Effect of deletion of the protein kinase PRKD1 on development of the mouse embryonic heart.

Congenital heart disease (CHD) is the most common congenital anomaly, with an overall incidence of approximately 1% in the United Kingdom. Exome sequencing in large CHD cohorts has been performed to provide insights into the genetic aetiology of CHD. This includes a study of 1891 probands by our group in collaboration with others, which identified three novel genes-CDK13, PRKD1, and CHD4, in patients with syndromic CHD. PRKD1 encodes a serine/threonine protein kinase, which is important in a variety of fundamental cellular functions. Individuals with a heterozygous mutation in PRKD1 may have facial dysmorphism, ectodermal dysplasia and may have CHDs such as pulmonary stenosis, atrioventricular septal defects, coarctation of the aorta and bicuspid aortic valve. To obtain a greater appreciation for the role that this essential protein kinase plays in cardiogenesis and CHD, we have analysed a Prkd1 transgenic mouse model (Prkd1em1 ) carrying deletion of exon 2, causing loss of function. High-resolution episcopic microscopy affords detailed morphological 3D analysis of the developing heart and provides evidence for an essential role of Prkd1 in both normal cardiac development and CHD. We show that homozygous deletion of Prkd1 is associated with complex forms of CHD such as atrioventricular septal defects, and bicuspid aortic and pulmonary valves, and is lethal. Even in heterozygotes, cardiac differences occur. However, given that 97% of Prkd1 heterozygous mice display normal heart development, it is likely that one normal allele is sufficient, with the defects seen most likely to represent sporadic events. Moreover, mRNA and protein expression levels were investigated by RT-qPCR and western immunoblotting, respectively. A significant reduction in Prkd1 mRNA levels was seen in homozygotes, but not heterozygotes, compared to WT littermates. While a trend towards lower PRKD1 protein expression was seen in the heterozygotes, the difference was only significant in the homozygotes. There was no compensation by the related Prkd2 and Prkd3 at transcript level, as evidenced by RT-qPCR. Overall, we demonstrate a vital role of Prkd1 in heart development and the aetiology of CHD.

Long-term exposure to traffic-related air pollution and non-accidental mortality: A systematic review and meta-analysis.

BACKGROUND: The health effects of traffic-related air pollution (TRAP) continue to be of important public health interest across the globe. Following its 2010 review, the Health Effects Institute appointed a new expert Panel to systematically evaluate the epidemiological evidence regarding the associations between long-term exposure to TRAP and selected health outcomes. This paper describes the main findings of the systematic review on non-accidental mortality. METHODS: The Panel used a systematic approach to conduct the review. An extensive search was conducted of literature published between 1980 and 2019. A new exposure framework was developed to determine whether a study was sufficiently specific to TRAP, which included studies beyond the near-roadway environment. We performed random-effects meta-analysis when at least three estimates were available of an association between a specific exposure and outcome. We evaluated confidence in the evidence using a modified Office of Health Assessment and Translation (OHAT) approach, supplemented with a broader narrative synthesis. RESULTS: Thirty-six cohort studies were included. Virtually all studies adjusted for a large number of individual and area-level covariates-including smoking, body mass index, and individual and area-level socioeconomic status-and were judged at a low or moderate risk for bias. Most studies were conducted in North America and Europe, and a few were based in Asia and Australia. The meta-analytic summary estimates for nitrogen dioxide, elemental carbon and fine particulate matter-pollutants with more than 10 studies-were 1.04 (95% CI 1.01, 1.06), 1.02 (1.00, 1.04) and 1.03 (1.01, 1.05) per 10, 1 and 5 µg/m3, respectively. Effect estimates are interpreted as the relative risk of mortality when the exposure differs with the selected increment. The confidence in the evidence for these pollutants was judged as high, because of upgrades for monotonic exposure-response and consistency across populations. The consistent findings across geographical regions, exposure assessment methods and confounder adjustment resulted in a high confidence rating using a narrative approach as well. CONCLUSIONS: The overall confidence in the evidence for a positive association between long-term exposure to TRAP and non-accidental mortality was high.

Long-term exposure to traffic-related air pollution and stroke: A systematic review and meta-analysis.

BACKGROUND: Stroke remains the second cause of death worldwide. The mechanisms underlying the adverse association of exposure to traffic-related air pollution (TRAP) with overall cardiovascular disease may also apply to stroke. Our objective was to systematically evaluate the epidemiological evidence regarding the associations of long-term exposure to TRAP with stroke. METHODS: PubMed and LUDOK electronic databases were searched systematically for observational epidemiological studies from 1980 through 2019 on long-term exposure to TRAP and stroke with an update in January 2022. TRAP was defined according to a comprehensive protocol based on pollutant and exposure assessment methods or proximity metrics. Study selection, data extraction, risk of bias (RoB) and confidence assessments were conducted according to standardized protocols. We performed meta-analyses using random effects models; sensitivity analyses were assessed by geographic area, RoB, fatality, traffic specificity and new studies. RESULTS: Nineteen studies were included. The meta-analytic relative risks (and 95% confidence intervals) were: 1.03 (0.98-1.09) per 1 µg/m3 EC, 1.09 (0.96-1.23) per 10 µg/m3 PM10, 1.08 (0.89-1.32) per 5 µg/m3 PM2.5, 0.98 (0.92; 1.05) per 10 µg/m3 NO2 and 0.99 (0.94; 1.04) per 20 µg/m3 NOx with little to moderate heterogeneity based on 6, 5, 4, 7 and 8 studies, respectively. The confidence assessments regarding the quality of the body of evidence and separately regarding the presence of an association of TRAP with stroke considering all available evidence were rated low and moderate, respectively. CONCLUSION: The available literature provides low to moderate evidence for an association of TRAP with stroke.

