Study of HLA class II loci reveals DQB1*03:03:02 as a risk factor for asthma in a Pakistani population.

Asthma, a chronic inflammatory disorder of the lungs and airways, typically results from a combination of multiple environmental and genetic factors. Human leucocyte antigen (HLA) region on chromosome 6p21 encodes the most highly polymorphic loci in the human genome, encoding genes with central roles in the immune function where HLA loci are strongly associated with various immune-mediated diseases such as autoimmunity, allergies and infection. The alleles of HLA class II genes such as DRB1 and DQB1 are the key genetic markers in the development of asthma and have been extensively studied in different ethnicities of the world population. However, the genetic screening of HLA class II alleles and haplotypes in Pakistani asthmatics has not been studied so far. The aim of the present study was to screen the HLA class II DRB1 and DQB1 alleles in asthma cases and controls in a Pakistani population. Seven hundred and two healthy controls and asthma patients were genotyped for HLA class II by sequence-specific polymerase chain reaction assays. The HLA-DRB1 and HLA-DQB1 allele and haplotype frequencies were calculated, and their risk or protective association with asthma was determined. Two-locus haplotypes of DRB1 and DQB1 alleles were imputed using Arlequin version 3.1 software. The signals of association with asthma were stronger at the DQB1 locus as compared to DRB1. HLA DQB1*03:03:02 (odds ratio [OR] = 2.42, 95% confidence interval [CI] = 1.34-4.25) was significantly associated with an increased risk of asthma, as was the haplotype comprised allele DRB1*07:01-DQB1*03:03:02 (OR = 2.40, 95% CI = 1.25-4.62). In contrast, DQB1*06 (OR = 0.39, 95% CI = 0.22-0.70) and DQB1*06:02 (OR = 0.27, 95% CI = 0.10-0.71) emerged as protective alleles for asthma. Our data concludes that the HLA DQB1*03:03:02 allele was a risk allele for asthma, whereas two DQB1 alleles, DQB1*06 and DQB1*06:02, were associated with asthma protection. Our findings highlight a prominent role for HLA-DQB1 alleles in asthma pathogenesis in studied Pakistani cases. More studies, especially with a larger study cohort are needed to confirm the utility of HLA DQB1*03:03:02 as a predictive marker.

Genetic association study of ERBB4 SNP rs1351592 with polycystic ovary syndrome in Pakistani population.

Polycystic ovary syndrome (PCOS) is an oligogenic condition characterised by hyperandrogenism, ovulatory dysfunction and polycystic ovarian morphology. Previously, European and Han Chinese populations identified different susceptibility loci, of which ERBB4 (rs1351592) was strongly associated with PCOS. Our study aimed to investigate the association of ERBB4 Single Nucleotide Polymorphism (SNP), rs1351592 with PCOS in Pakistani women of Hazara region. Fifty PCOS patients and 14 healthy women were recruited and SNP was replicated using ARMS-PCR and sequencing. The study showed that Luteinising Hormone (LH), Follicle Stimulating Hormone (FSH), and Testosterone (T) were significantly elevated in patients compared to controls (P <0.05). Overall, the frequency of G allele was higher than C allele and the SNP lacked significant association with PCOS. This is the first study demonstrating the association of ERBB4 SNP, rs1351592 with PCOS in Pakistani population. Further research using larger population size will help to estimate the role of ERBB4 SNP as potential biomarker for disease diagnosis.

Correlation of C-reactive protein levels, gene polymorphism and platelets count in Dengue infection.

