Maneesh Kumar Misra

Association of HLA‐G promoter and 14‐bp insertion–deletion variants with acute allograft rejection and end‐stage renal disease

The aim of this study was to investigate the HLA-G 14-bp insertion/deletion (I/D) polymorphism among end-stage renal disease (ESRD) patients. Cytomegalovirus (CMV) infection, acute allograft rejection (AR) and overall survival after renal transplantation was investigated in 300 ESRD patients and 302 age, sex and ethnicity-matched controls. Sequencing was performed to evaluate the impact of HLA-G promoter region single-nucleotide polymorphisms (SNPs) whereas semi-quantitative PCR method was used to determine the probable HLA-G expression pattern among ESRD and AR cases. Further, soluble human leukocyte antigen (HLA)-G (sHLA-G) expression levels were compared in AR vs non-AR cases in the light of HLA-G 14-bp I/D polymorphism. Increased risk was found for 14-bp D/D (deletion-DD) genotype and 14-bp D allele [DD: odds ratio (OR) = 1.46, 95% confidence interval (CI) = 1.03-2.06, P value = 0.0358; D: OR = 1.29, 95% CI = 1.03-1.62, P value = 0.0277], respectively for ESRD and CMV infection (DD: OR = 2.70, 95% CI = 1.45-5.05, P value = 0.0021; D: OR = 1.94, 95% CI = 1.22-3.08, P value = 0.0052). Nearly fourfold (OR = 3.62, 95%CI = 1.61-8.14, p = 0.0039) risk was observed for 14-bp I/I (insertion-II) genotype for AR. Survival analysis showed increased overall survival (OS) (AR or death) for 14-bp D/D genotype. HLA-G promoter region sequencing was carried out among 60 ESRD patients and 100 normal controls which showed increased risk for -964 G>A, -725 C>G/T and -486 A>C SNPs. -964 G>A and -725 C>G/T SNPs showed risk association for AR patients. High level of HLA-G transcripts was observed among non-AR patients. Further soluble HLA-G (sHLA-G) showed increased levels in ESRD patients (mean ± SEM; 62.16 ± 2.43 U/ml) as compared to controls (mean ± SEM; 21.06 ± 3.89 U/ml) (P = <0.0001). The 14-bp I/I, 14-bp I/D and 14-bp D/D genotypes showed significantly higher levels of sHLA-G among non-AR as compared to AR patients.

The transcription factor Forkhead Box P3 gene variants affect idiopathic recurrent pregnancy loss

INTRODUCTION Forkhead Box P3 (FOXP3) is an essential transcription factor for the induction and development of Tregs. It plays an important role in regulation and suppression of immune responses. We tested whether FOXP3 gene variants are associated with idiopathic recurrent miscarriages (IRM). METHODS We included 200 women with at least three unexplained spontaneous abortions before twentieth week of gestation and 300 healthy parous women. The detection of genetic variants of rs2232365, and rs5902434 SNPs were carried-out by using polymerase chain reaction (PCR) with sequence-specific primers, while rs3761548 and rs2294021 SNPs were genotyped by PCR followed by RFLP analysis. The logistic odds ratios (ORs) of idiopathic RM risk were estimated with a 95% confidence interval (CI) after maternal age adjustment. Multifactor dimension reduction (MDR) analysis was used to evaluate the potential SNP ∼ SNP interactions. RESULTS Single marker analysis revealed an increased risk ranged from almost 3-fold-2-fold for rs2232365, rs3761548, rs5902434 and rs2294021 SNPs in IRM cases. The mutant haplotype carriers of rs2232365, rs3761548, rs5902434 and rs2294021 SNPs showed an increased risk of 2.5-fold for IRM cases. Linkage disequilibrium analysis revealed moderate LD between rs2232365, rs3761548, rs5902434 and rs2294021 SNPs. The MDR analysis revealed 6-fold increased risk for IRM cases in four factor models of rs2232365, rs3761548, rs5902434 and rs2294021 SNPs. The maximum testing accuracy, highest cross validation consistency and greater significance was observed in four SNP model. DISCUSSION These results suggest that variants of FOXP3 SNPs namely; rs2232365, rs3761548, rs5902434 and rs2294021 may be associated with idiopathic RM.

HLA-G gene expression influenced at allelic level in association with end stage renal disease and acute allograft rejection.

