Use of next-generation sequencing to detect LDLR gene copy number variation in familial hypercholesterolemia

Abstract

Familial hypercholesterolemia (FH) is a heritable condition of severely elevated LDL cholesterol, caused predominantly by autosomal codominant mutations in the LDL receptor gene (LDLR). In providing a molecular diagnosis for FH, the current procedure often includes targeted next-generation sequencing (NGS) panels for the detection of small-scale DNA variants, followed by multiplex ligation-dependent probe amplification (MLPA) in LDLR for the detection of whole-exon copy number variants (CNVs). The latter is essential because ∼10% of FH cases are attributed to CNVs in LDLR; accounting for them decreases false negative findings. Here, we determined the potential of replacing MLPA with bioinformatic analysis applied to NGS data, which uses depth-of-coverage analysis as its principal method to identify whole-exon CNV events. In analysis of 388 FH patient samples, there was 100% concordance in LDLR CNV detection between these two methods: 38 reported CNVs identified by MLPA were also successfully detected by our NGS method, while 350 samples negative for CNVs by MLPA were also negative by NGS. This result suggests that MLPA can be removed from the routine diagnostic screening for FH, significantly reducing associated costs, resources, and analysis time, while promoting more widespread assessment of this important class of mutations across diagnostic laboratories.

Keywords: DNA variation; LDL; bioinformatics; coronary heart disease; diagnostic tools; genetic testing; lipid and lipoprotein metabolism; lipoprotein receptors; molecular biology/genetics; precision medicine.

Fig. 1.

Two methods of detection of a CNV deletion event in the LDLR gene in a patient with FH (subject GL15929) with a heterozygous deletion in LDLR exons 2–6. A: MLPA method output: heterozygous deletion in LDLR exons 2–6. Exon numbers are shown by “LDLR-N” (where N is the number of the exon, the first “LDLR-1” indicates the promoter, and “SMARCA4-35” is upstream of the promoter), and “*Reference” indicate reference probes bound to alternative chromosomes. For each probe target region, two separate plots are generated: 1) the normalized reference sample set is represented by 1-SD box plots, where “X” indicates the mean and the horizontal line the median probe-signal intensity; and 2) the normalized patient sample probe-signal ratio is overlaid as a dot and is surrounded by error bars depicting the 95% CI. The upper arbitrary border (blue line) and lower arbitrary border (red line) are placed ±0.3 from the reference sample mean of each probe. B: VarSeq CNV Caller method output: heterozygous deletion in LDLR exons 2–6. Different regions of the output are as follows. i: Normalized ratio metric computed for each LipidSeq target region in LDLR; depth of sequence coverage comparative to reference controls, where ∼1.0 indicates diploid (normal) copy number state and ∼0.50 indicates a heterozygous deletion event. ii: Normalized z-score metric; number of SDs the DOC is from the reference control mean coverage, where ≤−5.0 is the threshold set to indicate a deletion event. iii: CNV state, determined by ratio and z-score metrics together with supporting evidence from VAFs (not shown). Segmentation analysis has merged multiple affected target regions to call a contiguous heterozygous deletion event. iv: Exon map of LDLR gene. v: LipidSeq probe target regions.

Fig. 2.

Two methods of detection of a CNV duplication event in the LDLR gene in a patient with FH (subject GL12812) with a duplication in LDLR exon 7. See Fig. 1 legend for overall structure of the panels. A: MLPA method output: duplication in LDLR exon 7. B: VarSeq CNV Caller method output: duplication in LDLR exon 7. i: Normalized ratio metric computed for each LipidSeq target region in LDLR; depth of sequence coverage comparative to reference controls where ∼1.0 indicates diploid (normal) copy number state and ∼1.5 indicates a duplication event. ii: Normalized z-score metric; number of SDs the DOC is from the reference control mean coverage, where ≥5.0 is the threshold set to indicate a duplication event; other sections are as in Fig. 1.