Label free quantitative proteomic profiling of serum samples of intellectually disabled young patients revealed dysregulation of complement coagulation and cholesterol cascade systems.

Intellectual disability is a heterogeneous disorder, diagnosed using intelligence quotient (IQ) score criteria. Currently, no specific clinical test is available to diagnose the disease and its subgroups due to inadequate understanding of the pathophysiology. Therefore, current study was designed to explore the molecular mechanisms involved in disease perturbation, and to identify potential biomarkers for disease diagnosis and prognosis. A total of 250 participants were enrolled in this study, including 200 intellectually disabled (ID) subjects from the subgroups (mild, moderate, and severe) with age and gender matched healthy controls (n = 50). Initially, IQ testing score and biochemical profile of each subject was generated, followed by label-free quantitative proteomics of subgroups of IQ and healthy control group through nano-LC/MS- mass spectrometry. A total of 310 proteins were identified, among them198 proteins were common among all groups. Statistical analysis (ANOVA) of the subgroups of ID showed 142 differentially expressed proteins, in comparison to healthy control group. From these, 120 proteins were found to be common among all subgroups. The remaining 22 proteins were categorized as exclusive proteins found only in disease subgroups. Furthermore, the hierarchical cluster analysis (HCL) of common significant proteins was also performed, followed by PANTHER protein classification and GO functional enrichment analysis. Results provides that the datasets of differentially expressed proteins, belong to the categories of immune / defense proteins, transfer carrier proteins, apolipoproteins, complement proteins, protease inhibitors, hemoglobin proteins etc., they are known to involvein immune system, and complement and coagulation pathway cascade and cholesterol metabolism pathway. Exclusively expressed 22 proteins were found to be disease stage specific and strong PPI network specifically those that have significant role in platelets activation and degranulation, such as Filamin A (FLNA). Furthermore, to validate the mass spectrometric findings, four highly significant proteins (APOA4, SAP, FLNA, and SERPING) were quantified by ELISA in all the study subjects. AUROC analysis showed a significant association of APOA4 (0.830), FLNA (0.958), SAP (0.754) and SERPING (0.600) with the disease. Apolipoprotein A4 (APOA4) has a significant role in cholesterol transport, and in modulation of glucose and lipid metabolism in the CNS. Similarly, FLNA has a crucial role in the nervous system, especially in the functioning of synaptic network. Therefore, both APOA4, and FLNA proteins represent good potential for candidate biomarkers for the diagnosis and prognosis of the intellectual disability. Overall, serum proteome of ID patients provides valuable information of proteins/pathways that are altered during ID progression.

Association of hyperuricemia with metabolic syndrome and its components in an adult population of Faisalabad, Pakistan.

BACKGROUND AND AIMS: The rising prevalence of metabolic syndrome (MetS) is a matter of serious concern worldwide. Hyperuricemia has been observed as an independent risk factor in the development of MetS and each of its individual components in different populations. This study aims to determine the association of hyperuricemia with MetS and its individual components in a Pakistani cohort. METHODS AND RESULTS: A cross-sectional study was performed in a public sector hospital in Faisalabad, Pakistan. Total 204 participants were studied along with their anthropometric measurements and blood sample analysis for clinically important parameters. MetS was defined according to the NCEP-criteria. Independent sample t-test, Binomial logistic regression and Linear regression analyses were used to determine the association between hyperuricemia and metabolic syndrome. The prevalence of MetS and hyperuricemia in our study was 42.6% and 31.9% respectively. As compared to the normo-uricemic group, the hyperuricemic group had a significantly higher systolic blood pressure, BMI and lower HDL-C level (p < 0.05). After adjusting for age, gender, BMI and LDL-C, hyperuricemia was observed to increase the risk of MetS, increased systolic blood pressure and reduce HDL-C respectively by 1.34, 1.23 and 1.20 folds respectively. CONCLUSION: In this study, a significant association between hyperuricemia and metabolic syndrome, systolic hypertension, blood glucose and decreased HDL-C was observed.

Development of low-cost in-house tetra-ARMS-PCR assay for the screening of five CBS mutations found in Pakistani homocystinuria patients.

