Identification of ancient remains through genomic sequencing

Studies of ancient DNA have been hindered by the preciousness of remains, the small quantities of undamaged DNA accessible, and the limitations associated with conventional PCR amplification. In these studies, we developed and applied a genomewide adapter-mediated emulsion PCR amplification protocol for ancient mammalian samples estimated to be between 45,000 and 69,000 yr old. Using 454 Life Sciences (Roche) and Illumina sequencing (formerly Solexa sequencing) technologies, we examined over 100 megabases of DNA from amplified extracts, revealing unbiased sequence coverage with substantial amounts of nonredundant nuclear sequences from the sample sources and negligible levels of human contamination. We consistently recorded over 500-fold increases, such that nanogram quantities of starting material could be amplified to microgram quantities. Application of our protocol to a 50,000-yr-old uncharacterized bone sample that was unsuccessful in mitochondrial PCR provided sufficient nuclear sequences for comparison with extant mammals and subsequent phylogenetic classification of the remains. The combined use of emulsion PCR amplification and high-throughput sequencing allows for the generation of large quantities of DNA sequence data from ancient remains. Using such techniques, even small amounts of ancient remains with low levels of endogenous DNA preservation may yield substantial quantities of nuclear DNA, enabling novel applications of ancient DNA genomics to the investigation of extinct phyla.     

Phylogenetic analysis of mtDNA lineages in South American mummies

Some studies of mtDNA propose that contemporary Amerindians have descended from four haplotype groups, each defined by specific sets of polymorphisms. One recent study also found evidence of other potential founder haplotypes. We wanted to determine whether the four haplotypes in modern populations were also present in ancient South American aboriginals. We subjected mtDNA from Colombian mummies (470 to 1849 AD) to PCR amplification and restriction endonuclease analysis. The mtDNA D-loop region was surveyed for sequence variation by restriction analysis and a segment of this region was sequenced for each mummy to characterize the haplotypes. Our mummies exhibited three of the four major characteristic haplotypes of Amerindian populations denned by four markers. With sequence data obtained in the ancient samples and published data on contemporary Amerindians it was possible to infer the origin of these six mummies.     

Contamination detection in sequencing studies using the mitochondrial phylogeny

Within-species contamination is a major issue in sequencing studies, especially for mitochondrial studies. Contamination can be detected by analyzing the nuclear genome or by inspecting polymorphic sites in the mitochondrial genome (mtDNA). Existing methods using the nuclear genome are computationally expensive, and no appropriate tool for detecting sample contamination in large-scale mtDNA data sets is available. Here we present haplocheck, a tool that requires only the mtDNA to detect contamination in both targeted mitochondrial and whole-genome sequencing studies. Our in silico simulations and amplicon mixture experiments indicate that haplocheck detects mtDNA contamination accurately and is independent of the phylogenetic distance within a sample mixture. By applying haplocheck to The 1000 Genomes Project Consortium data, we further evaluate the application of haplocheck as a fast proxy tool for nDNA-based contamination detection using the mtDNA and identify the mitochondrial copy number within a mixture as a critical component for the overall accuracy. The haplocheck tool is available both as a command-line tool and as a cloud web service producing interactive reports that facilitates the navigation through the phylogeny of contaminated samples.

