Introduction

Glycogen storage diseases (GSDs) are inherited metabolic diseases caused by the deficiency of enzymes regulating glycogenolysis or gluconeogenesis. As glycogen primarily accumulates in the liver and muscle, the disorders of glycogen degradation affect the liver, muscles or both. Hypoglycemia is the main symptom of hepatic GSDs, whereas muscle weakness or elevated muscle enzyme is the main symptom of myopathic GSDs. Hepatic GSDs, except for GSD IXa, are autosomal recessive, and GSD IXa is an X-linked recessive disorder. GSD Ia, GSD III and GSD IXa account for 80% of hepatic GSDs.

GSD Ia (Mendelian Inheritance in Man (MIM) no. 232200) is caused by a deficiency of glucose-6-phosphatase (EC 3.1.3.9) in the endoplasmic reticulum. GSD Ib (MIM no. 232220) is caused by a deficiency of glucose-6-phosphate transporter, which leads to the dysfunction of glucose-6-phosphatase in the endoplasmic reticulum. GSD Ia is the most common GSD, and its frequency is 1/100 000 to 1/400 000 births in the general Caucasian population; GSD Ib is much less frequent than GSD Ia. The manifestations of GSD Ia are short stature, hypoglycemia, hepatomegaly, hyperlipidemia, hyperuricemia, hyperlactacidemia, hepatoadenoma, renal disorder1, 2 and hepatocellular carcinoma.3, 4 Most GSD Ib patients have neutropenia and neutrophil dysfunction in addition to these symptoms. GSD III (MIM no. 232400) is caused by a deficiency of the debranching enzyme, which consists of amylo-1,6-glucosidase (EC 3.2.1.33) and oligo-1,4-1,4-glucantransferase (EC 2.4.1.25). The incidence of GSD III has been reported to be 1 per 83 000 live births in Europe and 1 per 100 000 live births in North America.5 There are two major GSD III subtypes: GSD IIIa, which affects both the liver and muscle and accounts for 80% of all GSD III cases, and GSD IIIb, which affects only the liver and comprises approximately 15% of them.6 The manifestations of GSD III are similar to those of GSD Ia, and many patients with GSD IIIa have hypertrophic cardiomyopathy.7

GSD IV (MIM no. 232500) is caused by a deficiency of amylo-1,4 to 1,6-transglucosidase (EC 2.4.1.18), which leads to the absence of branched glycogen. GSD IV, which is the most severe type of GSD, represents 0.3% of all GSDs.8 This disease rapidly progresses to cirrhosis early in life and causes death between 3 and 5 years of age because of liver failure.9 If signs of GSD IV, such as cervical cystic hygroma, are detected,8 the patients are likely to die in the neonatal period. The effective treatment for progressive GSD IV is liver transplantation.10 GSD VI (MIM no. 232700), which is rarer and milder than the other hepatic GSDs, is caused by a deficiency of glycogen phosphorylase (EC 2.4.1.1) in the liver. GSD IXa (MIM no. 306000) is caused by a deficiency of phosphorylase kinase α2 (PHKA2)—a subunit of phosphorylase kinase (EC 2.7.11.19), which consists of four subunits, namely, α, β, γ and δ. The clinical course of GSD IXa is benign, and most adult patients are asymptomatic.11 With aging, clinical and biochemical abnormalities gradually disappear. The other subtypes of GSD IX include subtypes caused by a deficiency of phosphorylase kinase β, phosphorylase kinase γ or δ, or muscle phosphorylase kinase. The Fanconi–Bickel syndrome, GSD XI (MIM no. 227810), is caused by a deficiency of glucose transport 2 and is characterized by hepatorenal glycogen accumulation and proximal renal tubular dysfunction.12

The treatment for these hepatic GSDs comprises the prevention of hypoglycemia. The basic treatment is the consumption of frequent meals and uncooked cornstarch.13, 14, 15 Moreover, restriction of the intake of sugars, such as fructose, galactose, sucrose and lactose, is important mainly for GSD I.

