Case Report: Functional Investigation of an Undescribed Missense Variant Affecting Splicing in a Patient With Dravet Syndrome

Pathogenic variants in the SCN1A gene are associated with a spectrum of epileptic disorders ranging in severity from familial febrile seizures to Dravet syndrome. Large proportions of reported pathogenic variants in SCN1A are annotated as missense variants and are often classified as variants of uncertain significance when no functional data are available. Although loss-of-function variants are associated with a more severe phenotype in SCN1A, the molecular mechanism of single nucleotide variants is often not clear, and genotype-phenotype correlations in SCN1A-related epilepsy remain uncertain. Coding variants can affect splicing by creating novel cryptic splicing sites in exons or by disrupting exonic cis-regulation elements crucial for proper pre-mRNA splicing. Here, we report a novel case of Dravet syndrome caused by an undescribed missense variant, c.4852G>A (p.(Gly1618Ser)). By midigene splicing assay, we demonstrated that the identified variant is in fact splice-affecting. To our knowledge, this is the first report on the functional investigation of a missense variant affecting splicing in Dravet syndrome.

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Dravet syndrome and Dravet-like phenotype: a systematic review of the SCN1A and PCDH19 variants

10.5327/1516-3180.229 ◽

2021 ◽

Keyword(s):

Cognitive Deficit ◽

Significant Proportion ◽

Dravet Syndrome ◽

Motor Deficit ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Pathogenic Variants ◽

Voltage Dependent ◽

Uncertain Significance ◽

Similar Disease

Background: Dravet syndrome (DS) is a rare and severe epileptic syndrome of childhood with a prevalence around 1/40,000 people worldwide. Approximately 80% of patients with DS present SCN1A pathogenic variants, which encodes an alpha subunit of a neural voltage- dependent sodium channel. SCN1A variants were also related to DS. There is a correlation between PCDH19 pathogenic variants, encodes the protocadherin 19, and a similar disease to DS known as DS-like phenotype. Objectives: To clarify the differences between DS and DS-like phenotype according to the SCN1A and PCDH19 variants. Methodology: A review from March/2019 to November/2020 was conducted in PubMed and VHL databases, following PRISMA criteria. Results: 19 studies were included and a significant proportion of patients with DS carrying SCN1A was greater than patients with DS-like phenotype harboring PCDH19 variants (76.6% vs. 23.4%). Considering SCN1A and PCDH19, 47 variants were pathogenic and 12 of uncertain significance; 25% were deletions and 75% were single- nucleotide variants. Autism was predominantly observed in patients with DS-like carrying PCDH19 variants compared to SCN1A variants carriers (62.5% vs. 37.5%, p=0.044). In addition, it was noticed a significant predisposition to hyperthermia during seizures in patients with variants in the PCDH19 (p=0.003). There was no significance differences between both groups and cognitive deficit, ataxia, behavior problems, and motor deficit. Conclusions: The study is the first to point out differences between the DS and DS-like phenotype according to the SCN1A and PCDH19 variants.

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Unsuspected somatic mosaicism for FBN1 gene contributes to Marfan syndrome

Genetics in Medicine ◽

10.1038/s41436-020-01078-6 ◽

2021 ◽

Author(s):

Pauline Arnaud ◽

Hélène Morel ◽

Olivier Milleron ◽

Laurent Gouya ◽

Christine Francannet ◽

...

Keyword(s):

Marfan Syndrome ◽

Somatic Mosaicism ◽

Variant Calling ◽

Copy Number Variations ◽

Pathogenic Variant ◽

Single Nucleotide Variants ◽

Bioinformatics Analyses ◽

Single Nucleotide ◽

Fbn1 Gene ◽

Pathogenic Variants

Abstract Purpose Individuals with mosaic pathogenic variants in the FBN1 gene are mainly described in the course of familial screening. In the literature, almost all these mosaic individuals are asymptomatic. In this study, we report the experience of our team on more than 5,000 Marfan syndrome (MFS) probands. Methods Next-generation sequencing (NGS) capture technology allowed us to identify five cases of MFS probands who harbored a mosaic pathogenic variant in the FBN1 gene. Results These five sporadic mosaic probands displayed classical features usually seen in Marfan syndrome. Combined with the results of the literature, these rare findings concerned both single-nucleotide variants and copy-number variations. Conclusion This underestimated finding should not be overlooked in the molecular diagnosis of MFS patients and warrants an adaptation of the parameters used in bioinformatics analyses. The five present cases of symptomatic MFS probands harboring a mosaic FBN1 pathogenic variant reinforce the fact that apparently asymptomatic mosaic parents should have a complete clinical examination and a regular cardiovascular follow-up. We advise that individuals with a typical MFS for whom no single-nucleotide pathogenic variant or exon deletion/duplication was identified should be tested by NGS capture panel with an adapted variant calling analysis.

