Single Nucleotide Polymorphisms In Promoter Region Research Articles

Effect of SNPs in the Promoter Region on the Expression of Cytochrome P450 2E1 (CYP2E1) in Pig Liver.

Boar taint, an unfavorable odor in the meat of intact male pigs, is caused primarily by the accumulation of two compounds: androstenone and skatole. This multifactorial trait is regulated by numerous dietary, management and genetic factors. At the mechanistic level, there are many genes known to be involved in boar taint metabolism. Cytochrome P450 2E1 (CYP2E1) impacts boar taint through the phase I metabolism of skatole. The aim of this study was to identify single-nucleotide polymorphisms (SNPs) within the CYP2E1 gene promoter and explore their relationship with the expression of CYP2E1 mRNA and protein. Sequencing of the promoter region using pools of genomic DNA identified seven promoter region SNPs at -159, -586, -1693, -1806, -2322, -2369 and -2514 bp upstream of the ATG start site. Genomic DNA was obtained from 65 boars from the three major swine breeds: Duroc, Landrace and Yorkshire, and individual animals were genotyped for the identified SNPs. RNA was isolated from liver tissue and quantitative PCR was performed to measure CYP2E1 gene expression, while levels of CYP2E1 protein in liver were measured by Western blotting. Significant within-breed variation in CYP2E1 protein and mRNA expression was observed, indicating significant differences in gene expression among individuals. However, levels of CYP2E1 mRNA and protein were not significantly correlated. Two SNPs within the promoter were significantly associated with CYP2E1 mRNA expression, but not with protein expression. This study provides evidence of additional mutations affecting the gene expression of CYP2E1 and suggests that factors that affect the differences in translation of CYP2E1 mRNA may also be important in affecting skatole metabolism.

Association of single nucleotide polymorphisms in ITLN1 gene with ischemic stroke risk in Xi'an population, Shaanxi province.

Ischemic stroke (IS) is the main cause of death and adult disability. However, the pathogenesis of this complicated disease is unknown. The present study aimed to assess the relationship between ITLN1 single nucleotide polymorphisms (SNPs) and the susceptibility to IS in Xi'an population, Shaanxi province. In this study, we designed polymerase chain reaction (PCR) primers located at -3,308 bp upstream of the transcription initiation site within promoter region of the ITLN1 gene. The target fragment was amplified by PCR and identified by agarose gel electrophoresis. Sanger sequencing was then performed in the samples extracted from a cohort comprising 1,272 participants (636 controls and 636 cases), and the obtained sequences were compared with the reference sequences available on the National Center for Biotechnology Information (NCBI) website to detect SNPs in the ITLN1 gene promoter region. Logistic regression analysis was employed to assess the relationship between ITLN1 polymorphisms and IS risk, with adjustments for age and gender. Significant positive results were tested by false-positive report probability (FPRP) and false discovery rate (FDR). The interaction among noteworthy SNPs and their predictive relationship with IS risk were explored using the Multi-Factor Dimensionality Reduction (MDR) software. The results of Sanger sequencing were compared with the reference sequences on the NCBI website, and we found 14 SNPs in ITLN1 gene promoter satisfied Hardy-Weinberg equilibrium (HWE). Logistic regression analysis showed that ITLN1 was associated with a decreased risk of IS (rs6427553: Homozygous C/C: adjusted OR: 0.69, 95% CI [0.48-0.97]; Log-additive: adjusted OR: 0.83, 95% CI [0.70-0.98]; rs7411035: Homozygous G/G: adjusted OR: 0.66, 95% CI [0.47-0.94]; Dominant G/T-G/G: adjusted OR: 0.78, 95% CI [0.62-0.98]; Log-additive: adjusted OR: 0.81, 95% CI [0.69-0.96]; rs4656958: Heterozygous G/A: adjusted OR: 0.74, 95% CI [0.59-0.94]; Homozygous A/A: adjusted OR: 0.51, 95% CI [0.31-0.84]; Dominant G/A-A/A: adjusted OR: 0.71, 95% CI [0.57-0.89]; Recessive A/A: adjusted OR: 0.59, 95% CI [0.36-0.96]; Log-additive: adjusted OR: 0.73, 95% CI [0.61-0.88]), especially in people aged less than 60 years and males. In short, our study revealed a correlation between ITLN1 variants (rs6427553, rs7411035 and rs4656958) and IS risk in Xi'an population, Shaanxi province, laying a foundation for ITLN1 gene as a potential biomarker for predicting susceptibility to IS.

