Published ahead of print on May 25, 2006, doi:10.1164/rccm.200602-165OC Am. J. Respir. Crit. Care Med., Volume 174, Number 5, September 2006, 590-598 A more recent version of this article appeared on September 1, 2006
Submitted on February 3, 2006 High Frequency of BMPR2 Exonic Deletions/Duplications in Familial Pulmonary Arterial HypertensionJoy D Cogan1,1 Division of Medical Genetics, Vanderbilt University Medical Center, Nashville, TN, USA, 2 Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA, 3 Division of Pulmonary Biology, Vanderbilt University Medical Center, Nashville, TN, USA, 4 Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA * To whom correspondence should be addressed. E-mail: bill.nichols{at}cchmc.org.
Rationale: Previous studies have shown that ~55% of familial pulmonary arterial hypertension (PAH) patients have BMPR2 coding sequence mutations. However, direct sequencing does not detect other types of heterozygous mutations such as exonic deletions/duplications. Objective: To estimate the frequency of BMPR2 exonic deletions/duplications in FPAH. Methods: BMPR2 mRNA from lymphoblastoid cell lines of 30 PAH families and 14 idiopathic PAH patients was subjected to RT-PCR and sequencing. Sequencing of genomic DNA was used to identify point mutations in splice donor/acceptor sites. Multiplex Ligation-dependent Probe Amplification (MLPA) was used to detect exonic deletions/duplications with verification by real-time PCR. Measurements and Main Results: Eleven (37%) FPAH patients had abnormally-sized RT-PCR products. Four of the 11 were found to have splice site mutations resulting in aberrant splicing, and exonic deletions/duplications were detected in the remaining seven. Additionally, MLPA identified 3 deletions/duplications which were not detectable by RT-PCR. Coding sequence point mutations were identified in 11 of 30 (37%). Overall, mutations were identified in 21 of 30 (70%) FPAH patients with 10 of 21 mutations(48%) being exonic deletions/duplications. Additionally, 2 of 14 (14%) IPAH patients exhibited BMPR2 point mutations while none showed exonic deletions/duplications. Conclusions: Our study indicates that BMPR2 exonic deletions/duplications in FPAH patients account for a significant proportion of mutations (48%) which to now have not been screened for. Since the complementary approach used in this study is both rapid and cost effective, screening for BMPR2 deletions/duplications by MLPA and real-time PCR should accompany direct sequencing in all PAH testing. Key words: genetics, MLPA, dosage
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