PATIENT ENROLLMENT AND GENETIC ANALYSIS

The study had ethical and institutional approval (Moorfields Eye Hospital NHS Foundation Trust, London; the Newcastle Eye Centre, Royal Victoria Infirmary, Newcastle upon Tyne; the Royal Bolton Hospital, Bolton), and its design complied with the Declaration of Helsinki. The multicenter study cohort of 37 patients with a diagnosis of inherited optic neuropathy and WFS1 variants was ascertained in the Genetic Service at Moorfields Eye Hospital NHS Foundation Trust (London, UK), the Newcastle Eye Centre, Royal Victoria Infirmary (Newcastle upon Tyne, UK), and Bolton NHS Foundation Trust (Bolton, Greater Manchester, UK). WFS1 genetic testing was conducted in accredited molecular genetic laboratories.

External databases (Genome Aggregation Database [gnomAd, https://gnomad.broadinstitute.org ], ClinVar [ https://www.ncbi.nlm.nih.gov/clinvar ], and LOVD [ Leiden Open Variation Database, https://www.lovd.nl ]) and prediction algorithms (Polymorphism Phenotyping version 2 [PolyPhen-2, http://genetics.bwh.harvard.edu/pph2 ] and Mutation Taster [ http://www.mutationtaster.org ]) were used to assess WFS1 variant frequency and pathogenicity. ACMG (American College of Medical Genetics) classification for each variant is also provided.

For the purpose of comparative analyses, data from patients with DOA and disease-causing variants in OPA1 gene were included as detailed in the advanced psychophysics and metabolomic sections.

CLINICAL ASSESSMENT

Clinical data of the diagnostic and follow-up examinations of 37 patients were retrieved by retrospective review of the medical records from the different study sites. Thirteen of these patients were examined as part of the NIHR RD-TRC Inherited Optic Neuropathy study at Moorfields Eye Hospital NHS Foundation Trust (London, UK). Three of them underwent advanced psychophysical investigations. Blood samples were collected from 9 patients for metabolomic analysis.

The following demographic and clinical data were recorded: WFS1 genotype, detailed family history, sex, age at diagnosis of WON, extraocular abnormalities, best-corrected visual acuity (BCVA), visual field, red-green color discrimination using Ishihara pseudoisochromatic plates, optical coherence tomography (OCT) imaging of optic disc and macula, and visual electrophysiology. Visual fields were studied using either automated Humphrey visual field perimetry (Humphrey Visual Field Analyzer, Model 750; Humphrey Instruments) or Goldmann perimetry. The assessment profile for each patient in relation to the imaging, visual field, psychophysical, electrophysiological, and metabolomic analyses is indicated in Table 1 .

BCVA was recorded using either the Early Treatment Diabetic Retinopathy Study (ETDRS) or Snellen charts. The Snellen fractions were converted to logMAR units. For 2 elderly patients (P5, P7), no visual acuity data were available. Therefore, BCVA data of 35 patients, 18 of whom had AR and 17 AD WON, were included in the study. Follow-up BCVA data were available for 33 patients, 18 with AR and 15 with AD disease, with BCVA measurements obtained for at least 2 time points. The visual acuity values potentially affected by ocular diseases other than WON, or of the level of light perception, were not included in the analysis. Mean and median BCVA for the AR and AD subgroups were calculated using the first available BCVA value for each patient.

Altogether, 246 BCVA values were used for the longitudinal analysis. Hierarchical regression models were used to estimate longitudinal BCVA outcomes. Population-level effects, also known as fixed effects, were included to reflect the average response seen across all patients at all times (the intercept), separate gradients with respect to time in the AR and AD patient groups, and further nonlinear functions of time, as required. Group-level effects, also known as random effects, were used at the patient and eye levels to reflect that outcomes for an eye are correlated through time and that eyes for a patient are also highly correlated.

OCT IMAGING

High-resolution spectral-domain optical coherence tomography (SD-OCT) data were acquired with the Spectralis (Heidelberg Engineering Ltd) and Cirrus HD-OCT 4000 (Carl Zeiss Meditec, Inc) platforms. For peripapillary RNFL measurements, a 3.5-mm-diameter circular scan centered on the optic disc was used and the sectorial data were collected and compared to the normative data. Automated segmentation and thickness analyses of 10 retinal layers were performed for perifoveal volumetric retinal B-scans using the Heidelberg Engineering segmentation tool, included in the Spectralis Glaucoma Module software (version 6.0), as previously described. ^, Normative data were generated from SD-OCT images of 48 healthy eyes of 48 subjects.

