湖北汉族人群载脂蛋白A5遗传多态性分析

采用聚合酶链反应-限制性片断长度多态性(polymerase chain reaction restriction-fragment length polymorphism, PCR-RFLP)对257例湖北健康汉族人群APOA5 -1131T>C及56C>G基因多态性进行鉴定。结果发现: 湖北汉族人群中ApoA5 -1131T>C存在TT、TC、CC基因型, 3种基因型的频率分别为50.9%、32.9%及16.2%; 56C>G位点存在CC、CG基因型, 257名研究对象中, G等位基因分布频率小于5%; 各基因型频率和等位基因频率在不同种族和地域间分布存在显著性差异。结论: 湖北汉族人群中ApoA5基因-1131T>C位点存在单核苷酸多态性(single nucleotide polymorphism, SNP), 56C>G在该人群中应视为一个突变位点而不是多态性位点     

Influence of BLK polymorphisms on the risk of rheumatoid arthritis

B cell lymphocyte kinase (BLK) encodes a member of the Src kinase family and thus may influence the proliferation and differentiation of cells. A single nucleotide polymorphism (SNP) located in the first intron of BLK has shown that the risk C allele of rs2248932 is associated with lower levels of messenger RNA expression of BLK. We hypothesized that this polymorphism may contribute to rheumatoid arthritis (RA) susceptibility. We studied BLK rs2248932 T/C gene polymorphisms in 329 patients with RA and 697 controls in a Chinese population. Genotyping was done using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). When the BLK rs2248932 TT homozygote genotype was used as the reference group, the CC genotype was associated with a significantly increased risk of RA. In the recessive model, when the BLK rs2248932 TT/TC genotypes were used as the reference group, the CC homozygote genotype was associated with a significantly increased susceptibility to RA. In stratification analyses, a significantly increased risk for RA associated with the BLK rs2248932 CC genotype was evident among younger patients, CRP-negative patients and anti-CCP-positive patients compared with the BLK rs2248932 TT/TC genotype. The risk was also significantly evident among RF-positive patients, patients with lower ESR levels, patients with lower or higher DAS28 score and patients with a lower functional class. These findings suggested that the functional SNP BLK rs2248932 T/C variant allele was associated with RA development. However, our results were obtained from a moderate-sized sample, and therefore this is a preliminary conclusion. Validation in a larger study from a more diverse ethnic population is needed to confirm these findings.     

Ⅱ型糖尿病患者APOA5-1131T/C基因多态性与血脂代谢和胰岛素抵抗的关系研究

为了探讨载脂蛋白A5基因(APOA5)-1131T/C多态性在中国镇江地区的频率分布及其与血浆脂质代谢和Ⅱ型糖尿病患者胰岛素抵抗的关系, 采用聚合酶链反应-限制性片段长度多态性分析(PCR-RFLP)结合琼脂糖凝胶电泳技术检测152例健康人及71例Ⅱ型糖尿病患者APOA5 -1131T/C基因型及等位基因频率分布, 同时采用生化方法测定所有研究对象的血脂、血糖和胰岛素水平。结果显示: 糖尿病组APOA5 -1131C等位基因频率显著高于对照组(0.430 vs 0.296, P = 0.006)。CC纯合子患糖尿病的风险是TT纯合子的3.75倍(OR = 3.75, 95% CI: 1.57~8.92), 且经Logistic回归分析, 校正年龄、BMI和血浆HDL-c、LDL-c及ApoB水平等其他混杂因素影响后, 这种差异仍具有显著性意义(OR = 2.70, 95%CI: 1.24 ~ 5.86)。糖尿病组C等位基因携带者TG水平显著高于非C等位基因携带者(P < 0.01), TC水平和LDL-c水平亦明显升高(P < 0.05)。但是在两组中, 不同基因型患者 胰岛素抵抗相关指标均无显示差异。提示APOA5-1131T/C单核苷酸多态性对人群血浆TG水平有影响, -1131C等位基因与血浆TG、TC和LDL-c水平增高有关, 但是与糖尿病患者胰岛素相关指标无关; APOA5 -1131C等位基因可能与人群糖尿病的发生相关联。     

