Systemic sclerosis (SSc) is a life-threatening fibrotic connective tissue disorder that affects the skin and different internal organs 1. The 10-year survival of SSc patients reaches only 63%, with pulmonary involvement the leading cause of death 2. SSc is a complex disorder in which the environmental triggers and genetic susceptibility factors co-act in the development and maintenance of the disease 3. As a clearer picture of the genetic component of this disease is being revealed, interest in genetic markers for specific clinical features, especially lung involvement, is increasing. An SSc phenotype-restricted genome-wide analysis was carried out recently 4. Remarkably, two of the four new SSc genetic-susceptibility markers identified in the previously mentioned study might play a relevant role in the SSc-related fibrotic process, SOX5 and NOTCH4 4. However, no such strategy has yet been considered for pulmonary involvement, and only some studies have reported significant genetic association with SSc-related lung involvement 56789. Furthermore, only the association of CD226 with pulmonary fibrosis has been independently replicated 10. Wipff et al. 11 recently described the association of potassium voltage-gated channel shaker-related subfamily member 5 (KCNA5) with SSc-related pulmonary arterial hypertension (PAH).

Potassium voltage-gated channels (Kv channels) are homo- or heterotetramers of structural α-subunits and regulatory β-subunits, which control potassium (K) flux. K balance is known to regulate the apoptotic cell shrinkage, a main event in the apoptotic process 12. Specifically, KCNA5 participates in pulmonary artery smooth muscle cell (PASMC) apoptosis control 13. It has been reported that overexpression of the KCNA5 gene induces accelerated K efflux and increases caspase-3 proteolytic activity, promoting apoptosis 13.

KCNA5 is also involved in membrane-potential maintenance and vascular-tone regulation 14. Hypoxic conditions produce a specific inhibition of KCNA5 in the PASMCs, causing pulmonary vasoconstriction 151617. Moreover, it has been reported that primary pulmonary hypertension patients have an intrinsic reduced level of KCNA5 mRNA in their PASMCs, and this characteristic might play an important role in the pathogenesis of the disease 18.

The role of KCNA5 genetic polymorphisms in pulmonary disease also was explored. Remillard et al. 19 analyzed the influence of KCNA5 genetic variants in IPAH, and showed that different single-nucleotide polymorphisms (SNPs) were related to abnormal function or drug responsiveness. Recently, the importance of KCNA5 variants in SSc-related PAH was analyzed 11. In the previously mentioned study, the association of KCNA5 with SSc and specifically with PAH-positive (PAH⁺) patients was described 11. Moreover, the rs10744676 polymorphism was reported as the variant underlying the observed association 11.

In light of the previous evidence, the main goal of this report is to replicate the association of KCNA5 rs10744676 polymorphism with SSc and SSc-related PAH in an independent European population of Caucasian ancestry.

The power of our replication study in each stratified analysis is summarized in Table 1. We emphasize that the size of our pooled PAH⁺ patient subgroup represents the largest SSc-related PAH⁺ cohort analyzed to date (n = 179). The power of the study of this clinical feature in our population to detect an association equivalent to that previously reported by Wipff et al. is 95%, at the 5% significance level.

Table 2 shows the results of the comparison of the complete set of SSc and each of the previously defined subgroups with the healthy control group. As observed in the table, the analyzed polymorphism showed no significant association with either the disease or any of the subsets, not even the PAH-positive group. Furthermore, no significant association was observed in the individual population analyses, either in the whole disease comparison or in the different subphenotypes (see Additional file 1, Table S1). The different cohorts showed a high interpopulation combinability, as can be observed in the Breslow-Day test results (Table 2).

The minor allele in the Spanish and Italian control populations included in this study was less frequent than that in the pooled population described by the Wipff et al. (rs10744676*C Frequency_Spain = 10.48%; rs10744676*C Frequency_Italy = 10.00%; rs10744676*C Frequency_{pooled_Wipff} = 14.7%). However, these frequencies are in concordance with those reported for the HapMap CEU population (MAF_{HapMap_CEU} = 10.00%). Moreover, MAF differences have been reported in different European populations (MAF_{1000Genomes_CEU} = 17.64%, MAF_{1000Genomes_GBR} = 15.73%, MAF_{1000Genomes_FIN} = 13.98%, MAF_{1000Genomes_TSI} = 8.16%).

