The Genetics of Soft Connective Tissue Disorders.

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Review in Advance first posted online on May 18, 2015. (Changes may still occur before final publication online and in print.)

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Annu. Rev. Genom. Human Genet. 2015.16. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 05/24/15. For personal use only.

The Genetics of Soft Connective Tissue Disorders Olivier Vanakker,∗ Bert Callewaert,∗ Fransiska Malfait,∗ and Paul Coucke Center for Medical Genetics, Ghent University Hospital, 9000 Ghent, Belgium; email: [email protected]

Annu. Rev. Genomics Hum. Genet. 2015. 16:11.1–11.27 The Annual Review of Genomics and Human Genetics is online at genom.annualreviews.org This article’s doi: 10.1146/annurev-genom-090314-050039 c 2015 by Annual Reviews. Copyright All rights reserved ∗

These authors contributed equally to this article.

Keywords Ehlers-Danlos syndrome, pseudoxanthoma elasticum, cutis laxa, next-generation sequencing, gene discovery

Abstract Over the last few years, the field of hereditary connective tissue disorders has changed tremendously. This review highlights exciting insights into three prototypic disorders affecting the soft connective tissue: Ehlers-Danlos syndrome, pseudoxanthoma elasticum, and cutis laxa. For each of these disorders, the identification and characterization of several novel but related conditions or subtypes have widened the phenotypic spectrum. In parallel, the vast underlying molecular network connecting these phenotypes is progressively being uncovered. Identification and characterization (both clinical and molecular) of new phenotypes within the connective tissue disorder spectrum are often key to further unraveling the pathways involved in connective tissue biology and delineating the clinical spectrum and pathophysiology of the disorders. Although difficult challenges remain, recent findings have expanded our pathophysiological understanding and may lead to targeted therapies in the near future.

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INTRODUCTION


Heritable connective tissue disorders comprise a wide range of pleiotropic diseases that result from genetic defects that perturb extracellular matrix (ECM) assembly and/or homeostasis. Although individually rare, together they represent a significant proportion of human genetic diseases. They affect mainly the skin, the eyes, and the musculoskeletal, cardiovascular, and pulmonary systems, and they are associated with significant morbidity and mortality. The physical effects— which result in chronic and often severe handicaps—combined with the hereditary nature of these disorders represent a significant burden on the quality of life of patients and their families. The study of these entities contributes to understanding of normal connective tissue biology and could lead to etiologically directed therapies where, at present, only symptomatic treatments exist. Moreover, these disorders represent prototypes for the study of common health problems, such as osteoarthritis, osteoporosis, aortic aneurysms, stroke, emphysema, and premature aging. The first comprehensive effort to classify heritable connective tissue disorders was made by Victor McKusick (87) and was based on clusters of signs and symptoms, inheritance patterns, histological changes, and the limited information available on molecular defects. Over the last decade, the large phenotypic variability of these disorders, the emergence of spectra of related phenotypes, and the expanding knowledge of the vast underlying molecular network have demonstrated the limitations of this classification. Today, these disorders are divided into two classes based on the major constituent of the connective tissue involved: collagenopathies (disorders related to collagen) and elastinopathies (disorders related to elastin). The most common collagenopathies include Ehlers-Danlos syndrome (EDS), osteogenesis imperfecta, Alport syndrome, and the chondrodysplasias. Elastinopathies include Marfan syndrome and related disorders, pseudoxanthoma elasticum (PXE), and the cutis laxa (CL) syndromes. This article focuses on diseases of the soft connective tissue. In particular, we review the latest exciting findings from studies of three groups of prototypic heritable connective tissue disorders: EDS, PXE and PXE-associated disorders, and the CL syndromes. These disorders are examples of, respectively, collagenopathies, elastinopathies resulting from perturbed elastic fiber homeostasis, and elastinopathies resulting from defective elastic fiber assembly.

THE CONNECTIVE TISSUE AND EXTRACELLULAR MATRIX The connective tissue can be divided into three major types: loose or soft connective tissues (which embed organs and organ parts), hard connective tissues (bone and cartilage), and blood. Connective tissue is the most abundant tissue in the body and provides a supporting structure. Derived from the mesoderm, it includes but is not limited to cartilage, bone, ligaments, tendons, skin, lungs, parts of the eye, blood vessels, and blood. It consists of a diverse set of cellular and fibrous constituents embedded in a matrix containing four types of macromolecules: collagen, elastin embedded in elastic fibers, glycoproteins, and glycosaminoglycans (GAGs) (Figure 1). Collagens are the major components of the ECM and the most abundant protein in the body, accounting for approximately 25–30% of the total protein mass. Fibrillar collagens (collagen types I, II, III, V, and XI) are a heterogeneous group of glycoproteins in which three parallel polypeptide strands (the collagen α-chains) coil around each other to form a superhelix. The α-chains consist of a triplet repeat Gly-X-Y, where X and Y are often hydroxyproline and proline. Both glycine (the smallest amino acid, which is spaced every third position) and proline (which stabilizes the helix by its ring structure) are essential for helical conformation (92). In each α-chain, the triple helical domain is flanked at the N- and C-terminal ends by noncollagenous regions of variable sequence, size, and shape. These ends often contain recognizable peptide modules found in other ECM 11.2

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Epithelial cell Collagen Elastic fiber Macrophage Capillary Fibroblast


Glycosaminoglycans, proteoglycans, and glycoproteins

Mast cell

Figure 1 Simplified representation of the extracellular matrix.

molecules (37). Assembly of the three α-chains into trimeric procollagen monomers starts with the alignment of the C-terminal domains, which further initiates the formation of the triple helix progressing to the N terminus. Posttranslational modifications, including hydroxylation of specific proline and lysine residues and proteolytical cleavage of the N- and C-terminal propeptides, occur during the processing of the procollagen molecules into mature collagen (118). Further cross-linking strengthens the collagen fibrils into stiff, ropelike structures, providing the tensile strength of connective tissues. Elastic fibers provide resilience and elasticity to the connective tissue and contribute to tissue homeostasis by regulating cytokine signaling (63). Elastic fiber assembly, or elastogenesis, is a complex and incompletely understood multistep process that is subject to strict spatiotemporal regulation and further depends on proper growth factor signaling and mechanosensing. Elastic fibers are composed of parallel-oriented microfibrillar scaffolds on which elastin is deposited (2). Microfibrils consist largely of 150-kDa linear glycoproteins called fibrillins, which organize in a head-to-tail orientation through self-assembly and cysteine and transglutaminase cross-links. Microfibrils interact with many other nonfibrillar components, which can be integral parts of the microfibrils or may associate with them. These proteins have a structural role and/or may be functionally important in microfibril assembly; elastin deposition; interactions with other ECM proteins, including fibronectin and collagen; anchoring to basement membranes and cell surfaces (mainly through integrins); and growth factor sequestration (51, 64, 108). Elastin is synthesized as a monomer, tropoelastin, which is guided by chaperones (elastin-binding proteins) through the secretory pathway. During secretion, tropoelastin monomers undergo self-aggregation, a process called coacervation, into small globules. These globules associate with fibulins that control the coacervation process and interact with latent transforming growth factor β (TGFβ) binding proteins (LTBPs) to guide the globules to the microfibrils, where they coalesce into larger elastin aggregates. Lysyl oxidases and lysyl oxidase–like enzymes covalently cross-link the elastin monomers and ensure further maturation. Glycoproteins form a large group of noncollagenous matrix glycoproteins. As the ground substance of the ECM, they display a variety of functions in tissue morphogenesis and remodeling. Important proteins in this group include thrombospondins, tenascins, fibronectin, vitronectin, laminin, and osteopontin. GAGs mostly aggregate into proteoglycans by linking to a linear core protein. There are a large variety of proteoglycans that differ in their core protein structures and in the number, types, www.annualreviews.org • Genetics of Soft Connective Tissue Disorders


