Pancreatic islet-cell dysfunction, coupled with peripheral insulin resistance, is a primordial feature of the pathogenesis of non-insulin dependent diabetes mellitus (NIDDM). Hence, islet genes are likely to be involved in the genetic susceptibility to this complex disorder. Our knowledge concerning. genes expressed in human pancreatic islets is however very limited, largely due to the difficulties involved in the isolation of this tissue. This grant aims to identify, clone, sequence and place in the genetic linkage map a large number of genes specifically expressed in purified human islets. Using differential display of human islet and acinar mRNA we will identify and sequence .500 islet expressed sequence tags (ESTs). Our preliminary data has shown that, contrary to random sequencing from a human islet cDNA library, this is an expedient strategy to identify novel islet-specific genes, while avoiding common household and contaminating acinar-specific mRNAs. Semiquantitative reverse transcription PCR (RT-PCR) analysis will be used to assess the distribution of novel ESTs in multiple human tissues. [An estimated 300 novel islet ESTs will be mapped on human/rodent somatic hybrid panels for chromosomal localization. When regions of the genome are found to be linked to NIDDM, then subchromosomal localization by a variety of means will be completed for the subset of islet ESTs which map to that chromosome. Schematically, the following strategic hierarchy will be followed: a) P1 genomic clones will be isolated using the already synthesized PCR primers, and the islet ESTs further localized by fluorescence in situ hybridization (FISH), b) for selected ESTs, YACs will be isolated from CEPH libraries, and c) when necessary, the P1 genomic clones can also be used for development of microsatellite markers and linkage mapping in CEPH pedigrees.] In addition, the ESTs can be employed to discover new genes which will enhance our understanding of the molecular basis of islet-cell dysfunction. During this project we will thus select a minor subset (10-15) of islet-specific ESTs to isolate and characterize full-length human islet cDNAs. Sequences and mapping data will be deposited in public databases, and will be thus available for the entire research community. In summary, this project is intended to generate sequence and genetic linkage data from a selected group of novel human islet genes, which will prove invaluable in the long term goal of understanding the etiology and genetics of NIDDM, and will contribute to ongoing efforts to integrate expression, genetic and physical maps.
