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

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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The identification of novel gene mutations for degenerative lumbar spinal stenosis using whole-exome sequencing in a Chinese cohort

BMC Medical Genomics ◽

10.1186/s12920-021-00981-4 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Xin Jiang ◽

Dong Chen

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Spinal Stenosis ◽

Lumbar Spinal Stenosis ◽

Gene Mutations ◽

Specific Gene ◽

Single Nucleotide ◽

Degenerative Lumbar Spinal Stenosis ◽

Whole Exome ◽

Lumbar Spinal

Abstract Background Degenerative lumbar spinal stenosis (DLSS) is a common lumbar disease that requires surgery. Previous studies have indicated that genetic mutations are implicated in DLSS. However, studies on specific gene mutations are scarce. Whole-exome sequencing (WES) is a valuable research tool that identifies disease-causing genes and could become an effective strategy to investigate DLSS pathogenesis. Methods From January 2016 to December 2017, we recruited 50 unrelated patients with symptoms consistent with DLSS and 25 unrelated healthy controls. We conducted WES and exome data analysis to identify susceptible genes. Allele mutations firstly identified potential DLSS variants in controls to the patients’ group. We conducted a site-based association analysis to identify pathogenic variants using PolyPhen2, SIFT, Mutation Taster, Combined Annotation Dependent Depletion, and Phenolyzer algorithms. Potential variants were further confirmed using manual curation and validated using Sanger sequencing. Results In this cohort, the major classification variant was missense_mutation, the major variant type was single nucleotide polymorphism (SNP), and the major single nucleotide variation was C > T. Multiple SNPs in 34 genes were identified when filtered allele mutations in controls to retain only patient mutations. Pathway enrichment analyses revealed that mutated genes were mainly enriched for immune response-related signaling pathways. Using the Novegene database, site-based associations revealed several novel variants, including HLA-DRB1, PARK2, ACTR8, AOAH, BCORL1, MKRN2, NRG4, NUP205 genes, etc., were DLSS related. Conclusions Our study revealed that deleterious mutations in several genes might contribute to DLSS etiology. By screening and confirming susceptibility genes using WES, we provided more information on disease pathogenesis. Further WES studies incorporating larger DLSS patient cohorts are required to comprehend the genetic landscape of DLSS pathophysiology fully.

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NAPG mutation in family members with hereditary hemorrhagic telangiectasia in China

BMC Pulmonary Medicine ◽

10.1186/s12890-021-01524-4 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Yu Xu ◽

Yong-Biao Zhang ◽

Li-Jun Liang ◽

Jia-Li Tian ◽

Jin-Ming Lin ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Hereditary Hemorrhagic Telangiectasia ◽

Sanger Sequencing ◽

Conformational Stability ◽

Sequencing Analysis ◽

Large Pedigree ◽

Whole Exome ◽

Variant Filtering ◽

Genetic Contributions

Abstract Background Hereditary hemorrhagic telangiectasia (HHT) is a disease characterized by arteriovenous malformations in the skin and mucous membranes. We enrolled a large pedigree comprising 32 living members, and screened for mutations responsible for HHT. Methods We performed whole-exome sequencing to identify novel mutations in the pedigree after excluding three previously reported HHT-related genes using Sanger sequencing. We then performed in silico functional analysis of candidate mutations that were obtained using a variant filtering strategy to identify mutations responsible for HHT. Results After screening the HHT-related genes, activin A receptor-like type 1 (ACVRL1), endoglin (ENG), and SMAD family member 4 (SMAD4), we did not detect any co-segregated mutations in this pedigree. Whole-exome sequencing analysis of 7 members and Sanger sequencing analysis of 16 additional members identified a mutation (c.784A > G) in the NSF attachment protein gamma (NAPG) gene that co-segregated with the disease. Functional prediction showed that the mutation was deleterious and might change the conformational stability of the NAPG protein. Conclusions NAPG c.784A > G may potentially lead to HHT. These results expand the current understanding of the genetic contributions to HHT pathogenesis.

