National Heart, Lung, and Blood Institute Division of Intramural Research

BACKGROUND: Intima-media thickness of the walls of the common carotid artery and internal carotid artery may add to the Framingham risk score for predicting cardiovascular events. METHODS: We measured the mean intima-media thickness of the common carotid artery and the maximum intima-media thickness of the internal carotid artery in 2965 members of the Framingham Offspring Study cohort. Cardiovascular-disease outcomes were evaluated for an average follow-up of 7.2 years. Multivariable Cox proportional-hazards models were generated for intima-media thickness and risk factors. We evaluated the reclassification of cardiovascular disease on the basis of the 8-year Framingham risk score category (low, intermediate, or high) after adding intima-media thickness values. RESULTS: A total of 296 participants had a cardiovascular event. The risk factors of the Framingham risk score predicted these events, with a C statistic of 0.748 (95% confidence interval [CI], 0.719 to 0.776). The adjusted hazard ratio for cardiovascular disease with a 1-SD increase in the mean intima-media thickness of the common carotid artery was 1.13 (95% CI, 1.02 to 1.24), with a nonsignificant change in the C statistic of 0.003 (95% CI, 0.000 to 0.007); the corresponding hazard ratio for the maximum intima-media thickness of the internal carotid artery was 1.21 (95% CI, 1.13 to 1.29), with a modest increase in the C statistic of 0.009 (95% CI, 0.003 to 0.016). The net reclassification index increased significantly after addition of intima-media thickness of the internal carotid artery (7.6%, P<0.001) but not intima-media thickness of the common carotid artery (0.0%, P=0.99). With the presence of plaque, defined as intima-media thickness of the internal carotid artery of more than 1.5 mm, the net reclassification index was 7.3% (P=0.01), with an increase in the C statistic of 0.014 (95% CI, 0.003 to 0.025). CONCLUSIONS: The maximum internal and mean common carotid-artery intima-media thicknesses both predict cardiovascular outcomes, but only the maximum intima-media thickness of (and presence of plaque in) the internal carotid artery significantly (albeit modestly) improves the classification of risk of cardiovascular disease in the Framingham Offspring Study cohort. (Funded by the National Heart, Lung, and Blood Institute.).

IMPORTANCE: Interstitial lung abnormalities have been associated with lower 6-minute walk distance, diffusion capacity for carbon monoxide, and total lung capacity. However, to our knowledge, an association with mortality has not been previously investigated. OBJECTIVE: To investigate whether interstitial lung abnormalities are associated with increased mortality. DESIGN, SETTING, AND POPULATION: Prospective cohort studies of 2633 participants from the FHS (Framingham Heart Study; computed tomographic [CT] scans obtained September 2008-March 2011), 5320 from the AGES-Reykjavik Study (Age Gene/Environment Susceptibility; recruited January 2002-February 2006), 2068 from the COPDGene Study (Chronic Obstructive Pulmonary Disease; recruited November 2007-April 2010), and 1670 from ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints; between December 2005-December 2006). EXPOSURES: Interstitial lung abnormality status as determined by chest CT evaluation. MAIN OUTCOMES AND MEASURES: All-cause mortality over an approximate 3- to 9-year median follow-up time. Cause-of-death information was also examined in the AGES-Reykjavik cohort. RESULTS: Interstitial lung abnormalities were present in 177 (7%) of the 2633 participants from FHS, 378 (7%) of 5320 from AGES-Reykjavik, 156 (8%) of 2068 from COPDGene, and in 157 (9%) of 1670 from ECLIPSE. Over median follow-up times of approximately 3 to 9 years, there were more deaths (and a greater absolute rate of mortality) among participants with interstitial lung abnormalities when compared with those who did not have interstitial lung abnormalities in the following cohorts: 7% vs 1% in FHS (6% difference [95% CI, 2% to 10%]), 56% vs 33% in AGES-Reykjavik (23% difference [95% CI, 18% to 28%]), and 11% vs 5% in ECLIPSE (6% difference [95% CI, 1% to 11%]). After adjustment for covariates, interstitial lung abnormalities were associated with a higher risk of death in the FHS (hazard ratio [HR], 2.7 [95% CI, 1.1 to 6.5]; P = .03), AGES-Reykjavik (HR, 1.3 [95% CI, 1.2 to 1.4]; P < .001), COPDGene (HR, 1.8 [95% CI, 1.1 to 2.8]; P = .01), and ECLIPSE (HR, 1.4 [95% CI, 1.1 to 2.0]; P = .02) cohorts. In the AGES-Reykjavik cohort, the higher rate of mortality could be explained by a higher rate of death due to respiratory disease, specifically pulmonary fibrosis. CONCLUSIONS AND RELEVANCE: In 4 separate research cohorts, interstitial lung abnormalities were associated with a greater risk of all-cause mortality. The clinical implications of this association require further investigation.

Despite several observational studies showing that lipoprotein(a), or Lp(a), is associated with myocardial infarction (MI) 1, 2 , only circumstantial evidence exists regarding the causal nature of this association. Observational epidemiological studies, even with a sound prospective design, can provide hints to disease pathogenesis when the effect size is modest but cannot provide definitive evidence for causal relationships. Much of the current understanding of the causal factors in cardiovascular disease, such as the role of LDL, has been confirmed by randomized clinical trials (RCT) However, RCTs are not always feasible. In the case of Lp(a), the modest effect size and the lack of specific Lp(a) lowering therapy are major obstacles to obtaining causal evidence for its role in cardiovascular disease. In this issue of JAMA, Kamstrup and colleagues 5 provide insights using a "Mendelian randomization" approach and provide evidence for the causal role of Lp(a) in MI. This study elegantly demonstrates how Mendelian randomization can be used to improve the evidence for causality from observational studies and highlights the advantages and limitations of such an approach.

