A novel channelopathy in pulmonary arterial hypertension

Abstract

Background: Pulmonary arterial hypertension is a devastating disease with high mortality. Familial cases of pulmonary arterial hypertension are usually characterized by autosomal dominant transmission with reduced penetrance, and some familial cases have unknown genetic causes.

Methods: We studied a family in which multiple members had pulmonary arterial hypertension without identifiable mutations in any of the genes known to be associated with the disease, including BMPR2, ALK1, ENG, SMAD9, and CAV1. Three family members were studied with whole-exome sequencing. Additional patients with familial or idiopathic pulmonary arterial hypertension were screened for the mutations in the gene that was identified on whole-exome sequencing. All variants were expressed in COS-7 cells, and channel function was studied by means of patch-clamp analysis.

Results: We identified a novel heterozygous missense variant c.608 G→A (G203D) in KCNK3 (the gene encoding potassium channel subfamily K, member 3) as a disease-causing candidate gene in the family. Five additional heterozygous missense variants in KCNK3 were independently identified in 92 unrelated patients with familial pulmonary arterial hypertension and 230 patients with idiopathic pulmonary arterial hypertension. We used in silico bioinformatic tools to predict that all six novel variants would be damaging. Electrophysiological studies of the channel indicated that all these missense mutations resulted in loss of function, and the reduction in the potassium-channel current was remedied by the application of the phospholipase inhibitor ONO-RS-082.

Conclusions: Our study identified the association of a novel gene, KCNK3, with familial and idiopathic pulmonary arterial hypertension. Mutations in this gene produced reduced potassium-channel current, which was successfully remedied by pharmacologic manipulation. (Funded by the National Institutes of Health.)

Figure 1. Pedigrees of Families with Familial Pulmonary Arterial Hypertension

Segregation of KCNK3 mutations c.608 G→A (G203D) in the index family (Family 1) (Panel A), c.289G→A (G97R) in Family 2 (Panel B), and c.661G→C (V221L) in Family 3 (Panel C) is indicated. The sequence shown in Panel C is the sequence of the complementary strand. Arrows show the family members whose DNA was analyzed with the use of whole-exome sequencing. Genotypes of family members are shown under each symbol. NM/NM denotes nonmutated homozygote, and NM/M heterozygote with one copy of the KCNK3 mutation. The current age or the age at death, as well as the age at diagnosis (Dx), where applicable, is provided for each family member. Black squares denote affected males, black circles affected females, white squares unaffected males, and white circles unaffected females; slashes indicate deceased family members.

Figure 2. Topologic Analysis of the Human KCNK3 (hKCNK3) Channel and Sequence Alignment with Other Members of the KCNK Channel Family

Panel A shows a topologic analysis of the hKCNK3 channel, indicating the positions of the mutations that were identified in this study. Panel B shows the alignment of the amino acid sequences of KCNK3 with three other acid-sensitive members of the KCNK channel family and KCNK1. The positions of the mutations are indicated by the various colors. COOH denotes C-terminal.

Figure 3(facing page). Functional Consequences of hKCNK3 Mutations

Whole-cell patch-clamp procedures were used to measure expressed currents and their response to pH and pharmacologic agents. Panel A shows the representative pH dependence of the current in the nonmutant (NM) hKCNK3 channel. Dashed lines indicate current density at a pH of 7.4. For each point, 4 to 14 cells were studied. The solid curve shows the best fit for the dose–response values. Currents were measured at +60 mV and normalized to current measured at a pH of 10.4. Panel B shows current traces at a pH of 7.4 for the nonmutant hKCNK3 channel and the T8K, G97R, E182K, Y192C, G203D, and V221 mutants. Current density is measured as picoamperes per picofarad (pA/pF). For all current traces, the vertical scale is 10 pA/pF and the horizontal scale is 20 mV. The inset shows the ramp protocol (i.e., voltage steps or ramps). The vertical dashed lines represent the current at 60 mV. Panel C shows a summary of results illustrated in Panel B, according to mutation. Panel D shows a comparison between the homozygous nonmutant hKCNK3 channel and heterozygous channels incorporating the Y192C, G203D, or V221L mutant at a pH of 7.4. For every point, 7 to 25 cells were studied. In Panels C and D, data are shown as means; T bars indicate standard errors. Asterisks denote P<0.05 for the comparison between the nonmutant hKCNK3 channel and each mutant.

Figure 4(facing page). Pharmacologic Recovery of Mutant hKCNK3 Channels

The phospholipase A2 inhibitor ONO-RS-082 has been shown to activate nonmutant (NM) hKCNK3 channels. Panel A shows the representative recordings before the application of ONO-RS-082 (gray lines) and after the application (black lines) in nonmutant and mutant hKCNK3 channels. For all current traces, the vertical scale is 8 pA/pF. Panel B shows the time course of drug application before (gray squares), during (black circles), and after (gray triangles) application. Arrows indicate the current-density level before drug application. Panel C shows a summary of results of drug effects on nonmutant and mutant hKCNK3 channels. Light blue bars represent the current before drug application; dark blue bars represent the maximal drug response. Data are shown as means; T bars indicate standard errors. Asterisks indicate P<0.05 for the comparison between the current before drug application and the maximal drug response, as calculated by means of the paired Student’s t-test.