Skip to main content

Mitochondrial A12308G alteration in tRNALeu(CUN) in colorectal cancer samples



Colorectal cancer is the third most common type of cancer in men and women and the second leading cause of cancer-related deaths in the United States and UK. Colorectal cancer is strongly related to age, with almost three-quarters of cases occurring in people aged 65 or over. Pre-symptomatic screening is one of the most powerful tools for preventing colorectal cancer. Recently, the use of mitochondrial tRNA genes mutation or polymorphism patterns as a biomarker is rapidly expanding in different cancers because tRNA genes perform several functions including processing and translation which are essential components of mitochondrial protein synthesis. The aim of the present study was to find out the association of mitochondrial A12308G alteration in tRNALeu(CUN) in colorectal cancer and its usage as a new biomarker screening test.


A tumor tissues from 30 patients who had colorectal cancer were selected randomly. The A12308G alteration in tRNALeu (CUN) was screened in the 30 colorectal tumor tissues. For comparison, 100 blood samples of healthy controls using PCR-sequencing methods were selected and the following results were found.


The A12308G, a polymorphic mutation in V-loop tRNALeu(CUN), was found in 6 Colorectal tumor tissues and 3 healthy controls. A statistical significant difference was found between cases and control regarding the association of the A12308G mutation with the colorectal tumor (P < 0.05).


The A12308G, a polymorphic mutation in V-loop tRNALeu(CUN), could be considered as pathogenic mutation in combination with mitochondrial external conditions and other mitochondrial genes in developing different diseases especially cancers and could be used as one of the diagnostic tool. Also it seems that maybe there is relevance between A12308G mutation and other mutations that it can cause various phenotypes.


Worldwide, colorectal cancer (CRC) is the fourth most common cancer and affects both men and women equally and the American Cancer Society estimated that ~56,730 would die from this disease. The substantial mortality associated with this cancer makes it the leading cause of gastrointestinal cancer deaths [1]. Colorectal cancer is an uncontrolled cell division of the colon or rectal cells starting in the inner most layer and can grow through some or all of the other layers. These cells may also invade and destroy the tissue around them and spread to form new tumors in other parts of the body. Unfortunately, some colorectal cancers might be present without any signs or symptoms and often diagnosed late when the disease becomes more advanced. For this reason, it is very important to have regular colorectal screening tests for early detection when the disease is easier to cure/control. Screening has been found to be effective in reducing the incidence and mortality of colorectal cancer through the detection and removal of pre-cancerous lesions and through the detection of CRC in its early stages. Colonoscopy, sigmoidoscopy, and fecal occult blood tests are all recommended screening tests that have widespread availability [2]. Recently, Genetic testing is developed that offer more reliable options for colorectal cancer screening. Mitochondria play a central role in the regulation of cellular function, metabolism, free radical generation, and cell death. Defects in mitochondrial function have been speculated to have an impact on the development and progression of cancer [3].

Cancer development involves the accumulation of genetic changes that will happen in both nuclear and mitochondrial genes. In cancer cells, mutations in mtDNA were more readily detectable and 10 times abundant than nuclear DNA (nDNA), possibly due to the lack of introns, lack of histone protection, low efficiency of mtDNA repair systems and close proximity to damaging reactive oxygen species (ROS) [47].

Alterations in mitochondrial DNA (mtDNA) in the D-loop region as well as in other parts of the mitochondrial genome, including point mutations, deletions, insertions and genome copy number changes, are believed to be responsible for carcinogenesis in a variety of human cancers such as ovarian, colon, thyroid and endometrial cancer, salivary glands, liver, lung, gastric, brain, bladder, kidney, prostate, head and neck, breast cancer and leukemia [819].

Mutations in the Mt-tRNA genes have impact on the secondary and tertiary tRNA structure, and may cause transcriptional and translational defects and mitochondrial respiratory chain dysfunction consequently. More than half of mitochondrial mutations have been located in mt-tRNA genes which are hot spots for mitochondrial pathogenesis [20].

