Mitochondrial DNA polymorphism A4917G is independently associated with age-related macular degeneration.

The objective of this study was to determine if MTND2*LHON4917G (4917G), a specific non-synonymous polymorphism in the mitochondrial genome previously associated with neurodegenerative phenotypes, is associated with increased risk for age-related macular degeneration (AMD). A preliminary study of 393 individuals (293 cases and 100 controls) ascertained at Vanderbilt revealed an increased occurrence of 4917G in cases compared to controls (15.4% vs.9.0%, p = 0.11). Since there was a significant age difference between cases and controls in this initial analysis, we extended the study by selecting Caucasian pairs matched at the exact age at examination. From the 1547 individuals in the Vanderbilt/Duke AMD population association study (including 157 in the preliminary study), we were able to match 560 (280 cases and 280 unaffected) on exact age at examination. This study population was genotyped for 4917G plus specific AMD-associated nuclear genome polymorphisms in CFH, LOC387715 and ApoE. Following adjustment for the listed nuclear genome polymorphisms, 4917G independently predicts the presence of AMD (OR = 2.16, 95%CI 1.20-3.91, p = 0.01). In conclusion, a specific mitochondrial polymorphism previously implicated in other neurodegenerative phenotypes (4917G) appears to convey risk for AMD independent of recently discovered nuclear DNA polymorphisms.

Later Life Consequences of Developmental Mitochondrial DNA Damage in C. elegans

Mitochondria are responsible for producing the vast majority of cellular ATP, and are therefore critical to organismal health [1]. They contain thir own genomes (mtDNA) which encode 13 proteins that are all subunits of the mitochondrial respiratory chain (MRC) and are essential for oxidative phosphorylation [2]. mtDNA is present in multiple copies per cell, usually between 103 and 104 , though this number is reduced during certain developmental stages [3, 4]. The health of the mitochondrial genome is also important to the health of the organism, as mutations in mtDNA lead to human diseases that collectively affect approximately 1 in 4000 people [5, 6]. mtDNA is more susceptible than nuclear DNA (nucDNA) to damage by many environmental pollutants, for reasons including the absence of Nucleotide Excision Repair (NER) in the mitochondria [7]. NER is a highly functionally conserved DNA repair pathway that removes bulky, helix distorting lesions such as those caused by ultraviolet C (UVC) radiation and also many environmental toxicants, including benzo[a]pyrene (BaP) [8]. While these lesions cannot be repaired, they are slowly removed through a process that involves mitochondrial dynamics and autophagy [9, 10]. However, when present during development in C. elegans, this damage reduces mtDNA copy number and ATP levels [11]. We hypothesize that this damage, when present during development, will result in mitochondrial dysfunction and increase the potential for adverse outcomes later in life.

To test this hypothesis, 1st larval stage (L1) C. elegans are exposed to 3 doses of 7.5J/m2 ultraviolet C radiation 24 hours apart, leading to the accumulation of mtDNA damage [9, 11]. After exposure, many mitochondrial endpoints are assessed at multiple time points later in life. mtDNA and nucDNA damage levels and genome copy numbers are measured via QPCR and real-time PCR , respectively, every 2 day for 10 days. Steady state ATP levels are measured via luciferase expressing reporter strains and traditional ATP extraction methods. Oxygen consumption is measured using a Seahorse XFe24 extra cellular flux analyzer. Gene expression changes are measured via real time PCR and targeted metabolomics via LC-MS are used to investigate changes in organic acid, amino acid and acyl-carnitine levels. Lastly, nematode developmental delay is assessed as growth, and measured via imaging and COPAS biosort.

I have found that despite being removed, UVC induced mtDNA damage during development leads to persistent deficits in energy production later in life. mtDNA copy number is permanently reduced, as are ATP levels, though oxygen consumption is increased, indicating inefficient or uncoupled respiration. Metabolomic data and mutant sensitivity indicate a role for NADPH and oxidative stress in these results, and exposed nematodes are more sensitive to the mitochondrial poison rotenone later in life. These results fit with the developmental origin of health and disease hypothesis, and show the potential for environmental exposures to have lasting effects on mitochondrial function.

Lastly, we are currently working to investigate the potential for irreparable mtDNA lesions to drive mutagenesis in mtDNA. Mutations in mtDNA lead to a wide range of diseases, yet we currently do not understand the environmental component of what causes them. In vitro evidence suggests that UVC induced thymine dimers can be mutagenic [12]. We are using duplex sequencing of C. elegans mtDNA to determine mutation rates in nematodes exposed to our serial UVC protocol. Furthermore, by including mutant strains deficient in mitochondrial fission and mitophagy, we hope to determine if deficiencies in these processes will further increase mtDNA mutation rates, as they are implicated in human diseases.

PCR-Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage.

Because of the role that DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit, we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays.

Mitochondrial DNA damage induced autophagy, cell death, and disease.