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and lengths of the GAGs. Proteoglycans are highly sulfated and negatively charged and therefore are highly hydrated. This results in elastic properties, compressibility, and shock absorbance. In recent years, it has become clear that the perception of the connective tissue as a static structure is too limited. Given the complexity of this tissue, it has become apparent that it actively participates in several important physiological processes, including cell proliferation, differentiation, migration, development, and survival, acting through various cell signaling pathways, including mechanosensing. For example, Neptune et al. (94) showed that a structural deficiency of the fibrillin-1 protein not only is important in the development of Marfan syndrome but also leads to the dysregulation of the TGFβ pathway, one of the major biological pathways. This dysregulation has been shown to play a pivotal role in the development of aortic aneurysms, emphysema, and muscle hypotonia. These findings changed the concept of Marfan syndrome as a structural connective tissue disorder, and it is now viewed as a condition manifesting perturbed cytokine signaling with widespread developmental abnormalities (24, 38). Because connective tissue is ubiquitously present, it has important functions in blood coagulation (141), platelet adhesion (30, 111), and the immunological barrier (137).


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EHLERS-DANLOS SYNDROME EDS comprises a clinically and genetically heterogeneous group of heritable connective tissue disorders. Its principal clinical features reflect varying degrees of soft connective tissue fragility, affecting mainly the skin, ligaments, blood vessels, and internal organs. The prevalence of EDS is estimated to be approximately 1 in 5,000 births, with no racial predisposition; however, the incidence rises with increased physician awareness. The first attempts to classify EDS resulted in the 1988 Berlin nosology, which recognized 10 subtypes (7). Elucidation of the molecular basis of several types of EDS led to a revision of this classification, established in 1997 as the Villefranche nosology; this version recognized six subtypes, most of which had been identified in fibrillar collagens or their modifying enzymes (8) (Table 1). Over the last several years, the characterization of several new EDS variants has broadened insights into the molecular pathogenesis of EDS by implicating defects in other noncollagenous proteins involved in the biology of the ECM (29) (Table 1). With the ongoing identification of new genes in EDS-related phenotypes, the Villefranche classification is in need of an update, a task that will be performed by a new EDS consortium that was established during the first international meeting on EDS, which was held in Ghent in 2012 (17).

Defects in the Biosynthesis of Fibrillar Collagens Types I, III, and V For many years, EDS was assumed to be a disorder related exclusively to defects in the biosynthesis of fibrillar collagen types I, III, and V, as most of the initially recognized EDS subtypes were shown to result from mutations either in the genes encoding these collagens or in genes encoding enzymes involved in their posttranslational modification. The kyphoscoliotic subtype was the first EDS form to be solved at the biochemical level, and it represents the first human disorder linked to collagen biosynthesis (68, 105, 120). This autosomal recessive disorder, which is clinically characterized by skin hyperextensibility, joint hyperlaxity, early-onset and progressive kyphoscoliosis, and muscle hypotonia, is caused by a deficiency of lysyl hydroxylase 1 arising from homozygous or compound heterozygous mutations in its encoding gene, PLOD1 (40). This enzyme catalyzes hydroxylation of specific lysine residues in collagen to hydroxylysines, which act as precursors for the cross-linking process that is essential for the tensile strength of collagen. Whereas defects in the genes encoding type I procollagen generally result in osteogenesis imperfecta, specific alterations in this molecule have been associated with specific EDS subtypes. For 11.4

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Summary of Ehlers-Danlos syndrome (EDS) subtypes, modes of inheritance, and clinical features

EDS subtype

Inheritance

Gene

Clinical features


Villefranche nosology (8) Classic

AD

COL5A1/COL5A2

Joint hypermobility, skin hyperextensibility, atrophic scarring

Hypermobility

AD

Largely unknown; heterozygosity for TNXB mutations (rarely)

Joint hypermobility, minor skin manifestations

Vascular

AD

COL3A1

Thin, translucent skin; marked bruising; small-joint hypermobility; increased risk for rupture of arteries, bowel, and gravid uterus

Kyphoscoliotic

AR

PLOD1

Kyphoscoliosis, joint hypermobility, muscle hypotonia, skin hyperextensibility

Arthrochalasis

AD

COL1A1/COL1A2

Congenital bilateral hip dislocations, extreme joint hypermobility

Dermatosparaxis

AR

ADAMTS2

Soft, extremely fragile, and redundant skin; marked bruising; short stature; blue sclerae

Cardiac-valvular

AR

COL1A2

Cardiac valvular insufficiency, joint hypermobility, skin hyperextensibility

Vascular-like

AD

COL1A2 (Arg-to-Cys substitutions)

Propensity to rupture medium-sized arteries, joint hypermobility, skin hyperextensibility, osteoporosis

Tenascin-X deficient

AR

TNXB

Skin hyperextensibility, marked bruising, joint hypermobility, normal scarring, muscle weakness, progressive joint contractures

Progeroid

AR

B4GALT7

Loose, elastic skin; marked joint hypermobility; thin scars; hair loss; hypotonia; aged appearance

B3GALT6 deficient

AR

B3GALT6

Skin fragility, joint hypermobility, progressive contractures, spondyloepimetaphyseal dysplasia with bone fragility, severe kyphoscoliosis, intellectual disability

Musculocontractural

AR

CHST14, DSE

Hyperextensible thin skin; congenital contractures of hands and feet in conjunction with joint hypermobility; kyphoscoliosis; dysmorphic features; ocular, gastrointestinal, urogenital, and/or cardiac malformations

FKBP14 related

AR

FKBP14

Marked kyphoscoliosis, myopathy, hearing loss

Spondylocheirodysplastic

AR

SLC39A13

Spondyloepiphyseal dysplasia, mild short stature, hyperelastic thin skin with easy bruising, wrinkled palms, tapered fingers with contractures

EDS with periventricular heterotopia

XL

FLNA

Periventricular heterotopia, joint hypermobility

Periodontitis

AD

Unknown locus at 12p13

Periodontal loss, joint hypermobility, soft skin with anterior tibial plaques

Other

Abbreviations: AD, autosomal dominant; AR, autosomal recessives; XL, X-linked.

