ORIGINAL RESEARCH ARTICLE © American College of Medical Genetics and Genomics Expanding the clinical and mutational spectrum of the Ehlers–Danlos syndrome, dermatosparaxis type Tim Van Damme, MD1, Alain Colige, PhD2, Delfien Syx, PhD1, Cecilia Giunta, PhD3, Uschi Lindert, PhD3, Marianne Rohrbach, MD, PhD3, Omid Aryani, MD4, Yasemin Alanay, MD, PhD5, Pelin Özlem Simsek-Kiper, MD, PhD6, Hester Y. Kroes, MD, PhD7, Koen Devriendt, MD, PhD8, Marc Thiry, PhD9, Sofie Symoens, PhD1, Anne De Paepe, MD, PhD1 and Fransiska Malfait, MD, PhD1 Purpose: The Ehlers–Danlos syndrome (EDS), dermatosparaxis heterozygous mutation (c.2T>C, p.? and c.884_887delTGAA, type, is a recessively inherited connective tissue disorder caused by p.(Met295Thrfs26*)) in ADAMTS2 in five patients from four unre- deficient activity of ADAMTS-2, an enzyme that cleaves the amino- lated families. Three of these displayed a phenotype that was strik- terminal propeptide domain of types I, II, and III procollagen. Only ingly milder than that of previously reported patients. 10 EDS dermatosparaxis patients have been reported, all presenting Conclusion: This study expands the clinical and molecular a recognizable phenotype with characteristic facial gestalt, extreme ­spectrum of the dermatosparaxis type of EDS to include a milder skin fragility and laxity, excessive bruising, and sometimes major phenotypic variant and stresses the importance of good clinical complications due to visceral and vascular fragility. ­criteria. To address this, we propose an updated set of criteria that Methods: We report on five new EDS dermatosparaxis patients and accurately captures the multisystemic nature of the dermatosparaxis provide a comprehensive overview of the current knowledge of the type of EDS. natural history of this condition. Genet Med advance online publication 14 January 2016 Results: We identified three novel homozygous loss-of-function mutations (c.2927_2928delCT, p.(Pro976Argfs*42); c.669_670dupG, Key Words: ADAMTS2; ADAMTS-2; dermatosparaxis type; Ehlers– p.(Pro224Argfs*24); and c.2751-2A>T) and one compound Danlos syndrome; genotype; phenotype INTRODUCTION extremely fragile skin (dermatosparaxis means “tearing of the Ehlers–Danlos syndrome (EDS) comprises a clinically and skin”), resulting in large skin lacerations during delivery or genetically heterogeneous group of heritable connective-tissue early in life, with subsequent infections and premature death. disorders characterized by skin hyperextensibility and fragility, Early ultrastructural studies showed alterations in the dermis abnormal wound healing, joint hypermobility, and generalized of these animals, with loosely packed, thin, and twisted rib- fragility of the soft connective tissues. Genetic defects affecting bon-like collagen fibrils that displayed a typical “hieroglyphic the biosynthesis and/or structure of the fibrillar collagens have aspect” on cross-sections, pointing toward impaired collagen been implicated in the classic (type V collagen, OMIM 130000), biosynthesis and fibrillogenesis. Subsequent biochemical stud- vascular (type III collagen, OMIM 130050), arthrochalasia ies performed using dermatosparactic cattle revealed that these (type I collagen, OMIM 130060), kyphoscoliosis (lysylhydroxy- abnormal collagen molecules are composed of incompletely lase 1, OMIM 225400), and dermatosparaxis (ADAMTS-2, processed type I procollagen precursor molecules in which the OMIM 225410) forms of EDS. Major and minor clinical diag- ­amino(N)-terminal propeptide (pN) is insufficiently cleaved.2 nostic criteria for each of these subtypes were established in Deficient activity of ADAMTS-2, an endopeptidase that excises 1997 and are known as the Villefranche nosology.1 the N-propeptide of procollagen chains, was eventually dem- The dermatosparaxis subtype of EDS is an autosomal reces- onstrated.8 Although animal dermatosparaxis was the first sive condition that to date has been described in no more than recognized collagen disorder, it took more than 20 years to con- 10 humans. It was first reported in cattle2,3 and subsequently firm the existence of a human counterpart for the disorder. In in sheep,4 cats,5,6 and dogs.7 Affected animals display loose and 1992, three infants were reported with clinical signs resembling 1Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium; 2Laboratory of Connective Tissues Biology, Tour de Pathologie, GIGA-Cancer, University of Liège, Liège, Belgium; 3Division of Metabolism, Connective Tissue Unit, University Children's Hospital and Children's Research Centre, Zurich, Switzerland; 4Department of Medical Genetics, Special Medical Center, Tehran, Iran; 5Pediatric Genetics Unit, Department of Pediatrics, Acibadem University School of