**6.2 Familial linkage**

In the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) study in a cohort of 1209 patients mixed in ethnicity in the USA, 829 (69%) white, 240 (20%) black, and 99 (8%) Hispanic, family history of keratoconus was reported in 13.5% of the cases [65]. After follow-up for 8 years, the inheritance patterns were not established [66]. In another study from the USA, more females reported family history than males. But it was unclear whether it was a difference in attitude on reporting or a genuine gender difference in familial link [57]. In the New Zealand study, familial aggregation analysis showed keratoconus familial rate of 23.5% [59]. In Scotland, family history occurred in 5% of 186 white patients [67]. In a report from North India among 120 patients, 6 (5%) had family history [68]. In a review of keratoconus in Asians, family history ranged from 4.4 to 23.5% [40]. Overall, reported family history of keratoconus has widely ranged from 6% up to about 25% [37, 65].

A recent systemic review and meta-analysis on 29 eligible reports from different parts of the world selected from 3996 articles revealed family history as the strongest risk factor (odds ratio 6.42; 95% CI:2.59–10.24) among other established risk factors: eye rubbing (odds ratio 3.09; 95% CI:2.17–4.00), eczema (odds ratio 2.95; 95% CI:1.30–4.59), asthma (odds ratio 1.94; 95% CI:1.30–2.58), and allergy (odds ratio 1.42; 95% CI:1.06–1.79) [69]. Overall, no obvious differences exist in clinical or ophthalmic presentations between familial and sporadic keratoconus. As a genetic disease, keratoconus has shown weak penetrance with variable expressions. While family aggregation and linkage studies showed familial inheritance, the majority of reported keratoconus patients are sporadic.

#### **7. Mapping the keratoconus genes**

Candidate gene analysis, family linkage analysis, and more recently genomewide association study with support by candidate gene association studies and next generation sequencing, have contributed to identification of genetic loci or gene variants that are in association with keratoconus [70]. All are genetic associations. No direct keratoconus causing mutation has been identified.

#### **7.1 Linkage analysis**

Single nucleotide polymorphisms (SNPs) and microsatellite markers covering the whole genome have been attempted to find keratoconus loci or even genes in family pedigrees and sib pairs. But even in families, penetrance of keratoconus is low and clinical presentations are variable. A large number of samples have to be available. Vigorous research among various ethnic groups have identified a number of keratoconus loci which are replicable and of maximum LOD score greater than 3, in a collection of 67 sib pair Hispanic families, two-stage genome-wide analysis of 380 microsatellite markers in totally 351 subjects, among them 110 were affected by keratoconus which has led to identification of a multitude of loci in chromosomes *2q, 3p, 4q, 5q31, 5p, 9p, 9q34, 11p, 12p, 14q*, and *17q* [71]. In a collection of 25 Italian families, genome-wide scan of microsatellite markers in 77 affected and 57 unaffected members have mapped chromosomal regions for keratoconus in *5q32-q33; 5q21.2, 14q11.2,* and *15q2.32* [72]. Some of these loci had been replicated or refined in further investigations: *2p24* [73]; *3p14-q13* [74]; *5q14.3-q21.1* [75]; 5q31.1-q35.3 [72]; *13q32* [76]; *16q22.3-q23.1* [77]; and *20q12* [78]. These studies were mostly on European populations. The large number of associated loci exemplified the genetic heterogeneity of keratoconus. No disease-causing mutation has been identified from these loci.

#### **7.2 Genome-wide association studies (GWAS)**

Most GWAS in connection with keratoconus were conducted on the two quantitative traits of central corneal thickness (CCT) and corneal curvature (CC). These two are characteristic, but not exclusive, traits of keratoconus, with the corresponding morphological features of central corneal thinning and corneal steepness.

#### *7.2.1 Central corneal thickness (CCT)*

There were nine reported GWAS on central corneal thickness (CCT) up to July 2017. They were summarized in an excellent review [70]. All involved meta-analysis

**61**

*7.2.3 Other approaches*

*Molecular Genetics of Keratoconus: Clinical Implications DOI: http://dx.doi.org/10.5772/intechopen.90623*

the highest among all reported SNPs [79].

and 4.0 × 10<sup>−</sup><sup>5</sup>

P-values of 2.5 × 10<sup>−</sup><sup>7</sup>

*7.2.2 Corneal curvature (CC)*

ethnicities [86].

in initial and validation cohorts. More than 40 SNPs in 30 genes were reported. The biggest sample size was with totally 13,057 European and 6963 Asian samples, while the primary cohort was comprised of 874 patients and 6085 controls. A multitude of keratoconus associated SNPs in 26 loci was identified. SNP rs9938149 in the *BANP-ZNF469* gene attained GWAS significance of P-value = 2.4 × 10<sup>−</sup>49, which was

