Hyperinsulinism and hyperammonemia syndrome: report of twelve unrelated patients

Hyperinsulinism and hyperammonemia syndrome has been reported as a cause of moderately severe hyperinsulinism with diffuse involvement of the pancreas. The disorder is caused by gain of function mutations in the GLUD1 gene, resulting in a decreased inhibitory effect of guanosine triphosphate on the glutamate dehydrogenase (GDH) enzyme. Twelve unrelated patients (six males, six females) with hyperinsulinism and hyperammonemia syndrome have been investigated. The phenotypes were clinically heterogeneous, with neonatal and infancy-onset hypoglycemia and variable responsiveness to medical (diazoxide) and dietary (leucine-restricted diet) treatment. Hyperammonemia (90-200 micromol/L, normal <50 micromol/L) was constant and not influenced by oral protein, by protein- and leucine-restricted diet, or by sodium benzoate or N-carbamylglutamate administration. The patients had mean basal GDH activity (18.3 +/- 0.9 nmol/min/mg protein) not different from controls (17.9 +/- 1.8 nmol/min/mg protein) in cultured lymphoblasts. The sensitivity of GDH activity to inhibition by guanosine triphosphate was reduced in all patient lymphoblast cultures (IC(50), or concentrations required for 50% inhibition of GDH activity, ranging from 140 to 580 nM, compared with control IC(50) value of 83 +/- 1.0 nmol/L). The allosteric effect of ADP was within the normal range. The activating effect of leucine on GDH activity varied among the patients, with a significant decrease of sensitivity that was correlated with the negative clinical response to a leucine-restricted diet in plasma glucose levels in four patients. Molecular studies were performed in 11 patients. Heterozygous mutations were localized in the antenna region (four patients in exon 11, two patients in exon 12) as well as in the guanosine triphosphate binding site (two patients in exon 6, two patients in exon 7) of the GLUD1 gene. No mutation has been found in one patient after sequencing the exons 5-13 of the gene.     

Hyperinsulinism in infancy--genetic aspects

Hyperinsulinism in infancy (HI) is a heterogeneous disorder with respect to clinical presentation, genetics, histology and response to therapy. Advances in the understanding of the molecular basis of the disease have given the pediatric endocrinologists a better insight into the diagnosis and therapeutic choice. In 50-60% of cases, a genetic etiology is unraveled. Mutations in the genes encoding SUR1 (ABCC8) and KIR6.2 (KCNJ11) are the most frequent genetic causes of HI followed by mutations in the GLUD1 gene which encodes glutamate dehydrogenase (GDH) enzyme. The patients with GLUD1 mutations also have hyperammonemia (HA). Activating dominant mutations in glucokinase (GCK) gene which result in HI are rare. In GLUD1 and GCK mutations the disease is usually mild, has a late onset and is responsive to diazoxide. However, studies so far have failed to show a clear genotype phenotype relation in KATP channel mutations. In conclusion the genetic analysis of HI has provided valuable information to the clinicians about the beta cell.     

Novel compound heterozygous ATP6V0A4 mutations in an infant with distal renal tubular acidosis

A Japanese infant presenting with vomiting, failure to thrive, metabolic acidosis, and hyperammonemia was finally diagnosed with autosomal recessive distal renal tubular acidosis (dRTA). Hyperchloremic metabolic acidosis, hypokalemia, a normal serum anion gap, a positive urine anion gap, nephrocalcinosis, and high urine pH despite systemic acidemia were consistent with the cardinal manifestations in dRTA. Mutational analysis of the ATP6V0A4 gene revealed novel compound heterozygous mutations: Ile549fsX580 and Ile557Leu558del. The father was found to be heterozygote for the former mutation, the mother heterozygote for the latter. This is the first case of dRTA with hyperammonemia in which the ATP6V0A4 mutations were identified. dRTA should be considered in the differential diagnosis of children presenting with hyperammonemia. Additionally, in a possible case of autosomal recessive dRTA with normal hearing, mutational analysis of ATP6V0A4 gene may be recommended first to confirm the diagnosis.     

