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Heritable Collagen Disorders: The Paradigm of the Ehlers—Danlos Syndrome

      Introduction

      The heritable disorders called Ehlers-Danlos syndrome (EDS) are as disparate as they are fascinating. Their discovery and description has been an intimate part of the growth of our understanding of matrix biology. Joint hypermobility, skin extensibility, abnormal scarring, and tissue friability are the hallmark diagnostic features; however,
      • McKusick V.A.
      Heritable disorders of connective tissue.
      recognized that EDS is under-recognized because when the physical signs are not “classic” the diagnosis may be elusive. The medical and scientific history of EDS can be seen in three phases: clinical characterization, biochemical and molecular genetic analysis, and the use of high throughput genomic analysis to extend the phenotypes.

      Creating the Clinical Spectrum of Eds

      Hippocrates described the features of this disorder in 400 BC; however, the first medical description of some of the characteristics is credited to

      van Meek’ren Ja (1682) De Dilatabilitate Extra-ordinaria Cutis (observationes medico-chirugicae).

      . Tschernogubow’s presentation in 1892 seems to have been largely overlooked in Western Europe, probably because it was written in Russian.
      • Weber F.P.
      Ehlers-Danlos Syndrome.
      is credited with christening the disorder as EDS in 1936 after
      • Ehlers E.
      Cutis laxa, neigung zu haemorrhagien in der haut, lockerung meherer artikulationen.
      and
      • Danlos M.
      Un cas de cutis laxa avec tumeurs par contusion chronique des coudes et des genoux (xanthome juvénile pseudo-diabé-tique de MM Hallopeau et Macé de Lépinay).
      , two dermatologists who separately described affected patients in 1901 and 1908, respectively. The same year, Georg Sack, a German physician, described a condition that Barabas later identified as the arterial type.
      • McKusick V.A.
      Heritable disorders of connective tissue.
      provided the first synthesis of the clinical literature on the multisystemic and variable nature of EDS in his 1956 hallmark work on heritable connective tissue disorders. Over a decade later,
      • Barabas A.P.
      Heterogeneity of the Ehlers-Danlos syndrome: description of three clinical types and a hypothesis to explain the basic defect(s).
      proposed at least three distinct subtypes: classical, varicose, and arterial on the basis of his experience with 27 affected individuals. He suggested that the clinical subtypes reflected discrete etiologies, not simply variable expressions of one disorder. A short time later, Peter Beighton published a series of papers from a landmark clinical investigation of 100 individuals with EDS. He recognized and expanded Barabas’ work to a classification of five types (
      • Beighton P.
      • Price A.
      • Lord J.
      • et al.
      Variants of the Ehlers-Danlos syndrome. Clinical, biochemical, haematological, and chromosomal features of 100 patients.
      ). The first group included individuals similar to those initially described with skin hyperextensibility and fragility, with abnormal scarring and bruising, and striking joint hypermobility (the gravis form). The second was similar but the skin findings were less dramatic (mitis). A hypermobility form in which the joint findings were striking almost to the exclusion of skin changes was the third type. The Sack-Barabas group (the fourth type) had very severe vascular fragility and was at risk of arterial rupture and bowel rupture. The fifth group was thought to be X-linked and was characterized by joint hypermobility and intramuscular hemorrhage (Table 1).
      Table 1Classification of EDS
      Numerical typeDescriptive typeGenesOMIMInheritanceClinical features and notes
      I (Gravis)Classical
      Specific mutations in COL1A1 (substitutions of cysteine for arginine residues within the triple helical domain of proa1(I) chains) have been recognized to cause a form of EDS reminiscent of classical type with joint hypermobility, skin hyperextensibility, bruising and abnormal scarring, and predisposition to aortic aneurysm.
      COL5A1130000ADMarked joint hypermobility, skin hyperextensibility, bruising, and abnormal scarring
      II (Mitis)COL5A2130010
      IIIHypermobility
      One family with a specific mutation in COL3A1 is said to have only joint hypermobility with none of the other consequences seen in EDS type IV.
      TNXB Largely unknown130020ADMarked joint hypermobility, minor skin findings
      IVVascularCOL3A1130050ADThin translucent skin, marked bruising, small joint hypermobility, high risk for rupture of arteries, bowel, and gravid uterus
      VIAKyphoscoliosisPLOD1225400ARJoint hypermobility and kyphoscoliosis recalcitrant to surgical intervention, risk for arterial rupture
      VIBMusculocontracturalCHST14601776ARCongenital contractures of digits, dysmorphic features, kyphoscoliosis and hypermobility, hyperextensible thin skin, ocular involvement
      VIIAArthrochalasia multiplex congenitaCOL1A1130060ADMarked joint hypermobility, bilateral congenital hip dislocation
      VIIBCOL1A2
      VIICDermatosparaxisADAMTS2225410ARSoft, very fragile skin with late onset skin redundancy, blue sclerae, joint hypermobility
      VIIIPeriodontitisProbably heterogeneous; one locus at 12p13130080ADPeriodontal loss, joint hypermobility, soft skin with characteristic plaque on anterior tibial region
      OtherProgeroidB4GALT7130070AR
      Cardiac valvularCOL1A2225320AR (null)Cardiac valvular insufficiency, joint hypermobility, skin hyperextensibility
      FKBP14 relatedFKBP14614557ARMarked kyphoscoliosis, hearing loss, myopathy, short stature, joint hypermobility
      Spondylocheiro dysplasticSLC39A13612350ARSpondyloepiphyseal dysplasia with mild short stature, hyperelastic thin skin with easy bruising, protuberant eyes, bluish sclerae, fine wrinkling on palms
      Tenascin-X deficientTNXB606408ARJoint hypermobility, hyperextensible and sleeve-like character to skin, marked bruising, normal scarring
      Periventricular heterotopiaFLNA300537XLPeriventricular heterotopia, joint hypermobility
      Poorly characterized, disputed, retired, or reclassified
      We have included these previously identified “types” of EDS for historical perspective. They are not included in most classifications.
      VX linkedUnknown305200XLJoint hypermobility with muscle hemorrhage
      VIBBrittle Cornea syndromeZNF46922920ARBlue sclerae, corneal rupture, keratoconus, hyperextensible skin, joint hypermobility
      PRDM5614170
      IXOccipital Horn syndromeATP7A304150XLHyperextensible skin and bruising, hernias and bladder diverticuli, joint hypermobility
      XFibronectin deficientUnknown225310?One reported family with platelet dysfunction and mild features of EDS
      XIFamilial Joint LaxityUnknown147900ADRetired
      Abbreviations: AD, autosomal dominant; AR, autosomal recessive; EDS, Ehlers–Danlos syndrome; XL, X linked.
      1 Specific mutations in COL1A1 (substitutions of cysteine for arginine residues within the triple helical domain of proa1(I) chains) have been recognized to cause a form of EDS reminiscent of classical type with joint hypermobility, skin hyperextensibility, bruising and abnormal scarring, and predisposition to aortic aneurysm.
      2 One family with a specific mutation in COL3A1 is said to have only joint hypermobility with none of the other consequences seen in EDS type IV.
      3 We have included these previously identified “types” of EDS for historical perspective. They are not included in most classifications.

