Novel Biallelic DNAH1 Variations Cause Multiple Morphological Abnormalities of the Sperm Flagella

Abstract

Sperm motility is vital to human reproduction, and malformed sperm flagella can cause male infertility. Individuals with multiple morphological abnormalities of the flagella mostly have absent, short, coiled, bent, and/or irregular-caliber flagella. In this study, a patient with male infertility underwent a physical examination along with his wife. Genetic testing was performed by whole-exome sequencing of the couple, and Sanger sequencing was performed for validation. Novel biallelic variations in the DNAH1: (NM_015512.4) gene consisting of c.1336G>C (p.E446Q) and c.2912G>A (p.R971H) were identified. In silico structural analysis revealed that the amino acid residues affected by the variation were evolutionarily conserved, and the variant p.R971H influenced the stability of the DNAH1 protein. Morphological studies of the patient's sperm showed defects in its flagella. Results of Papanicolaou staining and scanning electron microscopy demonstrated coiled and short flagella with multiple anomalies. Transmission electron microscopy of the sperm flagella showed that the inner dynein arm and radial spoke were absent, and the dense fiber and microtubule doublets were displaced. Quantitative PCR of the mRNA of the patient's sperm showed that the expression of DNALI1 was dramatically reduced. Collectively, these findings elucidated the genetic cause of the family's infertility and provided insight into the functioning of the DNAH1 gene.

Introduction

Male infertility is one of the major issues of human health and affects >20 million people worldwide (Boivin et al., 2007). Eight to twelve percent of all couples suffer from infertility, and male partners account for approximately half of the cases (Agarwal et al., 2021). Sperm motility is vital to human reproduction, and malformations of the sperm flagella can cause infertility in males (Chemes and Rawe, 2003).

The DNAH1 gene encodes the heavy chain of an inner dynein arm that provides structural support between the radial spokes and the outer doublet of the flagella or cilia (Ben Khelifa et al., 2014). Spermatozoa of mice with a homozygous knockout of the orthologous DNAH1 gene had dramatically reduced velocity and motility, resulting in infertility (Neesen et al., 2001). Mutations in the DNAH1 were determined to cause primary ciliary dyskinesia (PCD; MIM #617577) (Imtiaz et al., 2015), and also contribute to multiple malformations of the sperm flagella leading to a spermatogenic failure (MIM #617576) (Ben Khelifa et al., 2014; Amiri-Yekta et al., 2016; Wang et al., 2017). PCD is a genetically heterogeneous ciliopathy caused by ultrastructural defects in ciliary or flagellar structures, characterized mainly by recurrent respiratory infections progressing to permanent lung damage and infertility (Leigh et al., 2009). However, some PCD patients showed sperm abnormalities only, suggesting analogous molecular mechanisms, which only affect sperm flagella. Since this phenotype is restricted to fertility, multiple morphological abnormalities of the flagella (MMAF) was used to describe this novel clinical phenomenon (Ben Khelifa et al., 2014).

Individuals with MMAF generally have a series of malformations of the sperm flagella, which include absent, short, coiled, bent, and/or irregular-caliber flagella (Ben Khelifa et al., 2014; Coutton et al., 2015; Yang et al., 2015). Since 2014, several groups of researchers have reported patients affected by MMAF, which showed absent, short, bent, coiled, and/or irregular sperm tails (Ben Khelifa et al., 2014; Amiri-Yekta et al., 2016; Sha et al., 2017; Wang et al., 2017; Coutton et al., 2019; Li et al., 2019; Tu et al., 2019). MMAF was speculated to be an autosomal recessive hereditary condition (Ben Khelifa et al., 2014). These studies suggested that MMAF was caused by genetically heterogeneous mutations, but further genetic pathologies need to be explored to understand the origin of MMAF completely.

