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B.O. Wolthers et al.
Validation of results
In validating our top SNP in the NFATC2 locus (rs62228256) and top SNPs in the PRSS1-PRSS2 locus (rs13228878 and rs10273639) we used two cohorts of chil- dren with ALL: one treated according to the DFCI ALL Consortium 87−01, 91−01, 95−01 and 00−01 protocols,27,28 and the other according to Children’s Oncology Group AALL0232 protocol8 (Online Supplement pages 6−12). Whereas the association between AAP and rs6228256 genotype was not replicated in the DFCI ALL Consortium cohort (P=0.77, Online Supplement page 10), both rs13228878 (hazard ratio, 0.68; 95% CI: 0.48 to 0.96; P=0.03) and rs10273639 (hazard ratio, 0.69; 95% CI: 0.49 to 0.98; P=0.04) were significantly associated with the risk of developing pancreatitis in the AALL0232 cohort (Online Supplement page 8).
Two previous studies have investigated SNPs associated with pancreatitis in children with ALL. Using different diagnostic criteria for pancreatitis and including controls with less than 5 weeks of asparaginase therapy, Liu et al. associated the rare variant (general population minor allele frequency=0.009%) rs199695765 in carboxypeptidase A2 encoding CPA2 (hazard ratio, 587; 95% CI: 66.8 to 5166; P=9 x 10-9) with AAP.8 We did not directly genotype this SNP, and none of the genotyped SNPs in the CPA2 region was in LD with rs199695765. Of 32 SNPs 10 kb up- and down-stream of CPA2, rs66839817 (T>C, OR, 1.28; 95% CI: 1.03 to 1.57; P=0.02) showed the strongest association.
In a NOPHO GWA study we previously found the ULK2 variant rs281366 (P=5.8 x 10-7) and RGS6 variant rs17179470 (P=1.3 x 10-6) to be most associated with AAP.13 Excluding cases and controls from the NOPHO study, we failed to validate these results in 184 cases and 712 controls. Both rs281366 and rs17179470 were directly genotyped in this cohort with non-significant P-values (P=0.84 and P=0.32, respectively).
Genotype-phenotype associations
Table 2 shows associations between PRSS1-PRSS2 genotype and amount of PEG-asparaginase given prior to AAP, days from PEG-asparaginase injection to AAP, com- plications of pancreatitis, and risk of a second episode of
AAP after re-exposure to asparaginase. The risk allele was not associated with number of PEG-asparaginase injec- tions prior to AAP or time from injection of PEG-asparag- inase to diagnosis of AAP. Furthermore, the risk of devel- oping acute complications was not found to be associated with PRSS1-PRSS2 genotype (Table 2). In the Nordic sub- set of cases (n=92) and controls (n=1024) we found no association between PRSS1-PRSS2 genotype and 5-year event-free survival (Online Supplementary Figure S9; P=0.4). Out of 46 children who were re-exposed to asparaginase, 17 (37%) developed a second episode of AAP; although not in a statistically significant manner, the PRSS1-PRSS2 minor allele indicated the same protective effect as in the risk of initial AAP (OR, 0.49; 95% CI: 0.15 to 1.41; P=0.20).
Discussion
This study is the first to find and validate variants in the PRSS1-PRSS2 locus associated with the risk of AAP in children with ALL. In doing so, we found that activation of trypsin within pancreatic acinar cells is a key initiating event in the pathogenesis of pancreatitis, regardless of the exposure i.e. alcohol, hyperlipidemia, or asparaginase. The role of trypsin activation in the pathogenesis of pan- creatitis had long been suspected, but an underlying genet- ic susceptibility was not documented until 1996 when Whitcomb et al. documented mutations in the PRSS1 gene causing hereditary pancreatitis,29 and later associated a common genetic variant in the PRSS1-PRSS2 locus (rs10273639) with the risk of alcohol-related and sporadic pancreatitis.30 This association was recently validated in larger European and Asian cohorts, and the haplotype has been studied in detail.31–33 rs10273639 is located 408 base pairs upstream of the translation initiation codon of cationic trypsinogen. A recent functional study document- ed that the proximal rs4726576 (C>A) variant (204 kb upstream of the translation initiation codon) drives the association.34 The rs4726576 and rs10273639 variants are in high LD (r2 >99%) in European and Asian populations but have a r2 = 0.8 in the African meta-population, and
Figure 2. Regional associa- tion plot of the PRSS1-2 locus on chromosome 7. Regional asssociation plot showing sin- gle nucleotide polymorphisms (SNPs) associated with asparaginase-associated pan- creatitis in 244 cases and 1320 controls. The x axis rep- resents genomic location, and the y axis represents the P val- ues for the SNP associations
calculated using regression adjusting for age and ancestry. rs13228878 (P=7.1 x 10-6) is represented in purple and rs10273639 (P=1.1 x 10-5) in red. The color of the dots reflects linkage disequilibrium (LD) of the genotyped SNPs. LD is based on 1000 genomes European samples, November 2014. The human assembly GRCh37 was used for reference.
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haematologica | 2019; 104(3)