Refinement of variant selection for the LDL cholesterol genetic risk score in the diagnosis of the polygenic form of clinical familial hypercholesterolemia and replication in samples from 6 countries.

BACKGROUND: Familial hypercholesterolemia (FH) is an autosomal-dominant disorder caused by mutations in 1 of 3 genes. In the 60% of patients who are mutation negative, we have recently shown that the clinical phenotype can be associated with an accumulation of common small-effect LDL cholesterol (LDL-C)-raising alleles by use of a 12-single nucleotide polymorphism (12-SNP) score. The aims of the study were to improve the selection of SNPs and replicate the results in additional samples. METHODS: We used ROC curves to determine the optimum number of LDL-C SNPs. For replication analysis, we genotyped patients with a clinical diagnosis of FH from 6 countries for 6 LDL-C-associated alleles. We compared the weighted SNP score among patients with no confirmed mutation (FH/M-), those with a mutation (FH/M+), and controls from a UK population sample (WHII). RESULTS: Increasing the number of SNPs to 33 did not improve the ability of the score to discriminate between FH/M- and controls, whereas sequential removal of SNPs with smaller effects/lower frequency showed that a weighted score of 6 SNPs performed as well as the 12-SNP score. Metaanalysis of the weighted 6-SNP score, on the basis of polymorphisms in CELSR2 (cadherin, EGF LAG 7-pass G-type receptor 2), APOB (apolipoprotein B), ABCG5/8 [ATP-binding cassette, sub-family G (WHITE), member 5/8], LDLR (low density lipoprotein receptor), and APOE (apolipoprotein E) loci, in the independent FH/M- cohorts showed a consistently higher score in comparison to the WHII population (P < 2.2 × 10(-16)). Modeling in individuals with a 6-SNP score in the top three-fourths of the score distribution indicated a >95% likelihood of a polygenic explanation of their increased LDL-C. CONCLUSIONS: A 6-SNP LDL-C score consistently distinguishes FH/M- patients from healthy individuals. The hypercholesterolemia in 88% of mutation-negative patients is likely to have a polygenic basis.

Whole exome sequencing of familial hypercholesterolaemia patients negative for LDLR/APOB/PCSK9 mutations.

BACKGROUND: Familial hypercholesterolaemia (FH) is an autosomal dominant disease of lipid metabolism, which leads to early coronary heart disease. Mutations in LDLR, APOB and PCSK9 can be detected in 80% of definite FH (DFH) patients. This study aimed to identify novel FH-causing genetic variants in patients with no detectable mutation. METHODS AND RESULTS: Exomes of 125 unrelated DFH patients were sequenced, as part of the UK10K project. First, analysis of known FH genes identified 23 LDLR and two APOB mutations, and patients with explained causes of FH were excluded from further analysis. Second, common and rare variants in genes associated with low-density lipoprotein cholesterol (LDL-C) levels in genome-wide association study (GWAS) meta-analysis were examined. There was no clear rare variant association in LDL-C GWAS hits; however, there were 29 patients with a high LDL-C SNP score suggestive of polygenic hypercholesterolaemia. Finally, a gene-based burden test for an excess of rare (frequency <0.005) or novel variants in cases versus 1926 controls was performed, with variants with an unlikely functional effect (intronic, synonymous) filtered out. CONCLUSIONS: No major novel locus for FH was detected, with no gene having a functional variant in more than three patients; however, an excess of novel variants was found in 18 genes, of which the strongest candidates included CH25H and INSIG2 (p<4.3×10(-4) and p<3.7×10(-3), respectively). This suggests that the genetic cause of FH in these unexplained cases is likely to be very heterogeneous, which complicates the diagnostic and novel gene discovery process.

Analysis of the frequency and spectrum of mutations recognised to cause familial hypercholesterolaemia in routine clinical practice in a UK specialist hospital lipid clinic.

