Diabetes Publish Ahead of Print, published online October 27, 2010 Congenital hyperinsulinism and glucose hypersensitivity in homozygous and heterozygous carriers of Kir6.2 (KCNJ11) mutation V290M mutation: KATP channel inactivation mechanism and clinical management *Karen J. Loechner2, *Alejandro Akrouh1, Harley T. Kurata1, Carlo Dionisi-Vici3, Arianna Maiorana3, Milena Pizzoferro4, Vittoria Rufini5, Jean de Ville de Goyet6, Carlo Colombo7, Fabrizio Barbetti7, 8, Joseph C. Koster1, and Colin G. Nichols1 *K. J. L. and A. A. contributed equally (1) Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110; (2) Department of Pediatrics, 3341 MBRB, UNC School of Medicine, Chapel Hill, NC 27599; (3) Unit of Metabolic Diseases, Department of Pediatrics, Bambino Gesù Children’s Hospital, Rome, Italy; (4) Unit of Nuclear Medicine, Department of Radiology, Bambino Gesù Children's Hospital, Rome, Italy; (5) Department of Nuclear Medicine, Catholic University of the Sacred Heart, Rome, Italy; (6) Dept. of Surgery, Bambino Gesù Children’s Hospital, Rome, Italy; (7) Laboratory of Monogenic Diabetes, Bambino Gesù Children's Hospital IRCCS, Rome, Italy; (8) Dept. of Internal Medicine, University of Tor Vergata, and Laboratory of Monogenic Diabetes, Bambino Gesù Children's Hospital IRCCS, Rome. Running title: Hyperinsulinism and glucose hyper-responsivity resulting from inactivating mutation in KCNJ11 Address correspondence and reprint requests to: Colin G. Nichols [email protected] or Fabrizio Barbetti [email protected] Submitted 22 May 2010 and accepted 15 October 2010. This is an uncopyedited electronic version of an article accepted for publication in Diabetes. The American Diabetes Association, publisher of Diabetes, is not responsible for any errors or omissions in this version of the manuscript or any version derived from it by third parties. The definitive publisher-authenticated version will be available in a future issue of Diabetes in print and online at http://diabetes.diabetesjournals.org. Copyright American Diabetes Association, Inc., 2010 Hyperinsulinism and glucose hyper-responsivity resulting from inactivating mutation in KCNJ11 + Objective: The ATP-sensitive K -channel (KATP) controls insulin secretion from the islet. Gain- or loss-of-function mutations in channel subunits underlie human neonatal diabetes mellitus (NDM) and congenital hyperinsulinism (HI), respectively. In this study we sought to identify the mechanistic basis of KATP-induced HI in two probands, and characterize the clinical course. Research Design and Methods: We analyzed HI in two probands and characterized the course of clinical treatment in each, as well as properties of mutant KATP channels expressed in COSm6 cells using Rb efflux and patch-clamp methods. Results: We identified mutation V290M in the pore-forming Kir6.2 subunit in each proband. In vitro expression in COSm6 cells supports the mutation resulting in an inactivating phenotype, which leads to significantly reduced activity in intact cells when expressed homomerically, and to a lesser extent when expressed heteromerically with WT subunits. In one heterozygous proband, fluoro-DOPA scan revealed a causal focal lesion, indicating uniparental disomy with loss of heterozygosity. In a second family, the proband, homozygous for the mutation, was diagnosed with severe diazoxide-unresponsive hypersinsulinism at 2 weeks of age. The patient continues to be treated successfully with octreotide and amlodipine. The parents and a male sibling are heterozygous carriers without overt clinical HI. Interestingly, both the mother and the sibling exhibit evidence of abnormally enhanced glucose tolerance. Conclusions: V290M results in inactivating KATP channels that underlies HI. Homozygous individuals may be managed medically, without pancreatectomy. Heterozygous carriers also show evidence of enhanced glucose sensitivity, consistent with incomplete loss of KATP channel activity. + he ATP-sensitive K -channel (KATP) Importantly, the V290M mutation is present regulates insulin secretion from the in the homozygous state in one of the HI- T pancreatic β-cell by coupling changes affected probands, and is heterozygous in the in metabolism to changes in electrical unaffected parents and one sibling. Oral activity. KATP overactivity suppresses insulin glucose tolerance tests on the heterozygous release and causes neonatal diabetes mellitus mother and sibling suggest hyper-responsivity (NDM) (1;2), whereas KATP underactivity in both individuals. causes hypersecretion and congenital hyperinsulinemia (HI) (3;4;5). MATERIALS AND METHODS HI mutations can cause aberrant channel Genetics and Molecular Biology. Genomic synthesis or trafficking, or altered channel DNA was isolated from whole blood using gating (5;6). Mature KATP channels are hetero- the DNeasy Tissue Isolation kit (Qiagen, octomers of four pore-forming Kir6.2 subunits Valencia, CA, USA). KCNJ11 was amplified (KCNJ11) and four sulfonylurea receptor by PCR and directly sequenced. The subunits (ABCC8) (7;8;9). We report a novel identified V290M mutation was engineered Kir6.2 mutation (V290M), identified in two into mouse Kir6.2 cDNA in pCMV6B using unrelated HI probands. V290M reduces the Quikchange site-directed mutagenesis kit channel activity by causing an inactivating (Stratagene, La Jolla, CA), and confirmed by phenotype, explaining the HI outcome. direct sequencing. 