Two DNAH5 mutations in a Kartagener syndrome patient with azoospermia

Dear Editor,

A 34-year-old male presented to West China Hospital due to a 6-year history of infertility. The patient had experienced recurrent respiratory tract infections since early childhood and had been previously diagnosed with primary ciliary dyskinesia (PCD) at an external institution. CT imaging confirmed situs inversus totalis, establishing a diagnosis of Kartagener syndrome [1]. At our facility, a semen analysis was performed following a 7-day abstinence period, which revealed a complete absence of spermatozoa (azoospermia). Hormonal evaluation demonstrated the following peripheral blood levels: luteinizing hormone 7.88 IU/L, follicle-stimulating hormone 4.3 IU/L, estradiol 117 pg/mL, prolactin 9.42 ng/mL, progesterone 0.58 ng/mL, and testosterone 6.75 ng/mL. Inhibin B concentration was 128.25 pg/mL. These indicate that only the estradiol level was significantly elevated compared to healthy males. In addition, Doppler ultrasonography revealed no abnormalities in the testes, epididymis, or spermatic cord. Transrectal ultrasonography also detected no abnormalities in the seminal vesicles. While the diagnosis of azoospermia was preliminarily confirmed, the underlying etiology remains undetermined and requires further investigation.

Further peripheral blood chromosomal karyotype analysis revealed a 46,X,Y (qh⁺) chromosomal pattern. Polymerase chain reaction confirmed intact loci in all azoospermia factor (AZF) regions: sY84 and sY86 (AZFa), sY127 and sY134 (AZFb), sY254 and sY255 (AZFc), along with the sex-determining region Y gene, demonstrating no evidence of Y-chromosome microdeletions. Bilateral testicular tissue biopsy revealed severe seminiferous tubular epithelial atrophy with complete absence of germ cells. Histopathological examination showed markedly thickened basement membranes. As PCD represents an inherited ciliopathy affecting both mucociliary clearance and sperm flagellar motility [2], whole-exome sequencing was recommended to investigate potential pathogenic variants in cilia-related genes. Genetic counseling was advised given the syndromic nature of the condition and its implications for assisted reproductive technologies. This patient was of East Asian descent, specifically from the Han Chinese ethnic group.

Following informed consent acquisition, genomic DNA was isolated from peripheral blood mononuclear cells using standard protocols. Libraries were prepared through enzymatic fragmentation and polymerase chain reaction amplification, with exome capture performed using the Roche KAPA HyperExome® kit (Roche, Pleasanton, CA, USA) targeting coding regions and splice junctions (±20 bp). High-throughput sequencing was conducted on the MGISEQ-2000 platform (MGI Tech, Shenzhen, China) (150 bp paired-end reads). The sequencing reads were aligned to the Genome Reference Consortium Human Build 37 reference genome via Burrows-Wheeler Aligner (Broad Institute, Cambridge, MA, USA), and duplicate reads were removed. The Genome Analysis Toolkit (Broad Institute, Cambridge, MA, USA) was used for detecting single nucleotide variants, insertion and deletion, and genotyping. The whole-exome sequencing identified two heterozygous mutations in the exon region of the Dynein Axonemal Heavy Chain 5 (DNAH5) gene, namely NM_001369.3: c.5658C>G (p.Y1886∗) and NM_001369.3: c.6305G>A (p.R2102H). The two identified mutations were further verified by Sanger sequencing (Fig. 1A and B). In gnomAD v4 exome, the allele frequency is 0.000003420 (0.00002520 in East Asian) for NM_001369.3: c.5658C>G (p.Y1886∗) and 0.000002052 (0.00002519 in East Asian) for NM_001369.3: c.6305G>A (p.R2102H).

Figure 1.

Whole-exome sequencing identification of two DNAH5 variants. (A) A heterozygous nonsense mutation in DNAH5 (NM_001369.3: c.5658C>G, p.Y1886∗) located in coding exon 34, with a C-to-G substitution at nucleotide position 5658 converting the codon for tyrosine (UAC) to a stop codon (UAG) and terminating protein synthesis; (B) A heterozygous missense mutation in DNAH5 (NM_001369.3: c.6305G>A, p.R2102H) located in coding exon 38, with a G-to-A substitution at nucleotide position 6305 converting the codon for arginine (CGC) to the codon for histidine (CAC).

