Study of variants associated with ventricular septal defects (VSDs) highlights the unique genetic structure of the Pakistani population.

BACKGROUND: Ventricular septal defects (VSDs) are one of the leading causes of death due to cardiac anomalies during the first months of life. The prevalence of VSD in neonates is reported up to 4%. Despite the remarkable progress in medication, treatment and surgical procedure for VSDs, the genetic etiology of VSDs is still in infancy because of the complex genetic and environmental interactions. METHODS: Three hundred fifty subjects (200 VSD children and 150 healthy controls) were recruited from different pediatric cardiac units. Pediatric clinical and demographic data were collected. A total of six variants, rs1017 (ISL1), rs7240256 (NFATc1), rs36208048 (VEGF), variant of HEY2, rs11067075 (TBX5) and rs1801133 (MTHFR) genes were genotyped by tetra-ARMS PCR and PCR-RFLP methods. RESULTS: The results showed that in cases, the rs1017 (g.16138A > T) variant in the ISL1 gene has an allele frequency of 0.42 and 0.58 respectively for the T and A alleles, and 0.75 and 0.25 respectively in the controls. The frequencies of the AA, TA and TT genotypes were, 52%, 11% and 37% in cases versus 21%, 8% and 71% respectively in the controls. For the NFATc1 variant rs7240256, minor allele frequency (MAF) was 0.43 in cases while 0.23 in controls. For the variant in the VEGF gene, genotype frequencies were 0% (A), 32% (CA) and 68% (CC) in cases and 0.0%, 33% and 67% respectively in controls. The allele frequency of C and A were 0.84 and 0.16 in cases and 0.83 and 0.17 respectively in controls. The TBX5 polymorphism rs11067075 (g.51682G > T) had an allelic frequency of 0.44 and 0.56 respectively for T and G alleles in cases, versus 0.26 and 0.74 in the controls. We did not detect the presence of the HEY2 gene variant (g.126117350A > C) in our pediatric cohort. For the rs1801133 (g.14783C > T) variant in the MTHFR gene, the genotype frequencies were 25% (CC), 62% (CT) and 13% (TT) in cases, versus 88%, 10% and 2% in controls. The ISL1, NFATc1, TBX5 and MTHFR variants were found to be in association with VSD in the Pakistani pediatric cohort whilst the VEGF and HEY2 variants were completely absent in our cohort. CONCLUSION: We propose that a wider programme of genetic screening of the Pakistani population for genetic markers in heart development genes would be helpful in reducing the risk of VSDs.

Lack of Association of LPA Gene Polymorphisms with Coronary Artery Disease in Pakistani Subjects.

Coronary artery disease (CAD) is the leading cause of death worldwide. Pakistan faces a high epidemic of CAD, and the disease burden is increasing with the passage of time. Several genetic markers have been reported to be significantly associated with CAD; one of them is the lipoprotein A gene. The aim of the current investigation was to genotype the LPA gene SNPs, rs3798220 and rs10455872, in Pakistani subjects with CAD in a case control study design. The genotyping was done by TaqMan allelic discrimination assay. The results showed that the cases had significantly higher prevalence of diabetes (64.6%), hypertension (62.1%), and smoking habits (29.5%). The level of cholesterol in cases was higher than in controls (208.25 ± 54.11 vs. 175.34 ± 43.51, p ≤ 0.0001). The LDL-C was higher in cases than in controls (104.62 ± 37.94 vs. 77.05 ± 21.17, p ≤ 0.0001). Similarly, triglycerides were also higher in cases than in controls (214.51 ± 74.60 vs. 190.54 ± 70.26, p ≤ 0.0001), whereas HDL-C was lower in cases than in controls (45.13 ± 11.63 vs. 67.9 ± 17.57, p ≤ 0.0001). For rs3798220, the risk allele (C) frequency was 0.005 in cases and 0.002 in controls. For rs10455872, the risk allele (G) frequency was 0.017 in cases and 0.014 in controls. The risk allele frequencies were not significantly different between cases and controls (p > 0.05). In conclusion, these two LPA SNPs do not contribute significantly to CAD progression and cannot be used as independent risk factors for CAD in Pakistani population.