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PCNT point mutations and familial intracranial aneurysms

Neurology ◽

10.1212/wnl.0000000000006614 ◽

2018 ◽

Vol 91 (23) ◽

pp. e2170-e2181 ◽

Cited By ~ 6

Author(s):

Oswaldo Lorenzo-Betancor ◽

Patrick R. Blackburn ◽

Emily Edwards ◽

Rocío Vázquez-do-Campo ◽

Eric W. Klee ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Intracranial Aneurysms ◽

Point Mutations ◽

Missense Mutations ◽

Loss Of Function ◽

Interaction Domain ◽

Knockout Mouse Model ◽

Whole Exome ◽

Primordial Dwarfism

ObjectiveTo identify novel genes involved in the etiology of intracranial aneurysms (IAs) or subarachnoid hemorrhages (SAHs) using whole-exome sequencing.MethodsWe performed whole-exome sequencing in 13 individuals from 3 families with an autosomal dominant IA/SAH inheritance pattern to look for candidate genes for disease. In addition, we sequenced PCNT exon 38 in a further 161 idiopathic patients with IA/SAH to find additional carriers of potential pathogenic variants.ResultsWe identified 2 different variants in exon 38 from the PCNT gene shared between affected members from 2 different families with either IA or SAH (p.R2728C and p.V2811L). One hundred sixty-four samples with either SAH or IA were Sanger sequenced for the PCNT exon 38. Five additional missense mutations were identified. We also found a second p.V2811L carrier in a family with a history of neurovascular diseases.ConclusionThe PCNT gene encodes a protein that is involved in the process of microtubule nucleation and organization in interphase and mitosis. Biallelic loss-of-function mutations in PCNT cause a form of primordial dwarfism (microcephalic osteodysplastic primordial dwarfism type II), and ≈50% of these patients will develop neurovascular abnormalities, including IAs and SAHs. In addition, a complete Pcnt knockout mouse model (Pcnt−/−) published previously showed general vascular abnormalities, including intracranial hemorrhage. The variants in our families lie in the highly conserved PCNT protein-protein interaction domain, making PCNT a highly plausible candidate gene in cerebrovascular disease.

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Identification of Variants in Primary and Recurrent Glioblastoma Using a Cancer-Specific Gene Panel and Whole Exome Sequencing

PLoS ONE ◽

10.1371/journal.pone.0124178 ◽

2015 ◽

Vol 10 (5) ◽

pp. e0124178 ◽

Cited By ~ 12

Author(s):

Selene M. Virk ◽

Richard M. Gibson ◽

Miguel E. Quinones-Mateu ◽

Jill S. Barnholtz-Sloan

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Recurrent Glioblastoma ◽

Gene Panel ◽

Specific Gene ◽

Whole Exome

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Identification of a Novel EXT Mutation in A Kindred With Hereditary Multiple Exostoses Using Whole-Exome Sequencing and Overview of Mutation Spectrum

10.21203/rs.3.rs-770260/v1 ◽

2021 ◽

Author(s):

yanhan deng ◽

yujian liu ◽

wei tu ◽

liu yang

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Mutation Spectrum ◽

Loss Of Function ◽

Site Mutation ◽

Hereditary Multiple Exostoses ◽

Multiple Osteochondromas ◽

Online Databases ◽

Whole Exome ◽

Multiple Exostoses

Abstract Background: Hereditary Multiple Osteochondromas(HMO) is a rare genetic musculoskeletal disorder characterized by multiple osteochondromas that form near to the growth plates of many bones. Loss-of-function mutations in EXT1 or EXT2 that encode glycosyltrasferases are the causal mutations for most HMO patients.Methods: After collecting the family history and clinical information, we used Whole-Exome Sequencing to find the pathogenic mutations in one Chinese Hereditary Multiple Exostoses pedigree. Sanger sequencing and relevant online databases were used to validate the screened variants. Lollipop plots were drew to map the reported mutations from online databases (Multiple Osteochondroma Mutation Database and clinvar)on a linear protein domains by MutationMapper.Results: A novel heterozygous splicing-site mutation in gene EXT1 (NM_000127:exon5:c.1417+1G>C，chr8:118834703) was found in this pedigree and mutation spectrum of genes EXT1 and EXT2 were demonstrated.Conclusions: Our results help this pedigree to identify the pathogenic variant and guide the prenatal diagnosis, also expand the mutation spectrum in Hereditary Multiple Osteochondromas.

