The T414G mitochondrial DNA mutation: a biomarker of ageing in human eye

Abstract

The mitochondrial mutation T414G (mtDNAT414G) has been shown to accumulate in aged and sun-exposed skin. The human eye is also exposed to solar harmful rays. More precisely, the anterior structures of the eye (cornea, iris) filter UV rays and the posterior portion of the eye (retina) is exposed to visible light. These rays can catalyse mutations in mitochondrial DNA such as the mtDNAT414G, but the latter has never been investigated in the human ocular structures. In this study, we have developed a technique to precisely assess the occurrence of mtDNAT414G. Using this technique, we have quantified mtDNAT414G in different human ocular structures. We found an age-dependent accumulation of mtDNAT414G in the corneal stroma, the cellular layer conferring transparency and rigidity to the human cornea, and in the iris. Since cornea and iris are two anterior ocular structures exposed to solar UV rays, this suggests that the mtDNAT414G mutation is resulting from cumulative solar exposure and this could make the mtDNAT414G a good marker of solar exposure. We have previously shown that the mtDNACD4977 and mtDNA3895 deletions accumulate over time in photo-exposed ocular structures. With the addition of mtDNAT414G mutation, it becomes feasible to combine the levels of these different mtDNA mutations to obtain an accurate assessment of the solar exposure that an individual has accumulated during his/her lifetime.

Figure 1.

Schematic representation of the Q-PCR technique used to evaluate the mtDNA^T414G/mtDNA ratio. Primer set F5/R4 is designed to amplify total mtDNA. The primer set R4/MutF3 specifically detects mtDNA molecules carrying the T414G mutation. The primer contains the T414G mutation in order to amplify only the mtDNA containing a guanine in position 414.

Figure 2.

mtDNA^T414G/total mtDNA ratio analysis in different ocular structures. Total DNA was harvested from the cornea, iris and neural retina isolated from 7 human subjects. The mtDNA^T414G/total mtDNA ratio was derived from each ocular structure by Q-PCR. A higher mtDNA^T414G level has been found in the iris (0.051%), when compared to the cornea (0.021%) and the neural retina (0.012%). According to Wilcoxon matched-paired rank test, the mtDNA^T414G/total mtDNA ratio was significantly different between the iris and the cornea and between the iris and the neural retina (*P value < 0.02) but not between the cornea and the neural retina.

Figure 3.

Age-related accumulation of mtDNA^T414G mutation in the iris. Total DNA was harvested from the iris of 13 subjects ranging from 38 to 70 years old and 13 subjects ranging from 70 to 94 years old. mtDNA^T414G/total mtDNA ratio was determined using Q-PCR. We found a significant higher mtDNA^T414G/total mtDNA ratio in the older group (70–94 years old), when compared to the younger 38- to 70-year-old group, with median values of 0.091% and 0.052%, respectively. A Mann–Whitney test was used (*P value < 0.02).

Figure 4.

mtDNA^T414G/total mtDNA ratio analysis in the different corneal cellular layers. The mtDNA^T414G/total mtDNA ratio was analysed by Q-PCR in the corneal epithelium, stroma and endothelium of 9 human subjects with a median age of 78 years old. We found that the mtDNA^T414G mutation is concentrated in the stroma (0.043%), when compared to the epithelium (0.003%) and the endothelium (0.013%). According to Wilcoxon test, the mtDNA^T414G/total mtDNA ratio is significantly different between the corneal stroma and the epithelium and between the stroma and the endothelium (*P value < 0.04).

Figure 5.

Age-related accumulation of mtDNA^T414G in the corneal stroma. The mtDNA^T414G/total mtDNA was analysed in total DNA harvested from the stroma of 14 subjects ranging from 38 to 70 years old and 23 subjects ranging from 70 to 94 years old. mtDNA^T414G/total mtDNA ratio was determined by Q-PCR. We found significantly more mtDNA^T414G mutation in the older group (0.017%) than in the younger one (0.007%). A Mann–Whitney test has been used (*P value < 0.01).

Figure 6.

mtDNAT^414G/total mtDNA ratio analysis in the macular and peripheral regions of the neural retina and the RPE. After dissection of the neural retina (RET) and the RPE of 7 patients with a median age of 60, the macular (mac) and peripheral (periph) regions were separated. Total DNA was extracted and mtDNA^T414G/total mtDNA ratio was determined using Q-PCR. Analysis of the mtDNAT^414G/total mtDNA ratio shows no significant difference between the macular and peripheral regions of both neural retina (0.027% and 0.025%, respectively) and RPE (0.011% and 0.014%, respectively).