Bi-allelic AEBP1 mutations in two patients with Ehlers–Danlos syndrome

Abstract

The Ehlers–Danlos syndromes (EDSs) are a clinically and molecularly diverse group of heritable connective tissue disorders caused by defects in a wide range of genes. Recently, bi-allelic loss-of-function mutations in the adipocyte enhancer-binding protein 1 (AEBP1) gene were reported in three families with an autosomal recessive EDS-like condition characterized by thin and hyperextensible skin, poor wound healing with prominent atrophic scarring, joint hypermobility and osteoporosis. Using whole exome sequencing, we identified novel bi-allelic AEBP1 variants in two unrelated adult patients, previously diagnosed with an undefined EDS type, which shows important clinical resemblance to several other EDS subtypes. Our patients present with similar cutaneous and musculoskeletal features as the previously reported patients. They also show unreported clinical features, including pectus deformity, premature aged appearance, sparse and frizzled hair, fatigue and pain. AEBP1 is ubiquitously expressed and encodes the secreted aortic carboxypeptidase-like protein (ACLP) that can bind fibrillar collagens and assist in collagen polymerization. Transmission electron microscopy studies on the patients’ skin biopsies show ultrastructural alterations in collagen fibril diameter and appearance, underscoring an important role for ACLP in collagen fibril organization. This report further expands the clinical, molecular and ultrastructural spectrum associated with AEBP1 defects and highlights the complex and variable phenotype associated with this new EDS variant.

Introduction

The Ehlers–Danlos syndromes (EDSs) comprise a group of clinically and genetically heterogeneous heritable connective tissue disorders (HCTDs), hallmarked by skin hyperextensibility and fragility, joint hypermobility and generalized connective tissue fragility. Over the years, distinct EDS subtypes have been delineated and defects were identified in an array of genes with diverse biological functions linked to collagen biosynthesis, assembly and organization, all leading to structural and functional alterations of the extracellular matrix (ECM). The latest EDS classification recognizes 13 distinct EDS subtypes caused by defects in 19 different genes and provides a regrouping based on the underlying genetic and pathogenetic mechanisms (1). Known EDS-associated genes affect either (a) the primary structure and processing of fibrillar collagens (COL1A1, COL1A2, COL3A1, COL5A1, COL5A2 and ADAMTS2); (b) collagen folding and cross-linking (PLOD1 and FKBP14); (c) myomatrix function and organization (TNXB and COL12A1); (d) proteoglycan biosynthesis (B3GALT6, B4GALT7, CHST14 and DSE); (e) the complement pathway (C1S and C1R) and (f) intracellular processes (SLC39A13, ZNF469 and PRDM5). Although our understanding of the molecular basis of EDS and other HCTDs has increased tremendously over the past decades, the underlying genetic defect remains elusive for a proportion of patients with an EDS phenotype, suggesting the involvement of other, presently unidentified genes.

Recently, whole exome sequencing (WES) resulted in the genetic elucidation of a novel autosomal recessive condition in four patients from three independent families presenting with joint hypermobility, skin hyperextensibility, poor wound healing with abnormal scarring, osteoporosis and other features reminiscent of EDS. In these patients, bi-allelic loss-of-function mutations were identified by WES in the adipocyte enhancer-binding protein 1 (AEBP1) gene (2,3), encoding the aortic carboxypeptidase-like protein (ACLP), which is secreted to the ECM. The protein is strongly expressed in collagen-rich tissues, including dermis, vasculature and developing skeleton (4,5), consistent with its important role in embryonic development and adult tissue repair.

In this study, we report the identification, through reanalysis of available WES data, of novel bi-allelic pathogenic variants in AEBP1 in two adult patients with a clinical diagnosis of EDS. We describe the clinical, molecular and ultrastructural consequences of these defects and demonstrate that AEBP1-associated EDS is a complex multisystemic disorder that shows extensive clinical similarity to other EDS subtypes.

Results

Clinical reports

Patient 1

Patient 1 (AN_005858) is the second child of a consanguineous couple of Caucasian descent. His father suffered from myasthenia gravis, and his mother is healthy. He has three siblings whom are all healthy.

He was born 6 weeks preterm after an uneventful pregnancy and delivery. Anthropomorphic information from the time of birth is not available. He was able to walk unsupported around the age of 18 months. At the age of 3 years, he was diagnosed with EDS because of hyperextensibility and fragility of the skin. He bruised easily and had slow wound healing with the formation of thin, cigarette paper scars, especially on the knees and shins. As a child, he also presented joint hypermobility and by the age of 5 years, he had suffered 3 dislocations of the elbow and multiple ankle distortions. There was no history of fractures. He had a spontaneous pneumothorax at the age of 22 years, herniation of fat in the right armpit at the age of 35 years and right-sided tinnitus from the age of 47 years.

