Investigating dysregulation of TGF-ß1/SMAD3 signaling in Atopic Dermatitis: A Molecular and Immunohistochemical Analysis".

Atopic Dermatitis (AD) is a persistent and recurring inflammatory condition affecting the skin. An expanding corpus of evidence indicates the potential participation of TGF-ß1 in the modulation of inflammation and tissue remodeling in AD. The primary objective of this study was to examine the aberrant modulation of TGF-ß1/SMAD3 signaling through a comprehensive analysis of their molecular and protein expression profiles. The study encompassed an aggregate of 37 participants, which included 25 AD patients and 12 controls. The assessment of mRNA and protein levels of TGF-ß1 and SMAD3 was conducted utilizing quantitative real-time PCR and immunohistochemistry, whereas serum IgE and vitamin D levels were estimated by ELISA and chemiluminescence, respectively. Quantitative analysis demonstrated a 2.5-fold upregulation of TGF-ß1 mRNA expression in the lesional AD skin (p<0.0001). Immunohistochemistry also exhibited a comparable augmented pattern, characterized by moderate to strong staining intensities. In addition, TGF-ß1 mRNA showed an association with vitamin D deficiency in serum (p<0.02), and its protein expression was linked with the disease severity (p<0.01) Furthermore, a significant decrease in the expression of the SMAD3 gene was observed in the affected skin (p = 0.0004). This finding was further confirmed by evaluating the protein expression and phosphorylation of SMAD3, both of which exhibited a decrease. These findings suggest that there is a dysregulation in the TGF-ß1/SMAD3 signaling pathway in AD. Furthermore, the observed augmentation in mRNA and protein expression of TGF-ß1, along with its correlation with the disease severity, holds considerable clinical significance and emphasizes its potential role in AD pathogenesis.

Analysis of intronic SNP (rs4147358) and expression of SMAD3 gene in Atopic Dermatitis: A case-control study.

BACKGROUND: Atopic Dermatitis (AD) is a multifactorial cutaneous disorder associated with chronic inflammation of the skin. Growing evidence points to TGF-ß/SMAD signaling as a key player in mediating inflammation and the subsequent tissue remodeling, often resulting in fibrosis. This study investigates the role of a core transcription factor involved in TGF-ß signaling i.e., SMAD3 genetic variants (rs4147358) in AD predisposition and its association with SMAD3 mRNA expression, serum IgE levels, and sensitization to various allergens in AD patients. METHODS: A total of 246 subjects including 134 AD cases and 112 matched healthy controls were genotyped for SMAD3 intronic SNP by PCR-RFLP. mRNA expression of SMAD3 was determined by quantitative Real-Time PCR (qRT-PCR), Vitamin-D levels by chemiluminescence, and total serum IgE levels by ELISA. In-vivo allergy testing was performed for the evaluation of allergic reactions to house dust mites (HDM) and food allergens. RESULTS: A significantly higher frequency of mutant genotype AA (cases: 19.4% vs controls: 8.9%) (OR = 2.8, CI = 1.2 - 6.7, p = 0.01) was observed in AD cases. The mutant allele 'A' also showed a 1.9-fold higher risk for AD compared to the wild allele 'C' indicating that the carriers of the A allele have a higher risk for AD predisposition (OR-1.9, CI = 1.3-2.8, p < 0.001). In addition, quantitative analysis of SMAD3 mRNA in peripheral blood showed 2.8-fold increased expression in AD cases as compared to healthy controls. Stratification analysis revealed the association of the mutant AA genotype with deficient serum Vitamin D levels (p = 0.02) and SMAD3 mRNA overexpression with HDM sensitization (p = 0.03). Furthermore, no significant association of genotypes with SMAD3 mRNA expression was observed. CONCLUSION: Our study indicates that SMAD3 intronic SNP bears a significant risk of AD development. Moreover, overexpression of SMAD3 mRNA and its association with HDM sensitization highlights the possible role of this gene in AD pathogenesis.

Unveiling the TGF- ß1 paradox: Significant implication of TGF- ß1 promoter variants and its mRNA and protein expression in atopic dermatitis.

