Department of Medical Genetics, University of Antwerp, and Antwerp University Hospital, Edegem, Belgium.
Gene. 2012 Jan 15;492(1):148-59. doi: 10.1016/j.gene.2011.10.034. Epub 2011 Oct 19.
Mutations in Exostosin-1 (EXT1) or Exostosin-2 (EXT2) cause the autosomal dominant disorder multiple osteochondromas (MO). This disease is mainly characterized by the appearance of multiple cartilage-capped protuberances arising from children's metaphyses and is known to display clinical inter- and intrafamilial variations. EXT1 and EXT2 are both tumor suppressor genes encoding proteins that function as glycosyltransferases, catalyzing the biosynthesis of heparan sulfate. At present, however, very little is known about the regulation of these genes. Two of the most intriguing questions concerning the pathogenesis of MO are how disruption of a ubiquitously expressed gene causes this cartilage-specific disease and how the clinical intrafamilial variation can be explained. Since mutations in the EXT1 gene are responsible for ~65% of the MO families with known causal mutation, our aim was to isolate and characterize the EXT1 promoter region to elucidate the transcriptional regulation of this tumor suppressor gene.
In the present study, luciferase reporter gene assays were used to experimentally confirm the in silico predicted EXT1 core promoter region. Subsequently, we evaluated the effect of single nucleotide polymorphisms (SNP's) on EXT1 promoter activity and transcription factor binding using luciferase assays, electrophoretic mobility shift assays (EMSA), and enzyme-linked immunosorbent assays (ELISA). Finally, a genotype-phenotype study was performed with the aim to identify one or more genetic modifiers influencing the clinical expression of MO.
Transient transfection of HEK293 cells with a series of luciferase reporter constructs mapped the EXT1 core promoter at approximately -917 bp upstream of the EXT1 start codon, within a 123 bp region. This region is conserved in mammals and located within a CpG-island containing a CAAT- and a GT-box. A polymorphic G/C-SNP at -1158 bp (rs34016643) was demonstrated to be located in a USF1 transcription factor binding site, which is lost with the presence of the C-allele resulting in a ~56% increase in EXT1 promoter activity. A genotype-phenotype study was suggestive for association of the C-allele with shorter stature, but also with a smaller number of osteochondromas.
We provide for the first time insight into the molecular regulation of EXT1. Although a larger patient population will be necessary for statistical significance, our data suggest the polymorphism rs34016643, in close proximity of the EXT1 promoter, to be a potential regulatory SNP, which could be a primary modifier that might explain part of the clinical variation observed in MO patients.
外切聚糖-1（EXT1）或外切聚糖-2（EXT2）基因突变导致常染色体显性遗传疾病多发性骨软骨瘤（MO）。这种疾病的主要特征是儿童干骺端出现多个软骨帽状突起，已知具有临床内外家族变异。EXT1 和 EXT2 都是肿瘤抑制基因，编码作为糖基转移酶发挥作用的蛋白质，催化肝素硫酸的生物合成。然而，目前对这些基因的调控知之甚少。关于 MO 发病机制的两个最有趣的问题是，普遍表达的基因的突变如何导致这种软骨特异性疾病，以及如何解释家族内的临床变异。由于 EXT1 基因突变负责已知致病突变的 65%的 MO 家族，我们的目的是分离和表征 EXT1 启动子区域，以阐明该肿瘤抑制基因的转录调控。
在本研究中，使用荧光素酶报告基因检测实验证实了 EXT1 核心启动子区域的计算机预测。随后，我们使用荧光素酶检测、电泳迁移率变动分析（EMSA）和酶联免疫吸附测定（ELISA）评估单核苷酸多态性（SNP）对 EXT1 启动子活性和转录因子结合的影响。最后，进行了基因型-表型研究，旨在确定一个或多个遗传修饰因子影响 MO 的临床表达。
瞬时转染 HEK293 细胞的一系列荧光素酶报告构建体将 EXT1 核心启动子映射到 EXT1 起始密码子上游约-917bp 处，位于 123bp 区域内。该区域在哺乳动物中保守，位于包含 CAAT 和 GT 盒的 CpG 岛中。在-1158bp 处的多态性 G/C-SNP（rs34016643）被证明位于 USF1 转录因子结合位点内，该结合位点缺失导致 C-等位基因的存在，从而导致 EXT1 启动子活性增加约 56%。基因型-表型研究表明，C-等位基因与身材矮小有关，但也与骨软骨瘤数量较少有关。
我们首次深入了解 EXT1 的分子调控。尽管需要更大的患者群体才能具有统计学意义，但我们的数据表明，位于 EXT1 启动子附近的多态性 rs34016643 可能是一个潜在的调节 SNP，它可能是主要的修饰因子，可以解释 MO 患者观察到的部分临床变异。
