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

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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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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Biallelic variants in MAATS1 encoding CFAP91, a calmodulin-associated and spoke-associated complex protein, cause severe astheno-teratozoospermia and male infertility

Journal of Medical Genetics ◽

10.1136/jmedgenet-2019-106775 ◽

2020 ◽

Vol 57 (10) ◽

pp. 708-716 ◽

Cited By ~ 4

Author(s):

Guillaume Martinez ◽

Julie Beurois ◽

Denis Dacheux ◽

Caroline Cazin ◽

Marie Bidart ◽

...

Keyword(s):

Male Infertility ◽

Sperm Cells ◽

Gene Encoding ◽

Infertile Men ◽

Transmission Electron ◽

Complex Protein ◽

Radial Spokes ◽

Gene Defects ◽

Primary Male ◽

And Function

BackgroundMultiple morphological abnormalities of the flagella (MMAF) consistently lead to male infertility due to a reduced or absent sperm motility defined as asthenozoospermia. Despite numerous genes recently described to be recurrently associated with MMAF, more than half of the cases analysed remain unresolved, suggesting that many yet uncharacterised gene defects account for this phenotypeMethodsExome sequencing was performed on 167 infertile men with an MMAF phenotype. Immunostaining and transmission electron microscopy (TEM) in sperm cells from affected individuals were performed to characterise the ultrastructural sperm defects. Gene inactivation using RNA interference (RNAi) was subsequently performed in Trypanosoma.ResultsWe identified six unrelated affected patients carrying a homozygous deleterious variants in MAATS1, a gene encoding CFAP91, a calmodulin-associated and spoke-associated complex (CSC) protein. TEM and immunostaining experiments in sperm cells showed severe central pair complex (CPC) and radial spokes defects. Moreover, we confirmed that the WDR66 protein is a physical and functional partner of CFAP91 into the CSC. Study of Trypanosoma MAATS1’s orthologue (TbCFAP91) highlighted high sequence and structural analogies with the human protein and confirmed the axonemal localisation of the protein. Knockdown of TbCFAP91 using RNAi impaired flagellar movement led to CPC defects in Trypanosoma as observed in humans.ConclusionsWe showed that CFAP91 is essential for normal sperm flagellum structure and function in human and Trypanosoma and that biallelic variants in this gene lead to severe flagellum malformations resulting in astheno-teratozoospermia and primary male infertility.

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

Journal of Medical Genetics ◽

10.1136/jmedgenet-2019-106011 ◽

2019 ◽

Vol 57 (1) ◽

pp. 31-37 ◽

Cited By ~ 13

Author(s):

Chunyu Liu ◽

Mingrong Lv ◽

Xiaojin He ◽

Yong Zhu ◽

Amir Amiri-Yekta ◽

...

Keyword(s):

Male Infertility ◽

Animal Studies ◽

Intraflagellar Transport ◽

Spontaneous Mutations ◽

Sperm Tail ◽

Morphological Abnormalities ◽

Functional Studies ◽

Whole Exome ◽

Homozygous Mutations ◽

Functional Analyses

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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Identification of DNAH6 mutations in infertile men with multiple morphological abnormalities of the sperm flagella

Scientific Reports ◽

10.1038/s41598-019-52436-7 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 8

Author(s):

Chaofeng Tu ◽

Hongchuan Nie ◽

Lanlan Meng ◽

Shimin Yuan ◽

Wenbin He ◽

...

Keyword(s):

Compound Heterozygous ◽

Missense Mutations ◽

Transmission Electron Microscopy Analysis ◽

Chinese Families ◽

Morphological Abnormalities ◽

Recessive Mutations ◽

Transmission Electron ◽

Whole Exome ◽

Electron Microscopy Analysis ◽

Reproductive Counseling

Abstract Male infertility due to spermatogenesis defects affects millions of men worldwide. However, the genetic etiology of the vast majority remains unclear. Here we describe three men with primary infertility due to multiple morphological abnormalities of the sperm flagella (MMAF) from two unrelated Han Chinese families. We performed whole-exome sequencing (WES) and Sanger sequencing on the proband of family 1, and found that he carried novel compound heterozygous missense mutations in dynein axonemal heavy chain 6 (DNAH6) that resulted in the substitution of a conserved amino acid residue and co-segregated with the MMAF phenotype in this family. Papanicolaou staining and transmission electron microscopy analysis revealed morphological and ultrastructural abnormalities in the sperm flagella in carriers of these genetic variants. Immunostaining experiments showed that DNAH6 was localized in the sperm tail. This is the first report identifying novel recessive mutations in DNAH6 as a cause of MMAF. These findings expand the spectrum of known MMAF mutations and phenotypes and provide information that can be useful for genetic and reproductive counseling of MMAF patients.

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Bi-allelic DNAH8 Variants Lead to Multiple Morphological Abnormalities of the Sperm Flagella and Primary Male Infertility

The American Journal of Human Genetics ◽

10.1016/j.ajhg.2020.06.004 ◽

2020 ◽

Vol 107 (2) ◽

pp. 330-341 ◽

Cited By ~ 6

Author(s):

Chunyu Liu ◽

Haruhiko Miyata ◽

Yang Gao ◽

Yanwei Sha ◽

Shuyan Tang ◽

...

Keyword(s):

Male Infertility ◽

Morphological Abnormalities ◽

Primary Male

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Genes predisposing to syndromic and nonsyndromic infertility: a narrative review

Egyptian Journal of Medical Human Genetics ◽

10.1186/s43042-020-00088-y ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Tajudeen O. Yahaya ◽

Usman U. Liman ◽

Haliru Abdullahi ◽

Yahuza S. Koko ◽

Samuel S. Ribah ◽

...

Keyword(s):

Male Infertility ◽

Sperm Morphology ◽

Sex Reversal ◽

Ovarian Failure ◽

Main Body ◽

Treatment Procedure ◽

Genetic Etiology ◽

Morphological Abnormalities ◽

The Common ◽

Spermatogenic Failure

Abstract Background Advanced biological techniques have helped produce more insightful findings on the genetic etiology of infertility that may lead to better management of the condition. This review provides an update on genes predisposing to syndromic and nonsyndromic infertility. Main body The review identified 65 genes linked with infertility and infertility-related disorders. These genes regulate fertility. However, mutational loss of the functions of the genes predisposes to infertility. Twenty-three (23) genes representing 35% were linked with syndromic infertility, while 42 genes (65%) cause nonsyndromic infertility. Of the 42 nonsyndromic genes, 26 predispose to spermatogenic failure and sperm morphological abnormalities, 11 cause ovarian failures, and 5 cause sex reversal and puberty delay. Overall, 31 genes (48%) predispose to male infertility, 15 genes (23%) cause female infertility, and 19 genes (29%) predispose to both. The common feature of male infertility was spermatogenic failure and sperm morphology abnormalities, while ovarian failure has been the most frequently reported among infertile females. The mechanisms leading to these pathologies are gene-specific, which, if targeted in the affected, may lead to improved treatment. Conclusions Mutational loss of the functions of some genes involved in the development and maintenance of fertility may predispose to syndromic or nonsyndromic infertility via gene-specific mechanisms. A treatment procedure that targets the affected gene(s) in individuals expressing infertility may lead to improved treatment.

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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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Novel Variants in DNAH9 Lead to Nonsyndromic Severe Asthenozoospermia

10.21203/rs.3.rs-197024/v1 ◽

2021 ◽

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.6294T>A, p.Phe2098Leu / c.10571T>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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