Cause Of Familial Hypercholesterolemia Research Articles

Variant Analysis in LDLR Gene Uncovers Genetic Basis of Familial Hypercholesterolemia: A Case Report

Familial Hypercholesterolemia (FH) is a hereditary disorder characterised by elevated blood cholesterol levels, predominantly Low-density Lipoprotein Cholesterol (LDL-C). This condition poses a significant risk for early-onset atherosclerotic cardiovascular diseases. A critical step toward effective clinical management is the precise identification of pathogenic variants responsible for FH. The present study aimed to unravel the genetic cause of FH through comprehensive variant effect prediction and comparison with clinical manifestations in a nine-year-old girl with hyperlipidemia. Whole Exome Sequencing (WES) was performed on the proband, and a set of three key genes associated with hyperlipidemia {Apolipoprotein E (APOE), Low -density Lipoprotein Receptors (LDLR), Proprotein Convertase Subtilisin /Kexin type 9 (PCSK9)} were evaluated for the presence of pathogenic mutations. The data were meticulously analysed based on the American College of Medical Genetics (ACMG) guidelines for variant classification. The analysis revealed two pathogenic variations in the LDLR gene: c.1A>C (p.Met1Leu) in exon 1 and a splice site variant c.1187-10G>A in intron 8. Sanger sequencing of family members confirmed the presence of one mutation each in the father and mother, while a younger sibling also carried both pathogenic variants. Genetic testing confirmed Heterozygous FH (HeFH) in the parents and Homozygous FH (HoFH) in both siblings. Proper classification of genetic variants is crucial for informed clinical decision-making and patient management. The study provides valuable insights into the molecular basis of FH in an Indian patient and contributes to the growing knowledge of the LDLR gene mutation spectrum.

Familial Hypercholesterolemia: A Literature Review of the Pathophysiology and Current and Novel Treatments.

Familial hypercholesterolemia (FH) is a genetically transmitted disorder. It shows an autosomal dominant mode of inheritance. It is a metabolic disorder. Mutation in chromosome 19 leads to this disorder. Chromosome 19 codes data for low-density lipoprotein (LDL) receptor (LDLR). LDLR either reduces increased LDL levels from the circulation or maintains a normal level of LDL. It precipitates the risk of cardiovascular disease at an early age. Characteristic features of FH are elevated levels of LDL in the blood because of sudden changes in LDLR, which causes a decrease in the clearance of LDL from the blood. Plaque gets deposited in the lumen of the arteries, called atherosclerosis, which occurs at an early young age. If both genes are affected then it is homozygous FH (HoFH); such a case is very rare. When a single gene is affected then that condition is known as heterozygous FH (HeFH). HoFH comes up with more severe cardiac disease than HeFH at an early age. The major cause of FH is a mutation in the LDLR gene while other causes include mutation in various genes like apolipoprotein B (apo B), proprotein convertase subtilisin/kexin type 9 (PCSK9), LDLR adaptor protein 1 (LDLRAP 1). In order to prevent cardiovascular crises due to FH, it must be diagnosed early and treated effectively. With increasing research and advances in medical sciences, many kinds of current and novel therapies are emerging that aim to reduce the level of LDL in blood.

Identification of c.683_684insCTGCAAGGACAAATCTGACGA pathogenic variant of the low-density lipoprotein receptor gene in a patient with familial hypercholesterolemia: a case report

Familial hypercholesterolemia (FH) is one of the most common monogenic diseases that leads to the early development of atherosclerosis and is characterized by a poor prognosis. However, only about 1% of FH cases are diagnosed in Russia. The aim of this study was to determine the genetic defect in the FH family and conduct DNA diagnostics in the proband relatives. The study was performed on blood samples obtained with the informed consent of the patients. Polymerase chain reaction and polyacrylamide gel electrophoresis were used. We report c.683_684insCTGCAAGGA CAAATCTGACGA pathogenic variant of the low-density lipoprotein receptor (LDLR) gene for the first time in Russia and demonstrate its cosegregation in a family with high blood cholesterol. The c.683_684in sCTGCAAGGACAAATCTGACGA insertion is considered as a probable cause of FH.

