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D A Marchuk

Publications and source records attributed to D A Marchuk.

At least 37 records · Page 2Linked to original sources
PubMed1999

Expression analysis of four endoglin missense mutations suggests that haploinsufficiency is the predominant mechanism for hereditary hemorrhagic telangiectasia type 1.

ENDOGLIN codes for a homodimeric membrane glycoprotein that interacts with receptors for members of the TGF-beta superfamily and is the gene mutated in the autosomal dominant vascular disorder hereditary hemorrhagic telangiectasia type 1 (HHT1). We recently demonstrated that functional endoglin was expressed at half levels on human umbilical vein endothelial cells (HUVECs) and peripheral blood activated monocytes from HHT1 patients. Two types of mutant protein were previously analyzed, the product of an exon 3 skip which was expressed as a transient intracellular species and prematurely truncated proteins that were undetectable in patient samples. Here we report the analysis of four proteins resulting from point mutations, with missense codons G52V and C53R in exon 2, W149C in exon 4 and L221P in exon 5. Metabolic labeling of activated monocytes from confirmed, clinically affected patients revealed reduced expression of fully processed normal endoglin in all cases. Pulse-chase analysis with HUVECs from a newborn with the C53R substitution indicated that mutant endoglin remained intracellular as a precursor form and did not impair processing of the normal protein. Biotinylation of cell surface proteins, metabolic labeling and pulse-chase analysis revealed that none of the engineered missense mutants was significantly expressed at the surface of COS-1 transfectants. Thus, these four HHT1 missense mutations lead to transient intracellular species which cannot interfere with normal endoglin function. These data suggest that haploinsufficiency, leading to reduced levels of one of the major surface glyco-proteins of vascular endothelium, is the predominant mechanism underlying the HHT1 phenotype.

Animals↗
PubMed1999

Mutations in the gene encoding KRIT1, a Krev-1/rap1a binding protein, cause cerebral cavernous malformations (CCM1).

Cerebral cavernous malformations (CCM) are congenital vascular anomalies of the brain that can cause significant neurological disabilities, including intractable seizures and hemorrhagic stroke. One locus for autosomal dominant CCM ( CCM1 ) maps to chromosome 7q21-q22. Recombination events in linked family members define a critical region of approximately 2 Mb and a shared disease haplotype associated with a presumed founder effect in families of Mexican-American descent points to a potentially smaller region of interest. Using a genomic sequence-based positional cloning strategy, we have identified KRIT1, encoding a protein that interacts with the Krev-1/rap1a tumor suppressor, as the CCM1 gene. Seven different KRIT1 mutations have been identified in 23 distinct CCM1 families. The identical mutation is present in 16 of 21 Mexican-American families analyzed, substantiating a founder effect in this population. Other Mexican-American and non-Hispanic Caucasian CCM1 kindreds harbor other KRIT1 mutations. Identification of a common Mexican-American mutation has potential clinical significance for presymptomatic diagnosis of CCM in this population. In addition, these data point to a key role for the Krev-1/rap1a signaling pathway in angiogenesis and cerebrovascular disease.

Blood Vessels↗
PubMed1999

Allelic and locus heterogeneity in inherited venous malformations.

Venous malformations are low-flow vascular lesions consisting of disorganized thin-walled vascular channels. These can occur sporadically but also as an autosomal dominant condition termed venous malformations, cutaneous and mucosal (VMCM; OMIM 600195). In two large unrelated kindreds mapping to chromosome 9, the identical R849W missense mutation was identified in the first kinase domain of Tie2, an endothelial cell-specific receptor tyrosine kinase. We report here the identification of four new kindreds with inherited venous malformations. Unlike the initial two families described, these four families demonstrate allelic and locus heterogeneity. In one of these families, the R849W mutation co-segregates with the disease phenotype. Three other families with venous malformations lack this mutation. One of these families is linked to markers near TIE2 on chromosome 9. In this family, we identified a novel mutation within the first kinase domain of Tie2 resulting in a Y897S change. Results from COS-1 cell transfections using expression constructs containing either the R849W or the Y897S mutation suggest that the receptors containing either mutation show ligand-independent hyperphosphorylation. These results suggest a gain-of-function mechanism for development of venous malformations in these families. Of the two remaining families, one excludes linkage to the TIE2 locus, establishing the existence of at least one additional locus for dominantly inherited venous malformations.

