Mutation analysis of VSX1 and SOD1 in Iranian patients with keratoconus

Abstract

Purpose: To evaluate mutations in the visual system homeobox gene 1 (VSX1) and superoxide dismutase 1 (SOD1) genes with keratoconus (KTCN), direct sequencing was performed in an Iranian population.

Methods: One hundred and twelve autosomal dominant KTCN patients and fifty-two unaffected individuals from twenty-six Iranian families, as well as one hundred healthy people as controls were enrolled. Genomic DNA was extracted from whole blood sample. Then to study the possible linkage between KTCN and six known loci linkage analysis was performed using 12 short tandem repeat (STR) markers. Also, the entire coding region and intron-exon boundaries of VSX1 and SOD1 were amplified by the PCR technique in each proband. Subsequently, PCR products were subjected to direct sequencing. Co-segregation analysis of the identified mutation was conducted in the family members. An Amplification Refractory Mutation System PCR (ARMS-PCR) was additionally employed for detection of the identified mutation in healthy controls.

Results: Linkage analysis of aforementioned loci did not detect evidence for linkage to KTCN. Direct PCR sequencing revealed two single nucleotide polymorphisms (SNPs; g.1502T>G and g.9683C>T), as well as two missense mutations that have been previously reported (R166W and H244R) in VSX1. We also found three undescribed SNPs (g.4886G>A, g.4990C>G, and g.9061T>A) in SOD1. The R166W and H244R mutations were co-segregated in affected family members but not in those that were unaffected. Moreover, the ARMS-PCR strategy did not detect the identified mutations in controls.

Conclusions: Our data suggest a significant association between KTCN patients and VSX1 genetic alterations (p.R166W and p.H244R). Although our findings support VSX1 as a plausible candidate gene responsible for keratoconus, other chromosomal loci and genes could be involved in KTCN development. Taken together, our results suggest that p.R166W and p.H244R could have possible pathogenic influences on KTCN.

Figures

Figure 1

Pedigree analysis and molecular study of Family 1. A: DNA sequencing revealed heterozygous missense mutation in the codon 244 VSX1 in which A→G (arrow indicates the position of nucleotide substitution). B: Amplification refractory mutation system (ARMS) for H244R VSX1 genotyping showing the co-segregation of the H244R VSX1 mutation among family members including two KTCN patients (III:1 and II:6) as well as in two individuals without KTCN clinical features (III:2 and II:5). PCR products of the internal control primer pair (383 bp), PCR product of the wild-type (WT) and mutant primer pairs (236 bp) are indicated. M, 50-bp ladder is present. C: The pedigree of Family 1 show four affected patients (arrow indicates the proband) and segregation of p.H244R through the family. Each individual was reported by age (in years), genotype and topography images. Filled symbols represent KTCN patient and open symbols reveal individuals without clinical KTCN.

Figure 2

Pedigree analysis and molecular study of Family 2. A: DNA sequencing chromatogram from heterozygous mutant of proband (II:1) showed missense mutation in codon 166 in which Arg was replaced by Trp (R166W C>T; arrow indicates the position of nucleotide substitution). B: The Pedigree of Family 2 indicates two affected patients (arrow indicates the proband) as well as the segregation of p.R166W in the family. The each family's member was presented by age (in years), genotype and topography images. Filled symbols show KTCN patient while open symbols represent persons without clinical KTCN.