Exome sequencing reveals novel causes as well as new candidate genes for human globozoospermia

Abstract STUDY QUESTION Can exome sequencing identify new genetic causes of globozoospermia? SUMMARY ANSWER Exome sequencing in 15 cases of unexplained globozoospermia revealed deleterious mutations in seven new genes, of which two have been validated as causing globozoospermia when knocked out in mouse models. WHAT IS KNOWN ALREADY Globozoospermia is a rare form of male infertility characterised by round-headed sperm and malformation of the acrosome. Although pathogenic variants in DPY19L2 and SPATA16 are known causes of globozoospermia and explain up to 70% of all cases, genetic causality remains unexplained in the remaining patients. STUDY DESIGN, SIZE, DURATION After pre-screening 16 men for mutations in known globozoospermia genes DPY19L2 and SPATA16, exome sequencing was performed in 15 males with globozoospermia or acrosomal hypoplasia of unknown aetiology. PARTICIPANTS/MATERIALS, SETTING, METHOD Targeted next-generation sequencing and Sanger sequencing was performed for all 16 patients to screen for single-nucleotide variants and copy number variations in DPY19L2 and SPATA16. After exclusion of one patient with DPY19L2 mutations, we performed exome sequencing for the 15 remaining subjects. We prioritised recessive and X-linked protein-altering variants with an allele frequency of <0.5% in the population database GnomAD in genes with an enhanced expression in the testis. All identified candidate variants were confirmed in patients and, where possible, in family members using Sanger sequencing. Ultrastructural examination of semen from one of the patients allowed for a precise phenotypic characterisation of abnormal spermatozoa. MAIN RESULTS AND ROLE OF CHANCE After prioritisation and validation, we identified possibly causative variants in eight of 15 patients investigated by exome sequencing. The analysis revealed homozygous nonsense mutations in ZPBP and CCDC62 in two unrelated patients, as well as rare missense mutations in C2CD6 (also known as ALS2CR11), CCIN, C7orf61 and DHNA17 and a frameshift mutation in GGN in six other patients. All variants identified through exome sequencing, except for the variants in DNAH17, were located in a region of homozygosity. Familial segregation of the nonsense variant in ZPBP revealed two fertile brothers and the patient’s mother to be heterozygous carriers. Paternal DNA was unavailable. Immunohistochemistry confirmed that ZPBP localises to the acrosome in human spermatozoa. Ultrastructural analysis of spermatozoa in the patient with the C7orf61 mutation revealed a mixture of round heads with no acrosomes (globozoospermia) and ovoid or irregular heads with small acrosomes frequently detached from the sperm head (acrosomal hypoplasia). LIMITATIONS, REASONS FOR CAUTION Stringent filtering criteria were used in the exome data analysis which could result in possible pathogenic variants remaining undetected. Additionally, functional follow-up is needed for several candidate genes to confirm the impact of these mutations on normal spermatogenesis. WIDER IMPLICATIONS OF THE FINDINGS Our study revealed an important role for mutations in ZPBP and CCDC62 in human globozoospermia as well as five new candidate genes. These findings provide a more comprehensive understanding of the genetics of male infertility and bring us closer to a complete molecular diagnosis for globozoospermia patients which would help to predict the success of reproductive treatments. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by The Netherlands Organisation for Scientific Research (918–15-667); National Health and Medical Research Council of Australia (APP1120356) and the National Council for Scientific Research (CONICET), Argentina, PIP grant 11220120100279CO. The authors have nothing to disclose.

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Investigation of monogenic causes of familial breast cancer: data from the BEACCON case-control study

npj Breast Cancer ◽

10.1038/s41523-021-00279-9 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Na Li ◽

Belle W. X. Lim ◽

Ella R. Thompson ◽

Simone McInerny ◽

Magnus Zethoven ◽

...

