somatic mtDNA mutations with adult onset leukaemia

Mitochondria, the power house of the cell are under the control of genomic DNA (mtDNA) and nuclear DNA.The mtDNA is a 16.6 kb molecule, encodes 13 subunits of respiratory chain complexes, as well as 22 tRNA and two ribosomal RNA for intramitochondrial synthesis. Mutations in mitochondrial DNA (mtDNA) are frequent in cancers but it is not yet clearly established whether they are modifier events involved in cancer progression or whether they are a consequence of tumorigenesis. We therefore decided to determine the spectrum of somatic mtDNA mutation in adult-onset leukaemia by comparing directly the entire mitochondrial genome sequence from normal and leukemic cells obtained from 24 patients with both chronic and acute presentations. This comparison gives the change in nucleotide composition. On this basis we can repair those sequences and modify it by gene therapy. By doing this the problems of graft rejection and bone marrow replacement are solved and the disease leukaemia can be treated in a more efficient way.










Introduction: Mitochondria are structures within cells that convert the energy from food into a form that cells can use. Although most DNA is packaged in chromosomes within the nucleus, mitochondria also have a small amount of their own DNA. This genetic material is known as mitochondrial DNA or mtDNA. In humans, mitochondrial DNA spans about 16,500 DNA building blocks (base pairs), representing a fraction of the total DNA in cells.
Mitochondrial DNA contains 37 genes, all of which are essential for normal mitochondrial function. Thirteen of these genes provide instructions for making enzymes involved in oxidative phosphorylation. Oxidative phosphorylation is a process that uses oxygen and simple sugars to create adenosine triphosphate (ATP), the cell's main energy source. The remaining genes provide instructions for making molecules called transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), which are chemical cousins of DNA. These types of RNA help assemble protein building blocks (amino acids) into functioning proteins.
Genes
ATP synthase: MT-ATP6, MT-ATP8
cytochrome c oxidase: MT-CO1, MT-CO2, MT-CO3, MT-CYB
NADH dehydrogenase: MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-ND6
12S, 16S: MT-RNR1, MT-RNR2
tRNA: MT-TA, MT-TC, MT-TD, MT-TE, MT-TF, MT-TG, MT-TH, MT-TI, MT-TK, MT-TL1, MT-TL2, MT-TM, MT-TN, MT-TP, MT-TQ, MT-TR, MT-TS1, MT-TS2, MT-TT, MT-TV, MT-TW, MT-TY, MT1X


Mitochondrial DNA is prone to noninherited (somatic) mutations. Somatic mutations occur in the DNA of certain cells during a person’s lifetime and typically are not passed to future generations. Somatic mutations in mitochondrial DNA have been reported in some forms of cancer, including breast, colon, stomach, liver, and kidney tumours. These mutations also have been associated with cancer of blood-forming tissue (leukaemia) and cancer of immune system cells (lymphoma).
Somatic mutations in mitochondrial DNA may increase the production of potentially harmful molecules called reactive oxygen species. Mitochondrial DNA is particularly vulnerable to the effects of these molecules and has a limited ability to repair itself. As a result, reactive oxygen species easily damage mitochondrial DNA, causing a buildup of additional somatic mutations. Researchers continue to investigate how these mutations may lead to uncontrolled cell division and the growth of cancerous tumours.
The mtDNA is inherited from mother’s body. In case of sperm the maximum mtDNA stays in the tail which is isolated during fertilization. As a result the zygote gets only maternal mtDNA and no paternal DNA.

Materials and methods
Patients
24 adult patients (aged 18–88 years), six with acute lymphatic leukaemia (ALL), six with acute myeloid leukaemia (AML), six with chronic lymphatic leukaemia (CLL) and six with chronic myeloid leukaemia (CML) had been studied. All had been given their informed consent for these investigations, which were approved by the local Ethics Committee. Tumour samples were represented by bone marrow (ALL and AML), blood with high white blood cell count (CLL) or blood during therapy failure (CML). For each patient, buccal epithelial cells were collected from mouthwashes by centrifugation as representative of normal tissue.
mtDNA sequencing
DNA was extracted by standard procedures and the entire mitochondrial genome was amplified in 28 overlapping fragments of between 600 and 700 bp using M13-tagged oligodeoxynucleotide primers to facilitate the direct sequencing of the PCR-amplified products. A complete list of the primer pairs and sequencing protocol has been published previously.26 Briefly, following PCR amplification, samples were purified to remove unincorporated primers and sequenced directly using BigDye terminator cycle sequencing chemistries (PE Biosystems) on an ABI 377 automated DNA sequencer. The sequences obtained were compared directly to the revised Cambridge reference sequence (rCRS)27 using Sequence Navigator and Factura software (PE Biosystems) to identify sequence variants. Somatic mutations were identified by directly comparing the sequence differences detected in buccal and tumour samples from individual patients.