Systematic data-querying of large pediatric biorepository identifies novel Ehlers-Danlos Syndrome variant

Ehlers Danlos Syndrome (EDS) describes a group of heritable disorders where collagen synthesis and fibril formation is disrupted. It affects approximately 1 in 5000 to 1 in 100,000 individuals, depending on subtype [1, 2]. Predominant phenotypes include articular hypermobility, lax joints, and fragile tissue, skin, and blood vessels. EDS can be extremely debilitating, requiring preventative and protective measures from birth or onset of symptoms. A large number of subtypes have been described, including six major classifications: 1) Classical (Types I&II); 2) Hypermobile (Type III); 3) Vascular (Type IV); 4) Kyphoscoliosic (Type VI); 5) Arthrochalasic (Type VIIA &VIIB); and 6) Dermatosparaxic (Type VIIC) [3]. These can be transmitted through autosomal dominant, autosomal recessive, or x-linked inheritance. Mutations in a number of genes involved in collagen synthesis have been associated with different EDS subtypes, including COL1A1, COL1A2, COL5A1, COL5A2 [2, 3], and ADAMTS2 ²¹. All EDS Type VIIC mutations identified to date have been autosomal recessive. Here, we report on an EDS patient, with symptomology that includes dermatosparaxis (literally, “tearing of the skin”), which is characteristic of EDS Type VIIC.

The whole genome analysis of proband (2113) yielded 3,546,583 variants. We identified about 248,772 SNV’s that are shared by the father, proband, and not present in the mother as we assumed it to be dominant model of inheritance. These were analyzed for non-synonymous mutations, which alter the amino acid sequence of a protein. We used ANNOVAR [12] for annotation of variants and used standard variant reduction method to reduce the number of candidate variants. As such we excluded variants that were in intronic regions, synonymous changes, and those identified by the dbSNP131 and the 1000 Genome Project [14]. This filtering resulted in a subset of novel non-synonymous candidate variants (Table 2). None of the known EDS genes (Table 3) showed mutations in the proband. However, manual curation and literature survey of the 52 variants resulted in the discovery of a mutation (c.1261G > A, p. Gly421Ser) in the ADAMTSL2 gene (Table 4). As none of the other 52 variants resided in genes that were putatively candidates for causing EDS, we considered the (c.1261G > A, p. Gly421Ser) variant in the ADAMTSL2 gene to be the most likely causative source of EDS in the family as it segregated in keeping with a disease-causing variant in this family. This heterozygous mutation (c.1261G > A, pGly421Ser) was detected in the proband (2113), affected father (1966) and, affected sister (0281). The mother (8380) and unaffected maternal half-brother (4376) were homozygous for the wild type allele. The mutation was confirmed by Sanger sequencing (Fig. 3). This process is illustrated in (Fig. 4).

Mutation Characteristics SIFT Polyphen Scores and AA Change. We identified a nonsynomous mutation resulting in an amino acid change—G1261A—at position 13641800 on chromosome 9.

This study illustrates the success of the research strategy where by systematic data querying of phenotype data from a large biorepository can be cross-referenced with disease codes to identify the most likely cause of a rare disease. Because we are a research/treatment hub for children with rare disease such as EDS, our biorepository is disproportionately enriched for phenotypes such as these. As such, we anticipate this approach will resolve other rare and unidentified diseases in the future.

The A Disintegrin and Metalloproteinase with thrombospondin motifs (ADAMTS) family is associated with a range of biological processes, including formation/regulation of connective tissue structure, coagulation, arthritis, angiogenesis, and cell migration [15]. The ADAMTS superfamily includes 19 distinct members that are secreted enzymes and at least seven ADAMTS-like proteins without enzymatic activity [15‐18]. The ADAMTS have two domains – the catalytic domain at the N terminus and the ancillary domain at their C terminus. These are required for interaction and are distinct from A Disintegrin and Metalloproteinases by 1+ type I repeats. They are synthesized as inactive zymogens, and post-translational modification is activated by their N-terminal propeptide, which is cleaved by furin [15]. ADAMTS-like proteins are different from ADAMTS ancillary domains in that they are lacking catalytic activity, due to the lack of protease domain.