The human mitochondrial DNA (mtDNA) is an extranuclear DNA molecule of ∼16.6 kb in length (Andrews et al. 1999). It is inherited exclusively through the maternal line, facilitating the reconstruction of the human maternal phylogeny and female (pre-)historical demographic patterns worldwide. The strict maternal inheritance of mtDNA results in a natural grouping of haplotypes into monophyletic clusters, referred to as haplogroups (Kivisild et al. 2006; Kloss-Brandstätter et al. 2011). Furthermore, second-generation sequencing enables the detection of heteroplasmy over the complete mitochondrial genome. Heteroplasmy is the occurrence of at least two different haplotypes of mtDNA in the investigated biological samples (e.g., cells or tissues). Depending on the sequencing coverage, heteroplasmic positions are reliably detectable down to the 1% variant level (Ye et al. 2014; Weissensteiner et al. 2016a).It has been shown that external or cross-contamination (Yao et al. 2007; Just et al. 2014, 2015; Yin et al. 2019; Brandhagen et al. 2020), artificial recombination (Bandelt et al. 2004), or index hopping (Van Der Valk et al. 2019) can generate polymorphic sites that can be erroneously interpreted as heteroplasmic sites (He et al. 2010; Bandelt and Salas 2012; Just et al. 2014, 2015; Ye et al. 2014).Sample contamination is still a major issue in both nuclear DNA (nDNA) and mtDNA sequencing studies that must be prevented to avoid mistakes as they occurred with Sanger sequencing studies in the past (Salas et al. 2005). Because of the accuracy and sensitivity of second-generation sequencing combined with the availability of improved computational models, within-species contamination is traceable down to the 1% level in whole-genome sequencing (WGS) studies (Jun et al. 2012).Several approaches exist to detect contamination in mtDNA sequencing studies. We and others previously showed that a contamination approach based on the coexistence of phylogenetically incompatible mitochondrial haplotypes observable as polymorphic sites is feasible (Li et al. 2010, 2015; Avital et al. 2012; Weissensteiner et al. 2016a). The method of Dickins et al. (2014) facilitates the check for contamination by building neighbor joining trees. Mixemt (Vohr et al. 2017) incorporates the mitochondrial phylogeny and estimates the most probable haplogroup for each sequence read. The implemented algorithm reveals advantages for contamination detection by detecting several haplotypes within one sample and is independent of variant frequencies. However, it is too computationally expensive when applied to thousands of samples. For ancient DNA studies, schmutzi (Renaud et al. 2015) uses sequence deamination patterns and fragment-length distributions to estimate contamination. Additionally, specific laboratory protocols were designed for eliminating contamination, for example, double-barcode sequencing approaches (Yin et al. 2019).For contamination detection in mitochondrial studies, mostly DNA cross-contamination is investigated (Ding et al. 2015; Wei et al. 2019; Yuan et al. 2020) by applying VerifyBamID (Jun et al. 2012; Zhang et al. 2020). Nevertheless, it becomes apparent that a tool for mitochondrial studies that rapidly and accurately detects contamination in thousands of samples is still missing. Because mtDNA is also present hundredfold to several thousandfold per cell depending on the cell type, also WGS data sets specifically targeting the autosomal genome result in a high coverage over the mitochondrial genome.In this study, we systematically evaluate the approach of using the mtDNA phylogeny for contamination detection and present haplocheck, a tool to report contamination in mtDNA targeted sequencing and WGS studies. In general, haplocheck works by identifying polymorphic sites down to 1% within an input sample. By grouping polymorphic sites into haplotypes, haplocheck identifies contamination using the mitochondrial phylogeny and the concept of haplogroups. Overall, this work should show the merits of the mitochondrial genome as an instrument for additional quality control in sequencing studies. It additionally presents haplocheck, a fast and accurate tool that takes advantage of a solid well-known mitochondrial phylogeny for detecting contamination.     

Evidence of mitochondrial DNA diversity in South American aboriginals

The absence in South American aboriginals of an Asian-specific marker, a 9-bp deletion between the genes for the second subunit of cytochrome oxidase II and lysine transfer RNA in region V, has been interpreted as a bottlenecking effect at the Isthmus of Panama during the peopling of the Americas. We screened mitochondrial DNA (mtDNA) for this 9-bp tandem repeat and for polymorphisms in specific regions of the mtDNA in 2 ancient and 31 contemporary samples from South American aboriginals. We found additional (mtDNA) diversity in South American aboriginals in three ways. First, an Asian-specific marker not previously reported in South American aboriginals was identified by a sequencing analysis in both the contemporary Andean and Amazonian aboriginal peoples. Second, two new haplotypes so far unique to South American aboriginals were found. Additionally, we show that South American aboriginals fall into discrete populations. These results suggest that the prehistoric colonization of South America is the outcome of multiple migrations; the data do not support a bottlenecking effect at the Isthmus of Panama.     