BACKGROUND: Limited data exist regarding the use of a genetic risk score (GRS) for predicting risk of incident cardiovascular disease (CVD) in US-based samples. METHODS AND RESULTS: By using findings from recent genome-wide association studies, we constructed GRSs composed of 13 genetic variants associated with myocardial infarction or other manifestations of coronary heart disease (CHD) and 102 genetic variants associated with CHD or its major risk factors. We also updated the 13 single-nucleotide polymorphism (SNP) GRSs with 16 SNPs recently discovered by genome-wide association studies. We estimated the association, discrimination, and risk reclassification of each GRS for incident cardiovascular events and prevalent coronary artery calcium (CAC). In analyses adjusted for age, sex, CVD risk factors, and parental history of CVD, the 13 SNP GRSs were significantly associated with incident hard CHD (hazard ratio, 1.07; 95% CI, 1.00-1.15; P=0.04), CVD (hazard ratio per allele, 1.05; 95% CI, 1.01-1.09; P=0.03), and high CAC (defined as >75(th) age- and sex-specific percentile; odds ratio per allele, 1.18; 95% CI, 1.11-1.26; P=3.4×10(-7)). The GRS did not improve discrimination for incident CHD or CVD but led to modest improvements in risk reclassification. However, significant improvements in discrimination and risk reclassification were observed for the prediction of high CAC. The addition of 16 newly discovered SNPs to the 13 SNP GRSs did not significantly modify these results. CONCLUSIONS: A GRS composed of 13 SNPs associated with coronary disease is an independent predictor of cardiovascular events and of high CAC, modestly improves risk reclassification for incident CHD, and significantly improves discrimination for high CAC. The addition of recently discovered SNPs did not significantly improve the performance of this GRS.

DNA sequencing technologies continue to make progress in increased throughput and quality, and decreased cost. As we transition from whole exome capture sequencing to whole genome sequencing (WGS), our ability to convert machine-generated variant calls, including single nucleotide variant (SNV) and insertion-deletion variants (indels), into human-interpretable knowledge has lagged far behind the ability to obtain enormous amounts of variants. To help narrow this gap, here we present WGSA (WGS annotator), a functional annotation pipeline for human genome sequencing studies, which is runnable out of the box on the Amazon Compute Cloud and is freely downloadable at (https://sites.google.com/site/jpopgen/wgsa/). Functional annotation is a key step in WGS analysis. In one way, annotation helps the analyst filter to a subset of elements of particular interest (eg, cell type specific enhancers), in another way annotation helps the investigators to increase the power of identifying phenotype-associated loci (eg, association test using functional prediction score as a weight) and interpret potentially interesting findings. Currently, there are several popular gene model based annotation tools, including ANNOVAR,1 SnpEff2 and the Ensembl Variant Effect Predictor (VEP).3 These can annotate a variety of protein coding and non-coding gene models from a range of species. It is well known among practitioners that different databases (eg, RefSeq4 and Ensembl5) use different models for …

Transcripts in platelets are largely produced in precursor megakaryocytes but remain physiologically active as platelets translate RNAs and regulate protein/RNA levels. Recent studies using transcriptome sequencing (RNA-seq) characterized the platelet transcriptome in limited number of non-diseased individuals. Here, we expand upon these RNA-seq studies by completing RNA-seq in platelets from 32 patients with acute myocardial infarction (MI). Our goals were to characterize the platelet transcriptome using a population of patients with acute MI and relate gene expression to platelet aggregation measures and ST-segment elevation MI (STEMI) (n = 16) vs. non-STEMI (NSTEMI) (n = 16) subtypes. Similar to other studies, we detected 9565 expressed transcripts, including several known platelet-enriched markers (e.g. PPBP, OST4). Our RNA-seq data strongly correlated with independently ascertained platelet expression data and showed enrichment for platelet-related pathways (e.g. wound response, hemostasis, and platelet activation), as well as actin-related and post-transcriptional processes. Several transcripts displayed suggestively higher (FBXL4, ECHDC3, KCNE1, TAOK2, AURKB, ERG, and FKBP5) and lower (MIAT, PVRL3, and PZP) expression in STEMI platelets compared to NSTEMI. We also identified transcripts correlated with platelet aggregation to TRAP (ATP6V1G2, SLC2A3), collagen (CEACAM1, ITGA2), and ADP (PDGFB, PDGFC, ST3GAL6). Our study adds to current platelet gene expression resources by providing transcriptome-wide analyses in platelets isolated from patients with acute MI. In concert with prior studies, we identify various genes for further study in regards to platelet function and acute MI. Future platelet RNA-seq studies examining more diverse sets of healthy and diseased samples will add to our understanding of platelet thrombotic and non-thrombotic functions.

