Utility of Genome Sequencing and Group-Enrichment to Support Splice Variant Interpretation in Marfan Syndrome

Purpose: To quantify the impact of non-canonical FBN1 splice site variants in undiagnosed Marfan syndrome (MFS), a connective tissue disorder associated with skeletal abnormalities and Familial Thoracic Aortic Aneurysm Disease (FTAAD). Methods: A systematic analysis of ultra-rare FBN1 variants was performed using genome sequencing data from the 100,000 Genomes Project. Variants were annotated with SpliceAI and the significance of enrichment among individuals with FTAAD was assessed using Fishers exact test. Experimental validation utilised RNAseq, RT-PCR, minigene constructs and replication analysis was with data from UK Biobank. Results: Using aggregate data for 78,195 individuals, we identified 13,864 singleton SNVs in FBN1 of which 21 were predicted to impact splicing (SpliceAI >0.5). Incidence of candidate splice variants in individuals recruited with FTAAD (9/703) was significantly elevated compared with that seen in non-FTAAD participants (12/77,492; OR=84, p=9.7x10-14). Additional analysis uncovered a further 14 families harbouring 11 different FBN1 splice variants. A total of 20 candidate splice variants in 23 families were identified, of which 70% lay beyond the ±8 splice regions. RNA testing confirmed the predicted splice aberration in 14/20 and for 9/20, pseudoexonization was the likely splicing-anomaly. Conclusion: Our findings indicate that non-canonical splice variants may account for ~3% of families with undiagnosed FTAAD, highlighting the importance of incorporating analysis of introns and confirmatory RNA testing into genetic testing for MFS. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was supported by the National Institute for Health and Care Research Oxford and Exeter Biomedical Research Centres. The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. Additional funding was from the Medical Research Council (MR/W01761X/1) and the National Institute for Health and Care Research Manchester Biomedical Research Centre (NIHR203308). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics approval was from Cambridge South REC (14/EE/1112). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data from the 100kGP and the GMS is held in the National Genomic Research Library, Genomics England, https://doi.org/10.6084/m9.figshare.4530893.v7. Data from UK Biobank cannot be shared publicly because of data availability and data return policies. Data are available from the UK Biobank for researchers who meet the criteria for access to datasets to UK Biobank (www.ukbiobank.ac.uk).