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ssociation of genetic polymorphisms and risk of lateost-transplantation infection in pediatric heart recipientsrin L. Ohmann, BS,a Maria M. Brooks, PhD,b Steven A. Webber, MBChB,a

iana M. Girnita, MD,c Robert E. Ferrell, PhD,d Gilbert J. Burckart, PharmD,e

ichard Chinnock, MD,f Charles Canter, MD,g Linda Addonizio, MD,h

aniel Bernstein, MD,i James K. Kirklin, MD,j David C. Naftel, PhD,j anddriana Zeevi, PhDc

From the Department of Pediatrics, Division Cardiology, bDepartment of Epidemiology, cDepartment of Pathology, andDepartment of Human Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania; eOffice of Clinical Pharmacology,ffice of Translational Science, U.S. Food and Drug Administration, Silver Spring, Maryland; fDepartment of Pediatrics,oma Linda University, Loma Linda, California; gDepartment of Pediatric Cardiology, Washington University School ofedicine, St. Louis Children’s Hospital, St. Louis, Missouri; hDepartment of Pediatrics, Division Cardiology, Columbianiversity, New York Presbyterian Hospital, New York, New York; iDivision of Pediatric Cardiology, Stanford University,ucile Packard Children’s Hospital, Palo Alto, California; and jDepartment of Surgery, University of Alabama at

irmingham, Birmingham, Alabama.

BACKGROUND: Late infections are common causes of morbidity and mortality after pediatric hearttransplantation. In this multicenter study from 6 centers, we investigated the association between geneticpolymorphisms (GPs) in immune response genes and late post-transplantation infections in 524 patients.METHODS: Late infection was defined as a clinical infectious process occurring �60 days after transplan-tation and requiring hospitalization, intravenous antimicrobial therapy, or a life-threatening infection requir-ing oral therapy. All patients provided a blood sample for GP analyses of 18 GPs in cytokine, growth factor,and effector molecule genes by single specific primer-polymerase chain reaction and/or sequencing. Sig-nificant associations in univariable analyses were tested in multivariable Cox regression models.RESULTS: Late infection was common, with 48.7% of patients experiencing �1 late infection, 25.2% had�1 late bacterial infection, and 30.5% had �1 late viral infection. Older age at transplantation was aprotective factor for late infection, both bacterial and viral (hazard ratio [HR] 0.89–0.92 per 1-year ageincrease, p � 0.001). Adjusting for age, race, and transplant etiology, late bacterial infection was associatedwith HMOX1 A�326G AG and GG genotypes (HR, 2.41, 95% confidence interval [CI] 1.35–4.30; p �0.003) and GZMB A-295G AA genotype (HR, 1.47; 95% CI; 1.03–2.1; p � 0.036). Late viral infection wasassociated with FAS A-670G GG genotype (HR, 1.42; 95% CI, 1.00–2.00; p � 0.050) in the adjusted model andwith CTLA4 A�49G AA and AG genotypes (HR, 1.49; 95% CI, 1.02–2.19; p � 0.041) in univariable analysis.CONCLUSION: We found an association between late bacterial infection and GP of HMOX1, which maycontrol macrophage activation. A weaker association was also found between late viral infection and GP ofCTLA4, a regulator of T-cell activation. This represents progress toward understanding the clinical andgenetic risk factors of outcomes after transplantation.J Heart Lung Transplant 2010;29:1342–51© 2010 International Society for Heart and Lung Transplantation. All rights reserved.

KEY WORDS:pediatric hearttransplant;bacteria infection;viral infection;genetic polymorphism

Reprint requests: Adriana Zeevi, PhD, Division of Transplant Pathology, Department of Pathology, W1551 Biomedical Science Tower, Pittsburgh, PA5261. Telephone: 412-624-1073. Fax: 412-624-6666.

E-mail address: zeevi@pitt.edu

053-2498/$ -see front matter © 2010 International Society for Heart and Lung Transplantation. All rights reserved.oi:10.1016/j.healun.2010.07.013

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1343Ohmann et al. Genetic Polymorphism and Infection in Pediatric Heart Transplantation

Although significant advances have been made in im-roving outcomes in pediatric heart transplant recipients,nfections are still a common and important cause of post-ransplantation morbidity and mortality.1,2 Immunosuppres-ion therapy and young patient age at transplantation haveeen associated with increased risk and rates of infection.2–4

patient’s genetic profile may influence his or her immuneesponse and susceptibility to infection and may also ex-lain some of the inter-individual variability seen in post-ransplantation infections.

