Unveiling the Role of the Most Impactful Cardiovascular Risk Locus through Haplotype Editing.

The 9p21.3 cardiovascular disease locus is the most influential common genetic risk factor for coronary artery disease (CAD), accounting for ∼10%-15% of disease in non-African populations. The ∼60 kb risk haplotype is human-specific and lacks coding genes, hindering efforts to decipher its function. Here, we produce induced pluripotent stem cells (iPSCs) from risk and non-risk individuals, delete each haplotype using genome editing, and generate vascular smooth muscle cells (VSMCs). Risk VSMCs exhibit globally altered transcriptional networks that intersect with previously identified CAD risk genes and pathways, concomitant with aberrant adhesion, contraction, and proliferation. Unexpectedly, deleting the risk haplotype rescues VSMC stability, while expressing the 9p21.3-associated long non-coding RNA ANRIL induces risk phenotypes in non-risk VSMCs. This study shows that the risk haplotype selectively predisposes VSMCs to adopt a cell state associated with CAD phenotypes, defines new VSMC-based networks of CAD risk genes, and establishes haplotype-edited iPSCs as powerful tools for functionally annotating the human genome.

Heterogeneous expression of alternatively spliced lncRNA mediates vascular smooth cell plasticity.

9p21.3 locus polymorphisms have the strongest correlation with coronary artery disease, but as a noncoding locus, disease connection is enigmatic. The lncRNA ANRIL found in 9p21.3 may regulate vascular smooth muscle cell (VSMC) phenotype to contribute to disease risk. We observed significant heterogeneity in induced pluripotent stem cell-derived VSMCs from patients homozygous for risk versus isogenic knockout or nonrisk haplotypes. Subpopulations of risk haplotype cells exhibited variable morphology, proliferation, contraction, and adhesion. When sorted by adhesion, risk VSMCs parsed into synthetic and contractile subpopulations, i.e., weakly adherent and strongly adherent, respectively. Of note, >90% of differentially expressed genes coregulated by haplotype and adhesion and were associated with Rho GTPases, i.e., contractility. Weakly adherent subpopulations expressed more short isoforms of ANRIL, and when overexpressed in knockout cells, ANRIL suppressed adhesion, contractility, and αSMA expression. These data suggest that variable lncRNA penetrance may drive mixed functional outcomes that confound pathology.

The 9p21.3 coronary artery disease risk locus drives vascular smooth muscle cells to an osteochondrogenic state.

Genome-wide association studies have identified common genetic variants at ~400 human genomic loci linked to coronary artery disease (CAD) susceptibility. Among these genomic regions, the most impactful is the 9p21.3 CAD risk locus, which spans a 60 kb gene desert and encompasses ~80 SNPs in high linkage disequilibrium. Despite nearly two decades since its discovery, the functional mechanism of this genomic region remains incompletely resolved. To investigate the transcriptional gene programs mediated by 9p21.3 risk locus, we applied a model of induced pluripotent stem cells (iPSCs) from risk and non-risk donors at 9p21.3, as well as isogenic lines with a full haplotype deletion. Upon differentiation to vascular smooth muscle cells (VSMC), single-cell transcriptomic profiling demonstrated iPSC-VSMC phenotypes resembling those from native human coronary arteries, confirming the robustness of this model. Remarkably, our analyses revealed that VSMCs harboring the 9p21.3 risk haplotype preferentially adopt an osteochondrogenic state. Importantly, we identified LIMCH1 and CRABP1 as signature genes critical for defining this transcriptional program. Our study provides new insights into the mechanism at the 9p21.3 risk locus and defines its role in driving a disease-prone transcriptional state in VSMCs.

High shear stress enhances endothelial permeability in the presence of the risk haplotype at 9p21.3.

Single nucleotide polymorphisms (SNPs) are exceedingly common in non-coding loci, and while they are significantly associated with a myriad of diseases, their specific impact on cellular dysfunction remains unclear. Here, we show that when exposed to external stressors, the presence of risk SNPs in the 9p21.3 coronary artery disease (CAD) risk locus increases endothelial monolayer and microvessel dysfunction. Endothelial cells (ECs) derived from induced pluripotent stem cells of patients carrying the risk haplotype (R/R WT) differentiated similarly to their non-risk and isogenic knockout (R/R KO) counterparts. Monolayers exhibited greater permeability and reactive oxygen species signaling when the risk haplotype was present. Addition of the inflammatory cytokine TNFα further enhanced EC monolayer permeability but independent of risk haplotype; TNFα also did not substantially alter haplotype transcriptomes. Conversely, when wall shear stress was applied to ECs in a microfluidic vessel, R/R WT vessels were more permeable at lower shear stresses than R/R KO vessels. Transcriptomes of sheared cells clustered more by risk haplotype than by patient or clone, resulting in significant differential regulation of EC adhesion and extracellular matrix genes vs static conditions. A subset of previously identified CAD risk genes invert expression patterns in the presence of high shear concomitant with altered cell adhesion genes, vessel permeability, and endothelial erosion in the presence of the risk haplotype, suggesting that shear stress could be a regulator of non-coding loci with a key impact on CAD.

