Role of Genetics in Lung Transplant Complications

Role of genetics in lung transplant complications

D. Ruttens1, E. Vandermeulen1 , S.E. Verleden1, H. Bellon1, R. Vos1, D.E. Van Raemdonck1, L. Dupont, B.M. Vanaudenaerde1, G.M. Verleden1

1KULeuven,and UZ Leuven, Dept of Clinical and experimental Medicine, lab of pneumology, lung transplant unit

Running title: Genetics and outcome after LTx

Grants: Glaxo Smith Kline (Belgium) chair in respiratory pharmacology at the KU Leuven; grants from the Research Foundation Flanders (FWO): G.0723.10, G.0679.12 and G.0705.12; grant from the KU Leuven: OT10/050. SEV is funded by the research fund FWO. RV is supported by FWO and KOF UZ Leuven.

Address for correspondence: Prof. Dr. Geert Verleden Lab of Pneumology, Lung Transplantation Unit KU Leuven Herestraat 49, B-3000 Leuven, Belgium Tel: + 32 16 330 195 Fax: + 32 16 347124 E-mail: [email protected] ABSTRACT

There is increasing knowledge that patients can be predisposed to a certain disease by genetic variations in their DNA. Extensive genetic variation has been described in molecules involved in short AND long term complications after lung transplantation (LTx), such as primary graft dysfunction (PGD), acute rejection, respiratory infection, CLAD and mortality. Several of these studies could or were not confirmed or reproduced in other cohorts. However, large multi-centric prospective studies need to be performed to define the real clinical consequence and significance of genotyping the donor and receptor of a LTx. The current review presents an overview of genetic polymorphisms (SNP) investigating an association with different complications after LTx. Finally, the major drawbacks, clinical relevance and future perspectives will be discussed. Key words:

Lung transplantation - outcome - mortality - primary graft dysfunction- acute rejection - chronic rejection - chronic lung allograft dysfunction- single nucleotide polymorphisms

Key message:

- Genetic background may play an important role in the outcome after lung transplantation.

- Some genetic polymorphisms were associated with functional changes influencing outcome after lung transplantation.

- There is a stringent need for multicentric prospective studies to reveal the clinical consequence of SNP’s. Introduction

Lung transplantation (LTx) is the ultimate treatment option for selected patients suffering from specific end-stage pulmonary disorders. However, after LTx, mortality rates remain relatively high, mainly due to the occurrence of chronic rejection (1). Chronic rejection, also defined as chronic lung allograft dysfunction (CLAD), with bronchiolitis obliterans syndrome

(BOS) and restrictive CLAD being the most frequent presentations, is characterized by an irreversible lung function and threatens over 50% of the lung transplant recipients within 5 years after LTx Chronic rejection is characterized by an irreversible lung function decline in forced expiratory volume in 1 second of at least 20% compared to the 2 best post-operative values (2). Lately, it became increasingly clear that different phenotypes of chronic rejection exist, which has important clinical and scientific implications. As a consequence, the term chronic lung allograft dysfunction (CLAD) has been introduced, which encompasses all forms of chronic rejection. This led to a new classification system which takes the different manifestations of chronic rejection into account (3), including restrictive CLAD (or restrictive allograft dysfunction, RAS) and the classical form of CLAD which is obstructive and is best known as BOS (4). Many of the old studies cited in this paper used the old terminology and hence when they were investigating the prevalence of BOS, they were most likely investigating the incidence of CLAD.BOS CLAD is accepted to be both an alloantigen dependent and independent process for which many risk factors have been identified, including acute rejection, lymphocytic bronchiolitis, the presence of auto-antibodies against collagen V, colonization with micro-organisms and air pollution (2-4). These insults will activate the immune system and increase airway neutrophilia, which will lead to epithelial damage, excessive airway wall repair and finally fibrosis/obliteration of the airways (1;5).

The underlying mechanisms of CLAD remain to be elucidated. Not only CLAD but also primary graft dysfunction (PGD), acute rejection and respiratory infections are risk factors for mortality after LTx (6).

These studies so far ignored the vast genetic diversity within transplant recipients and donors.

