Introduction Hereditarypulmonary arterial hypertension (HPAH) and hereditary hemorrhagic telangiectasia(HHT) are two rare genetic diseases affecting the pulmonary vasculature. Theyare both characterized by haploinsufficiency in different components of the bonemorphogenetic protein receptor type 2 (BMPR2) pathway. Despite shared genetics, the vascular phenotype differs. HPAH causes plexiform lesions, convolutes ofvascular channels that are considered a pathological hallmark of PAH. Incontrast, HHT can lead to pulmonary arteriovenous malformations (pAVMs), directconnections between arteries and veins. The pathobiology of both plexiformlesions and pAVMs is incompletely understood. In HHT-associated vascularmalformations in the skin and some solid organs, there is evidence that loss ofheterozygosity caused by somatic loss-of-function mutations in the functionalallele of the HHT gene results in clonally expanding endothelial cells. Inplexiform lesions, clonal vascular expansion has been described as well. Wetherefore aimed at detecting somatic mutations in pulmonary vascular lesions ofpatients with HPAH and a patient with HHT and PAH (HHT/PAH). Methods Genomic DNA wasextracted from vascular lesions of 2 patients with HPAH and 1 patient with HHT/PAH.Whole exome sequencing was performed on 2 plexiform lesions of the HPAHpatients. A targeted deep sequencing panel including 3 HHT causing genes, and 11VM associated genes was applied to 4 pAVMs and 14 plexiform lesions of thepatient with HHT/PAH. Results Germline variants in BMPR2 andSMAD9 were confirmed in the 2 HPAH patients, and a germline variant in Endoglinin the HHT/PAH patient. Interestingly, a somatic mutation in BMPR2 was found in theplexiform lesion of the patient with HPAH due to haploinsufficiency in BMPR2, whereas a somatic mutationin SMAD9 was present in the plexiformlesion of the SMAD9 patient. In the HHT/PAH patient, somatic mutations in VMassociated genes occurred in 4 out of 14 plexiform lesions. These included asomatic activating mutation in Phosphatidylinositol-4,5-Bisphosphate 3-KinaseCatalytic Alpha (PIK3CA) that was previously described in breast cancer andfunctionally validated as a moderately potent oncogenic mutation. Furthermore, no somatic mutations were found in pAVMs of this patient. Conclusions We identified localsomatic mutations in PAH causing genes in plexiform lesions of HPAH patientsand a somatic activating mutation in PIK3CA in a plexiform lesion of an HHT/PAHpatient. Both events likely result inaltered signaling that might contribute to changed endothelial cell behaviorand excessive proliferation in plexiform lesions.

Historically, the factor(s) that stimulate vascular malformation genesis in hereditary hemorrhagic telangiectasia (HHT) has been hotly debated. Once heterozygous loss-of-function germline mutations in ENG, ACVRL1, or SMAD4 were discovered in individuals with HHT, haploinsufficiency, a 50% reduction in the encoded protein, was proposed as the molecular mechanism of HHT. However, the focal and discrete nature of HHT-associated vascular malformations suggested to others that vascular malformation genesis requires an additional, local trigger. In this review, we discuss the evidence for the Knudsonian two-hit mutation mechanism of vascular malformation pathogenesis in HHT, where the inherited, heterozygous mutation is augmented by an acquired somatic mutation in the remaining normal copy of the gene. We consider the mechanisms of HHT–vascular malformation development in the broader context of the emerging role of somatic mutations in both sporadic and inherited vascular malformations. We discuss different mechanisms of biallelic gene inactivation in HHT, difficulties with the detection of all possible mechanisms of biallelic inactivation, and issues related to the somatic mosaic nature of the lesion. We then discuss the critical importance of non-genetic factors on the pathogenesis of HHT-associated vascular malformations. Finally, we discuss the implications of the two-hit mutation mechanism for the design of novel treatments for HHT.

