Glycogen storage disease type VI can progress to cirrhosis: ten Chinese patients with GSD VI and a literature review

Introduction

Glycogen storage disease type VI (GSD VI; OMIM #232700) is an autosomal recessive genetic disease caused by the deficiency of hepatic glycogen phosphorylase. PYGL gene containing 20 exons is the only gene responsible for encoding hepatic glycogen phosphorylase. The best estimation for the incidence of GSD VI is 1 of 100,000; however, it is generally believed that the true incidence is underestimated because of nonspecific and variable phenotypes [1]. Patients with GSD VI often have disease onset during infancy or childhood, with common phenotypes including hepatomegaly, poor growth, elevated hepatic transaminases, hyperlipidemia, ketosis, and hypoglycemia, and rare cases of severe phenotypes including preprandial and postprandial lactic acidosis, recurrent hypoglycemia, or marked hepatomegaly have been reported [2], [3]. GSD VI usually has a benign disease course, with most children having intact intellectual development. Clinical and biochemical abnormalities have a tendency to improve with age, and there is no consensus whether patients with GSD VI should be treated with uncooked cornstarch and high-protein diet [4]. In recent years, fibrosis, cardiomyopathy, focal nodular hyperplasia, and hepatocellular carcinoma have been reported in some patients [4], [5], [6], [7]. However, cirrhosis has not been reported in GSD VI. Molecular genetic testing can accurately diagnose GSD VI, and invasive liver biopsy may be avoided [2]. Although a GSD VI murine model recapitulating the phenotypes of human GSD VI was generated, there is a paucity of patients with GSD VI confirmed by molecular genetic testing in the literature including only one patient in Hong Kong, China [8], [9]. Considering the full genotypic and phenotypic spectrum of GSD VI continue to evolve, we reported 10 Chinese patients with GSD VI confirmed by next-generation sequencing (NGS) and reviewed literature to give a systematic description of patients with GSD VI in Chinese population. We believe that our study supports an expanding genotypic and phenotypic spectrum of GSD VI.

Methods

Results

Liver biopsy

The age of liver biopsy and histological features of our nine patients are summarized in Table 2. The age of liver biopsy was 30 ± 21 months (range: 13–73 months). The liver biopsy of patient 11 showed an extensive deposition of glycogen in the hepatocytes, with no cirrhosis or amylopectin found [9]. Excepted patient 6, all patients underwent liver biopsy and four patients (patients 7–10) underwent electron microscopy examination (Figure 1). Of the nine patients who underwent liver biopsy, all showed swollen hepatocytes (Figure 1A), and electron microscope examinations of four patients (patients 7–10) showed an extensive deposition of glycogen and lipid droplets in the swollen hepatocytes, which confirmed the diagnosis of GSDs (Figure 1B). Periodic Acid-Schiff (PAS) staining and Diastase-Periodic Acid-Schiff (D-PAS) staining showed liver sections stained with PAS-positive material and then subjected to diastase digestion no longer contained PAS-positive material (Figure 1C,D). Two patients (patient 1 and patient 2) revealed hepatic fibrosis. Four patients (patients 7–10) showed that the structure of normal liver lobules disappeared, and Reticular and Masson staining confirmed that extensive fibrosis encircled regenerative nodule (Figure 1E,F), indicating stage 4 (cirrhosis) [15]. For all these four patients with cirrhosis, serum albumin, prothrombin time, and international normalized ratio were normal and splenomegaly was not found under hepatic ultrasound. Except patient 7 who showed marked elevated transaminase and hepatomegaly under hepatic ultrasound (100 mm below costal), there was no significant difference from other patients. Group 1 included three patients (patients 3–5), and group 2 included six patients (patients 1–2 and patients 7–10). The age of liver biopsy in group 1 was significantly earlier than group 2 (p=0.048).

Table 2:

Histological features of nine Chinese patients with GSD VI.

