Genome-wide association studies significantly increased the number of hypertension risk variants; however, most of them focused on European societies. There is lack of such studies in developing countries, including Pakistan. The lack of research studies and the high prevalence of hypertension in the Pakistani community prompted us to design this study. Aldosterone synthase (CYP11B2) was thoroughly studied in different ethnic groups; however, no such study has been conducted in the Pashtun population of Khyber Pakhtunkhwa, Pakistan. In essential hypertension, the aldosterone synthase gene (CYP11B2) plays a significant role. Aldosterone synthesis is affected by both hereditary and environmental factors. Aldosterone synthase (encoded by the CYP11B2 gene) controls the conversion of deoxycorticosterone to aldosterone and, thus, has genetic influences. Polymorphisms in the CYP11B2 gene are linked to an increased risk of hypertension. Previous research on the polymorphism of the aldosterone synthase (CYP11B2) gene and its relationship to hypertension produced inconclusive results. The present study investigates the relationship between CYP11B2 gene polymorphism and hypertension in Pakistan’s Pashtun population. We used the nascent exome sequencing method to identify variants associated with hypertension. The research was divided into two phases. In phase one, DNA samples from 200 adult hypertension patients (of age ≥ 30 years) and 200 controls were pooled (n = 200/pool) and subjected to Exome Sequencing. In the second phase, the WES reported SNPs were genotyped using the Mass ARRAY technique to verify and confirm the association between WES-identified SNPs and hypertension. WES identified a total of eight genetic variants in the CYP11B2 gene. The chi-square test and logistic regression analysis were used to estimate the minor allele frequencies (MAFs) and chosen SNPs relationships with hypertension. The frequency of minor allele T was found to be higher in cases compared to the control (42% vs. 30%: p = 0.001) for rs1799998 of CYP11B2 gene, while no significant results (p > 0.05) were observed for the remaining SNPs; rs4536, rs4537, rs4545, rs4543, rs4539, rs4546 and rs6418 showed no positive association with HTN in the studied population (all p > 0.05). Our study findings suggest that rs1799998 increases susceptibly to HTN in the Pashtun population of KP, Pakistan.

Objective. Recent GWAS largely conducted in European populations have successfully identified multiple genetic riskvariants associated with Type 2 Diabetes Mellitus (T2DM). However, the effects conferred by these variants in thePakistani population have not yet been fully elucidated. The objective of this study was to examine European GWAS-identified T2DM risk variants in the Pakistani Pashtun population to better understand the shared genetic basis of T2DM in the European and Pakistani cohorts. Methodology. A total of 100 T2DM patients and 100 healthy volunteers of Pashtun ethnicity were enrolled in this study.Both groups were genotyped for 8 selected single nucleotide polymorphisms (SNPs) using the Sequenom MassARRAY®platform. The association between selected SNPs and T2DM was determined by using appropriate statistical tests. Results. Of the 8 studied SNPs, 5 SNPs, SLC30A8/ rs13266634 (p=0.031, OR=2.13), IGF2BP2/ rs4402960 (p=0.001,OR=3.01), KCNJ11/ rs5219 (p=0.042, OR=1.78), PPARG/ rs1801282 (p=0.042, OR=2.81) and TCF7L2/ rs7903146(p=0.00006, 3.41) had significant association with T2DM. SNP GLIS3/ rs7041847 (p=0.051, OR=2.01) showed nosufficient evidence of association. SNPs KCNQ1/ rs2237892 (p=0.140, OR=1.61) and HHEX/IDE/s1111875 (p=0.112,OR=1.31) showed opposite allelic effects and were not validated for T2DM risk in the study population. Among the studied SNPs, TCF7L2/ rs7903146 showed the most significant association. Conclusion. Our study finding indicates that selected genome-wide significant T2DM risk variants previously identifiedin European descent also increase the risk of developing T2DM in the Pakistani Pashtun population.

