Developments in Ocular Genetics: Annual Review.

ANNUAL REVIEW

Developments in Ocular Genetics: Annual Review Benjamin T. Whigham, BS and R. Rand Allingham, MD

Purpose: The purpose of this study was to summarize major developments in ocular genetics over the past year. Design: A literature review was performed for articles relating to the genetics of eye diseases and morphology. The search focused on articles published between September 15, 2011, and September 15, 2012. Methods: PubMed and Google Scholar search tools were used to search for ocular genetics articles in the desired date range. Results: Major advances have been reported in numerous areas including glaucoma, age-related macular degeneration, and keratoconus. Numerous novel associations have been identified through large genomewide association studies. In addition, numerous disease genes have been identified through next-generation sequencing technologies. Conclusions: Ocular genetics continues to advance at a rapid pace and benefit from new technologies. Numerous discoveries in the past year point toward areas for continued research. Key Words: eye, ocular, genetics (Asia-Pac J Ophthalmol 2013;2: 177Y186)

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he body of knowledge surrounding the genetics of ocular diseases has been growing at an ever-faster pace over the past decade. In the following pages, we summarize some of the most noteworthy discoveries published in the English language that have occurred over the past year, which for this review extends from September 15, 2011, through September 15, 2012. To provide useful context, several other advances that flank this period are also included. The speed and volume of information in ophthalmic genetics is so great that a full catalog is beyond the scope of this review. Rather, we have highlighted those discoveries with greatest impact in the field and emphasized current genetic research strategies, including genomewide association studies (GWAS) and next-generation sequencing technologies. There have been numerous GWAS and GWAS metaanalyses performed during this review interval (Table 1). These studies continue recent efforts that have uncovered multiple novel associations for a variety of ocular phenotypes, including central corneal thickness (CCT), corneal curvature, astigmatism, intraocular pressure (IOP), axial length, glaucoma of various types, keratoconus, and age-related macular degeneration (AMD).1Y11 Several of these GWAS were performed for traits or conditions that had been previously targeted by an earlier, smaller-sized GWAS. Others were the first to examine a particular disease or trait or were the first to examine a disease or trait in a different ethnic group or population. The

From the Department of Ophthalmology, Duke University Eye Center, Durham, NC. Received for publication December 13, 2012; accepted March 29, 2013. The authors have no funding or conflicts of interest to declare. Reprints: R. Rand Allingham, MD, DUMC 3802, Duke University Mailing Center, Durham, NC 27710. E-mail: [email protected]. Copyright * 2013 by Asia Pacific Academy of Ophthalmology ISSN: 2162-0989 DOI: 10.1097/APO.0b013e318294b837

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observed associations in these GWAS generally reflect the advantage of larger sample sizes, novel traits or conditions, and untested ethnic groups. The various associations will be discussed individually based on the disease process or trait of interest. Multiple reports highlight the impact of current DNA sequencing technologies on disease gene discovery and familybased studies (Table 2). Specifically, next-generation sequencing technology has enabled sequencing of genomic regions that were previously too large and expensive using traditional methods. This expanded capability has catalyzed the identification of causative mutations in numerous pedigree-based studies.12Y17 Identification of mutations has also benefitted from wholeexome sequencing. This technology allows for screening of all coding regions in the genome and has been used to identify many novel disease-causing mutations over the past year.18Y22 This review also includes articles that describe how recent advances in the field are contributing to the management of ocular disease. These contributions include genetic models of disease risk23,24; prediction of treatment response25; and new therapies.26 In addition, we have included articles that describe discoveries that inform how various clinical entities are related to each other.7,24,27 PubMed and Google Scholar search tools were used to search for ocular genetics articles published in English between September 15, 2011, and September 15, 2012. Both general and disease-specific search terms were used. Articles were considered if they described research relating to how genetic variation influences human eye disease or traits. The Human Genome Nomenclature Committee was used as a reference for gene symbols and names.28 For articles that used unofficial gene names, the Human Genome Nomenclature Committee symbol follows in parenthesis. Disease names were standardized to the Online Mendelian Inheritance in Man database.29 An initial search for ‘‘eye’’ + ‘‘gene’’ in PubMed returned 2000 articles. Additional search terms were added to focus on studies with greater focus on human eye disease and clinical entities. With successive searches that focused on individual diseases and structures of the eye, the number of articles was reduced. Ultimately, we summarized the findings of approximately 70 of the most important articles during our date range. Several additional articles were included to provide context to current discoveries. Articles are presented below grouped by disease.

OPEN-ANGLE GLAUCOMA Primary open-angle glaucoma (POAG) is a blinding disorder that is defined as characteristic glaucomatous optic atrophy with associated visual field loss in the absence of a secondary cause. Elevated IOP is a major risk factor, but it is not part of the definition. Primary open-angle glaucoma is a complex inherited disorder, and known genetic factors only account for a small fraction of disease risk and do not yet provide clinical utility.30 Still, research continues to identify new genetic factors that associate with both POAG and traits that increase POAG risk.

