mutation pattern of paired immunoglobulin heavy and light … · 2018. 10. 9. · and iglv segments...

1 1 8 8 | G H I O T T O E T A L . | M O L M E D 1 7 ( 1 1 - 1 2 ) 1 1 8 8 - 1 1 9 5 , N O V E M B E R - D E C E M B E R 2 0 1 1

INTRODUCTIONB-cell chronic lymphocytic leukemia

(CLL) is a clonal expansion of an antigen-experienced B cell (1,2) expressing CD5,CD19, CD23 and low levels of surfacemembrane immunoglobulin (smIg). De-spite the fact that CLL is the most com-mon leukemia in the western hemi-sphere, its biology and pathogenesis are

still poorly understood. Furthermore,CLL is a remarkably heterogeneous dis-order, with some patients surviving fordecades, often without therapy andeventually dying of unrelated causes,and others have a rapidly evolving, fataldisease despite aggressive therapy (3,4).

Several studies of the smIgs of CLLcells showed that the immunoglobulin

heavy variable (IGHV ) and immunoglobu-lin kappa/lambda variable (IGK/LV) had un-dergone the process of somatic hypermu-tation (5–8) and that this process couldbe ongoing during disease evolution,thereby generating intraclonal IGHV andIGK/LV diversification (9–11). Further-more, patients whose leukemic clone ex-hibit mutated IGHVs usually have lessaggressive disease and live longer thanpatients with unmutated IGHVs (12,13).However, none of these studies ad-dressed the molecular features of pairedIGHV and IGK/LV segments of individ-ual CLL clones.

Here we analyzed the mutation patternof paired IGHV–diversity-joining (IGHV-

Mutation Pattern of Paired Immunoglobulin Heavy and LightVariable Domains in Chronic Lymphocytic Leukemia B Cells

Fabio Ghiotto,1* Paolo Marcatili,2* Claudya Tenca,1 Maria Grazia Calevo,3 Xiao-Jie Yan,4 Emilia Albesiano,4

Davide Bagnara,4 Monica Colombo,5 Giovanna Cutrona,5 Charles C Chu,4 Fortunato Morabito,6

Silvia Bruno,1 Manlio Ferrarini,5 Anna Tramontano,7,8 Franco Fais,1 and Nicholas Chiorazzi4,9,10

1Department of Experimental Medicine, University of Genoa, Genoa, Italy; 2Department of Biochemical Sciences “Rossi Fanelli,”University of Rome “La Sapienza,” Rome, Italy; 3Epidemiology and Biostatistics Service, Scientific Directorate, G. Gaslini Institute,Genoa, Italy; 4The Feinstein Institute for Medical Research, North Shore–LIJ Health System, Manhasset, New York, United States ofAmerica; 5Division of Medical Oncology C, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy; 6Unità Operativa di Ematologia;Dipartimento di Medicina Interna; Azienda Ospedaliera di Cosenza; Cosenza, Italy; 7Department of Physics, Sapienza University ofRome, Rome, Italy; 8Istituto Pasteur Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy; 9Department ofMedicine, North Shore University Hospital, Manhasset, New York, and Albert Einstein School of Medicine, Bronx, New York, UnitedStates of America; and 10Department of Cell Biology, Albert Einstein School of Medicine, Bronx, New York, United States of America

