Aara Patel1, Jane D Hayward1, Vijay Tailor2, Rodney Nyanhete3, Helena Ahlfors3, Camila Gabriel1, Tommaso B Jannini1, Yassir Abbou-Rayyah4, Robert Henderson4, Ken K Nischal5, Lily Islam6, Maria Bitner-Glindzicz1, Jane Hurst1, Leonardo E Valdivia7, Mario Zanolli8, Mariya Moosajee9, John Brookes10, Maria Papadopoulos11, Peng T Khaw12, Thomas Cullup3, Lucy Jenkins13, Annegret Dahlmann-Noor2, Jane C Sowden14. 1. UCL Great Ormond Street Institute of Child Health, and NIHR Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London, London, United Kingdom. 2. NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust, and UCL Institute of Ophthalmology, London, United Kingdom. 3. North East Thames Regional Genetics Laboratory, Great Ormond Street Hospital for Children NHS Trust, London, United Kingdom. 4. Department of Ophthalmology, Great Ormond Street Hospital for Children NHS Trust, London, United Kingdom. 5. Department of Ophthalmology, Great Ormond Street Hospital for Children NHS Trust, London, United Kingdom; UPMC Eye Center, Children's Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. 6. UCL Great Ormond Street Institute of Child Health, and NIHR Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London, London, United Kingdom; Birmingham Women's Hospital, Birmingham, United Kingdom. 7. Centre for Integrative Biology, Facultad de Ciencias, Universidad Mayor, Santiago, Chile. 8. Facultad de Medicina Clínica Alemana de Santiago-Universidad del Desarrollo, Santiago, Chile. 9. NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust, and UCL Institute of Ophthalmology, London, United Kingdom; Department of Ophthalmology, Great Ormond Street Hospital for Children NHS Trust, London, United Kingdom. 10. Department of Ophthalmology, Great Ormond Street Hospital for Children NHS Trust, London, United Kingdom; Glaucoma Service, Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom. 11. Glaucoma Service, Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom. 12. NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust, and UCL Institute of Ophthalmology, London, United Kingdom; Glaucoma Service, Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom. 13. UCL Great Ormond Street Institute of Child Health, and NIHR Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London, London, United Kingdom; North East Thames Regional Genetics Laboratory, Great Ormond Street Hospital for Children NHS Trust, London, United Kingdom. 14. UCL Great Ormond Street Institute of Child Health, and NIHR Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London, London, United Kingdom. Electronic address: j.sowden@ucl.ac.uk.
Abstract
PURPOSE: To develop a comprehensive next-generation sequencing panel assay that screens genes known to cause developmental eye disorders and inherited eye disease and to evaluate its diagnostic yield in a pediatric cohort with malformations of the globe, anterior segment anomalies, childhood glaucoma, or a combination thereof. DESIGN: Evaluation of diagnostic test. PARTICIPANTS: Two hundred seventy-seven children, 0 to 16 years of age, diagnosed with nonsyndromic or syndromic developmental eye defects without a genetic diagnosis. METHODS: We developed a new oculome panel using a custom-designed Agilent SureSelect QXT target capture method (Agilent Technologies, Santa Clara, CA) to capture and perform parallel high-throughput sequencing analysis of 429 genes associated with eye disorders. Bidirectional Sanger sequencing confirmed suspected pathogenic variants. MAIN OUTCOME MEASURES: Collated clinical details and oculome molecular genetic results. RESULTS: The oculome design covers 429 known eye disease genes; these are subdivided into 5 overlapping virtual subpanels for anterior segment developmental anomalies including glaucoma (ASDA; 59 genes), microphthalmia-anophthalmia-coloboma (MAC; 86 genes), congenital cataracts and lens-associated conditions (70 genes), retinal dystrophies (RET; 235 genes), and albinism (15 genes), as well as additional genes implicated in optic atrophy and complex strabismus (10 genes). Panel development and testing included analyzing 277 clinical samples and 3 positive control samples using Illumina sequencing platforms; more than 30× read depth was achieved for 99.5% of the targeted 1.77-Mb region. Bioinformatics analysis performed using a pipeline based on Freebayes and ExomeDepth to identify coding sequence and copy number variants, respectively, resulted in a definitive diagnosis in 68 of 277 samples, with variability in diagnostic yield between phenotypic subgroups: MAC, 8.2% (8 of 98 cases solved); ASDA, 24.8% (28 of 113 cases solved); other or syndromic, 37.5% (3 of 8 cases solved); RET, 42.8% (21 of 49 cases solved); and congenital cataracts and lens-associated conditions, 88.9% (8 of 9 cases solved). CONCLUSIONS: The oculome test diagnoses a comprehensive range of genetic conditions affecting the development of the eye, potentially replacing protracted and costly multidisciplinary assessments and allowing for faster targeted management. The oculome enabled molecular diagnosis of a significant number of cases in our sample cohort of varied ocular birth defects.
PURPOSE: To develop a comprehensive next-generation sequencing panel assay that screens genes known to cause developmental eye disorders and inherited eye disease and to evaluate its diagnostic yield in a pediatric cohort with malformations of the globe, anterior segment anomalies, childhood glaucoma, or a combination thereof. DESIGN: Evaluation of diagnostic test. PARTICIPANTS: Two hundred seventy-seven children, 0 to 16 years of age, diagnosed with nonsyndromic or syndromic developmental eye defects without a genetic diagnosis. METHODS: We developed a new oculome panel using a custom-designed Agilent SureSelect QXT target capture method (Agilent Technologies, Santa Clara, CA) to capture and perform parallel high-throughput sequencing analysis of 429 genes associated with eye disorders. Bidirectional Sanger sequencing confirmed suspected pathogenic variants. MAIN OUTCOME MEASURES: Collated clinical details and oculome molecular genetic results. RESULTS: The oculome design covers 429 known eye disease genes; these are subdivided into 5 overlapping virtual subpanels for anterior segment developmental anomalies including glaucoma (ASDA; 59 genes), microphthalmia-anophthalmia-coloboma (MAC; 86 genes), congenital cataracts and lens-associated conditions (70 genes), retinal dystrophies (RET; 235 genes), and albinism (15 genes), as well as additional genes implicated in optic atrophy and complex strabismus (10 genes). Panel development and testing included analyzing 277 clinical samples and 3 positive control samples using Illumina sequencing platforms; more than 30× read depth was achieved for 99.5% of the targeted 1.77-Mb region. Bioinformatics analysis performed using a pipeline based on Freebayes and ExomeDepth to identify coding sequence and copy number variants, respectively, resulted in a definitive diagnosis in 68 of 277 samples, with variability in diagnostic yield between phenotypic subgroups: MAC, 8.2% (8 of 98 cases solved); ASDA, 24.8% (28 of 113 cases solved); other or syndromic, 37.5% (3 of 8 cases solved); RET, 42.8% (21 of 49 cases solved); and congenital cataracts and lens-associated conditions, 88.9% (8 of 9 cases solved). CONCLUSIONS: The oculome test diagnoses a comprehensive range of genetic conditions affecting the development of the eye, potentially replacing protracted and costly multidisciplinary assessments and allowing for faster targeted management. The oculome enabled molecular diagnosis of a significant number of cases in our sample cohort of varied ocular birth defects.
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