Can population-level genomic screening identify those at risk for disease?BRCA1 and BRCA2 variants were found in a higher proportion of patients than was previously reported.In this cross-sectional study of an unselected population cohort of 50 726 adults who underwent exome sequencing, pathogenic and likely pathogenic BRCA1/2 variant carriers may not be sufficient as a screening tool; population genomic screening for hereditary breast and ovarian cancer may better identify patients at high risk and provide an intervention opportunity to reduce mortality and morbidity.Current methods to identify  BRCA1 and BRCA2 (BRCA1/2) genes allows for cancer prevention and early diagnosis in high-risk individuals.Detection of disease-associated variants in the BRCA1/2 variants in an unselected research cohort, and to characterize the features associated with P/LP variants.To identify pathogenic and likely pathogenic (P/LP) BRCA1/2.This is a cross-sectional study of adult volunteers (n = 50 726) who underwent exome sequencing at a single health care system  from January 1, 2014, to March 1, 2016. Participants are part of the DiscovEHR cohort and were identified through the Geisinger MyCode Community Health Initiative. They consented to a research protocol that included sequencing and return of actionable test results. Clinical data from electronic health records and clinical visits were correlated with variants. Comparisons were made between those with (cases) and those without (controls) P/LP variants in BRCA1/2 variants in cohort, proportion of variant carriers not previously ascertained through clinical testing, and personal and family history of relevant cancers among BRCA1/2 variant carriers and noncarriers.Prevalence of P/LP BRCA1/2 variants and 267 (0.5%) were BRCA1/2 carriers. Of the 267 cases (148 [55.4%] were women and 119 [44.6%] were men with a mean [range] age of 58.9 [23-90] years), 183 (68.5%) received clinically confirmed results in their electronic health record. Among the 267 participants with P/LP BRCA1/2 variants, 219 (82.0%) had no prior clinical testing, 95 (35.6%) had BRCA1 variants, and 172 (64.4%) had BRCA2 variants. Syndromic cancer diagnoses were present in 11 (47.8%) of the 23 deceased BRCA1/2 carriers and in 56 (20.9%) of all 267 BRCA1/2 carriers. Among women, 31 (20.9%) of 148 variant carriers had a personal history of breast cancer, compared with 1554 (5.2%) of 29 880 noncarriers . Ovarian cancer history was present in 15 (10.1%) of 148 variant carriers and in 195 (0.6%) of 29 880 variant noncarriers . Among 89 BRCA1/2 carriers without prior testing but with comprehensive personal and family history data, 44 (49.4%) did not meet published guidelines for clinical testing.Of the 50 726 health system patients who underwent exome sequencing, 50 459 (99.5%) had no expected pathogenic BRCA1/2 variants. These findings suggest that genomic screening may identify BRCA1/2-associated cancer risk that might otherwise remain undetected within health care systems and may provide opportunities to reduce morbidity and mortality in patients.This study found that compared with previous clinical care, exome sequencing–based screening identified 5 times as many individuals with P/LP  BRCA1/2 variants among US adults enrolled in the MyCode Community Health Initiative.This cross-sectional study investigates the ascertainment of pathogenic and likely pathogenic  BRCA1 (OMIM 113705) and BRCA2 (OMIM 600185) genes were first associated with familial breast cancer risk in the mid-1990s.2 Associated risks for ovarian cancer, prostate cancer, pancreatic cancer, and melanoma have since been documented; these clinical associations are grouped as hereditary breast and ovarian cancer (HBOC) syndrome.3Pathogenic variants in the BRCA1 and BRCA2  gene became available in 1995.4 Risk-assessment strategies, based on personal and family history cancer thresholds, have been built, validated, and incorporated into clinical testing guidelines. These guidelines are routinely used to identify individuals with increased pretest probability of pathogenic BRCA1/2 variants.6 Current established guidelines have been issued by the National Comprehensive Cancer Network (NCCN),7 the US Preventive Services Task Force,8 and the American College of Medical Genetics and Genomics together with the National Society of Genetic Counselors.9Clinical testing for pathogenic variants in the BRCA1/2 variants may be associated with the failure to apply testing guideline criteria and the failure of criteria-based strategies to identify all true positives.11 Such underascertainment has been documented even in women with existing cancer diagnoses13 and has prompted calls for DNA sequence–based population screening.14Clinical underascertainment of individuals with pathogenic BRCA1/2 variants identified by exome sequencing. Sequencing-based screening findings were ascertained independent of participants’ age, sex, or cancer history.15 The cases and controls are part of the ongoing DiscovEHR cohort and were identified through the Geisinger MyCode Community Health Initiative. This large exome-sequenced research cohort has a median of 14 years of linked electronic health records (EHRs), and the patients consented to a research protocol that included sequencing and return of actionable test results.16 The genomic findings were confirmed by clinical laboratory testing and entered into the EHR.In this article, we report the pathogenic variants prevalence, HBOC syndrome–associated cancer history, and comparison of exome sequencing–based screening with indication-based ascertainment in a cross-sectional study of predominantly white (European ancestry) adults who underwent exome sequencing at a single US health care system  from January 1, 2014, to March 1, 2016. These individuals had “expected pathogenic”  STROBE) reporting guideline.This cross-sectional study received approval from Geisinger Institutional Review Board. Written informed consent was obtained from participants. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology  variant in either the BRCA1 or BRCA2 gene. We referred to these cases as BRCA1/2 carriers to be consistent with published literature. Controls are defined as the participants who underwent sequencing in the biobank  and screened negative for P/LP variants in both the BRCA1 and BRCA2 genes. We further subdivided the case population on the basis of (1) prior identification of a variant, (2) personal or family history that would have met established clinical criteria for referral or testing, and (3) gene involvement.BRCA1/2 carriers whose genetic risk was associated with personal history of relevant cancer at any age). The relevant cancers included breast, ovarian, prostate, pancreatic, and melanoma.For the purposes of this article, an HBOC syndrome diagnosis was assigned in cases in which both genotype and phenotype were present. Therefore, HBOC syndrome diagnosis refers to cases in which a relevant cancer occurred  classification in ClinVar with a *2 or *3 status, indicating strong evidence for pathogenicity18; (2) predicted loss of function; or (3) both , this application is not sufficient. Therefore, we employed a previously described diagnostic framework for incidental or secondary genomic findings. The framework’s 5 diagnostic groups include 3 groups with relevant cancer diagnoses  and in which the conventional diagnosis of HBOC is made as well as 2 groups in which relevant clinical findings are absent in the participants  encouraged to complete a targeted 3-generation family history (either in person or online). Each pedigree was independently analyzed by at least 2 licensed genetic counselors , who assessed threshold criteria for referral and testing according to the guidelines of the NCCN,7 US Preventive Services Task Force,8 and American College of Medical Genetics and Genomics–National Society of Genetic Counselors.9 Discordance between the reviewers was resolved by joint review and consensus. A total of 122 relevant 3-generation pedigrees were analyzed, of which 89 (72.9%) were from participants who had not undergone prior BRCA1/2 clinical testing  carriers  between  testing .Twenty participants died before their result became available, and 3 died after the return of result . Medical record review was carried out by 3 clinicians  independently and then jointly to determine the cancer diagnosis and cause of death  and 95% CIs. Two-sided P < .05 was considered statistically significant. All analyses were performed using SAS, version 9.4 (SAS Institute Inc).Data were summarized using means and ranges for continuous variables as well as frequencies and percentages for categorical variables. Comparisons between groups were performed using Wilcoxon rank sum test, Pearson χBRCA1/2 variants and 267 (0.5%) were BRCA1/2 carriers. Of the 267 cases (148 [55.4%] were women and 119 [44.6%] were men with a mean [range] age of 58.9 [23-90] years), 183 (68.5%) received clinically confirmed results in their EHR. Compared with the health system’s overall population, the biobank cohort was older , had a greater percentage of women , had a higher representation of white patients , and was enriched for relevant cancers .Of the 50 726 health system patients who underwent whole exome sequencing, 50 459 (99.5%) had no expected pathogenic BRCA1/2 variants was 1:190, and 1:180 when controlling for relatedness  had BRCA2 variants. There were 118 P/LP variants in BRCA1/2 in the 267 carriers  of the existing clinical significance designations in ClinVar; the single difference was 1 listed in ClinVar as variant of unknown significance that was designated as LP on the basis of updated information.The prevalence of participants with a Sanger sequencing–confirmed P/LP BRCA1 and 66 BRCA2 variants, 107 (90.67%) were putative loss-of-function variants  and only 5 (4.2%) were novel predicted loss-of-function variants. Eleven of 118 variants (9.3%) were missense, and no novel missense variants were classified as P/LP, in accordance with the criteria set forth by the American College of Medical Genetics and Genomics for variants interpretation.17Of the 52 BRCA1/2 carriers and ruled out 1 candidate carrier as a false-positive. The number of BRCA1/2 carriers with any given variant ranged from 1 to 24 (eTable 1 in the BRCA2 pathogenic variants (ClinVar variation ID: 38082 and 9320)22 accounted for 42 BRCA1/2 carriers, and 1 of 3 Ashkenazi Jewish founder mutations was found in 19 BRCA1/2 carriers.3Sanger sequencing confirmed 267 bioinformatically identified BRCA1/2 carriers were already aware of their result through prior clinical BRCA1/2 testing; these were patients who were diagnosed on the basis of traditional detection methods such as personal and family history. Newly diagnosed BRCA1/2 carriers  were more likely than controls (without BRCA1/2 variants) to have EHR evidence of personal or family history of HBOC syndrome–associated cancer diagnoses (28.0% vs 53.4%) , indicating significantly increased cancer risk associated with the BRCA1/2 carriers ascertained in this fashion.Forty-eight (17.9%) of the 267 s 53.4%) . New cars 53.4%) B-D. In 2BRCA1/2 carriers and in 56 (20.9%) of all 267 BRCA1/2 carriers. Among women, 31 (20.9%) of 148 variant carriers had a personal history of breast cancer, compared with 1554 (5.2%) of 29 880 noncarriers . Ovarian cancer history was present in 15 (10.1%) of 148 variant carriers and in 195 (0.6%) of 29 880 variant noncarriers .Syndromic cancer diagnoses were present in 11 (47.8%) of the 23 deceased BRCA1/2 carriers, results were returned to 183 eligible adults (68.5%) and their health care practitioner through a secure EHR portal; this was followed up by a letter and telephone call to the participants. Clinical care based on NCCN guidelines7 was offered, and 3 examples of early cancer detection were described in a report.21 A case of a woman in her 40s with subclinical disease detected on screening was identified after that report  of 78 BRCA2 carriers met the NCCN criteria. In nearly every category, women were more likely than men to meet criteria thresholds  of all cases .The participants’ final diagnostic category was based on all relevant clinical information reviewed, including the EHR and participant self-report when available . The HBOBRCA1/2-associated cancer in 9 people (39.1%), unrelated to BRCA1/2 in 11 (47.8%), and indeterminate in 3 (13.0%). In contrast to the deceased cases, 45 (18.4%) of 244 living patients have a personal history of a relevant cancer (eTables 3 and 4 in the Twenty-three (8.6%) of 267 participants were deceased