HLA-matched unrelated donors and patients share the same HLA alleles, however they may differ for MHC variation that is linked to their HLA haplotypes (8
). We hypothesized that the success of unrelated HCT might be influenced by undetected variation within the MHC, given the extreme density of immune-related genes in this region (1
). We used a discovery-validation study design to identify non-HLA transplantation determinants and confirm putative variants in large homogeneous transplant populations. We found that HLA-matched patients and unrelated transplant donors can differ for SNPs across the 4.9 megabase MHC region, indicating that haplotype content can differ among individuals who share the same HLA alleles. Two SNPs were validated as robust markers for disease-free survival and acute GVHD risk. These results demonstrate that the transplant barrier is defined by not only by classical HLA genes, but also by non-HLA genetic variation within the MHC.
A hallmark of the human genome is the positive association of two or more genetic markers together (linkage disequilibrium) (13
). The associated markers can be complex genes, simple bi-allelic SNPs, or other forms of variation (13
). Nowhere in the human genome is linkage disequilibrium as strong and long-range as in the MHC, a phenomenon that hampers the identification of true causal variants in disease (1
). To surmount these potential methodologic challenges, we studied only HLA-A, -C, -B, -DRB1, -DQB1-matched pairs (6
). After excluding the possibility that SNPs were markers for leukemia or myelodysplastic syndrome (Bonferroni P
> 0.00004), we tested but did not find any effect on outcome of HLA-C alleles in positive linkage disequilibrium with rs887464. Associations between SNP genotype and outcome were elucidated in pairs matched for rs887464 and rs2281389; no association of rs887464 with disease-free survival, or of rs2281389 with risk of acute GVHD was evident. Finally, adjustment for HLA-DPB1 mismatching, a known risk factor for GVHD (16
), yielded clear association of rs2281389 mismatching to GVHD risk. These results collectively demonstrate that undetected patient-donor mismatching for SNPs is an independent risk factor for transplant outcome after HLA-matched unrelated transplantation.
There are several limitations to the current study. In traditional disease-association mapping, the over (or under) representation of specific SNP genotypes in a disease cohort compared to healthy individuals signals the presence of potential causative genes (13
). In transplantation, not only could causative genes be defined by the genotype of SNPs carried by the patient or the donor, but if the causative gene functions in ways that are similar to the recognition of mismatched HLA antigens, then donor mismatching or patient mismatching for the SNP could be useful for mapping causative genes in transplantation. The vector of SNP incompatibility could serve as a useful tool for not only identifying the causative gene(s), but also could shed light on the mechanisms through which those genes provoke GVHD. However, if those genes are monomorphic then a vector analysis would not be enlightening. Fine mapping of the MHC near the SNPs of interest will help clarify the nature of the genetic variants responsible for risks after HLA-matched transplantation, and their potential mechanisms. Finally, certain world populations are distinguished by a few unique and highly conserved HLA haplotypes, resulting in a high frequency of HLA homozygosity with low SNP diversity (17
). The presence (or absence) of such conserved haplotypes may themselves be associated with clinical outcome (17
). The exceedingly low frequency (2%) of HLA homozygous patient-donor pairs in our predominately Caucasian study population precluded a definitive analysis of the clinical significance of specific HLA haplotypes. A larger transplant experience of HLA homozygous pairs will help to clarify the nature of risks associated with specific extended HLA haplotypes.
