Pathology & Oncology Research

, Volume 25, Issue 2, pp 503–512 | Cite as

Mutational Analysis of the Mitochondrial DNA Displacement-Loop Region in Human Retinoblastoma with Patient Outcome

  • Lata SinghEmail author
  • Neeru Saini
  • Neelam Pushker
  • Sameer Bakhshi
  • Seema Sen
  • Tapas C. Nag
  • Seema KashyapEmail author
Original Article


Alteration in mitochondrial DNA plays an important role in the development and progression of cancer. The Displacement Loop (D-loop) region of mitochondrial DNA (mtDNA) is the regulatory region for its replication and transcription. Therefore, we aimed to characterize mutations in the D-loop region of mitochondrial DNA along with the morphological changes and analyzed their impact on survival in retinoblastoma patients. mtDNA D-loop region was amplified by Nested-Polymerase Chain Reaction (Nested-PCR) and mutations were analyzed in 60 tumor samples from retinoblastoma patients by DNA sequencing. Transmission electron microscopy was performed on 5 retinoblastoma specimens. Mutations were correlated with clinical, histopathological parameters and patient survival. D-loop mutations were found in total of 52/60 (86.6%) patients. The most common mutations were T to C and C to T followed by A to G. There were 5.81% mutations which were not previously reported in the MITOMAP database. A73G (83.33%) were the most frequent mutations found in our cases and it was statistically significant with poor tumor differentiation and age. In addition, this study was further analyzed for morphological changes in retinoblastoma that had disorganized, swollen and less numbers of mitochondria on electron microscopy. This is the first study showing high frequency of mtDNA mutation which might be due to abnormal morphology of mitochondria in retinoblastoma. Our results indicate that pathogenic mtDNA D-loop mutations may be involved in tumorigenesis of retinoblastoma tumor.


Retinoblastoma D-loop Mitochondrial DNA DNA sequencing Electron microscopy 


Author Contribution

LS was responsible for designing and executing the experiment. NS helps in sequencing analysis. SK and SS did the histopathological evaluation. TCN did the electron microscopy evaluation. SB was responsible for providing follow up. NP was responsible proving samples.


This study was funded by Indian Council of Medical Research, New Delhi, INDIA (Award no: 5/4/6/09/Oph/2012-NCD II).

Compliance with Ethical Standards

Conflict of Interest

No potential conflicts of interest with any author.

Ethical Approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed Consent

Informed consent was obtained from all individual participants included in the study.