Therefore, mtDNA mutation pattern is a great molecular cancer biomarkers and it could increase the specificity of cancer detection and prediction. Here, we are studying about the human mitochondrial A12308G alteration in tRNALeu(CUN) in tumoral tissues from colorectal cancer patients.


Tumor tissues samples, from thirty Iranian colorectal cancer patients were collected at the cancer institute of Imam Khomeini Tehran hospital. For comparative purposes, blood samples from100 healthy controls of matched age and sex were collected too. The DNA from tumoral tissues was extracted using QIAamp DNA FFPE kit (QIAGENE) while, DNA from blood samples obtained from healthy control was extracted using DNA fast kit (Genefanavaran, Tehran, Iran). The A12308G alteration in tRNALeu(CUN) was screened by sequencing the PCR products from both patients and control samples. Primer sequences are as described in Table 1 or Additional file 1. PCR was carried out in a total volume of 25 μl, containing 2.5 mM Mgcl2, 200 μM of each dNTP, 10 Pm of each primer, 100 ng total DNA and 1U taq DNA polymerase in thermal cyclers (Eppendrof, Master cyclers, 5330). Thermocycling conditions were 94 °C for 5 min, followed by 32 cycles of 95 °C for 1 min, annealing for1 min at 50 °C and extension at 72 °C for 45 s, and finally 72 °C for 10 min for 32 cycles. The PCR products were examined for specificity using 1.5 % agarose gel electrophoresis. Double-stranded automated sequencing was performed using an ABI 3100 sequencing machine (Applied Biosystems, Kavosh Fanavaran Kawsar Company, Iran). All fragments were sequenced in both forward and reverse directions. Sequence of tumoral tissues were analyzed using a Finch TV program (chromatogram viewer which displays DNA sequence traces) and compared to the Human Mitochondrial Reference Sequence NC_012920 provided by the National Center for Biotechnology Information (NCBI). The Chi-square test was used with SPSS (Statistical Package for the Social Sciences, version: 13) to examine the association between the presence of mutation/polymorphisms in colorectal tumoral tissues and the blood of healthy controls. P values < 0.05 were regarded as statistically significant.

Table 1 Mitochondrial Primers for PCR-Sequencingof tRNALeu(CUN)


Homoplasmic A12308G, a polymorphic mutation in V-loop (tRNALeu(CUN)), was found in 6 colorectal tumor (20 %) and 3 healthy controls (3 %). This difference is statistically significant (P = 0.05).


Various human diseases have been associated with mtDNA mutations, indicating that dysfunction of the components of oxidative phosphorylation encoded by the mitochondrial genome can be deleterious [21]. Abnormalities in mtDNA have proven to be associated with leber’s hereditary optic neuropathy (LHON) [22], Primary open-angle glaucoma (POAG) [23, 24], pseudoexfoliation glaucoma (PEG), primary angle closure glaucoma (PACG), other spontaneous optic neuropathies [2527] and male infertility [28]. Moreover, 25-80 % of somatic mutations in mitochondrial DNA are found in various neoplasms [29]. Also, in 2012 the role of the mitochondrial tRNA genes was analyzed in patients with asthma compared with a set of healthy controls. They suggested that the mitochondrial tRNA genes play a key role in asthma development [30]. The use of mtDNA mutation patterns as a biomarker is rapidly expanding in rare metabolic diseases, aging, cancer, tracing of human migration patterns, population characterization and human identification in forensic science [31]. It seems that the mitochondrial genome is more useful in detecting tumor cells in body fluids and cytological specimens than mutations in nuclear DNA had been confirmed.

