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Clinical and Experimental Ophthalmology 2014; 42: 13–24 doi: 10.1111/ceo.12152

Review Genomics and anterior segment dysgenesis: a review Yoko A Ito PhD and Michael A Walter PhD Department of Medical Genetics, University of Alberta, Edmonton, Canada

ABSTRACT Anterior segment dysgenesis refers to a spectrum of disorders affecting structures in the anterior segment of the eye including the iris, cornea and trabecular meshwork. Approximately 50% of patients with anterior segment dysgenesis develop glaucoma. Traditional genetic methods using linkage analysis and familybased studies have identified numerous diseasecausing genes such as PAX6, FOXC1 and PITX2. Despite these advances, phenotypic and genotypic heterogeneity pose continuing challenges to understand the mechanisms underlying the complexity of anterior segment dysgenesis disorders. Genomic methods, such as genome-wide association studies, are potentially an effective tool to understand anterior segment dysgenesis and the individual susceptibility to the development of glaucoma. In this review, we provide the rationale, as well as the challenges, to utilizing genomic methods to examine anterior segment dysgenesis disorders. Key words: Axenfeld–Rieger syndrome, genome-wide association, linkage analysis, Peters anomaly, phenotypic and genotypic heterogeneity.

INTRODUCTION Anterior segment dysgenesis encompasses a group of developmental disorders that affect structures in the anterior segment of the eye. The anterior segment refers to the portion of the eye in front of the vitreous surface (Fig. 1). The cornea, iris and the lens are all

part of the anterior segment of the eye. The anterior segment also consists of the anterior chamber and posterior chamber spaces that are separated by the iris. The chambers are filled with aqueous humour fluid that provides nutrients to tissues in the anterior segment, including the avascular cornea and lens. The aqueous humour is produced by the ciliary body and partially drained by the trabecular meshwork and Schlemm’s canal. These structures that are involved in regulating the aqueous humour flow pathway are also structures that can be affected in patients with anterior segment dysgenesis. Dysregulation of aqueous humour flow can lead to elevated intraocular pressure (IOP), which is a major risk factor for developing glaucoma. Glaucoma is a progressively blinding condition that results from the death of retinal ganglion cells located in the posterior segment of the eye (Fig. 1). In general, approximately 50% of individuals with anterior segment dysgenesis develop glaucoma. In the normal population, glaucoma is an agerelated disease with significantly increased incidence after age 40.1,2 However, patients with anterior segment dysgenesis tend to develop earlier-onset glaucoma.3 Phenotypic and genotypic heterogeneity, plus overlap related to anterior segment dysgenesis clinical presentations, have created challenges for proper diagnosis and classification of disease. Several attempts have been made to categorize anterior segment dysgenesis to simplify diagnosis.4–7 However, a system of classification based solely on phenotype, genetics or affected cell type during development appears to be insufficient to fully describe the complexity of anterior segment dysgenesis.

䊏 Correspondence: Dr Michael A Walter, Department of Medical Genetics, University of Alberta, 8-32 Medical Sciences Building, Edmonton, AB T6G2H7, Canada. E-mail: [email protected] Received 20 April 2013; accepted 5 May 2013. Competing/conflicts of interest: No stated conflict of interest. Funding sources: Y.A.I. is supported by the Sir Frederick Banting and Dr. Charles Best Canada Graduate Scholarship provided by the Canadian Institutes of Health Research. © 2013 Royal Australian and New Zealand College of Ophthalmologists

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ANTERIOR SEGMENT

Cornea Schlemm’s canal

Aqueous humor

ANTERIOR CHAMBER

Trabecular Meshwork

Iris

POSTERIOR CHAMBER

Lens

POSTERIOR SEGMENT

Ciliary body

Suspensory ligaments

Vitreous humor

Figure 1. Schematic diagram of eye. The eye is divided into the anterior segment and the posterior segment. The anterior segment consists of the anterior and posterior chambers, which are both filled with aqueous humour. Structures located in the anterior segment of the eye, including the cornea, iris, trabecular meshwork and Schlemm’s canal, are affected in the anterior segment dysgeneis spectrum of disorders.

Retina (retinal ganglion cells)

Optic nerve

Recent advances in technology have made it possible to examine the genome as a whole and thus, the contribution of the genomic architecture to disease. Genome-wide association (GWA) methods have been used to uncover genetic factors that cause complex diseases,8,9 which were not possible to identify using conventional linkage and familybased genetic methods. Genomic approaches can also be utilized to further enhance our understanding of the complicated etiology of anterior segment dysgenesis. At present, no GWA studies for anterior segment dysgenesis disorders are published. The use of GWA approaches is an exciting, potentially untapped strategy to understand the complexity of anterior segment dysgenesis. In this review, a general overview of the phenotypes and specific disorders that are part of the anterior segment dysgenesis spectrum will be outlined. Then, the complex nature of anterior segment dysgenesis will be examined through various examples of phenotypic and genetic heterogeneity observed in patients. The various uses of genomic methods will be examined. Finally, the challenges of applying GWA methods to the analysis of anterior segment dysgenesis will be explored. The potential limitations of GWA in examining complex diseases in general, including anterior segment dysgenesis, will be considered.

