Identification of contributing factors, microorganisms and antimicrobial resistance involved in the complication of diabetic foot ulcer treatment.

Diabetic foot ulcer (DFU) is a neurological and peripherical complication of diabetes with unknown etiology that is often associated with polymicrobial infections. The present study was conducted to investigate the contributing factors in 285 DFU patients, which included 200 patients with diabetic foot infections (DFI). Identification and characterization of infecting bacterial isolates were done followed by assessment of their pattern of susceptibility to commonly used antibiotics. Among the studied subjects, type 2 diabetes mellitus (T2DM), ulcer type, depth, grade, loss of sensation, infection type, affected foot, recurrence, smoking status, Body Mass Index (BMI), and obesity levels revealed significant disease risk association. Ulcer grades 1 and 2 were more common in males while grade 3 in females. Recurrent infections were significantly higher in females (P = 0.03). Diabetic duration, hyperglycemia, ulcer type, infection type and BMI were positively correlated with delayed wound healing. In DFI samples, 40.2% consisted of gram-negative bacteria, with Pseudomonas aeruginosa (37.5%) being the most common, while in the 60% gram-positive isolates Staphylococcus aureus (40.5%) was the predominant species. Staphylococcus epidermidis was found more frequently in females (P = 0.05). The isolated bacterial strains presented higher resistance against the tested antibiotics; however, ceftriaxone was effective against most of the pathogens. In the current study T2DM along with diabetes duration, obesity, ulcer severity with polymicrobial infection was found to play a strong role in DFI development, where gender predisposition was also observed in ulcer grade and infection. DFI was correlated with loss of sensation, infection type, affected foot, smoking status, BMI and obesity levels.

Association of IL-17A promoter region SNP-rs2275913 with urinary bladder cancer.

Objective: Urinary bladder cancer (UBC) is the fourth most common cancer among men and tenth most common cancer in women. This study investigated an association of interleukins -17A promoter region single nucleotide polymorphism (SNP)-rs2275913 with UBC in Pakistani population. Methods: Population-based study was designed with 127 UBC patients and 100 healthy individuals. Only UBC Patients were included and other diseases hepatitis or any other malignancy/cancer were excluded from the study. Polymerase chain reaction Restriction fragment length polymorphism technique was used to genotype the rs2275913 SNP in patients and control. Linear regression analysis was performed on the genotype data and allelic frequency data. Online statistical tool was used to calculate ratio of odds. Results: Linear regression analysis showed that there was no association between rs2275913 SNP and UBC patients in the dominant model (OR = 0.815, CI = 0.415-1.6), recessive model (OR = 0.389, CI = 0.014-5.565), codominant model (OR = 0.376, CI=0.013-5.420) and (OR = 0.855, CI = 0.427-1.713). Moreover, among the UBC samples, low-grade non-muscle invasive UBC samples dominant model (OR = 0.722, CI = 0.316-1.637), recessive model (OR = 0.000, CI = 0.000-5.864), codominant model (OR = 0.864, CI = 0.030-12.668), and (OR = 0.788, CI = 0.341-1.806) did also not show any association. When same analysis was performed for high-grade muscle invasive UBC, dominant (OR = 0.936, CI = 0.403-2.155), recessive model (OR = 0.875, CI = 0.031-12.696), and codominant model (OR = 0.864, CI = 0.030-12.668,), and (OR = 0.942, CI = 0.394-2.232) did not show any association. Conclusion: Results revealed that rs2275913 did not show any associated with the high risk of UBC in Pakistani population. Some limitations of the studies are firstly, the samples size and other are detailed information on UBC and role of inflammation.

A common missense variant rs874881 of PADI4 gene and rheumatoid arthritis: Genetic association study and in-silico analysis.

