UTR Single Nucleotide Polymorphisms Research Articles

Porcine Single Nucleotide Polymorphism (SNP) Development and Population Structure of Pigs Assessed by Validated SNPs

In this study, we identified porcine single nucleotide polymorphisms (SNPs) by aligning eight sequences generated with two approaches: amplification of 665 intronic regions using one sample from each of eight breeds, including three East Asian pigs, and amplification of 289 3'-UTR regions using two samples from each of four major commercial breeds. The 1,760 and 599 SNPs were validated using two 384-sample DNA panels by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The phylogenetic tree and Structure analyses classified the pigs into two large clusters: Euro-American and East Asian populations. The membership proportions, however, differed between inferred clusters for K = 2 generated by the two approaches. With intronic SNPs, Euro-American breeds constituted about 100% of the Euro-American cluster, but with 3'-UTR SNPs, about 17% of the East Asian cluster comprised five Euro-American breeds. The differences in the SNP discovery panels may affect population structure found in study panels of large samples.

Clinical Significance of CD33 Non-Synonymous Single Nucleotide Polymorphisms (SNPs) in Pediatric Patients with Acute Myeloid Leukemia Treated with Gemtuzumab Ozogamicin-Containing Chemotherapy,

Abstract 3489 Introduction:Acute myeloid leukemia (AML) remains a difficult-to-treat disease, and outcome with standard conventional chemotherapeutics is often poor. An emerging approach uses monoclonal antibodies as a means to deliver targeted therapy. The antigen currently most exploited is CD33, in particular with gemtuzumab ozogamicin (GO), an immunoconjugate with a toxic calicheamicin derivative that causes DNA strand breaks, apoptosis, and cell death. However, while efficacious in some patients with untreated or relapsed/refractory disease, clinical response to GO is heterogeneous, emphasizing the need for studies to determine the subsets of patients that will benefit from GO. Although response to therapy is generally multi-factorial in origin, there is often a significant contribution of host genetic factors. Since GO targets CD33, we sought to determine the clinical impact of CD33 single nucleotide polymorphisms (SNPs) in a larger cohort of pediatric AML patients enrolled on Children's Oncology Group (COG) AAML03P1 pilot Phase III study, which explored the safety of GO addition to standard induction chemotherapy for newly-diagnosed AML. Methods:We genotyped four CD33 SNPs [rs35112940 (G>A; Arg304Gly), rs12459419 (C>T; Ala14Val), rs2455069 (A>G; Arg69Gly), and rs1803254 (G>C; 3’UTR)] occurring with an allelic frequency greater than 10% in genomic DNA samples in 242 pediatric AML patients. These were treated on trial COG-AAML03P1, and determined the associations between CD33 SNPs with CD33 protein expression levels in diagnostic leukemic cells and clinical outcome. Results:CD33 coding SNPs, rs35112940 (G>A; Arg304Gly) and rs12459419 (C>T; Ala14Val), and the 3’UTR SNP rs1803254 demonstrated significant associations with outcome or CD33 expression levels. Specifically, among white patients (which consisted of the major group 166/242=68.6%) homozygous for wild-type allele (GG) for the SNP rs35112940, a lower 3-year relapse risk from the end of course 1 was observed as compared to other genotypes (GG vs. AG vs. AA; 24±9% vs. 41±17% vs. 67±38%; p=0.018 Figure. 1A). Similarly, the 3-year overall survival from end of course 1 for among white patients homozygous for G allele in complete remission was 84±8% versus 68±15% for other genotypes (p=0.036; Figure. 1B). Patients homozygous for variant allele (TT) for SNP rs12459419 had a 3-year overall survival of 88±13% compared to 69±6% for other genotypes (p=0.056). Among white patients, the homozygous TT genotype also had a lower mean CD33 expression compared to other genotypes at this SNP (mean fluorescence intensity, 42 vs. 145, p=<0.001). Additionally, we observed an increased proportion of patients homozygous for variant allele (TT) for rs12459419 in the favorable risk group compared to genotypes CC and CT (52% vs. 31 %, p=0.034). [Display omitted] Conclusion:In conclusion, our findings suggest that CD33 SNPs are associated with clinical disease features such as CD33 expression levels and risk group as well as outcome after GO-containing chemotherapy in patients with newly diagnosed AML. The recently completed COG-AAML0531 randomized Phase III trial will allow validation of our findings. If their usefulness as a prognostic marker for treatment outcome of GO-based therapy is confirmed in an independent cohort, CD33 SNPs may serve to optimize the clinical use of this immunoconjugate; by extrapolation, prognostic information provided by CD33 SNPs may also be applicable to novel, second-generation CD33-targeting immuno-conjugates that have entered preclinical and clinical testing. Disclosures:No relevant conflicts of interest to declare.