In addition, the analysis of PAH⁺ versus PAH^- patients revealed no phenotype-restricted association in this subgroup (P_MH = 0.59; OR, 0.91; 95% CI, 0.64 to 1.28).

Despite the previous findings of an association of the rs10744676 KCNA5 genetic variant with PAH⁺ SSc patients, our data do not corroborate the reported protective effect of the minor allele of this polymorphism on SSc-related PAH.

It is worth mentioning that the prevalence of right-heart catheterization-confirmed PAH in our pooled population (7.64%) is consistent with previous reports 11. However, SSc is a chronic progressive disease, and some patients classified as PAH^- might develop PAH in the future. Therefore, we consider this issue a limitation of our study and similar reports. In this context, we point out that the mean disease duration for the patient group included in this study (mean disease duration, 13.9 ± 14.4 years, as reported in Bossini-Castillo et al. 23) was higher than that in Wipff et al. 11 (11.9 ± 9.4 years in the discovery cohort and 13.9 ± 14.4 years in the replication), which might influence the observed differences.

The PAH⁺ cohort studied in this report reached a 95% estimated statistical power to detect an association equivalent to that observed by Wipff et al. within this subgroup (OR, 0.47, in the comparison between PAH⁺ SSc patients and controls of the discovery phase and two replication steps 11). Hence, we consider that the lack of replication observed in our study is unlikely to be caused by a type II error (false negative) because of a reduced statistical power. However, autoimmune-associated variants usually show modest degrees of risk, especially in the case of non-HLA loci 24. Wipff et al. reported an OR for KCNA5 rs10744676 polymorphism that was remarkably more protective than the SSc genetic-association standards for non-HLA loci (that is, modest ORs between 0.70 and 1). Thus, we reflect that if the influence of rs10744676 in the SSc-patient genetic predisposition to PAH is modest, the statistical power to detect a possible association in the PAH⁺ cohort analyzed in the present article might be insufficient, and a possible modest effect of KCNA5 rs10744676 might be overlooked (Table 1).

In addition, it is established that PAH is more frequent in ACA⁺ patients 1, and despite the suggestive association with PAH⁺ patients, no significant association with the ACA⁺ subphenotype was identified in the previous SSc study 11 or in this report. This fact is also consistent with a lack of association of the selected polymorphism with PAH development.

Although rs10744676 might have a functional role in KCNA5 expression because of its location in its putative promoter, no evidence confirms a functional role for this variant. Therefore, we speculate that the previously mentioned association with SSc could be the consequence of a tagged causal variant yet to be discovered. Moreover, because PAH onset time has not been considered for the analyses, it might act as a confounding factor in the discrepant results.

In summary, our data do not support an important role of rs10744676 as a PAH genetic marker in SSc patients.

ACA: anti-centromere autoantibodies; ATA: anti-topoisomerase antibodies; BD test: Breslow-Day test; CD226: cluster of differentiation 226; CEU: Utah residents with ancestry from northern and western Europe; dcSSc: diffuse cutaneous systemic sclerosis; hRCT: High-resolution computed tomography; HWE: Hardy-Weinberg equilibrium; K: potassium; KCNA5: potassium voltage-gated channel shaker-related subfamily member 5; Kv channels: potassium voltage-gated channels; lcSSc: limited cutaneous systemic sclerosis; MAF: minor allele frequency; NOTCH4: Notch (Drosophila) homologue 4; OR: odds ratio; PAH: pulmonary arterial hypertension; PASMC: pulmonary artery smooth muscle cell; PCR: polymerase chain reaction; SNP: single-nucleotide polymorphism; SOX5: SRY (sex-determining region Y)-box 5; SSc: systemic sclerosis.

The authors declare that they have no competing interests.

LBC contributed to the analysis and interpretation of data and to the drafting of the manuscript. CPS, LB, and JB participated in the acquisition of data and the drafting of the manuscript. CF, TRDJR, and JM contributed to the conception and design of the study and critically revised the manuscript. MCV, JLC, PC, LRR, RGP, MAGG, IC, MTC, CT, EVR, MVE, AJS, RH, CL, JMvL, PS, AH, JW, CD, and the Spanish Scleroderma Group were involved in the acquisition of data and the revision of the manuscript. All authors read and approved the final manuscript.