Gene 536 (2014) 362–365
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Gene journal homepage: www.elsevier.com/locate/gene
A novel homozygous no-stop mutation in G6PC gene from a Chinese patient with glycogen storage disease type Ia Lei-Lei Gu a,b,1, Xin-Hua Li a,b,1, Yue Han a,b, Dong-Hua Zhang a,b, Qi-Ming Gong a,⁎, Xin-Xin Zhang a,b,⁎ a b
Department of Infectious Diseases, Institute of Infectious and Respiratory Diseases, Rui Jin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Sino-French Laboratory of Life Science and Genomics, Rui Jin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
a r t i c l e
i n f o
Article history: Accepted 27 November 2013 Available online 16 December 2013 Keywords: Glycogen storage disease type Ia Glucose-6-phosphatase Direct sequencing
a b s t r a c t Glycogen storage disease type Ia (GSD-Ia) is an autosomal recessive genetic disorder resulting in hypoglycemia, hepatomegaly and growth retardation. It is caused by mutations in the G6PC gene encoding Glucose-6phosphatase. To date, over 80 mutations have been identified in the G6PC gene. Here we reported a novel mutation found in a Chinese patient with abnormal transaminases, hypoglycemia, hepatomegaly and short stature. Direct sequencing of the coding region and splicing-sites in the G6PC gene revealed a novel no-stop mutation, p.*358Yext*43, leading to a 43 amino-acid extension of G6Pase. The expression level of mutant G6Pase transcripts was only 7.8% relative to wild-type transcripts. This mutation was not found in 120 chromosomes from 60 unrelated healthy control subjects using direct sequencing, and was further confirmed by digestion with Rsa I restriction endonuclease. In conclusion, we revealed a novel no-stop mutation in this study which expands the spectrum of mutations in the G6PC gene. The molecular genetic analysis was indispensable to the diagnosis of GSD-Ia for the patient. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Glucose-6-phosphatase (G6Pase, EC 3.1.3.9) mainly exists in gluconeogenic tissues, the liver, kidney and intestine. G6Pase couples functionally with glucose-6-phosphate transporter (G6PT) to hydrolyze glucose-6-phosphate (G6P) in the endoplasmic reticulum (ER). The G6PT/G6Pase complex is responsible for glucose production through glycogenolysis and gluconeogenesis, which maintains glucose homeostasis. G6Pase is encoded by the glucose-6-phosphatase gene (G6PC, GDB 231927) containing five exons. Mutations in the G6PC gene result in glycogen storage type Ia (GSD-Ia, MIM #232200), an autosomal recessive disorder characterized by metabolic impairment of terminal step of glycogenolysis and gluconeogenesis. Patients with GSD-Ia have a wide variety of biochemical abnormalities and symptoms, primarily including fasting hypoglycemia, lactic acidemia, hyperlipidemia, Abbreviations: ACE, angiotensin-converting enzyme; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CMV, cytomegalovirus; DBil, direct bilirubin; EBV, Epstein–Barr virus; ER, endoplasmic reticulum; GGT, γ-glutamyltranspeptidase; GSD-I, glycogen storage disease type I; GSD-Ia, glycogen storage disease type Ia; G6P, glucose6-phosphate; G6Pase, glucose-6-phosphatase; G6PC, glucose-6-phosphatase, catalytic; G6PT, glucose-6-phosphate transporter; HBsAg, hepatitis B surface antigen; HBcAg, hepatitis B core antigen; HIV, human immunodeficiency virus; PT, prothrombin time; SNP, single nucleotide polymorphism; TBil, total bilirubin. ⁎ Corresponding authors at: No. 197 Rui Jin Er Road, Shanghai 200025, China. Tel.: +86 21 64370045x681088; fax: +86 21 64668720. E-mail addresses: [email protected] (Q.-M. Gong), [email protected] (X.-X. Zhang). 1 The author contributed equally. 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.11.059
hyperuricemia, hepatomegaly, nephromegaly, and growth retardation (Chou et al., 2002, 2010). GSD-Ia represents over 80% of glycogen storage disease type I (GSDI) cases. The diagnosis of GSD-Ia was traditionally made on the basis of clinical symptoms and biochemical abnormalities, histologic analyses and measurements of G6Pase activity in liver biopsy tissues. After the cloning of the G6PC gene, DNA sequencing-based diagnostic methods are now used for definitive diagnosis of GSD-Ia, genetic counseling of families at risk and prenatal diagnosis. To date, over 80 mutations in the G6PC gene were identified from GSD-Ia patients all over the world. Although GSD-Ia is not restricted to any ethnic population, mutations unique to a specific race were identified. The c.648GNT mutation is the prevalent mutation found in East Asians, including Chinese, Japanese and Koreans. Besides, p.R83H is also a common mutation identified in Chinese population (Chou and Mansfield, 2008; Chou et al., 2010). In this study, we reported a novel no-stop mutation (p.*358Yext*43) in the G6PC gene in a Chinese patient with GSD-Ia. 2. Patient and methods 2.1. Patient characteristics A 12 year-old female patient with hepatocellular dysfunction and growth retardation was referred to the Department of Infectious Diseases at Rui Jin Hospital, Shanghai, China. She weighed 17 kg, her height was 106 cm (According to the data from The Physical Fitness and Health Surveillance of Chinese School Students in 2005, the mean weight and
L.-L. Gu et al. / Gene 536 (2014) 362–365
height of 12 year-old Chinese female children are 40.77 kg and 152.4 cm, respectively). The patient was born in a consanguineous marriage family, her parents are first degree cousins and her maternal grandparents are also first degree cousins (Fig. 1). There was no family history of note. She was developing normally until 1 year-old. Her liver function was found abnormal at age of 1 year. No episode of hypoglycemia was recorded in her medical history.
