UTR SNPs Research Articles

TranSNPs: A class of functional SNPs affecting mRNA translation potential revealed by fraction-based allelic imbalance

SummaryFew studies have explored the association between SNPs and alterations in mRNA translation potential. We developed an approach to identify SNPs that can mark allele-specific protein expression levels and could represent sources of inter-individual variation in disease risk. Using MCF7 cells under different treatments, we performed polysomal profiling followed by RNA sequencing of total or polysome-associated mRNA fractions and designed a computational approach to identify SNPs showing a significant change in the allelic balance between total and polysomal mRNA fractions. We identified 147 SNPs, 39 of which located in UTRs. Allele-specific differences at the translation level were confirmed in transfected MCF7 cells by reporter assays. Exploiting breast cancer data from TCGA we identified UTR SNPs demonstrating distinct prognosis features and altering binding sites of RNA-binding proteins. Our approach produced a catalog of tranSNPs, a class of functional SNPs associated with allele-specific translation and potentially endowed with prognostic value for disease risk.

38766 Massively Parallel Reporter Assay Reveals Functional Impact of 3™-UTR SNPs Associated with Neurological and Psychiatric Disorders

ABSTRACT IMPACT: Screening the effect of thousands of non-coding genetic variants will help identify variants important in the etiology of diseases OBJECTIVES/GOALS: Massively parallel reporter assays (MPRAs) can experimentally evaluate the impact of genetic variants on gene expression. In this study, our objective was to systematically evaluate the functional activity of 3’-UTR SNPs associated with neurological disorders and use those results to help understand their contributions to disease etiology. METHODS/STUDY POPULATION: To choose variants to evaluate with the MPRA, we first gathered SNPs from the GWAS Catalog that were associated with any neurological disorder trait with p-value < 10-5. For each SNP, we identified the region that was in linkage disequilibrium (r2 > 0.8) and retrieved all the common 3’-UTR SNPs (allele-frequency > 0.05) within that region. We used an MPRA to measure the impact of these 3’-UTR variants in SH-SY5Y neuroblastoma cells and a microglial cell line. These results were then used to train a deep-learning model to predict the impact of variants and identify features that contribute to the predictions. RESULTS/ANTICIPATED RESULTS: Of the 13,515 3’-UTR SNPs tested, 400 and 657 significantly impacted gene expression in SH-SY5Y and microglia, respectively. Of the 84 SNPs significantly impacted in both cells, the direction of impact was the same in 81. The direction of eQTL in GTEx tissues agreed with the assay SNP effect in SH-SY5Y cells but not microglial cells. The deep-learning model predicted sequence activity level correlated with the experimental activity level (Spearman’s corr = 0.45). The deep-learning model identified several predictive motifs similar to motifs of RNA-binding proteins. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study demonstrates that MPRAs can be used to evaluate the effect of non-coding variants, and the results can be used to train a machine learning model and interpret its predictions. Together, these can help identify causal variants and further understand the etiology of diseases.

Association of - 757T > C polymorphism of C-reactive protein gene with chronic periodontitis of South Indian population.

CRP gene polymorphism is common in inflammatory diseases, but such association has not been reported in periodontitis. Our objective was to interrogate SNPs of crp in chronic periodontitis in a case-control manner. DNAs were extracted from mouthwash samples of 116 volunteers using salting-out method. Selected 12 5'UTR SNPs of crp were genotyped using ARMS-PCR. TC genotype of - 757T > C polymorphism (rs3093059) showed protective association (OR- 0.29, 95%CI-0.12-0.68, and p-0.004), and wild type - 757TT showed susceptible association with a p value of 0.008 (OR-3.09, 95%CI-1.33-7.15). The observation of protective and susceptible association of crp - 757T > C polymorphism may be useful for better management and prophylaxis of periodontitis.

Genetic 3'UTR variation is associated with human pigmentation characteristics and sensitivity to sunlight.