PTEN hamartoma tumor syndrome (PHTS) is a rare tumor risk disorder caused by germline loss-of-function mutations in PTEN. Half of these patients develop vascular malformations, a hamartoma characterized by overgrowth of vessels. In this study, we harness biopsies and patient-derived endothelial cells (EC) to study the genetic etiology of PHTS-related vascular malformations. We discover that these lesions are generated by somatic loss of the PTEN wild-type allele through copy-neutral loss of heterozygosity, leading to somatic uniparental disomy of the PTEN-mutated allele in ECs. We established a mouse model of PHTS-related vascular malformations and identified that the mTOR inhibitor rapamycin and AKT inhibitor capivasertib block vascular lesion growth. As proof-of-concept for clinical activity, off-label treatment with rapamycin of two patients with PHTS reduced vascular overgrowth and abrogated lesion-associated pain. Overall, our results uncover the genetic cause of vascular malformations in patients with PHTS and open new avenues for therapeutic intervention. SIGNIFICANCE:Somatic loss of PTEN in ECs causes vascular malformations in patients with the tumor risk syndrome PHTS. These lesions respond to PI3K signaling inhibition. See related commentary by Del Prior and Toker, p. 1306.

Background Hereditary haemorrhagic telangiectasia (HHT) is an inherited disorder of vascular malformations. It is caused by inherited loss-of-function mutations in one of three genes, ENG , ACVRL1 or SMAD4 . We recently showed that HHT-associated vascular malformations from liver, lung, brain and skin develop via a two-hit genetic mechanism resulting from biallelic loss-of-function mutations in either ENG or ACVRL1 . Second-hit somatic mutations in SMAD4 have not been reported in HHT-associated vascular malformations. Here, we investigate a large, aggressively growing craniofacial arteriovenous malformation (AVM) from an individual with juvenile polyposis-HHT caused by a germline mutation in SMAD4 . Methods We sequenced DNA from the AVM using a targeted gene sequencing panel to at least 1000X to identify somatic mutations that might contribute to the development of the AVM. We analysed whole genome SNP genotyping data using the algorithm Mosaic Chromosomal Alterations (MoChA) to identify somatic loss of heterozygosity. Results We confirmed the germline mutation in SMAD4 (c.1610A>T, p.Asp537Val) and identified a second-hit somatic mutation also in SMAD4 (c.350dup, p.Tyr117*) that occurred in trans relative to the germline mutation. We also identified somatic loss of heterozygosity on the q arm of chromosome 18, including SMAD4 . Additionally, we confirmed that the loss of heterozygosity causes loss of the wild-type allele. Thus, we identified two independent somatic alterations in SMAD4 causing biallelic loss of SMAD4 function in the AVM tissue. Conclusion We identified biallelic loss of function of SMAD4 in a craniofacial AVM, evidence that SMAD4 also follows the two-hit mutation mechanism of HHT-associated vascular malformation pathogenesis.

Introduction: Circulating plasma protein profiling in individuals with brain vascular disorders may aid in the identification of robust diagnostic biomarkers, stratification of high-risk patients for treatment, and monitoring of disease progression or treatment response. This Brain Vascular Malformation Consortium (BVMC) study aimed to identify circulating inflammatory and angiogenic proteins that associate with familial Cerebral Cavernous Malformation (FCCM), Hereditary Hemorrhagic Telangiectasia (HHT), or Sturge-Weber Syndrome (SWS). Methods: We used the Angiome multiplex ELISA biomarker panel to assess the circulating plasma levels of 22 proteins related to inflammation and angiogenesis in 234 individuals enrolled in the BVMC, including 114 FCCM, 101 HHT and 19 SWS cases. Protein levels were measured in duplicate and absolute measurements obtained. We tested for biomarker associations between disease states using linear regression models adjusting for age at blood collection and sex. We calculated the intraclass correlation coefficient (ICC) to describe the similarity in replicates, and report proportional increase (PI) for protein levels for statistically significant results with Bonferroni-corrected P-values <0.05 (adjusted for 22 markers). Results: All 22 proteins were successfully measured in the 114 FCCM (39% male, median age 52 years), 101 HHT (49% male, median age 42 years, 36% with brain arteriovenous malformation), and 19 SWS (53% male, median age 15 years). The ICC was high for all markers (range 0.904-0.990). As expected, HHT cases with endoglin mutations had ~50% lower endoglin. We observed increased levels of endoglin (PI=1.38, 95% CI:1.27-1.50, P=6.50E-13) and IL10 (PI=1.48, 95% CI:1.16-1.91, P=0.04) in CCM compared to HHT cases, and decreased IL8 (PI=0.60, 95% CI:0.43-0.83, P=0.04) and OPN (PI=0.63, 95% CI:0.47-0.84, P=0.04) in CCM compared to SWS cases. We also observed higher levels of endoglin (PI=1.45, 95% CI:1.24-1.71, P=1.47E-04), GP130 (PI=1.24, 95% CI:1.11-1.38, P=2.91E-03), and OPN (PI=1.80, 95% CI:1.36-2.40, P=1.13E-03), and lower levels of IL1β (PI=0.30, 95% CI:0.15-0.59, P=0.01) in SWS compared to HHT cases. Conclusions: We identified circulating plasma biomarkers associated with FCCM, HHT and SWS. Larger ongoing work will confirm whether these inflammatory/angiogenic markers are relevant as potential diagnostic or therapeutic targets or have broader implications as clinical biomarkers in other brain vascular diseases.

