Lentivirus-mediated gene transfer to the rat, ovine and human cornea

Gene therapy of the cornea shows promise for modulating corneal transplant rejection but the most appropriate vector for gene transfer has yet to be determined. We investigated a lentiviral vector (LV) for its ability to transduce corneal endothelium. A lentivector expressing enhanced yellow fluorescent protein (eYFP) under the control of the Simian virus type 40 early promoter (LV-SV40-eYFP) transduced 80-90% of rat, ovine and human corneal endothelial cells as detected by fluorescence microscopy. The kinetics of gene expression varied among species, with ovine corneal endothelium showing a relative delay in detectable reporter gene expression compared with the rat or human corneal endothelium. Vectors containing the myeloproliferative sarcoma virus promoter or the phosphoglycerate kinase promoter were not significantly more effective than LV-SV40-eYFP. The stability of eYFP expression in rat and ovine corneas following ex vivo transduction of the donor cornea was assessed following orthotopic corneal transplantation. Following transduction ex vivo, eYFP expression was maintained in corneal endothelial cells for at least 28 days after corneal transplantation in the sheep and >60 days in the rat. Thus, rat, ovine and human corneal endothelial cells were efficiently transduced by the LV, and gene expression appeared stable over weeks in vivo.     

Optimizing aerosol gene delivery and expression in the ovine lung.

We have developed the sheep as a large animal model for optimizing cystic fibrosis gene therapy protocols. We administered aerosolized gene transfer agents (GTAs) to the ovine lung in order to test the delivery, efficacy, and safety of GTAs using a clinically relevant nebulizer. A preliminary study demonstrated GTA distribution and reporter gene expression throughout the lung after aerosol administration of plasmid DNA (pDNA):GL67 and pDNA:PEI complexes. A more comprehensive study examined the dose-response relationship for pDNA:PEI and assessed the influence of adjunct therapeutic agents. We found that the sheep model can differentiate between doses of GTA and that the anticholinergic, glycopyrrolate, enhanced transgene expression. Dose-related toxicity of GTA was reduced by aerosol administration compared to direct instillation. This large animal model will allow us to move toward clinical studies with greater confidence.     

血管紧张素转换酶基因多态性及其与心脑血管病的关系

目的:对近年来关于肾素-血管紧张素系统中血管紧张素转换酶及其多态性的研究现状进行综述。资料来源:应用计算机检索PUBMED1994-01/2006-10期间的相关文章,检索词为“angiotensin I-converting enzymegene”,并限定文章语言种类为English。同时计算机检索万方数据库1994-01/2006-10期间的相关文章,检索词为“血管紧张素转换酶,基因多态性”,并限定文章语言种类为中文。资料选择:对资料进行初审,并查看每篇文献后的引文。纳入标准:文章所述内容应与血管紧张素转换酶基因多态性及同心脑血管病的关系研究相关。排除标准:重复研究或Meta分析类文章。资料提炼:共收集到100篇相关文献,32篇文献符合纳入标准,排除的62篇文献为内容陈旧或重复。符合纳入标准的32篇文献中,3篇涉及肾素-血管紧张素系统的生理作用,10篇涉及血管紧张素转换酶的生物学特征,1篇涉及血管紧张素转换酶基因及其多态性,10篇涉及正常人群血管紧张素转换酶基因I/D多态性,10篇涉及血管紧张素转换酶基因I/D多态性与疾病的相关性。资料综合:肾素-血管紧张素系统是体内重要的体液调节系统,在水盐的代谢平衡和血管张力调节中有重要的作用。血管紧张素转换酶I/D多态性,可能与心脑血管疾病的发生发展相关;且在不同种族不同民族等正常人群中的分布有差异性,说明亦与种族和遗传背景有关;此外,血管紧张素转换酶I/D多态性与心脑血管病相关,在不同种族、不同民族甚至相同种族、相同民族的研究结果是不完全相同的。结论:血管紧张素转换酶是肾素-血管紧张素系统的关键酶,其基因I/D多态性在不同种族、不同民族等正常人群中分布存在差异,与某些心脑血管疾病的易感性,严重性及预后相关。     

Simultaneous detection of eight single nucleotide polymorphisms in the ovine prion protein gene

