Sequencing of modern Lepus VDJ genes shows that the usage of VHn genes has been retained in both Oryctolagus and Lepus that diverged 12 million years ago

Among mammals, the European rabbit (Oryctolagus cuniculus) has a unique mechanism of generating the primary antibody repertoire. Despite having over 200 VH genes, the VH1 gene, the most d-proximal VH gene, is used in 80-90 % of VDJ rearrangements, while the remaining 10-20 % is encoded by the VHn genes that map at least 100 Kb upstream of VH1. The maintenance of the VHn genes usage in low frequency in VDJ rearrangements has been suggested to represent a relic of an ancestral immunologic response to pathogens. To address this question, we sequenced VDJ genes for another leporid, genus Lepus, which separated from European rabbit 12 million years ago. Approximately 25 VDJ gene sequences were obtained for each one of three Lepus europaeus individuals. We found that Lepus also uses the VHn genes in 5-10 % of its VDJ rearrangements. Our results show that the VHn genes are a conserved ancestral polymorphism that has been maintained in the leporids genome and is being used for the generation of VDJ rearrangements by both modern Lepus and Oryctolagus.The Portuguese Foundation for Science and Technology supported the doctoral fellowship of Ana Pinheiro (SFRH/BD/71252/2010) and the postdoctoral fellowship of Pedro J. Esteves (SPRH/BPD/27021/2006). Isabel G. Fernández de Mera is supported by a Juan de la Cierva contract from the Spanish Ministry for Economy and Competitiveness. This work was also supported by a project of the Portuguese Foundation for Science and Technology (PTDC/BIA-BEC/103158/2008). Project “Genomics Applied To Genetic Resources” cofinanced by North Portugal Regional Operational Programme 2007/2013 (ON.2 – O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF) supported this work.Peer Reviewe