The peptidylarginine-deiminase 4 (PADI4) is involved in the post-translational catalytic conversion of arginine into citrulline. The autoantibodies including anti-citrullinated protein antibodies (ACPAs) produced in response to hypercitrullinated proteins are a hallmark of rheumatoid arthritis (RA) autoimmunity. Therefore, the role of a missense variant rs874881 (Gly112Ala) of PADI4 in RA susceptibility was analyzed, along with in-silico analysis of structural and functional impacts of this substitution. We did a case-control association study and in-silico analysis. For the case-control study, confirmed RA cases and healthy controls were recruited. Genotyping for rs874881 (n = 750) was performed through polymerase chain reaction-restriction fragment length polymorphism. Multivariate logistic regression analysis was employed to determine association. The in-silico analysis was carried out through HOPE, VarMap, MutationAssessor, MutPred2, SIFT, PolyPhen, CADD, REVEL and MetaLR. In the case-control study, the rs874881 exhibited a strong association with increased RA susceptibility (G vs C odds ratio = 3.85, 95 % confidence interval = 2.81-5.27). Interaction analysis revealed significant interaction of genotype with smoking and gender (p < 0.05). Significant results (p < 0.05) were also obtained in stratified analysis by presence/absence of comorbidities and radiographic damage. According to in-silico pathogenicity prediction analysis, this Gly112Ala substitution does not exert a major effect on protein structure and function including its enzymatic activity. We report a significant association of PADI4 rs874881 with overall RA susceptibility. To our knowledge, this is the first study to do the interaction and stratified analyses on the PADI4 rs874881 in RA. Similar detailed studies should also be performed in other populations.

A 3' untranslated region polymorphism rs2304277 in the DNA repair pathway gene OGG1 is a novel risk modulator for urothelial bladder carcinoma.

Altered DNA repair capacity may affect an individual's susceptibility to cancers due to compromised genomic integrity. This study was designed to elucidate the association of selected polymorphisms in DNA repair genes with urothelial bladder carcinoma (UBC). OGG1 rs1052133 and rs2304277, XRCC1 rs1799782 and rs25487, XRCC3 rs861539, XPC rs2228001, and XPD rs13181 were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 200 UBC cases and 200 controls. We found association of OGG1 rs2304277 [odds ratio (OR)GG = 3.55, 95% confidence interval (CI) = 1.79-7.06] and XPC rs2228001 (ORAC = 2.38, 95% CI = 1.43-3.94) with UBC. In stratified analysis with respect to smoking status, OGG1 rs2304277 and XPC rs2228001 exhibited increased risk in smokers [(rs2304277 ORGG = 4.96, 95% CI = 1.51-16.30) (rs2228001 ORAC = 2.19, 95% CI = 1.02-4.72)] as well as nonsmokers [(rs2304277 ORGG = 2.95, 95% CI = 1.26-6.90) (rs2228001 ORAC = 2.57, 95% CI = 1.31-5.04)]. These polymorphisms were also associated with both low-grade [(rs2304277 ORGG = 3.73, 95% CI = 1.72-8.09) (rs2228001 ORAC = 2.18, 95% CI = 1.21-3.92)] and high-grade tumors [(rs2304277 ORGG = 3.45, 95% CI = 1.52-7.80) (rs2228001 ORAC = 2.81, 95% CI = 1.48-5.33)] as well as with non-muscle-invasive bladder cancer [(rs2304277 ORGG = 4.03, 95% CI = 1.87-8.67) (rs2228001 ORAC = 2.14, 95% CI = 1.20-3.81)] and muscle-invasive bladder cancer [(rs2304277 ORGG = 3.06, 95%CI = 1.31-7.13) (rs2228001 ORAC = 2.95, 95%CI = 1.51-5.75)]. This is the first study on DNA repair gene polymorphisms and UBC in the Pakistani population. It identifies OGG1 rs2304277 and replicates XPC rs2228001 as significant modulators of UBC susceptibility.

Identification of novel potential genetic predictors of urothelial bladder carcinoma susceptibility in Pakistani population.

Urothelial bladder carcinoma (UBC) is the most common among urinary bladder neoplasms. We carried out a preliminary study to determine the genetic etiology of UBC in Pakistani population, for this 25 sequence variants from 17 candidate genes were studied in 400 individuals by using polymerase chain reaction-based techniques. Multivariate logistic regression analysis was performed for association analysis of the overall data as well as the data stratified by smoking status, tumor grade and tumor stage. Variants of GSTM1, IGFBP3, LEPR and ACE were found to be associated with altered UBC risk in the overall comparison. CYP1B1 and CDKN1A variants displayed a risk modulation among smokers; IGFBP3 and LEPR variants among non-smokers while GSTM1 polymorphism exhibited association with both. GSTM1 and LEPR variants conferred an altered susceptibility to low grade UBC; GSTT1, IGFBP3 and PPARG variants to high grade UBC while ACE polymorphism to both grades. GSTM1 and LEPR variants exhibited risk modulation for non-muscle-invasive bladder cancer (NMIBC); GSTT1 and PPARG variants for muscle-invasive bladder cancer (MIBC), and ACE variant for NMIBC as well as MIBC. In general, the susceptibility markers were common for low grade and NMIBC; and distinct from those for high grade and MIBC indicating the distinct pathologies of both groups. In brief, our results conform to reports of previously associated variants in addition to identifying novel potential genetic predictors of UBC susceptibility.