Mortality-Air Pollution Associations in Low Exposure Environments (MAPLE): Phase 2.

INTRODUCTION: Mortality is associated with long-term exposure to fine particulate matter (particulate matter ≤2.5 µm in aerodynamic diameter; PM2.5), although the magnitude and form of these associations remain poorly understood at lower concentrations. Knowledge gaps include the shape of concentration-response curves and the lowest levels of exposure at which increased risks are evident and the occurrence and extent of associations with specific causes of death. Here, we applied improved estimates of exposure to ambient PM2.5 to national population-based cohorts in Canada, including a stacked cohort of 7.1 million people who responded to census year 1991, 1996, or 2001. The characterization of the shape of the concentration-response relationship for nonaccidental mortality and several specific causes of death at low levels of exposure was the focus of the Mortality-Air Pollution Associations in Low Exposure Environments (MAPLE) Phase 1 report. In the Phase 1 report we reported that associations between outdoor PM2.5 concentrations and nonaccidental mortality were attenuated with the addition of ozone (O3) or a measure of gaseous pollutant oxidant capacity (Ox), which was estimated from O3 and nitrogen dioxide (NO2) concentrations. This was motivated by our interests in understanding both the effects air pollutant mixtures may have on mortality and also the role of O3 as a copollutant that shares common sources and precursor emissions with those of PM2.5. In this Phase 2 report, we further explore the sensitivity of these associations with O3 and Ox, evaluate sensitivity to other factors, such as regional variation, and present ambient PM2.5 concentration-response relationships for specific causes of death. METHODS: PM2.5 concentrations were estimated at 1 km2 spatial resolution across North America using remote sensing of aerosol optical depth (AOD) combined with chemical transport model (GEOS-Chem) simulations of the AOD:surface PM2.5 mass concentration relationship, land use information, and ground monitoring. These estimates were informed and further refined with collocated measurements of PM2.5 and AOD, including targeted measurements in areas of low PM2.5 concentrations collected at five locations across Canada. Ground measurements of PM2.5 and total suspended particulate matter (TSP) mass concentrations from 1981 to 1999 were used to backcast remote-sensing-based estimates over that same time period, resulting in modeled annual surfaces from 1981 to 2016.Annual exposures to PM2.5 were then estimated for subjects in several national population-based Canadian cohorts using residential histories derived from annual postal code entries in income tax files. These cohorts included three census-based cohorts: the 1991 Canadian Census Health and Environment Cohort (CanCHEC; 2.5 million respondents), the 1996 CanCHEC (3 million respondents), the 2001 CanCHEC (3 million respondents), and a Stacked CanCHEC where duplicate records of respondents were excluded (Stacked CanCHEC; 7.1 million respondents). The Canadian Community Health Survey (CCHS) mortality cohort (mCCHS), derived from several pooled cycles of the CCHS (540,900 respondents), included additional individual information about health behaviors. Follow-up periods were completed to the end of 2016 for all cohorts. Cox proportional hazard ratios (HRs) were estimated for nonaccidental and other major causes of death using a 10-year moving average exposure and 1-year lag. All models were stratified by age, sex, immigrant status, and where appropriate, census year or survey cycle. Models were further adjusted for income adequacy quintile, visible minority status, Indigenous identity, educational attainment, labor-force status, marital status, occupation, and ecological covariates of community size, airshed, urban form, and four dimensions of the Canadian Marginalization Index (Can-Marg; instability, deprivation, dependency, and ethnic concentration). The mCCHS analyses were also adjusted for individual-level measures of smoking, alcohol consumption, fruit and vegetable consumption, body mass index (BMI), and exercise behavior.In addition to linear models, the shape of the concentration-response function was investigated using restricted cubic splines (RCS). The number of knots were selected by minimizing the Bayesian Information Criterion (BIC). Two additional models were used to examine the association between nonaccidental mortality and PM2.5. The first is the standard threshold model defined by a transformation of concentration equaling zero if the concentration was less than a specific threshold value and concentration minus the threshold value for concentrations above the threshold. The second additional model was an extension of the Shape Constrained Health Impact Function (SCHIF), the eSCHIF, which converts RCS predictions into functions potentially more suitable for use in health impact assessments. Given the RCS parameter estimates and their covariance matrix, 1,000 realizations of the RCS were simulated at concentrations from the minimum to the maximum concentration, by increments of 0.1 µg/m3. An eSCHIF was then fit to each of these RCS realizations. Thus, 1,000 eSCHIF predictions and uncertainty intervals were determined at each concentration within the total range.Sensitivity analyses were conducted to