Compound Heterozygous REC114 Variants in Dizygotic Twins Causes Meiotic Arrest and Non-Obstructive Azoospermia

doi:10.21203/rs.3.rs-7093595/v1

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Case Report

Compound Heterozygous REC114 Variants in Dizygotic Twins Causes Meiotic Arrest and Non-Obstructive Azoospermia

https://doi.org/10.21203/rs.3.rs-7093595/v1

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published 30 Oct, 2025

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Background: Meiosis is essential for gametogenesis and the preservation of fertility. Central to this process is meiotic recombination, a mechanism that ensures accurate chromosome segregation and drives genetic diversity. Non-obstructive azoospermia (NOA), a severe form of male infertility, often results from meiotic arrest. Although monogenic mutations in genes critical for meiosis have been identified as a cause, the etiology of approximately 60–70% of NOA cases remains unresolved, highlighting a significant gap in our understanding of its genetic basis.

Case presentation: We utilized whole-exome sequencing (WES) to identify novel compound heterozygous variants in REC114 (c.523_524del: p.Lys175GlufsTer50 and c.640_641del: p.Leu214SerfsTer11) in a pair of Chinese dizygotic twin brothers with NOA. These variants are predicted to generate a truncated REC114 protein and disrupt its interaction with MEI4, which is essential for the formation of double-strand breaks (DSBs) during meiosis. Testicular histopathology and meiotic chromosome spread analyses confirmed meiotic arrest at the zygotene stage, which was consistent with a DSB formation defect.

Conclusion: Our findings expand the knowledge of the genetic factors contributing to NOA and suggest that the maintenance of DSB homeostasis is critical to the pathophysiology of meiotic arrest. This research offers new insights into the genetic foundations of male infertility and holds potential for the development of improved diagnostic tools and targeted therapeutic strategies.

Non-obstructive azoospermia

Meiosis

REC114 gene

Double-strand break

Male infertility

Human genetics

Non-obstructive azoospermia (NOA) is the most severe cause of male infertility and is characterized by impaired spermatogenesis that prevents the production of mature spermatozoa [1]. NOA can be classified as Sertoli-cell-only syndrome (SCOS), maturation arrest (MA), or hypospermatogenesis. MA is defined by the cessation of spermatogenesis at a specific stage, most commonly during meiosis at the spermatocyte stage. Meiosis is a meticulously orchestrated process involving a single round of DNA replication followed by two successive rounds of cell division. The common causes of MA include Y chromosome microdeletions, chromosomal abnormalities, and monogenic defects [2]. Through whole-exome sequencing (WES), numerous monogenic mutations associated with MA have been identified in genes such as Spermatogenesis Associated 22 (SPATA22) [3], Meiotic nuclear divisions 1 (MND1) [4], Testis Expressed 11 (TEX11) [5], MutS Homolog 4 (MSH4) [6], MutS Homolog 5 (MSH5) [7], and Shortage In Chiasmata 1 (SHOC1) [8]. Nevertheless, the genetic causes for the majority of human MA cases remain poorly understood.

During meiosis, the formation of programmed DNA double-strand breaks (DSBs) is regulated by the SPO11-TOPOVIBL complex, which is located on the chromosome axis. This process is mediated by the pre-DSB recombinosome, an assembly of evolutionarily conserved, meiosis-specific proteins, including evolutionarily conserved, meiosis-specific proteins such as REC114 meiotic recombination protein (REC114), Meiotic double-strand break formation protein 1 (MEI1), Meiotic double-strand break formation protein 4 (MEI4), Ankyrin repeat domain 31 (ANKRD31), and Interactor of HORMAD1 1 (IHO1). These factors are thought to target the SPO11-TOPOVIBL complex to PRDM9-dependent DSB hotspots, thereby ensuring DSB formation. Studies in mice have demonstrated that the knockout of any single gene within this DSB formation complex is sufficient to disrupt DSB generation, leading to meiotic arrest and infertility. Pathogenic variants in DSB-related genes, including REC114, MEI1, ANKRD31 and MEI4, have been identified as causes of NOA and MA in humans [9–13]. Previous studies have reported both a biallelic loss‑of‑function (LoF) variant in REC114 (c.568C > T) and a compound heterozygous mutation (c.123C > A/c.659_706dup) associated with NOA [14, 15]; however, additional compound heterozygous LoF alleles in this gene remain to be characterized.

