中国汉族人HLA—DQA1^＊0601基因转录启动子区显示新的核苷酸序列

本文测定了中国汉族人ＨＬＡ纯合细胞ＳＭＹ－４３Ａ的ＤＱＡ１＾＊０６０１基因启动子区（ＱＡＰ）核苷酸序列，发现顺式作用元件Ｗ ｂｏｘ中－２１５和－２１６位核苷酸组成与已报道的白种人ＤＱＡ１＾＊０６０１的ＤＡＰ序列不同，由于ＳＭＹ－４３Ａ的Ⅱ类基因单倍型组成为中国人特有，提示相应的ＱＡＰ多态性可能具有人种差异。     

三个中国汉族人纯合细胞DQA1基因启动子多态性的分子生物学研究

近年来国外发现参与Ⅱ类基因转录调控的启动子区也存在多态性，运用聚合酶链反应（ＰＣＲ）和末端终止法，对三个中国汉族人ＨＬＡ纯合细胞的ＤＱＡ１基因启动子区（ＱＡＰ）３１８ｂｐ的核苷酸（包括顺式作用元件Ｘ、Ｙ、Ｘ２和Ｗｂｏｘ）进行了扩增和测序，发现等位基因ＤＱＡ１＊０３０１１（纯合细胞ＳＭＹ２３Ａ）的ＱＡＰ结合和白种人的同一等位基因ＤＱＡ１＊０３０１１的ＱＡＰ不同，而与ＤＱＡ１＊０３０１２的ＱＡＰ结构相同，表明在ＤＱＡ１与ＱＡＰ之间可能发生了重组     

新的HLA-DRB1等位基因DRB1*1212的核苷酸序列分析

目的验证一个新的HLA等位基因HLA—DRB1＊1212的序列。方法采用盐析法抽提样本基因组DNA，利用HLA—DRB1组特异性引物PCR扩增先证者HLA—DRB1等位基因的第2外显子，PCR产物经割胶回收后进行测序分析，通过聚合酶链反应-序列特异性寡核苷酸探针方法验证测序发现突变点。结果先证者有两个HLA—DRB1等位基因，其中一个为HLA—DRB1＊090102，另一个HLA—DRB1等位基因，经BLAST验证为新的等位基因，新的等位基因序列已递交GenBank（AY899825）。与最接近的DRB1＊120101等位基因序列相比，新的等位基因仅在第2外显子上有1个核苷酸不同，即第199位A→C，导致第67位氨基酸Ile—Leu。结论该等化基因为新的HLA—DRB1等位基因，被世界卫生组织HLA因子命名委员会正式命名为HLA-DRB1＊1212。     

三个中国汉族人纯合细胞DQA1基因启动子多态性的分子生物学 …

近年来国外发现参与Ⅱ类基因转录调控的启动子也存在多态性，运用聚合酶链反应（ＰＣＲ）和末端终止法，对三个中国旅游人ＨＬＡ纯合细胞的ＤＱＡ１基因启动子区（ＱＡＰ）３１８ｂｐ的核苷酸（包括顺式作用元件Ｘ、Ｙ、Ｘ２和Ｗｂｏｘ）进行了扩增产测序，发现等位基因ＤＱＡ１＊０３０１１（纯合细胞ＳＭＹ２３Ａ）的ＱＡＰ结合和白种人的同一等位基因ＤＱＡ１＊０３０１１的ＱＡＰ不同，而与ＤＱＡ１＊０３０１２的ＱＡＰ结构相同     

鼻咽癌STGC3基因转录调控元件分析与鉴定*

目的：分析并鉴定STGC3基因转录调控元件,探讨鼻咽癌STGC3基因的转录调控分子机制。方法：运用 生物信息学,预测并分析STGC3基因核心启动子区的转录因子结合位点;将相关转录因子结合位点,制备3 种探 针即生物素标记野生型、突变型及未标记野生型;提取CNE2细胞核蛋白,利用电泳迁移率变动分析（ EMSA） 及染色质免疫共沉淀（ ChIP）分析,体内外鉴定转录因子结合位点。结果：在STGC3基因核心启动子区存在21 个转录因子结合位点,其中9 个为Sp1 转录因子结合位点;进一步严格限定参数,STGC3基因核心启动子区域含 有转录因子结合位点5 个,其中Sp1 转录因子结合位点2 个;EMSA和ChIP 实验结果显示,STGC3基因中存在转 录因子Sp1 特异性结合位点,从而鉴定出STGC3基因转录调控元件。结论： STGC3基因核心启动子区含有Sp1 特异性结合位点,为该基因的转录调控元件。     

p21WAF1/CIP1基因上游主要转录调控序列区及其相关功能

p21蛋白作为周期依赖性蛋白激酶抑制因子,调节细胞周期的进程,参与细胞生长、增殖、分化、衰老等多种活动.p21/WAF1/CIP1基因上游启动子中含有多个转录调控序列,包括p53,Sp1,Ap2,VDR及RAR,STAT,C/EBPα,β,E2A,MyoD和E2F等转录因子的顺式结合元件.在细胞的生长、分化,凋亡,衰老及疾病的发生发展中,这些转录因子通过与p21上游相应调控区相互作用,调节p21基因的表达.     

