Glucose-6-phosphatase (G6PC1) promoter polymorphism associated with glycogen storage disease type 1a among the Indian population

Abstract

Glycogen storage disease type 1a (GSD-1a) is an autosomal recessive inherited metabolic disorder caused by mutations in the G6PC1 gene leading to a deficiency of the glucose-6-phosphatase enzyme. To date more than 110 mutations have been identified in the coding regions as well as splice junctions, however only two polymorphisms have been so far identified in the promoter of the G6PC1 gene. Thirteen unrelated Indian patients (9 boys and 4 girls) with GSD-1a were screened for mutations through PCR amplification of genomic DNA for the promoter region of G6PC1 gene, followed by direct sequencing. Mutation analysis had identified a heterozygous polymorphism (g.-225C>G; rs559748047) with two unrelated GSD-1a patients, which had yielded a target site for the restriction enzyme HpyCH4III, hence RFLP was done for further confirmation. One hundred healthy Indian subjects were genotyped for this variant rs559748047 with an allele frequency of 13.5%. This polymorphism was found located in the AE-II region that consists of binding sites for several transcription factors including CRE. As a result, the transcriptional activity of the promoter construct containing mutant G allele exhibited an 8.43 fold decrease in activity than wild-type C allele as determined functionally by the transfection of luciferase reporter constructs containing the promoter region in HepG2 cells. Further, hormonally inductive transcriptional activity was significantly reduced for dexamethasone, dbcAMP and insulin with mutant G allele in the G6PC1 promoter. Thus, the promoter polymorphism (g.-225C>G) of G6PC1 gene was characterized to be associated with the decrease in both basal and hormonal transcriptional activity of G6Pase. These findings suggest that g.-225C>G, rs559748047 polymorphism of the G6PC1 promoter may be crucial in the disease development and progression of GSD-1a.

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1. Introduction

Glycogen Storage Disease type 1a (GSD-1a; MIM #232200), is an inherited metabolic disorder caused by autosomal recessive mutations in G6PC1 gene encoding glucose-6-phosphatase-α (G6Pase-α) enzyme that catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and phosphate in the terminal step of gluconeogenesis and glycogenolysis. G6Pase is an endoplasmic reticulum (ER) associated enzyme, normally produced in the liver, kidney, and intestinal mucosa, and thus the deficiency of this enzyme activity due to the defective G6PC1 gene is associated with increased accumulation of glycogen in those organs. GSD-1a is generally characterized by fasting hypoglycemia, growth retardation and hepatomegaly due to the accumulation of glycogen and fat in the liver.^1,2 Other metabolic consequences associated with elevated cytoplasmic G6P like hyperlactacidemia, hyperlipidemia and hyperuricemia are the most characteristic metabolic abnormalities of GSD-1.³ Long-term complications include gout, hepatic adenomas, osteoporosis, renal disease and short stature.^1,4,5 GSD-1a is the most prevalent of GSD representing about 80% among GSD-1 types, which has an estimated annual incidence of around 1/100 000 births⁴ with the carrier frequency of 1 in 150.⁶

G6PC1 is a single copy gene located on chromosome 17q21.31 (RefSeqGene: LRG_147) in humans^7,8 with a genomic size of 12.5 kb consisting of five exons, four introns and a proximal promoter region. The G6Pase catalytic unit contains 357 amino acids with a molecular mass of ∼35 kDa, which is anchored to the ER by nine transmembrane helices with its NH– and COOH– termini facing the lumen and cytoplasm, respectively.⁹ Cloning and functional analysis of 5′-UTR of first 1.2 kb promoter region revealed a three folds increase in the transcriptional activity than control and contains many hormone responsible elements, which are regulated by dexamethasone, dbcAMP and insulin.¹⁰ Two insulin response sequences are identified in the G6PC1 promoter from human, rat and mice, which is shown to regulate the insulin response activity.^10–12 Furthermore, two cAMP response elements (CRE1 and CRE2) are identified in the human G6PC1 promoter and functionally characterized to induce G6PC1 gene transcription through protein kinase-A mediated phosphorylation of transcription factor, cAMP-response element binding factor (CREB).^13,14