SNP exploring in the middle and terminal regions of the IGF-1 gene and association with production and reproduction traits in Holstein cattle

Five primer sets were designed in order to identify single nucleotide polymorphisms (SNPs) in middle and terminal exons (2 to 6) and in some flanking intronic regions of the bovine insulin-like growth factor 1 (IGF-1) gene. Sequencing results of PCR products for 10% of animals showed no variant in exons but a SNP at intron 4 was occurred. Both polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) and high resolution melting (HRM) methods were developed to genotype samples. The PCR–RFLP results showed the presence of three fragments on agarose gel for the C allele due to two cleavage sites while two fragments for the T allele were observed. Melting curves of 123 bp fragments in HRM analysis showed a difference between temperature melting (T_m) of two homozygous genotypes as the CC genotypes had higher T_m than the TT genotypes. Melting curve of the CT genotype was different and crossed two parallel patterns of homozygous genotypes. The frequencies of the CC, CT and TT genotypes were 0.6, 0.37 and 0.03, respectively. Also, the estimated allele frequencies were 0.785 and 0.215 for the C and T alleles, respectively. Results showed higher accuracy of the HRM analysis compared to the PCR–RFLP method. Least square means (LSMs) comparison of the different genotypes in the SNP showed significant association with milk fat yield trait in the first lactation and open days after the second calving. The polymorphism did not have a significant effect on other milk production or reproduction traits. It seems that other variants or QTLs known in this region underlie genetic variation in the production and reproduction of dairy cattle.     

Allele-specific extension allows base-pair neutral homozygotes to be discriminated by high-resolution melting of small amplicons

Not all single-nucleotide polymorphisms (SNPs) can be determined using high-resolution melting (HRM) of small amplicons, especially class 3 and 4 SNPs. This is due mainly to the small shift in the melting temperature (Tm) between two types of homozygote. Choosing rs1869458 (a class 4 SNP) as a sample, we developed a modified small amplicon HRM assay. An allele-specific extension (ASE) primer, which ended at an SNP site and matched only one of the alleles, was added to the reaction as well as additional thermal steps for ASE. Following asymmetric polymerase chain reaction and melting curve analysis, heterozygotes were easily identified. Two types of homozygote were also distinguishable, indicating that extension primers 11 to 13 bases in length worked efficiently in an allele-specific way. Modification of the limiting amplification primer with locked nucleic acid increased the Tm difference between extension and amplification peaks and facilitated subsequent genotyping. In addition, 194 human genomic DNA samples were genotyped with the developed assay and by direct sequencing, with the different methods providing identical genotyping results. In conclusion, ASE-HRM is a simple, inexpensive, closed-tube genotyping method that can be used to examine all types of SNP.     

Mitogen/extracellular signal-regulated kinase kinase-5 promoter region polymorphisms affect the risk of sporadic colorectal cancer in a southern Chinese population

