Defect in Dermatan Sulfate in Urine of Patients with Ehlers-Danlos Syndrome Caused by a CHST14/D4ST1 Deficiency

Title Defect in dermatan sulfate in urine of patients with Ehlers-Danlos syndrome caused by a CHST14/D4ST1 deficiency

Mizumoto, Shuji; Kosho, Tomoki; Hatamochi, Atsushi; Honda, Tomoko; Yamaguchi, Tomomi; Okamoto, Nobuhiko; Author(s) Miyake, Noriko; Yamada, Shuhei; Sugahara, Kazuyuki

Clinical Biochemistry, 50(12), 670-677 Citation https://doi.org/10.1016/j.clinbiochem.2017.02.018

Issue Date 2017-08

Doc URL http://hdl.handle.net/2115/68359

Rights(URL) http://creativecommons.org/licenses/by-nc-nd/4.0/

Type article (author version)

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Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP 1 2 Clinical Biochemistry 161110MZ 3 4 Defect in dermatan sulfate in urine of patients with Ehlers-Danlos syndrome 5 6 7 caused by a CHST14/D4ST1 deficiency 8 9 10 a* b c d 11 Shuji Mizumoto , Tomoki Kosho , Atsushi Hatamochi , Tomoko Honda , Tomomi 12 13 b e f a 14 Yamaguchi , Nobuhiko Okamoto , Noriko Miyake , Shuhei Yamada , 15 16 a,d* 17 and Kazuyuki Sugahara 18 19 20 21 a Department of Pathobiochemistry, Faculty of Pharmacy, Meijo University, 150 22 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan. 23 b Department of Medical Genetics, Shinshu University School of Medicine, 3-1-1 Asahi, 24 Matsumoto, Nagano 390-8621, Japan. 25 c Department of Dermatology, Dokkyo Medical University, School of Medicine, 880 26 Kitakobayashi, Mibu, Tochigi 321-0293, Japan. 27 d Laboratory of Proteoglycan Signaling and Therapeutics, Graduate School of Life Science 28 Hokkaido University, Sapporo 001-0021, Japan. 29 30 e Department of Medical Genetics, Osaka Medical Center and Research Institute for Maternal 31 and Child Health, Osaka 594-1101, Japan 32 f Department of Human Genetics, Yokohama City University Graduate School of Medicine, 33 Yokohama 236-0004, Japan. 34 35 36 *Corresponding authors at: Department of Pathobiochemistry, Faculty of Pharmacy, Meijo 37 38 University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan, Tel.: +81-52-839-2652; 39 40 Fax: +81-52-834-8090. 41 42 E-mail address: [email protected] (S. Mizumoto) or [email protected] (K. 43 44 Sugahara). 45 46 47 48 49 Running title: Urinary dermatan sulfate in Ehlers-Danlos syndrome 50 51 52 53 Keywords: Carbohydrate sulfotransferase 14; Chondroitin sulfate; Dermatan sulfate; 54 55 Dermatan 4-O-sulfotransferase; Ehlers-Danlos syndrome; Urine. 56 57 58 59 1 60 61 62 Abstract 63 64 Purpose: Dermatan sulfate (DS) plays a number of roles in a wide range of biological 65 66 67 activities such as cell signaling and tissue morphogenesis through interactions with various 68 69 extracellular matrix proteins including collagen. Mutations in the carbohydrate 70 71 sulfotransferase 14 gene (CHST14) encoding CHST14/dermatan 4-O-sulfotransferase-1 72 73 (D4ST1), which is responsible for the biosynthesis of DS, cause a recently delineated form of 74 75 Ehlers-Danlos syndrome (EDS, musculocontractural type 1), an autosomal recessive 76 77 connective tissue disorder characterized by congenital malformations (specific craniofacial 78 79 features, and congenital multiple contractures) and progressive fragility-related complications 80 81 (skin hyperextensibility, bruisability, and fragility with atrophic scars; recurrent dislocations; 82 83 84 progressive talipes or spinal deformities; and large subcutaneous hematomas). In an attempt 85 86 to develop a diagnostic screening method for the various types of EDS, the amount of DS in 87 88 the urine of patients was analyzed. 89 90 Methods: Urinary DS was quantified by an anion-exchange chromatography after 91 92 treatment with DS-specific degrading enzyme. 93 94 Results: DS was not detected in the urine of patients with homo- or compound 95 96 heterozygous mutations in CHST14. These results suggest that the quantification of DS in 97 98 urine is applicable to an initial diagnosis of DS-defective EDS. 99 100 101 Conclusions: This is the first study to perform a urinary disaccharide compositional 102 103 analysis of chondroitin sulfate (CS)/DS chains in patients with EDS caused by a 104 105 CHST14/D4ST1 deficiency, and demonstrated the absence of DS chains. This result suggests 106 107 systemic DS depletion in this disorder, and also proposes the usefulness of a urinary 108 109 disaccharide compositional analysis of CS/DS chains as a non-invasive screening method for 110 111 this disorder. 112 113 114 115 116 117 118 119 2 120 121 122 1. Introduction 123 124 Dermatan sulfate (DS) is a linear polysaccharide that is covalently attached to specific 125 126 127 core proteins forming DS-proteoglycans (DS-PGs), which are widely expressed at cell 128 129 surfaces and extracellular matrices [1]. DS is abundantly distributed in the skin, cartilage, and 130 131 aorta, and consists of alternating disaccharide units of L-iduronic acid (IdoUA) and N-acetyl- 132 133 D-galactosamine (GalNAc) with 50-200 repeats [2]. DS chains are irregularly modified by 134 135 sulfation at the hydroxy groups of C-2 on IdoUA and the C-4 positions of GalNAc residues, 136 137 respectively, and are involved in the regulation of a number of biological functions such as 138 139 the assembly of extracellular matrices, signal transduction, wound healing, and 140 141 anticoagulation through interactions with growth factors [3, 4]. 