Archives of Dermatological Research (2019) 311:265–275 https://doi.org/10.1007/s00403-019-01894-0 REVIEW Understanding the basis of Ehlers–Danlos syndrome in the era of the next-generation sequencing Francesca Cortini1,2 · Chiara Villa3 · Barbara Marinelli1 · Romina Combi3 · Angela Cecilia Pesatori1 · Alessandra Bassotti4 Received: 29 July 2018 / Revised: 26 November 2018 / Accepted: 12 February 2019 / Published online: 2 March 2019 © Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract Ehlers–Danlos syndrome (EDS) is a clinically and genetically heterogeneous group of heritable connective tissue disorders (HCTDs) defined by joint laxity, skin alterations, and joint hypermobility. The latest EDS classification recognized 13 sub- types in which the clinical and genetic phenotypes are often overlapping, making the diagnosis rather difficult and strength- ening the importance of the molecular diagnostic confirmation. New genetic techniques such as next-generation sequencing (NGS) gave the opportunity to identify the genetic bases of unresolved EDS types and support clinical counseling. To date, the molecular defects have been identified in 19 genes, mainly in those encoding collagen, its modifying enzymes or other constituents of the extracellular matrix (ECM). In this review we summarize the contribution of NGS technologies to the current knowledge of the genetic background in different EDS subtypes. Keywords Ehlers–Danlos syndrome · Heterogeneity · Heritable connective tissue disorders Introduction in 1988, represents the first attempt to classify EDS, recog- nizing 11 EDS subtypes [4], defined by Roman numerals and Ehlers–Danlos syndrome (EDS) comprises a clinically and classified according to clinical findings and the inheritance heterogeneous group of heritable connective tissue disor- pattern. With the elucidation of the clinical and molecular ders (HDCTs). The principal clinical features involve skin basis of many of these EDS subtypes, a revised classifica- fragility, easy bruising, and joint hypermobility [13]. The tion, the “Villefranche classification”, was proposed in 1997 incidence of EDS is estimated to be 1:5000 births and the [5]. This nosology recognizes six EDS subtypes, for which frequency is independent of sex and ethnic background. major and minor criteria were identified, with the inclu- To date, different EDS classifications exist due to its sion of biochemical and molecular basis, when known. A clinical and genetic heterogeneity. The “Berlin nosology”, descriptive name, which captures the main characteristics of each EDS phenotype, substituted the Roman numerals. How- Francesca Cortini and Chiara Villa have contributed equally to this ever, the recent advent of advanced genetic techniques such work. as next-generation sequencing (NGS) gives the opportunity to identify new EDS subtypes, leading thus to the revision of * Francesca Cortini the Villefranche classification. The latest 2017 EDS nosol- [email protected] ogy delineates 13 subtypes, as outlined in Table 1 [32], in 1 Department of Clinical Sciences and Community Health, which the clinical classification conserves the descriptive University of Milan, IRCCS Ca’ Granda Foundation, Via names that are widely used in the medical, scientific, and San Barnaba 8, Milan, Italy patient community. For the new EDS subtypes, a novel 2 Department of Medicine Preventive Services, UOC descriptor that captures the characteristic manifestations of Occupational Medicine, IRCCS Ca’ Granda Foundation, Via the phenotype is proposed. San Barnaba 8, Milan, Italy Molecular mechanisms explaining EDS subtypes are 3 School of Medicine and Surgery, University mainly characterized by causal mutations in genes encoding of Milano-Bicocca, Via Cadore 48, Monza, Italy collagen, its modifying enzymes or other proteins involved 4 Regional Center of Ehlers-Danlos Syndrome, IRCCS Ca’ in the biology of the extracellular matrix (ECM) [5, 13]. Granda Foundation, Via San Barnaba 8, Milan, Italy Vol.:(0123456789)1 3 266 Archives of Dermatological Research (2019) 311:265–275 Table 1 The 2017 EDS EDS subtypes Inheritance Gene classification (Malfait et al., 2017) Classical EDS (cEDS) AD COL5A1, COL5A2 Classical-like EDS (clEDS) AR TNXB Cardiac valvular EDS (cvEDS) AR COL1A2 (biallelic mutations that lead to COL1A2 NMD and the absence of pro α2(I) collagen chains) Vascular EDS (vEDS) AD COL3A1 (rare: COL1A1, c.934C > T, p. Arg312Cys, c.1720C > T, p.Arg574Cys, c.3227C > T, p.Arg1093Cys) Hypermobility EDS (hEDS) AD Unknown Arthrochalasia EDS (aEDS) AD COL1A1, COL1A2 Dermatosparaxis EDS (dEDS) AR ADAMTS2 Kyphoscoliotic EDS (kEDS) AR PLOD1, FKBP14 Brittle cornea syndrome (BCS) AR ZNF469, PRMD5 Spondylodysplastic EDS (spEDS) AR B4GALT7, B3GALT6, SLC39A13 Muscolocontractural