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example, COL1A1 or COL1A2 mutations leading to the loss of the cleavage site for procollagen type I amino-proteinase result in the arthrochalasis type of EDS, an autosomal dominant condition characterized primarily by congenital hip dislocation and extreme joint hyperlaxity (121). By contrast, decreased enzyme activity of the procollagen type I amino-proteinase itself, caused by biallelic mutations in its gene, ADAMTS2, is responsible for the recessive dermatosparaxis type of EDS (25). Although both conditions result from abnormal cleavage of the N-terminal propeptides of type I procollagen and incorporation of collagen precursors in the collagen fibrils, the dermatosparaxis type of EDS differs from the arthrochalasis type in its extremely fragile, bruisable, redundant, almost CL-like skin and markedly less severe joint hypermobility (82). Cabral et al. (18) recently showed that some mutations in the most N-terminal part of the type I collagen helical domain also interfere with removal of the N-terminal propeptide, even though they leave the N-proteinase cleavage site intact. These mutations result in a distinct EDS/osteogenesis imperfecta overlap phenotype. Type I collagen has also been associated with two other forms of EDS. The first is a rare autosomal recessive condition referred to as cardiac-valvular EDS, which is caused by total absence of the α2(I) collagen chain, resulting in the production of [α1(I)]3 homotrimers (116). This condition presents in childhood with mild skin and joint hypermobility, osteopenia, and muscular hypotonia but is complicated in adulthood by the development of severe cardiac valve insufficiency, which may require cardiac valve replacement. The second comprises a group of missense mutations in COL1A1 that result in the substitution of an arginine (R) residue with a cysteine (C) residue in the triple-helical domain, leading to the production of α1(I) dimers. Some of these substitutions [p.(R312C), p.(R574C), and p.(R1093C)] were found in patients with an unexpected propensity for arterial rupture (84). Other pro-α1(I) arginine-to-cysteine substitutions [p.(R1036C) and p.(R1066C)] were reported in families with an EDS/osteogenesis imperfecta overlap phenotype without signs of vascular fragility (19). Intriguingly, one specific α1(I) p.(R1014C) substitution was reported in several families with autosomal dominant infantile cortical hyperostosis (Caffey disease), a benign and self-limiting disorder of early childhood that is characterized by systemic inflammation and subperiosteal new bone formation but does not show signs of connective tissue fragility (35). Mutations in the COL5A1 and COL5A2 genes, encoding the pro-α1 and pro-α2 chains of type V procollagen, respectively, are present in the vast majority of individuals with classic EDS, a disorder characterized mainly by the triad of joint hypermobility, skin hyperextensibility, and atrophic scarring (8). Type V collagen is a minor fibrillar collagen that coassembles with type I collagen and acts as a regulator of collagen fibril diameter through the retention of a noncollagenous N-terminal domain of the pro-α1(V) collagen chain (10, 11, 78). Most mutations result in decreased availability of type V collagen in the ECM, caused by either decreased production or decreased secretion of normal type V collagen into the ECM, establishing functional type V collagen haploinsufficiency as the key factor in the pathogenesis of classic EDS (81, 115, 125, 140). The vascular type of EDS, which is characterized mainly by thin, translucent skin; easy bruising; and a high risk for rupture of the arteries, bowel, and gravid uterus, results from mutations in the COL3A1 gene, encoding the pro-α1-chain of type III procollagen. A wide spectrum of COL3A1 mutations have been identified, the majority of which are point mutations leading to substitutions for the obligatory glycine in the triple helical region of the collagen molecule (101, 102, 128).


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Defects in the Biosynthesis of Proteoglycans Recent research has demonstrated that proteoglycans play an important role in the pathogenesis of EDS. Proteoglycans rank among the most important components of cell plasma membranes 11.6

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and extracellular matrices of connective tissues, have a widespread tissue distribution, and are implicated in a wide variety of biological processes, including ECM organization, tissue repair, cell differentiation, proliferation, adhesion, and migration. They are composed of a protein core to which one or more GAG chains are attached, such as chondroitin sulfate (CS), dermatan sulfate (DS), and heparan sulfate (HS). The biosynthesis of these GAGs is a tightly regulated process that is orchestrated by the concerted action of several enzymes (Figure 2). The recent identification of defects in some of these enzymes in patients with EDS-like conditions has highlighted a role for defective GAG synthesis in the pathogenesis of EDS. A rare progeroid form of EDS was the first to be associated with GAG biosynthesis (43, 107). This EDS variant, which is characterized by a progeroid appearance in addition to typical EDS features, is caused by homozygous mutations in B4GALT7, which encodes galactosyltransferase I (or β4GalT7), the enzyme responsible for the addition of a galactose residue to the O-linked xylose on the proteoglycan core protein (31, 97). Biallelic mutations were recently identified in B3GALT6 [which encodes galactosyltransferase II (or β3GalT6), the third enzyme involved in the biosynthesis of the tetrasaccharide linker region] in patients with pleiotropic EDS-like disorders that also present with muscle hypotonia, kyphoscoliosis, spondyloepimetaphyseal dysplasia, bone fragility, and progressive contractures (83, 93). Deficiency of galactolystransferase I and II affects the initial steps in the formation of the GAG chains. Studies on fibroblast cultures from patients with biallelic B3GALT6 mutations provided evidence that these mutations lead to defects in the synthesis of both CS/DS and HS chains. Studies have also identified biallelic mutations in CHST14, encoding dermatan 4-Osulfotransferase 1 (D4ST1), in patients with musculocontractural EDS (85, 90). This form of EDS is characterized by a unique set of clinical features related to generalized connective tissue fragility affecting the skin, joints, and internal organs as well as developmental problems leading to typical dysmorphic features and congenital malformations of multiple organ systems (117). More recently, Muller et al. (91) found a homozygous missense variant in the DSE gene, encoding DS ¨ epimerase 1 (DS-epi1), in a few patients with a phenotype overlapping with musculocontractural EDS. Both D4ST1 and DS-epi1 deficiencies represent defects specifically affecting biosynthesis of DS (55). These defects alter the disaccharide composition of CS/DS GAG chains, eventually resulting in a lack of DS and excess of CS in DS proteoglycans such as versican, thrombomodulin, and the small leucine-rich proteoglycans decorin and biglycan. As a consequence, the functional and structural integrity of these DS proteoglycans—which display a widespread tissue distribution and are important in many processes, including organization of the ECM, wound repair, anticoagulant processes, and cell adhesion—may be compromised. In particular, loss of the normal hybrid CS/DS configuration in decorin is thought to decrease its capacity to regulate the interfibrillar spacing of collagen fibrils and thus lead to disorganized collagen bundle organization (67).

Defects in Other Proteins Tenascin X (TNX) was the first noncollagenous molecule implicated in EDS pathogenesis. Biallelic mutations in the TNXB gene result in a recessive EDS phenotype resembling, but still distinct from, classic EDS (16, 113). TNX is part of a family of ECM proteins with a complex multidomain structure that allows interaction with many other ECM components—including fibrillar collagens (types I, III, and V), fibril-associated collagens (types XII and IV), decorin, and many others— and is considered an important player in the organization of the ECM. The recent observation that complete TNX deficiency may result in a myopathic phenotype, characterized primarily by muscle weakness, argues for a molecular basis for the existence of an EDS-myopathy phenotypic spectrum (100). Studies have shown that TNX regulates the expression of type VI collagen (89) www.annualreviews.org • Genetics of Soft Connective Tissue Disorders


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GAG chain polymerization

Tetrasaccharide linkage region synthesis

Core protein Endoplasmic reticulum

Golgi apparatus

N

Heparan sulfate O n

EXT1/2 EXT1/2 complex

EXTL1/2/3 EXTL1/2/3


Multiple hereditary exostoses I/II

B3GAT3 Glucoronosyltransferase I

B4GALT7 Galactosyltransferase I

B3GALT6 Galactosyltransferase II

XYLT1 XYLT2 Xylosyltransferase I/II

C N

Chondroitin sulfate

S * 6 O n

Temtamy preaxial brachydactyly syndrome Autosomal recessive Larsen syndrome

CHSY/CHPF complex

*CHST3 Chondroitin 6-sulfotransferase

EDS progeroid type EDS β3GalT6-deficient type C

Glucoronyl C5 epimerization

Dermatan sulfate

S ** 4

Larsen-like syndrome B3GAT3 type

N

O

**CHST14 D4ST1

EDS musculocontractural type C

Glucuronic acid (GlcA)

Xylose (Xyl)

N-Acetylgalactosamine (GalNAc)

Amino acid Serine residue

Galactose (Gal)

N-Acetylglucosamine (GlcNAc)

Iduronic acid (IdoA)

S Sulfate

Figure 2 GAG synthesis. Synthesis is initiated by the attachment of a common tetrasaccharide linker region to a specific serine residue of the core protein. This linker region is synthesized by the stepwise action of specific enzymes: xylosyltransferase I and II (encoded by XYLT1 and XYLT2, respectively), galactosyltransferase I and II (encoded by B4GALT7 and B3GALT6, respectively), and glucuronyltransferase I (encoded by B3GAT3). After completion of the linker region, CS is formed by the alternating addition of GalNAc and GlcA residues, subsequently modified by several sulfotransferases. The formation of DS requires the epimerization of GlcA toward IdoA, which is accomplished by DS-epi1 and DS-epi2 (encoded by DSE and DSEL, respectively). This allows D4ST1 (encoded by CHST14) to catalyze the 4-O-sulfation of GalNAc, which prevents back-epimerization of the adjacent IdoA. Abbreviations: CS, chondroitin sulfate; D4ST1, dermatan 4-O-sulfotransferase 1; DS, dermatan sulfate; DS-epi1 and -2, dermatan sulfate epimerase 1 and 2; EDS, Ehlers-Danlos syndrome; GAG, glycosaminoglycan; GalNAc, N-acetylgalactosamine; GlcA, glucuronic acid; IdoA, iduronic acid.

