FBN1: The disease-causing gene for Marfan syndrome and other genetic disorders

Highlights

• FBN1 encodes fibrillin-1, a structural macromolecule for extracellular microfibrils.

• Mutations in FBN1 cause the Marfan syndrome and related disorders.

• Mutations in FBN1 also cause acromelic dysplasias and stiff skin syndrome.

• Abnormal growth factor signaling is implicated in these fibrillinopathies.

Abstract

FBN1 encodes the gene for fibrillin-1, a structural macromolecule that polymerizes into microfibrils. Fibrillin microfibrils are morphologically distinctive fibrils, present in all connective tissues and assembled into tissue-specific architectural frameworks. FBN1 is the causative gene for Marfan syndrome, an inherited disorder of connective tissue whose major features include tall stature and arachnodactyly, ectopia lentis, and thoracic aortic aneurysm and dissection. More than one thousand individual mutations in FBN1 are associated with Marfan syndrome, making genotype–phenotype correlations difficult. Moreover, mutations in specific regions of FBN1 can result in the opposite features of short stature and brachydactyly characteristic of Weill–Marchesani syndrome and other acromelic dysplasias. How can mutations in one molecule result in disparate clinical syndromes? Current concepts of the fibrillinopathies require an appreciation of tissue-specific fibrillin microfibril microenvironments and the collaborative relationship between the structures of fibrillin microfibril networks and biological functions such as regulation of growth factor signaling.

Introduction

FBN1 encodes the gene for fibrillin-1. In humans, there are three different genes (FBN1, FBN2,and FBN3) encoding fibrillins. Fibrillins are large (~ 350,000 MW) structural macromolecules that contribute to the integrity and function of all connective tissues. They are considered to be “structural macromolecules” because, like the collagens, the fibrillins form fibers that are visible in transmission electron micrographs. Unlike the collagens, fibrillins form “microfibrils” with uniform diameters (10–12 nm) that are not periodically cross-striated or “banded”. Fibrillin microfibrils display a characteristic morphology consisting of light and dark or hollow areas that give the appearance of railroad tracks. Fibrillin microfibrils exist as large bundles of microfibrils, as short individual microfibrils (usually in close proximity to basement membranes, for example on the endothelial cell side of the glomerular basement membrane), or as the peripheral microfibril mantle around elastin in all elastic fibers. Typical morphological features of fibrillin microfibrils are shown in Fig. 1. In the various types of connective tissue, fibrillin microfibrils are organized to best suit the functional integrity of the tissue: for example, in skin, elastic fibers form a loose network of interconnecting highways; in the dermis, these highways run parallel to the epidermis with turn-offs coursing perpendicularly up from the deeper elastic fibers to the basement membrane at the dermal-epidermal junction, where bundles of microfibrils intersect the lamina densa; in tendons and perichondrium/periosteum, elastic fibers run parallel to the long axis; in muscular arteries, elastic fibers encircle the lumen.

Although “10 nm microfibrils” had been described as ultrastructural entities, the molecular components of these microfibrils were not known until 1986. Protocols to extract microfibrillar molecules used harsh denaturing conditions as well as disulfide bond reducing agents (Ross and Bornstein, 1969). Reductive guanidine extractions of fetal bovine nuchal ligament, an elastic fiber rich tissue, yielded a 31,000 MW glycoprotein, which was named MAGP (“microfibril associated glycoprotein”) (Gibson et al., 1986). MAGP antiserum localized to elastin-associated microfibrils (Gibson et al., 1986). Today, a number of additional molecules are known to be associated with microfibrils. These molecules have been both immunolocalized to microfibrils and shown to bind directly to fibrillin. These include the fibulins (Reinhardt et al., 1996a, El-Hallous et al., 2007), the LTBPs (Latent TGFβ Binding Proteins) (Dallas et al., 1995, Isogai et al., 2003, Ono et al., 2009), and members of the Adamtslike (Tsutsui et al., 2010, Gabriel et al., 2012, Bader et al., 2012) and Adamts (Kutz et al., 2011) family of proteins.