  1. 1.
    Larsson NG, Clayton DA (1995) Molecular genetics aspects of human mitochondrial disorders. Annu Rev Genet 29:151–178CrossRefGoogle Scholar
  2. 2.
    Lane N (2006) Mitochondrial disease: powerhouse of disease. Nature 440:600–602CrossRefGoogle Scholar
  3. 3.
    Falk MJ (2010) Neurodevelopmental manifestations of mitochondrial disease. J Dev Behav Pediatr 31:610CrossRefGoogle Scholar
  4. 4.
    Taanman JW (1999) The mitochondrial genome: structure, transcription, translation and replication. Biochimi Biophys Acta (BBA)-Bioenergetics 9(1410):103–123CrossRefGoogle Scholar
  5. 5.
    Anderson S, Bankier AT, Barrell BG et al (1981) Sequence and organization of the human mitochondrial genome. Nature 290:457–465CrossRefGoogle Scholar
  6. 6.
    Vives-Bauza C, Gonzalo R, Manfredi G et al (2006) Enhanced ROS production and antioxidant defenses in cybrids harbouring mutations in mtDNA. Neurosci Lett 391:136–141CrossRefGoogle Scholar
  7. 7.
    Stoneking M (2000) Hypervariable sites in the mtDNA control region are mutational hotspots. Am J Hum Genet 67:1029–1032CrossRefGoogle Scholar
  8. 8.
    Van Oven M, Kayser M (2009) Updated comprehensive phylogenetic tree of global human mitochondrial DNA variation. Hum Mutat 30:E386–E394CrossRefGoogle Scholar
  9. 9.
    Mims MP, Hayes TG, Zheng S et al (2006) Mitochondrial DNA G10398A polymorphism and invasive breast cancer in African-American women. Cancer Res 66:1880–1881CrossRefGoogle Scholar
  10. 10.
    Janovjak H, Sapra KT, Kedrov A et al (2008) From valleys to ridges: exploring the dynamic energy landscape of single membrane proteins. Chem Phys Chem 9:954–966CrossRefGoogle Scholar
  11. 11.
    Arismendi-Morillo G (2011) Electron microscopy morphology of the mitochondrial network in gliomas and their vascular microenvironment. Biochim Biophys Acta (BBA)-Bioenergetics 1807:602–608CrossRefGoogle Scholar
  12. 12.
    Lohmann DR (1999) RB1 gene mutations in retinoblastoma. Hum Mutat 14:283–288CrossRefGoogle Scholar
  13. 13.
    Wallace DC, Chalkia D (2013) Mitochondrial DNA genetics and the heteroplasmy conundrum in evolution and disease. Cold Spring Harb Perspect Biol 5:a021220CrossRefGoogle Scholar
  14. 14.
    Alonso A, Martin P, Albarran C et al (1997) Detection of somatic mutations in the mitochondrial DNA control region of colorectal and gastric tumors by heteroduplex and single-strand conformation analysis. Electrophoresis 18:682–685CrossRefGoogle Scholar
  15. 15.
    Jarrett SG, Lewin AS, Boulton ME (2010) The importance of mitochondria in age-related and inherited eye disorders. Ophthalmic Res 44:179–190CrossRefGoogle Scholar
  16. 16.
    Schrier SA, Falk MJ (2011) Mitochondrial disorders and the eye. Curr Opin Ophthalmol 22:325–331CrossRefGoogle Scholar
  17. 17.
    Wallace DC, Singh G, Lott MT et al (1998) Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy. Science 242:1427CrossRefGoogle Scholar
  18. 18.
    Barreau E, Brossas JY, Courtois Y, Treton JA (1996) Accumulation of mitochondrial DNA deletions in human retina during aging. Invest Ophthalmol Vis Sci 37:384–391Google Scholar
  19. 19.
    Kowluru RA (2005) Diabetic retinopathy: mitochondrial dysfunction and retinal capillary cell death. Antioxid Redox Signal 7:1581CrossRefGoogle Scholar
  20. 20.
    Saraswathy S, Rao NA (2009) Mitochondrial proteomics in experimental autoimmune uveitis oxidative stress. Invest Ophthalmol Vis Sci 50:5559–5566CrossRefGoogle Scholar
  21. 21.
    Tezel G (2006) Oxidative stress in glaucomatous neurodegeneration: mechanisms and consequences. Prog Retin Eye Res 25:490–513CrossRefGoogle Scholar
  22. 22.
    DiMauro S, Garone C (2010) Historical perspective on mitochondrial medicine. Dev Disabil Res Rev 16:106–113CrossRefGoogle Scholar
  23. 23.
    Suzuki M, Toyooka S, Miyajima K et al (2003) Alterations in the mitochondrial displacement loop in lung cancers. Clin Cancer Res 9:5636–5641Google Scholar
  24. 24.
    Habano W, Sugai T, Yoshida T et al (1999) Mitochondrial gene mutation, but not large-scale deletion, is a feature of colorectal carcinomas with mitochondrial microsatellite instability. Int J Cancer 2683:625–629CrossRefGoogle Scholar
  25. 25.
    Liu VW, Shi HH, Cheung AN et al (2001) High incidence of somatic mitochondrial DNA mutations in human ovarian carcinomas. Cancer Res 61:5998–6001Google Scholar
  26. 26.
    Okochi O, Hibi K, Uemura T et al (2002) Detection of mitochondrial DNA alterations in the serum of hepatocellular carcinoma patients. Clin Cancer Res 8:2875–2878Google Scholar
  27. 27.
    Rosson D, Keshgegian AA (2004) Frequent mutations in the mitochondrial control region DNA in breast tissue. Cancer Lett 215:89–94CrossRefGoogle Scholar
  28. 28.
    Tamura G, Nishizuka S, Maesawa C et al (1999) Mutations in mitochondrial control region DNA in gastric tumours of Japanese patients. Eur J Cancer 35:316–319CrossRefGoogle Scholar
  29. 29.
    Levin BC, Cheng H, Reeder DJ (1999) A human mitochondrial DNA standard reference material for quality control in forensic identification, medical diagnosis, and mutation detection. Genomics 55:135–146CrossRefGoogle Scholar
  30. 30.
    Miyazono F, Schneider PM, Metzger R et al (2002) Mutations in the mitochondrial DNA D-Loop region occurs frequently in adenocarcinoma in Barrett’s esophagus. Oncogene 21:3780–3783CrossRefGoogle Scholar
  31. 31.
    Hibi K, Nakayama H, Yamazaki T et al (2001) Mitochondrial DNA alteration in esophageal cancer. Int J Cancer 92:319–321CrossRefGoogle Scholar
  32. 32.
    Sharma H, Singh A, Sharma C et al (2005) Mutations in the mitochondrial DNA D-loop region is frequent in cervical cancer. Cancer Cell Int 16:34CrossRefGoogle Scholar
  33. 33.
    Chatterjee A, Mambo E, Sidransky D (2006) Mitochondrial DNA mutations in human cancer. Oncogene 25:4663–4674CrossRefGoogle Scholar
  34. 34.
    Sharawat SK, Bakhshi R, Vishnubhatla S, Bakhshi S (2010) Mitochondrial D-loop variations in paediatric acute myeloid leukaemia: a potential prognostic marker. Br J Haematol 149:391–398CrossRefGoogle Scholar
  35. 35.
    Richard SM, Bailliet G, Paez GL et al (2000) Nuclear and mitochondrial genome instability in human breast cancer. Cancer Res 60:4231–4237Google Scholar

Copyright information

© Arányi Lajos Foundation 2018

Authors and Affiliations

  1. 1.Department of Ocular Pathology, Dr. R. P. Centre for Ophthalmic SciencesAll India Institute of Medical SciencesNew DelhiIndia
  2. 2.Functional Genomics UnitInstitute of Genomics and Integrative BiologyNew DelhiIndia
  3. 3.Department of Ophthalmology, Dr. R. P. Centre for Ophthalmic SciencesAll India Institute of Medical SciencesNew DelhiIndia
  4. 4.Department of Medical Oncology, IRCHAll India Institute of Medical SciencesNew DelhiIndia
  5. 5.Department of AnatomyAll India Institute of Medical SciencesNew DelhiIndia

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