An Unbiased Lipid Phenotyping Approach To Study the Genetic Determinants of Lipids and Their Association with Coronary Heart Disease Risk Factors.

Direct infusion high-resolution mass spectrometry (DIHRMS) is a novel, high-throughput approach to rapidly and accurately profile hundreds of lipids in human serum without prior chromatography, facilitating in-depth lipid phenotyping for large epidemiological studies to reveal the detailed associations of individual lipids with coronary heart disease (CHD) risk factors. Intact lipid profiling by DIHRMS was performed on 5662 serum samples from healthy participants in the Pakistan Risk of Myocardial Infarction Study (PROMIS). We developed a novel semi-targeted peak-picking algorithm to detect mass-to-charge ratios in positive and negative ionization modes. We analyzed lipid partial correlations, assessed the association of lipid principal components with established CHD risk factors and genetic variants, and examined differences between lipids for a common genetic polymorphism. The DIHRMS method provided information on 360 lipids (including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, and sterol lipids), with a median coefficient of variation of 11.6% (range: 5.4-51.9). The lipids were highly correlated and exhibited a range of associations with clinical chemistry biomarkers and lifestyle factors. This platform can provide many novel insights into the effects of physiology and lifestyle on lipid metabolism, genetic determinants of lipids, and the relationship between individual lipids and CHD risk factors.

Identification of new susceptibility loci for type 2 diabetes and shared etiological pathways with coronary heart disease.

To evaluate the shared genetic etiology of type 2 diabetes (T2D) and coronary heart disease (CHD), we conducted a genome-wide, multi-ancestry study of genetic variation for both diseases in up to 265,678 subjects for T2D and 260,365 subjects for CHD. We identify 16 previously unreported loci for T2D and 1 locus for CHD, including a new T2D association at a missense variant in HLA-DRB5 (odds ratio (OR) = 1.29). We show that genetically mediated increase in T2D risk also confers higher CHD risk. Joint T2D-CHD analysis identified eight variants-two of which are coding-where T2D and CHD associations appear to colocalize, including a new joint T2D-CHD association at the CCDC92 locus that also replicated for T2D. The variants associated with both outcomes implicate new pathways as well as targets of existing drugs, including icosapent ethyl and adipocyte fatty-acid-binding protein.

Human knockouts and phenotypic analysis in a cohort with a high rate of consanguinity.

A major goal of biomedicine is to understand the function of every gene in the human genome. Loss-of-function mutations can disrupt both copies of a given gene in humans and phenotypic analysis of such 'human knockouts' can provide insight into gene function. Consanguineous unions are more likely to result in offspring carrying homozygous loss-of-function mutations. In Pakistan, consanguinity rates are notably high. Here we sequence the protein-coding regions of 10,503 adult participants in the Pakistan Risk of Myocardial Infarction Study (PROMIS), designed to understand the determinants of cardiometabolic diseases in individuals from South Asia. We identified individuals carrying homozygous predicted loss-of-function (pLoF) mutations, and performed phenotypic analysis involving more than 200 biochemical and disease traits. We enumerated 49,138 rare (<1% minor allele frequency) pLoF mutations. These pLoF mutations are estimated to knock out 1,317 genes, each in at least one participant. Homozygosity for pLoF mutations at PLA2G7 was associated with absent enzymatic activity of soluble lipoprotein-associated phospholipase A2; at CYP2F1, with higher plasma interleukin-8 concentrations; at TREH, with lower concentrations of apoB-containing lipoprotein subfractions; at either A3GALT2 or NRG4, with markedly reduced plasma insulin C-peptide concentrations; and at SLC9A3R1, with mediators of calcium and phosphate signalling. Heterozygous deficiency of APOC3 has been shown to protect against coronary heart disease; we identified APOC3 homozygous pLoF carriers in our cohort. We recruited these human knockouts and challenged them with an oral fat load. Compared with family members lacking the mutation, individuals with APOC3 knocked out displayed marked blunting of the usual post-prandial rise in plasma triglycerides. Overall, these observations provide a roadmap for a 'human knockout project', a systematic effort to understand the phenotypic consequences of complete disruption of genes in humans.

Apolipoprotein(a) isoform size, lipoprotein(a) concentration, and coronary artery disease: a mendelian randomisation analysis.