Sunlight exposure induces signalling pathways leading to the activation of melanin synthesis and tanning response. MicroRNAs (miRNAs) can regulate the expression of genes involved in pigmentation pathways by binding to the complementary sequence in their 3'untranslated regions (3'UTRs). Therefore, 3'UTR SNPs are predicted to modify the ability of miRNAs to target genes, resulting in differential gene expression. In this study, we investigated the role in pigmentation and sun-sensitivity traits, as well as in melanoma susceptibility, of 38 different 3'UTR SNPs from 38 pigmentation-related genes. A total of 869 individuals of Spanish origin (526 melanoma cases and 343 controls) were analysed. The association of genotypic data with pigmentation traits was analysed via logistic regression. Web-based tools for predicting the effect of genetic variants in microRNA-binding sites in 3'UTR gene regions were also used. Seven 3'UTR SNPs showed a potential implication in melanoma risk phenotypes. This association is especially noticeable for two of them, rs2325813 in the MLPH gene and rs752107 in the WNT3A gene. These two SNPs were predicted to disrupt a miRNA-binding site and to impact on miRNA-mRNA interaction. To our knowledge, this is the first time that these two 3'UTR SNPs have been associated with sun-sensitivity traits. We state the potential implication of these SNPs in human pigmentation and sensitivity to sunlight, possibly as a result of changes in the level of gene expression through the disruption of putative miRNA-binding sites.

Immunomodulatory Role of HLA-G in Hematopoietic Stem Cell Transplantation Outcome

Introduction: HLA-G, the non-classical HLA class I molecule, is encoded by the human MHC complex at chromosome 6. Limited polymorphism, restricted expression and presence as isoforms are unique features of this immunomodulatory molecule that interacts with receptors ILT-2/ILT-4/KIR2DL4 on immune cells and creates a tolerogenic microenvironment. HLA-G expression and function is influenced by the polymorphism in 3’UTR region where different miRNA interact and impact mRNA stability. Objectives: The present study aimed to evaluate the clinical relevance of HLA-G in Hematopoietic Stem Cell Transplantation (HSCT). Material and Methods: We enrolled 50 North Indian patients (diagnosed with acute leukemia or aplastic anemia) who underwent HSCT and 202 healthy controls belonging to same ethnicity. We investigated HLA-G 3’UTR exon8 polymorphism, 14 bp insertion/deletion (ins/del) and SNPs. Exon8-14 bp ins/del was detected using sequence specific primers and SNPs were identified by sequencing. The soluble HLA-G levels and Treg cell frequency were estimated at pre and post transplant days 15,30,60,90,GvHD. Results: In this cohort, although the 14 bp ins/del genotype was predominant among controls, the sHLA-G levels were highest in those carrying14bp ins/ins genotype. Based on SNP linkage, 8 UTR haplotypes were identified. The sHLA-G levels were higher in individuals with 14 bp-INS linked haplotypes UTR2 and UTR4, in comparison to those with UTR 1, 6 and 7 (intermediate levels) and with 14 bp-DEL linked haplotypes UTR3 and UTR5 (low level). 40% of HSCT recipients experienced graft versus host disease (GvHD). Recipients with 14 bp ins/ins genotype had lower incidence of GvHD compared to those with del/del or ins/del genotype. Higher percentage of recipients carrying HLA-G 3’UTR SNPs +3027 CC vs CA/AA, +3035 CC vs TT/TC experienced GvHD. Majority of recipients carrying UTR1 and UTR3 developed GvHD. Further, sHLA-G levels were significantly lower at the time of GvHD, compared to the pre and post-transplant time points. The Treg cell frequency also correlated with sHLA-G levels. Discussion: GvHD is the major cause of morbidity and mortality post HSCT. Investigating HLA-G 3’UTR polymorphism, sHLA-G levels and Treg cell frequency may be beneficial for transplant monitoring. HLA-G molecule could be proposed as possible biomarker for predicting GvHD and HSCT outcome. All India Institute of Medical Sciences, New Delhi. Indian Council of Medical Research, India.

OR34 Examination of HLA DP inton/exon variation identifies two DPB1 evolutionary groups including intron 2 STR variants that may regulate DPB1 expression levels

