We describe a new syndrome characterized by early-onset diabetes associated with bone marrow failure, affecting mostly the erythrocytic lineage. Using whole-exome sequencing in a remotely consanguineous patient from a family with two affected siblings, we identified a single homozygous missense mutation (chr15.hg19:g.48,626,619A>G) located in the dUTPase (DUT) gene (National Center for Biotechnology Information Gene ID 1854), affecting both the mitochondrial (DUT-M p.Y142C) and the nuclear (DUT-N p.Y54C) isoforms. We found the same homozygous mutation in an unrelated consanguineous patient with diabetes and bone marrow aplasia from a family with two affected siblings, whereas none of the >60,000 subjects from the Exome Aggregation Consortium (ExAC) was homozygous for this mutation. This replicated observation probability was highly significant, thus confirming the role of this DUT mutation in this syndrome. DUT is a key enzyme for maintaining DNA integrity by preventing misincorporation of uracil into DNA, which results in DNA toxicity and cell death. We showed that DUT silencing in human and rat pancreatic β-cells results in apoptosis via the intrinsic cell death pathway. Our findings support the importance of tight control of DNA metabolism for β-cell integrity and warrant close metabolic monitoring of patients treated by drugs affecting dUTP balance.

Diabetes may be caused by rare monogenic mutations, accounting for 1%–5% of all cases of the disease (1,2), i.e., >2 million individuals worldwide. These mutations have been identified in the context of familial or atypical clinical presentations, which may affect various organs (1,2). Some of these monogenic diabetes entities remain unrecognized as such and are currently misdiagnosed as type 1 diabetes (T1D) or type 2 diabetes (T2D). Rare syndromic associations may be particularly challenging to recognize as specific entities due to the high prevalence of diabetes. The identification and study of familial cases is of critical importance in this situation. The recognition of these rare monogenic entities and their clinical and genetic characterization is important for correct diagnosis and to improve patient’s treatment, besides providing information on disease mechanisms. Here, we studied two index patients from two unrelated families having two siblings affected by a novel syndrome associating diabetes and bone marrow failure. Through genetic studies of these families, we identified the same mutation in the dUTPase (DUT) gene (National Center for Biotechnology Information Gene ID 1854) as responsible for this syndrome. We then performed DUT silencing in human and rat pancreatic β-cells to investigate further the mechanisms responsible for diabetes resulting from DUT deficiency.

Patients and Families

We studied two unrelated families with patients with diabetes affected by various degrees of bone marrow failure, ranging from dyserythropoiesis to bone marrow aplasia. Family 1 (patients 1 and 2) was a French family with healthy second cousin consanguineous parents. Family 2 (patients 3 and 4) was an Egyptian family with first cousin consanguineous parents. At the time of the genetic study, only patient 1 (French) and patient 3 (Egyptian, living in France) were available. Detailed clinical information from patient 2 (deceased) was available but no biological material. Limited clinical information was available from patient 4 (Egyptian, alive but living in Egypt), who was not available for study. Participating subjects or their families gave their written informed consent to participate to the study, which was approved by the ethics committees of Saint-Antoine Hospital, Saint-Louis Hospital, or the Hospice Civils de Lyon. Genomic DNA was extracted from peripheral blood using standard procedure.

Genome-Wide Linkage Analysis

Genome-wide linkage analysis was used to detect regions homozygous identical by descent (IBD) in patient 1 (family 1). This was performed using a subset of 7,676 autosomal common variants from the Human Exome BeadChip (Illumina, San Diego, CA), selected to be evenly distributed over the genome, with high minor allele frequencies (MAFs) (mean 0.41) and no linkage disequilibrium between single nucleotide polymorphism (SNP) pairs. For genotyping, samples processing and labeling were performed on the Infinium assay according to the manufacturer’s instructions. In addition to family 1 (patient 1), 132 independent Caucasian families (324 subjects) were genotyped for the same SNP array, and these data were used for quality controls and estimation of allele frequencies. Absence of pairwise linkage disequilibrium was confirmed using PLINK (3) and Haploview (4) software. Allele frequencies were estimated by maximization of likelihood. In family 1, parametric multipoint linkage analysis was performed using MERLIN (v1.1.2) (5) under an autosomal recessive model with complete penetrance, using a disease allele frequency of 0.001 and no phenocopy. Genetic map distance was approximated by the physical position of SNPs. Regions reaching the theoretical maximum (logarithm of the odds [LOD] score of 1.78, based on family structure of family 1) were considered linked (i.e., homozygous IBD).