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and interacts with type XII collagen, a member of the FACIT (fibril-associated collagens with interrupted triple helices) group (136). Mutations in genes encoding type VI collagen cause Ullrich congenital muscular dystrophy and Bethlem myopathy, which typically present with moderate to severe muscle weakness, joint hypermobility, and distal joint contractures (12), and mutations in genes encoding type XII collagen were recently shown to result in an EDS/myopathy overlap phenotype (148). Further evidence for a molecular link between EDS and myopathies has come from proteomics studies that identified an interaction between the conserved α1(V)-N-propeptide and type VI collagen (124). Two other novel autosomal recessive EDS variants were recently identified that show extensive clinical overlap with the kyphoscoliotic type of EDS and with forms of EDS resulting from abnormal GAG synthesis. One of these autosomal recessive variants is the spondylocheirodysplastic form of EDS, characterized by skin hyperextensibility, small-joint hypermobility with tapered fingers and a tendency toward contractures, and a mild skeletal dysplasia (36). Biallelic mutations were identified in the SLC39A13 gene, encoding the transmembrane zinc transporter ZIP13, which controls intracellular Zn2+ distribution. Jeong et al. (57) have postulated that loss of function of this transporter leads to sequestration of Zn2+ within the vesicles and decreased availability of Zn2+ for multiple Zn2+ -dependent processes. The other novel autosomal recessive EDS variant is characterized by severe progressive kyphoscoliosis, muscle hypotonia and myopathy, joint hypermobility, hyperelastic skin, and sensorineural hearing impairment. This condition is caused by mutations in FKBP14, encoding an endoplasmic reticulum (ER)–resident protein, FKBP22, belonging to the family of FK506-binding peptidyl-prolyl cis-trans isomerases (4). ER-resident FKBPs have been suggested to act as folding catalysts by accelerating cis-trans isomerization of peptidyl-prolyl bonds and to act occasionally as chaperones. The wide connective tissue involvement in the affected patients is attributed to a disturbance of protein folding in the ER that affects one or more components of the ECM.

PSEUDOXANTHOMA ELASTICUM PXE is an autosomal recessive disease resulting from mineralization and fragmentation of elastic fibers (132). Patients present with a triad of skin (papular lesions in flexural areas), ocular (angioid streaks with a propensity for subretinal neovascularization and hemorrhage), and cardiovascular (occlusive peripheral vessel disease) symptoms that can vary significantly in severity (Figure 3a). Since the discovery 15 years ago that mutations in the ABCC6 gene cause PXE (75), a continuous effort has been made to elucidate the molecular features and processes underlying this enigmatic disease. Although the precise mechanisms through which elastic fiber mineralization occurs in PXE remain to be clarified, its molecular etiology has become increasingly complex, evolving from strictly monogenic to a polygenic spectrum of diseases. Furthermore, recent insights into cellular pathways involved in PXE have begun to integrate observations from the last two decades.

From Monogenic Disease to a Polygenic Spectrum of Phenotypes The first causal gene for PXE, ABCC6, was identified in 2001 (74). The mutation spectrum was characterized over the next several years, with particular interest in genotype-phenotype correlations, although none could be described at that point (104). Methodologies such as multiplex ligation-dependent probe amplification and next-generation sequencing were introduced to optimize diagnostic efficiency (26, 47). The yield of an ABCC6 mutation analysis reaches 90–95%, leaving 5–10% of patients with an incomplete or undefined genotype. Both observations indicated that other genes may be involved in PXE. www.annualreviews.org • Genetics of Soft Connective Tissue Disorders


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In 2007, a first part of the PXE phenotypic spectrum was unveiled with the identification of a PXE-like disease with coagulation factor deficiencies (134). It not only introduced a second gene, GGCX, encoding a gamma-carboxylase in the vitamin K (VK) cycle, but also stipulated the importance of VK-dependent inhibitors of mineralization such as matrix Gla protein (MGP) in PXE. Mutations in GGCX cause several phenotypes, from isolated coagulation deficiencies to multisystem diseases, the latest of which is characterized by the unique combination of PXE with retinitis pigmentosa (61, 138). The mechanisms behind this plethora of phenotypes are unknown.


a

PXE phenotype

i

ii

iii

iv

v

vi

b

PXE pathophysiology

Oxidative stress Neovascularization

VEGF

Apoptosis

MMP9 Caspases

Liver and fibroblasts

Fibroblasts cMGP

cMGP

BMP2 pERK

Liver ?

VK ucMGP

TCF/LEF β-Catenin

ATP pE

TNAP

ENPP1

RK

Pi

PPi

PPi + AMP AMP

OPN OC

WNT DLX5

MSX2

CD73 Adenosine + Pi

Smads

TGFβ2 Smads

Fetuin A

CTGF

Elastic fiber mineralization 11.10

ABCC6

GGCX

Smads

RUNX2

?

