RNA SEQUENCING APPLICATIONS IN FORENSIC GENETICS
Jack Ballantyne1Cordula Haas2, Erin Hanson1
1University of Central Florida, National Center for Forensic Science, Orlando, Florida, United States, 2University of Zurich, Zurich Institute of Forensic Medicine, Zurich, Switzerland
The application of RNA profiling in forensic genetics has experienced tremendous growth and development in the past decade. The earliest studies, and main applications, have been applied to body fluid and tissue identification, using tissue-specific RNA transcripts and, principally, reverse transcription endpoint PCR and subsequent capillary electrophoretic (CE) separation. Several markers have been identified for the forensically most relevant body fluids and tissues and the method has been successfully used in casework. The introduction of Massively Parallel Sequencing (MPS) methodology has provided several benefits that continue to advance the field. Specifically, RNA sequencing permits a more quantitative nuanced approach to gene expression analysis compared to CE since transcripts are counted via the number of reads, resulting in a digital gene expression profile that is amenable to sophisticated statistical methods. Additionally, more targets can be tested in the same sample and RNA sequence variation within transcripts can be interrogated and used for high-resolution assignment of body fluids to donors in mixed in body fluid/tissue samples. This presentation will give an overview on forensic transcriptome analyses and applications, including whole transcriptome sequencing (WTS) as well as targeted MPS approaches. Using data from the authors’ laboratories, detailed examples will include RNA biomarker selection, body fluid and organ tissue identification and, via the recent development of an improved high-resolution MPS assay, the assignment of DNA donors to body fluids via coding region SNPs. Recent development in other applications will be briefly mentioned including the potential for determination of the age of stains, the age of the donor, the post-mortem interval and to aid post-mortem death investigations.
SINGLE-CELL PROFILING OF THE HUMAN M1 AND DLPFC IN ALS AND FTLD
Sebastian Pineda1, Erica Cook2, Hyeseung Lee3, Brent Fitzwalter3, Shahin Mohammadi3, Luc Pregent2, Bjorn Oskarsson4, Jaimin Shah4, Ronald Petersen4, Neil Graff-Radford4, Bradley Boeve4, David Knopman4, Keith Josephs4, Michael DeTure2, Melissa Murray2, Dennis Dickson2, Myriam Heiman3, Veronique Belzil2, Manolis Kellis1
1Department of Electrical Engineering and Computer Science, MIT, Cambridge, MA, USA, 2Department of Neuroscience, Mayo Clinic, Jacksonville, Florida, USA, 3Broad Institute of MIT and Harvard, Cambridge, MA, USA, 4Department of Neurology, Mayo Clinic, Jacksonville, Florida, USA 5Department of Neuroscience, Mayo Clinic, Jacksonville, Florida, USA
Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two devastating and fatal neurodegenerative conditions. While distinct, they share many clinical, genetic, and pathological characteristics, and both show selective vulnerability of layer 5b extratelencephalic-projecting cortical populations, including Betz cells in ALS and von Economo neurons (VENs) in FTLD. Here, we report the first high-resolution single-cell atlas of the human primary motor cortex (M1) and dorsolateral prefrontal cortex (DLPFC) and their transcriptional alterations in ALS and FTLD across 75 individuals, including 17 control samples and 58 sporadic and C9orf72-associated ALS and FTLD patient samples. We identify 47 transcriptionally distinct cellular subtypes including two Betz-cell subtypes, and we observe a previously unappreciated molecular similarity between Betz cells and VENs. Many of the dysregulated genes and pathways are shared across excitatory neurons, with Betz cells and VENs being the most transcriptionally affected. Our results suggest that transcriptional similarity between Betz cells and VENs underline the cell-specific vulnerability observed in ALS and FTLD, and explain their concomitant diagnoses.
NEWS FROM THE NEOLITHIC
University of Mainz, Mainz, Germany
The transition from foraging to a sedentary and agricultural way of life is the decisive step in human history, without which most of the human populations living today would not exist. This breathtaking cultural transition began 11,000 years ago and has been called the “Neolithic Revolution”. It has been researched for over 100 years, in the last 17 years also with the help of palaeogenetic data. Here I present our recent analysis of high-quality palaeogenomes from predominantly Neolithic contexts. For the first time, we have explicitly modeled the population history of late hunter-gatherers and early farmers. In this way, we have created a “demogenomic model” of human populations in southwest Asia and parts of Europe between 30,000 and 7,000 years ago. The study provides new insights into pre-Neolithic population dynamics during the Late Ice Age in Europe and Anatolia. It also clarifies basic demographic processes that led to the differentiation of early Neolithic populations (and present-day Western Mediterraneans). But what do we learn from this for the Neolithic itself?
IMPACT OF OMICS ON FORENSICS
Institute of Forensic Sciences, University of Santiago de Compostela, Santiago de Compostela, Spain
Genomics is revolutionizing many areas of forensic science at a similar extend as it is happening in clinical medicine and in a similar way as clinical genetics is moving towards genomics medicine; fforensic genetics is slowly transitioning into forensic genomics. The evolution of next generation or massively parallel sequencing is allowing the use of genomic, transcriptomic, and epigenomics to address various forensic questions that cannot be answered, or only in a limited way, using classical forensic genetics approaches. Forensic genetics has directly benefited of the advances in genomics and new applications as the determination of external physical characteristics, the geographic origin of samples or the estimation of the age from minute biological material are now possible. Human transcriptome data of different tissues generated are allowing the identification of RNA markers to determine the cellular source of crime scene samples. This is forensically relevant for reconstructing the course of events that may have happened at the scene of crime and to support the use of DNA at the activity level of evidence interpretation. Non-human genomic and transcriptomic are useful in different forensic contexts and forensic microbiome is now an important emerging field. Proteomics has also some important specific applications. Integration of data is challenging but key for the progress of the field. Forensic toxicology and forensic pathology are also benefiting of the advances in genomics with a special impact in the genetic diagnosis of cardiac sudden death and the so-called molecular autopsy is essential complement to classical autopsy.
THERAPEUTIC TARGETING OF THE FOXO3 CIS-REGULOME IN ANAPLASTIC THYROID CANCER
John “Al” Copland,Tamas Ordog
Mayo Clinic, Cancer Biology Department, San Pablo Road, Jacksonville, Florida, USA
Anaplastic thyroid cancer (ATC) is the most aggressive and deadliest human cancer (median survival: 4 months). Metastatic ATC is uniformly fatal. ATC is a rare disease (800-1000 U.S. cases/year) that develops in less than 2% of differentiated thyroid cancers (DTCs). Most DTC patients have excellent prognosis in response to surgery, thyroid stimulating hormone suppression, radioiodine and sometimes radiation therapy; and even metastatic disease is often curable. Several mutations have been found to contribute to the striking transformation of DTCs to ATC. However, therapies based upon these mutations have only improved outcomes in specific cases. We previously found that FOXO3, a transcription factor (TF) with an established tumor suppressor function in DTCs, becomes an oncogene in ATC, reflecting increased nuclear retention arising from dysregulated AKT signaling. However, the spectrum of FOXO3 transcriptional targets and downstream pathways, and the epigenetic mechanisms underlying these effects remain unclear. Therefore, our goal is to identify the ATC-specific cis-regulome of FOXO3, discover the epigenetic mechanisms recruited by FOXO3 to regulate these target genes, and investigate the therapeutic utility of agents rationally selected based on their targets’ involvement in FOXO3 actions and for their ability to kill ATC cells in vitro and in vivo. Our overall hypothesis is that due to increased DNA binding, FOXO3 becomes a master TF critical for establishing the ATC phenotype, making ATC cells specifically vulnerable to pharmacological agents targeting FOXO3-regulated mechanisms, genes and pathways. We are combining multi-omics with epigenetic pharmacology to define the ATC-specific FOXO3 cis-regulome, to investigate the role of FOXO3-induced chromatin decondensation and reconfiguration in enabling and augmenting the expression of oncogenic gene networks, and to determine the utility of pharmacological agents targeting FOXO3-regulated mechanisms. Our multiomics-supported, mechanistically informed approach may identify key ATC vulnerabilities and validate the therapeutic potential of their targeting with focus on combinations of clinically relevant agents to ultimately transform the therapy of ATC, the deadliest of cancers.
MOLECULAR BASIS OF EPIGENETIC CONTROL: EPIGENETIC REGULATION BY POST-SYNTHETIC DNA AND RNA MODIFICATIONS
Mayo Clinic, Rochester, Minnesota, USA
Post translational modifications of DNA and RNA are critical components regulating chromatin and RNA processes. In this presentation, I will present what we know about the chemistry underlying these modifications, some thoughts on how they may have evolved, and touch upon how perturbations of these mechanisms can impact health.
NON-INVASIVE PRENATAL TESTING FOR THE BETA-HEMOGLOBINOPATHIES USING NEXT-GENERATION SEQUENCING AND PROBE CAPTURE
Children’s Hospital Oakland Research Institute and UCSF Benioff Children’s Hospital Oakland, Oakland, CA, USA
The presence of fetal DNA in maternal plasma and the massively parallel and clonal features of Next Generation Sequencing (NGS) have made non-invasive prenatal testing (NIPT) a reality. The analysis by NGS of fetal DNA in maternal plasma has been applied to the diagnosis of chromosomal aneuploidies but the NIPT of autosomal recessive diseases has been more challenging. We have applied NGS to the NIPT of the autosomal recessive diseases, sickle cell anemia (SCA) and b-thalassemia, by using a capture probe panel that covers 4 Kb of the b-globin gene and linked SNPs as well as >450 genomic polymorphisms used to estimate the fetal fraction. The hybrid capture method is well suited to the analysis of the small DNA fragments present in the maternal plasma. The fetal fraction is estimated by counting paternally transmitted sequence reads in the plasma library for alleles present in the fetus but absent in the mother. If the mother and father’s mutations differ, the paternally transmitted mutation can be detected qualitatively as a sequence in the plasma that is absent in the mother but the mother’s transmitted allele is determined by quantitative analysis of the sequence read proportions in the plasma. If the maternal and paternal mutations are the same, as in a pregnancy at risk for SCD, the fetal b-globin genotype is inferred by counting sequence reads corresponding to the mutation and wild type alleles. The observed proportions are compared to those expected for each of the three possible fetal genotypes (Mut/Mut; Mut/WT; WT/WT) to infer fetal genotype. The expected values are calculated based on the fetal fraction estimate. An algorithm assigns probability values to each of the potential fetal genotypes. We have used the bioinformatic strategy of in silico size selection for the maternal plasma reads to increase the fetal fraction. We have also used haplotype information, when available, to consider the observed ratios at linked SNPs to help predict the fetal genotype at the mutation site. This probe capture/NGS system promises to provide a robust non-invasive test for sickle cell anemia and b-thalassemia and represents a model approach for other autosomal recessive diseases.