Keyword(s):

Breast Cancer ◽

High Risk ◽

Candidate Genes ◽

Familial Aggregation ◽

Loss Of Function ◽

Cancer Data ◽

Pathogenic Variants ◽

New Genes ◽

Predisposing Genes ◽

Predisposition Genes

AbstractBreast cancer (BC) has a significant heritable component but the genetic contribution remains unresolved in the majority of high-risk BC families. This study aims to investigate the monogenic causes underlying the familial aggregation of BC beyond BRCA1 and BRCA2, including the identification of new predisposing genes. A total of 11,511 non-BRCA familial BC cases and population-matched cancer-free female controls in the BEACCON study were investigated in two sequencing phases: 1303 candidate genes in up to 3892 cases and controls, followed by validation of 145 shortlisted genes in an additional 7619 subjects. The coding regions and exon–intron boundaries of all candidate genes and 14 previously proposed BC genes were sequenced using custom designed sequencing panels. Pedigree and pathology data were analysed to identify genotype-specific associations. The contribution of ATM, PALB2 and CHEK2 to BC predisposition was confirmed, but not RAD50 and NBN. An overall excess of loss-of-function (LoF) (OR 1.27, p = 9.05 × 10−9) and missense (OR 1.27, p = 3.96 × 10−73) variants was observed in the cases for the 145 candidate genes. Leading candidates harbored LoF variants with observed ORs of 2–4 and individually accounted for no more than 0.79% of the cases. New genes proposed by this study include NTHL1, WRN, PARP2, CTH and CDK9. The new candidate BC predisposition genes identified in BEACCON indicate that much of the remaining genetic causes of high-risk BC families are due to genes in which pathogenic variants are both very rare and convey only low to moderate risk.

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Next Generation Exome Sequencing of paediatric Asthma Identifies Rare and Novel Variants in Candidate Genes

10.21203/rs.3.rs-31295/v1 ◽

2020 ◽

Author(s):

Neda Bogari ◽

Amr A Amin ◽

Husni H Rayes ◽

Ahmed Abdelmotelb ◽

Mohiuddin M Taher ◽

...

Keyword(s):

Exome Sequencing ◽

Candidate Genes ◽

Association Studies ◽

Reference Sequence ◽

Exome Capture ◽

Sequencing Analysis ◽

Genetic Determinants ◽

Paediatric Asthma ◽

Novel Variants ◽

The Impact

Abstract Background: Multiple genes have been implicated to have a role in asthma predisposition by association studies. Paediatric patients often manifest more extensive disease and a particularly severe disease course. It is likely that genetic predisposition could play a more substantial role in this group. This study aims to identify the spectrum of rare and novel variation in known paediatric asthma susceptibility genes using whole exome sequencing-analysis in nine individual cases of childhood onset allergic asthma. Data were processed through an analytical pipeline to align sequence reads, conduct quality checks, identify and annotate variants where patient sequence differed from the reference sequence. Results: DNA samples from the nine children with a history of bronchial asthma diagnosis underwent targeted exome capture and sequencing. For each patient, the entire complement of rare variation within strongly associated candidate genes was catalogued. The analysis showed 21 variants in the subjects, 13 had been previously identified and 8 were novel. Also, amongst of which, nineteen were non-synonymous and 2 were nonsense. With regard to the novel variants, the 2 non-synonymous variants in the PRKG1 gene (PRKG1: p.C519W and PRKG1: p.G520W) were presented in 4 cases, and a non-synonymous variant in the MAVS gene (MAVS: p.A45V) was identified in 3 cases. The variants we found in this study will enrich the variants spectrum and build up the database in the Saudi population. Novel eight variants were identified in the study which provides more evidence in the genetic susceptibility in asthma among Saudi children.Conclusion: Screening a cohort of Saudi children for molecular identification of polymorphisms associated with allergic asthmatic response. These, together with the clinical phenotypes, revealed genetic determinants for paediatric asthma and also we compared to the similar previous reports. Providing a genetic screening map for the molecular genetic determinants of allergic disease in Saudi children, with the goal of reducing the impact of chronic diseases on the health and the economy. We belief that the advanced specified statistical filtration/annotation programs used in this study succeeded to release such results in preliminary study, exploring the genetic map of that disease in Saudi children.

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Exome sequencing identifies novel AD-associated genes.

10.1101/2020.07.22.20159251 ◽

2020 ◽

Cited By ~ 1

Author(s):

Henne Holstege ◽

Marc Hulsman ◽

Camille Charbonnier ◽

Benjamin Grenier-Boley ◽

Olivier Quenez ◽

...

