I probably wasn’t the only person who was roped into a fair share of board games this holiday season. I was reintroduced to the classic whodunnit mystery game Clue by my older son. As were were going around board taking turns, round after round of process of elimination that began aimlessly and gradually triangulated on the remaining possibilities, I confess my mind began to wonder.

I couldn’t shake the feeling that rare disease research is like a game of Clue — but much harder. Instead of solving a fictitious murder, the goal of Rare Clue is to identify the true mastermind behind a rare disease in a lineup of genetic suspects. This of course assumes there’s a lone wolf out there. For rare diseases caused by mutations in a single gene, that simplifying assumption is reasonable, even if it turns out that the biology of said gene is complex.

But a chromosomal disease is a tangled web of conspiracy: dozens or hundreds of so-called disease driver genes on an affected chromosome, some of which could cause a disease if mutated alone, not to mention the shadowy interactions between genes on 8p and genes on other chromosomes. It wasn’t just Miss Scarlett with a wrench in the ballroom. Or Professor Plum with a lead pipe in the study. It’s as though all the suspects were in cahoots, armed to the teeth with all the weapons, lurking in every room of the house.

Frustratingly, some of the suspects are red herrings, erecting roadblocks and diversions meant to throw researchers off the trail. And if we’re being completely candid, there are actually two intertwined conspiracies animating chromosomal diseases, as I mused earlier this year in a review on Substack.

The genes that are physically altered — losing or gaining a copy, or forcibly relocated to a different genomic address — constitute a network of so-called disease driver genes, or drivers for short. Elucidating the nodes, structure and hierarchy of that network is a prime focus for Project 8p. Unfortunately, the scientific challenge doesn’t end there.

The accompanying genomic imbalance that arises from megabase-scale chromosomal changes constitutes a second locus of disease pathology that is separate from the pathophysiological sequelae of tweaking the dosage of specific drivers. And just to make things even more interesting, consider the strong possibility that these two networks of conspirators could be active or lurking depending on the cell type, developmental timing, and conditional environmental stresses.

Below, I’ll highlight the incredible progress made by Project 8p sponsored research projects, highlighting four areas of discovery. We’ve worked with academic labs, CROs and biotech startups. We’ve interrogated 2-D and 3-D disease models in the dish. And with the successful launch of the 8p Health Canvas study at the 2024 Science and Family conference, we’re starting to bridge the gap between the lab and the real world.

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Project 8p launched a collaboration with Prof Hiruy Meharena’s lab at UCSD to characterize induced pluripotent stem cell (iPSC) derived neuronal models using a suite of '“omics” approaches. We originally recruited the Meharena lab to the 8p cause based on intriguing results from studies of trisomy 21 cell models, which we hypothesized could be relevant for 8p. The Meharena lab was set up to investigate the convergences between chromosomal diseases, which often engender a similar set of symptoms in affected patients.

Note that two years ago we saw the first Reelin over-expression data from 3-D cortical brain organoids grown in Prof Alysson Muotri’s lab at UCSD that hinted at the possibility that the 8p disease state is not unique to 8p. So going into the collaboration with Meharena lab, we were already poised to see more examples of convergence between 8p and neurodevelopmental chromosomal diseases like Edwards Syndrome (Trisomy 18) and Patau Syndrome (Trisomy 13).

I don’t want to steal Meharena lab’s thunder since validation experiments on more 8p hero cell lines are needed for a peer-reviewed publication, and hopefully a preprint on the n-of-1 findings is in the works. So I’ll briefly recap here.

Gene expression analysis — aka transcriptomics or RNAseq — of neural progenitor cells (NPCs) derived from the iPSCs of a pioneer 8p invdupdel hero revealed a massively statistically significant overlap with NPCs derived from Trisomy 21 iPSCs even though two completely different chromosomes are affected. I don’t think I’ve ever seen a p-value that low.

Since that initial provocative data point, Meharena lab completed ATACseq and HiSeq, which tells us about genome-wide chromatin accessibility and 3-D genome architecture, respectively. They’ve also zoomed in on senescence, a zombie-like physiological state that cells enter when they’re sufficiently damaged or chronically stressed, which can occur when there’s genomic imbalance.

Meharena lab observes evidence of senescence in 8p NPCs, as well as concomitant metabolic changes that indicate that 8p NPCs are on the knife’s edge when it comes to cellular energy requirement. If these changes are happening because of 8p genomic imbalance per se rather than due to the effects of one or more 8p drivers, perhaps NPCs are uniquely vulnerable to aneuploidy in ways that other cell types aren’t.

The most obvious way to assess how much genomic imbalance is a conspiratorial force in 8p chromosomal diseases is to see what’s happening in other parts of the body and at different stages of life.

In parallel to the time-consuming and expensive disease modeling efforts in 2-D neurons and 3-D brain organoids, Project 8p booted up a research plank focused on the simplest and most cost effective human-derived cell model: fibroblasts, or skin cells. Prof Nicoleta Moisoi’s lab in the UK was recruited by Project 8p to characterize a set of four 8p hero fibroblast lines through a cell morphology lens using a high-content-imaging-based approach that built on the preliminary fibroblast phenotyping work started in the late Prof Angelika Amon’s lab at MIT.

The take-home message from the Moisoi lab’s work is that 8p hero fibroblast lines do indeed have cell morphological features that are distinct from age-matched and sex-matched healthy control fibroblasts. What’s more, 8p fibroblasts lines have increased sensitivity to DNA damage and reductions in mitochondrial membrane potential, which together suggest a chronic stress response to genomic imbalance, or aneuploidy, in 8p skin cells. But the intensity of these image-based phenotypes varied across the four 8p fibroblast lines.

With the Moisoi dataset in hand, we knew we had to expand the 8p fibroblast characterization campaign to a larger sample size as four was too small to draw meaningful conclusions. Fortuitously, I met Dr Max Wilson, one of the founders of a company called Integrated Biosciences, at a biotech reception in San Francisco a year ago. We knew each from our days at Princeton, he an undergraduate and I a postdoctoral fellow. If biotech’s a small world, the rare disease world is even smaller.

Integrated Biosciences is named after the integrated stress response, which was shown to be activated in fibroblasts from patients with Trisomy 21 (Down Syndrome), and led to an induction of premature senescence. The Meharena lab was independently investigating senescence in the brain, so it made sense for us to find a research partner that could examine the question of senescence using orthogonal, high-throughput cell-based assays and a totally different cell type.

Meeting Max was a case of perfect timing. Within a few weeks of reconnecting, Max, his cofounder Dr Felix Wong, and I hatched a scheme to characterize all of the 8p fibroblasts line in the Project 8p biobank. That number ended up being 13, and a similar number of healthy controls for comparison.

In the new year, I’ll write up a summary of the findings from the Integrated Biosciences partnership, so a teaser must suffice. Based on the Meharena lab’s result convergence in NPCs, we naively expected there to be similar convergence in fibroblasts, or at least robust integrated stress response pathway changes in all 8p fibroblasts, as was observed in Trisomy 21 fibroblasts.

Much to our surprise, 8p fibroblasts did not manifest any classical signs of integrated stress response (ISR) pathway over-activation or under-activation. All of the standard ISR modulators had no effect, good or bad, on 8p fibroblasts. For example, if 8p fibroblasts were experiencing classical senescence their growth would be sensitive to senolytics. Yet none of the senolytics tested on 8p fibroblast had any effect on 8p fibroblast viability.

Although a few 8p fibroblast lines shockingly stained positive for beta-gal, a colorimetric indictor of senescence, only a minority of cells in the culture actually turned blue. So we’re not sure whether the spotty senescence that was observed is an artifact of how that particular cell line was isolated and established in the lab. Staining for p21, a canonical marker of senescence, did not corroborate the beta-gal staining results.

Curiously, the majority of 8p fibroblasts exhibited decreased resistance to the mTOR inhibitor (and hoped for fountain of youth) rapamycin. Meaning a lower dose of rapamcyin was required to kill the same fraction of 8p fibroblasts compared to healthy fibroblasts. What it means is still anyone’s guess, but it’s steering us in the direction of metabolic interventions.

Some may call it a “negative result” but we learn from every well-designed experiment, including those with “negative” findings. Scientists assign a positive emotional valence when they get the result they expect or want, but assign a negative emotional valence when they get a no-effect, unexpected or confusing result. If we’re objectively sitting with the data, maybe the mental model of two interlocking conspiracies is not too far off the mark.

To a first approximation, we can conclude that the genomic imbalance caused by alterations and copy number changes to 8p is not equivalent of the genomic imbalance caused by having a third 21st chromosome, at least not in fibroblasts.

Not all genomic imbalance is equal, at least for any given cell type. But genomic imbalance, and the exacerbating effects of specific 8p (and non-8p) driver genes, do seem to matter when we’re talking about brain cells, specifically the neural progenitor cells (NPCs) whose descendants populate the brain with all of its neurons.

Perhaps 8p disease can be conceptualized by the two-hit model akin to tumorigenesis. An activating mutation in a single oncogene is not enough to convert a cell to cancerous, but activating mutations in two oncogenes, or an inactivating mutation in a tumor suppressor at the same time, is required for runaway growth. Extending that framework to chromosomal diseases, specific 8p driver genes alone or 8p genomic imbalance alone don’t drive most cells off the cliff, but the convergence of both forces in certain cells at certain times is the problem.

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What if there were a way to decapitate the conspiracy without having to bring all the receipts and sort out who did what where? As a hedge but also as a nod to rapidly advancing genome editing technologies, Project 8p funded a proof-of-concept chromosome engineering project in Prof Jason Sheltzer’s lab at Yale, the first baby step in the lab toward what will one day hopefully mature into chromosome therapies in the clinic.

If we acknowledge the view that specific 8p driver genes and 8p genomic imbalance are colluding, then the most curative fix would be to surgically remodel or remove the entire damaged rearranged chromosome 8 in the cells of 8p heroes. Of course there will be cellular or tissue damage that won’t be immediately reversed by chromosome therapy especially the later in life such an intervention were to be administered. However, in theory, chromosome therapy alleviates the problem at its source, and the earlier in life the better. So it behooves Project 8p to place this speculative bet while simultaneously unraveling the web of conspiracies one strand at a time.

In the spirit of open science that animates Project 8p, Sheltzer lab has already posted a preprint summarizing their results. Briefly, Sheltzer lab was able to select for chromosomally corrected clones derived from a pioneer 8p invdupdel hero iPSC line. Chromosomally corrected means a derived clone from an 8p invdupdel hero iPSC line in which the rearranged maternal chromosome 8 was lost and replaced by a second copy of the un-rearranged paternal chromosome was gained.

To start, chromosomally corrected (also referred to as an isogenic control) iPSC lines are invaluable research assets because they are the best reference point or benchmark for an 8p hero iPSC line. Comparing a chromosomally corrected 8p iSPC line to its ancestral 8p hero iPSC line allows us to determine which changes, in this instance gene expression patterns, are the result of the 8p rearrangement because all other variables have essentially been held constant.

Thinking ahead many steps, there certainly will be delivery challenges associated with assembling the molecular equivalent of a chromosome surgical unit that can be safely deployed in actual humans. Would every cell in the body need to be chromosomally corrected or just the stem cells that give rise to differentiated daughter cells? It’s all pure science fiction today. But at least we’ve proved that it’s possible in a petri dish.

Lest you think all of Project 8p’s attention is solely focused on disease models that are ultimately still abstractions of actual 8 heroes, we started this year with an ambitious plan to involve 8p heroes and their families directly in research. We eventually named this initiative the 8p Health Canvas study, which I wrote about right after the 8p 2024 conference in Denver in July.

Since the conference, the science team at Project 8p has been sifting through many bytes of 8p hero data. Last month, a Project 8p Research Roundtable featured a discussion about the gut microbiome test called GI-MAP which was taken by at least 30 8p heroes. Reminiscent of the results from disease modeling in the lab, there is no universal 8p leaky gut or gut dysbiosis signature shared by all 8p heroes.

Instead we’re seeing a more fractal picture, where a few 8p heroes cluster together in subgroups whose defining features we still can’t discern. That said, we do indeed see evidence for distressed microbiomes and inflamed guts in 8p heroes, which are both clinically actionable in terms of diet optimization (i.e., removing gluten) and supplement stacks. A similar archipelago pattern of subgroups of 8p heroes is observed when we examine the MitoSwab results, which assess mitochondrial dysfunction.

Obviously, we’re working on a massive multi-variate analysis to start to untangle cause from effect and make the case for biologically justified 8p hero stratification. And we’re still awaiting results from Somalogic on 8p serum proteomics as well as genome-level assessments from Illumina. Slowly but surely, a first-of-its-kind and the most comprehensive genotype-phenotype database ever created for a chromosomal disease is coalescing.

I’ll end by spotlighting the 8p Health Canvas results from our collaboration with Unravel Biosciences. Working with Unravel’s rareSHIFT arm, we performed RNAseq on nasal swab samples collected from 8p heroes and a sex-matched sibling or parent for over 30 8p heroes to date (with more still in the queue). The goals of this segment of the 8p Health Canvas were twofold: stratify 8p heroes in buckets based on similar gene expression profiles and identify drug repurposing candidates in silico that normalize the 8p gene expression profile.

A fuller explication of the Unravel dataset is forthcoming in the new year. Suffice it to say, RNAseq on 8p hero nasal swab samples revealed that 8p heroes bucket into subgroups more so than other rare disease patient cohorts. In other words, there is no universal 8p disease signature or profile. However, there are a number of drug repurposing candidate predictions shared by almost all 8p heroes. Interestingly, these compounds include nutraceuticals and over-the-counter supplements that are thought to boost metabolism.

As for look ahead to 2025, the 8p conspiracy seems less scary and full of unknowns. We still need to understand if 8p heroes are fundamentally suffering from the same disease or whether 8p is a collection of n-of-1’s. All of the data we’ve amassed so far indicates that both mental models are two sides of the same coin. In the end, studying all 8p heroes together will lead to deeper, cross-cutting insights faster. Each 8p hero will contribute invaluable clues that will helps us crack the case for the benefit of all 8p heroes.

Happy New Year! Here’s to a 2025 full of new scientific discoveries and their real-world implications in the lives of 8p heroes and their families.

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In the Summer Research Newsletter published last week, I previewed the agenda and invited speakers for the 2024 8p Family & Science Conference, which kicks off today in Denver Colorado. The entire Project 8p team has meticulously planned this meeting since the start of the year, assembling a special group of stakeholders in one location. Notably, over 30 8p heroes and their families will be in attendance, the largest 8p community gathering to date.

On behalf of the Project 8p science team, I present the rationale for leveraging the 2024 8p Family & Science Conference as a popup clinical research site for a hypothesis generating, cross-sectional study to assess nutritional status, bioenergetics (mitochondrial) dysfunction, protein markers of inflammation, gene expression patterns, metabolite levels, and gut microbiome composition in 8p heroes.

Or what we have dubbed the 8p Health Canvas.

Technically, the 8p Health Canvas is a turbocharging of the ongoing IRB-approved, Project 8p-sponsored protocol that established a patient registry (database) of a geographically dispersed population of 8p heroes, the ability to contact (and recontact) 8p families with medical surveys, and a biobank containing cryopreserved cell lines and biosamples derived from 8p heroes.

This protocol, originally approved in 2019, can be considered the precursor to a proper natural history study. Project 8p previously leveraged the 2021 Moving Mountains conference as a popup site for biospecimens collection.

The 8p Health Canvas core team is comprised of three individuals. The Principal Investigator is Bina Maniar, the founder and CEO of Project 8p and mom to 8p hero Karina. I serve as the Study Director. Kaiti Syverson, Project 8p’s patient engagement manager and mom to 8p hero Chloe, serves as the Study Coordinator. Dr. Richard Frye, M.D., Geri Brewster, RD MPH CDN, serve as clinical advisors with a focus on mitochondrial dysfunction and nutritional optimization, respectively.

Stepping back, the 8p Health Canvas project has three strategic objectives: (1) perform a real-world IRB-approved clinical research study that prepares the 8p community for repurposed and novel therapies currently in discovery and preclinical development; (2) generate hypotheses about disease-driver mechanisms and biomarkers and how best to stratify 8p heroes based on measurable genotype-phenotype relationships; (3) promulgate best-practices guidelines for the use of over-the-counter nutritional interventions that can ultimately be published in a peer-reviewed journal.

What does that practically mean for 8p heroes? We hope to identify the first treatment options for 8p through drug repurposing, the process of finding a new use for an old medicine. In parallel, we hope to discover biomarkers (outcome measures) that will prove that a given intervention is working or not. We also hope to match 8p heroes with appropriate treatments based on their likelihood of responding aka hero stratification.

In the run-up to Denver, Project 8p disseminated medication and supplement surveys to the 161 8p families in the Project 8p registry. These survey results will constitute nutritional baselines which will be reviewed by Project 8p’s science team in consultation with the 8p Health Canvas clinical advisors. Over the span of two days at the conference, we will collect consented biospecimens from 8p heroes for a suite of assessments that I will describe in greater detail below.

After the conference, what began as a single wide-angled snapshot will transition into a time-lapse panorama: a longitudinal natural history study led by physician scientists that will track an expanding cohort of 8p heroes over time. This next phase will be done in collaboration with the Colorado Children’s Hospital’s 8p clinical care team.

If data from the cross-sectional study support it, an optional third phase (potentially requiring both IRB and IND approvals) would consist of an open label, baseline-controlled, multi-week interventional study in the population of 8p heroes that demonstrates measurable phenotypic defects that can be rationally expected to be modified by existing nutraceuticals or probiotics or over-the-counter repurposed drugs.

We are in part modeling the Health Canvas after Dr Richard Frye’s experience with Phelan-McDermid Syndrome (PMS) with the hopes of identifying over-the-counter, supplement-based interventions that could be rapidly translated to the clinic. Several mitochondrial genes are deleted in PMS, creating a rational basis for investigating mitochondrial function in living patients using easy-to-use buccal swabs.

In addition to the fact that several mitochondrial genes are also affected in 8p chromosome disorders, in vitro studies on four 8p fibroblasts lines by Prof Nicoleta Moisoi’s lab previously revealed a decrease in mitochondrial membrane potential, providing further evidence of secondary mitochondrial dysfunction as a potential contributor to pathophysiology in 8p chromosome disorders.

We also draw inspiration from our friends in the Angelman Syndrome (AS) community. Below is an example of AS longitudinal natural history study data. Each of the traces represents a single AS patient. Longitudinal data like this will ultimately enable 8p hero stratification and allow us to deeply understand each 8p hero’s biology so we can tailor therapeutic opportunities accordingly. Those data will also enable the generation of a synthetic placebo group for comparisons to active treatment groups in future clinical trials.

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The 8p Health Canvas will assess 8p heroes across seven dimensions using validated testing platforms. This systematic workup mirrors the flow of biology’s Central Dogma from DNA to RNA to protein to metabolites. It also captures physiological outputs like mitochondria function and gut microbiome composition.

Illumina will perform whole-genome sequencing (WGS) and and gene expression profiling (RNAseq) on peripheral blood mononuclear cells (PBMCs), and Alamya Health will complete DNA methylation analysis. Unravel Biosciences will perform RNAseq on nasal swabs. Somalogic’s SomaScan platform enables proteomics on over 7,000 serum proteins. Metabolomics on blood will be conduced by Metabolon. Religen’s buccal (cheek) MitoSwab test measures the activity of mitochondrial electron-transport chain complexes. Finally, the GI MAPS test by Diagnostic Solutions Laboratory (DSL) will reveal gut microbiome composition from stool samples.

All of these assessments are in the service of 8p hero stratification. Unravel Biosciences, Religen and DSL have the potential to yield drug repurposing candidates. Somalogic and Metabolon have the potential to surface quantitative biomarkers.

In conclusion, we’d like to thank our generous corporate and in-kind sponsors, in particular Somalogic, Unravel Biosciences and Illumina. We also appreciate discounted nonprofit pricing offered by Somalogic, Religen and DSL.

Onward toward the clinic for 8p!

