Expression of the glutathione peroxidase gene lacking its 3′ untranslated region

In order to investigate the expression of human cellular glutathione peroxidase (GPx), we mutated the gene encoding GPx by deleting either the 5 or 3 untranslated region (utr), subcloned the deleted fragments into plasmid pSVL followed by transfection into COS-7 cells and measured the amount of GPx expressed. When the 5 utr of the gene was deleted, GPx was not expressed. However, the deletion of the 3 utr resulted in some expression of GPx. Deletion of the poly A region of the GPx gene resulted in the expression of GPx but the level was lower than that of the full-length cGPx. The complete deletion of the 3 utr resulted in a half of the expression of the poly A deletion mutant. Thus, the expression of GPx increased according to the length of the 3 utr. These results suggest that the GPx gene carrying one SECIS on 5 utr (FEBS Lett. 312(1992)10-14) is essential for GPx expression. SECIS on 3 utr might not play a key role of GPx expression. Expression of GPx by COS-7 cells was not observed when a plasmid harboring an antisense gene was transfected.     

Identification of association between rs1057317 polymorphism in TLR4 3′-untranslated region and the susceptibility to osteoporosis

It has been proved that the expression of TLR4 is associated with a reduced risk of osteoporosis (OP). One single-nucleotide polymorphism located within the 3′-untranslated region (3′-UTR) of TLR4 may “generate” binding site of miR-34a and thereby associated with risk of OP. Bioinformatics analysis and luciferase reporter assay were used to specify the effect of polymorphisms on the interaction between miR-34a and TLR4 gene. Western blot analysis and real-time polymerase chain reaction were used to study the expressions of miR-34a, TLR4 in different groups or cells transfected with miR-34a mimics or inhibitor. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to estimate the effect of miR-34a on the apoptosis of osteoblast. TLR4 was identified as a target of miR-34a, with negative regulatory relationship predicted. The expression levels of miR-34a was comparable with each other between CC, CA, and AA groups, and the expression levels of TLR4 was evidently lower in CC compared with GG and GC groups. Also, TLR4 level in culture osteoblast (genotyped as CC) treated with miR-34a mimics was substantially downregulated compared with scramble control, while those cells (genotyped as CC) treated with miR-34a inhibitors showed increased expression of TLR4. Additionally, the apoptosis of osteoblast genotyped as CC was decreased following transfection with miR-34a mimics, while evidently promoted subsequent to transfect with miR-34a inhibitor. The regulatory association between rs1057317 polymorphism in TLR4 3′-UTR led to an inhibitory effect on the expression of TLR4 by miR-34a, which may explain the observed association between the polymorphism and the susceptibility to OP.     

PRND 3′UTR polymorphism may be associated with behavioral disturbances in Alzheimer disease

The etiology of behavioral and psychological symptoms of dementia (BPSD) is complex, including putative biological, psychological, social and environmental factors. Recent years have witnessed accumulation of data on the association between genetic factors and behavioral abnormalities in Alzheimer disease (AD). In this research paper, our aim is to evaluate the association between the APOE, CYP46, PRNP and PRND genes and the profile of neuropsychiatric symptoms in Polish subjects with AD and mild cognitive impairment (MCI). We studied 99 patients with AD and 48 subjects with MCI. The presence and profile of BPSD were evaluated at baseline and prospectively with the Neuropsychiatric Inventory (NPI). Patients were dichotomized into those having ever experienced a particular symptom and those who did not over the whole disease period. Genotyping was performed using previously described standard protocols. The prevalence of comorbid behavioral symptoms and the overall level of behavioral burden were significantly greater in AD compared with the MCI group. In AD patients, carrier status of the T allele of the 3′UTR (untranslated region) PRND polymorphism was associated with an increased cumulative behavioral load and an elevated risk for delusions, anxiety, agitation/aggression, apathy and irritability/emotional ability. Among MCI subjects, APOE ε4 carriers demonstrated a reduced risk for nighttime behavior change. No other statistically significant genotype-phenotype correlations were observed, including the APOE, CYP46 and PRNP genes. A precise estimation of the exact significance of particular polymorphisms in BPSD etiology requires future studies on large populations.     

