CLRN1 minigene splice assay. (A) Schematic to scale overview of the genomic CLRN1 structure including the 5′ and 3′ untranslated regions (5′ UTR and 3′ UTR, respectively). bp, basepairs. Exons (ex) are shown as colored boxes. (B) CLRN1 to scale minigene used for the splice experiments shown in (C,D). The position of the c.254–649 T > G mutation is indicated by an arrowhead and the novel exon generated by the mutation (referred to as “aberrant exon”) is displayed as a dashed purple box. The dashed lines represent a schematic magnification of the 3′ and 5′ splice sites flanking the aberrant exon. The consensus sequences for the putative branch point (BP), the polypyrimidine tract (PPT), the 3′ acceptor splice site (ASS), and the 5′ donor splice site (DSS) of the aberrant exon are underlined. The putative lariat-forming adenosine in the PPT is shown in upper case. The binding positions of the primers used for the RT-PCR shown in (C) are indicated by arrows. (C) Representative RT-PCR analysis from HEK293 cells transiently transfected with the single constructs as indicated. As control, RT-PCR from non-transfected HEK293 cells was performed. The length and exon composition of the two splice products for WT (432 bp) and the c.254–649T > G mutant (432 bp and 662 bp) are shown on the right panel. The position of the primer used for Sanger sequencing shown in (D) is indicated by an arrow. (D) Representative electropherograms of the exon-exon boundaries for both RT-PCR products of the c.254–649T > G mutant.