Targeted next-generation sequencing reveals MODY in up to 6.5% of antibody-negative diabetes cases listed in the Norwegian Childhood Diabetes Registry.

AIMS/HYPOTHESIS: MODY can be wrongly diagnosed as type 1 diabetes in children. We aimed to find the prevalence of MODY in a nationwide population-based registry of childhood diabetes. METHODS: Using next-generation sequencing, we screened the HNF1A, HNF4A, HNF1B, GCK and INS genes in all 469 children (12.1%) negative for both GAD and IA-2 autoantibodies and 469 antibody-positive matched controls selected from the Norwegian Childhood Diabetes Registry (3882 children). Variants were classified using clinical diagnostic criteria for pathogenicity ranging from class 1 (neutral) to class 5 (pathogenic). RESULTS: We identified 58 rare exonic and splice variants in cases and controls. Among antibody-negative patients, 6.5% had genetic variants of classes 3-5 (vs 2.4% in controls; p = 0.002). For the stricter classification (classes 4 and 5), the corresponding number was 4.1% (vs 0.2% in controls; p = 1.6 × 10-5). HNF1A showed the strongest enrichment of class 3-5 variants, with 3.9% among antibody-negative patients (vs 0.4% in controls; p = 0.0002). Antibody-negative carriers of variants in class 3 had a similar phenotype to those carrying variants in classes 4 and 5. CONCLUSIONS/INTERPRETATION: This is the first study screening for MODY in all antibody-negative children in a nationwide population-based registry. Our results suggest that the prevalence of MODY in antibody-negative childhood diabetes may reach 6.5%. One-third of these MODY cases had not been recognised by clinicians. Since a precise diagnosis is important for treatment and genetic counselling, molecular screening of all antibody-negative children should be considered in routine diagnostics.

Glycogenin-2 is dispensable for liver glycogen synthesis and glucagon-stimulated glucose release.

CONTEXT: The synthesis of glycogen is initiated by glycogenin. In humans, glycogenin-1 is expressed ubiquitously, whereas glycogenin-2 (GN2) is highly expressed in liver. It has therefore been suggested that GN2 is a liver isoform of glycogenin. In a search for possible copy number variations associated with monogenic diabetes, we identified a 102-kb deletion of the X chromosome involving the entire GYG2 gene (encoding GN2) in 2 families. OBJECTIVE: The purpose of this study was to test whether male GYG2 deletion carriers had abnormal glucose metabolism and/or glycogen synthesis. DESIGN, SETTING, AND PATIENTS: Two families with diabetes and a GYG2 deletion were investigated with medical history and examination, glucagon stimulation tests, and liver biopsies. RESULTS: We identified a GYG2 deletion in 3 members of family 1, 8 members of family 2, and 1 blood donor. The deletion showed no clear cosegregation with diabetes. Deletion carriers reported no symptoms related to fasting. Results of cardiac examination and abdominal ultrasound imaging were normal. A glucagon stimulation test in 4 male deletion carriers showed a mean rise in plasma glucose of 3.6 mmol/L (95% confidence interval, 2.9-4.2) compared with 2.8 mmol/L (95% confidence interval, 2.2-3.4) in control subjects. Liver biopsy specimens did not show clear morphologic changes by light microscopy and showed the presence of both α- and ß-glycogen by electron microscopy. We detected GYG1 but not GYG2 mRNA expression in the liver biopsy specimens. CONCLUSIONS: This is the first evaluation of humans without GN2 expression. Our data indicate that GN2 is not required for liver glycogen synthesis and glucagon-stimulated glucose release.

GCK-MODY diabetes as a protein misfolding disease: the mutation R275C promotes protein misfolding, self-association and cellular degradation.

