Homozygous mutations in SPEF2 induce multiple morphological abnormalities of the sperm flagella and male infertility

BackgroundMale infertility due to multiple morphological abnormalities of the sperm flagella (MMAF) is a genetically heterogeneous disorder. Previous studies revealed several MMAF-associated genes, which account for approximately 60% of human MMAF cases. The pathogenic mechanisms of MMAF remain to be illuminated.Methods and resultsWe conducted genetic analyses using whole-exome sequencing in 50 Han Chinese probands with MMAF. Two homozygous stop-gain variants (c.910C>T (p.Arg304*) and c.3400delA (p.Ile1134Serfs*13)) of the SPEF2 (sperm flagellar 2) gene were identified in two unrelated consanguineous families. Consistently, an Iranian subject from another cohort also carried a homozygous SPEF2 stop-gain variant (c.3240delT (p.Phe1080Leufs*2)). All these variants affected the long SPEF2 transcripts that are expressed in the testis and encode the IFT20 (intraflagellar transport 20) binding domain, important for sperm tail development. Notably, previous animal studies reported spontaneous mutations of SPEF2 causing sperm tail defects in bulls and pigs. Our further functional studies using immunofluorescence assays showed the absence or a remarkably reduced staining of SPEF2 and of the MMAF-associated CFAP69 protein in the spermatozoa from SPEF2-affected subjects.ConclusionsWe identified SPEF2 as a novel gene for human MMAF across the populations. Functional analyses suggested that the deficiency of SPEF2 in the mutated subjects could alter the localisation of other axonemal proteins.

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Loss-of-function mutations in SPEF2 cause multiple morphological abnormalities of the sperm flagella (MMAF)

Journal of Medical Genetics ◽

10.1136/jmedgenet-2018-105952 ◽

2019 ◽

Vol 56 (10) ◽

pp. 678-684 ◽

Cited By ~ 18

Author(s):

Wensheng Liu ◽

Yanwei Sha ◽

Yang Li ◽

Libin Mei ◽

Shaobin Lin ◽

...

Keyword(s):

Essential Role ◽

Gene Variants ◽

Loss Of Function ◽

Tem Analysis ◽

Sperm Tail ◽

Morphological Abnormalities ◽

Tail Development ◽

Transmission Electron ◽

Whole Exome ◽

Experimental Findings

BackgroundMultiple morphological abnormalities of the sperm flagella (MMAF) is a kind of severe teratozoospermia. Patients with the MMAF phenotype are infertile and present aberrant spermatozoa with absent, short, coiled, bent and/or irregular flagella. Mutations in several genes can explain approximately 30%–50% of MMAF cases and more genetic pathogenies need to be explored. SPEF2 was previously demonstrated to play an essential role in sperm tail development in mice and pig. Dysfunctional mutations in SPEF2 impair sperm motility and cause a short-tail phenotype in both animal models.ObjectiveBased on 42 patients with severe infertility and MMAF phenotype, we explored the new genetic cause of human MMAF phenotype.Methods and resultsBy screening gene variants in 42 patients with MMAF using whole exome sequencing, we identified the c. 12delC, c. 1745-2A > G, c. 4102 G > T and c. 4323dupA mutations in the SPEF2 gene from two patients. Both of these mutations are rare and potentially deleterious. Transmission electron microscope (TEM) analysis showed a disrupted axonemal structure with mitochondrial sheath defects in the patients’ spermatozoa. The SPEF2 protein level was significantly decreased in the spermatozoa of the patients revealed by Western blot (WB) and immunofluorescence (IF) analyses.ConclusionOur experimental findings indicate that loss-of-function mutations in the SPEF2 gene can cause the MMAF phenotype in human.

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Genetic Defects in DNAH2 Underlie Male Infertility With Multiple Morphological Abnormalities of the Sperm Flagella in Humans and Mice

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.662903 ◽

2021 ◽

Vol 9 ◽

Author(s):

Jae Yeon Hwang ◽

Shoaib Nawaz ◽

Jungmin Choi ◽

Huafeng Wang ◽

Shabir Hussain ◽

...

