A novel homozygous frameshift mutation in MCM8 causes primary gonadal dysgenesis in both genders

Abstract

Purpose

We aimed to demonstrate the genetic factors of primary gonadal dysgenesis in a consanguineous family characterized by underdeveloped testes and non-obstructive azoospermia (NOA) in a male and primary amenorrhoea and primary ovarian insufficiency (POI) in a female.

Methods

DNA was extracted from the male proband with infertility from the consanguineous family for whole-exome sequencing and bioinformatics analysis to screen for potential pathogenic genes and mutations. Sanger sequencing was used for further validation of his family pedigree. The effects of the identified novel mutation were evaluated in the male testes tissue by immunohistochemistry and in HEK293T cells by Western blot.

Results

A homozygous frameshift mutation c.998delG (p. Gly333Glufs*50) in MCM8 was identified in the two siblings. The testes tissue sections in the male showed a Sertoli cell–only syndrome (SCOS). Functional analysis in vitro suggested that the mutation results in a truncated protein of MCM8 in HEK293T cells, and immunohistochemistry in vivo showed decreased expression of MCM8 protein.

Conclusion

We identified a novel homozygous frameshift mutation of MCM8 in two siblings diagnosed with primary gonadal dysgenesis from a consanguineous family. Functional analysis confirmed the pathogenicity of this mutation. Our study not only further reveals the essential role of MCM8 in human gonadal development, but also expands the mutational spectrum of MCM8 involved in male NOA and female POI and provides a new molecular marker for genetic counseling of infertility.

Introduction

Primary gonadal dysgenesis is a genetic heterogeneous condition of complete or partial loss of gonadal development, which is featured by high levels of serum gonadotropin and a lack of spontaneous pubertal development in the clinic [1]. In males, it presents as testicular insufficiency characterized by underdeveloped testes, oligospermia, or non-obstructive azoospermia (NOA). In females, it manifests as primary or secondary amenorrhea, uterine hypoplasia, and primary ovarian insufficiency (POI) [1].

NOA is defined by the complete absence of spermatozoa in the ejaculate semen and is considered the most clinically severe form of male infertility [2, 3]. Spermatogenesis is a precisely regulated and highly ordered process, comprising mitotic proliferation of spermatogonia, meiosis of spermatocytes, and differentiation of haploid spermatids. Any defects in this process will prevent the production of sperm cells and lead to NOA, which is a heterogeneous sickness with a broad genetic basis [4, 5]. Besides chromosomal defects, a vast number of mutations in genes involved in meiosis (e.g., C14ROF39, DMC1, HFM1, MCM8, MCMDC2, MEIOB, MSH5, REC8, and TERB1), Piwi pathway and/or DNA repair (e.g., FANCM, GCNA, HENMT1, TDRD9, and TDRKH) and post-meiotic maturation (e.g., DDX25 and PDHA2) have been identified to be responsible for NOA [3, 6–8].

Primary ovarian insufficiency (POI) is due to ovarian dysgenesis caused by the depletion of the primordial follicular pool or diminished size of the pool results from accelerated atresia, which is one major cause of female infertility [1, 9]. Apart from chromosome abnormalities, the premutation of the FMR1 gene is the most common genetic factor of 46, XX POI [10]. Besides, other pathogenetic genes are mainly involved in homologous recombination and DNA damage repair (e.g., STAG3, MCM8, MCM9, RAD51, and BRCA1/2), follicle activation (e.g., NOBOX and SOHLH1), or follicle development and maturation (e.g., GDF9, BMP5, FSHR, and FOXL2) [8, 11–13].

Noteworthy, few genes have been reported to be responsible for both genders affected with primary gonadal dysgenesis in a family, and their genetic cause is still poorly understood.

MCM8 (MIM: 608,187) is one of the highly conserved mini-chromosome maintenance proteins (MCM) and plays an indispensable role in the regulation of gonadal development and maintenance by regulating the proliferation of mitotic primordial germ cells and meiotic homologous recombination–mediated DNA repair [14–16]. Mcm8 knockout mice are sterile due to the meiotic prophase I arrest in male spermatocytes and primary follicles arrest in females [17]. A recent study showed that MCM8-deficient mice impair the proliferation of primordial germ cells (PGCs) and ultimately result in infertility [15]. Several mutations in MCM8 have been identified to be responsible for female POI [1, 9, 18–21], but only two homozygous mutations (c.1954-1G > A and c.482A > C, p. His161Pro) have been reported to be associated with small testes and NOA [1, 3].

