Detection of low-level parental somatic mosaicism for clinically relevant SNVs and indels identified in a large exome sequencing dataset

Abstract Background Due to the limitations of the current routine diagnostic methods, low-level somatic mosaicism with variant allele fraction (VAF) < 10% is often undetected in clinical settings. To date, only a few studies have attempted to analyze tissue distribution of low-level parental mosaicism in a large clinical exome sequencing (ES) cohort. Methods Using a customized bioinformatics pipeline, we analyzed apparent de novo single-nucleotide variants or indels identified in the affected probands in ES trio data at Baylor Genetics clinical laboratories. Clinically relevant variants with VAFs between 30 and 70% in probands and lower than 10% in one parent were studied. DNA samples extracted from saliva, buccal cells, redrawn peripheral blood, urine, hair follicles, and nail, representing all three germ layers, were tested using PCR amplicon next-generation sequencing (amplicon NGS) and droplet digital PCR (ddPCR). Results In a cohort of 592 clinical ES trios, we found 61 trios, each with one parent suspected of low-level mosaicism. In 21 parents, the variants were validated using amplicon NGS and seven of them by ddPCR in peripheral blood DNA samples. The parental VAFs in blood samples varied between 0.08 and 9%. The distribution of VAFs in additional tissues ranged from 0.03% in hair follicles to 9% in re-drawn peripheral blood. Conclusions Our study illustrates the importance of analyzing ES data using sensitive computational and molecular methods for low-level parental somatic mosaicism for clinically relevant variants previously diagnosed in routine clinical diagnostics as apparent de novo.

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Detection of Low-Level Parental Somatic Mosaicism for Clinically Relevant Snvs and Indels Identified in a Large Exome Sequencing Dataset

10.21203/rs.3.rs-1020672/v1 ◽

2021 ◽

Author(s):

Daniel David Domogala ◽

Tomasz Gambin ◽

Roni Zemet ◽

Chung Wah Wu ◽

Katharina Schulze ◽

...

Keyword(s):

Exome Sequencing ◽

Peripheral Blood ◽

De Novo ◽

Somatic Mosaicism ◽

Hair Follicles ◽

Clinical Diagnostics ◽

Diagnostic Methods ◽

Single Nucleotide Variants ◽

Bioinformatics Pipeline ◽

Low Level

Abstract Background Due to the limitations of the current routine diagnostic methods, low-level somatic mosaicism with variant allele fraction (VAF) <10% is often undetected in clinical settings. To date, only a few studies have attempted to analyze tissue distribution of low-level parental mosaicism in a large clinical exome sequencing (ES) cohort. Methods Using a customized bioinformatics pipeline, we have analyzed apparent de novo single nucleotide variants or indels identified in the affected probands in ES trio data at Baylor Genetics clinical laboratories. Clinically relevant variants with VAFs between 30-70% in probands and lower than 10% in one parent were studied. DNA samples extracted from saliva, buccal cells, redrawn peripheral blood, urine, hair follicles, and nail, representing all three germ layers, were tested using PCR amplicon next generation sequencing (amplicon NGS) and droplet digital PCR (ddPCR). Results In a cohort of 592 clinical ES trios, we have found 61 trios, each with one parent suspected of low-level mosaicism. In 21 parents, the variants were validated using amplicon NGS and seven of them by ddPCR in peripheral blood DNA samples. The parental VAFs in blood samples varied between 0.08–9%. The distribution of VAFs in additional tissues ranged from 0.03% in hair follicles to 9% in re-drawn peripheral blood. Conclusions Our study illustrates the importance of analyzing ES data using sensitive computational and molecular methods for low-level parental somatic mosaicism for clinically relevant variants previously diagnosed in routine clinical diagnostics as apparent de novo.

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Case Report: Low-Level Maternal Mosaicism of a Novel CREBBP Variant Causes Recurrent Rubinstein-Taybi Syndrome in Two Siblings of a Chinese Family

Frontiers in Genetics ◽

10.3389/fgene.2021.640992 ◽

2021 ◽

Vol 12 ◽

Author(s):

Shaobin Lin ◽

Zhiming He ◽

Linhuan Huang ◽

Jialiu Liu ◽

Ting Lei ◽

...