OBJECTIVE: To determine the correlation of polymorphism in C-reactive protein gene with variation in serum levels in dengue patients. METHODS: The cross-sectional study was conducted at Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Pakistan, from October 2017 to October 2018, and comprised blood samples from dengue patients which were used to measure the serum levels of C-reactive protein. Deoxyribonucleic acid extraction followed by tetra amplification-refractory mutation system polymerase chain reaction was used to analyse the genotype variation T>G for single nucleotide polymorphism rs199953854 using allele-specific primers. Correlation of serum C-reactive protein levels with the C-reactive protein polymorphism in dengue patients was explored. Data was analysed using SPSS 21. RESULTS: Of the 200 patients, 108(54%) had very high C-reactive protein levels, 48(24%) had levels slightly higher than the upper limit, 14(7%) had low and 30(15%) had normal levels. Also, 162(81%) patients had low platelets count. Amplification of only T alleles was noted. CONCLUSIONS: C-reactive protein levels were found to be increased with suppressed platelets count in dengue patients. Single nucleotide polymorphism rs199953854 appeared to have no polymorphism.

Phytochemistry, antioxidant and antibacterial activities of fruit extracts of Myrsine africana L.

Myrsine africana L. a commonly consumed medicinal plant grows in forest of mountains region located at North East of Pakistan. In current study, the fruit extracts were chemically characterized and their bioactivities were determined. Higher quantity of total phenols, total flavonoids and tannins were obtained from methanolic fruit extracts. The HPLC analysis provided higher level of quercetin followed by rutin and p-coumaric acid. Whereas the GC-MS quantification had given significant level of ten saturated and unsaturated fatty acids and some of them were not reported earlier. In vitro study, lower cytotoxic behavior of fruit extracts but higher antioxidant values as well as higher zone of inhibition versus S. aureus, E. coli, K. pneumonia and B. subtilis and Mycobacterium tuberculosis were observed. The organic compounds found in fruit extracts of M. africana correlated well with its used in ethno medicines.

Significant association of DRD2 enhancer variant rs12364283 with heroin addiction in a Pakistani population.

The dopamine D2 receptor encoded by DRD2 has been implicated in multiple psychiatric disorders, mediated at least in part by two intronic variants affecting mRNA splicing, rs1076560 and rs2283265, and a less frequent enhancer variant, rs12364283, which increases DRD2 mRNA expression. This study tests whether these functionally validated variants confer susceptibility toward heroin addiction in a Pakistani population. A total of 540 heroin addicts and 467 healthy controls were genotyped, basic allele and genotype tests were performed. Neither rs1076560 nor rs2283265 significantly associated with heroin addiction. The enhancer rs12364283 occurs more frequently in heroin-dependent cases than controls (MAF 13% vs. 7%, respectively), revealing significant association with heroin addiction (p = 3.0E-06, OR 2.1). This study identifies rs12364283 of DRD2 as a potential risk factor for heroin addiction in the Pakistani study population. This enhancer variant had been shown to increase DRD2 mRNA expression, a possible factor in increased vulnerability to heroin addiction. Further studies are needed to validate this association of rs12364283.

Contributions of Interactions Between Lifestyle and Genetics on Coronary Artery Disease Risk.

PURPOSE OF THE REVIEW: To summarize current knowledge on interactions between genetic variants and lifestyle factors (G×L) associated with the development of coronary artery disease (CAD) and prioritize future research. RECENT FINDINGS: Genetic risk and combined lifestyle factors and behaviors have a log-additive effect on the risk of developing CAD. First, we describe genetic and lifestyle factors associated with CAD and then focus on G×L interactions. The majority of G×L interaction studies are small-scale candidate gene studies that lack replication and therefore provide spurious results. Only a few studies, of which most use genetic risk scores or genome-wide approaches to test interactions, are robust in number and analysis strategy. These studies provide evidence for the existence of G×L interactions in the development of CAD. Further G×L interactions studies are important as they contribute to our understanding of disease pathophysiology and possibly provide insights for improving interventions or personalized recommendations.

Mitochondrial Genetic Diversity and Phylogeography of Mus musculus castaneus in Northern Punjab, Pakistan.