EPIGENOMICS OF INFLAMMATORY BOWEL DISEASE
William A. Faubion
Mayo Clinic, Rochester, Minnesota, USA
Inflammatory bowel disease is a chronic intestinal inflammatory condition affecting 1 in 300 individuals in the developing world for which there is insufficient insight into pathogenicity or precision therapeutics. While there are genetic traits associated with disease, the rise in incidence over the last 3 decades supports an environmental influence to disease susceptibility and behavior. We will review environmental signals received by particular immune subsets (FOXP3+ CD4+ lymphocytes) that couple to epigenetic complexes regulating immune responses in the human IBD, Crohn’s disease.
SINGLE-CELL MULTIOMIC ANALYSIS TO IDENTIFY CELL-SPECIFIC TRANSCRIPTIONAL DEPENDENCIES IN CANCER AND COVID-19 INFLAMMATORY RESPONSE
Center for Individualized Medicine, Mayo Clinic, Rochester, MN, USA
The human body is composed of an estimated 37 trillion cells that live harmoniously among their neighbors. An equilibrium between differentiated cells, progenitor cells and stem cells has to be achieved at any point of development to enable homeostasis and proper tissue organization. However, in cancer, a single cell can lead to the downfall of an entire organism. To ensure that all the signals and processes are coordinated, cells need to regulate the expression of genes using a multitude of factors (transcription factors that bind to the DNA itself) and chromatin regulators (proteins that surround the DNA). Novel single cell technologies have enabled us to define both the expression and the chromatin landscape. Here, we will discuss how single cell multiomic analysis has enable us to define cell-specific transcriptional dependencies in cancer and in inflammatory response upon COVID19 infection.
3D GENOMIC STRATEGIES TO UNDERSTAND COMPLEX TRAIT GENETIC ARCHITECTURE
Struan F. A. Grant
Children’s Hospital of Philadelphia Research Institute, Philadelphia, PA, USA
We are employing cutting-edge 3D genomic approaches to facilitate understanding of genetic loci for common complex disease. There is a significant need to discover and validate new genetic targets that influence such traits to advance therapies to prevent and treat disease. We are focused on the functional significance of genome wide association study (GWAS) signals associated with various complex traits. While numerous GWAS efforts have been successful in discovering key genetic variants, this approach only reports genomic signals associated with a given phenotypic trait and not necessarily the precise localization of culprit effector genes. Approaches are now emerging to make these determinations; however, they typically suffer from low-resolution and inaccuracies. Using bone mineral density (BMD) as an example, we recently published our high-resolution genome-wide ‘variant to gene mapping’ efforts, where we integrated RNA-seq, ATAC-seq and chromatin conformation capture (promoter-focused Capture C) in primary human osteoblasts to implicate culprit effector genes for osteoporosis, including validating two novel effector genes, EPDR1 and ING3. ~30% of GWAS signals were found to reside in enhancers with direct physical contact with genes expressed in osteoblasts, totaling 86 leads – many being novel and warranting functional follow-up. However, this also means that many GWAS loci remain to be resolved, so in order to uncover additional aspects of the genetic architecture of bone density determination we are now studying temporally specific roles that are dependent on the stage of differentiation Crucially, our pipeline does not involve large sample sizes, but rather uses primary healthy human cells to triangulate key enhancers coinciding with, and signposted by, putatively causal variants. The ultimate aim is to provide the community with new, high value targets to aid in understanding mechanism, and eventually therapies, for common complex diseases.
POPULATION GENETICS, KINSHIP PRACTISES AND SOCIAL ORGANISATION IN PREHISTORIC SOCIETIES OF EUROPE
Max Planck Institute for Evolutionary Anthropology, Department of Archaeogenetics, Leipzig, Germany
The continuously growing record of ancient human genomic data reveals ever more detailed insights into the population history of prehistoric societies. By focussing on regional transects, closing spatial and temporal gaps, and through a proper integration of the archaeological context, archaeogenetic studies now exceed simplistic migration scenarios and can provide nuanced accounts of genetic and cultural transformations. Increasing numbers of intra-site studies add further details by shedding light on kinship practices and forms of social organisation in prehistoric societies. I will present a selection of recent case studies from various regions in Neolithic and Early Bronze Age Europe, which further advance our understanding of prehistoric societies and the formation of the European gene pool.
STONE AGE ENCOUNTERS: NEANDERTAL ANCESTRY OF EARLY HUMANS IN EURASIA
1Ancient Genomics Laboratory, The Francis Crick Institute, London, United Kingdom, 2Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
Our closest evolutionary relatives, the Neandertals, appeared in the European fossil record around 430,000 years ago (~430ka). Before their disappearance, Neandertals lived throughout Europe, western and central Asia, and the Near East. The ancestors of all modern humans emerged in Africa at least ~300ka and, sometime between ~100ka and ~50ka, a subset of those humans migrated out-of-Africa and spread across the world. However, by ~40ka Neandertals disappeared. The reasons of Neandertal disappearance, the extent to which they overlapped with modern humans, and the nature of interactions between the two hominin groups have been intensively debated for decades. Comparisons between the Neandertal genomes and the genomes of modern humans showed that Neandertals contributed ~2% to the genomes of all people living outside of sub-Saharan Africa. Based on the size of Neandertal segments in modern human genomes, the time of this admixture was narrowed to between 58ka and 52ka, and most likely in the Near East. However, where, when and how often these two groups came into contact was not well understood. Despite genomic data being recovered from more than 6,500 ancient humans to date, genome-wide data of individuals close in time when modern humans could have met some of the last Neandertals are still extremely sparse. Through the combination of minimally destructive sampling, decontamination with mild hypochlorite solution and hybridisation captures, we obtained genome-wide data of sixteen new and improved coverage of further nine previously published modern humans from western Eurasia older than 30ka. In addition to shedding light on the genetic diversity of past populations, these data allow us to start reconstructing the fine-scale dynamics of the interactions between late Neandertals and early humans in Eurasia. Our data suggest that small groups of first humans to arrive in Eurasia interacted intimately with Neandertals, having had close Neandertal ancestors, and were eventually either absorbed into their populations or became extinct. Moreover, our new data further raise the possibility of Neandertal populations becoming assimilated in more numerous human populations that arrived later, indicating a far more dynamic population history and turnovers during this time period than previously appreciated.
A FORENSIC GENOMICS APPROACH FOR THE IDENTIFICATION OF SISTER MARIJA KRUCIFIKSA KOZULIĆ OF RIJEKA
Charla Marshall1,2,3, Kimberly Sturk-Andreaggi1,2, Erin M. Gorden1,2, Jennifer Daniels-Higginbotham1,2, Sidney Gaston Sanchez1,2, Željana Bašić4, Ivana Kružić4, Šimun Anđelinović5,6, Alan Bosner7, Miran Čoklo8, Anja Petaros9, Timothy P. McMahon1, Dragan Primorac3,5,10,11,12,13,14,15,16,17, Mitchell M. Holland3
1Armed Forces Medical Examiner System (AFMES), Dover Air Force Base, Dover, DE, USA, 2SNA International, Contractor Supporting the AFMES, Alexandria, VA, USA, 3Department of Biochemistry & Molecular Biology, Forensic Science Program, The Pennsylvania State University, University Park, PA, USA, 4University Department of Forensic Sciences, University of Split, Split, Croatia, 5Medical School, University of Split, Split, Croatia, 6Clinical Department for Pathology, Legal Medicine and Cytology, Clinical Hospital Center Split, Split, Croatia, 7Department of Forensic Medicine and Criminalistics, University of Rijeka School of Medicine, Rijeka, Croatia, 8Institute for Anthropological Research, Center for Applied Bioanthropology, Zagreb, Croatia, 9National Board of Forensic Medicine, Department of Forensic Medicine, Linköping, Sweden, 10St. Catherine Specialty Hospital, Zagreb, Croatia, 11School of Medicine, University of Osijek, Osijek, Croatia, 12Faculty of Dental Medicine and Health, University of Osijek, Osijek, Croatia, 13The Henry C. Lee College of Criminal Justice and Forensic Sciences, University of New Haven, New Haven, CT, USA, 14School of Medicine, University of Rijeka, Rijeka, Croatia, 15Medical School REGIOMED, Coburg, Germany, 16University of Split, University Department of Forensic Sciences, Split, Croatia, 17The National Forensic Sciences University, Gandhinagar, Gujarat, India
Sister Marija Krucifiksa Kozulić (1852-1922) was a nun who dedicated herself to helping the poor and less fortunate. In light of her generous and virtuous life, Sister Kozulić is being considered for sainthood by the Vatican. However, this process could not proceed without the identification of her remains, which has required the skills of many experts, including pathologists, anthropologists, and molecular biologists. This presentation will highlight the efforts made to support the identification of Sister Kozulić with a forensic genomics approach; targeting both mitochondrial and nuclear DNA markers using capture and massively parallel sequencing (MPS) methods. Sister Kozulić was buried in a tomb in Rijeka with her biological sister, Tereza Kozulić, along with the commingled remains of other nuns from the Society of Sisters of the Sacred Heart of Jesus. In search for Sister Kozulić amongst the many skeletal remains recovered from the tomb, mitochondrial genome (mitogenome) sequencing was performed on femoral samples using capture and MPS methods developed for degraded DNA. The obtained mitogenome sequences revealed DNA averaging less than 100 bp in length that is typical of historical samples. The results revealed two individuals sharing the same H1 haplotype with an uncommon 13327 G/A heteroplasmy. This finding was taken as a preliminary indicator of the biological sisters’ remains, who were the only known maternal relatives buried in the tomb. Additionally, one of the haplotypes exhibited a private 12337 C insertion at ~30% frequency, which distinguished the two haplotypes from one another. Due to the uniqueness of the haplotype, combined with the rarity of the shared heteroplasmy, it was assumed that these remains represented the sisters. The next step was to perform autosomal DNA testing on the remains, along with a buccal swab from a known paternal niece, to evaluate their genetic relationship. The reference sample of the now-deceased niece was collected in 2011, and the extracted DNA was low quality. Due to the degree of DNA degradation, kinship analysis was attempted after MPS of identity informative SNPs and autosomal STRs using the Precision ID Identity SNP and GlobalFiler NGS STR panels. Although only partial SNP and STR profiles were obtained from the sisters, comparison of their genotypes with that of the paternal niece supported the expected kinship scenario. Based on the SNP and STR results, the likelihood ratio (LR) exceeded 93,000 for a full sibling relationship between the presumed remains of Marija and Tereza versus being unrelated, with a posterior probability of 98.1% when considering the degrees of relatedness tested. In addition, the findings were >574,000 times more likely in favor of the sisters as 2nd degree relatives of the niece than from individuals who were unrelated. Given the supported kinship relationship with a known family reference, the two sets of skeletal remains are believed to belong to Sisters Marija and Tereza Kozulić. In the absence of direct reference samples from the two sisters, it is not possible to identify which set of remains belongs to Sister Marija. Nonetheless, the findings have allowed for the Vatican to move forward with the beatification process. The points of view in this abstract are those of the authors and do not reflect the views of their respective agencies. In addition, this publication in no way reflects an endorsement of products, instruments, or software.
