Classification of Ehlers-Danlos Syndromes
The Ehlers-Danlos syndrome (EDS) is a heterogeneous group of heritable connective tissue disorders affecting as many as 1 in 5000 individuals, characterized by joint and skin laxity, and tissue fragility . The fundamental mechanisms of EDS are known to consist of dominant-negative effects or haploinsufficiency of mutant procollagen α-chains and deficiency of collagen-processing-enzymes . In a revised nosology established in the nomenclature conference held in June 1997 at Villefranche-sur-Mer, France, Beighton et al.  classified EDS into six major types (Table 1): classical type (OMIM#130000), hypermobility type (OMIM#130020), vascular type (OMIM#130050), kyphoscoliosis type (OMIM#225400), arthrochalasia type (OMIM#130060), and dermatosparaxis type (OMIM#225410). Additional minor variants of EDS have been identified with molecular and biochemical abnormalities: dermatan 4-O-sulfotransferase-1 (D4ST1)-deficient type/musculocontractural type (OMIM#601776), Brittle cornea syndrome (OMIM#229200), EDS-like syndrome due to tenascin-XB deficiency (OMIM#606408), EDS with progressive kyphoscoliosis, myopathy, and hearing loss (OMIM#614557); the spondylocheiro dysplastic form (OMIM#612350), cardiac valvular form (OMIM#225320), and progeroid form (OMIM#130070)  (Table 1). This chapter focuses on a recent breakthrough in EDS: discovery and delineation of D4ST1-deficient EDS (DD-EDS).
2. History of D4ST1-deficient EDS
DD-EDS, caused by loss-of-function mutations in the carbohydrate sulfotransferase 14 (CHST14) gene coding D4ST1, has been identified independently as a rare type of arthrogryposis syndrome, “adducted thumb–clubfoot syndrome (ATCS)” ; as a specific form of EDS, “EDS, Kosho Type” (EDSKT) ; and as a subset of kyphoscoliosis type EDS without evidence of lysyl hydroxylase deficiency, “Musculocontractural EDS” (MCEDS) .
2.1. Adducted thumb–Clubfoot syndrome
The original report of ATCS was written by Dündar et al.  from Erciyes University, Turkey, presenting two cousins, a boy aged 3.5 years and a girl aged 1.5 years, from a consanguineous Turkish family. In common, they had moderate to severe psychomotor developmental delay, ocular anterior chamber abnormality, facial characteristics, generalized joint laxity, arachnodactyly, camptodactyly, and distal arthrogryposis with adducted thumbs and clubfeet. They reported another patient with ATCS, a boy aged 3 months, from a consanguineous Turkish family including three affected siblings who died of unknown etiology between the ages of 1 and 4 months . The patient also had bilateral nephrolithiasis, a unilateral inguinal hernia, and bilateral cryptorchidism. The authors suggested that two brothers, aged 22 months and 7 months, from a Japanese consanguineous family reported by Sonoda and Kouno  would also fit the diagnosis of ATCS. The brothers had multiple distal arthrogryposis, characteristic facial features, cleft palates, short stature, hydronephrosis, cryptorchidism, and normal intelligence. Dündar et al.  also showed follow-up observations of the original patients: the intelligence quotient (IQ) was roughly 90 in one subject at age 7 years and 2 months and the other died of unknown cause at 5 years of age. Janecke et al.  from Innsbruck Medical University, Austria, reported two brothers with ATCS from a consanguineous Austrian family, one of whom died shortly after birth because of respiratory failure. The authors concluded that all these patients represented a new type of arthrogryposis with central nervous system involvement, congenital heart defects, urogenital defects, myopathy, connective tissue involvement (generalized joint laxity), and normal or subnormal mental development. In 2009, Dündar et al. reported that CHST14 was the causal gene for ATCS through homozygosity mapping using samples from four previously published consanguineous families. The authors mentioned some follow-up clinical findings including generalized joint laxity, delayed wound healing, ecchymoses, hematomas, and osteopenia/osteoporosis; and categorized ATCS as a generalized connective tissue disorder .
