Using Whole Exome Sequencing to Identify Genetic Variation and Polymorphisms Associated with Graft Versus Host Disease in Allogeneic Stem Cell Transplant Recipients

Objective: Allogeneic stem cell transplant (ASCT) is a potentially curative treatment for many hematologic malignancies. HLA match is the principal factor in donor selection but the total antigenic landscape as seen by the donor immune cells extends beyond the HLA and minor HLA antigens. Thus, despite "perfect" HLA match graft versus host disease (GVHD) caused by alloreactive donor immune cells reacting to disparate antigens in the recipient, remains a significant cause of morbidity and mortality. Whole exome sequencing (WES) which assays all the protein coding nucleotide sequences provides a powerful means to assess the entire antigenic makeup of the individual. We examined the WES of sibling donor-recipient pairs undergoing BMT to assess the influence of the extent of genetic disparity and specific gene polymorphisms on GVHD.

Methods: Sixteen matched sibling donor-recipient pairs receiving allogeneic stem cell transplants between June 2008 and April 2013 had samples available for retrospective WES. Transplant data including conditioning regimen, age of donor and recipient at the time of transplant, development and type of GVHD and cause of death were recorded. The sequencing was performed using Illumina HiSeq2000 and the single nucleotide variants (SNVs) and small insertions and deletions (INDELs) were identified using the haplotype caller within the Genome Analysis Toolkit GATK v2.7. Functional variants including non-synonymous, frame-shift, truncating, and splice site SNVs and INDELs identified were compared between donor and recipient and among those with GVHD and those without GVHD (non-GVHD). One-sided Fisher's exact test was used to determine the statistical significance of the SNPs in GVHD and non-GVHD. Candidate genes with a p value of <0.05 were further evaluated for their biochemical pathway and possible role in inflammatory or immune responses.

Results: Nine of 16 patients had a diagnosis of AML or high grade myelodysplastic syndrome, 3 had a diagnosis of non- Hodgkin lymphoma, 1 had Hodgkin lymphoma, 1 had multiple myeloma, and 2 had CML. Six patients were conditioned with a myeloablative regimen, others with a reduced intensity conditioning regimen. Eight of 16 patients were female and 7 of 16 donors were female. Nine of 16 patients developed stage II-IV acute and/or chronic GVHD with 7 of them developing GVHD of the skin. A total of 12,623 genes with 43,670 functional variants were identified using WES. 134 genes had a p value < 0.05 and enrichment analysis was performed on these genes. 12 out of 134 genes were associated with gene ontology (GO) term "immune system development" with an unadjusted enrichment p-value 0.0015. Three (IL31RA, OSMR, SIRPA) out of 134 genes were associated with "inflammation_Jak-STAT pathway" with an adjusted enrichment p-value 0.07. Variants of IL31RA, OSMR, and PTX3 were identified as genes with polymorphisms associated with GVHD (p= 0.003, 0.01, and 0.01, respectively) with 5 variants identified in IL31RA, 7 variants in OSMR, and 2 variants in PTX3. Further investigation of these three genes revealed their role in inflammatory or immune responses.

Discussion: GVHD is a cytokine mediated immune-inflammatory process that can occur despite "perfect" HLA matching. WES identified the extensive genetic variability between donors and recipients implying large allo-reactivity potential that can lead to GVHD. Quantification of genetic variation may allow for more precise determination of GVHD risk and better selection of donors. In our cohort, WES identified polymorphisms in IL31RA, OSMR, and PTX3 as statistically significantly associated with development of GVHD. The protein products of these three genes have each been shown to be involved in inflammatory processes and are influenced by other cytokines. IL31RA and OSMR are found in many tissues including the skin and lungs. They function both independently and as a heterodimer receptor capable of binding multiple cytokines including IL-31, IL-6, and TNF-α. PTX3 is a reactive protein that increases in response to cytokine release related to systemic inflammation. Levels of this protein have been used as a prognostic marker in many disease processes with high levels correlating with poor outcomes. The small sample size of our cohort precludes any firm conclusions but provides promising hypotheses for further evaluation in larger studies.