| Recent linkage studies and association analyses suggest the presence of at least one type 2 diabetes susceptibility gene in human region 20q12–13.1. A high-resolution 6.0 Mb transcript map of this interval was constructed using two parallel, complementary strategies. A series of BAC contigs were assembled from 56 overlapping BAC clones, using STS/marker screening of 42 genes, 43 ESTs, 38 STSs, 22 polymorphic and 3 BAC-end sequence markers. GraphMap, a novel software program that employs a greedy path searching algorithm with local heuristics, was designed to automate map assembly. The BAC contigs were anchored and oriented within a YAC scaffold built from a panel of 21 YAC clones. Concurrently, a sequence-based map was assembled from genomic sequence data released by the Human Genome Project, using a seed-and-walk approach. The map provides near-continuous coverage between markers SGC32867 and WI-17676 (∼6.0 Mb). EST database searches and genomic sequence alignments of ESTs, mRNAs, and UniGene clusters enabled the annotation of the sequence interval with experimentally confirmed and putative transcripts. The combination of BAC transcript map, YAC-to-BAC scaffold, and reference Human Genome Project sequence provides a powerful integrated resource for future genomic analysis of this region.; Using the sequence-based transcript map as a framework, 13 candidate genes and 10 novel ESTs were evaluated for functional allelic variants associated with type 2 diabetes, including adenosine deaminase (ADA), hepatocyte nuclear factor 4α (HNF-4α), and a novel protein kinase-C binding protein, PRKCBPl. No evidence of allelic association was identified.; A survey of the genomic sequence data within the transcript map facilitated the identification of GLUT10, a novel human facilitative glucose transporter. The GLUT10 cDNA encodes a 541 amino acid protein that shares between 23 and 32% amino acid identity with human glucose transporters 1–8. When expressed in Xenopus oocytes, GLUT10 exhibited 2-deoxy-D-glucose transport with an apparent Kin of 280 μM. Preincubation with insulin stimulated glucose transport by approximately 2-fold. Single strand conformation polymorphism analysis identified two coding single nucleotide polymorphisms within the second exon of GLUT10. Preliminary analysis has suggested that one of the allelic variants reduces the response of insulin stimulated glucose transport in micro-injected Xenopus oocytes. |