Some of the gene variants were analyzed in keratoconus and control cohorts. Possibly there was ethnic specificity. Rs96067 in COL8A2 was identified in a Singaporean cohort of 2538 Indians and 2542 Malays [80] but not in a separate GWAS study 0f European study subjects with 3931 German and 1418 Dutch study subjects. However, SNPs in *BANP-ZNF469* and COL5A1 were replicated [81]. In a cohort of Australian white study subjects with 933 keratoconus patients and more than 4000 controls, keratoconus susceptibility was detected at the *HGF* locus [82]. The risk factor allele rs3735520 was associated with keratoconus in a Czech cohort of 165 patients and 193 controls [83] and Australian whites of 157 patients and 673 controls [84]. Another study involved two independent cohorts of keratoconus patients, involving 222 Caucasian patients, 687 African Americans, 3324 Caucasian controls and 307 individuals from 70 keratoconus families reported strong association of *Lysyl Oxidase* gene (*LOX)* polymorphisms with keratoconus, with meta

respectively [85]. In a meta-analysis of 14 studies comprising 17,803 individuals of European ancestry 44 loci associated with CCT were found, two of them were LTBP1 and WNT10A 42 European specific while the rest occurred also in Asian

that contributed to identification of susceptibility genes. Eight SNPs in MTOR/ FRAP1 and 7 SNPs in PDGFRA were found in 10,008 samples from three population groups in Singapore: Malays, Chinese and Indians [87]. Another big cohort of 12,660 Asians included Japanese in addition to Malays, Chinese, and Indians, [88] Associations of CMPK1 rs17103186 and RBP3 rs11204213 with CC, and RBP3 rs11204213 with axial length were discovered. In the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort of 2023 individuals of white European descent, rs6554163 in *PDGFRA* was associated with both CC and axial length [89]. In a cohort of 1013 Australian individuals, 1788 twins and their families, CC was associate with rs2114039 near *PDGFRA* and rs2444240 which is 31 kb upstream to *TRIM29*, but not with any SNP at the FRAP1 locus [90]. In totally 15,168 study samples that included Japanese, Chinese or European ethicity, rs10453441 in WNT7B was strongly associated with both CC and axial length [91]. WNT7B rs10453441 is the only SNP associated with both traits of CCT and CC [92]. While these SNPs have no consistent and strong association with keratoconus, an exome sequencing analysis identified a *WNT10A* variant that was associated with corneal thickness and keratoconus [93]. In contrast, a GWAS on direct association of

Six GWAS on corneal curvature (CC) have been reported in multi-ethnic cohorts

keratoconus patients and controls involving 222 patients and 3324 controls found no

Apart from corneal morphologic features, a recent GWAS investigated corneal biomechanical properties in an European cohort of 6645 participants and 2384 participants from the British TwinsUK study [36]. The second stage of the association

GWAS significance of associated gene variants [94].

for LOX SNPs rs2956540 and rs10519694

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

in Asians, family history ranged from 4.4 to 23.5% [40]. Overall, reported family history of keratoconus has widely ranged from 6% up to about 25% [37, 65].

reported keratoconus patients are sporadic.

No direct keratoconus causing mutation has been identified.

**7.2 Genome-wide association studies (GWAS)**

*7.2.1 Central corneal thickness (CCT)*

**7. Mapping the keratoconus genes**

**7.1 Linkage analysis**

A recent systemic review and meta-analysis on 29 eligible reports from different parts of the world selected from 3996 articles revealed family history as the strongest risk factor (odds ratio 6.42; 95% CI:2.59–10.24) among other established risk factors: eye rubbing (odds ratio 3.09; 95% CI:2.17–4.00), eczema (odds ratio 2.95; 95% CI:1.30–4.59), asthma (odds ratio 1.94; 95% CI:1.30–2.58), and allergy (odds ratio 1.42; 95% CI:1.06–1.79) [69]. Overall, no obvious differences exist in clinical or ophthalmic presentations between familial and sporadic keratoconus. As a genetic disease, keratoconus has shown weak penetrance with variable expressions. While family aggregation and linkage studies showed familial inheritance, the majority of

Candidate gene analysis, family linkage analysis, and more recently genomewide association study with support by candidate gene association studies and next generation sequencing, have contributed to identification of genetic loci or gene variants that are in association with keratoconus [70]. All are genetic associations.

Single nucleotide polymorphisms (SNPs) and microsatellite markers covering the whole genome have been attempted to find keratoconus loci or even genes in family pedigrees and sib pairs. But even in families, penetrance of keratoconus is low and clinical presentations are variable. A large number of samples have to be available. Vigorous research among various ethnic groups have identified a number of keratoconus loci which are replicable and of maximum LOD score greater than 3, in a collection of 67 sib pair Hispanic families, two-stage genome-wide analysis of 380 microsatellite markers in totally 351 subjects, among them 110 were affected by keratoconus which has led to identification of a multitude of loci in chromosomes *2q, 3p, 4q, 5q31, 5p, 9p, 9q34, 11p, 12p, 14q*, and *17q* [71]. In a collection of 25 Italian families, genome-wide scan of microsatellite markers in 77 affected and 57 unaffected members have mapped chromosomal regions for keratoconus in *5q32-q33; 5q21.2, 14q11.2,* and *15q2.32* [72]. Some of these loci had been replicated or refined in further investigations: *2p24* [73]; *3p14-q13* [74]; *5q14.3-q21.1* [75]; 5q31.1-q35.3 [72]; *13q32* [76]; *16q22.3-q23.1* [77]; and *20q12* [78]. These studies were mostly on European populations. The large number of associated loci exemplified the genetic heterogeneity of keratoconus. No disease-causing mutation has been identified from these loci.