Whole-Exome Sequencing

Exome sequencing was performed on the genomic platform of IntegraGen (Evry, France). Exons of DNA sample (patient 1) were captured with in-solution enrichment methodology (SureSelect Human All Exon Kits Version 2; Agilent, Massy, France) with the company’s biotinylated oligonucleotide probe library (Human All Exon 50 Mb, version 2; Agilent). Genomic DNA was then sequenced on a sequencer as paired-end 75 bases (Illumina HiSeq 2000; Illumina, San Diego, CA). Image analysis and base calling were performed with real-time analysis Pipeline version 1.14 with default parameters (Illumina). The bioinformatic analysis of sequencing data was based on a pipeline (Consensus Assessment of Sequence and Variation [CASAVA] 1.8, Illumina). CASAVA performs alignment against human reference genome (build 137), calls the SNPs based on the allele calls and depth, and detects variants (SNPs and indels). Genetic variation annotation was performed by the company’s pipeline (IntegraGen), and results were provided per sample in tabulated text files. Mean sequencing depth was 51× per base. Exome variant analysis was then performed using an in-house python pipeline on genetic variation annotation results. Variants were filtered consecutively based on their quality (variant quality [Phred Q score] >20 and depth ≥5×), their genotype (homozygous status), the predicted consequence on coding capacity (missense, nonsense, splice-site, and coding insertion/deletion—frameshift or inframe), and their rare status based on information available in public databases (ExAC, release 0.3 (6); Exome Variant Server [EVS, release ESP6500SI-V2]; and Single Nucleotide Polymorphism database [dbSNP, v.138]) and in an in-house database (control subjects, IntegraGen). Variants that were found in the homozygous status or with a MAF >0.005 in any public or in-house database were excluded.

Mutation Confirmation and Screening by DNA Sequencing

Resequencing of the DUT mutation identified by exome sequencing (patient 1), sequencing of DUT coding regions (patient 3), and sequencing of DUT exons and regulatory regions (18 additional selected patients and families: four with diabetes and bone marrow failure and 14 with diabetes of likely monogenic origin compatible with linkage to the DUT chromosome region [see Supplementary Data]) was performed on PCR-amplified DNA using an Applied Biosystems 3730 DNA Analyzer (Foster City, CA). PCR and sequencing primers are shown in Supplementary Table 1.

Sequencing of 95 additional selected patients with bone marrow failure or myelodysplastic syndrome (Supplementary Data) was performed by amplicon-based targeted resequencing using a TruSeq Custom Amplicon kit v1.5 (Illumina) containing a custom pool of eight oligonucleotides pairs designed to amplify the DUT gene coding exons and exon-intron boundaries (Supplementary Table 2). After amplification, the final libraries were sequenced to a depth of at least 50× on a Illumina MiSeq platform. DNA of patient 3 was used as positive control.

Population of Origin Study and Relationship Inference

High-density genotyping was performed using Illumina Infinium Omni2.5-8 SNP array (>2.5 million SNPs) on the Centre National de Génotypage Infinium high-throughput genotyping platform according to the manufacturer’s instructions. To determine population clusters, principal component analysis (PCA) was performed using EIGENSTRAT and SmartPCA (POPGEN) software from the EIGENSOFT 5.0.2 package. Control populations used for the analysis consisted on approximately 7,000 subjects from a large range of populations including Africans (Yoruban), East Asians (Chinese and Japanese), and various European groups. We estimated the kinship coefficient between the two patients using KING software version 2.0, option kinship, robust to population structure (7).

Protein Structural Modeling

We used the X-ray crystal structure of human nuclear isoform of DUT (DUT-N) (Tyr54, wild-type), in complex with α,β-imido-dUTP and Mg2+ (Protein Data Bank 3EHW) as reference. We modeled the mutated DUT-N (Cys54, mutated) based on 3EHW structure using MODELER 9.11 package (8) to satisfy the spatial constraints of the 3 monomer (A, B, C chains) and all chemical component ligands. Intra- and interprotein chain interactions were calculate using Protein Interaction Calculator server on the wild-type and mutated structures (9) using default parameters. Specifically, for hydrophobic interactions, residues Ala, Val, Leu, Ile, Met, Phe, Trp, Pro, and Tyr were considered as interacting if they fell within 5 Å of each other; for hydrogen bond definition, a donor-acceptor distance cutoff of 3.5 Å was applied; for aromatic interaction, pairs of phenyl ring centroids were separated by a distance of 4.5 to 7 Å; cation-pi interactions were defined by a cationic chain (Lys or Arg) within 6 Å of an aromatic side chain (Phe, Tyr, or Trp). Supplementary Fig. 4 was generated using RasMol v2.7.5.2 (10).

