Loss of heterozygosity in CCM2 cDNA revealing a structural variant causing multiple cerebral cavernous malformations.

Loss-of-function variants in CCM1/KRIT1, CCM2/MGC4607, and CCM3/PDCD10 genes are identified in the vast majority of familial cases with multiple cerebral cavernous malformations. However, genomic DNA sequencing combined with large rearrangement screening fails to detect a pathogenic variant in 5% of the patients. We report a family with two affected members harboring multiple CCM lesions, one with severe hemorrhages and one asymptomatic. No causative variant was detected using DNA sequencing of the three CCM genes, CNV detection analysis, and RNA sequencing. However, a loss of heterozygosity in CCM2 was observed on cDNA sequences in one of the two affected members, which strongly suggested that this locus might be involved. Whole genome sequencing (WGS) identified a balanced structural variant on chromosome 7 with a breakpoint interrupting the CCM2 gene, preventing normal mRNA synthesis. These data underline the importance of WGS in undiagnosed patients with typical multiple CCM.

An AluYa5 Insertion in the 3'UTR of COL4A1 and Cerebral Small Vessel Disease.

Importance: Cerebral small vessel diseases (CSVDs) account for one-fifth of stroke cases. Numerous familial cases remain unresolved after routine screening of known CSVD genes. Objective: To identify novel genes and mechanisms associated with familial CSVD. Design, Setting, and Participants: This 2-stage study involved linkage analysis and a case-control study; linkage analysis and whole exome and genome sequencing were used to identify candidate gene variants in 2 large families with CSVD (9 patients with CSVD). Then, a case-control analysis was conducted on 246 unrelated probands, including probands from these 2 families and 244 additional probands. All probands (clinical onset

Age-related loss of Notch3 underlies brain vascular contractility deficiencies, glymphatic dysfunction, and neurodegeneration in mice.

Vascular aging affects multiple organ systems, including the brain, where it can lead to vascular dementia. However, a concrete understanding of how aging specifically affects the brain vasculature, along with molecular readouts, remains vastly incomplete. Here, we demonstrate that aging is associated with a marked decline in Notch3 signaling in both murine and human brain vessels. To clarify the consequences of Notch3 loss in the brain vasculature, we used single-cell transcriptomics and found that Notch3 inactivation alters regulation of calcium and contractile function and promotes a notable increase in extracellular matrix. These alterations adversely impact vascular reactivity, manifesting as dilation, tortuosity, microaneurysms, and decreased cerebral blood flow, as observed by MRI. Combined, these vascular impairments hinder glymphatic flow and result in buildup of glycosaminoglycans within the brain parenchyma. Remarkably, this phenomenon mirrors a key pathological feature found in brains of patients with CADASIL, a hereditary vascular dementia associated with NOTCH3 missense mutations. Additionally, single-cell RNA sequencing of the neuronal compartment in aging Notch3-null mice unveiled patterns reminiscent of those observed in neurodegenerative diseases. These findings offer direct evidence that age-related NOTCH3 deficiencies trigger a progressive decline in vascular function, subsequently affecting glymphatic flow and culminating in neurodegeneration.

Phenotype and imaging features associated with APP duplications.

BACKGROUND: APP duplication is a rare genetic cause of Alzheimer disease and cerebral amyloid angiopathy (CAA). We aimed to evaluate the phenotypes of APP duplications carriers. METHODS: Clinical, radiological, and neuropathological features of 43 APP duplication carriers from 24 French families were retrospectively analyzed, and MRI features and cerebrospinal fluid (CSF) biomarkers were compared to 40 APP-negative CAA controls. RESULTS: Major neurocognitive disorders were found in 90.2% symptomatic APP duplication carriers, with prominent behavioral impairment in 9.7%. Symptomatic intracerebral hemorrhages were reported in 29.2% and seizures in 51.2%. CSF Aß42 levels were abnormal in 18/19 patients and 14/19 patients fulfilled MRI radiological criteria for CAA, while only 5 displayed no hemorrhagic features. We found no correlation between CAA radiological signs and duplication size. Compared to CAA controls, APP duplication carriers showed less disseminated cortical superficial siderosis (0% vs 37.5%, p = 0.004 adjusted for the delay between symptoms onset and MRI). Deep microbleeds were found in two APP duplication carriers. In addition to neurofibrillary tangles and senile plaques, CAA was diffuse and severe with thickening of leptomeningeal vessels in all 9 autopsies. Lewy bodies were found in substantia nigra, locus coeruleus, and cortical structures of 2/9 patients, and one presented vascular amyloid deposits in basal ganglia. DISCUSSION: Phenotypes associated with APP duplications were heterogeneous with different clinical presentations including dementia, hemorrhage, and seizure and different radiological presentations, even within families. No apparent correlation with duplication size was found. Amyloid burden was severe and widely extended to cerebral vessels as suggested by hemorrhagic features on MRI and neuropathological data, making APP duplication an interesting model of CAA.

