Functional analysis of six Kir6.2 (KCNJ11) mutations causing neonatal diabetes

ATP-sensitive potassium (K_ATP) channels, composed of pore-forming Kir6.2 and regulatory sulphonylurea receptor (SUR) subunits, play an essential role in insulin secretion from pancreatic beta cells. Binding of ATP to Kir6.2 inhibits, whereas interaction of Mg-nucleotides with SUR, activates the channel. Heterozygous activating mutations in Kir6.2 (KCNJ11) are a common cause of neonatal diabetes (ND). We assessed the functional effects of six novel Kir6.2 mutations associated with ND: H46Y, N48D, E227K, E229K, E292G, and V252A. K_ATP channels were expressed in Xenopus oocytes and the heterozygous state was simulated by coexpression of wild-type and mutant Kir6.2 with SUR1 (the beta cell type of SUR). All mutations reduced the sensitivity of the K_ATP channel to inhibition by MgATP, and enhanced whole-cell K_ATP currents. Two mutations (E227K, E229K) also enhanced the intrinsic open probability of the channel, thereby indirectly reducing the channel ATP sensitivity. The other four mutations lie close to the predicted ATP-binding site and thus may affect ATP binding. In pancreatic beta cells, an increase in the K_ATP current is expected to reduce insulin secretion and thereby cause diabetes. None of the mutations substantially affected the sensitivity of the channel to inhibition by the sulphonylurea tolbutamide, suggesting patients carrying these mutations may respond to these drugs.     

Update of mutations in the genes encoding the pancreatic beta-cell K(ATP) channel subunits Kir6.2 (KCNJ11) and sulfonylurea receptor 1 (ABCC8) in diabetes mellitus and hyperinsulinism

The beta-cell ATP-sensitive potassium (K(ATP)) channel is a key component of stimulus-secretion coupling in the pancreatic beta-cell. The channel couples metabolism to membrane electrical events bringing about insulin secretion. Given the critical role of this channel in glucose homeostasis it is therefore not surprising that mutations in the genes encoding for the two essential subunits of the channel can result in both hypo- and hyperglycemia. The channel consists of four subunits of the inwardly rectifying potassium channel Kir6.2 and four subunits of the sulfonylurea receptor 1 (SUR1). It has been known for some time that loss of function mutations in KCNJ11, which encodes for Kir6.2, and ABCC8, which encodes for SUR1, can cause oversecretion of insulin and result in hyperinsulinism of infancy, while activating mutations in KCNJ11 and ABCC8 have recently been described that result in the opposite phenotype of diabetes. This review focuses on reported mutations in both genes, the spectrum of phenotypes, and the implications for treatment on diagnosing patients with mutations in these genes.     

Mutations in the genes encoding the pancreatic beta-cell KATP channel subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) in diabetes mellitus and hyperinsulinism

The beta-cell ATP-sensitive potassium channel is a key component of stimulus-secretion coupling in the pancreatic beta-cell. The channel couples metabolism to membrane electrical events, bringing about insulin secretion. Given the critical role of this channel in glucose homeostasis, it is not surprising that mutations in the genes encoding for the two essential subunits of the channel can result in both hypo- and hyperglycemia. The channel consists of four subunits of the inwardly rectifying potassium channel Kir6.2 and four subunits of the sulfonylurea receptor 1. It has been known for some time that loss of function mutations in KCNJ11, which encodes for Kir6.2, and ABCC8, which encodes for SUR1, can cause oversecretion of insulin and result in hyperinsulinemia (HI) of infancy; however, heterozygous activating mutations in KCNJ11 that result in the opposite phenotype of diabetes have recently been described. This review focuses on reported mutations in both genes, the spectrum of phenotypes, and the implications for treatment when patients are diagnosed with mutations in these genes.     

Sulfonylurea therapy in two Korean patients with insulin-treated neonatal diabetes due to heterozygous mutations of the KCNJ11 gene encoding Kir6.2

Permanent neonatal diabetes (PND) is a rare form of diabetes characterized by insulin-requiring hyperglycemia diagnosed within the first three months of life. In most cases, the causes are not known. Recently, mutations in the KCNJ11 gene encoding the Kir6.2 subunit of the ATP-sensitive K+ channel have been described in patients with PND. We report the first two Korean cases with PND due to a lysineto- arginine substitution at position 170 (K179R) and a valine-to-methionine substitution at position 59 (V59M) mutations of KCNJ11 encoding Kir6.2, respectively. After several years of insulin therapy, these patients were managed by oral glibenclamide therapy at a daily dose of 0.8-0.9 mg/kg. Their basal c-peptide levels increased after one week of glibenclamide therapy, and one month later, the insulin and c-peptide levels were in the normal ranges without any episodes of hyper- or hypoglycemia. These cases demonstrate that oral sulfonylurea may be the treatment of choice in PND patients with KCNJ11 mutations even at a young age.     

