A novel splicing variant in DNAH8 causes asthenozoospermia

Abstract

Purpose: To identify the genetic factors responsible for asthenozoospermia, which is a major cause of male infertility characterized by immotile and malformed spermatozoa.

Methods: Whole-exome sequencing was performed in two brothers from a family with asthenozoospermia to identify pathogenic variants. The functional effect of the identified variant was investigated in HEK293T cells using a minigene assay.

Results: We identified a novel homozygous splicing variant c.6311-2A>G in DNAH8 from two affected brothers belonging to the same consanguineous family. The splicing variant altered a consensus splice acceptor site of DNAH8 intron 44, which led to the deletion of exon 45 and resulted in a frameshift and a predicted truncated protein (p.G2104Efs*19). Although most spermatozoa from the patients presented with reduced sperm motility, intracytoplasmic sperm injection was able to overcome the inability of the spermatozoa to reach the ovum and thus produce a healthy child for the proband.

Conclusions: This finding expands the mutational spectrum of DNAH8, making it a potential genetic diagnostic marker for those suffering from primary male infertility.

Keywords: Asthenozoospermia; DNAH8; Male infertility; Variant.

Fig. 1

Identification of the variant in DNAH8. a The pedigree affected by male infertility with Sanger sequencing confirmations to the right. Both affected males had a homozygous variant in DNAH8. Squares denote male members, circles denote females, diamonds denote unknown gender, and solid squares refer to affected individuals. Question marks indicate unavailable DNA samples, and the double lines indicate consanguinity. b Homozygosity mapping of individuals II-1 (upper panel) and II-3 (lower panel) in family 1. Red regions suggest homozygous regions harboring strong signals. The asterisk (*) denotes the area in or around where DNAH8 is located

Fig. 2

The location and conservation of the mutated residue in DNAH8. a The location of variants in the DNAH8 exons and the predicted protein domains in DNAH8 according to Pfam (https://pfam.xfam.org/). Variants described in the previous study [11, 12] are marked in purple and novel variant identified in this study is marked in green. b The conservation analysis of the mutated residue in the conserved AAA_6 domain of DNAH8 among mammalian species was performed using Multiple Sequence Alignment (https://www.ebi.ac.uk/Tools/msa/muscle/). AAA_6, hydrolytic ATP binding site of dynein motor region. c Agarose gel electrophoresis illustrating the effect of the DNAH8 c.6311-2A>G variant. Compared with the wild-type lane, the c.6311-2A>G lane showed a smaller band. d A possible scheme illustrating of the effect of the DNAH8 c.6311-2A>G variant. In this case, the c.6311-2A>G skips the canonical receptor site leading to the entire deletion of exon 45 in cDNA and resulting in premature termination of the protein