GCKR, TR1B1, MAU2/TM6SF2, APOE, and PNPLA3). We also report a potentially causal effect of lower LPL expression in adipose tissue on NAFLD susceptibility and an effect of the FTO genotype on NAFLD. Positive genetic correlations between NAFLD and cardiometabolic diseases and risk factors such as body fat accumulation/distribution, lipoprotein-lipid levels, insulin resistance, and coronary artery disease and negative genetic correlations with parental lifespan, socio-economic status, and acetoacetate levels are observed. This large GWAS meta-analysis reveals insights into the genetic architecture of NAFLD.Non-alcoholic fatty liver disease (NAFLD) is a complex disease linked to several chronic diseases. We aimed at identifying genetic variants associated with NAFLD and evaluating their functional consequences. We performed a genome-wide meta-analysis of 4 cohorts of electronic health record-documented NAFLD in participants of European ancestry . We identify 5 potential susceptibility loci for NAFLD .To gain insight into the genetic architecture of non-alcoholic fatty liver disease (NAFLD), Ghodsian et al. performed a genome-wide meta-analysis of electronic health record-documented NAFLD and identify 7 potential susceptibility loci for this disease (located at or near  The NAFLD GWAS of the UK Biobank included 1,664 NAFLD cases and 400,055 controls identified only 2 regions robustly associated with NAFLD (PNPLA3 and PBX4/TM6SF2). The UK Biobank analysis did not exclude participants with secondary causes of NAFLD  and used a rather vague definition of NAFLD . Genetic variation at these 2 loci is also associated with NAFLD in the data freeze #4 of the FinnGen cohorts .To better understand the etiology of complex diseases such as NAFLD and to develop therapies that may help patients with this disease living longer and healthier, the genetic architecture of NAFLD needs to be better understood. Although genome-wide association studies (GWASs) have identified genetic variants associated with liver fat accumulation,Here, we present the results of a meta-analysis of electronic health record (EHR)-based GWASs to identify genetic variants associated with NAFLD. This analysis included GWAS summary statistics from the eMERGE and FinnGen cohorts, an updated NAFLD GWAS in the UK Biobank , and a new GWAS performed in the Estonian Biobank , for a total of 8,434 NAFLD cases and 770,180 controls.−8 . −8. The associated quantile-quantile plot is presented in GCKR, LPL, and FTO) associated with NAFLD , totaling 8,434 NAFLD cases, all identified through EHRs, and 770,180 controls. We identified 4 genetic loci that harbored at least 1 SNP that passed the genome-wide significance threshold of p £ 5 × 10th NAFLD . Figure th NAFLD B, ratherth NAFLD C or postth NAFLD D  became associated with NAFLD, with a p value below the GWAS significance threshold of £5 × 10−8. This analysis did not reveal any new NAFLD susceptibility loci beyond the variant at the GCKR locus.To determine whether these 7 SNPs were associated with NAFLD independently of obesity, we performed another GWAS meta-analysis using the same models described in the GCKR is a missense variant (p.P446L). The rs1260326 at APOE is also a missense variant (p.R130C). The lead variant at MAU2/TM6SF2 rs73001065 is in linkage disequilibrium (r2 = 0.90) with the missense variant p.E167K at TM6SF2, and the lead variant at PNPLA3 is in high linkage disequilibrium (r2 = 0.98) with the missense variant p.I148M at PNPLA3. MTARC1, a splice variant HSD17B13, and another variant at MBOAT7). This analysis confirmed previous NAFLD functional variants at MTARC1 and MBOAT7, but not at HSD17B13. Genetic variation at the PNPLA3, TM6SF2, and GCKR have been linked with NAFLD-related traits in previous studies.,,APOE, TR1B1, and FTO as potential new loci for liver enzymes.,LPL as a potential new susceptibility locus for NAFLD. Interestingly, the minor allele (C) at rs13702 associated here with protection against NAFLD has been predicted to disrupt a microRNA recognition element seed site for human microRNA miR-410, resulting in higher LPL expression.LPL expression was associated with NAFLD. We performed a transcriptome-wide association study for NAFLD to map genetically regulated genes from the Genotype Tissue Expression  consortiumPNPLA3 and TM6SF2 (data not shown). Genetically predicted LPL expression could be estimated in 11 tissues. The association between genetically predicted LPL expression in these 11 tissues and NAFLD is presented in LPL expression in subcutaneous adipose tissue and NAFLD (p = 3.1e−4). The LocusCompare plot  or NAFLD (diagnosed by computed tomography and/or MRI) were compared to 26,404 controls. The direction of the effects of the 7 SNPs were concordant with those observed in the GWAS meta-analysis. All SNPs were significantly associated with NAFLD in the Mass General Brigham Biobank, with the exception of the variants at the LPL loci . Liver fPL locus . FinallyWe performed cross-trait genetic correlation analyses between NAFLD and 240 human traits centralized in the LD Hub database. LD Hub includes GWAS publicly available summary statistics on hundreds of human traits and enables the assessment of LD score regression among those traits. The results presented in TM6SF2 and PNPLA3) and 5 potentially new candidate genetic regions for a clinical diagnosis NAFLD based on EHRs .We performed 2 genome-wide association studies for NAFLD in the UK Biobank and in the Estonian Biobank and combined these results with those of 2 publicly available NAFLD GWASs (from the eMERGE network and FinnGen). This GWAS meta-analysis included 8,434 NAFLD cases available via EHRs and 770,180 controls, making it the largest genome-wide analysis for a clinical diagnosis of NAFLD. In combination with a risk factor-informed bGWAS, this analysis identified 2 known susceptibility loci for NAFLD  that may be associated with NAFLD through their effects on NAFLD risk factors (BMI and triglycerides). A recent preprint identified a variant at the FTO locus as a susceptibility locus for having high ALT levels in the Million Veteran ProgramFTO locus is still a matter of debate, FTO is a well-characterized genetic locus for obesity.FTO locus was no longer significantly associated with NAFLD, confirming that the effect of this variant on NAFLD is dependent on its effect on body weight. Although variants at the GCKR locus were not associated with NAFLD in the main analysis, the bGWAS analysis and the conventional GWAS adjusted for BMI identified GCKR as a susceptibility locus for NAFLD. Other studies reported an association of variants at the GCKR loci and liver fat accumulationGCKR locus may modulate NAFLD risk associated with obesity and/or elevated triglyceride levels. The same may be true for variants at the LPL locus, the gene that encodes lipoprotein lipase (LPL). LPL is a key enzyme that regulates the catabolism of triglycerides-rich lipoproteins such as chylomicrons and very-low-density lipoproteins in adipose tissue, skeletal muscle, and the heart. Gain-of-function mutations in LPL were associated with lower triglyceride levels and lower risk of CAD.LPL expression in subcutaneous adipose tissue and NAFLD. These results are in line with the recent study of Maltais et al.,LPL function) met the criteria of NAFLD independently of their BMI. It should be noted that although the variant at the LPL locus linked with higher NAFLD was associated with higher liver enzymes levels in the UK Biobank, it was not associated with liver fat accumulation in the UK Biobank or with NAFLD in the Mass General Brigham Biobank. In addition, although these results did not reach the level of genome-wide significance, we found significant associations at the MTARC1 and MBOAT7 loci, thereby confirming the role of these genes in the etiology of NAFLD.Our conventional GWAS analyses (adjusted for BMI or not) report that variation at the LPL as being associated with lower lipid levels and risk of CAD, our analysis suggests that targeting the LPL pathway may prevent NAFLD as well as other diseases such as hyperlipidemia and CAD without increasing the risk of other human diseases. Drugs targeting the LPL pathway under investigation for NAFLD include the angiopoietin-like protein-3 (ANGPTL3) inhibitors,Previous studies have shown that NAFLD could be associated with or predict the risk of chronic diseases such as CVD or T2D. Our genetic correlation analyses revealed associations with these diseases as well as risk factors for these diseases such as obesity and insulin resistance. We also report interesting negative correlations between NAFLD and the ketone body acetoacetate ,FTO and LPL, 2 potentially new NAFLD loci. However, although the top variants at these loci were associated with liver fat accumulation and/or liver enzymes in the UK Biobank, these variants did not replicate in a smaller NAFLD GWAS. It should also be re-emphasized that variation at these loci act on NAFLD through selected risk factors and therefore may lead to NAFLD via indirect mechanisms. Although our study reports 2 conventional GWAS analyses (adjusting or not adjusting for BMI), we could not perform a GWAS meta-analysis adjusting for triglyceride levels. Therefore, studies with larger sample sizes and accounting for triglyceride levels will be needed to document whether variation at the LPL locus are strongly associated with NAFLD and whether their effects are entirely mediated by triglyceride levels.Our study has limitations. For instance, although we have excluded secondary causes of NAFLD whenever possible, an EHR-based diagnosis of complex diseases such as NAFLD may be prone to misclassification of cases and controls. Our analysis revealed LPL and NAFLD that will need confirmation by other, larger studies.In conclusion, we conducted a large NAFLD GWAS based on EHRs from 4 cohorts to identify genetic variants of NAFLD susceptibility. We identified known NAFLD variants and show that variants associated with liver fat accumulation and liver enzymes may also be associated with the presence of NAFLD. Our analysis revealed a potentially causal effect of lower adipose-tissue expression of benoit.arsenault@criucpq.ulaval.ca).Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Benoit Arsenault In the eMERGE study, logistic regression analysis was performed on over 7 million SNPs with MAF > 1% adjusted for age, gender, body mass index, genotyping site and the first three ancestry based principal components. In the UK Biobank genome-wide genotyping was available for over 28 million genetic markers directly genotyped or imputed by the Haplotype Reference Consortium (HRC) panel. In FinnGen, GWAS was performed using over 16 million genetic markers genotyped with the Illumina or Affymetrix arrays or imputed using the population specific SISu v3 reference panel. Variables included in the models were gender, age, the 10-main ancestry-based principal components and genotyping batch.We used the SAIGE  method to perform the GWAS in the UK Biobank and in the Estonian BiobankWe used bGWAS to identify more SNPs associated with NAFLD.,Locuscompare function from the LocuscompareR R packageLPL locus. Locuscompare enables visualization of the strengths of eQTLs and outcomes associations by plotting p values for each within a given genomic location, thereby contributing to distinguish candidates from false-positive genes.Tissues from the GTEx consortium (version 8) with less than 70 samples were not used to provide sufficient statistical power for eQTL discovery, resulting in a set of 48 tissues. Only non-gender-specific tissues (N = 43) were analyzed. Alignment to the human reference genome hg28/GRCh38 was performed using STAR v2.6.1d, based on the GENCODE v30 annotation. RNA-seq expression outliers were excluded using a multidimensional extension of the statistic described by Wright et al.In this cohort, genotyping was performed using the Illumina MEGA array. Association of each of the seven variants associated with NAFLD was assessed using logistic regression of disease status with age, gender and five principal components of ancestry as covariates.aberrant, resulting in a total of 32,976 individuals. We did not remove related individuals from this analysis as we used a linear mixed model able to account for cryptic relatedness in common variant association studies.As part of the study protocol of the UK Biobank, a subset of individuals who underwent detailed imaging between years 2014 and 2019 including abdominal MRI.https://biobank.ndph.ox.ac.uk/showcase/showcase/docs/serum_biochemistry.pdf.Age, gender and ancestry-based principal components-adjusted GWAS summary statistics on ALT, AST, GGT and ALP concentrations in 361,194 participants of the UK Biobank of European ancestry were obtained from the Neale lab. Details on the protocols used to measure these biomarkers is available on the UK Biobank website:


