Maternal association and influence of DHFR 19 bp deletion variant predisposes foetus to anencephaly susceptibility: a family-based triad study.

OBJECTIVE: Previous studies have not used family-based methods to evaluate maternal-paternal genetic effects of the folate metabolizing enzyme, dihydro folate reductase (DHFR) essential during embryogenesis. Present study focuses on evaluating the association and influence of parental genetic effects of DHFR 19 bp deletion in the development of foetal neural tube defects (NTDs) using family-based triad approach. MATERIALS AND METHODS: The study population (n = 924) including 124 NTD case-parent trios (n = 124 × 3 = 372) and 184 healthy control-parent trios (n = 184 × 3 = 552) from Telangana, India, was genotyped for DHFR 19 bp deletion. Statistical analysis was used by SPSS and parent-of-origin effects (POE). RESULTS: Foetuses with deletion genotype (DD) were at risk of developing anencephaly (OR = 3.26, p = 0.020). Among parents, increased maternal risk of having an anencephaly foetus (OR = 2.66, p = 0.028) was observed in mothers with DD genotype. In addition, POE analysis also demonstrated higher risk of maternal transmission of the deletion allele to anencephaly foetus compared with paternal transmission (OR = 6.00, p = 0.016). Interestingly, maternal-paternal-offspring genotype incompatibility revealed maternal deletion genotype (DD) in association with paternal heterozygous deletion genotype (WD) significantly increased risk for NTDs (OR = 5.29, p = 0.013). CONCLUSIONS: This study, using family-based case-parent and control-parent triad approach, is the first to report influence of maternal transmission of DHFR 19 bp deletion in the development of anencephaly in the foetus.

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 interleukin-10 gene promoter polymorphism in spontaneous abortions: a family-based triad study.

OBJECTIVE: The present study is a triad study designed to determine the co-relation of IL-10 -819C/T promoter polymorphism with the risk of spontaneous abortions. MATERIALS: A total of 50 families with spontaneous abortions and 60 families with medically terminated pregnancies were considered for the present study. Fetal tissue of less than 20 weeks of gestation along with peripheral blood from all the couples was collected in this study. METHODS: A standard amplification refractory mutation system-polymerase chain reaction was carried out to determine the IL 10 genotype in all the subjects. Odd's ratio and their respective 95% confidence intervals were used to determine the strength of association between IL-10 promoter gene polymorphism and spontaneous abortions. RESULTS: The study revealed a statistically significant association of IL-10 -819C/T polymorphism between the two family groups among fetuses (p=0.0000003) and mothers (p=0.0000001). No significant difference was observed in the genotype distribution of IL-10 among fathers. CONCLUSION: An increased frequency of TT genotype and T allele was observed in spontaneously aborted fetuses and their mothers compared to respective controls. In conclusion, IL-10 C -819T gene promoter polymorphism may act as a major genetic regulator in the etiology of spontaneous abortions.

Seroprevalence and Influence of Torch Infections in High Risk Pregnant Women: A Large Study from South India.