PCSK9 in extrahepatic tissues: What can we expect from its inhibition?

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme that belongs to the serine protease family and plays a key role in regulating low-density lipoprotein cholesterol (LDL-C) levels in the blood. PCSK9 binds to the LDL receptor (LDLR), targeting it for degradation, resulting in an increase in circulating LDL-C levels. Loss-of-function mutations in the PCSK9 gene are associated with lower LDL-C levels and lower cardiovascular risk; in contrast, gain-of-function mutations are a cause of familial hypercholesterolaemia. The identification of PCSK9 as a pharmacological target led to the development of inhibitors for the treatment of hypercholesterolaemia. To date, the monoclonal antibodies evolocumab and alirocumab (which target plasma PCSK9) and the small-interfering RNA inclisiran (which targets hepatic PCSK9 mRNA) have been approved for the treatment of hypercholesterolaemia. Although hepatic PCSK9 plays a central role in regulating plasma LDL-C levels, this protein is also expressed in other tissues, including the brain, pancreas, heart, kidney, intestine and adipose tissue. In extrahepatic tissues, the functions of PCSK9 are both dependent and independent of LDLR and not necessarily harmful. For this reason, it is essential to uncover any potentially harmful effects of therapies that inhibit PCSK9, beyond their known LDL-C-lowering and CV risk-reducing effects.

An integration-free iPSC line ZZUNEUi029-A derived from peripheral blood mononuclear cells of a patient with familial hypercholesterolemia carrying a mutation in LDLR gene

Familial hypercholesterolemia is a hereditary disorder that causes severely elevated low-density lipoprotein levels, which can lead to an increased risk for premature cardiovascular disease. Mutations in the LDLR gene are the most common cause of familial hypercholesterolemia. In this study, we report the generation of ZZUNEUi029-A, a human induced pluripotent stem cell line (hiPSC) from a male patient with c. 622 G → A in LDLR gene using non-integrative Sendai viral reprogramming technology. This cell line expressed pluripotency markers, had a normal male karyotype (46XY) and maintained the ability to differentiate into the three germ layers in vitro.

Age, Origin and Functional Study of the Prevalent LDLR Mutation Causing Familial Hypercholesterolaemia in Gran Canaria.

The p.(Tyr400_Phe402del) mutation in the LDL receptor (LDLR) gene is the most frequent cause of familial hypercholesterolaemia (FH) in Gran Canaria. The aim of this study was to determine the age and origin of this prevalent founder mutation and to explore its functional consequences. For this purpose, we obtained the haplotypic information of 14 microsatellite loci surrounding the mutation in one homozygous individual and 11 unrelated heterozygous family trios. Eight different mutation carrier haplotypes were identified, which were estimated to originate from a common ancestral haplotype 387 (110-1572) years ago. This estimation suggests that this mutation happened after the Spanish colonisation of the Canary Islands, which took place during the fifteenth century. Comprehensive functional studies of this mutation showed that the expressed LDL receptor was retained in the endoplasmic reticulum, preventing its migration to the cell surface, thus allowing us to classify this LDLR mutation as a class 2a, defective, pathogenic variant.

Possible explanations for the common clinical familial hypercholesterolemia phenotypes in the Faroe Islands

The prevalence of clinical familial hypercholesterolemia (FH) is very high in the Faroe Islands, but the possible causes are unknown. We aimed to describe potential genetic causes of FH in the Faroe Islands and to investigate whether levels of lipoprotein(a) and measures of dietary habits were associated with clinical FH in the Faroe Islands. In this case-control study, we identified potential clinical FH cases aged 18-75 years registered within a nationwide clinical laboratory database in the Faroe Islands and invited them for diagnostic evaluation according to clinical FH scoring systems. Controls were identified in the background population. Lipoprotein(a) was measured in plasma, while the fatty acid composition was determined in adipose tissue. The habitual diet of the participants was assessed using a food frequency questionnaire. Genetic testing for FH and polygenic variants was performed in a selection of clinical FH cases. A total of 121 clinical FH cases and 123 age- and sex-matched controls were recruited. We found a very low frequency of monogenic FH (2.5%), but a high level of polygenic FH (63%) in those genetically tested (67%). High levels of plasma lipoprotein(a) were associated with high odds of clinical FH. Clinical FH cases had a lower intake of saturated fatty acids (SFAs) measured by a high fat-score and a lower content of SFAs in adipose tissue compared with controls. The high prevalence of FH in the Faroe Islands may be due to polygenic causes of hypercholesterolemia and to a lesser extent other genetic factors and elevated plasma lipoprotein(a) levels.

