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Journal of Pediatric Endocrinology and Metabolism

Editor-in-Chief: Kiess, Wieland

Ed. by Bereket, Abdullah / Darendeliler, Feyza / Dattani, Mehul / Gustafsson, Jan / Luo, Fei Hong / Mericq, Veronica / Toppari, Jorma

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Volume 29, Issue 5


Improved molecular diagnosis of patients with neonatal diabetes using a combined next-generation sequencing and MS-MLPA approach

Gorka Alkorta-Aranburu / Madina Sukhanova / David Carmody
  • Section of Adult and Pediatric Endocrinology, Diabetes and Metabolism, Department of Medicine, The University of Chicago, Chicago, IL, USA
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Trevor Hoffman / Latrice Wysinger / Jennifer Keller-Ramey / Zejuan Li / Amy Knight Johnson / Frances Kobiernicki / Shaun Botes / Carrie Fitzpatrick / Soma Das / Daniela del Gaudio
Published Online: 2016-02-19 | DOI: https://doi.org/10.1515/jpem-2015-0341


Background: We evaluated a methylation-specific multiplex-ligation-dependent probe amplification (MS-MLPA) assay for the molecular diagnosis of transient neonatal diabetes mellitus (TNDM) caused by 6q24 abnormalities and assessed the clinical utility of using this assay in combination with next generation sequencing (NGS) analysis for diagnosing patients with neonatal diabetes (NDM).

Methods: We performed MS-MLPA in 18 control samples and 42 retrospective NDM cases with normal bi-parental inheritance of chromosome 6. Next, we evaluated 22 prospective patients by combining NGS analysis of 11 NDM genes and the MS-MLPA assay.

Results: 6q24 aberrations were identified in all controls and in 19% of patients with normal bi-parental inheritance of chromosome 6. The MS-MLPA/NGS combined approach identified a genetic cause in ~64% of patients with NDM of unknown etiology.

Conclusions: MS-MLPA is a reliable method to identify all known 6q24 abnormalities and comprehensive testing of all causes reveals a causal mutation in ~64% of patients.

Keywords: imprinting; methylation-specific multiplex ligation dependent probe amplification; neonatal diabetes mellitus; next-generation sequencing


  • 1.

    Greeley SA, Tucker SE, Naylor RN, Bell GI, Philipson LH. Neonatal diabetes mellitus: a model for personalized medicine. Trends Endocrin Met 2010;21:464–72.Web of ScienceGoogle Scholar

  • 2.

    Grulich-Henn J, Wagner V, Thon A, Schober E, Marg W, et al. Entities and frequency of neonatal diabetes: data from the diabetes documentation and quality management system (DPV). Diabetic Med 2010;27:709–12.Web of ScienceGoogle Scholar

  • 3.

    Iafusco D, Massa O, Pasquino B, Colombo C, Iughetti L, et al. Minimal incidence of neonatal/infancy onset diabetes in Italy is 1:90,000 live births. Acta Diabetol 2012;49:405–8.Web of ScienceGoogle Scholar

  • 4.

    Wiedemann B, Schober E, Waldhoer T, Koehle J, Flanagan SE, et al. Incidence of neonatal diabetes in Austria-calculation based on the Austrian Diabetes Register. Pediatr Diabetes 2010;11:18–23.Web of ScienceGoogle Scholar

  • 5.

    Metz C, Cave H, Bertrand AM, Deffert C, Gueguen-Giroux B, et al. Neonatal diabetes mellitus: chromosomal analysis in transient and permanent cases. J Pediatr 2002;141:483–9.Google Scholar

  • 6.

    Gardner RJ, Mackay DJ, Mungall AJ, Polychronakos C, Siebert R, et al. An imprinted locus associated with transient neonatal diabetes mellitus. Hum Mol Genet 2000;9:589–96.Google Scholar

  • 7.

    Pearson ER, Flechtner I, Njølstad PR, Malecki MT, Flanagan SE, et al. Switching from insulin to oral sulfonylureas in patients with diabetes due to Kir6.2 mutations. N Engl J Med 2006;355:467–77.Google Scholar

  • 8.

    Sagen JV, Raeder H, Hathout E, Shehadeh N, Gudmundsson K, et al. Permanent neonatal diabetes due to mutations in KCNJ11 encoding Kir6.2: patient characteristics and initial response to sulfonylurea therapy. Diabetes 2004;53:2713–8.Google Scholar

  • 9.

    Hattersley A, Bruining J, Shield J, Njolstad P, Donaghue KC. The diagnosis and management of monogenic diabetes in children and adolescents. Pediatr Diabetes 2009;10(Suppl 12):33–42.Google Scholar

  • 10.

    Flanagan SE, Patch AM, Mackay DJ, Edghill EL, Gloyn AL, et al. Mutations in ATP-sensitive K+ channel genes cause transient neonatal diabetes and permanent diabetes in childhood or adulthood. Diabetes 2007;56:1930–7.Web of ScienceGoogle Scholar

  • 11.

