Elucidation of molecular mechanism for colistin resistance among Gram-negative isolates from tertiary care hospitals.

Antimicrobial resistance is a growing concern of global public health. The emergence of colistin-resistance among carbapenem-resistant (CPR) Gram-negative bacteria causing fear of pan-resistance, treatment failure, and high mortality across the globe. AIM: To determine the genotypic colistin-resistance mechanisms among colistin-resistant (CR)Gram-negative clinical isolates along with genomic insight into hypermucoviscous(hv)-CR-Klebsiella pneumoniae. METHODS: Phenotypic colistin-resistance via broth-microdilution method. PCR-based detection of plasmid-mediated colistin resistance genes(mcr-1,2,3). Characterization of selected hvCR-K. pneumoniae via Whole-genome sequencing. RESULTS: Phenotypic colistin-resistance was 28% among CPR-Gram-negative isolates of which 90% of CR-isolates displayed MDR profile with overall low plasmid-mediated colistin resistance (mcr-2 = 9.4%;mcr-3 = 6%). Although K. pneumoniae isolates showed the highest phenotypic colistin-resistance (51%) however, relatively low plasmid-mediated gene-carriage (mcr-2 = 11.5%;mcr-3 = 3.4%) pointed toward other mechanisms of colistin-resistance. mcr-negative CR-K. pneumoniae displaying hv-phenotype were subjected to WGS. In-silico analysis detected 7-novel mutations in lipid-A modification genes includes eptA(I38V; V50L; A135P), opgE(M53L; T486A; G236S), and arnD(S164P) in addition to several non-synonymous mutations in lipid-A modification genes conferring resistance to colistin. Insertion of 6.6-kb region harboring putative-PEA-encoding gene(yjgX) was detected for the first time in K. pneumoniae (hvCRKP4771). In-silico analysis further confirmed the acquisition of not only MDR determinants but several hypervirulent-determinants displaying a convergent phenotype. CONCLUSION: overall high prevalence of phenotypic colistin resistance but low mcr-gene carriage suggested complex chromosomal mediated resistance mechanism especially in K. pneumoniae isolates. The presence of novel mutations in lipid-A modification genes and the acquisition of putative-PEA-encoding gene by hvCR-K. pneumoniae points toward the role of chromosomal determinants conferring resistance to colistin in the absence of mcr-genes.

The Kalash genetic isolate: ancient divergence, drift, and selection.

The Kalash represent an enigmatic isolated population of Indo-European speakers who have been living for centuries in the Hindu Kush mountain ranges of present-day Pakistan. Previous Y chromosome and mitochondrial DNA markers provided no support for their claimed Greek descent following Alexander III of Macedon's invasion of this region, and analysis of autosomal loci provided evidence of a strong genetic bottleneck. To understand their origins and demography further, we genotyped 23 unrelated Kalash samples on the Illumina HumanOmni2.5M-8 BeadChip and sequenced one male individual at high coverage on an Illumina HiSeq 2000. Comparison with published data from ancient hunter-gatherers and European farmers showed that the Kalash share genetic drift with the Paleolithic Siberian hunter-gatherers and might represent an extremely drifted ancient northern Eurasian population that also contributed to European and Near Eastern ancestry. Since the split from other South Asian populations, the Kalash have maintained a low long-term effective population size (2,319-2,603) and experienced no detectable gene flow from their geographic neighbors in Pakistan or from other extant Eurasian populations. The mean time of divergence between the Kalash and other populations currently residing in this region was estimated to be 11,800 (95% confidence interval = 10,600-12,600) years ago, and thus they represent present-day descendants of some of the earliest migrants into the Indian sub-continent from West Asia.

A study into the genetic basis of aspirin resistance in Pakistani patients with coronary artery disease.