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The last time 8p families and researchers gathered together to break bread over science and shared lived experience was at the Moving Mountains conference in Denver Colorado in the Summer of 2021, two years after Project 8p was launched by Bina Maniar. Project 8p was joined by two other foundations focused on a neurodevelopmental chromosomal disorder, Dup15q Alliance and Ring14 USA.

In spite of the headwinds of the pandemic, which ground so many rare disease projects to a halt, Project 8p was successfully able to nucleate a small but passionate community thanks in measure to securing CZI’s Rare As One grant funding. Project 8p made the first seed investments in a tiny portfolio of sponsored projects. A few months after the Moving Mountains, Project 8p brought me on as Science Director and we were off to the races.

Three years later, I’m thrilled to report that the 8p community — expanding in number of families and researchers and dollars fundraised, and more organized than ever — returns to Denver to inaugurate the next exciting era in 8p research. If the preceding chapter could be encapsulated as going zero to one, this next phase will be defined by acceleration: follow-on investments in successfully seeded projects will be deployed alongside fresh investments in emerging research opportunities. Overarching scientific goals are to cement discoveries into drug candidates and initiate the first clinical studies.

My tenure as Science Director is coming to end next month and I will transition into my new role on Project 8p’s Medical and Scientific Advisory Board (MSAB). There’s no person better suited to take the helm as Science Director than Dr. Barbara Celona, with whom I’ve had the pleasure of working on 8p and other rare diseases with at Perlara for the last few years. Barbara is joined by Project 8p’s new Executive Director, Dr. Lauren Chaby, who officially begins in her new role this week.

With that preamble out of the way, we want to thank our sponsors, Somalogic, Integrated Biosciences, Unravel Biosciences, and the Family Care Center for their support.

The science agenda supports facilitating information exchange, creative brainstorming and collaborative networking. Project 8p has two specific deliverables we want to emerge in the wake of the conference: (1) collaborative publication of peer-reviewed 8p Research Roadmap; (2) multi-PI grant proposals to advance inter-disciplinary research. The original 8p research roadmap was published in October 2022. A research roadmap refresh was published earlier this year.

Project 8p’s founder and CEO Bina Maniar will kick off the festivities with a morning keynote presentation called Early Onset Greatness on Thursday August 1, followed by a formal introduction of Dr. Lauren Chaby as Executive Director. I’ll take the stage next along with Dr. Barbara Celona to present a research roadmap update. Then we’ll hear a caregiver story to ground us to the daily challenges experienced by 8p families.

Dr. Terry Jo Bichell, founder of CombinedBrain, will present the 8p disease concept model, revealing connections between symptoms, concerns, and daily life impacts. The 8p disease concept study offers insights for research, clinical trials, and improving the lives of 8p heroes and caregivers.

Wrapping up the morning science track, Patient Engagement Manager, Kaiti Syverson, will summarize the My Hero Initiative, while Tobias Bruenger, Ph.D., will delve into the genotype and phenotype characterizations of 8p heroes. They will unveil the Chromosome 8p Insights Portal, an interactive portal for researchers dedicated to understanding Chromosome 8p disorders.

In the afternoon of Thursday August 1, there will be two Think Tank sessions. The first Think Tank will be a moderated discussion where we will delve into knowledge highlights and gaps. This session will also explore convergences between 8p and other chromosomal disorders (e.g. Down Syndrome). We’ll be joined by special guest Julia Vitarello, who will share her “Mila to Millions” vision as inspiration. The second Think Tank will feature discussion of grant opportunities with the aim of formulating a collaborative grant strategy among principal investigators.

Day Two of the conference starts in the morning of Friday August 2 with what we’re calling the Project 8p Medical Fair. Presenting companies and organizations will have booths where attendees will have a hands-on opportunity to explore a diverse array of therapies, devices, and interventions, all focused on cutting-edge technologies such as neuromodulation.

Building on insights from the previous day’s 8p Think Tanks, this twofer session will be dedicated to ideating and drafting specific aims for grant submissions. The second goal is to consolidate insights and connections into an outline for a peer-reviewed 8p Research Roadmap and define authorship roles.

Friday August 2 in the afternoon is devoted to family friendly and scientifically accessible presentations by Project 8p-funded researchers, with speaking slots given to the junior scientists who actually performed the work at the lab bench. Here’s the presenter lineup:

• Chromosome Engineering to Restore Euploidy in Cells with invdupdel 8p

  Presented By: Sophia Lee, M.S. (Dr. Jason Sheltzer’s lab, Yale University)

• Exploring Shared Symptoms in Genomic Imbalance Disorders to Uncover Underlying Causes

  Presented By: Ashley Watson, Ph.D. (Dr. Hiruy Meharena’s lab, UCSD)

• Studying the Molecular Mechanism of Chromosome 8p Disorder Using Brain Organoids Derived from 8p Heroes

  Presented By: Yanning Zuo, Ph.D. (Dr. Francesca Telese’s lab, UCSD)

• Understanding the Cellular Stress Responses in 8p Cells

  Presented By: Felix Wong, Ph.D. & Max Wilson, Ph.D.

  (Integrated Biosciences)

• Chromosome Dosage Modeling and Correction in Viable Human Aneuploidy & 8p CNVs

  Presented By: Stefan Pinter, Ph.D. (University of Connecticut)

• Health Canvas: A Natural History Study Expansion

  Presented By: Ethan Perlstein, Ph.D, (Project 8p Foundation)

You might be wondering what the 8p Health Canvas study is? My final Substack that will drop first thing next week will describe in greater detail what the 8p Health Canvas study entails. In short, we are expanding the scope of the Project 8p sponsored, IRB-approved patient registry and biosample collection protocol for assessing genotype-phenotype relationships in 8p heroes. The purpose of the study is to bring the 8p community one step closer to data-driven and hypothesis-driven interventional clinical trials. You don’t often get 30+ 8p heroes in the same room at the same time, so we’re taking advantage of the conference to gather as much precious 8p “in hero” data as possible.

We wrap up Day Two with a panel discussion with Parth Amin, Co-Founder and COO, Eysz, called The Digital Frontier: Exploring the Promise of Digital Biomarkers. We’ll tap into the digital frontier and explore the transformative potential of digital biomarkers to revolutionize diagnosis, treatment monitoring, and patient care in neurological settings.

Day Three on Saturday August 3 will be focused on families and all things clinical. Patient Engagement Manager, Kaiti Syverson, will debut Project 8p’s Patient Navigator and explore the transformative potential of the Chromosome 8p TeleEcho, a global learning program for providers and educators dedicated to advancing 8p knowledge and care. The afternoon will end with a Rapid Connect in small groups with the Multidisciplinary Clinic team from Children’s Hospital Colorado, who will first provide an update on their clinical outcomes and advancements towards care recommendations:

• Neurological Symptoms in 8p Heroes

  Presented By: Dr. Scott Demarest & Dr. Megan Abbott

• Development and Behavior in 8p Heroes

  Presented By: Dr. Andrea Miele

• Optimizing Care for 8p Heroes

  Presented By: Dr. Margarita Saenz & Dr. Kourtney Santucci

We’re estimating that 150+ people, 30+ 8p families and nearly two dozen researchers will be attending the conference in-person, and others will be joining virtually. See you all next week in the Mile High City, Denver.

💬 Research Question of the Month

There’s a perennial thorny question we keep returning to over and over again in our sponsored research projects: how similar are 8p heroes to each other? Initially, all of our studies focused on a single pioneer invdupdel 8p family trio — mom, dad, and affected child — for practical reasons. Over time, Project 8p’s biobank has grown to include additional 8p hero cell lines, so we now have a more representative census of the 8p hero population. Highlighted below are results from Professor Aryeh Warmflash’s lab at Rice University that start to chip away at this question about similarities (and differences) between 8p heroes.

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🔬Research Highlight

Project 8p funded Professor Warmflash’s lab to study early fetal development in the lab using induced pluripotent stem cells (iPSCs) derived from 8p heroes. The Warmflash lab specializes in a technique called the germ layer micropatterning assay, which offers a window into how the growing embryo organizes itself.

A quick refresher on developmental biology. All organ systems of the body originate from one of the three germ layers: ectoderm (outer layer), mesoderm (middle layer), and endoderm (inner layer). The endoderm gives rise to digestive and respiratory systems. The mesoderm gives rise to the skeleton, muscle, heart and bones. The ectoderm births the nervous system and the skin.

Prof Warmflash previously presented data at one of our monthly Research Roundtables on WES557, the pioneer invdupdel 8p hero that kickstarted all of our research projects. Project 8p sponsored this project in order to shed light on the earliest changes that occur in human development as a result of complex 8p rearrangements. Although this research won’t immediately translate to therapies, it’s the foundation on top of which medicines might one day be made.

As shown in the summary figure below, the WES557 has three distinct phenotypes that are easily discerned by eye: overgrowth of the mesoderm layer (blue), expansion of the extra-embryonic layer (red), and contraction of pluripotent cells (green).

Are these phenotypes affecting the 3-D organization and size of the germ layers unique to invdupdel 8p heroes? What would another invdupdel 8p hero sample look like? And what about deletion-only 8p heroes? Or duplication-only 8p heroes?

Below are results of a micropatterning assay that compared an unaffected control (ESI) to a second invdupdel 8p hero line (DD508A) and a deletion-only 8p hero line (DD504B). DD508A looks strikingly similar to WES557. We obviously need to process more invdupdel samples to make a firm conclusion but we can say a trend is emerging. Fascinatingly, the deletion-only 8p hero line DD504B does not look like a invdupdel 8p hero sample. In fact, DD504B appears more similar to the unaffected control.

TNKS1 is a candidate 8p driver gene that we’ve had on a radar. It’s typically in the deleted 8p segment resulting in haploinsufficiency, i.e., having just one copy of the gene versus the normal complement of two copies. The Warmflash lab sought to isolate the effects of TNKS1 haploinsufficiency (TNKS+/-) and compare them to the effects of knocking out both copies of TNKS1 (TNKS-/-). These experiments were not performed in 8p cell lines. Rather, one or both copies of TNKS1 were purposefully deleted in an unaffected wildtype control (WT) line. The results below show that TNKS1 haploinsufficiency (TNKS+/-) causes a reduction of the mesoderm phenotype, i.e., lower BRA signal intensity, which is further intensified in the TNKS-/- knockout.

The deletion-only 8p hero line DD504B shows a reduced ring of primitive streak mesoderm marked by BRA, to use an embryologist’s parlance. This result is consistent with the data on the TNKS1 knockout, which shows a similar phenotype. Therefore, deletion of TNKS1 may partially explain the DD504B germ-layer phenotype.

📄 Recent Articles and Publications

Let us know if you’ve read or published a paper or article recently that you think the 8p community should know about! 

1. Earlier this month, we published a Substack about the manhunt for 8p driver genes. We introduced the concept of driver genes that are not physically located on the short arm of chromosome 8 aka backseat drivers.

2. Chromosome Transplantation: Opportunities and Limitations. La Grua et al. Cells. April 2024. https://www.mdpi.com/2073-4409/13/8/666
   Nice review article that summarizes the state of play in what we’ve always referred to as chromosome therapy.

3. An asymptomatic male individual carrying a 5.72 Mb de novo deletion in 8p23.2‑p23.3: A case report. Keramida et al. Experimental and Therapeutic Medicine. June 2024. https://www.spandidos-publications.com/10.3892/etm.2024.12529

   Interesting case report of a 30-year-old asymptomatic male with a 5.721 Mb deletion in the 8p23.2‑p23.3 region, including ARHGEF10, CLN8, CSMD1, DLGAP2, ERICH1, FBXO25, KBTBD11, MYOM2, TDRP, and ZNF596. The normal phenotype of the patient supports the hypothesis that there is incomplete penetrance of 8p23.2‑p23.3 deletions, which means there may be “anti-drivers” out there: genes that prevent or blunt the effects of genomic imbalance.

💜 Family Corner

Last week, the Project 8P team hosted our Q2 Family Update, unveiling the expansion of our natural history study. Together, we're venturing into exciting new opportunities! Missed the update? Don't worry! Email kaiti@project8p.org to catch up on all the details.

📆 Upcoming Events

• Project 8p Research Roundtable, May 30th, at 12pm EST on Zoom. Email barbara@project8p.org to attend.

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Megabase alterations to the short arm of chromosome 8 are a plot to overthrow the organism, not the actions of a lone wolf. A whodunit orchestrated by a shadowy web of co-conspirators, double agents, and unwitting patsies. Innocent bystanders get swept up in the confusion. Everyone in a defined radius is under suspicion.

The evidence collected is often circumstantial, no obvious smoking gun. There are too many leads to chase down, most of them dead ends, and not enough investigators assigned to the case. The manhunt to round up driver genes feels like a race against time.

While dramatic, this analogy accurately portrays the challenges of studying 8p chromosomal disorders and other complex cases of genomic imbalance that do not obey the deterministic laws of classical aneuploidy. Classical aneuploidy works best in cases where a single driver gene that is amplified in a duplication or lost in a deletion is solely responsible for pathophysiology.

One example is Phelan-McDermid Syndrome, which may cause 1% of autism spectrum disorders. Deletions as small as one kilobase and up to 9 megabases on the long arm of chromosome 22 — 22q13del — knock out one copy of the SHANK3 gene, a situation that more closely matches the classical aneuploidy framework.

The genes located on 8p are not the only drivers of interest because the whole genome may be in on it. In the Grand Unified Theory of Neurodevelopmental Chromosome Disorders, the Project 8p science team introduced the concept of a probabilistic description of chromosomal mechanics that seeks to explain and predict the interactions within and between chromosomes modeled as dynamically phase-transitioning polymers.

However, unlike in the atomic world where things get weirder the smaller you go, chromosome behavior gets even funkier as you go from nucleotides to nucleosomes to the megabase scale.

Returning to the driver gene manhunt analogy, coordinating among different law enforcement agencies and jurisdictions that don’t always see eye to eye is no small feat. Molecular and cell biologists tend to focus on form: chromatin structure. Geneticists tend to focus on function: effects in cis or in trans. Systems biologists like to think they unite form and function: insert trendy genomics technique here.

Given that hundreds of genes — not to mention noncoding genes and other conserved functional elements — are directly physically affected by megabase-sized 8p alterations, the problem isn’t issuing indictments for suspected driver genes but convincing the court to convict.

Booth didn’t act alone, but Stanton had to prove there was a grand conspiracy to topple the Union government that was directly ordered by Jefferson Davis and carried out by a network of co-conspirators.

Back to chromosomes. It may not be the dosage of a particular driver gene located on the short arm of chromosome 8 that is driving disease or a disease phenotype. Instead, the levels of a protein encoded by an 8p gene might affect the dosage of a gene located on, say, chromosome 7p.

The 7p gene may be activated (de-repressed) or inhibited by the 8p gene, and then this change causes a disease phenotype or phenotypes. The levels of the 7p protein go on, in turn, to affect the dosage of another gene located on, say, chromosome 6p. And so on.

Who is really driving the car? Perhaps we should refer to the driver genes that are not located on 8p as backseat drivers.

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The foundational paper Okur et al., 2021 describes the first clinical cohort of 8p heroes. In that study, the authors noted six 8p hero genotypes and four minimal critical regions. Independently, two overlapping minimal critical regions in the 8p inverted duplications have been reported by Sajan et al., 2013 and Vibert et al., 2021.

The six 8p hero genotypes are shown in the figure below, which includes chromosome 8 genome coordinates:

1. Deletion with inverted duplication, or invdupdel (most common genotype)

2. Duplication

3. Distal deletion

4. Distal & proximal deletion

5. Proximal deletion

6. Proximal duplication

8p invdupdel heroes almost all have similar albeit not exactly the same chromosomal breakpoints. In the figure below, notice the yellow highlighted gap segment sandwiched between the purple deleted segments and the blue duplicated segments in the figure below. Two clusters of defensin genes line both sides of that gap.

Also note the brackets. The top four rows are the Okur et al minimal critical regions (#1-4). The bottom two rows are the Sajan et al minimal critical region (#5) and the Vibert et al minimal critical region (#6).

The yellow highlighted region is blown out in the next figure. The locations of defensin genes are also highlighted in yellow. A future Substack will go into greater detail about why these defensin gene clusters create a hotspot for inverted duplication events in meiosis.

For now, this genome browser resolution view provides a high-level explanation for the pattern of recurrent breakpoints:

The Supplemental Data section of Okur et al does an excellent job spotlighting potential driver genes of interest in each of the 8p minimal critical regions. In lieu of a recitation of genes located in each of the 8p minimal critical regions they identified, let’s take a closer look at the inverted duplication segment that affects almost all 8p invdupdel heroes:

The yellow-highlighted blue segment is referred to as the agenesis of the corpus callosum (ACC) minimal critical region, which is located in inverted duplications. The orange box delineates a 5.1-megabase segment of interest — a search perimeter — that includes a small portion of the Okur et al inverted duplication minimal critical region as well as the Sajan et al and Vibert et al inverted duplication minimal critical regions.

A long-suspected driver gene of interest RHOBTB2 resides in this 5.1-megabase region, along with neighboring genes that have something in common.

Credit to Project 8p researcher Prof Stefan Pinter who made the following insightful observations about potential driver genes in this 5.1-megabase stretch. There are six genes with high triplosensitivity scores (pTS > 0.9), meaning having three copies of the gene is likely to be pathogenic: DPYSL2, PPP2R2A, EBF2, PTK2B, KCTD9, RHOBTB2. Three of those triplosensitive genes (in bold) are also CUL3 targets.

Two other CUL3 targets live a few floors down on 8p: NEFL (neurofilament light chain) and NEFM (neurofilament medium chain). CUL3 is a ubiquitin E3 ligase that regulates protein degradation. A third copy of a triplosensitive gene may overwhelm the protein degradation capacity of the cell. Consistent with that model, two of the CUL3 targets in the 8p duplicated interval (PPP2R2A & RHOBTB2) can give rise to neurodevelopmental disorders on their own.

But like any grand conspiracy, there’s always another layer.

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Neurodevelopmental disorders and neuropsychiatric disorders may be two sides of the same coin because the same underlying neuronal pathways are implicated by human genetics. This is also borne out by our Reelin/RELN over-expression findings.

The RELN gene plays a neurogenic role in early brain development, and neuromaintenance and neuroprotective roles in adulthood. Project 8p is focused on neurodevelopment because most 8p heroes known to medicine are still children. As families deal with the demanding day-to-day realities of a special needs child, what will happen to the brains of 8p heroes as they age?

8p can learn from the experiences of researchers who have studied a 3-megabase heterozygous deletion of one copy of the long arm of chromosome 22, or 22q11.2.

22q11.2 deletions are a risk factor not just for schizophrenia but also autism spectrum disorders and intellectual disability. It just so happens to be the equivalent of a commuter train ride down the chromosome from 22q11.3, the location of the deletions that cause Phelan-McDermid Syndrome.

What makes 22q11.2 and 8p similar is that genetic variation outside of 22q11.2 plays a role in determining the type, severity and penetrance of neurological presentation. A paper published a few years ago — Nehme et al., 2022 — attempted to untangle the knotted web of genetic interactions between multiple haploinsufficient genes on 22q11.2 and backseat driver genes elsewhere in the genome. A quick review of their study is instructive for Project 8p.

The authors generated iPSCs lines from twenty 22q.11.2 deletion carriers and 29 unaffected controls. Next they performed bulk RNAseq on cells at three different times points during a neuronal differentiation protocol: undifferentiated pluripotent stem cells on Day 0; partially differentiated neural progenitor cells at Day 4; terminally differentiated excitatory neurons at Day 28.

The Venn diagram below reveals a striking result. Across the three timepoints and cell differentiation states, 90% of the differentially expressed genes (n=386) in 22q11.2 deletion cell lines are located outside of 22q11.2. Reassuringly, the 27 genes that are down-regulated in all three datasets are located in the 22q11.2 deletion region. They also reproduced the RNAseq results from the 22q11.2 deletion carrier cell lines in a critical isogenic control experiment by generating the 22q11.2 heterozygous deletion in a wild-type cell using CRISPR engineering.

