Molecular Basis of the Disease State

Genetics of GSD-I

GSD I is an autosomal recessive disorder which affect 1:100,000 individuals worldwide with ~80 genetic mutations identified. Although there are many known mutations to G6PC and SLC37A4, many ethnic groups demonstrate ethnic-specific mutations which account for ~90% of the known disease genes. Depending on the specific ethnic group, these common mutations can account for 100% of disease alleles. The human G6Pase gene, G6PC (OMIM #613742), is a 12.5 kb long single-copy gene with 5 coding exons located on the 17q21 chromosome. This gene has been successfully cloned and reproduced in 1993 by Lei et al. and was a significant breakthrough because it allowed scientists to accurately investigate the genetic basis for G6Pase and discover how it correlates with GSD-Ia (Lei et. al., 1993). Lei et al. continued research into G6PC paid off in 1994 when they identified several mutations causing GSD-Ia and produced a G6Pase-deficient mouse model with symptoms characteristic of GSD-Ia (Lei et al., 1994). Similarly, the human G6PT gene, SLC37A4 (OMIM #602671), is a 5.3 kb long single copy with 9 coding exons located on the 11q23 chromosome. G6PT has been implicated in GSD-Ib and was also successfully cloned. (Gerrin et al., 1997)

Common GSD-Ia mutations:

cDNA change:

Amino Acid change:

247C-> T

Arg83Cys

248G->A

Arg83His

648G->T

Leu216Leu (new splice site)

1039C->T

Gln347 (stop codon)

Janecke et al., 2001

Common GSD-Ib mutations:

cDNA change Amino Acid change
352T->C Trp118Arg
1015G->T Gly339Cys
1042C & 1043T deletion Leu348Val

Janecke et al., 2001

GSD-1 Genetics and Structure Relationship

The reason why these mutations results in protein deficiency is because with the mutation from one amino acid to another, the protein structure is altered. In some cases, the shift of a protein structure may not affect the protein function at all; however, in the case of G6Pase and G6PaseT, the mutations are significant enough to alter the structure in a way that the protein’s function is impacted. As discussed in prior section, (Hass and Bryne, 1960) determined a phosphoenzyme complex was being created and (Feldman et al., 1972) determined that the phosphoenzyme bond was a phosphohistidine bond. The crystal structure of G6Pase was predicted in 1997 by Hemrika and Wever by performing X-ray crystallography on a vanadium-containing chloroperoxidase (similar functional as G6Pase) and revealed 9 helical transmembrane domains which contradicted the previously held notion that G6Pase had 6 helical transmembrane domains. In addition, the proposed active site for G6Pase was predicted by X-ray crystallography to contain the residues Lys76, Arg83, His119, Arg170, and His176 (Hemrika and Wever, 1997).

Figure 1: Predicted G6Pase topography. A. Previously believed G6Pase structure with 6 transmembrane domain. B. Modified G6Pase structure with 9 transmembrane domains. Source: Hemrika and Wever, 1999
Figure 1: Predicted G6Pase topography. A. Previously believed G6Pase structure with 6 transmembrane domain. B. Modified G6Pase structure with 9 transmembrane domains.
Source: Hemrika and Wever, 1997

In 2001, Chou et al. built off of Hemrika and Wever by testing 48 G6PC mutations for G6Pase for structural changes and tested the activity assay of each mutant. Chou et al. confirmed that unlike mutating amino acids predicted to be in nonhelical regions, mutating amino acids in predicted helical regions destabilized the helical structure of G6Pase’s transmembrane domain which eliminated G6Pase activity (Chou et al, 2001). It was also confirmed through Western Blot analysis that these helically disrupted G6Pases were rapidly degraded via the proteasome pathway (Chou et al., 2001). This suggests that the helical integrity of the transmembrane domain is essential for activity and disruption of this domain results in the loss of function. Furthermore, when the Hemrika and Wever predicted G6Pase active site residues were mutated, G6Pase was completely inactivated (Chou et al., 2001).

Figure 2: Known G6Pase mutations and predicted G6Pase structure. Source: Chou et al., 2001
Figure 2: Known G6Pase mutations and predicted G6Pase structure.
Source: Chou et al., 2001

Similarly with previous publication, Chou et al.were attempting to solve G6PT structure. Instead of using X-ray crystallography, G6PT was tagged at the amino- and carboxy-terminal regions and subjected to proteolytic digestion. The proteolytic digestion revealed that both terminal ends were located within the cytoplasm which suggested either a 10 or 12 transmembrane domain regions (Chou et al., 1999). Chou et al. generated two G6PT mutants, T53N and S55N, in order to solve this dilemma. They decided to mutate these residues because in the 10 transmembrane domain model the residue would be outside the membrane while in the 12 membrane domain it would be within the membrane. These mutants were subjected to Asn-linked glycosylation where only residues outside the membrane would be glycosylated. Chou et al. determined that both the T53N and S53N where glycosylated which demonstrated that the 10 transmembrane domain model was reliable (Chou et al., 1999). Investigating further, the sequence alignment with known homolog UhpT suggests that R28 and R275 are involved in substrate binding and D388 and K391 are involved in forming intra-helical salt bridge. Chou et al. generated R28C and R28H mutants and demonstrated complete abolishment of G6PT function and confirmed the long held belief that G6PT was had 10 transmembrane domains (Chou et al, 2009).

Figure 3: Predicted structure of G6PT with selected mutations of predicted critical residues. Source: Chou et al, 2009
Figure 3: Predicted structure of G6PT with selected mutations of predicted critical residues.
Source: Chou et al, 2009

Disease Affects in GSD-I Patients

GSD-I has been implicated in a great deal of complications ranging such as hypoglycemia, hyperuricemia, hypertriglyceridemia, and lactic acidosis as the most frequently observed to stunted growth and malignant liver tumors. Due to the defects associated with G6Pase, the conversion of G6P to glucose is impaired. Thus, because of this inability to convert G6P to glucose, the body is unable to maintain the blood glucose levels and homeostasis. This results in the condition called hypoglycemia. Furthermore, the excessive amount of G6P is then fluxed into the alternative pathways with produce triglycerides, lactate, and uric acid or converted back to glycogen which then accumulates in the kidneys and liver. This accumulation in the kidneys and liver can then result in enlarged kidneys, and kidney tumors (benign and malignant). The development of malignant liver tumors has been a research interest. Seeking to characterize the molecular profile of GSD-associated malignant liver tumors (HCAs), Calderaro et al. analyzed 25 HCAs developed in 15 patients with GSD by gene expression and DNA sequence of HNF1A, CTNNB1, IL6ST, GNAS, and STAT3 genes which are various forms of HCAs. Also, Calderaro et al. investigated all the GSD non-tumor livers for glycolysis, gluconeogenesis, and fatty acid synthesis alterations then compared the these observations with those observed in other H-HCA and non-GSD liver samples. What they observed was a particular molecular profile in GSD-related HCA characterized by a lack of HNF1A inactivation which is observed in with HNF1A inactivation and glucose-6-phosphatase deficiency (Calderaro et al., 2013).

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