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Ancient mtDNA sequences in the human nuclear genome: a potential source of errors in identifying pathogenic mutations
D C Wallace1, C Stugard, D Murdock
1Center for Molecular Medicine, Emory University, Atlanta, GA 30322, USA.
Summary
Nuclear pseudogenes containing Alzheimer's-associated mutations explain findings in patient mtDNA. These ancient variants, preserved in nuclear DNA, suggest mutations may not originate from mitochondrial DNA in Alzheimer's disease.
Area of Science:
- Genetics
- Molecular Biology
- Neuroscience
Background:
- A previous study reported heteroplasmic mitochondrial DNA (mtDNA) with five linked missense mutations in Alzheimer's disease (AD) patients.
- These mutations were found in contiguous mtDNA CO1 and CO2 genes.
- Nuclear-localized mtDNA pseudogenes were hypothesized to explain these findings.
Purpose of the Study:
- To investigate the hypothesis that nuclear-localized mtDNA pseudogenes are the source of reported mtDNA mutations in Alzheimer's disease.
- To determine if the CO1 and CO2 sequences, including the five missense mutations, are present in nuclear DNA.
- To analyze the evolutionary origin of these nuclear pseudogenes.
Main Methods:
- Utilized PCR primers from the original report to amplify CO1 and CO2 sequences from mtDNA-less (rho degrees) cell lines.
- Tested amplified nuclear pseudogene sequences for the presence of the five specific missense mutations using restriction endonuclease site variant assays.
- Cloned and sequenced overlapping nuclear CO1 and CO2 PCR products.
- Performed phylogenetic analysis of nuclear CO1 and CO2 sequences.
Main Results:
- CO1 and CO2 sequences were successfully amplified from rho degrees cells, confirming their presence in nuclear DNA.
- All five reported missense mutations were found in the nuclear CO1 and CO2 sequences from rho degrees cells, but not in cells with normal mtDNA.
- Sequencing revealed the five missense mutations, a synonymous mutation, 32 additional base substitutions, including in tRNAs, and a two base pair deletion in CO2.
- Phylogenetic analysis indicated nuclear CO1 and CO2 sequences diverged from modern mtDNA early in hominid evolution (~770,000 years ago).
Conclusions:
- The five missense mutations proposed to be linked to Alzheimer's disease are present in nuclear mtDNA pseudogenes, not pathogenic mtDNA.
- These nuclear pseudogenes represent ancient mtDNA variants preserved through evolution.
- The findings suggest that the reported mutations may be artifacts of nuclear pseudogene sequences rather than causative agents of Alzheimer's disease.