Suzanne de Bruijn

290 Chapter 6 needed to be interrogated for putative pathogenic variants. Also today, where more and more sequencing data is generated, these approaches could still be valuable and might even be necessary in order to keep data analyses feasible. Nevertheless, this advantage also immediately represents the most important pitfall of these old techniques. When focusing on a specific genomic region, a genetic defect located outside the region of interest is easily overlooked. In this thesis, an important example is described in chapter 3 , where the genetic defect underlying DFNA21 was found to be located 0.9 Mb centromeric of the determined DFNA21 locus. In this case, the true locus within the family was masked by the presence of both phenocopies (individuals mimicking the HL phenotype, with other (non-genetic) causes underlying) and non-penetrance (asymptomatic individuals carrying the putative genetic defect). Because of this, the DFNA21 locus was falsely delimited in the past and the underlying pathogenic variant could not be identified by targeted sequencing of genes locatedwithin the locus. 40 Now, twenty years later, the introduction of WES and decreased sequencing costs allowed us to sequence the complete coding regions of the genome of multiple affected family members. Only by combining the genetic approaches (WES and linkage analysis) with the detailed phenotypic data that were collected over the years, we were able to finally resolve the genetic defect of DFNA21: an in-frame deletion in the RIPOR2 gene. Second generation sequencing Second generation sequencing, or next generation sequencing, allows the analysis of all genetic variants in the coding elements (WES) or of the human genome sequence (WGS) in a parallelized fashion. The first reports of RD- and HL-associated genes discovered by WES appeared in 2010 (e.g. TSPAN12 41 associated with familial exudative vitreoretinopathy or GPSM2 42 associated with non-syndromic HL). The genetic solve rate of inherited HL and RD by WES ranges from 30% to 80%, depending on the specific phenotype studied. 4-9 In general, the added diagnostic value of WGS for inherited disorders is estimated to be ~21% (i.e. 28%of cases solved byWES versus 49%byWGS). 43 In a first study in which the application of WES and WGS was compared for diagnostic purposes of inherited RD specifically, the added value of WGS was calculated to be 31%. 44 Considering the higher diagnostic yield, improved read coverage of both coding and non-coding regions and decreasing sequencing costs of WGS, it can be anticipated that soon WGS will replace WES in genetic diagnostics. Some important limitations that were hampering the field for a long time can be resolved by the implementation ofWGS. Most evidently,WGS allows the identification of non-coding variants that can have splice altering or regulatory effects. Additionally, the technique can identify SVs at a base-pair resolution. Although micro-array technologies