BACKGROUND AND OBJECTIVES:Despite many publications about cavernous malformations (CMs), controversy remains regarding diagnostic and management strategies. To update evidence-based guidelines for the clinical management of brain and spinal cord CMs. METHODS:The Alliance to Cure CMs, the patient support group in the United States advocating on behalf of patients and research in CM, convened a multidisciplinary writing group comprising expert CM clinicians to help summarize the existing literature related to the clinical care of CM, focusing on 5 topics: (1) epidemiology and natural history, (2) genetic testing and counseling, (3) diagnostic criteria and imaging standards, (4) neurosurgical considerations, and (5) neurological considerations. Building on prior evidence-based recommendations reflecting literature review through October 2014, the group conducted a systematic review of the more recent literature, identified references for mandatory citation, rated evidence, developed recommendations, and established consensus according to a prespecified protocol. Finally, the writing group outlined remaining knowledge gaps and controversies to guide future research. RESULTS:From 2672 publications published between October 1, 2014, and March 15, 2023, and meeting key word criteria, 234 were selected based on prearticulated criteria for mandatory consideration in evidence-based recommendations. Topic authors used these and other supporting references to summarize current knowledge and arrive at 53 management recommendations, with unanimous consensus based on a Delphi process. These were rated by class (strength of recommendation) and level (quality of evidence) per the American Heart Association/American Stroke Association criteria. Eighteen recommendations were class 1 (34%), class 2 in 31 (58%), and class 3 in 4 (8%). Three were level A (6%), 19 (36%) were level B, and 31 (58%) were level C. CONCLUSION:Current evidence supports prior and new recommendations for the management of CMs, but many reflect moderate classes and low levels, mandating further research to better inform clinical practice.

Background:Ferroptosis is an iron-dependent form of regulated cell death driven by lipid peroxidation. This process has been implicated in various diseases, including ischemic stroke. Ischemic stroke leads to oxidative stress, iron overload, and reactive oxygen species (ROS) accumulation, which collectively may trigger ferroptotic neuronal cell death. However, the regulatory mechanisms of ferroptosis in stroke remain poorly understood. Previous studies have identified ataxia telangiectasia mutated (ATM), a DNA damage kinase, as a critical regulator of ferroptosis. However, the therapeutic potential of this discovery remains unknown. Methods:We investigated the effect of ATM inhibitors, including the brain-penetrant AZD1390, on ferroptosis using in vitro, ex vivo , and in vivo models of ischemic stroke. Our analysis included assessments of cell viability, lipid peroxidation, ferroptosis marker expression, and infarct volume. Result:ATM inhibitors significantly alleviated ferroptosis-induced cell death in cultured cells and ex vivo murine brain slice cultures. In the oxygen-glucose deprivation (OGD) stroke model, treatment with AZD1390 reduced the expression of ferroptosis markers (xCT and PTGS2) and diminished neuronal cell death in rat and mouse brain slices. Furthermore, in a mouse model of ischemic stroke, AZD1390 decreased infarct volume confirming its therapeutic efficacy in vivo . Conclusions:This study identifies ferroptosis as a critical mechanism in ischemic stroke-induced neuronal cell death and highlights ATM inhibition, particularly with AZD1390, as a promising therapeutic candidate for mitigating stroke-associated damage. Targeting ferroptosis may provide a translationally relevant strategy to mitigate neuronal injury and improve clinical outcomes for stroke patients.