Initially, it was thought that the main function of microfibrils is to serve as the scaffold for elastic fiber formation. This function was based on morphological studies of developing elastic tissues, which documented that microfibrils appeared first in the embryo, followed by the deposition of amorphous elastin onto the microfibril scaffold (Fahrenbach et al., 1966). Biochemical investigations as well as genetic evidence from both humans and mice have uncovered many more functions of fibrillin microfibrils. Today we know that fibrillin microfibrils perform important tissue-specific architectural functions, beyond serving as scaffolds for elastin deposition. For example, fibrillin microfibrils are specifically required for the structural integrity of both the aortic wall (which contains elastin) and the suspensory ligament of the lens (which does not contain elastin). In addition, over the last decade, a novel and highly significant function of fibrillin microfibrils has emerged: fibrillin microfibrils target and sequester members of the TGFβ superfamily of growth factors. Because this superfamily of growth factors includes > 30 different members, this function diversifies the biological roles performed by fibrillin microfibrils, even though the microfibrils themselves are ubiquitous elements of all connective tissues. Using tissue-specific architectures, fibrillin microfibrils pattern the targeting and sequestration of a variety of growth factors and contribute to organ formation and repair. In this manner, the structures of fibrillin microfibrils collaborate with biological functions to shape and maintain connective tissues, and mutations in fibrillins exert powerful, even opposing, forces on tissue growth and homeostasis.

In the early 1980s, the era of protein discovery was in full swing. Monoclonal antibody technology, developed in 1975 by Kӧhler and Milstein (who shared the 1984 Nobel Prize), was employed to generate specific antibodies that yielded novel immunofluorescence staining patterns on human fetal membrane (amnion and chorion) and skin sections. In a large screen of antibody clones, two yielded patterns suggestive of the microfibrillar component of elastic fibers. These were selected and used to immunoprecipitate a large intra-chain disulfide bonded molecule from the medium of cultured human fibroblasts. The molecule was named “fibrillin” because electron microscopic immunolocalization experiments demonstrated periodic labeling along the lengths of 10 nm diameter microfibrils (Sakai et al., 1986). Monomeric fibrillin molecules, purified from fibroblast medium, were shown to be flexible extended strings with lengths of 148 nm and diameters of 2.2 nm (Fig. 2) (Sakai et al., 1991).

After rotary shadowing and electron microscopy, microfibrils extracted from tissues were visualized as “beaded strings” (Fig. 2) (Keene et al., 1991). These images suggested that monomeric fibrillin molecules likely contribute to the many strings present in the beaded string structure. Based on epitope mapping studies, it was proposed that single molecules of fibrillin-1 are organized in a parallel, head-to-tail fashion within individual microfibrils (Reinhardt et al., 1996b). Eight fibrillin molecules were estimated in each individual microfibril (Baldock et al., 2001).

Since monomeric fibrillin molecules assembled very quickly into disulfide-bonded aggregates (Reinhardt et al., 2000), it is possible that fibrillin also contributes to the globular bead structure. However, other proteins, particularly small globular molecules like MAGP, might contribute to the bead. Immunolocalization of MAGP was shown to be periodic along the length of individual microfibrils, suggesting that it might also be a structural component of the microfibril (Hanssen et al., 2004). Extraction of connective tissue microfibrils with collagenase resulted in extended periods between beads (Baldock et al., 2001). These extended lengths were sometimes greater than the length of single fibrillin molecules. Therefore, molecules in addition to fibrillins may be required to form the backbone structure of microfibrils. Alternatively, a model of staggered fibrillin molecules folding back on themselves was proposed to fit the ultrastructural images (Baldock et al., 2001). However, collagenase cleaved fibrillin molecules at specific sites, and extraction of tissue microfibrils with guanidine yielded microfibrils with much shorter periods (Kuo et al., 2007), suggesting that the long periods seen between beads in collagenase digested microfibrils were due to cleavage events rather than to unfolding or an unwinding of the microfibril structure. Together with additional antibody epitope mapping, a working model with fibrillin-1 molecules arranged head-to-tail and staggered in the beaded string microfibril was proposed (Kuo et al., 2007). Fig. 3A depicts this model.