Patient	Group	Age	Genotype	Microscopy				Electron microscope
				Enlarged hepatocyte	Glycogen condensation	Inflammation	Conclusion	Glycogen deposition	Lipid droplet
1	2	50 months	c.772+1G>A/c.1289C>A, p.(Ser430Tyr)	+	+	−	Fibrosis	NA	NA
2	2	27 months	c.697G>A, p.(Gly233Ser) (hom)	+	+	+	Fibrosis	NA	NA
3	1	18 months	c.772+1G>A (hom)	+	+	+	Glycogenosis	NA	NA
4	1	13 months	c.1768+1G>A (hom)	+	+	+	Glycogenosis	NA	NA
5	1	16 months	c.1768+1G>A/c.107T>G, p.(Leu36Arg)	+	+	+	Glycogenosis	NA	NA
7	2	17 months	c.244−1G>A/c.730C>T, p.(Leu244Phe)	+	+	−	Cirrhosis	+	+
8	2	42 months	c.772+1G>A/c.2467C>T, p.(Gln823Ter)	+	+	−	Cirrhosis	+	+
9	2	73 months	c.1366G>A, p.(Val456Met)/c.2417_2418delTA, p.(Ieu806Serfs*9)	+	+	−	Cirrhosis	+	+
10	2	17 months	c.1519−1G>C/c.1475G>A, p.(Trp492Ter)	+	+	−	Cirrhosis	+	+

1. +, positive result; −, negative result; NA, not available.

Figure 1:

Histological findings in four patients with GSD VI and cirrhosis. (A) Hematoxylin-eosin staining showed diffuse swollen hepatocytes resembling a lake with infiltration of inflammatory cells (200×); (B) Electron microscopy showed accumulation of glycogen and lipid droplets in swollen hepatocytes (scale bar 20 μm); (C and D) PAS staining and D-PAS staining showed liver sections stained with PAS-positive material and then subjected to diastase digestion no longer contained PAS-positive material (200x); (E and F) Reticular and Masson staining revealed that extensive fibrosis encircled regenerative nodule indicating cirrhosis (100×).

Literature review

Totally, we found 41 patients from the literature review [3], [4], [7], [16], [17], [18], [19], [20], [21], [22] (Table 4). One patient in Hong Kong, China, was included in our cohort as mentioned previously [9]. With available data, the average age of initial presentation of 40 non-Chinese patients from 36 families, 18 males and 22 females, was 20 ± 12 months (range: 1–54 months). The initial symptoms included hepatomegaly in 16 (73%) patients, distended abdomen in four (18%) patients, fasting miserable in one (4.5%) patient, and feeding difficulty in one (4.5%) patient. The clinical phenotypes were dominated by hepatomegaly (100%) and elevated transaminase (90%). Hyperlipidemia (82%) and short stature (74%) were common, and hypoglycemia (43%) was less common. The liver biopsy was performed in 15 patients, and six (40%) patients showed hepatic fibrosis. The genotypic spectrum of PYGL gene in Chinese and non-Chinese population is shown in Figure 2. Except one patient who carried only one PYGL gene variant, c.1900G>C, p.(Asp634His) [3], all patients carried biallelic PYGL gene variants. Totally, we identified 43 variants in non-Chinese population including 27 (63%) missense variants, eight (19%) splice variants, four (9%) nonsense variants, two (5%) large insertion and deletion variants, one (2%) synonymous variant, and one (2%) in-frame deletion variant. c.1900G>C, p.(Asp634His) and c.1366G>A, p.(Val456Met) were recurrent in four and three unrelated families, respectively. When we conducted a closer scrutiny to c.1900G>C, p.(Asp634His), we found that it carried an allele frequency of 0.003509 in general population and an allele frequency of 0.005469 in European population in the ExAC database, and it was predicted to be deleterious by using PolyPhen-2, SIFT, and CADD. All variants distributed over PYGL gene with no variant was found on exon 2, exon 13, exon 15, exon 18, and exon 19, so far.

Table 4:

The phenotype and genotype of patients with GSD VI published in the literature.

