June 29, 2020 – 12:15 – 4:45pm EDT
Chairs: John Moran, PhD and Kathy Burns, MD, PhD

Meeting Goals:

1. Identify opportunities and challenges for fundamental investigations: Define the “hard problems” or pressing questions on the drivers (biological and non-biological) and consequences of somatic mosaicism (SM) and Retrotransposition on human development and disease
2. Identify opportunities and challenges for translation – biomarkers and clinical applications
   This workshop will identify key opportunities that would advance the field of TE/SM with a goal of translating basic research to clinical applications. Overarching questions: Do we need additional fundamental science investigations or development of new technologies and resources?

Agenda

12:15 to 12:30 Speakers, discussants, and Chairs join

12:30 to 12:40 Opening Remarks — NIH Staff

Goals of the workshop and expected outcomes - Chairs: Kathy Burns & John Moran

12:40 to 1:00 Introductory Talk — Current and emerging science in Somatic Mosaicism and Retrotransposition
– John Moran and Kathy Burns

Introduction to SM and the types of SM mechanisms including various TEs in the human genome, mechanisms of mobility, TE-based intra- and inter-individual human genetic diversity, consequences of SM human development and disease.

1:00 to 1:40 Drivers of Somatic Mosaicism and Retrotransposition across Cell-types and Developmental Times Periods

Lead: Prescott Deininger
Discussants: Mike McConnell; Scott Devine Summarizer: Kenneth Ramos

• What are the mechanisms that drive somatic mosaicism (e.g., TE/ERV activation, de novo CNV formation) across human cell types and tissues?
• What are the critical periods of human development for TE activation (e.g., de-repression) and induction of somatic mosaicism for different organ or tissue types? How do these mechanisms vary by life stage (germ cell maturation, embryonic development, aging?)
• What are the contributing factors that drive TE expression or inhibition and result in mosaicism (genetic, epigenetic, biological, environmental exposures, medications, infection, immune activation, aging etc.)?
• Are there specific cell-types or genomic regions that are more permissive/sensitive to TE/SM expression/accumulation?

1:50 to 2:30 pm Consequences on the “Normal” Development and Aging — Current and Emerging science

Lead: Rusty Gage
Discussants: Astrid Haase; Flora Vaccarino
Summarizer: Todd Macfarlan

• How do TEs and SM (e.g., CNVs) impact molecular (e.g., gene regulation, epigenomic landscape) and cellular processes (e.g., signaling) in various cell types across development and disease?
• Are there consequences of TE/SM on normal development and aging process? Do they confer advantages or disadvantages (i.e., adaptation to changing cellular or tissue environments)?
• Is there a threshold of SM and TE events that affect cellular or tissue functions (i.e., the proportion of affected cells)? If so, how can those effects be measured?
• What is the variation in rates of transposition at the population level? Are there differences in maternal and paternal TE/SM alleles? Are there databases that collect this information?

2:35 to 3:20 pm Consequences of SM and Retrotransposition: the Progression of Disease, Disease Susceptibility

Lead: Vera Gorbunova
Discussants: Christine Beck, Cris Bragg
Summarizer: Alysson R. Muotri, Ph.D.

• To what extent are chronic diseases driven by TEs/SM and through what mechanisms or pathways?
• Is there any enrichment for TEs/SM in disease states? Are there measures of retrotransposon activity for an individual and are these enriched in some patient populations (e.g. circulating markers)?
• What is prevalence of TE/SM across diseases in in various populations?
• Are there good models to study the biological/physiological consequences of TE activity/SM during development or in the context of disease? • What are the effects of TEs/SM on inflammation, immune function or other biological processes? How does the immune system recognize and respond to mobilized TEs? 

3:30 to 4:00 Clinical Implications

Lead: Chris Walsh
Discussants: Avi Nath; J Ambati
Summarizer: Mary Crow

• How can current understanding of TEs/SM be applied to the diagnosis and treatment of disease? How can this research be applied to analyzing gene by environment interactions in common and rare diseases?
• Are there clinically relevant biomarkers of TE mobilization and SM? Can we establish TE “signatures” for conditions, cell states, diseases, etc.
• What are some of the co-morbidity factors in clinical contexts (viral infections, other disease states or underlying conditions) that interact with TE activation/SM to affect disease susceptibility?

4:05 to 4:35 Cross-cutting Opportunities & Outstanding Questions in the Field — Group Think Tank Discussion

Moderator: Haig Kazazian

• How do we apply/translate our understanding of TE/SM to disease biology in human?
• What computational approaches, technologies, and data resources are needed to address the questions on the drives and consequences?
• What are the best model systems for studying development / aging / emergence of disease related to TEs / SM?
• What are the opportunities for clinical translation and how do we accelerate that path?

