Familial Hypercholesterolemia Mutations Research Articles

Can Patients be Accurately Assessed for Familial Hypercholesterolaemia in Primary Care?

Familial Hypercholesterolaemia (FH) is the most prevalent monogenic condition causing premature coronary artery disease, although the majority of individuals remain undiagnosed. We sought to investigate whether individuals with FH could be accurately identified in primary care. The Dutch Lipid Clinic Network Criteria scores (DLCNCS) assessed by general practitioners (GPs) were compared with DLCNCS assessed by specialists using primary care data in 153 individuals. Thirty individuals with DLCNCS ≥4 underwent specialist review and genetic testing. Clinical FH was defined as DLCNCS ≥6, encompassing the probable and definite FH categories. GPs correctly classified 39 (86.7%) individuals with 'clinical FH', and 32 (94%) with 'unlikely FH' relative to specialists. Lin's concordance correlation coefficient was high (0.832 (0.783 - 0.881), p< 0.001) between specialist and GPs, with an overall agreement of 83.6%, κ 0.744 (0.642 - 0.831). After specialist review, 15 individuals (50%) were diagnosed with clinical FH, four (26.7%) had FH mutations. GPs correctly classified 12 (80%) of these individuals with clinical FH. GPs can accurately identify individuals at high and low risk of FH using the DLCNCS, which may augment opportunistic FH detection in the community. Increased education may enhance the diagnostic accuracy of FH in primary care.

Targeted genetic testing for familial hypercholesterolaemia using next generation sequencing: a population-based study.

BackgroundFamilial hypercholesterolaemia (FH) is a common Mendelian condition which, untreated, results in premature coronary heart disease. An estimated 88% of FH cases are undiagnosed in the UK. We previously validated a method for FH mutation detection in a lipid clinic population using next generation sequencing (NGS), but this did not address the challenge of identifying index cases in primary care where most undiagnosed patients receive healthcare. Here, we evaluate the targeted use of NGS as a potential route to diagnosis of FH in a primary care population subset selected for hypercholesterolaemia.MethodsWe used microfluidics-based PCR amplification coupled with NGS and multiplex ligation-dependent probe amplification (MLPA) to detect mutations in LDLR, APOB and PCSK9 in three phenotypic groups within the Generation Scotland: Scottish Family Health Study including 193 individuals with high total cholesterol, 232 with moderately high total cholesterol despite cholesterol-lowering therapy, and 192 normocholesterolaemic controls.ResultsPathogenic mutations were found in 2.1% of hypercholesterolaemic individuals, in 2.2% of subjects on cholesterol-lowering therapy and in 42% of their available first-degree relatives. In addition, variants of uncertain clinical significance (VUCS) were detected in 1.4% of the hypercholesterolaemic and cholesterol-lowering therapy groups. No pathogenic variants or VUCS were detected in controls.ConclusionsWe demonstrated that population-based genetic testing using these protocols is able to deliver definitive molecular diagnoses of FH in individuals with high cholesterol or on cholesterol-lowering therapy. The lower cost and labour associated with NGS-based testing may increase the attractiveness of a population-based approach to FH detection compared to genetic testing with conventional sequencing. This could provide one route to increasing the present low percentage of FH cases with a genetic diagnosis.

Abstract 395: Identification and Characterization of a Novel Mutation in the Low-Density Lipoprotein Receptor Gene in an Omani Arab Family With Familial Hypercholesterolemia

