Protein causes hyperinsulinemia: a Chinese patient with hyperinsulinism/hyperammonaemia syndrome due to a glutamate dehydrogenase gene mutation

Glucose is derived from three sources: intestinal absorption, glycogenolysis, and gluconeogenesis. Hypoglycemia in child is often attributed to depletion of glycogen stores. However, recently, congenital hyperinsulinism becomes an important cause of hypoglycaemia in early infancy. Mutations in the genes encoding SUR1 and KIR6.2 are the most frequent genetic causes of hyperinsulinism followed by mutations in the glutamate dehydrogenase (GDH) gene which encodes hyperinsulinism/hyperammonaemia (HI/HA) syndrome.1 HI/HA syndrome was first reported by Zammarchi et al2 in 1996. In 1998, Stanley et al3 found that the mutations in the GDH gene are the reason of HI/HA syndrome. Here we describe a boy with a severe form of HI/HA syndrome with a GDH gene mutation, which had never been reported in China before, and evaluated the efficacy of oral carbohydrate- protein-adipose tolerance test in the diagnosis. We also designed a test to choose a proper ratio between carbohydrate and protein in its treatment, whereas six-month follow-up showed that dietary therapy could be a useful treatment. METHODS Patient A Chinese patient was a boy aged 8 years and 7 months. At the age of 1 year, he was fed with milk and had an attack of crying and loss of consciousness. Blood glucose was subnormal and intravenous glucose infusion resulted in a rapid improvement. At the age of 3–4 years, he had the former manifestations almost once a day. He suffered from seizure several times and was diagnosed as epilepsy, but the treatment for epilepsy was useless. When he was 6 years old, the attacks became decreasing frequent because of drinking glucose water every day. He felt confusion, fatigue and weakness after ingestion of egg, salmon and pizza with thick cheese and he did not develop neuroglycopenic symptoms with fasting or exercise. He denied headaches, visual problems, cough, abdominal pain or any known medical problems. The family history indicated his parents and his younger sister were not be related. He had never taken any oral antidiabetic drugs. His weight was 25 kg (10th centile), his height was 123 cm (25th centile) with neither hepatomegaly nor splenomegaly observed. Results of an initial laboratory workup included blood routine test, hepatic and renal function were normal. The levels of pituitary hormones, cortisol, thyroid hormones were unremarkable. Enhancement computed tomography (CT) scan of the pancreas, insulin antibodies and insulin receptor antibodies showed no abnormality. Analysis of urinary organic acids demonstrated normal. Molecular genetic analysis Genomic DNA was extracted from a whole blood sample. The exon of the GDH gene was amplified by polymerase chain reaction (PCR) with 13 sets of primers. The PCR primer sequences and conditions were reported by Santer et al.4 Exon 7 is: forward, 5′-CCTACACTCTATGA- GATATTTACAAC-3′ and reverse, 5′-GGTATTGACCC- AAAGGAACTG-3′. The temperature (°C) was 55°;C. Oral carbohydrate-protein-adipose tolerance test Clinical laboratory tests revealed hypoglycemia (blood glucose: 2.3 mmol/L), hyperinsulinism (plasma insulin level 43.72 μU/ml) and hyperammonaemia (NH3 114.1 μmol/L) when he had an episode. The ratio of insulin/glucose was high (1.06; normal <0.4). His daily dietary menu showed a piece of belt fish and five meat wontons could cause hypoglycemia attack. So, we designed an oral carbohydrate-protein-adipose tolerance test for him. The heat quantity and composition of the test meal were based on the The List of Chinese Food Composition 2002 published by the Peking University Medical Press. The test meal for oral protein tolerance test was egg 50 g, cheese 10 g and spiced beef 50 g which contained 90 mg/kg leucine and 979 kJ (234 kcal). On the contrary, the oral carbohydrate and adipose tolerance tests were conducted using steamed bread 100 g and butter 26 g which both contained 979 kJ (234 kcal) as the test meal, respectively. Oral protein dose choice test We also designed a test to choose a proper ratio between carbohydrate and protein to avoid hypoglycemia. The test meal was strawberry yoghurt 250 ml and spiced beef 10 g which contained 35.0 mg/kg leucine and 803.7 kJ (192.1 kcal) (Type 1); strawberry yoghurt 125 ml and spiced beef 5 g (17.5 mg/kg leucine and 402.1 kJ (96.1 kcal)) (Type 2); strawberry yoghurt 125 ml, spiced beef 5 g and steamed bread 50 g (17.5 mg/kg leucine and 889.5 kJ (212.6 kcal)) (Type 3). RESULTS The carbohydrate and adipose test meal showed that the blood glucose remained in 3.4–5.3 mmol/L even after 3 hours, and the boy felt no complaints. However, the oral protein tolerance test ceased when the boy