Mutation detection in Croatian patients with Familial Hypercholesterolemia

Familial hypercholesterolemia (FH) is caused by mutations in the genes for LDLR, APOB or PCSK9, and identification of the causative mutation provides definitive diagnosis so that the patient can be treated, their relatives tested and, therefore, premature heart disease prevented. DNA of eight unrelated individuals with clinically diagnosed FH were analyzed using a High‐Resolution Melting method (HRM) for the LDLR gene (coding region, promoter and intron/exon boundaries), the APOB gene (part exon 26) and the PCSK9 gene (exon7). Variations found were sequenced and the effect on function of confirmed variants examined using predictive algorithms. Gross deletions and insertions were analysed using MLPA. Three novel LDLR variants were found, p.(S470C), p.(C698R) and c.2312–2A>C. All were predicted to be pathogenic using predictive algorithms. Three previously reported disease‐causing mutations were identified (p.(G20R), p.(N272T) and p.(S286R); the latter was also carried by a hypercholesterolaemic relative. One patient carried the pathogenic APOB variant p.(R3527Q). No large LDLR deletions nor insertions were found, neither were any PCSK9 variants identified. HRM is a sensitive method for screening for mutations. While the causative mutation has been identified in 88% of these clinically defined FH patients, there appears to be a high degree of allelic heterogeneity in Croatian patients.     

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.     

Molecular Spectrum of Autosomal Dominant Hypercholesterolemia in France

Autosomal Dominant Hypercholesterolemia (ADH), characterized by isolated elevation of plasmatic LDL cholesterol and premature cardiovascular complications, is associated with mutations in 3 major genes: LDLR (LDL receptor), APOB (apolipoprotein B) and PCSK9 (proprotein convertase subtilisin‐kexin type 9). Through the French ADH Research Network, we collected molecular data from 1358 French probands from eleven different regions in France. Mutations in the LDLR gene were identified in 1003 subjects representing 391 unique events with 46.0% missense, 14.6% frameshift, 13.6% splice, and 11.3% nonsense mutations, 9.7% major rearrangements, 3.8% small in frame deletions/insertions, and 1.0% UTR mutations. Interestingly, 175 are novel mutational events and represent 45% of the unique events we identified, highlighting a specificity of the LDLR mutation spectrum in France. Furthermore, mutations in the APOB gene were identified in 89 probands and in the PCSK9 gene in 10 probands. Comparison of available clinical and biochemical data showed a gradient of severity for ADH‐causing mutations: FH=PCSK9>FDB>‘Others’ genes. The respective contribution of each known gene to ADH in this French cohort is: LDLR 73.9%, APOB 6.6%, PCSK9 0.7%. Finally, in 19.0% of the probands, no mutation was found, thus underscoring the existence of ADH mutations located in still unknown genes. © 2010 Wiley‐Liss, Inc.     

Mutation spectrum and polygenic score in German patients with familial hypercholesterolemia

Autosomal-dominant familial hypercholesterolemia (FH) is characterized by increased plasma concentrations of low-density lipoprotein cholesterol (LDL-C) and a substantial risk to develop cardiovascular disease. Causative mutations in three major genes are known: the LDL receptor gene (LDLR), the apolipoprotein B gene (APOB) and the proprotein convertase subtilisin/kexin 9 gene (PCSK9). We clinically characterized 336 patients suspected to have FH and screened them for disease causing mutations in LDLR, APOB, and PCSK9. We genotyped six single nucleotide polymorphisms (SNPs) to calculate a polygenic risk score for the patients and 1985 controls. The 117 patients had a causative variant in one of the analyzed genes. Most variants were found in the LDLR gene (84.9%) with 11 novel mutations. The mean polygenic risk score was significantly higher in FH mutation negative subjects than in FH mutation positive patients (P < .05) and healthy controls (P < .001), whereas the score of the two latter groups did not differ significantly. However, the score explained only about 3% of the baseline LDL-C variance. We verified the previously described clinical and genetic variability of FH for German hypercholesterolemic patients. Evaluation of a six-SNP polygenic score recently proposed for clinical use suggests that it is not a reliable tool to classify hypercholesterolemic patients.     

