Mitochondrial Physiology Network 10.9: 119-120 (2005) - download pdf

A novel approach for rapid screening of mitochondrial D310 polymorphism.

Cenk Aral¹, H Kaya², Ç Ataizi-Çelikel², M Akkiprik¹, Ö Sönmez¹, BM Güllüoğlu³, A Özer¹

Marmara University, School of Medicine, ¹Departments of Medical Biology, ²Pathology and ³Surgery, Istanbul, Turkey. – caral@marmara.edu.tr

    Mitochondrial DNA alterations have been suspected to play an important role in the development and progression of cancer. Several mutations have been identified in a wide variety of human tumors including breast, colorectal, ovarian, gastric, hepatic and esophageal cancers as well as hematological malignancies [1]. D-loop region of the mtDNA is the most potent accumulation site for many of these mutations and numerous polymorphisms have also been reported in this region. The sequence alterations of this region may contribute to altered replication or transcription properties.

    Recently, Sanches-Cespedes and colleagues [2] have identified a polyC mononucleotide repeat located between 303 and 315 nucleotides within the D-loop region as a mitochondrial hot spot of deletion or insertion mutations. This region is part of the conserved sequence block (CSB) II and consists of a stretch of cytosines interrupted by a thymine nucleotide (CCCCCCCTCCCCC). Although the number of the cytosine residues at the first stretch of polyC is accepted as 7-C (GeneBank NC_001807), it is highly polymorphic ranging between 6-C to 9-C [3,4]. It is still questionable if there is any correlation between the number of the cytosine residues and development and progression of the cancer. Typically, time and money consuming methods such as  sequencing and radioactivity based gel electrophoresis are required in order to evaluate this polymorphism among individuals. Also, gel electrophoresis remains ineffective unless confirmed with sequencing and these limidations are especially obvious with studying large populations. 

    In this study we established a restriction fragment length polymorphism (RFLP) assay  for the first step rapid screening of the individuals if they carrying 7-C at their mitochondrial D310 region. We tested a total of 141 tissue samples including normal and cancerous tissues of 25 breast and 25 colorectal cancer patients and 41 blood samples of healty individuals.  By using this simple approach, 41 % of the studied samples were found that have 7-C in their mtDNA D310 region without need for sequencing and/or radioactive labelling.  Furthermore, we compared the cases and normal samples for their RFLP status and found a statistically significant difference between colorectal cancer samples and healty individuals.

1.    Penta JS, Johnson FM, Wachsman JT, Copeland WC (2001) Mitochondrial DNA in human malignancy. Mutat. Res. 488: 119-133.

2.    Sanches-Cespedes M, Parrella P, Nomoto S, Cohen D, Xiao Y, Etseller M, Jeronimo C, Jordan RCK, Nicol T, Koch WM, Schoenberg M, Mazzarelli P, Fazio VM, Sidransky D (2001) Identification of a mononucleotide repeat as a major target for mitochondrial DNA alteration in human tumors. Cancer Res. 61: 7015-7019.

3.    Parrella P, Xiao Y, Fliss M, Sanchez-Cespedes M, Mazzarelli P, Rinaldi M, Nicol T, Gabrielson E, Cuomo C, Cohen D, Pandit S, Spencer M, Rabitti C, Fazio VM, Sidransky D (2001) Detection of mitochondrial DNA mutations in primary breast cancer and fine-needle aspirates. Cancer Res. 61: 7623-7626.

4.    Parrella P, Xiao Y, Fliss M, Sanches-Cespedes M, Mazzarelli P, Gravina C, Gallucci M, Altomare V, Flammia G, Casalino B, Benedetti-Panicini PL, Fazio VM (2003) Mutations of the D310 mitochondrial mononucleotide repeat in primary tumors and cytological specimens. Cancer Lett. 190: 73-77.