Figure 2

Alternative splicing caused by the c.245–42 G > A variant creating the “AG” splice acceptor site. (a) Normal splicing identified in the affected boys (III-1 and III-2). Normal splicing has occurred using the wildtype “AG” splice acceptor motif, pyrimidine (Y)-rich sequence, and the branch site, generating wildtype mRNA, whereas aberrant splicing has taken place using the newly created “AG” splice acceptor motif that is associated with pyrimidine (Y)-rich sequence and two putative branch sites, producing mRNA with retention of a 40 bp intronic segment (highlighted with red) between exon 6 (highlighted with green) and exon 7 (highlighted with blue). Red asterisks indicate the highly conserved “A” nucleotide at branch sites. Exonic and transcribed intronic nucleotides are written with large letters, and intronic nucleotides with small letters. (b) Schematic representation of AR exons on the genome, and wildtype (WT) and mutant (MT) mRNA and protein. AF-1: activation function 1; NTD: N-terminal transactivation domain; DBD: DNA binding domain; LBD: ligand binding domain; and AF-2: activation function 2. The coding regions are shown with solid boxes, and the untranslated regions are depicted with open boxes; exon 6, exon 7, the transcribed intronic sequence, and the frameshifted sequence before the premature termination are shown in green, blue, red, and gray, respectively. It is predicted that the mutant mRNA primarily undergoes nonsense mediated decay (NMD), while it could escape NMD, producing a small amount of a truncated protein with a loss-of-function (LOF) activity. (c) RT-PCR analysis for mRNA samples extracted from the LCLs of the non-affected father (II-1), the affected boys (III-1 and III-2), and a healthy control adult male. The band intensity ratio has been obtained between the wildtype (WT)-specific and the mutant (MT)-specific bands produced by the P1/P2 primers in the affected boys. The WT mRNA expression ratio has been calculated with the data of quantitative PCR performed for CHX-treated LCLs using P_W/Pc or P_M/Pc; the black and the gray bars have been obtained using GAPDH and ACTB as internal controls, respectively.