Published ahead of print on July 19, 2007, doi:10.1164/rccm.200701-164OC Am. J. Respir. Crit. Care Med., Volume 176, Number 8, October 2007, 819-824 A more recent version of this article appeared on October 15, 2007
Submitted on January 31, 2007 Characterization of the BMPR2 5'-untranslated Region and a Novel Mutation in Pulmonary HypertensionMicheala A Aldred1,1 Division of Medical Genetics, Departments of Genetics and Cardiovascular Sciences, University of Leicester, Leicester, United Kingdom; Genomic Medicine Institute, Lerner Research Institute, and Taussig Cancer Center, Cleveland Clinic, Cleveland, OH, USA; Department of Genetics, Case Western Reserve University School of Medicine, Cleveland, OH, USA, 2 Division of Medical Genetics, Departments of Genetics and Cardiovascular Sciences, University of Leicester, Leicester, United Kingdom; Division of Genetics and Molecular Medicine, Kings College London, London, United Kingdom, 3 Division of Medical Genetics, Departments of Genetics and Cardiovascular Sciences, University of Leicester, Leicester, United Kingdom, 4 Division of Respiratory Medicine, Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrookes and Papworth Hospitals, Cambridge, United Kingdom * To whom correspondence should be addressed. E-mail: richard.trembath{at}genetics.kcl.ac.uk.
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. Objective: To identify mutations in untranslated sequence regulating BMPR2 transcription. Methods: DNA upstream of the coding region was analyzed by direct sequencing in 16 families. RT-PCR analysis and RACE of normal human lung RNA was 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, 1148bp 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 nonsensemediated 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 1 characterize the BMPR2 promoter and the importance of analyzing non-coding regions in cases with pulmonary arterial hypertension that are negative for mutations within the coding region and intron-exon junctions. Key words: Pulmonary Hypertension, BMPR2, 5'-UTR mutation, Kozak Sequence, Cryptic Translation Initiation Site
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