Association of anti-oxidized LDL and candidate genes with severity of coronary stenosis in the Women's Ischemia Syndrome Evaluation study

Atherosclerosis is the major cause of coronary artery disease (CAD), and oxidized LDL (oxLDL) is believed to play a key role in the initiation of the atherosclerotic process. Recent studies show that inflammation and autoimmune reactions are also relevant in atherosclerosis. In this study, we examined the association of antibodies against oxLDL (anti-oxLDL) with the severity of CAD in 558 Women's Ischemia Syndrome Evaluation (WISE) study samples (465 whites; 93 blacks) determined by coronary stenosis (< 20%, 20%-49%, > 50% stenosis). We also examined the relationship of anti-oxLDL with serum lipid levels and nine candidate genes including APOE, APOH, APOA5, LPL, LRP1, HL, CETP, PON1, and OLR1. IgM anti-oxLDL levels were significantly higher in the >20% stenosis group than in the ≥ 20% stenosis group in whites (0.69 ± 0.02 vs. 0.64 ± 0.01, respectively; P = 0.02). IgM anti-oxLDL levels correlated significantly with total cholesterol (r² = 0.01; P = 0.03) and LDL cholesterol (r² = 0.017; P = 0.004) in whites. Multiple regression analysis revealed a suggestive association of LPL/S447X single-nucleotide polymorphism (SNP) with both IgG anti-oxLDL (P = 0.02) and IgM anti-oxLDL (P = 0.07), as well as between IgM anti-oxLDL and the OLR1/3'UTR SNP (P = 0.020). Our data suggest that higher IgM anti-oxLDL levels may provide protection against coronary stenosis and that genetic variation in some candidate genes are determinants of anti-oxLDL levels.

TLR7 single-nucleotide polymorphisms in the 3' untranslated region and intron 2 independently contribute to systemic lupus erythematosus in Japanese women: a case-control association study

IntroductionThe Toll-like receptor 7 (TLR7) gene, encoded on human chromosome Xp22.3, is crucial for type I interferon production. A recent multicenter study in East Asian populations, comprising Chinese, Korean and Japanese participants, identified an association of a TLR7 single-nucleotide polymorphism (SNP) located in the 3' untranslated region (3' UTR), rs3853839, with systemic lupus erythematosus (SLE), especially in males, although some difference was observed among the tested populations. To test whether additional polymorphisms contribute to SLE in Japanese, we systematically analyzed the association of TLR7 with SLE in a Japanese female population.MethodsA case-control association study was conducted on eight tag SNPs in the TLR7 region, including rs3853839, in 344 Japanese females with SLE and 274 healthy female controls.ResultsIn addition to rs3853839, two SNPs in intron 2, rs179019 and rs179010, which were in moderate linkage disequilibrium with each other (r2 = 0.53), showed an association with SLE (rs179019: P = 0.016, odds ratio (OR) 2.02, 95% confidence interval (95% CI) 1.15 to 3.54; rs179010: P = 0.018, OR 1.75, 95% CI 1.10 to 2.80 (both under the recessive model)). Conditional logistic regression analysis revealed that the association of the intronic SNPs and the 3' UTR SNP remained significant after we adjusted them for each other. When only the patients and controls carrying the risk genotypes at the 3' UTR SNPpositionwere analyzed, the risk of SLE was significantly increased when the individuals also carried the risk genotypes at both of the intronic SNPs (P = 0.0043, OR 2.45, 95% CI 1.31 to 4.60). Furthermore, the haplotype containing the intronic risk alleles in addition to the 3' UTR risk allele was associated with SLE under the recessive model (P = 0.016, OR 2.37, 95% CI 1.17 to 4.80), but other haplotypes were not associated with SLE.ConclusionsThe TLR7 intronic SNPs rs179019 and rs179010 are associated with SLE independently of the 3' UTR SNP rs3853839 in Japanese women. Our findings support a role of TLR7 in predisposition for SLE in Asian populations.