Mammalian mitochondria contain multiple small genomes. While these organelles have efficient base excision removal of oxidative DNA lesions and alkylation damage, many DNA repair systems that work on nuclear DNA damage are not active in mitochondria. What is the fate of DNA damage in the mitochondria that cannot be repaired or that overwhelms the repair system? Some forms of mitochondrial DNA damage can apparently trigger mitochondrial DNA destruction, either via direct degradation or through specific forms of autophagy, such as mitophagy. However, accumulation of certain types of mitochondrial damage, in the absence of DNA ligase III (Lig3) or exonuclease G (EXOG), can directly trigger cell death. This review examines the cellular effects of persistent damage to mitochondrial genomes and discusses the very different cell fates that occur in response to different kinds of damage.

Are the native giant tortoises from the Seychelles really extinct? A genetic perspective based on mtDNA and microsatellite data.

The extinction of the giant tortoises of the Seychelles Archipelago has long been suspected but is not beyond doubt. A recent morphological study of the giant tortoises of the western Indian Ocean concluded that specimens of two native Seychelles species survive in captivity today alongside giant tortoises of Aldabra, which are numerous in zoos as well as in the wild. This claim has been controversial because some of the morphological characters used to identify these species, several measures of carapace morphology, are reputed to be quite sensitive to captive conditions. Nonetheless, the potential survival of giant tortoise species previously thought extinct presents an exciting scenario for conservation. We used mitochondrial DNA sequences and nuclear microsatellites to examine the validity of the rediscovered species of Seychelles giant tortoises. Our results indicate that the morphotypes suspected to represent Seychelles species do not show levels of variation and genetic structuring consistent with long periods of reproductive isolation. We found no variation in the mitochondrial control region among 55 individuals examined and no genetic structuring in eight microsatellite loci, pointing to the survival of just a single lineage of Indian Ocean tortoises.

Binding of MetJ repressor to specific and nonspecific DNA and effect of S-adenosylmethionine on these interactions.

We have used analytical ultracentrifugation to characterize the binding of the methionine repressor protein, MetJ, to synthetic oligonucleotides containing zero to five specific recognition sites, called metboxes. For all lengths of DNA studied, MetJ binds more tightly to repeats of the consensus sequence than to naturally occurring metboxes, which exhibit a variable number of deviations from the consensus. Strong cooperative binding occurs only in the presence of two or more tandem metboxes, which facilitate protein-protein contacts between adjacent MetJ dimers, but weak affinity is detected even with DNA containing zero or one metbox. The affinity of MetJ for all of the DNA sequences studied is enhanced by the addition of SAM, the known cofactor for MetJ in the cell. This effect extends to oligos containing zero or one metbox, both of which bind two MetJ dimers. In the presence of a large excess concentration of metbox DNA, the effect of cooperativity is to favor populations of DNA oligos bound by two or more MetJ dimers rather than a stochastic redistribution of the repressor onto all available metboxes. These results illustrate the dynamic range of binding affinity and repressor assembly that MetJ can exhibit with DNA and the effect of the corepressor SAM on binding to both specific and nonspecific DNA.

An Exploration into Fern Genome Space.

Ferns are one of the few remaining major clades of land plants for which a complete genome sequence is lacking. Knowledge of genome space in ferns will enable broad-scale comparative analyses of land plant genes and genomes, provide insights into genome evolution across green plants, and shed light on genetic and genomic features that characterize ferns, such as their high chromosome numbers and large genome sizes. As part of an initial exploration into fern genome space, we used a whole genome shotgun sequencing approach to obtain low-density coverage (∼0.4X to 2X) for six fern species from the Polypodiales (Ceratopteris, Pteridium, Polypodium, Cystopteris), Cyatheales (Plagiogyria), and Gleicheniales (Dipteris). We explore these data to characterize the proportion of the nuclear genome represented by repetitive sequences (including DNA transposons, retrotransposons, ribosomal DNA, and simple repeats) and protein-coding genes, and to extract chloroplast and mitochondrial genome sequences. Such initial sweeps of fern genomes can provide information useful for selecting a promising candidate fern species for whole genome sequencing. We also describe variation of genomic traits across our sample and highlight some differences and similarities in repeat structure between ferns and seed plants.

Bayesian variable selection in structured high-dimensional covariate spaces with applications in genomics

We consider the problem of variable selection in regression modeling in high-dimensional spaces where there is known structure among the covariates. This is an unconventional variable selection problem for two reasons: (1) The dimension of the covariate space is comparable, and often much larger, than the number of subjects in the study, and (2) the covariate space is highly structured, and in some cases it is desirable to incorporate this structural information in to the model building process. We approach this problem through the Bayesian variable selection framework, where we assume that the covariates lie on an undirected graph and formulate an Ising prior on the model space for incorporating structural information. Certain computational and statistical problems arise that are unique to such high-dimensional, structured settings, the most interesting being the phenomenon of phase transitions. We propose theoretical and computational schemes to mitigate these problems. We illustrate our methods on two different graph structures: the linear chain and the regular graph of degree k. Finally, we use our methods to study a specific application in genomics: the modeling of transcription factor binding sites in DNA sequences. © 2010 American Statistical Association.

