Azoospermia and reciprocal translocations: should whole-exome sequencing be recommended?

Abstract Background Although chromosome rearrangements are responsible for spermatogenesis failure, their impact depends greatly on the chromosomes involved. At present, karyotyping and Y chromosome microdeletion screening are the first-line genetic tests for patients with non-obstructive azoospermia. Although it is generally acknowledged that X or Y chromosome rearrangements lead to meiotic arrest and thus rule out any chance of sperm retrieval after a testicular biopsy, we currently lack markers for the likelihood of testicular sperm extraction (TESE) in patients with other chromosome rearrangements. Results We investigated the use of a single nucleotide polymorphism comparative genome hybridization array (SNP-CGH) and whole-exome sequencing (WES) for two patients with non-obstructive azoospermia and testicular meiotic arrest, a reciprocal translocation: t(X;21) and t(20;22), and an unsuccessful TESE. No additional gene defects were identified for the t(X;21) carrier - suggesting that t(X;21) alone damages spermatogenesis. In contrast, the highly consanguineous t(20;22) carrier had two deleterious homozygous variants in the TMPRSS9 gene; these might have contributed to testicular meiotic arrest. Genetic defect was confirmed with Sanger sequencing and immunohistochemical assessments on testicular tissue sections. Conclusions Firstly, TMPRSS9 gene defects might impact spermatogenesis. Secondly, as a function of the chromosome breakpoints for azoospermic patients with chromosome rearrangements, provision of the best possible genetic counselling means that genetic testing should not be limited to karyotyping. Given the risks associated with TESE, it is essential to perform WES - especially for consanguineous patients.

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Novel variants in helicase for meiosis 1 lead to male infertility due to non-obstructive azoospermia

Reproductive Biology and Endocrinology ◽

10.1186/s12958-021-00815-z ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Dongdong Tang ◽

Mingrong Lv ◽

Yang Gao ◽

Huiru Cheng ◽

Kuokuo Li ◽

...

Keyword(s):

Male Infertility ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Genetic Factors ◽

Testicular Biopsy ◽

Severe Form ◽

Testicular Sperm Extraction ◽

Obstructive Azoospermia ◽

Sperm Retrieval ◽

Whole Exome

Abstract Background Non-obstructive azoospermia (NOA) is the most severe form of male infertility; more than half of the NOA patients are idiopathic. Although many NOA risk genes have been detected, the genetic factors for NOA in majority of the patients are unknown. In addition, it is difficult to retrieve sperm from these patients despite using the microsurgical testicular sperm extraction (microTESE) method. Therefore, we conducted this genetic study to identify the potential genetic factors responsible for NOA and investigate the sperm retrieval rate of microTESE for genetically deficient NOA patients. Methods Semen analyses, sex hormone testing, and testicular biopsy were performed to categorize the patients with NOA. The chromosome karyotypes and Y chromosome microdeletion analyses were used to exclude general genetic factors. Whole exome sequencing and Sanger sequencing were performed to identify potential genetic variants in 51 patients with NOA. Hematoxylin and eosin staining (H&E) and anti-phosphorylated H2AX were used to assess the histopathology of spermatogenesis. Quantitative real time-polymerase chain reaction, western blotting, and immunofluorescence were performed to verify the effects of gene variation on expression. Results We performed whole exome sequencing in 51 NOA patients and identified homozygous helicase for meiosis 1(HFM1) variants (NM_001017975: c.3490C > T: p.Q1164X; c.3470G > A: p.C1157Y) in two patients (3.9%, 2/51). Histopathology of the testis showed that spermatogenesis was completely blocked at metaphase in these two patients carrying the HFM1 homozygous variants. In comparison with unaffected controls, we found a significant reduction in the levels of HFM1 mRNA and protein expression in the testicular tissues from these two patients. The patients were also subjected to microTESE treatment, but the sperms could not be retrieved. Conclusions This study identified novel homozygous variants of HFM1 that are responsible for spermatogenic failure and NOA, and microTESE did not aid in retrieving sperms from these patients.

