FAM177A1 Cure Odyssey
After her two kids completed a diagnostic odyssey with the UDN, Jill Hawkins created the FAM177A1 Research Foundation. The inaugural (virtual) FAM177A1 Research Roundtable will take place next month.
In collaboration with
Register for the inaugural FAM177A1 Virtual Family & Research Roundtable June 10, 2023 from 8AM-11AM Pacific Time here!
This focused CureMap for FAM177A1 encapsulates the scientific literature and opportunity landscape for the purpose of finding treatment options for patients living with FAM177A1-related disorders. Given the current limited FAM177A1 knowledgebase, we recommend addressing basic science gaps while simultaneously prioritizing unbiased drug repurposing screens using FAM177A1 disease models and gene therapy in vitro proof-of-concept experiments given the payload-accommodating size of the gene over the next 6-12 months.
Helen W. Hernandez, M.S., Cure Guide of Perlara PBC.
Johannes Morstein, PhD., Cure Guide of Perlara PBC.
Jerome Korzelius, PhD., Cure Guide of Perlara PBC.
Ethan O. Perlstein, PhD., CEO of Perlara PBC & Maggie’s Pearl LLC
FAM177A1 Gene and Disease Overview
The FAM177A1 (family with sequence similarity 177 member A1) gene is located on chromosome 14q13.2 and contains 8 exons. The FAM177A1 gene is ubiquitously expressed across multiple tissues.
The function or functions of the protein encoded by the FAM177A1 gene is currently unknown. AlphaFold predicts a disordered protein with a recognizable helix that could confer Golgi localization (see Figure 1). The impact of loss-of-function of FAM177A1 on transcriptomics in zebrafish and humans, as well as some detailed investigation of specific interactions of FAM177A1 with other proteins in the cell, are already coming together to shed light on what FAM177A1 does in the cell.
While there are several proteins identified by the String Interaction Network as potentially physically interacting with FAM177A1 (VPS13B, RHOJ, SLC27A2, and 22 more), only one of these connections is being investigated in more depth by Dr. Berrak Ugur in the laboratory of Dr. Pietro De Camilli, and this work is unpublished as of this writing. We strongly recommend further exploration of the genetic interaction network and the protein-protein interaction network of FAM177A1.
Unpublished transcriptomics from Dr. Solnica-Krezel and Dr. Shin (Washington University, St Louis) have revealed hundreds of differentially expressed transcripts in knockout zebrafish embryos that suggest dysfunction in the immune system, apoptosis, and cholesterol synthesis. However, those results do not speak to the exact function of this protein, nor do they clarify if disruptions in the function of these biological systems are causative of particular disease phenotypes or if they are downstream effects.
The published work that most convincingly elucidates FAM177A1 function focuses on the immune system and is corroborated by preliminary “omics” insights. Specifically, it has been shown that FAM177A1 inhibits IL-1B by interacting directly with TRAF-6 (Liao et al., 2021). While the focus of that paper was specifically the impact on IL-1B and the interaction between TRAF-6 (TNF receptor associated factor 6) and ubc-13, it stands to reason that the direct binding between TRAF-6 and FAM177A1 may also disrupt other signaling pathways that TRAF6 mediates. Influence of FAM177A1 on interactions between TRAF-6 and CD40, TNFSF11/RANCE, IL-1, IRAK1/IRAK, SRC, and PKCzeta deserve further investigations. Another open question is if JNK activation is disrupted in FAM177A1-related disease.
In 2015, a frameshift mutation in the FAM177A1 gene was identified in connection with macrocephaly, intellectual disability, dolichocephaly, and mild obesity in a study by Alazami and coworkers, and it was suggested as a candidate disease gene (Alazami et al., 2015). Several additional variants in FAM177A1 were discovered by the Undiagnosed Disease Network (UDN) and are associated with macrocephaly, developmental delay, and intellectual disability (Legro et al., 2020). Several of the individuals with biallelic loss-of-function mutations in the gene also experience seizures, have abnormal gait, have evidence of white matter abnormalities including delayed myelination, and either T2 hyperintensities or T2 hypointensities. Given that this UDN case only identified this gene in 2018, the field has advanced quite rapidly due to the work of several dedicated experts who are engaged with the FAM177A1 Research Fund (Legro et al., 2020).