2.2. Clinical examination Physical examination revealed a non-tender hepatomegaly with a liver span of 10 cm below the right costal margin, and 12 cm below the left costal margin. There were no red palms, telangiectases and splenomegaly. Fasting glucose, cholesterol, triglycerides and lactic acid were abnormal. Investigation of liver function showed elevated transaminases, γ-glutamyltranspeptidase (GGT), and bilirubin (Table 1). Albumin, prothrombin time (PT) were normal. Complete blood count revealed normal hemoglobin, neutrophil count, and a slightly elevated platelet count. Abdominal ultrasound examination revealed an enlarged liver with enhanced echo texture. The liver biopsy showed that hepatocytes were distended with glycogen. Moreover, vascular septa and regenerative nodules were seen in the biopsy sample, which suggested cirrhosis caused by glycogenosis. Neither hepatitis B surface antigen (HBsAg) nor hepatitis B core antigen (HBcAg) was detected. Copper staining was also negative. Viral hepatitis serology screen was negative, including hepatitis A antibody, HBsAg, hepatitis B core antibody, hepatitis C antibody, hepatitis D antibody, and hepatitis E antibody. The detection results for cytomegalovirus (CMV), Epstein–Barr virus (EBV) and human immunodeficiency virus (HIV) were also negative. Circulating autoantibodies tests were no positive findings. Serum copper and ceruloplasmin were at normal level.
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Table 1 Results of blood biochemical tests of the patient. Parameter
Tested value
Reference range
Glucose (mmol/L) Cholesterol (mmol/L) Triglycerides (mmol/L) Lactic acid (mmol/L) ALT (IU/L) AST (IU/L) GGT (IU/L) TBil (μmol/L) DBil (μmol/L)
2.70 12.26 14.01 10.5 249 337 438 86.2 50.4
3.90–6.10 2.33–5.70 0.56–1.70 b2.4 10–64 8–40 7–64 4.7–24 0–6.8
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, γ-glutamyltranspeptidase; TBil, total bilirubin; DBil, direct bilirubin.
2.3. Genomic DNA analysis of the G6PC gene Genomic DNA was extracted from peripheral leukocytes using QIAamp® DNA Blood Mini Kit (Qiagen, Germany). Five exons of the G6PC gene (GenBank accession no. U01120) and flanking splicing-sites were amplified using polymerase chain reaction (PCR) with previously reported primers (Angaroni et al., 2004; Lei et al., 1993). Each PCR reaction was performed in a total volume of 50 μl containing 10 μl of GoTaq® 5 × Reaction Buffer, 0.25 μl of GoTaq® DNA Polymerase (Promega, USA), 1 μl of 10 mM dNTP Mix, 1 μl of 30 μM each forward and reverse primers, 4 μl of 25 ng/μl genomic DNA template, and 32.75 μl of nuclease-free water. The PCR were carried out on GeneAmp® 60-well PCR system 9700 (Applied Biosystems, USA) using the following cycling conditions: 95 °C for 5 min, 35 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, and elongation at 72 °C for 40 s followed by a final elongation of 5 min. The PCR products were directly sequenced with the Big-Dye Termination Chemistry Kit (Applied Biosystems, USA) and analyzed on the ABI377 automated DNA sequencer (Applied Biosystems, USA). The sequencing results were analyzed using AlignX module in Vector NTI Advance 11.5.1 (Invitrogen, USA) and further inspection of chromatograms manually. Mutation nomenclature was referred to the last modified nomenclature recommendations of the Human Genome Variation Society (HGVS) (den Dunnen and Antonarakis, 2000). 2.4. Restriction endonuclease digestion assay To further determine the presence of the mutation in the patient, a primer set was designed (forward primer, 5′-GCGGTAGTGCCCCTGGCA TC-3′; reverse primer, 5′-TGTGCTGAGGGCAGGCTGGA-3′) and PCR was performed followed by digestion with Rsa I restriction endonuclease (New England Biolabs, USA). The digested DNA fragments were analyzed by using agarose gel electrophoresis. 2.5. Pedigree study and healthy control sequencing Direct sequencing was adopted for pedigree study to confirm the new mutation. 60 unrelated healthy control individuals (120 chromosomes) were screened using direct sequencing to exclude the novel mutation from a single nucleotide polymorphism (SNP). In the pedigree study and control screening, a new primer set was designed (forward primer, 5′-TCTTTGACTCCTTGAAACCC-3′; reverse primer, 5′-ATTAGG ATTGTAAAAGAGGACCA-3′) for the mutational analysis. 2.6. Construction of G6Pase mutants
Fig. 1. Pedigree of the family and chromatograms of direct sequencing results. The black arrow indicates the proband. The direct sequencing results of the family members, if available, are shown below each symbol. The red triangles indicate the alleles which the novel mutation (p.*358Yext*43) emerged.
