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

Abstract

Individuals with heritable thoracic aortic disease (HTAD) face a high risk of deadly aortic dissections, but genetic testing identifies causative variants in only a minority of cases. We explored the contribution of non-canonical splice variants (NCVAS) to thoracic aortic disease (TAD) using SpliceAI and sequencing data from diverse cohorts, including 551 early-onset sporadic dissection cases and 437 HTAD probands with exome sequencing, 57 HTAD pedigrees with whole genome sequencing, and select sporadic cases with clinical panel testing. NCVAS were identified in syndromic HTAD genes such as FBN1, SMAD3, and COL3A1, including intronic variants in FBN1 in two Marfan syndrome (MFS) families. Validation in the Penn Medicine BioBank and UK Biobank showed enrichment of NCVAS in HTAD-associated genes among dissections. These findings suggest NCVAS are an underrecognized contributor to TAD, particularly in sporadic dissection and unsolved MFS cases, highlighting the potential of advanced splice prediction tools in genetic diagnostics.

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).

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.

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.