Deregulation of a possible tumour suppressor gene, <i>ZC3H12D</i>, by translocation of <i>IGK@</i> in transformed follicular lymphoma with t(2;6)(p12;q25)

Abstract

Follicular lymphoma (FL) is characterized by the t(14;18) (q32;21) translocation, which juxtaposes the BCL2 gene (BCL2)at 18q21 with the immunoglobulin heavy locus gene (IGH@) at 14q32 (Knutsen, 1997). The resultant deregulation of BCL2 with subsequent inhibition of apoptosis is thought to contribute to the onset of FL. FL has an initial indolent course, but more rapid progression by transformation to diffuse large B-cell lymphoma (DLBCL) occurs in many cases. Although other additional chromosomal abnormalities, such as +7, del(1)(p36), del(6)(q) and del(10)(q22-24), have been shown to be associated with the morphological progression of FL (Horsman et al, 2001), their molecular significance remains to be completely elucidated. Deletions of the long arm of chromosome 6 (6q) have been assumed to play an important role in the pathogenesis of tumours including lymphoma, lung cancer, breast cancer and ovarian cancer (Tilly et al, 1994; Zhang et al, 1998; Bailey-Wilson et al, 2004). FL with 6q23-26 deletions was reported to have a high risk of transformation to DLBCL and to be a poor prognostic indicator (Tilly et al, 1994). We previously reported a novel translocation t(2;6)(p12;q23) that appeared during the transformation of FL to DLBCL (Yamamoto et al, 2003). We suggested that t(2;6)(p12;q23) played a crucial role in the transformation, and we speculated that an unknown gene at 6q23 may be deregulated as a consequence of IGK@ translocation. Therefore, we initially performed Southern blot analysis of genomic DNA extracted from lymph nodes of the patient with t(2;6)(p12;q23) described previously (Yamamoto et al, 2003) by using Jκ and Cκ probes (Fig 1). Whereas there was no detectable rearranged band with the Jκ probe, two extra bands in addition to a germline band were detected with the Cκ probe. Southern blot analyses of IGK@ for the case with t(2;6)(p12;q23). Genomic DNA extracted from lymph nodes of the patient with t(2;6)(p12;q23) described previously (Yamamoto et al, 2003) was subjected to Southern blot analyses by using DNA probes derived from a 1·8 kb genomic fragment containing the joining region (Jκ) of IGK@, and a 323 bp fragment containing the constant region (Cκ) as shown in Fig 2. Hybridization with a Cκ probe shows two rearranged bands (arrows) as well as a germline band (arrowhead). To clone the gene, tentatively called ‘transformed follicular lymphoma (TFL)’ at the translocation breakpoint of t(2;6)(p12;q23), the genomic DNA was completely digested by BamHI, and electrophoresed on a 0·8% agarose gel. A fraction between 7 and 12 kb, corresponding to the rearranged bands, was collected. The extracted DNA was ligated into the ZAP Express Predigested Vector. This genomic library was screened by plaque hybridization using a Cκ probe. Finally, three independent clones were obtained (Fig 2). Structure of the chimera genes at t(2;6)(p12;q25). Restriction maps of the germline IGK@ on chromosome 2, clone I; the rearranged IGK@ on chromosome 2, clone II; fusion of IGK@ and ZC3H12D on the der(6)t(2;6)(p12;q25), clone III; fusion of IGK@, ZC3H12D and DUSP1, and ZC3H12D on chromosome 6. Thin lines under the IGK@ indicate probes used for Southern blot analyses (Fig 1). Open horizontal arrows indicate the direction of transcription, and black vertical arrows indicate the breakpoint junction. IGK@, ZC3H12D and DUSP1 are shown in black, white and grey respectively. Exons are indicated by boxes. Shaded boxes indicate the putative coding regions of ZC3H12D. The nucleotide sequences encompassing the breakpoint junction and its alignment to the corresponding germline counterparts of chromosomes 2 and 6 are also shown. Clone I was a 7·6 kb fragment containing the physiologically rearranged IGK@ on chromosome 2. Clones II was a 6·7 kb fragment composed of the partially deleted clone I and a 865 bp fragment derived from 6q25, i.e. this clone contained the translocation breakpoint with IGK@. Thus, the real breakpoint was located at 6q25 but not at 6q23. The nucleotide sequence of the 865 bp fragment was a part of the second intron of a gene designated as ZC3H12D. The breakpoint of IGK@ was located between Vκ1−33 and Vκ3−34. ZC3H12D was fused to the IGK@ in a ‘head-to-head’ configuration on the der(6)t(2;6)(p12;q25). One additional nucleotide (T) was found to be inserted between two genes (Fig 2). Clone III was a 5·3 kb fragment composed of a partially deleted clone II and a 1946 bp fragment derived from the dual specificity phosphatase 1 gene (DUSP1) at 5q34. This study identified the translocation of IGK@ with ZC3H12D at 6q25 in transformed FL. The 5′ region of ZC3H12D, which included exon 1 and exon 2, was replaced by Vκ and Cκ regions of IGK@. The gene truncated by translocation might be expressed similarly to the genes observed in other B-cell lymphomas, with the translocations involving one of the immunoglobulin loci and a proto-oncogene, such as BCL2, BCL6 and MYC (Küppers, 2005). It is assumed that dysfunction or loss of function by the gene translocation may be implicated in the transformation of FL. It is unclear whether the transformed lymphoma of the patient expressed the truncated gene or not. At least, it is unlikely that aberrant expression of a fusion protein occurs in transformation, because the IGK@-ZC3H12D fusion gene was generated in a ‘head-to-head’ configuration. Furthermore, clone III contained the DUSP1 fragment in addition to the IGK@-ZC3H12D fusion gene. This rearrangement is thought to occur in a minor population of transformed cells by an additional translocation between der(6)t(2;6)(p12;q25) and chromosome 5, because the translocation could not be detected by G-banding analyses. Recently, ZC3H12D was shown to be involved in sporadic lung cancer by loss of heterozygosity (LOH) (Wang et al, 2007). This gene carries an A/G non-synonymous single nucleotide polymorphism at codon 106. Overexpression of the A allele of ZC3H12D exerted a tumour suppressor function in vitro as well as in vivo. Our case had already lost one allele of ZC3H12D by the formation of i(6)(p10) before the transformation to DLBCL (Yamamoto et al, 2003). Thus, there is a possibility that the remaining A allele of ZC3H12D was inactivated by t(2;6)(p12;q25), following LOH in lymphoma. It would be interesting to analyse gene expression in other cases of FL and transformed FL. ZC3H12D has a zinc finger motif, C-x8-C-x5-C-x3-H type, which generally plays a role in DNA-binding and occasionally binds to an AU rich element (ARE) in the 3′ untranslated region of mRNA. Interestingly, many ARE binding proteins (ABP) are associated with cell cycle or cell growth phase-related regulation (Barreau et al, 2006). These findings raise the possibility that ZC3H12D regulates cell growth as an ABP. Moreover, a homologue of ZC3H12D, ZC3H12A, led to cardiac myocyte apoptosis (Zhou et al, 2006). Taken together, ZC3H12D might play a crucial role regulating lymphoma cell growth and/or survival. However, it remains to be elucidated whether deregulation of the possible tumour suppressor by translocation or deletion is relevant to transformation of FL. Further investigation of the physiological function of the gene would probably contribute to a better understanding of the multiple steps that give rise to malignant lymphoma. This work was supported in part by grants-in-aid for scientific research from the Ministry of Health, Welfare and Labor and from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank S. Tronick for editing this manuscript.