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Copy number variation in the CCL4L gene is associated with susceptibility to acute rejection in lung transplantation.

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Lung transplantation (LT) has become an accepted therapy for selected patients with advanced lung disease. One of the main limitations to successful LT is rejection of the transplanted organ where chemokines are pivotal mediators. Here, we test the
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  ORIGINAL ARTICLE Copy number variation in the CCL4L gene is associated with susceptibility to acute rejection in lung transplantation  R Colobran 1,2 , N Casamitjana 1,3 , A Roman 4 , R Faner 1 , E Pedrosa 1,2 , JI Arostegui 5 , R Pujol-Borrell 1,2 ,M Juan 1,2,5 and E Palou 1 1 Laboratori d’Immunobiologia per a la Recerca i Aplicacions Diagno`stiques, Banc de Sang i Teixits, Institut d’Investigacio´  en Cie`ncies dela Salut Germans Trias i Pujol, Badalona, Barcelona, Spain;  2 Departament de Biologia Cellular, Fisiologia i Immunologia, Universitat Auto`noma de Barcelona, Bellaterra, Barcelona, Spain;  3 Laboratori de Seguretat Transfusional, Banc de Sang i Teixits, Barcelona, Spain; 4 Servei de Pneumologia, CIBERES, Hospital Universitari Vall d’Hebron, Barcelona, Spain and  5 Servei d’Immunologia, Centre deDiagno`stic Biome`dic, Hospital Clı´ nic, Institut d’Investigacions Biome`diques August Pi i Sunyer, Barcelona, Spain Lung transplantation (LT) has become an accepted therapy for selected patients with advanced lung disease. One of the main limitations to successful LT is rejection of the transplanted organ where chemokines are pivotal mediators. Here, we test the relationship between copy number variation (CNV) in the CCL4L chemokine gene and rejection risk in LT patients (n  ¼ 161).Patients with no acute rejection showed a significantly lower mean number of CCL4L copies than patients that showed acute rejection (1.66 vs 1.96, P  ¼ 0.014), with an even greater number of gene copies seen in patients with more than one episode of acute rejection (1.66 vs 2.30, P  ¼ 0.001). Additionally, patients with  X 2 CCL4L copies had a significantly higher risk of acute rejection compared with patients that had 0–1 CCL4L copies (odds ratio 2.65; 95% confidence interval, 1.33–5.28; P  ¼ 0.0046).A combined analysis of CCL4L CNV and the rs4796195 CCL4L single nucleotide polymorphism demonstrated that the effect of CCL4L copy number in acute rejection is mainly because of the number of copies of the CCL4L1 allelic variant. This finding constitutes the first report of CNV as a correlate factor in allograft rejection. Genes and Immunity advance online publication, 15 January 2009; doi:10.1038/gene.2008.96 Keywords:  human; rejection; lung transplantation; chemokines; copy number variation; SNP  Introduction Lung transplantation (LT) is an accepted therapeuticoption for a variety of end-stage pulmonary diseases.However, the application of LT is hindered by multiplefactors and long-term survival following LTremains lowprimarily because of immune-mediated complicationssuch as acute rejection and chronic rejection (CR). 1 Acutecellular rejection (AR) is defined by perivascular and bronchiolar inflammation characterized by accumula-tions of mononuclear cells and eosinophils. 2 But, acuteantibody-mediated rejection is not well defined. In CR, bronchiolitis obliterans, which is the most specificpulmonary manifestation, represents the end result of immunological injury to the allograft. 3 Leukocyte migration through vessels and beyond thevascular compartment is dependent, in addition toadhesion molecules, on small chemoattractant proteinscalled chemokines. 4 Indeed, chemokine-driven accumu-lation of leukocytes in the graft is a key feature of transplant rejection. 5 Specifically, recent research inanimal LT models and human LT recipients has demon-strated the important contribution of chemokines to lungallograft rejection. 6 Previous works have demonstrated that differences inindividual genetic background may affect propensity fortransplant rejection. 