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Featured researches published by Janne Molnes.


Nature Genetics | 2013

Assessing the phenotypic effects in the general population of rare variants in genes for a dominant Mendelian form of diabetes.

Jason Flannick; Nicola L. Beer; Alexander G. Bick; Vineeta Agarwala; Janne Molnes; Namrata Gupta; Noël P. Burtt; Jose C. Florez; James B. Meigs; Herman A. Taylor; Valeriya Lyssenko; Henrik Irgens; Ervin R. Fox; Frank Burslem; Stefan Johansson; M. Julia Brosnan; Jeff Trimmer; Christopher Newton-Cheh; Tiinamaija Tuomi; James G. Wilson; Christopher J. O'Donnell; Sekar Kathiresan; Joel N. Hirschhorn; Pål R. Njølstad; Tim Rolph; Jonathan G. Seidman; Stacey B. Gabriel; D. R. Cox; Christine E. Seidman; Leif Groop

Genome sequencing can identify individuals in the general population who harbor rare coding variants in genes for Mendelian disorders and who may consequently have increased disease risk. Previous studies of rare variants in phenotypically extreme individuals display ascertainment bias and may demonstrate inflated effect-size estimates. We sequenced seven genes for maturity-onset diabetes of the young (MODY) in well-phenotyped population samples (n = 4,003). We filtered rare variants according to two prediction criteria for disease-causing mutations: reported previously in MODY or satisfying stringent de novo thresholds (rare, conserved and protein damaging). Approximately 1.5% and 0.5% of randomly selected individuals from the Framingham and Jackson Heart Studies, respectively, carry variants from these two classes. However, the vast majority of carriers remain euglycemic through middle age. Accurate estimates of variant effect sizes from population-based sequencing are needed to avoid falsely predicting a substantial fraction of individuals as being at risk for MODY or other Mendelian diseases.


The EMBO Journal | 2005

Kir6.2 mutations causing neonatal diabetes provide new insights into Kir6.2–SUR1 interactions

Paolo Tammaro; Christophe Girard; Janne Molnes; Pål R. Njølstad; Frances M. Ashcroft

ATP‐sensitive K+ (KATP) channels, comprised of pore‐forming Kir6.2 and regulatory SUR1 subunits, play a critical role in regulating insulin secretion. Binding of ATP to Kir6.2 inhibits, whereas interaction of MgATP with SUR1 activates, KATP channels. We tested the functional effects of two Kir6.2 mutations (Y330C, F333I) that cause permanent neonatal diabetes mellitus, by heterologous expression in Xenopus oocytes. Both mutations reduced ATP inhibition and increased whole‐cell currents, which in pancreatic β‐cells is expected to reduce insulin secretion and precipitate diabetes. The Y330C mutation reduced ATP inhibition both directly, by impairing ATP binding (and/or transduction), and indirectly, by stabilizing the intrinsic open state of the channel. The F333I mutation altered ATP binding/transduction directly. Both mutations also altered Kir6.2/SUR1 interactions, enhancing the stimulatory effect of MgATP (which is mediated via SUR1). This effect was particularly dramatic for the Kir6.2‐F333I mutation, and was abolished by SUR1 mutations that prevent MgATP binding/hydrolysis. Further analysis of F333I heterozygous channels indicated that at least three SUR1 must bind/hydrolyse MgATP to open the mutant KATP channel.


PLOS ONE | 2012

Exome Sequencing and Genetic Testing for MODY

Stefan Johansson; Henrik Irgens; Kishan K. Chudasama; Janne Molnes; Jan Aerts; Francisco S. Roque; Inge Jonassen; Shawn Levy; Kari Lima; Per M. Knappskog; Graeme I. Bell; Pål R. Njølstad

Context Genetic testing for monogenic diabetes is important for patient care. Given the extensive genetic and clinical heterogeneity of diabetes, exome sequencing might provide additional diagnostic potential when standard Sanger sequencing-based diagnostics is inconclusive. Objective The aim of the study was to examine the performance of exome sequencing for a molecular diagnosis of MODY in patients who have undergone conventional diagnostic sequencing of candidate genes with negative results. Research Design and Methods We performed exome enrichment followed by high-throughput sequencing in nine patients with suspected MODY. They were Sanger sequencing-negative for mutations in the HNF1A, HNF4A, GCK, HNF1B and INS genes. We excluded common, non-coding and synonymous gene variants, and performed in-depth analysis on filtered sequence variants in a pre-defined set of 111 genes implicated in glucose metabolism. Results On average, we obtained 45 X median coverage of the entire targeted exome and found 199 rare coding variants per individual. We identified 0–4 rare non-synonymous and nonsense variants per individual in our a priori list of 111 candidate genes. Three of the variants were considered pathogenic (in ABCC8, HNF4A and PPARG, respectively), thus exome sequencing led to a genetic diagnosis in at least three of the nine patients. Approximately 91% of known heterozygous SNPs in the target exomes were detected, but we also found low coverage in some key diabetes genes using our current exome sequencing approach. Novel variants in the genes ARAP1, GLIS3, MADD, NOTCH2 and WFS1 need further investigation to reveal their possible role in diabetes. Conclusion Our results demonstrate that exome sequencing can improve molecular diagnostics of MODY when used as a complement to Sanger sequencing. However, improvements will be needed, especially concerning coverage, before the full potential of exome sequencing can be realized.


