Fig. 5

Genomic localization of variants and their functional consequence. 1. Germ-line variants located in the gene regulatory domains such as promoters or locus control regions will affect the level of gene transcription. In most instances variants in the promoters disrupt the binding of trans-acting factors thereby reducing expression of the gene. The composition of regulatory motifs is in many instances incompletely understood and it is in general difficult to predict the consequence of these variants. A few diseases exhibit unstable trinucleotide repeat sequences in the promoter, that when expanded is known to impair transcription. The functional significance of promoter variants is normally demonstrated by loss of expression (LOE) via RNA sequencing or measurement of the encoded protein. Repeat expansions may also be directly discerned from the WGS data 2. Variants located at the canonical splice donor (GT) or acceptor (AG) sites or at a known A – branch-site are in general pathogenic since these strongly conserved sequences are essential for splicing. Variants located deeper in the intron or in the connecting exons can also disrupt splicing due to disruption of enhancer or silencer motifs but the significance of these variants is more difficult to predict. The evaluation of these variants in general requires minigene analysis and/or RNA sequencing. 3. Coding nonsense or frameshift variants lead to premature translation termination and shortening of the encoded protein. In most cases this can lead to loss of function (LOF). Missense variants and small indels may disrupt protein function in a number of different ways such as reducing enzymatic activity, stability, localization or structure and macromolecular assembly. Consequently, the evaluation of these variants requires deep insight into the proteins function and in many instances various kinds of functional analysis is necessary in order to classify the variants as pathogenic. Since the functional significance of a particular variant may be difficult to predict - even for canonical splice mutations and LOF variants - it recommended that all classes of variants undergo evaluation according to ACMG/AMP criteria in order to determine pathogenicity