In this study, we recruited a patient with male infertility caused by MMAF. A combination of clinical and laboratory techniques was used to find the cause. We identified novel biallelic DNAH1 variants consisting of c.1336G>C (p.E446Q) and c.2912G>A (p.R971H). In silico analysis and electron microscopy were performed to provide additional evidence for the pathogenicity of the identified variants. Our study strengthened the significance of DNAH1 genotyping for patients with MMAF-associated infertility.

Materials and Methods

Patient selection

This study was approved by the Ethics Committee of Beijing Jiaen Hospital. All participants had signed written informed consent for clinical examination, genetic analysis, and data publication.

The selected patient was a 34-year-old man, and his wife was 32 years old. They had a 4-year history of infertility. The physical features and sexual functions of both were normal (Supplementary Tables S4 and S5). The couple denied any PCD-related symptoms in their family. This couple received a comprehensive clinical and laboratory examination. Control subjects were healthy males whose wives had given birth to healthy babies. Parameters of semen were evaluated according to the World Health Organization (WHO) laboratory manual (fifth edition) for the examination and processing of human semen. Fixation and staining of sperm were performed according to the Papanicolaou method to highlight their details. Computer-aided sperm analysis system (CASA) and computer-aided sperm morphometric assessment (CASMA) were used to evaluate the various parameters of sperm.

Genetic detection

Conventional G-banding karyotyping was performed on peripheral blood samples from the couple according to the AGT (American Genetic Technician) cytogenetics manual (Arsham et al., 2017). Genomic DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Germany). Chromosomal microarray analysis was performed on the CYTOSCAN HD platform (Affymetrix) to analyze genome-wide copy-number variations (CNVs).

Whole-exome sequencing (WES) was applied to identify point mutations and InDels (insertions and deletions) responsible for human MMAF. DNA fragments were hybridized and captured by the xGen Exome Research Panel (Integrated DNA Technologies). The NovaSeq 6000 platform (Illumina), along with ∼150 bp pair-end reads, was used for the genomic sequencing of DNA with ∼300 pM per sample using NovaSeq Reagent Kit. Sequenced raw reads (Quality level: Q30 > 90%) were aligned to the human reference genome (accession no. hg19/GRCh37; ftp://hgdownload.cse.ucsc.edu/golden-Path/hg19/chromosomes/) using the Burrows-Wheeler Aligner tool, and the PCR duplicates were removed using Picard v1.57. Variant calling was performed with the Verita Trekker^® Variants Detection system (v2.0; Berry Genomics, Inc.) and Genome Analysis Toolkit. Variants were then annotated and interpreted using ANNOVAR (v2.0) and Enliven^® Variants Annotation Interpretation systems (Berry Genomics, Inc.) (Wang and Hakonarson, 2010), based on the common guidelines of the American College of Medical Genetics and Genomics (ACMG) (Richards et al., 2015).

For the interpretation of pathogenicity, we referred to three frequency databases (1000G_2015aug_eas, ExAC_EAS, gnomAD_exome_EAS) and the HGMD proV2019 (Human Gene Mutation Database). Revel score (Ioannidis et al., 2016) (a combined method of pathogenicity prediction) and pLI score (representing the tolerance for truncating variants) were also used. Sanger sequencing was performed as a confirmatory test using the 3500DX Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific). The PCR primers for sequencing are listed in Supplementary Table S1.

In silico structural analysis

The evolutionary conservation of the affected amino acid (AA) residues was analyzed using MEGA7 with default parameters. WEBLOGO was used to analyze residue consensus. SWISS-MODEL was used to model the domains containing the two variants.

Analysis using scanning electron microscopy and transmission electron microscopy

For the scanning electron microscopy (SEM) analysis, the sperm specimens were immersed, rinsed, post-fixed, and dehydrated using conventional methods. Then, the specimens were analyzed by SEM (Stereoscan 260, United Kingdom) at an accelerating voltage of 20 kV.

