Background The genetic causes of the majority of male and female infertility caused by human non-obstructive azoospermia (NOA) and premature ovarian insufficiency (POI) with meiotic arrest are unknown.
Objective To identify the genetic cause of NOA and POI in two affected members from a consanguineous Chinese family.
Methods We performed whole-exome sequencing of DNA from both affected patients. The identified candidate causative gene was further verified by Sanger sequencing for pedigree analysis in this family. In silico analysis was performed to functionally characterise the mutation, and histological analysis was performed using the biopsied testicle sample from the male patient with NOA.
Results We identified a novel homozygous missense mutation (NM_007068.3: c.106G>A, p.Asp36Asn) in DMC1, which cosegregated with NOA and POI phenotypes in this family. The identified missense mutation resulted in the substitution of a conserved aspartic residue with asparaginate in the modified H3TH motif of DMC1. This substitution results in protein misfolding. Histological analysis demonstrated a lack of spermatozoa in the male patient’s seminiferous tubules. Immunohistochemistry using a testis biopsy sample from the male patient showed that spermatogenesis was blocked at the zygotene stage during meiotic prophase I.
Conclusions To the best of our knowledge, this is the first report identifying DMC1 as the causative gene for human NOA and POI. Furthermore, our pedigree analysis shows an autosomal recessive mode of inheritance for NOA and POI caused by DMC1 in this family.
- non-obstructive azoospermia
- premature ovarian insufficiency
- whole-exome sequencing
- dmc 1 gene
- meiotic arrest
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- non-obstructive azoospermia
- premature ovarian insufficiency
- whole-exome sequencing
- dmc 1 gene
- meiotic arrest
Infertility is a worldwide reproductive health problem. Approximately 15% of couples are unable to conceive after a prolonged period,1 with men and women being affected equally.2 3 Male infertility is usually due to a reduced number of sperm cells (oligozoospermia) or the complete absence of sperm cells (azoospermia, including obstructive azoospermia and non-obstructive azoospermia) in semen.4 5 Female infertility is usually caused by tubal infertility, endometriosis, uterine and cervical causes, folliculogenesis or ovulation disorders, such as premature ovarian insufficiency, polycystic ovary syndrome and resistant ovarian syndrome.6
Non-obstructive azoospermia (NOA) is the most severe form of male infertility,7 which affects ~0.6% of men from the general population and ~10% of infertile men,4 and its aetiology remains largely unknown. Spermatogenesis involves over 1000 genes, with mouse models identifying over 400 genes specifically linked to azoospermia.8 9 With the exception of candidate genes within the AZF region of the Y-chromosome,10 substantial efforts over the last few decades have verified only a small number of the genes associated with azoospermia proposed by mouse models in humans (TDRD9, TAF4B, ZMYND15 and TEX11).11–13 The genetic factors associated with the majority of human azoospermia cases remain unclear.
Premature ovarian insufficiency (POI) is the depletion or loss of normal ovarian function in women before 40 years of age, and it is an important cause of female infertility.14 This disorder classically manifests with at least a half-year history of amenorrhoea, with either primary amenorrhoea without pubertal development in the early peripubertal period or secondary amenorrhoea in adult women. Furthermore, patients with POI also present with elevated plasma levels of the circulating gonadotropin follicle-stimulating hormone (FSH; >25 mIU/mL).15–17 Genetic defects and anomalies have been described in non-syndromic and syndromic POI. However, variants in only a few X-linked (BMP15 and FMR1) and autosomal (ATM, FSHR, NOBOX, AIRE, FOXL2 and STAG3) genes have been identified to be responsible for POI. Furthermore, the vast majority of POI cases are idiopathic, and only 25% have been associated with definitive genetic aetiology.18
Gametogenesis, including spermatogenesis and oogenesis, share a common key meiotic prophase in which diploid germ cells undergo two reducing divisions to form haploid gametes (sperms or oocytes). Meiosis-related genes, such as Tex11, Spo11, Rad51 and Dmc1, 19–22 are involved in the regulation of this process in model animals. Among these, no definitely deleterious variant in DMC1 gene had been found to be responsible for human NOA or POI. Although Dmc1 knockout (KO) mice have demonstrated Dmc1 to be essential for meiotic homologous recombination, and Dmc1 mutant mice present with both NOA and POI,22 23 whether mutations in DMC1 could affect the fertility of men and women was not known.