Cerebral cavernous malformation (CCM) is a hemorrhagic cerebrovascular disease where lesions develop in the setting of endothelial mutations of CCM genes, with many cases also harboring somatic PIK3CA gain of function (GOF) mutations. Rapamycin, an mTORC1 inhibitor, inhibited progression of murine CCM lesions driven by Ccm gene loss and Pik3ca GOF, but it remains unknown if rapamycin is beneficial in the absence of induction of Pik3ca GOF. We investigated the effect of rapamycin at three clinically relevant doses on lesion development in the Ccm3−/−PDGFb-icreERPositive murine model of familial CCM disease, without induction of Pik3ca GOF. Lesion burden, attrition, and acute and chronic hemorrhaging were compared between placebo and rapamycin-treated mice. Plasma miRNome was compared to identify potential biomarkers of rapamycin response. Outlier, exceptionally large CCM lesions (> 2 SD above the mean lesion burden) were exclusively observed in the placebo group. Rapamycin, across all dosages, may have prevented the emergence of large outlier lesions. Yet rapamycin also appeared to exacerbate mean lesion burden of surviving mice when outliers were excluded, increased attrition, and did not alter hemorrhage. miR-30c-2-3p, decreased in rapamycin-treated mouse plasma, has gene targets in PI3K/AKT and mTOR signaling. Progression of outlier lesions in a familial CCM model may have been halted by rapamycin treatment, at the potential expense of increased mean lesion burden and increased attrition. If confirmed, this can have implications for potential rapamycin treatment of familial CCM disease, where lesion development may not be driven by PIK3CA GOF. Further studies are necessary to determine specific pathways that mediate potential beneficial and detrimental effects of rapamycin treatment, and whether somatic PIK3CA mutations drive particularly aggressive lesions.

BackgroundRadiation treatment for diseases of the brain can result in hemorrhagic adverse radiation effects. The underlying pathologic substrate of brain bleeding after irradiation has not been elucidated, nor potential associations with induced somatic mutations.MethodsWe retrospectively reviewed our department's pathology database over 5 years and identified 5 biopsy specimens (4 patients) for hemorrhagic lesions after brain irradiation. Tissues with active malignancy were excluded. Samples were characterized using H&E, Perl's Prussian Blue, and Masson's Trichrome; immunostaining for B-cells (anti-CD20), T-cells (anti-CD3), endothelium (anti-CD31), macrophages (anti-CD163), α-smooth muscle actin, and TUNEL. DNA analysis was done by two panels of next-generation sequencing for somatic mutations associated with known cerebrovascular anomalies.ResultsOne lesion involved hemorrhagic expansion among multifocal microbleeds that had developed after craniospinal irradiation for distant medulloblastoma treatment. Three bleeds arose in the bed of focally irradiated arteriovenous malformations (AVM) after confirmed obliteration. A fifth specimen involved the radiation field distinct from an irradiated AVM bed. From these, 2 patterns of hemorrhagic vascular pathology were identified: encapsulated hematomas and cavernous-like malformations. All lesions included telangiectasias with dysmorphic endothelium, consistent with primordial cavernous malformations with an associated inflammatory response. DNA analysis demonstrated genetic variants in PIK3CA and/or PTEN genes but excluded mutations in CCM genes.ConclusionsDespite pathologic heterogeneity, brain bleeding after irradiation is uniformly associated with primordial cavernous-like telangiectasias and disruption of genes implicated in dysangiogenesis but not genes implicated as causative of cerebral cavernous malformations. This may implicate a novel signaling axis as an area for future study.

