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.

Nanopore Deep Sequencing as a Tool to Characterize and Quantify Aberrant Splicing Caused by Variants in Inherited Retinal Dystrophy Genes.

The contribution of splicing variants to molecular diagnostics of inherited diseases is reported to be less than 10%. This figure is likely an underestimation due to several factors including difficulty in predicting the effect of such variants, the need for functional assays, and the inability to detect them (depending on their locations and the sequencing technology used). The aim of this study was to assess the utility of Nanopore sequencing in characterizing and quantifying aberrant splicing events. For this purpose, we selected 19 candidate splicing variants that were identified in patients affected by inherited retinal dystrophies. Several in silico tools were deployed to predict the nature and estimate the magnitude of variant-induced aberrant splicing events. Minigene assay or whole blood-derived cDNA was used to functionally characterize the variants. PCR amplification of minigene-specific cDNA or the target gene in blood cDNA, combined with Nanopore sequencing, was used to identify the resulting transcripts. Thirteen out of nineteen variants caused aberrant splicing events, including cryptic splice site activation, exon skipping, pseudoexon inclusion, or a combination of these. Nanopore sequencing allowed for the identification of full-length transcripts and their precise quantification, which were often in accord with in silico predictions. The method detected reliably low-abundant transcripts, which would not be detected by conventional strategies, such as RT-PCR followed by Sanger sequencing.

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.

Atonal homolog 7 (ATOH7) loss-of-function mutations in predominant bilateral optic nerve hypoplasia.

Optic nerve hypoplasia (ONH) is a congenital optic nerve abnormality caused by underdevelopment of retinal ganglion cells (RGCs). Despite being a rare disease, ONH is the most common optic disk anomaly in ophthalmological practice. So far, mutations in several genes have been identified as causative; however, many cases of ONH remain without a molecular explanation. The early transcription factor atonal basic-helix-loop-helix (bHLH) transcription factor 7 (ATOH7) is expressed in retinal progenitor cells and has a crucial role in RGC development. Previous studies have identified several mutations in the ATOH7 locus in cases of eye developmental diseases such as non-syndromic congenital retinal non-attachment and persistent hyperplasia of the primary vitreous. Here we present two siblings with a phenotype predominated by bilateral ONH, with additional features of foveal hypoplasia and distinct vascular abnormalities, where whole-exome sequencing identified two compound heterozygous missense mutations affecting a conserved amino acid residue within the bHLH domain of ATOH7 (NM_145178.3:c.175G>A; p.(Ala59Thr) and c.176C>T; p.(Ala59Val)). ATOH7 expression constructs with patient single nucleotide variants were cloned for functional characterization. Protein analyses revealed decreased protein amounts and significantly enhanced degradation in the presence of E47, a putative bHLH dimerization partner. Protein interaction assays revealed decreased heterodimerization and DNA-binding of ATOH7 variants, resulting in total loss of transcriptional activation of luciferase reporter gene expression. These findings strongly support pathogenicity of the two ATOH7 mutations, one of which is novel. Additionally, this report highlights the possible impact of altered ATOH7 dimerization on protein stability and function.

Functional Characterization of an In-Frame Deletion in the Basic Domain of the Retinal Transcription Factor ATOH7.

Basic helix-loop-helix (bHLH) transcription factors are evolutionarily conserved and structurally similar proteins important in development. The temporospatial expression of atonal bHLH transcription factor 7 (ATOH7) directs the differentiation of retinal ganglion cells and mutations in the human gene lead to vitreoretinal and/or optic nerve abnormalities. Characterization of pathogenic ATOH7 mutations is needed to understand the functions of the conserved bHLH motif. The published ATOH7 in-frame deletion p.(Arg41_Arg48del) removes eight highly conserved amino acids in the basic domain. We functionally characterized the mutant protein by expressing V5-tagged ATOH7 constructs in human embryonic kidney 293T (HEK293T) cells for subsequent protein analyses, including Western blot, cycloheximide chase assays, Förster resonance energy transfer fluorescence lifetime imaging, enzyme-linked immunosorbent assays and dual-luciferase assays. Our results indicate that the in-frame deletion in the basic domain causes mislocalization of the protein, which can be rescued by a putative dimerization partner transcription factor 3 isoform E47 (E47), suggesting synergistic nuclear import. Furthermore, we observed (i) increased proteasomal degradation of the mutant protein, (ii) reduced protein heterodimerization, (iii) decreased DNA-binding and transcriptional activation of a reporter gene, as well as (iv) inhibited E47 activity. Altogether our observations suggest that the DNA-binding basic domain of ATOH7 has additional roles in regulating the nuclear import, dimerization, and protein stability.