Avil encodes for the actin-binding protein ADVILLIN, is expressed in neural crest-derived cells, and was therefore used as a target in this study to knock out OXTR expression in neural-crest derived cells. Here, we tested if OXTRs specifically expressed in Avil positive neural crest-derived cells are necessary for species-typical adult social behaviors using a Cre-LoxP strategy. Genetically modified male and female mice lacking OXTR in Avil expressing cells (OXTRAvil KO) were tested for sociability and preference for social novelty. Males were also tested for resident intruder aggression. OXTRAvil KO males and females had reduced sociability compared to OXTRAvil WT controls. Additionally, OXTRAvil KO males had increased aggressive behaviors compared to controls. These data indicate that OXTRs in cells of neural crest origin are important regulators of typical social behaviors in C57BL/6J adult male and female mice and point to needed directions of future research.Social behaviors are foundational to society and quality of life while social behavior extremes are core symptoms in a variety of psychopathologies and developmental disabilities. Oxytocin (OXT) is a neuroactive hormone that regulates social behaviors through its receptor (OXTR), with all previously identified social behavior effects attributed to the central nervous system, which has developmental origins in the neural tube. However, OXTR are also present in neural crest-derived tissue including sensory ganglia of the peripheral nervous system.  A diverse body of evidence supports a role for the oxytocin (OXT) and the OXT receptor (OXTR) in modulating the brain and social behaviors across species , 2. StudAvil-expressing cells, which are neural-crest derived cells primarily located in the PNS, on adult social behavior in male and female C57BL/6J mice. Avil encodes the actin-binding protein ADVILLIN and is expressed in all dorsal root ganglion neurons, the trigeminal ganglion, enteric, parasympathetic and sympathetic neurons as well as their peripheral projections, particularly projections innervating blood vessels and Merkel skin cells [Studies focusing on site-specific knock out of the OXTR thus far indicate dissociable roles of OXTR in sociability, social novelty preference, and aggression –10. Howein cells . In addiin cells .Avil expressing cells, we used a Cre-LoxP strategy in well-established lines of mice to generate OXTRAvil WT and OXTRAvil KO subjects. OXTRAvil KO and WT adult male and female mice were tested for sociability and social novelty preference, and males were tested for aggression  but no effect of the Cre transgene on sociability . We tested both males and females and found no sex-specific effect on sociability . Thus, while whole body OXTR loss impacts adult sociability to a greater extent than just neural-crest derived OXTR loss, life-long loss of OXTR in Avil cells is sufficient to disrupt sociability levels in males and females and may account for half of the impaired sociability reported previously in congenital OXTR KO male mice. Other studies that have used Cre strategies to identify a role for brain OXTR in sociability have not observed a disruption with brain specific OXTR loss. For example, selective deletion of OXTR from CaMKIIα -expressing neurons [Previous studies have documented a large effect size of reduced sociability in conventional adult OXTR KO males, compared to WT (d = 1.2–1.8) , 6, 7, w neurons  or by vi neurons  or the h neurons  did not Avil expressing cells may be specific to regulating sociability levels but might not be involved in preference for social novelty or its social recognition prerequisite. In contrast, brain OXTR do seem to be needed for these behavioral components as Cre-based deletion of OXTR from the lateral septum does impair social novelty preference [Avil KO mice, suggests that central OXTR but not neural-crest derived OXTR are necessary for the behavioral display of adult social novelty preference.OXTR in eference . Consideeference , 7, 23, Avil KO adults may be a result of a developmental and/or activational effect. Future experiments involving temporal regulation of OXTR in sensory ganglia will resolve this question for adult sociability. While we did not observe differences in social novelty preferences in adults, differences might be present in younger ages. Future studies can address this by testing males and females for sociability and the developmental shift in preference for familiarity to social novelty.Reduced sociability in OXTRAvil cells on social behaviors. Because OXT injections rescue sociability and preference for social novelty deficits in congenital OXTR KO males [Avpr1a) mRNA is present in sensory ganglia and receptor binding sites are present in various peripheral tissues of neonatal mice [Compensatory mechanisms may have blunted or negated potential effects of the absence of OXTR in KO males  and OXT KO males , 24. Thital mice , 26.Avil expressing cells to reduce sociability in mice is reminiscent of mouse models of neurodevelopmental disorders which also exhibit reduced sociability that has been attributed to altered tactile discrimination and developmental loss of the autism spectrum disorder (ASD)-associated genes, Mecp2 or Gabrb3, in Avil cells [Avil cells may interact with ASD-associated genes during development to contribute to ASD core symptoms and subtypes. Peripheral changes alone may hinder the sensory-dependent development of forebrain regions involved in regulating adult social behavior, consistent with a developmental diaschisis model of neurodevelopmental disorders [The contribution of OXTR in il cells . Bluntedisorders .Avil KO males initiated aggression than OXTRAvil WT males and had shorter clinch attack latencies and higher number of attacks compared to WT  is similar to several studies using congenital OXTR KO mice (estimated Cohen’s d ~ 1.2) [The magnitude of the effect of OXTRd ~ 1.2) –7. This CaMKIIα promoter did not show an aggression phenotype. They concluded that OXTR in the pre-weaning stage is important in the development of typical aggression. Additionally, Takayanagi et al., (2005) reported heightened aggression in the RIT by OXTR KO and OXT KO males [The literature on OXTR in aggression hints strongly at a developmental role for OXTR. Dhakar et al., (2012) found increased aggression in congenital OXTR KO compared to OXTR WT mice in the RIT . SurprisKO males . HoweverKO males , 29, 30.Avil expressing cells in adult social behaviors. The role of OXTR in regulating specific social behaviors by acting in various Avil cell populations remains to be disentangled considering that Avil expression has been reported in both the somatic and autonomic PNS and tissue targets, as well as the dorsal habenula of the epithalamus and trigeminal nuclei of the brainstem [Avil positive cells may alter brain region-specific developmental trajectories to alter social behavior, through developmental diaschisis. We recently reported that full body congenital OXTR KO mice have impaired Oxt gene expression in the paraventricular nucleus of the hypothalamus beginning in the second postnatal week which persists in males into adulthood [Altogether, these data implicate OXTR in rainstem , 31–34. dulthood . Experiedulthood . Investiad libitum access to standard rodent chow  and water. For all subjects, the day of birth was considered postnatal day 0 (P0). Experimental mice were bred in house and weaned and ear tagged at P21. Upon weaning, a small tail snip (<1mm) was collected for genotyping and males and females were housed with same sex siblings until testing at 8–10 weeks old.All procedures were conducted after approval by the Institutional Animal Care and Use Committee of Florida State University. All mice had Oxtr gene flanked by LoxP recombinase binding sequences (fxOXTR) [fxOXTRfx/fx) and sires that were hemizygous for fxOXTR (fxOXTRfx/+) and Avil-Cre recombinase [Oxtr from Avil expressing cells; OXTRAvil HET). Male and female experimental subjects were selected from fxOXTRfx/fx offspring that were either Avil-Cre-  or Avil-Cre+ .Experimental subjects were adult male and female offspring from crosses of dams with the (fxOXTR)  at both ombinase  transgenAvil WT males, 15 OXTRAvil KO males, 19 OXTRAvil WT females, and 15 OXTRAvil KO females tested for sociability and preference for social novelty. Only males were tested for aggression in the RIT. One OXTRAvil WT male that went through sociability and preference for social novelty testing died from malocclusion before the RIT. To rule out a deleterious effect of the Cre-recombinase transgene on social behaviors, male and female Avil-Cre- and Avil-Cre+ littermates were also tested. Mice for these control experiments were generated by breeding wild-type (WT) dams with Avil-Cre+ sires. Twelve Avil-Cre- and 17 Avil-Cre+ control males as well as 20 Avil-Cre- and 16 Avil-Cre+ control females were tested.There were 20 OXTRStimulus mice were C57BL/6J male and female mice received from Jackson Labs at approximately 5–7 weeks old and allowed to habituate to our mouse colony for at least 2 weeks prior to testing. Stimulus animals were ear tagged for identification purposes. After testing, stimulus mice were placed into a temporary holding cage to avoid social transmission of the test to cage mates that had not gone through testing yet. Stimulus animals were placed back together into their home cages after testing was completed for the day.fxOXTR and Avil-cre genotypes. The fxOXTR forward primer 5’-GCT GAG TCT TGG AAG CAG GA and reverse primer 5’-GGT ACC TCC TTT GAG CTT CTG generated a 375 bp product for the fxOXTRfx/fx genotype and a 305 bp product for the fxOXTR+/+ genotype. Heterozygous