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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.
Abstract:
Nuclear-localized mtDNA pseudogenes might explain a recent report describing a heteroplasmic mtDNA molecule containing five linked missense mutations dispersed over the contiguous mtDNA CO1 and CO2 genes in Alzheimer's disease (AD) patients. To test this hypothesis, we have used the PCR primers utilized in the original report to amplify CO1 and CO2 sequences from two independent rho degrees (mtDNA-less) cell lines. CO1 and CO2 sequences amplified from both of the rho degrees cells, demonstrating that these sequences are also present in the human nuclear DNA. The nuclear pseudogene CO1 and CO2 sequences were then tested for each of the five "AD" missense mutations by restriction endonuclease site variant assays. All five mutations were found in the nuclear CO1 and CO2 PCR products from rho degrees cells, but none were found in the PCR products obtained from cells with normal mtDNA. Moreover, when the overlapping nuclear CO1 and CO2 PCR products were cloned and sequenced, all five missense mutations were found, as well as a linked synonymous mutation. Unlike the findings in the original report, an additional 32 base substitutions were found, including two in adjacent tRNAs and a two base pair deletion in the CO2 gene. Phylogenetic analysis of the nuclear CO1 and CO2 sequences revealed that they diverged from modern human mtDNAs early in hominid evolution about 770,000 years before present. These data would be consistent with the interpretation that the missense mutations proposed to cause AD may be the product of ancient mtDNA variants preserved as nuclear pseudogenes.
Insights
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.