In spite of this being a very recently discovered gene and protein, the small number of published studies in the literature points to disruption of the immune system which aligns with unpublished transcriptomics insights. As more research unfolds, the possibility for translational science becomes increasingly promising. To set the best foundation for this we recommend simultaneously advancing basic science, animal model development for drug screening, proof-of-concept in vitro gene therapy studies, and clinical biomarker discovery.
Dr. Solnika-Krezel’s group (WUSTL) has established a zebrafish model. Zebrafish have two paralogs of FAM177A1, namely FAM177A1A and FAM177A1B; a double knockout (KO) fish model was created. Unpublished immunostaining experiments revealed localization to the Golgi complex and likely endoplasmic reticulum (ER). The KO fish grow into healthy adults that are morphologically indistinguishable from wild-type animals and only exhibit mild developmental phenotypes in early development.
There is an unpublished way to exacerbate the developmental phenotype which may provide the foundation for a two-part screen wherein an initial stressed state is induced, and then compound libraries could be screened to see if any compounds reverse the effect. The time to full recovery of normal morphology of a particular cellular compartment (unpublished) is a screenable phenotype, which can be leveraged either in a primary screen (challenging but doable from an assay development standpoint) or more confidently in low-throughput hit validation studies. RNA sequencing has been done in these models and a publication is forthcoming.
Dr. Berrak Ugur in Dr. Pietro De Camilli’s group (Yale) has developed a FAM177A1 knock-out cell model and her work shows that FAM177A1 localizes to Golgi/ER. While the FAM177A1 knockout cell line on its own is still being characterized, the knockout with the addition of specific stressing conditions are well-poised to provide insights into both the basic science of FAM177A1 and set the foundation for an assay to be created for high throughput screening to determine what may reverse this exacerbated phenotype. Furthermore, the specifics of the phenotype observed (unpublished) are reminiscent of a knockout of different gene in the same interaction network as FAM177A1, for which high-throughput screening has already been performed which has yielded several promising hits.
The fly model has several benefits including reagent availability (RNAi and over-expression constructs) and known verified, low-cost assays for neurodegeneration phenotypes. Fly stocks for over-expression or knockdown by RNAi for the fly FAM177A1 gene CG8300 are easily available from several Drosophila stock centers at low costs (<10 USD/strain for most strains). These strains can be used to manipulate the CG8300/FAM177A gene in any cell type or tissue (such as brain, muscle) we would wish to study. Climbing assays may reflect gait issues, neurodevelopmental issues, possibly malaise, and it may actually represent a physiologically relevant convolution of multiple phenotypes. If screening can result in hits that improve exercise and coordination this may be related to one or more mechanisms. The feasibility of tissue-specific RNAi knockdown or over-expression specifically in neurons or glia is an additional benefit of a fly model over other models such as zebrafish or mouse and will help to further pinpoint the tissue of action.
Although the sequence similarity is not particularly high (44% similarity, 24% identity), the fly version of FAM177A1 (CG8300) is marked as orthologous to human FAM177A1 by 10/16 of the tools listed on Marrvel/Flybase. The use of a fly model could be a cheaper alternative to zebrafish but it has not been generated to date. Phenotypic characterization and drug screening efforts would be discussed with a Perlara partner such as the lab of Dr. Clement Chow at the University of Utah.
Mouse models are underway at Jackson Laboratory and are being generated with a less common background strain (FVB/NJ) since it is one that has only a single full copy of the FAM177A1 gene as opposed to more commonly utilized strains such as C57BL/6J which have multiple copies. Characterization of this mouse should be quite insightful and can hopefully give clues as to the role of FAM177A1 in the organs in which it is most highly expressed.
While examination of the brain (with particular attention to areas that according to the Allen Brain Institute display high FAM177A1 expression) will be of paramount interest given the neurological manifestations of the disease, we want to ensure we make use of this opportunity to examine tissues throughout the body with high FAM177A1 expression. The lungs, arteries (e.g. tibial), prostate, and the tibial nerve may, for example, be insightful to examine in the knockout mouse.
