Somatic hypermutation leads to diversification of the heavy chain immunoglobulin repertoire in cattle

The availability of unique variable (VH), diversity (D), and joining (JH) gene segments in the vertebrate germline determines the extent to which a primary immunoglobulin (Ig) repertoire can be generated through combinatorial rearrangement. Although bovine D segments possess unusual properties, the diversity of the primary Ig heavy chain (IgH) repertoire in cattle is restricted by the dominance of a single family of germline VH genes of limited number and diversity. Cattle therefore must employ other diversification strategies in order to generate a functional IgH repertoire, the main candidates being gene conversion and somatic hypermutation. In considering these possibilities, we predicted that if somatic hypermutation was active during B lymphocyte development, the process would introduce nucleotide substitutions to the VDJ exon and also non-coding region lying downstream of the rearranged JH segment. In contrast, our expectation was that gene conversion would show a greater tendency to confine modification to the IgH coding sequence, leaving intron regions substantially unmodified. An analysis of rearranged IgH sequences from cattle of different ages revealed that the diversification of germline sequences could be observed in very young calves and that substitution frequency increased with age. The age-dependent accumulation of mutations was particularly apparent in the second IgH complementarity-determining region (CDR2). Single base substitutions were found to predominate, with purines targeted more frequently than pyrimidines and transitions favoured over transversions. In non-coding regions, mutations were detected at a normalised frequency that was indistinguishable from that observed in CDR2. These data are consistent with a process of IgH diversification driven predominantly by somatic hypermutation.