FAMILIAL GLUCOSE/GALACTOSE MALABSORPTION

葡萄糖-半乳糖吸收不良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状缘的缺陷造成的 Na+/葡萄糖协同转运蛋白。 这次调查的目的是找出 编码这种膜转运的基因（SGLT 1）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，以识别受影响的患者，以及 作为杂合子携带者，（2）利用自然实验 代表有害的错义突变，以更有效地解决 Na+/葡萄糖协同转运蛋白的结构与功能关系 蛋白 为了实现这些目标，我们建议：（1）鉴定 通过检测PCR扩增的DNA片段中的单个 链构象多态性（SSCP），然后对SSCP进行测序 片段和（2）定位SGLT 1基因。 我们将PCR扩增a）分离的cDNA B）从肠粘膜活组织检查制备的cDNA， 来自淋巴细胞中基因的表达RNA，和c）编码 SGLT 1外显子。 基因定位将产生每个外显子侧翼的序列数据 允许通过PCR简单、快速地分析纯合子或杂合子携带者 随后进行SSCP、测序或适当时进行限制性内切酶分析。 突变的功能意义将使用 体外和体内表达系统。 在卵母细胞中，我们将测量 突变体RNA表达功能性Na+/葡萄糖共转运蛋白的能力 在细胞膜上。 将cRNA注入卵母细胞后， 通过示踪剂测定Na+依赖的糖转运， 电生理技术。 体外翻译实验将 让我们可以检查翻译，膜插入和糖基化。 定量PCR，原位杂交和免疫细胞化学将 用于检查突变对转录，翻译， 膜插入和处理。 这项研究将使我们能够开发工具，以帮助诊断葡萄糖- 半乳糖吸收不良，以确定突变的携带者。 的 结果还将提供关于结构的独特信息， Na+/葡萄糖协同转运蛋白的功能。

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.