Autosomal dominant familial hypercholesterolemia (FH) is caused due to mutations in the low-density lipoprotein receptor (LDLR), proprotein convertase subtilisin/kexin type 9 (PCSK9) and apolipoprotein B (ApoB) genes. Little is known regarding its prevalence in the Arab population, which not only exhibits relative genetic homogeneity, but also has a lengthy tradition of consanguinity. In this study we screened for mutations in FH associated gene in an Omani Arab family, where FH was detected in the index patients according to the Simon Broome criteria. Effect of the identified mutations on different protein structural levels was investigated in silico. The index patient (9-year-old female) was presented to the lipid clinic with eye xanthelasmata and thickening of both Achilles tendons, and showed off treatment total cholesterol of 896 mg/dL (23.2 mmol/L), low-density lipoprotein cholesterol (LDL-C) of 853 mg/dL (22.1 mmol/L), APOB of 4.5 g/L, triglyceride of 71 mg/dL (0.8 mmol/L), and high-density lipoprotein cholesterol of 0.74 mmol/L. She was treated with a combination of rosuvastatin/ezetimibe and LDL apheresis. Genetic analysis of the LDLR gene showed a homozygous frame-shift deletion mutation (272delG) at exon 3, which truncates the protein after 90 amino acid residues (versus ~840 amino acids in the wild-type). The mode of inheritance of this novel mutation was also studied to construct a family tree. Additionally it was also confirmed that FH in this family is due to mutation only in the LDLR and not PCSK9 and ApoB genes. Further, the effect of the mutation was also appraised in silico on the tertiary structure of LDLR. A model of the mutant LDLR was constructed using the coordinates of the wild-type LDLR extracellular domain. Based on the model, we present a mechanistic justification behind the observed detrimental effect of the mutation on LDL-C levels.

Detecting familial hypercholesterolaemia in the community: Impact of a telephone call from a chemical pathologist to the requesting general practitioner

ObjectiveTo determine whether a telephone call from a chemical pathologist to the requesting general practitioner (GP) of individuals at high risk of familial hypercholesterolaemia (FH) increases specialist referral and detection of FH. MethodIndividuals with an LDL-cholesterol ≥6.5 mmol/L without secondary causes were identified from a community laboratory; 100 cases and 96 historical controls.All laboratory reports (cases and controls) received interpretative comments highlighting FH. In addition, the cases' GPs received a telephone call from the chemical pathologist to highlight their patient's risk of FH and suggest specialist referral, whereas with the controls' GPs were not telephoned. ResultsAfter 12 months follow-up, 27 (27%) cases were referred to clinic compared with 4 (4%) controls (p < 0.0001). 25 cases were reviewed at clinic, 12 (48%) had definite FH and 18 (72%) had probable or definite FH according to the Dutch Lipid Clinic Network Criteria, 2 cases did not attend their clinic appointments. Genetic testing was performed in 23 individuals: 7 (30%) had pathogenic FH mutations. Genotypic cascade screening of 4 kindreds from the intervention group detected an additional 7 individuals with FH and excluded 5 mutation-negative family members. ConclusionsA telephone call from a chemical pathologist to the requesting GP of patients at high risk of FH was associated with significantly higher rates of FH detection and specialist referral. Over 70% of individuals with an LDL-cholesterol ≥6.5 mmol/L were diagnosed with FH. However, further investigation is required to improve the relatively low referral rate.

Use of targeted exome sequencing in genetic diagnosis of Chinese familial hypercholesterolemia.

Familial hypercholesterolemia is an autosomal dominant inherited disease characterized by elevated plasma low-density lipoprotein cholesterol (LDL-C). It is mainly caused by mutations of the low-density lipoprotein receptor (LDLR) gene. Currently, the methods of whole genome sequencing or whole exome sequencing for screening mutations in familial hypercholesterolemia are not applicable in China due to high cost. We performed targeted exome sequencing of 167 genes implicated in the homozygous phenotype of a proband pedigree to identify candidate mutations, validated them in the family of the proband, studied the functions of the mutant protein, and followed up serum lipid levels after treatment. We discovered that exon 9 c.1268 T>C and exon 8 c.1129 T>G compound heterozygous mutations in the LDLR gene in the proband derived from the mother and father, respectively, in which the mutation of c.1129 T>G has not been reported previously. The mutant LDL-R protein had 57% and 52% binding and internalization functions, respectively, compared with that of the wild type. After 6 months of therapy, the LDL-C level of the proband decreased by more than 50% and the LDL-C of the other family members with heterozygous mutation also reduced to normal. Targeted exome sequencing is an effective method for screening mutation genes in familial hypercholesterolemia. The exon 8 and 9 mutations of the LDLR gene were pedigree mutations. The functions of the mutant LDL-R protein were decreased significantly compared with that of the wild type. Simvastatin plus ezetimibe was proven safe and effective in this preschool-age child.

