Foram N. Ashar

Transcriptome analysis reveals dysregulation of innate immune response genes and neuronal activity-dependent genes in autism.

Recent studies of genomic variation associated with autism have suggested the existence of extreme heterogeneity. Large-scale transcriptomics should complement these results to identify core molecular pathways underlying autism. Here we report results from a large-scale RNA sequencing effort, utilizing region-matched autism and control brains to identify neuronal and microglial genes robustly dysregulated in autism cortical brain. Remarkably, we note that a gene expression module corresponding to M2-activation states in microglia is negatively correlated with a differentially expressed neuronal module, implicating dysregulated microglial responses in concert with altered neuronal activity-dependent genes in autism brains. These observations provide pathways and candidate genes that highlight the interplay between innate immunity and neuronal activity in the aetiology of autism.

Ribosomal biogenesis genes play an essential and p53-independent role in zebrafish pancreas development

Mutations in the human Shwachman-Bodian-Diamond syndrome (SBDS) gene cause defective ribosome assembly and are associated with exocrine pancreatic insufficiency, chronic neutropenia and skeletal defects. However, the mechanism underlying these phenotypes remains unclear. Here we show that knockdown of the zebrafish sbds ortholog fully recapitulates the spectrum of developmental abnormalities observed in the human syndrome, and further implicate impaired proliferation of ptf1a-expressing pancreatic progenitor cells as the basis for the observed pancreatic phenotype. It is thought that diseases of ribosome assembly share a p53-dependent mechanism. However, loss of p53 did not rescue the developmental defects associated with loss of zebrafish sbds. To clarify the molecular mechanisms underlying the observed organogenesis defects, we performed transcriptional profiling to identify candidate downstream mediators of the sbds phenotype. Among transcripts displaying differential expression, functional group analysis revealed marked enrichment of genes related to ribosome biogenesis, rRNA processing and translational initiation. Among these, ribosomal protein L3 (rpl3) and pescadillo (pes) were selected for additional analysis. Similar to knockdown of sbds, knockdown or mutation of either rpl3 or pes resulted in impaired expansion of pancreatic progenitor cells. The pancreatic phenotypes observed in rpl3- and pes-deficient embryos were also independent of p53. Together, these data suggest novel p53-independent roles for ribosomal biogenesis genes in zebrafish pancreas development.

Association between Mitochondrial DNA Copy Number in Peripheral Blood and Incident CKD in the Atherosclerosis Risk in Communities Study

Mitochondrial dysfunction in kidney cells has been implicated in the pathogenesis of CKD. Mitochondrial DNA (mtDNA) copy number is a surrogate measure of mitochondrial function, and higher mtDNA copy number in peripheral blood has been associated with lower risk of two important risk factors for CKD progression, diabetes and microalbuminuria. We evaluated whether mtDNA copy number in peripheral blood associates with incident CKD in a population-based cohort of middle-aged adults. We estimated mtDNA copy number using 25 high-quality mitochondrial single nucleotide polymorphisms from the Affymetrix 6.0 array. Among 9058 participants, those with higher mtDNA copy number had a lower rate of prevalent diabetes and lower C-reactive protein levels and white blood cell counts. Baseline eGFR did not differ significantly by mtDNA copy number. Over a median follow-up of 19.6 years, 1490 participants developed CKD. Higher mtDNA copy number associated with lower risk of incident CKD (highest versus lowest quartile: hazard ratio 0.65; 95% confidence interval, 0.56 to 0.75; P<0.001) after adjusting for age, sex, and race. After adjusting for additional risk factors of CKD, including prevalent diabetes, hypertension, C-reactive protein level, and white blood cell count, this association remained significant (highest versus lowest quartile: hazard ratio 0.75; 95% confidence interval, 0.64 to 0.87; P<0.001). In conclusion, higher mtDNA copy number associated with lower incidence of CKD independent of traditional risk factors and inflammation biomarker levels in this cohort. Further research on modifiable factors influencing mtDNA copy number may lead to improvement in the prevention and treatment of CKD.