mice (fxOXTRfx/+) produced both PCR products. PCR thermal cycling conditions for fxOXTR were as follows: 94°C for 2 minutes followed by 10 cycles of 94°C for 20 seconds, 65°C for 15 seconds for the first cycle with a 0.5°C decrease per cycle, and 68°C for 10 seconds and finally 28 cycles of 94°C for 15 seconds, 60°C for 15 seconds, and 72°C for 10 seconds, followed by 1 cycle of 72°C for 2 minutes. The Avil-cre forward primer (5’‐GCA CTG ATT TCG ACC AGG TT) and reverse primer (5’‐GAG TCA TCC TTA GCG CCG TA) generated a product approximately 420 base pairs for transgene positive and no band for transgene negative. PCR thermal cycling conditions for Avil-cre were: 95°C for 2 minutes followed by 10 cycles of 95°C for 30 seconds, 65°C for 30 seconds for the first cycle with a 1°C decrease per cycle, and 72°C for 30 seconds and finally 35 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, followed by 1 cycle of 72°C for 10 minutes. Post-PCR reactions were visualized by gel electrophoresis using a 2% agarose gel at 140 volts for 20 minutes for fxOXTR genotype determination and a 1% agarose gel at 140 volts for 20 minutes for Avil-cre identification. PCR products were visualized by ethidium bromide assisted imaging. All PCRs and gels were run with appropriate positive, negative, and no template controls.Tail samples were genotyped via polymerase chain reaction (PCR) to determine Oxtr in our Cre-LoxP strategy, we compared the expression levels of Oxtr mRNA in brain and peripheral tissues of adult OXTRAvil WT (n = 2) and OXTRAvil KO mice (n = 2). Specifically, we dissected olfactory bulbs and piriform cortex as representative neural tube derived central nervous system tissue, and trigeminal ganglia as a representative neural crest derived peripheral nervous system tissue. Because Oxtr is expressed at low levels, to calibrate the expected difference between WT and KO, we also compared the expression level of Oxtr mRNA in olfactory bulbs and piriform cortex from WT mice (n = 2) to conventional OXTR KO [2, tissues were extracted and immediately fresh frozen. Samples were stored at -80°C until RNA extraction by the Trizol method. RNA quality was confirmed by gel electrophoresis and quantity was measured by Nanodrop. Total RNA was DNAse digested and reverse transcribed. RT-qPCR was performed with primers for Gapdh , Oxtr  and Avil . SYBR dye was used in a cycling program: 94°C 5 minutes, 40 cycles of 94°C for 30 seconds, 60°C for 30 seconds, 55°C for 5 seconds. Melt curve analysis was used to confirm the appearance of a single amplicon. Non DNAse-treated RNA templates for each sample were used to confirm the absence of genomic DNA contamination and water was used as a no template control. Both controls performed as expected (zero to negligible amplification). These data are available in the To validate the selective deletion of  OXTR KO , 11, 35 In all procedures, the experimenter wore a disposable lab coat, gloves, and a facemask. Tests were conducted between 9am and 5pm with mice housed on a standard 12:12 light cycle with lights on at 8am. Mice were housed in a single room in cages with wire tops and wood chip bedding. Cages were cleaned once per week. Both male and female adults were tested in the sociability and preference for social novelty test. Males only were tested in the RIT after 2 weeks of social isolation. The sociability and social preference tests are conducted in a three-chambered testing apparatus described by Moy et al., 2004), wherein a rectangular testing apparatus is equally divided into two outer chambers and a center chamber . During , whereinBehavioral testing was conducted in the three-chambered social apparatus . The dimensions of each chamber were 20 × 40 × 22 cm(h). The walls of the chamber were made of clear Perspex. The bottom of the apparatus consisted of a non-reflective gray colored floor. Two sliding doors, also made of clear Perspex, were 5 × 8 cm(h) and when in place confined the subject to the center chamber. The top and bottom of the two-cylinder shaped stimulus holding towers were made of gray PVC and the grid bars were metal, 25 cm tall, 3 mm in diameter, and spaced 7 mm apart. Two lids made of clear Perspex with holes were placed on the outermost chambers to prevent the subject from climbing on top of the stimulus towers and escaping the apparatus during the test. Subjects were isolated for at least 1 hour in the testing room prior to testing in standard cages identical to those used in housing, with access to food and water. Prior to testing, the entire three chambered apparatus was wiped down with soft absorbent paper towels and a 10% bleach solution in filtered, deionized water and then a final wipe with sufficient water to remove residual odor before finally being dried with paper towels. Prior to testing, holding towers were also sprayed and wiped down with 10% bleach, then water, and then dried using soft absorbent paper towels. The subject habituated to the testing apparatus with the doors to the outermost chambers opened for 10 minutes. At the end of the 10-minute habituation period, an empty holding tower was placed in one outer chamber and a holding tower containing an age-, sex- and weight- matched conspecific was placed in the opposite chamber. The sociability test lasted for 10 minutes. The social novelty test then immediately commenced by placing an unfamiliar age-, sex-, and weight- matched conspecific into the previously empty tower. The social novelty test lasted for 10 minutes and included a familiar conspecific in one chamber (from the sociability test) and the unfamiliar conspecific in the opposite chamber. The familiar and unfamiliar stimulus animals were housed separately prior to arrival in our colony and had never interacted with each other or with the subjects. Stimulus animals were kept in different rooms from the subject mice on the day of testing. Stimulus mice were habituated to the holding towers and testing apparatus for at least three days prior to testing for 40 minutes per day. Stimulus mice were used once a day for a maximum of two days.Avil WT and 13/15 OXTRAvil KO males and 8/12 Avil-Cre- and 10/17 Avil-Cre+ males.In the RIT, an “intruder” or male stranger is placed into the home cage of an adult “resident” male that has been socially isolated or has had mating experience. Aggressive behaviors are measured including attack duration and frequency as well as latency to first attack. Only males were tested in the RIT. After the preference for social novelty test was completed, virgin male subjects were singly housed for two weeks before the RIT. Subjects’ cages were not cleaned for at least a week prior to the RIT (ranging from 7–13 days). On the day of the RIT, subjects’ home cages were transferred to the testing room at least 1 hour prior to testing (range was 1–2 hours). The RIT commenced when a male intruder weighing slightly less than the subject was placed into the subject’s (resident) home cage. The test lasted 15 minutes and ended with the intruder being removed from the home cage of the resident. A researcher monitored the entire RIT to prevent possible serious injuries to the intruder if necessary. Because we did not pre-screen intruders for absence of aggression, to analyze the effects of resident genotype on aggression, we only analyzed data from tests where the C57BL/6J stimulus intruder did not initiate aggression in the RIT. This was determined by scoring instances of the intruder starting a clinch attack without knowledge of resident genotype. After removing subjects with intruder-initiated aggression from the dataset, we analyzed data from 14/19 OXTRAll tests were video recorded with cameras positioned to achieve a bird’s eye view and side views of the entire testing apparatus. Experimenters blind to the genotype of the subjects later scored behaviors using JWatcher software (UCLA). For the sociability and preference for social novelty tests, the duration of time spent in each chamber, and the duration and frequency of tower sniffing were quantified. In addition, tower approach latencies were measured and defined as the time it took the subject from the start of the test to approach a tower. For the RIT, the latency, duration and frequency of clinch attacks, as well as social  and non-social  behaviors were scored. These data are available in the Avil WT males from the same litter , 1 OXTRAvil KO male and 1 OXTRAvil KO female with low total tower investigation times ranging from 0–37 seconds over the 10-minute test were removed from all further analyses for the sociability and social novelty tests. Importantly, removing these outliers resulted in homogeneity of variance and a normal distribution and did not change the outcome of significant test results but met the required variance and distribution parameters for confident interpretation of parametric tests. In addition, the low investigation duration of the removed outliers questions the validity of their test results since they had minimal participation. Tower approach latencies were square root transformed to reach normality and 1 Avil-Cre+ female outlier was removed with a latency of 566 seconds to approach the empty tower and 23 seconds to approach the tower with the unfamiliar mouse. 2 Avil-Cre+ males, 1 Avil-Cre- female, and 1 Avil-Cre+ female were identified as outliers based on deviation from the grand mean of total tower sniffing in the sociability test and removed from further sociability and preference for social novelty analyses. These subjects spent a range of 3–17 seconds sniffing both towers combined during the entire 10-minute test. Statistical analyses were conducted in SPSS (Version 25.0) and details can be found in the results section text and S1 Table in To determine if genotype influences the ability to display sociability and social novelty preferences, we analyzed tower investigations, chamber durations, and tower approach frequencies with a repeated measures two-way (sex and genotype) analysis of variance (RM ANOVA). Data for the sociability and preference for social novelty tests are presented as individual data points with averages ± standard error of the mean and as difference scores, in line with other studies , 20, to d) from prior work by extracting means and SEMs (converted to standard deviations) from plots and entering this information in the effect size calculator at https://lbecker.uccs.edu/.To compare our results with published studies, we estimated effect sizes (Cohen’s S1 File(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Situs inversus totalis (SIT) is a rare congenital abnormality in which the thoracic and abdominal organs are reversed or mirrored from their usual positions. We herein report the first case of robot-assisted transhiatal lower esophagectomy and proximal gastrectomy with esophagogastrostomy for treatment of Siewert type II advanced esophagogastric junction (EGJ) cancer with SIT.A 62-year-old man with SIT and intestinal malrotation was diagnosed with T3N0M0 Stage IIA EGJ cancer. Three-dimensional reconstruction of a computed tomography angiogram showed that the common hepatic artery was absent, the proper hepatic artery was derived from the superior mesenteric artery through the gastroduodenal artery, and an accessary left hepatic artery arose from the left