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Whole exome sequencing identifies novel DYT1 dystonia-associated genome variants as potential disease modifiers

10.1101/2020.03.15.993113 ◽

2020 ◽

Author(s):

Chih-Fen Hu ◽

G. W. Gant Luxton ◽

Feng-Chin Lee ◽

Chih-Sin Hsu ◽

Shih-Ming Huang ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Genetic Modifiers ◽

Loss Of Function ◽

Dyt1 Dystonia ◽

Whole Exome ◽

Important Regulator ◽

Blood Leukocyte ◽

Aaa Protein ◽

Neurological Movement Disorder

AbstractBackgroundDYT1 dystonia is a neurological movement disorder characterized by painful sustained muscle contractions resulting in abnormal twisting and postures. In a subset of patients, it is caused by a loss-of-function mutation (ΔE302/303; or ΔE) in the luminal ATPases associated with various cellular activities (AAA+) protein torsinA encoded by the TOR1A gene. The low penetrance of the ΔE mutation (∼30-40%) suggests the existence of unknown genetic modifiers of DYT1 dystonia.MethodsTo identify these modifiers, we performed whole exome sequencing of blood leukocyte DNA isolated from two DYT1 dystonia patients, three asymptomatic carriers of the ΔE mutation, and an unaffected adult relative.ResultsA total of 264 DYT1 dystonia-associated variants (DYT1 variants) were identified in 195 genes. Consistent with the emerging view of torsinA as an important regulator of the cytoskeleton, endoplasmic reticulum homeostasis, and lipid metabolism, we found DYT1 variants in genes that encode proteins implicated in these processes. Moreover, 40 DYT1 variants were detected in 32 genes associated with neuromuscular and neuropsychiatric disorders.ConclusionThe DYT1 variants described in this work represent exciting new targets for future studies designed to increase our understanding of the pathophysiology and pathogenesis of DYT1 dystonia.

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Whole exome sequencing identified a homozygous novel mutation in SUOX gene causes extremely rare autosomal recessive isolated sulfite oxidase deficiency and literature review

10.21203/rs.3.rs-74199/v3 ◽

2021 ◽

Author(s):

Rui Zhang ◽

Yajing Hao ◽

Ying Xu ◽

Jiale Qin ◽

Yanfang Wang ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Autosomal Recessive ◽

Novel Mutation ◽

Sulfite Oxidase ◽

Loss Of Function ◽

Oxidase Deficiency ◽

Neurometabolic Disorders ◽

Whole Exome ◽

Sulfite Oxidase Deficiency

Abstract Background: Isolated sulfite oxidase deficiency (ISOD) is the rarest types of life-threatening neurometabolic disorders characterized by neonatal intractable seizures and severe developmental delay with an autosomal recessive mode of inheritance. ISOD is extremely rare and till date only 32 mutations have been identified and reported worldwide. Germline mutation in SUOX gene causes ISOD. Methods: Here, we investigated a 5-days old Chinese female child, presented with intermittent tremor or seizures of limbs, neonatal encephalopathy, subarachnoid cyst and haemorrhage, dysplasia of corpus callosum, neonatal convulsion, respiratory failure, cardiac failure, hyperlactatemia, severe metabolic acidosis, hyperglycemia, hyperkalemia, moderate anemia, atrioventricular block and complete right bundle branch block. Results: Whole exome sequencing identified a novel homozygous transition (c.1227G>A) in exon 6 of the SUOX gene in the proband. This novel homozygous variant leads to the formation of a truncated sulfite oxidase (p.Trp409*) of 408 amino acids. Hence, it is a loss-of-function variant. Proband’s father and mother is carrying this novel variant in a heterozygous state. This variant was not identified in 200 ethnically matched normal healthy control individuals. Conclusions: Our study not only expand the mutational spectrum of SUOX gene associated ISOD, but also strongly suggested the application of whole exome sequencing for identifying candidate genes and novel disease-causing mutations.

