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

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 microfibrils^4,5,7; 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.^5,10,11,12

The LTBPs associate with transforming growth factor‐β (TGFβ), 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 TGFβ 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 TGFβ 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 TGFβ 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 (​(figfig 1​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)X_m(D/N)*X_n(Y/F), that mediates calcium binding in the N‐terminal region of the module (m and n are variable, and an asterisk denotes β‐hydroxylation).¹⁶

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.

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.^118,119,120 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 studies^123,126 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​3).). A decision as to whether mutation analysis is indicated when such phenotypes are suspected needs to be made on an individual basis.

In addition to the more or less well delineated disorders listed in table 3​3,, 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.

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.

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 (MIM154705) and shares some of the cardiovascular and skeletal features of classic MFS.

Identification of a chromosomal breakpoint disrupting the gene encoding the TGFβ 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 TGFβ 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; MIM609192), was shown to be associated with mutations in the genes for either TGFβ receptor type 1 or TGFβ receptor type 2 resulting in perturbations of TGFβ 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 cancer^166,167 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.

While CCA shares some clinical characteristics with MFS (table 4​4),), it does not share the usually shortened life expectancy.

Putnam et al¹¹⁰ were the first to identify mutations in two unrelated individuals with CCA; both were cysteine substitutions. It is important to note that in contrast to the distribution of FBN1 mutations causing MFS throughout the coding region, the FBN2 mutations so far identified in CCA appear to cluster between exons 23 and 34. The homologous region of FBN1, the so called neonatal region, contains the greatest percentage of mutations from MFS patients at the most severe end of that disorder's clinical spectrum.¹¹⁷ Virtually all of the known FBN2 mutations are of the calcium binding epidermal growth factor‐like (cbEGF) domains.^{174,175,176,177,178}

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

Initial studies showed that first degree relatives of probands with non‐syndromic TAAD have a higher risk of thoracic aortic aneurysms and sudden death compared with a control group.^198,199 In addition, these studies support the hypothesis that genetic factors play a role in the aetiology of TAADs in patients who do not have an identified syndrome causing aortic disease. Milewicz and colleagues described six families with aortic aneurysms and dissections, all of whom demonstrated autosomal dominant inheritance associated with decreased penetrance and variable age of onset of the aortic disease.¹⁸⁹ Various studies indicate that the aortic disease in the majority of these families is not due to a mutation in the FBN1 gene or other genes encoding vascular proteins, such as COL3A1.^189,200,201

More recently, several loci for non‐syndromic TAAD have been mapped. The first locus, termed TAAD1, was mapped to chromosome 5q13–14 with a maximum LOD score of 4.74 with the marker D5S2029.²⁰² This locus was confirmed by an independent study in a Finnish population where approximately one half of the families studied show evidence of linkage to TAAD1.²⁰³ The critical interval containing the defective gene maps to a 7.8 cM region. Another locus for familial aortic aneurysms and dissections has been mapped to the long arm of chromosome 11 (11q23–24) using a single large family.²⁰⁴ In contrast to the families linked to TAAD1, the clinical phenotype of the family linked to the FAA1 locus indicated a diffuse vascular aetiology. Apart from dilatation in the sinuses of Valsalva, involvement of other aortic segments and arteries was also observed, such as dilatation in the abdominal aorta and left subclavian artery. In addition, the disease was fully penetrant with aortic imaging in the family described. The FAA1 locus is a rare cause of the vascular condition as indicated by the fact that no other families demonstrate linkage of the phenotype to markers at this locus.