PRND 3′UTR polymorphism may be associated with behavioral disturbances in Alzheimer disease

The etiology of behavioral and psychological symptoms of dementia (BPSD) is complex, including putative biological, psychological, social and environmental factors. Recent years have witnessed accumulation of data on the association between genetic factors and behavioral abnormalities in Alzheimer disease (AD). In this research paper, our aim was to evaluate the association between the APOE, CYP46, PRNP and PRND genes and the profile of neuropsychiatric symptoms in Polish subjects with AD and mild cognitive impairment (MCI). We studied 99 patients with AD and 48 subjects with MCI. The presence and profile of BPSD were evaluated at baseline and prospectively with the Neuropsychiatric Inventory (NPI). Patients were dichotomized into those having ever experienced a particular symptom and those who did not over the whole disease period. Genotyping was performed using previously described standard protocols. The prevalence of comorbid behavioral symptoms and the overall level of behavioral burden were significantly greater in AD compared with the MCI group. In AD patients, carrier status of the T allele of the 3′UTR (untranslated region) PRND polymorphism was associated with an increased cumulative behavioral load and an elevated risk for delusions, anxiety, agitation/aggression, apathy and irritability/emotional ability. Among MCI subjects, APOE ε4 carriers demonstrated a reduced risk for nighttime behavior change. No other statistically significant genotype-phenotype correlations were observed, including the APOE, CYP46 and PRNP genes. A precise estimation of the exact significance of particular polymorphisms in BPSD etiology requires future studies on large populations.Key words: Alzheimer disease, mild cognitive impairment, behavioral symptoms, APOE, CYP46, PRNP, PRND, polymorphisms     

Characterization of fibroblast cytoplasmic proteins that bind to the 3′ UTR of human catalse mRNA

The excessive expression of catalase protein and its activity in cultured skin fibroblast from Zellweger Syndrome (ZS), a disorder of peroxisomal biogenesis, was found to be regulated at the translational level (J. Neurochem. 67: 2373-2378, 1996). Overall there is a considerable increase in the association of catalase mRNA with polysomes in ZS cell lines as compared to control indicating translational upregulation. To investigate the possibility that RNA-protein interactions are involved in the mediation of this increase in translation, the interaction between 3 untranslated region of human catalase mRNA and human fibroblast cytoplasmic proteins were investigated by RNA gel shift assay technique. Competition experiments demonstrated that all the 600 bases of 3 UTR (of human catalase gene) was required for efficient binding. Catalase RNA- protein interaction was sensitive to the altered redox state in these in vitro assays and this RNA-protein interaction could be enhanced by the addition of -mercaptoethanol in cytoplasm from control fibroblast but not in cytoplasm from ZS fibroblast. UV cross linked RNA-protein complexes on SDS polyacrylamide gel electrophoresis revealed the presence of at least four protein bands with approximate molecular masses of 38 kDa, 50 kDa, 66 kDa and 80 kDa. The potential role of these mRNA binding proteins in the regulation of catalase gene expression is discussed.     

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.Key words: BRCA1, haplotype, microRNA, SNP, 3′UTR, breast cancer, triple negative breast cancer     

Association of the SDF1-3′A polymorphism with susceptibility to myocardial infarction in Chinese Han population

SDF-1 has been demonstrated to be involved in the pathophysiology of atherosclerosis. This study was aimed to investigate whether the SDF1-3′A polymorphism (rs1801157) is associated to myocardial infarction (MI) in a sample of Chinese Han population. A total of 560 patients with MI and 532 controls were enrolled in the study. The SDF1-3′A polymorphism was determined by polymerase chain reaction -restriction fragment length polymorphism (PCR-RFLP) analysis. A significant difference in genotype distribution and allele frequency was observed between patients and controls (P = 0.003 and P = 0.001, respectively). The A allele carriers had a significantly reduced MI risk compared with the GG homozygotes (OR, 0.69; 95% CI, 0.52–0.92; adjusted P = 0.007) in a logistic regression model after controlling conventional risk factors. The present study showed a significant association between the SDF1-3′A polymorphism and MI in Chinese Han population.     

