Identification Genetic Variations (GVs) Causing Splicing of TNF Family Members and Adaptor Proteins That Modulate NFkB Pathways in Waldenstrom’s Maroglobulinemia (WM).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2516-2516
Author(s):  
Sophia Adamia ◽  
Lian Xu ◽  
Antonio Sacco ◽  
Steven Treon
Keyword(s):  
B Cell ◽  
Heavy Chain ◽  
Splice Variants ◽  
Family Members ◽  
Adaptor Protein ◽  
Immunoglobulin Heavy Chain ◽  
Genetic Variations ◽  
Aberrant Splice ◽  
Class Switching ◽  
Cd40 Gene

Abstract The TNF ligand-receptor superfamily and their adaptor proteins regulate important B-cell signaling pathways, including CD40L-CD40 and APRIL/BLYS-TACI through adaptor protein TRAF2. These pathways promote B-cell differentiation and immunoglobulin heavy chain class switching. Defects in immunoglobulin heavy chain class switching and presence of constitutive IgA and IgG hypogammaglobulinemia in patients with WM have previously been reported by us (Hunter et al, ASH2006). In WM patients we identified several novel splice variants of TNF-family members CD40 and BLYS, and their adaptor protein TRAF2. Cloning, sequencing and alignment analysis document that aberrant splice transcripts of CD40, CD40-Va, Vb and Vc, and BLYS, BLYS-Va and Vb, result from partial exon skipping (CD40Va and Vc) and entire or partial intron retention (CD40-Vb, BLYS-Va and Vb), while the TRAF2 variant is a result of exon skipping only. Using RT-PCR DNA fragment analysis, malignant and normal B-cells from the bone marrow of 25 WM patients and 6 healthy donors (HDs) were screened for the expression of CD40, BLYS and TRAF2 splice transcripts. This analysis identified overexpression of CD40-Vb (15/25WM vs. 0/6HD; P=0.01), CD40-Vc (21/25WM vs. 0/6HD; P=0.0003), BLYS-Vb (18/25WM vs. 0/6HD; P=0.002) and TRAF2-V (21/25WM vs. 1/6HD; P=0.004) in WM patients. The expression of other splice variants CD40-Va (18/25WM vs.2/4HD; P=0.09) and BLYS-Va (23/25WM vs. 4/6HD P=0.2) in the same group of WM patients were not significant. We hypothesized that these aberrations are consequences of genetic variations (GVs) distributed in the vicinity of splicing elements of these genes, as well as, alterations may have occurred in the repertoire of splicing factors (SFs) with respect of their expression levels. To address these issues, we started sequencing CD40, BLYS, and TRAF2 gene segments that are subjected to aberrant splicing. Sequencing analysis of the CD40 gene from WM B cells revealed 6 recurrent genetic variations (GVs-defined as mutations occurring more than one patients) that include 2 missense (on exon 3) and 3 silent substitutions (on exons 3-4-5), and 1 frame-shift deletion on exon 5. These substitutions lead to amino acid changes on CD40 gene, while the frame-shift deletion may cause truncation of wild-type CD40 protein. We also identified recurrent GVs on introns 4 and 5. All these GVs detected on exons and introns are distributed in the vicinity of key splicing elements that create and/or activate a new splice site in precisely the position required for the splicing events to create CD40 variants. Also, using TaqMan low density array (TaqMan-LDA) we evaluated levels of major SFs and other TNF family members involved in CD40 and BLYS signaling. These analyses showed that patients expressing aberrant splice variants of CD40 and BLYS overexpress not only other members of TNF family but also major SFs: SF2/ASF (a proto-oncogene), U2, hNRPA1 and SRP55. TaqMan-LDA analysis suggests that these SFs may play a significant role in CD40, BLYS and TRAF2 splicing in WM patients since these transcripts were upregulated (1.2-2.2 fold higher) only in those patients which expressed CD40, BLYS and TRAF2 variants. In conclusion, presence of GVs in the vicinity of splicing elements and upregulation of SFs collectively may promote aberrant CD40, BLYS and TRAF2 splicing and thus modulate TNF family pathways supportive of B-cell differentiation and immunoglobulin heavy chain class switching in patients with WM.

