The Ehlers–Danlos Syndromes (EDS) are a group of hereditary connective tissue disorders (HCTDs) characterized by skin hyperextensibility, joint hypermobility, and tissue fragility. They were classified into 13 subtypes based on symptoms and causative genes in the 2017 International Classification (Malfait et al., 2017). In 2018, Blackburn et al. (2018) identified biallelic variants in the adipocyte enhancer binding protein 1 (AEBP1) gene in patients displaying EDS-like features that were considered to represent a new subtype of EDS and were tentatively named classical-like type 2 (clEDS2; MIM #618000) (Malfait et al., 2020). To date, 10 patients from eight families have been described (Alazami et al., 2016; Blackburn et al., 2018; Hebebrand et al., 2019; Ritelli et al., 2019; Syx et al., 2019; Maddirevula et al., 2020; Vishwanath et al., 2020; Di Giosaffatte et al., 2022).

We report here an additional patient with clEDS2 who had novel variants in AEBP1 and was complicated by a critical vascular event.

The patient, a 45-year-old Japanese woman, was the second child of non-consanguineous parents. No skin hyperextensibility, fragility, or joint hypermobility were noted in her mother, elder sister, or two daughters. She was a preterm and low-birth-weight (1,980 g) infant. The patient had bilateral congenital hip dislocation for which she underwent fixation with a brace, experienced repetitive episodes of skin lacerations and subcutaneous hemorrhage after minor trauma, and had marked joint laxity, with repetitive sprains caused by unstable ankle joints. In her early 20 s, she was suspected to have EDS. Intractable hair loss has been her major physical concern since around that age. She also had spinal disc herniation. At the age of 36 years, she developed massive intraabdominal hemorrhages caused by rupture of the superior mesenteric artery, which were associated with multiple aneurysms and were treated with catheter embolization. Thin translucent skin was noted (Figure 1A-a).

When the patient was referred to us at the age of 45 years, she exhibited the following characteristics: hair with a kinky texture and generalized thinning; a high palate and multiple dental caries; hyperextensible and translucent skin (Figure 1A-a, b); skin striae in the lower extremities, with atrophic scars (Figure 1A-c); soft soles; an umbilical hernia (Figure 1A-d); pes planus (Figure 1A-e); and generalized joint hypermobility (Beighton score 8/9) (Figure 1A-f). Radiological examination showed no spinal deformities. There had been no episodes of dislocations or musculoskeletal pain. No aortic root dilatation or valve abnormalities were detected on echocardiography. She had high myopia, but no hearing impairment.

Genomic DNA was extracted from peripheral blood using a QIAamp DNA Blood Mini Kit on a QIAcube (Qiagen, Valencia, CA, United States). A next-generation sequencing (NGS) panel-based analysis was performed on an Ion Torrent system (Ion Chef and Ion GeneStudio S5, Thermo Fisher Scientific, Waltham, MA, United States) using an Ion AmpliSeq custom panel for 52 genes associated with EDS and other HCTDs (Supplementary Table S1). Detected variants were annotated by SnpEff and SnpSift (https://snpeff.sourceforge.net/) using the processed vcf file of the Genome Aggregation Database (gnomAD) v2.1.1 (https://gnomad.broadinstitute.org/downloads), ToMMo 8.3KJPN Genotype Frequency Panel (v20200831) (https://jmorp.megabank.tohoku.ac.jp/202008/downloads#variant) (Tadaka et al., 2019), ClinVar (ftp://ftp.ncbi.nlm.nih.gov/pub/clinvar/vcf_GRCh37/clinvar_20220328), dbNSFP3.4c and dbscSNV1.1 (https://sites.google.com/site/jpopgen/dbNSFP). Detected variants were evaluated in accordance with the 2015 American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines (Richards et al., 2015) and the ClinGen Sequence Variant Interpretation Working Group recommendations (SVI recommendations). Integrative Genomics Viewer (IGV) was used to visualize read alignments (Broad Institute, Cambridge, MA, United States). The NGS panel-based analysis revealed a non-sense variant c.2296G>T,p.(Glu766*) and a frameshift variant c.2383dup,p.(Glu795Glyfs*3) in AEBP1 (NM_001129.5), which were confirmed by Sanger sequencing (Figure 1B). The IGV revealed that the two variants were observed in trans (Figure 1C). The nonsense variant was registered in 8.3KJPN (1/16758, MAF = 0.0001, no homozygote) and the frameshift variant was registered in 8.3KJPN (1/16754, MAF = 0.0001, no homozygote). Both variants were classified as pathogenic (PVS1, PM2_Supporting, and PM3), in accordance with the 2015 ACMG/AMP guidelines and SVI recommendations.

Hematoxylin and eosin staining of a skin specimen obtained by a biopsy performed at the age of 36 years showed increased spaces between collagen fibers and disorganized orientations of these fibers in the lower and middle layer of the dermis (Figure 1D-b) compared with control (Figure 1D-a). Masson’s trichrome staining revealed decreased collagen fibers (Figure 1D-d) compared with control (Figure 1D-c). Elastica van Gieson staining showed prominent elastic fibers due to the decreased numbers of collagen fibers (Figure 1D-f) compared with control (Figure 1D-e). An ultrastructural analysis using transmission electron microscopy of the skin specimen revealed the presence of collagen fibrils with irregular contours (flower-like appearance) and small size under the cross-sectional view (Figure 1E). Measurement of procollagen production from cultured skin fibroblasts was performed as described previously (Shimaoka et al., 2010). Briefly, dermal fibroblasts from the patient were incubated with ³H-proline for 24 h. Labeled proteins secreted into the culture medium were digested with pepsin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/fluorography. Amounts of type I and type III procollagen were both reduced compared with an age- and sex-matched individual who served as a control (Figure 1F).

