001). No clonally
related sequences were identified in the Australian samples. For subsequent mutation analyses, clonally related sequences were removed from the data sets. After their removal, 1004 unique PNG sequences remained, including 118 IgE sequences, 445 IgG1 sequences, 276 IgG2 sequences, 49 IgG3 sequences and 116 IgG4 sequences. The average mutation count for the IgE-associated IGHV genes was 23.0. The average number of mutations seen in PNG sequences associated with the different IgG subclasses correlated with the position of the various constant region gamma genes in the constant PF-562271 region locus. IgG3, which is encoded by the most 5′ IGHG gene, had the lowest number of mutations (mean: 17.7). The IGHG1 gene is located downstream of the IGHG3 gene, and IgG1 sequences had an average 21.0 mutations. The IGHG2 gene is found downstream of IGHG1, and IgG2 sequences had an average 22.0 mutations. IgG4, which is encoded by the most 3′ IGHG gene, had the highest number of mutations (mean: 27.1). Differences between PNG isotypes were significant (one-way anova: P < 0.001) with IgG4 being significantly higher than all other isotypes including IgE (Dunn multiple comparison: P < 0.05). Perhaps surprisingly, there was no significant difference seen between the level of mutations FG-4592 order in the Australian IgG1 sequences (mean: 19.2) and in the PNG IgG1 sequences.
Mean numbers of mutations for PNG IgG subclasses and IgE are shown as Fig. 1, and the frequency distributions of IGHV mutation numbers are shown as Fig. 2A–F. Chi-squared analysis of the frequency distribution of
IGHV mutations showed a significant difference between isotypes (P < 0.01). Striking differences were seen in the proportion of sequences that were relatively unmutated (<10 mutations). Eight per cent of IgE sequences had fewer than 10 mutations, but very few IgG4 sequences were relatively unmutated, with only two of 116 IgG4 sequences having fewer than 10 mutations. In contrast, 31% of IgG3 sequences carried fewer than 10 mutations, with two sequences having no mutations at all. These differences between IgG4 and the other isotypes, including Forskolin research buy differences between IgG4 and IgE, were all significant (χ2 tests; in all cases P < 0.05). The percentages of PNG sequences in each sequence data set that showed evidence for selection are shown in Fig. 3, and plots of replacement mutations in the CDR (RCDR) against total IGHV mutations (Mv) are shown for IgE and the IgG subclasses as Fig. 4. The IgE sequences showed evidence of antigen selection in only 12% of sequences, which was significantly less than in the IgG sequences (χ2 test: P < 0.001). Amongst the IgG sequences, the percentage of sequences showing evidence of antigen selection were 28% (IgG1), 39% (IgG2), 22% (IgG3) and 27% (IgG4). All subclasses showed significantly elevated levels of selection in comparison with IgE (P < 0.