ATP

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Because of the striking similarities between their respective phenotypes, an interaction between ABCC6 and GGCX seemed plausible, which was corroborated by the report of a patient harboring a functional GGCX single-nucleotide polymorphism (SNP) along with two causal ABCC6 mutations (133). His phenotype was a perfect blend of PXE and PXE-like features at the clinical, histological, and biochemical levels and suggested that GGCX variants act as modifiers of PXE. The importance of GGCX as a causal gene was further underlined when families were described in whom PXE patients harbored one ABCC6 and one GGCX mutation, introducing the concept of digenic inheritance (76). Knowledge of the genetic etiology of PXE was further expanded by the discovery of mutations in the ENPP1 gene (encoding the enzyme ectonucleotide pyrophosphatase phosphodiesterase 1), which cause rare cases of PXE (95). ENPP1 mutations were long known to cause generalized arterial calcification of infancy (GACI), in which extensive vessel calcification and cell proliferation lead to early demise in infancy owing to a lack of the potent calcification inhibitor inorganic pyrophosphate (PPi ) (95). In addition to providing a molecular diagnosis in a small proportion of PXE patients with a wild-type ABCC6 genotype, the description of ENPP1 mutations gave fresh impetus to PXE research, leading investigators to explore the relevance of PPi metabolism in multisystemic mineralization, as detailed below. Besides the expansion of causal mutations, a growing interest in modifier genes has emerged in an attempt to explain the marked clinical variability and make patient management more personalized and effective (49). The hunt for genetic modifiers is difficult, not least because of the inconsistencies in defining phenotypes and the resemblance to susceptibility factors. Whereas the latter evoke mostly the risk of developing a disease, a truly useful genetic modifier enables one to ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 3 Phenotypic characteristics and pathophysiological pathways in PXE. (a) Examples of phenotypic characteristics. The main clinical features include (i ) papular lesions in flexural regions, (ii ) formation of additional skin folds, and (iii ) yellowish mucosal lesions. Fundoscopic findings include (iv) peau d’orange and angioid streaks (white circle and arrows, respectively), (v) comet tails (white arrows), and (vi ) subretinal hemorrhage. (b) Schematic and simplified overview of the current knowledge regarding the cellular mechanisms underlying PXE and its associated disorders. Upregulated mediators are in green boxes; downregulated mediators are in orange boxes. In the liver vasculature, decreased levels of ATP, possibly caused by an ABCC6-mediated mechanism, result in a lower production of the calcification inhibitor PPi by ENPP1. GGCX, TNAP, and CD73 can be expressed both in the liver and in peripheral tissues. Although loss of function of GGCX is present in the PXE-like disease with coagulation factor deficiency, in PXE mainly peripheral carboxylation of proteins such as MGP is disturbed owing to low serum VK levels. Together with chronic oxidative stress, the decreased levels of cMGP induce increased BMP2 expression in peripheral tissues. Via Smad and pERK signaling, BMP2 increases expression of the transcription factor RUNX2, leading to transcriptional activation of VEGF (resulting in neovascularization in the eyes), MMP9 and caspases (resulting in an increased apoptosis rate), and the pro-osteogenic mediators OPN and OC. Together, these effects facilitate precipitation of calcium on elastic fibers. RUNX2 expression is also increased through MSX2-WNT signaling via stabilization of β-catenin, which after nucleation increases TCF-1/LEF-1-dependent gene expression. A specific upregulation of TGFβ2 has been noted, which results in an increased MSX2 expression and higher CTGF activity, inducing ectopic mineralization. Finally, the systemic inhibitor of mineralization fetuin A is decreased in PXE patients, creating an environment in which mineralization can occur more easily. Abbreviations: cMGP, carboxylated matrix Gla protein; CTGF, connective tissue growth factor; ENPP1, ectonucleotide pyrophosphatase phosphodiesterase 1; GGCX, gamma-carboxylase; LEF, lymphoid enhancer-binding factor; MGP, matrix Gla protein; MMP9, matrix metalloproteinase 9; OC, osteocalcin; OPN, osteopontin; pERK, phosphorylated ERK; PPi , inorganic pyrophosphate; PXE, pseudoxanthoma elasticum; TCF, T cell factor; TGFβ2, transforming growth factor β2; TNAP, tissue nonspecific alkaline phosphatase; ucMGP, uncarboxylated matrix Gla protein; VEGF, vascular endothelial growth factor; VK, vitamin K. www.annualreviews.org • Genetics of Soft Connective Tissue Disorders


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better predict the course of a given symptom and/or the effectiveness of treatment. Many of the genes initially thought of as genetic modifiers of PXE—e.g., OPN, XYLT1, and XYLT2—can only be considered susceptibility genes, with little or no utility in clinical management (41, 114, 145). In addition to the GGCX SNPs mentioned above, which can elicit a more severe skin phenotype, variants in the VEGFA gene (encoding vascular endothelial growth factor A) are considered to have a modifying effect on retinopathy; two independent studies reported several VEGFA SNPs to be related to a higher need for anti-VEGF treatment and/or an increased propensity for vision loss (144).

The ABCC6 Transporter Annu. Rev. Genom. Human Genet. 2015.16. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 05/24/15. For personal use only.

Although the identification of the ABCC6 gene was a significant advance in the understanding of PXE, it also led almost immediately to two enigmas that have dominated PXE research ever since and have still not been completely solved. First, ABCC6 encodes MRP6, an ATP-dependent membrane transporter that has an obscure function and unknown substrates and is seemingly unrelated to the ECM changes seen in PXE. Second, this transporter is most abundant in the liver and kidney—both unaffected in PXE—and nearly absent in the tissues involved in the PXE phenotype (106). Despite numerous attempts, the identification of the ABCC6 substrates remains troublesome. A recent suggestion favored VK—based on the low levels of VK in patients’ serum— but was abandoned because VK supplementation did not affect the PXE phenotype in Abcc6−/− mice (135). Careful review of the results detailed by Brampton et al. (13) shows that VK serum levels in Abcc6−/− mice given VK supplements reach a maximum that is considerably lower than that of wild-type mice, suggesting that an abolished ABCC6 protein does influence VK metabolism. This was also shown in a recent molecular docking study, which in silico determined the presence of two substrate binding sites using previously reported low-affinity substrates of ABCC6 (48). Both binding sites were able to bind VK1 and VK2 but not VK3, which is the only form of VK that was specifically demonstrated not to be transported by ABCC6. Despite a list of possible substrates derived from docking experiments using the human metabolome database, the precise physiological spectrum of substrates remains a mystery. The role of ABCC6 in more common disorders, such as stroke, chronic kidney disease, and lipid metabolism diseases, placed the need for further insights in a broader perspective (45, 70, 73).

The Pseudoxanthoma Elasticum Pathophysiology: Involvement of TGFβ Signaling and Inorganic Pyrophosphate Insights into the mechanisms underlying elastic fiber calcification in PXE have long been dominated by an abundance of separate histological, cellular, and biochemical findings. Only in the last few years have attempts been made to integrate this knowledge, with mixed success. For a long time, the pathophysiology of PXE has been artificially divided into the metabolic and cellular hypotheses (58, 99). The first departs from the prime hepatic location of ABCC6, stating that inefficient transport of one or more substrates into the vasculature triggers promineralizing events. The second focuses on the role of local factors in the tissues affected by PXE, primarily fibroblasts, their altered behavior, and susceptibility to chronic oxidative stress. Several experiments have supported the metabolic hypothesis, but very recent data from a conditional, liver-specific Abcc6 knockout model seemed to put this hypothesis into question, because soft tissues of this murine model do not calcify at all (147). A more consistent integration of previous findings came with the identification of three pro-osteogenic pathways upregulated in tissues of PXE knockout mice and patients (46) (Figure 3b). All three were previously described 11.12

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in vascular mineralization, to date the best-studied example of soft tissue mineralization. The prominent role of the so-called BMP2-Smad-RUNX2 pathway has been confirmed independently in different Abcc6-related mice models (119). Upstream, MGP plays an important role as an inhibitor of BMP2; in PXE, the presence of uncarboxylated MGP caused by VK deficiency has been demonstrated in several studies and can (partially) explain the increased BMP2 expression (123). Likewise, oxidative stress enhances BMP2 expression (46). Downstream of BMP2, several target genes of the transcription factor RUNX2 are upregulated in PXE mice and patients, including those encoding osteopontin, osteocalcin, matrix metalloproteinase 9 (MMP9), tissue nonspecific alkaline phosphatase (TNAP), and VEGFA (46) (Figure 3b). Two other signaling cascades that are upregulated in PXE—TGFβ2-CTGF and MSX2-WNT—were more surprising, as was the noninvolvement of many other pathways previously described in vessel mineralization (46). Undoubtedly, these cellular events are the tip of the proverbial iceberg in PXE pathogenesis. Not only is each of the three highly regulated both up- and downstream, but they also have numerous interactions with each other as well as with other pathways. The way that they were identified depended strongly on previous knowledge from studies of the vasculature, thereby limiting the chance of finding pathways that are present exclusively in multisystem calcification or PXE. This bias can be tackled by non-hypothesis-driven methodologies. At the protein level, metabolomics studies have pointed toward the importance of ECM remodeling and oxidative stress as well as a role for ectonucleotides, with particular interest in PPi (71). Jansen et al. (56) showed that Abcc6−/− mice and PXE patients have low PPi serum levels and that ABCC6 dysfunction—by an unidentified mechanism—negatively influences PPi efflux from hepatocytes into the hepatic vasculature. Although this certainly confirms PPi as a novel player in the already complex PXE pathophysiology, its place among the other pathophysiological observations and its relative importance are not well understood. Indeed, similar low PPi levels have been described in GACI and other ectopic mineralization diseases, yet their phenotypes are strikingly different from those of PXE in age of onset, histological and clinical characteristics, natural history, and outcome. Conflicting results on PPi serum levels may reflect the technical challenges of accurate PPi measurement, but they have also brought more questions than answers. More than 100 years after PXE’s initial description, every novel piece of the puzzle seems to make its pathophysiology even more puzzling. Although the cohesion between all findings remains unclear, significant advances have been made in the clinical management, diagnostic efficiency, and pathophysiological understanding of PXE.

