Effect of splice site mutations. (A) RT-PCR using primers in exon 2 and 5 on cDNA from patient II-10 (c.308+1G>T) and two controls (c1 and c2). Wild-type amplicon, 250 bp; mutant, 152 bp. (B) (Sequencing of lower band unraveled skipping of exon 3 resulting in a premature stop-codon (r.211_308del: p.Thr71Argfs*8). (C) Sequencing of cDNA of patient V-12 (c.790-3A>G) for an amplicon covering exon 7/8 splice site revealed double-sequence. (D) Sequencing of cloned amplicon fragments revealed insertion of 2 nucleotides (AG) from the intronic acceptor site (r.790_791insAG) in mutant clones. This leads to a premature stop-codon (p.Val264Argfs*26).