Genetic Variation in NFATC2 Is Associated with a Higher Risk of Asparaginase Allergy
Authors: Fernandez Christian A. , Smith Colton , Yang Wenjian , Mullighan Charles G. , Qu Chunxu , Larsen Eric C. , Bowman William Paul , Liu Chengcheng , Ramsey Laura B. , Chang Tamara , Karol Seth E. , Loh Mignon L. , Raetz Elizabeth A. , Winick Naomi J. , Hunger Stephen , Carroll William L. , Jeha Sima , Pui Ching-Hon , Evans William E. , Devidas Meenakshi , Relling Mary V. 
Published: October 2019

Abstract
Asparaginase is an important drug for the treatment of acute lymphoblastic leukemia (ALL); however, hypersensitivity reactions to asparaginase can lead to suboptimal asparaginase exposure and to a higher risk of disease recurrence. Factors influencing the frequency of asparaginase hypersensitivity include asparaginase formulation, the schedule of asparaginase administration, and concurrent chemotherapy. In addition, using a candidate gene approach, our group previously identified a genetic association between HLA-DRB1*07:01 and asparaginase hypersensitivity in patients of European ancestry.[PMID 24970932] The objective of our current study was to use a genome-wide approach to identify additional genetic loci associated with hypersensitivity and to assess the relative importance of HLA-DRB1 as a genetic risk factor.
Asparaginase hypersensitivity was assessed in children with ALL enrolled on SJCRH protocols Total XIIIA (n = 154), Total XV (n = 498) and Total XVI (n = 271) or Children’s Oncology Group (COG) protocols POG 9906 (n = 222) and AALL0232 (n = 2,163). Patients enrolled on protocols Total XIIIA, Total XV and POG 9906 received native E. coli asparaginase during treatment, whereas patients on Total XVI and AALL0232 received PEGylated E. coli asparaginase. Hypersensitivities were graded according to the NCI common toxicity criteria, and reactions of grade 2 and above were considered cases. Germline DNA was genotyped using the Affymetrix 500K, Affymetrix 6.0, or the Illumina Exome Beadchip array. Univariate analysis of risk factors associated with asparaginase hypersensitivity using the combined cohort of patients (n = 3,308) identified a significant positive association with treatment arm (P < 2.2x10-16; highest risk in patients enrolled on Total XV), native E coli asparaginase formulation (P < 2.2x10-16), age < 10 years (P = 3.3x10-4), male gender (P = 0.06), and racial ancestry (P = 2.1x10-4; highest risk among African ancestry). In multivariate analysis, treatment arm (P < 2.2x10-16), gender (P = 0.04), racial ancestry (P = 0.07), and ALL lineage (P = 0.003) remained significant risk factors.
A multivariate logistic regression model adjusted for treatment arm, racial ancestry, gender, age, and ALL lineage was used to identify genomic variants associated with asparaginase hypersensitivity in the combined patient cohort. The minor allele of an intronic polymorphism in NFATC2 (rs6021191) was associated with hypersensitivity at the genome-wide significance threshold (P = 2.6x10-8, OR = 3.17). The minor allele frequency (MAF) for the NFATC2 high-risk variant was highest in non-European ancestries (MAF European = 0.001, MAF African = 0.142, MAF Hispanic = 0.043, MAF Asian = 0.151, MAF Other = 0.034). Using RNA-seq data available from the ALL tumor samples of 65 SJCRH patients, we found that patients carrying the variant had higher expression of NFATC2 compared to non-carriers (P = 1.0x10-3). NFATC2 is known to play a critical role in inducing T-cell gene transcription during immune responses.
The strongest association detected using SNPs from the Illumina Exome array was found in HLA-DRB1 (rs17885382, P = 4.0x10-6, OR = 1.65), and the SNP is in near complete linkage disequilibrium with the HLA-DRB1*07:01 allele, which we previously found associated with asparaginase hypersensitivity. A gene-level analysis was performed using SNP-set Kernel Association Test (SKAT), and the gene with the strongest association with hypersensitivity was PPT2, located in the HLA class III region on chromosome 6 (P = 3.70x10-6). To determine potential biological pathways differentially represented by the genes identified by our analysis, we used Ingenuity Pathway Analysis on the top 1% of genes associated with hypersensitivity as identified by our single SNP (68 genes) and gene level analysis (18 genes). We found an enrichment of genes (8 out of 21 genes, P = 2.6x10-5) within the top network involved in humoral immunity, suggesting that other genes regulating immune responses may also contribute to the risk of developing asparaginase hypersensitivity.
In conclusion, the top inherited genetic loci (NFATC2, HLA-DRB1*0701, PPT2) associated with asparaginase hypersensitivity are directly involved in human immune response, and pathway analysis suggests that inherited variations in other genes involved in humoral immunity likely play a role in the development of asparaginase hypersensitivity.

Disclosures
Evans: St. Jude: In accordance with institutional policy (St. Jude), I and/or my spouse have in the past received a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms as clinical diagnostics. Patents & Royalties. Relling:St. Jude: In accordance with institutional policy (St. Jude), I and/or my spouse have in the past received a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms as clinical diagnostics. Patents & Royalties.