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REVIEW |
1 Institute of Medical Genetics, Charité University Hospital, Humboldt University, Berlin, Germany
2 Department of Pediatrics, Mount Sinai School of Medicine-New York University, One Gustave L. Levy Place, New York, NY 10029, USA
3 Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, UK
4 Laboratoire de Génétique Moléculaire, Institut Universitaire de Recherche Clinique, Montpellier, France
5 Institute of Integrative and Comparative Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK
6 Centre for Medical Genetics, Ghent University Hospital, Ghent, Belgium
7 Departments of Pediatrics, Medicine and Molecular Biology and Genetics, and Howard Hughes Medical Institute, John Hopkins University School of Medicine, Baltimore, MD, USA
8 Division of Molecular and Cellular Biochemistry, Department of Biochemistry, University of Oxford, Oxford, UK
9 Department of Medicine, Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
10 Department of Internal Medicine, University of Texas Medical School at Houston, Houston, TX, USA
11 Laboratory of Genetics and Organogenesis, Research Division of the Hospital for Special Surgery and Department of Physiology and Biophysics at the Weill Medical College of Cornell University, New York, NY, USA
12 McGill University, Department of Anatomy and Cell Biology and Faculty of Dentistry, Montreal, Canada
13 Center for Human Molecular Genetics, University of Nebraska Medical Center, Omaha, NE, USA
Correspondence to:
Dr Peter N Robinson
Institute of Medical Genetics, Charité University Hospital, Humboldt University, Augustenburger Platz 1, 13353 Berlin, Germany; peter.robinson{at}charite.de]
Received 7 March 2006
9 March 2006
 | ABSTRACT |
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Abbreviations: BMP, bone morphogenetic protein; cbEGF, calcium binding epidermal growth factor; CCA, congenital contractural arachnodactyly; CMN, cystic medial necrosis; CSGE, conformation sensitive gel electrophoresis; DHPLC, denaturing high performance liquid chromatography; EBP, elastin-binding protein; ECM, extracellular matrix; HNPCC, hereditary non-polyposis colorectal cancer; LAP, latency-associated peptide; LDS, Loeys-Dietz aortic aneurysm syndrome; LLC, large latent complexes; LTBP, latent-TGFß-binding protein; MAGP-1, microfibril-associated glycoprotein-1; MFS, Marfan syndrome; MFS2, type 2 Marfan syndrome; MMP, matrix metalloproteinase; MMR, mismatch repair; MSI, microsatellite instability; NMR, nuclear magnetic resonance; PTC, premature termination codon; SSCP, single stranded conformation polymorphism; TAAD, thoracic ascending aortic aneurysms and dissections; TßRII, type II TGFß receptor; TGFß, transforming growth factor-ß; UMD, Universal Mutation Database; WMS, Weill-Marchesani syndrome
Keywords: fibrillin; Marfan syndrome; microfibril; TGFß
Marfan syndrome (MFS; MIM 154700 [OMIM] ) is a relatively common autosomal dominant hereditary disorder of connective tissue with prominent manifestations in the skeletal, ocular, and cardiovascular systems. MFS is caused by mutations in the gene for fibrillin-1 (FBN1). Many affected individuals have a characteristic habitus with tall stature, long slender limbs (dolichostenomelia), arachnodactyly, scoliosis, and pectus excavatum or carinatum. Ectopia lentis affects up to 80% of individuals with MFS and is almost always bilateral. The leading cause of premature death in untreated individuals with MFS is acute aortic dissection, which follows a period of progressive dilatation of the ascending aorta. Recent comprehensive treatments of the clinical aspects of MFS have been published.1,2
A review of the molecular genetics of MFS appeared in these pages in the year 2000.3 In the half decade since then, remarkable progress has been made in elucidating the molecular anatomy of both fibrillin and the fibrillin-rich microfibrils as well as in understanding the molecular pathogenesis of MFS. Initial ideas about the pathogenesis of MFS concentrated on a static dominant negative model based on the concept of fibrillin-rich microfibrils as purely architectural elements in the extracellular matrix. Recent findings greatly enhanced our understanding of the pathogenesis of MFS by demonstrating changes in growth factor signalling and other changes in matrix-cell interactions, which has set the stage for attempts to develop novel forms of treatment.