BACKGROUND: The lipoprotein(a) pathway is a causal factor in coronary heart disease. We used a genetic approach to distinguish the relevance of two distinct components of this pathway, apolipoprotein(a) isoform size and circulating lipoprotein(a) concentration, to coronary heart disease. METHODS: In this mendelian randomisation study, we measured lipoprotein(a) concentration and determined apolipoprotein(a) isoform size with a genetic method (kringle IV type 2 [KIV2] repeats in the LPA gene) and a serum-based electrophoretic assay in patients and controls (frequency matched for age and sex) from the Pakistan Risk of Myocardial Infarction Study (PROMIS). We calculated odds ratios (ORs) for myocardial infarction per 1-SD difference in either LPA KIV2 repeats or lipoprotein(a) concentration. In a genome-wide analysis of up to 17 503 participants in PROMIS, we identified genetic variants associated with either apolipoprotein(a) isoform size or lipoprotein(a) concentration. Using a mendelian randomisation study design and genetic data on 60 801 patients with coronary heart disease and 123 504 controls from the CARDIoGRAMplusC4D consortium, we calculated ORs for myocardial infarction with variants that produced similar differences in either apolipoprotein(a) isoform size in serum or lipoprotein(a) concentration. Finally, we compared phenotypic versus genotypic ORs to estimate whether apolipoprotein(a) isoform size, lipoprotein(a) concentration, or both were causally associated with coronary heart disease. FINDINGS: The PROMIS cohort included 9015 patients with acute myocardial infarction and 8629 matched controls. In participants for whom KIV2 repeat and lipoprotein(a) data were available, the OR for myocardial infarction was 0·93 (95% CI 0·90-0·97; p<0·0001) per 1-SD increment in LPA KIV2 repeats after adjustment for lipoprotein(a) concentration and conventional lipid concentrations. The OR for myocardial infarction was 1·10 (1·05-1·14; p<0·0001) per 1-SD increment in lipoprotein(a) concentration, after adjustment for LPA KIV2 repeats and conventional lipids. Genome-wide analysis identified rs2457564 as a variant associated with smaller apolipoprotein(a) isoform size, but not lipoprotein(a) concentration, and rs3777392 as a variant associated with lipoprotein(a) concentration, but not apolipoprotein(a) isoform size. In 60 801 patients with coronary heart disease and 123 504 controls, OR for myocardial infarction was 0·96 (0·94-0·98; p<0·0001) per 1-SD increment in apolipoprotein(a) protein isoform size in serum due to rs2457564, which was directionally concordant with the OR observed in PROMIS for a similar change. The OR for myocardial infarction was 1·27 (1·07-1·50; p=0·007) per 1-SD increment in lipoprotein(a) concentration due to rs3777392, which was directionally concordant with the OR observed for a similar change in PROMIS. INTERPRETATION: Human genetic data suggest that both smaller apolipoprotein(a) isoform size and increased lipoprotein(a) concentration are independent and causal risk factors for coronary heart disease. Lipoprotein(a)-lowering interventions could be preferentially effective in reducing the risk of coronary heart disease in individuals with smaller apolipoprotein(a) isoforms. FUNDING: British Heart Foundation, US National Institutes of Health, Fogarty International Center, Wellcome Trust, UK Medical Research Council, UK National Institute for Health Research, and Pfizer.

Two novel mutations in exon 3 and 4 of low density lipoprotein (LDL) receptor gene in patients with heterozygous familial hypercholesterolemia.

OBJECTIVE: To determine the common mutation of low density lipoprotein receptor in hypercholesterolemia patients requiring screening for heterozygous familial hypercholesterolemia (HeFH) in Karachi. STUDY DESIGN: Case-series. PLACE AND DURATION OF STUDY: Dr. Ziauddin Hospital Laboratory and Dr. Rubina Ghani's Pathological and Molecular Laboratories, Karachi, for the PCR bench work from June 2008 to October 2009. METHODOLOGY: All the patients selected for this study were from Dr. Ziauddin Hospital and National Institute of Cardiovascular Diseases. All the patients having high total cholesterol and LDL-cholesterol were included in this study with premature coronary artery diseases or a family history of hypercholesterolemia. Exclusion criteria included Diabetes mellitus, hypertension, renal disease, hypothyroidism and steroid therapy. After lipid profile with overnight fasting, DNA was extracted from whole blood collected in EDTA (ethylenediamine tetra acetic acid) tube and multiplex PCR (polymerase chain reaction) using forward and reverse primers of exons 3, 4, 9 and 14 of base pairs 162, 431, 550 and 496 respectively. RESULTS: Out of total of 120 hypercholesterolemia cases, 42 patients were classical cases of HeFH (heterozygous familial hypercholesterolemia) with xanthomas, xanthelasmas and LDL-C > 160 mg/dl. The total cholesterol (260± 57 mg/dL) and LDL-C (192 ± 39 mg/dL ) of cases was significantly high as compared to, controls having total cholesterol (184 ± 27 mg/dL) and LDL-C (105 ± 22 mg/dL), p > 0.001. Two novel point mutations were noted in exon 3 and exon 4. The other 78 cases were probable with raised LDL-C (low density lipoprotein cholesterol) and family history of premature coronary heart diseases. CONCLUSION: The frequency of HeFH was 35% classical and 65% probable cases out of total 120 hypercholesterolemia patients from two tertiary care hospitals in Karachi. The point mutation on exon 3 and exon 4 of LDLR gene was the most common. PCR is useful for the detection of large re-arrangements in the LDL-receptor gene and is a rapid and reliable method for diagnosis of familial hypercholesterolemia.

