Dutpase (DUT ) Is Mutated in a Novel Monogenic Syndrome with Diabetes and Bone Marrow Failure

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 parameters (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 syndrome (Sup- of likelihood. In family 1, parametric multipoint linkage anal- plementary Data) was performed by amplicon-based tar- ysis was performed using MERLIN (v1.1.2) (5) under an geted resequencing using a TruSeq Custom Amplicon kit autosomal recessive model with complete penetrance, using v1.5 (Illumina) containing a custom pool of eight oligo- a disease allele frequency of 0.001 and no phenocopy. Genetic nucleotides pairs designed to amplify the DUT gene cod- map distance was approximated by the physical position of ing exons and exon-intron boundaries (Supplementary SNPs. Regions reaching the theoretical maximum (logarithm Table 2). After amplification, the final libraries were se- of the odds [LOD] score of 1.78, based on family structure of quenced to a depth of at least 503 on a Illumina MiSeq family 1) were considered linked (i.e., homozygous IBD). platform. DNA of patient 3 was used as positive control. Whole-Exome Sequencing Population of Origin Study and Relationship Inference Exome sequencing was performed on the genomic plat- High-density genotyping was performed using Illumina form of IntegraGen (Evry, France). Exons of DNA sample Infinium Omni2.5-8 SNP array (.2.5 million SNPs) on the (patient 1) were captured with in-solution enrichment Centre National de Génotypage Infinium high-throughput 1088 DUT Mutation in Diabetes and Bone Marrow Failure Diabetes Volume 66, April 2017 genotyping platform according to the manufacturer’sin- autofluorescence-activated cell sorting (FACSAria; BD Biosci- structions. To determine population clusters, principal com- ence, San Jose, CA) and cultured for 48 h in Ham’sF-10 ponent analysis (PCA) was performed using EIGENSTRAT medium containing 10 mmol/L glucose, 2 mmol/L GlutaMAX, and SmartPCA (POPGEN) software from the EIGENSOFT 50 mmol/L 3-isobutyl-L-methyl-xanthine, 5% FBS, 0.5% FBS- 5.0.2 package. Control populations used for the analysis free albumin, 50 units/mL penicillin, and 50 mg/mL strepto- consisted on approximately 7,000 subjects from a large mycin, as previously described (13). The b-cell preparations range of populations including Africans (Yoruban), East used in the study contained mean 6 SEM 90 6 3% b-cells Asians (Chinese and Japanese), and various European (n = 5), with a viability of 89 6 2%. Rat insulin-producing groups. We estimated the kinship coefficient between the INS-1E cells (kindly provided by Dr. C. Wollheim, Centre two patients using KING software version 2.0, option kin- Medical Universitaire, Geneva, Switzerland) were cultured in ship, robust to population structure (7). RPMI 1640 GlutaMAX-I, 10 mmol/L HEPES, 1 mmol/L Protein Structural Modeling Na-pyruvate, 5% FBS, 50 mmol/L 2-mercaptoethanol, We used the X-ray crystal structure of human nuclear 50 units/mL penicillin, and 50 mg/mL streptomycin. isoform of DUT (DUT-N) (Tyr54, wild-type), in complex RNA Interference with a,b-imido-dUTP and Mg2+ (Protein Data Bank 3EHW) A list of the small interfering RNAs (siRNAs) used in this as reference. We modeled the mutated DUT-N (Cys54, mu- study is presented in Supplementary Table 3. Cells were tated) based on 3EHW structure using MODELER 9.11 package (8) to satisfy the spatial constraints of the 3 monomer cultured in antibiotic-free medium for at least 24 h before (A, B, C chains) and all chemical component ligands. Intra- transfection. Conditions for siRNA transfection as well as and interprotein chain interactions were calculate using optimal siRNA concentration (30 nmol/L) were established Protein Interaction Calculator server on the wild-type and as we previously described (14). Lipofectamine RNAiMAX mutated structures (9) using default parameters. Specifically, lipid reagent (Invitrogen, Carlsbad, CA) was used to trans- for hydrophobic interactions, residues Ala, Val, Leu, Ile, Met, fect cells, and the AllStars Negative Control siRNA (Qiagen, Phe, Trp, Pro, and Tyr were considered as interacting if they Venlo, the Netherlands) was used as a negative control fell within 5 Å of each other; for hydrogen bond definition, a (siCtrl) (15). We have previously shown that this siCtrl donor-acceptor distance cutoff of 3.5 Å was applied; for aro- does not affect b-cell gene expression, function, or viability matic interaction, pairs of phenyl ring