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.

Long-Range PCR-Based NGS Applications to Diagnose Mendelian Retinal Diseases.

The purpose of this study was to develop a flexible, cost-efficient, next-generation sequencing (NGS) protocol for genetic testing. Long-range polymerase chain reaction (PCR) amplicons of up to 20 kb in size were designed to amplify entire genomic regions for a panel (n = 35) of inherited retinal disease (IRD)-associated loci. Amplicons were pooled and sequenced by NGS. The analysis was applied to 227 probands diagnosed with IRD: (A) 108 previously molecularly diagnosed, (B) 94 without previous genetic testing, and (C) 25 undiagnosed after whole-exome sequencing (WES). The method was validated with 100% sensitivity on cohort A. Long-range PCR-based sequencing revealed likely causative variant(s) in 51% and 24% of proband from cohorts B and C, respectively. Breakpoints of 3 copy number variants (CNVs) could be characterized. Long-range PCR libraries spike-in extended coverage of WES. Read phasing confirmed compound heterozygosity in 5 probands. The proposed sequencing protocol provided deep coverage of the entire gene, including intronic and promoter regions. Our method can be used (i) as a first-tier assay to reduce genetic testing costs, (ii) to elucidate missing heritability cases, (iii) to characterize breakpoints of CNVs at nucleotide resolution, (iv) to extend WES data to non-coding regions by spiking-in long-range PCR libraries, and (v) to help with phasing of candidate variants.

Application of WES Towards Molecular Investigation of Congenital Cataracts: Identification of Novel Alleles and Genes in a Hospital-Based Cohort of South India.

Congenital cataracts are the prime cause for irreversible blindness in children. The global incidence of congenital cataract is 2.2-13.6 per 10,000 births, with the highest prevalence in Asia. Nearly half of the congenital cataracts are of familial nature, with a predominant autosomal dominant pattern of inheritance. Over 38 of the 45 mapped loci for isolated congenital or infantile cataracts have been associated with a mutation in a specific gene. The clinical and genetic heterogeneity of congenital cataracts makes the molecular diagnosis a bit of a complicated task. Hence, whole exome sequencing (WES) was utilized to concurrently screen all known cataract genes and to examine novel candidate factors for a disease-causing mutation in probands from 11 pedigrees affected with familial congenital cataracts. Analysis of the WES data for known cataract genes identified causative mutations in six pedigrees (55%) in PAX6, FYCO1 (two variants), EPHA2, P3H2,TDRD7 and an additional likely causative mutation in a novel gene NCOA6, which represents the first dominant mutation in this gene. This study identifies a novel cataract gene not yet linked to human disease. NCOA6 is a transcriptional coactivator that interacts with nuclear hormone receptors to enhance their transcriptional activator function.

[Ciliopathies]. / Retinale Ziliopathien.

Ciliopathies are disorders caused by ciliary dysfunction and can affect an organ system or tissues. Isolated or syndromic retinal dystrophies are the most common ocular manifestation of ciliopathies. The photoreceptor connecting cilium plays a leading role in these ciliopathy-related retinal dystrophies. Dysfunctional photoreceptor cilia cause the most severe type of retinal dystrophy: Leber's congenital amaurosis (LCA). The most common syndromic ciliopathies with an ocular manifestation are Bardet-Biedl syndrome (BBS) and Usher syndrome. Molecular-genetic analysis revealed a large number of cilia genes with a high phenotype heterogeneity. Diagnosis of ciliopathies require a multi-disciplinary approach. Causative treatment of ciliopathies is not yet available; therefore, rehabilitative and supportive treatment is mandatory.

Inhibition of G-protein-coupled receptor kinase 2 (GRK2) triggers the growth-promoting mitogen-activated protein kinase (MAPK) pathway.

