BACKGROUND:Tumor-derived DNA can be found in the plasma of cancer patients. In this study, we explored the use of shotgun massively parallel sequencing (MPS) of plasma DNA from cancer patients to scan a cancer genome noninvasively.

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The glycolytic enzyme glucokinase plays an important role in the regulation of insuln secretion and recent studies have shown that mutations in the human glucokinas gene are a common cause of an autosomal domint form of non-inssulin-dependent (type 2) diabetes meilitus (NIIDDM) that has an onset often during childhood. The majority of the mutations that have been identified are missense mutations that result in the syntheis of a glucokinas molecule with an altered amino acid sequence. To characterize the effect of these mutations on the catalytic properties of human 3-cell glucokinase, we have expressed native and mutant forms of this protein in Eschenchia coli. AU of the missense mutations show chnges in enzyme activity including a decrease in V.. and/or increase in K. for glucose. Using a model for the threedimensional structure of human glucokinas based on the crystal structure of the related enzyme yeast hexokinae B, the mutations map primarily to two regions of the protein. One group of mutations is lcated in the active site cleft separating the two domains of the enzyme as wefl as in surface loops leading into this cleft. These mutations usually result in large reductions in enzyme activity. The second group of mutations is located far from the active site in a region that is predicted to undergo a substrate-induced conformational change that results in closure of the active site cleft. These mutations show a smail -2-fold reduction in V., and a 5-to 10-fold increase in K. for glucose. The characterization of mutations in glucokinase that are asted with a distinct and readily recognizable form of NIDDM has led to the identification of key amino acids involved in glucokinas catalysis and locaized functionaUly important regions of the glucokinas molecule.

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We describe a codon 299 mutation in the glucokinase gene in a British pedigree with maturity-onset diabetes of the young (MODY) resulting in a substitution of glycine to arginine. One out of fifty patients diagnosed with classical late-onset type 2 diabetes mellitus was also found to have this mutation. All nine relatives of this patient who have inherited the mutation have type 2 diabetes, although six others without the mutation are also present with diabetes. The discovery that glucokinase mutations can cause MODY and was also found in ten affected members of a pedigree with type 2 diabetes in which MODY had not previously been considered indicates that diagnosis based on molecular pathology will be helpful in understanding the aetiology of type 2 diabetes.