142 143 144 The biosynthesis of repeating disaccharide regions of DS chains is initiated by the 145 146 formation of chondroitin as a precursor backbone, which is composed of alternating D- 147 148 glucuronic acid (GlcUA) and GalNAc. Chondroitin is synthesized on the carbohydrate- 149 150 protein linkage region tetrasaccharide sequence (GlcUA-galactose-galactose-xylose) attached 151 152 to the Ser residues of specific core proteins by various glycosyltransferases. DS-epimerase 153 154 (DSE) then converts GlcUA into IdoUA by epimerizing the C-5 position of GlcUA residues 155 156 after the formation of the chondroitin backbone [5]. Dermatan chains are subsequently 157 158 matured by sulfation reactions catalyzed by dermatan 4-O-sulfotransferase-1 (D4ST1), also 159 160 161 named carbohydrate sulfotransferase 14 (CHST14), and uronosyl 2-O-sulfotransferase, which 162 163 transfer a sulfate group from the sulfate donor 3’-phosphoadenosine 5’-phosphosulfate to the 164 165 C-4 position of GalNAc and C-2 position of IdoUA residues, respectively [6-8]. 166 167 Ehlers-Danlos syndrome (EDS) is a heterogenous group of heritable connective tissue 168 169 disorders characterized by skin hyperextensibility, joint hypermobility, and tissue fragility, 170 171 and has been classified into six major types: the classical type (MIM#130000), hypermobility 172 173 type (MIM#130020), vascular type (MIM#130050), kyphoscoliosis type (MIM#225400), 174 175 arthrochalasia type (MIM#130060), and dermatospraxis type (MIM#225410) [9, 10]. The 176 177 178 179 3 180 181 182 dominant negative effects of a haploinsufficiency in mutant procollagen -chain genes or 183 184 185 deficiency in collagen-processing enzymes have been identified as the basis for these types of 186 187 EDS [9]. Additional forms of EDS have also been identified in association with molecular 188 189 and biochemical abnormalities [10, 11]. EDS caused by biallelic mutations in CHST14 was 190 191 recently identified in three independently reported conditions: a rare type of arthrogryposis 192 193 syndrome, “adducted thumb-clubfoot syndrome” [12], a specific type of EDS, “EDS, Kosho 194 195 Type” [13, 14], and a subset of kyphoscoliosis-type EDS without a lysyl hydroxylase 196 197 deficiency, “musculocontractural EDS” [15]. All these conditions are now concluded to be a 198 199 single clinical entity, with the proposed names “D4ST1-deficient EDS (DDEDS)” [16], “EDS 200 201 202 caused by a CHST14/D4ST1 deficiency” [11], or “EDS, musculocontractural type 1 203 204 (EDSMC1) (MIM#601776) in order to distinguish a subsequently identified form of EDS 205 206 caused by biallelic loss-of-function mutations in DSE, which is registered as “EDS, 207 208 musculocontractural type 2 (EDSMC2)” (#615539) [17, 18]. To date, 40 patients from 27 209 210 families have been reported to have a CHST14/D4ST1 deficiency, manifesting multiple 211 212 congenital malformations (craniofacial characteristics, multiple congenital contractures, and 213 214 visceral or ophthalmological malformations) and progressive multisystem fragility-related 215 216 complications (skin hyperextensibility, bruisability, and fragility with atrophic scars; recurrent 217 218 219 dislocations; progressive talipes or spinal deformities; pneumothorax or pneumohemothorax; 220 221 large subcutaneous hematomas; and diverticular perforation) [10-15, 19-26]. 222 223 Sulfotransferase activity toward dermatan in the skin fibroblasts of affected EDS 224 225 patients with CHST14 mutations was previously shown to be significantly lower in a patient 226 227 (p.P281L/p.Y293C) (6.7%) and in another patient (p.P281L/p.P281L) (14.5%) than in each 228 229 age- and sex-matched control [14]. A disaccharide compositional analysis of chondroitin 230 231 sulfate (CS)/DS chains using the affected skin fibroblasts of these two patients showed a 232 233 negligible amount of DS and excess amount of CS [14], presumably due to an impaired 4-O- 234 235 236 sulfation lock and subsequent back-epimerization from IdoUA to GlcUA because of a 237 238 239 4 240 241 242 CHST14/D4ST1 deficiency [12, 14, 18]. Decorin is a major DS-PG in the skin and plays an 243 244 important role in the assembly of collagen fibrils, possibly through an electrostatic interaction 245 246 247 between decorin glycosaminoglycan (GAG) chains and adjacent collagen fibrils [27). CS, but 248 249 not DS dissacharides have been detected in the GAG chain of decorin from affected skin 250 251 fibroblasts, while DS disaccharides (approximately 95%) have mainly been found in the GAG 252 253 chain of decorin from control skin fibroblasts [14, 18]. Transmission electron microscopy 254 255 revealed that collagen fibrils in affected skin specimens were dispersed in the reticular dermis, 256 257 in contrast to the regularly and tightly assembled collagen fibrils observed in the controls [14]. 