Mohammad Ishaq

Factors Associated with Adherence to Anti-Hypertensive Treatment in Pakistan

Objectives Poor adherence is one of the biggest obstacles in therapeutic control of high blood pressure. The objectives of this study were (i) to measure adherence to antihypertensive therapy in a representative sample of the hypertensive Pakistani population and (ii) to investigate the factors associated with adherence in the studied population. Methods and Results A cross-sectional study was conducted on a simple random sample of 460 patients at the Aga Khan University Hospital (AKUH) and National Institute of Cardiovascular Diseases, Karachi, from September 2005–May 2006. Adherence was assessed using the Morisky Medication Adherence Scale (MMAS), with scores ranging from 0 (non-adherent) to 4 (adherent). In addition to MMAS, patient self-reports about the number of pills taken over a prescribed period were used to estimate adherence as a percentage. AKU Anxiety and Depression Scale (AKU-ADS) was incorporated to find any association between depression and adherence. At a cut-off value of 80%, 77% of the cases were adherent. Upon univariate analyses, increasing age, better awareness and increasing number of pills prescribed significantly improved adherence, while depression showed no association. Significant associations, upon multivariate analyses, included number of drugs that a patient was taking (P<0.02) and whether he/she was taking medication regularly or only for symptomatic relief (P<0.00001). Conclusions Similar to what has been reported worldwide, younger age, poor awareness, and symptomatic treatment adversely affected adherence to antihypertensive medication in our population. In contrast, monotherapy reduced adherence, whereas psychosocial factors such as depression showed no association. These findings may be used to identify the subset of population at risk of low adherence who should be targeted for interventions to achieve better blood pressure control and hence prevent complications.

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.

Causal Assessment of Serum Urate Levels in Cardiometabolic Diseases Through a Mendelian Randomization Study

BACKGROUND Although epidemiological studies have reported positive associations between circulating urate levels and cardiometabolic diseases, causality remains uncertain. OBJECTIVES Through a Mendelian randomization approach, we assessed whether serum urate levels are causally relevant in type 2 diabetes mellitus (T2DM), coronary heart disease (CHD), ischemic stroke, and heart failure (HF). METHODS This study investigated 28 single nucleotide polymorphisms known to regulate serum urate levels in association with various vascular and nonvascular risk factors to assess pleiotropy. To limit genetic confounding, 14 single nucleotide polymorphisms exclusively associated with serum urate levels were used in a genetic risk score to assess associations with the following cardiometabolic diseases (cases/controls): T2DM (26,488/83,964), CHD (54,501/68,275), ischemic stroke (14,779/67,312), and HF (4,526/18,400). As a positive control, this study also investigated our genetic instrument in 3,151 gout cases and 68,350 controls. RESULTS Serum urate levels, increased by 1 SD due to the genetic score, were not associated with T2DM, CHD, ischemic stroke, or HF. These results were in contrast with previous prospective studies that did observe increased risks of these 4 cardiometabolic diseases for an equivalent increase in circulating urate levels. However, a 1 SD increase in serum urate levels due to the genetic score was associated with increased risk of gout (odds ratio: 5.84; 95% confidence interval: 4.56 to 7.49), which was directionally consistent with previous observations. CONCLUSIONS Evidence from this study does not support a causal role of circulating serum urate levels in T2DM, CHD, ischemic stroke, or HF. Decreasing serum urate levels may not translate into risk reductions for cardiometabolic conditions.

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

Summary 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.

Association study of the angiotensin-converting enzyme (ACE) gene G2350A dimorphism with myocardial infarction

The angiotensin converting enzyme (ACE) is a strong candidate gene for myocardial infarction (MI). Insertion-deletion dimorphism in intron 16 of this gene has been inconclusively found to be associated with it. Several new polymorphisms in the ACE gene have been identified and among these, a dimorphism in exon 17, ACE G2350A, has a significant effect on plasma ACE concentrations. To assess the value of genotyping the ACE G2350A dimorphism in a genetically homogeneous population, we carried out a case-control study of dimorphism G2350A for a putative association with MI among Pakistani nationals. We investigated a sample population of 370 Pakistanis, comprising 163 controls, and 207 patients with clinical diagnosis of acute MI (AMI). ACE G2350A alleles were visualized by assays based on polymerase chain reaction and restriction endonuclease analysis. Frequencies of G alleles were 0.68 among controls and 0.72 among AMI patients. The ACE G2350A dimorphism showed no significant association with MI (χ2=0.90, 2 df, P=0.64), plasma levels of homocysteine (P=0.52) or with serum levels of folate (P=0.299). The results indicate that ACE G2350A polymorphism is not associated with risk of myocardial infarction in the Pakistani population investigated here.