Keyword(s):

Exome Sequencing ◽

Genetic Variants ◽

Rare Variants ◽

Mendelian Inheritance ◽

Loss Of Function ◽

App Processing ◽

Complex Disorders ◽

Pathogenic Variants ◽

Increased Risk ◽

The Impact

Background: With the development of next-generation sequencing technologies, it is possible to identify rare genetic variants that influence the risk of complex disorders. To date, whole exome sequencing (WES) strategies have shown that specific clusters of damaging rare variants in the TREM2, SORL1 and ABCA7 genes are associated with an increased risk of developing Alzheimers Disease (AD), reaching odds ratios comparable with the APOE-ε4 allele, the main common AD genetic risk factor. Here, we set out to identify additional AD-associated genes by an exome-wide investigation of the burden of rare damaging variants in the genomes of AD cases and cognitively healthy controls. Method: We integrated the data from 25,982 samples from the European ADES consortium and the American ADSP consortium. We developed new techniques to homogenise and analyse these data. Carriers of pathogenic variants in genes associated with Mendelian inheritance of dementia were excluded. After quality control, we used 12,652 AD cases and 8,693 controls for analysis. Genes were analysed using a burden analysis, including both non-synonymous and loss-of-function rare variants, the impact of which was prioritised using REVEL. Result: We confirmed that carrying rare protein-damaging genetic variants in TREM2, SORL1 or ABCA7 is associated with increased AD-risk. Moreover, we found that carrying rare damaging variants in the microglial ATP8B4 gene was significantly associated with AD, and we found suggestive evidence that rare variants in ADAM10, ABCA1, ORC6, B3GNT4 and SRC genes associated with increased AD risk. High-impact variants in these genes were mostly extremely rare and enriched in AD patients with earlier ages at onset. Additionally, we identified two suggestive protective associations in CBX3 and PRSS3. We are currently replicating these associations in independent datasets. Conclusion: With our newly developed homogenisation methods, we identified novel genetic determinants of AD which provide further evidence for a pivotal role of APP processing, lipid metabolism, and microglia and neuro-inflammatory processes in AD pathophysiology.

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OR06-6 Whole-Exome Sequencing of Patients with Pituitary Stalk Interruption Syndrome (PSIS) Reveals Probably Pathogenic Variants in Novel Candidate Genes.

Journal of the Endocrine Society ◽

10.1210/js.2019-or06-6 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

Author(s):

Fernanda Correa ◽

Marilena Nakaguma ◽

Alexander Jorge ◽

Mariana Funari ◽

Antonio Lerario ◽

...

Keyword(s):

Exome Sequencing ◽

Candidate Genes ◽

Whole Exome Sequencing ◽

Pituitary Stalk ◽

Pathogenic Variants ◽

Whole Exome

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DNAH11 compound heterozygous variants cause heterotaxy and congenital heart disease

PLoS ONE ◽

10.1371/journal.pone.0252786 ◽

2021 ◽

Vol 16 (6) ◽

pp. e0252786

Author(s):

Hong Xia ◽

Xiangjun Huang ◽

Sheng Deng ◽

Hongbo Xu ◽

Yan Yang ◽

...

Keyword(s):