Several studies have shown that genetic polymorphismsn immune response factors, including single nucleotideolymorphisms (SNPs) in cytokine, growth factor, and ef-ector molecule genes, are associated with clinical outcomesn infectious diseases5–9 and autoimmune disorders.10–13

olymorphisms of recipient genes involved in inflamma-ion, immune response, and tissue repair have also beenhown to be associated with worse outcomes after trans-lantation, including risk of rejection.14,15

We have previously demonstrated in large multicentertudies that the genotype distributions of cytokine androwth factor gene polymorphisms vary significantly byace and ethnicity16 and that high-expressing proinflamma-ory and low-expressing regulatory cytokine gene polymor-hism profiles are associated with an increased risk forcute rejection in pediatric heart transplant patients.15 In anngoing analysis of the same patient cohort, we have nowought to determine the influence of common polymor-hisms in cytokine, growth factor, and effector moleculeenes on the occurrence of late post-transplantation infec-ion in pediatric heart recipients and to assess whether anybserved associations are independent risk factors for latenfection.

ethods

articipants

his cohort consisted of pediatric heart transplant recipientsrom the 6 participating centers of the National Heart, Lungnd Blood Institute-sponsored Specialized Centers for Clin-cally Oriented Research (SCCOR) program for “Optimiz-ng Outcomes after Pediatric Heart Transplantation,” com-rising the University of Pittsburgh, Stanford University,oma Linda University, Washington University, Columbianiversity, and University of Alabama at Birmingham. Theatients were aged �18 years at transplantation and re-eived an allograft between January 1993 and December008. All patients in the study participated in the Pediatriceart Transplantation Study (PHTS), an international, pro-

pective, event-driven database study of risk factors forutcomes after listing for heart transplantation.17 Patientsere enrolled in the study after obtaining approval of the

nstitutional Review Boards of the participating centers andnformed consent from parents or guardians. All patients

ere maintained on immunosuppressive and anti-microbial

rophylaxis regimens according to individual institutionalrotocols.

ata collection

atients were monitored annually with additional data collec-ion forms completed at significant clinical events, includingejection, severe infection, post-transplant lymphoproliferativeisorder, retransplantation, and death. Demographic character-stics (gender, race/ethnicity, age at transplantation), transplanttiology, and clinical outcomes were collected prospectivelynd entered in the PHTS database. Late infection was defineds clinical evidence of an infectious process occurring �60ays after transplantation and requiring hospitalization, intra-enous antimicrobial therapy, or a potentially life-threateningnfection requiring oral therapy. For each infection episode, theate of infection, type of infection (bacterial, viral, fungal,rotozoan, or no organism identified), location of infection,herapy administered, and outcome were reported on standard-zed forms.

etection of genetic polymorphisms

sample of 3 to 6 ml anti-coagulated venous blood wasbtained from each patient enrolled in the SCCOR study.NA was extracted from peripheral blood mononuclear

ells by a standard phenol/chloroform procedure. Geno-ypes were assessed for the 18 SNPs in 12 immune responseenes listed in Table 1 by single specific primer-polymerasehain reaction (PCR) and/or sequencing, as previously de-cribed.15,16 PCRs were performed by a 7900HT v Fasteal-Time PCR System instrument (Applied Biosystems,oster City, CA), and data analysis was processed by SDS.2.2 software (Applied Biosystems).

Table 1 Genetic Polymorphisms of Interest

Class Gene Variant SNP ID

Cytokines IFN-� T�874A rs2430561IL-6 G-174C rs1800795IL-10 G-1082A rs1800896TNF-� A-308G rs1800629

Growth factors TGF�1 C�915G rs1800471VEGF A A-2578C rs699947

C�405G rs2010963Effector molecules CTLA4 A�49G rs231775

CT60 A/G rs3087243C-318T rs5742909

FAS A-670G rs1800682FASLG C-843T rs763110GZMB A-295G rs7144366

Q55R rs8192917HMOX1 A-489T rs2071746

A�326G rs11555832NOS2a Ser608Leu rs2297518

C�38G rs10459953

SNP, single nucleotide polymorphism.