    Mackay DJ, Temple IK. Transient neonatal diabetes mellitus type 1. Am J Med Genet C Semin Med Genet 2010;154C:335–42.Google Scholar

  • 12.

    Kamiya M, Judson H, Okazaki Y, Kusakabe M, Muramatsu M, et al. The cell cycle control gene ZAC/PLAGL1 is imprinted--a strong candidate gene for transient neonatal diabetes. Hum Mol Genet 2000;9:453–60.Google Scholar

  • 13.

    Arima T, Drewell RA, Oshimura M, Wake N, Surani MA. A novel imprinted gene, HYMAI, is located within an imprinted domain on human chromosome 6 containing ZAC. Genom 2000;67:248–55.Google Scholar

  • 14.

    Varrault A, Bilanges B, Mackay DJ, Basyuk E, Ahr B, et al. Characterization of the methylation-sensitive promoter of the imprinted ZAC gene supports its role in transient neonatal diabetes mellitus. J Biol Chem 2001;276:186536.Google Scholar

  • 15.

    Mackay DJ, Coupe AM, Shield JP, Storr JN, Temple IK, et al. Relaxation of imprinted expression of ZAC and HYMAI in a patient with transient neonatal diabetes mellitus. Hum Genet 2002;110:139–44.Google Scholar

  • 16.

    Bonnefond A, Durand E, Sand O, De Graeve F, Gallina S, et al. Molecular diagnosis of neonatal diabetes mellitus using next-generation sequencing of the whole exome. PloS one 2010;5:e13630.CrossrefGoogle Scholar

  • 17.

    Ellard S, Lango Allen H, De Franco E, Flanagan SE, Hysenaj G, et al. Improved genetic testing for monogenic diabetes using targeted next-generation sequencing. Diabetologia 2013;56:1958–63.Google Scholar

  • 18.

    Alkorta-Aranburu G, Carmody D, Cheng YW, Nelakuditi V, Ma L, et al. Phenotypic heterogeneity in monogenic diabetes: the clinical and diagnostic utility of a gene panel-based next-generation sequencing approach. Mol Genet Metab 2014;113:315–20.Google Scholar

  • 19.

    Dahl F, Gullberg M, Stenberg J, Landegren U, Nilsson M. Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments. Nucleic Acids Res 2005;33:e71.Google Scholar

  • 20.

    Berglund EC, Lindqvist CM, Hayat S, Övernäs E, Henriksson N, et al. Accurate detection of subclonal single nucleotide variants in whole genome amplified and pooled cancer samples using HaloPlex target enrichment. BMC Genom 2013;14:856.Google Scholar

  • 21.

    Alkorta-Aranburu G, Carmody D, Cheng YW, Nelakuditi V, Ma L, et al. Phenotypic heterogeneity in monogenic diabetes: The clinical and diagnostic utility of a gene panel-based next-generation sequencing approach. Mol Genet Metab 2014;113:315–20.Google Scholar

  • 22.

    Richards S, Aziz N, Bale S, Bick D, Das S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015;17:405–24.Web of ScienceGoogle Scholar

  • 23.

    Babenko AP, Polak M, Cave H, Busiah K, Czernichow P, et al. Activating mutations in the ABCC8 gene in neonatal diabetes mellitus. N Engl J Med 2006;355:456–66.Google Scholar

  • 24.

    Gloyn AL, Pearson ER, Antcliff JF, Proks P, Bruining GJ, et al. Activating mutations in the gene encoding the ATP-sensitive potassium-channel subunit Kir6.2 and permanent neonatal diabetes. N Engl J Med 2004;350:1838–49.Google Scholar

  • 25.

    Støy J, Edghill EL, Flanagan SE, Ye H, Paz VP, et al. Insulin gene mutations as a cause of permanent neonatal diabetes. Proc Natl Acad Sci U S A 2007;104:15040–4.Google Scholar

  • 26.

    Lopes JE, Torgerson TR, Schubert LA, Anover SD, Ocheltree EL, et al. Analysis of FOXP3 reveals multiple domains required for its function as a transcriptional repressor. J Immunol 2006;177:3133–42.Google Scholar

  • 27.

    Carmody D, Beca FA, Bell CD, Hwang JL, Dickens JT, et al. Role of noninsulin therapies alone or in combination in chromosome 6q24-related transient neonatal diabetes: sulfonylurea improves but does not always normalize insulin secretion. Diabetes Care 2015;38:e86–7.Web of ScienceGoogle Scholar

  • 28.

    Gardner RJ, Robinson DO, Lamont L, Shield JP, Temple IK. Paternal uniparental disomy of chromosome 6 and transient neonatal diabetes mellitus. Clin Genet 1998;54:522–5.Google Scholar

  • 29.

