ORIGINAL ARTICLE The role of HLA–G 14-bp polymorphism in allo-HSCT after short-term course MTX for GvHD prophylaxis P Chiusolo 1 , S Bellesi 1 , N Piccirillo 1 , S Giammarco 1 , S Marietti 1 , D De Ritis 1 , E Metafuni 1 , M Stignani 2 , OR Baricordi 2 , S Sica 1 , G Leone 1 and R Rizzo 2 1 Hematology Department, Istituto di Ematologia, Universita` Cattolica del Sacro Cuore, Rome, Italy and 2 Department of Experimental and Diagnostic Medicine, Section of Medical Genetics, University of Ferrara, Ferrara, Italy HLA-G molecules are HLA class Ib antigens characte- rized by tolerogenic and immunoinhibitory functions. The HLA-G 14-bp insertion/deletion (ins/del) polymorphism controls protein expression and seems to be implicated in both MTX treatment response and SCT outcome. The aim of our study is to evaluate the role of HLA-G 14 bp polymorphism in subjects affected by hematological malignancies undergoing allo-SCT and receiving MTX therapy for GvHD prophylaxis. We performed a retro- spective analysis of HLA-G 14 bp polymorphism using a specific PCR in 47 recipients and in their respective donors, and evaluated the correlation with the incidence of aGvHD, OS and disease-free survival (DFS) after allo- SCT. We did not observe any correlation between this polymorphism and the risk of aGvHD occurrence. On the contrary, we found that the recipients with a 14 bp ins/14 bp ins genotype were characterized by a lower OS and DFS in univariate and multivariate analysis (OS ¼ OR: 3.235; DFS ¼ OR: 3.302). These data indicate a role for recipient HLA-G 14 bp polymorphism in allo- SCT immunotolerance status and follow-up. Bone Marrow Transplantation advance online publication, 14 March 2011; doi:10.1038/bmt.2011.40 Keywords: human leukocyte antigen-G; allo-SCT; MTX; GvHD Introduction The human leukocyte antigen-G (HLA-G) is a non- classical HLA class I molecule characterized by immuno- modulatory, anti-inflammatory and tolerogenic activities. HLA-G has a restricted tissue distribution and a low allelic polymorphism in comparison with other HLA class I molecules (46 alleles). There are 15 expressed alleles that differ in the coding sequence and encode seven proteic isoforms, four membrane-bound (G1-G2-G3-G4) and three soluble (G5-G6-G7), obtained by mRNA alternative splicing. 1 The soluble isoforms retain the intron 4, which includes a stop codon and leads to the termination of the mRNA translation before the transmembrane domain. The HLA-G1 and HLA-G5 structures are characterized by three alpha domains, differently from the other isoforms, which lack one or more globular domains. The proteo- lytic cleavage of surface isoform HLA-G1 generates the soluble HLA-G1 form (sHLAG1). The expression of HLA-G was first identified on the surface of cytotrophoblast cells in where it is involved in establishing and maintaining maternal-fetal tolerance. 2 HLA-G antigens function as immunomodulatory mole- cules inhibiting natural killer cell activity, CD4 þ T lymphocyte and DC maturation, inducing apoptosis in activated CD8 þ CTLs and the development of regulatory T cells. 3,4 In physiological conditions, HLA-G molecules are expressed by peripheral blood CD14 þ monocytes and thymic epithelial cells. HLA-G antigens have been detected in pathological conditions such as in autoimmune and inflammatory diseases, viral infections, 5,6 tumours 7,8 and in transplantation. 9,10 In particular, the expression of HLA-G molecules by transplanted organs and the levels of soluble HLA-G in the sera of transplanted patients have a significant correlation with a reduced transplant rejection rate, confirming the immunosuppressive role of these molecules. 9–12 The HLA-G expression is regulated by a deletion (del)/insertion (ins) polymorphism of 14 base pairs (14 bp) at the 3 0 untranslated region of exon 8. 13 The 14 bp ins allele is associated with a decreased mRNA stability and a consequent lower HLA-G protein expression. 14–16 La Nasa et al. 17 have observed an association between the HLA-G 14 bp deletion allele and an increased risk of acute GvHD (aGvHD) development after SCT of thalassemic patients with a possible pharmacogenetic implication in MTX therapy. This hypothesis is in agreement with the results of Rizzo et al., 18 in which the authors showed that the secretion of HLA-G molecules by PBMCs after in vitro MTX treatment depends on 14 bp polymorphism. 18 They have observed that the HLA-G 14 bp deletion allele is responsible for a higher production of HLA-G molecules, and rheumatoid arthritis patients homozygous for the 14 bp Received 19 July 2010; revised 12 January 2011; accepted 14 January 2011 Correspondence: Dr P Chiusolo, Hematology Department, Istituto di Ematologia, Universita` Cattolica del Sacro Cuore, Largo A Gemelli 8, 00168 Rome, Italy. E-mail: p.chiusolo@rm.unicatt.it Bone Marrow Transplantation (2011), 1–5 & 2011 Macmillan Publishers Limited All rights reserved 0268-3369/11 www.nature.com/bmt