The current review will focus on the fibrillin-LTBP gene family and tissue organisation of microfibrils, mutations in genes associated with MFS and related phenotypes, the structure of fibrillin-1 domains, FBN1 mutations and proteolysis, and the genetics of MFS in mouse models. The current review will highlight advances published after the first review; interested readers are referred to the previous article for more information on other topics.3
| THE FIBRILLIN-LTBP GENE FAMILY |
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Members of the fibrillin-LTBP family have both structural and non-structural functions. Three closely related fibrillins have been described. Fibrillin-24 and the recently discovered fibrillin-35 have a domain organisation identical to that of fibrillin-1 and an overall level of amino acid identity of between 61% and 69%. Several lines of evidence suggest that the fibrillins have both overlapping and unique functions.5,6 All three fibrillins are structural components of microfibrils4,5,7; however, fibrillin-2 and fibrillin-3 are preferentially expressed in embryonic developmental stages,4,5,8 whereas fibrillin-1 is expressed from the gastrula to throughout adult life.9 In addition, distinct phenotypes are observed in fbn1 and fbn2 gene targeting experiments in mice; since microfibrils are assembled in both mice, some overlap of architectural functions appears likely.5,10–12
The LTBPs associate with transforming growth factor-ß (TGFß), thereby regulating its secretion and spatial and temporal activation; in humans, four members of the LTBP family are known, three of which undergo alternative splicing.13 TGFß is synthesised as a homodimeric proprotein, whereby the dimeric propeptide is cleaved intracellularly from the growth factor; the propeptide is called the latency-associated peptide (LAP) because TGFß cannot bind to its surface receptors when it is bound to the LAP. The LAP in turn is usually disulfide-bonded to an LTBP; this aggregate is referred to as the large latent complex (LLC). The LTBPs thus have a dual function: as structural components of the extracellular matrix and as modulators of TGFß availability (the reader is referred to Rifkin14 and Todorovic et al15 for recent reviews on this topic).
The fibrillins and LTBPs display a remarkably similar domain structure made up of repeated cysteine-rich structural modules including multiple copies of an epidermal growth factor-like module (EGF) and an 8-cysteine (8-Cys) module found only in the fibrillins and LTBPs (fig 1
). EGF modules are approximately 45 residues in length and are characterised by six conserved cysteine residues that form three intramodule disulfide bonds. Forty three of the 47 EGF modules in fibrillin-1 and many of those in the various LTBPs additionally conform to a consensus sequence, (D/N)-X-(D/N)(E/Q)Xm(D/N)*Xn(Y/F), that mediates calcium binding in the N-terminal region of the module (m and n are variable, and an asterisk denotes ß-hydroxylation).16
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| TISSUE ORGANISATION OF MICROFIBRILS |
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Microfibrils form loosely packed bundles in roughly parallel alignment. These bundles adopt tissue-specific architectures that are dictated by cells, and by the strength and direction of forces put upon the tissue. They are found in locations that are subject to repeated mechanical stresses, and in the proximity of basement membranes, and they serve a critical biomechanical anchoring role in dynamic connective tissues.25
Elastic fibre formation is a developmentally regulated process in which tropoelastin (the soluble precursor of mature elastin) is deposited on a preformed template of fibrillin microfibrils.26 Mature elastic fibres are a composite biomaterial with an outer microfibrillar mantle and an inner core of amorphous cross-linked elastin with some embedded microfibrils. The proportion of microfibrils to elastin appears to decline with age, with adult elastic fibres often having only sparse peripheral mantles of microfibrils.
A recent investigation by mass spectrometry of the composition of purified fibrillin-rich microfibrils from non-elastic and elastic tissues showed that in all microfibril preparations, fibrillin-1 was abundant and the only fibrillin isoform detected. Isolated microfibrils, extracted from tissues by enzyme digestions or homogenisation, have a "beads on a string" appearance with untensioned periodicity of 
56 nm.27,28 Unextracted hydrated zonular microfibrils appeared, by quick freeze deep etch microscopy, to be more tubular, suggesting that molecular components are lost or that there is a major molecular rearrangement on extraction.29 The molecular basis of the "beads", and fibrillin-1 alignment in microfibrils remain unclear.
Models of fibrillin-1 alignment
The details of the precise molecular architecture of fibrillin-1 alignment within microfibrils are not entirely clear, and several models have been proposed. An intermolecular fibrillin-1 transglutaminase cross-link30 provides molecular constraints, although mass spectrometry has shown that not all fibrillin-1 molecules within tissue microfibrils are cross-linked.31
The "hinge" model, based on detailed scanning transmission electron microscopy, mass mapping, automated electron tomography, and atomic force microscopy data,28,32–34 predicts maturation from an initial parallel head to tail alignment to an approximately one third stagger (100 nm) that would allow transglutaminase cross-link formation, and further packing into a more energetically favourable 56 nm untensioned form.