BACKGROUND Human leukocyte antigen (HLA)-G is a non-classical major-histocompatibility complex class-I molecule associated with immunosuppressive function. We have evaluated the impact of HLA-G allele associated with untranslated-region (UTR)-haplotype in end stage renal disease (ESRD) and acute allograft rejection (AR) cases. The mRNA levels of different HLA-G isoforms were evaluated in ESRD and AR cases. Subsequently, the total HLA-G mRNA levels and protein concentration were evaluated against its UTR-haplotype among ESRD and AR cases. METHODOLOGY Sequence based typing of the promoter region was carried-out to evaluate the impact of HLA-G haplotype in 350 ESRD cases and 300 controls. HLA-G gene expression was evaluated at the transcriptional level using semi-quantitative and quantitative PCR, whereas protein concentration was determined by ELISA among both cases and control. RESULTS Increased risk was observed for G*01:01:01:03, G*01:01:02, G*01:06 and G*01:05:N haplotypes while G*01:01:01:01 and G*01:04:01 haplotypes showed a protective effect in ESRD and AR cases. Higher level of soluble HLA-G isoforms (G5 and G6) was observed among ESRD cases. Reduced levels of soluble isoform (G5) and increased levels of membrane bound (G1 and G3) isoforms were found among AR cases, revealing risk association. Decreased HLA-G expression was observed at both mRNA and protein level for G*01:01:01:03 and G*01:05:N haplotypes in ESRD and AR cases. CONCLUSIONS These results suggest that the variation in the expression profile of membrane bound and soluble isoforms may modulate the risk for ESRD and AR. UTR-haplotypes appear to be involved in different HLA-G expression patterns at transcriptional and translational levels.

Association of CTLA-4 Gene Polymorphism with End-Stage Renal Disease and Renal Allograft Outcome

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is upregulated in effector-T-cells after activation that may alter signal transduction and subsequently cytokine production. The present study was designed to investigate the impact of CTLA-4+49 A>G (rs231775), -318 C>T (rs5742909), -658 C>T (rs11571317), -1147 C>T (rs16840252), -1661 A>G (rs4553808), +6230 A>G (rs3087243) SNPs, and microsatellite (AT)n repeat polymorphism among end-stage renal disease (ESRD), acute allograft rejection (AR), and delayed graft function (DGF) cases. In this regard, 350 ESRD patients and 350 controls were included. Polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis method was used for genotyping of CTLA-4 SNPs, while PCR-polyacrylamide gel electrophoresis method was adopted for studying CTLA4 (AT)n polymorphism. The mutant genotype GG of CTLA-4+49A>G,+6230 A>G, and longer alleles of (AT)n repeats polymorphisms were risk associated with ESRD, AR, and DGF cases. The distribution of haplotype+49G:+6230G and GCTTGG (constructed by using 6 studied SNPs) showed risk association for ESRD, DGF, and AR cases. Further, linkage analysis demonstrated strong to moderate linkage disequilibrium in our study populations. The meta-analysis also revealed risk associations for AR cases against GG genotype of CTLA-4+49A>G SNP, while CTLA-4 -318C>T polymorphism showed no correlation against TT genotype among AR cases. Subsequently, no correlation was established against the CTLA-4 -318C>T, -658 C>T, -1147 C>T, and -1661 A>G SNPs in the promoter region. Survival analysis revealed risk associations against GG genotype of CTLA-4+49A>G, +6230 A>G SNPs with overall survival (OS), and higher hazard for the OS. These results suggested that CTLA-4 variants might be involved in susceptibility to ESRD, AR, and DGF.

Association of functional genetic variants of transcription factor Forkhead Box P3 and Nuclear Factor-κB with end-stage renal disease and renal allograft outcome

BACKGROUND The transcription factor FOXP3 and NF-κB regulates the expression of various genes that play an important role in the regulation of renal inflammation. We investigated the association of FOXP3 (rs2232365, rs3761548, rs5902434 and rs2294021) and NF-κB1 (rs28362491 and rs696) gene variants in susceptibility and prognosis of end stage renal disease (ESRD) and renal allograft outcome. METHODS We genotyped four common polymorphisms of FOXP3 and two-tag SNPs of NF-κB1 genes in 350 ESRD cases and 350 controls. Single marker analysis and SNP-SNP interaction model (one to six way combinations) was used for determination of clinical outcome of ESRD and acute rejection episode (ARE). RESULTS We observed significantly higher occurrence of mutant genotypes of tag-SNPs of FOXP3 namely; rs2232365 and rs3761548 along with NF-kB1 namely; rs28362491 and rs696 in ESRD and ARE cases, suggested a risk association for ESRD and ARE. Interestingly, multifactor dimension reduction analysis suggested an increased risks of nearly 6-folds for ESRD and 23-folds for ARE cases under the six factors model which consists of tag-SNPs of FOXP3 (rs2232365, rs3761548, rs5902434 and rs2294021) and NF-kB1 (rs28362491 and rs696). Kaplan-Meier survival analysis showed the lowest overall survival for mutant genotypes compared with wild and heterozygous genotypes of rs2232365 and rs3761548 tag SNPs of FOXP3 as well as NF-kB1 tag-SNPs rs28362491 and rs696 in renal allograft recipients. The crude and adjusted hazard ratios in univariate and multivariate Cox regression models showed almost 2-folds to 3-folds risk for overall survival against mutant genotypes of tag-SNPs of FOXP3 (rs2232365 and rs3761548) and NF-kB1 (rs28362491 and rs696) genes. CONCLUSIONS These results suggest that variants of transcription factor FOXP3 and NF-kB1 might be associated with increased risk to the clinical outcome of ESRD and renal allograft survival.

