous sheaths. (B) Two axonemes in one section were probably due to a folded flagellum. The flagellum showed a “9+0” arrangement of microtubules and lacked the central pair of microtubules. (C) The cross section shows a thickened fibrous sheath and an absent central pair of microtubules. The outer dense fibers and peripheral microtubule are regularly arranged. (D) TEM shows the typical “9+2” microtubule structure in a normal testicular spermatozoon from a wild-type male mouse. (E and F) TEM analysis in Cfap43-deficient (E) and Cfap44-deficient (F) male mice shows a totally disorganized axoneme in a testicular spermatozoon. The central pair of microtubules are not apparent. The outer dense fibers and peripheral microtubules are misarranged. Abbreviations: CP, central pair of microtubules (green arrows); MT, peripheral microtubule doublet (white arrows); and ODF, outer dense fiber (red arrows).
Figure 4
Figure 4
Sperm Morphology in Cfap43- and Cfap44-Deficient Male Mice (A) A spermatozoon with normal morphology from a wild-type male mouse. (B–E) Almost all spermatozoa of Cfap43-deficient (B and C) and Cfap44-deficient (D and E) male mice had flagellar abnormalities. These flagella presented with short, coiled, or other distorted shapes, consistent with the clinical phenotypes in the subjects with MMAF.

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