MCP-1 −2518 A/G functional polymorphism is associated with increased susceptibility to active pulmonary tuberculosis in Tunisian patients

Monocyte chemoattractant protein-1 (MCP-1) plays crucial role in protective immunity against Mycobacterium tuberculosis (MT). In this study, we examined whether single nucleotide polymorphism (SNP) −2518 A/G (rs 1024611) of MCP-1 affect the susceptibility to active tuberculosis (TB) in Tunisian populations. Genomic DNA from patients with active TB (168 cases of pulmonary TB and 55 cases of extrapulmonary TB) and ethnically controls (150 cases) was genotyped for the MCP-1 −2518 A/G SNP by polymerase chain reaction fragment length polymorphism (PCR-RFLP). We observed that −2518 G allele and GG genotype (high MCP-1 producer) frequencies were significantly more elevated in active pulmonary TB group in comparison to control group [34 vs. 22%; P = 0.0007; 15 vs. 5%, P corrected for the number of genotypes (Pc) = 0.015; respectively]. Additionally, they were associated with increased risk development of this clinical form of TB [odds ratio (OR) = 1.83, 95% confidence intervals (CI) = 1.26–2.66; OR = 3.1, 95% CI = 1.28–7.76; respectively]. However, wild type allele −2518 A and AA genotype were over-represented in control group (78 and 62%) and seem to be protective factors against TB. Moreover, −2518 AA genotype was more frequent in control group and was associated with resistance against development of active pulmonary TB (OR = 0.56, 95% CI = 0.35–0.89, Pc = 0.03). Our findings confirm the key role of −2518 A/G SNP of MCP-1 and support its association with resistance/susceptibility to the development of active pulmonary TB in the Tunisian population.     

Clock gene PERIOD3 polymorphism is associated with susceptibility to Graves’ disease but not to Hashimoto’s thyroiditis

ABSTRACT

Circadian disruption has been linked with immune-related morbidities including autoimmune diseases. PERIOD3 (PER3) clock gene is a key player in the mammalian circadian system. This study evaluated the possible association of PER3 rs2797685 (G/A) polymorphism and susceptibility of autoimmune thyroid diseases (AITD) and assessed if this SNP contributes to disease characteristics and serum levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). The PER3 rs2797685 (G/A) polymorphism was assessed in 125 patients with AITD [Graves’ disease (GD), 69; Hashimoto’s thyroiditis (HT), 56] and 115 unrelated healthy controls. Subjects carrying at least one variant allele of PER3 rs2797685 (GA+AA) had increased risk for GD (OR 1.9, 95% CI 1–3.61, p= .05). There were no differences in the frequencies of genotypes and alleles of the PER3 rs2797685 polymorphism between HT patients and control subjects. No association was observed between genotypes of the studied SNP and any of the disease characteristics in GD and HT patients. The GA+AA genotype of PER3 rs2797685 was associated with lower levels of IL-6 in patients with Graves’ disease. There were no differences between genotypes of the studied SNP regarding TNF-α levels in GD, HT or control groups. In conclusion, this study provides the first evidence for a genetic association between GD and the PER3 gene, highlighting the possible relevance of polymorphisms in clock genes in the etiopathogenesis of AITD. However, functional studies to identify the underlying molecular mechanisms of this association are needed to translate these findings to clinical applications.     

p53 codon 72 polymorphism is associated with susceptibility to hepatocellular carcinoma in the Turkish population: a case–control study

