Systematic evidence review and meta-analysis of outcomes associated with cancer genetic counseling.

PURPOSE: Genetic counseling (GC) is standard of care in genetic cancer risk assessment (GCRA). A rigorous assessment of the data reported from published studies is crucial to ensure the evidence-based implementation of GC. METHODS: We conducted a systematic review and meta-analysis of 17 patient-reported and health-services-related outcomes associated with pre- and post-test GC in GCRA in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines and Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology. RESULTS: Twenty-five of 5393 screened articles met inclusion criteria. No articles reporting post-test GC outcomes met inclusion criteria. For patient-reported outcomes, pre-test GC significantly decreased worry, increased knowledge, and decreased perceived risk but did not significantly affect patient anxiety, depression, decisional conflict, satisfaction, or intent to pursue genetic testing. For health-services outcomes, pre-test GC increased correct genetic test ordering, reduced inappropriate services, increased spousal support for genetic testing, and expedited care delivery but did not consistently improve cancer prevention behaviors nor lead to accurate risk assessment. The GRADE certainty in the evidence was very low or low. No included studies elucidated GC effect on mortality, cascade testing, cost-effectiveness, care coordination, shared decision making, or patient time burden. CONCLUSION: The true impact of GC on relevant outcomes is not known low quality or absent evidence. Although a meta-analysis found that pre-test GC had beneficial effects on knowledge, worry, and risk perception, the certainty of this evidence was low according to GRADE methodology. Further studies are needed to support the evidence-based application of GC in GCRA.

Building Efficiency and Scaling With a Remote Genetic Counseling Program.

Purpose A third-party telemedicine (TM) genetic counseling program was initiated at a large community oncology practice spanning 35 clinical sites with 110 clinicians and 97 advanced practice providers throughout Tennessee and Georgia. Patients and Methods Appropriate patients were referred through the electronic health record (EHR) based on current National Comprehensive Cancer Network guidelines. A combination of TM and genetic counseling assistants enhanced convenience, broadened access, and decreased no-show rates. Physician education for mutation-positive screening recommendations was provided through deep integration of dedicated genetic counseling notes in the EHR. Results From 2019 to 2022, the program expanded from 1 to 20 clinics with referrals growing from 195 to 885. An average of 82% of patients completed genetic counseling consultations over TM with more than 70% completing genetic testing. The average was 4 to 6 days from referral to consultation. The no-show rate was maintained at less than 7%. In 2023, this model supported all 35 clinics across the state. Conclusion Our program illustrates how remote genetic counseling programs are an effective choice for scaling genetics care across a large community oncology practice. Deep integration of TM genetic counseling within the EHR helps identify patients who are high risk and improves test adoption, patient keep rate, and turnaround time, helping to achieve better patient outcomes.

International students' perspectives on the genetic counseling application process.

Between 2018 and 2023, one percent of matched applicants to North American genetic counseling graduate programs (GCGPs) have been international applicants (IAs). The COVID-19 pandemic led to changes in the GCGP application processes in 2020, most notably the incorporation of virtual interviews and GRE waivers, which uniquely impacted IAs. Twelve international genetic counseling (GC) students who matriculated into a U.S.-based GCGP in 2021 or 2022 participated in this qualitative study (42% of the total enrolled) to understand their application experience. Cost, location of the program, and rapport during interviews were the most important factors identified by IAs to apply to and rank the GCGPs. Shadowing and volunteer experiences relevant to GC were cited as important for applicants to learn about a genetic counseling career, but many had challenges finding opportunities in their home countries. Unique logistical challenges in taking the GRE, transcript evaluation services, and standardized English proficiency tests were described. Although virtual interviews offered the same experience as domestic applicants, the time difference was a major challenge, requiring IAs to interview through the night, creating additional stressors. Nine of 12 were re-applicants and shared that engaging with GCGPs early in the process was beneficial for improving applications and, at times, requesting waivers for transcript evaluation requirements and considering unique volunteering experiences. Participants suggested GCGPs can address barriers by providing more specific information on their websites as it pertains to IAs, and contact information for the international student office. Improving awareness of the applicants' backgrounds, home country experiences, and time zone differences would provide IAs with a more equitable application experience. Addressing these barriers could help promote diversity, equity, and inclusion allowing for more IAs and the growth of the genetic counseling profession.

