Bi-allelic Alterations in AEBP1 Lead to Defective Collagen Assembly and Connective Tissue Structure Resulting in a Variant of Ehlers-Danlos Syndrome
2018; Elsevier BV; Volume: 102; Issue: 4 Linguagem: Inglês
10.1016/j.ajhg.2018.02.018
ISSN1537-6605
AutoresPatrick R. Blackburn, Xu Zhi, Kathleen E. Tumelty, Rose Zhao, William J. Monis, Kimberly G. Harris, Jennifer Gass, Margot A. Cousin, Nicole J. Boczek, Mario Mitkov, Mark A. Cappel, Clair A. Francomano, Joseph E. Parisi, Eric W. Klee, Eissa Faqeih, Fowzan S. Alkuraya, Matthew D. Layne, Nazli B. McDonnell, Paldeep S. Atwal,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoAEBP1 encodes the aortic carboxypeptidase-like protein (ACLP) that associates with collagens in the extracellular matrix (ECM) and has several roles in development, tissue repair, and fibrosis. ACLP is expressed in bone, the vasculature, and dermal tissues and is involved in fibroblast proliferation and mesenchymal stem cell differentiation into collagen-producing cells. Aebp1−/− mice have abnormal, delayed wound repair correlating with defects in fibroblast proliferation. In this study, we describe four individuals from three unrelated families that presented with a unique constellation of clinical findings including joint laxity, redundant and hyperextensible skin, poor wound healing with abnormal scarring, osteoporosis, and other features reminiscent of Ehlers-Danlos syndrome (EDS). Analysis of skin biopsies revealed decreased dermal collagen with abnormal collagen fibrils that were ragged in appearance. Exome sequencing revealed compound heterozygous variants in AEBP1 (c.1470delC [p.Asn490_Met495delins(40)] and c.1743C>A [p.Cys581∗]) in the first individual, a homozygous variant (c.1320_1326del [p.Arg440Serfs∗3]) in the second individual, and a homozygous splice site variant (c.1630+1G>A) in two siblings from the third family. We show that ACLP enhances collagen polymerization and binds to several fibrillar collagens via its discoidin domain. These studies support the conclusion that bi-allelic pathogenic variants in AEBP1 are the cause of this autosomal-recessive EDS subtype. AEBP1 encodes the aortic carboxypeptidase-like protein (ACLP) that associates with collagens in the extracellular matrix (ECM) and has several roles in development, tissue repair, and fibrosis. ACLP is expressed in bone, the vasculature, and dermal tissues and is involved in fibroblast proliferation and mesenchymal stem cell differentiation into collagen-producing cells. Aebp1−/− mice have abnormal, delayed wound repair correlating with defects in fibroblast proliferation. In this study, we describe four individuals from three unrelated families that presented with a unique constellation of clinical findings including joint laxity, redundant and hyperextensible skin, poor wound healing with abnormal scarring, osteoporosis, and other features reminiscent of Ehlers-Danlos syndrome (EDS). Analysis of skin biopsies revealed decreased dermal collagen with abnormal collagen fibrils that were ragged in appearance. Exome sequencing revealed compound heterozygous variants in AEBP1 (c.1470delC [p.Asn490_Met495delins(40)] and c.1743C>A [p.Cys581∗]) in the first individual, a homozygous variant (c.1320_1326del [p.Arg440Serfs∗3]) in the second individual, and a homozygous splice site variant (c.1630+1G>A) in two siblings from the third family. We show that ACLP enhances collagen polymerization and binds to several fibrillar collagens via its discoidin domain. These studies support the conclusion that bi-allelic pathogenic variants in AEBP1 are the cause of this autosomal-recessive EDS subtype. Ehlers-Danlos syndrome (EDS) and EDS variants comprise a growing number of clinically heterogeneous connective tissue disorders with diverse molecular etiologies.1Murphy-Ryan M. Psychogios A. Lindor N.M. Hereditary disorders of connective tissue: a guide to the emerging differential diagnosis.Genet. Med. 2010; 12: 344-354Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 2Vanakker O. Callewaert B. Malfait F. Coucke P. The genetics of soft connective tissue disorders.Annu. Rev. Genomics Hum. Genet. 