The tumor suppressor p53 gene plays a crucial role in preventing carcinogenesis through its ability to induce cell cycle arrest and apoptosis following DNA damage and oncogene activation. A guanine (G)/cytosine (C) common single nucleotide polymorphism (SNP) at second position of codon 72 in exon 4 of p53 gene determines a arginine (Arg) to proline (Pro) (Arg72Pro) aminoacidic substitution within the proline-rich domain of p53 protein. Arg72 and Pro72 allele are different from a biochemical and biological point of view and many reports suggest that they can modulate individual cancer susceptibility. To determine the association of the p53 Arg72Pro polymorphism with the risk of hepatocellular carcinoma (HCC) development in a Turkish population, a hospital-based case–control study was designed consisting of 119 subjects with HCC and 119 cancer-free control subjects matched for age, gender, smoking and alcohol status. The genotype frequency of the p53 Arg72Pro polymorphism was determined by using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Our data shows that the Pro/Pro genotype of the p53 Arg72Pro polymorphism is associated with increased risk of HCC development in this Turkish population (OR = 3.20, 95% CI: 1.24–8.22, P = 0.02). Furthermore, according to stratified analysis, a significant association was observed between the homozygote Pro/Pro genotype and HCC risk in the subgroups of male gender (OR = 3.01, 95% CI: 1.14–7.97, P = 0.03) and patients with hepatitis B virus (HBV)-related HCC (OR = 4.04, 95% CI: 1.46–11.15, P = 0.007). Because our results suggest for the first time that the Pro/Pro homozygote of p53 Arg72Pro polymorphism may be a genetic susceptibility factor for HCC (especially in the male gender and HBV-infected patients) in the Turkish population, further independent studies are required to validate our findings in a larger series, as well as in patients of different ethnic origins.     

One functional variant in the 3′-untranslated region of TLR4 is associated with the elevated risk of ventilator-associated pneumonia in the patients with chronic obstructive pulmonary disease

The aim of this study was to identify the association polymorphism (rs11536889) in the 3′-untranslated region (3′-UTR) of Toll-like receptors 4 (TLR4) and the risk for ventilator-associated pneumonia (VAP). miRNA database online and luciferase assays were used to validate TLR4 as the target gene of miR-1236. Enzyme-linked immunosorbent assay analysis and western blot were used to analyze the level of TLR4 in different genotype groups. In the present study, miR-1236 was predicted to bind to the rs11536889 G allele rather than the rs11536889 C allele, which was further confirmed by the luciferase activity suppressed by a fragment of 3′-UTR containing the rs11536889 G allele induced by lipopolysaccharide (LPS) and interleukin-6 (IL-6). Bronchial epithelial cells isolated from participants genotyped as GG, GC, and CC, with no remarkable difference in TLR4 messenger RNA (mRNA) levels were observed among these genotype groups. After stimulating by LPS, a TLR4 ligand, the CC-genotyped cells expressed higher levels of IL-8, IL-6, and tumor necrosis factor alpha (TNF-α) on their surfaces than cells with the other genotypes. Finally, the western blot analysis results showed that the expression level of IL-8, IL-6, and TNF-α protein was much higher in the CC group than the GC and GG groups subsequent to stimulation by LPS, and the IL-8, IL-6, and TNF-α protein levels in the GC were grouped much lower compared with the GG group. These findings indicated the regulatory association of miR-1236 with TLR4 and the abnormal expression of TLR4 caused by the presence of rs11536889 in the 3′-UTR of mRNA, which interfere with its interaction with the miR-1236, contributing to the risk of VAP.     