Regions of Iran, Afghanistan, Pakistan and northwestern India have been proposed as the place of origin of Mus musculus castaneus. But despite the fact that Pakistan encompasses an important part of its range, M. m. castaneus populations in Pakistan have not been the subject of intensive genetic and biogeographic studies, except for a very small number of samples included in past studies. We studied genetic variation in M. m. castaneus (CAS) from northern Punjab Province, Pakistan, by using cytochrome b (Cytb) analysis in a sample of 98 individuals. Median-joining network revealed four well differentiated CAS sub-lineages coexisting within a small geographical region; these had previously been thought to have largely non-overlapping geographic distributions. Moreover, haplotypes from Pakistan occupied a central position in the network and all identified global haplotypes were also present in Pakistan. All identified CAS sub-lineages proved to be highly diverse on the basis of haplotype and nucleotide diversity indices. Tajima's D test and Fu's Fs tests of neutrality suggest recent population expansions in all sub-lineages. Expansion times were estimated as 21,760-134,930, 10,800-64,400 and 4950-30,665 ybp using substitution rates of 2.5%, 5% and 11%, respectively. Our results support the hypothesis that northern Punjab Province in Pakistan is the most likely source area for M. m. castaneus, and that the CAS sub-lineages in this region have undergone rapid population expansion events at different time periods, which appear to have benefitted from human-mediated transport, although one of them clearly predates the establishment of human settlements in this region.

Screening of cardiometabolic risks clustering in young Pakistani adults classified by anthropometric traits.

OBJECTIVE: To estimate frequencies of metabolic risk phenotypes and their associations in body mass index and waist circumference-based obesity categories. METHODS: The cross-sectional study was conducted at Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Pakistan, from August 2014 to March 2016. Anthropometric and clinical data of young adults was collected. All subjects were categorised into body mass index, waist circumference-based obesity categories and common metabolic risk phenotypes (hypertension, hyperglycaemia, dyslipidaemia) frequencies and their associations were estimated in age and gender adjusted models. Data was analysed using SPSS 21. RESULTS: Of the 2,000 participants, 800(40%) were females and 1,200(60%) were males. There were 500(25%) participants in each group, i.e. underweight, normal weight, overweight and obese. The overall mean age was 23.68±4.33 years (range: 16-30 years). All clinical parameters were significantly raised in general and abdominally obese class (p<0.05). Based on body mass index and waist circumference, the frequency of general obesity was 324(16.2%) and abdominal obesity was 994(49.7%). Co-morbid metabolic risk phenotypes were as follows: hypertension 1,098(54.9%) and 924(46.2%); hyperglycaemia 1,116(55.8%) and 550(27.5%); dyslipidaemia 300(15%) and 194(9.7%), respectively. The strongest associations of body mass index and waist circumference alone catergorised obesity were found with hyperglycaemia, (Odds ratio: 7.23, 6.49) followed by dyslipidemia (Odds ratio: 5.60, 5.67) and hypertension (Odds ratio: 3.28, 3.02). . CONCLUSIONS: Body mass index and waist circumference were found to be powerful, discriminating predictors of co-morbidities linked with general and abdominal obesity.

Identification of Metabolic risk phenotypes predisposing to Non-Alcoholic Fatty Liver Disease in a Pakistani Cohort.

OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) has emerged in the last two decades with worldwide prevalence of 25.24%. Due to its increasing frequency in Pakistan, it was aimed to identify disease predisposing metabolic risks and their association with NAFLD. METHODS: Anthropometric and biochemical investigations were collected from 1366 subjects with minor metabolic disturbances. Comparative analyses were performed to compute frequencies of common metabolic risk phenotypes while their associations with NAFLD were explored using regression analyses. The prevalence of NAFLD was also estimated in total, age, and gender-based population cohorts. RESULTS: Among metabolic risk phenotypes obesity, hyperglycemia, hypertension, and dyslipidemia significantly associated (p<0.001) with NAFLD risk irrespective of age, gender, and BMI. Prevalence of NAFLD in total study cohort was 14.8%, 16.1% in males, 13.4% in females, 19.9% in ≥40 years and 8.7% in ≤40 years respectively. CONCLUSION: General Pakistani populations experiencing common metabolic disturbances are at high risk of NAFLD development, especially male gender and advanced age. Based on these parameters the stratified NAFLD population could be treated accordingly.

