UTR SNP Research Articles

Sex- and genotype-dependent nicotine plus cue-primed reinstatement is enhanced in adolescent Sprague Dawley rats containing the human CHRNA6 3'-UTR polymorphism (rs2304297).

Large-scale human candidate gene studies have indicated that a genetic variant (rs2304297) in the alpha(α)6 nicotinic acetylcholine receptor (nAChR) subunit, encoded by the CHRNA6 gene, may play a key role in adolescent nicotine addictive behavior. We hypothesized that the polymorphism selectively enhances nicotine + cue-primed reinstatement, but not nicotine- or cue-reinstatement in α6 GG (risk) vs. α6 CC (non-risk) allele carriers, without having baseline effects on natural rewards. Using CRISPR-Cas9 genomic engineering, we developed a humanized rat line with the human gene variant of the CHRNA6 3'-UTR C 123 G polymorphism in Sprague-Dawley rats. Genetically modified adolescent male and female rats were food trained under a fixed-ratio (FR)1 schedule of reinforcement and progressively increased to FR5. Animals were implanted with catheters and began nicotine self-administration (15 μg/kg/infusion) at FR5. Upon reaching stable responding, reinforced behavior was extinguished by removal of drug and cues. Reinstatement testing began for cue only, nicotine only, and nicotine + cue in a Latin Square Design. Animals were returned to extinction conditions for 2 days minimum between testing. For natural food rewards, nicotine self-administration, progressive ratio, and extinction, adolescent male and female (α6 GG and α6 CC ) rats exhibited equivalent behaviors. Male α6 GG rats show enhanced nicotine + cue-primed reinstatement when compared with male α6 CC rats. This genotype effect on reinstatement was not seen in female rats. Our findings support the in vivo functional role of the human CHRNA6 3'-UTR SNP genetic variant in sex-dependently enhancing nicotine seeking behavior in adolescent rats. Overall, the findings support clinical and preclinical data highlighting a role of α6 nAChRs mediating sex heterogeneity in substance use and related phenotypes.

Acetylcholinesterase and Association 1 of ACHE 3’ UTR SNP rs17228602 with Psychiatric Disorders

Psychiatric disorders are complex mental conditions that cause significant emotional distress and impairment in a person’s ability to function normally. Globally, there is an alarming rise in the prevalence of psychiatric conditions. Genetic and environmental factors are involved in the pathophysiology of these disorders, but molecular underpinning is still elusive. Cholinergic dysregulation is one of the aetiology of psychiatric condition. This study was aimed to assess the status of hydrolyzing enzyme of cholinergic neurotransmitter, acetylcholinesterase (AChE) from blood and investigate the possible association of a single nucleotide polymorphisms in 3'UTR region of ACHE (rs17228602) with predisposition to psychiatric disorder. Methods: Ninety-five confirmed psychiatric and one hundred thirty healthy individuals were recruited for the study with due consents. AChE was determined by Elman’s method. SNP was studied by polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) and sanger sequencing on DNA samples. Results: The results showed notably reduced AChE in psychiatric cohorts with statistical significance (p≤0.05). Genotype and allelic association of the examined SNP was observed with the risk of psychiatric condition in patients.