by June 1, 2017, and the median age at death was 64.7 years  met current NCCN criteria for testing but had not received testing; conversely, 44 individuals (49.4%) did not meet NCCN testing criteria but had expected pathogenic variants. Of importance, those patients who did not meet the established thresholds for BRCA1/2 testing were not spared from relevant cancers, as evidenced by the diagnosis of early asymptomatic cancer in such individuals after the initiation of cancer risk management.21Findings demonstrate that routine exome sequencing–based screening may be a potential factor in long-term care improvement. This study found that only 17.9% of BRCA1/2 testing strategies to individuals with high pretest probabilities and the failure of such criteria-based strategies to be sufficiently sensitive to identify all true positives. The practical difficulties of incorporating sufficient family history risk analysis in routine health care settings are well described.24 The observed gap in relevant cancer history between all living BRCA1/2 carriers and deceased BRCA1/2 carriers signals an opportunity to prospectively identify and manage individuals with elevated risk to reduce the morbidity and mortality associated with their defined cancer risk. Early success in this area is demonstrated by the identification of 4 individuals with early-stage cancer during the screening and management recommended by the exome sequencing–based screening results process.Our evidence demonstrates that this clinical underascertainment may be associated with both the failure to apply criteria-based referral for 26 Underascertainment of these conditions, in which presymptomatic case identification is advantageous,25 should prompt prioritization of further research into genomic sequencing–based population screening.27Our findings show considerable clinical underascertainment of individuals with HBOC syndrome. Underascertainment has also been observed in familial hypercholesterolemia and Lynch syndrome, 2 conditions that  are designated as tier 1  by the Centers for Disease Control and Prevention Office of Public Health Genomics and are included in the Healthy People 2020 recommendations.BRCA1/2 expected pathogenic variants of 1:190, adjusted to 1:180 when controlling for relatedness up to the third degree.25 Consistent with other studies, HBOC syndrome–associated cancer rates were higher in women than in men and in BRCA1 carriers than in BRCA2 carriers.28 Unsurprisingly, those with prior clinical BRCA1/2 testing also had higher cancer rates than those without prior testing. For the subset of 23 deceased BRCA1/2 carriers, only 4 had prior clinical testing and the percentage with syndromic cancer diagnoses was 47.8%, which is similar to published observations of clinically ascertained cases.3Overall, ascertainment through sequencing-based screening in the biobank cohort showed an empirical prevalence for BRCA1 to BRCA2 case ratio of 1.0:1.8 is consistent with ratios in other relevant work.31 Previous research3 shows decreased penetrance for BRCA2 carriers, which may be a contributing factor in the underascertainment of these cases compared with BRCA1 cases; in our study, among those with prior testing and cancer diagnosis, more than 90% had BRCA1-associated cancer risk.The observed BRCA1/2 carriers who did not have prior clinical BRCA1/2 testing, the return of genomic result was the start of a diagnostic process aimed at deciding if the risk variants were associated with current or past disease.20 For those with a personal history of BRCA1/2-associated cancer, a diagnosis of HBOC syndrome was now clear and the recommended management was changed to align with NCCN guidelines and to offer cascade testing for at-risk family members. Those without BRCA1/2-associated cancer at results disclosure did not have a diagnosis of HBOC syndrome, and long-term risk-reduction measures were recommended. For individuals with risk variants but without disease, the variants result was listed in their EHR problem list as a test result and not as an HBOC syndrome diagnosis.32 This distinction is important because continued surveillance is recommended, but syndrome diagnosis may not ever be achieved.32Among the 82.1% of 32 Consistent with the vision of learning health care systems,35 this disclosure and follow-up approach is being informed and improved by ongoing feedback from biobank participants, health care practitioners, and researchers. In addition, the disclosure can be used as a template for other settings where the genome-first care model is implemented.Substantial infrastructure and health care practitioner collaboration were necessary to support the disclosure and follow-up care of the 183 participants whose biobank consent form allowed for clinical reporting.BRCA1/2-positive individuals,7 but patients are informed that important knowledge gaps exist. The capture and aggregation of health outcomes data from this and other cohorts is essential to developing evidence that guides precision management strategies for individuals identified through whole exome sequencing–based screening.36 These health outcomes data, coupled with analysis of the cost of care , will ultimately determine the value of exome sequencing–based screening and inform the implementation of screening strategies for the BRCA1/2 variants.Finally, optimal clinical management of patients identified through sequencing-based screening who do not otherwise meet the criteria for clinical testing is likely to evolve as more longitudinal outcomes data become available from this cohort and others. Given a near-term gap in evidence-based management, patients at Geisinger receive management based on previously developed protocols for BRCA1/2 variants as the control population, which mitigated this bias.In general, the participants in the biobank were older than the health system’s patient population; more important, this study skewed toward those who received regular health care, increasing their opportunity to be enrolled in the biobank research. The higher incidence of breast cancer in the biobank population was likely a reflection of frequency of care and age; however, in our risk analysis, we used the biobank patients without evidence of 3; (2) absence of attempts to assign significance to missense variants beyond that established in ClinVar, and (3) estimated underrepresentation of cases with early-onset disease in the study cohort as a result of HBOC syndrome–associated mortality, given the age difference between the BRCA1/2 cohort and the general health system population.37 The first 2 issues will be addressed in future work. We recognize that, because this study population is reflective of the local population and thus is overwhelmingly white,16 it may not be representative of other cohorts, particularly those from different racial/ethnic backgrounds. As with many areas of genomic medicine implementation, inclusion of greater numbers of individuals from underrepresented race/ethnicity is essential to a complete understanding of BRCA1/2 risk.38 Data from other cohorts will be needed to clarify whether this BRCA1 to BRCA2 ratio finding is generalizable for population screening.The study still likely underestimated prevalence in this health care system–based cohort because of the (1) limitations to the current variant identification strategy BRC1/2 variants in the general population may be substantially higher than was previously estimated, and reliance on personal and family history may be an inadequate measure to ascertain risk for BRCA1/2 variants.The prevalence of


Following the publication of  concernsFig 3D β-actin panel appears similar to Fig 5C β-actin panelThe authors have explained that an error was made during figure preparation. The authors accidentally used the duplicated micrograph image for the Fulvestrant-treated DU145 cells at 0h to represent the ETOH-treated cells at 0h. The authors have provided a replacement image for The lysate samples of the control and Fulvestrant-treated cells used in Western blot analysis were the same for generation of data presented in both Figs Please see the corrected S1 Dataset(ZIP)Click here for additional data file.


IGF1R variants in patients with SSC as compared to controls. Building upon this result, we used expression array data from calvarial osteoblasts isolated from infants with and without SSC to ascertain correlations between high IGF1 expression and expression of other osteogenic genes of interest. We identified a positive correlation between increased expression of IGF1 and RUNX2, a gene known to cause SSC with increased gene dosage. Subsequent phosphorylation assays revealed that osteoblast cell lines from cases with high IGF1 expression demonstrated inhibition of GSK3β, a serine/threonine kinase known to inhibit RUNX2, thus activating osteogenesis through the IRS1-mediated Akt pathway. With these findings, we have utilized established mouse strains to examine a novel model of polygenic inheritance (a phenotype influenced by more than one gene) of SSC. Compound heterozygous mice with selective disinhibition of RUNX2 and either overexpression of IGF1 or loss of function of GSK3β demonstrated an increase in the frequency and severity of synostosis as compared to mice with the RUNX2 disinhibition alone. These polygenic mouse models reinforce, in-vivo, that the combination of activation of the IGF1 pathway and disinhibition of the RUNX2 pathway leads to an increased risk of developing craniosynostosis and serves as a model of human SSC.Craniosynostosis is the premature fusion of the sutures of the calvaria and is principally designated as being either syndromic  or non-syndromic. While many forms of syndromic craniosynostosis are known to be caused by specific mutations, the genetic etiology of non-syndromic, single-suture craniosynostosis (SSC) is poorly understood. Based on the low recurrence rate (4–7%) and the fact that recurrent mutations have not been identified for most cases of SSC, we propose that some cases of isolated, single suture craniosynostosis may be polygenic. Previous work in our lab identified a disproportionately high number of rare and novel gain-of-function  Single-suture, non-syndromic craniosynostosis occurs in approximately 1/2500 live births with a familial recurrence rate of 4–7% [FGFR3 [TWIST1 [EFNB1 [TCF12 [A limited number of single gene mutations are known to cause single-suture craniosynostosis (SSC) , but th1 [TCF12 . These gIGF1R that were overrepresented in an SSC case population [COL1A2, ALP, and RUNX2, and matrix mineralization [Several genes have emerged as potential polygenic candidates through our RNA microarray and expression analysis, genomic sequencing, phosphorylation studies, and biomechanical assays. We first performed targeted candidate gene sequencing on 186 patients with SSC and identified rare sequence variants in pulation . Structupulation , 10, whilization –14. TakeIGF1R variants [Concurrently, our lab used correlation analysis of osteoblast transcriptomes from 199 human cases (including the 186 from the targeted sequencing project) and 50 controls to detect distinct patterns of gene expression that were associated with craniosynostosis , 16. Twovariants , demonstvariants . This acvariants , osteoblvariants , and chovariants .IGF1 expression. These experiments revealed increased contractility and decreased migration in cells with elevated levels of IGF1, supporting our previous results suggesting that enhanced differentiation is correlated with this subset of SSC cases [IGF1 expression, and gain-of-function mutations in IGF1R made the IGF1/IGF1R pathway an obvious choice for further examination.Subsequent biomechanical assays measuring contractility and migration were performed on primary SSC osteoblasts with and without elevated SC cases . The ideRUNX2 in two cases with familial metopic SSC and hypodontia [TWIST1 cause Saethre-Chotzen syndrome, a hereditary form of craniosynostosis [Using the same patient cohort, we performed copy number variant analysis and identified a duplication of podontia . This obpodontia . In addipodontia , 27, RUNpodontia , 29. Mutnostosis , 31, hownostosis .Twist1/Igf1 and Twist1/Gsk3β compound heterozygotes to test hypotheses generated from our previous human expression data. These mouse models demonstrate an increased frequency and severity of craniosynostosis and support the hypothesis that combined dysregulation of the TWIST1 and IGF1 pathways could contribute to an increase risk in humans.In this manuscript we present data from mouse cross-breeding