Spherotech-EDTA combined serum treatment reduces background more effectively as compared to One Lambda Adsorb Out™ and LIFECODES Serum Cleaner in Luminex-based solid-phase immunoassays for HLA antibody detection.

Spherotech (SPT) microparticles capture non-specific binding materials present in test serum, and EDTA removes the so called" prozone effect". This study presents a novel approach of combined SPT-EDTA serum treatment prior to Luminex HLA antibody testing to remove high background, and prozone effect in a single step process, and compared the efficacy of SPT-EDTA serum pre-treatment with AdsorbOut (ADS) and Serum Cleaner (SC) to reduce background in solid phase immunoassays (SPI). A total of 21 serum samples with a history of elevated negative control (NC) values ≥500, and 20 samples with normal NC values were included to assess the potential adverse effects. A problem of high background was noted in 25% of our samples in SPI. We observed 80% effectiveness in reducing NC values <500 with SPT-EDTA serum pre-treatment compared to 72%, and 67% for ADS and SC-treated sera, respectively. Interestingly, we found a strong correlation in antibody-binding levels between SPT versus ADS; and ADS versus SC-treated sera for both phenotype and single antigen bead assays (p < 0.001). No adverse effect was noted on NC, positive control (PC) values, PC/NC ratios in the upfront use of SPT-EDTA as compared to EDTA alone. Our data revealed that combined SPT-EDTA treated sera is more effective than ADS, and SC in reducing high background in SPI. Taken together, SPT-EDTA serum treatment prior to Luminex HLA Ab testing is cost-effective, our laboratory saves nearly 30% of the annual total cost for Ab testing and improved test turnaround time by two business days.

Identification of Candidate mRNA Genes and Their Potential MicroRNA Targets in Lung Cancer Induced by Smoking Tobacco.

BACKGROUND: Smoking is considered the single highest risk factor for lung cancer and has been suggested to be associated with accelerated somatic mutations in respiratory mucosa that lead to the development of lung cancer. MicroRNAs serve as modulators in smoking-induced mRNA gene expression changes in the human airway epithelium and are linked to the development of lung cancer. The thermodynamics in the microRNA (miRNA)-mRNA interactions may be affected in tobacco smokers, consequently, leading to phenotypic variations in lung cancer patients. Therefore, this study aimed to investigate the impact of smoking tobacco on somatic mutations in mRNA genes and assess their potential impact on miRNA-mRNA interactions in lung cancers. METHODS: The clinically significant pathogenic variants in mRNA genes in the dataset in lung cancer cases linked to smoking tobacco (n = 330) were obtained from the Cancer Atlas database (TCGA, http://cancergenome.nih.gov/) and used to assess the potential role of tobacco consumption in driving the genetic alterations in proto-oncogenes associated with lung cancer. The analysis of the miRNA interaction with the top five altered mRNA proto-oncogenes in lung cancer cases due to tobacco consumption was performed using the target prediction function in the miRDP program (Database version 5.2.3.1, https://mirdb.org/). RESULTS: We identified the top five mRNA proto-oncogenes enriched with simple somatic mutations (SSM) in lung cancer were TP53, EGFR, KRAS, FAT4, and KMT2D. Interestingly, we observed the highest incidence of SSM in the Tumor Protein p53 (TP53) gene at 63.64%. Similarly, the SSM incidence was 23.94% in the Epidermal Growth Factor Receptor (EGFR), 22.12% in the Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS), 18.48% in the FAT Atypical Cadherin 4 (FAT4), and 14.24% in the Lysine (K)-Specific Methyltransferase 2D (KMT2D) genes. Subsequently, we used a bioinformatics approach to assess the effect of miRNA-mRNA interactions in lung cancer among the top five SSM-enriched mRNA proto-oncogenes. Among the top 20 identified and selected miRNAs, we observed 18 unique microRNAs that bind specifically to TP53, KRAS, and FAT4 genes and 17 and 19 microRNAs that exclusively bind with the EGFR and KMT2D genes, respectively. CONCLUSIONS: Our study found that the top five SSM-enriched mRNA proto-oncogenes in lung cancers among tobacco smokers were TP53, EGFR, KRAS, FAT4, and KMT2D. Further, our results provide an important insight into the involvement of the intricate network of mRNA-miRNA interactions in the development of lung cancer.

Co-stimulatory CD28 and transcription factor NFKB1 gene variants affect idiopathic recurrent miscarriages.

Co-stimulatory CD28 and transcription factor NFKB1 genes are considered as a crucial player in the determination of inflammatory responses; genetic variability in these may modulate the risk for idiopathic recurrent miscarriages (IRM). We investigated the association of functional variants of CD28 (rs3116496 T/C) and NFKB1 (rs28362491 ins/del and rs696 A/G) with IRM cases. We recruited 200 IRM women with a history of at least three consecutive pregnancy losses before 20th week of pregnancy and 300 fertile control women. Determination of CD28 (rs3116496 T/C) and NFKB1 (rs28362491 ins/del and rs696 A/G) gene variants were based on the polymerase chain reaction pursued by restriction fragment length polymorphism analysis and validated with Sanger sequencing. Single marker analysis and multifactor dimensionality reduction (MDR) model used to predict the IRM risk. We observed nearly three- to twofold increased risk in single marker analysis for minor homozygous genotypes of rs3116496 T/C, rs28362491 ins/del and rs696 A/G tag-SNPs in IRM cases, suggesting the risk association. In MDR analysis, we observed 10.5-fold augmented risk among IRM women in three-SNP model (rs3116496 T/C, rs28362491 ins/del and rs696 A/G). The eQTL mapping analyses was performed to strengthen the results of our study. The eQTL mapping analysis revealed that the variations in CD28 and NFKB1 gene content might affect the abundance of transcripts of CD28 and Family with sequence similarity 177 member A1 (FAM177A1) genes, respectively. These results suggest that CD28 and NFKB1 gene variants may be associated with increased risks to IRM.