The evolutionary origin of Indian Ocean tortoises (Dipsochelys).

Today, the only surviving wild population of giant tortoises in the Indian Ocean occurs on the island of Aldabra. However, giant tortoises once inhabited islands throughout the western Indian Ocean. Madagascar, Africa, and India have all been suggested as possible sources of colonization for these islands. To address the origin of Indian Ocean tortoises (Dipsochelys, formerly Geochelone gigantea), we sequenced the 12S, 16S, and cyt b genes of the mitochondrial DNA. Our phylogenetic analysis shows Dipsochelys to be embedded within the Malagasy lineage, providing evidence that Indian Ocean giant tortoises are derived from a common Malagasy ancestor. This result points to Madagascar as the source of colonization for western Indian Ocean islands by giant tortoises. Tortoises are known to survive long oceanic voyages by floating with ocean currents, and thus, currents flowing northward towards the Aldabra archipelago from the east coast of Madagascar would have provided means for the colonization of western Indian Ocean islands. Additionally, we found an accelerated rate of sequence evolution in the two Malagasy Pyxis species examined. This finding supports previous theories that shorter generation time and smaller body size are related to an increase in mitochondrial DNA substitution rate in vertebrates.

Working at the interface of phylogenetics and population genetics: a biogeographical analysis of Triaenops spp. (Chiroptera: Hipposideridae).

New applications of genetic data to questions of historical biogeography have revolutionized our understanding of how organisms have come to occupy their present distributions. Phylogenetic methods in combination with divergence time estimation can reveal biogeographical centres of origin, differentiate between hypotheses of vicariance and dispersal, and reveal the directionality of dispersal events. Despite their power, however, phylogenetic methods can sometimes yield patterns that are compatible with multiple, equally well-supported biogeographical hypotheses. In such cases, additional approaches must be integrated to differentiate among conflicting dispersal hypotheses. Here, we use a synthetic approach that draws upon the analytical strengths of coalescent and population genetic methods to augment phylogenetic analyses in order to assess the biogeographical history of Madagascar's Triaenops bats (Chiroptera: Hipposideridae). Phylogenetic analyses of mitochondrial DNA sequence data for Malagasy and east African Triaenops reveal a pattern that equally supports two competing hypotheses. While the phylogeny cannot determine whether Africa or Madagascar was the centre of origin for the species investigated, it serves as the essential backbone for the application of coalescent and population genetic methods. From the application of these methods, we conclude that a hypothesis of two independent but unidirectional dispersal events from Africa to Madagascar is best supported by the data.

Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation.

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.

Interactions of chromatin context, binding site sequence content, and sequence evolution in stress-induced p53 occupancy and transactivation.

Cellular stresses activate the tumor suppressor p53 protein leading to selective binding to DNA response elements (REs) and gene transactivation from a large pool of potential p53 REs (p53REs). To elucidate how p53RE sequences and local chromatin context interact to affect p53 binding and gene transactivation, we mapped genome-wide binding localizations of p53 and H3K4me3 in untreated and doxorubicin (DXR)-treated human lymphoblastoid cells. We examined the relationships among p53 occupancy, gene expression, H3K4me3, chromatin accessibility (DNase 1 hypersensitivity, DHS), ENCODE chromatin states, p53RE sequence, and evolutionary conservation. We observed that the inducible expression of p53-regulated genes was associated with the steady-state chromatin status of the cell. Most highly inducible p53-regulated genes were suppressed at baseline and marked by repressive histone modifications or displayed CTCF binding. Comparison of p53RE sequences residing in different chromatin contexts demonstrated that weaker p53REs resided in open promoters, while stronger p53REs were located within enhancers and repressed chromatin. p53 occupancy was strongly correlated with similarity of the target DNA sequences to the p53RE consensus, but surprisingly, inversely correlated with pre-existing nucleosome accessibility (DHS) and evolutionary conservation at the p53RE. Occupancy by p53 of REs that overlapped transposable element (TE) repeats was significantly higher (p<10-7) and correlated with stronger p53RE sequences (p<10-110) relative to nonTE-associated p53REs, particularly for MLT1H, LTR10B, and Mer61 TEs. However, binding at these elements was generally not associated with transactivation of adjacent genes. Occupied p53REs located in L2-like TEs were unique in displaying highly negative PhyloP scores (predicted fast-evolving) and being associated with altered H3K4me3 and DHS levels. These results underscore the systematic interaction between chromatin status and p53RE context in the induced transactivation response. This p53 regulated response appears to have been tuned via evolutionary processes that may have led to repression and/or utilization of p53REs originating from primate-specific transposon elements.