Keratoconus is a complex condition with a multi-factorial etiology that normally occurs as a sporadic disorder [
25]. Recent studies have revealed the involvement of various genes in its development, such as
VSX1,
SOD1,
COL6A1,
TGFBI,
DOCK9,
STK24, and
IPO5 [
26‐
28]. Despite elucidate the etiology and the disease progression were conducted by numerous research,
VSX1 is the only gene indicated as a vital genetic factor in determining the keratoconus [
29]. Verma et al. ruled out the involvement of the
VSX1 gene in South Indian patients with sporadic keratoconus [
30]. However, Shetty et al. found that p.Leu268His, a novel missense variation in the coding region of VSX1, might be involved in the pathogenesis of sporadic keratoconus in Indians [
22]. These earlier studies indicated the need for further study on the relationship between
VSX1 pathogenic variations and sporadic keratoconus in different subsets of the keratoconus-affected population. Shetty et al. also summarized the
VSX1 coding variants that are associated with keratoconus in different ethnic groups, but the Chinese were not included in this work (Table
3) [
16‐
19,
22,
29,
31‐
38]. In the present study, PCR-SSCP and direct DNA sequencing techniques were used to examine nucleotide variations in the five exons and splicing regions of introns of the
VSX1 gene in 50 unrelated Chinese patients with keratoconus, which were compared with the sequences in 50 controls. The results showed the presence of four nucleotide variations in sporadic keratoconus patients, consisting of a missense p.Arg131Pro sequence variant (heterozygous), a missense p.Gly160Val sequence variant (heterozygous), and nucleotide c.8326G > A sequence variation (in both heterozygous and homozygous forms). p.Gly160Val was located in the second exon of the
VSX1 gene. In 2008, Mok et al. [
33] first reported that p.Gly160Val variation was found in 13 out of 249 cases (5.3%) of sporadic keratoconus in South Korea, being located in the homeobox DNA binding domain of
VSX1 gene coding regions and involving a polar amino acid being replaced by a neutral one. This may affect the binding of the
VSX1 protein to DNA and modify the transcription rate. Therefore, it was believed that p.Gly160Val of the
VSX1 gene increases the risk of the onset of keratoconus. In this study, we identified p.Gly160Val in two cases of 50 sporadic keratoconus patients, the sequence variant rate was 4.0%, but was absent in the control group. The findings suggested that p.Gly160Val may be the cause of two cases of sporadic keratoconus among these Chinese subjects. However, further biophysical studies are necessary to evaluate the precise molecular mechanism behind the effects of this variant form of
VSX1.
Table 3
Summary of
VSX1 coding variants identified in patients with keratoconus [
22]
p.Leu 17 Pro | Pathogenica
| Keratoconus | – | Italian | |
p.Leu 17 Val | Nonpathogenic | Keratoconus | + | Korean | |
p. Pro 58 Leu | Pathogenica, b
| Keratoconus | – | Caucasian | |
p. Leu 159 Met | Unknown | Keratoconus | – | Caucasian | |
p. Asn 151 Ser | Pathogenica
| Keratoconus | – | Korean | |
p. Gly 160 Val | Nonpathogenic | Keratoconus | + | Korean | |
p. Val 199 Leu | Nonpathogenic | Keratoconus | + | Korean | |
p. Arg 166 Trp | Unknown | Keratoconus | + | Caucasian, Iranian | |
p. Gln 175 His | Unknown | Keratoconus | – | Indian | |
p. Arg 217 His | Nonpathogenic | Keratoconus | + | Indian, Pakistan, European | |
p. Gly 239 Arg | Pathogenica, b
| Keratoconus | – | Italian | |
p. His 244 Arg | Unknown | Keratoconus | + | Caucasian, Iranian | |
p. Ser 251 Thr | Unknown | Keratoconus | – | Indian | |
p. Pro 247 Arg | Nonpathogenic | Keratoconus | + | Italian | |
p. Leu 268 His | Pathogenica, b
| Keratoconus | – | Indian | |
The c.8326G > A sequence variation occurred in the third intron of the
VSX1 gene. Abu-Amero et al. [
25] detected c.8326G > A heterozygous variation in the Saudi Arabian population. They identified this variation in keratoconus patients as well as in controls, with no difference in its incidence between the two groups. This variation is located within an intron and does not cause a change in the amino acid sequence encoded by the
VXS1 gene. Therefore, they considered that c.8326G > A variation belonged to SNP change of
VSX1 gene. In our study, the c.8326G > A variant of the
VSX1 gene was identified in both heterozygous and homozygous form, and it did not differ significantly in its incidence between the two groups. Overall, our results demonstrate the existence of SNPs in the
VSX1 gene in the Chinese population, and suggest that sporadic keratoconus patients from different ethnic groups may have the same pathogenesis.
In the present study, we identified one novel heterozygous variant c.5427G > C (p.Arg131Pro; rs267597889) of the VSX1 gene in a 20-year-old affected male, which was absent from all 50 controls. This patient had a history of keratoconus lasting more than 5 years. The slit lamp microscope examination showed conical protrusion (opacity at the top) of the cornea in both eyes, with corneal thinning and a vertical linear scar, as well as complete Fleischer’s ring. p.Arg131Pro (replacing the amino acid arginine with proline) has not been reported previously, so this work adds to the range of variants in the VSX1 gene and suggests the role of this novel variant in keratoconus. However, predictions made using the PolyPhen-2 and PROVEAN tools suggested that this variant would not have detrimental phenotypic effects. This is explained by the fact that the 131st amino acid arginine is located in a noncritical open reading frame region of the VSX1 gene, so its character is not particularly important and it has not been conserved among the gene’s orthologs. The presence of p.Arg131Pro in just 2% of the patients in this study reinforces the assertion that this is a minor gene, but its exact role during development needs to be clarified.