258 259 Each collagen fibril in the affected skin specimens was smooth and round, and did not vary in 260 261 size or shape, similar to that of the controls [14]. These findings indicate that skin fragility in 262 263 264 patients with a CHST14/D4ST1 deficiency is caused by the impaired assembly of collagen 265 266 fibrils through the replacement of a DS chain with a CS chain of decorin, which may alter the 267 268 electrostatic binding of decorin to collagen fibrils [10, 11, 14]. 269 270 We herein present our results on the first attempt to perform a disaccharide 271 272 compositional analysis on CS/DS chains in the urine samples of patients with the disorder, 273 274 which demonstrate the systemic effects of a CHST14/D4ST1 deficiency and also indicate the 275 276 potential of this analysis as a non-invasive screening method for this disorder. 277 278 279 280 281 2. Materials and methods 282 283 2.1. Patient materials 284 285 Urine samples were obtained from seven previously reported patients with EDS caused 286 287 by a CHST14/D4ST1 deficiency [13, 19, 20, 25], 15 healthy subjects (aged 6 months and 288 289 3−43 years old; 10 females, 5 males), and the parents (42 and 37 years old) and unaffected 290 291 sisters (3 and 11 years old) of patients #D7 and D8 (Table 1). 292 293 294 295 2.2. Materials 296 297 298 299 5 300 301 302 Six unsaturated standard disaccharides derived from CS, chondroitinase ABC (EC 303 304 4.2.2.20) from Proteus vulgaris, chondroitinase AC-I (EC 4.2.2.5) from Flavobacterium 305 306 307 heparinum, and chondroitinase AC-II (EC 4.2.2.5) from Arthrobacter auresens were 308

309 purchased from Seikagaku Biobusiness Corp. (Tokyo, Japan). Chondroitinase B (EC 4.2.2.19) 310 311 from F. heparinum was from IBEX Technologies (Montreal, Canada). All other chemicals 312 313 and reagents were of the highest quality available. 314 315 316 317 2.3. Quantification of urinary DS 318 319 The disaccharide compositions of the CS and DS chains in urine samples were assessed 320 321 as described previously [28]. Briefly, urine samples were concentrated and desalted using 322 323 324 Amicon Ultra-0.5 (10 k) centrifugal filter units (Millipore, Billerica, MA). After an aliquot 325 326 was individually digested with chondroitinase ABC, a mixture of chondroitinase AC-I and 327 328 AC-II, or chondroitinase B, each digest was labeled with 2AB, and excess 2AB reagents were 329 330 removed by extraction with chloroform. The 2AB-labeled digest was analyzed by anion- 331 332 exchange HPLC on a PA-G silica column (4.6 x 150 mm, YMC Co., Kyoto, Japan) using 333 334 isocratic conditions with 16 mM of NaH2PO4 for the first 10 min followed by a linear 335 336 gradient from 16 to 530 mM NaH2PO4 at room temperature over a 60-min period at a flow 337 338 rate of 0.5 ml/min. The eluates were monitored using a fluorometric detector with excitation 339 340 341 and emission wavelengths of 330 and 420 nm, respectively. The identification and 342 343 quantification of the resulting disaccharides were achieved by comparisons with the elution 344 345 positions of CS- or DS-derived authentic unsaturated disaccharides. The amounts of CS and 346 347 DS were normalized by urine creatinine levels, which were measured using the kit, 348 349 LabAssay™ Creatinine (Wako, Osaka, Japan). This study was approved by the local Ethics 350 351 Committees of Meijo University (Nagoya, Japan), Hokkaido University (Sapporo, Japan), and 352 353 Shinshu University (Matsumoto, Japan). 354 355 356 357 358 359 6 360 361 362 3. Results 363 364 The 4-O-sulfated disaccharide unit, ∆HexUA-GalNAc(4-O-sulfate), in which ∆HexUA 365 366 367 stands for 4,5-unsaturated hexuronic acid, in CS/DS was predominantly detected in the urine 368 369 samples of healthy controls and EDS patients (Fig. 1 and Table 2). The non-sulfated and 6-O- 370 371 sulfated disaccharide units, ∆HexUA-GalNAc and ∆HexUA-GalNAc(6-O-sulfate), 372 373 respectively, were also detected (Table 2). Furthermore, a small proportion of disulfated 374 375 disaccharide units including ∆HexUA(2-O-sulfate)-GalNAc(4-O-sulfate), ∆HexUA(2-O- 376 377 sulfate)-GalNAc(6-O-sulfate), and ∆HexUA-GalNAc(4-O-, 6-O-sulfate ) was found (Table 2). 378 379 The total amount of CS/DS in urine is known to vary depending on sex and age [29]. An 380 381 average value for the total amount of CS/DS disaccharides from healthy subjects (#N6 and 382 383 384 N7), who were approximately 30- and 20-year-old males, respectively, was 7.5 nmol/mg 385 386 creatinine (Table 2). In contrast, an average value of 14.0 nmol/mg creatinine was detected in 387 388 the urine of healthy subjects #N3 and N4 (29-year-old females). The average of total CS/DS 389 390 disaccharides from 3-year-old female controls (#N9 and N10) was 72.2 nmol/mg creatinine 391 392 (Table 2). Thus, in order to compare urinary CS and DS between a healthy control and patient, 393 394 sex- and age-matched controls are required. 