Keratoconus (KTCN; [OMIM] 148300) is a corneal ectatic disorder characterized by progressive thinning of the central cornea, which acquires a conical shape rather than its normal dome-shaped curve. This results in optical aberrations leading to distorted blurred vision, progressive high myopia and irregular astigmatism [1]. Keratoconus is mostly bilateral but can be unilateral, onset in teenage years and often progresses until the third or fourth decade of life [2]. The prevalence of keratoconus is estimated to be 1 in 2000 in the general population [2, 3]. It is more common in patients with atopic conditions, such as asthma, dermatitis, and found to be associated with many other genetic disorders like Down syndrome, posterior polymorphous corneal dystrophy, Leber congenital amaurosis, retinitis pigmentosa, Marfan syndrome, mitral valve prolapse and other documented associations [1, 2, 4]. It occurs usually in a sporadic form, though positive familial history has been reported in 6-10% of patients, with mostly autosomal dominant and sometime recessive or X-linked mode of inheritance [4–6]. Common histopathological features of KTCN include stromal thinning, iron deposition in the epithelial basement membrane, breaks in corneal Bowman’s layer and sometimes endothelial damage [2, 7]. The disease generally occurs with no gender preponderance; however, few evidence suggests a higher prevalence in either male or female [1, 6, 8]. At present, the milder forms of keratoconus are corrected by spectacles or contact lenses. In 10% to 20% of KTCN cases, corneal thinning may reach such a severity that it becomes very essential to undergo corneal transplantation or penetrating keratoplasty, which has emerged as a major medical burden in many countries [2, 9]. The exact etiology of KTCN is still unknown, and its pathogenesis may involve genetic as well as environmental or behavioural factors, such as ultra-violet radiation UVB, atopy/allergy, contact lens wear and mechanical eye rubbing, etc. [10–12]. Primarily, the genetic factors may play an important role in the development of keratoconus as evidenced by studies of familial inheritance, concordance between monozygotic twins and association with other known genetic disorders [12–14]. By linkage analysis and association studies, various loci have been mapped in families from different ethnic populations, well summarized by Nowak DM et al. [12]. Based on associated loci, genes such as SPARC, LOX, TIMP3, COL6A1, COL8A1, MMP9, and MMP2, etc., have been examined for their involvement [12, 15]. The SOD1 and CRB1 were also analyzed as possible candidate genes because of their role in keratoconus associated disease like Down syndrome and Leber congenital amaurosis respectively [16, 17]. However, genes under such studies, have not provided enough evidence for being an appropriate candidate gene in the pathogenesis of KTCN. One of the well-studied genes in genetic association with keratoconus is VSX1. Human VSX1 [OMIM 605020] is a member of the VSX1 group of vertebrate paired-like homeodomain transcription factors localized to human chromosome 20p11-q11. Heon et al., [18] first identified VSX1 mutations in patients with either keratoconus or posterior polymorphous corneal dystrophy (PPCD). This led to the assumption that mutations in the VSX1 gene may be involved in pathogenesis of keratoconus. A number of other studies, further showed the presence of VSX1 variants in keratoconus patients from different ethnic populations [19–24]. Among them, several variants were found in highly conserved residues of VSX1, and predicted to be pathogenic by the bioinformatics tools like PolyPhen, SIFT, etc. [21, 23]. The exonic and intragenic polymorphisms were frequently reported in such studies, and found to be associated with the disease in a few cases [22, 25]. On the other hand, many other studies excluded those previously reported VSX1 variations to be pathogenic, and showed a lack of mutation or association [26–30]. Therefore, the role of VSX1 in KTCN pathogenesis is ambiguous, and further genetic studies are required for any persuasive conclusions. In this study, we have undertaken the sequence analysis of coding regions of VSX1 gene in order to determine its genetic involvement in South Indian patients with sporadic form of keratoconus.

In our study, we have assessed the role of VSX1 by sequence analysis of its five exons in 117 sporadic KTCN patients. Our screening showed the absence of pathogenic variations whereas, four previously reported SNPs were observed. Since no pathogenic changes were detected, we compared the genotype and allele frequency of each of the identified polymorphisms between the disease cohort and healthy controls to access their possible disease involvement. However, allele frequencies of these identified SNPs were found in similar frequency between cases and controls confirming their non-pathogenicity. Other VSX1 gene variations such as p.D144E, p.L17P, p.N151S p.G160D, p.P247R, p.L159M, p.G160V, p.Q175H, p.R166W and p.H244R, reported in different previous VSX1 studies were not identified in our analysis [18–22, 24, 29]. There are few earlier studies, which have indicated the association of non pathogenic VSX1 variations. Stabuc-Silih et al., [25] found an absence of VSX1 pathogenic mutations but observed an association of c.650G>A polymorphism (p=0.043) in unrelated Slovenian patients diagnosed with the hereditary form of KTCN. Mok et al., [20] found a significant association of one VSX1 intragenic polymorphism ‘IVS1-11’ in unrelated Korean keratoconus patients (p=0.001). Mutational screening of 66 unrelated patients with keratoconus (27 familial cases; 39 sporadic cases) from the European population, showed a minor role of VSX1 in the pathogenesis of keratoconus [22]. However, several other studies have shown the absence of pathogenic variations or lack of association of VSX1 variants with KTCN. Tang YG et al., [29] ruled out the association of previously reported VSX1 variations in a case–control study of 77 white KTCN patients. Liskova et al., [28], in a study of 85 familial keratoconus pedigrees from different ethnic origins, found a lack of pathogenic variations in VSX1 and disqualified the previously reported c.432C>G (p.D144E) change to be pathogenic. Aldave et al., [27] found the absence of mutation in 100 unrelated KTCN subjects and concluded that VSX1 mutations are not associated with keratoconus. Overall, our study also rules out the possible involvement of VSX1 gene in sporadic, South Indian KTCN patients. However, few previous evidence of VSX1 pathogenic variations and their association with disease, suggest that it is more likely to be involved in a smaller subset of the KTCN population. The role of VSX1 variations in a minority of keratoconus patients may be influenced by its possible variable penetrance or pleiotropic effect in corneal tissue.

Our study supports the previous evidence of lack of pathogenic variations in VSX1, and corroborates the involvement of new genes, loci or any other genetic or environmental factors. Linkage analysis and association study are the two main approaches used to identify novel genes. Genome wide association study (GWAS) is a more useful approach as it is wide-ranging, unbiased and can be applied even in the absence of convincing indication regarding the function or location of the causal genes. The other genetic factors are needed to be investigated for KTCN pathogenesis. Abu-Amero et al., [32], analyzed VSX1 chromosomal copy number variations (deletions/duplications) in a group of sporadic patients, who were excluded for VSX1 mutations, and verified that such possible genetic changes are also not involved in keratoconus. Recent studies find that keratoconus corneas have signs of oxidative stress and high level of mitochondrial DNA damage [33]. More recent findings suggest that micro-RNA can be involved in the pathogenesis of keratoconus [34]. Therefore, mitochondrial genes and micro-RNA are the prospective emerging areas to explore in the context of other genetic factors related to keratoconus. Proteomic profiles in the KTCN corneas and tear have shown differential expression of several proteins, which may have possible role in the etiology of keratoconus [35–37]. Hence, genetic and proteomic approaches together can provide more useful information regarding disease etiology. For the genetic basis of keratoconus, other genetic factors, new chromosomal loci and genes are the subject of investigation to accomplish the better understanding of the pathogenesis of disease.