INTRODUCTION TO ANTERIOR SEGMENT DYSGENESIS The current section provides an overview on the diseases and phenotypes that are part of the anterior segment dysgenesis spectrum of disorders. First, syndromes that are associated with both ocular and

systemic phenotypes will be presented. Then, disorders that are primarily associated with just ocular phenotypes will be outlined. Finally, disorders that have been associated with anterior segment dysgenesis, but primarily present with non-ocular phenotypes, will be described. The goal of this section is not to provide a classification system for anterior segment disorders (for such a classification system, refer to previous studies4–7,10). Rather, through this overview, we will highlight the complex nature of anterior segment dysgenesis both from a phenotypic and genetic perspective.

Syndromes associated with both ocular and systemic anomalies Axenfeld–Rieger syndrome Axenfeld–Rieger Syndrome (ARS) is a rare autosomal dominant disorder that is part of the anterior segment dysgenesis group of developmental disorders. As many of the ARS phenotypes were independently described by various researchers and clinicians, many terms have been used to diagnose individuals with this disease (Table 1). ARS is a phenotypically heterogeneous disorder that is characterized by both ocular and systemic abnormalities. ARS patients exhibit a spectrum of anterior segment abnormalities that are highly penetrant, including iris hypoplasia, corectopia, polycoria, posterior embryotoxon (i.e. anteriorly displaced Schwalbe’s line), and peripheral anterior synechiae (i.e. iris strands that extend and attach to the trabecular meshwork) (Figs 2b,3b). In addition, ARS patients may present with various systemic abnormalities including facial dysmorphisms (e.g. hypertelorism,

© 2013 Royal Australian and New Zealand College of Ophthalmologists

Genomics and anterior segment dysgenesis Table 1. Summary of terms used to describe various anterior segment dysgenesis disorders Disease

Other terms used to describe disease

Axenfeld–Rieger Syndrome

Axenfeld anomaly, Axenfeld syndrome, Rieger(s) anomaly, Reiger(s) syndrome, Rieger(s) syndrome type I, Rieger(s) syndrome type II, Rieger(s) mesodermal dysgenesis, iridogoniodysgenesis anomaly Peters anomaly type I, Peters anomaly type II Krause–Kivlin syndrome Infantile congenital glaucoma, trabeculodysgenesis, goniodysgenesis

Peters anomaly Peters Plus syndrome Primary congenital glaucoma

prominent forehead, telecanthus), dental anomalies (e.g. hypodontia, microdontia) and a redundant preumbilical skin. Approximately 50% of ARS patients secondarily develop glaucoma.5,11,12 ARS patients tend to develop glaucoma in adolescence or in early adulthood (24 mm Hg >24 mm Hg Normal Normal Normal Normal Normal

Glaucoma Yes Yes No No Yes Yes Yes

Figure 2. Axenfeld–Rieger syndrome (ARS) family with FOXC1 S82T mutation. (a) Multiple individuals within the same family were diagnosed with ARS (blue fill). These patients have a S82T mutation in the FOXC1 transcription factor gene. Patient 1 was diagnosed with glaucoma, but no ARS phenotype was reported (grey fill). The number within the square or circle corresponds to the patient number in Figure 2c. (b) Patient 2 presented with a severe form of ARS with iris hypoplasia, peripheral anterior synechiae and a prominent Schwalbe’s line. (c) The summary of ARS-associated glaucoma show that there is great phenotypic heterogeneity in these patients with a FOXC1 S82T mutation. The five-point star denotes patient 2, whose ocular phenotype is shown in Figure 2b. IOP, intraocular pressure.

© 2013 Royal Australian and New Zealand College of Ophthalmologists

Genomics and anterior segment dysgenesis a

?

17

= Axenfeld-Rieger Syndrome

?

= no ocular phenotype; maxillary hypoplasia, dental anomalies

1

?

?

R69H ´

?

2

3

?

4

R69H

R69H

?

R69H

5

6

7

7

R69H

R69H

R69H

8

9

10

R69H

R69H

R69H

11

12

R69H

R69H b

c

Patient 1 2 3 4

Medication Yes Yes

Surgery Yes Yes

5 6 7

No -

Yes

´

8 No 9* 10 11 No 12 No (-) = not reported; (*) = no ocular phenotype

Glaucoma Yes Yes Yes Yes

-

IOP >24 mm Hg >24 mm Hg (Normal postsurgery) Normal >24 mm Hg (Normal postsurgery) Normal

-

Normal >24 mm Hg

No No No

No No Yes

No

Figure 3. Axenfeld-Rieger syndrome (ARS) family with PITX2 R69H mutation. (a) Multiple individuals within the same family were diagnosed with ARS (blue fill). These patients have a R69H mutation in the PITX1 transcription factor gene. A (?) depicts individuals with no availabe genotypic or phenotypic information. The number within the square or circle corresponds to the patient number in Figure 3b. (b) Iris hypoplasia is observed in patient 2, who carries the PITX2 R69H mutations. (c)The summary of ARS-associated glaucoma show that there is great phenotypic heterogeneity in these patients with a PITX2 R69H mutation. Note the differences in response to treatment. The five-point star denotes patient 2, whose ocular phenotype is shown in Figure 3b. IOP, intraocular pressure. © 2013 Royal Australian and New Zealand College of Ophthalmologists

