Detailed phenotyping of Tbr1-2A-CreER knock-in mice demonstrates significant impacts on TBR1 protein levels and axon development.

Spatiotemporal control of Cre-mediated recombination has been an invaluable tool for understanding key developmental processes. For example, knock-in of Cre into cell type marker gene loci drives Cre expression under endogenous promoter and enhancer sequences, greatly facilitating the study of diverse neuronal subtypes in the cerebral cortex. However, insertion of exogenous DNA into the genome can have unintended effects on local gene regulation or protein function that must be carefully considered. Here, we analyze a recently generated Tbr1-2A-CreER knock-in mouse line, where a 2A-CreER cassette was inserted in-frame just before the stop codon of the transcription factor gene Tbr1 . Heterozygous TBR1 mutations in humans and mice are known to cause autism or autism-like behavioral phenotypes accompanied by structural brain malformations, most frequently a reduction of the anterior commissure. Thus, it is critical for modified versions of Tbr1 to exhibit true wild-type-like activity. We evaluated the Tbr1-2A-CreER allele for its potential impact on Tbr1 function and complementation to Tbr1 loss-of-function alleles. In mice with one copy of the Tbr1-2A-CreER allele, we identified reduction of TBR1 protein in early postnatal cortex along with thinning of the anterior commissure, suggesting hypersensitivity of this structure to TBR1 dosage. Comparing Tbr1-2A-CreER and Tbr1 -null heterozygous and homozygous mice to Tbr1 -null complementation crosses showed reductions of TBR1 dosage ranging from 28.4% to 95.9%. Using these combinatorial genotypes, we found that low levels of TBR1 protein (∼16%) are sufficient to establish cortical layer positioning, while greater levels (>50%) are required for normal suppression of layer 5 identity. In total, these results strongly support the conclusion that Tbr1-2A-CreER is a hypomorphic allele. We advise caution when interpreting experiments using this allele, such as transcriptomic studies, considering the sensitivity of various corticogenic processes to TBR1 dosage and the association of heterozygous TBR1 mutations with complex neurodevelopmental disorders.

Population-Based Newborn Screening for Germline TP53 Variants: Clinical Benefits, Cost-Effectiveness, and Value of Further Research.

BACKGROUND: Identification of children and infants with Li-Fraumeni syndrome prompts tumor surveillance and allows potential early cancer detection. We assessed the clinical benefits and cost-effectiveness of population-wide newborn screening for TP53 variants (TP53-NBS). METHODS: We simulated the impact of TP53-NBS using data regarding TP53-associated pediatric cancers and pathogenic or likely pathogenic (P/LP) TP53 variants from Surveillance, Epidemiology, and End Results; ClinVar and gnomAD; and clinical studies. We simulated an annual US birth cohort under usual care and TP53-NBS and estimated clinical benefits, life-years, and costs associated with usual care and TP53-NBS. RESULTS: Under usual care, of 4 million newborns, 608 (uncertainty interval [UI] = 581-636) individuals would develop TP53-associated cancers before age 20 years. Under TP53-NBS, 894 individuals would have P/LP TP53 variants detected. These individuals would undergo routine surveillance after detection of P/LP TP53 variants decreasing the number of cancer-related deaths by 7.2% (UI = 4.0%-12.1%) overall via early malignancy detection. Compared with usual care, TP53-NBS had an incremental cost-effectiveness ratio of $106 009 per life-year gained. Probabilistic analysis estimated a 40% probability that TP53-NBS would be cost-effective given a $100 000 per life-year gained willingness-to-pay threshold. Using this threshold, a value of information analysis found that additional research on the prevalence of TP53 variants among rhabdomyosarcoma cases would resolve most of the decision uncertainty, resulting in an expected benefit of 349 life-years gained (or $36.6 million). CONCLUSIONS: We found that TP53-NBS could be cost-effective; however, our findings suggest that further research is needed to reduce the uncertainty in the potential health outcomes and costs associated with TP53-NBS.

Breast Cancer Screening Among Childhood Cancer Survivors Treated Without Chest Radiation: Clinical Benefits and Cost-Effectiveness.