Nehme et al went on to canvas those 386 potential backseat driver genes. How else are they connected to the 22q11.2 conspirators besides transcriptionally? What do those 386 genes have in common with each other? Are they directly associated with neurodevelopmental and/or neuropsychiatric disorders, or to the genes that give rise to said disorders?

The authors performed a number of analyses of answer those questions. They showed that differentially expressed genes in 22q11.2 pluripotent stem cells and 22q11.2 neural progenitor cells are more likely to interact genetically, and at the protein level, with a curated set of 295 genes associated with childhood neurodevelopmental disorders.

In parallel, the authors showed that differentially expressed genes in 22q11.2 neurons are more likely to interact genetically with known schizophrenia risk factor genes. If the same 22q11.2 deletion interacts with the rest the genome in context-dependent ways — affecting specific cell lineages at discrete times in life — it’s not unreasonable to assume megabase 8p alterations will behave similarly.

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To that end, Project 8p has been collaborating with Prof Hiruy Meharena’s lab on creating a multi-omics 8p brain cell atlas that includes RNAseq so we can perform the types of analyses presented in Nehme et al. Based on preliminary data from Meharena lab, the promoters of genes up-regulated in 8p neural progenitor cells are bound by members of four different families of transcription factors. The promoters of genes down-regulated in 8p neural progenitor cells are bound by members of three of those four families.

The gene expression levels of ten transcription factors belonging to those four families is shown below. Strikingly, one of the transcription factors is up-regulated — nearly doubled — in 8p neural progenitor cells relative to control neural progenitor cells. This transcription factor happens to be located on chromosome 6. Did we just discover a backseat driver of 8p?

In order to put candidate frontseat or backseat drivers to the test, we’ll need functional readouts (phenotypes) ideally in a rapid turnaround and cost-effective 2-D cell model. One potentially fruitful avenue is emerging. 8p neural progenitor cells have a proliferation defect, as shown below:

In our tireless search for 8p drivers, we have a growing appreciation for the who. But we still don’t understand the how and the when. Nehme et al speculate that 3-D genome architecture may provide an explanation.

How exactly 22q11.2del might regulate the expression of genes in trans remains a matter of great interest. One intriguing possibility is that 22q11.2del might impact chromatin architecture, thereby regulating the expression of genes outside of the region. Indeed, a recent study using 22q11.2del lymphoblastoid cell lines revealed changes in their genome architecture⁸⁷. It is thus possible that 22q11.2del spatially rearranges the genome of neuronal cells, resulting in mis-regulation of genes linked to neuropsychiatric disorders.

The Project 8p community will continue to work the problem with humility and an open mind. One day we will demystify the secret lives of chromosomes.

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💬 Research Question of the Month

Plots like the one above have become as commonplace in scientific journals as abstract expressionist paintings in modern art museums. Each dot represents an individual cell. Each color signifies an individual cell type. The multi-color murmurations are clusters of cells that express the same genes. What everyone struggles with is how to convert those arresting patterns into actionable insights.

Do you have pointers or tips for extracting concrete biological meaning from complex multidimensional datasets? Please drop feedback in the comments!

Leave a comment

🔬Research Highlight

Project 8p initiated a collaboration with Dr Francesca Telese at the UCSD to tackle that thorny question. Dr Yanning Zuo, a postdoctoral fellow in the Telese lab, joined the 8p Research Roundtable last month to present updates on a comprehensive bioinformatics analysis of a massive, first-of-its-kind, single-cell RNA sequencing (scRNASeq) experiment on a 8p family trio of cortical brain organoids.

The organoids were grown for up to nine months in culture by brain organoid pioneer Dr. Alysson Muotri, whose lab is also located at UCSD. Dr. Muotri’s 8p Research Roundtable talk from January 2023 can be viewed here. Recall that his lab performed a preliminary analysis on the 8p brain organoid scRNASeq dataset that revealed a striking result that comes with a
”single-proband, single-clone” proviso.

A peculiar population of Reelin-expressing neurons was present in the 8p hero brain organoids but not in brain organoids derived from either parent. Are these the fabled Cajal-Retzius neurons that are thought to exclusively express Reelin? Or are they something else entirely?

The goal of our collaboration with the Telese lab is to complete an unbiased, well-controlled analysis of the data and let the results inform the next set of experiments. Here’s a snapshot of the initial results from 75,111 high quality nuclei, from which 30 cell subtypes were identified after QC.

At 6 months, a population of glioblasts is significantly expanded in the 8p hero (JE3) brain organoids at the expense of neural progenitor cells (NPCs), radial glia, neuroblasts and interneurons. At 9 months, the proportions of Reelin-positive excitatory neurons and choroid cells are increased in the 8p hero brain organoids.

Zooming in on those Reelin-positive cells, Yanning unexpectedly found that their gene expression profile suggested that they are more similar to cerebellar granule cells than canonical Cajal-Retzius cells. If we were dealing with bona fide Cajal-Retzius cells, we’d expect the following genes to be expressed: RELN, NHLH2, CALB2, LHX5, BARHL2, NR2F2. However, the data show that none of those genes are co-expressed with RELN in the GC-1 and GC-2 (Granule Cell) populations of interest.

Instead, the genes that are co-expressed alongside RELN include: UNC5C, a marker of cerebellar origin; PAX6, a marker granule cell progenitors; NEUROD1, a marker of mature granule cells.

Watch Yanning’s talk on the Project 8p YouTube channel and be sure to stick around for the Q&A at the end.

📄 Recent Articles and Publications

Let us know if you’ve read or published a paper or article recently that you think the 8p community should know about! 

1. The variation and evolution of complete human centromeres. Logsdon G., et al. Nature. April 2024. https://www.nature.com/articles/s41586-024-07278-3
   The last unexplored tracts of the human genome are chromosomal centromeres, the megabases-long structural anchor point on a chromosome where microtubules attach and pull apart chromosomes pairs during cell division. Led by 8p researcher Dr. Glennis Logsdon, this study using long-read DNA sequencing to compare the centromeres of all chromosomes — including chromosome 8 — from two humans and found surprising variation in centromere length and composition even in this limited sample size.

2. Complete chromosome 21 centromere sequences from a Down syndrome family reveal size asymmetry and differences in kinetochore attachment. Mastrorosa F., et al. BioRxiv. February 2024. https://www.biorxiv.org/content/10.1101/2024.02.25.581464v1
   Drs. Glennis Logsdon and Evan Eichler are on a roll in 2024 when it comes to cutting-edge centromere research. This study focuses on the centromeres of chromosome 21 in the context of Down syndrome. As we learn more about the origins of genomic imbalance in meiosis, it becomes possible to imagine ways to prevent chromosomal mishaps from happening in the first place.

3. A high-resolution transcriptomic and spatial atlas of cell types in the whole mouse brain. Yao Z., et al. Nature. December 2023. https://www.nature.com/articles/s41586-023-06812-z

   This tour d’force foundational study performed by scientists at the Allen Institute for Brain Science shows what is possible with current gene expression profiling platform technologies. The Allen Brain Cell Atlas data can be accessed through this portal: https://knowledge.brain-map.org/abcatlas. Project 8p is funding a proof-of-concept scRNA-seq cell atlas project in Dr. Hiruy Meharena’s lab with plans in the works to expand to additional 8p hero samples.

💜 Family Corner

Join us at the 2024 Project 8p Family and Science Conference! 

Register now to explore the latest research and clinical recommendations while connecting with families, forging lasting connections and support networks. Let's come together for empowerment, education, and our 8p Heroes! See you there!

📆 Upcoming Events

• Project 8p Research Roundtable, April 22nd, 1:30 PM EST on Zoom. Email barbara@project8p.org to attend.

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💬 Research Question of the Month

Last month the Project 8p science team published a thinkpiece called A Grand Unified Theory of Neurodevelopmental Chromosomal Disorders. The purpose of the document was two-fold: highlight convergences between different chromosomal disorders, and demonstrate the advantages of studying 8p alongside other diseases of genomic imbalance.

For the researchers in the audience, can you share specific examples of biological convergences between chromosomal disorders? Please drop feedback in the comments!

Leave a comment

🔬Research Highlight

We were thrilled to host Drs Felix Wong and Max Wilson, the founders of Integrated Biosciences, on the February 8p Research Roundtable. Integrated Bioscience is a biotech company with a unique drug screening platform that enables discovery of small molecules that regulate the integrated stress response (ISR) pathway. In short, the team at Integrated Biosciences (IB) is able to artificially induce phantom stress inside of cells without actually exposing the cells to any harmful stressors.

This setup allows IB to identify small molecules that target the ISR sensor proteins directly rather than an indirect downstream or maladaptive response. They have already identified novel small molecules that can amplify or mute the ISR pathway. They also have a proprietary in-house library of several hundred thousand novel compounds.

Therapeutic applications of small molecule modulators of the ISR pathway are being directed at diseases of accelerated aging, which includes chromosomal disorders like 8p and its more well known cousin trisomy 21 aka Down Syndrome (DS). The first connections between genomic imbalance and chronic activation of the ISR pathway were documented five years ago by Drs. Peter Walter and Mauro Costa-Mattioli (Zhu et al., 2019) in multiple complementary models of trisomy 21, including a mouse model of DS, human induced pluripotent stem cells (iPSCs) from a DS patient and an euploid isogenic control, and postmortem human brain samples from 11 DS patients (compared to 11 unaffected controls).

As shown in the highlighted section above, the canonical marker of ISR pathway activation — the ratio of phosphorylated eIF2 protein to total eIF2 protein — is increased in DS mouse brain, DS human brain, and DS human iPSCs, indicative of ISR pathway activation. Intriguingly, analysis of other ISR pathway markers by Dr. Nicoleta Moisoi’s lab in a panel of four 8p hero fibroblast lines revealed that the ISR pathway is down-regulated.

We’re excited to launch a deep phenotyping collaboration with Integrated Biosciences where a full workup of the ISR pathway will be performed on a panel of ten or more 8p cell lines. The goal is to identify robust ISR-pathway-related disease phenotypes that can be rescued in a high-throughput drug screen. Stay tuned for an official announcement soon.

Learn more about the Integrated Biosciences approach and the vision for our collaboration from the founders themselves.

📄 Recent Articles and Publications

Let us know if you’ve read or published a paper or article recently that you think the 8p community should know about! 

1. Non-linear transcriptional responses to gradual modulation of transcription factor dosage. Domingo, J. et al. BioRxiv. March 2024. https://www.biorxiv.org/content/10.1101/2024.03.01.582837v1

   In this study, Domingo et al. used CRISPR activation and inactivation to modulate the expression of four master transcription factors associated with blood cell traits. They show that guide tiling around the transcription start site enables titration of gene dosage over a wide range of expression. Single-cell RNAseq showed that trans genes exhibited non-linear transcriptional responses to these changes in gene dosage.

2.  Autism genes converge on microtubule biology and RNA-binding proteins during excitatory neurogenesis. Sun, N. et al. BioRxiv. December 2023. https://www.biorxiv.org/content/10.1101/2023.12.22.573108v1

   In this study the authors used pooled CRISPR interference (CRISPRi) coupled with single cell RNA sequencing (CROP-Seq) to repress 87 high-confidence ASD genes in a human in vitro model of cortical neurogenesis (2D neural culture). Repression of 17 of these genes resulted in disruption of excitatory neurogenesis, further reinforcing the role of neurogenesis in ASD. 

3. A 25-year odyssey of genomic technology advances and structural variant discovery. Porubsky, D. and Eichler, EE. Cell. January 2024.  https://doi.org/10.1016/j.cell.2024.01.002

   A perspective on the technological advances in variant detection that have occurred over the last two decades, from low resolution g-banding to the base-pair resolution that is now possible with long-read sequencing, and the insights that this complete picture of human variation will make possible. 

💜 Family Corner

Participate in our latest survey in the Chromosome 8p Registry to help us gain insights into the sleep habits of our 8p Heroes.

For inquiries or enrollment in the My Hero Initiative, email kaiti@project8p.org today!

📆 Upcoming Events

• Project 8p Research Roundtable with Dr. Yanning Zuo and Dr. Francesca Telese from UCSD. March 25th, 1:30 PM EST on Zoom. Email barbara@project8p.org to attend.

🌐 Find us on the web

FACEBOOK | INSTAGRAM | TWITTER | LINKEDIN | YOUTUBE

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The p in 8p is short for petit but it could easily stand for puzzle.

In analogy to Newtonian mechanics failing to predict the composition and motion of subatomic particles, the classical frameworks of aneuploidy fail to pinpoint a minimal set of common driver genes that are responsible for the shared constellation of symptoms and disabilities experienced by all 8p heroes who suffered a pre-conception traumatic genome injury to the short arm of the maternal copy of chromosome 8.

Deletions, duplications and inversions — collectively referred to as copy number variants (CNVs) or complex chromosomal rearrangements — wreak havoc on a scale of one to forty megabases, embroiling hundreds of potential drivers on chromosome 8p alone, not to mention possible drivers lurking on other chromosomes.

Drivers, broadly defined, could be coding genes that make a protein, gene regulatory elements like promoters and enhancers, so-called noncoding genes that don’t encode protein, or other functional sequences.

Over half of 8p heroes suffer all three types of traumatic genome injury involving an enormous span of DNA equivalent to the entire 21st chromosome, which causes Down Syndrome when triplicated.

The typical 8p hero genotype is distal deletion at the tip of the short arm paired with an inverted duplication that variably extends down the short arm of chromosome 8 toward the centromere.

For the aficionados in the audience, the deletions tend to occur during maternal meiosis I between non-sister chromatids of homologous pairs. Duplications tend to occur because the repetitive defensin gene cluster creates fault lines along chromosome 8 where slippages and breakages are more likely to happen.

Unlike other chromosomal disorders that involve smaller CNVs or simpler rearrangements, visual inspection of each 8p hero’s chromosomal breakpoints reveals no single affected stretch of the short arm of chromosome 8 — also known as a critical region — shared by all 8p heroes.

Therein lies the puzzle. At no point along the short arm of chromosome 8 can a straight line intersect the affected segments of all 8p heroes.

In 2021, the first clinical cohort of ninety-seven (97) 8p heroes was described in a study recruited and funded by Project 8p — hereafter referred to as Okur et al — led by geneticist Dr. Wendy Chung, Chair of Pediatrics at Boston Children's Hospital.

A complex clinical presentation includes but is not limited to global developmental delays, intellectual disability, autism spectrum behavior, hypotonia, GI dysfunction, congenital heart defects and seizures.

Notably, those clinical features are shared with four other chromosomal disorders: trisomy 21 (Down Syndrome), trisomy 13, (Patau Syndrome), trisomy 18 (Edwards Syndrome), and idic15q (Dup15q Syndrome).

Okur et al identified four critical regions on 8p:

• a deleted segment and an inverted duplicated segment shared by invdupdel 8p heroes

• a distal deleted segment shared by some del only 8p heroes

• a proximal deleted segment shared by some del only 8p heroes

• an extended deleted segment shared by some del only 8p heroes

The authors noted the puzzling absence of top dog driver genes in the discussion section of their paper. The genomic equivalent of spooky action at a distance:

This variability of the distal breakpoints further confounds the identification of additional candidate genes beyond shared duplicated segment given that the same major clinical findings are reported in individuals with the smallest and largest duplications although the severity differs with the distal duplication breakpoints. Thus, in addition to the gene(s) within the shared duplicated segment in invdupdel(8p), involvement of additional genes beyond the shared duplicated segment such as CHRNA2 may add incrementally to the clinical spectrum.

Since 2022, Project 8p sponsorship of research has resulted in a foundational dataset for a pathfinding invdupdel family trio of biosamples obtained from 8p hero, mother and father.

We now have the biobank and operational playbook to scale up experiments that were prototyped on the inaugural 8p family trio. Over the course of 2024, Project 8p will fund new research that will assess the cellular and clinical convergences not only between 8p heroes, but also between 8p and other chromosomal disorders.

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In the history of science, solving a difficult puzzle often births a paradigm shift. Maybe the p in 8p actually stands for perspective? You see the number 8 but I see an inverted duplicated letter p — we’re both right.

Biologists may benefit from borrowing a few quantum mechanics concepts from physicists. Chromosomes are objects in motion, after all. As we learn more about the dynamic 3-D architectures of genomes, the rules of chromosomal shape-shifting appear to be both deterministic and stochastic.

Chromosomes exhibit quantum-like duality of matter: compressing into arrays of compact condensates at one moment, and then, in the snap of a finger, splaying out into conformations choreographed by DNA replication and chromatin remodeling machinery that demands hitting temporal and spatial marks, i.e., specific chromosome-to-chromosome contacts.

A century after physics, biology needs the equivalent of a quantum description of the genome, where the software and hardware of life converge into a remarkable set of computational polymers called chromosomes. The static, staged 2-D rendering of karyotypes is a convenient simplification for diagnostic purposes but misses the 3-D chromosomal choreography that takes places during replication, transcription and DNA damage repair.

Chromosomes condense and decondense, reversibly and conditionally, in specific locations and times inside the nuclei of specific cells. What if any disturbance along the length of chromosome 8p alters the contacts made between chromosome 8 and itself as well as with other chromosomes, which would in turn perturb transcriptional programs that require those long-distance physical interactions? The technologies exist to map the 3-D genome architecture of chromosomes inside the nucleus and we plan to apply them thoughtfully this year.

The fact that 8p heroes nearly universally present with global neurodevelopmental delay tells us that neurogenesis is particularly vulnerable to traumatic genome injuries involving 8p. Something is happening to the genomes of stem cells in the brain as they contort into neurons.

Are the physical pressures of gene regulation forcing chromosomes together and the physical compacting forces on chromosomes inside the nucleus conspiring to create sensitivity to genomic imbalance in brain cells more so than other cells of the body throughout life? A 2023 paper from the Deisseroth lab at Stanford documented dramatic 3-D genome architectural changes in cerebellar granule cells, an understudied neuronal population that actually accounts for the vast majority of neurons in the human brain (Tan et al., 2023).

Of course we must also consider the effects of genomic imbalance on the rest of the cell outside of the nucleus. We know that increased demands on cellular metabolism, over-consumption of rate-limiting factors required for genome maintenance, and chronic activation of cellular stress responses ensue in the aftermath of genomic imbalance.

We’re not sure how all the puzzle pieces fit together. We’re certainly missing a few pieces right now that will surface as our data corpus grows, or that will become visible when we shift perspective. However, we think we’ve found the four corner pieces, meaning at this moment in the arc of 8p research we feel confident that the puzzle is solvable.

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The p in 8p also stands for perseverance. We on the Science Team of Project 8p would like to think that we embody the spirit and the quest of the late Dr. Angelika Amon, who was a universally beloved scholar of aneuploidy.

Dr. Amon discovered the pillars of classical aneuploidy, which she viewed through the lens of cancer. Specifically, a dividing cell seeking a competitive growth advantage over its genetically identical neighbors, i.e., oncogenesis. Her work was so foundational and predictive that experiments performed in yeast cells (Saccharomyces cerevisiae) have been repeatedly corroborated in human cells and mouse embryos.

Had Dr. Amon not tragically passed away in 2020 her lab would surely be studying 8p as a means to deeper understanding of genomic imbalance, and just because she was such a kind-hearted person.

In fact, Dr. Amon received several 8p fibroblast lines from Dr. Chung’s lab. In what turned out to be a final parting gift, the Amon lab generated RNA-seq data comparing three invdupdel 8p fibroblast lines and three trisomy 21 fibroblast lines. These data are available on the online 8p data portal, which will ultimately be the home of all Project 8p-sponsored research.

In 2007, the Amon lab published a seminal paper describing a first-of-its-kind collection of yeast aneuploidy mutants harboring a gain or loss of each chromosome (Torres et al., 2007). On net, aneuploidy is a stressor. Aneuploid cells divide more slowly than euploid controls.