RNA-Seq optimization with eQTL gold standards

BackgroundRNA-Sequencing (RNA-Seq) experiments have been optimized for library preparation, mapping, and gene expression estimation. These methods, however, have revealed weaknesses in the next stages of analysis of differential expression, with results sensitive to systematic sample stratification or, in more extreme cases, to outliers. Further, a method to assess normalization and adjustment measures imposed on the data is lacking.ResultsTo address these issues, we utilize previously published eQTLs as a novel gold standard at the center of a framework that integrates DNA genotypes and RNA-Seq data to optimize analysis and aid in the understanding of genetic variation and gene expression. After detecting sample contamination and sequencing outliers in RNA-Seq data, a set of previously published brain eQTLs was used to determine if sample outlier removal was appropriate. Improved replication of known eQTLs supported removal of these samples in downstream analyses. eQTL replication was further employed to assess normalization methods, covariate inclusion, and gene annotation. This method was validated in an independent RNA-Seq blood data set from the GTEx project and a tissue-appropriate set of eQTLs. eQTL replication in both data sets highlights the necessity of accounting for unknown covariates in RNA-Seq data analysis.ConclusionAs each RNA-Seq experiment is unique with its own experiment-specific limitations, we offer an easily-implementable method that uses the replication of known eQTLs to guide each step in one’s data analysis pipeline. In the two data sets presented herein, we highlight not only the necessity of careful outlier detection but also the need to account for unknown covariates in RNA-Seq experiments.

Association of mitochondrial DNA copy number with cardiovascular disease

Importance Mitochondrial dysfunction is a core component of the aging process and may play a key role in atherosclerotic cardiovascular disease. Mitochondrial DNA copy number (mtDNA-CN), which represents the number of mitochondria per cell and number of mitochondrial genomes per mitochondrion, is an indirect biomarker of mitochondrial function. Objective To determine whether mtDNA-CN, measured in an easily accessible tissue (buffy coat/circulating leukocytes), can improve risk classification for cardiovascular disease (CVD) and help guide initiation of statin therapy for primary prevention of CVD. Design, Setting, and Participants Prospective, population-based cohort analysis including 21 870 participants (20 163 free from CVD at baseline) from 3 studies: Cardiovascular Health Study (CHS), Atherosclerosis Risk in Communities Study (ARIC), and Multiethnic Study of Atherosclerosis (MESA). The mean follow-up was 13.5 years. The study included 11 153 participants from ARIC, 4830 from CHS, and 5887 from MESA. Analysis of the data was conducted from March 10, 2014, to January 29, 2017. Exposures Mitochondrial DNA-CN measured from buffy coat/circulating leukocytes. Main Outcomes and Measures Incident CVD, which combines coronary heart disease, defined as the first incident myocardial infarction or death owing to coronary heart disease, and stroke, defined as the first nonfatal stroke or death owing to stroke. Results Of the 21 870 participants, the mean age was 62.4 years (ARIC, 57.9 years; MESA, 62.4 years; and CHS, 72.5 years), and 54.7% of participants were women. The hazard ratios for incident coronary heart disease, stroke, and CVD associated with a 1-SD decrease in mtDNA-CN were 1.29 (95% CI, 1.24-1.33), 1.11 (95% CI, 1.06-1.16), and 1.23 (95% CI, 1.19-1.26). The associations persisted after adjustment for traditional CVD risk factors. Addition of mtDNA-CN to the 2013 American College of Cardiology/American Heart Association Pooled Cohorts Equations for estimating 10-year hard atherosclerosis CVD risk was associated with improved risk classification (continuous net reclassification index, 0.194; 95% CI, 0.130-0.258; P < .001). Mitochondrial DNA-CN further improved sensitivity and specificity for the 2013 American College of Cardiology/American Heart Association recommendations on initiating statin therapy for primary prevention of ASCVD (net 221 individuals appropriately downclassified and net 15 individuals appropriately upclassified). Conclusions and Relevance Mitochondrial DNA-CN was independently associated with incident CVD in 3 large prospective studies and may have potential clinical utility in improving CVD risk classification.