CUTIS LAXA The CL syndromes are multisystem disorders that share loose redundant skin folds as a common hallmark clinical feature (Figure 4). In light microscopy, skin biopsies are characterized by severe elastic fiber fragmentation (23). The underlying molecular defects in these heterogeneous disorders involve all steps in elastic fiber assembly and therefore provide a basis for elucidating this complex process (Figure 5).

Mutations in Extracellular Matrix Components Mutations in genes encoding extracellular proteins that are structural components of the elastic fibers or are directly involved in their assembly typically affect several elastic tissues and cause pleiotropic manifestations, including aortic root dilatation, pulmonary emphysema, and gastrointestinal and bladder diverticula. As a prime example, autosomal dominant cutis laxa (ADCL) is caused by mutations in the ELN gene, which encodes elastin (20, 146). Patients with ADCL www.annualreviews.org • Genetics of Soft Connective Tissue Disorders


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XLCL

ARCL

ADCL

Occipital horn, genitourinary diverticula

ELN

ATP7A

Emphysema, genitourinary/ gastrointestinal diverticula


LTBP4

Arterial tortuosity, aneurysms FBLN4

FBLN5

SLC2A10

Skeletal malformation, delayed neuromotor development Osteoporosis, normal neuromotor development GORAB (SCYL1BP1)

ARCL type 1c

ARCL type 1a

ARCL type 1b

Progeroid, choreoathetosis, corpus callosum agenesis, cataract PYCR1

ALDH18A1

ARCL type 2b/ type 3a

ARCL type 3b

ATS

Cobblestone brain dysgenesis, Macrocephaly, glycosylation defects alopecia ATP6V0A2 (PYCR1)

RIN2

Geroderma osteodysplasticum

MACS/RIN2 ARCL type 2a (2b)

Figure 4 Clinical approach to the diagnosis of inherited forms of cutis laxa. The main features are shown in brown. Genes encoding proteins of the extracellular matrix are in green, genes involved in trafficking defects are in black, genes involved in metabolic pathways are in blue, and transporters are in orange. Abbreviations: ADCL, autosomal dominant cutis laxa; ARCL, autosomal recessive cutis laxa; ATS, arterial tortuosity syndrome; MACS, macrocephaly, alopecia, cutis laxa, and scoliosis; XLCL, X-linked recessive cutis laxa. Clinical photographs are from References 20–22, 33, 109, and 126.

manifest the disorder with variable severity and are prone to aortic root dilatation and emphysema (39). Typically, the mutations shift the reading frame at the 3 terminus, resulting in a readthrough in the 3 untranslated region. The resulting protein, stably deposited in the ECM, has a variably sized missense sequence and is elongated at its C terminus. The mutant protein has altered physicochemical properties that cause the tropoelastin monomers to coacervate early, precluding correct globule deposition on the microfibrils. This impairs its maturation to insoluble elastin (20). Moreover, the C terminus, rather that the N terminus, has been implicated in microfibril binding (96). This contrasts with the pathogenesis in supravalvular aortic stenosis and William-Beuren syndrome, which result from loss-of-function mutations and reduced elastin expression. In these disorders, reduced formation of normal elastic fibers induces increased smooth muscle cell proliferation and stenosis (130). Notably, some patients with acquired forms of CL harbor the p.(G773D) variant at the C terminus of the ELN gene, predisposing the elastin to inflammation-induced destruction (50). One patient with ADCL was reported to harbor a tandem duplication within the FBLN5 gene (encoding fibulin 5) that results in overexpression of the mutant protein (86).

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Biallelic mutations in FBLN5 cause autosomal recessive cutis laxa (ARCL) type 1a and cause a more severe phenotype, with life-threatening developmental emphysema that often manifests in infancy (80). Stenoses of the large arteries and diverticula of the bladder and bowel occur. Urban-Rifkin-Davis syndrome (also known as ARCL type 1c), a very similar CL disorder with perturbed lung, gastrointestinal, and genitourinary development, is caused by homozygous or compound heterozygous mutations in LTBP4 (21, 129). LTBP4 is a member of the LTBP family that shows structural overlap with the fibrillins. LTBP isoforms 1, 2, and 4 bind a small latent TGFβ complex consisting of a latency-associated protein containing an inactive TGFβ dimer, resulting in the sequestration of TGFβ (for a review on LTBPs, see 127). Dabovic et al. (27) have suggested that in lung tissue, LTBP4 guides the FBLN5-coated elastin globules to the microfibrils, and that this mechanism is responsible for the phenotypic overlap between FBLN5- and LTBP4related diseases. By contrast, biallelic mutations in FBLN4 have been associated with a primary cardiovascular phenotype with severe arterial tortuosity, arterial stenosis, and aneurysm formation (53, 109). Although both FBLN4 and FBLN5 are involved in elastic fiber formation and are able to bind N-terminal fibrillin-1, lysyl oxidases, and tropoelastin, the different disease manifestations in humans and mice must reflect some functional and spatiotemporal differences (for a review of these fibulins, see 98). Interestingly, FBLN4-, ELN-, and LTBP4-related forms of CL (but also ATP6V0A2-related CL—see below) all show evidence of increased TGFβ signaling (20, 21, 109). Deregulated TGFβ signaling was convincingly shown in the pathophysiology of thoracic aortic aneurysm disorders, including Marfan syndrome (caused by mutations in the FBN1 gene, encoding fibrillin-1) and Loeys-Dietz syndrome (caused by mutations in TGFBR1 and TGFBR2, encoding transforming growth factor receptors 1 and 2). In mice models for Marfan syndrome, a main pathomechanism pointed to increased TGFβ signaling in several manifestations of connective tissue pathology, including arterial aneurysms, emphysema, and muscular hypotrophy (24, 38, 94). Moreover, in mice, clinical manifestations can be attenuated by treatment with losartan, an angiotensin II receptor 1 inhibitor with TGFβ-antagonizing effects (15, 24, 38, 94). In Marfan syndrome, fibrillin-1 microfibrils sequester TGFβ in the ECM through interaction with the large latent complex, consisting of an LTBP molecule bound to a latency-associated protein containing a TGFβ dimer (127). As a consequence, mutations in the gene encoding fibrillin-1 would disturb the sequestration of TGFβ in the ECM and increase TGFβ release from the ECM. However, in Loeys-Dietz syndrome and several related thoracic aneurysm disorders, inactivating mutations found in TGFBR1, TGFBR2, SMAD3, and TGFB2 also result in increased TGFβ signaling, an intriguing paradox (77, 79, 131). As such, the exact spatiotemporal regulation of TGFβ signaling is still largely unsolved, and therefore the consequences of perturbed TGFβ signaling in different developmental stages and processes are poorly understood. In this context, the pathogenesis of arterial tortuosity syndrome (ATS), a disease closely resembling FBLN4-related CL, could be of interest. ATS is caused by mutations in SLC2A10, encoding the facilitative glucose transporter GLUT10 (69). Although the exact substrate(s) and localization of the transporter are still a matter of debate, studies on human aortic tissue have shown increased TGFβ signaling (69), whereas slc2a10 morpholino knockdown in a zebrafish model resulted in decreased TGFβ signaling in early stages (142), supporting the hypothesis that initial downregulation of TGFβ signaling is followed by an upregulation caused by defective ECM assembly. Furthermore, in ATS, it is hypothesized that defective GLUT10 impairs dehydroascorbic acid import into the ER and mitochondria, perturbing hydroxylation of ECM proteins as collagens and elastin as well as cellular respiration and reactive oxygen species production (142). These mechanisms may crosstalk with TGFβ signaling as well.



