KRIT1 in vascular biology and beyond.

KRIT1 is a 75 kDa scaffolding protein which regulates endothelial cell phenotype by limiting the response to inflammatory stimuli and maintaining a quiescent and stable endothelial barrier. Loss-of-function mutations in KRIT1 lead to the development of cerebral cavernous malformations (CCM), a disease marked by the formation of abnormal blood vessels which exhibit a loss of barrier function, increased endothelial proliferation, and altered gene expression. While many advances have been made in our understanding of how KRIT1, and the functionally related proteins CCM2 and PDCD10, contribute to the regulation of blood vessels and the vascular barrier, some important open questions remain. In addition, KRIT1 is widely expressed and KRIT1 and the other CCM proteins have been shown to play important roles in non-endothelial cell types and tissues, which may or may not be related to their role as pathogenic originators of CCM. In this review, we discuss some of the unsettled questions regarding the role of KRIT1 in vascular physiology and discuss recent advances that suggest this ubiquitously expressed protein may have a role beyond the endothelial cell.

Plasma water T2 detects age-stratified differences in cardiometabolic health among familial CCM patients with Hispanic CCM1 mutation.

Cerebral cavernous malformations (CCMs) are abnormal clusters of capillaries in the nervous system. This pilot study analyzed the cardiometabolic health status of individuals with familial CCMs caused by a rare mutation in the CCM1 gene (fCCM1). The aim was to compare plasma water T2 values from individuals with fCCM1 with values from metabolically unhealthy and healthy individuals with no known CCM mutations. This observational, cross-sectional study included 75 participants: 11 fCCM1 patients, 24 metabolically unhealthy and 40 metabolically healthy individuals. Plasma water T2, an early, global and practical marker of cardiometabolic health, was measured in the time domain using benchtop magnetic resonance relaxometry. The results were stratified by age (equal to or less than 45 vs. older than 45 years). Group means were compared using Welch's one-way ANOVA and post hoc Tukey-Kramer tests. Multivariable linear regression, with T2 as the outcome variable, was used to explore associations with age, gender, Hispanic ethnicity and fCCM1 status. In the younger age stratum, the fCCM1 group had a mean plasma water T2 value comparable to the metabolically healthy group (p = 0.6388), but higher than the unhealthy group (p < 0.0001). By contrast, in the older stratum, the mean plasma water T2 value for the fCCM1 group was comparable to the metabolically unhealthy group (p = 0.7819) and lower than the healthy group (p = 0.0005). Multivariable linear regression revealed that age and the interaction between age and fCCM1 status were significant predictors of T2, even after adjusting for gender and Hispanic ethnicity. Plasma water T2 shows potential as a biomarker for assessing the health status of individuals with fCCM1. Further research is needed to validate these preliminary observations and elucidate the association between CCMs and cardiometabolic health.

Towards a neurocognitive profile in familial cerebral cavernous malformations.

BACKGROUND: Familial cerebral cavernous malformations (FCCM) is a rare autosomal dominant disease, characterized by vascular malformations that can lead to macro and microhemorrhages. The neurocognitive impact of FCCM is still underrecognized. METHODS: We report the clinical, neurocognitive, imaging and genetic data of a three generation family with FCCM. RESULTS: A 63-year-old man (proband) had progressive memory impairment since the last year. Neurologic exam was unremarkable. Brain MRI showed multiple large cavernomas (mainly in the pons, left temporal, and right temporo-parietal) and scattered microhemorrhages. Neuropsychological assessment mainly revealed left frontal and right temporo-parietal dysfunction. A 41-year-old daughter, presented with headache, vertigo and memory complaints in the last 2 years. Neurological examination revealed left central facial paralysis. Brain MRI showed two small right parietal and internal capsule cavernomas, as well as microhemorrhages. Neuropsychological assessment showed moderate temporal neocortical left dysfunction. A 34-year-old daughter had recurrent headache and memory complaints, with unremarkable neurological exam. Brain MRI revealed two large cavernomas (left fronto-orbitary and inferior temporal), with few microhemorrhages. Neuropsychological assessment was normal. A granddaughter had mild headaches and a small right cerebellar cavernoma, without microhemorrhages. Neuropsychological assessment showed mild temporal neocortical left dysfunction. A nonsense variant, c.55C > T; p.R19* generating a premature stop codon in CCM2 gene shared by all affected family members was identified. CONCLUSIONS: Neuropsychological evaluation showed that memory complaints and cognitive impairment could be an important unrecognized finding in FCCM. Its pathophysiological mechanisms are still unknown but the role of recurrent microhemorrhages could provide an interesting hypothesis.

