WFS1 protein expression correlates with clinical progression of optic atrophy in patients with Wolfram syndrome

Abstract

Background: Wolfram syndrome (WFS) is a rare disorder characterised by childhood-onset diabetes mellitus and progressive optic atrophy. Most patients have variants in the WFS1 gene. We undertook functional studies of WFS1 variants and correlated these with WFS1 protein expression and phenotype.

Methods: 9 patients with a clinical diagnosis of WFS were studied with quantitative PCR for markers of endoplasmic reticulum (ER) stress and immunoblotting of fibroblast protein extracts for WFS1 protein expression. Luciferase reporter assay was used to assess ATF-6 dependent unfolded protein response (UPR) activation.

Results: 6 patients with compound heterozygous nonsense mutations in WFS1 had no detectable WFS1 protein expression; 3 patients with missense variants had 4%, 45% and 48% WFS1 protein expression. One of these also had an OPA1 mutation and was reclassified as autosomal dominant optic atrophy-plus syndrome. There were no correlations between ER stress marker mRNA and WFS1 protein expression. ERSE-luciferase reporter indicated activation of the ATF6 branch of UPR in two patients tested. Patients with partial WFS1 expression showed milder visual acuity impairment (asymptomatic or colour blind only), compared with those with absent expression (registered severe vision impaired) (p=0.04). These differences remained after adjusting for duration of optic atrophy.

Conclusions: Patients with WFS who have partial WFS1 protein expression present with milder visual impairment. This suggests a protective effect of partial WFS1 protein expression on the severity and perhaps progression of vision impairment and that therapies to increase residual WFS1 protein expression may be beneficial.

Keywords: diabetes mellitus; genetics, medical; neurodegenerative diseases.

Figure 1

Family pedigrees and functional data.(A) Pedigrees of the 7 families reported in this study. All patients included in the study marked in grey. The age of onset of diabetes mellitus (DM) and optic atrophy (OA) stated. S02 had a de-novo mutation. For patient S07, OPA1 and WFS1 variants were found in all three generations in this family: S07’s maternal grandfather had isolated OA; S07’s mother had isolated OA and Type 1 DM. OA and DM in S07’s family represented by quarter stripes (OA) and quarter black (DM). All other patients inherited recessive alleles from each parent.(B) Immunoblotting images and corresponding bar chart with standard error bars showing levels of WFS1 protein. WFS1 and beta-actin (BA) protein levels measured in fibroblast from patients with WFS and healthy controls. C1, C2, C3 = healthy controls; CAve: average of controls. WFS1 levels for CAve=100%. WFS1 protein was undetectable in patients: S03, S04, S06, S09, S10 and S11. WFS1 protein was reduced in S01, S02 and S07 by 96.2%, 53.3%, and 55.4% respectively in comparison to CAve. Analysis by Student's T-test.(C) Bar chart with standard error bars showing quantitative PCR analysis of WFS1 mRNA, as percentage change when standardised with control. C=control. (n= 4) Analysis by Student's T-test.(D) Bar chart with standard error bars showing quantitative PCR analysis of ER stress marker mRNA: BiP, CHOP and sXBP1, as percentage change, when standardised with control (C). (n=4). Dark grey bars indicate patient in the deficient WFS1 protein group, and the light grey bar indicates the patient is in the partial WFS1 protein group. Analysis by Student's T-test.(E) Bar chart with standard error bars showing quantification of ATF6-dependent UPR activation by ERSE luciferase reporters, for SO2 and S10, as a percentage change compared from control (C). (n=4) Analysis by Student's T-test.ns: P >0.05; * P≤0.05; ** P ≤0.01; *** P≤0.001 compared with control samples

Figure 2

Schematic representation of WFS1 protein and variant locations.

Figure 3

Comparison of visual acuity data between groups.(A) Box plot comparing LogMAR values between deficient (n=6) and partial WFS1 (n=2) protein groups; showing statistically significant difference (p=0.04). logMAR value (visual acuity logarithm of the minimum angle of resolution) is the magnification requirement, the higher the logMAR value, the worse the visual acuity (<1.0 mild-moderate visual impairment, 1.0-1.3 sight impaired (partial sighted) >1.3 severely sight impaired (blind). (B) Scatter graph showing logMAR values of each patient corresponding to the duration of optic atrophy from the diagnosis of OA (irrespective of the age of diagnosis). Plots for patients in deficient WFS1 protein group shown in grey triangles (n=6); regression line is drawn between the patients in deficient WFS1 protein group (correlation of determination is 0.93). Plots for patients in partial WFS1 protein group (SO1 + SO2) shown in white circles (n=2).