7,8 In fact, genetic polymorphismsinfluence the function or expression of key immuno-regulatory molecules that either directly or indirectlymediate graft rejection. 9,10 In past years, common geneticvariants of several chemokines have been associatedwith functional and biological effects, including suscept-ibility to a great variety of diseases, and there is evidencethat chemokine polymorphisms could be one of thefactors that define allograft rejection. 11–13 CCL4L chemokine is a nonallelic copy of CCL4/MIP-1 b  chemokine.  CCL4  and  CCL4L  genes are located atchromosome 17q12 and its mature proteins differ at onlya single amino acid.  CCL4L  gene displays copy numbervariation (CNV) between individuals. 14 CNV is wide-spread among normal individuals and is considered animportant form of genetic diversity among humans. 15,16 Variation in copy number can influence transcription ortranslation of overlapping or nearby genes, and sometypes of CNV are associated with differential suscept-ibility to both complex and common diseases. 17,18 The main aim of the current study was to evaluate theinfluence of   CCL4L  gene copy number in LT rejection. Received 5 September 2008; revised 13 November 2008; accepted 17November 2008Correspondence: Dr M Juan, Servei d’Immunologia, Centre deDiagno`stic Biome`dic, Hospital Clı´nic, Institut d’InvestigacionsBiome`diques August Pi i Sunyer, C/Villarroel 170, Barcelona08036, Spain.E-mail: mjuan@clinic.ub.es Genes and Immunity (2009),  1–6 & 2009 Macmillan Publishers Limited All rights reserved 1466-4879/09  $ 32.00 www.nature.com/gene  In addition to the  CCL4L  CNV, we also genotypedtwo functionally relevant  CCL4L  single nucleotidepolymorphisms (SNPs): rs4796195 and rs3744595.Both SNPs have been previously associated with HIVpathology 19,20 and, strikingly, the rs4796195 minor allele L2 , which encodes different  CCL4L  transcripts andproteins than the  L1  allele, shows among worldwidepopulations a strong positive correlation with  CCL4L copy number. 21 Our results showed a significant association betweenhigher genomic copy number for the  CCL4L  gene andrisk of AR in LT, an association mainly because of thenumber of   CCL4L1  copies. To our knowledge, this is thefirst evidence for the influence of gene copy number onthe susceptibility to allograft rejection. Results Copy number variation in CCL4L and LT rejection We evaluated a total of 161 LT patients and 80 healthycontrols. The mean number of   CCL4L  copies in thecontrol group was 1.86, a value consistent with apreviously published report quantifying  CCL4L  inother Caucasian populations. 21 In LT patients, the meannumber of   CCL4L  copies was 1.80. As expected, the meanand distribution of   CCL4L  copies in the LT patients werenot statistically different to the control group (Table 1and Supplementary Figure 1).For the purposes of the following analysis, LT patientswere classified in several nonmutually exclusive groups:patients with AR, patients with more than one episode of AR (AR 4 1) and patients with no acute rejection (NAR);and patients with CR and patients with no chronicrejection (NCR).The group of NAR patients showed a statisticallysignificantly lower mean number of   CCL4L  copiesthan AR patients (1.66 vs 1.96;  P ¼ 0.014; statisticalpower ¼ 80.4%). Moreover, the difference in mean copynumber between NAR patients and AR 4 1 patientswas even greater (1.66 vs 2.30;  P ¼ 0.001; statisticalpower ¼ 90.3%). Moreover, no significant difference wasobserved between the NCR and CR patients (1.83 vs 1.77, P ¼ NS; Table 1, Figure 1 and Supplementary Figure 1).After this initial analysis, we classified subjects intotwo groups according to the number of   CCL4L  copiesthat each individual possessed: those with 0  1  CCL4L copies, and those with  X 2  CCL4L  copies. LT patientswith X 2  CCL4L  copies had a significantly higher risk of AR than patients with 0–1  CCL4L  copies (odds ratio, OR,2.65; 95% confidence interval (CI), 1.33–5.28;  P ¼ 