ESPE Abstracts (2015) 84 P-1-6

Genetic Diagnosis of Congenital Primary Adrenal Insufficiency by Massive Parallel Sequencing

Florence Roucher Bouleza,b, Delphine Mallet Motaka, Mad-Helenie Guerrin Elsensohnb, Claire Bardelb, Pascal Royb & Yves Morela


aMolecular Endocrinology and Rare Diseases, CBPE, Hospices Civils de Lyon, Université Lyon 1, Bron-Lyon, France; bBiostatistics Department, Hospices Civils de Lyon, CNRS UMR 5558 LBBE, Pierre-Benite-Lyon, France


Background: Congenital primary adrenal insufficiency (PAI) can occur as three types: isolated glucocorticoid or mineralocorticoid deficiency, or global adrenal insufficiency, with combined mineralo and glucocorticoid deficiency. Once the most frequent genes (CYP21A2, ABCD1…) have been discarded by biological tests, many other genes may be involved in each type, and one gene may be responsible of different types. Consequently, there is no real decision tree in the gene analysis order.

Objective and hypotheses: The aim is to replace Sanger sequencing by massive parallel sequencing (MPS) in order to get a rapid, confident, and costless diagnosis. Instead of studying the genes one by one, they are analysed all at once. But do we get the same results?

Method: An AmpliSeq™ custom panel was designed including 17 genes: AAAS, AIRE, CDKN1C, Cited2, CYP11A1, CYP11B2, GPX1, MC2R, MCM4, MRAP, NNT, NR0B1, NR5A1, PBX1, PRDX3, StAR, and TXNRD2. To evaluate the MPS strategy, 20 patients, previously studied by Sanger sequencing (up to ten genes), were sequenced on an Ion Torrent PGM™ System. The bio-informatics pipeline used was the one implemented in the Torrent Suite™ Software. Statistical tools (log-linear and logit models) were used to compare the results, considering Sanger as the ‘gold standard’.

Results: 90% of our targets are well sequenced. The average read depth is 250×. The sensitivity is 80% and specificity 99.9%. No detectable variations were in GC rich regions within the 10% targets not covered or in CYP11B2, because mapping quality was at 0 due to its high homology with CYP11B1.

Conclusion: Complete molecular investigation by MPS was faster and cheaper. All regions insufficiently covered by MPS still need to be studied by Sanger sequencing. Taking this into account, no variations would have been missed except in CYP11B2. This underlines problem of pseudogenes or homologous genes in MPS.

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