Update of variants identified in the pancreatic β‐cell KATP channel genes KCNJ11 and ABCC8 in individuals with congenital hyperinsulinism and diabetes

The most common genetic cause of neonatal diabetes and hyperinsulinism is pathogenic variants in ABCC8 and KCNJ11. These genes encode the subunits of the β‐cell ATP‐sensitive potassium channel, a key component of the glucose‐stimulated insulin secretion pathway. Mutations in the two genes cause dysregulated insulin secretion; inactivating mutations cause an oversecretion of insulin, leading to congenital hyperinsulinism, whereas activating mutations cause the opposing phenotype, diabetes. This review focuses on variants identified in ABCC8 and KCNJ11, the phenotypic spectrum and the treatment implications for individuals with pathogenic variants.     

Update on mutations in glucokinase (GCK), which cause maturity-onset diabetes of the young,permanent neonatal diabetes,and hyperinsulinemic hypoglycemia

Glucokinase is a key regulatory enzyme in the pancreatic beta-cell. It plays a crucial role in the regulation of insulin secretion and has been termed the glucose sensor in pancreatic beta-cells. Given its central role in the regulation of insulin release it is understandable that mutations in the gene encoding glucokinase (GCK) can cause both hyper- and hypoglycemia. Heterozygous inactivating mutations in GCK cause maturity-onset diabetes of the young (MODY) subtype glucokinase (GCK), characterized by mild fasting hyperglycemia, which is present at birth but often only detected later in life during screening for other purposes. Homozygous inactivating GCK mutations result in a more severe phenotype presenting at birth as permanent neonatal diabetes mellitus (PNDM). A growing number of heterozygous activating GCK mutations that cause hypoglycemia have also been reported. A total of 620 mutations in the GCK gene have been described in a total of 1,441 families. There are no common mutations, and the mutations are distributed throughout the gene. The majority of activating mutations cluster in a discrete region of the protein termed the allosteric activator site. The identification of a GCK mutation in patients with both hyper- and hypoglycemia has implications for the clinical course and clinical management of their disorder. Hum Mutat 30: 1–15, 2009. © 2009 Wiley-Liss, Inc.     

Clinical and molecular characterization of neonatal diabetes and monogenic syndromic diabetes in Asian Indian children

Mutations in the pancreatic ATP sensitive K⁺ channel proteins [sulfonyluea receptor 1 (SUR1) and inward rectifier K⁺ channel Kir6.2 (Kir6.2), encoded by ATP‐binding cassette transporter subfamily C member 8 (ABCC8) and potassium channel J11 (KCNJ11), respectively], are the most common cause of neonatal diabetes. We describe the clinical presentation and molecular characterization of Asian Indian children with neonatal diabetes mellitus and monogenic syndromes of diabetes. We sequenced KCNJ11, ABCC8 and insulin (INS) genes in 33 unrelated Indian probands with onset of diabetes below one year of age. A total of 12 mutations were identified which included ABCC8 mutations in seven, KCNJ11 mutations in three and INS mutations in two children. The Asp212Tyr mutation in ABCC8 was novel. We also detected two novel mutations (Val67Met and Leu19Arg) in children with syndromic forms of diabetes like Berardinelli Seip syndrome [1‐acyl‐sn‐glycerol‐3‐phosphate acyltransferase beta (AGPAT2)] and Fanconi Bickel syndrome [solute carrier family 2A2 (SLC2A2)]. Children carrying the KCNJ11 (Cys42Arg, Arg201Cys) and ABCC8 (Val86Ala, Asp212Tyr) mutations have been successfully switched over from insulin therapy to oral sulfonylurea. Our study is the first large genetic screening study of neonatal diabetes in India.     

