A novel missense mutation of CCDC34 causes male infertility with oligoasthenoteratozoospermia in a consanguineous Pakistani family

Abstract

Male infertility is a worldwide health issue, affecting 8%–12% of the global population. Oligoasthenoteratozoospermia (OAT) represents a severe type of male infertility, characterized by reduced sperm count and motility and an increased frequency of sperm with aberrant morphology. Using whole-exome sequencing, this study identified a novel missense mutation (c.848C>A, p.A283E) in the coiled-coil domain-containing 34 gene (CCDC34) in a consanguineous Pakistani family. This rare mutation was predicted to be deleterious and to affect the protein stability. Hematoxylin and eosin staining of spermatozoa from the patient with OAT revealed multiple morphological abnormalities of the flagella and transmission electron microscopy indicated axonemal ultrastructural defects with a lack of outer dynein arms. These findings indicated that CCDC34 plays a role in maintaining the axonemal ultrastructure and the assembly or stability of the outer dynein arms, thus expanding the phenotypic spectrum of CCDC34 missense mutations.

INTRODUCTION

Infertility is a global health issue characterized by an inability to conceive after at least 12 months of regular, unprotected sexual intercourse.1 Half of infertility cases can be attributed to the male partners.2 Male infertility is a complicated disorder with heterogeneous clinical characteristics involving an abnormal sperm count, morphology, and motility, including azoospermia, asthenozoospermia, oligozoospermia, teratozoospermia, or a combination of these factors, such as asthenoteratozoospermia or oligoasthenoteratozoospermia (OAT).3 OAT is one of the most severe types of male infertility and is defined by a reduced number of spermatozoa and low percentages of both progressively motile and morphologically normal spermatozoa compared with the reference limits. Multiple morphological abnormalities of the sperm flagella, a typical feature of OAT, are mainly defined as the presence of abnormal spermatozoa with absence, short length, coiling, bending, and irregular-caliber flagella, resulting in significantly reduced sperm motility and count.4 Notably, however, the genetic factors responsible for OAT remain largely elusive and unexplained. Mutations in several cilia- and flagella-associated proteins (CFAP), such as CFAP91 and CFAP61, and centrosome proteins (CEPs) such as CEP128 have been proven to cause OAT in humans,5,6,7 but few genes in the coiled-coil domain-containing protein (CCDC) family have been connected with OAT in humans. A recent study identified two homozygous CCDC34 frameshift variants in infertile Chinese men with OAT phenotype;8 however, it remains unclear if missense mutations of CCDC34 may cause an OAT-like phenotype in humans. In the present study, we examined a consanguineous Pakistani family suffering from male infertility. Whole-exome sequencing (WES) analysis identified a novel missense mutation (c.848C>A, p.A283E) in CCDC34 accounting for the patient’s OAT phenotype. The mutation site was highly conserved and was predicted to be deleterious by several analysis tools. Transmission electron microscopy (TEM) analysis of the affected patient’s spermatozoa showed disorganized axonemal ultrastructure and impairment of the outer dynein arms (ODAs). In silico structural analysis of wild-type and mutant proteins indicated conformational changes around the mutant region, which may hinder the stability and functions of the CCDC34 protein. In summary, these results extend the phenotypic spectrum of the CCDC34 missense mutation (c.848C>A), including causing OAT in humans, and demonstrate a connection between CCDC34 mutations and ODA deficiency in axonemal ultrastructure.

PARTICIPANTS AND METHODS

Participants and clinical investigation

This study investigated a consanguineous Pakistani family (Register No. PK-INF-823; available at Human Reproductive Disease Resource Bank [https://mcg.ustc.edu.cn/bsc/newcase/]) with primary infertility. Blood samples were collected from accessible family members. Written informed consent was obtained from all accessible family members. The patient married but failed to conceive after years of trying, despite unprotected sexual intercourse with ejaculation. This study was supported by the Institutional Ethical Committee of the University of Science and Technology of China (Hefei, China; Approval No. USTCEC202000003).

