Knowledge, Attitudes and Perceptions towards COVID-19 Vaccinations: A Cross-Sectional Survey in Pakistan.

Background and Objectives: Several vaccines have been approved for the prevention of the coronavirus disease, discovered on 31 December in Wuhan, China. Pakistan procured vaccines from various countries. However, the lack of knowledge and reluctance of the general population to embrace the use of the vaccines are considered to be the major determinant of the slow vaccination rate. Hence, it is necessary to evaluate the willingness of the general population about their perception of the COVID-19 vaccination. Materials and Methods: A cross sectional survey based on a self-structured questionnaire comprising 18 questions was conducted (from 21 April-21 June) on 400 Pakistani participants to evaluate their knowledge, attitude, and perception towards the COVID-19 vaccination. Chi-square independent t-test and one-way Anova including a multiple step wise linear regression were used to draw conclusions about the results. p < 0.05 was considered significant. Results: A total of 400 participants responded in the knowledge, attitude, and perception (KAP) survey of which 46.5% were female and 53.5% were male. The mean age of participants was 36.08 years. This survey showed a poor knowledge (50.5%), a fair attitude (75.1%) and a poor perception (58.1%) towards the COVID-19 vaccination. Higher mean knowledge and attitude scores were reported in the age group 21-40, females, and unmarried urban citizens. Regression analysis showed that age, education, residence, and employment status influenced the knowledge and perception score to a considerable extent. Conclusions: The findings reflect an inadequate knowledge and perception on the one hand, but a better attitude towards the COVID-19 vaccination. This knowledge attitude and perception (KAP) survey will help in better understanding the opinion of the general population towards vaccination, and will be useful for policy makers and health care authorities aiming to increase the vaccination rate.

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.

Direct-acting antiviral agents in the treatment of chronic hepatitis C-Real-life experience from clinical practices in Pakistan.

This study aims to evaluate the clinical effectiveness in terms of sustained virological response (SVR), predictors of SVR and safety of available second-generation generic direct-acting antivirals in Pakistani chronic hepatitis C patients. This is a retrospective study conducted in multiple centers of Pakistan from January 2015 to January 2019. The samples include patients infected with chronic hepatitis C virus, regardless of virus genotype, cirrhosis, or prior treatment. A total of 993 patients were included in the present study, with the majority receiving sofosbuvir with daclatasvir (95%), sofosbuvir with daclatasvir and ribavirin (4%), and sofosbuvir with ribavirin (1%). There were 96% cases of chronic hepatitis, 3% cases compensated cirrhosis, and 1% cases of decompensated cirrhosis. Genotype 3 (99.6%) was the most common genotype. Overall SVR after 12 weeks was 98% for all treatment regimens. High SVR12 was observed with sofosbuvir in combination with daclatasvir (98.5%), then sofosbuvir in combination with daclatasvir and ribavirin (90.2%) and sofosbuvir in combination with ribavirin (75%). SVR rates were high in chronic hepatitis C patients (98.2%) as compared with cirrhotic patients (92.1%) and it was high in treatment-naive (98.8%) then interferon experienced patients (90.1%). In multivariate binary logistic regression analysis, patients' education status, treatment strategy, viral load, and alanine aminotransferase had a statistically significant association with SVR at 12 weeks. No major adverse events occurred which required treatment discontinuation. Generic oral direct acting antiviralss (sofosbuvir with daclatasvir) achieved higher SVR12 rates and were well tolerated in this large real-world cohort of genotype 3 infected patients.

REPORT - CYP2D6*4 null allele frequency in sixteen Pakistani ethnic groups.

CYP2D6 belongs to a family of Cytochrome P450 and is involved in metabolism of a number of commonly prescribed drugs. This study was designed to identify *4 allelic frequency of CYP2D6 in Pakistani population. The ethno-geographic variations in the CYP2D6 alleles are responsible for varied expression of this enzyme and thus influence the metabolic rate and efficacy of prescribed drugs. In total, 976 volunteers belonging to 16 different ethnic groups of Pakistan were screened for CYP2D6*4 polymorphism. The *4 allele was detected in all the ethnic groups with varied frequency ranging from 3.73%-13.64% and an overall average of 7.22% in different ethnic groups of the population. Maximum frequency was detected in northern Pakistani population including Meo (13.64%), Punjabi (11.96%) and Pathan (10.42%). Low frequency (<4%) of*4 polymorphism was observed in Kalash and Makrani groups, whereas an intermediate frequency (5-9%) was observed in all the other ethnic groups. The data indicates that despite ethnic diversity poor metabolizers in Pakistani population are expected to carry CYP2D6*4 allele at a relatively higher frequency than most other Asian populations. (Word count = 186).

