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Genes Associated With Myopia-Related Phenotypes

The HGF/cMET ligand-receptor axis

A widely studied candidate gene for myopia was Hepatocyte Growth Factor (HGF). HGF has been shown to have a biologically relevant role in ocular biology and is orthologous to Eye1, a major quantitative trait locus that contributes to a variable rate of eye growth in the mouse.20

HGF works by binding to its receptor cMET, a member of the tyrosine kinase receptor family.22 Once activated, the system triggers many cellular responses, including cell division, migration, differentiation, and survival of numerous cell types.23 In the eye, HGF and cMET are expressed in the lens, all three layers of the cornea, and in both fetal and adult retinal pigment epithelium.22,24–27

Natural genetic variants within HGF were first observed to be associated with high myopia in a family-based association study comprising 128 nuclear families of Chinese descent. One SNP in particular (HGF rs3735520) showed evidence of association with very high myopia (defined as SE ≤ –10.0 D), where the minor allele was observed to be conferring increased risk of disease (P = 0.003).21 However, a second follow-up study enrolling 288 high myopia cases and 208 controls, also of Chinese descent, failed to confirm association with this SNP.28 A third study, this time involving participants of European descent (the Duke– Cardiff cohort), identified the rs3735520 SNP as associated with mild to moderate myopia (SE from –0.50 to –5.00 D), but not with high (SE ≤ –5.00 D) or very high myopia (SE ≤ –10.0 D). Of note, evidence of association with high and very high myopia was observed with another SNP within HGF (rs2286194) in the Duke–Cardiff cohort.

Observed associations with cMET genetic variants and myopiarelated phenotypes were more recent.29 In a longitudinal cohort study, which enrolled school children aged 7–12 years (Singapore cohort study for risk factors of myopia; SCORM), a SNP within cMET (rs2073560) was found to be associated with an increased risk of myopia in general (SE ≤ –0.5 D). Longitudinal analysis showed that the variant allele also was associated with more rapid change in refractive error over time regardless of the initial refractive state. However, due to its location within the non-coding region of cMET, this SNP is unlikely to be the causative genetic variant responsible for the observed associations. Thus, further efforts involving direct sequencing of all coding and

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regulatory regions of cMET are needed to define the biologically relevant mutations.

Transforming growth factor-β (TGFB1)

Axial myopia is the commoner form of myopia compared to refractive myopia,30 and active scleral remodeling has been shown to play a crucial role in axial (globe) elongation, at least in animal models of myopia.31,32 One such gene that could be involved in the process of scleral remodeling is the one encoding for transforming growth factor-β (TGF-β , encoded by the TGFB1 gene). TGF-β is expressed in ocular tissues33 and its concentrations in the retinal pigmented epithelium, choroid, and the sclera have been found to be significantly reduced in myopic eyes. In addition, TGF-β is known to regulate the proliferation of fibroblasts, as well as the production of collagen, matrix metalloproteinases (MMP), and tissue inhibitors of MMP.34 All these processes contribute to the biology of scleral remodeling and consequent axial length change.35

Given all these prior biological information on the potential influence of TGF-β on axial length, it is unsurprising that natural genetic variation within TGFB1 has long been suspected to modify susceptibility to refractive errors, especially axial myopia. Indeed, a SNP within TGFB1 (rs1800470) has been found to associate with severe myopia in a study involving 201 severe myopia cases (SE ≤ –6.0 D) and 86 controls of Chinese descent from Taiwan. Here, the minor T allele was shown to be at reduced risk of severe myopia (OR = 0.55, 95%CI: 0.37 — 0.80; P = 0.001).36 This association was replicated in a second cohort involving 300 severe myopia cases (SE ≤ –8.0D) and 300 controls of Southern Chinese descent (OR = 0.72, 95%CI: 0.57 — 0.90; P = 0.004).37 However, in this second Chinese study,37 a previous SNP that was not assessed in the original study (rs4803455) was found to be much more strongly associated with decreased susceptibility towards severe myopia (OR = 0.66, 95%CI: 0.52–0.84; P = 4.9 × 10–4) compared with rs1800470. As such, SNP rs4803455 is a closer correlate for severe myopia compared to SNP rs1800470. Notably, a Japanese high myopia study (SE ≤ –9.25), which included 330 cases and 330 controls, failed to detect any association with 10 TGFB1 SNPs. However, neither rs1800470 nor rs4803455 were genotyped in the Japanese study. A second study showing non-replication of TGFB1 genetic variants also could not be adequately assessed, as only 1 SNP was typed; again, neither rs1800470 nor rs4803535 were analyzed.

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In the Singapore-based SCORM cohort, recessive carriage of the minor T allele at rs4803455 was found to be associated with decreased susceptibility to severe myopia (SE ≤ –5.0D)(OR = 0.46, 95%CI: 0.19–1.05; twotailed P = 0.046) (SCORM unpublished findings). These individuals also had on average shorter axial lengths (0.21 mm shorter on average, P = 0.05) compared to wild-type individuals, thus lending further support to the two previous Chinese studies and also supporting a role for TGFB1 and axial myopia.

Trabecular-meshwork inducible glucocorticoid

response (TIGR) gene

TIGR was first identified as a susceptibility gene for primary open angle glaucoma,38 but more recent data suggests that it could also be a susceptibility gene for high myopia.39,40 This is in keeping with clinical observations documenting an increased frequency of open-angle glaucoma in individuals with high-myopia. An increased prevalence of myopia in patients with glaucoma was also observed.39

The protein encoded by TIGR is myocillin (MYOC). It has been found to be expressed widely in ocular tissues (e.g. the choroid, cilliary bodies, sclera, and trabecular meshwork),41 and studies have shown that the expression of myocilin in the trebecular meshwork is affected by TGF-β (see previous section on TGFB1) and mechanical stretch.