WT1基因增强子构建位点对WT1基因启动子转录活性的影响

目的：以WT1基因启动子和增强子的功能片段为研究对象，探讨增强子构建在质粒的不同位点对基因启动子转录活性的影响。方法: 利用基因重组技术将WT1基因增强子的功能片段分别插入在已含有WT1基因启动子的质粒，pEWP的不同位点（MCS中的BamH I、EGFP 终止密码后的Not I和SV40polyA位点后的AflⅡ）中，将重组质粒转染慢粒白血病红白急变细胞株K562细胞、乳腺癌细胞株MCF-7细胞和人类胚胎肾转化细胞株293细胞，通过检测转基因细胞中EGFP的平均荧光强度来评估增强子对启动子的转录促进作用。结果: 通过基因重组技术分别构建了含有WT1基因启动子的载体，即pEWP，和含有WT1基因启动子和增强子的载体，即pEWPE、pEWPD和pEWPA。流式细胞仪检测EGFP的平均荧光强度，发现pEWPA在293细胞和K562细胞中能够增强WT1启动子的转录活性，而pEWPE和pEWPD则无增强作用。3者均不能在MCF7细胞中增强WT1启动子的转录活性。结论: SV40polyA位点后插入增强子能够有效地增强启动子的转录活性，且具有细胞特异性。     

人白细胞抗原DQB15''-调控区序列与I型糖尿病的关联

应用聚合酶链反应（PCR）、PCR/SSCP 和克隆测序等方法比较分析汉族1-型糖尿病患者与正常对照者HLA-DQB 1 5'-调控区多态性。结果显示携带不同等位基因的患者与对照者DQB 1 5'-调控区y s box 核苷酸序列相同，且与白种人基因结构一致；y box 核苷酸序列存在二种结构，CCTAGAGACAGATT 序列常常与DQB 1 *0302 等位基因在同一单倍型；转录起始位点至y box 间-44 至-61 位存在多态性，-59 至-61 位AAG等位基因可能与1-型糖尿病易感相关联，在2 例携带DQB 1 *0601 等位基因患者的-131 至-128 位间发现CACCACA A 单个碱基取代突变。表明HLA-DQB 1 基因5'-调控区主要作用元件旁侧核苷酸变异可能参与1-型糖尿病的病因学。     

NLRP1基因启动子区荧光素酶报告载体的构建与鉴定

目的构建人NLRP1(nucleotide-binding,leucine-rich repeat pyrin domain containing protein 1)基因启动子区SNPrs878329位点两种单倍体荧光素酶报告基因载体。方法分别以SNP rs878329的GG和CC基因型的人基因组DNA为模板,用PCR法扩增出包含该位点的长476 bp的NLRP1基因启动子区目的片段,用KpnⅠ/BglⅡ双酶切,切胶回收后分别与同样双酶切的荧光素酶报告基因载体PGL3-promoter相连接,构建pGL3-promoter-G和pGL3-promoter-C两个表达质粒,并测序验证其DNA序列。结果成功构建了人类NLRP1基因启动子SNP rs878329的两种纯合子基因型(GG型和CC型)的荧光素酶报告载体,且测序得以证实。结论成功构建出的荧光素酶报告载体,为研究NLRP1基因启动子SNP rs878329能否调控NLRP1基因表达提供了基本实验条件。     