Since the first description of G6Pase deficiency in GSD-1a and the elucidation of its molecular determinants of this genetic defect, a wide spectrum of around 110 different clinical mutations in the G6PC1 gene have been identified from approximately 550 GSD-1a patients and most of them are missense mutations accounting 64% that result in single-amino acid substitutions (Human Gene Mutation Database; http://www.hgmd.cf.ac.uk). One of the molecular abnormality found in the 5′-UTR region of G6PC1 gene was a common polymorphism at location of g.-77G>A from the infants of non sudden infant death syndrome (non-SIDS) and Scottish North European control population.¹⁵ Functional analysis of this novel polymorphism resulted in decreased basal G6PC1 promoter activity than the wild type promoter. Furthermore, basal G6PC1 transcriptional activity with wild type and -77G>A promoter polymorphism was hormonally regulated. Another registered SNP of G6PC1 promoter in the database is rs559748047 (c.-225C>G) with the global minor allele frequency of 0.0044 (0.44%) for G allele. Likewise, a promoter variant of serotonin receptor (HTR3A), rs1062613, has been previously reported to be associated with schizophrenia especially among Indian population.¹⁶ In this study, we present evidences for the occurrence of SNP: rs559748047 (-225C>G) in the G6PC1 promoter of Indian GSD-1a patients with reduced G6Pase activity as a functional consequence.

2. Subjects and methods

2.1. Study subjects and sample collection

The study group consisted of a total of 13 GSD-1a patients, who were periodically enrolled between 2010 and 2013 at the Kanchi Kamakoti CHILDS Trust Hospital, Chennai, India and one hundred healthy individuals without GSD (control). The subjects were exclusively unrelated Indians and recruited without restrictions on gender and age. The age distribution of the GSD-1a and non-GSD groups was 0.10–10 and 6–45 years, respectively. Among the 13 GSD-1a patients, 9 were male and 4 were female, and of the 100 non-GSD control individuals, 61 were male and 39 were female. Clinical diagnosis of GSD-1a was based on typical clinical and laboratory findings such as hepatomegaly, recurrent hypoglycemia, and hyperlactacidemia and further conformed by accumulation of glycogen in hepatocytes in liver biopsy. A one-time sample of approximately 3 ml of peripheral blood was collected from each participant. All subjects were provided with informed consent to participate in the study, as approved by the Institutional Ethical Committee of Madurai Kamaraj University, Madurai, India.

2.2. DNA extraction and genotyping

Genomic DNA was isolated from peripheral blood samples of all patients and healthy subjects using Blood Genomic DNA Miniprep Kit (Axygen, USA). Using genomic DNA as template, a 420 bp fragment of G6PC1 promoter (GenBank Accession no. U01120) region corresponding to the nucleotides -426 to -7, which is adjacent upstream sequence to the translational start site, was amplified using the primer sets 5′-CAGGCATAGAAAATCTGACA-3′ and 5′-TTCCTGAGGTGCCAAGGAA-3′.¹⁴ For screening mutations in the promoter region of G6PC1 gene, PCR was performed using genomic DNA (50 ng) from the blood samples of patients and control subjects. PCR conditions were as follows: denaturation at 94 °C for 4 min; 32 subsequent amplification cycles performed at 94 °C for 50 s, 56 °C for 50 s and 72 °C for 50 s; and a final step at 72 °C for 7 min. The amplified PCR products were gel purified using GenElute PCR Clean-up Kit (Sigma, USA) and nucleotide sequence of the amplicon was determined by direct sequencing of both the DNA strands. Further to confirm the mutant genotypes, the PCR products were subjected to RFLP analysis with the restriction enzyme HpycH4III and the digested products were analyzed on a polyacrylamide gel.

