1086 Diabetes Volume 66, April 2017 Reinaldo Sousa Dos Santos,1 Mathilde Daures,2 Anne Philippi,2 Sophie Romero,2 Lorella Marselli,3 Piero Marchetti,3 Valérie Senée,2 Delphine Bacq,4 Céline Besse,4 Baz Baz,5 Laura Marroquí,1 Sarah Ivanoff,6 Julien Masliah-Planchon,6 Marc Nicolino,7 Jean Soulier,6 Gérard Socié,8 Decio L. Eizirik,1 Jean-François Gautier,5 and Cécile Julier2 dUTPase (DUT ) Is Mutated in a Novel Monogenic Syndrome With Diabetes and Bone Marrow Failure Diabetes 2017;66:1086–1096 | DOI: 10.2337/db16-0839 We describe a new syndrome characterized by early- tight control of DNA metabolism for b-cell integrity and onset diabetes associated with bone marrow failure, warrant close metabolic monitoring of patients treated by affecting mostly the erythrocytic lineage. Using whole- drugs affecting dUTP balance. exome sequencing in a remotely consanguineous patient from a family with two affected siblings, we Diabetes may be caused by rare monogenic mutations, fi identi ed a single homozygous missense mutation accounting for 1%–5% of all cases of the disease (1,2), > (chr15.hg19:g.48,626,619A G) located in the dUTPase i.e., .2 million individuals worldwide. These mutations DUT ( ) gene (National Center for Biotechnology Informa- have been identified in the context of familial or atypical tion Gene ID 1854), affecting both the mitochondrial clinical presentations, which may affect various organs (DUT-M p.Y142C) and the nuclear (DUT-N p.Y54C) iso- (1,2). Some of these monogenic diabetes entities remain forms. We found the same homozygous mutation in an unrecognized as such and are currently misdiagnosed as unrelated consanguineous patient with diabetes and bone marrow aplasia from a family with two affected siblings, type 1 diabetes (T1D) or type 2 diabetes (T2D). Rare syn- whereas none of the >60,000 subjects from the Exome dromic associations may be particularly challenging to rec- fi Aggregation Consortium (ExAC) was homozygous for this ognize as speci c entities due to the high prevalence of fi mutation. This replicated observation probability was diabetes. The identi cation and study of familial cases is highly significant, thus confirming the role of this DUT of critical importance in this situation. The recognition of mutation in this syndrome. DUT is a key enzyme for main- these rare monogenic entities and their clinical and genetic taining DNA integrity by preventing misincorporation of characterization is important for correct diagnosis and to uracil into DNA, which results in DNA toxicity and cell improve patient’s treatment, besides providing informa- GENETICS/GENOMES/PROTEOMICS/METABOLOMICS death. We showed that DUT silencing in human and rat tion on disease mechanisms. Here, we studied two index pancreatic b-cells results in apoptosis via the intrinsic cell patients from two unrelated families having two siblings death pathway. Our findings support the importance of affected by a novel syndrome associating diabetes and bone 1ULB Center for Diabetes Research, Medical Faculty, Université Libre de Bruxelles, 7Hôpital Femme-Mère-Enfant, Division of Pediatric Endocrinology, Hospices Brussels, Belgium Civils de Lyon, Université Lyon 1, Lyon, France 2INSERM UMRS 958, Faculté de Médecine Paris Diderot, Université Paris Diderot- 8Hematology Transplantation, Department of Hematology, Immunology and On- Paris 7, Université Sorbonne Paris Cité, Paris, France cology, Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris, Paris, France 3 Department of Clinical and Experimental Medicine, Islet Cell Laboratory, Univer- Corresponding author: Cécile Julier, [email protected]. sity of Pisa, Pisa, Italy Received 10 July 2016 and accepted 5 January 2017. 4Centre National de Génotypage, Institut de Génomique, Commissariat à l’Energie Atomique, Evry, France This article contains Supplementary Data online at http://diabetes 5Hôpital Lariboisière, Assistance Publique-Hôpitaux de Paris, Department of .diabetesjournals.org/lookup/suppl/doi:10.2337/db16-0839/-/DC1. Diabetes and Endocrinology, Université Paris Diderot-Paris 7, Université Sorbonne R.S.D.S., M.D., and A.P. contributed equally to this work. D.L.E., J.