B-cell chronic lymphocytic leukemia (CLL) patients display leukemic clones bearing either germline or somatically mutated im-munoglobulin heavy variable (IGHV ) genes. Most information on CLL immunoglobulins (Igs), such as the definition of stereotypedB-cell receptors (BCRs), was derived from germline unmutated Igs. In particular, detailed studies on the distribution and nature ofmutations 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 immunoglobulinkappa/lambda variable-joining (IGK/LV-J ) rearrangements of 193 leukemic clones that displayed ≥2% mutations in at least one ofthe two immunoglobulin variable (IGV ) genes (IGHV and/or IGK/LV ). The relationship between the mutation frequency in IGHVand IGK/LV complementarity determining regions (CDRs) and framework regions (FRs) was evaluated by correlation analysis. Re-placement (R) mutation frequency within IGK/LV chain CDRs correlated significantly with mutation frequency of paired IGHV CDRsin λ but not κ isotype CLL clones. CDRs of IGKV-J rearrangements displayed a lower percentage of R mutations than IGHVs. The fre-quency/pattern of mutations in kappa CLL Igs differed also from that in κ-expressing normal B cells described in the literature. In-stead, the mutation frequency within the FRs of IGHV and either IGKV or IGLV was correlated. Notably, the amount of diversity in-troduced by replaced amino acids was comparable between IGHVs and IGKVs. The data indicate a different mutation patternbetween κ and λ isotype CLL clones and suggest an antigenic selection that, in κ samples, operates against CDR variation.© 2011 The Feinstein Institute for Medical Research, www.feinsteininstitute.orgOnline address: http://www.molmed.orgdoi: 10.2119/molmed.2011.00104

*FG and PM contributed equally to this work.

Address correspondence and reprint requests to Franco Fais, Department of ExperimentalMedicine, University of Genova, Via De Toni 14 16132 Genova, Italy. Phone: +39-010-

3537872, Fax: +39-010-3537881; E-mail: [email protected].

Submitted March 22, 2011; Accepted for publication July 13, 2011; Epub

(www.molmed.org) ahead of print July 13, 2011.

R E S E A R C H A R T I C L E

M O L M E D 1 7 ( 1 1 - 1 2 ) 1 1 8 8 - 1 1 9 5 , N O V E M B E R - D E C E M B E R 2 0 1 1 | G H I O T T O E T A L . | 1 1 8 9

D-J ) and IGK/LV-joining (IGK/LV-J ) re-arrangements of leukemic cells from 193CLL patients. Our data indicate that themutation patterns of κ and λ CLL clonesdiffer from each other. Moreover, by com-paring CLL cells to normal B cells, whichhave been so far studied with respect tothe κ isotype only (14), we identified adifferent somatic hypermutation pattern.

MATERIALS AND METHODS

Patients, Leukemic Cells,Immunoglobulin Variable (IGV )Sequences and Analyses

After obtaining informed consent, he-parinized venous blood was obtainedfrom patients with CLL, and peripheralblood mononuclear cells were isolated.The diagnosis of CLL was on the basis ofaccepted clinical and immunophenotypicfeatures (15). Rearranged IGHV-D-J andIGKV-J or IGLV-J paired segments weresequenced from cDNA of 218 CLL pa-tients as described (5,6); in addition, 148immunoglobulin (Ig) sequences (IGHV +IGK/LV) were retrieved from GenBank,bringing the total analyzed to 366. For oursequences, as well as sequences retrievedfrom GenBank, only samples with allelicexclusion of both IGHV and IGK/LV wereincluded in the study. Sequences were an-alyzed using the V-QUEST tool at the in-ternational ImmunoGeneTics (IMGT) In-formation System® (http://imgt.cines.fr)(initiator and coordinator: Marie-PauleLefranc, Montpellier, France [16]). The 3′end of IGK/LVs (coding for the light com-plementary determining region 3[LCDR3]) was inspected visually to ex-clude mutations identified by V-QUESTthat were actually due to the variable-joining (V-J) recombination process. Be-cause in this study both IGHV andIGK/LV segments were sequenced and an-alyzed, we defined as unmutated CLLcases those patients with leukemic clonesexhibiting


I G H A N D L C O M B I N E D M U T A T I O N P A T T E R N S I N C L L

categorical variables. The normality ofdata distribution was assessed by theKolmogorov-Smirnov test. Spearmanrank correlation was used to test the direction and strength of the relationshipbetween the number of IGHV andIGK/LV mutations. The Mann-Whitney Utest was used to compare sums of thescores obtained with the BLOSUM62 ma-trix analysis, computed separately forCDRs and FRs, between the IGHV andIGK/LV regions. Results were consideredstatistically significant when the P valuewas ≤0.05. All statistical tests were two-tailed. Statistical analyses were per-formed using the SPSS software (SPSS,Chicago, IL, USA) and the R software(http://www.R-project.org) (21).

All supplementary materials are availableonline at www.molmed.org.

