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.     

A single nucleotide polymorphism in the matrix metalloproteinase-2 promoter is associated with colorectal cancer

Matrix metalloproteinase-2 (MMP-2) is an enzyme with proteolytic activity against matrix proteins, particularly basement membrane constituents. A single nucleotide polymorphism C-->T transition at -1306, which disrupts an Sp1-type promoter site (CCACC box), displayed a strikingly lower promoter activity with T allele. Our study investigated whether the MMP-2 -1306 C-->T polymorphism contributed to the development and progression of colorectal cancer in the Chinese population. One hundred twenty-six colorectal cancer patients and 126 age- and sex-matched controls were included in this study. PCR-based denaturing high performance liquid chromatography analysis and sequencing were used to determine the MMP-2 genotypes. MMP-2 expression of each genotype was analyzed in four colorectal cancer cell lines by semi-quantitative RT-PCR. The correlation between the genotypes and clinicopathological parameters among colorectal cancer cases was investigated. The results showed that the levels of MMP-2 mRNA expression in cell lines containing CC genotype were much higher compared with cell with CT genotype. The frequency of MMP-2 CC genotype was significantly higher in colorectal cancer patients when compared with controls (OR, 1.959; 95% CI, 1.055-3.637). Colorectal cancers with CC genotype were more common with serosa/adventitia layer involvement compared with CT+TT genotypes. Our data suggest that MMP-2 -1306 C-->T polymorphism may be associated with colorectal cancer development and invasion in the Chinese population.     

A method of oligochip for single nucleotide polymorphism genotyping in the promoter region of the interleukin-1 beta gene and its clinical application

Single nucleotide polymorphisms (SNPs) can contribute to genetic predispositions or serve as genetic markers that are associated with complex diseases. So far, a few SNP arrays containing a limited number of SNPs have been used in routine genetic testing. This study described an oligochip-based method that genotypes two SNPs (-511 and -31) in the promoter region of the interleukin (IL)-1 beta gene. The sensitivity of this SNP genotyping method is derived from polymerase chain reaction (PCR)-amplified allele-specific primer-probes with a biotin label incorporated from the reverse primers. The amplified primer-probes can specifically hybridize with the oligonucleotides that are spotted on the oligochip. This oligochip-based method successfully discriminated the two biallelic SNPs with 9 different genotypes and all the genotyping results are in concordance with those from PCR restriction fragment length polymorphism (RFLP) analysis. Selective samples with various genotypes were also confirmed by direct sequencing. This method was applied in the genotyping of the patients with tuberculosis or gastric cancer and healthy controls. In the case control study, our genotyping data supported the reported association between gastric cancer and the genotypes of IL-1 beta -31 TT and -511 CC (p < 0.05). We also found that there is a significant difference of IL-1 beta -31 genotypes between 98 tuberculosis patients and healthy controls (p < 0.002). All of our results demonstrated that the oligochip can effectively and accurately identify SNP genotypes in the IL-1 beta promoter region.     

RANTES gene mRNA expression and its -403 G/A promoter polymorphism in coronary artery disease

Objective

Increased RANTES expression has been described to have a role in atherosclerosis plaque formation. Functional polymorphisms within RANTES promoter region have shown association with increased risk of coronary atherosclerosis (CAD). The aim of this study was to examine the RANTES mRNA expression in patients with CAD compared to patients without CAD and its association with RANTES − 403 G/A polymorphism in an Iranian population.

Methods

The study was performed on 319 patients who underwent coronary artery angiography and patients with > 50% stenosis in vessels considered as case groups (CAD+) N = 191 and normal vessels group as control (CAD−) N = 128. In each group 20 patients were examined for RANTES mRNA expression.RANTES mRNA expression was examined using quantitative real-time PCR. Genotyping of − 403 polymorphism was performed using PCR-RFLP technique.

Results

We found that RANTES mRNA expression was increased to 1.37 fold in CAD patients compared to the controls but the difference was not statistically significant. Also comparing the RANTES mRNA expression in patients with different RANTES − 403 G/A polymorphism showed that in patients carrying AA genotype RANTES mRNA expression was increased to 1.74 fold compared to patients carrying GG genotype and to 1.51 fold compared to patients carrying GA genotype. No significant difference for allele and genotype frequencies of RANTES − 403 polymorphism was found between cases and controls.

Conclusion

More studies on larger number of samples are required to further evaluate role of RANTES in pathogenesis of CAD.     

A single nucleotide polymorphism in the matrix metalloproteinase-1 (MMP-1) promoter influences amnion cell MMP-1 expression and risk for preterm premature rupture of the fetal membranes.

Interstitial collagen gives fetal membranes tensile strength, and membrane rupture has been attributed to collagen degradation. A polymorphism at -1607 in the matrix metalloproteinase-1 (MMP-1) promoter (an insertion of a guanine (G)) creates a core Ets binding site and increases promoter activity. We investigated whether this polymorphism is functionally significant for MMP-1 expression in amnion cells and whether it is associated with preterm premature rupture of the membranes (PPROM). The 2G promoter had >2-fold greater activity than the 1G allele in amnion mesenchymal cells and WISH amnion cells. Phorbol 12-myristate 13-acetate (PMA) increased mesenchymal cell nuclear protein binding with greater affinity to the 2G allele. Induction of MMP-1 mRNA by PMA was significantly greater in cells with a 1G/2G or 2G/2G genotype compared with cells homozygous for the 1G allele. When treated with PMA, the 1G/2G and 2G/2G cells produced greater amounts of MMP-1 protein than 1G/1G cells. A significant association was found between fetal carriage of a 2G allele and PPROM. We conclude that the 2G allele has stronger promoter activity in amnion cells, that it confers increased responsiveness of amnion cells to stimuli that induce MMP-1, and that this polymorphism contributes to the risk of PPROM.     

