Cytokine genes multi-locus analysis reveals synergistic influence on genetic susceptibility in Indian SLE - A multifactor-dimensionality reduction approach.

Systemic lupus erythematosus (SLE) is a prototype autoimmune disease with unclear etiology. Several loci associated with genetic susceptibility for lupus have been described. However, it lacks reports on cytokine gene-gene interactions among SLE patients from Asian population. Epistasis interaction among single nucleotide polymorphisms (SNPs) of cytokine genes in Indian SLE patients was tested using multifactor-dimensionality reduction (MDR) analysis. A total of fourteen SNPs lacking linkage disequilibrium among different cytokines genes were genotyped in a cohort of 200 SLE patients and 201 healthy individuals as controls of Indian origin. A high degree of synergism among Lymphotoxin-α (LT-α), Interleukin-1ß (IL-1ß) and Interleukin-10 (IL-10) gene polymorphisms was detected in our SLE patients. Furthermore, by virtue of biological inter-relations among different cytokines, a high strength of interactions was observed among pro-inflammatory (IL-1ß, IL-6) and anti-inflammatory (IL-10) cytokine gene SNPs. Also, among studied pro-inflammatory cytokines and chemokines, a strong synergistic effect among Tumor Necrosis Factor-α (TNF-α), LT-α and Monocyte Chemo-attractant Protein-1 (MCP-1) SNPs was occurred. A nature of strong interaction among the candidate cytokine genes may speculate a proactive role in causing genetic susceptibility to the disease in SLE patients with Indian origin.

Association of BCL11A genetic variant (rs11886868) with severity in ß-thalassaemia major & sickle cell anaemia.

BACKGROUND & OBJECTIVES: The amount of foetal haemoglobin that persists in adulthood affects the clinical severity of haemoglobinopathies including ß-thalassaemia major and sickle cell anaemia (SCA). The present study was undertaken to analyse ß-thalassaemia as well as SCA patients for the single nucleotide polymorphism (SNP), rs11886868 (T/C) in BCL11A gene and to evaluate the association between this polymorphism and severity of ß-thalassaemia major and SCA. METHODS: a total of 620 samples (420 ß-thalassaemia major and 200 SCA cases) were analysed before blood transfusion using basic screening tests like complete blood analysis and osmotic fragility and further confirmed by high performance liquid chromatography (HPLC), amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and reverse dot blot techniques. All patients were transfusion dependent. Patients with ß-thalassaemia and SCA were classified into mild, moderate, severe according to the severity score based on Hb levels, age of onset, age at which patients received their first blood transfusion, the degree of growth retardation and splenectomy. ß-thalassaemia as well as SCA patients were analysed for the SNP, rs11886868 (T/C) in BCL11A gene and association between this polymorphism and severity of ß-thalassaemia major as well as SCA was evaluated. RESULTS: There was a significant difference in genotypic and allelic frequencies of BCL11A gene polymorphism between mild and moderate and mild and severe cases in both the groups. A significant (P<0.001) difference was observed in the mean HbF levels between the three genotypes in different severity groups. HbF levels were found to be high in CC genotype bearing individuals followed by TC and TT in ß-thalassaemia major as well as SCA. INTERPRETATION & CONCLUSIONS: This study confirms that the T/C variant (rs11886868) of the BCL11A gene causing downregulation of BCL11A gene expression in adult erythroid precursors results in the induction of HbF and ameliorates the severity of ß-thalassaemia as well as SCA.

Association of Xmn1 -158 γG variant with severity and HbF levels in ß-thalassemia major and sickle cell anaemia.

Haemoglobinopathies including ß-thalassemia and sickle cell anaemia (SCA) are considered to be classical monogenic diseases. There is considerable clinical variability between patients inheriting identical ß-globin mutations. The reasons for this variability are not well understood. Previous studies have suggested that a variety of genetic determents influence different clinical phenotypes. The genetic variants that modulate HbF levels have a very strong impact on ameliorating the clinical phenotype. In the present study 6,500 blood samples from suspected cases were analysed using HPLC, ARMS-PCR, RDB techniques. Patients with ß-thalassemia and SCA were classified into mild, moderate, severe according to the severity score based on Hb levels, age of onset, age at which patients received their first blood transfusion, the degree of growth retardation and splenectomy. Patients with ß-thalassemia and SCA were analysed for Xmn1 polymorphism and association between this polymorphism and severity of ß-thalassemia and SCA was evaluated. We found a significant difference in genotypic and allelic frequencies of Xmn1 polymorphism between mild and moderate and mild and severe cases. There was a significant difference in high and low percentage of HbF in CC, CT and TT bearing individuals. The TT bearing individuals were found to have a high percentage of HbF in ß-thalassemia as well as SCA. This study confirms that increased γG-globin expression associated with Xmn1 polymorphism ameliorates the clinical severity in ß-thalassemia as well as SCA in the study population.

