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Donor Club Cell Secretory Protein G38A Polymorphism Is Associated With a Decreased Risk of Primary Graft Dysfunction in the French Cohort in Lung Transplantation

Hin, Angela MSc1; Kannengiesser, Caroline MD, PhD2; Roussel, Arnaud MD, MSc1,3; Renaud-Picard, Benjamin MD4; Roux, Antoine MD, PhD5; Reynaud-Gaubert, Martine MD, PhD6; Claustre, Johanna MD7; Tissot, Adrien MD8; Guillemain, Romain MD9; Mornex, Jean-François MD, PhD10; Mussot, Sacha MD11; Dromer, Claire MD12; Dahan, Marcel MD, PhD13; Brugière, Olivier MD, PhD1,14; Mercier, Olaf MD, PhD11; Borie, Raphaël MD, PhD1,15; Pretolani, Marina PharmD, PhD1; Castier, Yves MD, PhD1,3; Mordant, Pierre MD, PhD1,3 on behalf of the COLT Consortium

doi: 10.1097/TP.0000000000002143
Original Clinical Science—General
Free

Background Club Cell Secretory Protein (CCSP) G38A polymorphism has recently been involved in lung epithelial susceptibility to external injuries. Lung transplantation (LT) is currently limited by ischemia-reperfusion injury leading to primary graft dysfunction (PGD). We thus hypothesized that donor CCSP G38A polymorphism might impact the risk of PGD after LT.

Methods We focused on LT included in the French multicentric Cohort in Lung Transplantation (COLT), performed between January 2009 and December 2014, and associated with preoperative blood samples from the donor and the recipient. Characteristics of the donors, recipients, procedures, early and late outcomes were prospectively recorded in COLT. The CCSP serum concentration and CCSP gene G38A polymorphism were retrospectively determined in a blind manner. Their association with grade 3 PGD was studied in univariate and multivariate analysis.

Results The study group included 104 LT donors and recipients, 84 with grade 0 to 2 PGD and 20 with grade 3 PGD. Preoperative CCSP serum concentration was significantly higher in the donors (median, 22.54 ng/mL; interquartile range, 9.6-43.9) than in the recipients (median, 7.03 ng/mL; interquartile range, 0.89-19.2; P < 0.001) but none impacted the risk of grade 3 PGD (P = 0.93 and P = 0.69, respectively). Donor CCSP G38A polymorphism was associated with a decreased risk of grade 3 PGD in univariate (AG + AA 3/21 = 14.2% vs GG 10/26 = 38.4%, P = 0.044) and multivariate analysis (odds ratio associated with AG + AA, 0.22; 95% confidence interval, 0.041-0.88; P = 0.045), but recipient CCSP G38A polymorphism was not.

Conclusions Donor CCSP G38A polymorphism is associated with a decreased risk of severe PGD after LT in the COLT study. These findings should be confirmed in the frame of a prospective study.

Donor club cell secretory protein G38A polymorphism is associated with a decreased risk of severe primary graft dysfunction following lung transplantation in the French multicentric cohort in lung transplantation study.

1 INSERM UMR1152, Bichat Hospital, Paris-Diderot University, Paris, France.

2 Division of Genetics, Bichat Hospital, Paris-Diderot University, Paris. France.

3 Division of Vascular surgery, Thoracic Surgery, and Lung Transplantation, Bichat Hospital, Paris-Diderot University, Paris, France.

4 Division of Pulmonology, Strasbourg University Hospital, Strasbourg, France.

5 Division of Pulmonology, Foch Hospital, Versailles Saint-Quentin University, Suresnes, France.

6 Division of Pulmonology, Marseille University Hospital, Aix-Marseille University, Marseille, France.

7 Division of Pulmonology, INSERM U1055, Grenoble University Hospital, Grenoble University, Grenoble, France.

8 Division of Pulmonology, Nantes University Hospital, Nantes University, Nantes, France.

9 Division of Cardiac Surgery, Georges Pompidou European Hospital, Paris, France.

10 Division of Pulmonology, INRA UMR754, Hospices Civils de Lyon, Lyon University, Lyon, France.

11 Division of Thoracic surgery, Vascular Surgery, and Lung Transplantation, Marie Lannelongue Hospital, Paris-Sud University, Le Plessis Robinson, Orsay, France.