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1344 The Journal of Heart and Lung Transplantation, Vol 29, No 12, December 2010

linical outcome definitions

he primary end points included time to late infection andrequency of late infections.

tatistical analysis

erification of Hardy-Weinberg equilibrium of genotypesas performed using the chi-square test (1 degree of free-om). Kaplan-Meier estimates of the proportion of patientsree from late infection were compared among sub-groupsefined by age at transplantation, recipient race, transplanttiology, and genotype at each genetic polymorphism ofnterest using log rank statistics. The hazard ratio of latenfection for each SNP was calculated using univariableox proportional hazard regression models. Nelson-Aalenstimates of mean number of infections per patient at 5ears after transplantation by genotype were calculated witheans and 95% confidence intervals (CI) reported. Analy-

es compared genotypes dichotomized into known high andow expression phenotypes as established in the literature.or SNPs without an established phenotype relationship, theeterozygotes were combined with homozygous rare vari-nts. A multivariable model, adjusting for recipient race,ge at transplantation, and congenital heart disease as trans-lant etiology, was created for each SNP significantly as-ociated with late infection in univariable analyses (p �.10). Adjusted hazard ratios (HR) and 95% CIs are re-orted. All analyses were performed using SAS 9.1 soft-

Table 2 Characteristics of Multicenter Pediatric Heart TransplInfection After Transplantation

Characteristic

All patients

P

B(N � 524) (No. (%) N

Age at Htx, years�1 165 (31.5)1–4 120 (22.9)5–12 124 (23.7)�13 115 (22.0)

SexFemale 225 (42.9)Male 299 (57.1)

RaceWhite 409 (78.1) 1Black 80 (15.3)Other 35 (6.7)

Transplant etiologyCardiomyopathy 260 (49.6)Congenital heart disease 236 (45.0)Myocarditis 16 (3.1)Other 12 (2.3)

HTx, heart transplantation.aTotal number of patients with infection � 255.bPercentage of row total.

are (SAS Institute, Cary, NC). y

esults

articipants

he study included 524 pediatric heart recipients. Averagege at transplantation was 6.2 � 6.1 years. Populationemographics and characteristics are presented in Table 2.ollow-up information was available for participants for anverage of 5.6 years (interquartile range, 2.6–9.6 years) andata was censored at 10 years.

ate infection

verall, 255 patients (48.7%) experienced 753 late post-ransplantation infections. The most commonly diagnosedate infections were bacterial (302, 40.1% of all late infec-ions), followed by viral infections (274, 36.9%). Late fun-al and protozoan infections were rare events, with 182.4%) and 11 (1.5%) infections, respectively. No organismas identified in 148 infections (19.7%).Among 225 patients who experienced late infection, 160

62.7%) had �1 late viral infection, 132 (51.8%) had �1ate bacterial infection, and 146 (57.3%) had �2 late infec-ions. For the 366 patients with �3 years of follow-up, aingle infection episode was reported in 87 (23.8%), 4813.1%) had 2 infections, 30 (8.2%) had 3 infections, and 3910.7%) had �4 infections during the first 3 years. Thestimated cumulative count of all infections per patient at 5

n Cohort and Patients With 1 or More Late Bacterial or Viral

s with infection �60 days post-HTx

al

p-value

Viral

p-value32)a (n � 160)a

b No. (%)b

�0.001 �0.001.0) 74 (44.9).7) 48 (40.0).3) 24 (19.4).0) 14 (12.2)

0.500 0.893.7) 68 (30.2).08) 92 (30.8)

0.104 0.735.7) 128 (31.3).5) 23 (28.8).3) 9 (25.7)

0.006 0.206.2) 78 (30.0).6) 78 (33.1).8) 3 (18.8).7) 1 (8.3)

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1345Ohmann et al. Genetic Polymorphism and Infection in Pediatric Heart Transplantation

ated 0.74 � 0.08 bacterial infections per patient and anstimated 0.62 � 0.07 viral infections per patient (Figure 1).

emographics, clinical factors and risk of latenfection

lder age at transplantation was a consistent protectiveactor for late infection, both late bacterial and late viralnfections (HR between 0.89 and 0.92 per 1-year increase inge, p � 0.001). In univariable analyses, recipient race wasot significantly related to late bacterial or viral infectionp � 0.05). Transplant etiology was not significantly asso-iated with late viral infection (p � 0.10), although congen-tal heart disease as transplant etiology was associated withate bacterial infection (HR, 1.81; p � 0.001).