Complete blood glucose control by these measures is unlikely to ameliorate complications, such as hyperuricemia and hyperlipidemia.16 GSD patients are administered allopurinol for hyperuricemia and statin, fibrates or niacin formulations for hyperlipidemia.17, 18 Administration of angiotensin-converting enzyme inhibitor or/and angiotensin receptor blocker, which have a renoprotective effect, is recommended for GSDs with possible renal complications.19 Gene therapy can be an effective as a radical treatment measure for GSDs.20, 21 However, the definitive treatment of GSDs is only liver transplantation.22, 23, 24, 25

Many reports have been published overseas on the long-term outcome and treatment of GSD patients.11, 17, 26, 27, 28 However, no report has yet been published on the long-term outcome of GSDs in Japan, wherein GSD Ia with a mutation causing mild symptoms has been detected in many cases. We studied the current status of clinical manifestations, treatment, and long-term outcome of hepatic GSDs in Japan.

Materials and methods

Study patients

In 2009, we sent a questionnaire to 928 Japanese institutions, including the departments of pediatrics, endocrinology and metabolism, neonatology, genetics, and transplant surgery, asking doctors if they had diagnosed or provided medical care to hepatic GSDs patients. Each institution was the medical center for a locality and had 300 or more beds. Of the 928 institutions, 668 (72%) responded. Of these 668 institutions, 97 had treated patients with GSDs. A second questionnaire was then sent to these 97 institutions in 2009, and responses were received from 53 (55%) of them. On the basis of the received reports, 127 cases of GSDs diagnosed and treated between January 1999 and December 2009 were studied. We excluded patients who were not definitely diagnosed and considered patients visiting multiple institutions as single patients. The 127 cases of GSDs (types Ia, Ib, III, IV, VI, IXa and others) were diagnosed on the basis of clinical manifestations, family history, enzyme activity, metabolite analysis (75 g oral glucose tolerance test (OGTT) or/and glucagon test) and/or DNA analysis. This study was approved by the ethical committee of the Faculty of Life Science, Kumamoto University.

The definition of clinical manifestations of GSD applied in this study was the same as that proposed by Smit et al.27 In addition, we used the following definitions. Hyperlactacidemia was defined as a blood lactate level >2.2 mmol l–1. Hyperuricemia was defined by a history of receiving drugs for hyperuricemia and/or blood uric acid level >420 μmol l–1. Hyperlipidemia was defined by a history of medical treatment for hyperlipidemia, blood total cholesterol level >5.9 mmol l–1, or blood total triglyceride level >1.7 mmol l–1. Mental retardation was diagnosed if the patient’s intelligence quotient was <70, as per standardized tests, such as the Wechsler Intelligence Scale for Children and the Wechsler Adult Intelligence Scale. Proteinuria was defined by protein levels >30 mg dl–1 in 1 spot urea test or >500 mg day–1. Renal dysfunction was defined by blood creatinine levels >90 μmol l–1. Increased susceptibility to infection was defined as a neutrophil count of <1500/μl and/or hospitalization more than three times a year because of infection.

Statistical analysis

The age at onset of hepatic GSD patients was expressed in terms of the median and interquartile range, and the age of onset was analyzed by the Mann–Whitney U-test of IBM SPSS Statistics Version 19.29 A P-value of <0.05 was considered statistically significant. The height of hepatic GSD patients was expressed in terms of mean±s.d. values. Kaplan–Meier curves of estimated survival rate were generated by SPSS.

