Novel dominant KATP channel mutations in infants with congenital hyperinsulinism: Validation by in vitro expression studies and in vivo carrier phenotyping.

Inactivating mutations in the genes encoding the two subunits of the pancreatic beta-cell KATP channel, ABCC8 and KCNJ11, are the most common finding in children with congenital hyperinsulinism (HI). Interpreting novel missense variants in these genes is problematic, because they can be either dominant or recessive mutations, benign polymorphisms, or diabetes mutations. This report describes six novel missense variants in ABCC8 and KCNJ11 that were identified in 11 probands with congenital HI. One of the three ABCC8 mutations (p.Ala1458Thr) and all three KCNJ11 mutations were associated with responsiveness to diazoxide. Sixteen family members carried the ABCC8 or KCNJ11 mutations; only two had hypoglycemia detected at birth and four others reported symptoms of hypoglycemia. Phenotype testing of seven adult mutation carriers revealed abnormal protein-induced hypoglycemia in all; fasting hypoketotic hypoglycemia was demonstrated in four of the seven. All of six mutations were confirmed to cause dominant pathogenic defects based on in vitro expression studies in COSm6 cells demonstrating normal trafficking, but reduced responses to MgADP and diazoxide. These results indicate a combination of in vitro and in vivo phenotype tests can be used to differentiate dominant from recessive KATP channel HI mutations and personalize management of children with congenital HI.

Clinical heterogeneity of hyperinsulinism due to HNF1A and HNF4A mutations.

BACKGROUND: Dominant inactivating mutations in HNF1A and HNF4A have been described to cause hyperinsulinism (HI) before evolving to diabetes. However, information available in the literature regarding the clinical phenotype is limited. OBJECTIVE: To report the prevalence of HNF1A and HNF4A mutations in a large cohort of children with HI, and to describe their genotypes and phenotypes. DESIGN: Retrospective descriptive study. METHODS: Medical records were reviewed to extract clinical information. Mutation analysis was carried out for 8 genes associated with HI (ABCC8, KCNJ11, GLUD1, GCK, HADH, HNF4A, HNF1A, and UCP2). RESULTS: HNF1A and HNF4A mutations were identified in 5.9% (12 out of 204; HNF1A = 7, HNF4A = 5) of diazoxide-responsive HI probands. The clinical phenotypes were extremely variable. Two children showed evidence of ketone production during hypoglycemia, a biochemical profile atypical for hyperinsulinism. At the time of analysis, diazoxide was discontinued in 5 children at a median age of 6.8 years. None had developed diabetes mellitus at a median age of 7.0 years. CONCLUSIONS: Given the heterogeneous clinical phenotypes of HNF1A- and HNF4A-HI, all children with transient, diazoxide-responsive HI without clear history of perinatal stress, should be screened for HNF1A and HNF4A mutations as it predicts the clinical course and affects the subsequent management plan.

Pharmacological Correction of Trafficking Defects in ATP-sensitive Potassium Channels Caused by Sulfonylurea Receptor 1 Mutations.

ATP-sensitive potassium (KATP) channels play a key role in mediating glucose-stimulated insulin secretion by coupling metabolic signals to ß-cell membrane potential. Loss of KATP channel function due to mutations in ABCC8 or KCNJ11, genes encoding the sulfonylurea receptor 1 (SUR1) or the inwardly rectifying potassium channel Kir6.2, respectively, results in congenital hyperinsulinism. Many SUR1 mutations prevent trafficking of channel proteins from the endoplasmic reticulum to the cell surface. Channel inhibitors, including sulfonylureas and carbamazepine, have been shown to correct channel trafficking defects. In the present study, we identified 13 novel SUR1 mutations that cause channel trafficking defects, the majority of which are amenable to pharmacological rescue by glibenclamide and carbamazepine. By contrast, none of the mutant channels were rescued by KATP channel openers. Cross-linking experiments showed that KATP channel inhibitors promoted interactions between the N terminus of Kir6.2 and SUR1, whereas channel openers did not, suggesting the inhibitors enhance intersubunit interactions to overcome channel biogenesis and trafficking defects. Functional studies of rescued mutant channels indicate that most mutants rescued to the cell surface exhibited WT-like sensitivity to ATP, MgADP, and diazoxide. In intact cells, recovery of channel function upon trafficking rescue by reversible sulfonylureas or carbamazepine was facilitated by the KATP channel opener diazoxide. Our study expands the list of KATP channel trafficking mutations whose function can be recovered by pharmacological ligands and provides further insight into the structural mechanism by which channel inhibitors correct channel biogenesis and trafficking defects.