The use of next generation sequencing for detection of mutations in familial hypercholesterolaemia

The use of next generation sequencing for detection of mutations in familial hypercholesterolaemia

Familial hypercholesterolaemia (FH) in Wales is genetically heterogeneous

Familial hypercholesterolaemia (FH) in Wales is genetically heterogeneous

Quality Assessment of the Genetic Test for Familial Hypercholesterolemia in The Netherlands

Introduction. Familial hypercholesterolemia (FH) is an inherited disorder associated with a severely increased risk of cardiovascular disease. Although DNA test results in FH are associated with important medical and ethical consequences, data on accuracy of genetic tests is scarce. Methods. Therefore, we performed a prospective study to assess the overall accuracy of the DNA test used in the genetic cascade screening program for FH in The Netherlands. Individuals aged 18 years and older tested for one of the 5 most prevalent FH mutations, were included consecutively. DNA samples were analyzed by the reference and a counter-expertise laboratory following a standardized procedure. Results. 1003 cases were included. In the end, 317 (32%) carried an FH mutation, whereas in 686 (69%) samples no mutation was found. The overall accuracy of the reference laboratory was 99.8%, with two false positive results identified by the counter-expertise laboratory. Conclusion. The currently used mutation analysis is associated with a very low error rate. Therefore, we do not recommend routine use of duplicate testing.

Genetic Polymorphisms in LDLR, APOB, PCSK9 and Other Lipid Related Genes Associated with Familial Hypercholesterolemia in Malaysia

Familial hypercholesterolemia (FH) is an autosomal dominant disorder characterized by elevations in total cholesterol (TC) and low density lipoprotein cholesterol (LDLc). Development of FH can result in the increase of risk for premature cardiovascular diseases (CVD). FH is primarily caused by genetic variations in Low Density Lipoprotein Receptor (LDLR), Apolipoprotein B (APOB) or Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) genes. Although FH has been extensively studied in the Caucasian population, there are limited reports of FH mutations in the Asian population. We investigated the association of previously reported genetic variants that are involved in lipid regulation in our study cohort. A total of 1536 polymorphisms previously implicated in FH were evaluated in 141 consecutive patients with clinical FH (defined by the Dutch Lipid Clinic Network criteria) and 111 unrelated control subjects without FH using high throughput microarray genotyping platform. Fourteen Single Nucleotide Polymorphisms (SNPs) were found to be significantly associated with FH, eleven with increased FH risk and three with decreased FH risk. Of the eleven SNPs associated with an increased risk of FH, only one SNP was found in the LDLR gene, seven in the APOB gene and three in the PCSK9 gene. SNP rs12720762 in APOB gene is associated with the highest risk of FH (odds ratio 14.78, p<0.001). Amongst the FH cases, 108 out of 141 (76.60%) have had at least one significant risk-associated SNP. Our study adds new information and knowledge on the genetic polymorphisms amongst Asians with FH, which may serve as potential markers in risk prediction and disease management.

Atherosclerosis and Vascular Biology

Atherosclerosis and Vascular Biology

Duplication of exon 7–12 in the low-density lipoprotein receptor gene in three Danish patients with familial hypercholesterolemia

Familial hypercholesterolemia (FH) is one of the most frequent single-gene disorders; nevertheless, it is commonly underdiagnosed and undertreated. To increase the number of individuals diagnosed and treated for FH, an ongoing discovery of novel FH mutations is necessary as a prerequisite to implement good nationwide genetic FH screening strategies. Here we report on the finding of a seldom exon 7-12 duplication in the low-density lipoprotein receptor gene of three Danish patients with FH.