12 Division of Pulmonology, Bordeaux University Hospital, Bordeaux, France.

13 Division of Thoracic surgery, Toulouse University Hospital, Toulouse University, Toulouse, France.

14 Division of Pulmonology B and Lung Transplantation, Bichat Hospital, Paris-Diderot University, Paris, France.

15 Division of Pulmonology A and Rare Lung Diseases, Bichat Hospital, Paris-Diderot University, Paris, France.

Received 27 July 2017. Revision received 18 December 2017.

Accepted 20 December 2017.

This study has been funded by a research grant from Vaincre la Mucoviscidose.

A.H., C.K., and P.M. declare a patent application relevant to this work (SCGB1A1 polymorphism for the prediction and therapy or prevention of primary graft dysfunction, submitted to the European Patent Office on 28th March 2017, reference EP17305358.8). There are no other conflicts of interest.

A.H. participated in the data acquisition, data analysis, data interpretation. C.K. participated in the data analysis and data interpretation. A.R. participated in the data analysis and data interpretation. B.R.-P. participated in the study conception, data interpretation. A.R. participated in the study conception and data interpretation. M.R.-G. participated in the study conception, data interpretation. J.C. participated in the study conception, data interpretation. A.T. participated in the study conception, data interpretation. R.G. participated in the study conception and data interpretation. J.-F.M. participated in the study conception and data interpretation. S.M. participated in the study conception and data interpretation. C.D. participated in the study conception and data interpretation. M.D. participated in the study conception and data interpretation. O.B. participated in the study conception. O.M. participated in the study conception. R.B. participated in the study conception. M.P. participated in the study conception and study design. Y.C. participated in the study conception and data interpretation. P.M. participated in the study conception, study design, and data interpretation. COLT collaborators participated in the data acquisition.

Correspondence: Pierre Mordant, MD, Division of Vascular Surgery, Thoracic Surgery and Lung Transplantation Bichat Hospital, 46 rue Henri Huchard, 75018 Paris, France. (pierre.mordant@aphp.fr).

Lung transplantation (LT) is the only therapeutic option that can improve the pulmonary function of patients with end-stage respiratory failure due to chronic lung diseases. Early postoperative outcome is currently limited by primary graft dysfunction (PGD), a direct consequence of lung ischemia-reperfusion (IR) injury.1 From the clinical perspective, PGD is a complex inflammatory state associating hypoxemia, pulmonary oedema, systemic inflammatory response syndrome, and radiographic appearance of diffuse pulmonary opacities without other identifiable cause.2 From the biological standpoint, lung IR induces oxidative stress through the generation of reactive oxygen species, ultimately leading to not only leukocytes recruitment and inflammatory mediators release but also to epithelial apoptosis via mechanisms involving hypoxia-inducible factor, protein kinase C, and p53.3,4 Despite recent advances in lung graft management,5 PGD is still associated with a significant mortality, urging the need for a better prediction of graft response to IR.

Club cell secretory protein (CCSP) is produced by the nonciliated lung epithelium, has anti-inflammatory and immunomodulatory properties, and plays a significant role in host defence and control of oxidative stress.6 CCSP concentration has been reported to be decreased in patients with chronic obstructive pulmonary disease (COPD)6 and increased in patients with idiopathic pulmonary fibrosis (IPF).7 CCSP concentration has also been reported to be increased during mechanical ventilation8 or multiple organ failure,9,10 suggesting a role of this protein during acute lung injury.10 The single-nucleotide polymorphism (SNP) rs3741240 of Secretoglobin family 1A member 1 (SCGB1A1) gene encoding CCSP protein (nomenclature: c.-26G > A) is located in the 5′ untranslated region and was previously called as G38A describing a substitution at the 38th nucleotide (A to G) from the transcription initiation site.11 The A allele is associated with a 25% decrease in gene transcription levels as compared with the G allele.11,12CCSP G38A allele has been reported to be associated with COPD,7 asthma,13 sarcoidosis,14 and chronic lung allograft dysfunction (CLAD).15 More recently, in vitro experiments suggested that CCSP G38A polymorphism might influence CCSP response to external injury such as cigarette smoke exposure.16