Keyword(s):

Peripheral Blood ◽

De Novo ◽

Recurrence Risk ◽

Chinese Family ◽

Sequencing Analysis ◽

Mrna Levels ◽

Germline Mosaicism ◽

Low Level ◽

Normal Individuals ◽

High Depth

Familial Rubinstein-Taybi syndrome (RSTS) with recurrent RSTS siblings and apparently unaffected parents is rare; such cases might result from parental somatic and/or germline mosaicism. Parental low-level (<10%) germline mosaicism in the CREBBP-associated RSTS family has not been reported. Here, we present our studies of a Chinese family with two RSTS siblings and apparently unaffected parents. We detected the apparent de novo variant (DNV) c.3235C>T (p.Gln1079*) in CREBBP in the siblings via trio whole-exome sequencing. High-depth next-generation sequencing (NGS) for the parents revealed a low-level (<10%) mosaic variant in both the peripheral blood (3.64%) and buccal mucosa (1.94%) of the unaffected mother, indicating maternal somatic and germline mosaicism. Peripheral blood RNA-sequencing analysis for the patients and normal individuals indicated that the c.3235C>T (p.Gln1079*) non-sense variant did not trigger nonsense-mediated mRNA decay to reduce CREBBP mRNA levels. Transcriptome analysis revealed 151 downregulated mRNAs and 132 upregulated mRNAs between the patients and normal individuals. This study emphasizes that high-depth NGS using multiple specimens might be applied for a family with an affected sibling caused by an apparent CREBBP DNV to identify potential low-level parental mosaicism and provide an assessment of recurrence risk.

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Clinical and whole-exome sequencing findings in Individuals from Yunnan Province with Familial Exudative Vitreoretinopathy

10.21203/rs.2.20941/v1 ◽

2020 ◽

Author(s):

Zhen Zhang ◽

Yi-shuang Xiao ◽

Hong-chao Jiang ◽

Ru Sheng ◽

Li Tang ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Yunnan Province ◽

Illumina Miseq ◽

Wide Field ◽

Clinical Practices ◽

Single Nucleotide Variants ◽

Bioinformatics Pipeline ◽

Familial Exudative Vitreoretinopathy ◽

Whole Exome

Abstract Background : Familial exudative vitreoretinopathy (FEVR) is a rare inherited retinal disorder characterized by the failure of peripheral retinal vascularization at birth. FEVR can cause further pathological changes, such as neovascularization, exudation, haemorrhage, and retinal detachment, in turn. The molecular diagnosis enables a deeper understanding of this disease, so the differentiation of FEVR is important for the accuracy of genetic counselling. However, to date, only six genes have been identified as being responsible for this disease. Methods : Without a known FEVR gene mutation, six families were enrolled in our study between 2016 and 2017 from the clinical practices of ophthalmologists. The referring physician made a diagnosis for FEVR probands, and clinical data and DNA were collected from each participant. Whole-exome sequencing was used to detect the mutations present in the probands. The raw reads were obtained from Illumina Miseq. Then, an in-house bioinformatics pipeline was performed to detect both single nucleotide variants (SNVs) and small insertions/deletions (InDels). The pathogenic mutations were identified with stringent criteria and were further confirmed by conventional methods and cosegregation in families. Results : Using this approach, bioinformatic predictions showed that six mutations were found in our study: three mutations in the known genes of ZNF408, LRP5 and KIF11 and three mutations in the newly identified genes NR2E3,KRT3 and FOXL2 .To test the hypothesis that cases of FEVR are caused by NR2E3, KRT3 and FOXL2 , probands who were diagnosed as FEVR by a physician using wide-field fluorescein angiography were found to not have any mutations in any of the six known FEVR genes. Compared with previous reports, mutations in NR2E3, KRT3 and FOXL2 are believed to cause a broader spectrum of ocular disease. The NR2E3,KRT3 and FOXL2 genes likely play a role in retinal vascular development. Conclusions : This report is the first to describe FEVR mutations in Yunnan province children with FEVR. This study would provide information on the genetic forms of the disease and direct counselling by analysing the genetic testing and genotype-phenotype interaction.

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Mapping Autosomal Recessive Intellectual Disability: Combined Microarray and Exome Sequencing Identifies 26 Novel Candidate Genes in 192 Consanguineous Families

10.1101/092346 ◽

2016 ◽

Author(s):

Ricardo Harripaul ◽

Nasim Vasli ◽

Anna Mikhailov ◽

Muhammad Arshad Rafiq ◽

Kirti Mittal ◽

...

