Single nucleotide polymorphisms and risk associated genotypes

SNPs are the most studied and evaluated variants of the human genome. Mullally et al, combined knowledge from different studies and accumulated understanding of different structural variants (such as CNVs), and summarized that the dissimilarities between all individuals are much greater than previously thought (Mullally and Ritz, 2007). This insight into the diverse complexity of the genome was of great benefit to the field of HSCT on two levels: the generation of the transplant antigens and the individual susceptibility to transplant related toxicities. Advances in studies and techniques used for DNA sequencing made it easier to perform genome-wide analysis using high throughput standard procedures to test for genetic characteristics and details associated with patients and donors before performing the transplantation. Applying and incorporating these finding into clinically meaningful results will be the next challenge for transplant clinicians.

The extent of the human genome is apparent when studying SNPs. Indeed, the International Hapmap Project reported more than one million SNPs in the human genome in October 2005 (Altshuler et al., 2005). Different types of genome variations were described in the Hapmap project, including whole gene deletions, multiple copy gene duplication, inverted gene sequences, large-scale copy number variants and segmental duplications (hapmap.ncbi.nlm.nih.gov). Regarding SNPs, 11,500 were catalogued as non-synonymous coding SNPs. According to studies on copy number variations (CNVs) carried out in the following years, the normal human genome contains at least 600 structural variants, comprising at least 100 million bases of DNA sequence. These numbers continue to increase with new structural variants being discovered (Fredman et al., 2004; Sharp et al., 2005; Feuk et al., 2006)

Numerous studies on SNPs have shown that genes which bear genetic variation are to be enriched significantly during immune responses (Tuzun et al., 2005). This means that genes stimulated in, or responsible for, the immune response (e.g. cytokines) contain more structural rearrangements that other genes. Some genes were reported to be implicated in the adaptability and fitness of an organism in response to an external stimulus. Thus, the structural variations that occur in the genome represent the process of adoptive evolution. An example of these observations is the selection of gene copy number that has been reported for CCL3L1, an immune response gene,

28

where lower than average copy number is associated with HIV/AIDS (Gonzalez et al., 2005).

Understanding the human genetic diversity will help dissect the susceptibility and response to different diseases and specifically, these studies are of great significance to the field of HSCT. Indeed, along with deletions, non-synonymous SNPs can generate potentially immunogenic transplant antigens (Mullally et al., 2006). Non-HLA SNPs can influence the immune response. A study by Cho et al. suggested that significant variability in cytokine and chemokine expression after Toll-like receptor stimulation has been observed between individuals. This suggests that particular aspects of immune response, such as TLR stimulation in the case of this study, are closely associated with genetic variation (Cho et al., 2013).

To analyse the SNPs in relation to cGvHD, Clark et al suggested that SNPs in target genes can lead to better understanding of the biological basis of the different subtypes of cGvHD (Clark et al. 2010). Genes that could be subject to copy number variation include KIR, MHC, and the gene encoding Fc and immunoglobulin receptor. Genes involved in drug detoxification, which are also subject to structural variation leading to CNV, are of potential relevance to HSCT. These include genes relevant to the glutathione s-transferase gene family, the cyclophosphamide (cytochrome p450, GST family) and calcineurin inhibitor (cytochrome p450, UGT2B family metabolism). This suggests that normal gene structural variations could have an impact on individual outcomes during HSCT (Sebat et al., 2004).

The majority of the SNPs arise in non-coding regions including intronic, intergenic regions and untranslated regions (UTRs) (Engle et al., 2006). Those which are within genes, including genes affecting the immune response, can alter the expression of the gene or the structure of the encoded proteins (Dickinson and Norden, 2015a). Indeed, many of the genes which were associated with HSCT outcome, were also associated with autoimmune disease, however only few remained significant following genome wide association studies (GWAS) (Dickinson and Norden, 2015a).