2015; 16: 229-255Crossref PubMed Scopus (45) Google Scholar, 3Byers P.H. Murray M.L. Heritable collagen disorders: the paradigm of the Ehlers-Danlos syndrome.J. Invest. Dermatol. 2012; 132: E6-E11Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar EDS is estimated to affect approximately 1 in every 5,000 individuals and is often caused by pathogenic variation in fibrillar collagen genes (COL1A1 [MIM: 120150], COL1A2 [MIM: 120160], COL3A1 [MIM: 120180], COL5A1 [MIM: 120215], COL5A2 [MIM: 120190]), collagen-modifying proteins (PLOD1 [MIM: 153454], P4HA1 [MIM: 176710]4Zou Y. Donkervoort S. Salo A.M. Foley A.R. Barnes A.M. Hu Y. Makareeva E. Leach M.E. Mohassel P. Dastgir J. et al.P4HA1 mutations cause a unique congenital disorder of connective tissue involving tendon, bone, muscle and the eye.Hum. Mol. Genet. 2017; 26: 2207-2217Crossref PubMed Scopus (25) Google Scholar), or processing enzymes (ADAMTS2 [MIM: 604539]).2Vanakker O. Callewaert B. Malfait F. Coucke P. The genetics of soft connective tissue disorders.Annu. Rev. Genomics Hum. Genet. 2015; 16: 229-255Crossref PubMed Scopus (45) Google Scholar Several genes involved in the biosynthesis of proteoglycans (CHST14 [MIM: 608429], B4GALT7 [MIM: 604327], B3GALT6 [MIM: 615291]), when mutated, result in EDS subtypes.2Vanakker O. Callewaert B. Malfait F. Coucke P. The genetics of soft connective tissue disorders.Annu. Rev. Genomics Hum. Genet. 2015; 16: 229-255Crossref PubMed Scopus (45) Google Scholar Pathogenic variants in other noncollagenous protein-coding genes, such as TNXB (MIM: 600985), SLC39A13 (MIM: 608735), and FKBP14 (MIM: 614505), also cause rare EDS subtypes suggesting that additional genes with diverse biological functions related to collagen biosynthesis and assembly may be contributory to disease in the EDS spectrum.2Vanakker O. Callewaert B. Malfait F. Coucke P. The genetics of soft connective tissue disorders.Annu. Rev. Genomics Hum. Genet. 2015; 16: 229-255Crossref PubMed Scopus (45) Google Scholar Together these known genes and their encoded proteins regulate the expression, assembly, homeostasis, and remodeling processes that are essential for the development and maintenance of the extracellular matrix (ECM).2Vanakker O. Callewaert B. Malfait F. Coucke P. The genetics of soft connective tissue disorders.Annu. Rev. Genomics Hum. Genet. 2015; 16: 229-255Crossref PubMed Scopus (45) Google Scholar, 5Hynes R.O. Naba A. Overview of the matrisome--an inventory of extracellular matrix constituents and functions.Cold Spring Harb. Perspect. Biol. 2012; 4: a004903Crossref PubMed Scopus (673) Google Scholar, 6Mouw J.K. Ou G. Weaver V.M. Extracellular matrix assembly: a multiscale deconstruction.Nat. Rev. Mol. Cell Biol. 2014; 15: 771-785Crossref PubMed Scopus (801) Google Scholar Differences in ECM structure and composition in a variety of tissues throughout the body result in a broad spectrum of disease phenotypes in affected individuals.1Murphy-Ryan M. Psychogios A. Lindor N.M. Hereditary disorders of connective tissue: a guide to the emerging differential diagnosis.Genet. Med. 2010; 12: 344-354Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar Clinically, affected individuals present with connective tissue defects that may manifest as joint hypermobility, skin hyperextensibility and/or fragility, deficient wound healing, an increased risk of vascular rupture, and many other associated clinical features, and there may be considerable phenotypic overlap between individuals that carry distinct molecular diagnoses.2Vanakker O. Callewaert B. Malfait F. Coucke P. The genetics of soft connective tissue disorders.Annu. Rev. Genomics Hum. Genet. 