A forensic genomics approach for the identification of Sister Marija Crucifiksa Kozulić. C Marshall, K Sturk-Andreaggi, EM Gorden, J Daniels-Higginbotham, SG Sanchez, Ž Bašić, I Kružić, Š Andelinović, A Bosner, M Čoklo, A Petaros, TP McMahon, D Primorac, MM Holland (2020), Genes, 11, 938-950
MASSIVE EPIGENETIC REPROGRAMMING TRIGGERED BY A SINGLE GENETIC ALTERATION
Mayo Clinic, Rochester, MN, USA
SPOP is a Cullin3-based E3 ubiquitin ligase adaptor (substrate-binding) protein. Whole genome and exome sequencing studies including The Cancer Genome Atlas (TCGA) invariably show that SPOP is the most frequently mutated gene in human primary prostate cancers. In addition to the discovery that prostate cancers with SPOP mutations have highest androgen receptor (AR) transcriptional activity among all genotypically distinct subsets of prostate cancer, this subtype of tumors are reportedly associated with genome-wide DNA hypermethylation in prostate cancer although the underlying mechanisms were elusive. Recent studies from Dr. Haojie Huang’s laboratory at Mayo Clinic have shown that SPOP binds and promotes polyubiquitination and degradation of histone methyltransferase and DNMT interactor GLP. SPOP mutation induces stabilization of GLP and its partner protein G9a and aberrant upregulation of global DNA hypermethylation in cultured prostate cancer cell lines and primary specimens of patients. Genome-wide DNA methylome analysis shows that a subset of tumor suppressor genes (TSGs) including FOXO3, GATA5, and NDRG1, are hypermethylated and downregulated in SPOP-mutated prostate cancer cells. DNA methylation inhibitor 5-azacytidine effectively reverses expression of the TSGs examined, inhibits SPOP-mutated PCa cell growth in vitro and in mice, and enhances docetaxel anti-cancer efficacy. Together, these findings identify the GLP/G9a-DNMT module as a mediator of DNA hypermethylation in SPOP-mutated prostate cancer. These data also suggest that SPOP mutation could be a biomarker for effective treatment of prostate cancer with DNA methylation inhibitor alone or in combination with taxane chemotherapeutics.
RECOVERY OF NUCLEAR DNA FROM HAIR SHAFTS ASSOCIATED WITH THE ROMANOV FAMILY
Federal Bureau of Investigation (FBI), Quantico, VA, USA
To optimize the recovery of probative DNA from shed hair, an extraction protocol targeting ultrashort DNA fragments was applied to hair shafts found in items associated with the Romanov family. Published mitochondrial DNA genome sequences of Tsar Nicholas II and his wife, Tsarina Alexandra, made these samples ideal to assess DNA extraction techniques and evaluate the types of genetic information that can be recovered from aged hair. Using this method, the mtGenome of the Tsarina’s lineage was identified in hairs that were concealed in a pendant made by Karl Fabergé for Alexandra Feodorovna Romanov. In addition, to determine if the lock originated from more than one individual, two single hairs from the locket were extracted independently and the autosomal SNP data used to assess relatedness. Testing of hairs found in a second artifact, a framed photograph of Louise of Hesse-Kassel, Queen of Denmark and maternal grandmother of Tsar Nicholas II, revealed that the hair belonged to a woman who shared Tsar Nicholas’ maternal lineage, including the well-known point heteroplasmy at position 16169.
ARCHAIC AND MODERN HUMANS IN ISLAND SOUTHEAST ASIA
University of Uppsala, Uppsala, Sweden
Multiple lines of evidence show that modern humans interbred with archaic Denisovans. Denisovans were initially thought to have a simple shared demographic history with modern humans, through a single admixture event with the ancestor of Australasians, but later work suggest that Denisovan ancestry can also be detected in varying degree across Australasians and at lower levels in East Asian, South Asian, Siberian and Native American populations. I will discuss recent finds of additional admixture between Australasians and Denisovans distinctively in Island Southeast Asia and the Philippines. In total, 118 ethnic groups of the Philippines including 25 diverse self-identified Negrito populations were investigated and we show that some groups, for instance the Ayta Magbukon, possess the highest level of Denisovan ancestry in the world ~30-40% greater than the level in Australians and Papuans. This finding is consistent with an independent admixture event into Negritos from Denisovans. Together with the recently described fossils claimed to be a new species (Homo luzonensis), we propose diverse archaic groups inhabiting the Philippines prior to the arrival of modern humans, likely genetically similar to Denisovans. Altogether, our findings unveil a complex intertwined history of modern and archaic humans in the Asia-Pacific region, where distinct Islander Denisovan populations differentially admixed with incoming Australasians across multiple locations and at various points in time.
DYNAMIC ENHANCER-PROMOTER INTERACTIONS MEDIATE CHEMORESISTANCE IN PDAC
Steven A. Johnsen
Robert Bosch Center for Tumor Diseases, Stuttgart, Germany
Pancreatic ductal adenocarcinoma (PDAC) displays a remarkable propensity towards therapy resistance. However, molecular epigenetic and transcriptional mechanisms enabling this are poorly understood. We integrated epigenome, transcriptome, nascent RNA and chromatin topology data and identified a novel subgroup of enhancers that mediate transcriptional reprogramming and chemoresistance in PDAC. These Interactive Hubs (iHUBs) display characteristics typical for active enhancers (H3K27ac enrichment) in both therapy sensitive and resistant states but exhibit increased interactions and production of enhancer RNA (eRNA) in the resistant state. Notably, deletion of individual iHUBs was sufficient to decrease transcription of target genes and sensitize resistant cells to chemotherapy. Moreover, targeting either eRNA production or signaling pathways upstream of iHUB activation using clinically tested small molecule inhibitors decreased eRNA production and interaction frequency, and restored chemotherapy responsiveness in vitro and in vivo. Thus, our findings identify iHUBs as important regulators of chemotherapy response and demonstrate their targetability in sensitization to chemotherapy.
MULTI-OMICS TO MECHANISMS: THE ROAD TO MICROBIOME-DRIVEN PRECISION MEDICINE
Purna C. Kashyap
Mayo Clinic, Rochester, Minnesota, USA
The current treatment paradigm of one-size fit all does not consider interindividual variability in an individual’s exposome including diet, lifestyle and environment and genetics including host and microbial genes, which underlie the pathogenesis, susceptibility, and outcomes of most chronic diseases to varying levels. While there has been a significant interest in host genetics since with sequencing of the host genome, there is increasing realization that microbial genes also contribute to several pathophysiologic mechanisms. To move towards a personalized medicine approach, we need to consider nonlinear contributions from patient genetics, microbiome and exposome. In my presentation, I will discuss the contribution of the microbiome in the pathophysiology of functional gastrointestinal disorders like irritable bowel syndrome, in the context of host omics and physiological responses, as well as environmental factors. This approach can facilitate personalized treatment strategies by providing more meaningful mechanism-based stratification.
DEEP THINKING: WHERE MACHINE INTELLIGENCE ENDS AND HUMAN CREATIVITY BEGINS
Office of Garry Kasparov, New York, New York, USA
As our machines become capable of more complex tasks, they are evolving from tools to partners—if we use them wisely. There is little doubt that the combination of human plus machine is the key to unlocking the power of artificial intelligence. The key is finding ways to work together, to develop processes that get the best from both. Instead of being afraid of automation encroaching into the intellectual world, we must embrace the potential to discard rote cognitive tasks to focus on the uniquely human elements of life: leadership, creativity, and the pursuit of happiness. Much as a telescope extends human vision, artificial intelligence, or augmentedintelligence, as Kasparov prefers it, will extend our mental abilities. Also like a telescope, we must point our powerful new AI tools in the right direction, ambitiously and imaginatively.
PHARMACOGENOMICS IN CLINICAL PRACTICE: LESSONS FROM INDIVIDUALIZED MEDICINE CLINIC
Pharmacy Clinical Practice, Mayo Clinic Arizona, Phoenix, AZ, USA
Pharmacogenomics combines sciences of clinical pharmacology and human genomics to predict drug response phenotypes. This presentation will highlight current use of PGx in a clinical practice, its benefits and limitations, and future direction.
