UTR Single Nucleotide Polymorphisms Research Articles

Computational analysis of functional single nucleotide polymorphisms associated with the CYP11B2 gene.

Single nucleotide polymorphisms (SNPs) are the most common type of genetic variations in humans and play a major role in the genetics of human phenotype variation and the genetic basis of human complex diseases. Recently, there is considerable interest in understanding the possible role of the CYP11B2 gene with corticosterone methyl oxidase deficiency, primary aldosteronism, and cardio-cerebro-vascular diseases. Hence, the elucidation of the function and molecular dynamic behavior of CYP11B2 mutations is crucial in current genomics. In this study, we investigated the pathogenic effect of 51 nsSNPs and 26 UTR SNPs in the CYP11B2 gene through computational platforms. Using a combination of SIFT, PolyPhen, I-Mutant Suite, and ConSurf server, four nsSNPs (F487V, V129M, T498A, and V403E) were identified to potentially affect the structure, function, and activity of the CYP11B2 protein. Furthermore, molecular dynamics simulation and structure analyses also confirmed the impact of these nsSNPs on the stability and secondary properties of the CYP11B2 protein. Additionally, utilizing the UTRscan, MirSNP, PolymiRTS and miRNASNP, three SNPs in the 3′UTR region were predicted to exhibit a pattern change in the upstream open reading frames (uORF), and eight microRNA binding sites were found to be highly affected due to 3′UTR SNPs. This cataloguing of deleterious SNPs is essential for narrowing down the number of CYP11B2 mutations to be screened in genetic association studies and for a better understanding of the functional and structural aspects of the CYP11B2 protein.

Polymorphisms in human heat shock factor-1 and analysis of potential biological consequences.

The stress-activated transcription factor, heat shock factor-1 (HSF1), regulates many genes including cytoprotective heat shock proteins (HSPs). We hypothesized that polymorphisms in HSF1 may alter the level or function of HSF1 protein accounting for interindividual viability in disease susceptibility or prognosis. We searched for exomic variants in HSF1 by querying human genome databases and directly sequencing DNA from 80 anonymous genomic DNA samples. Overall, HSF1 sequence was highly conserved, with no common variations. We found 31 validated deviations from a reference sequence in the dbSNP database and an additional 5 novel variants by sequencing, with allele frequencies that were 0.06 or less. Of these 36, 2 were in 5'-untranslated region (5'UTR), 10 in 3'UTR, and 24 in the coding region. The potential effects of 5'UTR on secondary structure, protein structure/function, and 3'UTR targets of microRNAs were analyzed using RNAFold, PolyPhen-2, SIFT, and MicroSNiper. One of the 5'UTR variants was predicted to strengthen secondary structure. Eight of 3'UTR variants were predicted to modify microRNA target sequences. Eight of the coding region variants were predicted to modify HSF1 structure/function. Reducing HSF1 levels in A549 cells using short hairpin RNA (shRNA) increased sensitivity to heat-induced killing demonstrating the impact that genetic variants that reduce HSF1 levels might have. Using the pmirGLO expression system, we found that the wild-type HSF1 3'UTR suppressed translation of a firefly luciferase reporter plasmid by 65%. Introducing two of four 3'UTR single nucleotide polymorphisms (SNPs) increased HSF1 3'UTR translational suppression by 27-44% compared with the wild-type HSF1 3'UTR sequence while a third SNP reduced suppression by 25%. HSF1 variants may alter HSF1 protein levels or function with potential effects on cell functions, including sensitivity to stress.

Genome at juncture of early human migration: a systematic analysis of two whole genomes and thirteen exomes from Kuwaiti population subgroup of inferred Saudi Arabian tribe ancestry.