Evolution of MicroRNAs and the Diversification of Species

MicroRNAs (miRNAs) are ancient, short noncoding RNA molecules that regulate the transcriptome through post-transcriptional mechanisms. miRNA riboregulation is involved in a diverse range of biological processes, and misregulation is implicated in disease. It is generally thought that miRNAs function to canalize cellular outputs, for instance as “fail-safe” repressors of gene misexpression. Genomic surveys in humans have revealed reduced genetic polymorphism and the signature of negative selection for both miRNAs themselves and the target sequences to which they are predicted to bind. We investigated the evolution of miRNAs and their binding sites across cichlid fishes from Lake Malawi (East Africa), where hundreds of diverse species have evolved in the last million years. Using low-coverage genome sequence data, we identified 100 cichlid miRNA genes with mature regions that are highly conserved in other animal species. We computationally predicted target sites on the 3′-untranslated regions (3′-UTRs) of cichlid genes to which miRNAs may bind and found that these sites possessed elevated single nucleotide polymorphism (SNP) densities. Furthermore, polymorphic sites in predicted miRNA targets showed higher minor allele frequencies on average and greater genetic differentiation between Malawi lineages when compared with a neutral expectation and nontarget 3′-UTR SNPs. Our data suggest that divergent selection on miRNA riboregulation may have contributed to the diversification of cichlid species and may similarly play a role in rapid phenotypic evolution of other natural systems.

A Polymorphism In the 3'UTR Region of ABCB1 Is Associated with Increased Allele Activity with Corresponding Increases In P-Glycoprotein Expression and Function

AbstractAbstract 3990Multidrug resistance is one of the major prognostic factors in acute myeloid leukemia (AML). Expression of p-glycoprotein (p-gp), a member of the ABC transporter family and encoded by the ABCB1 gene, is associated with failure to respond to chemotherapy. Control of MDR1 expression is multifactorial and to-date emphasis has been on the 5'-promoter region of the gene. Evidence is now accumulating regarding a role for the 3'-untranslated region (3' UTR) of genes in controlling expression. We therefore studied a single nucleotide polymorphism (SNP) in the 3'UTR of ABCB1 (rs3842) and determined its effect on p-gp expression.Samples were available on 450 AML patients who had been entered into the UK LRF AML14 and NCRN AML15 trials. The distribution of the rs3482 A/G SNP was determined using the SNaPshot method; 10% of samples had their genotype confirmed by sequencing. P-gp protein expression and function were analysed by flow cytometry of CD45-gated cells; expression using MRK-16 and function by assessing the modulation of R123 accumulation by PSC833. To determine the activity of the two polymorphic alleles the 3'UTR of ABCB1 was cloned into a modified pGL3 luciferase vector and the normalised luciferase activity was assessed for both the A and G allele.The distribution of the rs3482 SNP in our AML patient cohort was 71% AA, 27% AG and 1.5% GG. These results are comparable with the published prevalence of the SNP in European populations. Approximately 60% of AML patients do not express p-gp so we therefore focused upon the 40% of patients who expressed p-gp. We found that in the p-gp-expressing patients a higher p-gp protein and function was associated with patients with the wild–type homozygous AA genotype when compared to the GA+GG genotypes. Furthermore when the ABCB1 3'UTR was cloned downstream of the firefly luciferase gene and transfected into HL-60 cells the ‘A’ construct demonstrated significantly increased activity when compared to the G construct (normalized ratio (firefly luciferase/renilla luciferase): A 7.7, G 1.9; n=4, p<0.05).Outcome data was available on 414 of the intensively treated trial patients. Whilst the ABCB1 3'UTR polymorphism had no impact on overall survival or relapse risk there was a trend towards an increased relapse free survival in patients with the wildtype AA genotype (RFS AA 37%; AG+GG 28%, p=0.08). This may correspond to higher p-gp expression in immunocompetent cells post-remission.In conclusion this work demonstrates that the ABCB1 rs3482 3'UTR SNP is functionally significant with the A allele associated with higher activity and a concomitant increase in p-gp expression and function in p-gp expressing patient samples. Disclosures:No relevant conflicts of interest to declare.

