A novel loss-of-function variant in DNHD1 linked to human asthenozoospermia
Article information
Abstract
Objective
Asthenozoospermia, characterized by diminished sperm motility, shows substantial genetic heterogeneity. Although many familial cases of asthenozoospermia have been documented, the genetic etiology for most affected males remains unknown.
Methods
This study used clinical and molecular approaches to characterize a hereditary form of asthenozoospermia. Two patients born to consanguineous parents exhibited impaired sperm motility. Whole-genome sequencing (WGS), followed by Sanger sequencing, was performed for molecular diagnosis and was coupled with three-dimensional protein modeling to characterize the identified variant.
Results
WGS revealed that both affected brothers carried a pathogenic variant (c.580C>T; p.Gln194*) in dynein heavy chain domain 1 (DNHD1). Homozygous variants in DNHD1 have previously been linked to spermatogenic failure 65 (Online Mendelian Inheritance in Man [OMIM] 619712). Three-dimensional protein modeling demonstrated significant alterations in DNHD1 secondary structure that could contribute to loss-of-function.
Conclusion
These findings support a role for DNHD1 in maintaining the structural and functional integrity of sperm. Accurate molecular diagnosis may facilitate genetic counseling and inform clinical management.
Introduction
Male infertility manifests as a broad spectrum of phenotypes and represents a complex condition with a substantial global burden. Approximately 10%–15% of couples attempting to conceive are affected by infertility, which has emerged as a global health concern [1]. Male factors account for roughly half of infertility cases and commonly present as azoospermia (complete absence of sperm), teratozoospermia (abnormal morphology), asthenozoospermia (reduced motility), or oligozoospermia (low sperm count). These sperm-related abnormalities may occur in combination. Asthenozoospermia is reported in approximately 60%–70% of male infertility cases [1,2].
Asthenozoospermia, a disorder of sperm motility, is a leading cause of male infertility [3]. Whole-genome and exome sequencing studies have shown that some cases of asthenozoospermia are caused by loss-of-function mutations in genes encoding key components of the sperm flagellum. The fibrous sheath (FS), centrosomes, axoneme (including the outer and inner dynein arms), intraflagellar transport (IFT) machinery, and radial spokes are all required for flagellar elongation and assembly. Deleterious mutations in genes involved in the biogenesis of these structures can lead to asthenozoospermia. Examples include the genes encoding leucine-rich repeat-containing 23, dynein axonemal heavy chain 1 and 2 (DNAH1 and DNAH2) [4], sperm-associated antigen 17 (SPAG17) [5], cilia- and flagella-associated protein 52 (CFAP52) and CFAP45 [6], sperm flagellar protein 2 (SPEF2) [7], and IFT-interacting proteins such as tetratricopeptide repeat domain 21A (TTC21A) and TTC29 [8,9]. Additional examples include the genes that encode FS components such as A-kinase anchoring protein 3 and 4 (AKAP3 and AKAP4) [10] and fibrous sheath-interacting protein 2 (FSIP2) [11]. Many of these genes have been identified in consanguineous families.
DNHD1, also known as coiled-coil domain-containing protein 35 (CCDC35), is located on chromosome 11p15.4 and spans 14,876 base pairs. DNHD1 encodes dynein heavy chain domain 1, a 4,753–amino-acid protein with five coiled-coil (CC) domains that is exclusively expressed in the sperm flagellum. The CCDC35 protein includes a highly conserved sequence with five CC domains (a superhelical protein motif), characterized by one or more α-helical peptides that form a supercoil by twisting around each other [11,12]. DNHD1 is a member of the CCDC family and is highly expressed in the testis. It is presumed to be important for normal axonemal function, and DNHD1 abnormalities have been linked to severe asthenoteratozoospermia, also known as spermatogenic failure 65 (Online Mendelian Inheritance in Man [OMIM] 619712) [12].
In this study, we recruited a consanguineous family that underwent genetic and clinical evaluation. Clinical assessment indicated impaired sperm motility in the affected members, consistent with asthenozoospermia. To investigate the genetic basis of this condition, we performed whole-genome sequencing (WGS) to identify candidate variants associated with male infertility. We identified a novel variant in DNHD1, a gene implicated in sperm flagellar structure, that was associated with asthenozoospermia in this family.
Methods
1. Patient recruitment and clinical assessments
For this investigation, we recruited a single family with two members affected by male infertility (Figure 1A). Detailed medical and family histories were obtained from both patients and participating controls. The affected individuals underwent clinical examination by an experienced clinician. Blood samples were collected from patients and controls for hormonal testing, karyotyping, and genetic analyses. Semen samples were also collected from both affected individuals for further evaluation. All participants provided written informed consent for clinical and genetic analyses and for the release of information and images, in accordance with the Declaration of Helsinki. The study was approved by the Institutional Review Board of the University of Management and Technology (UMT), Lahore, Pakistan (RC-2023/159). Informed consent was obtained from all subjects involved in the study.