Patient	Onset age, (month)	Race	Sex	Onset symptoms	Hepatomegaly	Short stature	Hypoglycemia	Elevated liver transaminase	Hyperlipidemia	Liver biopsy	Genotype
[16]	22	Mennonite kindred	Female	NA	+	+	−	NA	NA	Glycogenosis	c.1620+1G>A (hom)
Subject 1 [17]	24	Israeli Arab	Male	Hepatomegaly	+	+	−	+	+	NA	c.1768+1G>A (hom))
Subject 2 [17]	24	Suriname Hindustani	Male	Hepatomegaly	+	+	+	+	−	Glycogenosis	c.529−1G>C/c.1016A>G, p.(Asn339Ser)
Subject 3 [17]	12	Turkish	Female	Hepatomegaly	+	−	−	+	+	Glycogenosis	c.1131C>G,p.(Asn377Lys) (hom)
Subject 1 [3]	16	British Asian	Female	Fasting miserable	+	+	+	+	+	NA	c.2042A>C,p.(Lys 681Thr) (hom)
Subject 2 [3]	5	British	Female	Hepatomegaly	+	−	−	+	+	NA	c.1471C>T, p.(Arg491Cys)/c.1964_1969inv6;c.1969 + 1_+4delGTAC
Subject 3a [3]	14	French	Male	Hepatomegaly	+	+	+	+	+	NA	c.1366G>A, p.(Val456Met)/c.2024C>T, p.(Ser675Leu)
Subject 4a [3]	27	French	Male	Hepatomegaly	+	+	+	+	+	NA	c.1366G>A, p.(Val456Met)/c.2024C>T, p.(Ser675Leu)
Subject 5 [3]	25	Polish	Male	Abdominal distension	+	+	−	+	+	Glycogenosis	c.1895A>T, p.(Asn632Ile)/c.2023T>A, p.(Ser675Thr)
Subject 6 [3]	42	Polish	Male	Abdominal distension	+	−	−	+	−	Glycogenosis	c.38A>C, p.(Gln13Pro)/c.1900G>C, p.(Asp634His)
Subject 7 [3]	27	Polish	Female	Abdominal distension	+	+	−	+	+	Glycogenosis	c.1195C>T, p.(Arg399Ter)/c.2017G>A, p.(Glu673Lys)
Subject 8 [3]	1	Austrian	Female	Feeding difficulties	+	+	−	+	+	Glycogenosis	c.1900G>C, p.(Asp634His)/unknow
Subject 1 [18]	9	Algeria	Male	NA	+	NA	+	+	+	NA	c.501_502ins361b (hom)
Subject 2 [18]	10	Tunisia	Female	NA	+	NA	−	+	+	NA	c.1499 T>G, p.(Leu500Arg) (hom)
Subject 3 [18]	21	Europe	Male	NA	+	+	−	+	+	NA	c.2108-2110delAAG, p.(Glu703del)/c.528+2T>C
Subject 4 [18]	4	Europe	Female	NA	+	NA	−	+	+	NA	c.564C>A, p.(Asn188Lys)/c.2461T>C, p.(Tyr821His)
Subject 5 [18]	48	Turkey	Female	NA	+	+	+	+	+	NA	c.1131C>G, p.(Asn377Lys) (hom)
Subject 6 [18]	24	Europe	Male	NA	+	NA	−	+	+	NA	c.1145C>T, p.(Pro382Leu)/c.1472G>A, p.(Arg491His)
Subject 7 [18]	14	Europe	Male	NA	+	+	−	+	+	NA	c.1366G>A, p.(Val456Met)/c.2024C>T, p.(Ser675Leu)