Recent studies showed the existence of biologically relevant DPB1 expression variants that associate with two 3′UTR SNPs of DPB1. The knowledge of sequence variation in DPA1 and DPB1 alleles is limited to a few exons. Only a few alleles are fully sequenced resulting in limitations for DP typing by NGS. We developed robust and efficient methods for long range amplification and molecular cloning that allowed us to examine and characterize numerous alleles. Clones were sequenced by NGS and consensus was generated by a novel de novo assembly algorithm. The fragments generated span exon 1–4 and 2–4 of DPA1 and DPB1. We characterized 16 DPA1 and 40 DPB1 alleles defined but their unique sequences in 63 samples. We obtained novel sequence information in 11 DPA1 alleles including 7 extended sequences, 3 novel intron variants and 1 novel exon variant. In DPB1 we extended the coverage in 20 allleles and identified 12 novel intron variants and 2 novel exon variants. In addition to the well known variations in exons, the long DPA1 intron1 (3556–3590 nt) showed 201 SNPs and 17 indels. DPA1 intron 2 (340 nt) and intron 3 (214 nt) are short, not variable in size with low polymorphism (12 and 9 SNP, respectively). In DPB1 intron 3 is conserved in length (547 nt) and presents only 17 SNPs. Intron 2 of DPB1 (3952–4025 nt) has the largest variability with 176 SNPs and 9 indels; in addition this intron has one variable STR region located at 44 nt before the beginning of exon 3 with 4–14 repeats of the tetramer AAGG. Two groups of alleles that have only 4 repeats (short STR) and 8–14 repeats (long STR) were identified. Also, phylogenetic trees define two distinct groups at each, DPA1 and DPB1 loci. The DPB1 clusters are tighter when exon 2 is excluded from the analysis and correlate well with the intron 2 STR short and long variants. We observed that the 3′ UTR variants of DPB1 that correlate with high and low expression, also associate strictly in a biunivocal fashion with the short and long intron 2 STR variants, respectively. Our studies provide useful information for mapping reads in NGS typing. In addition our findings identify potential regulatory elements that may affect gene expression possibly by altering the rate of mRNA splicing. In transplantation, the expression levels of the mismatched allele appear to determine both immunogenicity and outcome.

In silico analysis of deleterious single nucleotide polymorphisms in human BUB1 mitotic checkpoint serine/threonine kinase B gene

One of the major challenges in the analysis of human genetic variation is to distinguish mutations that are functionally neutral from those that contribute to disease. BubR1 is a key protein mediating spindle-checkpoint activation that plays a role in the inhibition of the anaphase-promoting complex/cyclosome (APC/C), delaying the onset of anaphase and ensuring proper chromosome segregation. Owing to the importance of BUB1B gene in mitotic checkpoint a functional analysis using different in silico approaches was undertaken to explore the possible associations between genetic mutations and phenotypic variation. In this work we found that 3 nsSNPs I82N, P334L and R814H have a functional effect on protein function and stability. A literature search revealed that R814H was already implicated in human diseases. Additionally, 2 SNPs in the 5′ UTR region was predicted to exhibit a pattern change in the internal ribosome entry site (IRES), and eight MicroRNA binding sites were found to be highly affected due to 3′ UTR SNPs. These in silico predictions will provide useful information in selecting the target SNPs that are likely to have functional impact on the BUB1B gene.

3’UTR SNPs and Haplotypes in the GATA4 Gene Contribute to the Genetic Risk of Congenital Heart Disease

Introduction and objectivesSingle-nucleotide polymorphisms within a microRNA binding site can have different effects on gene expression, influencing the risk of disease. This study aimed to evaluate the association between single-nucleotide polymorphisms and haplotypes in the 3’UTR of the GATA4 gene and congenital heart disease risk. MethodsBioinformatics algorithms were used to analyze single-nucleotide polymorphisms in putative microRNA-binding sites of GATA4 3’UTR and to calculate the difference in free energy of hybridization (ΔFE, kcal/mol) for each wild-type vs the variant allele. ResultsThe study population comprised 146 Caucasian patients (73 males; 6.68 ± 7.79 years) and a 265 healthy newborn participants (147 males). The sum of all |ΔFE| was considered to predict the biological importance of single-nucleotide polymorphisms binding more microRNAs. Next, the 4 polymorphisms (+1158C > T, +1256 A > T, +1355 G > A, +1521C > G) with the highest predicted |ΔFEtot| (9.91, 14.85, 11.03, 21.66kcal/mol, respectively) were genotyped in a case-control study (146 patients and 250 controls). Applying a correction for multiple testing only the +1158 T allele was found to be associated with a reduced risk showing significant difference between patients and controls. Haplotype analysis showed that the T-T-G-C haplotype (more uncommon in congenital heart diseases than in controls) was associated with a significantly decreased risk (P = .03), while the rare C-A-A-C haplotype, which was very uncommon in controls (0.3%) compared with the disease (2.4%), was associated with a 4-fold increased risk of disease (P = .04). ConclusionsCommon variants in 3’UTR of the GATA4 gene jointly interact, affecting the congenital heart disease susceptibility, probably by altering microRNA posttranscriptional regulation.