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CARMIL2 Deficiency Presenting as Very Early Onset Inflammatory Bowel Disease

Inflammatory Bowel Diseases ◽

10.1093/ibd/izz103 ◽

2019 ◽

Vol 25 (11) ◽

pp. 1788-1795 ◽

Cited By ~ 8

Author(s):

Thomas Magg ◽

Anna Shcherbina ◽

Duran Arslan ◽

Mukesh M Desai ◽

Sarah Wall ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Early Onset ◽

Mononuclear Cells ◽

Severe Disease ◽

Effector Memory ◽

Loss Of Function ◽

Peripheral Blood Mononuclear ◽

Whole Exome ◽

Inflammatory Bowel

Abstract Background Children with very early onset inflammatory bowel diseases (VEO-IBD) often have a refractory and severe disease course. A significant number of described VEO-IBD-causing monogenic disorders can be attributed to defects in immune-related genes. The diagnosis of the underlying primary immunodeficiency (PID) often has critical implications for the treatment of patients with IBD-like phenotypes. Methods To identify the molecular etiology in 5 patients from 3 unrelated kindred with IBD-like symptoms, we conducted whole exome sequencing. Immune workup confirmed an underlying PID. Results Whole exome sequencing revealed 3 novel CARMIL2 loss-of-function mutations in our patients. Immunophenotyping of peripheral blood mononuclear cells showed reduction of regulatory and effector memory T cells and impaired B cell class switching. The T cell proliferation and activation assays confirmed defective responses to CD28 costimulation, consistent with CARMIL2 deficiency. Conclusion Our study highlights that human CARMIL2 deficiency can manifest with IBD-like symptoms. This example illustrates that early diagnosis of underlying PID is crucial for the treatment and prognosis of children with VEO-IBD.

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P1-170: WHOLE-EXOME SEQUENCING IN 20,197 INDIVIDUALS IDENTIFIES ULTRA-RARE SORL1 LOSS-OF-FUNCTION VARIANTS IN LATE-ONSET ALZHEIMER'S DISEASE

Alzheimer s & Dementia ◽

10.1016/j.jalz.2018.06.174 ◽

2006 ◽

Vol 14 (7S_Part_6) ◽

pp. P344-P344

Author(s):

Neha S. Raghavan ◽

Adam M. Brickman ◽

Howard Andrews ◽

Jennifer J. Manly ◽

Nicole Schupf ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Late Onset ◽

Loss Of Function ◽

Whole Exome

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Case report: targeted whole exome sequencing enables the first prenatal diagnosis of the lethal skeletal dysplasia Osteocraniostenosis

BMC Medical Genetics ◽

10.1186/s12881-019-0939-z ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Lara Pemberton ◽

Robert Barker ◽

Anna Cockell ◽

Vijaya Ramachandran ◽

Andrea Haworth ◽

...

Keyword(s):

Case Report ◽

Prenatal Diagnosis ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Skeletal Dysplasia ◽

Genetic Disorder ◽

Specific Gene ◽

Ultrasound Images ◽

Whole Exome ◽

Lethal Skeletal Dysplasia

Abstract Background Osteocraniostenosis (OCS) is a rare genetic disorder characterised by premature closure of cranial sutures, gracile bones and perinatal lethality. Previously, diagnosis has only been possible postnatally on clinical and radiological features. This study describes the first prenatal diagnosis of OCS. Case presentation In this case prenatal ultrasound images were suggestive of a serious but non-lethal skeletal dysplasia. Due to the uncertain prognosis the parents were offered Whole Exome Sequencing (WES), which identified a specific gene mutation in the FAMIIIa gene. This mutation had previously been detected in two cases and was lethal in both perinatally. This established the diagnosis, a clear prognosis and allowed informed parental choice regarding ongoing pregnancy management. Conclusions This case report supports the use of targeted WES prenatally to confirm the underlying cause and prognosis of sonographically suspected abnormalities.

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