WES, Sanger sequencing, and bioinformatic analysis

Genomic DNA was extracted from peripheral blood samples using a FlexiGene DNA kit, according to the manufacturer’s instructions (Qiagen, Hilden, Germany). For WES, libraries captured by AIExome Enrichment Kit V1 (iGeneTech, Beijing, China) were constructed for the proband (IV:3) and one of his fertile brothers (IV:5), following the manufacturer’s instructions. Sequencing was carried out using the HiSeq 2000 platform (Illumina, San Diego, CA, USA), and the raw data were processed as reported previously.9 The variant filtration details are listed in Supplementary Figure 1 ^{(86KB, tif)} . The function of the mutant protein was predicted using Sorting Intolerant from Tolerant (SIFT; http://sift.bii.astar.edu.sg/), Polymorphism Phenotyping version 2 (Polyphen-2; http://genetics.bwh.harvard.edu/pph2/), Combined Annotation-Dependent Depletion (CADD; https://cadd.gs.washington.edu/), and Functional Analysis through Hidden Markov Models-Multiple Kernel Learning software (FATHMN-MKL; http://fathmm.biocompute.org.uk/). The mutation was further validated by Sanger sequencing of patient IV:3 and his fertile brothers (IV:1 and IV:5). The following primers targeting the mutant sites were used for polymerase chain reaction (PCR): forward, 5′-CTATCGTGATTGGCGACGAC-3′; and reverse, 5′-AGACTGCTCCTCCAGACCCT-3′ (target product size 352 bp). For conservation analysis, CCDC34 protein sequences were extracted from the UniProt database and a phylogenetic tree of CCDC34 was constructed using Molecular Evolutionary Genetics Analysis Version 11 (MEGA11; https://www.megasoftware.net/)10 and visualized with the Tree Visualization by One Table tool (tvBOT; https://www.chiplot.online/tvbot.html).11

Sperm morphology analysis

Semen samples were collected from the patient by masturbation after 3–5 days of sexual abstinence, in accordance with the World Health Organization guidelines.3 Sperm morphology was examined by hematoxylin and eosin staining and at least 200 spermatozoa were examined to evaluate the percentages of morphologically abnormal spermatozoa.

TEM analysis

For TEM analysis, sperm samples from the patient and a fertile control were treated as described previously.12 Briefly, spermatozoa or tissues were fixed in 0.1 mmol l⁻¹ phosphate buffer (PB; pH 7.4) containing 4% paraformaldehyde, 8% glutaraldehyde, and 0.2% picric acid at 4°C overnight. After four washes with 0.1 mol l⁻¹ PB, the samples were postfixed with 1% OsO₄ and dehydrated, followed by infiltration with acetone and Epon resin mixture. The samples were embedded and cut into ultrathin sections (70 nm) before staining with uranyl acetate and lead citrate. The ultrastructure of the samples was examined and captured using an H-7650 microscope (Hitachi, Tokyo, Japan) at 100 kV.

Protein structure conformation and stability analysis

To evaluate the functional consequences of the variant, the three-dimensional structures of the wild-type and mutant proteins were created using Protein Homology/analogy Recognition Engine Version 2.0 (Phyre2; http://www.sbg.bio.ic.ac.uk/phyre2). To investigate the consequences of the mutation, we assessed the changes using the online Varsite tool (https://www.ebi.ac.uk/thornton-srv/databases/VarSite) with Q96HJ3 as the input UniProt accession ID. The protein structures were visualized using UCSF Chimera software (http://www.cgl.ucsf.edu/chimera/), according to the user guidelines. The DynaMut tool (http://biosig.unimelb.edu.au/dynamut/) was adopted to evaluate protein flexibility and interatomic interactions.13 The protein stability was evaluated comprehensively using the following tools: Mupro (http://mupro.proteomics.ics.uci.edu/) and mutation cut-off scanning matrix (mCSM; http://structure.bioc.cam.ac.uk/mcsm) based on machine learning approaches, Deletion Using Ensembles of Trees (DUET; https://biosig.lab.uq.edu.au/duet), iStable 2.0 (http://predictor.nchu.edu.tw/istable/), Multi Agent Stability Prediction web (MAESTROweb; https://pbwww.services.came.sbg.ac.at/maestro/web/), and Ensemble Learning Approach for Stability Prediction of Interface and Core Mutation (ELASPIC; http://elaspic.kimlab.org/) based on meta-approaches or mixed approaches.14,15,16,17,18