Common variants within the interleukin 4 receptor α gene (IL4R) are associated with susceptibility to osteoarthritis

Primary osteoarthritis (OA) is a common late-onset arthritis that demonstrates a complex mode of transmittance with both joint-site and gender-specific heterogeneity. We have previously linkage-mapped an OA susceptibility locus to a 12-cM interval at chromosome 16p12.3-p12.1 in a cohort of 146 affected female sibling-pair families ascertained by total hip replacement (female-THR families), with a maximum multipoint LOD score of 1.7. Despite the low LOD score, we were encouraged to investigate this interval further following the report of a linkage to the same interval in an Icelandic pedigree with an early-onset form of hip OA. Using public databases, we searched the interval for plausible candidates and concluded that the gene encoding the interleukin 4 receptor chain (IL4R) was a particularly strong candidate based on its known role in cartilage homeostasis. We genotyped nine common single nucleotide polymorphisms (SNPs) from within IL4R, including six non-synonymous SNPs, in the 146 probands from our female-THR families (stage 1) and in an independent cohort of 310 female-THR cases (stage 2). We compared allele frequencies with those of 399 age-matched female controls. All individuals were UK Caucasians. The minor alleles of two SNPs demonstrated association in both stages, with the most significant association having a P-value of 0.004 with an odds ratio (OR) of 2.1. These two SNPs defined two associated SNP groups. Inheriting a minor SNP allele from both groups was a particular risk factor (OR=2.4, P=0.0008). Our data suggest that functional variants within the IL4R gene predispose to hip OA in Caucasian females.     

Preferential translation of Hsp83 in Leishmania requires a thermosensitive polypyrimidine-rich element in the 3′ UTR and involves scanning of the 5′ UTR

Heat shock proteins (HSPs) provide a useful system for studying developmental patterns in the digenetic Leishmania parasites, since their expression is induced in the mammalian life form. Translation regulation plays a key role in control of protein coding genes in trypanosomatids, and is directed exclusively by elements in the 3′ untranslated region (UTR). Using sequential deletions of the Leishmania Hsp83 3′ UTR (888 nucleotides [nt]), we mapped a region of 150 nt that was required, but not sufficient for preferential translation of a reporter gene at mammalian-like temperatures, suggesting that changes in RNA structure could be involved. An advanced bioinformatics package for prediction of RNA folding (UNAfold) marked the regulatory region on a highly probable structural arm that includes a polypyrimidine tract (PPT). Mutagenesis of this PPT abrogated completely preferential translation of the fused reporter gene. Furthermore, temperature elevation caused the regulatory region to melt more extensively than the same region that lacked the PPT. We propose that at elevated temperatures the regulatory element in the 3′ UTR is more accessible to mediators that promote its interaction with the basal translation components at the 5′ end during mRNA circularization. Translation initiation of Hsp83 at all temperatures appears to proceed via scanning of the 5′ UTR, since a hairpin structure abolishes expression of a fused reporter gene.     

Polymorphisms in the 3′ UTR in the neurocalcin δ gene affect mRNA stability, and confer susceptibility to diabetic nephropathy

Using a large-scale genotyping analysis of gene-based single nucleotide polymorphisms (SNPs) in Japanese type 2 diabetic patients, we have identified a gene encoding neurocalcin δ (NCALD) as a candidate for a susceptibility gene to diabetic nephropathy; the landmark SNP was found in the 3′ UTR of NCALD (rs1131863: exon 4 +1340 A vs. G, P = 0.00004, odds ratio = 1.59, 95% CI 1.27–1.98). We also discovered two other SNPs in exon 4 of this gene (+999 T/A, +1307 A/G) that showed absolute linkage disequilibrium to the landmark SNP. Subsequent in vitro functional analysis revealed that synthetic mRNA corresponding to the disease susceptible haplotype (exon 4 +1340 G, +1307 G, +999 A) was degraded faster than mRNA corresponding to the major haplotype (exon 4 +1340 A, +1307 A, +999 T), and allelic mRNA expression of the disease susceptibility allele was significantly lower than that of the major allele in normal kidney tissues. In an experiment using a short interfering RNA targeting NCALD, we found that silencing of the NCALD led to a considerable enhancement of cell migration, accompanied by a significant reduction in E-cadherin expression, and by an elevation of α smooth muscle actin expression in cultured renal proximal tubular epithelial cells. We also identified the association of the landmark SNP with the progression of diabetic nephropathy in a 8-year prospective study (A vs. G, P = 0.03, odds ratio = 1.91, 95% CI 1.07–3.42). These results suggest that the NCALD gene is a likely candidate for conferring susceptibility to diabetic nephropathy. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The C/A polymorphism in intron 11 of the XPC gene plays a crucial role in the modulation of an individual’s susceptibility to sporadic colorectal cancer