Congenital Heart Disease ◽

Heart Disease ◽

Exome Sequencing ◽

Sanger Sequencing ◽

Congenital Heart ◽

Chinese Family ◽

Compound Heterozygous ◽

Internal Organs ◽

Pathogenic Variants ◽

Compound Heterozygous Variants

Heterotaxy (HTX), a condition characterized by internal organs not being arranged as expected relative to each other and to the left-right axis, is often accompanied with congenital heart disease (CHD). The purpose was to detect the pathogenic variants in a Chinese family with HTX and CHD. A non-consanguineous Han Chinese family with HTX and CHD, and 200 unrelated healthy subjects were enlisted. Exome sequencing and Sanger sequencing were applied to identify the genetic basis of the HTX family. Compound heterozygous variants, c.3426-1G>A and c.4306C>T (p.(Arg1436Trp)), in the dynein axonemal heavy chain 11 gene (DNAH11) were identified in the proband via exome sequencing and further confirmed by Sanger sequencing. Neither c.3426-1G>A nor c.4306C>T variant in the DNAH11 gene was detected in 200 healthy controls. The DNAH11 c.3426-1G>A variant was predicted as altering the acceptor splice site and most likely affecting splicing. The DNAH11 c.4306C>T variant was predicted to be damaging, which may reduce the phenotype severity. The compound heterozygous variants, c.3426-1G>A and c.4306C>T, in the DNAH11 gene might be the pathogenic alterations resulting in HTX and CHD in this family. These findings broaden the variant spectrum of the DNAH11 gene and increase knowledge used in genetic counseling for the HTX family.

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Detection and validation of six pathogenic variants in Chinese inherited heart disease patients using clinical whole-exome sequencing

10.22541/au.162566292.28770313/v1 ◽

2021 ◽

Author(s):

Lichao Cao ◽

Fei Ye ◽

Shuqi Xie ◽

Ying Ba ◽

Ying Zeng ◽

...

Keyword(s):

Heart Disease ◽

Next Generation Sequencing ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Sanger Sequencing ◽

Targeted Next Generation Sequencing ◽

Pathogenic Variants ◽

Whole Exome ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

The targeted next-generation sequencing (NGS) was employed in detecting the pathogenic mutations in inherited heart disease patients in the present study. Two main methods, the NGS and the classic Sanger sequencing, were used in this study. And, the whole-exome sequencing (WES) was specifically used in this study.

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Exome Sequencing and Epigenetic Analysis of Twins Who Are Discordant for Congenital Cataract

Twin Research and Human Genetics ◽

10.1017/thg.2015.34 ◽

2015 ◽

Vol 18 (4) ◽

pp. 393-398 ◽

Cited By ~ 4

Author(s):

Tanwei Wei ◽

Hui Sun ◽

Bo Hu ◽

Jie Yang ◽

Chen Qiao ◽

...

Keyword(s):

Exome Sequencing ◽

Candidate Genes ◽

Sanger Sequencing ◽

Congenital Cataract ◽

Genetic Factors ◽

Twin Pair ◽

Mutation Screening ◽

Sequencing Analysis ◽

Twin Sister ◽

Epigenetic Events

Purpose: To further understand genetic factors that contribute to congenital cataracts, we sought to identify early post-twinning mutational and epigenetic events that may account for the discordant phenotypes of a twin pair. Methods: A patient with a congenital cataract and her twin sister were assessed for genetic factors that might contribute to their discordant phenotypes by mutation screening of 11 candidate genes (CRYGC, CRYGD, CRYAA, CRYAB, CRYBA1, CRYBB1, CRYBB2, MIP, HSF4, GJA3, and GJA8), exome analysis followed by Sanger sequencing of 10 additional candidate genes (PLEKHO2, FRYL, RBP3, P2RX2, GSR, TRAM1, VEGFA, NARS2, CADPS, and TEKT4), and promoter methylation analysis of five representative genes (TRAM1, CRYAA, HSF4, VEGFA, GJA3, DCT) plus one additional candidate gene (FTL). Results: Mutation screening revealed no gene mutation differences between the patient and her twin sister for the 11 candidate genes. Exome sequencing analysis revealed variations between the twins in 442 genes, 10 of which are expressed in the eye. However, these differential variants could not be confirmed by Sanger sequencing. Furthermore, epigenetic discordance was not detected in the twin pair. Conclusions: The genomic DNA mutational and epigenetic events assessed in this study could not explain the discordance in the development of phenotypic differences between the twin pair, suggesting the possible involvement of somatic mutations or environmental factors. Identification of possible causes requires further research.

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O-115 Parental whole-exome sequencing allows the discovery of genetic causes of extreme IVF phenotypes such as oocyte/embryo developmental arrest and recurrent low fertilization

Human Reproduction ◽

10.1093/humrep/deab126.024 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

A Capalbo ◽

S Buonaiuto ◽

G Damaggio ◽

M Cetinkaya ◽

B Yuksel ◽

...