阴道毛滴虫LAG1基因启动子区克隆及分析

本研究旨在克隆并分析阴道毛滴虫LAG1基因启动子,为进一步研究在细胞衰老过程中LAG1基因的转录调控提供实验资料。预测启动子所在区域,用PCR技术扩增启动子区序列,并分别克隆入荧光素酶报告基因载体pGL3-Basic及增强型绿色荧光蛋白报告基因载体pEGFP-1,用脂质体介导的方法瞬时转染EC109细胞,然后测定荧光素酶表达活性及观察绿色荧光蛋白的表达情况。结果表明在构建的6种荧光素酶报告基因表达体系及2种增强型绿色荧光蛋白报告基因表达体系中,表达载体pGL3-455(-455~ 55bp)、pGL3-417(-417~ 55bp)、pGL3-280(-280~ 55bp)、pGL3-202(-202~ 55bp)、pGL3-81(-81~ 55bp)的荧光素酶表达活性相近,均明显高于表达载体pGL3-47(-47~ 55bp)荧光素酶表达活性,而pGL3-47表达载体荧光素酶表达活性极低,与阴性对照活性相近,提示-47~ 55bp区无启动子活性。绿色荧光蛋白的表达情况也类似,pEGFP-81(-81~ 55bp)有明显的绿色荧光蛋白表达,而pEGFP-47(-47~ 55bp)和空载体pEGFP-1未见表达。因此-81~-47bp区域含有阴道毛滴虫LAG1基因转录所必需的基本启动子序列。     

High-resolution genotyping of HLA-DQA1 in the GoKinD study and identification of novel alleles HLA-DQA1*040102, HLA-DQA1*0402 and HLA-DQA1*0404

In order to achieve high-resolution HLA-DQA1 genotyping, it is necessary to identify polymorphisms in exons 1, 2 and 3. We present a high-resolution sequence-based typing (SBT) strategy for genotyping exons 1, 2 and 3 of the polymorphic HLA-DQA1 locus. This method is an improvement upon previously presented methods, because it utilizes the minimum number of SSP-PCR assays to obtain clear DNA sequence in both the forward and reverse directions of all three exons. All known HLA-DQA1 alleles are resolved with the exception of HLA-DQA1*010101 and HLA-DQA1*010102 for which the distinguishing polymorphism is located in exon 4 and does not result in an amino acid change. This method has enabled our laboratory to identify three new HLA-DQA1 alleles - HLA-DQA1*040102, HLA- DQA1*0402 and HLA-DQA1*0404 - in the Genetics of Kidneys in Diabetes (GoKinD) study population. Additionally, we present single-allele amplification methods, which identify the coding sequences of HLA-DQA1 exons 1, 2, 3, intron 2 and 300 bp of the HLA-DQA1 promoter (QAP). This study, also describes the QAP for most of the known HLA-DQA1 alleles, three HLA-DQA2 promoter sequences and the intron 2 sequences for HLA-DQA1*040101, HLA-DQA1*040102, HLA-DQA1*0402 and HLA-DQA1*0404.     

DNA methylation and mRNA expression of HLA‐DQA1 alleles in type 1 diabetes mellitus

Type 1 diabetes (T1D) belongs among polygenic multifactorial autoimmune diseases. The highest risk is associated with human leucocyte antigen (HLA) class II genes, including HLA‐DQA1 gene. Our aim was to investigate DNA methylation of HLA‐DQA1 promoter alleles (QAP) and correlate methylation status with individual HLA‐DQA1 allele expression of patients with T1D and healthy controls. DNA methylation is one of the epigenetic modifications that regulate gene expression and is known to be shaped by the environment.Sixty one patients with T1D and 39 healthy controls were involved in this study. Isolated DNA was treated with sodium bisulphite and HLA‐DQA1 promoter sequence was amplified using nested PCR. After sequencing, DNA methylation of HLA‐DQA1 promoter alleles was analysed. Individual mRNA HLA‐DQA1 relative allele expression was assessed using two different endogenous controls (PPIA, DRA). We have found statistically significant differences in HLA‐DQA1 allele 02:01 expression (PPIA normalization, P_corr = 0·041; DRA normalization, P_corr = 0·052) between healthy controls and patients with T1D. The complete methylation profile of the HLA‐DQA1 promoter was gained with the most methylated allele DQA1*02:01 and the least methylated DQA1*05:01 in both studied groups. Methylation profile observed in patients with T1D and healthy controls was similar, and no correlation between HLA‐DQA1 allele expression and DNA methylation was found. Although we have not proved significant methylation differences between the two groups, detailed DNA methylation status and its correlation with expression of each HLA‐DQA1 allele in patients with T1D have been described for the first time.     

