Highly efficient genetic diagnostic testing in patients with inherited retinal dystrophies using Panel-based Next Generation Sequencing

Abstract

Purpose: Genetic heterogeneity is a well known feature in Retinal dystrophies (RD). Therefore next generation sequencing (NGS) technology is the most promising tool to identify mutations in RD. To date NGS is not routinely used in genetic diagnostic testing of RD patients. Our aim was to establish a diagnostic pipeline for RD patients using NGS.

Methods: 105 genes associated with RD were selected from literature or databases. The genes were subdivided into 8 subpanels according to the clinical diagnosis and the suspected mode of inheritance. For analysis we performed a customized target-in-solution-enrichment for exonic and flanking intronic region. The SOLiD 5500xl platform was used for NGS. Sequence reads were mapped to the human reference genome GRCh37/hg19. The variant calls generated by LifeScope were annotated using Ensembl, dbSNP and in-house variant databases. Indel calling was also supported. Variants with a global minor allele frequency below 5% (based on dbSNP and Exome Variant Server) were selected for further investigation. All mutations were validated by conventional Sanger sequencing. Regions with less than 10 reads per base were examined by Sanger sequencing.

Results: We analyzed 160 patients with different forms of RD. The majority of cases were diagnosed with retinitis pigmentosa (RP), where we obtained a diagnostic detection rate of 58%. Within the other monogenic forms of RD we detected 50% of solved cases. In the syndromic forms of RP (Usher and Bardet-Biedl syndome) we detected homozygous or compound heterozygous mutations in 74%. Among all analyzed patients the genes USH2A, EYS, ABCA4 and RPGR were more frequently affected. Furthermore, we found several mutations in x-linked and dominant genes among the cases with sporadic RD. These results imply consequences for counseling of patients and families.

Conclusions: We have developed a diagnostic NGS pipeline which allows us to detect the causative mutations in 50 to 74% of patients with RD. Thus, the NGS-based gene diagnostics is a reliable and cost efficient tool in genetically heterogeneous diseases.

Keywords: 539 genetics • 494 degenerations/dystrophies • 702 retinitis