Complex studies on gene polymorphisms of MMP2, MMP3, MMP9 matrix metalloproteinases and TIMP1, TIMP2 tissue inhibitors of metalloproteinases in the patients with primary open-angle glaucoma

Abnormal expression of matrix metalloproteinases (MMP) in watery moisture in patients with glaucoma may affect regulation of intraocular pressure (IOP). MMP activity is regulated by tissue metalloproteinase inhibitors (TIMP). The imbalance between tissue metalloproteinase inhibitors and matrix metalloproteinases may contribute to the development of glaucoma. Genetic factors, including polymorphism of matrix metalloproteinase genes and their inhibitors genes, can regulate the level of their expression, thereby affecting susceptibility to disease. Our aim was to perform comprehensive analysis of the MMP2 (rs243865), MMP3 (rs3025058), MMP9 (rs3918242) polymorphisms, and TIMP1 (rs4898), TIMP2 (rs8179090) tissue inhibitor genes polymorphisms in the patients with stage II (advanced) primary open-angle glaucoma.99 patients (52 men and 47 women) with a verified diagnosis of stage II primary open-angle glaucoma were examined. The comparison group consisted of 100 age-matched persons (81 women and 19 men) without ophthalmic disorders. The single-nucleotide polymorphisms in promoter regions of MMP2, TIMP1, TIMP2 genes were analyzed by the TaqMan method, the MMP3 and MMP9 genes, by means of restriction fragment length polymorphism technique. Statistical evaluation was carried out using the specialized package of IBM SPSS Statistics 23 programs. The critical level of significance was assumed to be 0.05.The differences in the distribution of MMP2 rs243865 allelotypes with decreased frequency of TT genotype were found in the patient group and, vice versa, increased heterozygosity rates were revealed among them. In addition, the frequency of TIMP1 rs4898 heterozygous genotype was decreased in this group as compared to control sample. Four MMP/TIMP complex genotypes are positively associated with the development of pathology. Two of them were of bilocus type, i.e., MMP2-1306TC:TIMP2-418GG, and MMP3-11715A6A:TIMP1 372CC whereas two three-locus constellations were revealed, i.e., MMP2-1306TC:MMP9-1562CC:TIMP2- 418GG, and MMP3-11715A6A:MMP9-1562CC:TIMP1 372CC. There are nine MMP/TIMP complexes, the frequency of which in patients with glaucoma was significantly reduced when compared with control group.Polymorphism of regulatory regions of MMP2, MMP3, MMP9 genes and distinct gene variants of their inhibitors (TIMP1, TIMP2 genes) can be considered potential markers of the POAG development associated with an imbalance of MMP/TIMP activities.

Analyses of P16INK4a gene promoter methylation relative to molecular, demographic and clinical parameters characteristics in non-small cell lung cancer patients: A pilot study.

The aim of this study was to examine the methylation status of p16INK4a promoter region in non small cell lung cancer (NSCLC) patients and their associations with single nucleotide polymorphisms (SNPs) of the epidermal growth factor receptor (EGFR) gene, as well as with demographic or clinical characteristics. Formalin-fixed and paraffin-embedded (FFPE) DNA samples extracted from 22 NSCLC patients were analyzed with methylation-specific polymerase chain reaction (PCR) method to obtain promoter methylation profile. The same cohort was genotyped for - 216G > T, -191C > A, and 181,946C > T EGFR SNPs. There was a significant association between methylated p16INK4a in patients prior therapy (p = 0.017) since a significantly higher frequency of methylated p16INK4a was detected in these patients (40.9%) in comparison to frequency in patients after therapy (31.8%). Also, a higher frequency of methylated p16INK4a was detected among patients with leucopenia (p = 0.056). No associations were observed between the methylation status of the p16INK4a promoter region and EGFR SNPs or other clinical and demographic data in this cohort. High frequency of methylation of the p16INK4a gene promoter was observed in NSCLC patients prior therapy and with leucopenia that can indicate their significance related to advanced clinical stage.