First report of polymorphisms in MTRR, GATA4, VEGF, and ISL1 genes in Pakistani children with isolated ventricular septal defects (VSD).

BACKGROUND: Ventricular septal defects (VSDs) are malformations in the septum separating the heart's ventricles. VSDs may present as a single anomaly (isolated/nonsyndromic VSD) or as part of a group of phenotypes (syndromic VSD). The exact location of the defect is crucial in linking the defect to the underlying genetic cause. The number of children visiting cardiac surgery units is constantly increasing. However, there are no representative data available on the genetics of VSDs in Pakistani children. METHODS: Two hundred forty-two subjects (121 VSD children and 121 healthy controls) were recruited from pediatric cardiac units of Lahore. The clinical and demographic data of the subjects were collected. A total of four SNPs, one each from MTRR, GATA4, VEGF, and ISL1 genes were genotyped by PCR-RFLP. RESULTS: The results showed that the minor allele (T) frequency (MAFs) for the MTRR gene variant rs1532268 (c.524C > T) was 0.20 and 0.41 in the controls and the cases, respectively, with the genotype frequencies 3, 35, 62% in the controls and 12, 59 and 29% in the cases for TT, CT, CC genotypes, respectively (allelic OR: 5.73, CI: 3.82-8.61, p-value: 5.11 × 10- 7). For the GATA4 variant rs104894073 (c.886G > A), the MAF for the controls and the cases was 0.16 and 0.37, respectively, the frequencies of AA, GA and GG genotypes were 2, 28, and 70% in the controls and 5, 64 and 31% of the cases (allelic OR: 3.08, CI: 2.00-4.74, p-value: 8.36 × 10- 8). The rs699947 (c.-2578C > A) of VEGF gene showed MAF 0.36 and 0.53 for the controls and cases, respectively, with the genotype frequencies 13, 42, and 45% in the controls and 22, 15, and 63% in the cases for the AA, CA, CC (allelic OR: 2.03, CI: 1.41-2.92, p-value: 0.0001). The ISL1 gene variant rs6867206 (g.51356860 T > C), the MAFs were 0.26 and 0.31 in the controls and cases, respectively. The genotype frequencies were 48, 52, 0% in the controls and 39, 61, 0% in the cases for TT, TC, CC genotypes (allelic OR: 0.27, CI: 0.85-1.89, p-value: 0.227). The MTRR, GATA4 and VEGF variants showed association while ISL1 variant did not appear to be associated with the VSD in the recruited cohort. CONCLUSION: This first report in Pakistani children demonstrates that single nucleotide polymorphisms in genes encoding transcription factors, signaling molecules and structural heart genes involved in fetal heart development are associated with developmental heart defects., however further work is needed to validate the results of the current investigation.

The abnormal lipid profile in obesity and coronary heart disease (CHD) in Pakistani subjects.