GCK-MODY, dominantly inherited mild hyperglycemia, is associated with more than 600 mutations in the glucokinase gene. Different molecular mechanisms have been shown to explain GCK-MODY. Here, we report a Pakistani family harboring the glucokinase mutation c.823C>T (p.R275C). The recombinant and in cellulo expressed mutant pancreatic enzyme revealed slightly increased enzyme activity (kcat) and normal affinity for α-D-glucose, and resistance to limited proteolysis by trypsin comparable with wild-type. When stably expressed in HEK293 cells and MIN6 ß-cells (at different levels), the mutant protein appeared misfolded and unstable with a propensity to form dimers and aggregates. Its degradation rate was increased, involving the lysosomal and proteasomal quality control systems. On mutation, a hydrogen bond between the R275 side-chain and the carbonyl oxygen of D267 is broken, destabilizing the F260-L271 loop structure and the protein. This promotes the formation of dimers/aggregates and suggests that an increased cellular degradation is the molecular mechanism by which R275C causes GCK-MODY.

SUMOylation of pancreatic glucokinase regulates its cellular stability and activity.

Glucokinase is the predominant hexokinase expressed in hepatocytes and pancreatic ß-cells, with a pivotal role in regulating glucose-stimulated insulin secretion, illustrated by glucokinase gene mutations causing monogenic diabetes and congenital hyperinsulinemic hypoglycemia. A complex tissue-specific network of mechanisms regulates this enzyme, and a major unanswered question in glucokinase biology is how post-translational modifications control the function of the enzyme. Here, we show that the pancreatic isoform of human glucokinase is SUMOylated in vitro, using recombinant enzymes, and in insulin-secreting model cells. Three N-terminal lysines unique for the pancreatic isoform (Lys-12/Lys-13 and/or Lys-15) may represent one SUMOylation site, with an additional site (Lys-346) common for the pancreatic and the liver isoform. SUMO-1 and E2 overexpression stabilized preferentially the wild-type human pancreatic enzyme in MIN6 ß-cells, and SUMOylation increased the catalytic activity of recombinant human glucokinase in vitro and also of glucokinase in target cells. Small ubiquitin-like modifier conjugation represents a novel form of post-translational modification of the enzyme, and it may have an important regulatory function in pancreatic ß-cells.

GCK-MODY diabetes associated with protein misfolding, cellular self-association and degradation.

GCK-MODY, dominantly inherited mild fasting hyperglycemia, has been associated with >600 different mutations in the glucokinase (GK)-encoding gene (GCK). When expressed as recombinant pancreatic proteins, some mutations result in enzymes with normal/near-normal catalytic properties. The molecular mechanism(s) of GCK-MODY due to these mutations has remained elusive. Here, we aimed to explore the molecular mechanisms for two such catalytically 'normal' GCK mutations (S263P and G264S) in the F260-L270 loop of GK. When stably overexpressed in HEK293 cells and MIN6 ß-cells, the S263P- and G264S-encoded mutations generated misfolded proteins with an increased rate of degradation (S263P>G264S) by the protein quality control machinery, and a propensity to self-associate (G264S>S263P) and form dimers (SDS resistant) and aggregates (partly Triton X-100 insoluble), as determined by pulse-chase experiments and subcellular fractionation. Thus, the GCK-MODY mutations S263P and G264S lead to protein misfolding causing destabilization, cellular dimerization/aggregation and enhanced rate of degradation. In silico predicted conformational changes of the F260-L270 loop structure are considered to mediate the dimerization of both mutant proteins by a domain swapping mechanism. Thus, similar properties may represent the molecular mechanisms for additional unexplained GCK-MODY mutations, and may also contribute to the disease mechanism in other previously characterized GCK-MODY inactivating mutations.

Familial occurrence of neonatal diabetes with duplications in chromosome 6q24: treatment with sulfonylurea and 40-yr follow-up.