Besides complex animal models, organoids offer some degree of three-dimensional structural complexity absent in two-dimensional cell culture models. However, they are not critical for initial screening and do not need to be prioritized over the other models outlined above at this point. If someone takes an interest in exploring FAM177A1 in organoids, sharing that FAM177A1 is highly expressed in the globus pallidus, a part of the basal ganglia, may spur research that sheds light on the motor aspect of FAM177A1 related disease. We acknowledge the Allen Brain Institute for providing that insight.
Thousands of small molecule drugs are clinically approved for the treatment of various diseases and pathways. Screening these libraries for drug repurposing to treat FAM177A1 would be the quickest way to identify potential drug candidates that could be used with minimal cost and time (Janes et al., 2018; Pushpakom et al., 2019).
Small Molecule Drug Discovery in Zebrafish
Small molecule drug screens in zebrafish have been pioneered by Prof. Randall Peterson and David Kokel at Harvard (Bruni et al., 2016; Kokel & Peterson, 2008, 2011). Peterson’s group is now located at the University of Utah; and Kokel is now an employee of the company Biosymetrics. Alternatively, Prof. Jason Sello (UCSF) is also conducting similar screens in zebrafish. In short, the method allows high-throughput drug screening in adult zebrafish with a neural phenotype, e.g., if seizures occur in adult fish.
The data available to Perlara indicates that most work around the zebrafish models from the Solnika-Krezel group has focused on zebrafish development. We recommend testing the adult zebrafish FAM177A1 KO model for a neural phenotype. If adults exhibit a similar phenotype to that observed in humans (namely, seizures) this could be tracked using standard Fish Box technology and would be an excellent system for a drug repurposing screen that will identify drugs that overcome the neural phenotype. The biggest practical consideration is cost. A primary screen in zebrafish of thousands of compounds will be expensive, on the order of $100,000. Given resource constraints, we recommend using the short stature phenotype in the zebrafish KO model to validate hits from a cell-based (or fly-based, if possible) primary drug repurposing screen, as described in the following section.
Small Molecule Drug Discovery in Cells
Out of respect for the ongoing and unpublished work the phenotype details will not be shared in this initial roadmap however we anticipate the ability to induce and quantify this to-be-published phenotype on a single-cell basis through automated high content imaging. While we acknowledge that there will need to be some adaptation of previous work to ensure reproducibility at scale and account for potential time effects, this system is very likely amenable to an unbiased imaging-based phenotypic screen (Chandrasekaran et al., 2021; Lin et al., 2020). In such a screen, cells would be stressed in a way that exacerbates a measurable phenotype and the test compound included in the library, and cells would be monitored to identify compounds that rescue the wildtype behavior. This work could also be combined with other readouts as research in the De Camilli lab progresses and identifies other screenable phenotypes, e.g., differences in lipid composition, indicators of apoptosis, or immune system dysfunction between KO cells and normal cells. Perlara and SF Biolabs are able to do optimization of the screening conditions and could work with the small molecule discovery center at UCSF (https://pharm.ucsf.edu/smdc) to conduct such an imaging based screen.
As an initial de-risking step, we recommend working with Muhammed Ansar and Berrak Ugur to test the hits from a potentially relevant screen in FAM177A1 KO cells to assess the degree of concordance between the two disease models. If there is significant overlap, it would serve to embolden FAM177A1 Research Fund to move forward with a focus on this phenotypic screen in FAM177A1 KO cells.
Gene therapy for FAM177A1 related disease is a promising strategy due to the relatively small size of the gene which makes it highly conducive to vector delivery. We should be able to leverage existing technology at several stages in the development of a gene therapy for FAM177A1 related disease. Antibodies for FAM177A1 do already exist so the presence of FAM177A1 could be readily measured before and after cellular delivery of a gene therapy construct.
There is a legitimate concern related to potential toxicity of AAV vectors and as the field advances this may or may not be addressed. In an aggregated study of 256 non-human primates from 33 separate studies with different methodologies (e.g. doses, route, capsids, promotors) showed that 83% of those individuals who received AAV through the CSF displayed dorsal root ganglion (DRG) pathology. The DRG pathology was observed at much lower rates, 32%, in those who received the AAV intravenously (Hordeaux et al., 2020).