0.0046;statistical power ¼ 87.9%; Table 2). Risk of AR inindividuals with  X 2  CCL4L  copies was even greaterwhen we considered only the subgroup of AR 4 1patients (OR 8.35; 95% CI, 1.83–38.07;  P ¼ 0.0007, statis-tical power ¼ 97.2%). Regarding CR, no significantlydifferent risk was observed between patients possessing0  1 copies or  X 2 copies of   CCL4L  (OR 1.06; 95% CI,0.49–2.31;  P ¼ 0.89). Thus there was evidence for associa-tion of   CCL4L  copy number with increased risk of AR inLT. Additionally, to assess the biological consequencesof having an even higher number of   CCL4L  copies,we classified subjects into two groups: those with 0  2 CCL4L  copies and those with X 3  CCL4L  copies (Supple-mentary Table 1). Interestingly, patients with X 3  CCL4L copies that had already suffered one episode of AR had asignificantly higher risk of suffering a second episodethan patients possessing 0  2 copies of   CCL4L  (OR 5.25;95% CI, 1.59–17.28;  P ¼ 0.0056; statistical power ¼ 86%). Possession of rs4796195 and rs3744595 CCL4L SNPs andrejection in LT patients We also genotyped two functionally relevant  CCL4L SNPs (rs4796195 and rs3744595) in LT patients andhealthy controls, assessing the exact number of   CCL4L copies belonging to each allelic variant. As expected, theallelic and genotypic frequencies between LT patientsand healthy controls were similar for both SNPs(Supplementary Tables 2 and 3). When we performedthe analysis among the different subsets of LT patients,we did not find any significant difference for the allelicand genotypic frequencies of both SNPs. Therefore,rs4796195 and rs3744595  CCL4L  SNPs are not signifi-cantly associated with LT rejection. Combined role of rs4796195 SNP and CCL4L copy number inLT rejection In previous work, 21 we demonstrated that there is astrong correlation between  CCL4L  copy number and theallelic frequencies of rs4796195  CCL4L  SNP (and not forthe rs3744595 SNP) in the Human Genome DiversityPanel. Specifically, the frequency of rs4796195  L2  alleleshows a strong positive correlation with  CCL4L  copynumber in worldwide populations. This phenomenon Table 1  Frequency distribution and mean number of   CCL4L  copies in control population and LT patient groups Group (number of samples)Number of copiesControl( N ¼ 80)Totaltransplanted( N ¼ 161)No acuterejection( N ¼ 79)Total acuterejection( N ¼ 78) Acute rejection 4 1 episode( N ¼ 23)No chronicrejection( N ¼ 104)Chronicrejection( N ¼ 39) 0 3.75% 3.73% 3.80% 3.85% 8.70% 3.85% 2.56%1 25% 31.06% 40.51% 19.23% 0.00% 30.77% 30.77%2 52.5% 48.45% 41.77% 57.69% 52.17% 46.15% 56.41%3 18.75% 14.91% 13.92% 15.38% 30.43% 17.31% 7.69%4 0% 1.86% 0% 3.85% 8.70% 1.92% 2.56%Mean number of copies 1.86 1.80 1.66 1.96 2.30 1.83 1.77 CCL4L  CNV and lung allograft rejection R Colobran  et al 2 Genes and Immunity  p = 0.014p = 0.001    M  e  a  n  n  u  m   b  e  r  o   f       C      C     L     4     L   c  o  p   i  e  s ControlTotal transplantedNo acute rejectionAcute rejectionAcute rejection> 1 episodeNo chronic rejectionChronic rejection    F  r  e  q  u  e  n  c  y   % No acute rejectionAcute rejectionAcute rejection> 1 episodeNumber of CCL4L  copies Figure 1  Association of   CCL4L  copy number and acute rejection in lung transplantation (LT). ( a ) Quantification of   CCL4L  copy number incontrol population and different groups of LT patients. ( b ) Distribution of   CCL4L  copy number in LT patients with or without acute rejectionand in the subset of patients with more than one episode of acute rejection. Table 2  Number of   CCL4L  gene copies and risk of acute and chronic rejection in LT patients No acute rejection Total acute rejection  P -value Odds ratio 95% CI  0–1 copies 35 18 0.0046 2.65 (1.33–5.28) X 2 copies 44 60 No acute rejection Acute rejection 1 episode  P- value Odds ratio  95% CI0–1 copies 35 160.072 1.94 (0.93–4.03) X 2 copies 44 39 No acute rejection Acute rejection 4 1 episode  P- value Odds ratio  95% CI0–1 copies 35 2 0.0007 8.35 (1.83–38.07) X 2 copies 44 21  Acute rejection 1 episode