Pharmacogenomics of Fludarabine in Pediatric HCT Recipients: Preliminary Results

Introduction: Total Body Irradiation (TBI) conditioning regimens are considered standard of care for pediatric hematopoietic cell transplant (HCT) recipients. However, TBI has many long-term effects, so avoiding it in young HCT recipients is critical. Fludarabine is frequently used in combination with other chemotherapeutic agents like busulfan and/or thiotepa in non-TBI based conditioning regimens. While multi-drug conditioning aims to avoid TBI-related late-effects, little is known about the effect of variations in genes that metabolize these drugs (pharmacogenomics/PGx) on clinical outcomes including engraftment, relapse, and acute drug-related toxicities. In this study, we investigated the pharmacogenomic effects on HCT outcomes such as event-free outcomes and re-transplantation outcomes, as well as fludarabine's pharmacokinetics (PK).Methods: This ongoing study included pediatric patients from a nationwide multi-center study who received fludarabine in combination with other agents as busulfan and thiotepa. From literature searches and PharmGKB databases, we selected 70 SNPs in 30 candidate pharmacological genes for these drug pathways. SNPs with a minor allele frequency of 10% were further selected for analysis with different endpoints such as: 1) Cumulative incidence with disease relapse with re-transplantation (using Gray's methods, controlling for competing risks); 2) fludarabine PK with cumulative area under the curve (cAUC); 3) 1-month-post-transplant chimerism levels defined as sufficient (CD3 ≥80% and CD14/15 ≥95% chimerism) and insufficient (CD3 <80% and CD14/15 <95% chimerism); and 4) event-free outcomes (comparing patients alive and well vs. others) OR alive re-transplantation outcomes (alive with vs. without re-transplantation). SNPs with an association p-value <0.05 were considered significant using generalized linear models from the SNPassoc R package.Results: Total of 87 patients included in the analysis had a median age of 3.5 (0.2 - 17.9) years, 63% were male, 48% were Caucasian, 85% had received allogenic HCT, and 43% had malignant hematologic disease (Table 1). For SNP rs2277119 in CYP39A1, presence of T allele was associated with greater cumulative incidence of re-transplantation (TC-TT vs. CC genotype: HR=3.64 (95% CI=1.14-11.62), p=0.02, Figure 1A); and SNP rs4715354 in GSTA5, presence of G allele was associated with lower cumulative incidence of re-transplantation (AG-GG vs. AA: HR=0.32 (95% CI=0.11-0.95), p=0.037, Figure 1B). In 79% of patients with available fludarabine cAUC (median of 3.72 mg•hr/L), we evaluated fludarabine pathway gene SNPs for association with cAUC. Within SNPs rs3925058 in CMPK1 and rs11853372 in the uptake transporter SLC28A1, presence of variant allele was associated with higher fludarabine cAUC (rs3925058: GA-AA vs. GG, p=0.049, Figure 2A, and rs11853372 GG-GT vs. TT, p=0.003, Figure 2B); and for a 3'-UTR SNP rs2037067 in TENM3/DCTD, the variant allele was associated with lower fludarabine cAUC levels (CT-CC vs. TT, p=0.005, Figure 2C). In an exploratory analysis with event-free outcomes, patients with variant allele for SNPs rs9937 in RRM1, rs2072671 in CDA, rs324148 in SLC29A1, and rs7533657 in CTPS1 had higher odds of having an event compared to patients with the reference allele; while rs11577910 in CTPS1 had a better outcome with presence of variant allele. With respect to patient's status being alive with re-transplantation outcomes, variants SNPs with rs507964 in SLC29A1, rs4244285 in CYP2C19, rs1021737 in CTH, rs1561876 in STIM1, and rs1130609 in RRM2 have high odds of re-transplantation. At 1-month follow-up, variant SNP rs12144160 in CTPS1 was associated with sufficient chimerism in CD3 and CD14/15.Conclusions: In this study, we have identified a number of SNPs that predict interpatient variability in clinical outcomes and fludarabine levels in pediatric HCT recipients. Results so far showed potential to predict outcomes and develop strategies that will consider pharmacogenomics when determining fludarabine doses in pediatric HCT recipients. Our ongoing study is focused on establishing and validating the pharmacogenetic markers predictive of pharmacokinetics of busulfan, fludarabine and thiotepa and clinical outcomes in pediatric HCT recipients.Acknowledgements: This study was supported by Florida Department of Health - Live Like Bella Discovery Award (9LA04). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

5'UTR polymorphism in the serotonergic receptor HTR3A gene is differently associated with striatal Dopamine D2/D3 receptor availability in the right putamen in Fibromyalgia patients and healthy controls-Preliminary evidence.