Here, we performed a genetic study using whole-exome sequencing (WES) of two affected members—one affected with NOA and the other with POI—from a consanguineous Chinese family. We detected a novel DMC1 variant with mouse orthologs known to be essential for normal meiotic homologous recombination during gametogenesis.22 This DMC1 variant has not previously been reported to be involved in men with NOA or females with POI.
Materials and methods
Study subjects and their families
A consanguineous Han Chinese family was recruited to identify the genetic causal factor of infertility at the Reproductive and Genetic Hospital of CITIC-Xiangya (Changsha, Hunan, China). The family includes normal parents (first cousins married to each other, family members III-1 and III-2), their son affected with NOA (proband, family member IV-2) and their daughter affected with POI (family member IV-1) (figure 1A). The healthy parents (family members III-1 and III-2) lived in the same village of YueYang, Hunan Province, with normal fertility. The proband’s wife, with normal fertility, failed to conceive after >2 years of marriage, despite unprotected sexual intercourse with ejaculation. The elder sister of the proband (IV-1) had also received a diagnosis of primary infertility after >3 years of marriage. Routine semen analyses of her husband revealed normal fertility.
Genomic DNA extraction and WES
Genomic DNA from peripheral blood samples was extracted using a QIAamp DNA blood midi kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The proband and his elder sister were subjected to WES. WES was performed using the HiSeq2000 sequencing platform (Illumina, San Diego, California, USA) by the Beijing Genome Institute at Shenzhen as described previously.24 WES data analysis was performed using the Genome Analysis Toolkit (GATK). Briefly, after removing adaptors, the WES raw reads were aligned to NCBI GRCh37 (Reference genome Hg19) using BWA,25 followed by removal of PCR duplicates and sorting using Picard (http://broadinstitute.github.io/picard/). Variant identification was performed using the GATK package26 following the recommended best practices, including base recalibration variant calling with Haplotype Caller, variant quality score recalibration and variant annotation using the ANNOVAR software.
Candidate genes were identified by the following inclusion criteria: (1) had a frequency below 5% in three public databases (1000 genomes variant database, NHLBI-GO exome sequencing project (GO-ESP) and Exome Aggregation Consortium); (2) predicted to be deleterious variants; (3) homozygous variants were considered with priority and (4) relevancy for phenotype shared in both patients using comprehensive expression data (expression in testis or ovary),27 Gene Ontology terms (biological process associated with gametogenesis)28 and model organism data (a male/female sterility phenotype presented in animal models).29 Meanwhile, homozygosity mapping was performed using HomozygosityMapper,30 and homozygous variants which located in homozygous regions >5.0 Mb were considered with priority.
Specific PCR primers flanking the homozygous mutation for the candidate gene DMC1 were designed and used to amplify the region, namely, DMC1-F: 5’-TGGGAAATCAGGGCACAGTC-3’ and DMC1-R: 5’-CCCTGCCAAGCTAAAACAGC-3. The amplified PCR products were analysed by 2.0% agarose gel electrophoresis to determine the band size, and then, bidirectional sequencing was carried using the ABI 3730 automated sequencer (Applied Biosystems, Forster City, California, USA).