Identification and Functional Analysis of APOB Variants in a Cohort of Hypercholesterolemic Patients.

Mutations in APOB are the second most frequent cause of familial hypercholesterolemia (FH). APOB is highly polymorphic, and many variants are benign or of uncertain significance, so functional analysis is necessary to ascertain their pathogenicity. Our aim was to identify and characterize APOB variants in patients with hypercholesterolemia. Index patients (n = 825) with clinically suspected FH were analyzed using next-generation sequencing. In total, 40% of the patients presented a variant in LDLR, APOB, PCSK9 or LDLRAP1, with 12% of the variants in APOB. These variants showed frequencies in the general population lower than 0.5% and were classified as damaging and/or probably damaging by 3 or more predictors of pathogenicity. The variants c.10030A>G;p.(Lys3344Glu) and c.11401T>A;p.(Ser3801Thr) were characterized. The p.(Lys3344Glu) variant co-segregated with high low-density lipoprotein (LDL)-cholesterol in 2 families studied. LDL isolated from apoB p.(Lys3344Glu) heterozygous patients showed reduced ability to compete with fluorescently-labelled LDL for cellular binding and uptake compared with control LDL and was markedly deficient in supporting U937 cell proliferation. LDL that was carrying apoB p.(Ser3801Thr) was not defective in competing with control LDL for cellular binding and uptake. We conclude that the apoB p.(Lys3344Glu) variant is defective in the interaction with the LDL receptor and is causative of FH, whereas the apoB p.(Ser3801Thr) variant is benign.

Opportunistic Genetic Screening for Familial Hypercholesterolemia in Heart Transplant Patients.

Heart transplantation remains the gold standard for the treatment of advanced heart failure (HF). Identification of the etiology of HF is mandatory, as the specific pathology can determine subsequent treatment. Early identification of familial hypercholesterolemia (FH), the most common genetic disorder associated with premature cardiovascular disease, has a potential important impact on clinical management and public health. We evaluated the genetic information in the genes associated with FH in a cohort of 140 heart-transplanted patients. All patients underwent NGS genetic testing including LDLR, APOB, and PCSK9. We identified four carriers of rare pathogenic variants in LDLR and APOB. Although all four identified carriers had dyslipidemia, only the one carrying the pathogenic variant LDLR c.676T>C was transplanted due to CAD. Another patient with heart valvular disease was carrier of the controversial LDLR c.2096C>T. Two additional patients with non-ischemic dilated cardiomyopathy were carriers of variants in APOB (c.4672A>G and c.5600G>A). In our cohort, we identified the genetic cause of FH in patients that otherwise would not have been diagnosed. Opportunistic genetic testing for FH provides important information to perform personalized medicine and risk stratification not only for patients but also for relatives at concealed high cardiovascular risk. Including the LDLR gene in standard NGS cardiovascular diagnostics panels should be considered.

Large-Scale CRISPR Screen of LDLR Pathogenic Variants.

Familial hypercholesterolemia (FH) is a frequently occurring genetic disorder that is linked to early-onset cardiovascular disease. If left untreated, patients with this condition can develop severe cardiovascular complications. Unfortunately, many patients remain undiagnosed, and even when diagnosed, the treatment is often not optimal. Although mutations in the LDLR gene are the primary cause of FH, predicting whether novel variants are pathogenic is not a straightforward task. Understanding the functionality of LDLR variants is crucial in uncovering the genetic basis of FH. Our study utilized CRISPR/Cas9 cytosine base editors in pooled screens to establish a novel approach for functionally assessing tens of thousands of LDLR variants on a large scale. A total of more than 100 single guide RNAs (sgRNAs) targeting LDLR pathogenic mutations were successfully screened with relatively high accuracy. Out of these, 5 sgRNAs were further subjected to functional verification studies, including 1 in the promoter, 1 in the antisense RNA, 1 in the exon, and 2 in the intron. Except for the variant caused by the sgRNA located at intron 16, the functionalities of the other LDLR variants were all downregulated. The high similarity of LDLR intron sequences may lead to some false positives. Overall, these results confirm the reliability of the large-scale screening strategy for functional analysis of LDLR variants, and the screened candidate pathogenic mutations could be used as an auxiliary means of clinical gene detection to prevent FH-induced heart disease.