Whole-Exome sequencing identifies GYS2 biallelic variants in individuals with suspected epilepsy.

BACKGROUND: Adequate glucose supply is essential for brain function, therefore hypoglycemic states may lead to seizures. Since blood glucose supply for brain is buffered by liver glycogen, an impairment of liver glycogen synthesis by mutations in the liver glycogen synthase gene (GYS2) might result in a substantial neurological involvement. Here, we describe the phenotypes of affected siblings of two families harboring biallelic mutations in GYS2. METHODS: Two suspected families - a multiplex Pakistani family (family A) with three affected siblings and a family of Moroccan origin (family B) with a single affected child who presented with seizures and reduced fasting blood glucose levels were genetically characterized. Whole exome sequencing (WES) was performed on the index patients, followed by Sanger sequencing-based segregation analyses on all available members of both families. RESULTS: The variant prioritization of WES and later Sanger sequencing confirmed three mutations in the GYS2 gene (12p12.1) consistent with an autosomal recessive pattern of inheritance. A homozygous splice acceptor site variant (NM_021957.3, c. 1646 -2A>G) segregated in family A. Two novel compound heterozygous variants (NM_021957.3: c.343G>A; p.Val115Met and NM_021957.3: c.875A>T; p.Glu292Val) were detected in family B, suggesting glycogen storage disorder. A special diet designed to avoid hypoglycemia, in addition to change of the anti-seizure medication led to reduction in seizure frequency. CONCLUSIONS: This study suggests that the seizures in patients initially diagnosed with epilepsy might be directly caused, or influenced by hypoglycemia due to pathogenic variants in the GYS2 gene.

Whole exome sequencing identifies variable expressivity of CLN6 variants in Progressive myoclonic epilepsy affected families.

Progressive myoclonic epilepsies (PMEs) are a group of neurodegenerative disorders, predominantly affecting adolescents and, characterized by generalized worsening myoclonus epilepsies, ataxia, cognitive deficits, and dementia. To date, several genes, having implications in diverse phenotypic expressions associated with PMEs, have been identified. Genetic diagnosis is available for most of the adolescence-onset myoclonic epilepsies. This study aimed to elucidate the genetic basis of PMEs in three multiplex Pakistani families exhibiting clinically variable phenotypes. Causative variant(s) in the studied families, and mode of segregation were identified by Whole Exome Sequencing (WES) of the probands, followed by bi-directional Sanger sequencing for final validation. We identified homozygous recessive CLN6 missense variant c.768 C>G (p.Asp256Glu) in Family 1, and c.889 C>A (p.Pro297Thr) variant in Family 2. While in Family 3, we found a homozygous variant (c.316dup) that caused a frameshift mutation, leading to a premature stop codon in the CLN6 protein, resulting in a truncated protein (p.Arg106ProfsTer26). Though CLN6 is previously identified to underlie late infantile and adolescent onset neuronal ceroid lipofuscinosis, this study supports and expands the phenotypic spectrum of CLN6 mutations and signifies diagnositc potential CLN6 variants for PMEs. Diverse pathological effects of variant c .768 C>G were observed in Family 1, with same genotypes, suggesting clinical heterogeneity and/or variable expressivity that might be the implication of pleiotropic effects of the gene in these cases.

Whole Exome Sequencing Reveals Clustering of Variants of Known Vitiligo Genes in Multiplex Consanguineous Pakistani Families.