VISUAL ELECTROPHYSIOLOGY

Twelve patients underwent visual electrophysiological testing according to the standard protocols of the International Society for Clinical Electrophysiology of Vision (ISCEV). ^, The tests included the pattern reversal visual evoked potential (PVEP), flash visual evoked potential (FVEP), and pattern electroretinogram (PERG). Pattern ERGs were recorded to a 0.8° check size using both a standard checkerboard field (12 × 15°) and additionally to a large field (24 × 30°). International-standard full-field ERGs were recorded using gold foil corneal electrodes in 9 patients. In addition to standard measurements, the photopic negative response (PhNR) component of the light-adapted single flash (LA 3) ERG was assessed. Two amplitude ratios, N95-P50 of PERG and PhNR–b-wave of the LA 3 ERG, were additionally computed, with the PhNR amplitude measured from the preceding b-wave peak to PhNR trough. The results were compared to reference normative data.

ADVANCED PSYCHOPHYSICAL INVESTIGATIONS

Three patients (P24, P25, and P30) underwent advanced psychophysical investigations using previously described methodology. The normal subjects for psychophysical tests were 15 individuals aged 17-78 years at the time of testing with normal BCVA and normal color vision as assessed by standard color vision tests. Data for 9 patients with OPA1 DOA were used for comparative analyses, and their details have previously been reported (patients P3, P4, P6, P9, P10, P11, P17, P18, and P23).

The test conditions for the following tests were as previously described by the same investigators. ^, Achromatic spatial contrast sensitivity was measured as a function of spatial frequency. The target stimuli were horizontally orientated Gabor patterns with spatial frequencies ranging from 0.5 to 16 cycles per degree (cpd) and with a spatial Gaussian window with a standard deviation of 6°. Chromatic discrimination was tested using the so-called trivector test procedure implemented as part of the Cambridge Colour Test, version 1.5 (Cambridge Research Systems Ltd).

The test conditions included the modification for observers with reduced visual acuity and the viewing distance of 62.6 cm so that the Landolt “C” opening subtended 5° of visual angle. L-cone temporal acuities (critical flicker fusion, cff) were measured using a Maxwellian-view optical system. The L-cone stimulus was produced by flickering a 650-nm circular target of 4° visual angle in diameter superimposed in the center of a steady 481-nm circular background field of 9° diameter. ^{, , ,}

STATISTICAL ANALYSIS

Independent samples Mann-Whitney U was used for comparison of distribution of continuous variables in the various patient subgroups and controls. Spearman rank correlation were used for the analysis of statistical dependence between various variables as indicated in the Results section. The SPSS, version 25 (IBM Corp), and R, version 3.6 (The R Foundation for Statistical Computing), were used for the analyses.

METABOLOMIC ANALYSIS

For the metabolomic analysis, blood samples were collected from 9 WON patients, 9 gender-matched OPA1 DOA patients, and 9 gender-matched healthy individuals as healthy controls (HC). The WON group (patients P1, P17, P18, P21, P27, P28, P30, P31, and P37) included 6 patients with AR and 3 patients with AD WON ( Table 1 ). Ages between WON patients, OPA1 DOA patients, and healthy controls were matched with means ± standard error of the mean (SEM) of 42 ± 4 years for both WFS1 and HC groups, and 44 ± 4 years for the OPA1 DOA group.

Serum samples were immediately processed after collection, aliquoted, and maintained at –80°C until required. As part of a wider metabolomic study, these samples were randomized among other patient cohorts with inherited optic neuropathies and assigned a unique identifier, prior to being sent to Metabolon Inc for processing. Proteins were precipitated with methanol under vigorous shaking for 2 minutes (Glen Mills GenoGrinder 2000) followed by centrifugation. The resulting extract was divided into 5 fractions: 2 for analysis by 2 separate reverse-phase (RP) / ultraperformance liquid chromatography (UPLC)–tandem mass spectrometry (MS/MS) methods with positive ion mode electrospray ionization (ESI), 1 for analysis by RP/UPLC-MS/MS with negative ion mode ESI, 1 for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and 1 sample was reserved for backup.

Samples were placed briefly on a TurboVap (Zymark) to remove the organic solvent. All samples were processed on an UPLC and Q-Exactive high resolution/accurate mass spectrometer (Thermo Scientific) interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The MS analysis alternated between MS and data-dependent MSn scans using dynamic exclusion. The scan range varied slighted between methods but covered 70-1000 m / z . Raw data were extracted, peak-identified, and QC processed using Metabolon’s hardware and software. Identification of known chemical entities was based on comparison to metabolomic library entries of purified standards. Using this protocol, 1319 metabolites were detected and 1018 metabolites were positively identified

MetaboAnalyst, version 4.0, and R, version 3.6 (The R foundation for Statistical Computing), were used in combination for data preprocessing, statistical analysis, and visualization of univariate and multivariate analyses. Metabolites that were present in less than 25% of all samples were filtered out. Blank data for the remaining metabolites were imputed as half of the minimum positive value. Data was then standardized using z scores. The number of metabolites detected in the final analysis was 694, with 585 metabolites being positively identified. Statistical significance when comparing WON, OPA1 DOA and HC groups was evaluated by 1-way analysis of variance with Benjamini-Hochberg correction for multiple hypothesis testing (FDR values), followed by Fisher least significant difference post hoc test for pairwise comparisons.

