Evaluation of somatic copy number variation detection by NGS technologies and bioinformatics tools on a hyper-diploid cancer genome.

BACKGROUND: Copy number variation (CNV) is a key genetic characteristic for cancer diagnostics and can be used as a biomarker for the selection of therapeutic treatments. Using data sets established in our previous study, we benchmark the performance of cancer CNV calling by six most recent and commonly used software tools on their detection accuracy, sensitivity, and reproducibility. In comparison to other orthogonal methods, such as microarray and Bionano, we also explore the consistency of CNV calling across different technologies on a challenging genome. RESULTS: While consistent results are observed for copy gain, loss, and loss of heterozygosity (LOH) calls across sequencing centers, CNV callers, and different technologies, variation of CNV calls are mostly affected by the determination of genome ploidy. Using consensus results from six CNV callers and confirmation from three orthogonal methods, we establish a high confident CNV call set for the reference cancer cell line (HCC1395). CONCLUSIONS: NGS technologies and current bioinformatics tools can offer reliable results for detection of copy gain, loss, and LOH. However, when working with a hyper-diploid genome, some software tools can call excessive copy gain or loss due to inaccurate assessment of genome ploidy. With performance matrices on various experimental conditions, this study raises awareness within the cancer research community for the selection of sequencing platforms, sample preparation, sequencing coverage, and the choice of CNV detection tools.

A Stem-like Patient-Derived Ovarian Cancer Model of Platinum Resistance Reveals Dissociation of Stemness and Resistance.

To understand chemoresistance in the context of cancer stem cells (CSC), a cisplatin resistance model was developed using a high-grade serous ovarian cancer patient-derived, cisplatin-sensitive sample, PDX4. As a molecular subtype-specific stem-like cell line, PDX4 was selected for its representative features, including its histopathological and BRCA2 mutation status, and exposed to cisplatin in vitro. In the cisplatin-resistant cells, transcriptomics were carried out, and cell morphology, protein expression, and functional status were characterized. Additionally, potential signaling pathways involved in cisplatin resistance were explored. Our findings reveal the presence of distinct molecular signatures and phenotypic changes in cisplatin-resistant PDX4 compared to their sensitive counterparts. Surprisingly, we observed that chemoresistance was not inherently linked with increased stemness. In fact, although resistant cells expressed a combination of EMT and stemness markers, functional assays revealed that they were less proliferative, migratory, and clonogenic-features indicative of an underlying complex mechanism for cell survival. Furthermore, DNA damage tolerance and cellular stress management pathways were enriched. This novel, syngeneic model provides a valuable platform for investigating the underlying mechanisms of cisplatin resistance in a clinically relevant context, contributing to the development of targeted therapies tailored to combat resistance in stem-like ovarian cancer.

Continuous Detection of Cobalt Ions and Pyrophosphates by NAC-CdTe Quantum Dots Fluorescence Probes.

In this work, a simple and sensitive N-acetyl-L-cysteine (NAC)-covered CdTe quantum dots (NAC-CdTe QDs) fluorescence probe for continuous detection of Co2+ and pyrophosphate ions (PPi, P2O74-) was synthesized. The fluorescence of the quantum dots was significantly quenched by Co2+ through the coordination of Co2+ and the carboxyl groups on the NAC-CdTe quantum dots. Interestingly, the combination of NAC-CdTe quantum dots with Co2+ yields a new fluorescence probe of Co2+-modified NAC-CdTe QDs (Co2+@NAC-CdTe). The addition of PPi restored the fluorescence due to the competition between PPi and carboxyl groups with Co2+ causing Co2+ to detach from the surface of Co2+@NAC-CdTe quantum dots. Thus, a sensitive and reversible detection of Co2+ and PPi had been successfully established. The Co2+@NAC-CdTe quantum dots fluorescence probe exhibits excellent selectivity and high sensitivity toward PPi detection with low detection limit of 0.28 µM. In addition, the novel fluorescence probe was successfully applied to detect the concentration of PPi in environmental water samples and in-vitro cells imaging.