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Rare Germline Variants in Chordoma-Related Genes and Chordoma Susceptibility

Cancers ◽

10.3390/cancers13112704 ◽

2021 ◽

Vol 13 (11) ◽

pp. 2704

Author(s):

Sally Yepes ◽

Nirav N. Shah ◽

Jiwei Bai ◽

Hela Koka ◽

Chuzhong Li ◽

...

Keyword(s):

Internal Control ◽

Susceptibility Gene ◽

Copy Number Variants ◽

Bone Cancer ◽

Chinese Patients ◽

European Ancestry ◽

Unknown Etiology ◽

Loss Of Function ◽

Single Nucleotide Variants ◽

Pathogenic Variants

Background: Chordoma is a rare bone cancer with an unknown etiology. TBXT is the only chordoma susceptibility gene identified to date; germline single nucleotide variants and copy number variants in TBXT have been associated with chordoma susceptibility in familial and sporadic chordoma. However, the genetic susceptibility of chordoma remains largely unknown. In this study, we investigated rare germline genetic variants in genes involved in TBXT/chordoma-related signaling pathways and other biological processes in chordoma patients from North America and China. Methods: We identified variants that were very rare in general population and internal control datasets and showed evidence for pathogenicity in 265 genes in a whole exome sequencing (WES) dataset of 138 chordoma patients of European ancestry and in a whole genome sequencing (WGS) dataset of 80 Chinese patients with skull base chordoma. Results: Rare and likely pathogenic variants were identified in 32 of 138 European ancestry patients (23%), including genes that are part of notochord development, PI3K/AKT/mTOR, Sonic Hedgehog, SWI/SNF complex and mesoderm development pathways. Rare pathogenic variants in COL2A1, EXT1, PDK1, LRP2, TBXT and TSC2, among others, were also observed in Chinese patients. Conclusion: We identified several rare loss-of-function and predicted deleterious missense variants in germline DNA from patients with chordoma, which may influence chordoma predisposition and reflect a complex susceptibility, warranting further investigation in large studies.

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Re-evaluation of single nucleotide variants and identification of structural variants in a cohort of 45 sudden unexplained death cases

International Journal of Legal Medicine ◽

10.1007/s00414-021-02580-5 ◽

2021 ◽

Author(s):

Jacqueline Neubauer ◽

Shouyu Wang ◽

Giancarlo Russo ◽

Cordula Haas

Keyword(s):

Sudden Death ◽

Cardiac Diseases ◽

Structural Variants ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Sudden Unexplained Death ◽

Unexplained Death ◽

Pathogenic Variants ◽

The Impact ◽

Death Cases

AbstractSudden unexplained death (SUD) takes up a considerable part in overall sudden death cases, especially in adolescents and young adults. During the past decade, many channelopathy- and cardiomyopathy-associated single nucleotide variants (SNVs) have been identified in SUD studies by means of postmortem molecular autopsy, yet the number of cases that remain inconclusive is still high. Recent studies had suggested that structural variants (SVs) might play an important role in SUD, but there is no consensus on the impact of SVs on inherited cardiac diseases. In this study, we searched for potentially pathogenic SVs in 244 genes associated with cardiac diseases. Whole-exome sequencing and appropriate data analysis were performed in 45 SUD cases. Re-analysis of the exome data according to the current ACMG guidelines identified 14 pathogenic or likely pathogenic variants in 10 (22.2%) out of the 45 SUD cases, whereof 2 (4.4%) individuals had variants with likely functional effects in the channelopathy-associated genes SCN5A and TRDN and 1 (2.2%) individual in the cardiomyopathy-associated gene DTNA. In addition, 18 structural variants (SVs) were identified in 15 out of the 45 individuals. Two SVs with likely functional impairment were found in the coding regions of PDSS2 and TRPM4 in 2 SUD cases (4.4%). Both were identified as heterozygous deletions, which were confirmed by multiplex ligation-dependent probe amplification. In conclusion, our findings support that SVs could contribute to the pathology of the sudden death event in some of the cases and therefore should be investigated on a routine basis in suspected SUD cases.