Aspirin is a key player in the management and prevention of stroke and myocardial infarction in patients with atherothrombosis. About 12% of Pakistanis suffering from coronary artery disease are resistant to aspirin's effects. Clinical, biochemical and genetic factors are known to be responsible for this phenomenon. We conducted this study to investigate whether previously studied polymorphisms in COX-1, GPIIIa, GPIa and P2RYI genes could be the cause of aspirin resistance in our population. Blood samples were collected from 29 aspirin non-responders and 60 ethnically matched responders. Aspirin response assay was performed on IMPACT-R and DNA prepared from blood using the phenol: chloroform method. Genotyping was carried out for four SNPS including COX-1 C50T (rs3842787), GPIIIA PIA1/A2 polymorphism (rs5918), GPIA C807T (rs1126643) and p2RY1 C893T (rs1065776). No statistically significant differences were observed in the allele or genotype frequencies between the aspirin non responders and responders indicating the possible involvement of different genetic determinants of aspirin resistance in our population. This study paves the way for further research into the field of aspirin resistance in Pakistan.

In silico analysis of SIGMAR1 variant (rs4879809) segregating in a consanguineous Pakistani family showing amyotrophic lateral sclerosis without frontotemporal lobar dementia.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting upper motor neurons in the brain and lower motor neurons in the brain stem and spinal cord, resulting in fatal paralysis. It has been found to be associated with frontotemporal lobar degeneration (FTLD). In the present study, we have described homozygosity mapping and gene sequencing in a consanguineous autosomal recessive Pakistani family showing non-juvenile ALS without signs of FTLD. Gene mapping was carried out in all recruited family members using microsatellite markers, and linkage was established with sigma non-opioid intracellular receptor 1 (SIGMAR1) gene at chromosome 9p13.2. Gene sequencing of SIGMAR1 revealed a novel 3'-UTR nucleotide variation c.672*31A>G (rs4879809) segregating with disease in this family. The C9ORF72 repeat region in intron 1, previously implicated in a related phenotype, was excluded through linkage, and further confirmation of exclusion was obtained by amplifying intron 1 of C9ORF72 with multiple primers in affected individuals and controls. In silico analysis was carried out to explore the possible role of 3'-UTR variant of SIGMAR1 in ALS. The Regulatory RNA motif and Element Finder program revealed disturbance in miRNA (hsa-miR-1205) binding site due to this variation. ESEFinder analysis showed new SRSF1 and SRSF1-IgM-BRCA1 binding sites with significant scores due to this variation. Our results indicate that the 3'-UTR SIGMAR1 variant c.672*31A>G may have a role in the pathogenesis of ALS in this family.

Monoamine Oxidase A gene polymorphisms and self reported aggressive behaviour in a Pakistani ethnic group.

OBJECTIVE: To investigate the association of monoamine oxidase Agene polymorphisms with aggression. METHODS: The study was conducted in an ethnic community in Lahore, Pakistan, from August 2008 to December 2009 on the basis of data that was collected through a questionnaire between August 2004 and September 2005. It analysed 10 single nucleotide polymorphisms of monoamine oxidase A in unrelated males from the same ethnic background who were administered a Punjabi translation of the Buss and Perry aggression questionnaire. SPSS 13 was used for statistical analysis. RESULTS: Of the total 133 haplotypes studied, 52(39%) were Haplotype A, 58(43.6%) B, 8(6%) C, 3(2.3%) D, 9(6.8%) E and 3(2.3%) F. The six haplotypes were analysed for association with scores of the four subscales of the aggression questionnaire and multivariate analysis of variance showed no significant differences (p>0.05 each) in the error variances of the total scores and scores for three of the sub-scales across the haplotypes. The variance was significantly different only for the anger sub-scale (p<0.05). CONCLUSIONS: The association of an extended haplotype with low levels of self-reported aggression in this study should assist in characterisation of functional variants responsible for non-aggressive behaviour in male subjects.

A study on the association of TNF-α(-308), IL-6(-174), IL-10(-1082) and IL-1Ra(VNTR) gene polymorphisms with rheumatic heart disease in Pakistani patients.

