Improved detection of low-level GNAQ mosaicism in Sturge-Weber syndrome through affected tissue testing, deep sequencing, and HRM-qPCR

Abstract Background Sturge-Weber syndrome (SWS) is a rare genetic neurocutaneous syndrome. While classical (type I) SWS is mainly caused by tissue-specific somatic mosaicism of a recurrent missense variant in the GNAQ gene, other genes have been implicated in SWS. Therefore, genetic testing at an early stage can support clinical management of affected children and genetic counselling of their families. Methods Thirteen type I SWS patients were enrolled in this study. In the majority, blood-based genetic testing did not lead to genetic confirmation. DNA isolated from tissue samples (lesional skin biopsies in n = 12 samples or affected brain tissue in n = 1 sample) was subjected to next-generation sequencing at high coverage. We also established and validated a high-resolution melt analysis quantitative PCR (HRM-qPCR) assay for specific detection of the recurrent GNAQ variant. Results In contrast to peripheral blood samples, tissue samples from all patients were found to harbor the hotspot GNAQ c.548G>A p.(Arg183Gln) variant at fractions ranging from 0.5% to 8.3%. Subsequently, presence of the variant was confirmed by HRM-qPCR, which was found to be sensitive enough to detect variants even at very low fractions (0.25%). Conclusion Data from our case series emphasize the importance of tissue-based analysis by deep sequencing to detect somatic mosaicism in SWS and suggest that HRM-qPCR is a powerful technique for the targeted confirmation of low-level somatic variants.