RESUMEN
The ATP-sensitive potassium (K(ATP)) channel controls insulin secretion by coupling glucose metabolism to excitability of the pancreatic beta-cell membrane. The channel comprises four subunits each of Kir6.2 and the sulphonylurea receptor (SUR1), encoded by KCNJ11 and ABCC8, respectively. Mutations in these genes that result in reduced activity or expression of K(ATP) channels lead to enhanced beta-cell excitability, insulin hypersecretion and hypoglycaemia, and in humans lead to the clinical condition congenital hyperinsulinism (CHI). Here we have investigated the molecular basis of the focal form of CHI caused by one such mutation in Kir6.2, E282K. The study led to the discovery that Kir6.2 contains a di-acidic ER exit signal, (280)DLE(282), which promotes concentration of the channel into COPII-enriched ER exit sites prior to ER export via a process that requires Sar1-GTPase. The E282K mutation abrogates the exit signal, and thereby prevents the ER export and surface expression of the channel. When co-expressed, the mutant subunit was able to associate with the wild-type Kir6.2 and form functional channels. Thus unlike most mutations, the E282K mutation does not cause protein mis-folding. Since in focal CHI, maternal chromosome containing the K(ATP) channel genes is lost, beta-cells of the patient would lack wild-type Kir6.2 to rescue the mutant Kir6.2 subunit expressed from the paternal chromosome. The resultant absence of functional K(ATP) channels leads to insulin hypersecretion. Taken together, we conclude that surface expression of K(ATP) channels is critically dependent on the Sar1-GTPase-dependent ER exit mechanism and abrogation of the di-acidic ER exit signal leads to CHI.
Asunto(s)
Hiperinsulinismo Congénito/genética , Retículo Endoplásmico/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Mutación Missense , Canales de Potasio de Rectificación Interna/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Línea Celular , Hiperinsulinismo Congénito/metabolismo , Retículo Endoplásmico/genética , Humanos , Insulina/metabolismo , Secreción de Insulina , Canales KATP/genética , Canales KATP/metabolismo , Datos de Secuencia Molecular , Proteínas de Unión al GTP Monoméricas/genética , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/metabolismo , Unión Proteica , Transporte de Proteínas , Alineación de SecuenciaRESUMEN
BACKGROUND: A common and well recognised feature of Turner's syndrome (partial or total monosomy X) is impaired glucose tolerance or type 2 diabetes mellitus. A small percentage of patients with Turner's syndrome have a complex mosaic karyotype with atypical clinical features and mental retardation. METHODS/PATIENT: We report the first case of a child with a complex mosaic Turner genotype and hyperinsulinaemic hypoglycaemia responsive to diazoxide therapy. RESULTS: Cytogenetic analysis showed four cell lines: one with 45,X; the others with an additional small ring chromosome, a small marker chromosome, and both the ring and marker chromosomes, respectively. FISH studies showed the abnormal chromosomes to originate from an X. The X inactivation locus (XIST) was present in the ring, but not in the marker chromosome. CONCLUSIONS: The recognition of hypoglycaemia in children with atypical Turner's syndrome is important as persistent hypoglycaemia may lead to brain damage in addition to the risk of mental retardation. Further studies are required to understand whether the mosaic over--or underexpression of unidentified X chromosome gene(s) in the pancreatic beta-cells leads to hyperinsulinaemic hypoglycaemia.