The human G6Pase cDNA from HepG2 cells was used as a template for wild-type and mutant construction by PCR. Five primers were designed for generation of wild-type and mutational constructs, some of which contained recognition sites of specific restriction endonucleases (Table 2). The PCR products were cloned into the pEGFP vector and the constructs were verified by DNA sequencing.
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Table 2 Oligonucleotide primers used for PCR in generation of G6Pase constructs. Primer
Sequence⁎
G6PaseNF-Xho I G6PaseNR-Xma I G6PaseMR G6PaseMF G6PaseMR-Xma I
5′-agatctcgagATGGAGGAAGGAATGAATGTTCTCC-3′ 5′-ggatcccgggtaTTACAACGACTTCTTGTGCGGCTGGC-3′ 5′-GTACAACGACTTCTTGTGCGGCTGGCCC-3′ 5′-CAGGGAGAGCTGCAAGGGG-3′ 5′-ggatcccgggtaTTAATTCCACGACGGCAGAATGGATGGC-3′
⁎ Lowercase letters indicate sequence contained recognition sites of specific restriction endonucleases.
2.7. Expression in Cos-7 cells and real-time qRT-PCR Cos-7 cells were grown at 37 °C in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum. Cells in 24-well cell culture plates were transfected with 1 μg wild-type or mutant construct by the XtremeGENE 9 DNA Transfection Reagent (Roche Diagnostics, Germany). RNA was isolated from approximately 4 × 105 Cos-7 cells per well using RNAprep Pure Cell/Bacteria Kit (Tiangen, Germany). After treating RNA with 1 unit DNase I (Thermo Scientific, USA) to remove contaminating genomic DNA, cDNA was synthesized from 1 μg RNA using Random Primers and M-MLV Reverse Transcriptase (Promega, USA). Glyceraldehyde 3-hosphate dehydrogenase (GAPDH) was used as the internal control gene. Expression of wild-type, mutant G6Pase and GAPDH transcripts was analyzed by real-time qRT-PCR using SYBR Premix Ex Taq (TaKaRa, Japan) on an Applied Biosystems 7500 RealTime System (Applied Biosystems, USA). Detection data were analyzed using the ΔΔCT method. 3. Results The patient is homozygous for a novel no-stop mutation (Fig. 1), p.*358Yext*43 (c.1074ANC), which changed the stop codon at position 358 to a codon for Tyrosine (Tyr, Y) and added a tail of 43 new amino acids (Fig. 2). Pedigree study revealed maternal grandfather, father, mother, and sister are heterozygous for this mutation (Fig. 1). Moreover, the mutant allele accompanied by a SNP, c.*23TNC (rs2229611), which locates at the sequence encoding the extended amino acids
Fig. 2. Sequencing result of the G6PC gene of the patient. The novel mutation, c.1074ANC (p.*358Yext*43) and SNP, c.*23TNC (rs2229611) are indicated with red triangles above the chromatogram of sequencing result. DNA and protein sequences of wild-type and mutant (patient) are compared. The extended amino acids due to the novel mutation are shown in green and the substitutions c.1074ANC, and c.*23TNC are highlighted in red.
(Fig. 2). The PCR products from samples without the mutation cannot be digested by Rsa I, which is the same as the undigested one, while the PCR products from the samples with the mutation can be cut by Rsa I (Fig. 3). The novel mutation was not found in 120 chromosomes from 60 healthy control individuals using direct sequencing. An obvious decrease in the expression level of the mutant G6Pase transcripts, 7.8% relative to wild-type G6Pase transcripts, was detected by qRT-PCR method (Fig. 4). 4. Discussion G6Pase is a 357-amino acid glycoprotein, anchored to the ER membrane by nine transmembrane helices with the enzymatic active site facing into the ER lumen. The amino-terminus (N-terminus) of G6Pase is located in the ER lumen, and the carboxyl-terminus (C-terminus) is in the cytoplasm (Chou and Mansfield, 2008; Chou et al., 2010). G6Pase is encoded by the G6PC gene. To date, over 80 G6PC gene mutations have been revealed, and Shieh et al. categorized these mutations into three groups: active site, helical, and non-helical, according to the predicted topological structure (Shieh et al., 2002). Based on the predicted structure, the last 11 amino acids at C-terminus of wild-type G6Pase are non-helical and facing into the cell cytoplasm. In this study, a novel no-stop mutation, p.*358Yext*43, was identified in a Chinese patient with GSD-Ia, which resulted in an extension of G6Pase by adding a 43 amino-acid tail at the C-terminus of the protein. Basing on the data from qRT-PCR, the novel mutation led to the distinct low expression of G6Pase transcripts (Fig. 4), which is likely to cause very limited expression of the G6Pase protein. Interestingly, the patient is also homozygous for a SNP, c.*23TNC (rs2229611), affecting the sequence of new added amino acids in mutant protein. Besides, the novel mutation (c.1074ANC, p.*358Yext*43) resulted in the emergence of a Rsa I recognition site (from GTAA to GTAC). Typical metabolic abnormalities of GSD-Ia are hypoglycemia, hyperlipidemia, lactic acidemia, and hyperuricemia. The main manifestations of patients include hepatomegaly and growth retardation, and the longterm complications are gout, hepatic adenomas, renal diseases, osteoporosis, and hepatocellular carcinoma (Chou and Mansfield, 2008; Matern et al., 2002). However, patients with GSD-Ia exhibit phenotypic heterogeneity. Severe cases with obvious hypoglycemic episodes were usually diagnosed in childhood, while milder cases may have no hypoglycemia and short stature, and may be diagnosed in adolescence or adulthood with late complications such as gouty arthritis, hepatic adenomas or even hepatocellular carcinoma (Akanuma et al., 2000; Shieh et al., 2012). The patient reported here had abnormal transaminases in infancy, and had obvious hepatomegaly, short stature, metabolic abnormalities and cirrhosis at the time of diagnosis. Although several studies suggested that some mutations may be more commonly associated with severe phenotypes, there are no adequate evidences for stringent genotype–phenotype relationship for each mutation. For example, the c.648GNT splicing mutation was suggested to be associated with hepatocellular carcinoma in some reports (Matern et al., 2002; Nakamura et al., 2001), but the appearance of
Fig. 3. Two percentage of agarose gel illustrating the digestion of PCR products with Rsa I. The wild-type PCR product does not exist the Rsa I recognition site, while the p.*358Yext*43 mutant PCR product has one Rsa I recognition site. Lane 1, 7: the proband's cousin and healthy control subject with wild-type band (282-bp). Lane 2–5: the proband's maternal grandfather, sister, mother and father with wild-type and mutant bands (282, 200-bp). Lane 6: the proband with mutant band (200-bp).