      The First Phase of Biochemical Characterization of Eds

      During the early phase of clinical characterization, clues to the molecular basis of any of the forms of EDS were sparse, with the exception that light and electron microscopy studies identified abnormalities in the structure of collagens in the dermal matrix (
      • Wechsler H.L.
      • Fisher
      Ehlers-Danlos syndrome. Pathologic, histochemical and electron microscopic observations.
      ,
      • Julkunen H.
      • Rokkanen P.
      • Inoue H.
      Scanning electron microscopic study of the collagen bundles of the skin in the Ehlers-Danlos syndrome.
      ). The dam broke in the early 1970s as a result of tissue analysis to assess interactions/cross-links between collagen molecules, animal studies, and use of cultured cells from selected individuals to examine collagen production. Detailed knowledge of the structure of cross-links and the awareness of their importance in tissue integrity provided the background in which the study of sisters with hypermobility, soft extensible skin, scoliosis recalcitrant to surgical intervention, and ocular globe fragility (
      • Krane S.M.
      • Pinnell S.R.
      • Erbe R.W.
      Lysyl-protocollagen hydroxylase deficiency in fibroblasts from siblings with hydroxylysine-deficient collagen.
      ,
      • Steinmann B.
      • Gitzelmann R.
      • Vogel A.
      • et al.
      Ehlers-Danlos syndrome in two siblings with deficient lysyl hydroxylase activity in cultured skin fibroblasts but only mild hydroxylysine deficit in skin.
      ) led to the identification of lysyl hydroxylase deficiency. In these sisters, hydroxylated complex cross-links were diminished and the virtual absence of hydroxylation of lysyl residues in triple helical collagen molecules resulted from deficiency of lysyl hydroxylase. Once the gene (PLOD1) was isolated, sequence analysis demonstrated inactivating mutations, the most common of which was a 7-exon duplication mediated by Alu–Alu recombination (
      • Heikkinen J.
      • Toppinen T.
      • Yeowell H.
      • et al.
      Duplication of seven exons in the lysyl hydroxylase gene is associated with longer forms of a repetitive sequence within the gene and is a common cause for the type VI variant of Ehlers-Danlos syndrome.
      ). This was one of the very early demonstrations of the role of repetitive elements in the genome as mediators of deletions or duplications. This disorder, a recessively inherited condition, was called EDS type VI and was the first established true disorder of collagen biosynthesis and structure in humans.
      Cattle with a curious and severe form of skin fragility provided the next step in the identification of human collagen disorders, as well as insight into the question of how an essentially insoluble molecule—collagen—could be synthesized and secreted from cells. The bovine disorder, dermatosparaxis, appeared to be inherited in an autosomal recessive manner, and biochemical studies demonstrated that affected animals failed to process an amino-terminal precursor peptide from all three chains of type I collagen (
      • Lenaers A.
      • Ansay M.
      • Nusgens B.V.
      • et al.
      Collagen made of extended -chains, procollagen, in genetically-defective dermatosparaxic calves.
      ). As a consequence, collagen fibrillogenesis was impaired and skin integrity compromised (
      • Piérard G.E.
      • Lapière M.
      Skin in dermatosparaxis.
      ). The animal study prompted investigation of tissues from a young woman born with bilateral hip dysplasia/dislocation and very marked joint laxity. Her skin contained a2(I) chains of type I collagen with an extension at the amino-terminal end (
      • Lichtenstein J.R.
      • Martin G.R.
      • Kohn L.D.
      • et al.
      Defect in conversion of procollagen to collagen in a form of Ehlers-Danlos syndrome.
      ,
      • Steinmann B.
      • Tuderman L.
      • Peltonen L.
      • et al.
      Evidence for a structural mutation of procollagen type I in a patient with the Ehlers-Danlos syndrome type VII.