MtDNA from extinct Tainos and the peopling of the Caribbean

Tainos and Caribs were the inhabitants of the Caribbean when Columbus reached the Americas; both human groups became extinct soon after contact, decimated by the Spaniards and the diseases they brought. Samples belonging to pre-Columbian Taino Indians from the La Caleta site (Dominican Republic) have been analyzed, in order to ascertain the genetic affinities of these groups in relation to present-day Amerinds, and to reconstruct the genetic and demographic events that took place during the peopling of the Caribbean.
Twenty-seven bone samples were extracted and analyzed for mtDNA variation. The four major Amerindian mtDNA lineages were screened through amplification of the specific marker regions and restriction enzymatic digestion, when needed. The HVRI of the control region was amplified with four sets of overlapping primers and sequenced in 19 of the samples. Both restriction enzyme and sequencing results suggest that only two (C and D) of the major mtDNA lineages were present in the sample: 18 individuals (75%) belonged to the C haplogroup, and 6 (25%) to the D haplogroup. Sequences display specific substitutions that are known to correlate with each haplogroup, a fact that helped to reject the possibility of European DNA contamination. A low rate of Taq misincorporations due to template damage was estimated from the cloning and sequencing of different PCR products of one of the samples. High frequencies of C and D haplogroups are more common in South American populations, a fact that points to that sub-continent as the homeland of the Taino ancestors, as previously suggested by linguistic and archaeological evidence. Sequence and haplogroup data show that the Tainos had a substantially reduced mtDNA diversity, which is indicative of an important founder effect during the colonization of the Caribbean Islands, assumed to have been a linear migratory movement from mainland South America following the chain configuration of the Antilles.     

The Etruscan timeline: a recent Anatolian connection

The origin of the Etruscans (the present day Tuscany, Italy), one of the most enigmatic non-Indo-European civilizations, is under intense controversy. We found novel genetic evidences on the mitochondrial DNA (mtDNA) establishing a genetic link between Anatolia and the ancient Etruria. By way of complete mtDNA genome sequencing of a novel autochthonous Tuscan branch of haplogroup U7 (namely U7a2a), we have estimated an historical time frame for the arrival of Anatolian lineages to Tuscany ranging from 1.1±0.1 to 2.3±0.4 kya B.P.     

True single-molecule DNA sequencing of a pleistocene horse bone

Second-generation sequencing platforms have revolutionized the field of ancient DNA, opening access to complete genomes of past individuals and extinct species. However, these platforms are dependent on library construction and amplification steps that may result in sequences that do not reflect the original DNA template composition. This is particularly true for ancient DNA, where templates have undergone extensive damage post-mortem. Here, we report the results of the first "true single molecule sequencing" of ancient DNA. We generated 115.9 Mb and 76.9 Mb of DNA sequences from a permafrost-preserved Pleistocene horse bone using the Helicos HeliScope and Illumina GAIIx platforms, respectively. We find that the percentage of endogenous DNA sequences derived from the horse is higher among the Helicos data than Illumina data. This result indicates that the molecular biology tools used to generate sequencing libraries of ancient DNA molecules, as required for second-generation sequencing, introduce biases into the data that reduce the efficiency of the sequencing process and limit our ability to fully explore the molecular complexity of ancient DNA extracts. We demonstrate that simple modifications to the standard Helicos DNA template preparation protocol further increase the proportion of horse DNA for this sample by threefold. Comparison of Helicos-specific biases and sequence errors in modern DNA with those in ancient DNA also reveals extensive cytosine deamination damage at the 3' ends of ancient templates, indicating the presence of 3'-sequence overhangs. Our results suggest that paleogenomes could be sequenced in an unprecedented manner by combining current second- and third-generation sequencing approaches.     

NDUFS1基因复合杂合变异致线粒体呼吸链复合物Ⅰ缺陷一家系

目的:探讨1个疑似线粒体病家系的遗传学病因。方法:收集患者及其家系成员的临床资料;抽取家系成员外周血,应用二代测序进行家系全外显子组、基因组拷贝数变异和线粒体基因组检测,对候选基因变异位点进行Sanger测序验证。结果:全外显子组家系检测发现患儿存在 NDUFS1基因父源c.64C>T(p.R22X)和母源...     

