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Nathaniel Kevin Mullin, Kristin R. Anfinson, Louisa M. Affatigato, Jeaneen L. Andorf, Megan J. Riker, Robert F Mullins, Edwin M Stone, Budd A. Tucker; Mutational burden of mt3243G in blood correlates with the age of onset of retinal symptoms.. Invest. Ophthalmol. Vis. Sci. 2021;62(8):1518.
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The mitochondrial mt3243A>G mutation is a common cause of retinal degeneration in patients with MIDD and MELAS. The relationship between mutational burden in peripheral tissues and severity of retinal disease is not currently well understood. We developed a digital PCR assay to screen blood and skin samples from 8 patients with clinically diagnosed and molecularly confirmed mt3243A>G disease. Using this platform, we correlated onset of visual symptoms with blood and skin mutational burden and explored the relationship between mitochondrial DNA copy number and mt3243A>G.
DNA was isolated from cultured dermal fibroblasts and peripheral blood mononuclear cells donated by 8 patients (ages 27-64) known to have retinal disease caused by the mt3243A>G mutation. The proportion of mutant (G) to wildtype (A) alleles was measured using digital PCR or TA-cloning with Sanger sequencing of resulting clones. Additionally, the mitochondrial DNA copy number was assessed by digital PCR. Patients were evaluated clinically by a single ophthalmologist. Molecular testing was performed masked to clinical information.
The proportion of mt3243G allele varied greatly both between individuals and between cell type (blood: range 7%-39%, mean 19.13%; skin: 13%-82%, mean 45.63%). However, there was a strong correlation between blood and skin mt3243G proportion within the same individual (R2 = 0.606, P value = 0.023). Additionally, a correlation between mt3243G proportion and mtDNA copy number was observed in cultured skin fibroblasts (R2 = 0.626, P value = 0.019), but not in blood. Finally, we found that age of onset of visual symptoms correlated with proportion of mt3243G in blood (R2 = 0.820, P value = 0.002) but not in skin (Figure 1).
Our results indicate that precise measurement of mt3243G mutational burden in peripheral tissues may be clinically relevant in retinal disease. Higher mutational burden of mt3243G correlated with an earlier onset of visual symptoms. Digital PCR offers a robust method to measure mutational burden in diseases caused by heteroplasmic mutations such as mt3243A>G. Future studies investigating the mechanisms by which mt3243 mutational burden is maintained in various cell types may shed light on the heterogenous phenotypes present in this family of disease.
This is a 2021 ARVO Annual Meeting abstract.
Correlation between age of first visual symptom onset and proportion of mutant mt3243G allele in blood.
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