2015; 16: 229-255Crossref PubMed Scopus (45) Google Scholar Recently, the International EDS Consortium proposed the 2017 International Classification for the Ehlers-Danlos Syndromes7Malfait F. Francomano C. Byers P. Belmont J. Berglund B. Black J. Bloom L. Bowen J.M. Brady A.F. Burrows N.P. et al.The 2017 international classification of the Ehlers-Danlos syndromes.Am. J. Med. Genet. C. Semin. Med. Genet. 2017; 175: 8-26Crossref PubMed Scopus (790) Google Scholar with a new classification schema that recognizes 13 distinct subtypes and provides significant updates to the Villefranche nosology.8Beighton P. De Paepe A. Steinmann B. Tsipouras P. Wenstrup R.J. Ehlers-Danlos National Foundation (USA) and Ehlers-Danlos Support Group (UK)Ehlers-Danlos syndromes: revised nosology, Villefranche, 1997.Am. J. Med. Genet. 1998; 77: 31-37Crossref PubMed Scopus (1366) Google Scholar This new classification system outlines the major and minor criteria that may be associated with each subtype and emphasizes the importance of molecular confirmation for the diagnosis of individuals and incorporation of next-generation sequencing approaches for the identification of novel EDS-associated genes. Importantly, in a significant proportion of suspected EDS-affected case subjects, no pathogenic variants in the known EDS genes are found. While our understanding of the molecular basis for the heritable connective tissue disorders has improved over the last few decades, the full extent and molecular underpinnings for several “unspecified” EDS-like conditions is the subject of intense ongoing study.1Murphy-Ryan M. Psychogios A. Lindor N.M. Hereditary disorders of connective tissue: a guide to the emerging differential diagnosis.Genet. Med. 2010; 12: 344-354Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 9Alazami A.M. Al-Qattan S.M. Faqeih E. Alhashem A. Alshammari M. Alzahrani F. Al-Dosari M.S. Patel N. Alsagheir A. Binabbas B. et al.Expanding the clinical and genetic heterogeneity of hereditary disorders of connective tissue.Hum. Genet. 2016; 135: 525-540Crossref PubMed Scopus (70) Google Scholar The adipocyte enhancer binding protein 1 (AEBP1 [MIM: 602981]) encodes a 1,158-amino acid secreted aortic carboxypeptidase-like protein (ACLP) that is associated with the extracellular matrix (ECM). ACLP consists of an N-terminal signal peptide, a lysine/proline/glutamic acid-rich motif, a discoidin domain, and a catalytically inactive metallocarboxypeptidase domain that shares ∼40% similarity with carboxypeptidase E (CPE [MIM: 114855]).10Reznik S.E. Fricker L.D. Carboxypeptidases from A to Z: implications in embryonic development and Wnt binding.Cell. Mol. Life Sci. 2001; 58: 1790-1804Crossref PubMed Scopus (134) Google Scholar, 11Kiedzierska A. Smietana K. Czepczynska H. Otlewski J. Structural similarities and functional diversity of eukaryotic discoidin-like domains.Biochim. Biophys. Acta. 2007; 1774: 1069-1078Crossref PubMed Scopus (75) Google Scholar, 12Carafoli F. Bihan D. Stathopoulos S. Konitsiotis A.D. Kvansakul M. Farndale R.W. Leitinger B. Hohenester E. Crystallographic insight into collagen recognition by discoidin domain receptor 2.Structure. 2009; 17: 1573-1581Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 13Carafoli F. Mayer M.C. Shiraishi K. Pecheva M.A. Chan L.Y. Nan R. Leitinger B. Hohenester E. Structure of the discoidin domain receptor 1 extracellular region bound to an inhibitory Fab fragment reveals features important for signaling.Structure. 