Acute rejection 4 1 episode  P- value Odds ratio  95% CI0–1 copies 16 20.037 4.31 (0.90–20.56) X 2 copies 39 21 No chronic rejection Chronic rejection  P- value Odds ratio  95% CI0–1 copies 36 130.89 1.06 (0.49–2.31) X 2 copies 68 26Significant  P  and OR values are denoted in bold. CCL4L  CNV and lung allograft rejection R Colobran  et al 3 Genes and Immunity  also occurs in our control population and in LT patients:individuals carrying the  L2  allele show a significantlyhigher number of   CCL4L  copies than those carrying onlythe  L1  allele (Supplementary Table 4). Then we tested forthe influence in LT rejection of   CCL4L  copy number of each allelic variant of rs4796195 SNP ( L1  and  L2 ).Interestingly, NAR patients had a significantly lowermean number of   CCL4L1  copies than AR patients (1.49 vs1.86;  P ¼ 0.0037; statistical power ¼ 91.8%), and consider-ably lower number of copies than AR 4 1 patients (1.49 vs2.41;  P ¼ 0.0005; statistical power ¼ 99.7%; Table 3). Wefound no significant differences between these groupswith respect to mean number of   CCL4L  copies in patientscarrying the  L2  allele. Moreover, we did not findsignificant differences in copy number betweenchronic-rejection patients possessing either  CCL4L1  or CCL4L2  copies. Thus, we conclude that there is astatistically significant association between  CCL4L1  copynumber and increased risk of AR in LT patients. Discussion Despite improvements in surgical technique, organ pre-servation, immunosuppression and infection manage-ment, acute organ rejection continues to present a majorobstacle to successful LT. The high rates of AR in LT andthe weak effect of HLA matching to predict the develop-ment of rejection after transplant suggest that genetic risksfor pulmonary allograft rejection are multifactorial. 22,23 In this study, we identified the  CCL4L  gene copynumber as a genetic factor correlated with AR in LT. The CCL4L  gene is located in a chromosome 17q12 copy-number-variable region that also includes the highlyrelated  CCL3L  gene. 24 Interestingly, although there areoften equivalent numbers of   CCL3L  and  CCL4L  genes,there are also haplotypes with different numbers of genecopies. This is significant because copy number of bothgenes can be different on a single chromosome. 14,25 However, the number of copies of   CCL3L  and  CCL4L  is,in general, positively correlated within populations. Thus,although in this study, we focused on the therapeuticcorrelates of   CCL4L  gene copy number,  CCL3L  genedosage may also contribute to the LT rejection effect.We demonstrated that the correlation between  CCL4L copy number and risk of acute LT rejection is mainlyexplained by the number of copies of the  CCL4L1  allelicvariant. This finding is consistent with the previous workshowing that CCL4L1 protein is fully functional  in vitro . 26 In contrast, computational data suggest that the CCL4L2protein, with a five amino-acid deletion, may show asignificant loss of function. 19 Regarding the relationship between  CCL4L  copynumber and the amount of CCL4L mRNA or proteinexpression, there is some, but still no conclusive, data.Although Townson  et al . 14 and Gonzalez  et al . 27 demon-strated that high  CCL3L  copy number correlates withincreased functional chemokine production, the study of Townson  et al . also analyzed the  CCL4L  gene and failedto reveal any consistent increase in CCL4L mRNAproduction from samples with high  CCL4L  copy number.However, these workers found that individuals withonly one copy of   CCL4L  consistently had a lowerexpression of CCL4L than those with higher copynumber. 14 We note that at the time of publication,Townson  et al . were not aware of the existence of the L1  and  L2  allelic variants of   CCL4L , two variants that canstrongly influence gene expression. 19 More recently, astudy by Melzer  et al . 