A 3′‐untranslated region variant is associated with impaired expression of CD226 in T and natural killer T cells and is associated with susceptibility to systemic lupus erythematosus

Costimulatory receptor CD226 plays an important role in T cell activation, differentiation, and cytotoxicity. This study was undertaken to investigate the genetic association of CD226 with susceptibility to systemic lupus erythematosus (SLE) and to assess the functional implications of this association. Twelve tag single-nucleotide polymorphisms (SNPs) in CD226 were typed in 1,163 SLE patients and 1,482 healthy control subjects from Europe or of European ancestry. Analyses of association were performed by single-marker Cochran-Mantel-Haenszel meta-analysis, followed by haplotype analysis. Gene expression was analyzed by quantitative real-time polymerase chain reaction analyses of RNA from peripheral blood mononuclear cells, and by fluorescence-activated cell sorter analysis. To study the functional impact of the associated variants, luciferase reporter constructs containing different portions of the 3'-untranslated region (3'-UTR) of the gene were prepared and used in transfection experiments. A 3-variant haplotype, rs763361;rs34794968;rs727088 (ATC), in the last exon of CD226 was associated with SLE (P = 1.3 × 10(-4) , odds ratio 1.24, 95% confidence interval 1.11-1.38). This risk haplotype correlated with low CD226 transcript expression and low CD226 protein levels on the surface of CD4+ and CD8+ T cells and natural killer T (NKT) cells. NK cells expressed high levels of CD226, but this expression was independent of the haplotype. Reporter assays with deletion constructs indicated that only the presence of rs727088 could account for the differences in the levels of luciferase transcripts. This study identified an association of CD226 with SLE in individuals of European ancestry. These data support the importance of the 3'-UTR SNP rs727088 in the regulation of CD226 transcription both in T cells and in NKT cells.

A 3′UTR SNP in NLRP3 Gene is Associated With Susceptibility to HIV-1 Infection

Innate immunity genes polymorphisms are known to be involved in the multifactorial susceptibility to HIV-1 infection. Recently it has been hypothesized that inflammasomes could play an important role in the host response to viruses. The aim of our study is to verify if single-nucleotide polymorphisms (SNPs) in genes encoding for NALPs-innate immune receptors that form molecular complexes leading to the production of IL-1beta and the activation of immune response-could influence the individual susceptibility to HIV-1. We performed an association study analyzing 2 NLRP1 and NLRP3 SNPs in HIV-1 vertically infected Brazilian children (n = 135), HIV-1-infected Brazilain adults (n = 192) and HIV-1-positive Italian seropositive subjects (n = 192). The 3'UTR NLRP3 rs10754558 SNP was associated with HIV-1 infection in all the studied groups. The frequency of rs10754558 G allele was differently distributed within seropositive subjects (HIV+) and controls, and in particular the GG genotype was less frequent in HIV+. susceptibility to HIV-1 infection is associated with a 3'UTR NLRP3 polymorphism. This is the first report linking SNPs in the NALPs with HIV-1 infection, and further epidemiologic and functional studies are needed to deeper investigate the role of inflammasome in the susceptibility to HIV-1 infection.

Functional glutathione peroxidase 3 polymorphisms associated with increased risk of Taiwanese patients with gastric cancer

Glutathione peroxidase 3 (GPX3) can enhance an antioxidant's capacity and reduce genomic damage caused by oxidants and thus influence tumorigenesis. We investigated the role of GPX3 as a risk of gastric cancer. We first conducted a case-control study to test for the association between 5 tagging single nucleotide polymorphisms (SNPs) of GPX3 and the risk of gastric cancer in Chinese. Multivariate logistic regression analysis was performed to estimate the genetic effect with adjustments for age and sex. Functional studies were performed by using the luciferase reporter assay to assess functional consequences of the significant SNPs. Among five SNPs (rs3763013, rs8177412, rs3805435, rs3828599, and rs2070593) genotyped in 227 cases and 844 controls, 3 SNPs were significant: intronic SNP rs3805435 (OR=0.70, P=0.037), intronic SNP 3828599 (OR=0.68, P=0.025), and 3' UTR SNP rs2070593 (OR=0.48, P=0.001). The two intronic SNPs rs3805435 and SNP rs3828599 were in linkage disequilibrium (D'=0.91). The reporter assays showed significant difference in the luciferase expression between protective and risk alleles of 2o intronic SNPs (P=0.004), whereas the 3'UTR SNP did not influence the luciferase expression. The intronic SNPs at GPX3 can influence gene expression leading to an alteration of gastric cancer risk.