In-Silico Computing of the Most Deleterious nsSNPs in HBA1 Gene.

Backgroundα-Thalassemia (α-thal) is a genetic disorder caused by the substitution of single amino acid or large deletions in the HBA1 and/or HBA2 genes.MethodUsing modern bioinformatics tools as a systematic in-silico approach to predict the deleterious SNPs in the HBA1 gene and its significant pathogenic impact on the functions and structure of HBA1 protein was predicted.Results and DiscussionA total of 389 SNPs in HBA1 were retrieved from dbSNP database, which includes: 201 non-coding synonymous (nsSNPs), 43 human active SNPs, 16 intronic SNPs, 11 mRNA 3′ UTR SNPs, 9 coding synonymous SNPs, 9 5′ UTR SNPs and other types. Structural homology-based method (PolyPhen) and sequence homology-based tool (SIFT), SNPs&Go, PROVEAN and PANTHER revealed that 2.4% of the nsSNPs are pathogenic.ConclusionsA total of 5 nsSNPs (G60V, K17M, K17T, L92F and W15R) were predicted to be responsible for the structural and functional modifications of HBA1 protein. It is evident from the deep comprehensive in-silico analysis that, two nsSNPs such as G60Vand W15R in HBA1 are highly deleterious. These “2 pathogenic nsSNPs” can be considered for wet-lab confirmatory analysis.

Genotype Distribution of Dihydrofolatereductase Variants and their Role in Disease Susceptibility to Acute Lymphoblastic Leukemia in Indian Population: An Experimental and Computational Analysis

Objectives: To establish the allele and genotype frequencies of dihydrofolate reductase (DHFR) -317A>G and -680C>A variants in Indian population, to find the association of these variants with the risk of acute lymphoblastic leukemia (ALL) and to analyze the effects of non-synonymous SNPs (nsSNPs) and 3’untranslated region (3’UTR)variants of DHFR gene on its structure and function using in-silico tools. Methods: A total of 235 unrelated healthy volunteers (controls) and 127 ALL patients (cases) were recruited for the study. DNA was extracted from peripheral leucocytes. Genotyping of DHFR polymorphisms was done by realtime PCR. We investigated the deleterious effect of nsSNPs and variants in 3’UTR of DHFR gene through computational platforms. Results: In the present study, the frequency of DHFR -317G and -680 A alleles was found to be 33.3% and 59.8% respectively. The studied DHFR variants (rs408626 and rs442767) did not confer a significant risk for ALL. Insilico analysis revealed that three nsSNPs potentially affect the structure, function and activity of the DHFR protein. Four microRNA binding sites were found to be highly affected due to 3’UTR SNPs. Further, docking simulation suggested that the order of binding affinity of methotrexate towards native and all three mutant forms of DHFR is D153V (rs121913223)>native>G18R (rs61736208)>D187Y (rs200904105). Conclusion: This is the first study to report the normative genotype distribution of DHFR variants in Indian population and also to report that DHFR (-317A>G and -680C>A) variants do not confer a potential risk for development of ALL. Inter-ethnic differences exist in the distribution of DHFR variant genotypes, and this can lead to variability in therapeutic response to DHFR substrates. Protein sequence analysis revealed rs200904105 influences the phosphorylation process (post-translational modification) of DHFR and docking simulation suggested methotrexate to have a higher affinity towards rs121913223 mutant form. Therefore, studies are warranted to explore the clinical impact of these variants in the Indian population.

HLA-G 3'UTR Polymorphisms Impact the Prognosis of Stage II-III CRC Patients in Fluoropyrimidine-Based Treatment.