Rare variants in ANO1, encoding a calcium-activated chloride channel, predispose to moyamoya disease.

Moyamoya disease, a cerebrovascular disease leading to strokes in children and young adults, is characterized by progressive occlusion of the distal internal carotid arteries and the formation of collateral vessels. Altered genes play a prominent role in the aetiology of moyamoya disease, but a causative gene is not identified in the majority of cases. Exome sequencing data from 151 individuals from 84 unsolved families were analysed to identify further genes for moyamoya disease, then candidate genes assessed in additional cases (150 probands). Two families had the same rare variant in ANO1, which encodes a calcium-activated chloride channel, anoctamin-1. Haplotype analyses found the families were related, and ANO1 p.Met658Val segregated with moyamoya disease in the family with an LOD score of 3.3. Six additional ANO1 rare variants were identified in moyamoya disease families. The ANO1 rare variants were assessed using patch-clamp recordings, and the majority of variants, including ANO1 p.Met658Val, displayed increased sensitivity to intracellular Ca2+. Patients harbouring these gain-of-function ANO1 variants had classic features of moyamoya disease, but also had aneurysm, stenosis and/or occlusion in the posterior circulation. Our studies support that ANO1 gain-of-function pathogenic variants predispose to moyamoya disease and are associated with unique involvement of the posterior circulation.

Extension of the Clinicoradiologic Spectrum of Newly Described End-Truncating LAMB1 Variations.

Objectives: To refine the clinical spectrum of a very recently identified phenotype associated with LAMB1 end-truncating pathogenic variations. Methods: Detailed clinical, neuropsychological, and MRI investigation of 6 patients from 2 unrelated families segregating end-truncating LAMB1 variations. Results: All patients harbored a LAMB1 end-truncating pathogenic variation. The specific association of a hippocampal type episodic memory dysfunction and a diffuse leukoencephalopathy was observed in all 4 patients aged older than 50 years, slightly worsening over time in 2 patients with several years of follow-up. Additional unspecific neurologic symptoms are reported, such as episodes of numbness, language troubles, or faintness in these 4 patients and the 2 younger ones. Discussion: The association of an extensive leukoencephalopathy with an episodic memory dysfunction of the hippocampal type is strongly suggestive of a LAMB1 end-truncating variation in adults older than 50 years. Early cognitive complaints and imaging abnormalities might exist decades before. Additional transient manifestations can be observed, and this association should lead to LAMB1 screening to avoid unnecessary invasive investigations.

Biallelic NPR1 loss of function variants are responsible for neonatal systemic hypertension.

BACKGROUND: Early-onset isolated systemic hypertension is a rare condition of unknown genetic origin. Renovascular, renal parenchymal diseases or aortic coarctation are the most common causes of secondary systemic hypertension in younger children and neonates. We investigated the genetic bases of early-onset isolated systemic hypertension. METHODS: Whole-exome sequencing (WES) was followed by variant filtering and Sanger sequencing for validation and familial segregation of selected variants in a large consanguineous family. mRNA expression was performed to evaluate the impact of the predicted pathogenic variant on gene expression. WES or Sanger sequencing was performed in additional unrelated affected individuals. RESULTS: In one consanguineous family with four children presenting with isolated neonatal-onset systemic hypertension, we identified homozygous stop-gain variant in the NPR1 gene (NM_000906.4:c.1159C>T (p.Arg387Ter)) in the affected individuals. This variant leads to a dramatic reduction of NPR1 RNA levels. NPR1 gene analysis of additional families allowed the identification of another family with two affected children carrying homozygous frameshift variant in NPR1 (NM_000906.4:c.175del (p.Val59TrpfsTer8)). CONCLUSION: We show for the first time that biallelic loss of function of NPR1 is responsible for isolated neonatal-onset systemic hypertension in humans, which represents a new autosomal recessive genetic cause of infantile systemic hypertension or cardiogenic shock. This is consistent with studies reporting early-onset systemic hypertension and sudden death in Npr1-deficient mice. NPR1 gene analysis should be therefore investigated in infants with early-onset systemic hypertension with or without cardiogenic shock of unknown origin.