Another locus for TAAD was mapped to a 25 cM region on chromosome 3p24–25 using another large family with multiple members with aneurysms and dissections of the thoracic aorta. The disease in the family was characterised as autosomal dominant with decreased penetrance and variable age of onset.²⁰⁵ Eighteen TAAD families described previously failed to show linkage to 3p24–25, indicating that TAAD2 is a minor locus for TAAD.^202,204 It was recently determined that mutations in the transforming growth factor beta receptor type II gene (TGFBR2) is the cause of disease at the TAAD2 locus.¹¹³ The TGFBR2 gene was screened for missense, nonsense, and exon splicing errors and mutations were found in four out of 80 unrelated families with familial TAAD, indicating that TGFBR2 mutations are a relatively rare cause of familial TAAD. Although most vascular disease in these families involved ascending aortic aneurysms leading to type A dissections, affected family members also had descending aortic disease and aneurysms of other arteries, including cerebral, carotid, and popliteal aneurysms. Strikingly, all four families carried mutations that affected arginine at amino acid 460 in the intracellular domain, suggesting a mutation hot spot for familial TAAD and establishing a strong genotype‐phenotype correlation between familial TAAD and mutations at this location. Structural analysis of the TGFBR2 serine/threonine kinase domain reveals that R460 is strategically located within a highly conserved region of this domain and that the amino acid substitutions resulting from these mutations will interfere with the receptor's ability to transduce signals. A surprising observation in these families was that there is no evidence of an increased susceptibility to cancer in families with germline TGFBR2 mutations, despite evidence in the literature that somatic TGFBR2 mutations occur in a variety of cancers.

The two predominant structural modules in fibrillin‐1 are the calcium‐binding epidermal growth factor‐like domain (cbEGF) and the transforming growth factor β binding protein‐like (TB or 8‐Cys) domain. High resolution structures of the fibrillin‐1 domain fragments cbEGF32–33, TB6, and cbEGF12–13 have been solved previously using nuclear magnetic resonance (NMR) which identifies the solution structure of the molecule.^17,35,206 More recently, x ray crystallography has been used to solve the structure of the triple domain fragment, cbEGF22‐TB4‐cbEGF23³⁶ and demonstrated a calcium‐stabilised tetragonal pyramidal conformation. An RGD integrin binding site localises to the tip of a β‐sheet within TB4 and is thus accessible to cell‐surface integrins. Comparative sequence alignments of the linker regions from cbEGF‐TB domains within fibrillin‐1 suggest that the relative orientation of cbEGF22‐TB4 is likely to be preserved at homologous sites within fibrillin‐1. In contrast, the variation in amino acid number and composition of TB‐cbEGF linker sequences suggests that these pairs will adopt different orientations with respect to one another within fibrillin‐1, and may contribute to the biomechanical properties of microfibrils. This is supported by the variability of K_d values of TB‐cbEGF pairs from fibrillin‐1 (see below). A model of a large region of fibrillin‐1 (cbEGF11‐TB5) has been generated from this combined structural information, which suggests that although the protein is in an extended conformation, it is not simply linear. A significant bend is introduced by the packing of cbEGF22 against TB4. Based on this structure, a staggered model for assembly of fibrillin‐1 into the microfibril has been suggested as an alternative to the proposed organisation based on electron microscopic studies.³⁶

Single cbEGF domains expressed from fibrillin‐1 and other proteins usually display low affinity binding in the mM range. However, in fibrillin‐1, and in many other proteins, the cbEGF domains are often arranged as repeating tandem arrays. On covalent linkage of an N‐terminal cbEGF, the affinity of the C‐terminal cbEGF increases.²⁰⁷ The bound calcium, together with the hydrophobic packing interaction, performs a key structural role in restricting interdomain flexibility^35,208 and therefore protects the modules against proteolytic cleavage.^209,210 Dynamics studies show that the most stable region of a cbEGF pair is in the vicinity of the interdomain calcium‐binding site.²⁰⁸ Analysis of different cbEGF domain pairs has, however, identified a range of affinities from 350 μM to 300 nM, suggesting that primary sequence variation, in addition to the pairwise domain interaction, must also influence affinity.^211,212 A study of heterologous TB‐cbEGF domain pairs³⁷ has shown that most of these domain pairs bind Ca²⁺ considerably more tightly than previously observed, with K_d values as low as 9 nM. These data suggest that under physiological conditions, many fibrillin‐1 cbEGF domains will be fully saturated and may impart rigidity to the native protein. However, the TB6‐cbEGF32 domain pair, with a K_d of 1.6 mM,²¹³ appears the most likely of the TB‐cbEGF domain pairs to be flexible and may contribute to the extensibility and elasticity of the microfibrils.