Extensive literature has investigated the role of serotonin (5-HT) in the control of the central dopamine (DA) systems, and their dysfunction in the pathological conditions. 5-HT stimulates the local DA release in striatal regions via activation of various receptors including serotonin receptor-3 (5-HT3). Several studies have related polymorphisms (SNPs) in the serotonin receptor-3 (HTR3) genes to be associated with the pain modulation and endogenous pain suppression. A few studies suggested a functional role of 5'UTR SNP in the serotonergic receptor HTR3A gene (rs1062613) in the development of the chronic pain and Fibromyalgia syndrome (FMS) in particular. Here, we investigated the effect of a 5'UTR SNP in the serotonergic receptor HTR3A gene (rs1062613) on striatal dopamine D2/D3 receptor (DRD2) availability and reward-associated DA release in response to unpredictable monetary rewards in 23 women with FMS and 17 age-matched healthy female controls. Furthermore, we aimed to examine if SNP rs1062613 is associated with thermal pain and pain tolerance thresholds. We used PET and [11 C]raclopride to assess the DRD2 availability. In the same participants we used the [11 C]raclopride PET bolus-plus-infusion method to measure the [11 C]raclopride receptor binding potential (ΔBP) between an unpredictable reward condition and a sensorimotor control condition. DRD2 availability and ΔBP were assessed in MRI-based striatal regions of interest. Thermal pain and pain tolerance thresholds were assessed outside the scanner. The frequency of SNP rs1062613 genotype differed significantly between groups, indicating that CC homozygotes were more frequent in FMS patients (82.6%) than in healthy controls (41.3%). Our results showed a significant main effect of SNP rs1062613 on [11 C]raclopride binding potential in the right caudate nucleus indicating a higher DRD2 receptor availability for CC-genotype of this SNP. Furthermore, we found a significant group × SNP interaction on [11 C]raclopride binding potential in the right putamen, indicating a higher DRD2 availability in T-carriers compared to CC genotype of SNP rs1062613 in FMS patients, whereas this effect was not present in healthy controls. However, we did not find an influence of SNP rs1062613 on reward-related DA release. In addition, there was no association between SNP rs1062613 and pain threshold or pain tolerance threshold in our data. These preliminary results indicate that SNP rs1062613 in the serotonergic receptor HTR3A gene possibly modulates the DRD2 receptor availability.

Genetic risk association of CDKN1A and RET gene SNPs with medullary thyroid carcinoma: Results from the largest MTC cohort and meta-analysis.

BackgroundMedullary thyroid carcinoma (MTC) is a rare subtype of thyroid cancer. Other than gain‐of‐function RET mutations, no other genetic, lifestyle or environmental risk associations have been established for MTC. Several case‐control studies and meta‐analysis have examined the risk association of different SNPs with MTC in different populations but with contradictory or inconclusive results.MethodsIn a large cohort of 438 Indian MTC cases and 489 gender and ethnicity matched healthy controls from 1000 genome project, a comprehensive risk association of 13 SNPs of three pathways—detoxification, cell cycle regulation and RET was performed along with meta‐analysis of RET SNPs.ResultsMultivariate logistic regression analysis identified a protective risk association of CDKN1ASer31Arg SNP with both hereditary (OR 0.26; 95% confidence interval [CI] 0.13‐0.55; P < .001) and sporadic MTC (OR 0.53; 95% CI 0.36‐0.78; P = .001). An increased risk association was identified for NAT2Y94Y SNP (OR 1.62, 95% CI 1.17‐2.25, P = .004) and CDKN2A3′UTR SNP (OR 1.89, 95% CI 1.19‐2.98, P = .006) with sporadic MTC and RET S904S with hereditary MTC (OR 2.82, 95% CI 1.64‐4.86, P < .001). Meta‐analysis of RET SNPs including our cohort identified increased risk association of all four RET SNPs with MTC.ConclusionIn this largest SNP risk association study for MTC and the only risk association study of the 13 most commonly studied MTC associated SNPs in a single cohort of this rare cancer, a significant protective risk association of CDKN1ASer31Arg SNP with MTC was shown for the first time. Meta‐analysis identified significant risk association of all four RET SNPs, not observed in previous meta‐analysis.

Comprehensive biomarker analyses identifies HER2, EGFR, MET RNA expression and thymidylate synthase 5'UTR SNP as predictors of benefit from S-1 adjuvant chemotherapy in Japanese patients with stage II/III gastric cancer.

Purpose: A comprehensive molecular analysis was conducted to identify prognostic and predictive markers for adjuvant S-1 chemotherapy in stage II/III Japanese gastric cancer (GC) patients and to evaluate their potential suitability for alternative cytotoxic or targeted drugs.Experimental Design: We investigated genetic polymorphisms of enzymes potentially involved in 5-fluoruracil (5-FU) metabolism as well as platinum resistance, previously identified genomic subtypes potentially predicting 5-FU benefit, and mRNA expression levels of receptor tyrosine kinases and KRAS as potential treatment targets in a single institution cohort of 252 stage II/III GC patients treated with or without S-1 after D2 gastrectomy.Results: 88% and 62% GC had a potentially 5-FU sensitive phenotype by SNP analyses of TS 3'UTR, and TS 5'UTR, respectively. 24%, 46%, 40%, 5%, and 44% GC had a potentially platinum sensitive phenotype by SNP analyses of GSTP1, ERCC1 rs11615, ERCC1 rs3212986, ERCC2, and XRCC1, respectively. High HER2, EGFR, FGFR2, or MET mRNA expression was observed in 49%, 66%, 72%, and 54% GC, respectively. High HER2 expression was the only significant prognosticator (HR=3.912, 95%CI: 1.706-8.973, p=0.0005). High HER2 (p=0.031), low EGFR (p=0.124), high MET (p=0.165) RNA expression, and TS 5'UTR subtype 2R/2R, 2R/3C, or 3C (p=0.058) were significant independent predictors for S-1 resistance.Conclusions: The present study suggests that platinum-based or RTK targeted agents could be alternative treatment options for a substantial subgroup of Japanese GC patients currently treated with S-1. HER2, EGFR, MET, and TS 5'UTR SNP appear to be promising predictive markers for S-1 resistance warranting validation in an independent GC series.