In this study, we found a consanguineous family characterized by underdeveloped testes and NOA in a male and primary amenorrhoea and primary ovarian insufficiency (POI) in a female. We performed whole-exome sequencing and identified a novel homozygous frameshift mutation c.998delG (p. Gly333Glufs*50) in the MCM8 gene. The testes tissue sections in males showed a SCOS. Functional analysis in vitro suggested that the mutation results in a truncated protein of MCM8 in HEK293T cells, and immunohistochemistry in vivo showed decreased expression of MCM8 protein. Overall, our findings expand the mutational spectrum of MCM8 and provide a new basis for genetic counseling of infertility.

Materials and methods

Clinical study subjects

The two siblings diagnosed with infertility from the consanguineous family were recruited from the Department of Reproductive Medicine of Jinling Hospital.

Ethical approval and consent to participate

This study was approved by the Research Ethics Committee of Nanjing Jinling Hospital. Informed consent was obtained from all participants prior to the start of our study.

Whole-exome sequencing, filtering, and variant analysis

Genomic DNA from the proband and his family were extracted from peripheral blood by following the instructions of the QIAamp DNA Blood Mini Kit (Qiagen). Whole-exome capture and sequencing analysis were performed using the Illumina HiSeq X10 platform. The raw FASTQ files were mapped to the human reference sequence (NCBI Genome build GRCh37), and variants were annotated with GRCh37 (dbSNP version 138). Candidate variants were filtered with the following criteria: (1) minor allele frequency < 0.1% or not found in the ExAC and gnomAD databases and (2) missense variants predicted to be damaging by at least one software such as Mutation taster (https://www.mutationtaster.org), Sorting Intolerant From Tolerant (SIFT) (https://sift.bii.a-star.edu.sg), or mutation taster (https://www.mutationtaster.org/), splice site and frameshift variants predicted to be loss of function.

Sanger sequencing

Sanger sequencing was used to further validate the mutation in the proband and other family members. And specific primers flanking the mutation in the MCM8 gene were shown as below: forward primer 5′-TCCTCAGCATCATGTGAACCC-3 ′; reverse primer 5′-TCCATGCTTACACCCATCCTC-3′.

Homozygosity mapping

Genome-wide homozygosity mapping was performed in Homozygosity Mapper. Priority was given to analyses of homozygous regions greater than 2.0 Mb.

Molecular modeling

SWISS-MODEL software (https://swismodel.expasy.org) was used to analyze the protein structure of wild-type or mutant MCM8, and PyMol software (http://www.pymol.org) (PDB ID: Q9UJA3) was used to visualize the protein structure of corresponding MCM8.

Cell lysate extraction and western blotting analysis

HEK293T cells were cultured for 36 h after transfection with MCM8 wild-type or mutant plasmid. Detailed methods of cell lysate extraction and Western blotting analysis were performed according to our previously published approaches [22]. The antibodies we used were as follows: mouse anti-Flag (1:3000 dilution, AE005, Abclonal), rabbit anti-β-actin (1:3000 dilution, AC038, Abclonal), goat anti-mouse (1:5000 dilution, 31,430, Thermo), and goat anti-mouse (1:5000 dilution, 31,460, Thermo).

Histopathological analyses  

Part tissue of testes from control and proband IV4-1 were collected and fixed in Bouin’s fixative solution at 4 °C overnight and then dehydrated and embedded in paraffin. The samples were cut into sections of 5 μm thickness for standard H&E staining. Histological images were obtained using an Olympus microscope (IX73, Olympus Corporation).

Immunohistochemistry analysis

Section of testes from control and proband IV4-1 were collected and performed immunohistochemistry according to our previously published approaches [23]. Images were obtained and analyzed using Slide Viewer software. The antibody was rabbit anti-MCM8 (1:200 dilution, 16,451–1-AP, proteintech).

Results

Clinical characteristics and phenotypes of patients

The proband IV-1 from a consanguineous family was 32 years old and had been diagnosed with underdeveloped testes and NOA. Bilateral testes showed a small volume (right, 30 × 16 × 21 mm, about 7.16 mL; left, 32 × 15 × 19 mm, about 6.48 mL), and no sperm was found in the routine examination of semen and even after centrifugation. His chromosome karyotype was normal (46, XY), and no microdeletions of the AZF (azoospermia factor) region were detected. He had high levels of follicle-stimulating hormone (FSH, 26.49 mIU/mL; normal range 1.40–18.10 mIU/mL) and low levels of inhibin B (INH B, 12.00 ng/L, normal range 18.22–311.27 ng/L). The patient further underwent surgery to perform a microdissection testicular sperm extraction (micro-TESE), but no living spermatozoa were extracted, and the histological analysis showed a Sertoli cell–only syndrome (Fig. 1C).

Fig. 1.