Association between mitochondrial DNA copy number and sudden cardiac death: Findings from the Atherosclerosis Risk in Communities study (ARIC)

Aims Sudden cardiac death (SCD) is a major public health burden. Mitochondrial dysfunction has been implicated in a wide range of cardiovascular diseases including cardiomyopathy, heart failure, and arrhythmias, but it is unknown if it also contributes to SCD risk. We sought to examine the prospective association between mtDNA copy number (mtDNA-CN), a surrogate marker of mitochondrial function, and SCD risk. Methods and results We measured baseline mtDNA-CN in 11 093 participants from the Atherosclerosis Risk in Communities (ARIC) study. mtDNA copy number was calculated from probe intensities of mitochondrial single nucleotide polymorphisms (SNP) on the Affymetrix Genome-Wide Human SNP Array 6.0. Sudden cardiac death was defined as a sudden pulseless condition presumed due to a ventricular tachyarrhythmia in a previously stable individual without evidence of a non-cardiac cause of cardiac arrest. Sudden cardiac death cases were reviewed and adjudicated by an expert committee. During a median follow-up of 20.4 years, we observed 361 SCD cases. After adjusting for age, race, sex, and centre, the hazard ratio for SCD comparing the 1st to the 5th quintiles of mtDNA-CN was 2.24 (95% confidence interval 1.58-3.19; P-trend <0.001). When further adjusting for traditional cardiovascular disease risk factors, prevalent coronary heart disease, heart rate, QT interval, and QRS duration, the association remained statistically significant. Spline regression models showed that the association was approximately linear over the range of mtDNA-CN values. No apparent interaction by race or by sex was detected. Conclusion In this community-based prospective study, mtDNA-CN in peripheral blood was inversely associated with the risk of SCD.

Genome‐Wide Associations of Global Electrical Heterogeneity ECG Phenotype: The ARIC (Atherosclerosis Risk in Communities) Study and CHS (Cardiovascular Health Study)

Background ECG global electrical heterogeneity (GEH) is associated with sudden cardiac death. We hypothesized that a genome‐wide association study would identify genetic loci related to GEH. Methods and Results We tested genotyped and imputed variants in black (N=3057) and white (N=10 769) participants in the ARIC (Atherosclerosis Risk in Communities) study and CHS (Cardiovascular Health Study). GEH (QRS‐T angle, sum absolute QRST integral, spatial ventricular gradient magnitude, elevation, azimuth) was measured on 12‐lead ECGs. Linear regression models were constructed with each GEH variable as an outcome, adjusted for age, sex, height, body mass index, study site, and principal components to account for ancestry. GWAS identified 10 loci that showed genome‐wide significant association with GEH in whites or joint ancestry. The strongest signal (rs7301677, near TBX3) was associated with QRS‐T angle (white standardized β+0.16 [95% CI 0.13–0.19]; P=1.5×10−26), spatial ventricular gradient elevation (+0.11 [0.08–0.14]; P=2.1×10−12), and spatial ventricular gradient magnitude (−0.12 [95% CI −0.15 to −0.09]; P=5.9×10−15). Altogether, GEH‐SNPs explained 1.1% to 1.6% of GEH variance. Loci on chromosomes 4 (near HMCN2), 5 (IGF1R), 11 (11p11.2 region cluster), and 7 (near ACTB) are novel ECG phenotype‐associated loci. Several loci significantly associated with gene expression in the left ventricle (HMCN2 locus—with HMCN2;IGF1R locus—with IGF1R), and atria (RP11‐481J2.2 locus—with expression of a long non‐coding RNA and NDRG4). Conclusions We identified 10 genetic loci associated with ECG GEH. Replication of GEH GWAS findings in independent cohorts is warranted. Further studies of GEH‐loci may uncover mechanisms of arrhythmogenic remodeling in response to cardiovascular risk factors.