Keyword(s):

Intellectual Disability ◽

Exome Sequencing ◽

Autosomal Recessive ◽

De Novo ◽

Clinical Diagnostics ◽

High Yield ◽

Disease Genes ◽

Missense Mutations ◽

Loss Of Function ◽

Whole Exome

Approximately 1% of the global population is affected by intellectual disability (ID), and the majority receive no molecular diagnosis. Previous studies have indicated high levels of genetic heterogeneity, with estimates of more than 2500 autosomal ID genes, the majority of which are autosomal recessive (AR). Here, we combined microarray genotyping, homozygosity-by-descent (HBD) mapping, copy number variation (CNV) analysis, and whole exome sequencing (WES) to identify disease genes/mutations in 192 multiplex Pakistani and Iranian consanguineous families with non-syndromic ID. We identified definite or candidate mutations (or CNVs) in 51% of families in 72 different genes, including 26 not previously reported for ARID. The new ARID genes include nine with loss-of-function mutations(ABI2, MAPK8, MPDZ, PIDD1, SLAIN1, TBC1D23, TRAPPC6B, UBA7,andUSP44),and missense mutations include the first reports of variants inBDNForTET1associated with ID. The genes identified also showed overlap withde novogene sets for other neuropsychiatric disorders. Transcriptional studies showed prominent expression in the prenatal brain. The high yield of AR mutations for ID indicated that this approach has excellent clinical potential and should inform clinical diagnostics, including clinical whole exome and genome sequencing, for populations in which consanguinity is common. As with other AR disorders, the relevance will also apply to outbred populations.

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Genetic landscape of Rett syndrome-like phenotypes revealed by whole exome sequencing

Journal of Medical Genetics ◽

10.1136/jmedgenet-2018-105775 ◽

2019 ◽

Vol 56 (6) ◽

pp. 396-407 ◽

Cited By ~ 10

Author(s):

Kazuhiro Iwama ◽

Takeshi Mizuguchi ◽

Eri Takeshita ◽

Eiji Nakagawa ◽

Tetsuya Okazaki ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Rett Syndrome ◽

De Novo ◽

Diagnostic Yield ◽

Single Nucleotide Variants ◽

Serine Threonine Kinase ◽

Whole Exome ◽

Genetic Landscape ◽

Strong Candidate

BackgroundRett syndrome (RTT) is a characteristic neurological disease presenting with regressive loss of neurodevelopmental milestones. Typical RTT is generally caused by abnormality of methyl-CpG binding protein 2 (MECP2). Our objective to investigate the genetic landscape of MECP2-negative typical/atypical RTT and RTT-like phenotypes using whole exome sequencing (WES).MethodsWe performed WES on 77 MECP2-negative patients either with typical RTT (n=11), atypical RTT (n=22) or RTT-like phenotypes (n=44) incompatible with the RTT criteria.ResultsPathogenic or likely pathogenic single-nucleotide variants in 28 known genes were found in 39 of 77 (50.6%) patients. WES-based CNV analysis revealed pathogenic deletions involving six known genes (including MECP2) in 8 of 77 (10.4%) patients. Overall, diagnostic yield was 47 of 77 (61.0 %). Furthermore, strong candidate variants were found in four novel genes: a de novo variant in each of ATPase H+ transporting V0 subunit A1 (ATP6V0A1), ubiquitin-specific peptidase 8 (USP8) and microtubule-associated serine/threonine kinase 3 (MAST3), as well as biallelic variants in nuclear receptor corepressor 2 (NCOR2).ConclusionsOur study provides a new landscape including additional genetic variants contributing to RTT-like phenotypes, highlighting the importance of comprehensive genetic analysis.

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Brain Tissue Low-Level Mosaicism for MTOR Mutation Causes Smith–Kingsmore Phenotype with Recurrent Hypoglycemia—A Novel Phenotype and a Further Proof for Testing of an Affected Tissue

Diagnostics ◽

10.3390/diagnostics11071269 ◽

2021 ◽

Vol 11 (7) ◽

pp. 1269

Author(s):

Krzysztof Szczałuba ◽

Małgorzata Rydzanicz ◽

Anna Walczak ◽

Joanna Kosińska ◽

Agnieszka Koppolu ◽

...