BACKGROUND: Classical homocystinuria is an inborn amino acid metabolism disorder resulting from mutations in the Cystathionine-ß-Synthase (CBS) gene. These mutations lead to elevated homocysteine and methionine levels and reduced cysteine levels in the blood. Typically, diagnosis occurs after patients display symptoms, and various lab methods confirm it. DNA sequencing is the best option for early detection of genetic variants in asymptomatic suspected individuals. Unfortunately, its high cost can hinder its use, especially in low-income countries like Pakistan. OBJECTIVE: Aim of this study was to devise a robust low-cost diagnostic/screening assay based on Tetra-ARMS-PCR for five prevalent genetic variants found in Pakistani classical homocystinuria patients. MATERIALS AND METHODS: In the current study, T-ARMS-PCR assays were developed for five mutations (c.975G > C, c.770C > T, c.752T > C, c.1039 + 1G > T, c.451 + 1GG > TA), which were characterized previously in classical homocystinuria patients. These low-cost T-ARMS-PCR assays were then used to screen the affected individuals and their family members to identify their genotypes for pathogenic variations in the asymptomatic patients and carriers in their respective families. RESULTS: The outcomes were entirely consistent with those obtained from Sanger DNA sequencing, confirming the sensitivity, specificity, and reliability of the T-ARMS-PCR assay for detecting CBS mutations. CONCLUSION: T-ARMS-PCR has wide applications for low-income countries for the screening and early diagnosis of asymptomatic patients and carriers in the homocystinuria affected families as well as other inherited diseases.

Mass-spectrometric analysis of APOB polymorphism rs1042031 (G/T) and its influence on serum proteome of coronary artery disease patients: genetic-derived proteomics consequences.

Genetic polymorphisms of apolipoprotein B gene (APOB) may result into serum proteomic perturbance in Coronary Artery Disease (CAD). The current case-control cohort of Pakistani subjects was designed to analyze the genetic influence of APOB rs1042031, (G/T) genotype on serum proteome. Subjects were categorized into two groups: CAD patients (n = 480) and healthy individuals (n = 220). For genotyping, tetra ARMS-PCR was carried out and validated through sequencing, whereas LC/MS-based proteomic analysis of serum samples was performed through label-free quantification. In initial step of genotyping, the frequencies of each genotype GG, GT, and TT were 70%, 27%, and 30% in CAD patients, while in control group, the subjects were 52%, 43%, and 5%, respectively, in CAD patients. The genotypic frequencies in patients vs. control groups found significantly different (p = 0.004), and a strong association of dominant alleles GG with the CAD was observed in both dominant (OR: 2.4 (1.71-3.34), p = 0.001) and allelic genetic models (OR: 2.0 (1.45-2.86), p = 0.001). In second step of label-free quantitation, a total of 40 significant proteins were found with altered expression in CAD patients. The enriched Gene Ontology (GO) terms of molecular functions and pathways of these protein showed upregulated pathways as follows: chylomicron remodeling and assembly, complement cascade activation, plasma lipoprotein assembly, apolipoprotein-A receptor binding, and metabolism of fat-soluble vitamins in G allele carrier of rs1042031 (G > T) vs. mutant T-allele carriers. This study provides better understanding of CAD pathobiology by proteogenomics of APOB. It evidences the influence of APOB rs1042031-dominant (GG) genotype with CAD patients.

Need and Challenges in Establishing Newborn Screening Programs for Inherited Metabolic Disorders in Developing Countries.

Even in this post genomic era, no national level newborn screening (NBS) programs for inborn errors of metabolism (IEMs) are yet available in several developing countries including Pakistan. Through NBS, various IEMs can be screened using minute quantities of biofluids. Targeted metabolomics and genomic techniques are the main approaches used for NBS. However, lack of technical expertise and such high-end "omics" based analytical facilities, and meager funding for healthcare in developing countries are the major reasons for unavailability of NBS programs. As it is reflected by only a handful reports about IEMs from Pakistan with population of ≈220 million and consanguinity rate of about 70%, which suggests an unmet need for an NBS program owing to reasonably high prevalence of inherited diseases. Around 200 IEMs are potentially treatable if diagnosed at an earlier stage through biochemical marker and genetic screening, so such patients can get benefit from the NBS program. This overview will help to persuade the stakeholders to setup NBS programs in developing countries including Pakistan, due to multitude of benefits for IEMs; timely diagnosis and early treatment can help the patients to live a nearly healthy life, reduced suffering of the family and minimal burden on society or national healthcare system.