Can IFNL3 polymorphisms predict response to interferon/ribavirin treatment in hepatitis C patients with genotype 3?

Favourable genotypes of IFNL3 polymorphism CC for rs12979860 and TT for rs8099917 are strongly associated with the interferon/ribavirin treatment outcome in hepatitis C virus (HCV) patients with genotypes 1 and 4. Contrarily, conflicting results have been reported for patients with HCV genotypes 2 and 3. Therefore, we sought to investigate the association between IFNL3 with sustained virological response (SVR) after treatment to ascertain the predictive value of IFNL3 single-nucleotide polymorphisms (SNPs) in HCV patients with genotype 3. For this purpose, we genotyped five IFNL3 SNPs, rs12980275, rs12979860, rs9109886, rs8099917 and rs7248668, in HCV patients with genotype 3 and assessed its association with SVR, individually and in haplotype. Interestingly, we report that the IFNL3 SNPs we genotyped have shown no association with SVR following treatment, either individually or in haplotype, indicating that genotyping IFNL3 SNPs have limited predictive value in HCV patients with genotype 3. Therefore, we propose that IFNL3 genotyping can be excluded from a patient's pre-treatment workup for subsequent treatment choice. This will greatly reduce the economic burden for HCV patients with genotype 3 in resource-limited regions, especially South Asia where genotype 3 is predominant.

Association of GSTM1 and GSTT1 deletion polymorphisms with Pakistani aplastic anemia patients and controls and meta-analysis.

Interaction of environmental and genetic elements plays a vital role in the pathogenesis of aplastic anemia (AA). Glutathione S-transferase (GST) is a key detoxifying enzyme. Absence or low levels of this enzyme may genetically predispose individuals to AA. GST genes GSTM1 and GSTT1 are polymorphic. The aim of this study was to screen Pakistani AA patients and controls for GSTM1 deletion GSTM0 and GSTT1 deletion GSTT0 and perform meta-analysis using our data and other published data regarding these polymorphisms. DNA samples from 137 patients and 220 controls were screened using multiplex polymerase chain reaction. GSTM0 emerged as susceptible genotype for AA in Pakistan with a percentage frequency of 49.6 % as compared to 30 % in controls with odds ratio (OR) of 2.25, 95 % confidence interval (CI) of 1.4-3.5 and corrected p = 0.006. The meta-analysis showed a significant association between the null genotype GSTT0 and AA in overall analysis with OR of 1.47, 95 % CI of 1.01-2.13 and p value of 0.04 in random effects model. Studies like these could play a role in understanding the underlying path in AA pathogenesis and therefore can help in designing means for prevention, diagnose and treatment.

A canonical splice site mutation in GIPC3 causes sensorineural hearing loss in a large Pakistani family.

With homozygosity mapping we have identified two large homozygous regions on chromosome 3q13.11-q13.31 and chromosome 19p13.3-q31.32 in a large Pakistani family suffering from autosomal recessive nonsyndromic hearing impairment (arNSHI). The region on chromosome 19 overlaps with the previously described deafness loci DFNB15, DFNB72 and DFNB95. Mutations in GIPC3 have been shown to underlie the nonsyndromic hearing impairment linked to these loci. Sequence analysis of all exons and exon-intron boundaries of GIPC3 revealed a homozygous canonical splice site mutation, c.226-1G>T, in GIPC3. This is the first mutation described in GIPC3 that affects splicing. The c.226-1G>T mutation is located in the acceptor splice site of intron 1 and is predicted to affect the normal splicing of exon 2. With a minigene assay it was shown to result in the use of an alternative acceptor site in exon 2, resulting in a frameshift and a premature stop codon. This study expands the mutational spectrum of GIPC3 in arNSHI.

Host immune players and their response to Hepatitis C therapies.

