Study Of Mutations On The D-Loop Region Of Mitochondrial DNA In Myelodysplastic Syndrome

Objective We undertook to systematically analyze the 2 hypermutation regions (HV1 and HV2) of D-loop in the mitochondrial genome by gene amplification and direct sequencing in 42 MDS and 11 acute leukemia patients, and study the character and rule of mtDNA mutation in different category of MDS patients, thereby to investigate it's meaning in the morbidity and progression of the disease.Methods 42 patients with MDS including 17 males and 25 females and 4 patients with acute leukeamia including 2 males and 2 females. Tumer samples were represented by bone marrow which was anticoagulated by 3.8% natrium citricum, and their oral epithelium cells were also collected for normal tissue comparison. Genomic DNA was extracted with an equal volume of phenol-chloroform. HV1 and HV2 region of D-loop was amplified by PCR. Then PCR products were gel purified and transferred to plasmid by T carrier. Whereafter the liquid that includes the aim segment were sent to sequencing. Results were compared with concomitantly studied normal tissues as well as mtDNA sequences in a standard database in revised Cambridge reference sequence (rCRS). Nucleotide changes that were both present in bone marrow and oral epithelium of the same patient were counted as polymorphisms, and which unwritten in the database(MITOMAP, mtDB and Genbank) were new polymorphisms. The changes that were only present in bone marrow were counted as mutation. Calculate the frequency of occurrence of polymorphisms.Results 1. Results of the agarose gel electrophoresis: There were clear straps at 470bp and 490bp, which confirmed that the 2 hypervariable regions of D-loop were amplified successfully.2. Polymorphisms of mtDNA in MDS patients: 199 polymorphisms were found in the 42 patients with MDS. There were 131 polymorphisms among the total in HV1 (360bp) region, and in single bone marrow prepare there were at lease 1 nucleotide change and at most 7 changes, and the frequency of occurrence of polymorphisms in HV1 region is 0.87%. In HV2 (316bp) region, there were 68 polymorphisms among the total and the number in single specimen was varied from 0 to 5, and the frequency of occurrence of polymorphisms is 0.51%. Over all, there was no new polymorphism in our study.3. Mutations of mtDNA in MDS patients: Mutations in the mtDNA D-loop region were detected in 14.29% (6 of 42) samples. 19 piont mutations were detected in these samples, including 5 microsatellite instabilities(4 in HV1 region, 1 in HV2 region). The others were base replacement between A, G or C, T(6 in HV1 region, 8 in HV2 region).4. Polymorphisms of mtDNA in acute leukemia developing from MDS: 21 polymorphisms were found in the 4 patients with acute leukemia. There were 13 polymorphisms among the total in HV1 region, and in single bone marrow prepare there were at lease 2 nucleotide change and at most 4 changes, and the frequency of occurrence of polymorphisms in HV1 region is 0.90%. In HV2 region, there were 8 polymorphisms among the total and the number in single specimen was varied from 1 to 3, and the frequency of occurrence of polymorphisms is 0.63%. No new polymorphism was found in our study.5. Mutations of mtDNA in acute leukemia developing from MDS: Mutations in the mtDNA D-loop region were detected in 75% (3 of 4) samples. 3 point mutations were detected in these samples, including microsatellite instability and base replacement between A, G or C, T. The differences of the mutational frequency between the two groups were statistically significant (P<0.05). Conclusion Excess apoptosis of bone marrow play a important role in ineffective hematopoiesis which occurred in MDS patients, while most of the signal inducing apoptosis may cause changes of mitochondrial function, increase the permeation of mitochondrial inner membrane, destroy the membrane potential and promote the release of Cytochrome C, and then trigger the caspase progress and eventually cause cell apoptosis. In our research, we found there were higher mutational rate in D-loop region, and as the promoter of transcription of H-chain and L-chain and the replication origin of H-chain were in D-loop region, the mutations happened in that place will influence the replication and transcription of mtDNA and impact the function of coding region, and then cause the whole functional disorder of mitochondrial, make it more sensitive to oxidative damage or directly result in apoptosis. Therefore, the functional defect caused by mutation of mtDNA D-loop region may be the reason of excessive apoptosis existed in MDS cells. In addition, the differences of mutational rate between MDS group and leukemia developing from MDS group were statistically significant. It indicated that mutations in D-loop region might play an important role in the advancement of MDS disease.