Molecular basis and characterization of the hyperinsulinism/hyperammonemia syndrome: predominance of mutations in exons 11 and 12 of the glutamate dehydrogenase gene. HI/HA Contributing Investigators.

Glutamate dehydrogenase (GDH) is allosterically activated by the amino acid leucine to mediate protein stimulation of insulin secretion. Children with the hyperinsulinism/hyperammonemia (HI/HA) syndrome have symptomatic hypoglycemia plus persistent elevations of plasma ammonium. We have reported that HI/HA may be caused by dominant mutations of GDH that lie in a unique allosteric domain that is encoded within GDH exons 11 and 12. To examine the frequency of mutations in this domain, we screened genomic DNA from 48 unrelated cases with the HI/HA syndrome for exon 11 and 12 mutations in GDH. Twenty-five (52%) had mutations in these exons; 74% of the mutations were sporadic. Clinical manifestations included normal birth weight, late onset of hypoglycemia, diazoxide responsiveness, and protein-sensitive hypoglycemia. Enzymatic studies of lymphoblast GDH in seven of the mutations showed that all had reduced sensitivity to inhibition with GTP, consistent with an increase in enzyme activity. Mutations had little or no effect on enzyme responses to positive allosteric effectors, such as ADP or leucine. Based on the three-dimensional structure of GDH, the mutations may function by impairing the binding of an inhibitory GTP to a domain responsible for the allosteric and cooperativity properties of GDH.

Genetic variation in trophectoderm function in parthenogenetic mouse embryos.

The developing oocyte constitutes the source of a unique and essential molecular legacy that supports embryo metabolism for a substantial period after fertilization and that also directs important epigenetic events that prepare the embryonic genome for transcription and faithful execution of the developmental program. Parthenogenetically activated embryos provide a useful tool with which to examine how maternally derived factors contribute to early development. They also provide a means for evaluating genetic effects on the maternal genomic imprinting process. We report here that the genetic background of the oocyte affects trophectoderm function at the blastocyst stage. Parthenogenetic embryos obtained from activated (B6D2)F1 oocytes hatch efficiently in culture, whereas parthenogenones from C57BL/6 oocytes hatch less efficiently. Fertilized embryos of both strains hatch efficiently. The (B6D2)F1 parthenogenones also undergo blastocoel re-expansion after treatment with cytoskeletal inhibitors more rapidly than do C57BL/6 parthenogenones and exhibit a moderately greater abundance of the Na+, K(+)-ATPase alpha 1 subunit mRNA. Surprisingly, parthenogenones of both strains undergo blastocoel re-expansion more rapidly than do their normal fertilized counterparts. Parthenogenones of both types are able to attach efficiently in culture after removal of the zona pellucida. These observations indicate that significant genetic effects of maternal genotype on trophectoderm function are revealed in the absence of a paternal genetic contribution and that trophectoderm function also differs between parthenogenetic embryos and fertilized embryos. The differences observed between parthenogenetic and fertilized embryos indicate a likely role for one or more imprinted genes in the development of hatching and blastocoel expansion ability. The effect of maternal genotype on parthenogenetic embryo phenotype is consistent with possible differences in maternal genome imprinting or differences in ooplasm composition that have long-term effects on development. The specific differences in hatching and blastocoel re-expansion between parthenogenones of the two strains may be the result of differences in the activity or expression of a hatching enzyme or other molecules that affect fluid accumulation within the blastocyst, such as components of junctional complexes or proteins that regulate Na+, K(+)-ATPase activity.