Human population genetic diversity as a function of SNP type from HapMap data

Data from the international HapMap project were mined to determine if the degree of genetic differentiation (Fst) is dependent on single nucleotide polymorphism (SNP) category. The Fst statistic was evaluated across all SNPs for each of 30 genes and for each of five chromosomes. A consistent decrease in diversity between Europeans and Africans was seen for nonsynonymous coding region SNPs compared to the three other SNP categories: synonymous SNPs, UTR, and intronic SNPs. This suggests an effect of balancing selection in reducing interpopulation genetic diversity at sites that would be expected to influence phenotype and therefore be subject to selection. This result is inconsistent with the concept of large population specific genetic differences that could have applications in "racialized medicine."

Identification of polymorphisms in the 3′-untranslated region of the human pregnane X receptor (PXR) gene associated with variability in cytochrome P450 3A (CYP3A) metabolism

Single nucleotide polymorphisms in the 3′-untranslated region (3′UTR) of the human pregnane X receptor (PXR) gene might contribute to interindividual variability in cytochrome P450 3A (CYP3A) activity.Genotype–phenotype associations involving PXR-3′UTR single nucleotide polymorphisms were investigated through in vitro (53 human livers from primarily White donors) and in vivo (26 mainly White or African-American volunteers) studies using midazolam 1′-hydroxylation and midazolam apparent oral clearance (CL/F), respectively, as CYP3A-specific probes.PXR-3′UTR resequencing identified twelve single nucleotide polymorphisms, including two that were novel. Although none of the single nucleotide polymorphisms evaluated were associated with altered midazolam 1′-hydroxylation in the liver bank, both rs3732359 homozygotes and rs3732360 carriers showed 80% higher (p < 0.05) CL/F compared with homozygous reference individuals. These differences in CL/F were even larger (100% and 120% higher, respectively; p < 0.01) when only African-American subjects (n = 14) were considered.Five major haplotypes were identified containing the PXR-3′UTR single nucleotide polymorphisms and previously identified intron single nucleotide polymorphisms. Although CL/F differences were not statistically significant within the entire study cohort, African-American carriers of Haplotype-1 (which includes both rs3732359 and rs3732360 variants) exhibited 70% higher median CL/F compared with African-American non-carriers (p = 0.036).The results identify rs3732359 and rs3732360 as PXR-3′UTR single nucleotide polymorphisms associated with higher CYP3A activity in vivo in African-Americans.

Polymorphisms in the Vitamin A Receptor and Innate Immunity Genes Influence the Antibody Response to Rubella Vaccination

Genetic polymorphisms play an important role in rubella vaccine-induced immunity. We genotyped 714 healthy children after 2 age-appropriate doses of rubella-containing vaccine for 142 potential single-nucleotide polymorphisms (SNPs). Specific polymorphisms in the vitamin A receptor, retinoic acid-inducible gene I (RIG-I), and tripartite motif 5 and 22 (TRIM5 and TRIM22) genes were significantly associated with rubella vaccine humoral immunity. The minor allele of the rs4416353 in the vitamin A receptor gene was associated with an allele dose-related decrease (P = .019) in rubella antibody response. The minor allele of rs6793694, in the vitamin A receptor gene, was associated with an allele dose-related antibody decrease (p = .039). The minor variant of nonsynonymous SNP rs10813831 (Arg7Cys) in the RIG-I gene was associated with an allele dose-related decrease in rubella antibody level from 37.4 to 28.0 IU/mL (P = .035), whereas increased representation of the minor allele of the 5'UTR SNP (rs3824949, P = .015) in the antiretroviral TRIM5 gene was associated with an allele dose-related increase in rubella antibody. It is of particular interest that the nonsynonymous SNP rs3740996 (His43Tyr) in the TRIM5 gene was associated with variations in rubella antibody response (P = .016) after having been previously found to play a significant functional role. These findings further expand our immunogenetic understanding of mechanisms of rubella vaccine-induced immunity.

