Mutations within the MGC4607 gene cause cerebral cavernous malformations.

Cerebral cavernous malformations (CCM) are hamartomatous vascular malformations characterized by abnormally enlarged capillary cavities without intervening brain parenchyma. They cause seizures and focal neurological deficits due to cerebral hemorrhages. CCM loci have already been assigned to chromosomes 7q (CCM1), 7p (CCM2), and 3q (CCM3) and have been identified in 40%, 20%, and 40%, respectively, of families with CCM. Loss-of-function mutations have been identified in CCM1/KRIT1, the sole CCM gene identified to date. We report here the identification of MGC4607 as the CCM2 gene. We first reduced the size of the CCM2 interval from 22 cM to 7.5 cM by genetic linkage analysis. We then hypothesized that large deletions might be involved in the disorder, as already reported in other hamartomatous conditions, such as tuberous sclerosis or neurofibromatosis. We performed a high-density microsatellite genotyping of this 7.5-cM interval to search for putative null alleles in 30 unrelated families, and we identified, in 2 unrelated families, null alleles that were the result of deletions within a 350-kb interval flanked by markers D7S478 and D7S621. Additional microsatellite and single-nucleotide polymorphism genotyping showed that these two distinct deletions overlapped and that both of the two deleted the first exon of MGC4607, a known gene of unknown function. In both families, one of the two MGC4607 transcripts was not detected. We then identified eight additional point mutations within MGC4607 in eight of the remaining families. One of them led to the alteration of the initiation codon and five of them to a premature termination codon, including one nonsense, one frameshift, and three splice-site mutations. All these mutations cosegregated with the disease in the families and were not observed in 192 control chromosomes. MGC4607 is so far unrelated to any known gene family. Its implication in CCMs strongly suggests that it is a new player in vascular morphogenesis.

Mutational analysis of GLUT1 (SLC2A1) in glut-1 deficiency syndrome; dong wang; pamela kranz-eble; darryl C. De vivo; (Article was originally published in human mutation 16:224-231, 2000)

An error was made in the reproduction of Figure 2. Therefore, the corrected version is being reprinted here.

Evaluation of DHPLC analysis in mutational scanning of Notch3, a gene with a high G-C content.

Notch3 mutations cause CADASIL, an increasingly recognized cause of subcortical ischemic stroke and vascular dementia in human adults. In the absence of any specific diagnostic criteria, CADASIL diagnosis is based on mutational scanning of Notch3, which is a large gene composed of 33 exons with a high G-C content. In this study we examined the sensitivity of denaturing high performance liquid chromatography (DHPLC). First we established the theoretical optimal parameters, then we examined a large collection of amplicons in which we had previously identified distinct pathogenic mutations or polymorphisms. We further performed Notch3 mutational scanning in five patients suspected of CADASIL diagnosis in which previous scanning, including SSCP and heteroduplexes analysis, failed to detect any pathogenic mutation. DHPLC resolved 97% of mutations previously detected by sequencing and allowed identification of two novel pathogenic mutations: R607C and F984C. These data indicate that DHPLC is a sensitive screening method particularly suitable for epidemio-genetic screening of CADASIL.

[CADASIL: genetics and physiopathology]. / CADASIL: génétique et physiopathologie.

CADASIL, an autosomal dominant adult onset arteriopathy causing stroke and dementia in humans, is underlaid by a non atherosclerotic non amyloid angiopathy involving mainly the media of small cerebral arteries; it is characterized by major lesions of vascular smooth muscle cells. Using a positional cloning approach, we mapped CADASIL locus on chromosome 19 and identified the mutated gene as being Notch3. This gene, previously unknown in humans, encodes for a large transmembrane receptor belonging to the Notch/lin12 gene family which are known to be involved in cell fate specification during development. Genetic analysis of more than 120 CADASIL unrelated families allowed us to show that these mutations are highly stereotyped and affect only the extra cellular domain of the protein. On the basis of these data, a molecular diagnostic test has been set up and is now widely required by clinicians involved in the diagnosis of vascular leukoencephalopathies. Using this test, we recently showed that CADASIL can also occur in patients who do not have any affected relative due to the existence of notch3 de novo mutations. As a first step to investigate the molecular and cellular mechanisms leading from Notch3 mutations to CADASIL phenotype, we analyzed by in-situ hybridization and immunohistochemistry the pattern of expression of this gene. Notch3 expression is highly restricted to the vascular smooth muscle cell in normal human adults. In CADASIL tissues there is a dramatic accumulation of the extracellular domain of the protein which suggests that one of the main mechanisms of CADASIL involves anomalies in the proteolytical cleavage and clearance of this protein. These data provide important clues to the mechanisms of this condition and current work should lead in the next future to a complete understanding of CADASIL and set up the basis of a rational therapeutical approach of this condition.