Amino Acid Sequence↗
PubMed1998

Cloning of the promoter region of human endoglin, the target gene for hereditary hemorrhagic telangiectasia type 1.

Endoglin (CD105) is a cell surface component of the transforming growth factor-beta (TGF-beta) receptor complex highly expressed by endothelial cells. Mutations in the endoglin gene are responsible for the hereditary hemorrhagic telangiectasia type 1 (HHT1), also known as Osler-Weber-Rendu syndrome (OMIM 187300). This is an autosomal dominant vascular disorder probably caused by a haploinsufficiency mechanism displaying low levels of the normal protein. To understand the mechanisms underlying the regulated expression of endoglin, a genomic DNA clone containing 3.3 kb of the 5'-flanking sequence of the human endoglin gene has been isolated. The 5'-flanking region of the endoglin gene lacks consensus TATA and CAAT boxes, but contains two GC-rich regions and consensus motifs for Sp1, ets, GATA, AP-2, NFkappaB, and Mad, as well as TGF-beta-, glucocorticoid-, vitamin D-, and estrogen-responsive elements. As determined by primer extension and 5' RACE experiments, a cluster of transcriptional start sites was found to be located 350 bp upstream from the translation initiation codon. To analyze the endoglin promoter activity, the upstream -400/+341 fragment was fused to the luciferase gene and transient transfections were conducted in several cell types. This construct displayed a tissue-specific activity in human and bovine endothelial cells. Analysis of various deletion constructs showed the existence of a basal promoter region within the -81/+350 fragment as well as major transcriptional regulatory elements within the -400/-141 fragment. Electrophoretic mobility shift assays demonstrated the specific interaction of a member of the ets family with a consensus motif located at position -68. A promoter construct mutated at this ets sequence showed a much reduced activity as compared with the wild-type construct, supporting the involvement of this ets motif in the basal activity of the promoter. The endoglin promoter exhibited inducibility in the presence of TGF-beta1, suggesting possible therapeutic treatments in HHT1 patients, in which the expression level of the normal endoglin allele might not reach the threshold required for its function. Isolation and characterization of the human endoglin promoter represents an initial step in elucidating the controlled expression of the endoglin gene.

Amino Acid Sequence↗
PubMed1998

Familial segregation of hemangiomas and vascular malformations as an autosomal dominant trait.

BACKGROUND: The pathogenesis of infantile hemangiomas is not yet understood. Growth factors and hormonal and mechanical influences have been thought to affect the focal abnormal growth of endothelial cells in these lesions. However, these influences may represent secondary responses to an underlying primary molecular event leading to the development of hemangiomas. OBSERVATIONS: We report the rare familial occurrence of hemangiomas and/or vascular malformations in 6 kindreds, suggesting autosomal dominant inheritance. In these families, multiple generations (2-4) were affected by hemangiomas or vascular malformations. In contrast to the generally accepted female-male ratio of 3:1 to 4:1 associated with sporadic hemangiomas, the families with hemangiomas in our study demonstrated a 2:1 ratio. Additionally, vascular malformations and hemangiomas were present in different members of the same family. The vascular lesions appeared to be transmitted in an autosomal dominant fashion with moderate to high penetrance. CONCLUSIONS: We have identified 6 families demonstrating autosomal dominant segregation of childhood hemangiomas. Additionally, family members with vascular malformations were identified in these kindreds. Physicians caring for children with hemangiomas and vascular malformations should include in their medical histories inquiries about vascular lesions in other family members, even when obvious lesions are not present in the parents. The identification of the mutation(s) underlying vascular lesions will provide insight into the pathogenesis of these familial hemangiomas and, potentially, common sporadic hemangiomas. In addition, such research would shed light on the regulation of angiogenic processes during development.

Arteriovenous Malformations↗
PubMed1998

Mutation and expression analysis of the endoglin gene in hereditary hemorrhagic telangiectasia reveals null alleles.