centroids were sepa- (14,15). b-Cells transfected with siRNAs were used after a – rated by a distance of 4.5 to 7 Å; cation-pi interactions were recovery period of 24 48 h. The different siRNAs used in defined by a cationic chain (Lys or Arg) within 6 Å of an this study inhibited mRNA expression by .40% under the aromatic side chain (Phe, Tyr, or Trp). Supplementary Fig. 4 different experimental conditions (Fig. 2A, C, E,andG and was generated using RasMol v2.7.5.2 (10). Supplementary Figs. 1A, C, E,andG and 2A). Culture of Human Islets, Primary Rat b-Cells, Cell Treatments and INS-1E Cells The following cytokine concentrations were used, based Human islets were isolated from three organ donors without on previous dose-response experiments performed by our diabetes (mean 6 SEM age 70 6 6years;BMI30.56 group (16,17): recombinant human interleukin-1b (R&D 2 3.0 kg/m ) in Pisa, Italy, with the agreement of the local Systems, Abingdon, U.K.) at 10 units/mL for INS-1E cells ethics committee in Pisa, Italy.Humanisletisolationwas or 50 units/mL for primary rat b-cells; recombinant rat performed by collagenase digestion and density-gradient pu- interferon-g (R&D Systems) at 100 units/mL for INS-1E fi ri cation and the isolated islets were cultured in M199 me- cells or 500 units/mL for primary rat b-cells. dium containing 5.5 mmol/L glucose (11). The human islets During cytokine exposure, primary rat b-cells were cul- – were then shipped to Brussels, Belgium, within 1 5daysof tured in FBS-free medium. For streptozotocin (STZ, Sigma- isolation. In Brussels, after overnight recovery, the human Aldrich, Bornem, Belgium) treatment, cells were incubated ’ islets were dispersed and cultured in Ham sF-10medium with STZ (0.5 or 1.0 mmol/L) in a FBS-free medium for containing 6.1 mmol/L glucose, 2 mmol/L GlutaMAX, 30 min. Afterward, cells were washed with PBS and incu- 50 mmol/L 3-isobutyl-1-methylxanthine, 10% FBS, 1% bated at 37°C with complete INS-1E culture medium. Treat- charcoal-absorbed bovine serum albumin, 50 units/mL peni- ment with hydrogen peroxide (H2O2) (50 or 100 mmol/L) cillin, and 50 mg/mL streptomycin. b-Cell content in the hu- (Sigma-Aldrich) was performed in INS-1E complete medium. man islet preparations was mean 6 SEM 53 6 8% (n =3)as determined by insulin immunocytochemistry (12), and their Assessment of Cell Viability viability, as assessed by the DNA-binding dyes Hoechst The percentage of living, apoptotic, and necrotic cells was 33342 and propidium iodide (see below), was 91 6 2%. determined after staining with DNA-binding dyes Hoechst Rats (Male Wistar; Charles River Laboratories, L’Arbresle 33342 and propidium iodide, as previously described Cedex, France) were housed and used in agreement with (13,18). At least 600 cells were counted per experimental the guidelines of the Belgian Regulations for Animal Care condition. Cell viability was determined by two different with approval of the local ethics committee. Islets were observers (one of them unaware of sample identity) with isolated from adult rats, and b-cells were purified by a .90% agreement between results achieved by them. In diabetes.diabetesjournals.org Dos Santos and Associates 1089

Figure 1—DUT gene identification. A: Whole-exome sequencing was performed in patient 1 (family 1), leading to the identification of a unique nonsynonymous mutation in the DUT gene, which was then found also in patient 3 (family 2). Affected individuals are designated by filled black symbols. DNA of patients marked in parentheses (2,4) was not available, and these patients were thus not genetically studied. B: Schematic representation of the DUT mutation on DUT-M and DUT-N isoforms. DUT-M and DUT-N share a common region (black), whereas their N-terminal sequence is specific to each isoform, with a mitochondrial targeting sequence present in the DUT-M isoform (gray) (28) and a nuclear localization signal (hatched) (53). some experiments, apoptosis was confirmed by Western a-tubulin (Cell Signaling) (used as the loading control). Mem- blot analysis of cleaved caspase-9 and -3. branes were exposed to secondary peroxidase-conjugated – – mRNA Extraction and Real-time PCR antibody (anti IgG (H+L)-HRP, Invitrogen) for 1 2hat Poly(A)+ mRNA extraction was done using Dynabeads room temperature. Protein signal was visualized using the mRNA DIRECT kit (Invitrogen) and reverse transcrip- SuperSignal West Femto chemiluminescent substrate (Thermo fi tion was carried out as previously described (13,19). Scienti c), detected using ChemiDoc XRS+ (Bio-Rad), and Quantitative real-time PCR was performed using SYBR quantified with the Image Lab software (Bio-Rad). Greenandcomparedwithastandardcurve(13,20).Ex- Statistical Analysis pression values were corrected by the housekeeping gene Data are presented as mean 6 SEM. Comparisons were glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and performed by two-tailed paired t test or by ANOVA followed b-actin for rat b-cells and human islet cells, respectively. by paired t test with Bonferroni correction, as indicated. The expression of these two housekeeping genes is not A P value ,0.05 was considered as statistically significant. modified under the conditions used in this study. The primers used herein are listed in Supplementary Table 4. RESULTS Western Blot Analysis Description of a Novel Syndrome Characterized by INS-1E cells were washed with cold PBS and lysed in Diabetes and Bone Marrow Failure in Two Unrelated Laemmli buffer, and equal amounts of total proteins were Consanguineous Families With Two Affected Siblings run in 12% SDS-polyacrylamide gel. After transference to We studied two unrelated families with patients with nitrocellulose membranes, immunoblot analysis was per- diabetes affected by various degrees of bone marrow failure, formed by overnight incubation with antibodies against ranging from dyserythropoiesis to bone marrow aplasia (Fig. cleavedcaspase-9and-3(CellSignaling,Danvers,MA)and 1A, Table 1, and Supplementary Data). Family 1 (patients 1090 DUT Mutation in Diabetes and Bone Marrow Failure Diabetes Volume 66, April 2017

Figure 2—DUT inhibition exacerbates apoptosis in pancreatic b-cells. INS-1E cells (A, B,andG–J), primary rat b-cells (C and D), or dispersed human islets (E and F) were transfected with siCtrl or with two different siRNAs targeting rat (siDUT#1 and siDUT#2, A–D and G–J)orhuman (siDUTh#1 and siDUTh#2, E and F) DUT and left to recover for 48 h. To confirm knockdown, DUT mRNA expression was evaluated by RT-qPCR in INS-1E cells (A, n =4;G, n = 5), primary rat b-cells (C, n = 5), and dispersed human islets (E, n = 3). DUT expression was normalized by the housekeeping genes GAPDH (A, C,andG)orb-actin (E) and then by the highest value of each experiment considered as 1. b-Cell apoptosis was evaluated in INS-1E cells (B, n = 4), primary rat b-cells (D, n = 5), and dispersed human islets (F, n = 3) using Hoechst 33342/PI staining. Results are means 6 SEM (n as indicated for each figure). H: Western blot showing expression of cleaved caspase-9, cleaved caspase-3, and a-tubulin (used as loading control) in INS-1E cells transfected with siCtrl or siDUT (siDUT#1 and siDUT#2). The figure is representative of five independent experiments. I and J:DensitometryanalysisoftheWesternblots(H) for cleaved caspase-9 (I) and caspase-3 (J) after normalization for the protein content using a-tubulin and then by the highest value of each experiment considered as 1. Results are means 6 SEM of five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with siCtrl, as analyzed by paired Student t test. diabetes.diabetesjournals.org Dos Santos and Associates 1091

1 and 2) was a French family with healthy second cousin affecting the DUT gene, located within a 33 Mb linkage consanguineous parents. Family 2 (patients 3 and 4) was region (homozygous IBD) on chromosome 15 (Supple- an Egyptian family with healthy first cousin consanguin- mentary Table 7). The mutation (chr15:g.48,626,619A.G, eous parents. Patient 1 had diabetes diagnosed as T2D hg19) was confirmed by Sanger sequencing and results in with an acute onset at 28 years without ketosis, initially a nonsynonymous substitution affecting both the mito- treated with oral antidiabetes drugs (OAD). In addition, chondrial (DUT-M p.Y142C) and the nuclear (DUT-N he had congenital macrocytosis with dyserythropoiesis, p.Y54C) isoforms (Fig. 1B and Supplementary Table 8). but without anemia except during two infectious epi- This variant, reported as rs373184762 in dbSNP, was pre- sodes. His sister (patient 2) also had congenital macro- sent in the EVS and ExAC (6) databases at the extremely cytosis that evolved to a more severe hematological low overall MAF of 0.000154 (2/12,990) and 0.000066 presentation, with recurring anemia and thrombocytope- (8/121,230), respectively, with no rare homozygous subject nia requiring transfusions, related to bone marrow failure identified (Supplementary Table 8). Across the multiethnic