Even in such a simple system as yeast, three tenets of classical aneuploidy explain what is seen in the lab, and therefore yeast cells are predictive of the behavior of cancer cells in the human body:

• An integrated stress response pathway that ramps up glycolysis, shuts down translation and increases protein degradation is chronically activated by imbalance of any chromosome (with a few exceptions based on the overall size of the chromosome).

• Genes that change in DNA copy number (dosage) lead to proportional increases in mRNA levels which can be buffered at the protein level on a protein by protein basis but on average: more DNA leads to more mRNA which leads to more protein.

• Specific dose-sensitive driver genes — sometimes just a single driver — are predominantly responsible for phenotypes rather than aneuploidy per se.

What constitutes a driver gene? Yeast and cancer cells behave similarly in “long term evolution” experiments, the former in the lab and the latter in the body. The mechanics of genome copying mean that even if one gene is solely responsible for a selective growth advantage, the cell can’t just selectively copy and paste parts of its genome on a strict gene-by-gene basis.

So, bystander genes down the street, chromosomally speaking, from a bona fide driver gene get carried along for the ride, say as part of a partial or whole-chromosome duplication event. The debate of what is a driver — and the extent to which aneuploidy itself is a driver of cellular or tissue pathology — has raged for decades in the oncology space.

Aneuploidy has been studied extensively in the context of cancer, which involves genome evolution of somatic cells. Genomic imbalance naturally occurs in individual cells of all humans as we age, usually taking decades to manifest as malignancy or decline in fitness.

Drs. Maitreya Dunham and David Botstein demonstrated in 2002 that yeast cells under selective pressure to grow faster experience the same kinds of chromosome copy number variations as precancerous cells in the human body (Dunham et al., 2002). In other words, aneuploidy occurs as a result of random errors in mitosis that increase in frequency as an organism ages.

But what happens when a traumatic genome injury occurs in meiosis, i.e., during the production of gametes (eggs or sperm), such that genomic imbalance is present in the first cell of a new life?

We all start out as a fertilized egg which multiplicatively blossoms into a fully formed fetus. In the case of 8p, the stresses of genomic imbalance and the shadowy actions of a conspiratorial network of drivers are experienced from the very first cell division, as one cell becomes two, two cells become four, and so on. Yet despite that congenital chromosomal wound, not all cells of the body are vulnerable to traumatic genome injuries involving 8p.

Dr. Amon’s lab showed that aneuploidy increases the rate of non-genetic individuality in cells, and this effect is amplified in whole organisms as they age (Beach et al., 2017). If you ask us, she was hot on the trail of a grand unified theory of chromosomal disorders. In our search for treatments and cures for 8p, we have taken up the banner in solidarity with a network of like-minded scientists in academia and industry.

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In October 2022, Project 8p published the first edition of the 8p Cure Roadmap, a two-year research plan with four focus areas. Having passed the halfway point a few months ago, the primary purpose of this document is to highlight and synthesize key discoveries made by 8p-sponsored researchers. We also honor our open science commitment by sharing scientific and operational learnings that we hope will benefit other foundations focused on chromosome disorders.

Some scientific bets paid off, while others approaches will need to be revised or postponed or deprioritized. We share our strategy for expanding the 8p researcher network to include mission-aligned industry partners, e.g., biotech platform companies.

Building on the chromosome structure analogy, we originally described four research segments, to which we added a fifth segment: Assistive/Augmentative Devices.

Almost all 8p heroes have an AAC device in their daily lives. The idea of “electroceuticals” whose clinical development path is uncoupled from target-based therapeutic modalities is attractive from the point of view of delivering meaningful interventions to 8p heroes in the here and now.

We’re still in the exploration phase of getting to know academics and startups in the space. For example, we hosted a company called Actipulse at the October 2023 8p Research Roundtable. At the 8p Conference this summer, we plan on hosting a medical carnival where companies like Actipulse can demo their wares and interact with families and researchers at the same time.

Project 8p has provided catalytic funding to an academic researcher network comprised of five active projects (Meharena, Pinter, Sheltzer, Warmflash and Telese) and four completed projects (Chung, Muotri, Moisoi, and Logsdon/Eichler) as of February 2024.

The Project 8p Science Team is:

• Ethan Perlstein, Ph.D. — Science Director

• Barbara Celona, Ph.D. — Programs Director

• Whitney Dolan, Ph.D. — Data Scientist

Segment 1: Drug Repurposing

Drug repurposing — finding a second or third (or nth) use for a drug already approved for one purpose — requires disease models that display pathological phenotypes that can be rescued in high-throughput assays.

We are primarily focusing on two complementary cell-based model systems: 8p skin fibroblasts, and 8p neural progenitor cells (NPCs) derived from reprogrammed iPSCs. We plan on deploying two unbiased assays: imaging of morphological features in cells by automated microscopy, and genome-wide measurements of mRNA expression levels in cells by automated sequencing.

Project 8p worked with Dr. Marta Dominguez-Prieto in Dr. Nicoleta Moisoi’s lab in the UK to perform a rigorous battery of image-based and sequencing-based tests on a set of four 8p fibroblast lines — two invdupdels and two dels only — alongside four control fibroblast lines. The readouts included the Cell Painting palette, integrated stress response gene expression profiling, mitochondrial function assessments, DNA damage assessments, and senescence marker analysis.

Moisoi lab observed biological variability between the four 8p hero-derived fibroblast lines across all assays. In spite of that variability, statistically significant trends were detected when the four 8p samples are pooled and compared to the average of four healthy control lines.

For example, mitochondrial membrane potential is consistently decreased in the 8p samples. As another example, lysosomal content is consistently increased in the 8p samples. Intriguingly, the percentage of senescent cells is consistently increased in the 8p samples. (We’ll return the subject of senescence later).

Thanks to the proof-of-concept data generated by the Moisoi lab in a university setting, we are currently in discussions with a platform startup that has the ability to perform automated, AI-assisted, image-based drug screening on fibroblasts using Cell Painting and senescence markers.

The scaled up (and already paid for) capacity of a venture-backed startup compared to a single academic lab funded by a seed grant will enable us to test at least 10 different 8p fibroblast lines alongside a comparable number of controls. Ultimately, we may need to test dozens of different 8p fibroblast lines to overcome the biological variability intrinsic to 8p cells.

In parallel, we’re taking a second stab at drug repurposing using RNA-seq as the readout. Working with the company Rarebase taught us three complementary scientific lessons. First, a primary drug screen needed to be performed on 8p cells, not on healthy cells followed by hit validation in 8p cells. Second, the genome-wide transcriptional profile of 8p cells needed to be rescued, not just the expression of individual suspected driver genes. Third, pluripotent neural progenitor cells (NPCs) are a more therapeutically relevant substrate for high-throughput drug screening than terminally differentiated glutamatergic neurons.

Based on those learnings, we are currently in discussions with a platform startup to perform RNA-seq-based drug repurposing screens on 8p NPCs with the goal of restoring the genome-wide 8p transcriptional profile to normal, or as close to normal as possible.

We will elaborate further in the next section, but promising results from Dr. Alysson Muotri’s lab on cortical brain organoids open up the possibility of drug repurposing on a 3-D model, not just 2-D fibroblasts and NPCs. We’ve had preliminary discussions with a platform startup that has developed technologies for high-throughput screening to rescue germ layer patterning defects in 3-D brain organoids.

Segment 2: Disease Modeling

As described above in the tribute to Dr. Angelika Amon, the first forays into 8p disease modeling combined the simplicity and ruggedness of skin fibroblasts with RNA-seq. Three 8p invdupdel lines were compared to three trisomy 21 lines. We offered a high-level summary of those findings, juxtaposed with the Moisoi lab phenotypic assessments, last summer on our Substack (see Skin Deep).

At the other extreme of disease model complexity, Dr. Alysson Muotri’s lab cultured in the lab 1-month, 3-month, 6-month and 9-month cortical brain organoids from the pioneering family trio. Muotri lab then performed single-cell RNA-seq on all organoids from all timepoints, yielding a fecund dataset that we’re still exploring.

Although Muotri lab tested just a single clone of the 8p hero, maternal and paternal lines, we were delighted by an unexpected result: an unusual population of Cajal-Retzius-like neurons that express high levels of the gene Reelin (RELN) was present in the 8p hero organoids at the 6-month and 9-month timepoints. Strikingly, terminally differentiated excitatory glutamatergic neurons cultured for 21 days — but not for 7 days — express high levels of RELN, too. Those high-level findings were posted last year on our Substack in the practice of open science (see Reelin Groovy).

Open science isn’t just a buzzword, it’s a proven research accelerant and an effective researcher recruitment tool. We would not have appreciated the Reelin convergence between 3-D cortical brain organoids and 2-D glutamatergic neurons had it not been for Project 8p’s monthly 8p Research Roundtables, where those data were shared hot off the presses.

Dr. Francesca Telese read the Reelin Groovy post and was inspired to contact us. That spark led to our current exciting bioinformatics collaboration with Dr. Yanning Zuo in the Telese lab. The goal of this project is to extract biological insights from the massive cortical organoid single-cell RNA-seq experiment completed by the Muotri lab. Where those insights will take us beyond Reelin and Cajal-Retzius neurons is anyone’s guess at this point, but that’s the nature of research on the frontier of knowledge.

We now have RNA-seq datasets from 8p fibroblasts, 8p neural progenitor cells, 8p differentiated excitatory neurons, and 8p cortical brain organoids for a pioneering invdupdel 8p hero. With our expanded Science Team in place and the launch of the 8p data portal in the weeks ahead, we will perform a comprehensive analysis and immediately share the results with our 8p researcher network.

Dr. Hiruy Meharena’s lab was recruited by Project 8p based on his wealth of experience with trisomy 21 and his integrative approach to studying chromosomal disorders that share a core set of clinical features.

So far, Co-Investigator Dr. Ashley Watson and teammates in the Meharena Lab have transcriptionally profiled 8p neural progenitor cells (NPCs) from the pioneer invdupdel proband side-by-side with trisomy 21 neural progenitor cells.

Incredibly, half of differentially expressed genes in 8p NPCs are identical in trisomy 21 NPCs! Cooler still, we observe the same degree of transcriptional convergence between 8p fibroblast lines and trisomy 21 fibroblast lines.

Rewinding the developmental clock, Project 8p sought out Dr. Aryeh Warmflash to study the first stages of brain development using induced pluripotent stem cells differentiated toward ectoderm, the embryonic tissue that in part gives rise to the brain.

The Warmflash lab collected data on the pioneer 8p invdupdel line showing a clear phenotype of increased mesoderm differentiation. His lab honed in on two 8p genes, FGF17 and TNKS, and assayed their effect on germ layer differentiation. The final results of this project will be published on our Substack soon.

What’s next on the disease modeling menu? We’ve got flies, zebrafish, frogs, and mice. All of those animal models have demonstrated their chops in monogenic disease modeling, but the lack of synteny across evolutionary timescales means human chromosome 8 is not the same as mouse chromosome 8, and forget about comparisons between human and the simpler model organisms. We’re in active discussions with academic labs and platform startups to determine the smartest proof-of-concept projects.

Segment 3: Chromosome Therapy

Acknowledging that chromosome therapies are on the distant horizon due to delivery challenges and the need for persistence over cell division, we are focusing on the practical: a toolkit for chromosome engineering.

The most important tool is a so-called isogenic control. In the case of a monogenic variant, CRISPR can be used to correct the disease-causing mutation while in theory not leaving a meaningful scratch anywhere else in the genome. But there is no gene editing machinery that can operate on the scale of megabases. The state of the art today, prime editing maxes out in the tens of kilobases. Instead we must rely on laboratory evolution.

Project 8p enlisted Dr. Jason Sheltzer to the 8p cause with the goal of isolating and characterizing an isogenic control of the pioneering invdupdel 8p hero induced pluripotent stem cell (iPSC) line. Dr. Sheltzer trained with Dr. Amon and his day job is the study of aneuploidy in the context of cancer.

Sophia Lee in the Sheltzer lab tried multiple approaches to restore disomy to the 8p iPSC line. We were looking for a needle in the haystack: a single 8p cell in which the rearranged chromosome 8 is lost and replaced by a second copy of the un-rearranged chromosome.

What seemed to work was the genomic equivalent of a Texas two-step. The un-rearranged copy of chromosome 8 got duplicated resulting in an unstable interim trisomic state that resolved back to disomy by subsequent shedding of the rearranged copy of chromosome 8. These results are published on our Substack (see Balancing Act).

Technically, the new ground state is called uniparental disomy, the closest we’ll get to an isogenic control short of performing reconstructive molecular surgery on the actual rearranged copy of chromosome 8. We’ll know if a presumptive isogenic corrected clone is free of any unwanted genomic alterations soon.

This point can’t be stressed enough: having an 8p isogenic control means in future experiments we’ll have a near-perfect comparison to the original 8p line, and won’t have to rely either on genetically unrelated age-matched and sex-matched controls or on parental controls. Eventually, Project 8p will support the generation of isogenic controls for all 8p hero iPSC lines.

To round out the chromosome engineering toolkit, we recruited Dr. Stefan Pinter to 8p based on his unique thoughtfulness and experience with another chromosomal disorder called Turner Syndrome. The Pinter lab has engineered a euploid (healthy) iPSC line and an 8p invdupdel iPSC line to express either a CRISPR interference (CRISPRi) construct enabling specific gene knockdown, or a CRISPR activation (CRISPRa) construct enabling specific gene activation.

The CRISPRi lines are ready for functional screens. The CRISPRa lines need further optimization but will hopefully be ready to deploy soon. An NGN2 cassette was added to said CRISPRi/a lines to permit differentiation of iPSCs into excitatory neurons. A proposal for an RNA-seq-based functional genomics screen that interrogates multiple candidate 8p drivers using the CRISPRi and CRISPRa lines is currently being evaluated. In parallel, plans are afoot to use XIST-mini genes to silence 1-2 megabase stretches at a time.

Many others are racing to solve the artificial chromosome synthesis problem and the artificial chromosome in vivo delivery problem. Where Project 8p could make a difference is by supporting proof-of-concept megabase-plus chromosome engineering in mouse stem cells and the eventual generation of the first bona fide 8p mouse model.

We think an ideal academic partner in this endeavor would be Dr. Jef Boeke’s lab at NYU based on their extensive success at chromosome engineering in yeast cells. Dr. Ran Brosh from the Dark Matter Project generated the figure below that shows conserved synteny blocks between human chromosome 8 and mouse chromosome 8 along the length of 8p.

A “Block 1” mouse may — we emphasize may — model 8p heroes with a distal deletion only. Even if we successfully obtain the deletion and observe a phenotype, interpreting and understanding whether it relates to 8p heroes will be challenging —and mice are expensive to maintain. We’re currently in early discussions to refine the scope of work and discharge as much risk upfront as possible.

We would also like to collaborate with Dr. Glennis Logsdon, now at University of Pennsylvania, who was funded by Project 8p during her time in Dr. Evan Eichler’s lab. She was eager to explore novel approaches on chromosome engineering directly in 8p cells.

Segment 4: In hero studies

An IRB approved longitudinal natural history study and biorepository has been expanding beyond the Chung study under the oversight of founder, Bina Maniar Shah, in dutiful service to the community as Principal Investigator. Now over 150 heroes are enrolled and caregivers with curated genetic breakpoints linked to clinical phenotypic data.

Much of the behind-the-scenes, unglamorous and often tedious work is attributed to Patient Engagement Manager, Kaiti Syverson, mom to 8p hero Chloe, and Dr. Whitney Dolan, staff data scientist. The diverse biosample collection is a powerful research asset with multiple clones and gender matched biological parent or sibling that will bring new stakeholders into the fold.

We are putting the finishing touches on the 8p Insights Portal where researchers will be able to compare de-identified genotype and phenotype data to form cohorts and hypotheses, as well as to request biosamples and experimental data to put their hypotheses to the test. This endeavor was partly inspired by the portals constructed by Dr. Dennis Lal’s lab for neurodevelopmental disorders, both monogenic and chromosomal. With every 8p hero affected differently and layering publicly available human phenotype ontology and eventually ML algos, you can see the endless possibilities of sleuthing for therapeutic targets.

In a collaboration with Illumina, an exciting data set and analysis is evolving and being made available to researchers. This genotype-phenotype set includes clinician reported data with whole genomes, RNA seq, transcriptomes, and DNA methylomes from peripheral blood mononuclear cells from 100 families across 8p, Dup15q, and Ring14 that will be interpreted and compared.

And none of this would be possible without our research partners’ commitment to open science making discovery data available.

In the concluding section below, we’ll share a synopsis of the first ever “in hero” interventional nutraceutical study that will be sponsored and managed by Project 8p.

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Duplicated 8p drivers of interest

We end this roadmap refresh by going a bit deeper on a few scientific concepts that we broached above, starting with driver genes that call 8p home. What follows is hypothesis and speculation, and a sprinkle of thought provocation.

Sajan et al., 2013 and Vibert et al., 2021 identified two overlapping duplicated 8p segments linked to anomalies or agenesis of the corpus callosum, which is observed at a frequency of 63% in a cohort of 27 8p heroes published just after Okur et al. A 5-megabase critical region includes 51 coding genes. One can make an argument for several of them.

First and foremost is the gene RHOBTB2. RHOBTB2 missense mutations are associated with a neurodevelopmental disorder similar in clinical presentation to 8p. Next on the list are the genes NEFL and NEFM, which encode neurofilament light chain and neurofilament medium chain, respectively. Mutations in NEFL and NEFM cause Charcot-Marie Tooth disease, which is a neurodegenerative condition. As a final example, the gene EBF2. EBF2 in mice controls the migration of Purkinje cells in the cerebellum, and GnRH neurons from the olfactory epithelium to the hypothalamus. Tying back to the RELN finding from the cortical brain organoids, EBF2 is also needed for the generation and migration of Cajal-Retzius cells.

We will use NGN2-induced 8p neurons with inducible CRISPRi to examine cellular and neuronal phenotypes of overexpression of candidate triplo-sensitive 8p driver genes like RHOBTB2, NEFL, NEFM and EBF2.

Genomic imbalance leads to genome dyshomeostasis

The prevailing wisdom about genomic imbalance is that out of the three — DNA, RNA, protein — the real culprit driving aneuploidy stress are out-of-whack protein levels. But what about the total levels of DNA? RNA?

The graduate work of Dr. Barbara Celona on our Science Team, where she studied chromatin remodeling and genome homeostasis, is coincidentally informative to our thinking about chromosomal disorders. We know from classical experiments that aneuploidy per se, i.e., extra and unplanned genomic DNA, is a universal cellular stress, or more accurately a universal collection of linked stresses.

All of that extra and unplanned DNA needs to be wrapped up into nucleosomes by proteins called histones. But it’s not as though the cell has an infinite supply of histones or the wherewithal to manufacture more histones on-demand without incurring unacceptable metabolic or biosynthetic tradeoffs. Also, a damaging loop ensues where DNA not bound by histones is more susceptible to damage and more likely to be inappropriately transcribed.

We’re just starting to wrap our minds around which features of 8p pathology are caused by discrete gene drivers and which features are caused by the cascading effects of genomic imbalance on cell health and survival. The truth is likely a complex interplay of both sets of features.

All segments point to neurogenesis

While the heart and skeletal systems are affected in 8p and other chromosomal disorders, the most affected organ is the brain, especially during fetal and early childhood neurodevelopmental. Why are neural progenitor cells (NPCs) so vulnerable to 8p genomic imbalance and more generally?

We can only make conjectures at this juncture. One consideration surely is the hard-wired metabolic shift from glycolysis to mitochondrial-dependent oxidative phosphorylation (OxPhos) that occurs in NPCs as they differentiate into committed neuronal or glial cell lineages.

Genomic imbalance is a metabolic imposition, an unplanned consumption of rate-limiting metabolites. Neurogenesis may be unique among all the 200+ cell fates possible in the human body because of the rarified combination of morphological and functional adaptations that only neurons seemingly possess. One of those is hypersensitivity to disruptions in metabolic supply chains.