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disorder characterized by multisystemic vascular dysplasia and recurrent hemorrhage from the sites of vascular lesions. Two genes have been identified for HHT. Endoglin, a TGF-beta binding protein which maps to chromosome 9q3, is the gene for HHT1. The type and location of most of the previously described mutations in the endoglin (ENG) gene suggested a dominant-negative model of receptor-complex dysfunction for the molecular basis of this disorder. In this article we describe 11 novel ENG mutations in HHT kindreds, which include missense and splice-site mutations. Two identical missense mutations in unrelated families disrupt the start codon of the gene. In addition, some frameshift and nonsense mutations lead to very low or undetectable levels of transcript from the mutant allele. These combined data suggest that the nature of most ENG mutations is to create a null (nonfunctional) allele, and that there is no requirement for the synthesis of a truncated endoglin protein in the pathogenesis of HHT.

Alleles↗
PubMed1998

C112R, W323S, N317K mutations in the vasopressin V2 receptor gene in patients with nephrogenic diabetes insipidus. Mutations in brief no. 165. Online.

Nephrogenic diabetes insipidus (NDI) is a rare, mostly X-linked recessive disorder characterized by renal tubular resistance to the antidiuretic effect of arginine vasopressin. The gene responsible for the X-linked NDI, the G-protein-coupled vasopressin V2 receptor, has been localized on the Xq28 region. In this study we present three NDI families from Hungary with three different missense mutations in the vasopressin V2 receptor gene. After the mutations in the affected probands in each family had been characterized, other family members were screened by restriction enzyme analysis. The N317K and W323S mutations have not been detected previously. The C112R is an already known mutation. The N317K was a de novo mutation in the patient. The C112R and the W323S were found in the mothers of the patients as carriers and in all other patients, but not in the unaffected members of the families. Segregation of the mutations was consistent with the clinically observed symptoms as well as their severity. As conclusion, these findings further evidence that X-linked NDI results from defects in the V2 receptor gene.

Diabetes Insipidus, Nephrogenic↗
PubMed1998

Quantitative DNA pooling to increase the efficiency of linkage analysis in autosomal dominant disease.

DNA pooling is an efficient method to rapidly perform genome-wide linkage scans in autosomal recessive diseases in inbred populations where affected individuals are likely to be homozygous for alleles near the disease gene locus. We wanted to examine whether this approach would detect linkage in autosomal dominant (AD) disorders where affected individuals may share one allele identical by descent at loci tightly linked to the disease. Two large outbred pedigrees in which the AD diseases familial venous malformation (FVM) and hereditary hemorrhagic telangiectasia (HHT1), linked to 9p and 9q, respectively, were investigated. Separate pools of DNA from affected (n = 21 for FVM and 17 for HHT1) and unaffected family members (n = 9 FVM and HHT1), and 25 unrelated population controls were established. Polymorphic markers spanning chromosome 9 at approximately 13.5-cM intervals were amplified using standard PCR. Allele quantitation was performed with a fluorimager. Visual inspection of allele intensities and frequency distributions suggested a shift in frequency of the most common allele in the affecteds lane when compared to control lanes for markers within 30 cM of the FVM and HHT1 loci. These subjective assessments were confirmed statistically by testing for the difference between two proportions (one-sided; P < or = 0.05). When using population controls, the true-positive rates for FVM and HHT1 were 5/5 and 2/5 markers, respectively. False-positive rates for FVM and HHT1 were 3/9 and 2/9, respectively. In both AD diseases investigated, quantitative DNA pooling detected shifts in allele frequency, thus identifying areas of known linkage in most cases. The utility of this technique depends on the size of the pedigree, frequency of the disease-associated allele in the population, and the choice of appropriate controls. Although the false-positive rate appears to be high, this approach still serves to reduce the amount of overall genotyping by about 60%. DNA pooling merits further investigation as a potential strategy in increasing the efficiency of genomic linkage scans.