BACKGROUND: Atopic Dermatitis (AD) is a chronic inflammatory skin disorder with evidence of lichenification in later stages. There is mounting evidence supporting the role of TGF- ß1 in mediating inflammation as well as subsequent tissue remodeling, often resulting in fibrosis. Given the role of genetic variants in the differential expression of TGF-ß1 in various diseases, this study seeks to ascertain the role of TGF-ß1 promoter variants (rs1800469 and rs1800468) in AD susceptibility, as well as their association with TGF- ß1 mRNA expression, TGF- ß1 serum levels and skin prick test positivity in Atopic Dermatitis patients. METHODS: An aggregate of 246 subjects including 134 AD cases and 112 matched healthy controls were genotyped for TGF-ß1 promoter polymorphisms by PCR-RFLP. TGF- ß1 mRNA was quantified by quantitative Real-Time PCR (qRT-PCR), Vitamin-D levels by chemiluminescence, and serum TGF- ß1, and total IgE levels were determined by ELISA. In-vivo allergy testing was performed for the evaluation of allergic reactions to house dust mites and food allergens. RESULTS: A higher frequency of TT genotypes of rs1800469 (OR = 7.7, p = 0.0001) and GA+AA genotypes of rs1800468 (OR-4.4, p < 0.0001) were observed in AD cases than those in controls. Haplotype analysis demonstrated that TG haplotype carriers had an increased risk of AD (p = 0.013). Quantitative analysis revealed a significant upregulation of both mRNA (p = 0.0002) and serum levels (p < 0.0001) of TGF- ß1 with a substantial positive correlation between them (Correlation coefficient=0.504; p = 0.01). Moreover, serum TGF-ß1 levels were associated with quality of life (p = 0.03), the severity of the disease (p = 0.03), and House dust mite allergy (p = 0.01) whereas TGF-ß1 mRNA levels positively correlated with disease severity(p = 0.02). Stratification analysis revealed that the TT genotype of rs1800469 was associated with higher IgE levels (p = 0.01) and eosinophil percentage(p = 0.007) whereas the AA genotype of rs1800468 correlated with elevated serum IgE levels (p = 0.01). Besides, no significant association of genotypes with mRNA and serum expression of TGF-ß1 was observed. CONCLUSION: Our study indicates that TGF-ß1 promoter SNPs bear a significant risk of AD development. Moreover, upregulation of TGF-ß1 mRNA and serum levels and their association with disease severity, quality of life, and HDM allergy suggests its role as a diagnostic/prognostic biomarker that could help in the development of new therapeutic and prevention strategies.

Implications of risk conferred by 5p15.33 loci genetic variants; human telomerase reverse transcriptase rs2736098 and rs2736100 in predisposition of bladder cancer.

Background: The polymorphic variations of human telomerase reverse transcriptase (hTERT) gene play an important role in predisposition to carcinogenesis. The current study aimed to elucidate the genetic predisposition to bladder cancer in two important variants, rs2736098 and rs2736100 of hTERT gene. Materials and methods: Confirmed 130 patients of bladder cancer and 200 healthy controls were genotyped by PCR-RFLP to determine different variants of hTERT rs2736098 and rs2736100. Results: hTERT rs2736098 homozygous variant AA genotype frequency was observed to significantly differ 2-fold between cases and controls (26.15% vs. 13.5%) (p = 0.02). In addition, rare 'A' allele significantly differed among two groups (cases: 47% versus controls: 39%: p = 0.03). hTERT rs2736098 was observed to be presented significantly more in high stage tumors (p = 0.02). hTERT rs2736100 genotype AA or variant allele A showed no significant difference between cases and controls. Haplotype CA displayed significantly different pattern of frequency as 0.5 in cases as compared to 0.16 in controls (p < 0.0001). Combination of variant A/G haplotype frequency implicated more in cases than in controls (0.34 vs. 0.14, p = 0.001). Conclusions: It is concluded that hTERT rs2736098 polymorphic variant has a vital role to confer a strong risk to bladder cancer in our population. Further, hTERT haplotypes CA and AG inhTERT could prove to be a promising tool to screen the risk for bladder cancer.

Role of human organic cation transporter-1 (OCT-1/SLC22A1) in modulating the response to metformin in patients with type 2 diabetes.

