Fig. 1.
Fig. 1. Expression of AID RNA transcripts in CLL and their relation in CSR and SHM process. / (A) Semiquantitative AID expression. The expression of AID transcripts was monitored by semiquantitative RT-PCR using AID and GAPDH-specific primers in the same RT-PCR tube reaction. Representative amplification for healthy B cells (00) and either unmutated (01 and 03) or mutated (05 and 07) B-CLLs are shown. Amounts of AID transcripts were determined by normalization with internal GAPDH expression. Relative units corresponding to AID and GAPDH transcript amplification levels were quantified by Quant software (Molecular Dynamics). (B) Presence of different AID RNA transcripts. Normal cDNA (00) and CLL cDNA (01) were amplified and migrated. Three different RNA forms of the AID gene were found (1, 2, 3). The figure depicts a schematic sequence of AID mRNA previously reported by Schroeder et al,4 corresponding to 198 amino acids. The other 2 variants are spliced forms, one consisting of 618 base pair (bp) with an open-reading frame containing a deletion of 10 amino acids and the other consisting of 495 bp containing a complete deletion of exons 4 and 5 (51 amino acids). Deletions are depicted as unfilled rectangles. (C) Clonal isotype switch transcripts. mRNA transcript amplifications with tumor-relatedVH primers in 5′ and Cμ, Cδ, Cγ, and Cα in 3′ from patients 1 and 3 with unmutated and patients 5 and 7 with mutated disease. Patient 1 expresses μ, δ, γ, and α transcripts, and patient 3 expresses μ, δ, and γ tumorally related transcripts, whereas patients 5 and 7 only express μ and δ transcripts related to the tumoral clone. The smearlike amplification observed for patient 7 corresponds to a polyclonal amplification of different γ transcripts as confirmed by sequence. After stimulation patient 5 expressed a tumorally related γ transcript, and patient 7 acquired tumorally related γ and α transcripts. (D) Distributions of mutations in the Iμ/Sμ region. A 1625-bp genomic fragment between the enhancer and the Sμ switch core was amplified with primers A and D for 4 healthy controls and 7 patients with CLL (Table1). Closed and open arrows indicate point mutations (30 in the 3′ subregion, 9 in the 5′ subregion). In addition, 4 deletions in the 3′ subregion, depicted as rectangles, were observed. Open arrows illustrate where repeated mutations took place. Primers B (5′-TGC CTG TCT CTT ACC ATG TCG GG-3′) and C (5′-GAC ATG GTA AGA GAC AGG CAG CCG-3′) were used as internal primers for sequencing reaction. Given that in all cases 3 independent sequences obtained from different PCRs with a high-fidelity Taq DNA polymerase (10−6 expected rate mutation) were carried out, Taq infidelity should play a minor, if any, role in the appearance of mutations.

Expression of AID RNA transcripts in CLL and their relation in CSR and SHM process.

(A) Semiquantitative AID expression. The expression of AID transcripts was monitored by semiquantitative RT-PCR using AID and GAPDH-specific primers in the same RT-PCR tube reaction. Representative amplification for healthy B cells (00) and either unmutated (01 and 03) or mutated (05 and 07) B-CLLs are shown. Amounts of AID transcripts were determined by normalization with internal GAPDH expression. Relative units corresponding to AID and GAPDH transcript amplification levels were quantified by Quant software (Molecular Dynamics). (B) Presence of different AID RNA transcripts. Normal cDNA (00) and CLL cDNA (01) were amplified and migrated. Three different RNA forms of the AID gene were found (1, 2, 3). The figure depicts a schematic sequence of AID mRNA previously reported by Schroeder et al,4 corresponding to 198 amino acids. The other 2 variants are spliced forms, one consisting of 618 base pair (bp) with an open-reading frame containing a deletion of 10 amino acids and the other consisting of 495 bp containing a complete deletion of exons 4 and 5 (51 amino acids). Deletions are depicted as unfilled rectangles. (C) Clonal isotype switch transcripts. mRNA transcript amplifications with tumor-relatedVH primers in 5′ and Cμ, Cδ, Cγ, and Cα in 3′ from patients 1 and 3 with unmutated and patients 5 and 7 with mutated disease. Patient 1 expresses μ, δ, γ, and α transcripts, and patient 3 expresses μ, δ, and γ tumorally related transcripts, whereas patients 5 and 7 only express μ and δ transcripts related to the tumoral clone. The smearlike amplification observed for patient 7 corresponds to a polyclonal amplification of different γ transcripts as confirmed by sequence. After stimulation patient 5 expressed a tumorally related γ transcript, and patient 7 acquired tumorally related γ and α transcripts. (D) Distributions of mutations in the Iμ/Sμ region. A 1625-bp genomic fragment between the enhancer and the Sμ switch core was amplified with primers A and D for 4 healthy controls and 7 patients with CLL (Table1). Closed and open arrows indicate point mutations (30 in the 3′ subregion, 9 in the 5′ subregion). In addition, 4 deletions in the 3′ subregion, depicted as rectangles, were observed. Open arrows illustrate where repeated mutations took place. Primers B (5′-TGC CTG TCT CTT ACC ATG TCG GG-3′) and C (5′-GAC ATG GTA AGA GAC AGG CAG CCG-3′) were used as internal primers for sequencing reaction. Given that in all cases 3 independent sequences obtained from different PCRs with a high-fidelity Taq DNA polymerase (10−6 expected rate mutation) were carried out, Taq infidelity should play a minor, if any, role in the appearance of mutations.

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