Most GWAS in connection with keratoconus were conducted on the two quantitative traits of central corneal thickness (CCT) and corneal curvature (CC). These two are characteristic, but not exclusive, traits of keratoconus, with the corresponding morphological features of central corneal thinning and corneal steepness.

There were nine reported GWAS on central corneal thickness (CCT) up to July 2017. They were summarized in an excellent review [70]. All involved meta-analysis

**60**

in initial and validation cohorts. More than 40 SNPs in 30 genes were reported. The biggest sample size was with totally 13,057 European and 6963 Asian samples, while the primary cohort was comprised of 874 patients and 6085 controls. A multitude of keratoconus associated SNPs in 26 loci was identified. SNP rs9938149 in the *BANP-ZNF469* gene attained GWAS significance of P-value = 2.4 × 10<sup>−</sup>49, which was the highest among all reported SNPs [79].

Some of the gene variants were analyzed in keratoconus and control cohorts. Possibly there was ethnic specificity. Rs96067 in COL8A2 was identified in a Singaporean cohort of 2538 Indians and 2542 Malays [80] but not in a separate GWAS study 0f European study subjects with 3931 German and 1418 Dutch study subjects. However, SNPs in *BANP-ZNF469* and COL5A1 were replicated [81]. In a cohort of Australian white study subjects with 933 keratoconus patients and more than 4000 controls, keratoconus susceptibility was detected at the *HGF* locus [82]. The risk factor allele rs3735520 was associated with keratoconus in a Czech cohort of 165 patients and 193 controls [83] and Australian whites of 157 patients and 673 controls [84]. Another study involved two independent cohorts of keratoconus patients, involving 222 Caucasian patients, 687 African Americans, 3324 Caucasian controls and 307 individuals from 70 keratoconus families reported strong association of *Lysyl Oxidase* gene (*LOX)* polymorphisms with keratoconus, with meta P-values of 2.5 × 10<sup>−</sup><sup>7</sup> and 4.0 × 10<sup>−</sup><sup>5</sup> for LOX SNPs rs2956540 and rs10519694 respectively [85]. In a meta-analysis of 14 studies comprising 17,803 individuals of European ancestry 44 loci associated with CCT were found, two of them were LTBP1 and WNT10A 42 European specific while the rest occurred also in Asian ethnicities [86].

### *7.2.2 Corneal curvature (CC)*

Six GWAS on corneal curvature (CC) have been reported in multi-ethnic cohorts that contributed to identification of susceptibility genes. Eight SNPs in MTOR/ FRAP1 and 7 SNPs in PDGFRA were found in 10,008 samples from three population groups in Singapore: Malays, Chinese and Indians [87]. Another big cohort of 12,660 Asians included Japanese in addition to Malays, Chinese, and Indians, [88] Associations of CMPK1 rs17103186 and RBP3 rs11204213 with CC, and RBP3 rs11204213 with axial length were discovered. In the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort of 2023 individuals of white European descent, rs6554163 in *PDGFRA* was associated with both CC and axial length [89]. In a cohort of 1013 Australian individuals, 1788 twins and their families, CC was associate with rs2114039 near *PDGFRA* and rs2444240 which is 31 kb upstream to *TRIM29*, but not with any SNP at the FRAP1 locus [90]. In totally 15,168 study samples that included Japanese, Chinese or European ethicity, rs10453441 in WNT7B was strongly associated with both CC and axial length [91]. WNT7B rs10453441 is the only SNP associated with both traits of CCT and CC [92]. While these SNPs have no consistent and strong association with keratoconus, an exome sequencing analysis identified a *WNT10A* variant that was associated with corneal thickness and keratoconus [93]. In contrast, a GWAS on direct association of keratoconus patients and controls involving 222 patients and 3324 controls found no GWAS significance of associated gene variants [94].

### *7.2.3 Other approaches*

Apart from corneal morphologic features, a recent GWAS investigated corneal biomechanical properties in an European cohort of 6645 participants and 2384 participants from the British TwinsUK study [36]. The second stage of the association

study involved 752 keratoconus patients as compared with 974 British TwinsUK or 13,828 EPIC-Norfolk. The results showed a likely role in development of keratoconus with 5 associated loci in CH, *ANAPC1*, *ADAMTS8*, *ADAMTS17*, *ABCA6*, and *COL6A1* [36].

It is notable that there are a lot more keratoconus genes that are identified through studies on quantitative traits, especially central cornea thickness and cornea curvature, than on direct association with keratoconus against controls (**Figure 1**).