      ). Although initially interpreted to result from enzymatic deficiency, studies later showed that the woman had a heterozygous splice donor mutation in intron 6 of one COL1A2 allele that led to exon skipping and loss of the sequence that contained both the propeptide cleavage site and the amino-terminal nonhelical cross-link site (
      • Steinmann B.
      • Tuderman L.
      • Peltonen L.
      • et al.
      Evidence for a structural mutation of procollagen type I in a patient with the Ehlers-Danlos syndrome type VII.
      ). Consistent with this finding, autosomal dominant inheritance was soon recognized and mutations in the COL1A1 gene that led to loss of the sequences of the homologous exon 6 were found in other affected individuals (
      • Byers P.H.
      • Duvic M.
      • Atkinson M.
      • et al.
      Ehlers-Danlos syndrome type VIIA and VIIB result from splice-junction mutations or genomic deletions that involve exon 6 in the COL1A1 and COL1A2 genes of type I collagen.
      ). Although the outcomes of mutations in the preceding acceptor site and succeeding donor site differ in detail, both lead to the same phenotype. Fewer individuals have COL1A1 mutations because COL1A1 donor site mutations lead to use of a cryptic donor site or intron inclusion, each of which leads to a frame shift, premature termination codon, mRNA instability, and an osteogenesis imperfecta phenotype. More than 20 years after the basis of dermatosparaxis was explained, a human form of the disorder (then called EDS VIIC) was identified (
      • Nusgens B.V.
      • Verellen-Dumoulin C.
      • Hermanns-Lê T.
      • et al.
      Evidence for a relationship between Ehlers-Danlos type VII C in humans and bovine dermatosparaxis.
      ,
      • Smith L.T.
      • Wertelecki W.
      • Milstone L.M.
      • et al.
      Human dermatosparaxis: a form of Ehlers-Danlos syndrome that results from failure to remove the amino-terminal propeptide of type I procollagen.
      ) and shown to result from biallelic mutations in ADAMTS2, which encodes the procollagen 1 N-proteinase (
      • Colige A.
      • Sieron A.L.
      • Li S.W.
      • et al.
      Human Ehlers-Danlos syndrome type VII C and bovine dermatosparaxis are caused by mutations in the procollagen I N-proteinase gene.
      ). Variation in the clinical picture reflected the presence of missense mutations rather than the early identified nonsense mutations (
      • Colige A.
      • Nuytinck L.
      • Hausser I.
      • et al.
      Novel types of mutation responsible for the dermatosparactic type of Ehlers-Danlos syndrome (Type VIIC) and common polymorphisms in the ADAMTS2 gene.
      ).
      The early biochemical discovery phase culminated with the demonstration that alteration in the amount of type III procollagen produced was the underlying cause of EDS type IV, the Sack Barabas type (
      • Pope F.M.
      • Martin G.R.
      • Lichtenstein J.R.
      • et al.
      Patients with Ehlers-Danlos syndrome type IV lack type III collagen.
      ). At first thought to be recessive because of its rarity, an isolated individual in a family, and apparent decrease in production of type III procollagen by cells from the parents (
      • Pope F.M.
      • Martin G.R.
      • McKusick V.A.
      Inheritance of Ehlers-Danlos type IV syndrome.
      ), subsequent analyses of the COL3A1 gene has shown that this is a dominant disorder (
      • Tsipouras P.
      • Byers P.H.
      • Schwartz R.C.
      • et al.
      Ehlers-Danlos syndrome type IV: cosegregation of the phenotype to a COL3A1 allele of type III procollagen.
      ). A single example of bi-allelic mutations has been identified out of more than 600 families studied (
      • Plancke A.
      • Holder-Espinasse M.
      • Rigau V.
      • et al.
      Homozygosity for a null allele of COL3A1 results in recessive Ehlers-Danlos syndrome.
      ). Some mutations result in failure to secrete type III procollagen from fibroblasts and accumulation of the protein in the rough ER and marked alteration in dermal structure that led to the idea that type III collagen formed a scaffold on which type I fibrillogenesis occurred (