experiments designed to explore possible polygenic inheritance of craniosynostosis using This study was reviewed and approved by the Institutional Review Board of Seattle Children’s Hospital . Written informed consent from parents or guardians of children with SSC was obtained and a consent waiver was obtained for the use of anonymous control samples. See reference [Post-surgical calvarial bone samples which would have otherwise been discarded were collected from 211 cases and 50 controls between the years of 2002 and 2006. Computed Tomography confirmed the diagnosis of isolated sagittal, metopic, or unilateral coronal synostosis in each case. All cases were screened for the presence of major medical conditions such as syndromic craniosynostosis, cardiac defects, seizure disorders, cerebral palsy, health conditions requiring surgical intervention, presence of three or more minor extra-cranial malformations; or presence of any other major malformations. No individuals with these conditions were enrolled. Control samples were obtained from patients undergoing a craniotomy for reasons other than craniosynostosis  or autopsies. Control samples were not obtained from individuals with disorders that affect bone such as skeletal dysplasias. Tissue samples were collected in Waymouth’s Media (WM)  and sent to the lab for processing.2, and 99% humidity. Upon confluence, cells were washed with PBS, trypsinized with 0.05% Trypsin-EDTA , and passaged into T75 flasks. Confluent cells were cryogenically stored in freezing media containing 90% FBS and 10% dimethyl sulfoxide in a liquid nitrogen freezer. As previously descried, alkaline phosphatase activity was used to assure osteoblast lineage and all samples demonstrated high ALP activity [Calvarial tissue was rinsed in WM and the surrounding soft tissue was removed. Using a sterile scalpel blade, the tissue was cut into 1-2mm pieces and 2 pieces per well were cultured in 12-well plates at 37°C, 5% COactivity .2. At 75% confluence, cells were trypsinized, washed in cold 1X PBS, and RNA was isolated using the Roche High Pure miRNA Isolation Kit  in accordance with the manufacturer’s instructions. Initial thawing of the cells for this process was done in batches, and batch effect was considered during expression analysis. RNA integrity was assessed using the Agilent 2100 Bioanalyzer and only samples with a RIN score of >8.0 were used for further analysis.Cell lines were cultured to sub-confluence in T25 flasks and passaged to a density of 175,000 cells per 25cmcDNA microarray assays were performed on 261 calvarial tissue samples (211 cases and 50 controls) using Affymetrix Human Gene 1.0 ST array technology. See full methods in reference .a priori based on their role in bone development and osteoblast differentiation. Transcript expression levels were assessed visually by applying conditional formatting to expression level values for each gene and analyzed by paired t-test.All microarray data are MIAME compliant and the raw dataset has been deposited in the MIAME compliant Gene Expression Omnibus (GEO) database under accession number GSE27976 . For theIgf1(+/tg)/Igf1(tg/tg) (transgenic overexpression of Igf1), Gsk3β(+/-) (hemizygous knockout of Gsk3β), and Twist1(+/-) (loss-of-function point mutation in Twist1 “twist-box” domain) from Jackson Laboratories, Bar Harbor, ME were bred to congenicity on a C57BL/6J background with a minimum of 9 backcross generations (≥99.5% identity). See official Jackson Laboratory nomenclature (Gsk3β(+/-) and Igf1(+/tg), and by Sanger sequencing for Twist1(+/-). Primers are listed in Twist1 and Gsk3β mice are known to be embryonic lethal and thus were maintained as heterozygotes [Gsk3β(+/-) mice have been shown to have significantly lower Gsk3β expression than their wildtype counterparts [Igf1 mice were maintained as both homozygotes and heterozygotes. Mouse IGF1 ELISA Kit  was used to validate overexpression of IGF1 in the serum of IGF1 transgenic animals . mice. Litters of interest were sacrificed at P28 by CO2 euthanasia and prepared for microCT scanning.All mouse work was approved by Seattle Children’s Institutional Animal Care and Use Committee and complies with NIH Guidelines for the Care and Use of Laboratory Animals. Commercially available mouse lines nclature . Compounnclature . The genozygotes , 35. Gskterparts  Igf1 micu, et al ). RandomA Skyscan 1076 micro Computed Tomography (microCT) scanner was used to render high-quality 3-dimensional CT images of skulls from P28 mice with our genotypes of interest. Scans were performed at a maximum of 35um slice thickness and with the following parameters; 55Kv, 179uA, 0.5mm aluminum filter, 360 ms exposure, rotation step of 0.7°, 180° scan, and 3 frame averaging. CT scans were reconstructed using NRecon software  and rendered in Drishti Volume Exploration and Presentation Tool   and Twist1(+/-) to Twist1(+/-)/Igf1(+/tg) and an n of 18 for the comparison of Gsk3β(+/-) and Twist1(+/-) to Twist1(+/-)/Gsk3β(+/-).Statistical analysis was performed using a chi-squared test for comparison of proportions. Percent of expected affected for each compound heterozygous genotype was calculated by the sum of observed affected in each component genotype. Proportions were then compared using an n of 17 for the comparison of RUNX2, a transcription factor critical for osteogenesis, was correlated with IGF1 expression and was significantly different between High IGF1 cases and unaffected controls  as well as affected controls .Our initial data review identified a striking association between IGF1 expression and many of the 399 candidate genes chosen a priori due to their role in osteogenesis and bone development. Comparison of the expression levels of our candidate genes in the 23 cases with the highest levels of IGF1 expression (High IGF1 Cases) and both the 50 unaffected controls  and the Gsk3β(+/-), Twist1(+/-), Igf1(+/tg), Igf1(tg/tg), and with the following compound- heterozygous variant genotypes; Gsk3β(+/-)/Twist1(+/-), Twist1(+/-)/Igf1(+/tg), and Gsk3β(+/-)/Igf1(+/tg) (Igf1(+/tg), Igf1(tg/tg), or wildtype mice. Fifteen percent of Gsk3β(+/-) mice had suture fusion, one of which had increased biparietal diameter suggestive of hydrocephalus. One Gsk3β(+/-) mouse without suture fusion also had increased biparietal diameter. Based on the suture morphology we suspect that this mouse had bilateral coronal suture fusion that was disrupted due to pressure from brain growth and concomitant hydrocephalus. This animal was classified as not having craniosynostosis (Twist1(+/-) mice had suture fusion.We analyzed 3D-rendered CT images of congenic, postnatal day 28 (P28) C57BL/6J mice with the following single-variant genotypes; f1(+/tg) , S1 Fig.Twist1(+/-) and 15% in Gsk3β(+/-) to 94% in Twist1(+/-)/Gsk3β(+/-), a significance of p = 0.0038 (accounting for the additive effect of these disease-causing genotypes). Of note, inclusion of the mouse with suspected craniosynostosis in our analysis did not change the interpretation of our results (p = 0.0121). There was no significant increase in the severity of craniosynostosis when comparing these groups. The incidence of fusion was not significantly increased in Twist1(+/-)/Igf1(+/tg) as compared to Twist1(+/-) mice, but the severity  increased from 6% of Twist1(+/-) to 29% of Twist1(+/-)/Igf1(+/tg) (p = 0.0766) /Igf1(+/tg) genotype than predicted by Hardy-Weinberg equilibrium (~10% as compared to the expected 25%), suggesting the possibility of prenatal lethality of this genotype. While none of the Gsk3β(+/-)/Igf1(+/tg) mice that survived to P28 showed suture fusion, the reduced number of offspring with this genotype precludes our ability to define the true craniosynostosis rate.We observed a disproportionally lower number of mice with the Igf1 and Gsk3β and disinhibition of Runx2, that result in a significantly increased rate or severity of craniosynostosis when compared to mice with each genotype in isolation.While there have been great advances in our understanding of the molecular genetic causes of syndromic craniosynostosis, the etiology of most cases of the more common single suture synostoses remains unknown. Its low recurrence rate suggests multifactorial, polygenic, or oligogenic inheritance. In this study, we describe compound heterozygous mutant mice with genetically altered expression of IGF1 and RUNX2 expression in a subgroup of osteoblasts derived from clinical cases of SSC. The IGF1/IGF1R pathway is critical for normal bone growth and density [Igf1(+/tg),Twist1(+/-), and Gsk3β(+/-) converge to reduce inhibition and increase the transcriptional activity of Runx2. The mouse models selected for our work mimicked activation of the Akt pathway and selective disinhibition of RUNX2 to determine the additive effects of pathway dysregulation  mouse is known to harbor a point mutation in the twist-box resulting in selective disinhibition of RUNX2 leading to coronal synostosis of variable penetrance and expressivity. This was validated in our study demonstrating that 35% of Twist1(+/-) mice developed coronal synostosis.The Igf1(+/tg) or Igf1(tg/tg) transgenic mice developed craniosynostosis, 23% of the Gsk3β(+/-) mice displayed craniosynostosis and/or presumed hydrocephalus, a previously undescribed phenotype with incomplete penetrance. With this unexpected result, we reviewed recent sequence data from our cohort and identified a missense mutation (c.T185C:p.I62T) in a single case of sagittal synostosis. This variant has not been identified in more than 250,000 alleles sequenced [Twist1(+/-)/Gsk3β(+/-) compound heterozygous mice. While is is known that C57B/6J mice naturally present with an “interfrontal bone” [While none of the equenced , 46, resal bone”  and thusTwist1(+/-)/Gsk3β(+/-) had a significantly higher frequency of craniosynostosis than predicted by the additive effects of each mutation alone (increasing from 35% in Twist1(+/-) and 15% in Gsk3β(+/-) to 94% in Twist1(+/-)/Gsk3β(+/-) compound heterozygotes). Furthermore, there was a near significant increase in the rate of bilateral coronal synostosis in Twist1(+/-)/Igf1(+/tg) compound heterozygotes when compared to the rate in the parental strains.Compound heterozygous These data suggest that a functional interaction between the TWIST1/RUNX2 and IGF1 pathways is integral to normal calvarial development. We have provided data supporting that combined dysregulation of these pathways result in the first example of polygenic inheritance of craniosynostosis in an animal model. While our data suggest that dysregulation of the TWIST1/RUNX2 and IGF1/Akt pathways may predispose humans to craniosynostosis, further translational research is necessary before the development of clinical utility.S1 Fignot having craniosynostosis. (**) Mouse died at P23 with potential hydrocephalus based on increased bi-parietal diameter. Postnatal age at the time of sacrifice is noted if other than P28.The phenotype of 91 mice was classified in this study. Each skull image is designated by genotype and pattern of suture fusion (U) Unilateral craniosynostosis, (Ub) Unilateral craniosynostosis with “bridging” phenotype, (B) Bilateral craniosynostosis, or (Bb) Bilateral craniosynostosis with “bridging” phenotype. (*) Irregular and wide suture margins and wide biparietal diameter of this mouse suggests the possibility bilateral coronal suture fusion that was disrupted due to concomitant hydrocephalus. This animal was classified as (PDF)Click here for additional data file.S1 TableIGF1 expression and 50 unaffected controls. (¥) indicates significant difference in expression levels between 23 cases with the highest levels of IGF1 expression and 188 affected control cases (e.g. cases with craniosynostosis with low IGF1 expression). (↑) indicates a the gene is positively correlated with IGF1 and (↓) indicates that a gene is inversely correlated with IGF1 for the group(s) it is significant in.Alpha list of 399 genes of interest chosen a priori for their role in osteoblast development and bone biology. (§) indicates significant difference in expression levels between 23 cases with the highest levels of (PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 TableIgf1 and Gsk3β sourced from Jackson Laboratories genotyping protocols (jax.org). Primers spanning Twist1 Ser192Pro designed in-house.Primer sequences for (PDF)Click here for additional data file.


Conserv Physiol 3(1): cov025; doi: 10.1093/conphys/cov025The original version of this manuscript was published with an incorrect figure in the place of figure 2. The manuscript has now been corrected.