Dysregulated miRNAs modulate tumor microenvironment associated signaling networks in pancreatic ductal adenocarcinoma.

The desmoplastic and complex tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC) has presented tremendous challenges for developing effective therapeutic strategies. Strategies targeting tumor stroma, albeit with great potential, have met with limited success due to the lack of knowledge on the molecular dynamics within the tumor microenvironment (TME). In pursuit of a better understanding of the influence of miRNAs on TME reprogramming and to explore circulating miRNAs as diagnostic and prognostic biomarkers for PDAC, using RNA-seq, miRNA-seq, and single-cell RNA-seq (scRNA-seq), we investigated the dysregulated signaling pathways in PDAC TME modulated by miRNAs from plasma and tumor tissue. Our bulk RNA-seq in PDAC tumor tissue identified 1445 significantly differentially expressed genes with extracellular matrix and structure organization as the top enriched pathways. Our miRNA-seq identified 322 and 49 abnormally expressed miRNAs in PDAC patient plasma and tumor tissue, respectively. We found many of the TME signaling pathways were targeted by those dysregulated miRNAs in PDAC plasma. Combined with scRNA-seq from patient PDAC tumor, our results revealed that these dysregulated miRNAs were closely associated with extracellular matrix (ECM) remodeling, cell-ECM communication, epithelial-mesenchymal transition, as well as immunosuppression orchestrated by different cellular components of TME. The findings of this study could assist the development of miRNA-based stromal targeting biomarkers or therapy for PDAC patients.

Reprogramming of human peripheral blood mononuclear cells into induced mesenchymal stromal cells using non-integrating vectors.

Mesenchymal stromal cells (MSCs) have great value in cell therapies. The MSC therapies have many challenges due to its inconsistent potency and limited quantity. Here, we report a strategy to generate induced MSCs (iMSCs) by directly reprogramming human peripheral blood mononuclear cells (PBMCs) with OCT4, SOX9, MYC, KLF4, and BCL-XL using a nonintegrating episomal vector system. While OCT4 was not required to reprogram PBMCs into iMSCs, omission of OCT4 significantly impaired iMSC functionality. The omission of OCT4 resulted in significantly downregulating MSC lineage specific and mesoderm-regulating genes, including SRPX, COL5A1, SOX4, SALL4, TWIST1. When reprogramming PBMCs in the absence of OCT4, 67 genes were significantly hypermethylated with reduced transcriptional expression. These data indicate that transient expression of OCT4 may serve as a universal reprogramming factor by increasing chromatin accessibility and promoting demethylation. Our findings represent an approach to produce functional MSCs, and aid in identifying putative function associated MSC markers.

Structural variant analysis of a cancer reference cell line sample using multiple sequencing technologies.

BACKGROUND: The cancer genome is commonly altered with thousands of structural rearrangements including insertions, deletions, translocation, inversions, duplications, and copy number variations. Thus, structural variant (SV) characterization plays a paramount role in cancer target identification, oncology diagnostics, and personalized medicine. As part of the SEQC2 Consortium effort, the present study established and evaluated a consensus SV call set using a breast cancer reference cell line and matched normal control derived from the same donor, which were used in our companion benchmarking studies as reference samples. RESULTS: We systematically investigated somatic SVs in the reference cancer cell line by comparing to a matched normal cell line using multiple NGS platforms including Illumina short-read, 10X Genomics linked reads, PacBio long reads, Oxford Nanopore long reads, and high-throughput chromosome conformation capture (Hi-C). We established a consensus SV call set of a total of 1788 SVs including 717 deletions, 230 duplications, 551 insertions, 133 inversions, 146 translocations, and 11 breakends for the reference cancer cell line. To independently evaluate and cross-validate the accuracy of our consensus SV call set, we used orthogonal methods including PCR-based validation, Affymetrix arrays, Bionano optical mapping, and identification of fusion genes detected from RNA-seq. We evaluated the strengths and weaknesses of each NGS technology for SV determination, and our findings provide an actionable guide to improve cancer genome SV detection sensitivity and accuracy. CONCLUSIONS: A high-confidence consensus SV call set was established for the reference cancer cell line. A large subset of the variants identified was validated by multiple orthogonal methods.