Glucokinase (GCK) mutations in hyper- and hypoglycemia: maturity-onset diabetes of the young, permanent neonatal diabetes, and hyperinsulinemia of infancy

Glucokinase is a key regulatory enzyme in the pancreatic beta-cell. It plays a crucial role in the regulation of insulin secretion and has been termed the pancreatic beta-cell sensor. Given its central role in the regulation of insulin release, it is understandable that mutations in the gene encoding glucokinase (GCK) can cause both hyperglycemia and hypoglycemia. Heterozygous inactivating mutations in GCK cause maturity-onset diabetes of the young (MODY), characterized by mild hyperglycemia, which is present at birth, but is often only detected later in life during screening for other purposes. Homozygous inactivating GCK mutations result in a more severe phenotype, presenting at birth as permanent neonatal diabetes mellitus (PNDM). Several heterozygous activating GCK mutations that cause hypoglycemia have also been reported. A total of 195 mutations in the GCK gene have been described, in a total of 285 families. There are no common mutations and the mutations are distributed throughout the gene. Mutations that cause hypoglycemia are located in various exons in a discrete region of the protein termed the heterotropic allosteric activator site. The identification of a GCK mutation in hyper- and hypoglycemia has implications for the clinical course and clinical management of the disorder.     

Cantú Syndrome Resulting from Activating Mutation in the KCNJ8 Gene

ATP‐sensitive potassium (K_ATP) channels, composed of inward‐rectifying potassium channel subunits (Kir6.1 and Kir6.2, encoded by KCNJ8 and KCNJ11, respectively) and regulatory sulfonylurea receptor (SUR1 and SUR2, encoded by ABCC8 and ABCC9, respectively), couple metabolism to excitability in multiple tissues. Mutations in ABCC9 cause Cantú syndrome (CS), a distinct multiorgan disease, potentially via enhanced K_ATP channel activity. We screened KCNJ8 in an ABCC9 mutation‐negative patient who also exhibited clinical hallmarks of CS (hypertrichosis, macrosomia, macrocephaly, coarse facial appearance, cardiomegaly, and skeletal abnormalities). We identified a de novo missense mutation encoding Kir6.1[p.Cys176Ser] in the patient. Kir6.1[p.Cys176Ser] channels exhibited markedly higher activity than wild‐type channels, as a result of reduced ATP sensitivity, whether coexpressed with SUR1 or SUR2A subunits. Our results identify a novel causal gene in CS, but also demonstrate that the cardinal features of the disease result from gain of K_ATP channel function, not from a Kir6‐independent SUR2 function.     

Relapsing diabetes can result from moderately activating mutations in KCNJ11

Neonatal diabetes can either remit and hence be transient or else may be permanent. These two phenotypes were considered to be genetically distinct. Abnormalities of 6q24 are the commonest cause of transient neonatal diabetes (TNDM). Mutations in KCNJ11, which encodes Kir6.2, the pore-forming subunit of the ATP-sensitive potassium channel (K(ATP)), are the commonest cause of permanent neonatal diabetes (PNDM). In addition to diabetes, some KCNJ11 mutations also result in marked developmental delay and epilepsy. These mutations are more severe on functional characterization. We investigated whether mutations in KCNJ11 could also give rise to TNDM. We identified the three novel heterozygous mutations (G53S, G53R, I182V) in three of 11 probands with clinically defined TNDM, who did not have chromosome 6q24 abnormalities. The mutations co-segregated with diabetes within families and were not found in 100 controls. All probands had insulin-treated diabetes diagnosed in the first 4 months and went into remission by 7-14 months. Functional characterization of the TNDM associated mutations was performed by expressing the mutated Kir6.2 with SUR1 in Xenopus laevis oocytes. All three heterozygous mutations resulted in a reduction in the sensitivity to ATP when compared with wild-type (IC(50) approximately 30 versus approximately 7 microM, P-value for is all <0.01); however, this was less profoundly reduced than with the PNDM associated mutations. In conclusion, mutations in KCNJ11 are the first genetic cause for remitting as well as permanent diabetes. This suggests that a fixed ion channel abnormality can result in a fluctuating glycaemic phenotype. The multiple phenotypes associated with activating KCNJ11 mutations may reflect their severity in vitro.     

KCNJ11 activating mutations are associated with developmental delay, epilepsy and neonatal diabetes syndrome and other neurological features