Keyword(s):

De Novo ◽

Focal Cortical Dysplasia ◽

Somatic Mosaicism ◽

Intractable Epilepsy ◽

Low Level ◽

Brain Anomalies ◽

Genes Encoding ◽

Affected Tissue ◽

The Brain ◽

Recurrent Hypoglycemia

De novo somatic variants in genes encoding components of the PI3K–AKT3–mTOR pathway, including MTOR, have been linked to hemimegalencephaly or focal cortical dysplasia. Similarly to other malformations of cortical development, this condition presents with developmental delay and intractable epilepsy, often necessitating surgical treatment. We describe a first patient with the Smith–Kingsmore syndrome phenotype with recurrent hypoglycemia caused by low-level mosaic MTOR mutation restricted to the brain. We provide discussion on different aspects of somatic mosaicism. Deep exome sequencing combined with a variant search in multiple tissues and careful phenotyping may constitute a key to the diagnosis of the causes of rare brain anomalies.

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Case Report: Identification of Maternal Low-Level Mosaicism in the Dystrophin Gene by Droplet Digital Polymerase Chain Reaction

Frontiers in Genetics ◽

10.3389/fgene.2021.686993 ◽

2021 ◽

Vol 12 ◽

Author(s):

Pengzhen Jin ◽

Xiaoyang Gao ◽

Miaomiao Wang ◽

Yeqing Qian ◽

Jingjin Yang ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Peripheral Blood ◽

De Novo ◽

Dystrophin Gene ◽

Chain Reaction ◽

Germline Mosaicism ◽

Blood Samples ◽

Low Level ◽

Polymerase Chain ◽

Digital Polymerase Chain Reaction

Germline mosaicism should be suspected when the same de novo mutations are identified in a second pregnancy with asymptomatic parents. Our study aims to find a feasible approach to reveal the existence of germline mosaicism. Multiplex Ligation-dependent Probe Amplification was performed on a Duchenne muscular dystrophy affected pedigree to detect deletion mutations. Then gap-polymerase chain reaction was performed to amplify the breakpoints junction sequence. Droplet digital polymerase chain reaction was utilized to identify the mutation frequencies in healthy parents. The same deletion in the exon 51 of the dystrophin gene, which was 50,035 bp in size, was detected in the proband and the fetus but not in their parents. Droplet digital polymerase chain reaction analysis of peripheral blood samples revealed mutant alleles of 3.53% in maternal blood cells. We here report a case of maternal low-level mosaicism confirmed by droplet digital polymerase chain reaction in peripheral blood samples, which reveals the existence of germline mosaicism. Gap-polymerase chain reaction combined with droplet digital polymerase chain reaction provide insights into the detection of germline mosaicism.

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Whole-Exome Sequencing Identifies One De Novo Variant in the FGD6 Gene in a Thai Family with Autism Spectrum Disorder

International Journal of Genomics ◽

10.1155/2018/8231547 ◽

2018 ◽

Vol 2018 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Chuphong Thongnak ◽

Areerat Hnoonual ◽

Duangkamol Tangviriyapaiboon ◽

Suchaya Silvilairat ◽

Apichaya Puangpetch ◽

...

Keyword(s):

Autism Spectrum Disorder ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

De Novo ◽

Autism Spectrum ◽

Spectrum Disorder ◽

Bioinformatics Pipeline ◽

Whole Exome ◽

De Novo Variant ◽

Genetic Analyzer

Autism spectrum disorder (ASD) has a strong genetic basis, although the genetics of autism is complex and it is unclear. Genetic testing such as microarray or sequencing was widely used to identify autism markers, but they are unsuccessful in several cases. The objective of this study is to identify causative variants of autism in two Thai families by using whole-exome sequencing technique. Whole-exome sequencing was performed with autism-affected children from two unrelated families. Each sample was sequenced on SOLiD 5500xl Genetic Analyzer system followed by combined bioinformatics pipeline including annotation and filtering process to identify candidate variants. Candidate variants were validated, and the segregation study with other family members was performed using Sanger sequencing. This study identified a possible causative variant for ASD, c.2951G>A, in the FGD6 gene. We demonstrated the potential for ASD genetic variants associated with ASD using whole-exome sequencing and a bioinformatics filtering procedure. These techniques could be useful in identifying possible causative ASD variants, especially in cases in which variants cannot be identified by other techniques.