examine associations between PM2.5 and mortality when in the presence of, or stratified by tertile of, O3 or Ox. Additionally, associations between PM2.5 and mortality were assessed for sensitivity to lower concentration thresholds, where person-years below a threshold value were assigned the mean exposure within that group. We also examined the sensitivity of the shape of the nonaccidental mortality-PM2.5 association to removal of person-years at or above 12 µg/m3 (the current U.S. National Ambient Air Quality Standard) and 10 µg/m3 (the current Canadian and former [2005] World Health Organization [WHO] guideline, and current WHO Interim Target-4). Finally, differences in the shapes of PM2.5-mortality associations were assessed across broad geographic regions (airsheds) within Canada. RESULTS: The refined PM2.5 exposure estimates demonstrated improved performance relative to estimates applied previously and in the MAPLE Phase 1 report, with slightly reduced errors, including at lower ranges of concentrations (e.g., for PM2.5 <10 µg/m3).Positive associations between outdoor PM2.5 concentrations and nonaccidental mortality were consistently observed in all cohorts. In the Stacked CanCHEC analyses (1.3 million deaths), each 10-µg/m3 increase in outdoor PM2.5 concentration corresponded to an HR of 1.084 (95% confidence interval [CI]: 1.073 to 1.096) for nonaccidental mortality. For an interquartile range (IQR) increase in PM2.5 mass concentration of 4.16 µg/m3 and for a mean annual nonaccidental death rate of 92.8 per 10,000 persons (over the 1991-2016 period for cohort participants ages 25-90), this HR corresponds to an additional 31.62 deaths per 100,000 people, which is equivalent to an additional 7,848 deaths per year in Canada, based on the 2016 population. In RCS models, mean HR predictions increased from the minimum concentration of 2.5 µg/m3 to 4.5 µg/m3, flattened from 4.5 µg/m3 to 8.0 µg/m3, then increased for concentrations above 8.0 µg/m3. The threshold model results reflected this pattern with -2 log-likelihood values being equal at 2.5 µg/m3 and 8.0 µg/m3. However, mean threshold model predictions monotonically increased over the concentration range with the lower 95% CI equal to one from 2.5 µg/m3 to 8.0 µg/m3. The RCS model was a superior predictor compared with any of the threshold models, including the linear model.In the mCCHS cohort analyses inclusion of behavioral covariates did not substantially change the results for both linear and nonlinear models. We examined the sensitivity of the shape of the nonaccidental mortality-PM2.5 association to removal of person-years at or above the current U.S. and Canadian standards of 12 µg/m3 and 10 µg/m3, respectively. In the full cohort and in both restricted cohorts, a steep increase was observed from the minimum concentration of 2.5 µg/m3 to 5 µg/m3. For the full cohort and the <12 µg/m3 cohort the relationship flattened over the 5 to 9 µg/m3 range and then increased above 9 µg/m3. A similar increase was observed for the <10 µg/m3 cohort followed by a clear decline in the magnitude of predictions over the 5 to 9 µg/m3 range and an increase above 9 µg/m3. Together these results suggest that a positive association exists for concentrations >9 µg/m3 with indications of adverse effects on mortality at concentrations as low as 2.5 µg/m3.Among the other causes of death examined, PM2.5 exposures were consistently associated with an increased hazard of mortality due to ischemic heart disease, respiratory disease, cardiovascular disease, and diabetes across all cohorts. Associations were observed in the Stacked CanCHEC but not in all other cohorts for cerebrovascular disease, pneumonia, and chronic obstructive pulmonary disease (COPD) mortality. No significant associations were observed between mortality and exposure to PM2.5 for heart failure, lung cancer, and kidney failure.In sensitivity analyses, the addition of O3 and Ox attenuated associations between PM2.5 and mortality. When analyses were stratified by tertiles of copollutants, associations between PM2.5 and mortality were only observed in the highest tertile of O3 or Ox. Across broad regions of Canada, linear HR estimates and the shape of the eSCHIF varied substantially, possibly reflecting underlying differences in air pollutant mixtures not characterized by PM2.5 mass concentrations or the included gaseous pollutants. Sensitivity analyses to assess regional variation in population characteristics and access to healthcare indicated that the observed regional differences inconcentration-mortality relationships, specifically the flattening of the concentration-mortality relationship over the 5 to 9 µg/m3 range, was not likely related to variation in the makeup of the cohort or its access to healthcare, lending support to the potential role of spatially varying air pollutant mixtures not sufficiently characterized by PM2.5 mass concentrations. CONCLUSIONS: In several large, national Canadian cohorts, including a cohort of 7.1 million unique census respondents, associations were observed between exposure to PM2.5 with nonaccidental mortality and several specific causes of death. Associations with nonaccidental mortality were observed using the eSCHIF methodology at concentrations as low as 2.5 µg/m3, and there was no clear evidence in the observed data of a lower threshold, below which PM2.5 was not associated with nonaccidental mortality.