It is well known that in large patients’ cohorts genetic background can be linked to human health and disease (7;8). Genetic predisposition has already proven to be important in many pulmonary diseases for example delta F508 mutation (9) in cystic fibrosis, MUC5B polymorphism in interstitial lung diseases (10), etc. Therefore, genetic predisposition may also play a role after LTx, although this has not yet been thoroughly investigated. The last decade, several groups provided evidence for the importance of the underlying genetic background regarding the outcome after LTx. Herein, we review current, but also historic evidence for the role of genetic predisposition in predicting the outcome after lung transplantation. We will specifically focus on complications after LTx, such as PGD, acute rejection, respiratory infection, CLAD and mortality. The major drawbacks, clinical relevance and future perspectives genotyping donor and/or recipient will also be discussed.

Primary graft dysfunction (PGD)

PGD, with an incidence of 10-30%, is the main cause of mortality and morbidity within the first 30 days after LTx (6). PGD is characterized by hypoxemia and radiographic infiltrates occurring within 72 h of LTx (11). PGD is subdivided in different grades (0, 1, 2, 3) according to the presence of diffuse alveolar infiltrates and the Pao2/FiO2 ratio; PGD grade 3 is defined as Pao2/FiO2 less than 200 with pulmonary infiltrates on X-ray (11). The primary outcome in all published genetic studies so far was any grade 3 PGD within 72 hours of reperfusion versus PDG <3. In such early phase after LTx, it seems logical that donor-related factors play a role in the development of PGD (12). The lung transplant outcome group

(LTOG) studies described specific pathways that are associated with the development of PGD, namely long pentraxin-3(PTX3) and the Prostaglandin E2 (PGE2) family. PTX3 is a phylogenetically conversed mediator of the innate immune response, shown to be involved in

PGD development (13). In this multi-centric study, blood samples of 654 LTx patients were included with genotyping of 10 haplotypes of PTX3. Two single nucleotid polymorphisms

(SNP) (rs2120243 and rs2305619) were associated with PGD. Because the levels of plasma

PTX3 demonstrated a wider variability among patients with idiopathic pulmonary fibrosis

(IPF) compared with those with chronic obstructive pulmonary disease, functional analysis of both SNPs focused on patients transplanted with IPF as underlying disease. The minor allele of the SNP (rs2305619) was functionally associated with higher levels of PTX3 before and

24h after LTx in patients with IPF (13 subjects with PGD compared to 34 without PGD) (14).

Secondly, they also studied the effect of PGE2 genetic polymorphisms on the development of

PGD in 680 lung recipients. Four SNPs in two genes of the PGE2 family, Prostaglandin E2 synthesis (rs13283456) and Prostaglandin E2 receptor (rs11957406, rs4434423, rs4133101), were associated with PGD. The Prostaglandin E2 receptor plays a central role in the immunomodulation and the control of inflammation mediated by PGE2 (15). Activation of the receptor inhibits activation and proliferation of T cells, driving cellular immunity (16). The immunosuppressive role of the PGE2 receptor was functionally demonstrated in 42 patients with increased Treg suppressor function in cells possessing the rs4434423 T allele, which was associated with lower PGD risk (24 subjects with PGD compared to 18 without PGD).

Thirteen other SNPs, coding for various other functions, from the in total about 1800 investigated genes in this LTOG study were associated with the development of PGD (17).

More details are demonstrated in table 1. Acute rejection

The diagnosis of acute rejection relies on the identification of lymphocytic infiltrates in lung tissue. Several studies have demonstrated that acute vascular (AR, acute rejection) or airway

(LB, lymphocytic bronchiolitis) rejection are the main risk factors for chronic rejection, the most common cause of death beyond the first year after LTx (18). AR and LB were defined on histopathology according to the ISHLT guidelines, and graded according to the severity of lymphocytic infiltration (A1-4/B1R-B2R) (19). The first study describing the association between genetic background and acute rejection (grade>A2) was done by the Pittburgh group.

Hundred nineteen LTx patients were analyzed for interleukin-10 (IL-10) genotype. IL-10 is an anti-inflammatory cytokine that is expressed in healthy airways (20) and has a possible protective effect in allografts (21;22). The genotype with functional increased IL-10 showed to be protective for the development of acute rejection. No direct correlations between acute rejection and specific genotypes for tumor necrosis factor α (TNF-α), transforming growth factor β1 (TGF- β1), IL-6 and Interferon-γ (INF- γ) were found (23). A few years before,

Jackson et al did not find an association with acute rejection (definition unclear) and 8 SNP’s of INF-γ, TNF-α, TGF- β1, IL-6 or IL-10 in 77 lung LTx patients (24).

