Supravalvular aortic stenosis: genetic and molecular dissection of a complex mutation in the elastin gene

We have identified two elastin gene (ELN) mutations located in cis in two r elated families with supravalvular aortic stenosis (SVAS). These mutations included an in-frame duplication in exon 18 (1034-1057dup) and a single bas e substitution in exon 26 (1829G -->A) predicted to result in the amino aci d substitution R610Q. Haplotype analysis in one of the families identified an individual with a recombination between exon 18 and 26 of the elastin ge ne. This individual was unaffected and carried the exon 18 insertion mutati on but not 1829G -->A. Skin fibroblasts were established from this recombin ant normal individual and from an affected individual carrying both of the mutations. Reverse transcription/polymerase chain reaction (RT-PCR) analysi s indicated that the expression of the mutant allele was reduced to 12%-27% of the normal allele in the affected but not in the unaffected individual. RNA-blot hybridization and immunoprecipitation experiments revealed reduce d steady-state elastin mRNA levels and tropoelastin synthesis in the affect ed individual. RT-PCR analysis of the mRNA rescued by cycloheximide treatme nt indicated that mutation 1829G -->A created a cryptic donor splice site w ithin exon 26, resulting in the deletion of four nucleotides at the 3'-end of exon 26 and a frameshift in the mRNA. This frameshift mutation generated a premature termination codon in the domain encoded by exon 28, clearly re sulting in nonsense-mediated decay (NMD) of this frameshift RNA product. De spite considerable variability in the molecular nature of mutations respons ible for SVAS, the unifying mechanism appears to be the generation of null alleles by NMD leading to elastin haploinsufficiency.