SNPs in 3′-UTR region of MBL2 increases susceptibility to recurrent vulvovaginal infections by altering sMBL levels

Recurrent vulvovaginal infections (RVVI), owing to their adverse health consequences, have become a serious dilemma worldwide. Low serum levels of Mannose-Binding Lectin (sMBL), a main component of innate immunity, was found to be associated with RVVI risk, though complete genetic bases are still elusive. To reveal unrecognised regulatory variants, 3′-UTR region of MBL2 with six putative functional SNPs i.e. rs10824792, rs2120132, rs2120131, rs2165813, rs2099903 and rs2099902 was sequenced and genotyped in the present study for 109 RVVI cases and age matched healthy controls. sMBL levels were measured by enzyme-linked immunosorbent assay. The homozygous CC genotype of rs10824792 polymorphism was found to be conferring risk (OR = 2.94) of developing RVVI. Significantly high frequency of corresponding CC genotype was found in Vulvovaginal Candidiasis (VVC) and Mixed Infections (MI) relative to controls. Significantly insufficient sMBL levels were observed in RVVI and its types (Bacterial Vaginosis, VVC and MI) than controls. sMBL levels varied for rs10824792 SNP as expected from the genetic analyses. Six marker haplotype analyses have shown CTTGCT, the haplotype containing only risk allele of rs10824792, conferred risk of RVVI and its types by lowering sMBL levels. In conclusion, a 3′-UTR SNP i.e. rs10824792 was identified as novel associated genetic marker for contributing low sMBL levels and RVVI risk. Our findings contribute to the novel future research directions for the development of emerging MBL substitution as effectual therapy for RVVI.

LIMK2 acts as an oncogene in bladder cancer and its functional SNP in the microRNA-135a binding site affects bladder cancer risk.

LIM kinases modulate multiple aspects of cancer development, including cell proliferation and survival. As the mechanisms of LIMK‐associated tumorigenesis are still unclear, we analyzed the tumorigenic functions of LIM kinase 2 (LIMK2) in human bladder cancer (BC) and explored whether the newly identified LIMK2 3´‐UTR SNP rs2073859 (G‐to‐A allele) is correlated with clinical features. Expression levels of LIMK2 in 38 human BC tissues and eight cell lines were examined using quantitative real‐time PCR and immunohistochemistry. LIMK2 was overexpressed in most BC tissues (27/38, 71%) and BC‐derived cell lines (6/8), and was more frequently overexpessed in high‐grade than low‐grade BC (80% vs. 47%). The effects of LIMK2 on BC cell proliferation, survival and migration, were studied by overexpression and RNA interference approaches in vitro and in vivo. LIMK2 overexpression promoted proliferation, migration and invasion of BC cells, while LIMK2 depletion inhibited cell invasion and viability and induced growth arrest in vitro and in vivo. PCR‐Restriction Fragment Length Polymorphism (RFLP) was used to genotype LIMK2 SNP rs2073859 and multivariate logistic regression applied to assess the relationship between allele frequency and clinical features in 139 BC patients. Functional analyses localized SNP rs2073859 within the microRNA‐135a seed‐binding region and revealed significantly lower LIMK2 G allele expression. The frequency of A genotypes (AG + AA) was higher in the BC group than normal controls and correlated with risks of high‐grade and high‐stage BC. In conclusion, LIMK2 may function as an oncogene in human BC, while allele‐specific regulation by microRNA‐135a may influence disease risk.

Polymorphism studies on microRNA targetome of thalassemia.