Host perspectives on international fieldwork placements for U.S.-based genetic counseling students.

Genetic counseling students from the United States are often interested in international summer fieldwork placements, but little is known about the hosts' perspectives when considering such requests. We sent out surveys to 132 international genetics providers (genetics clinics and genetics programs), to identify advantages, barriers and expectations for U.S.-based genetic counseling students seeking a fieldwork placement. Twenty-seven (20.4% response rate) participants from 14 different countries shared their experiences and views. Providers placed higher emphasis on teaching and benefits to students (95.2%) rather than intrinsic benefits to their programs (90.4%). Lack of American Board of Genetic Counseling's (ABGC) reciprocal recognition (30%) and cost of training (25%), were rated as the strongest barriers to hosting U.S.-based students. Surprisingly, 'Language Barrier' (20%), although mentioned in open-ended comments, was not ranked highly as a barrier. When asked about expectations of students, active participation in student-led counseling sessions under supervision was encouraged by a majority of participants (55.6%). Where most genetic counseling literature is U.S.-centric, this study reports on insights gathered from international genetics providers. Our study encourages U.S.-based programs to consider these findings when designing exchange programs and international fieldwork placements.

ß-Cyclodextrin-containing polymer treatment of cutaneous lupus and influenza improves outcomes.

Nucleic acid (NA)-containing damage- and pathogen-associated molecular patterns (DAMPs and PAMPs, respectively) are implicated in numerous pathological conditions from infectious diseases to autoimmune disorders. Nucleic acid-binding polymers, including polyamidoamine (PAMAM) dendrimers, have demonstrated anti-inflammatory properties when administered to neutralize DAMPs/PAMPs. The PAMAM G3 variant has been shown to have beneficial effects in a cutaneous lupus erythematosus (CLE) murine model and improve survival of mice challenged with influenza. Unfortunately, the narrow therapeutic window of cationic PAMAM dendrimers makes their clinical development challenging. An alternative nucleic acid-binding polymer that has been evaluated in humans is a linear ß-cyclodextrin-containing polymer (CDP). CDP's characteristics prompted us to evaluate its anti-inflammatory potential in CLE autoimmune and influenza infectious disease mouse models. We report that CDP effectively inhibits NA-containing DAMP-mediated activation of Toll-like receptors (TLRs) in cell culture, improves healing in lupus mice, and does not immunocompromise treated animals upon influenza infection but improves survival even when administered 3 days after infection. Finally, as anticipated, we observe limited toxicity in animals treated with CDP compared with PAMAM G3. Thus, CDP is a new anti-inflammatory agent that may be readily translated to the clinic to combat diseases associated with pathological NA-containing DAMPs/PAMPs.

Response Regulator CD1688 Is a Negative Modulator of Sporulation in Clostridioides difficile.