We present a Norwegian family, followed since 1967, with a chromosome 6q24 duplication in two siblings with neonatal diabetes, in their non-diabetic father, and in a female (third generation) with adult-onset diabetes. The parents (first generation) were healthy and non-consanguineous. After a miscarriage, the couple had two infants with birth weights of 1780 and 1620 g, respectively, both of whom died on their second day of life. Patient I (male, weight 1840 g at term) had a blood glucose level of 33 mmol/L on day 6. He was treated with insulin for 3 months. In adult life he had permanent diabetes, treated with oral hypoglycemic agents. At 43 yr of age, there were no diabetic late complications. Patient II (female, birth weight 1440 g at term) had an increasing blood glucose of 55 mmol/L on day 13. She received insulin treatment for 12.5 months. Subsequently, she was successfully treated with sulfonylurea (tolbutamide) for 10 yr. At 11 yr of age, insulin was again considered necessary. At 40 yr of age, no diabetic late complications were detected. Patient III had a birth weight of 2630 g at term and no diabetic symptoms as a neonate. She had insulin-requiring diabetes from age 19. We conclude that (i) neonatal diabetes with chromosome 6q24 duplications may become a permanent disease in adult life; (ii) this chromosome anomaly may also be associated with adult-onset diabetes; (iii) sulfonylurea treatment may be attempted, and (iv) late diabetic complications may be absent, even after more than 40 yr.

HNF1B mutation in a Turkish child with renal and exocrine pancreas insufficiency, diabetes and liver disease.

A small-for-gestational age female infant presented with bilateral hypoplastic kidneys at 3 months of age. She developed chronic renal insufficiency. Insulin-requiring, non-autoimmune diabetes was documented at 6 years of age. She had mild steatosis and iron deposition in the liver, and mal-development of pancreas. Genetic studies revealed a heterozygous mutation (S148L) of the HNF1B gene, compatible with an HNF1B-MODY phenotype (MODY5). This is the first case of HNF1B-MODY reported from Turkey and represents a particularly severe phenotype of the disease.

Binding of ATP at the active site of human pancreatic glucokinase--nucleotide-induced conformational changes with possible implications for its kinetic cooperativity.

Glucokinase (GK) is the central player in glucose-stimulated insulin release from pancreatic ß-cells, and catalytic activation by α-D-glucose binding has a key regulatory function. Whereas the mechanism of this activation is well understood, on the basis of crystal structures of human GK, there are no similar structural data on ATP binding to the ligand-free enzyme and how it affects its conformation. Here, we report on a conformational change induced by the binding of adenine nucleotides to human pancreatic GK, as determined by intrinsic tryptophan fluorescence, using the catalytically inactive mutant form T228M to correct for the inner filter effect. Adenosine-5'-(ß,γ-imido)triphosphate and ATP bind to the wild-type enzyme with apparent [L](0.5) (ligand concentration at half-maximal effect) values of 0.27±0.02 mm and 0.78±0.14 mm, respectively. The change in protein conformation was further supported by ATP inhibition of the binding of the fluorescent probe 8-anilino-1-naphthalenesulfonate and limited proteolysis by trypsin, and by molecular dynamic simulations. The simulations provide a first insight into the dynamics of the binary complex with ATP, including motion of the flexible surface/active site loop and partial closure of the active site cleft. In the complex, the adenosine moiety is packed between two α-helices and stabilized by hydrogen bonds (with Thr228, Thr332, and Ser336) and hydrophobic interactions (with Val412 and Leu415). Combined with enzyme kinetic analyses, our data indicate that the ATP-induced changes in protein conformation may have implications for the kinetic cooperativity of the enzyme.

[Progress in diabetes genetics]. / Fremskritt innen diabetesgenetikk.

BACKGROUND: Diabetes is classified as Type 1 diabetes, Type 2 diabetes, gestational diabetes and other types. Our goal was to provide an overview of new genetic knowledge of monogenic and type 2 diabetes. MATERIAL AND METHOD: The article is based on literature identified through a non-systematic search in PubMed and own experience concerning research in diabetes genetics and treatment of patients with monogenic diabetes. RESULTS: 18 genes have been found for which one single mutation may cause diabetes. The most common causes for such monogenic diabetes are mutations in the genes KCNJ11, ABCC8 and INS when the condition is diagnosed at the age 0 - 6 months, and in the genes HNF1A, GCK, HNF4A and HNF1B when the diagnosis is made later than six months of age. Genetic testing is appropriate in assessment of monogenic diabetes, because antidiabetic tablets rather that insulin injections can be used to treat patients with mutations in certain genes; i.e. KCNJ11, ABCC8, HNF1A and HNF4A. Genome-wide association studies have recently identified about 20 genetic variants that increase the risk of Type 2 diabetes, but which have a low predictive value for development of disease. How these genetic variants can cause Type 2 diabetes has not been assessed and clinical relevance remains to be shown. INTERPRETATION: So far, genetic findings only affect diagnosis and treatment of monogenic diabetes.