Genome-wide association studies have previously identified 23 genetic loci associated with circulating fibrinogen concentration. These studies used HapMap imputation and did not examine the X-chromosome. 1000 Genomes imputation provides better coverage of uncommon variants, and includes indels. We conducted a genome-wide association analysis of 34 studies imputed to the 1000 Genomes Project reference panel and including ∼120 000 participants of European ancestry (95 806 participants with data on the X-chromosome). Approximately 10.7 million single-nucleotide polymorphisms and 1.2 million indels were examined. We identified 41 genome-wide significant fibrinogen loci; of which, 18 were newly identified. There were no genome-wide significant signals on the X-chromosome. The lead variants of five significant loci were indels. We further identified six additional independent signals, including three rare variants, at two previously characterized loci: FGB and IRF1. Together the 41 loci explain 3% of the variance in plasma fibrinogen concentration.

White blood cell (WBC) count is a common clinical measure used as a predictor of certain aspects of human health, including immunity and infection status. WBC count is also a complex trait that varies among individuals and ancestry groups. Differences in linkage disequilibrium structure and heterogeneity in allelic effects are expected to play a role in the associations observed between populations. Prior genome-wide association study (GWAS) meta-analyses have identified genomic loci associated with WBC and its subtypes, but much of the heritability of these phenotypes remains unexplained. Using GWAS summary statistics for over 50 000 individuals from three diverse populations (Japanese, African-American and European ancestry), a Bayesian model methodology was employed to account for heterogeneity between ancestry groups. This approach was used to perform a trans-ethnic meta-analysis of total WBC, neutrophil and monocyte counts. Ten previously known associations were replicated and six new loci were identified, including several regions harboring genes related to inflammation and immune cell function. Ninety-five percent credible interval regions were calculated to narrow the association signals and fine-map the putatively causal variants within loci. Finally, a conditional analysis was performed on the most significant SNPs identified by the trans-ethnic meta-analysis (MA), and nine secondary signals within loci previously associated with WBC or its subtypes were identified. This work illustrates the potential of trans-ethnic analysis and ascribes a critical role to multi-ethnic cohorts and consortia in exploring complex phenotypes with respect to variants that lie outside the European-biased GWAS pool.

The study of genetic influences on drug response and efficacy ('pharmacogenetics') has existed for over 50 years. Yet, we still lack a complete picture of how genetic variation, both common and rare, affects each individual's responses to medications. Exome sequencing is a promising alternative method for pharmacogenetic discovery as it provides information on both common and rare variation in large numbers of individuals. Using exome data from 2203 AA and 4300 Caucasian individuals through the NHLBI Exome Sequencing Project, we conducted a survey of coding variation within 12 Cytochrome P450 (CYP) genes that are collectively responsible for catalyzing nearly 75% of all known Phase I drug oxidation reactions. In addition to identifying many polymorphisms with known pharmacogenetic effects, we discovered over 730 novel nonsynonymous alleles across the 12 CYP genes of interest. These alleles include many with diverse functional effects such as premature stop codons, aberrant splicesites and mutations at conserved active site residues. Our analysis considering both novel, predicted functional alleles as well as known, actionable CYP alleles reveals that rare, deleterious variation contributes markedly to the overall burden of pharmacogenetic alleles within the populations considered, and that the contribution of rare variation to this burden is over three times greater in AA individuals as compared with Caucasians. While most of these impactful alleles are individually rare, 7.6-11.7% of individuals interrogated in the study carry at least one newly described potentially deleterious alleles in a major drug-metabolizing CYP.

BACKGROUND: Ectopic fat density is associated with cardiovascular disease (CVD) risk factors above and beyond fat volume. Volumetric measures of ectopic fat have been associated with CVD risk factors and subclinical atherosclerosis. The aim of this study was to investigate the association between fat density and subclinical atherosclerosis. METHODS AND RESULTS: Participants were drawn from the Multi-Detector Computed Tomography (MDCT) substudy of the Framingham Heart Study (n=3079; mean age, 50.1 years; 49.2% women). Fat density was indirectly estimated by computed tomography attenuation (Hounsfield Units [HU]) on abdominal scan slices. Visceral fat (VAT), subcutaneous fat (SAT), and pericardial fat HU and volumes were quantified using standard protocols; coronary and abdominal aortic calcium (CAC and AAC, respectively) were measured radiographically. Multivariable-adjusted logistic regression models were used to evaluate the association between adipose tissue HU and the presence of CAC and AAC. Overall, 17.1% of the participants had elevated CAC (Agatston score [AS]>100), and 23.3% had elevated AAC (AS>age-/sex-specific cutoffs). Per 5-unit decrement in VAT HU, the odds ratio (OR) for elevated CAC was 0.76 (95% confidence interval [CI], 0.65 to 0.89; P=0.0005), even after adjustment for body mass index or VAT volume. Results were similar for SAT HU. With decreasing VAT HU, we also observed an OR of 0.79 (95% CI, 0.67 to 0.92; P=0.004) for elevated AAC after multivariable adjustment. We found no significant associations between SAT HU and AAC. There was no significant association between pericardial fat HU and either CAC or AAC. CONCLUSIONS: Lower VAT and SAT HU, indirect estimates of fat quality, are associated with a lower risk of subclinical atherosclerosis.

