Mafalda Bourbon

Characterization of a novel cellular defect in patients with phenotypic homozygous familial hypercholesterolemia

Familial hypercholesterolemia (FH) is characterized by a raised concentration of LDL in plasma that results in a significantly increased risk of premature atherosclerosis. In FH, impaired removal of LDL from the circulation results from inherited mutations in the LDL receptor gene or, more rarely, in the gene for apo B, the ligand for the LDL receptor. We have identified two unrelated clinically homozygous FH patients whose cells exhibit no measurable degradation of LDL in culture. Extensive analysis of DNA and mRNA revealed no defect in the LDL receptor, and alleles of the LDL receptor or apo B genes do not cosegregate with hypercholesterolemia in these families. FACS((R)) analysis of binding and uptake of fluorescent LDL or anti-LDL receptor antibodies showed that LDL receptors are on the cell surface and bind LDL normally, but fail to be internalized, suggesting that some component of endocytosis through clathrin-coated pits is defective. Internalization of the transferrin receptor occurs normally, suggesting that the defective gene product may interact specifically with the LDL receptor internalization signal. Identification of the defective gene will aid genetic diagnosis of other hypercholesterolemic patients and elucidate the mechanism by which LDL receptors are internalized.

Familial hypercholesterolaemia: A global call to arms.

Familial Hypercholesterolaemia (FH) is the commonest autosomal co-dominantly inherited condition affecting man. It is caused by mutation in one of three genes, encoding the low-density lipoprotein (LDL) receptor, or the gene for apolipoprotein B (which is the major protein component of the LDL particle), or in the gene coding for PCSK9 (which is involved in the degradation of the LDL-receptor during its cellular recycling). These mutations result in impaired LDL metabolism, leading to life-long elevations in LDL-cholesterol (LDL-C) and development of premature atherosclerotic cardiovascular disease (ASCVD) [1], [2] and [3]. If left untreated, the relative risk of premature coronary artery disease is significantly higher in heterozygous patients than unaffected individuals, with most untreated homozygotes developing ASCVD before the age of 20 and generally not surviving past 30 years [2], [3], [4] and [5]. Although early detection and treatment with statins and other LDL-C lowering therapies can improve survival, FH remains widely underdiagnosed and undertreated [1], thereby representing a major global public health challenge.

Determinants of Variable Response to Statin Treatment in Patients With Refractory Familial Hypercholesterolemia

Abstract—Interindividual variability in low density lipoprotein (LDL) cholesterol (LDL-C) response during treatment with statins is well documented but poorly understood. To investigate potential metabolic and genetic determinants of statin responsiveness, 19 patients with refractory heterozygous familial hypercholesterolemia were sequentially treated with placebo, atorvastatin (10 mg/d), bile acid sequestrant, and the 2 combined, each for 4 weeks. Levels of LDL-C, mevalonic acid (MVA), 7-&agr;-OH-4-cholesten-3-one, and leukocyte LDL receptor and hydroxymethylglutaryl coenzyme A reductase mRNA were determined after each treatment period. Atorvastatin (10 mg/d) reduced LDL-C by an overall mean of 32.5%. Above-average responders (&Dgr;LDL-C −39.5%) had higher basal MVA levels (34.4±6.1 &mgr;mol/L) than did below-average responders (&Dgr;LDL-C −23.6%, P <0.02; basal MVA 26.3±6.1 &mgr;mol/L, P <0.01). Fewer good responders compared with the poor responders had an apolipoprotein E4 allele (3 of 11 versus 6 of 8, respectively;P <0.05). There were no baseline differences between them in 7-&agr;-OH-4-cholesten-3-one, hydroxymethylglutaryl coenzyme A reductase mRNA, or LDL receptor mRNA, but the latter increased in the good responders on combination therapy (P <0.05). Severe mutations were not more common in poor than in good responders. We conclude that poor responders to statins have a low basal rate of cholesterol synthesis that may be secondary to a genetically determined increase in cholesterol absorption, possibly mediated by apolipoprotein E4. If so, statin responsiveness could be enhanced by reducing dietary cholesterol intake or inhibiting absorption.