Cerebral cavernous malformation (CCM) is a hemorrhagic neurovascular disease with no currently available therapeutics. Prior evidence suggests that different cell types may play a role in CCM pathogenesis. The contribution of each cell type to the dysfunctional cellular crosstalk remains unclear. Herein, RNA-seq was performed on fluorescence-activated cell sorted endothelial cells (ECs), pericytes, and neuroglia from CCM lesions and non-lesional brain tissue controls. Differentially Expressed Gene (DEG), pathway and Ligand-Receptor (LR) analyses were performed to characterize the dysfunctional genes of respective cell types within CCMs. Common DEGs among all three cell types were related to inflammation and endothelial-to-mesenchymal transition (EndMT). DEG and pathway analyses supported a role of lesional ECs in dysregulated angiogenesis and increased permeability. VEGFA was particularly upregulated in pericytes. Further pathway and LR analyses identified vascular endothelial growth factor A/ vascular endothelial growth factor receptor 2 signaling in lesional ECs and pericytes that would result in increased angiogenesis. Moreover, lesional pericytes and neuroglia predominantly showed DEGs and pathways mediating the immune response. Further analyses of cell specific gene alterations in CCM endorsed potential contribution to EndMT, coagulation, and a hypoxic microenvironment. Taken together, these findings motivate mechanistic hypotheses regarding non-endothelial contributions to lesion pathobiology and may lead to novel therapeutic targets.

Hereditary hemorrhagic telangiectasia (HHT) is an inherited disorder of vascular malformations characterized by mucocutaneous telangiectases and arteriovenous malformations (AVMs) in internal organs. HHT is caused by inheritance of a loss of function mutation in one of three genes. Although individuals with HHT are haploinsufficient for one of these genes throughout their entire body, rather than exhibiting a systemic vascular phenotype, vascular malformations occur as focal lesions in discrete anatomic locations. The inconsistency between genotype and phenotype has provoked debate over whether haploinsufficiency or a different mechanism gives rise to the vascular malformations. We previously showed that HHT-associated skin telangiectases develop by a two-hit mutation mechanism in an HHT gene. However, somatic mutations were identified in only half of the telangiectases, raising the question whether a second-hit somatic mutation is a necessary (required) event in HHT pathogenesis. Here, we show that another mechanism for the second hit is loss of heterozygosity across the chromosome bearing the germline mutation. Secondly, we investigate the two-hit mutation mechanism for internal organ AVMs, the source of much of the morbidity of HHT. Here, we identified somatic molecular genetic events in eight liver telangiectases, including point mutations and a loss of heterozygosity event. We also identified somatic mutations in one pulmonary AVM and two brain AVMs, confirming that mucocutaneous and internal organ vascular malformations undergo the same molecular mechanisms. Together, these data argue that bi-allelic loss of function in an HHT gene is a required event in the pathogenesis of HHT-associated vascular malformations.

Sturge-Weber Syndrome (SWS) is a sporadic (non-inherited) syndrome characterized by capillary vascular malformations in the facial skin, leptomeninges, or the choroid. A hallmark feature is the mosaic nature of the phenotype. SWS is caused by a somatic mosaic mutation in the GNAQ gene (p.R183Q), leading to activation of the G protein, Gαq. Decades ago, Rudolf Happle hypothesized SWS as an example of "paradominant inheritance", that is, a "lethal gene (mutation) surviving by mosaicism". He predicted that the "presence of the mutation in the zygote will lead to death of the embryo at an early stage of development". We have created a mouse model for SWS using gene targeting to conditionally express the Gnaq p.R183Q mutation. We have employed two different Cre-drivers to examine the phenotypic effects of expression of this mutation at different levels and stages of development. As predicted by Happle, global, ubiquitous expression of this mutation in the blastocyst stage results in 100% embryonic death. The majority of these developing embryos show vascular defects consistent with the human vascular phenotype. By contrast, global but mosaic expression of the mutation enables a fraction of the embryos to survive, but those that survive to birth and beyond do not exhibit obvious vascular defects. These data validate Happle's paradominant inheritance hypothesis for SWS and suggest the requirement of a tight temporal and developmental window of mutation expression for the generation of the vascular phenotype. Furthermore, these engineered murine alleles provide the template for the development of a mouse model of SWS that acquires the somatic mutation during embryonic development, but permits the embryo to progress to live birth and beyond, so that postnatal phenotypes can also be investigated. These mice could then also be employed in pre-clinical studies of novel therapies.

