Prostate cancer is the second most common cause of cancer death among men in the United States [
Mitochondria are found in all cells and are central to energy production, reactive oxygen species generation, and apoptosis, all altered in cancer. The mitochondrion is the site of cellular ATP production during the process of oxidative phosphorylation that involves the electron-transport chain (respiratory complexes I–IV) and the ATP synthase (complex V). The mitochondrion contains its own DNA (mtDNA), a 16.5 kb circular self-sufficient intron-free molecule that encodes two ribosomal RNAs (12S and 16S rRNAs), a complete complement of 22 transfer RNAs (tRNAs), and 13 polypeptides. mtDNA mutations have been found in breast, colorectal, ovarian, gastric, lung, pancreatic, brain, renal, thyroid, and many other solid tumors, including prostate [
MtDNA with its high copy number, is maternally transmitted, lacks recombination, and has a higher sequence evolution rate than the nuclear genome [
A major challenge lies in determining whether observed mutations are pathogenic. Amino acid altering mutations may affect protein function or may be essentially neutral [
Previously, we found that germline mutations in the mtDNA gene cytochrome c oxidase subunit 1 (COI) were associated with prostate cancer in Caucasian men [
Some of the study subjects were described previously [
The “no-cancer” control group was subjects at least 50 years old found to be free of prostate cancer as previously described [
Samples from prostate cancer patients were selected from Emory’s tissue bank, and peripheral blood mononuclear cell DNA was extracted using Qiagen FlexiGene (Valencia, CA, USA). For amplification of the mitochondrial COI region 5904–7445, the following sets of primers were used:
Sequencing was performed using BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) in 20
Evolutionary conservation is a strong predictor of pathogenicity with amino acid substitutions at evolutionarily conserved positions more likely to be pathogenic than those at less conserved positions. We compared the amino acid sequence of the COI gene of 61 nonhuman mammalian species to determine the conservation index of the 30 missense mutations identified in this study [
We used three programs to classify the pathogenicity of 30 missense mutations. (i) Polymorphism Phenotyping v2 (PolyPhen2) is software that uses eight sequence-based alignments and three structure-based criteria to predict the impact of amino acid substitutions on the structure and function of proteins using physical and evolutionary comparisons. A mutation is appraised as benign
We analyzed complete mtDNA COI gene sequences in 482 prostate cancer patients, including 250 Caucasians and 232 African Americans. We have also sequenced 46 Caucasian and 143 African American “no-cancer” controls. We found a total of 192 inherited mtDNA COI missense variants in 8.8% of Caucasian prostate cancer patients (versus 0.0% controls) and 72.8% of African American prostate cancer patients (versus 64.3% in controls; Table
Frequency of COI mutations in prostate cancer cases and controls.
|
COI mutant | Frequency (%) |
| |
---|---|---|---|---|
Cancer |
|
192 | 39.8 | |
CA | 250 | 22 | 8.8 | |
AA | 232 | 169 | 72.8 | |
No cancer |
|
92 | 48.7 | 0.109 |
CA | 46 | 0 | 0.0 | 0.028 |
AA | 143 | 92 | 64.3 | 0.256 |
CA: Caucasian American Ancestry; AA: African American Ancestry.
A total of 144 COI sequence differences with respect to the rCRS were identified including 30 missense variants (Table
Twelve of the 30 missense mutations are highly conserved with a CI of at least 97%–100%: A5935G, G5949A, G5973A, G6081A, T6124C, G6261A, G6285A, A6663G, G6924T, G7041A, T7080C, and A7305C (Table
Missense mutations in Caucasian and African American cases and controls.
