We successfully implemented a protocol for the full-length identification of HLA-DRB4*03:01N. Furthermore, we demonstrate that all samples that amplified successfully and fulfilled certain criteria after genotyping of HLA-DRB4 exons 2 and 3 (HLA-DRB1*07 present, no amplification of exon 2, exon 3 sequence concordant with HLA-DRB4*03:01N) were confirmed to be HLA-DRB4*03:01N. Consequently, we feel confident that in these cases HLA-DRB4*03:01N could be reported with high accuracy even without running an additional confirmation PCR. However, cases with very low amplification of exon 2 have been found to be caused by other reasons, including mutations in the primer binding regions.

Considering both DRB4-positive and -negative haplotypes, we determined an allele frequency of 0.0014% for HLA-DRB4*03:01N (46 cases) in the German population based on 1.67 million samples.