BACKGROUND: Obesity has become global epidemic in the last three decades, whereas Coronary Heart Disease (CHD) still remains the most important cause of mortality in the world. The study was aimed at determining the pattern of lipid profile for the obese and CHD population in Pakistan. As obesity is a strong predisposing risk factor for CHD, we aimed to analyze the lipid parameters in both conditions and compare them with the healthy controls of the same ethnicity. METHODS: Blood samples were collected from one thousand individuals (500 with CHD, 250 with obesity, 250 healthy controls). The lipid profile (total Cholesterol, triglycerides, HDL-C, LDL-C and VLDL) was measured using commercially available kits. The pattern of dyslipidemia was then studied by comparing the results in both groups with controls as well as population cutoffs. The quantitative variables were checked for normality and log transformation was done for variables where appropriate. Analysis of variance and logistic regression were done to check the association of lipid parameters with obesity and CHD. RESULTS: The obese and CHD groups showed a dyslipidemic profile than the healthy controls. CHD group had a higher proportion of CHD in any of the first degree blood relatives (36.0% vs. 1.8%), a similar trend was observed in the obese group, where 63.9% cases had positive family history. Among cases, 50.7% had combined lipid abnormalities, i.e., the values of TC, LDL-C, TG and HDL-C, all were deranged. Whereas 49.52% had TC more than normal cut off (> 200 mg/dl), 51.6% had LDL-C > 100 mg/dl. Similarly, 80.4% of patients had TG levels more than upper normal range (> 150 mg/dl) and 64% had HDL values in moderate CHD risk group (< 50 mg/dl). The results show that Pakistani cases are hyperlipidemic for lipid traits except for HDL which is lowered. Patients with comorbidities also had lipid profiles deviated from the normal range. CONCLUSION: The study provides information regarding the aberration of lipid profile in the metabolic disorders that can increase the predisposition to complications.

Genetic Contribution to Congenital Heart Disease (CHD).

Congenital heart defects (CHD) are the most common congenital problems in neonates. The basis for CHD is multifactorial, involving genetic and environmental components. The elucidation of genetic components remains difficult because it is a genetically heterogeneous disease. Currently, the major identified genetic causes include chromosomal abnormalities, large subchromosomal deletions/duplications, and point mutations. However, much more remains to be unraveled. An important insight from the research on the genetics of CHD is that any change at the genetic level that alters the dosage of genes required in any process during heart development results in a developmental defect. The use of conventional gene identification (linkage analysis and direct targeted sequencing) methods followed by the rapid advancements in high-throughput technologies (copy number variant platforms, SNP arrays, and next-generation sequencing) has identified an extensive list of genetic causes. However, the most common presentation of CHD is in the form of sporadic cases. Therefore, it is important to identify their underlying genetic cause. In this review, we revisit the causal genetic factors of CHD and discuss the clinical implications of research in the field.

The combined role of allelic variants of IRS-1 and IRS-2 genes in susceptibility to type2 diabetes in the Punjabi Pakistani subjects.

BACKGROUND: Diabetes mellitus is a multifactorial disorder characterized by a high level of glucose in the blood. Both genetic and environmental factors interact to cause diabetes. Insulin receptor substrate (IRS) proteins have a significant part in insulin signaling pathways. We aimed to investigate the relationship of type 2 diabetes with a Gly972Arg (G972R) variant of the IRS-1 gene and Gly1057Asp (G1057D) polymorphism of IRS-2 gene in the population of Punjab, Pakistan. METHODS: We collected 926 samples, 500 healthy controls (fasting blood sugar < 99 mg/dL, random blood sugar < 126 mg/dL) and 426 cases with diabetes (fasting blood sugar > 99 mg/dL, random blood sugar > 126 mg/dL). Several anthropometric measurements were measured. Statistical analysis was performed by using SPSS to determine the allele group/genotype frequency of the selected variants in the study population. RESULTS: The genotyping results of G972R by RLFP-PCR showed the allelic frequency of G = 0.68 and R = 0.32 in controls while G = 0.71 and R = 0.29 in the cases. The minor R allele had a slightly higher frequency in the cases than the controls (OR = 0.86, CI 0.706-1.052, p = 0.17). The genotyping results of G1057D showed allelic frequency G = 0.74 and D = 0.26 in the controls while G = 0.961 and D = 0.29 in the cases. The minor D allele appeared to be a risk allele for this SNP although the difference in the allele frequencies was not statistically significant (OR = 1.55, CI 0.961-1.41, p = 0.108). The combined genotype analysis showed that the difference in the allele and genotype frequencies reached statistical difference between the cases and the controls as well as the odds ratio substantially increased when the R allele (G972R) was present together with D allele (G1057D) in any combination. When the association of single variants with the lipid traits was observed, only D allele (G1057D) showed significant association with TG, HDL and LDL, however when the analysis was repeated for combined genotypes using general linear model, many more significant associations between the genotype where D allele and R allele are together, were seen with many lipid traits. CONCLUSION: In conclusion, the single nucleotide polymorphisms with low-modest effect size may not affect the phenotype individually but when in combination, the effect becomes stronger and more visible, therefore, for the SNP association studies, the more the number of SNPs included in the analysis, the more meaningful the results.

