Case report: Familial glycogen storage disease type IV caused by novel compound heterozygous mutations in a glycogen branching enzyme 1 gene

Glycogen storage disease type IV (GSD IV), caused by a mutation in the glycogen branching enzyme 1 (GBE1) gene, is a rare metabolic disorder with an autosomal recessive inheritance that involves the liver, neuromuscular, and cardiac systems. Here, we reported a case of familial GSD IV induced by novel compound heterozygous mutations in GBE1. The proband (at age 1) and her younger brother (at age 10 months) manifested hepatosplenomegaly, liver dysfunction, and growth retardation at onset, followed by progressive disease deterioration to liver cirrhosis with liver failure. During the disease course, the proband presented rare intractable asymptomatic hypoglycemia. The liver pathology was in line with GSD IV. Both cases carried pathogenic compound heterozygous mutations in GBE1 mutations, i.e., a missense mutation (c.271T>A, p. W91R) in exon 2 and a deletion mutation in partial exons 3–7. Both mutations are first reported. The internationally pioneered split-liver transplantation was performed during progression to end-stage liver disease, and the patients had normal liver function and blood glucose after. This study broadens the mutation spectrum of the GBE1 gene and the phenotypic spectrum of GSD IV.


Introduction
Glycogen storage disease type IV (GSD IV, OMIM 232500, Anderson disease) is a rare metabolic disorder with autosomal recessive inheritance. It is caused by a mutation in the glycogen branching enzyme 1 (GBE1) gene, which encodes the glycogen branching enzyme that catalyzes glycogen synthesis, with an incidence rate of 1/600,000-1/800,000 (Magoulas et al., 1993;Choi et al., 2018;Massese et al., 2022).
GBE1 catalyzes α-1,6-glucosidic bonds of the glycogen molecule, transferring the oligosaccharide chain containing six glucose residues to the adjacent fourth glucose molecule to form branches, thereby increasing glycogen solubility. Mutations in the GBE1 gene can lead to decreased or missed GBE1 activity, resulting in the accumulation of immature amylopectin-like polysaccharides in tissues (including the liver, skeletal muscle, cardiac muscle, brain, and peripheral neuropathy) (Magoulas et al., 1993;Li et al., 2010). The severity of the phenotype might depend on the residual activity of GBE1 (Massese et al., 2022).
With approval by the Medical Ethics Committee of the hospital and obtaining informed consent from the proband's parents, this study reported a case of familial GSD IV caused by compound heterozygous mutations in GBE1. The proband had rare intractable asymptomatic hypoglycemia.

Case presentation
The female proband was diagnosed with malnutrition and growth retardation at age 1 on physical examination (Figure 1), accompanied by moderate hepatosplenomegaly with a hard texture, transaminase elevation, and progressive liver function deterioration on routine physical examination.
At the age of 2.5 years, she manifested distension over the whole abdomen ( Figure 2A) with severe yellowing of the skin and sclera and umber urine and without any symptoms of hypoglycemia. On physical examination, her height, weight, and head circumference were 86 cm, 11 kg, and 46 cm, respectively ( Figure 1). Moreover, she had malnutrition. Her liver extended 1 cm above the umbilicus, and her spleen extended 2 cm below the umbilicus. Both the liver and spleen had a hard texture. Shifting dullness was positive. Moreover, she presented symmetrical pitting edema in bilateral lower limbs.

FIGURE 1
Growth and development of the proband within 3 years after birth. Note: weight and body mass index (BMI): normal nutrition and growth before the age of 1 year; moderate nutrition and insufficient weight gain after the age of 1 year; improved nutrition and growth after liver transplantation. Length/height: normal length/height and growth before the age of 1 year; inadequate growth after the age of 1 year; no improvement after liver transplantation, showing growth retardation. By now, the proband has a symmetrical short stature.
Frontiers in Genetics frontiersin.org eccentric nuclei. Histopathological investigation showed positive staining for periodic acid-Schiff (PAS). It also showed cholestasis, significant proliferation in interstitial fibrous tissues, and small bile ducts, with massive infiltration of lymphocytes. The disease progressed to decompensated cirrhosis with liver failure (Child-Pugh class C). Coagulation function was improved by plasma supplementation. Albumin infusion and diuresis were obtained to alleviate edema. Meanwhile, the patient was on dietary management and blood glucose monitoring. At the age of 3 years and 1 month, the patient received split-liver transplantation, followed by standard immunosuppressive therapy. She was regularly followed-up postoperatively for 1.5 years until now, presenting normal aminotransferase, peripheral blood cells, blood glucose, and a symmetrical short stature ( Figure 2D).

