Variants in autophagy genes MTMR12 and FAM134A are putative modifiers of the hepatic phenotype in α1-antitrypsin deficiency.

BACKGROUND AND AIMS: In the classical form of α1-antitrypsin deficiency, a misfolded variant α1-antitrypsin Z accumulates in the endoplasmic reticulum of liver cells and causes liver cell injury by gain-of-function proteotoxicity in a sub-group of affected homozygotes but relatively little is known about putative modifiers. Here, we carried out genomic sequencing in a uniquely affected family with an index case of liver failure and 2 homozygous siblings with minimal or no liver disease. Their sequences were compared to sequences in well-characterized cohorts of homozygotes with or without liver disease, and then candidate sequence variants were tested for changes in the kinetics of α1-antitrypsin variant Z degradation in iPS-derived hepatocyte-like cells derived from the affected siblings themselves. APPROACH AND RESULTS: Specific variants in autophagy genes MTMR12 and FAM134A could each accelerate the degradation of α1-antitrypsin variant Z in cells from the index patient, but both MTMR12 and FAM134A variants were needed to slow the degradation of α1-antitrypsin variant Z in cells from a protected sib, indicating that inheritance of both variants is needed to mediate the pathogenic effects of hepatic proteotoxicity at the cellular level. Analysis of homozygote cohorts showed that multiple patient-specific variants in proteostasis genes are likely to explain liver disease susceptibility at the population level. CONCLUSIONS: These results validate the concept that genetic variation in autophagy function can determine susceptibility to liver disease in α1-antitrypsin deficiency and provide evidence that polygenic mechanisms and multiple patient-specific variants are likely needed for proteotoxic pathology.

Janus Kinase 3 Deficiency Promotes Vascular Reendothelialization-Brief Report.

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LncRNA GAS5 attenuates fibroblast activation through inhibiting Smad3 signaling.

Transforming growth factor-ß (TGF-ß)-induced fibroblast activation is a key pathological event during tissue fibrosis. Long noncoding RNA (lncRNA) is a class of versatile gene regulators participating in various cellular and molecular processes. However, the function of lncRNA in fibroblast activation is still poorly understood. In this study, we identified growth arrest-specific transcript 5 (GAS5) as a novel regulator for TGF-ß-induced fibroblast activation. GAS5 expression was downregulated in cultured fibroblasts by TGF-ß and in resident fibroblasts from bleomycin-treated skin tissues. Overexpression of GAS5 suppressed TGF-ß-induced fibroblast to myofibroblast differentiation. Mechanistically, GAS5 directly bound mothers against decapentaplegic homolog 3 (Smad3) and promoted Smad3 binding to Protein phosphatase 1A (PPM1A), a Smad3 dephosphatase, and thus accelerated Smad3 dephosphorylation in TGF-ß-treated fibroblasts. In addition, GAS5 inhibited fibroblast proliferation. Importantly, local delivery of GAS5 via adenoviral vector suppressed bleomycin-induced skin fibrosis in mice. Collectively, our data revealed that GAS5 suppresses fibroblast activation and fibrogenesis through inhibiting TGF-ß/Smad3 signaling, which provides a rationale for an lncRNA-based therapy to treat fibrotic diseases.

Brain cytoplasmic RNA 1 suppresses smooth muscle differentiation and vascular development in mice.