Alleles↗
PubMed1998

Genetic abnormalities in hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT), or Rendu-Osler-Weber disease, is an autosomal dominant disorder of localized angiodysplasia, although it is sometimes mistakenly identified as a hemostatic disorder due to its associated characteristic bleeding. The vascular lesions that develop consist of direct arteriovenous connections without an intervening capillary bed. Germline mutations in one of two different genes, endoglin or ALK-1, can cause HHT. Both are members of the transforming growth factor (TGF)-beta receptor family of proteins, and are expressed primarily on the surface of endothelial cells. They are associated together in a receptor complex on the cell surface. Biochemical studies suggest that endoglin modulates TGF-beta signaling through ALK-1 and the type I TGF-beta receptor. Most mutations identified in endoglin and ALK-1 create null alleles, which lead to reduced message or protein levels. A model of haploinsufficiency is proposed, in which inheritance of a mutation predisposes an individual to develop HHT-associated vascular lesions. The factors that initiate lesion formation are unknown, but disruption of these genes in mice should provide animal models to address these and other important questions about the pathogenesis of HHT.

Activin Receptors↗
PubMed1997

Endoglin gene polymorphism as a risk factor for sporadic intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a common and serious type of stroke. Recent studies have shown that inherited factors that affect the development of the vessel wall can increase the risk of ICH. We studied endoglin as a candidate gene in patients with sporadic ICH, since mutations in this gene can cause telangiectasia formation. One hundred three patients with sporadic ICH and 202 controls were studied. The polymerase chain reaction and single-strand conformational polymorphism analysis were used to screen for mutations in exon 7 of the endoglin gene. No coding mutations in exon 7 were identified in the ICH patients or controls. A 6-base intronic insertion was found 26 bases beyond the 3' end of exon 7. The homozygous form of the insertion was present in 9 of 103 (8.7%) ICH patients compared with 4 of 202 (2.0%) controls, p = 0.012 (odds ratio 4.8 [95% confidence interval, 1.28, 21.60]). Analysis of the endoglin transcript around the insertion did not reveal any changes in the RNA sequence. There were no obvious clinical features that distinguished the ICH patients with the homozygous insertion from the other patients. The pathophysiologic mechanism underlying this association remains to be determined.

Adult↗
PubMed1997

The activin receptor-like kinase 1 gene: genomic structure and mutations in hereditary hemorrhagic telangiectasia type 2.

The activin receptor-like kinase 1 gene (ALK-1) is the second locus for the autosomal dominant vascular disease hereditary hemorrhagic telangiectasia (HHT). In this paper we present the genomic structure of the ALK-1 gene, a type I serine-threonine kinase receptor expressed predominantly in endothelial cells. The coding region is contained within nine exons, spanning < 15 kb of genomic DNA. All introns follow the GT-AG rule, except for intron 6, which has a TAG/gcaag 5' splice junction. The positions of introns in the intracellular domain are almost identical to those of the mouse serine-threonine kinase receptor TSK-7L. By sequencing ALK-1 from genomic DNA, mutations were found in six of six families with HHT either shown to link to chromosome 12q13 or in which linkage of HHT to chromosome 9q33 had been excluded. Mutations were also found in three of six patients from families in which available linkage data were insufficient to allow certainty with regard to the locus involved. The high rate of detection of mutations by genomic sequencing of ALK-1 suggests that this will be a useful diagnostic test for HHT2, particularly where preliminary linkage to chromosome 12q13 can be established. In two cases in which premature termination codons were found in genomic DNA, the mutant mRNA was either not present or present at barely detectable levels. These data suggest that mutations in ALK-1 are functionally null alleles.

Activin Receptors↗
PubMed1996

Vascular dysmorphogenesis caused by an activating mutation in the receptor tyrosine kinase TIE2.

Venous malformations (VMs), the most common errors of vascular morphogenesis in humans, are composed of dilated, serpiginous channels. The walls of the channels have a variable thickness of smooth muscle; some mural regions lack smooth muscle altogether. A missense mutation resulting in an arginine-to-tryptophan substitution at position 849 in the kinase domain of the receptor tyrosine kinase TIE2 segregates with dominantly inherited VM in two unrelated families. Using proteins expressed in insect cells, we demonstrate that the mutation results in increased activity of TIE2. We conclude that an activating mutation in TIE2 causes inherited VMs in the two families and that the TIE2 signaling pathway is critical for endothelial cell-smooth muscle cell communication in venous morphogenesis.