      • Holbrook K.A.
      • Byers P.H.
      Ultrastructural characteristics of the skin in a form of the Ehlers-Danlos syndrome type IV storage in the rough endoplasmic reticulum.
      ). A large clinical study proved it difficult to identify clear genotype phenotype correlations (
      • Pepin M.
      • Schwarze U.
      • Superti-Furga A.
      • et al.
      Clinical and genetic features of Ehlers-Danlos syndrome type IV, the vascular type.
      ). Recent studies, however, make it clear that heterozygosity for a null mutation in COL3A1 endows an individual with an extended natural history compared with the effects of missense and exon skipping mutations (
      • Leistritz D.F.
      • Pepin M.G.
      • Schwarze U.
      • et al.
      COL3A1 haploinsufficiency results in a variety of Ehlers-Danlos syndrome type IV with delayed onset of complications and longer life expectancy.
      ).
      EDS type I/II proved to be more difficult than expected to solve at the molecular level. Ultrastructural studies of the skin showed that there were dramatic alterations in the large dermal collagen fibrils, with variation in fibril diameter and aggregate formation (
      • Vogel A.
      • Holbrook K.A.
      • Steinmann B.
      • et al.
      Abnormal collagen fibril structure in the gravis form (type I) of Ehlers-Danlos syndrome.
      ). Despite this, linkage studies in families excluded type I collagen genes as candidates (
      • Sokolov B.P.
      • Prytkov A.N.
      • Tromp G.
      • et al.
      Exclusion of COL1A1, COL1A2, and COL3A1 genes as candidate genes for Ehlers-Danlos syndrome type I in one large family.
      ,
      • Wordsworth B.P.
      • Ogilvie D.J.
      • Sykes B.C.
      Segregation analysis of the structural genes of the major fibrillar collagens provides further evidence of molecular heterogeneity in type II Ehlers-Danlos syndrome.
      ). It was the finding of an X:9 chromosomal translocation in a woman with EDS type I and skin pigment alteration with identification of a breakpoint in one COL5A1 allele that shed light upon the molecular basis of the disorder (
      • Toriello H.V.
      • Glover T.W.
      • Takahara K.
      • et al.
      A translocation interrupts the COL5A1 gene in a patient with Ehlers-Danlos syndrome and hypomelanosis of Ito.
      ). At the same time, linkage studies and sequencing of both COL5A1 and COL5A2 led to the identification of mutations in individuals with EDS type I/II (
      • Burrows N.P.
      • Nicholls A.C.
      • Yates J.R.
      • et al.
      The gene encoding collagen alpha1(V)(COL5A1) is linked to mixed Ehlers-Danlos syndrome type I/II.
      ,
      • Nicholls A.C.
      • Oliver J.E.
      • McCarron S.
      • et al.
      An exon skipping mutation of a type V collagen gene (COL5A1) in Ehlers-Danlos syndrome.
      ,
      • Wenstrup R.J.
      • Langland G.T.
      • Willing M.C.
      • et al.
      A splice-junction mutation in the region of COL5A1 that codes for the carboxyl propeptide of pro alpha 1(V) chains results in the gravis form of the Ehlers-Danlos syndrome (type I).
      ). Most affected individuals studied had mutations that led to instability of mRNA derived from oneCOL5A1 allele (
      • Schwarze U.
      • Atkinson M.
      • Hoffman G.G.
      • et al.
      Null alleles of the COL5A1 gene of type V collagen are a cause of the classical forms of Ehlers-Danlos syndrome (types I and II).
      ,
      • Wenstrup R.J.
      • Florer J.B.
      • Willing M.C.
      • et al.
      COL5A1 haploinsufficiency is a common molecular mechanism underlying the classical form of EDS.
      ,
      • Malfait F.
      • Coucke P.
      • Symoens S.
      • et al.
      The molecular basis of classic Ehlers-Danlos syndrome: a comprehensive study of biochemical and molecular findings in 48 unrelated patients.
      ). A critical role of type V collagen in fibril nucleation was established in homozygous COL5A1-knockout mice that failed to survive embryogenesis because no large collagen fibrils were assembled (
      • Wenstrup R.J.
      • Florer J.B.
      • Brunskill E.W.