Thalassemia is one of the most prevalent hemoglobin disorders. It is caused by the decreased or absent synthesis of one globin chain that leads to moderate to severe hemolytic anemia in clinical complications. Some genetic factors cause these phenotypic variations by the alteration of gene expression. MicroRNAs (miRNAs) are post-transcriptional regulators in gene expression. Therefore, variations in 3'-untranslated region (3'-UTR) of target genes may affect gene expression. It is of interest to evaluate the impact of noncoding SNPs in thalassemia related genes on miRNA: mRNA interactions in the severity of thalassemia. Polymorphisms that alter miRNA: mRNA interactions were predicted using PolymiRTS and Mirsnpscore tools. Then, the effect of predicted target SNPs on thermodynamic stability, local RNA structure and regulatory elements was investigated using RNAhybrid, RNAsnp and RegulomeDB, respectively. The molecular functions and the Biological process of candidate genes were extracted and interaction network was created. Forty-six SNPs were predicted to affect 188 miRNA interactions. These results suggest that 3'-UTR SNP may affect gene expression and cause phenotypic variation in thalassemia patients.

Alpha-synuclein mRNA isoform formation and translation affected by polymorphism in the human SNCA 3'UTR.

BackgroundMultiple variants in SNCA, encoding alpha‐synuclein, a main component of Lewy bodies, are implicated in Parkinson's disease.MethodsWe searched for cis‐acting SNCA variants using allelic mRNA ratios in human brain tissues. In a SNCA 3′UTR (2,520 bp) luciferase reporter gene assay, translation in SH‐SY5Y cells in the presence of the rs17016074 G/A alleles was measured. To assess clinical impact, we queried neurocognitive genome‐wide association studies.ResultsAllelic ratios deviated up to twofold, measured at a marker SNP in the middle of a long 3′ untranslated region (3′UTR), but not at a marker at its start, suggesting regulation of 3′UTR processing. 3′UTR SNP rs17016074 G/A, minor allele frequency (MAF) <1% in Caucasians, 13% in Africans, strongly associates with large allelic mRNA expression imbalance (AEI), resulting in reduced expression of long 3′UTR isoforms. A second 3′UTR SNP (rs356165) associates with moderate AEI and enhances SNCA mRNA expression. The rs17016074 A allele reduces overall 3′UTR expression in luciferase reporter gene assays but supports more efficient translation, resolving previous contradictory results. We failed to detect significant genome‐wide associations for rs17016074, possibly a result of low MAF in Caucasians or its absence from most genotyping panels. In the “Genome Wide Association Study of Yoruba in Nigeria,” rs356165 was associated with reduced memory performance.ConclusionsHere, we identify two cis‐acting regulatory variants affecting SNCA mRNA expression, measured by allelic ratios in the 3′UTR. The rs17016074 minor A allele is associated with higher expression of luciferase protein activity. Resolving the genetic influence of SNCA polymorphisms requires study of the interactions between multiple regulatory variants with distinct frequencies among populations.

Allele frequency and genotype distribution of aldo keto reductase 1D1 (AKR1D1) rs1872930 genetic variant in a Macedonian population

AKR1D1 has been recently recognized as novel candidate gene regulating CYP450 coexpression. The AKR1D1 3’-UTR SNP (rs1872930) functional genetic variation was considered as important source of phenotypic variation in the activity of CYP450. The minor allele frequency of rs1872930 varies among populations suggesting population specific pharmacogenetic implications. The aim of the present study was to determine the AKR1D1 (rs1872930) minor allele frequencies and genotypes distribution in a population from Republic of Macedonia. Four hundred and fifty unrelated Macedonian subjects were studied. AKR1D1 variant allele was detected by real time polymerase chain reaction. The distribution of AKR1D1*1/*1, *1/*36 and *36/*36 genotypes was 0.569, 0.360 and 0.071, respectively. The overall frequency of AKR1D1*36 variant allele was 0.251. Our study is the first to assess the frequency distribution of the AKR1D1*36 (rs1872930) genetic variant in population from Republic of Macedonia and the findings are in accordance with the frequency reported for the Caucasian population. Keywords: AKR1D1, rs1872930, CYP450, variability, gene expression, enzyme activity

3\u2032UTR polymorphisms of carbonic anhydrase IX determine the miR-34a targeting efficiency and prognosis of hepatocellular carcinoma