AIM: To determine the frequency and spectrum of mutations causing Familial Hypercholesterolaemia (FH) in patients attending a single UK specialist hospital lipid clinic in Oxford and to identify characteristics contributing to a high mutation detection rate. METHODS: 289 patients (272 probands) were screened sequentially over a 2-year period for mutations in LDLR, APOB and PCSK9 using standard molecular genetic techniques. The Simon Broome (SB) clinical diagnostic criteria were used to classify patients and a separate cohort of 409 FH patients was used for replication. RESULTS: An FH-causing mutation was found in 101 unrelated patients (LDLR = 54 different mutations, APOB p.(Arg3527Gln) = 10, PCSK9 p.(Asp374Tyr) = 0). In the 60 SB Definite FH patients the mutation detection rate was 73% while in the 142 with Possible FH the rate was significantly lower (27%, p < 0.0001), but similar (14%, p = 0.06) to the 70 in whom there was insufficient data to make a clinical diagnosis. The mutation detection rate varied significantly (p = 9.83 × 10(-5)) by untreated total cholesterol (TC) levels (25% in those <8.1 mmol/l and 74% in those >10.0 mmol/l), and by triglyceride levels (20% in those >2.16 mmol/l and 60% in those <1.0 mmol/l (p = 0.0005)), with both effects confirmed in the replication sample (p for trend = 0.0001 and p = 1.8 × 10(-6) respectively). There was no difference in the specificity or sensitivity of the SB criteria versus the Dutch Lipid Clinic Network score in identifying mutation carriers (AROC respectively 0.73 and 0.72, p = 0.68). CONCLUSIONS: In this genetically heterogeneous cohort of FH patients the mutation detection rate was significantly dependent on pre-treatment TC and triglyceride levels.

Use of targeted exome sequencing as a diagnostic tool for Familial Hypercholesterolaemia.

BACKGROUND: Familial Hypercholesterolaemia (FH) is an autosomal dominant disease, caused by mutations in LDLR, APOB or PCSK9, which results in high levels of LDL-cholesterol (LDL-C) leading to early coronary heart disease. An autosomal recessive form of FH is also known, due to homozygous mutations in LDLRAP1. This study assessed the utility of an exome capture method and deep sequencing in FH diagnosis. METHODS: Exomes of 48 definite FH patients, with no mutation detected by current methods, were captured by Agilent Human All Exon 50Mb assay and sequenced on the Illumina HiSeq 2000 platform. Variants were called by GATK and SAMtools. RESULTS: The mean coverage of FH genes varied considerably (PCSK9=23x, LDLRAP1=36x, LDLR=56x and APOB=93x). Exome sequencing detected 17 LDLR mutations, including three copy number variants, two APOB mutations, missed by the standard techniques, two LDLR novel variants likely to be FH-causing, and five APOB variants of uncertain effect. Two variants called in PCSK9 were not confirmed by Sanger sequencing. One heterozygous mutation was found in LDLRAP1. CONCLUSIONS: High-throughput DNA sequencing demonstrated its efficiency in well-covered DNA regions, in particular LDLR. This highly automated technology is proving to be effective for heterogeneous diseases and may soon replace laborious conventional methods. However, the poor coverage of gene promoters and repetitive, or GC-rich sequences, remains problematic, and validation of all identified variants is currently required.

Low-density lipoprotein receptor gene familial hypercholesterolemia variant database: update and pathological assessment.

Familial hypercholesterolemia (FH) is caused predominately by variants in the low-density lipoprotein receptor gene (LDLR). We report here an update of the UCL LDLR variant database to include variants reported in the literature and in-house between 2008 and 2010, transfer of the database to LOVDv.2.0 platform (https://grenada.lumc.nl/LOVD2/UCL-Heart/home.php?select_db=LDLR) and pathogenicity analysis. The database now contains over 1288 different variants reported in FH patients: 55% exonic substitutions, 22% exonic small rearrangements (<100 bp), 11% large rearrangements (>100 bp), 2% promoter variants, 10% intronic variants and 1 variant in the 3' untranslated sequence. The distribution and type of newly reported variants closely matches that of the 2008 database, and we have used these variants (n= 223) as a representative sample to assess the utility of standard open access software (PolyPhen, SIFT, refined SIFT, Neural Network Splice Site Prediction Tool, SplicePort and NetGene2) and additional analyses (Single Amino Acid Polymorphism database, analysis of conservation and structure and Mutation Taster) for pathogenicity prediction. In combination, these techniques have enabled us to assign with confidence pathogenic predictions to 8/8 in-frame small rearrangements and 8/9 missense substitutions with previously discordant results from PolyPhen and SIFT analysis. Overall, we conclude that 79% of the reported variants are likely to be disease causing.