2.3. Construction of the G6PC1 promoter reporter plasmid and mutagenesis

The G6PC1 -225C and -225G constructs were generated by PCR amplification of nucleotides -426 to -7 of the G6PC1 gene promoter from human genomic DNA with the respective homozygous genotype. The amplified PCR products were cloned upstream of the firefly luciferase reporter gene into the pGL3-basic expression vector (Promega, USA). Constructs were subsequently sequence verified to confirm the presence or absence of mutations in both wild type and mutant constructs.

2.4. Cell culture and transient transfection

HepG2 (Human hepatocellular carcinoma) cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Himedia) containing 10% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, 100 μg ml⁻¹ penicillin and streptomycin at 37 °C under 5% CO₂ humidified, sterile conditions. HepG2 cells (1 × 10⁵) were transiently transfected at 70–80% confluency with 1 μg of G6PC1 wild-type and mutant promoter constructs using Lipofectamine 2000 (Invitrogen, USA). After 4 h of transfection the cells were added with fresh complete medium. Cells were collected following 48 h of incubation by centrifugation, washed with PBS, and lysed with 120 μl of passive lysis buffer (Promega, USA). To assess the hormonal influences, cells transiently transfected with promoter/reporter constructs were incubated for 4 h with the transfection medium. Cells were added with complete DMEM medium and incubated for 24 h. Subsequently the medium was replaced with DMEM containing dexamethasone, dbcAMP and insulin and further incubated for at least 24 h before assay.

2.5. Luciferase assay

Twenty microliters of the cell lysate was used to measure luciferase activity using luciferase assay system (Promega, USA) in a SIRIUS Luminometer (Berthold Detection System GmbH, Germany) according to the manufacturer's instructions. Five microliters of cell lysate was used to determine protein concentrations by Bradford method and the levels of firefly luciferase activity were normalized with protein content.

2.6. Statistical analysis

Statistical significance was determined by Student's t-test or one-way ANOVA followed by Tukey's honestly significant difference (HSD) test, with statistical significance being set at 0.05 (P < 0.05).

3. Results

3.1. G6PC1 mutational analysis

Genomic DNA isolated from the peripheral blood of 13 GSD-1a Indian patients were screened for the presence of sequence variants in ∼420 bp of the proximal promoter of G6PC1 gene by PCR analysis and direct sequencing. Sequencing of G6PC1 promoter region (-426 to -7) revealed a heterozygous substitution mutation in 2 of 26 alleles (7.7%) at -225th position (g.-225C>G; rs559748047). The sequence chromatograms for the promoter region of G6PC1 gene for both the patients are shown in the Fig. 1. DNA sequence analysis to find target sites for restriction enzymes in the G6PC1 region of the variant G allele (-225C>G) that had introduced a target site for the restriction enzyme HpycH4III when compared with wild-type sequence. PCR-RFLP analysis with HpycH4III digestion further confirmed heterozygosity of the identified variant in both the patients (Fig. 2). Clinical laboratory results of the patient-1 showed a normal blood cell count, glucose 40 mg dl⁻¹, SGOT 200 IU L⁻¹, SGPT 115 IU L⁻¹, lactate 4.2 mmol L⁻¹, triglyceride 198 mg dl⁻¹ and patient-2 had glucose 38 mg dl⁻¹, SGOT 117 IU L⁻¹, SGPT 124 IU L⁻¹, lactate 6.2 mmol L⁻¹ and triglyceride 1378 mg dl⁻¹. Both the patients were not presented with neutropenia. Further, screening of mutations in the exons and exon–intron junctions of G6PC1 gene from both the GSD-1a patients showed no other variations (data not shown). To confirm whether the identified substitution mutation (-225C>G) was a common polymorphism, G6PC1 promoter region of 100 unrelated healthy controls were sequenced and the results evidenced as a polymorphism that was found in 27 out of the 200 alleles (13.5%). This observed nucleotide substitution g.-225C>G was positioned in the consensus nucleotide region of the activation element-II (AE-II; -225 to -204, previously known as -146 to -125) for transcription of G6PC1 containing overlapping binding sites for the transcription factors (HNF3, CREB, and C/EBP) and the same location was also recognized as the conserved nucleotide motif of c-AMP response elements (CRE: GTAAATCACCCT) known to bind with CRE binding proteins (CREB) (Fig. 3A). Importantly, these binding sites were highly conserved in the equivalent location within the G6PC1 proximal promoter region of human, mice and rat (Fig. 3B).