Inflammation is an important contributor to the pathogenesis of rheumatic heart disease (RHD), a disorder of heart valves caused by a combination of immune, genetic and environmental factors. Cytokines are important mediators of inflammatory and immune responses. The aim of this study was to investigate the role of cytokine gene polymorphisms and their potential usefulness as biomarkers in RHD patients from Pakistan. We screened 150 RHD patients and 204 ethnically matched controls for tumor necrosis factor (TNF)-α(-308)G/A, interleukin (IL)-10(-1082) G/A, interleukin (IL)-6(-174) G/C and a variable number of tandem repeats (VNTRs) polymorphism of the IL-1Ra gene using polymerase chain reaction. The results showed that TNF-α(-308) A and IL-6(-174) G alleles were associated with susceptibility to RHD (p=0.000; OR=2.81; CI=1.5-5.14 and p=0.025; OR=1.50; CI=1.04-2.16 respectively). The TNF-α(-308) AA and GA genotypes were associated with susceptibility to RHD (p=0.012; OR=9.94; CI; 1.21-217.3 and p=0.046; OR=1.97; CI=0.98-3.97 respectively) while the GG genotype seemed to confer resistance (p=0.003; OR=0.39; CI=0.20-0.76). The GG genotype for IL-6(-174) was significantly associated with predisposition to RHD (p=0.015; OR=2.6; CI=1.17-5.85). The A1 (four repeats) and A2 (two repeats) alleles at the IL-1Ra VNTR polymorphism were associated with resistance and susceptibility to RHD respectively. However, this polymorphism deviated from Hardy-Weinberg equilibrium in both patients and controls in our population. TNF-α(-308) and IL-6(-174) polymorphisms may be useful markers for the identification of individuals susceptible to RHD in Pakistan. These individuals could be provided aggressive prophylactic intervention to prevent the morbidity and mortality associated with RHD.

HLA class I and II polymorphisms in the Gujjar population from Pakistan.

HLA polymorphisms at the HLA -A, -B, -C, -DRB and -DQB1 loci were investigated in the Gujjar population from the Punjab province of Pakistan. The Gujjars (n = 97) were genotyped using sequence specific primers for polymerase chain reaction. The allele and haplotype frequencies were calculated and a neighbor-joining (NJ) tree comparing the Gujjar with other populations was constructed. The class I allelic groups with a frequency greater than 10% include A*01, A*02, A*11, A*26 and A*31 at the HLA-A locus, B*08 and B*51 at the HLA-B locus and C*07 and C*14 at the HLA-C locus. Among the 12 allelic groups detected at the DRB1* locus, *03, *13, and *15 were present at frequencies higher than 10% whereas at the DQB1 locus, the allelic groups*06 and *02 accounted for over half of the Gujjar population. HLA-A*31-B*51-DRB1*13 was the most common (8.8%) haplotype in this population. A NJ tree revealed that the Pakistani Gujjar are closely related to the Golla tribe from Andhra Pradesh in India. The two populations are dedicated to the same profession, cattle breeding. HLA analyses of additional Punjab castes would provide valuable information for anthropological, organ transplantation and genetic disease studies.

Vitamin B6 based Pt(II) complexes: biomolecule derived potential cytotoxic agents for thyroid cancer.

Vitamin B6 is an essential vitamin that serves as a co-enzyme in a number of enzymatic reactions in metabolism of lipids, amino acids, and glucose. In the current study, we synthesized vitamin B6 derived ligand (L) and its complex Pt(L)Cl (C1). The ancillary chloride ligand of C1 was exchanged with pyridine co-ligand and another complex Pt(L)(py).BF4 (C2) was obtained. Both these complexes were obtained in excellent isolated yields and characterized thoroughly by different analytical methods. Thyroid cancer is one of the most common malignancies of the endocrine system, we studied the in vitro anticancer activity and mechanism of these vitamin B6 derived L and Pt(II) complexes in thyroid cancer cell line (FTC). Based on MTT assay, cell proliferation rate was reduced in a dose-dependent manner. According to apoptosis analysis, vitamin B6 based Pt(II) complexes treated cells depicted necrotic effect and TUNEL based apoptosis was observed in cancer cells. Furthermore, qRT-PCR analyses of cancer cells treated with C1 and/or C2 showed regulated expression of anti-apoptotic, pro-apoptosis and autophagy related genes. Western blot results demonstrated that C1 and C2 induced the activation of p53 and the cleavage of Poly (ADP-ribose) polymerase (PARP). These results suggest that these complexes inhibit the growth of FTC cells and induce apoptosis through p53 signaling. Thus, vitamin B6 derived Pt(II) complexes C1 and C2 may be potential cytotoxic agents for the treatment of thyroid cancer.

Structural and functional annotation of PR/SET Domain (PRDM) protein family: In-silico study elaborating role of PRDM12 mutation in congenital insensitivity to pain.

