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.

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.

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.

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.

Acetylation of TBX5 by KAT2B and KAT2A regulates heart and limb development.

TBX5 plays a critical role in heart and forelimb development. Mutations in TBX5 cause Holt-Oram syndrome, an autosomal dominant condition that affects the formation of the heart and upper-limb. Several studies have provided significant insight into the role of TBX5 in cardiogenesis; however, how TBX5 activity is regulated by other factors is still unknown. Here we report that histone acetyltransferases KAT2A and KAT2B associate with TBX5 and acetylate it at Lys339. Acetylation potentiates its transcriptional activity and is required for nuclear retention. Morpholino-mediated knockdown of kat2a and kat2b transcripts in zebrafish severely perturb heart and limb development, mirroring the tbx5a knockdown phenotype. The phenotypes found in MO-injected embryos were also observed when we introduced mutations in the kat2a or kat2b genes using the CRISPR-Cas system. These studies highlight the importance of KAT2A and KAT2B modulation of TBX5 and their impact on heart and limb development.

A genome-wide association study of congenital cardiovascular left-sided lesions shows association with a locus on chromosome 20.

Congenital heart defects involving left-sided lesions (LSLs) are relatively common birth defects with substantial morbidity and mortality. Previous studies have suggested a high heritability with a complex genetic architecture, such that only a few LSL loci have been identified. We performed a genome-wide case-control association study to address the role of common variants using a discovery cohort of 778 cases and 2756 controls. We identified a genome-wide significant association mapping to a 200 kb region on chromosome 20q11 [P= 1.72 × 10-8 for rs3746446; imputed Single Nucleotide Polymorphism (SNP) rs6088703 P= 3.01 × 10-9, odds ratio (OR)= 1.6 for both]. This result was supported by transmission disequilibrium analyses using a subset of 541 case families (lowest P in region= 4.51 × 10-5, OR= 1.5). Replication in a cohort of 367 LSL cases and 5159 controls showed nominal association (P= 0.03 for rs3746446) resulting in P= 9.49 × 10-9 for rs3746446 upon meta-analysis of the combined cohorts. In addition, a group of seven SNPs on chromosome 1q21.3 met threshold for suggestive association (lowest P= 9.35 × 10-7 for rs12045807). Both regions include genes involved in cardiac development-MYH7B/miR499A on chromosome 20 and CTSK, CTSS and ARNT on chromosome 1. Genome-wide heritability analysis using case-control genotyped SNPs suggested that the mean heritability of LSLs attributable to common variants is moderately high ([Formula: see text] range= 0.26-0.34) and consistent with previous assertions. These results provide evidence for the role of common variation in LSLs, proffer new genes as potential biological candidates, and give further insight to the complex genetic architecture of congenital heart disease.

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.

Tropomyosin 1: Multiple roles in the developing heart and in the formation of congenital heart defects.

Tropomyosin 1 (TPM1) is an essential sarcomeric component, stabilising the thin filament and facilitating actin's interaction with myosin. A number of sarcomeric proteins, such as alpha myosin heavy chain, play crucial roles in cardiac development. Mutations in these genes have been linked to congenital heart defects (CHDs), occurring in approximately 1 in 145 live births. To date, TPM1 has not been associated with isolated CHDs. Analysis of 380 CHD cases revealed three novel mutations in the TPM1 gene; IVS1+2T>C, I130V, S229F and a polyadenylation signal site variant GATAAA/AATAAA. Analysis of IVS1+2T>C revealed aberrant pre-mRNA splicing. In addition, abnormal structural properties were found in hearts transfected with TPM1 carrying I130V and S229F mutations. Phenotypic analysis of TPM1 morpholino-treated embryos revealed roles for TPM1 in cardiac looping, atrial septation and ventricular trabeculae formation and increased apoptosis was seen within the heart. In addition, sarcomere assembly was affected and altered action potentials were exhibited. This study demonstrated that sarcomeric TPM1 plays vital roles in cardiogenesis and is a suitable candidate gene for screening individuals with isolated CHDs.