requency of genetic polymorphisms

enotype distributions for all SNPs reached Hardy Wein-erg equilibrium in this population (p � 0.10). Table 3rovides the genotype frequencies of SNPs by recipientace.

enetic polymorphisms and late bacterialnfection

aplan-Meier and Cox regression unadjusted analyses ofate of late bacterial infection by genotype for the first 5ears after transplantation identified relationships betweenTLA4 A�49G, FAS A-670G, GZMB A-295G, HMOX1�326G, and NOS2a Ser60Leu and C�38G SNPs and lateacterial infection (Table 4). Figure 2 displays examples ofaplan-Meier curves of freedom from late bacterial infec-

ion by HMOX1 C�326G and GZMB A-295G genotypesxtending to 8 years of follow-up.

In multivariable Cox regression analyses adjusting forlack race, age at transplantation, and congenital heart dis-

Figure 1 Number of infections p

ase as transplant etiology, late bacterial infection was as- v

ociated with HMOX1 A�326G AG and GG genotypes,ith an adjusted HR of 2.41 (95% CI, 1.35–4.30, p �.003) and GZMB A-295G AA genotypes, with an adjustedR of 1.47 (95% CI, 1.03–2.10, p � 0.036), and showed a

rend with NOS2a C�38G C allele carriers, with an ad-usted HR of 2.26 (95% CI, 1.00–5.14, p � 0.051, Table 5).

Results obtained from an analysis of the frequency ofate bacterial infections per person by dichotomized geno-ypes were similar to those obtained from Kaplan-Meiernalyses (Figure 3A). We found relationships betweenGF�1 C�915G, CTLA4 A�49G, GZMB A-295G, andOS2a C�38G SNPs and the mean number of late bacterial

nfections per patient at 5 years after transplantation (p �.05). Notably, patients with GZMB A-295G AA genotypead more late bacterial infections than patients with GG andA genotypes (1.14 infections [95% CI, 0.98–1.31] vs..61 infections [95% CI, 0.52–0.69), and patients withOS2a C�38G CC and CG genotypes had more late bac-

erial infections than patients with GG genotype (0.79 in-ections [95% CI, 0.70–0.87] vs 0.39 infections [95% CI,.30–0.47].

enetic polymorphism and late viral infection

nadjusted Kaplan-Meier and Cox regression analyses ofate viral infection rates by genotype demonstrated asso-iations of late viral infection with CTLA4 A�49G andAS A-670G (Table 4; Figure 2). In Cox regressionnalyses adjusting for black race, age at transplantation,nd congenital heart disease as indication for transplan-ation, these same genetic polymorphisms showed a trendith late viral infection (p � 0.070 and p � 0.050,

espectively, Table 5).We also identified associations between CTLA4 A�49G

nd FAS A-670G SNPs and the estimated mean number ofate viral infections per patient at 5 years after transplanta-ion (p � 0.05, Figure 3B). Patients with CTLA4 A�49GA and AG variants experienced a greater number of late

nt over time, by type of infection.

iral infections than patients with the GG variant (0.70

1346 The Journal of Heart and Lung Transplantation, Vol 29, No 12, December 2010

Table 3 Frequencies of Genotypes in this Cohort by Race

SNP

Genotype frequency

p-value

Total White race Black race Other raceNo. (%) No. (%) No. (%) No. (%)(N � 524)a (n � 409)a (n � 80)a (n � 35)a

IFN� T�874A 0.169AA 193 (36.9) 141 (34.6) 36 (45.0) 16 (45.7)AT 247 (47.2) 196 (48.0) 37 (46.3) 14 (40.0)TT 83 (15.9) 71 (17.4) 7 (8.8) 5 (14.3)

IL-6 G-174C �0.001GG 274 (52.4) 179 (43.9) 66 (82.5) 29 (82.7)GC 183 (35.0) 164 (40.2) 14 (17.5) 5 (14.3)CC 66 (12.6) 65 (15.9) 0 (0.0) 1 (2.7)