Coronary Artery Diseases (CAD) remains the top among Non-communicable Diseases (NCDs). Variations in Apolipoprotein E (APOE) and Paroxonase 1 (PON1) have been associated with Myocardial Infarction (MI) in several populations. However, despite the high prevalence of CAD, no such study has been reported in the Pashtun ethnic population of Pakistan. We have conducted a two-stage (i.e., screening and validation) case-control study in which 200 cases and 100 control subjects have been recruited. In the first stage, Whole Exome Sequencing (WES) was used to screen for pathogenic variants of Myocardial Infarction (MI). In the second stage, selected variants of both APOE and PON1 genes (rs7412, rs429358, rs854560, and rs662) were analyzed through MassARRAY genotyping. Risk Allele Frequencies (RAFs) distribution and association of the selected SNPs with MI were determined using the Chi-square test and logistic regression analysis. WES identified a total of 12 sequence variants in APOE and 16 in PON1. Genotyping results revealed that APOE variant rs429358 (ɛ4 allele and ɛ3/ɛ4 genotype) showed significant association in MI patients (OR = 2.11, p value = 0.03; 95% CI = 1.25–2.43); whereas no significant difference (p˃ 0.05) was observed for rs7412. Similarly, the R allele of PON1 Q192R (rs662) was significantly associated with cases (OR = 1.353, p value = 0.048; 95% CI = 0.959–1.91), with particular mention of RR genotype (OR = 1.523, p value = 0.006; 95% CI = 1.087–2.132). Multiple logistic regression analysis showed that rs429358 (C allele) and rs662 (R allele) have a significantly higher risk of MI after adjustment for the conventional risk factors. Our study findings suggested that the rs429358 variant of APOE and PON1 Q192R are associated with MI susceptibility in the Pashtun ethnic population of Pakistan.

Genome-wide association studies have greatly increased the number of T2DM associated risk variants but most of them have focused on populations of European origin. There is scarcity of such studies in developing countries including Pakistan. High prevalence of T2DM in Pakistani population prompted us to design this study. We have devised a two stage (the discovery stage and validation stage) case-control study in Pashtun ethnic population in which 500 T2DM cases and controls each have been recruited to investigate T2DM genetic risk variants. In discovery stage Whole Exome Sequencing (WES) was used to identify and suggest T2DM pathogenic SNPs, based on SIFT and Polyphen scores; whereas in validation stage the selected variants were confirmed for T2DM association using MassARRAY genotyping and appropriate statistical tests. Results of the study showed the target positive association of rs1801282/PPARG (OR = 1.24, 95%Cl = 1.20–1.46, P = 0.010), rs745975/HNF4A (OR = 1.30, 95%Cl = 1.06–1.38, P = 0.004), rs806052/GLIS3 (OR = 1.32, 95%Cl = 1.07–1.66, P = 0.016), rs8192552/MTNR1B (OR = 1.53, 95%Cl = 0.56–1.95, P = 0.012) and rs1805097/IRS-2 (OR = 1.27, 95%Cl = 1.36–1.92, P = 0.045), with T2DM; whereas rs6415788/GLIS3, rs61788900/NOTCH2, rs61788901/NOTCH2 and rs11810554/NOTCH2 (P>0.05) showed no significant association. Identification of genetic risk factors/variants can be used in defining high risk subjects assessment, and disease prevention.

Human leukocyte antigen (HLA) system is the most polymorphic and gene dense region of human DNA that has shown many disease associations. It has been further divided into HLA classes I, II, and III. Polymorphism in HLA class II genes has been reported to play an important role in the pathogenesis of type 1 diabetes (T1D). It also showed association with T2D in different ethnic populations. However, a little is known about the relationship of HLA class I gene polymorphism and T2D. This study has evaluated the association of HLA-B (class I gene) variants with T2D in Pashtun ethnic population of Khyber Pakhtunkhwa. In the first phase of the study, whole exome sequencing (WES) of 2 pooled DNA samples was carried out, and DNA pools used were constructed from 100 diabetic cases and 100 control subjects. WES results identified a total of $n=17$ SNPs in HLA-B gene. In the next phase, first 5 out of $n=17$ reported SNPs were genotyped using MassARRAY® system in order to validate WES results and to confirm association of selected SNPs with T2D. Minor allele frequencies (MAFs) and selected SNPs×T2D association were determined using chi-square test and logistic regression analysis. The frequency of minor C allele was significantly higher in the T2D group as compared to control group (45.0% vs. 13.0%) ( $p=0.006$ ) for rs2308655 in HLA-B gene. No significant difference in MAF distribution between cases and controls was observed for rs1051488, rs1131500, rs1050341, and rs1131285 ( $p>0.05$ ). Binary logistic regression analyses showed significant results for SNP rs2308655 ( $\text{OR}=2.233$ , $\text{CI}\left(95\%\right)=1.223‐4.077$ , and $p=0.009$ ), while no considerable association was observed for the other 4 SNPs. However, when adjusted for these variants, the association of rs2308655 further strengthened significantly ( $\text{adjusted}\text{OR}=7.485$ , $\text{CI}\left(95\%\right)=2.353‐23.812$ , and $p=0.001$ ), except for rs1131500, which has no additive effect. In conclusion, the finding of this study suggests rs2308655 variant in HLA-B gene as risk variant for T2D susceptibility in Pashtun population.