For many hematological malignancies, allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative option. However, only 30–75% of patients find a fully matched human leukocyte antigen (HLA) donor . Hence, HLA-mismatched donors are used for HSCT although HLA mismatch is associated with increased incidence of graft versus host disease (GvHD) and inferior overall survival . GvHD is a severe, life-threating complication, which is even lethal for approximately 15% of patients . Besides the effect of HLA disparity, disease stage, type of conditioning prior to HSCT, age, immunosuppression, donor, and recipient sex, type of prophylaxis, prior exposure to viruses, and use of stem cells from peripheral blood influence the incidence of GvHD [3, 4]. Furthermore, different genetic polymorphisms affect the risk for GvHD, especially genes encoding for cytokines or chemokines, pharmacogenes and costimulatory molecules . For example, Kim et al. discovered an association between the presence of a specific single-nucleotide polymorphisms (SNP) in the NFKBIA or in the FAS gene of the recipient with increased risk of acute GvHD and the group suggested a SNP-based approach to improve the risk stratification of GvHD [6, 7]. In addition, various SNPs in donors’ or recipients’ genes were associated with an effect on the risk and/or the outcome of GvHD after HSCT, e.g., ABCB1, CYP3A, thrombomodulin, Interleukin-17, methylenetetrahydrofolate reductase, B-cell activating factor, Interleukin-2, tumor necrosis factor-α, and heparanase [8,9,10,11,12,13,14].
Recently, Bari et al. showed a correlation of the suppressor of fused (SUFU) polymorphism rs17114808 (C/T, 3′ untranslated region) and the incidence of acute GvHD . In total, they analyzed n = 168 pediatric patients after allogeneic hematopoietic stem cell transplantation (HSCT), divided into a discovery cohort of n = 68 patients and a validation cohort of n = 100 patients. The authors found a higher incidence of acute GvHD in both cohorts when patients were homozygous for CC, whereas heterozygous (CT) individuals developed less GvHD. Their genetic findings were supported by functional studies, which revealed lower expression of SUFU protein in dendritic cells of CC healthy donors that in turn increased HLA-DR expression and T-cell proliferation.
SUFU is a negative regulator of the hedgehog signaling pathway that interacts with the Glioma transcription factors (GLI) . The hedgehog signaling pathway is involved in cell proliferation, differentiation, and stem cell maintenance. Dysregulation or loss of function in the hedgehog signaling was detected in various types of tumors. It was shown that higher expression of GLI2 correlates with a negative effect on relapse-free survival and overall survival in patients with acute myeloid leukemia . Beyond, patients with B-cell chronic lymphocytic leukemia with poor clinical outcome exhibited an increased expression of GLI1 and SUFU . Henao-Martínez et al.  reported an association between polymorphisms in the SUFU gene with the immune protection in patients with Enterobacteriacea bacteremia.
Results and discussion
In a recent report, Bari et al. described a novel association between acute GvHD and the presence of the SNP rs17114808 in the SUFU gene. We performed a similar analysis in a cohort of n = 137 pediatric and n = 733 adult patients after HSCT of which n = 74 (54%) and n = 359 (49%) suffered from acute GvHD grade ≥1, respectively. Detailed information about the patient characteristics can be found in Table 1. For genotyping, we used the Taqman probe based SNP assay proposed by the authors and confirmed our genotyping assay by restriction fragment length polymorphism (RFLP) by the use of PsiI (TTATAA) (New England Biolabs, Ipswich, MA, USA). Digestion of the 93 bp amplicon of heterozygous individuals resulted in a 93 bp (TTACAA) and a 46 bp + 47 bp (TTATAA). Statistical analysis was performed using Fisher’s exact test and χ 2-test.
