Mutations in bone morphogenetic protein receptor type 2 (BMPR2) cause familial pulmonary arterial hypertension (FPAH), but the penetrance is reduced and females are significantly overrepresented. In addition, gene expression data implicating the oestrogen-metabolising enzyme CYP1B1 suggests a detrimental role of oestrogens or oestrogen metabolites. We examined genetic and metabolic markers of altered oestrogen metabolism in subjects with a BMPR2 mutation. Genotypes for CYP1B1 Asn453Ser (N453S) were determined for 140 BMPR2 mutation carriers (86 females and 54 males). Nested from those subjects, a case–control study of urinary oestrogen metabolite levels (2-hydroxyoestrogen (2-OHE) and 16α-hydroxyoestrone (16α-OHE1)) was conducted in females (five affected mutation carriers versus six unaffected mutation carriers). Among females, there was four-fold higher penetrance among subjects homozygous for the wild-type genotype (N/N) than those with N/S or S/S genotypes (p=0.005). Consistent with this finding, the 2-OHE/16α-OHE1 ratio was 2.3-fold lower in affected mutation carriers compared to unaffected mutation carriers (p=0.006). Our findings suggest that variations in oestrogens and oestrogen metabolism modify FPAH risk. Further investigation of the role of oestrogens in this disease with profound sex bias may yield new insights and, perhaps, therapeutic interventions.

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.

Familial pulmonary arterial hypertension (FPAH) is a progressive, fatal disease caused by mutations in the bone morphogenetic protein receptor type 2 gene (BMPR2). FPAH is inherited as an autosomal dominant trait and shows incomplete penetrance in that many with BMPR2 mutations do not develop FPAH suggesting a role for, as yet unidentified, modifier genes in disease penetrance. We hypothesized that variable level of expression of the wild type (WT) BMPR2 allele could act as a modifier and influence penetrance of FPAH. WT BMPR2 levels were determined by real-time PCR analysis in lymphoblastoid (LB) cell lines derived from normal controls and individuals with FPAH. The FPAH kindreds analyzed carried mutations that result in the activation of nonsense mediated decay (NMD) pathway, which leads to the degradation of the mutant RNA thus ensuring that only the WT BMPR2 transcripts will be detected in the real-time assay. Our data show that WT and mutant BMPR2 levels can be reproducibly measured in patient derived LB cell lines and that unaffected mutation carrier derived LB cell lines have higher levels of WT BMPR2 transcripts than FPAH patient derived LB cell lines (p≤0.005). Our findings suggest that the levels of expression of WT BMPR2 allele transcripts is important in the pathogenesis of FPAH caused by NMD + mutations. Furthermore, our study illustrates a novel application of lymphoblastoid cell lines in the study of PAH, especially important because the affected site, i.e. lung is not available for unaffected mutation carriers.