The one third staggered model was suggested on the basis of extrapolation of molecular dimensions,35 the crystal structure of fibrillin-1 cbEGF/TB/cbEGF domain arrays,36 and calcium binding studies of TB/cbEGF flexibility.37
Interested readers can find further discussion of issues surrounding fibrillin-1 alignment and microfibrillar architecture in Baldock et al,28 Davis et al,29, Kielty et al,34 and Lee et al.36
Early studies of isolated microfibrils revealed a number of highly stretched microfibrils with periodicities up to 150 nm, which suggested that microfibrils may have elastic properties.27 Stretching of isolated intact microfibrils has been achieved using surface tension forces and molecular combing.33 These studies showed that microfibrils behave as relatively stiff elastic filaments which can perform anchoring roles in ciliary zonules and other basement membrane interfaces. It was confirmed by x ray diffraction that hydrated microfibril bundles are elastic, and suggested that elasticity in microfibril-rich tissues may arise, in part, from reversible alterations in supra-microfibrillar arrangements. Proteolytic damage to microfibrils that may occur in MFS or ageing, may generate stretched microfibrils that may have lost their elastic properties.25,38
| ASSEMBLY OF MICROFIBRILS |
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Microfibril assembly in individuals with MFS
Early immunofluorescence studies with dermal fibroblasts or skin biopsies obtained from individuals with MFS showed reduced or qualitatively altered fibrillin networks as compared to controls.39–41 However, such altered patterns were not observed in all samples.41,42 One possible interpretation of these data is that a subset of mutations in fibrillin-1 compromises the assembly process and thus the formation of microfibril networks. Further evidence for this interpretation comes from pulse chase experiments using dermal fibroblasts from individuals with MFS. These analyses showed deficiencies at different levels such as fibrillin synthesis and secretion, as well as deposition into the extracellular matrix.43,44 Despite the differences in the secreted amount of fibrillin, a large portion of the fibroblasts studied showed impaired incorporation of the mutant fibrillin into the extracellular matrix, suggesting functional disturbances in early stages of the assembly mechanism.45,46 In cases where higher order assembly into beaded microfibrils was observed, the ultrastructural appearance showed several types of abnormalities including diffuse, frayed, or poorly defined interbead domains, or variable interbead periodicities.47–49 These consequences potentially could also emerge from functional problems in the assembly mechanism. In summary, although molecular evidence is still lacking, it seems clear that a certain subset of mutations in fibrillin-1 leading to MFS and other microfibrillopathies directly affects microfibril assembly mechanisms.
Role of propeptide processing in fibrillin assembly
Fibrillins are phylogenetically old proteins occurring in species from jellyfish to human. All known fibrillins possess highly conserved basic recognition sites (RX(K/R)R) for processing by endoproteinases of the furin/PACE type within the unique N- and C-terminal domains. It has been shown by several groups and methodologies that fibrillin-1 is indeed processed at these recognition sites.22,50–54 Processing produces a 17 or 20 residue N-terminal propeptide depending on the actual cleavage site for the signal peptide, and a 140 residue C-terminal propeptide. Due to the size of the propeptide, C-terminal processing is accessed much better experimentally and thus has received more attention. For fibrillin-1, it has been demonstrated that processing of the C-terminal propeptide is required for deposition into the extracellular matrix, suggesting that profibrillin-1 conversion to mature fibrillin-1 plays a regulatory role in assembly into higher order aggregates.43,50,53 On the molecular level, it is not clear how the presence of a C-terminal propeptide prevents matrix deposition and assembly. The propeptide may interact and mask self-assembly sites in fibrillin-1.24 A mutation in fibrillin-1 (R2726W) associated with isolated skeletal features of MFS was shown to interfere with normal processing at the C-terminal end and thus disturbed the incorporation of the mutated protein into the extracellular matrix.50 Other mutations close to processing sites may have similar consequences. Virtually no information is available as regards the functional role of the N-terminal processing. However, by analogy, it is predicted that the N-terminal propeptide also regulates assembly of fibrillins.
Self-assembly of fibrillins
As described in the "Tissue organisation of microfibrils" section, the molecular organisation of fibrillin-1 in microfibrils has been analysed by various groups resulting in a number of different models for the alignment of fibrillin in microfibrils.7,28,30,35,36,51 Despite the differences in these models in terms of stagger and molecular condensation of individual molecules, common to all models is a head to tail orientation of fibrillin-1 molecules as originally proposed by Sakai and coworkers in 1991.7 Another commonly accepted property of microfibrils is the involvement of six to eight fibrillin molecules per cross-section of the interbead region.28,55,56
