Recessive mutations in the INS gene result in neonatal diabetes through reduced insulin biosynthesis Intza Garina,b,c,1, Emma L. Edghilld,1, Ildem Akermanb,e,f,1, Oscar Rubio-Cabezasd,g,1, Itxaso Ricaa, Jonathan M. Locked, Miguel Angel Maestrob,e,f, Adnan Alshaikhh, Ruveyde Bundaki, Gabriel del Castilloj, Asma Deebk, Dorothee Deissl, Juan M. Fernandezm, Koumudi Godbolen, Khalid Hussaino, Michele O’Connellp, Thomasz Klupaq, Stanislava Kolouskovar, Fauzia Mohsins, Kusiel Perlmant, Zdenek Sumnikr, Jose M. Rialu, Estibaliz Ugartev, Thiruvengadam Vasanthiw, Neonatal Diabetes International Group2, Karen Johnstoned, Sarah E. Flanagand, Rosa Martíneza,b,c, Carlos Castañob,e,f, Ann-Marie Patchd, Eduardo Fernández-Rebolloa,b,c, Klemens Railel, Noel Morgand, Lorna W. Harriesd, Luis Castañoa,b,c, Sian Ellardd, Jorge Ferrerb,e,f, Guiomar Perez de Nanclaresa,b,c,3, and Andrew T. Hattersleyd,3 aEndocrinology and Diabetes Research Group, Hospital de Cruces, Barakaldo, 48903 Spain; bCentro de Investigación Biomédica en Red en Diabetes y Metabolismo; cCentro de Investigación Biomédica en Red en Enfermedades Raras, Instituto de Salud Carlos III, 48903 Spain; dInstitute of Biomedical and Clinical Science, Peninsula Medical School, University of Exeter, Exeter, United Kingdom; eHospital Clínic de Barcelona, Spain; fInstitut d’Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain; gDepartment of Endocrinology, Hospital Infantil Universitario Niño Jesús, Madrid, Spain; hKing Abdulaziz Medical City, National Guard Health Affairs, Jeddah, Kingdom of Saudi Arabia; iDepartment of Pediatric Endocrinology, Istanbul University Hospital, Istanbul, Turkey; jDepartment of Neonatology, Hospital Infantil Los Angeles, San Juan de Pasto, Columbia; kImperial College London Diabetes Centre, Abu Dhabi, United Arab Emirates; lDepartment of Pediatric Diabetology and Endocrinology, Charité Campus, Virchow Children’s Hospital, Berlin, Germany; mDepartment of Pediatric Endocrinology, Hospital Universitario San Cecilio, Granada, 18012 Spain; nDeenanath Mangehskar Hospital and Research Center, Erandawane, Pune, India; oGreat Ormond Street Hospital for Children NHS Trust, London, United Kingdom; pRoyal Children’s Hospital, Melbourne, Australia; qDepartment of Metabolic Disease, Jagiellonian University Medical College, Krakow, Poland; rDepartment of Pediatrics, Second Faculty of Medicine, Charles University, Prague, Czech Republic; sPediatric Diabetic Team, Bangladesh Institute of Research and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders, Dhaka, Bangladesh; tDivision of Endocrinology, Hospital for Sick Children, Toronto, Canada; uDepartment of Pediatric Endocrinology, Hospital Nuestra Señora de la Candelaria, Tenerife, 38010 Spain; vDepartment of Endocrinology, Hospital de Basurto, Bilbao, 48013 Spain; and wKanchi Kamakoti Childs Trust Hospital, Chennai, India MEDICAL SCIENCES Edited by Donald F. Steiner, University of Chicago, and approved December 22, 2009 (received for review September 15, 2009) Heterozygous coding mutations in the INS gene that encodes pre- chromosome 6q24 are the most common cause of TNDM (13), proinsulin were recently shown to be an important cause of perma- followed by mutations in the KCNJ11 and ABCC8 genes (14). nent neonatal diabetes. These dominantly acting mutations prevent Despite these advances, the etiology of neonatal diabetes is still normal folding of proinsulin, which leads to beta-cell death through not known in at least 30% of patients with PNDM, suggesting endoplasmic reticulum stress and apoptosis. We now report 10 dif- other genetic causes are still to be found (9). INS ferent recessive mutations in 15 probands with neonatal diabe- Insulin is secreted from islet beta cells of the pancreas. Insuffi- tes. Functional studies showed that recessive mutations resulted in cient secretion of insulin results in hyperglycemia and diabetes, diabetes because of decreased insulin biosynthesis through distinct whereas excessive secretion results in hypoglycemia. Insulin bio- mechanisms, including gene deletion, lack of the translation initia- synthesis and secretion are therefore tightly regulated to maintain tion signal, and altered mRNA stability because of the disruption of a polyadenylation signal. A subset of recessive mutations caused blood glucose levels within a narrow physiological range. Extensive cis INS abnormal INS transcription, including the deletion of the C1 and studies have dissected an array of sequence elements in the E1 cis regulatory elements, or three different single base-pair sub- promoter region and their cognate DNA binding factors, which stitutions in a CC dinucleotide sequence located between E1 and A1 together ensure the cellular specificity and rate of INS transcription