Congenital insensitivity to pain (CIP), classified as a type of hereditary sensory and autonomic neuropathies, is a rare disease in which the affected individuals fail to perceive sensation of pain. One of the PR/SET Domain Proteins, PRDM12, has been identified in recent past as a candidate gene for congenital insensitivity to pain. In the present study, we performed whole exome sequencing in a Pakistani family with CIP phenotype to ascertain the causative mutation. We identified a previously described alanine repeat duplication in PRDM12 (Ala353_Ala359dup) in this family. After this, we performed structural annotations for PR/SET Domain (PRDM) containing protein family to prognosticate the potential hypothetical structure of PRDM proteins with physical and chemical parameters. Out of nineteen members of this family, four members (PRDM5, PRDM8, PRDM12 and PRDM13) were specially focused because of their role in neurological disorders. Predictions about structure and interactions of these proteins revealed novel interacting molecules and pathways. Detailed in silico analysis of PRDM12 was performed to elaborate importance of its domain structure in interaction with other proteins and its role in pain insensitivity phenotype. These results have substantially enhanced our understanding regarding the etiology of congenital pain insensitivity and would stimulate further research on therapy and prevention.

Refined geographic distribution of the oriental ALDH2*504Lys (nee 487Lys) variant.

Mitochondrial aldehyde dehydrogenase (ALDH2) is one of the most important enzymes in human alcohol metabolism. The oriental ALDH2*504Lys variant functions as a dominant negative, greatly reducing activity in heterozygotes and abolishing activity in homozygotes. This allele is associated with serious disorders such as alcohol liver disease, late onset Alzheimer disease, colorectal cancer, and esophageal cancer, and is best known for protection against alcoholism. Many hundreds of papers in various languages have been published on this variant, providing allele frequency data for many different populations. To develop a highly refined global geographic distribution of ALDH2*504Lys, we have collected new data on 4,091 individuals from 86 population samples and assembled published data on a total of 80,691 individuals from 366 population samples. The allele is essentially absent in all parts of the world except East Asia. The ALDH2*504Lys allele has its highest frequency in Southeast China, and occurs in most areas of China, Japan, Korea, Mongolia, and Indochina with frequencies gradually declining radially from Southeast China. As the indigenous populations in South China have much lower frequencies than the southern Han migrants from Central China, we conclude that ALDH2*504Lys was carried by Han Chinese as they spread throughout East Asia. Esophageal cancer, with its highest incidence in East Asia, may be associated with ALDH2*504Lys because of a toxic effect of increased acetaldehyde in the tissue where ingested ethanol has its highest concentration. While the distributions of esophageal cancer and ALDH2*504Lys do not precisely correlate, that does not disprove the hypothesis. In general the study of fine scale geographic distributions of ALDH2*504Lys and diseases may help in understanding the multiple relationships among genes, diseases, environments, and cultures.

Y-chromosomal evidence for a limited Greek contribution to the Pathan population of Pakistan.

Three Pakistani populations residing in northern Pakistan, the Burusho, Kalash and Pathan claim descent from Greek soldiers associated with Alexander's invasion of southwest Asia. Earlier studies have excluded a substantial Greek genetic input into these populations, but left open the question of a smaller contribution. We have now typed 90 binary polymorphisms and 16 multiallelic, short-tandem-repeat (STR) loci mapping to the male-specific portion of the human Y chromosome in 952 males, including 77 Greeks in order to re-investigate this question. In pairwise comparisons between the Greeks and the three Pakistani populations using genetic distance measures sensitive to recent events, the lowest distances were observed between the Greeks and the Pathans. Clade E3b1 lineages, which were frequent in the Greeks but not in Pakistan, were nevertheless observed in two Pathan individuals, one of whom shared a 16 Y-STR haplotype with the Greeks. The worldwide distribution of a shortened (9 Y-STR) version of this haplotype, determined from database information, was concentrated in Macedonia and Greece, suggesting an origin there. Although based on only a few unrelated descendants, this provides strong evidence for a European origin for a small proportion of the Pathan Y chromosomes.

Human leukocyte antigen (HLA) class II association with rheumatic heart disease in Pakistan.