Temporal Mitochondrial DNA Variation in the Basque Country: Influence of Post-Neolithic Events

The Basque population has been considered an outlier in a large number of genetic studies, due to its hypothesized antiquity and greater genetic isolation. The present paper deals with an analysis of the mtDNA variability of the historical population of Aldaieta (VI–VII c. AD; Basque Country) which, together with genetic data existing for other prehistoric populations of the Basque Country (4,500–5,000 YBP), permits an appraisal of the hypotheses proposed for the origin of the genetic differentiation of the Basque population. Given that this is an aDNA study, application has been made both of standard precautions, to avoid contamination, and of authentication criteria (analysis of duplicates, replication in an independent laboratory, quantification of target DNA, sequencing and cloning of PCR products). The variability of the mtDNA haplogroups of the historical population of Aldaieta falls within the range of the present‐day populations of Europe's Atlantic fringe, whereas the prehistoric populations of the Basque Country display clear differentiation in relation to all others. Consequently, we suggest that between 5,000–1,500 YBP approximately, there may have been gene flow amongst the western European populations that homogenised mtDNA lineages.     

Genetic features of ancient West Siberian people of the Middle Ages, revealed by mitochondrial DNA haplogroup analysis

In order to investigate the genetic features of ancient West Siberian people of the Middle Ages, we studied ancient DNA from bone remains excavated from two archeological sites in West Siberia: Saigatinsky 6 (eighth to eleventh centuries) and Zeleny Yar (thirteenth century). Polymerase chain reaction amplification and nucleotide sequencing of mitochondrial DNA (mtDNA) succeeded for 9 of 67 specimens examined, and the sequences were assigned to mtDNA haplogroups B4, C4, G2, H and U. This distribution pattern of mtDNA haplogroups in medieval West Siberian people was similar to those previously reported in modern populations living in West Siberia, such as the Mansi, Ket and Nganasan. Exact tests of population differentiation showed no significant differences between the medieval people and modern populations in West Siberia. The findings suggest that some medieval West Siberian people analyzed in the present study are included in direct ancestral lineages of modern populations native to West Siberia.     

High-throughput sequencing of complete human mtDNA genomes from the Philippines

Because of the time and cost associated with Sanger sequencing of complete human mtDNA genomes, practically all evolutionary studies have screened samples first to define haplogroups and then either selected a few samples from each haplogroup, or many samples from a particular haplogroup of interest, for complete mtDNA genome sequencing. Such biased sampling precludes many analyses of interest. Here, we used high-throughput sequencing platforms to generate, rapidly and inexpensively, 109 complete mtDNA genome sequences from random samples of individuals from three Filipino groups, including one Negrito group, the Mamanwa. We obtained on average ～55-fold coverage per sequence, with <1% missing data per sequence. Various analyses attest to the accuracy of the sequences, including comparison to sequences of the first hypervariable segment of the control region generated by Sanger sequencing; patterns of nucleotide substitution and the distribution of polymorphic sites across the genome; and the observed haplogroups. Bayesian skyline plots of population size change through time indicate similar patterns for all three Filipino groups, but sharply contrast with such plots previously constructed from biased sampling of complete mtDNA genomes, as well as with an artificially constructed sample of sequences that mimics the biased sampling. Our results clearly demonstrate that the high-throughput sequencing platforms are the methodology of choice for generating complete mtDNA genome sequences.     

Multiple founder haplotypes of mitochondrial DNA in Amerindians revealed by RFLP and sequencing

