An Alternatively Spliced Form of CD79b Gene May Account for Altered B-Cell Receptor Expression in B-Chronic Lymphocytic Leukemia

Abstract

Several functional anomalies of B-chronic lymphocytic leukemia (B-CLL) cells may be explained by abnormalities of the B-cell receptor (BCR), a multimeric complex formed by the sIg homodimer and the noncovalently bound heterodimer Ig/Igβ (CD79a/CD79b). Because the expression of the extracellular Ig-like domain of CD79b has been reported to be absent in the cells of most CLL cases, we have investigated the molecular mechanisms that may account for this defect. Peripheral blood lymphocytes (PBL) from 50 patients and two cell lines (MEC1, MEC2) obtained from the PBL of one of them were studied. MEC1, MEC2, and 75% of CLL cases did not express detectable levels of the extracellular Ig-like domain of CD79b, which was nevertheless present in greater than 80% CD19+ cells from normal donors. In healthy subjects the expression of CD79b was equally distributed in CD5+ and CD5− B-cell subsets. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of CD79b RNA from all patients and from MEC1 and MEC2 cell lines consistently yielded two fragments of different size (709 bp and 397 bp). The 709-bp band corresponds to CD79b entire transcript; the 397-bp band corresponds to an alternatively spliced form lacking exon 3 that encodes the extracellular Ig-like domain. Both fragments were also visible in normal PBL. The expression of the 397-bp fragment was increased in normal activated B cells, while no difference was seen between CD5+ and CD5− B cells. To obtain a more accurate estimate of the relative proportions of the two spliced forms, a radioactive PCR was performed in 13 normal and 22 B-CLL samples and the results analyzed using a digital imager. The mean value of the CD79b to the CD79b internally deleted ratio was 0.64 ± 0.20 SD in normal donors and 0.44 ± 0.27 SD in B-CLL (P = .01). Direct sequencing of 397-bp RT-PCR products and of genomic DNA corresponding to exon 3 from MEC1, MEC2, their parental cells, and five fresh B-CLL samples did not show any causal mutation. Single-strand conformation polymorphism analysis of exon 3 performed in 18 additional B-CLL cases showed a single abnormal shift corresponding to a TGT → TGC polymorphic change at amino acid 122. We propose a role for the alternative splicing of CD79b gene in causing the reduced expression of BCR on the surface of B-CLL cells. As normal B cells also present this variant, the mechanism of CD79b posttranscriptional regulation might reflect the activation stage of the normal B cell from which B-CLL derives.

Defective assembly of the B‐cell receptor chains accounts for its low expression in B‐chronic lymphocytic leukaemia

“…In a previous work (Payelle-Brogard et al, 1999), we reported the absence of a genetic defect of the B29 gene in 10 CLL families, each of which included two affected members. In the present work, analysis of the CD79b molecule in 15 patients showed a constant low level of surface expression as described by others (Zomas et al, 1996;Alfarano et al, 1999a), which was not related to a defect in transcription. Indeed, B29 mRNA was detected in all except one of the patients, in variable amounts, and did not correlate with the very poor surface expression consistently observed in CLL patients.…”

“…We investigated whether a defect at the transcriptional or post-transcriptional level might account for this low expression by studying B29 mRNA expression, structural characteristics of the IgM transmembrane region that is associated with CD79a/CD79b and of the chaperone protein calnexin involved in folding and assembly of the BCR, protein synthesis, assembly and intracellular transport of the BCR components. We report here that B29 mRNA detection did not show an obvious correlation between its quantity and the low expression of membrane CD79b protein as reported by Alfarano et al (1999a), and that long transcripts of the B29 gene encoding for the complete membrane CD79b were variably expressed in CLL patients but not correlated to the consistent low expression of surface CD79b. Structural defects in membrane IgM and the chaperone protein calnexin were not detected in CLL patients.…”

Role of the microenvironment in chronic lymphocytic leukaemia

“…The BCR is central in regulating proliferation, differentiation and apoptosis of B cells and comprises sIg, which are the Ag-binding subunit, and the heterodimer CD79a/CD79b, which is the signalling subunit. In most CLL patients the extracellular domain of CD79b is absent (Zomas et al, 1996) and a CD79b truncated form has been detected (Alfarano et al, 1999;Payelle-Brogard et al, 1999). This form arises by alternative splicing of the CD79b gene (Hashimoto et al, 1995) and is able to inhibit signalling for apoptosis (Cragg et al, 2002).…”

Heterogeneous intracellular expression of B‐cell receptor components in B‐cell chronic lymphocytic leukaemia (B‐CLL) cells and effects of CD79b gene transfer on surface immunoglobulin levels in a B‐CLL‐derived cell line

“…In view of our observation of a selective lack of CD79b in a subset of B-CLL samples and of previous literature suggesting that abnormal CD79b expression could be responsible for the low sIg levels in these cells (Zomas et al, 1996;Thompson et al, 1997;Alfarano et al, 1999), we set out to explore whether gene transfer of CD79b may suffice to restore normal levels of surface IgM expression. To investigate this, we generated an Low III 82 445 97 480 61 348 97 487 53 321 94 392 18 Low III 55 378 88 597 35 385 96 485 4 636 90 358 19 Low III 88 509 94 478 57 381 92 441 25 353 90 362 20 Low III 89 430 99 567 54 362 98 439 33 407 84 327 21 Low III 53 445 98 471 25 348 99 487 21 389 82 343 22 Low III 76 443 85 453 77 401 97 492 58 362 87 370 Normal controls 1 79 593 68 418 88 474 91 462 76 428 88 373 2 81 560 75 414 89 431 74 330 82 473 74 355 MFI, mean fluorescence intensity; n.d.: not ...…”