This study aimed to investigate alterations in the expression of four key cytokines (IL-7, IL-11, IL-15, and IL-27) and assess differential FAM26F expression in response to Hepatitis C virus (HCV) infection. Additionally, it sought to analyze changes in these cytokines after treatment in 244 chronic HCV patients and 28 controls undergoing various treatments, including standard interferon, pegylated interferon, and Direct Acting Antivirals (DAAs). The objective was to compare immune system regulation between treatment groups. The expression levels of FAM26F and the cytokines (IL-7, IL-11, IL-15, and IL-27) were evaluated using Real-time qPCR in PBMCs of treatment groups. Results revealed significant downregulation of IL-7 and IL-27 in infected individuals compared to healthy controls, persisting even after treatment. This suggests the crucial roles of these immune modulators in facilitating the necessary T-cell response for viral clearance. IL-11 expression also remained suppressed post-treatment, supporting viral clearance by restoring the Th1 response. The decrease in IL-11 levels during treatment indicates the restoration of the Th1 response, vital for viral clearance. IL-15, the key cytokine regulating cytotoxic cells (NKT and NK cells), displayed consistent expression across all sample groups, indicating maintained IL-15-induced cytotoxicity in both control and infected individuals. Additionally, FAM26F expression was reduced in the HCV-infected group compared to controls, but higher in HCV-recovered cases, potentially due to reduced infection and enhanced immunity. In conclusion, this research unveils the relationship between FAM26F and HCV infection, highlighting the virus's tendency to suppress cytokine and FAM26F expression. An effective treatment strategy for establishing an ideal host immune response may involve restoring FAM26F and cytokine expression to their normal levels.

Novel mutation in AAA domain of BCS1L causing Bjornstad syndrome.

Bjørnstad syndrome is an extremely rare condition characterized by pilitorti and nerve deafness. Only few large families have been reported worldwide. Here we describe a large Pakistani family with five affected individuals. The hair fibers of all the patients were twisted around their axis and devoid of any pigment. In addition the patients had a moderate-to-severe degree of hearing impairment. Genotyping with high-density single-nucleotide polymorphism arrays showed homozygosity in two intervals on chromosome 2. Linkage with one of these regions (genomic position 218745685-221025443, hg19) was confirmed. This region encompasses the BCS1L gene. Mutations in this gene have previously been associated with Bjørnstad's syndrome. We sequenced the BCS1L gene for identification of the causative mutation in the family. A novel homozygous missense mutation c.901T>A was identified, which segregated with the disease in the family. This mutation results in the amino acid change p.Tyr301Asn and was predicted to be pathogenic by bioinformatics tools.

Study of PKRBD in HCV genotype 3a infected patients in response to interferon therapy in Pakistani population.

BACKGROUND: Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma and infects about 3% world population. Response to interferon therapy depends upon the genotype of the virus and factors associated with the host. Despite a good response to interferon therapy, a considerable number of genotype 3a infected patients remains unalleviated. RESULTS: In total forty-nine patients including twenty-five non-responders (non-SVR) and twenty-four responders (SVR) were recruited. Patients were tested for viral status at different intervals and the isolated RNA was sequenced for the NS5A region in both groups. The comparison of PKRBD of HCV between the SVR and non-SVR patients did not confirm any significant difference in the number of mutations. However, when the sequence downstream to the PKRBD of NS5A was compared, two important statistically significant mutations were observed; at positions 2309 (Ala to Ser) and 2326 (Gly to Ala). These mutations were then analysed for tertiary protein structure and important structural changes were observed. Statistically significant difference was also observed when age groups of patients were compared; younger patients showed better response than the older ones. CONCLUSIONS: The region between PKRBD and IRRDR may be important for prediction of response to IFN therapy for genotype 3a. ISDR and PKRBD have not shown any involvement in treatment response. Further functional analyses of these findings can help in understanding the involvement of the NS5A region in interferon treatment of HCV-3a infected patients.

Expression of IFN-Gamma is significantly reduced during severity of covid-19 infection in hospitalized patients.