THE FUNCTIONAL IMPACTS OF GENE FLOW BETWEEN ARCHAIC AND MODERN HUMANS
Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
The genomes of archaic and ancient modern humans offer a unique window into their histories. However, the sequencing and analysis of DNA from ancient humans is complicated by DNA degradation, chemical modifications and contamination. Recent technological advances have made it possible retrieve and sequence DNA from bones and other remains found at archaeological excavations, and we have been able to reconstruct the genomes of several Neandertals. We have also identified, based on their genome sequences, Denisovans, a previously unknown extinct Asian hominin group related to Neandertals.
By comparing these archaic genome sequences to the genome sequences of ancient and present-day modern humans we have shown that gene flow between archaic and modern humans occurred at multiple times, and that this gene flow has shaped the genomes of both Neandertals and of modern humans. For example, the ancestors of some modern humans interbred with Neandertals and Denisovans such that all present-day people outside of Africa carry approximately 2% Neandertal DNA, and that some populations, largely in Oceania, also carry DNA inherited from Denisovans. This introgressed DNA has been shown to have both positive and negative outcomes for present-day carriers: underlying apparently adaptive phenotypes such as high altitude adaptation, as well as influencing immunity and disease risk. In recent work we have identified Neandertal haplotypes that are likely of archaic origin and determined the likely functional consequences of these haplotypes using public genome, gene expression, and phenotype datasets.
THE GENETIC HISTORY AND ORIGIN OF THE BLACK DEATH
Max Planck Institute for the Science of Human History, Jena, Germany
High throughput DNA sequencing has revolutionized the field of archaeogenetics in the past decade, providing a better understanding of human genetic history, past population dynamics and host pathogen interactions through time. Targeted DNA capture approaches have allowed reconstructing complete ancient bacterial genomes providing direct insights into the evolution and origin of some of the most infamous bacterial pathogens known to humans such as Yersinia pestis, the causative agent of plague. Ancient Y. pestis genomes spanning over 5000 years of human history from the Stone Age to modern times provide novel insights into the evolution of one of the most infamous human pathogens. They provide direct evidence for the timing and emergence of major virulence factors essential for the transmission of Y. pestis by fleas. The oldest reconstructed genomes of Y. pestis fully capable of causing the bubonic form from the Eastern European Bronze Age provides evidence for prehistoric epidemics in prehistory. Suggesting that the emergence of this form of the disease happened more than 1000 years earlier than previously suggested. Temporal studies of pathogens might thus throw new light on the origin of human diseases and potentially allow predicting and preventing further transmissions and dissemination in the future.
THE ORIGINS, DISPERSAL, AND PATHOGEN HISTORY OF CHICKENS
University of Oxford, Oxford, UK
There are approximately 80 billion chickens on Earth which makes them by far the most numerous domestic animals. Despite their ubiquity there has been little consensus regarding the timing or location or circumstances of their domestication. In order to understand not just when, where, and how they first became associated with human societies, but also what happened next and how their close proximity to people has driven the evolution of Marek’s disease, a highly contagious viral neoplastic disease, I will discuss three recent studies. The first critically assessed the domestic status of chicken remains described in >600 sites in 89 countries, alongside an evaluation of zoogeographic, morphological, osteometric, stratigraphic, contextual, iconographic, and textual data. A second study predicated on the direct radiocarbon dating of >20 ancient European chickens reframes the arrival and dispersal of chickens across Eurasia, and a third study uses an ancient DNA approach to sequence the Marek’s virus to understand how decades of vaccines have driven the evolution and virulence of this disease. Combined, these studies establish a new and comprehensive foundation for understanding humanity’s most important bird.
GLYCANS AS BIOMARKERS AND FUNCTIONAL EFFECTORS IN CARDIOMETABOLIC DISEASES
1University of Zagreb Faculty of Pharmacy and Biochemistry, 2Genos Glycoscience Research Laboratory, Zagreb, Croatia
The majority of proteins that evolved after appearance of multicellular life are glycosylated and glycans significantly affect structure and function of these proteins. However, due to structural complexity of glycans and the absence of a direct genetic template, the analysis of protein glycosylation is much more complicated than the analysis of DNA or proteins. Consequently, the knowledge about the importance of individual variation in glycans for both normal physiological processes and diseases is still limited. By generating glycomic data for over 100,000 individuals from some of the best characterized clinical and epidemiological cohorts we enabled glycomics to meet other ‘omics. Changes in glycosylation have been observed in numerous diseases, often even before other symptoms of a disease appeared, indicating that they may reflect early steps in the molecular pathophysiology of many complex diseases. Initial data from intervention studies and animal models suggest that reversing changes in glycosylation may decrease the disease risk.
SENSITIZING CANCER CELLS TO DNA DAMAGE-INDUCING AGENTS AND ANTI-TUMOR IMMUNITY
Qin Zhou, Robert Mutter and Zhenkun Lou
Mayo Clinic, Rochester, MN, USA
Spleen-associated tyrosine kinase (Syk) is a non-receptor tyrosine kinase that regulates immunity, cell adhesion, and vascular development. Here we found a new role of Syk in DNA repair regulation. We discovered that Syk is activated by ATM and recruited to DNA double-strand breaks where Syk activates the key enzyme in homologous recombination (HR) to promote resection and HR. Furthermore, Syk upregulation and promotion of resection and HR is a potential mechanism of platinum and PARP inhibitor resistance in ovarian and breast cancer with high expression of Syk. This resistance can be overcome by Syk inhibition or deletion for Syk-high expressing cancer cells. In addition, Syk inhibition could promote anti-tumor immunity by inhibiting M2 macrophages. We propose that Syk is a new target to inhibit HR and sensitize resistant tumors to DNA targeted therapy and immunotherapy.
CROSSTALK BETWEEN HYPOXIA AND EPIGENETICS IN BREAST CANCER
University of Texas Southwestern Medical Center, Dallas, TX, USA
Hypoxia, a common feature of the tumor microenvironment, regulates various cancer biological processes to drive tumor progression. The hypoxia response is primarily controlled by the transcription factor hypoxia-inducible factor (HIF). HIF enhances thousands of downstream target genes and regulates many hypoxia-induced pathological processes in human cancers, including angiogenesis, metabolism, immune evasion, pH homeostasis, cell survival, maintenance of stem cells, and cell migration/invasion. The transcription activity of HIF is dynamically regulated by multiple epigenetic regulators including p300, JMJD2C, ZMYND8, G9a, GLP, CHD4, and BRD4 in cancer cells. HIF regulators are upregulated in breast cancer and contribute to breast cancer progression by augmenting hypoxia response. Apart from protein-coding genes, HIF also globally induces long non-coding RNAs in human breast cancer cells. These hypoxia-induced long non-coding RNAs represent another layer of mechanism of hypoxia-dependent breast cancer progression. Taken together, HIF and epigenetic regulators are mutually regulated and their crosstalk is crucial for breast cancer progression.
GENOME EDITING, STEM CELLS AND NEANDERTALS
Max Planck Institute for Evolutionary Anthropology, Department of Evolutionary Genetics, Leipzig, Germany
Neandertals are our closest extinct relatives and using their genomic sequences together with sequences of thousands of humans living today, we were able to define all genomic positions that are unique to modern humans. Out of those, only around one hundred positions have changed an amino acid and the biological significance of those is largely unknown. Since those changes are shared among all humans, we cannot study them in living humans and have to rely on other model systems, such as pluripotent stem cells (PSCs) and genome editing. This allows us to introduce the Neandertal substitution into a human PSC, and study the effect of the introduced substitution in differentiated tissue of interest, e.g., human brain organoids. In my talk, I will first present methodological improvements in genome editing in which we were able to increase editing efficiency and detect and reduce unwanted editing side effects. Then I will present our study of six amino acid changes in proteins that have key roles in kinetochore function and chromosome segregation. We could show that cells with Neandertal substitutions have shorter metaphase and more chromosome segregation errors when differentiated to human brain organoids, than cells without those substitutions. This suggests that one unique feature of modern humans is that the fidelity of chromosome segregation has improved during neocortex development.
DEVELOPMENT OF NEURONAL CELL DIVERSITY IN THE GUT REVEALED BY SINGLE-CELL TRANSCRIPTOMICS
Khomgrit Morarach, Anastassia Mikhailova, Viktoria Knoflach, Wei Li, Ziwei Liu, Fatima Memic, Ulrika Marklund
Karolinska Institute, Solna, Sweden
As largest part of the peripheral nervous system, the enteric nervous system (ENS) spans the entire gastrointestinal tract and organises into irregular ganglia of intermingled neural subtypes. Owing to these challenging anatomical features, research on ENS composition and development has lagged behind that of the central nervous system (CNS). Meticulous work over decades have identified enteric neural subtypes including motor-, inter- and sensory neurons but the ambiguous molecular markers have hampered elucidation of the full cellular complexity, and thus functionality, of the ENS. We have utilised single cell RNA-sequencing to establish unbiased molecular definitions of enteric neurons in the murine small intestine. Analysis of the myenteric plexus identified 12 enteric neuron classes (ENCs) that were validated in tissue by histochemical detection of unique marker combinations. Further transcriptome analysis of the fetal ENS presented a novel neural diversification mechanism. Unlike in the developing CNS, where spatial patterning of stem cells predominates cell fate decisions, myenteric neuron diversity seems primarily formed via identity conversion of postmitotic neurons. We anticipate that the mapping of enteric neuron classes may help to better define enteric neural circuits, while the developmental blueprint could pave the way for efficient derivation of specific enteric neuron types for the purpose of cell-based regenerative medicine or ENS disease modelling.