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Congenital hereditary endothelial dystrophy Similar to Peters anomaly, patients with corneal hereditary endothelial dystrophy (CHED) have corneal opacification (reviewed in Ciralsky & Colby32). Histopathological examination reveals degeneration of the corneal endothelium.33 In the autosomal-recessive form of CHED (=CHED 2), corneal opacification is present at birth and is not progressive. In contrast, in the autosomal-dominant CHED type (=CHED 1), corneal opacification becomes apparent in the first 2 years of life. Both CHED types are associated with severe impairments in visual acuity. Linkage analysis and homozygosity mapping have shown that CHED1 and CHED2, respectively, map to genetically distinct loci on chromosome 20.34 A candidate gene approach identified mutations in the sodium borate transporter, SLC4A11, in patients with CHED 2.35

Sclerocornea Sclerocornea can occur as an independent phenotype, as part of another disorder such as Peters anomaly, or in conjunction with other ocular phenotypes such as cornea plana, a condition in which the curvature of the cornea is reduced.10,36 In sclerocornea, there is no clear boundary between the sclera and the cornea as the cornea is scleralized and vascularized, resulting in partial corneal opacification. Although both autosomal-dominant and autosomalrecessive modes of inheritance have been observed for sclerocornea, the causative gene(s) have yet to be identified.

Megalocornea In megalocornea, patients have an enlarged corneal diameter, generally greater than 12.5 mm at birth, and a deep anterior chamber, but normal IOP. Using a combination of genomic techniques and fine mapping, mutations in Chordin-like 1 (CHRDL1) were determined to be associated with X-linked megalocornea.37 CHRDL1 encodes for the ventroptin protein, a bone morphogenic protein antagonist. Although the functional role of ventroptin had been previously studied in the brain and the retina, the functional relevance of ventroptin in the anterior segment of the eye was unexpected.37

Systemic syndromes associated with ocular phenotypes Many syndromes with major systemic consequences may also present with anterior segment anomalies of the eye. Alagille syndrome, SHORT (short stature,

hyperextensibility of joints of inguinal hernia, ocular depression, ARS-like eye anomalies, delay in dental eruption) syndrome and Pierson syndrome are just a few examples of systemic syndromes that may also have anterior segment abnormalities. However, it is unclear whether or not these syndromes are truly part of the anterior segment dysgenesis spectrum of disorders. A great majority of human genes (>90%) are expressed in the eye during or after development.38 In addition, about a third of disorders in the Online Mendelian Inheritance in Man database affect the structure or function of the eye.38 Thus, it is not surprising that many patients with systemic syndromes also have ocular phenotypes.

COMPLEXITY

OF ANTERIOR SEGMENT DYSGENESIS

As seen in the previous section, anterior segment dysgenesis has a strong genetic basis. Linkage analysis and positional cloning methods have successfully identified mutations in genes that cause many anterior segment dysgenesis diseases. The identification and functional analysis of specific disease-causing genes have given insight into the underlying pathological mechanism that contributes to the manifestation of the disease phenotype. Mutations in genes with a wide array of functions, including transcription factors, transporters and glycosylating proteins, have been identified in anterior segment dysgenesis disorders. At the same time, identification of specific anterior segment dysgenesis genes has underscored the highly genetically heterogeneous nature of anterior segment dysgenesis. Understanding the factors, such as genetic modifiers, that contribute to the phenotypically and genetically heterogeneous nature of anterior segment dysgenesis has proven to be challenging. Variable expressivity and incomplete penetrance are observed even between patients with the same mutation in a known causative gene. In this section, two ARS families with either a FOXC1 mutation or a PITX2 mutation are described to demonstrate the complex nature of anterior segment dysgenesis disorders.

ARS family with FOXC1 S82T mutation Mutations in the FOXC1 transcription factor are associated with ARS. Several studies have characterized the molecular consequence of various FOXC1 missense mutations. Although the resulting FOXC1 mutant protein has been shown to have impaired ability to function at the molecular level (e.g. localize to the nucleus and/or bind DNA), there appears to be no distinct genotype–phenotype correlation. In fact, the same FOXC1 missense mutation can result in a wide range of phenotypes. The FOXC1 S82T mutation was identified in multiple individuals diagnosed with ARS from the

© 2013 Royal Australian and New Zealand College of Ophthalmologists

Genomics and anterior segment dysgenesis same family (Fig. 2). Although members within this family carry the same FOXC1 S82T mutation, great variability in phenotype is observed (Fig. 2c). For example, patient 2 presents with a severe form of ARS with iris hypoplasia, peripheral anterior synechiae and a prominent Schwalbe’s line (Fig. 2b). Patient 2 also has elevated IOP and was diagnosed with earlier-onset glaucoma. Other individuals with the same FOXC1 S82T mutation, however, have a milder form of ARS. In contrast to patient 2, patients 3 and 4 have a normal IOP (without the use of medication or surgery) and do not have glaucoma.