Inhibition of G-protein-coupled receptor kinase 2 (GRK2) is an emerging treatment option for heart failure. Because GRK2 is also indispensable for growth and development, we analyzed the impact of GRK2 inhibition on cell growth and proliferation. Inhibition of GRK2 by the dominant-negative GRK2-K220R did not affect the proliferation of cultured cells. In contrast, upon xenograft transplantation of cells into immunodeficient mice, the dominant-negative GRK2-K220R or a GRK2-specific peptide inhibitor increased tumor mass. The enhanced tumor growth upon GRK2 inhibition was attributed to the growth-promoting MAPK pathway because dual inhibition of the GRK2 and RAF-MAPK axis by the Raf kinase inhibitor protein (RKIP) did not increase tumor mass. The MAPK cascade contributed to the cardioprotective profile of GRK2 inhibition by preventing cardiomyocyte death, whereas dual inhibition of RAF/MAPK and GRK2 by RKIP induced cardiomyocyte apoptosis, cardiac dysfunction, and signs of heart failure. Thus, cardioprotective signaling induced by GRK2 inhibition is overlapping with tumor growth promotion.

Identification and Characterization of ATOH7-Regulated Target Genes and Pathways in Human Neuroretinal Development.

The proneural transcription factor atonal basic helix-loop-helix transcription factor 7 (ATOH7) is expressed in early progenitors in the developing neuroretina. In vertebrates, this is crucial for the development of retinal ganglion cells (RGCs), as mutant animals show an almost complete absence of RGCs, underdeveloped optic nerves, and aberrations in retinal vessel development. Human mutations are rare and result in autosomal recessive optic nerve hypoplasia (ONH) or severe vascular changes, diagnosed as autosomal recessive persistent hyperplasia of the primary vitreous (PHPVAR). To better understand the role of ATOH7 in neuroretinal development, we created ATOH7 knockout and eGFP-expressing ATOH7 reporter human induced pluripotent stem cells (hiPSCs), which were differentiated into early-stage retinal organoids. Target loci regulated by ATOH7 were identified by Cleavage Under Targets and Release Using Nuclease with sequencing (CUT&RUN-seq) and differential expression by RNA sequencing (RNA-seq) of wildtype and mutant organoid-derived reporter cells. Additionally, single-cell RNA sequencing (scRNA-seq) was performed on whole organoids to identify cell type-specific genes. Mutant organoids displayed substantial deficiency in axon sprouting, reduction in RGCs, and an increase in other cell types. We identified 469 differentially expressed target genes, with an overrepresentation of genes belonging to axon development/guidance and Notch signaling. Taken together, we consolidate the function of human ATOH7 in guiding progenitor competence by inducing RGC-specific genes while inhibiting other cell fates. Furthermore, we highlight candidate genes responsible for ATOH7-associated optic nerve and retinovascular anomalies, which sheds light to potential future therapy targets for related disorders.

SwissGenVar: A Platform for Clinical-Grade Interpretation of Genetic Variants to Foster Personalized Healthcare in Switzerland.

Large-scale next-generation sequencing (NGS) germline testing is technically feasible today, but variant interpretation represents a major bottleneck in analysis workflows. This includes extensive variant prioritization, annotation, and time-consuming evidence curation. The scale of the interpretation problem is massive, and variants of uncertain significance (VUSs) are a challenge to personalized medicine. This challenge is further compounded by the complexity and heterogeneity of the standards used to describe genetic variants and the associated phenotypes when searching for relevant information to support clinical decision making. To address this, all five Swiss academic institutions for Medical Genetics joined forces with the Swiss Institute of Bioinformatics (SIB) to create SwissGenVar as a user-friendly nationwide repository and sharing platform for genetic variant data generated during routine diagnostic procedures and research sequencing projects. Its aim is to provide a protected environment for expert evidence sharing about individual variants to harmonize and upscale their significance interpretation at the clinical grade according to international standards. To corroborate the clinical assessment, the variant-related data will be combined with consented high-quality clinical information. Broader visibility will be achieved by interfacing with international databases, thus supporting global initiatives in personalized healthcare.

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.

Rapid onset hydroxychloroquine toxicity.

PURPOSE: Hydroxychloroquine (HCQ) can cause irreversible damage to the retina, especially when taken over longer periods. The American Academy of Ophthalmology recommends a regimen for dosing, screening and monitoring of patients treated with HCQ. We present an unusual case of a rapid development of severe HCQ-associated-retinopathy already after 2 years after commencing HCQ treatment. METHODS: Observational case report. Clinical examination, optical coherence tomography (OCT), fundus autofluorescence imaging (FAF), perimetry, full-field & multifocal electroretinography (ERG) were performed. Ancillary tests included neoplastic and paraneoplastic work-up, vitamin levels and whole exome sequencing, in order to rule out other potential causes of a panretinal degeneration. RESULTS: We report on a 58-year-old woman with rheumatoid arthritis, treated initially with 200mg HCQ daily for 1 year (daily dose 3.6mg/kg), then 400mg daily for 1 year (daily dose 7.2mg/kg), and a cumulative dose of 216 g. Her medical history was otherwise unremarkable. No family history for inherited retinal conditions. She was referred due to a rapid and sudden progressive and severe concentric visual field constriction, two years after commencing HCQ treatment. CONCLUSION: This case of a rapid-onset, severe panretinal degeneration shortly after start of HCQ treatment suggests underlying mechanisms and risk factors for HCQ toxicity in addition to those previously reported, and a potential need for supplementary screening tests to prevent HCQ toxicity. AAO dosing guidelines of 5 MG/KG should be strictly adhered to in patients receiving HCQ therapy.