4:35 to 4:45 Wrap up, Action Items, Next Steps — Introduction to Workshop 2. Kathy Burns and John Moran

4:45 pm Adjourn

July 2, 2020 – 12:15 – 4:45pm EDT
Chairs: Molly Gale Hammell, PhD and Alex Urban, PhD

Meeting Goals:

1. Define the capabilities and limitations of tools and resources available for detecting endogenous transposable elements, activation of transposable elements, evidence of transposition, structural variants, and somatic mutations;
2.  Identify new and emerging technologies that can be adapted to detect the events mentioned above.

Agenda

12:00 Speakers, discussants, and Chairs join

12:25 to 12:40 Opening Remarks — NIH Staff

12:40 to 12:55 Summary of Think Tank Meeting #1

Molly Gale Hammell, PhD and Alex Urban, PhD

12:55 to 1:25 Current and Emerging Technologies

Lead: Jef Boeke, PhD (6 minutes)
Discussants: Victoria Belancio, PhD, Charles Lee, PhD (3 minutes each)
Summary: Paul Flicek, PhD (4 minutes)
Questions for general discussion (14 minutes):

• Can we answer this profound question once and for all – retrotransposons – cause or consequence? In the contexts of “natural” aging, cancer, neurodegeneration? Could synthetic genomics help?
• Will long-read technology enable phased diploid genotypes for polymorphic TEs / SVs. Can this be done in the context of somatic mutation and made accessible at low cost?
• How can combination ‘omics inform TE biology in the context of human health, aging and disease?
• What are the definitive and best in class analysis packages for robust detection of sub-stoichiometric repetitive DNAs and SVs?
• Can we see TE activation in living cells in real time? Can we build a “Weather map” for the genome?
• What model mammalian systems are most informative to human health and disease biology?

1:30 to 2:00 Capturing Structural Variants and the Mobile DNA Genome

Lead: Scott Devine, PhD (6 minutes)
Discussants: Dixie Mager, PhD, Ting Wang, PhD (3 minutes each)
Summary: Karen Miga, PhD (4 minutes)
Questions for general discussion (14 minutes):

• What if we could develop an atlas of mobile element insertions and SVs using long-read technologies that spans: germline, multiple adult somatic tissues, subregions of the brain, cancers? This may require improved technologies that combine single cell genomics and long-read sequencing.
• Examine a “reference genome” from blood plus multiple somatic tissues/single cells from diverse heathy individuals
• What somatic tissues and cells are being mutagenized by mobile elements and SVs in healthy individuals? How does this vary across diverse populations? What are the patterns across tissues?
• This would provide a basis to understand when somatic mutagenesis by MEIs/SVs is abnormal and perhaps contributes to human disease.

2:10 to 2:40 Single Cell Analysis of TE/SV/SM

Lead: Chris Walsh, MD, PhD (6 minutes)
Discussants: Jenny Erwin, PhD, Mike McConnell, PhD (3 minutes each)
Summary: Johan Jakobsson, PhD (4 minutes)
Questions for general discussion (14 minutes):

• Clonal somatic mutations in disease—how to we define causality?
  • Recurrent driver mutations in some rare, relatively homogeneous disorders
  • Genetically heterogeneous diseases (ASD, SCZ): need burden of rare variants in disease versus normal
• Degree of genomic heterogeneity between cells
• Changes in genomes with age—SNV, TE, CNV
• Regulators of age-related changes (germline genome, environmental factors, intercurrent illness, etc)
• A forensic cell lineage map of the body exists in the pattern of clonal somatic mutation
• Recurrent patterns of lineage define recurrent mosaic diseases

2:45 to 3:15 Data Analysis: Opportunities and Challenges

Lead: Alexej Abyzov, PhD (6 minutes)
Discussants: Guillaume Bourque, PhD, Jeff Kidd, PhD (3 minutes each)
Summary: Alice Lee, PhD (4 minutes)
Questions for general discussion (14 minutes):

• What infrastructure for data collection would be needed for systems biology approaches and machine learning techniques to provide additional, significant insights?
• What is necessary to mine any existing datasets for additional insights into TE and SM (+SV)?
• What kinds of data-driven approaches will be necessary?
• What resources and technologies are needed to incorporate information about TE and SM (+SV) information into commonly used resources (e.g. the UCSC Genome Browser, Ensembl, etc.)? 

3:25 to 3:55 Current Technical Limitations and Reproducibility

Lead: Jan Korbel, PhD (6 minutes)
Discussants: Kristin Baldwin, PhD, Joanna Wysocka, PhD (3 min each)
Summary: Cedric Feschotte, PhD (4 minutes)
Questions:

• What strategies can be used to validate results of currently available technologies?
• What is the minimum acceptable level of reproducibility?
• Which assays need more work in model systems before being applied to human tissues?
• How can the reproducibility of different assays be improved?
• What are the variables and controls that need to be considered?