Family history
The proband is a Chinese girl. The parents and older sisters did not have liver disease, and the parents were nonconsanguineously married. Her younger brother presented hepatosplenomegaly with liver injury (ALT, 290 U/L; AST, 390 U/L) at the age of 10 months, and he experienced Frontiers in Genetics frontiersin.org progression to liver cirrhosis at the age of 1 year and 5 months accompanied by growth retardation. Normal FPG was detected by multiple tests. Split-liver transplantation followed by standard immunosuppressive therapy was performed at the age of 1 year and 7 months, and he presented normal liver enzyme levels during the 1-year follow-up until now.

Genetic testing and analysis Genetic testing
Peripheral blood samples (2 ml) were collected from the proband, her parents, and her younger brother. Whole-exome sequencing was performed to detect potential mutations. Sanger sequencing was used for point mutation validation. qPCR was used to determine copy number variation with the target sequences.
Compound heterozygous mutations in the GBE1 gene were detected in the proband and her younger brother, including a heterozygous missense mutation (c.271T>A, p. W91R) in exon 2 (chr3: 81754637, genome version: hg19, transcript: NM_000158) inherited from the father and a heterozygous deletion mutation in partial exons 3-7 (chr3: 81691916-81720106, genome version: hg19, transcript: NM_000158:c.314_991del) inherited from the mother (Figure 3), which were compatible with recessive inheritance. Both parents are heterozygous carriers. Cosegregation between the phenotype and genotype in the proband and her family members was revealed.

FIGURE 3
Genetic pedigree map and a GBE1 gene test result. Note: Ⅰ 1, father; Ⅰ 2, mother; Ⅱ 1, older sister 1; Ⅱ 2, older sister 2; Ⅱ 3, proband; and Ⅱ 4, younger brother; NP normal population. (A) Genetic pedigree map. The genotype of the proband's two older sisters is unknown. (B) qPCR peak figure for the partial exons of the GBE1 gene. The relative quantitation values of exons 3-7 (chr3:81691916-81720106) in the GBE1 gene of the proband, mother, and younger brother are about 1/2 of the normal control value, suggesting a heterozygous deletion mutation. (C) Sanger sequencing. Arrows point to the missense mutation. 271T>A (p.W91R) in the GBE1 gene carried by the proband, her father, and her younger brother. The mother has a wild-type genetic structure. (D) Homology alignment for the amino acid sequences corresponding to the GBE1 missense mutation across different species.
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The exon 3-7 deletion in the GBE1 gene is not included in the human databases DGV (http://dgv.tcag.ca/dgv/app/home) and gnomAD-SV. Moreover, deletions in a smaller range are recorded in the HGMD database, including the pathogenic exon 2-7 deletion mutation (Bruno et al., 2004) and the pathogenic exon 7 deletion mutation (Li et al., 2012). The exon 3-7 deletion mutation results in a frameshift mutation, leading to a premature termination codon and the production of a truncated protein.

Analysis of the spatial structure of variant proteins
Homology modeling of GBE1 was performed using AlphaFold 2 (https://alphafold.ebi.ac.uk), and the 3D structure of the protein was visualized using PyMOL 2.3 software ( Figure 4A). The tryptophan (Trp) residue at position 91 of GBE1 is located in the ligand-binding region and is highly conserved in the β-sheet secondary structure. The missense mutation c.271T>A changed Trp at position 91 to arginine (Arg), leading to local change from hydrophobic amino acids with aromatic side chains to strongly basic amino acids with positively charged side chains. As a consequence, the interactions between amino acid side chains of polypeptides and the electrostatic effects were altered, π-π interactions with P93 were lost, the hydrophobic interactions with amino acids E333, I334, Y310, C309, and P93 were lost, the locally hydrophilic property was enhanced, and the backbone was changed, leading to alteration in the spatial conformation of the protein ( Figure 4B).
GBE1 is a glycogen-branching enzyme containing 702 amino acids. Exon 3-7 deletion in the GBE1 gene brought about deletion of the amino acid at position 105-331. After transcriptional splicing between a nucleotide at the end of exon 2 (the first nucleotide of p.105) and a nucleotide at the front of exon 8 (the last nucleotide of p.331), frameshift mutation presented in the amino acids from exon 8, and a shift of four amino acids resulted in a premature termination codon UAA, production of a truncated protein, and residual amino acid sequences at position 1-107. Eventually, the catalytic domain at amino acid position 194-597 of GBE1 was lost ( Figure 4B).