Mitogen/extracellular signal-regulated kinase kinase-5 (MEK5), which belongs to a network of mitogen-activated protein kinase pathways, play a pivotal role in carcinogenesis. The purpose of this study was to investigate whether variants in the MEK5 gene promoter were involved in susceptivity of individuals to sporadic colorectal cancer (CRC). In the present hospital-based case-control study of 737 patients with sporadic CRC and 703 healthy control subjects in a southern Chinese population, the two polymorphisms of MEK5 promoter (i.e., rs7172582C>T and rs3743354T>C) were genotyped by TaqMan assay. There were significant differences between cases and controls in the genotype and allele distribution of the MEK5 gene rs3743354T>C polymorphism. The rs3743354 CC genotype was associated with a significantly decreased risk of CRC when compared with the TT genotype (adjusted odds ratios [ORs]=0.43; 95% confidence interval [CI], 0.24-0.77). Compared to the T allele, a significant correlation was detected between the presence of the C allele and decreased risk of CRC (adjusted OR=0.79; 95% CI, 0.61-0.94). The decreased risk of CRC associated with rs3743354 variant genotypes (i.e., CT+CC) was found in the smoker subgroup (adjusted OR=0.63; 95% CI=0.45-0.88). Further, environmental factors, including smoking and drinking, interacted with rs3743354C variant genotypes to reduce CRC risk. Western blot analysis showed that the levels of MEK5 protein in sporadic CRC neoplastic tissues and adjacent normal colorectal epithelium tissues were lower in the carriers of rs3743354 CC genotypes than that in those with rs3743354 TT genotypes or those with rs3743354 TC genotypes. However, no significant association was found between the rs7172582C>T polymorphism and risk of CRC. These data indicate that the rs3743354 polymorphism in the MEK5 promoter may affect the risk of developing CRC.     

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.     

IL-1b基因单核苷酸多态性与腰椎间盘疾病的相关性

为研究汉族人白细胞介素-1b（IL-1b）基因 ^-511T>C 和 ⁺³⁹⁵⁴C>T位点单核苷酸多态性与腰椎间盘疾病的关系, 采用聚合酶链反应技术, 扩增 81 例腰椎间盘疾病患者和 101 例正常对照者中分别包含IL-1b 基因 ^-511T>C 和 ⁺³⁹⁵⁴C>T 位点的片段, 酶切法鉴定 IL-1b基因 ^-511T>C和 ⁺³⁹⁵⁴C>T 位点单核苷酸多态性情况, 比较两组中基因多态性与腰椎间盘疾病的关系。同时, 利用 MRI 检测两组腰椎间盘退变的情况, 并分析其中小于45岁者IL-1b基因多态性与腰椎间盘退变严重程度的关系。结果显示, 腰椎间盘疾病病例组及对照组中均存在IL-1b基因 ^-511T>C 和 ⁺³⁹⁵⁴C>T 位点单核苷酸多态性。IL-1b 基因 ^-511T>C 位点 TT、TC 和 CC基因型, T、C 基因型差别与腰椎间盘疾病有关（P<0.01）, 与腰椎间盘退变严重程度无关(P>0.05), 但IL-1b基因 ⁺³⁹⁵⁴C>T 位点单核苷酸多态性与腰椎间盘退变严重程度及腰椎间盘疾病均无关(P>0.05)。表明在汉族人中, 存在 IL-1b 基因 ^-511T>C 和 ⁺³⁹⁵⁴C>T 位点单核苷酸多态性, 但仅 ^-511T>C 位点单核苷酸多态性与腰椎间盘疾病有关。     

Influence of apoA-V gene variants on postprandial triglyceride metabolism: impact of gender

Although apolipoprotein A-V (apoA-V) polymorphisms have been consistently associated with fasting triglyceride (TG) levels, their impact on postprandial lipemia remains relatively unknown. In this study, we investigate the impact of two common apoA-V polymorphisms (-1131 T>C and S19W) and apoA-V haplotypes on fasting and postprandial lipid metabolism in adults in the United Kingdom (n = 259). Compared with the wild-type TT, apoA-V -1131 TC heterozygotes had 15% (P = 0.057) and 21% (P = 0.002) higher fasting TG and postprandial TG area under the curve (AUC), respectively. Significant (P = 0.038) and nearly significant (P = 0.057) gender x genotype interactions were observed for fasting TG and TG AUC, with a greater impact of genotype in males. Lower HDL-cholesterol was associated with the rare TC genotype (P = 0.047). Significant linkage disequilibrium was found between the apoA-V -1131 T>C and the apoC-III 3238 C>G variants, with univariate analysis indicating an impact of this apoC-III single nucleotide polymorphism (SNP) on TG AUC (P = 0.015). However, in linear regression analysis, a significant independent association with TG AUC (P = 0.007) was only evident for the apoA-V -1131 T>C SNP, indicating a greater relative importance of the apoA-V genotype.     

