Whole-exome sequencing reveals TopBP1 as a novel gene in idiopathic pulmonary arterial hypertension

Abstract

Rationale: Idiopathic pulmonary arterial hypertension (IPAH) is a life-threatening disorder characterized by progressive loss of pulmonary microvessels. Although mutations in the bone morphogenetic receptor 2 (BMPR2) are found in 80% of heritable and ∼15% of patients with IPAH, their low penetrance (∼20%) suggests that other unidentified genetic modifiers are required for manifestation of the disease phenotype. Use of whole-exome sequencing (WES) has recently led to the discovery of novel susceptibility genes in heritable PAH, but whether WES can also accelerate gene discovery in IPAH remains unknown.

Objectives: To determine whether WES can help identify novel gene modifiers in patients with IPAH.

Methods: Exome capture and sequencing was performed on genomic DNA isolated from 12 unrelated patients with IPAH lacking BMPR2 mutations. Observed genetic variants were prioritized according to their pathogenic potential using ANNOVAR.

Measurements and main results: A total of nine genes were identified as high-priority candidates. Our top hit was topoisomerase DNA binding II binding protein 1 (TopBP1), a gene involved in the response to DNA damage and replication stress. We found that TopBP1 expression was reduced in vascular lesions and pulmonary endothelial cells isolated from patients with IPAH. Although TopBP1 deficiency made endothelial cells susceptible to DNA damage and apoptosis in response to hydroxyurea, its restoration resulted in less DNA damage and improved cell survival.

Conclusions: WES led to the discovery of TopBP1, a gene whose deficiency may increase susceptibility to small vessel loss in IPAH. We predict that use of WES will help identify gene modifiers that influence an individual's risk of developing IPAH.

Figure 1.

Proposed role of top three candidate genes identified by whole-exome sequencing in the pathogenesis of idiopathic pulmonary arterial hypertension. ECM = extracellular matrix; SMC = smooth muscle cell.

Figure 2.

Topoisomerase DNA binding II binding protein 1 (TopBP1) variants identified in patients with idiopathic pulmonary arterial hypertension (IPAH) are located within conserved protein domains. (A) Primary sequence of TopBP1 showing location of three identified variants. (B) Predicted and actual allele frequency of three TopBP1 SNVs in whole-exome sequencing cohort with the predicted amino acid (AA) switch. (C) Predicted functional impact of the three observed TopBP1 variant using MutationTaster, SIFT, LRT, Polyphen2 (PolyP2), and GERP. (D) Variants target conserved amino acids as seen when human TopBP1 protein sequence is aligned with that of other related organisms using MUSCLE. SNV = single-nucleotide variants.

Figure 3.

Topoisomerase DNA binding II binding protein 1 (TopBP1) nuclear abundance is reduced in idiopathic pulmonary arterial hypertension (IPAH). (A) Representative immunohistochemistry images of lung sections obtained from healthy donor (top panels) and patients with IPAH (bottom panels). Scale bar = 25 μm. (B) Quantitative polymerase chain reaction of TopBP1 mRNA expression in healthy donor and IPAH pulmonary microvascular endothelial cells (PMVECs). Bars represent mean SEM from experiments involving five patients per group. ***P < 0.0001, unpaired t test. (C) Representative nuclear extraction studies demonstrating TopBP1 expression in PMVECs from PMVECs purified from five healthy donors and patients with IPAH. Distribution of all three TopBP1 SNVs in IPAH PMVECs can be found in Table E4. Bars represent mean SEM from experiments involving five patients per group. ***P < 0.0001, unpaired t test. SNV = single-nucleotide variants.

Figure 4.

