Christina Gerth-Kahlert

Next generation sequencing based identification of disease-associated mutations in Swiss patients with retinal dystrophies.

Inherited monogenic diseases of the retina and vitreous affect approximately 1 in 2000 individuals. They are characterized by tremendous genetic heterogeneity and clinical variability involving mutations in approximately 250 genes and more than 20 different clinical phenotypes. Clinical manifestations of retinal dystrophies (RDs) range from mild retinal dysfunctions to severe congenital forms of blindness. A detailed clinical diagnosis and the identification of causative mutations are crucial for genetic counseling of affected patients and their families, for understanding genotype-phenotype correlations and developing therapeutic approaches. Using whole exome sequencing (WES) we have established a reliable and efficient high-throughput analysis pipeline to identify disease-causing mutations. Our data indicate that this approach enables us to genetically diagnose approximately 64% of the patients (n = 58) with variant(s) in known disease-associated genes. We report 20 novel and 26 recurrent variants in genes associated with RDs. We also identified a novel phenotype for mutations in C2orf71 and provide functional evidence for exon skipping due to a splice-site variant identified in FLVCR1. In conclusion, WES can rapidly identify variants in various families affected with different forms of RDs. Our study also expands the clinical and allelic spectrum of genes associated with RDs in the Swiss population.

Dental Phenotype in Jalili Syndrome Due to a c.1312 dupC Homozygous Mutation in the CNNM4 Gene

Jalili syndrome denotes a recessively inherited combination of an eye disease (cone-rod dystrophy) and a dental disorder (amelogenesis imperfecta), which is caused by mutations in the CNNM4 gene. Whereas the ophthalmic consequences of these mutations have been studied comprehensively, the dental phenotype has obtained less attention. A defective transport of magnesium ions by the photoreceptors of the retina is assumed to account for the progressive visual impairment. Since magnesium is also incorporated in the mineral of dental hard tissues, we hypothesized that magnesium concentrations in defective enamel resulting from mutations in CNNM4 would be abnormal, if a similar deficiency of magnesium transport also accounted for the amelogenesis imperfecta. Thus, a detailed analysis of the dental hard tissues was performed in two boys of Kosovan origin affected by Jalili syndrome. Retinal dystrophy of the patients was diagnosed by a comprehensive eye examination and full-field electroretinography. A mutational analysis revealed a c.1312 dupC homozygous mutation in CNNM4, a genetic defect which had already been identified in other Kosovan families and putatively results in loss-of-function of the protein. The evaluation of six primary teeth using light and scanning electron microscopy as well as energy-dispersive X-ray spectroscopy showed that dental enamel was thin and deficient in mineral, suggesting a hypoplastic/hypomineralized type of amelogenesis imperfecta. The reduced mineral density of enamel was accompanied by decreased amounts of calcium, but significantly elevated levels of magnesium. In dentin, however, a similar mineral deficiency was associated with reduced magnesium and normal calcium levels. It is concluded that the c.1312 dupC mutation of CNNM4 results in mineralization defects of both enamel and dentin, which are associated with significantly abnormal magnesium concentrations. Thus, we could not disprove the hypothesis that a disrupted magnesium transport is involved in the development of the dental abnormalities observed in Jalili syndrome.

A secreted WNT-ligand-binding domain of FZD5 generated by a frameshift mutation causes autosomal dominant coloboma