In the past 10 years, Palmer and colleagues were the most prominent researchers investigating the genetic background of acute rejection after LTx. They proposed that innate immune immunity is primarily responsible for developing acute lung rejection (25). Toll-like receptors (TLR) are a family of innate immune receptors critical for initiation of innate responses to microbial pathogens (26). In the lung, TLR-4 is highly expressed on the alveolar macrophages and on the airway epithelium. Activation of TLR-4 induces an increased production of proinflammatory cytokines and chemokines and also increases the expression of major histocompatibility complex and costimulatory molecules on alveolar macrophages, which facilitate recruitment of additional immune cells and promote an effective adaptive immune response (27). Two SNPs in the TLR-4 gene (Asp299Gly and Thr399Ile, respectively rs4986790 and rs4986791) were shown to be functionally associated with endotoxin hyporesponsiveness and reduced rate of acute allograft rejection (28). The presence of these TLR-4 polymorphisms in the genetic profile of 147 receptors, and not of the donors, was associated with reduced frequency, severity, and incidence of acute rejection (≥A1), without influencing chronic rejection. This finding confirmed that innate immunity contributes in the development of acute rejection after LTx (29). The second study of this group described a polymorphism of CD14, an innate pattern recognition receptor that binds to lipopolysaccharide and promotes signaling through TLR-4 (30). The TT genotype of the SNP, located in the promoter of CD14 gene, has been associated with enhanced transcriptional activity of this gene (30%) and increased levels of soluble CD14 in peripheral blood (31;32).

The TT genotype of this SNP (rs2569190) was associated with enhanced immune activation, exhibiting increased risk for developing acute rejection (A or B grade) in 226 recipients (33).

In the last two years the Leuven lung transplant unit published three studies of genetic polymorphisms with primary outcomes chronic rejection and mortality, and secondary endpoints like acute rejection, clearly subdivided in AR and LB. Caveolin-1 (CAV-1) is involved in tissue homeostasis, as it has anti-inflammatory and anti-oxidative effects and it also increases apoptosis and bacterial clearance (34). In 503 LTx recipients the polymorphism in

CAV gene (rs3807989), however, did not have an effect on AR, nor on LB (35). Also, a polymorphism in the immunoglobulin G receptor polymorphism (IgGR) demonstrated no association with AR (36). The only study of the Leuven Lung transplant group that showed a correlation between a genetic polymorphism and acute rejection was interleukin-17 receptor

(IL-17R, rs879574). IL-17 is an inducer of airway neutrophilia with a proven effect in acute rejection (≥A1) (37). The genetic polymorphism (AA and AT) of the IL-17R in 497 LTx recipients was associated with an increased risk of developing AR, but not LB, with a functional increased risk of BAL neutrophilia compared to the TT genotype (38) (table 2).

Infections

Mitsani et al described a polymorphism in 170 LTx recipients linked with elevated levels of

INF-γ which was associated with an increased risk of cytomegalovirus disease (CMV). CMV is an accepted risk factor for the development of CLAD (39). No association between TNF-α,

IL-10 and IL-6 SNP’s and CMV infections was found. Palmer et al did not find an association between SNPs in TLR4 and infectious complications (40). In the previously described studies of IL-17 and CAV-1 polymorphisms, no association was demonstrated with respiratory infections. There was, however, a genetic link between the IgG receptor SNP and respiratory infections. IgG is a protein representing approximately 75% of serum immunoglobulins. Low levels of IgG were associated with increased number of respiratory infections (41). The genotype (TT) at risk resulted in more respiratory infections and respiratory infections per patient compared to the other genotypes. The finding of the increased risk for respiratory infections was probably an indirect proof of the functionality of this SNP (rs12746613)(38).

