RNA Sequencing for Elucidating an Intronic Variant of Uncertain Significance (<i>SDHD</i> c.314+3A&gt;T) in Splicing Site Consensus Sequences

Gu, Hyunjung; Hong, Jinyoung; Lee, Woochang; Kim, Sung‐Bae; Chun, Sail; Min, Won‐Ki

doi:10.3343/alm.2022.42.3.376

Cited by 4 publications

(5 citation statements)

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“…Recent recommendations suggest utilizing SpliceAI as a splice prediction tool as it has been demonstrated to have a wide range of applicability 16–18 . As for functional tests, the gold standard is analysis of patient RNA from the tissue of interest to confirm alternative splicing 19 . However, specific analysis of each identified variant can be time‐consuming and difficult.…”

Section: Discussionmentioning

confidence: 99%

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Overcoming challenges associated with identifying FBN1 deep intronic variants through whole‐genome sequencing

Kim,

Jang,

Jang

et al. 2024

Clinical Laboratory Analysis

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BackgroundMarfan syndrome (MFS), caused by pathogenic variants of FBN1 (fibrillin‐1), is a systemic connective tissue disorder with variable phenotypes and treatment responsiveness depending on the variant. However, a significant number of individuals with MFS remain genetically unexplained. In this study, we report novel pathogenic intronic variants in FBN1 in two unrelated families with MFS.MethodsWe evaluated subjects with suspected MFS from two unrelated families using Sanger sequencing or multiplex ligation‐dependent probe amplification of FBN1 and/or panel‐based next‐generation sequencing. As no pathogenic variants were identified, whole‐genome sequencing was performed. Identified variants were analyzed by reverse transcription‐PCR and targeted sequencing of FBN1 mRNA harvested from peripheral blood or skin fibroblasts obtained from affected probands.ResultsWe found causative deep intronic variants, c.6163+1484A>T and c.5788+36C>A, in FBN1. The splicing analysis revealed an insertion of in‐frame or out‐of‐frame intronic sequences of the FBN1 transcript predicted to alter function of calcium‐binding epidermal growth factor protein domain. Family members carrying c.6163+1484A>T had high systemic scores including prominent skeletal features and aortic dissection with lesser aortic dilatation. Family members carrying c.5788+36C>A had more severe aortic root dilatation without aortic dissection. Both families had ectopia lentis.ConclusionVariable penetrance of the phenotype and negative genetic testing in MFS families should raise the possibility of deep intronic FBN1 variants and the need for additional molecular studies. This study expands the mutation spectrum of FBN1 and points out the importance of intronic sequence analysis and the need for integrative functional studies in MFS diagnosis.

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Section: Discussionmentioning

confidence: 99%

“… 16 , 17 , 18 As for functional tests, the gold standard is analysis of patient RNA from the tissue of interest to confirm alternative splicing. 19 However, specific analysis of each identified variant can be time‐consuming and difficult. Moreover, RNA analysis remains challenging given the need to obtain tissue samples.…”

Section: Discussionmentioning

confidence: 99%

Overcoming challenges associated with identifying FBN1 deep intronic variants through whole‐genome sequencing

Kim,

Jang,

Jang

et al. 2024

Clinical Laboratory Analysis

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“…For instance, c.5608G > A, p.Gly1870Arg detected in case 13 may have been elevated to LPV by adding the co-segregation score PP1 if the identical variant had been confirmed in two tall brothers. Functional studies can help strengthen the evidence of pathogenicity in borderline cases [34][35][36].…”

Section: Discussionmentioning

confidence: 99%

Re-evaluation of a Fibrillin-1 Gene Variant of Uncertain Significance Using the ClinGen Guidelines

