Biallelic Variants in DNAH12 Gene Linked to Male Infertility: Two New Cases and Literature Review

Abstract

Background/Objectives: Although biallelic pathogenic variants in different DNAH gene family members have been associated with infertility, the role of DNAH12 in this disorder is still incompletely understood. To date, few patients have been shown to have infertility due to biallelic variants in this gene. Here, we report two more unrelated patients with infertility who carry homozygous variants in DNAH12. Methods: This study included two male patients with primary infertility and oligoasthenoteratozoospermia (OAT). Patient 1 was a 32-year-old with 1.5 years of infertility and no chronic illnesses or prior assisted reproductive technologies (ARTs). Patient 2 was a 49-year-old with 24 years of infertility, a history of varicocelectomy, and the occasional use of PRN analgesics for bone pain. Using genome sequencing, we identified two homozygous variants: c.3757C>A, p. Pro1253Thr, and c.11086-1G>A, p.?, in patients 1 and 2, respectively. Results: Our findings add supportive evidence that DNAH12 is a gene implicated in rare cases of male infertility. The identification of these homozygous variants in two additional patients supports the association between DNAH12 variants and reproductive dysfunction. Conclusions: This study highlights the need for further research on the role of DNAH12, including functional studies to clarify the mechanisms contributing to infertility.

1. Introduction

Infertility is a medical condition where couples are unable to achieve a clinical pregnancy even after engaging in regular and unprotected sexual intercourse for 12 months. It is a globally prevalent issue, affecting approximately 8 to 12% of couples in their reproductive years [1]. According to the latest definition by the World Health Organization (WHO), infertility is a disease that generates disability as an impairment of function [2]. Worldwide, more than 186 million people suffer from infertility, the majority being residents of developing countries [3]. Sperm dysfunction is the most common cause of infertility in men. Male infertility is diagnosed based on the presence of “oligozoospermia” (reduced sperm count), “asthenospermia” (decreased sperm motility), and “abnormal sperm” (sperm with an abnormal morphology) [4]. Different genes play a role at the various stages of spermatogenesis. The main gene families include the Deleted in Azoospermia (DAZ) gene family [5], the cilia- and flagella-associated protein (CFAP) gene family [6], and the Dynein Axonemal Heavy Chain (DNAH) family [7]. The DNAH gene family is essential for the structure and function of cilia and flagella, which are primarily involved in cell motility and ATP hydrolysis. Currently, there are 13 identified members of the DNAH gene family, all of which encode axonemal dynein heavy chain proteins. Among the DNAH family members, the role of DNAH12 is less well characterized. Recent studies involving four unrelated families have demonstrated that biallelic variants in DNAH12 can also lead to male infertility [8,9]. In sperm cells, DNAH12 is crucial for the proper organization of the axoneme, the structural core of the sperm tail. Mutations in DNAH12 disrupt the recruitment of other important proteins, like DNAH1 and DNALI1 [10]. Moreover, a DNAH12 knockout mouse exhibited severe spermatogenesis failure, confirming a similar male infertility phenotype [8]. A recent preprint study identified DNAH12 variants in 11 individuals from six unrelated families; however, this study is still unpublished [10]. Here, we report two unrelated families with infertility and biallelic variants in DNAH12. Our findings provide additional cases that reinforce the emerging evidence supporting the role of DNAH12 in male infertility.

2. Materials and Methods

2.1. Patient Recruitment

Two unrelated families presenting with predominant infertility were referred for genome sequencing. Biological samples were collected after obtaining informed consent from the patients. The informed consent included permission for the publication of de-identified clinical and genetic data.

2.2. Genome Sequencing

To select the disease-causing variants in our probands, we performed genome sequencing based on the description below. The genetic variants are described following the Human Genome Variation Society (HGVS) recommendations (www.hgvs.org accessed on 12 December 2024). The selected variants were classified according to the ACMG/ClinGen guidelines.

