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

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."

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.

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.

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.

End-Truncated LAMB1 Causes a Hippocampal Memory Defect and a Leukoencephalopathy.

OBJECTIVE: The majority of patients with a familial cerebral small vessel disease (CSVD) referred for molecular screening do not show pathogenic variants in known genes. In this study, we aimed to identify novel CSVD causal genes. METHODS: We performed a gene-based collapsing test of rare protein-truncating variants identified in exome data of 258 unrelated CSVD patients of an ethnically matched control cohort and of 2 publicly available large-scale databases, gnomAD and TOPMed. Western blotting was used to investigate the functional consequences of variants. Clinical and magnetic resonance imaging features of mutated patients were characterized. RESULTS: We showed that LAMB1 truncating variants escaping nonsense-mediated messenger RNA decay are strongly overrepresented in CSVD patients, reaching genome-wide significance (p < 5 × 10-8 ). Using 2 antibodies recognizing the N- and C-terminal parts of LAMB1, we showed that truncated forms of LAMB1 are expressed in the endogenous fibroblasts of patients and trapped in the cytosol. These variants are associated with a novel phenotype characterized by the association of a hippocampal type episodic memory defect and a diffuse vascular leukoencephalopathy. INTERPRETATION: These findings are important for diagnosis and clinical care, to avoid unnecessary and sometimes invasive investigations, and also from a mechanistic point of view to understand the role of extracellular matrix proteins in neuronal homeostasis. ANN NEUROL 2021;90:962-975.

Early-Onset Cerebral Amyloid Angiopathy and Alzheimer Disease Related to an APP Locus Triplication.

BACKGROUND AND OBJECTIVE: To report a triplication of the amyloid-ß precursor protein (APP) locus along with relative messenger RNA (mRNA) expression in a family with autosomal dominant early-onset cerebral amyloid angiopathy (CAA) and Alzheimer disease (AD). METHODS: Four copies of the APP gene were identified by quantitative multiplex PCR of short fluorescent fragments, fluorescent in situ hybridization (FISH), and array comparative genomic hybridization. APP mRNA levels were assessed using reverse-transcription-digital droplet PCR in the proband's whole blood and compared with 10 controls and 9 APP duplication carriers. RESULTS: Beginning at age 39 years, the proband developed severe episodic memory deficits with a CSF biomarker profile typical of AD and multiple lobar microbleeds in the posterior regions on brain MRI. His father had seizures and recurrent cerebral hemorrhage since the age of 37 years. His cerebral biopsy showed abundant perivascular amyloid deposits, leading to a diagnosis of CAA. In the proband, we identified 4 copies of a 506-kb region located on chromosome 21q21.3 and encompassing the whole APP gene without any other gene. FISH suggested that the genotype of the proband was 3 copies/1 copy corresponding to an APP locus triplication, which was consistent with the presence of 2 APP copies in the healthy mother and with the paternal medical history. Analysis of the APP mRNA level showed a 2-fold increase in the proband and a 1.8 fold increase in APP duplication carriers compared with controls. DISCUSSION: Increased copy number of APP is sufficient to cause AD and CAA, with likely earlier onset in case of triplication compared with duplication.

Heterozygous HTRA1 nonsense or frameshift mutations are pathogenic.

Heterozygous missense HTRA1 mutations have been associated with an autosomal dominant cerebral small vessel disease (CSVD) whereas the pathogenicity of heterozygous HTRA1 stop codon variants is unclear. We performed a targeted high throughput sequencing of all known CSVD genes, including HTRA1, in 3853 unrelated consecutive CSVD patients referred for molecular diagnosis. The frequency of heterozygous HTRA1 mutations leading to a premature stop codon in this patient cohort was compared with their frequency in large control databases. An analysis of HTRA1 mRNA was performed in several stop codon carrier patients. Clinical and neuroimaging features were characterized in all probands. Twenty unrelated patients carrying a heterozygous HTRA1 variant leading to a premature stop codon were identified. A highly significant difference was observed when comparing our patient cohort with control databases: gnomAD v3.1.1 [P = 3.12 × 10-17, odds ratio (OR) = 21.9], TOPMed freeze 5 (P = 7.6 × 10-18, OR = 27.1) and 1000 Genomes (P = 1.5 × 10-5). Messenger RNA analysis performed in eight patients showed a degradation of the mutated allele strongly suggesting a haploinsufficiency. Clinical and neuroimaging features are similar to those previously reported in heterozygous missense mutation carriers, except for penetrance, which seems lower. Altogether, our findings strongly suggest that heterozygous HTRA1 stop codons are pathogenic through a haploinsufficiency mechanism. Future work will help to estimate their penetrance, an important information for genetic counselling.

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) - Still to be Considered in the Presence of Vascular Risk Factors.