Correlation of antibody epitopes in the fibrillin-1 molecule with the location of the corresponding epitopes in microfibrils clearly revealed that the terminal ends of the fibrillin molecules are situated in or close to the beads.7,28,51 Data obtained with recombinant fibrillin-1 fragments have established direct interaction in a homotypic N- to C-terminal fashion.57 These results established a linear head to tail self-assembly mechanism for fibrillin-1. Heterotypic interactions between fibrillin-1 and fibrillin-2 in an N- to C-terminal fashion suggested that both fibrillin isoforms can be organised within the same microfibril,57 and colocalisation of both isoforms has indeed been demonstrated by double immunogold labelling in tissue microfibrils.8 Electron microscopy after rotary shadowing of full length recombinant fibrillin-1 suggested that the interaction epitopes are relatively close to the terminal ends.57 These results were further substantiated by analyses of smaller overlapping fibrillin-1 fragments in various ligand interaction assays, positioning the interaction sites in the N-terminal region encoded by exons 1–8 and the C-terminal region encoded by exons 57–65.24
In addition to a linear head to tail self-interaction, there is evidence that lateral homotypic interactions also play a role in fibrillin assembly. Reducible homodimer formation early during biosynthesis was observed for recombinant fragments of fibrillin-1 and -2 spanning from the proline and glycine-rich domains, respectively, to the second 8-Cys/TB domain,58 as well as for smaller recombinant fragments of the proline-rich region of fibrillin-1 and the glycine-rich region of fibrillin-2 including flanking domains.59 Additionally, homotypic lateral interactions have been observed with an N-terminal recombinant fibrillin-1 fragment encoded by exons 1–8, as well as with an C-terminal fragment encoded by exons 57–65.24 The lateral homotypic interactions between N-terminally and C-terminally located fragments as well as the head to tail linear interactions between the N- and C-terminal ends are of high affinity with dissociation constants in the low nanomolar range, indicating that both types of mechanisms are highly relevant for initial assembly stages.24,57
Intermolecular cross-link formation in fibrillin assembly
Two types of intermolecular cross-links important for the stability of microfibrils have been identified: reducible disulfide bonds and non-reducible 
(
-glutamyl)lysine cross-links. Intermolecular disulfide bond formation is apparent early in the assembly of microfibrils since higher molecular weight disulfide-bonded aggregates containing fibrillin can be observed after a few hours in cell or organ cultures.22,60 Most of the highly conserved cysteine residues in fibrillins are predicted to stabilise individual domains through intramolecular disulfide bonds.17,35,36 One cysteine residue in the first hybrid domain of human fibrillin-1 and -2 has been shown to be available for intermolecular cross-links on the surface of the molecule.22
Additional data suggest that other cysteine residues, which are normally involved in intramolecular domain stabilisation, may be reshuffled to participate in intermolecular cross-links. Cysteine residues in the first and second 8-Cys/TB domain have been suggested for such a role based on the propensity of various recombinant fibrillin-1 and -2 fragments to form reducible homodimers.58,59 Such a mechanism would likely require the enzymatic activity of one or more protein disulfide isomerases on the cell surface or in the extracellular matrix.
Non-reducible (-glutamyl)lysine cross-links, catalysed by transglutaminases, have been identified in microfibrils extracted from various tissues.30,61,62 Detailed analyses of microfibrils from human tissues have identified transglutaminase cross-links in the N- and C-terminal regions of fibrillin-1, as well as a high overall content of these cross-links.30 In addition to fibrillin-1, another prominent protein in microfibrils, microfibril-associated glycoprotein-1 (MAGP-1), was also characterised as a substrate for transglutaminase.63 It is possible that besides homotypic fibrillin-1 transglutaminase cross-links, heterotypic fibrillin-1-MAGP-1 cross-links may be present in microfibrils. Zonular fibres in the eye have been demonstrated to be a target for transglutaminase 2.64 Biomechanical analyses of microfibrils suggested that transglutaminase cross-links play an important role in strengthening the microfibrils.65 Other potential roles of the transglutaminase cross-links may include correct lateral alignment of fibrillin or other molecules as a prerequisite for downstream assembly events. Mutations disrupting transglutaminase cross-link sites likely result in serious consequences for microfibril assembly and stability. To gain insight into such potential mechanisms in MFS and other microfibrillopathies, it will be important to identify the exact amino acid residues involved and the time course of transglutaminase cross-link formation.