Asunto(s)
Hiperinsulinismo/patología , Hipoglucemia/patología , Mosaicismo , Síndrome de Turner/genética , Síndrome de Turner/patología , Aberraciones Cromosómicas , Cromosomas Humanos X , Diazóxido/uso terapéutico , Femenino , Humanos , Hiperinsulinismo/complicaciones , Hiperinsulinismo/tratamiento farmacológico , Hipoglucemia/complicaciones , Hipoglucemia/tratamiento farmacológico , Recién Nacido , Cariotipificación , Resultado del Tratamiento , Síndrome de Turner/complicaciones , Vasodilatadores/uso terapéutico , Inactivación del Cromosoma X/genéticaRESUMEN
In this study, a second case of hyperinsulinemic hypoglycemia due to activation of glucokinase is reported. The 14-year-old proband had a history of neonatal hypoglycemia, treated with diazoxide. He was admitted with coma and convulsions due to nonketotic hypoglycemia. His BMI was 34 kg/m(2), and his fasting blood glucose ranged from 2.1 to 2.7 mmol/l, associated with inappropriately high serum levels of insulin, C-peptide, and proinsulin. An oral glucose tolerance test (OGTT) showed exaggerated responses of these peptides followed by profound hypoglycemia. Treatment with diazoxide and chlorothiazide was effective. His mother never had clinical hypoglycemic symptoms, even though her fasting blood glucose ranged from 2.9 to 3.5 mmol/l. Increases in serum insulin, C-peptide, and proinsulin in response to an OGTT suggested a lower threshold for glucose-stimulated insulin release (GSIR). Screening for mutations in candidate genes revealed a heterozygous glucokinase mutation in exon 10, substituting valine for alanine at codon 456 (A456V) in the proband and his mother. The purified recombinant glutathionyl S-transferase fusion protein of the A456V glucokinase revealed a decreased glucose S(0.5) (the concentration of glucose needed to achieve the half-maximal rate of phosphorylation) from 8.04 (wild-type) to 2.53 mmol/l. The mutant's Hill coefficient was decreased, and its maximal specific activity k(cat) was increased. Mathematical modeling predicted a markedly lowered GSIR threshold of 1.5 mmol/l. The theoretical and practical implications are manifold and significant.
Asunto(s)
Diabetes Mellitus/genética , Glucoquinasa/genética , Glucosa/metabolismo , Mutación Missense , Adolescente , Regulación Alostérica , Diabetes Mellitus/tratamiento farmacológico , Diabetes Mellitus/metabolismo , Diazóxido/uso terapéutico , Activación Enzimática , Glutatión Transferasa/genética , Homeostasis , Humanos , Recién Nacido , Recién Nacido Pequeño para la Edad Gestacional , Insulina/sangre , Insulina/metabolismo , Secreción de Insulina , Cinética , Masculino , FosforilaciónRESUMEN
OBJECTIVE: Activating glucokinase (GCK) mutations are a rarely reported cause of congenital hyperinsulinism (CHI), but the prevalence of GCK mutations is not known. METHODS: From a pooled cohort of 201 non-syndromic children with CHI from three European referral centres (Denmark, n=141; Norway, n=26; UK, n=34), 108 children had no K(ATP)-channel (ABCC8/KCNJ11) gene abnormalities and were screened for GCK mutations. Novel GCK mutations were kinetically characterised. RESULTS: In five patients, four heterozygous GCK mutations (S64Y, T65I, W99R and A456V) were identified, out of which S64Y was novel. Two of the mutations arose de novo, three were dominantly inherited. All the five patients were medically responsive. In the combined Danish and Norwegian cohort, the prevalence of GCK-CHI was estimated to be 1.2% (2/167, 95% confidence interval (CI) 0-2.8%) of all the CHI patients. In the three centre combined cohort of 72 medically responsive children without K(ATP)-channel mutations, the prevalence estimate was 6.9% (5/72, 95% CI 1.1-12.8%). All activating GCK mutations mapped to the allosteric activator site. The novel S64Y mutation resulted in an increased affinity for the substrate glucose (S(0.5) 1.49+/-0.08 and 7.39+/-0.05 mmol/l in mutant and wild-type proteins respectively), extrapolating to a relative activity index of approximately 22 compared with the wild type. CONCLUSION: In the largest study performed to date on GCK in children with CHI, GCK mutations were found only in medically responsive children who were negative for ABCC8 and KCNJ11 mutations. The estimated prevalence (approximately 7%) suggests that screening for activating GCK mutations is warranted in those patients.