Keyword(s):

Exome Sequencing ◽

Candidate Genes ◽

Whole Exome Sequencing ◽

False Positive ◽

High Impact ◽

Unexplained Infertility ◽

Developmental Arrest ◽

New Genes ◽

Fold Enrichment ◽

Whole Exome

Abstract Study question Do whole-exome sequencing (WES) data from infertile women provide valuable information for the discovery of genes/pathways involved in extreme IVF phenotypes, i.e. oocyte/embryo developmental arrest? Summary answer The development of a specific bioinformatic WES pipeline revealed known and new candidate genes/pathways for isolated oocyte/embryo developmental failure,providing the foundation to scale up research. What is known already The use of IVF has made it possible to identify extreme and isolated infertility phenotypes such as recurrent low oocytes maturity (LMR), recurrent low fertilization rate (LFR), or preimplantation developmental arrest (PDA) that would remain concealed in natural conception attempts. Recent applications of WES in families with such extreme adverse IVF phenotypes have led to the discovery of new genes and pathways affecting unique functions of gametes and exclusive mechanisms necessary for early embryo development. Here, we apply a tailored bioinformatic approach to WES from women displaying extreme IVF phenotypes to discover new causative genes/pathways involved in unexplained infertility. Study design, size, duration Twenty-two infertile consanguineous women(December 2018-September 2020) suffering from long-term unexplained infertility. Eight cases were classified as PDA (<20% normally developed embryos in > 2 IVF cycles), 8 as LMR (<20% mature oocytes in > 2 IVF cycles), 4 as LFR (<20% of normally fertilized oocytes in > 2 IVF cycles). Two women with recurrent IVF failure (>10 IVF cycles) were also included. A control set of 1660 WES from oocyte donors was used to control for false-positive discoveries. Participants/materials, setting, methods WES at 30X was performed on enrolled women’s gDNA using Illumina short-reads technology. Following annotation, variants were filtered to prioritize putative detrimental variants in genes relevant for oocyte/embryonic development using a previously developed and validated pipeline that minimizes false-positive discoveries. Runs of homozygosity (ROH) within each sample were identified using Refined IBD software. Individual-level single-cell RNAseq (scRNAseq) dataset from 18 human oocytes was used to verify the expression of the identified target genes. Main results and the role of chance The variant prioritization pipeline employed identified 1,160 unique variants in 1,017 genes (average per sample 59.9 sd 8.5). 453 variants were private to this study compared to the 1000 Genomes and gnomAD databases, 3% affecting splicing and/or the gene product length. Significant 5-fold enrichment of 41 genes involved in DNA-damage and repair pathways commonly associated with ovarian function/oocyte quality was observed (p < 0.001). TP53/AKT pathway also showed significant 5-fold enrichment for 45 genes (p < 0.001). This finding is consistent with the known relationship between infertility and cell-cycle/cancer genes. Overall, 66.4% (675/1,017; 95%CI:63.4-69.3) of the targeted genes were expressed in MII human oocytes. Two women (9%) were homozygous carriers of missense pathogenic variants in known candidate genes previously associated to oocyte/embryo developmental arrest (TRIP13, chr5_901344_C/T, CADD percentile 0.999; PADI6, chr1_17394384_C/G, CADD percentile 0.999). Remarkably, four additional women were carriers of high-impact variants in JAKMIP1, a member of a recently characterized family of proteins involved in various cellular processes, including cytoskeleton rearrangement, cell polarization, and intracellular transport. High-impact JAKMP1 variants were never observed in the oocyte donor control dataset. JAKMIP1 mRNA was detected in each individual biological replicate of scRNAseq analysis of MII oocytes with a mean of 6 transcripts per million. Limitations, reasons for caution Functional analysis is ongoing to validate the newly identified genes, data need to be verified in different ethnicities. Nevertheless, this study demonstrates the establishment of a specific and scalable analytical framework that can be employed for the identification of genetic causes in unexplained infertility cases characterized by defective developmental patterns. Wider implications of the findings Scaling up this investigative approach would provide an effective strategy for discovering new genes/pathways in what is considered idiopathic infertility, further defining precision reproductive medicine interventions. Importantly, this study revealed lesions in genetic patterns involved in chronic diseases providing a molecular footprint of the well-established link between infertility and comorbidities. Trial registration number none