Evolutionary conservation and in silico analyses
Evolutionary conservation analysis was performed by aligning the amino acid sequences of the DMC1 and RAD51 proteins among different species from the GenBank database (https://www.ncbi.nlm.nih.gov/homologene/). The potential pathogenicity of the DMC1 mutations was predicted by in silico analysis using four different tools: Polyphen-2 (http://genetics.bwh.harvard.edu/pph2), MutationTaster (http://www.mutationtaster.org/), SIFT (http://sift.bii.a-star.edu.sg/) and Combined Annotation Dependent Depletion (CADD) (cadd.gs.washington.edu/). Structural analysis of the DMC1 (NP_008999.2) and RAD51 (NP_002866.2) variant was performed using SWISS-MODEL software (https://swissmodel.expasy.org) based on the template of the RAD51 N-terminal domain structure (1B22.pdb). For protein structure visualisation, we used PyMol (http://www.pymol.org).
H&E staining and immunochemistry analysis
For H&E staining, testicular biopsy tissue from IV-2 and a normal control testis (from a patient with prostate cancer with normal fertility) were fixed in formalin-embedded paraffin, sectioned, processed and stained with H&E. For immunochemistry, sections of testicular tissue were stained with a primary anti-γH2AX antibody (goat polyclonal, 1:1000 dilution) (Abcam, Cambridge, UK), and then incubated with a secondary antibody (horseradish peroxidase-conjugated chicken antigoat secondary antibody, 1:200 dilution) (Santa Cruz Biotechnology, Santa Cruz County, California, USA). Staining was visualised using 3,3′-diaminobenzidine (Sigma-Aldrich, Missouri, USA) and haematoxylin was used as the counter stain.
The parents (family members III-1 and III-2) of the proband were married first cousins. Primary infertility was observed in both male and female offspring (family members IV-1 and IV-2 (the proband)) (figure 1A). For the proband aged 26 years, routine semen analyses revealed complete azoospermia with normal volume, and no abnormal hormone levels were detected (table 1). The testes were palpable and bilateral testicular size, as measured using B ultrasonography, was normal (table 1). H&E staining of a testis biopsy sample from the proband, revealed that only primary spermatocytes were present in the seminiferous tubules of the testis, in addition to the absence of round spermatids and elongated sperms (figure 1D). The proband has a normal 46, XY karyotype, and no abnormalities were observed on Y chromosome microdeletion detection. Other causes of infertility, such as drugs and exposure to toxic substances, were excluded. The physical examination showed normal results, including height, weight, hair distribution, mentality state and external genital organs.
The elder sister of the proband (IV-1), a woman aged 30 years, had normal growth and development, but was diagnosed with POI at the age of 20 years. She experienced menarche at the age of 13, but had an irregular menstrual cycle, with oligomenorrhoea to amenorrhoea before 27 years of age. Physical examination revealed normal breast development, normal vulva and perineum with sparse female pubic and axillary hair. Transvaginal ultrasound examination revealed that the body of the uterus was of normal size (38×29×38 mm) and a homogeneous echo pattern. The two ovaries were also of normal size (left ovary: 20×10×15 mm; right ovary: 19×8×13 mm), with only one small follicle visualised on the left. The endometrium was 4.7 mm thick and homogeneous. Her basal hormonal profile showed low levels of estradiol (7.18 pg/mL; normal range 21–251 pg/mL) and high levels of FSH (51.84 mIU/mL; normal range 3.03–8.08 mIU/mL) and luteinising hormone (29.08 mIU/mL; normal range 1.80–11.78 mIU/mL), with normal prolactin and thyroid-stimulating hormone levels. She was found to have a normal 46, XX karyotype and FMR1 CGG repeats in the normal polymorphic range. She had low anti-Müllerian hormone levels (0.065 ng/mL; normal range 0.24–11.78 ng/mL) and very low to undetectable inhibin B levels. The laboratory examination results are shown in table 1. No associated endocrinopathies or autoimmune disorders were detected. After hormonal therapy with oestrogen and a low-dose oestrogen/progesterone combined preparation, she had normal menses.