BACKGROUND: The increased complications to the mother and fetus during or after pregnancy and birth are often caused by a wide array of pathogenic organisms mostly belonging to the TORCH group [toxoplasmosis, rubella, cytomegalovirus (CMV), and herpes simplex virus (HSV)]. These agents cause asymptomatic or mild infection in the mother while serious consequences in fetus. The present study was aimed to find significant etiological pathogens in the causation of high risk pregnancy (HRP) in South Indian population. MATERIAL AND METHODS: A total of 1,158 HRP women (2010-2013) from Modern Government Maternity Hospital, Hyderabad were considered. Two milliliter of blood was obtained and the serum was analyzed for IgG and IgM antibodies against TORCH agents by ELISA. RESULTS: Twenty-five percent of the study group had fetal congenital malformation in the present pregnancy (Group 1; N = 291) while 75 % showed bad obstetric history (BOH) (Group 2; N = 867). Maternal age of ≤25 years, primi gravida, and consanguinity showed predisposing role for Group 1 while maternal age ≥30 years and ≥ 3 gravida were contributing risk for Group 2. The seropositvity in HRP women for toxoplasma, rubella, CMV, and HSV was 28, 84, 92, and 61 %, respectively for IgG while it was 6, 3, 4, and 3 % for IgG + IgM. Total seropositvity of toxoplasma, rubella, CMV, and HSV in Group 1 was 29, 97, 97, and 62 % while it was 36, 84, 97, and 65 % in Group 2, respectively. CONCLUSION: Maternal age of ≤25 years, primi gravida, and consanguinity contributed to fetal congenital malformation in the present pregnancy while maternal age of ≥30 years and ≥ 3 gravida towards BOH. Toxoplasma is protective while rubella and CMV are the infectious agents for HRP. Among the groups, toxoplasma and rubella conferred a predisposing risk towards Group 2 and Group 1, respectively. Sixty-one percent seropositvity of HSV in relation to bad obstetric outcome is the highest prevalence reported so far in India.

A functional polymorphism in the promoter region of interleukin-10 gene increases the risk for spontaneous abortions--a triad study.

PURPOSE: Spontaneous abortion or miscarriage is the natural death of an embryo or foetus in the early stages of prenatal development. Interleukin-10 is an anti-inflammatory cytokine, produced by human cytotrophoblasts, and defects in its production result in specific pathological conditions during pregnancy. The present study is aimed to evaluate the association of IL-10 -1082G/A polymorphism in spontaneous abortions by comparing foetal, maternal and paternal groups--a triad study. METHODS: A total of 50 families with spontaneous abortions and 60 families with medically terminated pregnancies were considered for the present study. DNA from foetal tissue and parental blood samples were extracted, and the genotype analysis of IL-10 -1082G/A promoter polymorphism was carried out by amplification refractory mutation system-polymerase chain reaction followed by agarose gel electrophoresis. A statistical analysis was applied to test for the significance of the results. RESULTS: There was a statistically significant difference in the distribution of AA genotypes and A allele of IL-10 -1082G/A between the two family groups among foetuses (P = 0.0002) and mothers (P = 0.00005). The paternal group showed no significant difference in the genotype distribution of IL-10 between cases and controls. CONCLUSION: In conclusion, IL-10 G-1082A gene promoter polymorphism may act as a major genetic regulator in the etiology of spontaneous abortions.

Heterozygosity of stromelysin-1 (rs3025058) promoter polymorphism is associated with gastric cancer.

BACKGROUND: Gastric cancer (GC) is the third most common cancer in India and is mediated by multiple genetic, epigenetic and environmental risk factors. A single nucleotide polymorphism rs3025058 at -1171 of the stromelysin-1 (matrix metalloproteinase [MMP]-3) promoter is resulting due to insertion/deletion of adenine thought to have an impact on increasing the risk for tumor formation. AIM: This study is aimed to understand the role of stromelysin-1 rs3025058 (-1171, 5A/6A) promoter polymorphism in the etiology of GC in Indian population. MATERIALS AND METHODS: Genomic DNA was isolated from blood samples of the GC patients and controls. The genotyping of stromelysin-1 rs3025058 (-1171, 5A/6A) promoter polymorphism was carried out by amplification refractory mutation system-polymerase chain reaction method followed by agarose gel electrophoresis. RESULTS: The frequency of 5A/5A, 5A/6A, and 6A/6A genotypes in GC patients were 7.69%, 76.92%, and 15.38%, while in controls were 5.31%, 86.73%, and 7.96%, respectively. There was a significant difference in the distribution of 5A/6A genotype in patients compared to the controls (P < 0.05). CONCLUSION: This study showed an increased frequency of heterozygotes for stromelysin-1 rs3025058 and thought to be involved in the etiology of GC.