395 396 The amount of CS/DS (6.2 nmol/mg creatinine) was markedly lower in the urine 397 398 sample of patient #D2 than in those of control samples #N3 and N4 (16.4 and 9.6 nmol /mg 399 400 401 creatinine, respectively) based on the results of chondroitinase ABC digestion (Table 2 and 402 403 Fig. 1). The amount of CS/DS in other patients, except for #D3 (p.P281L/p.P281L), was also 404 405 reduced (Table 2 and Fig. 2). However, the amount of CS/DS in urine samples from patient 406 407 #D3 was slightly higher than that of the corresponding control (Fig. 2). 408 409 The amount of CS disaccharides generated by digestion with chondroitinase AC 410 411 (Table 3 and Fig. 2B) was similar to that of CS/DS disaccharides obtained by digestion with 412 413 chondroitinase ABC (Table 2 and Fig. 2A). The concentration of CS disaccharides in the 414 415 urine of patient #D2 (p.P281L/p.P281L) was 5.6 nmol/mg creatinine (Table 3). On the other 416 417 418 419 7 420 421 422 hand, those of the corresponding healthy controls, #N3 and N4, were 18.1 and 8.8 nmol/mg 423 424 creatinine, respectively (Table 3). Thus, the amount of CS in urine samples was slightly lower 425 426 427 in the patient than in the healthy controls. 428 429 DS disaccharide was not detected in the urine of any patient, but was present in the 430 431 urine of healthy controls (0.2~1.2 nmol/mg creatinine) (Table 4). 432 433 The amounts of urinary CS and DS in family members of EDS patients #D7 and #D8 434 435 were also measured (Supplemental Table S1). The amounts of CS disaccharides from the 436 437 father (P281L/+) and mother (F209S/+) of patients #D7 and D8 (F209S/P281L) were 3.3 and 438 439 7.0 nmol/mg creatinine, respectively (Supplemental Table S1). The amounts of urinary CS 440 441 from the sisters, who have no phenotypes with EDS, of the patients were 22.8 and 47.8 442 443 444 nmol/mg creatinine, respectively (Supplemental Table S1). On the other hand, the amounts of 445 446 DS disaccharides were 0.2 ~ 1.6 nmol/mg creatinine in the father, mother, and sisters of 447 448 patients #D7 and D8 (Supplemental Table S1). The amounts of CS and DS from family 449 450 members of patients were similar to those from respective sex-matched and closely age- 451 452 matched normal subjects (Tables 3 and 4). Heterozygous carriers for CHST14 mutations, with 453 454 no phenotypic features of the disorder, showed a similar level of DS to healthy subjects with 455 456 no genotypic information available. 457 458 459 460 461 4. Discussion 462 463 In the present study in which a disaccharide compositional analysis was performed on 464 465 urinary CS and DS chains in patients with EDS caused by a CHST14/D4ST1 deficiency, DS 466 467 chains were not detected in any patient, which was significantly different from age- and sex- 468 469 matched healthy controls. These results suggest general DS depletion in this disorder; 470 471 previous findings only showed DS depletion in skin fibroblasts, and not in urine [12, 14, 18, 472 473 24]. These results also indicate the usefulness of a urinary disaccharide compositionnal 474 475 analysis of CS/DS chains as a non-invasive screening method for this disorder. 476 477 478 479 8 480 481 482 In the cultured skin fibroblasts of patients with EDS caused by a CHST14/D4ST1 483 484 deficiency, the DS side chain on DS-PG, decorin, was found to be replaced by CS, resulting 485 486 487 in the disruption of the assembly of collagen fibrils [14, 18]. The loss of decorin in mice was 488 489 previously reported to cause abnormal collagen fibrogenesis and skin fragility [30], and 490 491 affected the binding of fibroblast growth factor-7 (FGF7) and FGF2 to keratinocytes [31]. 492 493 Thus, post-translational modifications induced in decorin by DS play an important role in 494 495 collagen fibril assembly and FGF signaling. Several frameshift mutations in the decorin gene 496 497 (DCN), predicted to result in the loss of C-terminal amino acids, were found to cause 498 499 congenital stromal corneal dystrophy, an autosomal dominant eye disorder characterized by 500 501 diffuse bilateral corneal clouding with flake-like whitish opacities throughout the stroma [32]. 502 503 504 No patients with EDS caused by a CHST14/D4ST1 deficiency exhibited corneal dystrophy, 505 506 while no patients with congenital stromal corneal dystrophy caused by DCN mutations had 507 508 EDS-like systemic features. This result suggests that the decorin core protein, rather than the 509 510 DS side chain on decorin-PG is important for the formation of corneal collagen fibrils [32]. 511 512 On the other hand, spondyloepimetaphyseal dysplasia, which is characterized by anomalies in 513 514 the spine and epiphyses and metaphyses of long bones, resulting in a short stature and 515 516 osteoarthritic changes in joints, has been reported to be caused by mutations in BGN encoding 517 518 biglycan [33]. Biglycan is a DS-PG that is involved in skeletal growth and bone formation 519 520 521 through signaling pathways including transforming growth factor-, bone morphogenetic 522 523 protein 4, and Wnt [34, 35, 36]. Patients with EDS caused by a CHST14/D4ST1 deficiency 524 525 typically have congenital and progressive skeletal abnormalities [12-15, 18-25], suggesting 526 527 that alterations in DS side chains on biglycan and other DS-PG(s) affect bone development in 528 529 EDS patients. 