Unregulated insulin secretion by pancreatic beta cells in hyperinsulinism/hyperammonemia syndrome: role of glutamate dehydrogenase, ATP-sensitive potassium channel, and nonselective cation channel

The hyperinsulinism/hyperammonemia (HI/HA) syndrome is caused by "gain of function" of glutamate dehydrogenase (GDH). Several missense mutations have been found; however, cell behaviors triggered by the excessive GDH activity have not been fully demonstrated. This study was aimed to clarify electrophysiological mechanisms underlying the dysregulated insulin secretion in pancreatic beta cells with GDH mutations. GDH kinetics and insulin secretion were measured in MIN6 cells overexpressing the G446D and L413V. Membrane potentials and channel activity were recorded under the perforated-patch configuration that preserved intracellular environments. In mutant MIN6 cells, sensitivity of GDH to guanosine triphosphate (GTP) was reduced and insulin secretion at low glucose concentrations was enhanced. The basal GDH activity was elevated in L413V bearing a mutation in the antenna-like structure. The L413V cells were depolarized without glucose, often accompanying by repetitive Ca2+ firings. The depolarization was maintained in the presence of adenosine triphosphate (ATP) and disappeared by depleting ATP, suggesting that the depolarization depended on intracellular ATP. In L413V cells, the ATP-sensitive potassium channel (K(ATP) channel) was suppressed and the nonselective cation channel (NSCC) was potentiated, while sensitivity of the channels to their specific blockers or agonists was not impaired. These data suggest that the L413V cells increase the intracellular ATP/adenosine diphosphate (ADP) ratio, which in turn causes sustained depolarization not only by closure of the K(ATP) channel, but also by opening of the NSCC. The resultant activation of the voltage-gated Ca2+ channel appears to induce hyperinsulinism. The present study provides evidence that multiple channels cooperate in unregulated insulin secretion in pancreatic beta cells of the HI/HA syndrome.     

Two Novel Missense Mutations and a 5bp Deletion in the Erythroid‐Specific Promoter of the PKLR Gene in Two Unrelated Patients With Pyruvate Kinase Deficient Transfusion‐Dependent Chronic Nonspherocytic Hemolytic Anemia

We report two children with severe chronic hemolytic anemia, the cause of which was difficult to establish because of transfusion dependency. Reduced erythrocyte pyruvate kinase activity in their asymptomatic parents provided the diagnostic clues for mutation screening of the PKLR gene and revealed that one child was a compound heterozygote of a novel paternally derived 5‐bp deletion in the promoter region (c.‐88_‐84delTCTCT) and a maternally derived missense mutation in exon nine (c.1174G>A; p.Ala392Thr). The second child was a compound heterozygote of two novel missense mutations, namely a paternally derived exon ten c.1381G>A (p.Glu461Lys) and a maternally derived exon seven c.907‐908delCC (p.Pro303GlyfsX12) variant.     

Biochemical evaluation of an infant with hypoglycemia resulting from a novel de novo mutation of the GLUD1 gene and hyperinsulinism-hyperammonemia syndrome

Hyperinsulinism-hyperammonemia syndrome (HI/HA) (OMIM 606762), the second most common form of congenital hyperinsulinism (CHI) is associated with activating missense mutations in the GLUD1 gene, which encodes the mitochondrial matrix enzyme, glutamate dehydrogenase (GDH). Patients present with recurrent symptomatic postprandial hypoglycemia following protein-rich meals (leucine-sensitive hypoglycemia) as well as fasting hypoglycemia accompanied by asymptomatic elevations of plasma ammonia. In contrast to other forms of CHI, the phenotype is reported to be milder thus escaping recognition for the first few months of life. Early diagnosis and appropriate management are essential to avoid the neurodevelopmental consequences including epilepsy and learning disabilities which are prevalent in this disorder. We report an infant presenting with afebrile seizures secondary to hyperinsulinemic hypoglycemia resulting from a novel de novo mutation of the GLUD1 gene.     