Population of the State of Kuwait is composed of three genetic subgroups of inferred Persian, Saudi Arabian tribe and Bedouin ancestry. The Saudi Arabian tribe subgroup traces its origin to the Najd region of Saudi Arabia. By sequencing two whole genomes and thirteen exomes from this subgroup at high coverage (>40X), we identify 4,950,724 Single Nucleotide Polymorphisms (SNPs), 515,802 indels and 39,762 structural variations. Of the identified variants, 10,098 (8.3%) exomic SNPs, 139,923 (2.9%) non-exomic SNPs, 5,256 (54.3%) exomic indels, and 374,959 (74.08%) non-exomic indels are ‘novel’. Up to 8,070 (79.9%) of the reported novel biallelic exomic SNPs are seen in low frequency (minor allele frequency <5%). We observe 5,462 known and 1,004 novel potentially deleterious nonsynonymous SNPs. Allele frequencies of common SNPs from the 15 exomes is significantly correlated with those from genotype data of a larger cohort of 48 individuals (Pearson correlation coefficient, 0.91; p <2.2×10−16). A set of 2,485 SNPs show significantly different allele frequencies when compared to populations from other continents. Two notable variants having risk alleles in high frequencies in this subgroup are: a nonsynonymous deleterious SNP (rs2108622 [19:g.15990431C>T] from CYP4F2 gene [MIM:*604426]) associated with warfarin dosage levels [MIM:#122700] required to elicit normal anticoagulant response; and a 3′ UTR SNP (rs6151429 [22:g.51063477T>C]) from ARSA gene [MIM:*607574]) associated with Metachromatic Leukodystrophy [MIM:#250100]. Hemoglobin Riyadh variant (identified for the first time in a Saudi Arabian woman) is observed in the exome data. The mitochondrial haplogroup profiles of the 15 individuals are consistent with the haplogroup diversity seen in Saudi Arabian natives, who are believed to have received substantial gene flow from Africa and eastern provenance. We present the first genome resource imperative for designing future genetic studies in Saudi Arabian tribe subgroup. The full-length genome sequences and the identified variants are available at ftp://dgr.dasmaninstitute.org and http://dgr.dasmaninstitute.org/DGR/gb.html.

Genetic variants may play an important role in mRNA-miRNA interaction: evidence for haplotype-dependent downregulation of ABCC2 (MRP2) by miRNA-379.

The functional influence of single-nucleotide polymorphisms (SNPs) of the ATP-binding cassette (ABC) transporter ABCC2 (MRP2) has been characterized in numerous studies. The aim of this study was to address the question of whether distinct ABCC2 haplotypes, which differ in their mRNA secondary structures, show an influence on the degree of mRNA and protein downregulation through miRNA interaction. A model using human peripheral blood monocytic cells (PBMCs) isolated from healthy Caucasian volunteers, with three defined ABCC2 haplotypes comprising the 5'-UTR SNP -24C>T, the 1249G>A SNP (V417I), and the silent 3972C>T SNP, was outlined. Cells were transiently transfected with miRNA-379, already known to target ABCC2 in HepG2 cells. ABCC2 was downregulated through miR-379 in a haplotype-dependent manner: the wild-type CGC/CGC was modestly affected (mRNA: -12.7±4.2%, protein: -9.9±0.1%), whereas variant haplotypes were more strongly suppressed: CGT/CGT (mRNA: -36.7±2.4%, protein: -21.6±0.4%) and TGT/TGT (mRNA: -55.7±1.2%, protein: -46.3±4.0%). In addition, glutathione-methylfluorescein efflux was significantly reduced in miR-379-transfected peripheral blood monocytic cells corresponding to ABCC2 protein expression. This observation may suggest a differential suppression of ABCC2 by miR-379 caused by haplotype-dependent differences in mRNA secondary structures, resulting in changes in mRNA target accessibility or mRNA stability.

Common genetic variation in the glucokinase gene (GCK) is associated with type 2 diabetes and rates of carbohydrate oxidation and energy expenditure.

Aims/hypothesisGlucokinase (GCK) plays a role in glucose metabolism and glucose-stimulated insulin secretion. Rare mutations in GCK cause MODY. We investigated whether common variation (minor allele frequency ≥0.01) in GCK is associated with metabolic traits and type 2 diabetes.MethodsFour exonic single-nucleotide polymorphisms (SNPs) and three SNPs predicted to cause loss of promoter function were identified in whole-genome sequence data from 234 Pima Indians. These seven tag SNPs and rs4607517, a type 2 diabetes variant established in other studies, were analysed in 415 full-heritage non-diabetic Pima Indians characterised for metabolic traits, and 7,667 American Indians who had data on type 2 diabetes and BMI.ResultsA novel 3′ untranslated region (3′UTR) SNP, chr7:44184184-G/A, was associated with the rate of carbohydrate oxidation post-absorptively (β = 0.22 mg [kg estimated metabolic body size (EMBS)]−1 min−1, p = 0.005) and during a hyperinsulinaemic–euglycaemic clamp (β = 0.24 mg [kg EMBS]−1 min−1, p = 0.0002), the rate of carbohydrate oxidation in a respiratory chamber (β = 311 kJ/day, p = 0.03) and 24 h energy expenditure, which was attributable to the thermic effect of food (β = 520 kJ/day, p = 3.39 × 10−6). This 3′UTR SNP was also associated with diabetes (OR 1.36, 95% CI 1.11, 1.65, p = 0.002), where the A allele (allele frequency 0.05) was associated with a lower rate of carbohydrate oxidation, lower 24 h energy expenditure and higher risk for diabetes. In a Cox proportional hazards model, a rate of insulin-stimulated carbohydrate oxidation lower than the mean rate at baseline predicted a higher risk for developing diabetes than for those above the mean (hazard rate ratio 2.2, 95% CI 1.3, 3.6, p = 0.002).Conclusions/interpretationCommon variation in GCK influences the rate of carbohydrate oxidation, 24 h energy expenditure and diabetes risk in Pima Indians.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-014-3234-8) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