ARS family with PITX2 R69H mutation Mutations in the PITX2 transcription factor gene are also associated with ARS. The PITX2 R69H mutation was identified in at least 12 individuals within the same family39 (Fig. 3). Most of the individuals with the PITX2 R69H mutation have ocular abnormalities such as iris hypoplasia and peripheral anterior synechiae. However, one individual with the PITX2 R69H mutation (patient 9) does not have any ocular defects. This patient does have other ARS-associated systemic phenotypes including maxillary hypoplasia, hypodontia and microdontia. There is great variability in IOP measurements and development of glaucoma in these patients with the PITX2 R69H mutation (Fig. 3c). For example, while patient 7 has elevated IOP and glaucoma, her sibling with the same PITX2 mutation (patient 8) has normal IOP and has not been diagnosed with glaucoma. In addition, there appears to be variability in the effectiveness of treatments to control IOP. For example, while surgery successfully lowered IOP in patient 4, the same treatment was unable to lower IOP in her sibling, patient 3. The factors that contributed to differences in responsiveness to treatment between patient 3 and patient 4 are unclear. However, in addition to individual susceptibility to diseases, factors such as genetic modifiers may also affect the effectiveness of certain treatments including medication and surgery.

Heterogeneity in other anterior segment dysgenesis disorders Anterior segment dysgenesis disorders other than ARS are also heterogeneous. Mutations in PAX6 have been associated with both Peters anomaly and aniridia. Point mutations in PAX6 are most likely to cause the milder Peters anomaly phenotype while other mutations in PAX6, including deletions/ insertions and splicing mutations, tend to cause the more severe aniridia phenotype.40 However, this genotype–phenotype correlation is not always the

19 case, suggesting that additional genetic (and nongenetic factors) may contribute to the severity of the phenotype. Similarly, mutations in CYP1B1 have been associated with both Peters anomaly and PCG.

Complexity of anterior segment dysgenesis-associated glaucoma The pathophysiology of glaucoma is complicated as environmental, genetic and even stochastic factors all contribute to the pathology of glaucoma. As previously mentioned, approximately 50% of individuals with anterior segment dysgenesis develop glaucoma.5,11,12 Patients with anterior segment dysgenesis may have contributing factors that are specific to this subset of individuals. Abnormalities in the anterior segment structures are predicted to disrupt the regulation of aqueous humour flow, resulting in increased IOP, which is a major risk factor for developing glaucoma. As described previously, anterior segment dysgenesis refers to maldevelopment of anterior segment structures including the iris, lens, trabecular meshwork and Schlemm’s canal. There appears to be no correlation between the severity of clinically visible anterior segment anomalies (i.e. iris, lens and corneal anomalies) and the development of glaucoma.41 Similarly, in human ARS patients, the severity of clinically visible anterior segment abnormalities, such as iris strands, does not correlate with the occurrence of glaucoma.41 The iris strands apparently do not disrupt the flow of aqueous humour into the drainage structures. Instead, histopathological examination of ARS patients reveals the presence of abnormalities in the trabecular meshwork and Schlemm’s canal.41 Thus, ARS patients are postulated to develop glaucoma due to malformation of structures that are essential for proper aqueous humour drainage to occur. Unfortunately, determining the structural integrity of the trabecular meshwork and Schlemm’s canal is difficult without histopathological examination. While it remains a possibility that maldevelopment of the aqueous humour drainage structures also causes glaucoma in other anterior segment dysgenesis disorders including Peters anomaly and aniridia, there are likely other factors that contribute to the development of glaucoma. Elevation of IOP is not only dependent on proper development of the drainage structures. Extracellular matrix (ECM) occupies the intercellular space in the trabecular meshwork. The constant turnover of this ECM appears to play a role in maintaining proper aqueous humour resistance. Changes to the composition of the ECM or to the turnover rate have been suggested to increase resistance, which would ultimately result in increased IOP (reviewed in Acott & Kelley42). Another structure that is important for

© 2013 Royal Australian and New Zealand College of Ophthalmologists

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aqueous humour flow is the ciliary body, which is the site of aqueous humour production. Although an increase in aqueous humour production is not thought to be an underlying mechanism leading to elevated IOP, malfunctioning of the ciliary body could alter many aspects of aqueous humour including composition. Thus, the potential involvement of the ciliary body as a site of pathological importance should be considered. Finally, environmental stresses are thought to be a critical contributing factor to the development of glaucoma. Many of the anterior segment tissues are located in a dynamic environment where there is constant exposure to aqueous humour flow. Thus, these tissues are constantly exposed to various stresses including mechanical and oxidative stresses. Similar to other cells in the body, cells in the anterior segment tissues have multiple defence mechanisms that enable a fast and effective response to the specific stress. The antioxidant system protects cells against reactive oxygen species while the proteolytic system prevents the accumulation of potentially toxic protein aggregates within the cell. Also, a change in the global gene expression profile allows cells to produce an effective adaptive response to various stresses.43 However, a progressively less efficient defence mechanism combined with an accumulation of toxic biomaterials are likely to result in malfunction of anterior segment structures that are needed for maintaining proper aqueous flow. Clearly, such a scenario is not limited to anterior segment dysgenesis-related glaucoma. Thus, patients with anterior segment dysgenesis may have a compromised ability to respond to stress because of the structural anomalies or the presence of mutations in genes that function in the stress response pathway.

