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Dominant ER Stress–Inducing WFS1 Mutations Underlie a Genetic Syndrome of Neonatal/Infancy-Onset Diabetes, Congenital Sensorineural Deafness, and Congenital Cataracts Elisa De Franco,1 Sarah E. Flanagan,1 Takuya Yagi,2 Damien Abreu,2 Jana Mahadevan,2
Matthew B. Johnson,1 Garan Jones,3 Fernanda Acosta,4 Mphele Mulaudzi,5 Ngee Lek,6,7 Vera Oh,6
Oliver Petz,8 Richard Caswell,1 Sian Ellard,1,3 Fumihiko Urano,2 and Andrew T. Hattersley1
Diabetes 2017;66:2044–2053 | https://doi.org/10.2337/db16-1296
Neonatal diabetes is frequently part of a complex syndrome with extrapancreatic features: 18 genes causing syndromic neonatal diabetes have been identi- fied to date. There are still patients with neonatal diabetes who have novel genetic syndromes. We performed exome sequencing in a patient and his unrelated, unaffected parents to identify the genetic etiology of a syndrome characterized by neonatal diabetes, sensorineural deaf- ness, and congenital cataracts. Further testing was per- formed in 311 patients with diabetes diagnosed before 1 year of age in whom all known genetic causes had been excluded. We identified 5 patients, including the initial case, with three heterozygous missense mutations in WFS1 (4/5 confirmed de novo). They had diabetes diagnosed before 12 months (2 before 6 months) (5/5), sensorineural deaf- ness diagnosed soon after birth (5/5), congenital cataracts (4/5), and hypotonia (4/5). In vitro studies showed that these WFS1 mutations are functionally different from the known recessive Wolfram syndrome–causing mutations, as they tend to aggregate and induce robust endoplasmic reticulum stress. Our results establish specific dominant WFS1mutations as a cause of a novel syndrome including neonatal/infancy-onset diabetes, congenital cataracts,
and sensorineural deafness. This syndrome has a discrete pathophysiology and differs genetically and clinically from recessive Wolfram syndrome.
Neonatal diabetes is diagnosed before 6 months of age and reflects a severe reduction in b-cell number or function. A genetic diagnosis is possible in 82% of patients with 23 ge- netic causes identified to date (1,2). Thirty-nine percent of patients with neonatal diabetes have a genetic etiology that results in the development of at least one extrapancreatic feature in addition to diabetes (1). This subtype is pheno- typically and genetically heterogeneous with mutations in 18 genes known to cause syndromic forms of neonatal di- abetes (1). Fifteen percent of patients with syndromic neo- natal diabetes do not have a mutation in one of the known etiological genes, suggesting undescribed novel genetic syndromes.
The identification of novel etiological genes in patients with syndromic neonatal diabetes has been revolutionized by the introduction of next-generation sequencing. In outbred pedigrees, exome or whole-genome sequencing of an affected
1Institute of Biomedical and Clinical Science, University of Exeter Medical School, Exeter, U.K. 2Department of Medicine, Washington University School of Medicine in St. Louis, St. Louis, MO 3Department of Molecular Genetics, Royal Devon & Exeter NHS Foundation Trust, Exeter, U.K. 4Department of Pediatrics, Centro Médico Nacional 20 de Noviembre ISSSTE, Mexico City, Mexico 5Department of Paediatrics and Child Health, University of Pretoria Medical School, Pretoria, South Africa 6KK Women’s and Children’s Hospital, Singapore 7Duke-NUS Medical School, National University of Singapore, Singapore 8Praxis für Kinder-und Jugendmedizin, Diabetologische Schwerpunktpraxis, Coesfeld, Germany
Corresponding authors: Andrew T. Hattersley, firstname.lastname@example.org, and Fumihiko Urano, email@example.com.
Received 25 October 2016 and accepted 23 April 2017.
This article contains Supplementary Data online at http://diabetes .diabetesjournals.org/lookup/suppl/doi:10.2337/db16-1296/-/DC1.
E.D.F. and S.E.F. contributed equally to this work.
© 2017 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. More information is available at http://www.diabetesjournals .org/content/license.
2044 Diabetes Volume 66, July 2017
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case and their unaffected, unrelated parents is a powerful tool to identify de novo heterozygous mutations. We have successfully used this approach to identify two novel etiological genes for syndromic neonatal diabetes in our cohort (2,3).