EDS (mcEDS) AR CHST14, DSE Myopathic EDS (mEDS) AD/AR COL12A1 Periodontal EDS (pEDS) AD C1R, C1S EDS Ehlers–Danlos syndrome, AD autosomal dominant, AR autosomal recessive, NMD nonsense-mediated mRNA decay Given a great phenotypic and genetic EDS variability and Next‑generation sequencing: an overview the clinical overlap with the EDS subtypes or other HCTDs, a final diagnosis requires molecular confirmation with the The advent of high-throughput NGS technologies has identification of causative genetic variant. This overlap changed molecular approach in clinical laboratories by makes the diagnosis difficult for clinicians, so the use of accelerating the rate of sequence generation and reducing NGS can be very useful in this process. To date, the only the cost, as compared to the Sanger sequencing. genetically unsolved EDS form remains the hypermobile There are different NGS applications for the DNA subtype (hEDS), diagnosed only by clinical finding, that analysis: whole-exome sequencing (WES) amplifies only have extensively been revised in the latest classification. The protein-coding regions, whole-genome sequencing (WGS) clinical criteria are based on the co-presence of generalized amplifies the entire genome, and custom targeted sequenc- joint hypermobility, systemic manifestations of more gen- ing in which regions including only particular group of eralized connective tissue fragility, positive family history, genes (targeted approach) are amplified. WES [55] and and/or musculoskeletal complications [32]. Dissecting the custom targeted sequencing [17] become standard in genetic etiology in patients affected by hEDS is quite chal- clinical setting. To choose the correct strategy, clinicians lenging, probably due to clinical variability, genetic hetero- should take into account: (a) disease model, (b) the region geneity, and seeming sex-related penetrance. As well as the of interest, and (c) depth of coverage (the average number previously unrecognized EDS forms have been solved at the of times that a particular nucleotide is present in a deter- molecular level, it is likely that a better understanding of mined position in a collection of random of sequences). matrix biology will continue to expand. WES is the appropriate strategy for the identification of In this review, we describe the genetic and molecular fea- molecular defects in patients with suspected genetic disor- tures of EDS, focusing on the recent genetic breakthroughs ders [55]. Moreover, it represents the best NGS method in after the introduction of NGS techniques. The genetic analy- case of conventional Sanger sequencing or custom panel sis is mandatory to confirm or modify the clinical diagnosis which are not able to identify the causal gene due to the and is also essential for evaluating prognosis, making deci- genetic heterogeneity, atypical clinical presentation or lack sions on management and treatment strategies. 1 3 Archives of Dermatological Research (2019) 311:265–275 267 of causal gene [1]. Custom targeted resequencing is the The extracellular matrix (ECM): collagen, best NGS application to be used when there are only a elastic fibers, and glycoproteins few known disease-causing genes. The major advantage is the possibility to personalize the design (i.e., inclusion The extracellular matrix has relevant functions in regulating of complete gene sequence or specific intronic sequences). the development and homeostasis of all eukaryotic cells. The Moreover, it is also faster and cheaper than WES and WGS ECM is composed of different classes of macromolecules: [17]. On the other hand, when nothing has been detected fibrous proteins (including collagens and elastin), glycopro- in the exome, WGS should be useful to deliver a compre- teins, and glycosaminoglycans (GAGs) [45] (Fig. 1). hensive view of the entire genome, with coding as well as Collagens are the major component of the ECM and the non-coding sequences, which can be relied in regulatory most abundant proteins in the body, constituting approxima- regions. However, the limitations of its use in the clinical tively 25–30% of the total protein mass. To date, 29 types setting are related to a limited speed, high costs, and a of collagen are described, each composed of a domain con- complex data analysis as compared with WES. stituted of three α-chains intertwined as a triple helix. The Up to date, there are different platforms, such as MiSeq, α-chains consist of a triplet repeat Gly-Xaa-Yaa, where Xaa NextSeq (Illumina), Ion Torrent (Thermo Scientific) to and Yaa are often hydroxyproline and proline, respectively. load NGS genomic libraries, generating millions of reads The role of glycine (the smallest amino acid, found at every that are processed bioinformatically.