Lung transplantation is often offered as a therapeutic intervention for end-stage lung disease in young individuals. As such, in genetic epidemiology studies of such end-stage lung diseases, confounding of genotype results by lung donor DNA is a theoretical concern that could bias the results of genetic linkage and association studies if DNA samples are collected after lung transplantation. Our assessment of STR markers and SNPs in 10 individuals with severe, early-onset COPD before and after lung transplantation suggests that donor DNA chimerism does not interfere with routine STR and SNP genotyping.
Although the degree of peripheral chimerism associated with solid organ transplantation is not as high as chimerism associated with bone marrow transplant, low levels of donor cells have been detected in the skin, lymph nodes, and blood of liver transplant recipients even several years after transplantation (2
). In bronchoalveolar lavage samples from 10 patients who underwent lung transplant, donor lymphocytes were not detectable in any of 10 patients after 3 mo; however, low level of donor macrophages remained detectable 2 yr post-operatively. Wiebe and colleagues concluded that chimerism in the lung at the macrophage level may persist (3
). Using Y chromosome–specific DNA probes in female recipients of lungs from male donors, Kubit and coworkers quantified circulating donor cells in the blood, and observed that although donor cells could be detected in a variety of organs (such as heart, skin, kidney, lymph nodes, and spleen, as well as native lung) as a result of migration from the lung, the number of cells was relatively small (4
). Through investigation of donor-specific HLA-DRB1, Knoop and colleagues reported the detection of donor cells in single lung and heart-lung transplant recipients (5
). This group also noted that although blood chimerism as measured with HLA DRB1 was somewhat elevated in all patients in the immediate postoperative setting and during the first three post-transplant months, this decreased to a low level at 1 yr (6
). Because all of our study participants were at least 3 mo post-transplant, we cannot exclude chimerism in the very early postoperative period. Reinsmoen and colleagues observed blood microchimerism in 77% of lung and 91% of heart allograft recipients at a range of 12–18 mo after transplantation (7
). In another report they detected blood microchimerism in 47% of lung allograft recipients more than 11 mo post-transplant (8
). This same group has observed a positive correlation between blood microchimerism levels and FEV1
after lung transplantation (9
The assessment of chimerism may be most efficient through the use of techniques to assess donor versus recipient HLA loci. In the case of female recipients who receive lungs from male donors, this assessment may be undertaken by quantitative polymerase chain reaction amplification of SRY antigens (10
). STR markers have also been used to quantify mixed chimerism (11
). Concordance between pre- and post transplant results for STR genotypes of a panel of highly polymorphic STRs in our study suggests an absence of chimerism of a level that would influence large-scale genetic epidemiology studies. Kleeberger and coworkers demonstrated that for detection of microchimerism in liver transplant recipients, STR PCR assays may detect a chimeric state down to 5% (14
). One important advantage of the use of STRs is that they are independent of sex mismatching (which is necessary for use with X and Y chromosome specific antigen probes). This has relevance in genetic epidemiology studies, as the donor sex (or HLA information) is not readily collected as part of routine protocols in epidemiology studies that may include post-transplant individuals. Although microchimerism may exist, it is unlikely to influence the validity of genotyping results for genetic epidemiology studies of STR markers and SNPs assessed with more routine genotyping protocols.
Weaknesses of our approach to this hypothesis include lack of HLA donor and recipient information and lack of knowledge of the donor genotypes at each SNP locus. If we had been able to obtain donor genotype information, genotyping protocols with an increased PCR cycle number could have been used to determine if donor genotype could be detected. However, the probability of matching between a lung donor and recipient at each locus for all microsatellites and SNPs tested in this study is highly unlikely. Based on the estimated genotype frequencies for each SNP calculated from the allele frequencies observed in the parents (), the probability of an identical random donor match for all 23 SNPs was < 1 × 10−6. This exceedingly small probability suggests that all of the genotypes that we observed were likely those of transplant recipients uninfluenced by chimerism. As suggested above, one advantage of STR markers to detect chimerism is that they are likely independent of sex mismatching between lung donors and recipients. However, in our retrospective assessment, we had only one sex-mismatched recipient–donor pair, which limits our ability to directly compare STR and SRY approaches to detecting chimerism. Benefits of using STRs and SNPs to assess for chimerism include that the methods are readily automated at a low cost. Demonstrating the fidelity of these methods increases the possible number of participants in genetic epidemiology studies of end-stage lung diseases.
In conclusion, genetic epidemiology studies of end-stage lung disease can include post–lung transplant individuals, as the risk of bias due to contribution of donor DNA chimerism in investigations of STR markers and SNPs appears to be quite low. Expanding the pool of potential participants in genetic epidemiology studies by not excluding post-transplant individuals will provide the opportunity for larger scale studies with higher power to detect genetic associations relevant for advancing the understanding of a variety of severe lung diseases.