Single-nucleus DNA sequencing reveals hidden somatic loss-of-heterozygosity in Cerebral Cavernous Malformations.

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.

A novel KRIT1/CCM1 mutation accompanied by a NOTCH3 mutation in a Chinese family with multiple cerebral cavernous malformations.

Family cerebral cavernous malformations (FCCMs) are mainly inherited through the mutation of classical CCM genes, including CCM1/KRIT1, CCM2/MGC4607, and CCM3/PDCD10. FCCMs can cause severe clinical symptoms, including epileptic seizures, intracranial hemorrhage (ICH), or functional neurological deficits (FNDs). In this study, we reported a novel mutation in KRIT1 accompanied by a NOTCH3 mutation in a Chinese family. This family consists of 8 members, 4 of whom had been diagnosed with CCMs using cerebral MRI (T1WI, T2WI, SWI). The proband (II-2) and her daughter (III-4) had intracerebral hemorrhage and refractory epilepsy, respectively. Based on whole-exome sequencing (WES) data and bioinformatics analysis from 4 patients with multiple CCMs and 2 normal first-degree relatives, a novel KRIT1 mutation, NG_012964.1 (NM_194456.1): c.1255-1G > T (splice-3), in intron 13 was considered a pathogenic gene in this family. Furthermore, based on 2 severe and 2 mild CCM patients, we found an SNV missense mutation, NG_009819.1 (NM_000435.2): c.1630C > T (p.R544C), in NOTCH3. Finally, the KRIT1 and NOTCH3 mutations were validated in 8 members using Sanger sequencing. This study revealed a novel KRIT1 mutation, NG_012964.1 (NM_194456.1): c.1255-1G > T (splice-3), in a Chinese CCM family, which had not been reported previously. Moreover, the NOTCH3 mutation NG_009819.1 (NM_000435.2): c.1630C > T (p.R544C) might be a second hit and associated with the progression of CCM lesions and severe clinical symptoms.

Endothelial Differentiation of CCM1 Knockout iPSCs Triggers the Establishment of a Specific Gene Expression Signature.

Cerebral cavernous malformation (CCM) is a neurovascular disease that can lead to seizures and stroke-like symptoms. The familial form is caused by a heterozygous germline mutation in either the CCM1, CCM2, or CCM3 gene. While the importance of a second-hit mechanism in CCM development is well established, it is still unclear whether it immediately triggers CCM development or whether additional external factors are required. We here used RNA sequencing to study differential gene expression in CCM1 knockout induced pluripotent stem cells (CCM1-/- iPSCs), early mesoderm progenitor cells (eMPCs), and endothelial-like cells (ECs). Notably, CRISPR/Cas9-mediated inactivation of CCM1 led to hardly any gene expression differences in iPSCs and eMPCs. However, after differentiation into ECs, we found the significant deregulation of signaling pathways well known to be involved in CCM pathogenesis. These data suggest that a microenvironment of proangiogenic cytokines and growth factors can trigger the establishment of a characteristic gene expression signature upon CCM1 inactivation. Consequently, CCM1-/- precursor cells may exist that remain silent until entering the endothelial lineage. Collectively, not only downstream consequences of CCM1 ablation but also supporting factors must be addressed in CCM therapy development.

Circulating Plasma miRNA Homologs in Mice and Humans Reflect Familial Cerebral Cavernous Malformation Disease.