28 reported a new  cis -effect of a SNPlocated near the  CCL4L  gene (227kb) on CCL4L proteinproduction. They hypothesize that the effect is caused byvariation in copy number of the  CCL4L  gene that could be in linkage disequilibrium with the analyzed SNP.Although further studies are needed to assess the effectof   CCL4L  copies on gene expression, we surmise that CCL4L  copy number likely influences CCL4L mRNA/protein production. Given the functional redundancy of the human  CCL4  and  CCL4L  chemokine genes, 26 higherlevels of CCL4L chemokine, related to higher  CCL4L copy number, could attract more monocytes and Th1-cells, cells that are crucial to AR of LT. This concept isconsistent with the role of the CCL5 chemokine in acutelung allograft rejection:  in vivo  neutralization of CCL5attenuates acute lung allograft rejection and reducesallospecific responsiveness by markedly decreasingmononuclear cell recruitment. 29 In fact, CCL4L andCCL5 (together with CCL3, CCL3L and CCL4) are themain ligands of the chemokine receptor CCR5 and arepredominantly expressed on T cells and monocytes ormacrophages. Taken together, these data point to animportant role of CCR5 ligands in acute, rather thanchronic, lung allograft rejection, and suggest that Table 3  Role of copy number of   CCL4L1  and  CCL4L2  allelicvariants in LT rejection Genotype Mean no. of CCL4L copies  P -valueControls Total transplanted L1 1.78 1.64 0.1864L2 presence 2.32 2.41 0.5754 Genotype Mean no. of CCL4L copies  P -valueNo acute rejection Acute rejection L1 1.49 1.86  0.0037 L2 presence 2.42 2.39 0.8528 Genotype Mean no. of CCL4L copies  P -valueNo acute rejection Acute rejection 4 1 episode L1 1.49 2.41  0.0005 L2 presence 2.42 2.66 0.3850 Genotype Mean no. of CCL4L copies  P -valueNo chronic rejection Chronic rejection L1 1.68 1.59 0.5259L2 presence 2.39 2.36 0.9219Individuals were classified according their genotype. L1, indivi-duals homozygous for  CCL4L1  allele regardless of their copynumber; L2, individuals carrying at least one copy of   CCL4L2  alleleregardless of the presence of   L1  allele.Significant  P  values are denoted in bold. CCL4L  CNV and lung allograft rejection R Colobran  et al 4 Genes and Immunity  perhaps mild blockage of chemotaxis with CCR5antagonists could prevent AR in LT.In this study, we focused on the genetic analysis of therecipients as the source of CCL4L in the rejection processwas mainly their immune cells. However, we cannotcompletely rule out a possible contribution of donor cellson the global production of this chemokine.In summary, our results show that  CCL4L  CNV and,specifically, the copy number of the  CCL4L1  allelicvariant is a genetic factor that may influence the risk of acute LT rejection. Further studies will be necessary toconfirm these findings in independent larger series of LTpatients and to assess if this effect exists in other types of transplantation. Materials and methods Study subjects and main clinical features Appropriate Institutional Review Board approval wasobtained. The present study includes 161 consecutive adultpatients who underwent LT at the Hospital UniversitariVall d’Hebron (Barcelona, Spain) for treatment of end-stagelung disease between 1997 and 2004. The study alsoincludes 80 healthy blood donors from the Banc de Sang iTeixits (Barcelona, Spain). All study subjects were Cauca-sian individuals from Spain. LT recipients were selectedaccording to the clinical protocol of the Hospital Uni-versitari Vall d’Hebron in which HLA compatibility wasnot a selection criterium. The demographic characteristicsof the study population of LTrecipients are summarized inSupplementary Table 5. Additionally, characteristics of patients possessing low (0–1) or high ( X 2) number of  CCL4L  copies were compared. Both populations were notstatistically different in the number of males and females,age, native disease and type of transplant approach.Furthermore, the immunologic risk to both groups, asreflected in the degree of HLA mismatch or the number of patients with elevated panel reactive antibody, was similar(Supplementary Table 5).The protocol of immunosuppression included highdoses of steroids at the induction (first day) withoutany antilymphocyte