The DNA was extracted from Buccal swabs of the probands, and the TruSeq NanoDNA High Throughput Library Prep Kit (Illumina®; San Diego, CA, USA) was used to prepare the libraries, which were sequenced using the 150nt pair-end protocol on an Illumina platform to yield an average coverage depth of 30x for the nuclear genome and at least 1000x for the mitochondrial genome. Raw read alignments to the reference genome GRCh38 and variant calling, including single nucleotide substitutions (SNVs), small insertions/deletions (Indels), and structural variants (SVs) with default parameters, were performed using DRAGEN (version 4.2.4, Illumina). The SNV and Indel variant annotation was performed by Geneyx (https://geneyx.com accessed on 15 December 2024). The structural variants were annotated with ANNOTSV3.1 and in-house structural variant databases to obtain the allele frequencies. For the mitochondrial genome, variants with frequencies/heteroplasmy levels ≥ 5% were detected.

3. Results

3.1. Clinical Assessment

A clinical summary of the affected members from the two families is presented in

Table 1. Semen analysis case 1.

Occasions	First Time	Second Time	Third Time
Volume	5 mL	4 mL	6.5 mL
Sperm count	12 million/mL	5 million/mL	20 million/mL
Total motility	20%	30%	30%
Progressive motility	5%	10%	5%
Morphology	Normal: 1%	Normal: 1%	Normal: 1%
Occasions	First time	Second time	Third time
Volume	5 mL	4 mL	6.5 mL
Sperm count	12 million/mL	5 million/mL	20 million/mL
Total motility	20%	30%	30%
Progressive motility	5%	10%	5%

. The details of the clinical information of the patients are as follows.

3.2. Case 1

A 32-year-old male presented with primary infertility lasting for 1.5 years and was diagnosed with oligoasthenoteratozoospermia (OAT) based on his semen analysis (Table 1). The patient had no history of chronic illnesses or mumps infections, and his general physical examination was unremarkable. No assisted reproductive techniques (ARTs) had been utilized. There were no recurrent respiratory symptoms reported. His parents are cousins. On examination, the patient’s height was 170 cm, and his weight was 79 kg. Mild gynecomastia was noted, but his body hair distribution was normal. A genital examination was performed, which revealed normal genitalia and phallus. The testes were measured to be 25–30 cc each and showed bilateral small epididymal head cysts. Additionally, a bilateral grade II varicocele was observed. His hormone levels, including the total testosterone, LH, FSH, prolactin, and estradiol, were all within the reference ranges (

Table 2. Hormonal profile case 1.

Hormones	Result
Estradiol (E2)	27 pg/mL
Testosterone (T)	20.18 nmol/L
Luteinizing hormone (LH)	2.33 mIU/mL
Follicle-stimulating hormone (FSH)	3.42 mlU/mL
Prolactin (PRL)	6.03 ng/mL
Hormones	Result
Estradiol (E2)	27 pg/mL
Testosterone (T)	20.18 nmol/L
Luteinizing hormone (LH)	2.33 mIU/mL
Follicle-stimulating hormone (FSH)	3.42 mlU/mL
Prolactin (PRL)	6.03 ng/mL

).

3.3. Case 2

A 49-year-old male presented with primary infertility persisting for 24 years. He has been diagnosed with severe oligoasthenoteratozoospermia (OAT) based on his semen analysis (

Table 3. Semen analysis case 2.

Investigation	Results
Volume	4 mL
Concentration	1 million/mL
Motility	0% motile sperm
Morphology	90% abnormal forms

). He denies any recurrent respiratory symptoms or chronic illness. He is married to a 41-year-old woman with a normal gynecological workup. The couple has undergone three IVF/ICSI cycles, all using frozen sperm. Despite the embryo transfer in each cycle, no pregnancies have resulted. The family history reveals positive consanguinity and a significant occurrence of infertility affecting both males and females, including two brothers and three sisters. A physical examination revealed testes approximately 10 cc in volume, with a possible grade II varicocele on the left side but no other abnormalities. His hormone levels, including the total testosterone, LH, FSH, prolactin, and estradiol, are illustrated in (

Table 4. Hormonal profile case 2.