Culture of Human Islets, Primary Rat β-Cells, and INS-1E Cells

Human islets were isolated from three organ donors without diabetes (mean ± SEM age 70 ± 6 years; BMI 30.5 ± 3.0 kg/m2) in Pisa, Italy, with the agreement of the local ethics committee in Pisa, Italy. Human islet isolation was performed by collagenase digestion and density-gradient purification and the isolated islets were cultured in M199 medium containing 5.5 mmol/L glucose (11). The human islets were then shipped to Brussels, Belgium, within 1–5 days of isolation. In Brussels, after overnight recovery, the human islets were dispersed and cultured in Ham’s F-10 medium containing 6.1 mmol/L glucose, 2 mmol/L GlutaMAX, 50 μmol/L 3-isobutyl-1-methylxanthine, 10% FBS, 1% charcoal-absorbed bovine serum albumin, 50 units/mL penicillin, and 50 mg/mL streptomycin. β-Cell content in the human islet preparations was mean ± SEM 53 ± 8% (n = 3) as determined by insulin immunocytochemistry (12), and their viability, as assessed by the DNA-binding dyes Hoechst 33342 and propidium iodide (see below), was 91 ± 2%.

Rats (Male Wistar; Charles River Laboratories, L’Arbresle Cedex, France) were housed and used in agreement with the guidelines of the Belgian Regulations for Animal Care with approval of the local ethics committee. Islets were isolated from adult rats, and β-cells were purified by autofluorescence-activated cell sorting (FACSAria; BD Bioscience, San Jose, CA) and cultured for 48 h in Ham’s F-10 medium containing 10 mmol/L glucose, 2 mmol/L GlutaMAX, 50 μmol/L 3-isobutyl-L-methyl-xanthine, 5% FBS, 0.5% FBS-free albumin, 50 units/mL penicillin, and 50 mg/mL streptomycin, as previously described (13). The β-cell preparations used in the study contained mean ± SEM 90 ± 3% β-cells (n = 5), with a viability of 89 ± 2%. Rat insulin-producing INS-1E cells (kindly provided by Dr. C. Wollheim, Centre Medical Universitaire, Geneva, Switzerland) were cultured in RPMI 1640 GlutaMAX-I, 10 mmol/L HEPES, 1 mmol/L Na-pyruvate, 5% FBS, 50 μmol/L 2-mercaptoethanol, 50 units/mL penicillin, and 50 mg/mL streptomycin.

RNA Interference

A list of the small interfering RNAs (siRNAs) used in this study is presented in Supplementary Table 3. Cells were cultured in antibiotic-free medium for at least 24 h before transfection. Conditions for siRNA transfection as well as optimal siRNA concentration (30 nmol/L) were established as we previously described (14). Lipofectamine RNAiMAX lipid reagent (Invitrogen, Carlsbad, CA) was used to transfect cells, and the AllStars Negative Control siRNA (Qiagen, Venlo, the Netherlands) was used as a negative control (siCtrl) (15). We have previously shown that this siCtrl does not affect β-cell gene expression, function, or viability (14,15). β-Cells transfected with siRNAs were used after a recovery period of 24–48 h. The different siRNAs used in this study inhibited mRNA expression by >40% under the different experimental conditions (Fig. 2A, C, E, and G and Supplementary Figs. 1A, C, E, and G and 2A).

Cell Treatments

The following cytokine concentrations were used, based on previous dose-response experiments performed by our group (16,17): recombinant human interleukin-1β (R&D Systems, Abingdon, U.K.) at 10 units/mL for INS-1E cells or 50 units/mL for primary rat β-cells; recombinant rat interferon-γ (R&D Systems) at 100 units/mL for INS-1E cells or 500 units/mL for primary rat β-cells.

During cytokine exposure, primary rat β-cells were cultured in FBS-free medium. For streptozotocin (STZ, Sigma-Aldrich, Bornem, Belgium) treatment, cells were incubated with STZ (0.5 or 1.0 mmol/L) in a FBS-free medium for 30 min. Afterward, cells were washed with PBS and incubated at 37°C with complete INS-1E culture medium. Treatment with hydrogen peroxide (H2O2) (50 or 100 μmol/L) (Sigma-Aldrich) was performed in INS-1E complete medium.

Assessment of Cell Viability

The percentage of living, apoptotic, and necrotic cells was determined after staining with DNA-binding dyes Hoechst 33342 and propidium iodide, as previously described (13,18). At least 600 cells were counted per experimental condition. Cell viability was determined by two different observers (one of them unaware of sample identity) with a >90% agreement between results achieved by them. In some experiments, apoptosis was confirmed by Western blot analysis of cleaved caspase-9 and -3.

mRNA Extraction and Real-time PCR

Poly(A)+ mRNA extraction was done using Dynabeads mRNA DIRECT kit (Invitrogen) and reverse transcription was carried out as previously described (13,19). Quantitative real-time PCR was performed using SYBR Green and compared with a standard curve (13,20). Expression values were corrected by the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin for rat β-cells and human islet cells, respectively. The expression of these two housekeeping genes is not modified under the conditions used in this study. The primers used herein are listed in Supplementary Table 4.