On clinical evaluation at the age of 50 years, he had a weight of 87 kg, a height of 192.6 cm (lower segment, 100 cm), a head circumference of 57 cm and an arm span of 189.9 cm. He presented dysmorphic facial features, including an asymmetrical face, hypertelorism and posteriorly rotated, low-set ears with attached earlobes and webbed neck (Fig. 1A and B). Scleral hue, palate and uvula were normal. He had bilateral mild myopia and astigmatism. Teeth quality was bad with severe caries. His hair was thin and frizzled with partial alopecia, which had started around the age of 18 years, giving him a prematurely aged appearance (Fig. 1A and B). His skin was very thin and hyperextensible, with translucency on the thorax (Fig. 1D) and upper arms. Multiple cigarette paper scars and hemosiderotic plaques were present on elbows, knees and shins (Fig. 1C,G), and smaller atrophic scars were seen on the thorax and back. Marked varices were present on the lower limbs (Fig. 1C,G)) and healed decubitus wounds on the buttocks. Pronounced hyperlaxity of the finger joints, elbows and knees, foot deformities including pes planus, hallux valgus and hammertoes (Fig. 1H) and a pectus excavatum were also observed. Echocardiographic examination at the age of 50 years was grossly normal, with normal aortic dimensions but mild insufficiency of mitralis and tricuspid valves. He obtained a college degree, but mental health problems and depression, which developed later in life, forced him to refrain from occupational activities.

Figure 1

Clinical characteristics of patient 1 at the age of 50 years (A–D and H) or 58 years (E–G, I and J) and patient 2 at the age of 21 years (K and L). (A, B, E and F) Facial features of patient 1 include asymmetric face with hypertelorism, sparse, thin and frizzled hair and webbed neck. (C and G) Presence of severe and discoloured cigarette paper scars on the knees and the presence of varices on the lower limbs. (D) Thin and translucent skin on the thorax with visible venous pattern. (H) Foot deformities, including pes planus, hallux valgus and hammertoes. (I and J) Hands of patient 1 have an acrogeric aspect with a thin and wrinkled skin and show the presence of bruises. (K) Thin and translucent skin on the thorax of patient 2 with visible venous pattern. (L) Small atrophic scars on the knees.

Patient 2

Patient 2 (AN_005859) is the first child of healthy, non-consanguineous parents of Caucasian decent. Both her parents and younger sister are healthy.

She was born in breech position with a birth weight of 1650 g after a pregnancy of 35⁺² weeks following the diagnosis of HELLP (haemolysis, elevated liver enzymes and low platelets) syndrome in her mother. At the age of 3 months, she was diagnosed with left-sided hip dysplasia, which was treated with Pavlik harness. She walked unsupported at the age of 18 months. As a child, joint hypermobility was noted and she received physiotherapy treatment to improve her muscle strength and posture. She showed no manifest skin fragility but had somewhat delayed wound healing, a bleeding tendency with easy bruising and frequent episodes of petechiae on the feet. Following a cycling accident, she developed a severe vaginal hematoma, which required suprapubic catheterization for 1 week. Scoliosis (16°) was noted at the age of 12 years.

During adolescence, she developed progressive musculoskeletal complaints, including frequent subluxations of shoulders, knees, elbows, thumbs and temporomandibular joints and ankle sprains. She also had recurrent subluxations of the hips, and a spontaneous left hip subluxation at the age of 19 years compromised her mobility. In addition, she developed progressive bilateral weakness of the hip abductors and external rotators with a fast decline in strength over the course of 1.5 years. She gradually lost her ability to walk unsupported and by the age of 21 years she could walk only 5 m using a walker frame. She reported both fasciculations and involuntary movements in the legs. Trunk stability gradually declined to the point that she needed support of the hands to remain upright in a seated position. Neurological evaluation at the age of 21 years showed normal results on sensory testing and deep tendon reflex testing. Muscle strength and tone in legs were reduced (Medical Research Counsel grade: hip flexion, 3; knee flexion, 4). She was unable to walk unsupported, most likely as a result of both articular/joint and musculoskeletal disability. A muscle ultrasound showed mildly increased echo intensity, in line with previous observations in EDS (6). Needle biopsy of the lateral vastus muscle showed non-specific mildly reduced fibre type 1 size, but was otherwise normal. In addition to her musculoskeletal problems, she suffered from gastroesophageal reflux, esophageal spasms, dysphagia, bloated feeling, abdominal cramps/pain and episodes of constipation or diarrhoea. Urogenital problems include urinary retention requiring catheterization, bladder cramps and (often painful) urinary urgency. She also suffers from invalidating chronic fatigue. A reduced effect of local anaesthesia was reported. Echocardiographic evaluation at the age of 18 and 20 years, respectively, was normal. She was diagnosed with postural orthostatic tachycardia syndrome (POTS).

Clinical evaluation at the age of 21 years showed a normal, proportionate stature and absence of overt dysmorphic features. She had a height of 164 cm. Mild ptosis and mild myopia were observed. Her skin was very thin and translucent (Fig. 1K) with two small atrophic scars on the knees (Fig. 1L). Locomotor evaluation revealed generalized joint hypermobility with a Beighton score of 9/9, marked finger joint hyperlaxity, drooping shoulders and pes planus. Manual muscle testing (MMT) indicated bilateral weakness of the hip abductors and external rotators (left hip external rotation: MMT 3−; right hip external rotation: MMT 3+; quadriceps: 4−). The strength in the hip adductors (MMT, 4+), tibialis anterior (MMT 5) and gastrocnemius (MMT 5) was less affected. During walking, a bilateral Trendelenburg sign was present. She reported bad teeth quality with frequent caries and thinning of her hair over the past years. X-ray survey and head-to-pelvis magnetic resonance angiography did not show overt skeletal or vascular abnormalities, respectively. The patient shows normal intelligence and is currently pursuing a college degree, with an individualized trajectory adapted to cope with her fatigue and mobility problems.

