Suzanne de Bruijn

54 Chapter 1.2 insulators. 145,146 Regulatory elements are short DNA sequences (100-500 bp) that allow precise spatiotemporal control of gene expression levels. 145 Promoter and distant enhancer regions interact with each other via chromosomal looping, allowing the recruitment of the transcriptional machinery. Alternatively, insulators can block the interactions between promoters and enhancers. An enhancer element can be located up to 1 Mb away from its target gene and can serve as the transcriptional regulator of one or more genes. 145,147-149 Usually, an enhancer displays a spatiotemporal pattern of activity. Transcription factors, that bind enhancer or promoter elements, are the key regulators of these processes and modulate gene expression. Pathogenic variants in cis regulatory elements could alter the transcription factor binding sites or chromatin landscape and therefore the activity of the enhancer or promoter. 145,146 Databases such as JASPAR 150 that contain consensus sequences of transcription factor binding sites can be used to predict the effect of a potential regulatory variant on transcription factor binding. Regulatory variants that impact transcription initiation usually lead to subtle changes in gene expression and are difficult to assess. 146 Therefore, a context-specific profiling of the tissue- and cell type-specific cis regulatory architecture is essential. 151 Enhancer databases such as the ENCODE portal 152 , GeneHancer 153 and EnhancerAtlas 154 contain an overview of reported cis regulatory elements that are widespread through the genome. Potential interactions between promoter and enhancer elements can be assessed by evaluating available chromosome conformation capture data like Hi-C. Additionally, the presence of context-specific active enhancer hallmarks should be assessed. These include: (1) confirmed binding of transcription factors, (2) production of non-coding enhancer RNA, (3) an open chromatin conformation and (4) the presence of histone- modificationmarks that are associatedwith enhancer activity, such as histone 3 lysine 27 acetylation. 145,151 Figure 4 provides an overview of these hallmarks, suitable techniques to assess these, and a selection of available and relevant publicly available (epigenetic) datasets as used to interrogate the recently resolved autosomal dominant retinitis pigmentosa RP17 locus. 155 Once a candidate regulatory variant has been identified, experiments such as an in vitro luciferase reporter assay could be applied to confirm its effect on enhancer or promoter activity. 145 Structural variants SVs are defined as genomic rearrangements that are larger than 50 bp. 125 SVs include deletions and duplications, also referred to as copy number variations, as well as inversions, translocations and insertions. 147 In 2020, an amendment of the ACMG guidelines was published to aid in the classification of SVs. 156 SVs can have direct consequences on gene dosage levels when (partially) overlapping with coding regions