An important hallmark of CRC is the evasion of immune surveillance. HLA-G is a negative regulator of host’s immune response. Overexpression of HLA-G protein in primary tumour CRC tissues has already been associated to worse prognosis; however a definition of the role of immunogenetic host background is still lacking. Germline polymorphisms in the 3’UTR region of HLA-G influence the magnitude of the protein by modulating HLA-G mRNA stability. Soluble HLA-G has been associated to 3’UTR +2960 Ins/Ins and +3035 C/T (lower levels) and +3187 G/G (high levels) genotypes. HLA-G 3’UTR SNPs have never been explored in CRC outcome. The purpose of this study was to investigate if common HLA-G 3’UTR polymorphisms have an impact on DFS and OS of 253 stage II-III CRC patients, after primary surgery and ADJ-CT based on FL. The 3’UTR was sequenced and SNPs were analyzed for their association with survival by Kaplan-Meier and multivariate Cox models; results underwent internal validation using a resampling method (bootstrap analysis). In a multivariate analysis, we estimated an association with improved DFS in Ins allele (Ins/Del +Ins/Ins) carriers (HR 0.60, 95% CI 0.38–0.93, P = 0.023) and in patients with +3035 C/T genotype (HR 0.51, 95% CI 0.26–0.99, P = 0.045). The +3187 G/G mutated carriers (G/G vs A/A+A/G) were associated to a worst prognosis in both DFS (HR 2.46, 95% CI 1.19–5.05, P = 0.015) and OS (HR 2.71, 95% CI 1.16–6.63, P = 0.022). Our study shows a prognostic and independent role of 3 HLA-G 3’UTR SNPs, +2960 14-bp INDEL, +3035 C>T, and +3187 A>G.

Human aquaporin 4 gene polymorphisms in Chinese patients with neuromyelitis optica

ObjectiveTo investigate the association between aquaporin 4 (AQP4) gene polymorphisms and Chinese patients with neuromyelitis optica (NMO). Methods122 consecutive anti-AQP4 autoantibody (NMO-IgG) seropositive cases were enrolled for gene sequencing. Furthermore, ten SNPs were selected and genotyped for a case–control association analysis on 208 cases and 204 healthy subjects. Results14 novel SNPs were identified, while there were no nonsynonymous mutations and their frequency was low. The heterozygous genotype at two 3′ UTR SNPs was significantly higher in cases than controls: the A/T genotype of SNP rs1058424 (54.81% vs. 42.65%, padjusted=0.024, OR=1.670, 95% CI=1.071–2.605) and the C/T genotype of SNP rs3763043 (53.85% vs. 42.65%, padjusted=0.028, OR=1.638, 95% CI=1.054–2.545). Moreover, the 3′ UTR haplotype ATATGGAT may be protective for NMO (7.67% vs. 12.18%, p=0.042). ConclusionsPolymorphisms in the coding region of AQP4 are unlikely to confer NMO susceptibility. However, the 3′ UTR of this gene presents several polymorphic sites that may affect NMO risk in the Chinese.

Identification and analysis of a SMAD3 cis-acting eQTL operating in primary osteoarthritis and in the aneurysms and osteoarthritis syndrome

SummaryObjectiveThe TGF-β pathway plays a central role in joint development with polymorphism in TGF-β pathway genes implicated in osteoarthritis susceptibility. One association is to rs12901499, within intron 1 of SMAD3. Since rs12901499 is not in linkage disequilibrium with a non-synonymous polymorphism, it is likely the association is operating by influencing expression of SMAD3.DesignUsing tissues from the joints of primary osteoarthritis patients who had undergone joint replacement we measured the overall expression of SMAD3 by quantitative real-time PCR. We also measured allelic expression of SMAD3 using these tissues and vascular smooth muscle cells from patients with aneurysms and osteoarthritis syndrome, a rare condition featuring early-onset osteoarthritis. We tested the functional effect of SNPs in vitro using luciferase assays and assessed association with osteoarthritis using a large osteoarthritis case–control dataset.ResultsWe observed that genotype at rs12901499 did not correlate with overall SMAD3 expression or allelic expression. However, genotype at a 3′UTR SNP, rs8031440, did correlate with SMAD3 expression in cartilage (P = 0.005) which was supported by allelic expression data showing that the G allele correlated with decreased SMAD3 expression in joint tissues and vascular smooth muscle cells. This G allele was underrepresented in osteoarthritis cases vs controls (P = 0.027, odds ratio = 0.921). rs8031440 is in perfect linkage disequilibrium with five other SMAD3 3′UTR SNPs and our luciferase analysis identified rs3743342 and rs12595334 as being functional.ConclusionSMAD3 is subject to cis-acting regulatory polymorphism in the tissues of relevance to both primary osteoarthritis and the aneurysms-osteoarthritis syndrome.