Molecular analyses

Because the clinical presentation of both patients showed similarity to classical EDS [cEDS, (OMIM 130000 and 130010): skin fragility, atrophic scarring, easy bruising and generalized joint hypermobility] and vascular EDS [vEDS, (OMIM 130050): thin, translucent skin, easy bruising, varicose veins and pneumothorax], molecular analysis of the COL5A1, COL5A2 and COL3A1 genes was initially performed, but no causal sequence alterations could be detected in any of these genes. In addition, normal copper and ceruloplasmin levels in patient 1 excluded the X-linked connective tissue disorder occipital horn syndrome (OMIM 304150). Standard karyotyping was normal for patient 1 without structural or numerical aberrations (46,XY), and array comparative genomic hybridization (aCGH) analysis did not reveal genomic deletions or duplications.

We next performed WES for both patients. Data analysis initially focussed on all previously reported EDS-associated genes (COL5A1, COL5A2, COL3A1, COL1A1, COL1A2, ADAMTS2, TNXB, COL12A1, PLOD1, FKBP14, B4GALT7, B3GALT6, SLC39A13, CHST14, DSE, ZNF469, PRDM5, C1S and C1R) but could not demonstrate (a) causal variant(s) (Supplementary Material, Table S1). Following the recent identification of AEBP1 mutations resulting in a phenotype reminiscent of EDS (2,3), we revisited the WES data on AEBP1 and found a homozygous single-nucleotide duplication in exon 2 of the AEBP1 gene (c.362dupA) in patient 1, which results in a frameshift and is predicted to introduce a premature termination codon [p.(Glu122Glyfs*16)]. The variant was classified as likely pathogenic (class 4) according to the guidelines of the American College of Medical Genetics and Genomics (ACMG). Samples of the parents or siblings were not available for molecular testing.

For patient 2, WES reanalysis revealed compound heterozygosity for two indel variants in the AEBP1 gene. The first variant comprises a single-nucleotide duplication (c.443dupA) in exon 2 leading to a frameshift, predicted to introduce a premature termination codon [p.(Ala149Glyfs^*57)]. The second variant is a 4 bp deletion encompassing the donor splice site of exon 9 (c.1149_1150 + 2delCAGT). Splice site prediction programmes for the latter variant indicated a loss of the donor splice site of exon 9 and creation of an alternative splice site 4 bp upstream. The former variant was inherited from the mother, while the latter variant was inherited from the father. The unaffected sister is a heterozygous carrier of the maternal c.443dupA variant. Both variants were classified as pathogenic (class 5) according to the ACMG guidelines.

None of the identified variants were reported in public databases (queried on October 31, 2018).

Characterization of the identified AEBP1 mutations and expression analyses

The AEBP1 gene encodes the multi-domain ACLP protein, consisting of an N-terminal signal peptide, a lysine/proline/glutamic acid-rich (KPE) motif, a discoidin domain and a catalytically inactive metallocarboxypeptidase domain (Fig. 2A) (7). In contrast to the reported mutations, which are either located in the discoidin domain or the carboxypeptidase-like domain (2,3), the p.(Glu122Glyfs^*16) variant (patient 1) and the p.(Ala149Glyfs^*57) variant (patient 2) are located in the complex KPE motif. The c.1149_1150+2delCAGT variant (patient 2) resides just upstream of the discoidin domain (Fig. 2A). Reverse transcription polymerase chain reaction (RT-PCR) confirmed that the latter variant disrupts the donor splice site of exon 9 with activation of an alternative donor splice site 4 bp upstream. This results in the loss of the last 4 bp of exon 9 and a complete skip of exon 10 (110 bp), resulting in a loss of 114 bp corresponding to an in-frame deletion of 38 amino acids encompassing part of the discoidin domain (Fig. 2B).

Figure 2

Mutation identification and characterization. (A) Schematic representation of the ACLP protein structure with indication of the different domains. Newly identified mutations are indicated above the structure, whereas previously identified mutations are indicated below. SP denotes signal peptide. (B) Effect of the c.1149_1150+2delCAGT variant on splicing. The middle panel depicts the genomic structure of exons 8–11 of the AEBP1 gene. Dashed lines indicate the splicing events occurring. The upper panel represents the normal splicing mechanism, whereas the lower panel shows the consequences of the identified 4 bp deletion. RT-PCR showed normal splicing in control fibroblasts (upper panel), whereas in patient fibroblasts an alternative donor splice site is activated 4 bp upstream of the conventional site. This results in the deletion of the last 4 bp of exon 9 (indicated in dark grey) and a complete skip of exon 10 (lower panel). (C) RT-qPCR shows decreased expression of AEBP1 in dermal fibroblasts from patient 1 (P1) and patient 2 (P2) compared to controls (C1 and C2). Error bars denote standard error of the mean. ^***P < 0.001 (unpaired t-test). (D) Expression analysis in human tissues reveals ubiquitous AEBP1 expression. ACTB is used as a loading control.

