The candidate gene approach is based on the knowledge of the disease biochemistry and pathology and consists of identifying mutations in encoding genes for the proteins of the affected tissue. Several studies found enzymatic and biochemical anomalies in affected corneas [9, 10]. The association of keratoconus with osteogenesis imperfecta [11] and mitral valve disease [12, 13] indicates a potential role for collagen anomalies in its occurrence. Studies investigating the pathogenesis of keratoconus have attempted to identify mutations in genes coding for components of interleukin-I [14], proteases [15, 16], protease inhibitors [17, 18], and collagens. Type I, III, IV, V, VI, VII, and VIII collagens are present in the cornea. The first collagen-encoding gene tested was COL6A1, but no significant relationship with keratoconus was detected [19]. Similarly, other collagen-encoding genes of the cornea were tested and eliminated [10]. Negative results obtained from linkage analyses do not exclude the role of these encoding genes in certain types of keratoconus. Indeed, the degree of genetic heterogeneity of this disease is unknown and mutations of several encoding genes from families that have not been tested may yet play a role in the emergence of keratoconus. One candidate gene study led to the identification of related mutations among patients affected by keratoconus. Mutations affecting the gene VSX1 were identified. This gene codes for a putative transcription factor and is also involved in posterior polymorphous dystrophy [20]. VSX1, however, has a role in only 0.1–0.4 % of familial keratoconus and thus its importance in the pathogenesis of keratoconus remains low.

Keratoconus is occasionally associated with other genetic or ophthalmologic diseases such as Down’s syndrome , Leber congenital amaurosis , atopic diseases, conjunctivitis, some pigmentary retinopathies, mitral valve prolapse, collagen vascular diseases, and Marfan’s syndrome [9, 10, 21]. It remains difficult, however, to establish a direct relationship between these diseases and keratoconus. Interpretations of these relationships are indeed challenging because of the relatively high prevalence of keratoconus, which remains probably underestimated. As a result, it is difficult to determine if these diseases are associated as co-factors triggering a keratoconus genetic susceptibility or if they are directly involved in the disease pathogenesis. This is an issue of particular significance when considering associated genetic diseases that are the results of the alteration of known genes and where a genetic analysis would help identify the gene responsible for keratoconus.

Down’s syndrome is strongly related with keratoconus, with an estimated prevalence of 0.5–15 %, which is 10–300 times higher than the general population [10, 22, 23]. This suggests a link between keratoconus and chromosome 21. Numerous genetic studies have targeted this chromosome and some of them suggest its involvement in the pathogenesis of the disease [19]. This relationship, however, has also been attributed to some environmental factors such as eye rubbing, which may play a co-factor role, resulting in a strong prevalence of keratoconus [23].

Among all associated diseases, the direct cause of keratoconus is largely unknown and remains a matter of continued debate. This is predominantly due to the lack of basic information regarding the real prevalence of keratoconus and the impact of environmental factors.

An interesting global approach using ‘omic’ techniques aims to identify the origin of keratoconus without taking into account any preconceived ideas of the pathogenesis of the disease. In this approach, corneas affected by keratoconus are compared with healthy corneas during a particular cell machinery stage: the DNA (genomics ), the RNA (transcriptomics), or the protein (proteomics). Transcriptomics is used to analyse, at the mRNA level, which genes are being expressed and in what ratios, whereas proteomics looks at the proteins that are subsequently translated.

Unlike studies investigating candidate genes, genetic linkage analyses do not rely on any knowledge of the pathogenesis or biochemistry of the disease. Linkage analyses highlight genetic regions that contain genetic variants bound to a phenotype.

To date, 17 distinctive genetic regions have been identified [8], indicating the existence of a strong genetic heterogeneity in the development of keratoconus. Among these regions, only three have been independently verified (5q21, 5q32, and 14q11). These studies implicate two genes as potential minor candidates (MIR184 and DOCK9). More recently, global sequencing of the genome and/or exome has replaced linkage analyses.

The relative failure of previous approaches and the recent advancements in molecular biology have promoted new studies. Using the state-of-the-art technologies of high-throughput genotyping, recent studies have compared the frequency of hundreds of thousands of genetic variants distributed among chromosomes. The most convincing studies detected linkage with variants of the gene LOX (lysyl oxidase , involved in the cross-linking of stromal collagen) [24, 25]. Other authors have concentrated on comparing intermediary phenotypes rather than groups of individuals (case–control study). Lu et al., for example, identified a region associating keratoconus with central corneal thinning [26]. Complementary meta-analyses have identified several variants in or near this region (FOXO1, FNDC3B, RXRA-COL5A1, MPDZ-NFIB, COL5A1, and BANP-ZNF469). One of these variants, BANP-ZNF469, was also found in an independent Australian cohort [27]. The role played by variants of this particular gene was confirmed in a study by Lechner et al. [28]. ZNF469 is a gene that is also involved in another corneal syndrome (brittle cornea syndrome) and is linked to a thin and fragile cornea. This gene is currently the most important identified genetic factor in the pathogenesis of keratoconus.

Several studies have compared proteomes between patients affected by keratoconus and control patients. Comparability of results is difficult, due to differences in tissue type (tear fluid, corneal tissue: epithelium and/or stroma), age, disease severity, and development stage of keratoconus. A number of studies provide evidence that keratoconus is characterized by a cytokine imbalance in tear fluid and that these inflammatory mediators operate actively at the ocular surface. More than 1500 proteins have been identified in the analysis of the tear film. Higher levels of proteolytic activity and increased levels of proinflammatory cytokines, cell adhesion molecules , matrix metalloproteinases (MMP) , glycoproteins, and transporter proteins have been observed compared to controls [29–32]. In particular, studies have shown a strong concordance of elevated Interleukin 6 (IL6) , tumour necrosis factor α (TNFα) , and MMP9 in tears from keratoconus patients [29, 33–35]. MMP9 is one of the matrix-degrading enzymes produced by the human corneal epithelium and regulated by cytokine IL6. In addition to IL6, TNFα is considered a major pathogenic factor in systemic and corneal inflammation. TNFα induces the expression of MMP9. Balasubramanian et al. [29] found higher levels of MMP9 in tears from keratoconic eyes, but the difference was not statistically significant. They indicated that the observed discrepancy might be explained because they used antibodies to the active MMP9. Shetty et al. [35] observed that MMP9, IL6, and TNFα were strongly upregulated at the mRNA level in keratoconus patient epithelia. However, whereas tears of keratoconus patients demonstrated an acute increase in MMP9 and IL6 levels over controls, TNFα levels did not show any significant associations with different grades of keratoconus. They demonstrated that the administration of cyclosporine A strongly reduced the inflammatory stimulation and expression of MMP9 in tears of keratoconus patients and decreased the production of IL6, TNFα, and MMP9 by corneal epithelial cells, while follow-up of 20 keratoconus patients over 6 months demonstrated significant local topographical changes in the cornea measured by keratometry. Although overall change in keratometry may not be very significant in the study, the authors suggest that cyclosporine A may be a promising new treatment modality for keratoconus [35].