CAPTURE AND MPS FOR HUMAN IDENTIFICATION
Charla Marshall1,2, Kimberly Sturk-Andreaggi1,2, Jacqueline Tyler Thomas1,2, Courtney Cavagnino1,2, Katelyn Kjelland1,3, Jennifer Daniels-Higginbotham1,2, Suzanne Barritt-Ross1, Kerriann K. Meyers1,2, Ellen Greytak4, Janet Cady4, CeCe Moore4, Steven Armentrout4, Timothy P. McMahon1
1Armed Forces Medical Examiner System’s Armed Forces DNA Identification Laboratory (AFMES-AFDIL), Dover Air Force Base, Delaware, USA, 2SNA International, LLC, Alexandria, Virginia, USA 3Pacific Architects and Engineers, Falls Church, Virginia, 4Parabon NanoLabs, Inc., Reston, Virginia, USA
In 2015, the Armed Forces Medical Examiner System’s Armed Forces DNA Identification Laboratory (AFMES-AFDIL) became the first accredited forensic DNA laboratory worldwide to validate massively parallel sequencing (MPS) methods for routine casework. These MPS methods were needed to analyze DNA fragments from chemically treated, historical bone samples that were too degraded for traditional PCR enrichment, averaging only 70 base pairs in size. For example, MPS with hybridization capture (capture-MPS) enabled mitochondrial DNA (mtDNA) analysis of Korean War unknowns that were immersed in formalin baths in the 1950s as a post-mortem preservation treatment prior to burial. STR typing of these samples was not possible, and the success rates for Sanger sequencing of mtDNA were very low (~8%). However, with capture-MPS, mitochondrial genome analysis was successful for approximately 60% of all samples tested. This allowed for comparison of mtDNA between profiles obtained from unknowns and family reference specimens from maternal relatives. While the capability for mtDNA analysis led to hundreds of MPS-based identifications of missing U.S. service members from conflicts dating back to the mid-20th century, unsolved cases still remain. These include cases involving consistent mtDNA profiles, those of service members lacking mtDNA references, and others with hypothesized mutational events between the unknown and the family member’s mtDNA sequence. Therefore, SNP capture is necessary to expand the capability for DNA-based identifications. This presentation will broadly cover the SNP capture and MPS methods developed and/or evaluated at the AFMES-AFDIL. These targets include a large SNP panel (850,000 SNPs), two medium SNP panels (25,000 SNPs and 95,000 SNPs), a small SNP panel (5,000 SNPs), as well as whole genome enrichment approaches with some comparisons to untargeted whole genome sequencing.
Mayo Clinic, Department of Physiology and Biomedical Engineering, Rochester, Minnesota, USA
Type 2 diabetes mellitus (T2DM) is one of the major health challenges facing today’s society and projected to afflict nearly 1 in 3 people by year 2050. T2DM is associated with an increase in population morbidity/mortality, and more recently, has been shown to exacerbate adverse outcomes associated with COVID-19. The pathophysiology of T2DM is mediated by complex interactions among diverse environmental and genetic susceptibilities, which ultimately culminate in the development of pancreatic islet failure characterized by impaired insulin secretory function. Although underlying genetics contribute to the pathogenesis of T2DM, environmental and epigenetic factors appear to be the primary drivers of this disease. Specifically, recent evidence suggests that disruptions of circadian light/dark and fasting/feeding cycles have contributed to the induction of pancreatic islet failure and an overall increase in the predisposition to T2DM. The aim of the presentation is to describe emerging physiological, genetic and epigenetic insights into the role of the circadian system in regulating pancreatic islet function and failure in health and disease.
PROTEIN MONOAMINYLATION IN BRAIN: NOVEL MECHANISMS OF NEURAL DEVELOPMENT, PLASTICITY AND DISEASE
Icahn School of Medicine at Mount Sinai, Department of Neuroscience, New York, NY, USA
Histone H3 monoaminylations at glutamine (Q) 5 [e.g., serotonylation (H3Q5ser) and dopaminylation (H3Q5dop)] have recently been identified as epigenetic markers in neurons. These monoaminylation states appear to play critical roles in the mediation of permissive gene expression supported, in part, by adjacent lysine 4 (K4) methylation and have been implicated in diverse biological and disease processes, ranging from neuronal differentiation to the precipitation of drug-seeking behaviors in adult animals. Our previous work demonstrated that H3Q5ser and H3Q5dop are catalyzed by the Transglutaminase 2 (TGM2) enzyme, both in vitro and in vivo. Here, we discuss the identification of a new class of histone monoaminylation, H3Q5 histaminylation (H3Q5his), which displays dynamic expression in brain in the context of sleep-wake cycles. We further find that H3Q5his, unlike H3Q5ser, electrostatically inhibits recruitment of the chromatin reader protein WDR5 and attenuates MLL1 complex methyltransferase activity on H3K4. Importantly, we demonstrate that H3Q5 monoaminylation dynamics are determined by local concentrations of monoamines, which can be sensed by TGM2. This noncanonical erasure/’rewriting’ mechanism suggests a previously unreported biochemical process through which certain post-translational modifications can be established and removed by a single enzyme based upon its sensing of local cellular microenvironments.
THE GENESIS OF THE GENETIC LANDSCAPE IN NORTHEAST EUROPE
Mait Metspalu1, Lehti Saag1,2, Lena Kushniarevich1, Toomas Kivisild1,3, Aivar Kriiska4, Kristiina Tambets1
1Institute of genomics, University of Tartu, Tartu, Estonia, 2Research Department of Genetics, Evolution, and Environment, University College London, London, UK, 3Department of Human Genetics, KU Leuven, Leuven, Belgium, 4Institute of Archaeology, University of Tartu, Tartu, Estonia
It has been well established that postglacial peopling of Europe has involved three major demic expansions. Hunter-gatherers (HG) and early agriculturalists both from West Asia and pastoralists from the Pontic steppe. In addition, there is a more easterly influence in NE Europe detected in the Iron Age. Here we refine this broad narrative in NE Europe. We first explore the dynamics of western and eastern HGs in the Baltics, suggesting more genetic connections between people associated with different cultures/periods than previously thought. Then we document both abrupt and continuous patterns of genetic change during and after adoption of agriculture in the northern East European Plain. A genetic change with the arrival of steppe ancestry can be seen all over the region, whereas later changes are more subtle and region-specific. Finally, we explore patterns of genetic sharing between Estonians and Finns using large scale biobank datasets and novel methods. Despite substantive differentiation in allele frequencies, the two populations sport unexpectedly many segments of long shared allele intervals dating roughly to around 5th/6th century AD. This shows the importance of relatively recent events for the formation of contemporary populations.
IN THE ABSENCE OF SKELETAL REMAINS: STUDYING ANCIENT HUMAN DNA PRESERVATION IN SEDIMENT
Max Planck Institute for Evolutionary Anthropology, Evolutionary Genetics Department, Leipzig, Germany
In 2017, we reported the recovery of Neandertal and Denisovan mitochondrial DNA from Pleistocene cave sediments, suggesting that the analysis of sediment DNA may overcome our dependency on the scarce fossil record for investigating the human past. Yet, this work also raised a number of questions that are important to determine the relevance of this newly discovered source of DNA for future research: How common is ancient human DNA preservation in sediment? What are the temporal limits of ancient DNA preservation in sediment? What is the source of this DNA? Do single samples contain DNA from one or several individuals? Can human DNA move between layers? What are the limits of resolution that can be achieved in the analysis of DNA sequences from sediment? To address these questions, we have undertaken several large studies on different scales. On a global scale, we screened sediment samples from more than 200 archaeological sites, mostly in Eurasia, to assess the preservation of ancient human and faunal DNA. On a local scale, we reconstructed genetic time-series data from archaeological sites based on hundreds of sediment samples. For example, at Denisova Cave in Russia, we detected the DNA of Neandertals, Denisovans and modern humans in 175 out of 728 samples, enabling the reconstruction of the occupational history of the site at an unprecedented level of resolution. In Galería de las Estatuas in Spain, we expanded our search of DNA in sediment from mitochondrial to nuclear DNA, which allowed us to detect a turnover in the Neandertal populations that occupied the site. On a microscale, we analyzed DNA from blocks of sediment that had been impregnated in resin for micromorphological analysis, targeting microstructures derived from bone fragments, coprolites and minerals. Together, the results of these studies show that ancient human DNA is a very small yet frequently detectable component of DNA in Pleistocene cave sediment, that the isolation of the DNA from single individuals is possible from sediment, and that the movement of DNA across archaeological layers is not a common phenomenon. Current efforts focus on the improvement and simplification of methods for the recovery and analysis of DNA sequences from sediment, with the ultimate goal of making sediment DNA analysis a more widely used tool in prehistoric archaeology.
METABOLIC REPAIR: A NEW APPROACH TO THE TREATMENT OF ABNORMAL GLYCOSYLATION
Mayo Clinic, Department of Clinical Genomics, Rochester, Minnesota, USA
Congenital disorders of glycosylation are a group of genetic disorders that affect protein and lipid glycosylation and glycosylphosphatidylinositol synthesis. For most affected individuals, only symptomatic and preventive treatments are used. Innovative diagnostic approaches using functional glycomics, novel biomarkers, animal models have been developed and our emerging experience with organ transplantation led us to new therapeutic approaches. Most metabolic enzymes demonstrate substrate specificity, including most enzymes of the monosaccharide activation pathways, sugar nucleotide synthesis and transport; pathways, which are very important in the glycosylation process. In most of the cases, one compound is a much better substrate (usually 100- to 1000-fold) than the other structurally similar molecules. The study of inborn errors of metabolism thought us that enzyme specificity is not perfect. Numerous examples exist of “promiscuous” reactions in the context of inborn errors of metabolism, when the concentration of a substrate is highly elevated due to a metabolic block, leading to the production of unexpected metabolites. However, we can also make a use of such a flexibility in treating of inborn errors. The administration of a molecule (e.g., a specific monosaccharide) in high concentration can turn a molecule into a substrate for alternative pathways. In the lack of absolute specificity of metabolic enzymes increasing certain sugar metabolites can activate enzymes of decreased activity leading to suboptimal, but measurable kinetics. Proteins are flexible and amino acid side chains may alter their secondary structure to accept a molecule that is slightly different from the ideal substrate. Emerging metabolic therapies are using this concept in several glycosylation disorders (MPI-CDG, SLC35A2-CDG, SLC35C1-CDG and PGM1-CDG). High throughput drug screens led to unexpected discoveries like the demonstrating the efficacy of epalrestat in altering metabolic flux and enzyme activity in the most common congenital disorders of glycosylation, PMM2-CDG. Here we focus on recent advances in potential therapeutic approaches for CDG.