A novel DRB1*13 allele (DRB1*1327) on a DR17, DQ2 haplotype with a DRB1*0301 sequence motif in the 2nd hyperpolymorphic region

In a sample from a Netherlands Caucasian, we found a new DRB1*13 allele ( DRB1*1327 ). The nucleotide sequence of the second exon of the novel allele was identical to DRB1*1301 except for a single productive base substitution changing codon 26 from TTC to TAC, encoding phenylalanine and tyrosine, respectively. The new allele shares sequence with DRB1*03011 from codons 5 to 66. The haplotype carrying the new allele was, from known linkage disequilibria, deduced to be DRB1*1327, DRB3*0101, DQA1*05011, DQB1*0201 , i.e. similar to the DR17, DQ2 haplotype, which suggests that the DRB1*1327 allele has arisen by a double recombination event between a DR13 donor haplotype and a DR17, DQ2 recipient haplotype.     

Association of HLA-DQA1 (rs9272219) with susceptibility to rheumatoid arthritis in a Han Chinese population

HLA-DQA1 (rs9272219) has been previously reported that it is a susceptibility locus in rheumatoid arthritis (RA) of UK Caucasian population and North American; however, it has not reported in RA of Chinese population. Our study was to identify whether or not this relationship is reside between rs9272219 and RA in a Han Chinese population. 207 patients with RA and 199 control subjects were recruited. The single nucleotide polymorphism (SNP) of rs9272219 was tested in alleles and genotype frequencies and the data was analyzed by doing the statistic analysis of odds ratio (OR) and 95% confidence interval (CI) from multivariate unconditional logistic regression analyses after pairwise linkage disequilibrium (LD) was estimated. Finally, the Alleles and genotype frequencies distribution of rs9272219 locus among RA patients and control subjects were in accordance with Hardy-Weinberg equilibrium. We found significant association between rs9272219 and RA of Chinese population (OR 0.494, 95% confidence interval [95% CI] 0.354-0.688, P = 0 and OR 2.541, 95% CI 1.695-3.808, P = 0, respectively). In this study, we found that the SNP of rs9272219 in HLA-DQA1 is a potential susceptibility locus in RA of Han Chinese population; the results suggest that HLA-DQA1 may be related to the development of RA.     

Identification of Two Homozygous Sequence Variants in the COL7A1 Gene Underlying Dystrophic Epidermolysis Bullosa by Whole‐Exome Analysis in a Consanguineous Family

Dystrophic epidermolysis bullosa (DEB) is an inherited skin disorder with variable severity and heterogeneous genetic involvement. Diagnostic approaches for this condition include clinical evaluations and electron microscopy of patients’ skin biopsies, followed by Sanger sequencing (SS) of a large gene (118 exons) that encodes the alpha chain of type VII collagen (COL7A1) located on Chromosome 3p21.1. However, the use of SS may hinder diagnostic efficiency and lead to delays because it is costly and time‐consuming. We evaluated a 5‐generation consanguineous family with 3 affected individuals presenting the severe generalised DEB phenotype. Human whole‐exome sequencing (WES) revealed 2 homozygous sequence variants: the previously reported variant p.Arg578* in exon 13 and a novel variant p.Arg2063Gln in exon 74 of the COL7A1 gene. Validation by SS, performed on all family members, confirmed the cosegregation of the 2 variants with the disease phenotype. To the best of our knowledge, 2 homozygous COL7A1 variants have never been simultaneously reported in DEB patients; however, the upstream protein truncation variant is more likely to be disease‐causing than the novel missense variant. WES can be used as an efficient molecular diagnostic tool for evaluating autosomal recessive forms of DEB.     

Homozygous splice site variant affecting the first von Willebrand factor A domain of COL12A1 in a patient with myopathic Ehlers-Danlos syndrome

Myopathic Ehlers-Danlos syndrome (mEDS) is a subtype of EDS that is caused by abnormalities in COL12A1. Up-to-date, 24 patients from 15 families with mEDS have been reported, with 14 families showing inheritance in an autosomal dominant manner and one family in an autosomal recessive manner. We encountered an additional patient with autosomal recessive mEDS. The patient is a 47-year-old Japanese man, born to consanguineous parents with no related features of mEDS. After birth, he presented with hypotonia, weak spontaneous movements, scoliosis, and torticollis. He had soft palms but no skin hyperextensibility or fragility. Progressive scoliosis, undescended testes, and muscular torticollis required surgery. During adulthood, he worked normally and had no physical concerns. Clinical exome analysis revealed a novel homozygous variant in COL12A1 (NM_004370.6:c.395-1G > A) at the splice acceptor site of exon 6, leading to in-frame skipping of exon 6. The patient was diagnosed with mEDS. The milder manifestations in the current patient compared with previously reported patients with mEDS might be related to the site of the variant. The variant is located in the genomic region encoding the first von Willebrand factor A domain, which affects only the long isoform of collagen XII, in contrast to the variants in previously reported mEDS patients that affected both the long and short isoforms. Further studies are needed to delineate comprehensive genotype–phenotype correlation of the disorder.     

