Gene expression in BMPR2 mutation carriers with and without evidence of pulmonary arterial hypertension suggests pathways relevant to disease penetrance

Abstract

Background: While BMPR2 mutation strongly predisposes to pulmonary arterial hypertension (PAH), only 20% of mutation carriers develop clinical disease. This finding suggests that modifier genes contribute to FPAH clinical expression. Since modifiers are likely to be common alleles, this problem is not tractable by traditional genetic approaches. Furthermore, examination of gene expression is complicated by confounding effects attributable to drugs and the disease process itself.

Methods: To resolve these problems, B-cells were isolated, EBV-immortalized, and cultured from familial PAH patients with BMPR2 mutations, mutation positive but disease-free family members, and family members without mutation. This allows examination of differences in gene expression without drug or disease-related effects. These differences were assayed by Affymetrix array, with follow-up by quantitative RT-PCR and additional statistical analyses.

Results: By gene array, we found consistent alterations in multiple pathways with known relationship to PAH, including actin organization, immune function, calcium balance, growth, and apoptosis. Selected genes were verified by quantitative RT-PCR using a larger sample set. One of these, CYP1B1, had tenfold lower expression than control groups in female but not male PAH patients. Analysis of overrepresented gene ontology groups suggests that risk of disease correlates with alterations in pathways more strongly than with any specific gene within those pathways.

Conclusion: Disease status in BMPR2 mutation carriers was correlated with alterations in proliferation, GTP signaling, and stress response pathway expression. The estrogen metabolizing gene CYP1B1 is a strong candidate as a modifier gene in female PAH patients.

Figure 1

Ontology grouping of genes differentially regulated in BMPR2 mutation carriers with pulmonary hypertension compared to BMPR2 mutation carriers without disease. Study design aimed to avoid disease and drug effects, meaning that these should represent predisposition to disease rather than disease effect.

Figure 2

(A) Expression of selected genes in a larger sample set trends in the same direction as in arrays by quantitative RT-PCR, but both fold-changes and significance are reduced compared to the more limited sample set. The exception is CYP1B1, in which both fold change and significance are maintained. Expression is normalized to that of beta-actin and then to expression level in non-carriers; p-values are by Kruskal-Wallis. (B) Expression of CYP1B1 is lower in female, but not male, mutation carriers with PAH compared to unaffected carriers and non-carriers. n = number of individuals in category; * = p < .01 for sex-specific effect in disease by two-way ANOVA on log-transformed values.

Figure 3

GTP-related genes have large numbers of differentially regulated genes in all patient samples (registry # is outside of each circle). Increasing numbers of patients increases the significance of the ontology group, but produces decreasing numbers of genes changed in all patients. For instance, patient #723 and #266 have 25 GTP-related genes in common; this drops to 14 when overlap with patient #264 is added.