Ocular coloboma is a common eye malformation resulting from incomplete fusion of the optic fissure during development. Coloboma is often associated with microphthalmia and/or contralateral anophthalmia. Coloboma shows extensive locus heterogeneity associated with causative mutations identified in genes encoding developmental transcription factors or components of signaling pathways. We report an ultra-rare, heterozygous frameshift mutation in FZD5 (p.Ala219Glufs*49) that was identified independently in two branches of a large family with autosomal dominant non-syndromic coloboma. FZD5 has a single-coding exon and consequently a transcript with this frameshift variant is not a canonical substrate for nonsense-mediated decay. FZD5 encodes a transmembrane receptor with a conserved extracellular cysteine rich domain for ligand binding. The frameshift mutation results in the production of a truncated protein, which retains the Wingless-type MMTV integration site family member-ligand-binding domain, but lacks the transmembrane domain. The truncated protein was secreted from cells, and behaved as a dominant-negative FZD5 receptor, antagonizing both canonical and non-canonical WNT signaling. Expression of the resultant mutant protein caused coloboma and microphthalmia in zebrafish, and disruption of the apical junction of the retinal neural epithelium in mouse, mimicking the phenotype of Fz5/Fz8 compound conditional knockout mutants. Our studies have revealed a conserved role of Wnt-Frizzled (FZD) signaling in ocular development and directly implicate WNT-FZD signaling both in normal closure of the human optic fissure and pathogenesis of coloboma.

Optical Coherence Tomography and Magnetic Resonance Imaging in Multiple Sclerosis and Neuromyelitis Optica Spectrum Disorder

Irreversible disability in multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD) is largely attributed to neuronal and axonal degeneration, which, along with inflammation, is one of the major pathological hallmarks of these diseases. Optical coherence tomography (OCT) is a non-invasive imaging tool that has been used in MS, NMOSD, and other diseases to quantify damage to the retina, including the ganglion cells and their axons. The fact that these are the only unmyelinated axons within the central nervous system (CNS) renders the afferent visual pathway an ideal model for studying axonal and neuronal degeneration in neurodegenerative diseases. Structural magnetic resonance imaging (MRI) can be used to obtain anatomical information about the CNS and to quantify evolving pathology in MS and NMOSD, both globally and in specific regions of the visual pathway including the optic nerve, optic radiations and visual cortex. Therefore, correlations between brain or optic nerve abnormalities on MRI, and retinal pathology using OCT, may shed light on how damage to one part of the CNS can affect others. In addition, these imaging techniques can help identify important differences between MS and NMOSD such as disease-specific damage to the visual pathway, trans-synaptic degeneration, or pathological changes independent of the underlying disease process. This review focuses on the current knowledge of the role of the visual pathway using OCT and MRI in patients with MS and NMOSD. Emphasis is placed on studies that employ both MRI and OCT to investigate damage to the visual system in these diseases.

Biallelic Mutations in CRB1 Underlie Autosomal Recessive Familial Foveal Retinoschisis

PURPOSE To identify the genetic cause of autosomal recessive familial foveal retinoschisis (FFR). METHODS A female sibship with FFR was identified (Family-A; 17 and 16 years, respectively); panel based genetic sequencing (132 genes) and comparative genome hybridization (142 genes) were performed. Whole-exome sequencing (WES) was performed on both siblings using the Illumina-HiSeq-2500 platform. A sporadic male (Family-B; 35 years) with FFR underwent WES using Illumina NextSeq500. All three affected subjects underwent detailed ophthalmologic evaluation including fundus photography, autofluorescence imaging, spectral-domain optical coherence tomography (SD-OCT), and full-field electroretinogram (ERG). RESULTS Panel-based genetic testing identified two presumed disease causing variants in CRB1 (p.Gly123Cys and p.Cys948Tyr) in Family-A sibship; no deletion or duplication was detected. WES analysis in the sibship identified nine genes with two or more shared nonsynonymous rare coding sequence variants; CRB1 remained a strong candidate gene, and CRB1 variants segregated with the disease. WES in Family-B identified two presumed disease causing variants in CRB1 (p.Ile167_Gly169del and p.Arg764Cys) that segregated with the disease phenotype. Distance visual acuity was 20/40 or better in all three affected except for the left eye of the older subject (Family-B), which showed macular atrophy. Fundus evaluation showed spoke-wheel appearance at the macula in five eyes. The SD-OCT showed macular schitic changes in inner and outer nuclear layers in all cases. The ERG responses were normal in all subjects. CONCLUSIONS This is the first report to implicate CRB1 as the underlying cause of FFR. This phenotype forms the mildest end of the spectrum of CRB1-related diseases.

