Prácticas Situacionistas

Several studies have established the involvement of polymorphisms in non-HLA genes in determining clinical outcome after transplantation (Welniak et al., 2007). SNPs in genes essential for allogeneic immune responses and inflammatory reactions have been described as potential biomarkers for the severity of GvHD (Elmaagacli et al., 2008; Gruhn et al., 2009; Espinoza et al., 2011; Elbahlawan et al., 2012).

SNPs are an important variation to create diversity among individuals, as well as leading to different phenotypes, traits, and diseases (Shastry, 2009). Since miRNAs are key regulators of gene expression, miRNA-related SNPs including SNPs in miRNA genes and their target sites may function as regulatory SNPs, through modifying miRNA regulation to affect phenotypes and disease susceptibility (Dignam et al., 1983). Moreover, SNPs located in miRs are likely to have a complex influence by affecting miR maturation, functional strand selection and target selection (Dignam et al., 1983). Since 2005, several studies have systematically identified and analysed human polymorphisms in miRNAs and/or miRNA target sites (Iwai and Naraba, 2005; Saunders et al., 2007; Landi et al., 2008; Shastry, 2009; Ryan et al., 2010; Bhartiya et

al., 2011).

MiR-146a is an immediate early-response gene induced by various microbial components and proinflammatory mediators. The human genome contains two miR- 146 genes (miR-146a and miR-146b) on chromosomes 5 and 10, respectively, and their mature products differ only by 2 nucleotides in the 3′ region (Figure3.1) (Bentwich

et al., 2005; Cai et al., 2005).

Figure 3.1 Sequence alignment of the miR-146 family of miRNAs (adapted from Griffiths-Jones, 2004). All sequences are taken from the

MicroRNA Registry (release 7.1). Variable nucleotides are shown in red (Griffiths‐Jones, 2004).

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MiR-146a is highly expressed in Treg cells and is induced upon activation of effector T cells and myeloid cells (Lu et al., 2010). In the latter, miR-146a acts as a negative feedback regulator to limit TRAF6 and IRAK1-mediated signaling in inflammatory settings (further explained in Chapter 1, section 1.7.3.1) (Taganov et al., 2006a; Hou

et al., 2009), whereas in activated human T cells, miR-146a has been suggested to

oppose apoptosis and IL-2 production (Curtale et al., 2010).

miR-146a has been validated to target the expression of at least two genes, IRAK1 and TRAF6, and acts as a negative regulator in TLR and pro-inflammatory cytokine (IL-1) signaling pathway (further explained in Chapter 1, section 1.7.3.1) (Taganov et

al., 2006b). IRAK1 encodes for a key intracellular signaling protein that is activated by

ligands of Toll-like receptors. IRAK1 activation by interleukin-6 results in phosphorylation and activation of the transcription factor STAT3 and consequent transcriptional activation of the gene for C-reactive protein (Zhang et al., 1996). Specifically, IRAK1 plays significant role in TLR/IL-1 receptor (TIR) activation of NF-κB (Chatzikyriakidou et al., 2010). IRAK1 is considered as a linker of the TLR with the TRAF6 intracytoplasmic activator of transcription factor NF-κB, which subsequently increases the expression of many genes related to immunological reactions such as TNF-α and IL-8 (Dunne and O'Neill, 2003; Janssens and Beyaert, 2003). Subsequently, IRAK1 is subjected to negative feedback control by miR-146a, expression of which is also NF-κB dependent, leading to a concerted immunological response (Chatzikyriakidou et al., 2010).

Activation and nuclear translocation of NF-κB transcription factors is medicated by the TCR and Natural Killer Group 2D (NKG2D) receptor stimulation (Rajasekaran et al., 2011). Such stimulation is mediated by one of the most polymorphic NKG2D-ligands, MICA (Spear et al., 2013) (further explained in Chapter 1, section 1.9).

Thus, miR-146a, IRAK1 and MICA all participate in a network controlling diverse biological process. This complex network is further complicated by the presence pf SNPs in the miR-146a, IRAK1 and MICA encoding loci. A common polymorphism in

pre-miR-146a, designated rs2910164, causes a G to C change at position +60 relative

to the fist nucleotide of pre-miR-146a (Jazdzewski et al., 2008). This SNP leads to a miss-paired hairpin sequence within the precursor of miR-146a, which affects processing of the miRNA and consequently, lowers expression of the mature sequence (Onnis et al., 2012).

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MiR-146a rs2910164 has been previously investigated for its association with the severity of GvHD in allo-HSCT patients, where it was shown that the CC genotype is associated with severe aGvHD (Stickel et al., 2014). A study by Shen et al, showed that among 42 patients with familial breast cancer and 82 patients with ovarian cancer, those with at least one rs2910164(C) SNP tended to be diagnosed at an earlier age than those with only (G) alleles (Shen et al. 2008). Xu et al., also suggested that a functional polymorphism in the pre-miR-146a gene is associated with prostate cancer risk and mature miR-146a expression in vivo. The author reported that patients with the CC genotype of this SNP were at decreased risk for prostate cancer compared with those carrying the GG/GC genotype. In addition, the team also reported that the G-to-C change in the precursor of miR-146a resulted in reduced expression of mature miR-146a in prostate cancer tissue (Xu et al. 2010).

Another SNP in miR-146a is rs2431697. This occurs at position 5q35.1 of the miR- 146a gene and causes a T to C transition, resulting in the miR-146a (2) variant (SNPedia). Investigation of 20 patients with non-HLA psoriasis showed that miR-146a (2) is associated with susceptibility to psoriatic arthritis and psoriasis vulgaris in the Chinese population (Yang et al. 2013). There is currently no published information about the association of miR-146a (2) and HSCT outcome. However, a pilot study within our laboratory showed that presence of the T allele is associated with the incidence of relapse in a RIC cohort of HSCT patients with aGvHD grades II-IV (unpublished data).

Rs3027898 is a SNP in the 3'-UTR of the IRAK1 gene, which occurs at position Xq28. This SNP encodes an A->C tansversion. Because the gene is located on the X chromosome, men are more likely than woman to show an association between this SNP and diseases. Several studies have shown an association between both the A and C alleles with numerous diseases, such as atherothrombotic cerebral infarction (Yamada et al. 2008), rheumatoid arthritis (Chatzikyriakidou et al. 2010) and lupus erythematosus (Zhai et al. 2013).

There are currently more than 100 alleles known which encode for 79 protein variants for MICA (http://www.ebi.ac.uk/ipd/imgt/hla/). Interestingly, a SNP at position 454 (A→G, rs1051792) leads to an amino acid substitution of methionine by valine (Met→Val) at position 129 in the α2 domain of the MICA protein, that categorizes the

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MICA alleles into strong (MICA-129 Met) and weak binders (MICA-129 Val) binders of NKG2D (Raache et al,. 2012) (Figure 3.2).

Thus, it is important to pinpoint SNP-mRNA-miRNA regulatory network alterations and their contribution to the risks associated with HSCT. Such investigation will help elucidate the consequences of the interaction between these three genetic elements, deciphering the genetic risks of HSCT.

Figure 3.2 Ribbon diagram showing crystal structures of NKG2D bound to MICA (adapted from Li et al., 2001). The

NKG2D homodimer is colored in blue and magenta, MICA is green with domains labeled. NKG2D recognizes the alpha1 and alpha2 domains of MICA. rs1051792 occurs in the the α2 domain which is a binding site for NKG2D (Li et al., 2001b).

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