Results

Age at onset and methods for definitive diagnosis of hepatic GSDs

Table 1 indicates the age, onset age and methods used for definitive diagnosis in each of the 127 cases of hepatic GSD. GSD Ib and GSD IV patients manifested symptoms earlier than those with other types of GSD (GSD Ia vs GSD Ib, P=0.001; GSD Ia vs GSD IV, P=0.022; GSD Ia vs GSD XIa, P=0.002). Enzyme activity was measured in 50% (64/127) of the patients with GSDs, and genotype analysis was performed in 50% (63/127); genotypes could be identified in 40% (51/127) of the patients with GSDs. DNA analysis was performed in the case of 52 patients with GSD Ia, 7 patients with GSD Ib, 1 patient with GSD III, 1 patient with GSD VI, 5 patients with GSD IXa and 2 patients with GSD XI. Thereafter, identifiable mutations were detected at a rate of 79% (41/52) in GSD Ia patients, 86% (6/7) in GSD Ib patients, 40% (2/5) in GSD IXa patients and 100% (2/2) in GSD XI patients. Of the GSD Ia patients with recorded identifiable mutations, 81% (29/36) had g727t homozygote mutations and 17% (6/36) had compound heterozygotes with g727t mutations. Of the GSD Ib patients with recorded identifiable mutation, 83% (5/6) had homozygote or compound heterozygote mutations of W118R. Eight patients with GSD Ia, one patient with GSD IXa and one patient with GSD XI were diagnosed by DNA-based and enzymatic analyses.

Table 1 Age of onset, diagnosis and definitive diagnosis of hepatic GSD patients
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Clinical manifestations of hepatic GSD

Table 2 indicates the frequency of clinical manifestations in hepatic GSD patients. In GSD Ia patients, growth retardation (78%; 51/65), hypoglycemia (69%; 45/65), hyperuricemia (88%; 57/65) and hyperlipidemia (94%; 61/65) were observed at the frequency of >50% (Table 2a). Convulsions (9%; 6/65), mental retardation (9%; 6/65), liver tumors (22%; 14/65), proteinuria (26%; 17/65), renal dysfunction (11%; 7/65) and increased susceptibility to infection (5%; 3/65) were not frequently observed (Table 2b). Of the 14 GSD Ia patients with liver tumors, 4 had a single adenoma, 9 had 3 or more multifocal adenomas and 1 patient had hepatocellular carcinoma with multiple adenomas. Only one patient with GSD Ia developed acute pancreatitis.

Table 2 (a) Frequent manifestations of hepatic GSD; (b) Infrequent manifestations of hepatic GSD
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Height of hepatic GSD patients

Figures 1a–d show the height of male and female hepatic GSD patients. The height of 56% (14/25) of the male GSD Ia patients aged <18 years and 43% (6/14) of the male GSD Ia patients aged ⩾18 years was below the third percentile. The mean height of male GSD Ia patients aged ⩾18 years was 160.8±10.6 cm (n=14; Figure 1a). Fifty-seven percent (4/7) of the male GSD Ib patients, 50% (2/4) of the GSD III patients aged <18 years and 19% (6/32) of the male GSD IXa patients had heights below the third percentile (Figures 1b and c). One hundred percent (5/5) of the male GSD VI patients had height greater than the tenth percentile (Figure 1b). Thirty-three percent (5/15) of the female GSD Ia patients aged <18 years and 44% (4/9) of the female GSD Ia patients aged ⩾18 years had heights below the third percentile. The mean height of female GSD Ia patients aged ⩾18 years was 147.8±3.80 cm (n=9; Figure 1d).

Figure 1
figure 1

Stature of hepatic glycogen storage disease (GSD) patients. This figure was constructed with the age of GSD patients on the abscissa and the stature of patients with GSD on the ordinate. Percentiles are based on data from Japanese 2000 growth reports provided by the Ministry of Health, Labor and Welfare in Japan. (a) Stature of male patients with GSD Ia. The height of the male GSD Ia patient aged 7 years 11 months was measured after liver transplantation. ●: GSD Ia patients (n=39); ⊖: patients after liver transplant. (b) Stature of male patients with GSD Ib, GSD III and GSD VI. The heights of male GSD Ib patients aged 1 year 10 months and 14 years 10 months were measured after liver transplantation.▴: GSD Ib patients (n=7); ♦: GSD III patients (n=4); *: GSD VI patients (n=5); ⊖: patients after liver transplant. (c) Stature of male patients with GSD IXa. ●: GSD IXa patients (n=32). (d) Stature of female patients with GSD Ia, GSD Ib, GSD III and GSD VI. The heights of female GSD Ia patient aged 4 years 10 months and GSD Ib patients aged 4 years 7 months, 6 years 6 months and 11 years 11 months were measured after liver transplantation.●: GSD Ia patients (n=24), ▴: GSD Ib patients (n=4), ♦: GSD III patients (n=1), *: GSD VI (n=1), ⊖: patients after liver transplant.