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DMC1 mutation that causes human non-obstructive azoospermia and premature ovarian insufficiency identified by whole-exome sequencing

Journal of Medical Genetics ◽

10.1136/jmedgenet-2017-104992 ◽

2018 ◽

Vol 55 (3) ◽

pp. 198-204 ◽

Cited By ~ 24

Author(s):

Wen-Bin He ◽

Chao-Feng Tu ◽

Qiang Liu ◽

Lan-Lan Meng ◽

Shi-Min Yuan ◽

...

Keyword(s):

Exome Sequencing ◽

Missense Mutation ◽

Whole Exome Sequencing ◽

Male Patient ◽

Histological Analysis ◽

Pedigree Analysis ◽

Obstructive Azoospermia ◽

Ovarian Insufficiency ◽

Causative Gene ◽

Whole Exome

BackgroundThe genetic causes of the majority of male and female infertility caused by human non-obstructive azoospermia (NOA) and premature ovarian insufficiency (POI) with meiotic arrest are unknown.ObjectiveTo identify the genetic cause of NOA and POI in two affected members from a consanguineous Chinese family.MethodsWe performed whole-exome sequencing of DNA from both affected patients. The identified candidate causative gene was further verified by Sanger sequencing for pedigree analysis in this family. In silico analysis was performed to functionally characterise the mutation, and histological analysis was performed using the biopsied testicle sample from the male patient with NOA.ResultsWe identified a novel homozygous missense mutation (NM_007068.3: c.106G>A, p.Asp36Asn) in DMC1, which cosegregated with NOA and POI phenotypes in this family. The identified missense mutation resulted in the substitution of a conserved aspartic residue with asparaginate in the modified H3TH motif of DMC1. This substitution results in protein misfolding. Histological analysis demonstrated a lack of spermatozoa in the male patient’s seminiferous tubules. Immunohistochemistry using a testis biopsy sample from the male patient showed that spermatogenesis was blocked at the zygotene stage during meiotic prophase I.ConclusionsTo the best of our knowledge, this is the first report identifying DMC1 as the causative gene for human NOA and POI. Furthermore, our pedigree analysis shows an autosomal recessive mode of inheritance for NOA and POI caused by DMC1 in this family.

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Novel Variants in HFM1 Lead to Male Infertility Due to Non-obstructive Azoospermia

10.21203/rs.3.rs-431339/v1 ◽

2021 ◽

Author(s):

Dongdong Tang ◽

Mingrong Lv ◽

Yang Gao ◽

Huiru Cheng ◽

Kuokuo Li ◽

...

Keyword(s):