BACKGROUND AND AIM OF THE STUDY: Rheumatic heart disease (RHD) is widespread in Pakistan. Specific alleles of the human leukocyte antigen (HLA) system are associated with RHD in various world populations. The study aim was to investigate the involvement of HLA class II alleles in genetic susceptibility to RHD in patients with relatively homogeneous clinical manifestations, in Pakistan. METHODS: Blood samples were collected from 114 unrelated patients (94 females, 20 males) with rheumatic mitral valve disease, predominantly mitral stenosis, as assessed by echocardiography. The control group comprised 109 unrelated, ethnically matched, healthy individuals (60 females, 49 males) with normal echocardiograms. Genomic DNA was extracted from venous blood using a standard phenol/chloroform extraction procedure. HLA-DRB, -DQA1, and -DQB1 alleles were typed using polymerase chain reaction with sequence-specific primers. HLA allele and haplotypes frequencies were then calculated. RESULTS: A significantly higher frequency of DRB1*07 was observed in patients as compared to controls (one-way parametric analysis of variance, F = 4.84, p = 0.028; OR = 1.76, p = 0.039). No alleles for the HLA-DQA1 or -DQB1 loci were associated with the disease. HLA-DRB1*07-DQA1*0501-DQB1*02, the only haplotype that differed significantly between patients and controls (one-way parametric Anova, F = 4.866, p = 0.028; OR = 7.33, p = 0.06), did not exhibit significant linkage disequilibrium. CONCLUSION: These results show that HLA-DRB1*07, associated with RHD in various world populations, is also associated with RHD in the Pakistani population. The validation of HLA associations with RHD, which is observed in different world populations, may lead to the development of a cost-effective strategy in the primary prevention of this disease.

DC-SIGN interacts with Mycobacterium leprae but sequence variation in this lectin is not associated with leprosy in the Pakistani population.

The C-type lectin DC-SIGN is involved in early interactions between human innate immune cells and a variety of pathogens. Here we sought to evaluate whether DC-SIGN interacts with the leprosy bacillus, Mycobacterium leprae, and whether DC-SIGN genetic variation influences the susceptibility and/or pathogenesis of the disease. A case-control study conducted in a cohort of 272 individuals revealed no association between DC-SIGN variation and leprosy. However, our results clearly show that DC-SIGN recognizes M. leprae, indicating that mycobacteria recognition by this lectin is not as narrowly restricted to the Mycobacterium tuberculosis complex as previously thought. Altogether, our results provide further elucidation of M. leprae interactions with the host innate immune cells and emphasize the importance of DC-SIGN in the early interactions between the human host and the infectious agents.

Genetic instability in EBV-transformed lymphoblastoid cell lines.

Epstein Barr virus (EBV)-transformed lymphoblastoid cell lines are commonly used to provide an inexhaustible supply of DNA. We examined microsatellite instability in these cell lines in 35 individuals where DNA was available from the original blood samples and from cultured cell lines. Mutations were observed in 0.3% of the analyses, thus providing a quantitative measure of somatic mutation rate.

Separating the post-Glacial coancestry of European and Asian Y chromosomes within haplogroup R1a.

Human Y-chromosome haplogroup structure is largely circumscribed by continental boundaries. One notable exception to this general pattern is the young haplogroup R1a that exhibits post-Glacial coalescent times and relates the paternal ancestry of more than 10% of men in a wide geographic area extending from South Asia to Central East Europe and South Siberia. Its origin and dispersal patterns are poorly understood as no marker has yet been described that would distinguish European R1a chromosomes from Asian. Here we present frequency and haplotype diversity estimates for more than 2000 R1a chromosomes assessed for several newly discovered SNP markers that introduce the onset of informative R1a subdivisions by geography. Marker M434 has a low frequency and a late origin in West Asia bearing witness to recent gene flow over the Arabian Sea. Conversely, marker M458 has a significant frequency in Europe, exceeding 30% in its core area in Eastern Europe and comprising up to 70% of all M17 chromosomes present there. The diversity and frequency profiles of M458 suggest its origin during the early Holocene and a subsequent expansion likely related to a number of prehistoric cultural developments in the region. Its primary frequency and diversity distribution correlates well with some of the major Central and East European river basins where settled farming was established before its spread further eastward. Importantly, the virtual absence of M458 chromosomes outside Europe speaks against substantial patrilineal gene flow from East Europe to Asia, including to India, at least since the mid-Holocene.