Impaired retinoic acid signaling in cerebral cavernous malformations.

The capillary-venous pathology cerebral cavernous malformation (CCM) is caused by loss of CCM1/Krev interaction trapped protein 1 (KRIT1), CCM2/MGC4607, or CCM3/PDCD10 in some endothelial cells. Mutations of CCM genes within the brain vasculature can lead to recurrent cerebral hemorrhages. Pharmacological treatment options are urgently needed when lesions are located in deeply-seated and in-operable regions of the central nervous system. Previous pharmacological suppression screens in disease models of CCM led to the discovery that treatment with retinoic acid improved CCM phenotypes. This finding raised a need to investigate the involvement of retinoic acid in CCM and test whether it has a curative effect in preclinical mouse models. Here, we show that components of the retinoic acid synthesis and degradation pathway are transcriptionally misregulated across disease models of CCM. We complemented this analysis by pharmacologically modifying retinoic acid levels in zebrafish and human endothelial cell models of CCM, and in acute and chronic mouse models of CCM. Our pharmacological intervention studies in CCM2-depleted human umbilical vein endothelial cells (HUVECs) and krit1 mutant zebrafish showed positive effects when retinoic acid levels were increased. However, therapeutic approaches to prevent the development of vascular lesions in adult chronic murine models of CCM were drug regiment-sensitive, possibly due to adverse developmental effects of this hormone. A treatment with high doses of retinoic acid even worsened CCM lesions in an adult chronic murine model of CCM. This study provides evidence that retinoic acid signaling is impaired in the CCM pathophysiology and suggests that modification of retinoic acid levels can alleviate CCM phenotypes.

European Stroke Organisation (ESO) Guidelines on Moyamoya angiopathy Endorsed by Vascular European Reference Network (VASCERN).

The European Stroke Organisation (ESO) guidelines on Moyamoya Angiopathy (MMA), developed according to ESO standard operating procedure and Grading of Recommendations, Assessment, Development and Evaluation (GRADE) methodology, were compiled to assist clinicians in managing patients with MMA in their decision making. A working group involving neurologists, neurosurgeons, a geneticist and methodologists identified nine relevant clinical questions, performed systematic literature reviews and, whenever possible, meta-analyses. Quality assessment of the available evidence was made with specific recommendations. In the absence of sufficient evidence to provide recommendations, Expert Consensus Statements were formulated. Based on low quality evidence from one RCT, we recommend direct bypass surgery in adult patients with haemorrhagic presentation. For ischaemic adult patients and children, we suggest revascularization surgery using direct or combined technique rather than indirect, in the presence of haemodynamic impairment and with an interval of 6-12 weeks between the last cerebrovascular event and surgery. In the absence of robust trial, an Expert Consensus was reached recommending long-term antiplatelet therapy in non-haemorrhagic MMA, as it may reduce risk of embolic stroke. We also agreed on the utility of performing pre- and post- operative haemodynamic and posterior cerebral artery assessment. There were insufficient data to recommend systematic variant screening of RNF213 p.R4810K. Additionally, we suggest that long-term MMA neuroimaging follow up may guide therapeutic decision making by assessing the disease progression. We believe that this guideline, which is the first comprehensive European guideline on MMA management using GRADE methods will assist clinicians to choose the most effective management strategy for MMA.

Bi-allelic variants in the ESAM tight-junction gene cause a neurodevelopmental disorder associated with fetal intracranial hemorrhage.

