Frequency of HLA-DQA1 alleles in the Japanese population.

One of the HLA class II genes, HLA-DQA1, was typed from 290 unrelated healthy Japanese using the oligonucleotide typing method. The HLA-DQA1 gene was enzymatically amplified and typed by dot-blot hybridizations with 10 sequence-specific oligonucleotide probes labeled nonradioactively. Using this method, the HLA-DQA1 genotype was theoretically classified into 36 genotypes: 8 homozygous and 28 heterozygous ones. Actually, 26 genotypes were observed in the present study, and the gene frequency of each allele was calculated. The observed numbers were in accordance with the numbers expected under the Hardy-Weinberg equilibrium. The HLA-DQA1 genotype was also determined in aged bloodstains. Since the genotype is polymorphic in the Japanese population and a very small amount of blood is required for determination, this typing is particularly useful for forensic analysis.     

Myeloperoxidase promoter polymorphism -463G is associated with more severe clinical expression of cystic fibrosis pulmonary disease

The severity of cystic fibrosis (CF) pulmonary disease is not directly related to CFTR genotype but depends upon several parameters, including neutrophil-dominated inflammation. Identification of agents modulating inflammation constitutes a relevant goal. Myeloperoxidase (MPO) is involved in both microbicidal and proinflammatory neutrophil activities. The aim of this study was to evaluate whether the -463GA MPO promoter polymorphism is linked to clinical severity of CF-associated pulmonary inflammation. This polymorphism significantly affects the level of MPO gene expression in leukocytes and the G allele is more expressing than the A allele. We show that MPO genotype significantly influences the severity of pulmonary disease in early stages, prior to the development of chronic lung infections, with GG genotype being associated with more severe CF disease. Our findings indicate that the level of MPO gene expression influences the CF pathogenesis, presumably reflecting cellular damage by MPO-generated oxidants or other activity of MPO in airway inflammation.     

LIPG promoter polymorphism is associated with ischemic stroke in Korean population

Endothelial lipase (LIPG) is a member of the triglyceride lipase family which includes hepatic lipase and lipoprotein lipase. Its activity is related to clinically important parameters like blood lipid levels, hypertension, and obesity. In this work, we investigated the association of a LIPG promoter polymorphism, rs9958947C>T, with susceptibility to ischemic stroke in a Korean population. A total of 1,144 subjects (656 cerebral infarction patients and 488 controls) were enrolled on a voluntary basis. The rs9958947C>T polymorphism was genotyped using the single-base extension method. The association of rs9958947C>T with disease status was evaluated by statistical analyses. The frequencies of the rs9958947 C and T alleles were significantly different between the stroke patient group and control group (OR [95% CI], 1.300 [1.000?C1.691], P=0.0449). A significantly higher frequency of the CT+TT genotype was observed in the patient group compared to the control group (CC/CT+TT, OR [95% CI], 1.632 [1.094?C2.435], P=0.0164). The results suggest that the T allele of the LIPG promoter polymorphism rs9958947C>T should be considered as a genetic risk factor for ischemic stroke. Further association studies in other ethnic populations would help to generalize this hypothesis.     

Development of a high resolution melting method for genotyping of risk HLA-DQA1 and PLA2R1 alleles and ethnic distribution of these risk alleles

Recent studies have demonstrated that alleles at single nucleotide polymorphisms (SNPs) rs2187668 and rs4664308 within genes HLA-DQA1 and PLA2R1, respectively, had a significant impact on the susceptibility to idiopathic membranous nephropathy (IMN). Analysis of the two genomic loci could identify alleles for individuals at risk for IMN. Conventional methods for genotyping are labor intensive, expensive or time consuming. High resolution melting (HRM) is a new technique for genotyping and has the advantages of simplicity, speed, high sensitivity and low cost. Here, we describe genotyping of SNPs rs2187668 and rs4664308 using HRM. In this study, we identified polymorphisms of rs2187668 and rs4664308 in 480 healthy unrelated Chinese volunteers of two ethnic groups from three different geographical areas in China. The two genomic loci were genotyped by HRM using a saturating fluorescent dye SYTO® 9 on 7900 HT and RG 6000 instruments, and were further confirmed by direct DNA sequencing. Three different SNP genotypes were sufficiently distinguished by HRM with mean sensitivity of 98.8% and mean error rate of 1.9%. In addition, the allele frequencies varied greatly based on ethnic or geographic origins. In conclusion, HRM is a rapid, cost efficient, sensitive, suitable technique for genotyping, and simple enough to be readily implemented in a diagnostic laboratory. We believe this will be a valuable technique for determining the genotype of rs2187668 and rs4664308 and for assessing individual susceptibility to IMN.     