Personalized genome assembly for accurate cancer somatic mutation discovery using tumor-normal paired reference samples.

BACKGROUND: The use of a personalized haplotype-specific genome assembly, rather than an unrelated, mosaic genome like GRCh38, as a reference for detecting the full spectrum of somatic events from cancers has long been advocated but has never been explored in tumor-normal paired samples. Here, we provide the first demonstrated use of de novo assembled personalized genome as a reference for cancer mutation detection and quantifying the effects of the reference genomes on the accuracy of somatic mutation detection. RESULTS: We generate de novo assemblies of the first tumor-normal paired genomes, both nuclear and mitochondrial, derived from the same individual with triple negative breast cancer. The personalized genome was chromosomal scale, haplotype phased, and annotated. We demonstrate that it provides individual specific haplotypes for complex regions and medically relevant genes. We illustrate that the personalized genome reference not only improves read alignments for both short-read and long-read sequencing data but also ameliorates the detection accuracy of somatic SNVs and SVs. We identify the equivalent somatic mutation calls between two genome references and uncover novel somatic mutations only when personalized genome assembly is used as a reference. CONCLUSIONS: Our findings demonstrate that use of a personalized genome with individual-specific haplotypes is essential for accurate detection of the full spectrum of somatic mutations in the paired tumor-normal samples. The unique resource and methodology established in this study will be beneficial to the development of precision oncology medicine not only for breast cancer, but also for other cancers.

Single-nucleus chromatin accessibility and RNA sequencing reveal impaired brain development in prenatally e-cigarette exposed neonatal rats.

Although emerging evidence reveals that vaping alters the function of the central nervous system, the effects of maternal vaping on offspring brain development remain elusive. Using a well-established in utero exposure model, we performed single-nucleus ATAC-seq (snATAC-seq) and RNA sequencing (snRNA-seq) on prenatally e-cigarette-exposed rat brains. We found that maternal vaping distorted neuronal lineage differentiation in the neonatal brain by promoting excitatory neurons and inhibiting lateral ganglionic eminence-derived inhibitory neuronal differentiation. Moreover, maternal vaping disrupted calcium homeostasis, induced microglia cell death, and elevated susceptibility to cerebral ischemic injury in the developing brain of offspring. Our results suggest that the aberrant calcium signaling, diminished microglial population, and impaired microglia-neuron interaction may all contribute to the underlying mechanisms by which prenatal e-cigarette exposure impairs neonatal rat brain development. Our findings raise the concern that maternal vaping may cause adverse long-term brain damage to the offspring.

Whole genome and exome sequencing reference datasets from a multi-center and cross-platform benchmark study.

With the rapid advancement of sequencing technologies, next generation sequencing (NGS) analysis has been widely applied in cancer genomics research. More recently, NGS has been adopted in clinical oncology to advance personalized medicine. Clinical applications of precision oncology require accurate tests that can distinguish tumor-specific mutations from artifacts introduced during NGS processes or data analysis. Therefore, there is an urgent need to develop best practices in cancer mutation detection using NGS and the need for standard reference data sets for systematically measuring accuracy and reproducibility across platforms and methods. Within the SEQC2 consortium context, we established paired tumor-normal reference samples and generated whole-genome (WGS) and whole-exome sequencing (WES) data using sixteen library protocols, seven sequencing platforms at six different centers. We systematically interrogated somatic mutations in the reference samples to identify factors affecting detection reproducibility and accuracy in cancer genomes. These large cross-platform/site WGS and WES datasets using well-characterized reference samples will represent a powerful resource for benchmarking NGS technologies, bioinformatics pipelines, and for the cancer genomics studies.

Toward best practice in cancer mutation detection with whole-genome and whole-exome sequencing.

Clinical applications of precision oncology require accurate tests that can distinguish true cancer-specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole-genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection.

Establishing community reference samples, data and call sets for benchmarking cancer mutation detection using whole-genome sequencing.