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.     

The familial hypercholesterolaemia phenotype: Monogenic familial hypercholesterolaemia,polygenic hypercholesterolaemia and other causes

Familial hypercholesterolaemia (FH) is a monogenic disorder characterised by high low-density lipoprotein cholesterol (LDL-C) concentrations and increased cardiovascular risk. However, in clinically defined FH cohorts worldwide, an FH-causing variant is only found in 40%-50% of the cases. The aim of this work was to characterise the genetic cause of the FH phenotype in Portuguese clinical FH patients. Between 1999 and 2017, 731 index patients (311 children and 420 adults) who met the Simon Broome diagnostic criteria had been referred to our laboratory. LDLR, APOB, PCSK9, APOE, LIPA, LDLRAP1, ABCG5/8 genes were analysed by polymerase chain reaction amplification and Sanger sequencing. The 6-SNP LDL-C genetic risk score (GRS) for polygenic hypercholesterolaemia was validated in the Portuguese population and cases with a GRS over the 25th percentile were considered to have a high likelihood of polygenic hypercholesterolaemia. An FH-causing mutation was found in 39% of patients (94% in LDLR, 5% APOB and 1% PCSK9), while at least 29% have polygenic hypercholesterolaemia and 1% have other lipid disorders. A genetic cause for the FH phenotype was found in 503 patients (69%). All known causes of the FH phenotype should be investigated in FH cohorts to ensure accurate diagnosis and appropriate management.     

Mutational analysis of a cohort with clinical diagnosis of familial hypercholesterolemia: considerations for genetic diagnosis improvement

PurposeFamilial hypercholesterolemia (FH) is a common autosomal dominant disorder of lipid metabolism caused by mutations in LDLR, APOB, and PCSK9. To fulfill the World Health Organization recommendation, the Portuguese FH Study was established. Here, we report the results of the past 15 years and present practical considerations concerning the genetic diagnosis of FH based on our experience.MethodsOur approach comprises a biochemical and molecular study and is divided into five phases, including the study of whole APOB and functional assays.ResultsA total of 2,122 individuals were enrolled. A putative pathogenic variant was identified in 660 heterozygous patients: LDLR (623), APOB (33), and PCSK9 (4); 8 patients presented with homozygous FH. A detection rate of 41.5% was observed. A stricter biochemical criteria was shown to improve patient identification. Overall, we have identified 3.4% and 80% of all heterozygous and homozygous patients, respectively, estimated to exist in our country.ConclusionThe Portuguese FH Study has established the genetic diagnosis of FH in Portugal and is committed to continue the investigation of the genetic complexity of FH. Genetic diagnosis of FH should be expanded to include the study of all coding/flanking regions of APOB and functional in vitro studies, to improve the correct patient identification, and to avoid misdiagnosis.     

Genetic identification of familial hypercholesterolemia within whole genome sequences in 6820 newborns

Familial hypercholesterolemia (FH) is defined as a monogenic disease, characterized by elevated low-density lipoprotein cholesterol (LDL-C) levels. FH remains underdiagnosed and undertreated in Chinese. We whole-genome sequenced 6820 newborns from Qingdao of China to investigate the FH-related gene (LDLR, APOB, PCSK9) mutation types, carrier ratio and genotype–phenotype correlation. In this study, the prevalence of FH in Qingdao of China was 0.47% (95% CI: 0.32%–0.66%). The plasma lipid levels of FH-related gene mutation carriers begin to increase as early as infant. T-CHO and LDL-C of FH infants was higher by 48.1% (p < 0.001) and 42.9% (p < 0.001) relative to non-FH infants. A total of 22 FH infants and their parent participate in further studies. The results indicated that FH infant parent noncarriers have the normal plasma lipid level, while T-CHO and LDL-C increased in FH infants and FH infant parent carriers, but no difference between the groups. This highlights the importance of genetic factors. In conclusion, the spectrum of FH-causing mutations in the newborns of Qingdao, China was described for the first time. These data can serve as a considerable dataset for next-generation sequencing analysis of the Chinese population with FH and potentially helping reform regional policies for early detection and prevention of FH.     

