The HLA-DR and -DQ loci are close neighbors on chromosome 6 that are highly linked. We reported the finding of the unusual HLA DRB5*01:01~DRB1*15:01~DQA1*05:01~DQB1*02:01 haplotype in a German stem cell donor with East Frisian ancestry (Binder et al., Human Immunology, 2015). This unusual HLA class II haplotype was found in his daughter, and later in some more members of his extended family, but not in his son. His son seemed to be "homozygous" for the ordinary HLA DRB5*01:01~DRB1*15:01~DQA1*01:02~DQB1*06:02 haplotype. This "homozygosity" must be the result of a crossing over event in the spermatogenesis of his father, which (re-)converted the unusual into the ordinary HLA haplotype. The recombined extended paternal haplotype and the extended maternal haplotype were A*24:02~C*07:02~B*07:02~DRB5*01:01~DRB1*15:01~DQA1*01:02~DQB1*06:02. Our goal was to differentiate between both haplotypes and try to localize the recombination breakpoint.

We used different in house long range PCRs in combination with long (ONT) and short read sequencing systems (Illumina) to characterize the present haplotypes in highest possible resolution. Since all typing results of the standard HLA loci were identical in both parental haplotypes, we extended the genotype profiles with the following loci HLA-E, -F, -G, -DRA, -DPA1, -DPB1, -DOA, MICA, MICB and HFE. In addition we sequenced the intergenic XL9-SE (super enhancer) region between HLA-DRB1 and -DQA1.

NGS comparison of the extended parental haplotypes disclosed distinctions in only two loci leading to different alleles. In case of HFE we found an change from HFE*001:01:01 to HFE*002 and for HLA-DOA from DOA*01:01:02 to DOA*01:01:04. With these changes we could differentiate between parental haplotypes. Further on we were able to narrow down the crossing over breakpoint to a distinct XL9-SE region.

Only the combination of short reads with fully phased long read sequence data collected from an extended typing profile enabled us to distinguish the very similar parental HLA haplotypes. Localizing the recombination breakpoint within the intergenic region between HLA-DRB1 and -DQA1 might be a further step understanding the nature of recombination events since the XL9-SE as well as the DR-DQ-SE regions are involved in the regulation network of DRB1, DQA1 genes and perhaps some more HLA class II genes.

There is nothing to declare.