Patients with familial cerebral cavernous malformation (CCM) inherit germline loss of function mutations and are susceptible to progressive development of brain lesions and neurological sequelae during their lifetime. To date, no homologous circulating molecules have been identified that can reflect the presence of germ line pathogenetic CCM mutations, either in animal models or patients. We hypothesize that homologous differentially expressed (DE) plasma miRNAs can reflect the CCM germline mutation in preclinical murine models and patients. Herein, homologous DE plasma miRNAs with mechanistic putative gene targets within the transcriptome of preclinical and human CCM lesions were identified. Several of these gene targets were additionally found to be associated with CCM-enriched pathways identified using the Kyoto Encyclopedia of Genes and Genomes. DE miRNAs were also identified in familial-CCM patients who developed new brain lesions within the year following blood sample collection. The miRNome results were then validated in an independent cohort of human subjects with real-time-qPCR quantification, a technique facilitating plasma assays. Finally, a Bayesian-informed machine learning approach showed that a combination of plasma levels of miRNAs and circulating proteins improves the association with familial-CCM disease in human subjects to 95% accuracy. These findings act as an important proof of concept for the future development of translatable circulating biomarkers to be tested in preclinical studies and human trials aimed at monitoring and restoring gene function in CCM and other diseases.

Identification of a novel LATS1 variant associated with familial cerebral cavernous malformations in a Chinese family.

BACKGROUND: Cerebral cavernous malformations (CCMs) are common sporadic or hereditary vascular malformations in the central nervous system. CCM1-3 variants have been identified that are associated with the majority of familial cerebral cavernous malformations (FCCMs). However, there are still a few CCM1-3 wild-type FCCMs. The aim of the present study was to identify an additional pathogenic variant of FCCMs. METHODS: In this study, a large five-generation Chinese Han family affected by CCMs was recruited. Magnetic resonance imaging (MRI) was done for the detection of CCMs. Whole-exome sequencing (WES) was performed, and the identified variants were co-segregation analyzed by Sanger sequencing. The function of candidate variants was predicted in silico and experimental validated by angiogenesis assay in human umbilical vein endothelial cells (HUVECs) in vitro. RESULTS: Twenty-four family members and one healthy spouse were enrolled. We found that CCMs were exhibited on MRI in nine family members. Overall, twenty-seven candidate variants were identified using WES, and no CCM1-3 variants were detected. The missense variant in LATS1 (c.821C > T, p.Thr274Ile) was verified to be associated with the clinical and pathological phenotype of FCCMs. CONCLUSION: Our findings indicated that the LATS1 variant could be a potential pathogenic factor for FCCMs in this Chinese family.

Mesenteric cysts, lymphatic leak, and cerebral cavernous malformation in a proband with KRIT1-related disease.

Cerebral cavernous malformations (CCMs) of the central nervous system arise sporadically or secondary to genomic variation. Established genetic etiologies include deleterious variants in KRIT1 (CCM1), malcavernin (CCM2), and PDCD10 (CCM3). KRIT1-related disease has not been described in conjunction with lymphatic defects, although lymphatic defects with abnormal endothelial cell junctions have been observed in mice deficient in HEG1-KRIT1 signaling. We report a proband with CCMs, multiple chylous mesenteric cysts, and chylous ascites with leaky lymphatic vasculature. Clinical short-read exome sequencing detected a disease-associated KRIT1 variant (NM_194456.1:c.[1927C>T];[=], p.(Gln643*)). We postulate an expansion of KRIT1-related disease to include lymphatic malformations and lymphatic endothelial dysfunction.

Contribution of protein-protein interactions to the endothelial-barrier-stabilizing function of KRIT1.