Reverse transcription quantitative PCR (RT-qPCR) revealed a significantly decreased overall expression of AEBP1 in fibroblasts from both patient 1 and patient 2, indicating that the mutant transcripts are unstable and/or (partially) prone to nonsense-mediated mRNA decay (Fig. 2C).

In order to investigate the general expression profile of AEBP1, RT-PCR was performed on a range of commercially available human tissues. This demonstrated a ubiquitous expression of AEBP1 (Fig. 2D).

Biochemical collagen analyses

Fibroblast cultures were established from a skin biopsy from patient 1. SDS-PAGE analysis of metabolically labelled procollagens and pepsin-digested fluorescently labelled collagens extracted from the medium and the cell layer of dermal fibroblast cultures showed a normal electrophoretic pattern for the bands representing the α-chains of types I, III and V (pro)collagen proteins, without clear qualitative or quantitative alterations (data not shown).

Analysis of the urinary collagen cross-links from patient 1 revealed a lysylpyridinoline/hydroxylysylpyridinoline (LP/HP) ratio of 0.27, which is similar to that of normal controls (LP/HP, 0.19 ± 0.02; range, 0.12–0.25; n = 179).

Ultrastructural collagen analysis

We evaluated collagen fibril architecture in the reticular dermis of a skin biopsy from both patients by transmission electron microscopy (TEM). Cross-sectional TEM profiles in both patients demonstrated fibrils with variable diameters, and some fibrils displayed a small flower-like appearance with irregular contours (Fig. 3A). Morphometric analysis revealed mildly increased fibril diameters with a somewhat broader distribution of fibril diameters (patient 1, 97.8 ± 9.6 nm; patient 2, 98.7 ± 13.3 nm) compared to control (90.8 ± 7.1 nm). Interfibrillar spaces appeared grossly normal in patient 2 but were larger and filled with amorphous granulofilamentous deposits in patient 1 (Fig. 3A). In addition, the morphology of elastin showed an altered appearance with ruffled edges in both patients (Fig. 3B). Fibroblasts were morphologically normal and did not show dilated endoplasmic reticulum or other ultrastructural signs of activation of the unfolded protein response and/or autophagy (data not shown).

Figure 3

Ultrastructural studies. (A) Cross-sectional TEM of the reticular dermis of patients shows increased interfibrillar spacing with granulofilamentous deposits, some variability in the diameter of the fibrils as well as the presence of fibrils with irregular diameters (arrowheads) compared to control. Scale bar, 1 μm. Histogram of the size distribution of fibril areas measured on cross section (n = 500). C, control; P, patient. (B) Elastin deposited in the reticular dermis of patients shows a ruffled appearance on the edges. Scale bar, 1 μm.

Discussion

Bi-allelic AEBP1 mutations were recently identified in four individuals presenting with a complex EDS-like condition, originating from three unrelated families (2,3). We therefore re-evaluated available WES data in a cohort of patients with a molecularly unresolved EDS phenotype and identified novel homozygous or compound heterozygous AEBP1 mutations in two independent adult patients, bringing the total number of patients with AEBP1-associated EDS to six. While this is still a very small number, a phenotypic pattern becomes apparent (Table 1). Main characteristics include (a) marked cutaneous involvement, with skin hyperextensibility (6/6), delayed wound healing (6/6), skin fragility with easy bruising (5/6) and atrophic scarring (4/6), redundant skin with skin folds (4/6) and/or a thin translucent skin with a visible venous pattern especially over thorax and upper limbs (3/6) and (b) musculoskeletal involvement with generalized joint hypermobility (6/6), multiple joint dislocations and/or subluxations (5/6) frequently involving the hips (4/6) and the shoulders (3/6), foot deformities with pes planus (6/6), hallux valgus (5/6) and toe deformities (5/6), early-onset osteopenia (4/6) without history of low-impact fractures and scoliosis (2/6). Other, variably present features include cardiovascular problems [mitral valve prolapse (3/6), mild aortic dilatation (1/6) and stenosis of the carotids (1/6)]; gastrointestinal problems [motility issues (2/6), spontaneous bowel rupture (1/6) and umbilical hernia (1/6)]; and urogenital problems [cryptorchidism (1/4 males)], underscoring the multisystemic character and phenotypic variability of this condition. Some previously unreported features were also observed. Patient 1 presented a pectus excavatum and suffered a spontaneous pneumothorax in adulthood. He also showed a premature aged appearance with somewhat dysmorphic features, a frizzled aspect of the hair and early-onset partial alopecia and bad tooth quality. Premature hair loss and bad tooth quality was also observed in patient 2. She developed severe mobility problems at young adult age due to a combination of severe and intractable instability of the hip joints and progressive muscle weakness in the lower limbs, eventually leaving her wheelchair dependent.

