High Mitochondrial DNA Stability in B-Cell Chronic Lymphocytic Leukemia

BACKGROUND: Chronic Lymphocytic Leukemia (CLL) leads to progressive accumulation of lymphocytes in the blood, bone marrow, and lymphatic tissues. Previous findings have suggested that the mtDNA could play an important role in CLL. METHODOLOGY/PRINCIPAL FINDINGS: The mitochondrial DNA (mtDNA) control-region was analyzed in lymphocyte cell DNA extracts and compared with their granulocyte counterpart extract of 146 patients suffering from B-Cell CLL; B-CLL (all recruited from the Basque country). Major efforts were undertaken to rule out methodological artefacts that would render a high false positive rate for mtDNA instabilities and thus lead to erroneous interpretation of sequence instabilities. Only twenty instabilities were finally confirmed, most of them affecting the homopolymeric stretch located in the second hypervariable segment (HVS-II) around position 310, which is well known to constitute an extreme mutational hotspot of length polymorphism, as these mutations are frequently observed in the general human population. A critical revision of the findings in previous studies indicates a lack of proper methodological standards, which eventually led to an overinterpretation of the role of the mtDNA in CLL tumorigenesis. CONCLUSIONS/SIGNIFICANCE: Our results suggest that mtDNA instability is not the primary causal factor in B-CLL. A secondary role of mtDNA mutations cannot be fully ruled out under the hypothesis that the progressive accumulation of mtDNA instabilities could finally contribute to the tumoral process. Recommendations are given that would help to minimize erroneous interpretation of sequencing results in mtDNA studies in tumorigenesis

Combined fluorescent-chromogenic in situ hybridization for identification and laser microdissection of interphase chromosomes.

Chromosome territories constitute the most conspicuous feature of nuclear architecture, and they exhibit non-random distribution patterns in the interphase nucleus. We observed that in cell nuclei from humans with Down Syndrome two chromosomes 21 frequently localize proximal to one another and distant from the third chromosome. To systematically investigate whether the proximally positioned chromosomes were always the same in all cells, we developed an approach consisting of sequential FISH and CISH combined with laser-microdissection of chromosomes from the interphase nucleus and followed by subsequent chromosome identification by microsatellite allele genotyping. This approach identified proximally positioned chromosomes from cultured cells, and the analysis showed that the identity of the chromosomes proximally positioned varies. However, the data suggest that there may be a tendency of the same chromosomes to be positioned close to each other in the interphase nucleus of trisomic cells. The protocol described here represents a powerful new method for genome analysis

Schematic of the FISH-CISH procedure combined with microdissection and microsatellite allele genotyping.

<p><i>In situ</i> hybridization is performed on cells spread onto PEN membranes. The detection step is performed first with fluorescent antibodies followed by quality inspection under the fluorescence microscope. Then, a chromogenic detection step and the microdissection of selected chromosome pairs are performed under the bright-field microscope. DNA is amplified by whole genome amplification and chromosome identification is performed by allele-specific PCR.</p

Primers, addressed to microsatellites mapping to chromosome 21, used in the allele specific PCR of this study.

<p>Primers, addressed to microsatellites mapping to chromosome 21, used in the allele specific PCR of this study.</p

WGA efficiently amplified all 14 samples of microdissected chromosome 21 pairs.

<p>Lanes: M, molecular weight markers from 25–500 bp (far left) and 100–1000 bp (second to left); 1–14, chromosome samples.</p

WGA and allele-specific PCR analysis.

<p>(A) Allele-specific PCR with primers for the D21S11 and D21S1435 markers on WGA-DNA and genomic-DNA from trisomic cells yielded the same allele profiles. (B) Capillary electrophoresis in a Bioanalyzer of the PCR products for D21S11revealed the presence of two different alleles in two microdissected chromosome pairs. (C) PCRs with primers for the D21S1435 marker produced fragments of ∼200 bp in a 1.5% agarose gel (upper panel). Capillary electrophoresis in a Bioanalyzer revealed the presence of two alleles within this band (181 bp and 189 bp) in a limited number of microdissected chromosome pairs (lower panel).</p

Hybridization with HSA21 DNA probes on cells spread onto PEN membranes.

<p>(A) FISH signal (green) in interphase nuclei. Note the two juxtaposed chromosomes 21 are distant from a third chromosome in the nucleus of cell from human with DS. (B) Subsequent CISH (blue) on the same preparation. Chromosome pairs are visible as blue precipitate under the bright-field microscope. (C) A region of interest is computationally drawn and the membrane is cut with the laser microbeam (green). (D)The piece of membrane harboring the chromosomes is recovered by pressure catapulting (blank white space).</p

Variants in the 14q32 miRNA cluster are associated with osteosarcoma risk in the Spanish population.

Association studies in osteosarcoma risk found significant results in intergenic regions, suggesting that regions which do not codify for proteins could play an important role. The deregulation of microRNAs (miRNAs) has been already associated with osteosarcoma. Consequently, genetic variants affecting miRNA function could be associated with risk. This study aimed to evaluate the involvement of all genetic variants in pre-miRNAs described so far in relationship to the risk of osteosarcoma. We analyzed a total of 213 genetic variants in 206 pre-miRNAs in two cohorts of osteosarcoma patients (n = 100) and their corresponding controls (n = 256) from Spanish and Slovenian populations, using Goldengate Veracode technology (Illumina). Four polymorphisms in pre-miRNAs at 14q32 miRNA cluster were associated with osteosarcoma risk in the Spanish population (rs12894467, rs61992671, rs58834075 and rs12879262). Pathway enrichment analysis including target genes of these miRNAs pointed out the WNT signaling pathways overrepresented. Moreover, different single nucleotide polymorphism (SNP) effects between the two populations included were observed, suggesting the existence of population differences. In conclusion, 14q32 miRNA cluster seems to be a hotspot for osteosarcoma susceptibility in the Spanish population, but not in the Slovenian, which supports the idea of the existence of population differences in developing this disease