BACKGROUND: Early initiation of breast cancer screening is recommended for high-risk women, including survivors of childhood cancer treated with chest radiation. Recent studies suggest that female survivors of childhood leukemia or sarcoma treated without chest radiation are also at elevated early onset breast cancer risk. However, the potential clinical benefits and cost-effectiveness of early breast cancer screening among these women are uncertain. METHODS: Using data from the Childhood Cancer Survivor Study, we adapted 2 Cancer Intervention and Surveillance Modeling Network simulation models to reflect the elevated risks of breast cancer and competing mortality among leukemia and sarcoma survivors. Costs and utility weights were based on published studies and databases. Outcomes included breast cancer deaths averted, false-positive screening results, benign biopsies, and incremental cost-effectiveness ratios. RESULTS: In the absence of screening, the lifetime risk of dying from breast cancer among survivors was 6.8% to 7.0% across models. Early initiation of annual mammography with breast magnetic resonance imaging screening between ages 25 and 40 years would avert 52.6% to 64.3% of breast cancer deaths. When costs and quality-of-life impacts were considered, screening starting at age 40 years was the only strategy with an incremental cost-effectiveness ratio below the $100 000 per quality-adjusted life-year (QALY) gained cost-effectiveness threshold ($27 680 to $44 380 per QALY gained across models). CONCLUSIONS: Among survivors of childhood leukemia or sarcoma, early initiation of breast cancer screening at age 40 years may reduce breast cancer deaths by half and is cost-effective. These findings could help inform screening guidelines for survivors treated without chest radiation.

Universal newborn genetic screening for pediatric cancer predisposition syndromes: model-based insights.

PURPOSE: Genetic testing for pediatric cancer predisposition syndromes (CPS) could augment newborn screening programs, but with uncertain benefits and costs. METHODS: We developed a simulation model to evaluate universal screening for a CPS panel. Cohorts of US newborns were simulated under universal screening versus usual care. Using data from clinical studies, ClinVar, and gnomAD, the presence of pathogenic/likely pathogenic (P/LP) variants in RET, RB1, TP53, DICER1, SUFU, PTCH1, SMARCB1, WT1, APC, ALK, and PHOX2B were assigned at birth. Newborns with identified variants underwent guideline surveillance. Survival benefit was modeled via reductions in advanced disease, cancer deaths, and treatment-related late mortality, assuming 100% adherence. RESULTS: Among 3.7 million newborns, under usual care, 1,803 developed a CPS malignancy before age 20. With universal screening, 13.3% were identified at birth as at-risk due to P/LP variant detection and underwent surveillance, resulting in a 53.5% decrease in cancer deaths in P/LP heterozygotes and a 7.8% decrease among the entire cohort before age 20. Given a test cost of $55, universal screening cost $244,860 per life-year gained; with a $20 test, the cost fell to $99,430 per life-year gained. CONCLUSION: Population-based genetic testing of newborns may reduce mortality associated with pediatric cancers and could be cost-effective as sequencing costs decline.

Assessing the micro-scale environment using Google Street View: the Virtual Systematic Tool for Evaluating Pedestrian Streetscapes (Virtual-STEPS).

BACKGROUND: Altering micro-scale features of neighborhood walkability (e.g., benches, sidewalks, and cues of social disorganization or crime) could be a relatively cost-effective method of creating environments that are conducive to active living. Traditionally, measuring the micro-scale environment has required researchers to perform observational audits. Technological advances have led to the development of virtual audits as alternatives to observational field audits with the enviable properties of cost-efficiency from elimination of travel time and increased safety for auditors. This study examined the reliability of the Virtual Systematic Tool for Evaluating Pedestrian Streetscapes (Virtual-STEPS), a Google Street View-based auditing tool specifically designed to remotely assess micro-scale characteristics of the built environment. METHODS: We created Virtual-STEPS, a tool with 40 items categorized into 6 domains (pedestrian infrastructure, traffic calming and streets, building characteristics, bicycling infrastructure, transit, and aesthetics). Items were selected based on their past abilities to predict active living and on their feasibility for a virtual auditing tool. Two raters performed virtual and field audits of street segments in Montreal neighborhoods stratified by the Walkscore that was used to determine the 'walking-friendliness' of a neighborhood. The reliability between virtual and field audits (n = 40), as well as inter-rater reliability (n = 60) were assessed using percent agreement, Cohen's Kappa statistic, and the Intra-class Correlation Coefficient. RESULTS: Virtual audits and field audits (excluding travel time) took similar amounts of time to perform (9.8 versus 8.2 min). Percentage agreement between virtual and field audits, and for inter-rater agreement was 80% or more for the majority of items included in the Virtual-STEPS tool. There was high reliability between virtual and field audits with Kappa and ICC statistics indicating that 20 out of 40 (50.0%) items had almost perfect agreement and 13 (32.5%) items had substantial agreement. Inter-rater reliability was also high with 17 items (42.5%) with almost perfect agreement and 11 (27.5%) items with substantial agreement. CONCLUSIONS: Virtual-STEPS is a reliable tool. Tools that measure the micro-scale environment are important because changing this environment could be a relatively cost-effective method of creating environments that are conducive to active living.

