Familial Defective Apolipoprotein B-100 Research Articles

Assessing the Attitudes and Perceptions of the Old Order Amish toward Genetic Testing for Familial Defective Apolipoprotein B-100

Through a founder effect, one in eight Lancaster County, PA, Amish harbors the pathogenic APOB variant R3527Q, causing Familial Defective Apolipoprotein B-100 (FDB) that is rare in the general population. Affected individuals are at increased risk for high low density lipoprotein (LDL) cholesterol levels and cardiovascular disease and may benefit from early screening and treatment. A qualitative interview approach was used to investigate the genetic risk perceptions of the Amish and their attitudes towards genetic testing for FDB. The goal was to identify elements necessary for a successful program that would identify high risk individuals in the Lancaster County Amish community and provide them with potentially lifesaving education and interventions. Thirty interviews were conducted. Collected data were analyzed and coded for common themes. Participants perceived a great deal of importance in genetic risk and were aware of the significant impact genetics have on health. Most individuals perceived genetic risk as more significant than family history. Twenty-four of 30 individuals would consider testing; of these, all 24 reported they would take proactive measures to reduce risks if given a positive result. Most would prefer diet modification and natural health remedies, with prescription medication only as a last resort. Several participants stated that most Amish people would be unlikely to test if they were feeling healthy. Cost was the most significant barrier to pursuing testing. Implementing a successful screening program would require community outreach and awareness, risk reduction education and support, and minimal out-of-pocket expenses. The program’s success provides insights into the efficacy of predictive genetic screening for the general population. [Abstract by authors.]

Abstract 12043: The Genetic Variant P.(Lys3344Glu) in APOB Causes Familial Hypercholesterolemia

Introduction: Genetic variation in the APOB gene is frequent but the pathogenicity of most of the variations is unknown. Only the variant in APOB p.(Arg3527Gln), known as familial defective apoB100, unequivocally produces familial hypercholesterolemia (FH). Hypothesis: A new variant in APOB responsible for HF in three unrelated Spanish families is presented. Its pathogenesis has been demonstrated by family segregation and apoB-LDL receptor binding analysis. Methods: The three probands correspond to two women and one man referred for study of their hypercholesterolemia at the Lipid Unit of the Miguel Servet Hospital in Zaragoza, Spain. Secondary causes were ruled out and family history showed a dominant segregation of hypercholesterolemia in their families. Their clinical characteristics are described in the table. Results: Sequencing analysis of the LDLR, APOB, PCSK9 , and APOE genes showed the c.10030A>G; p.(Lys3344Glu) variant. No other pathogenic mutations were found.The family study included 14 first-degree relatives, with 8 of them being carriers of the APOB variant with a mean age of 42.5 years. The mean (range) of LDLc was 309 mg/dL (254-419) in carriers and 147 (98-179) in unaffected relatives. The apoB-LDL receptor binding study of the serum of the 3 probands (EC50) showed a reduction equivalent to that obtained with apoB p.(Arg3527Gln), and approximately 50% lower than that obtained in control serum. Conclusions: The p.(Lys3344Glu) variant in APOB is causal for FH and its mechanism is a defect in the affinity of the defective protein for the LDL receptor.

Familial Hypercholesterolemia: Real-World Data of 1236 Patients Attending a Czech Lipid Clinic. A Retrospective Analysis of Experience in More than 50 years. Part II. Clinical Characteristics