A ComprehensiveIn SilicoAnalysis of the Functional and Structural Impact of SNPs in theIGF1RGene

Insulin-like growth factor 1 receptor (IGF1R) acts as a critical mediator of cell proliferation and survival. Many single nucleotide polymorphisms (SNPs) found in the IGF1R gene have been associated with various diseases, including both breast and prostate cancer. The genetics of these diseases could be better understood by knowing the functions of these SNPs. In this study, we performed a comprehensive analysis of the functional and structural impact of all known SNPs in this gene using publicly available computational prediction tools. Out of a total of 2412 SNPs in IGF1R retrieved from dbSNP, we found 32 nsSNPs, 58 sSNPs, 83 mRNA 3′ UTR SNPs, and 2225 intronic SNPs. Among the nsSNPs, a total of six missense nsSNPs were found to be damaging by both a sequence homology-based tool (SIFT) and a structural homology-based method (PolyPhen), and one nonsense nsSNP was found. Further, we modeled mutant proteins and compared the total energy values with the native IGF1R protein, and showed that a mutation from arginine to cysteine at position 1216 (rs61740868) on the surface of the protein caused the greatest impact on stability. Also, the FASTSNP tool suggested that 31 sSNPs and 3 intronic SNPs might affect splicing regulation. Based on our investigation, we report potential candidate SNPs for future studies on IGF1R mutations.

Effects of two common polymorphisms in the 3' untranslated regions of estrogen receptor β on mRNA stability and translatability

BackgroundThe present study represents the first attempt to functionally characterize two common single nucleotide polymorphisms (SNPs) in the 3'untranslated regions (3'UTRs) of estrogen receptor β (ERβ), focusing on the differences between alleles with regard to mRNA stability and translatability. These two ERβ SNPs have been investigated for association with disease in a large number of reports.ResultsHere we examined allelic expression in breast tumor samples from heterozygous individuals. A significant difference in mRNA levels of the two alleles was observed for one of the SNPs. A cell model system was employed to further investigate potential molecular effects of the two SNPs. We used a modified plasmid, containing the ERβ promoter and ERβ 3'UTRs which include the different alleles of investigated SNPs. Quantitative Real-Time PCR was used to determine mRNA levels after inhibition of transcription by actinomycin D, and a luciferase assay was used to determine protein levels. The obtained results suggested that there was no difference in mRNA stability or translatability between the alleles of investigated SNPs.ConclusionOur results indicate that observed associations between ERβ 3'UTR SNPs and disease susceptibility are due to linkage disequilibrium with another gene variant, rather than the variant itself being the susceptibility factor.

Identification of Two Novel Mutations in the CYB5R3 Gene in Five Cases of Type I Methemoglobinemia

Congenital methemoglobinemia due to cytochrome b5 reductase (CYB5R3) deficiency are sporadic worldwide, although clusters of this disorder have been identified in certain ethnic groups. Here we describe the molecular basis of type I methemoglobinemia due to CYB5R3 deficiency in four patients representing three distinct ethnic backgrounds. Three subjects were of Indian ethnicity one being phenotypically normal and his two cousins diagnosed with type I methemoglobinemia. Causative mutations in the globin genes were excluded after sequencing. A fourth subject, a 12 year old Mexican child with Turner's syndrome and apparent congenital cyanosis was referred to us for evaluation. Her methemoglobin level was elevated and erythrocyte cytochrome b5 reductase was reduced (4% of the normal range). The fifth individual, a 36 year old woman of Greek ancestry, had a past history of childhood cyanosis and low O2 saturation (82%) post-surgery in 2007. In early 2008 she was diagnosed with methemoglobinemia due to dyspnea, disproportionate fatigue, and significant increase in methemoglobin levels (10%). Genomic DNA from all 5 subjects was isolated, and all nine exons of the CYB5R3 gene were PCR amplified and sequenced. A non-synonymous heterozygous mutation (G>A transversion) in exon 6 resulting in the change of amino acid 178 from alanine to threonine (A178T) was identified in both affected Indian siblings and not in their unaffected cousin. This mutation which is located in the NADH binding domain and in close proximity to the adenosine moiety, and may influence binding of the NADH coenzyme had been previously reported (Dekker et al., 2001). A novel homozygous mutation in an evolutionarily conserved position located in exon 3 (C>G transition) resulting in substitution of serine 53 for arginine (S53R) was found in the child of Mexican descent. This mutation, located within the FAD binding domain, is hypothesized to cause disruption of hydrogen bonding and steric hindrance due to the bulkier arginine side chain, consequently leading to protein instability. Five heterozygous mutations were detected in the subject with Greek ancestry. A synonymous G>A substitution (Pro44Pro) in exon 2, a previously reported type II mutation G>A (Val253Met) and two 3′-UTR single nucleotide polymorphisms (A>G at 29690 nt, and C>A at 29914 nt), all in exon 9, were found in a cis configuration. A novel heterozygous compound mutation in exon 6, a T>G transversion, resulting in substitution of phenylalanine 157 for cysteine (F>C) was identified in the same subject with Greek ancestry (Figure 1). PCR amplified CYB5R3 transcripts harboring either wild-type, S53R, or F157C sequences were cloned, in frame with the 10X histidine tag, into the pET16b expression vector. Sequence fidelity and in frame fusion of all constructs was verified by sequencing from both strands. Recombinant protein was purified using 1 mL niquel-affinity columns. Kinetic studies using recombinant CyB5R3 enzyme will follow. [Display omitted]