gastric artery. The patient underwent robot-assisted transhiatal lower esophagectomy and proximal gastrectomy with D2 lymph node dissection, including lower mediastinal lymphadenectomy. Intraoperative examination revealed minor vascular abnormalities, including three branches of the left gastric artery and two left gastric veins, that had not been recognized preoperatively. The surgery was performed safely, and the patient had an uneventful postoperative course.Robotic-assisted surgery is efficient even for complex conditions, such as Siewert type II advanced EGJ cancer with SIT. Situs inversus totalis (SIT) is a congenital abnormality in which the thoracic and abdominal organs are reversed or mirrored from their usual positions. It is a relatively rare condition that occurs in 1 of every 5000 to 20,000 people . Robot-aA 62-year-old man with SIT, intestinal malrotation, and type 2 diabetes underwent gastroduodenal endoscopy for investigation of epigastric discomfort. A 5-cm type 2 tumor was found at the cardia side of the EGJ Fig. . A biopsThe patient was placed in a spinal position and the port placement mirrored our conventional settings Fig. . The patAfter laparoscopic inspections, the lesser omentum was opened and suprapancreatic LN dissection was started. The two left gastric veins draining into the splenic vein (SPV) were clipped and cut Fig. a. The LGAfter checking the margin of softy on the back table, esophagogastrostomy was performed according to the side overlap with fundoplication by Yamashita (SOFY) method as follows . The cenThe surgical time was 296 min, and the amount of blood loss was small. Histopathological diagnosis revealed a Siewert type II tumor measuring 50 × 37 mm in diameter and moderately differentiated adenocarcinoma with subserosal invasion  it is not necessary for the surgeon to change standing positions , 15. (2)Notably, as shown in Table We have herein reported the first case of robot-assisted transhiatal lower esophagectomy and proximal gastrectomy with esophagogastrostomy (SOFY method) for treatment of advanced EGJ cancer with SIT. We believe that robotic surgery is efficient even for complex conditions, such as Siewert type II advanced EGJ cancer with SIT.


Cancer cells usually depend on the aberrant function of one or few driver genes to initiate and promote their malignancy, an attribute known as oncogene addiction. However, cancer cells might become dependent on the normal cellular functions of certain genes that are not oncogenes but ensure cell survival (non-oncogene addiction). The downregulation or silencing of DNA repair genes and the consequent genetic and epigenetic instability is key to promote malignancy, but the activation of the DNA-damage response (DDR) has been shown to become a type of non-oncogene addiction that critically supports tumour survival. In the present study, a systematic evaluation of DNA repair addiction at the pan-cancer level was performed using data derived from The Cancer Dependency Map and The Cancer Genome Atlas (TCGA). From 241 DDR genes, 59 were identified as commonly essential in cancer cell lines. However, large differences were observed in terms of dependency scores in 423 cell lines and transcriptomic alterations across 18 cancer types. Among these 59 commonly essential genes, 14 genes were exclusively associated with better overall patient survival and 19 with worse overall survival. Notably, a specific molecular signature among the latter, characterized by DDR genes like UBE2T, RFC4, POLQ, BRIP1, and H2AFX showing the weakest dependency scores, but significant upregulation was strongly associated with worse survival. The present study supports the existence and importance of non-oncogenic addiction to DNA repair in cancer and may facilitate the identification of prognostic biomarkers and therapeutic opportunities. However, it is known that tumours can also become dependent on the normal cellular functions of certain genes, which themselves are not classical oncogenes, an attribute known as non-oncogene addiction (NOA)2.Although tumours develop through a multistage process driven by the acquisition of genetic and epigenetic abnormalities, many of them become dependent on one or few genes to promote malignancy. As many of these genes were originally identified as oncogenes, this attribute was named oncogene addiction3. Genome instability is indeed a fundamental hallmark of cancer4, possibly linked to oncogene-induced DNA damage5. Accordingly, it has been demonstrated that DDR genes alterations are prevalent in multiple human cancer types7. Despite the need for this genomic instability, tumours require some degree of DNA repair proficiency to survive the damage induced by genotoxic stress, uncontrolled proliferation, and treatments. Thus, the activation of the DNA-damage response in cancer can be considered as a type of NOA that critically supports tumour survival8. NOA genes are especially associated with stress maintenance functions such as DNA double-stranded break repair, chromatid segregation, and DNA replication regulation9. Accordingly, several DNA repair genes have been shown to exhibit Copy Number Variation (CNV) gain in cancer, which is positively correlated with upregulation of DDR genes10.In order to achieve uncontrolled proliferation, tumour cells rely upon the downregulation or epigenetic silencing of DNA Damage Repair (DDR) genes and the consequent increase in genetic and epigenetic instability11. Additionally, two large pan-cancer CRISPR-Cas9 screens were also independently performed with the same goal, containing data from over 1000 screens of more than 900 cell lines13. Based on both approaches, a combined CRISPR-shRNA dependency score was later developed, providing a more sensitive measure to identify essential genes14. These loss-of-function genetic screens have allowed investigating cellular drug mechanism-of-action in cancer cell lines15 as well as the identification of potential therapeutic targets like the DNA repair helicase WRN, a synthetic lethal vulnerability and promising target in cancers with microsatellite instability16.Given the importance of both oncogene and non-oncogene addiction, different groups have developed a variety of approaches to identify and prioritize cancer dependencies and vulnerabilities to exploit them therapeutically. The Cancer Dependency Map was initially created by systematically identifying genetic dependencies using RNAi-based loss-of-function genetic screens in 501 cancer cell linesIn this study, I integrated data derived from The Cancer Dependency Map and transcriptomic data derived from The Cancer Genome Atlas (TCGA) to characterize the non-oncogene addiction to DNA repair across the pan-cancer scale. Next, I explored the relevance of DDR genes in terms of patient survival and potential therapeutic alternatives. Following this approach, a molecular signature of overexpressed DDR genes showing the weakest dependency scores was strongly associated with worse survival. The fact that cancer cells overexpress these genes despite the weak dependence on them (as growth promoters), suggests that they may act as late promoters of cell survival. Thus, as opposed to traditional cancer drivers, this supports the existence and importance of non-oncogenic addiction to DNA repair in cancer. This approach may facilitate the identification of prognostic biomarkers and therapeutic opportunities by targeting this non-oncogene addiction.17. In order to obtain an updated list, a literature search was done and the most comprehensive list found was used as a starting point6. This list comprised 276 DDR genes annotated in ten different DNA repair pathways, but genes involved in the nucleotide pool maintenance were excluded in this study as that is not truly a pathway. First, to identify the essentiality of these genes across different types of cancer cell lines, the efficacy and selectivity scores  were obtained from shinyDepMap14. These metrics are derived from a combined CRISPR-shRNA gene dependency score based on the Cancer Dependency Map dataset11. Efficacy scores represent the degree to which loss of a particular gene reduces cell growth in sensitive lines while selectivity represents the degree to which its essentiality varies across lines. After removing genes that were not found in the shinyDepMap dataset, 241 DDR genes  across 423 cancer cell lines were also obtained from the shinyDepMap dataset (Supplementary Table 18 was used to obtain the log2 fold change (log2 FC) of these genes from paired normal-tumour samples. TACCO includes the mRNAs and miRNA expression levels (Transcripts Per Million) of 26 types of cancer from the Broad GDAC Firehose. Gene expression data were obtained for 18 types of cancer  web serverSince it was noticed that genes with strongly negative dependency scores (more important for cancer cell growth) were not always highly upregulated, all variables were analyzed together to further explore clustering patterns. The efficacy and selectivity scores were included as continuous variables. The dependency scores of the 59 genes (for the whole set of 423 cancer cell lines) and the log2 FC (for the 18 types of cancer) were transformed from continuous to categorical  variables (see methods)  dataset20. The genes from each cluster were analyzed to determine if their expression was associated with worse or better overall survival in each of the three cancer groups  from the heatmap above (group B was subdivided into B1 and B2 as it was much larger and heterogeneous than A and C). Almost all 59 genes were statistically significant associated with worse or better survival at least in one cancer group  web serverFisher's exact test; Fig. As several genes have opposite effects for two or more cancer groups, genes that were exclusively associated with better survival and those exclusively associated with worse survival were analyzed as separate molecular signatures. Fourteen genes were classified as “better survival” (four from cluster 1 and five from cluster 3) whereas nineteen genes were classified as “worse survival” (five from cluster 1 and ten from cluster 3). Despite this trend, no statistically significant differences were found between the number of genes contributed by clusters 1 and 3 to each signature  was used to identify genes with similar expression patterns across the 18 cancer types. For each signature (better/worse), the Pearson’s correlation coefficient of 100 genes was obtained   (statistic score ≥ 0.50). In 10 of these genes , NAE1 (NEDD8 Activating Enzyme E1 Subunit 1) was the SLP with the highest score, and in 6 of them, it was the only gene with a statistic score ≥ 0.50  were strongly associated with better overall survival in some cancer groups. This was further confirmed when the 14 genes exclusively associated with better survival were considered a multi-gene signature and the higher expression of this signature was also associated with better survival across the 18 types of cancer. This is especially interesting considering that the lower expression and/or alterations in DDR genes have been associated with improved survival in some cancer types30. Nevertheless, a couple of studies have also reported that active DNA repair and higher expression of several DDR genes (including genes from clusters 