      • et al.
      Type V collagen controls the initiation of collagen fibril assembly.
      ).
      Although often cited to suggest that only about half of affected individuals with EDS type I or EDS type II (the previous gravis and mitis type and future “classical type”) have mutations in type V collagen genes (
      • Malfait F.
      • Coucke P.
      • Symoens S.
      • et al.
      The molecular basis of classic Ehlers-Danlos syndrome: a comprehensive study of biochemical and molecular findings in 48 unrelated patients.
      ), a recent study using more consistent clinical diagnosis places this number closer to 90% (
      • Symoens S.
      • Syx D.
      • Malfait F.
      • et al.
      Comprehensive molecular analysis demonstrates type V collagen mutations in over 90% of patients with classic EDS and allows to refine diagnostic criteria.
      ). Although genetic studies failed to demonstrate that mutations in type I collagen genes could cause EDS type I/ II (
      • Sokolov B.P.
      • Prytkov A.N.
      • Tromp G.
      • et al.
      Exclusion of COL1A1, COL1A2, and COL3A1 genes as candidate genes for Ehlers-Danlos syndrome type I in one large family.
      ,
      • Wordsworth B.P.
      • Ogilvie D.J.
      • Sykes B.C.
      Segregation analysis of the structural genes of the major fibrillar collagens provides further evidence of molecular heterogeneity in type II Ehlers-Danlos syndrome.
      ), biochemical analysis of type I collagens synthesized in culture succeeded. Several individuals with substitutions of arginine by cysteine within the triple helical domain of proa1(I) chains of type I collagen, encoded by COL1A1, had a clinical picture of EDS type I/II and also developed aortic aneurysms or dissection (
      • Nuytinck L.
      • Freund M.
      • Lagae L.
      • et al.
      Classical Ehlers-Danlos syndrome caused by a mutation in type I collagen.
      ,
      • Malfait F.
      • Symoens S.
      • De Backer J.
      • et al.
      Three arginine to cysteine substitutions in the pro-alpha (I)-collagen chain cause Ehlers-Danlos syndrome with a propensity to arterial rupture in early adulthood.
      ). In addition, homozygosity or compound heterozygosity for COL1A2-null mutations were found in several people who had a clinical presentation with polyvalvular cardiac involvement, moderate joint hypermobility, skin hyperextensibility, and limited bruising (
      • Schwarze U.
      • Hata R.
      • McKusick V.A.
      • et al.
      Rare autosomal recessive cardiac valvular form of Ehlers-Danlos syndrome results from mutations in the COL1A2 gene that activate the nonsense-mediated RNA decay pathway.
      ,
      • Malfait F.
      • Symoens S.
      • Coucke P.
      • et al.
      Total absence of the alpha2(I) chain of collagen type I causes a rare form of Ehlers-Danlos syndrome with hypermobility and propensity to cardiac valvular problems.
      ). Nonetheless, mutations in type I collagen genes account for only a small fraction of patients with EDS type I/II.
      By the mid 1990s there had been a profusion of single case reports that tried to define additional types of EDS, most of which relied largely on clinical differentiation in single systems and were not substantiated by comprehensive genetic or biochemical studies. Few survived the later purge done to help stabilize a classification. Notable among the survivors, but not included in the later classification, is what became known as a progeroid type of EDS in which features of early aging accompanied significant hypermobility and appeared to result from defective glycosaminoglycan addition to several proteoglycans (
      • Hernández A.
      • Aguirre-Negrete M.G.
      • González-Flores S.
      • et al.
      Ehlers-Danlos features with progeroid facies and mild mental retardation.
      ) (
      • Quentin E.
      • Gladen A.
      • Rodén L.
      • et al.
      A genetic defect in the biosynthesis of dermatan sulfate proteoglycan: galactosyltransferase I deficiency in fibroblasts from a patient with a progeroid syndrome.
      ), ultimately shown to result from mutations in B4GALT7 (
