Preferential targeting of somatic hypermutation to hotspot motifs and hypermutable sites and generation of mutational clusters in the IgVH alleles of a rheumatoid factor producing lymphoblastoid cell line

Epstein-Barr virus transforms human peripheral B cells into lymphoblastoid cell lines (LCL) that secrete specific antibodies. Our previous studies showed that a monoclonal LCL that secretes a rheumatoid factor expressed activation-induced cytidine deaminase (AID) and displayed an ongoing process of somatic hypermutation (SHM) at a frequency of 1.7×10^-3mut/bp in its productively rearranged IgVH gene. The present work shows that SHM similarly affects the nonproductive IgVH allele of the same culture. Sequencing of multiple cDNA clones derived from cellular subclones of the parental culture, showed that both alleles exhibited an ongoing mutational process with mutation rates of 2-3×10^-5mut/bp×generation with a high preference for C/G transition mutations and lack of a significant strand bias. About 50% of the mutations were targeted to the underlined C/G bases in the WRCH/DGYW and RCY/RGY hotspot motifs, indicating that they were due to the initial phase of AID activity. Mutations were targeted to the VH alleles and not to the Cμ or to the GAPDH genes. Genealogical trees showed a stepwise accumulation of only 1-3 mutations per branch of the tree. Unexpectedly, 27% of all the mutations in the two alleles occurred repeatedly and independently within certain sites (not necessarily the canonical hotspot motifs) in cellular clones belonging to different branches of the lineage tree. Furthermore, some of the mutations seem to arise as recurrent mutational clusters, independently generated in different cellular clones. Statistical analysis showed that it is very unlikely that these clusters were due to random targeting of equally accessible hotspots, indicating the presence of 'hypermutable sites' that generate recurring mutational clusters in the IgVH alleles. Intrinsic hypermutable sites may enhance affinity maturation and generation of effective mutated antibody repertoires against invading pathogens.