Bi-allelic variants in DNAH3 cause male infertility with asthenoteratozoospermia in humans and mice.

STUDY QUESTION: Are there other pathogenic genes for asthenoteratozoospermia (AT)? SUMMARY ANSWER: DNAH3 is a novel candidate gene for AT in humans and mice. WHAT IS KNOWN ALREADY: AT is a major cause of male infertility. Several genes underlying AT have been reported; however, the genetic aetiology remains unknown in a majority of affected men. STUDY DESIGN SIZE DURATION: A total of 432 patients with AT were recruited in this study. DNAH3 mutations were identified by whole-exome sequencing (WES). Dnah3 knockout mice were generated using the genome editing tool. The morphology and motility of sperm from Dnah3 knockout mice were investigated. The entire study was conducted over 3 years. PARTICIPANTS/MATERIALS SETTING METHODS: WES was performed on 432 infertile patients with AT. In addition, two lines of Dnah3 knockout mice were generated. Haematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), immunostaining, and computer-aided sperm analysis (CASA) were performed to investigate the morphology and motility of the spermatozoa. ICSI was used to overcome the infertility of one patient and of the Dnah3 knockout mice. MAIN RESULTS AND THE ROLE OF CHANCE: DNAH3 biallelic variants were identified in three patients from three unrelated families. H&E staining revealed various morphological abnormalities in the flagella of sperm from the patients, and TEM and immunostaining further showed the loss of the central pair of microtubules, a dislocated mitochondrial sheath and fibrous sheath, as well as a partial absence of the inner dynein arms. In addition, the two Dnah3 knockout mouse lines demonstrated AT. One patient and the Dnah3 knockout mice showed good treatment outcomes after ICSI. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This is a preliminary report suggesting that defects in DNAH3 can lead to asthenoteratozoospermia in humans and mice. The pathogenic mechanism needs to be further examined in a future study. WIDER IMPLICATIONS OF THE FINDINGS: Our findings show that DNAH3 is a novel candidate gene for AT in humans and mice and provide crucial insights into the biological underpinnings of this disorder. The findings may also be beneficial for counselling affected individuals. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants from National Natural Science Foundation of China (82201773, 82101961, 82171608, 32322017, 82071697, and 81971447), National Key Research and Development Program of China (2022YFC2702604), Scientific Research Foundation of the Health Committee of Hunan Province (B202301039323, B202301039518), Hunan Provincial Natural Science Foundation (2023JJ30716), the Medical Innovation Project of Fujian Province (2020-CXB-051), the Science and Technology Project of Fujian Province (2023D017), China Postdoctoral Science Foundation (2022M711119), and Guilin technology project for people's benefit (20180106-4-7). The authors declare no competing interests.

Identification of a missense variant of MND1 in meiotic arrest and non-obstructive azoospermia.

Meiotic arrest is a common pathologic phenotype of non-obstructive azoospermia (NOA), yet its genetic causes require further investigation. Meiotic nuclear divisions 1 (MND1) has been proved to be indispensable for meiotic recombination in many species. To date, only one variant of MND1 has been reported associated with primary ovarian insufficiency (POI), yet there has been no report of variants in MND1 associated with NOA. Herein, we identified a rare homozygous missense variant (NM_032117:c.G507C:p.W169C) of MND1 in two NOA-affected patients from one Chinese family. Histological analysis and immunohistochemistry demonstrated meiotic arrest at zygotene-like stage in prophase I and lack of spermatozoa in the proband's seminiferous tubules. In silico modeling demonstrated that this variant might cause possible conformational change in the leucine zippers 3 with capping helices (LZ3wCH) domain of MND1-HOP2 complex. Altogether, our study demonstrated that the MND1 variant (c.G507C) is likely responsible for human meiotic arrest and NOA. And our study provides new insights into the genetic etiology of NOA and mechanisms of homologous recombination repair in male meiosis.

Reclassification of DMD Duplications as Benign: Recommendations for Cautious Interpretation of Variants Identified in Prenatal Screening.

Duplications are the main type of dystrophin gene (DMD) variants, which typically cause dystrophinopathies such as Duchenne muscular dystrophy and Becker muscular dystrophy. Maternally inherited exon duplication in DMD in fetuses is a relatively common finding of genetic screening in clinical practice. However, there is no standard strategy for interpretation of the pathogenicity of DMD duplications during prenatal screening, especially for male fetuses, in which maternally inherited pathogenic DMD variants more frequently cause dystrophinopathies. Here, we report three non-contiguous DMD duplications identified in a woman and her male fetus during prenatal screening. Multiplex ligation probe amplification and long-read sequencing were performed on the woman and her family members to verify the presence of DMD duplications. Structural rearrangements in the DMD gene were mapped by long-read sequencing, and the breakpoint junction sequences were validated using Sanger sequencing. The woman and her father carried three non-contiguous DMD duplications. Long-read and Sanger sequencing revealed that the woman's father carried an intact DMD copy and a complex structural rearrangement of the DMD gene. Therefore, we reclassified these three non-contiguous DMD duplications, one of which is listed as pathogenic, as benign. We postulate that breakpoint analysis should be performed on identified DMD duplication variants, and the pathogenicity of the duplications found during prenatal screening should be interpreted cautiously for clinical prediction and genetic/reproductive counseling.

Novel MEIOB variants cause primary ovarian insufficiency and non-obstructive azoospermia.

Background: Infertility is a global health concern. MEIOB has been found to be associated with premature ovarian insufficiency (POI) and non-obstructive azoospermia (NOA), but its variants have not been reported in Chinese patients. The aim of this study was to identify the genetic aetiology of POI or NOA in three Han Chinese families. Methods: Whole-exome sequencing (WES) was used to identify candidate pathogenic variants in three consanguineous Chinese infertile families with POI or NOA. Sanger sequencing was performed to validate these variants in the proband of family I and her affected family members. In vitro functional analyses were performed to confirm the effects of these variants. Results: Two novel homozygous frameshift variants (c.258_259del and c.1072_1073del) and one novel homozygous nonsense variant (c.814C > T) in the MEIOB gene were identified in three consanguineous Han Chinese families. In vitro functional analyses revealed that these variants produced truncated proteins and affected their function. Conclusion: We identified three novel MEIOB loss-of-function variants in local Chinese patients for the first time and confirmed their pathogenicity using in vitro functional analyses. These results extend the mutation spectrum of the MEIOB gene and have important significance for genetic counselling in these families.