UTR SNPs Research Articles

Effects of polymorphisms in the 3′ untranslated region of the porcine PPARGC1A gene on muscle fiber characteristics and meat quality traits

Peroxisome proliferator-activated receptor γ coactivator 1 α (PPARGC1A) is a transcriptional coactivator that is involved in a variety of biological processes including muscle fiber type composition. Here, we identified two single nucleotide polymorphisms (SNPs; *2690T>C and *2864T>C) and one insertion/deletion in the 3' untranslated region of porcine PPARGC1A. These SNPs were genotyped by direct sequencing in a total of 439 pigs representing three different pig breeds (Berkshire, n=156; Yorkshire, n=163; Landrace, n=120). We evaluated the effects of diplotypes of individual PPARGC1A 3'UTR SNPs on muscle fiber characteristics and meat quality traits. The *2690T>C polymorphism was significantly associated with the percentage of type I and IIb fibers for both muscle fiber number and area composition (P<0.05), and also showed a significant association with muscle pH, a parameter of meat quality (P=0.0188). The *2864T>C polymorphism was also associated with meat quality traits including muscle pH (P=0.0071), drip loss (P=0.0006), and lightness (P=0.0702), but showed no significant association with muscle fiber characteristics. Interestingly, each SNP affected PPARGC1A expression significantly at the protein level but not at the mRNA level, thereby accounting for phenotypic variability among genotypes. Taken together, our data suggest that the *2690T>C and *2864T>C polymorphisms can be used as genetic markers for selection toward improved meat quality.

Polymorphism of 3′UTR region of TNFR2 coding gene and its role in clinical tuberculosis in Han Chinese pediatric population

Genetic factors of human susceptibility to tuberculosis (TB) are multiple and their effect may be ethnic- and age-dependent. TNFR2 encoded by the TNFRSF1B gene is one of the important TNF-α receptors; its polymorphisms were previously suggested as potential markers of host susceptibility to TB. Here, genotyping of three SNPs in TNFRSF1B 3′UTR (rs1061624, rs5030792, rs3397) was performed in Han Chinese pediatric population (229 TB patients and 233 control subjects). rs5030792 was found homozygous (TT genotype) in all studied individuals. The rs3397-T allele was almost equally represented in both gender groups in this study; in particular, it was detected in 33.9% and 35.2% in female cases and controls, respectively (P=0.8). This latter result differs strikingly from an African study where rs3397-T was found in only 12.8 and 16.2% of Ghanaian female cases and controls, respectively (P=0.007 [Möller et al., 2010. Am. J. Respir. Crit. Care. Med. 181, 388–393]). In contrast, rs1061624-A allele, acting recessively, was found to be a possible risk factor for clinical TB in females (P=0.03). The rs1061624 heterozygotes were overdominant in controls versus patients (P=0.015) that warrants further study of their hypothetical advantage in TB. Neither of the common haplotypes was associated with susceptibility to TB. Compared to the published contrasting data on African (7–15%) and European (57%) populations, GTT haplotype was found in an intermediate frequency (26%). Further studies on both adult and pediatric populations in ethnically diverse settings are needed to elucidate the functionality of these 3′UTR SNPs of the TNFR2 gene.

Polymorphisms of heat shock factor‐1: Genetic diversity in an evolutionarily conserved, multifunctional regulator gene

In the present study we analyzed heat shock factor‐1 (HSF‐1) for SNPs by mining the NCBI dbSNP database and directly sequencing the 13 HSF‐1 exons (2152 nt mRNA sequence) from 60 anonymous genomic DNA samples representing 30 Caucasians and 30 African Americans and comparing these with a reference human HSF‐1 sequence (NT_037704).ResultsMining the dbSNP database revealed 6 SNPs (3 in the 3′UTR and 3 in the coding sequence). One of the coding SNPs causes a proline‐to‐threonine missense at amino acid 365 adjacent to LZ3 and one causes a frame‐shift replacement of the 26‐amino acid C‐terminal transactivation domain. Direct sequencing confirmed the P365T SNP, identified a second novel nonsynonymous SNP (L25I) located in the DNA binding domain, 2 novel 5′UTR and 2 novel 3′UTR SNPs. Four of the five 3′UTR SNPs alter predicted miRNA target sequences (MicroSNiPer) and both 5′UTR SNPs alter predicted 5′UTR secondary structure (RNAFOLD).ConclusionsWe have identified several SNPs in both the coding and untranslated regions that are likely to alter HSF‐1 function and expression level, respectively. The functional consequences of these SNPs will be analyzed by overexpressing in MEFs from HSF‐1‐null mice.

