epidermal-growth-factor and Marfan-Syndrome

Fibrillin-1 is a large extracellular matrix glycoprotein which assembles to form 10-12 nm microfibrils in extracellular matrix. Mutations in the human fibrillin-1 gene (FBN-1) cause the connective tissue disease Marfan syndrome and related disorders, which are characterised by defects in the skeletal, cardiovascular and ocular systems of the body. Fibrillin-1 has a striking modular organisation which is dominated by multiple tandem repeats of the calcium binding epidermal growth factor-like (cbEGF) domain. This review focuses on recent studies which have investigated the structural and functional role of calcium binding to cbEGF domains in fibrillin-1 and 10-12 nm microfibrils.

Topics: Animals; Calcium; Calcium Chloride; Calcium-Binding Proteins; Egtazic Acid; Epidermal Growth Factor; Extracellular Matrix Proteins; Fibrillin-1; Fibrillins; Fibroblasts; Humans; Marfan Syndrome; Microfibrils; Microfilament Proteins; Mutation; Tandem Repeat Sequences

Topics: Amino Acid Sequence; Base Sequence; Chromosome Mapping; Chromosomes, Human, Pair 15; Cysteine; DNA Primers; Epidermal Growth Factor; Fibrillins; Genetic Variation; Humans; Marfan Syndrome; Microfilament Proteins; Molecular Sequence Data; Polymerase Chain Reaction

Marfan syndrome (MFS) is a highly variable genetic connective tissue disorder caused by mutations in the calcium binding extracellular matrix glycoprotein fibrillin-1. Patients with the most severe form of MFS (neonatal MFS; nMFS) tend to have mutations that cluster in an internal region of fibrillin-1 called the neonatal region. This region is predominantly composed of eight calcium-binding epidermal growth factor-like (cbEGF) domains, each of which binds one calcium ion and is stabilized by three highly conserved disulfide bonds. Crucially, calcium plays a fundamental role in stabilizing cbEGF domains. Perturbed calcium binding caused by cbEGF domain mutations is thus thought to be a central driver of MFS pathophysiology. Using steered molecular dynamics (SMD) simulations, we demonstrate that cbEGF domain calcium binding decreases under mechanical stress (i.e. cbEGF domains are mechanosensitive). We further demonstrate the disulfide bonds in cbEGF domains uniquely orchestrate protein unfolding by showing that MFS disulfide bond mutations markedly disrupt normal mechanosensitive calcium binding dynamics. These results point to a potential mechanosensitive mechanism for fibrillin-1 in regulating extracellular transforming growth factor beta (TGFB) bioavailability and microfibril integrity. Such mechanosensitive "smart" features may represent novel mechanisms for mechanical hemostasis regulation in extracellular matrix that are pathologically activated in MFS.

Topics: Biological Availability; Calcium; Disulfides; Epidermal Growth Factor; Extracellular Matrix; Fibrillin-1; Humans; Infant, Newborn; Marfan Syndrome; Mechanotransduction, Cellular; Microfibrils; Molecular Dynamics Simulation; Mutation; Protein Binding; Protein Domains; Transforming Growth Factor beta

In six index cases/families referred for Marfan syndrome (MFS) molecular diagnosis, we identified six novel mutations in the FBN1 gene: c.1753G>C (p.Gly585Arg), c.2456G>A (p.Gly819Glu), c.4981G>A (p.Gly1661Arg), c.5339G>A (p.Gly1780Glu), c.6418G>A (p.Gly2140Arg) and c.6419G>A (p.Gly2140Glu). These variants, predicted to result in Glycine substitutions are located at the third position of a 4 amino acids loop-region of calcium-binding Epidermal Growth Factor-like (cb-EGF) fibrillin-1 domains 5, 9, 24, 25 and 32. Familial segregation studies showing cosegregation with MFS manifestations or de novo inheritance in addition to in silico analyses (conservation, 3D modeling) suggest evidence for a crucial role of the respective Glycine positions. Extending these analyses to all Glycine residue at position 3 of this 4 residues loop in fibrillin-1 cb-EGF with the UMD predictor tool and alignment of 2038 available related sequences strongly support a steric strain that only allows Glycine or even Alanine residues for domain structure maintenance and for the fibrillin functions. Our data compared with those of the literature strongly suggest the existence of a cb-EGF domain subtype with implications for related diseases.

Topics: Adolescent; Adult; Aged; Calcium; Child; Epidermal Growth Factor; Female; Fibrillin-1; Fibrillins; Glycine; Humans; Male; Marfan Syndrome; Microfilament Proteins; Middle Aged; Models, Molecular; Mutation, Missense; Pedigree; Sequence Analysis, DNA; Young Adult

Human fibrillin-1 is an extra-cellular matrix glycoprotein with a modular organisation that includes 43 calcium-binding epidermal growth factor-like (cbEGF) domains arranged as multiple tandem repeats interspersed with transforming growth factor beta binding protein-like (TB) domains. We have studied Marfan syndrome-causing mutations which affect calcium binding to cbEGF13, and demonstrate that in human fibroblast cells they cause unexpected endoplasmic reticulum retention, indicative of a folding defect. Biochemical and biophysical studies of in vitro refolded fragments from the TB3-cbEGF14 region indicate long-range and unidirectional effects of these substitutions on the adjacent N-terminal domain cbEGF12. In contrast, only short-range effects of a pathogenic mutation affecting calcium binding to cbEGF19 are observed, and secretion of this mutant protein occurs. Further NMR studies on wild-type cbEGF12-13 and cbEGF12-14 identify a co-operative dependence of domain folding where calcium binding to cbEGF13 is required before cbEGF12 can adopt a native Ca(2+)-dependent fold. These data demonstrate that during biosynthesis of fibrillin-1, multiple tandem repeats of cbEGF domains may not necessarily fold independently and therefore missense mutations resulting in identical substitutions may have different effects on the fate of the mutant protein. Complex folding of modular proteins should therefore be considered when interpreting the molecular pathology of single-gene disorders.

Topics: Amino Acid Sequence; Amino Acid Substitution; Binding Sites; Calcium; Cells, Cultured; Endoplasmic Reticulum; Epidermal Growth Factor; Fibrillin-1; Fibrillins; Humans; Marfan Syndrome; Microfilament Proteins; Models, Biological; Molecular Sequence Data; Mutant Proteins; Mutation; Protein Binding; Protein Folding; Protein Processing, Post-Translational; Protein Structure, Tertiary

Mutations in the genes encoding fibrillin-1 (FBN1) and transforming growth factor beta receptor type II (TGFBR2) are known causes of Marfan syndrome (MFS) and related disorders. However, a sound correlation between the genotype and the cardiovascular phenotype has not yet been established. The objective of the present study was to identify novel mutations in FBN1 and TGFBR2 and to assess whether the type of mutation is linked to a particular clinical subtype of the cardiovascular condition.. The clinical records of 36 patients referred to us for molecular genetic diagnosis were reviewed to assess the course and severity of the vascular deterioration. A semiautomatic protocol was established enabling a rapid and cost-effective screening of the genes FBN1 and TGFBR2 by direct sequencing of all coding exons and flanking intronic regions.. Novel mutations in FBN1 and TGFBR2 were detected in 12 and 2 patients, respectively. Four individuals carried a recurrent mutation in FBN1. Throughout the study cohort, the incidence of aortic dissections per se did not depend on the type of mutation. However, we found that mutations affecting the calcium-binding epidermal growth factor-like domain were more frequently associated with a dissection of distal parts of the aorta than mutations that lead to a premature termination codon (chi(1)(2): p=0.013), suggesting that the spatio-temporal pattern of vascular deterioration may vary with the type of mutation.. Detecting a mutation in the genes FBN1 and TGFBR2 proves the genetic origin of vascular findings and allows the identification of family members at risk who should undergo preventive checkups. Routine genetic testing of patients with suspected MFS or thoracic aortic aneurysms/dissections could provide further insight into genotype/phenotype correlations related to aortic dissection.