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Prioritizing variants in complete Hereditary Breast and Ovarian Cancer (HBOC) genes in patients lacking known BRCA mutations

10.1101/039206 ◽

2016 ◽

Author(s):

Natasha G Caminsky ◽

Eliseos J Mucaki ◽

Ami M. Perri ◽

Ruipeng Lu ◽

Joan H.M. Knoll ◽

...

Keyword(s):

Binding Sites ◽

Rna Binding ◽

Wide Spectrum ◽

Pedigree Information ◽

Brca Mutations ◽

Pathogenic Variants ◽

Uncertain Significance ◽

Common Framework ◽

Flanking Sequences ◽

Coding Variants

BRCA1andBRCA2testing for HBOC does not identify all pathogenic variants. Sequencing of 20 complete genes in HBOC patients with uninformative test results (N=287), including non-coding and flanking sequences ofATM,BARD1,BRCA1,BRCA2,CDH1,CHEK2,EPCAM,MLH1,MRE11A,MSH2,MSH6,MUTYH,NBN,PALB2,PMS2,PTEN,RAD51B,STK11,TP53, andXRCC2, identified 38,372 unique variants. We apply information theory (IT) to predict novel functions for and prioritize non-coding variants of uncertain significance (VUS) in throughout regulatory, coding, and intronic regions based on changes in binding sites in these genesof these genes. Besides mRNA splicing, IT provides a common framework to evaluate potential affinity changes inin transcription factor (TFBSs), splicing regulatory (SRBSs), and RNA-binding protein (RBBSs) protein binding sites following mutationat mutated binding sites. We prioritized variants affecting the strengths of 10 variants affecting splice sites (4 natural, 6 cryptic), 148 SRBS, 36 TFBS, and 31 RBBS binding strength-affecting variantss. Three variants were also prioritized based on their predicted effects on mRNA secondary (2°) structure, and 17 for pseudoexon activation. Additionally, 4 frameshift, 2 in-frame deletions, and 5 stop-gain mutations were identified. When combined with pedigree information, complete gene sequence analysis can focus attention on a limited set of variants in a wide spectrum of functional mutation types for downstream functional and co-segregation analysis.

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A large deletion in the COL2A1 gene expands the spectrum of pathogenic variants causing bulldog calf syndrome in cattle

Acta Veterinaria Scandinavica ◽

10.1186/s13028-020-00548-w ◽

2020 ◽

Vol 62 (1) ◽

Cited By ~ 1

Author(s):

Joana Gonçalves Pontes Jacinto ◽

Irene Monika Häfliger ◽

Anna Letko ◽

Cord Drögemüller ◽

Jørgen Steen Agerholm

Keyword(s):