Abstract A105: Genetic variations in sonic pathway genes as predictors of bladder cancer risk

Abstract A105 BACKGROUND The hedgehog (Hh) signaling pathway plays a crucial role in normal embryonic development. However, abnormal activation of the pathway later in life has been demonstrated in a variety of human diseases, including bladder cancer. METHODS We genotyped 171 potentially functional single nucleotide polymorphisms (SNPs) and tagging SNPs among seven Sonic Hedgehog pathway-related genes in a case-control study including 803 Caucasian bladder cancer patients and 803 matched controls. RESULTS The homozygous variant genotype of a SNP located in the Intron of Gli2 (rs4848123) was associated with a significantly increased bladder cancer risk [odds ratios (OR), 4.21; 95% confidence interval (95% CI), 1.15-15.44]. Likewise, the homozygous variant genotype of a 3’ UTR SNP (rs3823720) and a SNP in the Intron (rs10951671) of GLI3 was associated with a significantly increased bladder cancer risk (OR, 1.58, 95% CI, 1.10-2.26; and OR, 1.87, 95% CI, 1.14-3.06; respectively). To assess the cumulative effects, we performed a combined unfavorable genotype analysis that included all SNPs showing at least a borderline statistical significance. We found that, compared with the low-risk reference group with less than two unfavorable genotypes, the medium-risk group with two unfavorable genotypes exhibited a 1.29-fold (0.92-1.81) increased risk and the high-risk group with more than two unfavorable genotypes exhibited a 1.92-fold (1.36-2.71) increased risk (P(trend) &amp;lt; 0.0001). CONCLUSION To our knowledge, this is the first epidemiologic study showing that sonic pathway-related genetic variants may affect bladder cancer risk individually and jointly. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A105.

CTLA-4 single-nucleotide polymorphisms in a Caucasian population with schizophrenia

Associations between a single-nucleotide polymorphism (SNP) in exon 1 of the cytotoxic T-lymphocyte antigen-4 ( CTLA4) gene and schizophrenia in a Korean population have been previously described. The current study investigated whether a similar link occurs in a Caucasian population with schizophrenia. One hundred and twenty-two age- and sex-matched pairs of people with DSM-III-R diagnosis of schizophrenia and healthy controls were included in this study. Three previously described SNPs (from the promoter, exon 1 and 3′ UTR) of the CTLA4 gene were analysed. In the entire sample, we detected no allelic or genotypic association for any of the three SNPs. Given documented gender differences in incidence of schizophrenia, we conducted separate analyses of male and female participants. In males, both the promoter region SNP (−318C/T) and the 3′ UTR SNP demonstrated nominally significant association with schizophrenia. The 3′ UTR SNP remained significant following correction for multiple testing (permuted P = 0.046). In addition, all possible haplogenotypes showed significant association with disease in males with two – both containing the 3′ UTR SNP – remaining significant following correction for the genotypic tests of all SNPs and haplogenotypes in males. These results suggest a role for the 3′ UTR SNP and/or variants in high linkage disequilibrium with this SNP in the pathogenesis of schizophrenia.

