Identification of p.HIS119leu mutation in the g6pc gene of a vietnamese patient with glycogen storage disease type IA

ACADEMIA JOURNAL OF BIOLOGY 2020, 42(2): 93–100 DOI: 10.15625/2615-9023/v42n2.14898 93 IDENTIFICATION OF p.HIS119LEU MUTATION IN THE G6PC GENE OF A VIETNAMESE PATIENT WITH GLYCOGEN STORAGE DISEASE TYPE Ia Nguyen Huy Hoang 1,2,* , Vu Chi Dung 3 , Nguyen Van Tung 1 , Nguyen Ngoc Lan 1 , Ha Thi Dung 1 1 Institute of Genome Research, VAST, Vietnam 2 Graduate University of Science and Technology, VAST, Vietnam 3 Vietnam National Hospital of Pediatrics, 18/879 La Thanh str., Dong Da, Ha Noi, Vietnam Received 17 March 2020, accepted 10 June 2020 ABSTRACT Glycogen storage disease type Ia (GSD Ia), a rare autosomal inherited disorder, is characterized by accumulation of excessive glycogen and fat in the liver. Primary symptoms of GSD Ia include hypoglycemia; metabolic acidosis; elevated levels of lactate, uric acid and lipids; hepatomagaly and growth retardation. Glycogen storage disease type Ia was caused by mutations in the G6PC gene. In this study, mutations in a Vietnamese patient with glycogen storage disease type Ia were analyzed using the whole exome sequencing method. A missense mutation c.356A>T (p.His119Leu) in the G6PC gene of the patient was identified in exon 3. Genetic analysis confirmed that this mutation was present under homozygous form In-silico analyses using SIFT and Mutation Taster confirmed the damaging effects of this mutations on the function of the proteins. This result enriches knowledge of the G6PC gene mutation spectrum and provides genetic data for further studies on glycogen storage disease type Ia in Viet Nam. Keywords: G6PC gene, Glycogen storage disease type Ia, mutation p.His119Leu, rare disease, whole exome sequencing. Citation: Nguyen Huy Hoang, Vu Chi Dung, Nguyen Van Tung, Nguyen Ngoc Lan, Ha Thi Dung, 2020. Identification of p.His119Leu mutation in the G6PC gene of a Vietnamese patient with glycogen storage disease type Ia. Academia Journal of Biology, 42(2): 93–100. https://doi.org/10.15625/2615-9023/v42n2.14898. *Corresponding author email: nhhoang@igr.ac.vn ©2020 Vietnam Academy of Science and Technology (VAST) Nguyen Huy Hoang et al. 94 INTRODUCTION Glycogen storage disease (GSD) is a rare group of genetic metabolic disorders that affects glycogen metabolism. In patients with GSD, while endogenous glucose production is suppressed in postprandial period, exogenous glucose is either metabolized to pyruvate or stored as glycogen in the liver and skeletal muscle (Saltik et al., 2000; Ozen, 2007). Glycogen stores must be metabolized by enzymes before being used. In the absence of enzymes needed for glycogen degradation, the glycogen will accumulate and cause disorders. Glycogen storage disease affect primarily liverand muscles. The incidence rate of GSD I is approximately 1/20.000–1/43.000 live births (Hicks et al., 2011). Depending on the level of enzyme deficiency and the affected tissues, glycogen storage diseases were classified into twelve type (Wolfsdorf & Weinstein, 2003; Rake et al. 2006). Different GSD types have different symptoms. Most types of GSD affect liver (type 0, I, III, IV, VI and IX). However, some types of GSD have complex signs and symptoms, affecting muscles, liver, and heart. These types of GSD (except GSD type 0) can cause the liver to enlarge due to glycogen being stored in the liver instead of being released as glucose into blood. Common symptoms of GSD are hypoglycemia, hyperlactatemia, hepatomegaly, hypertriglyceridemia. GSD type V and VII affect primarily the skeletal muscles, with muscle weakness and cramps being the most common