The cardiovascular system develops during the early stages of embryogenesis, and differentiation of smooth muscle cells (SMCs) is essential for that process. SMC differentiation is critically regulated by transforming growth factor (TGF)-ß/SMAD family member 3 (SMAD3) signaling, but other regulators may also play a role. For example, long noncoding RNAs (lncRNAs) regulate various cellular activities and events, such as proliferation, differentiation, and apoptosis. However, whether long noncoding RNAs also regulate SMC differentiation remains largely unknown. Here, using the murine cell line C3H10T1/2, we found that brain cytoplasmic RNA 1 (BC1) is an important regulator of SMC differentiation. BC1 overexpression suppressed, whereas BC1 knockdown promoted, TGF-ß-induced SMC differentiation, as indicated by altered cell morphology and expression of multiple SMC markers, including smooth muscle α-actin (αSMA), calponin, and smooth muscle 22α (SM22α). BC1 appeared to block SMAD3 activity and inhibit SMC marker gene transcription. Mechanistically, BC1 bound to SMAD3 via RNA SMAD-binding elements (rSBEs) and thus impeded TGF-ß-induced SMAD3 translocation to the nucleus. This prevented SMAD3 from binding to SBEs in SMC marker gene promoters, an essential event in SMC marker transcription. In vivo, BC1 overexpression in mouse embryos impaired vascular SMC differentiation, leading to structural defects in the artery wall, such as random breaks in the elastic lamina, abnormal collagen deposition on SM fibers, and disorganized extracellular matrix proteins in the media of the neonatal aorta. Our results suggest that BC1 is a suppressor of SMC differentiation during vascular development.

The long non-coding RNA GAS5 regulates transforming growth factor ß (TGF-ß)-induced smooth muscle cell differentiation via RNA Smad-binding elements.

Smooth muscle cell (SMC) differentiation is essential for vascular development, and TGF-ß signaling plays a critical role in this process. Although long non-coding RNAs (lncRNAs) regulate various cellular events, their functions in SMC differentiation remain largely unknown. Here, we demonstrate that the lncRNA growth arrest-specific 5 (GAS5) suppresses TGF-ß/Smad3 signaling in smooth muscle cell differentiation of mesenchymal progenitor cells. We found that forced expression of GAS5 blocked, but knockdown of GAS5 increased, the expression of SMC contractile proteins. Mechanistically, GAS5 competitively bound Smad3 protein via multiple RNA Smad-binding elements (rSBEs), which prevented Smad3 from binding to SBE DNA in TGF-ß-responsive SMC gene promoters, resulting in suppression of SMC marker gene transcription and, consequently, in inhibition of TGF-ß/Smad3-mediated SMC differentiation. Importantly, other lncRNAs or artificially synthesized RNA molecules that contained rSBEs also effectively inhibited TGF-ß/Smad3 signaling, suggesting that lncRNA-rSBE may be a general mechanism used by cells to fine-tune Smad3 activity in both basal and TGF-ß-stimulated states. Taken together, our results have uncovered an lncRNA-based mechanism that modulates TGF-ß/Smad3 signaling during SMC differentiation.

Janus Kinase 3, a Novel Regulator for Smooth Muscle Proliferation and Vascular Remodeling.

OBJECTIVE: Vascular remodeling because of smooth muscle cell (SMC) proliferation is a common process occurring in several vascular diseases, such as atherosclerosis, aortic aneurysm, post-transplant vasculopathy, restenosis after angioplasty, etc. The molecular mechanism underlying SMC proliferation, however, is not completely understood. The objective of this study is to determine the role and mechanism of Janus kinase 3 (JAK3) in vascular remodeling and SMC proliferation. APPROACH AND RESULTS: Platelet-derived growth factor-BB, an SMC mitogen, induces JAK3 expression and phosphorylation while stimulating SMC proliferation. Janex-1, a specific inhibitor of JAK3, or knockdown of JAK3 by short hairpin RNA, inhibits the SMC proliferation. Conversely, ectopic expression of JAK3 promotes SMC proliferation. Mechanistically, JAK3 promotes the phosphorylation of signal transducer and activator of transcription 3 and c-Jun N-terminal kinase in SMC, 2 signaling pathways known to be critical for SMC proliferation and vascular remodeling. Blockade of these 2 signaling pathways by their inhibitors impeded the JAK3-mediated SMC proliferation. In vivo, knockdown of JAK3 attenuates injury-induced neointima formation with attenuated neointimal SMC proliferation. Knockdown of JAK3 also induces neointimal SMC apoptosis in rat carotid artery balloon injury model. CONCLUSIONS: Our results demonstrate that JAK3 mediates SMC proliferation and survival during injury-induced vascular remodeling, which provides a potential therapeutic target for preventing neointimal hyperplasia in proliferative vascular diseases.