In this study, we performed WES on two brothers from a Chinese family who were affected by NOA. We identified novel compound heterozygous LoF variants in REC114 (c.523_524del: p.Lys175GlufsTer50 and c.640_641del: p.Leu214SerfsTer11). Our findings broaden the spectrum of known genetic variants underlying NOA, provide deeper insights into its genetic etiology, and may identify potential new targets for clinical therapy.

Participants

Two brothers with idiopathic NOA were enrolled at the Department of Andrology, Urologic Medical Center, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine. They had no identifiable causes for azoospermia, such as varicocele, orchitis, cryptorchidism, testicular cancer, or a history of radiation or chemotherapy. A detailed family history was taken, and clinical assessments included semen analysis (according to the WHO 6th edition guidelines) [16], hormonal evaluation, and genetic testing, which excluded Klinefelter syndrome and AZF microdeletions. Both patients consented to WES and testicular biopsies for histopathological examination. The study was approved by the Institutional Ethical Review Committee of Shanghai General Hospital (Permit Number 2020SQ199).

Whole-exome sequencing

Genomic DNA was extracted from peripheral blood samples via the DNeasy Blood and Tissue Kit (Qiagen). DNA integrity was evaluated via the Agilent 2100 Bioanalyzer, and libraries were prepared according to standard protocols. Paired-end sequencing (2 × 150 bp) was performed on an Illumina NovaSeq 6000 platform. The raw sequencing files were demultiplexed and converted to FASTQ format via bcl2fastq (v2.20). Quality control, including adapter removal and low-quality read trimming, was performed with fastp (v0.23.2). The sequences were aligned to the human reference genome (hg19/GRCh37) via the SpeedSeq pipeline. Potential artifacts such as PCR duplicates and sample contamination were assessed via VerifyBamID (v1.1.3). Variant calling for single nucleotide variants (SNVs) and small insertions/deletions (indels) was conducted via GATK’s UnifiedGenotyper (v3.8). ANNOVAR (v2019Oct24) was used to annotate variants with information from databases, including ClinVar, dbNSFP, and gnomAD (v2.1.1). Variants with a minor allele frequency exceeding 1% in any public database were excluded. Candidate variants were prioritized on the basis of predicted pathogenicity. Missense variants were evaluated via SIFT, PolyPhen-2, and MutationTaster, whereas LoF variants were defined as truncating mutations (nonsense/frameshift) or canonical splice-site alterations. Candidate genes were cross-referenced with testis-enriched expression profiles from the Human Protein Atlas and known infertility-associated genes from the OMIM and Mouse Genome Informatics databases. The final analysis was restricted to biallelic pathogenic variants following autosomal recessive or X-linked inheritance patterns.

Sanger sequencing

Sanger sequencing was used to validate the REC114 variants identified via WES. Genomic DNA was extracted from the peripheral blood of the brothers and their parents. The primers used for PCR and sequencing were as follows: REC114-F1: 5’-GCCTGTTTCGAGTACAGTTCAG-3’; REC114-R1: 5’- AAATGTCACACAGGTGCGTTC-3’; REC114-F2: 5’- CTATGTAAAGGAAGCCTGTAAAGC-3’; and REC114-R2: 5’- TTAATTTCTCAAACCCGCCAGC-3’. The PCR products were bidirectionally sequenced on a 3730xl DNA Analyzer (Applied Biosystems).

Hematein-eosin (H&E) staining

Testicular biopsies were obtained from the NOA-affected brothers and from a patient with obstructive azoospermia (OA) who served as a control. The tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 5 µm. The sections were dewaxed, rehydrated, and stained with hematoxylin and eosin following the manufacturer's protocol (ab245880, Abcam). Images were captured via a phase-contrast microscope (Leica).

Immunofluorescence (IF) staining

Testicular tissue sections were dewaxed and rehydrated. Antigen retrieval was performed in sodium citrate buffer (10 mM, pH 6.0) under high-temperature pressure. The sections were blocked with 10% BSA and incubated overnight at 4°C with the following primary antibodies: mouse anti-γH2AX (1:300, Millipore), rabbit anti-DMC1 (1:100, Santa Cruz), goat anti-SYCP3 (1:25, R&D Systems), and peanut agglutinin (PNA, 1:400, Thermo Fisher). After washing, the sections were incubated with species-specific Alexa Fluor® 488/594-conjugated secondary antibodies (1:1000, Thermo Fisher). Nuclei were counterstained with Hoechst 33342 (1:1000, Thermo Fisher). Images were acquired via a Leica SP8 confocal microscope.

