Sendai virus persistence questions the transient naive reprogramming method for iPSC generation.

Since the revolutionary discovery of induced pluripotent stem cells (iPSCs) by Shinya Yamanaka, the comparison between iPSCs and embryonic stem cells (ESCs) has revealed significant differences in their epigenetic states and developmental potential. A recent compelling study published in Nature by Buckberry et al.1 demonstrated that a transient-naive-treatment (TNT) could facilitate epigenetic reprogramming and improve the developmental potential of human iPSCs (hiPSCs). However, the study characterized bulk hiPSCs instead of isolating clonal lines and overlooked the persistent expression of Sendai virus carrying exogenous Yamanaka factors. Our analyses revealed that Sendai genes were expressed in most control PSC samples, including hESCs, which were not intentionally infected. The highest levels of Sendai expression were detected in samples continuously treated with naive media, where it led to overexpression of exogenous MYC, SOX2, and KLF4, altering both the expression levels and ratios of reprogramming factors. Our findings call for further research to verify the effectiveness of the TNT method in the context of delivery methods that ensure prompt elimination of exogenous factors, leading to the generation of bona fide transgene-independent iPSCs.

Testing multiplexed anti-ASFV CRISPR-Cas9 in reducing African swine fever virus.

African swine fever (ASF) is a highly fatal viral disease that poses a significant threat to domestic pigs and wild boars globally. In our study, we aimed to explore the potential of a multiplexed CRISPR-Cas system in suppressing ASFV replication and infection. By engineering CRISPR-Cas systems to target nine specific loci within the ASFV genome, we observed a substantial reduction in viral replication in vitro. This reduction was achieved through the concerted action of both Type II and Type III RNA polymerase-guided gRNA expression. To further evaluate its anti-viral function in vivo, we developed a pig strain expressing the multiplexable CRISPR-Cas-gRNA via germline genome editing. These transgenic pigs exhibited normal health with continuous expression of the CRISPR-Cas-gRNA system, and a subset displayed latent viral replication and delayed infection. However, the CRISPR-Cas9-engineered pigs did not exhibit a survival advantage upon exposure to ASFV. To our knowledge, this study represents the first instance of a living organism engineered via germline editing to assess resistance to ASFV infection using a CRISPR-Cas system. Our findings contribute valuable insights to guide the future design of enhanced viral immunity strategies. IMPORTANCE: ASFV is currently a devastating disease with no effective vaccine or treatment available. Our study introduces a multiplexed CRISPR-Cas system targeting nine specific loci in the ASFV genome. This innovative approach successfully inhibits ASFV replication in vitro, and we have successfully engineered pig strains to express this anti-ASFV CRISPR-Cas system constitutively. Despite not observing survival advantages in these transgenic pigs upon ASFV challenges, we did note a delay in infection in some cases. To the best of our knowledge, this study constitutes the first example of a germline-edited animal with an anti-virus CRISPR-Cas system. These findings contribute to the advancement of future anti-viral strategies and the optimization of viral immunity technologies.

Assessing contemporary Arctic habitat availability for a woolly mammoth proxy.

Interest continues to grow in Arctic megafaunal ecological engineering, but, since the mass extinction of megafauna ~ 12-15 ka, key physiographic variables and available forage continue to change. Here we sought to assess the extent to which contemporary Arctic ecosystems are conducive to the rewilding of megaherbivores, using a woolly mammoth (M. primigenius) proxy as a model species. We first perform a literature review on woolly mammoth dietary habits. We then leverage Oak Ridge National Laboratories Distributive Active Archive Center Global Aboveground and Belowground Biomass Carbon Density Maps to generate aboveground biomass carbon density estimates in plant functional types consumed by the woolly mammoth at 300 m resolution on Alaska's North Slope. We supplement these analyses with a NASA Arctic Boreal Vulnerability Experiment dataset to downgrade overall biomass estimates to digestible levels. We further downgrade available forage by using a conversion factor representing the relationship between total biomass and net primary productivity (NPP) for arctic vegetation types. Integrating these estimates with the forage needs of woolly mammoths, we conservatively estimate Alaska's North Slope could support densities of 0.0-0.38 woolly mammoth km-2 (mean 0.13) across a variety of habitats. These results may inform innovative rewilding strategies.