2012; 20: 688-697Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar ACLP is secreted by a number of different cell types including fibroblasts, myofibroblasts, and smooth muscle cells and is involved in vascular smooth muscle cell (VSMC) proliferation and tissue repair processes.14Layne M.D. Endege W.O. Jain M.K. Yet S.F. Hsieh C.M. Chin M.T. Perrella M.A. Blanar M.A. Haber E. Lee M.E. Aortic carboxypeptidase-like protein, a novel protein with discoidin and carboxypeptidase-like domains, is up-regulated during vascular smooth muscle cell differentiation.J. Biol. Chem. 1998; 273: 15654-15660Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 15Schissel S.L. Dunsmore S.E. Liu X. Shine R.W. Perrella M.A. Layne M.D. Aortic carboxypeptidase-like protein is expressed in fibrotic human lung and its absence protects against bleomycin-induced lung fibrosis.Am. J. Pathol. 2009; 174: 818-828Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 16Layne M.D. Yet S.F. Maemura K. Hsieh C.M. Bernfield M. Perrella M.A. Lee M.E. Impaired abdominal wall development and deficient wound healing in mice lacking aortic carboxypeptidase-like protein.Mol. Cell. Biol. 2001; 21: 5256-5261Crossref PubMed Scopus (76) Google Scholar, 17Layne M.D. Yet S.-F. Maemura K. Hsieh C.M. Liu X. Ith B. Lee M.E. Perrella M.A. Characterization of the mouse aortic carboxypeptidase-like protein promoter reveals activity in differentiated and dedifferentiated vascular smooth muscle cells.Circ. Res. 2002; 90: 728-736Crossref PubMed Scopus (56) Google Scholar Immunostaining of whole mouse embryos at E15.5 revealed high levels of ACLP expression in collagen-rich tissues including the dermal layer of the skin, the medial layer of blood vessels, the basement membrane of the lung, and the periosteum.16Layne M.D. Yet S.F. Maemura K. Hsieh C.M. Bernfield M. Perrella M.A. Lee M.E. Impaired abdominal wall development and deficient wound healing in mice lacking aortic carboxypeptidase-like protein.Mol. Cell. Biol. 2001; 21: 5256-5261Crossref PubMed Scopus (76) Google Scholar We previously generated Aebp1−/− mice that develop spontaneous skin ulcerations and show delayed wound healing that corresponds with a reduction in dermal fibroblast proliferation.16Layne M.D. Yet S.F. Maemura K. Hsieh C.M. Bernfield M. Perrella M.A. Lee M.E. Impaired abdominal wall development and deficient wound healing in mice lacking aortic carboxypeptidase-like protein.Mol. Cell. Biol. 2001; 21: 5256-5261Crossref PubMed Scopus (76) Google Scholar In an experimental fibrosis model, Aebp1−/− mice also exhibit a decreased fibroproliferative response in the lung with reduced collagen deposition and myofibroblast accumulation.15Schissel S.L. Dunsmore S.E. Liu X. Shine R.W. Perrella M.A. Layne M.D. Aortic carboxypeptidase-like protein is expressed in fibrotic human lung and its absence protects against bleomycin-induced lung fibrosis.Am. J. Pathol. 2009; 174: 818-828Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 18Tumelty K.E. Smith B.D. Nugent M.A. Layne M.D. Aortic carboxypeptidase-like protein (ACLP) enhances lung myofibroblast differentiation through transforming growth factor β receptor-dependent and -independent pathways.J. Biol. Chem. 2014; 289: 2526-2536Crossref PubMed Scopus (39) Google Scholar Given the emerging role of AEBP1 in ECM integrity, wound healing, and fibroproliferative pathways, it represents a highly plausible gene candidate for a human connective tissue disorder. We performed molecular analyses on individuals who were referred to one of the collaborating academic centers for diagnostic workup of a suspected connective tissue disorder, but for whom previous genetic testing had been unrevealing. Each research subject provided written informed consent for sample collection and subsequent analysis under a protocol approved by one of the local institutional review boards. Blood samples