Mutations in the ADAMTS family have been associated with EDS. Specifically ADAMTS2 mutations are associated with Type VIIC, which is characterized by dermatosparaxis. This manifests in extreme skin fragility, spontaneous ruptures of internal organs, as well as premature rupture of membranes during pregnancy [19]. Colige et al. [20] were the first to identify mutations responsible for EDS VIIC, which were found in the gene encoding the procollagen protease ADAMTS2, located on chromosome 5qter (5q35.3) (originally identified under the alias procollagen I –Proteinase). They identified six EDS VIIC individuals, five of whom were homozygous for a C → T transition that resulted in a premature termination codon, Q225X, of which four were further homozygous at three downstream polymorphic sites (His 286 (CAT), Asp 676 (GAT), and Asp 844 (GAT)). A sixth patient was homozygous for premature termination codon, Q225X. These are all consistent with recessive inheritance.

Three additional mutations have been identified in the same gene in two patients: one had a homozygous deletion of exons 205, a second was compound heterozygous for a missing exon 3 (maternal) as well as a deletion that caused in-frame skipping of exons 14–16 [21]. This second patient constituted the first example of compound heterozygosity as the cause for EDS VIIC. Three more patients from the same study had variations found in normals, which was paralleled by silent functionality. All five EDS VIIC patients presented with facial dysmorphisms, hypermobility, skin and muscle fragility, and (at least) four of were born prematurely (~32–34 weeks). Recently, a newborn with EDS VIIC was described as presenting with congenital skull fractures and skin lacerations [19].

Adapted from Le Goff & Cormier-Daire (2008), Relevant Diseases Associated with THE ADAMTS (L) family. Mutations in ADAMTS2 are associated EDS VIIC. A mutation in ADAMTSL2 has previously been associated with geleophysic dysplasia, whose phenotype includes joint limitation

A mutation in ADAMTSL2 has previously been associated with geleophysic dysplasia (GD) [OMIM: 231050], where the phenotype includes joint limitation. ADAMTSL2 encodes a secreted glycoprotein that binds the cell surface and extracellular matrix, and interacts with latent transforming growth factor beta binding protein 1. GD is autosomally recessive, and can also include severe short stature, facial dysmorphism, a ‘happy’ face, progressive cardiac valvular thickening, respiratory insufficiency, and lysosomal-like storage vacuoles in different tissues.

Le Goff et al. [22] used in situ hybridization to human fetal tissue to demonstrate ADAMTSL2 mRNA expression in cardiomyocytes, epidermis, dermal blood vessels, tracheal wall, skeletal muscle, pulmonary arteries, and bronchioles of the lung. Strong expression was also observed in chondrocyte columns in the hypertrophic and reserve zones of the proximal femoral growth plate. The group used a yeast 2-hybrid screen of a human muscle cDNA library, and found that ADAMTSL2 bound to latent TGFβ-binding protein-1, which is important to regulating TGFβ availability and storing TGFβ in the extracellular matrix. While unsubstantiated by current medical records, there is a possible relationship here with the proband’s antibody deficiency and history of recurrent infection.

Allali S et al.[23] recently identified 33 patients, of whom 19 had a mutation in ADAMTSL2. The cohort collectively shared 13 mutations. The authors compared phenotypes in those that did/did not carry an ADAMTSL2 mutation, finding that the main discriminating features were facial dysmorphism and tiptoe walking. These phenotypes were absent from the proband and his family. To our knowledge, ADAMTSL2 is not associated with any other rare disorder.

We note that Table 1 also includes a number of loci that may also be associated with EDS and other phenotypes reported here. Of particular interest is a heterozygous non-synonymous single nucleotide variation in A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), which is also known as von Willebrand factor-cleaving protease (VWFCP). Although this is an exonic splicing site, it may be of functional significance. ADAMTS13 is a zinc-containing metalloprotease enzyme that cleaves von Willebrand factor (vWf)—a large protein involved in platelet adhesion and in aggregating vascular lesions.

While this demonstrates a success for data-querying of de-identified EMR data, we stress the importance of clinical phenotyping, which can only be accomplished on a local level. Nevertheless, we demonstrate the utility of unbiased large-scale recruitment and biobanking in deciphering the genetic etiology of rare Mendelian diseases. Families with most other Mendelian disorders for which genetic variants have been found were biasedly recruited by clinical experts. The majority of rare Mendelian diseases may go unsequenced under such a recruitment model. With unbiased large-scale clinical recruitment we strive to sequence as many rare Mendelian diseases as possible, and this work in uncovering a new mutation underlying EDS serves as a successful proof of concept to that effect.

Although EDS type VII has been described as an autosomal-recessive disorder, the identification of heterozygous ADAMTSL2 mutations demonstrates a dominant form of EDS, strictly fulfilling the diagnostic criteria for EDS VII. Future studies delineating the specific function of the ADAMTSL2 protein will reveal how this novel mutation and others in N-glycan-rich module contribute to the disease phenotype.