Rare variants in NR2F2 cause congenital heart defects in humans.

Congenital heart defects (CHDs) are the most common birth defect worldwide and are a leading cause of neonatal mortality. Nonsyndromic atrioventricular septal defects (AVSDs) are an important subtype of CHDs for which the genetic architecture is poorly understood. We performed exome sequencing in 13 parent-offspring trios and 112 unrelated individuals with nonsyndromic AVSDs and identified five rare missense variants (two of which arose de novo) in the highly conserved gene NR2F2, a very significant enrichment (p = 7.7 × 10(-7)) compared to 5,194 control subjects. We identified three additional CHD-affected families with other variants in NR2F2 including a de novo balanced chromosomal translocation, a de novo substitution disrupting a splice donor site, and a 3 bp duplication that cosegregated in a multiplex family. NR2F2 encodes a pleiotropic developmental transcription factor, and decreased dosage of NR2F2 in mice has been shown to result in abnormal development of atrioventricular septa. Via luciferase assays, we showed that all six coding sequence variants observed in individuals significantly alter the activity of NR2F2 on target promoters.

High-content screening identifies small molecules that remove nuclear foci, affect MBNL distribution and CELF1 protein levels via a PKC-independent pathway in myotonic dystrophy cell lines.

Myotonic dystrophy (DM) is a multi-system neuromuscular disorder for which there is no treatment. We have developed a medium throughput phenotypic assay, based on the identification of nuclear foci in DM patient cell lines using in situ hybridization and high-content imaging to screen for potentially useful therapeutic compounds. A series of further assays based on molecular features of DM have also been employed. Two compounds that reduce and/or remove nuclear foci have been identified, Ro 31-8220 and chromomycin A3. Ro 31-8220 is a PKC inhibitor, previously shown to affect the hyperphosphorylation of CELF1 and ameliorate the cardiac phenotype in a DM1 mouse model. We show that the same compound eliminates nuclear foci, reduces MBNL1 protein in the nucleus, affects ATP2A1 alternative splicing and reduces steady-state levels of CELF1 protein. We demonstrate that this effect is independent of PKC activity and conclude that this compound may be acting on alternative kinase targets within DM pathophysiology. Understanding the activity profile for this compound is key for the development of targeted therapeutics in the treatment of DM.

Genome-wide association study identifies loci on 12q24 and 13q32 associated with tetralogy of Fallot.

We conducted a genome-wide association study to search for risk alleles associated with Tetralogy of Fallot (TOF), using a northern European discovery set of 835 cases and 5159 controls. A region on chromosome 12q24 was associated (P = 1.4 × 10(-7)) and replicated convincingly (P = 3.9 × 10(-5)) in 798 cases and 2931 controls [per allele odds ratio (OR) = 1.27 in replication cohort, P = 7.7 × 10(-11) in combined populations]. Single nucleotide polymorphisms in the glypican 5 gene on chromosome 13q32 were also associated (P = 1.7 × 10(-7)) and replicated convincingly (P = 1.2 × 10(-5)) in 789 cases and 2927 controls (per allele OR = 1.31 in replication cohort, P = 3.03 × 10(-11) in combined populations). Four additional regions on chromosomes 10, 15 and 16 showed suggestive association accompanied by nominal replication. This study, the first genome-wide association study of a congenital heart malformation phenotype, provides evidence that common genetic variation influences the risk of TOF.

Contribution of global rare copy-number variants to the risk of sporadic congenital heart disease.

