## 1 Introduction  
Keratoconus is cone shaped and occurs as bilateral corneal dystrophy, which is expressed as a progressive thinning of the corneal layers of stroma [1]. The condition has a multifactorial origin and is progressive but non-inflammatory. The initial corneal thinning leads to irregular astigmatism, a prominent level of myopia and a severe refractive error in the patient [2, 3], resulting in decreased visual acuity. At the initial stages of the disease, there are no obvious symptoms other than a mild loss of visual acuity. As the disease progresses, severe signs start to appear, such as [PERSON]'s ring, corneal protrusion, Vogt's striae and Munson [4]. Both eyes are affected in an asymmetric manner [5]. Globally the prevalence of keratoconus is 1 out of 2000 individuals, which varies with ethnicity having a prevalence rate in Caucasian populations of between 8.8-54.5 per 100,000 individuals [1], in a Malaysian population of 1 per 100 persons [6] and in an Iranian population 760 patients out of 100,000 individuals which is much higher than the prevalence of KC in the Western world population [7]. The same disease was studied in the United States for 48 years, and the data collected over this time reported the disease prevalence to be 54.5 patients per 100,000 individuals [8]. A study reported in 2017 showed the prevalence of disease in children in Saudi Arabia to be 4.8% [9].  
Manifestation of KC usually starts in the second decade of life and progresses in severity over the third and fourth [10], while the prevalence of this disease is higher in males compared to females in certain populations [11; 12]. The pattern of the disease prevalence is more sporadically oriented than familial linked, but a positive family history does contribute to the risk of the disease in the offspring. In familial linked cases, a history of the presence of inherited autosomal dominant mode is frequent when compared to X-linked or recessive inheritance [13; 14]. The exact etiological cause of KC is unclear due to the varying prevalence of the disease in different races and families, and it occurs sporadically. Nonetheless, bearing in mind KC is a multifactorial disease, it can be speculated that both the environmental as well as genetic factors play their individual and specific roles in the pathogenesis of the disease [15].  
Linkage analysis of the Keratoconus patient population of different ethnicities and Genome-wide association studies (GWAS) have indicated the involvement of various single nucleotide polymorphisms (SNPs) and loci in the pathogenesis of the disease [16], which includes matrix metalloproteinase (_MMP-9_), inhibitors of metalloproteinase 3 (_TIMP-3_), lysyyl oxidase (_LOX_), collagen type VI alpha-1 chain (_COL6A1_), hepatocyte growth factor (_HGF_), superoxide dismutase (_SOD_), crumbs homolog 1 (_CRBI_), zinc finger e-box binding homeobox 1 (_ZEBI_) and visual system homeobox 1 (_VSX1_). Among all these genes, proteins and factors, VSX1 is involved in human ocular development [17] but it is also expressed in embryonic craniofacial tissue [18], the inner layer of the retina, as well as corneal tissue in the adult [19].  
Despite the number of associated genes, the role of genetic deficits in the pathogenesis of keratoconus has not been explored in Pakistani families or the sporadic population. Moreover, bioinformatic information regarding the role of genetic variants and their impact on gene/protein function is severely lacking. The present study is designed to explore the contribution of the _VSX1_ genes in the recessive form of the disease in Pakistani KC patients and to perform bioinformatic analysis on identified variants to explore their potential contributions to KC development.