Kapoor, R.; (2010) Defining genotype-phenotype correlations in children with congenital hyperinsulinism. Doctoral thesis , UCL (University College London).

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Abstract

Background: Congenital hyperinsulinism (CHI) is a clinically heterogeneous condition. Mutations in seven genes (ABCC8, KCNJ11, GLUD1, GCK, HADH, SLC16A1 and HNF4A) are known to cause CHI, with mutations in HNF4A being the most recent identified genetic aetiology. Recessive mutations in ABCC8/KCNJ11 cause severe medically unresponsive hyperinsulinaemic hypoglycaemia (HH). Recently, dominant mutations in these genes have been described that cause mild, medically responsive HH. Controversy exists on whether these dominant ABCC8/KCNJ11 mutations predispose to diabetes mellitus in adulthood or not. The phenotype and prevalence of the genetic subgroups in a large cohort of patients has not been studied previously. Aims: The aims of this thesis include: 1. To investigate genotype/phenotype correlations in a large cohort of patients with CHI by comparing different genetic aetiologies 2. To examine the prevalence and phenotype of patients with HH resulting from HNF4A gene mutations 3. To study the phenotype of dominantly inherited KATP channel mutations causing CHI and functionally characterize the novel dominant mutations identified Methods: 300 patients with biochemically confirmed CHI were recruited. Detailed clinical information was collected prior to genotyping. ABCC8 and KCNJ11 genes were sequenced in all patients with CHI that were unresponsive to diazoxide, the mainstay of medical treatment in CHI. Mutations in the GCK, GLUD1 and HADH genes were sought in patients with diazoxide responsive CHI with hyperammonaemia (HI/HA) (or leucine sensitivity (GLUD1)), raised 3-hydroxybutyryl-carnitine (HADH) or positive family history and/or delayed presentation (GCK). If no mutations were identified and in all other patients with diazoxide responsive CHI (and where diazoxide responsiveness was not known); ABCC8, KCNJ11 and HNF4A genes were sequenced. The clinical characteristics of patients with the different genetic aetiologies identified were collated and the phenotypic characteristics of the patients found to have a HNF4A mutation were compared with the phenotypic characteristics of patients with transient and/ or diazoxide responsive CHI and a KATP mutation (n= 27), GLUD1 mutation (n= 13) or a HADH mutation (n=3). The one-way analysis of variance (ANOVA) test was used to compare the phenotypic data followed by LSD post-test to test for statistical significance. Protein sensitivity was investigated in patients with a HADH mutation (n=3). Upon confirmation of protein sensitivity, leucine tolerance test were conducted in these patients to understand the mechanism of protein sensitivity. The phenotype of ten families with dominant ABCC8/KCNJ11 mutations and the prevalence of diabetes mellitus in the adult mutation carriers were also studied in detail. Functional consequences of six novel dominant KATP channel mutations (five ABCC8 and one KCNJ11) were examined by reconstituting the KATP channel in HEK293 cells and evaluating the effect of drugs (diazoxide, glibenclamide) and metabolic poisoning on the channels using 86Rb flux assay. Results: Mutations were identified in 146/300 patients (48.6%). Mutations in the ABCC8/KCNJ11 were the commonest genetic cause identified (n=117, 39%). Among diazoxide unresponsive patients (n=105), mutations in these two genes were identified in 92 (87.6%); of whom 63 patients had recessively inherited mutations while four patients had three novel dominantly inherited ABCC8 mutations (G1485E, D1506E and M1514K). Among the diazoxide responsive patients (n=183), mutations were identified in 51 patients. These include mutations in the ABCC8(n=25), KCNJ11(n=3), HNF4A(n=7), GLUD1(n=16) and HADH(n=3). No mutations were identified in 132 (72%) patients in this group. Heterozygous missense mutations were detected in 15 patients with HI/HA, two of which are novel (N410D, D451V). In addition, a patient with a normal serum ammonia concentration (21μmol/l) was heterozygous for a novel missense mutation P436L. Functional analysis of this mutation confirmed that it is associated with a loss of GTP inhibition. Seizure disorder was common (43%) in our cohort of patients with a GLUD1 mutation. The study identified a novel homozygous missense mutation (M188V) in the HADH gene in a patient with normal acylcarnitines and urine organic acids. Hydroxyacyl-Coenzyme A dehydrogenase activity was significantly decreased compared with controls (index patient mean 26.8 ± SEM 4.8mU/mg protein vs. controls 48.0 ± 8.1; p=0.029) in skin fibroblasts. This patient and two other children with CHI due to HADH gene mutations were severely protein sensitive. The three children also demonstrated marked leucine sensitivity. HNF4A mutations were identified to cause persistent CHI in addition to transient CHI, reported previously. 3/8 children with an HNF4A mutation did not have a diabetic parent. Children with HNF4A mutations had increased birth weight (median +2.4 SDS) and presented early (median of day 1). Patients with a KATP channel mutation were also large at birth (birth weight SDS ranged between -1.96 to +4.66) with an early age of presentation (ranging from 1 day to 365 days). In contrast, patients with a GLUD1/ HADH mutation were diagnosed later (mean of 157 and 125 days respectively) and were of normal birth weight (mean birth wt SDS of -0.11 and -1.09 respectively). Study of the phenotype of the dominant ABCC8/KCNJ11 mutations identified an increased prevalence (57%) of diabetes in the adult mutation carriers. Functional studies on the novel ABCC8/KCNJ11 mutations showed no 86Rb efflux when the mutant channels were activated, thus confirming the pathogenicity of the mutations. Conclusions: A genetic diagnosis was possible in only 48.6% of patients with mutations in the ABCC8 gene being the commonest cause. Recessively inherited mutations in the ABCC8/ KCNJ11 are associated with diazoxide unresponsive disease. However, the phenotype associated with dominant ABCC8/ KCNJ11 mutations is variable, ranging from mild medically responsive CHI to severe early onset CHI requiring a near total pancreatectomy. In adults, dominant ABCC8/ KCNJ11 mutations may also be an important cause of dominantly inherited early onset diabetes mellitus. Patients with hyperinsulinism due to mutations in the GLUD1 gene have a high risk of epilepsy and may have normal serum ammonia concentrations. Hence GLUD1 mutational analysis may be indicated in patients with leucine sensitivity; even in the absence of hyperammonaemia. Mutations in the HADH gene are associated with protein induced HH due to leucine sensitivity, suggesting a novel biochemical pathway by which HADH regulates leucine induced insulin secretion. Patients with CHI due HADH gene mutations may have normal acylcarnitines and urine organic acids. Hence, sequencing of HADH must be considered in patients with diazoxide responsive HH from consanguineous families, even in the absence of these features. In this large series, HNF4A mutations were the third common cause of diazoxide responsive CHI causing both transient and persistent HH, even in the absence of a family history of diabetes. HNF4A sequence analysis must hence be considered in all patients diagnosed with HH in the first week of life, irrespective of a family history of diabetes mellitus. Future Work: The vast majority of patients with diazoxide responsive CHI had no mutations identified suggesting other novel mechanisms of insulin secretion. Understanding the genetic aetiology of CHI in this large cohort of patients will provide novel insights into pancreatic beta-cell physiology and have implications for hypoglycaemia and diabetes mellitus.

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