Table 1

Clinical summary of individuals with AEBP1 mutations identified so far

	Patient 1	Patient 2	A-II:1	B-II:1	C-IV:6	C-IV:4
	Present report	Present report	Blackburn et al. (3)	Blackburn et al. (3)	Alazami et al. (2)	Alazami et al. (2)
Age at the time of report (years)	58	21	35	33	12	24
Gender	Male	Female	Male	Male	Female	Male
Ethnicity	Caucasian	Caucasian	Caucasian	Caucasian	Middle Eastern	Middle Eastern
AEBP1 mutation (NM_001129.4)	c.[362dupA]; [362dupA]	c.[443dupA]; [1149_1150+2del]	c.[1470del]; [743C>A]	c.[1320_1326del]; [1320_1326del]	c.[1630+1G>A]; [1630+1G>A]	c.[1630+1G>A]; [1630+1G>A]
Protein change (NP_001120.3)	p.[(Glu122Glyfs*16)]; [(Glu122Glyfs*16)]	p.[(Ala149Glyfs*57)]; [?]	p.[(Asn490_Met495delins(40))]; [(Cys581*)]	p.[(Arg440Serfs*3)]; [(Arg440Serfs*3)]	p.[?]; [?]	p.[?]; [?]
Craniofacial
Facial dysmorphism	Asymmetrical face; hypertelorism; posteriorly rotated, low-set ears with attached earlobes; thin, frizzled hair and partial alopecia; webbed neck	Mild ptosis, thinning of hair	None	Micrognathia	Low posterior hairline, webbed neck, bilateral ptosis, excess redundant skin on face, large ears, narrow palate	Unknown
Dentition	Bad tooth quality with severe caries	Bad tooth quality	Retains a single baby tooth	Not reported	Abnormal dental alignment	Unknown
Cutaneous
Skin hyperextensibility	+	+	+	+	+	+
Thin, translucent skin	+	+	NA	+	NA	NA
Excessive skin/ skin folding	−	−	+ (Increase wrinkles on hands and feet)	+	+	+
Delayed wound healing	+	+ (Mild)	+	+	+	+
Abnormal scarring	Atrophic, widened scars	No excessive scarring	Atrophic, widened scars	Atrophic, widened scars with hyperpigmentation	Hyperpigmented atrophic scars, multiple keloids	Unknown
Easy bruising	+	+	+	+	+	Unknown
Musculoskeletal
Generalized joint hypermobility	+	+	+	+	+	+
Beighton score	Unknown	9/9	8/9	8/9	8/9	Unknown
Dislocations/ (sub)luxations	+	+ (Hip and shoulder subluxations)	+ (Hip subluxations)	+ (Congenital hip dislocation, shoulder dislocation)	+ (Hip and shoulder dislocations)	Unknown
Pectus deformity	+	−	Not reported	Not reported	Not reported	Not reported
Foot deformities	Pes planus, hallux valgus, hammertoes	Pes planus	Pes planus, hallux valgus, hammertoes	Pes planus, hallux valgus, hammertoes	Pes planus, hallux valgus, hammertoes	Pes planus, hallux valgus, toe deformities
(Kypho)scoliosis	−	+	−	+ (Thoracic)	Not reported	Not reported
Osteopenia	NA (no fractures)	− (No fractures)	+ (Hips) (no fractures)	+ (Requiring hip replacement) (Fractures, NA)	+ (Fractures, NA)	+ (Fractures, NA)

Table 1

Continued

	Patient 1	Patient 2	A-II:1	B-II:1	C-IV:6	C-IV:4
Other		Progressive decreased muscle strength		Upper thoracic scoliosis with degenerative disease and facet arthrosis of spine	Skull shows `copper beaten’ appearance, narrowing of the interpedicular distance of the lumbar spine distally, iliac bones are short and squared, long bones of the lower extremities are remodelled
Cardiovascular
Mitral valve prolapse	+ (Mild)	−	+	+	−	−
Aortic dilatation	−	−	−	+ (Mild)	−	−
Other		Vaginal hematoma after trauma		Bilateral stenosis of the carotids
Gastrointestinal
Other	−	Gastroesophageal reflux, esophageal spasms, dysphagia, bloated feeling, abdominal cramps/pain and episodes of constipation or diarrhoea	Motility issues	Bowel rupture	Not reported	Not reported
Urogenital
Cryptorchidism	Not reported	/	+	Not reported	/	Not reported
Other		Urinary retention requiring catheterization, bladder cramps and (often painful) urinary urgency
Other
Hernia	Not reported	Not reported	Not reported	Large ventral hernia developed at surgical sites secondary to ruptured bowel	Umbilical, ventral, inguinal	Unknown
Miscellaneous	Premature aged appearance, myopia, pneumothorax, decubitus wounds on buttocks	Mild myopia, chronic fatigue, chronic widespread pain, POTS	Delays in walking and acquisition of fine motor skills, impaired temperature sensation, keratoconjunctivitis sicca, piezogenic papules on feet, right distal radioulnar joint dislocation, surgically repaired	Elbow bursitis, piezogenic papules on feet, sacral dimple, hypertriglyceridemia	Uncontrolled diabetes mellitus, recurrent cellulitis	None

+ and − indicate the presence and absence of a clinical feature, respectively. NA, not assessed; POTS, postural orthostatic tachycardia syndrome.

