Novel and recurrent LDLR gene mutations in Pakistani hypercholesterolemia patients.

The majority of patients with the autosomal dominant disorder familial hypercholesterolemia (FH) carry novel mutations in the low density lipoprotein receptor (LDLR) that is involved in cholesterol regulation. In different populations the spectrum of mutations identified is quite different and to date there have been only a few reports of the spectrum of mutations in FH patients from Pakistan. In order to identify the causative LDLR variants the gene was sequenced in a Pakistani FH family, while high resolution melting analysis followed by sequencing was performed in a panel of 27 unrelated sporadic hypercholesterolemia patients. In the family a novel missense variant (c.1916T > G, p.(V639G)) in exon 13 of LDLR was identified in the proband. The segregation of the identified nucleotide change in the family and carrier status screening in a group of 100 healthy subjects was done using restriction fragment length polymorphism analysis. All affected members of the FH family carried the variant and none of the non-affected members nor any of the healthy subjects. In one of the sporadic cases, two sequence changes were detected in exon 9, one of these was a recurrent missense variant (c.1211C > T; p.T404I), while the other was a novel substitution mutation (c.1214 A > C; N405T). In order to define the allelic status of this double heterozygous individual, PCR amplified fragments were cloned and sequenced, which identified that both changes occurred on the same allele. In silico tools (PolyPhen and SIFT) were used to predict the effect of the variants on the protein structure, which predicted both of these variants to have deleterious effect. These findings support the view that there will be a novel spectrum of mutations causing FH in patients with hypercholesterolaemia from Pakistan.

Healthy individuals carrying the PCSK9 p.R46L variant and familial hypercholesterolemia patients carrying PCSK9 p.D374Y exhibit lower plasma concentrations of PCSK9.

BACKGROUND: We measured plasma PCSK9 concentrations in healthy men with a PCSK9 (proprotein convertase subtilisin/kexin type 9) loss-of-function variant (p.R46L), in statin-treated patients with a clinical diagnosis of familial hypercholesterolemia (FH) and carrying a PCSK9 gain-of-function mutation (p.D374Y), and in statin-treated patients with FH due to different genetic causes. METHODS: PCSK9 was measured with a previously described ELISA. RESULTS: In 81 healthy middle-aged Caucasian men, the PCSK9 concentration was significantly associated with the concentrations of total cholesterol (r = 0.42; P < 0.0001), LDL cholesterol (r = 0.34; P = 0.01), and triglycerides (r = 0.25; P = 0.02). In p.R46L carriers, mean (SD) concentrations of PCSK9 were 15% lower than in RR individuals [65.5 microg/L (21.6 microg/L) vs 77.5 microg/L (18.2 microg/L); P = 0.03]. In patients with the p.D374Y variant (n = 7), the mean PCSK9 concentration was significantly lower than in the combined group of patients with an LDLR (low density lipoprotein receptor) mutation (n = 25), an APOB [apolipoprotein B (including Ag(x) antigen)] variant encoding p.R3527Q (n = 6), or no detectable mutation (n = 14) [96.4 microg/L (42.5 microg/L) vs 151.6 microg/L (69.6 microg/L); P = 0.02]. Two of the 14 patients with no mutation had PCSK9 concentrations below the mean for p.D374Y carriers; sequencing of the PCSK9 gene and promoter revealed no mutations. Among 409 FH patients, we identified 6 carriers of the promoter variant -287G>A (1.5%), a frequency similar to that (1.0%) previously reported for 2772 healthy men in the UK. In neither group was the -287G>A variant associated with differences in lipid traits. CONCLUSIONS: The loss-of-function p.R46L variant is associated with the expected lower concentrations of circulating PCSK9; the gain-of-function p.D374Y mutation is also associated with lower concentrations, presumably because of the higher affinity of this variant for the LDL receptor and its more rapid clearance. In treated FH patients, a low plasma PCSK9 concentration does not appear to be a useful screening tool for identifying novel PCSK9 mutations.

Mutation scanning by meltMADGE: validations using BRCA1 and LDLR, and demonstration of the potential to identify severe, moderate, silent, rare, and paucimorphic mutations in the general population.

We have developed a mutation-scanning approach suitable for whole population screening for unknown mutations. The method, meltMADGE, combines thermal ramp electrophoresis with MADGE to achieve suitable cost efficiency and throughput. The sensitivity was tested in blind trials using 54 amplicons representing the BRCA1 coding region and a panel of 94 unrelated family breast cancer risk consultands previously screened in a clinical diagnostic laboratory. All 10 common polymorphisms, 15/15 previously identified disease-causing mutations, and three previously untested single base changes were identified. Assays of LDLR exons 3 and 8 were validated in 460 familial hypercholesteremics and detected 8/9 known variants. We then applied the exon 3 assay in several DNA banks representing approximately 8000 subjects with known cholesterol values and applied both assays in one DNA bank (n = 3600). In exon 3 we identified one previously reported moderate mutation, P84S (n = 1), also associated with moderate hypercholesteremia in this subject; an unreported silent variant, N76N (n = 1); and known severe hypercholesteremia splice mutation 313+1G-->A (n = 2). Around exon 8 we identified a paucimorphism (n = 35) at the splice site 1061-8T-->C (known to be in complete linkage disequilibrium with T705I) and unreported sequence variants 1186+11G-->A (n = 1) and D335N G-->A (n = 1). The cholesterol value for D335N was on the 96.2 percentile and for T705I, 2/35 carriers were above the 99th percentile. Thus, variants with predicted severe, moderate, and no effect were identified at the population level. In contrast with case collections, CpG mutations predominated. MeltMADGE will enable definition of the full population spectrum of rare, paucimorphic, severe, moderate (forme fruste), and silent mutations and effects.

Serum homocysteine concentrations, gemfibrozil treatment, and progression of coronary atherosclerosis.

The present study aimed to assess the effect of gemfibrozil on serum total homocysteine (tHcy) concentration and to evaluate the influence of tHcy on the angiographically determined progression of coronary atherosclerosis in a randomised, placebo-controlled trial of 395 post-coronary bypass men with low HDL cholesterol levels. The baseline levels of tHcy and those after 16 months of randomised therapy were measured by high-pressure liquid chromatography. Patients were genotyped for the thermolabile variant of N5,N10-methylenetetrahydrofolate reductase (MTHFR) (677C > T substitution). Gemfibrozil therapy was associated with a median 18% increase in tHcy levels (P < 0.01). In the gemfibrozil group increases in tHcy and HDL cholesterol were related (r = 0.217, P = 0.004), but changes in tHcy and triglycerides were not. Levels of tHCy were not associated with baseline extent or progression of coronary-artery disease. Subjects homozygous for the rare MTHFR T allele had 34% higher median tHcy concentrations than CC homozygotes or CT heterozygotes, but responses to gemfibrozil did not differ significantly among genotypes. The MTHFR genotype was not associated with extent or progression of coronary atherosclerosis. We conclude that gemfibrozil causes a significant elevation in tHcy levels, but the clinical relevance of this is unknown at present.