Western Blot Analysis

INS-1E cells were washed with cold PBS and lysed in Laemmli buffer, and equal amounts of total proteins were run in 12% SDS-polyacrylamide gel. After transference to nitrocellulose membranes, immunoblot analysis was performed by overnight incubation with antibodies against cleaved caspase-9 and -3 (Cell Signaling, Danvers, MA) and α-tubulin (Cell Signaling) (used as the loading control). Membranes were exposed to secondary peroxidase-conjugated antibody (anti–IgG (H+L)-HRP, Invitrogen) for 1–2 h at room temperature. Protein signal was visualized using the SuperSignal West Femto chemiluminescent substrate (Thermo Scientific), detected using ChemiDoc XRS+ (Bio-Rad), and quantified with the Image Lab software (Bio-Rad).

Statistical Analysis

Data are presented as mean ± SEM. Comparisons were performed by two-tailed paired t test or by ANOVA followed by paired t test with Bonferroni correction, as indicated. A P value <0.05 was considered as statistically significant.

Description of a Novel Syndrome Characterized by Diabetes and Bone Marrow Failure in Two Unrelated Consanguineous Families With Two Affected Siblings

We studied two unrelated families with patients with diabetes affected by various degrees of bone marrow failure, ranging from dyserythropoiesis to bone marrow aplasia (Fig. 1A, Table 1, and Supplementary Data). Family 1 (patients 1 and 2) was a French family with healthy second cousin consanguineous parents. Family 2 (patients 3 and 4) was an Egyptian family with healthy first cousin consanguineous parents. Patient 1 had diabetes diagnosed as T2D with an acute onset at 28 years without ketosis, initially treated with oral antidiabetes drugs (OAD). In addition, he had congenital macrocytosis with dyserythropoiesis, but without anemia except during two infectious episodes. His sister (patient 2) also had congenital macrocytosis that evolved to a more severe hematological presentation, with recurring anemia and thrombocytopenia requiring transfusions, related to bone marrow failure initially affecting the red and then the megakaryocytic lineages. She was diagnosed with T1D at 5 years and died at 11 years from brain hemorrhage related to severe thrombocytopenia. Bone marrow aspirate and bone marrow biopsies of patients 1 and 2 showed signs of dyserythropoiesis, with megaloblastic erythroblasts and some binucleated cells (Supplementary Data). Histology of the pancreas of patient 2 done at autopsy revealed that the pancreas was fibrotic, with rare and enlarged islets (data not shown). In addition, both patients had multiple naevi. The second index patient (patient 3) was diagnosed with T1D at age 17 years. He presented at age 21 years with bone marrow aplasia, which had progressed from infancy and required regular transfusions. At age 22, he developed a T-cell acute lymphoblastic leukemia. He was treated by chemotherapy and then bone marrow allograft and died shortly after from a severe graft-versus-host disease. His brother (patient 4) also had bone marrow aplasia successfully treated by a bone marrow allograft and T1D diagnosed at 12 years of age. Diabetes of these patients was easily equilibrated by insulin (patients 2 and 3) or by OAD later complemented by low insulin doses (patient 1). Four years after diabetes diagnosis, metabolic evaluation of patient 1 showed an important insulin secretory defect associated with a decrease in insulin sensitivity, in the range of T2D subjects (Supplementary Table 5). According to the family history, all four parents of these families did not have diabetes or any hematological disease, but clinical and genetic studies of the parents and other family relatives could not be performed.

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).

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).

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Table 1

Clinical description of the patients

Family
Family 1
Family 2
Consanguinity
Second cousin parents
First cousin parents
SubjectPatient 1Patient 2aPatient 3Patient 4b
Individual data
 
Sex
 
Male
 
Female
 
Male
 
Male
 
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 OAD and insulin
 
Insulin
 
Insulin
 
Insulin
 
Autoantibodies (GAD, IA2)
 
Negative
 
NA
 
Negative
 
NA
 
Hematology
 
Summary blood and bone marrow characteristics
 
Congenital macrocytosis. Dyserythropoiesis.
 
Congenital macrocytosis evolving to pancytopenia. Bone marrow failure mainly affecting the erythrocytic, then the megacaryocytic lineages.
 
Pancytopenia. Bone marrow aplasia.c
 
Bone marrow aplasia, treated by bone marrow transplantation.
 
