Transplant Immunology 17 (2007) 223–226www.elsevier.com/locate/trim

Brief communication

Prevalence of CCR5Δ32 polymorphism in long-term survivorsof heart transplantation

Manfred Hummel a,⁎, Christoph Bara b, Stephan Hirt c, Axel Haverich b, Roland Hetzer a

a German Heart Institute Berlin, Division of Cardiovascular Surgery, Augustenburger Platz 1, 13353 Berlin, Germanyb Division of Thoracic and Cardiovascular Surgery, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany

c Division of Cardiovascular Surgery, Kiel University Hospital, Arnold-Heller-Strasse 7, 24105 Kiel, Germany

Received 22 May 2006; accepted 13 November 2006

Abstract

Background: CC chemokine receptor 5 (CCR5) contributes to the alloimmune response following solid organ transplantation. In individualshomozygous for the CCR5Δ32 mutation, the receptor is inactive and lymphocyte recruitment and leukocyte trafficking during rejection areinhibited. A significant improvement in graft survival following renal transplantation has been observed in homozygous CCR5Δ32 patients,although conflicting data exist. To determine whether CCR5Δ32 homozygous heart transplant recipients may also benefit compared to those witha normally functioning CCR receptor, the proportion of patients with CCR5Δ32 mutation was examined in a large cohort of patients surviving fora long period after heart transplantation.Methods: The prevalence of CCR5 genotype was identified in patients who had survived ≥7 years after heart transplantation. Genotyping wasperformed centrally by polymerase chain reaction (PCR).Results: A total of 555 patients were recruited at three heart transplant centers in Germany. Of these, 442 patients (79.6%) were homozygous forthe wild-type allele, 106 (19.1%) were heterozygous for CCR5Δ32 and 7 (1.3%) were homozygous for CCR5Δ32. No statistically significantlydifferences were observed between the incidence of CCR5Δ32 homozygosity in the study population and the estimated incidence in the normalpopulation.Conclusions: In the absence of a control arm, it cannot be established if homozygous carriers of the CCR5Δ32 allele experience a long-termsurvival benefit following heart transplantation that may be masked by underrepresentation of the CCR5Δ32 allele in recipients of a hearttransplant. To answer this question, the prevalence of CCR5Δ32 homozygosity needs to be established in patients awaiting heart transplantation.© 2006 Elsevier B.V. All rights reserved.

Keywords: CCR5; Polymorphism; Homozygous; Heart transplantation; Survival

1. Introduction

Chemokines and chemokine receptors are critical for theselective recruitment and activation of T-lymphocytes in acute[1–3] and chronic [4] allograft rejection, and in cardiac allograftvasculopathy [5]. CC chemokine receptor 5 (CCR5) interacts witha range of chemokines including RANTES, MIP-1α and MIP-1β[6] and has been shown to contribute to the alloimmune responseboth in animal models of transplantation [7,8] and in the clinicalsetting [4,9]. In a mouse model of cardiac transplantation, bothCCR5-deficient animals and mice with normal CCR5 expression

⁎ Corresponding author. Tel.: +49 30 30008 116; fax: +49 30 30008 463.E-mail address: hummel@paulinenkrankenhaus.de (M. Hummel).

0966-3274/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.trim.2006.11.004

treatedwith a neutralizingmonoclonal antibody tomCCR5 showedenhanced graft survival compared to controls [7]. Recently, it hasalso been demonstrated in a non-human primate model that CCR5blockade in combination with cyclosporine can prolong survivaland delay cardiac allograft vasculopathy [10]. Clinically, aprospective analysis of biopsy data from 26 renal allograftrecipients has demonstrated significantly increased numbers ofCCR5-positive T-lymphocytes in the tubulointerstitium of patientswith acute rejection, and intrarenal RNA expression RANTES (aligand for CCR5) was significantly upregulated in the tissue ofrejecting grafts [9]. There are also preliminary data to suggest a rolefor CCR5 in chronic renal allograft rejection [4]. In cardiactransplant patients, serial endomyocardial biopsy samples obtainedover the first year post-transplant have revealed that expression of

Table 1Patient demographics and baseline characteristics (n=555)

Number of patients (%)

Male gender 459 (82.7%)Age at time of transplant (years) 44±14Time post-transplant (years) 13.5±3.1Primary indication for transplantationDilated cardiomyopathy 354 (63.8%)Coronary heart disease 159 (28.6%)Other 42 (7.6%)

Continuous variables are shown as mean±S.D.