Cytokines play an important role in SARS-CoV-2 infection progression and severity. A number of inflammatory cytokines have been directly associated with disease severity including IL-6 (interleukin-6), IL-10, TNF-α (tumor necrosis factor alpha), IFN-γ (interferon-gamma). Here, in this study, the aim was to better understand the interplay between host immune response mediated by cytokines and severity of SARS-CoV-2 infection by assessing cytokine expression. Therefore, we measured expression levels of a total of 12 genes (IFNA-1, IFN-γ, IL-1α, IL-1ß, IL-4, IL-6, IL-7, IL-10, IL-11, IL-13, IL-15, and IL-27) encoding inflammatory, anti-inflammatory and regulatory cytokines using QRT-PCR in hospitalized patients with severe infection compared to mildly infected. IFN-γ was identified as a potent marker of disease severity as indicated previously. Moreover, levels of IL-7 were also found to be partially reduced in patients compared to the healthy controls and linked negatively to disease severity. Identification of these cytokines may be helpful in not only understanding disease pathogenesis but also in better management of the patients after covid infection.

Occult HCV or delayed viral clearance from lymphocytes of Chronic HCV genotype 3 patients after interferon therapy.

BACKGROUND: A recently discovered occult HCV entity reported by various investigators seems to be highly controversial. Especially, the clinical significance of these findings remains uncertain. For optimal outcome of antiviral therapy, investigation of occult HCV needs a broad-based probe in order to investigate the results of viral therapy and its host/viral interaction. The current study was aimed at determining the prevalence of occult HCV in peripheral blood lymphocytes of predominantly genotype 3 HCV-infected patients after completion of antiviral therapy and to investigate long term outcomes in the presence or absence of PBMC positivity. METHOD: A total of 151 chronic, antiHCV and serum RNA-positive patients were enrolled in the study. Patients with a complete virological response at the end of treatment were screened for the presence of viral RNA in their PBMCs and were followed for up to one year for the presence of serum and PBMC viral genomic RNA. RESULTS: Out of 151 patients, 104 (70%) responded to the prescribed interferon treatment and showed viral-clearance from serum. These were screened for the presence of genomic RNA in their PBMCs. Sixteen samples were PBMC-positive for viral RNA at the end of treatment (EOT). All these patients had also cleared the virus from peripheral blood cells after the 6-12 month follow-up study. CONCLUSION: True occult hepatitis C virus does not exist in our cohort. Residual viremia at the EOT stage merely reflects a difference in viral kinetics in various compartments that remains a target of immune response even after the end of antiviral therapy and is eventually cleared out at the sustained viral response (SVR).

Patient HLA-DRB1* and -DQB1* allele and haplotype association with hepatitis C virus persistence and clearance.

Hepatitis C virus (HCV) infection is prevalent throughout the world and interferon (IFN)-based treatments are currently the only therapeutic option. However, depending upon variations in their human leukocyte antigen (HLA), some patients do not respond well to IFN therapy. The current study evaluated the HLA allele and haplotype distribution of 204 HCV-seropositive individuals from Islamabad, Pakistan, who were receiving standard IFN therapy. In this cohort, 150 patients (74%) showed a sustained virological response to IFN therapy, whereas 54 (26%) did not. In addition to the HCV patients, 102 unrelated healthy volunteers were used as controls. DNA was isolated from the blood of the patients and controls for HLA-DRB1 and HLA-DQB1 allele typing, whilst plasma was used for HCV detection and genotyping. HLA-DRB1*04 was found to impart a significant protective advantage [Bonferroni-corrected P value (pc)=0.047] against HCV infection. In patients on IFN therapy, HLA-DRB1*11 and -DQB1*0301 (pc=0.044) were found to be associated with viral clearance. In contrast, HLA-DRB1*07 (pc=0.008) individually or in combination with HLA-DQB1*02 was found to be associated with viral persistence. These associations of HLA with HCV persistence or clearance will be beneficial in deciding the therapeutic regimen for Pakistani patients infected with HCV genotype 3a.

MTHFR polymorphisms as risk for male infertility in Pakistan and its comparison with socioeconomic status in the world.

AIM: 5,10-MTHFR-single nucleotide polymorphisms are important for normal functioning of the enzyme that plays a key role in DNA synthesis, folate metabolism and methylation reactions. Methodology & results: Male infertility association of C665T and A1298C polymorphisms was explored, this topic is still debatable. Infertile men (232) and controls (114) were genotyped and statistically analyzed. Comparison of patients (6180) and controls (5744) of Caucasian populations was performed by meta-analysis. Pooled results showed A1298C minor allele and homozygous genotype to be of a significantly higher frequency in the low-income group. Increase in per capita income has shown an increasing trend in the minor allele frequency in various world populations, potentially due to dietry-folate compensation. CONCLUSION: A1298C seems more relevant marker than C665T for infertility association in Caucasian populations and may be addressed by improving dietary folate.