Previous studies have shown that copy-number variants (CNVs) contribute to the risk of complex developmental phenotypes. However, the contribution of global CNV burden to the risk of sporadic congenital heart disease (CHD) remains incompletely defined. We generated genome-wide CNV data by using Illumina 660W-Quad SNP arrays in 2,256 individuals with CHD, 283 trio CHD-affected families, and 1,538 controls. We found association of rare genic deletions with CHD risk (odds ratio [OR] = 1.8, p = 0.0008). Rare deletions in study participants with CHD had higher gene content (p = 0.001) with higher haploinsufficiency scores (p = 0.03) than they did in controls, and they were enriched with Wnt-signaling genes (p = 1 × 10(-5)). Recurrent 15q11.2 deletions were associated with CHD risk (OR = 8.2, p = 0.02). Rare de novo CNVs were observed in ~5% of CHD trios; 10 out of 11 occurred on the paternally transmitted chromosome (p = 0.01). Some of the rare de novo CNVs spanned genes known to be involved in heart development (e.g., HAND2 and GJA5). Rare genic deletions contribute ~4% of the population-attributable risk of sporadic CHD. Second to previously described CNVs at 1q21.1, deletions at 15q11.2 and those implicating Wnt signaling are the most significant contributors to the risk of sporadic CHD. Rare de novo CNVs identified in CHD trios exhibit paternal origin bias.

The Canadian healthy infant longitudinal development birth cohort study: biological samples and biobanking.

BACKGROUND: It is hypothesised that complex interactions between genetic and environmental factors give rise to allergy and asthma in childhood. The Canadian Healthy Infant Longitudinal Development (CHILD) study was designed to explore these factors. METHODS: CHILD is a longitudinal, general population birth cohort study following infants from mid-pregnancy to age 5 years. Over this time period, biological samples, questionnaires, clinical measures and environmental data are collected. RESULTS: A total of 3624 families have been recruited, and many thousands of samples and questionnaires have been collected, annotated, and archived. This report outlines the rationale and methodology for collecting and storing diverse biological samples from parents and children in this study, and the mechanisms for their release for analyses. CONCLUSIONS: The CHILD sample and data repository is a tremendous current and future resource and will provide a wealth of information not only informing studies of asthma and allergy, but also potentially in many other aspects of health relevant for Canadian infants and children.

Phenotype-specific effect of chromosome 1q21.1 rearrangements and GJA5 duplications in 2436 congenital heart disease patients and 6760 controls.

Recurrent rearrangements of chromosome 1q21.1 that occur via non-allelic homologous recombination have been associated with variable phenotypes exhibiting incomplete penetrance, including congenital heart disease (CHD). However, the gene or genes within the ~1 Mb critical region responsible for each of the associated phenotypes remains unknown. We examined the 1q21.1 locus in 948 patients with tetralogy of Fallot (TOF), 1488 patients with other forms of CHD and 6760 ethnically matched controls using single nucleotide polymorphism genotyping arrays (Illumina 660W and Affymetrix 6.0) and multiplex ligation-dependent probe amplification. We found that duplication of 1q21.1 was more common in cases of TOF than in controls [odds ratio (OR) 30.9, 95% confidence interval (CI) 8.9-107.6); P = 2.2 × 10(-7)], but deletion was not. In contrast, deletion of 1q21.1 was more common in cases of non-TOF CHD than in controls [OR 5.5 (95% CI 1.4-22.0); P = 0.04] while duplication was not. We also detected rare (n = 3) 100-200 kb duplications within the critical region of 1q21.1 in cases of TOF. These small duplications encompassed a single gene in common, GJA5, and were enriched in cases of TOF in comparison to controls [OR = 10.7 (95% CI 1.8-64.3), P = 0.01]. These findings show that duplication and deletion at chromosome 1q21.1 exhibit a degree of phenotypic specificity in CHD, and implicate GJA5 as the gene responsible for the CHD phenotypes observed with copy number imbalances at this locus.

Associations between bacterial communities of house dust and infant gut.