Genome-wide association studies, DNA sequencing studies, and other genomic studies are finding an increasing number of genetic variants associated with clinical phenotypes that may be useful in developing diagnostic, preventive, and treatment strategies for individual patients. However, few variants have been integrated into routine clinical practice. The reasons for this are several, but two of the most significant are limited evidence about the clinical implications of the variants and a lack of a comprehensive knowledge base that captures genetic variants, their phenotypic associations, and other pertinent phenotypic information that is openly accessible to clinical groups attempting to interpret sequencing data. As the field of medicine begins to incorporate genome-scale analysis into clinical care, approaches need to be developed for collecting and characterizing data on the clinical implications of variants, developing consensus on their actionability, and making this information available for clinical use. The National Human Genome Research Institute (NHGRI) and the Wellcome Trust thus convened a workshop to consider the processes and resources needed to: (1) identify clinically valid genetic variants; (2) decide whether they are actionable and what the action should be; and (3) provide this information for clinical use. This commentary outlines the key discussion points and recommendations from the workshop.

Background In addition to lowering low density lipoprotein cholesterol (LDL-C), statin therapy also raises high density lipoprotein cholesterol (HDL-C) levels. Inter-individual variation in HDL-C response to statins may be partially explained by genetic variation. Methods and results We performed a meta-analysis of genome-wide association studies (GWAS) to identify variants with an effect on statin-induced high density lipoprotein cholesterol (HDL-C) changes. The 123 most promising signals with p&lt;1×10 −4 from the 16 769 statin-treated participants in the first analysis stage were followed up in an independent group of 10 951 statin-treated individuals, providing a total sample size of 27 720 individuals. The only associations of genome-wide significance (p&lt;5×10 −8 ) were between minor alleles at the CETP locus and greater HDL-C response to statin treatment. Conclusions Based on results from this study that included a relatively large sample size, we suggest that CETP may be the only detectable locus with common genetic variants that influence HDL-C response to statins substantially in individuals of European descent. Although CETP is known to be associated with HDL-C, we provide evidence that this pharmacogenetic effect is independent of its association with baseline HDL-C levels.

Locally acting fat depots may contribute to obesity complications, in particular vascular disease, through direct paracrine effects (1,2). The carotid arteries are encased in fat, and total upper-body subcutaneous fat is estimated by neck circumference (NC). NC has been independently correlated with cardiometabolic risk factors above and beyond that of other adiposity measures (3,4). Carotid wall intima-media thickness (IMT) is a surrogate marker of subclinical atherosclerosis and has been associated with cardiovascular and stroke outcomes (5). The aim of this study was to evaluate the association of NC and carotid wall IMT above and beyond traditional cardiovascular risk factors and generalized adiposity. Framingham Heart Study offspring participants who underwent carotid ultrasonography and NC measurements were included ( n = 3,274; mean age 59 years; 52% women); NC, internal carotid artery (ICA) IMT, and common carotid artery (CCA) IMT were determined as previously described (4,5). Linear regression models evaluated the association between adiposity exposures and IMT. NC, BMI, and …

BACKGROUND: Upper body subcutaneous neck fat (UBSF) is a unique fat depot anatomically separate from visceral abdominal fat that appears to be associated with cardiometabolic risk above and beyond generalized adiposity. We sought to develop a protocol to quantify UBSF using multidetector computed tomography measurements. METHODS AND RESULTS: Protocol development was performed in participants from the Framingham Heart Study who had participated in the multidetector computed tomography scanning substudy, consisting of chest scans. Volumetric assessment of UBSF was defined by 40 contiguous 0.625‐mm slices superior to the body of the sternum. The reader manually traced the chest to identify total neck fat. Breast tissue exterior to the chest wall was excluded. Subcutaneous and visceral fat volumes were obtained using standard protocols. Age‐ and sex‐adjusted Pearson correlation coefficients were used to assess the association among UBSF, traditional adiposity measures, and cardiometabolic risk factors. Inter‐ and intrareader reproducibility was assessed using intraclass correlation coefficients. Volumetric assessments were obtained in 92 participants because 8 scans were not readable (51% women; mean age: 59 years [women], 58 years [men]). The mean volume of UBSF was 310 cm3 for women and 345 cm3 for men. Intra‐ and interreader class correlation coefficients were 0.99 and 0.99, respectively. UBSF was correlated with waist circumference (r=0.90), neck circumference (r=0.75), body mass index (r=0.89), subcutaneous adipose tissue (r=0.87), and visceral adipose tissue (r=0.86). CONCLUSIONS: UBSF can be quantified reproducibly using computed tomography in a community‐dwelling sample from the Framingham Heart Study.

This study investigated whether postmenopausal women on HRT would experience a greater reduction in oxidative stress after 24 weeks of aerobic exercise training compared to postmenopausal women not on HRT. Plasma thiobarbituric acid reactive substances (TBARS), an indicator of oxidative stress, was measured in 48 previously sedentary postmenopausal women on HRT (n = 21) and not on HRT (n = 27) before and after 24 weeks of aerobic exercise training. Baseline levels of TBARS differed significantly between groups after controlling for age, BMI, and fasting blood glucose (P = 0.03). There was a significant reduction in TBARS after 24 weeks of training in the overall group. When analyzed separately, both postmenopausal women on HRT and those not on HRT had a significant reduction in TBARS; however, there was no significant difference between groups (-0.71 +/- 0.14 nmol/ml in non-HRT users vs. -0.50 +/- 0.16 nmol/ml in HRT users; P = 0.33) even after controlling for age, BMI, and baseline levels of TBARS. Our results showed that aerobic exercise training significantly decreased oxidative stress in postmenopausal women; however, both HRT users and non-HRT users benefited equally.