1 and 2 from this study) were associated with better survival in gastric and ovarian cancer32. Taken together, this better survival signature might represent promising biomarkers for prognosis and further clinical studies should be performed to fully validate this.Since some of the common essential DDR genes in cancer cells may also be essential for normal cells, the implications of essentiality were analyzed at the transcriptomic level from paired normal-tumour samples and large differences were also found. Genes with strongly negative dependency scores (cluster 1), were upregulated in most cancer types suggesting that they might be essential for carcinogenesis. Some of them can even be considered as potential cancer drivers as previously suggested2. We can refer to this as the timing-dependent addiction, which means that although cancer cells have weak dependency on these genes (as growth promoters), these genes may act as late promoters of cell survival (given the evident overexpression). For instance, among genes from cluster 3, POLQ (Pol θ) might be one of the best examples of non-oncogene addiction to DNA repair (low efficacy and dependency scores but highly upregulated). In fact, it has been demonstrated that cancer cells with mutations in POLQ synthetic lethal (DDR) genes tend to become addicted to Theta Mediated End Joining (TMEJ) for survival, suggesting that POLQ becomes essential upon increased levels of endogenous and unrepaired DNA damage33.Another surprising finding was that genes from cluster 3 (weakest dependency and efficacy scores) like UBE2T, POLQ, BRIP1, H2AFX, and RTEL1, were also highly upregulated. This could indicate that cancer cells do not depend on these genes for their growth but survival, especially considering the persistent DNA damage and replication stress in cancer cells34. Additionally, it has been shown that the amplification and overexpression of several DDR genes across the pan-cancer scale are associated with reduced mutation burden, cell line drug resistance, and poor prognosis35. In contrast, a higher somatic tumour mutational burden has been associated with better overall survival in several types of cancer36. Taken together, the results from the present study support the notion that non-oncogene addiction to several DDR genes could reduce the tumour mutational burden and confer treatment resistance, suggesting an explanation for the worse overall survival associated with this signature.Remarkably, the present work also demonstrated that higher expression of UBE2T, RFC4, POLQ, BRIP1, and H2AFX is especially associated with worse overall survival (HR > 5) in several types of cancer (Group A and D). Additionally, when considered as part of the worse survival signature, these genes were associated with a worse prognosis in the 18 cancer types. This might be also linked to the correlation found between the expression levels of this signature and the "worse 100 signature", which is enriched with genes involved in DNA replication and cell cycle, probably suggesting a more aggressive phenotype. Previous findings have reported that genes involved in cell cycle phases and checkpoint genes had higher essentiality percentages both in embryonic stem cells and in cancer cell lines35. RFC4 (Replication factor C subunit 4) has been also identified as an upregulated DDR gene across the pan-cancer scale10. RFC4 protects colorectal cancer cells from X-ray-induced DNA damage and apoptosis through nonhomologous end joining (NHEJ)-mediated DNA repair37 and has also been associated with poorer prognosis in this malignancy38 and lung cancer39.The results discussed above also support previous findings reporting the key role of the DDR genes from cluster 3 in cancer. For instance, studying more than 10,000 TCGA pan-cancer tumours, Wu and colleagues found that tumours with amplifications in DDR genes like UBE2T (Ubiquitin Conjugating Enzyme E2 T) exhibited significantly reduced mutation burden, temozolomide resistance, and worse patient survival. In fact, UBE2T and BRIP1 are also in the top 10 amplified DDR genes in their studyPOLQ gene) is important in the repair of genomic double-strand breaks (DSBs) from many sources40. Upregulation of POLQ has been associated with poor clinical outcomes in breast and lung cancer44. Following the same line, the present study highlights the importance of POLQ as a prognostic factor associated with worse survival in different types of cancer . Recent reports have demonstrated that POLQ inhibitors selectively kill HR-deficient tumour cells in vitro and in vivo46. Additionally, it has been demonstrated that POLQ knockdown sensitizes several tumour cell lines to Ionizing Radiation and causes minimal effects on normal tissue radiosensitivity47.DNA polymerase theta (Pol θ)  has been linked to reduced or suppressed tumorigenesis in different types of cancer53. CHEK1  is essential to maintain cell viability in cancer cells54 and it has been shown that the combination of ATR and CHEK1 inhibitors results in cancer-specific synthetic lethality55. Notably, CHEK1 inhibition is synthetically lethal with loss of B-Family DNA Polymerases like POLE2 in lung cancer and colorectal cancer cells56. Taken together, inhibiting these genes from cluster 1 represents a promising therapeutic avenue in several types of cancer, especially focusing on the combined inhibition of CHEK1 and POLE2.RFC2, CHEK1, PCNA, and POLE2 were also associated with worse overall survival in several types of cancer (Group A and D). As they belong to cluster 1 (strongly negative dependency and efficacy scores and high expression levels), these genes might be more necessary for tumour growth and promotion. Upregulation of PCNA has been reported in several types of cancer and its inhibition is gaining more interest as a novel strategy in many cancers58. In fact, Neddylation is beginning to be considered as a key factor of the DNA repair process59. As NAE1 inhibition has been proposed as a new approach to treating cancer60, these results support the potential of the combined inhibition of NAE1 and several DDR genes. More research will be needed to validate the implication of the molecular signatures described in this study and to what extent they apply to different subtypes of cancer and the complexity of their genetic backgrounds.Finally, it was remarkable that NAE1 was identified as a synthetic lethal partner of ten genes from the worse survival signature. NAE1 is responsible for Neddylation, a post-translational modification that adds an ubiquitin-like protein (NEDD8) to multiple substrate proteins and it has been shown that its inhibition exerts anticancer effects mainly by triggering cell apoptosis, autophagy, and senescencehttps://www.rstudio.com/).All data processing steps, and statistical analyses were performed in the RStudio 1.4.1717 statistical environment  were obtained from shinyDepMap14 (https://labsyspharm.shinyapps.io/depmap). These values are derived from a combined CRISPR-shRNA gene dependency score based on the Cancer Dependency Map dataset11 . Additionally, for those genes with efficacy scores lower than -0.5 and selectivity scores lower than 0 , the combined  dependency scores across 423 cell lines were obtained. Likewise, the lineage-dependent essentiality of 17 cell lineages was also analyzed. For a lineage to be classified as dependent, all its cell lines must have dependency values that exceed the essentiality threshold obtained by analyzing all available genes.The list of DNA repair genes and their respective pathway annotation was obtained from one of the latest and more complete reports so far18 (http://tacco.life.nctu.edu.tw/) was used to identify the patterns of expression of essential DNA repair genes  in 18 cancer types. TACCO includes the mRNAs expression levels (Transcripts Per Million) for 26 cancer types from the Broad GDAC Firehose (https://gdac.broadinstitute.org/) (version stddata__2016_01_28). For this task, only the log2 fold change (log2_FC) of those genes with statistically significant (Benjamini-Hochberg) adjusted p values were included. For the heatmap elaboration, the ComplexHeatmap61 R package was used.For transcriptomic analysis, the TACCO (Transcriptome Alterations in CanCer Omnibus) webserverTo find clustering patterns among DDR genes, all variables were analyzed together. The efficacy and selectivity scores were included for each gene as continuous variables. Based on the median dependency score of each gene (for the whole set of 423 cell lines), dependency scores were transformed from continuous to categorical: weakly negative (from 0 to − 0.5), moderate (from − 0.5 to − 1.0), and strongly negative (from − 1.0 to − 1.5). The log2 FC was also transformed to categorical: downregulated (from 0 to − 1.25), upregulated (from 0 to 1.25), and highly upregulated (from 1.25 to 2.5 +). A distance matrix was created based on Gower's similarity coefficient, as it is suggested for mixed-type variables. The clustering algorithm used was Partitioning Around Medoids (PAM) and the silhouette plot was used to determine the number of clusters. The t-Distributed Stochastic Neighbor Embedding (tSNE) algorithm was used for dimensionality reduction . Hierarchical clustering was used as an additional algorithm for dimensionality reduction based on the same Gower's similarity coefficients calculated.19 (http://gepia.cancer-pku.cn/) was used to compare the different gene clusters in terms of patient overall survival across the cancer groups included in this study. GEPIA is based on The Cancer Genome Atlas (TCGA)20 and The Genotype-Tissue Expression (GTEx) project62. The thresholds for high/low gene expression levels between cohorts were adjusted to 50%. Log-rank P values, hazard ratios (HR), and 95% confidence intervals (CI) were obtained for each gene. Notably, as GEPIA calculations are run in R, some p values might be reported as cero since R does not report values lower than 2.220446e-16 (.Machine$double.eps). Therefore, p values of cero must be understood as “below machine precision”.The GEPIA  webserver21 (http://gepia2.cancer-pku.cn/#index) was used to identify genes that have a similar expression pattern for each signature across all cancer types. The Search Tool for the Retrieval of Interacting Genes/Proteins database (STRING v11.0) web server22 (https://string-db.org/) was used to construct the protein–protein network associated with the better/worse 100 signature). The stringApp23, a Cytoscape app that makes it easy to import STRING networks into Cytoscape24 was used for network visualization and analysis. Additionally, the g:Profiler web server25 (http://biit.cs.ut.ee/gprofiler/) was used to find statistically significant Gene Ontology terms, pathways, and other gene functions giving a list of DDR genes.The GEPIA2 web server27 (http://synlethdb.sist.shanghaitech.edu.cn/v2/#/) was used to search synthetic lethal partners given a specific gene. SynLethDB harbours a large set of synthetic lethality gene pairs collected from a variety of sources, including biochemical assays, other related databases, computational predictions, and text mining.The SynLethDB web serverSupplementary Table 1.Supplementary Table 2.Supplementary Table 3.Supplementary Table 4.Supplementary Table 5.Supplementary Table 6.Supplementary Table 7.