      • Okajima T.
      • Fukumoto S.
      • Furukawa K.
      • et al.
      Molecular basis for the progeroid variant of Ehlers-Danlos syndrome.
      ) (
      • Faiyaz-Ul-Haque M.
      • Zaidi S.H.
      • Al-Ali M.
      • et al.
      A novel missense mutation in the galactosyltransferase-I (B4GALT7) gene in a family exhibiting facioskeletal anomalies and Ehlers-Danlos syndrome resembling the progeroid type.
      ). The target matrix proteins are probably not limited to decorin and biglycan, but the lack of posttranslational modification presumably affects their function (
      • Götte M.
      • Kresse H.
      Defective glycosaminoglycan substitution of decorin in a patient with progeroid syndrome is a direct consequence of two point mutations in the galactosyltransferase I (beta4GalT-7) gene.
      ).
      The hope to restore order, similar to that brought by Beighton’s studies of the 1960s, led to a gathering of interested clinicians and geneticists at Villefranche in 1997 and the creation of a new set of clinical and biochemical criteria for the diagnosis of EDS (
      • Beighton P.
      • De Paepe A.
      • Steinmann B.
      • et al.
      Ehlers-Danlos syndromes: revised nosology, Villefranche, 1997. Ehlers-Danlos National Foundation (USA) and Ehlers-Danlos Support Group (UK).
      ). This new classification abolished the previous Roman numerical system and substituted a “descriptive” nomenclature, while at the same time banishing some previous types to an “other” category. Created before genetic testing for many disorders became widespread, this classification is showing its age and is in sore need of revision. However, any static classification system may be challenged by the expanding molecular characterization of disorders with some features of EDS. The tensions among a purely clinical classification, a purely genetic classification, and a mixed classification may be difficult to resolve and could satisfy neither clinicians nor molecular geneticists in the long run.
      The “post-Villefranche” era has experienced a proliferation of EDS types distinguished by clinical and genetic grounds but not yet incorporated into a coherent classification. The elegant studies by
      • Burch G.H.
      • Gong Y.
      • Liu W.
      • et al.
      Tenascin-X deficiency is associated with Ehlers-Danlos syndrome.
      of a child with 21-hydroxylase deficiency and a form of EDS led to identification of a new gene, the loss of function of which explained the connective tissue manifestations. The active 21-hydroxylase gene (CYP21A) is located centromeric to TNXB, which encodes tenascin X. A copy of the 3’ exons of TNXB (called TNXA) are located on the centromeric side of CYP21A and a deletion mediated by the almost exact sequence identity between the two tenascin X genes led to 21-hydroxylase deficiency and loss of function of both copies of TNXB. A member of the matrix tenascin protein family, tenascin X interacts with collagens and other matrix macromolecules. Tenascin X–deficient EDS is distinguished from classical EDS by autosomal recessive inheritance, absence of abnormal scarring in the presence of profound joint hypermobility, very hyperextensible skin, and striking bruising (
      • Schalkwijk J.
      • Zweers M.C.
      • Steijlen P.M.
      • et al.
      A recessive form of the Ehlers-Danlos syndrome caused by tenascin-X deficiency.
      ). The presence of significant hypermobility in heterozygous carriers of null mutations suggested TNXB as a candidate gene in EDS type III, the hypermobile type. Indeed, obligate heterozygotes demonstrated that about 80% of carrier women but only 20% of carrier men had significant joint hypermobility (
      • Zweers M.C.
      • Bristow J.
      • Steijlen P.M.
      • et al.
      Haploinsufficiency of TNXB is associated with hypermobility type of Ehlers-Danlos syndrome.
      ) but wider screening failed to document mutations at sufficient frequency to account for EDS type III. The genomic complexity of this region has proven a significant barrier to molecular diagnostics for this gene.