Dietary Supplementation of Microbial Dextran and Inulin Exerts Hypocholesterolemic Effects and Modulates Gut Microbiota in BALB/c Mice Models.

Microbial exopolysaccharides (EPSs), having great structural diversity, have gained tremendous interest for their prebiotic effects. In the present study, mice models were used to investigate if microbial dextran and inulin-type EPSs could also play role in the modulation of microbiomics and metabolomics by improving certain biochemical parameters, such as blood cholesterol and glucose levels and weight gain. Feeding the mice for 21 days on EPS-supplemented feed resulted in only 7.6 ± 0.8% weight gain in the inulin-fed mice group, while the dextran-fed group also showed a low weight gain trend as compared to the control group. Blood glucose levels of the dextran- and inulin-fed groups did not change significantly in comparison with the control where it increased by 22 ± 5%. Moreover, the dextran and inulin exerted pronounced hypocholesterolemic effects by reducing the serum cholesterol levels by 23% and 13%, respectively. The control group was found to be mainly populated with Enterococcus faecalis, Staphylococcus gallinarum, Mammaliicoccus lentus and Klebsiella aerogenes. The colonization of E. faecalis was inhibited by 59-65% while the intestinal release of Escherichia fergusonii was increased by 85-95% in the EPS-supplemented groups, respectively, along with the complete inhibition of growth of other enteropathogens. Additionally, higher populations of lactic acid bacteria were detected in the intestine of EPS-fed mice as compared to controls.

Histological and Microscopic Analysis of Fats in Heart, Liver Tissue, and Blood Parameters in Experimental Mice.

The intake of various types and amounts of dietary fats influences metabolic and cardiovascular health. Hence, this study evaluated the impact of routinely consumed Pakistani dietary fats on their cardiometabolic impact. For this, we made four groups of mice, each comprising 5 animals: (1) C-ND: Control mice on a normal diet, (2) HFD-DG: High-fat diet mice on a normal diet plus 10% (w/w) desi ghee, (3) HFD-O: Mice on normal diet plus 10% (w/w) plant oil (4) HFD-BG: Mice on normal diet plus 10% (w/w) banaspati ghee. Mice were fed for 16 weeks, and blood, liver, and heart samples were collected for biochemical, histological, and electron microscopic analysis. The physical factors indicated that mice fed on HFD gained more body weight than the C-ND group. Blood parameters do not show significant differences, but overall, the glucose and cholesterol concentrations were raised in the mice fed with a fat-rich diet, with the highest concentrations in the HFD-BG group. The mice fed with HFD-BG and HFD-O had more lipid droplets in the liver, compared to HFD-DG and C-ND.

Tetra-ARMS-PCR assay development for genotyping of AGT rs699 T/C polymorphism, its comparison with PCR-RFLP and application in a case-control association study of cardiovascular disease patients.

Genetic variations in the AGT gene play a significant role in controlling the plasma concentration of angiotensinogen (precursor protein of bioactive octapeptide angiotensin II) and the efficacy of antihypertensive drugs. In the current study, Tetra-Amplification Refractory Mutation System-Polymerase Chain Reaction (T-ARMS-PCR) was developed for genotyping of AGT rs699 T/C polymorphism and validated through Sanger DNA sequencing. Its efficiency was also tested using 474 human DNA samples [control, n = 181; cardiovascular disease (CVD) patients, n = 293]. Results showed that T-ARMS-PCR is superior to the commonly used PCR-Restriction Fragment Length Polymorphism (PCR-RFLP). Statistical analysis revealed that the AGT rs699 CC genotype is more prevalent in the CVD patient group (37% vs. 28%) and AGT rs699 C allele and CC genotype increased the risk of CVD by 1.4 and 1.9 fold, respectively. In summary, T-ARMS-PCR is the most suitable approach for quick and efficient genotyping of AGT rs699 T/C polymorphism in a large population in resource-limited countries, Furthermore, AGT rs699 T/C polymorphism is associated with the risk of CVD in the Punjabi Pakistani population.

