Novel CRYGC Mutation in Conserved Ultraviolet-Protective Tryptophan (p.Trp131Arg) Is Linked to Autosomal Dominant Congenital Cataract

Congenital cataract (CC), the most prevalent cause of childhood blindness and amblyopia, necessitates prompt and precise genetic diagnosis. The objective of this study is to identify the underlying genetic cause in a Swiss patient with isolated CC. Whole exome sequencing (WES) and copy number variation (CNV) analysis were conducted for variant identification in a patient born with a total binocular CC without a family history of CC. Sanger Sequencing was used to confirm the variant and segregation analysis was used to screen the non-affected parents. The first de novo missense mutation at c.391T>C was identified in exon 3 of CRYGC on chromosome 2 causing the substitution of a highly conserved Tryptophan to an Arginine located at p.Trp131Arg. Previous studies exhibit significant changes in the tertiary structure of the crystallin family in the following variant locus, making CRYGC prone to aggregation aggravated by photodamage resulting in cataract. The variant can be classified as pathogenic according to the American College of Medical Genetics and Genomics (ACMG) criteria (PP3 + PM1 + PM2 + PS2; scoring 10 points). The identification of this novel variant expands the existing knowledge on the range of variants found in the CRYGC gene and contributes to a better comprehension of cataract heterogeneity

Limited Added Diagnostic Value of Whole Genome Sequencing in Genetic Testing of Inherited Retinal Diseases in a Swiss Patient Cohort

The purpose of this study was to assess the added diagnostic value of whole genome sequencing (WGS) for patients with inherited retinal diseases (IRDs) who remained undiagnosed after whole exome sequencing (WES). WGS was performed for index patients in 66 families. The datasets were analyzed according to GATK’s guidelines. Additionally, DeepVariant was complemented by GATK’s workflow, and a novel structural variant pipeline was developed. Overall, a molecular diagnosis was established in 19/66 (28.8%) index patients. Pathogenic deletions and one deep-intronic variant contributed to the diagnostic yield in 4/19 and 1/19 index patients, respectively. The remaining diagnoses (14/19) were attributed to exonic variants that were missed during WES analysis due to bioinformatic limitations, newly described loci, or unclear pathogenicity. The added diagnostic value of WGS equals 5/66 (9.6%) for our cohort, which is comparable to previous studies. This figure would decrease further to 1/66 (1.5%) with a standardized and reliable copy number variant workflow during WES analysis. Given the higher costs and limited added value, the implementation of WGS as a first-tier assay for inherited eye disorders in a diagnostic laboratory remains untimely. Instead, progress in bioinformatic tools and communication between diagnostic and clinical teams have the potential to ameliorate diagnostic yields

Outcome of Pediatric Cataract Surgeries in a Tertiary Center in Switzerland

Purpose. To determine and to analyze the outcome of pediatric cataract surgery. Methods. A retrospective chart review of individuals aged up to 10 years who underwent cataract surgery between January 1, 2004, and December 31, 2014, at the UniversityHospital Zurich, Switzerland. Results. 63 children (94 affected eyes) with bilateral (68/94) or unilateral (26/94) cataract were identified. Surgery was performed at a median age of 1.5 months (IQR: 1.3–2.6 months) for the aphakic group (45/94) and of 50.7 months (IQR: 38.0–78.4 months) for the IOL group (49/94). At the last follow-up visit (median 31.1 months, IQR: 18.4–50.2 months), visual acuity was better in bilateral than in unilateral cataract cases. Posterior capsular opacification (PCO) was diagnosed in 30.9% of eyes without a significant difference in the IOL and aphakic groups (p=0.12). Aphakic glaucoma was diagnosed in 12/45 eyes at a median of 6.8 months (IQR 2.1–13.3 months) after surgery. Microcornea (5/12) and anterior segment anomalies (8/12) were associated with glaucoma development (p<0.05). Conclusion. Laterality and timing of surgery influence the outcome of pediatric cataract surgery. PCO was the most frequent postoperative complication. Aphakic glaucoma is often associated with ocular developmental abnormalities and a poor visual outcome