Cerebral Cavernous Malformations (CCMs) are vascular malformations of the central nervous system which can lead to moderate to severe neurological phenotypes in patients. A majority of CCM lesions are driven by a cancer-like three-hit mutational mechanism, including a somatic, activating mutation in the oncogene PIK3CA, as well as biallelic loss-of-function mutations in a CCM gene. However, standard sequencing approaches often fail to yield a full complement of pathogenic mutations in many CCMs. We suggest this reality reflects the limited sensitivity to identify low-frequency variants and the presence of mutations undetectable with bulk short-read sequencing. Here we report a single-nucleus DNA-sequencing approach that leverages the underlying biology of CCMs to identify lesions with somatic loss-of-heterozygosity, a class of such hidden mutations. We identify an alternative genetic mechanism for CCM pathogenesis and establish a method that can be repurposed to investigate the genetic underpinning of other disorders with multiple somatic mutations.

Background: Identifying genetic variants that affect the level of cell cycle reentry and establishing the degree of cell cycle progression in those variants could help guide development of therapeutic interventions aimed at effecting cardiac regeneration. We observed that C57Bl6/NCR (B6N) mice have a marked increase in cardiomyocyte S-phase activity after permanent coronary artery ligation compared with infarcted DBA/2J (D2J) mice. Methods: Cardiomyocyte cell cycle activity after infarction was monitored in D2J, (D2J×B6N)-F1, and (D2J×B6N)-F1×D2J backcross mice by means of bromodeoxyuridine or 5-ethynyl-2′-deoxyuridine incorporation using a nuclear-localized transgenic reporter to identify cardiomyocyte nuclei. Genome-wide quantitative trait locus analysis, fine scale genetic mapping, whole exome sequencing, and RNA sequencing analyses of the backcross mice were performed to identify the gene responsible for the elevated cardiomyocyte S-phase phenotype. Results: (D2J×B6N)-F1 mice exhibited a 14-fold increase in cardiomyocyte S-phase activity in ventricular regions remote from infarct scar compared with D2J mice (0.798±0.09% versus 0.056±0.004%; P <0.001). Quantitative trait locus analysis of (D2J×B6N)-F1×D2J backcross mice revealed that the gene responsible for differential S-phase activity was located on the distal arm of chromosome 3 (logarithm of the odds score=6.38; P <0.001). Additional genetic and molecular analyses identified 3 potential candidates. Of these, Tnni3k (troponin I-interacting kinase) is expressed in B6N hearts but not in D2J hearts. Transgenic expression of TNNI3K in a D2J genetic background results in elevated cardiomyocyte S-phase activity after injury. Cardiomyocyte S-phase activity in both Tnni3k-expressing and Tnni3k-nonexpressing mice results in the formation of polyploid nuclei. Conclusions: These data indicate that Tnni3k expression increases the level of cardiomyocyte S-phase activity after injury.

BACKGROUND: Cerebral cavernous malformations (CCMs) are vascular malformations that frequently cause stroke. CCMs arise due to loss of function in one of the genes that encode the CCM complex, a negative regulator of MEKK3-KLF2/4 signaling in vascular endothelial cells. Gain-of-function mutations in PIK3CA (encoding the enzymatic subunit of the PI3K (phosphoinositide 3-kinase) pathway associated with cell growth) synergize with CCM gene loss-of-function to generate rapidly growing lesions. METHODS: We recently developed a model of CCM formation that closely reproduces key events in human CCM formation through inducible CCM loss-of-function and PIK3CA gain-of-function in mature mice. In the present study, we use this model to test the ability of rapamycin, a clinically approved inhibitor of the PI3K effector mTORC1, to treat rapidly growing CCMs. RESULTS: We show that both intraperitoneal and oral administration of rapamycin arrests CCM growth, reduces perilesional iron deposition, and improves vascular perfusion within CCMs. CONCLUSIONS: Our findings further establish this adult CCM model as a valuable preclinical model and support clinical testing of rapamycin to treat rapidly growing human CCMs.