Identifying selection signatures for litter size in Guangxi Bama Xiang pigs.

Litter size is an important economic trait in pig production. However, the genetic mechanisms underlying varying litter size in Guangxi Bama Xiang pigs remain unknown. To identify selection signatures for litter size in Guangxi Bama Xiang pigs, we obtained 297 Illumina PorcineSNP50 BeadChip array data and the average born number (ABN) from parity one to nine in Guangxi Bama Xiang pigs. Fixation index (Fst) methods were used to identify the selection signature of the litter size, and three phenotypic gradient differential population pairs (according to the ABN) in individuals were used to reduce the false positives of signature selections. Single nucleotide polymorphisms (SNPs) were identified in the VEGFA promoter and exons. The general linear model was used to analyse the differences in distinct genotypes after they were typed using three-round multiplex PCR technology. Finally, the transcriptome factor and CpG island in the VEGFA promoter were predicted. A total of 328, 328 and 317 significant loci were identified in the 1st, 2nd and 3rd population pairs, respectively. After removing the false positives, 25 SNPs were defined as the selection signatures in relation to litter size. Ten (VEGFA, USP49, USP25, SRPK1, SLC26A8, RPL10A, PPARD, MAPK14, HMGA1 and CHRDL2) out of 52 genes in the selection regions were annotated as the candidate genes of litter size, respectively, VEGFA. There were no SNPs in the VEGFA exon region, but we obtained three SNPs (rs786889605, rs343769603 and rs323942424) in the VEGFA promoter regions. The ABN in CC was significantly higher than that in TT in rs786889605, and the ABN in TT was significantly lower than that in GG in rs323942424. Meanwhile, the mutation of the VEGFA promoter result in the loss of Sp1 and NF-1 and the formation of Oct-1. In summary, we obtained ten candidate genes, and two mutations in the VEGFA promoter that could be important potential molecular biomarkers for litter size in Bama Xiang pigs.

Genomic and Computational Analysis of Novel SNPs in TNP1 Gene Promoter Region of Bos indicus Breeding Bulls.

Transition nuclear proteins (TNPs), the principal proteins identified in the condensing spermatids chromatin, have been found to play a key role in histone displacement and chromatin condensation during mammalian spermatogenesis. One such gene belonging to the TNP family called TNP1 gene is abundantly expressed in the regulation of spermatogenesis, and its sequence is remarkably well conserved among mammals. Genomic analysis, by sequencing and computational approach, was used to identify the novel polymorphisms and to evaluate the molecular regulation of TNP1 gene expression in Sahiwal cattle breeding bulls. DNA samples were sequenced to identify novel single nucleotide polymorphisms (SNPs) in the TNP1 gene. Modern computational tools were used to predict putative transcription factor binding in the TNP1 promoter and CpG islands in the TNP1 promoter region. In the TNP1 gene, four SNPs, three TATA boxes, and one CAAT box were identified. One CAAT box was discovered at 89 bp upstream of start site ATG. The computational analyses indicated that the polymorphisms inside the promoter sequence results in an added HNF-1 transcription factor binding site. In contrast, the other variations may remove the naturally occurring SRF transcription factor binding site. The CpG islands in the TNP1 promoter region were predicted to be absent by the MethPrimer program before and after SNP site mutations. These findings pave the way for more research into the TNP1 gene's promoter activity and the links between these SNPs and reproductive attributes in the Sahiwal breeding bulls.

Аналіз клінічних, генетичних і гендерних детермінант неефективного лікування в пацієнтів з артеріальною гіпертензією

