Calling and Phasing of Single-Nucleotide and Structural Variants of the LDLR Gene Using Oxford Nanopore MinION

Abstract

The LDLR locus has clinical significance for lipid metabolism, Mendelian familial hypercholesterolemia (FH), and common lipid metabolism-related diseases (coronary artery disease and Alzheimer’s disease), but its intronic and structural variants are underinvestigated. The aim of this study was to design and validate a method for nearly complete sequencing of the LDLR gene using long-read Oxford Nanopore sequencing technology (ONT). Five PCR amplicons from LDLR of three patients with compound heterozygous FH were analyzed. We used standard workflows of EPI2ME Labs for variant calling. All rare missense and small deletion variants detected previously by massively parallel sequencing and Sanger sequencing were identified using ONT. One patient had a 6976 bp deletion (exons 15 and 16) that was detected by ONT with precisely located breakpoints between AluY and AluSx1. Trans-heterozygous associations between mutation c.530C>T and c.1054T>C, c.2141-966_2390-330del, and c.1327T>C, and between mutations c.1246C>T and c.940+3_940+6del of LDLR, were confirmed. We demonstrated the ability of ONT to phase variants, thereby enabling haplotype assignment for LDLR with personalized resolution. The ONT-based method was able to detect exonic variants with the additional benefit of intronic analysis in one run. This method can serve as an efficient and cost-effective tool for diagnosing FH and conducting research on extended LDLR haplotype reconstruction.

iPSC-Derived Endothelial Cells Reveal LDLR Dysfunction and Dysregulated Gene Expression Profiles in Familial Hypercholesterolemia

“…The first is considered pathogenic, the second as likely pathogenic, and both are transport-defective mutations that cause the immature LDLR to be retained in the endoplasmic reticulum and not transported to the Golgi apparatus for further maturation [42][43][44]. In patient FH 3.2.8, an extended deletion c.2141-966_2390-330del spans introns 14-16 and exons 15-16 and is considered to be pathogenic according to bioinformatic predictions, as it interferes with the production of the mature LDLR protein [40,45,46]. The second mutation, c.1327T>C to p.Trp443Arg (ClinVar ID 998052), which is considered likely pathogenic, causes a recycling defect, in which ligands are not released from the complex with LDLR in endosomes, and, as a result, LDLR is not recycled to the cell surface [38,40,45,46].…”

“…In patient FH 3.2.8, an extended deletion c.2141-966_2390-330del spans introns 14-16 and exons 15-16 and is considered to be pathogenic according to bioinformatic predictions, as it interferes with the production of the mature LDLR protein [40,45,46]. The second mutation, c.1327T>C to p.Trp443Arg (ClinVar ID 998052), which is considered likely pathogenic, causes a recycling defect, in which ligands are not released from the complex with LDLR in endosomes, and, as a result, LDLR is not recycled to the cell surface [38,40,45,46]. Our results on LDLR expression allow us to conclude that all LDLR mutations in theses iPSC lines and their iPSC-EC derivatives lead to a reduction in the mature form of the LDLR protein, whereas the combination of mutations in the second patient significantly reduces both mature and immature LDLR forms.…”

“…Two patient-specific iPSCs cell lines, called FH 1.3.1S and FH 3.2.8T, hPSCregs numbers ICGi036-A and ICGi038-A, were used in the study [38,39]. They were obtained previously from FH patients who were compound heterozygotes with pathogenic and likely pathogenic variants of both LDLR alleles, namely, p.Ser177Leu/p.Cys352Arg (db-SNP IDs: rs121908026/rs879254769) for FH 1.3.1S and p.Glu714_Ile796del/p.Trp443Arg (dbSNP IDs: no rs number/rs773566855) for FH 3.2.8T [38][39][40]. The control was the iPSC lines K7-4Lf and K6-4f from healthy donors, hPSCreg number ICGi022-A and ICGi021-A, respectively [70].…”

“…We reasoned that iPSC-derived endothelial cells lacking both native LDLR alleles would have more pronounced and potent effects of LDLR dysfunction and would therefore be more informative for studying gene function. Thus, to model the cellular and molecular mechanisms of FH in iPSC-ECs, we used two previously obtained lines of induced pluripotent stem cells (iPSCs) from patients who were compound heterozygotes for pathogenic and likely pathogenic allelic variants of the LDLR gene [38][39][40]. We obtained endothelial derivatives by direct differentiation of iPSC lines from a conditionally healthy donor and from patients with FH.…”

iPSC-Derived Endothelial Cells Reveals LDLR-Dysfunction and Dysregulated Gene Expression Profiles in Familial Hypercholesterolemia

“…Our study focused on identifying both the LDLR protein expression and transcriptomic patterns in endothelial cells with pathogenic allelic variants in the LDLR gene and discussed how this may potentially contribute to the FH progression. Previously, we obtained two lines of induced pluripotent stem cells (iPSCs) from patients who were compound heterozygotes for pathogenic and likely pathogenic allelic variants of the LDLR gene [7][8][9]. Both lines contained alterations within the LDLR gene that lead to impaired protein maturation, reducing the rate of LDLR transport to the membrane, and causing its partial retention in the endoplasmic reticulum.…”