The association of patient-donor SNP mismatching with clinical outcome signals the presence of linked genetic variation that is biologically meaningful (13
). Although the functional variants remain to be elucidated, the vector of the SNP mismatch is clinically relevant, suggesting genes or pathways that involve recognition of patient mismatching (rs2281389) or donor mismatching (rs887464). In this regard, it is intriguing that rs887464 is a putative expression quantitative locus (eQTL) for CCHCR1, TCF19, HLA-B, HLA-C, MICA and MICB genes within the MHC (18
). Interestingly, SNP rs887464 is a risk marker for autoimmune diseases including type 1 diabetes (19
). The association of rs887464 mismatching to disease-free survival in the current study raises the possibility that the level of HLA class I gene expression is an important determinant of transplant outcome, as well as the potential role for the non-classical class I genes MICA and MICB as ligands for the innate immune system (2
). Fine mapping of the regions 5′ and 3′ of rs887464 will clarify whether this SNP is a marker for a non-HLA genetic variant, or whether the SNP is effecting transplant outcome through differential expression of a target gene. If the latter is found, then linkage disequilibrium of rs887464 to the alleles of a target gene could help explain the association of rs887464 mismatching rather than genotype to outcome. If each of the two rs887464 alleles define haplotypes of polymorphic alleles of the target gene, then patient-donor mismatching at rs887464 could lead to clinical effects through allele-specific alterations in the expression of patient and/or donor alleles.
The association of rs2281389 mismatching with GVHD risk provides new information on the class II region. SNP rs2281389 maps 2 kb centromeric to the HLA-DPB1 3′ untranslated region and is a risk marker for pediatric asthma (21
), rheumatoid arthritis (22
), and Hodgkin lymphoma (23
). In the current study, mismatching for rs2281389 and not rs9277535 was statistically significantly associated with acute GVHD, although rs9277535 showed similar trends (HR 1.37 for GVH mismatch). SNP rs9277535 is a putative trans-eQTL and micro RNA binding site in the HLA-DPB1 3′ untranslated region (18
). In this context, our observations suggest a possible role of HLA-DPB1 haplotype-linked expression in GVHD, where we surmise that rs2281389 alleles define haplotypes with DPB1 alleles; depending on the specific DPB1 mismatch combination in a given patient and donor, differential expression of the mismatched alleles could place a patient at increased or lowered risk of GVHD. Given that GVHD is a highly inflammatory state (25
), it is not surprising that there may be pathways common to GVHD, autoimmunity, and infection.
Simple bi-allelic SNPs can be genotyped at high efficiency (13
). The results of the current analysis suggest that clinical outcome after HLA-matched unrelated donor transplantation can be improved through prospective donor evaluation of SNPs. The discovery-validation cohorts provide information on SNP match rates between the patient and the donor who was selected based on HLA match status but not on SNP criteria. Those data show that most HLA-matched patients and donors were matched for rs887464 whereas only 60% were matched for rs2281389, even among HLA homozygous pairs with common HLA haplotypes. Given a 60:40 match:mismatch rate for rs2281389, the results suggest that patients who prospectively identify three HLA-matched unrelated donors have a 90% chance that at least one donor will be rs2281389-matched. To demonstrate the feasibility of integrating SNP genotyping into donor selection, we analyzed patients who had identified two or more otherwise eligible HLA-matched donors on their search. The analysis of searchable donors provides information on rs2281389 match rates among a pool of individuals who share the same tissue type. The data demonstrate that SNP genotyping can be translated easily into donor selection. Furthermore, among patients with HLA-matched donors, the probability of having a choice of rs2281389-matched donors increases as the number of tested donors increases. Since patient-donor mismatching at HLA-DPB1 and rs2281389 each independently increase GVHD risk, future efforts to match donors for both genetic determinants are expected to lower risks for patients. With over 18 million unrelated donors registered worldwide (27
), the potential to benefit future patients in need of a life-saving transplant is anticipated to be significant.
In conclusion, the transplant barrier is comprised of classical HLA loci as well as non-HLA variation within the gene-dense MHC region. Two new genetic markers are informative for disease-free survival and acute GVHD after HLA-matched unrelated donor transplantation. The identification of MHC resident transplantation determinants provides clinicians with tools to lower post-transplant risks through comprehensive donor matching, and to identify patients at highest risk for complications who might benefit from directed preventive measures that include optimization of GVHD prophylaxis. This study provides the foundation for future fine-mapping approaches to identify the specific nature of the genes and their mechanisms in health and disease.