A severe case of hyperinsulinism due to hemizygous activating mutation of glutamate dehydrogenase.

Activating mutations in the GLUD1 gene, which encodes glutamate dehydrogenase (GDH), result in the hyperinsulinism-hyperammonemia syndrome. GDH is an allosterically regulated enzyme responsible for amino acid-mediated insulin secretion via the oxidative deamination of glutamate to 2-oxoglutarate, leading to ATP production and insulin release. This study characterizes a novel combination of mutations in GLUD1 found in a neonate who presented on the first day of life with severe hypoglycemia, hyperammonemia, and seizures. Mutation analysis revealed a novel frameshift mutation (c.37delC) inherited from the asymptomatic mother that results in a truncated protein and a de novo activating mutation (p.S445L) close to the GTP binding site that has previously been reported. GTP inhibition of GDH enzyme activity in 293T cells expressing the p.S445L or wild-type GDH showed that the half-maximal inhibitory concentration (IC50 ) for GTP was approximately 800 times higher for p.S445L compared to wild type. GTP inhibition of GDH activity in lymphoblasts from the patient, from a heterozygote for the p.S445L mutation, and in wild-type lymphoblasts showed that the IC50 for GTP of the patient was approximately 200 times that of wild type and 7 times that of heterozygote. However, while the patient had a loss of GTP inhibition of GDH that was more severe than that of heterozygotes, the patient's clinical phenotype is similar to typical heterozygous mutations of GDH. This is the first time we have observed a functionally homozygous activating mutation of GDH in a human.

Congenital hyperinsulinism in children with paternal 11p uniparental isodisomy and Beckwith-Wiedemann syndrome.

BACKGROUND: Congenital hyperinsulinism (HI) can have monogenic or syndromic causes. Although HI has long been recognised to be common in children with Beckwith-Wiedemann syndrome (BWS), the underlying mechanism is not known. METHODS: We characterised the clinical features of children with both HI and BWS/11p overgrowth spectrum, evaluated the contribution of KATP channel mutations to the molecular pathogenesis of their HI and assessed molecular pathogenesis associated with features of BWS. RESULTS: We identified 28 children with HI and BWS/11p overgrowth from 1997 to 2014. Mosaic paternal uniparental isodisomy for chromosome 11p (pUPD11p) was noted in 26/28 cases. Most were refractory to diazoxide treatment and half required subtotal pancreatectomies. Patients displayed a wide range of clinical features from classical BWS to only mild hemihypertrophy (11p overgrowth spectrum). Four of the cases had a paternally transmitted KATP mutation and had a much more severe HI course than patients with pUPD11p alone. CONCLUSIONS: We found that patients with pUPD11p-associated HI have a persistent and severe HI phenotype compared with transient hypoglycaemia of BWS/11p overgrowth patients caused by other aetiologies. Testing for pUPD11p should be considered in all patients with persistent congenital HI, especially for those without an identified HI gene mutation.

Clinical and Molecular Characterization of Hyperinsulinism in Kabuki Syndrome.