symptoms. In newborns, some GSD types lead to death within the first year of life, whereas other glycogen storage diseases are relatively asymptomatic or may cause only exercise intolerance (Hicks et al., 2011). Glycogen storage disease type I including type Ia (GSD Ia) and Ib (GSD Ib) characterized by hepatomegaly resulting from accumulation of glycogen in the liver. Among them, GSD type Ia is more common, accounting for about 80% of patients with GSD type I with an estimated annual incidence rate of about 1/100,000 live births (Chou et al. 2002). Glycogen storage type Ia is an autosomal recessive disorder cause by deficiencies in the activities of glucose-6- phosphatase (G6Pase), an integral resident endoplasmic reticulum (ER) protein. The G6PC gene is expressed primarily in the liver, kidneys, and intestines (Chou et al.. 2002). Patients with GSD Ia present many abnormal biochemical symptoms, mainly fasting hypoglycemia, lactic acidosis, hyperlipidemia, hyperuricemia, hepatomegaly, and growth retardation (Gu et al. 2014; Karthi et al. 2019). The G6PC gene is located on chromosome 17q21.31 which is the long arm of chromosome 17 at position 21.31. G6Pase which is a glycoprotein with 357 amino acid, is anchored in the membrane of the ER by 9 transmembrane helices (Pan et al.. 1998). Up to now, approximately 116 mutations in of the G6PC gene have been recorded among 550 patients in the Human Gene Mutation Database (HGMD). Almost all previously reported variants were missense. The active center of G6PC is proposed to comprise Lys-76, Arg-83, His-119, Arg-170 and His-176 (Stukey & Carman 1997; Hemrika & Wever, 1997). Mutations in active sites were shown to completely abolish G6PC enzymatic activity. In this study, whole exome sequencing was performed on a Vietnamese patient with GSD type Ia. A missense mutation p.His119Leu in G6PC gene was found in the patient and members of his family. Information about this mutation will contribute to a better understanding of the disease. MATERIALS AND METHODS Ethical Approval This study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Institute of Genome Research (No. 18/QD-NCHG on 22 March, 2018, Institute of Genome Research Institutional Review Board, Ha Noi, Vietnam). Identification of p.His119Leu mutation 95 Patient The patient with glycogen storage disease type Ia, a boy aged 7 years and 11 months, is the third child in the familiy whilethe second child died at 3 months of age due to unknown coma. The patient presented the first metabolic crisis at 3 months of age after immunization injection. At that time, he presented tachypnea, lethargy, metabolic acidosis (7.05), hypoglycemia (1.9 mmol/l, normal: 3.3−5.5 mmol/l ), hyperlactatemia (9.5 mmol/l, normal: 3.3−5.5 mmol/l), hypertriglyceridemia (7 mmol/l, normal: <1,65 mmol/l), ketonuria, elevated transaminase (ALT: 400, normal: <40). After diagnosis, the patient was treated with glucose infusion on metabolic crisis. Over the long term, the patient was treated with applied diet therapy with soymilk, cornstarch, medium- chain triglyceride oil and avoiding long fasting. He showed normal health until 6 years old. He was admitted to Vietnam National Hospital of Pediatrics because of tachypnea and lethargy. The patient presented hepatomegaly 7 cm under costal margin, hyperlactatemia (7.5 mmol/l), elevated transaminase (AST/ALT:1544/950 UI/l), hypertriglyceridemia (8.3 mmol/l). DNA extraction Peripheral blood samples from the patient and his family members were provided by Department of Endocrinology, Metabolism and Genetics, Vietnam National Hospital of Pediatrics. Genomic DNA was extracted from peripheral blood samples using