Vitiligo is an autoimmune complex pigmentation disease characterized by non-pigmented patches on the surface of the skin that affect approximately 0.5-2% population worldwide. The exact etiology is still unknown; however, vitiligo is hypothesized to be a multifactorial and genetically heterogeneous condition. Therefore, the current study is designed to investigate the anthropometric presentation and genetic spectrum of vitiligo in fifteen consanguineous Pakistani families. The clinical evaluation of participating individuals revealed varying degrees of disease severity, with 23 years as the average age of disease onset. The majority of the affected individuals had non-segmental vitiligo (NSV). Whole exome sequencing analysis revealed clustering of rare variants of known vitiligo-associated genes. For instance, in the affected individuals of family VF-12, we identified three novel rare variants of PTPN22 (c.1108C>A), NRROS (c.197C>T) and HERC2 (c.10969G>A) genes. All three variants replaced evolutionarily conserved amino acid residues in encoded proteins, which are predicted to impact the ionic interactions in the secondary structure. Although various in silico algorithms predicted low effect sizes for these variants individually, the clustering of them in affected individuals increases the polygenic burden of risk alleles. To our knowledge, this is the first study that highlights the complex etiology of vitiligo and genetic heterogeneity in multiplex consanguineous Pakistani families.

Differential Treatment Responses in Pakistani Schizophrenia Samples: Correlation with Sociodemographic Parameters, Drug Addiction, Attitude to the Treatment and Antipsychotic Agents.

Schizophrenia patients demonstrate variations in response to different therapies that are currently being used for the treatment of disorders, such as augmentation therapy (ECT or mood stabilizer) and combination therapy (with antipsychotics). These therapies are also used to treat schizophrenia patients in Pakistan; however, patients show poor overall response. Therefore, this study was conducted to investigate the association between the patients' response to treatment and the use of antipsychotic agents, with variability in overall response, within different groups of patients. Methods: We conducted a retrospective study that included schizophrenia subjects (N = 200) belonging to different age groups, ethnicities, and regions from different outpatient and inpatient departments in psychiatric institutes located in different cities of Pakistan. These patients were assessed for their response to treatment therapies and categorized into four groups (non-responders (N-R), slow response (S-R), patients with relapse, and completely recovered patients (C-R)) according to their responses. Results: The final analysis included 200 subjects, of which 73.5% were males. Mean age was 34 ± 10 years. Percentage of N-R was 5%, S-R was 42%, patients with relapse were 24%, and C-R was 1.5%. The generalized linear regression model shows a significant association between medication response and age (p = 0.0231), age of onset (p = 0.0086), gender (p = 0.005), and marital status (p = 0.00169). Variability within the medication responses was a result of the treatment regime followed. Antipsychotic agents were significantly associated with the treatment response (p = 0.00258, F = 4.981) of the patients. Significant variation was also observed in the treatment response (p = 0.00128) of the patients that were given augmentation therapy as well as combination therapy. Conclusion: The data suggests proper monitoring of patients' behavior in response to treatment therapies to implement tailored interventions. Despite several genetic studies supporting the heritability of schizophrenia, an insignificant association between characteristic features and family history might have been due to the limited sample size, suggesting collaborative work with massive sample sizes.

ß-Thalassemia in Pakistan: a pilot program on prenatal diagnosis in Multan.

Prenatal diagnosis (PND) of ß-thalassemia has been underutilized in Pakistan because of a number of social and economic factors. National Institute for Biotechnology and Genetic Engineering Faisalabad in collaboration with Multan Institute of Nuclear Medicine and Radiotherapy Multan introduced free PND service for carrier couples of Multan district. Multan has a population of about 4 million. More than 170 couples registered for retrospective PND and in 2 years 105 PND were carried out through first trimester chorionic villus sampling. Almost 90% of these couples were unable to afford the cost of PND and would not have undergone the test as free service was not available. Monoplex and Multiplex Amplification Refractory Mutation System-polymerase chain reaction and genomic DNA sequencing were used for detection of IVS (intervening sequence)-I-5 (G-C), FSC (frameshift codon)-8/9 (+G), FSC-41/42 (-TTCT), IVS-I-1 (G-T), 619 bp deletion, and CD-15 (G-A) ß-globin mutations. Eighty-one percent (85/105) couples analyzed were in a consanguineous marriage. Twenty-three fetuses were found homozygous mutant and all couples opted for discontinuation of affected pregnancies. More families are registering for PND after establishment of this free and accessible PND service.