In the present study, to the best of our knowledge, this is the first reported association between colorectal cancer and mtDNA A12308G alteration in tRNALeu(CUN). The A12308G change was introduced as a common polymorphism by Houshmand at the first time [14]. Several studies described the association of mt-tRNA mutations with human cancers. This mutation came to the attention of the breast cancer research communities as a plausible candidate marker for increased breast cancer susceptibility [29, 32]. In USA, the A12308G polymorphism was introduced as an important factor in kidney and prostate cancer risk [16]. In India, the A12308G mutation was seen as a significant change in the risk of oral cancer [33]. This alteration was, also, reported as a multiplier risk factor in advanced breast cancer tumors in European – American patients [34]. Increased prevalence of the A12308G mutation in mitochondrial tRNALeu(CUN)gene associated with Friedreich's ataxia in Iran, was reported [35]. In previous studies, A12308G alteration has occurred in association with another disease causing alteration in MELAS, myopathy and primary congenital glaucoma (PCG) where three such changes (G10398A, A12308G and G13708A) were present in the later [36, 37]. Moreover, the A12308G polymorphism in tRNALeu(CUN) increases the risk of developing stroke in patients with the A3243G mutation [38]. So, this polymorphism may act as a secondary mutation in this disease pathogenicity. The A12308G variation is also associated with increased ROS production [39]. Nine main European haplotypes (H, I, J, K, T, U, V, W and X) were analyzed in a series of patients with prostate and renal cancers studied by Booker et al. Using the A12308G substitution in tRNALeu as a marker of the mtDNA haplogroup U, it was found that patients carrying this haplogroup had an increased risk of renal and prostate cancer [16]. Some studies showed an increased frequency of the A12308G substitution in mitochondrial patients carrying mtDNA single macrodeletion. In this group of patients, A12308G substitution is associated with a higher relative risk of developing pigmentary retinal degeneration, short stature, dysphasia–dysarthria and cardiac conduction defects [40]. Moreover, the A12308G was found in 8 Alzheimer’s disease patients [41]. In the case of endometrial adenocarcinoma the presence of mitochondrial A12308G alteration in tRNALeu(CUN) was reported [42, 43]. Study in Italy stated that Mitochondrial DNA mutations have been causally linked with cardiomyopathies, both dilated (DCM) and hypertrophic. They identified the T12297C mutation in the mtDNA-tRNALeu(CUN) of a patient diagnosed with DCM. In the variable loop of the same tRNA, their patient also carried the A12308G transition [44].


In conclusion, the present study revealed that mitochondrial research will enable to establish biomarkers helping to identify individuals at high risk for developing specific cancer types and to develop screening approaches for early diagnosis of cancer. In addition, it seems that more research is essentially needed to understand the effect and role of the A12308G mutation as a common polymorphism or an inherited predisposition factor in the carcinogenesis. We believe that this mutation associated with other mutations and/or factors would lead to diverse phenotypes.


Written informed consent was obtained from the patients for the publication of this report and any accompanying images.


  1. 1.

    Amersi F, Agustin M, Ko CY. Colorectal cancer: epidemiology, risk factors, and health services. Clin Colon Rectal Surg. 2005;18:133–40.

    PubMed Central  PubMed  Article  Google Scholar 

  2. 2.

    Ned RM, Melillo S, Marrone M. Fecal DNA testing for Colorectal Cancer Screening: the ColoSure™ test. PLoS Curr. 2011;3, RRN1220.

    PubMed Central  PubMed  Google Scholar 

  3. 3.

    Verschoor ML, Ungard R, Harbottle A, Jakupciak JP, Parr RL, Singh G. Mitochondria and Cancer: Past, Present, and Future. Biomed Res Int. 2013;2013:612369.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  4. 4.

    Chatterjee A, Mambo E, Sidransky D. Mitochondrial DNA mutations in human cancer. Oncogene. 2006;25:4663–74.

    CAS  PubMed  Article  Google Scholar 

  5. 5.

    Modica-Napolitano JS, Singh K. Mitochondria as targets for detection and treatment of cancer. Expert Rev Mol Med. 2002;4:1–19.

    PubMed  Article  Google Scholar 

  6. 6.

    Modica-Napolitano JS, Singh KK. Mitochondrial dysfunction in cancer. Mitochondrion. 2004;4:755–62.

    CAS  PubMed  Article  Google Scholar 

  7. 7.

    Singh KK, Kulawiec M. Mitochondrial DNA polymorphism and risk of cancer. Methods Mol Biol. 2009;471:291–303.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  8. 8.

