Novel bi-allelic mutations in DNAH1 cause multiple morphological abnormalities of the sperm flagella resulting in male infertility

Abstract

Background: Male infertility is a major health concern and approximately 10-15% of cases are caused by genetic abnormalities. Defects in the sperm flagella are closely related to male infertility, since flagellar beating allows sperm to swim. The sperm of males afflicted with multiple morphological abnormalities of the flagella (MMAF) possess severe defects of the sperm flagella, may impair sperm motility and lead to male infertility. Currently, known genetic defects only account for MMAF in about 60% of patients and need more intensive efforts to explore the relationship between genes and MMAF.

Methods: The whole-exome sequencing (WES) was performed to analyze the genetic cause of the MMAF patient. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to observe the morphology of sperm cells and to identify the ultrastructural characteristics of the flagella in the patient. The expression of DNAH1 was analyzed by sperm immunofluorescence staining.

Results: We identified the negative effects produced by the DNAH1 mutations c. 8170.C>T (p. R2724*) and c. 4670C>T (p. T1557M) on DNAH1 expression and the development of sperm flagella.

Conclusions: Our findings suggest that DNAH1 is associated with the formation of sperm flagella and homozygous loss-of-function mutations in DNAH1 can impair sperm motility and cause male infertility.

Keywords: Multiple morphological abnormalities of the flagella (MMAF); male infertility; whole-exome sequencing (WES).

Figure 1

The MMAF phenotype was identified in the patient. (A) Papanicolaou staining of patient sperm cells showed short, absent, coiled flagella and irregular caliber (scale bars, 5 µm). (B) The patient sperm flagella showed typical MMAF phenotype by SEM (scale bars, 5 µm). (C) TEM results of the patient showed the abnormal ultrastructure of sperm flagellum: the central microtubule was missing, and the outer microtubule doublet was disordered arrangement in midpiece, principal piece, and end piece (scale bars, 100 nm). MMAF, multiple morphological abnormalities of the flagella; SEM, scanning electron microscopy; TEM, transmission electron microscopy.

Figure 2

The bi-allelic pathogenic mutations of DNAH1 in a consanguineous family. (A) Family pedigree. Squares represent male pedigree members, circles represent female pedigree members, and the black square represents the proband. Open symbols represent unaffected members. (B) Sanger sequencing of (c. 4607C>T) and (c. 8170C>T) were confirmed by PCR-sequencing in this family. (C) Multiple sequence alignment of the DNAH1 protein for different species. The black arrow denotes the position of the variants. PCR, polymerase chain reaction.

Figure 3

The influence of the bi-allelic mutations on DNAH1 protein. (A) Immunofluorescence staining in the sperm cells reveals an absence of DNAH1 protein in the patient compared to the normal control. (green, α-tubulin; red, DNAH1; blue, DAPI; scale bars, 5 µm). (B) The spatial structure of DNAH1 was totally changed by the c. 4670C>T (p. T1557M) mutation, including random coils, α-helix and β-sheet.