The mitochondrial DNA (mtDNA) of 139 individuals from eight tribes which belong to four linguistic groups of the Brazilian Amazon Region was studied both by RFLP and by sequencing of the D-loop region. RFLP analysis showed that 41 haplotypes (29%) belonged to haplogroup A, 39 (28%) to haplogroup B, 38 (27%) to haplogroup C., 19 (14%) to haplogroup D, and 2 (<2%) could not be assigned to any of the four haplogroups. Among the 92 individuals analyzed by direct sequencing of the D-loop region, we observed 43 different haplotypes defined by 48 polymorphic points, while one haplotype could not be assigned to any of the clusters previously described. Joint analysis of data obtained by RFLP and by sequencing of mtDNA demonstrated that, regardless of the method of analysis, the mtDNA haplotypes of contemporary Amerindians cluster into four groups, similar to those previously described, even though 7% of the total sample or 12% of the haplotypes have discrepancies between results obtained by RFLP and sequencing. In addition to supporting the prevalence of four major haplogroups among contemporary Amerindians, our data are compatible with multiple founder haplotypes in each haplogroup, based on: i) a high prevalence of unusual haplotypes; ii) presence of multiple polymorphic sites shared by different haplogroups; iii) relative differences in nucleotide diversity based on RFLP or sequencing within the different haplogroups.     

Selective enrichment of damaged DNA molecules for ancient genome sequencing

Contamination by present-day human and microbial DNA is one of the major hindrances for large-scale genomic studies using ancient biological material. We describe a new molecular method, U selection, which exploits one of the most distinctive features of ancient DNA—the presence of deoxyuracils—for selective enrichment of endogenous DNA against a complex background of contamination during DNA library preparation. By applying the method to Neanderthal DNA extracts that are heavily contaminated with present-day human DNA, we show that the fraction of useful sequence information increases ∼10-fold and that the resulting sequences are more efficiently depleted of human contamination than when using purely computational approaches. Furthermore, we show that U selection can lead to a four- to fivefold increase in the proportion of endogenous DNA sequences relative to those of microbial contaminants in some samples. U selection may thus help to lower the costs for ancient genome sequencing of nonhuman samples also.High-throughput DNA sequencing and the shift to library-based sample preparation techniques have greatly facilitated genetic research on human evolution in recent years. Increasing amounts of sequence data are becoming available not only from present-day humans but also from ancient human remains, helping to uncover the evolutionary histories of present-day human populations as well as their relationship to extinct archaic groups (Stoneking and Krause 2011). In ancient DNA studies, the most accessible target is mitochondrial (mt) DNA, which is present in several hundreds of copies per cell as opposed to the diploid-only nuclear genome. Consequently, complete mtDNA genomes have been sequenced from more than a dozen remains of ancient modern humans (Ermini et al. 2008; Gilbert et al. 2008; Green et al. 2010; Fu et al. 2013b; Raghavan et al. 2014) as well as representatives of two archaic hominin groups that went extinct during the Late Pleistocene, the Neanderthals (Green et al. 2008; Briggs et al. 2009; Prüfer et al. 2014), and Denisovans (Krause et al. 2010b; Reich et al. 2010). The recent recovery of mitochondrial sequences from an ∼400,000 yr-old hominin from Sima de los Huesos in Spain indicates that more comprehensive sequence data may soon become available even from Middle Pleistocene remains (Meyer et al. 2014). Retrieving nuclear sequences from ancient human material is generally more challenging, but such data have also been generated at various scales, ranging from a few megabases of sequences to full genome sequences determined with high accuracy (see Shapiro and Hofreiter 2014 for a recent summary).Despite these advances, sequencing ancient human DNA continues to be challenging for several reasons. First, only trace amounts of highly fragmented DNA are usually preserved in ancient bones and teeth, imposing limits on the number of sequences that can be recovered from ancient specimens. Second, DNA extracted from ancient material is in many cases dominated by microbial DNA, which often contributes to 99% or more of the sequences, making direct shotgun sequencing economically infeasible. This problem can be partially overcome by hybridization enrichment of hominin sequences, such as those of a small chromosome (Fu et al. 2013a) or, as recently proposed, of sequences from throughout the whole genome (Carpenter et al. 2013). Another approach is restriction digestion of GC-rich sequence motifs, which was performed to change the ratio between endogenous and microbial library molecules in the first study of the Neanderthal genome (Green et al. 2010). A third and particularly severe problem for working with ancient human samples is present-day human contamination, which is inevitably introduced during excavation and laboratory work. Fortunately, a solid framework for validating the authenticity of ancient human sequences can be established using the distinct pattern of substitutions caused by cytosine deamination in ancient DNA sequences (Hofreiter et al. 2001; Briggs et al. 2007). The deamination product of cytosine is uracil, which is read as thymine by most DNA polymerases. Resulting C to T substitutions (or G to A substitutions, depending on the orientation in which DNA strands are sequenced and the method used to prepare DNA libraries) are particularly frequent at the 5′ and 3′ ends of sequences due to the higher rate of cytosine deamination in single-stranded overhangs (Lindahl 1993). Importantly, the frequency of deamination-induced substitutions correlates with sample age (Sawyer et al. 2012) and is low in present-day human contamination (Krause et al. 2010a). These substitutions can thus be taken as evidence for the presence of authentic ancient human sequences. Deamination-induced substitutions have also been exploited for separating ancient sequences from present-day human contamination in silico (Skoglund et al. 2012, 2014a,b; Meyer et al. 2014; Raghavan et al. 2014). Although effective in principle, this approach is costly, because a large proportion of sequence data is excluded from downstream analysis. Furthermore, ancient DNA base damage is not determined directly and may occasionally be confounded with evolutionary sequence differences.Here we describe a novel laboratory technique, uracil selection (“U selection”), which enables physical separation of uracil-containing DNA strands from nondeaminated strands at the stage of DNA library preparation. Our method builds on a single-stranded library preparation method, which has been shown to be particularly efficient for retrieving sequences from highly degraded ancient DNA (Meyer et al. 2012; Gansauge and Meyer 2013). We apply U selection to several Neanderthal DNA extracts and show that it is a powerful tool for enriching Neanderthal DNA sequences against a background of present-day human contamination. We also report cases where U selection drastically increases the proportion of Neanderthal DNA relative to microbial DNA.     