Physical activity, smoking, and genetic predisposition to obesity in people from Pakistan: the PROMIS study.

BackgroundMultiple genetic variants have been reliably associated with obesity-related traits in Europeans, but little is known about their associations and interactions with lifestyle factors in South Asians.MethodsIn 16,157 Pakistani adults (8232 controls; 7925 diagnosed with myocardial infarction [MI]) enrolled in the PROMIS Study, we tested whether: a) BMI-associated loci, individually or in aggregate (as a genetic risk score - GRS), are associated with BMI; b) physical activity and smoking modify the association of these loci with BMI. Analyses were adjusted for age, age2, sex, MI (yes/no), and population substructure.ResultsOf 95 SNPs studied here, 73 showed directionally consistent effects on BMI as reported in Europeans. Each additional BMI-raising allele of the GRS was associated with 0.04 (SE = 0.01) kg/m2 higher BMI (P = 4.5 × 10−14). We observed nominal evidence of interactions of CLIP1 rs11583200 (Pinteraction = 0.014), CADM2 rs13078960 (Pinteraction = 0.037) and GALNT10 rs7715256 (Pinteraction = 0.048) with physical activity, and PTBP2 rs11165643 (Pinteraction = 0.045), HIP1 rs1167827 (Pinteraction = 0.015), C6orf106 rs205262 (Pinteraction = 0.032) and GRID1 rs7899106 (Pinteraction = 0.043) with smoking on BMI.ConclusionsMost BMI-associated loci have directionally consistent effects on BMI in Pakistanis and Europeans. There were suggestive interactions of established BMI-related SNPs with smoking or physical activity.

A novel interaction between the FLJ33534 locus and smoking in obesity: a genome-wide study of 14 131 Pakistani adults.

Background:Obesity is a complex disease caused by the interplay of genetic and lifestyle factors, but identification of gene–lifestyle interactions in obesity has remained challenging. Few large-scale studies have reported use of genome-wide approaches to investigate gene–lifestyle interactions in obesity.Methods:In the Pakistan Risk of Myocardial Infraction Study, a cross-sectional study based in Pakistan, we calculated body mass index (BMI) variance estimates (square of the residual of inverse-normal transformed BMI z-score) in 14 131 participants and conducted genome-wide heterogeneity of variance analyses (GWHVA) for this outcome. All analyses were adjusted for age, age2, sex and genetic ancestry.Results:The GWHVA analyses identified an intronic variant, rs140133294, in the FLJ33544 gene in association with BMI variance (P-value=3.1 × 10−8). In explicit tests of gene × lifestyle interaction, smoking was found to significantly modify the effect of rs140133294 on BMI (Pinteraction=0.0005), whereby the minor allele (T) was associated with lower BMI in current smokers, while positively associated with BMI in never smokers. Analyses of ENCODE data at the FLJ33534 locus revealed features indicative of open chromatin and high confidence DNA-binding motifs for several transcription factors, providing suggestive biological support for a mechanism of interaction.Conclusions:In summary, we have identified a novel interaction between smoking and variation at the FLJ33534 locus in relation to BMI in people from Pakistan.

Human knockouts 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.1 Null mutations can disrupt both copies of a given gene in humans and phenotypic analysis of such ‘human knockouts’ can provide insight into gene function. To date, comprehensive analysis of genes knocked out in humans has been limited by the fact that null mutations are infrequent in the general population and so, observing an individual homozygous null for a given gene is exceedingly rare.2,3 However, consanguineous unions are more likely to result in offspring who carry homozygous null mutations. In Pakistan, consanguinity rates are notably high.4 Here, we sequenced the protein-coding regions of 7,078 adult participants living in Pakistan and performed phenotypic analysis to identify homozygous null individuals and to understand consequences of complete gene disruption in humans. We enumerated 36,850 rare (<1 % minor allele frequency) null mutations. These homozygous null mutations led to complete inactivation of 961 genes in at least one participant. Homozygosity for null mutations at APOC3 was associated with absent plasma apolipoprotein C-III levels; at PLAG27, with absent enzymatic activity of soluble lipoprotein-associated phospholipase A2; at CYP2F1, with higher plasma interleukin-8 concentrations; and at either A3GALT2 or NRG4, with markedly reduced plasma insulin C-peptide concentrations. After physiologic challenge with oral fat, APOC3 knockouts displayed marked blunting of the usual post-prandial rise in plasma triglycerides compared to wild-type family members. These observations provide a roadmap to understand the consequences of complete disruption of a large fraction of genes in the human genome.