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Long-term survival of patients with hepatic GSD

Table 1 presents the number of hepatic GSD patients who survived and died. Two patients with GSD Ia (age of death: 6 years 10 months, male; 27 years, female), a male GSD Ib patient (13 years 5 months), a female GSD IIIa patient with cardiomyopathy (24 years 8 months) and a male GSD IV patient (1 year 11 months) died because of liver failure after liver transplantation. The other two patients with GSD IV died of liver failure 2 months after birth.

The long-term survival rate of GSD Ia patients at 20 years after birth was 97% for male patients and 100% for female patients (Figure 2). The survival rate of GSD Ib patients at 20 years after birth was 80% (Supplementary Figure 1).

Figure 2
figure 2

Long-term survival rates in patients with glycogen storage disease (GSD) Ia. The survival rates of 63 patients with different ages are shown by Kaplan–Meier survival curves. Two GSD Ia patients aged 6 years 10 months (male) and 27 years (female) died of liver failure after liver transplant. Male GSD Ia patients (black bold line), n=41; female GSD Ia patients (black fine line), n=22.

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Treatment for hepatic GSD

Table 3 indicates the treatment received by the hepatic GSD patients. Among the patients with GSD Ia, uncooked corn starch was administered to 98% (64/65) of the patients; allopurinol, to 74% (48/65); lipid-lowering drugs, to 42% (27/65); and angiotensin-converting enzyme inhibitor or angiotensin receptor blocker, to 15% (10/65). Dietary management with restriction of the intake of galactose, fructose and saccharose was used for 63% (41/62) of the patients. Most patients were not taking the GSD formula when they were taking corn starch. Lipid-lowering drugs were administered to 66% (18/27) of the GSD Ia patients with hyperlipidemia and aged 14 years or more. The youngest patient who received lipid-lowering drugs was 5 years old.

Table 3 Treatment for hepatic GSD
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Liver transplantation for hepatic GSD

Table 4 shows the ages at which liver transplants were performed for hepatic GSD patients. As many metabolic disorders, such as hypoglycemia, were improved in the two patients with GSD Ia who underwent successful liver transplantation, symptoms such as nasal bleeding and growth disorder were ameliorated. These two patients needed allopurinol, but not diet and corn starch treatment.

Table 4 Age at liver transplant for hepatic GSD
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Figure 3 and Supplementary Figure 2 present the comparison between the data obtained immediately before liver transplant and 1 year after liver transplant in five patients with GSD Ib. Blood levels of uric acid, total cholesterol and triglyceride in GSD Ib patients improved after liver transplantation, but the abnormalities in the neutrophil count were not ameliorated. All the five patients received granulocyte colony-stimulating factor after liver transplants; however, the frequency of granulocyte colony-stimulating factor administration after liver transplantation was lower than that before transplantation, as was the susceptibility to infection. No patients in this study received bone-marrow transplantation.

Figure 3
figure 3

Comparison of data immediately before and 1 year after liver transplantation in glycogen storage disease (GSD) Ib patients. The age at liver transplantation was 1 year 1 month in patient 1 (male), 3 years 6 months in patient 2 (female), 3 years 6 months in patient 3 (male), 3 years 11 months in patient 4 (female) and 8 years 6 months in patient 5 (female). Only patient 3 received allopurinol after liver transplant. T-chol, total cholesterol; TG, triglyceride; UA, uric acid.