The blood-brain barrier (BBB) is an essential gatekeeper for the central nervous system and incidence of neurodevelopmental disorders (NDDs) is higher in infants with a history of intracerebral hemorrhage (ICH). We discovered a rare disease trait in thirteen individuals, including four fetuses, from eight unrelated families associated with homozygous loss-of-function variant alleles of ESAM which encodes an endothelial cell adhesion molecule. The c.115del (p.Arg39Glyfs∗33) variant, identified in six individuals from four independent families of Southeastern Anatolia, severely impaired the in vitro tubulogenic process of endothelial colony-forming cells, recapitulating previous evidence in null mice, and caused lack of ESAM expression in the capillary endothelial cells of damaged brain. Affected individuals with bi-allelic ESAM variants showed profound global developmental delay/unspecified intellectual disability, epilepsy, absent or severely delayed speech, varying degrees of spasticity, ventriculomegaly, and ICH/cerebral calcifications, the latter being also observed in the fetuses. Phenotypic traits observed in individuals with bi-allelic ESAM variants overlap very closely with other known conditions characterized by endothelial dysfunction due to mutation of genes encoding tight junction molecules. Our findings emphasize the role of brain endothelial dysfunction in NDDs and contribute to the expansion of an emerging group of diseases that we propose to rename as "tightjunctionopathies."

Biallelic variants in NOS3 and GUCY1A3, the two major genes of the nitric oxide pathway, cause moyamoya cerebral angiopathy.

BACKGROUND: Moyamoya angiopathy (MMA) is a rare cerebrovascular condition leading to stroke. Mutations in 15 genes have been identified in Mendelian forms of MMA, but they explain only a very small proportion of cases. Our aim was to investigate the genetic basis of MMA in consanguineous patients having unaffected parents in order to identify genes involved in autosomal recessive MMA. METHODS: Exome sequencing (ES) was performed in 6 consecutive consanguineous probands having MMA of unknown etiology. Functional consequences of variants were assessed using western blot and protein 3D structure analyses. RESULTS: Causative homozygous variants of NOS3, the gene encoding the endothelial nitric oxide synthase (eNOS), and GUCY1A3, the gene encoding the alpha1 subunit of the soluble guanylate cyclase (sGC) which is the major nitric oxide (NO) receptor in the vascular wall, were identified in 3 of the 6 probands. One NOS3 variant (c.1502 + 1G > C) involves a splice donor site causing a premature termination codon and leads to a total lack of eNOS in endothelial progenitor cells of the affected proband. The other NOS3 variant (c.1942 T > C) is a missense variant located into the flavodoxine reductase domain; it is predicted to be destabilizing and shown to be associated with a reduction of eNOS expression. The GUCY1A3 missense variant (c.1778G > A), located in the catalytic domain of the sGC, is predicted to disrupt the tridimensional structure of this domain and to lead to a loss of function of the enzyme. Both NOS3 mutated probands suffered from an infant-onset and severe MMA associated with posterior cerebral artery steno-occlusive lesions. The GUCY1A3 mutated proband presented an adult-onset MMA associated with an early-onset arterial hypertension and a stenosis of the superior mesenteric artery. None of the 3 probands had achalasia. CONCLUSIONS: We show for the first time that biallelic loss of function variants in NOS3 is responsible for MMA and that mutations in NOS3 and GUCY1A3 are causing fifty per cent of MMA in consanguineous patients. These data pinpoint the essential role of the NO pathway in MMA pathophysiology.

Phenotypic variability in 446 CADASIL patients: Impact of NOTCH3 gene mutation location in addition to the effects of age, sex and vascular risk factors.

The recent discovery that the prevalence of cysteine mutations in the NOTCH3 gene responsible for CADASIL was more than 100 times higher in the general population than that estimated in patients highlighted that the mutation location in EGFr-like-domains of the NOTCH3 receptor could have a major effect on the phenotype of the disease. The exact impact of such mutations locations on the multiple facets of the disease has not been fully evaluated. We aimed to describe the phenotypic spectrum of a large population of CADASIL patients and to investigate how this mutation location influenced various clinical and imaging features of the disease. Both a supervised and a non-supervised approach were used for analysis. The results confirmed that the mutation location is strongly related to clinical severity and showed that this effect is mainly driven by a different development of the most damaging ischemic tissue lesions at cerebral level. These effects were detected in addition to those of aging, male sex, hypertension and hypercholesterolemia. The exact mechanisms relating the location of mutations along the NOTCH3 receptor, the amount or properties of the resulting NOTCH3 products accumulating in the vessel wall, and their final consequences at cerebral level remain to be determined.