Flicker electroretinogram in newborn infants

PURPOSE To develop and validate a flicker electroretinogram (ERG) protocol in term-born neonates as a potential tool for assessing preterm infants at risk of developing retinopathy of prematurity. METHODS A custom flicker ERG protocol was developed for use with the hand-held RETeval® electrophysiology device. Feasibility of measuring flicker ERG through closed eyelids and without mydriasis was established in a pilot study enabling optimisation of the test protocol. Following this, healthy term-born neonates (gestational age 37-42 weeks) were recruited at the Neonatology clinic of the University Hospital Zurich. Flicker ERG recordings were performed using proprietary disposable skin electrodes during the first four days of life when the infants were sleeping. Flicker stimuli were presented at 28.3 Hz for a stimulus series at 3, 6, 12, 30, and 50 cd·s/m$^{2}$, with two measurements at each stimulus level. Results were analysed offline. Flicker ERG peak times and amplitudes were derived from the averaged measurements per stimulus level for each subject. RESULTS 28 term-born neonates were included in the analysis. All infants tolerated the testing procedure well. Flicker ERG recording was achieved in all subjects with reproducible flicker ERG waveforms for 30 and 50 cd·s/m$^{2}$ stimuli. Reproducible ERGs were recorded in the majority of infants for the weaker stimuli (with detectable ERGs in 20/28, 25/28, and 27/28 at 3, 6, and 12 cd·s/m$^{2}$, respectively). Flicker ERG amplitudes increased with increasing stimulus strength, with peak times concurrently decreasing slightly. CONCLUSION Flicker ERG recording is feasible and reliably recorded in sleeping neonates through closed eyelids using skin electrodes and without mydriasis. Flicker ERG amplitude decreases for lower luminance flicker but remains detectable for 3 cd·s/m$^{2}$ flicker in the majority of healthy term-born neonates. These data provide a basis to study retinal function in premature infants using this protocol

Flicker electroretinogram in preterm infants

BACKGROUND Infants born prematurely are at risk of developing retinopathy of prematurity, which is associated with abnormalities in retinal function as measured using electroretinography. The aim of this study was to record non-invasive flicker electroretinograms (ERGs) in preterm infants and compare function of moderate and very or extremely preterm infants. METHODS In this non-randomized, cross-sectional study, 40 moderate preterm (gestational age (GA) 34 0/7 to 36 6/7 weeks, Group A) and 40 very or extremely preterm infants (GA ≤ 31 weeks, Group B) were recruited for flicker ERG recording through closed eyelids using the RETeval® device and skin electrodes. Group A was tested within the first week of life and Group B between 34th and 37th week postmenstrual age. Flicker stimuli were presented at 28.3 Hz with stimulus levels of 3, 6, 12, 30 and 50 cd•s/m$^{2}$. Primary endpoints were peak time (ms) and amplitude (µV). RESULTS Flicker ERGs were recordable in most infants with the highest proportion of reproducible ERGs at 30 cd•s/m$^{2}$. Amplitudes increased with stronger flicker stimulation, while peak times did not differ significantly between stimulus levels nor groups. Amplitudes were significantly greater in Group B at the strongest stimulus level (Mann-Whitney-U-Test=198.00, Z = 4.097, p = <0.001). CONCLUSIONS Feasibility of collecting flicker ERG data in most preterm infants was confirmed. We found no evidence of reduced retinal responses to flicker stimuli associated with extreme prematurity. Higher amplitudes in very and extremely preterm infants could indicate acceleration of retinal development following birth, triggered by visual stimulation

Functional Analysis of a Novel, Non-Canonical RPGR Splice Variant Causing X-Linked Retinitis Pigmentosa