Association of vitamin D receptor polymorphisms with cardiometabolic conditions in Pakistani population.

Apart from bone related effects, vitamin D has roles in immune modulation, hypertension, diabetes and cardiovascular diseases. Metabolic functions of vitamin D are mediated after binding with vitamin D receptor (VDR). VDR polymorphisms affect its physiological functions. Several VDR single nucleotide polymorphisms (SNPs) are reported previously. However, VDR polymorphisms causing influence on cardiovascular and metabolic disorders have not been investigated in Pakistani population so far. Therefore, present study was conducted to evaluate the role of VDR polymorphisms (rs2228570 and rs7975232) in the pathobiology of cardiometabolic disorders. In all, 400 cardiometabolic patients and 226 healthy control human adults were enrolled from Faisalabad, Pakistan. Biochemical parameters (serum glucose, liver function test, renal function test and lipid profile) were analyzed by standard kit methods. Genetic analysis was done by ARMS-PCR assay. Data was analyzed in SPSS v20. Regression analysis revealed that GG and AG genotypes of rs2228570 A>G polymorphism significantly increased the risk of hypertension in cardiovascular patients by 5.29 and 5.94 times respectively (GG: OR=5.29, 95% CI=1.63-17.2, p=0.005; AG: OR=5.94, 95% CI=1.70-20.7, p=0.005). However, rs7975232 C>A polymorphism was not correlated with cardiometabolic conditions. In conclusion, GG and AG genotypes of VDR SNP rs2228570 significantly contribute for hypertension in cardiovascular disease patients.

Comparative Biochemical Profiling of Aluminum Chloride and Sodium Azide Induced Neuroinflammation and Cardiometabolic Disturbance.

Comorbidities in human beings signify the numerous risk factors that increase the incidences of neuro- and cardio-metabolic disorders. Experimental models depicting comorbidities are important to explore the molecular pathophysiology that can help suggest appropriate treatment strategies. Tissue-accumulating potential and pathological effects of aluminium chloride (AlCl3)and sodium azide (NaN3) are well recognized. Hence, in the current work, we have for the first time aimed to investigate the unexplored potential of graded dose effects of AlCl3 and NaN3 in inducing early inflammation and cardiometabolic toxicity via comparative biochemical analysis of AlCl3 and/or NaN3. Rats were allocated into seven groups (n = 6). Group 1 was normal control. Remaining groups were given graded doses of AlCl3 and/or NaN3, as LD-AlCl3 (AlCl3 40 mg), MD-AlCl3 (AlCl3 45 mg), and HD-AlCl3 (AlCl3 50 mg) representing low dose, medium dose, and high dose of AlCl3, respectively, and the remaining as LD-NaN3 (NaN3 13 mg), MD-NaN3 (NaN3 15 mg), and HD-NaN3 (NaN3 17 mg) representing low dose, medium dose, and high dose of NaN3, respectively. Serum levels of glucose, insulin, lipid profile, inflammatory mediators like IL-6 and oxidative stress marker, and malondialdehyde (MDA) were analyzed. Likewise, subacute toxicity parameters were analyzed. Immunohistochemistry (IHC) and histopathology (H&E/Masson's trichrome staining) of brain, heart, and pancreatic tissues were done. ECG pattern of all groups was observed. HD-AlCl3 was associated with elevated levels of inflammatory biomarkers, MDA, and glycemic and lipid profiles, whereas it decreased the insulin levels. HD-NaN3 also showed the similar effects of aggravated inflammatory biomarkers, impaired glycemic and lipid profiles, but depicted the maximum mortality rate as compared to HD-AlCl3. IHC showed prominent amyloid plaques and neurofibrillary tangle formation with MD-AlCl3 and HD-AlCl3 as compared to NaN3-treated groups. Likewise, in brain tissues, vacuolation of white matter, vascular congestion, and hemorrhage were seen in HD-AlCl3 treated group, while HD-NaN3 induced death in animals. AlCl3 exposure resulted in an inverted QRS complex, while exposure to NaN3 showed ST depression but with increased mortality. AlCl3 has better controlled results as compared to NaN3 for induction of comorbid experimental animal model depicting early neuroinflammation and cardiometabolic disruption. These determined efforts facilitate the researchers for the development of clinically effective treatment strategies using such experimental models.