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TABLE 1. Genome-Wide Association Studies Over Past Year Year

Reference

Design

Population

Disease/Phenotype

Significant Associations*

2011 2012

55

GWAS meta-analysis GWAS

European US Siblings

10 known loci, FRK/COL10A1, VEGFA ARMS2/HTRA1

2012

11

GWAS meta-analysis

Asian

AMD Choroidal neovascularization subtype of AMD AL, high myopia

2012 2012 2012

8

GWAS GWAS GWAS meta-analysis

Asian European European

GWAS GWAS GWAS GWAS meta-analysis GWAS meta-analysis

Japanese European European Asian Asian + European

POAG IOP POAG + CCT CCT CCT, KTCN, POAG

GWAS GWAS meta-analysis GWAS GWAS

European Asian Asian European

KTCN Corneal astigmatism Corneal curvature Corneal curvature

2012 2012 2012 2012 2013

2012 2011 2011 2012

6

3 9

5 10 7 44 24

4 2 48 49

PACG POAG POAG, NTG

AL: ZC3H11A HM: ZC3H11A PLEKHA7, COLA11A1, PCMTD1/ST18 None POAG: CDKN2BAS, SIX1/SIX6* NTG: CDKN2BAS, 8q22 SIX6, CDKN2A-CDKN2B GAS7, TMCO1 NTM IBTK, CHSY1, 7q11.2, 9p23 CCT: 920 loci KTCN: FOXO1, FNDC3B POAG: FNDC3B RAB3GAP1 PDGFRA PDGFRA, FRAP1 (MTOR) PDGFRA, TRIM29

*Significant association in either discovery or replication cohort. AL indicates axial length; HM, high myopia; KTCN, keratoconus; NTG, normal tension glaucoma.

Over the past year, several GWAS have been performed on increasingly large data sets of POAG cases and controls. These studies have looked for association with POAG and associations with glaucoma-related endophenotypes, such as optic nerve cupping, IOP, and CCT. A GWAS performed in the United States found genetic loci that are associated with POAG and optic nerve degeneration, with some overlap between the loci. These associations were found through a combined analysis of 2 large GWAS studies: (1) the Glaucoma Genes and Environment (GLAUGEN) GWAS and (2) the National Eye Institute Glaucoma Human genetics collaBORation (NEIGHBOR) GWAS.9 Risk variants for POAG were found in 2 genomic regions: 9p21, containing CDKN2BAS and 14q23 located near the SIX1/SIX6 genes. These results were consistent with previous studies that reported strong association with vertical cup-disc ratio, a quantitative trait that is positively associated with glaucoma progression.31 The CDKN2BAS region was previously reported to be associated with POAG.1 The results of the GLAUGEN/ NEIGHBOR study have since been corroborated in a Japanese cohort, which found glaucoma associations in 9p21 and 14q23.5 In this study, the SIX1/SIX6 (14q23) association was analyzed with data from the 1000 Genomes Project,32 and a missense mutation in the SIX6 gene was identified as a potential functional variant.5 Subgroup analyses in these GWAS have examined the association of genetic variants with glaucoma endophenotypes.9 For example, in the NEIGHBOR/GLAUGEN study, unique associations were observed in a normal-tension glaucoma subgroup that were not seen among individuals with high-tension glaucoma. In the normal-tension subgroup, both CDKN2B (9p21) and the 8q22 loci were significant. Further study found that both the 9p21 and 8q22 loci were associated with the development of exfoliation glaucoma, a secondary form of glaucoma that develops in the setting of exfoliation syndrome. From these data it, appeared that the 9p21 and 8q22 loci may increase risk of glaucomatous optic nerve damage regardless of the specific insult.9

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Although the CDKN2B (9p21) and 8q22 loci appear to be related to optic nerve degeneration, other variants have been identified that associate with IOP. Elevated IOP is a heritable trait that is associated with glaucoma and optic nerve damage. In the past year, the first reported GWAS for IOP was performed in a set of European cohorts. It identified significant associations between IOP and variants located in GAS7 and TMCO1.10 TMCO1 had been associated to POAG in a previous GWAS,1 but the association between POAG and these loci observed in the IOP GWAS found only marginal association with POAG. Given the limited association to POAG in the IOP GWAS, it is possible that the GAS7 and TMCO1 variants are more strongly associated to IOP, a POAG endophenotype, than the POAG disease phenotype. Intraocular pressure is a highly complex trait with many contributing factors, so it is too early to draw specific conclusions at this time. Differences in genetic associations with traits or disease risk between populations and ethnicities are continuously being evaluated. As discussed earlier, a GWAS performed in the Japanese population confirmed several POAG-risk loci that were previously identified in Europeans. This study also observed association between POAG and the 7q31 region near CAV1-CAV2.5 The CAV1-CAV2 region has been implicated in POAG risk in the European and Chinese populations, but those variants were not polymorphic in Japanese populations.33 In the Japanese study, other single-nucleotide polymorphisms (SNPs) in the CAV1-CAV2 locus were associated with POAG at a comparable odds ratio to the original variants found in Europeans.5 However, the primary associated variant in Europeans was not associated with POAG in a Saudi cohort with POAG.34 Given these results, the CAV1-CAV2 locus appears to be associated with POAG in some, but perhaps not all, populations. A more complete understanding of the CAV1CAV2 locus will benefit from future study in other populations and larger data sets. Together, these studies highlight the value * 2013 Asia Pacific Academy of Ophthalmology



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Developments in Ocular Genetics

TABLE 2. Disease Gene Discovery Over the Past Year Year Study

Disease

2012

35Y38

2012

19

Complete CSNB

2011

20

Benign fleck retina

2012

13

Complete CSNB

2012

14

CRD, RP

2012

18

2011

12

2012

27

Fuchs endothelial corneal dystrophy (late-onset) Keratoconus, EDICT syndrome53,54 X-linked megalocornea

2012

72

Ocular coloboma

2011

15

Joubert syndrome

2012

16

Joubert syndrome

2012

21

Joubert syndrome

2012

75

Joubert Syndrome

2012

17

2012

22

Infantile cerebellar-retinal degeneration CRMCC

2012

77,78

LCA

Muscular dystrophy-dystroglycanopathy type A

Consanguineous Inheritance Pedigree With Disease?

