Non-canonical splice variants in thoracic aortic dissection cases and Marfan syndrome with negative genetic testing

Figures

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  Fig. 1. Pedigree and RT-PCR assay for sporadic dissection case with FBN1 c.2294-3 C > A variant ascertained through clinical testing. a Family members carrying the FBN1 variant are marked with a ‘+’, including the proband’s brother with a systemic score (SS) of 8. Individuals with normal aortic imaging are marked with a ‘*’. b Gel electrophoresis image of RT-PCR from skin fibroblasts of proband’s brother. Band 1 (blue) represents a small amount of two abnormal splice products generated by the rare use of two cryptic acceptor sites in the exon. Bands 2-5 (blue) are two heteroduplex pairs formed between normal and abnormal products. Ao: Aortic diameter at the sinuses of Valsalva; Z-score: normalized aortic diameter. CHX – sample treated with cycloheximide, an inhibitor of nonsense-mediated decay (NMD).

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  Fig. 2. Analysis strategy for identifying non-canonical variants predicted to alter splicing (NCVAS) in UTHealth, Penn Medicine BioBank, and UK Biobank cohorts. ES exome sequencing; WGS whole genome sequencing; ESTAD sporadic aortic dissection cohort ( < 60 years of age without family history or syndromic features); HTAD Families with multiple members affected by heritable thoracic aortic disease; TAA thoracic aortic aneurysm; HI haploinsufficiency; MAF minor allele frequency; LB/B likely benign or benign; NS not significant (p > 0.05) *number of HTAD pedigrees, 97 total individuals sequenced.

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  Fig. 3. MFS pedigrees with splice-altering variants identified on whole genome sequencing. Pedigree 1 (a) had extensive aortic disease and multiple individuals with a clinical diagnosis of MFS but negative genetic testing. WGS identified a novel FBN1 variant, c.6872-1003 C > T (SpliceAI – 0.39), in two affected brothers, predicted to activate a cryptic splice site within intron 56. Pedigree 2 (b) was similarly affected with aortic disease and clinical MFS diagnoses but negative molecular testing. WGS of the proband revealed a novel FBN1 variant, c.1148-16 T > A (SpliceAI – 0.9), predicted to cause intron retention and extension of exon 11 of the mRNA transcript. Systemic scores (SS), aortic diameters at the sinuses of Valsalva (Ao), and normalized aortic diameter Z-scores are shown for individuals who underwent clinical assessment. MFS – Marfan syndrome; WGS – whole genome sequencing.