Genetic risk score (GRS) constructed from polymorphisms in the PON1, IL-6, ITGB3, and ALDH2 genes is associated with the risk of coronary artery disease in Pakistani subjects.

BACKGROUND: Coronary artery disease (CAD) is a major killer in today's world. Pakistan is also affected by this non-communicable disease like other countries. It is a multifactorial disease and is influenced by many gene-gene and gene-environment interactions. METHODS: A total of 623 (219 controls, 404 cases) Pakistani subjects were genotyped for four SNPs, rs662 (PON1), rs5918 (ITGB3), rs671 (ALDH2), rs1800795 (IL-6) by PCR-RFLP. Various anthropometric parameters were noted and serum lipid profile was measured using commercially available kits. Statistical analysis was done by SPSS version 22. A Genetic Risk Score (GRS) was calculated from individual SNPs. The association of the SNPs and the GRS with CAD was checked using logistic regression. RESULTS: The results showed that the risk allele frequencies of all variants were higher in the cases than the controls, however the difference was not statistically significant association (p > 0.0125). The mean GRS in the controls was 3.99 ± 1.42 and in cases, it was 4.29 ± 1.39, the difference between the groups was significant (p = 0.0109). logistic regression of individual SNPs and GRS with the CAD showed that independent SNPs were not significantly associated with the CAD however, the GRS had a strong association (p = 1.4 × 10- 4). The subjects were divided into three groups based on GRS (Gp 1 with GRS 0-2, Gp 2 with GRS 3-5 and Gp 3 with GRS 6-8). The analysis of the effect of the individual SNPs and GRS groups on different lipid profile parameters revealed no significant association of any of the tested SNPs with any lipid parameter, however, the GRS groups showed marginally significant for TC and highly significant association for TG, LDL-c and HDL-c. CONCLUSION: In conclusion, use of a GRS can provide better information than individual SNPs. The larger the number of the SNPs included in the analysis, the better would be the risk prediction.

Use of a gene score of multiple low-modest effect size variants can predict the risk of obesity better than the individual SNPs.

BACKGROUND: Obesity is a complex disorder, the development of which is modulated by a multitude of environmental, behavioral and genetic factors. The common forms of obesity are polygenic in nature which means that many variants in the same or different genes act synergistically and affect the body weight quantitatively. The aim of the current study was to use information from many common variants previously identified to affect body weight to construct a gene score and observe whether it improves the associations observed. The SNPs selected were G2548A in leptin (LEP) gene, Gln223Arg in leptin receptor (LEPR) gene, Ala54Thr in fatty acid binding protein 2 (FABP2) gene, rs1121980 in fat mass and obesity associated (FTO) gene, rs3923113 in Growth Factor Receptor Bound Protein 14 (GRB14), rs16861329 in Beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1), rs1802295 in Vacuolar protein sorting-associated protein 26A (VPS26A), rs7178572 in high mobility group 20A (HMG20A), rs2028299 in adaptor-related protein complex 3 (AP3S2), and rs4812829 in Hepatocyte Nuclear Factor 4 Alpha (HNF4A). METHODS: A total of 475 subjects were genotyped for the selected SNPs in different genes using different genotyping techniques. The study subjects' age, weight, height, BMI, waist and hip circumference, serum total cholesterol, triglycerides, LDL and HDL were measured. A summation term, genetic risk score (GRS), was calculated using SPSS. RESULTS: The results showed a significantly higher mean gene score in obese cases than in non-obese controls (9.1 ± 2.26 vs 8.35 ± 2.07, p = 2 × 10- 4). Among the traits tested for association, gene score appeared to significantly affect BMI, waist circumference, and all lipid traits. CONCLUSION: In conclusion, the use of gene score is a better way to calculate the overall genetic risk from common variants rather than individual risk variants.