Long-term exposure to traffic-related air pollution and selected health outcomes: A systematic review and meta-analysis.

The health effects of traffic-related air pollution (TRAP) continue to be of important public health interest. Following its well-cited 2010 critical review, the Health Effects Institute (HEI) appointed a new expert Panel to systematically evaluate the epidemiological evidence regarding the associations between long-term exposure to TRAP and selected adverse health outcomes. Health outcomes were selected based on evidence of causality for general air pollution (broader than TRAP) cited in authoritative reviews, relevance for public health and policy, and resources available. The Panel used a systematic approach to search the literature, select studies for inclusion in the review, assess study quality, summarize results, and reach conclusions about the confidence in the evidence. An extensive search was conducted of literature published between January 1980 and July 2019 on selected health outcomes. A new exposure framework was developed to determine whether a study was sufficiently specific to TRAP. In total, 353 studies were included in the review. Respiratory effects in children (118 studies) and birth outcomes (86 studies) were the most commonly studied outcomes. Fewer studies investigated cardiometabolic effects (57 studies), respiratory effects in adults (50 studies), and mortality (48 studies). The findings from the systematic review, meta-analyses, and evaluation of the quality of the studies and potential biases provided an overall high or moderate-to-high level of confidence in an association between long-term exposure to TRAP and the adverse health outcomes all-cause, circulatory, ischemic heart disease and lung cancer mortality, asthma onsetin chilldren and adults, and acute lower respiratory infections in children. The evidence was considered moderate, low or very low for the other selected outcomes. In light of the large number of people exposed to TRAP - both in and beyond the near-road environment - the Panel concluded that the overall high or moderate-to-high confidence in the evidence for an association between long-term exposure to TRAP and several adverse health outcomes indicates that exposures to TRAP remain an important public health concern and deserve greater attention from the public and from policymakers.

Correction: Integrative analysis of genomic variants reveals new associations of candidate haploinsufficient genes with congenital heart disease.

[This corrects the article DOI: 10.1371/journal.pgen.1009679.].

Integrative analysis of genomic variants reveals new associations of candidate haploinsufficient genes with congenital heart disease.

Numerous genetic studies have established a role for rare genomic variants in Congenital Heart Disease (CHD) at the copy number variation (CNV) and de novo variant (DNV) level. To identify novel haploinsufficient CHD disease genes, we performed an integrative analysis of CNVs and DNVs identified in probands with CHD including cases with sporadic thoracic aortic aneurysm. We assembled CNV data from 7,958 cases and 14,082 controls and performed a gene-wise analysis of the burden of rare genomic deletions in cases versus controls. In addition, we performed variation rate testing for DNVs identified in 2,489 parent-offspring trios. Our analysis revealed 21 genes which were significantly affected by rare CNVs and/or DNVs in probands. Fourteen of these genes have previously been associated with CHD while the remaining genes (FEZ1, MYO16, ARID1B, NALCN, WAC, KDM5B and WHSC1) have only been associated in small cases series or show new associations with CHD. In addition, a systems level analysis revealed affected protein-protein interaction networks involved in Notch signaling pathway, heart morphogenesis, DNA repair and cilia/centrosome function. Taken together, this approach highlights the importance of re-analyzing existing datasets to strengthen disease association and identify novel disease genes and pathways.

Sources, variability and parameterizations of intra-city factors obtained from dispersion-normalized multi-time resolution factor analyses of PM2.5 in an urban environment.

Ambient fine particulate matter (PM2.5) data of similar continuously monitored species at two air monitoring sites with different characteristics within the City of Toronto were used to gauge the intra-city variations in the PM composition over a largely concurrent period spanning two years. One location was <8 m from the side of a major highway while the other was an urban background location. For the first time, multi-time resolution factor analysis was applied to dispersion-normalized concentrations to identify and quantify source contributions while reducing the influence of local meteorology. These factors were particulate sulphate (pSO4), particulate nitrate (pNO3), secondary organic aerosols (SOA), crustal matter (CrM) that were common to both sites, a hydrocarbon-like organic matter (HOM) exclusive to the urban background site, three black carbon related factors (BC, BC-HOM at the highway site, and a brown carbon rich factor (BC-BrC) at the urban background site), biomass burning organic matter (BBOM) and brake dust (BD) factors exclusive to the highway site. The PM2.5 composition was different between these two locations, over only a 10 km distance. The sum of SOA, pSO4 and pNO3 at the urban background site averaged 57% of the PM2.5 mass while the same species represented 43% of the average PM2.5 mass at the highway site. Local or site-specific factors may be of greater interest for control policy design. Thus, regression analyses with potential explanatory, site-specific variables were performed for results from the highway site. Three model approaches were explored: multiple linear regression (MLR), regression with a generalized reduced gradient (GRG) algorithm, and a generalized additive model (GAM). GAM gave the largest fraction of variance for the locally-found factors at the highway site. Heavy-duty vehicles were most important for explaining the black carbon (BC and BC-HOM) factors. Light-duty vehicles were dominant for the brake dust (BD) factor. The auxiliary modelling for the local factors showed that the traffic-related factors likely originated along the main roadways at their respective sites while the more regional factors, - pSO4, pNO3, SOA, - had sources that were both regional and local in origin and with contributions that varied seasonally. These results will be useful in understanding ambient particulate matter sources on a city scale that will support air quality management planning.

Systems genetics analysis identifies calcium-signaling defects as novel cause of congenital heart disease.

BACKGROUND: Congenital heart disease (CHD) occurs in almost 1% of newborn children and is considered a multifactorial disorder. CHD may segregate in families due to significant contribution of genetic factors in the disease etiology. The aim of the study was to identify pathophysiological mechanisms in families segregating CHD. METHODS: We used whole exome sequencing to identify rare genetic variants in ninety consenting participants from 32 Danish families with recurrent CHD. We applied a systems biology approach to identify developmental mechanisms influenced by accumulation of rare variants. We used an independent cohort of 714 CHD cases and 4922 controls for replication and performed functional investigations using zebrafish as in vivo model. RESULTS: We identified 1785 genes, in which rare alleles were shared between affected individuals within a family. These genes were enriched for known cardiac developmental genes, and 218 of these genes were mutated in more than one family. Our analysis revealed a functional cluster, enriched for proteins with a known participation in calcium signaling. Replication in an independent cohort confirmed increased mutation burden of calcium-signaling genes in CHD patients. Functional investigation of zebrafish orthologues of ITPR1, PLCB2, and ADCY2 verified a role in cardiac development and suggests a combinatorial effect of inactivation of these genes. CONCLUSIONS: The study identifies abnormal calcium signaling as a novel pathophysiological mechanism in human CHD and confirms the complex genetic architecture underlying CHD.

