The FASEB Journal • Letters to the Editor
Letters to the Editor: Genetic polymorphism and soluble urokinase plasminogen activator receptor regulation Line Jee Hartmann Rasmussen, Thomas Huneck Haupt, Ove Andersen, and Jesper Eugen-Olsen1 Clinical Research Centre, Copenhagen University Hospital Hvidovre, Hvidovre, Denmark Portelli et al. (1) reported experimental data in The FASEB Journal indicating that a single nucleotide polymorphism (SNP; rs4253238) in the kallikrein gene (KLKB1) promoter was associated with kallikrein activity and serum levels of soluble urokinase plasminogen activator receptor (suPAR). Kallikrein was found to act as a posttranslational regulator of suPAR by proteolytic cleavage and thereby abolished its suggested biologic effect. The enzymatic activity of kallikrein was influenced by rs4253238; presence of the major T:T genotype was associated with increased kallikrein activity and lower suPAR levels, whereas the C:C genotype was associated with reduced kallikrein activity and higher suPAR levels. The study investigated populations of patients with asthma (n = 514) and chronic obstructive pulmonary disease (COPD; n = 219) and found that these patients had lower kallikrein activity and higher suPAR levels than healthy controls (n = 96). In the entire cohort (n = 803), the frequency distribution of the genetic variant was reported as follows: C:C (minor allele) n = 188 (23.4%); T:C n = 401 (49.9%); and T:T (major allele) n = 214 (26.7%). In the COPD cohort, the median suPAR level was higher in patients with the C:C genotype (6.6 ng/ml, n = 57) compared with patients with the heterogeneous T:C genotype (5.4 ng/ml, n = 107) or patients who were homozygous for the T:T genotype (4.4 ng/ml, n = 55, P , 0.001). The same direction of effect was observed in the patients with asthma and healthy controls (1). We found these data to be highly interesting, as our work and the work of others have shown that suPAR is a strong marker of disease severity and prognosis. suPAR is a very unspecific marker of inflammation and immune activation, and its prognostic value has been shown to be significant across a wide range of diseases. Even in healthy individuals, elevated suPAR levels are associated with increased risk of developing diseases, such as type 2 diabetes, cardiovascular disease (CVD), and cancer (2). It has been widely debated whether suPAR has a pathologic function and plays an active role in disease or whether it merely reflects inflammation and disease without promoting disease progression in itself. In the kidney disease focal segmental glomerulosclerosis, suPAR has been shown to play an active role by activating b3 integrin and causing podocyte injury. Abrogation of suPAR function, via lowering of serum suPAR concentration with plasmapheresis or inhibition of the suPAR-b3 integrin interaction, prevented development of the disease (3). However, no conclusive evidence on the possibly pathologic role of suPAR in other diseases has been provided so far.
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Therefore, we conducted an SNP genotyping study of the rs4253238 polymorphism on DNA from the Danish general population cohort Inter99 (4). The rs4253238 polymorphism was analyzed and associated with serum suPAR levels and clinical endpoints in this cohort. The endpoints investigated were ischemic heart disease (IHD), stroke, CVD (both IHD and stroke), and mortality. Genotyping was performed on DNA from 6541 participants in the Inter99 cohort, whereas data on suPAR at baseline were available for 5538 participants. Genotype data and suPAR data were linked, and participants with missing suPAR (n = 1003) or genotype results (n = 98) were excluded. The final population included 5440 individuals. The frequency distribution of the genetic variant was as follows: C:C (minor allele) n = 1360 (25.0%); T:C n = 2686 (49.4%); and T:T (major allele) n = 1394 (25.6%), which was comparable to the distribution reported by Portelli et al. (1); however, contrary to the findings by Portelli et al. (1), there was hardly any difference in suPAR levels between genotype groups, with median suPAR levels of 3.3 ng/ml for the C:C genotype and 3.4 ng/ml for the T:C and T:T genotype groups. The association between suPAR, genotypes, and endpoints was analyzed with Cox proportional hazards model adjusted for age and sex. There was no association between the three rs4253238 genotypes and IHD (P = 0.88), stroke (P = 0.34), or CVD (P = 0.56). Neither was there any association between rs4253238 and death (P = 0.88). In this cohort, 448 (8.2%) participants had asthma. Among these patients, the frequency distribution of the genetic variant for rs4253238 resembled the distribution for the entire cohort: C:C n = 109 (24.3%); T:C n = 214 (47.8%); and T:T n = 125 (27.9%). In addition, as for the entire cohort, there were no differences in median suPAR levels between genotype groups: C:C 3.4 ng/ml; T: C 3.5 ng/ml; and T:T 3.5 ng/ml. We were unable to reproduce the genotype findings by Portelli et al. (1) in this large cohort of 5440 individuals. Differences in suPAR between genotype groups were minimal, and the direction of the effect could not be recovered in this cohort. Therefore, our results cannot support their findings. Furthermore, we found no association between rs4253238 and CVD or mortality. Thus, we find no support for rs4253238 as a posttranslational 1
Correspondence: Clinical Research Centre, Copenhagen University Hospital Hvidovre, Hvidovre, Denmark. E-mail:
[email protected] doi: 10.1096/fj.15-1201LTE