Two-component signal transduction systems (TCSs), consisting of a sensor histidine kinase (HK) and a response regulator (RR), sense environmental stimuli and then modulate cellular responses, typically through changes in gene expression. Our previous work identified the DNA binding motif of CD1586, an RR implicated in Clostridioides difficile strain R20291 sporulation. To determine the role of this RR in the sporulation pathway in C. difficile, we generated a deletion strain of cd1688 in the historical 630 strain, the homolog of cd1586. The C. difficile Δcd1688 strain exhibited a hypersporulation phenotype, suggesting that CD1688 negatively regulates sporulation. Complementation of the C. difficile Δcd1688 strain restored sporulation. In contrast, a nonphosphorylatable copy of cd1688 did not restore sporulation to wild-type (WT) levels, indicating that CD1688 must be phosphorylated to properly modulate sporulation. Expression of the master regulator spo0A, the sporulation-specific sigma factors sigF, sigE, sigG, and sigK, and a signaling protein encoded by spoIIR was increased in the C. difficile Δcd1688 strain compared to WT. In line with the increased spoIIR expression, we detected an increase in mature SigE at an earlier time point, which arises from SpoIIR-mediated processing of pro-SigE. Taken together, our data suggest that CD1688 is a novel negative modulator of sporulation in C. difficile and contributes to mediating progression through the spore developmental pathway. These results add to our growing understanding of the complex regulatory events involved in C. difficile sporulation, insight that could be exploited for novel therapeutic development. IMPORTANCE Clostridioides difficile causes severe gastrointestinal illness and is a leading cause of nosocomial infections in the United States. This pathogen produces metabolically dormant spores that are the major vehicle of transmission between hosts. The sporulation pathway involves an intricate regulatory network that controls a succession of morphological changes necessary to produce spores. The environmental signals inducing the sporulation pathway are not well understood in C. difficile. This work identified a response regulator, CD1688, that, when deleted, led to a hypersporulation phenotype, indicating that it typically acts to repress sporulation. Improving our understanding of the regulatory mechanisms modulating sporulation in C. difficile could provide novel strategies to eliminate or reduce spore production, thus decreasing transmission and disease relapse.

Multidimensional Immunophenotyping of Intraductal Papillary Mucinous Neoplasms Reveals Novel T Cell and Macrophage Signature.

BACKGROUND: Intraductal papillary mucinous neoplasms (IPMN) are the only radiographically identifiable precursor to pancreatic adenocarcinoma, yet little is known about how these lesions progress to cancer. Inflammation has been associated with dysplastic progression; however, the cause and composition of this inflammation remains poorly characterized. We sought to comprehensively profile immune cell infiltration using parallel spatial transcriptomic and flow cytometric techniques. METHODS: Twelve patients with resected IPMN exhibiting both high-grade dysplasia (HGD) and low-grade dysplasia (LGD) were selected for spatial transcriptomics (NanoString GeoMx). Immune (CD45+), epithelial (PanCK+), and stromal (SMA+) compartments were analyzed separately using the GeoMx NGS Pipeline. An additional 11 patients resected for IPMN of varying degrees of dysplasia underwent immunophenotyping using flow cytometry (DURAClone IM). RESULTS: Spatial transcriptomics revealed that T cells represent the dominant immune cell within IPMN stroma, which was confirmed by flow cytometry (56%). Spatial profiling found that the T-cell infiltrate was significantly higher in regions of LGD compared with HGD (62% vs. 50%, p = 0.038). Macrophages were the only other immune cell type with > 10% abundance, yet conversely, were generally more abundant in regions of HGD compared to LGD (19% vs. 11%, p = 0.058). Correspondingly, immune cells within regions of HGD demonstrated transcriptional upregulation of genes associated with macrophage activity including secretion (CXCL1) and phagocytosis (C1QA, C1S, C4B). CONCLUSIONS: IPMN immune infiltrate is primarily composed of T cells and macrophages. Regions of HGD appear to be relatively deplete of T cells and show a trend toward macrophage enrichment compared with regions of LGD.

Suppression of Fibrinolysis and Hypercoagulability, Severity of Hypoxemia, and Mortality in COVID-19 Patients: A Retrospective Cohort Study.