Introduction: Patients with familial hypercholesterolemia (FH) are at increased risk of premature atherosclerotic cardiovascular disease (ASCVD). Aim of study: To perform a retrospective analysis of data to assess the effects of individual lipoproteins and other risk factors (RFs) on the development of ASCVD and to compare these parameters in individuals with versus without ASCVD. Patients and methods: Our study group included a total of 1,236 patients with FH (395 men and 841 women with a mean age of 44.8 ± 16.7 years) attending a single lipid clinic. The diagnosis of FH was established using the Dutch Lipid Clinic Network score (DLCN). Among the 1236 FH patients, 1,008 of them [854 suspected with LDL receptor-mediated FH and 154 with familial defective apolipoprotein B-100 (FDB)] were genetically analysed. Their RFs were assessed based on the patients’ clinical characteristics. Results: While patients with ASCVD had higher baseline LDL-C, TC, TG and Lp(a) compared with patients without this diagnosis, this ratio was just the opposite by the follow-up. The highest statistically significant differences were seen in the baseline levels of Lp(a) and, quite surprisingly, TG. Except for Lp(a), the levels of all lipid parameters declined significantly over time. While the incidence of diabetes and arterial hypertension was not higher in our group compared with the general population, these patients were at a more significant risk of ASCVD. Conclusion: Familial hypercholesterolemia is a major RF for the development of ASCVD. While our analysis confirmed the important role of LDL-C, it also corroborated a strong correlation between ASCVD and other lipid parameters, and Lp(a) and TG in particular. Familial hypercholesterolemia is not the only RF and, to reduce cardiovascular risk of their patients, physicians have to search for other potential RFs. Patients diagnosed to have FH benefit from attending a specialized lipid clinic perse.

Familial defective apolipoprotein B-100 – the real world data of the group of patients from the center of preventive cardiology

Background and Aims: Atherosclerotic cardiovascular diseases (ASCVD) play the most important role in mortality of Czech patients. The only one causal risk factor of atherosclerosis is LDL-cholesterol (LDL-C), which is enhanced by genetic predispositions causing defects in LDL receptor, apolipoprotein B or PCSK9 enzyme. The aim of the study was to provide the clinical and laboratory characteristics of 105 patients with the diagnosis of familial defective apolipoprotein B-100 (FDB) from the large cohort of patients (n=1000) with familial hypercholesterolemia (FH). The data was obtained from the real clinical practice at the Center of preventive cardiology (CPC).

RARE PROTEIN-TRUNCATING VARIANTS IN APOB ASSOCIATE WITH LOWER LOW-DENSITY LIPOPROTEIN CHOLESTEROL, LOWER TRIGLYCERIDES, AND REDUCED RISK OF CORONARY HEART DISEASE

Familial hypobetalipoproteinemia (FHBL) is a genetic disorder caused by rare protein-truncating variants (PTV) in the gene encoding apolipoprotein B (APOB), the major protein component of low-density and triglyceride-rich lipoprotein particles. Whether heterozygous APOB deficiency is associated with

PCSK9 inhibitor therapy in homozygous familial defective apolipoprotein B-100 due to APOB R3500Q: A case report

Identification of a patient homozygous for familial defective apolipoprotein B-100(FDB) and successful treatment with PCSK9 inhibition.

A Case Report of a South Asian Family with Homozygous and Heterozygous Familial Defective APOB-100 Caused by p.(Arg3527Trp)

Familial hypercholesterolemia (FH) is an autosomal dominant disorder most commonly caused by mutations in the gene for the Low-Density Lipoprotein (LDL) receptor (LDLR), but about 5% of patients in the UK with a clinical diagnosis of FH have a mutation in the gene for apolipoprotein B (APOB). This disorder is called Familial Defective APOB-100 (FDB), and while plasma total- and LDL-cholesterol levels overlap between patients with FDB and those with LDLR mutations, usually those with FDB present with a milder form of the disease, especially in homozygous FDB compared to LDLR mutation-caused FH. The most common mutation in APOB is p.(Arg3527Gln), but another APOB mutation p.(Arg3527Trp) has previously been identified in a family of South Asian origin. Here we describe a consanguineous marriage of parents of South Asian origin with both homozygous and heterozygous offspring with the APOB p.(Arg3527Trp) mutation. The mean untreated levels of LDL-cholesterol in the three heterozygous, Father and Mother (age 45 years) and a girl (age 9 years) were 5.5 mmol/l, 4.5 mmol/l, and 4.2 mmol/l respectively, while the mean untreated levels of LDL-cholesterol in the two homozygous boys (age 15 years and 11 years) were 6.2 mmol/l and 7.0 mmol/l respectively and this was reduced by ~30% on statin treatment. This confirms the milder phenotype and good response to statin therapy even for homozygous FDB.