(369) - Candidate Gene Association Study in BOS

Bronchiolitis obliterans syndrome (BOS), a form of chronic lung allograft dysfunction, is the primary factor limiting long-term survival for lung transplant recipients. We sought to identify genetic variants in lung transplant recipients associated with increased risk of BOS. We performed a large-scale candidate gene association study utilizing recipients from the multicenter, prospective Lung Transplant Outcomes Group cohort enrolled between 2002-2011. The primary outcome was defined as time from transplant to BOS grade 1, conditional on surviving 180 days after transplant. Genotyping was performed using the Illumina HumanCVD BeadChip v2, containing SNPs from cardiovascular, metabolic and inflammatory gene loci. Cox proportional hazards models were used to test the association between each locus and time to BOS, in an additive mode, adjusting for recipient age, predisposing diagnosis, transplant type, and population admixture. A significance threshold of p<1x10-4 was utilized based on the candidate gene design of the chip. 695 subjects were included in the analysis, 159 of whom developed BOS. Median follow-up time was 914 days (IQR 526, 1495) with median time to BOS=696 days (IQR 376, 1175). Four single nucleotide polymorphisms (SNPs) were significantly associated with time to BOS. The minor allele at rs34926152 in activated leukocyte cell adhesion molecule (ALCAM; HR=2.9, p=2.2x10-6), a gene associated with tumor aggressiveness, is a coding non-synonomous variant. One 5’ UTR SNP in the gene encoding resistin like beta (RETNLB), a gene significantly associated with asthma and airway remodeling, was significantly associated with a lower risk of BOS (HR=0.5, p=4.0x10-5). SNPs in 4 genes were significantly associated with time to BOS in a multicenter cohort study. Variants in 2 genes, RETNLB and C3, are known to be related to airways disease and asthma. Important future directions will involve validating these findings and investigating the functional implications of these variants in lung transplant recipients.

MrSNP: Software to detect SNP effects on microRNA binding

BackgroundMicroRNAs (miRNAs) are short (19-23 nucleotides) non-coding RNAs that bind to sites in the 3’untranslated regions (3’UTR) of a targeted messenger RNA (mRNA). Binding leads to degradation of the transcript or blocked translation resulting in decreased expression of the targeted gene. Single nucleotide polymorphisms (SNPs) have been found in 3’UTRs that disrupt normal miRNA binding or introduce new binding sites and some of these have been associated with disease pathogenesis. This raises the importance of detecting miRNA targets and predicting the possible effects of SNPs on binding sites. In the last decade a number of studies have been conducted to predict the location of miRNA binding sites. However, there have been fewer algorithms published to analyze the effects of SNPs on miRNA binding. Moreover, the existing software has some shortcomings including the requirement for significant manual labor when working with huge lists of SNPs and that algorithms work only for SNPs present in databases such as dbSNP. These limitations become problematic as next-generation sequencing is leading to large numbers of novel variants in 3’UTRs.ResultIn order to overcome these issues, we developed a web-server named mrSNP which predicts the impact of a SNP in a 3’UTR on miRNA binding. The proposed tool reduces the manual labor requirements and allows users to input any SNP that has been identified by any SNP-calling program. In testing the performance of mrSNP on SNPs experimentally validated to affect miRNA binding, mrSNP correctly identified 69% (11/16) of the SNPs disrupting binding.ConclusionsmrSNP is a highly adaptable and performing tool for predicting the effect a 3’UTR SNP will have on miRNA binding. This tool has advantages over existing algorithms because it can assess the effect of novel SNPs on miRNA binding without requiring significant hands on time.

Association between polymorphisms in the thymidylate synthase gene and risk of breast cancer in a Mexican population.

