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Concept: Nuclear DNA

249

Ancient DNA is revealing new insights into the genetic relationship between Pleistocene hominins and modern humans. Nuclear DNA indicated Neanderthals as a sister group of Denisovans after diverging from modern humans. However, the closer affinity of the Neanderthal mitochondrial DNA (mtDNA) to modern humans than Denisovans has recently been suggested as the result of gene flow from an African source into Neanderthals before 100,000 years ago. Here we report the complete mtDNA of an archaic femur from the Hohlenstein-Stadel (HST) cave in southwestern Germany. HST carries the deepest divergent mtDNA lineage that splits from other Neanderthals ∼270,000 years ago, providing a lower boundary for the time of the putative mtDNA introgression event. We demonstrate that a complete Neanderthal mtDNA replacement is feasible over this time interval even with minimal hominin introgression. The highly divergent HST branch is indicative of greater mtDNA diversity during the Middle Pleistocene than in later periods.

Concepts: DNA, Human, Mitochondrion, Mitochondrial DNA, Neanderthal, Human evolution, Pleistocene, Nuclear DNA

165

Denisovans, a sister group of Neandertals, have been described on the basis of a nuclear genome sequence from a finger phalanx (Denisova 3) found in Denisova Cave in the Altai Mountains. The only other Denisovan specimen described to date is a molar (Denisova 4) found at the same site. This tooth carries a mtDNA sequence similar to that of Denisova 3. Here we present nuclear DNA sequences from Denisova 4 and a morphological description, as well as mitochondrial and nuclear DNA sequence data, from another molar (Denisova 8) found in Denisova Cave in 2010. This new molar is similar to Denisova 4 in being very large and lacking traits typical of Neandertals and modern humans. Nuclear DNA sequences from the two molars form a clade with Denisova 3. The mtDNA of Denisova 8 is more diverged and has accumulated fewer substitutions than the mtDNAs of the other two specimens, suggesting Denisovans were present in the region over an extended period. The nuclear DNA sequence diversity among the three Denisovans is comparable to that among six Neandertals, but lower than that among present-day humans.

Concepts: DNA, Human, Human genome, Genome, Mitochondrion, Mitochondrial DNA, DNA sequencing, Nuclear DNA

97

High throughput sequencing (HTS) has been used for a number of years in the field of paleogenomics to facilitate the recovery of small DNA fragments from ancient specimens. Recently, these techniques have also been applied in forensics, where they have been used for the recovery of mitochondrial DNA sequences from samples where traditional PCR-based assays fail because of the very short length of endogenous DNA molecules. Here, we describe the biological sexing of a ~4000-year-old Egyptian mummy using shotgun sequencing and two established methods of biological sex determination (RXand RY), by way of mitochondrial genome analysis as a means of sequence data authentication. This particular case of historical interest increases the potential utility of HTS techniques for forensic purposes by demonstrating that data from the more discriminatory nuclear genome can be recovered from the most damaged specimens, even in cases where mitochondrial DNA cannot be recovered with current PCR-based forensic technologies. Although additional work remains to be done before nuclear DNA recovered via these methods can be used routinely in operational casework for individual identification purposes, these results indicate substantial promise for the retrieval of probative individually identifying DNA data from the most limited and degraded forensic specimens.

Concepts: DNA, Molecular biology, Biology, Mitochondrion, Mitochondrial DNA, DNA sequencing, Sequence, Nuclear DNA

42

A unique assemblage of 28 hominin individuals, found in Sima de los Huesos in the Sierra de Atapuerca in Spain, has recently been dated to approximately 430,000 years ago. An interesting question is how these Middle Pleistocene hominins were related to those who lived in the Late Pleistocene epoch, in particular to Neanderthals in western Eurasia and to Denisovans, a sister group of Neanderthals so far known only from southern Siberia. While the Sima de los Huesos hominins share some derived morphological features with Neanderthals, the mitochondrial genome retrieved from one individual from Sima de los Huesos is more closely related to the mitochondrial DNA of Denisovans than to that of Neanderthals. However, since the mitochondrial DNA does not reveal the full picture of relationships among populations, we have investigated DNA preservation in several individuals found at Sima de los Huesos. Here we recover nuclear DNA sequences from two specimens, which show that the Sima de los Huesos hominins were related to Neanderthals rather than to Denisovans, indicating that the population divergence between Neanderthals and Denisovans predates 430,000 years ago. A mitochondrial DNA recovered from one of the specimens shares the previously described relationship to Denisovan mitochondrial DNAs, suggesting, among other possibilities, that the mitochondrial DNA gene pool of Neanderthals turned over later in their history.