High levels of blood glutamic acid and ornithine in children with intellectual disability.

Objectives: Aminoacidopathies are inborn errors of metabolism (IEMs) that cause intellectual disability in children. Luckily, aminoacidopathies are potentially treatable, if diagnosed earlier in life. The focus of this study was the screening of aminoacidopathies in a cohort of patients suspected for IEMs. Methods: Blood samples from healthy (IQ > 90; n = 391) and intellectually disabled (IQ < 70; n = 409) children (suspected for IEMs) were collected from different areas of Northern Punjab, Pakistan. An analytical HPLC assay was used for the screening of plasma amino acids. Results: All the samples (n = 800) were analyzed on HPLC and forty-three out of 409 patient samples showed abnormal amino acid profiles mainly in the levels of glutamic acid, ornithine and methionine. Plasma concentration (Mean ± SD ng/mL) were significantly high in 40 patients for glutamic acid (patients: 165 ± 38 vs. controls: 57 ± 8, p < 0.00001) and ornithine (patients: 3177 ± 937 vs. controls: 1361 ± 91, p < 0.0001). Moreover, 3 patients showed abnormally high (53.3 ± 8.6 ng/mL) plasma levels of methionine. Conclusion: In conclusion, biochemical analysis of samples from such patients at the metabolites level could reveal the underlying diseases which could be confirmed through advanced biochemical and genetic analyses. Thus, treatment to some of such patients could be offered. Thus burden of intellectual disability caused by such rare metabolic diseases could be reduced from the target populations.

Genetic Association of Beta-Myosin Heavy-Chain Gene (MYH7) with Cardiac Dysfunction.

Cardiac dysfunction accelerates the risk of heart failure, and its pathogenesis involves a complex interaction between genetic and environmental factors. Variations in myosin affect contractile abilities of cardiomyocytes and cause structural and functional abnormalities in myocardium. The study aims to find the association of MYH7 rs121913642 (c.1594 T>C) and rs121913645 (c.667G>A) variants with cardiac dysfunction in the Punjabi Pakistani population. Patients with heart failure (n = 232) and healthy controls (n = 205) were enrolled in this study. MYH7 variant genotyping was performed using tetra ARMS-PCR. MYH7 rs121913642 TC genotype was significantly more prevalent in the patient group (p < 0.001). However, MYH7 rs121913645 genotype frequencies were not significantly different between the patient and control groups (p < 0.666). Regression analysis also revealed that the rs121913642 C allele increases the risk of cardiac failure by ~2 [OR:1.98, CI: 1.31−2.98, p < 0.001] in comparison to the T allele. High levels of the cardiac enzymes cardiac troponin I (cTnI) and CK-MB were observed in patients. There was also an increase in total cholesterol, LDL cholesterol, and uric acid in patients compared to the healthy control group (p < 0.001). In conclusion, the MYH7 gene variant rs121913642 is genetically associated with cardiac dysfunction and involved in the pathogenesis of HF.

Toxicity assessment of emamectin benzoate and its commercially available formulations in Pakistan by in vivo and in vitro assays.

Emamectin benzoate (EMB) is generally considered a safe insecticide in agriculture and veterinary practices, yet, it can cause cytotoxic and genotoxic effects. Hence, the aim of this study was to evaluate toxic effects of 80% EMB and its commercially used formulations (Tycon 1.9% EC and Tycon plus 5% EW) in Pakistan and tested for acute toxicity in albino rats, rabbits and fish (Labeo rohita). Genotoxicity was investigated by in vivo comet assay and bone marrow micronucleues test in the rats. In vitro mutagenicity was tested in Salmonella typhimurium TA98 and TA100. The tested EMB formulations were found moderately toxic (oral LD50: 122-168 mg/kg), causing severe eye irritation in rabbits, highly toxic to fish (LC50: 9-43 µg/L) and found non mutagenic. Oral administrations of EMB (80% and 5%) at 100 mg/kg of body weight to male rats reduced red blood cells, hemoglobin, and slightly increased the blood glucose, urea and liver enzymes levels but had no significant damage to DNA. EMB induced bone marrow toxicity was observed as reduction of polychromatic erythrocytes. Overall, EMB exposure was highly toxic to fish, and caused hemo- and hepatotoxicity in rats. These findings warrant cautious use of EMB formulations in agrochemicals and veterinary medicine.