QIAamp DNA Blood Mini Kit-QIAGEN following the manufacturer’s guidelines. Whole exome sequencing The DNA library of patients was prepared using Agilent SureSelect Target Enrichment kit and whole exome sequencing was performed by applying Illumina platform. Bioinformatics analysis and variants screening After sequenced by Illumina platform, raw data was assessed and subjected to quality control using FastQC. The paired-end reads were aligned to the reference human genome (GRChr37/hg19) using BWA 0.7.10 (Li & Durbin, 2009). Picard tools ( was used to processed post-alignment data. Genome Analysis Toolkit v3.4 was used for variant calling (McKenna et al. 2010). The The effects of variants on genes such as amino acid changes were predicted using SnpEff v4.1 (Cingolani et al., 2012). In-silico analyses to confirm the effect of the mutations on the structure and function of the proteins was performed using SIFT (Ng & Henikoff, 2003) and Mutation Taster (Schwarz et al., 2014). The candidate variants were filtered using four conditions: (i) variants occurring in genes associated with GSD type I; (ii) all variants with a minor allele frequency of 0.1% were excluded; (iii) variants predicted as “Damaging” or “Disease causing” (iiii) all variants reported as benign in ClinVar database were excluded. Sanger sequencing to validation variants A fragment of G6PC gene was amplified using a specific primer designed using Primer blast (https://www.ncbi.nml.gov/tools/primer- blast/): G6PC-2F: 5 ’ -TTCCCAGAGCCTTGC ACAAT-3’ and G6PC-2R: 5’-AAGCCCT GCTGCTACTTCAC-3 ’ . PCR conditions used for the amplification were: 95 o C/12 min; (95 o C/45 s; 64 o C/45 s; 72 o C/45 s) x 35 cycles and 72 o C/8 min. The PCR product (639 bp) underwent electrophoresis in agarose 1%. Sanger sequencing was performed on DNA samples of the patient and members of his family for validating the variants of interest identified in bioinformatics analysis. RESULTS AND DISCUSSION Bioinformatics analysis revealeda homozygous missense variant c.356A>T (p.His119Leu) in exone 3 of G6PC gene. This mutation involves a change from Histidine (His) to Leucine (Leu) at residue Nguyen Huy Hoang et al. 96 119 (p.His119Leu). The mutation was first identified by Wu et al. (2000) in a Taiwan patient with glycogen storage disease type Ia. This mutation was reported in the dbSNP database (rs1401928680) but not in ClinVar. Sanger sequencing showed that the patient’s parents and sister carried a heterozygous c.356A > T mutation (Fig. 1). The second child of this family, who died at 3 months of age, was not reported in this study. Figure 1. Analysis of p.His119Leu mutation in the patient and his family. (A) G6PC gene is located on chromosome 17q21.31 which is the long (q) arm of chromosome 17 at position 21.31. (B) Exon–intron graph of G6PC gene. (C) Pedigree of the patient’s family and variant p.His119Leu in ther G6PC gene With a SIFT score of 0.012 (Fig. 2A) and MutationTaster2 result as disease-causing, this mutation is predicted to be deleterious. In addition, the His119 residue is located in a conserved amino acid across different species (Fig. 2B). In this study, the mutation p.His119Leu found in the patient changed hydrophilic amino acid (histidine) to hydrophobic amino acid (leucine). His-119 is an active site residue of G6Pase protein (Hemrika & Wever, 1997; Stukey & Carman, 1997), Identification of p.His119Leu mutation 97 providing the proton needed to liberate the glucose moiety (Chou & Mansfield, 2008). The mutation p.His119Leu has been identified in GSD-Ia patients and shown to completely abolish G6PC enzymatic activity (Shieh et al., 2002). The roles of His-119 were