All these tests were performed in 2016 when the proband and his sister were 25 and 29 years of age, respectively. Therefore, the proband was diagnosed with idiopathic NOA and the elder sister of the proband with idiopathic POI. In this family, the mode of inheritance and the presence of consanguinity are consistent with the autosomal recessive model in the affected patients.
Identification of the DMC1 mutation
WES was performed on the two affected siblings (family member IV-1 and IV-2). Raw data (average of 19.04 Gb) were generated with a mean depth of 210.97-fold for the target regions, indicating the high quality of sequencing (see online supplementary table S1). After mapping to the reference genome sequence (Hg19), approximately 99.66% of the targeted bases were covered sufficiently to pass quality assessment for calling SNPs and indels. Vast amounts of variations were identified for each individual (see online supplementary table S2). For rare inherited diseases, the frequency of the possible pathogenic variants in a healthy population should be very low. Therefore, the results were then filtered against MAF >5% in SNP and indel databases from public databases (see online supplementary table S3), a total of 20 565 and 20 714 variants were retained in IV-1 and IV-2, respectively. Among them, 433 and 418 variants in IV-1 and IV-2 were predicted to be deleterious or loss of function (see online supplementary table S3). For patients with fertility from a consanguineous family, homozygous variants were preferentially considered, and only 25 and 23 variants were retained, respectively. Finally, we focused on relevancy for phenotype shared in both affected patients. Only one novel homozygous variants (NM_007068.3: c.106G>A, p.Asp36Asn) in DMC1 shared by affected patients fulfilled these criteria. In addition, homozygosity mapping revealed that the variant in DMC1 also located in the absence of heterozygous regions with size bigger than 5 Mb shared in both affected individuals (figure 1B). Therefore, we speculated that the novel homozygous variant in DMC1 was likely to be the disease-causing candidate (see online supplementary table S4).
Supplementary file 1
Supplementary file 2
Supplementary file 3
Supplementary file 4
We then performed Sanger sequencing and cosegregation analysis using DNA samples available from the family members. A homozygous variant (NM_007068.3: c.106G>A, p.Asp36Asn) in DMC1 was identified in the affected patients (IV-1 and IV-2). Consistent with an autosomal recessive mode of inheritance, the unaffected parents (III-1 and III-2) were heterozygous carriers of this same DMC1 variant (figure 1C).
The impact of DMC1 mutations
The variant identified in the two affected patients was located on exon 4 of the DMC1 gene. This missense mutation causes the substitution of a conserved aspartic residue with an asparaginate residue in the modified H3TH motif of DMC1 (figure 1E,F). Since RAD51 and DMC1 are two homologues of RecA family recombinase, which originated from a gene duplication event in the presumed common ancestor of all eukaryotes and formed close partnership to process meiotic recombination,31 we complemented the conservative analysis of the equivalent amino acid in RAD51 of Asp36 in DMC1. Interestingly, the site of Asp37 is also highly conserved among different species (figure 1F). By in silico analysis, this mutation was predicted to be deleterious using four different tools (online supplementary table S4). A three-dimensional (3D) structure of the N-terminal structures of DMC1 (1–81 amino acid residues) was modelled with good confidence on the basis of the 43.94% identical N-terminal structures of RAD51 (1B22.pdb). We analysed the impact of p.Asp36Asn in DMC1 and the corresponding amino acid substitution in RAD51, p.Asp37Asn. A hydrogen bond between Asp36 and Leu40 was broken when Asp36 mutated to Asn in DMC1, and two redundant hydrogen bonds were formed between Asn37 and two amino acid (Asn34 and Gly32) when Asp37 was substituted by Asn in RAD51. Meanwhile, the 3D structure showed that the p.Asp36Asn mutation in DMC1 and p.Asp37Asn mutation in RAD51 change the shape of the proteins in the residues 37–40 and residues 57–62, respectively (figure 2A,B). These changes might decrease the stability of filament macrostructure of DMC1 and RAD51, and alter their ability to bind DNA. Based on this information, the Polyphen-2/MutationTaster/SIFT/CADD prediction of the novel mutation in DMC1 as deleterious is borne out by the structural analysis.