NEED

FOR GENOMICS IN ANTERIOR SEGMENT DYSGENESIS DISORDERS

The idea of genetic modifiers that alter individual susceptibility to disease has long been discussed.44 Studies using mouse models for anterior segment dysgenesis disorders have clearly support this idea. For example, a range of ocular abnormalities are observed in FOXC1 +/– mice with different genetic backgrounds. At 2–3 months of age, no clinically visible anterior segment abnormalities were present in FOXC1 +/– mice with a 129/SvEvTac background.45 In contrast, age-matched FOXC1 +/– mice with a B6, C57BL6J background had various ocular abnormalities including irregular pupils, misplaced pupils and iris strands that attached to the cornea.45 Thus, along with the mutation in FOXC1, different genetic modifiers present in the different mice strains are predicted to contribute to the variation in ocular abnormalities. However, identifying such genetic

modifiers has been less successful. In the past decade, GWA studies have identified hundreds of common, low-risk variants (i.e. present in more than 5% of the population) that are associated with human disease. The GWA approach is based on identifying loci that fit the ‘common disease, common variant’ hypothesis.46–48 Although anterior segment dysgenesis is a rare disease, GWA approaches may give insight into the genetic factors such as common variants that contribute to individual differences in disease susceptibility. A variety of factors may contribute to the development of glaucoma in anterior segment dysgenesis patients. The differences in individual susceptibility to glaucoma may be caused by common variants (i.e. genetic modifiers) that contribute to differential development of anterior segment structures. Variations during the development of structures such as the trabecular meshwork and Schlemm’s canal would inevitably alter the susceptibility to glaucoma. In addition, genetic variants could alter individual susceptibility to glaucoma by influencing the effectiveness of cellular response to stress postdevelopment. A myriad of proteins are part of the cellular stress response pathway. Variations in expression of functional ability of these stressresponsive proteins would also alter susceptibility to glaucoma. Thus, GWA is potentially an effective tool to understand the complexity underlying the development of glaucoma. GWA methods are mainly used to identify common, low-risk alleles that alter susceptibility to a complex disease. However, variants identified using GWA methods could be located within or in the vicinity of a causative gene. For example, using a genome-wide technique, B3GALTL was identified as a causative gene for Peters Plus syndrome. The genome of six patients with Peters Plus syndrome was analyzed using array-based comparative genomic hybridization (CGH) technology.26 An approximately 1.5 Mb interstitial deletion on chromosome 13, containing six genes, was identified in two related individuals. Lesnik Oberstein et al. sequenced the exons of the six genes and the regions flanking the genes to identify a splicing mutation in the B3GALTL gene.26 Several more mutations in the B3GALTL gene were identified by Lesnik Oberstein et al. and other groups in many more patients with Peters Plus syndrome.26,49 Thus, genome-wide techniques such as array CGH technology can successfully be used to identify single disease-causing genes. Also, prior knowledge of the function of a gene is not required with the GWA approach. None of the six genes located within the deleted region identified in the two related individuals with Peters Plus syndrome were obvious candidates for this disorder. B3GALTL was initially an unlikely candidate

© 2013 Royal Australian and New Zealand College of Ophthalmologists

Genomics and anterior segment dysgenesis gene for Peters Plus syndrome because of its function in glycosylation. With the identification of mutations in B3GALTL, Peters Plus syndrome is now considered to be a glycosylation disease. Thus, in this case, array CGH technology was able to narrow the region of interest to a manageable size that could then be sequenced. However, it should be noted that in many cases, large stretches of DNA in linkage disequilibrium results in many common variants within a particular region to be in association with a disease.8

CHALLENGES

OF

GWA

STUDIES

Challenges relating to GWA methodology The past decade has seen an explosion in the number of studies that utilize GWA methods to identify genetic factors that contribute to complex diseases, ranging from diabetes to schizophrenia to cardiovascular diseases. The increased use of GWA studies has also unexpectedly brought up new challenges in the methodology and interpretation of these studies. Although thousands of loci have been associated with a complex disease or trait, only a few of the identified variants have a clear functional consequence relating to the underlying mechanism of the disease.50 In fact, most identified loci are very lowrisk alleles. Many of these challenges are not unique to a specific disease or population and are thus applicable to potential GWA studies of anterior segment dysgenesis disorders. GWA studies rely on identifying common variants using a large sample size consisting of hundreds or thousands of patients. Increasingly, it has become evident that even larger sample sizes than previously thought are required to detect low-risk variants.8,51 Although each individual variant may only have a small effect, a large number of low-risk variants are predicted to contribute to the overall susceptibility to disease. Another important aspect to increase the confidence of any GWA study result is the ability to replicate in different populations. A major GWA study of POAG identified an SNP located in an intragenic region on chromosome 7q31 in an Icelandic population.52 The results were replicated in additional populations of European, Chinese and Caucasian US descent, strongly suggesting that the identified SNP is of functional importance.52,53 However, the results were not replicated in another ‘Iowa’ cohort.54 Thus, the fact that specific risk alleles may be specific to genetically different populations must be considered when conducting a GWA study. As anterior segment dysgenesis is a group of rare diseases, there may be challenges in obtaining sample sizes in different populations that are large enough to identify relevant variants. Imprecise phenotyping is another challenge of GWA studies, especially because thousands of