Amino acid polymorphisms in the prion protein gene (PrP) affect the susceptibility of sheep to scrapie, a transmissible spongiform encephalopathy (TSE). In particular, amino acid substitutions at codons 136, 154 and 171 of the ovine PrP gene are associated with different degrees of susceptibility to the classical form of scrapie, caused by 'typical' scrapie strains. Existing genotyping tests for scrapie susceptibility normally interrogate only the single nucleotide polymorphisms (SNPs) most relevant to 'typical' strains. Recently, however, a number of novel variants of the scrapie agent have been discovered. The ability of these new, 'atypical' scrapie variants to infect sheep that are resistant to 'typical' variants has raised concerns about the reduction in genetic variability that may result from intense selection for resistance to classical scrapie. Furthermore, a growing interest in a potential role for specific PrP genotypes in modulating performance traits is also driving a move toward more extensive characterization of haplotypes at the PrP locus. Here, we describe a single-tube method for the interrogation of eight SNPs within seven codons (112, 136, 141, 154, 171, 231 and 241) of the ovine PrP gene. This method is as accurate as sequencing, yet more affordable, and can easily be automated for high-throughput sample screening. Moreover, it can be modified to accommodate genetic variations that are found in local and heritage breeds.     

中国脑膜炎奈瑟菌nadA基因多态性分析

目的 了解我国脑膜炎奈瑟菌(Neisseria meningitidis,Nm)主要黏附蛋白(Neisseria adhesion A,NadA)编码基因nadA的多态性特征,以期研究我国主要流行Nm菌株NadA蛋白的流行病学意义及NadA蛋白作为脑膜炎球菌蛋白疫苗候选蛋白研发的可行性。 方法 选取我国26个省(市、自治区)1963-2012年间不同血清群代表性Nm菌株230株,采用多位点序列分型(muliti-locus sequence typing,MLST)、PCR、斑点杂交、基因测序等方法,检测Nm菌株基因型特征、nadA基因分布的多态性及其基因序列特征,利用BioEdit、Mega 4.0等软件对nadA基因序列进行比对分析。 结果 我国230株不同血清群的Nm菌株中,nadA+菌株55株,仅存在于ST-5 complex(序列群)的A群和ST-11 complex 的W135群Nm菌株中,B、C及其他血清群Nm菌株nadA-。nadA+菌株基因型以variant 3(V3)型为主(54/55),包括5种亚型(NadA V3.1~3.5),同源性高达99%以上。 结论 我国Nm菌株中,仅ST-5 complex的A群和ST-11 complex的W135群菌株存在NadA黏附蛋白,型别以variant 3型为主。B群菌株不含有NadA蛋白,variant 3型NadA蛋白不适合作为我国B群Nm(B meningococci,MenB)蛋白疫苗的候选蛋白。     

Polymorphism of interleukin-1 receptor antagonist gene and obesity

Background: Cytokines appear to be major regulators of adipose tissue metabolism. Interleukin-1 receptor antagonist (IL-1ra) serum concentrations are increased in human obesity, and are under strong genetic control. We tested the hypothesis that the IL-1ra gene might be a candidate for obesity. Methods: We investigated the frequency of a penta-allelic 86-bp tandem repeat (VNTR) in the intron 2 of IL-1ra gene in 52 lean (body mass index (BMI) <25 kg/m²), 133 overweight (BMI 25–29.9 kg/m²) and 76 obese (BMI ≥30.0 kg/m²) otherwise healthy Korean women. Total fat mass and percent body fat were determined by dual-energy X-ray absorptiometry. Genomic DNA was extracted and used for polymerase chain reaction-based genotyping of IL-1ra. Results: Carriers of the allele 2 did not show a significant difference in physical and clinical characteristics. The genotypic, or allelic distribution did not differ markedly between the three groups. The relative risk of being obese in comparison with lean group tended to be higher in allele 2 carriers, but not significantly. Conclusions: We found no relationship between the IL-1ra polymorphism and BMI in Korean women.     

Polymorphism screening in the cardiac K+ channel gene KCNA5

BACKGROUND: Common deoxyribonucleic acid polymorphisms that modulate normal cardiac electrophysiologic characteristics have previously been identified in long QT syndrome disease genes. In this study we screened an additional gene encoding the cardiac potassium channel KCNA5 (underlying I(Kur)) in 3 ethnic groups and evaluated the functional consequences of the variants identified. METHODS: The coding region was screened by single-stranded conformational polymorphism analysis and direct sequencing, and nonsynonymous variants were studied by patch-clamping transfected Chinese hamster ovary cells. Results Five synonymous and 6 nonsynonymous polymorphisms were found in KCNA5. None of these polymorphisms was present in greater than 7% of alleles screened or in all 3 ethnic groups. Expression of the nonsynonymous KCNA5 variants revealed normal gating. However, 2 variants (P532L and R578K, both in the C-terminus) were resistant to block by the prototypical inhibitor quinidine; the concentration required to block I(Kur) by 50% (IC(50)) was 8.4 micromol/L for wild type versus 54 micromol/L for R578K and 133 micromol/L for P532L (both P < .0001, versus wild type). CONCLUSION: KCNA5 displays little variability in its coding region. C-terminal KCNA5 variants displayed near-normal gating but striking resistance to drug block; thus these pharmacogenomic studies have identified a heretofore-unappreciated role of this region as a modulator of channel sensitivity to drugs. Resistance to I(Kur) blockers may be genetically determined.     