X-linked retinitis pigmentosa (XLRP) caused by mutations in the RPGR gene is one of the most severe forms of RP due to its early onset and intractable progression. Most cases have been associated with genetic variants within the purine-rich exon ORF15 region of this gene. RPGR retinal gene therapy is currently being investigated in several clinical trials. Therefore, it is crucial to report and functionally characterize (all novel) potentially pathogenic DNA sequence variants. Whole-exome sequencing (WES) was performed for the index patient. The splicing effects of a non-canonical splice variant were tested on cDNA from whole blood and a minigene assay. WES revealed a rare, non-canonical splice site variant predicted to disrupt the wildtype splice acceptor and create a novel acceptor site 8 nucleotides upstream of RPGR exon 12. Reverse-transcription PCR analyses confirmed the disruption of the correct splicing pattern, leading to the insertion of eight additional nucleotides in the variant transcript. Transcript analyses with minigene assays and cDNA from peripheral blood are useful tools for the characterization of splicing defects due to variants in the RPGR and may increase the diagnostic yield in RP. The functional analysis of non-canonical splice variants is required to classify those variants as pathogenic according to the ACMG’s criteria

A three-year longitudinal study of retinal function and structure in patients with multiple sclerosis

BACKGROUND Researchers have in recent years begun to investigate ophthalmological manifestations of multiple sclerosis (MS) other than optic neuritis (ON), and it is now clear that changes to retinal function (measured using the electroretinogram, ERG) and structure (measured using optical coherence tomography, OCT) are found in MS patients irrespective of prior ON episodes. ERG results are consistent with dysfunctional bipolar cells, as in other autoimmune diseases. To date, studies have presented only cross-sectional data regarding ERG and OCT. We, therefore, studied the longitudinal course of ERG and OCT in patients with MS, as well as the effect of disability changes and non-ON clinical relapses on these functional and structural measures. METHODS MS patients (n = 23) participating in an ongoing longitudinal observational study were invited to take part in a 3-year ophthalmological substudy. ERG and OCT were performed, and measures of MS-related disability and relapse history were obtained. Study visits were repeated annually. ERG peak times, rod b-wave amplitude, mixed rod/cone and cone b-/a-wave amplitude ratios, thickness of the peripapillary retinal nerve fibre layer, and volumes of the segmented retinal layers/complexes were analysed. Using generalised estimating equation models adjusted for age, ON, and MS treatment status, we assessed changes to ERG and OCT over the study duration, the effect of changes in disability and recent non-ON MS relapses on ERG and OCT, and the effect of selected OCT parameters on corresponding ERG parameters. RESULTS At the group level, small fluctuations of several ERG peak times were recorded, with OCT values remaining stable. Increased disability between visits was associated with significant prolongation of mixed rod-cone ERG b-wave peak times. No evidence of associations between OCT and ERG parameters was observed. CONCLUSIONS Retinal bipolar cell function may be affected by changes in disability in patients with MS; however, recent non-ON MS clinical relapses appear not to affect ERG or OCT results. As ERG changes in MS patients over 3 years are likely to be small and of uncertain clinical relevance, longitudinal studies of retinal function in MS should be planned over an extended period

Functional and Morphological Characteristics of the Retina of Patients with Drusen-like Deposits and Systemic Lupus Erythematosus Treated with Hydroxychloroquine: A Retrospective Study

Purpose: To evaluate the impact of drusen-like deposits (DLD) on retinal layer integrity and retinal function by optical coherence tomography (OCT) and multifocal electroretinography (mfERG) in patients with systemic lupus erythematosus (SLE). Methods: We identified 66 eyes of 33 SLE patients treated with hydroxychloroquine (HCQ) that were categorized into two groups according to whether DLDs were present (34 eyes, Group One) or absent (32 eyes, Group Two). The groups were matched for age, sex, HCQ treatment duration, daily, and cumulative dosage. OCT (retinal layer thicknesses, central retinal thickness, CRT) and mfERG concentric ring analysis were analyzed and compared. Results: CRT was significantly thicker in Group One compared to Group Two (273.21 ± 3.96 vs. 254.5 ± 7.62) (p = 0.023). Group One also demonstrated an overall thicker retinal pigment epithelium compared to Group Two; however, the other outer retinal layers, outer nuclear layer, and photoreceptor layer were found to be significantly thinner in Group One compared to Group Two. We found no differences in mfERG parameters between the two groups. Conclusions: DLDs in SLE patients lead to abnormal central retinal layer thickness, which has no measurable impact on cone-mediated retinal function assessed by mfERG