Breast cancer (BC) is the leading cause of cancer-related deaths among women in Mexico. Two single-nucleotide polymorphisms (SNPs) in the thymidylate synthase (TS) gene, the 28-base pair (bp) tandem repeat in the TS 5'-untranslated enhanced region (TSER) and the 6-bp insertion/deletion in the TS 3'-untranslated region (TS 3'-UTR), increase the rate of misincorporation of uridylate into DNA and may lead to chromosomal damage. We examined the association between these polymorphisms and BC risk in Mexican women according to menopause status. Mexican patients with initial BC diagnosis (N = 230) and 145 individuals from a reference general population group (RGP) were included. For statistical analysis, the BC group was divided into pre- and post-menopause groups (PRE and POST groups, respectively). We analyzed both TS polymorphisms (TSER and TS 3'-UTR) using polymerase chain reaction. Finetti analysis was used to evaluate inter-and intra-group differences. The results showed a high frequency for the 3R and ins6 alleles in the BC, RGP, PRE, and POST groups. No significant differences were observed for the TS and TSER genotype and allele frequency distributions between groups. We found that the TSER and TS 3'-UTR SNPs are not associated with BC risk in Mexican patients.

Genetic variants in C‐type lectin genes are associated with colorectal cancer susceptibility and clinical outcome

Inflammatory responses play a vital role at different stages of colorectal carcinogenesis. C-type lectins mediate inflammatory/immune responses and participate in immune escape of pathogens and tumors. Our study aimed to evaluate the correlation between polymorphisms in three C-type lectin genes, CD209, MBL2 and REG4, and colorectal cancer (CRC) risk and clinical outcome. We genotyped 15 potentially functional single nucleotide polymorphisms (SNPs) and assessed their associations with CRC risk in a case-control study of 1353 CRC cases and 767 healthy controls from the Czech Republic. We also analyzed these SNPs in relation to overall and event-free survival in 414 patients. Two CD209 SNPs were associated with CRC risk after adjustment for multiple comparison. Minor allele carriers of the promoter SNP rs2287886 had an increased risk of CRC (OR 1.30, 95% CI 1.08-1.56), while minor allele carriers of the 3'UTR SNP, rs7248637, had a decreased risk (OR 0.74, 95% CI 0.60-0.91). Multivariate survival analyses, including age, gender, TNM stage and grade, showed that patients without distant metastasis at the time of diagnosis and carrying the rs2994809 T allele had a decreased overall and event-free survival (HR 2.11, 95% CI 1.20-3.72 and HR 2.00, 95% CI 1.18-3.39, respectively). We show that SNPs in CD209 may affect CRC risk, while a SNP in REG4 may be a useful marker for CRC progression.

Germline hereditary, somatic mutations and microRNAs targeting-SNPs in congenital heart defects

Somatic mutations and dysregulation by microRNAs (miRNAs) may have a pivotal role in the Congenital Heart Defects (CHDs). The purpose of the study was to assess both somatic and germline mutations in the GATA4 and NKX2.5 genes as well as to identify 3′UTR single nucleotide polymorphisms (SNPs) in the miRNA target sites. We enrolled 30 patients (13 males; 13.4±8.3years) with non-syndromic CHD. GATA4 and NKX2.5 genes were screened in cardiac tissue of sporadic and in blood samples of familial cases. Computational methods were used to detect putative miRNAs in the 3′UTR region and to assess the Minimum Free Energy of hybridization (MFE, kcal/mol). Difference of MFEs (ΔMFE) ≥4kcal/mol between alleles was considered biologically relevant on miRNA binding. The sum of all ΔMFEs (|ΔMFEtot|=∑|ΔMFE|) was calculated in order to predict the biological importance of SNPs binding more miRNAs. No evidence of novel GATA4 and NKX2.5 mutations was found both in sporadic and familial patients. Bioinformatic analysis revealed 27 putative miRNAs binding to identified SNPs in the 3′UTR of GATA4. ΔMFE ≥4kcal/mol between alleles was obtained for the +354A>C (miR-4299), +587A>G (miR-604), +1355G>A (miR-548v, miR-139-5p) and +1521C>G (miR-583, miR-3125, miR-3928) SNPs. The +1521C>G SNP showed the highest ΔMFEtot (21.66kcal/mol). Luciferase reporter assays indicated that miR-583 was dose-dependently effective in regulating +1521 C allele compared with +1521 G allele. Based on the analysis of 100 CHD cases and 204 healthy newborns, the +1521 G allele was also associated with a lower risk of CHD (OR=0.5, 95% CI 0.3–0.9, p=0.03), likely due to the relatively low binding of the miRNA and high levels of protein. These results suggest that common SNPs in the 3′UTR of GATA4 alter miRNA gene regulation contributing to the pathogenesis of CHDs.