In silico analyses

The evolutionary conservation of the REC114 protein sites affected by the mutations was assessed via the UniProt database. The potential structural impact of the variants on the IHO1-REC114-MEI4 complex was modeled via AlphaFold3 and visualized via UCSF ChimeraX 1.4 [17]. The model was built via the canonical protein sequences for IHO1 (Q8IYA8), REC114 (Q7Z4M0), and MEI4 (A8MW99) from UniProt [18, 19]. Hydrogen bond analysis was performed to predict the conformational changes induced by the variants.

Clinical findings

The proband (P24615) and his dizygotic twin brother (P24614) were 32 years old at the time of analysis. The patient’s family history was negative for consanguinity and fertility problems, and the siblings had no history of chronic diseases. Physical examination revealed normal development of the penis, scrotum, vas deferens, and epididymis. Testicular volumes were 12 mL bilaterally for both individuals. Semen analysis confirmed a normal volume but complete azoospermia. Both brothers had a 46,XY karyotype with no Y chromosome microdeletions. They had no history of cryptorchidism, hypogonadism, cancer, drinking, or smoking. Both underwent microdissection testicular sperm extraction (mTESE), but no sperm were found. Histopathological analysis of testicular tissue yielded a Johnsen score of 5 for both brothers, indicating NOA with a meiotic defect. The detailed information is shown in Table 1.

Table 1
Clinical characteristics of NOA patients with mutations in *REC114.*
	Proband	Brother	Reference
Age at phenotyping	32	32	/
Height	175	170	/
Weight	70	70	/
Testis volume (left, mL)	12	12	12–15
Testis volume (right, mL)	12	12	12–15
Karyotype	46,XY	46,XY	46,XY
Y chromosome microdeletions	Normal	Normal	Normal
Semen volume (mL)	2.8	4.0	≥ 1.5
Centrifuged spermatozoa number(/ejaculate)	0	0	/
FSH (IU/L)	5.63	9.60	1.27–19.26
LH (IU/L)	2.05	2.41	1.24–8.62
T (µg/L)	3.07	2.38	1.75–7.81
NOA: non-obstructive azoospermia; FSH: follicle-stimulating hormone; LH: luteinizing hormone; T: testosterone.

Identification of the compound heterozygous variants of REC114 in the NOA-affected siblings

WES was performed on the proband and his brother. The pedigree is shown in Fig. 1A. Following data analysis, compound heterozygous variants in REC114 (NM_001042367: c.523_524del: p.Lys175GlufsTer50 and c.640_641del: p.Leu214SerfsTer11) were identified as the most likely pathogenic cause of their meiotic defects. Sanger sequencing confirmed these compound heterozygous LoF variants in both brothers (Fig. 1B). The frequencies of these two variants in the gnomAD database are 0.000029020 and 0.000004924, respectively, and predictive tools such as MutationTaster and CADD classify them as deleterious (Table 2). Consistent with an autosomal recessive inheritance pattern, the c.523_524del variant was inherited from their father, and the c.640_641del variant was inherited from their mother. These frameshift mutations are predicted to cause the absence of the MEI4-binding domain (amino acids 203–254), which is conserved across yeast, mice, and humans.

Table 2
Identification of compound heterozygous frameshift variants in the *REC114* gene among NOA patients.
	Genetic Variant 1	Genetic Variant 2
REC114 variant identified
DNA change	c.523_524del	c.640_641del
Protein alteration	p.Lys175GlufsTer50	p.Leu214SerfsTer11
Variant allele	Heterozygosity	Heterozygosity
Variant type	Frameshift	Frameshift
Reference SNP ID	rs769571961	rs781734668
Allele frequency in human population
All individuals in gnomAD V3.1.1	0.000029020	0.000004924
East Asians in gnomAD V3.1.1	NR	NR
1,000 Genomes Project	NR	NR
NCBI reference sequence number of REC114 in GenBank: NM_001042367.2
NOA: non-obstructive azoospermia; SNP: single-nucleotide polymorphism; NR, not reported.