Immunohistochemistry of the proband’s testis biopsy sample revealed that staining with γH2AX (a DNA double-stranded break (DSB) marker32) was only prominent in preleptotene to zygotene spermatocytes. In normal control seminiferous tubules, γH2AX staining was mainly focused on the sex body of zygotene spermatocytes (figure 3A,B). However, in biopsy specimens obtained from the proband, round spermatids and elongated spermatids were not observed, and γH2AX expression was prominent in preleptotene to zygotene spermatocytes, but completely absent in other germ cells stages and Sertoli cells (figure 3C,D). Furthermore, no sex bodies were observed in any spermatocyte from the proband (figure 3C,D). These results demonstrate that spermatogenesis was blocked in the zygotene stage during meiotic prophase I in the proband.
A homozygous missense mutation in DMC1 (p.M200V) has been reported in a French patient with sporadic POI.33 In vitro structural and functional analyses showed that DMC1-M200V was a disease-causing mutation.34 However, the pathogenicity of the variant has been later questioned owing to its high frequency in the African population, and has been found as homozygous 172 times in African and Latino populations according to the gnomAD database. Therefore, p.M200V should be considered a benign or likely benign polymorphism. Besides, only two known SNPs (rs11570392 and rs4987164 in an intron of DMC1) were discovered in 192 Chinese patients with POI, suggesting that mutations in DMC1-coding exons are not associated with Chinese women with POI.35 Therefore, to the best of our knowledge, this is the first report identifying an autosomal recessive mode of inheritance for a novel missense mutation in DMC1 as the cause of both POI and NOA in humans.
DMC1, a meiosis-specific gene located at 22q13.1 in humans, has 14 exons encoding a 340-amino acid protein.36 DMC1 is evolutionarily conserved and is essential for meiotic homologous recombination and DSB repair.36 37 Dmc1-KO mice and in vitro functional analyses confirmed that DMC1 is critical for the formation of synaptonemal complexes and chiasma in chromosomal crossovers during meiotic prophase I.10 22 38 In Dmc1-KO mice, oogenesis is blocked in the leptotene or zygotene stage of meiotic prophase I due to the inability to form synaptonemal complexes, and the phenotypes of Dmc1-KO mice are similar to the clinical features observed in women with POI.22 In our study, homozygous DMC1 mutation was found in the patient presenting with POI, which provided indirect evidence of the involvement of DMC1 in meiotic homologous recombination and in the regulation of ovarian function in humans.
In this study, we found a homozygous missense mutation (c.106G>A, p.Asp36Asn) in DMC1 in both a man with NOA and a woman with POI. The missense mutation is located on a modified H3TH motif, which is a non-specific DNA-binding domain near the DMC1 N-terminus and might be involved in DNA replication, repair and recombination.31 In particular, the N-terminal H3TH motif is only present in the RAD51 and DMC1, but not in other RecA superfamily members.31 DMC1 and RAD51 are two homologues of RecA family recombinase and form close partnership to process meiotic recombination in germline,31 and conservative analysis showed that the mutation in DMC1 (Asp36) and the equivalent amino acid in RAD51 (Asp37) are both highly conserved among different species. Therefore, these results confirmed that this residue is functionally important.