However, the impact of donor and recipient CCSP G38A polymorphism on lung IR injury has not been studied to date. We used the French COhort in Lung Transplantation (COLT) to determine the association between CCSP G38A polymorphism in donor and recipient, preoperative serum CCSP concentration in donor and recipient, and severe lung IR injury defined as the occurrence of severe PGD after LT.

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MATERIALS AND METHODS

Study Design

COLT is a French national, multicentric, prospective study initiated in 2009 with the goal to follow a cohort of LT candidates and recipients, and to identify biological risk factors of CLAD (http://clinicaltrials.gov/identification: NCT00980967). This cohort is associated with a collection of whole blood, serum, and DNA harvested from the donor and the recipient at various time points pretransplantation and posttransplantation. Our study included all donor and recipients involved in the COLT study, who underwent single or double LT between January 2009 and December 2014, with at least 1 pretransplant serum and DNA samples available from the donor and the recipient (n = 480). Patients with missing clinical data, missing donor or recipient pretransplant samples, heart-lung, or lobar transplant, were then excluded (n = 376). All together, 104 patients completed the study criteria and account for the study group. For each recipient included in the study, demographic characteristics, lung disease diagnosis and severity, and associated comorbidities were retrieved. For each donor, demographic characteristics, tobacco use, cause of death, and lung graft assessment were collected. For each procedure, the type of graft allocation (regular vs high emergency), and the type of LT performed (single vs double) were recorded. For each donor and recipient, peripheral blood was collected by participating centers preoperatively at the time of transplantation and centralized. The main outcome was severe PGD, defined as the occurrence of a grade 3 PGD at anytime during the first 72 hours after LT,2 and assigned by transplant physicians in each individual center. The study protocol has been approved by COLT scientific committee on June 17, 2015, and by the Greater Paris Committee for Patients’ Protection (Comité de Protection des Personnes-Ile de France 1), on December 13, 2015. Signed informed consent to participate to the COLT study was obtained from recipients. The Committee for Patients’ Protection waived need for specific consent authorizing genetic analysis from donors, but did not allow the record of donor and recipient genetic ancestry.

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CCSP G38A (rs3741240) Polymorphism Assessment

Genomic DNA was isolated from peripheral blood samples using a DNA extraction kit (Qiagen). Genomic DNA was normalized to a concentration of 20 ng/μL for a 500-μL volume and stored in 96-well plates. Specific PCR amplification of the CCSP coding gene SCGB1A1 (5′UTR of exon 1) was achieved as follows: 40 ng of genomic DNA was added to a PCR master mix containing H2O, MgCl2 25 mM, dNTP mix 5 mM, 10 pmol of forward and reverse primers, Buffer Gold 10× and Taq Gold (5 U/μL). Primers were designed with Primer3, further verified using BLAST and SNPCheck version: 3.2.1, Reference Genome Build version: 37.1 and dbSNP Build version: 141 for alternative unwanted amplifications and primer slipping. Primers used were: 5′-TCCCTTCACTGCCTCCAG-3′ (forward: 18 nt) and 5′-CTCCTCCCTCCAGGCTATTC-3′ (reverse: 20 nt). PCR thermocycler run after an initial denaturation at 95°C for 7 minutes the reactions were cycled 38 times through a temperature profile of 96°C for 30 seconds, 60°C for 1 minute, and 72°C for 1 minute. A final extension was performed at 72°C for 10 minutes. The products of the reaction were visualised by Caliper (LabChip). PCR yielded a fragment of 624 bp comprising the G38A SNP information of the patient. After confirming PCR amplification and clear blank controls, Exostar, enzymatic PCR clean up, was performed. Exostar thermocycler run following 37°C for 20 minutes and 80°C for 15 minutes followed with a FAST Sequencing reaction: adding H2O, big dye, buffer and 3,2pmol primers either forward or reverse. Product was centrifuged for 5 minutes at 2700 rpm on Sephadex G-50 Superfine (GE Healthcare) Millipore 96-well filtration plate, to enhance genotyping quality (preprepared a minimum of 3 hours before use) and collected on a sequencing plate. The sequences runs were performed on a 3130xl Genetic Analyzer (Applied Biosystems, Life Technologies) and the output of both forward and reverse sequence reactions were displayed on Chromas 1.4 and SeqScape Software allowing for G38A polymorphism assessment when compared with Reference sequence.