Context: Kabuki syndrome (KS) is associated with congenital hyperinsulinism (HI). Objective: To characterize the clinical and molecular features of HI in children with KS. Design: Retrospective cohort study of children with KS and HI evaluated between 1998 and 2023. Setting: The Congenital Hyperinsulinism Center of the Children's Hospital of Philadelphia. Patients: Thirty-three children with KS and HI. Main Outcome Measures: HI presentation, treatment, course, and genotype. Results: Hypoglycemia was recognized on the first day of life in 25 children (76%). Median age at HI diagnosis was 1.8 months (interquartile range [IQR], 0.6-6.1 months). Median age at KS diagnosis was 5 months (IQR, 2-14 months). Diagnosis of HI preceded KS diagnosis in 20 children (61%). Twenty-four children (73%) had a pathogenic variant in KMT2D, 5 children (15%) had a pathogenic variant in KDM6A, and 4 children (12%) had a clinical diagnosis of KS. Diazoxide trial was conducted in 25 children, 92% of whom were responsive. HI treatment was discontinued in 46% of the cohort at median age 2.8 years (IQR, 1.3-5.7 years). Conclusion: Hypoglycemia was recognized at birth in most children with KS and HI, but HI diagnosis was often delayed. HI was effectively managed with diazoxide in most children. In contrast to prior reports, the frequency of variants in KMT2D and KDM6A were similar to their overall prevalence in individuals with KS. Children diagnosed with KS should undergo evaluation for HI, and, because KS features may not be recognized in infancy, KMT2D and KDM6A should be included in the genetic evaluation of HI.

Phenotypic Characterization of Congenital Hyperinsulinism Due to Novel Activating Glucokinase Mutations.

The importance of glucokinase (GK) in the regulation of insulin secretion has been highlighted by the phenotypes of individuals with activating and inactivating mutations in the glucokinase gene (GCK). Here we report 10 individuals with congenital hyperinsulinism (HI) caused by eight unique activating mutations of GCK. Six are novel and located near previously identified activating mutations sites. The first recognized episode of hypoglycemia in these patients occurred between birth and 24 years, and the severity of the phenotype was also variable. Mutant enzymes were expressed and purified for enzyme kinetics in vitro. Mutant enzymes had low glucose half-saturation concentration values and an increased enzyme activity index compared with wild-type GK. We performed functional evaluation of islets from the pancreata of three children with GCK-HI who required pancreatectomy. Basal insulin secretion in perifused GCK-HI islets was normal, and the response to glyburide was preserved. However, the threshold for glucose-stimulated insulin secretion in perifused glucokinase hyperinsulinism (GCK-HI) islets was decreased, and glucagon secretion was greatly suppressed. Our evaluation of novel GCK disease-associated mutations revealed that the detrimental effects of these mutations on glucose homeostasis can be attributed not only to a lowering of the glucose threshold of insulin secretion but also to a decreased counterregulatory glucagon secretory response. ARTICLE HIGHLIGHTS: Our evaluation of six novel and two previously published activating GCK mutations revealed that the detrimental effects of these mutations on glucose homeostasis can be attributed not only to a lowering of the glucose threshold of insulin secretion but also to a decreased counterregulatory glucagon secretory response. These studies provide insights into the pathophysiology of GCK-hyperinsulinism and the dual role of glucokinase in ß-cells and α-cells to regulate glucose homeostasis.

Mosaic GLUD1 Mutations Associated with Hyperinsulinism Hyperammonemia Syndrome.

INTRODUCTION: The hyperinsulinemia-hyperammonemia syndrome (HIHA) is the second most common cause of congenital hyperinsulinism and is caused by activating heterozygous missense mutations in GLUD1. In the majority of HIHA cases, the GLUD1 mutation is found to be de novo. We have identified 3 patients in whom clinical evaluation was suggestive of HIHA but with negative mutation analysis in peripheral blood DNA for GLUD1 as well as other known HI genes. METHODS: We performed next-generation sequencing (NGS) on peripheral blood DNA from two children with clinical features of HIHA in order to look for mosaic mutations in GLUD1. Pancreas tissue was also available in one of these cases for NGS. In addition, NGS was performed on peripheral blood DNA from a woman with a history of HI in infancy whose child had HIHA due to a presumed de novo GLUD1 mutation. RESULTS: Mosaic GLUD1 mutations were identified in these 3 cases at percent mosaicism ranging from 2.7% to 10.4% in peripheral blood. In one case with pancreas tissue available, the mosaic GLUD1 mutation was present at 17.9% and 28.9% in different sections of the pancreas. Two unique GLUD1 mutations were identified in these cases, both of which have been previously reported (c.1493c>t/p.Ser445Leu and c.820c>t/p.Arg221Cys). CONCLUSION: The results suggest that low-level mosaic mutations in known HI genes may be the underlying molecular mechanism in some children with HI who have negative genetic testing in peripheral blood DNA.