initially affecting the red and then the megakaryocytic line- population panels of EVS (6,500 unrelated subjects, Euro- ages. She was diagnosed with T1D at 5 years and died at pean American and African American) and ExAC (.60,000 11 years from brain hemorrhage related to severe unrelated subjects from diverse ancestries worldwide strat- thrombocytopenia. Bone marrow aspirate and bone mar- ified in seven population subgroups), it was found only in row biopsies of patients 1 and 2 showed signs of dysery- the Caucasian populations recorded as European American thropoiesis, with megaloblastic erythroblasts and some in EVS and European (non-Finnish) (CEU) in ExAC, with a binucleated cells (Supplementary Data). Histology of MAF of 0.000120 (8/66,634) in ExAC (Supplementary Ta- the pancreas of patient 2 done at autopsy revealed that ble 8). We next sequenced the coding regions of DUT exons the pancreas was fibrotic, with rare and enlarged islets in patient 3 (family 2) and identified the same homozygous (data not shown). In addition, both patients had multiple mutation (data not shown). This patient originated from naevi. The second index patient (patient 3) was diagnosed Egypt, an admixed population of European/Near Eastern/ with T1D at age 17 years. He presented at age 21 years Maghrebi (all Caucasian) and sub-Saharan African origins with bone marrow aplasia, which had progressed from in- (21). Using the CEU MAF as a conservative (upper) esti- fancy and required regular transfusions. At age 22, he de- mate of the MAF in Egypt (allele frequency, P = 0.00012), veloped a T-cell acute lymphoblastic leukemia. He was and knowing that he was born to first cousin parents (in- treated by chemotherapy and then bone marrow allograft breeding coefficient, FP3 = 1/16), the probability of observ- and died shortly after from a severe graft-versus-host dis- ing homozygosity for the DUT-M p.Y142C variant in this ease. His brother (patient 4) also had bone marrow aplasia patient (replication probability) is pxFP3 = 0.00012 3 2 successfully treated by a bone marrow allograft and T1D (1/16), or 7.5 3 10 6, thus replicating the initial finding. diagnosed at 12 years of age. Diabetes of these patients was Overall, the joint probability of the observation of the easily equilibrated by insulin (patients 2 and 3) or by OAD DUT-M p.Y142C variant in the two unrelated patients later complemented by low insulin doses (patient 1). Four 1and3is(pxFP1) 3 (pxFP3) = (0.00012 3 [1/8]) 3 2 years after diabetes diagnosis, metabolic evaluation of pa- (0.00012 3 [1/16]), or 1.1 3 10 10, which strongly sug- tient 1 showed an important insulin secretory defect asso- gests that this mutation is responsible for the diabetes ciated with a decrease in insulin sensitivity, in the range of syndrome affecting these patients. Noteworthy, all the mis- T2D subjects (Supplementary Table 5). According to the sense or loss of function DUT variants reported in these family history, all four parents of these families did not databases were rare (MAF ,0.005) and never found homo- have diabetes or any hematological disease, but clinical zygous, supporting the highly conserved status of this gene. and genetic studies of the parents and other family relatives could not be performed. The DUT Mutation: Evidence for a Founder Effect in Two Patients From Distinct Population Backgrounds Identification of a Mutation in the DUT Gene To investigate if this mutation results from a founder as Responsible for the Disease effect and to confirm the distinct ancestry of the patients To identify the genetic defect responsible for the syn- based on medical records, we performed high-density dromic association of bone marrow failure and diabetes, we genotyping of both patients (.2.5 million SNPs) and per- performed whole-exome sequencing of patient 1 combined formed a PCA to compare them to various reference pop- with linkage analysis in this family (Fig. 1A). Because of ulation control groups. We found that both patients share remote consanguinity of his parents (second cousin), pa- a homozygous 1.9 Mb segment around the DUT mutation tient 1 was expected to have only 1/64 (1.56%) of his (from rs201088112 to rs8039142), and we estimated the genome homozygous IBD. Consistent with this, linkage frequency of this shared haplotype, using publically avail- analysis performed in family 1 identified three regions ho- able 1000 Genomes data, to be between 0 and 8.9 3 2 mozygous IBD over the whole genome, with a total length 10 15 depending on the population group (Supplementary of 50.0 Mb (Supplementary Table 6). Through whole- Table 9). This haplotype was estimated at a non-null fre- exome sequencing of patient 1, we identified a single rare quency only in populations from European ancestry or homozygous coding variant over the whole genome, admixed populations. PCA of the SNP genomic data of 1092 DUT uaini ibtsadBn arwFailure Marrow Bone and Diabetes in Mutation

