Produção Nacional
Revisado por pares
Mutations in Fibronectin Cause a Subtype of Spondylometaphyseal Dysplasia with “Corner Fractures”
2017; Elsevier BV; Volume: 101; Issue: 5 Linguagem: Inglês
10.1016/j.ajhg.2017.09.019
ISSN1537-6605
AutoresChae Syng Lee, He Fu, Nissan Vida Baratang, Justine Rousseau, Heena Kumra, V. Reid Sutton, Marcello Niceta, Andrea Ciolfi, Guilherme Lopes Yamamoto, Débora Romeo Bertola, Carlo Marcelis, Dorien Lugtenberg, Andrea Bartuli, Choel Kim, Julie Hoover‐Fong, Nara Sobreira, Richard M. Pauli, Carlos A. Bacino, Deborah Krakow, Jillian S. Parboosingh, Patrick Yap, Ariana Kariminejad, Marie McDonald, Mariana Aracena, Ekkehart Lausch, Sheila Unger, Andrea Superti‐Furga, James T. Lu, D.H. Cohn, Marco Tartaglia, Brendan Lee, Dieter P. Reinhardt, Philippe M. Campeau,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoFibronectin is a master organizer of extracellular matrices (ECMs) and promotes the assembly of collagens, fibrillin-1, and other proteins. It is also known to play roles in skeletal tissues through its secretion by osteoblasts, chondrocytes, and mesenchymal cells. Spondylometaphyseal dysplasias (SMDs) comprise a diverse group of skeletal dysplasias and often manifest as short stature, growth-plate irregularities, and vertebral anomalies, such as scoliosis. By comparing the exomes of individuals with SMD with the radiographic appearance of “corner fractures” at metaphyses, we identified three individuals with fibronectin (FN1) variants affecting highly conserved residues. Furthermore, using matching tools and the SkelDys emailing list, we identified other individuals with de novo FN1 variants and a similar phenotype. The severe scoliosis in most individuals and rare developmental coxa vara distinguish individuals with FN1 mutations from those with classical Sutcliffe-type SMD. To study functional consequences of these FN1 mutations on the protein level, we introduced three disease-associated missense variants (p.Cys87Phe [c.260G>T], p.Tyr240Asp [c.718T>G], and p.Cys260Gly [c.778T>G]) into a recombinant secreted N-terminal 70 kDa fragment (rF70K) and the full-length fibronectin (rFN). The wild-type rF70K and rFN were secreted into the culture medium, whereas all mutant proteins were either not secreted or secreted at significantly lower amounts. Immunofluorescence analysis demonstrated increased intracellular retention of the mutant proteins. In summary, FN1 mutations that cause defective fibronectin secretion are found in SMD, and we thus provide additional evidence for a critical function of fibronectin in cartilage and bone. Fibronectin is a master organizer of extracellular matrices (ECMs) and promotes the assembly of collagens, fibrillin-1, and other proteins. It is also known to play roles in skeletal tissues through its secretion by osteoblasts, chondrocytes, and mesenchymal cells. Spondylometaphyseal dysplasias (SMDs) comprise a diverse group of skeletal dysplasias and often manifest as short stature, growth-plate irregularities, and vertebral anomalies, such as scoliosis. By comparing the exomes of individuals with SMD with the radiographic appearance of “corner fractures” at metaphyses, we identified three individuals with fibronectin (FN1) variants affecting highly conserved residues. Furthermore, using matching tools and the SkelDys emailing list, we identified other individuals with de novo FN1 variants and a similar phenotype. The severe scoliosis in most individuals and rare developmental coxa vara distinguish individuals with FN1 mutations from those with classical Sutcliffe-type SMD. To study functional consequences of these FN1 mutations on the protein level, we introduced three disease-associated missense variants (p.Cys87Phe [c.260G>T], p.Tyr240Asp [c.718T>G], and p.Cys260Gly [c.778T>G]) into a recombinant secreted N-terminal 70 kDa fragment (rF70K) and the full-length fibronectin (rFN). The wild-type rF70K and rFN were secreted into the culture medium, whereas all mutant proteins were either not secreted or secreted at significantly