Identification of genetic basis of obesity and mechanistic link of genes and lipids in Pakistani population.

We aimed to identify the genetic causes of common forms of obesity in the Pakistani people and find out the mechanistic link by observing the relationship of genes and serum lipid traits. Four hundred and seventy-five subjects were genotyped for two mutations in (leptin:N103K and proopiomelanocortin:R236G) and ten common variants in different genes. Serum lipids were also measured. The prevalence of mutations was very low (one heterozygous subject each for both mutations), but fairly high minor/risk allele frequency (M/RAF) was observed for all SNPs. MAF of G2548A was 42.8% in obese and 30.1% in controls (P=5.7 × 10-5), it showed association with weight, body mass index (BMI), waist circumference (WC), high density lipoprotein cholesterol (HDL-c) and leptin, Gln223Arg had MAF 32% in obese and 18.7% in controls (P=5.4 × 10-6), it showed association with fasting plasma glucose (FPG) and all lipid traits, Ala54Thr had MAF 42.4% in obese and 33.1% (P=0.002), it showed association with none of the tested parameters. rs9939609 MAF was 26.6%, and showed association with none of the tested parameters. rs1802295 (P=0.002); rs7178572 (P=0.007); rs2028299 (P=0.04); rs4812829 (P=0.02) showed significant while rs3923113 and rs16861329 did not show a significant association (P=0.20 and P=0.3, respectively) with obesity. Major genetic contribution to common forms of obesity in Pakistan is from low/modest effect size common variants that act additively to affect body weight quantitatively and mechanism may involve modulating serum lipids.

GWAS implicated risk variants in different genes contribute additively to increase the risk of coronary artery disease (CAD) in the Pakistani subjects.

BACKGROUND: Coronary artery disease (CAD) remains the single most important cause of mortality worldwide. Many candidate and GWAS genetic variants have been identified in the recent years. In the current study, we selected six SNPs from various genes that have originally been identified in GWAS studies and examined the association of SNPs individually and as a genetic risk score (GRS) with CAD and blood lipid levels in the Pakistani subjects. METHODS: Six hundred twenty-four (404 cases and 219 controls) subjects were genotyped for variants rs10757274 in CDKN2A gene, rs17465637 in MIA3 gene, rs7025486 in DAB2IP gene, rs17228212 in SMAD3 gene, rs981887 in MRAS gene and rs1746048 in CXCL12 gene, by TaqMan and KASPar allele discrimination techniques. Serum lipid parameters were measured using commercially available kits. Statistical analyses were done using SPSS version 22. RESULTS: Individually, the single SNPs were not associated with CAD (p < 0.05). However, the combined GRS of 6 SNPs was significantly higher in cases than controls (4.89 ± 0.11 vs 4.58 ± 0.08, p = 0.024). Among blood lipids, GRS showed significant positive association with serum triglycerides levels (p = 0.022). CONCLUSION: The GRS was quantitatively associated with CAD risk and showed association with serum triglycerides levels, suggesting that the mechanism of these variants is likely to be in part at least through creating an atherogenic lipid profile in subjects carrying high numbers of risk alleles.

The SNP rs10911021 is associated with oxidative stress in coronary heart disease patients from Pakistan.