RAD51 structure has been well studied, and it is well known that DMC1 and RAD51 have very similar filament macrostructure with single-stranded DNA: the N-terminals in both proteins contribute to DNA binding and macrostructure formation.39–41 The N-terminal structure of DMC1 is very flexible conformations.42 Therefore, RAD51 could be used to simulate the DMC1 structure analyses.43 In this study, protein structure prediction showed that p.Asp36Asn in DMC1 might change the shape of protein in the N-terminal region. It has been reported that the N-terminal region of DMC1 is required for the formation of the octameric ring structure, which may support the proper DNA binding activity of the DMC1 protein.42 Thus, those changes might cause steric hindrance to decrease the stability of octameric ring structure, lead to macromolecular interaction perturbations and alter its ability to bind DNA. Besides, a previous study showed that DMC1-Ile37Asn, near the DMC1 N-terminal domain, can function as part of the DNA-binding path, resulting in improper meiotic recombination during meiosis.44 Interestingly, the homozygous DMC1 missense mutation (c.106G>A, p.Asp36Asn) identified in the affected patients is adjacent to DMC1-Ile37Asn, supporting our conclusion that DMC1 is the pathogenic gene in this family. Based on these results, it is reasonable for us to assume that DMC1-Asp36, similar to DMC1-Ile37, is important for the maintenance of the normal meiotic process.
Previously, the disruption of Dmc1 in Medaka was shown to produce abnormal sperm due to the failure to repair DSBs.45 In contrast, the male patient with homozygous DMC1 mutations presented with a clear NOA phenotype, indicating that mutations in DMC1 could lead to complex phenotypes. γH2AX, which recruits DSB repair factors, is a useful DNA DSB marker during meiosis prophase I.32 γH2AX staining on the testis biopsy sample from the proband showed a complete absence of sperm in the seminiferous tubule and no sex body was observed in any spermatocyte. These results demonstrate that spermatogenesis was blocked at the zygotene stage during meiotic prophase I, which is consistent with the results observed in Dmc1-KO mice during oogenesis.22
Meiotic prophase is a complex, sexually dimorphic process closely related to gametogenesis in both sexes. Several meiotic genes have been reported to be essential for meiosis, and loss of function of these genes in model animals results in male and/or female infertility due to abnormal meiotic recombination. For example, Stag3 is a crucial cohesin subunit essential for mammalian gametogenesis in both males and females.46 47 Additionally, spermatogenesis and oogenesis in Spo11-KO mice are blocked in prophase I due to the disruption of meiotic homologous recombination.20 However, with the exception of mutations in SYCP1,48 49 mutations in meiotic genes, such as STAG3,50 is only detected in women with POI, and no meiosis-related gene mutations leading to man with NOA and woman with POI have been detected in a family. Thus, our study provides evidence that a single mutation in a meiosis-related gene can cause both NOA in male and POI in female.
In conclusion, we discovered a novel missense mutation in DMC1. Furthermore, we examined a consanguineous Chinese family and confirmed that homozygosity for this mutation could lead to NOA in male and POI in female due to meiotic arrest. Further investigations are required to identify the pathological role of this mutation. Such investigations would involve screening large numbers of patients with meiotic arrest and the generation of KO mouse models using knock-in alleles that mimic the missense mutation found in patients with NOA and POI.
The authors would like to thank the families of the patients and the individuals who participated in this study. The authors would also like to thank the excellent technical support provided by Wei Zhen and Ruiling Tang, the genetic counselling team at the Reproductive and Genetic Hospital of CITIC-Xiangya, and the clinicians who referred the patients for the clinical study.
W-BH and C-FT contributed equally.
Contributors Contributors Y-QT designed the research; W-BH, C-FT performed the research; L-LM, S-MY, A-XL, F-SH, JS, JD and WL performed the bioinformatics analysis; L-QF, GL and G-XL analysed the data; Y-QT, W-BH and C-FT analysed the data and wrote the paper.
Funding This study was supported by grants from the National Natural Science Foundation of China (81771645 and 81471432 to Y-QT), the National Key Research and Development Program of China (2016YFC1000600 to L-QF) and Graduate Research and Innovation Projects of Central South University (grant 2017zzts071).
Competing interests None declared.
Patient consent Obtained.
Ethics approval This study was approved by the institutional ethics committees of the Reproductive and Genetic Hospital of CITIC Xiangya and Central South University. Written informed consent was obtained from all participating individuals.
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement The manuscript presents a mutation in a gene not previously associated with both NOA in males and POI in females in a family. No additional unpublished data are available from the study.
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