BACKGROUND: COVID-19 causes hypercoagulability, but the association between coagulopathy and hypoxemia in critically ill patients has not been thoroughly explored. This study hypothesized that severity of coagulopathy would be associated with acute respiratory distress syndrome severity, major thrombotic events, and mortality in patients requiring intensive care unit-level care. METHODS: Viscoelastic testing by rotational thromboelastometry and coagulation factor biomarker analyses were performed in this prospective observational cohort study of critically ill COVID-19 patients from April 2020 to October 2020. Statistical analyses were performed to identify significant coagulopathic biomarkers such as fibrinolysis-inhibiting plasminogen activator inhibitor 1 and their associations with clinical outcomes such as mortality, extracorporeal membrane oxygenation requirement, occurrence of major thrombotic events, and severity of hypoxemia (arterial partial pressure of oxygen/fraction of inspired oxygen categorized into mild, moderate, and severe per the Berlin criteria). RESULTS: In total, 53 of 55 (96%) of the cohort required mechanical ventilation and 9 of 55 (16%) required extracorporeal membrane oxygenation. Extracorporeal membrane oxygenation-naïve patients demonstrated lysis indices at 30 min indicative of fibrinolytic suppression on rotational thromboelastometry. Survivors demonstrated fewer procoagulate acute phase reactants, such as microparticle-bound tissue factor levels (odds ratio, 0.14 [0.02, 0.99]; P = 0.049). Those who did not experience significant bleeding events had smaller changes in ADAMTS13 levels compared to those who did (odds ratio, 0.05 [0, 0.7]; P = 0.026). Elevations in plasminogen activator inhibitor 1 (odds ratio, 1.95 [1.21, 3.14]; P = 0.006), d-dimer (odds ratio, 3.52 [0.99, 12.48]; P = 0.05), and factor VIII (no clot, 1.15 ± 0.28 vs. clot, 1.42 ± 0.31; P = 0.003) were also demonstrated in extracorporeal membrane oxygenation-naïve patients who experienced major thrombotic events. Plasminogen activator inhibitor 1 levels were significantly elevated during periods of severe compared to mild and moderate acute respiratory distress syndrome (severe, 44.2 ± 14.9 ng/ml vs. mild, 31.8 ± 14.7 ng/ml and moderate, 33.1 ± 15.9 ng/ml; P = 0.029 and 0.039, respectively). CONCLUSIONS: Increased inflammatory and procoagulant markers such as plasminogen activator inhibitor 1, microparticle-bound tissue factor, and von Willebrand factor levels are associated with severe hypoxemia and major thrombotic events, implicating fibrinolytic suppression in the microcirculatory system and subsequent micro- and macrovascular thrombosis in severe COVID-19.

Intravital optical imaging for immune cell tracking after photoimmunotherapy with plasmonic gold nanostars.

Bladder cancer has been ranked as one of the most commonly occurring cancers in men and women with approximately half of the diagnoses being the late stage and/or metastatic diseases. We have developed a novel cancer treatment by combining gold nanostar-mediated photothermal therapy with checkpoint inhibitor immunotherapy to treat bladder cancer. Experiment results with a murine animal model demonstrated that our developed photoimmunotherapy therapy is more efficacious than any individual studied treatment. In addition, we used intravital optical imaging with a dorsal skinfold window chamber animal model to study immune responses and immune cell accumulation in a distant tumor following our photoimmunotherapy. The mice used have the CX3CR1-GFP receptor on monocytes, natural killer cells, and dendritic cells allowing us to dynamically track their presence by fluorescence imaging. Our proof-of-principle study results showed that the photoimmunotherapy triggered anti-cancer immune responses to generate anti-cancer immune cells which accumulate in metastatic tumors. Our study results illustrate that intravital optical imaging is an efficient and versatile tool to investigate immune responses and mechanisms of photoimmunotherapy in future studies.

Controlling cancer-induced inflammation with a nucleic acid scavenger prevents lung metastasis in murine models of breast cancer.

Tumor cells release nucleic acid-containing proinflammatory complexes, termed nucleic acid-containing damage-associated molecular patterns (NA DAMPs), passively upon death and actively during stress. NA DAMPs activate pattern recognition receptors on cells in the tumor microenvironment leading to prolonged and intensified inflammation that potentiates metastasis. No strategy exists to control endogenous or therapy-induced inflammation in cancer patients. We discovered that the generation 3.0 polyamidoamine dendrimer (PAMAM-G3) scavenges NA DAMPs and mitigates their proinflammatory effects. In this study, we tested if the nucleic acid scavenger (NAS) PAMAM-G3 reduces lung metastasis in murine models of breast cancer. Our data indicate that PAMAM-G3 treatment decreases cell-free DNA levels and reduces lung metastasis in the experimental intravenous tumor-injection model and the postsurgical tumor-resection model of 4T1 breast cancer. Reduction in lung metastasis is associated with reduction in inflammatory immune cell subsets and proinflammatory cytokine levels in the tumor and the periphery. This study is the first example of NAS-mediated inhibition of metastasis to the lung. The study results provide a strong rationale for inclusion of NAS therapy in women with breast cancer undergoing standard-of-care surgery.