Insights into the role of a number of disease‐causing FBN1 mutations in the pathogenesis of MFS have been gained from NMR, calcium chelation, and limited proteolysis studies of recombinantly expressed fragments of fibrillin‐1. Reduction of calcium binding caused by substitution of a calcium ligand or destabilisation of the interdomain interface would be predicted to produce a less extended, more flexible structure within a region of fibrillin‐1. This may result in increased proteolytic susceptibility due to exposure of enzyme‐specific cryptic cleavage sites. The effect of a missense mutation on protease susceptibility of a cbEGF domain can, however, be influenced by a number of factors such as the particular residue mutated and the position of the mutant domain within the fibrillin‐1 peptide.

The structural effects of the pathogenic mutations C1977Y and C1977R which disrupt the 1–3 disulphide bond of cbEGF30 and are therefore predicted to cause misfolding, have been studied in a cbEGF29–31 triple construct using the combined methods of NMR, chelation, and limited proteolysis.²¹² The substitutions caused loss of Ca²⁺ binding to cbEGF30, consistent with intradomain misfolding and disrupted cbEGF29–30 domain‐domain packing. Surprisingly, the calcium binding properties of cbEGF29 and cbEGF31 were unaffected, suggesting these cysteine substitutions have relatively localised effects confined to the N‐terminal end of the mutant domain (fig 3A​3A).). However, a disruption of the 5–6 disulphide bond by a C750G substitution which affects the C‐5 residue of cbEGF7 in an EGF4‐TB3 fragment caused increased proteolytic susceptibility of cbEGF8.²¹⁴ This is presumably due to disruption of domain packing between cbEGF7 and 8 and hence reduction of the calcium binding affinity of cbEGF8. Cysteine substitutions are therefore likely to have different structural effects, which depend on the particular disulphide bond affected, and hence result in a variety of pathogenic mechanisms.

These studies, together with earlier reports, emphasise the structural heterogeneity that can be introduced into fibrillin‐1 by different FBN1 mutations. In the case of the folding substitution, G1127S, in cbEGF13, it was shown^215,216 that the mutant domain retained the ability to bind calcium (fig 3B​3B).). Studies^210,213 of the effects of the calcium binding substitution, N2144S, in domain pairs demonstrated that, while the structure of the mutant domain was unaffected, its ability to bind Ca²⁺ was reduced (fig 3C​3C).). Calcium binding substitutions which occur in the context of a cbEGF domain pair can, however, result in more significant structural changes.^210,217 For example, the protein engineered N2183S substitution in cbEGF33 (fig 3D​3D)) resulted in an increased proteolytic susceptibility of the cbEGF32–33 domain pair and the lack of calcium dependent protection indicated the absence of Ca²⁺ binding to the mutant domain.²¹⁰ Thus, as with cysteine substitutions, calcium‐binding substitutions may cause variable intramolecular effects dependent upon domain context.

It is evident that the structural effects of different FBN1 missense mutations are complex. In the neonatal region of fibrillin‐1, for example, missense mutations which affect structurally analogous calcium ligands in different cbEGF domains or cause substitution of different ligands coordinating the same Ca²⁺ (D1113G and N1131Y) produce varying phenotypes. Three missense mutations, K1043R, I1048T, and V1128I, which have no clear structural effect (although the I1048T substitution does introduce a glycosylation consensus sequence) are found to cluster on one face of a model constructed for the cbEGF11–15 region of fibrillin‐1. An unstructured, extended loop, present in cbEGF12 between cysteines 5 and 6, may also localise to this face of the model and be involved in intra‐ or intermolecular contacts.²⁰⁶ Analysis of the model shows that substitutions that may affect the calcium‐binding properties of cbEGF12 give rise to severe phenotypes. An increase in the intrinsic flexibility of this region resulting from defective calcium binding could distort a potential binding interface, which may be important for the microfibril assembly process and/or interactions with other microfibril components.