Two forms of familial hypercholesterolemia: differences in cardiovascular risk factors, cardiac and extracardiac atherosclerosis

The monogenetic hypercholesterolemias (HC) are associated with a very high risk of premature coronary heart disease (CHD). We sought to assess the influence of the genetic defect and the cardiovascular risk factors on the manifestation of atherosclerotic complications in two forms of genetic HC. Data of patients with genetically defined HC (54 LDL-receptor defective familial hypercholesterolemia (FH) and 54 familial defective apolipoprotein B (FDB)) were analysed retrospectively for cardiac and extracardiac atherosclerosis. Total and LDL-cholesterol were significantly higher in FH men than in FDB men, but not so in women. 41.8% of FH patients had CHD (mean age 41 years), 5.6% of FDB (mean age 52 years). Stenoses (>50% narrowing) of the internal carotid artery were verified in 15% of FH and 4% of FDB patients. Peripheral arterial disease was found in 3 FH and 2 FDB patients. Hypertension and active smoking were allotted almost equally, body weight was normal in most of the patients (BMI ≤ 25 kg/m(2)). Conlusion: Patients with genetic HC suffer from early manifestation of cardiac disease. Patients with FH seem to be affected more often than patients with FDB. Early diagnosis and early and lifelong treatment are essential and, according to the literature, lead to a delay of disease manifestation.

Plasma lipoprotein(a) levels in familial defective ApoB

Aims: Elevated lipoprotein(a) (Lp[a]) is a risk factor for coronary artery disease. Its concentrations depend on genetic and nongenetic factors, and vary considerably. Familial ligand-defective ApoB (FDB) characterized by the R3500Q mutation in the ApoB gene, has been controversially associated with elevated lipoprotein(a) levels. We report on the relation of Lp(a) and FDB in the largest existing population study. Materials & methods: Lipoprotein(a) levels in 100 FDB patients are compared with 2207 control patients. In addition, a subgroup of relatives of FDB patients was analyzed. Results: FDB patients had significantly higher lipoprotein(a), 18 mg/dl compared with 8 mg/dl for non-FDB patients. When plasma triglycerides, LDL, age and sex were taken into account there was no association between FDB and lipoprotein(a). In FDB families the defective ApoB 3500 mutation had no significant influence on Lp(a) levels. Conclusion: After correcting for the above factors there is no association of Familial defective ApoB and Lp(a).

Decreased Bone Mineral Density in Subjects Carrying Familial Defective Apolipoprotein B-100

Although numerous epidemiologic studies have documented associations between osteoporosis and cardiovascular disease, the mechanisms underlying this association remain to be clarified. One hypothesis is that hyperlipidemia may be a common predisposing factor to both atherosclerotic heart disease and bone fragility. To evaluate this, we compared bone mineral density (BMD) between subjects with and without the R3500Q APOB mutation, the cause of familial defective apolipoprotein B-100, which has been previously shown to markedly increase low-density lipoprotein cholesterol (LDL-C). We hypothesized that R3500Q carriers would have lower BMD due to lifetime, elevated LDL-C. This was a a cross-sectional study in the Old Order Amish (OOA) population. The R3500Q APOB mutation is present at a high frequency (∼6% vs <0.5%) in the OOA population due to a founder effect. Therefore, we conducted analysis on 1097 Amish individuals of whom 125 were R3500Q carriers. BMD was measured by dual-energy x-ray absorptiometry. After adjusting for age, age(2), sex, body mass index, and family structure, carriers for the Q risk allele had significantly lower BMD than noncarriers at the femoral neck (P = .037), lumbar spine (P = .035) and whole body (P = .016). Adjusting for LDL-C attenuated the association between R3500Q genotype and BMD but did not completely explain the relationship. Subgroup analyses showed no significant interactions with sex, age, or presence of metabolic syndrome. These results use the unique genetic architecture of the OOA population to provide a novel line of evidence supporting a causal role for elevated LDL-C in lowering BMD.