CDK12 inhibition reduces abnormalities in cells from patients with myotonic dystrophy and in a mouse model.

Myotonic dystrophy type 1 (DM1) is an RNA-based disease with no current treatment. It is caused by a transcribed CTG repeat expansion within the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Mutant repeat expansion transcripts remain in the nuclei of patients' cells, forming distinct microscopically detectable foci that contribute substantially to the pathophysiology of the condition. Here, we report small-molecule inhibitors that remove nuclear foci and have beneficial effects in the HSALR mouse model, reducing transgene expression, leading to improvements in myotonia, splicing, and centralized nuclei. Using chemoproteomics in combination with cell-based assays, we identify cyclin-dependent kinase 12 (CDK12) as a druggable target for this condition. CDK12 is a protein elevated in DM1 cell lines and patient muscle biopsies, and our results showed that its inhibition led to reduced expression of repeat expansion RNA. Some of the inhibitors identified in this study are currently the subject of clinical trials for other indications and provide valuable starting points for a drug development program in DM1.

The American English version of the validated French Flare Assessment in RA Questionnaire (FLARE-RA).

OBJECTIVE: To evaluate use of a British English version of the validated French FLARE-RA questionnaire among American English speaking patients. In addition, to create a culturally adapted American English (AmE) FLARE-RA questionnaire and to examine its attributes of patient-reported RA flare status. METHODS: Using standardized cultural adaptation guidelines, we cognitively debriefed 25 American English speaking rheumatoid arthritis (RA) outpatients and created AmE-FLARE-RA with their input. One hundred three additional RA patients were recruited. Patients completed the Routine Assessment of Patient Index Data 3 (RAPID3), patient global visual analogue scale (VAS), AmE-FLARE-RA, and self-reports of flare. Physician global VAS, physician-assessed flare, swollen and tender joint count (TJC), and clinical disease activity index (CDAI) were documented. AmE-FLARE-RA and disease activity measures were compared between patient-reported and physician-reported flare categories. RESULTS: Patients were female (89%), with mean (SD) age 51.1 (± 15.3) years and mean disease duration (SD) 11.9 (± 10.1) years, with 26% in remission/low disease activity. Total AmE-FLARE-RA scores, RAPID3, CDAI, and patient global VAS were significantly higher for both patient-reported flares and physician-reported flares compared with non-flaring patients by self- or physician report (p < 0.05). Total AmE-FLARE-RA scores correlated significantly with RAPID3 (corr = 0.50, p < 0.0001) and with CDAI (corr = 0.45, p < 0.0001). Across "no flares," "one flare," and "several flare" groups, there was a non-significant increase in AmE-FLARE-RA scores (p = 0.07). CONCLUSION: The British English FLARE-RA was successfully adapted for AmE-speaking RA patients. AmE-FLARE-RA significantly correlated with RAPID3 and CDAI and distinguished between patient-reported and physician-reported flares, making it useful to detect flares in American RA patients.Key Points• The American English FLARE-RA (AmE-FLARE-RA) questionnaire is the result of cognitive debriefing with American RA patients using the British English version of the validated French FLARE-RA and incorporates patient-recommended language modifications..• Patients self-reporting flares had significantly higher AmE-FLARE-RA scores, compared with those without flares at the time of visit. AmE-FLARE-RA scores correlate with RAPID3 and CDAI.• There was a non-statistically significant trend using the AmE-FLARE-RA scores when examining patients with no flare, one flare, or several flares.• AmE-FLARE-RA total scores are uniformly elevated (~ 6.0 on a 0-10 scale), regardless of discordance between patient and MD assessment of flare at time of visit (~ 30%).

HDAC4 and 5 repression of TBX5 is relieved by protein kinase D1.

TBX5 is a T-box family transcription factor that regulates heart and forelimb development in vertebrates and functional deficiencies in this protein result in Holt-Oram syndrome. Recently, we have shown that acetylation of TBX5 potentiates its activity and is important for heart and limb development. Here we report that class II histone deacetylases HDAC4 and HDAC5 associate with TBX5 and repress its role in cardiac gene transcription. Both HDAC4 and HDAC5 deacetylate TBX5, which promotes its relocation to the cytoplasm and HDAC4 antagonizes the physical association and functional cooperation between TBX5 and MEF2C. We also show that protein kinase D1 (PRKD1) relieves the HDAC4/5-mediated repression of TBX5. Thus, this study reveals a novel interaction of HDAC4/5 and PRKD1 in the regulation of TBX5 transcriptional activity.

Global Increase in Circular RNA Levels in Myotonic Dystrophy.