Subclinical atherosclerosis determined by coronary artery calcium deposition in patients with clinical familial hypercholesterolemia

Background and aimsLimited knowledge exists regarding the association between coronary artery calcium (CAC) deposition in patients with clinical familial hypercholesterolemia (FH) and FH subtypes such as polygenic causes. We studied CAC score in patients with clinical FH and subtypes including polygenic causes of FH compared to healthy controls. MethodsIn a case-control study, we identified potential clinical FH cases registered with an LDL-C >6.7 mmol/l within a nationwide clinical laboratory database on the Faroe Islands and invited them for diagnostic evaluation according to clinical FH scoring systems. Controls were identified in the background population. All subjects were aged 18–75 years and without a history of cardiovascular disease. FH mutation testing and genotypes of twelve LDL-C associated single nucleotide polymorphisms were determined using conventional methods in selected individuals. CAC scores were assessed by cardiac CT. Odds ratios obtained using multivariate logistic regression were used as measures of association. ResultsA total of 120 clinical FH patients and 117 age- and sex-matched controls were recruited. We found a very low frequency of monogenic FH (3%), but a high level of polygenic FH (60%) in those genetically tested (54%). There was a statistically significant association between the CAC score and a diagnosis of clinical FH with the highest observed odds ratio of 5.59 (95% CI 1.65; 18.94, p = 0.006) in those with a CAC score ≥300 compared to those with a CAC of zero. In supplemental analyses, there was a strong association between CAC scores and clinical FH of a polygenic cause. ConclusionWe found a statistically significant association between CAC levels and clinical FH with the highest observed risk estimates among clinical FH cases of a presumed polygenic cause.

Cold shock domain-containing protein E1 is a posttranscriptional regulator of the LDL receptor.

The low-density lipoprotein receptor (LDLR) controls cellular delivery of cholesterol and clears LDL from the bloodstream, protecting against atherosclerotic heart disease, the leading cause of death in the United States. We therefore sought to identify regulators of the LDLR beyond the targets of current therapies and known causes of familial hypercholesterolemia. We found that cold shock domain-containing protein E1 (CSDE1) enhanced hepatic LDLR messenger RNA (mRNA) decay via its 3' untranslated region and regulated atherogenic lipoproteins in vivo. Using parallel phenotypic genome-wide CRISPR interference screens in a tissue culture model, we identified 40 specific regulators of the LDLR that were not previously identified by observational human genetic studies. Among these, we demonstrated that, in HepG2 cells, CSDE1 regulated the LDLR at least as strongly as statins and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors. In addition, we showed that hepatic gene silencing of Csde1 treated diet-induced dyslipidemia in mice to a similar degree as Pcsk9 silencing. These results suggest the therapeutic potential of targeting CSDE1 to manipulate the posttranscriptional regulation of the LDLR mRNA for the prevention of cardiovascular disease. Our approach of modeling a clinically relevant phenotype in a forward genetic screen, followed by mechanistic pharmacologic dissection and in vivo validation, may serve as a generalizable template for the identification of therapeutic targets in other human disease states.