Age at follow-up (years)
 
6
 
32
 
Birth
 
5–6
 
9
 
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/ hyperplasia
 

 
Multiple naevi
 
Multiple naevi
 
T-cell ALL (21 years)
 
NA
 
Death Alive or cause of death (age, years) Alive (32) Brain hemorrhage (11) GVHD following bone marrow transplantation (22) Alive (24) 
Family
Family 1
Family 2
Consanguinity
Second cousin parents
First cousin parents
SubjectPatient 1Patient 2aPatient 3Patient 4b
Individual data
 
Sex
 
Male
 
Female
 
Male
 
Male
 
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 OAD and insulin
 
Insulin
 
Insulin
 
Insulin
 
Autoantibodies (GAD, IA2)
 
Negative
 
NA
 
Negative
 
NA
 
Hematology
 
Summary blood and bone marrow characteristics
 
Congenital macrocytosis. Dyserythropoiesis.
 
Congenital macrocytosis evolving to pancytopenia. Bone marrow failure mainly affecting the erythrocytic, then the megacaryocytic lineages.
 
Pancytopenia. Bone marrow aplasia.c
 
Bone marrow aplasia, treated by bone marrow transplantation.
 
Age at follow-up (years)
 
6
 
32
 
Birth
 
5–6
 
9
 
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/ hyperplasia
 

 
Multiple naevi
 
Multiple naevi
 
T-cell ALL (21 years)
 
NA
 
Death Alive or cause of death (age, years) Alive (32) Brain hemorrhage (11) GVHD following bone marrow transplantation (22) Alive (24) 

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 × 109/L (male) and >3.9 × 109/L (female).

iNormal values: 85–95 fL.

jNormal values: 25–100 × 106/L.

kNormal values >4 × 109/L (at birth >10 × 109/L).

lNormal values >150 × 109/L.

Identification of a Mutation in the DUT Gene as Responsible for the Disease

To identify the genetic defect responsible for the syndromic association of bone marrow failure and diabetes, we performed whole-exome sequencing of patient 1 combined with linkage analysis in this family (Fig. 1A). Because of remote consanguinity of his parents (second cousin), patient 1 was expected to have only 1/64 (1.56%) of his genome homozygous IBD. Consistent with this, linkage analysis performed in family 1 identified three regions homozygous IBD over the whole genome, with a total length of 50.0 Mb (Supplementary Table 6). Through whole-exome sequencing of patient 1, we identified a single rare homozygous coding variant over the whole genome, affecting the DUT gene, located within a 33 Mb linkage region (homozygous IBD) on chromosome 15 (Supplementary Table 7). The mutation (chr15:g.48,626,619A>G, hg19) was confirmed by Sanger sequencing and results in a nonsynonymous substitution affecting both the mitochondrial (DUT-M p.Y142C) and the nuclear (DUT-N p.Y54C) isoforms (Fig. 1B and Supplementary Table 8). This variant, reported as rs373184762 in dbSNP, was present in the EVS and ExAC (6) databases at the extremely low overall MAF of 0.000154 (2/12,990) and 0.000066 (8/121,230), respectively, with no rare homozygous subject identified (Supplementary Table 8). Across the multiethnic population panels of EVS (6,500 unrelated subjects, European American and African American) and ExAC (>60,000 unrelated subjects from diverse ancestries worldwide stratified in seven population subgroups), it was found only in the Caucasian populations recorded as European American in EVS and European (non-Finnish) (CEU) in ExAC, with a MAF of 0.000120 (8/66,634) in ExAC (Supplementary Table 8). We next sequenced the coding regions of DUT exons in patient 3 (family 2) and identified the same homozygous mutation (data not shown). This patient originated from Egypt, an admixed population of European/Near Eastern/Maghrebi (all Caucasian) and sub-Saharan African origins (21). Using the CEU MAF as a conservative (upper) estimate of the MAF in Egypt (allele frequency, P = 0.00012), and knowing that he was born to first cousin parents (inbreeding coefficient, FP3 = 1/16), the probability of observing homozygosity for the DUT-M p.Y142C variant in this patient (replication probability) is pxFP3 = 0.00012 × (1/16), or 7.5 × 10−6, thus replicating the initial finding. Overall, the joint probability of the observation of the DUT-M p.Y142C variant in the two unrelated patients 1 and 3 is (pxFP1) × (pxFP3) = (0.00012 × [1/8]) × (0.00012 × [1/16]), or 1.1 × 10−10, which strongly suggests that this mutation is responsible for the diabetes syndrome affecting these patients. Noteworthy, all the missense or loss of function DUT variants reported in these databases were rare (MAF <0.005) and never found homozygous, supporting the highly conserved status of this gene.