confirmed by Lei et al (1995) which substituted this amino acid with either alanine (His119Ala), isoleucine (His119Ile), lysine (His119Lys), methionine (His119Met), asparagine (His119Asn), arginine (His119Arg) and threonine (His119Thr). All of the His-119 mutant have shown a loss of activity in G6PC catalysis. Figure 2. In-silico analysis of the G6PC protein. (A) The mutation was predicted to be “Damaging” by SIFT. (B) Conservation of the amino acid changed by p.His119Leu in G6PC protein mutation across different species Signs and symptoms of glycogen storage disease type Ia include low blood sugar (hypoglycemia), which can lead to seizures. Patient can also have a buildup of lactic acid in the body (lactic acidosis), high blood levels of uric acid (hyperuricemia), and excess amounts of fats in the blood (hyperlipidemia). Patients with GSD IA have abnormal enlargement of the liver (hepatomegaly), they may have thinning of bones (osteoporosis), gout, kidney disease, and high blood pressure in the blood vessels (Rake et al., 2002; Froissart et al., 2011). The patient in this study presented hypoglycemia, hyperlactatemia, hepatomegaly, and hypertriglyceridemia ketonuria; biochemical indices were abnormal. Other studies in Chinese and Indian patients with GSD Ia showed similar symptoms (Gu et al., 2014; Zheng et al., 2015; Karthi et al., 2019). This suggests that patients presented with severe hypoglycemia can be clearly diagnosed in early childhood. However, in some studies on mild cases without hypoglycemia and growth retardation, patient can be diagnosed in adolescence or adulthood with complications such as gouty arthritis, hepatitis or tumors called adenomas forming in the liver (Akanuma et al., 2000; Shieh et al., 2012). Therefore, the early diagnosis and identification by genetic analysis is very important for treatment. Nguyen Huy Hoang et al. 98 Figure 3. G6Pase protein anchored in the endoplasmic reticulum (ER) by 9 transmembrane helices. The N terminus localized in the ER lumen and the C terminus in the cytoplasm. CONCLUSION In conclusion, by applying whole exome sequencing, we identified the p.His119Leu mutation in the G6PC gene in a Vietnamese patient with glycogen storage disease type Ia. This is the first report of this mutation in Vietnamese patients with GSD type Ia. The result of this study enriches knowledge of the G6PC gene mutation spectrum and provided genetic data for further studies on glycogen storage disease type Ia in Viet Nam. Acknowledgments: This research was funded by the Vietnam Academy of Science and Technology (VAST) under grant No. KHCBSS.02/18-20 and the senior researcher support program for 2020. The authors thank the patient and his family members for their time and support. REFERENCES Akanuma J., Nishigaki T., Fujii K., Matsubara Y., Inui K., Takahashi K., Kure S., Suzuki Y., Ohura T., Miyabayashi S., Ogawa E., Iinuma K., Okada S., Narisawa K., 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(2): 107–112. Bali D. S., Chen Y. T., Austin S., Goldstein J. L., 1993. Glycogen Storage Disease Type I. In: Adam M. P., Ardinger H. H., Pagon R. A., Wallace S. E., Bean L. J., Stephens K., Amemiya A., (Eds). GeneReviews®. Seattle (WA): University of Washington, Seattle. Identification of p.His119Leu mutation 99 Chou J. Y., Mansfield B. C. 2008. Mutations in the Glucose-6-Phosphatase-α (G6PC) Gene that Cause Type Ia Glycogen Storage Disease. Hum. Mutat., 29(7): 921–930. Chou J. Y., Matern D., Mansfield B. C., Chen Y. T. 2002. Type I glycogen storage diseases: disorders of the glucose-6- phosphatase complex. Curr. Mol. Med., 2(2): 121–143. Froissart R., Piraud M., Boudjemline