Mass Screening for Non-Synonymous SNPs Using Custom Cancer Microarrays Directly Reveals Possible Pathogenic Predisposition Factors in AA

Abstract 1333

While immune mechanisms are involved in the pathogenesis of idiopathic aplastic anemia (AA), identification of heritable predisposition/susceptibility traits has been difficult due to the impact of exogenous factors and the low prevalence of AA. The seemingly sporadic and likely complex and heterogeneous traits leading to AA are not easily amenable to genetic studies.

With the advent of whole genome scanning (WGS) technologies such as single nucleotide polymorphism arrays (SNP-A), large scale investigations in various disorders have been conducted. A systems level understanding of a particular disease may allow for the identification of candidate genetic variants as prognostic and diagnostic biomarkers. Previous studies utilized arrays with mostly tagging SNPs and thereby the identification of causative polymorphisms was only indirectly possible through the narrowing of LD intervals. We have applied a custom cancer chip (Illumina) containing 211,155 SNP probes designed for non-synonymous SNPs, allowing for a more direct discovery of pathogenic genes with the aim of identifying low prevalence genetic variants that contribute to the development, risk and therapy responsiveness in idiopathic AA. Our study involved 116 cases used for discovery and 120 cases to be used for confirmatory studies, as well as a cohort of 1964 controls to improve the power of detection. After exclusion of SNPs with a GenTrain score of <0.65 and those in violation of Hardy Weinberg equilibrium, 202,905 SNPs (96.0% of the initial set) were passed for further investigation. Single allele 2 statistics for all autosomal markers were performed; 853 SNPs with p<1×10-7 were selected. Subsequently, all 853 SNPs were screened for functional prediction (transcription factor binding site, affecting splicing, detrimental non-synonymous variant of proteins such as cytokines or cytokine receptors). An initial group of SNPs was expanded by all the SNPs being in linkage disequilibrium (>0.8) to a total of 7445 loci. Remarkably, informative LD blocks were identified, represented by multiple markers pointing to the presence of informative polymorphisms in the corresponding regions.

A total of 3 SNPs were prioritized for final investigation based on the frequency differential between patients/controls. Of great interest was rs2544773 located in MYT1L represented directly by a singular marker. The frequency of the heterozygous variant among patients was 41.9% vs. 6.5% in controls and 15.0% vs. 0.4% for the minor homozygous variant (p<1×10–17). Our results suggest that carriers of at least one copy of the G allele (GA/AA) are at a higher risk of developing AA (OR 17.6). Another interesting variant identified was a non-synonymous SNP (rs13405539) located in DPYSL5 represented directly by a singular marker. This gene was recently associated with autoimmune myelopathy and cancer. Our analysis showed that minor allele has a protective potential and was present at a frequency in patients; occurring at a homozygous (AA) frequency of 1.8% vs. 16.4% in patients and controls, respectively and a heterozygous (GA) frequency of 14.6% vs. 49.2% in patients and controls respectively (p<1×10–20).

Comparison of AA patients with healthy individuals has been a primary focus of GWAS. However, we have also compared subgroups defined by clinical criteria. We subdivided patients based on responsiveness to immunosuppressive therapy. CEBPZ was represented by rs3213746 through LD with rs12469082 (p<.0001). The heterozygous (CT) frequency observed amongst refractory patients was 12.4% vs. 1.4% and 8.2% vs. 0% for the minor homozygous (TT) variant, in refractory and responder, respectively. Odds ratio: Patients carrying at least one copy of the minor allele (CT/TT) are at much higher risk being a non-responder to immunosuppressive treatment (OR=26.3). Genotypic frequencies of patients that responded to immunosuppressive treatment were similar to the frequencies observed in healthy population. CEBPZ belongs to a family of CCAAT/enhancer proteins. It has been shown to interact with TP53 and therefore may play role in modulation of apoptosis.

In sum, our study represents novel, whole genome approach using custom designed, high density cancer microarray that unravels new gene targets responsible for disease susceptibility as well as response to immunosuppressive therapy.