BACKGROUND: Organic cation transporter 1 primarily governs the action of metformin in the liver. There are considerable inter-individual variations in metformin response. In light of this, it is crucial to obtain a greater understanding of the influence of OCT1 expression or polymorphism in the context of variable responses elicited by metformin treatment. RESULTS: We observed that the variable response to metformin in the responders and non-responders is independent of isoform variation and mRNA expression of OCT-1. We also observed an insignificant difference in the serum metformin levels of the patient groups. Further, molecular docking provided us with an insight into the hotspot regions of OCT-1 for metformin binding. Genotyping of these regions revealed SNPs 156T>C and 1222A>G in both the groups, while as 181C>T and 1201G>A were found only in non-responders. The 181T>C and 1222A>G changes were further found to alter OCT-1 structure in silico and affect metformin transport in vitro which was illustrated by their effect on the activation of AMPK, the marker for metformin activity. CONCLUSION: Taken together, our results corroborate the role of OCT-1 in the transport of metformin and also point at OCT1 genetic variations possibly affecting the transport of metformin into the cells and hence its subsequent action in responders and non-responders.

Association of epidermal differentiation complex (EDC) genetic variants with House Dust Mite sensitization in Atopic Dermatitis Patients.

The etiopathogenesis of AD is multifactorial and defects of the skin barrier, which physiologically constitutes the natural protection, are associated with the disease phenotype. The identification of the genetic and environmental factors paving the way for impaired barrier function is therefore important in developing new therapeutic and prevention strategies. MATERIAL AND METHODS: Confirmed 100 cases were tested against 106 controls for filaggrin mutation and LELP-1 polymorphism by PCR-RFLP and chain termination sequencing. Total IgE and Vitamin D were estimated by ELISA. House dust mite sensitization was assessed by an in-vivo skin prick test. RESULTS: FLG deletion (2282del4) was present in 4% of the patients and all these were heterozygous carriers, whereas FLG null mutation (R501X) was not present in any of the cases. In the control group, both the mutations were not found. CT genotype and T allele of LELP-1 (rs7534334) were significantly associated with elevated IgE levels, early-onset, HDM sensitization, and disease severity (P < 0.05). However, the genotypic and allelic distribution of LELP-1 among the cases and controls was found to be insignificant. CONCLUSION: The low frequency of 2282del4 deletion and the absence of R501X mutation suggest that filaggrin deficiency does not confer a major risk for AD in the Indian population. However, significant association of LELP-1 (rs7534334) variant allele with clinical variables may serve as a novel biomarker for the severity of Atopic Dermatitis as well as an indicator for the allergen-specific immunotherapy and hence bears important clinical implications and needs to study on larger sample size and diverse populations.

Interleukin 32 gene promoter polymorphism: A genetic risk factor for multiple sclerosis in Kashmiri population.

OBJECTIVE: Although the exact cause of multiple sclerosis is not known, there are a number of factors involved mainly environmental and genetic factors. The present study was done to determine association between IL-32 gene promoter polymorphism and IL-32 levels with multiple sclerosis. METHODS: 48 relapsing remitting multiple sclerosis patients and 60 healthy controls were compared for IL-32 gene promoter polymorphism and IL-32 levels. RESULTS: There was no significant difference in genotype CT between the MS patients and healthy controls (p 0.130) where as a significant difference in genotype (CC) frequencies among MS patients and healthy controls (p 0.039) was observed. The difference in C allele frequency was also statistically significant between two study groups (p 0.01). Multivariate regression analysis revealed that the CC genotype might impact the risk of disease susceptibility up to 3.71 times and the presence of C allele might increase the risk of susceptibility to multiple sclerosis by 2.26 fold. The serum IL-32 levels were not statistically different multiple sclerosis patients and healthy controls and between wild and mutant genotypes. CONCLUSIONS: IL-32 gene promoter polymorphism is a genetic risk factor for multiple sclerosis patients particularly women.

Effectiveness of Sublingual Immunotherapy in the Treatment of HDM-Induced Nasobronchial Allergies: A 3-Year Randomized Case-Control Study From Kashmir.