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Identification of de novo mutations for ARID1B haploinsufficiency associated with Coffin–Siris syndrome 1 in three Chinese families via array-CGH and whole exome sequencing

BMC Medical Genomics ◽

10.1186/s12920-021-01119-2 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Guanting Lu ◽

Qiongling Peng ◽

Lianying Wu ◽

Jian Zhang ◽

Liya Ma

Keyword(s):

Intellectual Disability ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Array Cgh ◽

De Novo ◽

Current Knowledge ◽

Comparative Genomic ◽

De Novo Mutations ◽

Single Nucleotide Variants ◽

Whole Exome

Abstract Background Coffin–Siris syndrome (CSS) is a multiple malformation syndrome characterized by intellectual disability associated with coarse facial features, hirsutism, sparse scalp hair, and hypoplastic or absent fifth fingernails or toenails. CSS represents a small group of intellectual disability, and could be caused by at least twelve genes. The genetic background is quite heterogenous, making it difficult for clinicians and genetic consultors to pinpoint the exact disease types. Methods Array-Comparative Genomic Hybridization (array-CGH) and whole exome sequencing (WES) were applied for three trios affected with intellectual disability and clinical features similar with those of Coffin–Siris syndrome. Sanger sequencing was used to verify the detected single-nucleotide variants (SNVs). Results All of the three cases were female with normal karyotypes of 46, XX, born of healthy, non-consanguineous parents. A 6q25 microdeletion (arr[hg19]6q25.3(155,966,487–158,803,979) × 1) (2.84 Mb) (case 1) and two loss-of-function (LoF) mutations of ARID1B [c.2332 + 1G > A in case 2 and c.4741C > T (p.Q1581X) in case 3] were identified. All of the three pathogenic abnormalities were de novo, not inherited from their parents. After comparison of publicly available microdeletions containing ARID1B, four types of microdeletions leading to insufficient production of ARID1B were identified, namely deletions covering the whole region of ARID1B, deletions covering the promoter region, deletions covering the termination region or deletions covering enhancer regions. Conclusion Here we identified de novo ARID1B mutations in three Chinese trios. Four types of microdeletions covering ARID1B were identified. This study broadens current knowledge of ARID1B mutations for clinicians and genetic consultors.

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Application of Trio-Whole Exome Sequencing in Genetic Diagnosis and Therapy in Chinese Children With Epilepsy

Frontiers in Molecular Neuroscience ◽

10.3389/fnmol.2021.699574 ◽

2021 ◽

Vol 14 ◽

Author(s):

Tiejia Jiang ◽

Jia Gao ◽

Lihua Jiang ◽

Lu Xu ◽

Congying Zhao ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Copy Number ◽

Pediatric Patients ◽

De Novo ◽

Treatment Options ◽

Copy Number Variants ◽

Genetic Diagnosis ◽

Single Nucleotide Variants ◽

Whole Exome

Epilepsy is one of the most common neurological disorders in pediatric patients with other underlying neurological defects. Identifying the underlying etiology is crucial for better management of the disorder. We performed trio-whole exome sequencing in 221 pediatric patients with epilepsy. Probands were divided into seizures with developmental delay/intellectual disability (DD/ID) and seizures without DD/ID groups. Pathogenic (P) or likely pathogenic (LP) variants were identified in 71/110 (64.5%) patients in the seizures with DD/ID group and 21/111 (18.9%) patients in the seizures without DD/ID group (P < 0.001). Eighty-seven distinct P/LP single nucleotide variants (SNVs)/insertion deletions (Indels) were detected, with 55.2% (48/87) of them being novel. All aneuploidy and P/LP copy number variants (CNVs) larger than 100 Kb were identifiable by both whole-exome sequencing and copy number variation sequencing (CNVseq) in 123 of individuals (41 pedigrees). Ten of P/LP CNVs in nine patients and one aneuploidy variant in one patient (Patient #56, #47, XXY) were identified by CNVseq. Herein, we identified seven genes (NCL, SEPHS2, PA2G4, SLC35G2, MYO1C, GPR158, and POU3F1) with de novo variants but unknown pathogenicity that were not previously associated with epilepsy. Potential effective treatment options were available for 32 patients with a P/LP variant, based on the molecular diagnosis. Genetic testing may help identify the molecular etiology of early onset epilepsy and DD/ID and further aid to choose the appropriate treatment strategy for patients.

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