4:00 to 4:30 Cross-cutting Opportunities and Outstanding Questions — Group Think Tank Discussion

Molly Gale Hammell, PhD and Alex Urban, PhD
Discussion Questions:

• Single-cell long read technologies are poised to answer many questions, but do we have the tools to make and analyze that data?
• What is the right balance of model organism biology, iPS cells in 2D/3D, and human biopsy/postmortem tissue?
  •  (mechanism, cataloging, exploration of functional consequence, translatability across species…)
• Inflammation and other drivers – are these predominantly driver vs. consequence, and can they be both?
• Do MEIs really decrease with age? Is that cell type/context specific? Is that only for disease-free individuals?
• Do we need to broaden scope to ask about disease associations properly? o (non-mutational impacts of TEs … and/or SNVs/TRs that  be more common in aging/disease)?

4:30 Wrap up and Adjourn

In the fall of 2019, the idea of a new Common Fund program to study the mechanisms of transposable elements (TEs), somatic mosaicism (SM), and somatic variation (SVs) was introduced to OSC staff, and the planning stage begun. In addition to OSC staff members (Richard Conroy, Tony Casco, Dena Procaccini), seventeen NIH staff members were recruited to become part of the workgroup from NCI, NIA, NIDA, NIDCR, NIMH, NICHD, NINDS, NIAMS, and NIEHS to ensure a broad range of expertise for the management of this potential program. The workgroup members convened and decided that the best way to develop a program that would have meaningful impact in the field of mobile elements would be to collect a group of experts and gather their feedback. The group decided that two “Think Tanks” – one on the drivers and consequences of mobile elements, and one on the tools, methods, and resources necessary to study mobile elements were necessary. Around 45 experts in the field were contacted and agreed to lead or participate in the workshops, and other members of the NIH community were welcome to listen to the discussion. Think Tank 1 was held on June 29, 2020, and chaired by John Moran, PhD and Kathy Burns, MD/PhD, and Think Tank 2 was held on July 2, 2020 and chaired by Molly Gale-Hammel, PhD and Alex Urban PhD. Approximately eighty people attended both workshops and said that they were very insightful and engaging.

June 29, 2020
Chairs: John Moran, PhD and Kathy Burns, MD, PhD

The goals of this Think Tank were to identify the mechanisms that drive retrotransposition and somatic mosaicism, and to discover the molecular, epigenetic, and cellular processes across cell-types and developmental time periods. Four sessions were held during the workshop to discuss 1) drivers of somatic mosaicism and retrotransposition, 2) “normal” development and tissue function, 3) inherited variants as contributors to aging and disease, and 4) clinical implications. During the sessions, the researchers suggested the areas of study that would need to be focused on in order to truly advance the field of transposition and somatic mosaicism. Two overarching ideas had almost universal backing. One was that any initiative would need to move beyond simply cataloging the elements to understanding the phenotypic changes they cause, and the other was that in order to study the effects of volatile repetitive elements, the initiative will require the union of cutting-edge genome and computational approaches. In particular, the participants identified seven key areas of development that would benefit from investment from the NIH, and thus could provide long-lasting and critical opportunities to elucidate the scope, mechanism, and impact on human health of repetitive volatile elements.

Key areas of study –

1. The impact of the biological processes of volatile repetitive elements on human health and disease, such as mobilization, how those mechanisms change during the lifespan, contributing factors that drive TE expression and inhibition, variation and frequency between individuals, how many TEs do individuals normally carry, is there a threshold level at which point transposition or mosaicism becomes an issue for the cell, or are there differences between maternal and paternal repetitive element alleles are just some of the biological questions that the researchers suggested should be asked into order to understand the function and processes of TEs.
2. The role epigenetic and environmental influences play in regulating volatile repetitive elements. Will need to develop new epigenomic assays in order to thoroughly study these influences, and bioinformatic techniques to characterize, analyze, and visualize the data from these new assays.
3. Determining which disorders volatile elements play a role in. They have been implicated in many different kinds of disorders, such as neurological, aging, immune system responses, infertility, developmental disorders, and cancer. It was suggested that the new initiative should launch a comprehensive survey of TE/SM related diseases by investigating their mechanisms, biological processes, prevalence of TE/SM in various populations, and co-morbidities that affect disease susceptibility as just a few facets that would need to be understood to fully comprehend the role of these elements in human disease.
4. Develop therapeutics (RNAi, AAVs to deliver shRNA, reverse transcriptase inhibitors) as well as repurposing abandoned compounds were all suggested as ways to moderate the activity and expression of TEs/SMs
5. Develop new high-throughput, multiplexed proteomics and single-cell assays to study both the structural and functional processes that are occurring in response to the TEs. It was mentioned by the researchers that this would be the area that the Common Fund could do the most good, as the current technology is not sufficient to thoroughly study repetitive elements. Current technology ignores repetitive sequences, and any technology that is developed will need to be synergistic with other efforts to be effective. Once efficient, high-throughput sequencing pipelines are developed, it will be necessary to study how the sequences vary from cell to cell, and tissue to tissue.
6. Develop model systems to investigate mobilization of TEs in situ across species in order to study species variation. While mice are commonly used as animal models for TEs, they are not the best animal models to study human TEs. Transgenic marmosets were suggested, as well as other non-human primates, Drosophila, Xenopus, C. elegans, and dogs. Stem cells and organoids were also suggested.
7. Finally, the researchers suggested that this initiative study the whole organism, not just genomic changes, as well as broaden the scope of the program to investigate CNVs as thoroughly as TEs.