Genetic variants of MicroRNA-related genes in susceptibility and prognosis of end-stage renal disease and renal allograft outcome among north Indians.

Background and aim MicroRNAs are important molecules of the innate and adaptive immune system, which may play an important role in maintaining normal immune homeostasis. The aim of this study was to investigate the impact of MIR146A C>G (rs2910164), MIR149 T>C (rs2292832), MIR196A2 T>C (rs11614913), and MIR499A A>G (rs3746444) single nucleotide polymorphisms (SNPs) among end-stage renal disease (ESRD) and acute allograft rejection (AR) cases. Materials and methods Genotyping of MicroRNA SNPs was performed using a PCR, followed by restriction fragment length polymorphism in 350 ESRD patients and 350 age-matched, sex-matched, and ethnically matched controls. Results We observed an increased risk of almost two-fold for ESRD and three-fold for AR cases under univariate and multivariate models for mutant genotypes of rs2910164, rs11614913, and rs3746444 SNPs. Subsequently, no susceptible/protective effect was observed for rs2292832 SNP with ESRD and AR cases. Interestingly, all the SNPs that were significant after multiple comparisons in ESRD and AR cases remained significant in the bootstrap analysis, providing internal validation to our initial observations. Survival analysis showed that the mutant genotypes of rs2910164, rs11614913, and rs3746444 SNPs were associated with the lowest overall survival compared with heterozygous and wild genotypes among renal allograft recipients. The crude and adjusted hazard ratios in univariate and multivariate Cox regression models showed an almost two-fold increased risk for overall survival against mutant genotypes of rs2910164, rs11614913, and rs3746444 SNPs in renal allograft recipients. Conclusion These results suggest that the variants of MicroRNA SNPs, namely, rs2910164, rs11614913, and rs3746444, might be involved in susceptibility to ESRD and AR.

The immunogenetics of neurological disease

Genes encoding antigen‐presenting molecules within the human major histocompatibility complex (MHC) account for the highest component of genetic risk for many neurological diseases, such as multiple sclerosis, neuromyelitis optica, Parkinsons disease, Alzheimers disease, schizophrenia, myasthenia gravis and amyotrophic lateral sclerosis. Myriad genetic, immunological and environmental factors may contribute to an individuals susceptibility to neurological disease. Here, we review and discuss the decades long research on the influence of genetic variation at the MHC locus and the role of immunogenetic killer cell immunoglobulin‐like receptor (KIR) loci in neurological diseases, including multiple sclerosis, neuromyelitis optica, Parkinsons disease, Alzheimers disease, schizophrenia, myasthenia gravis and amyotrophic lateral sclerosis. The findings of immunogenetic association studies are consistent with a polygenic model of inheritance in the heterogeneous and multifactorial nature of complex traits in various neurological diseases. Future investigation is highly recommended to evaluate both coding and non‐coding variation in immunogenetic loci using high‐throughput high‐resolution next‐generation sequencing technologies in diverse ethnic groups to fully appreciate their role in neurological diseases.

Genetic associations of killer immunoglobulin like receptors and class I human leukocyte antigens on childhood acute lymphoblastic leukemia among north Indians.

