John D. Curry

Genetic variants at 6p21.33 are associated with susceptibility to follicular lymphoma.

We conducted genome-wide association studies of non-Hodgkin lymphoma using Illumina HumanHap550 BeadChips to identify subtype-specific associations in follicular, diffuse large B-cell and chronic lymphocytic leukemia/small lymphocytic lymphomas. We found that rs6457327 on 6p21.33 was associated with susceptibility to follicular lymphoma (FL; N = 189 cases, 592 controls) with validation in another 456 FL cases and 2,785 controls (combined allelic P = 4.7 × 10−11). The region of strongest association overlapped C6orf15 (STG), located near psoriasis susceptibility region 1 (PSORS1).

Single-strand recombination signal sequence nicks in vivo: evidence for a capture model of synapsis

Variable (diversity) joining (V(D)J) recombination is initiated by the introduction of single-strand DNA breaks (nicks) at recombination signal sequences (RSSs). The importance and fate of these RSS nicks for the regulation of the V(D)J rearrangement and their potential contribution to genomic instability are poorly understood. Using two new methodologies, we were able to detect and quantify specific RSS nicks introduced into genomic DNA by incubation with recombination-activating gene proteins in vitro. In vivo, however, we found that nicks mediated by recombination-activating gene (RAG) proteins were detectable only in gene segments associated with RSSs containing 12–base pair spacers but not in those containing 23–base pair spacers. These data support a model of capture rather than synapsis for pairwise RSS cleavage during V(D)J recombination.

A functional TNFRSF5 gene variant is associated with risk of lymphoma.

CD40 and its ligand, CD154, are major costimulatory molecules whose interactions are important in humoral and cellular immunity. We hypothesized that single nucleotide polymorphisms (SNPs) in TNFRSF5 and TNFSF5 encoding the CD40 and CD154 proteins, respectively, influence lymphoma risk, particularly a functional TNFRSF5 SNP (-1C>T, rs1883832) associated with reduced B-cell CD40 expression. TNFRSF5 and TNFSF5 SNPs were examined in a population-based case-control study of non-Hodgkin lymphoma (376 cases/801 controls with DNA), and compelling findings were followed up in 2 independent populations. Pooled analyses of all 3 case-control studies (total N = 1776 non-Hodgkin lymphoma cases, N = 2482 controls) revealed an increased risk of follicular lymphoma (FL) associated with the TNFRSF5 -1TT genotype (odds ratio = 1.6; 95% confidence interval, 1.1-2.4). In addition, among women, an inverse association was found between the variant A allele for a TNFSF5 6809G>A SNP and FL risk (OR = .61; 95% CI, 0.36-0.98). In genotype-phenotype studies, significantly reduced circulating soluble CD40 was observed in TNFRSF5 -1TT compared with -1CC carriers. Further, dendritic cells from those with -1TT versus -1CC genotypes exhibited lower CD40 cell surface expression. These results suggest that the TNFRSF5 -1C>T polymorphism may increase FL susceptibility through mechanisms that hinder cellular immune responses. Further studies are needed to explore these findings.

Chromosome Translocations in Workers Exposed to Benzene

As benzene has been linked with elevated risk of both acute myeloid leukemia and lymphoma, we explored the effect of benzene exposure on levels of t(8;21), t(15;17), and t(14;18) translocations. Circulating lymphocytes of normal individuals also often contain t(14;18). Quantitative polymerase chain reaction analysis showed that 37 workers with benzene exposure had a decreased level of t(14;18) in their blood with only 16.2% having 10 or more copies of the t(14;18) BCL-2/IgH fusion gene/microg DNA, as opposed to 55% of 20 controls (P = .0063 by Fishers exact test). This decline may be related to the immunotoxicity to specific subtypes of circulating B-lymphocytes, but the data do not support the use of t(14;18) as a biomarker of increased lymphoma risk in benzene-exposed populations. None of 88 individuals (31 controls and 57 exposed) exhibited detectable t(8;21) transcripts, and while t(15;17) transcripts were detected in two individuals, the result is inconclusive as one was exposed and the other was unexposed.

RAG2's non-core domain contributes to the ordered regulation of V(D)J recombination.

Variable (diversity) joining [V(D)J] recombination of immune gene loci proceeds in an ordered manner with D to J portions recombining first and then an upstream V joins that recombinant. We present evidence that the non-core domain of recombination activating gene (RAG) protein 2 is involved in the regulation of recombinatorial order. In mice lacking the non-core domain of RAG2 the ordered rearrangement is disturbed and direct V to D rearrangements are 10- to 1000-times increased in tri-partite immune gene loci. Some forms of inter-chromosomal translocations between TCRβ and TCRδ D gene segments are also increased in the core RAG2 animals as compared with their wild-type (WT) counterparts. In addition, the concise use of proper recombination signal sequences (RSSs) appears to be disturbed in the core RAG2 mice as compared with WT RAG2 animals.