Immunoglobulin heavy-chain class switching in a pre-B cell line is accompanied by DNA rearrangement

Nature ◽  
1983 ◽  
Vol 306 (5940) ◽  
pp. 243-246 ◽  
Author(s):  
Peter D. Burrows ◽  
Gabriele B. Beck-Engeser ◽  
Matthias R. Wabl
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Heavy Chain ◽  
Immunoglobulin Heavy Chain ◽  
Dna Rearrangement ◽  
Class Switching

scholarly journals Rate of replication of the murine immunoglobulin heavy-chain locus: evidence that the region is part of a single replicon.

1987 ◽  
Vol 7 (1) ◽  
pp. 450-457 ◽  
Author(s):  
E H Brown ◽  
M A Iqbal ◽  
S Stuart ◽  
K S Hatton ◽  
J Valinsky ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Gene Cluster ◽  
Cell Lines ◽  
Heavy Chain ◽  
Dna Content ◽  
Immunoglobulin Heavy Chain ◽  
S Phase ◽  
C Value ◽  
Murine Erythroleukemia

We measured the temporal order of replication of EcoRI segments from the murine immunoglobulin heavy-chain constant region (IgCH) gene cluster, including the joining (J) and diversity (D) loci and encompassing approximately 300 kilobases. The relative concentrations of EcoRI segments in bromouracil-labeled DNA that replicated during selected intervals of the S phase in Friend virus-transformed murine erythroleukemia (MEL) cells were measured. From these results, we calculated the nuclear DNA content (C value; the haploid DNA content of a cell in the G1 phase of the cell cycle) at the time each segment replicated during the S phase. We observed that IgCH genes replicate in the following order: alpha, epsilon, gamma 2a, gamma 2b, gamma 1, gamma 3, delta, and mu, followed by the J and D segments. The C value at which each segment replicates increased as a linear function of its distance from C alpha. The average rate of DNA replication in the IgCH gene cluster was determined from these data to be 1.7 to 1.9 kilobases/min, similar to the rate measured for mammalian replicons by autoradiography and electron microscopy (for a review, see H. J. Edenberg and J. A. Huberman, Annu. Rev. Genet. 9:245-284, 1975, and R. G. Martin, Adv. Cancer Res. 34:1-55, 1981). Similar results were obtained with other murine non-B cell lines, including a fibroblast cell line (L60T) and a hepatoma cell line (Hepa 1.6). In contrast, we observed that IgCh segments in a B-cell plasmacytoma (MPC11) and two Abelson murine leukemia virus-transformed pre-B cell lines (22D6 and 300-19O) replicated as early as (300-19P) or earlier than (MPC11 and 22D6) C alpha in MEL cells. Unlike MEL cells, however, all of the IgCH segments in a given B cell line replicated at very similar times during the S phase, so that a temporal directionality in the replication of the IgCH gene cluster was not apparent from these data. These results provide evidence that in murine non-B cells the IgCH, J, and D loci are part of a single replicon.

Immunoglobulin heavy-chain enhancer is required to maintain transfected gamma 2A gene expression in a pre-B-cell line

1990 ◽  
Vol 10 (3) ◽  
pp. 1076-1083
Author(s):  
B Porton ◽  
D M Zaller ◽  
R Lieberson ◽  
L A Eckhardt
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Heavy Chain ◽  
Gene Activation ◽  
Immunoglobulin Heavy Chain ◽  
Transcription Unit ◽  
Enhancer Activity ◽  
Igh Genes ◽  
High Level

The immunoglobulin heavy-chain (IgH) enhancer serves to activate efficient and accurate transcription of cloned IgH genes when introduced into B lymphomas or myelomas. The role of this enhancer after gene activation, however, is unclear. The endogenous IgH genes in several cell lines, for example, have lost the IgH enhancer by deletion and yet continue to be expressed. This might be explained if the role of the enhancer were to establish high-level gene transcription but not to maintain it. Alternatively, other enhancers might lie adjacent to endogenous IgH genes, substituting their activity for that of the lost IgH enhancer. To address both of these alternatives, we searched for enhancer activity within the flanking regions of one of these IgH enhancer-independent genes and designed an experiment that allowed us to consider separately the establishment and maintenance of expression of a transfected gene. For the latter experiment we generated numerous pre-B cell lines stably transformed with a gamma 2a gene. In this gene, the IgH enhancer lay at a site outside the heavy-chain transcription unit, between DH and JH gene segments. After expression of the transfected gene was established, selective conditions were chosen for the outgrowth of subclones that had undergone D-J joining and thus IgH enhancer deletion. Measurements of gamma 2a expression before and after enhancer deletion revealed that the enhancer was required for maintenance of expression of the transfected gene. The implication of this finding for models of enhancer function in endogenous genes is discussed.