BACKGROUND Molecular interactions between KIRs and their cognate HLA class-I ligands, play a central role in the regulation of natural killer (NK) cell responses in malignancies. We aimed to determine the role of KIR genes and their HLA ligands in genetic predisposition of childhood acute lymphoblastic leukemia (ALL). METHODS Genotyping of 16 KIR genes, along with HLA class-I groups C1/C2 and Bw4 super-type ligands, was carried-out in 137 childhood ALL cases and 274 healthy controls. RESULTS We observed an increased incidence of activating KIRs namely; 2DS2 (OR=2.23, p=<0.001), 2DS3 (OR=1.74, p=0.011), 3DS1 (OR=2.22, p=<0.001), 2DS5 (OR=2.10, p=0.001), 2DS1 (OR=4.42, p=<0.001) and 2DS4 (OR=2.88, p=<0.001) genes in childhood ALL cases compared to controls. Frequency of BB genotype that possess 2-6 activating KIR genes was predominant in cases compared to controls (OR=2.55, p=<0.001). KIR-receptor/HLA-ligand combinations analysis revealed a moderate risk of almost 2-fold for activating KIR-ligand combinations namely; KIR2DS1-HLAC2, KIR2DS2-HLAC1 and KIR3DS1-HLABw4 in childhood ALL cases. CONCLUSION Our data suggests the role for KIR genes and their HLA ligands in aetiology of childhood ALL.

Cytotoxic T-lymphocyte antigen 4 gene polymorphism influences the incidence of symptomatic human cytomegalovirus infection after renal transplantation.

Background The role of CTLA4 gene polymorphisms in T-cell-mediated immunity in association with human cytomegalovirus (HCMV) infection after transplantation is poorly understood. In the present study, we have made an attempt to investigate the impact of CTLA4 single nucleotide polymorphisms (SNPs) (rs231775, rs5742909, rs11571317, rs16840252, rs4553808, rs3087243) and dinucleotide (AT)n repeat polymorphism on the incidence of symptomatic HCMV infection (disease) among 270 renal allograft recipients. Materials and methods Genotyping of CTLA4 SNPs was performed by a PCR, followed by a restriction fragment length polymorphism assay. The detection of the dinucleotide (AT)n repeat polymorphism was carried out by PCR-polyacrylamide gel electrophoresis. Results An almost three-fold increased risk was observed for the incidence of symptomatic HCMV infection in mutant genotype carriers of rs231775 and rs3087243 SNPs under additive and recessive models, respectively. The mutant haplotype carriers of six studied SNPs (rs231775, rs5742909, rs11571317, rs16840252, rs4553808 and rs3087243) showed an almost two-fold higher risk for symptomatic HCMV cases, whereas wild-type haplotype combinations of these six SNPs showed a protective effect. Subsequently, no correlation was observed in the promoter region SNPs of CTLA4, namely, rs5742909, rs11571317, rs16840252 and rs4553808 in symptomatic HCMV cases at the genotypic/allelic level. Survival analysis showed that the mutant genotypes of rs231775 and rs3087243 SNPs were associated with the lowest HCMV disease-free survival compared with heterozygous and wild genotypes. The crude and adjusted hazard ratios showed an almost three-fold and 2.5-fold increased risk in univariate and multivariate Cox regression models, respectively, for HCMV disease-free survival against mutant genotypes of rs231775 and rs3087243 SNPs. CTLA4 dinucleotide (AT)n repeat analysis showed that the smaller allele (102 bp) was associated with a protective effect, whereas the longer (110 and 116 bp) alleles showed a susceptible effect for symptomatic HCMV cases. Conclusion These results suggested that CTLA4 variants might be involved in the clinical manifestation of HCMV diseases.

Genetic variation in Micro-RNA genes of host genome affects clinical manifestation of symptomatic Human Cytomegalovirus infection

BACKGROUND Micro-RNAs are implicated in various physiological and pathologic processes. In this study, we tested whether Micro-RNA gene variants of host-genome affect clinical manifestation of symptomatic HCMV infection. METHODOLOGY HCMV infection was detected by fluorescent PCR and immuno-histochemistry. The detection of genetic variants of four studied Micro-RNA tag-SNPs was done through PCR-RFLP assay and validated with DNA sequencing. RESULTS We observed an increased risk ranged from 3-folds to 5-folds among symptomatic HCMV cases for mutant genotype of rs2910164 (crude OR=3.11, p=0.009 and adjusted OR=3.25, p=0.007), rs11614913 (crude OR=3.20, p=0.006 and adjusted OR=3.48, p=0.004) and rs3746444 (crude OR=4.91, p=0.002 and adjusted OR=5.28, p=0.002) tag-SNPs. Interestingly, all the tag-SNPs that were significant after multiple comparisons at a FDR of 5% in symptomatic HCMV cases remained significant even after bootstrap analysis, providing internal validation to these results. Multifactor Dimensionality Reduction (MDR) analysis revealed 5-folds increased risk for symptomatic HCMV cases under the four-factor model (rs2910164, rs2292832, rs11614913 and rs3746444). CONCLUSIONS These results suggest that Micro-RNA gene variants of host-genome may affect clinical manifestation of symptomatic HCMV infection.