In this work we analyze splicing mutations identified in propionic acidemia 
patients to clarify their functional effects and their involvement in the 
disease phenotype. Two mutations in the PCCA gene detected in homozygous 
patients and involving consensus splice sequences (IVS21+3del4 and IVS22-2A>G) 
were shown to produce some normal splicing in patients' cells, at very low 
levels, which were quantitated by real-time PCR methods, and which presumably 
are sufficient to moderate the phenotype. We have also analysed the effect of 
mutations c.653A>G and IVS10-11del6 in the PCCB gene present in heterozygous 
patients with mild phenotype. The c.653A>G mutation is located in the last codon 
of exon 6 and interferes with the correct spliceosomal assembly activating a 
cryptic splice site within exon 6, which leads to an in-frame six-nucleotide 
deletion (delV217-K218). Minigene analysis and sequence-specific hybridization 
probes using real-time PCR methods showed that no normally spliced transcript is 
detectable in the patients' fibroblasts. The IVS10-11del6 mutation shortens the 
polypyrimidine tract of the 3'-splice site of exon 11, resulting in exon 
skipping. Some normal transcript is detectable by allele-specific hybridization 
probes. These analyses suggest that, in some cases, the regulation of gene 
splicing can potentially play an important role in human disease influencing 
phenotypic parameters.