Potential partners in moving the gene therapy forward should be able to provide insights into how we will know which route of delivery is appropriate and any advances in this area that may mitigate this risk. The focus for the first year will be construct design and correction of disease phenotypes in cellular disease models.
Proposed Next Steps
Several strategies may be used in parallel to accelerate understanding of FAM177A1 basic function.
A specific example of an interaction (not shown in Figure 2) may shed light on apoptosis in FAM177A1 related disease. TRAF6 interacts with ASK1 (apoptosis signal-regulating kinase-1) which activates the SAPK pathway, but not NF-kB (Hoeflich et al., 1999). Activation of the SAPK pathway signals cell death and when TRAF6 is inhibited, this would negatively regulate this pathway and not signal for cell death. Since FAM177A1 binds to TRAF6, (Liao et al., 2021) it is possible that functional FAM177A1 negatively regulates the SAPK pathway at the level of TRAF6. So when FAM177A1 is not present in functional form, apoptosis may occur to a greater extent. This can easily be cross-referenced with omics data to support or refute this hypothesis.
Cross-referencing existing animal model RNA-seq insights with human cell results can strengthen the high-level understanding of what biological features (immune, cholesterol synthesis, apoptosis) are implicated in, contributing to, or arising from this disease state.
Find experts in several proteins identified by the String Interaction Network and alert them to the potential connection to the newly discovered FAM177A1 to catalyze deeper investigation into these connections by potentially interested parties (similar to what is ongoing by Dr. Ugur at Yale).
Prioritization of these proteins may also arise from consultation with the Allen Brain Institute to quantify the degree of co-localization in different brain structures between FAM177A1 and other suspected proteins of interest (e.g. VPS13B, RHOJ, SLC27A2 implicated in cholesterol synthesis) in the adult human brain based on data they have already generated. This can be done now visually (see Figure 5) but a simple mathematical comparison of data should strengthen evidence suggesting which potentially-interacting proteins we should steer research towards. This is under the assumption, albeit reasonable, that co-localization indicates the possibility of a meaningful interaction deserving further investigation.
FAM177A1 assay development: Validation of screen hits by Dr. Muhammad Ansar at the University of Lausanne in FAM177A1 KO cells will establish ifa screenable phenotype is indeed viable. If this is positive, we recommend going forward with assay development for cell-based screening (see below). Alternatively, these hits may be validated in zebrafish that could also serve as a model for drug repurposing (see below).
Exploring adult behavior of the zebrafish model created by Dr. Solnika-Krezel for neurological phenotype will allow to determine if this model is well-suited for fish box screening
Laboratories (Dr. Sello or Dr. Peterson) and companies (Dr. Kokel) specialized on the screening of neuroactive compounds in a fish box could be contacted to coordinate the exploration of neural phenotypes in adult fish. They are all equipped to identify and characterize a potential phenotype in addition to a medium-throughput drug screen.
Development of a high content imaging screening protocol would allow for rapid development of a drug repurposing screen. If Dr. De Camilli is willing to share the cell lines, this work could be carried out by Perlara at SF Biolabs or in the laboratory of Prof Muhammed Ansar.
Discuss potential secondary readout with Dr. De Camilli lab to validate and solidify potential hits.
Currently working with Dr Clement Chow’s lab to determine if there are screenable phenotypes.
Following the path paved by Dr David Fajgenbaum (author of Chasing my Cure and founder of Every Cure), the foundation will work with proteomics leader SomaLogic on a clinical biomarker discovery project.
The goal is to identify proteins whose expression is changed (in either direction) in FAM177A1 cases versus controls, starting with the Hawkins family.
Identify potential partners to work on developing a FAM177A1 gene therapy (likely self-complementary) and see if any can operate on a timeline aligned with the mouse model creation. Possible partners include:
UMass - Phillip W.L. Tai
U Iowa - Possible Contacts
SAB - Dr. Shangzhen Zhou
Selected partner to perform construct design and demonstrate correction of disease phenotypes in cellular disease models.
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