IL-10 G-1082A �0.001GG 86 (16.5) 78 (19.1) 8 (10.0) 0 (0,0)GA 231 (44.3) 190 (46.6) 29 (36.3) 12 (35.3)AA 205 (39.3) 140 (34.3) 43 (53.8) 22 (64.7)

TNF� A-208G 0.593AA 13 (2.5) 11 (2.7) 2 (2.5) 0 (0.0)AG 120 (22.9) 96 (23.5) 19 (23.8) 5 (14.3)GG 390 (74.6) 301 (73.8) 59 (73.8) 30 (85.7)

TGF�1 C�915G 0.379CC 2 (0.4) 1 (0.3) 1 (1.3) 0 (0.0)CG 61 (11.7) 52 (12.8) 7 (8.8) 2 (5.7)GG 460 (88.0) 355 (87.0) 72 (90.0) 33 (94.3)

VEGFA A-2578C �0.001AA 91 (17.5) 85 (21.0) 3 (3.8) 3 (8.6)AC 248 (47.8) 202 (49.9) 26 (32.9) 20 (57.1)CC 180 (34.7) 118 (29.1) 50 (63.3) 12 (34.3)

VEGFA C�405G 0.081CC 60 (11.6) 43 (10.6) 14 (18.0) 3 (8.8)CG 221 (42.7) 181 (44.7) 23 (29.5) 17 (50,0)GG 236 (45.6) 181 (44.7) 41 (52.6) 14 (41.2)

CTLA4 A�49G 0.102AA 148 (28.4) 124 (30.5) 15 (19.0) 9 (25.7)AG 240 (46.1) 189 (46.4) 36 (45.6) 15 (42.9)GG 133 (25.5) 94 (23.1) 28 (35.4) 11 (31.4)

CTLA4 CT60 �0.001AA 90 (17.3) 81 (20.0) 4 (5.1) 5 (14.3)GA 252 (48.5) 205 (50.5) 30 (38.0) 17 (48.6)GG (low) 178 (34.2) 120 (29.6) 45 (57.0) 13 (37.1)

CTLA4 C-318T 0.003CC 442 (84.8) 336 (82.6) 78 (98.7) 28 (80.0)CT 75 (14.4) 68 (16.7) 1 (1.3) 6 (17.1)TT 4 (0.8) 3 (0.7) 0 (0.0) 1 (2.7)

FAS A-670G �0,001AA 119 (22.8) 101 (24.8) 8 (10.1) 10 (28.6)AG 254 (48.8) 212 (52.1) 25 (31.7) 17 (48.6)GG 148 (28.4) 94 (23.1) 46 (58.2) 8 (22.9)

FASLG C-843T �0.001CC 181 (34.9) 165 (40.6) 1 (1.3) 15 (42.9)CT 242 (46.6) 192 (47.3) 35 (44.9) 15 (42.9)TT 96 (18.5) 49 (12.1) 42 (53.9) 5 (14.3)

GZMB A-295G 0.463AA 130 (25.0) 96 (23.6) 25 (31.7) 9 (25.7)AG 252 (48.4) 196 (48.2) 38 (48.1) 18 (51.4)GG 139 (26.7) 115 (28.3) 16 (20.3) 8 (22.9)

GZMB Q55R �0.001AA 307 (59.2) 252 (62.1) 38 (48.1) 17 (50.0)GA 184 (35.5) 139 (34.2) 29 (36.7) 16 (47.1)GG 28 (5.4) 15 (3.7) 12 (15.2) 1 (2.9)

Continued on page 1347.

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1347Ohmann et al. Genetic Polymorphism and Infection in Pediatric Heart Transplantation

nfections [95% CI, 0.61–0.79] vs 0.41 infections [95% CI,.33–0.50]); and patients with FAS A-670G GG genotypead more late viral infections than patients with theA�AG genotypes (0.85 infections [95% CI, 0.72–0.98]s 0.54 infections [95% CI, 0.46–0.62]).

Table 3 Continued from page 1346.