Identified Gene(s)

Comments

Autosomal recessive Autosomal recessive Autosomal recessive Autosomal recessive Autosomal recessive Autosomal dominant Autosomal dominant X-linked recessive Autosomal dominant Autosomal recessive Autosomal recessive Autosomal recessive Autosomal recessive

Yes

WES

NMNAT1

Yes

WES

GPR179

Yes

Homozygosity mapping + WES

PLA2G5

No

GPR179

Yes

Genetic mapping in mice model Y NGS Y human mutation screen Homozygosity mapping Y NGS

No

Previously mapped Y NGS

LOXHD1

No

Previously mapped Y NGS

MIR184

No

Yes

Comparative genomic hybridization CHRDL1 of X chromosome Positional cloning Y ABCB6 direct sequencing Homozygosity mapping Y NGS TMEM237

Yes

Linkage mapping Y NGS

No

WES

C5ORF42

Yes

Linkage mapping Y direct sequencing

TMEM138

Autosomal recessive Autosomal recessive Autosomal recessive

Yes

Homozygosity mapping + WES

No

WES

CTC1

Yes

Complementation assay Y NGS; SNP array + WES

ISPD

No

C8orf37

CEP41

TMEM216 ACO2

NGS indicates next-generation sequencing; WES, whole-exome sequencing.

of studying diverse ethnic groups in the analysis of genetic associations.

ANGLE-CLOSURE GLAUCOMA AND AXIAL LENGTH A GWAS was performed to investigate primary angleclosure glaucoma (PACG), identifying several genetic loci that associate with disease.8 This GWAS was conducted in a large Asian cohort with follow-up in a second, large replication data set that included cohorts of European and Middle Eastern descent. The presence of these diverse groups allowed for comparisons between populations for associated SNPs. In total, strong association with 3 SNPs was identified. Two SNPs have plausible functional implications. All 3 SNPs appeared to have consistent effects between populations, suggesting that these variants influenced PACG risk in multiple ethnic groups.8 One SNP was found in PLEKHA7 and functions in paracellular permeability. A second risk variant was located in COL11A1, which has previously been associated with nonprogressive axial myopia. Therefore, it is plausible that variants of this gene may cause hyperopia with shortened axial length, a risk factor for PACG. However, it remains to be determined if COL11A1 contributes to PACG by affecting axial length or through other * 2013 Asia Pacific Academy of Ophthalmology

mechanisms.8 COL11A1 was not identified in a separate GWAS that targeted axial length in Asians.11

LEBER CONGENITAL AMAUROSIS Mutations in NMNAT1 were found to cause Leber congenital amaurosis (LCA).35Y38 This result was obtained from exome sequencing performed in multiple unrelated families. Among affected members, a combination of missense, nonsense, and read-through mutations were found in the NMNAT1 gene. This gene is located in a suspected LCA-associated region, the LCA9 locus, a region previously identified in a consanguineous Pakistani family. NMNAT1 is known to promote neuroprotection and is involved in the biosynthesis of nicotinamide adenine dinucleotide (NAD+). Fibroblasts from an LCA proband with an NMNAT1 mutation had 16% less NAD+ than wild-type controls.37 In addition, NAD+ levels in fibroblasts carrying a missense mutation in NMNAT1 did not increase after exposure to nicotinic acid, a substrate that increases NAD+ by more than 50% in wild-type fibroblasts.37 These results were mirrored in red blood cells, where an individual carrying 2 mutated copies of NMNAT1 had significantly lower concentrations of NAD+ in his red blood cells compared with his mother with a single mutated copy.38 Based www.apjo.org


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on these results and studies that indicate a neuroprotective role for NMNAT1 in the mouse retina, a possible therapy is to target reduced levels of NAD+ in NMNAT1-associated LCA.35,36,38 In the future, NMNAT1-associated LCA may be treated with gene therapy, which is already being applied to LCA caused by mutations in previously identified genes. One is RPE65, which is a well-known cause of LCA and contains 80 documented pathogenic mutations spread across multiple ethnicities.39 Its gene product is found in the retinal pigment epithelium (RPE) and red-green cones, where it has a role in regeneration of photopigment.40 In carriers of RPE65 mutations, gene therapy has produced safe and stable visual improvement after viral delivery of the RPE65 gene. Improvement and safety have been documented out to 3 years.26 Patients also benefitted from a second round of gene therapy in the fellow, previously untreated eye.41 Treatment of the fellow eye was given between 1.7 and 3.3 years after the original injection. The result suggests that immunity is not induced by the original treatment, which would be a potential barrier to further treatment. Advances have also been reported in animal trials addressing LCA caused by mutations in AIPL1. This type of LCA is characterized by severely reduced visual acuity that manifest in the first decade of life. Currently, viral delivery of AIPL1 is being optimized in a murine model of AIPL1 deficiency.42 There are more than 15 genes that contain mutations that cause LCA, which has made comprehensive genetic screening for LCA difficult. Toward improving LCA-mutations screens, a massively parallel sequencing technique has been developed to test for all known LCA mutations.43 Of interest will be how effective these approaches will be and how these tools will assist in providing more reliable diagnostic and prognostic information for patients and families.