Genetic determinants of major blood lipids in Pakistanis compared with Europeans.

BACKGROUND: Evidence is sparse about the genetic determinants of major lipids in Pakistanis. METHODS AND RESULTS: Variants (n=45 000) across 2000 genes were assessed in 3200 Pakistanis and compared with 2450 Germans using the same gene array and similar lipid assays. We also did a meta-analysis of selected lipid-related variants in Europeans. Pakistani genetic architecture was distinct from that of several ethnic groups represented in international reference samples. Forty-one variants at 14 loci were significantly associated with levels of HDL-C, triglyceride, or LDL-C. The most significant lipid-related variants identified among Pakistanis corresponded to genes previously shown to be relevant to Europeans, such as CETP associated with HDL-C levels (rs711752; P<10(-13)), APOA5/ZNF259 (rs651821; P<10(-13)) and GCKR (rs1260326; P<10(-13)) with triglyceride levels; and CELSR2 variants with LDL-C levels (rs646776; P<10(-9)). For Pakistanis, these 41 variants explained 6.2%, 7.1%, and 0.9% of the variation in HDL-C, triglyceride, and LDL-C, respectively. Compared with Europeans, the allele frequency of rs662799 in APOA5 among Pakistanis was higher and its impact on triglyceride concentration was greater (P-value for difference <10(-4)). CONCLUSIONS: Several lipid-related genetic variants are common to Pakistanis and Europeans, though they explain only a modest proportion of population variation in lipid concentration. Allelic frequencies and effect sizes of lipid-related variants can differ between Pakistanis and Europeans.

Association of the 9p21.3 locus with risk of first-ever myocardial infarction in Pakistanis: case-control study in South Asia and updated meta-analysis of Europeans.

OBJECTIVE: To examine variants at the 9p21 locus in a case-control study of acute myocardial infarction (MI) in Pakistanis and to perform an updated meta-analysis of published studies in people of European ancestry. METHODS AND RESULTS: A total of 1851 patients with first-ever confirmed MI and 1903 controls were genotyped for 89 tagging single-nucleotide polymorphisms at locus 9p21, including the lead variant (rs1333049) identified by the Wellcome Trust Case Control Consortium. Minor allele frequencies and extent of linkage disequilibrium observed in Pakistanis were broadly similar to those seen in Europeans. In the Pakistani study, 6 variants were associated with MI (P<10(-2)) in the initial sample set, and in an additional 741 cases and 674 controls in whom further genotyping was performed for these variants. For Pakistanis, the odds ratio for MI was 1.13 (95% CI, 1.05 to 1.22; P=2 x 10(-3)) for each copy of the C allele at rs1333049. In comparison, a meta-analysis of studies in Europeans yielded an odds ratio of 1.31 (95% CI, 1.26 to 1.37) for the same variant (P=1 x 10(-3) for heterogeneity). Meta-analyses of 23 variants, in up to 38,250 cases and 84,820 controls generally yielded higher values in Europeans than in Pakistanis. CONCLUSIONS: To our knowledge, this study provides the first demonstration that variants at the 9p21 locus are significantly associated with MI risk in Pakistanis. However, association signals at this locus were weaker in Pakistanis than those in European studies.

The Pakistan Risk of Myocardial Infarction Study: a resource for the study of genetic, lifestyle and other determinants of myocardial infarction in South Asia.

The burden of coronary heart disease (CHD) is increasing at a greater rate in South Asia than in any other region globally, but there is little direct evidence about its determinants. The Pakistan Risk of Myocardial Infarction Study (PROMIS) is an epidemiological resource to enable reliable study of genetic, lifestyle and other determinants of CHD in South Asia. By March 2009, PROMIS had recruited over 5,000 cases of first-ever confirmed acute myocardial infarction (MI) and over 5,000 matched controls aged 30-80 years. For each participant, information has been recorded on demographic factors, lifestyle, medical and family history, anthropometry, and a 12-lead electrocardiogram. A range of biological samples has been collected and stored, including DNA, plasma, serum and whole blood. During its next stage, the study aims to expand recruitment to achieve a total of about 20,000 cases and about 20,000 controls, and, in subsets of participants, to enrich the resource by collection of monocytes, establishment of lymphoblastoid cell lines, and by resurveying participants. Measurements in progress include profiling of candidate biochemical factors, assay of 45,000 variants in 2,100 candidate genes, and a genomewide association scan of over 650,000 genetic markers. We have established a large epidemiological resource for CHD in South Asia. In parallel with its further expansion and enrichment, the PROMIS resource will be systematically harvested to help identify and evaluate genetic and other determinants of MI in South Asia. Findings from this study should advance scientific understanding and inform regionally appropriate disease prevention and control strategies.