The HNF1A mutant Ala180Val: Clinical challenges in determining causality of a rare HNF1A variant in familial diabetes.

AIMS: Heterozygous mutations in hepatocyte nuclear factor-1A (HNF1A) cause maturity-onset diabetes of the young type 3 (MODY3). Our aim was to compare two families with suspected dominantly inherited diabetes and a new HNF1A variant of unknown clinical significance. METHODS: The HNF1A gene was sequenced in two independently recruited families from the Norwegian MODY Registry. Both familes were phenotyped clinically and biochemically. Microsatellite markers around and within the HNF1A locus were used for haplotyping. Chromosomal linkage analysis was performed in one family, and whole-exome sequencing was undertaken in two affected family members from each family. Transactivation activity, DNA binding and nuclear localization of wild type and mutant HNF-1A were assessed. RESULTS: The novel HNF1A variant c.539C>T (p.Ala180Val) was found in both families. The variant fully co-segregated with diabetes in one family. In the other family, two subjects with diabetes mellitus and one with normal glucose levels were homozygous variant carriers. Chromosomal linkage of diabetes to the HNF1A locus or to other genomic regions could not be established. The protein functional studies did not reveal significant differences between wild type and variant HNF-1A. In each family, whole-exome sequencing failed to identify any other variant that could explain the disease. CONCLUSIONS: The HNF1A variant p.Ala180Val does not seem to cause MODY3, although it may confer risk for type 2 diabetes mellitus. Our data demonstrate challenges in causality evaluation of rare variants detected in known diabetes genes.

Prevalence of monogenic diabetes in the population-based Norwegian Childhood Diabetes Registry.

AIMS/HYPOTHESIS: Monogenic diabetes (MD) might be misdiagnosed as type 1 diabetes. The prevalence of MD among children with apparent type 1 diabetes has not been established. Our aim was to estimate the prevalence of common forms of MD in childhood diabetes. METHODS: We investigated 2,756 children aged 0-14 years with newly diagnosed diabetes who had been recruited to the nationwide population-based Norwegian Childhood Diabetes Registry (NCDR), from July 2002 to March 2012. Completeness of ascertainment was 91%. Children diagnosed with diabetes who were under12 months of age were screened for mutations in KCNJ11, ABCC8 and INS. Children without GAD and protein tyrosine phosphatase-like protein antibodies were screened in two ways. Those who had a parent with diabetes were screened for mutations in HNF1A, HNF4A, INS and MT-TL1. Children with HbA1c <7.5% (<58 mmol/mol) and no insulin requirement were screened for mutations in GCK. Finally, we searched the Norwegian MODY Registry for children with genetically verified MD. RESULTS: We identified 15 children harbouring a mutation in HNF1A, nine with one in GCK, four with one in KCNJ11, one child with a mutation in INS and none with a mutation in MT-TL1. The minimum prevalence of MD in the NCDR was therefore 1.1%. By searching the Norwegian MODY Registry, we found 24 children with glucokinase-MODY, 15 of whom were not present in the NCDR. We estimated the minimum prevalence of MD among Norwegian children to be 3.1/100,000. CONCLUSIONS/INTERPRETATION: This is the first prevalence study of the common forms of MD in a nationwide, population-based registry of childhood diabetes. We found that 1.1% of patients in the Norwegian Childhood Diabetes Registry had MD.

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.

Epidermal growth factor receptor efficiently activates mitogen-activated protein kinase in HeLa cells and Hep2 cells conditionally defective in clathrin-dependent endocytosis.

Epidermal growth factor (EGF)-induced signaling was investigated in cells conditionally defective in clathrin-dependent endocytosis by overexpression of K44A dynamin in HeLa cells and potassium depletion in Hep2 cells. Overexpression of mutant dynamin disrupts high-affinity EGF-EGF receptor (EGFR) interaction (T. Ringerike, E. Stang, L. E. Johannessen, D. Sandnes, F. O. Levy, and I. H. Madshus, 1998, J. Biol. Chem. 273, 16639-16642). However, the EGFR substrates Shc and c-Cbl were as efficiently tyrosine phosphorylated in endocytosis-deficient HeLa cells exhibiting only low-affinity EGFRs as in HeLa cells with intact endocytosis and with both high- and low-affinity EGFRs. Both Raf and mitogen-activated protein kinase (MAPK) were activated to the same extent and with the same kinetics. HeLa cells distributed equally in the cell cycle regardless of EGFR internalization. Upon potassium depletion of Hep2 cells, EGF-induced EGFR endocytosis was inhibited. However, the EGFR and MAPK were efficiently activated by EGF in both the absence and the presence of clathrin-dependent endocytosis. The EGFR was weakly tyrosine phosphorylated by potassium depletion even in the absence of EGF, and this activation resulted in detectable activation of MAPK. Our results demonstrate that internalization of EGFR by clathrin-dependent endocytosis is not required for activation of MAPK.