Topics: Adolescent; Adult; Aorta; Aortic Aneurysm; Aortic Dissection; Calcium-Binding Proteins; Cohort Studies; Epidermal Growth Factor; Female; Fibrillin-1; Fibrillins; Genotype; Heart Valves; Humans; Male; Marfan Syndrome; Microfilament Proteins; Middle Aged; Mutation; Phenotype; Pilot Projects; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta

Mutations in the fibrillin-1 (FBN1) gene cause Marfan syndrome (MFS) and have been associated with a wide range of overlapping phenotypes. Clinical care is complicated by variable age at onset and the wide range of severity of aortic features. The factors that modulate phenotypical severity, both among and within families, remain to be determined. The availability of international FBN1 mutation Universal Mutation Database (UMD-FBN1) has allowed us to perform the largest collaborative study ever reported, to investigate the correlation between the FBN1 genotype and the nature and severity of the clinical phenotype. A range of qualitative and quantitative clinical parameters (skeletal, cardiovascular, ophthalmologic, skin, pulmonary, and dural) was compared for different classes of mutation (types and locations) in 1,013 probands with a pathogenic FBN1 mutation. A higher probability of ectopia lentis was found for patients with a missense mutation substituting or producing a cysteine, when compared with other missense mutations. Patients with an FBN1 premature termination codon had a more severe skeletal and skin phenotype than did patients with an inframe mutation. Mutations in exons 24-32 were associated with a more severe and complete phenotype, including younger age at diagnosis of type I fibrillinopathy and higher probability of developing ectopia lentis, ascending aortic dilatation, aortic surgery, mitral valve abnormalities, scoliosis, and shorter survival; the majority of these results were replicated even when cases of neonatal MFS were excluded. These correlations, found between different mutation types and clinical manifestations, might be explained by different underlying genetic mechanisms (dominant negative versus haploinsufficiency) and by consideration of the two main physiological functions of fibrillin-1 (structural versus mediator of TGF beta signalling). Exon 24-32 mutations define a high-risk group for cardiac manifestations associated with severe prognosis at all ages.

Topics: Adolescent; Adult; Epidermal Growth Factor; Exons; Female; Fibrillin-1; Fibrillins; Humans; Male; Marfan Syndrome; Microfilament Proteins; Mutation; Phenotype; Prognosis; Protein Structure, Tertiary; Severity of Illness Index; Transforming Growth Factor beta

Homocystinuria is an inborn error of methionine metabolism that results in raised serum levels of the highly reactive thiol-containing amino acid homocysteine. Homocystinurics often exhibit phenotypic abnormalities that are similar to those found in Marfan syndrome (MFS), a heritable connective tissue disorder that is caused by reduced levels of, or defects in, the cysteine-rich extracellular matrix (ECM) protein fibrillin-1. The phenotypic similarities between homocystinuria and MFS suggest that elevated homocysteine levels may result in an altered function of fibrillin-1. We have used recombinant calcium binding epidermal growth factor-like (cbEGF) domain fragments from fibrillin-1, and an unrelated protein Notch1, to analyse the effects of homocysteine on the native disulphide (cystine) bonds of these domains. We show using analytical reverse phase, high performance liquid chromatography (HPLC), electrospray ionisation mass spectrometry (ESI-MS) and limited proteolysis that homocysteine attacks intramolecular disulphide bonds causing reduction of cystine and domain misfolding, and that the effects of homocysteine are dependent on its concentration. We also identify the importance of calcium binding to cbEGF domains for their stabilisation and protection against homocysteine attack. Collectively, these data suggest that reduction of intramolecular cbEGF domain disulphide bonds by homocysteine and the resulting disruption of this domain fold may contribute to the change in connective tissue function seen in homocystinuria. Furthermore, since we show that the effects of homocysteine are not unique to fibrillin-1, other cbEGF-containing proteins may be implicated in the pathogenic mechanisms underlying homocystinuria.

Topics: Calcium; Cystathionine beta-Synthase; Epidermal Growth Factor; Fibrillin-1; Fibrillins; Homocysteine; Homocystinuria; Humans; In Vitro Techniques; Marfan Syndrome; Microfilament Proteins; Models, Molecular; Oxidation-Reduction; Peptide Fragments; Protein Folding; Protein Structure, Tertiary; Receptor, Notch1; Receptors, Cell Surface; Recombinant Proteins; Spectrometry, Mass, Electrospray Ionization; Transcription Factors; Trypsin

Marfan Syndrome (MFS) is an autosomal dominant disorder caused by mutations in the fibrillin-1 gene (FBN1). Several calves, all sired by a phenotypically normal bull, were found to exhibit the major clinical and pathological characteristics of human MFS (aortic dissection, joint laxity, lens dislocation), and were recognized as potential models of the human disease. In this study, Fbn1 cDNA from affected animals was sequenced and a heterozygous c.3598G > A transition was detected in exon 29, which predicted the substitution of an evolutionarily conserved glutamic acid by lysine at position 1200 (p.E1200K). This residue is part of a calcium-binding epidermal growth factor-like (cbEGF-like) module, a domain that is frequently altered in human MFS. Analysis of genomic DNA from the original bull's sperm showed that less than 20% of the sperm harbored the mutation, consistent with the presence of germline mosaicism. This study validates the use of these animals as models of human MFS. These cows will be valuable for investigations into the molecular pathogenesis of MFS, and may lead to better therapeutic testing and evaluation of human Marfan patients.

Topics: Amino Acid Sequence; Animals; Calcium; Cattle; Disease Models, Animal; DNA, Complementary; Epidermal Growth Factor; Fibrillin-1; Fibrillins; Germ-Line Mutation; Humans; Marfan Syndrome; Microfilament Proteins; Molecular Sequence Data; Protein Structure, Tertiary

Homocystinuria, a disorder originating in defects in the methionine metabolism, is characterized by an elevated plasma concentration of homocysteine. Most patients have a defect in the cystathionine-beta-synthase, the key enzyme in the conversion of homocysteine to cysteine. Many abnormalities in the connective tissue of patients with homocystinuria resemble those seen in Marfan syndrome, caused by mutations in fibrillin-1. These observations led to the hypothesis that the structure and function of fibrillin-1 is compromised in patients with homocystinuria. To test this hypothesis we produced recombinant human fibrillin-1 fragments spanning the central portion of the molecule (8-Cys/transforming growth factor-beta binding domain 3 to calcium binding EGF domain 22) and extensively analyzed the potential of homocysteine to modify structural and functional properties of these proteins. Circular dichroism spectroscopy revealed moderate changes of their secondary structures after incubation with homocysteine. Equilibrium dialysis demonstrated a number of high affinity calcium binding sites in the tandemly repeated calcium binding epidermal growth factor-like domains 11-22. Calcium binding of homocysteine-modified fragments was completely abolished. Incubation of the recombinant proteins with homocysteine rendered the analyzed calcium binding EGF domains as well as the 8-Cys/transforming growth factor-beta binding domain 3 significantly more susceptible to proteolytic degradation. Furthermore, data were obtained demonstrating that homocysteine can covalently modify fibrillin-1 via disulfide bonds. These data strongly suggest that structural and functional modifications as well as degradation processes of fibrillin-1 in the connective tissues of patients with homocystinuria play a major role in the pathogenesis of this disorder.