Among the 137 pediatric patients, n = 102 (74.5%) were CC homozygous, n = 33 (24.1%) were CT and n = 2 (1.5%) were TT homozygous. For the 733 adult patients, n = 589 (80.4%) were CC homozygous, n = 139 (19%) were CT and n = 5 (0.7%) were TT homozygous. These data are in line with the genotype frequencies for European individuals available at NCBI (HapMap-CEU, ss52072255: CC 82.1%, CT 16.1%, TT 1.8%). Remarkably, in the study of Bari et al. the genotype frequencies in the discovery cohort (CC 81%, CT 19%, and TT 0%) and the validation cohort (CC 88%, CT 9%, and TT 3%) slightly differed. This might be explained by the fact that the validation cohort comprised more individuals with other races than White, Black, and Asian (3% vs. 17%). Considering the ethnic background is of central importance when studying SNPs and their relevance for diseases.
From all pediatric patients carrying the CC allele, n = 57 (55.9%) developed acute GvHD grade ≥ 1, whereas n = 45 (44.1%) did not. Within the group of CT/TT patients n = 17 (48.6%) suffered from acute GvHD compared to n = 18 (51.4%) without any signs of GvHD (p = 0.782, Fig. 1A). Furthermore, in the cohort of adult patients frequency of CC homozygous patients was comparable with (n = 289, 49.1%) and without GvHD (n = 300, 50.9%, p = 0.688, Fig. 1B). There is a considerable heterogeneity in our adult patient population. For this reason, additional statistics was performed analyzing the adult AML (n = 354) and adult ALL cohort (n = 92) as well as the pediatric AML (n = 22) and pediatric ALL (n = 34) cohort separately. We gradually reduced the number of co-variates (diagnosis, stem cell source, HLA mismatch, radiation, conditioning, … (Table 1)) in order to discover any genetic association with GvHD. In all of the additional comparative statistical analysis no association between SUFU polymorphism rs17114808 and acute GvHD was observed (Table 2).
In contrast to the work of Bari et al. with 42% HLA-mismatched transplantations our pediatric cohort comprised <2% and the adult cohort 26%. Instead our Tuebinger center performed haploidentical HSCTs (pediatric 45%, adults 8%) and other stem cell sources were preferred (adults 96% PBSC, children 56% PBSC in contrast to 34% in the cohorts of Bari et al.). Furthermore, the number of patients treated with radiation was less than in the cohorts of Bari and colleagues (47% vs. 27% for children and 43% for adults). GvHD prophylaxis comprised calcineurin inhibitors (CNI), antithymocyte globulin (ATG), muromonab-CD3 (OKT3), methotrexate (MTX), and mycophenolate mofetil (MMF) with different regimens for adults and children, respectively. Nevertheless, it has to be noticed that GvHD occurred with a similar frequency in our cohorts (children 54%, adults 49%) and both pediatric cohorts presented by Bari et al. (discovery cohort (57%), validation cohort (43%)). In addition, comparable numbers of patients were classified into GvHD grade I–IV in all four cohorts.
In summary, we could not confirm the association of SUFU rs17114808 with the higher risk for the development of acute GvHD in our pediatric and adult patients despite comparable GvHD rates with Bari’s publication. This might be explained by the differences in type of transplantation, stem cell source and graft manipulation. For this reason, careful consideration of the detailed patient characteristics is necessary when comparing different genetic association studies. Furthermore, other structural variations in the SUFU gene will be worthy of investigation, because functional analysis by Bari et al. supports the hypothesis that SUFU might be a valuable therapeutic target for GvHD management. It has to be evaluated in further studies whether new drugs that modulate the sonic hedgehog pathway could help to reduce GvHD reaction in patients undergoing HSCT.
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This work was supported by the Stiftung für krebskranke Kinder Tübingen e.V, the Stefan-Morsch-Stiftung, and the José Carreras Leukämie-Stiftung.
Conflict of interest
The authors declare that they have no conflict of interest.
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Katz, M.C., Michaelis, S., Siegmund, D.M. et al. Association analysis between SUFU polymorphism rs17114808 and acute graft versus host disease after hematopoietic stem cell transplantation. Bone Marrow Transplant 53, 377–382 (2018). https://doi.org/10.1038/s41409-017-0059-3
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