SNP

Genotype frequency

Total White racNo. (%) No. (%)(N � 524)a (n � 409

HMOX1 A-489TAA 167 (32.1) 150 (37.0AT 249 (47.9) 190 (46.8TT 104 (20.0) 66 (16.3

HMOX1 A�326GAA 469 (90.4) 395 (97.5AG 42 (8.1) 10 (2.5)GG 8 (1.5) 0 (0.0)

NOS2a Ser608LeuCC 382 (73.5) 281 (69.2CT 126 (24.2) 114 (28.1TT 12 (2.3) 11 (2.7)

NOS2a C�38GCC 251 (48.6) 200 (49.6CG 219 (42.4) 169 (41.9GG 46 (8.9) 34 (8.4)

SNP, single nucleotide polymorphism.aFor each SNP, a small number of patients (n � 1–8) did not have

Table 4 Selected Results (p � .10) From Unadjusted Associatand Late Viral Infections

Genetic polymorphism Genotype

Estimated prop�1 late infectyears post-HTx

Late bacterial infectionCTLA4 AA � AG 0.292A�49G GG 0.206FAS GG 0.341A-670G AA � AG 0.241GZMB AA 0.368A-295G GG � GA 0.232HMOX1 GG � GA 0.518A�326G AA 0.243NOS2a Ser60Leu CC 0.297

TT � TC 0.189NOS2a CC � CG 0.282C�38G GG 0.142

Late viral infectionCTLA4 AA � AG 0.358A�49G GG 0.28FAS GG 0.424A-670G AA � AG 0.303

CI, confidence interval; HR, hazard ratio.

iscussion

nfection after transplantation is associated with significantorbidity and mortality for pediatric heart recipients. In thisulticenter study, we report on severe infections after heart

p-value

Black race Other raceNo. (%) No. (%)(n � 80)a (n � 35)a

�0.00112 (15.2) 5 (14.3)37 (46.8) 22 (62.9)30 (38.0) 8 (22.9)

�0.00140 (50.6) 34 (97.1)31 (39.2) 1 (2.9)8 (10,1) 0 (0,0)

0.00171 (89.9) 30 (85.7)7 (8.9) 5 (14.3)1 (1.3) 0 (0.0)

0.74835 (44.9) 16 (45.7)36 (46.2) 14 (40.0)7 (9.0) 5 (14.3)

e information.

etween Genetic Polymorphisms and Rates of Late Bacterial

with5 Log rank

p-valueUnadjusted HR(95% CI) p-value

0.083 1.46 (0.95–2.24) 0.09

0.037 1.46 (1.02–2.09) 0.038

0.005 1.67 (1.17–2.38) 0.005

�0.001 2.38 (1.50–3.77) �0.001

0.061 1.50 (0.98–2.31) 0.063

0.051 2.21 (0.98–5.02) 0.058

0.04 1.49 (1.02–2.19) 0.041

0.075 1.35 (0.97–1.87) 0.076

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1348 The Journal of Heart and Lung Transplantation, Vol 29, No 12, December 2010

ransplantation in 524 pediatric patients. Overall, 48.7%xperienced late post-transplantation infection. Althoughhe number of late bacterial infections (40.1% of all latenfections) exceeded the number of late viral infections36.9%), more patients experienced late viral infection62.7% of patients with late infection) than late bacterialnfection (51.8%). Our observed infection rates and etiologyistribution differ slightly from the initial PHTS report onnfection by Schowengerdt et al2 that reported 41% ofatients with at least 1 infection, with bacterial (60%) and

igure 2 Kaplan-Meier freedom from (top panel) late bacterial-295G, (B) HMOX1 C�326G, (C) CTLA4 A�49G, and (D) FA

Table 5 Selected Results (p � .10) From AdjustedAssociations Between Genetic Polymorphisms and Rates ofLate Bacterial and Late Viral Infectiona

Geneticpolymorphism Genotype

Adjusted HR(95% CI) p-value

Late bacterialinfection

GZMB A-295G AA 1.47 (1.03–2.10) 0.036HMOX1A�326G GG � GA 2.41 (1.35–4.30) 0.003NOS2a C�38G CC � CG 2.26 (1.00–5.14) 0.051

Late viral infectionCTLA4 A�49G AA � AG 1.43 (0.97–2.10) 0.070FAS A-670G GG 1.42 (1.00–2.00) 0.050

CI, confidence interval; HR, hazard ratio.aCox regression models include age at transplantation, recipient

sblack race, and congenital heart disease as transplant etiology.

iral (31%) infections the most common etiologies of in-ection in pediatric heart transplant patients. However, theirtudy included early post-transplantation infections and wasimited by a mean follow-up time of 11.8 months, whereasur study exceeded 5 years of follow-up. As has beenreviously reported, we found younger age at transplanta-ion to be a strong risk factor for infection after transplan-ation.1,2 We also found an association of congenital heartisease as an indication for transplantation with an in-reased rate of late bacterial infection.