The caption of Fig. Table S1 in the Additional file of this original publication contained some errors. The updated Table S1 is published in this correction article.Additional file 1:Updated Table S1, the full supplementary materials can be downloaded from the original publication. (XLSX 10 kb)


Fusarium head blight caused by Fusarium graminearum is an important disease of wheat and barley. Previous studies have showed that all three MAP kinase genes, MGV1, FgHOG1, and GPMK1, are involved in regulating hyphal growth, sexual reproduction, plant infection, and stress responses in this pathogen. To determine the relationship between the Mgv1 and FgHog1 pathways, in this study, we generated and characterized the mgv1 Fghog1 double mutant. Deletion of FgHOG1 partially rescued the defects of the mgv1 mutant in vegetative growth and cell wall integrity but had no effects on its defects in plant infection and DON production. The mgv1 Fghog1 mutant grew faster and was more tolerant to cell wall stressors than the mgv1 mutant. Swollen compartments and cell burst were observed frequently in the mgv1 mutant but rarely in the mgv1 Fghog1 mutant when treated with fungicide fludioxonil or cell wall stressor Congo red. Conversely, the deletion of MGV1 also alleviated the hyperosmotic sensitivity of the Fghog1 mutant in vegetative growth. TGY assays indicated increased phosphorylation of FgHog1 in the mgv1 mutant, and TEY assays further revealed elevated activation of Gpmk1 in the mgv1 Fghog1 double mutant, particularly under cell wall stress conditions. Overall, our data showed that deletion of FgHOG1 partially suppressed the defects of the mgv1 mutant, possibly by affecting genes related to cell wall integrity and osmoregulation via the over-activation of Gpmk1 MAP kinase and avoiding intracellular turgor elevation. Fusarium head blight, a devastating disease of wheat and barley, is caused by the filamentous ascomycete Fusarium graminearum. In addition to severe yield losses, infested grains are often contaminated with mycotoxins, including deoxynivalenol (DON) and zearalenone  kinase cascades that are involved in regulating various developmental and infection processes in fungal pathogens  gene characterized in F. graminearum, is orthologous to the cell wall integrity (CWI) MAPK SLT2 in the budding yeast and MPS1 in Magnaporthe oryzae  and FgHOG1, also have been functionally characterized in F. graminearum  pathway and M. oryzae Osm1  significantly reduced the phosphorylation level of FgHog1  was generated and transformed into the mgv1 mutant. Transformants resistant to both hygromycin B and geneticin were isolated and screened by PCR for the deletion of FgHOG1  cultures were treated with a mixture of cell wall lytic enzymes for 30 min. In the mgv1 mutant, abundant protoplasts were produced, while germlings from the wild type strain PH-1 released few protoplasts , a compound interfering with the fungal cell wall  of the mgv1 mutant  is a commonly used stain to visualize the fungal cell wall. When germlings harvested from 12 h YEPD cultures were stained with 50 μg/ml CFW, fluorescence signals were evenly distributed along the cell wall and septa in the wild type and 1 mutant . Howeverartments . Weaker 1 mutant . These dmgv1 mutant was lower than 1%, while that of the mgv1 Fghog1 double mutant was increased to 40%  of CFW to treat the conidia, we found that all conidia of both the mgv1 and mgv1 Fghog1 mutants were unable to germinate and had empty conidium compartments  or became empty  in the wild type . In the me empty . The pre mutants . After ierminate . These rr turgor , the incmgv1Fghog1 mutant is more tolerant to hyperosmotic stress than the Fghog1 mutant in vegetative growth but not in conidial germination:The  Because FgHog1 MAPK is involved in regulating the response to hyperosmotic stress, we then assayed the effects of 0.2 M NaCl on the growth of the mgv1 and mgv1 Fghog1 mutants. As shown in Fghog1 and mgv1 Fghog1 mutants were significantly inhibited in vegetative growth on a CM plate with 0.2 M NaCl. Interestingly, the mgv1 Fghog1 double mutant formed a bigger colony than the Fghog1 mutant, which was contrary to the situation on a regular CM plate  and Fghog1 (94.6%)  . However1 mutant . These dFghog1 mutant . In yeast, a number of genes important for cell wall synthesis and assembly are affected by deletion of SLT2  also has higher resistance to fludioxonil than the mpk1 mutant  significantly inhibits the activation of FgHog1  in F. oxysporum   or CM   cultures and conidial germination in YEPD  were assayed as described  . Growth  pH 6.5) . Conidiaescribed . For promgv1 and Fghog1 single mutants were generated in previous studies  and incubated for 6 or 12 h at 25°C. In order to check the chitin distribution, germlings harvested from 12 h YEPD cultures were stained with 50 μg/ml CFW for 5 min. Germlings harvested from 18 h YEPD cultures grown at 25°C were shifted to 32°C and incubated for another 6 h to assay the effect of elevated temperature. For CWI assays, germlings harvested from 12 h YEPD cultures were digested with the cell wall lytic solution  at 30°C for 30 min before examination for protoplasts  to estimate the disease index. For each strain, a DON production assay in TBI cultures was performed as described (For plant infection assays, conidia harvested from 5-day-old CMC cultures by filtration through Miracloth were resuspended to a concentration of 1.0 × 10escribed . There wescribed , with a TM software. Quantitative changes in the phosphorylation levels of Gpmk1, Mgv1, and FgHog1 were analyzed with the Image LabTM software. Each experiment was repeated four times, independently.Total proteins were isolated from hyphae harvested from 18 h YEPD cultures as described . For assGW, CJ, and J-RX conceived and designed the experiments. JR, CL, and CG performed the experiments. GW and CJ analyzed the data. GW wrote the manuscript. J-RX and CJ improved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Centroscyllium fabricii. The data was acquired by metabarcoding using 16S rDNA. Centroscyllium fabricii, a deep sea shark found at depths below 275 m was sampled during Sagar Sampada cruise no 305 in the Indian Ocean and metagenomic DNA was isolated from the gut contents using QIAamp DNA stool minikit. V3 region of 16S rDNA region was amplified and the amplicons were sequenced on Illumina MiSeq system using 151 bp × 2 paired end reads. The data of this metagenome is available in the BioSample Submission Portal as Bio-Project PRJNA431407and Sequence Read Archive (SRA) accession number SRR6507004.This data article describes the bacterial diversity of the deep sea shark,  Specifications tableValue of the data•This is the first data report on bacterial diversity of deep sea fish gut from the Oxygen Minimum zone of Indian Ocean.•Centroscyllium fabricii gut can be regarded as a first step towards recognition of its bioactive potential.Generation of an inventory of bacterial diversity of •The significant proportion of hitherto unstudied bacterial phyla (25.30%) identified in the dataset indicates immense scope for Blue Biotechnology.•The data provided here can also be used to understand host-microbiome interactions and to shed light on the factors that influence the establishment of gut microbiota.1Centroscyllium fabricii are deep water schooling sharks found at depths ranging from 180–2250 m With 33,700 species described to date Actinobacteria (27.84%), Proteobacteria (18.99%) and Acidobacteria (10.89%). 25.30% of Operational Taxonomic Units (OTUs) did not have any significant hits against the taxonomic database and was categorized as unknown. 107 genera were identified in the dataset, but the abundance of each was found to be less than 1%. Acenitobacter (0.46%) was predominant at the genus level of taxonomic resolution. A vast majority of OTUs (92.4%) remained unclassified at this level. The top 10 genera including unknown are depicted in DNA metabarcoding centered on the 16S rDNA sequence is a high-throughput approach used to catalog taxonomic diversity of environmental samples. The data presented here was obtained by Illumina MiSeq sequencing of V3 region of 16SrDNA. A total of 23 bacterial phyla were identified in the dataset, as shown in 22.1Centroscyllium fabricii was sampled onboard the Fishery Oceanographic Research Vessel (FORV) SagarSampada cruise #305 in the Indian Ocean by using HSDT  net (8° 11.4” N and 75° 54.9” E). The fish was subjected to molecular identification by DNA isolation and PCR amplification of 5’ region of cytochrome c oxidase subunit I (cox1) gene from mitochondrial DNA using universal fish primers F2 and R2 KT905423.1).The deep sea shark, 2.2Metagenomic DNA was isolated from the gut contents using QIAamp DNA stool minikit  and the hypervariable V3 region of 16S rRNA gene was amplified using primers 341F 5’CCTACGGGAGGCAGCAG 3’ and 518R 5’ATTACCGCGGCTGCTGG 3’. The amplicons were sequenced on Illumina MiSeq system using 151 bp × 2 paired end reads. The raw sequence files were analyzed for base quality, base composition and GC content. V3 region was extracted from paired end reads by trimming of spacer and conserved region and by building a consensus V3 region from the trimmed paired end reads. High quality V3 sequences were extracted by passing the reads through filters for spacer region, conserved region, read quality and mismatch. Subsequent analyses were performed using Quantitative Insights Into Microbial Ecology (QIIME) pipeline.