Immunofluorescence assays

Semen smears from the patient and a fertile control were prepared by permeabilization for 30 min with 0.2% Triton X-100 in 1 × phosphate buffer saline (PBS; pH = 7.4) and incubated with blocking buffer (PBS containing 3% skim milk and 0.1% Triton X-100). The slides were then incubated with anti-intraflagellar transport protein 20 (IFT20) antibody (1:100 dilution; 13615-1-AP; Proteintech Group, Rosemont, IL, USA) and anti-α-tubulin antibody (1:200 dilution; T6074; Sigma, St. Louis, MO, USA) diluted in blocking buffer overnight at 4°C, followed by incubation with secondary mouse (Alexa-488; 1:100 dilution; A21121; Molecular Probes, Eugene, OR, USA) and rabbit antibodies (Alexa-555; 1:200 dilution; A31572; Molecular Probes) for 1.5 h at 37°C. Images were captured using an Olympus BX53 microscope (Olympus, Tokyo, Japan) with a Photometrics Prime BSI camera and cellSens Dimension software (Olympus).

RESULTS

Clinical investigation of the patient IV:3

The present study focused on a consanguineous family from Pakistan, in which the affected individual suffered from idiopathic infertility while his two brothers were fertile (Figure 1a). Routine semen analyses showed that the affected individual had a normal semen volume (mean±standard error of mean [s.e.m.]: 3.0±0.2 ml, reference range >1.5 ml) with reduced sperm concentration (mean±s.e.m.: 7.3 × 10⁶±1.2 × 10⁶ ml⁻¹, reference range >15.0 × 10⁶ ml⁻¹); however, nearly all the sperm from the patient were immotile (Table 1). We further investigated the possible reason for the severely compromised sperm motility by analyzing sperm morphology in semen smears from the patient and a fertile control. The patient’s sperm demonstrated various tail anomalies, including short, coiled, bent, absent, and irregular-caliber tails (Figure 2a). Collectively, these results indicated that the patient suffered from male infertility with OAT phenotype.

Figure 1.

Identification of a novel missense CCDC34 variant in a consanguineous Pakistani family. (a) Pedigree of PK-INF-823 with an infertile patient. Arrows indicate members selected for WES. Squares and circles denote males and females, respectively. Slashes denote deceased family members and double horizontal lines represent consanguineous marriages. (b) Electropherograms of Sanger sequencing for detecting the CCDC34 mutation in the genomic DNA from all the available family members. (c) The position of the identified CCDC34 mutation at transcript and protein levels. CCDC34 is located on chromosome (chr) 11, comprises 6 exons, and encodes a 373-amino-acid protein (reference transcript: ENST00000328697; UniProtKB: Q96HJ3). (d) Conservation of the affected amino acid across different species, the mutant amino acid Ala is highly conserved in the multiple sequence alignment. (e) The bar graph of the conservation property, while capital amino acids mean highly conservative across different species. WT: wild-type allele; MT: mutant allele c.848C>A; CCDC34: coiled-coil domain-containing 34 gene; WES: whole-exome sequencing; Ref: reference; Mut: mutation.

Table 1.

Clinical characteristics of patient IV:3

Parameter	Patient IV:3	Reference value
Fertility state	Infertile	-
Age at diagnosis (year)a	53	-
Duration of marriage (year)a	23	-
Height/weight (cm/kg)	161.0/79.0	-
Semen parameterb
Semen volume (ml), mean±s.d.	3.0±0.2	>1.5
Semen pH	Alkaline	Alkaline
Sperm concentration (×106 ml−1), mean±s.d.	7.3±1.2	>15.0
Motile sperm (%), mean±s.d.	1.0±0.2	>42.0
Immotile sperm (%), mean±s.d.	99.0±0.5	-
Flagellar defectsc, mean±s.d.
Normal (%)	2.7±0.4	-
Coiled (%)	43.0±0.8	-
Short (%)	6.7±0.7	-
Bent (%)	23.7±0.6	-
Absent (%)	18.3±1.8	-
Irregular caliber (%)	5.7±1.7	-

^aAt manuscript preparation (2023). ^bReference values were published in WHO 6^th (2021).3 ^cThree independent experiments were performed. -: no value; s.e.m.: standard error of mean; WHO: World Health Organization

Figure 2.