DNA hypomethylation, transient neonatal diabetes, and prune belly sequence in one of two identical twins.

One known genetic mechanism for transient neonatal diabetes is loss of methylation at 6q24. The etiology of prune belly sequence is unknown but a genetic defect, affecting the mesoderm from which the triad abdominal muscle hypoplasia, urinary tract abnormalities, and cryptorchidism develop, has been suggested. We investigated a family, including one twin, with transient neonatal diabetes and prune belly sequence. Autoantibody tests excluded type 1 diabetes. Microsatellite marker analysis confirmed the twins being monozygotic. We identified no mutations in ZFP57, KCNJ11, ABCC8, GCK, HNF1A, HNF1B, HNF3B, IPF1, PAX4, or ZIC3. The proband had loss of methylation at the 6q24 locus TNDM and also at the loci IGF2R, DIRAS3, and PEG1, while the other family members, including the healthy monozygotic twin, had normal findings. The loss of methylation on chromosome 6q24 and elsewhere may indicate a generalized maternal hypomethylation syndrome, which accounts for both transient neonatal diabetes and prune belly sequence.

[Molecular genetic diagnostics in syndromes associated with the RAS/MAPK signalling pathway]. / Mutasjonsdiagnostikk ved syndromer knyttet til RAS/MAPK-signalveien.

BACKGROUND: Mutations in genes of the mitogen-activated protein kinase (MAPK) cascade have recently been shown to cause several syndromes characterized by dysmorphic facial features, growth retardation, cognitive impairment, heart disease and cutaneous abnormalities. This signalling pathway involves RAS and RAF proteins, and is central in the regulation of normal growth and the development of cancer. MATERIAL AND METHODS: We have studied 23 Norwegian patients for whom there was a clinical suspicion of Costello, Noonan or cardio-facio-cutaneous syndrome. Patients suspected of having Noonan syndrome had previously tested negative for mutations in the tyrosine phosphatase gene PTPN11. The material was examined for mutations in the HRAS, KRAS, RAF1 and BRAF genes. Two patients are described to illustrate diagnostic challenges and the usefulness of genetic testing. RESULTS: Ten of 23 patients (43 %) had mutations affecting the RAS/MAPK signalling pathway. Mutations in HRAS were most common (five cases), while three patients had mutations in KRAS and two in RAF1. Spontaneous mutations were demonstrated in eight cases. Our data indicate an annual incidence of 1-2 new cases of congenital RAS/RAF mutations in Norway. INTERPRETATION: Upon clinical suspicion of syndromes of the RAS/MAPK signalling pathway, molecular genetic analyses may be essential for a correct diagnosis. Certain mutations are associated with an increased cancer risk, exemplifying that results of genetic laboratory testing may influence medical management.

De novo HRAS and KRAS mutations in two siblings with short stature and neuro-cardio-facio-cutaneous features.

Mutations in genes involved in Ras signalling cause Noonan syndrome and other disorders characterised by growth disturbances and variable neuro-cardio-facio-cutaneous features. We describe two sisters, who presented with dysmorphic features, hypotonia, retarded growth and psychomotor retardation. The patients were initially diagnosed with Costello syndrome, an autosomal recessive inheritance was assumed. Remarkably, however, we identified a germline HRAS mutation (G12A) in one sister and a germline KRAS mutation (F156L) in her sibling. Both mutations had arisen de novo. The F156L mutant K-Ras protein accumulated in the active, guanosine triphosphate-bound conformation and affected downstream signalling. The patient harbouring this mutation was followed for three decades, and her cardiac hypertrophy gradually normalised. However, she developed severe epilepsy with hippocampal sclerosis and atrophy. The occurrence of distinct de novo mutations adds to variable expressivity and gonadal mosaicism as possible explanations of how an autosomal dominant disease may manifest as an apparently recessive condition.