Investigation	Results
Estradiol (E2)	39 pg/mL
Testosterone (T)	18.03 nmol/L
Luteinizing Hormone (LH)	4.19 mIU/mL
Follicle-Stimulating Hormone (FSH)	14.2 mIU/mL
Prolactin (PRL)	8.23 ng/mL

).

4. Genetic Analysis

The DNA from both probands was subjected to genome sequencing. The TruSeq Nano DNA High Throughput Library Prep Kit (Illumina^®) was used to prepare the libraries, which were sequenced using the 150nt pair-end protocol on an Illumina platform to yield an average coverage depth of 30x for the nuclear genome. Raw read alignments to the reference genome GRCh38 were performed. No other genetic variants in the known genes associated with infertility were found in either proband. However, novel variants in the DNAH12 gene (NM_001366028.2) were identified. The proband in family 1 was found to carry a novel homozygous missense variant (c.3757C>A, p. Pro1253Thr), located in exon 25. This variant was found with a minor allele frequency (MAF) of 0.0000066 in gnomAD (10 carriers and no homozygotes). This variant is not listed in ClinVar. The CADD [11] and REVEL [12] scores for this variant were 25.8 and 0.63, respectively. The proband in family 2 was identified as carrying a novel homozygous splicing variant (c.11086-1G>A, p.?), affecting the canonical site, and the skipping of exon 69 out of 74 by this variant causes a frameshift. The variant is located in intron 68, and the minor allele frequency (MAF) is zero (absent) in gnomAD. The variant is not listed in ClinVar (see Figure 1).

5. Discussion

The DNAH gene family is essential for the structure and function of cilia and flagella. To date, 13 members of this gene family have been implicated in either male infertility or primary ciliary dyskinesia (PCD), although the role of DNAH12 has been less thoroughly characterized. Previously, Oud et al. reported a case of infertility associated with DNAH12 variants; however, the trans status was unconfirmed [13]. Li et al. later described a patient carrying biallelic DNAH12 variants with multiple morphological abnormalities of the sperm flagella (MMAF) [9]. Most recently, Geng et al. [8] demonstrated in three unrelated patients—and confirmed with knockout mouse models—that the DNAH12 loss causes male infertility. A very recent preprint by Yang et al. [10] identified biallelic DNAH12 variants in 11 individuals from six families, providing further functional support that DNAH12 is necessary for sperm flagellar organization but dispensable for respiratory ciliary function. Our study adds supportive evidence by describing two unrelated cases carrying novel homozygous variants in DNAH12. Both cases presented with severe oligoasthenoteratozoospermia but without recurrent respiratory infections, consistent with the previous reports suggesting that DNAH12 mutations primarily affect the sperm flagella rather than cilia. Importantly, our findings should be interpreted cautiously due to certain limitations. Functional validation—such as transmission electron microscopy to assess the flagellar ultrastructure, immunofluorescence staining, or RNA splicing analysis—was not performed. Similarly, segregation studies in family members were not conducted due to patient refusal, which constrains the ability to definitively confirm the pathogenicity of the identified variants. Although the association between DNAH12 and male infertility is increasingly supported, DNAH12 should currently be considered as a gene contributing to rare cases of male infertility, rather than a major, frequent cause. We recommend further studies with functional analyses and broader cohort investigations to establish DNAH12’s full clinical significance. Regarding assisted reproduction, it is noteworthy that in our study, one patient had undergone three unsuccessful ICSI attempts, whereas previous reports have shown successful pregnancies following ICSI in cases with DNAH-family gene mutations [8,10]. This highlights the need for larger series to determine the ICSI outcomes in DNAH12-associated infertility. Additionally, our observation that several female relatives in family 2 were infertile raises the possibility that DNAH12 might also play a role in female reproductive function, although further studies are needed to investigate this hypothesis.

6. Limitations

Due to the lack of available patient samples and patient consent, functional validation studies (e.g., sperm ultrastructural analysis, immunostaining, cDNA splicing assays) and family segregation analyses were not performed. These limitations reduce the strength of the causal inference between the identified DNAH12 variants and the observed infertility phenotype. Future studies addressing these gaps are necessary to further validate DNAH12’s pathogenic role.