Mitochondrial DNA analysis in primary congenital glaucoma

Abstract

Purpose: To screen mitochondrial DNA (mtDNA) for nucleotide variations in primary congenital glaucoma (PCG).

Methods: The entire coding region of the mitochondrial genome was amplified by polymerase chain reaction from 35 PCG patients and 40 controls. The full mtDNA genome except the D-loop was sequenced. All sequences were analyzed against mitochondrial reference sequence NC_012920.

Results: MtDNA sequencing revealed a total of 132 and 58 nucleotide variations in PCG and controls, respectively. Of 132 nucleotide variations, 42 (31.81%) were non-synonymous and 82 (62.12%) were synonymous changes, and 8 were in RNA genes. The highest number of nucleotide variations were recorded in complex I followed by complex IV, then complex V. Eight patients (22.85%) had potentially pathogenic mtDNA nucleotide changes and twenty (57.14%) had mtDNA sequence changes associated with elevated reactive oxygen species (ROS) production. Mitochondria not only constitute the energy-generating system in the cell, but are also critically involved in calcium signaling and apoptosis. Mitochondrial function can be affected by mutations in mitochondrial and nuclear DNA, chemical insults to components of the electron transport chain, and a lack of substrates such as oxygen. Mitochondrial dysfunction results in an excessive generation of free radicals and reduced mitochondrial respiration. Developing trabecular meshwork (TM) is deficient in antioxidant enzymes, and thus is more susceptible to oxidative stress (OS) induced damage. Previous studies have documented certain mtDNA sequence variations associated with elevated ROS levels and OS. Three such changes (G10398A, A12308G, and G13708A) were present in our patients. Elevated ROS may cause OS. This OS may further damage mtDNA and may cause decreased mitochondrial respiration. This may lead to impaired growth, development and differentiation of TM and consequently trabecular-dysgenesis, which is a characteristic feature of PCG. OS affects both TM and retinal ganglion cells (RGCs) and may be involved in the neuronal death affecting the optic nerve in glaucoma. There are several studies which point to mitochondrial dysfunction in different types of glaucoma and critically participate in RGC death. Recent studies also implicate mitochondrial dysfunction-associated OS as a risk factor for glaucoma patients. It has been reported that elevated hydrostatic pressure causes breakdown of the mitochondrial network by mitochondrial fission and induce cristae depletion and cellular ATP reduction in differentiated RGC-5 cells in vitro as well as in vivo.

Conclusions: A total of 44 novel mtDNA variations were identified in this study. Non-synonymous mtDNA variations may adversely affect respiratory chain, impair OXPHOS pathway result in low ATP production, high ROS production and impair growth, development and differentiation of TM lead to trabecular-dysgenesis and consequently RGC's death. Such cases with mtDNA variations and consequent OS may benefit by early diagnosis and prompt management by antioxidant therapy. This may delay OS induced injury to TM and RGCs and hence improve visual prognosis.

Figure 1

Bar diagram showing distribution of nucleotide variations in different mitochondrial genes in PCG. Abbrevations: ND1- NADH dehydrogenase subunit 1; ND2- NADH dehydrogenase subunit 2; ND3- NADH dehydrogenase subunit 3; ND4- NADH dehydrogenase subunit 4; ND4L- NADH dehydrogenase subunit 4L; ND5- NADH dehydrogenase subunit 5; CO1- cytochrome c oxidase I; CO2- cytochrome c oxidase II; CO3- cytochrome c oxidase III; ATPase6- ATP synthase subunit a (F-ATPase protein 6); ATPase8- ATP synthase protein 8; CYB- cytochrome B; tRNA- transfer ribo nucleic acid; rRNA- ribosomal ribonucleic acid.

Figure 2

Schematic representation of components of the OXPHOS pathway localized in inner mitochondrial membrane.

Figure 3

Possible role of mitochondrial sequence variations in trabecular-dysgenesis and RGC cell death in primary congenital glaucoma. Abbrevations: RGCs- Retinal ganglion cells; TM- trabecular meshwork; ROS- reactive oxygen species; ATP- adenosine tri phosphate; IOP- intra ocular pressure.

Figure 4

Role of elevated IOP in RGCs death in glaucoma. Abbrevations: RGCs- Retinal ganglion cells; IOP- intra ocular pressure; OPA1-optic nerve atrophy type-I.