Genetic Polymorphism of CYP2C19 in Pakistani Population.

CYP2C19 polymorphism is associated with pretreatment drug response prediction, metabolism, and disposition. Pakistan consists of a population comprising of various ethnic groups residing in different regions of the country each claiming diverse ethnic origins. The identification of CYP450 genotypic composition of these populations is therefore necessary to avoid adverse drug reactions in these individuals. The main objective of the study was to investigate the prevalence of CYP2C19*2 and CYP2C19*17 alleles in these ethnic groups. The study was conducted on one thousand and twenty-eight (n = 1028) healthy volunteers from nine ethnic groups of Pakistan namely Brusho (n = 28), Hazara (n = 102), Kalash (n = 64), Pathan (n = 170), Punjabi (n = 218), Saraiki (n = 59), Brahui (n = 118), Parsi (n = 90), and Sindhi (n = 179). DNA was extracted from leukocytes and analyzed by allele specific amplification polymerase chain reaction (ASA-PCR). Multi allelic polymorphism of CYP2C19 led to four distinct phenotypes identified as extensive metabolizer (EM), poor metabolizer (PM), intermediate metabolizer (IM), and ultra-rapid metabolizer (UM). Over all, the percentage of predicted poor metabolizer allele was 29.0% compared to UM allele (23.70%). Among the studied groups, Saraiki and Brahui showed highest percentage of PM allele (40%, 36%) whereas Parsi and Hazara had highest percentage of UM allele (37% and 30% respectively). In conclusion, the high allele frequency of PM (CYP2C19*2 and *17) in Pakistani population led to the recommendation of a pre-treatment test to monitor drug response and dosage (personalized medicine) to avoid post-treatment adverse drug reactions.

VNTR polymorphism of the DRD4 locus in different Pakistani ethnic groups.

The present study was designed to investigate the dopamine receptor D4 (DRD4) locus variable number of tandem repeat (VNTR) allelic distribution in different Pakistani ethnic groups. DNA samples from nine different ethnic groups of Pakistan were analyzed. Greek and Somali samples were included as representatives of the European and African populations, respectively. Pakistani, Greek, and Somali populations were also compared to the published data on different world populations. The allelic distribution revealed that the four-repeat allele was the most common allele in all the Pakistani ethnic groups as is in different other world populations, followed by the seven- and two-repeat alleles. To study the evolutionary relationship of the Pakistani ethnic groups among themselves and with a few other world populations, multidimensional scaling based on the allelic frequencies of the DRD4 VNTR was obtained. This analysis grouped most of the Pakistani ethnic groups together and closer to the European and Middle Eastern populations, except for the Mohanna from Sindh, who grouped with the African populations. In addition, the Somali and the Greek samples analyzed in this study grouped closer to the previous data obtained on the African and European populations, respectively.

Single nucleotide polymorphisms in asthma candidate genes TBXA2R, ADAM33 FCER1B and ORMDL3 in Pakistani asthmatics a case control study.

BACKGROUND: Genetic variations in different loci and genes are important in asthma pathogenesis. There is much importance of various immunological pathways in the IgE secretion regulation. Alterations in any main part of these pathways can increase the risk of asthma development. Polymorphisms in these genetic markers can effect certain pathways which predict the asthma susceptibility. In the present study, SNPs directly or indirectly affecting the immunological process pathways are selected. METHODS: This study was conducted to determine association of 16 SNPs in 10 candidate genes with asthma in Pakistani population in 333 asthmatic cases and 220 healthy controls. Genotyping was performed using the Sequenom Mass ARRAY iPLEX platform (14 SNPs) and TaqMan assay (2 SNPs). RESULTS: The minor allele at two of the SNPs showed association with protection from asthma, rs1131882 in TBXA2R gene (OR 0.73, 95% CI 0.52-1.01, P = 0.05) and rs2280091 in the ADAM33 gene (OR 0.69, 95% CI 0.50-0.97, P = 0.03). For FCER1B gene, rs2583476 the asthmatic male gender had higher TT genotype counts as compared to controls (OR = 1.86, 95% CI = 1.09-3.17, p = 0.01). In rs11650680 of ORMDL3 gene the CT genotype is more prevalent in female asthma cases in comparison with female controls (OR = 1.99, 95% CI = 1.02-3.89, p = 0.03). CONCLUSIONS: This data suggests that variations at TBXA2R and ADAM33 genes are found to be associated with asthma susceptibility in Pakistan. FCER1B gene is associated with male and ORMDL3 in female asthmatics. These genetic markers can be important source of asthma risk in Pakistani population.

