Molecular Genetics Of Heritable Human Disorders

Child Health And Human Development

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Abstract

INTRAMURAL RESEARCH PROJECT Z01 HD-00912-18 [unreadable] October 1, 2005 to September 30, 2006[unreadable] [unreadable] Molecular Genetics of Heritable Human Disorders[unreadable] [unreadable] The Section on Cellular differentiation conducts research to increase our understanding of the biology and pathogenesis of GSD-I and G6Pase-beta deficiency and to develop novel therapeutically approaches. GSD-Ia patients manifest a pro-atherogenic lipid profile but are not at elevated risk for atherosclerosis. We investigate cellular cholesterol efflux, the first step in reverse cholesterol transport, and antioxidant capacity, both are protective against atherosclerosis, in the sera of GSD-Ia patients. We show that sera from GSD-Ia patients are more efficient than sera from control subjects in promoting the scavenger receptor class B type I (SR-BI)-mediated cellular cholesterol efflux and have increased total antioxidant capacity compared to controls. The results suggest that the increase in SR-BI-mediated cellular cholesterol efflux and antioxidant capacity in the sera of GSD-Ia patients may contribute to protection against premature atherosclerosis.[unreadable] [unreadable] We hypothesized that disturbed glucose homeostasis manifested by GSD-Ia patients might affect myeloid functions. Using a mouse model of GSD-Ia that mimicking the human disorder, we show that GSD-Ia mice exhibit normal neutrophil activities but have elevated myeloid progenitor cells in the bone marrow and spleen. Interestingly, GSD-Ia mice exhibit a persistent increase in peripheral blood neutrophil counts along with elevated serum levels of G-CSF and chemokine KC. Taken together our results suggest that a loss of glucose homeostasis can compromise the immune system, resulting in neutrophilia. This may explain some of the unexpected clinical manifestations seen in GSD-Ia. [unreadable] [unreadable] GSD-Ib patients manifest disturbed glucose homeostasis as well as myeloid dysfunctions. However, whether G6PT deficiency in the bone marrow underlies myeloid dysfunctions in GSD-Ib remains controversial. To investigate this, we generated chimeric BM-G6PT-KO mice by transferring bone marrows from G6PT-deficient (G6PT-KO) mice to wild-type (WT) mice. While WT mice have normal myeloid functions, BM-G6PT-KO mice manifest myeloid abnormalities characteristic of G6PT-KO mice. Both have impaired neutrophil respiratory burst, chemotaxis, and calcium flux activities and exhibit neutropenia. In the bone marrow of BM-G6PT-KO and G6PT-KO mice, the numbers of myeloid progenitor cells are increased, while in the serum there is an increase in the levels of G-CSF and chemokine KC. These findings demonstrate that myeloid dysfunctions in GSD-Ib are intrinsically linked to G6PT deficiency in the bone marrow and neutrophils.[unreadable] [unreadable] G6PT is a hydrophobic protein anchored to the ER by ten-transmembrane helices. To evaluate the feasibility of gene replacement therapy for GSD-Ib, we have infused adenoviral (Ad) vector carrying human G6PT (Ad-hG6PT) into G6PT-/- mice. Ad-hG6PT-infusion restores significant levels of G6PT mRNA expression in the liver, bone marrow, and spleen and corrects metabolic as well as myeloid abnormalities in G6PT-/- mice. The G6PT-/- mice receiving gene therapy exhibit improved growth; normalized serum profiles for glucose, cholesterol, triglyceride, uric acid, and lactic acid; and reduced hepatic glycogen deposition. The therapy also corrects neutropenia and lowers the elevated serum levels of G-CSF. The development of bone and spleen in the infused G6PT-/- mice is improved and accompanied by increased cellularity and normalized myeloid progenitor cell frequencies in both tissues. This effective use of gene therapy to correct metabolic imbalances and myeloid dysfunctions in GSD-Ib mice holds promise for the future of gene therapy in humans.[unreadable] [unreadable] Neutropenia and neutrophil dysfunction are common in many diseases although the etiology is often unclear. Previous views held that there was a single ER enzyme, G6Pase-alpha, whose activity, limited to the liver, kidney and intestine, was solely responsible for the final stages of gluconeogenesis and glycogenolysis in which G6P is hydrolyzed to glucose for release to the blood. Recently, we characterized a second G6Pase activity, G6Pase-beta, which is also capable of hydrolyzing G6P to glucose, but ubiquitously expressed, and not implicated in interprandial blood glucose homeostasis. We now report the unexpected discovery that the absence of G6Pase-beta leads to neutropenia; defects in neutrophil respiratory burst, chemotaxis, and calcium flux; and increased susceptibility to bacterial infection. Consistent with this, neutrophils from G6Pase-beta-deficient mice undergo ER stress and enhanced rate of apoptosis. The results demonstrate that G6P translocation and metabolism in the ER are critical for normal neutrophil functions and define a molecular pathway to neutropenia and neutrophil dysfunction of previously unknown etiology, providing a potential model for the treatment of these conditions.