Homozygosity mapping and targeted sanger sequencing reveal genetic defects underlying inherited retinal disease in families from pakistan.

BACKGROUND: Homozygosity mapping has facilitated the identification of the genetic causes underlying inherited diseases, particularly in consanguineous families with multiple affected individuals. This knowledge has also resulted in a mutation dataset that can be used in a cost and time effective manner to screen frequent population-specific genetic variations associated with diseases such as inherited retinal disease (IRD). METHODS: We genetically screened 13 families from a cohort of 81 Pakistani IRD families diagnosed with Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), congenital stationary night blindness (CSNB), or cone dystrophy (CD). We employed genome-wide single nucleotide polymorphism (SNP) array analysis to identify homozygous regions shared by affected individuals and performed Sanger sequencing of IRD-associated genes located in the sizeable homozygous regions. In addition, based on population specific mutation data we performed targeted Sanger sequencing (TSS) of frequent variants in AIPL1, CEP290, CRB1, GUCY2D, LCA5, RPGRIP1 and TULP1, in probands from 28 LCA families. RESULTS: Homozygosity mapping and Sanger sequencing of IRD-associated genes revealed the underlying mutations in 10 families. TSS revealed causative variants in three families. In these 13 families four novel mutations were identified in CNGA1, CNGB1, GUCY2D, and RPGRIP1. CONCLUSIONS: Homozygosity mapping and TSS revealed the underlying genetic cause in 13 IRD families, which is useful for genetic counseling as well as therapeutic interventions that are likely to become available in the near future.

Exome sequencing identifies three novel candidate genes implicated in intellectual disability.

Intellectual disability (ID) is a major health problem mostly with an unknown etiology. Recently exome sequencing of individuals with ID identified novel genes implicated in the disease. Therefore the purpose of the present study was to identify the genetic cause of ID in one syndromic and two non-syndromic Pakistani families. Whole exome of three ID probands was sequenced. Missense variations in two plausible novel genes implicated in autosomal recessive ID were identified: lysine (K)-specific methyltransferase 2B (KMT2B), zinc finger protein 589 (ZNF589), as well as hedgehog acyltransferase (HHAT) with a de novo mutation with autosomal dominant mode of inheritance. The KMT2B recessive variant is the first report of recessive Kleefstra syndrome-like phenotype. Identification of plausible causative mutations for two recessive and a dominant type of ID, in genes not previously implicated in disease, underscores the large genetic heterogeneity of ID. These results also support the viewpoint that large number of ID genes converge on limited number of common networks i.e. ZNF589 belongs to KRAB-domain zinc-finger proteins previously implicated in ID, HHAT is predicted to affect sonic hedgehog, which is involved in several disorders with ID, KMT2B associated with syndromic ID fits the epigenetic module underlying the Kleefstra syndromic spectrum. The association of these novel genes in three different Pakistani ID families highlights the importance of screening these genes in more families with similar phenotypes from different populations to confirm the involvement of these genes in pathogenesis of ID.

Identification of a recurrent insertion mutation in the LDLR gene in a Pakistani family with autosomal dominant hypercholesterolemia.

Familial Hypercholesterolemia (FH) results in elevated levels of blood lipids including total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) with normal triglycerides (TG). This disease is one of the major contributors towards an early onset of coronary heart disease (CHD). The aim of the present study was to identify the genes responsible for causing FH in Pakistani population, for this purpose a large consanguineous FH family was selected for genetic analysis. Serum lipid levels, including TC, TG, LDL-C and high density lipoprotein cholesterol (HDL-C), were determined in patients and healthy controls. In order to find the causative mutation in this family, direct sequencing of the low density lipoprotein receptor (LDLR) gene was performed. In addition the part of the Apolipoprotein-B (APOB) gene containing the mutations R3500Q and R3500W was also sequenced. Affected individuals of the family were found to have raised TC and LDL-C levels. Sequencing revealed an insertion mutation (c.2416_2417InsG) in exon 17 of the LDLR gene in all the affected individuals of the family. Common FH causing APOB mutations were not present in this family. Heterozygous individuals had TC levels ranging from ~300-500 mg/dl and the only homozygous individual with typical xanthomas had TC levels exceeding 900 mg/dl. This is the first report of a known LDLR gene mutation causing FH in the Pakistani population. Despite a large heterogeneity of LDLR mutations there are still some common mutations which are responsible for FH throughout the world.