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

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.