antibody. This protocol was basedon triple therapy with cyclosporine or tacrolimus,azathioprine or mycophenolate mofetil and low dose of steroids without discontinuing.We diagnosed AR mainly by transbronchial biopsyfindings or, in a few cases, when the patient had clinicalworsening and all other conditions were ruled out. Wedid not perform surveillance bronchoscopies; instead,this procedure was considered only by clinical indica-tion. We classified AR in agreement with the interna-tional consensus criteria 30 and we consideredsymptomatic AR to occur when the criteria level wasA2 or higher. We treated AR with pulse intravenousinjection of methylprednisolone at a dose of 10mg/kg perday, over 3 days. In accord with the internationalconsensus, we definedCR by the loss of FEV1. Informationabout bronchiolitisobliterans syndrome(CR) was collectedat the end of the study. On the basis of immunologicalcomplications, we classified patients according to thepresence or absence of acute rejection or CR. For some of the analyses, we treated the AR group according to thenumber of rejections that subjects demonstrated (that is,one episode of rejection vs more than one). Estimation of CCL4L copy number We estimated the number of   CCL4L  copies per studysubject using methods described in detail elsewhere. 21 Briefly, the assay is based on quantification by real-timePCR in an ABI PRISM Sequence Detection System 7900Instrument (Applied Biosystems, Foster City, CA, USA).We designed a mix of PCR primers and a TaqManMGB to specifically amplify  CCL4L . Primers and probesequences were as follows: sense primer 5 0 -CATGGTCAGGCAGAGGAAGATG-3 0 ; antisense primer 5 0 -GCTTGCCTCTTTTGGTTTGGAAT-3 0 ; probe 5 0 -TACCACAGGCAAGGGAT-3 0 (FAM labeled). As a control(single copy number gene), we used TaqMan RNasePControl (Applied Biosystems) following the manufac-turer’s recommendations. Genotyping of CCL4L polymorphisms We conducted  CCL4L  genotyping for rs4796195 andrs3744595 SNPs as previously described. 21 This assay isperformed by real-time PCR with fluorescence resonanceenergytransfer(FRET) probesina LightCyclerInstrument(Roche, Mannheim, Germany). For each SNP, we used apair of specific primers for the  CCL4L  region where theSNP is located and a pair of FRET probes (SupplementaryTable 6). This assay, when applied to samples in which thenumber of   CCL4L  copies is already known, allows thespecific assessment of the exact number of   CCL4L  copiesthat belong to each allelic variant. Statistical methods We tested for association among  CCL4L  copy numbermeans and type of LTrejection using the Mann–Whitney U  -test. We performed analysis of association involving biallelic markers using SNPStats software. 31 ORs and95% CIs were estimated by unconditional logisticregression analysis. P -value lower than 0.05 indicated statistical signifi-cance. We corrected for multiple testing for the obtained P -values. A Bonferroni’s correction for multiple testing(three polymorphisms tested in each group) led to acorrected threshold of about 0.017.Power calculations were performed to give the prob-ability of finding the differences between the  CCL4L copy number means as statistically significant, giventhe sample size (http://www.dssresearch.com/toolkit/spcalc/power.asp). Our study typically considered anadequate statistical power X 80%. Acknowledgements We thank Justi Gil and Pepi Caro for their support in thegenotyping process and Maria del Pilar Armengol forgeneral support in the laboratory. Disclosure/conflict of interest This study was supported by a grant from the Fundacio´npara la Investigacio´n y la Prevencio´n del Sida en Espan˜a(project number 36487/05), by the Fondo de Investiga-ciones Sanitarias del Instituto de Salud Carlos III (PI02/0278, PI07/0329), by the Red de Investigacio´n enEnfermedades Renales (REDINREN) and by the PROFIT(FIT 010000-2006-38) of the Banc de Sang i Teixits R Faner CCL4L  CNV and lung allograft rejection R Colobran  et al 5 Genes and Immunity
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