Mutational Analysis of Mir-29 Family Members in Chronic Lymphocytic Leukemia

MicroRNAs (miRNAs) are small non-coding RNA molecules regulating gene expression by inhibition of mRNA translation and/or stability. Numerous miRNAs were described as tumor suppressor genes or oncogenes. In this respect, miR-29 family belongs to the most important miRNAs involved in CLL biology. Three different isoforms of the miR-29 (miR-29a-2, miR-29b-2 and miR-29c) were shown to be down-regulated in aggressive CLL (Calin et al., 2004; Mraz et al., 2009). Moreover, miR-29 was suggested to target the expression of MCL1, CDK6 and TCL1, a critical oncogene in aggressive CLL. Recently, the generation of transgenic mice over-expressing miR-29 in B-cells demonstrated its direct role in CLL pathogenesis. Additionally, mutations in miR-29c and miR-29b-2 have been also detected in CLL patients (Calin et al., 2005); however, their impact and frequency have yet to be elucidated.

In this study, we searched for novel sequence variations in three members of the miR-29 family (miR-29a, miR-29b-2 and miR-29c) using re-sequencing microarray and Sanger sequencing.

The pre-miRNAs (29a, 29b-2, 29c) and 107 other pre-miRNAs were analyzed by a custom re-sequencing microarray (Affymetrix, 50K) in 98 high-risk CLL patients (81.6% of patients had unmutated IgVH; 37.8% had a mutation in the TP53 gene). To cover the whole genomic area of pre-miRNAs, ∼20 nucleotides from both 5' and 3' ends of pri-miRNAs were also analysed. MiRNAs were amplified from genomic DNA isolated from peripheral blood cells (CLL lymphocytes or mononuclear cells) using long range PCR. Amplicons were pooled, fragmented and co-hybridized on the array according to the manufacturer's protocol. Sanger sequencing was used to confirm the presence of the mutations detected by microarray. The genomic regions (455-bp, 940-bp) of pri-miR-29c and pri-miR-29b-2, respectively, were further analyzed by Sanger sequencing in another 192 CLL patients (63.5% with unmutated IgVH; 21.4% with TP53 gene mutation).

Using resequencing microarray and confirmatory Sanger sequencing, one heterozygous variation (A/G +22 in 3') was found in the pri-miR-29a in one of 98 CLL patients. Screening of pri-miR-29c (455-bp genomic area) detected two substitutions in 4 of 192 CLL patients. In particular, the known G/A substitution (-31 in 5') was found as heterozygous in two patients. Additionally, novel heterozygous T/A substitution (+137 in 3') was found in two other patients. Screening of 940-bp genomic region of the pri-miR-29b-2 detected insertion (+A) (+107 in 3') in 22 of 192 patients. However, the known G/A substitution (+212 in 3') was not detected in our cohort. Furthermore, 2 novel heterozygous variations in pri-miR-29b-2 (C/G −169 in 5' and A/G − 256 in 5') were found in 5 and 2 patients, respectively. According to our results, pri-miR-29b-2 seems to be the most frequently mutated member of the miR-29 family, since three types of variations were detected in 29 of 192 CLL patients (15.1%). Two substitutions were found in pri-miR-29c in 4 of 192 CLL patients (2.1%) and only one substitution was detected in pri-miR-29a in one of 98 CLL patients (1%).

We herein confirm that both known mutations and novel sequence variations are present in three members of the miR-29 family which is already implicated in CLL pathogenesis or progression. Altogether, we have found 6 types of variations in 34 of 290 analyzed CLL patients (11.7%). The insertion (+A) (+107 in 3') in pri-miR-29b-2 was the most common variation detected in our cohort of CLL patients; however, its impact on CLL pathogenesis has to be elucidated.

Supported by the grants IGA-MZ-CR NT11218-6/2010, NS10439-3/2009, NS9858-3/2009, MPO-CR-FR-TI2/254 and MSMT-CR-MSM0021622430.

Mayer:Roche: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Astellas: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Novartis: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Fresenius Medical Care: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Pfizer: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Genzyme: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; GSK: Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Amgen:.