were collected from the affected subjects as well as their unaffected parents. Skin biopsies were obtained from both affected subjects and unaffected family members when possible for fibroblast generation and subsequent analysis. To determine the molecular diagnosis in each subject, whole-exome sequencing (WES) was performed on genomic DNA extracted from blood leukocytes derived from the proband and parents in each family and run on different platforms. Details on the WES methodologies are available in the Supplemental Note. Sanger sequencing was used to confirm variants of interest in each subject and their relatives as well as the expected mode of inheritance. Subject A-II:1 is a 35-year-old white male of German and Panamanian ancestry who was initially seen at the Mayo Clinic (Figures 1A–1E). Subject B-II:1 is a 36-year-old white male of Italian ancestry who was seen at the NIH (Figures 1F–1K and S2). Subjects C-IV:4 and C-IV:6 were seen at King Fahad Medical City in Saudi Arabia and have been reported previously.9Alazami A.M. Al-Qattan S.M. Faqeih E. Alhashem A. Alshammari M. Alzahrani F. Al-Dosari M.S. Patel N. Alsagheir A. Binabbas B. et al.Expanding the clinical and genetic heterogeneity of hereditary disorders of connective tissue.Hum. Genet. 2016; 135: 525-540Crossref PubMed Scopus (70) Google Scholar The clinical phenotype in affected individuals exhibits significant clinical overlap with several EDS subtypes (classical [MIM: 130000], vascular [MIM: 130050], and arthrochalasia type [MIM: 130060]) and includes the presence of both major and minor supporting criteria including severe joint and skin laxity, osteoporosis affecting the hips and spine, osteoarthritis, soft redundant skin that can be acrogeria-like, delayed wound healing with abnormal atrophic scarring, and shoulder, hip, knee, and ankle dislocations (Figure 1 and Table 1). While the subjects described to date share many similarities, there are also variable features including gastrointestinal and genitourinary manifestations (bowel rupture, gut dysmotility, cryptorchidism, and hernias), vascular complications (mitral valve prolapse and aortic root dilation), and skeletal anomalies (Table 1). Significant clinical heterogeneity with a complex, multisystemic presentation is characteristic of other EDS subtypes, suggesting that the observed phenotypes are consistent with an EDS-spectrum disorder. Detailed clinical descriptions can be found within the Supplemental Note.Table 1Summary of Clinical Characteristics of Individuals with Autosomal-Recessive Mutations in AEBP1A-II:1B-II:1C-IV:6C-IV:4Citationthis reportthis reportAlazami et al.9Alazami A.M. Al-Qattan S.M. Faqeih E. Alhashem A. Alshammari M. Alzahrani F. Al-Dosari M.S. Patel N. Alsagheir A. Binabbas B. et al.Expanding the clinical and genetic heterogeneity of hereditary disorders of connective tissue.Hum. Genet. 2016; 135: 525-540Crossref PubMed Scopus (70) Google ScholarAlazami et al.9Alazami A.M. Al-Qattan S.M. Faqeih E. Alhashem A. Alshammari M. Alzahrani F. Al-Dosari M.S. Patel N. Alsagheir A. Binabbas B. et al.Expanding the clinical and genetic heterogeneity of hereditary disorders of connective tissue.Hum. Genet. 2016; 135: 525-540Crossref PubMed Scopus (70) Google ScholarSexmalemalefemalemaleEthnicitywhitewhiteMiddle EasternMiddle EasternAge at last evaluation35 y33 y12 y24 yAEBP1 variant(s) (NM_001129.4)c.1470delC; c.1743C>A; compound hetc.1320_1326del; homozygousc.1630+1G>A; homozygousc.1630+1G>A; homozygousProtein change (NP_001120.3)p.Asn490_Met495delins(40); p.Cys581∗p.Arg440Serfs∗3p.?p.?Beighton score8 out of 98 out of 98 out of 9unknownGeneralized joint hypermobility++++Hip dislocationhip subluxations reportedcongenital hip dislocation, surgically corrected at 18 modislocations reportedunknownShoulder