Single, short in‐del, and copy number variations detection in monogenic dyslipidemia using a next‐generation sequencing strategy

Abstract

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.

PCSK9 Variants in Familial Hypercholesterolemia: A Comprehensive Synopsis

“…With the continuous development of NGS technology, new NGS-based detection is also gradually being applied to FH screening. In a single target NGS panel study, this new NGS is found to be an effective variant detection method, which can better help to understand the phenotype of FH, and is expected to become a personalized diagnosis method for dyslipidemia ( Marmontel et al, 2018 ). In addition, in the first study of capture-based NGS, this NGS method covering the entire LDLR genome region has improved the efficiency of structural variation detection.…”

Genetics of Familial Hypercholesterolemia: New Insights

“…These events can account for between 2 and 20% of all positive cases in some populations ( Marduel et al, 2010 ; Pirillo et al, 2017 ; Tichý et al, 2017 ; Sun et al, 2018 ). Although multiplex ligation-dependent probe amplification is still the gold standard for the identification of large intragenic rearrangements, the analysis of copy number variations (CNVs) from NGS data can also achieve very good results ( Marmontel et al, 2018 ) and has been gradually introduced into standard diagnostic schemes in routine labs. The opportunity to obtain information about the genomic sequence of many genes and about the potential presence of rearrangements in these genes at the same time in one step is the main advantage of the NGS approach.…”

Development of a new expanded next‐generation sequencing panel for genetic diseases involved in dyslipidemia

“…Whereas it is known that NGS emergence makes CNV detection easier, this study establish that CNV algorithm tool is a critical step. 2,6,8 Indeed, our pipeline identified a partial deletion of one exon only after working on a cut bed file. 9 Without this option, as for other pipelines, the minimum limit of CNV detection was at least the smallest wholeexon (lower limit about 300 bp).…”

“…The insertion of an Alu element in APOB, detected on the BAM file, was not called by the pipeline. NGS workflows do not usually assess the detection of mobile elements, 2,6,8,11 although the detection of such variant is challenging. 12 Indeed, this detection needs specific bioinformatics tools including ancillary calculations to decrease falsepositive rates.…”

“…detect variants in the most frequent dyslipidemia-related genes and allowed molecular diagnosis of familial hypercholesterolemia (FH), familial hypobetalipoproteinemia (FHBL) and familial chylomicronemia syndrome (FCS). 2 However, at least 27 monogenic dyslipidemia caused by variants affecting a total of 25 genes are from now described 3 and dyslipidemia can also be part of several syndromes with a genetic basis such as lipodystrophy, Smith-Lemli-Opitz or Shwachman-Diamond syndromes. 4 Furthermore, the often complex and multifactorial nature of these clinical relevant traits is becoming more evident.…”