As mentioned before, most of these studies looked at CLAD, without making a distinction between the different forms of CLAD. The first study to describe a link between genetic polymorphisms and CLAD after LTx was performed by Awad et al (42). A functional gene of

INF-γ, an inflammatory cytokine that has been implicated in the development of fibrosis in inflamed tissues (43), was associated, with increased alveolar graft fibrosis, in 82 transplant recipients. In a second study, this group, demonstrated one SNP (Major allele codon 25), to be associated with an increased production of TGF- β1, a profibrotic cytokine (44). This SNP was also associated with an increased risk of post-LTx alveolar graft fibrosis (45). The third study of this group confirmed the findings of the TGF- β1 SNP (Major allele codon 25) and demonstrated that a second TGF- β1 SNP (Cytosine dilatation) was also associated with allograft fibrosis (46). In 2002, Lu et al published a genetic polymorphism of INF-γ and concluded that there was an earlier development of CLAD after LTx, in recipients (n=93) with the genotype at risk. The same association was found with an IL-6 polymorphism.

Although in this study, no association was found between CLAD and genetic polymorphisms of TNF-α, TGF- β1 and IL-10 (47).

These positive studies for IL-6, INF-γ and TGF- β1 were not confirmed in other studies,

Jackson et al did not find an association between CLAD and SNP’s of INF-γ, TNF-α, TGF-

β1, IL-6 and IL-10, neither did Snyder et al in 2 independent cohorts (48).

The TLR-4 genotype (rs4986790, rs4986791) had an effect on the acute rejection rate as previously described, but had no effect on CLAD development (29;40), whereas in the study of Palmer et al regarding CD14 (rs2569190), an association with CLAD was indeed present.

LTx recipients with the TT-genotype had a higher incidence and earlier development of

CLAD (33). Six SNPs for mannose binding lectin (MBL), a recognition molecule for innate immunity (49), were studied in 181 donors and 198 LTx recipients a few years later. MBL deficiency has been associated with increased morbidity and mortality in other solid organ transplantations (50;51). The recipients who received a graft from a donor with a heterozygote variant of a MBL SNP located in a promotor region developed less CLAD compared to those with the homozygote variant (52). In 2010 and 2011 the group of Utrecht published several papers studying genetic polymorphisms and CLAD after LTx. The first study described 64 polymorphisms, in 10

TLRs genes (TLR1 to TLR10) in 110 LTx patients. They showed an association of TLR2

(rs1898830 and rs7656411), TLR 4 (rs1927911) and TLR9 (rs352162 and rs187084) with

CLAD: homozygotes of the major allele of rs187084, rs1898830 and rs352162 had an increased risk to develop CLAD compared to the carriers of the minor allele and the homozygotes of minor allele of rs7656411 and rs1927911 had an increased risk to develop

CLAD compared to the carriers of the minor allele (53). A second study in the same cohort, of a SNP of matrix metalloproteinases-7(MMP-7), important in lung repair (54), demonstrated an increased risk for CLAD development. The increased risk was found in patients with a homozygote variant for the major alleles of rs177098318, rs11568818 and rs12285347 and the minor allele of rs10502001. Two other studied SNPs revealed no association with CLAD.

Functionally, patients homozygous for the major alleles of rs11568818 and rs12285347, had lower concentrations of MMP-7 compared to homozygotes of the minor allele (55). In the third of the Utrecht group, 4 SNPs of the already described CAV-1 gene were genotyped.

Homozygosity of the minor allele of rs3807989 was associated with an increased risk for

CLAD and this SNP was also associated with increased levels blood of CAV-1 (56).

In 2012 D’Ovidio and colleagues published a study of a surfactant protein A (SP-A) SNP, playing an important role in the innate host defense and which may serve as cross-talk protein between innate and adaptive immune response (57). Patients with low SP-A mRNA levels associated with specific genetic phenotype in the donor lungs were detected, but there was no clear relation with CLAD (58). Compared to the study of two genetic polymorphisms of SP-

D, which has a comparable mechanism of SP-A (57), an association with CLAD was found in one SNP. In 191 LTx patients, the homozygote variant of a genetic polymorphism, altering amino acid in the mature protein N-terminal domain codon 11(Met11Thr) of donor DNA had an increased rate of CLAD compared to the heterozygote variant (59).

Bourdin and colleagues studied a donor and receptor clara cell secretory protein (CCSP) polymorphism. Clara cells are bronchiolar stem cells characterized by unique morphologic features and are crucial for to small airway repair processes and epithelium integrity (60). In

63 LTx patients, this polymorphism was associated with an increased risk of CLAD and a functional decrease in CCSP BAL levels was observed (61).