Association of serum trace elements and minerals with genetic generalized epilepsy and idiopathic intractable epilepsy.

Certain minerals and trace elements are essential for the development of healthy nervous system. Altered serum levels of these elements may lead to the development of various diseases including epilepsy. The present study was designed to evaluate the association of serum calcium, magnesium, zinc and copper in the development of genetic generalized epilepsy [GGE; erstwhile known as idiopathic generalized epilepsy (IGE)] as well as idiopathic intractable epilepsy (IIE), in which seizures persist despite treatment with at least two or three antiepileptic drugs tolerated at reasonable dosage. 200 GGE patients and equal number of healthy controls were recruited for study with their written informed consent. The patients were further divided into responders and non-responders based on their response to antiepileptic drugs. Copper and zinc levels were assayed by atomic absorption spectrophotometer whereas calcium and magnesium were analyzed by Human Star 600 fully automated biochemistry analyzer. The patients with GGE had significant low levels of calcium, magnesium and zinc (1.85 ± 0.33, 0.69 ± 0.13 mmol/L and 11.33 ± 3.32 µmol/L respectively) and the corresponding values for controls were 2.27 ± 0.22, 0.89 ± 0.15, 12.71 ± 3.24 (p < 0.05). Significant high levels of copper were found in patients as compared to controls (26.69 ± 8.79 µmol/L; 16.64 ± 3.64) (p < 0.05). Significantly decreased levels of zinc were noted in non-responders (10.38 ± 2.99) compared to responders (12.62 ± 3.30) (p < 0.05). No significant difference was observed in serum calcium, magnesium and copper levels between responders and non-responders. In conclusion, low levels of calcium, magnesium, zinc and high levels of copper were found to be associated with GGE. Further, the patients with IIE were also found to have low levels of zinc.

Association of GABRA6 1519 T>C (rs3219151) and Synapsin II (rs37733634) gene polymorphisms with the development of idiopathic generalized epilepsy.

The idiopathic generalized epilepsy (IGE) is a neurological disorder which accounts for approximately 30% of all epilepsy cases. Patients identified with IGE syndromes have pharmacoresponsive epilepsies without abnormal neurological symptoms, structural brain lesions and are of unknown origin. A genetic etiology to IGEs has been proposed. Gamma amino butyric acid (GABA), a major inhibitory neurotransmitter acts by binding to transmembrane GABAA and GABAB receptors of both pre- and postsynaptic neurons. Synapsin II (SynII), a neuron specific phosphoprotein plays a major role in synaptogenesis and neurotransmitter release. The present study was carried out with an aim to evaluate the association of GABRA6 (rs3219151) T>C and Syn II (rs37733634) A>G gene polymorphisms with IGE. Molecular analysis revealed that the frequency of 'CC' genotype and 'C'allele of GABRA6 (rs3219151) T>C gene polymorphism was significantly higher in IGE patients compared to healthy controls [CC vs. TT, χ2=26; p<0.001; Odds ratio=3.6 (95% CI; 2.1-5.9); C vs T, χ2=24.7; p<0.001; Odds ratio=1.78 (95% CI; 1.4-2.2)]. The frequency of 'GG' genotype and 'G' allele of the intronic polymorphism A>G in Syn II gene was also found to be significantly associated with the disease when compared to controls [GG vs AA, χ2=64.52; p<0.001; Odds ratio=7.37 (95% CI; 4.4-12.3); G vs. A, χ2=65.78; p<0.001; Odds ratio=2.57 (95% CI; 2.0-3.2)]. The generalized multifactor dimensionality reduction method was employed to detect gene-gene interactions. The gene-gene interaction at two loci involving GABRA6 and Syn II revealed a significant association [χ2=36.6, p<0.001, Odds ratio=3.17 (95% CI; 2.2-4.6)] with IGE. Therefore, the present study clearly indicates that both GABRA6 (rs3219151) T>C and Syn II (rs37733634) A>G polymorphisms are important risk factors for the development of IGE in the South Indian population from Andhra Pradesh. The gene-gene interaction studies demonstrated significant interactive effects of these two loci in the development of the disease.