The CCDC34 missense variant leads to male infertility with OAT and aberrant axonemal ultrastructure. (a) Representative images of spermatozoa from a fertile control and the affected patient IV:3. Spermatozoa from the patient showed severe tail abnormalities. Scale bars = 10 μm. (b) The natural CCDC34 protein structure and (c) the mutant p.A283E CCDC34 protein structure were constructed and visualized. Unfavored local conformation changes were observed in the mutant CCDC34 protein. The red arrows and dotted lines indicate the structure differences between the WT and MT structures. (d) Representative TEM micrographs of spermatozoa from a fertile control and the affected patient IV:3. Green and blue arrows show the intact IDAs and ODAs structures, respectively, while red arrowheads indicate the defective ODAs structures in the patient IV:3. Scale bars = 200 nm. WT: wild-type allele; MT: mutant allele c.848C>A; CCDC34: coiled-coil domain-containing 34 gene; IDAs: inner dynein arms; ODAs: outer dynein arms; OAT: oligoasthenoteratozoospermia.

WES identified a homozygous missense mutation c.848C>A in CCDC34 in the patient

To identify the genetic cause of the patient’s infertility, we carried out WES of the patient (IV:3) and his fertile brother (IV:5). After a series of variant filtration steps, a CCDC34 homozygous missense mutation (c.848C>A, p.A283E) was identified (Supplementary Figure 1 ^{(86KB, tif)} ). Sanger sequencing validation was then conducted for all the available family members, which indicated that the mutation segregated recessively with an infertility phenotype in this family (Figure 1b). Primary ciliary dyskinesia (PCD) is a genetic disease with symptoms caused by malfunction of motile cilia, such as chronic nasal discharge, ear, nose, and chest infections, and pulmonary disease. Half of the affected patients also experience situs inversus and, in many cases, male infertility.19 The current affected individual, however, had no visceral abnormalities and no PCD-related symptoms (Supplementary Figure 2 ^{(88KB, tif)} ). This mutation p.A283E was predicted to be deleterious by several tools and localized to the uncharacterized coiled-coil domain of CCDC34. We further assessed the conservation of the mutated amino acid, the result showed that the mutated amino acid Ala is highly conserved across different species (Figure 1c–1e, and Supplementary Figure 3 ^{(70.5KB, tif)} ). Collectively, these results indicated that the homozygous missense mutation c.848C>A in CCDC34 could account for the OAT phenotype in patient IV:3.

Homozygous CCDC34 missense mutation c.848C>A may damage protein structure and functions

Considering that the mutant p.A283E localized to the coiled-coil domain and was highly conserved through evolution, we evaluated the effects of changing Ala (with an aliphatic side chain) to Glu (with a negatively charged side chain, making it hydrophilic). Using the Varsite tool, we showed that Ala is an inert amino acid that tends to be buried in the interior of the protein, and changing Ala to Glu resulted in a large side chain and local charge alteration, thus hindering the protein from fulfilling its function (Figure 2b and 2c). The missense mutation c.848C>A was predicted to result in a dramatic decrease in thermodynamic stability, based on Mupro, mCSM, DUET, iStable 2.0, MAESTROweb, and ELASPIC analyses (Table 2). To clarify the subtle changes in three-dimensional protein structure, we explored the wild-type and mutant structures using Phyre2, which indicated that changing Ala to Glu led to local re-organization, which may damage protein flexibility and interactions under physiological conditions (Figure 2b and 2c, and Supplementary Figure 4 ^{(99.4KB, tif)} ). Immunofluorescence assays also showed that IFT20 signals, as a potential interactor of CCDC34, were absent in the patient IV:3, suggesting that the function of CCDC34 may have been impaired (Supplementary Figure 5 ^{(64.8KB, tif)} ). Together, these analyses suggested that the CCDC34 missense mutation c.848C>A may damage the protein stability, conformation, and function.

Table 2.