lower amounts. Immunofluorescence analysis demonstrated increased intracellular retention of the mutant proteins. In summary, FN1 mutations that cause defective fibronectin secretion are found in SMD, and we thus provide additional evidence for a critical function of fibronectin in cartilage and bone. Spondylometaphyseal dysplasias (SMDs), or bone dysplasias affecting the spine and growth plates, comprise a heterogeneous group of conditions from both a clinical and genetic perspective. Genetic mutations have been identified for several SMDs (in COL2A1 [MIM: 120140], TRPV4 [MIM: 605427], SBDS [MIM: 607444], GPX4 [MIM: 138322], PCYT1A [MIM: 123695], and ACP5 [MIM: 171640]), but rarer forms still escape molecular diagnosis.1Machol K. Jain M. Almannai M. Orand T. Lu J.T. Tran A. Chen Y. Schlesinger A. Gibbs R. Bonafe L. et al.Corner fracture type spondylometaphyseal dysplasia: Overlap with type II collagenopathies.Am. J. Med. Genet. A. 2017; 173: 733-739Crossref PubMed Scopus (6) Google Scholar One such condition is SMD with “corner fractures” (MIM: 184255). First recognized by Sutcliffe in 1966, fewer than 25 individuals or families have been reported.2Sutcliffe J. Metaphyseal dysostosis.Ann. Radiol. (Paris). 1966; 9: R215-R223Google Scholar These individuals generally show developmental coxa vara but no scoliosis (as was the case for several individuals in our cohort without FN1 [MIM: 135600] mutations; see Table S1).3Currarino G. Birch J.G. Herring J.A. Developmental coxa vara associated with spondylometaphyseal dysplasia (DCV/SMD): “SMD-corner fracture type” (DCV/SMD-CF) demonstrated in most reported cases.Pediatr. Radiol. 2000; 30: 14-24Crossref PubMed Scopus (19) Google Scholar, 4Langer Jr., L.O. Brill P.W. Ozonoff M.B. Pauli R.M. Wilson W.G. Alford B.A. Pavlov H. Drake D.G. Spondylometaphyseal dysplasia, corner fracture type: a heritable condition associated with coxa vara.Radiology. 1990; 175: 761-766Crossref PubMed Scopus (33) Google Scholar At the edges of the irregular metaphyses, flake-like, triangular, or curvilinear ossification centers simulate fractures. This specifically affects the distal tibia, the distal radius (ulnar aspect), the proximal humerus, and the proximal femur. The so-called “corner fractures” are unlikely to be true fractures but instead represent irregular ossification at the growth plates and secondary ossification centers.3Currarino G. Birch J.G. Herring J.A. Developmental coxa vara associated with spondylometaphyseal dysplasia (DCV/SMD): “SMD-corner fracture type” (DCV/SMD-CF) demonstrated in most reported cases.Pediatr. Radiol. 2000; 30: 14-24Crossref PubMed Scopus (19) Google Scholar These fractures tend to become larger in older children and disappear after growth has stopped. Corner fractures on radiographs can also be seen in Duetting-type SMD (or SMD type A4 [MIM: 609052]),5Duetting T. Schulze A. Troeger J. Spranger J. A rare form of spondylometaphyseal dysplasia-type A4.Am. J. Med. Genet. 1998; 78: 61-66Crossref PubMed Scopus (7) Google Scholar Schmid (MIM: 156500) and Jansen (MIM: 156400) types of metaphyseal chondrodysplasia, Strudwick-type spondyloepimetaphyseal dysplasia (MIM: 184250),6Walter K. Tansek M. Tobias E.S. Ikegawa S. Coucke P. Hyland J. Mortier G. Iwaya T. Nishimura G. Superti-Furga A. Unger S. COL2A1-related skeletal dysplasias with predominant metaphyseal involvement.Am. J. Med. Genet. A. 2007; 143A: 161-167Crossref PubMed Scopus (30) Google Scholar Blount disease (MIM: 188700), Menkes disease (MIM: 309400), nonaccidental injury, congenital contractures, rickets, and scurvy.7Kozlowski K. Beighton P. Gamut index of skeletal dysplasias: an aid to radiodiagnosis. Springer-Verlag, 1984Crossref Google Scholar Some individuals initially thought to have SMD with corner fractures were later identified to have type 2 collagenopathy.6Walter K. Tansek M. Tobias E.S. Ikegawa S. Coucke P. Hyland J. Mortier G. Iwaya T. Nishimura G. Superti-Furga A. Unger S. COL2A1-related skeletal dysplasias with predominant metaphyseal involvement.Am. J. Med. Genet. A. 2007; 143A: 161-167Crossref PubMed Scopus (30) Google Scholar Fibronectin is found in the human body in both a soluble form (∼300 μg/mL in plasma) and an insoluble form as a principal component of the fibrillar extracellular matrix (ECM) of virtually all tissues.8Mosher D.F. Plasma fibronectin concentration: a risk factor for arterial thrombosis?.