Allergen immunotherapy (AIT) is the only disease-modifying treatment for allergic disorders that induces immunological tolerance through administration of specific allergens. Studies on AIT for subcutaneous route are in abundance; however, the efficacy of AIT in tablet form through sublingual route has not been well elucidated. The present prospective, parallel-group, controlled study sought to compare the efficacy of sublingual immunotherapy (SLIT) tablets with pharmacotherapy (PT) in 332 house dust mite (HDM)-specific allergic asthma and/or rhinitis patients over a period of 3 years. Patients were followed up for a 6-month run-in period and then randomly stratified as those who would receive SLIT, SLIT in addition to PT (SLIT+PT), and PT alone. AIT was administered in the form of sublingual tablets. Symptom and medication scores were measured every 3 months. In vitro evaluation of serum total and HDM specific immunoglobulin E (HDM sIgE) levels was carried out every 3 months, whereas in vivo skin prick test was performed annually for 3 years. Our study demonstrated sustained clinical improvement, reduction in inhaled corticosteroid (ICS) dose and duration as well as prevention from development of neosensitization to other aero allergens in HDM-allergic asthmatics and/or rhinitis patients treated with 3 years SLIT. Despite a remarkable clinical improvement with AIT, we observed that SLIT did not significantly change the skin reactivity to HDM at 3 years and there was no significant change in the ratio of serum total and HDM sIgE. Given the immune and disease modifying effects of AIT in allergic diseases, the present study supports the notion of its sublingual mode being an effective long-term immunomodulator in HDM-sensitized nasobronchial allergies.

GSTT1 null and rs156697 Polymorphism in GSTO2 Influence the Risk and Therapeutic Outcome of B-Acute Lymphoblastic Leukemia Patients.

INTRODUCTION: Glutathione S-transferase (GST) gene deletion or polymorphic sequence variations lead to decreased enzyme activity that influences susceptibility and response to chemotherapy in acute lymphoblastic leukemia (ALL). This case-control study investigated the association of GST gene polymorphisms with the etiology and therapeutic outcome of B-ALL among Kashmiri population. METHODS: A total of 300 individuals including 150 newly diagnosed B-ALL patients and an equal number of age and gender matched controls were genotyped for five GST gene polymorphisms by polymerase chain reaction-restriction fragment length polymorphism technique (PCR-RFLP) and multiplex PCR techniques. RESULTS: Higher frequency of GSTT1 null, GSTO2-AG, and GSTO2-GG genotypes was observed in ALL cases compared to controls that associated significantly with ALL risk (GSTT1 null: OR = 2.93, p = 0.0001; GSTO2-AG: OR = 2.58, p = 0.01; GSTO2-GG: OR = 3.13, p = 0.01). GSTM1, GSTP1, and GSTO1 SNPs showed no significant association (p > 0.05). Combined genotype analysis revealed significant association of GSTT1 null/GSTM1 null (OR = 4.11, p = 0.011) and GSTT1 null/GSTP1-AG (OR = 4.93, p = 0.0003) with B-ALL susceptibility. Haplotype analysis of rs4925 and rs156697 revealed that carriers of CG haplotype had increased risk of B-ALL (p = 0.04). Kaplan-Meier plots revealed significantly inferior 3-year disease-free survival for GSTO2-GG carriers (p = 0.002). Multivariate analysis confirmed GSTO2-GG as an independent poor prognostic factor for DFS (HR = 4.5, p = 0.034). Among combined genotypes, only GSTT1 null/GSTP1-AG associated significantly with poorer DFS rates (p = 0.032). CONCLUSION: This study demonstrated that GSTT1 null individually or in combination with GSTM1null and GSTP1-AG genotypes associated with increased B-ALL risk. Also, rs156697 variant genotypes (AG and GG) associated with B-ALL, whereas the GG genotype of rs156697 influenced the treatment outcome.

Galectin-1 as a predictive biomarker in ovarian cancer.

AIM: There is an urgent need to set up a useful biomarker for ovarian cancer. Galectin-1 is a promising carbohydrate-binding protein which plays a remarkable role in various malignancies yet its clinical significance is questionable. In this study, we have tested the clinical implications of serum Galectin-1 levels in patients with ovarian tumours. MAIN METHODS: Serum Galectin-1 levels were quantified in 84 newly diagnosed ovarian tumour patients and 20 healthy controls by Enzyme Linked Immuno Sorbent Assay during the course of the disease. Therefore the samples were taken at diagnosis, after surgery and after chemotherapy. KEY FINDINGS: The Galectin-1 levels were found to be associated with various variables of Ovarian Cancer patients. The levels were found to be prominently high in postmenopausal patients. Galectin-1 levels were raised in epithelial ovarian tumours with significantly high levels in serous subtype. A decrease in Galectin-1 levels post-surgical intervention and after receiving chemotherapy was found. Galectin-1 levels evidently distinguished between normal, benign, malignant and metastatic cases as compared to CA125 levels. Galectin-1 demonstrated to be a better biomarker than CA125 according to the Receiver Operating Characteristic (ROC) curve analysis. SIGNIFICANCE: The study emphasizes that serum Galectin-1 may serve as a better surrogate biomarker in Ovarian Cancer for early detection, discriminating between malignant and benign abdominal masses and monitoring the progression of the disease and response to treatment.