dislocationnot reported++unknownFoot deformitiespes planus, hallux valgus, hammer toespes planus, hallux valgus, hammer toespes planus, hallux valgus, hammer toespes planus, hallux valgus, toe deformitiesSkin hyperextensibility++++Excess skin/skin foldingincreased wrinkles on hands and feet (acrogeria-like)+++Delayed wound healing++++Abnormal scarringatrophic, widened scarsatrophic, widened scars with hyperpigmentationhyperpigmented atrophic scars, multiple keloidsunknownEasy bruising+++unknownHernianot reportedlarge ventral hernia developed at surgical sites secondary to ruptured bowelumbilical, ventral, and inguinalunknownGenitourinary abnormalitiescryptorchidism, surgically corrected at 15 years of agenot reportednot reportednot reportedGastrointestinal abnormalitiesmotility issuesbowel rupturenot reportednot reportedVascular abnormalitiesmitral valve prolapsemitral valve prolapse, mildly dilated aortic root, bilateral stenosis of the carotids, aortic dilation requiring surgerynormal echonormal echoDentitionretains a single baby toothnot reportedabnormal dental alignmentunknownFacial dysmorphismsnonemicrognathialow posterior hairline, webbed neck, bilateral ptosis, excess redundant skin on face, large ears, narrow palateunknownSkeletal anomaliessevere osteopenia involving the hipship replacement for severe osteopenia, upper thoracic scoliosis with degenerative disease and facet arthrosis of spineskull shows ‘copper beaten’ appearance, severe osteopenia, narrowing of the interpedicular distance of the lumbar spines distally, iliac bones are short and squared, long bones of the lower extremities are remodeledsevere osteopeniaMRI findingsmild disc bulging at the C4-5 and C7-T1 levelsempty sellanot donenot doneOtherdelays in walking and acquisition of fine motor skills, impaired temperature sensation, keratoconjunctivitis sicca, piezogenic papules on feet, right distal radioulnar joint dislocation, surgically repairedelbow bursitis, piezogenic papules on feet, sacral dimple, and hypertriglyceridemiauncontrolled diabetes mellitus, recurrent cellulitisnone+ indicates clinical feature is present. Open table in a new tab + indicates clinical feature is present. Exome sequencing of subject A-II:1 revealed compound heterozygous frameshift and nonsense variants in AEBP1 comprising c.1470delC (chr7(GRCh37): g.44150393del; GenBank: NM_001129.4 for c.1470delC; GenBank: NP_001120.3 for p.Asn490Lysfs∗6) in exon 12 and c.1743C>A (chr7(GRCh37): g.44151132C>A; GenBank: NM_001129.4 for c.1743C>A; GenBank: NP_001120.3 for p.Cys581∗) in exon 15 (Figure 2). Testing of the subject’s parents revealed that the c.1470delC variant was maternally inherited while the c.1743C>A variant was paternally inherited (Figure 1L). The c.1470delC variant was predicted to cause a frameshift mutation, denoted p.Asn490Lysfs∗6. The c.1470delC variant was not observed in more than 123,136 exomes and 15,496 genomes in the Genome Aggregation Database (gnomAD).19Lek M. Karczewski K.J. Minikel E.V. Samocha K.E. Banks E. Fennell T. O’Donnell-Luria A.H. Ware J.S. Hill A.J. Cummings B.B. et al.Exome Aggregation ConsortiumAnalysis of protein-coding genetic variation in 60,706 humans.Nature. 2016; 536: 285-291Crossref PubMed Scopus (6555) Google Scholar The c.1743C>A (p.Cys581∗) variant was seen in only one individual in gnomAD (1/264,694 alleles, MAF: 3.778 × 10−6) (Table S1) and is predicted to cause loss of normal protein function either through protein truncation or nonsense-mediated decay. Exome sequencing of subject B-II:1 revealed a homozygous frameshift deletion variant in AEBP1 in exon 11 (chr7(GRCh37): g.44149865_44149871del; GenBank: NM_001129.4 for c.1320_1326del; GenBank: NP_001120.3 for p.Arg440Serfs∗3) (Figure 2). Sanger sequencing confirmed that both parents were carriers for the c.1320_1326del variant as well as an unaffected male sibling (Figure 1M). The variant was not observed in gnomAD (Table S1). Subjects C-IV:4 and C-IV:6 were previously found to have a homozygous splice site variant (chr7(GRCh37): g.44150657G>A; GenBank: NM_001129.4 for c.1630+1G>A) in intron 13 of AEBP1 (Figure 2).9Alazami A.M. Al-Qattan S.M. Faqeih E. Alhashem A. Alshammari M. Alzahrani F. Al-Dosari M.S. Patel N. Alsagheir A. Binabbas B. et al.Expanding the clinical and genetic heterogeneity of hereditary disorders of connective tissue.Hum. Genet. 