2 Hyperinsulinism and glucose hyper-responsivity resulting from inactivating mutation in KCNJ11 Clinical studies. Oral Glucose Tolerance basis to evaluate the safety and efficacy of the Testing (OGTT) D-glucose was given orally two treatment regimens. and blood drawn via peripheral IV at baseline Fluoro-DOPA analysis of pancreas. 18F-L- and then at hourly intervals. Samples were DOPA PET-CT study (4 MBq/Kg of 18F-L- assayed for serum glucose, insulin, and DOPA administered intravenously 45 minutes proinsulin at the Mayo Clinical Laboratory before acquisition) was performed using a (Rochester, MN). For the mother, the glucose hybrid machine (Gemini GXL, Philips Medical load (Glucola) was 75g. For the sibling child, Systems). PET scan was performed under the load was 1g/kg, and the duration of the general anesthesia and glucose infusion to OGTT was truncated to 3 hours due to age. Of maintain normoglycemia, after 6h-fasting, note, behavioral changes (e.g., hunger, without stopping medications. lethargy) that often followed a meal were Expression of KATP channels in COSm6 cells reported for the sibling. Given that . COSm6 cells were cultured in Dulbecco’s heterozygous KATP channel mutations have Modified Eagle Medium plus 10 mM glucose been identified in patients with HI (6), the (DMEM-HG), supplemented with fetal calf mother requested testing for her and her son. serum (FCS, 10%). Cells were transfected This case report was submitted to the IRB at with cDNA using FuGENE 6 Transfection UNC and declared “exempt”. Reagent (Roche Diagnostics, Indianapolis, Continuous Glucose Montoring System IN), and then plated on sterile glass coverslips (CGMS) Due to parental wishes to decrease the overnight prior to patch-clamp experiments. frequency of Octeotide injections, medical Electrophysiological methods. Patch-clamp therapy was adjusted while monitoring under experiments were performed at room CGMS as an off-label use. A sensor was placed temperature on COSm6 cells that fluoresced on three separate occasions for Proband #1 green under UV illumination, 3-5 days post- (CareLink(TM), Medtronic MiniMed, Inc) transfection. Membrane patches were voltage- after application of topical anesthetic. clamped using an Axopatch 1-D amplifier Medtronic (Caremark) provided training to the (Axon Instruments, Union City, CA). All parents on use and how to mark events such as currents were measured at a membrane medication and meals, as well as to corroborate potential of -50mV. Data were collected using hypo- (sensor set at <80) or hyperglycemic the pClamp8.2 software suite (Axon (>200) events detected by external blood Instruments, Union City, CA) and Microsoft glucose meter. Reference ranges were chosen Excel. Bath and pipette control solutions for the alarm settings to avoid hypoglycemia (KINT) contained (mM): 150 KCl, 10 HEPES, and minimize glycosuria, as well as avoid and 1 EGTA (pH 7.4). Where indicated, ATP excessive fingersticks for the child. Sensors was added to the bathing solution as were placed for a maximum of 5 days and dipotassium salts. Tolbutamide was dissolved corresponded to periods when treated with (1) in KINT from a 100 mM stock solution in 100 Octreotide alone, or (2) Octreotide + mM KOH. Amlodipine. Of note, the child was tapered off Macroscopic 86Rb+ efflux assays. COSm6 cells amlodipine while on CGMS for a period of 2 in 12-well plates were incubated for 24 hr in weeks. After monitoring with Octreotide alone, culture medium containing 86RbCl (1 μCi/mL) amlodipine (0.1mg/kg divided twice daily) was 2 days after transfection. Cells were washed re-introduced for 5 days prior to CGMS testing. twice with Ringer’s solution (Basal) (in mM: Baseline glucose levels and excursions (high 118 NaCl, 2.5 CaCl2, 1.2 KH2PO4, 4.7 KCl, 25 and low) were documented on a continuous NaHCO3, 1.2 MgSO4, 10 HEPES; pH 7.4) with or without metabolic inhibition (MI)(1 mM 2- 3 Hyperinsulinism and glucose hyper-responsivity resulting from inactivating mutation in KCNJ11 deoxy-D-glucose and 2.5 μg/mL oligomycin). H At selected time points, solution