A single nucleotide polymorphism in an exon dictates allele dependent differential splicing of episialin mRNA

The episialin gene (MUC1) encodes an epithelial mucin containing a variable number of repeats with a length of twenty amino acids, resulting in many different alleles that can be subdivided into two size classes. The episialin pre-mRNA uses either one of two neighbouring splice acceptor sites for exon 2, which mainly encodes the repeats. Using the genetic polymorphism of the episialin gene to identify different alleles, we show here that the splice site recognition is allele dependent and is based on a single A/G nucleotide difference in exon 2 eight nucleotides downstream of the second splice acceptor site. Transfection experiments confirm that this polymorphic nucleotide regulates the splice site selection. The identity of this nucleotide is in most cases correlated with one of the size classes of the alleles, indicating that mutations altering the number of repeats seldom arise by unequal cross-over between the repeat regions.     

Single nucleotide polymorphism (-468 Gly to A) at the promoter region of SREBP-1c associates with genetic defect of fructose-induced hepatic lipogenesis [corrected

To evaluate the genetic susceptibility to metabolic disorders induced by high fructose diet, we investigated the metabolic characteristics in 10 strains of inbred mice and found that they were separated into CBA and DBA groups according to the response to high fructose diet. The hepatic mRNA expression of the sterol regulatory element-binding protein-1 (SREBP-1) in CBA/JN was remarkably enhanced by high fructose diet but not in DBA/2N. Similar results were observed in primary hepatocytes after exposure to fructose. The nucleotide sequence at -468 bp from the putative starting point of the SREBP-1c gene was adenine in the DBA group while it was guanine in the CBA group. In hepatocytes from CBA/JN, the activity of CBA-SREBP-1c promoter was significantly increased by 2.4- and 2.2-fold, in response to 30 mm fructose or 10 nm insulin, respectively, whereas the activity of DBA-SREBP-1c promoter responded to insulin but not to fructose. In hepatocytes from DBA/2N, both types of SREBP-1c promoter activities in response to insulin were attenuated. Furthermore, electrophoretic mobility shift assay revealed an unidentified nuclear protein bound to the oligonucleotides made from the region between -453 to -480 bp of the SREBP-1c promoter of CBA/JN but not to the probe from DBA/2N. Thus, in DBA/2N, the reduced mRNA expression of SREBP-1 after fructose refeeding appeared to associate with two independent mechanisms, 1). loss of binding of unidentified proteins to the region between -453 to -480 bp of the SREBP-1c promoter and 2). impaired insulin stimulation of SREBP-1c promoter activity.     

Identification of a single nucleotide polymorphism in the TATA box of the CYP2A6 gene: impairment of its promoter activity

Human cytochrome P450 2A6 (CYP2A6) constitutes the major nicotine oxidase, and large interindividual differences are seen in the levels of this enzyme, to a great extent caused by the distribution of several different polymorphic gene variants mainly located in the open reading frame (ORF). In the present study, we report a common polymorphism located in the 5' flanking region of CYP2A6 affecting its expression. DHPLC analysis and complete sequence of the open reading frame of the gene from a Turkish individual revealed a -48T > G substitution disrupting the TATA box. Using dynamic allele-specific hybridization (DASH), genotyping of this novel variant (named CYP2A6*9) was carried out in 116 Swedish, 132 Turkish, and 102 Chinese subjects, and the allele frequencies were found to be 5.2, 7.2, and 15.7%, respectively. The significance of the polymorphism was investigated by the construction of luciferase reporter plasmids containing 135 or 500 bp of the 5'-upstream region of the gene transfected into human hepatoma B16A2 cells. The constructs carrying the -48T > G mutation were only expressed at about 50% of the wild-type alleles. It is concluded that the CYP2A6*9 allele might be one of the most common CYP2A6 variants in Caucasians that alters the levels of enzyme expression.     

Genetic association of CCR5 promoter single nucleotide polymorphism in seronegative and seropositive rheumatoid arthritis

The aim of this study was to investigate the possible role of the CCR5 59029 A→G promoter point mutation polymorphism in determining the susceptibility to rheumatoid factor-positive and rheumatoid factor-negative rheumatoid arthritis. This polymorphism was assessed in 85 seropositive and 39 seronegative rheumatoid arthritis patients and in 126 healthy individuals of the same geographic and ethnic origin. We found an increase in the genetic frequency of the A allele in the 59029 A→G promoter region of the CCR5 receptor in patients with rheumatoid arthritis compared with healthy controls (p = 0.01; OR = 1.5, 95% CI (1.0-2.2). Likewise, the homozygous state for the A allele was found to be more frequent in rheumatoid arthritis patients, again when compared with healthy controls (p = 0.03; OR = 1.8, 95% CI 1.0-3.0). The increased frequency of the A allele was more evident in the more benign, seronegative rheumatoid arthritis group when compared with controls (p = 0.003; OR 2.4 95% CI 1.3-4.4), and when combining the A homozygous and the AG heterozygous patients compared with healthy subjects. These results suggest that this CCR5 promoter polymorphism seems to play an important role in determining different clinical courses in both forms of rheumatoid arthritis.