Genetic causes of monogenic familial hypercholesterolemia in the Greek population: Lessons,mistakes, and the way forward

Familial hypercholesterolemia (FH) is a leading cause of premature atherosclerosis. Genetic defects in the LDLR, APOB and PCSK9 genes cause FH, and confirmation of a gene defect is essential for an indisputable diagnosis of the disease. FH is underdiagnosed and we aimed to revise the genetic defects that have been characterized in FH patients of Greek origin and define an effective, future strategy for genetic studies. A literature search was performed in MEDLINE and EMBASE on genetic studies with FH patients of Greek origin. To date, no APOB and PCSK9 mutations have been found in the Greek population. It must be noted however, that only a small number of patients has been screened for PCSK9 mutations. In total, 41 LDLR defects have been characterized, with 6 common mutations c.1646G>A (p.Gly546Asp), c.858C>A (p.Ser286Arg), c.81C>G (p.Cys27Trp), c.1285G>A (p.Val429Met), c.517T>C (p.Cys173Arg), and c.1775G>A (p.Gly592Glu) that account for >80% of all mutations. Due to geographic isolation, ​founder mutations exist in a subpopulation in North West Greece and the Greek Cypriot population but not in the general population. Genetic testing should focus primarily on LDLR, and subsequently on PCSK9 and APOB. The Greek population is genetically homogeneous, which allows for a quick molecular diagnosis of the disease. Cascade screening is feasible and will certainly facilitate the identification of additional patients.     

Mutational analysis in UK patients with a clinical diagnosis of familial hypercholesterolaemia: relationship with plasma lipid traits, heart disease risk and utility in relative tracing

As part of a randomised trial [Genetic Risk Assessment for Familial Hypercholesterolaemia (FH) Trial] of the psychological consequences of DNA-based and non-DNA-based diagnosis of FH, 338 probands with a clinical diagnosis of FH (46% with tendon xanthomas) were recruited. In the DNA-based testing arm (245 probands), using single-strand conformation polymorphism of all exons of the low-density lipoprotein receptor (LDLR) gene, 48 different pathogenic mutations were found in 62 probands (25%), while 7 (2.9%) of the patients had the R3500Q mutation in the apolipoprotein B (APOB) gene. Compared to those with no detected mutation, mean untreated cholesterol levels in those with the APOB mutation were similar, while in those with an LDLR mutation levels were significantly higher (None=9.15±1.62 vs LDLR=9.13±1.16 vs APOB=10.26±2.07 mmol/l p<0.001, respectively). Thirty seven percent of the detected mutations were in exon 3/4 of LDLR, and this group had significantly higher untreated cholesterol than those with other LDLR mutations (11.71±2.39 mmol/l vs 9.88±2.44 mmol/l, p=0.03), and more evidence of coronary disease compared to those with other LDLR or APOB mutations (36 vs 13% p=0.04). Of the probands with a detected mutation, 54 first-degree relatives were identified, of whom 27 (50%) had a mutation. Of these, 18 had untreated cholesterol above the 95th percentile for their age and gender, but there was overlap with levels in the non-carrier relatives such that 12% of subjects would have been incorrectly diagnosed on lipid levels alone. In the non-DNA-based testing arm (82 probands) only 19 of the 74 relatives identified had untreated cholesterol above the 95th percentile for their age and gender, which was significantly lower (p<0.0005) than the 50% expected for monogenic inheritance. These data confirm the genetic heterogeneity of LDLR mutations in the UK and the deleterious effect of mutations in exon 3 or 4 of LDLR on receptor function, lipids and severity of coronary heart disease. In patients with a clinical diagnosis of FH but no detectable mutation, there is weaker evidence for a monogenic cause compared with relatives of probands with LDLR mutations. This supports the usefulness of DNA testing to confirm diagnosis of FH for the treatment of hyperlipidaemia and for further cascade screening.     