SNP-Based Genome-Wide Association Mapping of Pollen Viability Under Heat Stress in Tropical Zea mays L. Inbred Lines.

Global environmental changes with more extreme episodes of heat waves are major threats to agricultural productivity. Heat stress in spring affects the reproductive stage of maize, resulting in tassel blast, pollen abortion, poor pollination, reduced seed set, barren ears and ultimately yield loss. As an aneamophelous crop, maize has a propensity for pollen abortion under heat stress conditions. To overcome the existing challenges of heat stress and pollen abortion, this study utilized a broad genetic base of maize germplasm to identify superior alleles to be utilized in breeding programs. A panel of 375 inbred lines was morpho-physiologically screened under normal and heat stress conditions in two locations across two consecutive planting seasons, 2017 and 2018. The exposure of pollen to high temperature showed drastic decline in pollen germination percentage. The average pollen germination percentage (PGP) at 35 and 45°C was 40.3% and 9.7%, respectively, an average decline of 30.6%. A subset of 275 inbred lines were sequenced using tunable genotyping by sequencing, resulting in 170,098 single nucleotide polymorphisms (SNPs) after filtration. Genome wide association of PGP in a subset of 122 inbred lines resulted in ten SNPs associated with PGP35°C (p ≤ 10-5), nine with PGP45°C (p ≤ 10-6-10-8) and ten SNPs associated with PGP ratio (p ≤ 10-5). No SNPs were found to be in common across PGP traits. The number of favorable alleles possessed by each inbred line for PGP35°C, PGP45°C, and the PGP ratio ranged between 4 and 10, 3-13 and 5-13, respectively. In contrast, the number of negative alleles for these traits ranged between 2 and 8, 3-13 and 3-13, respectively. Genetic mapping of yield (adjusted weight per plant, AWP-1) and flowering time (anthesis-silking interval, ASI) in 275 lines revealed five common SNPs: three shared for AWP-1 between normal and heat stress conditions, one for ASI between conditions, and one SNP, CM007648.1-86615409, was associated with both ASI and AWP-1. Variety selection can be performed based on these favorable alleles for various traits. These marker trait associations identified in the diversity panel can be utilized in breeding programs to improve heat stress tolerance in maize.

Demographic and Clinical Determinants of Tuberculosis and TB Recurrence: A Double-Edged Retrospective Study from Pakistan.

Objective: TB recurrence is the second episode of TB after initial treatment bringing about an additional 7% load in TB burden intensified by 17.7% of multidrug-resistant recurrent cases. It is necessary to curb recurrence so that attempts to deal with active disease can be made more effective. This study aimed to characterize sociodemographic and clinical factors associated with recurrent TB in a high-burden setting. Methodology. A retrospective case-control study was carried out at two hospitals in Rawalpindi, Pakistan. TB patients and controls were included in the study. Sociodemographic and clinical data were collected by questionnaire from all subjects. Multivariate regression analysis was performed to determine factors associated with TB and TB recurrence respectively. Results: In our study cohort, factors significantly associated with TB were low BMI (OR: 0.961 (CI 0.954-0.968), p < 0.001), female gender (OR: 2.065 (CI 1.922-2.219), p < 0.001), being single/unmarried (OR: 1.214 (CI 1.109-1.328), p=0.003), middle-income status (OR: 1.935 (CI 1.616-2.323), p < 0.001), smoking (OR: 1.567 (CI 1.435-1.710), p < 0.001), and diabetes mellitus (OR: 1.142 (CI 1.017-1.278), p=0.023). TB recurrence constituted 11.2% of patients presenting to the hospital. Compared with the first episode of TB, cases with recurrence were more likely to be older (OR: 1.011 (CI 1.004-1.017), p < 0.001), have disease awareness (OR: 1.906 (CI 1.486-2.437), p < 0.001), smear positive (OR: 2.384 (CI 1.650-3.536), p < 0.001), and be drug-resistant (OR: 5.615 (CI 4.265-7.386), p < 0.001). Conclusion: In the present study cohort, low BMI, female gender, being single, middle-income status, being unemployed, smoking, and being diabetic came out to be the sociodemographic and clinical risk factors for TB. Further exploring the TB cases increasing age, drug resistance and smear positivity stood out to be the major sociodemographic and clinical factors of TB recurrence despite high disease awareness.