Fig. 1 Sequence chromatogram of the G6PC1 promoter region of GSD-1a patients showing heterozygosity for the mutant allele at the position g.-225C>G.

Fig. 2 PCR-RFLP analysis of G6PC1 promoter (420 bp) of GSD-1a patients with HpyCH4III. The healthy control PCR product does not contain HpyCH4III target site, while the g.-225C>G variant PCR product has one HpyCH4III target site. Lane M – 100 bp DNA ladder, Lane 1 – control, Lane 2 – control digested, Lane 3 – patient 1 and Lane 4 – patient 2.

Fig. 3 (A) Nucleotides sequences (-426/-7) of the G6PC1 promoter region. The TATA-box and regulatory binding sites are under lined. AE-II region is marked in a box. The first identified promoter polymorphism -77 G is marked^#. The polymorphism identified in this study at nucleotide -225C is highlighted*. (B) Comparison of the human G6PC1 promoter sequence between -243 and -198 with the orthologous G6PC1 sequences from rat and mouse. AE-II region of G6PC1 promoter encompassing consensus transcriptional binding sites is highlighted.

3.2. Luciferase activity of wild type and mutant promoter construct

The effect of identified polymorphism (-225C>G) in the G6PC1 promoter region on its transcriptional activity was determined by generating wild type (-426 to -7) and variant (-426 to -7)-225C>G promoter constructs using a pGL3 basic vector. Lysates from HepG2 cells transiently transfected with the wild-type construct showed 18.83 folds increase in basal transcriptional activity relative to the basic pGL3 vector (P < 0.01).

In contrast, the (-426/-7)-225C>G variant exhibited only 10.4 folds increase in the expression of basal transcriptional activity relative to the basic pGL3 vector (P < 0.01) and 8.43 folds decreased transcriptional activity (P < 0.01) when compared with the wild-type G6PC1 promoter (Fig. 4A). These results primarily confirmed that g.-225C>G polymorphism leading a significant reduction in the G6Pase activity due to a loss of transcription from the G6PC1 promoter.

Fig. 4 Basal transcriptional activity on -426/-7 wild-type and mutant G6PC1 constructs. HepG2 cells were transfected using Lipofectamine 2000 with 1 μg of G6PC1 construct. The luciferase activity was measured 48 h after transfection and normalised relative to the protein content of cell lysate. (A) Luciferase activity of each construct is expressed as the mean ± SEM (n = 6), relative to basal expression of the pGL3 basic vector construct, which is set as 1. The -426/-7 mutant construct contains the -225C>G mutation. **P < 0.01 compared to wild-type basal activity. (B) The effect of hormones on -426/-7 wild-type and mutant G6PC1 constructs. Transfected cells were incubated in the presence of dexamethasone (1 μM), insulin (500 nM) and dbcAMP (500 μM) for 24 h. Luciferase activity of each construct is expressed as the mean ± SEM (n = 6), relative to basal expression of the construct, which is set as 1. **P < 0.01, +P < 0.01 compared to -426/-7 wild-type basal activity; #P < 0.01 compared to -426/-7 mutant basal activity; ϕP < 0.05 compared to -426/-7 wild-type activity in the presence of dexamethasone; ×P < 0.05 compared to -426/-7 wild-type activity in the presence of dbcAMP; $P < 0.05 compared to -426/-7 wild-type activity in the presence of insulin.