Significant Implications of APOA1 Gene Sequence Variations and Its Protein Expression in Bladder Cancer.

Apolipoprotein A1 (APOA1) is a potential biomarker because of its variable concentration in different types of cancers. The current study is the first of its kind to evaluate the association between the APOA1 genotypes of -75 G/A and +83 C/T in tandem with the APOA1 protein expression in urine samples to find out the risk and potential relationship for differentially expressed urinary proteins and APOA1 genotypes. The study included 108 cases of bladder tumors and 150 healthy controls that were frequency matched to cases with respect to age, sex, and smoking status. Genotyping was performed using PCR-RFLP and the urinary expression of the APOA1 protein was done using ELISA. Bladder tumor cases were significantly associated with the APOA1 -75 AA genotype (p < 0.05), while the APOA1 +83 C/T heterozygotes showed an association with cases (p < 0.05). The overall distribution of the different haplotypes showed a marked difference between the cases and controls in GT when compared with the wild type GC (p < 0.03). Bladder tumor cases that carried the variant genotype APOA1 -75AA were found more (70.0%) with a higher expression (≥20 ng/mL)of the APOA1 urinary protein and differed significantly against wild type GG (p = 0.03). Again, in low grade bladder tumors, urinary APOA1 protein was exhibited significantly more (52.4% vs. 15.4% high grade) with a higher expression (≥20 ng), while high grade tumor cases (84.6% vs. 47.5% low grade) showed a lower APOA1 expression (<20 ng/mL) (O.R = 6.08, p = 0.002). A strong association was observed between APOA1 -75G/A and risk for bladder tumor and its relation to urinary protein expression, which substantiates its possible role as a marker for the risk assessment of the disease and as a promising diagnostic marker for different grades of malignant bladder tumors.

Impact of ABCB1 Gene (C3435T/A2677G) Polymorphic Sequence Variations on the Outcome of Patients with Chronic Myeloid Leukemia and Acute Lymphoblastic Leukemia in Kashmiri Population: A Case-Control Study.

Inherited polymorphic sequence variations in drug transport genes like ABCB1 impact a portion of patients with hematologic malignancies that show intrinsic or acquire resistance to treatment. Keeping in view inter-individual sensitivities for such drugs, we through this case-control study tested whether ABCB1 C3435T and G2677T polymorphisms have any influence on the risk and treatment response in patients with chronic myeloid leukemia (CML) and B-acute lymphoblastic leukemia (B-ALL). Genotyping for ABCB1 polymorphisms was performed by polymerase chain reaction-restriction fragment length polymorphism in 100 CML and 80 B-ALL patients along with 100 age and gender matched healthy controls. ABCB1 C3435T and G2677T polymorphism showed no association with CML. Genotype distribution revealed significant higher frequency of TT genotype for both SNPs in B-ALL cases and associated with increased B-ALL risk (OR 2.5, p = 0.04 for 3435TT; OR 2.4, p = 0.04 for 2677TT). There was no significant difference in genotype frequency of 3435C > T and 2677G > T among resistant and responsive groups for the two leukemia types. Kaplan-Meier survival plots revealed significantly lower event free survival in CML and B-ALL patients that were carriers of 3435TT genotype (p < 0.05). Multivariate analysis considered 3435TT genotype as independent risk factor for imatinib resistance in CML cases (HR 6.24, p = 0.002) and increased relapse risk in B-ALL patients (HR 4.51, p = 0.03). The current study provides preliminary evidence of a significant association between variant TT genotype and increased B-ALL risk. Also, results suggest that ABCB1 3435TT genotype increases imatinib resistance in CML and influence therapeutic outcome in B-ALL.

Glutathione S-transferase gene polymorphic sequence variations: Association with risk and response to Imatinib among Chronic Myeloid Leukemia patients of Kashmir.

