**9. Molecular markers**

*Ocular Surface Diseases - Some Current Date on Tear Film Problem and Keratoconic Diagnosis*

104 patients and 157 controls, with 3 variants, P141L, D326Y, and G895G, in COL4A3 and 5 variants in COL4A4P482S, M1327 V, V1516 V, and F1644F differentiating patients and controls with statistical significance P < 0.005 [118]. Association was replicated in a Greek study [99]. In an Iranian cohort of 112 patients and 150 controls, The COL4A4 rs2229813 (M1327 V) An allele increased keratoconus risk for KC (P = 0.018, OR = 1.5), but COL4A4 rs2228555 (C1516V)

Another COL4A4 SNP, rs2228557 (F1644F) in 144 patients, 153 controls in Iran, showed a high association (P = 0.0001) [119]. *COL4A4* rs2229813 and rs2228557 are strongly associated with keratonconus in Americans (P = 1.3 × 10<sup>−</sup>12, OR = 2.38

showed weak association [120]. Although *COL4A3* and *COL4A4* plays biological roles in cornea structure and properties, there is no evidence that they directly

The pathophysiology of keratoconus is attributed to disruption of the cornea morphology in association with the corneal collagen. A review of published studies on proteins, collagen and risk factors of keratoconus between January 2016 and June 2018 has revealed altered regulations in more than 30 proteins and their genes. They belong to protein families including degradative enzymes, cellular proteases, and collagens [121]. Up- and downregulations of hosts of proteins in corneal epithelium and stroma have been reported in keratoconus, especially different types of collagen

Collagen is the major structural protein in the cornea. Decrease in collagen lamellae and fibers, together with reduced microfibers and fine granules, has been described in keratoconus [122]. Disruption in collagen contents and integrity affect corneal thinning and properties. Reduction of types I, III, and IV, which are major structural proteins in the basement membranes, have been shown in keratoconus [123]. Among the 6 main subtypes of COL4A, which plays important structural and functional roles, COLA4A1 is reportedly downregulated in cornea of keratoconus patients [124]. Expression studies also showed downregulation of many collagen types and subtypes in keratoconus, including COL8A1, COL8A2, COL12A1,

COL13A1, COL6A2, COL15A1, and COL18A1 [125–127]. They may be considered for use as biomarkers in keratoconus, [121] but practical protocols and validations are

MMP-1 and MMP-9, are upregulated in corneal tissue and affect collagen properites and dyregulate proteolysis [128]. In a pathways enrichment analysis of 19 keratoconus genes consistently reported as risk genes to keratoconus in 16 studies, interleukin-1 processing and assembly of collagen fibrils are associated with keratoconus pathology [129]. MMPs have been assayed in tears. It is noted that in one study there was no detectable MMP-1 in tears of healthy subjects [130]. Elevations in MMP-1 [131] and MMP-9 levels [132] has been shown in keratoconus tear samples. On study reported correlation between MMP-9 level and disease severity [133]. However, other studies did not find MMP-9 elevation [131, 134].

, OR = 0.54 respectively) [102]. Replication in a Chinese cohort

showed no association [98].

and P = 4.5 × 10<sup>−</sup><sup>7</sup>

cause keratoconus*.*

**8. Specific proteins**

and matrix metalloproteinases.

**8.1 Collagen**

still to be established.

**8.2 Matrix metalloproteinases**

**64**

After meta-analysis of 24 eligible studies selected after screening of 668 reports between 1950 and 2016, 16 SNPs in 14 genes/loci were found to be associated with keratoconus out of 53 polymorphisms in 28 genes/loci. Stratification analysis revealed strong association of 8 SNPs in 6 genes/loci with keratoconus in Whites, including *FOXO1* rs2721051, *RXRA-COL5A1* rs1536482, *FNDC3B* rs4894535, *IMMP2L* rs757219 and rs214884, and *BANP-ZNF469* rs9938149, and *COL4A4.*

rs2229813 and rs2228557 [95]. While they may be potential genetic markers for keratoconus in Whites, there is no further data of validation. Replications in Chinese and Arabic populations [120] revealed weak associations in *COL4A4* rs2229813 and rs2228557, which are strongly associated with keratonconus in Whites with statistical significance of P = 1.3 × 10<sup>−</sup>12, OR = 2.38 and P = 4.5 × 10−<sup>7</sup> , OR = 0.54 respectively. In Chinese, another SNP, rs1324183 in *MPDZ-NF1B*, is related to disease severity and corneal biomechanical properties, and may be a potential molecular marker [136].

In a review in 2001 keratoconus was reputed to be an inheritable disease [137]. Since then more evidences of familial links and susceptible genes or loci have been reported as a result of vigorous research in different populations and geographic locations. There are proven genetic and non-genetic risk factors [35]. In a recent