Nucleotide position | Amino acid | Conservation index | Grantham value | Allelic index | Controls ( |
Control frequency | Cases ( |
Case frequency |
---|---|---|---|---|---|---|---|---|
Count | % | Count | % | |||||
C5911T | A3V | 5/61 = 8% | 64 | 0.2 | 4 | 2.1 | 3 | 0.6 |
G5913A | D4N | 8/61 = 13% | 23 | 0.4 | 0 | 0.0 | 1 | 0.2 |
A5935G | N11S | 61/61 = 100% | 46 | Unique | 0 | 0.0 | 1 | 0.2 |
G5949A | G16X | 61/61 = 100% | — | Unique | 0 | 0.0 | 1 | 0.2 |
G5973A | A24T | 60/61 = 98% | 58 | 0.04 | 0 | 0.0 | 1 | 0.2 |
A6040G | N46S | 8/61 = 13% | 46 | 0.1 | 0 | 0.0 | 1 | 0.2 |
G6081A | A60T | 60/61 = 98% | 58 | Unique | 0 | 0.0 | 1 | 0.2 |
T6124C | M74T | 60/61 = 98% | 81 | Unique | 0 | 0.0 | 1 | 0.2 |
G6150A | V83I | 58/61 = 95% | 29 | 0.2 | 8 | 4.2 | 5 | 1.0 |
T6253C | M117T | 44/61 = 72% | 81 | 0.9 | 7 | 3.7 | 7 | 1.5 |
G6261A | A120T | 61/61 = 100% | 58 | 0.5 | 1 | 0.5 | 8 | 1.7 |
G6267A | A122T | 56/61 = 92% | 58 | 0.1 | 0 | 0.0 | 2 | 0.4 |
G6285A | V128I | 61/61 = 100% | 29 | 0.04 | 0 | 0.0 | 1 | 0.2 |
C6340T | T146I | 45/61 = 74% | 89 | 0.1 | 0 | 0.0 | 2 | 0.4 |
G6366A | V155I | 42/61 = 69% | 29 | 0.3 | 1 | 0.5 | 1 | 0.2 |
G6480A | V193I | 58/61 = 95% | 29 | 0.1 | 0 | 0.0 | 3 | 0.6 |
A6663G | I254V | 59/61 = 97% | 29 | 0.2 | 7 | 3.7 | 14 | 2.9 |
A6891G | S330G | 7/61 = 11% | 56 | 0.04 | 0 | 0.0 | 1 | 0.2 |
G6924T | A341S | 61/61 = 100% | 99 | Unique | 0 | 0.0 | 1 | 0.2 |
G7041A | V380I | 61/61 = 100% | 29 | 0.04 | 0 | 0.0 | 1 | 0.2 |
T7080C | F393L | 60/61 = 98% | 22 | 0.04 | 0 | 0.0 | 2 | 0.4 |
A7083G | I395V | 13/61 = 21% | 29 | 0.04 | 0 | 0.0 | 1 | 0.2 |
A7146G | T415A | 15/61 = 25% | 58 | 3.1 | 36 | 19.0 | 69 | 14.3 |
C7147T | T415I |
15/61 = 25% | 89 |
Unique | 0 | 0.0 | 2 | 0.4 |
A7158G | I419V | 9/61 = 15% | 29 | 0.1 | 0 | 0.0 | 3 | 0.6 |
A7299G | M466V | 39/61 = 64% | 21 | 0.07 | 1 | 0.5 | 0 | 0.0 |
T7354C | M484T | 11/61 = 18% | 81 | Unique | 1 | 0.5 | 0 | 0.0 |
A7305C | M468L | 60/61 = 98% | 95 | Unique | 0 | 0.0 | 1 | 0.2 |
T7389C | Y496H | 15/61 = 25% | 83 | 2 | 25 | 13.2 | 57 | 11.8 |
|
|
— | — | 0.4 | 1 | 0.5 | 1 | 0.2 |
| ||||||||
|
|
Missense mutations’ computation methods and prediction of phenotypic effect.