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CCSP Serum Measurements

Serum was isolated from peripheral blood samples and stored at −80°C. CCSP serum measurements were performed using the Human Uteroglobin (CCSP) DuoSet ELISA (R&D Systems).

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Statistical Analysis

Continuous variables with normal distribution were reported as mean and standard deviation, and compared using Student t test. Continuous variable with non-normal distribution were reported as mean, median, and interquartile range (IQR) and compared using Mann-Whitney test. Categorical variables were reported as count and proportion, and compared using Fisher or χ2 tests when appropriate. CCSP G38A genotype was modeled as dominant, as previously published.15,16 As donors and recipients were considered as paired binary data, matched analyses were performed when comparing variables between donors and recipients. Continuous variables were compared using paired Student test or paired Mann-Whitney test as appropriate and McNemar test was used for categorical variables. Primary outcome was severe PGD, defined as the occurrence of a grade 3 PGD at anytime during the first 72 hours after LT. Multivariate analysis was performed using Generalized Linear Models. Secondary outcome was overall survival, defined as the time interval between the date of operation and the date of death or the last follow-up visit for censored patients. Follow-up information was obtained from the hospital case records, or from a questionnaire completed by the chest physician. Median time of follow-up for the study group was 1687 days (IQR, 1110-1897). Actuarial survival curves were estimated by the Kaplan-Meier method. Statistical comparisons between survival distributions were made using the log-rank test. All data analyses were conducted with the two-sided test. A P value less than 0.05 was considered significant. Statistical analyses were conducted using Prism (GraphPad Prism version 6.00 for Windows; GraphPad Software, La Jolla, CA, www.graphpad.com) and R software (R Foundation for Statistical Computing, Vienna, Austria, www.r-project.org).

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RESULTS

Study Group

All together, 104 recipients paired with their respective donor were included, and account for the study group. Among them, 84 patients (81%) with grades 0, 1, and 2 PGD were included in group 1, whereas 20 patients (19%) with grade 3 PGD were included in group 2. Recipients, donors, and procedure characteristics as well as specific outcomes are summarized in Table 1. As compared with group 1—PGD0-2, group 2—PGD3 was characterized by a higher frequency of donor tobacco use (63% vs 33%, P = 0.023) and a nonsignificant increase in high-emergency graft allocation (21% vs 8%, P = 0.12). As compared with group 1—PGD0-2, group 2—PGD3 was also associated with a significantly worse outcome, as witnessed by a higher frequency of postoperative extracorporeal membrane oxygenation support (55% vs 0%, P < 0.001), a longer stay in intensive care unit (ICU) after transplantation (median, 42.5 vs 14 days, P < 0.001), a higher mortality 90 days after the surgery (20% vs 1%, P < 0.001), a nonsignificant increase in 1-year mortality (20% vs 7%, P = 0.080), and an adverse overall survival (Figure 1, P = 0.23).