Interleukin 10 gene promoter polymorphisms in women with early-onset pre-eclampsia.

Pre-eclampsia is one of the most serious disorders of human pregnancy and T helper type 1 (Th1)/Th2 imbalance plays a major role in its aetiology. The Th2 cytokine, interleukin (IL)-10, plays a significant role in the maintenance of pregnancy. The present study is aimed at understanding the role of IL-10 promoter polymorphisms (-1082 G/A; -592 A/C and -819 C/T) and their haplotypes in early-onset pre-eclampsia. A total of 120 patients and an equal number of women with normal pregnancy, from Government Maternity Hospital, Petlaburz, Hyderabad, India, were considered for the present study. A standard amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) was carried out for genotyping followed by agarose gel electrophoresis. Appropriate statistical methods were applied to test for the significance of the results. It was found that the IL-10 -819 C allele (P = 0·003) and -592 A (P = 0·005) allele frequencies increased significantly in patients compared to controls. No significant difference was found with regard to -1082 promoter polymorphism. Haplotype analysis of the IL-10 single nucleotide polymorphisms (SNPs) revealed a significant association with ACC haplotype with a twofold increased risk in patients compared to controls. The frequencies of two common IL-10 haplotypes (GCC and ATA) did not show any significant difference. Further, the diplotype analysis revealed five genotypes: -1082A with -819C (P = 0·0016); -1082G with -819C (P = 0·0018); -819C with -592C (P = 0·001); -1082A with -592C (P = 0·032); and -1082G with -592C (P = 0·005) associated with the disease. These findings support the concept of contribution of IL-10 gene polymorphisms in the pathogenesis of early-onset pre-eclampsia.

A single-nucleotide polymorphism in tumor necrosis factor-α (-308 G/A) as a biomarker in chronic pancreatitis.

OBJECTIVE: Chronic pancreatitis is a gradual, long-term inflammation of the pancreas that results in alteration of its normal structure and function. The study aims to investigate the role of -308 (G/A) polymorphism of TNF-α gene in chronic pancreatitis. MATERIAL AND METHODS: A total of 200 subjects were included in this case-control study. A total of 100 in patients admitted in the Gastroenterology Unit of Gandhi Hospital and Osmania General Hospital, Hyderabad were included in the present study. An equal number of healthy control subjects were randomly selected for the study. The genotyping of TNF-α gene was carried out by tetra-primer ARMS PCR followed by gel electrophoresis. The TNF-α levels were assayed by enzyme-linked immunosorbent assay. RESULTS: A significant variation with respect to the genotypic and allelic distribution in the disease group when compared to control subjects [OR=2.001 (1.33-3.005), p<0.0001**] was observed. Subjects homozygous for the A allele had higher TNF-α levels compared to G allele. CONCLUSION: The present study revealed a significant association of the TNF-α gene promoter polymorphism with chronic pancreatitis. Thus, TNF-α genotype can be considered as one of the biological markers in the etiology of chronic pancreatitis.

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.

Non-Robertsonian translocation t (2;11) is associated with infertility in an oligospermic man.

Infertility is a major health problem which affects approximately 22% of married couples in reproductive age. Chromosomal defects are the most common genetic abnormalities in infertile men, with an incidence of cytogenetic abnormalities ranging from 2.1% to 15.5%. We describe here the clinical and cytogenetic studies carried out in a couple with repeated abortions. Cytogenetic analysis of the couple showed a de novo chromosomal translocation t (2;11)(p14;q21) in the male partner and a normal 46, XX karyotype in the female counterpart. Such an autosomal translocation may lead to the disruption of genes responsible for spermatogenesis or impaired synaptic complex pairing during meiosis resulting in reproductive failure.

