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

Abstract

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.

Keywords: RNA-seq; eye diseases; genomics; molecular diagnostic techniques.

Figure 1

Distribution of solved cases, possibly solved cases and unsolved cases among the 25 clinical subgroups. ACHM, achromatopsia; ADRP, autosomal dominant retinitis pigmentosa; ARRP, autosomal recessive retinitis pigmentosa; BBS, Bardet-Biedl syndrome; BCM, blue cone monochromacy; BVMD, Best vitelliform macular dystrophy; CACD, central areolar choroidal dystrophy; CD, cone dystrophy; CHM, choroideraemia; CRD, cone-rod dystrophy; CSNB, congenital stationary night blindness; DOA, dominant optic atrophy; LCA, Leber congenital amaurosis; LHON, Leber hereditary optic neuropathy; MDS, macular dystrophy; MISC, miscellaneous diagnosis; NYS, nystagmus; OA, ocular albinism; SCHI, retinoschisis; SRP, simplex retinitis pigmentosa; STGD, Stargardt disease; UD, unclear diagnosis; USH I, Usher syndrome type I; USH II, Usher syndrome type 2; XRP, X linked retinitis pigmentosa.

Figure 2

Characteristics of identified disease-causal genomic variation. (A) Variants with HGMD entry are described as already described variants. Novel variants are those with no HGMD entry so far. The fraction of structural variants is shown for which no matching HGMD entries are listed, as most HGMD entries do not specify exact breakpoints. (B) Distribution of variant types among the 1097 unique variants identified in the cohort. (C) Subcategories of variants from subgroups ‘other types of variants’ (n=25) and ‘structural variants’ (n=71) from (B). snRNA, small nuclear RNA.

Figure 3

Illustrative example for biallelic structural variants. Genome sequencing with comprehensive bioinformatic analysis revealed a structural variant with breakpoints in intronic regions and only partial copy number alteration together with a deletion in trans in the EYS gene. The upper part shows the corresponding IGV screenshots, the lower part a schematic representation summarising the structural changes. The HGVS nomenclature is NC_000006.11:g.[65922918_66006755delinsGTTTTCTTTTTA]; [64832337_64839052delins64914341_64945399inv]. Biallelism of the structural variants was confirmed by carrier testing using shallow genome sequencing. HGVS, Human Genome Variation Society; IGV, Integrative Genomics Viewer.