    Docherty LE, Poole RL, Mattocks CJ, Lehmann A, Temple IK, et al. Further refinement of the critical minimal genetic region for the imprinting disorder 6q24 transient neonatal diabetes. Diabetologia 2010;53:2347–51.Web of ScienceGoogle Scholar

  • 30.

    Mackay DJ, Boonen SE, Clayton-Smith J, Goodship J, Hahnemann JM, et al. A maternal hypomethylation syndrome presenting as transient neonatal diabetes mellitus. Hum Genet 2006;120:262–9.Google Scholar

  • 31.

    Mackay DJ, Callaway JL, Marks SM, White HE, Acerini CL, et al. Hypomethylation of multiple imprinted loci in individuals with transient neonatal diabetes is associated with mutations in ZFP57. Nat Genet 2008;40:949–51.Web of ScienceGoogle Scholar

  • 32.

    Edghill EL, Flanagan SE, Patch AM, Boustred C, Parrish A, et al. Insulin mutation screening in 1,044 patients with diabetes: mutations in the INS gene are a common cause of neonatal diabetes but a rare cause of diabetes diagnosed in childhood or adulthood. Diabetes 2008;57:1034–42.Web of ScienceGoogle Scholar

  • 33.

    Edghill EL, Bingham C, Slingerland AS, Minton JA, Noordam C, et al. Hepatocyte nuclear factor-1 beta mutations cause neonatal diabetes and intrauterine growth retardation: support for a critical role of HNF-1beta in human pancreatic development. Diabetic Med 2006;23:1301–6.Google Scholar

  • 34.

    Naylor RN, Greeley SA, Bell GI, Philipson LH. Genetics and pathophysiology of neonatal diabetes mellitus. J Diabetes Investig 2011;2:158–69.Google Scholar

  • 35.

    De Franco E, Flanagan SE, Houghton JA, Lango Allen H, Mackay DJ, et al. The effect of early, comprehensive genomic testing on clinical care in neonatal diabetes: an international cohort study. Lancet 2015;386:957–63.Web of ScienceGoogle Scholar

  • 36.

    von Mühlendahl KE, Herkenhoff H. Long-term course of neonatal diabetes. N Engl J Med 1995;333:704–8.Google Scholar

  • 37.

    Massa O, Iafusco D, D’Amato E, Gloyn AL, Hattersley AT, et al. KCNJ11 activating mutations in Italian patients with permanent neonatal diabetes. Hum Mutat 2005;25:22–7.Google Scholar

  • 38.

    Rubio-Cabezas O, Flanagan SE, Damhuis A, Hattersley AT, Ellard S. KATP channel mutations in infants with permanent diabetes diagnosed after 6 months of life. Pediatr Diabetes 2012;13:322–5.Web of ScienceGoogle Scholar

  • 39.

    Pearson ER, Flechtner I, Njolstad PR, Malecki MT, Flanagan SE, Larkin B, et al. Switching from insulin to oral sulfonylureas in patients with diabetes due to Kir6.2 mutations. N Engl J Med 2006;355:467–77.Google Scholar

  • 40.

    Rafiq M, Flanagan SE, Patch AM, Shields BM, Ellard S, et al. Effective treatment with oral sulfonylureas in patients with diabetes due to sulfonylurea receptor 1 (SUR1) mutations. Diabetes Care 2008;31:204–9.Web of ScienceGoogle Scholar

  • 41.

    Sovik O, Aagenaes O, Eide SA, Mackay D, Temple IK, et al. Familial occurrence of neonatal diabetes with duplications in chromosome 6q24: treatment with sulfonylurea and 40-yr follow-up. Pediatric diabetes 2012;13:155–62.Web of ScienceGoogle Scholar

  • 42.

    Schimmel U. Long-standing sulfonylurea therapy after pubertal relapse of neonatal diabetes in a case of uniparental paternal isodisomy of chromosome 6. Diabetes Care 2009;32:e9.Google Scholar

  • 43.

    Stoy J, Greeley SA, Paz VP, Ye H, Pastore AN, et al. Diagnosis and treatment of neonatal diabetes: a United States experience. Pediatr Diabetes 2008;9:450–9.Web of ScienceGoogle Scholar

About the article

Corresponding author: Daniela del Gaudio, PhD, University of Chicago, 5841 S. Maryland Ave. MC.0077, Chicago, IL 60637, USA, Phone: +(773) 834-6751, Fax: +(773) 834-0556, E-mail:

Received: 2015-08-24

Accepted: 2016-01-04

Published Online: 2016-02-19

Published in Print: 2016-05-01

Citation Information: Journal of Pediatric Endocrinology and Metabolism, Volume 29, Issue 5, Pages 523–531, ISSN (Online) 2191-0251, ISSN (Print) 0334-018X, DOI: https://doi.org/10.1515/jpem-2015-0341.

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