Multiomic analyses implicate a neurodevelopmental program in the pathogenesis of cerebral arachnoid cysts.

Cerebral arachnoid cysts (ACs) are one of the most common and poorly understood types of developmental brain lesion. To begin to elucidate AC pathogenesis, we performed an integrated analysis of 617 patient-parent (trio) exomes, 152,898 human brain and mouse meningeal single-cell RNA sequencing transcriptomes and natural language processing data of patient medical records. We found that damaging de novo variants (DNVs) were highly enriched in patients with ACs compared with healthy individuals (P = 1.57 × 10-33). Seven genes harbored an exome-wide significant DNV burden. AC-associated genes were enriched for chromatin modifiers and converged in midgestational transcription networks essential for neural and meningeal development. Unsupervised clustering of patient phenotypes identified four AC subtypes and clinical severity correlated with the presence of a damaging DNV. These data provide insights into the coordinated regulation of brain and meningeal development and implicate epigenomic dysregulation due to DNVs in AC pathogenesis. Our results provide a preliminary indication that, in the appropriate clinical context, ACs may be considered radiographic harbingers of neurodevelopmental pathology warranting genetic testing and neurobehavioral follow-up. These data highlight the utility of a systems-level, multiomics approach to elucidate sporadic structural brain disease.

Genetic dysregulation of an endothelial Ras signaling network in vein of Galen malformations.

To elucidate the pathogenesis of vein of Galen malformations (VOGMs), the most common and severe congenital brain arteriovenous malformation, we performed an integrated analysis of 310 VOGM proband-family exomes and 336,326 human cerebrovasculature single-cell transcriptomes. We found the Ras suppressor p120 RasGAP ( RASA1 ) harbored a genome-wide significant burden of loss-of-function de novo variants (p=4.79×10 -7 ). Rare, damaging transmitted variants were enriched in Ephrin receptor-B4 ( EPHB4 ) (p=1.22×10 -5 ), which cooperates with p120 RasGAP to limit Ras activation. Other probands had pathogenic variants in ACVRL1 , NOTCH1 , ITGB1 , and PTPN11 . ACVRL1 variants were also identified in a multi-generational VOGM pedigree. Integrative genomics defined developing endothelial cells as a key spatio-temporal locus of VOGM pathophysiology. Mice expressing a VOGM-specific EPHB4 kinase-domain missense variant exhibited constitutive endothelial Ras/ERK/MAPK activation and impaired hierarchical development of angiogenesis-regulated arterial-capillary-venous networks, but only when carrying a "second-hit" allele. These results illuminate human arterio-venous development and VOGM pathobiology and have clinical implications.

Mutation of key signaling regulators of cerebrovascular development in vein of Galen malformations.

To elucidate the pathogenesis of vein of Galen malformations (VOGMs), the most common and most severe of congenital brain arteriovenous malformations, we performed an integrated analysis of 310 VOGM proband-family exomes and 336,326 human cerebrovasculature single-cell transcriptomes. We found the Ras suppressor p120 RasGAP (RASA1) harbored a genome-wide significant burden of loss-of-function de novo variants (2042.5-fold, p = 4.79 x 10-7). Rare, damaging transmitted variants were enriched in Ephrin receptor-B4 (EPHB4) (17.5-fold, p = 1.22 x 10-5), which cooperates with p120 RasGAP to regulate vascular development. Additional probands had damaging variants in ACVRL1, NOTCH1, ITGB1, and PTPN11. ACVRL1 variants were also identified in a multi-generational VOGM pedigree. Integrative genomic analysis defined developing endothelial cells as a likely spatio-temporal locus of VOGM pathophysiology. Mice expressing a VOGM-specific EPHB4 kinase-domain missense variant (Phe867Leu) exhibited disrupted developmental angiogenesis and impaired hierarchical development of arterial-capillary-venous networks, but only in the presence of a "second-hit" allele. These results illuminate human arterio-venous development and VOGM pathobiology and have implications for patients and their families.