Localized islet nuclear enlargement hyperinsulinism (LINE-HI) due to ABCC8 and GCK mosaic mutations.

Objective: Congenital hyperinsulinism (HI) is the most common cause of persistent hypoglycemia in children. In addition to typical focal or diffuse HI, some cases with diazoxide-unresponsive congenital HI have atypical pancreatic histology termed Localized Islet Nuclear Enlargement (LINE) or mosaic HI, characterized by histologic features similar to diffuse HI, but confined to only a region of pancreas. Our objective was to characterize the phenotype and genotype of children with LINE-HI. Design: The phenotype and genotype features of 12 children with pancreatic histology consistent with LINE-HI were examined. Methods: We compiled clinical features of 12 children with LINE-HI and performed next-generation sequencing on specimens of pancreas from eight of these children to look for mosaic mutations in genes known to be associated with diazoxide-unresponsive HI (ABCC8, KCNJ11, and GCK). Results: Children with LINE-HI had lower birth weights and later ages of presentation compared to children with typical focal or diffuse HI. Partial pancreatectomy in LINE-HI cases resulted in euglycemia in 75% of cases; no cases have developed diabetes. Low-level mosaic mutations were identified in the pancreas of six cases with LINE-HI (three in ABCC8, three in GCK). Expression studies confirmed that all novel mutations were pathogenic. Conclusion: These results indicate that post-zygotic low-level mosaic mutations of known HI genes are responsible for some cases of LINE-HI that lack an identifiable germ-line mutation and that partial pancreatectomy may be curative for these cases.

Case Report: Two Distinct Focal Congenital Hyperinsulinism Lesions Resulting From Separate Genetic Events.

Focal hyperinsulinism (HI) comprises nearly 50% of all surgically treated HI cases and is cured if the focal lesion can be completely resected. Pre-operative localization of the lesion is thus critical. Few cases of hyperinsulinism with multiple focal lesions have been reported, and assessment of the molecular mechanisms driving this rare occurrence has been limited. We present two cases of multifocal HI, each resulting from two independent, pancreatic focal lesions. 18Fluoro-dihydroxyphenylalanine positron emission tomography/computed tomography detected both lesions preoperatively in one patient, whereas identification of the second lesion was an incidental finding during surgical exploration in the other. Complete resection of the focal lesions resulted in cure of the HI in both cases. In each patient, genetic testing of the individual focal lesions revealed different regions of loss of heterozygosity for the maternal 11p15 allele, confirming that each lesion arose from independent somatic events in the setting of a paternally inherited germline ABCC8 mutation. These cases highlight the importance of a multidisciplinary and personalized approach to the management of infants with HI.

Congenital Hyperinsulinism in Infants with Turner Syndrome: Possible Association with Monosomy X and KDM6A Haploinsufficiency.

BACKGROUND: Previous case reports have suggested a possible association of congenital hyperinsulinism with Turner syndrome. OBJECTIVE: We examined the clinical and molecular features in girls with both congenital hyperinsulinism and Turner syndrome seen at The Children's Hospital of Philadelphia (CHOP) between 1974 and 2017. METHODS: Records of girls with hyperinsulinism and Turner syndrome were reviewed. Insulin secretion was studied in pancreatic islets and in mouse islets treated with an inhibitor of KDM6A, an X chromosome gene associated with hyperinsulinism in Kabuki syndrome. RESULTS: Hyperinsulinism was diagnosed in 12 girls with Turner syndrome. Six were diazoxide-unresponsive; 3 had pancreatectomies. The incidence of Turner syndrome among CHOP patients with hyperinsulinism (10 of 1,050 from 1997 to 2017) was 48 times more frequent than expected. The only consistent chromosomal anomaly in these girls was the presence of a 45,X cell line. Studies of isolated islets from 1 case showed abnormal elevated cytosolic calcium and heightened sensitivity to amino acid-stimulated insulin release; similar alterations were demonstrated in mouse islets treated with a KDM6A inhibitor. CONCLUSION: These results demonstrate a higher than expected frequency of Turner syndrome among children with hyperinsulinism. Our data suggest that haploinsufficiency for KDM6A due to mosaic X chromosome monosomy may be responsible for hyperinsulinism in Turner syndrome.