The phenotype associated with AEBP1 mutations shows considerable clinical similarity to several other EDS subtypes. The combination of generalized joint hypermobility with skin hyperextensibility and fragility, poor wound healing and cigarette paper scarring with hemosiderotic plaques (as observed in patient 1, present study), are reminiscent of cEDS (OMIM 130000 and 130010) due to type V collagen defects, whereas the marked skin translucency, easy bruising, bleeding tendency, pneumothorax and bowel rupture resemble vEDS (OMIM 130050), caused by a type III collagen defect. There is also substantial clinical similarity to some of the rare, autosomal recessive forms of EDS, especially with musculocontractural EDS (OMIM 601776), caused by deficiency of dermatan-4-sulfotransferase-1 (thin, translucent skin, bleeding tendency, cryptorchidism and gastrointestinal problems) and with spondylodysplastic EDS (OMIM 615349) due to deficiency of galactosyltransferase II (osteopenia, sparse hair, premature aged appearance and bad tooth quality). The identification of this novel EDS subtype underscores that, given the phenotypic variability within the different EDS subtypes and the important clinical resemblance between the EDS subtypes and with other HCTDs, molecular confirmation of the clinical diagnosis is warranted (1). Next-generation sequencing approaches have a high diagnostic value for this complex group of conditions, and since there is still a substantial group of EDS patients that remain genetically unresolved, WES provides the opportunity to simultaneously interrogate all known EDS genes in a timely manner as well as to identify novel EDS disease genes.

To date, a total of seven AEBP1 mutations have been identified, which are localized throughout the AEBP1 gene, including one nonsense variant, four indels and two variants affecting a splice site region, all of which are predicted to disrupt the function of the corresponding ACLP protein. Functional involvement of this protein in connective tissue development and homeostasis was already demonstrated in the Aebp1 knockout (Aebp1^−/−) mouse model. The majority of Aebp1^−/− mice die perinatally due to gastroschisis, a severe disruption of the anterior abdominal wall leading to herniation of the abdominal organs. Surviving Aebp1^−/− mice (~6.6%) develop spontaneous skin ulcerations and present with delayed wound healing that is correlated to a reduction in dermal fibroblast proliferation (4). These wound healing defects are reminiscent of the reported human phenotype and indicate a role for ACLP in wound repair. ACLP levels are indeed highly increased in injured dermis and vasculature (4). Moreover, it was demonstrated that ACLP can contribute to wound healing by promoting differentiation of (lung) fibroblasts to myofibroblasts (an important process during wound healing) through activation of transforming growth factor β (TGFβ) receptor signalling and this transition was diminished in Aebp1^−/− mice (8,9).

The different (organ) systems affected by ACLP deficiency are in line with the expression pattern of AEBP1 in adult human tissues. ACLP, a secreted protein that associates with the ECM, is mainly present in collagen-rich tissues, including dermis, vasculature and developing skeleton (4,5). Recombinant ACLP was shown to increase collagen expression and deposition in lung fibroblasts in a TGFβ receptor independent way (8,9). The lack of ACLP negatively influences collagen deposition, as demonstrated by the decreased dermal collagen content observed in patients (3). Interestingly, the discoidin domain of ACLP has been shown to directly interact with fibrillar collagens and preferentially binds to types I, III and V collagen, which are defective in other EDS subtypes that show significant phenotypic similarity to ACLP-deficient EDS. ACLP appears to promote type I collagen polymerization in vitro and is important for collagen fibrillogenesis in vivo (3). We did not observe any changes in the electrophoretic migration pattern of the bands corresponding to the different α-chains of the fibrillar collagen types in patient 1, suggesting normal posttranslational modification of these collagens, but we did see abnormalities in the dermal collagen fibril organization in our patients, with mild flower-like appearance of some collagen fibrils, and modest changes in the fibril diameter distribution. Interestingly, we also observed for the first time ultrastructural alterations in elastin associated with ACLP deficiency.

ACLP expression was first demonstrated in vascular smooth muscle cells of the blood vessels (4). ACLP is also highly expressed in developing skeleton (5), and it has been hypothesized to be involved in osteogenic differentiation (10). In four of the six reported patients, the presence of osteoporosis or osteopenia has been described. Intriguingly, ACLP was recently demonstrated to activate the canonical Wnt signalling pathway in hepatic stellate cells through binding frizzled-8 and low-density lipoprotein receptor-related protein 6 (LRP6) and trigger β-catenin-dependent gene expression (11). Wnt signalling plays an important role in bone development and homeostasis (12). Activation of the canonical Wnt pathway has been shown to lead to increased bone formation and bone mass and decreased bone resorption (13). Therefore, it is tempting to speculate that a defect in ACLP can contribute, at least in part, to a reduced β-catenin signalling in bone cells, which can result in reduced bone mass and in turn lead to osteoporosis as seen in affected individuals. Nevertheless, deregulation of other pathways that are potentially regulated by ACLP may contribute to this process as well.

A schematic representation of the mechanisms described above is depicted in Figure 4. However, it needs to be taken into account that mechanisms of action might differ between tissues and that distinct signalling pathways, activated by ACLP, are likely cell-type specific. In addition, the precise roles of the different ACLP domains need to be further determined and might explain the different effects of the identified mutations, depending on their remaining activity. Therefore, the extent and contribution of alterations in these and possibly other pathways and mechanisms that are disturbed because of ACLP deficiency, and how they contribute to this constellation of clinical features and ultrastructural findings require further investigation.