Amino Acid Sequence↗
PubMed1996

Chromosome breakpoint at 17q11.2 and insertion of DNA from three different chromosomes in a glioblastoma with exceptional glial fibrillary acidic protein expression.

A glioblastoma that retained glial fibrillary acidic protein (GFAP) in culture has a break in the long arm of chromosome 17 at band 17q11.2. DNA inserted at this breakpoint came from chromosome bands 3p21, 3q23, 16q11.2, and 22q11.2. These chromosome fragments were inserted in band 17q11.2 proximal to the neurofibromatosis-1 (NF-1) gene and neu (HER2; erbB2) oncogene loci. The glioblastoma also contained a reciprocal translocation between 16p12 and 20p12. These structural abnormalities, previously undescribed in gliomas, were demonstrated by high-resolution chromosome banding, microdissection, and fluorescence in situ hybridization (FISH). Numerical changes typical of glioblastoma were present: gain of chromosome 7 and losses of chromosomes 10, 13, and 22. The complex chromosome origin of DNA inserted in this glioma chromosome is described. The association of two infrequent events in this single glioblastoma line, this complex insertion and retention of GFAP expression, is not likely to be a chance occurrence. It raises the possibility of an association between the two events.

Cells, Cultured↗
PubMed1996

Mutations in the activin receptor-like kinase 1 gene in hereditary haemorrhagic telangiectasia type 2.

Hereditary haemorrhagic telangiectasia, or Osler-Rendu-Weber (ORW) syndrome, is an autosomal dominant vascular dysplasia. So far, two loci have been demonstrated for ORW. Linkage studies established an ORW locus at chromosome 9q3; endoglin was subsequently identified as the ORW1 gene. A second locus, designated ORW2, was mapped to chromosome 12. Here we report a new 4 cM interval for ORW2 that does not overlap with any previously defined. A 1.38-Mb YAC contig spans the entire interval. It includes the activin receptor like kinase 1 gene (ACVRLK1 or ALK1), a member of the serine-threonine kinase receptor family expressed in endothelium. We report three mutations in the coding sequence of the ALK1 gene in those families which show linkage of the ORW phenotype to chromosome 12. Our data suggest a critical role for ALK1 in the control of blood vessel development or repair.

Activin Receptors↗
PubMed1996

Clinical heterogeneity in hereditary haemorrhagic telangiectasia: are pulmonary arteriovenous malformations more common in families linked to endoglin?

Pulmonary arteriovenous malformations (PAVMs) occur in up to 27% of patients with hereditary haemorrhagic telangiectasia (HHT) and are associated with a rate of paradoxical cerebral embolism at presentation of up to 36%. At least two different loci have been shown for HHT. Mutations in endoglin have been found in some families and the locus designated ORW1. In other families this locus has been excluded. In this paper we confirm that in families linked to ORW1 there is a prevalence of PAVMs among affected members of 29.2%, compared to a prevalence of 2.9% in families in which this locus has been excluded (chi 2 = 19.2, p < 0.001). This information can be used to decide how to screen HHT patients for PAVMs.

Antigens, CD↗
PubMed1996

Identification and mapping of type 1 neurofibromatosis (NF1) homologous loci.

During the establishment of a YAC contig for the type 1 neurofibromatosis (NF1) region on human chromosome 17q11.2, several YAC clones were isolated which originated from a different chromosome but which retained strong homology to NF1 coding regions (Marchuk et al., 1992). Fluorescence in situ hybridization (FISH) using these clones has identified NF1-homologous loci on several human chromosomes, including 2, 14, 15, 21, and 22. PCR amplification using primers originally designed to amplify NF1 exons has confirmed these chromosome localizations and, in addition, has revealed NF1-homologous sequences on chromosomes 12 and 20. Sequence analysis of the amplified products has demonstrated that (1) most of these loci have > 90% identity with NF1 sequences; (2) most of these loci represent nonprocessed pseudogenes; and (3) for the chromosome 12 locus, the two described regions of homology with NF1 have open reading frames. Finally, we have identified two novel alpha-satellite DNA repeat units in proximity to NF1-homologous sequences on chromosome 14. Their association suggests a mechanism for dispersion of the NF1-homologous loci based on alpha-satellite-mediated exchange between nonhomologous chromosomes.

Base Sequence↗