De Novo ◽

Genetic Disorders ◽

Polymerase Chain Reaction Analysis ◽

Large Deletion ◽

Whole Genome Sequence ◽

De Novo Mutation ◽

Loss Of Function ◽

Single Nucleotide Variants ◽

Pathogenic Variants ◽

Base Pair Deletion

Abstract Background Congenital bovine chondrodysplasia, also known as bulldog calf syndrome, is characterized by disproportionate growth of bones resulting in a shortened and compressed body, mainly due to reduced length of the spine and the long bones of the limbs. In addition, severe facial dysmorphisms including palatoschisis and shortening of the viscerocranium are present. Abnormalities in the gene collagen type II alpha 1 chain (COL2A1) have been associated with some cases of the bulldog calf syndrome. Until now, six pathogenic single-nucleotide variants have been found in COL2A1. Here we present a novel variant in COL2A1 of a Holstein calf and provide an overview of the phenotypic and allelic heterogeneity of the COL2A1-related bulldog calf syndrome in cattle. Case presentation The calf was aborted at gestation day 264 and showed generalized disproportionate dwarfism, with a shortened compressed body and limbs, and dysplasia of the viscerocranium; a phenotype resembling bulldog calf syndrome due to an abnormality in COL2A1. Whole-genome sequence (WGS) data was obtained and revealed a heterozygous 3513 base pair deletion encompassing 10 of the 54 coding exons of COL2A1. Polymerase chain reaction analysis and Sanger sequencing confirmed the breakpoints of the deletion and its absence in the genomes of both parents. Conclusions The pathological and genetic findings were consistent with a case of “bulldog calf syndrome”. The identified variant causing the syndrome was the result of a de novo mutation event that either occurred post-zygotically in the developing embryo or was inherited because of low-level mosaicism in one of the parents. The identified loss-of-function variant is pathogenic due to COL2A1 haploinsufficiency and represents the first structural variant causing bulldog calf syndrome in cattle. Furthermore, this case report highlights the utility of WGS-based precise diagnostics for understanding congenital disorders in cattle and the need for continued surveillance for genetic disorders in cattle.

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Identification of a likely pathogenic structural variation in the LAMA1 gene by Bionano optical mapping

npj Genomic Medicine ◽

10.1038/s41525-020-0138-z ◽

2020 ◽

Vol 5 (1) ◽

Author(s):

Min Chen ◽

Min Zhang ◽

Yeqing Qian ◽

Yanmei Yang ◽

Yixi Sun ◽

...

Keyword(s):

Optical Mapping ◽

Pathogenic Variant ◽

Screening Methods ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Real Time Quantitative Pcr ◽

Pathogenic Variants ◽

Whole Exome ◽

Great Insight

Abstract Recent advances in Bionano optical mapping (BOM) provide a great insight into the determination of structural variants (SVs), but its utility in identification of clinical likely pathogenic variants needs to be further demonstrated and proved. In a family with two consecutive pregnancies affected with ventriculomegaly, a splicing likely pathogenic variant at the LAMA1 locus (NM_005559: c. 4663 + 1 G > C) inherited from the father was identified in the proband by whole-exome sequencing, and no other pathogenic variant associated with the clinical phenotypes was detected. SV analysis by BOM revealed an ~48 kb duplication at the LAMA1 locus in the maternal sample. Real-time quantitative PCR and Sanger sequencing further confirmed the duplication as c.859-153_4806 + 910dup. Based on these variants, we hypothesize that the fetuses have Poretti-Boltshauser syndrome (PBS) presenting with ventriculomegaly. With the ability to determine single nucleotide variants and SVs, the strategy adopted here might be useful to detect cases missed by current routine screening methods. In addition, our study may broaden the phenotypic spectrum of fetuses with PBS.

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Recent Advances in Understanding the Genetic Architecture of Autism

Annual Review of Genomics and Human Genetics ◽

10.1146/annurev-genom-121219-082309 ◽

2020 ◽

Vol 21 (1) ◽

pp. 289-304 ◽

Cited By ~ 1

Author(s):

Caroline M. Dias ◽

Christopher A. Walsh

Keyword(s):

Genetic Architecture ◽

De Novo ◽

Clinical Care ◽

Copy Number Variants ◽

Autism Spectrum ◽

Loss Of Function ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Recent Advances ◽

Insight Into

Recent advances in understanding the genetic architecture of autism spectrum disorder have allowed for unprecedented insight into its biological underpinnings. New studies have elucidated the contributions of a variety of forms of genetic variation to autism susceptibility. While the roles of de novo copy number variants and single-nucleotide variants—causing loss-of-function or missense changes—have been increasingly recognized and refined, mosaic single-nucleotide variants have been implicated more recently in some cases. Moreover, inherited variants (including common variants) and, more recently, rare recessive inherited variants have come into greater focus. Finally, noncoding variants—both inherited and de novo—have been implicated in the last few years. This work has revealed a convergence of diverse genetic drivers on common biological pathways and has highlighted the ongoing importance of increasing sample size and experimental innovation. Continuing to synthesize these genetic findings with functional and phenotypic evidence and translating these discoveries to clinical care remain considerable challenges for the field.