Fibrillin was first cloned from human placental cDNA libraries using a mixed pool of oligonucleotides representing all coding possibilities for a fibrillin peptide sequence (CEDIDEC) (Maslen et al., 1991). Fibrillin peptide sequences were determined by amino acid sequencing of pepsin-resistant peptides extracted from human tissues and identified using fibrillin monoclonal antibodies (Maddox et al., 1989). The complete deduced amino acid sequence revealed a modular domain structure consisting primarily of Epidermal Growth Factor (EGF)-like domains (with 6 cysteines per domain) and novel domains containing 8-cysteines (Maslen et al., 1991, Corson et al., 1993). Each fibrillin molecule is composed of 47 EGF-like domains, 43 of which are predicted to bind calcium (cbEGF), 7 8-cysteine containing domains (8-cys), 2 “hybrid” domains that share features of both the 8-cysteine domain and the EGF-like domain, a proline-rich domain, and amino- and carboxyl-terminal domains (Fig. 4).

Cloning methods led to the discovery of a second fibrillin (Lee et al., 1991). It turned out that the monoclonal antibodies that were used to first characterize and clone fibrillin had identified fibrillin-1 (Maslen et al., 1991, Maddox et al., 1989). Expression of the gene for fibrillin-2 was largely limited to fetal development (Zhang et al., 1994). Both fibrillin-1 and fibrillin-2 molecules were present within individual fetal microfibrils, indicating that fibrillin microfibrils are heteropolymers (Charbonneau et al., 2003). Fibrillin-2 molecules were found in postnatal tissues but epitopes were masked by fibrillin-1 (Charbonneau et al., 2010a), suggesting that mature microfibril structure involves the accretion of fibrillin-1 molecules around a core of fibrillin-2 molecules that were assembled during fetal development. Mutations in FBN2 were discovered to cause congenital contractural arachnodactyly (now called Distal Arthrogryposis, Type 9). These mutations clustered between exons 23 and 34 (Gupta et al., 2002).

A third fibrillin, fibrillin-3, was also found by cloning methods (Nagase et al., 2001, Corson et al., 2004). Expression of FBN3 was also limited to fetal development, and fibrillin-3 was immunolocalized to fetal microfibrils (Corson et al., 2004). All three fibrillins share the same overall organization of modular domains (Fig. 4). However, in place of the proline-rich domain in fibrillin-1, there is a glycine-rich domain in fibrillin-2 and a proline- and glycine-rich domain in fibrillin-3. Specific functions for these distinctive domains are not known. The second calcium-binding EGF-like domain is missing in fibrillin-3. In mouse, the gene for fibrillin-3 was inactivated during evolution.

Fig. 4 depicts the molecular structures of the three fibrillins and the LTBPs, molecules structurally and functionally related to fibrillins. There are 4 LTBPs, and all four are composed of domain modules found in fibrillins. The 8-cysteine module is present only in the LTBPs and in the fibrillins. Interestingly, the 8-cysteine module in LTBPs is used to form covalent bonds with the small latent complex of TGFβ (Saharinen et al., 1996, Gleizes et al., 1996). Unlike the fibrillins, which are restricted to a very similar size, the LTBPs vary in size and are smaller than fibrillin. LTBPs have been immunolocalized to microfibrils (Dallas et al., 1995, Isogai et al., 2003), and LTBP-1, -2 and -4 bind directly to fibrillin (Isogai et al., 2003, Ono et al., 2009, Hirani et al., 2007). Periodic localization of LTBPs along the lengths of microfibrils has not been reported. Furthermore, because the LTBPs are smaller than fibrillins and variable in size, it seems unlikely that these molecules contribute in the same way as fibrillins to the backbone beaded string structure of microfibrils.