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From neurodev to neuropsych

8p heroes with distal dups tend be to mild-to-moderate in terms of clinical presentation. Their neurological symptoms manifest more as autism-spectrum disorder and attention deficit disorder. Project 8p will begin this year to broaden our scope beyond early childhood neurodevelopment.

Based on hints and clues from other chromosomal disorders, we cannot ignore that as 8p heroes age there needs to be in place proactive surveillance for neuropsychiatric and possibly neurodegenerative symptoms. Over the next few months will we will have conversations with appropriate experts in the field.

Integrated Stress Response and Senescence

A landmark 2019 paper from the labs of Drs. Peter Walter and Mauro Costa-Mattioli implicated the integrated stress response (ISR) pathway is a driver of disease pathology in trisomy 21 aka Down Syndrome (Zhu et al., 2019). Combined with the knowledge of Dr. Meharena’s work on trisomy 21 NPCs, senescence was on our radar, too.

The Moisoi lab results consisting of four 8p fibroblast lines indicated that, on the mRNA level at least, levels of canonical ISR pathway markers are modestly decreased.

We are in contract discussions with a platform startup to discover novel small molecule modulators of ISR and senescence, which could serve as both 8p medicines and one day in the future anti-aging drugs.

Minimum Viable Clinical Trial

Collaborating with nutritionist Geri Brewster RD MPH CDN, we are putting in place the key ingredients of a patient-driven nutraceuticals trial that will establish a robust regiment to optimize development with over-the-counter supplements for all 8p heroes. That way all 8p heroes start out on a more level playing field for foundational health and nutrients. At the same time, we maximize benefit from interventions that don’t require FDA approval. Protocol drafting will commence next month.

Project 8p will act as the trial sponsor with respect to IRB approval. We anticipate recruiting a dozen 8p heroes to participate. We are in discussions with health tech companies and developers for an app or browser-based tool to help us collect trial data easily with a reduced burden on caregivers. It can include electronic health records and using simple mobile phone based video and survey assessments. This in itself will help us go deeper with some families on our natural history study but also invent tools for clinical outcome measures and endpoint analysis.

We’re not holding our breath that a nutraceutical cocktail will be a magic elixir. This exercise is about building the muscle memory and lived experience of 8p heroes and their families so that when a drug repurposing or novel drug candidate or an assistive device/drug combination therapy is ready for prime time, so is the 8p community. Let’s do this!

[The next edition of the 8p roadmap will be a team science co-authored effort written by the end of 2024 and take us through the end of the decade. It will serve as the “roadmap of record” in a peer-reviewed journal.]

💬 Research Question of the Month

In 2024 we plan to invite more scientists and clinicians from outside the 8p research network to present at our monthly research roundtable. What topics would you like to see covered? Do you know someone we should invite? Let us know in the comments!

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🔬Research Highlight

As 2023 wrapped up, so did the first Project 8p-Sponsored Research Project. The project, which was led by Dr. Nicoleta Moisoi at De Montfort University, and carried out by post-doctoral researcher Dr. Marta Domínguez-Prieto, began in July 2022, when we sent 4 fibroblast cell lines from 8p heroes across the pond to their lab in the UK.  

Mitochondrial dysfunction is a hallmark of neurodegenerative disease and we sought to understand if this is also the case in Chromosome 8p disorders. Fibroblasts are a good model system for studying cellular physiology because metabolic changes observed in neurons are often preserved in fibroblasts, yet they are easier and more cost-effective to work with than neurons (Olesen et al. 2022, Cordone et al. 2020). The first phase of the project focused on phenotyping mitochondrial function and cellular features of the fibroblasts. The experiments showed that 8p cells exhibit differences in mitochondrial resting membrane potential, lysosomal staining, staining of the plasma membrane and ER membrane, and cellular senescence. You can read more about those results on the Chromo 8p Substack. Further work will be necessary to understand how these changes may contribute to disease pathology, but in the meantime, we can take advantage of these differences to screen for drug candidates capable of correcting the morphological phenotypes of 8p cells such that they look more like the control cells. 

The second phase of the project, which was completed in December, aimed to assess the DNA damage and repair response in 8p fibroblasts, as accumulation of DNA damage is another common feature of neurodegeneration. To induce DNA damage, cells were treated with Bleomycin. They were then stained for the DNA double-strand break repair proteins H2AX and 53BP1 to assess their response. The image above shows the stained cells after treatment with 40 μg/mL of Bleomycin (Hoechst, blue, nuclear stain; γH2AX, red; 53bp1, green). 

In glucose media, two of the 8p lines accumulated fewer marker proteins (8p 1 and 8p 2), while the two accumulated similar levels as the controls (8p 3 and 8p 4). In galactose media, which enhances metabolism by mitochondrial respiration, all of the 8p lines showed a reduced response to Bleomycin treatment. The reduced accumulation of DNA damage/repair markers could mean that the cells are resistant to DNA damage, or that they are impaired in targeting the repair proteins to sites of DNA damage. These results are consistent with our earlier results (i.e., increased autophagy, lower mitochondrial membrane potential, enhanced senescence), which suggest that cellular stress is driving 8p cells into a state of senescence. Could these changes also happen in brain cells? If so, drugs that are able to alleviate the stress of aneuploidy in skin cells might also promote healthy neuronal development and function.

📄 Recent Articles and Publications

Let us know if you’ve read or published a paper or article recently that you think the 8p community should know about! 

1. Chromosome 8p engineering reveals increased metastatic potential targetable by patient-specific synthetic lethality in liver cancer. Huth, T. et al. Sci. Adv. Dec 2023. https://www.science.org/doi/10.1126/sciadv.adh1442 

   Deletions on chr8p are common in many cancers and correspond with poor prognosis. To study the effects of chr8p loss of heterozygosity on gene expression and metastatic potential, the authors used CRISPR-Cas9 to generate liver cancer cell lines with heterozygous deletion of a 33 Mbp region of chr8p (chr8pLOH). Furthermore, they performed a synthetic lethal CRISPR screen of the chr8pLOH lines, which identified NUDT17 (Chr 1) as a synthetic lethal paralog of the 8p gene NUDT18. The authors propose that these two proteins function redundantly to clear 8oxoG and show that loss of NUDT18 sensitizes cells to loss of NUDT17. As wild-type cells are minimally affected by loss of NUDT17, the gene could be used as a therapeutic target to specifically inhibit the growth of cancer cells harboring chr8p deletions.  

   Interestingly, NUDT18 is within the region that is often duplicated in Chr 8p disorders, suggesting that such cells may actually have an enhanced ability to respond to oxidative stress. 

2. Constructing human neural circuits in living systems by transplantation. Pașca, SP. Cell. Jan 2024. https://www.sciencedirect.com/science/article/pii/S0092867423013405?dgcid=author

   Human stem cell-derived cells transplanted into rodent brains allow the study of complex neural circuits not formed in in vitro models. This review describes the current approaches, applications, and limitations of such xenotransplantation techniques.

3. Building synthetic chromosomes from natural DNA. Coradini, ALV et al. Nat. Commun. Dec 2023. https://www.nature.com/articles/s41467-023-44112-2

   This article describes a new method for constructing synthetic chromosomes from natural DNA components, as opposed to traditional de novo assembly methods, which involve the synthesis of many DNA fragments. The method, named CReATiNG (Cloning, Reprogramming, and Assembling Tiled Natural Genomic DNA), involves cloning fragments of chromosomes and assembling them in yeast through homologous recombination. CReATiNG can be used to study the effects of genome structure and copy number by synthesizing chromosomes with inversions, duplications, and deletions. The method is cheaper and faster than existing methods, and the synthetic chromosomes generated can replace the native chromosomes in cells. Further development is likely to extend this method to mammalian DNA. 

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💜 Family Corner

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Email kaiti@project8p.org and add your piece to the puzzle today!

📆 Upcoming Events

• Research Roundtable with the Multidisciplinary Neurogenetics Clinic, Children’s Hospital Colorado. January 22, 2024 1:30 pm EST on Zoom. Email whitney@perlara.com to attend.

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🔬Research Highlight

This year was an exciting one for 8p research, with 9 sponsored research projects underway, 4 of which launched just this year. 

The best way to keep up with 8p research is by attending our monthly Research Roundtable calls, which bring together 8p families and researchers to discuss research projects, results, new technologies, what matters most to 8p families, and more. We are taking the month of December off for the holidays, but if you’d like to join us at our next meeting in January, please get in touch. In the meantime, you can catch up on any of the past roundtable discussions by watching them on our YouTube channel, or reading our monthly Newsletters on the Chromo 8p Substack.

In 2024 we have even more to look forward to, including more research results and gathering together at the Project 8p Family and Science Conference in Denver, Colorado in August. More on that to come soon! 

We are so thankful that you are a part of this community, and we can’t wait to see you in the new year! 

🧠 Project 8p at the 2023 American Society of Epilepsy Meeting

Project 8p Founder and CEO, Bina Shah, attended the 2023 American Society of Epilepsy Meeting in Orlando, Florida earlier this month, along with many of the members of the NDD-CNV Commision.

The Multidisciplinary Neurogenetics Clinic at the Children’s Hospital Colorado (CHCO), which serves patients and families affected by Chromosome 8p and related neurogenetic conditions, had three posters at the conference. You can view the posters on the Project 8p website here: https://project8p.org/clinic-publications/

The NDD-CNV Commission (co-founded by Project 8p, Dup15q Alliance, and Ring14 USA) held an Investigators Workshop on the “Value of a Shared Framework for Copy Number Variants and Structural Variants” and a Special Interest Group on “Genetics: Clinical Genetics in 2024: What You Need to Know!”

“Epilepsies as a Collection of Rare Diseases” was presented by Dr. Kelly Knupps at CHCO. This theme resonated for most, with attendees throughout the conference discussing how science innovation is prevalent, but clinical therapies are still not very efficacious. Why is that? Perhaps, this will improve with more patient-led groups now intimately involved in advancing their translational science programs into the clinic and actively partnering with industry. There is a lot of work to be done but we did have Dr. Walter Koroshetz, head of NINDS, and other funders listen intently about CNVs, including Chromosome 8p disorders.

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📄 Recent Articles and Publications

Let us know if you’ve read or published a paper or article recently that you think the 8p community should know about! 

1. Assembloid CRISPR screens reveal impact of disease genes in human neurodevelopment. Meng, X. et al. Nature. September 2023. https://www.nature.com/articles/s41586-023-06564-w. Open Access.

   In this study, Meng et al. performed a CRISPR screen of 425 NDD genes in two organoid systems. First, they used human subpallial organoids to screen for defects in interneuron generation, from which they identified 13 candidate genes, including 5 cell-cycle genes. Next, they integrated human subpallial organoids with human cortical organoids to form human forebrain assembloids, which they used to screen for defects in interneuron migration. This screen identified 33 candidate genes, including a number of cytoskeleton and cell migration genes, which they further validated. Efforts like these to map the function of NDD genes are crucial to understanding the complex biology of neurodevelopmental diseases and developing targeted therapeutics.

2. Atypical cortical networks in children at high-genetic risk of psychiatric and neurodevelopmental disorders. Doherty, JL et al. Neuropsychopharmacology. July 2023. https://www.nature.com/articles/s41386-023-01628-x. Open Access.

   In this study, Doherty et al. used magnetoencephalography to assess network function in 34 children with 22q11.2 deletion syndrome and 25 control children. They identified specific changes in local and long-range cortical circuitry that were associated with ASD symptoms and cognitive ability.

3. Unraveling the Link between CNVs, General Cognition, and Individual Neuroimaging Deviation Scores from a Reference Cohort. Fraza, C. et al. November 2023. https://doi.org/10.1101/2023.11.29.23298954. Open Access. This article is a pre-print and has not been peer-reviewed. 

   Franz et al. used normative modeling to map neuroimaging features from several large datasets and then used these models to assess the neurobiological profiles of CNV carriers in the UK Biobank. They found a high degree of heterogeneity between carriers of the same pathogenic CNVs. Their study highlights the impact of individual genetic variation in phenotypic outcomes and provides a means to assess this impact at the level of the individual. 

   Earlier this year, the same group published a tool that makes it easier for others to perform similar analyses: Predictive Clinical Neuroscience Portal (PCNportal). Barkema, P et al. Wellcome Open Research. July 2023. https://wellcomeopenresearch.org/articles/8-326/v2. Open Access.

   Read more about the normative modeling framework: https://www.nature.com/articles/s41596-022-00696-5

4. Dysfunctional sodium channel kinetics as a novel epilepsy mechanism in chromosome 15q11-q13 duplication syndrome. Elamin, M. et al. Epilepsia. September 2023. https://pubmed.ncbi.nlm.nih.gov/37329181/.

   Elamin et al. performed whole-cell patch-clamp electrophysiology on Dup15q neurons and CRISPR-corrected isogenic control neurons and identified specific changes in sodium channel kinetics previously linked to multiple forms of epilepsy. These results shed light on the etiology of seizures in Dup15q patients and will help to guide therapeutic development.

5. Efficacy, safety, and tolerability of soticlestat as adjunctive therapy for the treatment of seizures in patients with Dup15q syndrome or CDKL5 deficiency disorder in an open-label signal-finding phase II study (ARCADE). Demarest, S. et al. Epilepsy Behav. May 2023. https://pubmed.ncbi.nlm.nih.gov/37011526/. Open Access.

   Many commonly used anti-seizure medications show low efficacy in Dup15q and CDD patients. Soticlestat is a new drug in clinical development with a novel mechanism of action for controlling seizures. To determine if Soticlestat could effectively control seizures in these populations, Demarest et al. performed a Phase II clinical trial of Soticlestat in 8 individuals with Dup15q syndrome and 12 individuals with CDD. They found that treatment was associated with a decrease in overall seizure frequency in both groups, and a decrease in motor seizure frequency in the CDD group, but that motor seizure frequency increased in the Dup15q group.

6. Top 5 Insights from the American Epilepsy Society Meeting 2023. Mingorance, A. Dracaena Reports. December 2023. ​​https://www.draccon.com/dracaena-report/aes2023. 

   Dr. Ana Mingorance, Chief Development Officer of the LouLou Foundation and Co-Founder of Singularity Bio and Theracule, shares the trends that she is observing in rare epilepsy syndrome research.  

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💜 Family Corner

We are thrilled to share that we’re three-quarters of the way to achieving our target of 200 8p Heroes in the Chromosome 8p Registry. This achievement is a testament to the strength and unity of our community.

As we reflect on this achievement, we're also eagerly anticipating the advancements in our understanding of Chromosome 8p Disorders set to unfold in 2024. 

We invite you to join us in 2024 by adding your piece to the Chromosome 8p Puzzle. Visit the My Hero Initiative webpage to learn more.

Together Towards Treatment

📆 Upcoming Events

• No Research Roundtable for December. We hope you enjoy the holidays and we’ll see you in 2024!

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💬 Research Question of the Month

In December, 8p researchers and advisors will meet to review and update Project 8p’s two-year research plan. What would you like to see added to the research plan? 

Share your thoughts in the comments section!

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🔬Research Highlight

The most recent Segment to be added to the Project 8p Research Plan is the Fifth Segment focused on Augmentative Devices and Assistive Technologies. At the October Research Roundtable we talked with 8p families about their successes and struggles with various assistive technologies that they use or have tried with their 8p heroes.

Neuroaugmentative technologies are a particularly exciting and active area of research in this field. In the near future, such technologies have the potential to help control seizures, and improve speech and motor function. Project 8p is actively seeking out companies and researchers developing these technologies in order to bring their devices to 8p heroes. At the October Roundtable, we were joined by ActiPulse Neuroscience CEO Adrien Châtillon and CSO Gabriel Villafuerte, who shared their non-invasive brain technology that is being developed to treat Major Depressive Disorder, enhance drug delivery to the brain, and treat brain cancers, all by applying magnetic pulses to the brain. 

Watch the Roundtable on YouTube:

👩‍🔬Featured 8p Researchers

This month, we are highlighting more of the talented researchers who are a part of the Project 8p Research network. They each bring unique expertise and perspective to their fields and to Chromosome 8p disorder research. You can find the first and second installations of this series in the September and October Newsletters on the Chromo 8p Substack.

Dr. Aryeh Warmflash - Associate Professor, CPRIT Scholar in Cancer Research, Department of Biosciences, Rice University.

With a B.A. in Physics and Mathematics and a Ph.D. in Physics, Dr. Warmflash brings a particularly unique perspective to 8p Research. His research combines quantitative biology, physics, mathematics, and engineering to enable insights into early embryonic development and cell signaling. As a postdoctoral fellow working with Dr. Brivanlou and Dr. Siggia at The Rockefeller University, Dr. Warmflash demonstrated that stem cells confined to a small area self-organize into germ layers, reminiscent of the early embryo (Warmflash et al. 2014). Since then, his lab at Rice University has used this method and others to uncover fundamental aspects of cellular communication (Britton et al. 2019, Heemskerk et al. 2019, Nemashkalo et al. 2017, Liu et al. 2022) .

Project 8p has partnered with Dr. Warmflash to investigate the effects of Chromosome 8p rearrangements and candidate driver genes on early embryonic development. Micropatterning experiments using iPSCs from an 8p hero with inversion/duplication/deletion of 8p showed that the cells display distinct changes in patterning. His lab is now comparing these results with those from cells harboring different 8p rearrangements, or single-gene deletions, to test hypotheses about the contributions of different genes to the observed phenotypes.   

Dr. Warmflash is a recipient of the prestigious NSF CAREER Award, the Charles W. Duncan Jr. Achievement Award for Outstanding Faculty (Rice University), and was named a 2017 Simon’s Foundation Investigator. Recently, Dr. Warmflash was awared a $1.9 million dollar, five-year grant from the NIH to model human ectodermal development (Rice News, May 2023). 

Learn more about Dr. Warmflash’s research:

• The Warmflash Lab

• Modeling Development and Disorders with Human Embryonic Stem Cells - Project 8p Research Roundtable, August 2022

Dr. Glennis Logsdon - Postdoctoral Scholar, Department of Genome Sciences, University of Washington School of Medicine. 

Dr. Logsdon is an expert in centromere biology, and in developing and applying wet-lab techniques, long-read sequencing technologies, and computational methods to resolve complex DNA sequences. Using these techniques, she determined the first complete sequence of a human autosomal centromere (Logsdon et al. 2021, Logsdon and Eichler 2021), followed by those of all human centromeres (Altemose et al. 2022). This had been one of the major gaps in previous genome assemblies, and her work contributed to a large group effort to complete the human genome (Nurk et al. 2022, The New York Times). Dr. Logsdon’s interest in centromere biology also extends to the engineering of artificial chromosomes, of which centromere formation is a key challenge. In 2019, she created a streamlined approach to the construction of human artificial chromosomes that bypasses centromeric DNA (Logsdon et al. 2019).

After finishing the first telomere-to-telomere assembly of Chromosome 8 (Logsdon and Eichler 2021), Dr. Logsdon turned to doing the same for Chromosome 8 with a deletion and inversion/duplication of 8p, resolving the exact breakpoints of the rearrangement for the first time. Dr. Logsdon and Dr. Eichler plan to expand this work to more 8p heroes and their families to answer questions about how 8p rearrangements arise, as well as the molecular basis for the phenotypic variability among 8p heroes.

Dr. Logsdon is soon to be an Assistant Professor of Genetics at the University of Pennsylvania Perelman School of Medicine. Her lab will study centromere variation, evolution, and function, and will apply their findings to the engineering of artificial chromosomes. She is a recipient of the highly competitive NIH K99/R00 Pathway to Independence Award, as well as an NIH Ruth L. Kirschstein F32 Postdoctoral Fellowship Award, and NIH T32 Cell and Molecular Biology Training Grant.

Learn more about Dr. Logsdon’s research:

• The Logsdon Lab

• Creating the First Complete Sequence Assembly of Chromosome 8 From an 8p Hero - Project 8p Research Roundtable, July 2023

• Project 8p Research Newsletter August 2023

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💜 Family Corner

Our monthly research roundtables will be on a short hiatus during the months of November and December as we work on updating the 2024 Research Roadmap. Stay tuned for a Family-Friendly Research Update in January, where we will present the refreshed plan to you.