Krev-interaction trapped protein 1 (KRIT1) is an endothelial scaffold protein that promotes adherens junction (AJ) stability. The precise mechanism by which KRIT1 promotes barrier stabilization is unclear. We tested the ability of a panel of KRIT1 constructs containing mutations that inhibit Rap1 binding, ICAP1α binding, disrupt KRIT1's phosphotyrosine-binding (PTB) domain, or direct KRIT1 to the plasma membrane, either alone or in combination, to restore barrier function in KRIT1-deficient endothelial cells. We found that ablating the 192NPAY195 motif or disrupting the PTB domain was sufficient to restore AJ protein localization and barrier function to control levels, irrespective of the junctional localization of KRIT1 or Rap1 binding. The ability of our KRIT1 constructs to rescue AJ and barrier function in KRIT1-depleted endothelial cells correlated with decreased ß1 integrin activity and maintenance of cortical actin fibers. Taken together, our findings indicate that Rap1 binding, ICAP1α binding and junctional localization are not required for the ability of KRIT1 to stabilize endothelial contacts, and suggest that the ability of KRIT1 to limit integrin activity could be involved in barrier stabilization.

Description of Two Families with New Mutations in Familial Cerebral Cavernous Malformations Genes.

Cerebral cavernous malformations (CCMs) are dilated aberrant leaky capillaries located in the Central Nervous System. Familial CCM is an autosomal dominant inherited disorder related to mutations in KRIT1, Malcavernin or PDCD10. We show two unrelated families presenting familial CCM due to two new mutations in KRIT1 and PDCD10, producing truncated proteins. Clinical phenotype was highly variable among patients from asymptomatic individuals to diplopia, seizures or severe intracranial hemorrhage. PDCD10 patients usually show a more aggressive course and they frequently showed multiple meningiomas. This work provides evidence for the pathogenicity of two new mutations in CCM genes and supports previous findings regarding familial CCM and multiple meningiomas.

Children with cavernous malformations of the central nervous system.

BACKGROUND: Cavernous malformations (CM) of the central nervous system (CNS) are a rare pathology in the pediatric population that may present with an acute onset of severe neurological symptoms. OBJECTIVE: The aim of our study was to evaluate the clinical presentation, family history, genetic background, radiological features, treatment and outcome of children with CM. METHODS: This observational cohort study included all children with CM of the CNS diagnosed in the period 2000-2020 at University Children's Hospital in Ljubljana, Slovenia. Whole exome sequencing was utilized. RESULTS: We identified a cohort of 20 children with CM (mean age 9.3 years, range: 10 days-18.4 years). In our cohort, 16 patients were symptomatic and 4 were asymptomatic; 7 patients had a solitary lesion, and 13 had multiple lesions. Children with multiple lesions become symptomatic at an earlier age compared with children with solitary lesions. We identified five families with familiar cavernous malformation (FCM) syndrome affecting two or more generations; FCM represented 65% of all pediatric cases identified in our study. We confirmed a mutation in FCM associated genes in all but one patient with multiple lesions, with the KRIT1 mutation being the most common. CONCLUSION: Multiple CM lesions and symptomatic brainstem lesions are associated with worse neurological deficits in pediatric patients. Not all cases of multiple lesions can be linked to mutations in KRIT1, CCM2, or PDCD10, which may indicate that there are other as yet unidentified genes associated with FCM.

Fetal Familial Cerebral Cavernous Malformation With a Novel Heterozygous KRIT1 Variation.

BACKGROUND AND OBJECTIVES: To identify fetal familial cerebral cavernous malformation (CCM) and a novel variation. METHODS: A 37-year-old pregnant woman (G4P0) presented with right-handed numbness since 2 weeks at 31 weeks of gestation. Evaluation with brain MRI revealed multiple CCMs. As a result, fetal MRI, fetal whole exome sequencing, and maternal Sanger sequencing were performed. RESULTS: The mother's brain MRI demonstrated numerous CCMs involving the brain stem, cerebral hemispheres, and cerebellum. Fetal MRI showed a CCM located in the left frontal lobe in susceptibility-weighted imaging (SWI). The neuroimaging characteristics of the mother and the fetus suggested that their CCMs may be familial. Genetic analysis revealed a novel variation in KRIT1 (c.1A>G, p.0?), also called CCM1, in the mother and the baby. The mother delivered a daughter at 32 weeks of gestation with an Apgar score of 10 by cesarean section. DISCUSSION: This variation of the initial codon in the KRIT1 gene leads to a phenotype with an early-onset. To our knowledge, this is the first-ever reported case of fetal familial CCM and this novel variation. Brain MRI has excellent sensitivity and specificity, providing the best option for detecting CCMs, even in utero, primarily when SWI is used.