In silico analyses of the CCDC34 missense variant identified in the patient IV:3

Characteristic	Patient IV:3	Classification
Human gene symbol	CCDC34	-
cDNA alteration	c.848C>A	-
Protein alteration	p.A283E	-
Allele frequency in human population
1000 genomes	0	Rare
gnomAD (version 3.1)	0	Rare
Protein stability (ΔΔG, kcal mol−1)a
Mupro	−0.7	Destabilizing
mCSM	−1.3	Destabilizing
DUET	−0.8	Destabilizing
iStable 2.0	−0.9	Destabilizing
MAESTROweb	−0.8	Destabilizing
ELASPIC	−0.9	Destabilizing
Function prediction
SIFT	-b	Deleterious
PolyPhen-2	-	Probably damaging
CADD	-	Deleterious
FATHMN-MKL	-	Deleterious

^aChange in Gibbs free energy (ΔΔG) represents stability change upon mutation, ΔΔG ≤0.5 kcal mol⁻¹ was considered destabilizing. CCDC34: coiled-coil domain-containing 34 gene; -: no value; gnomAD: genome aggregation database; mCSM: mutation cutoff scanning matrix; DUET: deletion using ensembles of trees; MAESTROweb: multi agent stability prediction web; ELASPIC: ensemble learning approach for stability prediction of interface and core mutation; SIFT: sorting intolerant from tolerant; PolyPhen-2: polymorphism phenotyping version 2; CADD: combined annotation-dependent depletion; FATHMN-MKL: functional analysis through hidden Markov models-multiple kernel learning

Homozygous CCDC34 missense mutation c.848C>A caused OAT and axonemal ultrastructure defects with ODA deficiency

A previous study reported that patients carrying CCDC34 frameshift mutations presented with severe anomalous arrangements, but no details are available.8 To clarify the consequences of the current identified CCDC34 missense mutation, we further examined spermatozoa ultrastructure in our patient and a fertile control using TEM. Well-organized nine doublets of microtubules grouped circularly around the central pair complex were observed in the axoneme in the fertile control, while sperm axonemal ultrastructure was severely disorganized in the patient, with obvious ODA deficiency (Figure 2d). Together, these results indicated that the CCDC34 missense mutation p.A283E may have been responsible for OAT in the patient, along with axonemal ultrastructure defects.

DISCUSSION

In the present study, WES screening identified a novel CCDC34 missense variant (c.848C>A, p.A283E) leading to male infertility with OAT phenotype in a consanguineous Pakistani family. This mutation was further validated by Sanger sequencing and was shown to segregate recessively with male infertility in the family members. TEM analysis of the patient’s spermatozoa showed disorganized axonemal ultrastructure and severely affected ODAs in cross-sections, suggesting that CCDC34 may play an important role in maintaining axonemal organization and regulating the integrity of specific ODAs. These results thus identified CCDC34 as an OAT-related gene essential for human male fertility.

CCDC34 encodes a protein belonging to the CCDC family, which includes a distinctive helical domain, and is highly expressed in mammalian sperm.20 The CCDC family encompasses approximately 180 genes and participates in a wide range of cellular functions, such as intercellular transmembrane signal transduction, genetic signal transcription, and various other essential cellular functions.21 These proteins are also involved in flagella formation and the stability of the axonemal complex structure. Deficiencies in several CCDC family proteins, such as CCDC38 and CCDC62, are involved in male infertility due to sperm flagellum malformations.22,23 CCDC34 has been reported to be up-regulated in cancer cells, including hepatocellular carcinoma, and knockdown of CCDC34 potently inhibited the proliferation and metastasis of hepatocellular carcinoma cells, suggesting a role for CCDC34 in regulating cell proliferation and migration.24 However, although CCDC34 was predicted to be dominantly expressed in the testis, its role in reproduction remains largely unknown. Two CCDC34 frameshift variants (c.731dup, p. N244Kfs*3; and c.799_817del, p. F267Kfs*72) have recently been reported in patients with multiple morphological abnormalities of the sperm flagella displaying OAT phenotype, and a mutant Ccdc34 mouse model (c.562insT, p.Glu188fs*) also resembled the symptoms in humans.8 The current study demonstrated a novel missense mutation (c.848C>A, p.A283E) localized to the coiled-coiled domain of CCDC34, potentially resulting in decreased protein stability and unfavorable changes in the region’s conformation, leading to impaired protein function. Mutations like single-nucleotide polymorphisms and indels frequently occur either spontaneously or hereditarily, with the latter being especially common in consanguineous marriages. Some of these mutations are pathogenic and affect protein stability or reduce its activity. Notably, however, a lack of biological samples and experimental materials means that some variants remain unknown or poorly studied. Here, we used traditional experimental verification combined with bioinformatic algorithms to study the effects of a missense mutation within CCDC34, thus providing a different view of its potential causes. A similar strategy may be used to provide a comprehensive analysis of other disease-causing missense mutations.25,26