Актуальність. Поліморфізм промоторної ділянки гену альдостеронової синтази CYP11B2 -344C/T (rs1799998) вiдiграє одну з ключових ролей в модуляції об’єм- і натрій-залежних механізмів артерiальноï гiпертензiï (АГ). Пошук маркерів ефективності антигіпертензивної терапії (АГТ) обумовлений частим недосягненням цільових рівнів АТ незалежно від характеру, дози антигіпертензивних препаратів (АГП), прихильності пацієнта до лікування.Мета. Метою роботи було визначити роль поліморфізму -344С/Т гену CYP11B2 в поєднанні з традиційними факторами ризику (ФР) в контролі артеріального тиску у пацієнтів з АГ ІІ-ІІІ ст.Матеріали та методи. Обстежено 93 пацієнта з АГ ІІ-ІІІ ст. Пацієнти були розділені на 2 групи: 1 гр . (n=38) – пацієнти з цільовим рівнем АТ на тлі АГТ, 2 гр. (n = 55) – пацієнти з АТ≥135/85 мм рт. ст. Всім пацієнтам виконували стандартні клініко-лабораторні дослідження, оцінювали ФР, виконували генотипування методом полімеразної ланцюгової реакції з визначенням довжини рестрикційних фрагментів та визначенням гаплотипу -344С/Т CYP11B2.Результати. Групи пацієнтів з різними рівнями АТ були порiвнюванi за частотою модифікованих ФР, кількісним та якісним складом АГТ. У групі осіб з неконтрольованою АГ частка курців, а також пацієнтів з супутнім цукровим діабетом 2 типу була достовірно вище в порівнянні з пацієнтами 1 гр. Аналіз частот алель них варіантів гену -344С/Т CYP11B2 показав, що частка пацієнтів, що мають Т- монозиготний генотип, була достовірно вище серед пацієнтів 2 гр.Висновок. Наявність Т-алеліі TТ-генотипу -344С/Т CYP11B2 в поєднанні з множинними ФР асоціювалася з недостатнім контролем АТ , незалежно від АГТ.

AgReg-SNPdb: A Database of Regulatory SNPs for Agricultural Animal Species.

Simple SummaryRegulatory SNPs (rSNPs) are SNPs located within promoter regions that have a high potential to alter gene expression by changing the binding affinity of transcription factors to their binding sites. Such rSNPs are gaining importance in the life sciences due to their causality for specific traits and diseases. In this study, we present agReg-SNPdb, the first database comprising rSNP data of seven agricultural and domestic animal species: cattle, pig, chicken, sheep, horse, goat, and dog, and made it usable via a web interface.Transcription factors (TFs) govern transcriptional gene regulation by specifically binding to short DNA motifs, known as transcription factor binding sites (TFBSs), in regulatory regions, such as promoters. Today, it is well known that single nucleotide polymorphisms (SNPs) in TFBSs can dramatically affect the level of gene expression, since they can cause a change in the binding affinity of TFs. Such SNPs, referred to as regulatory SNPs (rSNPs), have gained attention in the life sciences due to their causality for specific traits or diseases. In this study, we present agReg-SNPdb, a database comprising rSNP data of seven agricultural and domestic animal species: cattle, pig, chicken, sheep, horse, goat, and dog. To identify the rSNPs, we constructed a bioinformatics pipeline and identified a total of 10,623,512 rSNPs, which are located within TFBSs and affect the binding affinity of putative TFs. Altogether, we implemented the first systematic analysis of SNPs in promoter regions and their impact on the binding affinity of TFs for livestock and made it usable via a web interface.

Role of C-X-C motif chemokine ligand 14 promoter region DNA methylation and single nucleotide polymorphism in influenza A severity

ObjectiveThe purpose of our experiment is to discuss the function of DNA methylation and single nucleotide polymorphism (SNP) in C-X-C motif chemokine ligand 14 (CXCL14) promoter region in influenza A (H1N1) severity. MethodsClinic data and blood samples from H1N1 patients were collected. Blood routine indexes were measured. Levels of T lymphocytes were assessed. Importantly, CXCL14 expression and methylation in H1N1 patients and A549 cells were detected through functional assays. Additionally, rs2237061, rs2237062 and rs2547 of CXCL14 were genotyped to analyze the relation of CXCL14 SNP and H1N1 severity. ResultsThe number of leukocytes, neutrophils and lymphocytes as well as T lymphocytes in H1N1 patients was lower than that in healthy subjects, and that was decreased in severe H1N1 patients compared with the mild H1N1 patients. In HIN1 patients, CXCL14 expression was decreased, while CXCL14 methylation was increased, and CXCL14 expression was further decreased and CXCL14 methylation was further increased in severe H1N1 patients. CXCL14 methylation was negatively correlated with T lymphocytes in H1N1 patients. CXCL14 methylation was elevated in H1N1-infected A549 cells. GA and AA genotypes of rs2547 in CXCL14 were risky genotypes for H1N1, and AA genotype was risky genotype for severe H1N1. Number of T lymphocytes was lower in H1N1 patients carrying AA genotype of rs2547 than that in GA + GG genotype. ConclusionCXCL14 promoter region DNA methylation and SNP were correlated with H1N1 severity.