EPIGENETIC SIGNATURES OF PSYCHOSOCIAL STRESS AND TRAUMA
Connie J. Mulligan
Department of Anthropology, Genetics Institute, University of Florida, Gainesville, FL, USA
The field of social and behavioral epigenetics examines how social and behavioral experiences, such as psychosocial stress, can lead to epigenetic changes. We investigate how psychosocial stress or trauma that is experienced during pregnancy may induce epigenetic marks in future generations. Specifically, we test for associations between maternal stress or trauma, changes in DNA methylation, and health outcomes. Results from two collaborative projects are reported: 1) associations of maternal stress, newborn birthweight, and newborn epigenetic changes in a longitudinal study of mother-infant dyads from the Democratic Republic of Congo (DRC) and 2) investigation of epigenetic signatures of violence trauma in three groups of three-generation Syrian families with contrasting exposures to war violence. DNA methylation (DNAm) data were generated using the Illumina MethylationEPIC BeadChip. In the DRC project, babies at birth (n=66), 6 months (n=7), 1 year (n=34), 2 years (n=31), and 3 years (n=16) were investigated. We identified a signal of epigenetic age acceleration that was significantly correlated with low birthweight and only emerged over time. There was no correlation of maternal stress with newborn DNAm. These results suggest that the impact of low birthweight on adult health may be mediated by epigenetic changes that emerge over the life course. In the Syria project, 45 individuals directly exposed to war violence, 30 prenatally exposed, 15 germ-line exposed, and 45 control individuals were analyzed. Methylation at multiple CpG sites was associated with violence trauma at all three levels of exposure even after strict Bonferroni correction for multiple testing. These results suggest that exposure to violence experienced during pregnancy may impact future generations at the epigenomic level.
Funding statement: This research was supported by NSF grants BCS-1231264, BCS-1719866, and BCS-1849379 and the University of Florida (UF) College of Liberal Arts and Science.
SINGLE-CELL OMICS FOR FORENSIC USAGE
Department of Cell Biology, Department of Genetic Identification, Erasmus University Medical Center Rotterdam (Erasmus MC), Rotterdam, The Netherlands
The separation of individuals from biological mixture and the subsequent genetic characterization and individual identification steps are crucial components of forensic investigation that still pose a significant challenge to the field despite several attempts. Single-cell (multi)omics approaches are an emerging set of tools that are providing an unprecedented level of understanding about cellular identity and processes. In the past few years, several single-cell techniques have emerged that can capture the transcriptome (scRNA-seq), epigenome (e.g., chromatin accessibility, scATAC-seq; DNA-methylation), and genome. These techniques have the potential to solve the challenges that are faced in forensic research, but their adaptation is still lagging behind. Thus, we are exploring the possibility of adapting existing single-cell approaches and develop novel ones for forensic research. First, by using existing scRNA-seq and by developing a novel SNP-based mixture deconvolution bioinformatics pipeline, we succeeded to separate individuals from multi-person blood mixtures according to the individual contributors. In subsequent steps, we were able to determine the sex and biogeographic ancestry (maternal, paternal, and bi-parental ancestry) of the separated individuals and the tissue of origin of the biological mixture. In addition, by comparing the individual SNP profile (from the scRNA-seq) with a reference set (exome-seq), we were able to achieve individual identification of the separated contributors. Next, in order to increase the number of SNPs that can be used for mixture deconvolution and increase the ability of separating more complex mixtures, we tested the possibility of using single-cell scATC-seq and obtained robust separation. At the current state, our novel approach has the potential to identify perpetrators of violent crime from blood mixtures found at crime scenes, while further adaptations may allow moving to other types of biological mixtures. Driven by this success, we are now developing other novel and affordable forensic-specific single-cell methods that will allow determining appearance, ancestry, sex, and other forensically important information.
IMPACT OF DISORDERED METABOLISM ON 3D GENE REGULATION IN DIABETES
Mayo Clinic, Rochester, Minnesota, USA
Diabetes mellitus involves systemic alterations in metabolic functions, which contribute to end-organ dysfunction leading to diabetic complications. We previously reported that reduced mitochondrial gene expression and mitochondrial density in type 1 and type 2 diabetic patients were associated with peripheral autonomic neuropathy and delayed gastric emptying. Investigating the underlying mechanisms, we found impaired oxidative metabolism to associate with reduced physiologimarical hypoxic signaling in human and mouse enteric neurons. Genetic and pharmacological manipulations of hypoxia-inducible factor 1 alpha (HIF1A) revealed a role for HIF1A in the regulation of expression of neuronal nitric oxide synthase (NOS1), the source of the gaseous inhibitory neurotransmitter nitric oxide, an established regulator of gastric emptying. Genome-wide analysis of HIF1A binding in conjunction with chromosome conformation capture identified a role for HIF1A in the regulation of target genes including Nos1 by modifying short- and long-range cis-regulatory interactions in part via cohesin recruitment to loop anchors. Pharmacological upregulation of HIF1A levels with a drug approved for use in humans reversed diabetic gastroparesis in female streptozotocin-diabetic mice. In a parallel line of studies, we investigated the role of mitochondrial dysfunction in interstitial cells of Cajal (ICC), electrical pacemaker and neuromediator cells of the gut, which have also been implicated in diabetic gastroparesis. Genetic deletion of the mitochondrial tricarboxylic acid cycle and electron transfer chain enzyme complex succinate dehydrogenase in ICC altered repressive histone and DNA modifications and Kit expression without any deleterious effects on gastric emptying in normal mice but dramatically increased the prevalence and severity of gastroparesis in female but not male diabetic mice. Together, our studies highlight transcriptional and epigenetic mechanisms downstream of impaired mitochondrial function in diabetic enteric neuropathy and gastroparesis.
SOLVING THE MYSTERY OF KASPAR HAUSER
Medical University of Innsbruck, Institute of Legal Medicine, Innsbruck, Austria
Molecular genetic identification of historic individuals, also known as Celebrity Genetics, has become a recognized discipline in forensics. These investigations are not only finding interest among a broad audience, the challenging biological material involved often requires the development of alternative technical solutions to yield successful results. Thus, new concepts stimulated by difficult cases have contributed to the field in significant ways. The molecular investigations on remains attributed to Kaspar Hauser perfectly add to this tradition. Kaspar Hauser was a celebrity, the center of curiosity of Germany’s Biedermann society, a feature in newspapers and an object of interest to visitors of the city of Nuremburg in the early 19th century. In May 1828, he appeared in Nuremberg seemingly out of nowhere, a lubberly appearance that was barely able to speak and walk. According to his own account, for as long as he could remember, he sat in a small, dark dungeon without ever getting to see anybody else. His only companions were two horses and a dog, all made of wood. While it still remains unclear whether or not his story holds true, the fate of a child growing up in lack of any social contact has been a focus of academic research throughout the past two centuries. As a matter of fact, evidence was brought forward that Kaspar Hauser could have been an abducted prince of the Grand Duchy of Baden, South Germany. In an attempt to shed more light on this assertion, forensic genetic analyses were conducted on samples attributed to him and samples from pedigree members of the House of Baden in the late 1990s and early 2000’s. These analyses led to contradictory results. Some of these results were scientifically published, others were only discussed in the media, which left the case unsolved and provided room for speculations. With the emergence of novel forensic genetic methods, including Massively Parallel Sequencing, the case was reopened again in 2019. Old and new samples were investigated using these methods. These analyses not only yielded significant results; they also serve as basis to explain the discrepant data obtained 20 years ago. The new methods and conclusions further provide foreground for the field of forensic genetics and answer some of the questions regarding Kaspar Hauser’s descent.
OSTEOARTHRITIS: FROM MOLECULAR PATHWAYS TO THERAPEUTIC ADVANCES
1St. Catherine Specialty Hospital, Zagreb, Croatia, 2Medical School, University of Zagreb, Zagreb, Croatia, 3University of Osijek Faculty of Dental Medicine & Health, Osijek, Croatia, 4University of Split, School of Medicine, Split, Croatia, 5Department of Biochemistry & Molecular Biology, The Pennsylvania State University, State College, PA, USA, 6The Henry C Lee College of Criminal Justice & Forensic Sciences, University of New Haven, West Haven, CT, USA, 7The National Forensic Sciences University, Gandhinagar, Gujarat, India, 8University of Rijeka, School of Medicine, Rijeka, Croatia, 9Medical School REGIOMED, Coburg, Germany, 10School of Medicine, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia, 11Medical School, University of Mostar, Mostar, Bosnia and Herzegovina
Being the most common musculoskeletal progressive condition, osteoarthritis is an interesting target for research. It is estimated that the prevalence of knee osteoarthritis (OA) among adults 60 years of age or older is approximately 10% in men and 13% in women, making knee OA one of the leading causes of disability in the elderly population. Today, it is clear that osteoarthritis is not a disease characterized by loss of cartilage due to mechanical loading only, but a condition that affects all tissues in the joint, causing detectable changes in tissue architecture, metabolism, and function. The pathogenesis of OA is primarily determined by the imbalance of pro-inflammatory and anti-inflammatory mediators, leading to low-grade inflammation, which is responsible for cartilage degradation, bone remodeling, and synovial proliferation. The pathogenesis of this degenerative process is not completely understood; however, a low-grade inflammation leading to an imbalance between anabolic and catabolic processes is a well-established factor. The complex network of cytokines regulating these processes and cell communication has a central role in the development and progression of osteoarthritis. In addition, concentrations of both proinflammatory and anti-inflammatory cytokines were found to be altered depending on the osteoarthritis stage and activity. At the moment, biological treatments such as platelet-rich plasma, bone marrow mesenchymal stem cells, and autologous micro-fragmented adipose tissue (MFAT) containing stromal vascular fraction (SVF) are ordinarily used. The cell-based treatment options seem to be the only methods so far that increase the quality of cartilage in osteoarthritis patients. Mesenchymal stem cell (MScs) research offers new opportunities for osteoarthritis treatment as their paracrine effect exhibits clinical improvement in osteoarthritis patients, providing much-needed minimally invasive treatment options. In my lecture, I will present several prospective, non-randomized, interventional, single-center, open-label clinical studies performed at St Catherine hospital where patients with OA, were treated with the intraarticular application of autologous micro-fragmented adipose tissue (MFAT) containing stromal vascular fraction (SVF). After the treatment, dGEMRIC sequencing was used to analyze the contents of cartilage glycosaminoglycans (GAGs) in specific areas of the treated knee joint as the anionic, negatively charged contrast gadopentetate dimeglumine (Gd-DTPA2−) infiltrates into the cartilage, thus indirectly showing the amount of GAGs in areas of interest at different time points. Our results showed a stable dGEMRIC index in the first 12 months after application of MFAT and a mild decrease in dGEMRIC index after 24 months of application. We believe that molecular changes in the cartilage in patients with OA are mediated by a complex interplay of pro-inflammatory and anti-inflammatory cytokines, chemokines, growth factors, and adipokines. Therefore, we recently launched one of the most comprehensive multi-omic study (Clinical and molecular phenotypization of OA: personalized approach to diagnostics and treatment) on OA patients, aiming to explore cytokines, chemokines, N-glycans, phenylalanine, and miRNA changes in the plasma and synovium before and after intraarticular application of MFAT with SVF in knees of the patients with OA. Simultaneously, we will observe changes in the glycosaminoglycans level (GAG) by using delayed gadolinium (Gd)-enhanced magnetic resonance imaging of cartilage (dGEMRIC), but also, we will perform a standard orthopedic physical examination including KOOS, WOMAC, VAS, CESD-R assessments as well as MRI Osteoarthritis Knee Score (MOAKS).