3.3. Hormonal activity of wild type and mutant construct

Transcriptional activity of G6PC1 promoter is known to be regulated by cellular levels of cAMP, glucocorticoids and hormones through several transcription factors including HNF, IRS, GRE and CRE. Thus, we further determined the impact of hormonal regulation on transcriptional activity with the promoter polymorphism that was confined in the consensus nucleotide region of the AE-II. For this, wild type (-426/-7) and variant (-426 -7)-225C>G promoter constructs of pGL3 basic vectors were transiently expressed in HepG2 cells followed by incubation in the presence of dexamethasone, dbcAMP and insulin, and the lysates were assayed for promoter activity (Fig. 4B). Basal transcriptional activity of wild-type construct was significantly elevated in the presence of dexamethasone (2.52 folds) and dbcAMP (4.14 folds), whereas insulin had not exhibited any significant difference in the transcriptional activity (1.34 folds). Compared to wild type, transcriptional activity of the promoter variant (-426/-7)-225C>G was significantly lower in the presence of dexamethasone, dbcAMP and insulin (1.07, 2.64, 0.64 folds, respectively). However, the basal transcriptional activity of (-426/-7)-225C>G variant construct was induced significantly by dexamethasone (1.64 folds) and dbcAMP (4.21 folds), but insulin had no significant changes in the transcriptional activity.

4. Discussion

Glucose-6-phosphatase (G6Pase-α) is an ER membrane residing enzyme that catalyzes the rate-limiting step of gluconeogenesis and glycogen breakdown in the liver, kidney and intestine. Deficiency of G6Pase results GSD-1a with the classical symptoms including hypoglycemia, lactic acidosis, hyperuricemia, hyperlipidemia and hepatomegaly. Though several disease causing mutations have been identified throughout the protein-coding sequences of G6PC1, the mutation g.-225C>G identified in this study is a heterozygous promoter polymorphism of G6PC1 gene (rs559748047), which is the second promoter variant of G6PC1, recognized for the first time as associated with two unrelated GSD-1a patients from Indian ethnicity and also has not been reported previously elsewhere. Hence, we genotyped 100 normal healthy Indian subjects for this SNP rs559748047 and identified this variant as a common polymorphism with an allele frequency of 13.5%. Reporter gene assays in HepG2 cells revealed a significant decrease in transcription (∼43%) of G6PC1 promoter region (-426/-7) containing plasmids carrying variant G allele at -225th position, compared with plasmids carrying wild-type C allele. Previously, a heterozygous a novel polymorphism -77 G>A in the 5′-UTR of G6PC1 had been reported with non-SIDS infant patients and healthy controls from Scottish North European population.¹⁵

Molecular mechanisms towards uncovering the transcriptional regulation of the G6PC1 gene at promoter levels are explored intensively.^11,13,14,17 The G6PC1 proximal promoter is ∼237 bp in length between nucleotides -234 to +3, which is shown to encompass three activation elements (AE) at nucleotides -234/-212 (AE-I), -146/-125 (AE-II), and -124/-71 (AE-III) that binds with transcription factors and transactivate G6Pase gene expression in hepatocytes. In addition, AE-II region is predicted to have overlapping binding sites for HNF3 (-139/-133), CREB (-136/-129), and C/EBP (-130/-125), which was demonstrated to bind with multiple proteins by EMSA analysis and functional assays further confirmed that HNF1, HNF3γ and CREB play important roles in regulating G6Pase expression.¹³ The g.-225C>G polymorphism observed in this study from a GSD-1a patient was found to be localized in the AE-II region that consists binding sites for several transcription factors, which exemplifies its significance in the regulation of G6Pase expression. This region has also been recently recognized as putative consensus nucleotide motif of c-AMP response elements (CRE2), which is known to bind with CREB.¹² Thus, it is not a surprise that G6PC1 promoter with the variant G allele at -225th position having abrogated transcriptional activity than the wild-type C allele, indicating that this polymorphism g.-225C>G is indispensable for basal G6PC1 gene transcription and play prominent role in regulation of G6Pase expression.