Identification of a novel homozygous frameshift mutation in MCM8. A The pedigree of the consanguineous family affected by the mutation in MCM8. The mutation was confirmed by Sanger sequencing and displayed a recessive inheritance pattern. Squares denote male members, and circles denote females. Solid squares and circles indicate affected individuals. Equal signs indicate infertility, and the double lines between couples represent consanguinity. B Homozygosity mapping of IV-1 in Family 1. Red indicates the homozygous region with the strongest signal. The “*” represents the area where the MCM8 gene is located. C The example of testes tissue sections from the normal and individual IV-1 with testicular defects (Sertoli cell–only syndrome). Scale bar is 20 µm

His sister (IV-2), a 30-year-old woman, had a delayed puberty, primary amenorrhea. Ultrasound examination revealed that her uterus and ovaries were not observed. Her karyotype was normal (46, XX). Her related hormone values were elevated, with low anti-Müllerian hormone (AMH, 0.02 ng/mL; normal range > 1.22 ng/mL) and estradiol levels (E₂, 14.00 pg/mL; normal range 21.00–251.00 pg/mL), high levels of FSH (68.87 mIU/mL; normal range 3.03–8.08 mIU/mL) and LH (20.39 mIU/mL; normal range 1.80–11.78 mIU/mL), and normal levels of pituitary prolactin (RPL). Additionally, her INH B (< 10 ng/L) levels were too low to be detected. The above indicators were consistent with primary ovarian failure or primary ovarian insufficiency (POI).

The detailed clinical examination data of the affected individuals in the family are shown in Table 1.

Table 1.

Clinical examinations of affected individuals in the family

Study participants	IV-1 (male)	IV-2 (female)	Normal value range
Age at diagnosis (y)	32	30	/
Hormone levels
T (nmol/L)	19.11	< 0.45	Male: 6.07–27.10 Female: 0–2.603
FSH (mIU/mL)	26.49	68.87	Male: 1.40–18.10 Female: 3.03–8.08
LH (mIU/mL)	9.30	20.39	Male: 1.10–8.70 Female: 1.80–11.78
PRL (ng/mL)	10.42	15.68	Male: 2.11–17.70 Female: 5.18–26.53
E2 (pg/mL)	43.05	14.00	Male: 0–56.00 Female: 21.00–251.00
AMH (ng/mL)	6.99	0.02	Male: > 1.22 Female: > 1.22
INH B (ng/L)	12.00	< 10	Male: 18.22–311.27 Female: 19.67–147.62
Ultrasound imaging	Small testes: right,7.16 mL; left, 6.48 mL	No ovaries and no uterus	/
Genetic investigation
Karyotype	46, XY	46, XX	/

T; testosterone, FSH; follicle-stimulating hormone, LH; luteinizing hormone, PRL; prolactin, E₂; estradiol, AMH; anti-Müllerian hormone, INH B; inhibin B

Identification of a novel mutation in MCM8

Whole-exome sequencing was used to identify the variants. After variants filtering and genetic analysis, we identified a homozygous frameshift mutation c.998delG (p. Gly333Glufs*50) in MCM8 (GenBank: NM_032485.6) in the proband IV-1 and his sister. Subsequently, the homozygous mutation was confirmed using Sanger sequencing and was inherited from their parents, respectively, showing a recessive inheritance pattern. The pedigrees of the individuals are shown in Fig. 1A. Meanwhile, homozygosity mapping of the proband IV-1 showed a strong signal in the region of chromosome 20 containing MCM8 (Fig. 1B).

The identified mutation c.998delG was located in exon 9 and N-terminal DNA-binding domain (Fig. 2A). And the altered amino acids of the mutation are highly evolutionarily conserved across different mammalian species (Fig. 2B). As shown in Fig. 2C, the mutation was predicted to result in the formation of a truncated protein of 383 amino acids, and the 3D structure was significantly impaired. Also, the mutation was predicted to be damaging or disease-causing to the function of MCM8 by in silico analysis in SIFT and Mutation taster (Table 2). In addition, the frequency of the mutation was absent in the gnomAD database (Table 2).

Fig. 2.

The location and conservation of mutated residue in MCM8. A The position of the novel mutation in MCM8 in the genomic and protein structure. B Conservation analysis of the altered amino acid in different species. C The 3D protein structure models of wild-type (left) and mutant (right) MCM8

Table 2.