INTRODUCTION: Glutathione S-transferase (GST) gene deletion or polymorphic sequence variations lead to decreased enzyme activity that influences susceptibility and response to tyrosine kinase inhibitors in chronic myeloid leukemia (CML). We aimed to analyze relation of different GST gene sequence variants with susceptibility and response to Imatinib in CML. MATERIAL AND METHODS: A total of 150 CML cases and equal number of age and gender matched healthy controls were genotyped for five GST polymorphisms by multiplex-PCR and PCR-RFLP techniques. BCR-ABL1 transcripts were quantified by quantitative Real Time PCR (qRT-PCR). RESULTS: GSTT1, GSTO1, and GSTO2 SNPs revealed no association, while as GSTM1null genotype was observed to protect against the development of CML (OR = 0.53, P = .01). GSTP1 variant genotypes AG (OR = 2.1, P = .003) and GG (OR = 5.6, P < .001), significantly associated with increased risk of CML. Combined genotype analysis showed protective impact of GSTT1present /GSTM1null (OR = 0.44, P = .003) while as GSTT1present /GSTP1-GG (OR = 6.92, P < .001) and GSTM1present /GSTP1-GG (OR = 6.33, P < .001), significantly increased CML risk. GSTM1null genotype individually and in combination with GSTT1present associated with superior rate of major molecular response (MMR) and event free survival (EFS) (log-rank P = .029). GSTO2-AG+GG genotype associated with significantly inferior MMR rates at 3, 6, and 12 months. Also, patients with GSTO2-GG genotype showed significantly reduced EFS (log-rank P = .025). Multivariate analysis confirmed GSTM1null as a better (HR:0.19, P = .029) and GSTO2-GG genotype as an independent poor prognostic factor (HR:2.29, P = .037). CONCLUSION: GSTM1null genotype seems to have a better prognostic role while GSTP1 variants significantly increase CML risk. Also, results support a correlation between disease outcome and GSTO2 polymorphism.

Improved pulmonary function test (PFT) after 1 one year of Sublingual Immunotherapy (SLIT) in unison with pharmacotherapy in mild allergic asthmatics.

BACKGROUND: Allergen immunotherapy (AIT) is a promising treatment for allergic disease that induces immunological tolerance through the administration of specific allergens. The study of AIT is in its early stage and its clinical effects are not well elucidated. The present study was aimed at determining the effect of AIT on pulmonary function and serum variables of mild allergic asthma patients. METHODS: A total of 80 patients with mild allergic asthma were recruited for the study. Allergen Specific Immunotherapy was administered in the form of Sublingual Immunotherapy and consisted of a build up phase followed by a maintenance phase (six months each respectively). Total serum IgE and vitamin D levels were quantified by ELISA. The percent eosinophill count was determined by cell analyzers. Pulmonary function test was performed at the baseline and after the end of study period. Subjective symptom score was recorded in the form of asthma control questionnaire score. RESULTS: There was a significant increase in the pre FEV1% and pre FEV1/FVC post AIT administration. A significant decrease in the total serum IgE was found post AIT. A decrease in Asthma control Questionnaire (ACQ) scores indicated an improvement in clinical symptoms. Besides there was a significant effect on ICS discontinuation after AIT. CONCLUSION: The study supports SLIT as an effective treatment for Immunomodulation in mild allergic asthmatics besides it gives us significant information regarding the safety and efficacy of SLIT in such patients.

Analysis of MNS16A VNTR polymorphic sequence variations of the TERT gene and associated risk for development of bladder cancer.

BACKGROUND: The MNS16A variable number tandem repeat (VNTR) polymorphism of the human telomerase reverse transcriptase (hTERT) gene acts as a regulator of hTERT promoter activity and has been shown to have a role in the predisposition toward various cancers. The current study aimed to investigate the association between MNS16A VNTR alleles and genetic predisposition to bladder cancer in the Kashmir region of northern India. MATERIALS AND METHODS: A total of 130 patients with bladder cancer and 170 age- and gender-matched healthy controls were included in this study. Primer-specific polymerase chain reaction was used to genotype the different variants of VNTR alleles of the MNS16A VNTR polymorphism. RESULTS: Short allele VNTR-243 (SS) genotype frequency significantly differed between cases (9.23%) and controls (3.52%) (OR = 3.08 [95% CI = 1.10-8.61], p = 0.042). The VNTR-243 short allele (S) was found significantly more frequent in bladder cancer cases (28.46%) than controls (20.88%) (OR = 1.50 [95% CI = 1.03-2.19], p = 0.034). Likewise, the long allele (LL) hTERT MNS16A genotype was distributed more frequently in low stage disease versus high stage disease (60.29% vs. 39.70%) (OR = 0.79 [95% CI = 0.39-1.60], p = 0.595). CONCLUSION: The MNS16A VNTR short allele (S) was associated with a higher risk for bladder cancer in our population as compared to long alleles.