Sequence and haplotypes of ankyrin 1 gene (ANK1) and their association with carcass and meat quality traits in yak.

Ankyrin 1 (ANK1) gene has been demonstrated to be a functional candidate gene for meat quality that helps to constitute and maintain the structure of the cell skeleton. In this study, three contiguous ANK1 regions from yak were analyzed using polymerase chain reaction-single-stranded conformational polymorphism (PCR-SSCP). As a result, nine single-nucleotide polymorphisms (SNPs) were identified, four of them in the coding region and three (c.179 C/A, c.250 G/C, and c.313 C/T) putatively resulting in amino acid changes (p. Ala 60 Glu, p. Asp 84 His, and p. Pro 105 Ser). Some SNPs in promoter region were located within or nearby the putative transcription factor binding sites, such as Sp1 and GATA, which might have an impact on the expression of the yak ANK1 gene. The presence of C1-D3 and C1-A3 were associated with an increased hot carcass weight (p = 0.0045) and a decreased drip loss rate (p = 0.0046). The presence of B1-B3, C1-A3 and C1-D3 had decreased Warner-Bratzler shear force (p = 0.0066, p = 0.0343 and p = 0.0004). The presence of one and two copies of B1-B3 and C1-A3 had decreased Warner-Bratzler shear force (p = 0.0005 and p = 0.0443), and C1-A3 had also decreased drip loss rate (p = 0.0164). These findings indicated that genetic variations of the ANK1 gene would be a preferable biomarker for the improvement of yak meat quality.

SIRT1 Gene SNP rs932658 Is Associated With Medication-Related Osteonecrosis of the Jaw

Medication-related osteonecrosis of the jaw (MRONJ) is a rare but serious adverse drug reaction. Our previous whole-exome sequencing study found SIRT1 intronic region single-nucleotide polymorphism (SNP) rs7896005 to be associated with MRONJ in cancer patients treated with intravenous (iv) bisphosphonates (BPs). This study aimed to identify causal variants for this association. In silico analyses identified three SNPs (rs3758391, rs932658, and rs2394443) in the SIRT1 promoter region that are in high linkage disequilibrium (r2 > 0.8) with rs7896005. To validate the association between these SNPs and MRONJ, we genotyped these three SNPs on the germline DNA from 104 cancer patients of European ancestry treated with iv BPs (46 cases and 58 controls). Multivariable logistic regression analysis showed the minor alleles of these three SNPs were associated with lower odds for MRONJ. The odds ratios (95% confidence interval) and p values were 0.351 (0.164-0.751; p = 0.007) for rs3758391, 0.351 (0.164-0.751; p = 0.007) for rs932658, and 0.331 (0.157-0.697; p = 0.0036) for rs2394443, respectively. In the reporter gene assays, constructs containing rs932658 with variant allele A had higher luciferase activity than the reference allele, whereas constructs containing SNP rs3758391 and/or rs2394443 did not significantly affect activity. These results indicate that the promoter SNP rs932658 regulates the expression of SIRT1 and presumably lowers the risk of MRONJ by increasing SIRT1 expression. © 2020 American Society for Bone and Mineral Research (ASBMR).

Functional Study of Polymorphism 1888 C>T in the Promoter Region of Human PLUNC Gene

BackgroundOur previous study showed that a single nucleotide polymorphisms (SNP) of 1888 C>T located at promoter region of human PLUNC gene might affect the susceptibility of nasopharyngeal carcinoma (NPC) in a Chinese population. This study aims to analyze the effect of the genetic variant on PLUNC promoter activity. Materials and methodsThe DNA fragments of the PLUNC promoter region including the SNP 1888 C>T were obtained by polymerase chain reaction (PCR). The recombinant plasmid of the fragment and the pGL3-Enhancer firefly luciferase reporter vector were cloned and identified. Relative luciferase activity (RLA) was measured and electrophoretic mobility shift assay (EMSA) was analyzed. ResultsLuciferase reporter assays demonstrated that luciferase activity of the 1888 T-allele was significantly higher, compared with the C-allele. EMSA experiment proved that the PLUNC gene promoter region SNP 1888 TT genotype had the ability to bind the nucleus protein with the human NPC CEN2 cell, whereas the CC genotype had not. ConclusionsSNP 1888 C>T in the promoter region of PLUNC gene might be a functional mutant locus, indicating that individuals carrying SNP 1888 C-C genotype might be more likely to develop NCP due to the reduced expression of the PLUNC gene. Further functional studies on PLUNC genetic variants are warranted to verify our findings.