The DUT Mutation: Evidence for a Founder Effect in Two Patients From Distinct Population Backgrounds

To investigate if this mutation results from a founder effect and to confirm the distinct ancestry of the patients based on medical records, we performed high-density genotyping of both patients (>2.5 million SNPs) and performed a PCA to compare them to various reference population control groups. We found that both patients share a homozygous 1.9 Mb segment around the DUT mutation (from rs201088112 to rs8039142), and we estimated the frequency of this shared haplotype, using publically available 1000 Genomes data, to be between 0 and 8.9 × 10−15 depending on the population group (Supplementary Table 9). This haplotype was estimated at a non-null frequency only in populations from European ancestry or admixed populations. PCA of the SNP genomic data of the two patients in comparison with various control populations showed that patient 1 clusters within the French population, as expected, whereas patient 3 clusters within a small Greek subgroup, compatible with his Egyptian ancestry, in the absence of referenced Egyptian control group (Supplementary Fig. 3A and B). The kinship coefficient of the two patients estimated using KING software was negative (−0.165), confirming that they are unrelated (7). Overall, this suggests that both patients share the same ancestral haplotype carrying the DUT mutation, originating from an ancestral European founder effect, despite their distinct population background.

Predicted Impact of the DUT Mutation

DUT is a key enzyme of nucleotide metabolism (22,23). It catalyses the hydrolysis of dUTP to dUMP+PPi, which is essential to maintain DNA integrity by two mechanisms: 1) dUMP is the substrate of thymidilate synthase to produce dTTP and 2) this maintains a low dUTP/dTTP, hence limiting the integration of uracil in DNA, whose accumulation leads to cell death due to DNA toxicity, as evidenced by knockout models in various prokaryotic and eukaryotic organisms and other cellular systems (24,25,26,27). The mutation is located near “conserved motif I” of the protein (22,23) and is predicted to be damaging (Supplementary Table 10). Tridimensional modeling of the homotrimeric wild-type and mutated protein predicted conformational changes, including the loss of interactions with several residues, supporting a functional impact of the mutation (Supplementary Fig. 4 and Supplementary Table 11).

Search for Additional DUT Mutated Patients in Extended Patient Collections

To search for additional patients with DUT mutations and to delineate the phenotype spectrum of the syndrome, we sequenced the DUT gene in a highly selected complementary set of patients presenting at least one of the distinctive clinical features observed in the patients of the two index families (i.e., diabetes or bone marrow failure) and whose genetic cause of their disease was unknown (Supplementary Data): 1) 4 unrelated patients with diabetes associated with bone marrow failure, all of whom however had additional clinical manifestations that were not described in the original patients (e.g., short stature, intellectual impairment, dysmorphologies); 2) 14 unrelated patients with juvenile-onset diabetes and their families with increased risk of monogenic causes based on family structure and/or atypical clinical presentation, which were selected to be compatible with linkage to the DUT chromosome region (C.J., unpublished data); and 3) 95 unrelated patients with bone marrow failure or myelodysplastic syndrome, excluding patients with Fanconi anemia (FA), who were selected to have increased risk of monogenic causes based on a young age at onset, physical abnormalities, and/or familial history. We did not identify any rare homozygous or compound heterozygous variant compatible with the mutation status in the coding or regulatory regions of the gene in any of these patients, supporting the rarity to this new syndrome and suggesting that DUT mutations are not a frequent cause of monogenic diabetes or monogenic bone marrow failure.

DUT Inhibition Exacerbates Apoptosis in Pancreatic β-Cells Through the Mitochondrial Cell Death Pathway

To evaluate DUT effects on β-cell viability, specific siRNAs were used to inhibit DUT expression in rat and human islet cells (Fig. 2). DUT inhibition by two independent siRNAs increased apoptosis in both rodent and human β-cells (Fig. 2B, D, and F). These results were confirmed by higher expression of cleaved caspase-9 and -3 in INS-1E cells following DUT inhibition, suggesting that apoptosis is triggered via the mitochondrial-mediated intrinsic pathway (Fig. 2H–J). Despite the observed increased apoptosis, insulin and PDX1 mRNA expression were not modified in DUT-inhibited cells (Supplementary Fig. 5). To determine whether DUT inhibition sensitizes β-cells to different stressors, we treated INS-1E cells transfected with siCtrl or siDUT with cytokines; streptozotocin (STZ), an alkylating agent that induces DNA strand breaks; and H2O2, an inducer of oxidative stress. These agents increased β-cell death following transfection with either siCtrl or siDUT, but there was no clear potentiation between DUT inhibition and cell death induced by these different stressors (Supplementary Fig. 1).

Figure 2

DUT inhibition exacerbates apoptosis in pancreatic β-cells. INS-1E cells (A, B, and GJ), primary rat β-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, AD and GJ) or human (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 β-cells (C, n = 5), and dispersed human islets (E, n = 3). DUT expression was normalized by the housekeeping genes GAPDH (A, C, and G) or β-actin (E) and then by the highest value of each experiment considered as 1. β-Cell apoptosis was evaluated in INS-1E cells (B, n = 4), primary rat β-cells (D, n = 5), and dispersed human islets (F, n = 3) using Hoechst 33342/PI staining. Results are means ± SEM (n as indicated for each figure). H: Western blot showing expression of cleaved caspase-9, cleaved caspase-3, and α-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: Densitometry analysis of the Western blots (H) for cleaved caspase-9 (I) and caspase-3 (J) after normalization for the protein content using α-tubulin and then by the highest value of each experiment considered as 1. Results are means ± SEM of five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with siCtrl, as analyzed by paired Student t test.