A homozygous FITM2 mutation causes a deafness-dystonia syndrome with motor regression and signs of ichthyosis and sensory neuropathy.

A consanguineous family from Pakistan was ascertained to have a novel deafness-dystonia syndrome with motor regression, ichthyosis-like features and signs of sensory neuropathy. By applying a combined strategy of linkage analysis and whole-exome sequencing in the presented family, a homozygous nonsense mutation, c.4G>T (p.Glu2*), in FITM2 was identified. FITM2 and its paralog FITM1 constitute an evolutionary conserved protein family involved in partitioning of triglycerides into cellular lipid droplets. Despite the role of FITM2 in neutral lipid storage and metabolism, no indications for lipodystrophy were observed in the affected individuals. In order to obtain independent evidence for the involvement of FITM2 in the human pathology, downregulation of the single Fitm ortholog, CG10671, in Drosophila melanogaster was pursued using RNA interference. Characteristics of the syndrome, including progressive locomotor impairment, hearing loss and disturbed sensory functions, were recapitulated in Drosophila, which supports the causative nature of the FITM2 mutation. Mutation-based genetic counseling can now be provided to the family and insight is obtained into the potential impact of genetic variation in FITM2.

Heterogeneous genomic locations within NS3, NS4A and NS4B identified for genotyping and subtyping of Hepatitis C virus: A simple genome analysis approach.

Hepatitis C virus (HCV) displays excessive genetic heterogeneity and exists in several genotypes and subtypes. Characterizing these genotypes and subtypes becomes extremely important for diagnostic and epidemiological reasons. Present study analyzed HCV genome using a simple genome analysis approach. We combined manual sectioning of a reference genome alignment (RA) followed by a comprehensive comparative phylogenetic analysis. The main aim was to identify heterogeneous locations on HCV genome suitable for genotyping/subtyping. HCV reference dataset, comprising of whole genome sequences from all HCV genotypes and subtypes, was aligned into an RA. The RA was manually clipped into overlapping sections of 500 bases, each 50 bases apart. Phylogeny for each section and RA was estimated using neighbor-joining phylogenetic method. Clustering pattern between section phylogenies and RA phylogeny was compared for similarity. Sections (locations on genome) with clustering similar to whole genome were selected since it displays comparable genetic heterogeneity making these sections suitable for genotyping/subtyping. Based on this conception, we identified new genomic locations on NS3, NS4A and NS4B suitable for genotyping and subtyping. Exact genomic positions for known genotyping locations, core and NS5B were also identified. Furthermore, phylogenetic analyses at such small genomic scale provided opportunities to explore evolutionary relationships usually overlooked.

An angiotensin I-converting enzyme insertion/deletion polymorphism is associated with Pakistani asthmatic cases and controls.

Asthma is a chronic disease due to inflammation of the airways of lungs that is clinically characterized by variable symptoms including wheezing, coughing and shortness of breath. Angiotensin I-converting enzyme (ACE) plays a major role in fibrous tissue formation and is highly expressed in lungs. The main aim of this research work was to study the role of ACE insertion/deletion (I/D) polymorphism, rs4646994, in asthma in Pakistani patients. A total of 854 subjects, including 333 asthma patients and 521 ethnically matched controls, were studied. The ACE (I/D) polymorphism was genotyped using polymerase chain reaction (PCR). Chi-square, Fisher's exact and Hardy-Weinberg equilibrium tests were used to compare groups. Homozygous insertion genotype II (p less than 0.0001, OR=3.38) and insertion allele (I) was significantly more frequent in Pakistani asthmatics than in healthy controls (p=0.0007, OR=1.40). The ID genotype (p less than 0.0001, OR=0.43) and the deletion allele (D) were associated with protection of disease in Pakistani patients (p=0.0007, OR=0.71). These data suggest the involvement of ACE I/D polymorphism in asthma risk in the Pakistani population. This marker may be an important indication in the molecular mechanism of asthma and can become a useful tool in risk assessment and help in designing strategy to combat disease.