Asunto(s)
Hiperinsulinismo Congénito/genética , Glucoquinasa/genética , Mutación , Estudios de Cohortes , Hiperinsulinismo Congénito/tratamiento farmacológico , Hiperinsulinismo Congénito/epidemiología , Dinamarca/epidemiología , Activación Enzimática/efectos de los fármacos , Frecuencia de los Genes , Genotipo , Glucoquinasa/metabolismo , Glucosa/metabolismo , Heterocigoto , Humanos , Noruega/epidemiología , Prevalencia , Especificidad por Sustrato , Reino Unido/epidemiologíaRESUMEN
OBJECTIVE: Congenital hyperinsulinism (CHI) is a cause of persistent and severe hypoglycaemia in infancy. Mutations in the genes ABCC8 and KCNJ11 encoding SUR1 and Kir6.2, respectively, are the commonest cause of CHI. We investigated whether the possession of two DNA variants leading to coding changes in a single allele of ABCC8 can affect the potential mechanism of disease pathogenesis. DESIGN AND PATIENTS: We studied two patients with complex mutations in the ABCC8 gene with CHI and used in vitro studies to explore the potential disease mechanism and the contribution of the various mutant allelles. RESULTS: The first case had diffuse disease and was homozygous for the mutations D1193V and R1436Q in SUR1. Channel complexes containing the D1193V mutant were delivered to the plasma membrane and were functional and those containing R1436Q were also present at the plasma membrane but were nonfunctional. Combining the two mutations (SUR1D1193V/R1436Q) led to intracellular retention of the channel complex. In a second family, the patient had histologically focal disease and was heterozygous for two mutations from his father (G228D and D1471N) and one from his mother (V1572I). SUR1 G228D and D1471N singly or in combination led to intracellular retention of the channel complex and loss of function. By contrast, V1572I is trafficked appropriately and is functional, consistent with a mechanism of reduction to hemizygosity of paternal ABCC8 in focal disease. V1572I is likely to be a benign DNA variant. CONCLUSION: In one patient the combination of two coding variants led to intracellular retention of channel complex. In a second patient, functional studies allowed us to unravel the DNA variants likely to be causing the abrogation of ATP-sensitive K(+) channel function.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Hiperinsulinismo Congénito/genética , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio/genética , Receptores de Droga/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adulto , Animales , Western Blotting , Células CHO , Estudios de Casos y Controles , Cricetinae , Cricetulus , Expresión Génica , Genotipo , Humanos , Recién Nacido , Masculino , Ratones , Mutagénesis Sitio-Dirigida , Mutación , Fenotipo , Canales de Potasio/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Receptores de Droga/metabolismo , Coloración y Etiquetado , Receptores de Sulfonilureas , Transfección/métodosRESUMEN
BACKGROUND: In severe, medically unresponsive congenital hyperinsulinism (CHI), the histological differentiation of focal versus diffuse disease is vital, since the surgical management is completely different. Genetic analysis may help in the differential diagnosis, as focal CHI is associated with a paternal germline ABCC8 or KCNJ11 mutation and a focal loss of maternal chromosome 11p15, whereas a maternal mutation, or homozygous/compound heterozygous ABCC8 and KCNJ11 mutations predict diffuse-type disease. However, genotyping usually takes too long to be helpful in the absence of a founder mutation. METHODS: In 4 patients, a rapid genetic analysis of the ABBC8 and KCNJ11 genes was performed within 2 weeks on request prior to the decision of pancreatic surgery. RESULTS: Two patients had no mutations, rendering the genetic analysis non-informative. Peroperative multiple biopsies showed diffuse disease. One patient had a paternal KCNJ11 mutation and focal disease confirmed by positron emission tomography scan and biopsies. One patient had a de novo heterozygous ABBC8 mutation and unexplained diffuse disease confirmed by positron emission tomography scan and biopsies. CONCLUSION: A rapid analysis of the entire ABBC8 and KCNJ11 genes should not stand alone in the preoperative assessment of patients with CHI, except for the case of maternal, or homozygous/compound heterozygous disease-causing mutations.