Comprehensive mutagenesis maps the effect of all single-codon mutations in the AAV2 rep gene on AAV production.

Recombinant adeno-associated viruses (rAAVs) are the predominant gene therapy vector. Several rAAV vectored therapies have achieved regulatory approval, but production of sufficient rAAV quantities remains difficult. The AAV Rep proteins, which are essential for genome replication and packaging, represent a promising engineering target for improvement of rAAV production but remain underexplored. To gain a comprehensive understanding of the Rep proteins and their mutational landscape, we assayed the effects of all 39,297 possible single-codon mutations to the AAV2 rep gene on AAV2 production. Most beneficial variants are not observed in nature, indicating that improved production may require synthetic mutations. Additionally, the effects of AAV2 rep mutations were largely consistent across capsid serotypes, suggesting that production benefits are capsid independent. Our results provide a detailed sequence-to-function map that enhances our understanding of Rep protein function and lays the groundwork for Rep engineering and enhancement of large-scale gene therapy production.

Cryosectioning-enabled super-resolution microscopy for studying nuclear architecture at the single protein level.

DNA-PAINT combined with total Internal Reflection Fluorescence (TIRF) microscopy enables the highest localization precisions, down to single nanometers in thin biological samples, due to TIRF's unique method for optical sectioning and attaining high contrast. However, most cellular targets elude the accessible TIRF range close to the cover glass and thus require alternative imaging conditions, affecting resolution and image quality. Here, we address this limitation by applying ultrathin physical cryosectioning in combination with DNA-PAINT. With "tomographic & kinetically-enhanced" DNA-PAINT (tokPAINT), we demonstrate the imaging of nuclear proteins with sub-3 nanometer localization precision, advancing the quantitative study of nuclear organization within fixed cells and mouse tissues at the level of single antibodies. We believe that ultrathin sectioning combined with the versatility and multiplexing capabilities of DNA-PAINT will be a powerful addition to the toolbox of quantitative DNA-based super-resolution microscopy in intracellular structural analyses of proteins, RNA and DNA in situ.

CRISPR-mediated germline mutagenesis for genetic sterilization of Anopheles gambiae males.

Rapid spread of insecticide resistance among anopheline mosquitoes threatens malaria elimination efforts, necessitating development of alternative vector control technologies. Sterile insect technique (SIT) has been successfully implemented in multiple insect pests to suppress field populations by the release of large numbers of sterile males, yet it has proven difficult to adapt to Anopheles vectors. Here we outline adaptation of a CRISPR-based genetic sterilization system to selectively ablate male sperm cells in the malaria mosquito Anopheles gambiae. We achieve robust mosaic biallelic mutagenesis of zero population growth (zpg, a gene essential for differentiation of germ cells) in F1 individuals after intercrossing a germline-expressing Cas9 transgenic line to a line expressing zpg-targeting gRNAs. Approximately 95% of mutagenized males display complete genetic sterilization, and cause similarly high levels of infertility in their female mates. Using a fluorescence reporter that allows detection of the germline leads to a 100% accurate selection of spermless males, improving the system. These males cause a striking reduction in mosquito population size when released at field-like frequencies in competition cages against wild type males. These findings demonstrate that such a genetic system could be adopted for SIT against important malaria vectors.

Genome streamlining to improve performance of a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973.