RESULTS

Sample DescriptionWe analyzed the sequences of the re-

arranged IGHVs and IGK/LVs of 366IgM+ CLL patients. The cohort com-prised 47.3% (173/366) unmutated CLLcases and 52.7% (193/366) mutated CLLcases. The 193 M-CLL cases were used toinvestigate the reciprocal mutation pat-terns of paired IGV genes. In this group,60.1% (116/193) and 39.9% (77/193) ofclones were κ or λ isotype expressing, re-spectively. The IGHV and IGK/LV reper-toires were analyzed separately andcompared with previously reported co-horts (6,8,22). No major differences wereevident (Supplementary Figures 1 and 2).

Analysis of MutationsThe M-CLL group comprised patients

displaying ≥2% mutations in at least oneof the two IGVs (IGHV and/or IGK/LV).Notably, whereas only 3.6% (7/193) ofthe samples had an IGHV with



heavy complementary determining re-gions (HCDRs); in addition, the numberof R mutations in FRs of the rearrangedIGK/LV was plotted as a function of thenumber of R mutations in FRs of theirpartner IGHV. This step was done for allsamples together (Figure 1A) and sepa-rately for the κ isotype (Figure 2A) and λisotype samples (Figure 2C). Interestingly,while a significant concordance in R mu-tations was found for both CDRs and FRsfor λ isotype samples, a correlation wasobserved for κ isotype samples only forFRs and not for CDRs. We asked whetherusing the absolute number of mutations,without considering CDR and FR lengths,would lead to biased results. To this pur-pose, CDR mutations were expressed as apercentage of the total number of CDRbase pairs and FR mutations as a percent-age of the total number of FR base pairs(see Materials and Methods). These re-sults were almost identical to those ob-tained by plotting the absolute number of

mutations (Supplementary Figure 3). Toallow comparison with published data(vide infra), the absolute number of muta-tions was used hereinafter.

To determine if the discordant patternof R mutations was due to a differentialaction of the mutation mechanism onIGHVs and IGK/LVs or to selective anti-genic pressure, we determined the distri-bution patterns of S mutations. For thispurpose, the number of S mutations inthe K/LCDRs was plotted as a function ofthe number of S mutations in the HCDRs;the same was done for the number of Smutations in FRs of the rearrangedIGK/LV as a function of the number of Smutations in FRs of their rearranged part-ner IGHV for all samples (Figure 1B) andseparately for κ isotype (Figure 2B) and λisotype samples (Figure 2D). We found astatistically significant correlation be-tween S mutations of IGHV and IGKVsegments in both CDRs and FRs in pooledκ and λ samples (P = 0.026 and P = 0.0001,

respectively). Significant correlation wasobserved between S mutations of IGHVand IGKV in both CDRs and FRs of κ iso-type samples (P = 0.03 and P = 0.001, re-spectively). In λ isotype samples, a signifi-cant correlation was detected for FRs (P =0.005), whereas a significant correlationwas not found in CDRs (P = 0.42), al-though the small number of silent muta-tions in the CDRs of λ isotype samplesshould be taken into account.

To date, only one study has addressedthe pairing of H and L chain variable re-gions among B cells of the normal reper-toire (14); this pairing was carried out foronly IGHV/IGKV pairs of IgM-expressingB cells from two healthy donors. Thesedata indicate that IGHV R mutations cor-related with IGKV R mutations in CDRsbut not in FRs, the opposite mutationpattern that we have uncovered in CLLcell clones (see Figure 3 for a comparisonof data).

While evaluating the percentage ofmutation of paired IGHV and IGK/LV,we observed, in a proportion of patients,a relevant difference between the twochains. Therefore, we asked whether theabsence of correlation of the number ofmutations between IGHV and IGK/LVcould be due to the presence of differentpopulations within the patient cohort(for example, one with a similar numberof mutations in both IGVs and the otherwith a notable difference in the numberof mutations between the paired IGHVand IGK/LV segments). To exclude thepossibility that two populations couldbias the analysis, κ and λ isotype sam-ples were divided into two groups usingthree units of percent difference (at leasteight nucleotides; see Materials andMethods). Samples with a difference inthe percentage of mutation of the twochains lower than three percentage unitswere considered “concordantly mutated”and assigned to group A (63 κ isotypesamples and 52 λ isotype samples),whereas samples with a differencegreater than three percentage units wereclassified as “discordantly mutated” andassigned to group B (53 κ isotype sam-ples and 25 λ isotype samples) (Figure 4).