Pi, a pre-B-cell-specific enhancer element in the immunoglobulin heavy-chain enhancer

1993 ◽  
Vol 13 (10) ◽  
pp. 5957-5969
Author(s):  
T A Libermann ◽  
D Baltimore
Keyword(s):  
B Cell ◽  
Heavy Chain ◽  
Mutational Analysis ◽  
Protein S ◽  
Immunoglobulin Heavy Chain ◽  
Chain Gene ◽  
Cell Stage ◽  
Enhancer Activity ◽  
Enhancer Element ◽  
Light Chain Gene

We have identified a new immunoglobulin heavy-chain enhancer element, designated pi, between the microE2 and microE3 elements. The pi enhancer element is transcriptionally active primarily during early stages of B-cell development but becomes virtually inactive during B-cell maturation at about the stage of immunoglobulin kappa light-chain gene rearrangement. Mutational analysis suggests that the pi element is crucial for immunoglobulin heavy-chain enhancer activity at the pre-B-cell stage but is almost irrelevant for enhancer activity at the mature B-cell or plasma-cell stage. The activity of the pi enhancer element correlates with the presence of an apparently pre-B-cell-specific protein-DNA complex. The similarity of the pi site to recognition sequences for members of the ets gene family suggests that the protein(s) interacting with the pi site most likely are ets-related transcription factors.

A second B cell-specific enhancer 3' of the immunoglobulin heavy-chain locus

Nature ◽  
1990 ◽  
Vol 344 (6262) ◽  
pp. 165-168 ◽  
Author(s):  
Sven Pettersson ◽  
Graham P. Cook ◽  
Marianne Brüggemann ◽  
Gareth T. Williams ◽  
Michael S. Neuberger
Keyword(s):  
B Cell ◽  
Heavy Chain ◽  
Immunoglobulin Heavy Chain ◽  
Chain Locus ◽  
Heavy Chain Locus

scholarly journals Changes in Locus-specific V(D)J Recombinase Activity Induced by Immunoglobulin Gene Products during B Cell Development

1997 ◽  
Vol 185 (4) ◽  
pp. 609-620 ◽  
Author(s):  
Andrei Constantinescu ◽  
Mark S. Schlissel
Keyword(s):  
B Cells ◽  
B Cell ◽  
Heavy Chain ◽  
Light Chain ◽  
Immunoglobulin Heavy Chain ◽  
Cell Development ◽  
B Cell Development ◽  
Allelic Exclusion ◽  
Chain Locus ◽  
Heavy Chain Locus

The process of V(D)J recombination is crucial for regulating the development of B cells and for determining their eventual antigen specificity. Here we assess the developmental regulation of the V(D)J recombinase directly, by monitoring the double-stranded DNA breaks produced in the process of V(D)J recombination. This analysis provides a measure of recombinase activity at immunoglobulin heavy and light chain loci across defined developmental stages spanning the process of B cell development. We find that expression of a complete immunoglobulin heavy chain protein is accompanied by a drastic change in the targeting of V(D)J recombinase activity, from being predominantly active at the heavy chain locus in pro-B cells to being exclusively restricted to the light chain loci in pre-B cells. This switch in locus-specific recombinase activity results in allelic exclusion at the immunoglobulin heavy chain locus. Allelic exclusion is maintained by a different mechanism at the light chain locus. We find that immature, but not mature, B cells that already express a functional light chain protein can undergo continued light chain gene rearrangement, by replacement of the original rearrangement on the same allele. Finally, we find that the developmentally regulated targeting of V(D)J recombination is unaffected by enforced rapid transit through the cell cycle induced by an Eμ-myc transgene.

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