BACKGROUND: rs10911021 (a single nucleotide polymorphism present upstream of the GLUL gene) affects glutamic acid metabolism, and was shown to be associated with coronary heart disease (CHD) in patients with T2DM but a definite mechanism is unknown. It may affect glutathione cycle, an important effector in the antioxidant defense mechanism, in the cells. We checked the association of this SNP with CHD and oxidative stress biomarkers, malondialdeheyde (MDA), GSH and GSSG in Pakistani patients. METHODS: A total of 650 subjects (425 CHD cases and 225 controls) were genotyped by TaqMan allelic discrimination technique. The levels of MDA, GSH and GSSG were measured by standard protocols. RESULTS: The risk allele frequency was higher in cases than controls, but the difference was insignificant (p = 0.55). The SNP was not associated with CHD (p = 0.053) but when the analysis was limited to CHD patients having DM, a significant association (p = 0.03) was observed. The blood levels of MDA and GSSG were higher while that of GSH was significantly lower in the cases than the controls (p < 0.05). Each risk allele increased MDA and GSSG by 0.29 (0.036) mmol/l and 0.4 (0.04) mmol/l, respectively, while decreased GSH by -0.36 (0.03) mmol/l. The SNP was not associated with any of the tested blood lipids. CONCLUSION: The SNP rs10911021 was associated with CHD only in patients having diabetes, but the SNP was associated with total oxidative stress biomarkers MDA and GSH and GSSG levels. As the SNP rs10911021 showed significant association with oxidative stress parameters and these parameters should an increased oxidative stress in the CHD subjects, it can be concluded that the SNP may have contributed to increase the risk of heart diseases in the diabetic subjects by increasing the oxidative stress.

Association Patterns of Endothelial Nitric Oxide Synthase Gene (NOS3) Variant Glu298Asp with Blood Pressure and Serum Lipid Levels in Subjects with Coronary Artery Disease from Pakistan.

Nitric oxide is an important antiatherosclerotic agent. The main determinant of nitric oxide levels is enzyme nitric oxide synthase encoded by the NOS3 gene, the common variants in this gene may be responsible for variations in plasma enzyme levels. The association of NOS3 variants with coronary artery disease (CAD) varies in different ethnicities. The current study aimed to determine the association of NOS3 Glu298Asp (rs1799983) with CAD and blood lipid levels in Pakistani subjects. Six hundred thirty-six samples (412 cases, 224 controls) were genotyped by TaqMan allelic discrimination assay and serum total cholesterol, and High Density Lipoprotein cholesterol (HDL-C)/Low Density Lipoprotein cholesterol (LDL-C) and triglycerides were measured. The genotype frequency was Glu/Glu = 64.6%, Glu/Asp = 30.1%, and Asp/Asp = 5.3% in cases, and Glu/Glu = 68.8%, Glu/Asp = 26.7%, and Asp/Asp = 4.5% in controls. The Asp298 (T) frequency was not significantly higher in cases than controls (20.4% vs 17.9%, P = 0.28) and risk allele was not associated with CAD (OR 1.15 (0.86-1.54), P = 0.33) and the tested lipid traits but had a strong association with blood pressure (for systolic and diastolic P = 1.9×10--56 and 4×10--40 , respectively). In conclusion, although Glu298Asp did not show association with CAD and lipid profile in the studied cohort, it may exert its effect through blood pressure; however, the mechanism of this effect needs to be explored in the future.

Common variants in the genes of triglyceride and HDL-C metabolism lack association with coronary artery disease in the Pakistani subjects.