Three most common nonsynonymous UGT1A6*2 polymorphisms (Thr181Ala, Arg184Ser and Ser7Ala) and therapeutic response to deferiprone in ß-thalassemia major patients.

Deferiprone is used as a chelation agent in chronic iron overload in ß-thalassemia patients. Patients on deferiprone therapy show variable response to this drug in terms of reduction in iron overload as well as adverse drug reactions (ADRs). The pharmacogenetic studies on deferiprone have not carried out in patients with blood disorders in India. Therefore, the present study was carried out to evaluate the three most common nonsynonymous UGT1A6 polymorphisms Thr181Ala (541 A/G), Arg184Ser (552 A/C) and Ser7Ala (19 T/G) and therapeutic response to deferiprone in ß-thalassemia major patients. Two hundred and eighty six (286) ß-thalassemia major patients were involved in the study. Serum ferritin levels were estimated periodically to assess the status of the iron overload and the patients were grouped into responders and non-responders depending on the ferritin levels. The UGT1A6 2 polymorphisms were detected by PCR-RFLP methods. The association between the genotypes and outcome as well as ADRs was evaluated by Open EPI software. A significant difference was observed in the genotypic distribution of UGT1A6 2 Thr181Ala polymorphism in responders and non-responders. However, there was no difference in the genotypic distribution between patients with and without ADRs. As far as the UGT1A6 2 Arg184Ser polymorphism is concerned, no significant difference was observed between responders and non-responders. Further, evaluating the association of UGT1A6 2 Ser7Ala polymorphism with drug response, there was no significant difference in the genotypic distribution between responders and non-responders. However, there was a significant difference between responders with and without ADRs and non-responders with and without ADRs. In addition to this haplotype analysis was also carried out. However, we did not find any specific haplotype to be significantly associated with the deferiprone response in ß-thalassemia major patients.

Association of ALOX5AP1 SG13S114T/A variant with ischemic stroke, stroke subtypes and aspirin resistance.

The important role of genetic variants in the etiology and pathophysiology of stroke is being increasingly recognized. Simultaneously, the influence of genetic factors in the clinical outcome of drug therapy cannot be ignored. 5-lipoxygenase activating (ALOX5AP) gene involved in the synthesis of leukotrienes, has been recognized as an important gene contributing towards susceptibility of stroke risk. Leukotrienes are involved in the physiological mechanism of atherosclerotic events and inflammation. The present study was designed to identify the association of SG13S114T/A polymorphism in ALOX5AP1 gene with risk of stroke, its subtypes and aspirin resistance. We studied six hundred and ten patients with ischemic stroke and six hundred and ten age and sex matched healthy controls. The ischemic stroke was classified according to Trial of Org 10172 in Acute stroke Treatment. ALOX5AP1 SG13S114T/A polymorphism was determined using PCR RFLP methods. Follow-up was done for all the patients for a period of 3 months, 6 months and 12 months. The patients were classified into two groups responders and non-responders. The non-responders were identified to have a poor clinical outcome defined as a score of more than 2 on modified Rankin Scale Score and less than 5 on extended Glassgow Outcome Scale from stroke onset. We found statistically significant difference in the genotypic distribution between patients and controls (for AA vs TT, χ2=9.894; p=0.001, odds ratio=1.68 (95% confidence interval (CI); 1.215, 2.326). Significant difference was observed in the frequency of A and T alleles in patients and controls (A vs T χ(2)=10.23; p=0.001, odds ratio=1.301 (95% CI; 1.107, 1.528). Multiple logistic regression analysis revealed, the most predictive risk factor for stroke was AA genotype [adjusted odds ratio=1.660 (95% CI; 1.167-2.361) and p=0.005], hypertension, smoking and diabetes (p<0.001 in each case). We also found a significant association of AA genotype with intracranial large artery atherosclerosis (p=0.002, odds ratio=2.04, (95%CI; 1.279-3.275) and cardioembolism (p<0.001, odds ratio=4.73 (95% CI; 2.661-8.439). The risk of aspirin resistance was significantly higher among patients with AA genotype in comparison to carriers of homozygous TT genotype (AA vs TT, χ2=22.25, odds ratio=2.983, 95% CI; 1.884- 4.723, p<0.001). The frequency of recurrence and death events was more in non-responders. We didn't find a significant association of the aspirin dose with outcome. Our results indicate that the individuals bearing AA genotype of ALOX5AP1 SG13S114T/A polymorphism are more prone to stroke and bad outcome as well as with aspirin resistance than TA and TT genotypes.