Specific quantification of human genomes from low copy number DNA samples in forensic and ancient DNA studies

We reviewed the current methodologies used for human DNA quantitation in forensic and ancient DNA studies, including sensitive hybridization methods based on the detection of nuclear alpha-satellite repetitive DNA regions or more recently developed fluorogenic real-time polymerase chain reaction (PCR) designs for the detection of both nuclear and mitochondrial DNA regions. Special emphasis has been put on the applicability of recently described different real-time PCR designs targeting different fragments of the HV1 mtDNA control region, and a segment of the X-Y homologous amelogenin gene. The importance of these quantitative assays is to ensure the consistency of low copy number DNA typing (STR profiling and mtDNA sequencing).     

More evidence for non-maternal inheritance of mitochondrial DNA?

Background: A single case of paternal co-transmission of mitochondrial DNA (mtDNA) in humans has been reported so far. Objective: To find potential instances of non-maternal inheritance of mtDNA. Methods: Published medical case studies (of single patients) were searched for irregular mtDNA patterns by comparing the given haplotype information for different clones or tissues with the worldwide mtDNA database as known to date—a method that has proved robust and reliable for the detection of flawed mtDNA sequence data. Results: More than 20 studies were found reporting clear cut instances with mtDNAs of different ancestries in single individuals. As examples, cases are reviewed from recent published reports which, at face value, may be taken as evidence for paternal inheritance of mtDNA or recombination. Conclusions: Multiple types (or recombinant types) of quite dissimilar mitochondrial DNA from different parts of the known mtDNA phylogeny are often reported in single individuals. From re-analyses and corrigenda of forensic mtDNA data, it is apparent that the phenomenon of mixed or mosaic mtDNA can be ascribed solely to contamination and sample mix up.     

Updating the East Asian mtDNA phylogeny: a prerequisite for the identification of pathogenic mutations