Cyanobacteria are photosynthetic organisms that have garnered significant recognition as potential hosts for sustainable bioproduction. However, their complex regulatory networks pose significant challenges to major metabolic engineering efforts, thereby limiting their feasibility as production hosts. Genome streamlining has been demonstrated to be a successful approach for improving productivity and fitness in heterotrophs but is yet to be explored to its full potential in phototrophs. Here, we present the systematic reduction of the genome of the cyanobacterium exhibiting the fastest exponential growth, Synechococcus elongatus UTEX 2973. This work, the first of its kind in a photoautotroph, involved an iterative process using state-of-the-art genome-editing technology guided by experimental analysis and computational tools. CRISPR-Cas3 enabled large, progressive deletions of predicted dispensable regions and aided in the identification of essential genes. The large deletions were combined to obtain a strain with 55-kb genome reduction. The strains with streamlined genome showed improvement in growth (up to 23%) and productivity (by 22.7%) as compared to the wild type (WT). This streamlining strategy not only has the potential to develop cyanobacterial strains with improved growth and productivity traits but can also facilitate a better understanding of their genome-to-phenome relationships.IMPORTANCEGenome streamlining is an evolutionary strategy used by natural living systems to dispense unnecessary genes from their genome as a mechanism to adapt and evolve. While this strategy has been successfully borrowed to develop synthetic heterotrophic microbial systems with desired phenotype, it has not been extensively explored in photoautotrophs. Genome streamlining strategy incorporates both computational predictions to identify the dispensable regions and experimental validation using genome-editing tool, and in this study, we have employed a modified strategy with the goal to minimize the genome size to an extent that allows optimal cellular fitness under specified conditions. Our strategy has explored a novel genome-editing tool in photoautotrophs, which, unlike other existing tools, enables large, spontaneous optimal deletions from the genome. Our findings demonstrate the effectiveness of this modified strategy in obtaining strains with streamlined genome, exhibiting improved fitness and productivity.

Progress and limitations in engineering cellular adhesion for research and therapeutics.

Intercellular interactions form the cornerstone of multicellular biology. Despite advances in protein engineering, researchers artificially directing physical cell interactions still rely on endogenous cell adhesion molecules (CAMs) alongside off-target interactions and unintended signaling. Recently, methods for directing cellular interactions have been developed utilizing programmable domains such as coiled coils (CCs), nanobody-antigen, and single-stranded DNA (ssDNA). We first discuss desirable molecular- and systems-level properties in engineered CAMs, using the helixCAM platform as a benchmark. Next, we propose applications for engineered CAMs in immunology, developmental biology, tissue engineering, and neuroscience. Biologists in various fields can readily adapt current engineered CAMs to establish control over cell interactions, and their utilization in basic and translational research will incentivize further expansion in engineered CAM capabilities.

Highly cooperative chimeric super-SOX induces naive pluripotency across species.

Our understanding of pluripotency remains limited: iPSC generation has only been established for a few model species, pluripotent stem cell lines exhibit inconsistent developmental potential, and germline transmission has only been demonstrated for mice and rats. By swapping structural elements between Sox2 and Sox17, we built a chimeric super-SOX factor, Sox2-17, that enhanced iPSC generation in five tested species: mouse, human, cynomolgus monkey, cow, and pig. A swap of alanine to valine at the interface between Sox2 and Oct4 delivered a gain of function by stabilizing Sox2/Oct4 dimerization on DNA, enabling generation of high-quality OSKM iPSCs capable of supporting the development of healthy all-iPSC mice. Sox2/Oct4 dimerization emerged as the core driver of naive pluripotency with its levels diminished upon priming. Transient overexpression of the SK cocktail (Sox+Klf4) restored the dimerization and boosted the developmental potential of pluripotent stem cells across species, providing a universal method for naive reset in mammals.

Comparative Transcriptome Analysis of Acne vulgaris, Rosacea, and Hidradenitis Suppurativa Supports High Dose Dietary Zinc as a Therapeutic Agent.

Acne vulgaris, rosacea, and hidradenitis suppurativa are enduring inflammatory skin conditions that frequently manifest with akin clinical attributes, posing a considerable challenge for their distinctive diagnosis. While these conditions do exhibit certain resemblances, they also demonstrate distinct underlying pathophysiological mechanisms and treatment modalities. Delving into both the molecular parallels and disparities among these three disorders can yield invaluable insights for refined diagnostics, effective management, and targeted therapeutic interventions. In this report, we present a comparative analysis of transcriptomic data across these three diseases, elucidating differentially expressed genes and enriched pathways specific to each ailment, as well as those shared among them. We also identified high dose dietary zinc as a potential therapeutic agent and validated its efficacy in an acne mouse model.

Impaired neural stress resistance and loss of REST in bipolar disorder.

