Molecular Genetics of Heritable Human Disorders

Janice Y. Chou, PhD, Head, Section on Cellular Differentiation
Wentao Peng, PhD, Staff Scientist
Shih-Yin Chen, PhD, Postdoctoral Fellow
Hyun-Sik Jun, PhD, Postdoctoral Fellow
Wai Han Yiu, PhD, Postdoctoral Fellow
Brian C. Mansfield, PhD, Guest Researcher
Chi-Jiunn Pan, BS, Senior Research Assistant
Yuk Yin Cheung, MS, Graduate Student
Paul A. Mead, BS, Postbaccalaureate Fellow

Glycogen storage disease type I (GSD-I) comprises a group of autosomal recessive disorders consisting of GSD-Ia, which are caused by a deficiency in the liver-/kidney-/intestine-restricted glucose-6-phosphatase-alpha (G6Pase-alpha), and GSD-Ib, which are caused by a deficiency in a ubiquitously expressed glucose-6-phosphate transporter (G6PT). Normally, G6Pase-alpha hydrolyzes G6P to glucose, and G6PT translocates G6P from the cytoplasm to the lumen of the endoplasmic reticulum (ER). Both diseases manifest a phenotype of disturbed glucose homeostasis, although GSD-Ib patients also suffer from neutropenia and neutrophil dysfunctions. There is no cure for GSD-I, and the current dietary therapy cannot prevent the development of long-term complications. The recent development of animal disease models presents an opportunity to delineate the disease more precisely and develop therapies that target the underlying disease process. Recently, we reported a second ubiquitously expressed G6P hydrolase, G6Pase-beta (G6PC3). Neutrophils express both G6Pase-beta and G6PT, suggesting that neutrophil homeostasis and function may require endogenous glucose production in the ER via the concerted action of G6Pase-beta and G6PT. To examine this hypothesis, we generated mouse lines deficient in either G6Pase-beta or G6PT and showed that both G6Pase-beta–deficient (G6pc3^−/−) and G6PT-deficient mice manifest neutropenia and neutrophil dysfunctions that mimic the symptoms of human GSD-Ib patients.

Mice lacking G6Pase-beta manifest neutropenia and neutrophil dysfunctions

Cheung, Kim,¹ Yiu, Pan, Jun, Ruef,² Mansfield, Chou; in collaboration with Lee, Westphal

Previous views held that a single ER enzyme, G6Pase-alpha, limited to the liver, kidney, and intestine, was solely responsible for hydrolysis of G6P to glucose for release into the blood. Recently, we characterized a second ubiquitously expressed G6Pase enzyme, G6Pase-beta (G6PC3), that shares kinetic properties and topological and active site structure with G6Pase-alpha and couples functionally with the G6PT to form an active G6Pase complex that hydrolyzes G6P to glucose. The G6Pase-beta-G6PT complex might be functional in neutrophils, and the myeloid defects in GSD-Ib might be attributable to the loss of activity of the G6Pase complex. To test such a hypothesis, we generated G6Pase-beta–deficient (G6pc3^−/−) mice and showed that the mice manifest neutropenia and neutrophil dysfunctions mimicking GSD-Ib. We further showed that neutrophils from G6pc3^−/− mice undergo ER stress and an enhanced rate of apoptosis. The results demonstrate that G6P translocation and metabolism in the ER are critical for normal neutrophil function and define a molecular pathway to neutropenia and neutrophil dysfunction of previously unknown etiology.

Cheung YY, Kim SY, Yiu WH, Pan CJ, Jun HS, Ruef RA, Lee EJ, Westphal H, Mansfield BC, Chou JY. Impaired neutrophil activity and increased susceptibility to bacterial infection in mice lacking glucose-6-phosphatase-beta. J Clin Invest 2007;117:784-793

Neutrophil stress and apoptosis underlie myeloid dysfunction in GSD-Ib

Kim,¹ Jun, Mead, Mansfield, Chou

GSD-Ib patients and mice manifest neutropenia and neutrophil dysfunctions of unknown mechanism. Neutrophils express both G6PT and G6Pase-beta that together transport G6P into the ER lumen and hydrolyze it to glucose. Given that G6PT-deficient neutrophils should be unable to produce endogenous glucose, we hypothesized that such an inability would lead to ER stress and increased apoptosis. Using GSD-Ib mice, we showed that GSD-Ib neutrophils exhibit increased production of ER chaperones and oxidative stress, consistent with ER stress, and increased Annexin V binding and caspase-3 activation, consistent with an increased rate of apoptosis. Bax activation, mitochondrial release of pro-apoptotic effectors, and caspase-9 activation demonstrate the involvement of the intrinsic mitochondrial pathway in these processes. The results show that G6P translocation and hydrolysis are required for normal neutrophil function, supporting the hypothesis that neutrophil dysfunction in GSD-Ib is attributable, at least in part, to ER stress and increased apoptosis.

Cheung YY, Kim SY, Yiu WH, Pan CJ, Jun HS , Ruef RA, Lee EJ, Westphal H, Mansfield BC, Chou JY. Impaired neutrophil activity and increased susceptibility to bacterial infection in mice lacking glucose-6-phosphatase-beta. J Clin Invest 2007;117:784-793.
Kim SY, Jun HS , Mead PA , Mansfield BC, Chou JY. Neutrophil stress and apoptosis underlie myeloid dysfunction in glycogen storage disease type Ib. Blood 2008;111:5704-5711.