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lar genetic analysis, which expands the spectrum of mutations in the G6PC gene. Eventually, the definitive diagnosis of GSD-Ia for the patient was confirmed by clinical manifestations, biochemical examinations and histological analysis of liver biopsy sample combined with mutational analysis of the G6PC gene.
Conflict of interest We declare no conflict of interest. Acknowledgments
Fig. 4. Relative quantitation (RQ) of wild-type and mutant G6Pase transcripts analyzed by the ΔΔCT method. The level of mutant G6Pase transcripts was only 7.8% relative to the wild-type G6Pase transcripts. The graph represents the mean of 3 separate experiments in which each sample was analyzed in triplicate. Error bars depict Standard Deviation (SD). * P = 0.0182, *** P = 0.0001 as determined by two-tailed paired t-test.
hepatic carcinoma varied greatly among the 40 GSD-Ia patients with homozygous c.648GNT mutation in a larger study (Akanuma et al., 2000). Recently, a novel mutation, p.M121V, resulting in a mild GSD-Ia was revealed in two adult patients with multiple benign and malignant hepatic tumors when diagnosed (Shieh et al., 2012). The patient, who carries homozygous p.*358Yext*43 mutation, had notable clinical symptoms and biochemical abnormalities of GSD-Ia in this study. Since the DNA-analysis of the G6PC gene was available, it has been recommended that diagnosis of GSD-Ia should be made on clinical presentation and biochemical abnormalities and confirmed by mutational analysis, rather than traditional measurements of enzyme activity in liver biopsy samples (Chou et al., 2010; Rake et al., 2000, 2002). Dietary therapies are able to achieve maintenance of near normal glucose levels, growth and development. Unfortunately, long-term complications still remain with current dietary therapies. Drug therapies, such as antioxidants and angiotensin-converting enzyme (ACE) inhibitors, may be beneficial for renal damage in patients with GSD-Ia. Liver or kidney transplantation was recommended to patients with end-stage liver disease or renal failure. Gene therapy remains an experimental stage in both mouse and dog models, and have shown promising results (Chou et al., 2010; Koeberl et al., 2007; Yiu et al., 2010). In summary, we revealed a novel no-stop mutation, p.*358Yext*43, in a Chinese patient with typical GSD-Ia presentation by using molecu-
This work was supported by the PhD candidates' Innovation Foundation of Shanghai Jiao-Tong University School of Medicine. References Akanuma, J., et al., 2000. Glycogen storage disease type Ia: molecular diagnosis of 51 Japanese patients and characterization of splicing mutations by analysis of ectopically transcribed mRNA from lymphoblastoid cells. Am. J. Med. Genet. 91, 107–112. Angaroni, C.J., et al., 2004. Glycogen storage disease type Ia in Argentina: two novel glucose-6-phosphatase mutations affecting protein stability. Mol. Genet. Metab. 83, 276–279. Chou, J.Y., Mansfield, B.C., 2008. Mutations in the glucose-6-phosphatase-alpha (G6PC) gene that cause type Ia glycogen storage disease. Hum. Mutat. 29, 921–930. Chou, J.Y., et al., 2002. Type I glycogen storage diseases: disorders of the glucose-6phosphatase complex. Curr. Mol. Med. 2, 121–143. Chou, J.Y., et al., 2010. Glycogen storage disease type I and G6Pase-beta deficiency: etiology and therapy. Nat. Rev. Endocrinol. 6, 676–688. den Dunnen, J.T., Antonarakis, S.E., 2000. Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum. Mutat. 15, 7–12. Koeberl, D.D., et al., 2007. Efficacy of helper-dependent adenovirus vector-mediated gene therapy in murine glycogen storage disease type Ia. Mol. Ther. 15, 1253–1258. Lei, K.J., et al., 1993. Mutations in the glucose-6-phosphatase gene that cause glycogen storage disease type 1a. Science 262, 580–583. Matern, D., et al., 2002. Glycogen storage disease type I: diagnosis and phenotype/ genotype correlation. Eur. J. Pediatr. 161 (Suppl. 1), S10–S19. Nakamura, T., et al., 2001. Glucose-6-phosphatase gene mutations in 20 adult Japanese patients with glycogen storage disease type 1a with reference to hepatic tumors. J. Gastroenterol. Hepatol. 16, 1402–1408. Rake, J.P., et al., 2000. Glycogen storage disease type Ia: recent experience with mutation analysis, a summary of mutations reported in the literature and a newly developed diagnostic flow chart. Eur. J. Pediatr. 159, 322–330. Rake, J.P., et al., 2002. Glycogen storage disease type I: diagnosis, management, clinical course and outcome. Results of the European Study on Glycogen Storage Disease Type I (ESGSD I). Eur. J. Pediatr. 161 (Suppl. 1), S20–S34. Shieh, J.J., et al., 2002. The molecular basis of glycogen storage disease type 1a: structure and function analysis of mutations in glucose-6-phosphatase. J. Biol. Chem. 277, 5047–5053. Shieh, J.J., et al., 2012. Misdiagnosis as steatohepatitis in a family with mild glycogen storage disease type 1a. Gene 509, 154–157. Yiu, W.H., et al., 2010. Complete normalization of hepatic G6PC deficiency in murine glycogen storage disease type Ia using gene therapy. Mol. Ther. 18, 1076–1084.