WFS1 GENOTYPE

The study cohort included 37 patients (22 females and 15 males) with WON. Their demographic, genetic, and clinical data are presented in Table 1 . The results of the assessment of the pathogenicity using external databases and the ACMG classification are presented for each variant in Table 2 . Nineteen patients from 10 families carried a single heterozygous pathogenic WFS1 variant compatible with AD WON ( Table 1 ). All dominant variants were missense.

Table 2

WFS1 Gene Variants in the Study Population

Genotype, WFS1 Variant	Patients(n)	Exon	Allele Frequency gnomAD(all)	Prediction PolyPhen2(score)	Prediction Mutation Taster	ClinVar Interpretation (No. of Submissions)	LOVD	ACMG Classification	First Published Report *
AD inheritance
c.2051C>T, p.Ala684Val	13	8	VNF	Probably damaging (1.0)	Disease causing	Pathogenic (8)	Pathogenic	Pathogenic: PS3, PM1, PM2, PP2, PP3, PP5	Tessa et al
c.2390A>T, p.Asp797Val	1	8	VNF	Possibly damaging (0.826)	Disease causing	—	—	Likely pathogenic: PM2, PM5, PP2, PP3	Rohayem et al ; patient reported in Majander et al ; not reported in AD disease in other cohorts
c.968A>G, p.His323Arg	2	8	VNF	Probably damaging (0.997)	Disease causing	—	—	Likely pathogenic: PM2, PP1, PP2, PP3, PP5,	Smith et al ; patients reported in Majander et al ; not reported in AD disease in other cohorts
c.2161A>T, p.Asn721Tyr	2	8	VNF	Possibly damaging (0.826)	Disease causing	—	—	Likely pathogenic: PM2, PP1, PP2, PP3	Patients reported in Majander et al ; not reported in other cohorts
De novo
c.937C>T, p.His313Tyr	1	8	VNF	Probably damaging (0.974)	Disease causing	Likely pathogenic (1)	—	Likely pathogenic: PS2, PM2, PP2, PP3, PP5	Hansen et al
AR inheritance
c.505G>A, p.Glu169Lys	2	5	0.00002483	Probably damaging (1.0)	Disease causing	Uncertain significance (1)	—	Likely pathogenic: PM2, PM5, PP1, PP2, PP3	Hardy et al
c.874C>A, p.Pro292Thr		8	0.000007966	Probably damaging (1.0)	Disease causing	—	—	Likely pathogenic: PM2, PP1, PP2, PP3, PP5	Astuti et al
c.2648_2651delTCTT, p.Phe883Serfs * 68	2	8	0.00009239	—	Disease causing	Pathogenic (2) Likely pathogenic (1)	—	Pathogenic: PVS1, PM2, PP1, PP3, PP5	Hardy et al
c.1597C>T, p.Pro533Ser		8	0.0007487	Probably damaging (1.0)	Disease causing	Benign (1) Likely benign (1) Uncertain significance (3)	VUS	Likely pathogenic: PM1, PM2, PM5, PP1, PP2, PM3, PP3	Crawford et al ; patients reported in Majander et al ; not reported in optic neuropathy or Wolfram syndrome in other cohorts
c.2643_2644delC, p.Phe883Leufs * 56 (homozygous)	1	8	VNF	—	Disease causing	—	—	Pathogenic: PVS1, PM2, PP3, PP5	Patient reported in Majander et al ; not reported in other cohorts
c.409_424dup16, p.Val142Glyfs * 110	1	4	0.00004386	—	Disease causing	Pathogenic (3)	—	Pathogenic: PVS1, PM2, PP5	Rohayem et al
c.2262_2263delCT, p.Cys755Serfs * 3		8	VNF	—	Disease causing	—	—	Pathogenic: PVS1, PM2, PP3	Khanim et al
c.1232_1233delCT, p.Ser411Cysfs * 131 (homozygous)	1	8	0.000003977	—	Disease causing	—	—	Pathogenic: PVS1, PM2, PP3	Giuliano et al
c.1433G>A, p.Trp478 *	1	8	0.00002003	—	Disease causing	—	—	Pathogenic: PVS1, PM2, PP3	Hardy et al
c.2648_2651delTCTT, p.Phe883Serfs * 68		8	0.00009239	—	Disease causing	Pathogenic (2) Likely pathogenic (1)	—	Pathogenic: PVS1, PM2, PP3, PP5	Hardy et al
