Genetic analysis and clinical assessment of four patients with Glycogen Storage Disease Type IIIa in China

Glycogen Storage Disease Type III (GSD III) is a rare autosomal recessive metabolic disorder. There are two major clinical types of GSD III. GSD IIIa involves liver and muscle while GSD IIIb only affects the liver. GSD III commonly presents with growth retardation, fasting hypoglycemia, hepatomegaly, and seizures during childhood. The neuromuscular manifestations of GSD IIIa are mainly reported in adults [1‐3]. GSD III is caused by AGL gene mutation leading a deficiency of glycogen debrancher enzyme activity. The human AGL gene is located on chromosome 1p21 and consists of 35 exons spanning ~ 85 kb of genomic DNA. AGL gene is expressed from the third exon [4]. Genetic analysis of the AGL gene in several ethnic populations has revealed over 150 different AGL gene mutations [5]. However, few AGL mutations have been reported in the Chinese population in mainland China. In this study, we reported four Chinese patients with GSD IIIa and analyzed the relationship between genotype and phenotype of GSD IIIa in the Chinese population.

Our study included three male and one female GSD IIIa Chinese patients from three families. Patients 3 and 4 were siblings from one family. When these patients were admitted to our hospital, their clinical information, such as age, gender, height, weight, liver function and clinical symptoms were collected. Then, routine laboratory test, echocardiography, electromyogram, abdominal ultrasound, muscle MRI and neuropsychological test were conducted. All of this clinical information of the first visit and two years follow-up were shown in Table 1. The guideline for the diagnosis of GSD III had been followed [5].

Our patients mainly complained physical weakness accompanied by elevated creatine kinase level. Although patients had not received high-protein and uncooked cornstarch (UCS) diet therapy in childhood, growth retardation was not observed after adolescence. Treatment with high-protein and UCS diet was implemented in all patients after the first visit, according to preprandial blood glucose and the level of abnormal alanine aminotransferase (ALT) and aspartate aminotransferase (AST). After high-protein and UCS diet therapy, clinical manifestations of weakness of our patients were significantly relieved at the last follow-up. Currently, their therapy followed standard treatment guidelines, with UCS in 1.5 g/kg/time for 1–3 times per day and protein in 2 g/kg/d.

The results of laboratory tests showed the level of creatine kinase (CK) was significantly increased among all patients, but ketonic hypoglycemia was only detected in patient 1. Serum cholesterol and triglyceride were normal in patient 1 but they were elevated in other patients. After a high-protein and UCS diet therapy for two years, the level of CK was still 5–10 times over the upper limit of normal, but preprandial blood glucose, serum cholesterol and triglyceride of all patients had nearly normalized at the last follow-up.

All our patients showed asymptomatic cardiac hypertrophy accompanied by normal ejection fraction and cardiac conduction (Table 1). Meanwhile, hepatomegaly accompanied by abnormal ALT and AST was common during our patients’ childhood. After a high-protein and UCS diet therapy for two years, cardiac hypertrophy of our patients did not deteriorated. Moreover, levels of AST and ALT had decreased and hepatic adenoma was not detected in our patients. We didn’t observe hepatic cirrhosis or hepatocellular carcinoma in our patients by liver ultrasound or laboratory tests. Indeed, we did not perform the liver biopsy which was invasive and potentially detrimental to the patients.

To better identify the neurological manifestations of GSD IIIa in our patients, nerve conduction studies (NCS), electromyogram (EMG), muscle MRI, brain MRI, electroencephalogram and neuropsychological tests were performed. All patients showed normal results in NCS, brain MRI and electroencephalogram, but the results of EMG were abnormal. Myopathic motor unit potential was detected in all patients, but the specificity and sensitivity were not satisfactory. Notably, Muscle MRI transverse sections from the mid-thigh and lower leg muscles of patient 2 presented mild T1 signal intensity in the long head of femoris and peroneus longus muscles (arrows), indicating an increase in adipose-like tissue (Fig. 1).What’s more, lateral heads of gastrocnemius muscles showed atrophy in patient 2 (Fig. 1). In general, the posterior and lateral muscles of the lower limbs were mostly affected (Fig. 1).

GSD III is caused by mutations in the AGL gene. Over 150 different mutations in the AGL gene have been identified. However, few AGL mutations have been reported in mainland China [4, 5]. Genetic analysis of our four Chinese GSD IIIa patients revealed three different mutations in AGL gene. One novel frameshift insert mutation c.206dupA (N69Kfs*8) is located in exon4. It is likely be classified as a pathogenic mutation by following the ACMG/AMP sequence variant pathogenicity classification guideline [11]. As to the relationship between genotype and phenotype, we found the patient with AGL c.206dupA mutation showed severe clinical manifestations compared with the other patients. The patient carrying the AGL c.206dupA mutation still suffered severe hypoglycemia symptoms even in adolescence, which were absent in other patients. These manifestations indicated AGL c.206dupA mutation might cause more severe functional deficiency of the glycogen debranching enzyme than other mutations. Therefore, the patient with AGL c.206dupA mutation required a high-protein and UCS diet therapy even in adults. Then, AGL c.1735 + 1G > T mutation, which is first reported in Japan [12], is predicted to impair normal splicing. AGL c.1735 + 1G > T mutation might be a recurrent mutation in Chinese patients and should be given priority when gene detection is conducted in Chinese GSD IIIa patients. Moreover, AGL c.2590 C>T,which is first reported by Caucasians [13], is predicted to lead to premature termination, which completely abolishes the enzyme activity. In summary, the patient carrying AGL c.206dupA homozygous mutation suffered more severe disease, whereas patient 2 carrying the splicing homozygous mutation had milder disease. This observation is in keeping with the notion that some residual functional protein may be produced from the splicing mutation.

We have collected detailed clinical information of four Chinese GSD IIIa patients from three families. We revealed clinic characteristics of Chinese GSD IIIa: Firstly, all of our patients mainly complained of weakness when running and climbing stairs, and a high-protein and UCS diet therapy could improve the symptom of muscle weakness effectively. Secondly, our patients showed growth retardation in childhood, but they can catch up to normal level in adolescence. Mental or physical development disabilities did not appear in most of patients. Thirdly, asymptomatic hepatomegaly is common in our patients. Liver and cardiac dysfunction is not significant in all of our patients. Finally, our study showed muscle MRI combined with genetic testing might be reliable, convenient and less invasive compared with muscle biopsy [14, 15].

In conclusion, our study provides a comprehensive overview of genetic and clinical features of GSD IIIa in China. Our results revealed that muscle weakness accompanied with high level of CK are the main clinical manifestations in adolescence. Cardiomyopathy and hepatic adenoma needs long term follow-up. AGL c.206dupA mutation is a novel mutation leading severe clinical manifestations and AGL C.1735 + 1G > T might be a recurrent mutation in Chinese patients with GSD IIIa. Moreover, clinical history, muscle MRI and genetic testing may provide greater benefit to the patient in diagnosing GSD IIIa.