A DNA Microarray for the Detection of Point Mutations and Copy Number Variation Causing Familial Hypercholesterolemia in Europe

To facilitate genetic cascade screening for familial hypercholesterolemia (FH) in Europe, two versions (7 and 9) of a DNA microarray were developed to detect the most frequent point mutations in the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), and proprotein convertase subtilisin/kexin 9 (PCSK9) genes. The design of these microarrays is based on LIPOchip, version 4, which detects 191 LDLR and APOB mutations identified in Spanish patients with FH. A major improvement of LIPOchip, versions 7 and 9, is the ability to detect copy number variation (deletions or duplications of entire exons) in LDLR, thus abolishing the need to perform multiplex ligase-dependent probe amplification in patients with FH. The aim of this study was to validate a tool capable of detecting point mutations and copy number variations simultaneously and to evaluate its use and the newly developed software for analysis in clinical practice by reanalysis of several patients with known mutations causing FH. With the help of these validations, several aspects were analyzed, improved, and implemented in a newer version, which was evaluated through an internal validation.

First Detection of Hypercholesterolemia Causing ApoB-100 R3527Q Mutation in a Family in Greece

Familial defective apolipoprotein B (FDB) is an autosomal dominant genetic disorder causing hypercholesterolemia in affected patients. It is occurring due to mutation of apoB gene leading to a decreased low density lipoprotein (LDL) particles clearance. The R3527Q mutation is one of the disease’s causative mutations. Data support mutation’s origin 6000-7000 years ago in Central Europe and its prevalence is decreasing in relation to the distance from where initially occurred. Until now, the presence of R3527Q mutation had not been confirmed in Greece. This is the first report of FDB due to R3527Q mutation in a family in Northwest Greece.

First report of APOB gene variations in Moroccans

The normal clearance of LDL by the LDL receptor is dependent upon normal function of both the LDL receptor and of apoB-100. Accordingly, mutations in the LDL receptor gene and in the apoB-100 gene have been found to cause autosomal dominant hypercholesterolemia. Mutations in the LDL receptor gene cause familial hypercholesterolemia, whereas mutations in the apoB-100 gene cause familial defective apoB-100. In Front lack of data on genotype and phenotype among ADH patients in Moroccan populations, we carried up this study to determine genotype of ADH among subjects of north of Morocco, who have increased rates of TC, LDLc, ApoB and TG. 46 probands were studied. Genomic DNA was isolated us described previously. Direct sequencing is used to determine the mutations on genes LDLR, APOB and PCSK9 and the genotype of the apoE. We have detected for the first time in the Moroccan population three known polymorphisms and V4238A mutation combined with increased TG levels in three probands. This mutation appears to be common polymorphism. Those patients were homozygous of alleles E3/E3. In conclusion, further studies are needed to elucidate the molecular basis of the dyslipidemia observed in the studied probands.

Genetic analysis of familial hypercholesterolaemia in Western Australia

ObjectiveTo determine the spectrum of mutations associated with familial hypercholesterolaemia (FH) and their detection rate in the FH Western Australia (FHWA) Program. MethodsMutation testing of the LDLR gene, plus select regions in APOB and PCSK9, was performed in the first 343 patients considered to be phenotypic index cases of FH and classified on the basis of the Dutch Lipid Clinic Network Criteria (DLCNC) score as “possible”, “probable”, or “definite” FH. ResultsOverall, 86 different pathogenic (or likely pathogenic) mutations were identified in 129 patients, including four compound heterozygotes manifesting a more severe clinical phenotype. Fourteen of these mutations were novel and twelve (9.6%) were large deletions/duplications of the LDLR. The most common mutations were the familial defective apoB-100 mutation APOB p.Arg3527Gln (7.2%) and an LDLR intron 3 splice site mutation c.313 + 1G > A (4.8%). While 70% of ‘definite’ FH patients were found to carry a mutation, only 29% of ‘probable’ and 11% of ‘possible’ FH patients were mutation-positive. ConclusionThis information provides a useful DNA database on which to base ongoing cascade screening for FH and future research into the genetic aetiology of FH in Western Australia. These findings suggest genetic testing should be prioritised to those with high DLCNC scores and offers a cost-effective family screening method from FH index cases, leading to detection of other previously undiagnosed and younger family members, enabling early instigation of intervention and preventative measures for premature coronary heart disease.