Common mutations of familial hypercholesterolemia patients in Taiwan: Characteristics and implications of migrations from southeast China

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in low-density lipoprotein receptor (LDLR), apolipoprotein B-100 (APOB), and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. This study investigated FH patients carrying common mutations in Taiwan and compared them to FH southeastern Asians. Causal FH mutations were identified by exon-by-exon sequencing with/without multiplex ligation-dependent probe amplification among 208 Taiwanese with clinically diagnosed FH. Haplotype analyses among probands and family members were undertaken using TaqMan® Assays. Totally, LDLR mutations were found in 118 probands, consisting of 61 different loci, and APOB 10579C>T mutations in 12 probands. Three mutations (64delG, 1661C>T, and 2099A>G) were novel. LDLR 986G>A (13.1%), 1747C>T (10.8%), and APOB 10579C>T (9.2%) were common mutations with no differences in phenotypes. LDLR 1747C>T associated with one haplotype (CAAGCCCCATGG/(dTA)n-112nt); LDLR 986G>A with two. APOB 10579C>T associated with the same LDLR binding-domain pattern in Taiwanese and southeastern Asians. We concluded that LDLR 986G>A, 1747C>T and APOB 10579C>T are common mutations, with combined frequency of approximately 33%. The presence of different haplotypes associated with FH common mutations in Taiwan indicates multiple historical migrations, probable multiple recurrent origins from southern China, and haplotype homologies reflect the presence of common ancestors in southern China.

The Arabic allele: A single base pair substitution activates a 10-base downstream cryptic splice acceptor site in exon 12 of LDLR and severely decreases LDLR expression in two unrelated Arab families with familial hypercholesterolemia

Familial hypercholesterolemia (FH) is a monogenic autosomal dominant disorder caused by defects in LDLR. Few reports describe FH mutations among Arabs. We describe a mutation in LDLR of two unrelated Arab families. We investigated 19 patients using DNA sequencing, RFLP, and real-time (RT) PCR. DNA sequencing showed a base pair substitution (c.1706-2 A>T) in the splice acceptor site of LDLR intron 11. Our results were confirmed by RFLP on 2% agarose gel. In silico analysis predicted a new cryptic splice site downstream of the original position generating a 10-base deletion from the beginning of exon 12; (c.1706-1715del.ATCTCCTCAG). cDNA sequencing of exon 12 confirmed the computational analysis. The deletion was visualized on 4% agarose gel. The deletion generates a frameshift and a premature termination codon (c.1991-1993; p.(Asp569Valfs*93). RT-PCR revealed that LDLR mRNA is 9.3% ± 6.5 and 17.9% ± 8.0 for FH homozygote and heterozygote individuals respectively, compared to a healthy family control. We predict a class II LDLR mutation that leads to a truncated receptor missing exons 14–18. We called this mutation “the Arabic allele”. We expect a significant contribution of this mutation to the prevalence of FH among Arabs. Also, we propose that the severe down regulation of LDLR mRNA expression is due to nonsense-mediated-decay

Founder mutations in the Netherlands: geographical distribution of the most prevalent mutations in the low-density lipoprotein receptor and apolipoprotein B genes

BackgroundIn the Netherlands, a screening programme was set up in 1994 in order to identify all patients with familial hypercholesterolaemia (FH). After 15 years of screening, we evaluated the geographical distribution, possible founder effects and clinical phenotype of the 12 most prevalent FH gene mutations.MethodsPatients who carried one of the 12 most prevalent mutations, index cases and those identified between 1994 and 2009 through the screening programme and whose postal code was known were included in the study. Low-density lipoprotein cholesterol (LDL-C) levels at the time of screening were retrieved. The prevalence of identified FH patients in each postal code area was calculated and visualised in different maps.ResultsA total of 10,889 patients were included in the study. Mean untreated LDL-C levels ranged from 4.4 to 6.4 mmol/l. For almost all mutations, a region of high prevalence could be observed. In total, 51 homozygous patients were identified in the Netherlands, of which 13 true homozygous for one of the 12 most prevalent mutations. The majority of them were living in high-prevalence areas for that specific mutation.ConclusionsPhenotypes with regard to LDL-C levels varied between the 12 most prevalent FH mutations. For most of these mutations, a founder effect was observed. Our observations can have implications with regard to the efficiency of molecular screening and physician’s perception of FH and to the understanding of the prevalence and distribution of homozygous patients in the Netherlands.

