Ancient mtDNA sequences in the human nuclear genome: a potential source of errors in identifying pathogenic mutations - PubMed (original) (raw)

Ancient mtDNA sequences in the human nuclear genome: a potential source of errors in identifying pathogenic mutations

D C Wallace et al. Proc Natl Acad Sci U S A. 1997.

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.

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Figures

Figure 1

Figure 1

Amplification of PCR products from total cellular DNAs extracted from two different human cell lines and their mtDNA-deficient ρ° counterparts using various pairs of primers homologous to the normal human mtDNA sequence (26). The cell lines are 143B-TK− (lanes 3, 8, 13, and 18), 143B-87 (ρ°) (lanes 4, 9, 14, and 19), WAL2A (lanes 5, 10, 15, and 20), and WAL2A-EB2 (ρ°) (lanes 6, 11, 16, and 21). The primer pairs used are 14430FOR/15461REV (lanes 2–6) and 15238FOR/15865REV (lanes 7–11), both encompassing positions of cytochrome b; 5803COIFOR/7570 CO1REV encompassing CO1 (lanes 12–16) and 7483CO2FOR/8383CO2REV encompassing CO2 (lanes 17–21). Negative controls involving amplification without template are shown in lanes 2, 7, 12, and 17. A size standard is shown in lane 1.

Figure 2

Figure 2

Detection of the five missense mutations in CO1 and CO2 genes amplified from DNA of 143B-TK− and 143B-87 ρ° cells by restriction endonuclease digestion. PCR products obtained from 143B-TK− DNA are shown in lanes 3 and 5 for CO1 and lanes 7, 9, and 11 for CO2. PCR products obtained from 143B-87 ρ° DNA are shown in lanes 4 and 6 for CO1 and lanes 8, 10, and 12 for CO2. Detection of the np 6366 G-to-A mutation in CO1 by_Bsm_AI digestion is presented in lanes 3 and 4. Detection of the np 7146 A-to-G mutation in CO1 by _Nru_I digestion is shown in lanes 5 and 6. The double band in lane 6 is most likely the result of partial digestion. Detection of the np 7650 C-to-T mutation by _Hph_I digestion is shown in lanes 7 and 8. Detection of the np 7868 C-to-T mutation by _Mse_I digestion is shown in lanes 9 and 10. Detection of the np 8021 A-to-G mutations by_Hpa_II digestion is shown in lanes 11 and 12. Lanes 1 and 2 are size standards: lane 1 is a ∅X174 _Hae_III digest and lane 2 is a 1-kb DNA ladder.

Figure 3

Figure 3

Unrooted NJ tree relating the human nuclear CO1 and CO2 sequences to the homologous sequences from the human, chimpanzee, gorilla, gibbon, and orangutan mtDNAs. This tree is based on genetic distances calculated using the maximum likelihood model (DNAML) (28). Boostrap analysis from 100 independent trees generated the diagrammed result in 100% of the comparisons between the human nuclear and cytoplasmic sequences and between the gibbon and orangutan sequences, in 99% of the comparisons between the two human sequences and the chimpanzee sequence, and in 82% of the comparisons between the chimpanzee and gorilla sequences and between the gorilla and the collective gibbon and orangutan sequences. Phylogenies with identical branching orders were obtained by using genetic distances calculated by the parsimony, Jukes-Cantor, and Kimura two-parameter methods.

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References

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