Analysis of variants in DNA damage signalling genes in bladder cancer

BackgroundChemicals from occupational exposure and components of cigarette smoke can cause DNA damage in bladder urothelium. Failure to repair DNA damage by DNA repair proteins may result in mutations leading to genetic instability and the development of bladder cancer. Immunohistochemistry studies have shown DNA damage signal activation in precancerous bladder lesions which is lost on progression, suggesting that the damage signalling mechanism acts as a brake to further tumorigenesis. Single nucleotide polymorphisms (SNPs) in DSB signalling genes may alter protein function. We hypothesized that SNPs in DSB signalling genes may modulate predisposition to bladder cancer and influence the effects of environmental exposures.MethodsWe recruited 771 cases and 800 controls (573 hospital-based and 227 population-based from a previous case-control study) and interviewed them regarding their smoking habits and occupational history. DNA was extracted from a peripheral blood sample and genotyping of 24 SNPs in MRE11, NBS1, RAD50, H2AX and ATM was undertaken using an allelic discrimination method (Taqman).ResultsSmoking and occupational dye exposure were strongly associated with bladder cancer risk. Using logistic regression adjusting for age, sex, smoking and occupational dye exposure, there was a marginal increase in risk of bladder cancer for an MRE11 3'UTR SNP (rs2155209, adjusted odds ratio 1.54 95% CI (1.13–2.08, p = 0.01) for individuals homozygous for the rare allele compared to those carrying the common homozygous or heterozygous genotype). However, in the hospital-based controls, the genotype distribution for this SNP deviated from Hardy-Weinberg equilibrium. None of the other SNPs showed an association with bladder cancer and we did not find any significant interaction between any of these polymorphisms and exposure to smoking or dye exposure.ConclusionApart from a possible effect for one MRE11 3'UTR SNP, our study does not support the hypothesis that SNPs in DSB signaling genes modulate predisposition to bladder cancer.

Influence of a novel polymorphism in the 3’ untranslated region of the gene PLK1 upon PLK1 mRNA expression and survival of patients with renal cell carcinoma

22005 Background: Polo-like kinases (PLK) belong to the family of serine/threonine kinases. They regulate the transit from the G2 phase to mitosis during the cell cycle. PLK1 is overexpressed in human tumors which correlates with cancer progression. Novel PLK1-inhibitors were developed to improve the efficacy of standard chemotherapeutics which interfere with the cell cycle. Because genetic polymorphisms can influence the transcription of genes, the importance of an A>G single nucleotide polymorphism (SNP) in the 3’ untranslated region (3’UTR) of PLK1 was examined for PLK1 expression and survival for patients with clear cell renal cell carcinoma (RCC). Methods: The examinedtumor collective comprised 141 patients who were treated for RCC at the Department of Urology of the University Hospital of Essen, Germany. DNA was genotyped using the restriction fragment length polymorphism method (RFLP). Kaplan- Meier plots and the log-rank test for trend were used to detect a potential association of overall survival with the 3’UTR SNP. To determine potential genotype-dependent mRNA expression quantitative real-time PCR (qRT-PCR) was applied. Results were compared using ANOVA. Results: Genotyping of the RCC collective showed that 41.1% of the patients were homozygous for the A-allele, 49.6% were homozygous for the G-allele and 9.3% were heterozygous. Acorrelation between the 3’UTR-polymorphism in PLK1 and overall survival was detected showing reduced overall survival for patients carrying the G allele (p=0.009). qRT-PCR analysis showed an increased mRNA expression of PLK1 associated with the G- allele compared to the A allele in tumor tissue from patients with RCC. Conclusion: It has already been described that PLK1 is overexpressed in human tumors and that the abundance of PLK1 correlates with their malignancy. The results presented here show that the abundance of PLK1 is associated with genotypes of the polymorphic 3’UTR. PLK1 genotypes could be a marker for survival of patients with RCC and could potentially be used to identify responders to PLK1 inhibitors. No significant financial relationships to disclose.

β3‐Adrenergic receptor gene polymorphisms: comparison of human with chimpanzee and Rhesus macaque

The Trp64Arg single nucleotide polymorphism (SNP) of the human β3‐adrenergic receptor is potentially associated with several disease states including obesity and type 2 diabetes. However, in vitro data with site‐directed mutagenesis have not consistently shown this SNP to yield an altered function. This raises the possibility that the SNP may be an indicator in linkage disequilibrium with functionally more relevant SNPs. To explore this and gain evolutionary insight, we have sequenced the β3‐adrenergic receptor gene from 91 humans, 12 chimpanzees and 12 Rhesus macaques. Four additional SNPs (G1219T, A2135G, G2502C, A3558T) and a tandem repeat polymorphism ΔTG3273/4) were detected in the intron and 3′‐UTR of the human gene. Their consistent association indicates the presence of a haploblock. Within the limited monkey and ape populations no polymorphisms were detected, but for the one coding and two 3′‐UTR human SNPs the human wild‐type differed from the monkey and ape wild‐type. We conclude that the intron polymorphisms of the β3‐adrenergic receptor gene have developed evolutionarily later than the coding and 3′‐UTR SNPs. Source of funding: none