Topics: Amino Acid Sequence; Calcium; Cells, Cultured; Chymotrypsin; Circular Dichroism; Dose-Response Relationship, Drug; Epidermal Growth Factor; Extracellular Matrix; Fibrillin-1; Fibrillins; Fibroblasts; Glycosylation; Homocysteine; Homocystinuria; Humans; Marfan Syndrome; Microfilament Proteins; Microscopy, Fluorescence; Molecular Sequence Data; Peptides; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins; Sequence Homology, Amino Acid; Transfection; Trypsin

The calcium-binding epidermal growth factor-like (cbEGF) module and the transforming growth factor beta-binding protein-like (TB) module are the two major structural motifs found in fibrillin-1, the extracellular matrix (ECM) protein defective in the Marfan syndrome (MFS). An MFS-causing mutation, N2144S, which removes a calcium ligand in cbEGF32, does not detectably affect fibrillin-1 biosynthesis, rate of secretion, processing, or deposition of reducible fibrillin-1 into the ECM. Since the residue at position 2144 is normally engaged in calcium ligation, it is unable to mediate intermolecular interactions. We have shown previously that this mutation does not affect the folding properties of the TB or cbEGF domains in vitro, but does decrease calcium-binding in cbEGF and TB-cbEGF domain constructs. Here, we use NMR spectroscopy to probe the effects of the N2144S mutation on backbone dynamic properties of TB6-cbEGF32. Analysis of the backbone (15)N relaxation data of wild-type TB6-cbEGF32 has revealed a flexible inter-domain linkage. Parallel dynamics analysis of the N2144S mutant has shown increased flexibility in the region joining the two domains as well as in the calcium-binding site at the N terminus of cbEGF32. This research demonstrates that a small change in peptide backbone flexibility, which does not enhance proteolytic susceptibility of the domain pair, is associated with an MFS phenotype. Flexibility of the TB-cbEGF linkage is likely to contribute to the biomechanical properties of fibrillin-rich connective tissue microfibrils, and may play a role in the microfibril assembly process.

Topics: Amino Acid Sequence; Amino Acid Substitution; Binding Sites; Calcium; Carrier Proteins; Diffusion; Epidermal Growth Factor; Fibrillin-1; Fibrillins; Humans; Intracellular Signaling Peptides and Proteins; Latent TGF-beta Binding Proteins; Magnetic Resonance Spectroscopy; Marfan Syndrome; Microfilament Proteins; Models, Molecular; Molecular Sequence Data; Motion; Mutation; Pliability; Protein Structure, Secondary; Protein Structure, Tertiary

Human fibrillin-1, an extracellular matrix glycoprotein, has a modular organization that includes 43 calcium-binding epidermal growth factor-like (cbEGF) domains arranged as multiple tandem repeats. A missense mutation that changes a highly conserved glycine to serine (G1127S) has been identified in cbEGF13, which results in a variant of Marfan syndrome, a connective tissue disease. Previous experiments on isolated cbEGF13 and a cbEGF13-14 pair indicated that the G1127S mutation caused defective folding of cbEGF13 but not cbEGF14. We have used limited proteolysis methods and two-dimensional NMR spectroscopy to identify the structural consequences of this mutation in a covalently linked cbEGF12-13 pair and a cbEGF12-14 triple domain construct. Protease digestion studies of the cbEGF12-13 G1127S mutant pair indicated that both cbEGF12 and 13 retained similar calcium binding properties and thus tertiary structure to the normal domain pair, because all identified cleavage sites showed calcium-dependent protection from proteolysis. However, small changes in the conformation of cbEGF13 G1127S, revealed by the presence of a new protease-sensitive site and comparative two-dimensional NOESY data, suggested that the fold of the mutant domain was not identical to the wild-type, but was native-like. Additional cleavage sites identified in cbEGF12-14 G1127S indicated further subtle changes within the mutant domain but not the flanking domains. We have concluded the following in this study. (i) Covalent linkage of cbEGF12 preserves the native-like fold of cbEGF13 G1127S and (ii) conformational effects introduced by G1127S are localized to cbEGF13. This study demonstrates that missense mutations in fibrillin-1 cbEGF domains can cause short range structural effects in addition to long range effects previously observed with a E1073K mutation in cbEGF12.

Topics: Amino Acid Sequence; Amino Acid Substitution; Binding Sites; Calcium; Cloning, Molecular; Conserved Sequence; Epidermal Growth Factor; Extracellular Matrix Proteins; Fibrillin-1; Fibrillins; Genetic Variation; Glycine; Humans; Marfan Syndrome; Microfilament Proteins; Models, Molecular; Molecular Sequence Data; Mutation, Missense; Nuclear Magnetic Resonance, Biomolecular; Protein Conformation; Protein Structure, Secondary; Recombinant Proteins; Serine

Calcium binding (cb) epidermal growth factor-like (EGF) domains are found in a wide variety of extracellular proteins with diverse functions. In several proteins, including the fibrillins (1 and 2), the low-density lipoprotein receptor, the Notch receptor and related molecules, these domains are organised as multiple tandem repeats. The functional importance of calcium-binding by EGF domains has been underscored by the identification of missense mutations associated with defective calcium-binding, which have been linked to human diseases. Here, we present (15)N backbone relaxation data for a pair of cbEGF domains from fibrillin-1, the defective protein in the Marfan syndrome. The data were best fit using a symmetric top model, confirming the extended conformation of the cbEGF domain pair. Our data demonstrate that calcium plays a key role in stabilising the rigidity of the domain pair on the pico- to millisecond time-scale. Strikingly, the most dynamically stable region of the construct is centred about the domain interface. These results provide important insight into the properties of intact fibrillin-1, the consequences of Marfan syndrome causing mutations, and the ultrastructure of fibrillins and other extracellular matrix proteins.