Association of E-selectin gene polymorphism (S128R) with ischemic stroke and stroke subtypes.

E-selectin is an important inflammatory cytokine involved in the pathogenesis of various diseases such as atherosclerosis and stroke. We investigated the association of E-selectin gene polymorphism (S128R) with ischemic stroke and its subtypes. We studied 610 patients with ischemic stroke and 610 age- and sex-matched healthy controls. The ischemic stroke was classified according to Trial of Org10172 in Acute Stroke Treatment (TOAST). E-selectin gene polymorphism (S128R) was determined by polymerase chain reaction-restriction fragment length polymorphism technique. We found statistically significant difference in the genotypic distribution between patients and controls (for AC vs. AA, χ(2) = 49.5; p < 0.001, odds ratio = 5.47(95 % CI, 3.25-9.21). A significant difference was observed in the frequency of C and A alleles in patients and controls (for C vs. A, χ(2) = 47.4; p < 0.001, odds ratio = 5.13 (95 % CI, 3.06-8.57). Multiple logistic regression analysis revealed that the most predictive risk factor for stroke was AC genotype (adjusted odds ratio = 1.450 (95 % CI, 1.23-2.75) and p = 0.001), hypertension, smoking, and diabetes (p = 0.001 in each case). We also found a significant association of AC genotype with intracranial large artery atherosclerosis (p < 0.01, odds ratio = 9.37, (95 % CI, 5.31-16.5) and small artery occlusion (p < 0.0001, odds ratio = 9.81 (95 % CI, 4.94-19.4). Our results indicate that the individuals bearing AC genotype of E-selectin gene polymorphism (S128R) are more prone to stroke than AA genotype.

Association of matrix metalloproteinase-7 (-181A/G) promoter polymorphism in chronic pancreatitis.

BACKGROUND & OBJECTIVES: Chronic pancreatitis is progressive and irreversible destruction of the pancreas. Matrix metalloproteinase-7 (MMP-7) is a secreted matrilysin, which contributes to angiogenesis and breakdown of basement membranes of pancreatic tissues. The present study was aimed to investigate the association of MMP-7 -181A/G (rs11568818) gene promoter polymorphism in patients with chronic pancreatitis. METHODS: A total of 100 chronic pancreatitis patients and 150 unrelated healthy individuals were included in this case control study. The genotyping of the MMP-7 gene (- 181 A/G) (rs11568818) was carried out based on PCR-RFLP. The serum levels of MMP-7 were determined by ELISA. Association between genotypes and chronic pancreatitis was examined by odds ratio (OR) with 95% confidence interval (CI). RESULTS: The frequencies of the genotypes in promoter of MMP-7 were AA 49 per cent, AG 25 per cent and GG 26 per cent in chronic pancreatitis patients and AA 53 per cent, AG 38 per cent and GG 9 per cent in control subjects. Frequency of MMP-7 -181GG genotype and - 181G allele was significantly associated with chronic pancreatitis compared to healthy subjects [OR = 1.58 (95% CI: 1.06 -2.36), p =0.019]. There was no significant difference in the serum MMP-7 levels in the patients compared to control subjects. INTERPRETATION & CONCLUSIONS: The present study revealed a significant association of MMP-7 -181A/G (rs11568818) GG genotype with chronic pancreatitis patients, indicating its possible association with the disease.

Significance of MDR1 gene polymorphism C3435T in predicting drug response in epilepsy.

Antiepileptic drug (AED) treatment in epilepsy is often compromised by the unpredictability of efficacy and inter-individual variability among patients, which at least in part is the result of genetic variation. The idea to determine an individual's response to a prescribed medicine came into inception around 29 years ago. Pharmacogenetics is used to predict the drug response and efficacy, as well as potential adverse effects. We investigated the functional significance of the C3435T polymorphism of the MDR1 gene in a South Indian population. The patients were divided into responders and non-responders based on their clinical outcome and AED response. The risk of drug resistance was significantly higher in patients bearing TT genotype in comparison to carriers of the homozygous CC genotype [TT vs. CC, χ(2)=12.52; p=0.001, Odds ratio=2.34 (95% CI: 1.942-11.32)]. We suggest that the influence of the C3435T polymorphism in predicting the drug-resistance in epilepsy, might be significant and further investigations focusing on carbamazepine and phenytoin, in various ethnic populations are necessary to clarify the effect of C3435T polymorphism on the multidrug resistance in epilepsy patients.