224 M. Hummel et al. / Transplant Immunology 17 (2007) 223–226

CCR5 increases significantly in patients experiencing rejectionepisodes, but that it normalizes after steroid treatment [11].Although clinical data concerning a possible relation betweenCCR5 expression and cardiac allograft vasculopathy (CAV) are notavailable, CCR5 expression is selectively and strongly increased incardiac rat transplants that developed vasculopathy [12].Moreover,in a murine model of CAV, blockade of the CCR5 receptor withMet-RANTES significantly reduced infiltration of CD4 and CD8T-lymphocytes into the transplant, inhibited intimal thickening andattenuated chronic rejection [13]. Other researchers have shownthat blockade of CCR5 downregulates local immune activation andT-lymphocyte recruitment resulting in prolonged cardiac allograftsurvival in mice [14].

The CCR5 receptor is functionally defective in individuals whoare homozygous for a 32-base pair deletion (CCR5Δ32). Theseindividuals have no apparent phenotype and are healthy; indeednon-functional CCR5 is associated with very high resistance toHIV infection because CCR5 is the major co-receptor for M-tropicHIV strains [15]. It has been proposed that CCR5Δ32 homozygoustransplant recipientsmay experience protection against lymphocyterecruitment during rejection because infiltrating mononuclear cellsdo not express CCR5 and leukocyte trafficking is impaired [16,17].In an observational study of 576 renal transplant recipients followedup to 10 years post-transplant, Fischereder et al. identified 21patients (1.7%) who were homozygous for CCR5Δ32 [16]. Graftsurvival was significantly higher in the homozygous CCR5Δ32patients: 1/21 (5%) homozygous patients experienced death-cen-sored graft loss over the 10-year period, compared with 78/555(14%) who were heterozygous or homozygous for the wild-typeallele. However, two smaller studies in renal transplant recipientsfound no significant difference in the incidence of rejection amongrecipients with or without the CCR5Δ32 mutation [18,19]. Otherresearchers [20] have proposed that the CCR5 genotype of theorgan donor can influence the recipient's immunological responseto a renal allograft. In liver allograft recipients, an epidemiologicalstudy in over 200 patients reported no association betweenCCR5Δ32 genotype and risk of acute rejection or graft survival[21]. Data in cardiac transplantation are scarce. Fildes et al. foundno correlation between recipient CCR5 genotype and outcomes in178 heart transplant patients, although there was an associationbetween donor genotype and mortality in patients transplanted fornon-ischemic conditions, and the authors suggested that donor-derivedCCR5 ismore likely to influenceT-lymphocyte recruitmentto the graft than recipient CCR5 status [22].

In the current study, we assessed the prevalence of recipienthomozygous and heterozygous genotypes for CCR5Δ32among long-term survivors of heart transplantation to examinethe hypothesis that expression of a functioning CCR5 receptormay have an adverse effect on survival following hearttransplantation, which would be indicated if the proportion ofpatients with the CCR5Δ32 mutation was higher than thatobserved in the normal population.

2. Objective

The primary objective of the study was to evaluate the pro-portion of CCR5Δ32 homozygous patients in heart transplant

patients surviving at least seven years after transplantation. Asecondary objective was to identify the proportion of patientsheterozygous for CCR5Δ32.

3. Material and methods

This was an exploratory study undertaken in patients who had survived for atleast seven years following heart transplantation. To minimize the effect ofgeographic variations in prevalence of the CCR5Δ32 allele [23], all patientswere recruited at heart transplant centers in Germany. All patients surviving atleast seven years after heart transplantation at participating centers were asked toprovide a blood sample for analysis of CCR5 receptor genotype. Blood sampleswere collected either as a 10 mL venous blood sample or a fingerprick sampleusing ISOCODE™ STIX (Schleicher and Schüll GmbH, Germany). Patient datawere extracted from hospital records. Written, informed consent was obtainedfrom all participants following approval from the Institutional Review Board orIndependent Ethics Committee at each center.

