16116Novel pathogenic mutations and skin biopsy analysis in Knobloch syndrome

[摘要] Purpose: To facilitate future diagnosisof Knobloch syndrome (KS) and better understand its etiology, we soughtto identify not yet described COL18A1 mutations in KS patients.In addition, we tested whether mutations in this gene lead to absenceof the COL18A1 gene product and attempted to bettercharacterize the functional effect of a previously reported missensemutation. Methods: Direct sequencing of COL18A1exons was performed in KS patients from four unrelated pedigrees. Weused immunofluorescent histochemistry in skin biopsies to evaluate thepresence of type XVIII collagen in four KS patients carrying twoalready described mutations: c.3277C>T, a nonsense mutation, andc.3601G>A, a missense mutation. Furthermore, we determined thebinding properties of the mutated endostatin domain p.A1381T(c.3601G>A) to extracellular matrix proteins using ELISA and surfaceplasmon resonance assays. Results: We identified four novelmutations in COL18A1, including a large deletion involving exon41. Skin biopsies from KS patients revealed lack of type XVIII collagenin epithelial basement membranes and blood vessels. We also found areduced affinity of p.A1381T endostatin to some extracellular matrixcomponents. Conclusions: COL18A1 mutationsinvolved in Knobloch syndrome have a distribution bias toward thecoding exons of the C-terminal end. Large deletions must also beconsidered when point mutations are not identified in patients withcharacteristic KS phenotype. We report, for the first time, lack oftype XVIII collagen in KS patients by immunofluorescent histochemistryin skin biopsy samples. As a final point, we suggest the employment ofthis technique as a preliminary and complementary test for diagnosis ofKS in cases when mutation screening either does not detect mutations orreveals mutations of uncertain effect, such as the p.A1381T change.

[学科分类] 生物化学/生物物理