Influence of single gene variants of FOXP3 on allergic asthma predisposition.

BACKGROUND: The role of FoxP3, a master regulator of T regulatory cells, in allergic diseases such as asthma is of immense importance yet the effect of its gene variants on the disease predisposition is not fully understood. We studied the association of FoxP3 polymorphisms (-2383C/T and -3279C/A) in allergic asthma patients and their correlation with serum IL-4, IL-13, Total IgE, and Vitamin D levels. METHODS: In this study 350 individuals were enrolled, 150 allergic asthma patients and 200 healthy controls. SNP analyses were performed by RFLP. IL-4, IL-13 vitamin D and Total IgE were measured by ELISA. RESULTS: The AA homozygous mutant of -3279C/A posed a three-fold risk [P < 0.005; OR, 3.52] whereas the -2383C/T variants TT genotype carried a fourfold risk [P = 0.002; OR, 4.04]. Haplotype analysis exhibited predisposition to allergic asthmawith CC/TT [P = 0.01; OR 5.93 (95%CI)], AA/CC [P = 0.01; OR 3.29] and AA/TT haplotypes [P = 0; OR 11.86 (1.31-85.87)]. A negative correlation between IgE and Vitamin D was found [r = -0.30p-value 0.001] but a negative correlation betweenIgE and Vit D was established in the haplotype CC/TT [r = -0.45P = 0.002] and CC/CT [r = -0.52P = 0.04]. In allergic patients, the eosinophils count was high [p = 0.003] and the mean levels of pro-inflammatory cytokines IL-4 and IL-13 were elevated [P < 0.001] as well. CONCLUSIONS: The study suggests SNP -3279 -AA genotype and, -2383-TT genotype in association with certain haplotypes pose a risk for allergy development. There was no correlation between different genotypes and serum levels of various cytokines.

Methylenetetrahydrofolate Reductase Gene C677T and A1298C Polymorphic Sequence Variations Influences the Susceptibility to Chronic Myeloid Leukemia in Kashmiri Population.

Background: Methylenetetrahydrofolate reductase (MTHFR) gene is a crucial regulator of folate metabolism and its two prominent polymorphic variants C677T and A1298C lead to decreased MTHFR enzyme activity. Aim of the Study: We planned this case-control study based on numerous studies supporting the association of MTHFR polymorphisms (C677T and A1298C) with CML risk in different ethnic populations. Therefore, the influence of these polymorphisms on CML susceptibility was investigated among Kashmiri population (North India). Materials and Methods: Polymerase chain reaction/restriction fragment length polymorphism (RFLP) technique was employed for genotyping MTHFR C677T and A1298C SNP's in 125 CML patients as against 150 age and gender matched healthy controls. Results: A significant difference was observed in frequency of 677CT genotype between cases and controls [46.4 vs. 27.3% (p = 0.0005)]. Similarly combined 677CT+TT genotype showed significant difference between cases and controls [50.4 vs. 28.6% (p = 0.0002)]. Both MTHFR 677CT and 677CT+TT genotypes imposed greater than 2-fold risk of developing CML (OR = 2.4, 95%CI: 1.46-4.05; OR = 2.5, 95%CI: 1.53-4.16). In case of A1298C SNP, the frequency of 1298AC genotype was higher in controls (64.0%) as compared to CML cases (48.8%) (p = 0.04) and imparted a significant protective role from CML predisposition. Furthermore, haplotype analysis revealed only "677CT/1298AA" haplotype significantly increased the risk of CML predisposition [(p = 0.008) (OR = 3.2, 95% CI: 1.3-7.4)]. Conclusion: We conclude that both MTHFR C677T and A1298C polymorphisms may be important genetic modifiers and seem to have a plausible role to confer risk of CML in Kashmiri population, where C677T SNP strongly increases the risk of CML while as A1298C SNP has a protective effect.

Influence of bcr-3 PML-RARα transcript on outcome in Acute Promyelocytic Leukemia patients of Kashmir treated with all-trans retinoic acid and/or arsenic tri-oxide.