Association between polymorphisms in the SLC27A1 gene and milk production traits in Chinese Holstein cattle

We assessed SLC27A1, a candidate gene for milk production traits in Chinese Holstein cattle. DNA was extracted from the blood of 48 top Chinese Holstein Cattle selected according to phenotypic character and mixed into DNA pool for SNP detection. We tested blood samples of these cattle for SNPs in exon 3 and the 3'-flanking region of the SLC27A1 gene by using polymerase chain reaction-single-stranded conformation polymorphism (PCR-SSCP) and DNA sequencing. We found 2 polymorphic sites: 112T>C, a synonymous mutation, in exon 3 (SNP(1)), and 64G>A in the 3'-UTR (SNP(2)). We also determined the genotypes of 330 Chinese Holstein cattle by using PCR-restriction fragment length polymorphism (RFLP). We found 3 genotypes each at SNP(1) (TT, TC, and CC) and SNP(2) (GG, GA, and AA). The association among the different genotypes at these 2 sites and milk production traits was analyzed using a least-squares procedure. The results showed that cows with genotype CC had higher milk yields than those with genotype TC (0.01 < p < 0.05). No significant difference was detected among the 3 SNP(2) genotypes in terms of milk production traits. Our results provide evidence that the C allele have potential effects on milk yield trait.     

Phenotype and genotype relationship of glutathione peroxidase1 (GPx1) and rs 1800668 variant: the homozygote effect on kinetic parameters

GPx1 is one of the most important enzymes involved in oxidative balance so that, we studied the phenotype and genotype relationship of GPx1 activity and rs 1800668 (C/T) site and also evaluated the changes of GPx1 kinetic parameters in the rs 1800668 homozygotes. One hundred fifty eight subjects were recruited after clinical exams. The rs 1800668 (C/T) genotype distribution was identified using RFLP-PCR method. The hemolysate GPx1 activity was spectrophotometrically measured in a reaction coupled with glutathione reductase (GR). The GPx1 enzyme was purified using gel filtration chromatography with Sephacryl S-300 column and, Km(app) was studied in the rs 1800668 TT and CC homozygotes. The results showed that the GPx1 activity is significantly associated to the rs 1800668 (C/T) genotype distribution (P<0.05) so that, the GPx1 activity was high among the CC homozygotes (P<0.03). In addition, Km(app) for TBHP substrate in the TT homozygote (8.48 μM) was higher than the CC homozygote (5.74 μM). We concluded that the C allele within rs 1800668 position is related to the GPx1 activity and may be a potential factor involved in development of inflammatory events.     

Case-control study on association of peroxisome proliferator-activated receptor-δ and SNP-SNP interactions with essential hypertension in Chinese Han population

The aim of this study was to investigate the association of peroxisome proliferator-activated receptor-δ (PPAR-δ) and additional SNP-SNP interaction with essential hypertension (EH) in Chinese Han population. A total of 1248 subjects (625 males, 623 females), including 620 EH patients and 628 normotension subjects, were included in the study. The mean age was 51.2?±?15.1 years old. Logistic regression model was used to examine the association between four SNP and EH; odds ratio (OR) and 95 % confident interval (95 %CI) were calculated. Generalized multifactor dimensionality reduction (GMDR) was employed to analyze SNP-SNP interaction. EH risk was significantly lower in carriers of C allele of the rs2016520 polymorphism than those with TT (TC?+?CC versus TT, adjusted OR (95 %CI)?=?0.61 (0.49–0.78)). In addition, we also found a significant association between rs9794 and EH; EH risk was also significantly lower in carriers of G allele of the rs9794 polymorphism than those with CC (CG?+?GG versus CC, adjusted OR (95 %CI)?=?0.65 (0.53–0.83)). We also found a potential SNP-SNP interaction between rs2016520 and rs9794; subjects with TC or CC of rs2016520 and CG or GG of rs9794 genotype have the lowest EH risk, compared to subjects with TT of rs2016520 and CC of rs9794 genotype; OR (95 %CI) was 0.32 (0.23–0.62) after covariate adjustment. Our results support an important association between rs2016520 and rs9794 minor allele of PPAR-δ and decreased risk of EH and additional interaction between rs2016520 and rs9794.     