530 531 EDSMC2 caused by a loss-of-function mutation in DSE has been reported in three 532 533 patients from two families [17, 18]. Patients showed characteristic facial features, congenital 534 535 536 contractures in the thumbs and feet, hypermobility of the finger, elbow, and knee joints, and a 537 538 539 9 540 541 542 susceptibility to atrophic scarring of the skin [17]. The enzymatic activity of DSE in 543 544 fibroblasts from these patients was significantly weaker than that of healthy subjects [17]. In 545 546 547 addition, the amount of DS from the fibroblasts of these patients was less than that in the 548 549 control [17]. It may be difficult at present, with no urinary DS data available on patients with 550 551 DSE deficiency, to identify which of the genes, CHST14 or DSE, are associated with DS- 552 553 defective EDS by measuring urinary DS. 554 555 The major characteristics of the kyphoscoliosis type of EDS are severe muscle 556 557 hypotonia, generalized joint laxity, and scoliosis [37]. This type of EDS is caused by 558 559 mutations in PLOD1 encoding lysyl hydroxylase 1 (procollagen-lysine 2-oxoglutarate 5- 560 561 dioxygenase 1), which hydroxylates lysyl residues on procollagen -chains. The ratio of 562 563 564 urinary lysyl pyridinoline to hydroxylysyl pyridinoline in these patients is abnormally high 565 566 [38]. D4ST1-defective EDS has been classified as the kyphoscoliosis type of EDS without a 567 568 lysyl hydroxylase deficiency (EDS-type VIB), based on similarities to the characteristic facial 569 570 and skeletal features of the kyphoscoliosis type of EDS (EDS-type VIA) [13, 19]. Thus, the 571 572 quantification of urinary DS is also a useful diagnostic test for identifing the kyphoscoliosis 573 574 type of EDS with lysyl hydroxylase or D4ST1 deficiencies. 575 576 Chst14/D4st1-deficient mice have smaller body weights, kinked tails, and more fragile 577 578 579 skin and are also less fertile than the wild type [39]. In addition, the impaired proliferation of 580 581 neural stem cells, reduced neurogenesis, and altered subpopulations of radial glial cells have 582 583 been demonstrated in Chst14/D4st1-deficient mice [40]. These phenotypes are partially 584 585 consistent with those of patients with EDS caused by a CHST14/D4ST1 deficiency. However, 586 587 the amount of the DS chain in Chst14—/—/D4st1—/— mice was not reported in detail. An 588 589 analysis of urinary DS in the knockout mice may support our results. 590 591 592 593 5. Conclusion 594 595 596 In conclusion, this is the first study to perform a urinary disaccharide compositional 597 598 599 10 600 601 602 analysis on CS/DS chains in patients with EDS caused by a CHST14/D4ST1 deficiency, and 603 604 demonstrate the absence of DS chains. This result suggests systemic DS depletion in this 605 606 607 disorder, and also proposes the usefulness of a urinary disaccharide compositional analysis of 608 609 CS/DS chains as a non-invasive screening method for this disorder. 610 611 612 Grant/funding support 613 614 This work was supported in part by a Grant-in-Aid for Young Scientists (B) 615 616 25860037 (to S.M.), for Scientific Research (C) 16K08251 (to S.M.), and Scientific Research 617 618 (B) 16H05357 (to N.M.) from the Japan Society for the Promotion of Science, Japan; by a 619 620 Grant-in-Aid for Challenging Scientific Research from the Research Institute of Meijo 621 622 University (to S.M.); by the Nakatomi Foundation (to S.M.); by the Practical Research Project 623 624 625 for Rare/Intratable Diseases #105 from the Japan Agency for Medical Research and 626 627 Development (AMED) (to T.K., S.M., A.H., N.M., and S.Y.); by a Grant-in-Aid for Scientific 628 629 Research on Innovative Areas (Transcription Cycle) (to N.M.) from the Ministry of 630 631 Education, Culture, Sports, Science and Technology of Japan; and by the Takeda Science 632 633 Foundation (to N.M.). 634 635 636 637 Conflict of Interests 638 639 The authors declare that there are no conflicts of interest regarding the publication of 640 641 642 this manuscript. 643 644 645 646 Acknowledgments 647 648 We thank Takako Hayashi, Ryu Son-Oku, and Tomoyo Tada for their technical 649 650 assistance. 651 652 653 654 Abbreviations 655 656 CHST14, carbohydrate sulfotransferase 14; C4ST, chondroitin 4-O-sulfotransferase; CS, 657 658 659 11 660 661 662 chondroitin sulfate; D4ST, dermatan 4-O-sulfotransferase; DS, dermatan sulfate; DSE, 663 664 dermatan sulfate epimerase; GAG, glycosaminoglycan; GalNAc, N-acetyl-D-galactosamine; 665 666 667 GlcUA, D-glucuronic acid; IdoUA, L-iduronic acid; PG, proteoglycan. 668 669 670 References 671 672 [1] Iozzo RV. 1998. 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Modulation of canonical Wnt signaling by the extracellular matrix component 954 955 biglycan. Proc Natl Acad Sci USA. 108:17022-17027. 956 957 958 959 16 960 961 962 [37] Beighton P, De Paepe A, Steinmann B, Tsipouras P, Wenstrup R. J. 1998. Ehlers-Danlos 963 964 syndromes. Am J Med Genet. 77:31-37. 965 966 967 [38] Steinmann B, Eyre D. R, Shao P. 1995. Urinary pyridinoline cross-links in Ehlers- 968 969 Danlos syndrome type VI. Am J Hum Genet. 57:1505-1508. 