Accessory molecules important for fibrillin assembly
In addition to self-assembly and cross-linking mechanisms, other molecules may be involved in the assembly process of microfibrils. Several regions in fibrillin-1 have been identified as interacting with heparin/heparan sulfate with high affinity.66–68 In cell culture assembly assays, these glycosaminoglycans inhibit the formation of microfibrillar networks,66,67 leading to the hypothesis that heparan sulfate or heparan sulfate containing proteoglycans may have regulatory functions in the assembly of microfibrils. Recently, the heparan sulfate containing proteoglycan perlecan was identified as a molecule which interacts with fibrillin-1 and with microfibrils close to basement membrane zones.69 Reduced amounts of microfibrils in basement membrane zones of perlecan null mice may reflect a potential role for perlecan in microfibril assembly.69
| MICROFIBRIL-ASSOCIATED PROTEINS |
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) and proteins that can associate with fibrillin-rich microfibrils but do not serve an integral structural function (table 2). We will not attempt a comprehensive review of the non-fibrillin proteins but rather will summarise the most important functions that have been attributed to these proteins and their interaction with fibrillin. Readers are referred to Gibson70 and the references in the tables 1 and 2 for further information. It appears likely that more interacting proteins remain to be discovered in light of a recent proteomics study of fibrillin-rich microfibrils showing copurification of a number of proteins in addition to fibrillin-1. For instance, -crystallin copurified with zonular microfibrils, suggesting an interaction that could contribute to zonule anchorage to the lens.31
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Interaction with other matrix components
Interactions between fibrillin-rich microfibrils and tropoelastin are important for the formation of elastic fibres. In addition to interactions between fibrillin-1 and tropoelastin,75 MAGP-1 interacts with tropoelastin76,77 in a way that is important for tropoelastin deposition.78 Microfibrils interact with a variety of other extracellular matrix structures and some of these interactions are mediated by non-fibrillin proteins. For instance, MAGP-1 binds to the 
3 chain of type VI collagen. Since type VI collagen microfibrils and fibrillin-rich microfibrils are often found near to one another in some extracellular matrices, it is plausible that MAGP-1 may mediate a molecular interaction between type VI collagen microfibrils and fibrillin-containing microfibrils.79
Interaction with cells
It has become increasingly clear that fibrillin-rich microfibrils have functions that are not directly related to structural integrity but rather have to do with growth factor metabolism and triggering cellular signals. As will be discussed in more detail below, the latent transforming growth factor-ß binding proteins (LTBP) are a family of secreted glycoproteins, three of which play an important role in the regulation of TGFß regulation.15 In addition to TGFß, microfibrils could conceivably be involved in the regulation of other growth factors, although little experimental evidence is available at this point.14 MAGP-2 can interact with Jagged1 and induce its shedding; it is thus conceivable that MAGP-2 may be able to modulate the Notch signalling pathway.80 There is evidence of interaction between fibrillin-1 and at least one bone morphogenetic protein (BMP),81,82 and fibrillin-2-rich microfibrils and BMP-7 have been shown to functionally interact in the regulation of limb patterning in a mouse model.12 Finally, an interesting area of research is the question whether novel cell signalling pathways are triggered in MFS or other diseases of the microfibrils, and whether fragments of the proteins of the microfibrils can acquire novel signalling properties through exposure of otherwise cryptic binding sites, a phenomenon that has been well described in several other disorders such as osteoarthritis.83 As will be discussed below, there is evidence that fibrillin fragments can induce matrix metalloproteinase (MMP) expression, and it is well known that elastin fragments can induce MMP expression.84,85
Enzymatic activity
The enzyme lysyl oxidase, which is involved in the cross-linking of tropoelastin monomers, has been localised to the interface between extracellular bundles of amorphous elastin and the microfibrils.86 Although there is no evidence of a direct interaction between fibrillin and lysyl oxidase to date, it is plausible that interactions with fibrillin or other microfibrillar components might be important for elastogenesis.70 Interestingly, ADAMTS10,87 an extracellular matrix protease, is mutated in the autosomal recessive form of Weill-Marchesani syndrome (WMS).88 The dominant form of WMS is caused by mutations in fibrillin-1, suggesting, perhaps, a potential interaction between ADAMTS10 and fibrillin-1. However, the substrates of ADAMTS10 or indeed other potential functions of this molecule remain to be elucidated.
Mutations in FBN1, FBN2, and other genes associated with MFS and related phenotypes
Mutations in FBN1 were discovered in individuals with MFS in 1991,109 and subsequently mutations in FBN2 were discovered in individuals with a phenotypically related disorder, congenital contractural arachnodactyly (CCA).110 More recently, mutations in the genes for TGFBR1 and TGFBR2 were found in several disorders with varying degrees of overlap with classic MFS.111–113 Genetic loci for other forms of isolated aortic dilatation and dissection have been identified. The identification of mutations in these genes has provided significant insight into the pathogenetic pathways involved in MFS and related disorders, and further insight is to be expected from characterising the full spectrum of mutations associated with these disorders and from identifying the full set of genes in which mutations cause related disorders of connective tissue. The following sections present the current state of knowledge on FBN1, FBN2, and TGFBR2 mutations and on loci involved in isolated aortic aneurysm and dissection.
| MUTATION ANALYSIS OF THE FBN1 GENE IN INDIVIDUALS WITH MFS: SENSITIVITY, METHODS, AND CLINICAL INDICATIONS |
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Therefore, if the Ghent criteria are fulfilled, then the diagnosis of MFS is certain. In adult patients presenting with a small number of non-specific skeletal manifestations often seen in MFS, the diagnosis is unlikely, but a full clinical evaluation including echocardiography and ophthalmologic examination is indicated.
In situations of clinical uncertainty, molecular analysis of the FBN1 gene therefore seems a logical aid to the clinical diagnosis of MFS. In practice, however, the large and complex structure of the FBN1 gene and the wide scope of FBN1 mutations have hampered clinical implementation of FBN1 testing. Moreover, literature data show great variation in detection rates of FBN1 mutations and methodologies used. This can be accounted for by a variety of factors such as the type of mutational analysis method, the substrate (genomic versus cDNA) and, most importantly, the accuracy of the clinical diagnosis. The studies that have looked at the sensitivity and specificity of FBN1 mutation analysis show substantial differences in one or several of these factors.
