Here we show that the c.2157+5_+6 gc>ag POLG allele is indeed pathogenic by affecting splicing of the POLG mRNA. This mutation causes the exon 12 - intron 12 splice junction to be bypassed, allowing the cell to utilize the next likely splice junction at c.2157+30. Within this 30 nucleotide insertion is an in-frame UAG PTC at c.2157+28–30. Transcripts with PTCs that are at least 50–55 nucleotides upstream of an intron are subjected to NMD. The message arising from the c.2157+5_+6 gc>ag POLG allele fits this criterion as POLG contains 23 exons, and the double mutation occurs in intron 12.
The degradation of the c.2157+5_+6 gc>ag POLG
message was confirmed by sequencing individual mature cDNA clones. Analysis revealed that 19% of the fully spliced transcripts were alternatively spliced. These alternatively spliced transcripts all contained the two intronic mutations. Sequence analysis of prespliced POLG
transcripts in the exon 7 region showed an equal proportion of wild-type message and message containing A467T. The discrepancy between the prespliced transcripts and the relatively low amount of c.2157+5_+6 gc>ag POLG
message indicates that the c.2157+5_+6 gc>ag message was degraded, due to the PTC which triggered the NMD pathway. This caused an effective haplotype insufficiency, where the bulk of the surviving transcripts contain the A467T mutation. Our previous biochemical analysis showed that the A467T mutation results in a pol γ with low intrinsic polymerase activity and a defect in functional association with the pol γ accessory subunit leading to poor mtDNA replication (Chan et al., 2005a
). However, this does not address why patients who are homozygous for the A467T mutation can develop one of the three major POLG
disease phenotypes, and have a longer survival time as compared with compound A467T/W748S heterozygotes (Tzoulis et al., 2006
). Our previous study of the first genotyped Alpers patient (A467T/E873X) (Chan et al., 2005b
), and this current study take us closer to answering this question. In the previous study, we showed that for a patient with A467T/E873X POLG
, most transcripts arise from the allele containing the A467T mutation, leading to mono-allelic expression of A467T pol γ, and is the most likely cause of early onset Alpers syndome in this patient (Chan et al., 2005b
). reveals a striking correlation of PTC mutations in trans
with either A467T or W748S and early onset Alpers or myocerebrohepatopathy syndrome, while other mutations in trans
with PTCs are associated with later onset PEO disease. This suggests that functional gene dose of the A467T allele could dictate age of onset.
We present the first analysis of the consequence of an intronic mutation that leads to alternative splicing in a POLG disease patient. Our analysis represents the second POLG case that causes one of the POLG alleles to be removed by NMD. Transcripts derived from POLG alleles containing any of the 18 known PTCs or splice-site mutations are also expected to undergo NMD. Thus, transcripts arising from alleles containing POLG PTC, frame-shift, or intronic mutations that are predicted to affect splicing should be regarded as possible targets for NMD. Consequently for patients and physicians, the mutation found in the other POLG allele may be a better predictor of disease state.