Analysis of GBE1 mutations via protein expression studies in glycogen storage disease type IV: A report on a non-progressive form with a literature review

Abstract

Background: Glycogen storage disease type IV (GSD IV), caused by GBE1 mutations, has a quite wide phenotypic variation. While the classic hepatic form and the perinatal/neonatal neuromuscular forms result in early mortality, milder manifestations include non-progressive form (NP-GSD IV) and adult polyglucosan body disease (APBD). Thus far, only one clinical case of a patient with compound heterozygous mutations has been reported for the molecular analysis of NP-GSD IV. This study aimed to elucidate the molecular basis in a NP-GSD IV patient via protein expression analysis and to obtain a clearer genotype-phenotype relationship in GSD IV.

Case presentation: A Japanese boy presented hepatosplenomegaly at 2 years of age. Developmental delay, neurological symptoms, and cardiac dysfunction were not apparent. Observation of hepatocytes with periodic acid-Schiff-positive materials resistant to diastase, coupled with resolution of hepatosplenomegaly at 8 years of age, yielded a diagnosis of NP-GSD IV. Glycogen branching enzyme activity was decreased in erythrocytes. At 13 years of age, he developed epilepsy, which was successfully controlled by carbamazepine.

Molecular analysis: In this study, we identified compound heterozygous GBE1 mutations (p.Gln46Pro and p.Glu609Lys). The branching activities of the mutant proteins expressed using E. coli were examined in a reaction with starch. The result showed that both mutants had approximately 50% activity of the wild type protein.

Conclusion: This is the second clinical report of a NP-GSD IV patient with a definite molecular elucidation. Based on the clinical and genotypic overlapping between NP-GSD IV and APBD, we suggest both are in a continuum.

Keywords: APBD, adult polyglucosan body disease; Epilepsy; Functional analysis; GBE, 1,4-alpha-glucan-branching enzyme; GSD; GSD IV, glycogen storage disease type IV; Glycogen storage disease type IV; Glycogenosis, glycogen branching enzyme 1; NP-GSD IV, non-progressive form of glycogen storage disease type IV; RT-PCR, reverse transcriptase-polymerase chain reaction; WT, wild type.

Fig. 1

Organization of the GBE1 gene, and disease-associated mutations hitherto reported. The number above each box indicates the exon number. References are denoted in parentheses. The mutations identified in our patient are indicated by an asterisk. Null mutations such as intragenic deleterious, nonsense, frameshift, and splice-site mutations are underlined. Herein, we gathered neonatal and perinatal forms in a mass because their diagnostic criteria are not strictly determined and clinical outcomes in these forms do not differ significantly. Null mutations, except for those located in exons 15 and 16, tend to associate with more severe forms of glycogen storage disease type IV (GSD IV), such as classic hepatic form or perinatal/neonatal neuromuscular forms. The same mutations are often reported in unrelated patients with milder forms, such as non-progressive-GSD IV (NP-GSD IV) and adult polyglucosan body disease (APBD).

Fig. 2

Light microscopic images from the liver biopsy specimen of our patient (bar: 100 μm). The hepatocyte cytoplasm was stained with periodic acid-Schiff (PAS) stain with a coarse granular pattern (A). PAS-positive deposits were partially diastase-resistant (B). A mild fibrotic change was observed via Azan staining (C).

Fig. 3

GBE1 mutation analysis. Electropherogram of GBE1 gDNA of our patient, showing heterozygous missense mutations (arrow). (B) Glutamine at position 46 (Gln46) and glutamate at position 609 (Glu609) in Homo sapiens GBE1 are conserved. (C) PolyPhen-2 and SIFT analyses of the p.Gln46Pro and p.Glu609Lys mutations. (D) A structural model of GBE protein generated via PyMOL. Hydrogen bonding is indicated with dashed lines and steric hindrance is indicated with discs. (D-1, D-3) The native GBE structure shows that the Gln46 and Glu609 interact with Asp44 and Asn456, respectively. (D-2, D-4) The modeled structure of the mutant proteins (Gln46Pro-GBE and Glu609Lys-GBE) is speculated to have structural changes caused by steric hindrance with Asp44 and Asn456, respectively.