AFTER MORE THAN 20 YEARS: UPDATE ON THE DNA IDENTIFICATION OF 9/11 VICTIMS
John Jay College of Criminal Justice, New York, NY, USA
The terrorist attack on the World Trade Center in New York City on September 11, 2001 took the lives of at least 2,753 individuals on the ground and on the two airplanes. As of March 2022, 1,647 or approximately 60% of the victims have been identified. This mass fatality incident was characterized by severe fragmentation, degradation, and destruction of the human remains, which explains why so many of the victims seem to have disappeared. With only 289 intact bodies, but over 20,000 fragments of human remains, the World Trade Center victim identification became a DNA driven effort. Short tandem repeat (STR) analysis was the main tool for typing muscle tissue and bone samples, as well as ante mortem personal effects and buccal references. The project triggered the development of DNA matching software tools and quality review and anthropological verification procedures. Victim identification took place in several distinct phases and is still ongoing today. Systematic resampling of previously unidentified remains has led to additional identifications, with the most recent ones having been reported in September 2021. The talk will provide an overview on lessons learned during the project and present the most recent developments.
CHARACTERIZING LOCUS SPECIFIC CHROMATIN STRUCTURE AND DYNAMICS WITH CORRELATIVE CONVENTIONAL AND SUPER-RESOLUTION IMAGING IN LIVING CELLS
Dushyant Mehra1,2, Santosh Adhikari1, Elias M. Puchner1
1School of Physics and Astronomy, University of Minnesota, Minnesota, USA, 2Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota, USA
The spatio-temporal organization of chromatin is critical for gene regulation. However, simultaneously mapping the structure of chromatin and its dynamics remains a challenge. Conventional fluorescence microscopy in combination with CRISPR/dCas9 labeling has been able to image the spatial distribution and dynamics of loci in living cells, but cannot resolve structural features below ~250 nm. The advent of super-resolution photoactivated localization microscopy (PALM) presented a breakthrough for resolving intracellular structures with up to ~20 nm resolution in fixed cells. However, the motion of chromatin during the long data acquisition time precludes any structural characterization of chromatin in living cells due to motion blurring. Here I will present our correlative conventional fluorescence and PALM imaging approach to quantitatively map time-averaged structure and dynamics of chromatin below the optical diffraction limit in living cells. By employing a repetitive telomere sequence as a well studied model system and by assigning localizations to a telomere as it moves, we reliably discriminate between bound and unbound dCas9 molecules, whose mobilities overlap. Our approach accounts for changes in DNA mobility and relates local chromatin motion to larger scale domain movement. In our experimental system, we show that compacted telomeres move faster and have a higher density of bound dCas9 molecules, but the relative motion of those molecules is more restricted than in less compacted telomeres. Correlative conventional and PALM imaging therefore improves the ability to analyze the mobility and time-averaged nanoscopic structural features of locus specific chromatin with single molecule sensitivity and yields unprecedented insights across length and time scales.
TARGETING EPIGENETIC REGULATOR MUTATIONS IN KIDNEY CANCER
Ryan T Wagner, Ryan A Hlady, Xiaoyu Pan, Liguo Wang, Sungho Kim, Jeong-Heon Lee, Thai Ho, Keith D Robertson
Mayo Clinic, Rochester, Minnesota, USA
Clear cell renal cell carcinoma (ccRCC) accounts for ~75% of kidney cancers and is the 8th leading cause of cancer death in the United States. After completion of The Cancer Genome Atlas (TCGA) Project, clinically actionable mutations were identified in virtually every solid tumor. One major exception, however, is RCC, where the current standard of care, checkpoint inhibitor and anti-VEGF therapy, does not take into account that ~50% of RCCs have mutations in chromatin regulators. After first-line therapy, response rates are 20% and there are no FDA-approved therapies that target chromatin regulators, highlighting the need to identify how epigenome regulator mutations can be therapeutically targeted. The epigenome is profoundly disrupted in cancers including ccRCC, including altered DNA and histone methylation patterns that promote oncogenic transcriptional patterns and elevated DNA damage. Aside from the near ubiquitous loss of VHL, the mutational landscape of ccRCC is dominated by loss-of-function mutations in epigenetic regulators, including SETD2, BAP1, and PBRM1. SETD2, the sole factor responsible for trimethylating the histone H3 lysine 36 position, has been firmly linked to poor outcome and the promotion of metastasis. SETD2 and its mark H3K36me3 have been linked to diverse processes ranging from transcriptional regulation, mRNA splicing, nucleosome positioning, and DNA repair, yet exactly how this regulator and its mark drive cancer phenotypes, particularly in ccRCC, remains unknown. Using a combination of engineered cell line models, biochemical methods, and primary patient tumors, coupled with transcriptome/epigenome analysis and interaction studies, we describe novel ways that SETD2 loss-of-function contributes to cancer initiation and progression. We also probe the interplay among multiple regulators of methylation at the H3K36 position to define novel pharmacologic paradigms that may lead to individualized therapies that target SETD2 mutant tumors.
MULTIOMICS REVEALS NEW PATHOBIOLOGIC TARGETS FOR ALCOHOL ASSOCIATED HEPATITIS
Vijay H. Shah
Mayo Clinic, Rochester, MN, USA
Alcoholic hepatitis (AH) is associated with liver neutrophil infiltration through activated cytokine pathways leading to elevated chemokine expression. Super-enhancers are expansive regulatory elements driving augmented gene expression. Here, we explore the mechanistic role of super-enhancers linking cytokine TNFα with chemokine amplification in AH. Our findings highlight the role of super-enhancer in propagating inflammatory signaling by inducing chemokine expression and the therapeutic potential of BET inhibition in AH treatment. Alcohol-associated liver disease (ALD) in its earliest form is evidenced as hepatic steatosis which may progress to liver cirrhosis. The mechanisms behind this initiating insult are poorly understood and therapeutics to treat ALD are limited. Liver is a specialized organ with cells exhibiting heterogeneity along the porto-central axis. Periportal preponderance of lipid droplet accumulation was noted in human ALD livers compared to other clinical causes of hepatic steatosis. Using single-cell multiomics technology, we studied transcriptional mechanisms across the hepatic lobule that could account for liver zonation of lipid droplets in a murine ALD model. We utilized multiomics data to provide novel insight into HNF4α and PPARα mediated, zone-specific regulation of HSD17β13. We conclude that mechanisms underlying ALD initiate in a zonated manner leading to spatially distinct establishment of hepatic steatosis and provide novel insight into disease pathogenesis.
THE ORIGINS OF HANSEN’S DISEASE (LEPROSY)
Adele Crane1, Kelly Blevins2,3, Felicia King1, Christopher Lum4, Kanako Furuta5, Luciano Nakazato6, Mrinalini Watsa7,8, Gideon Erkenswick7,9, Keolu Fox10, Anne Stone2,11
1School of Life Sciences, Arizona State University, Tempe, AZ, USA, 2Center for Bioarchaeological Research, Arizona State University, Tempe, AZ, USA, 3Department of Archaeology, Durham University, Lower Mount Joy South Road, Durham, UK, 4Department of Pathology, John A Burns School of Medicine, University of Hawaii, Honolulu, Hawaii, 5Hawaii Pathologists Laboratory, Honolulu, Hawaii, 6Department of Veterinary Medicine, Universidade Federal de Mato Grosso, Cuiabá, Mato Grosso, Brazil, 7Field Projects International, Escondido, CA, USA, 8San Diego Zoo Wildlife Alliance, San Diego, CA, USA, 9Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA, 10Department of Anthropology, University of California, San Diego, La Jolla, CA, USA, 11School of Human Evolution and Social Change, ASU, Tempe, AZ, USA
Hansen’s disease, also known as leprosy, is caused by the pathogens Mycobacterium leprae and the more recently discovered M. lepromatosis, which is primarily found in Mexico and the Caribbean. Hansen’s disease is one of the oldest known human diseases and remains a public health issue today, with over 200,000 new cases reported yearly. Ancient genome analyses show that M. leprae lineages have a most recent common ancestor approximately five thousand years ago. However, the global pattern of genomic variation in M. leprae is not well defined. This is particularly true in the Pacific Islands, where the origins of the pathogen in humans in relation to Colonialism are disputed, and in animals, which are poorly surveyed for this pathogen. To investigate this, we have extracted DNA from 98 formalin-fixed paraffin-embedded biopsy blocks collected between 1992 to 2016 from patients living in the Pacific and from PCR positive samples from 11 species of animals from Brazil. We have also survey small mammals in the Amazon basin of Peru. To date, we have successfully used whole-genome enrichment and next-generation sequencing to generate 12 Pacific M. leprae genomes ranging from 1.6 – 63x depth of coverage. Phylogenetic analyses place these strains in branches 0 and 5, the basal lineages of the M. leprae phylogeny. The phylogeographical patterning and evolutionary dating analysis of these strains support a pre-modern introduction of M. leprae into the Pacific Islands. We have also expanded this work by including time-series samples from patients during treatment and will use both empirical data and modelling to identify ongoing selection. In Peru, the 92 small mammals tested to date have been negative using the RLEP qPCR assay, while initial capture of the Brazilian samples is in progress. This research provides insight into the evolutionary history of M. leprae and the exchanges of this pathogen among species.
