Study Of Mutations In The D-LOOP Region Of Mitochondrial DNA In Acute Leukaemia

Objective Mitochondria is the only organelle that has DNA in the altitude zooblast. It generate cellular energy in the form of ATP (adenosine triphosphate) by the process of oxidative phosphorylation (OXPHOS). Human mitochondrial DNA (mtDNA) is composed of a 16.6-kb, double-stranded, closed-circular DNA molecule. Human mtDNA has a mutational rate that is at least 10 times higher than nuclear DNA. The D-LOOP, which is 1124 bp in size (positions 16028 - 577), is a noncoding region, and acts as a promoter for both the heavy and light strands of the mtDNA. The D-LOOP region regulates the transcription and replication of mtDNA, and alterations in this region might indeed alter the rate of mtDNA replication. This may be why various cancers frequently have mutations in the D-LOOP region. The D-LOOP region is a hot spot for mtDNA alterations. In recent years, numerous reports on somatic mtDNA mutations in cancers have supported the concept that somatic mtDNA alterations are an integral part of tumorigenesis. Consequently we decided to investigate the instabilities, polymorphisms and orther variations of mitochondrial D-LOOP region in acute leukaemia patients. Thus we can sum up the rule of the mutations in D-LOOP region of these people,explore the possible mechanism and results of these mutations,and provide molecule biological theories for the diagnoses and therapy of acute leukaemia.Method The bone marrow mononuclear cell (BMMNC) of the 20 acute leukaemia patients were extracted by lymphocyte separatory liquor. Genomic DNA was extracted with an equal volume of phenol-chloroform. The D-LOOP region of 20 acute leukaemia patients were amplified by PCR. Forward and reverse peimers for the D-LOOP region were: 5'-ATCATTGGACAAGTAGCATC-3'(ntl5791-ntl5810) and 5'-GGTGAACTCACTGGAACGGG-3'(nt725-nt706). The condition for PCR is designed as followed: 95° C for 5min, 1 cycle;94° C for 1 min, 55° C for 1 min, and 72° C for 1 min, 35 cycles;and a final extension at 72° C for 7 min. PCR products were gel purified and transferred to plasmid by T carrer. Whereafter the liquid that includes the aimy segment were sent to detect their sequence.We use the software of BLAST and MITOMAP to detect whether the mtDNA D-LOOP region of these patients's have arisesed any mutations and try to grasp their mutational disciplinarian.Result 1. The mitochondrial DNA D-LOOP regions of 20 specimens were amplified successfully. 2. Mutations in the mtDNA D-LOOP region were detected in 80%(16 of 20) samples. 62 piont mutations were detected in these samples,including 5 insertions, 11 deletions and 32 piont mutations. 75 % (19of 25) point mutations were G-C transition. 66.7%(3 of 5) insertions were A,and 71.4%(8 of 11) deletions were also A. 3. Big segment deletions were found in one samples,and microsatellites were included in it. 4. Poly (CA)3 and (A)8 were found to have deleted in two samples.Conclusion It was concluded that the D-LOOP region of mitochondrial DNA was a highly polymorphoric and mutable region and the mutation rate was relatively high in patients with acute leukaemia.And the mutations in D-LOOP region may provide a potential diagnosis marker for acute leukaemia.