Figure 2

DUT inhibition exacerbates apoptosis in pancreatic β-cells. INS-1E cells (A, B, and GJ), primary rat β-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, AD and GJ) or human (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 β-cells (C, n = 5), and dispersed human islets (E, n = 3). DUT expression was normalized by the housekeeping genes GAPDH (A, C, and G) or β-actin (E) and then by the highest value of each experiment considered as 1. β-Cell apoptosis was evaluated in INS-1E cells (B, n = 4), primary rat β-cells (D, n = 5), and dispersed human islets (F, n = 3) using Hoechst 33342/PI staining. Results are means ± SEM (n as indicated for each figure). H: Western blot showing expression of cleaved caspase-9, cleaved caspase-3, and α-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: Densitometry analysis of the Western blots (H) for cleaved caspase-9 (I) and caspase-3 (J) after normalization for the protein content using α-tubulin and then by the highest value of each experiment considered as 1. Results are means ± SEM of five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with siCtrl, as analyzed by paired Student t test.

Close modal

DUT-M is the constitutively expressed isoform of DUT (28,29), and we next knocked down specifically DUT-M using two independent siRNAs to evaluate its contribution for β-cell survival (Supplementary Fig. 2). Similar to the observations following knockdown of both DUT isoforms (Fig. 2), DUT-M–specific knockdown resulted in higher rates of β-cell apoptosis (Supplementary Fig. 2B) via the mitochondrial pathway of apoptosis, as demonstrated by the increased expression of cleaved caspase-9 and -3 in DUT-M–silenced cells (Supplementary Fig. 2C–E).

This is the first report of a DUT mutation in humans, showing that a specific nonsynonymous DUT mutation leads to a new syndrome characterized by diabetes and bone marrow failure.

Despite the extreme rarity of the mutation identified, our genetic study design was able to provide both identification power and validation power, as indicated by the following points.

  1. The consanguineous and multiplex nature of the families studied, with two siblings affected by the same atypical clinical association in each family, strengthens the hypothesis of a rare monogenic inheritance for this syndrome.

  2. The identification power was increased by the study of a child born to remotely consanguineous parents, leading to the reduction of the “targeted region” to 50 Mb (region homozygous IBD in the studied index patient) representing a >60× reduced size compared with the whole genome, leading to the identification of a single rare homozygous coding variant.

  3. The identification of the same mutation in an unrelated patient from a distinct population background was highly statistically significant based on the known frequency of this specific mutation in large multiethnic control populations.

The observed clinical presentation of patients shares similarities with several syndromes caused by impaired cell cycle dependent DNA repair mechanisms, such as FA, characterized by progressive bone marrow failure and increased risk of leukemia (30,31,32). Contrary to FA patients, however, blood and bone marrow cells from our patients did not show increased spontaneous or induced chromosome breakage compared with control subjects. dU repair mechanism uses base-excision repair, which is distinct from the FA DNA repair pathway (DNA interstrand crosslink repair) (31). The lack of synergistic effects between DUT inhibition and STZ (alkylation)-, cytokines-, or H2O2 (single-strand breaks)-induced cell death (present data) is consistent with different DNA repair pathways involved (33).

Diabetes is a cardinal feature of the DUT-mutated syndrome. It is nonautoimmune, as indicated by the lack of autoantibodies against pancreatic β-cells and starts either during childhood or early adulthood (Table 1). In all cases, the patients eventually became insulin requiring. This is in line with our present findings that DUT knockdown triggers apoptosis of human and rat β-cells, with the altered pancreatic islet histology of patient 2 and the very low insulin secretion in patient 1. Glucose metabolism and insulin secretion are frequently impaired in diseases associated with DNA repair defects, including FA (34,35) and Bloom syndrome (36). DNA damage and genomic instability have been shown to alter β-cell replication through cell-cycle inhibition, leading to impaired glucose metabolism and diabetes (36,37). Interestingly, cell-cycle pathway genes are over-represented among T2D susceptibility genes (38), and DNA repair genes were found to be downregulated in T2D patients (39), supporting the role of DNA damage, repair, and cell-cycle pathways in diabetes etiology. Disease severity was variable between patients who carried the same homozygous DUT mutation, suggesting the contribution of other genetic or environmental factors. In the 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 heterozygous state.

