Optimal molecular diagnosis of primary dyslipidemia is challenging to confirm the diagnosis, test and identify at risk relatives. The aim of this study was to test the application of a single targeted Show more
Optimal molecular diagnosis of primary dyslipidemia is challenging to confirm the diagnosis, test and identify at risk relatives. The aim of this study was to test the application of a single targeted next-generation sequencing (NGS) panel for hypercholesterolemia, hypocholesterolemia, and hypertriglyceridemia molecular diagnosis. NGS workflow based on a custom AmpliSeq panel was designed for sequencing the most prevalent dyslipidemia-causing genes (ANGPTL3, APOA5, APOC2, APOB, GPIHBP1, LDLR, LMF1, LPL, PCSK9) on the Ion PGM Sequencer. One hundred and forty patients without molecular diagnosis were studied. In silico analyses were performed using the NextGENe software and homemade tools for detection of copy number variations (CNV). All mutations were confirmed using appropriate tools. Eighty seven variations and 4 CNV were identified, allowing a molecular diagnosis for 40/116 hypercholesterolemic patients, 5/13 hypocholesterolemic patients, and 2/11, hypertriglyceridemic patients respectively. This workflow allowed the detection of CNV contrary to our previous strategy. Some variations were found in previously unexplored regions providing an added value for genotype-phenotype correlation and familial screening. In conclusion, this new NGS process is an effective mutation detection method and allows better understanding of phenotype. Consequently this assay meets the medical need for individualized diagnosis of dyslipidemia. Show less
Molecular diagnosis of cardiomyopathies remains difficult not only because of the large number of causative genes and the high rate of private mutations but also due to the large number of unclassifie Show more
Molecular diagnosis of cardiomyopathies remains difficult not only because of the large number of causative genes and the high rate of private mutations but also due to the large number of unclassified variants (UVs) found in patients' DNA. This study reports the functional splicing impact of nine novel genomic variations previously identified in unrelated patients with cardiomyopathies. To identify splice variants among these UVs, a combination of in silico and in vitro hybrid minigene tools was used as transcript is not available. Using this two-step approach, these UVs were reclassified as splicing mutations (MYBPC3-c.655-25A>G, MYBPC3-c.1790G>A (p.Arg597Gln), MYBPC3-c.2414-36G>T) or as mutations with a majority of abnormally spliced transcripts (MYBPC3-c.1182C>A, TNNT2-c.460G>A (p.Glu154Lys), and TNNT2-c.822-3C>A) or as variations with a weak splicing effect (TNNT2-c.1000-38C>A). For the two remaining variations in intron 11 of the TNNT2 gene in the vicinity of the acceptor splice site (c.571-7G>A, c.571-29G>A), a minigene assay was inconclusive as exon 12 is neither recognized as an exon by HeLa nor by H9c2 cells. Our study highlights the importance of the combined use of in silico and in vitro splicing assays to improve the prediction of the functional splicing impact of identified genetic variants if the RNA sample from the patient is not easily available. Show less