During this break, please check if your My Hero Initiative participation is up to date. We're thrilled to announce that we've reached 149 Heroes in the Chromosome 8p Registry, with nearly 100 participants completing all surveys. For assistance, please contact Kaiti Syverson at kaiti@project8p.org

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Since the dawn of life three billion years ago up to this very moment, chromosomes perform a timeless microscopic Cirque du Soleil inside the nucleus of every mother cell that is poised to divide into two daughter cells. This improbable performance has repeated trillions upon trillions of times across the tree of life over our collective evolutionary history. Critically, the genetic material in a mother cell — in the case of humans, 23 pairs of chromosomes — must be evenly bequeathed to her two daughter cells. Otherwise, the genome is out of balance and the consequences can be catastrophic.

For example, most miscarriages are caused by aneuploidy affecting one or more chromosomes. Just this week, a study of 948 individuals showed that chromosomes are constantly lost or gained or rearranged in tissues across the body, becoming the seeds of cancer that try to sneak past the immune system. Chromosome acrobats, flying near flawlessly every night without a net sometimes fall. There are cases where aneuploidy is not lethal in utero and allows for development of a fetus to term. The most well known case is trisomy 21 (T21), or Down Syndrome.

Trisomy is compatible with life in the cases of chromosome 18 (T18), Edward Syndrome, and chromosome 13 (T13), or Patau Syndrome. Not surprisingly, trisomy of a smaller chromosome that contains less genetic cargo is more likely to be tolerated than trisomy of a larger chromosome that contains more genetic cargo. Stem cells in particular may be particularly sensitive to aneuploidy because they need to function nearly flawlessly throughout our lives.

To wit, multi-month in vitro passaging of trisomic induced pluripotent stem cells (iPSCs) from patients with Down Syndrome, Edward Syndrome, and Patau Syndrome leads to the spontaneous generation of disomic cells that lose their toxic third chromosome and regain disomy, which confers a selective growth advantage over the trisomic founder cells. A study from 2019 on Down Syndrome iPSCs is dispositive:

What about 8p, where the duplicated segment can be as large as 30 megabases, which is the same size as the entire length chromosome 21? For inv/dup/del 8p heroes, it’s not just a duplication it’s also a 1-10 megabase deletion, and an inversion just for kicks. Is the rearranged 8p chromosome toxic to cells like the extra copies of chromosome 13, 18 or 21? Though keep in mind that in T21, T13, and T18, the extra genetic material is a whole freely segregating chromosomes, whereas in 8p it is not.

As stated in the Cure Roadmap published a year ago, Project 8p set out to answer that question and others in collaboration with Professor Jason Sheltzer’s lab at Yale University. This project is actually a twofer in the sense that it yields not only discoveries but also practical deliverables. Not only will we be able to assess the intrinsic cytotoxicity of the rearranged 8p chromosome in iPSCs, but in the process we will be able to isolate disomic but otherwise genetically identical (isogenic) 8p IPSC clones. In the business we call that an isogenic corrected control line, which itself is a non-negotiable asset for downstream research applications like chromosome therapy.

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Everyone at this point in time has probably heard of gene therapy: replacing a single broken gene with an intact working copy. When genetic lightning strikes a chromosome, imagine gene therapy but on anabolic steroids: replacing or fixing dozens if not hundreds of storm-tossed genes at once.

8p rearrangements are a complex coalition of copy number variant disorders. One cohort of 8p heroes has lost one copy of the genes located in deleted 8p segments, resulting in haploinsufficiency. Another cohort of 8p heroes has an additional copy of each gene — meaning three copies instead of the usual two — in 8p duplicated segments. The largest cohort of 8p heroes has both duplications and deletions and the aforementioned inverted segment.

Confusingly, the body sometimes doesn’t mind if some genes are present in half or double the normal amount, but cares deeply when the genome as a whole is out of balance. At the same time, some genes have the dubious honor of being “driver genes,” as appears to be the case with the gene called RELN aka Reelin. That means the body cares a lot whether a specific gene is in balance, i.e., present in two copies, one from mom and one from dad.

Building on their success in studying aneuploidy in the cancer space, Sophia Lee in the Sheltzer lab at Yale University applied those learnings to 8p. As a first step, the 8p pioneer induced pluripotent stem cell (iPSC) trio — mom, dad and 8p hero — was karyotyped. That’s the technical term for assessing how many copies of each chromosome are present in a sample from a patient. Normally, there are two copies of each of the 22 autosomal chromosomes. The 23rd pair of sex chromosomes — XX in girls, XY in boys — can also be detected.

Zooming in on the affected proband, which is genetics-speak for the affected child, we can clearly discern both the deleted (haploinsufficient) 8p segment at the half of the normal copy number as well as the duplicated (trisomic) 8p segment at double the normal copy number. The Y chromosome is undetectable, as expected since the proband is female. Crucially, besides 8p there are no other detectable chromosomal copy number variants (CNV) detected. That means the results won’t be compromised by an unwanted CNV outside of the 8p region.

Next, Sophia and Jason devised a devilishly simple quantitative PCR (qPCR) assay for tracking changes in chromosome 8 copy number in cells passaged in the lab over many weeks. It works by quantifying the amount of a marker gene as a proxy for the copy number of an affected segment of 8p. The CSMD1 gene resides in the proband’s deleted 8p segment, while the MICU3 gene is located in the proband’s duplicated 8p segment. As expected, only a single copy of CSMD1 is detected in the proband in contrast to the two copies detected in the parents and an unrelated healthy control line. Correspondingly, one extra copy of MICU3 is detected in the proband.

The next phase of the experiment is a waiting game. As cells divide in culture, there’s selection pressure to grow faster and faster. If the damaged 8p chromosome is intrinsically toxic, the selective pressure to jettison the damaged chromosome while at the same time making an entire extra copy of the unaffected chromosome 8 is enormous.

After more than 100 days of passaging in the lab, Sophia assessed the copy-number status of chromosome 8 using the CSMD1 deleted-segment marker and the MICU3 duplicated-segment marker. The results are fairly definitive: there was no spontaneous return to genomic balance. Therefore, we can likely conclude that the damaged 8p chromosome is not inherently cytotoxic — at least in undifferentiated iPSCs — like the three well-known trisomies. As a community we still have to digest this result and its implications, but for the purposes of generating a euploid 8p proband clone, just letting 8p cells divide over time wasn’t sufficient to do the trick.

In his original proposal, Jason mapped out several alternate selection strategies in the event that the spontaneous path didn’t pan out. The first trick up their sleeve was to treat proband cells with a compound called AZ3146, which inhibits the mitotic kinase MPS1. When that happens, the probability of what are called missegregation events increases significantly. Missegregation events mean errant cell division resulting in whole-chromosome gains and losses.

Dozens of clones were assessed for chromosome 8 copy number changes, in any direction. What we want to see are clones that have an identical profile to the control, which is diploid (two copies of each gene). Most of the clones did not exhibit any change in copy number; they stayed exactly the same. Clone 37 exhibited a quadrupling of the CSMD1 marker gene, implying that just the deleted 8p segment was present in tetrasomy, or four copies. Interestingly, several clones show an increase in copy number of both CSMD1 and MICU3, not just one or the other.

For example, clones 31 and 33 look promising. CSMD1 is clearly increased in copy number from 1 to 2. MICU3 also appears to be increased in copy number from 3 to 4, though the result is more striking for clone 33. These data suggest that clones 31 and 33 gained an entire copy of chromosome 8, or a trisomy comprised of the original unaffected paternal chromosome 8, a newly duplicated unaffected paternal chromosome, and the damaged maternal chromosome 8.

Whole genome sequencing of clones 31 and 33 confirmed the presence of a third copy of chromosome 8. The yellow arrows show the q (long) arm of chromosome 8 at > 1 copy number. Consistent with the interpretation of a gain of an unaffected full-length chromosome 8, the deleted 8p segment and the duplicated 8p segment also increase in copy number to the same extent.

In the next phase of this foundation-laying project, clones 31 and 33 will be further passaged in the hopes that the rearranged 8p chromosome is spontaneously lost during a cell division. Sheltzer lab has other tools available to target the rearranged 8p chromosome for elimination, e.g., KaryoCREATE.

Once we have proband iPSC clones with restored disomy, the final phase of the project will involve a head-to-head comparison to the original rearranged/damaged 8p chromosome proband iPSC line. Phenotypes that will be examined are doubling time, whole-transcriptome RNAseq profiling, and neural rosette formation. A similar workup was performed in the above-cited 2019 paper describing spontaneous recovery of disomy of Down Syndrome iPSCs in long-term cell culture.

Unfortunately, genomic imbalance is not just a threat to life starting in the embryo. Aneuploidy is a silent assassin that is constantly taking out individual cells in your body all the time, and the rate accelerates as we age faster than our immune system can keep up. Aneuploidy is one of the pillars of aging.

So when anyone asks why they should care about 8p, aneuploidy is why. Project 8p is leading this charge and making the case for why 8p will be an experimental sandbox for developing curative chromosome therapies one day in the future.

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💬 Research Question of the Month

Project 8p is looking for companies and researchers working on novel technologies for augmentative devices. How have augmentative devices helped your 8p hero? How do you imagine they could help? Do you know of any companies we should talk to?

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🔬Research Highlight  - The Project 8p Research Network 

This month, we are highlighting more of the talented researchers who are a part of the Project 8p Research network. They each bring unique expertise and perspective to their fields and to chromosome 8p disorder research. You can find the first installation of this series in the September Newsletter on the Chromo 8p Substack.

Dr. Nicoleta Moisoi - Associate Professor in Pharmacology and Director of Leicester Institute for Pharmaceutical Innovation, Leicester School of Pharmacy, De Montfort University. 

Dr. Moisoi is an expert in the field of mitochondrial homeostasis, a cellular process essential to healthy brain function (Savu and Moisoi 2022). Mitochondrial dysfunction is a hallmark of many neurodegenerative diseases, and Dr. Moisoi’s work has focused on elucidating the role of mitochondria and cellular stress signaling pathways in neurodegenerative diseases like Parkinson’s (Moisoi et al. 2014, Temelie, Savu, and Moisoi 2018, Temelie et al. 2023), using cell and animal models including Drosophila and mice. As a professor of pharmacology, Dr. Moisoi brings to the 8p research network valuable experience in identifying druggable pathways and a strong focus on translatability and therapeutic potential.  

In collaboration with Project 8p, Dr. Marta Domínguez-Prieto, a postdoctoral research fellow in Dr. Moisoi’s lab, has characterized mitochondrial function and cytological features of fibroblasts from 8p heroes (Skin deep - Project 8p Substack). The work they have done will serve as the basis for the first  high-throughput drug-repurposing screen for chromosome 8p disorders. Ongoing work in the Moisoi lab is looking at the function of DNA damage and repair pathways in 8p cells. Mitochondrial signaling plays an important role in responding to genotoxic stress, and when impaired, leads to the accumulation of DNA damage, which can harm cell function. This work will determine whether accumulation of DNA damage in 8p cells might be contributing to abnormal neuronal function.

Dr. Moisoi was named a Junior Associate of the International Centre for Theoretical Physics, Trieste Italy and a Royal Society/NATO Postdoctoral Fellow. Her research has been funded by groups such as the Michael J Fox Foundation. 

Learn more about Dr. Moisoi’s research:

• Dr. Nicoleta Moisoi 

• Phenotyping of Mitochondrial Dysfunction and DNA Damage and Repair in Chromosome 8p Disorder (Project 8p Research Roundtable - May 2022)

• Mitochondrial Dysfunction and DNA Damage Repair in 8p Fibroblasts (Project 8p Research Roundtable - January 2023) 

Dr. Hiruy Meharena - Assistant Professor, Department of Neurobiology and Molecular Biology, University of California, San Diego.

Dr. Meharena completed his postdoctoral training in the lab of Dr. Li-Huei Tsai at MIT. It was there he began studying memory formation, neurodegeneration, and Trisomy 21 using cutting edge genomics and imaging techniques to peek into the nucleus (Marco et al. 2020). In an important finding, Dr. Meharena discovered that trisomy 21 induces features of cellular senescence in neural progenitor cells, which could serve as a therapeutic intervention point (Meharena et al. 2022). Now, his lab at UCSD applies these techniques to iPSC-derived 2D and 3D models of the brain to understand how genomic imbalance gives rise to neurodegeneration and intellectual disabilities (Watson and Meharena 2023). 

Dr. Meharena’s experience studying Trisomy 21 serves as a valuable foundation for the study of chromosome 8p disorders. Currently, his lab is studying neural progenitor cells derived from the iPSCs of 8p heroes in order to identify cellular and molecular phenotypes, and whether they might be similar to those of trisomy 21 and other copy number variants. These phenotypes could be used for high throughput screening and inform possible therapeutic interventions, and will provide insights into the molecular underpinnings of 8p disorders.

Dr. Meharena is a recipient of the Massachusetts Institute of Technology (MIT) Infinite Expansion Award, and has been named an Alana Down syndrome Center (ADSC) Fellow, Rising STAR in Biomedical Sciences Fellow, UNCF/Merck Fellow, Massachusetts Institute of Technology (MIT) Impact Fellow, and Burroughs Wellcome Fund PDEP Fellow.

Learn more about Dr. Meharena’s research:

• The Developmental and Cognitive Genomics Research Group at UCSD

• Exploring the Molecular Etiology Underlying Neurodevelopmental & Cognitive Features of 8p Disorders (Project 8p Research Roundtable, October 2022)

• Provocative New Findings Suggest Surprising Cause of Down Syndrome (STAT news, January 2022)

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💜 Family Corner

Mark your calendars for the Family and Science Conference in Denver, Colorado from July 31 to August 3. Save the date and get ready for an inspiring gathering of knowledge and community.

📆 Upcoming Events

8p Research Roundtable, October 23rd, 1:30 PM EST. Email whitney@perlara.com if you would like to attend.

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💬 Research Question of the Month

Do you know a researcher who should be part of the Project 8p Research Network? 

Let us know in the Comments section below!

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🔬Research Highlight  - The Project 8p Research Network

This month we are highlighting some of the talented researchers who are a part of the Project 8p Research network. They each bring unique expertise and perspective to their fields and to chromosome 8p disorder research.

Dr. Alysson Muotri - Professor, Departments of Pediatrics and Cellular & Molecular Medicine, School of Medicine, University of California, San Diego. 

Dr. Muotri’s lab studies the evolution and development of the human brain using human stem cell models, brain organoids, electrophysiological recordings, and genomics techniques. He has made important contributions to the field of autism research and his partnerships with multiple rare diseases foundations, including the Loulou Foundation, Pitt-Hopkins Research Foundation, and the Rett Syndrome Research Trust, have led to the development of disease models that will help these communities better understand their disorders, and ultimately develop therapeutic treatments (Marchetto et al. 2010, Urresti et al. 2021, Wu et al. 2022). 

Currently, Dr. Muotri’s lab is using cortical organoids derived from 8p families to better understand which cell types drive 8p neurodevelopmental phenotypes. Already this work has uncovered a potential key player in the pathobiology of 8p, a gene called RELN.

Dr. Muotri was a 2002 Pew Fellow, and is the recipient of numerous NIH grants and awards, including the NIH Director’s New Innovator Award, NARSAD, Emerald Foundation Young Investigator Award, Surugadai Award, Rock Star of Innovation, NIH EUREKA Award, and two Telly Awards for Excellence in Science Communication. Dr. Mutori will co-lead the newly launched UCSD Gene Therapy Initiative.

Learn more about Dr. Muotri and his research:

• The Muotri Lab

• Modeling 8p with human brain organoids (Project 8p Research Roundtable, September 2022)

• Alysson Muotri: A Brain Begins - in a Dish (Science Clear+Vivid podcast with Alan Alda)

• STEM Capsule: Alysson Muotri

Dr. Jason Sheltzer - Assistant Professor of Surgery (Oncology) and Genetics, Yale School of Medicine.  

Dr. Sheltzer began studying aneuploidy in cancer as a postdoctoral fellow in the lab of the late Dr. Angelica Amon at MIT. Since then, he has made important contributions to the field of cancer research, identifying drivers of cancer resistance (Lukow et al. 2021) and prognosis (Smith and Sheltzer 2022), highlighting oversights in drug development (Lin et al. 2019, Bhattacharjee et al. 2022, Médard and Sheltzer 2023, The New York Times), and developing and employing novel CRISPR techniques to identify cancer’s genetic dependencies (Girish et al. 2023, Yale News). He has also published multiple papers on the topic of gender disparities in STEM (Sheltzer and Smith 2014, Sheltzer 2018, Greider et al. 2019, The Washington Post).

Now, Dr. Sheltzer is applying his deep knowledge of aneuploidy and experience with cutting-edge CRISPR techniques to chromosome 8p disorders. Using multiple techniques, his lab will attempt to restore disomy to 8p iPS cells in order to generate genetically matched 8p and control lines, which will serve as an important model for further research and drug screens. This work will also shed light on the possibility of chromosome engineering as a therapeutic strategy. 

Dr. Sheltzer has been awarded the Gilula Prize from the American Society for Cell Biology, the Presidential Early-Career Science and Engineering Award, and was named one of Forbes Magazine’s  30 under 30 in Science (2015). Dr. Sheltzer is also a co-founder of the biotech startup Meliora Therapeutics, which has raised $11M in seed funding to develop mechanism-focused oncology drugs.

Learn more about Dr. Sheltzer’s research:

• The Sheltzer Lab

• Chromosome Engineering to Restore Disomy in 8p iPS Cells (Project 8p Research Roundtable, June 2023)

• Project 8p Research Newsletter - July 2023

Dr. Stefan Pinter - Assistant Professor, Genetics and Genome Sciences, Institute for Systems Genomics, University of Connecticut. 

Dr. Pinter did his postdoctoral work in the Harvard lab of Dr. Jeannie Lee, a pioneer in the field of long noncoding RNA. There he became an expert in X chromosome inactivation (Pinter et al. 2012, Simon et al. 2013, Pinter 2016, Bansal et al. 2020, Bansal et al. 2021). Now, at the University of Connecticut, his lab is applying tools like XIST and CRISPR to study the developmental impact of changes in gene dosage in aneuploidy disorders like Trisomy 21 and Turner Syndrome (Ahern et al. 2022, Ahern et al. 2023, Bansal et al. 2023). His experience thinking about aneuploidy, complex modes of gene regulation, and in developing gold-standard iPSC-derived models are a huge asset to the 8p research network.

Dr. Pinter’s lab is currently working on a project to equip 8p and euploid iPS cells with inducible NGN2, to drive neuronal differentiation, and inducible CRISPR constructs capable of turning gene expression on (CRISPRa) or off (CRISPRi) in a targeted fashion. These tools will be used to dissect the impact of changes in gene dosage of individual 8p genes.  

Dr. Pinter is a recipient of the prestigious NIH MIRA Award. He has also received the Centennial Award for Molecular Genetics from The Genetics Society of America for his outstanding research article “Allelic Imbalance Is a Prevalent and Tissue-Specific Feature of the Mouse Transcriptome” (Pinter et al. Genetics 2015), and has received fellowships from Massachusetts General Hospital and the German Research Foundation (DFG). 

Learn more about Dr. Pinter’s research:

• The Pinter Lab

• CRISPRa/i tools to probe gene function in 8p research (Project 8p Research Roundtable, May 2023)

• Project 8p Research Newsletter - June 2023

💜 Family Corner 

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• 8p Research Roundtable, September 25th, 1:30 PM EST. Email whitney@perlara.com if you would like to attend.

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Project 8p is the world’s first nonprofit biotech whose mission is to lead the charge toward treatments and cures for complex chromosome diseases caused by megabase rearrangements. Pick your flavor combo: deletion, duplication, and even inversion. 8p heroes can have all three, including heroes who just have an 8p deletion or an 8p duplication.

For some perspective on scale, and as another reminder why 8p is particularly challenging: you can fit an entire copy of human chromosome 21, which encodes several hundred genes, inside a single typical 8p duplicated segment.