Co-occurrence of vitamin D-dependent rickets type 1 and phenylketonuria.

UNLABELLED: Vitamin D-dependent rickets type 1 (VDDR1) was diagnosed in a 15-month-old girl with well-controlled phenylketonuria (PKU). The patient was homozygous for the PAH mutation L249F. The PAH and CYP27B1 genes are both located on the long arm of chromosome 12 and could possibly have been inherited from a common ancestor. The parents were not aware of any ancestral relationship and the patient was compound heterozygous for two different CYP27B1 mutations (R389H and S416X). Her mutations were shown to originate from each of her four grandparents. In Norway, the co-occurrence of PKU and VDDR1 is expected to occur by chance one to two times per billion births. CONCLUSION: The extremely rare co-occurrence of VDDR1 and PKU requires careful genetic work-up and close attention to family information, but the combined treatment of the two metabolic disorders may not create special problems.

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.

Catalytic activation of human glucokinase by substrate binding: residue contacts involved in the binding of D-glucose to the super-open form and conformational transitions.

alpha-D-Glucose activates glucokinase (EC 2.7.1.1) on its binding to the active site by inducing a global hysteretic conformational change. Using intrinsic tryptophan fluorescence as a probe on the alpha-D-glucose induced conformational changes in the pancreatic isoform 1 of human glucokinase, key residues involved in the process were identified by site-directed mutagenesis. Single-site W-->F mutations enabled the assignment of the fluorescence enhancement (DeltaF/F(0)) mainly to W99 and W167 in flexible loop structures, but the biphasic time course of DeltaF/F(0) is variably influenced by all tryptophan residues. The human glucokinase-alpha-D-glucose association (K(d) = 4.8 +/- 0.1 mm at 25 degrees C) is driven by a favourable entropy change (DeltaS = 150 +/- 10 J.mol(-1).K(-1)). Although X-ray crystallographic studies have revealed the alpha-d-glucose binding residues in the closed state, the contact residues that make essential contributions to its binding to the super-open conformation remain unidentified. In the present study, we combined functional mutagenesis with structural dynamic analyses to identify residue contacts involved in the initial binding of alpha-d-glucose and conformational transitions. The mutations N204A, D205A or E256A/K in the L-domain resulted in enzyme forms that did not bind alpha-D-glucose at 200 mm and were essentially catalytically inactive. Our data support a molecular dynamic model in which a concerted binding of alpha-D-glucose to N204, N231 and E256 in the super-open conformation induces local torsional stresses at N204/D205 propagating towards a closed conformation, involving structural changes in the highly flexible interdomain connecting region II (R192-N204), helix 5 (V181-R191), helix 6 (D205-Y215) and the C-terminal helix 17 (R447-K460).

Mutations in the insulin gene can cause MODY and autoantibody-negative type 1 diabetes.