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
A novel homozygous no-stop mutation in G6PC gene from a Chinese patient with glycogen storage disease type Ia Lei-Lei Gu a,b,1, Xin-Hua Li a,b,1, Yue Han a,b, Dong-Hua Zhang a,b, Qi-Ming Gong a,⁎, Xin-Xin Zhang a,b,⁎ a b
Department of Infectious Diseases, Institute of Infectious and Respiratory Diseases, Rui Jin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Sino-French Laboratory of Life Science and Genomics, Rui Jin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
a r t i c l e
i n f o
Article history: Accepted 27 November 2013 Available online 16 December 2013 Keywords: Glycogen storage disease type Ia Glucose-6-phosphatase Direct sequencing
a b s t r a c t Glycogen storage disease type Ia (GSD-Ia) is an autosomal recessive genetic disorder resulting in hypoglycemia, hepatomegaly and growth retardation. It is caused by mutations in the G6PC gene encoding Glucose-6phosphatase. To date, over 80 mutations have been identified in the G6PC gene. Here we reported a novel mutation found in a Chinese patient with abnormal transaminases, hypoglycemia, hepatomegaly and short stature. Direct sequencing of the coding region and splicing-sites in the G6PC gene revealed a novel no-stop mutation, p.*358Yext*43, leading to a 43 amino-acid extension of G6Pase. The expression level of mutant G6Pase transcripts was only 7.8% relative to wild-type transcripts. This mutation was not found in 120 chromosomes from 60 unrelated healthy control subjects using direct sequencing, and was further confirmed by digestion with Rsa I restriction endonuclease. In conclusion, we revealed a novel no-stop mutation in this study which expands the spectrum of mutations in the G6PC gene. The molecular genetic analysis was indispensable to the diagnosis of GSD-Ia for the patient. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Glucose-6-phosphatase (G6Pase, EC 3.1.3.9) mainly exists in gluconeogenic tissues, the liver, kidney and intestine. G6Pase couples functionally with glucose-6-phosphate transporter (G6PT) to hydrolyze glucose-6-phosphate (G6P) in the endoplasmic reticulum (ER). The G6PT/G6Pase complex is responsible for glucose production through glycogenolysis and gluconeogenesis, which maintains glucose homeostasis. G6Pase is encoded by the glucose-6-phosphatase gene (G6PC, GDB 231927) containing five exons. Mutations in the G6PC gene result in glycogen storage type Ia (GSD-Ia, MIM #232200), an autosomal recessive disorder characterized by metabolic impairment of terminal step of glycogenolysis and gluconeogenesis. Patients with GSD-Ia have a wide variety of biochemical abnormalities and symptoms, primarily including fasting hypoglycemia, lactic acidemia, hyperlipidemia, Abbreviations: ACE, angiotensin-converting enzyme; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CMV, cytomegalovirus; DBil, direct bilirubin; EBV, Epstein–Barr virus; ER, endoplasmic reticulum; GGT, γ-glutamyltranspeptidase; GSD-I, glycogen storage disease type I; GSD-Ia, glycogen storage disease type Ia; G6P, glucose6-phosphate; G6Pase, glucose-6-phosphatase; G6PC, glucose-6-phosphatase, catalytic; G6PT, glucose-6-phosphate transporter; HBsAg, hepatitis B surface antigen; HBcAg, hepatitis B core antigen; HIV, human immunodeficiency virus; PT, prothrombin time; SNP, single nucleotide polymorphism; TBil, total bilirubin. ⁎ Corresponding authors at: No. 197 Rui Jin Er Road, Shanghai 200025, China. Tel.: +86 21 64370045x681088; fax: +86 21 64668720. E-mail addresses: [email protected] (Q.-M. Gong), [email protected] (X.-X. Zhang). 1 The author contributed equally. 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.11.059