Table 1—Clinical description of the patients Family Family 1 Family 2 Consanguinity Second cousin parents First cousin parents Subject Patient 1 Patient 2a Patient 3 Patient 4b Individual Sex Male Female Male Male data Origin French French Egyptian Egyptian Stature and BMI Normal Normal Normal Normal Birth Birth weight (g) 3,600 2,480 NA NA Gestational age (weeks) 38 36 NA NA Diabetes Age at onset (years) 28 5 17 12 Treatment OAD (4 years), then Insulin Insulin Insulin OAD and insulin Autoantibodies (GAD, IA2) Negative NA Negative NA Hematology Summary blood and bone Congenital Congenital macrocytosis evolving to Pancytopenia. Bone Bone marrow aplasia, treated by marrow characteristics macrocytosis. pancytopenia. Bone marrow failure mainly marrow aplasia.c bone marrow transplantation. Dyserythropoiesis. affecting the erythrocytic, then the megacaryocytic lineages. Age at follow-up (years) 6 32 Birth 5–69 21d NA Hemoglobine 12.1 14.5 12.2f 10–11 5.9 4.1 Fetal hemoglobing 1% NA NA 8–20% NA NA Red blood cell counth 3.2 3.7 2.8 NA 1.5 1.3 Mean erythrocyte volumei 112 114 139 120 112 76 Reticulocyte countj NA 46.7 185 68–78 NA None White blood cell countk 7.8 4.7 18.4 2.1–3.1 1.6 0.4 Platelet countl 334 164 Normal 16–36 7 19 Cancerology/ Multiple naevi Multiple naevi T-cell ALL (21 years) NA hyperplasia

Death Alive or cause of Alive (32) Brain hemorrhage (11) GVHD following bone Alive (24) Diabetes death (age, years) marrow transplantation (22) ALL, acute lymphoblastic leukemia; GVHD, graft-versus-host disease; NA, not available. aDNA of patient 2 was not available. bDNA and detailed clinical information of patient 4 were not available. cPatient 3 was first seen at age 19 years and already had bone marrow aplasia. dBefore acute lymphoblastic leukemia and bone marrow transplantation. eNormal values .12 g/L (.14 g/L at birth). fHemoglobin level decreased to 7.9 g/L 11 days after birth. g% of total hemoglobin. hNormal values .4.5 3 109/L (male) and .3.9 3 109/L (female). iNormal values: 85–95 fL. 2017 April 66, Volume jNormal values: 25–100 3 106/L. kNormal values .4 3 109/L (at birth .10 3 109/L). lNormal values .150 3 109/L. diabetes.diabetesjournals.org Dos Santos and Associates 1093 the two patients in comparison with various control pop- regions of the gene in any of these patients, supporting the ulations showed that patient 1 clusters within the French rarity to this new syndrome and suggesting that DUT mu- population, as expected, whereas patient 3 clusters within a tations are not a frequent cause of monogenic diabetes or small Greek subgroup, compatible with his Egyptian ances- monogenic bone marrow failure. try, in the absence of referenced Egyptian control group DUT Inhibition Exacerbates Apoptosis in Pancreatic (Supplementary Fig. 3A and B). The kinship coefficient of b-Cells Through the Mitochondrial Cell Death Pathway the two patients estimated using KING software was neg- To evaluate DUT effects on b-cell viability, specific siRNAs ative (20.165), confirming that they are unrelated (7). were used to inhibit DUT expression in rat and human Overall, this suggests that both patients share the same islet cells (Fig. 2). DUT inhibition by two independent ancestral haplotype carrying the DUT mutation, originat- siRNAs increased apoptosis in both rodent and human ing from an ancestral European founder effect, despite b-cells (Fig. 2B, D, and F). These results were confirmed their distinct population background. by higher expression of cleaved caspase-9 and -3 in INS-1E Predicted Impact of the DUT Mutation cells following DUT inhibition, suggesting that apoptosis DUT is a key enzyme of nucleotide metabolism (22,23). It is triggered via the mitochondrial-mediated intrinsic H–J catalyses the hydrolysis of dUTP to dUMP+PPi, which is pathway (Fig. 2 ). Despite the observed increased ap- essential to maintain DNA integrity by two mechanisms: optosis, insulin and PDX1 mRNA expression were not fi 1) dUMP is the substrate of thymidilate synthase to pro- modi ed in DUT-inhibited cells (Supplementary Fig. 5). duce dTTP and 2) this maintains a low dUTP/dTTP, To determine whether DUT inhibition sensitizes b-cells hence limiting the integration of uracil in DNA, whose to different stressors, we treated INS-1E cells transfected accumulation leads to cell death due to DNA toxicity, as with siCtrl or siDUT with cytokines; streptozotocin (STZ), evidenced by knockout models in various prokaryotic an alkylating agent that induces DNA strand breaks; and and eukaryotic organisms and other cellular systems H2O2, an inducer of oxidative stress. These agents in- (24,25,26,27). The mutation is located near “conserved creased b-cell death following transfection with either motif I” of the protein (22,23) and is predicted to be siCtrl or siDUT, but there was no clear potentiation be- damaging (Supplementary Table 10). Tridimensional mod- tween DUT inhibition and cell death induced by these dif- eling of the homotrimeric wild-type and mutated protein ferent stressors (Supplementary Fig. 1). predicted conformational changes, including the loss of DUT-M is the constitutively expressed isoform of DUT fi interactions with several residues, supporting a func- (28,29), and we next knocked down speci cally DUT-M tional impact of the mutation (Supplementary Fig. 4 using two independent siRNAs to evaluate its contribu- and Supplementary Table 11). tion for b-cell survival (Supplementary Fig. 2). Similar to the observations following knockdown of both DUT iso- Search for Additional DUT Mutated Patients forms (Fig. 2), DUT-M–specific knockdown resulted in in Extended Patient Collections higher rates of b-cell apoptosis (Supplementary Fig. 2B) DUT To search for additional patients with mutations and via the mitochondrial pathway of apoptosis, as demon- to delineate the phenotype spectrum of the syndrome, we strated by the increased expression of cleaved caspase-9 DUT sequenced the gene in a highly selected complemen- and -3 in DUT-M–silenced cells (Supplementary Fig. 2C–E). tary set of patients presenting at least one of the distinc- tive clinical features observed in the patients of the two DISCUSSION index families (i.e., diabetes or bone marrow failure) and This is the first report of a DUT mutation in humans, whose genetic cause of their disease was unknown (Sup- showing that a specific nonsynonymous DUT mutation plementary Data): 1) 4 unrelated patients with diabetes leads to a new syndrome characterized by diabetes and associated with bone marrow failure, all of whom however bone marrow failure. had additional clinical manifestations that were not de- Despite the extreme rarity of the mutation identified, our scribed in the original patients (e.g., short stature, intel- genetic study design was able to provide both identification lectual impairment, dysmorphologies); 2) 14 unrelated power and validation power, as indicated by the following patients with juvenile-onset diabetes and their families points. with increased risk of monogenic causes based on family structure and/or atypical clinical presentation, which were 1. The consanguineous and multiplex nature of the fam- selected to be compatible with linkage to the DUT chro- ilies studied, with two siblings affected by the same mosome region (C.J., unpublished data); and 3)95unre- atypical clinical association in each family, strengthens lated patients with bone marrow failure or myelodysplastic the hypothesis of a rare monogenic inheritance for this syndrome, excluding patients with Fanconi anemia (FA), syndrome. who were selected to have increased risk of monogenic 2. The identification power was increased by the study of causes based on a young age at onset, physical abnormal- a child born to remotely consanguineous parents, lead- ities, and/or familial history. We did not identify any rare ing to the reduction of the “targeted region” to 50 Mb homozygous or compound heterozygous variant compati- (region homozygous IBD in the studied index patient) ble with the mutation status in the coding or regulatory representing a .603 reduced size compared with the 1094 DUT Mutation in Diabetes and Bone Marrow Failure Diabetes Volume 66, April 2017

