LDLR gene rearrangements in Czech FH patients likely arise from one mutational event.

BACKGROUND: Large deletions and duplications within the low-density lipoprotein receptor (LDLR) gene make up approximately 10% of LDLR pathogenic variants found in Czech patients with familial hypercholesterolemia. The goal of this study was to test the hypothesis that all probands with each rearrangement share identical breakpoints inherited from a common ancestor and to determine the role of Alu repetitive elements in the generation of these rearrangements. METHODS: The breakpoint sequence was determined by PCR amplification and Sanger sequencing. To confirm the breakpoint position, an NGS analysis was performed. Haplotype analysis of common LDLR variants was performed using PCR and Sanger sequencing. RESULTS: The breakpoints of 8 rearrangements within the LDLR gene were analysed, including the four most common LDLR rearrangements in the Czech population (number of probands ranging from 8 to 28), and four less common rearrangements (1-4 probands). Probands with a specific rearrangement shared identical breakpoint positions and haplotypes associated with the rearrangement, suggesting a shared origin from a common ancestor. All breakpoints except for one were located inside an Alu element. In 6 out of 8 breakpoints, there was high homology (≥ 70%) between the two Alu repeats in which the break occurred. CONCLUSIONS: The most common rearrangements of the LDLR gene in the Czech population likely arose from one mutational event. Alu elements likely played a role in the generation of the majority of rearrangements inside the LDLR gene.

Low Density Lipoprotein Receptor Variants in the Beta-Propeller Subdomain and Their Functional Impact.

Background: Pathogenic variants in the low density lipoprotein receptor gene are associated with familial hypercholesterolemia. Some of these variants can result in incorrect folding of the LDLR protein, which is then accumulated inside the cell and cannot fulfill its function to internalize LDL particles. We analyzed the functional impact of 10 LDLR variants localized in the beta-propeller of epidermal growth factor precursor homology domain. The experimental part of the work was complemented by a structural analysis on the basis of 3D LDLR protein structure. Methods: T-Rex Chinese hamster ovary cells transfected with the human LDLR gene were used for live cell imaging microscopy, flow cytometry, and qRT-PCR analysis. Results: Our results showed that the analyzed LDLR protein variants can be divided into three groups. (1) The variants buried inside the 3D protein structure expressing proteins accumulated in the endoplasmic reticulum (ER) with no or reduced plasma membrane localization and LDL particle internalization, and associated with an increased gene expression of ER-resident chaperones. (2) The variants localized on the surface of 3D protein structure with slightly reduced LDLR plasma membrane localization and LDL particle internalization, and associated with no increased mRNA level of ER-resident chaperones. (3) The variants localized on the surface of the 3D protein structure but expressing proteins with cell responses similar to the group 1. Conclusion: All analyzed LDLR variants have been evaluated as pathogenic but with different effects on protein localization and function, and expression of genes associated with ER stress.

The molecular basis of familial hypercholesterolemia in the Czech Republic: spectrum of LDLR mutations and genotype-phenotype correlations.

BACKGROUND: Familial hypercholesterolemia (FH), a major risk for coronary heart disease, is predominantly associated with mutations in the genes encoding the low-density lipoprotein receptor (LDLR) and its ligand apolipoprotein B (APOB). RESULTS: In this study, we characterize the spectrum of mutations causing FH in 2239 Czech probands suspected to have FH. In this set, we found 265 patients (11.8%) with the APOB mutation p.(Arg3527Gln) and 535 patients (23.9%) with a LDLR mutation. In 535 probands carrying the LDLR mutation, 127 unique allelic variants were detected: 70.1% of these variants were DNA substitutions, 16.5% small DNA rearrangements, and 13.4% large DNA rearrangements. Fifty five variants were novel, not described in other FH populations. For lipid profile analyses, FH probands were divided into groups [patients with the LDLR mutation (LDLR+), with the APOB mutation (APOB+), and without a detected mutation (LDLR-/APOB-)], and each group into subgroups according to gender. The statistical analysis of lipid profiles was performed in 1722 probands adjusted for age in which biochemical data were obtained without FH treatment (480 LDLR+ patients, 222 APOB+ patients, and 1020 LDLR-/APOB- patients). Significant gradients in i) total cholesterol (LDLR+ patients > APOB+ patients = LDLR-/APOB- patients) ii) LDL cholesterol (LDLR+ patients > APOB+ patients = LDLR-/APOB- patients in men and LDLR+patients > APOB+ patients >LDLR-/APOB- patients in women), iii) triglycerides (LDLR-/APOB- patients > LDLR+ patients > APOB+ patients), and iv) HDL cholesterol (APOB+ patients > LDLR-/APOB- patients = LDLR+ patients) were shown. CONCLUSION: Our study presents a large set of Czech patients with FH diagnosis in which DNA diagnostics was performed and which allowed statistical analysis of clinical and biochemical data.

An APEX-based genotyping microarray for the screening of 168 mutations associated with familial hypercholesterolemia.