The angiotensin system mediates renal fibrosis in GSD-Ia nephropathy

Yiu, Pan, Ruef, Peng, Mansfield, Chou; in collaboration with Starost

GSD-Ia patients develop renal disease of unknown etiology despite intensive dietary therapies. We showed that the expression of angiotensinogen (Agt), angiotensin type 1 receptor, transforming growth factor-beta1 (TGF-beta1), and connective tissue growth factor (CTGF) was higher in the kidneys of GSD-Ia mice than in those of the controls. The increase in renal expression of Agt occurred one week earlier than the increase in TGF-beta1 and CTGF, consistent with the upregulation of TGF-beta1 and CTGF by angiotensin II. Renal fibrosis was characterized by a marked increase in the synthesis and deposition of extracellular matrix proteins in the renal cortex and histological abnormalities, including tubular basement membrane thickening, atrophy, dilation, and multifocal interstitial fibrosis. Our results suggest that activation of the angiotensin system plays an important role in the pathophysiology of renal disease in GSD-Ia.

Chou JY, Mansfield BC. Mutations in the glucose-6-phosphatase-α (G6PC) gene that cause type Ia glycogen storage disease. Hum Mutat 2008;29:921-930.
Yiu WH, Pan C-J, Ruef RA, Peng WT, Starost MF, Mansfield BC, Chou JY. The angiotensin system mediates renal fibrosis in glycogen storage disease type Ia nephropathy. Kid Internal 2008;73:716-723.

Hepatic injury correlates with increased neutrophil infiltration of the liver in GSD-Ia

Kim,² Mansfield, Chou; in collaboration with Weinstein, Starost

GSD-Ia patients manifest hepatocellular adenoma (HCA) of unknown etiology. We showed that both GSD-Ia patients and mice have an underlying immune abnormality characterized by subclinical neutrophilia and elevated serum chemokine IL-8 or KC. We further showed that the elevation in serum IL-8 was more prominent in HCA-bearing GSD-Ia patients. Correlated with this elevation, in the mouse model of GSD-Ia, we observed hepatic injury characterized by necrotic foci and increased chemokines KC and MIP-2 as well as elevated neutrophil infiltration of the liver, suggesting one mechanism by which adenoma may arise.

Kim SY, Chen L-Y, Weinstein DA , Chou JY. Neutrophilia and elevated serum cytokines are implicated in glycogen storage disease type Ia. FEBS Lett 2007;581:3833-3838.
Kim SY, Weinstein DA , Starost MF, Mansfield BC, Chou JY. Hepatic injury correlates with increased neutrophil infiltration of the liver in glycogen storage disease type Ia. J Hepatol 2008;48:479-485.

The G6PT is a phosphate-linked antiporter deficient in GSD-Ib and GSD-Ic

Chen, Pan, Mansfield, Chou; in collaboration with Nandigama, Ambudkar

It is well established that a deficiency in G6PT causes GSD-Ib. Interestingly, deleterious mutations in the G6PT gene were identified in clinical cases of GSD-Ic that were thought to result from a deficiency in an inorganic phosphate (P_i) transporter. We hypothesized that G6PT is both the G6P and P_i transporter. Using reconstituted proteoliposomes, we showed that both G6P and P_i were taken up efficiently into P_i-loaded G6PT-proteoliposomes but were not detectable in P_i-loaded proteoliposomes containing the p.R28H G6PT null mutant. The specific G6PT inhibitors chlorogenic acid and vanadate inhibited the G6PT-proteoliposome–mediated G6P or P_i uptake. Taken together, our results indicate that G6PT plays a dual role as a G6P and a P_i transporter and that GSD-Ib and GSD-Ic are attributable to a deficiency in the same G6PT gene.

Chen SY, Pan CJ, Nandigama K, Mansfield BC, Ambudkar SV, Chou JY. The glucose-6-phosphate transporter is a phosphate-linked antiporter deficient in glycogen storage disease type Ib and Ic. FASEB J 2008;22:2206-2213.
Chou JY, Mansfield BC. von Gierke disease. In: Lang F, ed. Encyclopedia of Molecular Mechanisms of Disease. Springer, 2008, in press.

¹ So Youn Kim, PhD, former Postdoctoral Fellow
² Robert A. Ruef, BS, former Postbaccalaureate Fellow

Collaborators

Suresh V. Ambudkar, PhD, Laboratory of Cell Biology, Center for Cancer Research, NCI, Bethesda, MD
Eric J. Lee, DVM, Program in Genomics of Differentiation, NICHD, Bethesda, MD
Krishnamachary Nandigama, PhD, Laboratory of Cell Biology, Center for Cancer Research, NCI, Bethesda, MD
Matthew F. Starost, PhD, DVR, Division of Veterinary Resources, NIH, Bethesda, MD
David A. Weinstein, MD, University of Florida College of Medicine, Gainesville, FL
Heiner Westphal, MD, Program in Genomics of Differentiation, NICHD, Bethesda, MD

For further information, contact chouja@mail.nih.gov.