Knowledge about the world phylogeny of human mitochondrial DNA (mtDNA) is essential not only for evaluating the pathogenic role of specific mtDNA mutations but also for performing reliable association studies between mtDNA haplogroups and complex disorders. In the past few years, the main features of the East Asian portion of the mtDNA phylogeny have been determined on the basis of complete sequencing efforts, but representatives of several basal lineages were still lacking. Moreover, some recently published complete mtDNA sequences did apparently not fit into the known phylogenetic tree and conflicted with the established nomenclature. To refine the East Asian mtDNA tree and resolve data conflicts, we first completely sequenced 20 carefully selected mtDNAs--likely representatives of novel sub-haplogroups--and then, in order to distinguish diagnostic mutations of novel haplogroups from private variants, we applied a 'motif-search' procedure to a large sample collection. The novel information was incorporated into an updated East Asian mtDNA tree encompassing more than 1000 (near-) complete mtDNA sequences. A reassessment of the mtDNA data from a series of disease studies testified to the usefulness of such a refined mtDNA tree in evaluating the pathogenicity of mtDNA mutations. In particular, the claimed pathogenic role of mutations G3316A, T3394C, A4833G and G15497A appears to be most questionable as those initial claims were derived from anecdotal findings rather than e.g. appropriate association studies. Following a guideline based on the phylogenetic knowledge as proposed here could help avoiding similar problems in the future.     

Mitochondrial haplogroup C in ancient mitochondrial DNA from Ukraine extends the presence of East Eurasian genetic lineages in Neolithic Central and Eastern Europe

Recent studies of ancient mitochondrial DNA (mtDNA) lineages have revealed the presence of East Eurasian mtDNA haplogroups in the Central European Neolithic. Here we report the finding of East Eurasian lineages in ancient mtDNA from two Neolithic cemeteries of the North Pontic Region (NPR) in Ukraine. In our study, comprehensive haplotyping information was obtained for 7 out of 18 specimens. Although the majority of identified mtDNA haplogroups belonged to the traditional West Eurasian lineages of H and U, three specimens were determined to belong to the lineages of mtDNA haplogroup C. This find extends the presence of East Eurasian lineages in Neolithic Europe from the Carpathian Mountains to the northern shores of the Black Sea and provides the first genetic account of Neolithic mtDNA lineages from the NPR.     

唐氏综合征患儿线粒体基因组突变的分析

目的 研究唐氏综合征中线粒体DNA突变情况.方法 采用高通量测序和焦磷酸测序检测7个唐氏综合征(Down's syndrome,DS)家系中的患儿和母亲的线粒体基因组序列,分析线粒体基因组序列的变化情况.结果 ①DS患儿中检测到36个与其母亲中不同的线粒体DNA突变,其中14个位点是首次在唐氏综合征样本中发现;②36个线粒体DNA突变主要发生于D-Loop区和线粒体复合物Ⅰ中;③ 线粒体基因组13个编码基因中,有11个基因检测到线粒体DNA的突变;④ 焦磷酸测序对线粒体基因组杂合突变频率的检测结果和高通量测序结果吻合.结论 DS患儿中广泛存在线粒体DNA的突变,这些突变可能与唐氏综合征的线粒体功能异常相关.     

Low frequency of mtDNA point mutations in patients with PEO associated with POLG1 mutations

Mitochondrial myopathy in progressive external ophthalmoplegia (PEO) has been associated with POLG1 mutations. POLG1 encodes the catalytic alpha subunit of polymerase gamma and is the only polymerase known to be involved in mtDNA replication. It has two functionally different domains, one polymerase domain and one exonuclease domain with proofreading activity. In this study we have investigated whether mtDNA point mutations are involved, directly or indirectly, in the pathogenesis of PEO. Muscle biopsy specimens from patients with POLG1 mutations, affecting either the exonuclease or the polymerase domain, were investigated. Single cytochrome c oxidase (COX)-deficient muscle fibers were dissected and screened for clonally expanded mtDNA point mutations using a sensitive denaturing gradient gel electrophoresis analysis, in which three different regions of mtDNA, including five different tRNA genes, were investigated. To screen for randomly distributed mtDNA point mutations in muscle, two regions of mtDNA including deletion breakpoints were investigated by high-fidelity PCR, followed by cloning and sequencing. Long-range PCR revealed multiple mtDNA deletions in all the patients but not the controls. No point mutations were identified in single COX-deficient muscle fibers. Cloning and sequencing of muscle homogenate identified randomly distributed point mutations at very low frequency in patients and controls (<1:50 000). We conclude that mtDNA point mutations do not appear to be directly or indirectly involved in the pathogenesis of mitochondrial disease in patients with different POLG1 mutations.