G6Pase regulates hepatic glucose production, whose expression is induced by cAMP, glucocorticoids, glucose and fatty acids^10,12,18 and inhibited by insulin, tumor necrosis factor-α, and interleukin-6.^11,19 Regulation of G6Pase expression at transcriptional levels is coordinated by the involvement of several transcription factors that bind to specific cis-regulatory elements on the G6PC1 promoter and mediate the effect of various hormones and other regulatory molecules.^13,15 G6PC1 promoter constructs (g.-231) generated individually with mutations in the cis-elements HNF-1, HNF-4, CRE1, and CRE2 leading to disruption of transcriptional binding sites have exhibited significant decrease in the transcription in the presence of dexamethasone than the wild-type, which have clearly evidenced the involvement of HNF and CRE binding sites in the G6PC1 promoter as accessory factor regulatory elements for glucocorticoid mediated induction of G6Pase gene transcription.²⁰ Furthermore, cAMP levels were shown induce G6Pase expression at mRNA levels in primary hepatocytes without altering the mRNA stability of G6Pase,²¹ which has explained the implications cAMP in the regulation of G6PC1 gene at a transcriptional level. Hence, we further determined that whether the SNP rs559748047 (g.-225C>G) identified in this study from GSD-1a patients has any functional role in the regulation of G6Pase gene transcription by hormones and glucocorticoids in terms of transcriptional activity, which is directly proportional to G6Pase activity. Dexamethasone and dbcAMP induced the basal transcriptional activity of the -426/-7 wild-type construct significantly, but insulin had no significant difference in the basal transcriptional activity. These results corroborated with the previous findings of hormonal influences on G6Pase transcription.^10,11,19 At the same time, transcriptional activity of the promoter with variant allele (-426/-7)-225C>G was significantly lower in the presence of dexamethasone, dbcAMP and insulin. Basal transcriptional activity of (-426/-7)-225C>G mutant constructs were induced significantly by dexamethasone and dbcAMP, while insulin showed no effects in the basal transcriptional activity. Altogether, these results substantiated that the g.-225C>G mutation abolished AE-II region that encompass overlapping binding sites for HNF, CRE, and C/EBP responsible for hormonal mediated regulation and exhibited impairment of G6PC1 expression resulting diminished G6Pase activity. Hepatic G6Pase catalyses the last step of glycogenolysis and gluconeogenesis and both the pathways are hampered when its expression is low. Normally, hepatic G6Pase activity in humans is low during gestation and increases dramatically at birth and becoming independent of the maternal source of glucose.²² Thus, diminished expression of G6PC1 associated with low G6Pase activity in GSD-1a patient having g.-225C>G polymorphism could be a failure to normally switch on hepatic G6Pase activity at birth and vulnerable to hypoglycemia. Further, it can also been hypothesized that hypoglycemic status of mother during pregnancy of infants possibly because of variation in maternal G6PC1 promoter could be responsible for the GSD1a-like phenotype observed in the probands even at the heterozygous conditions. However, a comprehensive study is needed to elucidate this hypothesis with large cohort of samples.

5. Conclusions

In this study, we have identified a promoter polymorphism rs559748047 (g.-225C>G) in G6PC1 and its occurrence is revealed for the first time among two unrelated GSD-1a patients from the Indian ethnicity. Additionally, we have also shown that the g.-225C>G variation might be a functionally lethal variant that negatively regulates the transcriptional activity of G6PC1.

Author contributions

BA and PV designed the study, performed the experiments and wrote the manuscript. SK, PM and KG performed the experiments and analyzed data. RG was involved with the management of the patients. All the authors read and approved the final manuscript.

Conflict of interest

Authors have no competing interests to declare in this research.

Acknowledgements

This research did not receive any specific grant from any funding agency. SK was supported by DST-INSPIRE Fellowship Program, India [F. no/39-289/2010 (SR)]. Authors gratefully acknowledge DST-PURSE program (INDIA), Madurai Kamaraj University for the infrastructure.

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