Since the original work regarding candidate gene associations published by Middleton et al, multiple studies investigating lager cohort gene associations have been reported on SNPs located in more than 20 genes that either code for cytokines or other molecules playing a significant role in the biology of HSCT (Dickinson and Norden,

29

2015a; Cavet et al., 1999; Socie et al., 2001; Mullighan et al., 2007; Espinoza et al., 2011). The relationship between SNPs in the NOD2 gene with GvHD and HSCT outcome has been extensively studied several groups. SNPs which were originally identified in the NOD2 gene for their association with Crohn’s disease have been associated with HSCT outcome (Holler et al., 2006; Grube et al., 2015). Patients carrying one variant of rs206684 (SNP8), rs2066845 (SNP12) or rs41450053 (SNP 13) have a 2 to 4-fold increase of developing Chron’s disease and this risk increases to 20-fold in patients who are homozygotes or compound heterozygotes (Economou et

al., 2008; Chien et al., 2012). NOD2 plays a major role in defense against infection as

it recognises pathogen-associated patters and thus induces cytokine cytokine response and it is also regulated by pro-inflammatory cytokines (Rosenstiel et al., 2003).

Another example is the FOXP3 gene region, within which more than 90 SNPs have been identified and several among these SNPs have been identified as risk factors for a number of malignant and autoimmune diseases (Eastell et al., 2007). Tregs, defined as CD4+_CD25+_FOXP3+ _{T cells are involved in the maintenance of immunological}

tolerance (Beres et al., 2013) and have been the focus of several HSCT studies due to their ability to supress alloreactivity during GVHD (Hoffmann et al., 2002). A SNP, rs3761548, in the promotor region of FOXP3, resulting in A->C base exchange was shown to cause loss of binding to E47 and c-Myb factors and thus, leading to defective transcription of the FOXP3 gene (Shen et al., 2010). In HSCT setting, this polymorphism was shown to be associated with the development of auto or alloimmune conditions, including type I diabetes, and graft rejection in renal transplantation (Noriega et al., 2015). In patients transplanted from donors carrying short alleles (≤(GT)15), this polymorphism was shown to be associated with a lower incidence of severe GvHD (grade 3-4) (Noriega et al., 2015). This polymorphism however had no effect on relapse, event free survival or overall survival in patients with aGvHD and cGvHD (Noriega et al., 2015).

Specific polymorphisms in genes for IL-10, IL-6, TNF- α and IFN-γ in a pediatric cohort of 57 HLA-identical sibling myeloablative transplants were reported by Goussetis and colleagues who retrospectively studied these polymorphisms and found a significant association between the IL10 promoter haplotype polymorphism at -1082, -819 and - 592 with the incidence of severe aGVHD (Goussetis et al., 2011). The authors reported that patients with the haplotype GCC showed a decreased risk of sever aGvHD in

30

comparison with other IL10 haplotypes (Goussetis et al., 2011). Chien et al, reported that 2 SNPs in the same gene IL10, rs1800872 and rs1800872, were associated with a 30% decrease of the risk of severe aGvHD (Chien et al., 2011). In the case of IL6, the donor genotype for rs1800795 in IL6, was associated with a 20% to 50% increase in the risk for aGvHD (II-IV) and the IL2 polymorphism rs2069762 in the donor genotype was showed to be associated with a 1.3-fold increase in the risk of grade III- IV aGvHD (Chien et al., 2011). The Ogawa group identified three new loci that were shown to be significantly associated with severe aGvHD (II to IV) including the SNP rs17473423 within the KRAS locus (Stao et al., 2015). In a study by Bair et al, two GvHD susceptibility loci (rs17114803 and rs17114808) in the suppressor of fused homolog (SUFU) gene have been found (Bari et al., 2015). The Incidence of aGvHD was shown to be higher in patients who were homozygous for CC at SUFU rs17114808 (Bari et al., 2015).

In microRNAs (miRNAs), SNPs can alter regulatory properties but elucidation of the functions of these SNPs is not straight forward (Hudson, 2003). Moreover, SNPs located in miRNAs can affect the miRNAs maturation, function, and target selection. To date, a number of studies have demonstrated that SNPs in target sites or miRNA genes are associated with diseases such as chronic lymphocytic leukaemia, non- small-cell lung cancer, papillary thyroid carcinoma and breast cancer (Chin et al., 2008; Jazdzewski et al., 2008; Sethupathy and Collins, 2008; Mencía et al., 2009).

1.7.3 Implication of microRNAs in the pathophysiology of graft versus host