CENTRAL CORNEAL THICKNESS AND ITS RELATIONSHIP TO KERATOCONUS AND GLAUCOMA Central corneal thickness is a quantitative eye metric that is highly heritable.44 Several GWAS studies in the past several years have identified CCT-associated loci in both Asian and European cohorts.44Y47 Most recently, a large meta-analysis of GWAS data from diverse Asian and European cohorts identified an additional 16 loci, bringing the total to 27 CCT-associated loci. Most of these variants have had consistent impact on CCT in both Asian and European samples. Collectively, these variants appear to account for 8.3% of CCT variance in Europeans and 7% in Asians, assuming additive variance.24 The same meta-analysis that investigated CCT also examined whether the CCT-associated variants were also associated with either keratoconus or POAG because both are associated with lower CCT. For keratoconus, 11 of the 27 CCTassociated loci were nominally associated, and 6 were significant. In 5 of the 6 significant loci, the CCT-reducing allele was associated with increased risk of keratoconus. The one exception was for a locus containing ZNF469, for which the allele associated with increased CCT was also associated with keratoconus. The authors speculate that this observation may result from ZNF469 either representing a false positive or having pleiotropic actions in the eye.24 In the case of POAG, only 1 CCTassociated locus, FNDC3B, was found to be significant from the meta-analysis of CCT-associated loci. Interestingly, the FNDC3B alleleYassociated low CCT was also associated with decreased POAG risk.24 Another gene, NTM, appears to confer POAG risk and influence CCT based on a separate study of samples from the

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NEIGHBOR and the GLAUGEN consortia.7 This study, like the previously mentioned CCT meta-analysis, identified common genetic factors shared between CCT variation and POAG risk. However, in both this study and the meta-analysis, most of CCT-associated variants were not associated with POAG. Because the data set was large, this negative result suggests that the effect size of genetic variants for CCT on POAG risk may be small or more complex than initially believed.7

CORNEAL CURVATURE AND ASTIGMATISM Several GWAS studies have searched for genes that influence corneal curvature, which is defined as the average radius of corneal curvature at the horizontal and vertical meridians. The first GWAS was performed in a diverse Asian cohort that included Indian, Chinese, and Malay individuals. Significant associations with variants in the genes FRAP1 (MTOR) and PDGFRA were found.48 More recently, these 2 loci were tested for association to corneal curvature in an Australian cohort of Northern European descent. In this group, FRAP1 (MTOR) was not associated with corneal curvature, suggesting that it might be an Asian-specific variant. In contrast, PDGFRA was associated with corneal curvature in the Australian population, with a minor-allele frequency that is similar to the original Asian GWAS result.49 PDGFRA variants have also been associated with corneal astigmatism. This result comes from a second GWAS in a diverse Asian cohort that included both Indian and Chinese samples.2 Interestingly, corneal astigmatism was minimally correlated with corneal curvature in the tested cohort, but both it and corneal curvature were associated with the gene PDGFRA, which suggests that PDGFRA exerts pleiotropic effects on corneal morphology.2 Of interest, PDGFRA was also previously implicated in refraction and myopia based on a significant association in Europeans and a nominal association in African Americans.50,51 However, no association was found between PDGFRA and myopia or refractive status in this Asian cohort.2

MEGALOCORNEA More than 20 years ago, X-linked megalocornea was linked to the region Xq12-q26. Within this region, however, no gene had been identified. Work using comparative genomic hybridization has now identified causative mutations in the CHRDL1 gene.27 Given that CHDRL1 mutations cause megalocornea, it might be expected that variants near this gene would influence general corneal diameter. However, CHRDL1 variants were not associated with corneal diameter outside of the disease state. This result suggests that this is not a quantitative trait locus; rather, it may be that functional CHRDL1 is necessary for normal anterior segment development.27 Mutations in CHRDL1 are also associated with several central nervous system findings that include decreased white matter volume and increased verbal intelligence.27

FUCHS ENDOTHELIAL CORNEAL DYSTROPHY Progress has been made toward understanding genetic architecture of Fuchs endothelial corneal dystrophy (FECD), a disorder of the corneal endothelium that is the second leading indication for corneal transplantation surgery. Fuchs endothelial corneal dystrophy has both early- and late-onset forms. Some early-onset cases are caused by mutations in COL8A2. The late-onset form appears to result from rare mutations in several other genes that include SLC4A11 and TCF8 (ZEB1).18 In addition to these genes, several chromosomal loci have been identified for FECD. In the past year, one of these loci located * 2013 Asia Pacific Academy of Ophthalmology



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on chromosome 18 was sequenced using next-generation sequencing technology, leading to identification of a causal mutation in the LOXHD1 gene.18 This result was obtained by using exome-capture technology to sequence all coding sequence among members of an affected family. This study highlights the impact of next-generation sequencing technology in family-based studies where causative genetic variants can be successfully identified from small numbers of affected persons or families, which was frequently not possible in the past using conventional genetic linkage studies. More support for LOXHD1 in FECD was obtained by screening for LOXHD1 mutations in patients with sporadic cases of FECD.18 In 200 FECD cases, 15 heterozygous missense mutations were found that did not occur in more than 800 control chromosomes. In addition, nonsynonymous variants that were found in both cases and controls were significantly more common in the cases.18 Efforts are underway to determine the role of LOXHD1 in the pathogenesis of FECD. It is known that the LOXHD1 gene is expressed in both corneal epithelial and endothelial cells and that mutation carriers have abnormal distribution of LOXHD1 protein in the endothelium and Descemet membrane.18 Mutations in LOXHD1 have also been found to cause a form of autosomal recessive deafness, which might be related to its role in FECD. Interestingly, several of LOXHD1 mutations also demonstrate both FECD and hearing loss.18 COL8A2, a previously identified FECD gene, is now being studied in a mouse model. The mouse model contains mutant COL8A2 alleles that result in Q455K, a known pathogenic human mutation. The mouse phenotype was found to be highly similar to the human FECD phenotype, with having both gradual loss of endothelial cells and appearance of basement membrane guttae.52 Importantly, these mice do not have evidence of a second eye disease, posterior polymorphous corneal dystrophy, that can occur in a subset of humans with Q445K. Of interest now is the molecular process that causes the FECD phenotype in these mice. Initial work found their corneal endothelial cells to have dilated endoplasmic reticulum and activation of unfolded protein response, which can lead to apoptosis.52 Investigations using this model will assist the study of FECD, which has been dependent on corneal transplant tissue obtained from patients in the late stages of disease. This research offers the opportunity to develop treatment approaches for preclinical disease in a tissue that is readily accessible and to reduce or eliminate corneal transplant surgery, the only management option currently available for this common blinding disease.52

