The phenylalanine hydroxylase c.30C>G synonymous variation (p.G10G) creates a common exonic splicing silencer

Abstract

PKU is caused by mutations in PAH. A c.30C>G synonymous variation in exon 1, previously reported as neutral, was observed in two patients. The variation creates a GGG triplet, which is part of several exonic splicing silencer (ESS) motifs. Because the 5'-splice site of PAH exon 1 is intrinsically weak and therefore could be responsive to a new flanking ESS, we hypothesized that c.30C>G could cause aberrant mRNA splicing. We demonstrate that c.30C>G causes aberrant mRNA splicing in two different reporter minigenes, and that this is abolished if a preexisting flanking GGG triplet is disrupted. GGG triplets are part of the consensus motif bound by splicing-inhibitory hnRNPH proteins and we observed a dramatic increase in hnRNPH binding to c.30C>G PAH RNA. We conclude that c.30C>G creates a hnRNPH-binding ESS, which can disrupt mRNA splicing. A disease-causing mutation in HEXB, which has previously been associated with exon skipping in patients also creates a GGG triplet. We show that the mutant HEXB motif causes exon skipping of a reporter minigene and that this is also influenced by a flanking GGG triplet. We suggest that aberrant splicing caused by creation/abolishment of GGG triplets located together with a preexisting flanking GGG triplet, may be an underreported cause of human disease. It is important to recognize that exonic sequence changes may disrupt mRNA splicing. This is particularly important in PAH, since PKU patients harboring such mutations are unlikely to respond to therapy with 6R-tetrahydrobiopterin (BH(4)), despite the fact that the genetic code indicates otherwise.