Genetic heterogeneity and absence of founder effect in a series of 36 French cerebral cavernous angiomas families.

Cerebral cavernous angiomas malformations (CCM) can be inherited as an autosomal dominant condition. CCM1, a yet unidentified gene mapping on 7q21-q22, was shown to be involved in all CCM Hispano-American families, with a strong founder effect. Genetic heterogeneity in non Hispano-American families was established in two families. We conducted a genetic linkage analysis on 36 French CCM families using eight microsatellite markers mapping within the CCM1 interval. Admixture analysis showed that 65% of these families were linked to the CCM1 locus. Haplotypes analysis of CCM1-linked families did not show any evidence for a strong founder effect.

Strong clustering and stereotyped nature of Notch3 mutations in CADASIL patients.

BACKGROUND: CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy) is commonly overlooked or misdiagnosed owing to its recent identification and its variable mode of presentation. The defective gene in CADASIL is Notch3, which encodes a large transmembrane receptor. To set up a diagnostic test and to delineate the Notch3 domains involved in CADASIL., we undertook mutations analysis in this gene in a group of CADASIL patients. METHODS: 50 unrelated patients with CADASIL and 100 healthy controls were screened for mutations along the entire Notch3 sequence, by means of single-strand conformation polymorphism, heteroduplex, and sequence analysis. FINDINGS: Strongly stereotyped mis-sense mutations, located within the epidermal-growth-factor-like (EGF-like) repeats, in the extracellular domain of Notch3, were detected in 45 patients. Clustering of mutations within the two exons encoding the first five EGF-like repeats was observed (32 patients). All these mutations lead to loss or gain of a cysteine residue and therefore to an unpaired number of cysteine residues within a given EGF domain. None of these mutations was found in the 100 controls. INTERPRETATION: Because of the strong clustering and highly stereotyped nature of the pathogenetic mutations detected in CADASIL patients, and easy and reliable diagnostic test for CADASIL is feasible. The findings suggest that aberrant dimerisation of Notch3, due to abnormal disulphide bridging with another Notch3 molecule or with another protein, may be involved in the pathogenesis of this disorder.

Notch3 mutations in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a mendelian condition causing stroke and vascular dementia.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited condition whose key features include recurrent subcortical ischemic events, migraine attacks and vascular dementia in association with diffuse white-matter abnormalities seen on neuroimaging. Pathologic examination shows multiple small deep cerebral infarcts, a leukoencephalopathy and a nonatherosclerotic nonamyloid angiopathy involving mainly the media of small cerebral arteries. To progress in understanding the pathophysiological mechanisms of this condition, we undertook the identification of the mutated gene. We mapped the CADASIL gene on chromosome 19p13.1. More than 120 families have been referred to our lab. Genetic linkage analysis of 33 of these families allowed us to reduce the size of the genetic interval to less than 1 cM and to demonstrate the genetic homogeneity of this condition. In the absence of any candidate gene, we undertook positional cloning of this gene. We identified, within the CADASIL critical region, the human Notch3 gene, whose sequence analysis revealed deleterious mutations in CADASIL families co-segregating with the affected phenotype. These data establish that this gene causes CADASIL. Identification of the CADASIL gene will provide a valuable diagnostic tool for clinicians and could be used to estimate the prevalence of this underdiagnosed condition. It should help in the understanding of pathophysiological mechanisms of CADASIL and vascular dementia.

Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia.