Carbonic anhydrase IX (CA9) expression level has been considered as a poor prognostic factor in hepatocellular carcinoma (HCC) patients. However, the judging criteria of CA9 level is hard to define for potential clinical applications. Unlike CA9 expression level, CA9 polymorphism is poorly documented in HCC. Here, we found that people carry A allele at CA9 rs1048638, a 3′UTR SNP, has higher risk of HCC. rs1048638-CA correlates with advanced stages, larger tumor sizes, more vascular invasion, and shorter survival of HCC patients. A allele at CA9 rs1048638 impairs miR-34a, a tumor suppressor miRNA in HCC, binding to CA9 3′UTR and desensitizes CA9 mRNA to miR-34a-dependent RNA degradation. CA9 expression levels were also correlated with miR-34a levels and rs1048638 genotypes in HCC patients. rs1048638 influences HCC risk and progression through effects on miR-34a-targeted CA9 expression in HCC. In conclusion, genetic variations of the CA9 3′UTR play important roles in regulating CA9 expression and cancer progression, which is a novel determinant and target for HCC metastasis and prognosis.

3′-UTR SNP rs2229611 in G6PC1 affects mRNA stability, expression and Glycogen Storage Disease type-Ia risk

The frequency of rs2229611, previously reported in Chinese, Caucasians, Japanese and Hispanics, was investigated for the first time in Indian ethnicity. We analyzed its role in the progression of Glycogen Storage Disease type-Ia (GSD-Ia) and breast cancer. Genotype data on rs2229611 revealed that the risk of GSD-Ia was higher (P=0.0195) with CC compared to TT/TC genotypes, whereas no such correlation was observed with breast cancer cases. We observed a strong linkage disequilibrium (LD) among rs2229611 and other disease causing G6PC1 variants (|D′|=1, r2=1). Functional validation performed in HepG2 cells using luciferase constructs showed significant (P<0.05) decrease in expression than wild-type 3′-UTR due to curtailed mRNA stability. Furthermore, AU-rich elements (AREs) mediated regulation of G6PC1 expression characterized using 3′-UTR deletion constructs showed a prominent decrease in mRNA stability. We then examined whether miRNAs are involved in controlling G6PC1 expression using pmirGLO-UTR constructs, with evidence of more distinct inhibition in the reporter function with rs2229611. These data suggests that rs2229611 is a crucial regulatory SNP which in homozygous state leads to a more aggressive disease phenotype in GSD-Ia patients. The implication of this result is significant in predicting disease onset, progression and response to disease modifying treatments in patients with GSD-Ia.

Functional Implications of the CLOCK 3111T/C Single-Nucleotide Polymorphism.

Circadian rhythm disruptions are prominently associated with bipolar disorder (BD). Circadian rhythms are regulated by the molecular clock, a family of proteins that function together in a transcriptional–translational feedback loop. The CLOCK protein is a key transcription factor of this feedback loop, and previous studies have found that manipulations of the Clock gene are sufficient to produce manic-like behavior in mice (1). The CLOCK 3111T/C single-nucleotide polymorphism (SNP; rs1801260) is a genetic variation of the human CLOCK gene that is significantly associated with increased frequency of manic episodes in BD patients (2). The 3111T/C SNP is located in the 3′-untranslated region of the CLOCK gene. In this study, we sought to examine the functional implications of the human CLOCK 3111T/C SNP by transfecting a mammalian cell line (mouse embryonic fibroblasts isolated from Clock−/− knockout mice) with pcDNA plasmids containing the human CLOCK gene with either the T or C SNP at position 3111. We then measured circadian gene expression over a 24-h time period. We found that the CLOCK3111C SNP resulted in higher mRNA levels than the CLOCK 3111T SNP. Furthermore, we found that Per2, a transcriptional target of CLOCK, was also more highly expressed with CLOCK 3111C expression, indicating that the 3′-UTR SNP affects the expression, function, and stability of CLOCK mRNA.

Variant in the RFWD3 gene associated with PATN1, a modifier of leopard complex spotting.

Leopard complex spotting (LP), the result of an incompletely dominant mutation in TRPM1, produces a collection of unique depigmentation patterns in the horse. Although the LP mutation allows for expression of the various patterns, other loci are responsible for modification of the extent of white. Pedigree analysis of families segregating for high levels of patterning indicated a single dominant gene, named Pattern-1 (PATN1), as a major modifier of LP. Linkage analysis in two half-sibling families segregating for PATN1 identified a 15-Mb region on ECA3p that warranted further investigation. Whole transcriptome sequencing of skin samples from horses with and without the PATN1 allele was performed to identify genic SNPs for fine mapping. Two Sequenom assays were utilized to genotype 192 individuals from five LP-carrying breeds. The initial panel highlighted a 1.6-Mb region without a clear candidate gene. In the second round of fine mapping, SNP ECA3:23 658 447T>G in the 3'-UTR of RING finger and WD repeat domain 3 (RFWD3) reached a significance level of P = 1.063 × 10(-39). Sequencing of RFWD3 did not identify any coding polymorphisms specific to PATN1 horses. Genotyping of the RFWD3 3'-UTR SNP in 54 additional LP animals and 327 horses from nine breeds not segregating for LP further supported the association (P = 4.17 × 10(-115)). This variant is a strong candidate for PATN1 and may be particularly useful for LP breeders to select for high levels of white patterning.