KERATOCONUS Keratoconus is a noninflammatory corneal dystrophy characterized by corneal thinning and deformation that often causes reduction in visual acuity caused by marked refractive error and corneal stromal opacity. Previous linkage studies have identified several genomic regions that are associated with keratoconus. To date, there have been no genes identified within these loci.4 The first keratoconus GWAS, reported in a European cohort, found an SNP in the region of RAB3GAP1 that, although not significant at a genome-wide level, is associated with keratoconus and was replicated in 2 different patient cohorts.4 Mutations in RAB3GAP1 cause Warburg Micro syndrome, a condition characterized by neurological and ocular abnormalities, including microcornea.4 Several other variants were observed in the original discovery cohort but were not replicated. It was suggested that because of the relative rarity of * 2013 Asia Pacific Academy of Ophthalmology


keratoconus and the population structure of the data sets, there was insufficient power to detect these associations.4 A novel keratoconus mutation was identified in the MIR184 microRNA, located in the chromosome 15q22-25 region.12 This region was previously linked to autosomal dominant keratoconus and early-onset anterior polar cataracts in a family of Northern European descent. The association in this region was then linked to a specific mutation through nextgeneration sequencing technology. In this case, candidate variants were identified based on their location inside known gene sequences and then filtered according to projected pathogenicity using current databases. Ultimately, 3 candidate variants were identified, with the 1 falling in MIR184 being considered the strongest biological candidate. MIR184 is the most highly expressed microRNA in corneal and lens epithelial tissues. This expression fits well with the keratoconus and anterior polar cataracts that were observed in the affected family. In vitro mutated MIR184 failed to suppress MIR205, a major target of MIR184.12 This loss of function suggests a functional mechanism for this variant of familial keratoconus as well as a future therapeutic target. This mutation in MIR184 has also been shown to cause a familial developmental disease termed EDICT syndrome (endothelial dystrophy, iris hypoplasia, congenital cataracts, and stromal thinning).53 Interestingly, EDICT syndrome and the MIR184-related keratoconus appear to share a causative mutation but have clinically distinct features. EDICT syndrome is characterized by multiple iris abnormalities that were not reported in the keratoconus family. EDICT syndrome is characterized by uniform and nonectatic stromal thinning, which is distinct from keratoconus. One similarity was anterior polar cataracts, which was seen in both EDICT and keratoconus families.53 The authors considered the possibility of other functional variants but found no second mutation in the MIR184 region.53 Accepting the current reports of these phenotypes, it is unclear why there is phenotypic variance. From available evidence, it appears that the MIR184 microRNA might have diverse phenotypic effects in the eye. It may also affect refraction, given a previous GWAS for myopia that found an association near MIR184.12 Further study of mutations in and around the MIR184 locus will help clarify the pathogenic mechanisms of these related phenotypes.54

AGE-RELATED MACULAR DEGENERATION Age-related macular degeneration is a common blinding condition that is influenced by both environmental factors, such as smoking and obesity, and numerous genetic factors. Many genetic associations have been identified through GWAS data. A large meta-analysis of previous GWAS data has confirmed 10 previously described genetic loci and identified 2 additional loci that are near FRK/COL10A1 and VEGFA.55 Altogether, these known risk variants accounted for approximately 39% of the risk for advanced AMD. Likely, there are additional genetic factors that contribute to AMD, as from previous twin studies the heritability of AMD is approximately 71%. Future studies with increased sample size and power are expected to identify loci of more modest impact.55 Eventually, a more complete understanding of risk factors may have diagnostic utility. Variants in complement factor H (CFH) have been associated with AMD in multiple studies and populations. A study has now identified a highly penetrant mutation in CFH that was found to associate with both AMD and various glomerulopathies including atypical hemolytic uremic syndrome.56 www.apjo.org


