Novel pathogenic mutations and skin biopsy analysis in Knobloch syndrome

Abstract

Purpose: 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.

Figure 1

Molecular modeling of wild type and p.A1381T endostatins. Analysis of the electrostatic surface of wild type (A) and p.A1381T (B) endostatins. Areas shown in red represent a negative potential while the blue areas are positive. The position 48 of the endostatin domain (Protein Data Bank entry 1BNL) corresponds to the position 1381 of the NC11–493 isoform (GenBank AF018081.1).

Figure 2

SPR sensorgrams of endostatin interactions with ECM components. Endostatin variants (wild type, mutant p.A1381T) were diluted to a concentration series in 0.02 M Tris-HCl, 0.11 M NaCl containing 0.05% P-20, and then injected into the sensor chips immobilized with laminin-1-nidogen-1 complex (A=wt, B=p.A1381T), fibulin (C=wt, D=p.A1381T), and perlecan (E=wt, F=p.A1381T) at 25 °C with a flow rate of 20 μl/min. Sensorgrams show binding of various concentrations of endostatin to the coated sensor surfaces. The association curves were monitored for 3 min, and the dissociation phases were recorded for 10 min but presented for 5 min. All the kinetics studies were performed three times independently at concentrations of 0–400 nM, and the data were analyzed with BIAevaluation software version 3.1 using the 1:1 Langmuir binding model.

Figure 3

Immunofluorescent localization of type XVIII collagen in KS and control skin samples. Differential interference contrast (DIC) images (A, F, K) show that the epithelial and connective tissues obtained from skin biopsies were intact and well preserved after being prepared using cryofixation and cryosubstitution techniques. Immunolocalization analyses of KS patients carrying the p.A1381T change (B, C) and the nonsense mutation c.3277C>T (NC11–493 isoform position p.G1273X, GenBank AF018081.1; G, H) were negative for expression of collagen XVIII as compared to controls (L, M). Pseudocolor, thermo images showed that collagen XVIII expression was present in control samples (M) but undetectable in KS samples (C, H). Although the distribution pattern of type IV collagen was similar in both control (N, O), and KS (D, E, I, J) groups, the immunostaining intensity was noticeably lower in KS samples. The thermo color bar to the right of panel E indicates immunofluorescence staining intensity: red, higher levels; blue/black, lower levels. Arrows point to basement membrane, and arrowheads mark blood vessels. Scale bars equal 100 μm.