Keyword(s):

Male Infertility ◽

Sperm Morphology ◽

Cellular Mechanisms ◽

Morphological Abnormalities ◽

Transmission Electron ◽

Whole Exome ◽

Synonymous Variant ◽

Primary Male ◽

Analysis Models ◽

Multiple Variants

Asthenozoospermia accounts for over 80% of primary male infertility cases. Reduced sperm motility in asthenozoospermic patients are often accompanied by teratozoospermia, or defective sperm morphology, with varying severity. Multiple morphological abnormalities of the flagella (MMAF) is one of the most severe forms of asthenoteratozoospermia, characterized by heterogeneous flagellar abnormalities. Among various genetic factors known to cause MMAF, multiple variants in the DNAH2 gene are reported to underlie MMAF in humans. However, the pathogenicity by DNAH2 mutations remains largely unknown. In this study, we identified a novel recessive variant (NM_020877:c.12720G > T;p.W4240C) in DNAH2 by whole-exome sequencing, which fully co-segregated with the infertile male members in a consanguineous Pakistani family diagnosed with asthenozoospermia. 80–90% of the sperm from the patients are morphologically abnormal, and in silico analysis models reveal that the non-synonymous variant substitutes a residue in dynein heavy chain domain and destabilizes DNAH2. To better understand the pathogenicity of various DNAH2 variants underlying MMAF in general, we functionally characterized Dnah2-mutant mice generated by CRISPR/Cas9 genome editing. Dnah2-null males, but not females, are infertile. Dnah2-null sperm cells display absent, short, bent, coiled, and/or irregular flagella consistent with the MMAF phenotype. We found misexpression of centriolar proteins and delocalization of annulus proteins in Dnah2-null spermatids and sperm, suggesting dysregulated flagella development in spermiogenesis. Scanning and transmission electron microscopy analyses revealed that flagella ultrastructure is severely disorganized in Dnah2-null sperm. Absence of DNAH2 compromises the expression of other axonemal components such as DNAH1 and RSPH3. Our results demonstrate that DNAH2 is essential for multiple steps in sperm flagella formation and provide insights into molecular and cellular mechanisms of MMAF pathogenesis.

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Germline variant in REXO2 is a novel candidate gene in familial pheochromocytoma

Genetics Research ◽

10.1017/s0016672320000038 ◽

2020 ◽

Vol 102 ◽

Cited By ~ 1

Author(s):

Yael Laitman ◽

Shay Tzur ◽

Ruben Attali ◽

Amit Tirosh ◽

Eitan Friedman

Keyword(s):

Allelic Imbalance ◽

Chromosomal Region ◽

Cancer Susceptibility ◽

Functional Studies ◽

Cancer Susceptibility Genes ◽

Whole Exome ◽

Familial Pheochromocytoma ◽

The Family ◽

Recombination Processes

Abstract Pheochromocytoma (PCC) is a rare, mostly benign tumour of the adrenal medulla. Hereditary PCC accounts for ~35% of cases and has been associated with germline mutations in several cancer susceptibility genes (e.g., KIF1B, SDHB, VHL, SDHD, RET). We performed whole-exome sequencing in a family with four PCC-affected patients in two consecutive generations and identified a potential novel candidate pathogenic variant in the REXO2 gene that affects splicing (c.531-1G>T (NM 015523.3)), which co-segregated with the phenotype in the family. REXO2 encodes for RNA exonuclease 2 protein and localizes to 11q23, a chromosomal region displaying allelic imbalance in PCC. REXO2 protein has been associated with DNA repair, replication and recombination processes and thus its inactivation may contribute to tumorigenesis. While the study suggests that this novel REXO2 gene variant underlies PCC in this family, additional functional studies are required in order to establish the putative role of the REXO2 gene in PCC predisposition.