The first mutational studies, performed on cDNA, showed low efficiency of FBN1 mutation detection using SSCP (single stranded conformation polymorphism) and yielded mutation rates of 9% to 23%.116,117 Subsequent studies, using genomic DNA as template for analysis of the 65 individual exons of the FBN1 gene, obtained higher detection rates but varied according to the type of screening method used.118–120 Initially the best results were obtained with CSGE (conformation sensitive gel electrophoresis), with detection rates ranging from 57% to 90%.42,121,122 Subsequent studies have shown that mutation detection by DHPLC (denaturing high performance liquid chromatography) is highly efficient,123 although a relatively high false-positive rate may be an issue.124 It now appears that DHPLC is the most efficient approach for mutation detection also because of its potential for automation when combined with robotic PCR.
The most important factor influencing the mutation detection rate appears to be the clinical homogeneity or heterogeneity of the patient population. Indeed, several studies have shown that the incidence of FBN1 mutations is significantly higher in patients who fulfil the MFS diagnostic criteria than in patients who do not. In a large study including a cohort of 94 MFS patients and 77 patients with MFS related phenotypes, Loeys et al125 found FBN1 detection rates of 66% versus 5%, respectively. They demonstrated that fulfilling the clinical diagnosis of MFS in itself is a good predictor of the outcome of FBN1 mutation analysis. Katzke et al120 supported these findings by their study which showed a much higher incidence of mutations in a group of MFS patients versus those with an MFS related condition, and concluded that clinical overdiagnosis is the most important explanation for low FBN1 mutation detection rates. Two other studies123,126 also showed that the majority of patients in whom an FBN1 mutation was found, met the clinical diagnosis of MFS, supporting the opinion that the robustness of selection criteria is the most important determinant of the outcome of mutational studies.
Several clinical situations can occur in which molecular studies of the FBN1 gene may be helpful. In patients who present with skeletal, cardiovascular, and/or possibly other manifestations of MFS but have no involvement of the ocular system, it can be difficult to establish the diagnosis strictly on clinical grounds, particularly in the absence of a positive family history. Here, however, MRI studies can reveal the presence of dural ectasia in which case the diagnostic criteria may still be met. Several mutational studies which report FBN1 mutations in Marfan-like patients have not verified the presence or absence of dural ectasia, so that the possibility remains that the diagnostic criteria are in fact met. In cases where no definitive conclusion can be reached with the clinical data, molecular analysis of the FBN1 gene is an alternative option.
Because of the evolving nature of the phenotype, particularly so for the cardiovascular and skeletal manifestations, children with suspected MFS may not yet fulfil the diagnostic criteria. In those instances, it is better to postpone a final diagnosis until later. The identification of an FBN1 mutation in children or young adults not (yet) fulfilling the diagnostic criteria can help to identify those who need to be clinically followed with particular attention.
FBN1 mutations have been identified in a range of phenotypes, the type 1 fibrillinopathies, with greater or lesser degrees of clinical overlap with MFS (table 3). A decision as to whether mutation analysis is indicated when such phenotypes are suspected needs to be made on an individual basis.
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, FBN1 mutations can be found in individuals with Marfan-like disorders who do not fulfil the criteria of the Ghent nosology.127 It is recommend that accepted clinical guidelines for the care and management of MFS are applied in these cases even if the criteria of the Ghent nosology are not fulfilled, because it is possible that complications such as aortic dilatation can emerge at any age. Finally, the availability of a molecular test also allows prenatal or preimplantation diagnosis for prospective parents. This is one option for which requests appear to be steadily increasing.
In summary, after stringent clinical selection, a detection rate of up to 90% is currently achievable in FBN1 molecular testing of patients with classic MFS.128 This allows the implementation of mutational studies in clinical practice.
| THE UMD FBN1 DATABASE: A DATABASE FOR FBN1 MUTATIONS IMPLICATED IN MFS AND RELATED PHENOTYPES |
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The mutation records of the database include point mutations, large and small deletions, insertions, and splice mutations in the FBN1 gene. Each record contains the molecular and clinical data for a given mutation in a standardised, easily accessible, and summary form; if available, data on fibrillin protein biosynthesis classification groups45 are included.
To date, 601 FBN1 mutations are available online. The mutations are spread throughout almost the entire gene without obvious predilection for any given region. Approximately 12% of mutations are recurrent.149
The mutation studies performed to date generally have concentrated on screening the 65 coding exons of FBN1. For the most part, methods capable of detecting larger deletions were not applied and it is not clear how much flanking sequence or other regions of the gene were investigated. It is also unclear whether, once a mutation was identified, the remaining gene regions were regularly and fully evaluated.