TABLE 1

TABLE 1

FIGURE 1

FIGURE 1

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Serum CCSP Concentration

Pretransplant CCSP serum concentration was significantly lower in recipients (mean, 19.85 ng/mL; median, 7.03; IQR, 0.89-19.2) than in donors (mean, 33.32 ng/mL; median, 22.54; IQR, 9.6-43.9; P < 0.001, Figure 2A). In recipients, pretransplant CCSP serum concentration was significantly higher in patients with interstitial lung disease (mean, 45.21 ng/mL; median, 43.02; IQR, 28.51-57.53) than in patients with COPD (mean, 8.19 ng/mL; median, 6.06; IQR, 1.31-37.14) and cystic fibrosis (mean, 18.47 ng/mL; median, 4.15 ng/mL; IQR, 0.11-8.70) when bronchial diseases were considered together (P < 0.001, Figure 2B). As compared with group 1—PGD0-2, group 2—PGD3 was not associated with any significant difference in CCSP serum concentration in donors (mean, 33.85 ng/mL and median, 22.29 ng/mL vs mean, 30.81 ng/mL and median, 23.24 ng/mL, respectively, P = 0.93) and recipients (mean, 21.52 ng/mL and median, 6.58 ng/mL vs mean, 11.85 ng/mL and median, 7.71 ng/mL, respectively, P = 0.69, Figure 2C).

FIGURE 2

FIGURE 2

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CCSP Polymorphism

CCSP G38A (rs3741240) polymorphism was analyzed in donors and recipients (Figure 3A). There was no significant difference in the distribution of CCSP G38A polymorphism between donors and recipients, with the A polymorphism being present in 47% of the donors and 61% of the recipient (P = 0.16, Table 2). Pretransplant CCSP serum concentrations were then deciphered according to the CCSP G38A polymorphism. In donors, the presence of the CCSP G38A genotype was associated with a nonsignificant decrease in the serum concentration of CCSP (GG, 43.76 ± 30.73 ng/mL; AG, 29.47 ± 24.38 ng/mL; AA, 29.49 ± 21.87 ng/mL; GG vs AG, P = 0.087; GG vs AG + AA, P = 0.070, Figure 3B). In recipients, there was no significant difference in the serum concentration of CCSP according to the CCSP genotype (GG, 16.23 ± 23.43 ng/mL; AG, 24.86 ± 43.02 ng/mL; AA, 27.59 ± 46.21; P = 0.73; Figure 3C). Interestingly, a very significant difference was observed in the serum concentration of CCSP between GG homozygotes donor versus recipient (P < 0.001) but not between AG heterozygotes donor versus recipient (P = 0.073) nor between AA homozygotes donors versus recipients (P = 0.33). When deciphering donors' genotypes by PGD outcomes, the presence of CCSP AG genotype was associated with a decreased frequency of PGD3 (1/18 = 5.5% of patients with AG genotype, 2/5 = 40% of patients with AA genotype, 10/26 patients = 38.4% of patients with GG genotype, P = 0.022 for multiple comparisons, Table 2), with a significant difference between GG and AG + AA genotypes (P = 0.044).

FIGURE 3

FIGURE 3

TABLE 2

TABLE 2

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Prognostic Analysis

In multivariate analysis, including donor smoking, donor CCSP G38A polymorphism significantly impacted the risk of severe PGD (odds ratio [OR] associated with AG + AA, 0.22; 95% confidence interval [CI], 0.041-0.88; P = 0.045; Table 3).

TABLE 3

TABLE 3

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DISCUSSION

Main Results Reminder

Studying donors and recipients CCSP G38A polymorphism, pretransplant CCSP serum concentration, and their impact on PGD, we found donor CCSP G38A polymorphism to be associated (i) with a decreased concentration of CCSP in the peripheral blood before LT; and (ii) with a decreased risk of severe PGD after LT.