Here, we set out to determine the identity of much debated BMP-treated hESCs by comparing their transcriptome to recently published single cell transcriptomic data from early human embryos (Human embryonic stem cells (hESCs) possess an immense potential to generate clinically relevant cell types and unveil mechanisms underlying early human development. However, using hESCs for discovery or translation requires accurately identifying differentiated cell types through comparison with their embryos . Our anaThis article has an associated First Person interview with the first author of the paper. Summary: We show that BMP-treated human embryonic stem cells (hESCs) are more likely to represent an amnion rather than a trophectoderm cell type. The identity of BMP-treated hESCs has been particularly controversial, with arguments made for three different extra-embryonic tissues – trophectoderm, amnion and extra-embryonic mesoderm (in vivo data.Human embryonic stem cells (hESCs) provide a unique window into early stages of human development. Over the last few years, they have been used to generate many medically relevant cell types and models of early human development . Howevermesoderm ,b. Basedmesoderm . HoweverObtaining data directly from human embryos is of paramount importance because there are significant differences between human embryos and those of mammalian model organisms such as the mouse, especially in the formation of amnion – the extra-embryonic tissue that covers the embryo in a protective sac . In humaIn a major breakthrough, a recent study  succeedein vivo identity of BMP-treated hESCs, we first reexamined whether the data in Xiang et al. support labeling the cells denoted as amnion as a distinct cell type as prior studies have hinted at a transcriptional similarity between amnion and trophectoderm cells. Monkey amniotic cells in vivo or purported human amnion cells in vitro express TFAP2A, GATA2/3, CDX2, and TP63, all well-known trophectoderm markers .We utilized the lineage-specific genes documented in the Fig. S1C,E,F). It also correctly placed the derived cell types with their respective parent lineages – syncytiotrophoblast (STB) and extra-villous cytotrophoblast (EVT) cells with cytotrophoblast cells (CTB) and primitive streak cells with epiblast cells.This known lineage marker gene set effectively separates the three lineages, even at the post-implantation stage, in both principal component and correlation analyses . Consistent with this, most D14 ‘AME cells do not express known pluripotency and primitive streak markers, thus questioning their placement in the PSA cluster  are transcriptionally correlated with CTB cells A. ConsisFig. 2A–C).To further discern the identity of ‘AME, we compared the transcriptomes of cells in the human embryo in the Xiang et al. study with cells in the post-implantation cynomolgus monkey embryo , which hFig. S2C). Most amnion cells, however, are also transcriptionally similar to trophectoderm-derived cells, consistent with the expression of trophectoderm-specific genes in the monkey amnion  are transcriptionally similar to epiblast cells, which is their parent lineage . Thus, expression of these genes can be utilized to determine whether human ‘AME represent epiblast derived amnion cells, as suggested by As this restricted lineage gene space does not distinguish the amnion and trophectoderm lineages, we repeated the analysis considering genes with variable expression across all monkey cells . In this space, the amnion cells retain transcriptional similarity with the epiblast but lose similarity with the trophectoderm  transcriptional signature of the monkey amnion . D12 ‘AMTo sum, the transcriptional similarity of ‘AME with monkey trophectoderm-derived cells and not monkey amnion cells, supports the notion that they represent mislabeled trophectoderm cells.We next sought to understand the reason that the analyses in Fig. S3).To determine the gene categories (Ensembl biotypes) that contribute the most to the two principal components in the above analyses, we plotted the normalized PC coefficient of different gene categories for each principal component. The top two contributors are protein coding genes and pseudogenes. Strikingly, the contribution of pseudogenes increases as the ‘AME-‘intermediate-primitive streak cluster moves to a distinct PC1 B. This cPseudogenes are homologous to protein coding genes but with a frameshift or stop codon, which renders them non-translational . While tTo further determine which trophoblast cell type ‘AME corresponds to, we repeated the principal component analyses using expressed genes with high variability  between D12,14 amnion, CTB, STB and EVT cells A. We remPCA reveals three distinct populations of ‘AME B. One ceConsistent with PCA, a correlation analyses of expression levels in same gene set also reveals three distinct populations of ‘AME. One cell (1/12) is transcriptionally correlated (Pearson correlation coefficient >0.4) with EVT cells, half of the cells (6/12) are correlated with STB cells, and the rest (5/12) show either low or no correlation with any of three trophoblast cell types E. To detTaken together, our results suggest that the data in In the absence of a unique human amnion transcriptome, we turned to the post implantation monkey embryo to resolve the identity of BMP-treated hESCs. We have previously shown that sparsely seeded hESCs treated with BMP4 ligands for 42 h transcriptionally resemble trophectoderm cells, and not extra-embryonic mesoderm cells . In thisWe first defined a set of lineage specific genes for the early post implantation monkey amnion, trophectoderm and epiblast (D11-14), and then compared the expression of those genes in monkey embryo with BMP-treated hESCs. We chose early stages (D11-14) of monkey post implantation development because the transcriptional similarity with corresponding human stages is higher at these stages compared to the later (D16–17) A.Table S1). These genes clearly separate the three lineages transcriptionally =0.01]. From this list, we excluded genes that are differentially expressed between different time points within that lineage [embryonic day (D)11–14] to reduce noise within the lineage and further removed genes with a low expression value [fragments per kilobase of transcript per million mapped reads (FPKM)<5 in at least two of the four time points for that lineage]. This gave a list of 571 lineage-specific genes .Correlation analyses of human lineage specific genes across different cell types in the Xiang et al. dataset revealed that two additional cell populations – ‘intermediate cells and inner cell mass (‘ICM) cells, are likely mislabeled , not epiblast cells. This trend is preserved at the level of single cells, where a majority of ‘Intermediate cells are not transcriptionally correlated with the epiblast or epiblast-derived primitive streak cells .However, in the lineage-specific gene space in our analyses, ‘intermediate cells exhibit maximum transcriptional similarity with primitive endoderm cells  are transcriptionally correlated with primitive endoderm (PrE) cells A. ConsisSimilar to ‘AME, the misclassification of ‘intermediate cells is also due to the inclusion of pseudogenes. When the principal component analysis is limited to protein coding genes, ‘intermediate cells share PC1 with PrE cells on both D12 and D14 in all the three gene sets analyzed A,C,D, inIn the variable gene space of the monkey embryo that separates the two primitive endoderm derived lineages – the EXMC and the VE/YE, most ‘intermediate cells  exhibit maximum transcriptional similarity with monkey EXMC cells. Consistent with this, ‘intermediate cells express known monkey EXMC genes like GATA4, GATA6, COL6A1, VIM, CDH2, SNAI2 (We relabel ‘intermediate cells as primitive endoderm derived if they exhibit maximum correlation with PrE lineage in the human embryo (condition1) A, and fuDuring implantation, the epiblast transitions between the naïve and primed pluripotent states, in both mouse and monkey. At the molecular level, this transition results in a reduced expression of naïve pluripotency genes, along with a sustained expression of core pluripotency genes . As precComparing expression of lineage specific genes in individual ‘ICM cells with average expression of those genes in different lineages in the embryo reveals that a majority of these cells (49/52) are transcriptionally correlated with CTB, not epiblast cells A. ConsisIn D6-9 human embryos, the absolute number of trophectoderm cells is higher than ICM/epiblast cells . Thus, iIt is worth noting that the cells of the D5 human embryo cannot be distinguished based on the known lineage markers as they co-express markers of the three lineages . HoweverTaken together, the above analyses suggest that 49/52 ICM cells – 30/32 D6, 17/18 D7 and 2/2 D9 ICM cells are mislabeled CTB cells on the corresponding days. Of the remaining 3 cells, 2 represent epiblast cells  and 1 represents primitive endoderm (D6A4S6) on the corresponding day, as indicated by the transcriptional similarity of known lineage genes A.hESCs offer a unique opportunity to probe early stages of human development. Their immense potential to differentiate into a variety of different cell types offers a valuable resource for both translational and fundamental research. However, to make accurate inferences, it is essential to determine the identity of cells obtained by differentiation of hESCs through careful comparisons with embryos.In this study, we examined the transcriptome of the much-debated BMP-treated hESCs, which have been variably considered similar to three extra-embryonic cell types – the trophectoderm, the extra-embryonic mesoderm and the amnion ,b. CompaComparison of hESC derived lineages to mouse development argues against a trophectoderm identification: The expression of mature trophectoderm markers by BMP-treated hESCs sparked off a debate on the physiological relevance of these cells as it contradicted previous results in the mouse literature . Mouse ein vivo. That is, under these conditions, hESCs differentiated into large multi-nucleated cells and secreted placental hormones including hCG, estradiol and progesterone  BMP-treated hESCs form cells that morphologically and molecularly resemble mature trophoblasts, as argued earlier. (2) BMP-treated hESCs are transcriptionally more similar to monkey amnion cells than monkey trophectoderm cells. Assuming transcriptional similarity between human and monkey amnion and trophoblast lineages at this stage of development, it can be reasoned that BMP-treated hESCs possess a unique ability to differentiate into mature trophectoderm cell types via an amnion-like transcriptional state.in vivo and whether the transcriptional similarity translates into functional plasticity between the two lineages where amnion cells can differentiate into trophoblast cell types and vice versa remains to be experimentally tested.A recently published preprint also comes to the same conclusion, although from a different starting point. The authors of the study show hESCs treated with a chemical cocktail devoid of BMP, activate endogenous BMP signaling and differentiate into mature trophectoderm subtypes via an amnion-like intermediate state preprint.Our analyses also revealed that the inclusion of pseudogenes leads to the mislabeling of amnion cells in the Xiang et al. dataset . In the in vivo identity, their placement with the epiblast and amnion cells implies that they presumably represent an epiblast derived cell population. However, we show that excluding pseudogenes changes their position in the principal component space and moves them closer to the primitive endoderm cluster, indicating that they likely represent primitive-endoderm derived cells ]. For all analyses except in


