ECE2022 Rapid Communications Rapid Communications 2: Adrenal and Cardiovascular Endocrinology 1 (8 abstracts)
A rapid genetic diagnosis for >80% individuals with non-CAH Primary Adrenal Insufficiency is achievable by candidate gene sequencing combined with WES
Chris Smith 1 , Jordan Read 1 , Charlotte Hall 1 , Avinaash Maharaj 1 , Lucia Marroquin Ramirez 1 , Younus Qamar 1 , Claire Hughes 1,2 , Adrian Clark 1 , Rathi Prasad 1 , Li Chan 1 , Salwa Musa 3 & Louise Metherell 1
1William Harvey Research Institute, United Kingdom; 2Royal London Hospital, United Kingdom; 3Gaafar Ibn Auf Children Hospital, Pediatric Endocrinology Unit, Sudan
Primary adrenal insufficiency in children can be due to mutations in >20 genes, most commonly CYP21A2, giving rise to 21-hydroxylase deficiency. Phenotypically these disorders overlap and present with conditions ranging from isolated (or familial) glucocorticoid deficiency (FGD) to syndromic disorders involving multiple tissues. Distinguishing between them can be problematic, especially where biochemical testing is not possible or not undertaken. Over the last 30 years 400 individuals with suspected FGD, from 31 different countries, have been referred to our centre for genetic testing. All cases had low/undetectable serum cortisol and, where measured, elevated plasma ACTH levels. Using a combined, two-step protocol we have sequenced 369 of the 400 individuals. In the first step we sequenced the small, frequently mutated, candidate genes; MC2R, MRAP, STAR and CYP11A1, by Sanger sequencing (CGS) before proceeding to whole exome sequencing (WES) if these were mutation free. For CGS, sequences were aligned to reference sequences using BioEdit software and WES variant call files were analysed using Ingenuity Variant Analysis package and/or examination of BAM files, using the Integrative Genomics Viewer, to detect exonic deletions. Rare, synonymous or predicted benign variants were subjected to an in vitro splicing assay using the pET01 vector (MoBiTec). In 308/369 individuals we found a definitive diagnosis in a causative gene for adrenal insufficiency, a success rate of 81%, and identified many novel mutations. The findings also highlighted a number of causal synonymous and predicted benign variants resulting in splice defects. The aetiologies of cases with a gene defect were as follows; MC2R (22%), MRAP (17%), NNT (15%), STAR (9%), CYP11A1 (7%), with the remaining 30% due to a further 13 genes. Previous founder effects were reinforced e.g S74I in MC2R and rs6161 in CYP11A1 in the UK population, P24Rfs*4 in MCM4 in Ireland and R188C in STAR in Canada, with new associations being discovered for T731= in NNT in Sudan and R222Q in SGPL1 in Saudi Arabia. In contrast, common MRAP splice mutations seen at the exon 3/intron 3 junction were present in individuals from many countries. The use of CGS/WES now permits a rapid genetic diagnosis for >80% individuals and is an invaluable, cost-effective tool to improve tailored patient management. For patients without a genetic diagnosis, it is unclear whether they have unconventional mutations in known genes or if there are further gene defects to be discovered.
Volume 81
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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