Polymorphism at the mucin-like protocadherin gene influences susceptibility to gallstone disease

BackgroundGallstone disease (GSD) is a common disease that can be caused by environmental influences, common genetic factors and their interactions. Mucin glycoproteins may be one important factor for GSD. We conducted a case–control study to investigate the relationship between the mucin-like protocadherin (MUPCDH) gene polymorphisms and GSD.MethodsThe study included 452 GSD cases and 491 healthy controls who had no evidence of gallstones by ultrasound examination. Two common tagging single nucleotide polymorphism (SNP) rs3758650 and rs7932167, and four non-synonymous SNPs rs34362213, rs2740375, rs7108757 and rs2740379 were genotyped. The genetic effects were evaluated using the multivariate regression model.ResultsThe genotypes of these SNPs were all in Hardy–Weinberg equilibrium. Three non-synonymous SNPs (rs34362213, rs7108757 and rs2740379) were monomorphic. The single SNP analysis showed two SNPs (rs7932167 and rs2740375) were not associated with GSD and only SNP rs3758650 had the association of the presence of GSD with an odds ratio (OR) of 1.59 (adjusted P = 0.013) for the AG genotype and 5.82 (adjusted P = 0.007) for the AA genotype when compared with the reference GG genotype. The haplotype analysis of the three polymorphic SNPs showed GCA was significant for GSD (adjusted p = 0.001) with an odds ratio (OR) of 1.41 when compared to other haplotypes.ConclusionsThe MUPCDH genetic polymorphism rs3758650 was considered a genetic marker to predict symptomatic GSD subjects. It may be of importance for GSD patients with the risk SNPs to be frequently checked because they may develop symptomatic GSD.     

中国人血管紧张素转换酶基因I/D多态性的研究

目的:探讨中国人血管紧张素转换酶(angiotensinconvertingenzyme,ACE)基因多态性分布。方法:利用聚合酶链反应(polymerasechainreaction,PCR)方法检测141例正常人和200例高血压患者的ACE基因型,同时检测其血清ACE水平,并对55例未服用过降压药物的高血压患者分别于治疗前及口服卡托普利25mg1h后测定其血清ACE活性的变化。结果:高血压组的I等位基因频率(0.65)显著高于正常对照组(0.57)(P<0.05)。DD型患者ACE活性最高,临床合并症多,预后差。未发现基因型与血管紧张素转换酶抑制剂(angiotensinconvertingenzymein-hibitor,ACEI)疗效有关。结论:①ACEI等位基因可能为中国人原发性高血压的危险因子。②ACE基因缺失多态性与血清ACE活性有关,并增加高血压合并靶器官损害的危险。③ACE基因多态性与ACEI疗效无关。     

维生素D受体基因多态与糖尿病肾病易感性研究进展

糖尿病肾病是常见的糖尿病并发症之一,近年来发现其发生机制与遗传背景有关。国内外大量研究表明维生素D受体(vitamin D receptor,VDR)基因多态性是糖尿病肾病的易感因素,糖尿病肾病并非完全由高血糖直接导致,将为糖尿病及糖尿病肾病的深入研究提供重要线索,本文对维生素D受体基因多态性与糖尿病肾病的易感性进行综述。     

汉族人强直性脊柱炎致病基因多态性的研究

目的:①探讨汉族人HLA-B27亚型基因结构特点。②合成HLA-B27基因检测试剂。方法:采用聚合酶链式反应、顺序特异性引物法和核苷酸探针杂交技术。对120例健康人和46例强直性脊柱炎患者的HLA-B27基因进行了分析。结果:采用本项目合成的HLA-B27DNA检测试剂,在120例正常人中进行了检测,有6例出现HLA-B27扩增带,HLA-B27基因携带者占5%;而65例疑为AS患者中有46例出现HLA-B27扩增带,占70.3%,(RR=46,P<0.01);制备的HLA-B27基因检测试剂,具有快速简便、准确性高和特异性强等优点。结论:①在中国汉族人中至少存在2种HLA-B27亚型基因,分别是B2704和B2705,同时B2704和B2705也是汉族人HLA-B27主要的亚型;HLA-B27的B2704和B2705是强直性脊柱炎的主要致病基因。②DNA分型检测HLA-B27基因完全可以取代目前的血清学方法,具有很大的应用价值。     