In our analyses, we distinguished early infection fromate infection (�60 days after transplantation). Several stud-es have demonstrated increased risk of infection within therst month after heart transplantation.3,18,19 By emphasizing

ate infection and excluding early infection in our analyses,e reduced the influence of the immediate post-transplan-

ation milieu of intense immunosuppressive therapy, post-urgical complications, and nosocomial infections on ournalyses.

In this multicenter study, we identified biologically plau-ible associations between late viral infection after trans-lantation and SNPs in CTLA4 and FAS, and we demon-trated associations between late bacterial infection andenetic polymorphisms of GZMB and HMOX1.

Cytotoxic T-lymphocyte associated protein 4, ex-ressed by CTLA4, limits the proliferative response of Tymphocytes. The CTLA4 A�49G SNP encodes a mis-

ion and (bottom panel) late viral infection curves by (A) GZMB70G genotypes.

infect

ense mutation in the first exon, with the GG genotype

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1349Ohmann et al. Genetic Polymorphism and Infection in Pediatric Heart Transplantation

ssociated with decreased expression of membrane-ound CTLA (mCTLA4).20,21 Decreased expression ofCTLA4 may disrupt the downregulation and apoptosis of

ctivated cytotoxic T lymphocytes. A recent study sug-ested that the CTLA4 A�49G GG genotype was alsossociated with increased interferon-� production after im-une stimulation.22 We found an association of the A allele

t this SNP with the risk of late viral infection. Conversely,e found the GG genotype, with its decreased downregu-

ation of cytotoxic T cells, was associated with reduced riskf viral infection after transplantation. Adjusting for race, agend transplant etiology, the association between CTLA4�49G genotype and late viral infection did not reach signif-

igure 3 (A) Selected results of (A) Nelson-Aalen unadjustedstimated mean number of late bacterial infections and (B) esti-ated mean number of late viral infections per patient at 5 years

fter transplantation by genetic polymorphism. The error barsndicate 95% confidence intervals. *Results with p � .05. Darkray bars represent TGF�1 C�915G CC�CG, CTLA4 A�49GA�AG, FAS A-670G AA�AG, GZMB A-295G GG�GA,MOX1 A�326G GG�GA, and NOS2a C�38G CC�CG. Lightrey bars represent TGF�1 C�915G GG, CTLA4 A�49G GG,AS A-670G GG, GZMB A-295G AA, HMOX1 A�326G AA, andOS2a C�38G GG.

cance (p � 0.07). However, we did find an association be- f

ween CTLA4 genotypes and the number of late viral infectionser patient, with patients with CTLA4 A�49G AA and AGariants experiencing more late viral infections than patientsith the GG variant.FAS encodes the CD95 receptor that forms the death-

nducing signaling complex (DISC) upon binding its ligand,asL. CD95 plays a key role in apoptotic signaling for manyells, including those of the immune system.23 The FASolymorphism investigated in this study, A-670G, alters theTAT1 transcription factor-binding site in the promoter andecreases CD95 receptor expression.24,25 Thus, the GG ge-otype of FAS A-670G induces a state of decreased apo-totic signaling, which could impair a host’s ability toliminate infected cells, increasing the likelihood of spreadf the infection to neighboring cells and permitting patho-en persistence. In this study, we found the FAS A-670GG genotype was associated with an increased risk of lateost-transplantation viral infections.

HMOX1 encodes heme oxygenase-1 (HO-1), an induc-ble enzyme essential for heme degradation to biliverdinhat is converted to carbon monoxide and bilirubin by biliv-rdin reductase. HO-1 has cytoprotective, anti-oxidant, andnti-inflammatory effects. A polymorphism in the promoterf HMOX1 associated with slower induction was found toe associated with susceptibility to pneumonia in an olderapanese population.26 Zampetaki et al27 suggested thatver-expression of HO-1 in the setting of lipopolysaccha-ide induction represses cytokine release and decreases leu-ocyte adhesion molecule expression, inhibiting leukocyteecruitment. With the anti-inflammatory effects of HO-1ependent on the local concentration, it is plausible that aenetic polymorphism of HMOX1 could influence HO-1nduction and expression and, thereby, susceptibility to bac-erial infection. The HMOX1 A�236G SNP investigated inhis study has a functional significance (FS) score of 0.5,ndicating probable influence of transcriptional regula-ion.28,29 We found an association between the G allelearriers of this SNP and late bacterial infection as well as arend in greater number of late bacterial infections.