Neurodevelopmental changes and impaired stress resistance have been implicated in the pathogenesis of bipolar disorder (BD), but the underlying regulatory mechanisms are unresolved. Here we describe a human cerebral organoid model of BD that exhibits altered neural development, elevated neural network activity, and a major shift in the transcriptome. These phenotypic changes were reproduced in cerebral organoids generated from iPS cell lines derived in different laboratories. The BD cerebral organoid transcriptome showed highly significant enrichment for gene targets of the transcriptional repressor REST. This was associated with reduced nuclear REST and REST binding to target gene recognition sites. Reducing the oxygen concentration in organoid cultures to a physiological range ameliorated the developmental phenotype and restored REST expression. These effects were mimicked by treatment with lithium. Reduced nuclear REST and derepression of REST targets genes were also observed in the prefrontal cortex of BD patients. Thus, an impaired cellular stress response in BD cerebral organoids leads to altered neural development and transcriptional dysregulation associated with downregulation of REST. These findings provide a new model and conceptual framework for exploring the molecular basis of BD.

Engineering Ca2+-Dependent DNA Polymerase Activity.

Advancements in synthetic biology have provided new opportunities in biosensing, with applications ranging from genetic programming to diagnostics. Next generation biosensors aim to expand the number of accessible environments for measurements, increase the number of measurable phenomena, and improve the quality of the measurement. To this end, an emerging area in the field has been the integration of DNA as an information storage medium within biosensor outputs, leveraging nucleic acids to record the biosensor state over time. However, slow signal transduction steps, due to the time scales of transcription and translation, bottleneck many sensing-DNA recording approaches. DNA polymerases (DNAPs) have been proposed as a solution to the signal transduction problem by operating as both the sensor and responder, but there is presently a lack of DNAPs with functional sensitivity to many desirable target ligands. Here, we engineer components of the Pol δ replicative polymerase complex of Saccharomyces cerevisiae to sense and respond to Ca2+, a metal cofactor relevant to numerous biological phenomena. Through domain insertion and binding site grafting to Pol δ subunits, we demonstrate functional allosteric sensitivity to Ca2+. Together, this work provides an important foundation for future efforts in the development of DNAP-based biosensors.

SeqVerify: An accessible analysis tool for cell line genomic integrity, contamination, and gene editing outcomes.

1Over the last decade, advances in genome editing and pluripotent stem cell (PSC) culture have let researchers generate edited PSC lines to study a wide variety of biological questions. However, abnormalities in cell lines such as aneuploidy, on-target and off-target editing errors, and microbial contamination can arise during PSC culture or due to undesired editing outcomes. Any of these abnormalities can invalidate experiments, so detecting them is crucial. The ongoing decline of next-generation sequencing prices has made whole genome sequencing (WGS) an effective quality control option, since WGS can detect any abnormality involving changes to DNA sequences or presence of unwanted sequences. However, this approach has suffered from a lack of easily usable data analysis software. Here, we present SeqVerify, a computational pipeline designed to take raw WGS data and a list of intended edits, and verify that the edits are present and that there are no abnormalities. We anticipate that SeqVerify will be a useful tool for researchers generating edited PSCs, and more broadly, for cell line quality control in general.

PAM-flexible genome editing with an engineered chimeric Cas9.

CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN (R = A or G, Y = C or T) PAM preference, with the N-terminus of Sc + +, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse PAMs and disease-related loci for potential therapeutic applications. In total, the approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning.

Reliable multiplex generation of pooled induced pluripotent stem cells.

Reprogramming somatic cells into pluripotent stem cells (iPSCs) enables the study of systems in vitro. To increase the throughput of reprogramming, we present induction of pluripotency from pooled cells (iPPC)-an efficient, scalable, and reliable reprogramming procedure. Using our deconvolution algorithm that employs pooled sequencing of single-nucleotide polymorphisms (SNPs), we accurately estimated individual donor proportions of the pooled iPSCs. With iPPC, we concurrently reprogrammed over one hundred donor lymphoblastoid cell lines (LCLs) into iPSCs and found strong correlations of individual donors' reprogramming ability across multiple experiments. Individual donors' reprogramming ability remains consistent across both same-day replicates and multiple experimental runs, and the expression of certain immunoglobulin precursor genes may impact reprogramming ability. The pooled iPSCs were also able to differentiate into cerebral organoids. Our procedure enables a multiplex framework of using pooled libraries of donor iPSCs for downstream research and investigation of in vitro phenotypes.

Geospatially-resolved public-health surveillance via wastewater sequencing.