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This result suggests that AMD might be more closely related to other complement-pathwayYrelated diseases than what was previously suspected and provides further support for CFH variants as AMD risk factors.56 The clinical significance of another CFH variant, Y402H, is a current area of research. In a cohort study over 20 years, individuals who carry the Y402H variant were found to be more likely to develop AMD and progress to more advanced stages.57 The effect of the Y402H variant on response to treatment for neovascular AMD was the subject of another large meta-analysis. This study looked at response to both antivascular endothelial growth factor (VEGF) and photodynamic therapies observed in 10 clinical studies. The Y402H variant was found to reduce response to anti-VEGF therapy by 1.6-fold in individuals homozygous for the risk allele (C allele) compared with those homozygous for the ancestral allele. Interestingly, risk of treatment failure in heterozygotes was not significantly higher than low-risk homozygotes.25 The effect on treatment response was most notable for anti-VEGF treatments. Another study determined that associations between CFH variants and early-onset AMD were influenced by the age of study participants. Specifically, they observed that high-risk CFH variants actually appeared protective in younger patients but reversed to a positive association with AMD in older age groups.58 In addition, the prevalence of low-risk homozygotes rose with each increasing age cohort. From these results, the authors concluded that associations between AMD and CFH variants must be considered in context with the tested age group. The reasons for this age dependence are unclear, but possible explanations include random chance, differences between AMD as seen in younger and older patients despite similar clinical presentations, and a survivorship bias caused by studies that are limited to the eyes of subjects who are still living and healthy enough to participate in the studies.58 Given the reported age dependence of AMD association with CFH variants, caution was advised when interpreting these data.58 Advances have been made on other AMD-related genes. PON1 has antioxidant functions and has been previously identified as an AMD-risk gene. Previous work has focused on coding region variants, with inconsistent and sometimes reversed associations observed among European and Japanese cohorts. Now, putative regulatory variants upstream of PON1 have been associated with neovascular AMD.59 Functional studies done in cultured human cell lines support a role for some of these variants in gene expression, albeit in a tissue-specific manner.59 The association of an antioxidant gene such as PON1 with neovascular AMD supports a role of oxidative stress in the disease process.59 Although progress continues into the genetics of AMD, current guidelines recommend that genetic testing should be reserved for research settings only.30 The American Academy of Ophthalmology recommends withholding genetic testing from clinical settings until there is sufficient evidence that it would change disease management. Progress has continued toward understanding the genetics of AMD and may eventually provide novel mechanisms for treatment or diagnosis of AMD.

RETINITIS PIGMENTOSA Retinitis pigmentosa (RP) is a genetically heterogenous group of diseases that affect both retinal pigment epithelial cells and photoreceptors. A promising treatment for RP is gene therapy, especially given the success of gene therapy for LCA caused by mutations in RPE65. Gene therapy is currently being

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pursued for multiple forms of RP. Gene therapy successfully improved vision in a canine model of X-linked RP caused by mutations in RPGR.60 Mutations in this gene account for more than 70% of RP cases in humans. However, before a human trial is initiated, additional studies are needed in large animal models including a larger sample size of canines carrying mutations in RPGR.60 Gene therapy is also being pursued for RP caused by CNGB1 mutations. In a CNGB1-deficient mouse strain, subretinal delivery of CNGB1a improved vision-guided behavior and delayed retinal degeneration.61 For another type of RP that results from CCDC66 mutations, a new knockout mouse strain has been generated. This knockout mouse model exhibits a progressive retinal atrophy and is hoped to facilitate research into new therapies.62 In addition to these animal models, other research has focused on the molecular roles of RP genes, which has produced numerous insights into the function of RPrelevant genes.63Y65

CONGENITAL RETINOPATHIES Congenital stationary night blindness (CSNB) is caused by mutations in several genes. A subset of complete CSNB (cCSNB) was found to result from mutations in the gene GPR179. This result was obtained by separate groups using 2 distinct methods.13,19 The first group identified a mouse phenotype consistent with CSNB, mapped the genetic locus, and sequenced the region using next-generation sequencing. This approach identified a mutation in GPR179. These investigators sequenced GPR179 in 44 patients with cCSNB, finding 2 patients to carry deactivating mutations on both copies of GPR179.13 A second group reached this conclusion by performing full-exome sequencing in patients lacking cCSNB mutations.19 The exome sequencing revealed 2 homozygous mutations in GPR179, one in the affected members of a consanguineous family and another in an unrelated patient. Efforts are underway to characterize the role of GPR179 protein in cCSNB. In a zebrafish model, knockdown of the GPR179 protein caused a reduction in the amplitude of the electroretinogram b-wave. Staining for the GPR179 protein was absent in the retina of mice with a mutation in GPR179 that results in cCSNB, whereas wild-type mice stained positive for GPR179.13 The subcellular localization of GPR179 in healthy retinas is also of interest because this should help determine its role in the transduction of visual signals in low-light conditions. Current studies suggest that GPR179 is expressed in the outer-plexiform layer of the retina. However, more refined localization studies are ongoing, with the cell type and subcellular localization still under investigation. Current reports suggest that GPR179 may localize to depolarizing bipolar cells such as previously known cCSNB genes, but its presence in other cell types, such as Mu¨ller cells, appear possible.13,19 The localization and function of GPR179 is important to determine how it fits with previously identified cCSNB genes in the retina and how these genes function in the transmission of visual signals. Work with early-onset cone-rod dystrophy (CRD) and RP has identified pathogenic mutations in a ciliary-expressed gene, C8ORF37.14 This gene was identified in a consanguineous family affected by an autosomal-recessive form of CRD and RP. Homozygosity mapping narrowed a causative mutation to a 46 megabase region, which was analyzed with next-generation sequencing. Further C8ORF37 mutations were found in other families. Immunohistochemical studies have localized C8ORF37 to the base of the primary cilium of RPE cells. This result is * 2013 Asia Pacific Academy of Ophthalmology