2.2. EDS, Kosho type
We encountered the first patient with a specific type of EDS in 2000 and the second with parental consanguinity in 2003. They were Japanese girls with strikingly similar symptoms: characteristic craniofacial features; skeletal features including multiple congenital contractures, malfanoid habitus, pectus excavatum, generalized joint laxity, recurrent dislocations, and progressive talipes and spinal deformity; skin hyperextensibility, bruisability, and fragility with atrophic scars; recurrent hematomas; and hypotonia with mild motor developmental delay . These symptoms overlapped those in the kyphoscoliosis type EDS (previously known as EDS type VI), which is typically associated with deficiency of lysyl hydroxylase (EDS type VIA) . A rare condition with the clinical phenotype of the kyphoscoliosis type EDS but with normal lysyl hydroxylase activity were reported and named as EDS type VIB . Therefore, we tentatively proposed that the two patients represented a clinically recognizable subgroup of EDS type VIB . Through their long-term clinical evaluation as well as four additional unrelated Japanese patients including one with parental consanguinity and another reported by Yasui et al. , we concluded that they—four female patients and two male patients aged 4–32 years, represented a new clinically recognized type of EDS with distinct craniofacial characteristics, multiple congenital contractures, progressive joint and skin laxity, and multisystem fragility-related manifestations . The disorder has been registered as EDS Kosho Type (EDSKT) in the London Dysmorphology Database (http://www.lmdatabases.com/index.html) and in POSSUM (http://www.possum.net.au/). In 2009, we identified CHST14 as causal for the disorder through homozygosity mapping using samples from two consanguineous families and all the other patients were also found to have compound heterozygous CHST14 mutations .
2.3. Musculocontractural EDS
Malfait et al.  from Ghent University, Belgium have found mutations in CHST14 through homozygosity mapping of two Turkish sisters and an Indian girl both presenting clinically with EDS VIB and with parental consanguinity. They had distinct craniofacial features, joint contractures, and wrinkled palms in addition to common features of kyphoscoliosis type EDS including kyphoscoliosis, muscular hypotonia, hyperextensible, thin, and bruisable skin, atrophic scarring, joint hypermobility, and variable ocular involvement. Malfait et al.  concluded that their series and ATCS, as well as EDSKT, formed a phenotypic continuum based on their clinical observations and identification of an identical mutation in both conditions; and proposed to coin the disorder as “musculocontractural EDS” (MCEDS).
3. Pathophysiology of D4ST1-deficient EDS
3.1. Glycobiological abnormalities in D4ST1-deficient EDS
D4ST1 is a regulatory enzyme in the glycosaminoglycan (GAG) biosynthesis that transfers active sulfate to position 4 of the N-acetyl-D-galactosamine residues of dermatan sulfate (DS) (Fig. 1) [16, 17]. DS, together with chondroitin sulfate (CS) and heparan sulfate, constitutes GAG chains of proteoglycans and is implicated in cardiovascular disease, tumorigenesis, infection, wound repair, and fibrosis via DS-containing proteoglycans such as decorin and biglycan .
Sulfotransferase activity toward dermatan in the skin fibroblasts derived from the patients was significantly decreased to 6.7% (patient 1 with a compound heterozygous mutation: P281L/Y293C) and 14.5% (patient 3 with a homozygous mutation: P281L) of each age- and sex-matched control) (Fig. 2A). Disaccharide composition analysis of CS/DS chains isolated from the skin fibroblasts showed a negligible amount of DS and a slight excess of CS (Fig. 2B). Subsequently, we focused on a major DS proteoglycan in the skin, decorin, consisting of core protein and one GAG chain and playing an important role in assembly of collagen fibrils (Nomura, 2006). No DS disaccharides were detected in the GAG chains of decorin from the patients, whereas the GAG chains of decorin from the controls were mainly composed of DS disaccharides (approximately 95%) (Fig. 2C) .
3.2. Pathological abnormalities in D4ST1-deficient EDS
Hematoxylin and eosin (H&E)-stained light microscopy on patients’ skin specimens showed that fine collagen fibers were present predominantly in the reticular to papillary dermis with marked reduction of normally thick collagen bundles (Fig. 3a, b). Electron microscopy showed that collagen fibrils were dispersed in the reticular dermis, compared with the regularly and tightly assembled ones observed in the control; whereas each collagen fibril was smooth and round, not varying in size and shape, similar to each fibril of the control (Fig. 3c, d) .