Nucleotide position | Amino acid | *PolyPhen | †nsSNP analyzer | ‡PMut | ‡PMut reliability |
---|---|---|---|---|---|
C5911T | A3V | Benign | Neutral | Neutral | 3 |
G5913A | D4N | Benign | Neutral | Neutral | 4 |
A5935G | N11S | Probably damaging | Disease | Pathological |
|
G5949A | G16X | — | — | — | — |
G5973A | A24T | Benign | Neutral | Pathological |
|
A6040G | N46S | Benign | Neutral | Pathological |
|
G6081A | A60T | Benign | Neutral | Pathological |
|
T6124C | M74T | Probably damaging | Disease | Pathological |
|
G6150A | V83I | Benign | Neutral | Neutral | 5 |
T6253C | M117T | Benign | Neutral | Pathological |
|
G6261A | A120T | Benign | Neutral | Pathological |
|
G6267A | A122T | Benign | Neutral | Pathological |
|
G6285A | V128I | Benign | Disease | Neutral | 2 |
C6340T | T146I | Benign | Disease | Pathological |
|
G6366A | V155I | Benign | Neutral | Neutral | 4 |
G6480A | V193I | Benign | Neutral | Neutral | 3 |
A6663G | I254V | Benign | Neutral | Neutral | 7 |
A6891G | S330G | Benign | Neutral | Neutral | 6 |
G6924T | A341S | Benign | Neutral | Pathological |
|
G7041A | V380I | Benign | Disease | Neutral | 4 |
T7080C | F393L | Benign | Disease | Pathological |
|
A7083G | I395V | — | — | — | — |
A7146G | T415A | Benign | Neutral | Neutral | 3 |
C7147T | T415I |
Benign | Neutral | Pathological |
|
A7158G | I419V | Benign | Neutral | Neutral | 8 |
A7299G | M466V | Benign | Neutral | Pathological |
|
T7354C | M484T | Benign | Neutral | Pathological |
|
A7305C | M468L | Benign | Neutral | Pathological |
|
T7389C | Y496H | Benign | Neutral | Neutral | 5 |
|
|
— | — | — | — |
We presented the first evidence in 2005 that inherited mutations in the mitochondrial COI gene predispose to prostate cancer in a predominantly Caucasian American population [
Amongst amino acid altering (missense) mutations (relative to Cambridge), one can begin to assess the likelihood that the substitution is biologically important by two main methods. The first is analysis of the degree to which the amino acid side chains differ in terms of chemical composition, polarity, and molecular volume. For the Grantham value (GV), lower numbers indicate a relatively mild chemical difference (thus less likely to be important biologically) and higher numbers indicate a relatively marked chemical difference (more likely to be biologically important; Table
The next level of analysis is to compare the rate of that specific mutation in various populations including prostate cancer cases and controls or a comparison of prostate cancer cases to larger population databases of sequences. The online mtDB database contains 2,704 individual complete mitochondrial DNA sequences and is readily searched [
We also compared the frequency of each mutation in cases and controls. Our data includes two distinct case-control comparisons. The first is the Caucasian American (CA) cases (
Our findings were both race specific and race independent. If one compares the overall frequency of missense COI mutations in all prostate cancer cases (192/482 = 39.8%) to controls (92/189 = 48.7%), there is no statistically significant difference (
Sequencing of CA prostate cancer cases revealed several COI mutations that were only found in CA cases. These included missense mutations at nucleotide positions (n.p.) 6253 (3 times), 6261 (4 times), 6663 (twice), and 7080 (twice). The frequencies of these mutations in AA men were quite different, and each of these mutations was found in the controls. When both CA and AA men were considered together, the n.p. 6253 mutation was found in 7 cases and 7 controls, the n.p. 6261 mutation was found in 8 cases and 1 one control, the n.p. 6663 in 16 cases and 7 controls, and the n.p. 7080 in just 2 cases and no controls. In addition, mutations associated with African lineage were discordant in cases and controls with the 7146 mutation found in 69 cases and 36 controls and the 7389 mutation in 57 cases and 25 controls. Thus, some mutations that appeared to be only found in Caucasian prostate cancer cases were found in the African American control population.