Targeting Tumor Acidosis and Regulatory T Cells Unmasks Anti-Metastatic Potential of Local Tumor Ablation in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an immunologically heterogenous disease that lacks clinically actionable targets and is more likely to progress to metastatic disease than other types of breast cancer. Tumor ablation has been used to increase response rates to checkpoint inhibitors, which remain low for TNBC patients. We hypothesized that tumor ablation could produce an anti-tumor response without using checkpoint inhibitors if immunosuppression (i.e., Tregs, tumor acidosis) was subdued. Tumors were primed with sodium bicarbonate (200 mM p.o.) to reduce tumor acidosis and low-dose cyclophosphamide (100-200 mg/kg i.p.) to deplete regulatory T cells, as has been shown independently in previous studies. A novel injectable ablative was then used to necrose the tumor, release tumor antigens, and initiate an immune event that could create an abscopal effect. This combination of bicarbonate, cyclophosphamide, and ablation, called "BiCyclA", was tested in three syngeneic models of TNBC: E0771 (C57BL/6), 67NR (BALB/c), and 4T1-Luc (BALB/c). In E0771 and 67NR, BiCyclA therapy significantly reduced tumor growth and cured 5/7 and 6/10 mice 50 days after treatment respectively. In the metastatic 4T1-Luc tumors, for which surgery and checkpoint inhibitors fail, BiCyclA cured 5/10 mice of primary tumors and lung metastases. Notably, CD4+ and CD8+ T cells were found to be crucial for the anti-metastatic response, and cured mice were able to resist tumor rechallenge, suggesting production of immune memory. Reduction of tumor acidity and regulatory T cells with ablation is a simple yet effective therapy for local and systemic tumor control with broad applicability as it is not limited by expensive supplies.

Dissecting the immune landscape of tumor draining lymph nodes in melanoma with high-plex spatially resolved protein detection.

BACKGROUND: In melanoma patients, microscopic tumor in the sentinel lymph-node biopsy (SLN) increases the risk of distant metastases, but the transition from tumor in the SLN to metastatic disease remains poorly understood. METHODS: Fluorescent staining for CD3, CD20, CD11c, and DNA was performed on SLN tissue and matching primary tumors. Regions of interest (ROI) were then chosen geometrically (e.g., tumor) or by fluorescent cell subset markers (e.g., CD11c). Each ROI was further analyzed using NanoString Digital Spatial Profiling high-resolution multiplex profiling. Digital counts for 59-panel immune-related proteins were collected and normalized to account for system variation and ROI area. RESULTS: Tumor regions of SLNs had variable infiltration of CD3 cells among patients. The patient with overall survival (OS) > 8 years had the most CD11c- and CD3-expressing cells infiltrating the SLN tumor region. All patients had CD11c (dendritic cell, DC) infiltration into the SLN tumor region. Selecting ROI by specific cell subtype, we compared protein expression of CD11c cells between tumor and non-tumor/normal tissue SLN regions. Known markers of DC activation such as CD86, HLA-DR, and OX40L were lowest on CD11c cells within SLN tumor for the patient with OS < 1 year and highest on the patient with OS > 8 years. CONCLUSION: We demonstrate the feasibility of profiling the protein expression of CD11c cells within the SLN tumor. Identifying early regulators of melanoma control when the disease is microscopically detected in the SLN is beneficial and requires follow-up studies in a larger cohort of patients.

Characterization of Sentinel Lymph Node Immune Signatures and Implications for Risk Stratification for Adjuvant Therapy in Melanoma.