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is a rare hereditary vasculopathy that primarily affects the brain, caused mostly by missense mutations of the NOTCH3 gene which is located on chromosome 19. Clinically, it manifests as transient ischemic attacks and strokes in individuals under the age of 60 years without vascular risk factors. We report a 46-year-old male with a 9 and 3-month history of progressive unilateral lower limb weakness and dysarthria, respectively. He had a history of diabetes mellitus but no hypertension, hyperlipidemia, or smoking history. Both parents had a stroke at the age of 65 years. Neurological examination was significant for moderate dysarthria and reduced right upper limb dexterity. Magnetic resonance imaging (MRI) of the brain revealed extensive white matter disease, lacunar infarcts, and a few microhemorrhages. Electron microscopy of his skin biopsy showed electron-dense deposits of extracellular osmiophilic granular material adjacent to smooth muscle cells. NOTCH3 gene analysis revealed a heterozygous typical mutation in exon 6. He was commenced on aspirin and atorvastatin. Over time, he became more dysarthric and demented. MRI revealed the progression of the white matter disease and a new right subcortical infarct. His aspirin was switched to clopidogrel, and donepezil was added. CADASIL should be considered among younger stroke patients with vascular risk factors, especially in the presence of widespread white matter disease. Genetic counselling may be needed after the diagnosis is made.

Further refinement of COL4A1 and COL4A2 related cortical malformations.

Mutations in COL4A1 have been reported in schizencephaly and porencephaly combined with microbleeds or calcifications, often associated with ocular and renal abnormalities, myopathy, elevated creatine kinase levels and haemolytic anaemia. In this study, we aimed to clarify the phenotypic spectrum of COL4A1/A2 mutations in the context of cortical malformations that include schizencephaly, polymicrogyria and/or heterotopia. METHODS: We screened for COL4A1/A2 mutations in 9 patients with schizencephaly and/or polymicrogyria suspected to be caused by vascular disruption and leading to a cerebral haemorrhagic ischaemic event. These included 6 cases with asymmetrical or unilateral schizencephaly and/or polymicrogyria and 3 cases with bilateral schizencephaly. RESULTS: One de novo missense COL4A1 mutation (c.3715 G > A, p.(Gly1239Arg)) and two COL4A2 mutations were found, respectively in one familial case (c.4129G > A, p.(Gly1377Arg)) and one sporadic patient (c.1776+1G > A). In three other cases, COL4A1 variants of unknown significance were identified. None of our patients demonstrated neuromuscular or hematological anomalies. Brain malformations included a combination of schizencephaly, mainly asymmetrical, with porencephaly or ventriculomegaly (3/3 mutated patients). We did not observe microbleeds or microcalcifications in any of our cases, hence we do not believe that they represent a distinctive feature of COL4A1/A2 mutations. CONCLUSIONS: Our study further emphasizes the need to search for both COL4A1 and COL4A2 mutations in children presenting with uni- or bilateral polymicrogyria with schizencephaly, even in the absence of intracranial microbleeds, calcification or associated systemic features.

Analytical evaluation of point of care cTnT and clinical performances in an unselected population as compared with central laboratory highly sensitive cTnT.

OBJECTIVES: To report the analytical performances of the Radiometer AQT90 FLEX® cTnT assay (Neuilly-Plaisance, France) and to evaluate the concordance with hs-cTnT results from central laboratory for the diagnosis of acute myocardial infarction (AMI) at baseline and during a short follow-up among unselected patients admitted in emergency room or cardiology department. DESIGN AND METHODS: Analytical performances of AQT90 FLEX® cTnT immunoassay included imprecision study with determination of a coefficient of variation at 10% and 20%, linearity, and limit of detection. The concordance study was based on samples obtained from 170 consecutive patients with chest pain suggestive of acute coronary syndrome (ACS) admitted in the emergency room or cardiology department. The kinetic study (within 62 additional samples 3h later) was based on absolute delta criterion and the combination of relative change of 30% with absolute change of 7ng/L. RESULTS: The cTnT assay from Radiometer was evaluated as clinically usable, although less sensitive than the Roche hs-cTnT assay as demonstrated by the concordance and the kinetic studies. CONCLUSIONS: In non-selected population, the cTnT AQT Flex© assay on AQT90© with kinetic change at 3h, provides similar clinical classification of patients, particularly for AMI group as compared to central laboratory hs-cTnT assay and could be suitable for clinical use.

Evaluation of NM-BAPTA method for plasma total calcium measurement on Cobas 8000®.

OBJECTIVES: A new method was developed by Roche for the measurement of plasma calcium using the chromophore 5-nitro-5'-methyl-(1,2-bis(o-aminophenoxy)ethan-N,N,N',N'-tetraacetic acid (NM-BAPTA) which could have several advantages over the CPC method. The aim of our study was to evaluate the analytical performances of the NM-BAPTA assay from Roche on c701/Cobas 8000® and to perform a comparison study of calcium values with CPC and Arsenazo III methods. METHODS: The analytical performance including imprecision study, linearity, and stability of the NM-BAPTA assay was tested on the c701/Cobas 8000®analyzer. The most frequent interferences such as magnesium and gadolinium-based contrast agents (Gd-CAs) were examined with spiked human plasma on the selected method. The calcium Arsenazo III method from Horiba (Montpellier, France) installed on ABX Pentra 400® was used as a reference method. Linear regression analysis was performed to compare data from the different methods. RESULTS: The CV of the NM-BAPTA assay showed good analytical performances with CV <1.5%, in agreement with the proposed and interim European biologic goals. We found no interference neither with gadobenate dimeglumine nor with gadoteric acid considering significant findings as interference greater than 5%. In the analytical range from 0.85 to 3.80 mmol/L, the NM-BAPTA method was closely correlated to the Arsenazo III method. CONCLUSIONS: Our data demonstrate that this new calcium NM-BAPTA method developed by Roche analyzers perform as well as the conventional method, especially for the outermost values. Thus, this new colorimetric assay could substitute the CPC method on Roche analyzers.