Supravalvar aortic stenosis (SVAS) is a characteristic feature of Williams-Beuren syndrome (WBS). SVAS is present in 67% of those with WBS, but severity varies; 21% have clinically significant SVAS requiring surgical intervention while 33% have no appreciable aortic disease. Little is known about genetic modifiers outside the 7q11.23 region that might contribute to SVAS severity. To investigate, we collaboratively phenotyped 473 individuals with WBS and performed the largest whole-genome-sequencing study to date. We developed a set of strategies for modifier discovery including extreme phenotyping (surgical SVAS vs. no SVAS) and prioritization of non-synonymous variants with increased predicted functional impact along with an allele frequency difference between the extreme phenotype groups. We identified pathways enriched in common or less frequent variants, followed by association testing of SVAS severity with the enriched pathways. The common variant analysis identified pathways including the extracellular matrix and the innate immune system, while pathways encompassing adaptive immunity, ciliary function, lipid metabolism and PI3KAKT were captured by both the common and less frequent variant analyses. Cell cycle and estrogen responsive pathways were among those identified through the less frequent variant analysis. Among the 69 genes reported in other large genome wide association studies assessing aortic traits, 11 genes, including PCSK9 and ILR6, were found in our study, suggesting overlapping disease mechanisms. In summary, this study presents novel strategies for identification of disease modifiers in rare conditions like WBS.

All authors have completed the ICMJE uniform disclosure form at www.icmje.org/coi_disclosure.pdf and declare: BPR, CAM, and CBM have received funding from the WSA in the past. As a parent advocacy group, the WSA does have an interest in the submitted work but does not stand to financially profit from the findings. Additional government support noted above. No financial relationships with any other organizations that might have an interest in the submitted work occurred in the previous three years; no other relationships or activities that could appear to have influenced the submitted work are noted.

The NIH effort was supported by the NHLBI Division of Intramural Research (BAK). BPR and CAM were supported by grants from the Williams Syndrome Association (WSA) and LRO received funding from the Canadian Institutes for Health Research (MOP77720. CBM received support from the National Institute of Neurological Disorders and Stroke (R01 NS35102) and the WSA (WSA 0104 and WSA 0111). CAM and LRO were also partially supported by subcontracts from the grant of R01 NS35102. The Genomic Disorder Biobank of the Telethon Network of Genetic Biobanks was supported by Telethon Italy grant GTB12001G, GM)

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Institutional Review Board of the National Institutes of Health, the University of Nevada School of Medicine Internal Review Board, the University of Toronto Health Sciences Research Ethics Board, the Boston Children Hospital Internal Review Board, and Fondazione IRCCS Casa Sollievo della Sofferenza Ethics Board gave ethical approval for this work.

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.

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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).

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I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.

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The summary statistics of the non-synonymous variants in no SVAS and surgical SVAS groups will be available upon reasonable request to the corresponding author, Dr. Beth Kozel via beth.kozel@nih.gov.