Splicing aberrations induced as a consequence of the sequestration of muscleblind-like splicing factors on the dystrophia myotonica protein kinase transcript, which contains expanded CUG repeats, present a major pathomechanism of myotonic dystrophy type 1 (DM1). As muscleblind-like factors may also be important factors involved in the biogenesis of circular RNAs (circRNAs), we hypothesized that the level of circRNAs would be decreased in DM1. To test this hypothesis, we selected 20 well-validated circRNAs and analyzed their levels in several experimental systems (e.g., cell lines, DM muscle tissues, and a mouse model of DM1) using droplet digital PCR assays. We also explored the global level of circRNAs using two RNA-Seq datasets of DM1 muscle samples. Contrary to our original hypothesis, our results consistently showed a global increase in circRNA levels in DM1, and we identified numerous circRNAs that were increased in DM1. We also identified many genes (including muscle-specific genes) giving rise to numerous (>10) circRNAs. Thus, this study is the first to show an increase in global circRNA levels in DM1. We also provided preliminary results showing the association of circRNA level with muscle weakness and alternative splicing changes that are biomarkers of DM1 severity.

Subependymal Giant Cell Astrocytoma Size Measurement in Tuberous Sclerosis Complex: Noncontrast vs Contrast-Enhanced 3-Dimensional T1-Weighted Magnetic Resonance Imaging (MRI).

PURPOSE: Tuberous sclerosis complex is a multisystem genetic disorder characterized by multiorgan hamartomas, seizures, and developmental delay. Current consensus recommendations for tuberous sclerosis complex include brain magnetic resonance imaging (MRI) without and with contrast every 1 to 3 years until age 25 years for subependymal giant cell astrocytoma screening, and more often in patients with known subependymal giant cell astrocytoma. Recent reports in the literature regarding gadolinium deposition have prompted us to investigate the added value of intravenous contrast in this setting. MATERIALS AND METHODS: Thirty-nine MRI examinations from 12 tuberous sclerosis complex patients with known subependymal giant cell astrocytoma were retrospectively reviewed for all caudothalamic groove lesions larger than 1 cm. Lesions were measured in 2 planes on both noncontrast and contrast-enhanced axial 3-dimensional (3D) T1-weighted imaging in a randomized fashion by 2 attending pediatric neuroradiologists. Differences in lesion measurements were compared to zero using a 1-sample t test. RESULTS: On average, radiologist A measured lesions 1.1 mm larger in long-axis diameter without contrast (P = .0003), whereas radiologist B measured lesions 0.1 mm larger without contrast (not significant). Differences in lesion measurement were smaller when comparing noncontrast to contrast-enhanced 3D T1-weighted imaging than when comparing radiologist A to radiologist B. CONCLUSIONS: Noncontrast axial 3D T1-weighted imaging is similar to contrast-enhanced axial 3D T1-weighted imaging for subependymal giant cell astrocytoma size measurement. It has high accuracy and may be a safer and more efficient imaging method for tuberous sclerosis complex patients who commonly undergo numerous MRI examinations throughout their lifetime. Further studies may be appropriate to determine whether contrast material could be beneficial in certain situations.

Advances in science and applications of air pollution monitoring: A case study on oil sands monitoring targeting ecosystem protection.

The potential environmental impact of air pollutants emitted from the oil sands industry in Alberta, Canada, has received considerable attention. The mining and processing of bitumen to produce synthetic crude oil, and the waste products associated with this activity, lead to significant emissions of gaseous and particle air pollutants. Deposition of pollutants occurs locally (i.e., near the sources) and also potentially at distances downwind, depending upon each pollutant's chemical and physical properties and meteorological conditions. The Joint Oil Sands Monitoring Program (JOSM) was initiated in 2012 by the Government of Canada and the Province of Alberta to enhance or improve monitoring of pollutants and their potential impacts. In support of JOSM, Environment and Climate Change Canada (ECCC) undertook a significant research effort via three components: the Air, Water, and Wildlife components, which were implemented to better estimate baseline conditions related to levels of pollutants in the air and water, amounts of deposition, and exposures experienced by the biota. The criteria air contaminants (e.g., nitrogen oxides [NOx], sulfur dioxide [SO2], volatile organic compounds [VOCs], particulate matter with an aerodynamic diameter <2.5 µm [PM2.5]) and their secondary atmospheric products were of interest, as well as toxic compounds, particularly polycyclic aromatic compounds (PACs), trace metals, and mercury (Hg). This critical review discusses the challenges of assessing ecosystem impacts and summarizes the major results of these efforts through approximately 2018. Focus is on the emissions to the air and the findings from the Air Component of the ECCC research and linkages to observations of contaminant levels in the surface waters in the region, in aquatic species, as well as in terrestrial and avian species. The existing evidence of impact on these species is briefly discussed, as is the potential for some of them to serve as sentinel species for the ongoing monitoring needed to better understand potential effects, their potential causes, and to detect future changes. Quantification of the atmospheric emissions of multiple pollutants needs to be improved, as does an understanding of the processes influencing fugitive emissions and local and regional deposition patterns. The influence of multiple stressors on biota exposure and response, from natural bitumen and forest fires to climate change, complicates the current ability to attribute effects to air emissions from the industry. However, there is growing evidence of the impact of current levels of PACs on some species, pointing to the need to improve the ability to predict PAC exposures and the key emission source involved. Although this critical review attempts to integrate some of the findings across the components, in terms of ECCC activities, increased coordination or integration of air, water, and wildlife research would enhance deeper scientific understanding. Improved understanding is needed in order to guide the development of long-term monitoring strategies that could most efficiently inform a future adaptive management approach to oil sands environmental monitoring and prevention of impacts. Implications: Quantification of atmospheric emissions for multiple pollutants needs to be improved, and reporting mechanisms and standards could be adapted to facilitate such improvements, including periodic validation, particularly where uncertainties are the largest. Understanding of baseline conditions in the air, water and biota has improved significantly; ongoing enhanced monitoring, building on this progress, will help improve ecosystem protection measures in the oil sands region. Sentinel species have been identified that could be used to identify and characterize potential impacts of wildlife exposure, both locally and regionally. Polycyclic aromatic compounds are identified as having an impact on aquatic and terrestrial wildlife at current concentration levels although the significance of these impacts and attribution to emissions from oil sands development requires further assessment. Given the improvement in high resolution air quality prediction models, these should be a valuable tool to future environmental assessments and cumulative environment impact assessments.

