BMPR2 haploinsufficiency as the inherited molecular mechanism for primary pulmonary hypertension

Abstract

Primary pulmonary hypertension (PPH) is a potentially lethal disorder, because the elevation of the pulmonary arterial pressure may result in right-heart failure. Histologically, the disorder is characterized by proliferation of pulmonary-artery smooth muscle and endothelial cells, by intimal hyperplasia, and by in situ thrombus formation. Heterozygous mutations within the bone morphogenetic protein type II receptor (BMPR-II) gene (BMPR2), of the transforming growth factor beta (TGF-beta) cell-signaling superfamily, have been identified in familial and sporadic cases of PPH. We report the molecular spectrum of BMPR2 mutations in 47 additional families with PPH and in three patients with sporadic PPH. Among the cohort of patients, we have identified 22 novel mutations, including 4 partial deletions, distributed throughout the BMPR2 gene. The majority (58%) of mutations are predicted to lead to a premature termination codon. We have also investigated the functional impact and genotype-phenotype relationships, to elucidate the mechanisms contributing to pathogenesis of this important vascular disease. In vitro expression analysis demonstrated loss of BMPR-II function for a number of the identified mutations. These data support the suggestion that haploinsufficiency represents the common molecular mechanism in PPH. Marked variability of the age at onset of disease was observed both within and between families. Taken together, these studies illustrate the considerable heterogeneity of BMPR2 mutations that cause PPH, and they strongly suggest that additional factors, genetic and/or environmental, may be required for the development of the clinical phenotype.

Figures

Figure 1

BMPR2 cDNA structure and location of all reported PPH mutations. Exons are numbered 1–13, and their boundaries are indicated by the solid black lines segmenting the cDNA schematic. Extracellular/ligand binding, transmembrane, kinase, and cytoplasmic tail domains of BMPR-II are defined as indicated. Mutations shown above the BMPR2 cDNA are newly reported in this study, whereas those shown below have been reported elsewhere (Deng et al. ;The International PPH Consortium et al. ; Thomson et al. 2000). Underlined mutations have been reported in patients with sporadic PPH (Thomson et al. 2000); asterisks (*) indicate mutations that were independently reported (Deng et al. 2000b). Frameshift mutations (fs) are denoted by the one-letter amino acid abbreviation and amino acid–position number at which the mutation occurs, followed by the number of extra amino acids before the next stop codon—for example, L168fs (+11).

Figure 2

Cosegregation and sequence analysis of BMPR2 mutations in two kindreds with PPH. Black symbols denote affected individuals, and white symbols denote unaffected individuals. Nucleotides are numbered according to the cDNA sequence, with the adenosine of the initiation codon assigned +1. Family US13 (top) carries a C→T substitution at nucleotide 994, as shown in the chromatogram (top, right). Segregation of the mutations was tracked through the family by TaqI digestion of exon 8 PCR products shown on the agarose gel below the pedigree. Gene carriers are detected by presence of a 288-bp band due to loss of a TaqI site. Gel lanes correspond to the individuals numbered in the pedigree. Family Nor01 (bottom) carries a T→A substitution at nucleotide 367, as shown in the chromatogram (bottom, right). MseI digestion of exon 3 PCR products was used to track inheritance of the mutations through the family, as shown on the agarose gel below the pedigree. The mutation is evidenced by gain of an MseI site, resulting in the generation of 227- and 87-bp fragments. Individuals are identified by numbers corresponding to those on gels.

Figure 3

Detection of heterozygous deletions of the BMPR2 gene. A, Fluorescent-dosage PCR of an affected individual in family UK01. The patient demonstrates a reduction of 50% in peak intensity for exon 1 of the BMPR2 gene, compared with both exon 4 of the SURF6 gene and exon 12 of the BMPR2 gene. This indicates a heterozygous deletion encompassing at least exon 1 of the BMPR2 gene. Also shown is an electropherogram from a normal control subject, in whom peaks for all three PCR products are approximately equal. B, Sequence analysis of BMPR2 lung mRNA RT-PCR products of UK sporadic 14 subject. An RNA product with nucleotide 50 contiguous to nucleotide 815 was detected by sequence analysis, as seen in the chromatogram. The normal-sized RT-PCR product was also identified, indicating that this patient is heterozygous for a deletion of nucleotides 51–814.

Figure 4

Sequence analysis of RT-PCR products for a heterozygous BMPR2 intron 7 splice-site mutant. The splice-site mutation leads to skipping of exon 7 in the mutant. Nucleotides are listed above the sequencing peaks and indicate the end of exon 6, joined with the beginning of exon 7 or exon 8 in the normal or mutant, respectively. The arrow indicates the splice junction between exon 6 and either exon 7 or 8. Exon 7 sequence can be seen as the higher peaks in the right half of the chromatogram, with exon 8 represented by the lower peaks.

Figure 5

In vitro expression analysis of wild-type and mutant BMPR2 in NMuMG cells. The control transfection using only the SBE reporter plasmid in the presence (+) or absence (−) of the ligand BMP4 measures activation of endogenous type II receptor function. Cotransfection of the SBE reporter with either wild-type or mutant BMPR2 constructs determines functional activity of the exogenous BMPR-II. Cotransfection of wild-type BMPR2 and the SBE reporter yielded a twofold increase in reporter activity. In contrast, cotransfection of mutant BMPR2 yielded functional activity equivalent to that of the endogenous receptor, indicating loss of function for both mutants.