Idiopathic pulmonary arterial hypertension (IPAH) pulmonary microvascular endothelial cells (PMVECs) demonstrate evidence of increased DNA damage and apoptosis in response to hydroxyurea (HU). (A) Representative immunofluorescence studies demonstrating nuclear topoisomerase DNA binding II binding protein 1 (TopBP1) (green) and p-H2AX (red) in PMVECs from healthy donors (upper six panels) and patients with IPAH (lower six panels) at baseline and after exposure to HU (2 mM) for 24 hours. Scale bar = 25 μm. (B) Caspase 3/7 activity assays of healthy donor and IPAH PMVECs at baseline and after HU exposure for 24 hours. Bars represent mean SEM from experiments involving five patients per group. LU = luminescence intensity. *P < 0.05 versus healthy donor −HU, one-way analysis of variance with Bonferroni post-test unpaired t test.

Figure 5.

Impact of candidate topoisomerase DNA binding II binding protein 1 (TopBP1) SNVs on susceptibility to hydroxyurea-mediated replication stress in healthy pulmonary microvascular endothelial cells. Representative immunofluorescence studies demonstrating nuclear TopBP1 (green) and p-H2AX (red) in healthy pulmonary microvascular endothelial cells transfected with either wild-type (WT) or mutant constructs containing each of the three TopBP1 candidate SNVs (rs55633281, rs17301766, and rs10935070) after exposure to hydroxyurea (2 mM) for 24 hours. Scale bar = 10 μm. SNV = single-nucleotide variants.

Figure 6.

Topoisomerase DNA binding II binding protein 1 (TopBP1) siRNA knockdown increases susceptibility to DNA damage and apoptosis in healthy pulmonary microvascular endothelial cells (PMVECs). (A) Western blot showing TopBP1 expression in PMVECs transfected with nontargeting (NT) or TopBP1-specific siRNA. Densitometry is measured relative to α-tubulin as a loading control. ***P < 0.0001, unpaired t test. (B) Representative immunofluorescence studies demonstrating nuclear TopBP1 (green) and p-H2AX (red) in PMVECs transfected with NT (top) or TopBP1 siRNA (bottom) at baseline after 24 hours. Scale bar = 10 μm. (C) Relative caspase 3/7 activity assays of NT and TopBP1 siRNA transfected PMVECs at baseline for 24 hours. Bars represent mean SEM from experiments performed in triplicate. *P < 0.05, unpaired t test. (D) Matrigel tube formation assay comparing NT and TopBP1 siRNA transfected PMVECs. Tube number was quantified 6 hours after seeding the cells. **P < 0.001, unpaired t test. Scale bar = 150 μm.

Figure 7.

Restoration of topoisomerase DNA binding II binding protein 1 (TopBP1) levels protect idiopathic pulmonary arterial hypertension (IPAH) pulmonary microvascular endothelial cells (PMVECs) against hydroxyurea-induced apoptosis and improve tube formation. (A) Western blot showing TopBP1 expression in IPAH PMVECs transfected with wild-type (WT) TopBP1 expression construct (0, 1, and 2 μg). Densitometry is measured relative to α-tubulin as a loading control. ***P < 0.0001 versus healthy donor. One-way analysis of variance (ANOVA) with Bonferroni post-test, n = 3. (B) Caspase 3/7 activity assays of IPAH PMVECs transfected with either empty vector or WT TopBP1 expressing construct following hydroxyurea exposure for 24 hours. Bars represent mean SEM from experiments performed in triplicate. ***P < 0.0001 versus healthy donor, one-way ANOVA with Bonferroni post-test. (C) Matrigel tube formation assay comparing healthy donor, IPAH+vector, and IPAH+WT TopBP1 PMVECs. Tube number was quantified six hours after seeding the cells. *P < 0.05 versus healthy donor, ***P < 0.0001 versus healthy donor, one-way ANOVA with Bonferroni post-test. Scale bar = 150 μm.

Figure 8.

Proposed model. Topoisomerase DNA binding II binding protein 1 (TopBP1) helps protect pulmonary microvascular endothelial cells against injury and promotes angiogenesis (top). Reduced TopBP1 may contribute to idiopathic pulmonary arterial hypertension (IPAH) by increasing susceptibility to DNA damage resulting in loss of pulmonary microvascular endothelial cells and impaired angiogenesis (bottom).