Genetic Variations of Mitochondrial Cytochrome B and Breast Cancer

Research Article

Austin Biomark Diagn. 2014;1(2): 5.

Genetic Variations of Mitochondrial Cytochrome B and Breast Cancer

Gazi Nurun Nahar Sultana1*, ADA Shahinuzzaman2, Rokeya Begum1, Iffat Jahan1 and M Mizanur Rahman3

1Centre for Advanced Research in Sciences, University of Dhaka, Bangladesh

2Bangladesh Council of Scientific and Industrial Research (BCSIR), Bangladesh

3National Institute of Cancer Research and Hospital, Bangladesh

*Corresponding author: Gazi Nurun Nahar Sultana, Centre for Advanced Research in Sciences (CARS), University of Dhaka, Dhaka-1000, Bangladesh.

Received: November 05, 2014; Accepted: December 09, 2014; Published: December 11, 2014


The role of mitochondria encoded cytochrome b (cyt b) gene mutations has been drawn a great interest to understand the function of this gene in various cancer including breast cancer. Although most cancer cells harbor somatic mutations in mitochondrial DNA (mtDNA) including cytochrome b gene, the question of whether such mutations in cyt b contribute to the promotion of breast cancer remains obscured. In this study, we investigated the frequency of mutations in cyt b gene in forty (n = 40) breast cancer patients of Bangladesh. The results were compared with forty eight age matched control samples (n = 48) from our database. Nine (9) different sequence variations were found on cyt b genein cancer samples. Detail study of cyt b gene of forty eight (n = 48) controls found eighteen (n = 18) different variations, which demonstrates the high variability of cyt b sequence. Three of these eighteen mutations 15055 (G>A), 15301 (G>A), and 16326 (A>G) are also found in cancer patients. Two mutations in breast cancer patients were homoplasmic and identified as major percentage compared to control samples. Two mutations have been identified at np 14766 (C>T) 100%, followed by 14783 (T>C) in 40%breast cancer samples. Two other mutations at np15479 (T>C) and 15497 (G>A) have been reported in 2% breast cancer patients but absent from control samples. Three phylogenetic variations at np15055 (T>C), 15301 (G>A) and 15326 (A>G) have been found in 18% cancer patients respectively but also found in control samples. We have identified two insertion polymorphisms at 15970 (A-ins) and 15980 (T-ins) in 2% cancer samples. Thus, we hypothesized that mutation at np 14766 (C>T) 100% in cyt b gene may cause defective assembly and function of complex- III which thus hampers ATP production in cancer cells in addition with others mechanisms of breast cancer development. As a result, cancer cells are forced to use glycolytic pathways for ATP production. Alternatively two mutations14766 (C>T) and 15326 (G>A) lead to frame shift in amino acids (T7I) and (T194A) of translated protein. However report from the software analysis suggests that this (T7I) change might not be tolerated in protein function with some deleterious effect.

Keywords: Breast cancer; Mitochondrial cytochrome; mtDNA; OXPHOS


The risk of mitochondrial DNA (mtDNA) mutations with breast cancer is just in the beginning of understanding. Not much investigation has been done related to mitochondrial cytochrome b gene mutation in breast cancer patients. Although there are several reports on cyt b gene mutations in different cancer but functional effect of these mutations in tumor development is yet to be reveled [1,2]. Mitochondria play a vital role for regulation of Oxidative Phosphorylation System (OXPHOS) producing cellular currency called ATP. The OXPOS system is composed of five complexes (I-V) and some of them are encoded by mitochondrial DNA (mtDNA) and others by nuclear DNA [3]. Only cyt b of mitochondria plays a vital role for the assembly and function of complex-III, and together with Cytochrome c1 and iron-sulfur protein, it forms the catalytic core of the enzymes [4]. In particular, mutations in the mtDNA encoded cyt bgene are associated with combine complex I+III deficiency or with only complex III deficiency [5]. As a result functional defect in the OXPHOS system may force to impede electron flow down the electron transport chain could increase ROS production and contribute to cancer [6,7]. It was shown in other study that over expression of cyt b generated increase ROS accompanied by increase oxygen consumption and lactate production. Cyt b over expression induced significant tumor growth in vitro and in vivo by triggering significant cell cycle progression through up regulation of nuclear factor NF?B2 signaling pathway and may set up cell for further cell cycle progression, invasion and inhibition of apoptosis [8,9]. In this study, we examined the hypothesis that accumulation of mutation in cyt b gene might be an additional risk factor for the promotion of tumor growth in breast cancer.

Materials and Methods

Sample collection

All approval has been taken from the local ethical committee of Bangladesh Medical Research Council (BMRC) and University of Dhaka. Forty (n = 40) breast cancer patients from Dhaka Medical College Hospital, Dhaka and National Institute of Cancer Research Hospital (NICRH), Dhaka, Bangladesh were included for the study in 2010-2012. We have collected personal history of those patients who were diagnosed as breast cancer. Blood and tumor tissue samples were collected from 37 patients during surgical operation before chemotherapy. Rest of the 3 samples was collected from patients who already had chemotherapy before surgery. A healthy cohort of 48 age matched female individuals from mainstream population was also included. All two populations share the same ethnicity and nationality and reside in Bangladesh. Subjects with breast cancer were interviewed. Blood samples 3-5 mL was collected in EDTA coated tubes from breast cancer patients who visited the hospital for treatment and tissue samples were collected in screw capped polypropylene vials during surgery. The samples were transported to laboratory and blood samples kept at -20°C and tissue samples at -40°C until analyzed.

DNA isolation, PCR and sequencing

Cancer and normal blood DNA was isolated by standard proteinase K treatment followed by phenol/ chloroform/ isoamyl alcohol extraction. Tissue samples were isolated by DNA extraction kit purchased from (Qiagen, Turn berry LaneValencia, California). DNA was precipitated with 0.3 M sodium acetate (pH 5.2) in 70% ethanol at -20°C overnight and suspended in Tris-EDTA (TE) buffer (pH 8.0). DNA quantification was performed by taking absorbance at 260 nm and visualized by 0.8% agarose gel electrophoresis [10]. Partial mitochondrial genome was amplified using two sets of primers Table 1 and the resultant amplicon were checked in 2% agarose gel electrophoresis [11]. Distinct PCR bands were observed for different primers amplifying Cytochrome b gene, along with diluted 1 Kb Plus DNA ladder purchased from life Technologies, USA (Cat no. 10787-018). 20 μl PCR reaction contained 10-20 ng DNA and 0.5 μM primers, 0.2 mM each of deoxynucleotide triphosphate (dNTP), 1U of TaqMan™ DNA Polymerase (Applied Biosystems, USA) and 2.5 mM MgCl2. The PCR amplification of specific regions of mtDNA was performed on the basis of following cycling conditions: initial denaturing at 95°C for 5 min followed by 94°C for 30 sec, 58°C for 30 sec, and 72°C for 2 min for 35 cycles and final extension step at 72°C for 7 min. In Sequencing PCR, the ABI-prism Big Dye Terminator V 3.1 containing ampliTaq polymerase, dye terminators (fluorescent label), deoxynucleotide triphosphate, magnesium chloride, was used for direct sequencing of PCR product for specific primers (forward/ reverse primer). The sequencing PCR was performed on the basis of following cycling conditions: initial denaturing at 95°C for 1 min followed by 94°C for 10 sec, 55°C for 30 sec, and 60°C for 4 min for 35 cycles.