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1193-1195Crossref PubMed Scopus (36) Google Scholar, 9Singh P. Carraher C. Schwarzbauer J.E. Assembly of fibronectin extracellular matrix.Annu. Rev. Cell Dev. Biol. 2010; 26: 397-419Crossref PubMed Scopus (616) Google Scholar Fibronectin contains binding sites for integrins, collagens, glycoproteins, and glycosaminoglycans, as well as self-association sites.10Pankov R. Yamada K.M. Fibronectin at a glance.J. Cell Sci. 2002; 115: 3861-3863Crossref PubMed Scopus (1465) Google Scholar It self-assembles in a cell-dependent manner upon binding to integrins and other cell-surface components9Singh P. Carraher C. Schwarzbauer J.E. Assembly of fibronectin extracellular matrix.Annu. Rev. Cell Dev. Biol. 2010; 26: 397-419Crossref PubMed Scopus (616) Google Scholar and initiates the assembly of the ECM.11Sabatier L. Chen D. Fagotto-Kaufmann C. Hubmacher D. McKee M.D. Annis D.S. Mosher D.F. Reinhardt D.P. Fibrillin assembly requires fibronectin.Mol. Biol. Cell. 2009; 20: 846-858Crossref PubMed Scopus (179) Google Scholar, 12Sottile J. Hocking D.C. Fibronectin polymerization regulates the composition and stability of extracellular matrix fibrils and cell-matrix adhesions.Mol. Biol. Cell. 2002; 13: 3546-3559Crossref PubMed Scopus (448) Google Scholar, 13Dallas S.L. Sivakumar P. Jones C.J. Chen Q. Peters D.M. Mosher D.F. Humphries M.J. Kielty C.M. Fibronectin regulates latent transforming growth factor-beta (TGF beta) by controlling matrix assembly of latent TGF beta-binding protein-1.J. Biol. Chem. 2005; 280: 18871-18880Crossref PubMed Scopus (238) Google Scholar We performed exome sequencing in individuals with SMD with corner fractures, who had been identified through the Texas Children’s Skeletal Dysplasia Program, International Skeletal Dysplasia Registry, Baylor-Hopkins Center for Mendelian Genetics, Skeldys emailing list of the International Skeletal Dysplasia Society (ISDS), Shriners Canada Skeletal Dysplasia Clinic, and existing collaborations. Families provided written informed consent for protocols approved by the institutional review board at Baylor College of Medicine or local institutions. The procedures followed were in accordance with the ethical standards of the relevant committees on human experimentation. Details on the exome sequencing libraries and alignment are presented in Table S2. In two of the first few individuals sequenced, who have been previously reported, COL2A1 mutations were identified.1Machol K. Jain M. Almannai M. Orand T. Lu J.T. Tran A. Chen Y. Schlesinger A. Gibbs R. Bonafe L. et al.Corner fracture type spondylometaphyseal dysplasia: Overlap with type II collagenopathies.Am. J. Med. Genet. A. 2017; 173: 733-739Crossref PubMed Scopus (6) Google Scholar In total, 13 individuals were exome sequenced, and a comparison of the rare or undescribed variants shared between these individuals revealed variants in FN1 in three of these affected individuals (two variants segregating with the disease, c.260G>T [p.Cys87Phe] [GenBank: NM_212482.2 and NP_997647.1, respectively] in family 1 and c.718T>G [p.Tyr240Asp] in family 4, and a de novo variant, c.2425_2427del [p.Thr809del] in family 6). The genetic cause of SMD is still undetermined for the other individuals. Subsequently, through the ISDS emailing list, the GeneMatcher tool, and the Shriners Canada Skeletal Dysplasia Clinic,14Sobreira N. Schiettecatte F. Valle D. Hamosh A. GeneMatcher: a matching tool for connecting investigators with an interest in the same gene.Hum. Mutat. 