Discussion
GSD IV involves multiple systems, based on which it can be classified into hepatic and neuromuscular subtypes with significant clinical heterogeneity (Magoulas et al., 1993;Li et al., 2010). Pediatric cases are mainly hepatic subtypes. Its clinical manifestations include hepatomegaly, liver dysfunction, and progressive liver cirrhosis, potentially with neuromuscular lesions, cardiomyopathy, and growth retardation. Linear glycogen molecules can be metabolized, so often without hypoglycemia (Magoulas et al., 1993;Massese et al., 2022). The proband (at age 1) and her younger brother (at age 10 months) both manifested hepatosplenomegaly, dysfunction, and growth retardation at onset, followed by disease progression to liver cirrhosis with liver failure, consistent with the pathology of GSD IV. During the disease course, the proband presented rare intractable asymptomatic hypoglycemia which is easily neglectable. Since long-term hypoglycemia impacts the energy supply to the brain tissues and then impairs brain development and even causes sudden death, the blood glucose of patients with GSD IV should be dynamically monitored.
The diagnosis of GSD IV is established by the demonstration of reduced GBE1 activity in the liver, muscle, or skin fibroblasts and/or the identification of biallelic pathogenic variants in GBE1 (Magoulas et al., 1993;Ozen, 2007;Massese et al., 2022). Both the proband and her younger brother carried previously unreported compound heterozygous mutations in the GBE1 gene, including an exon 2 missense mutation (c.271T>A) and partial exon 3-7 deletion mutation. Homology modeling revealed that the missense mutation c.271T>A changed Trp at position 91 to Arg, leading to potential changes in the spatial conformation and property of the protein. Additionally, it may also affect the catalytic domain stability due to loss of hydrophobic interactions with amino acids E333, I334, Y310, and C309, eventually interfering with enzyme catalytic activity. The exon 3-7 deletion mutation in the GBE1 gene led to loss of the enzyme catalytic domain and then decline in catalytic activity.
Symptomatic treatment is the mainstay for treatment of GSD IV now, and there is no available enzyme replacement therapy (Massese et al., 2022). A strict dietary regimen includes a high protein diet and carbohydrate restriction. This can maintain glucose and lipid homeostasis, to minimize glycogen accumulation and catabolism; is fundamental to prevent hypoglycemia in ketotic GSD IV; and can even improve growth and normalize serum aminotransferases (Derks et al., 2021). Most of the patients with a progressive GSD IV would die from liver failure or other complications of liver cirrhosis within 5 years of age, and some may deteriorate to liver cancer that can only be cured by liver transplantation. However, liver transplantation cannot alleviate the glycogen accumulation in other organs and tissues in general (Magoulas et al., 1993;Beyzaei et al., 2022;Massese et al., 2022). Therefore, the extent of organ involvement (neuromuscle, heart, etc.) is the primary prognostic factor for liver transplantation, especially cardiomyopathy (Willot et al., 2010;Liu and Sun, 2021;Beyzaei et al., 2022). It should be noted that a previous report demonstrated improvement with significant reduction in abnormal amylopectin in extrahepatic organs in patients receiving liver transplantation (Ozen, 2007). The two subtypes of GSD IV, including the hepatic and neuromuscular subtypes, can occur successively (Derks et al., 2021). Previous literature reported a case where the patient presented with hepatic GSD IV at the onset at the age of 2 years and then had neuromuscular involvement at the age of 45 years (Paradas et al., 2014). Although no extrahepatic organ involvement has been observed in the two cases here so far, the existing literature reported a case of neuromuscular involvement caused by deletion in a range smaller than exon 3-7 (Bruno et al., 2004;Li et al., 2012). A follow-up is required to detect extrahepatic involvement as they grow up, including electrocardiogram, echocardiography, neurologic assessment, and nutritional assessment.
In conclusion, the clinical phenotype and genotype of GSD IV are highly heterogeneous. Most patients have poor prognosis, and patients with suspected GSD IV should be aggressively managed by liver biopsy and test for GBE activity or GBE1 gene mutation to obtain a definite diagnosis.

Data availability statement
The datasets for this article are not publicly available due to concerns regarding participant/patient anonymity. Requests to Frontiers in Genetics frontiersin.org access the datasets should be directed to the corresponding author.

Ethics statement
The studies involving human participants were reviewed and approved by the Ethical committee of Shunde Women and Children's Hospital of Guangdong Medical University (Maternity and Child Healthcare Hospital of Shunde Foshan). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the individual(s) and minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.