In view of these glycobiological and pathological findings, skin fragility in this disorder is suggested to be caused by impaired assembly of collagen fibrils resulting from loss of DS in the GAG chain of decorin . Decorin DS regulates the interfibrillar distance in collagen fibrils and permits the extracellular matrix to resist physical stress, possibly through electrostatic interaction between decorin DS chains and adjacent collagen fibrils (Fig. 4A) . Collagen fibrils are dispersed in patients’ skin tissues where the decorin GAG chains are exclusively composed of CS (Fig. 4B), whereas collagen fibrils in controls’ skin specimens are tightly assembled through the GAG chains of decorin exclusively composed of DS (Fig. 4A).
4. Delineation of D4ST1-deficient EDS
Independently identified three conditions, ATCS, EDSKT, and MCEDS caused by loss-of-function mutations in CHST14, were supposed to be a single clinically recognizable type of connective tissue disorder [7, 21]. Shimizu et al.  presented detailed clinical information of two additional unrelated patients and a comprehensive review of all reported 20 patients, which could definitely unite the three conditions named as “D4ST1-deficient EDS (DD-EDS)”. Kosho et al.  concluded that categorization of the disorder into a form of “EDS” was appropriate clinically because the disorder satisfied all the hallmarks of EDS including skin hyperextensibility, joint hypermobility, and tissue fragility affecting the skin, ligaments, joints, blood vessels, and internal organs  and etiologically because multisystem fragility in the disorder was illustrated to be caused by impaired assembly of collagen fibrils resulting from loss of DS in the decorin GAG chains .
To date, 26 patients have been reported to have homozygous or compound heterozygous CHST14 mutations (Table 2) [24, 25, 26]. Clinical characteristics are summarized in Table 3, consisting of progressive multisystem fragility-related manifestations and various malformations .
Characteristic craniofacial features including large fontanelle, hypertelorism, short and downslanting palpebral fissures, blue sclerae, short nose with hypoplastic columella, low-set and rotated ears, high palate, long philtrum, thin upper lip vermilion, small mouth, and micro-retrognathia are noted at birth to early childhood (Fig. 5A, B). Slender and asymmetrical facial shapes with protruding jaws are noted from school age (Fig. 5C) [12. 15, 22].
Congenital multiple contractures, most specifically adduction-flexion contractures of thumbs and talipes equinovarus, were cardinal features (Fig. 5D, G, J, K, M). In childhood, peculiar fingers described as “tapering”, “slender”, and “cylindrical” are also common features (Fig. 5E, F, H, I). Talipes deformities (planus, valgus) (Fig. 5L, N) and spinal deformities (scoliosis, kyphoscoliosis) with tall vertebral bodies and decreased physiological curvature (Fig. 5O, P, Q, R, S, T) occur and progress. Malfanoid habitus, recurrent joint dislocations, and pectus deformities (flat and thin, excavatum, carinatum) are also evident [12. 15, 22].
Cutaneous features include hyperextensibility (Fig. 5U, V) to redundancy (Fig. 5W), bruisability, fragility leading to atrophic scars, acrogeria-like fine palmar creases or wrinkles (Fig. 5F, I), hyperalgesia to pressure, and recurrent subcutaneous infections with fistula formation (Kosho et al., 2005; Kosho et al., 2010; Shimizu et al., 2011).
Recurrent large subcutaneous hematomas are the most serious complication, which sometimes progress acutely and massively to be treated intensively (admission, blood transfusion, surgical drainage) and are supposed to be caused by rupture of subcutaneous arteries or veins (Fig. 5X) [12. 15, 22].
DD-EDS is a newly recognized and delineated form of EDS, characterized by progressive multisystem fragility-related manifestations (skin hyperextensibilty and fragility, progressive spinal and foot deformities, large subcutaneous hematoma) and various malformations (facial features, congenital eye/heart/gastrointestinal defects, congenital multiple contractures). The cause of multisystem connective tissue fragility is supposed to be impaired assembly of collagen fibrils resulting from loss of DS in the decorin GAG chains. It is the first human disorder affecting biosynthesis of DS, which emphasize a role for DS in human development and extracellular matrix maintenance .
The author is thankful to all the patients and their families for participating in this study. The authors also express the gratitude to all the collaborators. All the studies were supported by Research on Intractable Diseases from Japanese Ministry of Health, Welfare, and Labor.