Purpose: To determine mitochondrial (mt)DNA variants in AMD and age-matched normal retinas.
Methods: Total DNA was isolated from retinas (AMD, n = 13; age-matched normal, n = 13), choroid (AMD, n = 3), and blood (AMD, n = 138; normal, n = 133). Long-extension-polymerase chain reaction amplified the full-length ( approximately 16.2 kb) mtDNA genome. Retinal mtDNA was sequenced for nucleotide variants and length heteroplasmy. Pyrosequencing was performed on heteroplasmic mtDNA. PCR amplification and enzyme digestions were used to analyze for nucleotide changes.
Results: Retinal mtDNA had a greater number of rearrangements and deletions than did blood mtDNA in normal samples (9.3 +/- 1.78 vs. 3 +/- 1.18, P = 0.019), and AMD samples (14.33 +/- 1.96 vs. 5.2 +/- 0.80, P = 0.0031. Five (55%) of 9 AMD patients had unreported SNPs, and 2 (16.6%) of 12 of the normal group did. The mtDNA coding region had 20 SNPs that produced amino acid changes. The noncoding MT-Dloop region had nucleotide heteroplasmy and length heteroplasmy. There were more SNPs per person in the AMD population than in the older (P = 0.003) and younger (P = 0.05) normal subjects. The C12557T (T-I) in the MT-ND5 gene was present in two AMD subjects (2/138) but was absent in the normal (0/133). Common mutations for Leber's hereditary optic neuropathy (LHON: G11778A; T14484C; and G3460A) were not present in AMD samples.
Conclusions: AMD subjects have high levels of large mtDNA deletions/rearrangements in the retinas, unreported and amino acid-changing SNPs in the coding genome, and a greater number of SNPs per person in the noncoding MT-Dloop region. These mtDNA variants could diminish energy production efficiency, alter the mtDNA copy numbers and/or impact transcription in AMD retinas.