Dominant ER Stress-Inducing WFS1 Mutations Underlie a Genetic Syndrome of Neonatal/Infancy-Onset Diabetes, Congenital Sensorineural Deafness, and Congenital Cataracts

Abstract

Neonatal diabetes is frequently part of a complex syndrome with extrapancreatic features: 18 genes causing syndromic neonatal diabetes have been identified 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 deafness, and congenital cataracts. Further testing was performed 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 deafness 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 WFS1 mutations 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.

Figure 1

Phenotypic spectrum caused by autosomal dominant and autosomal recessive WFS1 mutations. The figure includes WFS1 mutations reported as disease causing in the Human Gene Mutation Database that have a minor allele frequency <0.00447 in the ExAC database (maximum allele frequency possible for Wolfram syndrome–causing mutations, assuming a disease frequency of 1/100,000, as calculated by the alleleFrequencyApp [http://cardiodb.org/allelefrequencyapp/]).

Figure 2

Comparative protein modeling of WFS1. The COOH-terminal lumenal domain of WFS1 (residues 653–869) was submitted to SWISS-MODEL for template identification; models were then constructed for residues 798–838 using the top-scoring template (PDB identifier 1ltl; MCM protein from Methanothermobacter thermautotrophicus; 27% sequence similarity over region modeled). Upper panels show the protein backbone with selected side chains as indicated in stick format for wild-type and variant WFS1, the predicted molecular surface is shown as a transparent layer, and broken green lines indicate predicted hydrogen bonds. Lower panels show the same structures but rotated ∼45° away from the viewer around a horizontal axis and with the predicted molecular surface shown as a solid layer. In all panels, amino acids are colored by type (red, acidic; blue, basic; yellow, uncharged polar; gray, nonpolar/hydrophobic); regions of significantly altered surface properties are indicated by red ovals in the lower panels. A830, p.Ala830; E809, p.Glu809; E830, p.Glu830; K809, p.Lys809.

Figure 3

Double immunofluorescence staining of COS7 cells transiently transfected with dominant and recessive variants of WFS1. COS7 cells were transiently transfected with 1) control (pcDNA3); 2) wild-type WFS1 tagged at its C-terminus with a FLAG epitope (WT); 3) the dominant mutations p.His313Tyr (H313Y), p.Glu809Lys (E809K), and p.Glu830Ala (E830A); 4) a dominant variant causing isolated adult-onset diabetes, p.Trp314Arg (W314R) (7); and 5) two recessive Wolfram syndrome–causing mutations, p.Pro724Leu (P724L) and p.Gly695Val (G695V) (20). The cells were stained with anti-FLAG (red fluorescence, left) and anti-calnexin (green fluorescence, center). Right panels: Merged images are shown. n = 4; magnification is 10×.

Figure 4

High–molecular weight complexes of WFS1 mutants in detergent-insoluble fractions. HeLa cells were transfected with control (pcDNA), FLAG-tagged wild-type WFS1 (WT), mutant WFS1 p.Pro724Leu (p.P724L), p.Glu809Lys (p.E809K), p.Glu830Ala (p.E830A), or mutant WFS1 p.His313Tyr (p.H313Y) expression plasmid and separated into detergent-soluble (Sol) and detergent-insoluble (Insol) fractions with anti-FLAG antibody. IB, immunoblot.

Figure 5

Luciferase reporter assays in HeLa cells transfected with the ERSE reporter together with control (pcDNA) (EV), wild-type WFS1 (WT), mutant c.2171C>T p.Pro724Leu (P724L), mutant c.937C>T p.His313Tyr (p.H313Y), mutant c.2489A>C p.Glu830Ala (p.E830A), or mutant c.2425G>A p.Glu809Lys (p.E809K) expression plasmid. Cells were untreated (UT) or treated with TG (100 nmol/L) for 8 h. Relative intensity of luciferase (Promega Dual-Luciferase Reporter Assay System) was then measured (n = 3; dashed lines represent mean). Transfections were normalized with the pRL-TK vector (Promega) as an internal control. Asterisks indicate a significant difference analyzed by one-way ANOVA followed by Dunnett test: *P < 0.01, **P < 0.001, ***P < 0.0001.