Abstract
Familial hypercholesterolemia (FH) is an autosomal dominant disease of lipid metabolism caused by defects in the genes LDLR, PCSK9, APOB y LDLRAP1. The prevalence of heterozygous familial hypercholesterolemia is estimated between 1/200 and 1/250, and the prevalence of homozygosis familial hypercholesterolemia is estimated in 1/160.000-300.000. It is a genetic disease characterized by high levels of cholesterol-low-density lipoprotein (c-LDL) in plasma, which increases the risk of premature coronary heart disease (CHD). Therefore, early detection of patients with FH allows the initiation of adequate treatment, reducing the risk of CHD. Genetic analysis was performed by Next Generation Sequencing (NGS) using a customized panel of 198 genes in DNA samples of 180 subjects with a clinical diagnosis of probable or definitive FH using the “Dutch Lipid Clinic Network” (DLCN) criteria. Performing a bioinformatic analysis with the aim of finding new interesting genetic variants of the PCSK9 and LDLR genes. Variants of the LDLR gene, and gain-of-function (GOF) variants of the PCSK9 gene are important in FH. As no new variants of the PCSK9 gene were found, only LDLR variants found in patients were validated functionally in hamster ovarian cells (CHO-ldlA7). Low-density lipoprotein receptor (LDLR) activity was measure by flow cytometry, LDLR expression was detected by immunofluorescence and western blot, and colocalization LDLR-Dil-LDL by immunofluorescence. Five variants of the LDLR gene were tested, two of them new, c.640 T>C;p.(Trp214Gly) and c.1369 G>C;p.(Asp457His), and three of them previously described, c.148 G>T;p.(Ala50Ser), c.889 A>C;p.(Asn297His), and c.967 G>A;p.(Gly323Ser). Finally, the variants c.640 T>C;p.(Trp214Gly), c.889 A>C;p.(Asn297His), and c.967 G>A;p.(Gly323Ser) were classified as pathogenic, and the variants c.148 G>T;p.(Ala50Ser) and c.1369 G>C;p.(Asp457His) as benign. The functional in vitro characterization of rare variants at the LDLR and PCSK9 is a useful tool to classify the new variants avoiding the classification as “uncertain significant variants” (VUS). This approach allows to confirm the genetic diagnosis of FH, and therefore, carry out cascade family screening.
