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Somatic point mutations provide glimpses into B-cell histories, and mutation numbers generally correlate with antibody affinity. We recently proposed a model of human isotype function, based in part on mutation analysis, in which the dominant pathway of isotype switching involves B cells moving sequentially through the four immunoglobulin (Ig) G subclasses. This should result in predictable differences in affinity between isotypes, and this helps explain how different isotypes work together. The model built on analysis of rearranged immunoglobulin heavy chain sequences amplified from Papua New Guinean villagers, which showed highly significant differences in the mean number of V-REGION mutations in sequences, associated with the different IgG subclasses. To determine whether this relationship between mutation levels and isotypes is a more general phenomenon, the present study was conducted in healthy, urban residents of Sydney, Australia. VDJ sequences were generated from eight individuals, using 454 pyrosequencing, from cells expressing all isotypes except IgD and IgE. This resulted in 35 118 unique, productive VDJ sequences for the study. The data confirm that VDJ genes associated with progressively more 3′ Ig heavy chain gamma (IGHG) constant region genes show increasing levels of point mutation. Mean V-REGION mutations in IgA1 and IgA2 sequences were similar. Patterns of mutations also differed between isotypes. Despite their association with T-independent responses, IgG2 sequences showed significantly more mutational evidence of antigen selection than other IgG isotypes. Antigen selection was also significantly higher in IgA2 than in IgA1 sequences, raising the possibility of a preferential switch pathway from IGHG2 to IGHA2. [ABSTRACT FROM AUTHOR]