Plasma fibrinogen is an acute phase protein playing an important role in the blood coagulation cascade having strong associations with smoking, alcohol consumption and body mass index (BMI). Genome-wide association studies (GWAS) have identified a variety of gene regions associated with elevated plasma fibrinogen concentrations. However, little is yet known about how associations between environmental factors and fibrinogen might be modified by genetic variation. Therefore, we conducted large-scale meta-analyses of genome-wide interaction studies to identify possible interactions of genetic variants and smoking status, alcohol consumption or BMI on fibrinogen concentration. The present study included 80,607 subjects of European ancestry from 22 studies. Genome-wide interaction analyses were performed separately in each study for about 2.6 million single nucleotide polymorphisms (SNPs) across the 22 autosomal chromosomes. For each SNP and risk factor, we performed a linear regression under an additive genetic model including an interaction term between SNP and risk factor. Interaction estimates were meta-analysed using a fixed-effects model. No genome-wide significant interaction with smoking status, alcohol consumption or BMI was observed in the meta-analyses. The most suggestive interaction was found for smoking and rs10519203, located in the LOC123688 region on chromosome 15, with a p value of 6.2 × 10(-8). This large genome-wide interaction study including 80,607 participants found no strong evidence of interaction between genetic variants and smoking status, alcohol consumption or BMI on fibrinogen concentrations. Further studies are needed to yield deeper insight in the interplay between environmental factors and gene variants on the regulation of fibrinogen concentrations.