A suitable sequence of bioinformatics tools for NGS diagnosis of familial hypercholesterolemia

Background and Aims : Familial hypercholesterolemia (FH) is the most common monogenic congenital metabolic disease with an estimated frequency of 1: 200 - 500 in the population. Aberrations of the LDL receptor gene (LDLR) or mutation in his ligand apolipoprotein B100 (FDB) are the most common cause of FH. To date, over 2000 different mutations have been described in the LDLR. More rarely the pathology could be detected in other genes (APOB, LDLRAP , PCSK9, etc.). Early diagnosis using molecular genetic methods enables appropriate treatment and belongs to the basic pillars of routine practise. Considering various types of possible mutations, next - generation sequencing (NGS) method using hybridization-based enriched libraries can provide a potent solution for required multilevel genetic analysis. Each level of data analysis requires specific bioinformatic tool although the sensitivity to detect certain types of variants may partially overlap.Methods: We present three bioinformatic tools (FreeBayes, Manta and CNVkit) as an example of suitable unique combination of optimal NGS data analysis pipeline - specifically for the custom Hyperlipoproteinemia 1 (HLP) SureSelect library panel (Agilent Technologies) covering 53 HLP genes and 55 SNPs.Results: The spectrum of causal mutations detected in our FH and HLP patients cohort were as follows: 81% of single-nucleotide variants, 5% of minor and 14% of gross (two and more exons) intragenic rearrangements. Mutations in LDLR covered 74,4% of causal variants, 11,6% of mutations were in APOB and 14% in another genes from the HLP panel.Conclusions: The presented combination of bioinformatic tools represents effective diagnostic algorithm with increase diagnostic yield.*This work was supported by the Cooperatio Program, research area "Metabolic Diseases 207037" Background and Aims : Familial hypercholesterolemia (FH) is the most common monogenic congenital metabolic disease with an estimated frequency of 1: 200 - 500 in the population. Aberrations of the LDL receptor gene (LDLR) or mutation in his ligand apolipoprotein B100 (FDB) are the most common cause of FH. To date, over 2000 different mutations have been described in the LDLR. More rarely the pathology could be detected in other genes (APOB, LDLRAP , PCSK9, etc.). Early diagnosis using molecular genetic methods enables appropriate treatment and belongs to the basic pillars of routine practise. Considering various types of possible mutations, next - generation sequencing (NGS) method using hybridization-based enriched libraries can provide a potent solution for required multilevel genetic analysis. Each level of data analysis requires specific bioinformatic tool although the sensitivity to detect certain types of variants may partially overlap. Methods: We present three bioinformatic tools (FreeBayes, Manta and CNVkit) as an example of suitable unique combination of optimal NGS data analysis pipeline - specifically for the custom Hyperlipoproteinemia 1 (HLP) SureSelect library panel (Agilent Technologies) covering 53 HLP genes and 55 SNPs. Results: The spectrum of causal mutations detected in our FH and HLP patients cohort were as follows: 81% of single-nucleotide variants, 5% of minor and 14% of gross (two and more exons) intragenic rearrangements. Mutations in LDLR covered 74,4% of causal variants, 11,6% of mutations were in APOB and 14% in another genes from the HLP panel. Conclusions: The presented combination of bioinformatic tools represents effective diagnostic algorithm with increase diagnostic yield. *This work was supported by the Cooperatio Program, research area "Metabolic Diseases 207037"

Evidence of novel APO B gene complex allele causing familial hypercholesterolaemia