Familial Defective Apolipoprotein B-100 and Increased Low-Density Lipoprotein Cholesterol and Coronary Artery Calcification in the Old Order Amish

Elevated low-density lipoprotein cholesterol (LDL-C) levels are a major cardiovascular disease risk factor. Genetic factors are an important determinant of LDL-C levels. To identify single nucleotide polymorphisms associated with LDL-C and subclinical coronary atherosclerosis, we performed a genome-wide association study of LDL-C in 841 asymptomatic Amish individuals aged 20 to 80 years, with replication in a second sample of 663 Amish individuals. We also performed scanning for coronary artery calcification (CAC) in 1018 of these individuals. From the initial genome-wide association study, a cluster of single nucleotide polymorphisms in the region of the apolipoprotein B-100 gene (APOB) was strongly associated with LDL-C levels (P < 10(-68)). Additional genotyping revealed the presence of R3500Q, the mutation responsible for familial defective apolipoprotein B-100, which was also strongly associated with LDL-C in the replication sample (P < 10(-36)). The R3500Q carrier frequency, previously reported to be 0.1% to 0.4% in white European individuals, was 12% in the combined sample of 1504 Amish participants, consistent with a founder effect. The mutation was also strongly associated with CAC in both samples (P < 10(-6) in both) and accounted for 26% and 7% of the variation in LDL-C levels and CAC, respectively. Compared with noncarriers, R3500Q carriers on average had LDL-C levels 58 mg/dL higher, a 4.41-fold higher odds (95% confidence interval, 2.69-7.21) of having detectable CAC, and a 9.28-fold higher odds (2.93-29.35) of having extensive CAC (CAC score ≥400). The R3500Q mutation in APOB is a major determinant of LDL-C levels and CAC in the Amish.

Mechanism of LDL binding and release probed by structure-based mutagenesis of the LDL receptor

The LDL receptor (LDL-R) mediates cholesterol metabolism in humans by binding and internalizing cholesterol transported by LDL. Several different molecular mechanisms have been proposed for the binding of LDL to LDL-R at neutral plasma pH and for its release at acidic endosomal pH. The crystal structure of LDL-R at acidic pH shows that the receptor folds back on itself in a closed form, obscuring parts of the ligand binding domain with the epidermal growth factor (EGF)-precursor homology domain. We have used a structure-based site-directed mutagenesis approach to examine 12 residues in the extracellular domain of LDL-R for their effect on LDL binding and release. Our studies show that the interface between the ligand binding domain and the EGF-precursor homology domain seen at acidic pH buries residues mediating both LDL binding and release. Our results are consistent with an alternative model of LDL-R whereby multiple modules of the extracellular domain interact with LDL at neutral pH, concurrently positioning key residues so that at acidic pH the LDL-R:LDL interactions become unfavorable, triggering release. After LDL release, the closed form of LDL-R may target its return to the cell surface.

A new method for measurement of total plasma PCSK9: clinical applications

The proprotein convertase subtilisin kexin-9 (PCSK9) circulates in plasma as mature and furin-cleaved forms. A polyclonal antibody against human PCSK9 was used to develop an ELISA that measures total plasma PCSK9 rather than only the mature form. A cross-sectional study evaluated plasma levels in normal (n = 254) and hypercholesterolemic (n = 200) subjects treated or untreated with statins or statin plus ezetimibe. In controls, mean plasma PCSK9 (89.5 +/- 31.9 ng/ml) correlated positively with age, total cholesterol, LDL-cholesterol (LDL-C), triglycerides, and fasting glucose. Sequencing PCSK9 from individuals at the extremes of the normal PCSK9 distribution identified a new loss-of-function R434W variant associated with lower levels of circulating PCSK9 and LDL-C. In hypercholesterolemic subjects, PCSK9 levels were higher than in controls (99.3 +/- 31.7 ng/ml, P < 0.04) and increased in proportion to the statin dose, combined or not with ezetimibe. In treated patients (n = 139), those with familial hypercholesterolemia (FH; due to LDL receptor gene mutations) had higher PCSK9 values than non-FH (147.01 +/- 42.5 vs. 127.2 +/- 40.8 ng/ml, P < 0.005), but LDL-C reduction correlated positively with achieved plasma PCSK9 levels to a similar extent in both subsets (r = 0.316, P < 0.02 in FH and r = 0.275, P < 0.009 in non-FH). The detection of circulating PCSK9 in both FH and non-FH subjects means that this assay could be used to monitor response to therapy in a wide range of patients.