GZMB encodes granzyme B, a cytotoxin and serine pro-ease that can be produced by macrophages.30 Increasedevels of granzyme B have been associated with gram-egative infections,31,32 and genetic polymorphisms inZMB may decrease its cytolytic effect on target cells.33,34

lthough one study demonstrated no variation in granzymeexpression by genotype of GZMB A-295G,35 this SNP

as a FS score of 0.5, indicating probable influence onranscription factor binding.28,29 Our study found an asso-iation between the AA genotype of GZMB A-295G and anncreased risk of late bacterial infection and number of lateacterial infections.

We did not find associations between late post-transplan-ation infection and polymorphisms of VEGF A, IL-6, orL-10, which we previously reported to be associated withncreased risk for rejection in pediatric heart transplantatients.15 Thus, the mechanisms underlying genetic predis-osition to post-transplant infection may differ from those

or rejection. Previously, we reported a disparate distribu-

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1350 The Journal of Heart and Lung Transplantation, Vol 29, No 12, December 2010

ion of SNPs in IFN-�, IL-6, VEGFA, FAS, FASLG, GZMB,BCB1, and CYP3A5 among racial and ethnic groups ofediatric heart transplant patients. Pediatric heart recipientsf black race, primarily, and Hispanic ethnicity, secondarily,ere more likely to have genetic profiles that may predispose

hem to a proinflammatory and low regulatory immune state asell as pharmacogenetic profiles that may reduce immunosup-ressive drug exposure and efficacy.16 In this study, we presentew data demonstrating disparate distribution of genotypes ofTLA4 CT60, HMOX-1 A�236G and A-489T, and NOS2aer608Leu among racial groups of pediatric heart recipients.

Our multicenter study may be limited by differences inmmunosuppressive and anti-microbial prophylaxis regi-ens. Also, the criteria for initiating hospitalization or in-

ravenous anti-microbial therapy were individualized by in-titution, and diagnostic evaluation, including cultures anderologic testing, were not uniformly performed. Further-ore, the pediatric heart transplantation population is in-

erently heterogeneous, and compliance with therapy isariable in this population.

Although our patient population is larger than prior studies,ome influences of gene polymorphisms and their combina-ions may still be missed. Moreover, we have investigated onlysmall set of selected candidate genetic polymorphisms, and it

s likely that other potential genetic variations may influenceransplant recipient outcomes. Functionality of some of theenetic polymorphism has not been demonstrated in in vitro orn vivo studies. In addition, because our analyses were notdjusted for multiple comparisons in this exploratory study, theisk of finding spurious associations is possible.

Despite these considerations, our study identified poten-ially biologically plausible associations between GPs inffector molecules and increased risk of late post-transplan-ation infection in pediatric heart recipients. Genetic poly-orphisms of effector molecules that may affect innate

mmunity through macrophage activation were associatedith risk of bacterial infections. In contrast, genetic poly-orphisms in mediators of adaptive immunity, such as

egulators of cytoxic T cells and apoptosis, were associatedith viral infections. This represents progress toward un-erstanding the clinical, demographic, and genetic risk fac-ors of post-transplantation outcomes in pediatric heart re-ipients. We hope that by integrating the results of this studynd other studies investigating the genetic risk factors forost-transplantation outcomes, in the future, recipient geno-yping will allow us accurately to predict a patient’s post-ransplant course, enhancing our ability to tailor immuno-uppressive regimens to individual patient needs.

isclosure statement

his work was supported by grant 5P50 HL 074 732-05rom the National Heart Lung and Blood Institute, Nationalnstitutes of Health. Erin L. Ohmann is supported by fund-ng from Dean Arthur S. Levine, MD, and the Clinicalcientist Training Program at the University of Pittsburghchool of Medicine, and by the Doris Duke Clinical Re-

earch Fellowship program.

None of the authors has a financial relationship with aommercial entity that has an interest in the subject of theresented manuscript or other conflicts of interest to dis-lose.

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