Topics: Amino Acid Sequence; Animals; Calcium; Cattle; Connective Tissue; Epidermal Growth Factor; Fibrillin-1; Fibrillins; Humans; Magnetic Resonance Spectroscopy; Marfan Syndrome; Microfibrils; Microfilament Proteins; Models, Chemical; Models, Molecular; Molecular Sequence Data; Motion; Pliability; Protein Structure, Tertiary; Time Factors

Most extracellular proteins consist of various modules with distinct functions. Mutations in one common type, the calcium-binding epidermal growth factor-like module (cbEGF), can lead to a variety of genetic disorders. Here, we describe as a model system structural and functional consequences of two typical mutations in cbEGF modules of fibrillin-1 (N548I, E1073K), resulting in the Marfan syndrome. Large (80-120 kDa) wild-type and mutated polypeptides were recombinantly expressed in mammalian cells. Both mutations did not alter synthesis and secretion of the polypeptides into the culture medium. Electron microscopy after rotary shadowing and comparison of circular dichroism spectra exhibited minor structural differences between the wild-type and mutated forms. The mutated polypeptides were significantly more susceptible to proteolytic degradation by a variety of proteases as compared with their wild-type counterparts. Most of the sensitive cleavage sites were mapped close to the mutations, indicating local structural changes within the mutated cbEGF modules. Other cleavage sites, however, were observed at distances beyond the domain containing the mutation, suggesting longer range structural effects within tandemly repeated cbEGF modules. We suggest that proteolytic degradation of mutated fibrillin-1 may play an important role in the pathogenesis of Marfan syndrome and related disorders.

Topics: Amino Acid Sequence; Calcium; Cell Line; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; Fibrillin-1; Fibrillins; Humans; Marfan Syndrome; Microfilament Proteins; Molecular Sequence Data; Mutation; Protein Conformation; Protein Folding; Structure-Activity Relationship

Calcium binding epidermal growth factor-like domains (cbEGFs) are present in many extracellular proteins, including fibrillin-1, Notch-3, protein S, factor IX and the low density lipoprotein (LDL) receptor, which perform a diverse range of functions. Genetic mutations that cause amino acid changes within these proteins have been linked to the Marfan syndrome (MFS), CADASIL, protein S deficiency, haemophilia B and familial hypercholesterolaemia, respectively. A number of these mutations disrupt calcium binding to cbEGFs, emphasising the critical functional role of calcium in these proteins. We have determined the calcium binding affinity of two sites within a cbEGF pair (cbEGF12-13) from human fibrillin-1 using two-dimensional nuclear magnetic resonance (NMR) and fluorescence techniques. Fibrillin-1 is a mosaic protein containing 43 cbEGF domains, mainly arranged as tandem repeats. Our results show that the cbEGF13 site in the cbEGF12-13 pair possesses the highest calcium affinity of any cbEGF investigated from fibrillin-1. A comparative analysis of these and previously reported calcium binding data from fibrillin-1 demonstrate that the affinity of cbEGF13 is enhanced more than 70-fold by the linkage of an N-terminal cbEGF domain. In contrast, comparison of calcium binding by cbEGF32 in isolation relative to when linked to a transforming growth factor beta-binding protein-like domain (TB6-cbEGF32) reveals that the same enhancement is not observed for this heterologous domain pair. Taken together, these results indicate that fibrillin-1 cbEGF Ca2+ affinity can be significantly modulated by the type of domain which is linked to its N terminus. The cbEGF12-13 pair is located within the longest contiguous section of cbEGFs in fibrillin-1, and a number of mutations in this region are associated with the most severe neonatal form of MFS. The affinities of cbEGF domains 13 and 14 in this region are substantially higher than in the C-terminal region of fibrillin-1. This increased affinity may be important for fibrillin assembly into 10-12 nm connective tissue microfibrils and/or may contribute to the biomechanical properties of the microfibrillar network.

Topics: Binding Sites; Calcium; Epidermal Growth Factor; Fibrillin-1; Fibrillins; Humans; Magnetic Resonance Spectroscopy; Marfan Syndrome; Microfilament Proteins; Mutation; Phenotype; Protein Binding; Protein Structure, Secondary; Spectrometry, Fluorescence; Tyrosine

We report 9 new mutations in German patients presenting with classical Marfan syndrome. All mutations occur in exons with calcium-binding (cb) epidermal growth factor-like (EGF) domains. Five mutations are missense involving exons 12, 27, 30, 44, and 52 with the resultant substitution of cysteine by phenylalanine (C504F), cysteine by tyrosine (C1129Y), tyrosine by cysteine (Y1261C), cysteine by serine (C1833S), and cysteine by tyrosine (C2142Y), respectively. The other four mutations are single base deletions in exons 39, 43, 48, and 58, at nucleotide A4826, C5311, T6018, and A7291, respectively, each resulting in frameshift with premature termination. Four mutations were detected in sporadic cases and are likely to be de novo.

Topics: Adult; Calcium-Binding Proteins; Epidermal Growth Factor; Exons; Female; Fibrillin-1; Fibrillins; Frameshift Mutation; Germany; Humans; Male; Marfan Syndrome; Microfilament Proteins; Mutation, Missense

Fibrillin-1 (FBN1) contains 47 epidermal growth factor (EGF)-like domains characterized by six conserved cysteine residues. Cysteine substitutions that disrupt one of the three disulfide bonds are frequent causes of Marfan syndrome (MFS). We identified 19 new substitutions involving cysteine residues in each of the six positions of EGF-like domains. Allele-specific mRNA assays revealed equal abundance of mutant and normal FBN1 transcripts in all 10 individuals studied. Quantitative pulse-chase analysis of fibrillin protein was performed on 25 mutant fibroblast strains with substitutions of 22 different cysteine residues in 18 different EGF-like domains spanning the entire gene. Normal synthesis and stability of mutant fibrillin molecules was seen in 20/25 individuals, 11 of whom showed delayed intracellular processing and/or secretion. In the remaining five cases, the mutant protein was apparently unstable. In four of these five cases, the second or third disulfide bond of EGF-like domains immediately preceding an 8-cysteine or hybrid domain was affected. All but two mutations caused severe reduction of matrix deposition, which was attributed to a dominant-negative effect of mutant molecules. For genotype/phenotype comparisons, clinical data on 25 probands and 19 mutation-positive family members were analyzed. Ocular manifestations were among the most consistent features (ectopia lentis in 86%, myopia in 80%). Nine mutations encoded by exons 26-32 resulted in early-onset classic MFS and, in one case, neonatal-lethal MFS. Mutations outside this region were associated with variable clinical phenotypes, including individuals with fibrillinopathies not meeting diagnostic criteria for MFS.

Topics: Adolescent; Adult; Age of Onset; Aged; Alleles; Amino Acid Substitution; Cells, Cultured; Child; Child, Preschool; Cysteine; Disulfides; DNA Mutational Analysis; Epidermal Growth Factor; Exons; Fibrillin-1; Fibrillins; Fibroblasts; Genes, Lethal; Genotype; Humans; Infant, Newborn; Marfan Syndrome; Microfilament Proteins; Middle Aged; Molecular Sequence Data; Mutation; Phenotype; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

The effects of the removal and replacement of divalent cations on the ultrastructure of 10 to 12 nm fibrillin-1-containing microfibrils have been studied, in order to investigate the conformation of fibrillin-1 calcium-binding epidermal growth factor-like (cbEGF-like) domains within the microfibril. The NMR structure of a covalently linked pair of cbEGF-like domains from fibrillin-1 recently identified a rigid rod-like conformation for the domain pair stabilised by interdomain calcium binding. This suggested that tandem arrays of fibrillin-1 cbEGF-like domains may adopt an extended conformation within a microfibril. If correct, then removal of bound calcium from fibrillin-1 would be expected to increase the flexibility of each cbEGF-like interdomain linkage, resulting in a decrease in the length of the interbead region of the microfibril (and thus a decrease in bead to bead periodicity), a concomitant increase in its diameter, and an overall increase in the flexibility of the microfibril. Our results show that removal of calcium by treatment with EGTA causes a large alteration of the microfibril structure, resulting in microfibrils with a reduced beaded periodicity, a disrupted interbead region and an increased overall flexibility. These effects are readily reversible by the re-addition of calcium (in the form of CaCl2), but not by the addition of magnesium (MgCl2). This is consistent with conformational changes in cbEGF-like domains causing the major structural effects on the microfibril. These results provide the first direct experimental evidence to support an extended rod-like conformation for multiple tandem repeats of fibrillin-1 cbEGF-like domains within the microfibril, as predicted by the NMR structure of an isolated fibrillin-1 cbEGF-like domain pair.