Transplantation is a remarkable treatment that gives a “second chance” of life to patients with end-stage organ failure. Unfortunately, this exposes the transplanted organ or allograft to the recipient’s immunity, predisposing the allograft to acute rejection, a dreadful complication, and a major risk factor for allograft loss. To preserve the allograft’s health, providers carefully monitor their patients to maintain immunosuppression adequacy, toward balancing the risk of rejection and infection. Monitoring approaches vary substantially among providers and transplant programs. Generally, monitoring encompasses biopsy of the allograft for histopathology and light microscopy to assess rejection, measurement of the allograft function (creatinine for kidney, pulmonary function test for lung, liver function test for liver, echocardiography for heart), measurement of immune activity with donor-specific antibody (DSA) testing, and assessment of immunosuppression adequacy by measuring immunosuppression drug blood concentrations. Assessment of allograft function or immunosuppression drug concentrations uses noninvasive testing approaches. Thus, these tests are generally implemented as surveillance, wherein patients undergo testing at predetermined posttransplant intervals independent of symptoms or clinical suspicion of allograft dysfunction. When patients present with signs for allograft dysfunction, additional clinical indication testing is performed. Biopsy, on the other hand, is an invasive procedure associated with procedure-related complications. Further, biopsy samples are analyzed by routine histopathology and light microscopy, which have limited sensitivity and precision to adequately detect and phenotype rejection and nonrejection complications. As a result, practice varies; some centers perform surveillance plus clinically indicated biopsies. Other centers only perform clinically indicated biopsies. In lung transplantation, for example, 70% of centers in the USA perform surveillance plus clinically indicated biopsies while 30% of centers perform only clinically indicated biopsies. The true benefit of these different monitoring approaches remains undefined. Fortunately, in the last few decades, novel molecular approaches have been introduced; many show promising results with benefits that address the limitations of biopsy plus conventional histopathology. In this Q&A, we focus on 2 prototype tools that recently received approval for patient use by the Centers for Medicare and Medicaid Services of the United States: plasma-based donor-derived cell-free DNA (dd-cfDNA) and “molecular biopsy,” the latter to indicate gene expression profiling of biopsy tissue samples using microarray and other platforms. Five experts discuss the potential use of these novel tools to monitor solid organ transplant patients. Michael Oellerich: dd-cfDNA is a minimally invasive quantitative biomarker for the detection of graft injury. As organ transplants are also genome transplants, dd-cfDNA opens up the possibility to monitor allograft health. In case of graft cell death, nucleosomes are released into the bloodstream as cfDNA. The clinical validity of dd-cfDNA to detect or exclude rejection and other graft injuries has been documented in more than 50 studies. Increases of dd-cfDNA levels were found in transplant recipients several days or even up to 3 months before clinical manifestation of acute rejection. There has been discussion that increased dd-cfDNA levels may be a trigger of inflammation. Early detection of in particular subclinical antibody-mediated rejection enables adapted therapeutic interventions and may improve outcomes. This needs to be further investigated in controlled studies with different dd-cfDNA tests. In kidney transplantation, dd-cfDNA may be useful to early detect antibody-mediated rejection in DSA positive patients. An adequate diagnostic performance of dd-cfDNA is suggested by currently published studies for detection of acute rejection [ROC area under curve (AUC) = 0.81]. Clinical sensitivity and specificity are around 80% and 76%, respectively. The high negative predictive value of 90% suggests that dd-cfDNA is useful to exclude rejection. Therefore, dd-cfDNA could be helpful to avoid unnecessary biopsies triggered by increased plasma creatinine. After successful rejection treatment, dd-cfDNA rapidly declined in liver, heart, or kidney transplant recipients. There are several limitations of dd-cfDNA. Increases of dd-cfDNA are not rejection specific. Other sources of graft injury associated with increase of dd-cfDNA include for example, pyelonephritis, acute tubular necrosis, and BK-virus nephropathy in kidney transplant recipients. In general, patients with advanced interstitial fibrosis tubular atrophy showed only a relatively small increase of dd-cfDNA and a discrimination of values from patients with normal histology was not possible. During stages of active disease progression, dd-cfDNA levels might be increased in interstitial fibrosis tubular atrophy. The detection of T cell-mediated rejection seems to be influenced by the employed test procedure. False-negative results may be due to the use of relatively long amplicons (100–130 bp) in the assay used. dd-cfDNA test results should be interpreted in context of all available relevant clinical data and diagnostic findings to achieve individualized transplant patient therapy with the potential to reduce graft loss and to save costs. Based on currently available evidence regarding clinical validity, dd-cfDNA seems to be useful for surveillance of transplant recipients and for clinically indicated testing. Further optimization of dd-cfDNA testing for effective clinical use is an ongoing challenge. Philip Halloran: Surveillance: no. Clinically indicated: cautious yes, if a probabilistic algorithm to guide the use of the information to avoid biopsy can be defined based on continuous numbers, not cutoffs. The utility of commercially available dd-cfDNA tests in surveillance is limited by high cost. Repeated use of an expensive surveillance test in stable patients is probably not justified without indications. dd-cfDNA has not been shown to actually reduce biopsies and to create savings, and it may be producing unnecessary biopsies. We need to know what to do with the many ambiguous values for dd-cfDNA in the commercial assays because they have the potential to trigger unnecessary biopsies and investigations. We need good algorithms that state the probability of rejection over the whole range of dd-cfDNA values. Michael Keller: Plasma dd-cfDNA is a noninvasive quantitative molecular biomarker that increases in the setting of allograft injury. Recent evidence suggests that the rise in dd-cfDNA may precede clinical manifestations of allograft injury by several months. Several observational cohort studies performed in kidney, heart, and lung transplant recipients indicate that dd-cfDNA displays acceptable performance characteristics for the detection of acute rejection—both acute cellular rejection and antibody-mediated rejection. In particular, dd-cfDNA demonstrates a high negative predictive value for acute rejection, providing the potential to effectively “rule out” the majority of acute rejection events. These characteristics make plasma dd-cfDNA appealing as a method of surveillance monitoring for underlying allograft injury. Despite a paucity of high-quality evidence supporting its use, most heart and lung transplant recipients undergo routine surveillance biopsy as standard of care to screen for acute rejection. As a screening tool, these biopsies provide procedural risks, are costly, and are often inconvenient. In addition, analysis of histopathology has high interobserver variability in the pathologic grading of biopsy samples. Dd-cfDNA provides a safe, accurate, and minimally invasive method of screening patients who may benefit most from proceeding to biopsy. A high-quality, randomized control trial comparing a dd-cfDNA method of surveillance monitoring in comparison to traditional surveillance biopsy in heart and lung transplant recipients would be especially helpful to further validate its use. In addition, further validation of specific threshold values to indicate the presence of acute rejection would be