Patients with the REC114 variant exhibit an MA phenotype

The MA phenotype in the affected patients was investigated via H&E staining, IF, and meiotic chromosomal spread analysis. Compared with the control testis from a patient with OA, H&E staining of the proband's testis revealed a reduced number of spermatocytes and a complete absence of spermatids and spermatozoa, whereas the numbers of Sertoli cells and spermatogonia were normal (Fig. 2A).

IF analysis revealed positive SYCP3 expression in the proband, but the XY body, typically labeled by γH2AX in pachytene, was not observed, indicating impaired DSB repair (Fig. 2B). Furthermore, the absence of a PNA signal confirmed that no spermatids or spermatozoa were present (Fig. 2C). In contrast, the expression of SYCP3, γH2AX, DMC1, and PNA appeared normal in the OA control tissue (Fig. 2B, C). These findings collectively indicate MA in patients.

To pinpoint the specific stage of arrest, meiotic chromosomal spread analysis was performed. In the proband's spermatocytes, γH2AX signals were observed at a relatively normal intensity but exhibited diffuse nuclear staining, and the X and Y chromosomes failed to condense to form the sex body. This pattern is indicative of MA at a zygotene-like stage (Fig. 3A). In contrast, complete progression of the meiotic stages from leptotene to pachytene was observed in the OA control (Fig. 3B). Therefore, brothers with compound heterozygous LoF variants in REC114 exhibit meiotic arrest at the zygotene-like stage.

Analysis of REC114 expression and the structural changes caused by the identified REC114 variant

To investigate REC114 expression during human spermatogenesis, we analyzed single-cell RNA sequencing (scRNA-seq) data from the Male Health Atlas (MHA) [20]. The analysis revealed that REC114 is highly expressed in meiotic prophase I spermatocytes, particularly at the zygotene stage, with transcripts persisting into the early spermatid stage (Supplementary Figure S1A-C). The AlphaFold model of the IHO1-REC114-MEI4 complex predicted that the truncating mutations in REC114 lead to structural alterations, accompanied by a significant reduction in contacts and hydrogen bonds with IHO1 and MEI4 (Supplementary Figure S2).

In this study, we identified compound heterozygous LoF variants in REC114 (c.523_524del and c.640_641del) in dizygotic twin brothers with NOA. These variants result in truncated REC114 proteins, leading to MA at the zygotene stage. This report provides additional evidence that REC114 is a causative gene for MA and NOA.

REC114, located at chromosome 15q24.1, is a meiosis-specific gene that plays a crucial role in the formation of programmed DSBs during meiotic prophase I [14]. These DSBs are essential for homologous recombination and proper chromosome segregation. Meiotic DSBs are generated by the SPO11-TOPOVIBL complex, and accessory proteins such as REC114, MEI4, and IHO1 are key to regulating the activity of this complex [11, 12, 21]. REC114 interacts directly with MEI4, forming a pre-DSB recombinosome that helps target the SPO11-TOPOVIBL complex to DSB hotspots [12].

Rec114-knockout mice exhibit significant defects in DSB formation, resulting in early prophase meiotic arrest and infertility [14, 21]. The REC114 variants identified in our study are predicted to produce a truncated protein lacking the C-terminal domain essential for interaction with MEI4, thereby disrupting REC114-MEI4 complex formation and leading to MA at the zygotene-like stage. Our IF staining results confirmed normal chromosome axis formation (SYCP3 expression) but diffuse nuclear γH2AX signals and a failure to form the sex body, which is consistent with this defect. Previous coimmunoprecipitation assays have shown that a similarly mutated REC114 protein fails to form a stable complex with MEI4, underscoring the importance of the REC114-MEI4-IHO1 axis in regulating DSB formation [14]. The conservation of these protein interactions across species highlights the evolutionary importance of REC114 in meiosis. Our findings align with results from mouse models and structural analyses, which together emphasize that the REC114-MEI4 interaction is critical for proper DSB formation and homologous recombination.

This study reports novel compound heterozygous variants in REC114 that cause MA and NOA, demonstrating the crucial role of REC114 in maintaining DSB homeostasis and ensuring proper meiotic progression. Our findings expand the mutational spectrum associated with male infertility and reinforce the genetic basis of this condition. Further research is needed to identify additional causative genes and elucidate their molecular mechanisms to better understand and manage NOA.