21 patients are required to conduct the study. Anterior segment dysgenesis encompasses a large number of phenotypes. Although many phenotypes such as aniridia and corectopia are easier to diagnose, others are not so obvious. In addition, it is difficult to interpret whether some phenotypes observed in anterior segment dysgenesis patients are pathological or not. Many ARS patients present with posterior embryotoxon, which is seen as an anteriorly displaced Schwalbe’s line. However, the pathological significance of posterior embryotoxon is unknown because it is not present in all ARS patients. More importantly, posterior embryotoxon is present in up to 15% of eyes in the normal population without any apparent consequences on vision.55 Also, the distinction between iris processes and peripheral anterior synechiae is difficult to make. Iris processes are present in about a third of normal eyes with no apparent disruption to aqueous humour flow.5 In contrast to iris processes, peripheral anterior synechiae are broader and more irregular. Thus, imprecise phenotyping of anterior segment dysgenesis patients and normal controls may occur and potentially affect the GWA results. As approximately 50% of anterior segment dysgenesis patients develop earlier-onset glaucoma, examining the phenotypes associated with glaucoma by GWA is another approach to identify genetic variants. GWA studies using ocular phenotypes such as central corneal thickness, vertical cup to disc ratio and optic disc areas have proven to be potentially useful in identifying glaucoma-associated variants (reviewed in Liu & Allingham56). A similar strategy including ocular phenotypes such as peripheral anterior synechiae and abnormalities in the trabecular meshwork and Schlemm’s canal, although difficult to observe, may be useful in studying anterior segment dysgenesis-associated glaucoma.

Challenges relating to GWA data interpretation The vast majority (>80%) of common variants that have been associated with a complex disease by GWA studies are located in non-coding regions.57 Although non-coding regions make up 99% of the genome, more research has been carried out in understanding the function of protein-coding regions. Thus, much of the functional significance of these non-coding variants is often unknown. For example, the common variant identified in a number of POAG GWA studies is located in an intragenic region between two caveolin genes at chromosome 7q31.52 Caveolins are the membrane proteins of caveolae, which form from invaginations of the plasma membrane.58 Caveolae have been proposed to function in lipid regulation and mechanosensing.58 However, the functional

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significance of the intragenic variant associated with POAG is currently unknown. Non-coding regions are predicted to contain essential regulatory elements that affect various processes including splicing, transcription and regulation of RNA. Great strides have been made to annotate the non-coding genome resulting in various maps including a chromatin-state map, functional genomic map, DNase hypersensitivity map and protein-binding map.59 Unfortunately, as these maps are specific to cell type,8,59 current maps may be of limited value for variants associated with anterior segment dysgenesis. Low-risk variants may be of little predictive value. However, pathway-based approaches, where these low-risk variants are examined as part of a group of related genes, could potentially determine pathways that contribute to disease susceptibility (reviewed in Wang et al.60). Based on prior knowledge of the biological function of genes, the GWA study data are applied to identify pathways (i.e. network of genes) that associate with the disease. Although prior knowledge of gene function is required for the pathway-based approach, the resulting associated pathway may not be previously associated with the disease. For example, the pathway-based approach has identified biological pathways such as blood lipids61,62 that have previously not been associated to be of importance when considering cardiovascular diseases. Most genes and their protein products exert its function through its interaction with other cellular compartments.63 Thus, a phenotypic defect does not result from malfunction of a single gene in which the mutation was identified, but rather, is due to disruptions to the network of genes of which the mutated gene is a part. A pathway-based approach could potentially result in identification of disease-causing variants because proteins that are involved in the same disease are also more likely to interact with each other.63–65 Interestingly, many of the identified causative genes for anterior segment dysgenesis disorders such as FOXC1, PITX2 and PAX6 encode for transcription factors, highlighting the idea that a complex network of genes is involved in the development and the functioning of the anterior segment of the eye. Also, the ARS-causing genes, FOXC1 and PITX2, have been shown to interact with each other and thus appear to be part of the same regulatory network.66,67

CONCLUSIONS Genomic approaches are an invaluable tool to potentially decipher the complexity of anterior segment dysgenesis disorders. However, there are many limitations and challenges relating to the GWA technology. The vast majority of loci identified through GWA studies have failed to explain the heritable

phenotypic variation for a trait.68 This ‘missing heritability’ issue has resulted in re-assessment of the advantages and disadvantages of GWA technology.68 Improvements to the GWA technology, allowing the identification of even rarer variants and structural variants, as well as improvements to the interpretation methodology, are in progress. Such improvements will likely open new avenues to gain insight into the functional importance of regulatory elements within the non-coding regions. Although the full potential of genomic technology has yet to be realized,68 genomic technology by itself is likely not sufficient to understand the pathology of complex diseases such as anterior segment dysgenesis. Thus, genomic approaches should be used in conjunction with other approaches such as genetic, molecular and animal model-based approaches. Such collaborative efforts are necessary to ultimately provide accurate and fast diagnosis, effective treatment options, and preventative measures for those patients with complex diseases.

ACKNOWLEDGEMENTS We would like to thank Dr O.J. Lehmann and Dr W.G. Pearce for contribution of the images in Figure 2b and Figure 3b, respectively. Y.A.I. is supported by the Sir Frederick Banting and Dr. Charles Best Canada Graduate Scholarship provided by the Canadian Institutes of Health Research.

