Glycogen storage disease type Ia in Argentina: two novel glucose-6-phosphatase mutations affecting protein stability

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Abstract

Glycogen storage disease type Ia (GSD-Ia) is caused by deleterious mutations in the glucose-6-phosphatase gene (G6PC). A molecular study of this gene was carried out in 11 Argentinean patients from 8 unrelated families. Four missense (p.Gln54Pro, p.Arg83Cys, p.Thr16Arg, and p.Tyr209Cys) and one deletion (c.79delC) mutations have been identified. Two novel mutations, p.Thr16Arg (c.47C > G) located within the amino-terminal domain and p.Tyr209Cys (c.626A > G) situated in the sixth transmembrane helix, were uncovered in this study. Site-directed mutagenesis and transient expression assays demonstrated that both p.Thr16Arg and p.Tyr209Cys mutations abolished enzymatic activity as well as reduced G6Pase stability.

Introduction

Glucose-6-phosphatase (G6Pase, EC 3.1.3.9), which is a key enzyme in the homeostatic regulation of blood glucose, catalyses the hydrolysis of glucose-6-phosphate (G6P) to glucose and phosphate in the terminal step of gluconeogenesis and glycogenolysis. A deficiency in G6Pase causes glycogen storage disease type Ia (GSD-Ia, MIM #232200), an autosomal recessive disorder characterized by fasting hypoglycemia, hepatomegaly, short stature, hyperlactacidemia, hyperuricemia, and hyperlipidemia [1], [2].

The human glucose-6-phosphatase gene (G6PC, GDB 231927) [3] contains five exons spanning approximately 12.5 kb DNA located on chromosome 17q21. The encoded enzyme is anchored to the ER by nine transmembrane helices with the amino (N)-terminus in the lumen and the carboxyl (C)-terminus in the cytoplasm [4]. At present, 82 separate mutations have been identified in the G6PC gene of GSD-Ia patients (http://uwcmml1s.uwcm.ac.uk/uwcm/mg/search/231927.html). Structure and function studies of 48 missense and the p.Phe327del mutations grouped as active site, helical and non-helical have suggested that the structural integrity of transmembrane helices is critical for the stability and enzymatic activity of G6Pase [5]. In this study, we report the results of the first molecular analysis of the G6PC gene in eleven Argentinean patients and functional characterization of two novel mutations that abolish enzymatic activity and affect protein stability.

Section snippets

Patients

During the last 22 years, 11 GSD-I patients (6 females and 5 males) from 8 unrelated families were investigated. The diagnosis of GSD-I was based on typical clinical and laboratory findings; in 8 patients (six families) the G6Pase activity in liver biopsy samples was determined by different methods: in 4 patients the activity was measured in fresh liver homogenate according to Ricketts’ procedure [6] (period 1982–1988), in other 4 probands the enzymatic assay was performed in disrupted and

Results and discussion

Four missense and one deletion mutations have been identified in the G6PC gene of 10 Argentinean GSD-Ia patients (Table 2). The genotypes of the 10 patients were: [p.Gln54Pro] + [p.Gln54Pro] (3 patients, 2 families), [p.Fs35Ter] + [p.Fs35Ter] (2 unrelated patients), [p.Arg83Cys] + [p.Arg83Cys] (one patient), [p.Gln54Pro] + [p.Thr16Arg] (2 sisters), and [p.Arg83Cys] + [p.Tyr209Cys] (2 brothers).

In one patient, no mutation was detected in the G6PC coding region, therefore a complete analysis of the

Acknowledgments

C.J.A. was supported by a travel fellowship from the Universidad Católica de Córdoba. We thank Dra. A.M. Smania for her invaluable help and Dra. Laura E. Larovere for critical reading of the manuscript. This work was supported in part by the Agencia Córdoba Ciencia.

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