A miR-151 binding site polymorphism in the 3′-untranslated region of the cyclin E1 gene associated with nasopharyngeal carcinoma

Genetic alterations in nasopharyngeal carcinoma (NPC) have been reported in previous works. However, it remains unclear whether polymorphisms within the miRNA-target binding sites are associated with individual NPC risk. In this study, new experimental and computational approaches were developed to assess the polymorphism frequency distribution within the miRNA sites in NPC patients, and to explore its association with NPC risk. We focused on 220 single-nucleotide polymorphisms (SNPs) in the 3′-untranslated regions (3′UTRs) of 32 genes carrying putative miRNA-binding sites by specialized algorithms. A total of 9 candidate genes were selected for further investigation, which were reportedly overexpressed in NPC, including EGFR, COX2, CCNE1, hTERT, MMP2, MMP9, NF-κB VEGF, and WNT3. SNPs in 3′UTRs were genotyped by direct polymerase chain reaction sequencing of the genomic DNA of 24 cases and 24 controls. Then, EGFR rs884225, CCNE1 rs3218073, and MMP2 rs7201 were screened with large samples. Based on the analysis of a series of 167 NPC cases and 171 controls from Guangdong Province, statistically significant associations were found between NPC risk and variant genotypes of CCNE1 rs3218073 for TC+TT (OR=1.585; 95% CI=1.023–2.458; P=0.046) and for T-allele (OR=1.464; 95% CI=1.012–2.118; P=0.042). In addition, a significant association among rs3218073 genotype TC (OR=1.959, P=0.043), T-allele (OR=2.123, P=0.006), and primary tumor (T3–T4) was retrieved. Genotype TC (OR=1.959, P=0.043) and T-allele (OR=2.123, P=0.006) of rs3218073 were correlated with increased risk of higher NPC stage (III to IV). In support of the postulation that the 3′UTR SNP directly affected miRNA-binding site, luciferase reporter assay indicated that CCNE1 was a direct target of miR-151, and the rs3218073 T>C change resulted in altered regulation of CCNE1 expression. By contrast, no statistically significant association with NPC risk was found for MMP2 rs7201 and EGFR rs884225 polymorphisms (P>0.05). In conclusion, our data demonstrate that CCNE1 rs3218073 polymorphism located at miRNA-151 binding site is associated with NPC susceptibility and is correlated with NPC stage. These results suggest that CCNE1 rs3218073 polymorphism can be exploited as a novel biomarker for future NPC diagnosis and prognosis.

Refining theUGT1AHaplotype Associated with Irinotecan-Induced Hematological Toxicity in Metastatic Colorectal Cancer Patients Treated with 5-Fluorouracil/Irinotecan-Based Regimens

Despite the importance of UDP-glucuronosyltransferase (UGT) 1A1*28 in irinotecan pharmacogenetics, our capability to predict drug-induced severe toxicity remains limited. We aimed at identifying novel genetic markers that would improve prediction of irinotecan toxicity and response in advanced colorectal cancer patients treated with folic acid (leucovorin), fluorouracil (5-FU), and irinotecan (camptosar)-based regimens. The relationships between UGT1A candidate markers across the gene (n = 21) and toxicity were prospectively evaluated in 167 patients. We included variants in the 3'untranscribed region (3'UTR) of the UGT1A locus, not studied in this context yet. These genetic markers were further investigated in 250 Italian FOLFIRI-treated patients. Several functional UGT1A variants, including UGT1A1*28, significantly influenced risk of severe hematologic toxicity. As previously reported in the Italian cohort, a 5-marker risk haplotype [haplotype II (HII); UGTs 1A9/1A7/1A1] was associated with severe neutropenia in our cohort [odds ratio (OR) = 2.43; P = 0.004]. The inclusion of a 3'UTR single-nucleotide polymorphism (SNP) permitted refinement of the previously defined HI, in which HIa was associated with the absence of severe neutropenia in combined cohorts (OR = 0.55; P = 0.038). Among all tested UGT1A variations and upon multivariate analyses, no UGT1A1 SNPs remained significant, whereas three SNPs located in the central region of UGT1A were linked to neutropenia grade 3-4. Haplotype analyses of these markers with the 3'UTR SNP allowed the identification of a protective HI (OR = 0.50; P = 0.048) and two risk haplotypes, HII and HIII, characterized by 2 and 3 unfavorable alleles, respectively, revealing a dosage effect (ORs of 2.15 and 5.28; P ≤ 0.030). Our results suggest that specific SNPs in UGT1A, other than UGT1A1*28, may influence irinotecan toxicity and should be considered to refine pharmacogenetic testing.

