RATIONALE: Familial pulmonary arterial hypertension results from heterozygous inactivating mutations of the BMPR2 gene. Traditional mutation analysis identifies pathogenic mutations in some 70% of linked families. We hypothesized that the apparent shortfall is due to mutations located in the promoter region of the gene, resulting in abnormal gene regulation. OBJECTIVES: To identify mutations in untranslated sequence regulating BMPR2 transcription. METHODS: DNA upstream of the coding region was analyzed by direct sequencing in 16 families. Reverse transcription-polymerase chain reaction analysis and rapid amplification of cDNA ends of normal human lung RNA were used to investigate transcription of this region. Transcript levels were assessed by allele-specific expression analysis and inhibition of nonsense-mediated decay in lymphoblastoid cell lines. MEASUREMENTS AND MAIN RESULTS: The wild-type transcriptional start site of BMPR2 was defined, 1,148 bp upstream of the ATG. Within this region, we identified a double-substitution mutation, predicted to form a cryptic translational start site, in one family. The mutant transcript contains a premature stop codon predicted to trigger nonsense-mediated decay. Expression analysis in the patient's cell line indeed showed reduced expression of the mutant transcript that could be restored to normal by inhibiting nonsense-mediated decay. CONCLUSIONS: Activation of a cryptic translation initiation site is a novel mutational mechanism in this disorder. These results demonstrate that the 5'-untranslated region of BMPR2 is considerably longer than previously thought, emphasizing the need to fully characterize the BMPR2 promoter and the importance of analyzing noncoding regions in patients with pulmonary arterial hypertension who are negative for mutations within the coding region and intron-exon junctions.
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