Male Infertility ◽

Y Chromosome ◽

Testicular Biopsy ◽

Severe Form ◽

Testicular Sperm Extraction ◽

Obstructive Azoospermia ◽

Sperm Retrieval ◽

Gene Variation ◽

Y Chromosome Microdeletions ◽

Whole Exome

Abstract Background Non-obstructive azoospermia (NOA) is the most severe form of male infertility. More than half of the NOA patients were idiopathic for their etiology, in whom it’s difficult to retrieve sperm despite the application of microsurgical testicular sperm extraction (microTESE). Therefore, we conducted to this study to identify the potential genetic factors responsible for NOA, and investigate the sperm retrieval rate of microTESE for the genetic defected NOA.Methods One NOA patient from a consanguineous family (F1-II-1) and fifty NOA patients from non-consanguineous families were included in the study. Semen analyses, chromosome karyotypes, screening of Y chromosome microdeletions, sex hormone testing, and subsequent testicular biopsy were performed to categorize NOA or obstructive azoospermia. Potentialgenetic variants were identified by whole exome sequencing (WES),and confirmed by Sanger sequencing in F1 II-1. The candidate genes were screened in the other fifty NOA patients. Further experiments including quantitative real time-polymerase chain reaction and western blotting were performed to verify the effects of gene variation on gene expression.Results Normal somatic karyotypes and Y chromosome microdeletions were examined in all patients. Hematoxylin and eosin staining (H&E) of the testicular tissues suggested meiotic arrest, and a novel homozygous HFM1 variant (c.3490C>T: p.Q1164X) was identified in F1 II-1. Furthermore, another homozygous HFM1 variant (c.3470G>A: p.C1157Y) was also verified in F2 II-1 from the fifty NOA patients. Significantly decreased expression levels of HFM1 mRNA and protein were observed in the testicular tissues of these two mutants compared with controls. MicroTESE was performed in these two patients, while no sperm were retrieved. Conclusions Our study identified two novel homozygous variants of HFM1 that are responsible for spermatogenic failure and NOA, even microTESE can not contribute to retrieve sperm in these patients.

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Whole exome sequencing in non-obstructive azoospermia allows the identification of a high-risk subgroup of infertile men for undiagnosed Fanconi Anemia, a cancer-prone disease

Endocrine Abstracts ◽

10.1530/endoabs.56.oc2.3 ◽

2018 ◽

Author(s):

Csilla Krausz ◽

Antoni Riera-Escamilla ◽

Chiara Chianese ◽

Daniel Moreno-Mendoza ◽

Osvaldo Rajmil ◽

...

Keyword(s):

High Risk ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Fanconi Anemia ◽

Obstructive Azoospermia ◽

Infertile Men ◽

Whole Exome

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Pathogenic variants of ATG4D in infertile men with non‐obstructive azoospermia identified using whole‐exome sequencing

Clinical Genetics ◽

10.1111/cge.13995 ◽

2021 ◽

Author(s):

Yanwei Sha ◽

Wensheng Liu ◽

Xiaoli Wei ◽

Xingshen Zhu ◽

Bowen Tang ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Obstructive Azoospermia ◽

Pathogenic Variants ◽

Infertile Men ◽

Whole Exome

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Combined Use of Whole Exome Sequencing and CRISPR/Cas9 to Study the Etiology of Non-Obstructive Azoospermia: Demonstration of the Dispensable Role of the Testis-Specific Genes C1orf185 and CCT6B

Cells ◽

10.3390/cells11010118 ◽

2021 ◽

Vol 11 (1) ◽

pp. 118

Author(s):

Caroline Cazin ◽

Yasmine Neirijnck ◽

Corinne Loeuillet ◽

Lydia Wehrli ◽

Françoise Kühne ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Specific Gene ◽

Obstructive Azoospermia ◽

Loss Of Function ◽

Whole Exome ◽

Variant Filtering ◽

Genetic Landscape ◽

Mouse Orthologs ◽

Human Spermatogenesis

The genetic landscape of male infertility is highly complex. It is estimated that at least 4000 genes are involved in human spermatogenesis, but only few have so far been extensively studied. In this study, we investigated by whole exome sequencing two cases of idiopathic non-obstructive azoospermia (NOA) due to severe hypospermatogenesis. After variant filtering and prioritizing, we retained for each patient a homozygous loss-of-function (LoF) variant in a testis-specific gene, C1orf185 (c.250C>T; p.Gln84Ter) and CCT6B (c.615-2A>G), respectively. Both variants are rare according to the gnomAD database and absent from our local control cohort (n = 445). To verify the implication of these candidate genes in NOA, we used the CRISPR/Cas9 system to invalidate the mouse orthologs 4930522H14Rik and Cct6b and produced two knockout (KO) mouse lines. Sperm and testis parameters of homozygous KO adult male mice were analyzed and compared with those of wild-type animals. We showed that homozygous KO males were fertile and displayed normal sperm parameters and a functional spermatogenesis. Overall, these results demonstrate that not all genes highly and specifically expressed in the testes are essential for spermatogenesis, and in particular, we conclude that bi-allelic variants of C1orf185 and CCT6B are most likely not to be involved in NOA and male fertility.