c.1558C>T, p.Gln520 *	1	8	0.000003991	—	Disease causing	—	—	Pathogenic: PVS1, PM2, PP3	Giuliano et al
c.1372G>A, p.Ala458Thr		8	0.00003188	Probably damaging (0.990)	Disease causing	—	—	VUS: PM2, PM3, PP3	Not reported
c.1283C>G, p.Pro428Arg	1	8	VNF	Probably damaging (1.0)	Disease causing	—	—	Likely pathogenic: PM1, PM2, PP3, BP1, PM3	Astuti et al
c.2319C>G, p.Tyr773 *		8	VNF	—	Disease causing	—	—	Pathogenic: PVS1, PM2, PP3	Astuti et al
c.1549delC, P.Arg517fs * 5	1	8	VNF	—	Disease causing	—	—	Likely pathogenic: PVS1, PM2	Hardy et al
c.1597C>T, p.Pro533Ser		8	0.0007487	Probably damaging (1.0)	Disease causing	Benign (1) Likely benign (1) Uncertain significance (2)	VUS	VUS: PM2, PP3, BP1	Crawford et al ; not reported in optic neuropathy or Wolfram syndrome in other cohorts
c.1309G>C, p.Gly437Arg	1	8	0.000003983	Benign (0.029)	Polymorphism	—	—	Likely benign: PM2, PP5	Hardy et al
c.977C>T, p.Ala326Val		8	0.00004951	Probably damaging (0.997)	Disease causing	Uncertain significance (1)	—	Likely benign: PM2, PP3, BP1, BP6	Toppings et al
c.409_424dup16, p.Val142Glyfs * 110	1	4	0.00004386	—	Disease causing	Pathogenic (3)	—	Pathogenic: PVS1, PM2, PP5	Gómez-Zaera et al
c.2194C>T, p.Arg732Cys		8	0.00006092	Probably damaging (1.0)	Disease causing	Uncertain significance (1)	VUS	VUS: PM2, PM3, PP3	La Morgia et al
c.1234_1237delCTGT, p.Val412Serfs * 29	1	8	0.000003976	—	Disease causing	—	—	Likely pathogenic: PVS1, PM2	Gasparin et al
c.1673G>A, p.Arg558His		8	0.00006383	Probably damaging (1.0)	Disease causing	Likely pathogenic (1)	—	Likely pathogenic: PM2, PM5, PP3, PP5	Smith et al
c.2213C>A, p.Ala738Asp	2	8	0.00005054	Benign (0.011)	Disease causing	—	—	Likely pathogenic: PM2, PM3, PP1	Not reported
c.2648_2651delTCTT, p.Phe883Serfs * 68		8	0.00009239	—	Disease causing	Pathogenic (2) Likely pathogenic (1)	—	Pathogenic: PVS1, PM2, PP5	Hardy et al
c.1243_1245del, p.Val415del	1	8	0.00004772	—	Disease causing	Pathogenic (2)	—	Likely pathogenic: PM1, PM2, PM4, PP3, PP5	Hardy et al
c.1885C>T, p.Arg629Trp		8	0.00001195	Probably damaging (0.996)	Polymorphism	—	—	VUS: PM2, PP5	Giuliano et al
c.505G>A, p.Glu169Lys	1	5	0.00002483	Probably damaging (1.0)	Disease causing	Uncertain significance (1)	—	Likely pathogenic: PM2, PM3, PP3, PP5	Hardy et al
c.2648_2651delTCTT, p.Phe883Serfs * 68		8	0.00009239	—	Disease causing	Pathogenic (2) Likely pathogenic (1)	—	Pathogenic: PVS1, PM2, PP3, PP5	Hardy et al

Only gold members can continue reading. Log In or Register to continue

Share this:

• Click to share on Twitter (Opens in new window)
• Click to share on Facebook (Opens in new window)

Related

Related posts:

1. Indocyanine Green Angiography Features in Acute Syphilitic Posterior Placoid Chorioretinitis
2. Self-Reported Driving Difficulty, Avoidance, and Negative Emotion With On-Road Driving Performance in Older Adults With Glaucoma
3. Factors Associated With Gonioscopy-Assisted Transluminal Trabeculotomy (GATT) Complications and Failure in Children
4. Clinical Outcomes in Patients After Duet Procedure for Reversible Trifocality Using a Supplementary Trifocal Intraocular Lens

Stay updated, free articles. Join our Telegram channel

Join