ORIGINAL ARTICLE Mutational Analysis of the Mitochondrial DNA Displacement-Loop Region in Human Retinoblastoma with Patient Outcome Lata Singh 1 & Neeru Saini 2 & Neelam Pushker 3 & Sameer Bakhshi 4 & Seema Sen 1 & Tapas C. Nag 5 & Seema Kashyap 1 Received: 5 June 2017 /Accepted: 21 February 2018 # Arányi Lajos Foundation 2018 Abstract 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. Keywords Retinoblastoma . D-loop . Mitochondrial DNA . DNA sequencing . Electron microscopy Introduction Mammalian cells possess nuclear and mitochondrial genome which comprises a dual genetic system. [1] The mitochondri- on is a key venue for regulation of cell metabolism and consider as the powerhouse of the cell. [2] It has also shown to play an important role in reactive oxygen species (ROS) generation, cell signaling, apoptosis, and metabolic pathways, including the Krebs cycle, ß-oxidation, and lipid and choles- terol synthesis. [3] Mitochondrial DNA (mtDNA) contains 16,569 base pair genome in 100-1000 copies per cell contains 37 genes encoding 13 genes for respiratory chain subunits, 22tRNA and 2 ribosomal RNAs essential for protein synthe- sis. [4] The mitochondrial genome is more susceptible to oxidative damage and prone to higher rate of mutation than the nuclear genome [5]. The mutation frequency in mtDNA is about 10- to 20-fold greater in comparison to nuclear DNA due to lack of protective histone proteins and DNA repair mechanism [6]. D-loop (1124bp) region is the only non-coding segment of mtDNA containing regulatory elements that are involved in its replication and thus mutations in the D-loop may affect its copy number. This region considered to be a mutational hot spot region in various human cancers [7]. There are two hyper variable (HV) sites known as HVR-I considered as Blow Lata Singh and Seema Kashyap contributed equally to this work. * Lata Singh lata.aiims@gmail.com * Seema Kashyap dr_skashyap@hotmail.com 1 Department of Ocular Pathology, Dr. R. P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India 2 Functional Genomics Unit, Institute of Genomics and Integrative Biology, Mall Road, New Delhi, India 3 Department of Ophthalmology, Dr. R. P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India 4 Department of Medical Oncology, IRCH, All India Institute of Medical Sciences, New Delhi, India 5 Department of Anatomy, All India Institute of Medical Sciences, New Delhi, India Pathology & Oncology Research https://doi.org/10.1007/s12253-018-0391-y