TEM can be used to study the connection between different sperm axonemal components; however, in the previous study,8 the entire cross-sections of the axoneme were disorganized and indistinct. To clarify the consequence of CCDC34 deficiency on the axonemal components, we therefore checked the TEM results and found that the axoneme structure was disordered in half the cross sections, while ODAs were missing in the other mildly affected axonemes. These results suggested an important role of CCDC34 in maintaining the integrity of the axoneme and in the assembly or stability of ODAs. Mutations of several coiled-coil domain-containing genes, such as Ccdc103,27,28 Ccdc114,29,30 and Ccdc151,31,32 have been shown to result in reduced or absent ODAs with reduced motility in flagella or cilia. Interestingly, most of these proteins are localized in the ODAs of the axoneme, and the fact that CCDC34 deficiency induces ODAs impairment suggests a relationship between CCDC34 and ODAs in terms of axonemal spatial ultrastructure. The precise function and mechanism of CCDC34 in regulating the assembly and proper organization of axonemal components like ODAs need further exploration.

In summary, the present study identified a novel homozygous CCDC34 missense mutation leading to male infertility with OAT phenotype. This pathogenic variant was shown to reduce protein stability and lead to unfavorable conformational changes. The sperm ultrastructure in the patient suggested that CCDC34 played an essential role in maintaining the proper organization of the axoneme and regulating the assembly or stability of specific ODAs. Our results provide more genetic evidence for the pathogenicity of CCDC34 mutations in OAT, and broaden the spectrum of CCDC34 missense mutations in humans, thus helping clinicians to treat or provide reproductive counseling to affected men and their families.

AUTHOR CONTRIBUTIONS

QHS conceived and designed the study. NA and MLY performed the experiments, and JTZ and MLY contributed to whole-exome sequencing and bioinformatic analyses. NA, MLY, AZ, MZ, and WS analyzed the data. NA and MLY wrote the manuscript. NA, MLY, WS, TA, HZ, XHJ, YWZ, and QHS modified the manuscript. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.

Supplementary Figure 1

WES data analysis flowchart. In the flowchart, variant filtration was performed using WES data from the affected individual (IV:3), a fertile brother (IV:5). CCDC34 nonsynonymous SNV c.848C>A was identified as the candidate variant. SNV: Single Nucleotide Variants; MAF: Minor Allele Frequency; ESP6500: NHLBI GO Exome Sequencing Project 6500; ExAC: Exome Aggregation Consortium; gnomAD: genome Aggregation Database; LoF: Loss-of-Function; RoH: Runs of Homozygosity.

Supplementary Figure 2

X-ray clinical investigation. X-ray clinical examination of the patient IV:3 presented situs solitus.

Supplementary Figure 3

Phylogenetic analysis of CCDC34 proteins across different species. The phylogenetic tree suggests a highly conserved role of CCDC34.

Supplementary Figure 4

The effects of CCDC34 c.848C>A mutation on protein flexibility and interaction. (a) ΔΔSVib ENCoM represents Δ Vibrational Entropy Energy Between wild-type and mutant, the value is −0.223 kcal. mol⁻¹. K⁻¹, indicating a decrease of molecule flexibility. The amino acids are colored according to the vibrational entropy change upon mutation. Blue represents a rigidification of the structure and red means a gain in flexibility. (b) CCDC34 wild-type and (c) CCDC34 mutant residues are colored in light green and are also represented as sticks alongside the surrounding residues which are involved in any type of interaction. The orange lines represent hydrogen bonds, the red represents water-mediated hydrogen bonds.

Supplementary Figure 5

The signals of IFT20 were absent in the sperm of the patient IV:3. Representative image of spermatozoa from a fertile control and patient IV:3 stained with IFT20 antibody, α-Tubulin antibody, and Hoechst 33342 (blue). Scale bars = 10 μm.