Development of a high-resolution melting method for mutation detection in familial hypercholesterolaemia patients

Current screening methods, such as single strand conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (dHPLC) that are used for detecting mutations in familial hypercholesterolaemia (FH) subjects are time consuming, costly and only 80-90% sensitive. Here we have tested high-resolution melt (HRM) analysis for mutation detection using the Rotor-Gene(6000) realtime rotary analyser. Methods and subjects Polymerase chain reaction and melt conditions (HRM) for 23 fragments of the LDL-receptor gene, a region of exon 26 in the APOB gene (including p.R3527Q) and exon 7 of the PCSK9 gene (including p.D374Y) were optimized. Two double stranded DNA saturating dyes, LC-Green and Syto9, were compared for sensitivity. Eighty-two samples with known mutations were used as positive controls. Twenty-eight Greek FH heterozygous patients and two homozygous patients from the UK and Croatia were screened. HRM was able to identify all the positive control mutations tested, with similar results with either dye. Eight different variations were found in 17 of the 28 Greek FH patients for an overall detection rate of 61%: c.41delT (1), p.W165X (1), p.C173R (3), p.S286R (2), p.V429M (4), p.G549D (4), p.V613I (1), and a previously unreported mutation p.F694V (1) which is predicted to be FH-causing by functional algorithms. Mutations were found in both the homozygous patients; p.Q92X (Croatia) and p.Y489C (UK); both patients were homozygous for their respective mutations. HRM is a sensitive, robust technique that could significantly reduce the time and cost of screening for mutations in a clinical setting.

Multiplex MassARRAY spectrometry (iPLEX) produces a fast and economical test for 56 familial hypercholesterolaemia‐causing mutations

Familial hypercholesterolaemia (FH) is a common single gene disorder, pre-disposing to cardiovascular disease, which is most commonly caused by mutations in the LDL-receptor (LDLR) gene. About 5% of patients carry the p.R3527Q (previously R3500Q) mutation in the apolipoprotein B (APOB) gene and 2% carry the p.D374Y mutation in the PCSK9 gene, but the lack of high-throughput methods make routine genetic diagnosis difficult. In this study, we developed an iPLEX MassARRAY Spectrometry mutation test to identify 56 mutations (54 in the LDLR gene, 1 in the APOB gene and 1 in the PCSK9 gene). The iPLEX test was verified by analysing 150 DNA samples from FH patients with a previously characterized mutation and 96 no-mutation control samples. Mutations were identified in all 150 FH mutation-positive samples using the iPLEX assay, with 96% directly called by the software. The false-positive rate in no-mutation control samples was 0.015%. The overall specific mutation assay failure rate was 2.1%. In the UK, this gives an average detection rate of 75%.The FH iPLEX test is not only designed for large-scale targeted population screening for FH mutations, such as lipid clinic patients, but can also be used for population screening. The assay can easily be developed further to include additional FH-causing mutations, thus increasing the sensitivity of the diagnostic assay.

Multiplex ARMS analysis to detect 13 common mutations in familial hypercholesterolaemia

DNA analysis and mutation identification is useful for the diagnosis of familial hypercholesterolaemia (FH), particularly in the young and in other situations where clinical diagnosis may be difficult, and enables unambiguous identification of at-risk relatives. Mutation screening of the whole of the three FH-causing genes is costly and time consuming. We have tested the specificity and sensitivity of a recently developed multiplex amplification refractory mutation system assay of 11 low-density lipoprotein receptor gene (LDLR) mutations, one APOB (p.R3527Q) and one PCSK9 (p.D374Y) mutation in 400 patients attending 10 UK lipid clinics. The kit detected a mutation in 54 (14%) patients, and a complete screen of the LDLR gene using single-stranded conformation polymorphism/denaturing high performance liquid chromatography identified 59 different mutations (11 novel) in an additional 87 patients, for an overall detection rate of 35%. The kit correctly identified 38% of all detected mutations by the full screen, with no false-positive or false-negative results. In the patients with a clinical diagnosis of definite FH, the overall detection rate was higher (54/110 = 49%), with the kit detecting 52% of the full-screen mutations. Results can be obtained within a week of sample receipt, and the high detection rate and good specificity make this a useful initial DNA diagnostic test for UK patients.