whole genome, leading to the identification of a single increased uracil content in DNA (42), as well as DNA dam- rare homozygous coding variant. age and cell-cycle arrest (43), suggesting a possible role of 3. The identification of the same mutation in an unrelated nuclear DNA integrity in megaloblastosis and the possible patient from a distinct population background was implication of DUT-N defect on this trait. However, hema- highly statistically significant based on the known fre- tological manifestations have also been reported in various quency of this specific mutation in large multiethnic mitochondrial diseases (44). In particular, megaloblastic control populations. anemia and bone marrow failure, together with endocrine and exocrine pancreas deficiency, are observed in Pearson The observed clinical presentation of patients shares syndrome, characterized by large mitochondrial DNA dele- similarities with several syndromes caused by impaired cell tions (45), which were not found in our patients (data not cycle dependent DNA repair mechanisms, such as FA, shown). Furthermore, in thiamine-responsive megaloblas- characterized by progressive bone marrow failure and tic anemia, caused by SLC19A2 mutations and character- increased risk of leukemia (30,31,32). Contrary to FA pa- ized by megaloblastic anemia, diabetes, and sensorineural tients, however, blood and bone marrow cells from our deafness, respiratory chain defect has been reported in a patients did not show increased spontaneous or induced unique patient, suggesting the role of mitochondrial dys- chromosome breakage compared with control subjects. dU function (46). The fact that DUT expression in adult b-cells repair mechanism uses base-excision repair, which is distinct is mainly contributed by the DUT-M isoform, constitutively from the FA DNA repair pathway (DNA interstrand cross- expressed (28,29), whereas DUT-N is expressed only in link repair) (31). The lack of synergistic effects between DUT actively dividing cells (adult human b-cells divide poorly, inhibition and STZ (alkylation)-, cytokines-, or H2O2 (single- if at all), together with the present observation of increased strand breaks)-induced cell death (present data) is consis- b-cell apoptosis in DUT-M–specific knockdown, suggest tent with different DNA repair pathways involved (33). that DUT-M is the main responsible isoform for diabetes. Diabetes is a cardinal feature of the DUT-mutated syn- In addition to their well-documented role for maintain- drome. It is nonautoimmune, as indicated by the lack of ing a tight control of cellular dUTP level, recent studies autoantibodies against pancreatic b-cells and starts either indicate that dUTPases are also involved in the regulation during childhood or early adulthood (Table 1). In all cases, other cellular processes that may contribute to autoimmu- the patients eventually became insulin requiring. This is nity and apoptosis (23). The presently described syndrome in line with our present findings that DUT knockdown further broadens the multifaceted spectrum of DUT’s func- triggers apoptosis of human and rat b-cells, with the al- tion to the regulation of biological processes of clinical sig- tered pancreatic islet histology of patient 2 and the very nificance, with impact on diabetes and bone marrow failure. low insulin secretion in patient 1. Glucose metabolism 5-fluorouracil, leading to dTTP depletion, is commonly and insulin secretion are frequently impaired in diseases used for cancer chemotherapy (47,48). Both myelosup- associated with DNA repair defects, including FA (34,35) pression and secondary diabetes are common side effects and Bloom syndrome (36). DNA damage and genomic in- of 5-fluorouracil therapy (49,50), and a case of fulminant stability have been shown to alter b-cell replication through T1D following 5-fluorouracil treatment has recently been cell-cycle inhibition, leading to impaired glucose metabo- reported (51). Although a recent first-phase study of a lism and diabetes (36,37). Interestingly, cell-cycle pathway dUTPase inhibitor indicates good tolerance (52), our find- genes are over-represented among T2D susceptibility genes ings warrant close monitoring of hematological and met- (38), and DNA repair genes were found to be downregu- abolic adverse events in these patients. lated in T2D patients (39), supporting the role of DNA Our observation highlights the importance of the tight damage, repair, and cell-cycle pathways in diabetes etiology. control of uracil level in DNA for numerous biological Disease severity was variable between patients who carried functions, including hematopoiesis and insulin produc- the same homozygous DUT mutation, suggesting the con- tion, and identifies a novel form of monogenic diabetes tribution of other genetic or environmental factors. In the