Diagnostic screening of MODY2/GCK mutations in the Norwegian MODY Registry

Background: Maturity‐onset diabetes of the young, type 2 (MODY2) is caused by mutations in the glucokinase gene (GCK). The aim of our study was to determine the prevalence of GCK mutations in the Norwegian MODY Registry and to delineate the clinical phenotype of identified GCK mutation carriers. Methods: We screened 122 probands referred to the MODY Registry for mutations in GCK and studied extended families with MODY2. Results: We found 2 novel (S76Y and N231S) and 13 previously reported (V62A, G72R, L146R, R191W, A208T, M210K, Y215X, M235T, R275C, E339G, R377C, S453L, and IVS5+1G>C) GCK mutations in 23 probands and in their 33 family members. The prevalence of MODY2 was 12% in the Norwegian MODY Registry. The subjects with GCK mutations had features of mild diabetes. Yet, 15 of 56 MODY2 subjects were treated with oral drugs or insulin. Three subjects had retinopathy and one had macrovascular disease. Also, a limited number of cases had elevated fasting serum triglyceride values. Moreover, two GCK mutation carriers were diagnosed with type 1 diabetes. Conclusions: According to our diagnostic screening of GCK in the MODY Registry, MODY2 is less prevalent than MODY3 in Norway but is likely to be underreported. Recognizing MODY2 in diabetic patients is important in order to prevent overtreatment. Finally, our study demonstrates the co‐occurrence of MODY2 in families with type 1 or type 2 diabetes.     

Convergent evolution of 11p allelic loss in multifocal Wilms tumors arising in WT1 mutation carriers

Wilms tumors in patients with constitutional WT1 mutations are examples of Knudson's tumor suppressor paradigm, with somatic inactivation of the second allele occurring through 11p loss of heterozygosity. The time point of this second hit has remained unknown. We analyzed seven Wilms tumors from two patients with constitutional WT1 mutations by whole exome sequencing and genomic array. All tumors exhibited wild type WT1 loss through uniparental isodisomy. Each tumor had a unique genomic breakpoint in 11p, typically accompanied by a private activating mutation of CTNNB1. Hence, convergent evolution rather than field carcinogenesis underlies multifocal tumors in WT1 mutation carriers.     

Glyburide ameliorates motor coordination and glucose homeostasis in a child with diabetes associated with the KCNJ11/S225T,del226‐232 mutation

Gain‐of‐function mutations of KCNJ11 can cause permanent neonatal diabetes mellitus, but only rarely after 6 months of age. Specific uncommon mutations KCNJ11give rise to a syndrome defined as developmental delay, epilepsy, and neonatal diabetes (DEND), or – more frequently – to a milder sub‐type lacking epilepsy, denoted as intermediate‐DEND (iDEND). Our aim was to consider a possible monogenic etiology in a 12‐yr‐old boy with early onset diabetes and mild neurological features. We studied a subject diagnosed with diabetes at 21 months of age, and negative to type 1 diabetes autoantibodies testing. He had learning difficulties during primary school, and a single episode of seizures at the age of 10 yr. We performed direct DNA sequencing of the KCNJ11 gene with subsequent functional study of mutated channels in COSm6 cells. The patient's clinical response to oral glyburide (Glyb) was assessed. Motor coordination was evaluated before and after 6 and 12 months of Glyb therapy. Sequencing of the KCNJ11 gene detected the novel, spontaneous mutation S225T, combined with deletion of amino acids 226–232. In vitro studies revealed that the mutation results in a K_ATP channel with reduced sensitivity to the inhibitory action of ATP. Glyb improved diabetes control (hemoglobin A1c on insulin: 52 mmol/mol/6.9%; on Glyb: 36 mmol/mol/5.4%) and also performance on motor coordination tests that were impaired before the switch of therapy. We conclude that KCNJ11/S225T, del226‐232 mutation caused a mild iDEND form in our patient. KCNJ11 should be considered as the etiology of diabetes even beyond the neonatal period if present in combination with negative autoantibody testing and even mild neurological symptoms.     