Many different kinds of mutation have been identified in FBN1. Point mutations are the most common mutational event, with nonsense and missense mutations comprising about 10% and 60% of all reported mutations. The most common missense mutations substitute cysteine residues that form disulfide bonds within one of the cbEGF or 8-Cys domains, but missense mutations creating novel cysteine residues in these modules are also common. The majority of the remaining mutations in these modules affect residues of the calcium consensus sequence. About a quarter of missense mutations affect modules other than cbEGF, and for the most part, the pathophysiological mechanisms of these mutations remain unclear.
Small insertions, deletions, or duplications represent about 13% of all reported mutations. The majority of these mutations create a premature termination codon (PTC). Another 13% of reported mutations consist of various classes of splicing errors, most commonly affecting canonical splice sequences at exon/intron boundaries. Many splice site mutations in FBN1 result in in-frame exon skipping, such that the mutant fibrillin-1 lacks an entire cbEGF domain. Such mutations can be associated with a particularly severe phenotype.151 Some exon-skipping mutations in FBN1 result in a frameshift152 with reduced mutant RNA levels through nonsense-mediated decay of the mutant transcript.153 A nonsense mutation136 and a silent exonic mutation154 in exon 51 have been reported as inducing in-frame skipping of the entire exon 51 and demonstrate the existence of an exonic splicing enhancer.155,156
Global analysis of FBN1 mutations reveals two classes of mutations. The first type, which represents more than one third of the mutations, contains mutations predicted to result in shortened fibrillin-1 molecules, including nonsense mutations, splicing errors, insertions, and duplications, as well as in-frame or out-of-frame deletions. These mutations are likely to result in nonsense-mediated decay resulting in reduction in the level of the mutant allele. The second type represents slightly less than two thirds of the mutations and contains missense mutations, mostly located in cbEGF-like modules. They can be subclassified into: (a) mutations creating or substituting cysteine residues potentially implicated in disulfide bonding and consequently in the correct folding of the monomer; (b) amino acids implicated in calcium binding and subsequently in interdomain linkage, structural integrity of affected domains, and increased protease susceptibility; and (c) other mutations that might affect the conformation of affected modules, interdomain packing, or other functions such as protein-protein interactions.
Elucidating the molecular basis of MFS and related fibrillinopathies is the major goal of the teams working on this subject.149,157 The extreme clinical variability, the difficulties associated with clinical diagnosis, and the low detection rate of mutations in this large gene all conspire to negatively impact on progress. At present it is not possible to predict the phenotype for a given FBN1 mutation. On the one hand, mutations affecting different positions within a given module may be associated with quite different phenotypes. On the other hand, mutations affecting an analogous residue within two different modules may also be associated with differing phenotypes. Therefore, it is apparent that neither the location of the affected structural module in the protein nor the position of the altered residue is, in itself, sufficient to predict potential genotype-phenotype correlations.158 The high degree of intrafamilial variability suggests that environmental and perhaps stochastic factors or modifying genes are important for the phenotypic expression of disease. The level of the expression of the normal fibrillin-1 allele159 and hyperhomocysteinaemia related to the C677T methylenetetrahydrofolate reductase polymorphism160 have been proposed as factors that modify the clinical severity of MFS. The elucidation of the full range of modifying factors in MFS represents an interesting area for further research.
| MUTATIONS IN TGFBR1 AND TGFBR2 |
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Linkage to chromosome 3p24.2–p25 was demonstrated for a large family with a Marfan-like phenotype for whom linkage to FBN1 and FBN2 had previously been excluded.162,163 This disorder has been termed MFS type II (MIM 154705 [OMIM] ) and shares some of the cardiovascular and skeletal features of classic MFS.
Identification of a chromosomal breakpoint disrupting the gene encoding the TGFß receptor 2 (TGFBR2) in a boy with short stature, dural ectasia, and several skeletal and cardiovascular manifestations of MFS led to the identification of three further missense mutations in four families or individuals with manifestations of MFS in whom FBN1 mutations had been ruled out (one mutation was found in two unrelated families).111 All the mutations were found in the serine-threonine kinase domain of the TGFß receptor 2.
More recently, a new aortic aneurysm syndrome with hypertelorism, bifid uvula or cleft palate, and generalised arterial tortuosity with ascending aortic aneurysm together with other findings such as craniosynostosis, mental retardation, and congenital heart disease was described; this disorder, Loeys-Dietz aortic aneurysm syndrome (LDS; MIM 609192 [OMIM] ), was shown to be associated with mutations in the genes for either TGFß receptor type 1 or TGFß receptor type 2 resulting in perturbations of TGFß signalling.112 As will be noted below, mutations of the arginine at position 460 of TGFBR2 have been identified in individuals with thoracic ascending aortic aneurysms and dissections (TAAD), although there appears to be some degree of phenotypic overlap with LDS in many affected individuals.