ARID1A (BAF250A), SMARCA2 (BRM), SMARCA4 (BRG1), and SMARCB1 (INI1). SMARCA1 (SNF2L) is another member of the chromatin remodelers, and has not yet been studied in neoplasia. As SMARCA1 is located on chromosome X, could be potentially inactivated by a single hit. We aimed to evaluate GAs and protein expression of SMARCA1 in soft tissue tumors.Vital cellular processes such as proliferation and differentiation are regulated by chromatin remodeling complexes. A variety of neoplasms have been discovered to have genomic alterations (GAs) and loss of immunohistochemical (IHC) expression of chromatin remodelers SMARCA1 GAs. Our institutional archives were queried to collect 26 cases of soft tissue tumors including 10 undifferentiated sarcomas, 5 leiomyosarcomas, 6 liposarcomas, and 5 malignant peripheral sheath tumors (MPNST). IHC for SMARCA1 with an SNF 2C4 monoclonal antibody was performed on whole tissue sections.The publically available cBioPortal.32e34 platform was queried to analyze data on soft tissue tumors from The Cancer Genome Atlas project (TCGA) related to SMARCA1 GAs were present in 8/261 soft tissue sarcomas (3%) in the TCGA dataset. Leiomyosarcomas had most common SMARCA1 GAs in 6/99 cases. SMARCA1 deletions existed in 1/56 dedifferentiated liposarcomas and 1/48 undifferentiated sarcomas. No SMARCA1 GAs occurred in other sarcoma subtypes. SMARCA1 IHC was studied in the sarcoma subtypes with potential SMARCA1 alterations in our institutional cases. SMARCA1 nuclear expression was lost in 3/10 cases (30%) of undifferentiated sarcoma, and 2/5 cases of MPNST (40%). SMARCA1 expression was intact in all cases of leiomyosarcoma and liposarcoma.SMARCA1 in the differentiation process and molecular mechanisms of SMARCA1 inactivation.This is the first study to demonstrate loss of expression of SMARCA1 in soft tissue sarcomas subtypes, including undifferentiated sarcoma. Our study highlights merit for further investigation on the role of  ARID1A (BAF250A), SMARCA2 (BRM), SMARCA4 (BRG1), and SMARCB1 (INI1) [SMARCA1 (SNF2L) is another member of the chromatin remodelers, and has not yet been studied in soft tissue tumors [SMARCA1 gene on chromosome X makes it susceptible to gene inactivation by the single hit mechanism [Chromatin remodeling complexes modify the nucleosomal architecture, which provides compact structure and regulates DNA accessibility ,2. VitalID1A BAF20A, SMARCID1A BAF20A, SMARCechanism . We aimeSMARCA1 in soft tissue tumors to analyze the available data. For performing IHC studies, our institutional records were queried for cases of soft tissue sarcoma diagnosed between 2008 and 2018. Twenty six cases were selected from our institutional archives including 10 undifferentiated sarcomas, 5 leiomyosarcomas, 6 liposarcomas, and 5 malignant peripheral sheath tumors (MPNST). IHC staining was performed on paraffin embedded whole tissue sections on Ventana Discovery with the rat monoclonal antibody SMARCA1  in 1:100 dilution. Normal testis tissue was used as a positive control. SMARCA1 expression was evaluated in tumor cell nuclei. When adjacent normal tissue was present for evaluation, SMARCA1 expression was evaluated in nuclei of adjacent nerve, smooth muscle, adipose tissue, endothelium and stromal cells. SMARCA1 expression on IHC was considered intact when expressed in >5% of nuclei of tissue of interest, and lost when not expressed in >5% of nuclei. Results were tabulated in an IRB approved database.In order to study genomic alterations we queried the publically available The Cancer Genome Atlas project (TCGA) platform cBioPortal.32e34 for SMARCA1 genomic alterations were found in eight (3%) of these cases. Leiomyosarcomas were the most common to harbor SMARCA1 genomic alterations, being found in 6 of 99 cases (6%). Of these deletions were found in 3 cases, missense mutation in 1 case, and amplification in 1 case. SMARCA1 deletions were present in only one case of dedifferentiated liposarcoma among 56 cases of liposarcomas (1.7%) and in only one of 48 (2%) undifferentiated sarcomas. None of the other sarcoma subtypes had SMARCA1 genomic alterations.The dataset from TCGA contained 261 soft tissue sarcomas. SMARCA1 nuclear expression was lost in 3 of 10 cases (30%) of undifferentiated sarcomas. There was no difference in morphology or nuclear features of tumor cells between the positive and negative cases of undifferentiated sarcoma. Representative images are shown in Expression of SMARCA1 was noted in 56% of adjacent normal smooth muscle, endothelium, and stromal cells. In adjacent normal adipose tissue, SMARCA1 was expressed patchy in only 1 case (5%). In these tissues, the expression was patchy and nuclear. SMARCA1 was not expressed in any of the adjacent normal peripheral nerves 0% (n=8). In the 3 cases of undifferentiated sarcoma with loss of SMARCA1, the adjacent normal tissue also did not express SMARCA1. Of the two cases of liposarcoma and MPNST with SMARCA1 loss, one case each had expression in adjacent normal tissue.SNF2L or SMARCA1, and SNF2H or SMARCA5 [Chromatin remodeling complexes maintain stability and compactness of the DNA through the nucleosomes and regulate accessibility of the DNA to transcription factors ,2. The c SMARCA5 ,5,7. Chr SMARCA5 ,8.SMARCA2 and SMARCA4 deficiency was noted in 10% of nonsmall cell lung cancer cases, 80% of SMARCA2/SMARCA4 deficient tumors was also TTF-1 negative, and recently SMARCA4 loss was found to be a predictor of response to platinum based chemotherapy [ARID1A mutations [SMARCB1  has been found altered in malignant extrarenal rhabdoid tumor (mutations), epithelioid sarcoma (deletions), epithelioid malignant peripheral nerve sheath tumor (loss of expression), myoepithelial carcinoma (loss of expression), and extraskeletal myxoid chondrosarcoma (loss of expression) [utations . The losutations . Among sression) . The mecression) .KRAS, BRAF [Synthetic lethality is co-occurrence of two genetic events that can be used for cell death, and exploited for cancer therapy . In contAS, BRAF .SMARCA1 (SNF2L), and none on SMARCA1 in soft tissue sarcoma [SMARCA1 is expressed in a broad range of normal tissues and has been reported to modulate the Wnt/ß catenin pathway [SMARCA1 silenced by aberrant methylation in gastric cancer cells [Our search of literature revealed there are occasional rare studies on  sarcoma ,5. SMARC pathway . A recenSMARCA1 genomic alterations may have different effect on protein expression. Loss of SMARCA1 IHC expression in MPNST with no genomic alterations in this subtype suggests that other mechanisms may drive SMARCA1 down regulation on the transcriptional or posttranslational level. Further investigation into loss of expression of SMARCA1 with more cases is required to study the association between SMARCA1 genomic alterations and protein expression, and the utility of SMARCA1 loss of expression for diagnostic and prognostic purposes. More studies on regulation and function of SMARCA1 are required to further understand its role in neoplasia and possibly elucidate interaction with other molecules and chromatin remodeling complexes for better therapy.We found genomic alterations in soft tissue sarcomas in TCGA dataset, namely leiomyosarcomas, liposarcomas and undifferentiated sarcomas. Data on methylation was not available in the dataset. The finding of SMARCA1 IHC loss in undifferentiated sarcoma is in keeping with TCGA molecular alterations. The fact that we did not find SMARCA1 IHC loss of expression in leiomyosarcomas and liposarcomas may be due to the different tumor cohorts and smaller number of cases in our study. In addition, SMARCA1 needs to be further explored in the differentiation process and neoplasia. The molecular mechanisms of SMARCA1 inactivation merit further investigation in order to understand and utilize its role in diagnostic, prognostic and therapeutic purposes.Our study is the first to demonstrate loss of expression of SMARCA1 in subtypes of soft tissue sarcomas, including undifferentiated sarcoma and malignant peripheral nerve sheath tumors. The role of


Preclinical data have shown that ERBB2 activating mutations are responsive to HER2 tyrosine kinase inhibitors. The aim of this study is to characterize the landscape of ERBB2 mutations in solid tumors and the potential efficacy of ERBB2 targeting.We analyzed the next-generation sequencing results from 17,878 patients with solid tumors and reported the outcome of 4 patients with advanced ERBB2-mutated tumors treated with a combination of trastuzumab and lapatinib.n = 282) were known as activating mutations. ERBB2 mutation was not mutually exclusive of ERBB2 amplification which occurred in up to 10% of cases. PI3KCA activating mutations were associated with ERBB2 mutations in 12.4% of cases mainly in breast and lung cancer. Four patients  were treated with a combination of trastuzumab and lapatinib. All of them experienced tumor shrinkage resulting in stable disease in three cases and partial response in one case. One patient developed secondary resistance. Sequencing of the progressing metastasis allowed the identification of the ERBB2 L869R mutation previously associated with resistance to lapatinib in vitro.ERBB2 mutations occurred in 510 patients (2.85%). The tumor types with the highest incidence of ERBB2 mutations were the following: bladder (16.6%), small bowel (8.6%), ampullar (6.5%), skin non-melanoma (6.1%), and cervical cancer (5.5%). 49.4%  contains supplementary material, which is available to authorized users. To the EditorERBB2 mutations in a large panel of tumors analyzed by next-generation sequencing (NGS) and reported the clinical impact of ERBB2 targeting in this setting as well as potential mechanisms of secondary resistance.We investigated the incidence of We analyzed the AACR Project Genomics Evidence Neoplasia Information Exchange (GENIE)  and the ERBB2 mutations involving all the domains of the receptor  of the mutations identified were described as oncogenic according to COSMIC and were more frequently detected in the bladder (9.4%), small bowel (7.1%), ampullar (6.5%), cervical cancer (4.1%), and nerve sheath tumor (2.9%), respectively , CDK12 amplification , and PI3KCA mutation .The three most frequent alterations co-occurring with ERBB2 amplification and mutation, 16 (32%) were breast ductal carcinoma, 8 (16%) lung adenocarcinoma, and 6 (12%) bladder urothelial carcinoma.Among the 39 tumors bearing both ERBB2 mutation-bearing tumor were treated with dual ERBB2 blockade with trastuzumab + lapatinib and experienced clinical benefit  may be dependent on the type of ERBB2 alteration and type of tumor. For instance, no clinical activity was observed in bladder and colorectal cancers . Of notePI3KCA mutations represent one of the most frequent co-alterations identified in ERBB2-mutated tumors. The PI3K/Akt/mTOR pathway has been shown to play a potential important role in resistance to ERBB2 targeting therapy in ERBB2-overexpressing breast cancer. Moreover, preclinical studies indicated that inhibitors of this pathway can act synergistically with trastuzumab in resistant models [ERBB2-targeted therapy in ERBB2-mutated tumors.Interestingly, t models . This fiERBB2/EGFR inhibitor such as neratinib in the clinical setting [Unfortunately, targeted therapies suffer from a major limitation, that is, the duration of any observed clinical benefit is invariably limited in length, owing to the relatively rapid acquisition of drug resistance. We report here for the first time a case of a ERBB2 activation loop mutation in a patient with non-amplified ERBB2 mutant colorectal cancer with acquired resistance to trastuzumab combined with lapatinib. The L869R mutation is located within the activation loop of the kinase domain and associated with gain of function activity. This mutation, which was identified in the secondary progressing brain lesion was not present in the primary tumor, was shown to confer resistance to lapatinib in vitro but to be sensitive to second-generation ERBB2 in several tumor types and the promising preliminary activity of dual ERBB2 targeting reported here deserved further clinical investigation aiming to demonstrate that ERBB2-mutational driven therapy can improve patient care irrespective of histology.The significant mutation rate of Additional file 1:Supplementary Methods and Results. (DOCX 622 kb)