Identification and Characterization of a Novel Retinoic Acid Response Element in Zebrafish cyp26a1 Promoter

Cyp26A1 is a major enzyme that controls retinoic acid (RA) homeostasis by metabolizing RA into bio‐inactive metabolites. Previously, we demonstrated that zebrafish cyp26a1 promoter possesses two conserved RA response elements (RAREs; proximal R1 and distal R2) in response to RA. Here, we report that it contains a novel RARE (R3) lying between R1 and R2. Mutagenesis analysis reveals that R3 works together with R1 and R2 to ensure the maximum RA inducibility of cyp26a1 promoter. Performing electrophoretic mobility shift assay and chromatin immunoprecipitation assay, we show that RA receptor alpha can bind the novel RARE. Creating and analyzing transgenic zebrafish of Tg(cyp26a1‐R3mut:eYFP)nju3/+ that harbor enhanced yellow fluorescent protein reporter gene (eYFP) driven by cyp26a1 promoter with mutated R3, we demonstrate that the reporter is mainly expressed in tissues of endogenous RA independent but not regions of RA dependent. Like Tg(cyp26a1:eYFP)nju1/+, which harbor eYFP driven by wild‐type cyp26a1 promoter, the reporter in Tg(cyp26a1‐R3mut:eYFP)nju3/+ responds to excessive RA dose dependently. However, it is expressed in a significantly lower level than the reporter in Tg(cyp26a1:eYFP)nju1/+ in response to exogenous RA. Taken together, our results demonstrate that zebrafish cyp26a1 promoter contains a novel RARE that plays crucial roles in regulating cyp26a1 expression during early development of zebrafish. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.     

Ulinastatin Preconditioning Attenuates Inflammatory Reaction of Hepatic Ischemia Reperfusion Injury in Rats via High Mobility Group Box 1(HMGB1) Inhibition

Objective It has been found that ulinastatin (UTI) can attenuate hepatic injury in a rat model of ischemia reperfusion (IR), but the specific mechanism is unclear. This study aims to investigate possible pathomechanism of ulinastatin in reducing the inflammatory response after hepatic IR.Methods A male sprague-dawley(SD) rat model of hepatic ischemia reperfusion injury was used. The rats were randomly divided into 4 groups on average, which were 0.9% saline and IR group as control, ulinastatin preconditioning (UPC) group, UPC+rHMGB1 (recombinant HMGB1) group and UPC +anti-HMGB1 group. Serum aminotransferases, TNF-α, IL-1 and Myeloperoxidase (MPO) levels were measured. Histopathology examination and apoptotic cell detection and the different expression of HMGB1 protein were also assessed.Results Serum levels of aminotransferases, cytokines and hepatic MPO in UPC and UPC+anti-HMGB1 groups were significantly lower than those in control group (p<0.05). Decreased histologic damage and apoptosis were also seen in these two groups (p<0.05).Conclusions HMGB1 expressions in UPC and UPC+anti-HMGB1 groups were significantly lower than those in the two control groups (p<0.05), pretreatment with ulinastatin attenuated liver IR injury by reducing HMGB1 expression through its anti-inflammatory effects.     

Genetic variants in pseudogene E2F3P1 confer risk for HBV-related hepatocellular carcinoma in a Chinese population

Recent studies showed that pseudogenes can regulate the expression of their coding gene partners by competing for miRNAs. The E2F family plays a crucial role in the control of cell cycle checkpoint. E2F3P1 is a pseudogene of E2F3. Few studies focused on genetic variations on pseudogenes. In this study, we performed a case-control study to assess the association between single nucleotide polymorphisms (SNPs) in E2F3P1 and hepatocellular carcinoma (HCC) risk in 1050 hepatitis B virus (HBV)-positive HCC cases and 1050 chronic HBV carriers. Logistic regression analysis was applied to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the associations between genotypes and HCC risk. We found that the variant CT/TT genotypes of rs1838149 were associated with a significantly decreased risk of HCC (adjusted OR = 0.66, 95% CIs = 0.51-0.86, P = 0.002) compared to those with wildtype CC homozygote. Furthermore, the AA genotype of rs9909601 had an increased HCC risk with an adjusted OR of 1.41 (95% CIs = 1.07-1.86), and the A allele of rs9909601 was significantly associated with HCC risk compared to those with the G allele (adjusted OR = 1.17, 95% CIs = 1.03-1.33, P = 0.017). These results indicate that genetic variations in the pseudogene E2F3P1 may confer HCC risk.