Figure 4

Schematic representation of (possible) pathophysiologic mechanisms associated with ACLP deficiency. (A) Effect on wound healing. Under normal circumstances, ACLP is involved in fibroblast to myofibroblast transition, an important process during wound healing. ACLP interacts with and activates TGFβ receptors and stimulates downstream Smad3 signal transduction pathways to enhance the expression of the pro-contractile, cytoskeletal protein smooth muscle α-actin (9). ACLP defects contribute to defective wound repair through diminished (TGFβ) receptor signalling and reduced fibroblast to myofibroblast transition. (B) Effect on ECM integrity. In normal conditions, ACLP affects the expression of collagen. Moreover, the central discoidin domain of ACLP was shown to preferentially bind fibrillar types I, III and V collagens and assist in proper type I collagen polymerization, suggesting a role for ACLP in the modulation of collagen fibrillogenesis and the organization and remodelling of the ECM (3,9). As such, the absence of functional ACLP can lead to abnormal collagen fibril assembly and organization, as shown by ultrastructural analyses in the skin of patients [(3) and present study]. The compromised ECM can also influence myofibroblast proliferation and differentiation via biochemical and mechanical signalling and as such affect proper wound repair (8). (C) Effect on bone homeostasis. ACLP has been shown to interact with frizzled-8 and LRP6 and activates the canonical (β-catenin-dependent) Wnt signalling pathway (11). As this is also an important pathway for bone development and homeostasis, ACLP defects might compromise Wnt signalling thereby contributing to the observed osteoporosis in patients.

In summary, the identification of loss-of-function mutations in AEBP1 expands the genetic heterogeneity associated with EDS. Defects in AEBP1 result in a variable, multisystemic phenotype, mainly characterized by a hyperextensible and translucent skin, delayed wound healing with abnormal scar formation, easy bruising, generalized joint hypermobility frequently complicated by dislocations and/or subluxations, foot deformities and premature osteoporosis. Our study further highlights the variability in phenotypic presentation associated with ACLP deficiency, both at the clinical and ultrastructural level. Furthermore, this study underscores the importance of WES to determine the molecular diagnosis in patients with an (unclassified) EDS phenotype.

Materials and Methods

Patient information and material

Blood samples were taken from the patients, parents and siblings when available. Genomic DNA (gDNA) was extracted from the blood sample using the Gentra Puregene extraction method (Qiagen, Hilden, Germany). This study was approved by the Ethics Committee of Ghent University Hospital, Belgium. Informed consent was obtained from the patients.

Molecular analyses

All coding exons and flanking intronic regions of the COL5A1 (NM_000093.3), COL5A2 (NM_000393.3) and COL3A1 (NM_000090.3) genes were PCR amplified followed by bidirectional sequencing using the BigDye® Terminator Cycle Sequencing kit and the ABI 3730XL DNA Analyzer (Applied Biosystems, Foster City, CA). To evaluate if both COL5A1 and COL3A1 alleles were expressed, heterozygosity for four single-nucleotide polymorphisms was examined at the gDNA and cDNA level as previously described (14).

WES was performed using the SureSelect XT Human All Exon V6 enrichment kit (Agilent Technologies, Santa Clara, CA) followed by paired-end sequencing (2 × 150 bp) on Illumina’s HiSeq3000 sequencer (Illumina, San Diego, CA). Reads were mapped and variants were called and annotated with the BCbio pipeline. Variants were subsequently analysed and filtered using our custom analysis platform Seqplorer. The identified AEBP1 variants were confirmed with bidirectional sequencing as described above. Nucleotide numbering reflects cDNA numbering, with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence (NM_001129.4). Amino acid residues are numbered from the first methionine residue of the reference sequence (NP_001120.3). Occurrence of the variant was assessed using the Genome Aggregation Database (gnomAD v2.1; http://gnomad.broadinstitute.org). Variant nomenclature follows the Human Genome Variation Society (HGVS) guidelines (http://www.hgvs.org/mutnomen) and was checked with the Mutalyzer software (https://mutalyzer.nl) (15). All variants were submitted to the EDS Variant Database (https://eds.gene.le.ac.uk/). A unique identifier was obtained for each patient following submission to the EDS Variant Database and was included in the text. Variants were classified according to the standards and guidelines of the ACMG (16). Splice site prediction was performed using the Alamut^® Visual software (v2.10; Interactive Biosoftware, Rouen, France).

Cytogenetic analyses

Conventional karyotyping of G-banded metaphase chromosomes was performed on short-term cultured lymphocytes using standard procedures. For aCGH, gDNA was isolated from total blood using the QiaAmp purification kit (Qiagen) according to the manufacturer’s recommendations. gDNA was hybridized on the Agilent SurePrint G3 Human CGH Microarray 60 K (AMADID#021924; Agilent Technologies), according to the manufacturer’s instructions with minor modifications. Array CGH data was analysed with arrayCGHbase (17) for copy number analysis.

Cell culture

Dermal fibroblast cultures were established from a skin biopsy from patient 1 and grown and maintained in Dulbecco’s Modified Eagle Medium (Gibco; Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum Good (PAN-Biotech, Aidenbach, Germany), 1% non-essential amino acids (Gibco), 1% penicillin/streptomycin (Gibco), 1% kanamycin (Gibco) and 0.1% amphotericin B (Gibco) and incubated at 37°C with 5% CO₂.