Figure 3. Comparison of the pattern of R mutations in CDRs (upper row) and FRs (lowerrow) of paired IGHVs and IGKVs between κ-expressing normal B-cell Igs (left column) andκ isotype CLL Igs (right column). Left: The scatter plots related to normal κ isotype B-cellrepertoire are derived with permission from Brezinschek et al. (14). Right: scatter plots ob-tained from our data on κ isotype CLL samples (see Figure 1B). rsp (ρsp), Spearman rank.



The degree of concordance betweenthe number of R mutations in the CDRsand FRs of the two chains in the twogroups was then computed for κ and λseparately (Figure 5A). For κ isotypesamples, no significant correlation be-tween the number of IGHV and IGKV Rmutations was present in CDRs, neitherin group A nor in group B (see Figure 5A,upper row). However, for λ isotype sam-ples, the concordance previously ob-served in R mutations of both CDRs andFRs, when the two groups were analyzedtogether, was lost in group B but not ingroup A (Figure 5A, lower row).

To evaluate the mechanism underlyingthe R distribution pattern in the two sub-

groups, the number of S mutations in theK/LCDRs was plotted as a function ofthe number of S mutations in the HCDRs;the same was done for the number ofS mutations in FRs of the rearrangedIGK/LV as a function of the number ofS mutations in FRs of their rearrangedpartner IGHV for both groups A and B(Figure 5B). We found a statistically sig-nificant correlation between S mutationsof IGHV and IGKV in both CDRs and FRsfor group A (P = 0.01 and P < 0.0001, re-spectively), whereas there was no signifi-cant correlation in both CDRs and FRs forgroup B (P = 0.61 and P = 0.06, respec-tively). Analyzing the S mutation distri-

bution in λ chains, we observed a signifi-cant correlation between IGLV and IGHVonly in FRs of group A.

The results of these analyses were fur-ther verified by comparing them withdata obtained by plotting percentages in-stead of absolute numbers. No differ-ences were observed between the twoanalyses (Supplementary Figure 4).

We next investigated if the differentpatterns of mutation observed in IGKVand IGHV genes could be explained by adifferent pattern of R amino acid substi-tutions. For this reason, the amount ofdiversity introduced by R mutations wasdetermined for CDRs and FRs of both

Figure 4. Splitting CLL samples into groupsA and B on the basis of different mutationpercentages between IGHVs and IGK/LVs.Scatter plot of the percentage of muta-tions in IGHV and IGK/LV pairings for sam-ples displaying a



IGHV and IGK/LV. This step was doneusing the BLOSUM62 matrix as a refer-ence to measure the nature of each muta-tion, with a positive score indicating aconservative change and a negative scoreindicating a nonconservative change,with values ranging from +4 to –6 (seeMaterials and Methods). Values obtainedfor R mutations in the CDRs and FRs ofeach IGHV and IGK/LV were added andanalyzed separately for samples ofgroups A and B (Figure 6). The resultsdid not indicate a significant differencein the nature of R changes betweenIGHV and IGKV of group A, both inCDRs and FRs. However, in group B,changes in the kappa complementary de-termining regions (KCDRs) were moreconservative than those in the HCDRs.The opposite was true for FRs, wherethere were more conservative changes inIGHVs. For λ isotype Igs, conservation inboth CDRs and FRs was higher in IGHVthan in IGLV in group A, whereas therewas more conservation in the LCDRsthan HCDRs in group B.

A schematic representation summa-rizing all of the above data is shown(Figure 7).

DISCUSSIONOur analyses demonstrate that the pat-

tern of somatic hypermutation in the re-arranged IGV genes expressed on thesurface of leukemic B cells from CLL pa-tients is different for κ versus λ isotypeIgs and that the mutation pattern ofκ IGVs is also different from that of nor-mal B cells. These findings point to a se-lective pressure acting on mutations inKCDRs of CLL Igs.