A. M., Vianey.Saban C., Petit F., Hubert. Buron A., Eberschweiler P. T., Gajdos V., Labrune P., 2011. Glucose-6-phosphatase deficiency. Orphanet. J. Rare Dis. 6: 27. Gu L. L., Li X. H., Han Y., Zhang D. H., Gong Q. M., Zhang X. X. 2014. A novel homozygous no-stop mutation in G6PC gene from a Chinese patient with glycogen storage disease type Ia. Gene, 536(2): 362–365. Hemrika W., Wever R., 1997. A new model for the membrane topology of glucose- 6-phosphatase: the enzyme involved in von Gierke disease. FEBS Lett., 409(3): 317–319. Hicks J., Wartchow E., Mierau G., 2011. Glycogen storage diseases: a brief review and update on clinical features., genetic abnormalities., pathologic features., and treatment. Ultrastruct. Pathol., 35(5): 183–196. Karthi S., Manimaran P., Varalakshmi P., Ganesh R., Kapoor S., Goyal M., Ashokkumar B., 2019. Mutational spectrum and identification of five novel mutations in G6PC1 gene from a cohort of Glycogen Storage Disease Type 1a. Gene, 700: 7–16. Lei K. J., Pan C. J., Liu J. L., Shelly L. L., Chou, J. Y., 1995. Structure Function Analysis of Human Glucose6phosphatase, the Enzyme Deficient in Glycogen Storage Disease Type 1a. J. Biol. Chem., 270: 11882–11886. Li H., Durbin R., 2009. Fast and accurate short read alignment with Burrows- Wheeler transform. Bioinforma. Oxf. Engl., 25(14): 1754–1760. McKenna A., Hanna M., Banks E., Sivachenko A., Cibulskis K., Kernytsky A., Garimella K., Altshuler D., Gabriel S., Daly M., DePristo M. A., 2010. The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res., 20(9): 1297–1303. Ng P. C., Henikoff S., 2003. SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res., 31(13): 3812–3814. Ozen H., 2007. Glycogen storage diseases: new perspectives. World J. Gastroenterol.. 13(18): 2541–2553. Pan C. J., Lei K. J., Annabi B., Hemrika W., Chou J. Y., 1998. Transmembrane topology of glucose-6-phosphatase. J. Biol. Chem., 273(11): 6144–6148. Rake J. P., Visser G., Labrune P., Leonard J. V., Ullrich K., Smit G. P. A., 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: S20-34. Rake J. P., Visser G., Smit G. P. A., 2006. Disorders of Carbohydrate and Glycogen Metabolism. In: Blau N., Leonard J., Hoffmann G. F., Clarke J. T. R., editors. Physician’s Guide Treat Follow-Metab Dis Roach PJ. 2002. Glycogen and its metabolism. Curr. Mol. Med., 2(2): 101–120. Saltik I. N., Ozen H., Ciliv G., Koçak N., Yüce A., Gürakan F., Dinler G., 2000. Glycogen storage disease type Ia: frequency and clinical course in Turkish children. Indian J. Pediatr., 67(7): 497–501. Schwarz J. M., Cooper D. N., Schuelke M., Seelow D., 2014. MutationTaster2: mutation prediction for the deep- sequencing age. Nat. Methods., 11(4): 361–362. Nguyen Huy Hoang et al. 100 Shieh J. J., Lu Y. H., Huang S. W., Huang Y. H., Sun C. H., Chiou H. J., Liu C., Lo M. Y., Lin C. Y., Niu D. M., 2012. Misdiagnosis as steatohepatitis in a family with mild glycogen storage disease type 1a. Gene, 509(1): 154–157. Shieh J. J., Terzioglu M., Hiraiwa H., Marsh J., Pan C. J., Chen L. Y., Chou J. Y., 2002. The molecular basis of glycogen storage disease type 1a: structure and function analysis of mutations in glucose- 6-phosphatase. J. Biol. Chem., 277(7): 5047–5053. Stukey J., Carman G. M., 1997. Identification of a novel phosphatase sequence motif. Protein Sci. Publ. Protein Soc., 6(2): 469–472. Wolfsdorf J. I., Weinstein D. A, 2003. Glycogen storage diseases. Rev. Endocr. Metab. Disord., 4(1): 95–102. Wu M. C., Tsai F. J., Lee C. C., Tsai C. H., Wu J. Y., 2000. A novel missense mutation (H119L) identified in a Taiwan Chinese family with glycogen storage disease type 1a (von Gierke disease) Human Mutat., 16: 447.