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Mutations Affecting Elastin-Modifying Enzymes


Copper, a cofactor for lysyl oxidase, is imported into the Golgi complex by the copper-transporting P-type ATPase, encoded by the ATP7A gene. Mutations in ATP7A result in occipital horn syndrome, formerly known as EDS type 9 (60). This X-linked disorder presents with variable neurological involvement and is part of a wider disease spectrum, with Menkes diseases at its severe end. In congruence with lysyl oxidase function, occipital horn syndrome affects both elastic fiber and collagen production. It is characterized by wrinkly skin, bladder diverticula, tortuosity of intracranial arteries, autonomous nervous system dysfunction, and pathognomonic formation of

RER

NUCLEUS

i. COG7 SER

Retrograde transport

viii. Cu2+ transporter ATPase

ii. ATP6V0A2

GOLGI APPARATUS

xi. GLUT10

MITOCHONDRION

SECRETORY VESICLE

iv. P5CS iii. PYCR1 Coacervating elastin

x. GORAB

Elastin globules

ENDOSOME

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bony exostoses (including occipital horns at the insertion of the trapezius muscle). Pili torti are typically seen on light microscopy of hair follicles. It is interesting that mutations in this copper transporter also induce manifestations beyond the connective tissue, including the central and peripheral nervous system and pigmentation (59, 62, 103). Some of the findings can be explained by decreased availability of copper for several copper-dependent enzymes. For example, deficiency of peptidyl-α-amidating mono-oxygenase accounts for reduced bioactivity of a variety of neuroendocrine peptides; deficiency of tyrosinase causes pigmentory abnormalities; and deficiency of dopamine-β-hydroxylase, which is necessary for the conversion of dopamine to norepinephrine in norepinephrinergic neurons, may induce dysautonomia. However, the pathophysiology of this spectrum of disorders also implies a role for ATP7A in axonal outgrowth, synapse integrity, and neuronal activation. For instance, impaired translocation of ATP7A from the Golgi network to the plasma membrane may reduce copper release from axons and the neuromuscular junction, providing a possible mechanism for the dyingback axonopathy in isolated X-linked motor neuropathy, an allelic disorder that results from a different set of mutations within the ATP7A gene (62; for an overview, see 59).

Mutations Affecting Cellular Trafficking Mutations that affect cellular trafficking result in CL syndromes with neurological and skeletal involvement. ATP6V0A2-related CL (ARCL type 2a), comprising the phenotypic continuum of the historically denominated Debré type and the milder wrinkly skin syndrome, shows typical craniofacial characteristics, variable skin features (from obvious CL to confined areas of wrinkly skin), variable neurological manifestations (microcephaly, neuromotor delay, cobblestone cerebral dysgenesis, and epilepsy), and skeletal involvement (congenital hip dislocation, scoliosis, and even soft tissue calcification) (66). All patients have N-glycosylation anomalies that seem to be specific to this type of CL. Although the mechanisms are still poorly understood, it appears that ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 5 Molecular targets in cutis laxa syndromes. Elastin is synthesized as tropoelastin monomers that undergo hydroxylation and coacervation through the secretory pathway. Small elastin globules are linearly deposited on a preexisting microfibrillar scaffold. These aggregates are further cross-linked by lysyl hydroxylases to mature insoluble elastin. (i ) COG7 and (ii ) the H+ -transporting protein ATP6V0A2 interfere with vesicle trafficking, mostly of retrograde Golgi transport, and lead to congenital disorders of glycosylation with cutis laxa. In addition, ATP6V0A2 defects reduce acidification of the secretory vesicles containing elastin. This enhances coacervation, precluding regular and linear deposition of small globules of elastin on the microfibrillar fibers. (iii ) PYCR1 and (iv) P5CS (encoded by PYCR1 and ALDH18A1, respectively) are mitochondrial enzymes that are important in glutamate and proline metabolism in the Krebs cycle and are localized in the mitochondria. Defects in PYCR1 result in ARCL types 2b or 3a, whereas defects in ALDH18A1 result only in ARCL type 3b. (v) FBLN4 interacts with LOXL1 and elastin, guiding deposition on the microfibrillar scaffold. FBLN4 deficiency results in ARCL type 1b. FBLN5-elastin-LOX complexes interact with LTBP4, which directs the complex to the microfibrils. (vi ) FBLN5 and (vii ) LTBP4 defects result in ARCL types 1a and 1c, respectively. LOX and LOXL1 further cross-link the elastin to mature insoluble elastin. (viii ) LOX and LOXL1 are copper-dependent enzymes, and a deficiency in the Cu2+ transporter ATPase (encoded by ATP7A) leads to low copper levels in the serum and Golgi apparatus, causing defective cross-linking of elastin and collagens that results in the X-linked form of cutis laxa. (ix) Defects in RIN2 interfere with early endosome formation. (x) GORAB (encoded by SCYL1BP1) is a Rab-interacting kinesin involved in vesicle trafficking to the plasma membrane. (xi ) The facilitative glucose transporter GLUT10 (encoded by SLC2A10) is a transporter with affinity for dehydroascorbic acid that is likely localized to the mitochondria and endoplasmic reticulum. This has been suggested to result in decreased elastin hydroxylation and impaired mitochondrial functioning, with upregulation of the reactive oxygen species pathway. Abbreviations: ARCL, autosomal recessive cutis laxa; ATP6V0A2, ATPase 6 V0 domain subunit A2; COG7, component of oligomeric Golgi complex subunit 7; FBLN4 and -5, fibulin 4 and 5; GLUT10, glucose transporter 10; GORAB, Rab6-interacting golgin; LOX, lysyl oxidase; LOXL1, lysyl oxidase–like 1; LTBP4, latent transforming growth factor β binding protein 4; P5CS, 1 -pyrroline-5-carboxylate synthase; PYCR1, pyrroline-5-carboxylate reductase 1; RER, rough endoplasmic reticulum; RIN2, Ras and Rab interactor 2; SER, smooth endoplasmic reticulum. www.annualreviews.org • Genetics of Soft Connective Tissue Disorders


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mutations in ATP6V0A2 (encoding the α2 subunit of the V0 complex of the H+ ATPase) cause reduced acidification of trafficking vesicles in dermal fibroblasts. Increased pH induces increased coacervation of elastin, and hence interferes with proper cross-linking and maturation of elastin and may affect the functioning of glycosylation enzymes (52). Furthermore, vacuolar-type ATPase may affect membrane fusion of vesicles and retrograde Golgi transport, as shown by the delayed Golgi collapse following brefeldin A treatment of ATP6V0A2 mutant fibroblasts (32). Patients with mutations in COG7 (encoding component of oligomeric Golgi complex subunit 7) present a more severe tableau, with severe neurological manifestations, intrauterine growth restriction, and cholestatic liver disease, often causing early demise (143). COG function is important for Golgi function and structure and for vesicular trafficking. Retrograde Golgi transport is more affected than anterograde transport, which may cause glycosylation enzymes to be mislocalized, explaining the glycosylation defects in COG7-deficient patients (143). Patients with MACS/RIN2 (macrocephaly, alopecia, cutis laxa, and scoliosis/Ras and Rab interactor 2) syndrome present with distinct facial characteristics, hyperextensible or lax skin, macrocephaly, paucity of hair growth, and scoliosis (3, 126). The RIN2 gene encodes a guanine nucleotide exchange factor that interacts with R-RAS and Rab5 during early endocytosis (112). Gerodermia osteodysplastica, characterized primarily by a bone phenotype (osteoporosis and fractures), CL or wrinkled skin, lipodystrophy, and periodontitis, is caused by mutations in SCYL1BP1, encoding GORAB, a Rab6-interacting Golgi protein (42). This golgin is important in recruiting kinesins required for the trafficking of secretory vesicles to the plasma membrane (42).