      Genetic and Genomic Analysis Identifies More Types of Eds and Their Genetic Bases

      Astute observation of distinctive clinical features in consanguineous families coupled with genomic searches led to recognition of several forms of EDS that fall outside the Villefranche classification system. These include the spondylocheiro dysplastic form that results from mutations in SLC39A13 (
      • Fukada T.
      • Civic N.
      • Furuichi T.
      • et al.
      The zinc transporter SLC39A13/ZIP13 is required for connective tissue development; its involvement in BMP/TGF-beta signaling pathways.
      ,
      • Giunta C.
      • Elçioglu N.H.
      • Albrecht B.
      • et al.
      Spondylocheiro dysplastic form of the Ehlers-Danlos syndrome—an autosomal-recessive entity caused by mutations in the zinc transporter gene SLC39A13.
      ). The gene encodes an endoplasmic reticulum–located zinc transporter but additional studies suggested that transforming growth factor-b signaling may also be defective, although the relationship to phenotype is not clear. Mutations in CHST14 (
      • Malfait F.
      • Syx D.
      • Vlummens P.
      • et al.
      Musculocontractural Ehlers-Danlos Syndrome (former EDS type VIB) and adducted thumb clubfoot syndrome (ATCS) represent a single clinical entity caused by mutations in the dermatan-4-sulfotransferase 1 encoding CHST14 gene.
      ,
      • Miyake N.
      • Kosho T.
      • Mizumoto S.
      • et al.
      Loss-of-function mutations of CHST14 in a new type of Ehlers-Danlos syndrome.
      ), which encodes dermatan-4-sulfotransferase 1, confirms the importance of this modification as a part of matrix stability. The mutations result in a musculocontractural form of EDS. Very recently a new form of EDS characterized by kyphoscoliosis, myopathy, and hearing impairment was identified that results from biallelic mutations in FKBP14 (
      • Baumann M.
      • Giunta C.
      • Krabichler B.
      • et al.
      Mutations in FKBP14 cause a variant of Ehlers-Danlos syndrome with progressive kyphoscoliosis, myopathy, and hearing loss.
      ), a member of the prolyl cis-trans isomerase family. Mutations in a family member, FKBP10, result in a form of osteogenesis imperfecta (
      • Alanay Y.
      • Avaygan H.
      • Camacho N.
      • et al.
      Mutations in the gene encoding the RER protein FKBP65 cause autosomal-recessive osteogenesis imperfecta.
      ), perhaps because of effects on folding of either the proa chains of type I procollagen or of lysyl hydroxylases. This type of mechanism might help explain the clinical overlaps among one form of osteogenesis imperfecta, EDS type VI, Bruck syndrome, and this new form of EDS. The genetic etiology of another rare form of EDS associated with periventricular heterotopia was established by recognition of the similarity of the neurology features with that of periventricular heterotopia due to FLNA mutations (
      • Sheen V.L.
      • Jansen A.
      • Chen M.H.
      • et al.
      Filamin A mutations cause periventricular heterotopia with Ehlers-Danlos syndrome.
      ). To date, this is the only confirmed X-linked form of EDS. Most reported individuals are female, consistent with an embryonic lethal effect in males.