BACKGROUND: Although sentinel lymph node (SLN) biopsy is a standard procedure used to identify patients at risk for melanoma recurrence, it fails to risk-stratify certain patients accurately. Because processes in SLNs regulate anti-tumor immune responses, the authors hypothesized that SLN gene expression may be used for risk stratification. METHODS: The Nanostring nCounter PanCancer Immune Profiling Panel was used to quantify expression of 730 immune-related genes in 60 SLN specimens (31 positive [pSLNs], 29 negative [nSLNs]) from a retrospective melanoma cohort. A multivariate prediction model for recurrence-free survival (RFS) was created by applying stepwise variable selection to Cox regression models. Risk scores calculated on the basis of the model were used to stratify patients into low- and high-risk groups. The predictive power of the model was assessed using the Kaplan-Meier and log-rank tests. RESULTS: During a median follow-up period of 6.3 years, 20 patients (33.3%) experienced recurrence (pSLN, 45.2% [14/31] vs nSLN, 20.7% [6/29]; p = 0.0445). A fitted Cox regression model incorporating 12 genes accurately predicted RFS (C-index, 0.9919). Improved RFS was associated with increased expression of TIGIT (p = 0.0326), an immune checkpoint, and decreased expression of CXCL16 (p = 0.0273), a cytokine important in promoting dendritic and T cell interactions. Independent of SLN status, the model in this study was able to stratify patients into cohorts at high and low risk for recurrence (p < 0.001, log-rank). CONCLUSIONS: Expression profiles of the SLN gene are associated with melanoma recurrence and may be able to identify patients as high or low risk regardless of SLN status, potentially enhancing patient selection for adjuvant therapy.

Tetanus toxoid and CCL3 improve dendritic cell vaccines in mice and glioblastoma patients.

After stimulation, dendritic cells (DCs) mature and migrate to draining lymph nodes to induce immune responses. As such, autologous DCs generated ex vivo have been pulsed with tumour antigens and injected back into patients as immunotherapy. While DC vaccines have shown limited promise in the treatment of patients with advanced cancers including glioblastoma, the factors dictating DC vaccine efficacy remain poorly understood. Here we show that pre-conditioning the vaccine site with a potent recall antigen such as tetanus/diphtheria (Td) toxoid can significantly improve the lymph node homing and efficacy of tumour-antigen-specific DCs. To assess the effect of vaccine site pre-conditioning in humans, we randomized patients with glioblastoma to pre-conditioning with either mature DCs or Td unilaterally before bilateral vaccination with DCs pulsed with Cytomegalovirus phosphoprotein 65 (pp65) RNA. We and other laboratories have shown that pp65 is expressed in more than 90% of glioblastoma specimens but not in surrounding normal brain, providing an unparalleled opportunity to subvert this viral protein as a tumour-specific target. Patients given Td had enhanced DC migration bilaterally and significantly improved survival. In mice, Td pre-conditioning also enhanced bilateral DC migration and suppressed tumour growth in a manner dependent on the chemokine CCL3. Our clinical studies and corroborating investigations in mice suggest that pre-conditioning with a potent recall antigen may represent a viable strategy to improve anti-tumour immunotherapy.

Recombinant Poliovirus for Cancer Immunotherapy.

Mechanisms to elicit antiviral immunity, a natural host response to viral pathogen challenge, are of eminent relevance to cancer immunotherapy. "Oncolytic" viruses, naturally existing or genetically engineered viral agents with cell type-specific propagation in malignant cells, were ostensibly conceived for their tumor cytotoxic properties. Yet, their true therapeutic value may rest in their ability to provoke antiviral signals that engage antitumor immune responses within the immunosuppressive tumor microenvironment. Coopting oncolytic viral agents to instigate antitumor immunity is not an easy feat. In the course of coevolution with their hosts, viruses have acquired sophisticated strategies to block inflammatory signals, intercept innate antiviral interferon responses, and prevent antiviral effector responses, e.g., by interfering with antigen presentation and T cell costimulation. The resulting struggle of host innate inflammatory and antiviral responses versus viral immune evasion and suppression determines the potential for antitumor immunity to occur. Moreover, paradigms of early host:virus interaction established in normal immunocompetent organisms may not hold in the profoundly immunosuppressive tumor microenvironment. In this review, we explain the mechanisms of recombinant nonpathogenic poliovirus, PVSRIPO, which is currently in phase I clinical trials against recurrent glioblastoma. We focus on an unusual host:virus relationship defined by the simple and cytotoxic replication strategy of poliovirus, which generates inflammatory perturbations conducive to tumor antigen-specific immune priming.