The molecular basis of familial hypercholesterolemia in Lebanon: Spectrum of LDLR mutations and role of PCSK9 as a modifier gene

Autosomal dominant hypercholesterolemia (ADH), a major risk for coronary heart disease, is associated with mutations in the genes encoding the low‐density lipoproteins receptor (LDLR), its ligand apolipoprotein B (APOB) or PCSK9 (Proprotein Convertase Subtilin Kexin 9). Familial hypercholesterolemia (FH) caused by mutation in the LDLR gene is the most frequent form of ADH. The incidence of FH is particularly high in the Lebanese population presumably as a result of a founder effect. In this study we characterize the spectrum of the mutations causing FH in Lebanon: we confirm the very high frequency of the LDLR p.Cys681X mutation that accounts for 81.5 % of the FH Lebanese probands recruited and identify other less frequent mutations in the LDLR. Finally, we show that the p.Leu21dup, an in frame insertion of one leucine to the stretch of 9 leucines in exon 1 of PCSK9, known to be associated with lower LDL‐cholesterol levels in general populations, is also associated with a reduction of LDL‐cholesterol levels in FH patients sharing the p.C681X mutation in the LDLR. Thus, by studying for the first time the impact of PCSK9 polymorphism on LDL‐cholesterol levels of FH patients carrying a same LDLR mutation, we show that PCSK9 might constitute a modifier gene in familial hypercholesterolemia. © 2009 Wiley‐Liss, Inc.     

Multiplex ligation-dependent probe amplification analysis to screen for deletions and duplications of the LDLR gene in patients with familial hypercholesterolaemia

The most common genetic defect in patients with autosomal dominant hypercholesterolaemia is a mutation of the low-density lipoprotein receptor ( LDLR ) gene. An estimate of the frequency of major rearrangements has been limited by the availability of an effective analytical method and testing of large cohorts. We present data from a cohort of 611 patients referred with suspected heterozygous familial hypercholesterolaemia (FH) from five UK lipid clinics, who were initially screened for point mutations in LDLR and the common APOB and PCSK9 mutations. The 377 cases in whom no mutation was found were then screened for large rearrangements by multiplex ligation-dependent probe amplification (MLPA) analysis. A rearrangement was identified in 19 patients. This represents 7.5% of the total detected mutations of the cohort. Of these, the majority of mutations (12/19) were deletions of more than one exon, two were duplications of more than one exon and five were single exon deletions that need interpreting with care. Five rearrangements (26%) are previously unreported. We conclude that MLPA analysis is a simple and rapid method for detecting large rearrangements and should be included in diagnostic genetic testing for FH.     

Widening the spectrum of genetic testing in familial hypercholesterolaemia: Will it translate into better patient and population outcomes?

Familial hypercholesterolaemia (FH) is caused by pathogenic variants in LDLR, APOB or PCSK9. Impaired low-density lipoprotein (LDL) receptor function leads to decreased LDL catabolism and premature atherosclerotic cardiovascular disease (ASCVD). Thousands of LDLR variants are known, but assignation of pathogenicity requires accurate phenotyping, family studies and assessment of LDL receptor function. Precise, genetic diagnosis of FH using targeted next generation sequencing allows for optimal treatment, distinguishing FH from pathogenically distinct disorders requiring different treatment. Polygenic hypercholesterolaemia resulting from an accumulation of LDL cholesterol-raising single nucleotide polymorphisms (SNPs) could also be suspected by this approach. Similarly, ASCVD risk could be estimated by broader sequencing of cholesterol and non-cholesterol-related genes. Both of these areas require further research. The clinical management of FH, focusing on the primary or secondary prevention of ASCVD, has been boosted by PCSK9 inhibitor therapy. The efficacy of PCSK9 inhibitors in homozygous FH may be partly predicted by the LDLR variants. While expanded genetic testing in FH is clinically useful in providing an accurate diagnosis and enabling cost-effective testing of relatives, further research is needed to establish its value in improving clinical outcomes.     