Effect of APOB polymorphism rs562338 (G/A) on serum proteome of coronary artery disease patients: a "proteogenomic" approach.

In the current study, APOB (rs1052031) genotype-guided proteomic analysis was performed in a cohort of Pakistani population. A total of 700 study subjects, including Coronary Artery Disease (CAD) patients (n = 480) and healthy individuals (n = 220) as a control group were included in the study. Genotyping was carried out by using tetra primer-amplification refractory mutation system-based polymerase chain reaction (T-ARMS-PCR) whereas mass spectrometry (Orbitrap MS) was used for label free quantification of serum samples. Genotypic frequency of GG genotype was found to be 90.1%, while 6.4% was for GA genotype and 3.5% was for AA genotypes in CAD patients. In the control group, 87.2% healthy subjects were found to have GG genotype, 11.8% had GA genotype, and 0.9% were with AA genotypes. Significant (p = 0.007) difference was observed between genotypic frequencies in the patients and the control group. The rare allele AA was found to be strongly associated with the CAD [OR: 4 (1.9-16.7)], as compared to the control group in recessive genetic model (p = 0.04). Using label free proteomics, altered expression of 60 significant proteins was observed. Enrichment analysis of these protein showed higher number of up-regulated pathways, including phosphatidylcholine-sterol O-acyltransferase activator activity, cholesterol transfer activity, and sterol transfer activity in AA genotype of rs562338 (G>A) as compared to the wild type GG genotype. This study provides a deeper insight into CAD pathobiology with reference to proteogenomics, and proving this approach as a good platform for identifying the novel proteins and signaling pathways in relation to cardiovascular diseases.

Adult GAMT deficiency: A literature review and report of two siblings.

Guanidinoacetate methyltransferase (GAMT) deficiency is a creatine deficiency disorder and an inborn error of metabolism presenting with progressive intellectual and neurological deterioration. As most cases are identified and treated in early childhood, adult phenotypes that can help in understanding the natural history of the disorder are rare. We describe two adult cases of GAMT deficiency from a consanguineous family in Pakistan that presented with a history of global developmental delay, cognitive impairments, excessive drooling, behavioral abnormalities, contractures and apparent bone deformities initially presumed to be the reason for abnormal gait. Exome sequencing identified a homozygous nonsense variant in GAMT: NM_000156.5:c.134G>A (p.Trp45*). We also performed a literature review and compiled the genetic and clinical characteristics of all adult cases of GAMT deficiency reported to date. When compared to the adult cases previously reported, the musculoskeletal phenotype and the rapidly progressive nature of neurological and motor decline seen in our patients is striking. This study presents an opportunity to gain insights into the adult presentation of GAMT deficiency and highlights the need for in-depth evaluation and reporting of clinical features to expand our understanding of the phenotypic spectrum.

Paper-based analytical devices for colorimetric detection of S. aureus and E. coli and their antibiotic resistant strains in milk.