hyperuricemia, hepatomegaly, nephromegaly, and growth retardation (Chou et al., 2002, 2010). GSD-Ia represents over 80% of glycogen storage disease type I (GSDI) cases. The diagnosis of GSD-Ia was traditionally made on the basis of clinical symptoms and biochemical abnormalities, histologic analyses and measurements of G6Pase activity in liver biopsy tissues. After the cloning of the G6PC gene, DNA sequencing-based diagnostic methods are now used for definitive diagnosis of GSD-Ia, genetic counseling of families at risk and prenatal diagnosis. To date, over 80 mutations in the G6PC gene were identified from GSD-Ia patients all over the world. Although GSD-Ia is not restricted to any ethnic population, mutations unique to a specific race were identified. The c.648GNT mutation is the prevalent mutation found in East Asians, including Chinese, Japanese and Koreans. Besides, p.R83H is also a common mutation identified in Chinese population (Chou and Mansfield, 2008; Chou et al., 2010). In this study, we reported a novel no-stop mutation (p.*358Yext*43) in the G6PC gene in a Chinese patient with GSD-Ia. 2. Patient and methods 2.1. Patient characteristics A 12 year-old female patient with hepatocellular dysfunction and growth retardation was referred to the Department of Infectious Diseases at Rui Jin Hospital, Shanghai, China. She weighed 17 kg, her height was 106 cm (According to the data from The Physical Fitness and Health Surveillance of Chinese School Students in 2005, the mean weight and
L.-L. Gu et al. / Gene 536 (2014) 362–365
height of 12 year-old Chinese female children are 40.77 kg and 152.4 cm, respectively). The patient was born in a consanguineous marriage family, her parents are first degree cousins and her maternal grandparents are also first degree cousins (Fig. 1). There was no family history of note. She was developing normally until 1 year-old. Her liver function was found abnormal at age of 1 year. No episode of hypoglycemia was recorded in her medical history.
2.2. Clinical examination Physical examination revealed a non-tender hepatomegaly with a liver span of 10 cm below the right costal margin, and 12 cm below the left costal margin. There were no red palms, telangiectases and splenomegaly. Fasting glucose, cholesterol, triglycerides and lactic acid were abnormal. Investigation of liver function showed elevated transaminases, γ-glutamyltranspeptidase (GGT), and bilirubin (Table 1). Albumin, prothrombin time (PT) were normal. Complete blood count revealed normal hemoglobin, neutrophil count, and a slightly elevated platelet count. Abdominal ultrasound examination revealed an enlarged liver with enhanced echo texture. The liver biopsy showed that hepatocytes were distended with glycogen. Moreover, vascular septa and regenerative nodules were seen in the biopsy sample, which suggested cirrhosis caused by glycogenosis. Neither hepatitis B surface antigen (HBsAg) nor hepatitis B core antigen (HBcAg) was detected. Copper staining was also negative. Viral hepatitis serology screen was negative, including hepatitis A antibody, HBsAg, hepatitis B core antibody, hepatitis C antibody, hepatitis D antibody, and hepatitis E antibody. The detection results for cytomegalovirus (CMV), Epstein–Barr virus (EBV) and human immunodeficiency virus (HIV) were also negative. Circulating autoantibodies tests were no positive findings. Serum copper and ceruloplasmin were at normal level.
363
Table 1 Results of blood biochemical tests of the patient. Parameter
Tested value
Reference range
Glucose (mmol/L) Cholesterol (mmol/L) Triglycerides (mmol/L) Lactic acid (mmol/L) ALT (IU/L) AST (IU/L) GGT (IU/L) TBil (μmol/L) DBil (μmol/L)
2.70 12.26 14.01 10.5 249 337 438 86.2 50.4
3.90–6.10 2.33–5.70 0.56–1.70 b2.4 10–64 8–40 7–64 4.7–24 0–6.8
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, γ-glutamyltranspeptidase; TBil, total bilirubin; DBil, direct bilirubin.