The studies published by our own group on the CAV-1 and IgG polymorphisms did not find an association with development of CLAD (35;36). The study of the IL-17R polymorphism

(rs879574), not only an inducer of acute but also CLAD (37), in a cohort of 497 LTx patients revealed that the allele at risk (AA/AT compared to TT) was associated with an increased susceptibility to CLAD. As already mentioned before, this polymorphism had a functional increased risk of BAL neutrophilia (38). For a summary and more details see table 3.

Mortality

A lot of the previously described studies do not only have CLAD (and more specifically

BOS) as an endpoint, they also described the association between genetic polymorphisms and mortality. The Manchester group was the first to described an association between genetic polymorphisms and mortality in 91 LTx patients. While only one SNP of TGF-β1 had an effect on CLAD, LTx recipients who were homozygous for both one non-functional variant and the functional genetic variant of TGF-β1 showed poor survival (45). The TLR4 study of

Palmer and colleagues in 2004 could not find and association with mortality (40). In the first cohort (n=76) of Snyder et al, the IL-6 polymorphism (GG and GC) was associated with a worse survival, while in the second bigger cohort (n=198) this association could not be confirmed (48). The TT genotype of a CD14 SNP (rs2569190) was associated with enhanced immune activation, exhibiting not only an increased risk for developing acute/ CLAD, but the

TT genotype was also associated with increased mortality in 226 recipients (33). The same observation was seen in the study of donor MBL promotor SNP, whereas the recipients who received a graft from a donor with a homozygote variant of an MBL SNP had higher mortality compared to recipients who received a graft from a donor with the wild type variant

(52). D’Ovidio et al demonstrated that several SP-A2 polymorphisms were associated with lower SP-A mRNA expression and with increased mortality (58). In addition to this study, a homozygote variant of a genetic polymorphism SP-D, altering amino acid in the mature protein N-terminal domain codon 11(Thr11Thr) of donor DNA in 191 LTx patients, not only resulted in an increased rate of CLAD, but also in a worse survival compared to the heterozygote variant (59). In 63 LTx patients, the functional donor CCSP SNP was associated with an increased risk of mortality (61).

In contrast to CLAD, for which no association was found with CAV-1 and the IgGR polymorphism, mortality was affected by these 2 genetic polymorphisms. The (AA+AG) genotypes of rs3807989 (CAV-1 SNP) resulted in a worse survival compared to the GG genotype (35). The IgGR (rs12746613) polymorphism was associated with a higher risk of mortality in the TT-genotype compared with the CC-genotype in 418 patients (36). In the IL-

17 polymorphism study, no significant association was found with mortality (38).

Some of the studies showed a difference in CLAD, but not in mortality. This is possibly due to the fact that mortality after lung transplantation is related to different causes such as infection, post-operative complications and not always due to CLAD. Even late post-operative mortality can be totally unrelated to CLAD but due to for example cancer or infection. It would be interesting to look at CLAD-related mortality, but unfortunately this was not done in the majority of the cited studies.

Major drawbacks and clinical relevance

Genetic influences on LTx outcomes belong to complex disease pathways, where not only the patient (receptor) but also the donor should be considered. This statement already indicates the first concern in the interpretation of genetic studies. Most of the studies were performed on the receptor DNA without taking the genetic profile of the donors into account. There is also an important lack of reproducibility of some results which may be due to several reasons:

1) inadequate (too low) power, 2) poorly defined endpoints, 3) failure to correct for confounders, 4) retrospective studies, 5) short follow-up time and 6) no replication cohort.

Another problem is the historical effect, whereas some cohorts go back to 1990 for inclusion of the first patients. Since then, lots of improvements were made in donor preservation, selection recipients, operative techniques, post-operative management, medications, etc and it is very difficult to correct for this. Nevertheless, the genetic background of a recipient or donor lung can be considered to be important for later post-LTx outcome. Knowing the genetic profile of the donor and receptor could be used in a preventive way, indicating that closer follow-up might prevent complications. For example if one knows that there is a higher risk for infections, broader prophylactic precautions may be warranted.