The Association Between <Emphasis Type="Italic">APOA5</Emphasis> Gene Polymorphisms and Plasma Lipids in the Turkish Cypriot Population: A Possible Biomarker for Preventing Cardiovascular Diseases

Apolipoprotein A5 (APOA5 or APO A-V) polymorphisms have long been reported to be associated with cardiovascular disease and plasma lipid levels. The present study was undertaken to investigate the relationship between the rs662799, rs3135507, and rs2075291 with biochemical parameters in the Turkish Cypriot population. A total of 100 Turkish Cypriot volunteer subjects (53 female and 47 male), with a mean age of 40.8, participated in the study. A basic biochemical analysis, including serum glucose, total serum cholesterol, HDL-C, LDL-C, and triglycerides, was performed for each participant. Genotyping for the APOA5 three polymorphisms was performed by polymerase chain reaction followed by restriction fragment length polymorphism analysis. Biochemical parameters except the low-density lipoprotein cholesterol (LDL-C) were all within the normal limits. LDL-C was found to be slightly elevated in participants according to WHO guidelines. With respect to the genotype and allele distributions of APOA5 rs662799 T>C polymorphism, TT genotypes are more frequent (62%) in the population and the frequency of T allele is 0.78. The TT genotype for APOA5 rs2075291 G<T was not observed in the study population. Ancestral GG is the only genotype present in the study population. Minor Allele Frequency of APOA5 rs3135507 G>A variant is 0.12 for the A allele. No association between the two studied APOA5 polymorphisms (rs662799 and rs3135507) and the biochemical components of glucose, total cholesterol, and triglyceride were observed. On the other hand, a strong statistical association between the high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) clinical parameters and APOA5 rs662799 CC and rs3135507 AA genotypes was found (p = 0.014 and p = 0.017, respectively). APOA5 polymorphisms rs662799 and rs3135507, with the CC and the AA genotypes, respectively, are associated with increased levels of both high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) in the Turkish Cypriot population.     

Potential impact of ADAM-10 genetic variants with the clinical features of oral squamous cell carcinoma

A disintegrin and metalloproteinase domain-containing protein 10 (ADAM-10) involves in the tumour progression, but the impacts of single-nucleotide polymorphism (SNP) of ADAM-10 on oral squamous cell carcinoma (OSCC) remain unclear. The aim of this study was to investigate the influence of SNP of ADAM-10 on the clinical features of OSCC in male Taiwanese. Five loci of ADAM-10 SNPs including rs653765 (C/T), rs2305421 (A/G), rs514049 (A/C), rs383902 (T/C) and rs2054096 (A/T) were genotyped by TaqMan allelic discrimination in 1138 OSCC patients and 1199 non-OSCC individuals. The ADAM-10 SNP rs2305421 GG (AOR: 1.399, 95% CI: 1.045–1.874, p = 0.024) and G allele (AOR: 1.170, 95% CI: 1.012–1.351, p = 0.034) illustrated a significantly higher genotypic frequencies in the OSCC group compared to the distribution of the ADAM-10 SNP rs2305421 AA wild type. In the subgroup analysis, the ADAM-10 SNP rs383902 TC+CC was significantly correlated to tumour size larger than T2 in betel quid chewer (AOR: 1.375, 95% CI: 1.010–1.872, p = 0.043), while the ADAM-10 SNP rs653765 CT+TT was significantly associated with tumour size larger than T2 in cigarette smoker (AOR: 1.346, 95% CI: 1.023–1.772, p = 0.034). The results from The Cancer Genome Atlas revealed highest ADAM-10 mRNA level in T2 stage of current smokers with head and neck squamous cell carcinoma (HNSCC). In conclusions, the ADAM-10 SNP rs2305421 G allele is associated with the presence of OSCC, and the ADAM-10 SNP rs383902 TC+CC and ADAM-10 SNP rs653765 CT+TT correlates to large tumour size in specific conditions.     