Introductions to the Community: Early-Career Researchers in the Time of COVID-19.

COVID-19 has unfortunately halted lab work, conferences, and in-person networking, which is especially detrimental to researchers just starting their labs. Through social media and our reviewer networks, we met some early-career stem cell investigators impacted by the closures. Here, they introduce themselves and their research to our readers.

Metabolic Dysregulation of the Lysophospholipid/Autotaxin Axis in the Chromosome 9p21 Gene SNP rs10757274.

BACKGROUND: Common chromosome 9p21 single nucleotide polymorphisms (SNPs) increase coronary heart disease risk, independent of traditional lipid risk factors. However, lipids comprise large numbers of structurally related molecules not measured in traditional risk measurements, and many have inflammatory bioactivities. Here, we applied lipidomic and genomic approaches to 3 model systems to characterize lipid metabolic changes in common Chr9p21 SNPs, which confer ≈30% elevated coronary heart disease risk associated with altered expression of ANRIL, a long ncRNA. METHODS: Untargeted and targeted lipidomics was applied to plasma from NPHSII (Northwick Park Heart Study II) homozygotes for AA or GG in rs10757274, followed by correlation and network analysis. To identify candidate genes, transcriptomic data from shRNA downregulation of ANRIL in HEK-293 cells was mined. Transcriptional data from vascular smooth muscle cells differentiated from induced pluripotent stem cells of individuals with/without Chr9p21 risk, nonrisk alleles, and corresponding knockout isogenic lines were next examined. Last, an in-silico analysis of miRNAs was conducted to identify how ANRIL might control lysoPL (lysophosphospholipid)/lysoPA (lysophosphatidic acid) genes. RESULTS: Elevated risk GG correlated with reduced lysoPLs, lysoPA, and ATX (autotaxin). Five other risk SNPs did not show this phenotype. LysoPL-lysoPA interconversion was uncoupled from ATX in GG plasma, suggesting metabolic dysregulation. Significantly altered expression of several lysoPL/lysoPA metabolizing enzymes was found in HEK cells lacking ANRIL. In the vascular smooth muscle cells data set, the presence of risk alleles associated with altered expression of several lysoPL/lysoPA enzymes. Deletion of the risk locus reversed the expression of several lysoPL/lysoPA genes to nonrisk haplotype levels. Genes that were altered across both cell data sets were DGKA, MBOAT2, PLPP1, and LPL. The in-silico analysis identified 4 ANRIL-regulated miRNAs that control lysoPL genes as miR-186-3p, miR-34a-3p, miR-122-5p, and miR-34a-5p. CONCLUSIONS: A Chr9p21 risk SNP associates with complex alterations in immune-bioactive phospholipids and their metabolism. Lipid metabolites and genomic pathways associated with coronary heart disease pathogenesis in Chr9p21 and ANRIL-associated disease are demonstrated.

Phylogenetic comparison of huntingtin homologues reveals the appearance of a primitive polyQ in sea urchin.

Huntingtin is a completely soluble 3,144 amino acid (aa) protein characterized by the presence of an amino-terminal polymorphic polyglutamine (polyQ) tract, whose aberrant expansion causes the progressively neurodegenerative Huntington's disease (HD). Biological evidence indicates that huntingtin (htt) is beneficial to cells (particularly to brain neurons) and that loss of its neuronal function may contribute to HD. The exact protein domains involved in its neuroprotective function are unknown. Evolutionary analyses of htt primary aa have so far been limited to a few species, but its thorough assessment may help to clarify the functions emerging during evolution. We made an extensive comparative analysis of the available htt protein homologues from different organisms along the metazoan phylogenetic tree and defined the presence of 3 different conservative blocks corresponding to human htt aa 1-386 (htt1), 683-1,586 (htt2), and 2,437-3,078 (htt3), in which HEAT (Huntingtin, Elongator factor3, the regulatory A subunit of protein phosphatase 2A, and TOR1) repeats are well conserved. We also describe the cloning and sequencing of sea urchin htt mRNA, the oldest deuterostome homologue so far available. Multiple alignment shows the first appearance of a primitive polyQ in sea urchin, which predates an ancestral polyQ sequence in a nonchordate environment and defines the polyQ characteristic as being typical of the deuterostome branch. The fact that glutamines have conserved positions in deuterostomes and the polyQ size increases during evolution suggests that the protein has a possibly Q-dependent role. Finally, we report an evident relaxing constraint of the N-terminal block in Ciona and drosophilids that correlates with the absence of polyQ and which may indicate that the N-terminal portion of htt has evolved different functions in Ciona and protostomes.