This study demonstrates that the tetraspanins Tspan5 and Tspan15 which directly associate with the metalloprotease ADAM10 have an opposite impact on its endocytosis and half-life. ADAM10 is a transmembrane metalloprotease that is essential for development and tissue homeostasis. It cleaves the ectodomain of many proteins, including amyloid precursor protein, and plays an essential role in Notch signaling. ADAM10 associates with six members of the tetraspanin superfamily referred to as TspanC8 , which regulate its exit from the endoplasmic reticulum and its substrate selectivity. We now show that ADAM10, Tspan5, and Tspan15 influence each other’s expression level. Notably, ADAM10 undergoes faster endocytosis in the presence of Tspan5 than in the presence of Tspan15, and Tspan15 stabilizes ADAM10 at the cell surface yielding high expression levels. Reciprocally, ADAM10 stabilizes Tspan15 at the cell surface, indicating that it is the Tspan15/ADAM10 complex that is retained at the plasma membrane. Chimeric molecules indicate that the cytoplasmic domains of these tetraspanins contribute to their opposite action on ADAM10 trafficking and Notch signaling. In contrast, an unusual palmitoylation site at the end of Tspan15 C-terminus is dispensable. Together, these findings uncover a new level of ADAM10 regulation by TspanC8 tetraspanins. Many cell and developmental processes are regulated by a proteolytic cleavage of membrane-anchored proteins in their extracellular region, a process referred to as ectodomain shedding. Several proteases have been shown to be involved in this process, including several members of the ADAM  family of membrane-anchored metalloproteases . ADAM10 The activity of ADAM10 is regulated by both intrinsic properties and extrinsic factors. ADAM metalloproteases are synthesized as zymogens that remain catalytically inactive until the prodomain is released after cleavage by pro-protein convertases during transport to the cell surface . The recCaenorhabditis elegans and the three Drosophila TspanC8 tetraspanins regulate ADAM10 subcellular localization in vivo  undergoes faster internalization than ADAM10Tspan15 after reaching the plasma membrane, an anti-ADAM10 mAb added to the medium at 37°C should accumulate more rapidly inside the cells in Tspan5-positive/Tspan15-negative cells. We incubated U2OS cells at 37°C with the DyLight 650–labelled ADAM10 mAbs for different times. After detachment, the cells were incubated with a PE-labelled secondary reagent, and the fluorescence was measured by flow-cytometry. DyLight 650 fluorescence measures the total pool of ADAM10 having passed through the plasma membrane (either still present or endocytosed) and the PE fluorescence the surface ADAM10 fraction. As shown in Antibodies to the ectodomain of proteins that traffic through the plasma membrane can bind to their target, even if the passage at the plasma membrane is transient  and are To validate the previous findings in a different cell model, Tspan5 and Tspan15 were also expressed in HeLa cells. Both tetraspanins induced a similar twofold increase in ADAM10 surface expression levels . Again, So far, the data show that Tspan15 expression is associated with both a higher stability and a diminished endocytosis of ADAM10. We reasoned that if the higher stability of the protein is a consequence of the stabilization at the cell membrane, the surface pool of ADAM10 should be more stable in the presence of Tspan15. To address this question, surface proteins were labelled with biotin, and the cells were incubated at 37°C for 15 h before lysis, or lysed directly to determine the initial level of surface protein. ADAM10 and CD81 were then immunoprecipitated, and the level of biotin-labelled protein was determined using streptavidin blotting . As a coThe preceding data showing that Tspan5 and Tspan15 differentially regulate ADAM10 trafficking and turn-over were obtained using transfected cells. To analyze the impact of endogenous Tspan5 and Tspan15, we generated Tspan5 and Tspan15 KO cells using the CRISPR/Cas9 gene editing technology.In a first model, ablation of Tspan5 in U2OS cells did not modify ADAM10 surface levels, but reduced its internalization rate by 50%, further suggesting that Tspan5 stimulates ADAM10 endocytosis . In PC3 The anti-Tspan5 mAbs TS5-2 immunoprecipitated only a small fraction of mature ADAM10 in parental cells. In contrast, most mature Tspan5 is associated with ADAM10, as shown by the comparison of the ADAM10 and TS5-2 IPs and by the lack of immunoprecipitation of the upper forms with TS5-1r .There was more Tspan5 in the Tspan5 IP from Tspan15 KO cells , consistTetraspanins are palmitoylated at several conserved juxtamembrane intracellular cysteines. Tspan15, in contrast to Tspan5, lacks such typical tetraspanin intracellular cysteines . BecauseTo test whether Tspan5 and Tspan15 are palmitoylated proteins, the cells were incubated with azido palmitic acid and palmitoylated proteins were labelled with biotin using click chemistry. As expected, Tspan5 efficiently incorporated palmitic acid and the Tspan5plm mutant, in which all four cysteines that are putative palmitoylation sites were changed to alanines, did not , indicatBoth the Tspan5plm mutant and the Tspan15 CCLC mutant were expressed at the cell surface similarly to the WT proteins (data not shown). However, these mutations did not affect the influence of Tspan5 and Tspan15 on ADAM10 surface expression levels in U2OS cells or Notch activity .To get further insights into the mechanisms whereby Tspan5 and Tspan15 differentially regulate ADAM10 endocytosis and Notch signaling, we generated a series of Tspan5/Tspan15 chimeras  and exprA major difference between Tspan5 and Tspan15 is the presence in Tspan15 of a cytoplasmic intracellular C-terminal domain unusually long for a tetraspanin . Replacing the N- and C-cytoplasmic domains of Tspan5 by that of Tspan15 (T5NC15) yielded a chimera that strongly inhibited ADAM10 endocytosis in HeLa cells . ChimeraWhen expressed in U2OS cells, all chimeras in which the cytoplasmic domains of Tspan5 and Tspan15 were exchanged stimulated an increase in ADAM10 surface expression level (about twofold) that was lower than that induced by Tspan15 (Tspan5 overexpression did not result in an increase in ADAM10 surface levels) . At leasWe also analyzed the effect of these chimeras on Notch signaling . The repThe above data show that Tspan5 and Tspan15 have a positive and a negative impact, respectively, on ADAM10 endocytosis. Consistent with this finding, the Tspan5 mAb was efficiently internalized after 1 h at 37°C in HeLa/Tspan5 cells, and the internalized fraction colocalized with ADAM10, suggesting again that both proteins were internalized together , top. HoTo validate the hypothesis that ADAM10 stabilizes Tspan15 at the cell surface, we generated ADAM10 KO PC3 cells. Although there was no change in Tspan15 mRNA levels (data not shown), there was a 60% decrease in Tspan15 surface expression levels  in theseThis finding may explain why transfection of Tspan5 in PC3 cells led to a ∼40% decrease of Tspan15 surface expression . The oveThe trafficking and function of the metalloprotease ADAM10 is regulated by the members of the TspanC8 subgroup of tetraspanins. Whereas all TspanC8 members allow ADAM10 to exit from the ER, Tspan10 and Tspan17 were shown to target ADAM10 to late endosomes, and the remaining four members target ADAM10 to the plasma membrane . Here, wAlthough both Tspan5 and Tspan15 target ADAM10 to the plasma membrane, Tspan15 is associated with higher ADAM10 surface expression levels: indeed, transfection of Tspan15 in a Tspan5/Tspan14–positive cell line (U2OS) induced a large increase in ADAM10 surface expression levels, whereas transfection of Tspan5 in a Tspan5/Tspan15–positive cell line (PC3) decreased the ADAM10 surface level. In addition, we observed that the surface staining of PC3 cells with a Tspan15 mAb was nearly ten times higher than that observed with a Tspan5 mAb, suggesting a much higher expression level of Tspan15 at the protein level. Accordingly, Tspan5 knockout had little effect on ADAM10 expression, whereas Tspan15 knockout reduced by 75% the surface expression of ADAM10 suggesting that most surface ADAM10 molecules are associated with Tspan15 at the steady state level. In both the U2OS and PC3 models, we have shown that the higher ADAM10 surface levels observed in the presence of Tspan15 reflects a higher total expression level, which is explained at least in part by a longer ADAM10 half-life.Contrasting with U2OS and PC3 cells, Tspan5 transfection in HeLa cells yields a twofold increase in the ADAM10 surface level. This is most likely because in this cell line, ADAM10 is retained to a large extent in the ER because of a limiting amount of TspanC8 . Thus, tTspan15 is at least in part the consequence of a longer residence time at the cell surface and that the analysis of ADAM10 cell surface expression at the steady state does not reflect the overall amount of ADAM10 having passed through the plasma membrane. Indeed, we have shown in several cellular models that ADAM10Tspan5 undergoes faster endocytosis than ADAM10Tspan15, and that after endocytosis, ADAM10 is targeted to CD63-positive compartments which include late endosomes and lysosomes. Notably, incubation of the cells with an anti-ADAM10 mAb showed a time-dependent accumulation of the mAbs inside the cells, which was inhibited by the expression of Tspan15 (either in U2OS-transfected cells or WT PC3 cells). After 20-h incubation with the anti-ADAM10 mAb at 37°C, staining of Tspan15 KO PC3 cells is comparable with that of parental cells or Tspan5 KO cells, indicating that although the ADAM10 level is strongly reduced in Tspan15 KO PC3 cells, the amount of ADAM10 trafficking through the plasma membrane is similar in all cell types. Thus, the fact that ADAM10 is mainly associated with Tspan15 at a steady state in PC3 cells is not due to a lower Tspan5 level but mainly if not exclusively to the shorter residence time of ADAM10Tspan5 at the plasma membrane.Our data indicate that the longer half-life of ADAM10Tspan5 and ADAM10Tspan15. Because Tspan14 is the major TspanC8 tetraspanin expressed by HeLa and U2OS cells (besides Tspan5 in this latter case), it is possible that ADAM10Tspan14 is endocytosed at a rate intermediate between that of ADAM10Tspan5 and ADAM10Tspan15. Further work will be required to test this hypothesis when mAbs to Tspan14 are available.Tspan15 was shown to stabilize ADAM10 and to inhibit its endocytosis in all cell lines tested and using all assays performed. This, together with the finding that its deletion is associated with a decrease of the mature form of ADAM10 in the brain , suggestThe endocytosis assays were performed using an antibody uptake assay, raising the question of the influence of the mAbs on ADAM10 endocytosis. The finding that incubation at 37°C with the anti-ADAM10 mAbs led to a decrease in ADAM10 at the surface of cells lacking Tspan15, that is, in U2OS and U2OS/Tspan5 cells and in tspan15 KO PC3 cells , indicates that the mAb accelerates to some extent the rate of ADAM10 endocytosis. Nevertheless, several lines of evidence indicate that the internalization of the mAb mirrors the endocytosis of ADAM10 under normal conditions. First, the decrease at the cell surface of U2OS cells is for the most part observed when comparing the labelling after cell detachment with that observed after 1 h at 37°C, suggesting that cell detachment may accelerate endocytosis of antibody-bound ADAM10 in these cells. This could explain why we observe after 1-h incubation ∼20% endocytosis by confocal microscopy and >50% using the flow cytometry protocol. Second, at time points later than 1 h, both in U2OS cells and Tspan15 KO PC3 cells, the mAbs continue to undergo effective internalization, accumulating into the cells at a much higher level than the initial surface level, whereas the surface level decreases proportionally much less.The finding that overexpression of Tspan5 in U2OS cells does not induce an increase in the ADAM10 surface level contrasts with our previous study using GFP-tagged Tspan5 and Tspan15, which induce a three and five times increase in ADAM10 surface levels, respectively . This isWe have started to study how Tspan5 and Tspan15 differentially affect ADAM10 surface levels by analyzing the activity of Tspan5/15 chimeras. Our data indicate a role for the cytosolic domains of Tspan5 and Tspan15 but also indicate that other domains contribute to their effect on ADAM10 endocytosis. This complex situation contrasts with the recent finding that replacing the C-terminus of Tspan15 by that of Tspan33 was sufficient to target Tspan15 and ADAM10 to apical junctions .It has been suggested that the intracellular domain of ADAM10 interacts with the clathrin adaptor AP2 through a non-canonical AP2-binding motif that was shown to regulate to some extent the internalization of an IL2Rα/ADAM10 chimera . BecauseTspan15 at the plasma membrane could be due to a different recycling rate. In this regard, it was shown that Rab14 and its Guanine nucleotide exchange factor FAM116A play a role in recycling membrane proteins, and that depletion of these molecules in A549 cells, which strongly express Tspan15 (data not shown), induced the accumulation of ADAM10 in an intracellular transferrin-positive compartment  yielded chimeras which had an intermediate effect on ADAM10 surface levels in U2OS cells and