Microsatellite instability (MSI) is a prominent feature in hereditary non-polyposis colorectal cancer (HNPCC) and some forms of acquired colon cancer. Defects in mismatch repair (MMR) genes and associated coding region MSI can cause frameshift mutations with functional inactivation of affected genes, thereby providing a growth advantage to MMR deficient cells. TGFBR2 is one of the most commonly affected genes with mutations in a polyadenine tract in exon 3 being found in up to 90% of cases of microsatellite-instable sporadic and HNPCC associated colon cancer.164 Germline mutations in TGFBR2, however, are a rare cause of HNPCC.165 Interestingly, one mutation (R528H) was found both as a somatic mutation in colon cancer166,167 and as a germline mutation in LDS.112 At present, there is no evidence that individuals with LDS are at increased risk for colon cancer.
It should be noted that there has been some controversy as to whether certain TGFBR2 mutations lead to a phenotype that is identical or at least very similar to that of classic MFS, thus justifying the diagnosis of type 2 MFS (MFS2). The question about locus heterogeneity for MFS has been addressed by several studies. Historical linkage data in MFS families showed a cumulative LOD score for the FBN1 locus in excess of 100, which provides evidence for a single predominant locus for MFS. On the other hand, none of the individuals with LDS112 fulfilled the clinical diagnostic criteria for MFS. In a previous study, Loeys et al128 identified 86 FBN1 mutations in a cohort of 93 patients with classic MFS. None of the remaining patients had a TGFBR2 or TGFBR1 mutation. Overall, this suggests strongly that FBN1 is the predominant if not sole locus for MFS.128 However, there have been reports of individuals with TGFBR2 mutations diagnosed with MFS2 without features characterising LDS.168 Given that the aneurysms in LDS appear to be more aggressive than those in MFS and the fact that the cardiovascular involvement is also characterised by arterial aneurysms throughout the arterial tree and marked arterial tortuosity, it is clinically very important to be aware of the potential differences between these two syndromes. Detailed clinical characterisation of individuals with TGFBR2 mutations will be required to determine if a subset of these mutations is associated with MFS2 or whether the diagnosis of LDS is more appropriate.
| MUTATIONS IN FBN2 AND CONGENITAL CONTRACTURAL ARACHNODACTYLY |
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While CCA shares some clinical characteristics with MFS (table 4), it does not share the usually shortened life expectancy.
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Molecular studies of only one individual with severe/lethal CCA have been performed.179 This individual had an exon splicing mutation that caused the skipping of exon 34, a cbEGF-like domain. Significantly, this individual’s mother was a somatic mosaic with one third of her fibroblasts also harbouring the same exon 34 mis-splicing mutation. Therefore, one can speculate that there is a threshold for certain mutations causing skeletal perturbations versus severe developmental abnormalities in the cardiovascular and gastrointestinal systems.
Although individuals with CCA usually do not have aortic involvement, aortic root dilatation does occur in some cases and screening for aortic involvement should be performed in individuals with this disorder.180
Fibrillin-2 in development and animal models
The temporal and spatial expression of fibrillin-2 has been examined in several species. In human fetal aorta, antibodies to fibrillin-2 were found to stain most intensely in the media, where elastic fibres are most abundant. In human elastic cartilage, fibrillin-2 localised to the cartilaginous core while fibrillin-1 localised primarily to the surrounding connective tissue.4 Fibrillin-1 and fibrillin-2 demonstrate a similar spatial and temporal distribution in most tissues during early human embryonic development. Exceptions included the kidney, liver, rib anlagen, and notochord.181 Similar studies in the developing mouse showed that in most tissues fibrillin-2 was expressed earlier than fibrillin-1.182
Studies in the chick have shown that fibrillin-2 (called JB-3 in the early literature) is expressed very early in development and is found in the regions of heart development.183 The early expression of the fibrillins has led to speculation that they may mediate the tensile forces that shape the early embryo.184 A possible role for fibrillin-2 in lung development has been shown in a rat model. Studies of fetal lung explants demonstrated abnormal branch morphogenesis when the explants were incubated with antisense oligonucleotides to fibrillin-2.185
Browning et al186 described a mouse with syndactyly (sne) that was derived from chemical mutagenesis of murine embryonic stem cells. They showed that sne (now renamed syfp-3J) was an allele of the sy locus. sy is the shaker-with-syndactylism mouse, a radiation mutant with a chromosome 18 (syntenic to human chromosome 5) contiguous gene deletion syndrome.187 The deleted region contains the gene encoding fibrillin-2. Some spontaneously occurring mouse models with syndactyly also mapped to the sy locus (syfp and syfp-2J). All three syfp mutations are FBN2 mutations.186,188 Additional evidence that absence of FBN2 leads to syndactyly came from gene targeting studies.12 The FBN2–/– knockout mouse displayed the same type of syndactyly observed in the sy mice. Interestingly, two of the fibrillin-2 mutations in the syfp mice were outside the neonatal region, that is, the area in which all of the human CCA mutations have been found.188 These findings have led to the obvious speculation that fibrillin-2 mutations outside the neonatal region
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ABSTRACT
THE FIBRILLIN-LTBP GENE FAMILY