2016; 135: 525-540Crossref PubMed Scopus (70) Google Scholar This variant has been observed in 9 individuals in gnomAD (9/271,654 individuals, no homozygotes, MAF: 3.313 × 10−5). Sanger sequencing confirmed that one allele was inherited from their mother and the other from their father who are double first cousins.9Alazami A.M. Al-Qattan S.M. Faqeih E. Alhashem A. Alshammari M. Alzahrani F. Al-Dosari M.S. Patel N. Alsagheir A. Binabbas B. et al.Expanding the clinical and genetic heterogeneity of hereditary disorders of connective tissue.Hum. Genet. 2016; 135: 525-540Crossref PubMed Scopus (70) Google Scholar No other clinically reportable variants were noted in any of the affected individuals, including in known connective tissue disorder genes. RT-PCR analyses performed previously in individuals C-IV:4 and C-IV:6 showed that the c.1630+1G>A splice site variant results in the loss of the last 22 bp of exon 13 in AEBP1, resulting in a frameshift and truncation of the transcript.9Alazami A.M. Al-Qattan S.M. Faqeih E. Alhashem A. Alshammari M. Alzahrani F. Al-Dosari M.S. Patel N. Alsagheir A. Binabbas B. et al.Expanding the clinical and genetic heterogeneity of hereditary disorders of connective tissue.Hum. Genet. 2016; 135: 525-540Crossref PubMed Scopus (70) Google Scholar To determine the functional consequence of the variants found in subjects A-II:1 and B-II:1, we compared AEBP1 expression in dermal fibroblasts from the affected probands using RT-PCR and examined ACLP expression by western blotting as previously described.16Layne M.D. Yet S.F. Maemura K. Hsieh C.M. Bernfield M. Perrella M.A. Lee M.E. Impaired abdominal wall development and deficient wound healing in mice lacking aortic carboxypeptidase-like protein.Mol. Cell. Biol. 2001; 21: 5256-5261Crossref PubMed Scopus (76) Google Scholar In subject A-II:1, the c.1470delC variant was expected to induce a frameshift in the mRNA leading to an amino acid substitution at position 490 of the encoded protein followed by a stop codon after 6 amino acids (p.Asn490Lysfs∗6). The c.1743C>A nonsense variant also generates a premature stop codon (p.Cys581∗). Cell lysates from this individual, along with human U2OS and mouse 10T1/2 control cell lines, were examined by western blot analysis using an antibody against ACLP.16Layne M.D. Yet S.F. Maemura K. Hsieh C.M. Bernfield M. Perrella M.A. Lee M.E. Impaired abdominal wall development and deficient wound healing in mice lacking aortic carboxypeptidase-like protein.Mol. Cell. Biol. 2001; 21: 5256-5261Crossref PubMed Scopus (76) Google Scholar Unexpectedly, a band >170 kDa was detected in subject A-II:1, which was slightly larger than the band observed in either the U2OS and 10T1/2 controls (Figure 3A). To determine the nature of this protein, we isolated mRNA from these cells along with wild-type human dermal fibroblasts and generated cDNA which was amplified by PCR using primer sets that spanned both the c.1470delC and c.1743C>A variants. Interestingly, the PCR product spanning the c.1470delC variant was approximately 100 bp larger than the wild-type sample (Figure 3B, indicated by an asterisk). This band was isolated and subjected to DNA sequencing. The expected single-nucleotide deletion