金华地区男性肝脂酶多态性分布研究

目的探讨肝脂酶（HL）基因启动子－250G／A、－514C／T、－710T／C、－763A／G多态性在浙江金华地区的分布及与脂类代谢间的关系。方法采用聚合酶链反应一限制性片段长度多态性技术检测中国金华地区355例健康男性的HI。基因启动子－250G／A、－514C／T、－710T／C和一763A／G的多态性,并探讨基因多态性与血脂指标间的关系。355例研究对象按年龄分组,6～7岁组74例,18～〈20岁组109例,20～〈40岁组67例,40～73岁组105例。结果4组间HI,基因启动子250G／A的基因型分布和等位基因频率差异均无统计学意义（P〉O．05）。HL－514C／T多态性的基因型频率分布显示,18～〈20岁组与6～7岁组及40～73岁组间的差异均有统计学意义（P〈0．05）。HL－710T／C、－763A／G多态性的基因型和等位基因频率分布显示,40～73岁组与6～7岁组及18～〈20岁组间的差异均有统计学意义（P〈O．05）。HL基因多态性与血液脂代谢有关。HL－250G／A、－514C／T、－710T／C和一763A／G间存在强连锁不平衡。结论HL－250G／A、－514C／T、－710T／C和－763A／G间存在强连锁不平衡,HL基因启动子多态性可能与脂类的代谢有关。     

Polymorphism of the angiotensin-converting enzyme gene affects the outcome of acute respiratory distress syndrome

OBJECTIVE: There has been increasing evidence that angiotensin II may play an important role in the pathogenesis and in the evolution of acute lung injury. It was therefore hypothesized that polymorphisms of the angiotensin-converting enzyme gene affects the risk and outcome of acute respiratory distress syndrome (ARDS). DESIGN: Prospective, observational study. PATIENTS AND SETTINGS: The ARDS group consisted of 101 patients treated at the medical intensive care unit; the control groups consisted of 138 "at-risk" patients treated at the medical intensive care unit due to acute respiratory failure but did not meet the ARDS criteria throughout the hospital course, and 210 non-at-risk subjects. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: The ARDS patients and control subjects were genotyped for the insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme gene. Association of the polymorphism and the risk and the outcome of ARDS was analyzed. There was no significant difference in the frequencies of the genotypes between the ARDS, at-risk, and non-at-risk groups. The 28-day mortality rates were significantly different between the three angiotensin-converting enzyme genotypes (42%, 65%, and 75% for II, ID, and DD, respectively; p = .036). Survival analysis showed that the II genotype favorably affected 28-day survival (hazard ratio, 0.46; 95% confidence interval, 0.26-0.81; p = .007), whereas ARDS caused by hospital-acquired pneumonia had a negative effect (hazard ratio, 2.34; 95% confidence interval, 1.25-4.40; p = .008). The II genotype (hazard ratio, 0.53; 95% confidence interval, 0.32-0.87; p = .012) and ARDS caused by hospital-acquired pneumonia (hazard ratio, 2.13; 95% confidence interval, 1.24-3.68; p = .006) were also significant prognostic factors for the in-hospital mortality. CONCLUSIONS: The angiotensin-converting enzyme I/D polymorphism is a significant prognostic factor for the outcome of ARDS. Patients with the II genotype have a significantly better chance of survival. This study did not show an increased risk for ARDS in Chinese patients with the D allele.     

Polymorphism of vascular angiotensin II receptor gene and cardiovascular disorders

AIM: To study polymorphism of the gene of vascular angiotensin II receptor. MATERIALS AND METHODS: Polymorphism that consists in variability of adenine (A) and cytosine (C) residues at position 1166 of the gene for vascular angiotensin II receptor (AT1R) was analyzed in a Moscow population (n = 98) and three groups of affected patients with myocardial infarction (n = 32, MI), left ventricular hypertrophy (LVH, n = 38) and essential hypertension (EH, n = 178). Polymorphic region of the AT1R gene was amplified using the polymerase chain reaction (PCR) and genomic DNAs from human whole blood as template. PCR products were electrophoresied in a gel after digestion with BstDEI restriction nuclease. Significance of differences in distribution of both allele and genotype frequencies at the population sample and in affected patients were estimated via exact Fisher's test. RESULTS: A significant decrease in the frequency of the A genotype was detected in all the three affected groups compared to healthy controls. Besides, the frequency of the A allele was significantly decreased in EH group with a corresponding increase in the frequency of both the AC genotype and the C allele. CONCLUSION: The A1166C polymorphism of the AT1R gene is associated with EH, MI and LVH in a Moscow population. The association is stronger with EH. The A allele and the AA genotype protect against development of disorders at early onset while the other genotypes and the C-allele are risk factors. A protective role of the AA genotype is more significant than predisposition action of the CC homozygote.