Whole Exome Sequencing Reveals the Major Genetic Contributors to Nonsyndromic Tetralogy of Fallot.

RATIONALE: Familial recurrence studies provide strong evidence for a genetic component to the predisposition to sporadic, nonsyndromic Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease phenotype. Rare genetic variants have been identified as important contributors to the risk of congenital heart disease, but relatively small numbers of TOF cases have been studied to date. OBJECTIVE: We used whole exome sequencing to assess the prevalence of unique, deleterious variants in the largest cohort of nonsyndromic TOF patients reported to date. METHODS AND RESULTS: Eight hundred twenty-nine TOF patients underwent whole exome sequencing. The presence of unique, deleterious variants was determined; defined by their absence in the Genome Aggregation Database and a scaled combined annotation-dependent depletion score of ≥20. The clustering of variants in 2 genes, NOTCH1 and FLT4, surpassed thresholds for genome-wide significance (assigned as P<5×10-8) after correction for multiple comparisons. NOTCH1 was most frequently found to harbor unique, deleterious variants. Thirty-one changes were observed in 37 probands (4.5%; 95% CI, 3.2%-6.1%) and included 7 loss-of-function variants 22 missense variants and 2 in-frame indels. Sanger sequencing of the unaffected parents of 7 cases identified 5 de novo variants. Three NOTCH1 variants (p.G200R, p.C607Y, and p.N1875S) were subjected to functional evaluation, and 2 showed a reduction in Jagged1-induced NOTCH signaling. FLT4 variants were found in 2.4% (95% CI, 1.6%-3.8%) of TOF patients, with 21 patients harboring 22 unique, deleterious variants. The variants identified were distinct to those that cause the congenital lymphoedema syndrome Milroy disease. In addition to NOTCH1, FLT4 and the well-established TOF gene, TBX1, we identified potential association with variants in several other candidates, including RYR1, ZFPM1, CAMTA2, DLX6, and PCM1. CONCLUSIONS: The NOTCH1 locus is the most frequent site of genetic variants predisposing to nonsyndromic TOF, followed by FLT4. Together, variants in these genes are found in almost 7% of TOF patients.

Mortality-Air Pollution Associations in Low-Exposure Environments (MAPLE): Phase 1.