was observed, but the cDNA fragment retained all 103 bp of intron 12 with in-frame continuation of exon 13 (Figure 3C). The predicted amino acid sequence derived from the mRNA sequence of the c.1470delC variant allele has loss of the last six amino acids encoded by exon 12 due to the shift in reading frame and inclusion of 40 aberrant amino acids before returning to the in-frame translation of exon 13, which we designated p.Asn490_Met495delins(40) (Figure 3D). Notably, this alteration occurs in the critical collagen-binding discoidin-like domain of ACLP, which we predict disrupts ACLP function. Similar studies were performed on fibroblasts derived from subject B-II:1. No ACLP protein was detected by western blot, suggesting that the homozygous c.1320_1326delGACCCAG variant identified in this individual leads to nonsense-mediated decay of the mRNA product and is a null variant (Figure 3E). We next aimed to determine the functional consequence of ACLP loss in affected tissues derived from the research subjects. Ultrastructural examination of the skin, performed by transmission electron microscopy (TEM), revealed the presence of irregular disrupted collagen fibrils or “collagen flowers,” with moderate variation in collagen size in subject A-II:1 (Figures 4A and 4B ). Longitudinal sections revealed that these fibrils had a ragged or frayed appearance. Disrupted collagen fibrils can be found in several EDS variants and their presence can support a diagnosis of EDS but cannot differentiate between related subtypes. Trichome staining of skin derived from subject B-II:1 and examination under a light microscope revealed decreased dermal collagen when compared to heterozygous carriers and unaffected control subjects from the same family (Figures 4C–4E). ACLP has several distinct domains that are thought to mediate different protein-protein interactions within the ECM. The central discoidin domain of ACLP shares significant homology and structural similarity with the discoidin domain-containing receptors (DDR1 [MIM: 600408] and DDR2 [MIM: 191311]). Within DDRs, the discoidin domain contains a collagen-binding region and helps mediate binding and specificity for fibrillar collagens, and pathogenic variants in DDR2 are known to cause autosomal-recessive spondylometaepiphyseal dysplasia, short limb-hand type (SMED-SL [MIM: 271665]).20Curat C.A. Eck M. Dervillez X. Vogel W.F. Mapping of epitopes in discoidin domain receptor 1 critical for collagen binding.J. Biol. Chem. 2001; 276: 45952-45958Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 21Leitinger B. Molecular analysis of collagen binding by the human discoidin domain receptors, DDR1 and DDR2. Identification of collagen binding sites in DDR2.J. Biol. Chem. 2003; 278: 16761-16769Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 22Abdulhussein R. McFadden C. Fuentes-Prior P. Vogel W.F. Exploring the collagen-binding site of the DDR1 tyrosine kinase receptor.J. Biol. Chem. 2004; 279: 31462-31470Crossref PubMed Scopus (58) Google Scholar, 23Xu H. Raynal N. Stathopoulos S. Myllyharju J. Farndale R.W. Leitinger B. Collagen binding specificity of the discoidin domain receptors: binding sites on collagens II and III and molecular determinants for collagen IV recognition by DDR1.Matrix Biol. 2011; 30: 16-26Crossref PubMed Scopus (130) Google Scholar, 24Fu H.-L. Valiathan R.R. Arkwright R. Sohail A. Mihai C. Kumarasiri M. Mahasenan K.V. Mobashery S. Huang P. Agarwal G. Fridman R. Discoidin domain receptors: unique receptor tyrosine kinases in collagen-mediated signaling.J. Biol. Chem. 2013; 288: 7430-7437Crossref PubMed Scopus (145) Google Scholar, 25Bargal R. Cormier-Daire V. Ben-Neriah Z. Le Merrer M. Sosna J. Melki J. Zangen D.H. Smithson S.F
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