Detail Characterization of 22q11 Deletion and Mir-650 Role in CLL Patients.

Chromosomal abnormalities are present in the majority of CLL cases (>80%) and recently, a novel deletion of 22q11 was described in CLL patients. This locus encodes lambda immunoglobulin light chain (IgL) subgenes and several protein-coding genes, which distinguishes IgL from kappa Ig or heavy Ig locus. Gunn et al. (2008) previously characterized the minimally deleted region of 22q11 in CLL as 0,34Mb (containing PRAME, ZNF280A, ZNF280B, GGTLC2) and described the PRAME as a candidate gene with prognostic impact in CLL. Interestingly, the IgL variable segment (V2-8) located at 22q11 includes an annotated miR gene miR-650. According to the performed BLAST search also other subgenes of V2 family include homologues for miR-650, which overlay the leader exon of the V2 subgenes. This could be of particular importance because the expression of miRNAs was previously shown to be associated with known prognostic markers in CLL: 13q deletion, IgVH status (Calin, 2002, 2005) and p53 deletion/mutation (Mraz, 2009). Moreover, miRNA pathways are involved in the regulation of immune system functions including IgG production and V(D)J recombination (Vigorito, 2007; Koralov, 2008).

We analyzed genomes of 40 CLL patients using array-CGH (CGH Microarray 4×44 K, Agilent) and FISH (del13q14, del17p13, del11q23, +12), and in particular focused on detailed characterization of 22q11 deletion. An expression analyses (RT-qPCR, TaqMan miRNA Assay, ABI) was performed for protein-coding genes and miR-650 located at 22q11.

Using array-CGH additional aberrations to FISH were found in the majority of samples and among them, the deletion of 22q11 locus was observed most often (ranging in size up to ∼ 0,77 Mb). Altogether, 40% of patients (16/40) had detectable deletion in this locus and according to our finding, all observed deletions were a consequence of a VJ rearrangement event, which lead to an elimination of specific genes located between VJ elements. These rearrangements were characterized in detail by PCR detection (BIOMED-2 protocol) and sequencing. In our cohort, deletions of 22q11 containing PRAME gene constituted only ∼44% (n=7) and 56% patients (n=9) harbored smaller deletions not involving PRAME. To analyze the gene expression, we performed a Real-Time PCR analyses for previously mentioned protein-coding genes and miR-650. The Real-Time PCR assay for miR-650 reported higher expression of miR-650 in CLL cells utilizing V2-8, V2-5, V2-14, V2-23 subgenes for IgL (n=5) compared to samples utilizing different V lambda family or expressing kappa Ig (n=36). It remains to be elucidated how and whether all the V2 subgenes give rise to a mature and functional miRNA. The overlap between miR-650 and the leader exon of IgL gene could provide a unique mechanism for coordinated IgL and miRNA expression. To assess the function of miR-650 we performed a microarray (Human OneArray, Phalanx) expression profiling 24/48 hours post transfection of CLL cells with a short RNA mimicing miR-650 (miRIDIAN Mimic, Dharmacon). The transfection led to a significant down-regulation of dozens of mRNAs that could be considered as potential targets for miR-650. Interestingly, the predicted targets for miR-650 include early B cell factor (EBF3; target with highest total score – TargetScan) and VPREB1 (pre B lymphocyte gene 1) whose expression is important for immature phenotype of pre-B lymphocytes prior to light chain recombination. The coupled activation of miR-650 and lambda Ig expression following productive rearrangement of IgL locus could potentially affect the expression of VPREB1/EBF3.

Deletion of 22q11 is frequently detected in CLL patients and we suggest that it is caused by physiological process of lambda immunoglobulin variable locus rearrangement. This leads to the elimination of protein-coding genes located between VJ elements. Interestingly, the V2 family of variable segments for lambda light chain at 22q11 includes an annotated miR gene miR-650 and its homologues. The productive lambda locus rearrangement utilizing V2 family could be also coupled with activation of miR-650 expression with consequent relevance for B cell biology. This work is in progress and in further analyses we expect complex characterization of miR-650 functions in B cells and CLL. Supported by IGA MZ CR NR9293-3/2007 and IGA MZ CR NS10439-3/2009.

Mayer:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy; Fresenius: Consultancy; Roche: Research Funding; Pfizer: Research Funding.