Medicine, Istanbul, Turkey; 6Department of Pediatric Genetics, Faculty of Medicine, Hacettepe University, Ankara, Turkey;7Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands; 8Center for Human Genetics, University Hospitals Leuven, KU Leuven, Leuven, Belgium; 99Laboratory of Cell and Tissue Biology, GIGA-Neurosciences, University of Liège, Liège, Belgium. Correspondence: Fransiska Malfait ([email protected]) Submitted 1 September 2015; accepted 2 November 2015; advance online publication 14 January 2016. doi:10.1038/gim.2015.188 882 Volume 18 | Number 9 | September 2016 | GeneticS in medicine Ehlers-Danlos syndrome, dermatosparaxis type | VAN DAMME et al ORIGINAL RESEARCH ARTICLE dermatosparaxis.8,9 Ultrastructural studies of the dermis demon- primer sequences, and thermal cycling conditions are available strated the same “hieroglyphic” pattern of collagen fibrils as that on request. PCR products were bidirectionally sequenced using observed in dermatosparactic animals, and biochemical stud- the BigDye Terminator Cycle Sequencing kit protocol (Applied ies on cultured human fibroblasts confirmed deficient cleavage Biosystems, Life Technologies, Carlsbad, CA) and analyzed on an of the proα1(I) and proα2(I) N-propeptides. Seven additional ABI 3730XL Genetic Analyzer (Applied Biosystems). Nucleotide patients were identified subsequently, all of whom displayed numbering reflects cDNA numbering, with +1 corresponding a severe phenotype with extremely fragile and lax skin, severe to the A of the ATG translation initiation codon in the reference bruising, and characteristic dysmorphic face, which usually lead sequence. Amino acid residues are numbered from the first methi- to a diagnosis within the first few months of life.10–17 The clini- onine residue of the reference sequence (NP_055059.2). The out- cal findings observed in this small group of patients served as a come of splice site variants was checked at the cDNA level (primers basis for establishing diagnostic criteria, with severe skin fragil- and conditions available on request). Variant nomenclature fol- ity, sagging, and redundant skin as major clinical criteria, and lows the HGVS guidelines (http://www.hgvs.org/mutnomen). All a soft, doughy skin texture, easy bruising, premature rupture variants were checked with Mutalyzer software (https://mutalyzer. of fetal membranes, and large hernias (umbilical, inguinal) as nl/) and submitted to the EDS Variant Database (https://eds.gene. minor criteria.1 It took until 1999 to identify the first biallelic le.ac.uk/). A unique identifier was obtained for each proband fol- mutations in ADAMTS2, the gene encoding ADAMTS-2 (a dis- lowing submission to the EDS Variant Database and is included in integrin and metalloproteinase with thrombospondin motifs 2), the text. Occurrence of the identified variants was assessed using in several patients with the dermatosparaxis type of EDS and in the 1000 Genomes project browser (http://www.1000genomes. a strain of dermatosparactic calf.17 org/), the NHLBI Exome Sequencing Project Exome Variant In this paper, we present clinical, biochemical, ultrastructural, Server database (release ESP6500, http://evs.gs.washington.edu/ and molecular data for five new patients with the dermato- EVS/), the Exome Aggregation Consortium (http://exac.broadin- sparaxis type of EDS. We expand the clinical phenotype of stitute.org/), and the EDS Variant database (https://eds.gene.le.ac. this disorder by including a much milder clinical and ultra- uk/home.php?select_db=ADAMTS2). structural variant that may go undiagnosed in early life, and we review and summarize the current clinical data on human Expression analysis dermatosparaxis to provide the necessary foundation to update Quantitative PRC (qPCR) primers were designed for ADAMTS2 the clinical criteria. and three reference genes (HPRT1, RPL13A, and YWHAZ). Total RNA was extracted in triplicate as described, and cDNA MATERIALS AND METHODS was synthesized with the iScript reverse-transcriptase kit (Bio- Patient cohort Rad, Hercules, CA). Assays were performed in triplicate for We studied five individuals from four unrelated families who each sample on a Roche Lightcycler 480 system (Roche, Basel, were referred to a medical genetics center because of clinical Switzerland) using Realtime Ready DNA probes Mastermix suspicion of EDS. All patients were investigated by a trained supplemented with ResoLight Dye (Roche) according to the clinical geneticist (F.M., Y.A., M.R., O.A., P.O.S.K., K.D.). manufacturer’s protocol. Data were analyzed using qbase+ 3.0 Informed consent was obtained from the patients and/or par- (Biogazelle, Zwijnaarde, Belgium). ents participating in this study, and all patients and/or parents consented to the publication