The lack of samples for generating standardized DNA datasets for setting up a sequencing pipeline or benchmarking the performance of different algorithms limits the implementation and uptake of cancer genomics. Here, we describe reference call sets obtained from paired tumor-normal genomic DNA (gDNA) samples derived from a breast cancer cell line-which is highly heterogeneous, with an aneuploid genome, and enriched in somatic alterations-and a matched lymphoblastoid cell line. We partially validated both somatic mutations and germline variants in these call sets via whole-exome sequencing (WES) with different sequencing platforms and targeted sequencing with >2,000-fold coverage, spanning 82% of genomic regions with high confidence. Although the gDNA reference samples are not representative of primary cancer cells from a clinical sample, when setting up a sequencing pipeline, they not only minimize potential biases from technologies, assays and informatics but also provide a unique resource for benchmarking 'tumor-only' or 'matched tumor-normal' analyses.

A multicenter study benchmarking single-cell RNA sequencing technologies using reference samples.

Comparing diverse single-cell RNA sequencing (scRNA-seq) datasets generated by different technologies and in different laboratories remains a major challenge. Here we address the need for guidance in choosing algorithms leading to accurate biological interpretations of varied data types acquired with different platforms. Using two well-characterized cellular reference samples (breast cancer cells and B cells), captured either separately or in mixtures, we compared different scRNA-seq platforms and several preprocessing, normalization and batch-effect correction methods at multiple centers. Although preprocessing and normalization contributed to variability in gene detection and cell classification, batch-effect correction was by far the most important factor in correctly classifying the cells. Moreover, scRNA-seq dataset characteristics (for example, sample and cellular heterogeneity and platform used) were critical in determining the optimal bioinformatic method. However, reproducibility across centers and platforms was high when appropriate bioinformatic methods were applied. Our findings offer practical guidance for optimizing platform and software selection when designing an scRNA-seq study.

Correction: PDGFB-expressing mesenchymal stem cells improve human hematopoietic stem cell engraftment in immunodeficient mice.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Unique anabolic action of stem cell gene therapy overexpressing PDGFB-DSS6 fusion protein in OVX osteoporosis mouse model.

In the present study we sought to improve the efficacy and safety of our Sca1+ PDGFB stem cell gene therapy for osteoporosis in ovariectomized (OVX) mouse model. This therapy is administered by marrow transplantation. We established the promise of this approach by previously showing that this therapy in normal mice increase bone density, increased endosteal cortical and trabecular bone formation, caused de novo trabecular bone formation, increased cortical thickness and improve bone strength. In the current study we produced a fusion gene, PDGFB-DSS6. We reasoned that the DSS6, calcium binding protein would trap the PDGFB at the bone surface and thereby limit the amount of PDGFB required to produce an optimal bone formation response, i.e. efficacy with a lower engraftment. The result shows that indeed with a very low level of engraftment we achieved a large increase in bone formation in the OVX model of bone loss. Serum analysis for biochemical marker of new bone formation showed an approximate 75% increase in alkaline phosphatase levels in Sca1+PDGFB-DSS6 group as compared to other groups. Quantitative analysis of bone by microCT showed a massive increase in trabecular bone density and trabecular connectivity of the femur in the metaphysis in Sca1+ PDGFB-DSS6 group. The increased cortical porosity produced by OVX was replaced by the Sca1+ PDGFB-DSS6 therapy but not by the positive control Sca1+ PDGFB. Additionally, an increase in the femur bone strength was also observed specifically in Sca1+ PDGFB-DSS6 as compared to other treatment groups, emphasizing the functional significance of the observed anabolic action is on bone formation. In future work we will focus on nontoxic preconditioning of our marrow transplantation procedure and also on transcriptional control of therapeutic gene expression to avoid excess bone formation.

PDGFB-expressing mesenchymal stem cells improve human hematopoietic stem cell engraftment in immunodeficient mice.