Anna A Artaryan: paediatric neurosurgeon who created the first chair in the specialty

<h3>Background</h3> Multiple Sclerosis (MS) is the most common demyelinating disorder. Still today its pathogenesis is not well known, but there are several drugs to slow down disease progression. Interferon-B (IFN-B)...

Promoter region single nucleotide polymorphism of siglec-8 gene associates with susceptibility to allergic asthma.

Aim: Siglec-8 is exclusively expressed on mast cells, eosinophils and basophils. Possible association of six siglec-8 single nucleotide polymorphisms (SNPs) with susceptibility to allergic asthma in the Azeri population of Iran was investigated in this study. Materials & methods: A total of 194 patients and 190 normal subjects were enrolled. PCR single strand conformation polymorphism (PCR-SSCP) was used to determine the genotypes of the studied SNPs. Results: The rs36498 showed significant association with allergic asthma (odds ratio [OR]: 0.65; p=0.022) and the T allele was found as a protective allele (OR: 0.61; p=0.008). Also, eosinophil count in the CC genotype was significantly higher than that in the other genotypes (p=0.026). Conclusion: The rs36498 is thought to influence the expression level of siglec-8. Siglec-8 could be a potential therapeutic target for allergic asthma.

High-throughput functional analysis of lncRNA core promoters elucidates rules governing tissue specificity.

Transcription initiates at both coding and noncoding genomic elements, including mRNA and long noncoding RNA (lncRNA) core promoters and enhancer RNAs (eRNAs). However, each class has a different expression profile with lncRNAs and eRNAs being the most tissue specific. How these complex differences in expression profiles and tissue specificities are encoded in a single DNA sequence remains unresolved. Here, we address this question using computational approaches and massively parallel reporter assays (MPRA) surveying hundreds of promoters and enhancers. We find that both divergent lncRNA and mRNA core promoters have higher capacities to drive transcription than nondivergent lncRNA and mRNA core promoters, respectively. Conversely, intergenic lncRNAs (lincRNAs) and eRNAs have lower capacities to drive transcription and are more tissue specific than divergent genes. This higher tissue specificity is strongly associated with having less complex transcription factor (TF) motif profiles at the core promoter. We experimentally validated these findings by testing both engineered single-nucleotide deletions and human single-nucleotide polymorphisms (SNPs) in MPRA. In both cases, we observe that single nucleotides associated with many motifs are important drivers of promoter activity. Thus, we suggest that high TF motif density serves as a robust mechanism to increase promoter activity at the expense of tissue specificity. Moreover, we find that 22% of common SNPs in core promoter regions have significant regulatory effects. Collectively, our findings show that high TF motif density provides redundancy and increases promoter activity at the expense of tissue specificity, suggesting that specificity of expression may be regulated by simplicity of motif usage.

The -3279C>A and -924A>G polymorphisms in the FOXP3 Gene Are Associated With Viral Load and Liver Enzyme Levels in Patients With Chronic Viral Liver Diseases.

