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

Abstract Background Marfan 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. Methods We 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. Results We 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. Conclusion Variable 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.