Geographically separate increases in the frequency of the derived ADH1B*47His allele in eastern and western Asia.

The ADH1B Arg47His polymorphism has been convincingly associated with alcoholism in numerous studies of several populations in Asia and Europe. In a review of literature from the past 30 years, we have identified studies that report allele frequencies of this polymorphism for 131 population samples from many different parts of the world. The derived ADH1B*47His allele reaches high frequencies only in western and eastern Asia. To pursue this pattern, we report here new frequency data for 37 populations. Most of our data are from South and Southeast Asia and confirm that there is a low frequency of this allele in the region between eastern and western Asia. The distribution suggests that the derived allele increased in frequency independently in western and eastern Asia after humans had spread across Eurasia.

Detection of novel Y SNPs provides further insights into Y chromosomal variation in Pakistan.

Biallelic polymorphisms on the Y chromosome have been extensively used to study the history, evolution, and migration patterns of world populations. In this study we screened 8.5 kb of Y chromosomal DNA for single nucleotide polymorphisms (SNPs) in a panel of 95 male individuals belonging to different haplogroups. Five novel Y-SNPs (PK1-5) were identified, four in the Pakistani sample and one in an African sample. The ancestral state of each SNP was determined in two chimpanzee samples and a variety of Pakistani ethnic groups. In addition to these novel Y-SNPs 77 additional markers on the Y chromosome were analyzed to place the SNPs on the phylogenetic tree of Y chromosomal lineages and to further investigate extant human Y chromosomal variation within Pakistan. BATWING analysis gave an estimate of between 2,500 and 7,300 YBP for population expansion in Pakistan which coincides with the period of the Indus Valley civilizations.

Mutation screening of Pakistani families with congenital eye disorders.

To map the disease loci several Pakistani families suffering from autosomal recessive retinitis pigmentosa with preserved para-arteriolar retinal pigment epithelium and Leber congenital amaurosis (LCA) were analyzed. Analysis revealed close genetic linkage between the disease phenotype of some of the families (3330RP, 111RP and 010LCA) and the microsatellite markers on chromosome 1q31. Mutation screening of the candidate gene CRB1 revealed a G to A transversion in exon 7 in arRP family 330RP and a T to C substitution in another arRP family, 111RP. In exon 9 of the CRB1 gene a T to C transversion was found in the family suffering from LCA (010LCA). The LCA phenotype of another family (011LCA) in which the CRB1 locus was excluded, showed linkage with microsatellite markers D17S1294 and D17S796 on chromosome 17p13.1. The association of the candidate gene GUCY2D (17p13.1) with the disease phenotype was excluded as no disease-associated mutation was found in any of its exons. Mutation screening of another candidate gene, AIPL1 located in the same region, showed a novel homozygous C to A substitution in exon 2. These sequence changes are unique for the Pakistani families and some of these have not been reported previously.

Y-chromosomal DNA variation in Pakistan.

Eighteen binary polymorphisms and 16 multiallelic, short-tandem-repeat (STR) loci from the nonrecombining portion of the human Y chromosome were typed in 718 male subjects belonging to 12 ethnic groups of Pakistan. These identified 11 stable haplogroups and 503 combination binary marker/STR haplotypes. Haplogroup frequencies were generally similar to those in neighboring geographical areas, and the Pakistani populations speaking a language isolate (the Burushos), a Dravidian language (the Brahui), or a Sino-Tibetan language (the Balti) resembled the Indo-European-speaking majority. Nevertheless, median-joining networks of haplotypes revealed considerable substructuring of Y variation within Pakistan, with many populations showing distinct clusters of haplotypes. These patterns can be accounted for by a common pool of Y lineages, with substantial isolation between populations and drift in the smaller ones. Few comparative genetic or historical data are available for most populations, but the results can be compared with oral traditions about origins. The Y data support the well-established origin of the Parsis in Iran, the suggested descent of the Hazaras from Genghis Khan's army, and the origin of the Negroid Makrani in Africa, but do not support traditions of Tibetan, Syrian, Greek, or Jewish origins for other populations.