OBJECTIVE: Mutations in the insulin (INS) gene can cause neonatal diabetes. We hypothesized that mutations in INS could also cause maturity-onset diabetes of the young (MODY) and autoantibody-negative type 1 diabetes. RESEARCH DESIGN AND METHODS: We screened INS in 62 probands with MODY, 30 probands with suspected MODY, and 223 subjects from the Norwegian Childhood Diabetes Registry selected on the basis of autoantibody negativity or family history of diabetes. RESULTS: Among the MODY patients, we identified the INS mutation c.137G>A (R46Q) in a proband, his diabetic father, and a paternal aunt. They were diagnosed with diabetes at 20, 18, and 17 years of age, respectively, and are treated with small doses of insulin or diet only. In type 1 diabetic patients, we found the INS mutation c.163C>T (R55C) in a girl who at 10 years of age presented with ketoacidosis and insulin-dependent, GAD, and insulinoma-associated antigen-2 (IA-2) antibody-negative diabetes. Her mother had a de novo R55C mutation and was diagnosed with ketoacidosis and insulin-dependent diabetes at 13 years of age. Both had residual beta-cell function. The R46Q substitution changes an invariant arginine residue in position B22, which forms a hydrogen bond with the glutamate at A17, stabilizing the insulin molecule. The R55C substitution involves the first of the two arginine residues localized at the site of proteolytic processing between the B-chain and the C-peptide. CONCLUSIONS: Our findings extend the phenotype of INS mutation carriers and suggest that INS screening is warranted not only in neonatal diabetes, but also in MODY and in selected cases of type 1 diabetes.

Management of neonatal and infancy-onset diabetes mellitus.

Diabetes mellitus is a rare disorder during the first 2 years of life, amounting to about 3-5% of all cases diagnosed before the fifteenth birthday. However, in spite of low numerical values, this is an important diagnosis, since we are dealing with a vulnerable age group with major and special problems related to diagnosis, treatment and psychosocial follow-up. Efforts should be made to establish a molecular genetic diagnosis as early as possible (e.g. homozygous glucokinase deficiency, defects of the ATP-sensitive potassium channel, chromosome 6 imprinting abnormalities). This is particularly important, since patients with Kir6.2 and SUR1 defects can now be treated with oral sulfonylureas. Major advancements have been obtained and continue to be made with respect to diagnosis and classification. Differentiation between transient and permanent neonatal diabetes can only be done after long-term follow-up. Patients should be scrutinized for comorbidity (e.g. celiac disease, Wolcott-Rallison syndrome). Type 1 diabetes is probably the most prevalent subtype, particularly after the first year of life. Insulin treatment in infancy continues to represent major technical, medical and psychological challenges. Family support is mandatory and close attention should be paid to psychosocial issues.

Studies in 3,523 Norwegians and meta-analysis in 11,571 subjects indicate that variants in the hepatocyte nuclear factor 4 alpha (HNF4A) P2 region are associated with type 2 diabetes in Scandinavians.

OBJECTIVE: Recent publications have found an association between common variants near the hepatocyte nuclear factor 4 alpha (HNF4A) P2 promoter and type 2 diabetes in some populations but not in others, and the role for HNF4A in type 2 diabetes has remained unclear. In an attempt to address these inconsistencies, we investigated HNF4A single nucleotide polymorphisms (SNPs) in a large population-based sample and included a meta-analysis of published studies. RESEARCH DESIGN AND METHODS: We genotyped 12 SNPs in the HNF4A region in a Norwegian population-based sample of 1,644 individuals with type 2 diabetes and 1,879 control subjects (the Nord-Trøndelag Health Study [HUNT] 2). We combined our data with all previously published case/control studies and performed a meta-analysis. RESULTS: Consistent with initial studies, we found a trend toward association for the SNPs rs1884613 (odds ratio [OR] 1.17 [95% CI 1.03-1.35]) and rs2144908 (1.21 [1.05-1.38]) in the P2 region and for rs4812831 (1.21 [1.02-1.44]), located 34 kb downstream of the P2 promoter. Meta-analysis, comprising 12,292 type 2 diabetic case and 15,519 control subjects, revealed a nonsignificant OR of 1.05 (95% CI 0.98-1.12) but with significant heterogeneity between the populations. We therefore performed a subanalysis including only the data for subjects from Scandinavia. Among the 4,000 case and 7,571 control Scandinavian subjects, a pooled OR of 1.14 (1.06-1.23), P = 0.0004, was found for the SNP rs1884613. CONCLUSIONS: Our results suggest that variation in the HNF4A region is associated with type 2 diabetes in Scandinavians, highlighting the importance of exploring small genetic effects in large, homogenous populations.