Thomsen-Friedenreich antigen expression in gastric carcinomas is associated with MUC1 mucin VNTR polymorphism

Aberrant glycosylation of mucins is a common phenomenon associated with oncogenic transformation. We investigated the association between expression of the tumor-associated antigens T, Tn, and sialyl-Tn and polymorphism in the length of the MUC1 mucin tandem repeat in a series of gastric carcinomas. We further evaluated the relevance of MUC1 tandem repeat length on the expression of these tumor-associated carbohydrate antigens (TACAs) using a gastric carcinoma cell line model expressing recombinant MUC1 constructs carrying 0, 3, 9, and 42 repeats. Gastric carcinomas showed a high prevalence of Tn and sialyl-Tn antigens, whereas T antigen was less frequently expressed. The expression of T antigen was significantly higher in gastric carcinomas from patients homozygous for MUC1 large tandem repeat alleles. No significant associations were found for Tn and sialyl-Tn antigens. This novel association was reinforced by the gastric carcinoma cell line model experiments, where de novo expression of T antigen was detected in clones transfected with larger VNTR regions. Our results indicate that polymorphism in the MUC1 tandem repeat influences the expression of TACAs in gastric cancer cells and may therefore allow the identification of subgroups of patients that develop more aggressive tumors expressing T antigen.     

Association of MCP-1 promoter polymorphism with susceptibility to nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is prevalent among populations from southern China and is influenced by both genetic and environmental risk factors. The monocyte chemoattractant protein-1 (MCP-1), a member of cysteine-cysteine chemokine family, plays critical roles in cancers. A polymorphism within the MCP-1 promoter, rs1024611, has been shown to be significantly associated with the risk of several cancers. Our purpose was to assess the role of rs1024611 in NPC susceptibility. By polymerase chain reaction-restriction fragment length polymorphism method, we genotyped rs1024611 in 593 patients with NPC (cases) and 480 cancer-free subjects (controls) among Guangxi population from southern China. We observed that the G allele of rs1024611 was significantly associated with the increased risk of NPC in an additive model and dominant model, respectively (P = 0.018 and 0.010, odds ratio = 1.25 and 1.41, respectively). No appreciable variation of the effects was found across the subgroups stratified by age, sex, nationality, smoking and drinking status, and smoking level. In addition, significantly higher messenger RNA (mRNA) expression level of MCP-1 was observed in NPC tissues than that in normal nasopharyngeal tissues, and the G allele of rs1024611 was significantly associated with elevated mRNA expression level of MCP-1 in Epstein-Barr virus-transformed lymphocytes. In conclusion, our findings suggested that rs1024611 at the MCP-1 promoter may be a risk factor for NPC. Further studies with larger sample size are necessary to confirm these findings.     

A single nucleotide polymorphism at the Vrn-D1 promoter region in common wheat is associated with vernalization response

Facultative wheat varieties adapt to a particular environment. But the molecular basis for the facultative growth habit is not clear relative to winter and spring growth habit. Two sets of wheat varieties were chosen for this study. Set 1 comprised ten spring accessions and Set 2 comprised ten facultative accessions. All accessions had been tested by the previously described allele-specific markers and shown having the same allelic composition of vrn-A1 vrn-B1 Vrn-D1 and vrn-B3. Here we examined whether differences in growth habit might be associated with as yet unidentified sequence variation at Vrn-D1 locus. A region including the intron 1 deletion, the entire reading frame from a cDNA template and a part of promoter region of the dominant Vrn-D1 gene in each of the accessions was sequenced, and a single nucleotide polymorphism was found between facultative accessions and spring accessions in the CArG-box at the promoter region. The novel allele in facultative accessions was designated as Vrn-D1b. The investigation of an F₂ population segregating for Vrn-D1b and Vrn-D1a (previously, Vrn-D1) in the greenhouse under long days without vernalization showed that the plants with Vrn-D1b homozygous allele headed 32?days later and had about three more leaves than the plants with Vrn-D1a homozygous allele. As Vrn-D1b has the same deletion in intron 1 as Vrn-D1a, and, in addition, a single nucleotide mutation at promoter region, and is associated with facultative growth habit, we suggest that the promoter mutation may modify the basal activity level of an allele of VRN1 that is already active (due to the loss of segments in intron 1). Our finding further supports that both the promoter and intron 1 regulatory affect vernalization response and work independently.     