DSBs	Double-strand breaks
H&E	Hematein-eosin
IF	Immunofluorescence
LoF	Loss-of-function
MA	Maturation arrest
mTESE	Microdissection testicular sperm extraction
NOA	Non-obstructive azoospermia
SCOS	Sertoli-cell-only syndrome
scRNA-seq	Single-cell RNA sequencing
SNP	Single-nucleotide polymorphism
SNVs	Single nucleotide variants
WES	Whole-exome sequencing

Ethics approval and consent to participate

This study was approved by the Institutional Ethical Review Committee of Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine (Permit Number 2020SQ199), and informed consent for the use of clinical data and testicular tissues for research was obtained from the donors.

Consent for publication

Written informed consent for the publication of this case report, including all associated data, images, and details, was obtained from the individuals described.

Availability of data and materials

The data that support the findings of this study are available upon request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

Competing interests

All the authors declare that they have no competing financial interests.

Funding

This study was supported by the National Key R&D Program of China (2022YFC2702701); the Inner Mongolia Academy of Medical Sciences Public Hospital Joint Science and Technology Project (2023GLLH0045); the National Natural Science Foundation of China (82371616); the National Natural Science Foundation of China (82401869); the Specific Project of Shanghai Jiao Tong University for "Invigorating Inner Mongolia through Science and Technology" (2022XYJG001-01-19); the Shanghai Hospital Development Center Foundation (SHDC12023121); and the Emerging Advanced Technology Joint Research Project of SHDC (SHDC12023121).

Authors' contributions

All the authors participated in the development and design of the study. Yuxiang Zhang, Peng Li, and Zheng Li designed the research; Wanze Ni and Chenwang Zhang performed the research; Clinical data were collected by Shuai Xub and Haowei Bai. Yifan Sun, Zizhou Meng, Dewei Qian and Qian Sun collected and prepared clinical samples. Wanze Ni and Shuai Xu performed the bioinformatics analysis; Peng Li and Chencheng Yao analyzed the data; Wanze Ni, Yuxiang Zhang, Chencheng Yao and Zheng Li wrote and revised the manuscript. All authors read and approved the final manuscript.

Acknowledgments

The authors would like to thank the families for their participation in this study.

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No competing interests reported.

FigureS1.tiff
Figure S1 REC114 mRNA expression patterns in human testicular tissue.(A) UMAP plot of germ cell clusters detected by single-cell sequencing analyses in adult human testes. (B) UMAP expression pattern of REC114 in human adult testicular germ cells. (C) Expression patterns (violin plot) of REC114across different human germ cell clusters. SSC1: spermatogonial stem cell I; SSC2: spermatogonial stem cell II; SSC3: an aligned spermatogonial III; diff_ing_SPG: differentiating spermatogonia; diff_ed_SPG: differentiated spermatogonia; L_SPC: leptotene spermatocyte; Z_SPC: zygotene spermatocyte; P_SPC: pachytene spermatocyte; D_SPC: diplotene spermatocyte; SPC7: a mixture of diakines, metaphase, anaphase, telophase, and secondary spermatocytes; progenitor, spermatogonial progenitor.
FigureS2.tiff
Figure S2 Modeling of the human 4:4:2 IHO1-REC114-MEI4 complex and conformational changes caused by the REC114 variant. (A) The structure of the 4:4:2 IHO1‐REC114‐MEI4 complex predicted by AlphaFold3. The green chain represents IHO1, the purple chain represents MEI4, and the orange/red chain represents REC114 (the red chain indicates the chain after Lys175). The black dashed box indicates hydrogen-bonding interactions. (B) (C) (D) Magnified images of the black dashed boxes in Figure (A) and Figure (B). The red dashed lines represent hydrogen bonds. The black arrow indicates the variant affected residue (Lys173). (E) The structure of the mutant REC114 (p.Lys175GlufsTer50) complex. (F) Magnified image of the black, dashed box in Figure (D). The black dotted lines represent hydrogen bonds. The black arrows indicate missense amino acids (Lys175). Red arrows indicate premature termination codons. (G) The structure of the mutant REC114 (p.Leu214SerfsTer11) complex. (H) Magnified image of the black, dashed box in Figure (G). The red dotted lines represent hydrogen bonds. The black arrows indicate missense amino acids (Leu214). Red arrows indicate premature termination codons.

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Compound Heterozygous REC114 Variants in Dizygotic Twins Causes Meiotic Arrest and Non-Obstructive Azoospermia

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