HLA genotyping by NGS in cases with delayed hypersensitivity and cases with immediate hypersensitivity to penicillins
The clinical characteristics of the 24 patients with delayed hypersensitivity to penicillins who were subjected to HLA NGS/ NGS-based HLA typing are detailed in Supplemental Table 1 . Taking into account the results of skin testing, subjects classified as group A (i.e., positive only to ampicillin and amoxicillin, n = 14) were more numerous than those with less selective responses classified as group B (i.e., positive to benzylpenicillin, ampicillin and amoxicillin, n = 6) and C (i.e., positive to penicillins and aminocephalosporins, n = 4). On the HLA alleles retrieved in the 24 Italian patients, two HLA alleles,DRB3*02:02:01:02 and DRB3*02:02:01:01 , had a dramatic enrichment among the seven alleles with an allelic frequency >20%, compared to the control population of the Allele Frequency Net Database (Table 1 and Supplemental Tables 2 and 3 ). The alleles DRB3*02:02:01:02 and DRB3*02:02:01:01predicted a very high risk of delayed reaction, which could not be evaluated precisely because such alleles had not been previously reported in the general European population. 83% of the patients (20/24) harbored at least one of the two reported HLA-DRB3alleles, and 71% (11/24) were homozygous for one of the twoHLA-DRB3 alleles or had a compound heterozygous status (Table 2 and Supplemental Table 2 ). We observed a higher frequency of DRB3*02:02:01:02 and DRB3*02:02:01:01homozygous genotypes in group A (n=12, 69%) than in group B and C patients (n=3, 30%). However, no difference of allele frequency was observed among the 3 groups (Table 2 ).
The allele frequency of the DRB3*02:02 alleles (DRB3*02:02:01:02 and DRB3*02:02:01:01 ) was significantly higher in patients with delayed hypersensitivity than in those with immediate hypersensitivity to penicillins (77% vs. 28%, respectively). These figures corresponded to an increased risk of delayed reactions in penicillin hypersensitivity cases harboring DRB3*02:02 alleles, with an OR of 8.9 (95% CI, 3.4–23.3; P <0.0001) and an absolute risk difference of 49% (29%–64%; P<0.0001).