Identification of a functional SNP in the 3'UTR of CXCR2 that is associated with reduced risk of lung cancer.

Global changes in gene expression accompany the development of cancer. Thus, inherited variants in miRNA-binding sites are likely candidates for conferring inherited susceptibility. Using an in silico approach, we compiled a comprehensive list of SNPs predicted to modulate miRNA binding in genes from several key lung cancer pathways. We then investigated whether these SNPs were associated with lung cancer risk in two independent populations. In general, SNPs in miRNA-binding sites are rare. However, some allelic variation was observed. We found that rs1126579 in CXCR2 was associated with a reduced risk of lung cancer in both European American [ORTT vs. CC 0.56 (0.37-0.88); P = 0.008] and Japanese [ORTT vs. CC 0.62 (0.38-1.00); P = 0.049] populations. Furthermore, we found that the SNP disrupted a novel binding site for miR-516a-3p, led to a moderate increase in CXCR2 mRNA and protein expression, and increased MAPK signaling. Moreover, analysis of rs1126579 with serum levels of IL8, its endogenous ligand, supported an interaction whereby rs1126579-T and high serum IL8 conferred synergistic protection from lung cancer. Our findings demonstrate a function for a 3'UTR SNP in modulating CXCR2 expression, signaling, and susceptibility to lung cancer.

Interferon-γ (IFNG) microsatellite repeat and single nucleotide polymorphism haplotypes of IFN-α receptor (IFNAR1) associated with enhanced malaria susceptibility in Indian populations

Pro-inflammatory cytokines IFNγ and IFNα function through their cellular receptors IFNγR1 and IFNαR1, respectively to mediate immune processes during malaria infection. A total of 21 SNPs, 2 ins/del polymorphisms and a microsatellite repeat, selected on the basis of their reported association with infectious diseases including malaria in world populations, were analysed for association with Plasmodium falciparum malaria susceptibility in a case-control study with adult patients and ethnically-matched controls drawn from a disease meso- to hyperendemic and a nonendemic region of India. Among the five IFNG SNPs tested, an intron 3 and a 3′UTR SNP associated with disease in the endemic region. In addition, large (CA)n repeats of IFNG intron 1 associated with protection from severe malaria in the endemic region (severe vs. control, odds ratio=0.21, 95% CI=0.08–0.52, P=1.3×10−4). The TA11CAG haplotype (rs2069705 T/C, rs2430561 A/T, rs3138557 (CA)n, rs2069718 T/C, rs2069727 A/G, rs2069728 G/A) carrying a short CA11 repeat also exhibited very strong association with severe malaria, particularly in the endemic region (severe vs. control, OR=14.56, 95% CI=3.39–85.81, P=3×10−5). One SNP each from the IFNA8 and IFNA17 of IFNA gene cluster had a protective effect in the non-endemic region but not in the endemic region. A promoter and an intron 2 SNP of IFNAR1 were risk factors for disease and the IFNAR1 haplotype GCCAGG (rs2843710 C/G, rs2850015 C/T, +6993 C/T, rs2243594 A/G, rs1012335 G/C, rs2257167 G/C) carrying both the risk alleles strikingly associated with disease manifestation in the endemic region (severe vs. control, OR=27.14, 95% CI=3.12–1254, P=2×10−5; non-severe vs. control, OR=61.87, 95% CI=10.08–2521, P=1×10−8). The data indicates dissimilar contribution of cytokine and cytokine receptor variants to disease in populations residing in areas of differential malaria endemicity.

Allelic association, DNA resequencing and copy number variation at the metabotropic glutamate receptor GRM7 gene locus in bipolar disorder.