Chromosomal reinsertion of broken RSS ends during T cell development

The V(D)J recombinase catalyzes DNA transposition and translocation both in vitro and in vivo. Because lymphoid malignancies contain chromosomal translocations involving antigen receptor and protooncogene loci, it is critical to understand the types of “mistakes” made by the recombinase. Using a newly devised assay, we characterized 48 unique TCRβ recombination signal sequence (RSS) end insertions in murine thymocyte and splenocyte genomic DNA samples. Nearly half of these events targeted “cryptic” RSS-like elements. In no instance did we detect target-site duplications, which is a hallmark of recombinase-mediated transposition in vitro. Rather, these insertions were most likely caused by either V(D)J recombination between a bona fide RSS and a cryptic RSS or the insertion of signal circles into chromosomal loci via a V(D)J recombination-like mechanism. Although wild-type, p53, p53 x scid, H2Ax, and ATM mutant thymocytes all showed similar levels of RSS end insertions, core-RAG2 mutant thymocytes showed a sevenfold greater frequency of such events. Thus, the noncore domain of RAG2 serves to limit the extent to which the integrity of the genome is threatened by mistargeting of V(D)J recombination.

Alterations in serum immunoglobulin levels in workers occupationally exposed to trichloroethylene

Trichloroethylene (TCE) has been associated with a variety of immunotoxic effects and may be associated with an increased risk of non-Hodgkin lymphoma (NHL). Altered serum immunoglobulin (Ig) levels have been reported in NHL patients and in animals exposed to TCE. Recently, we reported that occupational exposure to TCE is associated with immunosuppressive effects and immune dysfunction, including suppression of B-cell counts and activation, even at relatively low levels. We hypothesized that TCE exposure would also affect Ig levels in humans. We measured serum levels of IgG, IgM and IgE, by enzyme-linked immunosorbent assay, in TCE-exposed workers (n = 80) and unexposed controls (n = 45), matched by age and gender, in a cross-sectional, molecular epidemiology study of occupational exposure to TCE in Guangdong, China. Exposed workers had about a 17.5% decline in serum levels of IgG compared with unexposed controls (P = 0.0002). Similarly, serum levels of IgM were reduced by about 38% in workers exposed to TCE compared with unexposed controls (P < 0.0001). Serum levels of both IgG and IgM were significantly decreased in workers exposed to TCE levels below 12 p.p.m., the median exposure level. Adjustment for B-cell counts had minimal impact on our findings. IgE levels were not significantly different between exposed and control subjects. These results provide further evidence that TCE is immunotoxic at relatively low exposure levels and provide additional biologic plausibility for the reported association of TCE with NHL.

Real‐time reverse transcription polymerase chain reaction detection and quantification of t(1;19) (E2A–PBX1) fusion genes associated with leukaemia

A real‐time reverse transcription polymerase chain reaction (RT‐PCR) method is described that enabled the detection and quantification of E2A–PBX1 fusion gene transcripts associated with t(1;19). The method was highly reproducible and offered exceptional sensitivity at 5 fg of fusion transcript per reaction, without the need for a nested PCR primer design. To illustrate the usefulness of this new technology the E2A–PBX1 fusion gene transcript expression level for several human leukaemia cell lines that are positive and negative for cytogenetically detectable t(1;19) was determined. The RCH–ACV had a threefold higher expression of E2A–PBX1 transcripts (600 transcripts per cell) than the other t(1;19) positive 697 (150 transcripts per cell). The only other cell line with detectable E2A–PBX1 was CEM, but the level of expression was < 1 transcript per cell.

Association of HLA-DQB1 alleles with risk of follicular lymphoma

In a recent genome-wide association study of follicular lymphoma (FL), we identified novel risk alleles on chromosome 6p21.33 that appeared to be part of an extended haplotype including HLA-DRB1*0101, DQA1*0101, and DQB1*0501. To follow up on these findings, we obtained 2–4 digit HLA-DQB1 allelotypes on a subset of 265 cases of FL and 757 controls using a novel assay that applies multiplexed ligation-dependent probe amplification (MLPA). We confirmed a positive association between FL and the HLA-DQB1*05 allele group (OR = 1.70, 95% CI 1.28–2.27; adjusted p-value = 0.013) and also identified an allele group inversely associated with FL risk, HLA-DQB1*06 (OR = 0.51, 95% CI 0.38–0.69; adjusted p-value = 4.46 × 10−5). Although these findings require verification, the role of HLA class II proteins in B-cell survival and proliferation makes this a biologically plausible association.

Measurement of SIL–TAL1 fusion gene transcripts associated with human T-cell lymphocytic leukemia by real-time reverse transcriptase-PCR

TAL1 disruption at 1p32 [del(1p)] is a common rearrangement in the development of T-cell acute lymphocytic leukemia (T-ALL). The del(1p) are usually interstitial 90kb deletions placing TAL1 under control of the SCL interrupting locus (SIL) gene forming the SIL-TAL1 fusion product. A reverse transcriptase real-time PCR assay to quantify SIL-TAL1 fusion genes is described. A SIL-TAL1 fusion gene RNA transcript was built that permitted absolute standard curves to be generated. Sensitivity of the RT-PCR assay was determined to be 10 cells (CEM cell line) in 10(6) human lymphocytes. Peripheral blood lymphocytes from 10 healthy adults and 10 neonates were assayed. None of the samples showed any SIL-TAL1 expression. However, when lymphocytes from three adults were cultured in vitro the SIL-TAL1 transcript was detectable in the RNA isolates. No RAG2 expression was detected in these expanded samples, suggesting that the clones bearing the SIL-TAL1 fusion gene may have existed at low levels prior to the ex vivo expansion.