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Discussion

Most patients with hepatic GSD, except for GSD VI and IXa, which were mild types, manifested symptoms before 2 years of age. Further, the age of onset for GSD Ib and IV was lower than that for the other hepatic GSDs. However, two male GSD Ia patients presented with symptoms at 11 years and 9 years, thereby indicating that GSD may be detected at any age. Enzyme activity in the erythrocytes or leukocytes was measured in patients with GSD III, VI, IXa and XI, without performing invasive liver biopsy. Genome sequencing for GSD III, VI and IXa was difficult and not likely to be performed. Among the GSD I patients, the g727t mutation of the glucose-6-phosphatase gene has been detected in almost 90% alleles of GSD Ia,30 and the W118R mutation of glucose-6-phosphate transporter gene is highly frequent in GSD Ib patients.31 Therefore, we performed DNA analysis rather than enzyme assay, which requires invasive liver biopsy in GSD I patients. As this study focused on GSD patients younger than 18 years, we did not include many GSDs patients older than 18 years. Thus, the exclusion of GSD patients older than 18 years and GSD III patients may have introduced a bias in the results.

We investigated the statures of patients with hepatic GSD. Among the hepatic GSDs, GSD I commonly presents with short stature. Height <3 percentile were noted in 56% of the male GSD Ia patients and 33% of female GSD Ia patients aged <18 years. Mean stature in patients with GSD Ia aged >18 years was 160.8±10.6 cm (n=14) and 147.8±3.80 cm (n=9) for male and female patients, respectively. Therefore, we can expect that the final stature of patients with GSD Ia ranges from 3 to 10 percentile of the Japanese height.

Liver tumor and renal dysfunction, which are not frequently observed, are important determinants of the prognosis in patients with GSD.1 It has been reported that liver adenomas are detected in 22 to 75% of patients with GSD Ia,28, 32 and some of these adenomas developed to hepatocellular carcinoma.33, 34 In this study, liver tumors, which have been reported to be less frequent overseas, were detected in 22% (14/65) of the patients with GSD Ia and in 18% (2/11) of patients with GSD Ib. The youngest GSD Ia patient with liver adenoma was a male patient aged 13 years 4 months, and 41% (14/34) of GSD Ia patients older than this patient had liver adenoma. Nakamura et al.35 reported that 57.9% (11/19) of adult GSD Ia patients with the g727t homozygote mutation had liver adenomas, and 16% (3/19) of them had hepatocellular carcinoma. In this study, only one patient developed hepatocellular carcinoma, which was treated by percutaneous ethanol injection therapy and radiofrequency ablation, and did not recur.

Proteinuria, which is detected in many patients with GSD I, may progress to renal dysfunction or renal failure. In this study, two of the seven GSD Ia patients with renal dysfunction underwent hemodiafiltration. Chen et al. reported that 70% of GSD Ia patients aged >10 years presented with renal dysfunction and that 40% of GSD Ia patients with renal dysfunction developed progressive renal failure. The incidence of renal dysfunction, which was 11% (7/65) in GSD Ia patients of this study and 19% (7/36), in GSD Ia patients >10 years old, was very low.

As GSD Ia with g727t mutation is considered to be a mild type of GSD Ia, patients with the g727t mutation may develop only proteinuria but are not likely to develop renal dysfunction. It has been reported that transforming growth factor-β expression increases in the tubular epithelial cells and is involved in the pathophysiology of renal interstitial fibrosis, which results from the increase in the expression of extracellular matrix proteins in GSD I patients.36 Angiotensin receptor blocker, angiotensin-converting enzyme inhibitor and alloprinol have been considered drugs with the highest potential of interfering with transforming growth factor-β expression because the renin angiotensin–aldosterone system and uric acid have been known to involved in the expression of transforming growth factor-β.37, 38 Moreover, it has been recognized that the small, dense low-density lipoprotein and modified low-density lipoprotein induce the development of glomerular sclerosis and renal dysfunction.39

