MCCA and B missense mutations that alter splicing. (a) MCCA D532H. The 1594G→C transversion of the last bp of exon 13 results in the missense mutation D532H. The 3′ base of an exon also contributes to donor splice site recognition and, as shown in the lower panel, RT-PCR of MCCA cDNA in this patient with primers corresponding to the 5′ and 3′ UTR resulted in a product smaller than in wild-type. Sequence analysis of this product showed that exon 13 (217 bp) is skipped, which shifts the reading frame. Thus, the deleterious consequences of this missense mutation appear to be entirely due to the splicing defect. (b) MCCB I437V. As shown in the upper panel, the 1309A→G transition in exon 14 results in the replacement of isoleucine by valine, a conservative change. However, the mutation also activates a cryptic splice donor. Use of this new donor splice site deletes the last 64 bp of exon 14 from the mature transcript. As shown in the lower panel, direct sequencing of the RT-PCR product shows that virtually all the transcript present uses the new, more 5′ splice donor. The second allele of this compound heterozygous patient does not produce detectable RNA. wt, wild-type; mut, mutant.