The bone marrow (BM) niche regulates multiple hematopoietic stem cell (HSC) processes. Clinical treatment for hematological malignancies by HSC transplantation often requires preconditioning via total body irradiation, which severely and irreversibly impairs the BM niche and HSC regeneration. Novel strategies are needed to enhance HSC regeneration in irradiated BM. We compared the effects of EGF, FGF2, and PDGFB on HSC regeneration using human mesenchymal stem cells (MSCs) that were transduced with these factors via lentiviral vectors. Among the above niche factors tested, MSCs transduced with PDGFB (PDGFB-MSCs) most significantly improved human HSC engraftment in immunodeficient mice. PDGFB-MSC-treated BM enhanced transplanted human HSC self-renewal in secondary transplantations more efficiently than GFP-transduced MSCs (GFP-MSCs). Gene set enrichment analysis showed increased antiapoptotic signaling in PDGFB-MSCs compared with GFP-MSCs. PDGFB-MSCs exhibited enhanced survival and expansion after transplantation, resulting in an enlarged humanized niche cell pool that provide a better humanized microenvironment to facilitate superior engraftment and proliferation of human hematopoietic cells. Our studies demonstrate the efficacy of PDGFB-MSCs in supporting human HSC engraftment.

Thalidomide Reduces Hemorrhage of Brain Arteriovenous Malformations in a Mouse Model.

BACKGROUND AND PURPOSE: Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. Current treatments for bAVM are all associated with considerable risks. There is no safe method to prevent bAVM hemorrhage. Thalidomide reduces nose bleeding in patients with hereditary hemorrhagic telangiectasia, an inherited disorder characterized by vascular malformations. In this study, we tested whether thalidomide and its less toxic analog, lenalidomide, reduce bAVM hemorrhage using a mouse model. METHODS: bAVMs were induced through induction of brain focal activin-like kinase 1 (Alk1, an AVM causative gene) gene deletion and angiogenesis in adult Alk1-floxed mice. Thalidomide was injected intraperitoneally twice per week for 6 weeks, starting either 2 or 8 weeks after AVM induction. Lenalidomide was injected intraperitoneally daily starting 8 weeks after AVM induction for 6 weeks. Brain samples were collected at the end of the treatments for morphology, mRNA, and protein analyses. The influence of Alk1 downregulation on PDGFB (platelet-derived growth factor B) expression was also studied on cultured human brain microvascular endothelial cells. The effect of PDGFB in mural cell recruitment in bAVM was explored by injection of a PDGFB overexpressing lentiviral vector to the mouse brain. RESULTS: Thalidomide or lenalidomide treatment reduced the number of dysplastic vessels and hemorrhage and increased mural cell (vascular smooth muscle cells and pericytes) coverage in the bAVM lesion. Thalidomide reduced the burden of CD68+ cells and the expression of inflammatory cytokines in the bAVM lesions. PDGFB expression was reduced in ALK1-knockdown human brain microvascular endothelial cells and in mouse bAVM lesion. Thalidomide increased Pdgfb expression in bAVM lesion. Overexpression of PDGFB mimicked the effect of thalidomide. CONCLUSIONS: Thalidomide and lenalidomide improve mural cell coverage of bAVM vessels and reduce bAVM hemorrhage, which is likely through upregulation of Pdgfb expression.

Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency.

CRISPR-Cas9 is a powerful genome editing technology, yet with off-target effects. Truncated sgRNAs (17nt) have been found to decrease off-target cleavage without affecting on-target disruption in 293T cells. However, the potency of 17nt sgRNAs relative to the full-length 20nt sgRNAs in stem cells, such as human mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), has not been assessed. Using a GFP reporter system, we found that both 17nt and 20nt sgRNAs expressed by lentiviral vectors induce ~95% knockout (KO) in 293T cells, whereas the KO efficiencies are significantly lower in iPSCs (60-70%) and MSCs (65-75%). Furthermore, we observed a decrease of 10-20 percentage points in KO efficiency with 17nt sgRNAs compared to full-length sgRNAs in both iPSCs and MSCs. Off-target cleavage was observed in 17nt sgRNAs with 1-2nt but not 3-4nt mismatches; whereas 20nt sgRNAs with up to 5nt mismatches can still induce off-target mutations. Of interest, we occasionally observed off-target effects induced by the 17nt but not the 20nt sgRNAs. These results indicate the importance of balancing on-target gene cleavage potency with off-target effects: when efficacy is a major concern such as genome editing in stem cells, the use of 20nt sgRNAs is preferable.