Future prospectives LTx is a rather infrequent treatment option which makes acquiring a high number of patients for genetic analysis difficult. As a consequence, there is certainly need for multi-centric studies of patients with comparable genetic background, as nowadays performed by the

LTOG, to increase the number of patients. Secondly, it would be ideal to confirm findings in a comparable, replication cohort. Thirdly, the genetic variations that are studied should ideally be functional, for example as is obvious from other solid organ transplantations, or functionality should at least be confirmed, for example by measuring protein levels that are affected by the investigated gene. Fourthly, genetic analysis could be very interesting in the ongoing discussion of the different phenotypes of CLAD. Genetic information could be important in the search for a potential differential mechanisms driving BOS and RAS as both forms of CLAD are likely to have different genetic risks. The progress in genotyping techniques makes it now possible to test large cohorts with a large set of genetic variations in terms of genome-wide association studies (GWAS). The strength of the genome wide screening is its ability to reveal not only the gene that would be expected to play a role, but also other genes leading to new insights into pathophysiology. There may also possible be a role for epigenetic studies in LTx. This is the study of changes in gene expression caused by certain base pairs in DNA, or RNA, being "turned off" or "turned on" again, through chemical reactions. Epigenetics is mostly the study of heritable changes that are not caused by changes in the DNA sequence; to a lesser extent, epigenetics also describe the study of stable, long- term alterations in the transcriptional potential of a cell that are not necessarily heritable (62).

In the hope that epigenetics reveal differences in the pathophysiological mechanisms of the phenotypes of CLAD and that altering epigenetics in transplanted organs will ultimately lead to a higher quality of life for transplant patients (63). Conclusion

Several SNPs have been associated with various outcome parameters after LTx. However, there is a stringent need for prospective multi-centric studies and replication of these findings, in order to make current data more robust and to reveal the clinical consequences. Despite the limitations of using data obtained from genetic studies in LTx, the challenge of incorporating this research into clinical care must be pursued in order to improve our understanding of pathogenesis of post-LTx complications and, more important, to achieve improved treatment or prevention, resulting in better outcome after LTx. Reference List

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Reference n P Age Male DLTx ID Gene G (%) (%) number D Diamond(14 654 Y 52 337(52) NA rs2120243 Long pentraxin-3(PTX3) ) e rs2305619 Long pentraxin-3(PTX3) s rs9289983 Long pentraxin-3(PTX3) Y rs1456099 Long pentraxin-3(PTX3) e rs35948036 Long pentraxin-3(PTX3) s rs3816527 Long pentraxin-3(PTX3) N rs55757068 Long pentraxin-3(PTX3) o rs3845978 Long pentraxin-3(PTX3) N rs35415718 Long pentraxin-3(PTX3) o rs4478039 Long pentraxin-3(PTX3) 680 N 53 359(53) NA rs1328345 PTGES2 Diamond(18 o 6 PTGER4 ) N rs1195740 PTGER4 o 6 PTGER4 N rs4434423 TBC1D1 o rs4133101 TBC1D1 N rs2996044 TBC1D1 o rs2925956 PMCH N rs1313218 F13A1 o 4 GAA N rs7973796 CAV3 o rs3024388 COL4A1 Y rs1245261 CASP8 e 6 IRX4 s rs237865 ITGB5 Y rs1758859 PRKG1 e 1 FTCD s rs1683696 Y 5 e rs260400 s rs3772843 Y rs1881597 e rs1700450 s 4 Y e s Y e s Y e s Y e s Y e s Y e s Y e s Y e s Y e s Y e s Y e s Y e s Y e s Table 1: Overview of all studies with possible associations between genetic background and PGD after LTx. PGD= primary graft dysfunction, DLTx= bilateral lung transplantation, D/R= material from donor/receptor, funct= functionality, NA= Not available/ stated. Yes/no describes the possible association between the SNP and PGD. Functionally gives an indication whether the SNP is associated with changes in gene related protein expression or activity. Reference n rejection

ID number

Gene function

Location

Jackson(25) Zheng(24)

NA 55(46)

41(34)

R -308

Codon 20

Codon 25

TNF-α

TGF-β1

INF-γ TNF-α

TGF-β1

Cell death

Cell growth and differentiation

Immunoregulation

Acute phase response

Pro-inflammatory

Cell death

Cell growth and differentiation

Immunoregulation

Promotor

Promotor Promotor

Intron

Palmer(30) Palmer(34)