A correlation between the in vitro structural effects of amino acid substitutions with their cellular behaviour and consequences for intracellular trafficking and secretion is important for understanding the pathogenesis of MFS. Fibrillin‐1 biosynthesis, processing, and matrix deposition have been studied by pulse‐chase analyses of patient fibroblast cell cultures.^44,45,218 The interpretation of such pulse‐chase studies, however, is complicated by the presence of normal fibrillin‐1 produced from the wild type allele, which cannot be distinguished from the mutant product. In order to study the fate of mutant fibrillin‐1, a recombinant system has been developed using a fibroblast host cell.²¹⁹

A number of FBN1 mutations have been shown to increase the susceptibility of recombinant fibrillin‐1 polypeptides to in vitro proteolysis (table 5​5).). Many of the FBN1 mutations investigated in in vitro assays to date are predicted to reduce the calcium binding affinity of the cbEGF module harbouring the mutation. It has been shown by methods including two‐dimensional NMR spectroscopy²¹⁶ and N‐terminal sequencing of mutant polypeptide proteolytic fragments,²¹⁷ that FBN1 mutations can cause short and long range conformational deformations. The fact that FBN1 mutations can produce protease‐sensitive sites can be explained by reduced steric hindrance for the proteases caused by a missing calcium ion or other conformational changes.²¹⁷ The molecular effects of calcium‐binding mutations may be influenced by the calcium‐binding affinity of affected cbEGF modules and by the location of the cbEGF module in the protein as well as the neighbouring modules.²¹⁰ This, as well as the observation that different mutations within one cbEGF module can have a differential effect on susceptibility to proteolysis,²²⁸ provides potential explanations for some genotype‐phenotype correlations in MFS. Two mutations affecting the 1–3 disulfide bond of cbEGF30 of fibrillin‐1 had structural effects localised to the N‐terminal end of the mutant domain, with impaired domain packing with the preceding module.²¹² On the other hand, C750G, which affects the 5–6 disulfide bond of cbEGF7, increases proteolytic susceptibility of cbEGF8.²¹⁴

These data can be interpreted to mean that both the residues affected, the localisation within the cbEGF module, the localisation within the polypeptide chain of fibrillin‐1, and the nature of the neighbouring residues and the strength or flexibility of interdomain linkages all can be important for determining the effects of FBN1 mutations on proteolytic susceptibility.

While the above observations suggest that proteolysis may play a role in the pathogenesis of MFS, it should be noted that there is no formal proof of this available at the moment. Assuming that increased susceptibility to proteases does play a role in the pathogenesis of MFS, then there are at least two mechanisms that could be important. Proteolytic degradation of mutant fibrillin‐1 monomers could take place following secretion into the extracellular space but before incorporation into the microfibrils, thus reducing the total amount of fibrillin‐1 available for assembly of microfibrils. Alternatively, mutant fibrillin‐1 monomers could be incorporated into microfibrils and then represent a sort of Achilles' heel causing increased susceptibility of the entire microfibrillar structure to proteolysis and fragmentation.²²⁹ These issues, as well as a formal demonstration of the presence of fibrillin‐1 proteolytic fragments in tissues of MFS patients, represent potentially fruitful areas for future research.

