Mutation pattern of paired immunoglobulin heavy and light variable domains in chronic lymphocytic leukemia B cells

Abstract

B-cell chronic lymphocytic leukemia (CLL) patients display leukemic clones bearing either germline or somatically mutated immunoglobulin heavy variable (IGHV ) genes. Most information on CLL immunoglobulins (Igs), such as the definition of stereotyped B-cell receptors (BCRs), was derived from germline unmutated Igs. In particular, detailed studies on the distribution and nature of mutations in paired heavy- and light-chain domains of CLL clones bearing mutated Igs are lacking. To address the somatic hyper-mutation dynamics of CLL Igs, we analyzed the mutation pattern of paired IGHV-diversity-joining (IGHV-D-J ) and immunoglobulin kappa/lambda variable-joining (IGK/LV-J ) rearrangements of 193 leukemic clones that displayed ≥ 2% mutations in at least one of the two immunoglobulin variable (IGV ) genes (IGHV and/or IGK/LV ). The relationship between the mutation frequency in IGHV and IGK/LV complementarity determining regions (CDRs) and framework regions (FRs) was evaluated by correlation analysis. Replacement (R) mutation frequency within IGK/LV chain CDRs correlated significantly with mutation frequency of paired IGHV CDRs in λ but not κ isotype CLL clones. CDRs of IGKV-J rearrangements displayed a lower percentage of R mutations than IGHVs. The frequency/pattern of mutations in kappa CLL Igs differed also from that in κ-expressing normal B cells described in the literature. Instead, the mutation frequency within the FRs of IGHV and either IGKV or IGLV was correlated. Notably, the amount of diversity introduced by replaced amino acids was comparable between IGHVs and IGKVs. The data indicate a different mutation pattern between κ and λ isotype CLL clones and suggest an antigenic selection that, in κ samples, operates against CDR variation.

Figure 1

Scatter plot of the number of R and S mutations in CDRs and FRs of paired IGHV and IGLV segments of all CLL samples. (A) R mutations. (B) S mutations. Each dot represents one or more CLL samples; color ranges from light gray (one sample) to dark gray (four or more samples). r_sp (ρ_sp), Spearman rank.

Figure 2

Scatter plot of the number of R and S mutations in CDRs and FRs of paired IGHVs and IGK/LVs. (A) R mutations of the κ isotype samples. (B) S mutations of κ isotype samples. (C) R mutations of λ isotype samples. (D) S mutations of λ isotype samples. Each dot represents one or more CLL samples; color ranges from light gray (one sample) to dark gray (four or more samples). r_sp (ρ_sp), Spearman rank.

Figure 3

Comparison of the pattern of R mutations in CDRs (upper row) and FRs (lower row) of paired IGHVs and IGKVs between κ-expressing normal B-cell Igs (left column) and κ isotype CLL Igs (right column). Left: The scatter plots related to normal κ isotype B-cell repertoire are derived with permission from Brezinschek et al. (14). Right: scatter plots obtained from our data on κ isotype CLL samples (see Figure 1B). r_sp (ρ_sp), Spearman rank.

Figure 4

Splitting CLL samples into groups A and B on the basis of different mutation percentages between IGHVs and IGK/LVs. Scatter plot of the percentage of mutations in IGHV and IGK/LV pairings for samples displaying a <3% difference between IGHV and IGK/LV mutation percentages (group A) or ≥3% difference (group B), respectively (see Materials and Methods for details). (A) All CLL samples. (B) κ Isotype samples. (C) λ Isotype samples. Each dot represents one or more CLL samples; color ranges from light gray (one sample) to dark gray (four or more samples).

Figure 5

Scatter plot of the number of R (A) and S (B) mutations in CDRs and FRs of IGHV and IGK/LV pairings in group A (<3% difference) and group B (≥3% difference). Each single dot represents one or more CLL samples; color ranges from light gray (one sample) to dark gray (four or more samples). (A) Upper row: κ isotype samples; lower row: λ isotype samples. (B) Upper row: κ isotype samples; lower row: λ isotype samples. r_sp (ρ_sp), Spearman rank.

Figure 6

Comparison of the sums of the BLOSUM scores between IGHVs and IGK/LVs in groups A and B. Sums of the BLOSUM scores, obtained with the BLOSUM62 matrix analysis, were computed separately for CDRs and FRs. In each graph, the box represents the interquartile range (25–75th percentile) and the line within this box is the median value. Bottom and top bars of the whisker indicate the variation range. P values are shown.

Figure 7

Schematic summarizing of mutation frequency concordance and chemical features of R mutations in paired IGHV and IGK/LV of mutated CLL clones. The different findings obtained from the analysis of the correlation of mutations and from the analysis of trait conservation for the paired IGHV and IGK/LV of the CLL cohort are shown. The left column summarizes the data shown in Figure 1 and indicates concordance (yes) or not (no) in the number of R mutations in paired IGHVs and IGK/LVs. The central column summarizes data presented in Figures 4A and 5 and displays the presence or absence of R mutation concordance as well as the comparison of diversification features generated by R mutations in IGHV and IGK/LV segments. The right column summarizes the data in Figure 4B and displays concordance of S mutations between paired IGHV and IGK/LV.