Ruttens(39)

Vandermeulen(36)

Ruttens(37)

497 503

81(54)

130(58)

263(53) 268(53)

208(50)

116(79)

190(84)

374(75)

373(74)

314(75)

133(89) 204(90)

485(98)

491(98)

408(98)

NA rs4986790 rs4986791 rs2569190 rs879574 rs2201841 rs10489628 rs2066808 rs1343151 rs1569922 rs3807989 rs12746613

INF-γ

CD14 IL-17R

IL-23R

IL-23A

IL-23R

FCGRA2

Acute phase response

Pro-inflammatory

Innate immune activity

Tissue homeostasis IgG receptor/ metabolisme

Coding

5′ UTR

Intron

1q23.3

Yes NA

Table 2: overview of all studies with possible associations between genetic background and acute rejection. n= n-value, DLTx= bilateral lung transplantation,D/R= donor/ receptor, funct= functionality , NA= Not available/ stated. Yes/no describes the possible association between the SNP and acute rejection. Functionally gives an indication whether the SNP is associated with changes in gene related protein expression or activity.

Reference n Rejection Age Male DLTx Race ID Gene Function Location Funct (%) (%) White(%) numb er

Reference n CLAD Age DLTx Race (%) White(%) Awad(45) 82 Yes NA NA NA El-Gamel(48) 91 No 39 50(55) NA Yes Awad(49) 95 No NA NA NA No Yes No Yes Jackson(25) 77 No NA NA NA No No No No No No No Lu(50)(64)(65)(67) 93 Yes NA NA NA Yes No No No Palmer(30) 170 No 49 139(82) 153(90) No Snyder(51) 78 No 44 38(49) 69(88) 198 No 49 165(83) 180(91) No No No Palmer(34) 226 Yes 49 190(84) 204(90) Munster(55) 277 No 43 216(78) NA Yes No No No No

Kastelijn(56) 110 Yes 50 93(85) NA Yes Yes Yes Yes No Kastelijn(58) 110 Yes 50 93(85) NA Yes Yes Yes No No Kastelijn(59) 110 No 50 93(85) NA No Yes No D’Ovidio(61) 42 No 50 33(79) NA No Aramini(62) 191 Yes 57 141(74) 155(51) No Bourin(64) 63 Yes 49 49(78) NA Ruttens(39) 497 Yes 48 374(75) 485(98) No No No No No Vandermeulen(36) 503 No 48 373(74) 491(98) Ruttens(37) 418 No 48 314(75) 408(98)

Reference n CLAD Age Male SSLTx Race White

Table 3: different genetic background studies with an association with CLAD after lung transplantation after LTx. CLAD= chronic lung allograft dysfunction, DLTx= bilateral lung transplantation, D/R= material from donor/receptor, funct= functionality, NA= Not available/ stated. Yes/no describes the possible association between the SNP and chronic rejection. Functionally gives an indication whether the SNP is associated with changes in gene related protein expression or activity.

Reference n CLAD Age Male Race D/R (%) White(%) El-Gamel(48) 91 Yes 39 NA NA R Yes Palmer(30) 170 No 49 94(55) 153(90) R No Snyder(51) 78 No 44 40(51) 69(88) R 198 No 49 110(56) 180(91) R No No No Palmer(34) 226 Yes 49 130(58) 204(90) R Munster(55) 277 No 43 150(54) NA D+R Yes No No No No D’Ovidio(61) 42 No 50 23(55) NA D Yes Aramini(62) 191 Yes 57 90(47) 155(51) D No Bourin(64) 63 Yes 49 30(48) NA D+R Ruttens(39) 497 No 48 263(53) 485(98) R No No No No No Vandermeulen(36) 503 Yes 48 268(53) 491(98) R Ruttens(37) 418 Yes 48 208(50) 408(98) R

Reference n MOR Age Male DLTx Race (%) (%) White

Table 4: different genetic background studies with an association with mortality after lung transplantation after LTx. MOR= mortality, DLTx= bilateral lung transplantation, D/R= material from donor/receptor, funct= functionality, NA= Not available/ stated. N-value(%).Yes/no describes the possible association between the SNP and mortality. Functionally gives an indication whether the SNP is associated with changes in gene related protein expression or activity.