Role of miRNA binding site SNPs in candidate genes in a North Indian schizophrenia cohort

Schizophrenia (SZ) is a debilitating neuropsychiatric disorder with ~80% heritability. Despite several genetic studies including linkage and candidate gene association and more recently GWAS, which have identified several risk variants, the total heritability of SZ remains elusive. In addition, a number of gene expression studies have reported dysregulation of candidate genes both in brain and blood of SZ cases compared to controls. Although, the role of coding, promoter, intergenic and UTR SNPs, have been demonstrated, very little is known about the role of miRNA binding site SNPs. In this study, we undertook to investigate the association, if any, of this important class of regulatory variants with SZ. Using in silico prediction tools, 27 functionally relevant SNPs from around 150 candidate genes were prioritized and genotyped in a north Indian SZ cohort (n=507 cases; n=522 controls). Test of association of these SNPs showed only one variant rs7430 in PPP3CC to be associated (p=0.01) with SZ. Analysis of genotype data in a subset of patients (TD positive n=89; TD negative n=160) with Tardive dyskinesia (TD), an iatrogenic disorder of SZ, showed association of rs4846049 in MTHFR (p=0.04) & rs17881908 in GCLM (p= 0.05 ) with this condition. Further regression analysis of the genotype data with neurocognitive measures in a subset (cases n=152; controls n=290) of the study cohort, showed significant association of nine SNPs (p< 0.05) with different domains of cognition. Based on this moderately powered study, the contribution of miRNA binding site SNPs in candidate genes to SZ and to TD seems negligible. However, their promising contribution to cognitive parameters warrants additional investigations.

Targeted Re-Sequencing Identified rs3106189 at the 5′ UTR of TAPBP and rs1052918 at the 3′ UTR of TCF3 to Be Associated with the Overall Survival of Colorectal Cancer Patients

Recent studies have demonstrated the power of deep re-sequencing of the whole genome or exome in understanding cancer genomes. However, targeted capture of selected genomic whole gene-body regions, rather than the whole exome, have several advantages: 1) the genes can be selected based on biology or a hypothesis; 2) mutations in promoter and intronic regions, which have important regulatory roles, can be investigated; and 3) less expensive than whole genome or whole exome sequencing. Therefore, we designed custom high-density oligonucleotide microarrays (NimbleGen Inc.) to capture approximately 1.7 Mb target regions comprising the genomic regions of 28 genes related to colorectal cancer including genes belonging to the WNT signaling pathway, as well as important transcription factors or colon-specific genes that are over expressed in colorectal cancer (CRC). The 1.7 Mb targeted regions were sequenced with a coverage ranged from 32× to 45× for the 28 genes. We identified a total of 2342 sequence variations in the CRC and corresponding adjacent normal tissues. Among them, 738 were novel sequence variations based on comparisons with the SNP database (dbSNP135). We validated 56 of 66 SNPs in a separate cohort of 30 CRC tissues using Sequenom MassARRAY iPLEX Platform, suggesting a validation rate of at least 85% (56/66). We found 15 missense mutations among the exonic variations, 21 synonymous SNPs that were predicted to change the exonic splicing motifs, 31 UTR SNPs that were predicted to occur at the transcription factor binding sites, 20 intronic SNPs located near the splicing sites, 43 SNPs in conserved transcription factor binding sites and 32 in CpG islands. Finally, we determined that rs3106189, localized to the 5′ UTR of antigen presenting tapasin binding protein (TAPBP), and rs1052918, localized to the 3′ UTR of transcription factor 3 (TCF3), were associated with overall survival of CRC patients.

Independent effects of 5′ and 3′ functional variants in the serotonin transporter gene on suicidal behavior in the context of childhood trauma

The serotonin transporter, encoded by the SLC6A4 gene, influences the synaptic actions of serotonin and is responsive to stress hormones. We hypothesized that 5-HTTLPR, a functional SLC6A4 promoter polymorphism, and two tightly-linked, putatively functional 3′ UTR SNPs (rs3813034, rs1042173) might have independent effects on suicidal behavior in the context of childhood trauma (CT).DNA and Childhood Trauma Questionnaire scores were available for a total of 474 African Americans, including 112 suicide attempters and 362 non-suicide attempters. Genotyping was performed for the triallelic 5-HTTLPR polymorphism, 14 SLC6A4 haplotype-tagging SNPs, and 186 ancestry informative markers.There were independent G × E interactive effects of 5-HTTLPR (p = 0.017) and the rs3813034–rs1042173 diplotype (p = 0.011) on suicidal behavior. In individuals exposed to high CT the risk of suicide attempt was 0.52 in carriers of the low activity 5-HTTLPR variant and 0.32 in medium/high activity variant carriers. Likewise, CT exposed carriers of the major rs3813034–rs1042173 ATAT diplotype had an increased risk of suicidal behavior relative to the ATCG/CGCG diplotype carriers (0.40 vs 0.31). Neither the 5′ nor the 3′ functional variants had an effect in individuals without CT: suicide attempt risk = 0.12–0.22. In individuals exposed to high CT the prevalence of suicide attempt was 0.56 in carriers of both 5′ and 3′ risk variants, 0.39 in carriers of one risk variant and 0.25 in individuals without either risk variant.Our findings suggest that the 5′ and 3′SLC6A4 functional variants have independent effects on the risk for suicidal behavior in CT exposed individuals.

