Identification of New Copy Number Variation and the Evaluation of a CNV Detection Tool for NGS Panel Data in Polish Familial Hypercholesterolemia Patients.

Familial hypercholesterolemia (FH) is an inherited, autosomal dominant metabolic disorder mostly associated with disease-causing variant in LDLR, APOB or PCSK9. Although the dominant changes are small-scale missense, frameshift and splicing variants, approximately 10% of molecularly defined FH cases are due to copy number variations (CNVs). The first-line strategy is to identify possible pathogenic SNVs (single nucleotide variants) using multiple PCR, Sanger sequencing, or with more comprehensive approaches, such as NGS (next-generation sequencing), WES (whole-exome sequencing) or WGS (whole-genome sequencing). The gold standard for CNV detection in genetic diagnostics are MLPA (multiplex ligation-dependent amplification) or aCGH (array-based comparative genome hybridization). However, faster and simpler analyses are needed. Therefore, it has been proposed that NGS data can be searched to analyze CNV variants. The aim of the study was to identify novel CNV changes in FH patients without detected pathogenic SNVs using targeted sequencing and evaluation of CNV calling tool (DECoN) working on gene panel NGS data; the study also assesses its suitability as a screening step in genetic diagnostics. A group of 136 adult and child patients were recruited for the present study. The inclusion criteria comprised at least "possible FH" according to the Simon Broome diagnostic criteria in children and the DLCN (Dutch Lipid Clinical Network) criteria in adults. NGS analysis revealed potentially pathogenic SNVs in 57 patients. Thirty selected patients without a positive finding from NGS were subjected to MLPA analysis; ten of these revealed possibly pathogenic CNVs. Nine patients were found to harbor exons 4−8 duplication, two harbored exons 6−8 deletion and one demonstrated exon 9−10 deletion in LDLR. To test the DECoN program, the whole study group was referred for bioinformatic analysis. The DECoN program detected duplication of exons 4−8 in the LDLR gene in two patients, whose genetic analysis was stopped after the NGS step. The integration of the two methods proved to be particularly valuable in a five-year-old girl presenting with extreme hypercholesterolemia, with both a pathogenic missense variant (c.1747C>T) and exons 9−10 deletion in LDLR. This is the first report of a heterozygous deletion of exons 9 and 10 co-occurring with SNV. Our results suggest that the NGS-based approach has the potential to identify large-scale variation in the LDLR gene and could be further applied to extend CNV screening in other FH-related genes. Nevertheless, the outcomes from the bioinformatic approach still need to be confirmed by MLPA; hence, the latter remains the reference method for assessing CNV in FH patients.

Determining the accuracy of next generation sequencing based copy number variation analysis in Hereditary Breast and Ovarian Cancer.

BACKGROUND: Copy number variations (CNVs) are commonly associated with malignancies, including hereditary breast and ovarian cancers. Next generation sequencing (NGS) provides solutions for CNV detection in a single run. This study aimed to compare the accuracy of CNV detection by NGS analyzing tool against Multiplex Ligation Dependent Probe Amplification (MLPA). RESEARCH DESIGN AND METHODS: In total, 1276 cases were studied by targeted NGS panels and 691 cases (61 calls in 58 NGS-CNV positive and 633 NGS-CNV negative cases) were validated by MLPA. RESULTS: Twenty-eight (46%) NGS-CNV positive calls were consistent, whereas 33 (54%) calls showed discordance with MLPA. Two cases were detected as SNV by the NGS and CNV by the MLPA analysis. In total, 2% of the cases showed an MLPA confirmed CNV region in BRCA1/2. The results of this study showed that despite the high false positive call rate of the NGS-CNV algorithm, there were no false negative calls. The cases that were determined to be negative by the NGS and positive by the MLPA were actually carrying SNVs that were located on the MLPA probe binding sites. CONCLUSION: The diagnostic performance of NGS-CNV analysis is promising; however, the need for confirmation by different methods remains.