2015; 36: 928-930Crossref PubMed Scopus (830) Google Scholar four other individuals with de novo FN1 variants (c.367T>C [p.Cys123Arg] in families 2 and 7, c.675C>G [p.Cys225Trp] in family 3, and c.778T>G [p.Cys260Gly] in family 5) and SMD with corner fractures were identified. In one of them (individual 3, identified with the ISDS emailing list), only FN1 was sequenced on the basis of the initial findings. Thus, in this cohort, previously unreported FN1 variants were found in 7 of 16 families affected by SMD with corner fractures. The clinical phenotypes of the individuals with FN1 variants are detailed in Table 1, and the variants are presented in Table 2. Figure 1 shows pedigrees, Figure 2 shows photographs, Figure 3 shows selected radiographs, and Figure S1 shows additional radiographs.Table 1Clinical FeaturesFamily 1Family 2Family 3Family 4Family 5Family 6Family 7SMD Corner-Fracture Type or Sutcliffe Type (from Currarino et al.;3Currarino G. Birch J.G. Herring J.A. Developmental coxa vara associated with spondylometaphyseal dysplasia (DCV/SMD): “SMD-corner fracture type” (DCV/SMD-CF) demonstrated in most reported cases.Pediatr. Radiol. 2000; 30: 14-24Crossref PubMed Scopus (19) Google Scholar n = 18)Individual in pedigreeII-2 (mother)III-1 (first child)III-2 (second child)II-1II-1III-2 (child)II-1II-1II-1NAGenderfemalemalemalemalefemalefemalefemalefemalefemaleNAAge at last assessment29 years13 years9 years14 years3 years, 9 months2 years, 1 month5 years, 9 months16 years, 11 months4 yearsNAHeight (cm)147 (−3 SD)113 (−5.7 SD)107 (−4.6 SD)136 (−3.38 SD)92 (−2.5 SD)83.7 (−0.9 SD)97 (−3.3 SD)137 (−4.66 SD)89 (<3.0 SD)NAOvoid vertebral bodiesNA+−−+−+++16/18Scoliosis+ (operated)+ (operated)+ (operated)+−−+ (operated)++1/18Developmental coxa vara−−−−−+−++18/18Irregular metaphysesNA++++++++16/18“Corner fractures”−++++++++15/18Knee anomalies−−genu varum (operated)−genu varum−genu varum (operated)−genu varumone genu varum, one genum valgumChest or rib anomaly (e.g., pectus)pectus carinatumpectus carinatumpectus carinatum−NA−pectus carinatum−−NAOtherhip surgery at 18 years, pregnancy-induced hypertensionborn at term, weight 1,673 g (−4.3 SD), length 38 cm (−2.7 SD), hyponatremia at 1 month (unknown cause), hypoplasia of T12 vertebra and triangular S1, back and leg painborn at term, weight 1,729 g (−3.9 SD), length 39 cm (−2.6 SD), leg painfacial asymmetry and dysmorphisms (dysplastic left ear), missing teeth 34 and 44 (island of compact bone instead), intradural lipoma and megacisterna magna on MRIborn at 33 weeks, weight 1,754 gfemoral roddingborn at 36 weeks, weight 2,060 g (3rd–10th percentile), length 43.5 cm (3rd–10th percentile), OFC 29.5 cm (3rd percentile), normal renal sonography and kidney function, no signs of proteinuria or microalbuminuria, leg painborn at 39 weeks, weight 2,280 g (<3rd percentile), length 45 cm ( Ac.260G>Tp.Cys87Phedominant2chr2: g.216298095A>Gc.367T>Cp.Cys123Argde novo3chr2: g.216295448G>Cc.675C>Gp.Cys225Trpde novo4chr2: g.216293029A>Cc.718T>Gp.Tyr240Aspdominant5chr2: g.216292969A>Cc.778T>Gp.Cys260Glyde novo6chr2: g.216273022_216273024delc.2425_2427delp.Thr809delde novo7chr2: g.216298095A>Gc.367T>Cp.Cys123Argde novoa All variants are absent from the ExAC Browser. Open table in a new tab Figure 2Photographs of Some of the Individuals with FN1 MutationsShow full caption(A) Photographs in a figure reproduced with permission from Sutton et al.30Sutton V.R. Hyland J.C. Phillips W.A. Schlesinger A.E. Brill P.W. A dominantly inherited spondylometaphyseal dysplasia with “corner fractures” and congenital scoliosis.Am. J. Med. Genet. A. 2005; 133A: 209-212Crossref PubMed Scopus (4) Google Scholar (copyright © 2005 Wiley-Liss, Inc.). On the left are the three affected individuals who have an FN1 mutation and the maternal grandmother. In the middle is the older child at age 5 years, and on the right is the younger child at age 3 years.(B) Affected child from family 2 at age 13 years. Note the short trunk, facial asymmetry, dysplastic left ear, and normal hands and feet.(C) Affected child from family 5 at age 2 years and 11 months. Note the scoliosis, genu varum, and normal hands and feet.(D) Affected child from family 6 at age 8 years. Note the short trunk and scoliotic posture.