A year ago, Project 8p commissioned the first edition of a CureMap, which identified five interlocking research priorities that assemble into a superstructure, much like the segments of a chromosome. Disease modeling is the centromere, the core scaffold around which the rest of the chromosome is constructed.

Project 8p placed a bet on Dr Alysson Muotri’s lab at UCSD to conduct a first-of-its-kind neurodevelopmental disease modeling study on a pioneer 8p family trio. Specifically, induced pluripotent stem cells (iPSCs) from mom, dad and affected child were differentiated into 1-month, 3-month, 6-month, and 9-month-old brain organoids. Brain organoids were disaggregated and then single-cell RNA-seq was performed.

The bet paid off with the surprising discovery of Reelin (RELN) over-expression in a large population of differentiated glutamatergic neurons in the proband’s 9-month 8p brain organoids that appear to be Cajal-Retzius cells. There’s also a second but smaller population of Reelin-positive glutamatergic neurons in the proband’s 9-month 8p brain organoids that are actively dividing. Followup electrophysiology studies are already underway in Dr Muotri’s lab. Project 8p is also working with Dr Hiruy Meharena’s lab at UCSD, which has been studying aneuploidy from the trisomy 21 perspective.

In this update, we’ll highlight a complementary approach to 8p disease modeling using 8p hero-derived skin cells aka fibroblasts. At first blush, fibroblasts may pale in comparison to brain organoids but fibroblasts have a few tricks up their sleeve. Like enabling drug repurposing screens faster and cheaper than any other 8p disease model available. And serving as a launchpad to studies in slower and costlier 8p disease models.

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The technical term for complex chromosome rearrangements is aneuploidy. The legendary trailblazing aneuploidy scientist and yeast geneticist Dr Angelika Amon, whom the world tragically lost to cancer in October 2020, once defined aneuploidy as:

A change in chromosome number that is not the exact multiple of the haploid karyotype is known as aneuploidy. This condition interferes with growth and development of an organism and is a common characteristic of solid tumors.

Because of her expertise and reputation, one of the first people contacted by Project 8p’s founder was Dr Amon, whose lab was located at the Massachusetts Institute of Technology. Years ago, using the budding yeast Saccharomyces cerevisiae as a model system, Dr Amon’s lab published numerous studies showing that proteotoxic stress, increased glucose uptake and other changes in basic cell physiology occur when a chromosome is out of balance. (Her lab was the first to identify suppressor mutations that allow cells to tolerate aneuploidy!)

However, a general theory of aneuploidy that explains the vulnerabilities of specific cells to specific chromosome imbalances at specific times throughout the life of an organism has so far eluded scientists. There are so many gears and mechanisms in constant motion. And yet there’s hope.

Despite having unique chromosomal breakpoints and non—overlapping sets of affected genes, 8p heroes share a constellation of symptoms. Is Nature telling us that if you break the short arm of the 8th chromosome in any manner, the outputs converge into 8p disease?

Even if there are indeed individual culpable driver genes on 8p — or lurking on other chromosomes — that are responsible for one of more aspects of disease, does the collective action of these drivers explain everything, or is chromosome imbalance itself a primary disease driver? If the answer tends toward the latter, then the implication is that complex chromosome diseases may be united by one or more common mechanisms of disease onset and progression.

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Before the brain organoid bet, the first 8p disease models were actually a pioneer 8p family trio of skin fibroblasts studied in the Amon lab at MIT in 2019 and 2020. Specifically, bulk RNA-seq was performed on said 8p trio in order to assess how the expression of each gene in the genome is altered in: i) 8p fibroblasts; ii) unaffected age-matched controls; iii) fibroblasts derived from people living with trisomy 21 aka Down Syndrome.

The following analysis was performed by Perlara Cure Guide Dr Arun Ramani. In keeping with Project 8p’s open science credo, these data will soon be available for download on the 8p online data portal.

Hierarchical clustering shows that three different 8p fibroblast lines and three different T21 fibroblast lines form sister clades. Three out of four wildtype fibroblast lines co-cluster and form an out-group relative to 8p and T21. One of the four wildtype fibroblast lines wedged itself into the T21 cluster.

Another way to flatten high-dimensional dataset into a meaningful 2-D figure is principal component analysis (PCA). Mirroring the results of hierarchical clustering, 8p and wildtype fibroblasts form distinct groups separated in PCA space. The three 8p fibroblast lines are huddled closely together. The three wildtype fibroblast lines are a bit more spaced out. The T21 fibroblast lines are stretched out along both PCA1 and PCA2 axes, but overall more similar to the 8p lines than to the wildtype lines.

So how similar are 8p fibroblasts and T21 fibroblasts to each other? We generated lists of differentially expressed genes and visualized the results as Venn diagrams. Let’s start with genes whose expression goes down. There are 161 genes that are down-regulated in both 8p and T21 relative to wildtype, which translates to 60% of all 8p-down-regulated genes and 40% of all T21-down-regulated genes. That quite a bit of overlap between 8p and T21!

Now let’s examine the genes whose expression goes up. The association gets even stronger. There are 404 genes that are up-regulated in both 8p and T21 relative to wildtype, which translates to 69% of all 8p-down-regulated genes and 52% of all T21-down-regulated genes.

Shifting gears to focus on the comparison between 8p and wildtype, a volcano plot was generated comparing gene expression of wildtype fibroblasts versus 8p fibroblasts. Some of the differentially expressed genes are labeled. There are hundreds of differentially expressed genes above the p-value and 2-fold change thresholds, so we turned to pathway analysis to glean insights — too many to process individually.

Gene set enrichment shows that known aneuploidy-sensitive cell physiological processes like protein translation, organelle biogenesis and mRNA splicing are affected in 8p. Following up on the organelle biogenesis and maintenance gene ontology, mitochondrial genes are strongly down-regulated in 8p fibroblasts relative to wildtype fibroblasts.

We look closer at a single cellular component called the spliceosome, which as its name suggests in the site of action for processing mRNA into its final form. The building blocks of spliceosomes are strongly down-regulated in 8p fibroblasts relative to wildtype fibroblasts.

While we’re still some ways from a general theory of aneuploidy, it’s clear from studying gene expression changes in skin cells from 8p heroes that chromosome imbalance itself can be considered a primary disease driver. Keep reading to find out what we do with this insight.

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It’s not practical to use RNA-seq as a readout for a high-throughput drug repurposing screen. Based on precedents with skin fibroblasts in perhaps the most well-studied complex chromosome disease — trisomy 21 (T21) aka Down Syndrome — Project 8p sponsored a cell biological workup on 8p fibroblasts in Dr Nicoleta Moisoi’s lab at De Montfort University in the UK.

Dr. Marta Dominguez-Prieto in the Moisoi lab put 8p hero skin fibroblasts through a series of stress tests alongside age-matched control fibroblasts from healthy donors. Fibroblast lines from four 8p heroes were compared to fibroblast lines from four age-matched controls, as well as the two parental lines that form a trio with one of the 8p hero lines.

Both control and 8p fibroblasts were subjected to a battery of image-based cellular assays, including the Cell Painting panel of vital dyes that was originally developed in Dr Anne Carpenter’s lab at the Broad Institute. Mitochondrial function in particular was examined given that aneuploidy is known to affect cellular metabolism. Growth in media supplemented with glucose was also compared to growth in media supplemented with galactose, which forces cells to use their mitochondria for energy production.

Mitochondrial resting membrane potential was assessed using the fluorescent probes TMRM and TMRE. Results for TMRE are shown below (and the same result was observed using TMRM). In both glucose and galactose conditions, 8p fibroblasts have lower mitochondrial resting membrane potential than control fibroblasts. These results are reassuringly consistent with Arun’s analysis of the 8p fibroblast RNA-seq dataset.

Next, each of the Cell Painting dyes was evaluated in either glucose or galactose media. 8p cells exhibited a phenotype, i.e., looked different than control fibroblasts, more robustly and reproducibly in galactose media than in glucose media. Sometimes the 8p lines showed a reduction in signal intensity, other times the 8p lines showed an increase in signal intensity. In the series of figures below, a stock image of a generic cell stained with the corresponding dye or probe is displayed for reference.

For example, wheat germ agglutinin (WGA), which stains glycoproteins and glycolipids in the plasma membrane, stains 8p fibroblasts more intensely than control fibroblasts only in galactose media in two independent experiments.

Using Lysotracker to stain lysosomes, we observed the same pattern of effect only in galactose media. Increased Lysotracker staining is indicative of autophagic flux and increased lysosomal degradation.

Conversely, concanavalin A, which primarily stains glycoproteins and glycolipids in the endoplasmic reticulum, has the opposite effect. It increases signal intensity in 8p fibroblasts grown in glucose media but not in galactose media.

All in all, these exploratory assay optimization experiments give us confidence that Cell Painting features will distinguish 8p fibroblasts vs control fibroblasts. However, no single readout above resulted in complete statistical separation of 8p fibroblasts vs control fibroblasts, justifying the use of the Cell Painting panel instead of relying on rescue of a single phenotype.

Finally, we assessed whether 8p fibroblasts exhibited higher rates of senescence because there’s a vast literature on the relationship between aneuploidy and senescence. We observed a modest increase in the fraction of senescent 8p cells grown in either glucose media or galactose media.

Stay tuned for a refresh of the 8p CureMap as we approach the midway point of the inaugural two-year research cycle.

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💬 Research Question of the Month

How do the unique breakpoints and genetic variation of each 8p hero shape their disorder? New sequencing and DNA assembly technologies are enabling unprecedented resolution of genome sequences. With this information, we can start to better understand the genotype-phenotype relationship in chromosome 8p disorders.

Let us know what you think in the comments section below!

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🔬Research Highlight 

Chromosome 8p inversions, duplications, and deletions arise due to the presence of a gene cluster called the beta-defensin locus (Floridia et al. AJHG 1996). This locus is flanked by repetitive sequences on both ends, which have the potential to misalign and undergo recombination during meiosis I, causing the chromosome to break. Where the chromosome breaks can be different each time, resulting in breakpoints that are unique to each 8p hero.      

Most of the genetic information that we have on 8p heroes comes from microarray assays, which lack the resolution to determine chromosomal breakpoints at the single-nucleotide level. Even most whole-genome sequencing methods cannot resolve complex or repetitive stretches of DNA. In Dr. Evan Eichler’s lab at the University of Washington, Dr. Glennis Logsdon and her colleagues have been working on new computational methods to assemble complete chromosome sequences from telomere-to-telomere (end-to-end) (Rautiainen et al. Nat Biotechnol 2023) 

Now, Dr. Logsdon has applied these approaches to the sequencing and assembly of a chromosome with an 8p inverted duplication and deletion, allowing the accurate identification of the breakpoints for the first time. An additional, somewhat surprising, finding was that the chromosome has a normal length telomere. When the chromosome is broken during meiosis I, the telomere is lost, but the cells have apparently added a new one. Telomere defects can lead to a number of diseases, so this is good news for 8p heroes! 

Next, Dr. Logsdon will look at gene expression and DNA methylation, a mechanism that cells use to alter gene expression, across chromosome 8 to better understand how the cells adjust to changes in gene dosage brought on by the inverted duplication and deletion. Understanding these changes will also help inform targets for therapeutic development. Soon, this work will be expanded to additional 8p variants, allowing for more comparative analysis.

Dr. Logsdon is also using these findings to inform the engineering of artificial chromosomes that could be used to study the effects of CNVs in cells or animal models.  It’s possible that, one day, artificial chromosomes could even be used to replace the deleted genes in 8p heroes. 

Featured image: Confirmation of 8p breakpoints by fluorescence in-situ hybridization (FISH). Top: A diagram of the 8p hero’s maternal chromosome 8 with the inverted duplication and deletion. The left and right facing arrows indicate the duplicated region, and the beta-defensin locus is highlighted in blue. The small blue, green, and red bars indicate the binding sites of the fluorescent probes. Bottom left: The complete set of metaphase chromosomes isolated from the 8p hero. Bottom right: The paternal (left) and maternal (right) copies of chromosome 8 with FISH probes bound. On the maternal chromosomes, the blue probe and the green probe that correspond to the duplication are seen to bind in two places, whereas on the paternal chromosomes, they only bind in one location. Image from Dr. Glennis Logsdon.

You can watch Dr. Logsdon’s recent Research Roundtable on the Project 8p YouTube Channel.

📄 Articles and Publications

1. Gene copy number variation and pediatric mental health/neurodevelopment in a general population. Zarrei, M. et al. August 2023. Hum Mol Genet. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10360394/. Open Access. 

   Of the 7,100 unrelated children sampled in this study, 807 individuals had a rare CNV on chromosome 8 and 222 had a loss-of-function variant (SNV or Indel) on chromosome 8. The study identified CSMD1, MCPH1, and COPS5 as novel loci on chromosome 8 that are associated with ADHD and/or inattention traits.

2. Aneuploidy effects on human gene expression across three cell types. Liu, S. et al. May 2023. PNAS. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10214149/. Open Access.

   In this study, Liu et al. examined the effects of chromosome X, Y, and 21 dosage on gene expression in lymphoblastoid cell lines, fibroblasts, and induced pluripotent stem cell-derived excitatory neurons. They found that changes in gene expression on the affected chromosomes are generalizable across cell types, but that changes in gene expression beyond the affected chromosomes are not. 

3. Mechanisms of copy number variants in neuropsychiatric disorders: From genes to therapeutics. Forrest, MP and Penzes, P. July 2023. Curr Opin Neurobiol. https://pubmed.ncbi.nlm.nih.gov/37515924/. 

   This review describes models of gene interactions within CNVs, approaches to rodent and stem cell disease models, recent successes in genetic and pharmacological approaches to rescuing CNV-mediated pathophysiology, and proposes using a ‘rescue approach’ for more precise mechanistic understanding and to inform therapeutic development. 

4. Assortative mating and parental genetic relatedness drive the pathogenicity of variably expressive variants. Smolen, C. et al. May 2023. medRxiv 2023.05.18.23290169 https://www.medrxiv.org/content/10.1101/2023.05.18.23290169v1. Open Access. This article is a pre-print and has not been peer reviewed. 

   Smolen et al. studied 38,000 spouse pairs from four neurodevelopmental disease cohorts and the UK Biobank to identify associations with neurodevelopmental disease risk in children. Based on their analysis, they propose that spousal assortment on neuropsychiatric features drives disease risk by increasing genome-wide homozygosity in children. 

Let us know if you’ve read or published a paper or article recently that you think the 8p community should know about! 

💜 Family Corner

No one understands 8p heroes better than themselves and the ones who love them most. The My Hero Initiative invites families to share their 8p hero’s journey and add their piece to the Chromosome 8p Puzzle.

This puzzle of 8p reflects many questions such as:

• What does this diagnosis mean? 

• What symptoms do I look out for? 

• What can I do to improve these symptoms? 

• What treatments have worked and what did not work?

• What should I expect in the next 1 year, 3 years, 10 years, and future? 

• Who is experiencing the same challenges I am? 

• Is there an 8p expert to help me? 

• Is there a path to a cure?

By participating consistently, we can work as a community to answer these questions for you.

📆 Upcoming Events

No Research Roundtable for the month of August. We’ll see you again in September!

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💬 Research Question of the Month

We know that complex rearrangements of the short arm of chromosome 8 cause a multi-system disease, but is the damaged 8p chromosome itself toxic to cells the way a third copy (trisomy) of chromosome 21 or 18 is toxic to cells in Down and Edwards Syndrome, respectively?

Prof Jason Sheltzer's lab conclusively showed that patient-derived 8p cells (specifically, iPS cells) grown over many weeks in the lab do not spontaneously lose the damaged 8p chromosome. This result tells us that the damaged 8p chromosome is not acutely toxic. It also strongly suggests that not all cells are equally vulnerable to 8p rearrangements, and may explain why the brain is particularly sensitive to any changes to 8p.

Let us know what you think in the Comments section below!

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🔬Research Highlight 

This month’s Research Roundtable featured 8p researcher Dr. Jason Sheltzer from the Yale University School of Medicine. Dr. Sheltzer and his group are working on a project aimed at restoring normal chromosome copy number (disomy) in 8p iPS cells. The primary goal of this work is to create pairs of cell lines where the only difference between them is the 8p rearrangement (so-called “isogenic lines”). This will allow researchers to separate the impacts of the 8p CNV from those of the background genetic variation when it comes to studying the cellular biology of 8p or performing drug screens. In addition, this research will also help pave the way for chromosome therapies that can eventually be applied in vivo.  

The research plan involves taking several parallel approaches to restoring disomy. The first approach they tested was to culture the cells over a long time period to see if the cells would spontaneously lose the affected copy of chromosome 8, as has been observed in cells with trisomy 21 or 18. However, after culturing for 80 days, the cells still contained the affected chromosome. Although it is not clear why this was the case, it points to potentially interesting differences between 8p and the trisomic syndromes. 

Another ongoing approach is to treat the cells with an inhibitor of Mps1, a protein that is essential for chromosome alignment during mitosis, to promote loss of the affected chromosome 8. Other, more technically involved, approaches will include CRISPR-based editing techniques like ReDACT (recently developed by the Sheltzer lab) and KaryoCreate. 

Although chromosome engineering still has a long way to go before reaching the clinic, experiments like these will provide important insights into 8p biology and begin to shed light on possible avenues for chromosome therapy.

Featured image above: 

DNA copy number (vertical axis) across the genome (horizontal axis, binned by chromosome) derived from whole-genome DNA sequencing of an 8p hero (proband) with an 8p inversion/duplication deletion and their sex-matched parent. Copy number of 2 indicates a duplication and a copy number of 0.5 indicates a deletion, as seen in the yellow box for the 8p hero. Figure modified from Dr. Jason Sheltzer. 

You can watch the meeting on Project 8p’s YouTube channel: 

🔧Open Access Research Tools

This month, we are highlighting a few open access research tools that could be useful to the 8p research community. 

1. CRISPRbrain - Data Commons for functional genomics screens in differentiated human cell types, including astrocytes, glutamatergic neurons, microglia, T-cells, pluripotent stem cells and hematopoietic stem cells. Explore, visualize, and compare data from (at present) 34 different screens. 

2. CZ CELLxGENE - Visually explore and download over 800 standardized single-cell datasets representing 678 cell types. 

3. CNV-ClinViewer - Tool for the clinical evaluation and visual exploration of user-supplied CNV coordinates. Developed by 8p Researcher Dr. Dennis Lal and his group in collaboration with the Broad Institute. 

4. NDD-CNV Portal - Similar to the CNV-ClinViewer but focuses on a curated set of data pertaining to 8p, Ring14, and Dup15q. You can learn more from Dr. Lal’s Research Roundtable presentation last year. 

5. National Brain Gene Registry - An NIH-funded collaboration led by researchers at Washington University in St. Louis, Boston Children’s Hospital and Harvard Medical School, and the University of North Carolina at Chapel Hill, aimed at identification of genes involved in neurodevelopment. 

Do you know of an open access resource or tool that could be useful to the 8p community? Please share it in the comments or send us an email!

📄 Articles and Publications

1. Triplication of the interferon receptor locus contributes to hallmarks of Down syndrome in a mouse model. Waugh, K.A. et al. Nature Genetics. June 5th, 2023. https://www.nature.com/articles/s41588-023-01399-7 Open Access. Read the Research Briefing: https://www.nature.com/articles/s41588-023-01400-3

   Previously, Dr. Espinosa’s group showed that interferon signaling is constitutively activated in trisomy 21 cells, likely due to triplication of the Interferon Receptor (INFR) gene cluster located on chromosome 21 (Sullivan et al. 2016). Here, they used CRISPR to restore the copy number of the INFR locus in a mouse model of Down Syndrome and showed that by doing so, many of the clinical features of Down Syndrome were ameliorated. Clinical trials studying the effects of drugs that inhibit interferon signaling in individuals with Down Syndrome are already in progress. While this research does not have direct implications for 8p, it demonstrates how CNV research can go from hypothesis all the way to the clinic. 

