Genomic Answers for Kids: Toward more equitable access to genomic testing for rare diseases in rural populations.

Next-generation sequencing has revolutionized the speed of rare disease (RD) diagnoses. While clinical exome and genome sequencing represent an effective tool for many RD diagnoses, there is room to further improve the diagnostic odyssey of many RD patients. One recognizable intervention lies in increasing equitable access to genomic testing. Rural communities represent a significant portion of underserved and underrepresented individuals facing additional barriers to diagnosis and treatment. Primary care providers (PCPs) at local clinics, though sometimes suspicious of a potential benefit of genetic testing for their patients, have significant constraints in pursuing it themselves and rely on referrals to specialists. Yet, these referrals are typically followed by long waitlists and significant delays in clinical assessment, insurance clearance, testing, and initiation of diagnosis-informed care management. Not only is this process time intensive, but it also often requires multiple visits to urban medical centers for which distance may be a significant barrier to rural families. Therefore, providing early, "direct-to-provider" (DTP) local access to unrestrictive genomic testing is likely to help speed up diagnostic times and access to care for RD patients in rural communities. In a pilot study with a PCP clinic in rural Kansas, we observed a minimum 5.5 months shortening of time to diagnosis through the DTP exome sequencing program as compared to rural patients receiving genetic testing through the "traditional" PCP-referral-to-specialist scheme. We share our experience to encourage future partnerships beyond our center. Our efforts represent just one step in fostering greater diversity and equity in genomic studies.

Components from the Human c-myb Transcriptional Regulation System Reactivate Epigenetically Repressed Transgenes.

A persistent challenge for mammalian cell engineering is the undesirable epigenetic silencing of transgenes. Foreign DNA can be incorporated into closed chromatin before and after it has been integrated into a host cell's genome. To identify elements that mitigate epigenetic silencing, we tested components from the c-myb and NF-kB transcriptional regulation systems in transiently transfected DNA and at chromosomally integrated transgenes in PC-3 and HEK 293 cells. DNA binding sites for MYB (c-myb) placed upstream of a minimal promoter enhanced expression from transiently transfected plasmid DNA. We targeted p65 and MYB fusion proteins to a chromosomal transgene, UAS-Tk-luciferase, that was silenced by ectopic Polycomb chromatin complexes. Transient expression of Gal4-MYB induced an activated state that resisted complete re-silencing. We used custom guide RNAs and dCas9-MYB to target MYB to different positions relative to the promoter and observed that transgene activation within ectopic Polycomb chromatin required proximity of dCas9-MYB to the transcriptional start site. Our report demonstrates the use of MYB in the context of the CRISPR-activation system, showing that DNA elements and fusion proteins derived from c-myb can mitigate epigenetic silencing to improve transgene expression in engineered cell lines.

Design, Construction, and Validation of Histone-Binding Effectors in Vitro and in Cells.

Chromatin is a system of nuclear proteins and nucleic acids that plays a pivotal role in gene expression and cell behavior and is therefore the subject of intense study for cell development and cancer research. Biochemistry, crystallography, and reverse genetics have elucidated the macromolecular interactions that drive chromatin regulation. One of the central mechanisms is the recognition of post-translational modifications (PTMs) on histone proteins by a family of nuclear proteins known as "readers". This knowledge has launched a wave of activity around the rational design of proteins that interact with histone PTMs. Useful molecular tools have emerged from this work, enabling researchers to probe and manipulate chromatin states in live cells. Chromatin-based proteins represent a vast design space that remains underexplored. Therefore, we have developed a rapid prototyping platform to identify engineered fusion proteins that bind histone PTMs in vitro and regulate genes near the same histone PTMs in living cells. We have used our system to build gene activators with strong avidity for the gene silencing-associated histone PTM H3K27me3. Here, we describe procedures and data for cell-free production of fluorescently tagged fusion proteins, enzyme-linked immunosorbent assay-based measurement of histone PTM binding, and a live cell assay to demonstrate that the fusion proteins modulate transcriptional activation at a site that carries the target histone PTM. This pipeline will be useful for synthetic biologists who are interested in designing novel histone PTM-binding actuators and probes.

Complex trait associations in rare diseases and impacts on Mendelian variant interpretation.

Emerging evidence implicates common genetic variation - aggregated into polygenic scores (PGS) - impacting the onset and phenotypic presentation of rare diseases. In this study, we quantified individual polygenic liability for 1,151 previously published PGS in a cohort of 2,374 probands enrolled in the Genomic Answers for Kids (GA4K) rare disease study, revealing widespread associations between rare disease phenotypes and PGSs for common complex diseases and traits, blood protein levels, and brain and other organ morphological measurements. We observed increased polygenic burden in probands with variants of unknown significance (VUS) compared to unaffected carrier parents. We further observed an enrichment in overlap between diagnostic and candidate rare disease genes and large-effect PGS genes. Overall, our study supports and expands on previous findings of complex trait associations in rare disease phenotypes and provides a framework for identifying novel candidate rare disease genes and in understanding variable penetrance of candidate Mendelian disease variants.