A cleavable signal peptide enhances cell surface delivery and heterodimerization of Cerulean-tagged angiotensin II AT1 and bradykinin B2 receptor.

Heterodimerization of the angiotensin II AT1 receptor with the receptor for the vasodepressor bradykinin, B2R, is known to sensitize the AT1-stimulated response of hypertensive individuals in vivo. To analyze features of that prototypic receptor heterodimer in vitro, we established a new method that uses fluorescence resonance energy transfer (FRET) and applies for the first time AT1-Cerulean as a FRET donor. The Cerulean variant of the green fluorescent protein as donor fluorophore was fused to the C-terminus of AT1, and the enhanced yellow fluorescent protein (EYFP) as acceptor fluorophore was fused to B2R. In contrast to AT1-EGFP, the AT1-Cerulean fusion protein was retained intracellularly. To facilitate cell surface delivery of AT1-Cerulean, a cleavable signal sequence was fused to the receptor's amino terminus. The plasma membrane-localized AT1-Cerulean resembled the native AT1 receptor regarding ligand binding and receptor activation. A high FRET efficiency of 24.7% between membrane-localized AT1-Cerulean and B2R-EYFP was observed with intact, non-stimulated cells. Confocal FRET microscopy further revealed that the AT1/B2 receptor heterodimer was functionally coupled to receptor desensitization mechanisms because activation of the AT1-Cerulean/B2R-EYFP heterodimer with a single agonist triggered the co-internalization of AT1/B2R. Receptor co-internalization was sensitive to inhibition of G protein-coupled receptor kinases, GRKs, as evidenced by a GRK-specific peptide inhibitor. In agreement with efficient AT1/B2R heterodimerization, confocal FRET imaging of co-enriched receptor proteins immobilized on agarose beads also detected a high FRET efficiency of 24.0%. Taken together confocal FRET imaging revealed efficient heterodimerization of co-enriched and cellular AT1/B2R, and GRK-dependent co-internalization of the AT1/B2R heterodimer.

Whole Exome Sequencing in Coloboma/Microphthalmia: Identification of Novel and Recurrent Variants in Seven Genes.

Coloboma and microphthalmia (C/M) are related congenital eye malformations, which can cause significant visual impairment. Molecular diagnosis is challenging as the genes associated to date with C/M account for only a small percentage of cases. Overall, the genetic cause remains unknown in up to 80% of patients. High throughput DNA sequencing technologies, including whole-exome sequencing (WES), are therefore a useful and efficient tool for genetic screening and identification of new mutations and novel genes in C/M. In this study, we analyzed the DNA of 19 patients with C/M from 15 unrelated families using singleton WES and data analysis for 307 genes of interest. We identified seven novel and one recurrent potentially disease-causing variants in CRIM1, CHD7, FAT1, PTCH1, PUF60, BRPF1, and TGFB2 in 47% of our families, three of which occurred de novo. The detection rate in patients with ocular and extraocular manifestations (67%) was higher than in patients with an isolated ocular phenotype (46%). Our study highlights the significant genetic heterogeneity in C/M cohorts and emphasizes the diagnostic power of WES for the screening of patients and families with C/M.

Genotype-phenotype spectrum in isolated and syndromic nanophthalmos.