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Radiographs Showing the “Corner Fractures” and Other Radiological ChangesShow full caption(A) Individual from family 7; note the significant scoliosis.(B) Individual from family 3; note the absence of coxa vara, the presence of irregular metaphyses with corner fractures, and the presence of ovoid vertebral bodies.Additional radiographs from all families are available in Figure S1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The following abbreviations are used: OFC, occipitofrontal circumference; and NA, not available. (A) Photographs in a figure reproduced with permission from Sutton et al.30Sutton V.R. Hyland J.C. Phillips W.A. Schlesinger A.E. Brill P.W. A dominantly inherited spondylometaphyseal dysplasia with “corner fractures” and congenital scoliosis.Am. J. Med. Genet. A. 2005; 133A: 209-212Crossref PubMed Scopus (4) Google Scholar (copyright © 2005 Wiley-Liss, Inc.). On the left are the three affected individuals who have an FN1 mutation and the maternal grandmother. In the middle is the older child at age 5 years, and on the right is the younger child at age 3 years. (B) Affected child from family 2 at age 13 years. Note the short trunk, facial asymmetry, dysplastic left ear, and normal hands and feet. (C) Affected child from family 5 at age 2 years and 11 months. Note the scoliosis, genu varum, and normal hands and feet. (D) Affected child from family 6 at age 8 years. Note the short trunk and scoliotic posture. (A) Individual from family 7; note the significant scoliosis. (B) Individual from family 3; note the absence of coxa vara, the presence of irregular metaphyses with corner fractures, and the presence of ovoid vertebral bodies. Additional radiographs from all families are available in Figure S1. All FN1 variants discovered in this study are absent from the ExAC Browser and affect highly conserved residues (Figure 4). The majority (4/6) affect cysteine residues, all of which form disulfide bonds that are critical for the three-dimensional structure of the type I fibronectin domains.15Baron M. Norman D. Willis A. Campbell I.D. Structure of the fibronectin type 1 module.Nature. 1990; 345: 642-646Crossref PubMed Scopus (92) Google Scholar Five of the mutations (c.260G>T [p.Cys87Phe], c.367T>C [p.Cys123Arg], c.675C>G [p.Cys225Trp], c.718T>G [p.Tyr240Asp], and c.778T>G [p.Cys260Gly]) are located in the N-terminal assembly domain (spanning fibronectin domains I-1 through I-5), which is important to initiating assembly on the cell surface.9Singh P. Carraher C. Schwarzbauer J.E. Assembly of fibronectin extracellular matrix.Annu. Rev. Cell Dev. Biol. 2010; 26: 397-419Crossref PubMed Scopus (616) Google Scholar The remaining mutation (c.2425_2427del [p.Thr809del]) was found in the III-2 domain, which contains a fibronectin binding site and is involved in conformational changes promoting fibronectin assembly.9Singh P. Carraher C. Schwarzbauer J.E. Assembly of fibronectin extracellular matrix.Annu. Rev. Cell Dev. Biol. 2010; 26: 397-419Crossref PubMed Scopus (616) Google Scholar The Tyr240 residue has previously been mutated into a Ser residue in vitro and shown to be critical for fibronectin binding to fibroblasts.16Sottile J. Schwarzbauer J. Selegue J. Mosher D.F. Five type I modules of fibronectin form a functional unit that binds to fibroblasts and Staphylococcus aureus.J. Biol. Chem. 1991; 266: 12840-12843Abstract Full Text PDF PubMed Google Scholar Heterozygous FN1 mutations have previously been reported in type 2 autosomal-dominant glomerulopathy with fibronectin deposits (MIM: 601894).17Ohtsubo H. Okada T. Nozu K. Takaoka Y. Shono A. Asanuma K. Zhang L. Nakanishi K. Taniguchi-Ikeda M. Kaito H. et al.Identification of mutations in FN1 leading to glomerulopathy with fibronectin deposits.Pediatr. Nephrol. 