elements. In keeping with an earlier and more severe beta-cell (15–22). In addition, insulin biosynthesis is strongly dependent on defect, patients with recessive INS mutations had a lower birth posttranscriptional regulatory mechanisms, including the modu- weight (−3.2 SD score vs. −2.0 SD score) and were diagnosed earlier lation of translation and stability (23–25). The latter is largely (median 1 week vs. 10 weeks) compared to those with dominant INS mediated through sequences located in the untranslated regions of mutations. Mutations in the insulin gene can therefore result in INS transcripts (26–28). neonatal diabetes as a result of two contrasting pathogenic mech- anisms. Moreover, the recessively inherited mutations provide a genetic demonstration of the essential role of multiple sequence Author contributions: O.R.-C., K.R., N.M., L.W.H., L.C., S.E., J.F., G.P.d.N., and A.T.H. de- elements that regulate the biosynthesis of insulin in man. signed research; I.G., E.L.E., I.A., O.R.-C., I.R., J.M.L., M.A.M., A.A., R.B., G.d.C., A.D., D.D., J.M.F., K.G., K.H., M.O., T.K., S.K., F.M., K.P., Z.S., J.M.R., E.U., T.V., N.D.I.G., K.J., S.E.F., R. gene regulation | genetic testing | gene expression regulation | RNA M., C.C., A.-M.P., E.F.-R., and K.R. performed research; I.G., I.A., O.R.-C., L.W.H., S.E., J.F., and G.P.d.N. analyzed data; and E.L.E., O.R.-C., J.F., G.P.d.N., and A.T.H. wrote the paper. instability | promoter regions The authors declare no conflict of interest. This article is a PNAS Direct Submission. eonatal diabetes is diagnosed within the first 6 months of life Freely available online through the PNAS open access option. N(1, 2) and there are two main clinical subtypes: the persistent, 1 I.G., E.L.E., I.A., and O.R.-C. contributed equally to this work. permanent neonatal diabetes (PNDM) and the remitting and 2Members of the Neonatal Diabetes International Group: Firdevs Bas, Faculty of Medicine, frequently relapsing, transient neonatal diabetes (TNDM). Istanbul University, Istanbul, Turkey; Ondrej Cinek, Motol University Hospital, Czech Re- Recently there have been considerable advances in the under- public; Maciek Malecki, Department of Metabolic Disease, Jagiellonian University Med- standing of the genetics of neonatal diabetes (3). Most patients ical College, Krakow, Poland; and Marianna Rachmiel, Division of Endocrinology, Hospital for Sick Children, Toronto, Canada. with PNDM have activating mutations in KCNJ11 or ABCC8, the 3To whom correspondence may be addressed. E-mail: [email protected] or genes encoding the potassium ATP-sensitive (KATP) channel [email protected]. – subunits Kir6.2 (4) and SUR1 (5 7), or heterozygous mutations in This article contains supporting information online at www.pnas.org/cgi/content/full/ the preproinsulin (INS) gene (8–12). In contrast, abnormalities in 0910533107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0910533107 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 Heterozygous missense mutations in the coding region of the important for the INS promoter activity in transient transfection INS gene have recently been described as a cause of neonatal studies (15, 18, 33–35). The c.-331(C > G, C > A) and c.-332C > G diabetes (8–12). Most of the reported mutations are predicted to mutations were located between the E1 and A1 elements (Fig. 3A). disrupt the folding of the proinsulin molecule. The resulting This sequence is conserved among a subset of mammalian species misfolded protein accumulates in the endoplasmic reticulum (Fig. 3A) and mutagenesis of multiple bases neighboring this (ER), resulting in ER stress and beta-cell apoptosis (29, 30). An dinucleotide impairs INS promoter activity (36). We constructed alternative potential genetic mechanism would be reduced insulin promoter fragments carrying the point mutations c.-331(C > insulin secretion because of a disruption of the INS coding G, C > A) and c.-332C > G. The point mutations induced up to 90% sequence, as seen in the double Ins1 and Ins2 knockout mouse reduction in transcriptional activity, while a control mutation, c.- (31), or of the sequences that regulate insulin biosynthesis. 339G > A, did not alter the transcriptional activity in pancreatic However, as yet this has not been demonstrated in humans. beta-cell lines (Fig. 3B). Thus, the CC dinucleotide that is mutated We now report recessively acting mutations within the INS gene in eight unrelated probands with neonatal diabetes forms part of an in a series of patients with neonatal diabetes. In contrast to the essential positive cis regulatory sequence of the INS promoter. previously described dominant mutations, these mutations reduce Mutated or absent translational start site. The two point mutations insulin