-F.G., and C.J. Paris Cité, Paris, France contributed equally to this work. 6Aplastic Anemia Reference Centre, Hematology Laboratory, Hôpital Saint-Louis, © 2017 by the American Diabetes Association. Readers may use this article as Assistance Publique-Hôpitaux de Paris, INSERM U944, Université Paris long as the work is properly cited, the use is educational and not for profit, and the Diderot-Paris 7, Université Sorbonne Paris Cité, Paris, France work is not altered. More information is available at http://www.diabetesjournals .org/content/license. diabetes.diabetesjournals.org Dos Santos and Associates 1087 marrow failure. Through genetic studies of these families, methodology (SureSelect Human All Exon Kits Version 2; we identified the same mutation in the dUTPase (DUT) Agilent, Massy, France) with the company’s biotinylated oligo- gene (National Center for Biotechnology Information Gene nucleotide probe library (Human All Exon 50 Mb, version 2; ID 1854) as responsible for this syndrome. We then per- Agilent). Genomic DNA was then sequenced on a sequencer as formed DUT silencing in human and rat pancreatic b-cells paired-end 75 bases (Illumina HiSeq 2000; Illumina, San to investigate further the mechanisms responsible for di- Diego, CA). Image analysis and base calling were performed abetes resulting from DUT deficiency. with real-time analysis Pipeline version 1.14 with default pa- rameters (Illumina). The bioinformatic analysis of sequencing RESEARCH DESIGN AND METHODS data was based on a pipeline (Consensus Assessment of Se- Patients and Families quence and Variation [CASAVA] 1.8, Illumina). CASAVA per- We studied two unrelated families with patients with diabetes forms alignment against human reference genome (build 137), affected by various degrees of bone marrow failure, ranging calls the SNPs based on the allele calls and depth, and detects from dyserythropoiesis to bone marrow aplasia. Family 1 (pa- variants (SNPs and indels). Genetic variation annotation was tients 1 and 2) was a French family with healthy second cousin performed by the company’s pipeline (IntegraGen), and results consanguineous parents. Family 2 (patients 3 and 4) was an were provided per sample in tabulated text files. Mean se- Egyptian family with first cousin consanguineous parents. At quencing depth was 513 per base. Exome variant analysis the time of the genetic study, only patient 1 (French) and was then performed using an in-house python pipeline on patient 3 (Egyptian, living in France) were available. Detailed genetic variation annotation results. Variants were filtered clinical information from patient 2 (deceased) was available consecutively based on their quality (variant quality [Phred but no biological material. Limited clinical information was Q score] .20 and depth $53), their genotype (homozygous available from patient 4 (Egyptian, alive but living in Egypt), status), the predicted consequence on coding capacity who was not available for study. Participating subjects or their (missense, nonsense, splice-site, and coding insertion/ families gave their written informed consent to participate to deletion—frameshift or inframe), and their rare status the study, which was approved by the ethics committees of based on information available in public databases Saint-Antoine Hospital, Saint-Louis Hospital, or the Hospice (ExAC, release 0.3 (6); Exome Variant Server [EVS, re- Civils de Lyon. Genomic DNA was extracted from peripheral lease ESP6500SI-V2]; and Single Nucleotide Polymorphism blood using standard procedure. database [dbSNP, v.138]) and in an in-house database (control subjects, IntegraGen). Variants that were found Genome-Wide Linkage Analysis . Genome-wide linkage analysis was used to detect regions in the homozygous status or with a MAF 0.005 in any homozygous identical by descent (IBD) in patient 1 (family public or in-house database were excluded. 1). This was performed using a subset of 7,676 autosomal Mutation Confirmation and Screening common variants from the Human Exome BeadChip (Illu- by DNA Sequencing mina, San Diego, CA), selected to be evenly distributed over Resequencing of the DUT mutation identified by exome the genome, with high minor allele frequencies (MAFs) sequencing (patient 1), sequencing of DUT coding regions (mean 0.41) and no linkage disequilibrium between single (patient 3), and sequencing of DUT exons and regulatory nucleotide polymorphism (SNP)pairs.Forgenotyping,sam- regions (18 additional selected patients and families: four ples processing and labeling were performed on the Infinium with diabetes and bone marrow failure and 14 with di- assay according to the manufacturer’s instructions. In addi- abetes of likely monogenic origin compatible with linkage tion to family 1 (patient 1), 132 independent Caucasian fam- to the DUT chromosome region [see Supplementary Data]) ilies (324 subjects) were genotyped for the same SNP array, was performed on PCR-amplified DNA using an Applied and these data were used for quality controls and estimation Biosystems 3730 DNA Analyzer (Foster City, CA). PCR and of allele frequencies. Absence of pairwise linkage disequilib- sequencing primers are shown in Supplementary Table 1. rium was confirmed using PLINK (3) and Haploview (4) Sequencing of 95 additional selected patients with software. Allele frequencies were estimated by maximization bone marrow failure or myelodysplastic