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CCSP and LT

Preclinical studies underlined the role of CCSP in the response of lung epithelium to ischemia and reperfusion. In vitro, CCSP protein increased epithelial cell proliferation while protecting against cell death by oxidative stress.17 In vivo, CCSP deprivation has been associated with an increased susceptibility to oxidative stress.18 The first clinical study exploring the role of CCSP after LT focused on bronchiolitis obliterans syndrome (BOS), the most frequent pattern of CLAD characterized by a fibrosing process of the small airways causing irreversible airway obstruction. After 22 LT recipients over 2 years, Nord et al19 found that levels of CCSP in serum and BAL were lowered in BOS patients, suggesting that recipient serum CCSP concentration could be an early marker for BOS. Ten years later, Diamond et al20 focused on PGD on a prospective cohort of 104 LT recipients, and determined levels of plasma CCSP at 3 time points: pretransplant, 6 hours posttransplant, and 24 hours posttransplant. Interestingly, elevated CCSP levels at 6 hours posttransplant were associated with increased odds of PGD in univariate and multivariate analysis. Concomitantly, Gilpin et al21 studied the kinetics of CCSP(+) bone marrow cells in the first days after LT in 30 recipients, and found a significant increase in these progenitors at 24 hours, with a decrease by 48 hours. More recently, Shah et al22 studied the impact of preoperative recipient levels of 5 biomarkers (CCSP, sRAGE, ICAM-1, IL-8, and protein C) on the occurrence of severe PGD in 714 patients and found preoperative recipient levels of CCSP to be associated with PGD in non-IPF subjects (OR for highest quartile of CCSP, 2.87; 95% CI, 1.37-6.00, P = 0.005) but not in subjects with IPF (OR, 1.38; 95% CI, 0.43-4.45; P = 0.59). All together, these data suggest that lung IR leads to increased levels of CCSP that correlate with altered alveolar epithelial permeability, whereas increased CCSP(+) peripheral blood mononuclear cell mobilization after LT may have beneficial effects on lung oxygenation and recovery time, pointing CCSP and CCSP(+) peripheral blood mononuclear cell as a key determinant of early graft function. Unfortunately, these studies did not take into account the genetic variability of donor and recipients.

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CCSP G38A Polymorphism

The gene encoding CCSP is indeed subject to a frequent polymorphism that may alter its expression at baseline or its response to external injuries. In humans, the gene encoding CCSP has 3 short exons and 2 introns for a total length of 4.1 kb. It is located on chromosome 11q12.3-13.1 in the vicinity of other genes associated with inflammatory and immune processes.23,24 Studies of the noncoding region of the exon 1 of CCSP gene have identified a number of binding sites for transcription factors.16 This region is also subject to an SNP located 38-bp downstream of the transcription initiation site, defined as dbSNP rs3741240, and characterized by an adenine/guanine substitution.25,26 This CCSP G38A polymorphism is found in 34% of the population and associated with 25% reduced transcription levels as compared with the G allele.11,12 Interestingly, we report similar proportions of CCSP G38A polymorphism in donors and recipients, but associated decrease in serum CCSP levels were found in donors but not in recipients. These results should be interpreted cautiously. It could suggest that in healthy individuals, such as lung donors, CCSP polymorphism is associated with decreased serum CCSP levels, but that in patients with end-stage lung disease such as lung recipients, CCSP polymorphism is associated with sustained serum CCSP levels—that is, that CCSP G38A polymorphism could be associated with a resistance to cell signals associated with end-stage respiratory disease. In the literature, CCSP G38A polymorphism has been studied mostly in sarcoidosis and asthma. The association between the presence of an A allele and the development of asthma in the general population is still questioned,13,27 whereas serum CCSP levels have been independently related to small airway hyperresponsiveness in asymptomatic individuals,28 COPD patients,6 and asthmatic patients.29 The fact that CCSP polymorphism has not been formally associated with the development of asthma, but serum CCSP levels has been associated with small airway hyperresponsiveness in various situations, could be explained by confounding factors. Individual exposure to cigarette smoke, air pollution, and professional toxics could interact with individual genetic susceptibility to impact the serum concentration of CCSP and the development of symptoms.16

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Gene-environment Interaction