The PCSK9 gene R46L variant is associated with lower plasma lipid levels and cardiovascular risk in healthy U.K. men.

In the present study, we have determined the relative frequency of the R46L, I474V and E670G variants in the PCSK9 (protein convertase subtilisin/kexin type 9) gene and its association with plasma lipid levels and CHD (coronary heart disease) in healthy U.K. men and patients with clinically defined definite FH (familial hypercholesterolaemia). Genotypes were determined using PCR and restriction enzyme digestion in 2444 healthy middle-aged (50-61 years) men from the prospective NPHSII (Second Northwick Park Heart Study), with 275 CHD events (15 years of follow-up), and in 597 U.K. FH patients from the Simon Broome Register. In the NPHSII healthy men, the R46L genotype distribution was in Hardy-Weinberg equilibrium and the frequency of 46L was 0.010 [95% CI (confidence interval), 0.007-0.013], with one man homozygous for the 46L allele. There was significant association of the 46L allele with lower mean (S.D.) total cholesterol [5.74 (1.01) mmol/l for RR compared with 5.26+/-1.03 mmol/l for RL; P=0.001], apolipoprotein B [0.87 (0.24) g/l for RR compared with 0.75 (0.26) g/l for RL; P<0.0001] and low-density lipoprotein cholesterol [4.01 (0.95) mmol/l for RR compared with 3.62 (0.97) mmol/l for RL; P=0.02]) levels, after adjustment for age, general medical practice, smoking, body mass index and systolic blood pressure. As expected, 46L carriers had a low risk of definite or possible CHD [hazard ratio, 0.46 (95% CI, 0.11-1.84)], but this was not statistically significant (P=0.27). Two other common PCSK9 variants I474V [V allele frequency, 0.179 (95% CI, 0.17-0.19)] and E670G [G allele frequency, 0.034 (CI, 0.03-0.04)] were not associated with any significant effects on lipid levels or CHD risk. In FH patients, the frequency of 46L was 0.003 (95% CI, 0.00-0.01), which was significantly lower (P=0.037) than the healthy subjects. In the four FH patients carrying 46L, mean untreated total cholesterol levels were not different (P=0.91) in carriers and non-carriers (median, 10.3 mmol/l compared with 10.2 mmol/l respectively, after adjustment for age, gender and mutation type). In conclusion, the PCSK9 46L allele is more frequent in healthy U.K. men than in FH patients and is strongly associated with a protective plasma lipid profile risk for CHD. Its low frequency (approx. 2% carriers) means that it does not make a major contribution to determining population CHD risk in the U.K.

The A370T variant (StuI polymorphism) in the LDL receptor gene is not associated with plasma lipid levels or cardiovascular risk in UK men.