The impact of variant classification on the clinical management of hereditary cancer syndromes.

PURPOSE: The reclassification of genetic variants poses a significant challenge for laboratories and clinicians. Variant review has resulted in the reclassification of variants of unknown significance as well as the reclassification of previously established pathogenic and likely pathogenic variants. These reclassifications have the potential to alter the clinical management of patients with hereditary cancer syndromes. METHODS: Results were reviewed for 1694 patients seen for hereditary cancer evaluation between August 2012 and May 2017 to determine the frequency and types of variant reclassification. Patients with reclassifications with high potential for impact were monitored for alterations in organ surveillance, prophylactic surgery, and cascade testing. RESULTS: One hundred forty-two variants were reclassified representing 124/1694 (7.3%) patients; 11.3% of reclassifications (16/142) had a high potential for clinical impact with 94% (15/16) altering clinical management of patients with 56% (9/16) changing multiple areas of management. CONCLUSION: While reclassifications are rare, the impact on clinical management is profound. In many cases, patients with downgraded pathogenic/likely pathogenic variants had years of unnecessary organ surveillance and underwent unneeded surgical intervention. In addition, cascade testing misidentified those at risk for developing cancers, thereby altering the management across generations. The frequency and types of alterations to clinical management highlight the need for timely variant reclassification.

Role of the highly conserved G68 residue in the yeast phosphorelay protein Ypd1: implications for interactions between histidine phosphotransfer (HPt) and response regulator proteins.

BACKGROUND: Many bacteria and certain eukaryotes utilize multi-step His-to-Asp phosphorelays for adaptive responses to their extracellular environments. Histidine phosphotransfer (HPt) proteins function as key components of these pathways. HPt proteins are genetically diverse, but share a common tertiary fold with conserved residues near the active site. A surface-exposed glycine at the H + 4 position relative to the phosphorylatable histidine is found in a significant number of annotated HPt protein sequences. Previous reports demonstrated that substitutions at this position result in diminished phosphotransfer activity between HPt proteins and their cognate signaling partners. RESULTS: We report the analysis of partner binding interactions and phosphotransfer activity of the prototypical HPt protein Ypd1 from Saccharomyces cerevisiae using a set of H + 4 (G68) substituted proteins. Substitutions at this position with large, hydrophobic, or charged amino acids nearly abolished phospho-acceptance from the receiver domain of its upstream signaling partner, Sln1 (Sln1-R1). An in vitro binding assay indicated that G68 substitutions caused only modest decreases in affinity between Ypd1 and Sln1-R1, and these differences did not appear to be large enough to account for the observed decrease in phosphotransfer activity. The crystal structure of one of these H + 4 mutants, Ypd1-G68Q, which exhibited a diminished ability to participate in phosphotransfer, shows a similar overall structure to that of wild-type. Molecular modelling suggests that the highly conserved active site residues within the receiver domain of Sln1 must undergo rearrangement to accommodate larger H + 4 substitutions in Ypd1. CONCLUSIONS: Phosphotransfer reactions require precise arrangement of active site elements to align the donor-acceptor atoms and stabilize the transition state during the reaction. Any changes likely result in an inability to form a viable transition state during phosphotransfer. Our data suggest that the high degree of evolutionary conservation of residues with small side chains at the H + 4 position in HPt proteins is required for optimal activity and that the presence of larger residues at the H + 4 position would cause alterations in the positioning of active site residues in the partner response regulator.

Regulatory Targets of the Response Regulator RR_1586 from Clostridioides difficile Identified Using a Bacterial One-Hybrid Screen.