Breast cancer brain metastases (BCBM) continue to represent a challenge for clinicians as systemic treatment options remain limited and are associated with poor prognosis. Around 10–16% of patients with metastatic breast cancer will develop clinically significant central nervous system (CNS) involvement, including parenchymal or leptomeningeal disease (LMD) . The medThere are known differences in the incidence of brain metastases in specific breast cancer subtypes. Hormone receptor positive (HR+) is the most common breast cancer subtype ; howeverp < 0.001) and also led to a significant improvement in OS . Patients with CNS disease were also excluded from this trial [p = 0.000002]). Again, patients with breast cancer brain metastases were excluded from this study [The cyclin dependent 4 and 6 (CDK4/6) inhibitors, palbociclib, ribociclib and abemaciclib, have changed the treatment paradigm of HR+ advanced breast cancer. Abemaciclib was initially approved by the United States Food and Drug Administration in 2017 in combination with endocrine therapy and as monotherapy. MONARCH 1 was a phase 2 study in which 132 women with heavily pretreated HR+/HER2- metastatic breast cancer received abemaciclib monotherapy (200 mg every 12 hours). The objective response rate (ORR) was 19.7%, with a median progression-free survival (PFS) of 6 months, and median overall survival (OS) of 17.7 months. Patients with brain metastases were excluded from this study . MONARCHis trial . Finallyis study .There is growing interest in expanding the use of targeted therapies, such as CDK4/6 inhibitors to patients with BCBM. Drug delivery into the CNS is a common barrier for the use of systemic therapy in brain metastases. Abemaciclib has shown blood brain barrier (BBB) penetration in animal models  and in aA phase 2 trial was designed to evaluate the intracranial objective response rate (iORR) of patients with HR+ BCBM treated with abemaciclib . PatientOne question is would abemaciclib have activity among patients with BCBM who have previously been treated with a CDK 4/6 inhibitor? Patients with metastatic HR+ disease often develop CNS metastases later in their disease course , and CDKAnother question that remains is could there have been activity for abemaciclib for HR+/HER2+ BCBM if perhaps endocrine therapy and HER2-directed therapy with CNS activity had been utilized? Data from monarcHER suggested that among patients with HR+/HER2+ disease, adding endocrine therapy to abemaciclib and trastuzumab led to improved efficacy compared to chemotherapy and trastuzumab, whereas abemaciclib with trastuzumab without endocrine therapy performed very similarly to chemotherapy and trastuzumab . These fFinally, a remaining question is could utilization of abemaciclib in the early disease setting help prevent CNS metastases? Data looking at adjuvant abemaciclib with endocrine therapy in monarchE among patients with high risk early stage HR+/HER2- breast cancer demonstrated that with a median follow up of 19.1 months there was an absolute benefit of 3% in invasive disease-free survival favoring the abemaciclib arm . Longer In summary, there are preclinical and now clinical data showing CNS penetrance and clinical activity of abemaciclib in patients with BCBM. Further studies are needed to explore the safety and efficacy of abemaciclib with other targeted agents, particularly for patients with HR+/HER2+ BCBM.


Can increasing awareness about deprescribing prior to primary care visits reduce the use of potentially inappropriate long-term medications for individuals with cognitive impairment?In this pragmatic cluster randomized clinical trial of deprescribing education for 3012 older adults with cognitive impairment taking 5 or more medications and their primary care clinicians, patients from intervention clinics and control clinics were taking a similar mean number of medications (6.4 vs 6.5) at 6 months, and similar proportions of patients were taking 1 or more potentially inappropriate medications after 6 months.Educating patients and clinicians about deprescribing in primary care did not have an effect on the number of long-term medications or percentage of potentially inappropriate medications for older adults taking 5 or more long-term medications; findings suggest such interventions should target older adults taking relatively more medications. Individuals with dementia or mild cognitive impairment frequently have multiple chronic conditions  and take multiple medications, increasing their risk for adverse outcomes. Deprescribing  may decrease their risk of adverse outcomes.To examine the effectiveness of increasing patient and clinician awareness about the potential to deprescribe unnecessary or risky medications among patients with dementia or mild cognitive impairment.This pragmatic, patient-centered, 12-month cluster randomized clinical trial was conducted from April 1, 2019, to March 31, 2020, at 18 primary care clinics in a not-for-profit integrated health care delivery system. The study included 3012 adults aged 65 years or older with dementia or mild cognitive impairment who had 1 or more additional chronic medical conditions and were taking 5 or more long-term medications.An educational brochure and a questionnaire on attitudes toward deprescribing were mailed to patients prior to a primary care visit, clinicians were notified about the mailing, and deprescribing tip sheets were distributed to clinicians at monthly clinic meetings.The number of prescribed long-term medications and the percentage of individuals prescribed 1 or more potentially inappropriate medications (PIMs). Analysis was performed on an intention-to-treat basis.P = .14). The estimated percentages of patients in the intervention and control groups taking 1 or more PIMs were similar . In preplanned subgroup analyses, adjusted differences between the intervention and control groups were –0.16  for individuals prescribed 7 or more long-term medications at baseline (n = 1434) and –0.03  for those prescribed 5 to 6 medications (n = 1578) .This study comprised 1433 individuals  in 9 intervention clinics and 1579 individuals  in 9 control clinics who met the eligibility criteria. At baseline, both groups were prescribed a similar mean (SD) number of long-term medications (7.0 [2.1] in the intervention group and 7.0 [2.2] in the control group), and a similar proportion of individuals in both groups were taking 1 or more PIMs . At 6 months, the adjusted mean number of long-term medications was similar in the intervention and control groups (6.4  vs 6.5 ; This large-scale educational deprescribing intervention for older adults with cognitive impairment taking 5 or more long-term medications and their primary care clinicians demonstrated small effect sizes and did not significantly reduce the number of long-term medications and PIMs. Such interventions should target older adults taking relatively more medications.NCT03984396ClinicalTrials.gov Identifier:  This cluster randomized clinical trial examines the effectiveness of educating patients and clinicians about the potential to deprescribe unnecessary or risky medications among patients with dementia or mild cognitive impairment. The goal of the intervention was to increase patient and clinician awareness about the potential to deprescribe unnecessary or risky medications. We hypothesized that the intervention would reduce the number of long-term medications prescribed and the percentage of individuals taking 1 or more PIMs at 6 months’ follow-up.32 It was conducted as a pragmatic trial at Kaiser Permanente Colorado (KPCO), a not-for-profit integrated health care delivery system. This study was conducted in 18 KPCO adult primary care clinics (9 intervention clinics and 9 control clinics) in the Denver-Boulder geographic area. Clinics were randomized by the study biostatistician using a uniform distribution in blocks of 2 by clinic size to ensure comparable numbers of patients and clinicians. Clinic patient populations ranged from 170 to 1125. The study protocol has been published and was registered on ClinicalTrials.gov (NCT03984396)18  reporting guideline.The OPTIMIZE intervention was designed to be patient centered and easily incorporated into primary care practice.International Statistical Classification of Diseases and Related Health Problems, Tenth Revision–coded diagnosis of ADRD or MCI from any in-person encounter during the preceding year or from the medical record problem list  received the clinician component of the intervention.The intervention targeted adults aged 65 years or older receiving outpatient care from an established primary care clinician at 1 of the 18 KPCO primary care clinics. Eligibility criteria were health plan enrollment for at least 1 year prior to the intervention, an 35 These materials were intended to educate patients and family members about deprescribing because the education of patients and family members is found to be most effective when coupled with the activation achieved through meaningful questions around specific topics.38 Patients and family members were asked to complete the questionnaire and return it in an included stamped, self-addressed envelope.The intervention ran from April 1, 2019, to March 31, 2020. It had 2 components: (1) a patient and family component comprising materials mailed in advance of primary care visits and (2) a clinician component of monthly educational materials on deprescribing plus notification in the electronic health record schedule about upcoming visits with participating patients. For the patient intervention, eligible individuals were identified every weekday by study personnel using electronic health record data and visit schedules. Those with upcoming, in-person primary care visits were mailed a brochure (“Managing Medication”) providing information on deprescribing and a 9-question, validated, revised Patients’ Attitudes Toward Deprescribing questionnaire version for cognitive impairment.Because family members are often engaged in the care of patients with cognitive impairment, the cover letter encouraged patients to share materials with family members. If patients had a second visit more than 8 weeks after their first visit, they received another mailing. Individuals meeting eligibility criteria at control clinics were similarly identified and tracked for outcome measures but did not receive materials.32 Clinicians from control clinics did not receive any intervention materials. Usual care at KPCO includes routine medication reconciliation at primary care visits and electronic health record alerts for potentially high-risk prescribing . Patient and family materials and clinician intervention materials are available on the US Deprescribing Research Network website.39Clinicians at intervention clinics received a 3-part clinician intervention. First, an initial educational presentation was delivered by the KPCO principal investigator at a monthly clinician meeting. Second, 11 tip sheets and 1 poster on deprescribing topics were handed out (hard copy) to clinicians at monthly clinician meetings. Third, clinicians were notified with an appointment note in the electronic health record (placed by study staff) when they were scheduled to see a patient who had received the intervention brochure. The