Ehlers-Danlos syndrome (EDS) is a clinically and genetically heterogenous group of heritable connective tissue disorders. Typical clinical signs include joint hypermobility, skin hyperextensibility, and tissue fragility [1
]. Different types of EDS can be distinguished according to the underlying pathomechanisms, such as defective primary structure and processing of collagen, collagen folding and cross-linking, structure and function of the myomatrix, glycosaminoglycan biosynthesis, intracellular processes, or complement pathway [2
]. In humans, variants in 20 genes have so far been shown to cause different forms of EDS (Table 1
In dogs, EDS was first described as cutaneous asthenia more than 70 years ago [3
]. To date, several reports of dogs with connective tissue disorders, such as EDS have been published [4
]. However, only for a few of them, the underlying genetic variant has been identified. EDS in a Doberman Pinscher was caused by a homozygous nonsense variant in ADAMTS2
(OMIA 000328-9615) [18
], which encodes the procollagen I N-proteinase that excises the N-propeptide of type I and type II procollagens [19
Two compound heterozygous missense variants in TNXB
were reported in a single mixed-breed dog with EDS. However, the evidence for pathogenicity was extremely weak and it is not fully clear whether this dog really suffered from a TNXB
-related form of EDS (OMIA 002203-9615) [20
]. The encoded tenascin-X is a large extracellular matrix protein, which is an essential regulator of collagen deposition by dermal fibroblasts [21
Two independent variants in COL5A1
have been identified in a Labrador Retriever and a mixed-breed dog affected with EDS (OMIA 002165-9615) [24
encodes the α1 chain of type V collagen, which is important for correct collagen fibrillogenesis [25
In this study, we investigated the clinical and histopathological phenotype of an EDS-affected Chihuahua and the underlying causative genetic defect.
2. Materials and Methods
2.1. Animal Selection
This study included an EDS-affected Chihuahua and its unaffected parents. For the whole genome sequencing data analysis, we used 783 control dogs from different breeds and 9 wolves (Table S1
). The control dogs and wolves had already been used in earlier studies, e.g., [26
2.2. Clinical and Histopathological Examinations
The affected dog and both its parents underwent clinical examination. A piece of skin flap from a trauma-induced wound at the dorsum of the affected dog was fixed in 10% neutral-buffered formalin, processed routinely, and sections were stained with hematoxylin-eosin and saffron (HES).
2.3. DNA Extraction
Genomic DNA was isolated from EDTA blood of the affected dog using the Maxwell® 16 Blood DNA Purification Kit and from buccal cells of both parents using the Maxwell® 16 Buccal Swab LEV DNA Purification Kit, both using the Maxwell® 16 Instrument (Promega, Dübendorf, Switzerland). Genomic DNA was frozen at −20 °C until further use.
2.4. Whole-Genome Sequencing and Variant Calling
An Illumina TruSeq PCR-free DNA library with ~340 bp insert size of the affected dog was prepared and sequenced on a NovaSeq 6000 instrument with 25× coverage (Illumina, San Diego, CA, USA). The sequence data were submitted to the European Nucleotide Archive with the study accession PRJEB16012 and the sample accession SAMEA8797073. Mapping, alignment, and variant calling were performed as described [26
]. Private variant filtering was performed with a hard filtering approach requiring the genotype 0/1 for heterozygous or 1/1 for homozygous variants in the affected dog and simultaneously a homozygous reference or missing genotype in the control dogs (0/0 or ./.).
2.5. Gene Analysis
All references within the canine COL5A2 gene correspond to the NCBI RefSeq accession numbers XM_005640393.3 (mRNA) and XP_005640450.1 (protein). We used the CanFam3.1 reference genome assembly and NCBI annotation release 105.
2.6. PCR and Sanger Sequencing
Sanger sequencing was used to validate the candidate variant COL5A2:c.3388_3414del in the affected dog and to genotype both parents. PCR products were amplified from 10 ng genomic DNA using GoTaq® G2 Flexi DNA Polymerase in a total volume of 25 µL including 0.2 µL Taq polymerase, 5 µL 5× buffer, 1.5 µL MgCl2 solution at 25 mM (Promega, Madison, WI, USA), 0.5 µL dNTP mix at 10 mM each (MP Biomedicals, Irvine, CA, USA) together with 0.5 µL each forward primer 5′-TAGCGTTCAGGCTTCCACTG-3′ and reverse primer 5′-CTCCAACACCTACGTGAGCC-3′ (primer concentrations were 10 µM). PCR amplification comprised 32 cycles (denaturation 30 s at 94 °C, annealing 40 s at 60 °C, and elongation 40 s at 72 °C) followed by 5 min at 72 °C. Electrophoresis was performed on a 2% agarose gel. After DNA gel extraction using the QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany), amplicons were sequenced on an ABI 3730XL DNA Analyzer (Thermo Fisher Scientific, Reinach, Switzerland). Sanger sequences were analyzed using the Chromas 2.6.6 software (Technelysium, Pty, Ltd., South Brisbane, Australia).