AIMS: Distinct types of PML-RARα hybrid transcripts viz bcr-1, bcr-2 and bcr-3 result from translocation between chromosomes 15 and 17 t(15;17) in Acute Promyelocytic Leukemia patients. We aimed to determine the frequencies of the PML-RARα transcripts and FLT3-ITD mutations in APL patients to evaluate their prognostic implications and also to analyze their impact on disease outcome. MAIN METHOD: RT-PCR and Rq-PCR were adopted for transcript typing and quantitation of PML-RARα transcripts while FLT3-ITD was detected by PCR in APL patients. KEY FINDINGS: PML-RARα bcr-1, bcr-2 and bcr-3 transcripts were found in 26, 3 and 16 cases respectively. 64.4% patients achieved complete remission, 22.2% expired early wherein majority of the cases expressed bcr-3 transcript (p = 0.03). 50% relapse rate was observed in patients with bcr-3 transcripts. Multivariate analysis showed expression of bcr-3 transcript associated with early death (p = 0.027) and increased relapse risk (P = 0.046). Patients expressing bcr-3 hybrid transcript showed lowest OS of 28.0 months (±â€¯5.26) (p = 0.027). FLT3-ITD mutation was detected in 5 (11.1%) patients and presence of these mutations was not associated either with PML-RARα transcripts or with disease outcome. SIGNIFICANCE: bcr-3 transcript has a more lethal outcome and is also associated with frequent relapse risk in APL patients of our region.

Real-time quantitative PCR: a reliable molecular diagnostic and follow-up tool for 'minimal residual disease' assessment in chronic myeloid leukemia.

Molecular monitoring of BCR-ABL transcript levels by real-time quantitative PCR is increasingly being used to diagnose the disease and assess treatment response in patients with chronic myeloid leukemia (CML). This has become particularly relevant when residual levels of leukemia usually fall below the level of detection by cytogenetic analysis. Forty-two CML patients, including 18 males (42.86%) and 24 females (57.14%) aged 7-75 years, were enlisted for the study and followed-up for the response to imatinib treatment. Patients were subjected to Multiplex RT-PCR (reverse-transcriptase PCR) and were all found to harbor either e13a2 or the e14a2, which could be analyzed by a single Taqman probe based quantitation kit (Geno-Sen's) to quantitate the BCR-ABL transcript load. The Multiplex RT-PCR and peripheral blood cytogenetics providing specific and sensitive detection of BCR-ABL fusion transcripts and metaphase signal load respectively were used as parallel reference tools to authenticate the q-PCR findings. There was 100% concordance between the multiplex RT-PCR and the q-PCR as every positive RT-PCR assay for a transcript reflected as q-PCR load of above 0% for that transcript. q-PCR also demonstrated a strong Pearson correlation with the cytogenetic response.

Prognostic Implication of BCR-ABL Fusion Transcript Variants in Chronic Myeloid Leukemia (CML) Treated with Imatinib. A First of Its Kind Study on CML Patients of Kashmir

Background: The prognostic significance of the common BCR-ABL transcripts like e13a2 (b2a2) and e14a2 (b3a2) in Chronic myeloid leukemia (CML) has been reported from patients treated with different tyrosine kinase inhibitors but its impact on clinical response and overall survival remains still unexplored. The aim of this study was to evaluate the prognostic significance of different transcript types in a cohort of CML patients treated with imatinib. Methods: A total 42 confirmed cases of Chronic Myeloid Leukemia (CML) patients were recruited into our cohort study and a multiplex Reverse Transcriptase-Polymerase Chain Reaction technique (RT-PCR) was used to detect 3 main transcript types 'e1a2', 'e13a2', and 'e14a2' found in CML. Results: Only two types of transcripts e13a2 (b2a2) and e14a2 (b3a2) were detected in our CML patients and none had the e1a2 type. All the patients were RT-PCR positive for either e13a2 or e14a2 fusion transcript demonstrating 100% concordance with their Ph+ve cytogenetic status at baseline. TLC count (range of 201-600x103/µl) and platelet count (range of 201-900x103/µl) at baseline were found to be associated more with the e14a2 (b3a2) than the e13a2 (b2a2) transcript type (p-value: 0.001). The two transcripts found did not relate significantly towards sex, age-group or indicated spleen size ranges as well as percentage ranges of blast cells. Conclusion: We conclude that there is no overall prognostic implication of either the e13a2 or the e14a2 transcript type across the spectrum of indicated clinical parameters evaluated. Even the overall survival analysis of the two transcript types revealed no prognostic association whatsoever.