Background and Aims : APOB variants are a rare cause of familial hypercholesterolaemia (FH). Most of APOB variants remain of uncertain significance (VUS) for the absence of functional studies. We aim to characterize a novel APOB complex allele causing a severe form of heterozygous FH.Methods: Two unrelated patients with clinical suspect of heterozygous FH were screened by next-generation sequencing (NGS). Variant confirmation and relative analysis were done by direct sequencing. Variant pathogenicity was assessed by American College of Medical Genetics and Genomics (ACMG) guidelines. Functional characterization was carried-out evaluating the proliferation of U937 cells incubated with patient or control plasma or measuring LDL uptake on a hepatoma cell line. Solid-phase immunoassay was used to evaluate the affinity of variant APOB for LDLR.Results: In both patients, sequencing identified 3 rare variants at heterozygous status in the exon 21 and 26 of APOB gene: c.3220G>A - p.(Gly1074Arg), c.10031A>T - p.(Lys3344Ile) and c.11087T>C – (p.Ile3696Thr). Family analysis showed that 1. the variants were on the same allele; 2. relatives carrying the variants were hypercholesterolemic. The proliferation assay revealed a decreased cell growth (<65% of wild-type). Results were confirmed by decreased uptake of patient’s LDL (<25% of control) and by the increased EC50 of patient’s LDL for LDLR (approximately four times greater than wild-type).Conclusions: A complex APOB allele was identified and functionally characterized as affecting the binding of APOB to LDLR. NGS allows to analyze all FH-causing genes improving the variant detection. Background and Aims : APOB variants are a rare cause of familial hypercholesterolaemia (FH). Most of APOB variants remain of uncertain significance (VUS) for the absence of functional studies. We aim to characterize a novel APOB complex allele causing a severe form of heterozygous FH. Methods: Two unrelated patients with clinical suspect of heterozygous FH were screened by next-generation sequencing (NGS). Variant confirmation and relative analysis were done by direct sequencing. Variant pathogenicity was assessed by American College of Medical Genetics and Genomics (ACMG) guidelines. Functional characterization was carried-out evaluating the proliferation of U937 cells incubated with patient or control plasma or measuring LDL uptake on a hepatoma cell line. Solid-phase immunoassay was used to evaluate the affinity of variant APOB for LDLR. Results: In both patients, sequencing identified 3 rare variants at heterozygous status in the exon 21 and 26 of APOB gene: c.3220G>A - p.(Gly1074Arg), c.10031A>T - p.(Lys3344Ile) and c.11087T>C – (p.Ile3696Thr). Family analysis showed that 1. the variants were on the same allele; 2. relatives carrying the variants were hypercholesterolemic. The proliferation assay revealed a decreased cell growth (<65% of wild-type). Results were confirmed by decreased uptake of patient’s LDL (<25% of control) and by the increased EC50 of patient’s LDL for LDLR (approximately four times greater than wild-type). Conclusions: A complex APOB allele was identified and functionally characterized as affecting the binding of APOB to LDLR. NGS allows to analyze all FH-causing genes improving the variant detection.

Familial Hypercholesterolemia: Real-World Data of 1236 Patients Attending a Czech Lipid Clinic. A Retrospective Analysis of Experience in More than 50 years. Part I: Genetics and Biochemical Parameters

Introduction: The cause of familial hypercholesterolemia (FH) is defect in LDL receptor or familial defect of apolipoprotein B-100 (FDB) or, rarely, defect in proprotein convertase subtilisin/kexin type 9. Identification and treatment of patients with FH improves their prognosis. Our data represent retrospective analysis of 50 years of specialised care in our center. Patients and Methods: A group of 1236 FH patients (841 women, 395 men; 993 study subjects and 243 relatives; mean age 44.8 ± 16.7 years) included 154 FDB patients followed at the Lipid Clinic of the General University Hospital in Prague since the mid-1960s to the present. Clinical diagnosis was based on the Dutch Lipid Clinic Network Criteria. Genetic analysis was performed using PCR-RFLP to detect FDB and apolipoprotein E (APOE) polymorphism. Biochemical data were collected and statistically analysed. Results: At baseline, mean LDL-C and total cholesterol (TC) levels of all FH patients combined were 6.49 ± 1.92 mmol/L and 8.95 ± 1.95 mmol/L, respectively. Their LDL-C levels decreased to 3.26 ± 1.57 mmol/L and TC levels to 5.43 ± 1.69 mmol/L during follow-up. In the subgroup of LDL receptor-mediated FH (non-FDB) patients, baseline LDL-C and TC levels of 6.61 ± 1.95 mmol/L and 9.09 ± 1.97 mmol/L declined to 3.21 ± 1.60 mmol/L and 5.39 ± 1.72 mmol/L, respectively, during follow-up. In the FDB subgroup of patients, baseline levels of LDL-C and TC were 5.57 ± 1.46 mmol/L and 7.88 ± 1.58 mmol/L decreasing to 3.45 ± 0.24 mmol/L and 5.58 ± 1.37 mmol/L, respectively, during follow-up. Differences were also found in the effects of various APOE isoforms on lipid lowering. A significant decrease in lipid parameters was observed with the E2E2 isoform whereas a minimal decrease was seen with the E4E4 and E3E3 isoforms. Conclusion: Whereas, overall, non-FDB patients had higher baseline lipid levels, these levels declined more appreciably compared with FDB patients during follow-up. Our retrospective analysis also found different effects of APOE isoforms on the decrease in lipid levels.