Subject 8 [18]	NA	Indian	Female	NA	+	NA	−	+	N	NA	c.1620+1G>C (hom)
Subject 9 [18]	12	Algeria	Female	NA	+	NA	+	+	+	NA	c.682G>T, p.(Asp288Tyr) (hom)
Subject 10 [18]	10	Europe	Female	NA	+	+	NA	+	+	NA	c.2313-1G>T/c.1768+1G>A
Subject 11 [18]	26	Africa	Male	NA	+	+	+	+	+	NA	c.856-29_c.1518 + 614del (hom)
Subject 1b [4]	8	Canadian	Female	Hepatomegaly	+	−	NA	+	NA	Fibrosis	c.1729C>T, p.(Gln577Ter)/c.856−9G>A
Subject 2b [4]	13	Canadian	Female	Hepatomegaly	+	+	NA	+	NA	Fibrosis	c.1729C>T, p.(Gln577Ter)/c.856−9G>A
Subject 3 [4]	22	Canadian	Male	Hepatomegaly	+	−	NA	+	NA	Fibrosis	c.1198C>T, p.( His400Tyr) (hom)
Subject 4 [4]	54	Canadian	Male	Hepatomegaly	+	−	NA	+	NA	Fibrosis	c.1518G>A, p.(Glu506Glu) (hom)
[19]	12	Indian	Female	Abdominal distension	+	+	+	+	+	Fibrosis	c.1297G>T, p.(Glu433Ter) (hom)
Subject 1c [7]	12	Caucasian	Female	Hepatomegaly	+	NA	NA	−	−	Glycogenosis	c.777T>A, p.(Asn259Lys)/c.1900G>C, p.(Asp634His)
Subject 2c [7]	36	Caucasian	Male	Hepatomegaly	+	NA	NA	+	−	NA	c.777T>A, p.(Asn259Lys)/c.1900G>C, p.(Asp634His)
Subject 3c [7]	13	Caucasian	Female	Hepatomegaly	+	NA	NA	−	+	NA	c.777T>A, p.(Asn259Lys)/c.1900G>C, p.(Asp634His)
Subject 4 [7]	12	Caucasian	Female	Hepatomegaly	+	+	NA	+	+	NA	c.43A>G, p.( Ser15Gly) (hom)
Subject 5 [7]	28	Caucasian	Female	Hepatomegaly	+	NA	NA	+	−	Hepatic fibrosis	c.1901A>G, p.(Asp634Gly)/c.2017G>A, p.(Glu673Tyr)
Subject 6 [7]	16	Caucasian	Female	Hepatomegaly	+	NA	NA	−	+	Glycogenosis	c.2435T>C, p.(Phe812Ser)/c.2446C>T, p.(Arg816Ter)
Subject 1 [20]	NA	NA	Male	NA	NA	NA	NA	NA	NA	NA	c.385G>A p.(Asp129Asn)/c.2446C>T, p.(Arg816Ter)
Subject 2 [20]	NA	NA	Male	NA	NA	NA	NA	NA	NA	NA	c.418C>G, p.(Leu140Val)/c.1366G>A, p.(Val456Met)
Subject 3 [20]	NA	NA	Male	NA	NA	NA	NA	NA	NA	NA	c.131G>A, p.(Arg44His)/c.1900G>C, p.(Asp634His)
[21]	38	NA	Male	NA	NA	NA	+	NA	NA	NA	c.1727G>A, p.(Arg576Gln) (hom)
Subject 1 [22]	NA	Brazilian	Female	NA	NA	NA	NA	NA	NA	NA	c.697G>A, p.(Gly233Ser) (hom)
Subject 2 [22]	NA	Brazilian	Female	NA	NA	NA	NA	NA	NA	NA	c.131G>A, p.(Arg44His) (hom)