The unique role of proprotein convertase subtilisin/kexin 9 in cholesterol homeostasis

The LDL receptor (LDLR) plays an essential role in the regulation of plasma (LDL) cholesterol concentrations by virtue of its ability to clear plasma LDL. Down-regulation of the LDLR by proprotein convertase subtilisin/kexin 9 (PCSK9) has recently emerged as a regulatory mechanism that controls plasma LDL cholesterol concentrations. Studies in which PCSK9 is over-expressed in mice, have demonstrated that PCSK9, by enhancing hepatic LDLR degradation, decreases the availability of the LDLR for LDL uptake, resulting in increased plasma LDL cholesterol levels. However, PCSK9 has also recently been shown to mediate down-regulation of surface receptors other than the LDLR, suggesting that it may have much broader roles than initially thought.

Abstract: P847 THE PREVALENCE OF APOLIPOPROTEIN B-100 GENE MUTATIONS IN RUSSIAN FAMILIAL HYPERCHOLESTEROLEMIA PATIENTS

Familial hypercholesterolemia (FH) and familial defective apolipoprotein B-100 (FDB) are relatively common lipid disorders caused by mutations in the low-density lipoprotein receptor (LDLR) and apolipoprotein B (apo B) genes, respectively. Molecular analysis at these loci was performed in eight New Zealand subjects with clinical features of heterozygous FH. Utilization of an in vitro lymphocyte receptor assay demonstrated normal receptor function in four patients, three of whom screened positive for the founder-type apo B mutation, R3500Q, causing FDB. Four patients with reduced LDLR function, consistent with heterozygous FH, revealed three previously documented mutations in exons 3 (W66X), 6 (C292Y) and 7 (G322S) of the LDLR gene and, a novel 2-bp deletion (TC or CT) after nucleotide 1204 (or 1205) in exon 9. The remaining patient was found to be FH/FDB negative after extensive mutation screening using both denaturing gradient gel electrophoresis and heteroduplex-single strand conformation polymorphism analysis. Haplotype analysis at the LDLR and apo B loci finally excluded the likelihood that mutations in these two genes underlie the FH phenotype in the molecularly uncharacterized New Zealand family originating from the United Kingdom. This family represents a valuable source of material for future genetic dissection of autosomal dominant hypercholesterolemia (ADH), shown to be a heterogeneous disease through molecular analysis.

Oft unterschätzte Probleme in der Lipidologie: Verspätung in der Diagnose und Tachyphylaxie in der Therapie

Summary Frequently underestimated problems in lipidology: late diagnosis and tachyphylaxis in treatment ( We report the case of a 44-year-old patient with severe hypercholesterol- aemia, referred to our outpatient clinic . On the basis of the clinical findings we performed a molecular genetic analysis which confirmed the suggested point mutation at amino acid position 3,500 (R3,500Q) in the gene encoding apolipo- protein B-100. The diagnosis of familial defective apolipoprotein B-100 (FDB) was thus molecularly proven. Unfortunately, at the time of diagnosis, a hitherto unknown three-vessel coronary artery disease was already present. ( In addition, this case confronted us with the problem of a potential tachyphy- laxis phenomenon when using atorvastatin. Replacing atorvastatin by rosu- vastatin resulted in a marked and persistent subsequent reduction of the total and LDL-cholesterol concentrations lasting for 40 months at least. ( The frequent problems of late diagnosis in patients with familial forms of hypercholesterolaemia and an insufficient long-term treatment are discussed.