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.

Binding modes and pharmacophoric features of muscarinic antagonism and ß2 agonism (MABA) conjugates.

ß2 agonists and anticholinergics are two major classes of bronchodilators which form first line of drugs recommended in symptomatic treatment of asthma and COPD. Combinational therapy involving ß agonists and anticholinergics prove more effective in treating airway disease than use of either agent alone. In present investigation, binding modes of Muscarinic Antagonism and ß 2 Agonism (MABA) conjugates designed by Lyn et al. were revealed on structural grounds adopting molecular docking techniques. The conjugates tether ß 2 motif onto M3 motif which makes it a single molecule that acts as both ß 2 agonist and antimuscarinic agent. Series of conjugates were docked against ß 2 adrenergic receptor and modeled M3 muscarinic acetylcholine receptor and pharmacophoric features were identified. Upon screening the conjugates on the basis of receptor ligand free energy, hydrogen bonding and internal electrostatic interaction, conjugate 11 demonstrated superior interactions with the receptors compared to remaining conjugates in the series. While, in vitro results and in silico outcomes are in agreement to reveal that conjugate 11 to possess best pharmacological profile, binding modes obtained in docking can be utilized to design new conjugates with improved biological activity. A close study of receptor residues in binding site and atoms, groups and substructures of conjugates may be used to develop favourable secondary valence forces towards receptor-ligand interactions.

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.

Influence of matrix metalloproteinase-1 gene -1607 (1G/2G) (rs1799750) promoter polymorphism on circulating levels of MMP-1 in chronic pancreatitis.

This study investigated the role of -1607 (1G/2G) (rs1799750) polymorphism of the MMP-1 gene in chronic pancreatitis. We genotyped 100 patients with chronic pancreatitis and 100 control subjects using tetra-primer ARMS-PCR followed by agarose gel electrophoresis. Serum levels of MMP-1 were determined by Elisa. Statistical analysis was applied to test the significance of the results. The genotypic and allelic distribution varied significantly between the disease group and the control subjects [OD = 1.981 (1.236-3.181), p = 0.004]. MMP-1 levels were higher in subjects homozygous for the 2G allele than in subjects with the 1G allele. The present study revealed a significant association of the MMP-1 -1607 1G/2G (rs1799750) gene promoter polymorphism with chronic pancreatitis, and it can be considered a biological marker in the etiology of chronic pancreatitis.

Matrix metalloproteinase-9, transforming growth factor-ß1, and tumor necrosis factor-α plasma levels in chronic pancreatitis.

OBJECTIVE: The aim of the study was to investigate the plasma levels of matrix metalloproteinase-9 (MMP-9), transforming growth factor-ß 1 (TGF-ß1), and tumor necrosis factor-α (TNF-α) in chronic pancreatitis (CP). METHODS: Blood samples were obtained from 71 patients with CP and 100 control subjects, and plasma levels of MMP-9, TGF-ß1, and TNF-α were determined by enzyme-linked immunosorbent assay. RESULTS: The plasma levels of MMP-9 (18.3 ± 3.0 ng/mL, p < 0.0001), TGF-ß1 (215.4 ± 178.1 ng/mL, p = 0.0301), and TNF-α (111.2 ± 69.3 ng/mL, p < 0.001) were significantly elevated in CP compared to the control group. CONCLUSION: The role of elevated plasma MMP-9, TGF-ß1, and TNF-α in CP requires further evaluation.