      Final Considerations

      The genomic era promises to shed additional light on unsolved forms of EDS. For example, a form of EDS characterized by joint hypermobility, easy bruising, and early periodontal loss without significant inflammation was defined as EDS type VIII. This was shown not to result from mutations in COL3A1 even though some features were shared. Linkage to a locus on the short arm of chromosome 12 (12p13) was identified in a large Swedish family but excluded in others, consistent with locus heterogeneity (
      • Rahman N.
      • Dunstan M.
      • Teare M.D.
      • et al.
      Ehlers-Danlos syndrome with severe early-onset periodontal disease (EDS-VIII) is a distinct, heterogeneous disorder with one predisposition gene at chromosome 12p13.
      ). To date, the molecular etiology of this form remains to be established. Similarly, the genetic etiology in the majority of persons affected with hypermobile EDS (type III) remains to be determined. There are a variety of challenges to dissecting the genetic causes of hypermobile EDS, including but not limited to the clinical variability, seeming sex-related penetrance, and likely genetic heterogeneity. Identifying and understanding the clinical
      diversity, genetic etiology, and pathophysiologic mechanisms of various forms of EDS will undoubtedly continue to expand the work of the last 50 years toward understanding the biology of the extracellular matrix and the role of the constituent macromolecules in human disease.

      Conflict of Interest

      The authors state no conflict of interest.

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