The heat penalty of walkable neighbourhoods.

"Walkability" or walking-friendliness is generally considered a favourable attribute of a neighbourhood that supports physical activity and improves health outcomes. Walkable neighbourhoods tend to have high-density infrastructure and relatively high amounts of concrete and pavement for sidewalks and streets, all of which can elevate local urban temperatures. The objective of this study was to assess whether there is a "heat penalty" associated with more walkable neighbourhoods in Montréal, Québec, Canada, using air temperature measurements taken in real time at street level during a heat event. The mean temperature of "Car-Dependent" neighbourhoods was 26.2 °C (95% CI 25.8, 26.6) whereas the mean temperature of "Walker's Paradise" neighbourhoods was 27.9 °C (95% CI 27.8, 28.1)-a difference of 1.7 °C (95% CI 1.3, 2.0). There was a strong association between higher walkability of Montréal neighbourhoods and elevated temperature (r = 0.61, p < 0.01); suggestive of a heat penalty for walkable neighbourhoods. Planning solutions that support increased walking-friendliness of neighbourhoods should consider simultaneous strategies to mitigate heat to reduce potential health consequences of the heat penalty.

Elucidation of the substrate specificity of the C1s protease of the classical complement pathway.

The complement system is a central component of host defense but can also contribute to the inflammation seen in pathological conditions. The C1s protease of the first complement component, the C1 complex, initiates the pathway. In this study we have elucidated the full specificity of the enzyme for the first time using a randomized phage display library. It was found that, aside from the crucial P(1) position, the S(3) and S(2) subsites (in that order) played the greatest role in determining specificity. C1s prefers Leu or Val at P(3) and Gly or Ala residues at P(2). Apart from the S(2)' position, which showed specificity for Leu, prime subsites did not greatly affect specificity. It was evident, however, that together they significantly contributed to the efficiency of cleavage of a peptide. A peptide substrate based on the top sequence obtained in the phage display validated these results and produced the best kinetics of any C1s substrate to date. The results allow an understanding of the active site specificity of the C1s protease for the first time and provide a basis for the development of specific inhibitors aimed at controlling inflammation associated with complement activation in adverse pathological situations.

Importance of the prime subsites of the C1s protease of the classical complement pathway for recognition of substrates.

The classical complement pathway, which plays a vital role in preventing infection, is initiated by the action of the serine proteases C1r and C1s. We have examined the hydrolysis of substrates representing cleavage sequences in the physiological substrates for C1s, C2 and C4. These studies showed that the P(1)'-P(4)' substrate residues of C2 and C4 conferred greater affinity of substrate for enzyme and also induced a sigmoidal dependence of enzyme velocity on substrate concentration. This indicates that the substrate gave rise to homotropic positive cooperative behavior in the enzyme. When C1s was in complex with C1q and C1r, as would occur under physiological conditions, the same behavior was observed, indicating that this mechanism is relevant in the complement pathway in vivo. We further investigated the requirements of C1s for prime side amino acids by examining a substrate library in which each of the P(1)'-P(4)' positions had been substituted by different classes of amino acids. This revealed that the P(1)' position was a major determinant of the selectivity of the enzyme, while certain substitutions at the P(1)'-P(4)' positions abolished the allosteric behavior, indicating that contact residues at these positions in the C1s enzyme must mediate the cooperativity. The studies reported here highlight the importance of prime subsites in C1s for interaction with its cognate substrates in the complement pathway and therefore yield greater understanding of the mechanism of interaction between this vital protease and its physiological substrates.