High frequency of BMPR2 exonic deletions/duplications in familial pulmonary arterial hypertension

Abstract

Rationale: Previous studies have shown that approximately 55% of patients with familial pulmonary arterial hypertension (FPAH) 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 families with PAH and 14 patients with idiopathic PAH (IPAH) was subjected to reverse transcriptase-polymerase chain reaction (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%) patients with FPAH had abnormally sized RT-PCR products. Four of the 11 patients were found to have splice-site mutations resulting in aberrant splicing, and exonic deletions/duplications were detected in the remaining seven patients. MLPA identified three deletions/duplications that were not detectable by RT-PCR. Coding sequence point mutations were identified in 11 of 30 (37%) patients. Mutations were identified in 21 of 30 (70%) patients with FPAH, with 10 of 21 mutations (48%) being exonic deletions/duplications. Two of 14 (14%) patients with IPAH exhibited BMPR2 point mutations, whereas none showed exonic deletions/duplications.

Conclusions: Our study indicates that BMPR2 exonic deletions/duplications in patients with FPAH account for a significant proportion of mutations (48%) that until now have not been screened for. Because the complementary approach used in this study is rapid and cost effective, screening for BMPR2 deletions/duplications by MLPA and real-time PCR should accompany direct sequencing in all PAH testing.

Figure 1.

BMPR2 cDNA sequence and multiplex ligation-dependent amplification (MLPA) analysis of Family 12 showing deletion of exon 2. (A) The absence of exon 2 in the mutant allele with splicing of exon 1 directly to exon 3 detected by sequencing of the gel-purified mutant reverse transcriptase–polymerase chain reaction product. (B) MLPA tracing for an affected individual from Family 12 (top tracing) demonstrating a reduction in the exon 2 peak (asterisk) as compared with a normal control (bottom tracing). This approximate 50% reduction in peak intensity is indicative of a heterozygous deletion of exon 2 at the genomic DNA level.

Figure 2.

Splicing mutations in Family 68 and Family 76 resulting in partial deletions of exon 2 due to use of cryptic donor sites. (A) Heterozygous G-to-T substitution at the 6th position of intron 2 (247+6T > G) detected in the genomic DNA of an affected individual of Family 68. (B) Heterozygous deletion of the 2nd base of intron 2 (247+1delC) detected in Family 76. (C) The sequence of exon 2 (uppercase) and a portion of the sequence of IVS2 (lowercase). The normal IVS2 splice donor site (gc) is indicated by the green arrow. The positions of the IVS2 donor splice-site mutations are boxed for Families 68 (+6T > G) and 76 (+2delC). The exon 2 cryptic donor splice sites used as a result of the splice donor mutations are marked by red arrows and blue arrows. There is a cryptic donor site with a score of 0.85 (blue arrow) that seems to be used over the mutated donor splice site. A second cryptic donor site upstream of the first with a score of 0.65 (red arrow) also seems to be used, though to a lesser extent. Cryptic GT splice sites were scored using NNSPLICE (www.fruitfly.org/seq_tools/splice.html).

Figure 3.

BMPR2 cDNA (A) and genomic DNA sequence (B) of Family 67. As seen in A, cDNA sequencing identified a mutant transcript lacking exons 8 and 9. Genomic DNA sequencing (B) revealed a heterozygous G > T substitution at the first base of intron 8 (1128+1G > T), causing the aberrant splicing.

Figure 4.

MLPA analysis of Family 57 indicating a heterozygous deletion of BMPR2 exons 2–13. The MLPA tracing of an affected individual from Family 57 (top) shows a reduction in peak height/area of all exons except exon 1 as compared with the tracing from a normal individual (bottom).

Figure 5.

Distribution of BMPR2 mutations identified in 30 families with FPAH. The types of mutations identified are represented by colored circles within the outer circle representing the families analyzed. The shaded circle represents the 12 families previously analyzed. The area outside of the shaded circle represents the 18 families not previously analyzed. Numbers in parentheses indicate the total number of patients in each labeled category. Numbers in the circles show the distribution within each mutation type.

Figure 6.

Recommended workflow for identification of BMPR2 mutations in patients with PAH. Steps using genomic DNA are shown in blue, and those requiring RNA are indicated in red. Labeled circles indicate actions within the workflow. Text accompanying arrows indicates rationale for flow from one step to the next. Possible outcomes from actions within the labeled circles are indicated in text at the ends of arrows.