Functional analysis of new variants at the low‐density lipoprotein receptor associated with familial hypercholesterolemia

Familial hypercholesterolemia is an autosomal dominant disease of lipid metabolism caused by defects in the genes LDLR, APOB, and PCSK9. The prevalence of heterozygous familial hypercholesterolemia (HeFH) is estimated between 1/200 and 1/250. Early detection of patients with FH allows initiation of treatment, thus reducing the risk of coronary heart disease. In this study, we performed in vitro characterization of new LDLR variants found in our patients. Genetic analysis was performed by Next Generation Sequencing using a customized panel of 198 genes in DNA samples of 516 subjects with a clinical diagnosis of probable or definitive FH. All new LDLR variants found in our patients were functionally validated in CHO‐ldlA7 cells. The LDLR activity was measured by flow cytometry and LDLR expression was detected by immunofluorescence. Seven new variants at LDLR were tested: c.518 G>C;p.(Cys173Ser), c.[684 G>T;694 G>T];p.[Glu228Asp;Ala232Ser], c.926C>A;p.(Pro309His), c.1261A>G;p.(Ser421Gly), c.1594T>A;p.(Tyr532Asn), and c.2138delC;p.(Thr713Lysfs*17). We classified all variants as pathogenic except p.(Ser421Gly) and p.(Ala232Ser). The functional in vitro characterization of rare variants at the LDLR is a useful tool to classify the new variants. This approach allows us to confirm the genetic diagnosis of FH, avoiding the classification as “uncertain significant variants”, and therefore, carry out cascade family screening.     

A presumptive new locus for autosomal dominant hypercholesterolemia mapping to 8q24.22

Cenarro A, García‐Otín A‐L, Tejedor MT, Solanas M, Jarauta E, Junquera C, Ros E, Mozas P, Puzo J, Pocoví M, Civeira F. A presumptive new locus for autosomal dominant hypercholesterolemia mapping to 8q24.22. Molecular testing of patients with autosomal dominant hypercholesterolemia (ADH) fails to detect a causal functional mutation in 15.25% of subjects. We studied an ADH pedigree in which known ADH‐causing genes (LDLR, APOB and PCSK9) were excluded. Genome‐wide analysis on 15 family members detected significant association for ADH and dbSNP RS ID rs965814 (G/A), located in 8q24.22 cytoband. ADH was significantly associated to rs965814 G allele (p < 0.05) in a case–control study based on 200 unrelated ADH subjects without LDLR or APOB gene defects and 198 normolipidemic controls. We chose 24 markers for a detailed analysis of 8q24.22 cytoband, now based on an extended set of family members (21 individuals). One particular 24 marker haplotype was significantly associated to both higher total and low‐density lipoprotein‐cholesterol concentrations. Similar results were found for a shorter haplotype, composed of the distal six markers from the complete haplotype. Therefore, a presumptive new locus for ADH could be located in 8q24.22 cytoband, a region not previously linked or associated to ADH.     

Genetic causes of familial hypercholesterolaemia in patients in the UK: relation to plasma lipid levels and coronary heart disease risk

Aims

To determine the relative frequency of mutations in three different genes (low‐density lipoprotein receptor (LDLR), APOB, PCSK9), and to examine their effect in development of coronary heart disease (CHD) in patients with clinically defined definite familial hypercholesterolaemia in UK.

Patients and methods

409 patients with familial hypercholesterolaemia patients (158 with CHD) were studied. The LDLR was partially screened by single‐strand conformational polymorphism (SSCP) (exons 3, 4, 6–10 and 14) and by using a commercial kit for gross deletions or rearrangements. APOB (p.R3500Q) and PCSK9 (p.D374Y) were detected by specific assays. Coding exons of PCSK9 were screened by SSCP.