Functional polymorphisms of xenobiotics metabolizing enzymes—a research topic

The human genome harbors an impressive number of genes encoding enzymes that primarily metabolize or transport drugs or other xenobiotics (XMEs). Genetic and functional variation in these genes is tremendous and has complex consequences, depending, for example, on whether enzyme structure or expression is affected, or whether the produced metabolite is pharmacologically or toxicologically active or not. Despite numerous impressive examples of the impact of genetic variation on pharmacokinetics and drug response, today's knowledge is incomplete regarding most XME genes and fragmentary even for many well-investigated XMEs. This is one of the reasons why clinical pharmacogenetic studies are often controversial and clinical application in personalized medicine is presently limited. Advanced technology and ongoing large-scale projects are rapidly uncovering the existing genetic variation in all populations on earth, ultimately enabling the personal genome in the very near future. A wealth of mostly rare novel variants is awaiting functional characterization either by high-throughput expression/phenotyping techniques or by prediction using improved algorithms to estimate functional relevance. With this Research Topic we would like to give an up-to-date overview about the current knowledge in this field by covering both, known hard facts as well as cutting-edge advancement in novel genetic and genomic variation of XMEs and their functional consequences. Five major subtopics which include 20 research or review papers are included in this E-book. These are the following: History and current knowledge of XMEs Clinical application of CYP2C19 pharmacogenetics toward more personalized medicine (Lee, 2013, review). Pharmacogenetics of cytochrome P450 2B6 (CYP2B6): advances on polymorphisms, mechanisms, and clinical relevance (Zanger and Klein, 2013, review). Pharmacogenetics of human ABC transporter ABCC11: new insights into apocrine gland growth and metabolite secretion (Ishikawa et al., 2013, review). Pharmacogenomics of cytochrome P450 3A4: recent progress toward the “missing heritability” problem (Klein and Zanger, 2013, review). Clinical implications of XME gene variants ABCB1 4036A>G and 1236C>T polymorphisms affect plasma efavirenz levels in South African HIV/AIDS patients (Swart et al., 2012, research article). Genetic variations in drug-induced liver injury (DILI): resolving the puzzle (Stephens et al., 2012, opinion). MDMA, methamphetamine, and CYP2D6 pharmacogenetics: what is clinically relevant? (de la Torre et al., 2012, review). Molecular interactions between NAFLD and xenobiotic metabolism (Naik et al., 2013, review). Toward a clinical practice guide in pharmacogenomics testing for functional polymorphisms of drug-metabolizing enzymes. Gene/drug pairs and barriers perceived in Spain (Agundez et al., 2012, perspective). Inter/intraethnic variability of XME gene variants Characterization of the genetic variation present in CYP3A4 in three South African populations (Drogemoller et al., 2013, research article). Frequencies of 23 functionally significant variant alleles related with metabolism of antineoplastic drugs in the Chilean population: comparison with Caucasian and Asian populations (Roco et al., 2012, research article). Pharmacogenomic diversity among Brazilians: influence of ancestry, self-reported color, and geographical origin (Suarez-Kurtz et al., 2012, review). Regulation of XME gene expression Impact of the interaction between 3′-UTR SNPs and microRNA on the expression of human xenobiotic metabolism enzyme and transporter genes (Wei et al., 2012, research article). Molecular mechanisms of genetic variation and transcriptional regulation of CYP2C19 (Helsby and Burns, 2012, review). Pharmacogenetics in cancer therapy Impact of genetic polymorphisms on chemotherapy toxicity in childhood acute lymphoblastic leukemia (Gervasini and Vagace, 2012, review). Multilocus genotypes of relevance for drug metabolizing enzymes and therapy with thiopurines in patients with acute lymphoblastic leukemia (Stocco et al., 2013, review). Functional polymorphisms in xenobiotic metabolizing enzymes and their impact on the therapy of breast cancer (Vianna-Jorge et al., 2013, review). High-resolution melting analysis of the common c.1905+1G>A mutation causing dihydropyrimidine dehydrogenase deficiency and lethal 5-fluorouracil toxicity (Borras et al., 2013, research article). Polymorphisms of phase I and phase II enzymes and breast cancer risk (Justenhoven, 2012, review). Analysis of the functional polymorphism in the cytochrome P450 CYP2C8 gene rs11572080 with regard to colorectal cancer risk (Ladero et al., 2012, research article).