This concept of gene-environment interaction could be of tremendous importance when considering lung grafts subjected to IR. In our study, the AG genotype in the donor was associated with a decreased risk of severe PGD both in univariate and multivariate analyses. Tentative explanation might include the resistance of the A allele to p53, as recently suggested.16 Lung IR has been associated with increased levels of p53 in the airway epithelium,3 that correlates with increased apoptosis of airway epithelial cells.30 Interestingly, Knabe et al16 recently reported that p53 binds to the promoter of CCSP and causes a decrease in CCSP gene expression in vitro. In humans, this binding site is located at the position of the G38A polymorphism site, thus explaining the resistance of CCSP G38A to p53-mediated CCSP downregulation in vitro. Specifically, BEAS-2B cells were transfected by either the CCSP 38G or 38A construct, in the presence/absence of cigarette smoke extract and modulating transcription factor p53. Baseline CCSP transcription levels were similar between the wild and variant constructs. Cigarette smoke extract decreased more profoundly the CCSP transcription level of 38A-transfected cells, but p53 decreased the CCSP transcription level more intensely with the wild than with the variant construct.16 Such experiments have not been performed after IR injury. Our hypothesis is that in lung grafts with the G allele of CCSP polymorphism, IR causes an increase in p53 associated with an increase in apoptosis and a decrease in the production of CCSP in lung epithelial cells, thus constituting a vicious circle that may alter epithelial function and lead to PGD. Conversely, in lung grafts with the A allele of CCSP G38A polymorphism, IR-induced p53 increase has no effect on CCSP expression and CCSP levels (Figure 4), and is thus associated with sustained lung epithelial function. This p53 resistance of G38A binding site may therefore explain the decrease frequency of PGD in grafts harboring the CCSP G38A genotype. Unfortunately, no blood sample has been collected immediately after transplant in the COLT study. As a consequence, we have not been able to determine the levels of CCSP posttransplant and to study the impact of donor CCSP polymorphism on the evolution of CCSP levels immediately after transplant. Even though it has been indirectly corroborated by recent publications,3,16,30 our hypothesis should therefore be confirmed both experimentally and clinically.

FIGURE 4

FIGURE 4

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Limitations

Our study has some limitations, including its retrospective design, the absence of information on genetic background, the limited number of patients, borderline P value, lack of long-term analysis after the 1-year timepoint, and lack of validation cohort. These limitations might be balanced by the multicentric and prospective design of COLT, the determination of PGD status in each participating center, and the centralized analysis of CCSP concentrations and CCSP polymorphism blinded from the clinical characteristics and studied outcome. However, the borderline P value of the multivariate analysis and the lack of validation cohort still constitute major limitations of this study.

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CONCLUSIONS

In this study, donor CCSP G38A gene polymorphism is associated with a decreased concentration of CCSP in the peripheral blood before LT, and a decreased risk of severe PGD after LT. These findings should be confirmed in further analyses performed on another retrospective series or in the frame of a prospective study. If confirmed, these findings would identify donor CCSP G38A status as an important risk factor and potential therapeutic target to predict and prevent the occurrence of PGD.

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ACKNOWLEDGMENTS

The authors would like to thank Aurore Foureau for administrative assistance, the Nantes University Hospital Biologic Resources Centre for DNA extraction (BRIF : BB-0033-00040), the members of the COLT consortium for their involvement into the study, and the patients and families for their participation to the COLT study.

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Appendix

Members of the COLT consortium.

