Leukemia (2001) 15, 1772–1778 2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu Somatic hypermutation targeting to intrinsic hotspots of immunoglobulin genes in follicular lymphoma and multiple myeloma C Belessi1, K Stamatopoulos2, N Stavroyianni2, K Zoi2, T Papadaki3 and C Kosmas4 1Hematology Laboratory, General Hospital of Nikea, Piraeus; 2First Department of Medicine, Athens University School of Medicine, Laikon General Hospital, Athens; 3Hemopathology Unit, Evangelismos Hospital, Athens; and 4Department of Medicine, Helena Venizelou Hospital, Athens, Greece In this study, we analyzed the targeting of the somatic hyper- of the secondary lymphoid organs where contact with and mutation (SHM) mechanism at specific hotspot sequence 3 ␬ selection by antigen takes place. The molecular hallmark of motifs in the VH and V genes of 10 follicular lymphoma (FL) cases and the V␬ and V␭ genes of 11 ␬- and six ␭-light chain this phase is the introduction of mutations within rearranged expressing multiple myeloma (MM) cases. These sequences IgV genes, at a rate much higher than usual, a phenomenon were analyzed for targeting of specific motifs, ie certain highly described as somatic hypermutation.4 mutable trinucleotides (3-NTPs), the tetranucleotide (4-NTP) Antigen selection in B cell ontogeny is evidenced by non- RGYW and its complementary, WRCY (where R = purine, random distribution of somatic mutations in lg HC and LC V = = Y pyrimidine and W A or T). Comparisons were carried out genes, a feature providing useful information concerning the between mutation frequencies in RGYW vs WRCY and the inci- 5 dence of mutations in complementarity determining region ontogenetic assignment of B cell neoplastic disorders. In this (CDR)-1 vs CDR2 vs CDR3. Statistically significant differences context, an increased ratio of replacement (R) to silent (S) were obtained when comparing: (1) the ratio of mutations in 4- mutations in the complementarity determining regions (CDRs) NTPs (RGYW, WRCY, RGYW+WRCY)/mutations in the whole V of IgV genes, has been considered as the most reliable surro- sequence in MM-V␬ vs MM-V␭; (2) the total number of mutated gate marker of selection by antigen for higher avidity.6 How- ␬ ␬ 4-NTPs in MM-V vs FL-V ; (3) the number of mutated RGYW ever, irrespective of subsequent selection, somatic hypermut- 4-NTPs in MM-V␬ vs FL-V␬ and FL-VH vs FL-V␬; (4) the number of mutated WRCY 4-NTPs in MM-V␬ vs FL-V␬ (P = 0.006) and ation is primarily targeted at specific hotspots within V genes, FL-V vs FL-V␬; (5) the targeting of RGYW vs WRCY in the ie tri-or tetra-nucleotidesequence motifs, such as the RGYW H = = = CDRs of FL-VH genes. Similar results (regarding statistical motif (R purine, Y pyrimidine, W A or T) and its significance) were obtained when undertaking intergroup com- complementary, WRCY.7 parisons for 3-NTPs. These findings conform well with relevant While the exact mechanism of somatic hypermutation data derived from normal peripheral B cells. The differences remains elusive, this phenomenon is characterized by certain observed in favor of 4-NTP (RGYW and WRCY) targeting in FL- 8 ␬ ␬ ␬ unique features. The nature of mutations indicates a prefer- VH vs FL-V and MM-V vs FL-V may implicate differences in the evolution of SHM coupled with selection in different stages ence for transitions over transversions with purines being of B cell ontogeny. Several explanations can be offered for the more frequently targeted than pyrimidines, suggesting strand fact that hotspot sequences were not always targeted by SHM bias; mutations are concentrated mainly in the CDRs and most in FL and MM: (1) other unrecognized motifs may be targets of often are single nucleotide substitutions rather than deletions SHM; (2) ‘inappropriately’ introduced mutations were fixed and or insertions; certain codons are targeted more often by the propagated by the neoplastic process; (3) certain FL and MM cases might have lost their ability to correct mutations intro- mutational process, while others are less likely to tolerate duced in classic hotspots due to deficient mismatch-repair changes; finally, a striking bias exists for G and C over A and (MMR) mechanisms; conversely, in other cases with intact T nucleotide mutations.9 MMR function, the hotspot to non-hotspot targeting of somatic In the present study, we analyzed the distribution of somatic hypermutation is balanced. Leukemia (2001) 15, 1772–1778. hypermutation and its targeting at specific mutational hotspots Keywords: immunoglobulin genes; hypermutation; follicular