The human gut is host to a diverse and abundant community of bacteria that influence health and disease susceptibility. This community develops in infancy, and its composition is strongly influenced by environmental factors, notably perinatal anthropogenic exposures such as delivery mode (Cesarean vs. vaginal) and feeding method (breast vs. formula); however, the built environment as a possible source of exposure has not been considered. Here we report on a preliminary investigation of the associations between bacteria in house dust and the nascent fecal microbiota from 20 subjects from the Canadian Healthy Infant Longitudinal Development (CHILD) Study using high-throughput sequence analysis of portions of the 16S rRNA gene. Despite significant differences between the dust and fecal microbiota revealed by Nonmetric Multidimensional Scaling (NMDS) analysis, permutation analysis confirmed that 14 bacterial OTUs representing the classes Actinobacteria (3), Bacilli (3), Clostridia (6) and Gammaproteobacteria (2) co-occurred at a significantly higher frequency in matched dust-stool pairs than in randomly permuted pairs, indicating an association between these dust and stool communities. These associations could indicate a role for the indoor environment in shaping the nascent gut microbiota, but future studies will be needed to confirm that our findings do not solely reflect a reverse pathway. Although pet ownership was strongly associated with the presence of certain genera in the dust for dogs (Agrococcus, Carnobacterium, Exiguobacterium, Herbaspirillum, Leifsonia and Neisseria) and cats (Escherichia), no clear patterns were observed in the NMDS-resolved stool community profiles as a function of pet ownership.

Mutation in myosin heavy chain 6 causes atrial septal defect.

Atrial septal defect is one of the most common forms of congenital heart malformation. We identified a new locus linked with atrial septal defect on chromosome 14q12 in a large family with dominantly inherited atrial septal defect. The underlying mutation is a missense substitution, I820N, in alpha-myosin heavy chain (MYH6), a structural protein expressed at high levels in the developing atria, which affects the binding of the heavy chain to its regulatory light chain. The cardiac transcription factor TBX5 strongly regulates expression of MYH6, but mutant forms of TBX5, which cause Holt-Oram syndrome, do not. Morpholino knock-down of expression of the chick MYH6 homolog eliminates the formation of the atrial septum without overtly affecting atrial chamber formation. These data provide evidence for a link between a transcription factor, a structural protein and congenital heart disease.

Low-frequency intermediate penetrance variants in the ROCK1 gene predispose to Tetralogy of Fallot.

BACKGROUND: Epidemiological studies indicate a substantial excess familial recurrence of non-syndromic Tetralogy of Fallot (TOF), implicating genetic factors that remain largely unknown. The Rho induced kinase 1 gene (ROCK1) is a key component of the planar cell polarity signalling pathway, which plays an important role in normal cardiac development. The aim of this study was to investigate the role of genetic variation in ROCK1 on the risk of TOF. RESULTS: ROCK1 was sequenced in a discovery cohort of 93 non-syndromic TOF probands to identify rare variants. TagSNPs were selected to capture commoner variation in ROCK1. Novel variants and TagSNPs were genotyped in a discovery cohort of 458 TOF cases and 1331 healthy controls, and positive findings were replicated in a further 209 TOF cases and 1290 healthy controls. Association between genotypes and TOF was assessed using LAMP.A rare SNP (c.807C > T; rs56085230) discovered by sequencing was associated with TOF risk (p = 0.006) in the discovery cohort. The variant was also significantly associated with the risk of TOF in the replication cohort (p = 0.018). In the combined cohorts the odds ratio for TOF was 2.61 (95% CI 1.58-4.30); p < 0.0001. The minor allele frequency of rs56085230 in the cases was 0.02, and in the controls it was 0.007. The variant accounted for 1% of the population attributable risk (PAR) of TOF. We also found significant association with TOF for an uncommon TagSNP in ROCK1, rs288979 (OR 1.64 [95% CI 1.15-2.30]; p = 1.5x10⁻5). The minor allele frequency of rs288979 in the controls was 0.043, and the variant accounted for 11% of the PAR of TOF. These association signals were independent of each other, providing additional internal validation of our result. CONCLUSIONS: Low frequency intermediate penetrance (LFIP) variants in the ROCK1 gene predispose to the risk of TOF.