Pediatric Diabetes | 2008

Diagnostic screening of MODY2/GCK mutations in the Norwegian MODY Registry.

Jørn V. Sagen; Lise Bjørkhaug; Janne Molnes; Helge Ræder; Louise Grevle; Oddmund Søvik; Pål R. Njølstad

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.


FEBS Journal | 2008

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

Janne Molnes; Lise Bjørkhaug; Oddmund Søvik; Pål R. Njølstad; Torgeir Flatmark

α‐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 α‐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 (ΔF/F0) mainly to W99 and W167 in flexible loop structures, but the biphasic time course of ΔF/F0 is variably influenced by all tryptophan residues. The human glucokinase–α‐d‐glucose association (Kd = 4.8 ± 0.1 mm at 25 °C) is driven by a favourable entropy change (ΔS = 150 ± 10 J·mol−1·K−1). Although X‐ray crystallographic studies have revealed the α‐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 α‐d‐glucose and conformational transitions. The mutations N204A, D205A or E256A/K in the L‐domain resulted in enzyme forms that did not bind α‐d‐glucose at 200 mm and were essentially catalytically inactive. Our data support a molecular dynamic model in which a concerted binding of α‐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).


Journal of Biological Chemistry | 2007

Allosteric Activation of Human Glucokinase by Free Polyubiquitin Chains and Its Ubiquitin-dependent Cotranslational Proteasomal Degradation

Lise Bjørkhaug; Janne Molnes; Oddmund Søvik; Pål R. Njølstad; Torgeir Flatmark

Human glucokinase (hGK) is a monomeric enzyme highly regulated in pancreatic β-cells (isoform 1) and hepatocytes (isoforms 2 and 3). Although certain cellular proteins are known to either stimulate or inhibit its activity, little is known about post-translational modifications of this enzyme and their possible regulatory functions. In this study, we have identified isoforms 1 and 2 of hGK as novel substrates for the ubiquitin-conjugating enzyme system of the rabbit reticulocyte lysate. Both isoforms were polyubiquitinated on at least two lysine residues, and mutation analysis indicated that multiple lysine residues functioned as redundant acceptor sites. Deletion of its C-terminal α-helix, as part of a ubiquitin-interacting motif, affected the polyubiquitination at one of the sites and resulted in a completely inactive enzyme. Evidence is presented that poly/multiubiquitination of hGK in vitro serves as a signal for proteasomal degradation of the newly synthesized protein. Moreover, the recombinant hGK was found to interact with and to be allosterically activated up to ∼1.4-fold by purified free pentaubiquitin chains at ∼100 nm (with an apparent EC50 of 93 nm), and possibly also by unidentified polyubiquitinated proteins assigned to their equilibrium binding to the ubiquitin-interacting motif site. The affinity of pentaubiquitin binding to hGK is regulated by the ligand (d-glucose)-dependent conformational state of the site. Both ubiquitination of hGK and its activation by polyubiquitin chains potentially represent physiological regulatory mechanisms for glucokinase-dependent insulin secretion in pancreatic β-cells.


Journal of Biological Chemistry | 2013

SUMOylation of Pancreatic Glucokinase Regulates Its Cellular Stability and Activity

Ingvild Aukrust; Lise Bjørkhaug; Maria Negahdar; Janne Molnes; Bente B. Johansson; Yvonne Müller; Wilhelm Haas; Steven P. Gygi; Oddmund Søvik; Torgeir Flatmark; Rohit N. Kulkarni; Pål R. Njølstad

Background: Glucokinase is a key player in carbohydrate metabolism, but how this enzyme is regulated by post-translational modifications is largely unknown. Results: Glucokinase is SUMO-modified in vitro and in pancreatic β-cells, increasing its activity and stability. Conclusion: SUMOylation of glucokinase is a novel form of modification, regulating its cellular stability and activity. Significance: SUMO conjugation of glucokinase may have an important regulatory function in pancreatic β-cells. Glucokinase is the predominant hexokinase expressed in hepatocytes and pancreatic β-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 β-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 β-cells.


Diabetologia | 2017

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

Bente B. Johansson; Henrik Irgens; Janne Molnes; Paweł Sztromwasser; Ingvild Aukrust; Pétur Benedikt Júlíusson; Oddmund Søvik; Shawn Levy; Torild Skrivarhaug; Geir Joner; Stefan Johansson; Pål R. Njølstad

Aims/hypothesisMODY 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.MethodsUsing 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).ResultsWe 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/interpretationThis 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.


Clinical Genetics | 2013

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

S Jahnavi; Poovazhagi; Mohan; D Bodhini; P Raghupathy; A Amutha; P Suresh Kumar; P Adhikari; M Shriraam; T Kaur; Anamika Das; Janne Molnes; Pål R. Njølstad; Ranjit Unnikrishnan; Radha

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.


FEBS Journal | 2011

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

Janne Molnes; Knut Teigen; Ingvild Aukrust; Lise Bjørkhaug; Oddmund Søvik; Torgeir Flatmark; Pål R. Njølstad

Glucokinase (GK) is the central player in glucose‐stimulated insulin release from pancreatic β‐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′‐(β,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.

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Henrik Irgens

Haukeland University Hospital

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Ingvild Aukrust

Haukeland University Hospital

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