Stroke is the third leading cause of death, and vascular dementia the second cause of dementia after Alzheimer's disease. CADASIL (for cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) causes a type of stroke and dementia whose key features include recurrent subcortical ischaemic events and vascular dementia and which is associated with diffuse white-matter abnormalities on neuroimaging. Pathological examination reveals multiple small, deep cerebral infarcts, a leukoencephalopathy, and a non-atherosclerotic, non-amyloid angiopathy involving mainly the small cerebral arteries. Severe alterations of vascular smooth-muscle cells are evident on ultrastructural analysis. We have previously mapped the mutant gene to chromosome 19. Here we report the characterization of the human Notch3 gene which we mapped to the CADASIL critical region. We have identified mutations in CADASIL patients that cause serious disruption of this gene, indicating that Notch3 could be the defective protein in CADASIL patients.

[Familial hemiplegic migraine. Localization of a responsible gene on chromosome 19]. / Migraine hémiplégique familiale. Localisation d'un gène responsable sur le chromosome 19.

Familial hemiplegic migraine is an autosomal dominant disorder of unknown pathogenesis in which the migrainous attacks are marked by the occurrence of a transient hemiplegia during the aura. The aim of our study was the identification of the affected gene. The first step was the chromosomal mapping of the affected gene, for which we used a "candidate gene" strategy. The first candidate gene was the gene responsible for CADASIL. While investigating CADASIL, mapped previously to chromosome 19, we observed that some patients had recurrent attacks of migraine with aura. Although the clinical and neuroimaging features of familial hemiplegic migraine differ markedly from CADASIL, we hypothesized that the same gene could be involved in the pathogenesis of both conditions. We chose two large pedigrees for linkage analysis of familial hemiplegic migraine. A maximum lodsore > 8 was found with two markers that are strongly linked to CADASIL. Multilocus linkage analysis located the affected gene within an interval of about 30 cM on chromosome 19, containing the gene responsible for CADASIL. At this step it's not possible to conclude that CADASIL and familial hemiplegic migraine are due to the same mutated gene. It will be necessary to analyse other familial hemiplegic migraine and CADASIL families in order to reduce the size of their respective interval and ultimately identify the mutated gene(s).

A gene for familial hemiplegic migraine maps to chromosome 19.

Familial hemiplegic migraine is an autosomal dominant disorder of unknown pathogenesis in which the migrainous attacks are marked by the occurrence of a transient hemiplegia during the aura. While investigating CADASIL, mapped previously to chromosome 19, we observed that some patients had recurrent attacks of migraine with aura. Although the clinical and neuroimaging features of familial hemiplegic migraine differ markedly from CADASIL, we hypothesized that the same gene could be involved in the pathogenesis of both conditions. We chose two large pedigrees for linkage analysis of familial hemiplegic migraine. A maximum lod score > 8 was found with two markers that are also strongly linked to CADASIL. Multilocus linkage analysis suggested that the loci responsible for the two diseases reside within an interval of about 30 cM on chromosome 19.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy maps to chromosome 19q12.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) has been recently reported as a cause of stroke. It is characterized, in the absence of hypertension, by recurrent subcortical ischaemic strokes, starting in early or midadulthood and leading in some patients to dementia. Magnetic resonance imaging and pathological examination show numerous small subcortical infarcts and a diffuse leukoencephalopathy underlaid by a non-arteriosclerotic, non-amyloid angiopathy. We performed genetic linkage analysis in two unrelated families and assigned the disease locus to chromosome 19q12. Multilocus analysis with the location scores method established the best estimate for the location of the affected gene within a 14 centimorgan interval bracketed by D19S221 and D19S222 loci.

T cell response to myelin basic protein epitopes in multiple sclerosis patients and healthy subjects.

T cell lines and clones specific for human myelin basic protein (BP) were selected from three multiple sclerosis (MS) patients and two healthy subjects and tested for their proliferative responses to a battery of synthetic peptides, 9 to 21 amino acid residues long. The combined amino acid sequence of the peptides spanned the complete sequence of the human BP. The results suggest the development of T cells sensitized to at least four independent regions of the human BP, indicating some diversity of the human T cell repertoire to BP. However, an immunodominant T cell epitope was located in the C-terminal region, defined by residues 149-162. This epitope was recognized by T cells from three subjects out of five (one MS patient and both healthy controls) in the context of different DR specificities. Another epitope (located in the 57-75 region) which triggered one MS patient's T cell response was also recognized by a mycobacteria-specific T cell clone cross-reacting with BP.