The first new insight regarding the pathogenesis of cardiovascular disease derived from the study of mouse strains homozygous for hypomorphic (low expressing) fibrillin‐1 (Fbn1) alleles. They included the mgΔ/mgΔ mice, which express a centrally deleted protein at about 5% of the normal level and die shortly after birth, and mgR/mgR mice, which express full length protein at about 15% of the normal level and die at 3–6 months of age.^10,11,241 That mgΔ/mgΔ mice exhibit only focal disruptions in elastic fibre organisation demonstrated that minimal (if any) residual microfibrillar function is sufficient to support the deposition of extended elastic structures.¹⁰ On the other hand, the longer life span and normal aortic wall architecture of mgR/mgR mice allowed demonstration of the pathogenic sequence that culminates in aneurysm in these animals.¹¹ The secondary events include regional elastic fibre calcification, intimal hyperplasia, and adventitial inflammation, findings that were generally spatially coincident and which were subsequently confirmed in MFS patients as well.²⁴¹ Loss of physical connections between vascular smooth muscle cells and neighbouring elastic fibres that are normally mediated by fibrillin‐1 are believed to contribute to the initiation of this destructive process.²⁴¹ As a result, the morphology and synthetic program of the cells are altered, perhaps in an abortive attempt to reconstitute a normal extracellular environment, and the ensuing remodelling process is characterised by overproduction of multiple structural matrix components and mediators of elastolysis, including MMPs 2 and 9.²⁴¹ Subsequent breach of the internal or external elastic laminae allows infiltration of inflammatory cells into the media, resulting in intense elastolysis that correlates temporally with aneurysm formation and dissection. The critical insights derived from this work are that a normal complement of fibrillin‐1 is needed for elastic fibre homeostasis rather than formation, and that there may be important opportunities to alter the natural history of disease through modification of secondary events including inflammation and matrix destruction.

A full understanding of the pathogenesis of MFS will require further advances in many different areas. We are beginning to understand a range of pathophysiological processes involving protein folding and stability, microfibrillar assembly, proteolysis of mutant fibrillin monomers, secondary effects of fragmentation, loss of smooth muscle cell attachments, and alterations in TGFβ signalling. The challenge for the coming years will be to sort out the relative contributions of these defects to the pathogenesis of the various manifestations of MFS. There is no reason to believe that the molecular pathogenesis of ectopia lentis must be the same as that of long bone overgrowth or aortic dissection, and indeed the fact that the various manifestations of MFS do not always occur together in the same patient suggests that there may be important differences in detailed pathogenetic mechanisms.

We still do not know enough about the molecular mechanisms in MFS to develop a model that would explain the sequence of events leading from a mutation in FBN1 to clinical disease. Clinical observations and experimental studies using in vitro systems, cell culture models, or animal models have demonstrated multiple abnormalities, but it is not entirely clear how the sequence of pathogenetic events seen in MFS occurs. One interpretation of the available evidence is that FBN1 mutations lead to either a microfibrillar assembly defect with reduced levels of fibrillin‐rich microfibrils in tissues, or that mutant fibrillin monomers are incorporated into microfibrils and cause a slow but progressive loss of microfibrils. By whatever mechanism this occurs, the loss of fibrillin‐rich microfibrils then has a series of other secondary effects, including a disturbance of the ability to sequester TGFβ and modulate its activity appropriately. Disturbances in the ability of microfibrils to adequately sequester other growth factors may also be important. Increased levels of MMPs may be a result of increased TGFβ activity, of phenotypic alteration of vascular smooth muscle cells owing to loss of cell attachments that are normally mediated by fibrillin‐1, or of effects of the fibrillin fragments themselves. Other secondary effects of fibrillin or elastin fragments could include recruitment of macrophages into the aortic wall by chemotactic stimulation, which could contribute to further progression of aneurysm formation.

The study of mutations and genotype‐phenotype correlations in MFS and related disorders may provide clues that will help elucidate the above issues. At present, there are no plausible molecular explanations for most of the genotype‐phenotype correlations that have been observed with FBN1 mutations. For instance, we are not able to explain why a mutation in exon 59 is associated with isolated ectopia lentis¹³² and why other mutations are associated with the severe neonatal form of MFS.¹²⁹

Although our knowledge of the molecular pathogenesis of MFS is still incomplete, the new paradigms emerging from recent studies have the potential to improve treatment for individuals with MFS. The most promising new direction is the pharmacological manipulation of the TGFβ signalling pathway with angiotensin II type 1 receptor blockers such as losartan, which both lowers blood pressure and attenuates TGFβ signalling.²⁴⁸