Cohort Of Lung Transplantation-COLT (associating surgeons; anesthetists-intensivists; physicians, research staff). Bordeaux: J. Jougon, J.-F. Velly; H. Rozé; E. Blanchard, C. Dromer; Grenoble : E. Arnaud-Crozat, O. Chavanon, S. Guigard, R. Hacini, C. Martin, A. Pirvu, P. Porcu; P. Albaladejo, C. Allègre, A. Bataillard, D. Bedague, E. Briot, M. Casez-Brasseur, D. Colas, G. Dessertaine, M. Durand, G. Francony, A. Hebrard, M.R. Marino, D. Protar, D. Rehm, S. Robin, M. Rossi-Blancher; C. Augier, P. Bedouch, A. Boignard, H. Bouvaist, E. Brambilla, A. Briault, B. Camara, J. Claustre, S. Chanoine, M. Dubuc, S. Quétant, J. Maurizi, P. Pavèse, C. Pison, C. Saint-Raymond, N. Wion; C. Chérion; Lyon : R. Grima, O. Jegaden, J.-M. Maury, F. Tronc; C. Flamens, S. Paulus; J.-F. Mornex, F. Philit, A. Senechal, J.-C. Glérant, S. Turquier; D. Gamondes; L. Chalabresse, F. Thivolet-Bejui; C Barnel, C. Dubois, A. Tiberghien; Paris, Hôpital Européen Georges Pompidou : F. Le Pimpec-Barthes, A. Bel, P. Mordant, P. Achouh; V. Boussaud; R. Guillemain, D. Méléard, M.O. Bricourt, B. Cholley; V. Pezella; Marseille: G. Brioude, X.B. D’Journo, C. Doddoli. P. Thomas, D. Trousse; S. Dizier, M. Leone, L. Papazian; F. Bregeon, B. Coltey, N. Dufeu, H. Dutau, S. Garcia, JY. Gaubert, C. Gomez, S. Laroumagne, G. Mouton, A. Nieves, − Ch. Picard, M. Reynaud-Gaubert, JM. Rolain, E. Sampol, V. Secq; Nantes : P. Lacoste, C. Perigaud, J.C. Roussel, T. Senage, A Mugniot; I. Danner, A Haloun A. Magnan, A Tissot, S Abbes, C Bry, FX Blanc; T. Lepoivre, K. Botturi-Cavaillès, S. Brouard, R. Danger, J. Loy, M. Bernard, E. Godard, P.-J. Royer, E. Durand, K. Henrio, M. Durand, C. Brosseau, A.Foureau; Le Plessis Robinson, Hôpital Marie Lannelongue : Ph. Dartevelle, D. Fabre, E. Fadel, O. Mercier, S. Mussot; F. Stephan, P. Viard; J. Cerrina, P. Dorfmuller, S. Feuillet M. Ghigna, Ph. Hervé, F. Le Roy Ladurie, J. Le Pavec, V. Thomas de Montpreville; L. Lamrani; Paris, Hôpital Bichat : Y. Castier, P. Mordant, P. Cerceau, P. Augustin, S. Jean-Baptiste, S. Boudinet, P. Montravers; O. Brugière, G. Dauriat, G. Jébrak, H. Mal, A. Marceau, A.-C. Métivier, G. Thabut, E. Lhuillier, C. Dupin, V. Bunel; Strasbourg : P. Falcoz, G. Massard, N. Santelmo; A. Olland, J. Reeb, J. Seitlinger, G. Ajob, O. Collange O. Helms, J. Hentz, A. Roche; T. Degot, A. Dory, S. Hirschi, S. Ohlmann-Caillard, L. Kessler, R. Kessler, A. Schuller;B. Renaud-Picard, M. Porzio, Sarah Idris-Khodja, Julien Stauder; Suresnes, Hôpital Foch : P. Bonnette, A. Chapelier, P. Puyo, E. Sage; J. Bresson, V. Caille, C. Cerf, J. Devaquet, V. Dumans-Nizard, ML. Felten, M. Fischler, AG. Si Larbi, M. Leguen, L. Ley, N. Liu, G. Trebbia; S. De Miranda, B. Douvry, F. Gonin, D. Grenet, A.M. Hamid, H. Neveu, F. Parquin, C. Picard, A. Roux, M. Stern; F. Bouillioud, P. Cahen, M. Colombat, C. Dautricourt, M. Delahousse, B. D’Urso, J. Gravisse, A. Guth, S. Hillaire, P. Honderlick, M. Lequintrec, E. Longchampt, F. Mellot, A. Scherrer, L. Temagoult, L. Tricot; M. Vasse, C. Veyrie, L. Zemoura; Toulouse : M Dahan, M Murris, H Benahoua, J Berjaud, A Le Borgne Krams, L Crognier, L Brouchet, O Mathe, A Didier.

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