TNFR1 promoter -329G/T polymorphism results in allele-specific repression of TNFR1 expression

Tumor necrosis factor (TNF) and the TNF receptor (TNFR) superfamily play very important roles for cell death as well as normal immune regulation. Dysregulation of TNF-TNFR superfamily gene expression will influence many biological processes, and contributes to human diseases, including cancer. We investigated the genetic alterations of the TNF-TNFR superfamily genes in hepatocellular carcinoma (HCC). Several genetic alterations were detected in the 44 TNF-TNFR superfamily genes by sequencing hepatocellular carcinoma DNA samples. In particular, we found that the TNFR1 promoter −329G/T polymorphism was strongly associated with primary HCC (odds ratio [OR] = 5.22, p = 0.0007). We also observed frequent loss of heterozygosity at the polymorphic TNFR1 −329G/T site in the primary tumor tissues, indicating that the polymorphic TNFR1 −329G/T site is very susceptible to genetic alterations in HCC. Furthermore, in the polymorphic TNFR1 −329G/T site, the T allele resulted in the repression of TNFR1 expression. Therefore, our results suggest that TNFR1 −329G/T polymorphism may play an important role in the development of HCC.     

A single nucleotide polymorphism on the promoter of eotaxin1 associates with its mRNA expression and asthma phenotypes

Eotaxin1 plays a pivotal role in eosinophil-associated inflammation. Previously, we demonstrated 14 single-nucleotide polymorphisms (SNPs) in the human eotaxin1 gene and the association between the EOT+67G>A allele and the level of IgE. In this study, we investigated the association between the SNPs and plasma eotaxin1 levels, peripheral blood eosinophil counts, and PC20 methacholine values in normal and asthmatic subjects, and the effects of SNPs on the process of eotaxin1 production. The EOT-576C>T and EOT-384A>G polymorphisms and haplotypes (ht1 and ht4) were significantly associated with plasma eotaxin1 levels in the asthmatics (p < 0.001-0.040). The log [plasma eotaxin1] values correlated with the log [serum total IgE] values in the asthmatics and the normal controls (p = 0.012 and p = 0.004, respectively), and with the log [PC20 methacholine] values in the asthmatics (p = 0.014). A DNA-protein complex was formed with EOT-384A>G, but not with the other SNPs of the promoter. The interaction was stronger with the minor allele than with the common allele, and was reduced upon TNF-alpha exposure. TNF-alpha-stimulated PBMCs from the asthmatics with the minor allele homozygote expressed significantly lower levels of eotaxin1 mRNA than those from individuals with the common allele. The EOT+67G>A polymorphism, which substitutes alanine with threonine, did not affect eotaxin1 production or activity. Our data suggest that the EOT-384A>G SNP participates in the regulation of eotaxin1 expression by providing a potential binding site for a repressor, and that the ANOVA of EOT-384A>G may predict asthma phenotypes.     

Functional polymorphism located in MMP-9 gene promoter is strongly associated with obesity

The adipose tissue expansion is accompanied by remodeling of extracellular matrix performed by matrix metalloproteinases (MMPs). Higher plasma and tissue MMP-9 levels are found in obese; therefore, we evaluated if the functional C(-1562)T polymorphism (rs3918242) located in promoter region of the MMP-9 gene is associated with obesity in women. We studied 112 lean and 114 obese women. Plasma MMP-9 and tissue inhibitor of MMP-9 (TIMP)-1 were measured using enzyme-linked immunosorbent assay. We found different genotype frequencies between lean and obese women (p=0.008), prevailing T-allele in obese (2.3-fold). However, although obese women present higher levels of plasma MMP-9, lack of modulation by the polymorphism was found (all p>0.05). Our findings suggest that C(-1562)T polymorphism may contribute to pathogenetic mechanisms involved in the development of obesity in women.     

A visfatin promoter polymorphism is associated with low-grade inflammation and type 2 diabetes

Visfatin (also known as pre-B cell colony-enhancing factor, or PBEF) is a pro-inflammatory adipokine expressed predominantly in visceral fat. We investigated whether polymorphisms at the visfatin/PBEF locus influence the risk of type 2 diabetes (T2D). Linkage disequilibrium analysis of 52 single nucleotide polymorphisms spanning the entire gene (34.7 kb) plus 20.5 kb of the upstream region and 25.5 kb of the downstream region revealed a single haplotype block that could be tagged by seven single nucleotide polymorphisms. These seven tags were typed in a group of T2D patients (n = 814) and a group of non-diabetic controls (n = 320) of white origin. A significant association was observed at -948C>A, with minor allele frequencies of 0.157 in T2D cases and 0.119 in non-diabetic controls (p = 0.021). In a non-diabetic population (n = 630), the same -948 allele that conferred increased risk of T2D was significantly associated with higher plasma levels of fibrinogen and C-reactive protein (p = 0.0022 and 0.0038, respectively). However, no significant associations were observed with BMI, waist circumference, serum glucose levels, or fasting insulin levels. Our findings suggest that the visfatin/PBEF gene may play a role in determining T2D susceptibility, possibly by modulating chronic, low-grade inflammatory responses.