Genetic diagnosis of familial hypercholesterolaemia: the importance of functional analysis of potential splice-site mutations

Familial hypercholesterolemia (FH) results from defective low-density lipoprotein receptor (LDLR) activity, mainly due to LDLR gene defects. Of the many different LDLR mutations found in patients with FH, about 6% of single base substitutions are located near or within introns, and are predicted to result in exon skipping, retention of an intron, or activation of cryptic sites during mRNA splicing. This paper reports on the Portuguese FH Study, which found 10 such mutations, 6 of them novel. For the mutations that have not been described before or those whose effect on function have not been analysed, their effect on splicing was investigated, using reverse transcriptase PCR analysis of LDLR mRNA from freshly isolated blood mononuclear cells. Two of these variants (c.313+6 T→C, c.2389G→T (p.V776L)) caused exon skipping, and one caused retention of an intron (c.1359−5C→G), whereas two others (c.2140+5 G→A and c.1061−8T→C) had no apparent effect. Any effect of c.1185G→C (p.V374V) on splicing could not be determined because it was on an allele with a promoter mutation (−42C→G) that was probably not transcribed. Variants in four patients lost to follow-up could not be tested experimentally, but they almost certainly affect splicing because they disrupt the invariant AG or GT in acceptor (c.818−2A→G) or donor (c.1060+1G→A, c.1845+1delG and c.2547+1G→A) spice sites. These findings emphasise that care must be taken before reporting the presence or absence of a splice-site mutation in the LDLR gene for diagnostic purposes. The study also shows that relatively simple, quick and inexpensive RNA assays can evaluate putative splicing mutations that are not always predictable by available software, thereby reducing genetic misdiagnosis of patients with FH.

How good is controlled attenuation parameter and fatty liver index for assessing liver steatosis in general population: correlation with ultrasound

Liver steatosis measurement by controlled attenuation parameter (CAP) is a non‐invasive method for diagnosing steatosis, based on transient elastography. Its usefulness as screening procedure for hepatic steatosis in general population has not been previously evaluated. The aim of this study was to evaluate the diagnostic accuracy of CAP and fatty liver index (FLI) for detection and quantification of steatosis in general population.

Characterization of Autosomal Dominant Hypercholesterolemia Caused by PCSK9 Gain of Function Mutations and its Specific Treatment with Alirocumab, a PCSK9 Monoclonal Antibody

Background—Patients with PCSK9 gene gain of function (GOF) mutations have a rare form of autosomal dominant hypercholesterolemia. However, data examining their clinical characteristics and geographic distribution are lacking. Furthermore, no randomized treatment study in this population has been reported. Methods and Results—We compiled clinical characteristics of PCSK9 GOF mutation carriers in a multinational retrospective, cross-sectional, observational study. We then performed a randomized placebo-phase, double-blind study of alirocumab 150 mg administered subcutaneously every 2 weeks to 13 patients representing 4 different PCSK9 GOF mutations with low-density lipoprotein cholesterol (LDL-C) ≥70 mg/dL on their current lipid-lowering therapies at baseline. Observational study: among 164 patients, 16 different PCSK9 GOF mutations distributed throughout the gene were associated with varying severity of untreated LDL-C levels. Coronary artery disease was common (33%; average age of onset, 49.4 years), and untreated LDL-C concentrations were higher compared with matched carriers of mutations in the LDLR (n=2126) or apolipoprotein B (n=470) genes. Intervention study: in PCSK9 GOF mutation patients randomly assigned to receive alirocumab, mean percent reduction in LDL-C at 2 weeks was 62.5% (P<0.0001) from baseline, 53.7% compared with placebo-treated PCSK9 GOF mutation patients (P=0.0009; primary end point). After all subjects received 8 weeks of alirocumab treatment, LDL-C was reduced by 73% from baseline (P<0.0001). Conclusions—PCSK9 GOF mutation carriers have elevated LDL-C levels and are at high risk of premature cardiovascular disease. Alirocumab, a PCSK9 antibody, markedly lowers LDL-C levels and seems to be well tolerated in these patients. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique Identifier: NCT01604824.

Update of the Portuguese Familial Hypercholesterolaemia Study

The main aim of the Portuguese Familial Hypercholesterolaemia Study is to identify the genetic cause of hypercholesterolaemia in individuals with a clinical diagnosis of Familial Hypercholesterolaemia (FH). A total of 1340 blood samples were collected from 482 index patients and 858 relatives with the collaboration of clinicians from several hospitals all over the country. The genetic diagnosis of FH in this study is based on the analyses of three genes: LDLR, APOB and PCSK9. In the last 10 years, the Portuguese FH Study identified a genetic defect in a total of 171 index patients, corresponding to an overall of 48% of the total received cases with clinical diagnosis of FH. Although the Simon Broome FH register criteria have been adapted to our study, 59 patients that did not fulfil all criteria were included in the study and a mutation causing disease was identified in 8 of these patients. In the LDLR gene were found 80 different mutations in 165 unrelated index patients: 159 heterozygous, 3 compounds heterozygous and 3 true homozygous. The APOB p.Arg3527Gln and the PCSK9 p.Asp374His mutation were not found in any of our patients since our last report, but a novel mutation in the APOB gene, predicted to cause a single amino acid substitution p.Tyr3560Cys, was found in one patient. The cascade screening in relatives of these 171 index patients allowed the identification and genetic characterization of a total of 404 FH patients in Portugal.

