Zeinab Fadaie

effect on the splice donor site of exon 7. However, it disturbs the balance between the enhancer and silencer motifs in this position and thus leads to a splice defect. This chapter highlights the important role of conserved motifs in the splicing processes.
The emphasis of Chapter 3.3 is on the effect of variants in cis in the resulting protein function. We described two unrelated families with adult-onset vitelliform macula dystrophy carrying a complex allele in IMPG2 (c.[3023-NST>AIPO2PG>A]) upon analysis of WES data. The c.PO2PG>A variant is not only a putative pathogenic missense mutation by itself but acts as a modifier element for c.3023-NST>A as well by enhancing its splice defect. In both families, individuals carrying this complex allele in a heterozygous manner did not always develop macular dystrophy which highlights the importance of other genetic and environmental factors that contribute to this disease.

Chapter 3.4 sheds light on the pathogenic role of a branchpoint variant (c.592-2NA>T) in BBSN in four unrelated families with non-syndromic retinitis pigmentosa, who carried c.NNS9T>G, the most frequent variant in BBSN, in the other allele. The branchpoint variant was identified through WGS analysis and its complex splice defect was revealed using in vitro midigene splice assays. This chapter characterizes the first pathogenic branchpoint variant reported in IRDs.

Chapter 4 describes a comprehensive study in which 100 individuals with different IRDs were assessed using WGS-based sequence analysis. The probands were previously screened by WES or targeted sequencing approaches and were genetically unsolved. The WGS analysis was performed with special attention to the non-coding variants and the putative causal variants were investigated by in vitro functional assays to prove the pathogenicity. We provide a molecular diagnosis for 27% of the pre-screened cases and suggest employing improved sequencing technologies and variant interpretation to increase the diagnostics yield for IRDs.

Chapter 5 illustrates the implementation of a novel strategy for identification of the genetic defect in IRDs with a strong genotype-phenotype correlation. In a family with choroideremia, previous RNA analysis of an affected proband showed CHM exon 12 skipping, but the underlying DNA defect remained elusive for 18 years. Using optical genome mapping and long-read WGS, an inverted duplication in CHM was identified. We provided a model that demonstrates the origin of the SV through fork stalling and a template switching/microhomology-mediated break-induced replication mechanism. Additionally, we speculated on the mechanism of the splice defect and proposed an altered RNA secondary structure that disrupted the splicing of exon 12. This chapter emphasizes the great opportunities of optical genome mapping and long-read WGS to unravel the hidden SVs in unsolved cases as well as the underappreciated role of an altered RNA secondary structure as a disease mechanism.

Finally, Chapter 6 provides a general discussion of the main findings in this thesis and their implications towards an improved diagnosis of IRDs. The application of different sequencing technologies and their advantages and disadvantages are explained. The importance of branchpoint variants, complex alleles, and variants which affect the RNA secondary structure were discussed. Additionally, the need of accurate splicing tools was explained by comparing different prediction algorithms and highlighting their importance in in vitro functional splice assays. Finally, potential reasons for missing pathogenic variants in the studied cohort were discussed, and it was postulated that application of long-read sequencing will be an important technology in the future of IRDs and other genetic conditions. Moreover, the different cellular systems for in vitro functional analysis can shed light on the currently missed causes of IRDs in the future.