Neonatal diabetes has not been described in patients with recessive loss-of-function mutations in WFS1, the ge- netic cause of Wolfram syndrome, even though early-onset diabetes is a cardinal feature of this multisystem disease. In these patients, diabetes onset occurs in the first decade of life (median age at onset 6 years, range 1–32 years [4–6]). Additional extrapancreatic features (optic atrophy, diabetes insipidus, and hearing loss) usually develop sequentially be- tween the first and third decade of life.
Heterozygous mutations in WFS1 have been found to cause less severe phenotypes than Wolfram syndrome– associated recessive mutations. In particular, dominant WFS1 mutations have been reported as causing isolated adult- onset diabetes (7), isolated low-frequency hearing loss (8,9), optic atrophy and hearing impairment (10–12), and isolated congenital nuclear cataracts (13) (Fig. 1).
We used a trio-based exome sequencing strategy to identify dominant WFS1 mutations as the cause of a novel syn- drome characterized by permanent neonatal/infancy-onset
diabetes, congenital or early-onset sensorineural deafness, and congenital cataracts.
RESEARCH DESIGN AND METHODS
Genetic Analysis We performed exome sequencing on samples from an affected proband and parents using SureSelect Human All Exon Kit V4 (Agilent). Paired-end sequencing was per- formed on an Illumina HiSeq2500, using 100 bp reads. The resulting reads were aligned to the hg19 reference genome with BWA. Variants were called with GATK UnifiedGenotyper and annotated using ANNOVAR and SeattleSeq Annotation server as previously described (3).
Replication studies were performed in a cohort of 311 patients diagnosed with diabetes before 12 months of age in whom mutations in the 22 known neonatal diabetes genes had been excluded. All the patients in the cohort were analyzed using our targeted next-generation sequencing assay that includes baits for WFS1 as previously described (14).
The bioinformatics tools SIFT, PolyPhen-2, and Align GVGD were accessed through the Alamut software (In- teractive Biosoftware, Rouen, France) to predict the effect of novel variants on the WFS1 protein. If the variant was deemed likely pathogenic or of unknown significance,
Figure 1—Phenotypic spectrum caused by autosomal dominant and autosomal recessive WFS1 mutations. The figure includes WFS1 muta- tions reported as disease causing in the Human Gene Mutation Database that have a minor allele frequency
mutation testing was performed in the patient (to confirm the mutation) and the parents (when available) to assess whether the mutation had arisen de novo. Microsatellite analysis of parent/proband trios using the PowerPlex kit (Promega, Southampton, U.K.) was used to confirm family relationships for the putative de novo mutations. In silico modeling of the WFS1 p.Glu809Lys and p.Glu830Ala mu- tations was carried out using the SWISS-MODEL server (15). Additionally, as templates of only limited sequence similarity could be identified, the PredictProtein server was used to predict secondary structure and functional mo- tifs from primary sequence alone (16).
Immunostaining COS7 cells were transiently transfected with dominant and recessive WFS1 mutations for 48 h. The cells were fixed in 2% paraformaldehyde for 30 min at room temperature and then permeabilized with 0.1% Triton X-100 for 2 min. The fixed cells were washed with PBS/Tween 0.1%, blocked with Image-iT FX signal enhancer (Invitrogen) for 30 min, and incubated in primary antibody overnight at 4°C. The cells were washed three times in PBS/Tween 0.1% and incubated with secondary antibody for 1 h at room temperature. Im- ages were obtained with an FSA 100 microscope (Olympus). Anti-FLAG antibody and anti-calnexin antibody were obtained from Cell Signaling Technology (Danvers, MA) and Proteintech Group (Chicago, IL), respectively.
INS-1 cells were plated in eight-chamber slides (Thermo Fisher Scientific) at a density of 50,000 cells per chamber and transiently transfected with dominant and recessive GFP- tagged WFS1 mutant constructs for 48 h. The cells were fixed in 4% paraformaldehyde/PBS for 30 min at room temperature, washed three times with PBS, and then per- meabilized with 0.1% Triton X-100/PBS for 10 min at room temperature. Cells were blocked overnight in a 2% BSA so- lution of 0.1% Triton X-100/PBS and washed three times the next day with PBS. Slides were prepared with antifade mounting medium containing DAPI (Vector Laboratories), and images were obtained with an FSA 100 microscope.
Immunoblotting HeLa cells were transfected with full-length human WFS1 cDNA tagged with a FLAG epitope