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regulator of suPAR; however, whether suPAR plays a causal role via other unidentified mechanisms so far remains an unanswered question. REFERENCES 1. Portelli, M. A., Siedlinski, M., Stewart, C. E., Postma, D. S., Nieuwenhuis, M. A., Vonk, J. M., Nurnberg, P., Altmuller, J., Moffatt, M. F., Wardlaw, A. J., Parker, S. G., Connolly, M. J., Koppelman, G. H., and Sayers, I. (2014) Genome-wide protein QTL mapping identifies human plasma kallikrein as a post-translational regulator of serum uPAR levels. FASEB J. 28, 923–934 2. Eugen-Olsen, J., Andersen, O., Linneberg, A., Ladelund, S., Hansen, T. W., Langkilde, A., Petersen, J., Pielak, T., Møller, L. N., Jeppesen, J., Lyngbaek, S., Fenger, M., Olsen, M. H., Hildebrandt, P. R., Borch-Johnsen, K., Jørgensen, T., and Haugaard, S. B. (2010) Circulating soluble urokinase plasminogen activator receptor predicts cancer, cardiovascular disease, diabetes and mortality in the general population. J. Intern. Med. 268, 296–308 3. Wei, C., El Hindi, S., Li, J., Fornoni, A., Goes, N., Sageshima, J., Maiguel, D., Karumanchi, S. A., Yap, H.-K., Saleem, M., Zhang, Q., Nikolic, B., Chaudhuri, A., Daftarian, P., Salido, E., Torres, A., Salifu, M., Sarwal, M. M., Schaefer, F., Morath, C., Schwenger, V., Zeier, M., Gupta, V., Roth, D., Rastaldi, M. P., Burke, G., Ruiz, P., and Reiser, J. (2011) Circulating urokinase receptor as a cause of focal segmental glomerulosclerosis. Nat. Med. 17, 952–960 4. Jørgensen, T., Jacobsen, R. K., Toft, U., Aadahl, M., Gl¨umer, C., and Pisinger, C. (2014) Effect of screening and lifestyle counselling on incidence of ischaemic heart disease in general population: Inter99 randomised trial. BMJ 348, g3617
Response by Ian Sayers, Michael A. Portelli,2 Mateusz Siedlinski We thank Dr. Rasmussen and colleagues for their comments on our work outlined in “Genome-wide protein QTL mapping identifies human plasma kallikrein as a posttranslational regulator of serum uPAR levels” (FASEB J. 28, 923–934) and the opportunity to respond to key points. The authors have attempted to replicate some of our findings by investigating the association between the KLKB1 rs4253238 single nucleotide polymorphism (SNP) and soluble urokinase plasminogen activator receptor (suPAR) protein levels in serum in a Danish general population cohort called Inter99. The authors did not identify an association in this large population, which is of interest as replication of association findings is the gold standard for genetic studies. We appreciate their work to further develop our understanding of the regulation of this disease-relevant protein; however, we would like to take the opportunity to reply with a few comments. First, it is important to note that the initial genetic association finding was a relatively small component of our research paper, which went further to validate the observation using a range of biochemical analyses and cellular approaches that ultimately confirmed the importance of rs4253238 as a marker of KLKB1 activity in the same human serum samples and the role of KLKB1 in regulating urokinase plasminogen activator receptor (uPAR) cleavage and activity in cell and cell-free systems. In the absence of equivalent serum analyses in the 2 Correspondence: Division of Respiratory Medicine, University of Nottingham, Queens Medical Centre, Nottingham, United Kingdom. E-mail:
[email protected]
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Danish sample groups, the lack of genetic association between rs4253238 and suPAR serum levels is difficult to interpret. Is the rs4253238 SNP related to KLKB1 activity in the Danish cohort as we demonstrated for our subject serum samples? Second, it is unclear how closely our study has been replicated (i.e., was the same uPAR enzyme-linked immunosorbent assay used to provide comparable data?). This is essential as uPAR exists in several soluble forms generated from both mRNA splicing and proteolytic cleavage (1). Did the authors measure the same suPAR species using the same monoclonal antibody as we did? In our study, soluble uPAR was determined using detection and antibodies available through R&D Systems (Minneapolis, MN, USA). We and others have confirmed variable detection of uPAR isoforms and cleavage products depending on the antibody used (epitope recognized). The good concordance between rs4253238 genotype frequencies in both the Danish and our cohorts is reassuring with regard to comparable technologies. Third, the study population is somewhat different from our study [i.e., our study was enriched for patients with obstructive lung disease (both asthma and chronic obstructive pulmonary disease (COPD)], and although we were able to detect association between rs4253238 and suPAR levels in subjects with no disease, the magnitude of effect was less pronounced compared with subjects with COPD, for example. The authors began to address this point by examining the association in a subset of subjects with asthma, and again they did not observe an association between rs4253238 and uPAR levels. A key question is how comparable these subjects with asthma are to our asthma cohort? Were suPAR levels elevated in the asthma subjects versus control subjects in the Danish cohort, as we had identified in our cohorts? Finally, the authors go on to test for association between the KLKB1 rs4253238 SNP and a range of disease states, including ischemic heart disease, stroke, and cardiovascular disease. We are unsure of the rationale for these analyses in the absence of evidence that the KLKB1 rs4253238 SNP is a driver of suPAR serum levels and that suPAR is a driver of these diseases. In our original paper, we did not formally test the association between rs4253238 and asthma or COPD diagnosis because we observed the genotype effects of the rs4253238 SNP on suPAR levels in both health and disease, and numbers were limited. In conclusion, we appreciate the work done by Dr. Rasmussen and colleagues to replicate the genetic association between KLKB1 rs4253238 and suPAR levels in serum using a large general population cohort. This study has several strengths, including the large population size; however, the limited scope of the study, and for the reasons outlined, makes it difficult to interpret findings in the context of our own work. We remain confident in our findings using 3 independent cohorts (control, asthma, and COPD) that KLKB1 rs4253238 SNP is a regulator of KLKB1 activity and influences suPAR levels in these subjects and that the reason for the lack of association in the Danish population remains to be resolved. There is accumulating evidence that uPAR is an important functional receptor involved in several
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