Rare BRCA1 haplotypes including 3’UTR SNPs associated with breast cancer risk

Genetic markers identifying women at an increased risk of developing breast cancer exist, yet the majority of inherited risk remains elusive. While numerous BRCA1 coding sequence mutations are associated with breast cancer risk, BRCA1 mutations account for less then 5% of breast cancer risk. Since 3' untranslated region (3'UTR) polymorphisms disrupting microRNA (miRNA) binding can be functional and can act as genetic markers of cancer risk, we tested the hypothesis that such polymorphisms in the 3'UTR of BRCA1 and haplotypes containing these functional polymorphisms may be associated with breast cancer risk. We sequenced the BRCA1 3'UTR from breast cancer patients to identify miRNA disrupting polymorphisms. We further evaluated haplotypes of this region including the identified 3'UTR variants in a large population of controls and breast cancer patients (n=221) with known breast cancer subtypes and ethnicities. We identified three 3'UTR variants in BRCA1 that are polymorphic in breast cancer populations, and haplotype analysis including these variants revealed that breast cancer patients harbor five rare haplotypes not generally found among controls (9.50% for breast cancer chromosomes, 0.11% for control chromosomes, p=0.0001). Three of these rare haplotypes contain the rs8176318 BRCA1 3'UTR functional variant. These haplotypes are not biomarkers for BRCA1 coding mutations, as they are found rarely in BRCA1 mutant breast cancer patients (1/129 patients= 0.78%). These rare BRCA1 haplotypes and 3'UTR SNPs may represent new genetic markers of breast cancer risk.

A genome-wide SNP-association study confirms a sequence variant (g.66493737C&gt;T) in the equine myostatin (MSTN) gene as the most powerful predictor of optimum racing distance for Thoroughbred racehorses

BackgroundThoroughbred horses have been selected for traits contributing to speed and stamina for centuries. It is widely recognized that inherited variation in physical and physiological characteristics is responsible for variation in individual aptitude for race distance, and that muscle phenotypes in particular are important.ResultsA genome-wide SNP-association study for optimum racing distance was performed using the EquineSNP50 Bead Chip genotyping array in a cohort of n = 118 elite Thoroughbred racehorses divergent for race distance aptitude. In a cohort-based association test we evaluated genotypic variation at 40,977 SNPs between horses suited to short distance (≤ 8 f) and middle-long distance (> 8 f) races. The most significant SNP was located on chromosome 18: BIEC2-417495 ~690 kb from the gene encoding myostatin (MSTN) [Punadj. = 6.96 × 10-6]. Considering best race distance as a quantitative phenotype, a peak of association on chromosome 18 (chr18:65809482-67545806) comprising eight SNPs encompassing a 1.7 Mb region was observed. Again, similar to the cohort-based analysis, the most significant SNP was BIEC2-417495 (Punadj. = 1.61 × 10-9; PBonf. = 6.58 × 10-5). In a candidate gene study we have previously reported a SNP (g.66493737C>T) in MSTN associated with best race distance in Thoroughbreds; however, its functional and genome-wide relevance were uncertain. Additional re-sequencing in the flanking regions of the MSTN gene revealed four novel 3' UTR SNPs and a 227 bp SINE insertion polymorphism in the 5' UTR promoter sequence. Linkage disequilibrium was highest between g.66493737C>T and BIEC2-417495 (r2 = 0.86).ConclusionsComparative association tests consistently demonstrated the g.66493737C>T SNP as the superior variant in the prediction of distance aptitude in racehorses (g.66493737C>T, P = 1.02 × 10-10; BIEC2-417495, Punadj. = 1.61 × 10-9). Functional investigations will be required to determine whether this polymorphism affects putative transcription-factor binding and gives rise to variation in gene and protein expression. Nonetheless, this study demonstrates that the g.66493737C>T SNP provides the most powerful genetic marker for prediction of race distance aptitude in Thoroughbreds.