beneficial. Based on the available evidence, it is reasonable to utilize dd-cfDNA for surveillance monitoring in select patient populations such as heart and lung transplant recipients at high risk for procedural complications from biopsy or during periods of time when the performance of surveillance biopsy is limited, such as during periods of the COVID-19 pandemic. Iwijn De Vlaminck: One promising feature of dd-cfDNA is that it is an early marker of graft injury. It’s therefore reasonable to integrate dd-cfDNA in surveillance testing and not just clinically indicated testing. There are practical limitations that need be overcome, however, including the current high cost of the assay. Michael Oellerich: Both approaches, fractional and absolute determination of dd-cfDNA in kidney transplant recipients, have strengths and weaknesses. Fractional determination has the advantage that it is less sensitive to preanalytical variables (e.g., DNA extraction efficiency). Measurements can be easily compared between different studies. Fractional determination is insensitive to changes in the rate of degradation of cfDNA in blood circulation. It has, however, the disadvantage that it is affected by changes in recipient cfDNA (e.g., by infection or exercise). Leukocytosis or leukopenia can alter dd-cfDNA fraction as recipient cfDNA accounts for the major part of the denominator in fractional quantification. This can result in false-negative or false-positive results. During long-term surveillance dd-cfDNA fraction increases due to a decrease of total cfDNA so that the threshold would have to be adapted. The decline of total cfDNA with time after kidney transplantation is presumably due to a decrease in apoptosis rate for white blood cells as immunosuppressant drug doses are tapered off. Absolute quantification (cp/mL) has the advantage that it is not affected by changes in recipient cfDNA by other factors and that there is no influence on the threshold due to total cfDNA decline over time. In clinical practice, the combination of fractional and absolute determination seems to provide the most comprehensive diagnostic information in kidney transplant recipients. Philip Halloran: Absolutely. The use of ratios is never desirable when both the numerator and the denominator can change. Michael Keller: Plasma dd-cfDNA is traditionally reported as a percentage, representing the fraction of donor to donor + recipient cfDNA. While this method may help account for differences between the relative sizes of recipients and donor allografts, there are inherent limitations to this approach. Due to its presence in the denominator, levels of recipient cfDNA in the plasma will influence %dd-cfDNA. Recipient cfDNA may rise in a number of conditions irrespective of the status of the allograft including in the setting of sepsis, multiorgan failure, extreme exercise, and leukocytosis. This may make the interpretation of %dd-cfDNA difficult under conditions that alter the levels of recipient cfDNA. It also raises questions surrounding the validity of specific threshold values of %dd-cfDNA in the detection and diagnosis of various types of allograft injury. Established threshold values for %dd-cfDNA may carry more weight if validated in stable controls and performed on otherwise stable outpatient transplant recipients. However, it may be difficult to interpret %dd-cfDNA in the setting of systemic illness or nonallograft related organ failure, such as in hospitalized patients. Measurements of the absolute amount of plasma dd-cfDNA are not influenced by recipient cfDNA. However, differences in the sizes of allografts—particularly in lung transplant where total tissue mass may differ between individuals to a considerable degree—may impact the ability to interpret prespecified threshold values for the detection of allograft injury. Observational cohort studies in kidney transplant patients have demonstrated similar performance characteristics between absolute and %dd-cfDNA for the detection of acute rejection. There are strengths and weaknesses of both methods, however, developing a diagnostic algorithm using a combination of the 2 methods may be the optimal approach. Iwijn De Vlaminck: It’s clear that a readout of the relative proportion of donor DNA can be confounded by changes in recipient DNA that are not related to the health of the transplant organ. The absolute burden of donor DNA in blood does not have that issue, and may therefore provide a more robust readout of transplant health, but this will need to be confirmed in clinical studies. Measurements of the absolute amount of donor DNA may prove more difficult to standardize and may be sensitive to changes in the rate of clearance of cfDNA from the blood circulation. Perhaps models that integrate both the absolute and relative burden of cfDNA will prove to be optimal. Philip Halloran: Surveillance: no for kidney, surveillance biopsies are not justified for kidney; yes for heart because histology kappa values are poor. Clinically indicated: cautious yes. The clinician needs the quantitative assessments that only molecular assessment can provide, although we need a probabilistic algorithm to guide the use of the information to avoid biopsy based on continuous numbers not cutoffs. Iwijn De Vlaminck: The molecular biopsy is a great technology that enables detailed profiling of different pathways of transplant rejection. It represents an important advance in transplant medicine, but because it relies on invasive tissue biopsies it has a limited role to play as a surveillance tool, and is more relevant for clinically indicated testing. An ideal test would achieve the resolution in cellular pathology of the molecular biopsy but from blood rather than from tissue. Angela Wu: Most of our work has been focused on monitoring the interactions between microbes and their hosts in a clinical setting, whether these microbes are pathogenic or not. We have found that in addition to monitoring immunosuppression, the metagenomic composition of “molecular biopsy” samples can also be highly informative as to the health status of the patient. When used in combination with dd-cfDNA, host-associated biomarkers, and other clinical measures, this metagenomic information can enhance the accuracy of diagnosis and outcome prediction. Based on our investigations, “molecular biopsy” results can be strongly affected by different practices at different centers. In particular, if considering additionally the microbial component, the environmental microbes can be hugely different at different sites, affecting analysis outcomes and downstream decision-making. To ensure robustness of the sample handling and results, it is preferable to perform sample processing at a central location. Additional molecular based methods for preserving sample integrity and reducing loss/degradation of cfDNA are also being developed and streamlined to improve the logistical challenges associated with sending samples to central facilities. Michael Oellerich: For routine implementation of dd-cfDNA testing there are a number of challenges. The laboratory requires specific instrumentation, such as a ddPCR or a next generation sequencing instrument as well as specialized laboratory professionals. Standard clinical chemistry assay validation is necessary for these tests and the development of external quality management programs is recommended. Furthermore, clinical practice recommendations have to be developed. Centralized reference laboratories may have the advantage of lower testing costs and high quality. Individual laboratories in transplant centers have the advantage of shorter turnaround times of dd-cfDNA test results for hospitalized patients if the test would have to be used for actual decision-making. For out-patients, services from centralized reference laboratories would allow for a broader A monitoring should be in clinical outcome reasonable the cost for dd-cfDNA monitoring would be relatively reducing graft it is to be that costs for a to or could be substantially using dd-cfDNA testing and other relevant approaches the molecular biopsy. Philip Halloran: if at The performance of molecular assays as has been in as shown by the measurement of DSA using and and (e.g., This is true when the results use algorithms