MirSNP, a database of polymorphisms altering miRNA target sites, identifies miRNA-related SNPs in GWAS SNPs and eQTLs.

BackgroundNumerous single nucleotide polymorphisms (SNPs) associated with complex diseases have been identified by genome-wide association studies (GWAS) and expression quantitative trait loci (eQTLs) studies. However, few of these SNPs have explicit biological functions. Recent studies indicated that the SNPs within the 3’UTR regions of susceptibility genes could affect complex traits/diseases by affecting the function of miRNAs. These 3’UTR SNPs are functional candidates and therefore of interest to GWAS and eQTL researchers.DescriptionWe developed a publicly available online database, MirSNP (http://cmbi.bjmu.edu.cn/mirsnp), which is a collection of human SNPs in predicted miRNA-mRNA binding sites. We identified 414,510 SNPs that might affect miRNA-mRNA binding. Annotations were added to these SNPs to predict whether a SNP within the target site would decrease/break or enhance/create an miRNA-mRNA binding site. By applying MirSNP database to three brain eQTL data sets, we identified four unreported SNPs (rs3087822, rs13042, rs1058381, and rs1058398), which might affect miRNA binding and thus affect the expression of their host genes in the brain. We also applied the MirSNP database to our GWAS for schizophrenia: seven predicted miRNA-related SNPs (p < 0.0001) were found in the schizophrenia GWAS. Our findings identified the possible functions of these SNP loci, and provide the basis for subsequent functional research.ConclusionMirSNP could identify the putative miRNA-related SNPs from GWAS and eQTLs researches and provide the direction for subsequent functional researches.

Two single nucleotide polymorphisms in the human nescient helix-loop-helix 2 (NHLH2) gene reduce mRNA stability and DNA binding

Nescient helix-loop-helix-2 (NHLH2) is a basic helix-loop-helix transcription factor, which has been implicated, using mouse knockouts, in adult body weight regulation and fertility. A scan of the known single nucleotide polymorphisms (SNPs) in the NHLH2 gene revealed one in the 3′ untranslated region (3′UTR), which lies within an AUUUA RNA stability motif. A second SNP is nonsynonymous within the coding region of NHLH2, and was found in a genome-wide association study for obesity. Both of these SNPs were examined for their effect on NLHL2 by creating mouse mimics and examining mRNA stability, and protein function in mouse hypothalamic cell lines. The 3′UTR SNP causes increased instability and, when the SNP-containing Nhlh2 3′UTR is attached to luciferase mRNA, reduced protein levels in cells. The nonsynonymous SNP at position 83 in the protein changes an alanine residue, conserved in NHLH2 orthologs through the Drosophila sp. to a proline residue. This change affects migration of the protein on an SDS-PAGE gel, and appears to alter secondary structure of the protein, as predicted using in silico methods. These results provide functional information on two rare human SNPs in the NHLH2 gene. One of these has been linked to human obese phenotypes, while the other is present in a relatively high proportion of individuals. Given their effects on NHLH2 protein levels, both SNPs deserve further analysis in whether they are causative and/or additive for human body weight and fertility phenotypes.

Genotypes and haplotypes in the 3′ untranslated region of the HLA‐G gene and their association with clinical outcome of hematopoietic stem cell transplantation for beta‐thalassemia

Polymorphisms in the 3' untranslated region (3'UTR) of HLA-G, an important player in immunological tolerance, could be involved in post-transcriptional expression control, and their association with different clinical immune-related conditions including autoimmunity and transplantation is of mounting interest. Most studies have focused on a 14 base pair (bp) insertion/deletion (ins/del), while additional single-nucleotide polymorphisms (SNPs) in the HLA-G 3'UTR have been described but not extensively investigated for their clinical relevance. Here we have comparatively studied the association between 3'UTR haplotypes of HLA-G, or the 14 bp ins/del, with clinical outcome of HLA-identical sibling hematopoietic stem cell transplantation (HSCT) in 147 Middle Eastern beta-thalassemia patients. Sequence based typing of 3'UTR HLA-G polymorphisms in the patients and in 102 healthy Italian blood donors showed strong linkage disequilibrium between the 14 bp ins/del and five 3'UTR SNPs, which together could be arranged into eight distinct haplotypes based on expectation-maximization studies, with four predominant haplotypes (UTRs1-4). After HSCT, we found a moderate though not significant association between the presence of UTR-2 in double dose and protection from acute graft versus host disease (hazard ratio (HR) 0.45, 95% confidence intervals (CI): 0.14-1.45; P = 0.18), an effect that was also seen when the corresponding 14 bp ins/ins genotype was considered alone (HR 0.42, 95% CI: 0.16-1.06; P = 0.07). No association was found with rejection or survival. Taken together, our data show that there is no apparent added value of considering entire 3'UTR HLA-G haplotypes for risk prediction after allogeneic HSCT for beta-thalassemia.