Sequencing the Exon.4 of the LDL Receptor Gene in Patients with Familial Hypercholesterolemia in the Population of Bushehr, Southwestern Iran: the Possible New Mutations

Background &amp; Objective: Familial hypercholesterolemia (FH) is the most common genetic disease in the world and an autosomal dominant disease characterized by increased plasma cholesterol and low-density lipoprotein (LDL) concentrations. The clinical diagnosis of the disease is based on family history, the findings of medical examinations, and the measurement of cholesterol levels. The most important cause of FH is the mutation in one of the LDL-R, APOB, and PSCK9 genes. About 90% of mutations occur in the LDL-R gene, which accounts for a total of 2,000 different mutations. Different types of mutations have been observed on different exons of the LDL-R gene, but most of the mutations have been reported on Exon 4. The aim of this study was to sequence and analyze Exon 4 in patients with familial hypercholesterolemia in Bushehr province in Iran.&#x0D; Materials &amp; Methods: In this study, 32 patients were selected based on global criteria for diagnosing the disease, and a portion of LDL-R containing complete sequence of exon 4 was amplified using LDLRE4F1/ LDLRE4R1 Primers and blood genomic DNA as a template. PCR products were sequenced and compared with reference sequence to find probable muta­tions.&#x0D; Results: The results of sequencing and comparison with the reference sequence showed that no mutation was found in the exon 4 LDL-R gene. Therefore, this exon did not play a role in FH in the population under study.&#x0D; Conclusion: Therefore, the cause of FH may be due to the mutations in other areas of the LDL-R gene or other genes, such as APOB and PSCK9.

RNA Studies Demonstrate that the LDLR c.2389+4A>G Intronic Variant Causes Familial Hypercholesterolemia through an RNA Splicing Impact

Lead Author's Financial Disclosures H.Z. is an employee of Ambry Genetics. Study Funding None. Background/Synopsis Familial hypercholesterolemia (FH) is an inherited disorder characterized by high plasma levels of low-density lipoprotein cholesterol (LDL-C). Mutations in the low-density lipoprotein receptor (LDLR) gene are the most common cause of FH. However, not all alterations in LDLR are pathogenic; some represent benign genetic variation, and the clinical significance of any given variant is not always clear. Variants of uncertain significance (VUS) require additional studies to elucidate their pathogenicity. Objective/Purpose We sought to further characterize an intronic LDLR alteration originally classified as a VUS in a large family with clinical FH. Methods We performed multigene panel testing for four genes associated with FH (APOB, LDLR, LDLRAP1, and PCSK9) on the proband of a large family with a strong family history of high cholesterol and premature death; additional affected and unaffected family members were genotyped by Sanger sequencing for an identified intronic LDLR variant. The splicing impact of the LDLR variant was ascertained by RT-PCRseq performed on three affected individuals. Results Panel testing detected LDLR c.2389+4A>G in the proband. No other pathogenic variants, likely pathogenic variants, or VUSs were identified. Subsequent familial testing indicated that LDLR c.2389+4A>G cosegregates with disease. RNA studies demonstrated that this alteration causes skipping of exon 16, which is predicted to result in the in-frame deletion of 26 amino acids. This deletion removes part of the transmembrane domain and is expected to disrupt the ability of LDLR to insert into the membrane, resulting in loss of receptor function. Other pathogenic alterations impacting the same donor splice site have been shown to have a similar impact on splicing (LDLR c.2389G>T and LDLR c.2389+1G>T; Bourbon M et al. J. Med. Genet., 2009 May;46:352-7; Holla ØL et al. Mol. Genet. Metab., 2009 Apr;96:245-52). Conclusions The LDLR c.2389+4A>G intronic variant is a pathogenic mutation that results in a splice defect and cosegregates with disease. In this case, RNA and family studies resulted in a reclassification from a VUS to a pathogenic mutation and confirmed the molecular cause of the multigenerational FH. Clinical management recommendations exist for individuals with a confirmed FH diagnosis, and an accurate genetic diagnosis can improve patient outcomes through tailored treatments (Brown E et al. J. Clin. Lipidol.,2020 Jul-Aug;14:398-413). Our findings highlight the utility of familial testing and RNA studies in clarifying the classification of DNA variants. H.Z. is an employee of Ambry Genetics.