Somatic PIK3CA Mutations in Sporadic Cerebral Cavernous Malformations.

BACKGROUND: Cerebral cavernous malformations (CCMs) are common sporadic and inherited vascular malformations of the central nervous system. Although familial CCMs are linked to loss-of-function mutations in KRIT1 (CCM1), CCM2, or PDCD10 (CCM3), the genetic cause of sporadic CCMs, representing 80% of cases, remains incompletely understood. METHODS: We developed two mouse models harboring mutations identified in human meningiomas with the use of the prostaglandin D2 synthase (PGDS) promoter. We performed targeted DNA sequencing of surgically resected CCMs from patients and confirmed our findings by droplet digital polymerase-chain-reaction analysis. RESULTS: We found that in mice expressing one of two common genetic drivers of meningioma - Pik3ca H1047R or AKT1 E17K - in PGDS-positive cells, a spectrum of typical CCMs develops (in 22% and 11% of the mice, respectively) instead of meningiomas, which prompted us to analyze tissue samples from sporadic CCMs from 88 patients. We detected somatic activating PIK3CA and AKT1 mutations in 39% and 1%, respectively, of lesion tissue from the patients. Only 10% of lesions harbored mutations in the CCM genes. We analyzed lesions induced by the activating mutations Pik3ca H1074R and AKT1 E17K in mice and identified the PGDS-expressing pericyte as the probable cell of origin. CONCLUSIONS: In tissue samples from sporadic CCMs, mutations in PIK3CA were represented to a greater extent than mutations in any other gene. The contribution of somatic mutations in the genes that cause familial CCMs was comparatively small. (Funded by the Fondation ARC pour la Recherche contre le Cancer and others.).

Polymorphisms in genes related to oxidative stress and inflammation: Emerging links with the pathogenesis and severity of Cerebral Cavernous Malformation disease.

Cerebral Cavernous Malformation (CCM) is a cerebrovascular disease of genetic origin affecting 0.5% of the population and characterized by abnormally enlarged and leaky capillaries that predispose to seizures, neurological deficits, and intracerebral hemorrhage (ICH). CCM occurs sporadically or is inherited as dominant condition with incomplete penetrance and highly variable expressivity. Three disease genes have been identified: KRIT1 (CCM1), CCM2 and CCM3. Previous results demonstrated that loss-of-function mutations of CCM genes cause pleiotropic effects, including defective autophagy, altered reactive oxygen species (ROS) homeostasis, and enhanced sensitivity to oxidative stress and inflammatory events, suggesting a novel unifying pathogenetic mechanism, and raising the possibility that CCM disease onset and severity are influenced by the presence of susceptibility and modifier genes. Consistently, genome-wide association studies (GWAS) in large and homogeneous cohorts of patients sharing the familial form of CCM disease and identical mutations in CCM genes have led to the discovery of distinct genetic modifiers of major disease severity phenotypes, such as development of numerous and large CCM lesions, and susceptibility to ICH. This review deals with the identification of genetic modifiers with a significant impact on inter-individual variability in CCM disease onset and severity, including highly polymorphic genes involved in oxidative stress, inflammatory and immune responses, such as cytochrome P450 monooxygenases (CYP), matrix metalloproteinases (MMP), and Toll-like receptors (TLR), pointing to their emerging prognostic value, and opening up new perspectives for risk stratification and personalized medicine strategies.

Cancer-secreted exosomal miR-21-5p induces angiogenesis and vascular permeability by targeting KRIT1.

Cancer-secreted exosomes are critical mediators of cancer-host crosstalk. In the present study, we showed the delivery of miR-21-5p from colorectal cancer (CRC) cells to endothelial cells via exosomes increased the amount of miR-21-5p in recipient cells. MiR-21-5p suppressed Krev interaction trapped protein 1 (KRIT1) in recipient HUVECs and subsequently activated ß-catenin signaling pathway and increased their downstream targets VEGFa and Ccnd1, which consequently promoted angiogenesis and vascular permeability in CRC. A strong inverse correlation between miR-21-5p and KRIT1 expression levels was observed in CRC-adjacent vessels. Furthermore, miR-21-5p expression in circulating exosomes was markedly higher in CRC patients than in healthy donors. Thus, our data suggest that exosomal miR-21-5p is involved in angiogenesis and vascular permeability in CRC and may be used as a potential new therapeutic target.