Association of functional genetic variants of CTLA4 with reduced serum CTLA4 protein levels and increased risk of idiopathic recurrent miscarriages.

OBJECTIVE: To determine whether idiopathic recurrent miscarriages (IRM) are associated with the alteration in serum CTLA4 protein levels and to evaluate their correlation with CTLA4 tag single-nucleotide polymorphisms (SNPs). DESIGN: Retrospective case-control study. SETTING: Tertiary-care referral hospital. PATIENT(S): Three hundred women with IRM (mean age: 28.6 ± 5.4 years) and 600 age-matched (mean age: 29.2 ± 6.8) control women. INTERVENTION(S): Detection of genetic variants of CTLA4 markers rs231775, rs5742909, rs11571317, rs16840252, rs4553808, and rs3087243 by polymerase chain reaction followed by restriction fragment length polymorphism analysis and validated through DNA sequencing, and CTLA4 serum levels measured by enzyme-linked immunosorbent assay. MAIN OUTCOME MEASURE(S): Serum CTLA4 levels, genotypes, and haplotype frequencies compared in IRM cases versus controls. RESULT(S): We observed statistically significantly higher occurrence of minor allele homozygous of rs231775 and rs3087243 tag-SNPs in IRM cases, which suggests a risk association. A statistically significantly reduced level of CTLA4 protein was seen for mutant genotypes of rs231775 and rs3087243 tag-SNPs in women with IRM, revealing a risk association. Serum CTLA4 levels were statistically significantly reduced in women with IRM as compared with the control women. The mutant haplotype carriers of six studied tag-SNPs showed 2.34-fold higher frequencies in IRM cases. In silico analyses strengthened our observations and suggested that variation in CTLA4 gene content may influence the expression of this gene and directly or indirectly influence the function of other genes in the protein-protein interaction pathway. CONCLUSION(S): These results suggest an effect of CTLA4 gene variants, with reduced sCTLA4 secretion and an increased risk for IRM. Reduced CTLA4 secretion and specific CTLA4 variants may contribute to the pathogenesis of IRM.

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 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.

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.

Implication of HLA-G 5' upstream regulatory region polymorphisms in idiopathic recurrent spontaneous abortions.

The effect of HLA-G 5'-upstream regulatory region (URR) single nucleotide polymorphisms (SNP) in idiopathic recurrent spontaneous abortion (RSA) was evaluated. Parental genotype combination analysis and HLA-G expression at transcriptional level was evaluated for 5'URR SNP, which have shown increased risk for idiopathic RSA. If a fetus were aneuploid, attributing causation to a HLA-G 5'-URR SNP would be illogical; therefore couples with abnormal parental karyotypes and also those with abortus material that revealed chromosomal abnormalities were excluded. One hundred women who had experienced idiopathic RSA, along with their respective male partners and 100 pairs of control couples, were studied. HLA-G 5'-URR SNP were evaluated through sequencing. Quantitative polymerase chain reaction was used for HLA-G expression analysis. An increased risk for idiopathic RSA cases among women carriers of mutant genotypes of -1179G>A(rs1233335), -725C>G/T(rs915670) and -486A>C(rs114252012) SNP. The parental genotype combination analysis revealed a 3.5-fold increased risk for -1179G>A and 4.3-fold increased risk for -725C>G/T SNP among carriers of mutant parental genotypes in couples who have experienced idiopathic RSA. Down-regulation in HLA-G expression was seen at transcriptional level for -1179G>A and -725C>G/T SNPs in cases of idiopathic RSA. Transmission of a mutant allele from single carrier parents may, therefore, affect pregnancy outcome.

Platelet-specific collagen receptor glycoprotein VI gene variants affect recurrent pregnancy loss.

OBJECTIVE: To determine whether platelet-specific collagen receptor glycoprotein VI (GP6) gene variants are associated with recurrent miscarriages (RM). DESIGN: Genetic association study. SETTING: Tertiary care referral hospital. PATIENT(S): A total of 200 women with at least three unexplained spontaneous abortions before 20 weeks of gestation and 300 healthy parous women. INTERVENTION(S): Determination of variants of GP6 single-nucleotide polymorphisms (SNPs) namely; rs1671153, rs1654410, rs1654419, and rs1613662 was based on polymerase chain reaction-restriction fragment-length polymorphism. MAIN OUTCOME MEASURE(S): Genotypes and haplotypes frequencies were compared in RM case subjects versus control subjects. RESULT(S): We observed significantly higher occurrence of rare alleles of SNPs in GP6, namely, rs1671153, rs1654410, rs1654419, and rs1613662, among RM cases, revealing risk association for fetal losses. The synergistic effects of haplotype combinations were also evaluated and showed that four haplotypes G-T-G-G, T-C-A-A, G-C-G-A, and G-T-A-A were more prevalent among RM cases, revealing increased risk for fetal losses. In silico analysis revealed that GP6 has an impact on biologic pathways and significant influence in collagen binding. Gene-gene interaction network analysis revealed that GP6 consisted of a total of 25 interactions with 13 genes in the human genome. CONCLUSION(S): These results suggest that variants of GP6 SNPs, namely, rs1671153, rs1654410, rs1654419, and rs1613662, may be associated with risk of recurrent miscarriage. In silico analyses demonstrated the influence of GP6 in biologic pathways, molecular function, including collagen binding, and gene-gene interaction in the human genome.

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 SNP's 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.