Morris Brian J, Markus AndreaM, Glenn Cheryl L, Adams David J, Colagiuri S, Wang Li
Basic & Clinical Genomics Laboratory, Department of Physiology and Institute for Biomedical Research, The University of Sydney, New South Wales, 2006, Australia.
J Mol Med (Berl). 2002 Feb;80(2):96-104. doi: 10.1007/s00109-001-0287-1. Epub 2001 Oct 5.
Complications of diabetes have a genetic influence. Since increased inducible nitric oxide synthase (iNOS) gene ( NOS2A) expression can contribute to tissue damage, NOS2A is a worthy candidate for such a role. We therefore tested a 4-bp insertion/deletion (+/-) polymorphism 0.7 kb upstream of NOS2A for association with complications in type 2 diabetes patients, and also performed transient transfection experiments to examine the effect of this variant on promoter activity in kidney cells in culture. We investigated 379 Caucasian type 2 diabetes patients of British/European descent, 93 of whom had microalbuminuria, 26 overt nephropathy, 46 retinopathy, and 73 clinical neuropathy. Genotyping for the variant was carried out by PCR and automated Genescan analysis. Transient transfection studies involved the renal HEK 293 cell line and luciferase reporter gene constructs containing 1.1 kb of 5'-flanking DNA from '+' or '-' allele homozygotes. We found that the '+' allele frequency in patients without microalbuminuria was 12%, but was 23% in those with microalbuminuria ( P=0.0005), and was 26% in those with nephropathy ( P=0.0007), 22% in those with retinopathy ( P=0.037), and 23% in those with neuropathy ( P=0.045). The odds ratios for homozygote +/+ to have microalbuminuria or nephropathy were 2.4 (95% CI 1.4-4.2, P=0.0023) and 5.4 (95% CI 1.8-16, P=0.0009), respectively. Luciferase reporter gene constructs containing 1 kb of NOS2A promoter DNA for each allele were made and sequence analysis confirmed that the +/- variation was the only sequence difference present. Transient transfection of these into HEK 293 cells revealed 25 times higher reporter gene activity for the '+' allele compared with the '-' allele. Gel shift analysis with 30mer oligonucleotides corresponding to each allele showed specific binding to nuclear extracts, being greater for the '+' allele. Thus the '+' allele of the NOS2A promoter variant may confer higher iNOS expression, and could contribute to complications of type 2 diabetes, especially in the approximately 5% of patients homozygous for this variant.
糖尿病并发症受遗传因素影响。由于诱导型一氧化氮合酶（iNOS）基因（NOS2A）表达增加会导致组织损伤，因此NOS2A是承担这一作用的一个值得研究的候选基因。我们检测了NOS2A上游0.7 kb处一个4碱基插入/缺失（+/-）多态性与2型糖尿病患者并发症的关联性，并进行了瞬时转染实验，以研究该变异对培养的肾细胞中启动子活性的影响。我们调查了379名具有英国/欧洲血统的白种人2型糖尿病患者，其中93人有微量白蛋白尿，26人有显性肾病，46人有视网膜病变，73人有临床神经病变。通过聚合酶链反应（PCR）和自动基因扫描分析对该变异进行基因分型。瞬时转染研究使用了肾HEK 293细胞系和含有来自“+”或“-”等位基因纯合子1.1 kb 5'侧翼DNA的荧光素酶报告基因构建体。我们发现，无微量白蛋白尿患者中“+”等位基因频率为12%，有微量白蛋白尿患者中为23%（P = 0.0005），肾病患者中为26%（P = 0.0007），视网膜病变患者中为22%（P = 0.037），神经病变患者中为23%（P = 0.045）。纯合子“+ / +”发生微量白蛋白尿或肾病的优势比分别为2.4（95%可信区间1.4 - 4.2，P = 0.0023）和5.4（95%可信区间1.8 - 16，P = 0.0009）。构建了包含每个等位基因1 kb NOS2A启动子DNA的荧光素酶报告基因构建体，序列分析证实+/-变异是唯一存在的序列差异。将这些构建体瞬时转染到HEK 293细胞中发现，“+”等位基因的报告基因活性比“-”等位基因高25倍。用与每个等位基因对应的30聚体寡核苷酸进行凝胶迁移分析表明，其与核提取物有特异性结合，“+”等位基因的结合更强。因此，NOS2A启动子变异的“+”等位基因可能导致更高的iNOS表达，并可能导致2型糖尿病的并发症，尤其是在约5%的该变异纯合子患者中。