Glucokinase diabetes in 103 families from a country‐based study in the Czech Republic: geographically restricted distribution of two prevalent GCK mutations

Pruhova S, Dusatkova P, Sumnik Z, Kolouskova S, Pedersen O, Hansen T, Cinek O, Lebl J. Glucokinase diabetes in 103 families from a country‐based study in the Czech Republic: geographically restricted distribution of two prevalent GCK mutations. Background: Glucokinase diabetes, also called GCK‐MODY or maturity‐onset diabetes of the young type 2 (MODY2), is caused by heterozygous mutations in the gene encoding glucokinase (GCK). Objective: The aim of study was to investigate the current prevalence of GCK mutations in a large cohort of Czech patients with typical clinical appearance of GCK‐MODY. In addition, we reanalyzed the negative results obtained previously by screening using the denaturing high‐performance liquid chromatography (dHPLC). Methods: We studied 140 unrelated Czech probands with clinical picture of GCK‐MODY who were referred to our center from the whole of the Czech Republic between the years 1999–2009 by direct sequencing of GCK gene. Results: A mutation in GCK was identified in 103 of 140 probands (74%). We identified 46 different GCK mutations of which 13 were novel. Several mutations were detected in multiple families: p.Glu40Lys (20 families), p.Gly318Arg (12), p.Leu315His (7) and p.Val33Ala (six families). Direct sequencing detected a GCK mutations in 9 of 20 previously dHPLC‐negative samples; the sensitivity of the dHPLC screening was calculated as 84%. Conclusions: The study shows a relatively high proportion of GCK mutations among individuals with GCK‐like phenotype, confirming the effectiveness of carefully applied clinical criteria prior to genetic testing. In the Czech MODY registry, GCK‐MODY represents the biggest subgroup of MODY (35%). We report several prevalent GCK mutations with a likely founder effect in the Czech population. Furthermore, our results provide ground for a possible recommendation to reinspect all negative results previously obtained by screening using dHPLC.     

A CACNA1D mutation in a patient with persistent hyperinsulinaemic hypoglycaemia,heart defects,and severe hypotonia

Congenital hyperinsulinaemic hypoglycaemia (HH) can occur in isolation or it may present as part of a wider syndrome. For approximately 40%‐50% of individuals with this condition, sequence analysis of the known HH genes identifies a causative mutation. Identifying the underlying genetic aetiology in the remaining cases is important as a genetic diagnosis will inform on recurrence risk, may guide medical management and will provide valuable insights into β‐cell physiology. We sequenced the exome of a child with persistent diazoxide‐responsive HH, mild aortic insufficiency, severe hypotonia, and developmental delay as well as the unaffected parents. This analysis identified a de novo mutation, p.G403D, in the proband's CACNA1D gene. CACNA1D encodes the main L‐type voltage‐gated calcium channel in the pancreatic β‐cell, a key component of the insulin secretion pathway. The p.G403D mutation had been reported previously as an activating mutation in an individual with primary hyper‐aldosteronism, neuromuscular abnormalities, and transient hypoglycaemia. Sequence analysis of the CACNA1D gene in 60 further cases with HH did not identify a pathogenic mutation. Identification of an activating CACNA1D mutation in a second patient with congenital HH confirms the aetiological role of CACNA1D mutations in this disorder. A genetic diagnosis is important as treatment with a calcium channel blocker may be an option for the medical management of this patient.     