    Aikhionbare FO, Mehrabi S, Kumaresan K, Zavareh M, Olatinwo M, Odunsi K, et al. Mitochondrial DNA sequence variants in epithelial ovarian tumor subtypes and stages. J Carcinog. 2007;6:7–14.

    Article  Google Scholar 

  9. 9.

    Ksiȩzakowska-Łakoma K, Zyła M, Wilczyński JR. Mitochondrial dysfunction in cancer. Przeglad Menopauzalny. 2014;18:136–44.

    Google Scholar 

  10. 10.

    Mithani SK, Shao C, Tan M, Smith IM, Califano JA, El-Naggar AK, et al. Mitochondrial mutations in adenoid cystic carcinoma of the salivary glands. PLoS ONE. 2009;4, e8493.

    PubMed Central  PubMed  Article  Google Scholar 

  11. 11.

    Hsu CC, Lee HC, Wei YH. Mitochondrial DNA alterations and mitochondrial dysfunction in the progression of hepatocellular carcinoma. World J Gastroentero. 2013;19:8880–86.

    CAS  Article  Google Scholar 

  12. 12.

    Yang Ai SS, Hsu K, Herbert C, Cheng Z, Hunt J, Lewis CR, et al. Mitochondrial DNA mutations in exhaled breath condensate of patients with lung cancer. Resp Med. 2013;107:911–8.

    Article  Google Scholar 

  13. 13.

    Lee HC, Huang KH, Yeh TS, Chi CW. Somatic alterations in mitochondrial DNA and mitochondrial dysfunction in gastric cancer progression. World J Gastroentero. 2014;20:3950–9.

    CAS  Article  Google Scholar 

  14. 14.

    Houshmand M, Larsson NG, Holme E, Oldfors A, Tulinius MH, Andersen O. Automatic sequencing of mitochondrial tRNA genes in patients with mitochondrial encephalomyopathy. BBA-Mol Basis Dis. 1994;1226:49–55.

    CAS  Article  Google Scholar 

  15. 15.

    Petros JA, Baumann AK, Ruiz-Pesini E, Amin MB, Sun CQ, Hall J, et al. mtDNA mutations increase tumorigenicity in prostate cancer. P Natl Acad Sci USA. 2005;102:719–24.

    CAS  Article  Google Scholar 

  16. 16.

    Booker LM, Habermacher GM, Jessie BC, Sun QC, Baumann AK, Amin M, et al. North American white mitochondrial haplogroups in prostate and renal cancer. J Urology. 2006;175:468–73.

    CAS  Article  Google Scholar 

  17. 17.

    Ha PK, Tong BC, Westra WH, Sanchez-Cespedes M, Parrella P, Zahurak M, et al. Mitochondrial C-tract alteration in premalignant lesions of the head and neck a marker for progression and clonal proliferation. Clin Cancer Res. 2002;8:2260–5.

    CAS  PubMed  Google Scholar 

  18. 18.

    Bai RK, Leal SM, Covarrubias D, Liu A, Wong LJC. Mitochondrial genetic background modifies breast cancer risk. Cancer Res. 2007;67:4687–94.

    CAS  PubMed  Article  Google Scholar 

  19. 19.

    Clayton DA, Vinograd J. Circular dimer and catenate forms of mitochondrial DNA in human leukaemic leucocytes. Nature. 1967;216:652–7.

    CAS  PubMed  Article  Google Scholar 

  20. 20.

    Sternberg D, Danan C, Lombès A, Laforêt P, Girodon E, Goossens M, et al. Exhaustive scanning approach to screen all the mitochondrial tRNA genes for mutations and its application to the investigation of 35 independent patients with mitochondrial disorders. Hum Mol Genet. 1998;7:33–42.

    CAS  PubMed  Article  Google Scholar 

  21. 21.

    Wallace DC. Diseases of the mitochondrial DNA. Annu Rev Biochem. 1992;61:1175–212.

    CAS  PubMed  Article  Google Scholar 

  22. 22.

    Abu-Amero KK, Bosley TM. Mitochondrial abnormalities in patients with LHON-like optic neuropathies. Invest Ophth Vis Sci. 2006;47:4211–20.