(A) Pedigree of the family illustrating autosomal recessive inheritance; Sanger sequencing results are shown below each symbol. (B) Schematic representation of the dynein heavy chain domain 1 (DNHD1) protein and its domains; dotted lines indicate the location of the identified novel variant. (C) Partial DNHD1 amino acid sequence demonstrating conservation of Gln194 across species. (D) MetaDome analysis assessing tolerance at the variant position; the site was classified as intolerant, supporting a disease-causing effect. ATP, adenosine triphosphate.
2. Karyotyping and DNA extraction
Blood samples were obtained for karyotyping and DNA extraction. DNA extraction and quantification were performed using conventional techniques [13].
3. Y-chromosome microdeletion
To investigate Y-chromosome microdeletions across four azoospermia factor (AZF) regions (AZFa, AZFb, AZFc, AZFd), polymerase chain reaction was performed using primer pairs designed to amplify the targeted loci [14].
4. Whole-genome sequencing
WGS was performed using DNA from both affected individuals (IV-2 and IV-4). Sequencing procedures and variant filtering were performed as previously described [15]. We prioritized variants with predicted functional impact and variants previously reported in association with similar phenotypes, using standard screening procedures. Genes described in OMIM and PubMed were given priority.
5. In silico analysis
Several online tools were used to assess the pathogenic potential of the identified variant, including MutationTaster, VarSome, and Franklin. To determine whether the variant has been reported in the general population, the Exome Aggregation Consortium and Genome Aggregation Database were searched. Amino acid conservation was evaluated using the National Center for Biotechnology Information HomoloGene. The DNHD1 protein sequence was retrieved from UniProt (https://www.uniprot.org), and structural modeling was performed using SWISS-MODEL (https://swissmodel.expasy.org). PyMOL version 3.0.4 (The PyMOL Molecular Graphics System, Schrödinger) was used for figure visualization and processing.
6. Sanger sequencing
Segregation of the variant was assessed using standard Sanger sequencing. Variant-specific primers were designed, and Sanger sequencing was performed for all available family members [16].
Results
1. Clinical description
A consanguineous family with two patients affected by asthenozoospermia (IV-2, 24 years; and IV-4, 27 years) was recruited from Punjab Province, Pakistan (Figure 1A). Both individuals were married to reproductively healthy women and reported engaging in unprotected intercourse. The two affected males (IV-2 and IV-4) were born to a first-cousin consanguineous union.
Both affected males had average height and weight, normal external genitalia, and normal secondary sexual characteristics. Intellectual function was reported as normal, and there were no clinical features suggestive of ciliopathy, such as primary ciliary dyskinesia. Semen analysis was performed according to the World Health Organization 2021 criteria and indicated impaired sperm motility consistent with asthenozoospermia. Follicle-stimulating hormone and luteinizing hormone levels were unremarkable in both patients (Table 1). Karyotyping showed a normal male karyotype (46,XY), and no Y-chromosome microdeletions (AZFa, AZFb, AZFc, AZFd) were detected.
Clinical data of affected individuals (IV-2, IV-4)
2. Molecular investigation
Whole-genome sequencing and variant filtering were performed using previously published procedures [15]. After screening homozygous and compound heterozygous candidates, we identified a novel homozygous stop-gain variant in DNHD1 (c.580C>T; p.Gln194*) (Figure 1A, 1B). This variant converts glutamine (Gln) at position 194 to a premature stop codon, resulting in truncation of the DNHD1 protein (Figure 2). In Figure 2, the cyan region indicates the portion of the protein predicted to be lost due to truncation. This variant may also result in nonsense-mediated mRNA decay, potentially causing complete loss of DNHD1 function. More than 2,000 control exomes were screened, and the affected residue was conserved across the evaluated species (Figure 1C). MetaDome tolerance landscape analysis further indicated that c.580C>T (p.Gln194*) lies within an intolerant (functionally constrained) region, suggesting low tolerance to variation at this position and supporting a pathogenic role (Figure 1D). According to the American College of Medical Genetics and Genomics classification, this variant was categorized as pathogenic (PM3, PP5, PM2).
Three-dimensional representation of the wild-type(WT) and variant dynein heavy chain domain 1 (DNHD1) proteins. (A) WT DNHD1 protein model. (B) Variant DNHD1 protein model; the cyan region is absent, and the remaining translated region is shown in red. The truncation is predicted to impair normal function and may result in protein degradation.