Glucocorticoid minimizes local anesthetic infusion requirement through adductor canal block and improves perioperative prosthetic joint range of motion in total knee arthroplasty.

INTRODUCTION: The use of glucocorticoid as local anesthetic adjuvant in single-injection adductor canal block (ACB) is well-documented but its effects in the presence of an indwelling catheter is unclear. The purpose of this study was to determine the impacts of one-time perineural glucocorticoid injection on continuous adductor canal block in patients undergoing total knee arthroplasty. METHODS: A single center retrospective study of 95 patients undergoing unilateral total knee arthroplasty (TKA) was performed. Patients were divided into three groups based on adjuvant received through ACB before continuous catheter placement: a control group with no adjuvant (N = 41), a treatment group with dexamethasone (DEX) as adjuvant (N = 33) and another treatment group with DEX/ Methylprednisolone acetate (MPA) as adjuvant (N = 21). The primary outcome was the amount of ropivacaine administered via patient controlled ACB catheter. Secondary outcomes included numeric pain score, perioperative opioid usage, immediately postoperative prosthetic knee joint active range of motion (AROM), opioid usage at 6 weeks and 3 months, length of stay and discharge disposition. RESULTS: Patients in both treatment groups demonstrated a statistically significant decrease in the requirement of self-administered ropivacaine than the control group on postoperative day (POD) 1 (p<0.001) and POD 2 (p<0.001). There was no significant difference in opioid consumption and pain scores between either treatment group vs. control. Compared to control (66%), more home disposition was observed in the DEX (88%, p = 0.028) and DEX/MPA group (95%, p = 0.011). CONCLUSION: This study suggested that single dose perineural glucocorticoid injection with DEX or DEX/MPA significantly decreased the dose of local anesthetic ropivacaine infusion required through continuous ACB for TKA while maintaining comparable level of pain score and opioid consumption, and significantly more patients were discharged home.

Site-directed targeting of transcriptional activation-associated proteins to repressed chromatin restores CRISPR activity.

Previously, we used an inducible, transgenic polycomb chromatin system to demonstrate that closed, transcriptionally silenced chromatin reduces Cas9 editing. Here, we investigated strategies to enhance Cas9 editing efficiency by artificially perturbing closed chromatin. We tested UNC1999, a small molecule inhibitor that blocks enhancer of zeste homolog 2, an enzyme that maintains closed polycomb chromatin. We also tested DNA-binding, transiently expressed activation-associated proteins (AAPs) that are known to support an open, transcriptionally active chromatin state. When cells that carried a polycomb-repressed transgene (luciferase) were treated with UNC1999 or the AAP fusion Gal4P65, we observed loss of histone 3 lysine 27 trimethylation (H3K27me3), a silencing-associated chromatin feature, at the transgene. Only Gal4P65 treatment showed full restoration of luciferase expression. Cas9 activity, determined by insertion deletion mutations, was restored in Gal4P65-expressing cells, while no CRISPR enhancement was observed with UNC1999 treatment. CRISPR activity was also restored by other Gal4-AAP fusions that did not activate luciferase expression. Our results demonstrate the use of DNA-binding, activator-associated fusion proteins as an effective method to enhance Cas9 editing within polycomb-repressed chromatin.

Characterization of diverse homoserine lactone synthases in Escherichia coli.

Quorum sensing networks have been identified in over one hundred bacterial species to date. A subset of these networks regulate group behaviors, such as bioluminescence, virulence, and biofilm formation, by sending and receiving small molecules called homoserine lactones (HSLs). Bioengineers have incorporated quorum sensing pathways into genetic circuits to connect logical operations. However, the development of higher-order genetic circuitry is inhibited by crosstalk, in which one quorum sensing network responds to HSLs produced by a different network. Here, we report the construction and characterization of a library of ten synthases including some that are expected to produce HSLs that are incompatible with the Lux pathway, and therefore show no crosstalk. We demonstrated their function in a common lab chassis, Escherichia coli BL21, and in two contexts, liquid and solid agar cultures, using decoupled Sender and Receiver pathways. We observed weak or strong stimulation of a Lux receiver by longer-chain or shorter-chain HSL-generating Senders, respectively. We also considered the under-investigated risk of unintentional release of incompletely deactivated HSLs in biological waste. We found that HSL-enriched media treated with bleach were still bioactive, while autoclaving deactivates LuxR induction. This work represents the most extensive comparison of quorum signaling synthases to date and greatly expands the bacterial signaling toolkit while recommending practices for disposal based on empirical, quantitative evidence.