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Whole-exome sequencing of a large Chinese azoospermia and severe oligospermia cohort identifies novel infertility causative variants and genes

Human Molecular Genetics ◽

10.1093/hmg/ddaa101 ◽

2020 ◽

Vol 29 (14) ◽

pp. 2451-2459

Author(s):

Shitao Chen ◽

Guishuan Wang ◽

Xiaoguo Zheng ◽

Shunna Ge ◽

Yubing Dai ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Molecular Characteristics ◽

Obstructive Azoospermia ◽

Sequencing Data ◽

Chinese Han ◽

Functional Studies ◽

Variants Of Unknown Significance ◽

Pathogenic Variants ◽

Whole Exome

Abstract Rare coding variants have been proven to be one of the significant factors contributing to spermatogenic failure in patients with non-obstructive azoospermia (NOA) and severe oligospermia (SO). To delineate the molecular characteristics of idiopathic NOA and SO, we performed whole-exome sequencing of 314 unrelated patients of Chinese Han origin and verified our findings by comparing to 400 fertile controls. We detected six pathogenic/likely pathogenic variants and four variants of unknown significance, in genes known to cause NOA/SO, and 9 of which had not been earlier reported. Additionally, we identified 20 novel NOA candidate genes affecting 25 patients. Among them, five (BRDT, CHD5, MCM9, MLH3 and ZFX) were considered as strong candidates based on the evidence obtained from murine functional studies and human single-cell (sc)RNA-sequencing data. These genetic findings provide insight into the aetiology of human NOA/SO and pave the way for further functional analysis and molecular diagnosis of male infertility.

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Whole exome sequencing identifies genes associated with non-obstructive azoospermia

10.1101/2020.05.29.20116558 ◽

2020 ◽

Author(s):

Hongguo Zhang ◽

Hui Huang ◽

Xinyue Zhang ◽

Wei Li ◽

Yuting Jiang ◽

...

Keyword(s):

Linkage Analysis ◽

Association Study ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Rare Variant ◽

Biological Study ◽

Obstructive Azoospermia ◽

Novel Genes ◽

Rare Variant Association ◽

Whole Exome

AbstractBackgroundGenetic etiology is the main cause of non-obstructive azoospermia, but little is known about the landscape of the disease causative genes.ObjectiveTo identify the association of non-obstructive azoospermia and the putative causative genetic factors.Design, setting, and participantsA single-center perspective case-control study of 133 patients, with clinicopathologic non-obstructive azoospermia and 495 fertile men control was performed. Eleven trio families were available and enrolled from the 133 patients’ families.Outcome measurements and statistical analysisWhole exome sequencing based rare variant association study between the cases and controls was performed by means of gene burden association testing. Linkage analysis on the trio family was also described to screen the causative genes.Results and limitationsTotally 80 genes (p < 0.05) were identified associated with non-obstructive azoospermia (2 of which were previously reported), meanwhile 5 novel genes out of which were also found potentially causative through the linkage analysis on the trio families. The pathway enrichment analysis was also provided to assess the potential interaction between genes identified in this study and previously reported together. The 5 novel identified overlap genes by both above mentioned test with the rare mutations account for an overall 20% (26 /133 patients) incidence, together with the 2 known genes together would account for an overall 20% incidence for non-obstructive azoospermia in this study. The study is limited by the lack of functional biological study.ConclusionsFive novel genes were identified associated with non-obstructive azoospermia by means of both rare variant association study and linkage analysis through trio families. They could account for about 20% clinical incidence among the patients in our study.Patient summary133 infertile patients (11 of them with parents enrolled) with idiopathic non-obstructive azoospermia and 300 fertile male controls were recruited from single clinic center.All patients underwent semen analyses at least on three different occasions.

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