felt unconsciousness after 75 minutes. As shown in Table 1, a high insulin level (44.9 μU/ml) and a low blood glucose level (1.8 mmol/L) were observed in combination with hyperammonaemia. Table 2 shows the results of oral protein dose choice test, revealing that the combination of carbohydrate and protein with lower level of leucine could prevent episode of hypoglycemia. And the proper ratio of the heat quantity between protein and carbohydrate might be about 1:1 (402 kJ (96 kcal): 490 kJ (117 kcal)). Direct DNA sequence analysis revealed the heterozygous mutation c.978G>A(R269H) in exon 7 of the GDH gene compared with the normal gene as follows (Figure). And this genetic mutation which had been reported by Santer et al4 in 2001 can lead to arginine replaced by histidine and activated GDH. During six-month follow-up, the boy was treated with the food of the combination of protein and carbohydrate, and the ratio of heat quantity between protein and carbohydrate was 1:1. His blood glucose level was almost higher than 4 mmol/L, and he has suffered from hypoglycemia only once in six months.Table 1: Oral protein tolerance testTable 2: Oral protein dose choice test (blood glucose, mmol/L)DISCUSSION This patient was diagnosed as hypoglycemia due to the symptoms and the blood glucose level. The blood glucose level and the plasma insulin level showed hypoglycemia resulted from hyperinsulinemia. It showed no correlation to insulinoma and autoimmune hypoglycemia in that CT scan of pancreas and insulin autoantibody were negative. Further food analysis revealed most episodes of hypoglycemia resulted from ingestion of protein food, and the oral protein tolerance test was positive. The blood ammonia level was also persistently high. These tests clinically showed HI/HA syndrome. And the diagnosis was confirmed by the further direct DNA sequence analysis which showed the mutation c.978G>A(R269H) in exon 7 of the GDH gene.Figure.: The molecular genetic analysis of the patient shows the heterozygous mutation c.978G >A(R269H) in exon 7 of GDH gene compared with the normal.HI/HA syndrome is a recently identified inborn error of metabolism. Affected child suffers hypoglycemia with moderate hyperammonemia. The mechanism of hyperinsulinism and hyperammonemia is an activating mutation of GDH, resulting in excessive intracellular α-ketoglutarate and blood ammonia levels, leading to inappropriate deamination of glutamate. GDH can also make N-acetylglutamate down-regulation by reducing ammonia detoxification in the urea cycle. Firstly, in pancreatic β cells, the levels of ATP elevated for the high intracellular α-ketoglutarate, and then, the closure of the K+-ATP channels mediated by ATP leads to hyperinsulinemia and hypoglycemia. Secondly, the decrease in intracellular N-acetylglutamate leads to inactivity of carbamyl phosphate synthetase which can cause an overproduction of ammonia. Leucine acts as an activator of GDH.4,5 So, HI/HA syndrome typically is observed in child and the patients often have the episode after ingestion of protein especially comprising a great quantity of leucine. It can be explained by the former theory that this patient often has the episode after eating eggs, salmon and pizza. Hsu et al6 reported that the postprandial blood glucose response to a protein meal is more sensitive than prolonged fasting for detecting hypoglycemia in the HI/HA syndrome. Toriumi et al7 performed an oral protein tolerance test to diagnose HI/HA syndrome. We designed an oral carbohydrate-protein-adipose tolerance test to detect the reason of hypoglycemia. It showed the difference between ingestion of carbohydrate, protein and adipose and revealed the protein, especially leucine, led to hypoglycemia induced by hyperinsulinism. Bahi-Buisson et al8 reported seizures were commonly observed in HI/HA patients, and a mutation in the guanosine triphosphate binding site (exon 6, exon 7) was highly related to the seizure. The genetic mutation of this boy was also in exon 7, so it might be the reason that he had been misdiagnosed as seizure. The food analysis revealed the combination of protein and carbohydrate could prevent episode of hypoglycemia. And we designed a test to show that even very low level of leucine could make the boy have the episode of hypoglycemia, whereas ingestion of the combination of protein and carbohydrate with a proper ratio could avoid this manifestation. The goal of dietary therapy for child with HI/HA syndrome is to maintain blood glucose concentrations in a safe range. So, we choose the dietary therapy for the boy and six-month follow-up showed a satisfactory result, too. Recently a new chemical compound, which was potent inhibitors of GDH, was reported and it may be a better drug for HI/HA patients.9