Results

Mutations were detected in 253 (61.9%) patients: 236 (57.7%) carried LDLR, 10 (2.4%) carried APOB p.Q3500 and 7 (1.7%) PCSK9 p.Y374. No additional mutations were identified in PCSK9. After adjusting for age, sex, smoking and systolic blood pressure, compared to those with no detectable mutation, the odds ratio of having CHD in those with an LDLR mutation was 1.84 (95% CI 1.10 to 3.06), for APOB 3.40 (0.71 to 16.36), and for PCSK9 19.96 (1.88 to 211.5; p = 0.001 overall). The high risk in patients carrying LDLR and PCSK9 p.Y374 was partly explained by their higher pretreatment cholesterol levels (LDLR, PCSK9 and no mutation, 10.29 (1.85), 13.12 and 9.85 (1.90) mmol/l, respectively, p = 0.001). The post‐statin treatment lipid profile in PCSK9 p.Y374 carriers was worse than in patients with no identified mutation (LDL‐C, 6.77 (1.82) mmol/l v 4.19 (1.26) mmol/l, p = 0.001, HDL‐C 1.09 (0.27) mmol/l v 1.36 (0.36) mmol/l, p = 0.03).

Conclusions

The higher CHD risk in patients carrying PCSK9 p.Y347 or a detected LDLR mutation supports the usefulness of DNA testing in the diagnosis and management of patients with familial hypercholesterolaemia. Mutations in PCSK9 appear uncommon in patients with familial hypercholesterolaemia in UK.Familial hypercholesterolaemia is an autosomal dominant disorder associated with increased risk of coronary heart disease (CHD), with an estimated prevalence in the UK of 1 in 500 to 1 in 600.¹ Roughly half of the men with familial hypercholesterolaemia, if untreated, will have developed clinically evident CHD by the age of 55 years. Affected women from the same families typically develop CHD about 9 years later than their affected male relatives, but again, often remarkably prematurely.² The proportion of patients with familial hypercholesterolaemia identified and being treated in lipid clinics to date in the UK is, at best, 15% of the predicted number, with most of these being young people.¹ Because lipid‐lowering drug treatment with statins substantially reduces coronary morbidity and mortality,³ identification of affected people by screening is crucially important. To this end, the Department of Health has recently funded five pilot sites in the UK to determine the efficiency of cascade testing in the current social structure and the framework of the National Health Service. Cascade testing is a cost‐effective method of finding additional patients with familial hypercholesterolaemia,⁴ and has been used extensively in other countries in Europe, most notably in Holland,⁵ for the past 5 years.

Key points

• Patients with familial hypercholesterolaemia with a detectable LDLR mutation have a higher risk of early CHD than those in whom no mutation was detected.
• Patients with the pD374Y mutation in PCSK9 have the highest pretreatment and post‐treatment levels of plasma cholesterol and the highest risk of early CHD.
• Mutations in PCSK9 appear to be uncommon in patients with familial hypercholesterolaemia in UK.