    Article  Google Scholar 

  23. 23.

    Abu-Amero KK, Morales J, Bosley TM. Mitochondrial abnormalities in patients with primary open-angle glaucoma. Invest Ophth Vis Sci. 2006;47:2533–41.

    Article  Google Scholar 

  24. 24.

    Collins DW, Gudiseva HV, Trachtman BT, Jerrehian M, Gorry T, Merritt III WT, et al. Mitochondrial Sequence Variation in African-American Primary Open-Angle Glaucoma Patients. PloS one. 2013;8, e76627.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  25. 25.

    Abu-Amero KK, Bosley TM, Morales J. Analysis of nuclear and mitochondrial genes in patients with pseudoexfoliation glaucoma. Mol Vis. 2008;14:29–36.

    CAS  PubMed Central  PubMed  Google Scholar 

  26. 26.

    Abu-Amero KK, Morales J, Osman MN, Bosley TM. Nuclear and mitochondrial analysis of patients with primary angle-closure glaucoma. Invest Ophth Vis Sci. 2007;48:5591–6.

    Article  Google Scholar 

  27. 27.

    Bosley TM, Constantinescu CS, Tench CR, Abu-Amero KK. Mitochondrial changes in leukocytes of patients with optic neuritis. Mol Vis. 2007;13:1516–28.

    CAS  PubMed  Google Scholar 

  28. 28.

    Kumar R, Venkatesh S, Kumar M, Tanwar M, Shasmsi M, Gupta N, et al. Oxidative stress and sperm mitochondrial DNA mutation in idiopathic oligoasthenozoospermic men. Indian J Biochem Biophys. 2009;46:172–7.

    CAS  PubMed  Google Scholar 

  29. 29.

    Grzybowska-Szatkowska L, Slaska B. Polymorphisms in genes encoding mt-tRNA in female breast cancer in Poland. Mitochondr DNA. 2012;23:106–11.

    CAS  Article  Google Scholar 

  30. 30.

    Zifa E, Daniil Z, Skoumi E, Stavrou M, Papadimitriou K, Terzenidou M, et al. Mitochondrial genetic background plays a role in increasing risk to asthma. Mol Biol Rep. 2012;39:4697–708.

    CAS  PubMed  Article  Google Scholar 

  31. 31.

    Wallace DC. A mitochondrial paradigm of metabolic and degenerative diseases, aging and cancer: a dawn for evolutionary medicine. Annu Rev Genet. 2005;39:359.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  32. 32.

    Czarnecka AM, Krawczyk T, Zdrożny M, Lubiński J, Arnold RS, Kukwa W, et al. Mitochondrial NADH-dehydrogenase subunit 3 (ND3) polymorphism (A10398G) and sporadic breast cancer in Poland. Breast Cancer Res Tr. 2010;121:511–8.

    CAS  Article  Google Scholar 

  33. 33.

    Datta S, Majumder M, Biswas NK, Sikdar N, Roy B. Increased risk of oral cancer in relation to common Indian mitochondrial polymorphisms and Autosomal GSTP1 locus. Cancer. 2007;110:1991–9.

    CAS  PubMed  Article  Google Scholar 

  34. 34.

    Amend K, Hicks D, Ambrosone CB. Breast cancer in African-American women: differences in tumor biology from European-American women. Cancer Res. 2006;66:8327–30.

    CAS  PubMed  Article  Google Scholar 

  35. 35.

    HEIDARI MM, Khatami M, Houshmand M, Mahmoudi E, Nafissi S. Increased Prevalence 12308 A > G mutation in Mitochondrial tRNALeu (CUN) Gene Associated with earlier Age of Onset in Friedreich Ataxia. Iran J Child Neurol. 2011;5:25–31.

    Google Scholar 

  36. 36.

    Jaksch M, Klopstock T, Kurlemann G, Dörner M, Hofmann S, Kleinle S, et al. Progressive myoclonus epilepsy and mitochondrial myopathy associated with mutations in the tRNASer (UCN) gene. Ann Neurol. 1998;44:635–40.