Abstract 916: Functional significance of sulfotransferase 1A1 polymorphisms in the 3’ untranslated region in determination of enzymatic activity

Abstract SULT1A1 is the most highly expressed hepatic sulfotransferase and is involved in the biotransformation of a wide variety of endo-and xenobiotics. The inter-individual variation of SULT1A1 enzyme activity can markedly influence drug toxicity and/or treatment efficacy in the clinical setting. We and others have examined a common SNP in the coding region of SULT1A1 (Arg213His, SULT1A1*1/2), as well as several SNPs in the proximal promoter that were associated with altered enzymatic activity. However, the SNPs account for only a small proportion of variation of SULT1A1 activity in human populations. In order to identify SNPs that markedly affect SULT1A1 activity, we resequenced the 3’UTR of SULT1A1 in 97 liver samples. Direct sequencing revealed 2 SNPs (902A&amp;gt;G, 973C&amp;gt;T) in the 3’UTR and 1 SNP (1307G&amp;gt;A) in the 3’ flanking region. Interestingly, we found these SNPs are in absolute linkage disequilibrium and are closely associated with SULT1A1 expression and enzymatic activity. To further elucidate the important role of SNPs in 3’UTR, we performed haplotype analysis to predict the collective effects of functional genetic variants in the 3’ UTR regions, the SULT1A1*1/2 polymorphism and copy number variation on SULT1A1 enzymatic activity. Notably, two major haplotypes (ACGG and GTAA) were significantly correlated with SULT1A1 activity, Copy number did not influence on SULT1A1 activity in liver samples. 3’UTR SNPs are in linkage disequilibrium with SULT1A1*1/2 polymorphism, so to examine the effect of the SULT1A1*1/2 polymorphism, we performed haplotype-activity assay in 499 Caucasian and 129 African American subjects. Haplotype and regression analysis both strongly suggest that the 3’UTR SNPs are the pivotal predictor of SULT1A1 activity. MicroInspector predicts 3116C&amp;gt;T influences the binding of miR-631 to SULT1A1 3’UTR. Furthermore, in vitro luciferase reporter assays and overexpression of miRNA mimics and inhibitors indicated that SULT1A1 is a direct target of miR-631 and miR-631 differentially regulates the C and T alleles of rs1042157. The results suggest, for the first time, that allele-specific regulation of SULT1A1 by miR-631 contributes to the activity variation of SULT1A1 in population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 916.

Analysis of inherited genetic variations at theUGT1locus in the French-Canadian population

The UDP-glucuronosyltransferase UGT1 locus is composed of nine exon 1s, each flanked by a unique promoter region, and common exons (2, 3, 4, and the alternatively spliced exons 5a and 5b). Here, we characterized the genetic architecture of the UGT1 gene in a Caucasian sample. Overall, 98 variations in regulatory domains, exons and exon-intron boundaries were genotyped in 254 unrelated subjects, including 12 unreported UGT1 polymorphisms. We determined allele frequencies, computed pairwise linkage disequilibrium (LD), and inferred haplotypes; this thorough analysis yielding a limited number of common UGT1 haplotypes. Moreover, only 17 haplotype tagging single nucleotide polymorphisms (htSNPs) are required to capture most of the allelic diversity of the locus. Four haplotype blocks were inferred: Block 9/6 (UGT1A9, UGT1A7 and UGT1A6), Block 4 (UGT1A4), Block 3/1 (UGT1A3 and UGT1A1), and Block C (3'UTR). A high level of linkage exists between Blocks 9/6 and 3/1, while the 3'UTR SNPs are genetically isolated. The most common haplotype (16.5%) presents multiple deleterious alleles, mainly 1A1*28, 1A3*2, 1A6*2, and 1A7*4. More interestingly, we reveal the co-occurrences of multiple deleterious variations, some of which could be associated with interindividual differences in glucuronidation. Comparison with the HapMap data set demonstrated differences in haplotypic diversity between ethnic samples, but similarity between Caucasian cohorts, as observed previously. This report provides relevant data for further pharmacogenomic studies.

On the association of the oxidised LDL receptor 1 (OLR1) gene in patients with acute myocardial infarction or coronary artery disease

The human oxidised low-density lipoprotein receptor 1 (OLR1) gene is a functional candidate for atherosclerosis. An association of the OLR1 gene with acute myocardial infarction (AMI) or coronary artery disease (CAD) has recently been reported. In the present study a total of 677 Italian subjects, 327 CAD-free, 350 CAD, of which 190 with AMI and 160 AMI-free, was genotyped for the following four OLR1 single nucleotide polymorphisms: exon 4 K167N, IVS4 -73C>T, IVS4 -14A>G, and 3'UTR 188 C>T. No statistically significant difference was observed in allele or genotype distribution of the exon 4, intron 4, or 3'UTR SNPs in CAD patients compared to CAD-free subjects, or within CAD, in AMI patients compared to AMI-free patients. A correlation was found between the K167N G/G genotype and the increased number of obstructed vessels. Even if the OLR1 genotype frequency distribution data in CAD or AMI subjects here reported do not fully confirm the positive results of some other association studies, an association with a marker of CAD severity was observed.