The extent to which DNA testing for familial hypercholesterolaemia complements cholesterol measurement in cascade screening to identify affected patients is unclear, as is its role in determining the risk of CHD and response to treatment. In the current study, we carried out molecular genetic testing in patients recruited from the Simon Broome familial hypercholesterolaemia register⁶ in the UK as a cross‐sectional cohort study to identify risk factors for premature CHD in patients with familial hypercholesterolaemia.⁷ Primary results from this study indicated that the conventional cardiovascular risk factors of age, sex, smoking, pretreatment cholesterol levels and low levels of high‐density lipids (especially in women) were all associated with higher risk of CHD,⁷ confirming associations reported in other studies, for example.⁸ When this UK study was started, mutations at two loci causing familial hypercholesterolaemia had been identified, with mutations in the low‐density lipoprotein receptor gene (LDLR) accounting for most of the identified mutations,⁹ whereas one particular mutation in the gene encoding the ligand for the low density lipoprotein (LDL) receptor—namely apolipoprotein B (FDB)—occurs in about 5% of patients in the UK.¹⁰ This mutation, which alters a single amino acid (p.R3500Q), has been shown to reduce the affinity of the LDL cholesterol particle,¹⁰ where ApoB is the single‐protein component for the receptor. For the LDLR, currently >100 mutations have been reported in UK patients to date^9,11 (see also www.ucl.ac.uk/FH). A commercially available kit for screening for deletions and rearrangements of the LDLR gene has become available, and it is known that up to 5% of patients with familial hypercholesterolaemia in patients in the UK may have such a deletion.¹²Recently, defects in a third gene causing monogenic hypercholesterolaemia have been identified.¹³ The gene protein convertase subtilisin/kexin type 9 (PCSK9) codes for an enzyme that has also been called “neural apoptosis regulated convertase 1”, which has recently been proposed to be involved in degrading the LDLR protein in the lysosome of the cell and preventing it from recycling.¹⁴ Gain‐ of‐ function mutations in the PCSK9 gene could therefore cause increased degradation of LDLRs, reduced numbers of receptors on the surface of the cell, and monogenic hypercholesterolaemia. An alternative mechanism has also been proposed for the hypercholesterolaemic effect, whereby the gain of function causes increased secretion of apoB‐containing lipoproteins from the liver, with this being supported by in vivo turnover studies in patients carrying PCSK9 missense mutations¹⁵ and by in vitro studies in transiently transfected rat liver cells.¹⁶ One mutation in this gene, p.D374Y, has been reported in several independent families^16,17,18 and we therefore also tested for this cause of familial hypercholesterolaemia in this group of patients, as well as using single‐strand conformational polymorphism (SSCP) analysis and direct sequencing to screen all coding exons of the gene.Although patients with no identified mutation may have a monogenic cause of the disorder in an as yet undiscovered gene, it is also possible that some may have polygenic hypercholesterolaemia and have been misclassified using current clinical diagnostic criteria. These patients would be expected to have a milder degree of hyperlipidaemia, possibly not present from birth but only developing in later life, and would therefore be predicted to have a lower risk of CHD. The hypothesis we set out to test was that patients with identified mutations in the LDLR, PCSK9 or APOB genes would be at greater risk of CHD than patients with no identified mutation.     

Single,short in‐del,and copy number variations detection in monogenic dyslipidemia using a next‐generation sequencing strategy

Optimal molecular diagnosis of primary dyslipidemia is challenging to confirm the diagnosis, test and identify at risk relatives. The aim of this study was to test the application of a single targeted next‐generation sequencing (NGS) panel for hypercholesterolemia, hypocholesterolemia, and hypertriglyceridemia molecular diagnosis. NGS workflow based on a custom AmpliSeq panel was designed for sequencing the most prevalent dyslipidemia‐causing genes (ANGPTL3, APOA5, APOC2, APOB, GPIHBP1, LDLR, LMF1, LPL, PCSK9) on the Ion PGM Sequencer. One hundred and forty patients without molecular diagnosis were studied. In silico analyses were performed using the NextGENe software and homemade tools for detection of copy number variations (CNV). All mutations were confirmed using appropriate tools. Eighty seven variations and 4 CNV were identified, allowing a molecular diagnosis for 40/116 hypercholesterolemic patients, 5/13 hypocholesterolemic patients, and 2/11, hypertriglyceridemic patients respectively. This workflow allowed the detection of CNV contrary to our previous strategy. Some variations were found in previously unexplored regions providing an added value for genotype‐phenotype correlation and familial screening. In conclusion, this new NGS process is an effective mutation detection method and allows better understanding of phenotype. Consequently this assay meets the medical need for individualized diagnosis of dyslipidemia.     