were not inhibitors of Notch signaling. Based on these data and the previous demonstration that Tspan5 and Tspan15 differentially regulate ADAM10 membrane compartmentalization , we suggTspan15 at the plasma membrane is a consequence of the interaction with yet unknown proteins that stabilize this complex at lateral contacts, where it could process certain cadherins and perhaps other substrates. Concerning Tspan5, the short membrane residence time of ADAM10Tspan5 may prevent ADAM10 from processing discrete substrates until appropriate, for example, after it is stabilized upon proper signaling. This may also allow ADAM10 levels to be increased in a transient manner in response to a particular signal. This may be especially true in the nervous system where Tspan5 is abundantly expressed . Labelling of antibodies with DyLight 650 or 550 N-Hydroxysuccinimide Esters was performed according to the manufacturer’s instructions (Thermo Fisher Scientific). All fluorochrome-labelled anti-Ig antibodies were from Thermo Fisher Scientific. The plasmids encoding Tspan5 and Tspan15 fused to GFP were previously described (GICLAQN-Tspan5 for T15C5 and Tspan5-ALLQIFGVLLTLL-Tspan15 for T5C15. The sequences of the swap sites at the N-terminus were Tspan15-FSYLWLKYFIFGF-Tspan5 for T5N15 and T5NC15 and Tspan5-PEVSCCIKFSLII-Tspan15 for T15N5 and T15NC5 (the conserved amino acid where the swap was made is shown in boldface and is underlined).The mAb directed to human ADAM10 (11G2), CD9 (TS9), CD81 (TS81), CD63 (TS63), and Tspan5  were generated in our laboratory . The mAbescribed . After Pescribed . The seq6 cells using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s instructions. cDNA synthesis was performed from 1 μg of total RNA using 200u of SuperScript III Reverse Transcriptase (Invitrogen) primed with random hexamer (Promega), in a 20-μl reaction volume. Quantitative real-time PCRs (qPCRs) were then carried out on duplicates in a final volume of 25 μl containing 2× GoTaq qPCR Master Mix (SYBR Green) from Promega, 0.4 μM each forward and reverse primers and 1 μl cDNA. Quantification was performed with the Mx3005P QPCR Systems and MxPro software (Agilent). The values were normalized to rpl38 expression according to the ΔCt method. The oligonucleotides used for amplification have been previously described   were culhttps://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design). The corresponding guide DNA sequences were cloned into the lentiCRISPRv2 plasmid  according to the instructions of the Zhang laboratory (https://www.addgene.org/52961/)  and the/52961/) . The plaCells were detached with Trypsin–EDTA, washed in complete DMEM, and incubated for 1 h at 4°C with 10 μg/ml primary antibody or hybridoma supernatant. After three washings, the cells were incubated for 30 min at 4°C with an Alexa647-conjugated goat anti-mouse antibody. The cells were analyzed using an Accuri C6 flow-cytometer (Becton Dickinson).2. The cells were transfected 24 h later with the Notch luciferase reporter and Renilla plasmids using FuGene HD (Promega). 24 h later, the cells were co-cultured with OP9 or OP9-DLL1 at 35,000 cells/cm2. The activities of firefly and Renilla luciferases were determined using a dual luciferase reporter assay (Promega) according to the manufacturer’s instructions.This analysis was performed as previously described : U2OS ce2 and MgCl2 was added to the buffer for maximal tetraspanin/tetraspanin interaction . Western blotting on lysates was performed using appropriate primary and Alexa Fluor 680-labelled secondary antibodies. Western blotting on immunoprecipitations was performed using biotin-labelled antibodies and fluorescent streptavidin except for the anti-Tspan15 and the CD81 mAbs. All acquisitions were performed using the Odyssey Infrared Imaging System (LI-COR Biosciences).Immunoprecipitations were performed as previously described with minor modifications . For immeraction . After 34, 1, 5 μl reaction buffer additive 1, and after 3 min 3 μl click-it additive 2. After 1 h at room temperature, 20 μl 3× Laemmli buffer was added to the reaction. The samples were analyzed by Western blot using Alexa680-labelled streptavidin.All reagents used for the analysis of Tspan5 and Tspan15 palmitoylation by click chemistry were obtained from Life technologies. U2OS cells expressing GFP-tagged Tspan5, Tspan15, or the mutants were incubated overnight with 50 μM click-it palmitic acid (15-azidopentadecanoic acid) in serum-free medium. After three washes, the cells were lysed in PBS containing 1% Triton X-100, and the proteins were immunoprecipitated using GFP-trap beads (ChromoTek). After three washing, the beads were resuspended in 15 μl of a solution containing 50 mM Tris HCl, pH 8 and 0.5% SDS. Biotinylation of the proteins labelled with the modified palmitic acid was performed by subsequent addition of 15 μl 40 μM biotin alkyne in click-it reaction buffer, 1, 5 μl CuSO4Cl in PBS. For surface labelling, the cells were then incubated directly for 1 h with 10 μg/ml of antibodies in PBS supplemented with 0.1% BSA at room temperature. For intracellular labelling, the cells were incubated with antibodies in the same buffer supplemented with 0.1% saponin. The binding of primary antibodies was revealed using appropriate secondary reagents. The cells were mounted in Prolong Gold (Thermo Fisher Scientific) supplemented with DAPI and examined with a Leica SP5 confocal microscope .The cells grown in complete medium were fixed for 15 min with 4% paraformaldehyde at room temperature, washed in PBS and then incubated for 15 min in 50 mM NHhttp://icy.bioimageanalysis.org). The purpose of this protocol is to use the surface staining (“surface image”) to generate a mask allowing removing the surface labelling from the total staining . An inherent difficulty of this approach is that the results may vary considerably according to the threshold applied to the images. By definition, the internalized pool is not labelled by the secondary reagent or minimally in rare cases of partial permeabilization upon fixation. The surface and internalized pool can, therefore, be distinguished by the ratio of the two fluorescence intensities, independently of any threshold applied. Thus, one of the first steps in our protocol is to divide the “total image” by the “surface image.” A threshold image is then generated, which allows removing from the total image the fraction of the signal that overlaps with the surface labelling, yielding a new image with only the intracellular labelling . The intracellular signal is quantified using the spot detector plugin. The total and surface images are quantified after applying a threshold determined using the K-mean method. In HeLa cells, the relative signals of the internalized fraction to the surface fraction were so different in the various transfectants that using the K-mean method on each cell type was not reliable. We, therefore, applied on all HeLa transfectants a threshold calculated on parental cells using the K-mean method. The percentage of endocytosed mAbs was obtained by dividing the value of the internal signal by that of the total signal. Three to eight fields were acquired for each experiment and averaged. At least three independent experiments were performed for each cell type. All images of internalization assays are maximum-intensity projections of the z-stacks.The cells were incubated for 1 h at 37°C with the anti-ADAM10 mAb 11G2 coupled to DyLight 650. After fixation or cooling to 4°C, the surface pool is then labelled with an anti-mouse polyclonal antibody coupled to Alexa Fluor 568 or Alexa Fluor 488. Images were acquired every 0.25 μm throughout the height of the cells using a Leica SP5 confocal microscope . Quantification was performed using a protocol developed in Icy imaging software   in the presence of saponin to reveal the internalized mAb. Quantification using Icy was performed as above, except that an endocytosis index was obtained by dividing the value of the internal image by that of the surface image.i) as follows: FL4i = [FL4n-(FL2n × FL40/FL20)], where FL20 or FL2n and FL40 or FL4n are the PE and DyLight 650 mean fluorescence intensity of nontreated cells (labelled after detachment) and cells treated for n hours. The ratio FL4i/FL40 was then calculated to express the data relatively to the amount present at the cell surface at the steady state level.The cells were incubated at 37°C with 10 μg/ml DyLight 650–labelled ADAM10 mAb for different times. After detachment using accutase, the cells were incubated at 4°C with a PE-labelled secondary reagent, and the fluorescence was measured by flow-cytometry. In each experiment, a fraction of cells was not incubated with the ADAM10 mAb at 37°C, but at 4°C after detachment, and subsequently with the PE-labelled secondary reagent. This allows determining the ratio of PE (in FL2 channel) to DyLight 650 (in FL4 channel) fluorescence when the ADAM10 mAb is only at the cell surface, and thus an estimation of the surface fraction of ADAM10 from the DyLight 650 fluorescence in the cells incubated with the mAb at 37°C. The mean fluorescence intensity in the FL4 channel corresponds to the sum of the fluorescence of the mAb present at the cell surface and internalized. We estimated the fluorescence intensity corresponding to the internalized mAb (FL4Statistical analysis was performed with GraphPad Prism on independent experiments as indicated in the legend to the figures. All graphs show the mean ± SEM.


Statins are an important class of medications in reducing the risk of cardiovascular events as well as overall mortality. However, a well-known adverse effect of statins is skeletal muscle toxicity, which may lead to abrupt discontinuation of the statin. In turn, patients may miss out on the benefits of statin therapy. An important factor to consider is a patient's solute carrier organic anion transporter 1B1 (SLCO1B1) gene T521C polymorphism status. Herein, an overview of the pharmacogenetics of SLCO1B1 is provided as well as recommendations for use in practice. Statins are, by and far, the number one prescribed class of medication worldwide . Their mThe most common side effect associated with statins is skeletal muscle toxicity that includes pain without evidence of muscle degradation , pain with evidence of muscle degradation (myopathy), and severe muscle damage with acute kidney injury (rhabdomyolysis) . ControlRegarding this association between statins and skeletal muscle toxicity, an important pharmacogenetic marker to consider is the SLCO1B1 gene. The solute carrier organic anion transporter family member 1B1 (SLCO1B1) protein facilitates the hepatic uptake of statins . The mosThe Clinical Pharmacogenetics Implementation Consortium (CPIC) has published dosing recommendations regarding simvastatin and the activity of the SLCO1B1 enzyme (Table CPIC also published information regarding the percent increase in statin exposure in individuals with the SLCO1B1 c.521 CC genotype [Pharmacogenetic testing for the SLCO1B1 gene can be helpful to clinicians who may have a suspicion that a patient’s statin therapy is causing skeletal muscle toxicity. Also, utilizing SLCO1B1 genotyping can help in deciding which statin may be best for providers initiating statin therapy. Adjustments to the dose of a statin or choosing an alternative can be the difference for a patient especially in those cases where prematurely stopping a statin places the patient at risk of losing the cardiovascular benefits of this therapy. Consideration of following an algorithm where SLCO1B1 gene testing is incorporated in practice can help patients initiating a statin or considering an alternative statin . Provide