Generation of Mesenchymal Stem Cells by Blood Cell Reprogramming.

Mesenchymal stem cells (MSCs) have been successfully used to treat multiple diseases in animal studies and clinical trials. Currently, the commonly used MSCs are derived from bone marrow and adipose tissue. Alternative approaches include differentiation of induced pluripotent stem cells (iPSCs) into MSCs, or direct reprogramming of blood cells into MSCs. This review summarizes recent progresses concerning how to generate MSCs by blood cell reprogramming and how studies in cellular reprogramming may help identify new factors to expand or even rejuvenate adult MSCs.

PDGFB-based stem cell gene therapy increases bone strength in the mouse.

Substantial advances have been made in the past two decades in the management of osteoporosis. However, none of the current medications can eliminate the risk of fracture and rejuvenate the skeleton. To this end, we recently reported that transplantation of hematopoietic stem/progenitor cells (HSCs) or Sca1(+) cells engineered to overexpress FGF2 results in a significant increase in lamellar bone matrix formation at the endosteum; but this increase was attended by the development of secondary hyperparathyroidism and severe osteomalacia. Here we switch the therapeutic gene to PDGFB, another potent mitogen for mesenchymal stem cells (MSCs) but potentially safer than FGF2. We found that modest overexpression of PDGFB using a relatively weak phosphoglycerate kinase (PGK) promoter completely avoided osteomalacia and secondary hyperparathyroidism, and simultaneously increased trabecular bone formation and trabecular connectivity, and decreased cortical porosity. These effects led to a 45% increase in the bone strength. Transplantation of PGK-PDGFB-transduced Sca1(+) cells increased MSC proliferation, raising the possibility that PDGF-BB enhances expansion of MSC in the vicinity of the hematopoietic niche where the osteogenic milieu propels the differentiation of MSCs toward an osteogenic destination. Our therapy should have potential clinical applications for patients undergoing HSC transplantation, who are at high risk for osteoporosis and bone fractures after total body irradiation preconditioning. It could eventually have wider application once the therapy can be applied without the preconditioning.

Novel brain arteriovenous malformation mouse models for type 1 hereditary hemorrhagic telangiectasia.

Endoglin (ENG) is a causative gene of type 1 hereditary hemorrhagic telangiectasia (HHT1). HHT1 patients have a higher prevalence of brain arteriovenous malformation (AVM) than the general population and patients with other HHT subtypes. The pathogenesis of brain AVM in HHT1 patients is currently unknown and no specific medical therapy is available to treat patients. Proper animal models are crucial for identifying the underlying mechanisms for brain AVM development and for testing new therapies. However, creating HHT1 brain AVM models has been quite challenging because of difficulties related to deleting Eng-floxed sequence in Eng(2fl/2fl) mice. To create an HHT1 brain AVM mouse model, we used several Cre transgenic mouse lines to delete Eng in different cell-types in Eng(2fl/2fl) mice: R26CreER (all cell types after tamoxifen treatment), SM22α-Cre (smooth muscle and endothelial cell) and LysM-Cre (lysozyme M-positive macrophage). An adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) was injected into the brain to induce focal angiogenesis. We found that SM22α-Cre-mediated Eng deletion in the embryo caused AVMs in the postnatal brain, spinal cord, and intestines. Induction of Eng deletion in adult mice using R26CreER plus local VEGF stimulation induced the brain AVM phenotype. In both models, Eng-null endothelial cells were detected in the brain AVM lesions, and formed mosaicism with wildtype endothelial cells. However, LysM-Cre-mediated Eng deletion in the embryo did not cause AVM in the postnatal brain even after VEGF stimulation. In this study, we report two novel HHT1 brain AVM models that mimic many phenotypes of human brain AVM and can thus be used for studying brain AVM pathogenesis and testing new therapies. Further, our data indicate that macrophage Eng deletion is insufficient and that endothelial Eng homozygous deletion is required for HHT1 brain AVM development.