The CDR1 sequences of a major proportion of human germline Ig VH genes are inherently susceptible to amino acid replacement

Abstract

The variable (V) genes of antigen-selected antibodies are known to exhibit a higher frequency of amino acid replacement mutations in the sequences encoding the antigen-contacting complementarity-determining regions (CDRs) than in those encoding the 'structural' framework regions (FRs). Here, Bernard Chang and Paolo Casali analyse the impact of regional differences in the codon composition of human germline Ig V H and V L genes on regional differences in the frequency of replacement mutations in the gene products (i.e. the antigen-binding sites of antibody molecules). This analysis reveals that CDR and FR sequences can differ significantly in their inherent susceptibility to amino acid replacement given any single nucleotide change. Thus, the CDR sequences of all the Ig V H genes analysed comprise a higher frequency of codons susceptible to replacement mutations than would be expected for a random sequence. Conversely, the FR sequences comprise codons less susceptible to replacement mutations than expected. Random accumulation of nucleotide changes throughout the coding sequence of an Ig V-gene segment containing CDRs inherently more prone to replacement mutations than the respective FRs would inevitably yield a higher rate of amino acid replacements in the CDRs than in the FRs. This would provide a fertile structural substrate of hypervariability for antigen selection while still maintaining the structural integrity of the FRs.The production of antibodies with progressively higher affinity for antigen is an important feature of B-cell clonotypes recruited by repeated antigenic challenge, and is referred to as affinity maturation 1 . The molecular basis of this process was first unveiled by the analysis of the binding affinity and primary structure of monoclonal antibodies (mAbs) generated at successive stages of murine immune responses to different conjugated haptens and antigens [2][3][4][5][6][7][8][9][10] . The first exposure to antigen results in recruitment of B-cell clonotypes that bind antigen by virtue of the combinatorial and junctional specificity of their unmutated surface receptors (Ig V segments). Subsequent exposures to antigen lead to accumulation of somatic point mutations in the antibody V segments and antigen selection of the highaffinity mutated B-cell clonotypes. Sequential rounds of mutation and selection eventually result in restriction of the response to those B cells with the best 'fit' for antigen 11,12 . Somatic point mutations in Ig V-gene segmentsIn the absence of negative or positive selective pressure on the gene product, a random mutational process would entail an even distribution of nucleotide changes yielding amino HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript acid replacements (R mutations) and nucleotide changes not yielding amino acid replacements (S, or silent, mutations) throughout the coding sequence. However, antigenselected antibodies have been shown to include a higher frequency of R mutations in the Ig V-gene complemen...

IgV and bcl6 somatic mutation analysis reveals the heterogeneity of cutaneous B-cell lymphoma, and indicates the presence of undisclosed local antigens

“…The usage of V H 2-70 gene in three cases (1 FL and 2 DLBCLs) seems to indicate a preferential usage of this gene in primary cutaneous BCL, hypothetically correlated with the local presence of specific antigens. 25 Presence of IgV H mutations was observed in all cases, with a lower rate (o2%) in primary MZLs and in the single case analyzed here of cutaneous involvement by disseminated FL. This seems to confirm the heterogeneity of cutaneous BCL.…”

“…25 Significant evidence of positive selection was observed in one case (case 8, primary DBCL), where there were more replacement mutations in CDR regions than expected by comparison with FR regions.…”

“…25 For each case, the intraclonal variability was recorded, and the sequence of dominant clone, the consensus sequence, was considered the most frequent sequence among clones from each case. Only confirmed sequences were taken as evidence of clonal heterogeneity; the unconfirmed cases were disregarded because they could have been due to Taq polymerase error.…”

Lineage tree analysis of immunoglobulin variable-region gene mutations in autoimmune diseases: Chronic activation, normal selection

“…Replacement and silent mutations were enumerated and statistically analyzed with a computer program developed in our Lab (Zuckerman et al, unpublished) using the Lossos et al multinomial correction [23] to the Chang & Casali method [24].…”

Immunoglobulin gene analysis as a tool for investigating human immune responses

“…The initial hypothesis was that statistical comparison of replacement and silent mutation distribution across the IGHV gene would differ in an antigen‐selected gene compared to the mutation expected if it were completely random with no selection pressure. Such that an antigen‐selected gene would have more replacements than silent mutations in the CDRs which encode the antibody binding site, and conversely more silent than replacement mutations in the framework region of the antibody that is needed for antibody structural integrity 27. Calculations then had to be modified to account for our discovery that even in the absence of selection, in out‐of‐frame gene rearrangements there were more mutations in CDRs than framework regions 28.…”