Heterozygous activating mutations in the gene encoding for the ATP-sensitive potassium channel subunit Kir6.2 (KCNJ11) have recently been shown to be a common cause of permanent neonatal diabetes. Kir6.2 is expressed in muscle, neuron and brain as well as the pancreatic beta-cell, so patients with KCNJ11 mutations could have a neurological phenotype in addition to their diabetes. It is proposed that some patients with KCNJ11 mutations have neurological features that are part of a discrete neurological syndrome termed developmental Delay, Epilepsy and Neonatal Diabetes (DEND), but there are also neurological consequences of chronic or acute diabetes. We identified KCNJ11 mutations in four of 10 probands with permanent neonatal diabetes and one affected parent; this included the novel C166F mutation and the previously described V59M and R201H. Four of the five patients with mutations had neurological features: the patient with the C166F mutation had marked developmental delay, severe generalised epilepsy, hypotonia and muscle weakness; mild developmental delay was present in the patient with the V59M mutation; one patient with the R201H mutation had acute and chronic neurological consequences of cerebral oedema and another had diabetic neuropathy from chronic hyperglycaemia. In conclusion, the clinical features in these patients support the existence of a discrete neurological syndrome with KCNJ11 mutations. The severe DEND syndrome was seen with the novel C166F mutation and mild developmental delay with the V59M mutation. These features differ markedly from the neurological consequences of acute or chronic diabetes.     

The Glu23Lys polymorphism in KCNJ11 and impaired hypoglycaemia awareness in patients with type 1 diabetes

Impaired awareness of hypoglycaemia affects approximately 25% of all patients with type 1 diabetes (T1D). Duration of diabetes and tight glycaemic control represent the main risk factors for hypoglycaemia unawareness. However, even among patients with good glycaemic control and longstanding T1D, awareness of hypoglycaemia may be intact. Genetic factors might explain some of this remaining variability, and genes involved in central glucose sensing should represent plausible candidates. Some evidence indicates that ventromedial hypothalamus glucose-responsive neurons require the potassium inward rectifier (KIR) 6.2 subunit of the K_ATP channel to sense glucose. Therefore, the effects of the Glu23Lys polymorphism in the KCNJ11 (KIR6.2) gene (potassium inwardly rectifying channel, subfamily J, member 11) on impaired hypoglycaemia awareness in 217 patients with T1D were studied. Hypoglycaemia awareness status was determined using standardized questionnaires. Genotyping of the Glu23Lys in a cohort of T1D subjects was done using the TaqMan allelic discrimination assay (frequency of the Lys-allele=0.35; P=0.57 for Hardy–Weinberg equilibrium). The study confirms that diabetes duration, C-peptide, and HbA_1c represent risk factors for impaired hypoglycaemia awareness. However, no significant effect of the Glu23Lys polymorphism on impaired hypoglycaemia awareness was observed with or without adjustment for age, diabetes duration, C-peptide, and HbA_1c. Even though the study provides a relatively large dataset, it is possible that small differences may have been missed.     

Novel mutation in the KCNJ10 gene in three siblings with seizures,ataxia and no electrolyte abnormalities

We report a consanguineous family with three affected siblings with novel mutation in the KCNJ10 gene. All three presented with central nervous system symptoms in the form of infantile focal seizures, ataxia, slurred speech with early developmental delay and intellectual disability in two siblings. None had any associated electrolyte abnormalities and no symptomatic hearing deficits were observed.     

SNPs in the KCNJ11-ABCC8 gene locus are associated with type 2 diabetes and blood pressure levels in the Japanese population

Many genetic association studies support a contribution of genetic variants in the KCNJ11-ABCC8 gene locus to type 2 diabetes (T2D) susceptibility in Caucasians. In non-Caucasian populations, however, there have been only a few association studies, and discordant results were obtained. Herein, we selected a total of 31 SNPs covering a 211.3-kb region of the KCNJ11-ABCC8 locus, characterized the patterns of linkage disequilibrium (LD) and haplotype structure, and performed a case-control association study in a Japanese population consisting of 909 T2D patients and 893 control subjects. We found significant associations between eight SNPs, including the KCNJ11 E23K and ABCC8 S1369A variants, and T2D. These disease-associated SNPs were genetically indistinguishable because of the presence of strong LD, as found previously in Caucasians. For the KCNJ11 E23K variant, the most significant association was obtained under a dominant genetic model (OR 1.32, 95% CI 1.09–1.60, P = 0.004). A meta-analysis of East Asian studies, comprising a total of 3,357 T2D patients (77.4% Japanese) and 2,836 control subjects (77.8% Japanese), confirmed the significant role of the KCNJ11 E23K variant in T2D susceptibility. Furthermore, we found evidence suggesting that the KCNJ11 E23K genotype is independently associated with higher blood-pressure levels. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genotype-phenotype correlations of KCNJ2 mutations in Japanese patients with Andersen-Tawil syndrome