The CA controls had multiple negative prostate core biopsies while the AA controls were not biopsied but had only a negative digital rectal exam of the prostate. Examination alone is significantly less specific than biopsy, so there is a greater chance that undiagnosed cases entered the AA control population.
There are many mutations that were more common in cases than controls even in the combined group. These include inherited missense mutations at nucleotide positions 5913, 5953, 5949, 5973, 6040, 6081, 6124, 6267, 6285, 6340, 6891, 6924, 7041, 7080, 7083, 7158, and 7305 that were
The mtDNA sequencing chromatogram shows the presence of the G→A mutation at position 6261 in one patient compared to the revised Cambridge Reference Sequence (rCRs).
The G6261A mutation is unique with a conversation index of 100% and an allelic index of 0.5. This means that all 61 non-human mammalian species have the wildtype amino acid at this location and only 0.5% of the 2704 sequences in mtDB had this alteration. This was seen in a higher rate in both CA and AA cases than their respective control groups. This mutation therefore fulfills all of our criteria for being potentially important in the pathogenesis of prostate cancer. The G6261A has also been reported in bladder cancer patients [
In another study of specific human mtDNA clades and adaptation to different climates, the G6261A mutation was reported in one Asian and one European [
We used Sift, PolyPhen2, nsSNP Analyzer, and PMut programs to analyze our 30 missense mutations and found that mutations C6340T and T7080C were both predicted as disease (nsSNP analyzer) and pathological (PMut) with a PMut reliability index of 9 and 6, respectively. These numbers are deemed as having a high level of confidence. G5973A, A6040G, G6081A, T6253C,
Overall, PolyPhen2 predicted only two mutations as “Probably Damaging”, (false positive rate of 20%). The nsSNP Analyzer predicted only five of the missense mutations as “Disease,” (false positive rate of 38%, false negative rate of 28%) [
Inherited missense mutations in the mitochondrially encoded COI gene are present in both Caucasian and African American prostate cancer patients, and to a lesser extent in controls. There are two missense mutations in this gene that occur with exceptionally high frequency in African Americans because they arose early in human evolution and have become “fixed” in a high proportion of the population. While pathogenicity cannot be assigned to those founder mutations, the 7389 mutation was significantly associated with disease. In a combined analysis of both Caucasians and African Americans, 20 of the 30 missense mutations occur only in men with cancer and are never found in 189 controls. Of these 20 mutations, 3 (A5935G, G5949A, and G6924T) have not been observed in 2,704 complete sequences in the mtDB database and have an interspecies (
The G6261 (A120T) mutation was found in 8 cancer patients compared to a single control for a 3-fold increase in frequency in cases (1.7% versus 0.5%). Furthermore, this mutation is associated with prostate cancer in both ethnic groups, is found in only 0.5% of the online 2704 sequences, and also has 100% interspecies conservation.
Inherited COI gene missense mutations are significantly associated with prostate cancer in both Caucasians and African Americans. Some significant mutations appear to be race specific while others are race independent. Specific disease-associated mutations may warrant further study in the laboratory to determine possible mechanisms of disease association. It is possible that some of the racial disparity in prostate cancer may be due to inherited mitochondrial DNA mutations.
Mitochondrial DNA
Reactive oxygen species
Oxidative phosphorylation
Single nucleotide polymorphisms
Caucasian American
African American
Conservation index
Grantham value
Allelic index
Revised Cambridge Reference Sequence.
This work was supported by a Veterans Administration MERIT Award (J. A. Petros) and NIH Grant PO1CA98912 as well as the Evans County CARES foundation and Mr. Larry C. Williams, The Breckenridge Group, Atlanta, GA, USA.
No authors have any financial relationship with any commercial entities mentioned in this paper.
The authors wish to thank Dr. Fray F. Marshall for his significant effort to support this research both financially and with patient recruitment and materials. Dr. Kathleen A. Cooney, Anna M. Ray and Kimberly A. Zuhlke (University of Michigan) contributed African American cases and controls, without which this study would not have been possible.