Specifically, we found that the muta-tion pattern of κ-expressing CLL andnormal B lymphocytes (14) are quitedifferent. Normal B cells display a cor-relation between the number of IGHVand IGKV R mutations in CDRs but notin FRs (14), whereas the opposite wasobserved in CLL, where there was nocorrelation between the number ofR mutations in HCDRs and KCDRs butthere was a relationship between thenumber of R mutations in H and K FRs.

This result held true also when sampleswere divided into two groups (“A”[concordantly mutated] and “B” [dis-cordantly mutated]) and were analyzedseparately. This scenario may reflectantigen-driven selection that protectsKCDRs from R mutations, which wouldmore likely change amino acid struc-ture and antigen reactivity. Such a pos-sibility was reinforced by analyses ofthe distribution patterns of S mutationsin IGHV and IGKV segments. Indeed,the concordance between the relativenumbers of S mutations of IGHV andIGKV in group A further suggests selec-tive pressure acting on KCDRs. As ex-pected, in group B, no correlation wasobserved because of the arbitrary muta-tion cut off applied.

Differently from κ isotype samples, thesmIgs in λ isotype CLLs show a muta-tional pattern in accordance with the 3%cutoff criteria: in group A, the number ofIGHV mutations correlated with IGLVmutations in both CDRs and FRs,whereas in group B, no correlation wasfound in either CDRs or FRs. The latterdata support the hypothesis of Hersh-berg and Shlomchik (23) regarding strat-egies for κ and λ L chain variation. Theseauthors observed differences in germlineκ and λ L chain responses to mutationand attributed these differences to a needto balance diversity and stability in theimmune response.

Since the low amount of R mutationsobserved in KCDRs may be balanced byimpactful nonconservative amino acid

Figure 6. Comparison of the sums of the BLOSUM scores between IGHVs and IGK/LVs ingroups 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 in-terquartile range (25–75th percentile) and the line within this box is the median value. Bot-tom and top bars of the whisker indicate the variation range. P values are shown.



changes, we verified the degree of con-servation of R mutations in κ isotypesamples divided into A and B groups.This analysis showed that there were nosignificant differences between IGHV andIGKV in either CDRs or FRs of group A;in group B, there were more changes inHCDRs than in KCDRs, whereas the con-trary was observed in FRs.

Comparing the analysis of trait conser-vation of κ and λ isotype samples di-vided into the A and B groups, a differ-ent behavior between the two isotypeswas evident. Of note, the FR conserva-

tion trait of κ and λ isotypes in group Bwas opposite that of group A, with muchmore diversification of κ than λ FRs,again in agreement with published data(23). Although we cannot compare ourdata with corresponding λ Igs of λ-expressing B lymphocytes from normalsubjects, because the latter have not beeninvestigated in sufficient detail to pro-vide correlative information, collectivelyour results suggest an important role forthe KCDRs in binding of antigen, as op-posed to LCDRs. This result indicatesdifferent B-cell receptor (BCR)/antigen

interaction modalities between B lym-phocytes expressing different L chain iso-types.

It is of note that the combined κ and λCLL samples belonging to group B repre-sent ~40% of the cohort. These samplesare characterized by an evident differ-ence in the amount of mutations betweenIGHV and IGK/LV segments. In particu-lar, in 75% of the cases, a mutated chain(≥2%) is associated with an unmutatedchain (



cal Research Center grant RR018535),and from The Karches Foundation, ThePrince Family Foundation, The MarksFoundation, The Jerome Levy Founda-tion, The Leon Levy Foundation and theJoseph Eletto Leukemia Research Fund.G Cutrona and F Fais would like tothank the Fondazione Internazionale inMedicina Sperimentale (FIRMS) for fi-nancial and administrative assistance.

DISCLOSUREThe authors declare that they have no

competing interests as defined by Molec-ular Medicine, or other interests thatmight be perceived to influence the re-sults and discussion reported in thispaper.

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