associated with bone marrow failure. absence of clinical study of the parents and other heterozygous carriers from these families, we cannot conclude on the clinical consequences of this DUT mutation in the het- Acknowledgments. The authors thank the patients and their families for erozygous state. their participation. The authors thank Claire Vandiedonck from INSERM UMRS The DUT mutation identified affects both nuclear and 958 for expert methodological advice and helpful discussions. The authors thank mitochondrial isoforms, but their contributions to the ob- the Centre National de Génotypage (CNG) for providing access to their genomic served clinical manifestations remain to be established. platform and Céline Derbois, Christophe Caloustian, and Sylvana Pavek (from the CNG) for expert technical assistance. The authors thank INSERM, Université Paris Megaloblastic anemia occurs in several instances of alter- Diderot-Paris 7, and the Assistance Publique-Hôpitaux de Paris for their support. ation of the pyrimidine biosynthesis pathway. It may be Funding. This work was supported by grants from the Agence Nationale de la fi caused by genetic defects, such as dUMP de ciency be- Recherche (ANR-09-GENO-021), the European Foundation for the Study of Diabetes/ cause of uridine monophosphate synthase (UMPS) muta- JDRF/Novo Nordisk (“Identification of genes with monogenic contribution”), the As- tions (40) or by vitamin B12 or folate deficiency (41). sistance Publique-Hôpitaux de Paris (Programme Hospitalier de Recherche Clinique Megaloblastic anemia erythroblasts were reported to have DIAGENE), the GIS Maladies Rares, France Génomique (DIAPED) (to C.J.), the diabetes.diabetesjournals.org Dos Santos and Associates 1095

European Union Framework Programme Horizon 2020 (project T2DSystems), and the 15. Moore F, Naamane N, Colli ML, et al. STAT1 is a master regulator of Fonds National de la Recherche Scientifique, Belgium, and Actions de Recherche pancreatic beta-cell apoptosis and islet inflammation. J Biol Chem 2011;286: Concertée de la Communauté Française, Belgium (to D.L.E.). R.S.D.S. was supported 929–941 by a postdoctoral fellowship from the Conselho Nacional de Desenvolvimento 16. Eizirik DL, Mandrup-Poulsen T. A choice of death–the signal-transduction of Científico e Tecnológico, Brazil. M.D. was supported by a doctoral fellowship immune-mediated beta-cell apoptosis. Diabetologia 2001;44:2115–2133 from the Ministère de l’Éducation Nationale, de l’Enseignement Supérieur et de 17. Kutlu B, Cardozo AK, Darville MI, et al. Discovery of gene networks regu- la Recherche, France. L.Marr. was supported by a scientific postdoctoral fel- lating cytokine-induced dysfunction and apoptosis in insulin-producing INS-1 lowship from the Fonds National de la Recherche, Belgium. cells. Diabetes 2003;52:2701–2719 Duality of Interest. No potential conflicts of interest relevant to this article 18. Marroquí L, Santin I, Dos Santos RS, Marselli L, Marchetti P, Eizirik DL. were reported. BACH2, a candidate risk gene for type 1 diabetes, regulates apoptosis in pan- Author Contributions. R.S.D.S., L.Marr., and D.L.E. performed the creatic b-cells via JNK1 modulation and crosstalk with the candidate gene functional experiments and analyses of pancreatic islets. M.D., A.P., S.R., V.S., PTPN2. Diabetes 2014;63:2516–2527 D.B., and C.J. performed the genetic experiments and analyses. A.P. performed 19. Chen MC, Proost P, Gysemans C, Mathieu C, Eizirik DL. Monocyte che- the protein modeling analysis. L.Mars. and P.M. contributed human islets material moattractant protein-1 is expressed in pancreatic islets from prediabetic NOD for the functional studies. C.B. performed the SNP array genotyping. B.B., G.S., and mice and in interleukin-1 beta-exposed human and rat islet cells. Diabetologia J.-F.G. identified the two index patients and families and performed the clinical 2001;44:325–332 investigations. S.I., M.N., and J.S. identified and selected additional patients with 20. Overbergh L, Valckx D, Waer M, Mathieu C. Quantification of murine cy- bone marrow failure. J.M.-P. performed next-generation sequencing targeted tokine mRNAs using real time quantitative reverse transcriptase PCR. Cytokine sequencing. D.L.E., J.-F.G., and C.J. conceived and designed the study. D.L.E. and 1999;11:305–312 C.J. wrote the article, with contributions from R.S.D.S., M.D., A.P., S.R., and J.-F.G. 21. Henn BM, Botigué LR, Gravel S, et al. 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