July 2, 2020
Chairs: Molly Gale Hammell, PhD and Alex Urban, PhD

The goal of this Think Tank was to define the capabilities and limitations of tools and resources available for detecting: somatic activation and insertion of endogenous transposable elements (TEs), and somatic structural variant (SV) mutations of all types (e.g. deletions, duplications, inversions). Five sessions were held to discuss (1) Current and Emerging Technologies, (2) Capturing the Mobile DNA Genome, (3) Single Cell Analysis of TE/SVs, (4) Data Analysis of TE/SVs, and (5) Current Technical Limitations and Reproducibility. During the sessions, obstacles were identified that currently limit our ability to reliably detect somatic mosaicism from TE-insertions (TEIs) and other SV-generating mechanisms. However, all participants agreed that many emerging tools and technologies are at the cusp of being ready to quantitatively and reproducibly detect somatic mosaicism constituted by TEIs/SVs. In particular, the participants identified 12 key areas of development that, with sufficient and timely support from the NIH, could provide transformative opportunities to fundamentally understand TE/SV somatic mosaicism: its scope, mechanisms of formation, and importantly, its implications for human disease.

Key Areas of Development:

1. Improved experimental protocols and computational tools to enable reliable, efficient, scalable, and affordable generation of genome sequencing data from single-cells (including from linked-reads and long-reads).
2. Improved experimental protocols and computational tools to generate long-reads (and/or linked reads) data from single-cells for other data types, such as transcriptomes and epigenomes, which will enable both an exploration of the mechanisms that lead to somatic mutation as well as a readout of their molecular consequences (also as a tool of validation).
3. Computational tools to combine and integrate data from multiple detection platforms to boost reproducibility and bridge across the limits of individual detection strategies.
4. Computational tools to combine and integrate multi-omics data to facilitate an understanding of the genetic, epigenetic, and environmental cues that drive new mutations in somatic cells.
5. The development of benchmark, ‘gold standard’, references (both experimental and computational) to validate new technologies and analysis methods. This could include reference cell lines with known variants (created by titration/cell-mixing), reference cell lines clonally propagated with known, possibly engineered-in, somatic variants from the parental lines, and others.
6. A comprehensive reference atlas of TE/SV somatic mutations across key developmental timepoints and relevant tissues that would provide expected frequencies of TE/SV somatic mutations, and reveal potential hotspots, to use as a reference and baseline when analyzing disease-associated rates and patterns in patient tissues (combined with developing a stringent set of standards for quality control and experimental validation).
7. The adoption of a comprehensive reference map of known TE and SV locations, and relevant polymorphisms in the germline (e.g. active/hot LINE-1s and all their alleles, SegDup configurations predisposing to SV formation), and the use of a graph-based genome modeling format to represent areas of known variation that new software packages would be motivated to adopt for omics analysis.
8. The comprehensive TE/SV mapping of one or two model organisms (e.g. marmosets, naked mole rats) that could then be used as a platform for exploring mechanisms that drive somatic mutation and its effects on disease states and lifespan.
9. Imaging technologies to visualize TE activity / de novo SV formation, ideally in living cells, to better understand questions about interactions with cell cycle, the likelihood that increased mutations lead to cell death, and the potential interactions with immune cells and/or innate immune pathways that detect products of TE/SV activity.
10. Support for a comprehensive effort to combine the integration of existing resources (e.g. GTEx, ENTEx, TCGA, PsychENCODE, the Brain Somatic Mosaicism Network, The telomere-to-telomere (T2T) consortium, the Human Cell Atlas, and others). These could then be enhanced and filled-in as needed and where opportune, with newly generated resources.
11. Development of technologies to detect TE activity and SV formation in liquid biopsies (e.g. leaked cytoplasmic LINE-1-cDNA, leaked circular genomic DNA).
12. Carefully calibrated but sufficient support for key mechanistic and functional/basic biology studies that could provide pivotal additional resources, e.g. a crystal structure for the LINE-1 ORF2 proteins.