BACKGROUND: Serum Triglyceride (TG) and High Density Lipoprotein (HDL-C) levels are modifiable coronary artery disease (CAD) risk factors. Polymorphisms in the genes regulating TG and HDL-C levels contribute to the development of CAD. The objective of the current study was to investigate the effect of four such single nucleotide polymorphism (SNPs) in the genes for Lipoprotein Lipase (LPL) (rs328, rs1801177), Apolipoprotein A5 (APOA5) (rs66279) and Cholesteryl ester transfer protein (CETP) (rs708272) on HDL-C and TG levels and to examine the association of these SNPs with CAD risk. METHODS: A total of 640 subjects (415 cases, 225 controls) were enrolled in the study. The SNPs were genotyped by KASPar allelic discrimination technique. Serum HDL-C and TG were determined by spectrophotometric methods. RESULTS: The population under study was in Hardy Weinberg equilibrium and minor allele of SNP rs1801177 was completely absent in the studied subjects. The SNPs were association with TG and HDL-C levels was checked through regression analysis. For rs328, the effect size of each risk allele on TG and HDL-C (mmol/l) was 0.16(0.08) and -0.11(0.05) respectively. Similarly, the effect size of rs662799 for TG and HDL-C was 0.12(0.06) and -0.13(0.0.3) and that of rs708272 was 0.08(0.04) and 0.1(0.03) respectively. The risk allele frequencies of the SNPs were higher in cases than controls, but the difference was not significant (p > 0.05) and SNPs were not associated with CAD risk (p > 0.05). The combined gene score of four SNPs significantly raised TG and lowered HDL-C but did not increase CAD risk. CONCLUSION: The studied SNPs were associated with TG and HDL-C levels, but not with CAD in Pakistani population under study.

Genetic risk analysis of coronary artery disease in Pakistani subjects using a genetic risk score of 21 variants.

BACKGROUND AND AIMS: Conventional coronary artery disease (CAD) risk factors like age, gender, blood lipids, hypertension and smoking have been the basis of CAD risk prediction algorithms, but provide only modest discrimination. Genetic risk score (GRS) may provide improved discrimination over and above conventional risk factors. Here we analyzed the genetic risk of CAD in subjects from Pakistan, using a GRS of 21 variants in 18 genes and examined whether the GRS is associated with blood lipid levels. METHODS: 625 (405 cases and 220 controls) subjects were genotyped for variants, NOS3 rs1799983, SMAD3 rs17228212, APOB rs1042031, LPA rs3798220, LPA rs10455872, SORT1 rs646776, APOE rs429358, GLUL rs10911021, FTO rs9939609, MIA3 rs17465637, CDKN2Ars10757274, DAB2IP rs7025486, CXCL12 rs1746048, ACE rs4341, APOA5 rs662799, CETP rs708272, MRAS rs9818870, LPL rs328, LPL rs1801177, PCSK9 rs11591147 and APOE rs7412 by TaqMan and KASPar allele discrimination techniques. RESULTS: Individually, the single SNPs were not associated with CAD except APOB rs1042031 and FTO rs993969 (p = 0.01 and 0.009 respectively). However, the combined GRS of 21 SNPs was significantly higher in cases than controls (19.37 ± 2.56 vs. 18.47 ± 2.45, p = 2.9 × 10-5), and compared to the bottom quintile, CAD risk in the top quintile of the GRS was 2.96 (95% CI 1.71-5.13). Atherogenic blood lipids showed significant positive association with GRS. CONCLUSIONS: The GRS was quantitatively associated with CAD risk and showed association with blood lipid levels, suggesting that the mechanism of these variants is likely to be, in part at least, through creating an atherogenic lipid profile in subjects carrying high numbers of risk alleles.

rs3751812, a common variant in fat mass and obesity-associated (FTO) gene, is associated with serum high- and low-density lipoprotein cholesterol in Pakistani individuals.

OBJECTIVES: The aim of the present study was to investigate the effect of a common variant of the fat mass and obesity associated (FTO) gene, rs3751812 in obese Pakistani individuals, compare this effect with nonobese controls of the same ethnicity, and then correlate it with serum lipid profile and anthropometric parameters. METHODS: We genotyped 475 samples using TaqMan allelic discrimination assay. Serum total cholesterol, triacylglycerols, and high- (HDL-C) and low-density lipoprotein cholesterol (LDL-C) concentrations were measured. Statistical analysis was done by SPSS version 22 and the results were analyzed for any significant associations. RESULTS: Results demonstrated that the variant is significantly associated with obesity in Pakistan. The study found, for the first time, that the variant has a significant effect on lowering HDL-C and increasing LDL-C. Among anthropometric measures, the variant showed significant association with body mass index and weight. CONCLUSION: The study concludes that the FTO variant is consistently associated with obesity in the Pakistani population and its association with anthropometric and lipid parameters show that it may mediate its role by altering fat deposition and disturbing serum lipid profile. However, future studies with larger sample size are needed to validate the results of the present study.