Epidemiological data show that colorectal cancer (CRC) is the second most frequent malignancy worldwide. The involvement of “minor impact genes” such as XME and DNA-repair genes in the etiology of sporadic cancer has been postulated by other authors. We focused on analyzing polymorphisms in DNA-repair genes in CRC. We considered the following genes involved in DNA-repair pathways: base excision repair (OGG1 Ser326Cys, XRCC1 Trp194Arg and Arg399Gln); nucleotide excision repair [XPA (−4)G/A, XPC C/A (i11) and A33512C (Lys939Gln), XPD Asp312Asn and A18911C (Lys751Gln), XPF Arg415Gln, XPG Asp1104His, ERCC1 C118T]; homologous recombination repair [NBS1 Glu185Gln, Rad51 135G/C, XRCC3 C18067 (Thr241Met)]. The study group consisted of 133 patients diagnosed with sporadic CRC, while the control group was composed of 100 age-matched non-cancer volunteers. Genotyping was performed by PCR and PCR-RFLP. Fisher’s exact test with a Bonferroni correction for multiple testing was used. We found that: (i) XPC C/A (i11) heterozygous variant is associated with increased risk of CRC [OR is 2.07 (95% CI 1.1391, 3.7782) P = 0.038], (ii) XPD A18911C (Lys751Gln) is associated with decreased risk of CRC [OR = 0.4497, (95% CI 0.2215, 0.9131) P = 0.031] for an individual with at least one A allele at this locus. (1) The XPC C/A (i11) genotype is associated with an increased risk of sporadic colorectal cancer. (2) The NER pathway has been highlighted in our study, as a most important in modulation of individual susceptibility to sCRC.     

Characterization of a polymorphism in the 3′ part of the chicken vitellogenin gene

Summary An allele giving rise to a polymorphism within the 3 part of the chicken vitellogenin gene was cloned, sequenced, and compared to the previously cloned allele. The polymorphism is formed by a perfect copy of 343 bp from intron 32 in tandem array with a perfect copy of 244 bp from intron 33; this 587-bp element is inserted in a head-to-tail arrangement in intron 33. We propose a mechanism in which an unequal crossing-over resulted in a vitellogenin gene with two exons 33, one of which was subsequently deleted. Thus, intron 33 was enlarged by the tandem repeats without affecting the protein-encoding sequence of the gene. At the boundaries of the repeated elements, two short direct repeats are found that resemble the recombination signals of immunoglobulin genes. They may have had a key role in the formation of the new allele.     

A 5′UTR SNP of GHRHR locus is associated with body weight and average daily gain in Chinese cattle

Growth hormone-releasing hormone receptor (GHRHR) has important functions in the regulation of the growth hormone axis and the development and proliferation of pituitary somatotropes. Moreover, some mutations in mouse GHRHR can induce the dwarfism. The objective of this paper is to reveal the association of GHRHR with growth traits in three Chinese cattle breeds, including Nanyang cattle (NY, 220), Qinchuan cattle (QC, 114), and Jiaxian cattle (JX, 142). A novel single nucleotide polymorphism (NM_181020:c.102C>T) in 5′UTR of GHRHR was identified using PCR–SSCP and DNA sequencing. The frequency of NM_181020:c.102C allele ranged from 0.926 to 0.956. We found that the locus was significantly associated with NY cattle’s body weight (BW) of 6?months, with average daily gain (ADG) of 0–6?months, and as well as with ADG of 6–12?months (p?<?0.05). The data suggested that the polymorphism (NM_181020:c.102C>T) of the GHRHR could be a molecular marker candidate for breeding of NY cattle in favor of BW.