高分辨率熔解曲线法检测CYP4A118590T＞C单核苷酸多态性

目的 建立CYP4A11 8590T＞C单核苷酸多态性（single nucleotide polymorphism,SNP）的高分辨率熔解曲线（high resolution melting,HRM）检测方法.方法先采用温度梯度PCR,确定适宜的退火温度;再利用正交试验,优化引物、DNA模板量和Mg2＋量,最终确定PCR反应体系和反应条件.通过对607例无血缘关系的受试者基因组DNA进行HRM分析,并随机选择50例产物测序.结果 引物最适退火温度为57.8 ℃;PCR最佳反应体系为20 μl,包括2×conc dNTP mix 10 μl,上下游引物（10 μmol/L）各0.5 μl,DNA溶液（30 ng/μl）1.0 μl,Mg2＋（2.5 mmol/L）1.5 μl和灭菌水6.5 μl.607例受试者中CYP4A11 8590TT、TC和CC基因型频率分别为54.7 %、37.6 %和7.7 %.结论该正交试验优化的HRM技术可用于检测CYP4A11 8590T＞C单核苷酸多态性,且其分析结果和测序结果一致.     

LALBA基因SNP与内蒙古白绒山羊经济性状的关联

利用PCR-SSCP和DNA测序技术检测452份内蒙古白绒山羊α-乳白蛋白(LALBA)基因单核苷酸多态性(SNP), 并分析SNP与产绒量、绒厚、绒长和体重性状的关联。结果表明, 仅P2引物位点存在SSCP多态, 其外显子3区域存在1个突变位点: M63868:g.1897T>C。内蒙古白绒山羊群体LALBA基因M63868:g.1897位点以TT型为主, T等位基因频率为0.983, 且处于Hardy-Weinberg平衡状态(P>0.05)。方差分析表明, LALBA基因M63868:g.1897位点多态仅与产绒量存在显著相关(P=0.017); 1897位点TC基因型个体产绒量比TT基因型个体多产绒142.68 g, 高26.21%, 且差异显著(P<0.05)。因此, TC基因型可作为山羊产绒性状标记辅助选择的有效DNA标记。     

Toll-like receptor 4 Asp299Gly and Thr399Ile polymorphisms in cancer: a meta-analysis

Toll-like receptor 4 (TLR4) is critical in the recognition of Gram-negative bacteria serving as a key immune system effector. Recently, a number of case-control studies were conducted to investigate the association between TLR4 gene polymorphism and cancer risk, especially Asp299Gly and Thr399Ile polymorphisms. However, published data were still conflicting. In this paper, we summarized 9463 cancer cases and 10,825 controls from 22 studies and attempted to assess the susceptibility of TLR4 gene polymorphism to cancers by a synthetical meta-analysis. Odds ratios (ORs) with 95% confidence intervals (CIs) were estimated to assess the relationship. Our results suggested that Asp299Gly represented a risk factor on cancers in digestive system (G allele versus A allele, OR=1.64, 95% CI: 1.02-2.64; GA+GG versus AA, OR=1.64, 95% CI: 1.00-2.71) but tend to have a protective effect on prostate cancer (GG versus AA, OR=0.37, 95% CI: 0.14-0.98; GG versus GA+AA, OR=0.37, 95% CI: 0.14-0.98). Thr399Ile polymorphism was significantly associated with an elevated cancer risk in overall analysis (T allele versus C allele, OR=1.72, 95% CI: 1.27-2.33; TC versus CC, OR=1.63, 95% CI: 1.18-2.26; TT+TC versus CC, OR=1.70, 95% CI: 1.24-2.34) and especially in gastrointestinal subgroup (T allele versus C allele, OR=2.01, 95% CI: 1.40-2.89; TC versus CC, OR=1.86, 95% CI: 1.26-2.74; TT+TC versus CC, OR=1.97, 95% CI: 1.35-2.88). Further prospective researches with larger numbers of worldwide participants are warranted to draw comprehensive and true conclusions.     

