High-coverage next-generation sequencing (NGS) for retinal dystrophies and Usher syndrome: High diagnostic yield, CNV detection, novel disease mechanisms and therapy targets

Abstract

Purpose: Comprehensive genetic analysis for genetically heterogeneous retinal dystrophies (RD): RP, early-onset severe RD (EOSRD/LCA), macular dystrophies (MD), Usher syndrome (USH).

Methods: With NGS on the Illumina MiSeq system, we targeted the exons of 31 autosomal recessive (ar)RP (413), 23 autosomal dominant (ad)RP (248), 16 EOSRD (215) and 10 USH genes (373) and analyzed 126 RD and 57 USH patients. A bioinformatic pipeline was established for quantitative analysis of NGS data to detect copy number variations (CNVs). NGS was subsequent to Sanger sequencing (SaS) and MLPA in USH2A-negative USH2, and the initial analysis in atypical USH or USH1.

Results: High coverage of Ø300-400 reads per exon was obtained, and mutations identified in most RD patients: arRP: 72%; adRP: 81%; MD: 57%; EOSRD: 53%. CNVs were confirmed by MLPA analysis. Heterozygous CNVs unmasked apparently homozygous mutations as hemizygous. CNVs and point mutations in non-coding exon 1 of EYS were identified in trans to coding mutations. Heterozygous PRPF31 CNVs in sporadic patients shifted diagnosis from arRP to adRP with incomplete penetrance. RP1 was the major arRP gene. Truncating C-terminal RP1 alleles appeared to be non-pathogenic in ar families with biallelic mutations in secondary genes. SaS and NGS identified mutations in 95% of USH2 and 94% of atypical/USH1. CNVs accounted for 11% of USH2A alleles. We show PTC124-induced read-through for a novel USH2A hot spot mutation (23% of alleles), p.Trp3955*. Combined homozygous mutations in OTOA and NR2E3 phenocopied USH1 in one patient.

Conclusions: NGS of RD genes dramatically increases the diagnostic yield, improving genetic counseling. CNV detection from NGS data highlights the method’s potential beyond sequencing. It avoids pitfalls of SaS in consanguineous families with hemizygous mutations, identifies the “missing hit” in recessive RD with monoallelic mutations and uncovers novel candidate disease exons, illustrating the benefit of 5’-UTR inclusion in NGS of disease gene or exome panels. The discovery of non-pathogenic truncating RP1 alleles in RDs due to mutations in other genes underscores the necessity to consider the variant load of all genes to avoid false interpretation. A new USH2A hot spot mutation was accessible to PTC124-induced readthrough and represents a major therapy target for USH2.

Keywords: 539 genetics • 696 retinal degenerations: hereditary • 537 gene screening