T-bet polymorphisms are associated with asthma and airway hyperresponsiveness.

T-bet (TBX21 or T-box 21) is a critical regulator of T-helper 1 lineage commitment and IFN-gamma production. Knockout mice lacking T-bet develop airway hyperresponsiveness (AHR) to methacholine, peribronchial eosinophilic and lymphocytic inflammation, and increased type III collagen deposition below the bronchial epithelium basement membrane, reminiscent of both acute and chronic asthma histopathology. Little is known regarding the role of genetic variation surrounding T-bet in the development of human AHR. To assess the relationship between T-bet polymorphisms and asthma-related phenotypes using family-based association. Single nucleotide polymorphism discovery was performed by resequencing the T-bet genomic locus in 30 individuals (including 22 patients with asthma). Sixteen variants were genotyped in 580 nuclear families ascertained through offspring with asthma from the Childhood Asthma Management Program clinical trial. Haplotype patterns were determined from this genotype data. Family-based tests of association were performed with asthma, AHR, lung function, total serum immunoglobulin E, and blood eosinophil levels. We identified 24 variants. Evidence of association was observed between c.-7947 and asthma in white families using both additive (p = 0.02) or dominant models (p = 0.006). c.-7947 and three other variants were also associated with AHR (log-methacholine PC(20), p = 0.02-0.04). Haplotype analysis suggested that an AHR locus is in linkage disequilibrium with variants in the 3'UTR. Evidence of association of AHR with c.-7947, but not with other 3'UTR SNPs, was replicated in an independent cohort of adult males with AHR. These data suggest that T-bet variation contributes to airway responsiveness in asthma.

Linkage disequilibrium between polymorphisms in the human TNFRSF1B gene and their association with bone mass in perimenopausal women.

Osteoporosis is a multifactorial disease with a strong genetic component characterized by reduced bone density and increased fracture risk. A candidate locus for regulation of hip bone mineral density (BMD) has been identified on chromosome 1p36 by linkage analysis. One of the positional and functional candidate genes located within this region is the tumour necrosis factor receptor superfamily member 1B (TNFRSF1B). In order to investigate whether allelic variation in TNFRSF1B contributes to regulation of bone mass, we studied several polymorphisms of this gene in a population based cohort study of 1240 perimenopausal women from the UK. We studied a T676G change in exon 6 (196: Met-Arg) and three SNPs (G593A, T598G, and T620C) in the 3'UTR of the gene. The 3'UTR SNPs were in strong linkage disequilibrium (LD) with each other (P<0.00001), and the exon 6 SNP was in LD with G593A and T598G (P<0.00001). We found no association between T676G alleles and BMD at the spine or hip. However, haplotype analysis showed that subjects homozygous for the A593-T598-C620 haplotype (n=85) had femoral neck BMD values 5.7% lower than those who did not carry the haplotype (n=1155; P<0.00008) and this remained significant after correcting for confounding factors and multiple testing (P<0.0009). Regression analysis showed that the ATC haplotype accounted for 1.2% of the population variance in hip BMD and was the second strongest predictor after body weight. In summary, our work supports the view that allelic variation in the 3'UTR of TNFRSF1B gene contributes to the genetic regulation of bone mass, with effects that are specific for femoral neck BMD.

Linkage disequilibrium pattern in the L-myc gene in Italian and Japanese non-small-cell lung-cancer patients.

Italian and Japanese non-small-cell lung-cancer patients were genotyped for an intragenic L-myc EcoRI restriction site polymorphism previously reported to be associated with lung-tumor prognosis in Asian populations but not in Caucasians. Screening of the L-myc sequence in Italian samples allowed identification of 2 additional 3'-UTR SNPs, located 2.3-3.0 kb from the EcoRI polymorphism, but no coding polymorphism was found. No significant association was found between any of the 3 SNPs and lung-tumor prognosis in Italian patients, consistent with the reported difference between Caucasian and Asian populations. Moreover, the newly discovered polymorphisms in the Italian group were not present in Japanese patients. Significant LD between EcoRI and the 2 other SNPs was detected in the Italian population, whereas no significant LD between the 2 3'-UTR markers was detected despite their close proximity (0.7 kb). Thus, the disparate conclusions about the role of L-myc polymorphism in tumor prognosis among different populations may rest in population-specific LD between the functional gene and the L-myc polymorphism.