We have identified and described a 11th patient (9th family) with clEDS2, who was found to have novel compound heterozygous pathogenic variants in AEBP1. Detailed and comprehensive clinical and molecular features of all previously reported patients and the current patient are shown in Table 1.

Dental abnormalities, skin hyperextensibility, atrophic scars, easy bruising, and neuromuscular abnormalities were observed in all patients whose data were available. Excessive skin/skin folding (90.9%), delayed wound healing (90.9%) generalized joint hypermobility (90.9%), dislocations/(sub)luxations (90.9%), pes planus (90.9%), translucent skin (87.5%), piezogenic papules (80.0%), hernia (77.8%), osteopenia (71.4%), spine deformities (60.0%), prematurely aged appearance (57.1%), hallux valgus (54.5%), and hammertoes (54.5%) were observed in more than half of the patients whose data were available. Decreased hair described as “thinning” or “(partial) alopecia” was observed in five patients, and was a major physical concern in the current patient. The current patient developed multiple aneurysms and a rupture in the superior mesenteric artery, which was treated with catheter embolization. Cardiovascular complications reported in the previous patients included mitral valve prolapse/regurgitation, tricuspid valve regurgitation, pulmonary valve regurgitation, varicose veins, and aortic root dilation requiring surgery. A bowel rupture occurred in one patient, requiring repeated attempts to re-anastomose the bowel and colostomy. This is the first report of skin lacerations, which occurred in the current patient, whereas skin fragility was only noted in two other patients. Heterozygous individuals appear to have no relevant symptoms.

Most reported AEBP1 variants were null variants, including nonsense, frameshift and splice site variants, predicted to lead to nonsense-mediated mRNA decay (NMD) (Table 1; Figure 1G). Some variants were experimentally confirmed to affect the gene product. In Patients 1 and 2, a homozygous splice site variant (c.1630 + 1G>A) led to activation of the cryptic 5′splice site within exon 13 and skipping of the last 22 bp of exon 13. The shift in reading frame (p.Val537Leufs*31) was predicted to lead to NMD. In Patient 3, a 1-bp deletion (c.1470del) in exon 12 in one allele led to the retention of intron 12 (p.Asn490_Met495delinsLysAlaMetArgLysTrpTrpAlaProCysProGlySerTrpLeuCysSerHisCysLeuGlyGluGlyTrpAlaLeuArgGlyAlaGlySerThrAlaLeuArgProAlaSerProGln) (Blackburn et al., 2018). Its protein was retained intracellularly and not secreted (Vishwanath et al., 2020). A non-sense variant (c.1743C>A,p.Cys581*) in the other allele in Patient 3 was predicted to lead to NMD. In Patient 4, a homozygous frameshift variant (c.1320_1326del) led to a shift in reading frame (p.Arg440Serfs*3) (Blackburn et al., 2018). No ACLP protein was detected by western blotting, suggesting NMD. In Patient 6, a 4-bp deletion (c.1149_1150+2del) in one allele led to the loss of the last 4 bp of exon 9 and skipping of exon 10, resulting in an in-frame deletion (p.Val383_Gln420del) (Syx et al., 2019). A frameshift variant (c.443dup) in the other allele in Patient 6 was predicted to lead to NMD. The mRNA expression was significantly decreased, indicating that the AEBP1 transcript was unstable and/or prone to NMD.

In the current patient, light microscopic analyses showed increased interfibrillar spaces in the reticular dermis, a disorganized arrangement of collagen fibers and decreased collagen content, and a biochemical analysis showed reduced secretion of type I and type III procollagen. Electron microscopic analysis showed the presence of collagen fibrils with irregular contours (flower-like appearance) and small size. Blackburn et al. (2018) reported that light microscopy showed decreased collagen, while electron microscopy revealed the presence of irregular disrupted collagen fibrils. In their report, the discoidin domain, a highly conserved structural motif of ACLP, preferentially bound to collagen types I, III and V, and ACLP promoted the polymerization of type I collagen in vitro. Syx et al. (2019) reported electron microscopic observations that corresponded with those of Blackburn et al. (2018) and the current study, and a biochemical analysis that showed a normal electrophoretic pattern of procollagen types I, III and V. This biochemical analysis was performed on the medium of cultured skin fibroblasts to detect procollagen secreted from these fibroblasts. The discrepant results might be attributable to some functional differences between the variants reported by Syx et al. and the variants in the current patient, which could be related to the difference in the transcription status of these procollagen genes or in the secretion status of these types of procollagen. In view of all of these findings, ACLP protein is likely an important player in collagen fibrillogenesis.

In conclusion, the clinical findings and disease course in the current patient, together with the review of previously reported patients, paints a picture of the clinical similarities and variations in clEDS2. Furthermore, the biochemical and pathological findings in the current patient, in addition to the relevant findings in the previous patients, suggest the importance of ACLP in collagen fibrillogenesis. Further clinical, molecular, and pathophysiological studies are required to produce a more detailed and comprehensive delineation of this disorder.