Wastewater, which contains everything from pathogens to pollutants, is a geospatially-and temporally-linked microbial fingerprint of a given population. As a result, it can be leveraged for monitoring multiple dimensions of public health across locales and time. Here, we integrate targeted and bulk RNA sequencing (n=1,419 samples) to track the viral, bacterial, and functional content over geospatially distinct areas within Miami Dade County from 2020-2022. First, we used targeted amplicon sequencing (n=966) to track diverse SARS-CoV-2 variants across space and time, and we found a tight correspondence with clinical caseloads from University students (N = 1,503) and Miami-Dade County hospital patients (N = 3,939 patients), as well as an 8-day earlier detection of the Delta variant in wastewater vs. in patients. Additionally, in 453 metatranscriptomic samples, we demonstrate that different wastewater sampling locations have clinically and public-health-relevant microbiota that vary as a function of the size of the human population they represent. Through assembly, alignment-based, and phylogenetic approaches, we also detect multiple clinically important viruses (e.g., norovirus ) and describe geospatial and temporal variation in microbial functional genes that indicate the presence of pollutants. Moreover, we found distinct profiles of antimicrobial resistance (AMR) genes and virulence factors across campus buildings, dorms, and hospitals, with hospital wastewater containing a significant increase in AMR abundance. Overall, this effort lays the groundwork for systematic characterization of wastewater to improve public health decision making and a broad platform to detect emerging pathogens.

CRISPR-mediated germline mutagenesis for genetic sterilization of Anopheles gambiae males.

Rapid spread of insecticide resistance among anopheline mosquitoes threatens malaria elimination efforts, necessitating development of alternative vector control technologies. Sterile Insect Technique (SIT) has been successfully implemented in multiple insect pests to suppress field populations by the release of large numbers of sterile males, yet it has proven difficult to adapt to Anopheles vectors. Here we outline adaptation of a CRISPR-based genetic sterilization system to selectively ablate male sperm cells in the malaria mosquito Anopheles gambiae. We achieve robust mosaic biallelic mutagenesis of zero population growth (zpg, a gene essential for differentiation of germ cells) in F1 individuals after intercrossing a germline-expressing Cas9 transgenic line to a line expressing zpg-targeting gRNAs. Approximately 95% of mutagenized males display complete genetic sterilization, and cause similarly high levels of infertility in their female mates. Using a fluorescence reporter that allows detection of the germline leads to a 100% accurate selection of spermless males, improving the system. These males cause a striking reduction in mosquito population size when released at field-like frequencies in competition cages against wild type males. These findings demonstrate that such a genetic system could be adopted for SIT against important malaria vectors.

Fertility restoration in mice with chemotherapy induced ovarian failure using differentiated iPSCs.

BACKGROUND: Treatment options for premature ovarian insufficiency (POI) are limited to hormone replacement and donor oocytes. A novel induced pluripotent stem cell (iPSC) transplant paradigm in a mouse model has potential translational applications for management of POI. METHODS: Mouse ovarian granulosa cell derived-iPSCS were labelled with green fluorescent protein (GFP) reporter and differentiated in vitro into oocytes. Differentiated cells were assayed for estradiol and progesterone secretion by enzyme-linked immunosorbent assays. After Fluorescence-Activated Cell Sorting (FACS) for the cell surface marker anti-Mullerian hormone receptor (AMHR2), enriched populations of differentiated cells were surgically transplanted into ovaries of mice that had POI secondary to gonadotoxic pre-treatment with alkylating agents. A total of 100 mice were used in these studies in five separate experiments with 56 animals receiving orthotopic ovarian injections of either FACS sorted or unsorted differentiated iPSCSs and the remaining animals receiving sham injections of PBS diluent. Following transplantation surgery, mice were stimulated with gonadotropins inducing oocyte development and underwent oocyte retrieval. Nine transplanted mice were cross bred with wild-type mice to assess fertility. Lineage tracing of resultant oocytes, F1 (30 pups), and F2 (42 pups) litters was interrogated by GFP expression and validation by short tandem repeat (STR) lineage tracing. FINDINGS: [1] iPSCs differentiate into functional oocytes and steroidogenic ovarian cells which [2] express an ovarian (GJA1) and germ cell (ZP1) markers. [3] Endocrine function and fertility were restored in mice pretreated with gonadotoxic alkylating agents via orthotopic transplantation of differentiated iPSCS, thus generating viable, fertile mouse pups. INTERPRETATION: iPSC-derived ovarian tissue can reverse endocrine and reproductive sequelae of POI. FUNDING: Center for Infertility and Reproductive Surgery Research Award, Siezen Foundation award (RMA). Reproductive Scientist Development Program, Marriott Foundation, Saltonstall Foundation, Brigham Ovarian Cancer Research Fund (K.E).