Animal derived milk which is an important part of human diet due to its high nutritional value not only supports humans but also presents a growth environment for pathogenic bacteria. Milk may become contaminated with bacteria through udder infections or through contact within the dairy farm environment. Infections are treated with antibiotics, with ß-lactams most commonly used in veterinary medicine. However, their frequent use leads to the emergence of ß-lactam resistant bacterial strains, which causes difficulties in the treatment of infections in both humans and animals. Detection of pathogens as well as their antibiotic sensitivity is a pre-requisite for successful treatment and this is generally achieved with laboratory-based techniques such as growth inhibition assays, enzyme-linked immunosorbent assays (ELISA) or polymerase chain reactions (PCRs), which are unavailable in resource-limited settings. Here, we investigated paper-based analytical devices (µPADs) for the presumptive detection of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) and their antibiotic resistant bacterial strains in milk samples. The µPADs were fabricated on filter paper using wax printing, and then impregnated with chromogenic substrates, which reacted with bacterial enzymes to form coloured products. Limits of detection of S. aureus and E. coli and their antibiotic resistant strains in milk samples were found to be 106 cfu mL-1. Enrichment of milk samples in a selective medium for 12 h enabled detection as low as 10 cfu mL-1. The paper devices tested on a set of 640 milk samples collected from dairy animals in Pakistan demonstrated more than 90% sensitivity and 100% selectivity compared to PCR, showing promise to provide inexpensive and portable diagnostic solutions for the detection of pathogenic bacteria in resource-limited settings.

Biochemical investigation of rs1801282 variations in PPAR-γ gene and its correlation with risk factors of diabetes mellitus in coronary artery disease.

We aimed to investigate the association of single nucleotide polymorphism of Pro/Ala (rs1801282) in peroxisome proliferator-activated receptor-gamma (PPAR-γ) gene with risk factors of diabetes mellitus (DM) in cardiovascular disease (CVD) patients. We recruited 244 participants from Faisalabad Institute of Cardiology and Department of Cardiology, Sargodha District Head Quarter Teaching Hospital, Pakistan. Out of 244 participants, 144 cases were CVD patients and 100 were healthy controls. CVD patients were further divided into 111 coronary artery disease (CAD) and 33 cardiomyopathy (CMP) patients. Assessment of variant specific polymorphism/mutation of Pro/Pro and Pro/Ala genotypes was done through amplification refractory mutation system polymerase chain reaction (ARMS-PCR). Further, serum biomarkers were measured to investigate the association among risk factors of DM and Pro/Ala polymorphism in PPAR-γ gene. About 31.5% Pro/Ala genotype was found in CVD patients out of which 22.5% were CAD patients and 9% were CMP patients. As a result, obesity, hypertension and smoking (35%, 23%, 21%, respectively) were observed to be the most critical risk factors accompanying Pro/Ala mutation in PPAR-γ particularly in CAD patients as compared to that in CMP patients. A similar pattern of association was observed among the elevated levels of glucose, cholesterol, triglyceride and ALT with Pro/Ala mutation in CAD patients. Further, CAD patients using ACE inhibitors (18%) and ß-blockers (13%) were found to be the carriers of Pro/Ala genotype and also showed significant increase in glucose level. This study suggests that hyperglycaemia in CAD patients particularly obese, smokers and hypertensives having Pro/Ala polymorphism in PPAR-γ gene are at high risk of developing DM as clearly observed by hyperglycaemia in CAD patients.

Pharmacogenetic study of ACE, AGT, CYP11B1, CYP11B2 and eNOS gene variants in hypertensive patients from Faisalabad, Pakistan.

OBJECTIVE: To investigate the association of genetic variants of renin angiotensin aldosterone system, endothelial nitric oxide synthase and 11-beta-hydroxylase genes, and the drug efficacy of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker. METHODS: This two time-point study was conducted from April to November 2016 at Allied Hospital, Faisalabad and National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, and comprised of hypertensive patients taking angiotensin-converting enzyme inhibitor and angiotensin receptor blocker who were followed up for 12 weeks. Baseline and follow-up clinical and biochemical parameters were measured for all patients. Total 11 polymorphisms were genotyped by polymerase chain reaction, polymerase chain reaction-restriction fragment length polymorphism and amplification-refractory mutation system-polymerase chain reaction assays. Data was divided into baseline and follow-up groups, while the latter group was further divided into responding and non-responding subgroups on the basis of patient response to angiotensin-converting enzyme inhibitor and angiotensin receptor blocker drugs. Data was analysed using SPSS 20. RESULTS: Of the 45 patients, 25(55.5%) were females and 20(44.5%) were males. There was a significant reduction in the systolic blood pressure (p=0.004) and low-density lipoprotein cholesterol (p<0.001) from the baseline to the follow-up. Systolic blood pressure was significantly reduced in the responding group (p=0.003), while diastolic blood pressure (p=0.121) was not significantly different. There was no effect of angiotensin-converting enzyme, angiotensinogen, 11-beta-hydroxylase, aldosterone synthase and endothelial nitric oxide synthase gene polymorphisms on angiotensin converting enzyme inhibitor and angiotensin receptor blocker efficacy. CONCLUSIONS: Inter-individual response to angiotensin converting enzyme inhibitor and angiotensin receptor blocker was found to be independent of genetic polymorphisms in renin angiotensin aldosterone system, endothelial nitric oxide synthase and 11-beta-hydroxylase genes.