9p21 Locus Polymorphism Is A Strong Predictor of Metabolic Syndrome and Cardiometabolic Risk Phenotypes Regardless of Coronary Heart Disease.

The world population is genetically predisposed to metabolic syndrome (MetS) and its components, also known as cardiometabolic risk phenotypes, which can cause severe health complications including coronary heart disease (CHD). Genetic variants in the 9p21 locus have been associated with CHD in a number of populations including Pakistan. However, the role of the 9p21 locus in MetS and cardiometabolic risk phenotypes (such as obesity, hypertension, hyperglycemia, and dyslipidemia) in populations with CHD or no established CHD has not been explored. Therefore, the present study was designed to explore the association of the minor/risk allele (C) of 9p21 locus SNP rs1333049 with MetS or its risk phenotypes regardless of an established CHD, in Pakistani subjects. Genotyping of rs1333049 (G/C) was performed on subjects under a case-control study design; healthy controls and cases, MetS with CHD (MetS-CHD+) and MetS with no CHD (MetS-CHD-), respectively. Genotype and allele frequencies were calculated in all study groups. Anthropometric and clinical variables (Means ± SD) were compared among study groups (i.e., controls, MetS + CHD and MetS-CHD) and minor/risk C allele carriers (GC + CC) vs. non-carriers (Normal GG genotype). Associations of the risk allele of rs1333049 SNP with disease and individual metabolic risk components were explored using adjusted multivariate logistic regression models (OR at 95% CI) with a threshold p-value of ≤0.05. Our results have shown that the minor allele frequency (MAF) was significantly high in the MAF cases (combined = 0.63, MetS-CHD+ = 0.57 and MetS-CHD- = 0.57) compared with controls (MAF = 0.39). The rs1333049 SNP significantly increased the risk of MetS, irrespective of CHD (MetS-CHD+ OR = 2.36, p < 0.05 and MetS-CHD- OR = 4.04, p < 0.05), and cardiometabolic risk phenotypes; general obesity, central obesity, hypertension, and dyslipidemia (OR = 1.56-3.25, p < 0.05) except hyperglycemia, which lacked any significant association (OR = 0.19, p = 0.29) in the present study group. The 9p21 genetic locus/rs1333049 SNP is strongly associated with, and can be a genetic predictor of, MetS and cardiometabolic risks, irrespective of cardiovascular diseases in the Pakistani population.

Whole-Exome Sequencing of Pakistani Consanguineous Families Identified Pathogenic Variants in Genes of Intellectual Disability.

Intellectual disability (ID) is a condition of significant limitation of cognitive functioning and adaptive behavior, with 50% of etiology attributed to genetic predisposition. We recruited two consanguineous Pakistani families manifesting severe ID and developmental delay. The probands were subjected to whole exome sequencing (WES) and variants were further prioritized based on population frequency, predicted pathogenicity and functional relevance. The WES data analysis identified homozygous pathogenic variants in genes MBOAT7 and TRAPPC9. The pathogenicity of the variants was supported by co-segregation analysis and in silico tool. The findings of this study expand mutation spectrum and provide additional evidence to the role of MBOAT7 and TRAPPC9 in causation of ID.