Novel Hypoglycemia Phenotype in Congenital Hyperinsulinism Due to Dominant Mutations of Uncoupling Protein 2.

Context: The rarest genetic form of congenital hyperinsulinism (HI) has been associated with dominant inactivating mutations in uncoupling protein 2 (UCP2), a mitochondrial inner membrane carrier that modulates oxidation of glucose vs amino acids. Objective: To evaluate the frequency of UCP2 mutations in children with HI and phenotypic features of this form of HI. Design: We examined 211 children with diazoxide-responsive HI seen at The Children's Hospital of Philadelphia (CHOP) between 1997 and October 2016. Setting: CHOP Clinical and Translational Research Center. Results: Of 211 cases of diazoxide-responsive HI, we identified 5 unrelated children with UCP2 mutations (5 of 211; 2.4%). All 5 were diagnosed with HI before 6 months of age; diazoxide treatment was only partly effective in 3 of the 5. Among the 5 cases, 4 unique mutations (3 missense and 1 splicing) were identified. Three mutations were novel; 1 was previously reported. In vitro functional assays showed 30% to 75% decrease in UCP2 activity. Two of the children, when not taking diazoxide, developed hypoketotic-hypoglycemia after fasting 15 to 20 hours; a similar trend toward hypoglycemia after fasting 24 hours occurred in 4 adult carriers. In contrast, both children and 2 of the 4 carriers developed symptomatic hypoglycemia 4 hours following oral glucose. Unusual oscillating glucose and insulin responses to oral glucose were seen in both cases and carriers. Conclusions: These data indicate that dominant UCP2 mutations are a more important cause of HI than has been recognized and that affected individuals are markedly hypersensitive to glucose-induced hypoglycemia.

Functional and Metabolomic Consequences of KATP Channel Inactivation in Human Islets.

Loss-of-function mutations of ß-cell KATP channels cause the most severe form of congenital hyperinsulinism (KATPHI). KATPHI is characterized by fasting and protein-induced hypoglycemia that is unresponsive to medical therapy. For a better understanding of the pathophysiology of KATPHI, we examined cytosolic calcium ([Ca2+] i ), insulin secretion, oxygen consumption, and [U-13C]glucose metabolism in islets isolated from the pancreases of children with KATPHI who required pancreatectomy. Basal [Ca2+] i and insulin secretion were higher in KATPHI islets compared with controls. Unlike controls, insulin secretion in KATPHI islets increased in response to amino acids but not to glucose. KATPHI islets have an increased basal rate of oxygen consumption and mitochondrial mass. [U-13C]glucose metabolism showed a twofold increase in alanine levels and sixfold increase in 13C enrichment of alanine in KATPHI islets, suggesting increased rates of glycolysis. KATPHI islets also exhibited increased serine/glycine and glutamine biosynthesis. In contrast, KATPHI islets had low γ-aminobutyric acid (GABA) levels and lacked 13C incorporation into GABA in response to glucose stimulation. The expression of key genes involved in these metabolic pathways was significantly different in KATPHI ß-cells compared with control, providing a mechanism for the observed changes. These findings demonstrate that the pathophysiology of KATPHI is complex, and they provide a framework for the identification of new potential therapeutic targets for this devastating condition.

Seizures and diagnostic difficulties in hyperinsulinism-hyperammonemia syndrome.