3. Three-dimensional modeling of DNHD1
A partial DNHD1 amino acid sequence was modeled using SWISS-MODEL to evaluate the predicted impact of the stop-gain variant (p.Gln194*). The analysis suggested that Gln at position 194 contributes to local structural stability through interactions with surrounding residues (Figure 2). Introduction of a premature stop codon at this position would eliminate the downstream sequence and disrupt these interactions, yielding a truncated protein. This truncation is expected to result in an unstable protein that may be degraded or unable to perform its normal function, consistent with the severe phenotype observed in the affected individuals.
Discussion
In this study, we identified a novel homozygous stop-gain variant in DNHD1 in two affected brothers. The variant converts Gln (Q) at position 194 to a premature stop codon (p.Gln194*), which may lead to nonsense-mediated decay. Because this loss-of-function variant occurs in the first half of the DNHD1 protein, it may result in a marked reduction or absence of functional DNHD1 protein (Figure 1B). To date, 14 DNHD1 variants have been reported in association with asthenoteratozoospermia (Table 2).
Men with biallelic DNHD1 variants have been reported to exhibit classic asthenoteratozoospermia, including reduced sperm motility and abnormal sperm flagella. DNHD1 variants may be associated with inter-individual variability in semen parameters, including sperm quantity and motility. In the present study, both patients showed asthenozoospermia with reduced sperm motility, consistent with previous reports [12,17].
Several genes implicated in asthenoteratozoospermia—including CFAP47 (OMIM 301059), DNAH10 (OMIM 605884), TTC29 (OMIM 618735), DNAH8 (OMIM 603337), DNAH17 (OMIM 610063), and DNAH2 (OMIM 603333)—have also been linked to similar sperm phenotypes. Males with DNHD1 variants may exhibit reduced sperm motility for multiple reasons, including variant type and location, affected domains, phenotypic heterogeneity, lifestyle factors, and environmental exposures [12].
The flagellar axoneme is the core motility apparatus of spermatozoa and has a ‘9+2’ structure consisting of a central pair (CP) surrounded by nine doublet microtubules, each composed of A- and B-tubules [18]. Previous studies have shown that abnormalities in proteins involved in the axoneme and periaxoneme are associated with asthenoteratozoospermia [19]. In a prior study, transmission electron microscopy showed that, among individuals with biallelic DNHD1 variants, ultrastructural abnormalities were present in nearly 95% of axonemal cross-sections; the most common defects were axonemal disarray and absence of the CP. Rawe et al. [19] further reported that immunofluorescence analyses showed that SPAG6 and SPEF2 were nearly absent in sperm from men with biallelic DNHD1 variants, suggesting that DNHD1 may act in concert with axonemal proteins and is required for normal flagellar assembly.
Similarly, male Dnhd1−/− mice were reported to be infertile, with multiple flagellar abnormalities and immotile sperm. In one report, >95.5% of sperm from Dnhd1−/− mice exhibited flagellar defects, compared with 6.0% in Dnhd1+/+ mice. Motility analyses indicated reduced motility in Dnhd1−/− males; these animals also showed lower sperm concentration. Similar findings have been reported in mouse models involving other genes, including Dnah10, Cfap58, and Cfap47 [19,20].
There is no established pharmacologic therapy that reliably improves semen parameters in asthenozoospermia, and pregnancy is typically achieved using intracytoplasmic sperm injection (ICSI). ICSI was recommended for seven individuals with biallelic DNHD1 variants in a prior report; however, four of these individuals reportedly conceived naturally. Accordingly, Tan et al. [12] suggested that males with DNHD1-associated asthenozoospermia should consider ICSI.
In conclusion, we identified a novel homozygous pathogenic variant in DNHD1 in a family with male infertility characterized by asthenozoospermia. Genetic findings from the affected individuals support the involvement of DNHD1 in sperm motility and advance understanding of the molecular mechanisms underlying asthenozoospermia in humans.
Notes
Conflict of interest
No potential conflict of interest relevant to this article was reported.
Acknowledgments
Genetic data have been uploaded to the Leiden Open Variation Database and will be made publicly available upon acceptance of the manuscript. Additional data will be provided upon reasonable request.
The authors are thankful to the family for their cooperation and participation in the study.
Author contributions
Conceptualization: SN, RK, MU. Methodology: SN, SS. Formal analysis: AN(Abdul Nasir), HK. Data curation: AN(Abdul Nasir), HK. Validation: SN, AN(Anam Nayab). Supervision: RK, MU. Writing-original draft: SN, SS, AN(Anam Nayab). Writing-review & editing: RK, MU. Approval of final manuscript: RK, MU.