Findings from previous studies investigating the association between genetic variants at the TIGR locus and severe myopia have yielded slightly inconsistent results. In a family-based association study conducted in Hong Kong involving 162 Chinese nuclear families (overall n = 557), two microsatellite polymorphisms flanking TIGR and two SNPs (rs2421853 and rs235858) within the 3′ untranslated region of the gene were shown to be associated with high myopia (SE ≤ –6.0 D) (lowest P = 4 × 10–6).39 A second, smaller case-control study (n = 70 severe myopia cases and 69 non-myopic controls) from Hong Kong was unable to demonstrate evidence of association with one of the microsatellite markers reported (–339(GT)12–16; NGA17).42 Notably, this second Hong Kong-based study did not assess the remaining three polymorphisms (microsatellite NGA19, and SNPs rs2421853 and rs235858), thus rendering it of limited value in demonstrating lack of association.

A subsequent reassessment of these four genetic markers consisting of over 1000 Caucasian subjects (the Duke–Cardiff cohort) also did not

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replicate evidence of association with these four TIGR polymorphisms.40 Instead, the Duke–Cardiff cohort showed that SNP rs1684720 (which was previously not tested in the Hong Kong study but is in linkage disequilibrium (LD) with the other two SNPs in East Asians) was consistently associated with high myopia in both the case control and family-based approach. In the Singaporean SCORM cohort, association was observed for SNP rs1684720 with a more negative population-wide SE (P = 0.032) in general, and severe myopia in particular (SE ≤ –5.0 D)(P = 0.048; SCORM cohort). (SCORM unpublished findings.) Although inconsistent with regards to the exact SNPs involved in the association signal, these findings are nonetheless supportive of a role for TIGR genetic variants and increased susceptibility to severe myopia.

The collagen family of genes

Collagen makes up about 30 percent of total body protein and is a basic building block for human connective tissue. Colagen provides structure and tensile strength to many important body components (e.g. skin, bone, cartilage, and finer structures, such as blood vessels and the sclera). As 90 percent of the human sclera is primarily an ECM of collagen fibrils (mainly type I fibrils),32 genes encoding for the collagen family of proteins are natural candidates for study in myopia pathogenesis and development.

Scleral thinning has been attributed to a general loss of ECM (see previous section on TGFB1),43 and studies in animal models such as tree shrews have revealed that myopic eyes typically suffer from thinned and weakened sclerae, which are less capable of withstanding the expansive forces of a positive intraocular pressure. It is thus hardly surprising that one characteristic feature of axial myopia is an elongated eyeball.44

At the cellular level, amounts of collagen mRNA at steady state before and after monocular visual form-deprivation has been well correlated; deprived-eye scleras are myopic and have less collagen mRNA, whereas recovering eye scleras have more.45 Such findings are in keeping with the observation that visual form-deprivation produces a more extensible sclera, and that recovery from form-deprivation reduces the extensibility of the sclera.46,47 Currently, the two most well-studied collagen genes in myopia pathogenesis are COL1A1 and COL2A1, encoding for the α1 and α2 chains of type I collagen respectively.

Rare function-altering genetic mutations in COL1A1 have been found to be associated with two distinct connective tissue diseases: osteogenesis

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imperfecta and Ehlers–Danlos syndrome. For both disorders, the clinical phenotype is a result of compromised type-I collagen structure. Studies assessing the role of genetic polymorphisms in COL1A1 and susceptibility to myopia first appeared with Asian cohorts. Conflicting results were observed; a Taiwan-based study with 471 severe myopia (SE ≤ –6.0 D) cases and 623 controls of Chinese ethnicity could not detect evidence of association with COL1A1 genetic variants after assessing 10 representative ‘tagging’ markers.48 In contrast, a Japanese study with 330 severe myopia cases (SE ≤ –9.25 D) and 330 controls managed to detect association with COL1A1 SNPs rs2075555 and rs2269336.49 However, a second Japanese study using 427 severe myopia cases (SE ≤ –5.0 D) and 420 controls failed to find association with these two SNPs.50 Data from the SCORM cohort involving Singaporean Chinese also failed to confirm evidence of association (SCORM unpublished findings). Thus, the current evidence with COL1A1 is not conclusive of association with myopia.

Moving on to COL2A1, where severe gene disruptions during foetal development could result in Stickler’s syndrome, a different picture is observed. Stickler’s syndrome (congenital, progressive arthro-ophthal- mopathy) is an autosomal dominant form of collagenopathy (disease due to abnormal synthesis of collagen), whereby affected children have a distinct facial appearance, eye abnormalities, hearing loss, and joint problems. It has been observed that many patients with COL2A1 Stickler syndrome suffer from a mono-genic form of severe ‘nearsightedness’ (described as having high myopia) due to the inherently abnormal shape of the eye. In light of these findings with these highly penetrant genetic mutations, it was hypothesized that commoner and less penetrant genetic variants within COL2A1 could also modify individual susceptibility to less severe forms of myopia.

In a family-based study involving 123 nuclear families of mixed (but predominantly Caucasian; 62%) ethnicity, strong evidence of association with common myopia (SE ≤ –0.75 D) was detected for one COL2A1 SNP (rs1635529, P = 7.0 × 10–5).51 A second study (the Duke–Cardiff cohort) also showed supporting evidence of association for this particular SNP with common (SE ≤ –0.5 D; P < 0.05 in Duke) and high-grade myopia (SE ≤ –5.0 D; P < 0.05 in Duke). Further replication was achieved in the Cardiff cohort (P = 0.007 for common myopia, and P = 0.004 for highgrade myopia).52 Compared to COL1A1, the evidence of association for COL2A1 and the myopia trait is more robust. Although SNP rs1635529