The DUT mutation identified affects both nuclear and mitochondrial isoforms, but their contributions to the observed clinical manifestations remain to be established. Megaloblastic anemia occurs in several instances of alteration of the pyrimidine biosynthesis pathway. It may be caused by genetic defects, such as dUMP deficiency because of uridine monophosphate synthase (UMPS) mutations (40) or by vitamin B12 or folate deficiency (41). Megaloblastic anemia erythroblasts were reported to have increased uracil content in DNA (42), as well as DNA damage and cell-cycle arrest (43), suggesting a possible role of nuclear DNA integrity in megaloblastosis and the possible implication of DUT-N defect on this trait. However, hematological manifestations have also been reported in various mitochondrial diseases (44). In particular, megaloblastic anemia and bone marrow failure, together with endocrine and exocrine pancreas deficiency, are observed in Pearson syndrome, characterized by large mitochondrial DNA deletions (45), which were not found in our patients (data not shown). Furthermore, in thiamine-responsive megaloblastic anemia, caused by SLC19A2 mutations and characterized by megaloblastic anemia, diabetes, and sensorineural deafness, respiratory chain defect has been reported in a unique patient, suggesting the role of mitochondrial dysfunction (46). The fact that DUT expression in adult β-cells is mainly contributed by the DUT-M isoform, constitutively expressed (28,29), whereas DUT-N is expressed only in actively dividing cells (adult human β-cells divide poorly, if at all), together with the present observation of increased β-cell apoptosis in DUT-M–specific knockdown, suggest that DUT-M is the main responsible isoform for diabetes.

In addition to their well-documented role for maintaining a tight control of cellular dUTP level, recent studies indicate that dUTPases are also involved in the regulation other cellular processes that may contribute to autoimmunity and apoptosis (23). The presently described syndrome further broadens the multifaceted spectrum of DUT’s function to the regulation of biological processes of clinical significance, with impact on diabetes and bone marrow failure.

5-fluorouracil, leading to dTTP depletion, is commonly used for cancer chemotherapy (47,48). Both myelosuppression and secondary diabetes are common side effects of 5-fluorouracil therapy (49,50), and a case of fulminant T1D following 5-fluorouracil treatment has recently been reported (51). Although a recent first-phase study of a dUTPase inhibitor indicates good tolerance (52), our findings warrant close monitoring of hematological and metabolic adverse events in these patients.

Our observation highlights the importance of the tight control of uracil level in DNA for numerous biological functions, including hematopoiesis and insulin production, and identifies a novel form of monogenic diabetes associated with bone marrow failure.

Acknowledgments. The authors thank the patients and their families for their participation. The authors thank Claire Vandiedonck from INSERM UMRS 958 for expert methodological advice and helpful discussions. The authors thank the Centre National de Génotypage (CNG) for providing access to their genomic platform and Céline Derbois, Christophe Caloustian, and Sylvana Pavek (from the CNG) for expert technical assistance. The authors thank INSERM, Université Paris Diderot-Paris 7, and the Assistance Publique-Hôpitaux de Paris for their support.

Funding. This work was supported by grants from the Agence Nationale de la Recherche (ANR-09-GENO-021), the European Foundation for the Study of Diabetes/JDRF/Novo Nordisk (“Identification of genes with monogenic contribution”), the Assistance Publique-Hôpitaux de Paris (Programme Hospitalier de Recherche Clinique DIAGENE), the GIS Maladies Rares, France Génomique (DIAPED) (to C.J.), the European Union Framework Programme Horizon 2020 (project T2DSystems), and the Fonds National de la Recherche Scientifique, Belgium, and Actions de Recherche Concertée de la Communauté Française, Belgium (to D.L.E.). R.S.D.S. was supported by a postdoctoral fellowship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil. M.D. was supported by a doctoral fellowship from the Ministère de l’Éducation Nationale, de l’Enseignement Supérieur et de la Recherche, France. L.Marr. was supported by a scientific postdoctoral fellowship from the Fonds National de la Recherche, Belgium.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. R.S.D.S., L.Marr., and D.L.E. performed the functional experiments and analyses of pancreatic islets. M.D., A.P., S.R., V.S., D.B., and C.J. performed the genetic experiments and analyses. A.P. performed the protein modeling analysis. L.Mars. and P.M. contributed human islets material for the functional studies. C.B. performed the SNP array genotyping. B.B., G.S., and J.-F.G. identified the two index patients and families and performed the clinical investigations. S.I., M.N., and J.S. identified and selected additional patients with bone marrow failure. J.M.-P. performed next-generation sequencing targeted sequencing. D.L.E., J.-F.G., and C.J. conceived and designed the study. D.L.E. and C.J. wrote the article, with contributions from R.S.D.S., M.D., A.P., S.R., and J.-F.G. All co-authors read and approved the manuscript. C.J. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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