Genetic analysis of Japanese patients with persistent hyperinsulinemic hypoglycemia of infancy - Nucleotide-binding fold-2 mutation impairs cooperative binding of adenine nucleotides to sulfonylurea receptor 1

To elucidate the genetic etiology of persistent hyperinsulinemic hypoglycem ia of infancy (PHHI) in the Japanese population, we conducted a polymerase chain reaction-single-strand conformation polymorphism analysis of the sulf onylurea receptor 1 (SUR1) and Kir6.2 genes in 17 Japanese PHHI patients, i ncluding a pair of siblings from a consanguineous family. me also analyzed the glutamate dehydrogenase gene for the exons encoding an allosteric regul atory domain of the enzyme. In the SUR1 gene, we identified one frameshift (I446fsdelT) and two missense (R1420C, R1436Q) mutations. None of these mut ations mere found in control Japanese subjects. Siblings homozygous for the R1420C mutation had a mild form, whereas two patients heterozygous for the I446fsdelT and R1436Q mutations, respectively, exhibited a severe form of PHHI. Functional consequences of these mutations on K-ATP function mere eva luated using Rb-86(+) efflux studies in COS-7 cells. SUR1-446fsdelT and SUR 1-1436Q did not form a functional K-ATP. Western blot analysis after transi ent expression in COS-7 cells revealed the expression of SUR1-1436Q protein to be markedly reduced, suggesting SUR1-1436Q to be unstable in these cell s. K-ATP (SUR1-1420C) showed reduced responses to metabolic inhibition by o ligomycin and 2-deoxyglucose. K-ATP channels are under complex regulation b y intracellular ATP and ADP. ATP both inhibits and activates these channels . The inhibition is probably mediated through direct ATP interaction with a pore-forming subunit Kir6.2, whereas the activation is likely to be throug h a regulatory subunit SUR1. There is a cooperative regulation of ATP and A DP binding to SUR1, and this cooperativity may be involved in regulating th e K-ATP channel. In SUR1-1420C, high-affinity binding of ATP to the nucleot ide-binding fold (NBF)-1 was indistinguishable from that of wild-type SUR1. However, stabilization of ATP binding to NBF-1 by MgATP or MgADP mas impai red, suggesting that this defect may account for impaired K-ATP(SUR1-1420C) function. This is the first direct biochemical evidence that the cooperati vity of nucleotide binding to SUR1 is impaired in a SUR1 mutant causing PHH I. No mutations mere identified in the Kir6.2 and glutamate dehydrogenase g enes. The genetic etiology of PHHI appears to be heterogeneous. SUR1 mutati ons may account for no more than 20% of PHHI cases in Japanese patients. Mu tations of Kir6.2 and glutamate dehydrogenase genes are likely to be even l ess common.