Association of Single Nucleotide Polymorphisms in VDR and DBP Genes with HBV-Related Hepatocellular Carcinoma Risk in a Chinese Population

Background

Polymorphisms of genes encoding components of the vitamin D pathway including vitamin D receptor (VDR) and vitamin D binding protein (DBP) have been widely investigated because of the complex role played by vitamin D in cancer tumorogenesis. In this study, we investigated the association between VDR and DBP gene polymorphisms and HBV-related HCC risk in a Chinese population.

Methods

Study subjects were divided into three groups: 184 HBV patients with HCC, 296 HBV patients without HCC, and 180 healthy controls. The VDR rs2228570, and rs3782905 and the DBP rs7041 polymorphisms were genotyped using PCR-RFLP and the VDR rs11568820 polymorphism was genotyped by PCR-SSP, respectively. DNA sequencing was performed to validate the genotype results.

Results

We found that there were significant differences in the genotype and allele frequencies of the VDR rs2228570 and DBP rs7041 polymorphisms between HBV patients with HCC and healthy controls. The rs2228570 T allele was associated with a significant increased HBV-related HCC risk as compared with the C allele. The rs2228570 TT and TT/TC genotypes were correlated with a significant increased HBV-related HCC risk when compared with the wild-type CC homozygote. Similarly, the rs7041 G allele was associated with a significant increased HBV-related HCC risk as compared with the T allele. The rs7041 GG and GG/TG genotypes were correlated with a significant increased HBV-related HCC risk when compared with the wild-type TT homozygote. However, we did not observe any significant effect of VDR rs11568820, and rs3782905 polymorphisms on HBV-related HCC risk in this population. In haplotype analysis, we also did not find any significant differences in haplotype frequencies of the VDR gene between HBV patients with HCC and the healthy controls.

Conclusions

We conclude that the VDR rs2228570 and DBP rs7041 polymorphisms may contribute to increased susceptibility to HBV-related HCC in the Chinese population. Due to the marginal significance, further large and well-designed studies in diverse ethnic populations are needed to confirm our results.     

Identification of ryanodine receptor 1 single-nucleotide polymorphisms by high-resolution melting using the LightCycler 480 System

High-resolution melting (HRM) allows single-nucleotide polymorphism (SNP) detection/typing using inexpensive generic heteroduplex-detecting double-stranded DNA (dsDNA) binding dyes. Until recently HRM has been a post-PCR process. With the LightCycler 480 System, however, the entire mutation screening process, including post-PCR analysis, can be performed using a single instrument. HRM assays were developed to allow screening of the ryanodine receptor gene (RYR1) for potential mutations causing malignant hyperthermia (MH) and/or central core disease (CCD) using the LightCycler 480 System. The assays were validated using engineered plasmids and/or genomic DNA samples that are either homozygous wild type or heterozygous for one of three SNPs that lead to the RyR1 amino acid substitutions T4826I, H4833Y, and/or R4861H. The HRM analyses were conducted using two different heteroduplex-detecting dsDNA binding dyes: LightCycler 480 HRM dye and LCGreen Plus. Heterozygous samples for each of the HRM assays were readily distinguished from homozygous samples with both dyes. By using engineered plasmids, it was shown that even homozygous sequence variations can be identified by using either small amplicons or the addition of exogenous DNA after PCR. Thus, the LightCycler 480 System provides a novel, integrated, real-time PCR/HRM platform that allows high throughput, inexpensive SNP detection, and genotyping based on high-resolution amplicon melting.