Lysyl hydroxylase 3-mediated post-translational modifications are required for proper biosynthesis of collagen α1α1α2(IV).

Collagens are the most abundant proteins in the body and among the most biosynthetically complex. A molecular ensemble of over 20 endoplasmic reticulum resident proteins participates in collagen biosynthesis and contributes to heterogeneous post-translational modifications. Pathogenic variants in genes encoding collagens cause connective tissue disorders, including osteogenesis imperfecta, Ehlers-Danlos syndrome, and Gould syndrome (caused by mutations in COL4A1 and COL4A2), and pathogenic variants in genes encoding proteins required for collagen biosynthesis can cause similar but overlapping clinical phenotypes. Notably, pathogenic variants in lysyl hydroxylase 3 (LH3) cause a multisystem connective tissue disorder that exhibits pathophysiological features of collagen-related disorders. LH3 is a multifunctional collagen-modifying enzyme; however, its precise role(s) and substrate specificity during collagen biosynthesis has not been defined. To address this critical gap in knowledge, we generated LH3 KO cells and performed detailed quantitative and molecular analyses of collagen substrates. We found that LH3 deficiency severely impaired secretion of collagen α1α1α2(IV) but not collagens α1α1α2(I) or α1α1α1(III). Amino acid analysis revealed that LH3 is a selective LH for collagen α1α1α2(IV) but a general glucosyltransferase for collagens α1α1α2(IV), α1α1α2(I), and α1α1α1(III). Importantly, we identified rare variants that are predicted to be pathogenic in the gene encoding LH3 in two of 113 fetuses with intracranial hemorrhage-a cardinal feature of Gould syndrome. Collectively, our findings highlight a critical role of LH3 in α1α1α2(IV) biosynthesis and suggest that LH3 pathogenic variants might contribute to Gould syndrome.

Elderly CADASIL patients with intact neurological status.

BACKGROUND AND PURPOSE: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is one of the most devastating cerebral small vessel diseases. However, despite its progression with aging, some patients remain neurologically intact (Nint) even when they get older. Their main characteristics are poorly known. We aimed to delineate their clinical, imaging, and molecular features. METHODS: Individuals aged over 65 years were selected from a cohort of 472 CADASIL patients. Subjects who had no focal deficit, cognitive impairment, or disability were considered Nint. Their demographic, genetic, clinical, and imaging features were compared to those with permanent neurological symptoms (Nps). RESULTS: Among 129 patients, 23 (17.8%) individuals were considered Nint. The frequency of vascular risk factors and NOTCH3 cysteine mutations in epidermal growth factor-like repeat (EGFr) domains 7-34 did not differ between Nint and Nps patients but Nint patients had less stroke events and were more likely to have migraine with aura. The number of lacunes and microbleeds and degree of brain atrophy were lower in the Nint group, but the volume of white matter hyperintensities did not differ between the two groups. CONCLUSIONS: Nearly one in five CADASIL patients can remain Nint after the age of 65 years. Their clinical and imaging profile differed from that of other age-matched CADASIL patients. The location of NOTCH3 mutation inside or outside EGFr domains 1-6 cannot fully explain this discrepancy. The factors involved in their relative preservation of brain tissue from severe damage despite aging remain to be determined.

Moyamoya disease: diagnosis and interventions.

Moyamoya disease is a rare cause of stroke, radiologically characterised by progressive stenosis of the terminal portion of the internal carotid arteries and compensatory capillary collaterals. The discovery that RNF213, which encodes an unconventional E3 ubiquitin ligase, is the major susceptibility gene for moyamoya disease in people from east Asia has opened new avenues for investigation into the mechanisms of disease and potential treatment targets. The Arg4810Lys variant of the gene is most strongly associated with moyamoya disease, but the penetrance is lower than 1%, suggesting a synergistic relationship with additional environmental and genetic risk factors. White people carry less common non-Arg4810Lys variants of RNF213, which partly explains the lower prevalence of moyamoya disease in European countries and in the USA than in east Asian countries. Several monogenic moyamoya syndromes possess the radiological characteristics of moyamoya disease and have been associated with multiple genes and pathways involved in moyamoya angiopathy pathogenesis. Further clarification of the genetic and environmental factors that contribute to the emergence of moyamoya angiopathy could enable development of new treatment strategies for moyamoya disease.