Novel functional APOB mutations outside LDL-binding region causing familial hypercholesterolaemia

Familial hypercholesterolaemia (FH) is characterized by increased circulating low-density lipoprotein (LDL) cholesterol leading to premature atherosclerosis and coronary heart disease. Although FH is usually caused by mutations in LDLR, mutations in APOB and PCSK9 also cause FH but only a few mutations have been reported, APOB p.R3527Q being the most common. However, 30-80% of clinical FH patients do not present an identifiable mutation in any of the described genes. To identify the genetic cause of the hypercholesterolaemia in 65 patients without mutations in LDLR, PCSK9 or in fragments of exon 26 and 29 of APOB currently analysed, we performed whole sequencing of APOB by pyrosequencing. A total of 10 putative mutations in APOB were identified. Flow cytometry with fluorescently labelled LDL from patients and relatives showed that p.Arg1164Thr (exon 22) and p.Gln4494del (exon 29) presented a 40% decrease in internalization in lymphocytes and HepG2 cells, very similar to APOB3527. The proliferation assays with U937 cells showed reduced growth for both cases. The variant p.Tyr1247Cys was found to be neutral and other three alterations were considered polymorphisms. Our results emphasize the need to study the whole APOB in routine protocols to improve patient identification and cardiovascular risk assessment.

Pooling and expanding registries of familial hypercholesterolaemia to assess gaps in care and improve disease management and outcomes: Rationale and design of the global EAS Familial Hypercholesterolaemia Studies Collaboration

BACKGROUND The potential for global collaborations to better inform public health policy regarding major non-communicable diseases has been successfully demonstrated by several large-scale international consortia. However, the true public health impact of familial hypercholesterolaemia (FH), a common genetic disorder associated with premature cardiovascular disease, is yet to be reliably ascertained using similar approaches. The European Atherosclerosis Society FH Studies Collaboration (EAS FHSC) is a new initiative of international stakeholders which will help establish a global FH registry to generate large-scale, robust data on the burden of FH worldwide. METHODS The EAS FHSC will maximise the potential exploitation of currently available and future FH data (retrospective and prospective) by bringing together regional/national/international data sources with access to individuals with a clinical and/or genetic diagnosis of heterozygous or homozygous FH. A novel bespoke electronic platform and FH Data Warehouse will be developed to allow secure data sharing, validation, cleaning, pooling, harmonisation and analysis irrespective of the source or format. Standard statistical procedures will allow us to investigate cross-sectional associations, patterns of real-world practice, trends over time, and analyse risk and outcomes (e.g. cardiovascular outcomes, all-cause death), accounting for potential confounders and subgroup effects. CONCLUSIONS The EAS FHSC represents an excellent opportunity to integrate individual efforts across the world to tackle the global burden of FH. The information garnered from the registry will help reduce gaps in knowledge, inform best practices, assist in clinical trials design, support clinical guidelines and policies development, and ultimately improve the care of FH patients.

Variable phenotypic expression of homozygous familial hypobetalipoproteinaemia due to novel APOB gene mutations

Homozygous familial hypobetalipoproteinaemia (Ho‐FHBL) is a rare co‐dominant disorder characterized by extremely low levels of low‐density lipoprotein cholesterol (LDL‐C) and apolipoprotein B (apoB). Most patients with Ho‐FHBL have mutations in APOB gene resulting in truncated apoBs. Some patients are asymptomatic, while others have fatty liver, intestinal fat malabsorption and neurological dysfunctions. We investigated three adult subjects with severe hypobetalipoproteinaemia and a family history of FHBL. Proband FHBL‐47 had liver cirrhosis with hepatocarcinoma and a renal carcinoma but no clinical manifestations related to FHBL. He was a compound heterozygote for a 7‐bp deletion in exon 21 and a base insertion in exon 26 resulting in truncated apoBs (apoB‐22.46/apoB‐66.51). Proband FHBL‐53, with severe hepatic steatosis and fibrosis, had a nonsense mutation in exon 19 resulting in a truncated apoB (apoB‐20.61) and a rare nucleotide substitution in intron 14 (c.2068‐4T>A). The latter was also present in her daughter, found to have low plasma LDL‐C and apoB. Proband FHBL‐82 had chronic diarrhoea and steatorrhoea. She was found to be homozygous for a nonsense mutation in exon 24 resulting in a truncated apoB (apoB‐26.65). In adult subjects, the presence of chronic liver disease and chronic diarrhoea, when associated with severe hypobetalipoproteinaemia, may lead to the diagnosis of Ho‐FHBL.