Ancient mtDNA sequences in the human nuclear genome: A potential source of errors in identifying pathogenic mutations
Ancient mtDNA sequences in the human nuclear genome: A potential source of errors in identifying pathogenic mutations
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DOI：
10.1073/pnas.94.26.14900
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发表时间：
1997-12-23
影响因子：
11.1
通讯作者：
Brown, MD
中科院分区：
文献类型：
--
作者：
Wallace, DC;Stugard, C;Brown, MD
核定位的mtDNA假基因可以解释最近的一份报告，该报告描述了阿尔茨海默病(AD)患者中一种含有五个连锁错义突变的异质mtDNA分子，该分子分散在连续的mtDNACO1和CO2基因上。为了验证这一假设，我们使用原始报告中使用的PCR引物从两个独立的Rho度(mtDNA-less)细胞系中扩增出CO1和CO2序列，从两个Rho度细胞中扩增出CO1和CO2序列，证明这些序列也存在于人类核DNA中，然后通过限制性内切酶酶切位点变异分析检测核假基因CO1和CO2序列中的每一个，在Rho度细胞的核CO1和CO2扩增产物中都发现了这五个突变？但从线粒体DNA正常的细胞中获得的PCR产物中没有发现任何一个，而且，当克隆和测序重叠的核CO1和CO2PCR产物时，发现了全部五个错义突变，以及一个连锁的同义突变，与原始报告的发现不同，还发现了另外32个碱基替换，包括相邻tRNA中的两个碱基替换和二氧化碳基因中的两个碱基对缺失，对核CO1和CO2序列的系统发育分析表明，它们与大约77万年前人类进化早期的mtDNA有所不同。这些数据将与以下解释一致，即导致AD的错义突变可能是作为核假基因保存的古代mtDNA变体的产物。
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 he the product of ancient mtDNA variants preserved as nuclear pseudogenes.