Incidence of retinopathy of prematurity (ROP) and ROP treatment in Switzerland 2006-2015: a population-based analysis

Background Retinopathy of prematurity (ROP) is a severe complication of preterm birth and can lead to severe visual impairment or even blindness if untreated. The incidence of ROP requiring treatment is increasing in some developed countries in conjunction with higher survival rates at the lower end of gestational age (GA). Material and methods The incidence of ROP and severe ROP (sROP) requiring treatment in Switzerland was analysed using the SwissNeoNet registry. We conducted a retrospective cohort analysis of very preterm infants with a GA below 32 weeks who were born between 2006 and 2015 in Switzerland. Patient characteristics were stratified according to GA. Results 9.3% and 1.8% of very preterm infants in Switzerland developed ROP of any stage and sROP, respectively. The incidence of ROP treatment was 1.2%. Patients with 24 and 25 weeks GA had the highest proportion of ROP treatment at 14.5% and 7.3%, respectively, whereas the proportion of treated infants at or above a GA of 29 weeks was 0.06%. Similarly, the risk of sROP declined strongly with increasing GA. During the observation period of 10 years, the incidence of ROP treatment ranged between 0.8% and 2.0%. Incidences of sROP or ROP treatment did not increase over time. Conclusion The incidence of ROP treatment in Switzerland is low and was stable over the analysed period. The low incidence of sROP in patients with a GA of 29 weeks or more leaves room for a redefinition of ROP screening criteria.

C2orf71 Mutations as a Frequent Cause of Autosomal-Recessive Retinitis Pigmentosa: Clinical Analysis and Presentation of 8 Novel Mutations

Purpose To define the phenotype of C2orf71 associated retinopathy and to present novel mutations in this gene. Methods A retrospective multicenter study of patients with retinopathy and identified C2orf71 mutations was performed. Ocular function (visual acuity, visual fields, electroretinogram [ERG] responses); retinal morphology (fundus, optical coherence tomography); and underlying mutations were analyzed. Results Thirteen patients from 11 families, who were aged 7 to 63 years (mean: 32.1 years) at their first examination with presumed compound heterozygous (6/13 patients) or homozygous (7/13 patients) C2orf71 mutations were identified. Eight of the mutations were novel. Truncation mutations were responsible in all cases. Nyctalopia was observed in less than 50% of patients. Visual acuity ranged from 20/20 to light perception. Severe visual loss was associated with atrophic maculopathy. Full-field ERG responses showed severe progressive cone-rod or rod-cone dysfunction. Typical fundus changes were progressive symmetrical retinopathy with an early mild maculopathy and patchy circular midperipheral RPE atrophy. Normal retinal lamination was preserved despite early disruption of the ellipsoid zone and RPE irregularities. Outer retinal tubulations were associated with better-preserved visual acuity. Conclusions On the basis of our multicenter analysis, C2orf71 might represent a more frequently mutated gene in autosomal recessive retinitis pigmentosa in some populations. The phenotype analysis over a wide age range showed a variable and progressive retinal degeneration with early onset maculopathy and a better visual potential before the age of 30 years.

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.

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

BackgroundWe 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.MethodsWe 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.ResultsWe 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.ConclusionWe 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.

Teaching NeuroImages: Recurrent oculomotor palsies caused by neurosarcoidosis

A 53-year-old man presented with a recurrent left fourth nerve and acute left third nerve palsy (figure 1). Brain MRI showed leptomeningeal and oculomotor nerve contrast enhancement (figure 2). CSF examination indicated chronic lymphocytic meningitis with massively increased soluble interleukin (IL)-2 receptor and IL-6.1 Extensive infectious disease workup remained unremarkable. [18F]-Fluorodeoxyglucose (FDG)-PET/CT revealed active deep cervical lymph nodes containing noncaseating granulomas (figure 2). No pulmonary or other systemic manifestation was found, consistent with clinically isolated neurosarcoidosis.2 Upon immunosuppressive treatment,1,2 the oculomotor palsies rapidly remitted. Neurosarcoidosis is a rare condition lacking specific biomarkers.2 When suspected, FDG-PET/CT can guide diagnosis and prevent CNS biopsy.1