Overview of the MCM8 mutation in the affected family

Gene	Genomic position (bp)	Exon	cDNA change	Protein change	Variant type	Genotype	SIFT	Mutation Taster	gnomADa
MCM8	chr20:5,948,204	9	c.998delG	p. Gly333Glufs*50	Frameshift Deletion	Homozygous	Damaging	Disease-causing	NA

NA; not available

^aFrequency of the corresponding variant in the gnomAD database

The influence of the homozygous frameshift mutation on MCM8

To evaluate the effect of the frameshift mutation on the function of MCM8, the vector with wild-type or mutant MCM8 fused with a Flag tag was constructed and transfected into HEK293T cells. Western blot analysis showed that the frameshift mutation led to premature termination codons in MCM8 and formed a truncated protein (Fig. 3A). And compared to normal men, immunohistochemistry showed the decreased expression of MCM8 protein in the section of testes tissue of the proband IV-1 (Fig. 3B).

Fig. 3.

Effects of the frameshift mutation c.998delG on MCM8 in vitro and in vivo. A Western blotting analysis of the size and level of MCM8 protein in HEK293T cells transfected with wild-type or mutant vector carrying Flag tag. B Immunohistochemistry analysis of the level of MCM8 protein in the section of testes tissues of the normal and individual IV-1. Scale bar: 100 μm (top), 20 μm (bottom)

Discussion

In summary, we identified a novel homozygous frameshift mutation c.998delG (p. Gly333Glufs*50) in MCM8 in two siblings with primary gonadal dysgenesis from a consanguineous family, which presented as male infertility characterized by NOA and female infertility characterized by POI. The novel mutation c.998delG (p. Gly333Glufs*50) caused a truncated protein of MCM8 in HEK293T cells in vitro and decreased the level of MCM8 protein in testes tissue in vivo.

Previous studies have demonstrated that both male and female MCM8-deficient mice are infertile due to defects in meiotic homologous recombination double-strand break repair. Male spermatocyte arrest at meiotic prophase I and female ovaries have only arrested primary follicles [17]. A recent study published by Wen et al. found that knockout mice for MCM8 also led to proliferation defects of primordial germ cells (PGCs) by impairing the maintenance of genome integrity. Further mechanism study clarified that disabling MCM8 decreased the interaction with two known helicases DDX5 and DHX9 and further caused the increasing R-loop levels by reducing retention of the helicases at loops, thus resulting in the unstable genome [15].

MCM8 has emerged as a core gene in regulating human ovarian aging [24], and the single nucleotide polymorphism rs16991615 (SNP) is reported to be significantly associated with the age of menopause [25, 26]. In addition, MCM8 is also crucial for meiotic homologous recombination repair. Several studies have reported that a large number of MCM8 mutations, including missense, insertion, deletion, and splicing mutations, are responsible for female POI. These mutations were identified as impairing DNA repair and causing chromosomal instability [9, 18–21]. Only two homozygous missense mutations c.1954-1G > A and c.482A > C (p. His161Pro) were reported to be associated with testicular insufficiency characterized by small testes and NOA [1, 3]. In addition, the proband IV-1 diagnosed with underdeveloped testes and NOA in our study show the same phenotypes (Sertoli cell–only syndrome) as the previously reported mutation. All of these genetic studies reveal an indispensable role for MCM8 in human reproduction including gonadal development, gametogenesis, and infertility.

Also, our study has some limitations. Firstly, only one case has been identified, and more cases need to be verified for the pathogenicity of this novel mutation; secondly, we have only verified the disruption of this novel mutation on the MCM8 protein. The exact molecular mechanism identified in previous studies needs to be confirmed, and more potential original signal pathways need to be explored using knock-in mice.

In conclusion, we have identified a novel homozygous mutation c.998delG (p. Gly333Glufs*50) in the MCM8 gene underlying primary gonadal dysgenesis in humans of both sexes that expanded the mutational spectrum and enriched phenotypic variability. Our findings strongly support the major role of MCM8 in primary gonadal development and maintenance and provide new diagnostic markers for genetic counseling of clinical patients with female and male infertility.

Author contribution

JD, JM, and BY conceived and designed the research study. ZZ, LC, and XG collected the clinical samples. ZZ and LC organized the medical records. JD, RM, and JJ analyzed the whole-exome data and performed the genome sequencing. JD and WW performed the corresponding experiments. JD and JM drafted and revised the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (82301869, 82471651, 82274651), the China Postdoctoral Science Foundation grant (2023M734294), Jiangsu Funding Program for Excellent Postdoctoral Talent (2022ZB759), and the 2022 Annual Foundation of Nanjing Jinling Hospital (22JCYYZD7).

Data availability

The data and material underlying this article will be shared on reasonable request to the corresponding author.

Declarations

Ethical approval and consent to participate

Our study was approved by the Ethics Committee of the Medical College of Ethics Committees of the hospital (2018NZK-005–02). All blood samples were donated for the investigation after informed consent.

Conflict of interest

The authors declare no competing interests.