Analysis of Mitochondrial DNA Mutations and Altered Protein Expression in Human Retinoblastoma

Abstract

Purpose: Mitochondria are critical for cellular function in cancer and play an important role in cell differentiation and survival. Displacement loop (D-loop) is a control site for expression of the mitochondrial genome. Mutations in D-loop region may alter the rate of mtDNA transcription and replication. Genetic variability in D-loop region has been suggested to affect the function of mitochondrial complexes and could contribute to tumor initia tion. The purpose of this study was to determine if mutations in the mitochondrial D-loop region could cause or effect the expression of mitochondrial complex (s) and the morphological changes of mitochondria in human primary retinoblastoma tissues.

Methods: In the present study, the entire D-loop region of mtDNA was amplified in two overlapping polymerase chain reaction fragments (Nested-PCR) and variations were evaluated in 24 primary retinoblastoma patients by direct DNA sequencing methods. Morphology of mitochondria was studied by transmission electron microscopy (TEM). Expression of mitochondrial complex I was performed in all the 24 cases by immunohistochemistry and then validated by western blotting on representative cases.

Results: A total of 301 mutations were observed at 170 positions in the mitochondrial D-Loop region. The most common variations were 73A-G (83.3%), 263A-G (70.8%) and 16223C-T (66.6%) followed by 30 novel mutations. Loss of mitochondrial complex I was seen in 19/24 (79.16%) cases by immunohistochemistry. Western blotting was performed to confirm the immunoreactivity results. Electron microscopy showed numerous degenerated and swollen mitochondria in tumor cells. On statistical analysis, the expression of mitochondrial complex I correlated significantly with poorly differentiated retinoblastoma and tumor invasion.

Conclusions: This is the first study to show a high frequency in mt D-loop variations and deficiency of mitochondrial complex I in retinoblastoma tumor. Electron microscopy revealed morphological changes in mitochondria which may be due to damage in mtDNA genome. D-loop variations could probably cause alteration in mitochondrial complex (s) which is still being investigated. Exploring mtDNA alterations might be helpful for developing biomarkers in the management of retinoblastoma patients.

Keywords: 703 retinoblastoma • 600 mitochondria • 533 gene/expression