Biochemical collagen analyses

For steady-state (pro)collagen analyses, fibroblasts were plated in petri dishes and grown in medium supplemented with 25 μg/ml ascorbic acid for 24 h. Next, confluent cell layers were incubated for 24 h with medium containing L-proline or ¹⁴C-proline for collagen or procollagen analyses, respectively. For collagen analyses, procollagens were harvested from cell layers and conditioned media and precipitated with ammonium sulphate. Next, collagens were prepared from procollagens by pepsin digestion (50 μg/ml), followed by precipitation in 0.9 M sodium chloride (NaCl). Precipitates were subsequently resuspended in 0.1 M sodium carbonate, 0.5 M NaCl, pH 9.3 and labelled with a freshly prepared solution of monoreactive Cy5 NHS ester (GE Healthcare, Chicago, IL) in N,N-dimethylformamide, incubated for 30 min at room temperature with gentle shaking. The Cy5 labelled samples were separated on 5% polyacrylamide mini gels. Gels were directly scanned with the Typhoon Trio imager system (GE Healthcare). To perform steady-state procollagen analysis, metabolically (¹⁴C-proline-) labelled procollagen proteins were precipitated from medium fractions with 66% ethanol and dissolved in 0.5 M acetic acid. Next, aliquots were lyophylized and dissolved in electrophoresis sample buffer and separated on 5% polyacrylamide gels. Gels were fixed in 10%/10% (v/v) methanol/acetic acid, 96% acetic acid, impregnated with 20% 2,5-diphenyloxazole, washed in deionized water and dried. Dried gels were exposed to BAS Storage Phosphor Screens (GE Healthcare) for 3–5 days, which were subsequently scanned with the Typhoon Trio imager system (GE Healthcare).

The levels of LP and HP excretion in urine were quantified with high-performance liquid chromatography as described previously (18).

Expression analysis

Total RNA was extracted in triplicate from fibroblast cultures using the RNeasy^® kit (Qiagen) with DNase digestion, followed by cDNA synthesis using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA) and RT-qPCR for AEBP1. Data analysis was performed with the qBasePlus software (Biogazelle, Ghent, Belgium) using reference genes HPRT1 and YWHAZ for normalization. Tissue-specific expression of AEBP1 was assessed using the FirstChoice Human Total RNA Survey Panel (Life Technologies, Carlsbad, CA), human tendon (OriGene Technologies, Rockville, MD) and adult skin RNA (United States Biological, Salem, MA) and pooled RNA from three human dermal fibroblast cultures. cDNA was synthesized with the iScript cDNA Synthesis Kit (Bio-Rad Laboratories). RT-PCR was performed using two primer pairs (only one is shown) and an aliquot of each reaction was analysed on the Fragment Analyzer capillary electrophoresis instrument (Advanced Analytical, Agilent Technologies) and PROSize^® Data Analysis software (Advanced Analytical, Agilent Technologies). ACTB was included as loading control. Band intensities correspond to the expression of the gene of interest in a tissue.

Evaluation of effect on splicing

RNA was extracted from fibroblast cultures as described above and converted to cDNA using Moloney Murine Leukemia Virus reverse transcriptase (Life Technologies) according to the manufacturer's instructions. A fragment encompassing exon 6–12 was PCR amplified and bidirectionally sequenced as described above.

TEM

For ultrastructural studies, small fragments of a skin biopsy from patient 1 and patient 2 were immersed in a fixative solution of 2.5% glutaraldehyde and 3% formaldehyde in 0.1 M Na-cacodylate buffer, placed in a vacuum oven for 30 min and left rotating for 3 h at room temperature. This solution was later replaced with fresh fixative, and samples were left rotating overnight at 4°C. After washing, samples were postfixed in 1% OsO₄ with K₃Fe(CN)₆ in 0.1 M Na-cacodylate buffer, pH 7.2. After washing in ddH₂O, samples were subsequently dehydrated through a graded ethanol series, including a bulk staining with 2% uranyl acetate at the 50% ethanol step followed by embedding in Spurr’s resin. To select the area of interest on the block and in order to have an overview of the phenotype, semi-thin sections were first cut at 0.5 μm and stained with toluidine blue. Ultrathin sections of a gold interference colour were cut using an ultra microtome (Leica EM UC6), followed by a post-staining in a Leica EM AC20 for 40 min in uranyl acetate at 20°C and for 10 min in lead stain at 20°C. Sections were collected on formvar-coated copper slot grids. Grids were viewed with a JEM-1400plus transmission electron microscope (JEOL, Tokyo, Japan) operating at 60 kV or a Zeiss LEO 960 electron microscope (Carl Zeiss Microscopy, Thornwood, NY). The reticular dermis was evaluated. Morphometric analyses were performed using the Fiji software (19) and included quantification of the area of individual fibrils (n = 500) from different areas in the reticular dermis, followed by calculation of the fibril diameters. Abnormally shaped collagen fibrils were excluded from this analysis.

Statistical analysis

Unpaired t-test was used for individual comparisons. Analyses were performed using the GraphPad Prism software (v5; GraphPad Software, LaJolla, CA, USA).

Acknowledgements

We thank the patients and their families for their contribution to the study. We are grateful to Cecilia Giunta and her team at the University Children’s Hospital (Zurich, Switzerland) for urinary collagen cross-links analysis.

Conflict of Interest statement. None declared.

Funding

Research Foundation Flanders (12Q5917N to D.S., 1842318N to F.M.); Ghent University (BOFMET2015000401 to A.D.P.).