Computational Genomics in the Era of Precision Medicine: Applications to Variant Analysis and Gene Therapy.

Rapid methodological advances in statistical and computational genomics have enabled researchers to better identify and interpret both rare and common variants responsible for complex human diseases. As we continue to see an expansion of these advances in the field, it is now imperative for researchers to understand the resources and methodologies available for various data types and study designs. In this review, we provide an overview of recent methods for identifying rare and common variants and understanding their roles in disease etiology. Additionally, we discuss the strategy, challenge, and promise of gene therapy. As computational and statistical approaches continue to improve, we will have an opportunity to translate human genetic findings into personalized health care.

LncRNA GAS5 regulates vascular smooth muscle cell cycle arrest and apoptosis via p53 pathway.

Vascular remodeling is a pathological process following cardiovascular intervention. Vascular smooth muscle cells (VSMC) play a critical role in the vascular remodeling. Long noncoding RNAs (lncRNA) are a class of gene regulators functioning through various mechanisms in physiological and pathological conditions. By using cultured VSMC and rat carotid artery balloon injury model, we found that lncRNA growth arrest specific 5 (GAS5) serves as a negative regulator for VSMC survival in vascular remodeling. By manipulating GAS5 expression via adenoviral overexpression or short hairpin RNA knockdown, we found that GAS5 suppresses VSMC proliferation while promoting cell cycle arrest and inducing cell apoptosis. Mechanistically, GAS5 directly binds to p53 and p300, stabilizes p53-p300 interaction, and thus regulates VSMC cell survival via induction of p53-downstream target genes. Importantly, local delivery of GAS5 via adenoviral vector suppresses balloon injury-induced neointima formation along with an increased expression of p53 and apoptosis in neointimal SMCs. Our study demonstrated for the first time that GAS5 negatively impacts VSMC survival via activation the p53 pathway during vascular remodeling.

Serologic assessment of the risk of developing chronic Q fever in cohorts of acutely infected individuals.

OBJECTIVE: The aim of this study was to assess the clinical significance of serological profiles suggestive of chronic Q fever after acute infection. METHODS: A prospective follow-up study consisting of two separate cohorts was conducted to monitor the serological evolution of Q fever. The first cohort comprised subjects with acute Q fever diagnosed in 2004-2007 and the second enrolled subjects whose infection occurred in 2009. The indirect immunofluorescence assay was used for serological monitoring, with serum PCR testing added for subjects whose serological profiles revealed high titers of anti-phase I IgG≥800, titers suggestive of chronic Q fever. RESULTS: In the first cohort of 92 persons, seventeen (18%) subjects had serological profiles suggestive of chronic Q fever (titers of anti-phase I IgG: 1280-5120, median: 1280) after a median follow-up period of 606.5 days. After a further follow-up (median period: 592 days) exclusively for those seventeen subjects, serological resolution with fourfold decrease of titers of anti-phase I IgG was noted in five of them. In the second cohort, only one (4%) of the twenty-eight subjects had high levels of anti-phase I IgG 180 days after acute infection. All the eighteen subjects with high levels of anti-phase I IgG were asymptomatic and had negative serum PCR testing. The different prevalence of subjects with high titers of anti-phase I IgG in the two cohorts was associated with duration of follow-up period (P < .01). CONCLUSIONS: Subjects with high titers of anti-phase I IgG≥800 was not uncommon and might not be detected until more than six months after acute Q fever infection. Asymptomatic subjects with high levels of anti-phase I IgG alone should not be treated as chronic Q fever and might not need continued serological monitoring in the absence of predisposing factors to chronic Q fever.