2016; 31: 1459-1467Crossref PubMed Scopus (22) Google Scholar Remarkably, all FN1 mutations previously implicated in this glomerulopathy cluster in more C-terminally-located regions important for heparin binding and integrin binding (Figure 4).17Ohtsubo H. Okada T. Nozu K. Takaoka Y. Shono A. Asanuma K. Zhang L. Nakanishi K. Taniguchi-Ikeda M. Kaito H. et al.Identification of mutations in FN1 leading to glomerulopathy with fibronectin deposits.Pediatr. Nephrol. 2016; 31: 1459-1467Crossref PubMed Scopus (22) Google Scholar, 18Castelletti F. Donadelli R. Banterla F. Hildebrandt F. Zipfel P.F. Bresin E. Otto E. Skerka C. Renieri A. Todeschini M. et al.Mutations in FN1 cause glomerulopathy with fibronectin deposits.Proc. Natl. Acad. Sci. USA. 2008; 105: 2538-2543Crossref PubMed Scopus (99) Google Scholar, 19Ertoy Baydar D. Kutlugun A.A. Bresin E. Piras R. A case of familial glomerulopathy with fibronectin deposits caused by the Y973C mutation in fibronectin.Am. J. Kidney Dis. 2013; 61: 514-518Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar In vitro, the mutations cause decreased heparin and integrin binding, reduced endothelial cell spreading, and cytoskeletal reorganization, which has been hypothesized to affect glomerular size selectivity and protein trafficking. Importantly, none of the SMD individuals in our cohort had any evidence of renal disease. As stated above, all cysteine residues affected by mutations in our cohort are involved in disulfide bonds in fibronectin type I domains (bridges form between Cys87 and Cys76, between Cys123 and Cys135, between Cys225 and Cys213, and between Cys260 and Cys231). We selected one of these (p.Cys87Phe) to model the consequence of the mutation on the three-dimensional structure of fibronectin domains (Figure 5A). The p.Cys87Phe substitution was predicted to destabilize the structure of the I-1 domain by breaking the disulfide bond with residue Cys76 and by displaying a hydrophobic residue at the surface. A similar disruptive impact was predicted for the other missense changes involving Cys123, Cys225, and Cys260. For the two other amino acid residues affected by mutations (Tyr240 and Thr809), 3D structures adequate for modeling the mutations were available only for p.Tyr240Asp (Thr809 was found only at the C-terminal end of a solution structure of III-2,20Vakonakis I. Staunton D. Rooney L.M. Campbell I.D. Interdomain association in fibronectin: insight into cryptic sites and fibrillogenesis.EMBO J. 2007; 26: 2575-2583Crossref PubMed Scopus (70) Google Scholar which limits the interpretation of its interactions). Modeling predicted that the p.Tyr240Asp variant would disrupt the pi-stacking (or π-π stacking) of the side chains of Tyr240 and Trp246 and thus destabilize the I-5 domain (Figure 5B). We next aimed to analyze the functional consequences of selected FN1 mutations on the fibronectin protein. Because cells from affected individuals were not available for this study, we generated a recombinant 70 kDa N-terminal fragment of fibronectin (rF70K), spanning the region where five of the six missense variants identified in this study are located, and a full-length recombinant fibronectin (rFN) (Figure 6A). Expression vectors for the human wild-type rF70K and rFN, as well as selected mutants, were generated by standard cloning procedures in the pcDNA3.1+ plasmid (Thermo Fisher Scientific, V79020) with the sequence for a C-terminal V5 epitope tag to facilitate detection and a hexa-histidine tag intended for chromatographic purification. At the 5′ ends, the vectors contained the coding sequence for either the BM40 signal peptide (rF70K) or the native fibronectin signal peptide (rFN) to ensure secretion of the recombinant protein through the secretory pathway. The rFN expression plasmid was constructed with the sequence for the alternatively spliced EDA and EDB domains. For this analysis, we selected two cysteine missense variants (p.Cys87Phe and p.Cys260Gly) because they represent the major group of the identified mutations, as well as a non-cysteine missense variant (p.Tyr240Asp). These point mutants were generated from the wild-type plasmids via the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, 200519). The plasmids