Plasmodium falciparum we inducibly knocked down the expression of EXP2. Reduction in EXP2 expression strongly reduced parasite growth proportional to the degree of protein knockdown and tended to stall development about half way through the asexual cell cycle. Once the knockdown inducer was removed and EXP2 expression restored, parasite growth recovered dependent upon the length and degree of knockdown. To establish EXP2 function and hence the basis for growth reduction, the trafficking of an exported protein was monitored following EXP2 knockdown. This resulted in severe attenuation of protein export and is consistent with EXP2, and PTEX in general, being the conduit for export of proteins into the host compartment.Malaria parasites remodel their host erythrocytes to gain nutrients and avoid the immune system. Host erythrocytes are modified by hundreds of effector proteins exported from the parasite into the host cell. Protein export is mediated by the PTEX translocon comprising five core components of which EXP2 is considered to form the putative pore that spans the vacuole membrane enveloping the parasite within its erythrocyte. To explore the function and importance of EXP2 for parasite survival in the asexual blood stage of  Plasmodium spp. parasites. In 2016 there was an estimated 216 million cases reported, resulting in 445,000 deaths, mostly of children under 5 [Plasmodium parasites invade erythrocytes and remodel them to obtain supplementary nutrition from the blood plasma and to evade the immune system. Symptomatic malaria disease is caused by intracellular blood stage parasites which are enveloped in a parasitophorous vacuole membrane (PVM) within the erythrocyte. Residing on the PVM is an essential protein translocon called PTEX (Plasmodium translocon for exported proteins) [Almost half the world’s population is at risk of malaria, the disease caused by infection with roteins) , 4.Plasmodium genus. The first is HSP101, a AAA+ heat shock protein chaperone which is predicted to form a hexameric structure to unfold proteins for export [Plasmodium berghei since its gene can be deleted, however its loss reduces export efficiency and virulence [P. berghei resulted in reduced sequestration and virulence [P. falciparum resulted in reduced binding to the endothelial receptor CD36 [PTEX consists of five core components, HSP101, PTEX150, EXP2, TRX2 and PTEX88 [irulence , 6, 7. Pirulence , 10, andThe final core PTEX protein is EXP2 which lacks predicted transmembrane spanning domains typical of membrane pores, yet it is the most membrane associated protein of the PTEX complex , 12. VerToxoplamsa gondii [In addition to a full size PTEX complex of >1200 kDa, we have shown EXP2 forms homo-oligomers of approximately 600 and 700 kDa in size by blue native polyacrylamide gel electrophoresis raising the question of what the smaller forms could be doing [a gondii , 16, 17.a gondii .Export of effector proteins can be inhibited by disrupting the PTEX complex by inducibly knocking down expression of HSP101, PTEX150 and EXP2 , 14 or bP. falciparum we have inducibly knocked down EXP2 expression. We show that EXP2 knockdown is detrimental for parasite growth and the extent of growth arrest is correlated with the degree of knockdown. Upon restoration of EXP2 expression parasite growth can partially recover dependent on the length and strength of knockdown. Upon EXP2 knockdown, growth arrest was probably due to the inhibition of export of essential effector proteins since the export of a protein marker was also curtailed.To explore the role of EXP2 in the asexual blood stages of P. falciparum genomic DNA, the 3’ end of the EXP2 coding region was amplified with forward primer EXP2_1F: AGGAGATCTGGTCACGTATGTGGTGGGTA and reverse primer EXP2_2R A’: CTTATACTGCAGCTTCTTTATTTTCATCTTTTTTTTCATTTTTAAATAAATCTCCACTGGCA. After digestion with BglII and PstI the exp2 DNA fragment was ligated into pPTEX150_HAglmS after the ptex150 flank was removed by digestion with the same enzymes to produce pEXP2-HAglmS.From 100 ng of 3D7 P. falciparum was cultured in human RBCs  at 4% haematocrit (HCT) in AlbuMaxII media  at 37°C as described previously [P. falciparum strain 3D7 was subsequently cultured [exp2 locus. Once western blot validation with anti-HA IgG indicated the exp2 locus had been appended with the HAglmS construct, clonal parasite lines were isolated by limiting dilution. Successful integration was validated by performing diagnostic PCRs with genomic DNA isolated from each clonal parasite line using the primers: A TGCAGAAACAACTTTGCCACA; A’ TGGCATCTTCTTCTTCAACGGT; B’ TCCTTACGGCTGTGATCTGC.eviously . 100 μg cultured . TransfeTo knockdown protein expression in EXP2-HAglmS parasites 1M glucosamine (Sigma) was added at the desired concentration to 5 mL of 1% synchronous ring stage parasites in 4% hematocrit. Parasites were grown for 1 cell cycle (~48 h) and then harvested by centrifugation. The cell pellet was treated with 0.09% saponin in PBS with Complete Protease Cocktail Inhibitors (Roche) to remove the haemoglobin. Cell proteins were solubilised in reducing SDS sample buffer and fractionated on 4–12% acrylamide Bis-Tris gels (Invitrogen) and transferred via iBlot to nitrocellulose membranes (Invitrogen). Membranes were blocked in 1% casein and probed with mouse EXP2 monoclonal, rabbit anti-ERC IgG and chicken anti-HA IgY (Abcam) as per [For parasite growth and recovery assays 5 mL ring stage parasites at 4% hematocrit and 3% parasitemia were treated with the desired GlcN concentration for 1 day. Thin blood smears were then made and 3 x 100 μL aliquots of culture removed for LDH assays. The remaining culture was treated with 0.09% saponin with Complete Protease Cocktail Inhibitors (Roche) to remove the haemoglobin. Parasites to be sampled 2 and 3 days after GlcN treatment were set up as above but diluted 1/5 in 4% hematocrit and those to be sampled after 5 days were diluted 1/25. At days 2 and 3 post GlcN treatment samples were removed for analysis as above. Parasites that were to be grown for 5 days were fed on days 2 and 3 with fresh media and washed to remove GlcN or fresh GlcN was added back. After completion of the time course lactate dehydrogenase (LDH) assays that correlate with parasitaemia were performed as described previously  and absoIFAs were performed as described previously . BrieflyP. falciparum in in vitro culture [exp2 null parasites, an alternative approach was adopted where an inducible knockdown system based on the glmS ribozyme was employed [exp2 was substituted with a heterologous 3' UTR containing glmS (exp2 coding sequence for antibody-based detection of the protein. In this knockdown system the glmS ribozyme self cleaves the mRNA upon addition of glucosamine (GlcN) to the parasite culture, leading to removal of the poly-A tail, destabilization of the mRNA and subsequent reduction in protein production. The EXP2-HAglmS construct was introduced into the exp2 locus of the wildtype 3D7 parasite line by homologous recombination , before expression of EXP2 peaks at approximately 20–30 hpi , 26. ParIt was expected that a reduction in EXP2 levels would have major effects on the growth and protein export of the parasites on the basis that reducing the function of the other two main PTEX components, PTEX150 and HSP101, had a significant effect , 4. To iNext, parasite proliferation using lactate dehydrogenase (LDH) activity as a marker of biomass was measured and indicated that GlcN-induced knockdown of EXP2 proportionally reduced proliferation after 3 days (cycle 1) . Growth To further explore the relationship between EXP2 levels and parasite growth, 0.5 and 2 mM GlcN that had been added to EXP2-HAglmS and 3D7 rings in cycle 0 was washed out in cycle 1 at rings (2 day GlcN treatment) or trophozoites (3 day GlcN treatment). The parasites were then further grown until cycle 2 (day 5) and compared with untreated parasites (0 mM GlcN) and with parasites continuously treated with GlcN for 5 days. Parasite samples from days 1, 2, 3 and 5 were retained for analysis and LDH assays indicated that in parasites treated with 0.5 mM GlcN for 5 days significant growth inhibition was only observed for EXP2-HAglmS. For parasites in which 0.5 mM GlcN was removed after 2 and 3 days, growth almost fully recovered by day 5 . For 2 mThe EXP2-HAglmS parasites were also monitored by microscopy throughout the knockdown and recovery period. In 0.5 mM GlcN the parasites appeared to lag behind the untreated parasites so that by day 5 the trophozoite stage parasites were smaller than the untreated trophozoites and younger rings stages were also present . By day Western blot analysis was next performed on EXP2-HAglmS to determine how protein levels responded during knockdown and recovery. A second 5 day knockdown and recovery assay was performed and the LDH growth results were similar as before . WesternPfEMP1 export [In light of EXP2's putative PTEX pore function we expected that the growth arrest observed upon reduction of EXP2 levels would be due to the parasite's inability to export critical proteins into the erythrocyte. To determine if protein export in the EXP2 knockdown parasites was curtailed we performed microscopy on skeleton binding protein 1 (SBP1), an early exported protein, in ring stage parasites before growth arrested. Once exported, SBP1 accumulates at Maurer's clefts which are parasite-formed vesicular structures that play a role in 1 export , 29. ImmThe exposure times required to produce satisfactory images of the untreated 3D7 parasites were used for all subsequent imaging and likewise all brightness and contrast settings were identically adjusted for the SBP1 and EXP2 fluorescence channels. Imaging of EXP2-HAglmS parasites indicated that EXP2 levels were markedly reduced following GlcN treatment . FurtherAlthough we have previously reported GlcN mediated export trapping in PTEX150-HAglmS parasites we repeated this here for comparison with EXP2-HAglmS . GlcN trglmS ribozyme onto EXP2 and reduced its expression by up to 84% following treatment with 1 mM GlcN. This appeared to arrest parasite growth in late rings one cycle after adding GlcN to degrade the protein’s mRNA. The EXP2-HA knockdown parasites export SBP1 less efficiently than parasites with normal levels of EXP2 suggesting growth arrest could be due to the failure to export functionally crucial proteins other than SBP1, which itself is not essential [To investigate the role of EXP2 in the PTEX protein export complex we attempted to knockdown EXP2 and examine subsequent phenotypes. Our knockdown strategy appended a HA tag and ssential , 29. Durssential . Both thssential , 14.glmS mediated knockdown of EXP2-HA was responsible for growth reduction is provided by the fact that the HAglmS tag only appeared to append to EXP2 and no other genes since no other HA bands apart from EXP2 were present in western blots. The degree of knockdown of EXP2-HA protein was critically dependent on the concentration of GlcN added to the parasite culture and GlcN had no substantial effect on 3D7 parasites except at high concentrations (≥ 2 mM) for 3 or more days. Furthermore, when GlcN was removed EXP2 expression and parasite growth were partially restored. Specifically, when 0.5 mM GlcN was washed out after 2 and 3 days treatment, EXP2 protein levels and parasite proliferation almost fully recovered by day 5. At a higher dose of 2 mM GlcN for 2 days, only partial recovery occurred by day 5. GlcN mediated knockdown of EXP2 also greatly reduced export of SBP1 consistent with EXP2 being part of the PTEX complex [That the  complex , 29. Con complex .Our initial data suggested that growth of EXP2-HAglmS parasites was arrested by 0.5 mM GlcN treatment after 2–3 days, however closer investigation revealed that growth was more likely just slowed given that the parasites recovered well once GlcN was removed and EXP2 expression resumed. These parasites had about a half to a third the normal amount of EXP2-HA after 2–3 days treatment and could probably still export enough proteins and obtain sufficient nutrients to survive and hence recovered following GlcN removal. Whether slowed growth also resulted in fewer merozoites being formed was not examined, but nutrient restriction via knockdown of the new permeability pathway protein RhopH2 has been noted to reduce merozoite numbers per schizont . This coParasites seem to be only able to tolerate the strong knockdown of EXP2 caused by 2 mM GlcN treatment for a few days since after this, growth recovery was poor and dead parasites began to be observed after 3 days of 2 mM GlcN treatment. After 2 days of 2 mM GlcN treatment EXP2 levels had fallen to 20% of normal levels and this enabled many parasites to survive since they partially recovered by day 5 if the GlcN was washed out after 2 days. After 3 days 2 mM GlcN treatment when levels of EXP2 had fallen to ~10%, recovery was poor probably because most parasites had died by then. The ability of ring stage arrested parasites to partially recover growth as long as 2 days after arrest has been previously detected for another PTEX protein HSP101, and is consistent with our EXP2 observations here [P. berghei also reduced export of several different PEXEL proteins and in P. falciparum knockdown of HSP101 function reduced the export of several PEXEL and PNEP proteins [PfEMP1 display and the trafficking of other exported proteins, the precise functional role for most exported proteins is not understood, with a few exceptions [P. falciparum genome with piggyBac transposons has helped determine which genes might be unmutable and essential and are therefore a priority for future study [Both our study and Garten et al (2018)  showed tproteins , 4. A knproteins , 31–33. ceptions . A recenre study . AlthougP. falciparum blood stages probably because it is required for the efficient export of functionally important proteins into the erythrocyte compartment. Although this finding has been recently published [In conclusion, we have shown that EXP2 is crucial for survival of ublished  our studublished , still lS1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.