For the transmission electron microscopy (TEM) analysis, sperm cells were fixed routinely. After the cells were embedded, ultrathin sections were stained with uranyl acetate and lead citrate, and observed and photographed by TEM (TECNAI-10, Philips) at an accelerating voltage of 80 kV.

Quantitative PCR analysis

To investigate the impact of the variation of gene expression in sperms, sperm mRNA was extracted, and the relative levels of expression of DNAH1, DNAH12, DNALI1, and DNAI2 were measured by Quantitative PCR (QPCR) using a 7500 Fast Real-Time PCR instrument (Applied Biosystems) (primer sequences listed in Supplementary Table S2).

Results

Genetic findings

Both patients II-1 and II-2 had normal karyotypes, and no CNV of clinical significance was identified (Supplementary Fig. S1).

Compound heterozygous variants of DNAH1 (NM_015512.4) were identified in patient II-1 (Fig. 1B), namely exon9: c.1336G>C (p.E446Q) and exon18: c.2912G>A (p.R971H). Details of the variants are provided in Table 1.

FIG. 1.

Pedigree diagrams and genetic variants of the family. (A) The pedigree was diagrammed for the family. Traits carried by parents are displayed in different colors. (B) Raw data of detected variants from WES were visualized using IGV. (C) The detected variants in the family are shown in sequence and peak patterns in forward complementary sequence. Different color blocks of the variants corresponded to their inheriting pattern in the pedigree. IGV, Integrative Genome Browser; WES, whole-exome sequencing.

Table 1.

Data of Diagnostic Variations Detected in This Study

Carriers ID.	Gene^*	Chromosome location^#	Exon	cDNA alteration	Protein alteration	Frequency in three databases^a	Mutation type	HGMD^b	LRT	Mutation Taster	SIFT	Polyphen2	ACMG^c
I-1, II-1	DNAH1	chr3:52378555	exon9	c.1336G>C	p.E446Q	0; 0; 0	Missense het variant	—	Deleterious	Disease causing	Tolerated	Benign	Likely pathogenic
I-2, II-1	DNAH1	chr3:52386608	exon18	c.2912G>A	p.R971H	0.000199681; 0.000200; 0.000186923	Missense het variant	—	Neutral	Polymorphism	Damaging	Probably damaging	Likely pathogenic

Gene^*: DNAH1 (NM_015512.4); Chromosome location ^#: GRCh37/hg19 position.

1000g2015aug (https://www.internationalgenome.org/); ExAC (http://exac.broadinstitute.org); gnomAD_exome (http://gnomad.broadinstitute.org/).

HGMD^®: Human Gene Mutation Database (Professional Version 2019.4).

ACMG: American College of Medical Genetics and Genomics.

Results of Sanger sequencing showed that the parents of the proband were both carriers of one variant each (Fig. 1A, C). Specifically, the father carried variant c.1336G>C, whereas the mother carried variant c.2912G>A.

In silico analysis

The location of the two variants is shown in Figure 2A. The affected AA residues, E⁴⁴⁶ and R⁹⁷¹ of the DNAH1 peptide, are evolutionarily conserved with high consensus across eight species (Fig. 2B). Only the domain containing the p.R971H variant was credibly modeled using the PDB: 6sc2.1 file as its wild-type template (Seq identity: 25.11%; Qualitative Model Energy Analysis: −4.63). The R971H variant, which replaced the arginine with a histidine, changed the number of hydrogen bonds from two to one at the side chain with E974, thus reducing the stability of the protein (Fig. 2C).

FIG. 2.

(A) Diagnostic variants are illustrated at gene exon and protein domain levels. The protein domains are shown in different colors. (B) The conservation of two affected amino acid residues were compared among eight mammalian species at sequence level using MEGA7and WEBLOGO, including DNAH1: p.E446 and p.R971. (C) The structural changes between DNAH1 wild-type (upper box) and DNAH1^R971H (lower box).