were transfected into human HEK293 cells, which synthesize and secrete endogenous fibronectin and represent an efficient system for producing a range of correctly folded and posttranslationally modified ECM proteins (subfragments as well as mutants).21Kirschner R. Hubmacher D. Iyengar G. Kaur J. Fagotto-Kaufmann C. Brömme D. Bartels R. Reinhardt D.P. Classical and neonatal Marfan syndrome mutations in fibrillin-1 cause differential protease susceptibilities and protein function.J. Biol. Chem. 2011; 286: 32810-32823Crossref PubMed Scopus (43) Google Scholar, 22McKee K.K. Harrison D. Capizzi S. Yurchenco P.D. Role of laminin terminal globular domains in basement membrane assembly.J. Biol. Chem. 2007; 282: 21437-21447Crossref PubMed Scopus (133) Google Scholar, 23Fox J.W. Mayer U. Nischt R. Aumailley M. Reinhardt D. Wiedemann H. Mann K. Timpl R. Krieg T. Engel J. et al.Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV.EMBO J. 1991; 10: 3137-3146Crossref PubMed Scopus (380) Google Scholar After the plasmids were stably transfected into human HEK293 cells as described previously,24Lin G. Tiedemann K. Vollbrandt T. Peters H. Batge B. Brinckmann J. Reinhardt D.P. Homo- and heterotypic fibrillin-1 and -2 interactions constitute the basis for the assembly of microfibrils.J. Biol. Chem. 2002; 277: 50795-50804Crossref PubMed Scopus (120) Google Scholar the secreted wild-type recombinant proteins were readily identified in the cell-culture medium (conditioned for 2 days) with fibronectin-specific or anti-V5-specific antibodies (Figure 6B). Surprisingly, all three rF70K and rFN mutants were either undetectable in the culture medium or detectable at much lower levels than the wild-type protein. Analysis of recombinant mRNA with primers excluding the endogenous fibronectin mRNA revealed that the amount of mutant mRNA was similar to that of wild-type mRNA (Figure 6C). Immunofluorescence analysis demonstrated very low amounts of the wild-type proteins within the stably transfected cells because the proteins secreted into the culture medium (Figures 6D and 6E). In contrast to this finding, all three rF70K and rFN mutants consistently showed strong accumulation within the cells. These data clearly demonstrate that p.Cys87Phe, p.Tyr240Asp, and p.Cys260Gly exert a common molecular defect in fibronectin secretion. To assess whether the intracellular retention induced the unfolded protein response in HEK293 cells, we analyzed XBP1 splicing (IRE1 pathway) and the mRNA expression levels of CHOP and ATF4 (PERK pathway). However, no indication of increased XBP1 splicing or changes in the CHOP or ATF4 mRNA expression levels was detected (Figure S2). In addition, the mutant rFN cell clones did not undergo caspase-3-mediated apoptosis (Figure S3). Fibronectin is present throughout cartilage differentiation and persists in mature cartilaginous tissue.25Singh P. Schwarzbauer J.E. Fibronectin and stem cell differentiation - lessons from chondrogenesis.J. Cell Sci. 2012; 125: 3703-3712Crossref PubMed Scopus (144) Google Scholar However, little is known about the functional role of fibronectin in the development and homeostasis of cartilage. Given that fibronectin has been established as a key ECM protein that guides fibrillogenesis of several other ECM proteins, including proteins present in cartilage, such as fibrillin-1,26Keene D.R. Jordan C.D. Reinhardt D.P. Ridgway C.C. Ono R.N. Corson G.M. Fairhurst M. Sussman M.D. Memoli V.A. Sakai L.Y. Fibrillin-1 in human cartilage: developmental expression and formation of special banded fibers.J. Histochem. Cytochem. 