Role of SLC10A1 SNPs in regulating cytochrome P450 expression

Cytochrome's P450 (CYPs) are a group of enzymes that have a major role in metabolizing drugs, and their variable expression can lead to pharmacokinetic and pharmacodynamic differences within the population. Genetic variants in CYPs have been extensively investigated, but they only explain a part of the interindividual variation in hepatic CYP expression. A recent study has proposed SLC10A1, a bile acid uptake carrier, to be a novel regulator of CYP expression. Hence, we hypothesized that functional SNPs (single nucleotide polymorphisms) in SLC10A1 may have an effect on CYP expression. Towards this end we resequenced all exons, promoter, and 3′‐UTR of SLC10A1 in 98 human livers (43 over‐ and 55 under‐expressing) to look for functional SNPs. We found a total of eight SNPs. An exon 1 synonymous SNP (rs4646285) occurred with a higher frequency in livers overexpressing SLC10A1, whereas a 3′‐UTR SNP (rs45593332) occurred with a higher frequency in the livers underexpressing SLC10A1. The genotypes of rs4646285 correlated with total mRNA of SLC10A1 as well as the downstream target CYP3A4 (p=0.02). An allelic expression imbalance assay demonstrated samples having imbalanced SLC10A1 expression, although none of the SLC10A1 SNPs explained the cause of this imbalance. In conclusion, an exonic SLC10A1 SNP led to increased SLC10A1 mRNA expression and increased CYP3A4 expression and may help explain some of the variation in the pharmacokinetics of CYP3A4 substrate drugs. This work was funded by the Pediatric Oncology Education (POE) summer program at St. Jude Children's Hospital (O.V.).

A 3′ UTR SNP in COL18A1 Is Associated with Susceptibility to HBV Related Hepatocellular Carcinoma in Chinese: Three Independent Case-Control Studies

BackgroundAccumulated evidences indicate that single nucleotide polymorphisms (SNP) in angiogenesis and tumorigenesis related genes are associated with risk of Hepatocellular carcinoma (HCC). COL18A1 encodes the precursor of endostatin, which is a broad-spectrum angiogenesis inhibitor, and we speculate that SNPs in COL18A1 may be associated with susceptibility to HCC.Methods and FindingsWe carried out a 2-stage association study in 3 independent case-control groups in a total of 1067 chronic hepatitis B (CHB) patients and 808 hepatitis B virus (HBV) related HCC patients in Han Chinese. Four SNPs which can represent all potential functional SNPs with MAF>0.1 recorded in HapMap database were genotyped using TaqMan methods. Levels of total COL18A1 mRNA were also examined using quantitative real-time RT-PCR. We found that rs7499 located in 3′-UTR to be strongly associated with HBV related HCC (Pcombined = 0.0000005, OR = 0.72, 95%CI = 0.63–0.82). COL18A1 mRNA expression was significantly decreased as the disease progressed (P = 0.000026).ConclusionThese findings indicate that COL18A1 rs7499 may contribute to the risk of HCC in Han Chinese.

Identification of single nucleotide polymorphisms in the bovine Toll-like receptor 1 gene and association with health traits in cattle