Hyperinsulinism/hyperammonemia (HI/HA) syndrome is a rare disorder presented with recurrent hypoglycemia and elevated serum ammonia, which may lead to development delays, permanent neurologic damages, if it remains underdiagnosed. It is caused by activating mutations in the GLUD1 gene which encodes the intra-mitochondrial enzyme glutamate dehydrogenase (GDH). HI/HA syndrome is considered the second most common form of hyperinsulinism (HI), and usually associated with epileptic seizures, mental retardation and generalized dystonia. We reported a patient who was diagnosed as HI/HA with multiple episodes of seizures; and previously had been diagnosed and treated for epilepsy. She has heterozygous mutation in GLUD1 gene. Treatment with diazoxide enabled complete resolution of the seizures. One year later, when her brother was six months old, he was also diagnosed with HI/HA. Later, the same mutation of GLUD1 was detected in both her father and brother too.

Histologic and Molecular Profile of Pediatric Insulinomas: Evidence of a Paternal Parent-of-Origin Effect.

CONTEXT: Acquired insulinomas are rare causes of hyperinsulinemic hypoglycemia in children and are much less common than focal lesions of congenital hyperinsulinism. The latter are known to be associated with isodisomy for paternally transmitted ATP-sensitive potassium channel mutations on 11p15; however, the molecular basis for pediatric insulinomas is not well characterized. OBJECTIVE: The purpose of this study was to characterize the histopathological and molecular defects in a large group of 12 pediatric insulinomas seen at The Children's Hospital of Philadelphia. RESULTS: Twelve children with insulinomas were seen between 1971 and 2013, compared to 201 cases with focal congenital hyperinsulinism seen between 1997 and 2014. The age of insulinoma patients ranged from 4-16 years at the time of surgery. Features of MEN1 syndrome were present in five of the 12, including four cases with heterozygous mutations of MEN1 on 11q. Immunohistochemical analysis revealed nuclear loss of p57 staining consistent with loss of the maternal 11p15 allele in 11 of the 12 insulinomas, including all five MEN1-associated tumors. Imbalance of the paternal 11p allele was confirmed by single nucleotide polymorphism genotyping and methylation assays of the 11p imprinting control loci in four of five MEN1-associated tumors and six of seven sporadic insulinomas. In addition, single nucleotide polymorphism genotyping revealed extensive tumor aneuploidy beyond chromosome 11. CONCLUSIONS: These data indicate that MEN1 mutations are more common in insulinomas in children than in adults. Aneuploidy of chromosome 11 and other chromosomes is common in both MEN1 and non-MEN1 insulinomas. The novel observation of a paternal parent-of-origin effect in all MEN1 and most non-MEN1 tumors suggests a critical role for imprinted growth-regulatory genes in the 11p region in the genesis of ß-cell endocrine tumors in children.

Dominant form of congenital hyperinsulinism maps to HK1 region on 10q.

BACKGROUND/AIMS: In a family with congenital hyperinsulinism (HI), first described in the 1950s by McQuarrie, we examined the genetic locus and clinical phenotype of a novel form of dominant HI. METHODS: We surveyed 25 affected individuals, 7 of whom participated in tests of insulin dysregulation (24-hour fasting, oral glucose and protein tolerance tests). To identify the disease locus and potential disease-associated mutations we performed linkage analysis, whole transcriptome sequencing, whole genome sequencing, gene capture, and next generation sequencing. RESULTS: Most affecteds were diagnosed with HI before age one and 40% presented with a seizure. All affecteds responded well to diazoxide. Affecteds failed to adequately suppress insulin secretion following oral glucose tolerance test or prolonged fasting; none had protein-sensitive hypoglycemia. Linkage analysis mapped the HI locus to Chr10q21-22, a region containing 48 genes. Three novel noncoding variants were found in hexokinase 1 (HK1) and one missense variant in the coding region of DNA2. CONCLUSION: Dominant, diazoxide-responsive HI in this family maps to a novel locus on Chr10q21-22. HK1 is the more attractive disease gene candidate since a mutation interfering with the normal suppression of HK1 expression in beta-cells could readily explain the hypoglycemia phenotype of this pedigree.