Sperm morphology

Parameters of semen from the patient and control were analyzed in-depth (indexed in Table 2). Basic parameters showed normal semen volume and normal sperm concentration (>15 × 10⁶/mL) but severe asthenozoospermia with sperm motility of 0.67% (compared with sperm cells of the control). Approximately 90% of the flagella of the patient's sperm showed morphological abnormalities (∼20% flagella are abnormal in the control). The abnormalities comprised coiled flagella (53.33%) and short flagella (18.00%), and a multiple anomalies index of 12.00%. Papanicolaou staining and SEM analysis confirmed the various types of abnormalities of the flagella (Fig. 3A, B).

FIG. 3.

Sperm morphology and RNA expression. (A) Optical microscopy analysis of sperm morphology from patient triply. Sperm samples were spread over a slide and dried at room temperature for Papanicolaou staining. (B) Sperm of the patient presented with dysmorphologies in sperm flagella, such as short and coiled flagella, flagella of irregular caliber, and other malformations diagnosed as MMAF. (C) Electron microscopy analysis of sperm flagellum of the patient and control. Cross-section from the patient's sample showing numerous defects: absence of inner dynein arm and radial spoke, displacement of dense fiber and microtubules doublets. (D) and (E) Relative mRNA expression of DNAH1, DNAH12, DNALI1, and DNAI2 in the sperm of patient and control. ****p < 0.0001 indicates significantly difference. MMAF, multiple morphological abnormalities of the flagella; ns, no statistical difference.

Table 2.

Semen Parameters for Patient and Control

Type of analysis	Control^a	Patient
Type of analysis	Control^a	1st	2nd	3rd	Mean ± SD
Basic parameters
Semen volume (mL)	3 (1.5–4.0)	2.7	3.3	1.9	2.63 ± 0.70
Sperm concentration ( × 10⁶/mL)	40 (20–55)	12	19	18	16.33 ± 3.79
Progressive motility (%)	55 (50–70)	1	0	1	0.67 ± 0.58
Sperm morphology
Normal flagella (%)	80	10	8	14	10.67 ± 3.06
Short flagella (%)	5	17	14	23	18.00 ± 4.58
Coiled flagella (%)	2	55	63	42	53.33 ± 10.60
Absent flagella (%)	1	7	1	3	3.67 ± 3.06
Bent flagella (%)	3	4	2	0	2.00 ± 2.00
Flagella of irregular caliber (%)	1	1	0	0	0.33 ± 0.58
Multiple anomalies index (%)	8	6	12	18	12.00 ± 6.00

Average of 10 control subjects.

Results of the TEM analysis of the sperm flagella revealed that not only were the inner dynein arm and radial spoke absent, but the dense fiber and microtubule doublets were also displaced (Fig. 3C).

Expression level changes

We initially tested the level of RNA expression of DNAH1 and its closest paralogs DNAH12 (MIM *603340), in the sperm of the patient and the control. There was no significant difference in the level of RNA expression between the patient and the control (Fig. 3D).

DNALI1 and DNAI2 are both well-established diagnostic markers of the inner and outer dynein arms (Rashid et al., 2006; Loges et al., 2008). So, the RNA expression of DNALI1 and DNAI2 in the sperm between the patient and the control was also tested. Expression of DNAI2 was comparable between the samples of the patient and the control, whereas the expression of DNALI1was dramatically reduced in the patient (Fig. 3E).

Discussion

The human DNAH1 gene is located on chromosome 3p21.1 and comprises 79 exons, covering a 13,126 bp genomic region. The DNAH1 gene encodes the heavy-chain of the inner-arm dynein, which is an axonemal component. The heavy chains are responsible for dynein motor activity (Perrone et al., 2000; Toba et al., 2011). The absence of DNAH1 is deleterious for the organization and biogenesis of the axoneme of sperm, which could lead to MMAF (Ben Khelifa et al., 2014). To date, about 40 mutations in the DNAH1 gene have been reported to be associated with MMAF (Supplementary Table S3).