Isolation of Oxyberberine and ß-Sitosterol from Berberis lycium Royle Root Bark Extract and In Vitro Cytotoxicity against Liver and Lung Cancer Cell Lines.

Berberis lycium Royle has been traditionally used to cure rheumatism, eye and ear diseases, malarial fever, diabetes, stomach disorders, and skin diseases. There is a least amount of data available on cytotoxic capacity of Berberis lycium from Pakistani origin, so on this basis, the present study was aimed to screen Berberis lycium root bark extracts for cytotoxicity against cancer cell lines and isolation of chemical constituents from the most cytotoxic extract. Initial screening of extracts was performed on HepG2 cells at 100 µg/mL for 72 hours of treatment by using an MTT assay. Active fractions were subjected to a series of column chromatographies for the isolation of cytotoxic compounds. Molecular structures were elucidated by using combined data from 1H-NMR, 13C-NMR, and ESI-MS graphs. Assessment of reduction in cell proliferation by isolated compounds was performed on three human cancer cell lines (SK-Hep-1, HepG2, and NCI-H1299). Both n-hexane and chloroform fractions were found active with percent cell viabilities of 8.41 ± 2.23 and 22.31 ± 9.11 in HepG2 cells compared with lupeol 35.43 ± 3.35 percent viability. A protoberberine alkaloid identified as oxyberberine was isolated from chloroform fraction while ß-sitosterol was isolated from n-hexane fraction. Oxyberberine inhibited SK-Hep-1 cell proliferation under a dose-dependent manner with an IC50 value of 34.26 ± 3.34 µM while HepG2 cells showed 50% inhibition at 62.96 ± 4.12 µM. ß-Sitosterol showed reduction in cell viability in SK-Hep-1 cells and HepG2 cells with IC50 values of 123.12 ± 3.51 µM and 140 ± 4.21 µM. This is the first report on the isolation of oxyberberine and ß-sitosterol from Berberis lycium root bark and their cytotoxic evaluation against SK-Hep-1 and NCI-H1299 cells. The cytotoxic potential of Berberis lycium Royle extracts and isolated compounds is suggesting that it is a promising candidate for anticancer drug discovery.

Sequence variants in three genes underlying leukodystrophy in Pakistani families.

Leukodystrophies (LDs) are a heterogeneous group of rare and progressive genetic diseases that affect brain, spinal cord, and often the peripheral nerves. They are characterized by abnormal development or destruction of the myelin sheath of the brain. This study was aimed to search for the causative variants in three unrelated consanguineous families presented with LD. Detailed clinical investigations were carried out on probands in three unrelated consanguineous families of Pakistani origin. Targeted gene sequencing and Whole Exome Sequencing (WES) were performed for variant identification. Candidate variants were checked for co-segregation with the phenotype using Sanger sequencing. Public databases including ExAC, gnomAD, dbSNP, and the 1,000 Genome Project were searched to determine frequencies of the alleles. Conservation of the missense variants was ensured by aligning orthologous protein sequences from diverse vertebrate species. Targeted gene sequencing identified a novel homozygous missense mutation [c.2135G > A, p.(Arg712His) in the ATP Binding Cassette Subfamily D Member 1 (ABCD1; OMIM# 300371) in three affected siblings in family A.WES followed by validation by Sanger sequencing revealed previously reported homozygous missense variants [c.162C > A; p.(Asn54Lys)] in ASPA (OMIM# 608034) in family B and [c.361G > C,p.(Gly121Arg)] in ARSA (OMIM# 607574) in family C. Investigation of three families underlies importance of WES as an amazing diagnostic tool for conclusive determination of a specific type of LD. Further, the study would assist in carrier testing and prenatal diagnosis of the affected families. In addition, searching for common variants in the genes causing LD would help in designing low-cost targeted variation testing in patients.