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consistent with an important role for ciliary process dysfunction in retinal dystrophies.14 The strategy of analyzing consanguineous families with next-generation sequencing technologies was also used to identify mutations in PLA2G5 that cause familial benign fleck retina.20 Specifically, 2 consanguineous families were studied. Exome sequencing was used to identify all of the coding variants in each family member. To filter this, each genome was mapped for regions of homozygosity that enabled identification of the homozygous candidate disease-causing mutations. Variants in these regions were filtered using data from the 1000 Genomes data set32; in this manner a small subset of candidate variants were identified including 1 missense change in PLA2G5. PLA2G5 appeared to be the most plausible candidate gene, and Sanger sequencing of PLA2G5 in 4 unrelated patients found 3 patients that were either homozygous for a PLA2G5 mutation or a compound heterozygote. In all, 4 mutations in PLA2G5 were identified, and these mutations appear to cause autosomal recessive benign fleck syndrome.20 Research into retinal dystrophies has led to advances in understanding how molecular pathways influence retinal pathology, and how these networks can be interrogated to understand the genetics of eye disease. This was explored in a study of the transcription factor NRL, which determines rod versus cone fate.66 In the study, several genome-wide techniques were used to study NRL interactions, including 2 platforms of chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) and global expression profiling. Three hundred genes were found to interact with NRL. Of those identified, 22 were associated with retinal dystrophies, and 95 were in genetic loci associated with retinal dystrophies.66 Knockdown of NRL target genes caused rod photoreceptor death or dysfunction for 16 genes that interacted with NRL. From this study, KDM5B was also identified as a regulator of rodexpressed genes.66 Encouragingly, the 2 different ChIP-Seq platforms used in this study achieved similar results despite unique chemistries, supporting ChIP-Seq technology in the study of transcription factors.66

BASIC RESEARCH INTO RETINA AND RETINAL PIGMENT EPITHELIUM For many of the articles discussed in this review, the primary focus was on known clinical entities, whether it was a rare congenital disorder or a common later-onset form of disease. In contrast, other efforts have focused predominately on understanding fundamental aspects of eye physiology. Some of these studies point to the importance of particular genes. For instance, one study has identified MEGF10 and MEGF11 as necessary for mosaic spacing of retinal neurons.67 Another has uncovered the roles of the MTOR and STAT3 pathways in optic nerve regeneration.68 In yet another, the PAX6 regulatory circuit has been investigated for its role in the generation of both retinal and retinal pigment epithelial cells.69 This expanding body of knowledge has great potential to fuel advances in treatment by identifying critical, shared molecular pathways. This could provide the foundation for treatment of multiple forms of 1 disease or several diseases that share a common disease pathway.

AXIAL LENGTH AND HIGH MYOPIA Axial length of the eye is an important quantitative trait that is a critical component of refraction. Ocular elongation is the most common cause of myopia or near-sightedness, a leading cause of visual impairment in the developed world. Axial length is highly heritable and can be precisely measured. * 2013 Asia Pacific Academy of Ophthalmology


Therefore, identifying genetic factors that control axial length can aid in the study of genes that lead to the development of myopia.11 A meta-analysis of 3 GWAS in Asian subjects has found that variants in region 1q41 influence ocular axial length. This was then confirmed in 2 Japanese cohorts.11 Not only did the region associate with axial length, but the minor allele of a regional SNP also associated with decreased risk of high myopia (odds ratio, 0.75). This SNP was located in the gene ZC3H11B that encodes a zinc finger protein, a class of proteins implicated in high myopia.70 A study to examine axial length as a quantitative trait identified multiple variants that associate with high myopia in the 1q41 locus. This region was previously reported as a locus for refraction in the Beaver Dam Eye Study through microsatellite markers. However, this association did not survive reanalysis with SNP markers in this study.71 An Asian GWAS identified a locus on chromosome 1q41 that is associated with axial length through association with refraction (spherical equivalent). There was a weaker but still significant association in 2 Chinese GWAS cohorts but not in a Malay GWAS cohort. This observation agrees with previous data that suggest that axial length and spherical equivalents are less strongly correlated in the Malay cohort possibly because of a higher burden of lens opacity.11 Although the 1q41 locus appears to be significant for axial length and high myopia in Asians, studies in other populations will also be important to determine if it has consistent effects in multiple populations. Asian populations are known to have higher rates of myopia than Europeans. Furthermore, in East Asian children, axial length is a greater determinant of refraction than in European children.11 In addition to these clinical differences between Asians and other groups, it also appears that allele frequencies in 1q41 vary widely between populations. The effects of 1q41 in other populations will require further study.11

OCULAR COLOBOMA AND OCULAR MALFORMATION Ocular colobomas result from unsuccessful fusion of the optic fissure during embryonic development. Approximately 50% of coloboma cases are explained by known mutations.72 These mutations are found in a diverse set of genes. The past year has seen multiple advances in understanding of these mechanisms.73,74 A newly identified disease gene, ABCB6, was found to harbor coloboma-causing mutations.72 The discovery of ABCB6 resulted from work in a Chinese family that carried an autosomal dominant form of ocular coloboma. The relevant mutations were located through genome-wide linkage analysis followed by direct sequencing of 76 genes in a candidate region. More mutations were confirmed in several sporadic Indian cases, and further work in zebrafish suggests that pathogenic mutations reduce the function of ABCB6.72

SYSTEMIC DISEASES WITH OCULAR INVOLVEMENT Numerous genetic diseases with systemic involvement cause pathology in the eye. Several of these diseases have been traced to specific genetic mutations in the past year. One example is Joubert syndrome, a systemic ciliopathy with numerous physical findings that include RP. Over the past year, mutations in multiple genes have been identified for Joubert syndrome.15,16,21,75,76 These genes include TMEM138, TMEM216, TMEM237, CEP41, TCTN1, and C5ORF42. Most of these genes were identified through studies that examined 1 or multiple families. Techniques used to identify these genes included www.apjo.org