INTRODUCTION: Fine particulate matter (particulate matter ≤2.5 µm in aerodynamic diameter, or PM2.5) is associated with mortality, but the lower range of relevant concentrations is unknown. Novel satellite-derived estimates of outdoor PM2.5 concentrations were applied to several large population-based cohorts, and the shape of the relationship with nonaccidental mortality was characterized, with emphasis on the low concentrations (<12 µg/m3) observed throughout Canada. METHODS: Annual satellite-derived estimates of outdoor PM2.5 concentrations were developed at 1-km2 spatial resolution across Canada for 2000-2016 and backcasted to 1981 using remote sensing, chemical transport models, and ground monitoring data. Targeted ground-based measurements were conducted to measure the relationship between columnar aerosol optical depth (AOD) and ground-level PM2.5. Both existing and targeted ground-based measurements were analyzed to develop improved exposure data sets for subsequent epidemiological analyses.Residential histories derived from annual tax records were used to estimate PM2.5 exposures for subjects whose ages ranged from 25 to 90 years. About 8.5 million were from three Canadian Census Health and Environment Cohort (CanCHEC) analytic files and another 540,900 were Canadian Community Health Survey (CCHS) participants. Mortality was linked through the year 2016. Hazard ratios (HR) were estimated with Cox Proportional Hazard models using a 3-year moving average exposure with a 1-year lag, with the year of follow-up as the time axis. All models were stratified by 5-year age groups, sex, and immigrant status. Covariates were based on directed acyclical graphs (DAG), and included contextual variables (airshed, community size, neighborhood dependence, neighborhood deprivation, ethnic concentration, neighborhood instability, and urban form). A second model was examined including the DAG-based covariates as well as all subject-level risk factors (income, education, marital status, indigenous identity, employment status, occupational class, and visible minority status) available in each cohort. Additional subject-level behavioral covariates (fruit and vegetable consumption, leisure exercise frequency, alcohol consumption, smoking, and body mass index [BMI]) were included in the CCHS analysis.Sensitivity analyses evaluated adjustment for covariates and gaseous copollutants (nitrogen dioxide [NO2] and ozone [O3]), as well as exposure time windows and spatial scales. Estimates were evaluated across strata of age, sex, and immigrant status. The shape of the PM2.5-mortality association was examined by first fitting restricted cubic splines (RCS) with a large number of knots and then fitting the shape-constrained health impact function (SCHIF) to the RCS predictions and their standard errors (SE). This method provides graphical results indicating the RCS predictions, as a nonparametric means of characterizing the concentration-response relationship in detail and the resulting mean SCHIF and accompanying uncertainty as a parametric summary.Sensitivity analyses were conducted in the CCHS cohort to evaluate the potential influence of unmeasured covariates on air pollution risk estimates. Specifically, survival models with all available risk factors were fit and compared with models that omitted covariates not available in the CanCHEC cohorts. In addition, the PM2.5 risk estimate in the CanCHEC cohort was indirectly adjusted for multiple individual-level risk factors by estimating the association between PM2.5 and these covariates within the CCHS. RESULTS: Satellite-derived PM2.5 estimates were low and highly correlated with ground monitors. HR estimates (per 10-µg/m3 increase in PM2.5) were similar for the 1991 (1.041, 95% confidence interval [CI]: 1.016-1.066) and 1996 (1.041, 1.024-1.059) CanCHEC cohorts with a larger estimate observed for the 2001 cohort (1.084, 1.060-1.108). The pooled cohort HR estimate was 1.053 (1.041-1.065). In the CCHS an analogous model indicated a HR of 1.13 (95% CI: 1.06-1.21), which was reduced slightly with the addition of behavioral covariates (1.11, 1.04-1.18). In each of the CanCHEC cohorts, the RCS increased rapidly over lower concentrations, slightly declining between the 25th and 75th percentiles and then increasing beyond the 75th percentile. The steepness of the increase in the RCS over lower concentrations diminished as the cohort start date increased. The SCHIFs displayed a supralinear association in each of the three CanCHEC cohorts and in the CCHS cohort.In sensitivity analyses conducted with the 2001 CanCHEC, longer moving averages (1, 3, and 8 years) and smaller spatial scales (1 km2 vs. 10 km2) of exposure assignment resulted in larger associations between PM2.5 and mortality. In both the CCHS and CanCHEC analyses, the relationship between nonaccidental mortality and PM2.5 was attenuated when O3 or a weighted measure of oxidant gases was included in models. In the CCHS analysis, but not in CanCHEC, PM2.5 HRs were also attenuated by the inclusion of NO2. Application of the indirect adjustment and comparisons within the CCHS analysis suggests that missing data on behavioral risk factors for mortality had little impact on the magnitude of PM2.5-mortality associations. While immigrants displayed improved overall survival compared with those born in Canada, their sensitivity to PM2.5 was similar to or larger than that for nonimmigrants, with differences between immigrants and nonimmigrants decreasing in the more recent cohorts. CONCLUSIONS: In several large population-based cohorts exposed to low levels of air pollution, consistent associations were observed between PM2.5 and nonaccidental mortality for concentrations as low as 5 µg/m3. This relationship was supralinear with no apparent threshold or sublinear association.

Quantitative Methods to Monitor RNA Biomarkers in Myotonic Dystrophy.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are human neuromuscular disorders associated with mutations of simple repetitive sequences in affected genes. The abnormal expansion of CTG repeats in the 3'-UTR of the DMPK gene elicits DM1, whereas elongated CCTG repeats in intron 1 of ZNF9/CNBP triggers DM2. Pathogenesis of both disorders is manifested by nuclear retention of expanded repeat-containing RNAs and aberrant alternative splicing. The precise determination of absolute numbers of mutant RNA molecules is important for a better understanding of disease complexity and for accurate evaluation of the efficacy of therapeutic drugs. We present two quantitative methods, Multiplex Ligation-Dependent Probe Amplification and droplet digital PCR, for studying the mutant DMPK transcript (DMPKexpRNA) and the aberrant alternative splicing in DM1 and DM2 human tissues and cells. We demonstrate that in DM1, the DMPKexpRNA is detected in higher copy number than its normal counterpart. Moreover, the absolute number of the mutant transcript indicates its low abundance with only a few copies per cell in DM1 fibroblasts. Most importantly, in conjunction with fluorescence in-situ hybridization experiments, our results suggest that in DM1 fibroblasts, the vast majority of nuclear RNA foci consist of a few molecules of DMPKexpRNA.

The effect of individual radiographers on rates of attendance to breast screening: a 7-year retrospective study.

AIM: To establish whether individual radiographers had significantly different rescreening rates whilst controlling for other known confounding factors. MATERIALS AND METHODS: Women aged 50-69 years were identified from a state-wide screening database at their first screening attendance during the study period (2007-2013). The radiographer performing this index screen and potential confounding factors were recorded and subsequent screening behaviour was assessed. Clients with abnormal screens and those known to have died during the time period were excluded. A univariate analysis of the data from 160,028 women was assessed using the chi-square test to compare those women who attended their next mammography with non-re-attenders. Logistic regression was used to calculate the likelihood of "re-attendance success" across a range of variables. The probability of re-attendance for 11 randomly selected radiographers was determined from the logistic regression model, whilst controlling for other variables. RESULTS: Comparison of non-re-attenders (n=49,698) with 110,330 (69%) women attending the next round of screening revealed significant differences, including radiographer (Wald statistics=1188, p<0.000) even when all other known factors were controlled. CONCLUSION: This large, population-level study demonstrates that individual radiographer factors appear to influence a women's decision to return for their next screening round. Further research is required to identify reasons for differing rescreen rates and provide education and retraining of individual radiographers as appropriate.