Figure 1. Representation of reasoning to follow upon identification of candidate sequence variants.

Figure 2. Schematic representation of minigene splicing assays. (a) The most basic minigene is composed of a plasmid containing a promoter and a poly(A) signal with a multiple cloning site (MCS) between the two. The region of the gene in which the suspected mutation is found is inserted into the MCS. A minimum of three exons/two introns must be inserted into the MCS together with the exon of which the pre‐mRNA processing is thought to be affected by the mutation (be it intronic or exonic) as the central exon. The minigene is transfected into the appropriate cell line, and RT‐PCR analysis is then performed using primers specific to the minigene, usually one in an exon and another in an untranslated region of the plasmid (small black arrows). The product of RT‐PCR analysis is then visualized on an agarose gel, where the effect of the UV on mRNA processing may be observed. (b) PTB hybrid minigene composed of a α‐globin gene promoter and SV40 enhancer sequences (indicated by the arrow at the start of the gene) to allow polymerase II transcription in the transfected cell lines. This is followed by a series of exonic and intronic sequences (indicated by boxes and lines, respectively) that derive from α‐globin (white boxes) and fibronectin exons (black boxes), while at the 3′ end a functional polyadenylation site, derived from the α‐globin gene, is present. The genomic DNA region of interest that contains a putative splicing mutation is introduced into the minigene in a unique restriction site (NdeI). In the case of deep intronic mutations, hybrid minigenes are created in which the two exons flanking the intron carrying the mutation and the intron itself (or a shortened version of it) are inserted into the minigene at the NdeI site. The primers used in RT‐PCR are at the junction of the hybrid exon, and are therefore specific to the minigene; (c) Schematic representation of the hybrid minigene SXN13. This minigene consists of a 34 nt alternative exon flanked by duplicated intron 1 from human α‐globin, such that the first and third exons are globin exons 1 and 2. In the absence of a splicing enhancer, this element is predominately skipped due to its small size and a non‐canonical 5′SS. Regions of exonic DNA suspected of having enhancer activity can be cloned into the alternative exon and tested for their effect on splicing.