In this study, we performed a multiplatform genetic detection on a patient with MMAF-related infertility. Biallelic DNAH1 variations consisting of two novel missense variants, c.1336G>C (p.E446Q) and c.2912G>A (p.R971H), were identified. The c.1336G>C variant caused a G to C substitution at 1336 of the exon9, leading to the replacement of glutamic acid with glutamine in the translated sequence. This variant was not included in the 1000G_2015aug_eas, ExAC_EAS, or the orgnomAD_exome_EAS. Moreover, the HGMD had no records of the gene variant. The c.2912G>A variant caused a G to A substitution at 2912 of the exon18, resulting in a change from arginine to histidine in the translated sequence. This variant was present at low frequencies in the databases (Table 1); however, there were no records of it in the HGMD.

The two affected AA residues were both located in the tail and linker domains. The N-terminal part, which is important for maintaining the structure of the dynein arm, binds to the cargo and interacts with other dynein components. Both the affected AA residues were highly conserved across species, which supported the pathogenicity of these identified variants. The DNAH1: p.E446Q variant caused the replacement of a strongly acidic AA (glutamic acid) with a neutral AA (glutamine), which probably resulted in the disruption of the function of the protein. The DNAH1: p.R971H caused the replacement of a strongly basic AA (arginine) with a weakly basic AA (histidine) and led to a decrease in the hydrogen bonding with E974, resulting in a reduction in the stability of the protein. Hence, structural analysis supported that the biallelic variations in DNAH1 affected protein function. Functional predictions (LRT, Mutation Taster, SIFT, and Polyphen2) of variations showed contradictory results (Table 1). According to the variant interpretation criteria by ACMG (Richards et al., 2015), they were classified as variations that are likely pathogenic (with the evidence of PS2+PS3+PP2+PP3+PP4; Table 1).

Semen analysis revealed that the patient had asthenospermia (reduced sperm motility). Papanicolaou staining, SEM, and TEM analyses subsequently revealed anomalies of the sperm flagella in the patient with DNAH1 variants, which supported the hypothesis that disorganization of microtubules of the axoneme was the major cause of the MMAF phenotype (Coutton et al., 2015). RNA expression levels indicated no significant alteration in DNAH1 and DNAH12 expression in the sperm of the patient, suggesting that the missense variants might not have affected the expression of DNAH1 and its paralogous gene. The significant decrease of DNALI1 (markers of the inner dynein arms) expression levels indicated that the DNAH1 variation probably led to the disruption of the inner dynein arms in the flagella. To sum up, sperm morphological studies suggested that the asthenozoospermia of the patient was caused by structural abnormalities of the flagella.

It is noteworthy that although early consultation ruled out the possibility of PCD, late onset of the symptoms of PCD is still possible. On a different note, although there have been no successful reports of spontaneous pregnancy or conventional in vitro fertilization ((IVF)) for individuals with MMAF, there have been positive results after intracytoplasmic sperm injection in some cases (Wambergue et al., 2016). Combining the results of the patient's clinical and genetic conditions, we suggested the couple try IVF in the next cycle. The couple chose IVF and successfully gave birth to a healthy baby (detailed data to be inquired).

In summary, novel compound heterozygous variants of the DNAH1 gene were determined to cause MMAF-related male infertility. These findings provide insights into the mechanism of dysmorphology of sperm flagella and may benefit future families with such complications.

Data Availability Statement

The data sets for this article are not publicly available due to concerns regarding participant/patient anonymity. Requests to access the data sets should be directed to the corresponding author on reasonable grounds.

Ethics Statement

The studies involving human participants were reviewed and approved by The Ethics Committee of Beijing Jiaen Hospital. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

Footnotes

Acknowledgments

We thank the patients and their families for participating in this study.

Disclosure Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding Information

No funding was received for this article.

Supplementary Material

Supplementary Figure S1

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

Supplementary Table S4

Supplementary Table S5

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