Molecular Genetic Features of Cerebral Cavernous Malformations (CCM) Patients: An Overall View from Genes to Endothelial Cells.

Cerebral cavernous malformations (CCMs) are vascular lesions that affect predominantly microvasculature in the brain and spinal cord. CCM can occur either in sporadic or familial form, characterized by autosomal dominant inheritance and development of multiple lesions throughout the patient's life. Three genes associated with CCM are known: CCM1/KRIT1 (krev interaction trapped 1), CCM2/MGC4607 (encoding a protein named malcavernin), and CCM3/PDCD10 (programmed cell death 10). All the mutations identified in these genes cause a loss of function and compromise the protein functions needed for maintaining the vascular barrier integrity. Loss of function of CCM proteins causes molecular disorganization and dysfunction of endothelial adherens junctions. In this review, we provide an overall vision of the CCM pathology, starting with the genetic bases of the disease, describing the role of the proteins, until we reach the cellular level. Thus, we summarize the genetics of CCM, providing a description of CCM genes and mutation features, provided an updated knowledge of the CCM protein structure and function, and discuss the molecular mechanisms through which CCM proteins may act within endothelial cells, particularly in endothelial barrier maintenance/regulation and in cellular signaling.

KRIT1 Gene in Patients with Cerebral Cavernous Malformations: Clinical Features and Molecular Characterization of Novel Variants.

Cerebral cavernous malformations (CCMs) are vascular malformations that may result in headaches, seizures, focal neurological deficits, and hemorrhage. CCMs occur sporadically (80%) or in familial form (20%), with autosomal dominant inheritance. Among the three CCM-related genes, mutations in KRIT1 account for 53-65% of familial cases and more than 100 different mutations have been identified so far. In the present work, we describe the clinical, neuroradiological, and genetic findings of sixteen CCM Italian patients, 13 belonging to 4 unrelated families and 3 sporadic cases. Six distinct KRIT1 gene variants, two novel (c.1730+1_1730+3del, c.1664 C>T) and four previously described (c.966G>A, c.1255-1G>A c.1197_1200del, c.1255-1_1256del), were identified, including a possible de novo mutation. All the variants resulted in a premature stop codon. Cerebral 1.5 T magnetic resonance imaging showed multiple CCMs in all the mutation carriers for whom it was available, including sporadic cases. One patient had also cutaneous angiomas. Among the mutation carriers, symptomatic patients constituted 66% and a variable phenotypic expression was observed. Our data confirms phenotypic variability and incomplete penetrance of neurological symptoms in KRIT1-positive families, expands the mutational spectrum of this gene, and highlights how sporadic cases with multiple lesions need an approach similar to individuals with familial CCM.

Improving clinical interpretation of five KRIT1 and PDCD10 intronic variants.

Cerebral cavernous malformation (CCM) is a vascular malformation of the central nervous system which may occur sporadically or segregate within families due to heterozygous variants in KRIT1/CCM1, MGC4607/CCM2 or PDCD10/CCM3. Intronic variants are not uncommon in familial CCM, but their clinical interpretation is often hampered by insufficient data supporting in silico predictions. Here, the mRNA analysis for two intronic unpublished variants (KRIT1 c.1147-7 T > G and PDCD10 c.395 + 2 T > G) and three previously published variants in KRIT1 but without data supporting their effects was carried out. This study demonstrated that all variants can induce a frameshift with the lack of residues located in the C-terminal regions and involved in protein-protein complex formation, which is essential for vascular homeostasis. These results support the introduction of mRNA analysis in the diagnostic pathway of familial CCM and expand the knowledge of abnormal splicing patterning in this disorder.