The Clostridioides difficile R20291 genome encodes 57 response regulator proteins that, as part of two-component signaling pathways, regulate adaptation to environmental conditions. Genomic and transcriptomic studies in C. difficile have been limited, due to technical challenges, to the analysis of either high-throughput screens or high-priority targets, such as primary regulators of toxins or spore biology. We present the use of several technically accessible and generally applicable techniques to elucidate the putative regulatory targets of a response regulator, RR_1586, involved in sporulation of the hypervirulent C. difficile strain R20291. A DNA-binding specificity motif for RR_1586 was determined using a bacterial one-hybrid assay originally developed for Drosophila transcription factors. Comparative bioinformatics approaches identified and in vitro experiments confirmed RR_1586 binding sites upstream of putative target genes, including those that encode phosphate ion transporters, spermidine/putrescine biosynthesis and transport pathways, ABC type transport systems, known regulators of sporulation, and genes encoding spore structural proteins. Representative examples of these regulatory interactions have been tested and confirmed in Escherichia coli-based reporter assays. Finally, evidence of possible regulatory mechanisms is also presented. A working model includes self-regulation by RR_1586 and phosphorylation-dependent and -independent DNA binding at low- and high-fidelity binding sites, respectively. Broad application of this and similar approaches is anticipated to be an important catalyst for the study of gene regulation by two-component systems from pathogenic or technically challenging bacteria.IMPORTANCEClostridioides difficile spores survive under harsh conditions and can germinate into actively dividing cells capable of causing disease. An understanding of the regulatory networks controlling sporulation and germination in C. difficile could be exploited for therapeutic advantage. However, such studies are hindered by the challenges of working with an anaerobic pathogen recalcitrant to genetic manipulation. Although two-component response regulators can be identified from genetic sequences, identification of their downstream regulatory networks requires further development. This work integrates experimental and bioinformatic approaches, which provide practical advantages over traditional transcriptomic analyses, to identify the putative regulon of the C. difficile response regulator RR_1586 by first screening for protein-DNA interactions in E. coli and then predicting regulatory outputs in C. difficile.

Crystal structure and DNA binding activity of a PadR family transcription regulator from hypervirulent Clostridium difficile R20291.

BACKGROUND: Clostridium difficile is a spore-forming obligate anaerobe that can remain viable for extended periods, even in the presence of antibiotics, which contributes to the persistence of this bacterium as a human pathogen during host-to-host transmission and in hospital environments. We examined the structure and function of a gene product with the locus tag CDR20291_0991 (cdPadR1) as part of our broader goal aimed at elucidating transcription regulatory mechanisms involved in virulence and antibiotic resistance of the recently emergent hypervirulent C. difficile strain R20291. cdPadR1 is genomically positioned near genes that are involved in stress response and virulence. In addition, it was previously reported that cdPadR1 and a homologue from the historical C. difficile strain 630 (CD630_1154) were differentially expressed when exposed to stressors, including antibiotics. RESULTS: The crystal structure of cdPadR1 was determined to 1.9 Å resolution, which revealed that it belongs to the PadR-s2 subfamily of PadR transcriptional regulators. cdPadR1 binds its own promoter and other promoter regions from within the C. difficile R20291 genome. DNA binding experiments demonstrated that cdPadR1 binds a region comprised of inverted repeats and an AT-rich core with the predicted specific binding motif, GTACTAT(N2)ATTATA(N)AGTA, within its own promoter that is also present in 200 other regions in the C. difficile R20291 genome. Mutation of the highly conserved W in α4 of the effector binding/oligomerization domain, which is predicted to be involved in multi-drug recognition and dimerization in other PadR-s2 proteins, resulted in alterations of cdPadR1 binding to the predicted binding motif, potentially due to loss of higher order oligomerization. CONCLUSIONS: Our results indicate that cdPadR1 binds a region within its own promoter consisting of the binding motif GTACTAT(N2)ATTATA(N)AGTA and seems to associate non-specifically with longer DNA fragments in vitro, which may facilitate promoter and motif searching. This suggests that cdPadR1 acts as a transcriptional auto-regulator, binding specific sites within its own promoter, and is part of a broad gene regulatory network involved, in part, with environmental stress response, antibiotic resistance and virulence.