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various combinations homozygosity mapping, genome-wide exome sequencing, targeted next-generation sequencing, and direct sequencing to rule out known mutations and identify novel mutations. The protein product for one of the genes, CEP41, was localized to the cilia and centrioles of retinal pigment epithelial cells.16 These findings should yield insights into how ciliary dysfunction contributes to retinal dystrophies such as RP. Other systemic disorders with retinal involvement were also studied during the past year. It was found that cerebroretinal microangiopathy with calcifications and cysts (CRMCC), or Coats plus disease, can result from mutations in CTC1.22 This syndrome involves retinal telangiectasias and exudates along with other manifestations in the central nervous, skeletal, and gastrointestinal systems. Interestingly, the CTC1 mutations were identified in nonconsanguineous families. Because of the lack of consanguinity in these families, an early attempt at homozygosity mapping failed to generate useful data. Ultimately, a genome-wide exome-sequencing strategy was used to identify disease variants. Consistent with the lack of consanguinity, the individuals with CRMCC were found to be compound heterozygotes, meaning that they carried unique mutations on each of their 2 copies of CTC1.22 Mutations in another gene, ISPD, have been found to cause muscular dystrophy-dystroglycanopathy type A (MDDGA), a congenital disease that is characterized by muscle dysfunction, central nervous system malformation, and eye manifestations. Severe cases of this disease are often referred to as Walker-Warburg syndrome. Its eye manifestations include iridocorneal malformation, membrane-like structure of the lens, and retinal dysplasia. Muscular dystrophy-dystroglycanopathy type A had previously been traced to mutations in 6 genes that together explained half of all cases.77 In the past year, 2 groups have independently identified causative mutations in the ISPD gene in a subset of previously unexplained MDDGA cases. One group examined a cohort of unexplained cases with SNP arrays, which identified 2 patients who were homozygous for deletions that involved the ISPD gene.78 Another group created a complementation assay from the fibroblasts of MDDGA patients who lacked any known mutations. From the results of this assay, linkage analysis, and sequencing, the group identified mutations in ISPD.77 Like other MDDGA genes, ISPD appears necessary for functional glycosylation of >-dystroglycan.77,78 Further research into this and other MDDGA genes will hopefully reveal more about the disease mechanism. In addition, this finding should benefit diagnostic screening because ISPD mutations accounted for 10% of cases in 1 MDDGA cohort.78 Progress has also been reported in a congenital disorder termed infantile cerebellar-retinal degeneration. Several cases were traced to a mutated gene involved in the citric acid cycle.17 The study examined 8 cases from 2 unrelated families with common findings. Individuals experienced a continuous deterioration in visual tracking caused by optic nerve atrophy and severe retinal dystrophy determined by reduced electroretinogram responses. Homozygosity mapping was first used to determine shared regions of homozygosity among the affected individuals. Regions of homozygosity that appeared specific to affected cases were then sequenced with exome sequencing. This revealed homozygous mutations in ACO2, encoding mitochondrial aconitase involved in the citric acid cycle, which yielded new insight into the disease and how it causes anterior segment malformations and retinal dystrophy.17

NEW TECHNOLOGIES AND RESOURCES Review of the studies highlights the advantages presented by next-generation sequencing technologies. In several studies,

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homozygosity mapping was used to narrow regions of interest that would otherwise have been too large to sequence using traditional sequencing methods. Next-generation technologies allow larger regions of the genome to be screened quickly and are frequently used to screen loci that are too large for conventional sequencing.12Y17 Exome sequencing is now commonly used to screen for rare variants in coding regions of the genome.18Y22 This technology is increasingly being used to identify coding variants that contribute to disease phenotypes. Future work will benefit from further advances in sequencing technology. A recent report describes the capacity for accurate whole-genome sequencing and haplotyping from 10 to 20 human cells.79 Beyond just identifying alleles, this method used a long fragment read technology that revealed the context (haplotype) of every allele. It also coupled this with an extremely low error rate of approximately 1 incorrect base pair per 10 megabases.79 With the rapid improvement in sequencing technologies, the capacity to look for clinically relevant variants will greatly increase and enter the domain of clinical care. In addition to new technologies, genetic research has been accelerated by accumulated data regarding linkage and populationspecific allele frequencies. Multiple studies here combined freshly obtained data with known allele frequencies and linkage patterns to analyze regions regarding suspected variation.5,11,20 As genetic research progresses, it will be of great interest to see how accumulated knowledge can be organized to aid in new analyses and discovery.

CONCLUSIONS The past year has witnessed numerous advances in ocular genetics. Many of the reported discoveries have been enabled by our increasing ability to extract information from the human genome. As genetic information becomes less expensive and more widely available, research related to ocular genetics will continue its rapid advance, fueled by expanding and newer technologies. Also important, genetic studies have collectively sampled an increasing portion of human diversity. Work across multiple ethnic groups has proven essential for reasonable interpretation of identified variants. Given these successes, current work continues to seek out new populations for study. In conclusion, numerous discoveries over the past year have expanded our understanding of ocular genetics and point to new and continued areas of research for future study.

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Developments in Ocular Genetics: 2013 Annual Review.

Genetics and ocular disorders: a focused review.

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New developments in the genetics of bipolar disorder.

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Prostate Cancer Genetics: A Review.

Current developments in breeding, genetics, genomics, and molecular biology applied to sweetpotato improvement.

New developments in the genetics, pathogenesis, and therapy of IgA nephropathy.

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Posterior Capsule Opacification After Phacoemulsification: Annual Review.

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Recent developments in age-related macular degeneration: a review.

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