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Novel variants in DNAH9 lead to nonsyndromic severe asthenozoospermia

Reproductive Biology and Endocrinology ◽

10.1186/s12958-021-00709-0 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Dongdong Tang ◽

Yanwei Sha ◽

Yang Gao ◽

Jingjing Zhang ◽

Huiru Cheng ◽

...

Keyword(s):

Sanger Sequencing ◽

Sperm Morphology ◽

Immunofluorescence Staining ◽

Compound Heterozygous ◽

Sperm Tail ◽

Transmission Electron ◽

Whole Exome ◽

Detailed Medical History ◽

Novel Variants ◽

Common Causes

Abstract Background Asthenozoospermia is one of the most common causes of male infertility, and its genetic etiology is poorly understood. DNAH9 is a core component of outer dynein arms in cilia and flagellum. It was reported that variants of DNAH9 (OMIM: 603330) might cause primary ciliary dyskinesia (PCD). However, variants in DNAH9 lead to nonsyndromic severe asthenozoospermia have yet to be reported. Methods Whole exome sequencing (WES) was performed for two individuals with nonsyndromic severe asthenozoospermia from two non-consanguineous families, and Sanger sequencing was performed to verify the identified variants and parental origins. Sperm routine analysis, sperm vitality rate and sperm morphology analysis were performed according the WHO guidelines 2010 (5th edition). Transmission electron microscopy (TEM, TECNAI-10, 80 kV, Philips, Holland) was used to observe ultrastructures of sperm tail. Quantitative realtime-PCR and immunofluorescence staining were performed to detect the expression of DNAH9-mRNA and location of DNAH9-protein. Furthermore, assisted reproductive procedures were applied. Results By WES and Sanger sequencing, compound heterozygous DNAH9 (NM_001372.4) variants were identified in the two individuals with nonsyndromic severe asthenozoospermia (F1 II-1: c.302dupT, p.Leu101fs*47 / c.6956A > G, p.Asp2319Gly; F2 II-1: c.6294 T > A, p.Phe2098Leu / c.10571 T > A, p.Leu3524Gln). Progressive rates less than 1% with normal sperm morphology rates and normal vitality rates were found in both of the two subjects. No respiratory phenotypes, situs inversus or other malformations were found by detailed medical history, physical examination and lung CT scans etc. Moreover, the expression of DNAH9-mRNA was significantly decreased in sperm from F1 II-1. And expression of DNAH9 is lower in sperm tail by immunofluorescence staining in F1 II-1 compared with normal control. Notably, by intracytoplasmic sperm injection (ICSI), F1 II-1 and his partner successfully achieved clinical pregnancy. Conclusions We identified DNAH9 as a novel pathogenic gene for nonsyndromic severe asthenospermia, and ICSI can contribute to favorable pregnancy outcomes for these patients.

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Deficiency of the Tbc1d21 gene causes male infertility with morphological abnormalities of the sperm mitochondria and flagellum in mice

PLoS Genetics ◽

10.1371/journal.pgen.1009020 ◽

2020 ◽

Vol 16 (9) ◽

pp. e1009020

Author(s):

Ya-Yun Wang ◽

Chih-Chun Ke ◽

Yen-Lin Chen ◽

Yu-Hua Lin ◽

I-Shing Yu ◽

...

Keyword(s):

Male Infertility ◽

Morphological Abnormalities

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NovelCFAP43 andCFAP44 mutations cause male infertility with multiple morphological abnormalities of the sperm flagella (MMAF)

Reproductive BioMedicine Online ◽

10.1016/j.rbmo.2018.12.037 ◽

2019 ◽

Vol 38 (5) ◽

pp. 769-778 ◽

Cited By ~ 10

Author(s):

Huan Wu ◽

Weiyu Li ◽

Xiaojin He ◽

Chunyu Liu ◽

Youyan Fang ◽

...