Topics: Actin Cytoskeleton; Binding Sites; Calcium; Cations, Divalent; Cell Line; Chelating Agents; Edetic Acid; Egtazic Acid; Epidermal Growth Factor; Fibrillin-1; Fibrillins; Humans; Magnetic Resonance Spectroscopy; Marfan Syndrome; Microfilament Proteins; Microscopy, Electron; Protein Conformation

Congenital contractural arachnodactyly (CCA) is an autosomal dominant disorder of connective tissue and is characterized by multiple congenital contractures, arachnodactyly, and external ear malformations. Recent investigations indicate that mutations in the fibrillin-2 gene (FBN2) cause CCA. Here, we report a G-->C transversion at nucleotide 3340 (G3340C) of FBN2 in a family with phenotypic characteristics of CCA. The G3340C mutation predicts the substitution of histidine for aspartic acid at amino acid residue 1114 (Asp1114His) and also alters the 5' donor splice site consensus sequence of exon 25. Reverse transcription/polymerase chain reaction and DNA sequence analyses demonstrate that this missense mutation also causes low level in-frame mis-splicing of exon 25 (del exon 25). Consequently, this single point mutation produces a heterogeneous population of mutant fibrillin-2 molecules in a single individual. Despite the complex manifestation of the mutation, it is associated with a relatively mild phenotype. Analysis of FBN2 allele expression in cultured dermal fibroblasts derived from the proband has shown that the mutant allele is preferentially expressed, contributing about 84% of the total transcript. This indicates that an overabundance of mutant transcript does not necessarily correlate with a more severe CCA phenotype.

Topics: Aged; Amino Acid Sequence; Amino Acid Substitution; Aspartic Acid; Epidermal Growth Factor; Exons; Female; Fibrillin-2; Fibrillins; Histidine; Humans; Male; Marfan Syndrome; Microfilament Proteins; Molecular Sequence Data; Point Mutation; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Sequence Alignment; Sequence Deletion; Sequence Homology, Amino Acid; Transcription, Genetic

Congenital contractural arachnodactyly (CCA) is an autosomal dominant disorder that is phenotypically related to the Marfan syndrome. CCA has recently been shown to result from mutations in the FBN2 gene, which encodes an elastin-associated microfibrillar protein called fibrillin-2. Two siblings are reported here with classic manifestations of CCA with unaffected parents. Analysis of the FBN2 cDNA from dermal fibroblasts from one of the affected siblings revealed a heterozygous exon splicing error deleting nt 3722-3844 of the FBN2 mRNA. This cDNA deletion resulted in selective removal of one of the 43 calcium-binding EGF-like domains of the fibrillin-2 protein. Analysis of the FBN2 gene in the affected siblings' DNA indicated that the splicing error resulted from an A-to-G transition 15 nt upstream from the 3' splice site of the intron. The genomic mutation resulting in the splicing error alters a putative branch point sequence important for lariat formation, an intermediate structure of normal splicing. The mutation was detectable in DNA from the father's hair bulbs and buccal cells but not his white blood cell DNA, indicating that the father was a somatic mosaic. Analysis of transcript levels by use of dermal fibroblasts from the proband demonstrated that the FBN2 allele containing the exon deletion was expressed at a higher level than the allele inherited from the mother. These results indicate that FBN2 exon splicing errors are a cause of CCA, furthering the understanding of the molecular basis of this disorder. In addition, the demonstration of gonadal mosaicism in the FBN2 gene is important for accurate genetic counseling of families with sporadic cases of CCA. Finally, the preferential expression of the mutated FBN2 allele in dermal fibroblasts may have implications for understanding the pathogenesis and rarity of CCA.

Topics: Alleles; Calcium-Binding Proteins; Dinucleotide Repeats; Epidermal Growth Factor; Female; Fibrillin-2; Fibrillins; Fibroblasts; Humans; Male; Marfan Syndrome; Microfilament Proteins; Mosaicism; Mutation; Pedigree; Polymerase Chain Reaction; RNA Splicing; RNA, Messenger; Sequence Analysis, DNA; Skin

The anatomical substrate of Marfan's syndrome is a degeneration of elastic fibres and disorganization of the collagen. It is now known that these lesions are due to mutation of genes localised on chromosome 15. The first of them (FBN1) codes for the main constitutive protein of the elastic tissue: fibrillin 1, present mainly in structures which must resist load and stress (aortic adventitia, the suspending ligament of the lens, skin); the second (FBN2) codes for fibrillin 2: responsible for the orientation of the elastin and mainly present in cartilage, the aortic media, the bronchi, and all tissues rich in elastin. Mutations of FBN1 are very common and are associated not only with Marfan's syndrome but also fibrillinopathies: incomplete forms, neonatal forms, ectopic lens, isolated aneurysms of the thoracic aorta. The widespread distribution of fibrillin explains the pleiotropic nature of Marfan's syndrome and its clinical presentation. The variability of interfamilial expression is due to genetic heterogeneity (at least two genes) and alletic differences (different mutations of FBN1 from one family to another), also explaining mild forms due to quantitative reduction in normal fibrillin and severe forms by "negative dominance" where the fibrillin is structurally abnormal because of alteration of the polymerisation mechanism. The biologic diagnosis of fibrillopathy can be made by a protein test analysing fibrillin on a culture of the patient's fibroblast obtained by skin biopsy. At present, molecular diagnosis of the mutation within the FBN1 gene is not feasible as a routine procedure.

Topics: Abnormalities, Multiple; Amino Acid Sequence; Child; Child, Preschool; Chromosomes, Human, Pair 15; Epidermal Growth Factor; Fibrillin-1; Fibrillin-2; Fibrillins; Humans; Infant; Infant, Newborn; Marfan Syndrome; Microfilament Proteins; Molecular Sequence Data; Mutation; Pedigree

The nuclear magnetic resonance structure of a covalently linked pair of calcium-binding (cb) epidermal growth factor-like (EGF) domains from human fibrillin-1, the protein defective in the Marfan syndrome, is described. The two domains are in a rigid, rod-like arrangement, stabilized by interdomain calcium binding and hydrophobic interactions. We propose a model for the arrangement of fibrillin monomers in microfibrils that reconciles structural and antibody binding data, and we describe a set of disease-causing mutations that provide the first clues to the specificity of cbEFG interactions. The residues involved in stabilizing the domain linkage are highly conserved in fibrillin, fibulin, thrombomodulin, and the low density lipoprotein receptor. We propose that the relative orientation of tandem cbEGF domains in these proteins is similar, but that in others, including Notch, pairs adopt a completely different conformation.