MLb-LDLr: A Machine Learning Model for Predicting the Pathogenicity of LDLr Missense Variants

Untreated familial hypercholesterolemia (FH) leads to atherosclerosis and early cardiovascular disease. Mutations in the low-density lipoprotein receptor (LDLr) gene constitute the major cause of FH, and the high number of mutations already described in the LDLr makes necessary cascade screening or invitro functional characterization to provide a definitive diagnosis. Implementation of high-predicting capacity software constitutes a valuable approach for assessing pathogenicity of LDLr variants to help in the early diagnosis and management of FH disease. This work provides a reliable machine learning model to accurately predict the pathogenicity of LDLr missense variants with specificity of 92.5% and sensitivity of 91.6%.

Splice correction therapies for familial hypercholesterolemic patients with low-density lipoprotein receptor mutations.

Purpose of reviewAntisense oligomers (ASOs) have been available for decades: however, only recently have these molecules been applied clinically. This review aims to discuss the possible development of antisense-mediated splice correction therapies as precision medicines for familial hypercholesterolemic patients carrying mutations that compromise normal splicing of the low-density lipoprotein receptor (LDLR) gene transcript.Recent findingsThree antisense drugs are currently being assessed in ongoing clinical trials for dyslipidemias, aiming to lower the plasma concentrations of lipoproteins that lead to end-organ damage, principally coronary artery disease. Although a handful of drugs may be applicable to many patients with familial hypercholesterolemia (FH), mutation-specific personalised antisense drugs may be even more effective in selected patients. Currently, there is no therapy that effectively addresses mutations in the LDLR, the major cause of FH. Many mutations in the LDLR that disrupt normal pre-mRNA processing could be applicable to splice correction therapy to restore receptor activity.SummaryPrecision medicine could provide long-term economic and social benefits if they can be implemented effectively and sustainably. Many mutations found in the LDLR gene could be amendable to therapeutic splice correction and we should consider developing a therapeutic ASO platform for these mutations.

Genetic Analysis in a Taiwanese Cohort of 750 Index Patients with Clinically Diagnosed Familial Hypercholesterolemia

Aim: Familial hypercholesterolemia (FH) is underdiagnosed in most countries. The genetic heterogeneity of FH requires an algorithm to efficiently integrate genetic testing into clinical practice. We aimed to report the spectrum of genetic mutations from patients with clinically diagnosed FH in Taiwan. Methods: Patients with LDL-C＞190 mg/dL or those with probable or definite FH according to the Taiwan Lipid Guidelines underwent genetic testing. Samples from 750 index patients from the Taiwan FH registry were screened using custom-made mass spectrometry, followed by targeted next generation sequencing (NGS) and/or multiplex ligation-dependent probe amplification (MLPA) if found negative. Results: The mean age of the patients was 52.4±15.1 years and 40.9% were male. Mutations were detected in 445 patients (59.3%). The distribution of mutations was as follows:LDLR (n=395),APOB (n=58),PCSK9 (n=0), andABCG5 (n=3). The most common mutations wereAPOB c.10579 C＞T (p.R3527W) (12.6%),LDLR c.986 G＞A (p.C329Y) (11.5%), andLDLR c.1747 C＞T (p.H583Y) (10.8%).LDLR c.1187-10 G＞A (IVS 8-10) andAPOB c.10580 G＞A (p.R3527Q) were detected using targeted NGS in Taiwan for the first time. Four novel mutations were identified, includingLDLR c.1060＋2 T＞C (IVS 7＋2),LDLR c.1139 A＞C (p.E380A),LDLR c.1322 T＞C (p.A431T)＋c.1867 A＞G (p.I623V), and ABCG5 c.1337 G＞A (p.R447Q). Conclusion: LDLR andAPOB, but notPCSK9, mutations were the major genetic causes of FH. Four novel mutations inLDLR orABCG5 were identified. This genetic screening method using mass spectrometry, targeted NGS, and MLPA analysis provided an efficient algorithm for genetic testing for clinically diagnosed FH in Taiwan.