The transcription factor FOXP3 is an essential marker of the development and activation of regulatory T cells (Tregs), which are cells specialized in the regulation and normal tolerance of the immune response. In the context of chronic viral liver diseases, Tregs participate in the maintenance of infections by promoting histopathological control and favor the immune escape of viral agents by suppressing the antiviral response. Single nucleotide polymorphisms (SNPs) may influence the function of FOXP3 in a number of pathological conditions. The present study sought to evaluate the influence of SNPs in the FOXP3 gene promoter region in patients with chronic viral liver diseases. Three SNPs (−3279C>A, −2383C>T, and −924A>G) were analyzed in groups of patients with chronic hepatitis C (CHC), active chronic hepatitis B (CHB-A), inactive chronic hepatitis B (CHB-I), and a healthy control group (CG) using real-time PCR. The frequencies of the polymorphic variants were compared between groups and correlated with liver histopathological characteristics and enzyme levels [i.e., alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl transpeptidase (GGT)] obtained via biopsy and from the clinical records of the participating patients, respectively. For the −2338C>T SNP, no significant differences were found in the frequencies of variants between groups or in the histological findings. Significant associations between the polymorphisms and the CHB-I group were not established. The −3279C>A SNP was associated with altered viral loads (log10) and GGT levels in CHC patients with advanced stages of inflammatory activity and liver fibrosis. The −924A>G SNP was associated with altered viral loads (log10) and liver enzyme levels among CHB-A patients with milder inflammation and fibrosis. However, the frequencies of the −3279C>A and −924A>G polymorphisms were not directly associated with the histopathological profiles of the analyzed patients. These polymorphic variants may influence hepatic function in patients with chronic viral liver diseases but are not directly associated with the establishment of the degree of inflammatory activity and liver fibrosis.

Two novel SNPs in the promoter region of PKR gene in hepatitis C patients and their impact on disease outcome and response to treatment

Background and study aimsThe double-stranded RNA dependent protein kinase (PKR) plays a vital role in the immune system. During HCV infection, PKR has antiviral effect by inhibition of protein synthesis of the HCV. The functional single nucleotide polymorphisms (SNPs) in PKR promoter region might have a relation to HCV disease outcome and response to treatment. The objective of the present work was threefold. First, it proposed an optimized protocol for PCR amplification of PKR promoter. Second, it screened the promoter region of PKR gene in HCV Egyptian patients to detect the possible SNPs’ function. Third, to study the association between the detected SNPs and the response to treatment. Patients and methodsThe functional SNPs in PKR promoter region were detected using DNA sequencing in 40 HCV infected patients; 20 sustained virologic response (SVR) patients and 20 nonresponse (NR) patients after combined interferon/ribavirin therapy. Twenty healthy subjects were included as a control. ResultsTwo functional SNPs were detected: rs62133148T>G and rs12992188C>T within our target PKR promoter region. In rs62133148 polymorphism, there is a significant difference between patients and control subjects for TT and TG genotypes (p < 0.0001). In addition, the G allele is more predominant in HCV patients. In rs12992188 polymorphism, the CC genotype is significantly different between patients and healthy control subjects (OR/95% CI: 0.033/0.006–0.172, p < 0.0001). The presence of C allele was significantly associated with the NR patients (OR/95%CI: 0.25/0.097–0.643, p = 0.006). The TT genotype is significantly different between SVR and NR (OR/95%CI: 8.5/1.54–46.871, p = 0.014). ConclusionThis study is a pioneer clinical study on these two functional SNPs (rs62133148T>G and rs12992188 C>T). The rs62133148 polymorphism does not show any association with response to treatment. The TT genotype in rs12992188 polymorphism shows association with response to treatment. Therefore, patients with TT genotypes were more likely to achieve SVR.

Do Intracerebral Cytokine Responses Explain the Harmful Effects of Dexamethasone in Human Immunodeficiency Virus-associated Cryptococcal Meningitis?

BackgroundThe CryptoDex trial showed that dexamethasone caused poorer clinical outcomes and slowed fungal clearance in human immunodeficiency virus–associated cryptococcal meningitis. We analyzed cerebrospinal fluid (CSF) cytokine concentrations from participants over the first week of treatment to investigate mechanisms of harm and test 2 hypotheses: (1) dexamethasone reduced proinflammatory cytokine concentrations, leading to poorer outcomes and (2) leukotriene A4 hydrolase (LTA4H) genotype influenced the clinical impact of dexamethasone, as observed in tuberculous meningitis.MethodsWe included participants from Vietnam, Thailand, and Uganda. Using the Luminex system, we measured CSF concentrations of the following: interferon γ, tumor necrosis factor (TNF) α, granulocyte-macrophage colony-stimulating factor, monocyte chemoattractant 1, macrophage inflammatory protein 1α, and interleukin 6, 12p70, 8, 4, 10, and 17. We determined the LTA4H genotype based on the promoter region single-nucleotide polymorphism rs17525495. We assessed the impact of dexamethasone on cytokine concentration dynamics and the association between cytokine concentration dynamics and fungal clearance with mixed effect models. We measured the influence of LTA4H genotype on outcomes with Cox regression models.ResultsDexamethasone increased the rate TNF-α concentration’s decline in (−0.13 log2pg/mL/d (95% confidence interval, −.22 to −.06 log2pg/mL/d; P = .03), which was associated with slower fungal clearance (correlation, −0.62; 95% confidence interval, −.83 to −.26). LTA4H genotype had no statistically significant impact on outcome or response to dexamethasone therapy. Better clinical outcomes were associated with higher baseline concentrations of interferon γ.ConclusionsDexamethasone may slow fungal clearance and worsen outcomes by increasing TNF-α concentration’s rate of decline.