1997; 45: 1069-1082Crossref PubMed Scopus (70) Google Scholar it is conceivable that a reduced amount of secreted fibronectin from chondrocytes could result in a deficient matrix network in cartilage. It is well known that mutations affecting another cartilage protein, cartilage oligomeric matrix protein (COMP [MIM: 600310]), lead to pseudoachondroplasia (MIM: 177170) and multiple epiphyseal dysplasia (MIM: 132400) through accumulation of mutant COMP in the endoplasmic reticulum of chondrocytes.27Maddox B.K. Keene D.R. Sakai L.Y. Charbonneau N.L. Morris N.P. Ridgway C.C. Boswell B.A. Sussman M.D. Horton W.A. Bächinger H.P. Hecht J.T. The fate of cartilage oligomeric matrix protein is determined by the cell type in the case of a novel mutation in pseudoachondroplasia.J. Biol. Chem. 1997; 272: 30993-30997Crossref PubMed Scopus (91) Google Scholar It is thought that this protein accumulation impairs secretion of other matrix proteins and leads to abnormal ECM and chondrocyte death.28Hecht J.T. Montufar-Solis D. Decker G. Lawler J. Daniels K. Duke P.J. Retention of cartilage oligomeric matrix protein (COMP) and cell death in redifferentiated pseudoachondroplasia chondrocytes.Matrix Biol. 1998; 17: 625-633Crossref PubMed Scopus (80) Google Scholar, 29Hashimoto Y. Tomiyama T. Yamano Y. Mori H. Mutation (D472Y) in the type 3 repeat domain of cartilage oligomeric matrix protein affects its early vesicle trafficking in endoplasmic reticulum and induces apoptosis.Am. J. Pathol. 2003; 163: 101-110Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar It is possible that a similar mechanism exists for retained mutant fibronectin and leads to the SMD phenotype observed in our cohort. Conditional double-knockout experiments of fibronectin in mouse cartilage and liver or knockin of some of the mutations are currently underway to further elucidate the molecular basis of fibronectin in the development and maintenance of cartilage. This project was supported in part by operating grants from the Canadian Institutes for Health Research (clinician-scientist award RN315908 to P.M.C.; MOP-137091 to D.P.R.), the Fonds de Recherche du Québec - Santé clinical research scholar award 30647 to P.M.C., the Quebec Network for Oral and Bone Health Research (RSBO Emerging Collaborating Project 2014-2015 to P.M.C. and D.P.R.), the NIH (UM1 HG006542 to the Baylor Hopkins Center for Mendelian Genomics), the Heart and Stroke Foundation of Canada (G-16-00014634 to D.P.R.), the Italian Ministry of Health (Ricerca Corrente 2016 to M.T. and M.N.), and Fondazione Bambino Gesù (Vite Coraggiose grant to M.T.). This work was also supported by NIH P01 HD070394, by HD024064 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development granted to the Baylor College of Medicine (BCM) Intellectual and Developmental Disabilities Research Center (for processing samples obtained and managing clinical protocols), by the BCM Advanced Technology Cores through funding from the NIH (AI036211, CA125123, and RR024574), by the Rolanette and Berdon Lawrence Bone Disease Program of Texas, and by the BCM Center for Skeletal Medicine and Biology (to B.H.L). Analysis of individual 5 was supported by the São Paulo Research Foundation (FAPESP 2015/21783-9; Centros de Pesquisa, Inovação, e Difusão [CEPID] 2013/08028-1) and National Council for Scientific and Technological Development (CNPq 302605/2013-4 and 304130/2016-8 to D. B). E.L. is supported by grants from the German Research Foundation (CRC 1140), the German Ministry for Education and Research (MaTrOC), and the European Union (SYBIL grant agreement no. 602300; RARENET). We thank Dr. Deane Mosher and Dr. Douglas Annis for providing the FN1 pAcGP67A plasmid. We thank Dr. Reggie Hamdy for referring individual 7 to Dr. Campeau and Dr. Amélie Damphousse for the interpretation of the radiographs. The accession numbers for the variants identified in this study are ClinVar: SCV000574553–SCV000574558. Download .pdf (4.65 MB) Help with pdf files Document S1. Figures S1–S3 and Table S1 and S2 Baylor-Hopkins Center for Mendelian Genetics (BH-CMG), http://bhcmg.org/ExAC Browser, http://exac.broadinstitute.org/GenBank, http://www.ncbi.nlm.nih.gov/genbank/GeneMatcher, https://www.genematcher.orgInternational Skeletal Dysplasia Society (ISDS), http://www.isds.ch/International Skeletal Dysplasia Registry (ISDR), http://ortho.ucla.edu/isdrOMIM, http://www.omim.org/RCSB Protein Data Bank, https://www.rcsb.org/pdb/home/home.doUniProt, http://www.uniprot.org/uniprot/
Referência(s)