2.3. Genomic DNA analysis of the G6PC gene Genomic DNA was extracted from peripheral leukocytes using QIAamp® DNA Blood Mini Kit (Qiagen, Germany). Five exons of the G6PC gene (GenBank accession no. U01120) and flanking splicing-sites were amplified using polymerase chain reaction (PCR) with previously reported primers (Angaroni et al., 2004; Lei et al., 1993). Each PCR reaction was performed in a total volume of 50 μl containing 10 μl of GoTaq® 5 × Reaction Buffer, 0.25 μl of GoTaq® DNA Polymerase (Promega, USA), 1 μl of 10 mM dNTP Mix, 1 μl of 30 μM each forward and reverse primers, 4 μl of 25 ng/μl genomic DNA template, and 32.75 μl of nuclease-free water. The PCR were carried out on GeneAmp® 60-well PCR system 9700 (Applied Biosystems, USA) using the following cycling conditions: 95 °C for 5 min, 35 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, and elongation at 72 °C for 40 s followed by a final elongation of 5 min. The PCR products were directly sequenced with the Big-Dye Termination Chemistry Kit (Applied Biosystems, USA) and analyzed on the ABI377 automated DNA sequencer (Applied Biosystems, USA). The sequencing results were analyzed using AlignX module in Vector NTI Advance 11.5.1 (Invitrogen, USA) and further inspection of chromatograms manually. Mutation nomenclature was referred to the last modified nomenclature recommendations of the Human Genome Variation Society (HGVS) (den Dunnen and Antonarakis, 2000). 2.4. Restriction endonuclease digestion assay To further determine the presence of the mutation in the patient, a primer set was designed (forward primer, 5′-GCGGTAGTGCCCCTGGCA TC-3′; reverse primer, 5′-TGTGCTGAGGGCAGGCTGGA-3′) and PCR was performed followed by digestion with Rsa I restriction endonuclease (New England Biolabs, USA). The digested DNA fragments were analyzed by using agarose gel electrophoresis. 2.5. Pedigree study and healthy control sequencing Direct sequencing was adopted for pedigree study to confirm the new mutation. 60 unrelated healthy control individuals (120 chromosomes) were screened using direct sequencing to exclude the novel mutation from a single nucleotide polymorphism (SNP). In the pedigree study and control screening, a new primer set was designed (forward primer, 5′-TCTTTGACTCCTTGAAACCC-3′; reverse primer, 5′-ATTAGG ATTGTAAAAGAGGACCA-3′) for the mutational analysis. 2.6. Construction of G6Pase mutants
Fig. 1. Pedigree of the family and chromatograms of direct sequencing results. The black arrow indicates the proband. The direct sequencing results of the family members, if available, are shown below each symbol. The red triangles indicate the alleles which the novel mutation (p.*358Yext*43) emerged.
The human G6Pase cDNA from HepG2 cells was used as a template for wild-type and mutant construction by PCR. Five primers were designed for generation of wild-type and mutational constructs, some of which contained recognition sites of specific restriction endonucleases (Table 2). The PCR products were cloned into the pEGFP vector and the constructs were verified by DNA sequencing.
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Table 2 Oligonucleotide primers used for PCR in generation of G6Pase constructs. Primer
Sequence⁎
G6PaseNF-Xho I G6PaseNR-Xma I G6PaseMR G6PaseMF G6PaseMR-Xma I
5′-agatctcgagATGGAGGAAGGAATGAATGTTCTCC-3′ 5′-ggatcccgggtaTTACAACGACTTCTTGTGCGGCTGGC-3′ 5′-GTACAACGACTTCTTGTGCGGCTGGCCC-3′ 5′-CAGGGAGAGCTGCAAGGGG-3′ 5′-ggatcccgggtaTTAATTCCACGACGGCAGAATGGATGGC-3′
⁎ Lowercase letters indicate sequence contained recognition sites of specific restriction endonucleases.
2.7. Expression in Cos-7 cells and real-time qRT-PCR Cos-7 cells were grown at 37 °C in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum. Cells in 24-well cell culture plates were transfected with 1 μg wild-type or mutant construct by the XtremeGENE 9 DNA Transfection Reagent (Roche Diagnostics, Germany). RNA was isolated from approximately 4 × 105 Cos-7 cells per well using RNAprep Pure Cell/Bacteria Kit (Tiangen, Germany). After treating RNA with 1 unit DNase I (Thermo Scientific, USA) to remove contaminating genomic DNA, cDNA was synthesized from 1 μg RNA using Random Primers and M-MLV Reverse Transcriptase (Promega, USA). Glyceraldehyde 3-hosphate dehydrogenase (GAPDH) was used as the internal control gene. Expression of wild-type, mutant G6Pase and GAPDH transcripts was analyzed by real-time qRT-PCR using SYBR Premix Ex Taq (TaKaRa, Japan) on an Applied Biosystems 7500 RealTime System (Applied Biosystems, USA). Detection data were analyzed using the ΔΔCT method. 3. Results The patient is homozygous for a novel no-stop mutation (Fig. 1), p.*358Yext*43 (c.1074ANC), which changed the stop codon at position 358 to a codon for Tyrosine (Tyr, Y) and added a tail of 43 new amino acids (Fig. 2). Pedigree study revealed maternal grandfather, father, mother, and sister are heterozygous for this mutation (Fig. 1). Moreover, the mutant allele accompanied by a SNP, c.*23TNC (rs2229611), which locates at the sequence encoding the extended amino acids
Fig. 2. Sequencing result of the G6PC gene of the patient. The novel mutation, c.1074ANC (p.*358Yext*43) and SNP, c.*23TNC (rs2229611) are indicated with red triangles above the chromatogram of sequencing result. DNA and protein sequences of wild-type and mutant (patient) are compared. The extended amino acids due to the novel mutation are shown in green and the substitutions c.1074ANC, and c.*23TNC are highlighted in red.