Mechanical activation of noncoding-RNA-mediated regulation of disease-associated phenotypes in human cardiomyocytes.

How common polymorphisms in noncoding genome regions can regulate cellular function remains largely unknown. Here we show that cardiac fibrosis, mimicked using a hydrogel with controllable stiffness, affects the regulation of the phenotypes of human cardiomyocytes by a portion of the long noncoding RNA ANRIL, the gene of which is located in the disease-associated 9p21 locus. In a physiological environment, cultured cardiomyocytes derived from induced pluripotent stem cells obtained from patients who are homozygous for cardiovascular-risk alleles (R/R cardiomyocytes) or from healthy individuals who are homozygous for nonrisk alleles contracted synchronously, independently of genotype. After hydrogel stiffening to mimic fibrosis, only the R/R cardiomyocytes exhibited asynchronous contractions. These effects were associated with increased expression of the short ANRIL isoform in R/R cardiomyocytes, which induced a c-Jun N-terminal kinase (JNK) phosphorylation-based mechanism that impaired gap junctions (particularly, loss of connexin-43 expression) following stiffening. Deletion of the risk locus or treatment with a JNK antagonist was sufficient to maintain gap junctions and prevent asynchronous contraction of cardiomyocytes. Our findings suggest that mechanical changes in the microenvironment of cardiomyocytes can activate the regulation of their function by noncoding loci.

Influence of donor age on induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are being pursued as a source of cells for autologous therapies, many of which will be aimed at aged patients. To explore the impact of age on iPSC quality, we produced iPSCs from blood cells of 16 donors aged 21-100. We find that iPSCs from older donors retain an epigenetic signature of age, which can be reduced through passaging. Clonal expansion via reprogramming also enables the discovery of somatic mutations present in individual donor cells, which are missed by bulk sequencing methods. We show that exomic mutations in iPSCs increase linearly with age, and all iPSC lines analyzed carry at least one gene-disrupting mutation, several of which have been associated with cancer or dysfunction. Unexpectedly, elderly donors (>90 yrs) harbor fewer mutations than predicted, likely due to a contracted blood progenitor pool. These studies establish that donor age is associated with an increased risk of abnormalities in iPSCs and will inform clinical development of reprogramming technology.

Selective conversion of fibroblasts into peripheral sensory neurons.

Humans and mice detect pain, itch, temperature, pressure, stretch and limb position via signaling from peripheral sensory neurons. These neurons are divided into three functional classes (nociceptors/pruritoceptors, mechanoreceptors and proprioceptors) that are distinguished by their selective expression of TrkA, TrkB or TrkC receptors, respectively. We found that transiently coexpressing Brn3a with either Ngn1 or Ngn2 selectively reprogrammed human and mouse fibroblasts to acquire key properties of these three classes of sensory neurons. These induced sensory neurons (iSNs) were electrically active, exhibited distinct sensory neuron morphologies and matched the characteristic gene expression patterns of endogenous sensory neurons, including selective expression of Trk receptors. In addition, we found that calcium-imaging assays could identify subsets of iSNs that selectively responded to diverse ligands known to activate itch- and pain-sensing neurons. These results offer a simple and rapid means for producing genetically diverse human sensory neurons suitable for drug screening and mechanistic studies.

An evolutionary recent neuroepithelial cell adhesion function of huntingtin implicates ADAM10-Ncadherin.

The Huntington's disease gene product, huntingtin, is indispensable for neural tube formation, but its role is obscure. We studied neurulation in htt-null embryonic stem cells and htt-morpholino zebrafish embryos and found a previously unknown, evolutionarily recent function for this ancient protein. We found that htt was essential for homotypic interactions between neuroepithelial cells; it permitted neurulation and rosette formation by regulating metalloprotease ADAM10 activity and Ncadherin cleavage. This function was embedded in the N terminus of htt and was phenocopied by treatment of htt knockdown zebrafish with an ADAM10 inhibitor. Notably, in htt-null cells, reversion of the rosetteless phenotype occurred only with expression of evolutionarily recent htt heterologues from deuterostome organisms. Conversely, all of the heterologues that we tested, including htt from Drosophila melanogaster and Dictyostelium discoideum, exhibited anti-apoptotic activity. Thus, anti-apoptosis may have been one of htt's ancestral function(s), but, in deuterostomes, htt evolved to acquire a unique regulatory activity for controlling neural adhesion via ADAM10-Ncadherin, with implications for brain evolution and development.