Topics: Calcium-Binding Proteins; Connective Tissue Diseases; Epidermal Growth Factor; Humans; Image Processing, Computer-Assisted; Marfan Syndrome; Microfilament Proteins; Molecular Sequence Data; Mutation; Protein Conformation; Protein Structure, Tertiary; Sequence Homology, Amino Acid

Marfan syndrome (MFS), a heritable connective tissue disorder, is caused by mutations in the gene coding for fibrillin-1 (FBN1), an extracellular matrix protein. One of the three major categories of FBN1 mutations involves exon-skipping. To rapidly detect such mutations, we developed a long RT-PCR method. Either three segments covering the entire FBN1 coding sequence or a single 8.9 kb FBN1 coding segment were amplified from reverse-transcribed total fibroblast RNA. Restriction fragment patterns of these RT-PCR products were compared and abnormal fragments were directly sequenced. Six exon-skipping mutations were identified in a panel of 60 MFS probands. All skipped exons encode calcium binding epidermal growth factor (EGF)-like domains and maintain the reading frame. In five probands, exon-skipping was due to point mutations in splice site sequences, and one had a 6 bp deletion in a donor splice site. Pulse-chase analysis of labelled fibrillin protein revealed normal levels of synthesis but significantly reduced matrix deposition. This dominant-negative effect of the mutant monomers is considered in the light of current models of fibrillin assembly. Probands with this type of FBN1 mutation include the most severe forms of MFS, such as neonatally lethal presentations.

Topics: Epidermal Growth Factor; Exons; Fibrillin-1; Fibrillins; Humans; Marfan Syndrome; Microfilament Proteins; Mutation; Polymerase Chain Reaction

Congenital contractural arachnodactyly (CCA) is an autosomal dominant disorder that is phenotypically similar to Marfan syndrome (MFS) and characterized by arachnodactyly, dolichostenomelia, scoliosis, multiple congenital contractures and abnormalities of the external ears. In contrast to MFS, CCA does not affect the aorta or the eyes. Two closely related genes, FBN1 located on chromosome 15q15-21.3 and FBN2 located at 5q23-31, encode large fibrillin proteins found in extracellular matrix structures called microfibrils. The MFS is caused by mutations in FBN1, while CCA has been genetically linked to FBN2 (refs 2, 5, 6). We now describe a pair of FBN2 missense mutations in two CCA patients that cause substitution of distinct cysteine residues in separate epidermal growth-factor-like (EGF) repeats. Our study provides final proof of the association between FBN2 mutations and CCA pathology, thus establishing the role of the fibrillin-2 in extracellular matrix physiology and pathology.

Topics: Base Sequence; Cell Line; Cysteine; DNA Mutational Analysis; Epidermal Growth Factor; Fibrillin-1; Fibrillin-2; Fibrillins; Fibroblasts; Humans; Marfan Syndrome; Microfilament Proteins; Molecular Sequence Data; Point Mutation; Polymorphism, Single-Stranded Conformational; Protein Structure, Tertiary; Repetitive Sequences, Nucleic Acid

Ascending aortic disease, ranging from mild aortic root enlargement to aneurysm and/or dissection, has been identified in 10 individuals of a kindred, none of whom had classical Marfan syndrome (MFS). Single-strand conformation analysis of the entire fibrillin-1 (FBN1) cDNA of an affected family member revealed a G-to-A transition at nucleotide 3379, predicting a Gly1127Ser substitution. The glycine in this position is highly conserved in EGF-like domains of FBN1 and other proteins. This mutation was present in 9 of 10 affected family members and in 1 young unaffected member but was not found in other unaffected members, in 168 chromosomes from normal controls, and in 188 chromosomes from other individuals with MFS or related phenotypes. FBN1 intragenic marker haplotypes ruled out the possibility that the other allele played a significant role in modulating the phenotype in this family. Pulse-chase studies revealed normal fibrillin synthesis but reduced fibrillin deposition into the extracellular matrix in cultured fibroblasts from a Gly1127Ser carrier. We postulate that the Gly1127Ser FBN1 mutation is responsible for reduced matrix deposition. We suggest that mutations such as this one may disrupt EGF-like domain folding less drastically than do substitutions of cysteine or of other amino acids important for calcium-binding that cause classical MFS. The Gly1127Ser mutation, therefore, produces a mild form of autosomal dominantly inherited weakness of elastic tissue, which predisposes to ascending aortic aneurysm and dissection later in life.

Topics: Adult; Aged; Amino Acid Sequence; Aortic Aneurysm; Aortic Dissection; Base Sequence; Epidermal Growth Factor; Female; Fibrillin-1; Fibrillins; Haplotypes; Humans; Male; Marfan Syndrome; Microfilament Proteins; Middle Aged; Models, Molecular; Molecular Sequence Data; Mutation; Netherlands; Nucleic Acid Hybridization; Phenotype; Polymorphism, Genetic; Risk Factors

Topics: Adult; Amino Acid Sequence; Animals; Biglycan; Bone and Bones; Cartilage; Cattle; Cell Adhesion; Chimera; Collagen; Cytokines; Decorin; Embryo, Mammalian; Endothelial Growth Factors; Epidermal Growth Factor; Exostoses, Multiple Hereditary; Extracellular Matrix; Extracellular Matrix Proteins; Fetus; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Heparin; Heparin-binding EGF-like Growth Factor; Humans; Integrins; Intercellular Signaling Peptides and Proteins; Lymphokines; Marfan Syndrome; Membrane Glycoproteins; Mice; Molecular Sequence Data; Neoplasms; Osteogenesis; Proteoglycans; Proto-Oncogene Proteins; Rats; Syndecan-4; Tissue Extracts; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Topics: Alternative Splicing; Amino Acid Sequence; Cells, Cultured; Chromatography, Affinity; Cysteine; Disulfides; Epidermal Growth Factor; Extracellular Matrix Proteins; Fibrillins; Fluorescent Antibody Technique; Humans; Marfan Syndrome; Microfilament Proteins; Microscopy, Electron; Molecular Sequence Data; Mutation; Protein Conformation

The extracellular matrix protein fibrillin-1 is a major component of elastic microfibrils, which are complex assemblies of several proteins and are found in most connective tissues, frequently associated with elastin. Fibrillin-1 contains 43 precursor epidermal growth factor-like (pEGF) domains that have a consensus sequence for calcium binding. The calcium binding potential of a fibrillin-1 pepsin fragment (PF2) was quantitatively analyzed using microvolume equilibrium dialysis. Peptide sequence data and pepsin fragment size determination indicate that PF2 contains seven pEGF domains, each with the calcium binding consensus sequence. Scatchard plot analysis of the calcium binding data shows that PF2 has six to seven high affinity binding sites with a Kd = 250 microM at pH 7.5. There is a second overlapping consensus sequence in the pEGF domains for beta-hydroxylation of a specific Asp/Asn residue. Five partially hydroxylated Asn residues have been identified by protein sequence analysis of fibrillin-1 fragments. This is the first demonstration of this modification in a connective tissue protein. The calcium binding consensus sequence also contains a conserved Ser residue with an apparently novel modification, which causes the Ser residue to behave like an Asp residue during protein sequencing. Marfan syndrome, a heritable disorder of connective tissue, is known to be associated with mutations in the FBN1 gene. Most of these mutations have been found in pEGF domains, frequently substituting Cys for another amino acid, destroying the pEGF motif secondary structure along with its calcium binding potential. Other mutations cause the substitution of single amino acids in the calcium binding consensus sequence, which could affect calcium binding but also the hydroxylation of Asp/Asn residues or the modification of Ser residues.