Kim,

Kim

et al. 2023

Ann Lab Med

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Background: Marfan syndrome (MFS) is caused by fibrillin-1 gene (FBN1) variants. Mutational hotspots and/or well-established critical functional domains of FBN1 include cysteine residues, calcium-binding consensus sequences, and amino acids related to interdomain packaging. Previous guidelines for variant interpretation do not reflect the features of genes or related diseases. Using the Clinical Genome Resource (ClinGen) FBN1 variant curation expert panel (VCEP), we re-evaluated FBN1 germline variants reported as variants of uncertain significance (VUSs).Methods: We re-evaluated 26 VUSs in FBN1 reported in 161 patients with MFS. We checked the variants in the Human Genome Mutation Database, ClinVar, and VarSome databases and assessed their allele frequencies using the gnomAD database. Patients' clinical information was reviewed.Results: Four missense variants affecting cysteines (c.460T > C, c.1006T > C, c.5330G > C, and c.8020T > C) were reclassified as likely pathogenic and were assigned PM1_strong or PM1. Two intronic variants were reclassified as benign by granting BA1 (stand-alone). Four missense variants were reclassified as likely benign. BP5 criteria were applied in cases with an alternate molecular basis for disease, one of which (c.7231G > A) was discovered alongside a pathogenic de novo COL3A1 variant (c.1988G > T, p.Gly633Val).Conclusions: Considering the high penetrance of FBN1 variants and clinical variability of MFS, the detection of pathogenic variants is important. The ClinGen FBN1 VCEP encompasses mutational hotspots and/or well-established critical functional domains and adjusts the criteria specifically for MFS; therefore, it is beneficial not only for identifying pathogenic FBN1 variants but also for distinguishing these variants from those that cause other connective tissue disorders with overlapping clinical features.

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“…Tumor suppressor genes are the most widely sequenced genes in the clinical environment and are appropriate targets for RNA sequence analysis because of their loss-of-function disease mechanisms and blood expression ( Landrith et al, 2020 ; Lee et al, 2021 ; Gu et al, 2022 ; Na et al, 2022 ). In addition, the widespread adoption of state-of-the-art in silico tools for aberrant splicing prediction, such as SpliceAI ( Jaganathan et al, 2019 ), has increased the need to confirm the consequences of pre-mRNA splicing.…”

Section: Introductionmentioning

confidence: 99%

Reclassification of variants of tumor suppressor genes based on Sanger RNA sequencing without NMD inhibition

Ha,

Jang,

Kim

et al. 2023

Front. Genet.

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Introduction: RNA sequence analysis can be effectively used to identify aberrant splicing, and tumor suppressor genes are adequate targets considering their loss-of-function mechanisms. Sanger sequencing is the simplest method for RNA sequence analysis; however, because of its insufficient sensitivity in cases with nonsense-mediated mRNA decay (NMD), the use of cultured specimens with NMD inhibition has been recommended, hindering its wide adoption.Method: The results of Sanger sequencing of peripheral blood RNA without NMD inhibition performed on potential splicing variants of tumor suppressor genes were retrospectively reviewed. For negative cases, in which no change was identified in the transcript, the possibility of false negativity caused by NMD was assessed through a review of the up-to-date literature.Results: Eleven potential splice variants of various tumor suppressor genes were reviewed. Six variants were classified as pathogenic or likely pathogenic based on the nullifying effect identified by Sanger RNA sequencing. Four variants remained as variants of uncertain significance because of identified in-frame changes or normal expression of both alleles. The result of one variant was suspected to be a false negative caused by NMD after reviewing a recent study that reported the same variant as causing a nullifying effect on the affected transcript.Conclusion: Although RNA changes found in the majority of cases were expected to undergo NMD by canonical rules, most cases (10/11) were interpretable by Sanger RNA sequencing without NMD inhibition due to incomplete NMD efficiency or allele-specific expression despite highly efficient NMD.

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RNA Sequencing for Elucidating an Intronic Variant of Uncertain Significance (SDHD c.314+3A>T) in Splicing Site Consensus Sequences

Cited by 4 publications

References 10 publications

Overcoming challenges associated with identifying FBN1 deep intronic variants through whole‐genome sequencing

Overcoming challenges associated with identifying FBN1 deep intronic variants through whole‐genome sequencing

Re-evaluation of a Fibrillin-1 Gene Variant of Uncertain Significance Using the ClinGen Guidelines

Reclassification of variants of tumor suppressor genes based on Sanger RNA sequencing without NMD inhibition

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