title: Novel pathogenic mutations and skin biopsy analysis in Knobloch syndrome.
abstract: OBJECTIVE
  To facilitate future diagnosis of Knobloch syndrome (KS) and better understand its etiology, we sought to identify not yet described COL18A1 mutations in KS patients. In addition, we tested whether mutations in this gene lead to absence of the COL18A1 gene product and attempted to better characterize the functional effect of a previously reported missense mutation.
  
  
  METHODS
  Direct sequencing of COL18A1 exons was performed in KS patients from four unrelated pedigrees. We used immunofluorescent histochemistry in skin biopsies to evaluate the presence of type XVIII collagen in four KS patients carrying two already described mutations: c.3277C>T, a nonsense mutation, and c.3601G>A, a missense mutation. Furthermore, we determined the binding properties of the mutated endostatin domain p.A1381T (c.3601G>A) to extracellular matrix proteins using ELISA and surface plasmon resonance assays.
  
  
  RESULTS
  We identified four novel mutations in COL18A1, including a large deletion involving exon 41. Skin biopsies from KS patients revealed lack of type XVIII collagen in epithelial basement membranes and blood vessels. We also found a reduced affinity of p.A1381T endostatin to some extracellular matrix components.
  
  
  CONCLUSIONS
  COL18A1 mutations involved in Knobloch syndrome have a distribution bias toward the coding exons of the C-terminal end. Large deletions must also be considered when point mutations are not identified in patients with characteristic KS phenotype. We report, for the first time, lack of type XVIII collagen in KS patients by immunofluorescent histochemistry in skin biopsy samples. As a final point, we suggest the employment of this technique as a preliminary and complementary test for diagnosis of KS in cases when mutation screening either does not detect mutations or reveals mutations of uncertain effect, such as the p.A1381T change.
authors.affiliation: Centro de Estudos do Genoma Humano, Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Oscar
authors.initials: O
authors.lastname: Suzuki
authors.forename: Erika
authors.initials: E
authors.lastname: Kague
authors.forename: Kelly
authors.initials: K
authors.lastname: Bagatini
authors.forename: Hongmin
authors.initials: H
authors.lastname: Tu
authors.forename: Ritva
authors.initials: R
authors.lastname: Heljasvaara
authors.forename: Lorenza
authors.initials: L
authors.lastname: Carvalhaes
authors.forename: Elisandra
authors.initials: E
authors.lastname: Gava
authors.forename: Gisele
authors.initials: G
authors.lastname: de Oliveira
authors.forename: Paulo
authors.initials: P
authors.lastname: Godoi
authors.forename: Glaucius
authors.initials: G
authors.lastname: Oliva
authors.forename: Gregory
authors.initials: G
authors.lastname: Kitten
authors.forename: Taina
authors.initials: T
authors.lastname: Pihlajaniemi
authors.forename: Maria-Rita
authors.initials: MR
authors.lastname: Passos-Bueno
mesh_terms: D043170:Collagen Type XVIII / Q000235:genetics* / Q000378:metabolism; D043169:Endostatins / Q000235:genetics* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004848:Epithelium / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D015785:Eye Diseases, Hereditary / Q000235:genetics*; D007150:Immunohistochemistry; D008958:Models, Molecular; D009154:Mutation; D017422:Sequence Analysis, DNA; D012867:Skin / Q000378:metabolism*; D013577:Syndrome
chemical_list: D043170:Collagen Type XVIII; D043169:Endostatins; D016326:Extracellular Matrix Proteins