Exploring the Genetic Architecture of Spontaneous Coronary Artery Dissection Using Whole-Genome Sequencing.

BACKGROUND: Spontaneous coronary artery dissection (SCAD) is a cause of acute coronary syndrome that predominantly affects women. Its pathophysiology remains unclear but connective tissue disorders (CTD) and other vasculopathies have been observed in many SCAD patients. A genetic component for SCAD is increasingly appreciated, although few genes have been robustly implicated. We sought to clarify the genetic cause of SCAD using targeted and genome-wide methods in a cohort of sporadic cases to identify both common and rare disease-associated variants. METHODS: A cohort of 91 unrelated sporadic SCAD cases was investigated for rare, deleterious variants in genes associated with either SCAD or CTD, while new candidate genes were sought using rare variant collapsing analysis and identification of novel loss-of-function variants in genes intolerant to such variation. Finally, 2 SCAD polygenic risk scores were applied to assess the contribution of common variants. RESULTS: We identified 10 cases with at least one rare, likely disease-causing variant in CTD-associated genes, although only one had a CTD phenotype. No genes were significantly associated with SCAD from genome-wide collapsing analysis, however, enrichment for TGF (transforming growth factor)-ß signaling pathway genes was found with analysis of 24 genes harboring novel loss-of-function variants. Both polygenic risk scores demonstrated that sporadic SCAD cases have a significantly elevated genetic SCAD risk compared with controls. CONCLUSIONS: SCAD shares some genetic overlap with CTD, even in the absence of any major CTD phenotype. Consistent with a complex genetic architecture, SCAD patients also have a higher burden of common variants than controls.

Transposon clusters as substrates for aberrant splice-site activation.

Transposed elements (TEs) have dramatically shaped evolution of the exon-intron structure and significantly contributed to morbidity, but how recent TE invasions into older TEs cooperate in generating new coding sequences is poorly understood. Employing an updated repository of new exon-intron boundaries induced by pathogenic mutations, termed DBASS, here we identify novel TE clusters that facilitated exon selection. To explore the extent to which such TE exons maintain RNA secondary structure of their progenitors, we carried out structural studies with a composite exon that was derived from a long terminal repeat (LTR78) and AluJ and was activated by a C > T mutation optimizing the 5' splice site. Using a combination of SHAPE, DMS and enzymatic probing, we show that the disease-causing mutation disrupted a conserved AluJ stem that evolved from helix 3.3 (or 5b) of 7SL RNA, liberating a primordial GC 5' splice site from the paired conformation for interactions with the spliceosome. The mutation also reduced flexibility of conserved residues in adjacent exon-derived loops of the central Alu hairpin, revealing a cross-talk between traditional and auxilliary splicing motifs that evolved from opposite termini of 7SL RNA and were approximated by Watson-Crick base-pairing already in organisms without spliceosomal introns. We also identify existing Alu exons activated by the same RNA rearrangement. Collectively, these results provide valuable TE exon models for studying formation and kinetics of pre-mRNA building blocks required for splice-site selection and will be useful for fine-tuning auxilliary splicing motifs and exon and intron size constraints that govern aberrant splice-site activation.

Functional genomics and gene-environment interaction highlight the complexity of congenital heart disease caused by Notch pathway variants.

Congenital heart disease (CHD) is the most common birth defect and brings with it significant mortality and morbidity. The application of exome and genome sequencing has greatly improved the rate of genetic diagnosis for CHD but the cause in the majority of cases remains uncertain. It is clear that genetics, as well as environmental influences, play roles in the aetiology of CHD. Here we address both these aspects of causation with respect to the Notch signalling pathway. In our CHD cohort, variants in core Notch pathway genes account for 20% of those that cause disease, a rate that did not increase with the inclusion of genes of the broader Notch pathway and its regulators. This is reinforced by case-control burden analysis where variants in Notch pathway genes are enriched in CHD patients. This enrichment is due to variation in NOTCH1. Functional analysis of some novel missense NOTCH1 and DLL4 variants in cultured cells demonstrate reduced signalling activity, allowing variant reclassification. Although loss-of-function variants in DLL4 are known to cause Adams-Oliver syndrome, this is the first report of a hypomorphic DLL4 allele as a cause of isolated CHD. Finally, we demonstrate a gene-environment interaction in mouse embryos between Notch1 heterozygosity and low oxygen- or anti-arrhythmic drug-induced gestational hypoxia, resulting in an increased incidence of heart defects. This implies that exposure to environmental insults such as hypoxia could explain variable expressivity and penetrance of observed CHD in families carrying Notch pathway variants.

Spliceogen: an integrative, scalable tool for the discovery of splice-altering variants.

MOTIVATION: In silico prediction tools are essential for identifying variants which create or disrupt cis-splicing motifs. However, there are limited options for genome-scale discovery of splice-altering variants. RESULTS: We have developed Spliceogen, a highly scalable pipeline integrating predictions from some of the individually best performing models for splice motif prediction: MaxEntScan, GeneSplicer, ESRseq and Branchpointer. AVAILABILITY AND IMPLEMENTATION: Spliceogen is available as a command line tool which accepts VCF/BED inputs and handles both single nucleotide variants (SNVs) and indels (https://github.com/VCCRI/Spliceogen). SNV databases with prediction scores are also available, covering all possible SNVs at all genomic positions within all Gencode-annotated multi-exon transcripts. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

High-density lipoproteins augment hypoxia-induced angiogenesis via regulation of post-translational modulation of hypoxia-inducible factor 1α.

Increasing evidence suggests that high-density lipoproteins (HDLs) promote hypoxia-induced angiogenesis. The hypoxia-inducible factor 1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway is important in hypoxia and is modulated post-translationally by prolyl hydroxylases (PHD1-PHD3) and E3 ubiquitin ligases (Siah1 and Siah2). We aimed to elucidate the mechanisms by which HDLs augment hypoxia-induced angiogenesis. Preincubation (16 h) of human coronary artery endothelial cells with reconstituted high-density lipoprotein (rHDL) containing apolipoprotein A-I (apoA-I) and phosphatidylcholine (20 µM, final apoA-I concentration), before hypoxia, increased Siah1 (58%) and Siah2 (88%) mRNA levels and suppressed PHD2 (32%) and PHD3 (45%) protein levels compared with hypoxia-induced control levels. After Siah1/2 small interfering RNA knockdown, rHDL was unable to suppress PHD2/3 and failed to induce HIF-1α, VEGF, and tubulogenesis in hypoxia. Inhibition of the upstream phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway also abrogated the effects of rHDL. Furthermore, knockdown of the scavenger receptor SR-BI attenuated rHDL-induced elevations in Siah1/2 and tubulogenesis in hypoxia, indicating that SR-BI plays a key role. Finally, the importance of VEGF in mediating the ability of rHDL to drive hypoxia-induced angiogenesis was confirmed using a VEGF-neutralizing antibody. In summary, rHDL augments the HIF-1α/VEGF pathway via SR-BI and modulation of the post-translational regulators of HIF-1α (PI3K/Siahs/PHDs). HDL-induced augmentation of angiogenesis in hypoxia may have implications for therapeutic modulation of ischemic injury.