Somatic Mutations in Circulating Cell-Free D-Loop Region of Mitochondrial DNA: A Study from North-East India

Head and neck Squamous cell carcinoma (HNSCC) is highly prevalent in Northeast India. The widespread use of tobacco exposure is a known risk factor, makingmitochondrial DNA (mtDNA) more susceptible to damage by oxidative stress incomparison to nuclear DNA. Mitochondrial dysfunction being a hallmark of cancer, thestudy aims to evaluate liquid biopsy involving circulating cell-free mitochondrial DNA(cfmtDNA) as an early diagnostic marker by reducing the dependability over tumor tissuebiopsy specimen. A total of 50 HNSCC cases reported at Cancer Hospital, Guwahati MedicalCollege from January 2018 to August 2018 were included in this study. Cell-free DNA wasisolated using QIAamp Circulating Nucleic Acid Kit. PCR based amplification ofmitochondrial D-loop, followed by direct sequencing. Our result indicated the presence ofsomatic mutations (73(A/G), 93(G/A), 146(T/C) and 207 (G/A)). Polymorphism was alsoobserved in the sequences (263A>G, 275G>A, 318T>C, 16034T>C, 16257C>A and16519T>C) upon comparison with reference sequence. Analysis of c-tract region showedthe presence of an additional cytosine nucleotide at position 309.Identifying somaticmutations in cfmtDNA using liquid biopsy approach will certainly minimize thedependency of clinicians and molecular biologist over the availability of tumor tissuespecimens. The identified somatic variations from our study will help in theimplementation of preventive measure. Therefore, our study provides an early mtDNAdiagnostic marker using liquid biopsy approach.

The Genotyping by the Nucleotide, Length, and Quantitative Polymorphism of C-stretch of HVR I of mtDNA D-Loop / 대한법의학회지

To analyze the nucleotide polymorphism and length polymorphism of C-stretch of HVR 1 (Hypervariable Region I) of mtDNA D-loop in the maternal lineages in Koreans, sequencing of C-stretch, GeneScan analysis and cloning of a cases were performed in 266 random objects and 128 families which were confirmed by STR analysis of autosome, X chromosome and nucleotide polymorphism of mitochondrial DNA D-loop. C- stretch was classified into two groups. First group (the group of nucleotide polymorphism 74.2%) has 14 bases which show nucleotide polymorphism composed of adenine, cytosine and thymine without length polymorphism and second group (poly C tract 25.8%) shows length polymorphism by the changes of the number of adenine and cytosine. The patterns of nucleotide polymorphism were as follows: A4C5TC4 (64.8%), A4C5TC2TC (0.8%), A4C4TTC2TC (0.8%), A4C3TCTC4 (5.5%), A4C2TC2TC4 (0.8%), A4CTC3TC4 (0.8%), A4TC4TC4 (0.8%). The pattern of length polymorphism of poly C tract were as follows : LP10-16 (3.0%), LP11-16 (6.1%), LP11-17 (27.2%), LP11-18 (6.1%), LP12-16 (12.1%), LP12- 17 (39.4%), LP12-18 (3.0%), LP13-16 (3.0%) The copy number ratios of each fragment length in the same pattern of length polymorphism were various, and this data were valuable in individual identification because of the quantitative polymorphism of each fragment length. The correlation coefficients of copy number ratio of each fragment length in the families were more than 0.98 in 84.8% (28 of 33 families) of cases, which was interpreted as maternal inheritance of the copy number ratio of fragment length. However, in some cases (9.1%, 3 of 33 families), the correlation value of fragment length were less than 0.96 and in 2 cases of 33 families (6.1%), were 0.89 and 0.75 which suggested a possibility of mutation in the quantitative polymorphism although the length polymorphism were maternally inherited. From the above results, sequencing analysis of nucleotide polymorphism and GeneScan analysis of C- stretch must be combined to clearly identify the nucleotide polymorphism, length polymorphism, and quantitative polymorphism of C-stretch of HVR 1 of mtDNA D-loop.

Interpretation of C tract in the D-Loop of Mitochondrial DNA / 대한법의학회지

The D-loop of mitochondrial DNA contain C tracts(homopoymeric cytosines) in the positions of 16183 -16193, 303 -315, 568 -573 of GI 1944628 of NCBI. When the sequence of these positions are analyzed, the sequencing reactions or their signals after the C tract were abruptly decreased and cannot clarify the nucleotide signals in many cases. It is hard to characterize of the C tract. To characterize the C tract, we tried to analyze the sequence of clone and amp-length polymorphism with silver stain after amplification of small fragments including C tract of 16183 -16193 and 303-315.These results were compared with the results of characterization of nucleotides after separation of each nucleotide signal from the electropherogram which were increased in the Y axis to intensify the small overlapped signals using CHROMAS program. All methods we tried showed similar pattern of heteroplasmies of homopolymeric cytosines. Conclusively, it is not necessary to try resequencing reaction after cloning or amp-length polymorphism using silver stain. Intensification of the small overlapped signals in Y axis and separation of each signal is enough method to characterize the C tract.