Mutational screening of VSX1, SPARC, SOD1, LOX, and TIMP3 in keratoconus

Patrizia De Bonis, Antonio Laborante, Costantina Pizzicoli, Raffaella Stallone, Raffaela Barbano, Costanza Longo, Emilio Mazzilli, Leopoldo Zelante, Luigi Bisceglia, Patrizia De Bonis, Antonio Laborante, Costantina Pizzicoli, Raffaella Stallone, Raffaela Barbano, Costanza Longo, Emilio Mazzilli, Leopoldo Zelante, Luigi Bisceglia

Abstract

Purpose: To evaluate the involvement of Visual System Homeobox 1 (VSX1), Secreted Protein Acidic and Rich in Cysteine (SPARC), Superoxide Dismutase 1 (SOD1), Lysyl Oxidase (LOX), and Tissue Inhibitor of Metalloproteinase 3 (TIMP3) in sporadic and familial keratoconus.

Methods: Mutational analysis of the five genes was performed by sequencing and fragment analysis in a large cohort of 302 Italian patients, with a diagnosis of keratoconus based on clinical examination and corneal topography. The variants identified in VSX1 and SPARC were also assessed in the available relatives of the probands.

Results: A novel mutation p.G239R and previously reported mutations were found in VSX1. Novel and already reported variants were identified in SPARC and SOD1, whose pathogenic significance has not been established. No pathogenic variants have been identified in LOX and TIMP3.

Conclusions: Molecular analysis of the five genes in a cohort of 225 sporadic and 77 familial keratoconus cases confirms the possible pathogenic role of VSX1 though in a small number of patients; a possible involvement of LOX and TIMP3 could be excluded; and the role played by SOD1 and SPARC in determining the disease as not been definitively clarified. Further studies are required to identify other important genetic factors involved in the pathogenesis and progression of the disease that in the authors' opinion, and according with several authors, should be considered as a complex disease.

Figures

Figure 1
Figure 1
Analysis of the c.715G>C (p.G239R) sequence variant in VSX1 exon 4. A: DNA sequence electropherogram of the c.715G>C (p.G239R) sequence variant in VSX1 exon 4 (NM_014588). B: multiple sequence alignment of the amino acid sequences of VSX1 in different species. Alignments were performed using the program Clustal (provided in the public domain by European Bioinformatics Institute, European Molecular Biology Laboratory, Heidelberg, Germany). C: segregation of p.G239R in family K264. Each individual was reported by age (in years), genotype and videokeratographs. Filled symbols refer to keratoconus individual, whereas open symbols are individuals without clinical keratoconus.
Figure 2
Figure 2
Segregation analysis of the VSX1 mutation p.P247R in family K361. Each individual was reported by age (in years), genotype and videokeratographs. Filled symbols refer to keratoconus individuals, whereas open symbols are individuals without clinical keratoconus.
Figure 3
Figure 3
A: DNA sequence chromatogram of the SPARC variations: c.187G>A (p.E63K), c.276G>A (p.M92I), and c.657C>A (p.D219E; NM_003118). B: multiple sequence alignment of the amino acid sequences of SPARC in different species. Alignments were performed using the program Clustal.
Figure 4
Figure 4
SOD1 variation c.169+50delTAAACAG: fragments and sequencing analysis, in the patients K151 and K283, and normal control. A: DHPLC chromatograms showing the presence of the deleted allele of 211 bp in patients. B: DNA sequence electropherogram of the c.169+50delTAAACAG sequence variant in SOD1 intron 2 (NM_000454).

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