Bovine mastitis remains the most common and costly disease of dairy cattle worldwide. A complementary control measure to herd hygiene and vaccine development would be to selectively breed cattle with greater resistance to mammary infection. Toll-like receptor 1 (TLR1) has an integral role for the initiation and regulation of the immune response to microbial pathogens, and has been linked to numerous inflammatory diseases. The objective of this study was to investigate whether single nucleotide polymorphisms (SNPs) within the bovine TLR1 gene (boTLR1) are associated with clinical mastitis (CM).Selected boTLR1 SNPs were analysed within a Holstein Friesian herd. Significant associations were found for the tagging SNP -79 T > G and the 3'UTR SNP +2463 C > T. We observed favourable linkage of reduced CM with increased milk fat and protein, indicating selection for these markers would not be detrimental to milk quality. Furthermore, we present evidence that some of these boTLR1 SNPs underpin functional variation in bovine TLR1. Animals with the GG genotype (from the tag SNP -79 T > G) had significantly lower boTLR1 expression in milk somatic cells when compared with TT or TG animals. In addition, stimulation of leucocytes from GG animals with the TLR1-ligand Pam3csk4 resulted in significantly lower levels of CXCL8 mRNA and protein.SNPs in boTLR1 were significantly associated with CM. In addition we have identified a bovine population with impaired boTLR1 expression and function. This may have additional implications for animal health and warrants further investigation to determine the suitability of identified SNPs as markers for disease susceptibility.

Impact of the Interaction between 3′-UTR SNPs and microRNA on the Expression of Human Xenobiotic Metabolism Enzyme and Transporter Genes

Genetic variation in the expression of human xenobiotic metabolism enzymes and transporters (XMETs) leads to inter-individual variability in metabolism of therapeutic agents as well as differed susceptibility to various diseases. Recent expression quantitative traits loci (eQTL) mapping in a few human cells/tissues have identified a number of single nucleotide polymorphisms (SNPs) significantly associated with mRNA expression of many XMET genes. These eQTLs are therefore important candidate markers for pharmacogenetic studies. However, questions remain about whether these SNPs are causative and in what mechanism these SNPs may function. Given the important role of microRNAs (miRs) in gene transcription regulation, we hypothesize that those eQTLs or their proxies in strong linkage disequilibrium (LD) altering miR targeting are likely causative SNPs affecting gene expression. The aim of this study is to identify eQTLs potentially regulating major XMETs via interference with miR targeting. To this end, we performed a genome-wide screening for eQTLs for 409 genes encoding major drug metabolism enzymes, transporters and transcription factors, in publically available eQTL datasets generated from the HapMap lymphoblastoid cell lines and human liver and brain tissue. As a result, 308 eQTLs significantly (p < 10−5) associated with mRNA expression of 101 genes were identified. We further identified 7,869 SNPs in strong LD (r2 ≥ 0.8) with these eQTLs using the 1,000 Genome SNP data. Among these 8,177 SNPs, 27 are located in the 3′-UTR of 14 genes. Using two algorithms predicting miR-SNP interaction, we found that almost all these SNPs (26 out of 27) were predicted to create, abolish, or change the target site for miRs in both algorithms. Many of these miRs were also expressed in the same tissue that the eQTL were identified. Our study provides a strong rationale for continued investigation for the functions of these eQTLs in pharmacogenetic settings.

Variants in the 3′ untranslated region of the KCNQ1-encoded Kv7.1 potassium channel modify disease severity in patients with type 1 long QT syndrome in an allele-specific manner

AimsHeterozygous mutations in KCNQ1 cause type 1 long QT syndrome (LQT1), a disease characterized by prolonged heart rate-corrected QT interval (QTc) and life-threatening arrhythmias. It is unknown why disease penetrance and expressivity is so variable between individuals hosting identical mutations. We aimed to study whether this can be explained by single nucleotide polymorphisms (SNPs) in KCNQ1's 3′ untranslated region (3′UTR).Methods and resultsThis study was performed in 84 LQT1 patients from the Academic Medical Center in Amsterdam and validated in 84 LQT1 patients from the Mayo Clinic in Rochester. All patients were genotyped for SNPs in KCNQ1's 3′UTR, and six SNPs were found. Single nucleotide polymorphisms rs2519184, rs8234, and rs10798 were associated in an allele-specific manner with QTc and symptom occurrence. Patients with the derived SNP variants on their mutated KCNQ1 allele had shorter QTc and fewer symptoms, while the opposite was also true: patients with the derived SNP variants on their normal KCNQ1 allele had significantly longer QTc and more symptoms. Luciferase reporter assays showed that the expression of KCNQ1's 3′UTR with the derived SNP variants was lower than the expression of the 3′UTR with the ancestral SNP variants.ConclusionOur data indicate that 3′UTR SNPs potently modify disease severity in LQT1. The allele-specific effects of the SNPs on disease severity and gene expression strongly suggest that they are functional variants that directly alter the expression of the allele on which they reside, and thereby influence the balance between proteins stemming from either the normal or the mutant KCNQ1 allele.