In this study, we describe a Chihuahua with Ehlers-Danlos syndrome. EDS is a heterogeneous group of diseases affecting connective tissue. In human patients, 13 clinically different EDS subtypes with known causal variants in 20 different genes are recognized. A recently introduced additional clinical subtype has not yet been characterized at the molecular level [2
In humans, the diagnosis of an EDS subtype is made by identifying clinical signs that can be categorized into major or minor criteria and by complementary molecular analysis. For the so-called classical EDS, the major and minor criteria are listed in Table 3
Minimal criteria suggestive for classical EDS are the presence of either both major criterions or one major criterion and at least three minor criteria [1
]. As this classification of EDS subtypes has been developed in humans, it must be used with caution in animals. Nevertheless, given the clinical, histopathological, and molecular similarities between animal models of EDS and human EDS, it is realistic to use it to define an animal EDS subtype.
The dog described here presented in its clinical history the first major criterion, namely (1) hyperextensibility of the skin and atrophic scarring, as well as three clinical signs listed as minor criteria, namely (1) easy bruising, (3) skin fragility and traumatic splitting, and (6) hernia. Thus, by referring to the human classification, the dog falls into the clinical category of classical EDS. As in humans, clinical diagnosis must be supplemented by molecular analysis. In our case, the genetic analysis confirmed the clinical hypothesis of a classical EDS.
Suspicion of EDS in animals usually leads to performing a skin biopsy and histopathological analysis of the dermis. Classically, histopathological descriptions report a dermis of normal thickness but present disorganized, smaller, curved collagen fibers of variable length and unequal diameter [9
]. However, these characteristics are not always found and some affected animals show no abnormality on histological analysis. In some cases, a dermis of reduced thickness has been identified in affected animals, notably cats and dogs [5
], as it is in our case.
Using a whole-genome sequencing approach, we identified a single heterozygous protein-changing variant in a known EDS candidate gene in the investigated EDS-affected Chihuahua. The variant is an in-frame deletion of 27 bases in COL5A2, encoding the α2 chain of type V collagen.
Type V collagen is present mainly as heterotrimers of two α1(V) chains (encoded by COL5A1
) and one α2(V) chain [30
]. Homotrimers of three α1(V) chains or heterotrimers comprised of an α1(V), α2(V), and α3(V) chain (encoded by COL5A3
) also exist; however, their physiological function is largely unknown [2
]. Type V collagen co-assembles with type I collagen into heterotypic fibrils in the extracellular matrix, with type V collagen being crucial for initial fibril formation [25
]. Correct fibril formation and integrity play a key role in maintaining the physical properties of skin and other tissues [2
The three chains of type V collagen are assembled into a triple helix. The sequence of each procollagen chain is characterized by extended Gly-Xaa-Yaa repeats. The presence of glycine (which has no side chain) in every third position permits the formation of the triple-helical structure. The Xaa and Yaa are often proline and hydroxyproline but can be any amino acid [2
-associated EDS mostly results from COL5A1
haploinsufficiency, as type V procollagen molecules cannot accommodate more than a single proα2(V) chain, and the reduction of available proα1(V) chains results in the production of about half the normal amount of type V collagen [33
]. By contrast, proα1(V) chains can form functional homotrimers [34
]. Thus, no heterozygous COL5A2
null alleles have been identified in EDS patients so far [35
-associated EDS is mostly related to structural variants located in the triple helix domain, resulting in the production of mutant proα2(V) chains which are expected to be incorporated in defective type V collagen molecules [35
In accordance with [35
], the COL5A2
variant identified in the EDS-affected dog from this study is predicted to remove three Gly-Xaa-Yaa triplet repeats and thus induce a structural alteration of the synthesized mutant proα2(V) chains. We assume that this causes aberrant heterotrimer formation and a defective type V collagen structure corresponding to a dominant-negative gain-of-function in the mutant allele.
In domestic animals, only one other COL5A2
variant, p.Gly789Val, has been reported to cause EDS in Holstein cattle [28