Liver tumor is related to constant stimulation by hormones, such as insulin and glucagon, by persistent peripheral hypoglycemia. Therefore, the expression of renal dysfunction and liver tumor negatively correlates with metabolic control.40 Important treatment strategies are restriction of the intake of galactose, fructose, and saccharose and blood glucose control by consumption of frequent meals and uncooked cornstarch.40 Moreover, allopurinol, lipid-lowering drugs, and angiotensin receptor blocker or angiotensin-converting enzyme inhibitor have been reported to be significantly important in delaying the progression of kidney disease in GSD I patients.19, 39, 41

Recent reports have indicated that GSD patients may present with diabetes. Two GSD Ia patients who were brothers and had the g727t homozygote mutation developed type II diabetes and received therapy involving an α-glucosidase inhibitor and an insulin secretagogue. They monitored themselves for hypoglycemia attacks and corrected the same by consuming food or glucose. As shown in Table 1 and Figure 2, patients with hepatic GSD, except for those with GSD IV, can survive in the long term. Further, reports have also shown that GSD Ib and GSD III patients developed type II diabetes.42, 43 Therefore, physicians must pay attention to the development of obesity- and lifestyle-related diseases in GSD patients.

Table 3 indicates the treatments received by patients with hepatic GSD. As treatment after liver transplantation was recorded in Table 3, none of patients with GSD IV take dietary treatment and corn starch treatment. Use of lipid-lowering drugs has been recommended for adult GSD patients overseas.18 Although definitive criteria for the use of lipid-lowering drugs in Japan have not yet been established, the youngest patient who received hypoglycemic medication was 5 years old.

Fourteen patients with hepatic GSD received liver transplants. According to overseas reports, the indications for liver transplantation in GSD patients are the progression of adenomatous lesions or multiple adenomas, suspicion or detection of malignant transformation of an adenoma, unresponsiveness to medical therapy, insufficient control of hypoglycemia, and growth or sexual retardation.17, 24, 44 In Japan, the definitive criteria for liver transplants are controversial; many pediatricians and transplant surgeons follow the same indications reported overseas for liver transplantation. GSD I patients with uncontrolled hypoglycemia, which leads to convulsions and mental retardation, should receive liver transplants. Ninety-one percent (10/11) of patients with GSD I received liver transplants because of insufficient control of hypoglycemia and metabolic disorders, despite medical therapy. GSD III and GSD IV patients received liver transplants because of liver failure, which was considered an indication of liver transplant, as per the pediatric end-stage liver disease scores. In this study, all GSD I patients with multiple liver adenomas underwent hepatectomy, and only one patient with GSD I received a liver transplant because of adenoma recurrence after adenoma resection. Five of 14 GSD patients died because of liver failure <2 months after liver transplantation. The other nine patients survived and improved such that they did not develop hypoglycemia without medication and showed better increase in height. The frequency of infection decreased in GSD Ib patients after transplantation, as described previously.45 Liver transplants contributed to an improved quality of life (QOL) in GSD patients. We believe that liver transplants should be proactively performed in patients with GSD Ib. Although the success rate of liver transplantation for hepatic GSD in this study was lower than that reported abroad,24, 46, 47, 48, 49 the low success rate of liver transplants may be attributed to the severe liver failure in the fatal GSD cases before transplantation.

In conclusion, we discussed the diagnosis, treatment and long-term outcome of hepatic GSDs and the present status of hepatic GSD patients in Japan. We found a characteristic genetic pattern with many GSD Ia patients presenting with the g727t mutation and GSD Ib patients showing the W118R mutation. Although patients with hepatic GSD, except for those with GSD IV, develop a variety of symptoms, they can survive in the long-term by diet therapy, corn starch treatment and supportive care. Liver transplantation is an important therapeutic strategy for hepatic GSD and can help improve the patients’ QOL.