Concepts: DNA, Gene, Human, Biology, Mitochondrion, Mitochondrial DNA, Pleistocene, Nuclear DNA

35

Claims of extreme survival of DNA have emphasized the need for reliable models of DNA degradation through time. By analysing mitochondrial DNA (mtDNA) from 158 radiocarbon-dated bones of the extinct New Zealand moa, we confirm empirically a long-hypothesized exponential decay relationship. The average DNA half-life within this geographically constrained fossil assemblage was estimated to be 521 years for a 242 bp mtDNA sequence, corresponding to a per nucleotide fragmentation rate (k) of 5.50 × 10(-6) per year. With an effective burial temperature of 13.1°C, the rate is almost 400 times slower than predicted from published kinetic data of in vitro DNA depurination at pH 5. Although best described by an exponential model (R(2) = 0.39), considerable sample-to-sample variance in DNA preservation could not be accounted for by geologic age. This variation likely derives from differences in taphonomy and bone diagenesis, which have confounded previous, less spatially constrained attempts to study DNA decay kinetics. Lastly, by calculating DNA fragmentation rates on Illumina HiSeq data, we show that nuclear DNA has degraded at least twice as fast as mtDNA. These results provide a baseline for predicting long-term DNA survival in bone.

Concepts: DNA, Mitochondrion, Mitochondrial DNA, Radioactive decay, Fossil, Exponential growth, Nuclear DNA, Moa

13

Determining the levels of human mitochondrial heteroplasmy is of utmost importance in several fields. In spite of this, there are currently few published works that have focused on this issue. In order to increase the knowledge of mitochondrial DNA (mtDNA) heteroplasmy, the main goal of this work is to investigate the frequency and the mutational spectrum of heteroplasmy in the human mtDNA genome. To address this, a set of nine primer pairs designed to avoid co-amplification of nuclear DNA (nDNA) sequences of mitochondrial origin (NUMTs) was used to amplify the mitochondrial genome in 101 individuals. The analysed individuals represent a collection with a balanced representation of genders and mtDNA haplogroup distribution, similar to that of a Western European population. The results show that the frequency of heteroplasmic individuals exceeds 61%. The frequency of point heteroplasmy is 28.7%, with a widespread distribution across the entire mtDNA. In addition, an excess of transitions in heteroplasmy were detected, suggesting that genetic drift and/or selection may be acting to reduce its frequency at population level. In fact, heteroplasmy at highly stable positions might have a greater impact on the viability of mitochondria, suggesting that purifying selection must be operating to prevent their fixation within individuals. This study analyses the frequency of heteroplasmy in a healthy population, carrying out an evolutionary analysis of the detected changes and providing a new perspective with important consequences in medical, evolutionary and forensic fields.

Concepts: DNA, Mitochondrion, Mitochondrial DNA, Population genetics, Human mitochondrial genetics, Mitochondrial disease, Nuclear DNA, Paternal mtDNA transmission

7

Mitochondrial DNA (mtDNA) typing can be a useful aid for identifying people from compromised samples when nuclear DNA is too damaged, degraded or below detection thresholds for routine short tandem repeat (STR)-based analysis. Standard mtDNA typing, focused on PCR amplicon sequencing of the control region (HVS I and HVS II), is limited by the resolving power of this short sequence, which misses up to 70% of the variation present in the mtDNA genome.

Concepts: DNA, Bacteria, Human genome, Virus, Mitochondrion, Mitochondrial DNA, DNA polymerase, Nuclear DNA