for results on a different assays should generally be central the of and practices are and for on an ongoing Michael Keller: the assays and plasma dd-cfDNA should be performed in centralized to maintain assay reduce in results, and The and of the technology to the assays may its implementation in many transplant centers. in methods and assay also a when implemented at centers. While the assay at a centralized may increase turnaround this may not present an in several clinical such as surveillance monitoring or to Iwijn De Vlaminck: The molecular biopsy is a great technology that enables detailed profiling of different pathways of transplant rejection. It represents an important advance in transplant medicine, but because it relies on invasive tissue biopsies it has a limited role to play as a surveillance and is more relevant for clinically indicated testing. An ideal test would achieve the resolution in cellular pathology of the molecular biopsy but from blood rather than from tissue. Angela Wu: As our work has focused on the metagenomic of which both donor and microbes and Monitoring of microbial and in cfDNA has been demonstrated as a to immunosuppression adequacy and also the need for There are 2 major that can be further investigated in this detection and monitoring of specific microbes of and surveillance of microbial and as a for the For the it is that are and could be from donor to recipient the transplanted in some In this or detection with methods may be detection of cfDNA or from these of and lower cost. However, assays from other such as in the case of or to The of an between total cfDNA sequencing and can be further sequencing or sequencing that uses to potential by to specific could be a to ensure both specificity and In addition, current commercial assays which have relatively and long if cfDNA can be into the detection by also DNA this could also improve detection Michael Oellerich: dd-cfDNA may be helpful to assess necessary with and to provide information for optimization of It can guide in patients to immune of immunosuppressant is often necessary to and dd-cfDNA may be helpful to detect and improve immunosuppressant DSA development that is a risk factor for antibody-mediated rejection and graft loss in kidney concentrations were associated in patients with increased dd-cfDNA values immune dd-cfDNA is to drug monitoring that but is a of graft due to conventional have In kidney transplant recipients, for example, a of graft can be present by the time a rise in plasma is plasma is not specific for allograft injury. Due to a of validation studies for monitoring immune activity or expression of are not used. biopsies are clinically and associated with a major with dd-cfDNA seems to be useful in patients on who are at increased risk of complications from biopsies. dd-cfDNA is more practical than biopsies to detect in immune This is especially important in kidney transplant patients with high and high immune DNA is a diagnostic for monitoring transplant patients immunosuppression Philip Halloran: that they detect rejection. Monitoring would be and and these can be in indication biopsies by central molecular testing. dd-cfDNA active rejection, so in a that this a of Michael Keller: such as the and are generally to achieve concentrations in solid organ transplant recipients. Due to the of various factors and drug the doses to achieve these concentrations may vary between levels may provide adequate immunosuppression for some patients other patients may of acute rejection at the variability and in concentrations In a ability to detect of acute rejection as a marker of immunosuppression however, of the of immunosuppression is to the of acute rejection in the As a quantitative biomarker of underlying allograft dd-cfDNA may allow for the of to achieve a of immunosuppression that is individualized to the recipient and the of acute rejection. While promising in further studies are to and validate its role for this use. Iwijn De Vlaminck: no that quantitative of immunosuppression are in transplant dd-cfDNA, however, provides only an indication of that the immune cell of immune cells or the burden of in blood may provide a more readout of immunosuppression adequacy in the Angela Wu: studies of dd-cfDNA that focus not only on the of the DNA but also additional such as or microbial composition are all additional information that the utility of cfDNA as a for organ health, disease and immune In the combination of these approaches will allow to assay for monitoring and as well as There is no that the of these types of assays and assessments are informative and can improve but the of information needs to be with the clinical need and costs to be in a Additional that can this information from cfDNA at cost and with of will be to mass Michael Oellerich: dd-cfDNA will be helpful to achieve immunosuppression in transplant recipients with the potential to reduce graft loss. Additional is to of dd-cfDNA with other such as and as well as a gene to further improve diagnostic randomized clinical utility studies are necessary to show that dd-cfDNA changes in treatment will improve outcomes. Philip Halloran: The for the commercial assays is that values indicate probability of active rejection, and high values are due to active antibody-mediated rejection or T cell-mediated rejection are values are and resolution on such values should guide is we need more probabilistic information a result is the time in a patient with no and in a patient with for biopsy and suspicion of rejection. Michael Keller: dd-cfDNA is an with the potential to provide a of useful clinical including the detection of allograft an assessment of to treatment of acute rejection, monitoring of the adequacy of immunosuppression, and Further validation of its clinical utility and into routine clinical care may the of solid organ transplant into an of precision and care The with other molecular approaches demonstrates further that expression of such as molecular diagnostic may molecular that in the detection and discrimination of various types of allograft may also provide further into in which in dd-cfDNA are not by evidence of allograft injury. While the may be further is necessary to several to its interpretation and clinical Iwijn De Vlaminck: dd-cfDNA is a marker of graft tissue injury. In this a major but also a major donor DNA does not provide into the of the graft injury. molecular approaches can be that provide more detailed information the cell and tissue types that are and this in may between different of graft including infection and and different pathways of transplant rejection. Recent in assays that and specific cfDNA may a confirmed they have to the of this and have the to the and of or analysis and interpretation of or the for approval of the published and to be for all of the that questions related to the accuracy or integrity of part of the are investigated and all the potential of De De One of and De the of of the Clinical the a from the and the One De at the Clinical of for

Abstract Lymphangioleiomyomatosis (LAM) is a rare progressive disease, characterized by mutations in the tuberous sclerosis complex genes ( Tsc1 or Tsc2 ), and hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1). The effectiveness of mTORC1 inhibitors is limited by their lack of cytotoxic effects. Here, we report that E26 transformation specific (ETS) Variant Transcription Factor 2 (ETV2) is a critical regulator of Tsc2-deficient cell survival. Nuclear localization of ETV2 in Tsc2-deficient cells is mTORC1-independent and is enhanced by spleen tyrosine kinase (Syk) inhibition. In the nucleus, ETV2 transcriptionally regulates poly(ADP-ribose) polymerase 1 binding protein (PARPBP), a coregulator of transcription, mRNA and protein expression. Silencing of ETV2 or PARPBP in Tsc2-deficient cells induced ER-stress and increased cell death in vitro and in vivo . We also found ETV2 expression in human cells with loss of heterozygosity for TSC2 lending support to the translational relevance of our findings. In conclusion, we report a novel signaling axis unique to Syk-inhibition is mTORC1-independent and promotes a cytocidal response in Tsc2-deficient cells, and therefore, maybe a potential alternative therapeutic target in LAM.