Linkage between TIM3 genotype and human HER2 vaccine response in Diversity Outbred (DO) HER2 transgenic (Tg) mice

Abstract Regulatory T cell (Treg) depletion boosts HER2 vaccine response in human HER2 transgenic (Tg) mice. To test whether polymorphisms of Treg-associated genes impact vaccine response, individually distinct F1(DOxB6 HER2 Tg) mice were vaccinated 2× with Adv/HER2 to generate highly variable responses. Single nucleotide polymorphisms (SNP) of 16 candidate genes were analyzed by MonsterPlex sequencing or TaqMan assay, with TIM3 showing an association with vaccine response. There are14 SNPs in the 2kb proximal promoter region and 10 SNPs in a 1kb region containing TIM3 exon 7. Four distinct TIM3 genotypes were identified for either region with one genotype consistently matching between the promoter and exon 7 loci, i.e. the CAST/EiJ strain genotype. Analysis of 84 vaccinated DOxB6 HER2 Tg mice using a tag SNP rs28230613 showed that mice with homozygous CC produce HER2 Ab at 26.9±28.9 μg/mL, and those with heterozygous CA (CAST/Eij genotype) 12.8±19.4 μg/mL (p=0.0075). T cell response was absent in most CA heterozygotes. Relative transcript levels of splenocyte TIM3 over CD4 showed heterozygotes at 2.06±2.27 and homozygotes 0.54±0.51 (p=0.0073, n=24), indicating higher TIM3 transcript in CA heterozygotes. Thus, CAST/EiJ TIM3 genotype is associated with higher TIM3 transcript level and lower vaccine response to self HER2. This correlation is absent in mice depleted of CD25+ Treg, implicating Treg and their TIM3 in regulating HER2 vaccine response. The segregation of TIM3 genotype with vaccine response is not absolute, suggesting additional molecular regulation in HER2 vaccine response. CA76340

CISH promoter polymorphism effects on T cell cytokine receptor signaling and type 1 diabetes susceptibility

BackgroundImpaired regulatory T cell immunity plays a central role in the development of type 1 diabetes (T1D). Interleukin-2 receptor (IL-2R) signaling is essential for regulatory T cells (TREG), and cytokine-inducible SH2-containing protein (CIS) regulates IL-2R signaling as a feedback inhibitor. Previous studies identified association of CISH promoter region single nucleotide polymorphisms (SNPs) with susceptibility to infectious diseases.MethodsHere we analyzed allele frequencies of three CISH SNPs (i.e., rs809451, rs414171, rs2239751) in a study of T1D patients (n = 260, onset age < 5 years, duration > 10 years). Minor allele frequencies were compared to a control cohort of the 1000 Genomes Project. Assigned haplotypes were determined for effects on T1D manifestation and severity. Finally, the CISH haplotype influence on cytokine signaling and function was explored in T cells from healthy donors.ResultsWe detected similar minor allele frequencies between T1D patients and the control cohort. T1D onset age, residual serum C-peptide level, and insulin requirement were comparable between different haplotypes. Only minor differences between the haplotypes were found for in vitro cytokine (i.e., IL-2, IL-7)-induced CIS mRNA expression. STAT5 phosphorylation was induced by IL-2 or IL-7, but no differences were found between the haplotypes. TREG purified from healthy donors with the two most common haplotypes showed similar capacity to inhibit heterologous effector T cells.ConclusionsThis study provides no evidence for an association of CISH promoter SNPs with susceptibility to T1D or severity of disease. In contrast to previous studies, no influence of different haplotypes on CIS mRNA expression or T cell-mediated functions was found.