Genetic Variants in the HLA-genes Are Associated with PEG-asparaginase Allergy - a Genome Wide Association Study on the NOPHO ALL2008 Protocol

Introduction: Asparaginase is a cornerstone in treatment of childhood acute lymphoblastic leukemia (ALL). However, balancing positive anti-leukemic effect against toxicities has proven difficult. A frequent toxicity is allergy; 13% of children treated according to the Nordic NOPHO ALL2008 protocol develop an allergic reaction to pegylated asparaginase (PEG-asparaginase) causing acute morbidity and necessitating truncation of therapy. (Henriksen et al, PBC, 2014 and 2017) .

Background: Children with allergic reactions have no enzyme activity at any time-point during their treatment (Henriksen et al, PBC, 2017, Tong et al, Blood, 2014), thus they do not benefit from asparaginase administered prior to the allergic reaction. These allergic reactions are true allergic type 1 IgE-mediated reactions as opposed to allergy-like reactions and silent inactivation. We speculate that a subset of children with ALL have a genetic predisposition to developing allergy to PEG-asparaginase.

Objectives and Methods: To investigate a genetic predisposition to PEG-asparaginase allergy, we performed a genome-wide association study (GWAS) on 1288 children (age 1-17.9 years) with ALL and treated according to the NOPHO ALL2008 protocol from July 2008 - December 2014. Of these, 178 (13.8%) were registered with an allergic reaction to PEG-asparaginase. Registration relied solely on the physician's ability to recognize symptoms of an allergic reaction, which can be difficult because of the diverse clinical manifestations. To define allergy and increase specificity, we used asparaginase enzyme measurements, only including children with available enzyme measurements; after quality control, 63 children with clinical allergy and without enzyme activity (< 25 IU/l in all samples) and 360 children with enzyme activity (≥ 25 IU/l in any sample) were included as cases and controls. Germline DNA was genotyped on Illumina Omni2.5exome-8 BeadChip arrays. Logistic regression analyses were performed in PLINK, adjusting for age and genetic ancestry, and excluding single nucleotide polymorphisms (SNPs) with a minor allele frequency < 0.01.

Results: Median age was 3 years (IQR: 2-6) for cases and 4 years (IQR: 3-8) for controls, and the frequency of males was 44.4% and 55.3% in cases and controls, respectively. Cases developed allergy after a median of 3 (IQR: 2-4) injections of PEG-asparaginase, and 49% of the cases had a severe allergic reaction grade 3 or 4, of those 5 cases had an anaphylactic reaction. Thevariant rs3998159 on 6p21.32 showed the strongest association to PEG-asparaginase allergy (P=1.23∙10^-6; OR=3.7, 95% CI [2.2, 6.4]). This SNP is located 27kb upstream of the HLA-DQA2 gene. 6/7 (86%), 22/75 (29%) and 34/318 (11%) of children respectively homozygote, heterozygote and without any risk allele developed PEG-allergy. Our top 17 SNPs are all located in close proximity to the top SNP and are in high linkage disequilibrium (R²=1).

HLA-DQA2 is a part of the MCH class II genes. These genes play an important role in the antigen-specific IgE synthesis since they are responsible for the antigen presenting process and mediation of humoral immunity. HLA-DQA2, HLA-DQB1 and HLA-DRB1 have been reported to be associated with total IgE-levels (Granada, J Allergy Clin Immunol, 2012), all are within close proximity (< 30Kb) to the top SNPs in our study. Fernandez et al. (Blood 2015) have previously associated NFACT2 with PEG-asparaginase allergy. A finding we fail to validate (top snp rs2426294 , P= 0,00988). This could be explained by a different route of administration and asparaginase dosage and formulation. Furthermore, to our knowledge our study is the first to include enzyme activity enabling to better distinguish between the real reactions and allergy like reactions.

Conclusion: This study associates variants in the HLA -region with PEG-asparaginase allergy, a region highly involved in the immune responses. These results fit into the exiting literature in the field on allergic pathways and expand our knowledge about the genetic background for PEG-asparaginase allergy.