PURPOSE: To (i) describe a series of patients with isolated or syndromic nanophthalmos with the underlying genetic causes, including novel pathogenic variants and their functional characterization and (ii) to study the association of retinal dystrophy in patients with MFRP variants, based on a detailed literature review of genotype-phenotype correlations. METHODS: Patients with nanophthalmos and available family members received a comprehensive ophthalmological examination. Genetic analysis was based on whole-exome sequencing and variant calling in core genes including MFRP, BEST1, TMEM98, PRSS56, CRB1, GJA1, C1QTNF5, MYRF and FAM111A. A minigene assay was performed for functional characterization of a splice site variant. RESULTS: Seven patients, aged between three and 65 years, from five unrelated families were included. Novel pathogenic variants in MFRP (c.497C>T, c.899-3C>A, c.1180G>A), and PRSS56 (c.1202C>A), and a recurrent de novo variant in FAM111A (c.1706G>A) in a patient with Kenny-Caffey syndrome type 2, were identified. In addition, we report co-inheritance of MFRP-related nanophthalmos and ADAR-related Aicardi-Goutières syndrome. CONCLUSION: Nanophthalmos is a genetically heterogeneous condition, and the severity of ocular manifestations appears not to correlate with variants in a specific gene. However, retinal dystrophy is only observed in patients harbouring pathogenic MFRP variants. Furthermore, heterozygous carriers of MFRP and PRSS56 should be screened for the presence of high hyperopia. Identifying nanophthalmos as an isolated condition or as part of a syndrome has implications for counselling and can accelerate the interdisciplinary care of patients.

Genetic Analysis in a Swiss Cohort of Bilateral Congenital Cataract.

IMPORTANCE: Identification of geographic population-based differences in genotype and phenotype heterogeneity are important for targeted and patient-specific diagnosis and treatment, counseling, and screening strategies. OBJECTIVE: To report disease-causing variants and their detailed phenotype in patients with bilateral congenital cataract from a single center in Switzerland and thereby draw a genetic map and perform a genotype-phenotype comparison of this cohort. DESIGN, SETTING, AND PARTICIPANTS: This clinical and molecular-genetic cohort study took place through the collaboration of the Department of Ophthalmology at the University Hospital Zurich and the Institute of Medical Molecular Genetics, University of Zurich, Schlieren, Switzerland. Thirty-seven patients from 25 families with different types of bilateral congenital cataract were included. All participating family members received a comprehensive eye examination. Whole exome sequencing was performed in the index patients, followed by a filtering process to detect possible disease-associated variants in genes previously described in association with congenital cataract. Probable disease-causing variants were confirmed by Sanger sequencing in available family members. All data were collected from January 2018 to June 2020, and the molecular-genetic analyses were performed from January 2019 to July 2020. MAIN OUTCOMES AND MEASURES: Identification of the underlying genetic causes of bilateral congenital cataract, including novel disease-causing variants and phenotype correlation. RESULTS: Among the 37 patients (18 [49%] male and 19 [51%] female; mean [SD] age, 17.3 [15.9] years) from 25 families, pathogenic variants were detected in 20 families (80% detection rate), which included 13 novel variants in the following genes: BCOR, COL4A1, CRYBA2, CRYBB2, CRYGC, CRYGS, GJA3, MAF, NHS, and WFS1. Putative disease-causing variants were identified in 14 of 20 families (70%) as isolated cases and in 6 of 20 families (30%) with syndromic cases. A recessive variant in the CRYBB2 gene in a consanguineous family with 2 affected siblings showing a nuclear and sutural cataract was reported in contrast to previously published reports. In addition, the effect on splicing in a minigene assay of a novel splice site variant in the NHS gene (c.[719-2A>G]) supported the pathogenicity of this variant. CONCLUSIONS AND RELEVANCE: This study emphasizes the importance of genetic testing of congenital cataracts. Known dominant genes need to be considered for recessive inheritance patterns. Syndromic types of cataract may be underdiagnosed in patients with mild systemic features.

De Novo Assembly-Based Analysis of RPGR Exon ORF15 in an Indigenous African Cohort Overcomes Limitations of a Standard Next-Generation Sequencing (NGS) Data Analysis Pipeline.

RPGR exon ORF15 variants are one of the most frequent causes for inherited retinal disorders (IRDs), in particular retinitis pigmentosa. The low sequence complexity of this mutation hotspot makes it prone to indels and challenging for sequence data analysis. Whole-exome sequencing generally fails to provide adequate coverage in this region. Therefore, complementary methods are needed to avoid false positives as well as negative results. In this study, next-generation sequencing (NGS) was used to sequence long-range PCR amplicons for an IRD cohort of African ancestry. By developing a novel secondary analysis pipeline based on de novo assembly, we were able to avoid the miscalling of variants generated by standard NGS analysis tools. We identified pathogenic variants in 11 patients (13% of the cohort), two of which have not been reported previously. We provide a novel and alternative end-to-end secondary analysis pipeline for targeted NGS of ORF15 that is less prone to false positive and negative variant calls.