Topics: Amino Acid Sequence; Calcium; Calcium-Binding Proteins; Carbohydrate Sequence; Consensus Sequence; Dialysis; Epidermal Growth Factor; Extracellular Matrix Proteins; Fibrillin-1; Fibrillins; Glycosylation; Humans; Hydroxylation; Marfan Syndrome; Microfilament Proteins; Models, Molecular; Molecular Sequence Data; Pepsin A; Peptide Fragments; Protein Precursors; Protein Processing, Post-Translational; Protein Structure, Tertiary; Sequence Analysis; Sequence Homology, Amino Acid

Topics: Adult; Base Sequence; DNA Mutational Analysis; Epidermal Growth Factor; Exons; Female; Fibrillin-1; Fibrillins; Genes; Humans; Male; Marfan Syndrome; Microfilament Proteins; Molecular Sequence Data; Point Mutation; Polymorphism, Genetic; Protein Structure, Tertiary

We present here a family with a clinical phenotype resembling Marfan syndrome (MFS), and displaying joint contracture and episodes of knee joint effusions, but lacking the cardiovascular features of the syndrome. The phenotype of this family represents a unique mixture of connective tissue symptoms, some of which are found in classical MFS and some of which are typical of dominant ectopia lentis. Linkage analyses suggested a linkage (LOD score 2.4; theta = 0) between the phenotype of the family and a polymorphic marker in the vicinity of the fibrillin locus on chromosome 15 (FBN1). Furthermore, a novel transition mutation was identified in the FBN1 gene in all the affected members of the family. In contrast to the majority of fibrillin mutations reported so far, this mutation substitutes a cysteine for arginine, producing an extra cysteine in one of the non-calcium-binding EGF-like motifs of the fibrillin polypeptide, most probably disturbing the formation of one of the three disulfide bridges known to be essential for the normal conformation of this motif.

Topics: Adult; Base Sequence; Calcium; Chromosomes, Human, Pair 15; Cysteine; Epidermal Growth Factor; Fibrillin-1; Fibrillins; Genetic Linkage; Humans; Male; Marfan Syndrome; Microfilament Proteins; Molecular Sequence Data; Mutation; Pedigree

Fibrillin is an important structural protein of the extracellular matrix. It is a large cysteine-rich glycoprotein with extensive intrachain disulfide bonds, likely contributed by multiple EGF-like repeats. We have previously published 6.9 kb of FBN1 cDNA sequence. FBN1 cDNA clones that extend the sequence 3089 bp in the 5' direction are described in this report. The deduced primary structure suggests that fibrillin is composed of multiple domains. The most predominant feature is the presence of 43 calcium binding EGF-like repeats. We demonstrate here that fibrillin molecules bind calcium. In addition, three alternatively spliced exons at the 5' end are described. Analysis of 5.8 kb of surrounding genomic sequence revealed a 1.8-kb CpG island spanning the alternatively spliced exons and the next downstream exon. Since FBN1 is the gene responsible for Marfan syndrome, the information presented here will be useful in identifying new mutations and in understanding the function of fibrillin in the pathogenesis of the disease.

Topics: Alternative Splicing; Amino Acid Sequence; Base Sequence; Binding Sites; Blotting, Northern; Calcium; Cloning, Molecular; DNA, Complementary; Epidermal Growth Factor; Exons; Female; Fibrillin-1; Fibrillins; Fibroblasts; Gene Library; Genomic Library; Humans; Marfan Syndrome; Microfilament Proteins; Molecular Sequence Data; Oligodeoxyribonucleotides; Placenta; Pregnancy; Repetitive Sequences, Nucleic Acid; RNA; Sequence Homology, Amino Acid; Skin

The Marfan syndrome, an autosomal dominant connective tissue disorder, is manifested by abnormalities in the cardiovascular, skeletal, and ocular systems. Recently, fibrillin, an elastin-associated microfibrillar glycoprotein, has been linked to the Marfan syndrome, and fibrillin mutations in affected individuals have been documented. In this study, genetic linkage analysis with fibrillin specific markers was used to establish the prenatal diagnosis in an 11-wk-gestation fetus in a four-generation Marfan kindred. At birth, skeletal changes suggestive of the Marfan syndrome were observed. Reverse transcription-PCR amplification of the fibrillin gene mRNA detected a deletion of 123 bp in one allele in affected relatives. This deletion corresponds to an exon encoding an epidermal growth factor-like motif. Examination of genomic DNA showed a G-->C transversion at the +1 consensus donor splice site.

Topics: Adult; Base Sequence; Blotting, Southern; Deoxyribonucleases, Type II Site-Specific; DNA Mutational Analysis; Epidermal Growth Factor; Exons; Female; Fibrillins; Humans; Marfan Syndrome; Microfilament Proteins; Molecular Sequence Data; Pedigree; Point Mutation; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Pregnancy; Prenatal Diagnosis; Repetitive Sequences, Nucleic Acid; RNA Splicing; Sequence Analysis, RNA

Dermal fibroblasts from nine Marfan syndrome patients with missense mutations in the fibrillin-1 gene (FBN1) produced nearly normal amounts of fibrillin as determined by quantitative pulse-chase experiments. However, six of the seven mutations involving substitutions of highly conserved cysteine residues exhibited lower rates of intracellular transport and secretion. This effect is likely due to improper folding, since intracellular fibrillin processing was also affected by the reducing agent dithiothreitol. Normal secretion patterns were seen in three mutations that either change the conformation of EGF-like domains or change consensus amino acids required for Ca(++)-binding. In all nine fibroblasts strains, however, the deposition of fibrillin in the extracellular matrix was reduced to 50% of normal in two and to less than 30% in seven of the nine samples studied. The protein alterations caused by these missense mutations are associated with moderate to severe features of Marfan syndrome and a dominant negative mechanism is suggested to play a major role in their pathogenesis.