As a heterozygous nonsense mutation, NM_001369.3: c.5658C>G (p.Y1886∗) is located in coding exon 34 of the DNAH5 gene. This variant results in a C-to-G substitution at nucleotide position 5658, converting the codon for tyrosine (UAC) into a stop codon (UAG). Consequently, premature termination of protein synthesis occurs. In accordance with the American College of Medical Genetics and Genomics (ACMG) guidelines [3], this variant is classified as likely pathogenic (criteria: PVS1 [Pathogenic Very Strong] + PM2 [Pathogenic Moderate]). Additionally, a heterozygous missense mutation, NM_001369.3: c.6305G>A (p.R2102H), is located in coding exon 38 of the DNAH5 gene. This variant involves a G-to-A substitution at nucleotide position 6305, altering the codon for arginine (CGC) to the codon for histidine (CAC). Computational predictions using the Sorting Intolerant From Tolerant software (SIFT Analytics, San Francisco, CA, USA) suggest that this substitution is likely to impair the function of the DNAH5 protein (score: 0.001; pathogenic supporting). To enhance the reliability of this prediction, we further validated the variant using three additional computational tools: Polymorphism Phenotyping version 2 (Sunyaev Lab, Boston, MA, USA; score: 1.000, probably damaging), Combined Annotation Dependent Depletion (Kircher Lab, Seattle, Washington, USA; score: 33, pathogenic strong), and Rare Exome Variant Ensemble Learner (Narla Lab, Ann Arbor, MI, USA; score: 0.742, pathogenic supporting). All tools consistently supported a damaging effect of this variant. Specifically, the mutation affects a highly conserved coiled-coil domain, and molecular dynamics simulations indicate that it disrupts the stability of the α-helix structure. Based on the ACMG guidelines [3], this variant is also classified as likely pathogenic (criteria: PM2 + PM3_Supporting + PM5 + PP3 [Pathogenic Supporting] + BP1 [Benign Supporting]).

This mutational profile satisfies the ACMG pathogenic criteria, consistent with autosomal recessive transmission of PCD-associated mutations. The presence of two likely pathogenic variants supports their causative role in PCD and azoospermia. The compound heterozygosity pattern may explain the association between genotype and phenotype, but further evidence from whole-genome sequencing or Sanger sequencing is still needed. However, the patient's parents declined additional testing, citing a lack of therapeutic necessity. The informed consent for treatment and also for publication was obtained from the patient.

To date, mutations in more than 50 genes have been implicated in PCD, including DNAH5, DNAH11, and DNAI1 [4]. Among these, DNAH5 is the most frequently mutated gene, located on chromosome 5 (5p15–p14) [5]. DNAH5 is a critical gene encoding a component of the axonemal dynein complex, which is essential for the proper function of motile cilia [5]. These cilia play a vital role in various physiological processes, including sperm motility and embryonic development [6]. In the context of male infertility, DNAH5 mutations can disrupt the structure and function of sperm flagella, leading to asthenozoospermia or obstructive azoospermia. Notably, DNAH5 mutations have been implicated in impairing efferent duct ciliary motility, in addition to their possible role in sperm flagellar dysfunction, thereby contributing to the pathogenesis of male infertility [1,7]. These defects highlight the critical role of DNAH5 in male reproductive health. The five most commonly affected exons of the DNAH5 gene are 34, 50, 63, 76, and 77 [8]. In this study, we identified two mutations in exons 34 and 38 of DNAH5, which have not been previously reported in the literature. These findings expand the genetic variant landscape of PCD and its association with infertility, providing new insights into the molecular mechanisms and paving the way for improved diagnostic and therapeutic strategies.

Author contributions

Study concept and design: Yang Xiong, Mingyu Hu.

Data acquisition: Yang Xiong, Mingyu Hu.

Data analysis: Qing Jiang, Wei Wang, Jiuhong Yuan.

Drafting of manuscript: Yang Xiong, Yangchang Zhang.

Critical revision of the manuscript: Yangchang Zhang, Feng Qin.

Conflicts of interest

The authors declare no conflict of interest.