Clinical resistance associated with a novel MAP2K1 mutation in a patient with Langerhans cell histiocytosis

Patients with Langerhans cell histiocytosis (LCH) harbor BRAF V600E and activating mutations of MAP2K1/MEK1 in 50% and 25% of cases, respectively. We evaluated a patient with treatment‐refractory LCH for mutations in the RAS‐RAF‐MEK‐ERK pathway and identified a novel mutation in the MAP2K1 gene resulting in a p.L98_K104 > Q deletion and predicted to be auto‐activating. During treatment with the MEK inhibitor trametinib, the patient's disease showed significant progression. In vitro characterization of the MAP2K1 p.L98_K104 > Q deletion confirmed its effect on cellular activation of the ERK pathway and drug resistance.     

Microcephaly,epilepsy, and neonatal diabetes due to compound heterozygous mutations in IER3IP1: insights into the natural history of a rare disorder

Neonatal diabetes mellitus is known to have over 20 different monogenic causes. A syndrome of permanent neonatal diabetes along with primary microcephaly with simplified gyral pattern associated with severe infantile epileptic encephalopathy was recently described in two independent reports in which disease‐causing homozygous mutations were identified in the immediate early response‐3 interacting protein‐1 (IER3IP1) gene. We report here an affected male born to a non‐consanguineous couple who was noted to have insulin‐requiring permanent neonatal diabetes, microcephaly, and generalized seizures. He was also found to have cortical blindness, severe developmental delay and numerous dysmorphic features. He experienced a slow improvement but not abrogation of seizure frequency and severity on numerous anti‐epileptic agents. His clinical course was further complicated by recurrent respiratory tract infections and he died at 8 years of age. Whole exome sequencing was performed on DNA from the proband and parents. He was found to be a compound heterozygote with two different mutations in IER3IP1: p.Val21Gly (V21G) and a novel frameshift mutation p.Phe27fsSer*25. IER3IP1 is a highly conserved protein with marked expression in the cerebral cortex and in beta cells. This is the first reported case of compound heterozygous mutations within IER3IP1 resulting in neonatal diabetes. The triad of microcephaly, generalized seizures, and permanent neonatal diabetes should prompt screening for mutations in IER3IP1. As mutations in genes such as NEUROD1 and PTF1A could cause a similar phenotype, next‐generation sequencing approaches—such as exome sequencing reported here—may be an efficient means of uncovering a diagnosis in future cases.     

高胰岛素血症高氨血症综合征诊治研究进展

高胰岛素血症-高氨血症综合征（hyperinsulinism-hyperammonemia syndrome，HI/HA syndrome）是先天性高胰岛素血症（HI）中第二大常见亚型。患有该综合征的儿童有空腹和高蛋白质饮食诱发的低血糖，并伴有持续高氨血症。编码谷氨酸脱氢酶的基因GLUD1致病变异是HI/HA综合征的病因。谷氨酸脱氢酶在肝、肾、脑和胰腺细胞中表达。即使在血糖正常的情况下，HI/HA综合征患者出现神经系统损害较常见。服用ATP敏感的钾离子通道开放剂二氮嗪可以很好地控制HI/HA综合征患者的低血糖，但高氨血症无法改善。     

Truncation and microdeletion of EVC/EVC2 with missense mutation of EFCAB7 in Ellis‐van Creveld syndrome

Ellis‐van Creveld syndrome (EvC) is a ciliopathy with cardiac anomalies, disproportionate short stature, polydactyly, dystrophic nails and oral defects. To obtain further insight into the genetics of EvC, we screened EVC/EVC2 mutations in eight Vietnamese EvC patients. All the patients had a congenital heart defect with atypical oral and/or skeletal abnormalities. One had compound heterozygous EVC2 mutations: a novel mutation c.769G > T‐p.E177X in exon 6 inherited from father and another previously reported c.2476C > T‐p.R826X mutation in exon 14 inherited from mother. The EVC2 mRNA expression level was significantly lower in the patient and her parents compared to controls. Another case had a novel heterozygous EVC mutation (c.1717C > G‐p.S572X) in exon 12, inherited from his father. Of note, the mother without any EVC mutation on Sanger sequencing showed a lower expression level of EVC mRNA compared with controls. SNP array analysis revealed that the patient and mother had a heterozygous 16.4 kb deletion in EVC. This patient also had a heterozygous novel variant in exon 9 of EFCAB7 (c.1171 T > C‐p.Y391H), inherited from his father. The atypical cardiac phenotype of this patient and the father suggested that EFCAB7 may modify the phenotype by interacting with EVC. In conclusion, we detected two novel nonsense mutations and a partial deletion of EVC/EVC2 in two Vietnamese families with EvC. Moreover, we found in one family a missense mutation of EFCAB7, a possible modifier gene in EvC and its related disorders.     