1. ^{a, b, c}: Siblings in each family.

Figure 2:

Genotypic spectrum of PYGL gene in Chinese and non-Chinese population. Gray box: exons of PYGL gene (exons were not drawn in the scale); the variants above exons are PYGL gene variants identified in Chinese population; the variants below exons are variants identified in PYGL gene in non-Chinese population; red variants: novel PYGL gene variants identified in our study; blue variants: large PYGL gene variants of insertion and deletion.

Discussion

We reported genotypic and phenotypic spectrum of 10 patients with GSD VI in mainland China. Hepatomegaly is the most common symptom caused by increased glycogen storage in hepatocytes in Chinese patients with GSD VI, and hypoglycemia may not be present as gluconeogenesis is intact. The most common abnormal laboratory examination finding is elevated transaminase due to the damage of hepatocytes. Elevated transaminase can reflect the degree of hepatocyte damage to some extent because there are not enough hepatocytes to release transaminase in the later stage. Liver cirrhosis is the final usual outcome of various chronic liver diseases, with most patients progressing into decompensated stage silently. The prevalence of cirrhosis varies among the different types of GSDs with unclear pathogenesis. Patients with glycogen storage disease type I (GSD I) had the most severe metabolic phenotype, with only mildly elevated transaminase. However, hepatic fibrosis or cirrhosis was not observed in animal models and patients with GSD I [23], [24]. Transaminase levels elevated most obviously in glycogen storage disease type III (GSD III), and hepatic fibrosis is a common long-term complication of GSD III, with live biopsy showing the deposition of abnormally structured glycogen, resembling limit dextrin [1]. Although dietary treatment can prevent hypoglycemia and improve growth, it was found that the improvement of metabolism cannot prevent progressive hepatic fibrosis in animal models and patients with GSD III [25], [26], [27]. Patients with glycogen storage disease type IV showing the deposition of abnormally structured glycogen, resembling plant-like fibers of amylopectin in live biopsy, can rapidly progress to cirrhosis [1]. A high-protein diet and cornstarch cannot prevent progression of the liver disease, and liver transplantation is the primary treatment [28]. All these accumulations of abnormally structured glycogen may be a critical factor for hepatocyte damage and result in a rapid progression to hepatic fibrosis and cirrhosis. Cirrhosis had a high frequency in glycogen storage disease type IXc (GSD IXc), with 40% of patients with GSD IXc affected by cirrhosis, and although information about treatment and prognosis was rare, it was found that some patients did not develop cirrhosis under treatment of uncooked cornstarch and protein supplementation [29]. Cirrhosis was also reported in some cases with glycogen storage disease type IXa, and a structured treatment regimen can improve metabolic control and cirrhosis [30], [31]. Hepatic fibrosis is caused by chronic liver injury because of viral infection, drugs, toxins, or metabolic disorders [32]. Activated hepatic stellate cells (HSCs) play an important role in pathogenesis of hepatic fibrosis [8], [32]. Under the stimulation of cytokines and chemokines released by immune cells and damaged hepatocytes, HSCs can be activated, which then differentiate into myofibroblasts that secrete various extracellular matrix proteins causing hepatic fibrosis [32]. Cirrhosis can always develop silently, and liver biopsy is an effective way to identify cirrhosis. Here, for the first time, we reported that four patients showed cirrhosis featured by the formation of regenerative nodule in liver biopsy, revealing a risk of progression to end-stage liver disease. Because most liver biopsies in literature review were conducted without exact time and visual confirmation, we cannot conduct a systematic analysis for hepatic histopathology. However, in our study, we analyzed the time of liver biopsy between two groups to explain cirrhosis. We found that the age of liver biopsy conducted in patients without hepatic fibrosis and cirrhosis was significantly earlier. Thus, we cannot rule out the possibility that hepatic fibrosis and even cirrhosis may present over time. A recently constructed GSD VI murine model also revealed that the damage, inflammation, and fibrosis of hepatocytes aggravated with aging [8]. So far, no cirrhosis was reported in patients with GSD VI, and this may be a result of the lower number of liver biopsy that has been investigated. Moreover, cirrhosis is a complicated process where genetic and metabolic factor may be involved in, and it has been demonstrated that cirrhosis was a pathway of hepatocellular carcinoma transformation [25]. Here, our study combined with published literature further confirms that GSD VI can also be in another extreme of the disease severity. Considering mounting evidence supported that long-term complications can be prevented by optimizing metabolic control, we recommend an aggressive therapy for GSD VI [30]. Systematic follow-up and specific biomarkers are also needed to monitor this silent course although the improvement of elevated transaminase was observed in most of our patients with GSD VI [15], [, 33].

Kobayashi et al. concluded that variants with an allele frequency higher than 0.01% in the ExAC database were generally already well characterized in the literature as known founder mutations [34]. When we reviewed the literature, we found that c.1900G>C, p.(Asp634His) was recurrent in four European families, and we speculated that this variant may be a founder mutation in European population. Notably, this variant contributed a large burden of disease with an allele frequency of 0.003509 in general population and an allele frequency of 0.005469 in European population in the ExAC database. Considering this variant alone can contribute to a world-wide prevalence of 1.2 of 100,000 in the genetic context based on the Hardy-Weinberg equilibrium (the prevalence is equal to the square of the minor allele frequency), the prevalence of GSD VI is further confirmed to be underestimated. Inconsistent with an autosomal recessive pattern, only one patient is female in our cohort; this may be due to boys getting more attention than girls in the family, and their parents have a stronger inclination to bring them to tertiary hospital in China. It is also suggested that some female patients are underdiagnosed or unreported in China. As c.772+1G>A was recurrent in three families in Chinese population, it is suggested that the genotypic spectrum of PYGL gene may vary among the population. Furthermore, we identified seven novel variants, bringing the total number of PYGL gene variants to 53, and we considered that all these novel variants were pathogenic based on the type of variant, in silico analysis, and allele frequency in the public database.