This large, multinational, sex-based analysis among individuals with HHT showed that pulmonary AVM frequency, physical characteristics, presentation, complications and management do not generally significantly differ between males and females https://bit.ly/3TNLA6v.

Ischemic stroke, caused by vessel blockage, results in cerebral infarction, the death of brain tissue. Previously, quantitative trait locus (QTL) mapping of cerebral infarct volume and collateral vessel number identified a single, strong genetic locus regulating both phenotypes. Additional studies identified RAB GTPase-binding effector protein 2 (Rabep2) as the casual gene. However, there is yet no evidence that variation in the human ortholog of this gene plays any role in ischemic stroke outcomes. We established an in vivo evaluation platform in mice by using adeno-associated virus (AAV) gene replacement and verified that both mouse and human RABEP2 rescue the mouse Rabep2 knockout ischemic stroke volume and collateral vessel phenotypes. Importantly, this cross-species complementation enabled us to experimentally investigate the functional effects of coding sequence variation in human RABEP2. We chose four coding variants from the human population that are predicted by multiple in silico algorithms to be damaging to RABEP2 function. In vitro and in vivo analyses verify that all four led to decreased collateral vessel connections and increased infarct volume. Thus, there are naturally occurring loss-of-function alleles. This cross-species approach will expand the number of targets for therapeutics development for ischemic stroke.

Cerebral cavernous malformations (CCM) are vascular malformations consisting of collections of enlarged capillaries occurring in the brain or spinal cord. These vascular malformations can occur sporadically or susceptibility to develop these can be inherited as an autosomal dominant trait due to mutation in one of three genes. Over a decade ago, we described a 77.6 Kb germline deletion spanning exons 2–10 in the CCM2 gene found in multiple affected individuals from seemingly unrelated families. Segregation analysis using linked, microsatellite markers indicated that this deletion may have arisen at least twice independently. In the ensuing decades, many more CCM patients have been identified with this deletion. In this present study we examined 27 reportedly unrelated affected individuals with this deletion. To investigate the origin of the deletion at base pair level resolution, we sequenced approximately 10 Kb upstream and downstream from the recombination junction on the deleted allele. All patients showed the identical SNP haplotype across this combined 20 Kb interval. In parallel, genealogical records have traced 11 of these individuals to five separate pedigrees dating as far back as the 1600-1700s. These haplotype and genealogical data suggest that these families and the remaining “unrelated” samples converge on a common ancestor due to a founder mutation occurring centuries ago on the North American continent. We also note that another gene, NACAD , is included in this deletion. Although patient self-reporting does not indicate an apparent phenotypic consequence for heterozygous deletion of NACAD, further investigation is warranted for these patients.

Cerebral cavernous malformations (CCMs) are a neurovascular anomaly that may occur sporadically or be inherited due to autosomal dominant mutations in KRIT1, CCM2 or PDCD10 (refs. 1–4). Individual lesions are caused by somatic mutations that have been identified in KRIT1, CCM2, PDCD10, MAP3K3 and PIK3CA5–11. However, the interactions between mutations and their relative contributions to sporadic versus familial cases are unclear. We show that mutations in KRIT1, CCM2, PDCD10 and MAP3K3 are mutually exclusive but may co-occur with mutations in PIK3CA. We also find that MAP3K3 mutations may cause sporadic but not familial CCM. Furthermore, we find identical PIK3CA mutations in CCMs and adjacent developmental venous anomalies (DVAs), a common vascular malformation frequently found in the vicinity of sporadic CCMs12–14. However, somatic mutations in MAP3K3 are found only in the CCM. This suggests that sporadic CCMs are derived from cells of the DVA that have acquired an additional mutation in MAP3K3. Snellings et al. show that an identical PIK3CA mutation is found in both developmental venous anomalies (DVAs) and associated cerebral cavernous malformations (CCMs). However, an activating MAP3K3 mutation appears only in CCMs, supporting a mechanism where DVAs develop as the result of a PIK3CA mutation.