A modified conformation sensitive gel electrophoresis (CSGE) method for rapid and accurate detection of low density lipoprotein (LDL) receptor gene mutations in Familial Hypercholesterolemia

Objectives: A conformation sensitive gel electrophoresis (CSGE) method was modified for detection of LDL receptor gene mutations. Design and methods: Usage of a temperature gradient against running time was tested with 33 identified mutations in the LDL receptor gene. Most of the mutations were missense. Results: 32 mutations were detected using this method (sensitivity: 97%). The duration required for running the test for each of the exons was reduced to 3–4 h. Conclusions: This modified CSGE method may be used as a screening procedure in genetic diseases where a variety of mutations may cause illness.

Global Defects in the Expression and Function of the Low Density Lipoprotein Receptor (LDLR) Associated with Two Familial Hypercholesterolemia Mutations Resulting in Misfolding of the LDLR Epidermal Growth Factor-AB Pair

The low density lipoprotein (LDL) receptor is a modular protein involved in the endocytosis of cholesterol-rich lipoproteins from the circulation. Mutations to the receptor result in familial hypercholesterolemia, and over 60 of these occur in the calcium-binding epidermal growth factor-like domain pair. Two selected mutations in this region (G322S and R329P) were introduced into the domain pair and analyzed by in vitro refolding. Both exhibited differing levels of protein misfolding with R329P being the most pronounced. Solution NMR studies of the mutant domain pairs after purification established that a fraction of protein maintains a native-like fold and that this fraction contains two intact calcium-binding sites. An in vivo analysis of intact receptors containing these binding sites showed significantly reduced cell-surface expression compared with the native LDL receptor levels, again with R329P showing the most severe decrease. The sum of these results suggests that either local changes in structure or domain misfolding may be associated with the mutations. There is also the possibility that the misfolding of the calcium-binding epidermal growth factor-like pair region is propagated to other regions of the intact receptor, resulting in more global defects. Surprisingly, for both mutants, those full-length receptors that fold and reach the cell surface retain the ability to bind LDL and release the ligand upon exposure to low pH. This analysis provides significant insight into the protein defect resulting from each of the two mutations and allows their classification to be 2B (partially transport-defective). The results also highlight a range of misfolding defects that may be associated with familial hypercholesterolemia and may enable the prediction of the consequences of homologous disease-causing mutations to other proteins.

Simultaneous detection of multiple familial hypercholesterolemia mutations facilitates an improved diagnostic service in South african patients at high risk of cardiovascular disease.

DNA testing can provide a definitive diagnosis of familial hypercholesterolemia (FH), even in the absence of the clinical characteristics of this inherited cardiovascular disease (CVD) subtype. Our aim was to design a rapid diagnostic assay capable of simultaneously analyzing seven point mutations in the low-density lipoprotein receptor (LDLR) gene, which occur at high frequency in South African FH patients. The test is based on multiplex DNA amplification and hybridization to membrane strips presenting a parallel array of immobilized allele-specific oligonucleotide probes. A reverse-hybridization assay for genotyping LDLR point mutations D154N, D200G, D206E, C356Y, G361V, V408M, and P664L was set-up and validated using pretyped human DNA samples, as well as recombinant plasmid clones containing mutant alleles. The procedure is rapid (6 hours) and may be automated to a large extent. The new FH strip-assay forms an important part of the comprehensive cardiovascular genetic screen offered routinely to high-risk population groups in South Africa. A genetic approach based on FH testing in conjunction with other 'genetic' CVD risk factors is feasible and justified, since the spectrum of disease-related mutations have been defined to a large extent in the genetically distinct population groups of South Africa. Knowledge of a significantly increased CVD risk due to the presence of gene variations, which can be targeted for risk reduction by the avoidance of relevant environmental risk factors and the appropriate treatment, provides a powerful message to motivate people into implementing preventative measures based on their genetic profile.