    CAS  PubMed  Article  Google Scholar 

  37. 37.

    Kumar M, Tanwar M, Faiq MA, Pani J, Shamsi MB, Dada T, et al. Mitochondrial DNA nucleotide changes in primary congenital glaucoma patients. Mol Vis. 2013;19:220–30.

    CAS  PubMed Central  PubMed  Google Scholar 

  38. 38.

    Pulkes T, Sweeney M, Hanna M. Increased risk of stroke in patients with the A12308G polymorphism in mitochondria. The Lancet. 2000;356:2068–9.

    CAS  Article  Google Scholar 

  39. 39.

    Ross OA, McCormack R, Curran MD, Alistair Duguid R, Barnett YA, Maeve Rea I, et al. Mitochondrial DNA polymorphism: its role in longevity of the Irish population. Exp Gerontol. 2001;36:1161–78.

    CAS  PubMed  Article  Google Scholar 

  40. 40.

    Crimi M, Del Bo R, Galbiati S, Sciacco M, Bordoni A, Bresolin N, et al. Mitochondrial A12308G polymorphism affects clinical features in patients with single mtDNA macrodeletion. Eur J Hum Genet. 2003;11:896–8.

    CAS  PubMed  Article  Google Scholar 

  41. 41.

    Sheybaninia S, Azadfar P, Akbari L, Assarzadegan F, Houshmand M. New Mutations in 22 Mitochondrial tRNA Genes in Alzheimer’s Disease. Genetics in the 3rd millennium. 2011;9:2367–72.

    Google Scholar 

  42. 42.

    Li H, Zhong S, Li C. Study on the mitochondrion DNA mutation in tumor tissues of gynecologic oncology patients. Zhonghua fu chan ke za zhi. 2003;38:290–3.

    PubMed  Google Scholar 

  43. 43.

    Xu L, Hu Y, Chen B, Tang W, Han X, Yu H, et al. Mitochondrial polymorphisms as risk factors for endometrial cancer in southwest China. Int J Gynecol Cancer. 2006;16:1661–7.

    CAS  PubMed  Article  Google Scholar 

  44. 44.

    Grasso M, Diegoli M, Brega A, Campana C, Tavazzi L, Arbustini E. The mitochondrial DNA mutation T12297C affects a highly conserved nucleotide of tRNA (Leu (CUN)) and is associated with dilated cardiomyopathy. Eur J Hum Genet. 2001;9:311–5.

    CAS  PubMed  Article  Google Scholar 

Download references

Author information



Corresponding author

Correspondence to Ali Reza Rezaee khorasany.

Additional information

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

AR carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. FM participated in its design and helped to draft the manuscript. MH conceived of the study, participated in its design, coordination, helped to draft the manuscript and Corresponding author. All authors read and approved the final manuscript.

Authors’ information

FM: MSc. Cellular and Molecular Biology, University of Mazandaran, Babolsar, Iran, 2012. BSc. Cellular and Molecular genetic, Shahed university, Tehran, Iran, 2010.

MH: PhD. Medical Molecular Genetic, Gothenburg University, Gothenburg, Sweden, 1999. MSc. Molecular Genetic, Gothenburg University, Gothenburg, Sweden, 1992. BSc. in Medical Laboratory, Gothenburg University, Gothenburg, Sweden, 1990.

AR: PhD student. Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran, 2012. MSc. Animal breeding and genetic- Biotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Iran, 2008. BSc. Animal breeding and genetic- Biotechnology, Faculty of Agriculture, Gorgan University, Gorgan, Iran, 2004.

Additional file

Additional file 1: Table 1.

Mitochondrial Primers for PCR-Sequencing of tRNALeu(CUN).

Rights and permissions

Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.

The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this licence, visit

The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

MA Mohammed, F., Rezaee khorasany, A.R., Mosaieby, E. et al. Mitochondrial A12308G alteration in tRNALeu(CUN) in colorectal cancer samples. Diagn Pathol 10, 115 (2015).

Download citation


  • Colorectal cancer
  • Mutation
  • Mitochondrial tRNALeu(CUN)
  • A12308G mutation