REFERENCES 1. Quigley HA, Broman AT. The number of people with glaucoma worldwide in 2010 and 2020. Br J Ophthalmol 2006; 90: 262–7. 2. Rudnicka AR, Mt-Isa S, Owen CG, Cook DG, Ashby D. Variations in primary open-angle glaucoma prevalence by age, gender, and race: a Bayesian metaanalysis. Invest Ophthalmol Vis Sci 2006; 47: 4254–61. 3. Strungaru MH, Dinu I, Walter MA. Genotypephenotype correlations in Axenfeld-Rieger malformation and glaucoma patients with FOXC1 and PITX2 mutations. Invest Ophthalmol Vis Sci 2007; 48: 228–37. 4. Wilson ME. Congenital iris ectropion and a new classification for anterior segment dysgenesis. J Pediatr Ophthalmol Strabismus 1990; 27: 48–55. 5. Idrees F, Vaideanu D, Fraser SG, Sowden JC, Khaw PT. A review of anterior segment dysgeneses. Surv Ophthalmol 2006; 51: 213–31. 6. Waring GO,3rd, Rodrigues MM, Laibson PR. Anterior chamber cleavage syndrome. A stepladder classification. Surv Ophthalmol 1975; 20: 3–27. 7. Alward WL. Axenfeld-Rieger syndrome in the age of molecular genetics. Am J Ophthalmol 2000; 130: 107–15. 8. McPherson R. From genome-wide association studies to functional genomics: new insights into cardiovascular disease. Can J Cardiol 2013; 29: 23–9.

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Genomics and anterior segment dysgenesis 9. Klein RJ, Zeiss C, Chew EY et al. Complement factor H polymorphism in age-related macular degeneration. Science 2005; 308: 385–9. 10. Nischal KK. A new approach to the classification of neonatal corneal opacities. Curr Opin Ophthalmol 2012; 23: 344–54. 11. Sowden JC. Molecular and developmental mechanisms of anterior segment dysgenesis. Eye (Lond) 2007 Oct;21: 1310–8. 12. Tumer Z, Bach-Holm D. Axenfeld-Rieger syndrome and spectrum of PITX2 and FOXC1 mutations. Eur J Hum Genet 2009; 17: 1527–39. 13. D’haene B, Meire F, Claerhout I et al. Expanding the spectrum of FOXC1 and PITX2 mutations and copy number changes in patients with anterior segment malformations. Invest Ophthalmol Vis Sci 2011; 52: 324– 33. 14. Phillips JC, Del Bono EA, Haines JL et al. A second locus for Rieger syndrome maps to chromosome 13q14. Am J Hum Genet 1996; 59: 613–9. 15. Semina EV, Reiter R, Leysens NJ et al. Cloning and characterization of a novel bicoid-related homeobox transcription factor gene, RIEG, involved in Rieger syndrome. Nat Genet 1996; 14: 392–9. 16. Mears AJ, Jordan T, Mirzayans F et al. Mutations of the forkhead/winged-helix gene, FKHL7, in patients with Axenfeld-Rieger anomaly. Am J Hum Genet 1998; 63: 1316–28. 17. Mears AJ, Mirzayans F, Gould DB, Pearce WG, Walter MA. Autosomal dominant iridogoniodysgenesis anomaly maps to 6p25. Am J Hum Genet 1996; 59: 1321–7. 18. Nishimura DY, Swiderski RE, Alward WL et al. The forkhead transcription factor gene FKHL7 is responsible for glaucoma phenotypes which map to 6p25. Nat Genet 1998; 19: 140–7. 19. Harissi-Dagher M, Colby K. Anterior segment dysgenesis: Peters anomaly and sclerocornea. Int Ophthalmol Clin 2008; 48: 35–42. 20. Hanson IM, Fletcher JM, Jordan T et al. Mutations at the PAX6 locus are found in heterogeneous anterior segment malformations including Peters’ anomaly. Nat Genet 1994; 6: 168–73. 21. Doward W, Perveen R, Lloyd IC, Ridgway AE, Wilson L, Black GC. A mutation in the RIEG1 gene associated with Peters’ anomaly. J Med Genet 1999; 36: 152–5. 22. Vincent A, Billingsley G, Priston M et al. Phenotypic heterogeneity of CYP1B1: mutations in a patient with Peters’ anomaly. J Med Genet 2001; 38: 324–6. 23. Maillette de Buy Wenniger-Prick LJ, Hennekam RC. The Peters’ plus syndrome: a review. Ann Genet 2002; 45: 97–103. 24. Zaidman GW, Flanagan JK, Furey CC. Long-term visual prognosis in children after corneal transplant surgery for Peters anomaly type I. Am J Ophthalmol 2007; 144: 104–8. 25. Heinonen TY, Maki M. Peters’-plus syndrome is a congenital disorder of glycosylation caused by a defect in the beta1,3-glucosyltransferase that modifies thrombospondin type 1 repeats. Ann Med 2009; 41: 2–10.