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Mutations Affecting Metabolism PYCR1-related CL (ARCL type 2b) causes a variable phenotype, including intrauterine growth retardation, skeletal problems (hip dislocation), and central nervous system involvement (mental retardation, microcephaly, corpus callosum agenesis, and epilepsy) (110). More typical findings pointing to the diagnosis are choreoathetosis and corneal clouding resulting from rupture of the Descemet’s membrane. If the latter feature is present, the disorder is usually classified as de Barsy syndrome (ARCL type 3a) (65). ALDH18A1-related CL (ARCL type 3b) reveals a more severe phenotype within the same spectrum, and typical findings include thin, translucent skin (5, 9). Arterial malformations may be present (33). ALDH18A1 and PYCR1 encode, respectively, the mitochondrial enzymes 1 -pyrroline-5carboxylate synthase (PC5S), involved in glutamate synthesis, and pyrroline-5-carboxylate reductase 1 (PYCR1), involved in proline synthesis. Although reduced levels of intermediary products of the urea cycle may occur in ALDH18A1-related CL (5), the absence of these reduced levels in most patients points to another pathogenesis. Indeed, patient fibroblasts show increased sensitivity to oxidative stress (69), which could connect to the mitochondrial anomalies identified in ATS, as mentioned above (142).

NEXT-GENERATION SEQUENCING IN THE DIAGNOSTICS OF CONNECTIVE TISSUE DISORDERS Over the last few years, large-scale DNA sequencing using next-generation sequencing technology has transformed the field of clinical genetic diagnostics tremendously. Especially for genetically heterogeneous disorders, the traditional gene-by-gene Sanger sequencing methodology, which has always been the gold standard for detecting mutations in human genes, is significantly less efficient, more expensive, and more time-consuming compared with next-generation sequencing. 11.18

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This powerful technology provides a platform to analyze a panel of genes known to be related to a specific phenotype in a single test. Through whole-exome sequencing and whole-genome sequencing, it also allows the identification of the responsible mutation(s) in genes not previously associated with the disease. Whole-exome sequencing is currently a state-of-the-art technology in most molecular genetic labs. An important drawback of the implementation of next-generation sequencing in genetic diagnostics is the detection of a considerable number of variants of unknown significance (VUSs), especially when analyzing larger gene panels. The majority of these VUSs are missense mutations or potential splice-site changes for which the functional outcome is less clear. These VUSs have become a novel challenge in reporting genetic results. There are several ways to investigate the possible pathogenicity of VUSs, such as mining of disorder-specific databases, verification of conservation between species, in silico prediction of the severity of amino acid changes, the use of splice prediction programs, and segregation analysis. These approaches often do not yield convincing results, however, leaving the final interpretation of VUSs in clinically relevant genes to rely on a functional assay. These assays often require evaluations at the protein level or in cellular or in vivo systems, and often no reliable and useful models can be easily generated. Therefore, these functional tests are often expensive and labor intensive and are not routinely available in a diagnostic setting. Recently, the zebrafish has been introduced as a model system for variant validation, mainly in congenital and pediatric disorders (28). The relatively small evolutionary distance from the zebrafish genome to the human genome (compared with invertebrate models), the similarity of several organs and signaling processes, the low cost, the transparency, and the ability to manipulate its genome efficiently make the zebrafish a powerful model. A straightforward approach is to rescue a morpholino knockdown phenotype with both wild-type and mutant human mRNA. The absence of rescue following injection of mutant mRNA but not of wild-type mRNA would then suggest pathogenicity. Recent advances have also been made in the ability to rapidly generate stable mutant lines with the use of zinc-finger nucleases (ZFNs), transcription activator–like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated system (CRISPR/Cas) (6, 54, 88). Therefore, zebrafish models might become a powerful tool to investigate possible disease-causing variants, even in a diagnostic setting, although one should be aware of the limitations of this particular approach.

FUTURE PERSPECTIVES The comprehensive mapping of the genetic defects in several connective tissue disorders now allows investigators to address the phenotypic spectrum and natural history of several entities in more detail. This will help to differentiate overlapping phenotypes and redefine confusing clinical classifications. Furthermore, the study of connective tissue disorders has led to many new insights into the assembly and homeostasis of the ECM, including TGFβ signaling, intracellular trafficking, and proper Golgi and mitochondrial functioning. Many of these mechanisms have repercussions beyond the connective tissue and may also impair skeletal, neurological, and eye development. A thorough understanding of how these novel mechanisms interfere with matrix biology may help to reveal the genetic contribution to the pathogenesis of more common health problems, such as pulmonary emphysema (14, 44, 139), myopathic states (34), age-related macular degeneration (122), motor neuropathy (1, 62), and arterial calcification and stroke (132), thereby providing anchor points for etiologically directed therapies. The functional overlap between the different defects and the extensive crosstalk between involved pathways will represent a huge challenge in finding specific disease modifiers. Indeed, the promise of losartan as a treatment of www.annualreviews.org • Genetics of Soft Connective Tissue Disorders


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Marfan syndrome demonstrated in mouse models did not fully replicate in all human studies, adding to the complexity of ECM biology and interspecies variability (72). Significant advances have been made in the clinical management, diagnostic efficiency, and pathophysiological understanding of soft connective tissue disorders, although the cohesion between all pathophysiological mechanisms remains somewhat blurred. Several therapeutic approaches lure just around the corner, and beyond any doubt, the coming years hold promise for more intriguing and surprising discoveries in the study of EDS, PXE, CL, and related disorders.

DISCLOSURE STATEMENT Annu. Rev. Genom. Human Genet. 2015.16. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 05/24/15. For personal use only.

The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.

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Genetic mapping in the connective tissue disorders.

The red face revisited: connective tissue disorders.

Pneumococcal soft-tissue infections: possible association with connective tissue diseases.

Cardiovascular manifestations of connective tissue disorders.

Esophageal disorders in mixed connective tissue diseases.

[Marfan syndrome and related connective tissue disorders].

Pectus excavatum and heritable disorders of the connective tissue.

Molecular nosology of heritable disorders of connective tissue.

Neurologic manifestations of inherited disorders of connective tissue.

Heritable disorders of connective tissue: Ehlers-Danlos syndrome.

Research perspectives in heritable disorders of connective tissue.

Eating disorders and connective tissue disease. Etiologic and treatment considerations.

Connective tissue disorders: systemic lupus erythematosus, Sjögren's syndrome, and scleroderma.

The connective tissue of lung.

Tissue antinuclear antibodies in renal biopsies of patients with systemic connective tissue disorders.

Pathogenesis of some traumatic and degenerative disorders of soft tissue.

Connective tissue graft combined with autogenous bone graft in the treatment of peri-implant soft and hard tissue defect.

Exome analysis of connective tissue dysplasia: death and rebirth of clinical genetics?

epidermal junction in connective tissue disorders.

Connective tissue disorders associated with vasculitis and vaso-occlusive disease of the hand.

Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate.

Piezogenic papules of the feet in healthy children and their possible relation with connective tissue disorders.

The Clinical Value of Soluble Urokinase Plasminogen Activator Receptor (suPAR) Levels in Autoimmune Connective Tissue Disorders.

Connective tissue disorders and cardiovascular complications: the indomitable role of transforming growth factor-beta signaling.