Keyword(s):

Male Infertility ◽

Morphological Abnormalities

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Effects of Environmental and Pathological Hypoxia on Male Fertility

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.725933 ◽

2021 ◽

Vol 9 ◽

Author(s):

Zhibin Li ◽

Sumin Wang ◽

Chunli Gong ◽

Yiyang Hu ◽

Jiao Liu ◽

...

Keyword(s):

Male Infertility ◽

Animal Studies ◽

Sperm Quality ◽

Reproductive Toxicity ◽

Causative Factor ◽

Reproductive Hormones ◽

Epigenetic Changes ◽

Causal Association ◽

Spermatogenic Cells ◽

Male Population

Male infertility is a widespread health problem affecting approximately 6%–8% of the male population, and hypoxia may be a causative factor. In mammals, two types of hypoxia are known, including environmental and pathological hypoxia. Studies looking at the effects of hypoxia on male infertility have linked both types of hypoxia to poor sperm quality and pregnancy outcomes. Hypoxia damages testicular seminiferous tubule directly, leading to the disorder of seminiferous epithelium and shedding of spermatogenic cells. Hypoxia can also disrupt the balance between oxidative phosphorylation and glycolysis of spermatogenic cells, resulting in impaired self-renewal and differentiation of spermatogonia, and failure of meiosis. In addition, hypoxia disrupts the secretion of reproductive hormones, causing spermatogenic arrest and erectile dysfunction. The possible mechanisms involved in hypoxia on male reproductive toxicity mainly include excessive ROS mediated oxidative stress, HIF-1α mediated germ cell apoptosis and proliferation inhibition, systematic inflammation and epigenetic changes. In this review, we discuss the correlations between hypoxia and male infertility based on epidemiological, clinical and animal studies and enumerate the hypoxic factors causing male infertility in detail. Demonstration of the causal association between hypoxia and male infertility will provide more options for the treatment of male infertility

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Homozygous TRPV4 mutation causes congenital distal spinal muscular atrophy and arthrogryposis

10.1101/402388 ◽

2018 ◽

Author(s):

Jose Velilla ◽

Michael Mario Marchetti ◽

Agnes Toth-Petroczy ◽

Claire Grosgogeat ◽

Alexis H Bennett ◽

...

Keyword(s):

Spinal Muscular Atrophy ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

In Silico ◽

Muscular Atrophy ◽

Structural Modeling ◽

Recessive Mutation ◽

Homozygous Mutation ◽

Functional Studies ◽

Whole Exome

AbstractObjectiveThe objective of this study is to identify the genetic cause of disease in a congenital form of congenital spinal muscular atrophy and arthrogryposis (CSMAA).MethodsA 2-year-old boy was diagnosed with arthrogryposis multiplex congenita, severe skeletal abnormalities, torticollis, vocal cord paralysis and diminished lower limb movement. Whole exome sequencing was performed on the proband and family members. In silico modeling of protein structure and heterologous protein expression and cytotoxicity assays were performed to validate pathogenicity of the identified variant.ResultsWhole exome sequencing revealed a homozygous mutation in the TRPV4 gene (c.281C>T; p.S94L). The identification of a recessive mutation in TRPV4 extends the spectrum of mutations in recessive forms of the TRPV4-associated disease. p.S94L and other previously identified TRPV4 variants in different protein domains were compared in structural modeling and functional studies. In silico structural modeling suggests that the p.S94L mutation is in the disordered N-terminal region proximal to important regulatory binding sites for phosphoinositides and for PACSIN3, which could lead to alterations in trafficking and/or channel sensitivity. Functional studies by western blot and immunohistochemical analysis show that p.S94L reduces TRPV4 protein stability because of increased cytotoxicity and therefore involves a gain-of-function mechanism.ConclusionThis study identifies a novel homozygous mutation in TRPV4 as a cause of the recessive form of congenital spinal muscular atrophy and arthrogryposis.

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