OBJECTIVE: Familial hypercholesterolemia (FH) is an inborn disorder of lipid metabolism characterised by elevated plasma concentrations of low-density lipoprotein cholesterol and total cholesterol. This imbalance results in accelerated atherosclerosis and premature coronary heart disease. The early identification and treatment of FH patients is extremely important because it leads to significant reduction of both coronary morbidity and mortality. FH is transmitted in an autosomal dominant manner and associated predominantly with mutations in the genes encoding the low-density lipoprotein receptor (LDLR) and its ligand apolipoprotein B (APOB). To date, more than 1000 sequence variants have been described in the LDLR gene. In marked contrast to LDLR, only one APOB mutation is prevalent in Europe. METHODS AND RESULTS: The aim of this study was, on the basis of data obtained by the molecular genetic analysis of 1945 Czech FH probands, to propose, generate, and validate a new diagnostic tool, an APEX (Arrayed Primer EXtension)-based genotyping DNA microarray called the FH chip. The FH chip contains the APOB mutation p.Arg3527Gln, all 89 LDLR point mutations and small DNA rearrangements detected in Czech FH patients, and 78 mutations frequent in other European and Asian FH populations. The validation phase revealed the sensitivity and specificity of this platform, 100% and 99.1%, respectively. CONCLUSIONS: This FH chip is a rapid, reproducible, specific, and cost-effective tool for genotyping, and in combination with MLPA (multiple ligation-dependent probe amplification) represents a reliable molecular genetic protocol for the large-scale screening of FH mutations in the Czech population.

Genomic characterization of large rearrangements of the LDLR gene in Czech patients with familial hypercholesterolemia.

BACKGROUND: Mutations in the LDLR gene are the most frequent cause of Familial hypercholesterolemia, an autosomal dominant disease characterised by elevated concentrations of LDL in blood plasma. In many populations, large genomic rearrangements account for approximately 10% of mutations in the LDLR gene. METHODS: DNA diagnostics of large genomic rearrangements was based on Multiple Ligation dependent Probe Amplification (MLPA). Subsequent analyses of deletion and duplication breakpoints were performed using long-range PCR, PCR, and DNA sequencing. RESULTS: In set of 1441 unrelated FH patients, large genomic rearrangements were found in 37 probands. Eight different types of rearrangements were detected, from them 6 types were novel, not described so far. In all rearrangements, we characterized their exact extent and breakpoint sequences. CONCLUSIONS: Sequence analysis of deletion and duplication breakpoints indicates that intrachromatid non-allelic homologous recombination (NAHR) between Alu elements is involved in 6 events, while a non-homologous end joining (NHEJ) is implicated in 2 rearrangements. Our study thus describes for the first time NHEJ as a mechanism involved in genomic rearrangements in the LDLR gene.

Identification and characterization of large deletions in the phenylalanine hydroxylase (PAH) gene by MLPA: evidence for both homologous and non-homologous mechanisms of rearrangement.

Large gene deletions and duplications were analyzed in 59 unrelated phenylketonuria (PKU) patients negative for phenylalanine hydroxylase (PAH) mutations on one or both alleles from previous exon by exon analysis. Using the novel multiplex ligation-dependent probe amplification (MLPA) method, a total of 31 partial PAH deletions involving single exons were identified in 31 PKU patients. Nineteen cases exhibited deletion of exon 5, and 12 cases provided evidence for the deletion of exon 3. Subsequently, using restriction enzyme digestion and DNA sequencing, three different large deletions, EX3del4765 (12 cases), EX5del955 (2 cases) and EX5del4232ins268 (17 cases) were identified and confirmed by long-range PCR and by the analysis of aberrant transcripts. Altogether, the 31 large deletions presented account for 3% of all PAH mutant alleles investigated in Czech PKU patients. Bioinformatic analysis of three breakpoints showed that the mutation EX3del4765 had arisen through an Alu-Alu homologous recombination, whereas two other mutations-the EX5del955 and EX5del4232ins268, had been created by a non-homologous end joining (NHEJ). We conclude that MLPA is a convenient, rapid and reliable method for detection of intragenic deletions in the PAH gene and that a relatively high number of alleles with large deletions are present in the Slavic PKU population.

Spectrum of low density lipoprotein receptor mutations in Czech hypercholesterolemic patients.

The aim of our study was to define mutations causing familial hypercholesterolemia (FH) phenotype in Czech hypercholesterolemic individuals. A combination of heteroduplex analysis, SSCP, DGGE, DNA sequencing and PCR/restriction analysis was used for this purpose. Molecular searching in the promoter region and coding sequence of the low density lipoprotein receptor (LDLR) gene in 130 patients from 68 unrelated families resulted in the identification of 37 sequence variations. Thirty of them are most likely disease causing mutations. Nineteen mutations were novel (two nonsense, five missense, six nucleotide(s) insertions and six nucleotide(s) deletions). Their pathological effect can be predicted on the basis of their position with respect to previously reported mutations with an estimated reduction of the receptor activity and/or premature termination of translation. These results expand our knowledge of mutations responsible for FH. Seven nucleotide variations were characterized as silent polymorphisms.