Description of a Large Family with Autosomal Dominant Hypercholesterolemia Associated with the APOE p.Leu167del Mutation

Apolipoprotein (apo) E mutants are associated with type III hyperlipoproteinemia characterized by high cholesterol and triglycerides levels. Autosomal dominant hypercholesterolemia (ADH), due to the mutations in the LDLR, APOB, or PCSK9 genes, is characterized by an isolated elevation of cholesterol due to the high levels of low‐density lipoproteins (LDLs). We now report an exceptionally large family including 14 members with ADH. Through genome‐wide mapping, analysis of regional/functional candidate genes, and whole exome sequencing, we identified a mutation in the APOE gene, c.500_502delTCC/p.Leu167del, previously reported associated with sea‐blue histiocytosis and familial combined hyperlipidemia. We confirmed the involvement of the APOE p.Leu167del in ADH, with (1) a predicted destabilization of an alpha‐helix in the binding domain, (2) a decreased apo E level in LDLs, and (3) a decreased catabolism of LDLs. Our results show that mutations in the APOE gene can be associated with bona fide ADH.     

Genetic diagnosis of familial hypercholesterolemia in Han Chinese

Familial hypercholesterolemia (FH) is an inherited autosomal dominant disorder of lipoprotein metabolism resulting in elevated serum levels of low-density lipoprotein cholesterol (LDL-C), which lead to increased risk for premature cardiovascular disease. The recognized cause is mutations of the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), or proprotein convertase subtilisin/kexin type 9 genes. This study reviewed the literature in Han Chinese to investigate the frequency and spectrum of mutations that are recognized by molecular genetics as causes of FH, the clinical characteristics, and mutation detection rates of FH. MEDLINE, EMBASE, BIOSIS, Wanfang, CNKI, and FH websites, were reviewed through December 2014. Sixty-six studies met inclusion criteria. Totally, 143 different LDLR mutations were identified, including 134 point mutations and 9 large rearrangements; functional characteristics of 46 point mutations were studied. The 5 most frequent mutations included APOB 10579C>T, LDLR 986G>A, 1747C>T, 1879G>A, and 268G>A. Most of these mutations were reported in Southeast China, Hong Kong, and Taiwan. DNA detection rates of heterozygous FH were 6.5% to 77.5%, depending on the inclusion criteria and chosen screening method. With the economic growth and Western-like diet patterns being adopted over the past decade in municipalities in mainland China and Taiwan, the mean pretreatment concentration of LDL-C is higher among heterozygous FH patients reported since 2005 than in patients reported before 2005 (231 vs 196 mg/dL, P < .001). This review of DNA data for Han Chinese patients with FH updates the frequency and spectrum of FH scenarios. Large-scale investigations are needed to determine the interactions between mutations and LDL-C level in relation to cardiovascular risk assessment and management.     

Phenotypic and genotypic characterization of familial hypercholesterolemia in French adult and pediatric populations

BackgroundFamilial hypercholesterolemia (FH) is the most common genetic disorder associated with a high risk for premature atherosclerotic cardiovascular disease attributable to increased levels of LDL-cholesterol (LDL-C) from birth. FH is both underdiagnosed and undertreated.ObjectiveWe describe the clinical, biological, and genetic characteristics of 147 patients in France with clinical FH (including a group of 26 subjects aged < 20 years); we explore how best to detect patients with monogenic FH.MethodsWe retrospectively reviewed all available data on patients undergoing genetic tests for FH from 2009 to 2019. FH diagnoses were based on the Dutch Lipid Clinics Network (DLCN) scores of adults, and elevated LDL-C levels in subjects < 20 years of age. We evaluated LDLR, APOB, and PCSK9 status.ResultsThe mutations of adults (in 25.6% of all adults) were associated with DLCN scores indicating “possible FH,” "probable FH, and “definitive FH” at rates of 4%, 16%, and 53%, respectively. The areas under the ROC curves of the DLCN score and the maximum LDL-C level did not differ (p = 0.32). We found that the pediatric group evidenced more monogenic etiologies (77%, increasing to 91% when an elevated LDL-C level was combined with a family history of hypercholesterolemia and/or premature coronary artery disease).ConclusionDiagnosis of monogenic FH may be optimized by screening children in terms of their LDL-C levels, associated with reverse-cascade screening of relatives when the children serve as index cases.