Genomic DNA (approximately 10–30 ng) was isolated from venous bloodsamples by mixing 10 μL aliquots with 20 μL lysis solution (REDExtract-N-AmpBlood PCR Kit; Sigma GmbH, Germany), incubating at room temperature for5 min, adding 180 μL neutralization solution from the Sigma kit, and mixing theneutralized lysate thoroughly. Fingerprick blood samples were placed in a sterile1.5 mL microcentrifuge tube and rinsed in 500 μL sterile deionized water(Millipore water) by vortexing thoroughly three times for at least 5 s. Sampleswerethen transferred into a new1.5mLmicrocentrifuge tube and 50μL sterile deionizedwater was added. After incubation for 30 min at 95 °C, the lysate was mixedthoroughly. Genotype analysis was undertaken by polymerase chain reaction(PCR) using 2 μL aliquots of lysate combined with 4 μLwater, 10 μL REDExtractPCRMix, 2 μL hCCR5 for3 primers (10 pmol/μL) and 2 μL hCCR5 rev3 primers(10 pmol/μL) in a 250 μL thin-walled PCR tube. The sequence of the PCR primershas been published previously [16]. The relevant portion of the CCR5 gene wasamplified in a RoboCycler (Stratagene GmbH, Germany) for 3 min at 94 °C (1cycle), 1 min at 94 °C, 1 minute at 58 °C, 2 min at 72 °C (40 cycles) and 10 min at72 °C (1 cycle). PCR products were analyzed on 2% agarose gels. The accuracy ofgel electrophoresis analysis was validated by DNA sequence analysis in a smallnumber of samples. All blood samples were analyzed centrally.

The sample size calculation was based on an assumption that the proportionof CCR5Δ32 homozygous carriers in the German population is 1.0–1.7%including the corresponding confidence interval and that the proportion ofhomozygous carriers in the heart transplant recipients is similar to that of thegeneral German population i.e. that the likelihood of heart transplantation is notcorrelated with the CCR5Δ32 homozygosity. On this basis, a population of 500patients would have a power of 60–80% and a confidence level of 90% (Type Ierror 0.10) to detect a difference between various null hypothesis scenarios (inwhich the proportion of CCR5Δ32 homozygous patients ranged from 1.0–1.7%) and observed scenarios (in which the proportion ranged from 0.18–0.30%) using the one-sided one-sample χ2-Test.

4. Results

In total, 555 patients were recruited at three heart transplant centersin Germany (Berlin n=300, Hannover n=166, Kiel n=89). The

Table 2CCR5 genotype in patients surviving for at least 7 years after heart transplantation

n Homozygouswild-type allele

Heterozygousfor CCR5Δ32

Homozygousfor CCR5Δ32

Hannover 166 127 (76.5%) 36 (21.7%) 3 (1.8%)Berlin 300 243 (81.0%) 53 (17.7%) 4 (1.3%)Kiel 89 72 (80.9%) 17 (19.1%) 0Total 555 442 (79.6%) 106 (19.1%) 7 (1.3%)

Figures in brackets indicate percentage of patients.

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demographics of the study population and causes of heart failure areshown in Table 1. The majority of patients were male and the meantime post-transplant was 13.5 years. CCR5 genotype analysis showedthat 442 patients (79.6%) were homozygous for the wild-type allele,106 (19.1%) were heterozygous for CCR5Δ32 and 7 (1.3%) werehomozygous for CCR5Δ32 (Table 2). Statistical analysis found nosignificant differences between the incidence of CCR5Δ32 homozy-gosity in the study population (1.3%) and the estimated incidence in thenormal population (1.0–1.7%).

5. Discussion

The prevalence of CCR5Δ32 homozygosity in this popula-tion of long-term survivors after heart transplantation (1.3%) issimilar to that reported previously among renal transplantpatients (1.7%) [16], heart transplant patients with a wide rangeof survival times (1.7%) [22], and in a non-transplant Germanpopulation (1.1%) [23]. The proportion of patients who wereheterozygous for CCR5Δ32 was 14.1%, slightly higher thanthat reported in a UK population of heart transplant recipientswith no minimum follow-up time (11.2%) [22].

This trial had no control arm, and therefore interpretation ofthese findings is necessarily limited. A prospective cohort studyfollowing newly-transplanted homozygous and non-homozy-gous heart transplant recipients for at least 10 years with similarimmunosuppressive therapy would represent the most robuststudy design to confirm or exclude the hypothesis thathomozygous carriers of the CCR5Δ32 experience a survivalbenefit. Such a benefit would probably be related to improvedimmunologic graft tolerance, with fewer episodes of acute andchronic rejection. However, the required sample size and theduration of the study as well as frequent changes toimmunosuppressive drug protocols make this study designimpracticable. A case-control trial would be unreliable becausethe control group could not be adequately matched i.e. datawould not be available for short-term survivors of transplan-tation who died prior to seven years post-transplant.

In the absence of a control arm, two hypotheses are worthy ofconsideration. First, homozygous carriers of the CCR5Δ32allele do not experience any benefit in terms of survivalfollowing heart transplantation, such that the proportion oflong-term heart transplant survivors homozygous forCCR5Δ32 is not different to that in short-term survivors or inthe non-transplant population.