970 971 [39] Akyüz N, Rost S, Mehanna A, Bian S, Loers G, Oezen I, Mishra B, Hoffmann K, 972 973 Guseva D, Laczynska E, Irintchev A, Jakovcevski I, Schachner M. 2013. Dermatan 4- 974 975 O-sulfotransferase1 ablation accelerates peripheral nerve regeneration. Exp Neurol. 976 977 247:517-530. 978 979 [40] Bian S, Akyüz N, Bernreuther C, Loers G, Laczynska E, Jakovcevski I, Schachner M. 980 981 2011. Dermatan sulfotransferase Chst14/d4st1, but not chondroitin sulfotransferase 982 983 984 Chst11/C4st1, regulates proliferation and neurogenesis of neural progenitor cells. J 985 986 Cell Sci. 124:4051–4063. 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 17 1020 1021 1022 Figure legends 1023 1024 Fig. 1. HPLC profiles of digests of CS and DS prepared from urine samples following 1025 1026 1027 treatments with three kinds of chondroitinases. 1028 1029 CS and DS in the urine of a healthy subject, #N3 (A, C, E) and EDS patient, #D2 (B, D, F) 1030 1031 were digested with chondroitinase ABC (A, B), a mixture of chondroitinases AC-I and AC-II 1032 1033 (C, D), and chondroitinase B (E, F) into disaccharides for analyses of CS and DS together, CS 1034 1035 alone, and DS alone, respectively. Each digest was labeled with 2AB, and 2AB-labeled 1036 1037 CS/DS disaccharides were separated by anion-exchange HPLC on an amine-bound silica PA- 1038 1039 G column using a linear gradient of NaH2PO4, as indicated by the dashed line. The elution 1040 1041 positions of authentic 2-AB-labeled CS disaccharides are indicated by the numbered arrows: 1, 1042 1043 1044 ∆HexUA-GalNAc; 2, ∆HexUA-GalNAc(6S); 3, ∆HexUA-GalNAc(4S); 4, ∆HexUA(2S)- 1045 1046 GalNAc(6S); 5, ∆HexUA(2S)-GalNAc(4S); 6, ∆HexUA-GalNAc(4S,6S). The longitudinal 1047 1048 axis of chromatograms in panels E and F are magnified (4-fold). 1049 1050 1051 1052 Fig. 2. Comparison of CS and DS amounts in healthy controls and EDS patients. 1053 1054 The amount of CS/DS (A), CS (B), or DS (C) disaccharides in the urine of EDS patients is 1055 1056 depicted in the bar graphs based on Tables 2-4. Sex- and age-matched urine from healthy 1057 1058 control subjects (shaded bars) was utilized for comparisons (black bars). The error bars 1059 1060 indicate standard errors. 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 18 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 19 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 20 1200 1201 1202 1203 Table 1. Urine preparations from healthy subjects and patients with a CHST14/D4ST1 1204 1205 deficiency. 1206 1207 1208 No. Sex Age CHST14 mutations Others 1209 D1 F 11y P281L/Y293C Patient 1 in Ref. 17 1210 D2 F 29y P281L/P281L Patient 2 in Ref. 17 1211 D3 M 32y P281L/P281L Patient 3 in Ref. 17 1212 D4 F 20y P281L/C289S Patient 5 in Ref. 17 1213 D5 F 4y P281L/Y293C Patient 6 in Ref. 17 1214 D6 F 41y F209S/P281L Ref. 25 1215 D7 M 10y F209S/P281L Patient 2 in Ref. 20 1216 D8 M 3m F209S/P281L Brother of patient D7 1217 1218 Father M 42y P281L/WT Father of D7 & D8 1219 Mother F 37y F209S/WT Mother of D7 & D8 1220 Sister #1 F 11y WT/WT Sister #1 of D7 & D8 1221 Sister #2 F 3y P281L/WT Sister #2 of D7 & D8 1222 N1 F 12y N.E. Normal subject 1223 N2 F 10y N.E. Normal subject 1224 N3 F 29y N.E. Normal subject 1225 N4 F 29y N.E. Normal subject 1226 N5 M 30y N.E. Normal subject 1227 1228 N6 M 31y N.E. Normal subject 1229 N7 F 18y N.E. Normal subject 1230 N8 F 21y N.E. Normal subject 1231 N9 F 3y N.E. Normal subject 1232 N10 F 3y N.E. Normal subject 1233 N11 F 39y N.E. Normal subject 1234 N12 F 43y N.E. Normal subject 1235 N13 M 10y N.E. Normal subject 1236 N14 M 11y N.E. Normal subject 1237 N15 M 6m N.E. Normal subject 1238 1239 WT, wild-type. 1240 1241 N.E., not examined (no features of EDS caused by a CHST14/D4ST1 deficiency). 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254 1255 1256 1257 1258 1259 21 1260 1261 1262 1263 Table 2. Disaccharide composition of CS and DS chains in urine of healthy subjects and EDS patients. 1264 1265 1266 Urine samples were individually digested with chondroitinase ABC for the analysis of CS and DS together, and each digest was treated with 2-AB 1267 1268 to label the yielded CS/DS-derived disaccharides, which were analyzed by anion-exchange HPLC (Fig. 1). The amount of resultant disaccharides in 1269 1270 each sample was calculated based on the peak area in each chromatogram. 1271 1272 Normal #N1 Normal #N2 Normal #N3 Normal #N4 Normal #N5 Normal #N6 Normal #N7 Normal #N8 1273 nmol/mg creatinine (mol%) 1274 a 2.8 (24.3) 4.2 (17.0) 3.4 (18.3) 2.8 (28.9) 2.8 (37.1) 2.8 (37.2) 2.6 (25.1) 2.2 (29.8) 1275 ∆O 1276 ∆C 3.8 (32.9) 7.7 (31.4) 7.0 (38.3) 3.1 (31.6) 2.1 (27.9) 2.5 (34.0) 3.5 (34.2) 2.2 (29.3) 1277 ∆A 4.7 (40.0) 12.1 (49.3) 7.3 (39.7) 3.5 (36.5) 2.5 (32.8) 2.0 (26.8) 3.8 (37.5) 2.8 (38.4) 1278 ∆D 0.09 (0.8) 0.2 (0.8) 0.2 (0.9) 0.05 (0.5) 0.04 (0.5) 0.05 (0.7) 0.07 (0.6) 0.04 (0.6) 1279 ∆B 0.01 (0.1) 0.04 (0.2) N.D. N.D. N.D. N.D. N.D. N.D. 1280 ∆E 0.2 (1.8) 0.3 (1.3) 0.5 (2.9) 0.2 (2.4) 0.1 (1.6) 0.1 (1.3) 0.3 (2.6) 0.1 (2.0) 1281 b 1282 ∆T N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1283 Total 11.6 (100) 24.5 (100) 18.4 (100) 9.6 (100) 7.5 (100) 7.5 (100) 10.3 (100) 7.3 (100) 1284 1285 