Genetic markers at the GRM7 gene have shown allelic association with bipolar disorder (BP) in several case–control samples including our own sample. In this report, we present results of resequencing the GRM7 gene in 32 bipolar samples and 32 random controls selected from 553 bipolar cases and 547 control samples (UCL1). Novel and potential etiological base pair changes discovered by resequencing were genotyped in the entire UCL case–control sample. We also report on the association between GRM7 and BP in a second sample of 593 patients and 642 controls (UCL2). The three most significantly associated SNPs in the original UCL1 BP GWAS sample were genotyped in the UCL2 sample, of which none were associated. After combining the genotype data for the two samples only two (rs1508724 and rs6769814) of the original three SNP markers remained significantly associated with BP. DNA sequencing revealed mutations in three cases which were absent in control subjects. A 3′-UTR SNP rs56173829 was found to be significantly associated with BP in the whole UCL sample (P = 0.035; OR = 0.482), the rare allele being less common in cases compared to controls. Bioinformatic analyses predicted a change in the centroid secondary structure of RNA and alterations in the miRNA binding sites for the mutated base of rs56173829. We also validated two deletions and a duplication within GRM7 using quantitative-PCR which provides further support for the pre-existing evidence that copy number variants at GRM7 may have a role in the etiology of BP. © 2014 The Authors. American Journal of Medical Genetics Part B: Neuropsychiatric Published by Wiley Periodicals, Inc.

Genetic variation in aldo‐keto reductase 1D1 (AKR1D1) affects expression and activity of multiple cytochrome P450s (292.1)

Human liver gene regulatory network analysis identified a cytochrome P450 subnetwork with AKR1D1 as its key regulatory driver. This study assessed the biologic importance of AKR1D1 [key enzyme in synthesis of bile acids, ligand activators of FXR, PXR and CAR, known P450 regulators] to hepatic P450 expression. Overexpression of AKR1D1 in primary human hepatocytes led to increased expression of CYP3A4, 2C8, 2C9, 2C19, and 2B6. Conversely, AKR1D1 knockdown decreased expression of these P450s. Resequencing AKR1D1 from donor livers identified a 3'‐UTR (rs1872930) SNP that was significantly associated with higher AKR1D1 mRNA. AKR1D1 3'‐UTR‐luciferase reporter studies showed that variant allele resulted in higher luciferase activity, suggesting that the SNP increases AKR1D1 mRNA stability and/or translation efficiency. AKR1D1 3'‐UTR SNP was significantly associated with increased hepatic mRNA of multiple P450s (CYP3A4, 2C8, 2C9, 2C19, and 2B6) and CYP3A4, 2C8, 2C19, and 2B6 activities. After adjusting for multiple testing, association remained significant for AKR1D1, CYP2C9, and CYP2C8 mRNA and CYP2C8 activity. These results provide new insights into the variation in expression and activity of P450s that can account for interindividual differences in drug metabolism/efficacy. In conclusion, we provide the first experimental evidence supporting a role for AKR1D1 as a key genetic regulator of P450 network.

Genetic variation in aldo‐keto reductase 1D1 (AKR1D1) affects expression and activity of multiple cytochrome P450s (LB595)

Human liver gene regulatory network analysis identified a cytochrome P450 subnetwork with AKR1D1 as its key regulatory driver. This study assessed the biologic importance of AKR1D1 [key enzyme in synthesis of bile acids, ligand activators of FXR, PXR and CAR, known P450 regulators] to hepatic P450 expression. Overexpression of AKR1D1 in primary human hepatocytes led to increased expression of CYP3A4, 2C8, 2C9, 2C19, and 2B6. Conversely, AKR1D1 knockdown decreased expression of these P450s. Resequencing AKR1D1 from donor livers identified a 3'‐UTR (rs1872930) SNP that was significantly associated with higher AKR1D1 mRNA. AKR1D1 3'‐UTR‐luciferase reporter studies showed that variant allele resulted in higher luciferase activity, suggesting that the SNP increases AKR1D1 mRNA stability and/or translation efficiency. AKR1D1 3'‐UTR SNP was significantly associated with increased hepatic mRNA of multiple P450s (CYP3A4, 2C8, 2C9, 2C19, and 2B6) and CYP3A4, 2C8, 2C19, and 2B6 activities. After adjusting for multiple testing, association remained significant for AKR1D1, CYP2C9, and CYP2C8 mRNA and CYP2C8 activity. These results provide new insights into the variation in expression and activity of P450s that can account for interindividual differences in drug metabolism/efficacy. In conclusion, we provide the first experimental evidence supporting a role for AKR1D1 as a key genetic regulator of P450 network.