2. Oncogene-like addiction to aneuploidy in human cancers. Girish, V. et al. Science. July 6th, 2023. https://www.science.org/doi/10.1126/science.adg4521

   8p researcher Dr. Jason Sheltzer and his group describe a new molecular approach for studying aneuploidy called ReDACT (Restoring Disomy in Aneuploid cells using CRISPR Targeting). Here, they use this tool to show that cancer cells are dependent on certain aneuploidies for malignant growth. You can hear him describe this method in his June Research Roundtable presentation. 

Let us know if you’ve read or published a paper or article recently that you think the 8p community should know about!

💜 Family Corner

Please join Project 8p for our second annual 8000 Steps for 8p event, an awareness and fundraising campaign; taking place the entire month of August 2023. 

All funds raised support Project 8's mission to empower a unified community for chromosome 8p heroes for a meaningful life today while accelerating treatments for tomorrow.

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📆 Upcoming Events

8p Research Roundtable, Dr. Glennis Logsdon, July 24th, 1:30 PM EST. Email whitney@perlara.com if you would like to attend.

🌐 Find us on the web

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💬 Research Question of the Month

How are chromosomal syndromes like trisomy 21 similar to, or different from, chromosome 8p disorder? What can we learn about one from the other? What tools can be applied to the study of both? 

Let us know what you think in the Comments section below!

🔬Research Highlight 

Large chromosomal copy number variants, such as trisomy 21 and 8p duplication/deletion, affect the expression of a large number of genes. A fundamental question is how do these changes in gene expression give rise to the cellular and developmental phenotypes that are characteristic of these syndromes?   

At this month’s Research Roundtable, Dr. Stefan Pinter (University of Connecticut) shared how his lab has used XIST as a tool to answer some of these questions in trisomy 21. XIST is a long non-coding RNA that silences the expression of the second X chromosome present in female cells. By transplanting the XIST gene onto chromosome 21 in cells with trisomy 21, the extra copy of Chr21 is similarly silenced (Jiang et al. 2014). Dr. Pinter’s lab further developed the capabilities of this tool by creating an inducible version of XIST that can be turned on at different points of cellular development, including in terminal neural lineages, enabling them to probe questions such as which cell types, phenotypes, and developmental windows are most amenable to therapy? (Bansal et al. 2022)

8p disorder differs from trisomy 21 in that the whole of chromosome 8 is not duplicated, rather large portions of the p arm are deleted and/or duplicated and inverted. The complexity of these variants means that a tool like XIST, which is only able to silence large swaths of chromosomes, has more limited utility for the study of 8p. To address this, Dr. Pinter is working on combining CRISPR constructs that are capable of either repressing (CRISPRi) or activating (CRISPRa) gene expression with an inducible construct that drives iPSC differentiation into neurons. These molecular tools can then be used to interrogate gene function in 8p in two ways: 

1. In a top-down approach, these constructs can be integrated into 8p cells and used to increase the expression of monogenic genes, or to decrease the expression of trisomic genes, until the effects of 8p on the cells are ameliorated. 

2. In a bottom-up approach, these constructs can be integrated into control cells and then genes on 8p can be turned on or off until 8p cellular phenotypes are recapitulated. 

Using these approaches, we can start to identify the genes on 8p that are responsible for various cellular phenotypes and to generate and test hypotheses about therapeutic interventions. 

Featured image above: Euploid cells (left) and trisomy 21 cells with a dox-inducible XIST construct (middle and right) at day 28 of neural differentiation. Staining: Blue - Hoechst; Green - astroglial s100b; Red - neuronal MAP2; White - H2AK119ub. In T21 cells without doxycycline treatment (middle) the extra copy of chromosome 21 is expressed and the cells preferentially differentiate into astroglial cells (green), whereas the euploid cells show more differentiation into neurons (left, red). When the T21 cells are treated with dox (right), XIST is turned on and silences the extra copy of chromosome 21, correcting the balance of differentiation towards neurons. Image courtesy of Dr. Pinter, Bansal et al. bioRxiv 2022.05.11.491519.

You can watch the meeting on Project 8p’s YouTube channel: 

💰 Open Grant Opportunities

Chan Zuberberg Initiative, Collaborative Pairs Pilot Project -  LOI due June 22nd

📄 Articles and Publications

Let us know if you’ve read or published a paper or article recently that you think the 8p community should know about! 

1. From neurodevelopment to neurodegeneration: utilizing human stem cell models to gain insight into Down syndrome. Watson, LA and Meharena, HM.  Front. Genet., 30 May 2023. https://www.frontiersin.org/articles/10.3389/fgene.2023.1198129/full (Open Access)

   8p Researchers Dr. Hiruy Meharena and Dr. Ashley Watson review the neurological symptoms of Down Syndrome and the cellular and animal models that have been developed to study the underlying molecular mechanisms. Some of these approaches are already being implemented in 8p research, but many still require development. 

2. Organoid intelligence (OI): the new frontier in biocomputing and intelligence-in-a-dish. Smirnova, L. et al. Front. Sci., 28 Feb 2023 https://www.frontiersin.org/journals/science/articles/10.3389/fsci.2023.1017235 (Open Access) 

   This article is part of an Article Hub on Organoid Intelligence, which lays forth a roadmap for the development of organoid intelligence as a scientific discipline. 8p Researcher Dr. Alysson Muotri is a contributing author. 

3. CRISPRi-based screens in iAssembloids to elucidate neuron-glia interactions. Li, E. et al. bioRxiv 2023.04.26.538498. https://www.biorxiv.org/content/10.1101/2023.04.26.538498v1.abstract (Open Access, This is a pre-print and has not been peer reviewed.) 

   In this publication, the Kampmann lab describes a new 3D co-culture system, termed iAssembloids (induced multi-lineage assembloids), that enables the rapid generation of homogenous neuron-glia spheroids. Like cortical organoids, iAssemboids can be used to study the interactions between different cell types, however they can be generated much faster. Using these iAssembloids, Li et al. performed a neuronal survival screen using CRISPRi and identified that GSK3β inhibits neuroprotective NRF2, which was not identified in previous screens using 2D monocultures. 

💜 Family Corner

Data Freeze - Monday, June 19th, 2023 

Participate in the Chromosome 8p Registry 

Our team will be publishing Care Guidelines in 2023. Include your 8p hero by enrolling, completing surveys, and uploading your genetic report. 

Community Benefits:

Care Guidelines will summarize the present clinical and patient-reported knowledge of Chromosome 8p Disorders for all areas of symptoms including the brain, vision, heart, muscular, gut, and more. It will include recommendations for clinical tests needed and how doctors and caregivers can communicate as a team. 

We need to know the nuances and unique experiences for every 8p hero so we don’t miss anything. As we learn more about Chromosome 8p, we anticipate these recommendations will evolve, and future editions will be published.

📆 Upcoming Events

8p Research Roundtable, Dr. Jason Sheltzer, June 26th, 1:30 PM EST. Email whitney@perlara.com if you would like to attend.

🌐 Find us on the web

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In collaboration with

As discussed in the 8p CureMap v1 published last year, the driver gene concept defines the way we think about chromosome diseases.

For example, in trisomy 21 aka Down Syndrome, there is an extra third copy of chromosome 21. Curiously, only some genes on chromosome 21 are dosage-sensitive, e.g., Dyrk1a, meaning the cell stresses out because of the over-expression (or under-expression in the case of chromosomal deletions) of some genes, not others. Put another way, the body doesn’t seem to care if all chromosome 21 genes are in excess, just a select handful of driver genes.

An alternative conceptual framework for dissecting the genetic basis of chromosome diseases is that while there may be driver genes responsible for specific life-altering symptoms or congenital malformations, rather it’s the cumulative burdens of aneuploidy experienced by every cell sum up to 8p disease.

An exacerbating complication is that genes physically located on other chromosomes — ostensibly beyond the blast radius of the damaged 8p chromosome — get hit in the cross-fire either because of transcriptional network entanglement or via long-distance physical interactions between genetic elements on different chromosomes.

Of course, the messy reality for 8p disease probably involves contributions from all three mental models: one or more driver genes on the damaged chromosome; one or more driver genes on unaffected chromosomes; aging-related cell stresses caused by aneuploidy.

As part of the disease modeling segment of the Two-Year Research Plan, Project 8p has funded two complementary approaches using iPSCs from each member — mom, dad, child — of a pioneer 8p family trio. Hereafter the child is referred to as the proband, and parents are referred to as controls.

One set of vials was shipped to a biotech startup in the Bay Area, and another set was sent to a translationally focused academic lab in SoCal. The first approach was performed in Palo Alto by Rarebase PBC, whose platform Function leverages 2-D glutamatergic excitatory cortical neurons (“iNeurons”) cultured for 1-3 weeks in the lab. Here’s quality control data indicating successful differentiation of iPSCs into iNeurons.

The second approach was performed in the lab of Professor Alysson Muotri at UCSD. The Muotri lab has many years of expertise in long-term culturing of 3-D brain organoids derived from patient iPSC lines. As a first of its-kind-experiment, the Muotri lab grew up brain organoids from all three members of a family trio for up to 9 months!

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Let’s examine the Rarebase data first. As a sanity check, we first confirmed that the 8p genes in the deleted and duplicated regions behave as expected.

In other words, the mRNA expression of 8p genes located in the deleted region should be ~50% of normal, e.g., CSMD1 (orange box), while the mRNA expression of 8p genes located in the duplicated region, e.g., FGFR1 (blue box), should be ~200% of normal. So far, so good.

Next, a genome-wide analysis below shows the expression of genes across all 23 pairs of chromosomes. Note the absence of any signal from the Y chromosome since the child/proband is female.

Chromosome 8 is highlighted (second row, center), as is chromosome 7 to the left of chromosome 8, and chromosome 20 (fourth row). We’ll explain in a moment why chromosome 7 is highlighted. Chromosome 20 is more concerning potentially.

There’s a duplication of the long arm of chromosome 20 that recurs in the generation of iPSC lines, as our 8p researcher Professor Stefan Pinter pointed out in last month’s 8p Research Newsletter. This duplication confers a growth advantage during iPSC generation and/or passage in culture, and therefore may be unavoidable in this model system. As a remedy, that’s why we generate independent clones of the same iPSC line. One clone is not enough!

The following plots show bulk RNAseq data from 8p proband iNeurons cultured for one week (Day 7) or three weeks (Day 21) and normalized to the maternal line. Data from the paternal line will be processed soon and will hopefully replicate these results.

Reassuringly, the genes located in chromosome 8p behave as expected. For example, you can see CSMD1 is under-expressed at both Day 7 and Day 21. CSMD1 has been on our radar as an 8p driver gene since the Okur paper (Okur, V. et al 2021).

OPRM1, which encodes the mu opioid receptor, is statistically significantly over-expressed at both Day 7 and Day 21. Two genes are over-expressed only at Day 21: CARTPT and RELN, which is short for Reelin. CARTPT stands for “Cocaine- and amphetamine-regulated transcript.”

Zooming in on the differences between the two timepoints yields this plot. Clearly all three genes — OPRM1, CARTPT and RELN/Reelin — pass the sniff test in terms of genes that may be playing a physiological role in 8p disease.

However, without unbiased confirmation that any of those three genes is dysregulated in a different 8p disease model — ideally a more complex model like brain organoids differentiated from the same pioneer 8p family trio — all we have is a preliminary result.

Sometimes in science, lightning strikes twice. That’s usually the sign of a discovery. In the same week in late January, we learned that Reelin/RELN was the star of the brain organoid dataset.

Here’s the summary slide carefully prepared by Prof Muotri who presented at the January 8p Research Roundtable, the kickoff for the 2023 season. Busier than the New York City subway map, only connoisseurs of UMAP plots will appreciate the finer details.

For our purposes, let’s zoom in on the plot summarizing the cell proportions over time.

At the 1-month and 3-month timepoints, the cell composition of the family trio of organoids are mostly the same. Early data suggested increased apoptosis in the proband, the brain organoid version of microcephaly. But brain organoids of one of the controls (JE2) were smaller than the other control (JE1), so best not to over-interpret on the basis of a single trio.

Going back to the cell composition breakdown at 9 months, there’s a striking population of glutamatergic RELN+ neurons that are not present in mom or dad brain organoids. The late-onset of Reelin expression in both the brain organoids and iNeurons is also noteworthy.

However, like any result from a 9-month brain organoid from a single clone of a single proband’s iPSC line, we need to replicate this result in another 8p family trio. Reelin over-expression must also be confirmed by at the protein level and also by immunohistochemistry. If the Reelin result is real, we’re just at the beginning.

Of course it could be very well be a false positive, or a mischievous experimental artifact. An alternative framing is that we know Reelin meets the definition of a dosage-sensitive gene to a tee. It’s been most studied in the context of its under-expression or loss-of-function.

But what happens when there’s overactive Reelin or too much Reelin at the wrong time, say too much in fetal development? If you get past the neurodevelopmental window and the slide toward neurodegeneration begins, could there actually be a protective advantage to increased Reelin activity?

Data published in 2011 from a Reelin-overexpressing mouse model suggests a protective effect. Data published just yesterday shows that a rare gain-of-function Reelin variant protected a person against early-onset Alzheimer’s.

This is why we’re Reelin groovy.

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💬 Research Question of the Month

How should the 8p community be preparing for drug repurposing and biomarker discovery? Those are two 8p-hero-powered initiatives that can deliver clinically actionable results quickly relative to the pace of academic research. While disease modeling results start to flow in from the projects that Project 8p funded last year, the 8p community can itself advance research and take matters in their own hands by self-organizing around drug repurposing and biomarker discovery.

Let us know what you think in the Comments section below!

🔬Research Highlight 

This month we tried something different at the Research Roundtable. Instead of having one or two researchers present their work, we posed several research questions to the group and then opened the floor for discussion. The experiment clearly worked, as there was a great exchange of ideas and knowledge, from both researchers and families.

The first question discussed was around the re-analysis of transcriptomic data. Transcriptomic data provides a snapshot of information about the genes being expressed at a given point in time in a cell or group of cells. To date, we have collected RNAseq data on 8p fibroblasts, glutamatergic neurons derived from iPSCs, and cortical (brain) organoids. In addition to using these data to understand the expression of genes on chromosome 8p, we can also interrogate the effects of 8p CNVs/SVs on gene expression more globally in order to identify cellular pathways or specific gene drivers that might contribute to disease pathology, or that could even be targeted pharmacologically. One of our first approaches will be to use the fibroblast RNAseq data to inform ongoing efforts to identify cellular phenotypes that could be used for drug repurposing screens. If any of these data could be useful to your 8p research project, please get in touch.

The second question the group discussed was how we can start to assess the risk of accelerated aging in 8p heroes. The limited clinical data that we have on adult 8p heroes suggests that some may experience features of accelerated aging, similar to individuals with Down’s Syndrome. Many of our experiments are done using iPSCs; however, the process of creating iPSCs resets the age of the cells such that epigenetic markers of aging are lost (Horvath, S. 2013). One alternative is to use a process of direct differentiation to go from a collected biospecimen to the cell type of interest, such as neurons, skipping the iPSC stage altogether, but this can be a long and technically challenging process. The group agreed that the first step should be to determine whether cells from 8p heroes have alterations in their epigenetic clocks by comparing DNA methylation in samples collected from 8p heroes of different ages. These experiments are relatively straightforward, and can be done from a number of different types of biospecimens. Based on the results of such experiments, we could then decide whether direct differentiation is worth pursuing. 

You can watch the whole discussion on Project 8p’s YouTube channel: 

💫 Coming Soon: The Project 8p Insights Portal 

Project 8p’s various sponsored research projects are beginning to produce a lot of exciting data. To make the most of this data, and in keeping with our Open Science policy, we aim to make the data as widely available to the community as possible. To achieve this, we are creating The Project 8p Insights Portal. The portal will include experimental data and patient survey data collected through our registries, as well information about available biospecimens. From this online portal researchers and clinicians will be able to perform cohort analyses, visualize data, and request samples and raw data. The portal will also serve as a way for patient families to more easily see and contribute their data.  

We plan to roll out the first iteration of the portal later this year.   

📄 Articles and Publications

Let us know if you’ve read or published a paper or article recently that you think the 8p community should know about! 

1. KaryoCreate: A CRISPR-based technology to study chromosome-specific aneuploidy by targeting human centromeres. Bosco, N et al. Cell. Apr 2023 https://doi.org/10.1016/j.cell.2023.03.029. 

   KaryoCreate is a molecular technique that can be used to create arm-level and chromosome-level gains and losses. The technique could be employed to study the effects of loss or duplication of 8p in isolation. Thanks to Dr. Matt Lalli for sharing this paper with us!

2. A Critical Review of the Impact of Candidate Copy Number Variants on Autism Spectrum Disorders. Abedini, SS et al. arXiv:2302.03211v2 [q-bio.GN]. Mar 2023. https://doi.org/10.48550/arXiv.2302.03211 Open Access. *This a pre-print and has not yet been peer reviewed.

   Abedini et al. review the protein-coding genes and long noncoding RNAs found within 47 autism spectrum disorder (ASD)-associated CNV regions. To identify ASD candidate genes, they looked at their expression in the brain and during iPSC to neuron differentiation, as well as whether mutant mice showed any nervous system phenotypes. On chromosome 8p they looked at deletions on 8p23.1 and identified TNKS, PINX1, MSRA, XKR6, FAM167A, NEIL2, RP1L1, GATA4, and lncRNA RP11-1E4.1 as possible contributors to ASD.

3. Targeting RNA with small molecules: lessons learned from Xist RNA. Nickbarg, EB. et al. RNA. Apr  2023 https://pubmed.ncbi.nlm.nih.gov/36725318/ Open Access.

   Most drugs on the market target disease causing proteins, however long noncoding RNAs are becoming increasingly appreciated as drivers of disease. RNAs are more difficult to target though due to the numerous confirmations they can adopt. This perspective discusses recent success with a structure agnostic way to screen for RNA-binding small molecules using affinity-selection mass spectrometry.

4. New 8p Case Reports:

   Expanding the Phenotype of 8p23.1 Deletion Syndrome: Eight New Cases Resembling the Clinical Spectrum of 22q11.2 Microdeletion. Montenegro, MM. et al. J Pediatr. Jan 2023. https://doi.org/10.1016/j.jpeds.2022.08.051

   Insight into the 8p23.1 duplication syndrome: Case report of a young woman with infertility. El Karaaoui, AK. et al. Heliyon. Apr 2023. https://doi.org/10.1016/j.heliyon.2023.e15515 Open Access.

   8p Microdeletion Syndrome Presenting as Congenital Scoliosis and Facial Dysmorphism with Novel Anomalies. Ansari, FF. et al. SAS J Med. Mar 2023. https://saspublishers.com/article/14392/ Open Access.

💜 Family Corner

My Hero Initiative is seeking adult 8p heroes!

Demographic diversity is a critical component of natural history studies; this month, we want to highlight the importance of age cohorts. We need representation from all age groups to understand how the disease progresses over time. Participation from our adult 8p heroes is low; our research team is recruiting 8p heroes aged 16+. To learn more about participating and enroll your hero, please email kaiti@project8p.org

All My Hero Initiative Participants:

We are excited to announce that our Partners at Rare-X are utilizing mobile phlebotomy for biorepository blood sample collection. At no cost to you, a phlebotomist can come to your home and collect a sample. Participation in the Rare-X 8p Data Collection Program is required. Email kaiti@project8p.org to get started.

📆 Upcoming Events

• 8p Research Roundtable, Dr. Stefan Pinter, May 22nd 1:30 PM EST. Email whitney@perlara.com or kaiti@project8p.org if you would like to attend. 

• Drug Repurposing Town Hall, May 26th 11:30 AM EST. Email kaiti@project8p.org if you would like to attend.

• 8p Scientific Meeting coming in October!

🌐 Find us on the web

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Founded in November 2018 as a 501(c)(3) nonprofit, Project 8p Foundation works to empower a unified community for chromosome 8p heroes for a meaningful life today while accelerating treatments for tomorrow.