Germ-line mutation of KCNQ2, p.R213W, in a Japanese family with benign familial neonatal convulsion

Background: Benign familial neonatal convulsion (BFNC) is an autosomal‐dominantly inherited epilepsy of neonates. The KCNQ2 and KCNQ3 genes have been cloned as the responsible genes for BFNC. Detection of mutations in these genes is helpful for confirmation of BFNC or differential diagnosis of convulsive disorders in the neonatal period. Methods: A Japanese family with BFNC was investigated. Two siblings were clinically diagnosed as having BFNC. KCNQ2 and KCNQ3 were screened for mutations using a combination of polymerase chain reaction and denaturing high‐performance liquid chromatography. Nucleotide substitutions were confirmed by direct sequencing. Results: In the affected siblings a C‐to‐T heterozygous substitution was detected at nucleotide 683 (c.683C>T) in KCNQ2, leading to substitution of arginine with tryptophan at amino acid position 213 (p.R213W) in the S4 voltage‐sensing domain of the KCNQ2 protein. The detected mutation may disrupt this highly conserved region among potassium channel proteins. The c.683C>T substitution in KCNQ2 was not present in the parents. KCNQ3 was also analyzed and a single nucleotide polymorphism, c.1241A>G (National Center for Biotechnology Information (NCBI), SNP ID: rs2303995), was detected in the index family. Conclusions: Two siblings with BFNC had a novel heterozygous missense mutation, p.R213W, in KCNQ2. This mutation may affect potassium gating, leading to neuronal excitability or convulsions in the patients. Furthermore, neither of the parents had the p.R213W mutation, indicating that it was a germ‐line mutation. The possibility of recurrence of such a germ‐line mutation in the next siblings should be explained during genetic counseling.     

Synonymous mutation in TP53 results in a cryptic splice site affecting its DNA‐binding site in an adolescent with two primary sarcomas

Pathologic variants in TP53 are known risk factors for the development of cancer. We report a 17‐year‐old male who presented with two primary sarcomas. Germline sequencing revealed a novel TP53 c.672 G>A mutation. Sequencing revealed wild‐type TP53 in the parents, and there was no history of cancer in first‐degree relatives. This de novo synonymous germline mutation results in a 5′ cryptic splice site that is bound by U1, resulting in a shift of the splice site by 5 base pairs. The frame shift results in a truncated protein at residue 246, which disrupts the DNA‐binding domain of p53.     

Congenital nephrotic syndrome with a novel NPHS1 mutation

Congenital nephrotic syndrome of the Finnish type (CNF) is a rare autosomal recessive disorder. The incidence of CNF is relatively high in Finland but considerably lower in other countries. We encountered a male newborn with CNF, associated with compound heterozygous mutations in nephrosis 1, congenital, Finnish type (NPHS1). The patient was admitted to hospital as a preterm infant. Physical and laboratory findings fulfilled the diagnostic criteria of nephrotic syndrome, and were compatible with a diagnosis of CNF, but there was no family history of the disease. On genetic analysis of NPHS1 a paternally derived heterozygous frame‐shift mutation caused by an 8 bp deletion, resulting in a stop codon in exon 16 (c.2156‐2163 delTGCACTGC causing p.L719DfsX4), and a novel, maternally derived nonsense mutation in exon 15 (c.1978G>T causing p.E660X) were identified. Early genetic diagnosis of CNF is important for proper clinical management and appropriate genetic counseling.