Antenna Modification in a Fast-Growing Cyanobacterium Synechococcus elongatus UTEX 2973 Leads to Improved Efficiency and Carbon-Neutral Productivity.

Our planet is sustained by sunlight, the primary energy source made accessible to all life forms by photoautotrophs. Photoautotrophs are equipped with light-harvesting complexes (LHCs) that enable efficient capture of solar energy, particularly when light is limiting. However, under high light, LHCs can harvest photons in excess of the utilization capacity of cells, causing photodamage. This damaging effect is most evident when there is a disparity between the amount of light harvested and carbon available. Cells strive to circumvent this problem by dynamically adjusting the antenna structure in response to the changing light signals, a process known to be energetically expensive. Much emphasis has been laid on elucidating the relationship between antenna size and photosynthetic efficiency and identifying strategies to synthetically modify antennae for optimal light capture. Our study is an effort in this direction and investigates the possibility of modifying phycobilisomes, the LHCs present in cyanobacteria, the simplest of photoautotrophs. We systematically truncate the phycobilisomes of Synechococcus elongatus UTEX 2973, a widely studied, fast-growing model cyanobacterium and demonstrate that partial truncation of its antenna can lead to a growth advantage of up to 36% compared to the wild type and an increase in sucrose titer of up to 22%. In contrast, targeted deletion of the linker protein which connects the first phycocyanin rod to the core proved detrimental, indicating that the core alone is not enough, and it is essential to maintain a minimal rod-core structure for efficient light harvest and strain fitness. IMPORTANCE Light energy is essential for the existence of life on this planet, and only photosynthetic organisms, equipped with light-harvesting antenna protein complexes, can capture this energy, making it readily accessible to all other life forms. However, these light-harvesting antennae are not designed to function optimally under extreme high light, a condition which can cause photodamage and significantly reduce photosynthetic productivity. In this study, we attempt to assess the optimal antenna structure for a fast-growing, high-light tolerant photosynthetic microbe with the goal of improving its productivity. Our findings provide concrete evidence that although the antenna complex is essential, antenna modification is a viable strategy to maximize strain performance under controlled growth conditions. This understanding can also be translated into identifying avenues to improve light harvesting efficiency in higher photoautotrophs.

Modeling of mitochondrial genetic polymorphisms reveals induction of heteroplasmy by pleiotropic disease locus 10398A>G.

Mitochondrial (MT) dysfunction has been associated with several neurodegenerative diseases including Alzheimer's disease (AD). While MT-copy number differences have been implicated in AD, the effect of MT heteroplasmy on AD has not been well characterized. Here, we analyzed over 1800 whole genome sequencing data from four AD cohorts in seven different tissue types to determine the extent of MT heteroplasmy present. While MT heteroplasmy was present throughout the entire MT genome for blood samples, we detected MT heteroplasmy only within the MT control region for brain samples. We observed that an MT variant 10398A>G (rs2853826) was significantly associated with overall MT heteroplasmy in brain tissue while also being linked with the largest number of distinct disease phenotypes of all annotated MT variants in MitoMap. Using gene-expression data from our brain samples, our modeling discovered several gene networks involved in mitochondrial respiratory chain and Complex I function associated with 10398A>G. The variant was also found to be an expression quantitative trait loci (eQTL) for the gene MT-ND3. We further characterized the effect of 10398A>G by phenotyping a population of lymphoblastoid cell-lines (LCLs) with and without the variant allele. Examination of RNA sequence data from these LCLs reveal that 10398A>G was an eQTL for MT-ND4. We also observed in LCLs that 10398A>G was significantly associated with overall MT heteroplasmy within the MT control region, confirming the initial findings observed in post-mortem brain tissue. These results provide novel evidence linking MT SNPs with MT heteroplasmy and open novel avenues for the investigation of pathomechanisms that are driven by this pleiotropic disease associated loci.