The second hypothesis is that homozygosity for CCR5Δ32does confer a survival benefit compared to other CCR5genotypes. This could only be the case if the CCR5Δ32 allele

is underrepresented among patients requiring heart transplan-tation because the allele is protective against cardiovasculardisease. Thus, if the CCR5Δ32 mutation was associated withreduced risk of acute rejection or CAV, long-term survivors ofheart transplantation would be expected to show a higher re-lative prevalence of CCR5Δ32 homozygosity than those whodied early even though the absolute prevalence might be closeto that seen in renal or non-transplant populations.

Two arguments suggest that the effect of CCR5 polymor-phism on graft survival may be organ-specific, supporting thehypothesis that heart transplant recipients – in contrast to renaltransplant patients – derive no survival benefit from theCCR5Δ32 allele. The role of CCR5 in the immune responseto grafts appears to vary between organ types [8,24], a findingthat would be compatible with the conflicting results ofpreviously published studies concerning the impact of CCR5polymorphism in renal [16,18,19], liver [21] and cardiac [22]transplant populations. A second consideration is that theintensity of immunosuppression differs according to organ type.Heart transplant recipients receive the most intensive immuno-suppressive regimens, and it is conceivable that these potentregimens mask any effect of CCR5Δ32 on cardiac allograftrejection. In these circumstances, CCR5 mutation may even bedisadvantageous, because patients with CCR5Δ32 may be morevulnerable to over-immunosuppression such that a higher riskof infection-related death could outweigh the immunologicaladvantage of improved allograft tolerance.

The second hypothesis, that CCR5Δ32 homozygotes do infact experience a survival benefit following heart transplanta-tion, is feasible if the CCR5Δ32 allele is associated withreduced cardiovascular risk and therefore that the proportion ofheart transplant recipients who are homozygous for CCR5Δ32is lower than that observed in the general population.Alternatively, this hypothesis is possible if the causes of deathin CCR5Δ32 patients compared to those with the wild typeallele differ significantly in terms of acute or chronic rejectionand infection.

Szalai and colleagues compared the occurrence of theCCR5Δ32 mutation in 318 patients referred for coronarybypass surgery with healthy controls [25]. They found that noneof the 318 patients awaiting bypass surgery were CCR5Δ32homozygotes, compared to 6/320 (1.9%) in the control group,and concluded that individuals who are homozygous for theCCR5Δ32 allele are at reduced risk for severe coronary arterydisease. The frequency of the CCR5Δ32 allele has also beenshown to be significantly lower in patients experiencingmyocardial infarction (MI) at an early age (b55 years) than incontrols [26]. This is consistent with results from murine studiesin which CCR5 deficiency was associated with reducedneointima formation and T-cell infiltration following carotidartery allografting, suggesting a role for CCR5 in transplantarteriosclerosis [24] which may extend to general arterioscle-rotic development. The same researchers also showed thatCCR5-deficient animals had less tissue remodeling andimproved graft survival after cardiac transplantation [24].However only about 50% of patients who are accepted forheart transplantation suffer from ischemic heart disease,

226 M. Hummel et al. / Transplant Immunology 17 (2007) 223–226

whereas the majority of end-stage heart failure is caused bydilated cardiomyopathy of various origins (e.g. myocarditis).

Without an examination of the prevalence of CCR5polymorphism at the time of heart transplantation regardingthe origin of heart disease, the possibility that CCR5Δ32confers a similar survival advantage in heart transplantation tothat reported in renal transplant recipients cannot be ruled out[16].

The results of this study must be interpreted against thebackground of a heterogeneous study population with anunknown incidence of CCR5Δ32 homozygosity and heterozy-gosity before transplantation, different heart diseases and avariety of immunosuppressive regimens during a period of atleast seven years since transplantation. To confirm or excludethe hypothesis that the CCR5Δ32 mutation is advantageous forlong-term graft survival after heart transplantation, withconsequent implications for selective blockade of the CCR5receptor, a trial would be required in which a minimum of 500patients awaiting heart transplantation were screened for theCCR5Δ32 allele in order to identify any significant differencesin CCR5Δ32 homozygosity and heterozygosity versus ourstudy population. Such a comparison could not only answerwhether the CCR5Δ32 mutation is advantageous for hearttransplant recipients, as has been observed after renaltransplantation, but may also contribute to the knowledgebase concerning the role of CCR5 in the development of severecardiovascular disease.

Acknowledgements

The authors gratefully acknowledge the contribution ofAngelika Costard-Jäckle, Silvia Chilla, Michael Fischereder,Detlef Schlöndorff, Klaus Pfeffer, Ulrike Huffstadt, RudiBalling, Wolfgang Wurst, Hermann-Josef Gröne and Eva Kiss.

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