Positive selection on the osteoarthritis-risk and decreased-height associated variants at the GDF5 gene in East Asians.

GDF5 is a member of the bone morphogenetic protein (BMP) gene family, and plays an important role in the development of the skeletal system. Variants of the gene are associated with osteoarthritis and height in some human populations. Here, we resequenced the gene in individuals from four geographically separated human populations, and found that the evolution of the promoter region deviated from neutral expectations, with the sequence evolution driven by positive selection in the East Asian population, especially the haplotypes carrying the derived alleles of 5′ UTR SNPs rs143384 and rs143383. The derived alleles of rs143384 and rs143383, which are associated with a risk of osteoarthritis and decreased height, have high frequencies in non-Africans and show strong extended haplotype homozygosity and high population differentiation in East Asian. It is concluded that positive selection has driven the rapid evolution of the two osteoarthritis osteoarthritis-risk and decreased height associated variants of the human GDF5 gene, and supports the suggestion that the reduction in body size during the terminal Pleistocene and Holocene period might have been an adaptive process influenced by genetic factors.

Genetic Architecture of MicroRNA Expression: Implications for the Transcriptome and Complex Traits

We sought to comprehensively and systematically characterize the relationship between genetic variation, miRNA expression, and mRNA expression. Genome-wide expression profiling of samples of European and African ancestry identified in each population hundreds of miRNAs whose increased expression is correlated with correspondingly reduced expression of target mRNAs. We scanned 3' UTR SNPs with a potential functional effect on miRNA binding for cis-acting expression quantitative trait loci (eQTLs) for the corresponding proximal target genes. To extend sequence-based, localized analyses of SNP effect on miRNA binding, we proceeded to dissect the genetic basis of miRNA expression variation; we mapped miRNA expression levels-as quantitative traits-to loci in the genome as miRNA eQTLs, demonstrating that miRNA expression is under significant genetic control. We found that SNPs associated with miRNA expression are significantly enriched with those SNPs already shown to be associated with mRNA. Moreover, we discovered that many of the miRNA-associated genetic variations identified in our study are associated with a broad spectrum of human complex traits from the National Human Genome Research Institute catalog of published genome-wide association studies. Experimentally, we replicated miRNA-induced mRNA expression inhibition and the cis-eQTL relationship to the target gene for several identified relationships among SNPs, miRNAs, and mRNAs in an independent set of samples; furthermore, we conducted miRNA overexpression and inhibition experiments to functionally validate the miRNA-mRNA relationships. This study extends our understanding of the genetic regulation of the transcriptome and suggests that genetic variation might underlie observed relationships between miRNAs and mRNAs more commonly than has previously been appreciated.

Mutagen-specific mutation signature determines global microRNA binding.

Micro-RNAs (miRNAs) are small non-coding RNAs that regulate gene products at the post-transcriptional level. It is thought that loss of cell regulation by miRNAs supports cancer development. Based on whole genome sequencing of a melanoma tumor, we predict, using three different computational algorithms, that the melanoma somatic mutations globally reduce binding of miRNAs to the mutated 3′UTRs. This phenomenon reflects the nature of the characteristic UV-induced mutation, C-to-T. Furthermore, we show that seed regions are enriched with Guanine, thus rendering miRNAs prone to reduced binding to UV-mutated 3′UTRs. Accordingly, mutation patterns in non UV-induced malignancies e.g. lung cancer and leukemia do not yield similar predictions. It is suggested that UV-induced disruption of miRNA-mediated gene regulation plays a carcinogenic role. Remarkably, dark-skinned populations have significantly higher GC content in 3′UTR SNPs than light-skinned populations, which implies on evolutionary pressure to preserve regulation by trans-acting oligonucleotides under conditions with excess UV radiation.