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Identification of novel pathogenic ABCA4 variants in a Han Chinese family with Stargardt disease

Bioscience Reports ◽

10.1042/bsr20180872 ◽

2019 ◽

Vol 39 (1) ◽

Cited By ~ 2

Author(s):

Qin Xiang ◽

Yanna Cao ◽

Hongbo Xu ◽

Yi Guo ◽

Zhijian Yang ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Sanger Sequencing ◽

Retinal Pigment ◽

Han Chinese ◽

Chinese Family ◽

Compound Heterozygous ◽

Stargardt Disease ◽

Pathogenic Variants ◽

Whole Exome

Abstract Stargardt disease (STGD1, OMIM 248200) is a common hereditary juvenile or early adult onset macular degeneration. It ultimately leads to progressive central vision loss. Here, we sought to identify gene mutations associated with STGD1 in a three-generation Han Chinese pedigree by whole exome sequencing and Sanger sequencing. Two novel potentially pathogenic variants in a compound heterozygous state, c.3607G>T (p.(Gly1203Trp)) and c.6722T>C (p.(Leu2241Pro)), in the ATP binding cassette subfamily A member 4 gene (ABCA4) were identified as contributing to the family’s STGD1 phenotype. These variants may impact the ABCA4 protein structure and reduce the retinal-activated ATPase activity, leading to abnormal all-trans retinal accumulation in photoreceptor outer segments and in retinal pigment epithelium cells. The present study broadens the mutational spectrum of the ABCA4 responsible for STGD1. A combination of whole exome sequencing and Sanger sequencing is likely to be a time-saving and cost-efficient approach to screen pathogenic variants in genetic disorders caused by sizable genes, as well as avoiding misdiagnosis. These results perhaps refine genetic counseling and ABCA4-targetted treatments for families affected by STGD1.

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Human X chromosome exome sequencing identifies BCORL1 as contributor to spermatogenesis

Journal of Medical Genetics ◽

10.1136/jmedgenet-2019-106598 ◽

2020 ◽

Vol 58 (1) ◽

pp. 56-65

Author(s):

Chuncheng Lu ◽

Yan Zhang ◽

Yufeng Qin ◽

Qiaoqiao Xu ◽

Ran Zhou ◽

...

Keyword(s):

Male Infertility ◽

Exome Sequencing ◽

Candidate Genes ◽

X Chromosome ◽

Knockout Mice ◽

Expression Patterns ◽

Critical Role ◽

Obstructive Azoospermia ◽

Independent Population

BackgroundInfertility affects approximately 15% of couples worldwide with male infertility being responsible for approximately 50% of cases. Although accumulating evidence demonstrates the critical role of the X chromosome in spermatogenesis during the last few decades, the expression patterns and potential impact of the X chromosome, together with X linked genes, on male infertility are less well understood.MethodsWe performed X chromosome exome sequencing followed by a two-stage independent population validation in 1333 non-obstructive azoospermia cases and 1141 healthy controls to identify variant classes with high likelihood of pathogenicity. To explore the functions of these candidate genes in spermatogenesis, we first knocked down these candidate genes individually in mouse spermatogonial stem cells (SSCs) using short interfering RNA oligonucleotides and then generated candidate genes knockout mice by CRISPR-Cas9 system.ResultsFour low-frequency variants were identified in four genes (BCORL1, MAP7D3, ARMCX4 and H2BFWT) associated with male infertility. Functional studies of the mouse SSCs revealed that knocking down Bcorl1 or Mtap7d3 could inhibit SSCs self-renewal and knocking down Armcx4 could repress SSCs differentiation in vitro. Using CRISPR-Cas9 system, Bcorl1 and Mtap7d3 knockout mice were generated. Excitingly, Bcorl1 knockout mice were infertile with impaired spermatogenesis. Moreover, Bcorl1 knockout mice exhibited impaired sperm motility and sperm cells displayed abnormal mitochondrial structure.ConclusionOur data indicate that the X-linked genes are associated with male infertility and involved in regulating SSCs, which provides a new insight into the role of X-linked genes in spermatogenesis.

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