Novel biallelic loss-of-function mutations in CFAP43 cause multiple morphological abnormalities of the sperm flagellum in Pakistani families

Abstract

Multiple morphological abnormalities of the sperm flagella (MMAF) is a specific type of asthenoteratozoospermia, presenting with multiple morphological anomalies in spermatozoa, such as absent, bent, coiled, short, or irregular caliber flagella. Previous genetic studies revealed pathogenic mutations in genes encoding cilia and flagella-associated proteins (CFAPs; e.g., CFAP43, CFAP44, CFAP65, CFAP69, CFAP70, and CFAP251) responsible for the MMAF phenotype in infertile men from different ethnic groups. However, none of them have been identified in infertile Pakistani males with MMAF. In the current study, two Pakistani families with MMAF patients were recruited. Whole-exome sequencing (WES) of patients and their parents was performed. WES analysis reflected novel biallelic loss-of-function mutations in CFAP43 in both families (Family 1: ENST00000357060.3, p.Arg300Lysfs*22 and p.Thr526Serfs*43 in a compound heterozygous state; Family 2: ENST00000357060.3, p.Thr526Serfs*43 in a homozygous state). Sanger sequencing further confirmed that these mutations were segregated recessively in the families with the MMAF phenotype. Semiquantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was carried out to detect the effect of the mutation on mRNA of the affected gene. Previous research demonstrated that biallelic loss-of-function mutations in CFAP43 accounted for the majority of all CFAP43-mutant MMAF patients. To the best of our knowledge, this is the first study to report CFAP43 biallelic loss-of-function mutations in a Pakistani population with the MMAF phenotype. This study will help researchers and clinicians to understand the genetic etiology of MMAF better.

Keywords: cilia and flagella-associated proteins; male infertility; multiple morphological abnormalities of the sperm flagella; whole-exome sequencing.

Figure 1

Pedigree of (a) Family 1 and (b) Family 2. Two Pakistani families with three infertile patients were recruited. I, II, III, and IV represent generation 1, 2, 3, and 4, respectively. Squares represent males, circles represent females, diamonds indicate offspring, and the inside numerals indicate the number of offspring. The slashes denote deceased family members. Solid squares indicate patients. Parallel slash lines indicate consanguineous marriage. Red arrows indicate the members selected for WES. WES: whole-exome sequencing.

Figure 2

Morphology and transmission electron microscopic analysis of spermatozoa from normal control and infertile patients. (a) Most spermatozoa of patients (middle and right panels) presented abnormal sperm flagella (^*), compared with control spermatozoa (left panel). Scale bars = 10 μm. (b) Cross-section of fertile male spermatozoa (left panel). An axoneme of a fertile male’s spermatozoa comprised DMTs circularly arranged around a CPC of microtubules (9 + 2 organization), surrounded by ODFs and FS. Cross-section of the patient II:1 of Family 1 (CFAP43-deficient), see right panel. Spermatozoa display totally disorganized axoneme; outer dense fibers and peripheral microtubules are misarranged. The central pair is displaced. Scale bars = 500 nm. DMTs: doublets of microtubules; CPC: central pair complex; ODF: outer dense fiber; FS: fibrous sheath; CFAP: cilia and flagella-associated protein.

Figure 3

Sanger sequencing results of CFAP43 mutations in DNA and mRNA levels. Chromatograms of the CFAP43 mutations from (a) Family 1 and (b) Family 2. Red/Blue arrows show the genomic position of CFAP43 mutations. (c) SqRT-PCR analysis of CFAP43 mRNA levels in male control and Family 2-IV:3 sperm samples. SqRT-PCR: semiquantitative reverse-transcriptase polymerase chain reaction; CFAP: cilia and flagella-associated protein; bp: base pair; Ref: reference; Het: heterozygous; chr10: chromosome 10; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; del: deletion.

Figure 4

The identified mutations in CFAP43 gene and predicted mutant proteins. CFAP43 gene structure (Ensembl transcript ID: ENST00000357060) is shown with mutations identified in both families. Vertical bars indicate exons and slashed lines represent introns. CFAP43 (1665 AA) comprises two domains: WD (tryptophan-aspartic acid (W-D) repeat domain and SMC_N coil domain. CFAP: cilia and flagella-associated protein; AA: amino acid; SMC_N: N-terminus of structural maintenance of chromosome; del: deletion.

Figure 5

Summary of all reported CFAP43 mutations in MMAF patients. (a) All compound heterozygous mutations are listed above the gene map; horizontal connections represent two mutations identified in one patient. All homozygous mutations are listed below the gene map. Red ones indicate the mutations identified in current study. (b) Statistic of all CFAP43 mutations. CFAP: cilia and flagella-associated protein; MMAF: multiple morphological abnormalities of the sperm flagella; del: deletion; WD: tryptophan-aspartic acid (W-D).