FAMILIAL GLUCOSE-GALACTOSE MALABSORPTION

葡萄糖半乳糖吸收不良(GGM)是一种常染色体隐性遗传病 我们已经证明这是由于肠道刷毛边缘的缺陷造成的 Na+/葡萄糖共转运蛋白。这项调查的目标是确定 编码这种膜运输的基因(SGLT1)的突变 蛋白。我们的主要兴趣包括使用这些新数据来(1) 开发诊断遗传学方法来识别患有疾病的患者 作为杂合子携带者，以及(2)利用大自然的实验 表现为有害的错义突变，以更有效地解决 钠/葡萄糖共转运蛋白的结构/功能关系 蛋白。为了实现这些目标，我们建议(1)在 应用聚合酶链式反应扩增单链DNA片段诊断GGM患者 链构象多态(SSCP)及其测序 (2)对SGLT1基因进行定位。我们将用聚合酶链式反应扩增分离到的a)cdna。 从肠粘膜活检组织中，b)非法从 在淋巴细胞中表达该基因的RNA和c)编码 SGLT1外显子。基因作图将产生每个外显子两侧的序列数据 允许用聚合酶链式反应对纯合子或杂合子携带者进行简单、快速的分析 然后是SSCP、测序或适当的限制分析。 这些突变的功能意义将通过在 体外和体内表达系统。在卵母细胞中，我们将测量 突变RNA表达功能性Na+/葡萄糖共转运蛋白的能力 在质膜中。在将CRNA注入卵母细胞后，我们将 用示踪剂测定Na~+依赖的糖转运 电生理技术。体外翻译实验将 允许我们检查翻译、膜插入和糖基化。 定量PCR、原位杂交和免疫细胞化学将 用来检测突变对转录、翻译、 膜的插入和加工。 这项研究将使我们能够开发工具来帮助诊断葡萄糖- 半乳糖吸收不良，以确定突变的携带者。这个 结果还将提供关于结构和 Na+/葡萄糖共转运蛋白的功能。

Glucose-galactose malabsorption (GGM) is an autosomal recessive disease which we have shown to be due to a defect in the intestinal brush border Na+/glucose cotransporter. The goal of this investigation is to identify the mutations in the gene (SGLT1) coding for this membrane transport protein. Our principle interests include the use of these new data to (1) develop diagnostic genetic methods to identify afflicted patients as well as heterozygote carriers, and (2) exploit the experiments of nature represented in deleterious missense mutations to more effectively address the structure/function relationships of the Na+/glucose cotransporter protein. To achieve these ends we propose to (1) identify mutations in GGM-diagnosed patients by examining PCR-amplified DNA fragment for single strand conformational polymorphisms (SSCPs) followed by sequencing the SSCP fragments and (2) map the SGLT1 gene. We will PCR-amplify a) cDNA isolated from intestinal mucosal biopsies, b) cDNA prepared from illegitimately expressed RNA from the gene in lymphocytes and c) genomic DNA coding for SGLT1 exons. Gene mapping will yield sequence data flanking each exon permitting simple, rapid analysis of homo- or heterozygote carriers by PCR followed by SSCP, sequencing, or, where appropriate, restriction analysis. The functional significance of the mutations will be examined using in vitro and in vivo expression systems. In oocytes we will measure the ability of the mutant RNA to express functional Na+/glucose cotransporters in the plasma membrane. After injection of cRNA into oocytes we will measure Na+-dependent sugar transport by tracer by tracer and electrophysiological techniques. in vitro translation experiments will allow us to examine translation, membrane insertion and glycosylation. Quantitative PCR, in situ hybridization and immunocytochemistry will be employed to examine the effect of mutations on transcription, translation, membrane insertion and processing. This study will enable us to develop tools to aid in diagnosis of glucose- galactose malabsorption, to identify carriers of the mutations. The results will also provide unique information about the structure and function of the Na+/glucose cotransporter.