1286 Normal #N9 Normal #N10 Normal #N11 Normal #N12 Normal #N13 Normal #N14 Normal #N15 1287 nmol/mg creatinine (mol%) 1288 ∆O 11.7 (21.7) 11.1 (12.3) 4.0 (19.8) 2.0 (33.5) N.D. N.D. N.D. 1289 ∆C 12.4 (23.0) 21.7 (24.1) 7.2 (35.7) 1.7 (28.5) 3.4 (19.9) 3.9 (17.0) 11.2 (16.0) 1290 1291 ∆A 28.6 (52.9) 53.3 (59.0) 7.8 (39.0) 2.0 (34.9) 13.8 (80.1) 18.9 (83.0) 58.0 (82.2) 1292 ∆D 0.2 (0.3) 0.6 (0.7) N.D. N.D. N.D. N.D. 0.7 (1.0) 1293 ∆B 0.3 (0.5) 1.5 (1.6) 0.4 (1.8) N.D. N.D. N.D. N.D. 1294 ∆E 0.9 (1.6) 2.1 (2.3) 0.7 (3.6) 0.2 (3.0) N.D N.D 0.6 (0.8) 1295 ∆T N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1296 Total 54.1 (100) 90.3 (100) 20.1 (100) 5.9 (100) 17.3 (100) 22.7 (100) 70.5 (100) 1297 1298 1299 1300 22 1301 1302 1303 1304 1305 1306 Patient #D1 Patient #D2 Patient #D3 Patient #D4 Patient #D5 Patient #D6 Patient #D7 Patient #D8 1307 nmol/mg creatinine (mol%) 1308 ∆O 2.9 (18.3) 1.5 (24.5) 2.4 (19.0) 1.6 (16.3) 5.9 (16.2) 2.0 (26.7) N.D. N.D. 1309 1310 ∆C 5.4 (33.9) 2.3 (36.9) 5.2 (40.4) 5.1 (52.5) 11.3 (31.1) 2.0 (26.8) 4.9 (20.6) 9/0 (13.0) 1311 ∆A 7.1 (44.3) 2.1 (34.2) 4.6 (35.6) 2.6 (27.0) 18.1 (50.0) 3.3 (43.8) 18.7 (79.4) 59.1 (85.1) 1312 ∆D 0.2 (1.4) 0.1 (1.4) 0.2 (1.5) 0.2 (2.1) 0.5 (1.3) N.D. N.D. 0.8 (1.2) 1313 ∆B N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1314 ∆E 0.3 (1.8) 0.2 (3.0) 0.5 (3.5) 0.2 (2.1) 0.5 (1.4) 0.2 (2.7) N.D. 0.5 (0.7) 1315 ∆T N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1316 Total 15.9 (100) 6.2 (100) 12.7 (100) 9.7 (100) 36.3 (100) 7.5 (100) 23.6 (100) 69.4 (100) 1317 a, ∆O,∆C, ∆A, ∆D, ∆B, ∆E, and ∆T represent the unsaturated disaccharides, ∆HexUA-GalNAc, ∆HexUA-GalNAc(6S), ∆HexUA-GalNAc(4S), 1318 1319 ∆HexUA(2S)-GalNAc(6S), ∆HexUA(2S)-GalNAc(4S), ∆HexUA-GalNAc(4S,6S), and ∆HexUA(2S)-GalNAc(4S,6S), respectively. ∆HexUA, 1320 1321 GalNAc, 2S, 4S, and 6S stand for 4,5-unsaturated hexuronic acid, N-acetyl-D-galactosamine, 2-O-, 4-O-, and 6-O-sulfate, respectively. 1322 1323 1324 b, not detected (<0.01 nmol/mg creatinine). 1325 1326 1327 1328 1329 1330 1331 1332 1333 1334 1335 1336 1337 1338 1339 1340 1341 23 1342 1343 1344 1345 Table 3. Disaccharide composition of CS chains in urine of healthy subjects and EDS patients. 1346 1347 1348 Urine samples were individually digested with a mixture of chondroitinases AC-I and AC-II for the analysis of CS only, and each digest was 1349 1350 treated with 2-AB to label the yielded CS-derived disaccharides, which were analyzed by anion-exchange HPLC (Fig. 1). The amount of resultant 1351 1352 disaccharides in each sample was calculated based on the peak area in each chromatogram. 1353 1354 Normal #N1 Normal #N2 Normal #N3 Normal #N4 Normal #N5 Normal #N6 Normal #N7 Normal #N8 1355 nmol/mg creatinine (mol%) 1356 ∆O 1.8 (16.1) 4.4 (16.7) 3.3 (18.4) 2.6 (29.4) 2.8 (36.8) 2.6 (37.2) 2.6 (25.2) 2.1 (30.9) 1357 ∆C 4.1 (37.3) 8.5 (31.7) 7.6 (41.8) 2.9 (32.8) 2.2 (28.5) 2.5 (35.7) 3.6 (34.3) 2.1 (31.4) 1358 ∆A 4.8 (44.1) 13.3 (49.9) 6.6 (36.4) 3.1 (35.1) 2.5 (32.8) 1.8 (25.5) 3.9 (37.4) 2.4 (35.7) 1359 ∆D 0.06 (0.5) 0.1 (0.5) 0.1 (0.5) 0.03 (0.3) 0.03 (0.3) 0.03 (0.5) 0.04 (0.4) 0.02 (0.3) 1360 1361 ∆B N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1362 ∆E 0.2 (2.0) 0.3 (1.2) 0.5 (2.9) 0.2 (2.4) 0.1 (1.5) 0.08 (1.1) 0.3 (2.7) 0.1 (1.7) 1363 ∆T N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1364 Total 11.0 (100) 26.6 (100) 18.1 (100) 8.8 (100) 7.6(100) 7.0 (100) 10.4 (100) 6.7 (100) 1365 1366 1367 Normal #N9 Normal #N10 Normal #N11 Normal #N12 Normal #N13 Normal #N14 Normal #N15 1368 nmol/mg creatinine (mol%) 1369 ∆O 13.3 (29.6) 15.9 (27.6) 6.9 (41.7) 2.5 (15.5) N.D. N.D. N.D. 1370 ∆C 12.4 (27.4) 17.0 (29.5) 6.5 (39.5) 9.5 (57.9) 7.4 (25.6) 8.1 (25.0) 21.0 (22.1) 1371 ∆A 19.2 (42.7) 23.8 (41.2) 2.9 (17.7) 4.4 (26.6) 21.5 (74.4) 24.4 (75.0) 73.5 (77.2) 1372 1373 ∆D N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1374 ∆B N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1375 ∆E 0.1 (0.3) 1.0 (1.7) 0.2 (1.1) N.D. N.D. N.D. 0.7 (0.7) 1376 ∆T N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1377 Total 45.0 (100) 57.7 (100) 16.5 (100) 16.4 (100) 28.8 (100) 32.5 (100) 95.2 (100) 1378 1379 1380 1381 1382 24 1383 1384 1385 1386 1387 1388 Patient #D1 Patient #D2 Patient #D3 Patient #D4 Patient #D5 Patient #D6 Patient #D7 Patient #D8 1389 nmol/mg creatinine (mol%) 1390 ∆O 2.7 (17.9) 1.3 (23.4) 2.3 (18.5) 1.2 (14.7) 4.7 (15.1) 1.6 (33.3) N.D. N.D. 1391 1392 ∆C 5.2 (34.5) 2.2 (39.2) 5.2 (41.9) 4.4 (54.9) 9.7 (31.4) 1.4 (28.2) 9.5 (29.1) 21.9 (17.3) 1393 ∆A 6.8 (45.4) 1.9 (34.0) 4.4 (35.6) 2.2 (27.6) 15.9 (51.6) 1.4 (28.4) 23.1 (70.9) 103.2 (81.6) 1394 ∆D 0.05 (0.3) 0.02 (0.4) 0.04 (0.4) 0.05 (0.6) 0.1 (0.4) N.D. N.D. 0.3 (0.3) 1395 ∆B N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1396 ∆E 0.3 (1.8) 0.2 (3.0) 0.5 (3.6) 0.2 (2.1) 0.4 (1.4) 0.5 (10.1) N.D. 1.0 (0.8) 1397 ∆T N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1398 Total 15.1 (100) 5.6 (100) 12.4 (100) 8.0 (100) 30.8 (100) 4.9 (100) 32.6 (100) 126.4 (100) 1399 1400 1401 1402 1403 1404 1405 1406 1407 1408 1409 1410 1411 1412 1413 1414 1415 1416 1417 1418 1419 1420 1421 1422 1423 25 1424 1425 1426 1427 Table 4. Disaccharide composition of DS chains in urine of healthy subjects and EDS patients. 