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We report on a method to analyze length heteroplasmy within the human mitochondrial genome in which there are polycytosine [poly(C)] stretches. These poly(C) tracts induce heteroplasmy with the resultant inherent problems of accurate sequence designations. In this study, 20 samples that exhibited length heteroplasmy due to variation in the C-tracts within hypervariable region I (HVI) were treated with bisulfite, and one or more cytosine bases in these C-tracts were converted randomly to uracil. This resulted in an accurate sequence designation for nearly all samples. The only exceptions in which the DNA sequence could still not be determined occurred when there was total conversion, or a lack of conversion, of the cytosine bases. Replicate tests on the same samples showed that individual cytosine bases were randomly converted to uracil. This simple method was useful for investigating length heteroplasmy due to 16189C and 310C transitions in the mitochondrial-DNA control region. It is valuable for medical and forensic investigations.

Introduction: Alterations inside Polycytosine tract (C-tract) of mitochondrial DNA (mtDNA) have been described in many different tumor types. The Poly-Cytosine region is located within the mtDNA D-loop region which acts as point of mitochondrial replication origin. A suggested pathogenesis is that it interferes with the replication process of mtDNA which in turn affects the mitochondrial functioning and generates disease. Methodology: 100 premalignant cases (50 leukoplakia & 50 oral submucous fibrosis) were selected and the mitochondrial DNA were isolated from the lesion tissues and from the blood samples. Polycytosine tract in mtDNA was sequenced by direct capillary sequencing. Results: 40 (25 leukoplakia & 15 oral submucous fibrosis) patients harbored lesions that displayed one additional cytosine after nucleotide thymidine (7CT6C) at nt position 316 in C-tract of mtDNA which were absent in corresponding mtDNA derived from blood samples. Conclusion: Our results show an additional cytosine in the mtDNA at polycytosine site in oral precancer cases. It is postulated that any increase/decrease in the number of cytosine residues in the Poly-Cytosine region may affect the rate of mtDNA replication by impairing the binding of polymerase and other transacting factors. By promoting mitochondrial genomic instability, it may have a central role in the dysregulation of mtDNA functioning, for example alterations in energy metabolism that may promote tumor development. We, therefore, report and propose that this alteration may represent the early development of oral cancer. Further studies with large number of samples are needed in to confirm the role of such mutation in carcinogenesis.

Accurate sequencing of the control region of the mitochondrial genome is notoriously difficult due to the presence of polycytosine bases, termed C-tracts. The precise number of bases that constitute a C-tract and the bases beyond the poly cytosines may not be accurately defined when analyzing Sanger sequencing data separated by capillary electrophoresis. Massively parallel sequencing has the potential to resolve such poor definition and provides the opportunity to discover variants due to length heteroplasmy. In this study, the control region of mitochondrial genomes from 20 samples was sequenced using both standard Sanger methods with separation by capillary electrophoresis and also using massively parallel DNA sequencing technology. After comparison of the two sets of generated sequence, with the exception of the C-tracts where length heteroplasmy was observed, all sequences were concordant. Sequences of three segments 16184–16193, 303–315 and 568–573 with C-tracts in HVI, II and III can be clearly defined from the massively parallel sequencing data using the program SEQ Mapper. Multiple sequence variants were observed in the length of C-tracts longer than 7 bases. Our report illustrates the accurate designation of all the length variants leading to heteroplasmy in the control region of the mitochondrial genome that can be determined by SEQ Mapper based on data generated by massively parallel DNA sequencing. [ABSTRACT FROM AUTHOR]

We report on a method to analyze length heteroplasmy within the human mitochondrial genome in which there are polycytosine [poly(C)] stretches. These poly(C) tracts induce heteroplasmy with the resultant inherent problems of accurate sequence designations. In this study, 20 samples that exhibited length heteroplasmy due to variation in the C-tracts within hypervariable region I (HVI) were treated with bisulfite, and one or more cytosine bases in these C-tracts were converted randomly to uracil. This resulted in an accurate sequence designation for nearly all samples. The only exceptions in which the DNA sequence could still not be determined occurred when there was total conversion, or a lack of conversion, of the cytosine bases. Replicate tests on the same samples showed that individual cytosine bases were randomly converted to uracil. This simple method was useful for investigating length heteroplasmy due to 16189C and 310C transitions in the mitochondrial-DNA control region. It is valuable for medical and forensic investigations. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Alterations inside Polycytosine tract (C-tract) of mitochondrial DNA (mtDNA) have been described in many different tumor types. The Poly-Cytosine region is located within the mtDNA D-loop region which acts as point of mitochondrial replication origin. A suggested pathogenesis is that it interferes with the replication process of mtDNA which in turn affects the mitochondrial functioning and generates disease. Methodology: 100 premalignant cases (50 leukoplakia & 50 oral submucous fibrosis) were selected and the mitochondrial DNA were isolated from the lesion tissues and from the blood samples. Polycytosine tract in mtDNA was sequenced by direct capillary sequencing. Results: 40 (25 leukoplakia & 15 oral submucous fibrosis) patients harbored lesions that displayed one additional cytosine after nucleotide thymidine (7CT6C) at nt position 316 in C-tract of mtDNA which were absent in corresponding mtDNA derived from blood samples. Conclusion: Our results show an additional cytosine in the mtDNA at polycytosine site in oral precancer cases. It is postulated that any increase/decrease in the number of cytosine residues in the Poly-Cytosine region may affect the rate of mtDNA replication by impairing the binding of polymerase and other transacting factors. By promoting mitochondrial genomic instability, it may have a central role in the dysregulation of mtDNA functioning, for example alterations in energy metabolism that may promote tumor development. We, therefore, report and propose that this alteration may represent the early development of oral cancer. Further studies with large number of samples are needed in to confirm the role of such mutation in carcinogenesis. [Copyright &y& Elsevier]