Andersen-Tawil syndrome (ATS) is a rare inherited disorder characterized by periodic paralysis, mild dysmorphic features, and QT or QU prolongation with ventricular arrhythmias in electrocardiograms (ECGs). Mutations of KCNJ2, encoding the human inward rectifying potassium channel Kir 2.1, have been identified in patients with ATS. We aimed to clarify the genotype-phenotype correlations in ATS patients. We screened 23 clinically diagnosed ATS patients from 13 unrelated Japanese families. Ten different forms of KCNJ2 mutations were identified in the 23 ATS patients included in this study. Their ECGs showed normal QTc intervals and abnormal U waves with QUc prolongation and a variety of ventricular arrhythmias. Especially, bidirectional ventricular tachycardia (VT) was observed in 13 of 23 patients (57%). Periodic paralysis was seen in 13 of 23 carriers (57%), dysmorphic features in 17 (74%), and seizures during infancy in 4 (17%). Functional assays for the two novel KCNJ2 mutations (c. 200G>A (p. R67Q) and c. 436G>A (p. G146S)) displayed no functional inward rectifying currents in a heterologous expression system and showed strong dominant negative effects when co-expressed with wild-type KCNJ2 channels (91% and 84% reduction at -50 mV respectively compared to wild-type alone). Immunocytochemistry and confocal imaging revealed normal trafficking for mutant channels. In our study, all of the clinically diagnosed ATS patients had KCNJ2 mutations and showed a high penetrance with regard to the typical cardiac phenotypes: predominant U wave and ventricular arrhythmias, typically bidirectional VT.     

Association of eNOS (G894T,rs1799983) and KCNJ11 (E23K,rs5219) gene polymorphism with coronary artery disease in North Indian population

BackgroundEndothelial nitric oxide synthase (eNOS) and potassium voltage-gated channel subfamily J member 11 (KCNJ11) could be the candidate genes for coronary artery disease (CAD). This study investigated the relationship of the eNOS (rs1799983) and KCNJ11 (rs5219) polymorphisms with the presence and severity of CAD in the North Indian population.MethodsThis study included 300 subjects, 150 CAD cases and 150 healthy controls. Single nucleotide polymorphism was evaluated by Polymerase chain reaction and Restriction fragment length polymorphism (PCR-RFLP). Analysis was performed by SPSS (version 21.0).ResultsWe observed that KK genotype of KCNJ11E23K (rs5219) polymorphism (P=0.0001) genotypes and K allele (P=0.0001) was found to be a positive risk factor and strongly associated with CAD. In the case of eNOSG894T (rs1799983) there was no association found with CAD.ConclusionThese results illustrate the probability of associations between SNPs and CAD although specific genetic polymorphisms affecting ion channel function and expression have still to be clarified by further investigations involving larger cohorts.     

Novel glucokinase mutations in patients with monogenic diabetes - clinical outline of GCK-MD and potential for founder effect in Slavic population

Glucokinase (GCK) gene mutations are the causative factor of GCK-MD (monogenic diabetes) characterized by a mild clinical phenotype and potential for insulin withdrawal. This study presents the results of a nationwide genetic screening for GCK-MD performed in Poland. A group of 194 patients with clinical suspicion of GCK-MD and 17 patients with neonatal diabetes were subjected to GCK sequencing. Patients negative for GCK mutations were subjected to multiplex ligation-dependent probe amplification (MLPA) to detect deletions or insertions. A total of 44 GCK heterozygous mutations were found in 68 probands (35%). Among those, 20 mutations were novel ones: A282fs, D198V, E158X, G246V, G249R, I348N, L165V, L315Q, M115I, N254S, P284fs, Q338P, R377L, R43C, R46S, S212fs, S212P, T255N, V406A and Y214D. No abnormalities were detected in MLPA analysis. Homozygous D278E mutation was found in one patient with neonatal diabetes. The most frequently observed combinations of symptoms typical for GCK-MD were mild diabetes and/or fasting hyperglycaemia (98.3%), positive C-peptide at diagnosis (76%) and dominant mode of inheritance (59%). This study outlines numerous novel mutations of the GCK gene present in white Caucasians of Slavic origin. Thorough clinical assessment of known factors associated with GCK-MD may facilitate patient selection.     

Klippel-Trenaunay-Weber syndrome associated with a 5:11 balanced translocation

We report on a case of Klippel-Trenaunay Weber syndrome (KTWS) associated with a reciprocal translocation [46,XX,t(5;11)(q13.3;p15.1)]. The patient had developmental delay and minor anomalies in addition to classic findings of KTWS. These data support the notion that Klippel-Trenaunay-Weber syndrome may be due to a single gene defect and suggests the possible localization of a Klippel-Trenaunay-Weber gene(s) to 5q or 11p. © 1995 Wiley-Liss, Inc.