Prenatal Diagnosis of COL4A1 Mutations in Eight Cases: Further Delineation of the Neurohistopathological Phenotype.

BACKGROUND: Increasing number of mutations responsible for vascular lesions, leading to ischemic or hemorrhagic stroke in young adults, has been identified in the recent years. It has been demonstrated in both mice and humans, that mutations in COL4A1 gene promote cerebral hemorrhages. In humans, both adults and children may be affected, and the spectrum has been broadened recently to neonates and fetuses. METHODS: We present a cohort of eight COL4A1 mutated fetuses in which cerebral hemorrhages were detected by ultrasound leading to elective terminations of pregnancy. RESULTS: Our neuropathological studies demonstrated a strikingly similar pathological pattern, dominated by supra- and infratentorial multifocal hemorrhagic lesions of various abundance and age in the vicinity of enlarged small vessels having a discontinuous wall. This was constantly associated with a spectrum of supratentorial post-ischemic damages of the grey and white matters. Morphometric studies of brain vessels confirmed vascular dilation and hypervascularization in both grey and white matters and severe attenuation of the smooth-muscle actin staining in the white matter. CONCLUSION: These observations add to the rare human neuropathological phenotype of COL4A1 mutations. Its recognition is mandatory to enhance the number of tested patients in the future, as well as the genetic counseling of parents.

RAVAQ: An integrative pipeline from quality control to region-based rare variant association analysis.

Next-generation sequencing technologies have opened up the possibility to sequence large samples of cases and controls to test for association with rare variants. To limit cost and increase sample sizes, data from controls could be used in multiple studies and might thus be generated on different sequencing platforms. This could pose some problems of comparability between cases and controls due to batch effects that could be confounding factors, leading to false-positive association signals. To limit batch effects and ensure comparability of datasets, stringent quality controls are required. We propose an integrative five-steps pipeline, RAVAQ, that (a) performs a specific three-step quality control taking into account the case-control status to ensure data comparability, (b) selects qualifying variants as defined by the user, and (c) performs rare variant association tests per genomic region. The RAVAQ pipeline is wrapped in an R package. It is user-friendly and flexible in its arguments to adapt to the specificity of each research project. We provide examples showing how RAVAQ improves rare variant association tests. The default RAVAQ quality control outperformed the widely used Variant Quality Score Recalibration method, removing inflation due to spurious signals. RAVAQ is open source and freely available at https://gitlab.com/gmarenne/ravaq.

COL4A1/COL4A2 and inherited platelet disorder gene variants in fetuses showing intracranial hemorrhage.

BACKGROUND: Variants of COL4A1/COL4A2 genes have been reported in fetal intracranial hemorrhage (ICH) cases but their prevalence and characteristics have not been established in a large series of fetuses. Fetal neonatal alloimmune thrombocytopenia is a major acquired ICH factor but the prevalence and characteristics of inherited platelet disorder (IPD) gene variants leading to thrombocytopenia are unknown. Herein, we screened COL4A1/COL4A2 and IPD genes in a large series of ICH fetuses. METHODS: A cohort of 194 consecutive ICH fetuses were first screened for COL4A1/COL4A2 variants. We manually curated a list of 64 genes involved in IPD and investigated them in COL4A1/COL4A2 negative fetuses, using exome sequencing data from 101 of these fetuses. RESULT: Pathogenic variants of COL4A1/COL4A2 genes were identified in 36 fetuses (19%). They occurred de novo in 70% of the 32 fetuses for whom parental DNA was available. Pathogenic variants in two megakaryopoiesis genes (MPL and MECOM genes) were identified in two families with recurrent and severe fetal ICH, with variable extraneurological pathological features. CONCLUSION: Our study emphasizes the genetic heterogeneity of fetal ICH and the need to screen both COL4A1/COL4A2 and IPD genes in the etiological investigation of fetal ICH to allow proper genetic counseling.