1428 1429 1430 Urine samples were individually digested with chondroitinase B for the analysis of DS only, and each digest was treated with 2-AB to label the 1431 1432 yielded DS-derived disaccharides, which were analyzed by anion-exchange HPLC (Fig. 1). The amount of resultant disaccharides in each sample was 1433 1434 calculated based on the peak area in each chromatogram. 1435 1436 Normal #N1 Normal #N2 Normal #N3 Normal #N4 Normal #N5 Normal #N6 Normal #N7 Normal #N8 1437 nmol/mg creatinine (mol%) 1438 ∆O N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1439 ∆C N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1440 ∆A 0.3 (100) 0.6 (100) 0.9 (100) 0.4 (100) 0.2 (95.9) 0.2 (100) 0.4 (92.3) 0.2 (100) 1441 ∆D N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1442 1443 ∆B N.D. N.D. N.D. N.D. 0.01 (4.2) N.D. 0.03 (7.8) N.D. 1444 ∆E N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1445 ∆T N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1446 Total 0.3 (100) 0.6 (100) 0.9 (100) 0.4 (100) 0.21 (100) 0.2 (100) 0.43 (100) 0.2 (100) 1447 1448 1449 Normal #N9 Normal #N10 Normal #N11 Normal #N12 Normal #N13 Normal #N14 Normal #N15 1450 nmol/mg creatinine (mol%) 1451 ∆O N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1452 ∆C N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1453 ∆A 1.1 (91.9) 2.6 (90.2) 0.6 (91.1) 0.2 (61.8) 0.4 (100) 0.6 (100) 2.3 (80.6) 1454 1455 ∆D N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1456 ∆B 0.1 (8.1) 0.3 (9.8) 0.06 (8.9) 0.1 (38.2) N.D. N.D. 0.5 (19.4) 1457 ∆E N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1458 ∆T N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1459 Total 1.2 (100) 2.9 (100) 0.66 (100) 0.3 (100) 0.4 (100) 0.6 (100) 2.8 (100) 1460 1461 1462 1463 1464 26 1465 1466 1467 1468 1469 1470 Patient #D1 Patient #D2 Patient #D3 Patient #D4 Patient #D5 Patient #D6 Patient #D7 Patient #D8 1471 nmol/mg creatinine (mol%) 1472 ∆O N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1473 1474 ∆C N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1475 ∆A N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1476 ∆D N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1477 ∆B N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1478 ∆E N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1479 ∆T N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 1480 Total — — — — — — — — 1481 1482 1483 1484 1485 1486 1487 1488 1489 1490 1491 1492 1493 1494 1495 1496 1497 1498 1499 1500 1501 1502 1503 1504 1505 27 1506 1507 1508 1509 Supplemental Table S1. Disaccharide composition of CS and DS chains in urine of family members of EDS patients. 1510 1511 1512 Urine samples from the family of EDS patient #D7 were individually digested with chondroitinases ABC, AC, or B for analyses of CS/DS, CS, or 1513 1514 DS, respectively, and each digest was treated with 2-AB to label the yielded CS/DS-derived disaccharides, which were analyzed by anion-exchange 1515 1516 HPLC (data not shown). The amount of resultant disaccharides in each sample was calculated based on the peak area in each chromatogram. 1517 1518 1519 1520 CS/DS disaccharide analysis by chondroitinase ABC 1521 1522 Father Mother Sister #1 Sister #2 1523 (42y) (37y) (11y) (3y) 1524 nmol/mg creatinine (mol%) 1525 ∆O N.D. N.D. N.D. N.D. 1526 1527 ∆C 0.4 (21.2) 0.7 (20.6) 1.9 (15.8) 3.8 (12.5) 1528 ∆A 1.4 (78.8) 2.5 (73.5) 9.9 (81.0) 26.0 (85.4) 1529 ∆D N.D. N.D. 0.1 (1.2) 0.3 (1.1) 1530 ∆B N.D. N.D. N.D. N.D. 1531 ∆E N.D. 0.2 (5.9) 0.2 (2.0) 0.3 (1.0) 1532 1533 ∆T N.D. N.D. N.D. N.D. 1534 Total CS 1.8 (100) 3.4 (100) 12.3 (100) 30.5 (100) 1535 1536 1537 1538 1539 1540 1541 1542 1543 1544 1545 1546 28 1547 1548 1549 1550 CS disaccharide analysis by chondroitinase AC 1551 1552 1553 Father Mother Sister #1 Sister #2 1554 (42y) (37y) (11y) (3y) 1555 nmol/mg creatinine (mol%) 1556 ∆O N.D. N.D. N.D. N.D. 1557 ∆C 1.0 (29.8) 2.0 (28.4) 5.1 (22.4) 8.3 (17.3) 1558 ∆A 2.3 (70.2) 4.7 (67.7) 17.3 (75.8) 39.1 (81.8) 1559 1560 ∆D N.D. N.D. N.D. N.D. 1561 ∆B N.D. N.D. N.D. N.D. 1562 ∆E N.D. 0.3 (3.9) 0.4 (1.9) 0.9 (0.9) 1563 ∆T N.D. N.D. N.D. N.D. 1564 Total CS 3.3 (100) 7.0 (100) 22.8 (100) 47.8 (100) 1565 1566 1567 1568 DS disaccharide analysis by chondroitinase B 1569 1570 Father Mother Sister #1 Sister #2 1571 (42y) (37y) (11y) (3y) 1572 nmol/mg creatinine (mol%) 1573 ∆O N.D. N.D. N.D. N.D. 1574 ∆C N.D. N.D. N.D. N.D. 1575 1576 ∆A 0.2 (100) 0.5 (100) 0.7 (80.9) 1.3 (83.2) 1577 ∆D N.D. N.D. N.D. N.D. 1578 ∆B N.D. N.D. 0.2 (19.1) 0.3 (16.8) 1579 ∆E N.D. N.D. N.D. N.D. 1580 ∆T N.D. N.D. N.D. N.D. 1581 1582 Total DS 0.2 (100) 0.5 (100) 0.9 (100) 1.6 (100) 1583 1584 1585 1586 1587 29 1588 Highlight (Mizumoto et al., Defect in dermatan sulfate in urine of patients with Ehlers-Danlos syndrome caused by a CHST14/D4ST1 deficiency)

・ CHST14/D4ST1 deficiency causes a specific type of Ehlers-Danlos syndrome (EDS)

・ No urinary dermatan sulfate was detected in the EDS patients with CHST14 deficiency

・ Measurement of urinary dermatan sulfate could be a non-invasive screening of the EDS with CHST14 deficiency