1. Research project objectives/ Research hypothesis Since 2000, at the Department of Biology and Genetics of the Medical University of Gdansk, mutational analysis of LDLR, APOB and PCSK9 genes is conducted in a group of patients with familial hypercholesterolemia (FH). For this purpose, MLPA, Sanger sequencing and more recently next generation sequencing (NGS) techniques are applied. To date, approximately 1500 probands and their family members have been tested which allowed to form the National Centre of Diagnostics and Treatment of Familial Hypercholesterolemia (NCD&TFH). The aim of the project is to perform: a) in vitro studies in order to analyze the expression and activity of 20 selected LDLR genetic variants. In addition, the results will be compared with clinical data of FH patients harboring the particular LDLR mutation, b) mutational analysis of the coding sequences of APOB, STAP1 and LDLRAP1 genes in a group of 200 patients with FH.

2. Research project methodology a) in vitro studies of LDLR Wild type LDLR and LDLR with the investigated specific alterations will be cloned into expression vectors and introduced into HepG2 cells. HA tag will be added to C-end of the proteins. It will allow to detect the presence of the LDLR-HA fusion protein in the cells by specific antibodies. Subsequently, the quantity of altered proteins will be determined and compared to wild type LDLR by western blotting. In addition, quantity of LDLR variants in a fraction of membrane and cytoplasmic proteins will be analyzed. Finally, to detect localization of LDLR variants in a cells, immunofluorescence and confocal microscopy will be applied. To examine whether studied mutations could alter LDL binding, assays with fluorescently labeled LDL and FACS (fluorescence-activated cell sorting) analysis will be employed. b) mutational analysis of APOB, STAP1 and LDLRAP1 genes In total, 200 FH patients registered in our database NCD&TFH, with no mutation within LDLR, PCSK9 and fragment of exon 26 of APOB, will be enrolled to the study. DNA previously isolated from peripheral blood lymphocytes will be analyzed by TruSeq Custom Amplicon and MiSeq (Illumina). To analyze the NGS data, Illumina Variant Studio and Geneious R9 software will be applied. Presence of the detected mutations will be confirmed by bidirectional Sanger sequencing.

3. Expected impact of the research project on the development of science, civilization and society To date, over 1700 pathogenic mutations of LDLR have been described. The presence of the mutation alter the structure and/or activity of the receptor. Defects in the LDL receptor are functionally divided into five classes depending on the impact of the mutation on the presence of mRNA, receptor maturation, disparity between LDL and immunoglobulin binding on the cell surface, LDL receptor degradation and trafficking. In vitro studies will allow to characterize the selected variants of unknown significance in LDLR. In addition, the results from functional studies will be compared with clinical data of FH patients. In contrast to LDLR mutations, approximately only few genetic alterations of APOB are linked to FH. Most of alterations are located within the fragment of APOB exon 26, nearby codon R3500. However, more recent studies revealed the presence of genetic variants located within the other exons of the APOB. Therefore, the mutational analysis of the coding sequence of APOB will possibly allow to detect a novel pathogenic APOB variants responsible for FH. More recently, STAP1 mutations have been detected in a group of FH patients. However, the number of the tested individuals was limited and genetic alterations were found only in several patients from the Netherlands and Germany. In a vast majority, familial hypercholesterolemia is an autosomal dominant disease. However, autosomal recessive familial hypercholesterolemia (ARH) has been also described. To date, only LDLRAP1 mutations were detected in a group of patients with ARH. The mutational analysis of LDLRAP1 will be performed in a current study. To summarize, the study will allow to get new insights into pathogenesis of familial hypercholesterolemia. In addition, it can allow to increase a number of individuals with molecularly confirmed FH and may allow to update an algorithm of molecular diagnosis of FH patients and members of their families.