GENES, CULTURE, AND HUMAN EVOLUTION
1Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany, 2Université Lyon 1, CNRS Laboratoire de Biométrie et Biologie Evolutive, Villeurbanne, France
A commonly-held view is that humans have stopped evolving because we rely on culture to adapt to changing circumstances. However, an alternative view is that culture can also influence human evolution. I will show, by way of examples, that some cultural practices have directly impacted specific genes, while others have indirectly influenced patterns of genetic variation. Furthermore, given that some cultural practices have genetic consequences, we can use genetic analyses to learn more about such cultural practices; as an example, I will discuss a genetic approach to dating the origin of clothing.
INFERRING GENETIC RELATIONSHIPS FROM WHOLE GENOME SEQUENCES – A FORENSIC PERSPECTIVE
1National Board of Forensic Medicine, Department of Forensic Genetics and Forensic Toxicology, Linköping, Sweden, 2Linköping University, Faculty of Medicine and Health Sciences, Department of Biomedical and Clinical Sciences, Linköping, Sweden
Being able to determine genetic relationships between individuals has long been important in legal medicine and forensic applications, like paternity testing and missing person identification. Current practice typically involves DNA analysis of a small number (around 20) of short tandem repeat markers, which usually is sufficient to establish close relationships. Recent advances in DNA typing technologies have however made it possible to a relatively low cost, with increased sensitivity and with high quality, obtain much more DNA information from a single DNA analysis. This opens up new possibilities for inferring distant relationships but also for introducing new forensic applications such as investigative genetic genealogy. Large DNA datasets may require new methods for the relationship inference and for the assessment of the statistical weight. This presentation will provide an overview of such methods and show how large DNA datasets, obtained from whole genome sequencing, could be used to infer genetic relationships in legal and forensic settings.
REGULATION AND DEREGULATION OF DNA EPIGENETIC MARKS
University of Minnesota, Minneapolis, MN, USA
Reversible DNA methylation allows for the precise activation and inactivation of genes in a tissue-specific manner by mediating DNA-protein interactions and influencing chromatin structure. 5-Methylcytosine (5mC) marks are introduced by DNA methyltransferases (DNMT), while DNA demethylation is initiated by ten eleven translocation (TET) dioxygenases which oxidize 5mC to hydroxymethyl-C (hmC), formyl-C (fC) and carboxy-C. While DNA methylation patterns are stable in healthy somatic tissues, they can become scrambled as a result of inflammation, environmental stress and chemical exposures, contribution to cancer initiation. Recent studies revealed that many other diseases including asthma, Alzheimer’s disease, and autism have epigenetic drivers. My laboratory investigates the potential role of DNMT and TET proteins in inflammation-mediated colon cancer and smoking induced lung cancer. We have conducted animal studies to show that chronic inflammation and chronic exposure to cigarette smoke cause dramatic changes in DNA hydroxymethylation, gene expression changes, and aberrant protein expression. Mechanistic studies with recombinant proteins probes potential mechanisms for the observed epigenetic changes, while CRISPR-cas 9 gene editing and RNA interference studies are starting to reveled the functional roles of the affected genes in lung cancer. We employed structure-based design to develop small molecule inhibitors of TET proteins, which can be used as chemical probes to investigate the functions of DNA demethylating enzymes in lung cancer and could comprise initial leads for future drug design. Finally, we are investigating reversible cross-linking between fC in DNA and histone proteins as a potential novel mechanism of epigenetic regulation.
DEVELOPMENTS TOWARDS PERSONALIZED EPIGENOMIC PROFILING IN FORENSICS
Erasmus MC, University Medical Center Rotterdam, Department of Genetic Identification, Rotterdam, The Netherlands
Human genetic variation is a major resource in forensics, but does not always allow us to answer crucial forensically relevant questions. Since the epigenome acts as an interphase between the fixed genome and our dynamic environment, it offers possibilities to address many questions regarding an individual’s phenotype. Back in 2017, we proposed that, together with genetic prediction of appearance and biogeographical ancestry, DNA methylation-based ageing and lifestyle habit prediction is expected to increase the ability of narrowing down suspect pools. However, there are still various challenges to be addressed prior to implementing such approach in forensics. During this talk, I aim to present our recent developments towards offering a personalized epigenomic fingerprint. First, to consider the issue of dealing with heterogeneous material, we focused our efforts investigating age-associated patterns of the Y-chromosome, in both blood and sperm. We envision that a future Y-chromosome based age prediction tool would allow us to estimate the age of males from mixed samples, often encountered in sexual assault cases. Secondly, to apply already existing knowledge on lifestyle-associated DNA methylation, we built and thoroughly validated statistical models for the prediction of both tobacco smoking and alcohol consumption habits. In the case of smoking, we also developed a targeted lab method based on next-generation sequencing, which we optimized and tested in a population cohort. Thirdly, to improve DNA methylation detection towards standardization, we developed and validated a novel, patent-pending tool for assessing the initial step of bisulfite conversion, which is currently the golden standard. Finally, to deal with the limited amount of available human biological material, we are currently focusing in developing a new technology, CpGtracer that will soon allow us to simultaneously analyze hundreds of DNA methylation markers from trace amounts of DNA. Overall, while there are still additional considerations to tackle, including privacy, ethical and legal concerns, we are confident that a broadened DNA-based forensic intelligence including epigenomic profiling will soon become valuable, especially during criminal investigations with unknown suspect(s).
CANCER PHARMACOGENOMICS: DISCOVERY AND TRANSLATION
Mayo Clinic, Rochester, MN, USA
A major challenge facing genomics science is understanding and predicting how sequence variation in noncoding regions of the genome might contribute to variation in gene regulation, variation that could result in variations in various phenotypes either cellular or clinical phenotypes. This challenge is highlighted by the fact that approximately 90% of genome-wide association study (GWAS) single nucleotide polymorphism (SNP) signals map outside of protein coding genes (1-3). A significant advance has been our recognition that many of these GWAS SNP signals locate in the non-coding regions, regulating gene expression through enhancer, so called “expression quantitative trait loci” (eQTLs), SNPs that are associated with variation in gene expression (3, 4). Here, we will present a novel mechanism that we have identified repeatedly for these SNPs using breast cancer clinical trial DNA samples to understand how the SNP might influence drug response, both efficacy and toxicity. What we have found is that the SNP effect on gene expression, leading to different clinical outcome, is dependent on the presence of drugs/hormones mediated though nuclear receptors such as the estrogen and glucocorticoid receptors. This type of SNP-gene expression relationship is often not present or is much less significant at baseline—ie before ligand (either agonists or antagonists) exposure, and occurs only in the presence of a drug or hormone. This type of SNP- gene expression relationship highly depends on the presence of individual endogenous or exogenous compounds, so called PGx eQTL, which can occur often and can be highly significant functionally. As a result, we could potentially take advantage of specific SNP genotypes to manipulate gene expression by exposure to various compounds. These SNPs could be also used as biomarkers associated with various clinical phenotypes.
SIGNAL-DEPENDENT CHROMATIN REMODELING IN METABOLISM AND INFLAMMATION
Liu Wang, SM Bukola Obayomi, Zong Wei
Mayo Clinic, Department of Physiology and Biomedical Engineering, Scottsdale, AZ, USA
In type 2 diabetes (T2D), inflammation induces massive changes in the transcriptome and epigenome of islet endocrine and immune cells, resulting in eventual dysfunction of islets. We identified a novel mechanism of vitamin D-dependent chromatin accessibility dynamics, orchestrated by the balance between two SWI/SNF chromatin remodeling complexes, BRD9-BAF and BRD7-PBAF, in regulating islet dysfunction. In beta cells, the balance between BAF-BRD9 and PBAF-BRD7 determines the VDR-driven anti-inflammatory and pro-survival response. Pharmacologically potentiated VDR signaling by a synthetic ligand in combination with a BRD9 inhibitor can partially restore beta cell function and glucose homeostasis in various T2D mouse models. Tissue specific genetic models further demonstrated the functional role of VDR and BRD9 in beta cell stress response in vivo. Recently, we also identified BRD9 as a modulator for glucocorticoid responses in macrophages. Pharmacologic inhibition of BRD9 potentiated the anti-inflammatory responses of dexamethasone. Mechanistically, BRD9 co-localized at a subset of GR genomic binding sites, and depletion of BRD9 enhanced GR occupancy primarily at inflammatory-related genes to potentiate GR-induced repression. Together, our results revealed the context-dependent function of specific SWI/SNF subunits on VDR/GR activity, and demonstrated the therapeutic potential of targeting bromodomain readers to synergistically enhance NR function.
NEANDERTAL GENE VARIANTS AND COVID-19
1Karoliniska Institutet, Department of Neuroscience, Stockholm, Sweden, 2Max Planck Institute for Evolutionary Anthropology, Department of Evolutionary Genetics, Leipzig, Germany
Early in the COVID-19 pandemic, it was clear that SARS-CoV-2 infection tend to have drastically different outcome in different people. Whereas some patients suffer only mild disease, others become critically ill. Some risk factors, such as high age and metabolic syndrome, can explain some of the variability but far from all of it. Therefore, host genetic risk factors have been investigated by several large studies, which have been successful in identifying more than 20 risk genetic risk factors influencing the outcome of COVID-19. In this talk I will describe the evolutionary history and the Neandertal origin of two of the major genetic variants influencing the outcome of COVID-1