lym- in the clonotypic V and V␬ genes of follicular lymphoma (FL) phoma; multiple myeloma H as well as the V␬ and V␭ genes of multiple myeloma (MM), tumors corresponding to antigen-selected intra-germinal center (GC) and post-GC stages of B cell ontogeny. Introduction The antigen-independent phase of B cell ontogeny takes place Materials and methods in the bone marrow and is characterized by ordered immuno- globulin (lg) gene rearrangements leading to the assembly of Sequence analysis distinct variable (V), diversity (D) (for heavy chains only) and joining (J) gene segments into a V(D)J gene complex, a process Included in the present study were the clonotypic V and V␬ 1 H known as V(D)J recombination. Successful rearrangement of gene sequences of 10 FL cases10 as well as the clonotypic V␬ heavy chains (HC) lg genes and subsequently that of light and V␭ gene sequences of 11␬-and six ␭-light chain express- ␬ ␭ chain (LC) lg genes ( or ) will enable the developing B cell ing MM cases analyzed previously by our group.11,12 The to later express on its surface a fully functional lg receptor sequences have been submitted to the EMBL database 2 with unmutated V region sequences. (http://www.ebi.ac.uk/embl/index.html) with the following The second, antigen-dependent, phase will start when the accession numbers: for FL-VH, AJ410896 to AJ410905; for FL- ‘naı¨ve’ B cell exiting the bone marrow enters into the follicles V␬, AJ410886 to AJ410895; for MM-V␬ AJ410906 to AJ410916; and for MM-V␭, AJ410917 to AJ410922. The analysis aimed at determining whether specific nucleo- Correspondence: C Kosmas, Department of Medicine-Oncology Unit, Helena-Venizelou Hospital, 21 Apolloniou Street, 163 41 Athens, tide motifs, ie the tetranucleotide RGYW and its complemen- Greece; Fax: 30.1.9962917 tary WRCY were targeted by the somatic hypermutation Received 31 January 2001; accepted 28 June 2001 machinery. A further objective was to identify whether the Hypermutation in Ig V genes of FL and MM C Belessi et al 1773 Table 1 Numbers of mutations in and mutated tetra-and tri-nucleotidesand their distributions in each CDR, FWR (framework region), entire V sequence, and RGYW/WRCY motifs FWR1 FWR2 FWR3 CDR1 CDR2 CDR3 FWRS CDRS Total Mutations/4NTPS FL-kappa 1/4 4/12 9/38 15/28 2/20 10/27 14/54 27/75 41/129 FL-heavy 9/43 7/29 23/83 17/25 50/101 39/155 67/126 106/281 MM-kappa 0/9 17/30 29/109 36/57 9/34 20/48 46/148 65/139 111/287 MM-lambda 1/9 10/32 13/76 16/35 5/23 11/40 27/117 32/98 60/215 4NTPS/Mutated FL-kappa 1/10 4/63 8/66 13/42 2/11 10/28 13/139 25/81 38/220 FL-heavy 10/56 5/22 24/53 16/28 36/52 39/131 52/80 91/211 MM-kappa 0/11 21/72 24/72 33/53 6/17 12/24 45/155 51/94 96/249 MM-lambda 1/22 9/31 13/42 16/25 5/7 6/17 23/95 27/49 50/144 Mutations/3NTPS FL-kappa 0/0 6/13 14/33 17/29 6/20 16/29 20/46 39/78 59/124 FL-heavy 6/36 5/28 41/80 16/25 66/99 52/144 82/124 134/268 MM-kappa 4/6 14/29 42/120 35/58 20/38 28/48 60/155 83/144 143/299 MM-lambda 1/6 15/31 18/75 13/37 8/23 12/37 34/112 33/97 67/209 3NTPS/Mutated FL-kappa 0/39 5/66 17/113 12/73 6/25 14/44 22/218 32/142 54/360 FL-heavy 7/66 4/39 39/153 17/27 57/107 50/256 76/134 126/390 MM-kappa 4/42 18/72 40/123 84/92 11/30 23/49 62/137 68/171 130/308 MM-lambda 1/34 12/29 16/57 12/32 8/13 9/20 29/120 29/65 58/185 Mutations/RGYW FL-kappa 0/4 1/12 4/38 8/28 0/20 6/27 5/54 14/75 19/129 FL-heavy 3/43 7/29 11/83 12/25 27/101 21/155 39/126 60/281 MM-kappa 0/9 10/30 5/42 26/57 3/34 10/48 23/149 39/139 62/287 MM-lambda 0/9 4/32 9/76 9/35 3/23 9/40 13/117 21/98 34/215 RGYW/Mutated FL-kappa 0/1 1/30 4/30 8/28 0/4 6/18 5/61 14/50 19/111 FL-heavy 3/30 5/21 10/21 8/15 18/34 18/72 26/49 44/121 MM-kappa 0/2 10/33 13/38 21/33 2/10 8/16 23/72 31/59 54/131 MM-lambda 0/7 4/16 8/24 8/14 3/5 4/10 12/42 15/27 27/72 Mutations/WRCY FL-kappa 1/4 4/12 5/38 5/28 2/20 4/27 10/54 11/75 21/129 FL-heavy 7/43 0/29 15/83 9/25 31/101 22/155 40/126 62/281 MM-kappa 0/9 12/30 1/9 13/57 5/34 8/48 24/149 26/139 55/287 MM-lambda 1/9 8/32 7/76 11/35 2/23 4/40 16/117 17/98 33/215 WRCY/Mutated FL-kappa 1/9 3/33 4/36 5/15 2/6 4/10 8/78 11/31 19/109 FL-heavy 7/26 0/1 14/32 9/13 18/18 21/59 26/31 44/90 MM-kappa 0/9 11/39 11/35 12/20 4/7 4/8 19/83 20/35 39/118 MM-lambda 1/17 5/15 5/18 8/11 2/4 2/7 11/50 12/22 23/72 Mutations/4NTPs, mutations found in tetranucleotides, Mutations/3NTPs, mutations found in trinucleotides, 4NTPs/Mutated, number of tetran- ucleotides mutated; 3NTPs/Mutated, number of trinucleotides mutated, Mutations/RGYW or WRCY, mutations found in RGYW or WRCY; RGYW or WRCY/mutated, number of mutated RGYW or WRCY motifs.