Homozygosity for a Novel DOCK7 Variant Due to Segmental Uniparental Isodisomy of Chromosome 1 Associated with Early Infantile Epileptic Encephalopathy (EIEE) and Cortical Visual Impairment

Early infantile epileptic encephalopathy (EIEE) is a severe neurologic and neurodevelopmental disease that manifests in the first year of life. It shows a high degree of genetic heterogeneity, but the genetic origin is only identified in half of the cases. We report the case of a female child initially diagnosed with Leber congenital amaurosis (LCA), an early-onset retinal dystrophy due to photoreceptor cell degeneration in the retina. The first examination at 9 months of age revealed no reaction to light or objects and showed wandering eye movements. Ophthalmological examination did not show any ocular abnormalities. The patient displayed mildly dysmorphic features and a global developmental delay. Brain MRI demonstrated pontine hypo-/dysplasia. The patient developed myoclonic epileptic seizures and epileptic spasms with focal and generalized epileptiform discharges on electroencephalogram (EEG) at the age of 16 months. Genetic screening for a potentially pathogenic DNA sequence variant by whole-exome sequencing (WES) revealed a novel, conserved, homozygous frameshift variant (c.5391delA, p.(Ala1798LeufsTer59)) in exon 42 of the DOCK7 gene (NM_001271999.1). Further analysis by SNP array (Karyomapping) showed loss of heterozygosity (LOH) in four segments of chromosome 1. WES data of the parents and the index patient (trio analysis) demonstrated that chromosome 1 was exclusively inherited from the mother. Four LOH segments of chromosome 1 alternately showed isodisomy (UPiD) and heterodisomy (UPhD). In WES data, the father was a noncarrier, and the mother was heterozygous for this DOCK7 variant. The DOCK7 gene is located in 1p31.3, a region situated in one of the four isodisomic segments of chromosome 1, explaining the homozygosity seen in the affected child. Finally, Sanger sequencing confirmed maternal UPiD for the DOCK7 variant. Homozygous or compound heterozygous pathogenic variants in the DOCK7 (dedicator of cytokinesis 7) gene are associated with autosomal recessive, early infantile epileptic encephalopathy 23 (EIEE23; OMIM #615,859), a rare and heterogeneous group of neurodevelopmental disorders diagnosed during early childhood. To our knowledge, this is the first report of segmental uniparental iso- and heterodisomy of chromosome 1, leading to homozygosity of the DOCK7 frameshift variant in the affected patient

Characterization of two novel intronic OPA1 mutations resulting in aberrant pre-mRNA splicing.

BACKGROUND We report two novel splice region mutations in OPA1 in two unrelated families presenting with autosomal-dominant optic atrophy type 1 (ADOA1) (ADOA or Kjer type optic atrophy). Mutations in OPA1 encoding a mitochondrial inner membrane protein are a major cause of ADOA. METHODS We analyzed two unrelated families including four affected individuals clinically suspicious of ADOA. Standard ocular examinations were performed in affected individuals of both families. All coding exons, as well as exon-intron boundaries of the OPA1 gene were sequenced. In addition, multiplex ligation-dependent probe amplification (MLPA) was performed to uncover copy number variations in OPA1. mRNA processing was monitored using RT-PCR and subsequent cDNA analysis. RESULTS We report two novel splice region mutations in OPA1 in two unrelated individuals and their affected relatives, which were previously not described in the literature. In one family the heterozygous insertion and deletion c.[611-37_611-38insACTGGAGAATGTAAAGGGCTTT;611-6_611-16delCATATTTATCT] was found in all investigated family members leading to the activation of an intronic cryptic splice site. In the second family sequencing of OPA1 disclosed a de novo heterozygous deletion c.2012+4_2012+7delAGTA resulting in exon 18 and 19 skipping, which was not detected in healthy family members. CONCLUSION We identified two novel intronic mutations in OPA1 affecting the correct OPA1 pre-mRNA splicing, which was confirmed by OPA1 cDNA analysis. This study shows the importance of transcript analysis to determine the consequences of unclear intronic mutations in OPA1 in proximity to the intron-exon boundaries