Diagnostic genome sequencing improves diagnostic yield: a prospective single-centre study in 1000 patients with inherited eye diseases.

PURPOSE: Genome sequencing (GS) is expected to reduce the diagnostic gap in rare disease genetics. We aimed to evaluate a scalable framework for genome-based analyses 'beyond the exome' in regular care of patients with inherited retinal degeneration (IRD) or inherited optic neuropathy (ION). METHODS: PCR-free short-read GS was performed on 1000 consecutive probands with IRD/ION in routine diagnostics. Complementary whole-blood RNA-sequencing (RNA-seq) was done in a subset of 74 patients. An open-source bioinformatics analysis pipeline was optimised for structural variant (SV) calling and combined RNA/DNA variation interpretation. RESULTS: A definite genetic diagnosis was established in 57.4% of cases. For another 16.7%, variants of uncertain significance were identified in known IRD/ION genes, while the underlying genetic cause remained unresolved in 25.9%. SVs or alterations in non-coding genomic regions made up for 12.7% of the observed variants. The RNA-seq studies supported the classification of two unclear variants. CONCLUSION: GS is feasible in clinical practice and reliably identifies causal variants in a substantial proportion of individuals. GS extends the diagnostic yield to rare non-coding variants and enables precise determination of SVs. The added diagnostic value of RNA-seq is limited by low expression levels of the major IRD disease genes in blood.

Impact of OCT scan-patterns in identifying morphological features of lamellar macular holes and macular pseudoholes.

AIM: To evaluate the impact of the optical coherence tomography (OCT) scan patterns on the detection of the features associated with lamellar macular hole (LMH) and macular pseudohole (MPH). METHODS: This is a retrospective analysis of 100 consecutive eyes with LMH (n=41) and MPH (n=59) having at least three of the following OCT features, which include mandatory criteria for the diagnosis of LMH and MPH: Epiretinal membrane, epiretinal proliferation, verticalization, intraretinal cystoid spaces, foveoschisis, irregular foveal contour, foveal cavity with undermined edges, and ellipsoid line disruption. Primary outcome measurement was the detection frequency of the features in three different OCT scan patterns: 1) volume scan; 2) six radial scans (R6); and 3) vertical and horizontal radial scans (R2). RESULTS: Of the total eight features, the maximal detection frequency was found as 4.45±1.45, 4.35±1.47, and 3.70±1.59, by the volume, R6 and R2, respectively. R2 was inferior to the other patterns in detection of the total features (P<0.001), whereas R6 and volume patterns were found comparable (P=0.312). CONCLUSION: The physician should be aware that the selection of the OCT-scan pattern may influence the detection of mandatory morphological criteria for the diagnosis of LMH and MPH.

ABCA4-associated disease as a model for missing heritability in autosomal recessive disorders: novel noncoding splice, cis-regulatory, structural, and recurrent hypomorphic variants.

PURPOSE: ABCA4-associated disease, a recessive retinal dystrophy, is hallmarked by a large proportion of patients with only one pathogenic ABCA4 variant, suggestive for missing heritability. METHODS: By locus-specific analysis of ABCA4, combined with extensive functional studies, we aimed to unravel the missing alleles in a cohort of 67 patients (p), with one (p = 64) or no (p = 3) identified coding pathogenic variants of ABCA4. RESULTS: We identified eight pathogenic (deep-)intronic ABCA4 splice variants, of which five are novel and six structural variants, four of which are novel, including two duplications. Together, these variants account for the missing alleles in 40.3% of patients. Furthermore, two novel variants with a putative cis-regulatory effect were identified. The common hypomorphic variant c.5603A>T p.(Asn1868Ile) was found as a candidate second allele in 43.3% of patients. Overall, we have elucidated the missing heritability in 83.6% of our cohort. In addition, we successfully rescued three deep-intronic variants using antisense oligonucleotide (AON)-mediated treatment in HEK 293-T cells and in patient-derived fibroblast cells. CONCLUSION: Noncoding pathogenic variants, novel structural variants, and a common hypomorphic allele of the ABCA4 gene explain the majority of unsolved cases with ABCA4-associated disease, rendering this retinopathy a model for missing heritability in autosomal recessive disorders.