Molecular Genetics Of Heritable Human Disorders

Child Health And Human Development

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Abstract

INTRAMURAL RESEARCH PROJECT Z01 HD-00912-18 HDB October 1, 2002 to September 30, 2003 Molecular Genetics of Heritable Human Disorders Glycogen storage disease type I (GSD-I) is caused by deficiencies in the endoplasmic reticulum (ER)-bound glucose-6-phosphatase (G6Pase) system comprised of G6Pase and glucose-6-phosphate transporter (G6PT). G6PT translocates glucose-6-phosphate (G6P), the product of gluconeogenesis and glycogenolysis, from cytoplasm to the lumen of the ER and G6Pase hydrolyses the intraluminal G6P to glucose and phosphate. Together, G6Pase and G6PT maintain glucose homeostasis. Deficiencies in G6Pase and G6PT cause GSD-1a and GSD-1b, respectively. Both manifest the symptoms of G6Pase deficiency characterized by growth retardation, hypoglycemia, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic acidemia. GSD-Ib patients also present with unique symptoms of neutropenia and myeloid dysfunction, not obviously related to G6P metabolism in the gluconeogenic tissues. Over the last 20 years dietary therapy has alleviated some metabolic abnormalities of GSD-I patients and delayed disease progress. However, the underlying disease remains untreated and the efficacy of dietary treatment is frequently limited by poor compliance. Therefore, long-term complications still develop in adult patients. An understanding of the molecular genetics and pathogenesis of GSD-1 is needed to lead to therapies that can rectify the long-term complications of GSD-1. The G6PT is a 10-transmembrane domain ER protein. To date, 69 G6PT mutations, including 28 missenses and 2 codon-deletions, have been identified in GSD-Ib patients. We previously characterized 15 of the missense and one codon-deletion mutations using a pSVL-based expression assay. A lack of sensitivity in this assay limited the discrimination between mutations that lead to loss of function and mutations that leave a low residual activity. We now report an improved G6PT assay, based on an adenoviral vector-mediated expression system, and its use in the functional characterization of all 30 codon mutations found in GSD-Ib patients. Twenty of the naturally occurring mutations completely abolish microsomal G6P uptake activity while the other 10 mutations, including 5 previous characterized ones, partially inactivate the transporter. This information should greatly facilitate genotype-phenotype correlation. We also report a structure-function analysis of G6PT. In addition to the 3 destabilizing mutations reported previously, we now show that the G50R, C176R, V235del, G339C, and G339D mutations also compromise the G6PT stability. Mutation analysis of the amino-terminal domain of G6PT shows that it is required for optimal G6P uptake activity. Finally, we show that degradation of both wild-type and mutant G6PT is inhibited by a potent proteasome inhibitor, lactacystin, demonstrating that G6PT is a substrate for proteasome-mediated degradation. Amino acid sequence alignments has identified a signature motif shared by G6PT and a family of transporters of phosphorylated metabolites. Two null signature motif mutations have been identified in the G6PT gene of GSD-Ib patients. We now characterize the activity of seven additional mutants within the motif. Five mutants lack microsomal G6P uptake activity and one retains residual activity, suggesting that in G6PT the signature motif is a functional element required for microsomal glucose-6-phosphate transport. In addition to disrupted glucose homeostasis, GSD-Ib patients have unexplained and unexpected defects in neutrophil respiratory burst, chemotaxis, and calcium flux, in response to the bacterial peptide f-Met-Leu-Phe, as well as intermittent neutropenia. We generated a G6PT knockout (G6PT-/-) mouse that mimics all known defects of the human disorder and used the model to further our understanding of the pathogenesis of GSD-Ib. We demonstrate that the neutropenia is caused directly by the loss of G6PT activity; that chemotaxis and calcium flux, induced by the chemokines KC and macrophage inflammatory protein-2, are defective in G6PT-/- neutrophils; and that local production of these chemokines and the resultant neutrophil trafficking in vivo are depressed in G6PT-/- ascites during an inflammatory response. The bone and spleen of G6PT-/- mice are developmentally delayed and accompanied by marked hypocellularity of the bone marrow, elevation of myeloid progenitor cell frequencies in both organs and a corresponding dramatic increase in granulocyte colony stimulating factor levels in both GSD-Ib mice and humans. So, in addition to transient neutropenia, a sustained defect in neutrophil trafficking due to both the resistance of neutrophils to chemotactic factors, and reduced local production of neutrophil-specific chemokines at sites of inflammation, may underlie the myeloid deficiency in GSD-Ib. These findings demonstrate that G6PT is not just a G6P transport protein but also an important immunomodulatory protein whose activities need to be addressed in treating the myeloid complications in GSD-Ib patients. G6Pase is a highly hydrophobic protein anchored to the ER by 9-transmembrane helices. The protein can not be expressed in a soluble form and must embed correctly in the ER membrane and couple with other proteins to be functional. Therefore, enzyme replacement therapy is not an option, but somatic gene therapy, targeting G6Pase to the liver and the kidney, is an attractive possibility. Using a G6Pase knockout (G6Pase-/-) mouse model generated in this laboratory, we have shown that a combined adeno (Ad-mG6Pase) and adeno-associated virus (AAV) serotype 2 (AAV2-mG6Pase)-mediated gene transfer leads to sustained G6Pase expression in both the liver and the kidney and corrects the murine GSD-Ia disease for at least 12 months. The most critical clinical presentation in GSD-Ia is the life threatening hypoglycemia. Because AAV2-mG6Pase-mediated transgene expression increases slowly, typically peaking 5 to 7 weeks post-infusion, we co-infused AAV2-mG6Pase with Ad-mG6Pase which allows the infused animals to survive weaning. We now demonstrate that neonatal G6Pase-/- mice infused only with AAV1-mG6Pase, a recombinant AAV of a different serotype, not only survived weaning but also lived to 12 months of age. Our results suggest that human GSD-Ia would be treatable by gene therapy.