Over 800 different missense mutations in the low density lipoprotein (LDL) receptor gene (LDLR) have been identified in patients with familial hypercholesterolaemia (FH). Only two of them, including the Alanine to Threonine change at position 370 (A370T), have been discovered in FH patients but do not cause FH. The frequency of the 370T allele has been reported worldwide to be between 0.022 and 0.070, with no clear association with high cholesterol levels or risk for coronary heart disease (CHD) and stroke. To explore this relationship in more detail we have determined this genotype in 2,659 healthy middle-aged (50-61 years) men participating in the prospective Second Northwick Park Heart Study, with 236 CHD and 67 stroke incident events. The genotype distribution was in Hardy-Weinberg equilibrium and in the no-event group the frequency of 370T was 0.046 (95% CI 0.040-0.052). Overall, there was no significant association of the 370T allele with any measured plasma lipid trait, and there was no difference in genotype distribution or allele frequency between the no-event and CHD (0.059; 95% CI 0.040-0.085) or stroke (0.037; 95% CI 0.012-0.085) groups ( p= 0.18 and 0.65, respectively). There was evidence for significant interaction ( p= 0.006) between body mass index (BMI) and genotype on CHD risk, with 370A homozygotes showing the expected higher CHD risk for those with higher BMI, whilst risk for 370T allele carriers was highest in men in the lowest tertile of BMI. The explanation for this association is unclear, and may simply be chance. Thus, these data confirm the absence of a significant impact of the A370T polymorphism on LDL receptor function, at least as measured by the effect on plasma lipid levels and CHD risk.

Three novel mutations in the apolipoprotein E gene in a sample of individuals with type 2 diabetes mellitus.

BACKGROUND: Apolipoprotein E (apoE) is found in association with triglyceride-rich lipoproteins and is the ligand for the removal of these particles from the plasma. Genetic variations in exon 4 lead to three common gene variants: E2, E3, and E4. METHODS: We performed apoE genotyping in 765 individuals with type 2 diabetes. RESULTS: We identified three new variant heteroduplex patterns. Sequencing of these variants revealed three novel mutations that were related to biochemical and clinical characteristics. One mutation produced a frameshift at amino acid position 166, which predicted termination of protein synthesis. This individual had a heteroduplex pattern and sequence of E3E3, which was associated with a change in the plasma isoelectric focusing pattern and a 70% lower plasma concentration of apoE compared with healthy individuals. The other mutations were both single base changes. A CGC>CAC change at amino acid position 150 predicted a substitution of Arg>His. This individual had a heteroduplex pattern and sequence of E2E2, which was not associated with major changes in plasma lipids or apoE concentration. The third individual had a CGC>CCC base change at amino acid position 114, which predicted an Arg>Pro change. This person had a heteroduplex pattern and sequence of E3E3, higher plasma total cholesterol, and moderately decreased plasma apoE. CONCLUSIONS: The frequency of new mutations in this sample (1 in 255) is higher than that of a healthy population (1 in 7900). Further screening for common apoE gene variants in individuals at risk for dyslipidemia may reveal abnormal heteroduplex patterns and uncover further mutations in this important lipid-regulating gene.

R643G polymorphism in PECAM-1 influences transendothelial migration of monocytes and is associated with progression of CHD and CHD events.

The 643R allele of R643G polymorphism (also known as R670G in the premature protein) in PECAM-1 has been associated with risk of myocardial infarction (MI), while the 643G allele has been associated with risk of coronary artery stenosis (CAS). The aim of this study was to investigate this apparently conflicting association. The association of R643G with risk of MI was determined in the second Northwick Park Heart study (2037 men with 138 CHD events; mean age: 56 years). Smokers homozygous for the 643R allele showed increased risk of MI with a hazard ratio of 2.47 (95% CI: 1.23-4.97; P=0.01) compared to smokers homozygous for the 643G allele. Progression of disease was determined in the Lopid Coronary Angiography Trial (279 men; mean age: 58.9 years). The 643G homozygotes showed greater focal (-0.08 +/- 0.02 mm) and diffuse (-0.01 +/- 0.01 mm) progression of CAS compared to 643R homozygotes (-0.02 +/- 0.02 mm and 0.001 +/- 0.01 mm, respectively; P=0.04). While there was no genotype effect on platelet aggregation, PECAM-1 tyrosine phosphorylation in HUVECs of GG genotype was 2.4-fold greater (P <0.01) than cells of RR genotype, and the level of transendothelial migration of monocytes of GG genotype was greater than that of monocytes of RR genotype following stimulation with either IL-1beta (12% higher, P <0.01) or TNF-alpha (10% higher, P=0.05). These data confirm the association of the R643G polymorphism with MI and CAS and suggest that greater influx of monocytes in individuals homozygous for the 643G may explain the association with CAS.