(Fig. 2). The PCR products from samples without the mutation cannot be digested by Rsa I, which is the same as the undigested one, while the PCR products from the samples with the mutation can be cut by Rsa I (Fig. 3). The novel mutation was not found in 120 chromosomes from 60 healthy control individuals using direct sequencing. An obvious decrease in the expression level of the mutant G6Pase transcripts, 7.8% relative to wild-type G6Pase transcripts, was detected by qRT-PCR method (Fig. 4). 4. Discussion G6Pase is a 357-amino acid glycoprotein, anchored to the ER membrane by nine transmembrane helices with the enzymatic active site facing into the ER lumen. The amino-terminus (N-terminus) of G6Pase is located in the ER lumen, and the carboxyl-terminus (C-terminus) is in the cytoplasm (Chou and Mansfield, 2008; Chou et al., 2010). G6Pase is encoded by the G6PC gene. To date, over 80 G6PC gene mutations have been revealed, and Shieh et al. categorized these mutations into three groups: active site, helical, and non-helical, according to the predicted topological structure (Shieh et al., 2002). Based on the predicted structure, the last 11 amino acids at C-terminus of wild-type G6Pase are non-helical and facing into the cell cytoplasm. In this study, a novel no-stop mutation, p.*358Yext*43, was identified in a Chinese patient with GSD-Ia, which resulted in an extension of G6Pase by adding a 43 amino-acid tail at the C-terminus of the protein. Basing on the data from qRT-PCR, the novel mutation led to the distinct low expression of G6Pase transcripts (Fig. 4), which is likely to cause very limited expression of the G6Pase protein. Interestingly, the patient is also homozygous for a SNP, c.*23TNC (rs2229611), affecting the sequence of new added amino acids in mutant protein. Besides, the novel mutation (c.1074ANC, p.*358Yext*43) resulted in the emergence of a Rsa I recognition site (from GTAA to GTAC). Typical metabolic abnormalities of GSD-Ia are hypoglycemia, hyperlipidemia, lactic acidemia, and hyperuricemia. The main manifestations of patients include hepatomegaly and growth retardation, and the longterm complications are gout, hepatic adenomas, renal diseases, osteoporosis, and hepatocellular carcinoma (Chou and Mansfield, 2008; Matern et al., 2002). However, patients with GSD-Ia exhibit phenotypic heterogeneity. Severe cases with obvious hypoglycemic episodes were usually diagnosed in childhood, while milder cases may have no hypoglycemia and short stature, and may be diagnosed in adolescence or adulthood with late complications such as gouty arthritis, hepatic adenomas or even hepatocellular carcinoma (Akanuma et al., 2000; Shieh et al., 2012). The patient reported here had abnormal transaminases in infancy, and had obvious hepatomegaly, short stature, metabolic abnormalities and cirrhosis at the time of diagnosis. Although several studies suggested that some mutations may be more commonly associated with severe phenotypes, there are no adequate evidences for stringent genotype–phenotype relationship for each mutation. For example, the c.648GNT splicing mutation was suggested to be associated with hepatocellular carcinoma in some reports (Matern et al., 2002; Nakamura et al., 2001), but the appearance of
Fig. 3. Two percentage of agarose gel illustrating the digestion of PCR products with Rsa I. The wild-type PCR product does not exist the Rsa I recognition site, while the p.*358Yext*43 mutant PCR product has one Rsa I recognition site. Lane 1, 7: the proband's cousin and healthy control subject with wild-type band (282-bp). Lane 2–5: the proband's maternal grandfather, sister, mother and father with wild-type and mutant bands (282, 200-bp). Lane 6: the proband with mutant band (200-bp).
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lar genetic analysis, which expands the spectrum of mutations in the G6PC gene. Eventually, the definitive diagnosis of GSD-Ia for the patient was confirmed by clinical manifestations, biochemical examinations and histological analysis of liver biopsy sample combined with mutational analysis of the G6PC gene.
Conflict of interest We declare no conflict of interest. Acknowledgments
Fig. 4. Relative quantitation (RQ) of wild-type and mutant G6Pase transcripts analyzed by the ΔΔCT method. The level of mutant G6Pase transcripts was only 7.8% relative to the wild-type G6Pase transcripts. The graph represents the mean of 3 separate experiments in which each sample was analyzed in triplicate. Error bars depict Standard Deviation (SD). * P = 0.0182, *** P = 0.0001 as determined by two-tailed paired t-test.
hepatic carcinoma varied greatly among the 40 GSD-Ia patients with homozygous c.648GNT mutation in a larger study (Akanuma et al., 2000). Recently, a novel mutation, p.M121V, resulting in a mild GSD-Ia was revealed in two adult patients with multiple benign and malignant hepatic tumors when diagnosed (Shieh et al., 2012). The patient, who carries homozygous p.*358Yext*43 mutation, had notable clinical symptoms and biochemical abnormalities of GSD-Ia in this study. Since the DNA-analysis of the G6PC gene was available, it has been recommended that diagnosis of GSD-Ia should be made on clinical presentation and biochemical abnormalities and confirmed by mutational analysis, rather than traditional measurements of enzyme activity in liver biopsy samples (Chou et al., 2010; Rake et al., 2000, 2002). Dietary therapies are able to achieve maintenance of near normal glucose levels, growth and development. Unfortunately, long-term complications still remain with current dietary therapies. Drug therapies, such as antioxidants and angiotensin-converting enzyme (ACE) inhibitors, may be beneficial for renal damage in patients with GSD-Ia. Liver or kidney transplantation was recommended to patients with end-stage liver disease or renal failure. Gene therapy remains an experimental stage in both mouse and dog models, and have shown promising results (Chou et al., 2010; Koeberl et al., 2007; Yiu et al., 2010). In summary, we revealed a novel no-stop mutation, p.*358Yext*43, in a Chinese patient with typical GSD-Ia presentation by using molecu-
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