Effect of SORT1, APOB and APOE polymorphisms on LDL-C and coronary heart disease in Pakistani subjects and their comparison with Northwick Park Heart Study II.

BACKGROUND: Many SNPs have been identified in genes regulating LDL-C metabolism, but whether their influence is similar in subjects from different ethnicities is unclear. Effect of 4 such SNPs on LDL-C and coronary heart disease (CHD) was examined in Pakistani subjects and was compared with middle aged UK men from Northwick Park Heart Study II (NPHSII). METHODS: One thousand nine hundred sixty-five (1770 non CHD, 195 CHD) UK and 623 (219 non CHD, 404 CHD) Pakistani subjects were enrolled in the study. The SNPs SORT1 rs646776, APOB rs1042031 and APOE rs429358, rs7412 were genotyped by TaqMan/KASPar technique and their gene score was calculated. LDL-C was calculated by Friedewald equation, results were analyzed using SPSS. RESULTS: Allele frequencies were significantly different (p= <0.05) between UK and Pakistani subjects. However, the SNPs were associated with LDL-C in both groups. In UK non CHD, UK CHD, Pakistani non CHD and Pakistani CHD respectively, for rs646776, per risk allele increase in LDL-C(mmol/l) was 0.18(0.04), 0.06(0.11), 0.15(0.04) and 0.27(0.06) respectively. For rs1042031, per risk allele increase in LDL-C in four groups was 0.11(0.04), 0.04(0.14), 0.15(0.06) and 0.25(0.09) respectively. For APOE genotypes, compared to Ɛ3, each Ɛ2 decreased LDL-C by 0.11(0.06), 0.07(0.15), 0.20(0.08) and 0.38(0.09), while each Ɛ4 increased LDL-C by 0.43(0.06), 0.39(0.21), 0.19(0.11) and 0.39(0.14) respectively. Overall gene score explained a considerable proportion of sample variance in four groups (3.8%, 1.26% 13.7% and 12.3%). Gene score in both non-CHD groups was significantly lower than CHD subjects. CONCLUSIONS: The SNPs show a dose response association with LDL-C levels and risk of CHD in both populations.

Role of a common variant of Fat Mass and Obesity associated (FTO) gene in obesity and coronary artery disease in subjects from Punjab, Pakistan: a case control study.

BACKGROUND: The FTO gene has recently become one of the most extensively investigated genes associated with body mass and has been shown to play a role in cardiovascular diseases as well. The aim of the current study was to investigate the effect of a common variant of FTO gene, rs9939609 in obese and coronary heart disease (CHD) patients of Pakistan and investigate whether it has any influence on the serum biochemical parameters. METHODS: A total of 970 samples (295 obese, 425 CHD and 250 controls) were genotyped using TaqMan allelic discrimination assay. Serum total cholesterol, HDL-C and triglycerides were measured using spectrophotometric methods. LDL-C was calculated by Friedwalds equation. Statistical analysis was done by SPSS version 22. RESULTS: Results showed moderately high minor allele frequency (MAF) in obese and CHD cases as compared to controls (obese = 0.381 CAD = 0.361 and controls = 0.286). The variant was significantly associated with obesity and CAD (obesity odds ratio (OR) = 1.54, confidence interval (CI) = 1.07-2.21, p = 0.0009; CHD OR = 1.43, CI = 1.02-2.01, p = 0.004) in Pakistan. The risk allele did not show a significant association with any of the lipid trait tested (p > 0.05) but a strong association was observed with plasma glucose levels (obese p = 0.001, CAD p = 0.014, controls p = 0.019). CONCLUSION: In conclusion, the variant was associated with obesity and CAD in the studied subjects and its possible effect may involve the blood sugar metabolism but not serum lipid chemistry.