Topics: Amino Acid Sequence; Animals; Binding Sites; Cattle; Cells, Cultured; Conserved Sequence; Epidermal Growth Factor; Factor X; Female; Fibrillin-1; Fibrillins; Fibroblasts; Humans; Male; Marfan Syndrome; Microfilament Proteins; Molecular Sequence Data; Phenotype; Point Mutation; Protein Folding; Protein Processing, Post-Translational; Protein Structure, Secondary; Reference Values; Sequence Homology, Amino Acid; Skin

Marfan syndrome results from mutations in an extracellular matrix glycoprotein, fibrillin. Previous studies have characterized approximately 6.9-kb of the estimated 10-kb fibrillin transcript. We have now completed the primary structure of fibrillin, elucidated the exon/intron organization of the gene and derived a physical map of the genetic locus. Pre-fibrillin consists of 2,871 amino acids which, excluding the signal peptide, are arranged into five structurally distinct regions. The largest of these regions comprises about 75% of the entire protein and consists of numerous repeated cysteine-rich sequences homologous to the peptide motifs of the epidermal growth factor (EGF) and transforming growth factor-beta binding protein (TGF-bp). Forty-three of the forty-six EGF-like repeats contain a calcium binding consensus sequence (EGF-CB) conceivably mediating protein-protein interactions. Fibrillin exhibits a few additional cysteine-rich modules that are apparently unique to this macromolecule and may represent evolutionary variants of the EGF-CB and TGF-bp motifs. Almost all of the cysteine-rich repeats are encoded by single exons; consequently, the fibrillin gene is relatively large (approximately 110-kb) and highly fragmented (65 exons). This study provides the first comprehensive analysis of the fibrillin gene and relevant information for the full characterization of Marfan syndrome mutations.

Topics: Amino Acid Sequence; Base Sequence; Carrier Proteins; Chromosome Mapping; Chromosomes, Fungal; Cloning, Molecular; Cysteine; DNA; Epidermal Growth Factor; Exons; Fibrillins; Gene Library; Genome, Human; Humans; Intracellular Signaling Peptides and Proteins; Latent TGF-beta Binding Proteins; Marfan Syndrome; Microfilament Proteins; Molecular Sequence Data; Repetitive Sequences, Nucleic Acid; Transforming Growth Factor beta

Defects of fibrillin (FBN1), a glycoprotein component of the extracellular microfibril, cause Marfan syndrome. This disorder is characterized by marked inter- and intrafamilial variation in phenotypic severity. To understand the molecular basis for this clinical observation, we have screened the fibrillin gene (FBN1) on chromosome 15, including the newly cloned 5' coding sequence, for disease-producing alterations in a panel of patients with a wide range of manifestations and clinical severity. All the missense mutations identified to date, including two novel mutations discussed here, are associated with classic and moderate to severe disease and occur at residues with putative significance for calcium binding to epidermal growth factor (EGF)-like domains. In contrast, two new mutations that create premature signals for termination of translation of mRNA and are associated with reduction in the amount of mutant allele transcript produce a range of phenotypic severity. The patient with the lowest amount of mutant transcript has the mildest disease. These data support a role for altered calcium binding to EGF-like domains in the pathogenesis of Marfan syndrome and suggest a dominant negative mechanism for the pathogenesis of this disorder.

Topics: Alleles; Amino Acid Sequence; Base Sequence; Binding Sites; Calcium; Chromosomes, Human, Pair 15; DNA; Epidermal Growth Factor; Female; Fibrillin-1; Fibrillins; Humans; Macromolecular Substances; Male; Marfan Syndrome; Microfilament Proteins; Models, Structural; Molecular Sequence Data; Oligodeoxyribonucleotides; Oligonucleotides, Antisense; Pedigree; Phenotype; Point Mutation; Polymerase Chain Reaction; Protein Biosynthesis; Protein Conformation; RNA, Messenger; Transcription, Genetic

Topics: Adult; Amino Acid Sequence; Base Sequence; Binding Sites; Calcium; DNA; DNA Mutational Analysis; Epidermal Growth Factor; Female; Fibrillins; Humans; Male; Marfan Syndrome; Microfilament Proteins; Molecular Sequence Data; Pedigree; Point Mutation

The Marfan syndrome is an autosomal dominant heritable disorder of connective tissue with prominent involvement of the ocular, skeletal, and cardiovascular systems. The gene on chromosome 15 encoding fibrillin (FBN1), a 350-kDa glycoprotein component of the extracellular microfibril, is the site of defect in most, if not all cases. Complementary DNA sequence reveals a gene composed largely of epidermal growth factor-like repeats, each containing six predictably spaced cysteine residues. To date, two FBN1 gene missense mutations have been reported. Here we describe the identification of three new missense mutations in the FBN1 gene in patients with the Marfan syndrome. All of the 5 characterized missense mutations occur within the epidermal growth factor-like repeats of the FBN1 gene. In addition, 4 of 5 involve the substitution of cysteine residues and 3 of 5 substitute the third cysteine in the epidermal growth factor-like motif consensus sequence. These data suggest that defined residues within EGF-like domains of FBN1 have particular significance and, when altered, play a pivotal role in expression of the Marfan phenotype.

Topics: Amino Acid Sequence; Base Sequence; Cysteine; DNA; DNA Mutational Analysis; Epidermal Growth Factor; Fibrillin-1; Fibrillins; Humans; Marfan Syndrome; Microfilament Proteins; Molecular Sequence Data; Molecular Structure; Phenotype; Polymerase Chain Reaction; Polymorphism, Genetic; Repetitive Sequences, Nucleic Acid

To examine the associations among fibrillin gene mutations, protein function, and Marfan syndrome phenotype, we screened for alterations in the fibrillin coding sequence in patients with a range of manifestations and clinical severity. A cysteine to serine substitution at codon 1409 (C1409S) was identified in an epidermal growth factor (EGF)-like motif from one fibrillin allele which segregates with the disease phenotype through three generations of a family affected with the Marfan syndrome. This alteration was not observed in 60 probands from other families or in 88 unrelated normal individuals. The altered cysteine is completely conserved in all EGF-like motifs identified in fibrillin, and in all proteins that contain this motif. These observations strongly indicate that C1409S is the disease-producing mutation in this family. The phenotype of individuals carrying C1409S varied widely with respect to onset of disease, organ-system involvement, and clinical severity; certain affected adults were unaware of their status before being diagnosed through this investigation. We conclude that fibrillin gene defects cause familial Marfan syndrome, that mutations in the EGF-like motif of the fibrillin gene are not uniformly associated with severe disease, and that fibrillin genotype is not the sole determinant of Marfan phenotype.

Topics: Amino Acid Sequence; Base Sequence; Consensus Sequence; DNA; Epidermal Growth Factor; Fibrillins; Humans; Marfan Syndrome; Microfilament Proteins; Molecular Sequence Data; Mutation; Oligodeoxyribonucleotides; Pedigree; Sequence Alignment

Fibrillin is a large (relative molecular mass 350,000) glycoprotein which can be isolated from fibroblast cell cultures and is a component of the microfibrils that are ubiquitous in the connective tissue space. The microfibrils of the suspensory ligament of the lens as well as the elastic fibre microfibrils of the blood vessel wall are composed of fibrillin. The ocular and cardiovascular manifestations of the Marfan syndrome are consistent with a defect in the gene coding for a structural constituent of these connective tissues. Immunohistological experiments have recently implicated fibrillin microfibrils in the pathogenesis of the Marfan syndrome. Genetic linkage data localizing the Marfan gene to chromosome 15 and the in situ hybridization of fibrillin complementary DNA to 15q21.1 together support fibrillin as a candidate Marfan gene. As a first step towards investigating the function of fibrillin in the architecture and development of connective tissues and its relationship to the Marfan syndrome, we report the cloning and partial sequencing of fibrillin cDNA.

Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; Epidermal Growth Factor; Fibrillins; Humans; Marfan Syndrome; Microfilament Proteins; Molecular Sequence Data; Sequence Homology, Nucleic Acid