tip sheets were developed with clinician input, contained suggested language for deprescribing discussions, and addressed high-priority deprescribing topics and situations.40 plus opioid medications and were defined as an individual having a days’ supply overlapping the identified dates  excluding selected 2-digit Generic Product Identifier codes . We selected 6 months as a sufficient time to permit ongoing discussions between patients, family members, and clinicians about medication discontinuation while minimizing substantial changes in goals of care for most patients. Each individual’s index date was the first clinic appointment eligible for a mailed brochure between March 2019 and early March 2020. Six-month follow-up dates extended through September 2020. We estimated medication counts at 6 months using linear regression analysis of covariance models accounting for baseline medication count and a random clinic effect. Models also included self-reported race and ethnicity, which differed between groups, as well as age and sex. We examined normality of residuals to confirm adequate model fit. The percentage of individuals with 1 or more PIMs was modeled using logistic regression with the same specification.18 We estimated that we would be able to detect a decrease in medications of –0.70 or more  with 80% power based on differences of 2 Poisson rates in a cluster randomized design. By the fall of 2019, participant numbers were more than 30% lower than expected primarily owing to a high proportion of clinic appointments scheduled less than 7 days in advance. At that time, we recalculated power for change in medications, conservatively assuming an estimated number of 2590 with a mean number of 145 per clinic. The power remained comparable (–0.70 medications difference detectable) as can occur in cluster randomized trials in which power is driven by the number of clusters, which remained unchanged.42 Statistical analysis was conducted with SAS Studio software, release 3.7, enterprise edition (SAS Institute Inc). All P values were from 2-sided tests, and results were deemed statistically significant at P < .05.We initially projected a sample size of 3671, with mean numbers per clinic of approximately 204 and an intraclass correlation coefficient of 0.01.43Several prespecified additional analyses of the 2 primary outcomes were conducted. Sensitivity analyses examined whether effect estimates changed when restricting the cohorts to those with 90 days or more of follow-up . We also compared subgroups with 5 to 6 long-term medications vs 7 or more long-term medications at baseline, those with ADRD vs MCI, and those who received 1 brochure mailing vs 2 brochure mailings via intervention subgroup interactions. In analysis of covariance models of the medication outcome, an indicator for 7 or more medications would be highly correlated with the baseline medication counts included in the model. Therefore, comparisons of the interactions of 7 or more medications vs 5 to 6 medications by intervention vs delayed control were estimated using constrained repeated-measures models.Safety monitoring was conducted under guidance of a data safety monitoring board. Safety monitoring compared hospitalization and mortality rates between intervention and control clinics and manually adjudicated medical records for all mortality events and every third hospitalization preceded by a primary care encounter with a medication discontinuation.Of 11 722 individuals aged 65 years or older with cognitive impairment and 1 or more additional chronic conditions in the 18 primary care clinics, 3012 met all eligibility criteria . EligiblP = .14) (P = .08).At 6 months’ follow-up, the estimated mean number of long-term medications was 6.4  in the intervention group vs 6.5  in the control group  . The estP = .08), and the difference in the percentage of individuals taking 1 or more PIMs was –3.2 percentage points  (P = .08) (P = .28 for interaction) (P = .19 for interaction) , the adjusted estimated mean difference in prescribed long-term medications was –0.1  (P = .08) . In prepraction) . Among praction) . There wraction) .Safety monitoring did not reveal any differences in hospitalization or mortality rates between the intervention or control clinics for individuals in the full cohort during the 4 months after the index date . Record adjudication did not reveal any pattern of adverse drug withdrawal events associated with the intervention.This large cluster randomized pragmatic intervention of patient, family, and clinician education and activation about deprescribing conducted in 18 primary care clinics did not significantly decrease the number of long-term medications prescribed or the percentage of individuals taking 1 or more PIMs among older adults with cognitive impairment, multiple chronic conditions, and 5 or more long-term medications. There were also no significant interactions between prespecified subgroups. However, these analyses suggest that further investigations should explore whether individuals taking more long-term medications may have a greater potential to benefit from deprescribing. These results have implications for future large-scale pragmatic interventions to decrease the number of unnecessary or risky medications prescribed in vulnerable populations of older adults with cognitive impairment, multimorbidity, and polypharmacy.44 Polypharmacy is often defined as taking 5 or more long-term medications owing to associations with increased risk of medication-related harms above this threshold.47 Accordingly, many deprescribing trials in community populations (including OPTIMIZE) have used this definition to identify target populations.51 However, other investigations have proposed that individuals prescribed more than 5 medications are at greater risk for adverse effects and are more appropriate targets for deprescribing interventions.55 Educational deprescribing interventions, such as OPTIMIZE, may be more fruitful among older adult populations with dementia or cognitive impairment and higher levels of polypharmacy. Individuals taking more long-term medications may have more opportunities for medication refinement and may also be more likely to be prescribed PIMs that would be particularly appropriate for discontinuation. This was true for the OPTIMIZE trial, in which 23% of those taking 5 to 6 long-term medications and 38% of those taking 7 or more long-term medications at baseline had 1 or more PIMs. Deprescribing PIMs is potentially beneficial regardless of total medication burden.56Polypharmacy reflects the use of more medications than are medically necessary. Persons with dementia are more likely than noncognitively impaired persons to experience polypharmacy, even accounting for their morbidity burden.57 Given how many older adults experience adverse medication effects resulting in detrimental outcomes, such as admission to a skilled nursing facility, if found to be effective in other investigations, small effect sizes may be acceptable for pragmatic interventions efficiently and safely delivered at scale to targeted populations in primary care.59The effect sizes of the OPTIMIZE intervention in the full cohort as well as in prespecified subgroups were small. These effect sizes reflect not only the low-intensity intervention but also the potential for absolute risk reduction of the cohorts and the difference from usual care at KPCO. Other community deprescribing interventions have reported a wide range of effect sizes (0.01-0.95), reflecting a range of approaches and target populations.Our primary trial outcomes of number of long-term medications and percentage of individuals taking PIMs reflect the direct effect of deprescribing. Not all medication discontinuations will affect downstream clinical outcomes, such as potentially reduced falls, improved quality of life, or preserved cognition, which are multifactorial and may take longer to manifest. However, reductions in medication burden alone can still be considered a positive outcome of deprescribing if individuals are able to take fewer medications with no detriment to their clinical status.19 Future interventions should explore other approaches relevant to different practice settings. Identifying target populations in other settings using data sources relevant to those settings could, for example, assess dementia severity or target caregivers—information that was not available in OPTIMIZE. Although the intervention was delivered during the 12 months ending in March 2020, the follow-up period included the COVID-19 pandemic. Fewer opportunities for clinic visits and deprescribing discussions during the COVID-19 pandemic period in both the intervention and control clinics may have reduced the effect of the intervention on outcomes. However, any pandemic-related care changes likely biased results toward the null. Finally, the list of PIMs was selected for the OPTIMIZE study population and was selected from, and expanded on, the Beers list. We could only identify the medications that were prescribed and dispensed, which excluded over-the-counter medications and likely underascertained PIMs. Any list of PIMs is not a perfect proxy for appropriate medication use, which ultimately reflects clinical need.40This study had several limitations. Mailing intervention brochures to patients required that appointments be made at least 7 days in advance—an approach selected to avoid interfering with primary care clinic workflow. Although patients and care partners found paper mailings acceptable in the pilot study, this approach was relatively labor intensive and reached only slightly more than half of potentially eligible patients and family members .18,19 Fu11 To our knowledge, no published trials have focused on addressing polypharmacy or PIM use in populations with cognitive impairment and multiple chronic conditions in primary care settings.17 Finally, large-scale pragmatic interventions may promote subtle, long-lasting outcomes, such as creating a culture of thoughtful deprescribing as part of high-quality care. Focused, intensive deprescribing initiatives within systems already accustomed to deprescribing could produce additional effects in high-priority target populations.This study also has some strengths. A substantial strength of the intervention was the large-scale pragmatic trial design, adding to the generalizability and potential sustainability. All deprescribing decisions and activities were conducted by primary care clinicians and were not made through a deprescribing algorithm or involvement of research personnel. Clinicians were alerted when encountering patients who had been mailed the brochure so that the intervention cohort incorporated clinician activation along with the patient activation—an addition to previous interventions targeting clinicians in which education alone was unlikely to result in substantial deprescribing.This pragmatic cluster randomized clinical trial demonstrated that a large-scale patient and clinician education and activation deprescribing intervention can be delivered safely in primary care within an integrated delivery system. The intervention had small effect sizes that did not reach statistical significance; however, subgroup results suggest that similar interventions should be explored in populations with higher levels of polypharmacy. If found to be effective, such approaches could improve downstream clinical deprescribing outcomes and support medication management strategies focused on patient-centered treatment goals.