Association of ACE I/D Polymorphism with Risk of Diabetes in Cardiovascular Disease Patients.

Angiotensin converting enzyme (ACE), as part of renin angiotensin aldosterone system, is involved in blood pressure regulation and control several physiological functions. Insertion/Deletion (I/D) polymorphism of ACE has pronounced effects on development of metabolic diseases like diabetes, cardiovascular diseases (CVDs) and hypertension. However, association of I/D polymorphism with risk of diabetes in CVD patients is not known. The aim of present study was to check the association of ACE I/D polymorphism with risk of diabetes in subjects with CVD. For this, 531 subjects were sampled and divided into 3 groups; G1-H: (healthy controls, n=117), G2-CN: (cardiac patients without diabetes, n=271) and G3-CD: (cardiac patients with diabetes, n=143). Genotyping of ACE I/D polymorphism was done by polymerase chain reaction. Allelic and genotypic frequencies were in Hardy Weinberg Equilibrium (χ2=0.11, p>0.05) and revealed high prevalence of I allele (55%) among all groups. However, II genotype was more common (37%) in G3-CD: group. Level of glucose was also higher in subjects with II genotype than DD genotype (12.6±6.3 mmol/L vs. 9.7±5.1 mmol/L). Logistic regression analysis revealed that ACE II genotype increase the risk of diabetes in CVD patients by ~2 times [OR=1.94, CI: 1.24-3.01, p=0.03]; however, this association did not reach the significance level when adjusted for age and gender. In conclusion, ACE I/D polymorphism influence the risk of diabetes in CVD patients and ACE II increases this risk by ~2 fold.

Biochemical Screening of Intellectually Disabled Patients: A Stepping Stone to Initiate a Newborn Screening Program in Pakistan.

Inborn errors of metabolism (IEMs) are rare group of genetic disorders comprising of more than 1,000 different types. Around 200 of IEMs are potentially treatable through diet, pharmacological and other therapies, if diagnosed earlier in life. IEMs can be diagnosed early through newborn screening (NBS) programs, which are in place in most of the developed countries. However, establishing a NBS in a developing country is a challenging task due to scarcity of disease related data, large population size, poor economy, and burden of other common disorders. Since, not enough data is available for the prevalence of IEMs in Pakistan; therefore, in this study, we set out to find the prevalence of various treatable IEMs in a cohort of intellectually disabled patients suspected for IEMs, which will help us to initiate a NBS program for the most frequent IEMs in Pakistan. Therefore, a total of 429 intellectually disabled (IQ <70) patient samples were collected from Pakistan. A subset of 113 patient samples was selected based on the clinical information for the detailed biochemical screening. Advance analytical techniques like, Amino Acid Analyzer, GC-MS, UHPLC-MS, and MS/MS were used to screen for different treatable IEMs like aminoacidopathies, fatty acid ß-oxidation disorders and mucopolysaccharidoses (MPS) etc. A total of 14 patients were diagnosed with an IEM i.e., 9 with homocystinuria, 2 with MPS, 2 with Guanidinoacetate methyltransferase (GAMT) deficiency and 1 with sitosterolemia. These IEMs are found frequent in the collected patient samples from Pakistan. Thus, present study can help to take an initiative step to start a NBS program in Pakistan, especially for the homocystinuria having highest incidence among aminoacidopathies in the studied patients, and which is amenable to treatment. This endeavor will pave the way for a healthier life of affected patients and will lessen the burden on their families and society.