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Oligogenic inheritance of congenital heart disease involving a NKX2-5 modifier

10.1101/266726 ◽

2018 ◽

Cited By ~ 1

Author(s):

Casey A. Gifford ◽

Sanjeev S. Ranade ◽

Ryan Samarakoon ◽

Hazel T. Salunga ◽

T. Yvanka de Soysa ◽

...

Keyword(s):

Congenital Heart Defect ◽

Congenital Heart ◽

Nuclear Family ◽

Induced Pluripotent Stem Cell ◽

Missense Variant ◽

Left Ventricular ◽

Single Nucleotide Variants ◽

Experimental Proof ◽

Single Nucleotide ◽

Heart Defect

AbstractComplex genetic inheritance is thought to underlie many human diseases, yet experimental proof of this model has been elusive. Here, we show that a human congenital heart defect, left ventricular non-compaction (LVNC), can be caused by a combination of rare, inherited heterozygous missense single nucleotide variants. Whole exome sequencing of a nuclear family revealed novel single nucleotide variants of MYH7 and MKL2 in an asymptomatic father while the offspring with severe childhood-onset LVNC harbored an additional missense variant in the cardiac transcription factor, NKX2-5, inherited from an unaffected mother. Mice bred to compound heterozygosity for the orthologous missense variants in Myh7 and Mkl2 had mild cardiac pathology; the additional inheritance of the Nkx2-5 variant yielded a more severe LVNC-like phenotype in triple compound heterozygotes. RNA sequencing identified genes associated with endothelial and myocardial development that were dysregulated in hearts from triple heterozygote mice and human induced pluripotent stem cell–derived cardiomyocytes harboring the three variants, with evidence for NKX2-5’s contribution as a modifier on the molecular level. These studies demonstrate that the deployment of efficient gene editing tools can provide experimental evidence for complex inheritance of human disease.One sentence summaryA combination of three inherited heterozygous missense single nucleotide variants underlying a familial congenital heart defect.

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Targeted long-read sequencing resolves complex structural variants and identifies missing disease-causing variants

10.1101/2020.11.03.365395 ◽

2020 ◽

Author(s):

Danny E. Miller ◽

Arvis Sulovari ◽

Tianyun Wang ◽

Hailey Loucks ◽

Kendra Hoekzema ◽

...

Keyword(s):

Copy Number ◽

Genetic Diagnosis ◽

Clinical Testing ◽

Structural Variants ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Pathogenic Variants ◽

Long Read ◽

Repeat Expansions ◽

Complex Structural

ABSTRACTBACKGROUNDDespite widespread availability of clinical genetic testing, many individuals with suspected genetic conditions do not have a precise diagnosis. This limits their opportunity to take advantage of state-of-the-art treatments. In such instances, testing sometimes reveals difficult-to-evaluate complex structural differences, candidate variants that do not fully explain the phenotype, single pathogenic variants in recessive disorders, or no variants in specific genes of interest. Thus, there is a need for better tools to identify a precise genetic diagnosis in individuals when conventional testing approaches have been exhausted.METHODSTargeted long-read sequencing (T-LRS) was performed on 33 individuals using Read Until on the Oxford Nanopore platform. This method allowed us to computationally target up to 100 Mbp of sequence per experiment, resulting in an average of 20x coverage of target regions, a 500% increase over background. We analyzed patient DNA for pathogenic substitutions, structural variants, and methylation differences using a single data source.RESULTSThe effectiveness of T-LRS was validated by detecting all genomic aberrations, including single-nucleotide variants, copy number changes, repeat expansions, and methylation differences, previously identified by prior clinical testing. In 6/7 individuals who had complex structural rearrangements, T-LRS enabled more precise resolution of the mutation, which led, in one case, to a change in clinical management. In nine individuals with suspected Mendelian conditions who lacked a precise genetic diagnosis, T-LRS identified pathogenic or likely pathogenic variants in five and variants of uncertain significance in two others.CONCLUSIONST-LRS can accurately predict pathogenic copy number variants and triplet repeat expansions, resolve complex rearrangements, and identify single-nucleotide variants not detected by other technologies, including short-read sequencing. T-LRS represents an efficient and cost-effective strategy to evaluate high-priority candidate genes and regions or to further evaluate complex clinical testing results. The application of T-LRS will likely increase the diagnostic rate of rare disorders.

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