Serum albumin levels in ischemic stroke and its subtypes: correlation with clinical outcome.

OBJECTIVE: Previous studies have associated low serum albumin levels with poor outcome in ischemic stroke. Animal studies also demonstrated neuroprotective effects of serum albumin in focal ischemia. However, there are very limited studies on the association of serum albumin levels with stroke outcome in ischemic stroke divided into subtypes. The present study was carried out to investigate the association of serum albumin levels with outcome in ischemic stroke and its subtypes. METHODS: The study involved 560 patients. Serum albumin levels were estimated and follow-up interviews were conducted at 3 mo postevent to determine stroke outcome. The association between serum albumin levels and stroke outcome was evaluated by multiple logistic regression analysis after adjustment for potential confounders. RESULTS: Low levels of albumin associated significantly with poor outcome (score of >3 on the modified Rankin Scale). The adjusted odds ratio was 1.972 (95% confidence interval, 1.103-4.001; P < 0.001). The recurrence of stroke and death rate also was high in patients with low levels of albumin compared with patients with elevated levels of albumin. The reduced level of serum albumin associated significantly with poor outcome in all the stroke subtypes classified according to TOAST (Trial of ORG 10172 in Acute Stroke Treatment). CONCLUSIONS: Relatively high serum albumin levels in acute stroke decrease poor outcome.

Association of C3435T multi drug resistance gene-1 polymorphism with aspirin resistance in ischemic stroke and its subtypes.

Aspirin is the most commonly used antiplatelet drug for treatment of a serious vascular event, most notably myocardial infarction and stroke. Significant fraction of aspirin treated patients is resistant to the antiplatelet effects of the drugs. Previous studies have suggested that a genetic basis for aspirin resistance exists. Therefore the present study was taken up to investigate the role of C3435T polymorphism (rs 1045642) of multiple drug resistance-1 (MDR-1) gene with aspirin resistance in stroke patients. Five hundred and sixty ischemic stroke patients and 560 age and sex matched healthy controls were involved in the study. Baseline clinical data were collected and follow-up telephone interviews were conducted with patients at 3, 6 and 12 months post event to determine stroke outcome. Blood samples were collected and genotypes determined. Significant difference was observed in the genotype distribution and allele frequency between patients and controls. The results were confirmed by a step wise multiple logistic regression analysis controlling all other confounding risk factors [adjusted Odds ratio=3.132 (95% CI; 2.043-4.800; p<0.001)]. There was a significant difference in genotype distribution between drug responders and non-responders. The risk of aspirin resistance was significantly high in patients with TT genotype in comparison to those with CC genotype [(TT vs. CC, χ(2)=6.268; p=0.012, Odds ratio=1.85) (95% CI; 1.142-3.017) (adjusted Odds ratio=2.465; 95% CI; 1.895-4.625 and p<0.001)]. As far as the stroke subtypes are concerned TT genotype associated significantly with aspirin resistance in intracranial large artery atherosclerosis. Our results indicate that the risk of aspirin resistance is more in patients with 3435TT genotype than in those with CC genotype. However, this is a preliminary study and a large study of replication is needed to confirm our results.

Lack of association of G779A ZHX-2 gene variant with HbF levels in ß-thalassemia major.

The inherited disorders of hemoglobin synthesis are the most common monogenic disorders worldwide. They include thalassemias, hemoglobin variants, and hereditary persistence of fetal hemoglobin. ß-thalassemia is the most common monogenic disorder in India. Clinical manifestations of ß-thalassemia are extremely variable in severity. The reasons for this heterogeneity are not very well understood. Previous studies have shown that the genetic variants that modulate HbF levels have a very strong impact on ameliorating the clinical phenotype. In the present study, 5570 blood samples from suspected cases were analyzed using HPLC, amplification refractory mutation system-PCR and reverse dot blot techniques. Of 5570 individuals, we found 676 cases of ß-thalassemia disease. Molecular analysis revealed the presence of different ß-thalassemia mutations in the population under study. Patients with ß-thalassemia were classified into mild, moderate, and severe according to severity score based on Hb level, age of onset, age at which patients received their first blood transfusion, degree of growth retardation and splenectomy. Patients with ß-thalassemia were analyzed for zinc finger and homeoboxes 2 (ZHX2) G779A polymorphism, and the association between ZHX2 gene polymorphism and severity of ß-thalassemia was evaluated. We did not find a significant difference in genotypic and allelic frequency of ZHX2 gene between mild and moderate, mild and severe, and moderate and severe cases. There was no significant difference in high and low percentage of HbF in GG, GA, and AA bearing individuals showing that ZHX2 gene variant has no role in ameliorating the severity of ß-thalassemia major in the South Indian population from Andhra Pradesh.