6

Gut microbial communities are key mediators of health and disease and have the capacity to drive the pathogenesis of diverse complex diseases including metabolic and chronic inflammatory diseases as well as aging. Host genetics is also a major determinant of disease phenotypes, whereby two different genomes play a role, the nuclear (nDNA)- and mitochondrial genome (mtDNA). We investigated the impact of mutations in mtDNA on the gut microbiota using conplastic mouse strains exhibiting distinct mutations in their mtDNA on an identical nDNA. Each of three strain tested harbors a distinct gut microbiota, ranging from differences at the phylum- to operational taxonomic units level. The C57BL/6J-mt (FVB/NJ) strain, carrying a mutation in the mitochondrial ATP8 synthase gene, exhibits higher Firmicutes abundance than Bacteroidetes, indicating a possible indicative for metabolic dysfunctions. In line with this, the C57BL/6J-mt (FVB/NJ) displays a variety of different phenotypes, including increased susceptibility to metabolic-related and inflammatory disorders. Furthermore, we discuss the cross-talk between mitochondrial genome/mitochondria and commensal microbiota in relation to clinical phenotypes. In summary, we demonstrate that mutations in mtDNA lead to significant differences in the composition of gut microbial communities in mice. Such differences may facilitate the emergence of metabolic disease and therefore constitute potential therapeutic targets.

Concepts: DNA, Gene, Genetics, Bacteria, Gut flora, Mitochondrion, Mitochondrial DNA, Nuclear DNA

4

PurposeDiagnosing primary mitochondrial diseases (MDs) is challenging in clinical practice. The mitochondrial disease criteria (MDC) have been developed to quantify the clinical picture and evaluate the probability of an underlying MD and the need for a muscle biopsy. In this new genetic era with next-generation sequencing in routine practice, we aim to validate the diagnostic value of MDC.MethodsWe retrospectively studied MDC in a multicenter cohort of genetically confirmed primary MD patients.ResultsWe studied 136 patients (61 male, 91 nuclear DNA (nDNA) mutations). Forty-five patients (33%) had probable MD and 69 (51%) had definite MD according to the MDC. A muscle biopsy was performed in 63 patients (47%). Patients with nDNA mutations versus mitochondrial DNA mutations were younger (6.4 ± 9.7 versus 19.5 ± 17.3 y) and had higher MDC (7.07 ± 1.12/8 versus 5.69 ± 1.94/8). At a cutoff of 6.5/8, the sensitivity to diagnose patients with nDNA mutations is 72.5% with a positive predictive value of 69.5%. In the nDNA mutation group, whole-exome sequencing could diagnose patients with lower scores (MDC (6.84 ± 1.51/8) compared to Sanger sequencing MDC (7.44 ± 1.13/8, P = 0.025)). Moreover 7/8 patients diagnosed with possible MD by MDC were diagnosed by whole-exome sequencing.ConclusionMDC remain very useful in the clinical diagnosis of MD, in interpreting whole-exome results and deciding on the need for performing muscle biopsy.

Concepts: DNA, Genetics, Mutation, Mitochondrion, Mitochondrial DNA, Diagnosis, Nuclear DNA

4

DNA sequencing identifies common and rare genetic variants for association studies, but studies typically focus on variants in nuclear DNA and ignore the mitochondrial genome. In fact, analyzing variants in mitochondrial DNA (mtDNA) sequences presents special problems, which we resolve here with a general solution for the analysis of mtDNA in next-generation sequencing studies. The new program package comprises 1) an algorithm designed to identify mtDNA variants (i.e., homoplasmies and heteroplasmies), incorporating sequencing error rates at each base in a likelihood calculation and allowing allele fractions at a variant site to differ across individuals; and 2) an estimation of mtDNA copy number in a cell directly from whole-genome sequencing data. We also apply the methods to DNA sequence from lymphocytes of ~2,000 SardiNIA Project participants. As expected, mothers and offspring share all homoplasmies but a lesser proportion of heteroplasmies. Both homoplasmies and heteroplasmies show 5-fold higher transition/transversion ratios than variants in nuclear DNA. Also, heteroplasmy increases with age, though on average only ~1 heteroplasmy reaches the 4% level between ages 20 and 90. In addition, we find that mtDNA copy number averages ~110 copies/lymphocyte and is ~54% heritable, implying substantial genetic regulation of the level of mtDNA. Copy numbers also decrease modestly but significantly with age, and females on average have significantly more copies than males. The mtDNA copy numbers are significantly associated with waist circumference (p-value = 0.0031) and waist-hip ratio (p-value = 2.4×10-5), but not with body mass index, indicating an association with central fat distribution. To our knowledge, this is the largest population analysis to date of mtDNA dynamics, revealing the age-imposed increase in heteroplasmy, the relatively high heritability of copy number, and the association of copy number with metabolic traits.

Concepts: DNA, Gene, Genetics, Mitochondrion, Mitochondrial DNA, Body mass index, Nuclear DNA, Paternal mtDNA transmission