23 26. Lesnik Oberstein SA, Kriek M, White SJ et al. Peters Plus syndrome is caused by mutations in B3GALTL, a putative glycosyltransferase. Am J Hum Genet 2006; 79: 562–6. 27. Sarfarazi M, Akarsu AN, Hossain A et al. Assignment of a locus (GLC3A) for primary congenital glaucoma (Buphthalmos) to 2p21 and evidence for genetic heterogeneity. Genomics 1995; 30: 171–7. 28. Stoilov I, Akarsu AN, Sarfarazi M. Identification of three different truncating mutations in cytochrome P4501B1 (CYP1B1) as the principal cause of primary congenital glaucoma (Buphthalmos) in families linked to the GLC3A locus on chromosome 2p21. Hum Mol Genet 1997; 6: 641–7. 29. Lee H, Khan R, O’Keefe M. Aniridia: current pathology and management. Acta Ophthalmol 2008; 86: 708– 15. 30. Fantes JA, Bickmore WA, Fletcher JM, Ballesta F, Hanson IM, van Heyningen V. Submicroscopic deletions at the WAGR locus, revealed by nonradioactive in situ hybridization. Am J Hum Genet 1992; 51: 1286– 94. 31. Ito YA, Footz TK, Berry FB et al. Severe molecular defects of a novel FOXC1 W152G mutation result in aniridia. Invest Ophthalmol Vis Sci 2009; 50: 3573–9. 32. Ciralsky J, Colby K. Congenital corneal opacities: a review with a focus on genetics. Semin Ophthalmol 2007; 22: 241–6. 33. Kirkness CM, McCartney A, Rice NS, Garner A, Steele AD. Congenital hereditary corneal oedema of Maumenee: its clinical features, management, and pathology. Br J Ophthalmol 1987; 71: 130–44. 34. Callaghan M, Hand CK, Kennedy SM, FitzSimon JS, Collum LM, Parfrey NA. Homozygosity mapping and linkage analysis demonstrate that autosomal recessive congenital hereditary endothelial dystrophy (CHED) and autosomal dominant CHED are genetically distinct. Br J Ophthalmol 1999; 83: 115–9. 35. Vithana EN, Morgan P, Sundaresan P et al. Mutations in sodium-borate cotransporter SLC4A11 cause recessive congenital hereditary endothelial dystrophy (CHED2). Nat Genet 2006; 38: 755–7. 36. Mataftsi A, Islam L, Kelberman D, Sowden JC, Nischal KK. Chromosome abnormalities and the genetics of congenital corneal opacification. Mol Vis 2011; 17: 1624–40. 37. Webb TR, Matarin M, Gardner JC et al. X-linked megalocornea caused by mutations in CHRDL1 identifies an essential role for ventroptin in anterior segment development. Am J Hum Genet 2012; 90: 247–59. 38. Sheffield VC, Stone EM. Genomics and the eye. N Engl J Med 2011; 364: 1932–42. 39. Pearce WG, Mielke BC, Kulak SC, Walter MA. Histopathology and molecular basis of iridogoniodysgenesis syndrome. Ophthalmic Genet 1999; 20: 83–8. 40. Tzoulaki I, White IM, Hanson IM. PAX6 mutations: genotype-phenotype correlations. BMC Genet 2005; 6: 27. 41. Shields MB. Axenfeld-Rieger syndrome: a theory of mechanism and distinctions from the iridocorneal

© 2013 Royal Australian and New Zealand College of Ophthalmologists

24

42. 43.

44. 45.

46. 47.

48.

49.

50.

51.

52.

53.

54.

Ito and Walter endothelial syndrome. Trans Am Ophthalmol Soc 1983; 81: 736–84. Acott TS, Kelley MJ. Extracellular matrix in the trabecular meshwork. Exp Eye Res 2008; 86: 543–61. de Nadal E, Ammerer G, Posas F. Controlling gene expression in response to stress. Nat Rev Genet 2011; 12: 833–45. Nadeau JH. Modifier genes in mice and humans. Nat Rev Genet 2001; 2: 165–74. Smith RS, Zabaleta A, Kume T et al. Haploinsufficiency of the transcription factors FOXC1 and FOXC2 results in aberrant ocular development. Hum Mol Genet 2000; 9: 1021–32. Reich DE, Lander ES. On the allelic spectrum of human disease. Trends Genet 2001; 17: 502–10. Collins FS, Guyer MS, Charkravarti A. Variations on a theme: cataloging human DNA sequence variation. Science 1997; 278: 1580–1. Pritchard JK. Are rare variants responsible for susceptibility to complex diseases. Am J Hum Genet 2001; 69: 124–37. Reis LM, Tyler RC, Abdul-Rahman O et al. Mutation analysis of B3GALTL in Peters Plus syndrome. Am J Med Genet A 2008; 146A: 2603–10. Manolio TA. Genomewide association studies and assessment of the risk of disease. N Engl J Med 2010; 363: 166–76. Visscher PM, Medland SE, Ferreira MA et al. Assumption-free estimation of heritability from genome-wide identity-by-descent sharing between full siblings. Plos Genet 2006; 2: e41. Thorleifsson G, Walters GB, Hewitt AW et al. Common variants near CAV1 and CAV2 are associated with primary open-angle glaucoma. Nat Genet 2010; 42: 906–9. Wiggs JL, Kang JH, Yaspan BL et al. Common variants near CAV1 and CAV2 are associated with primary open-angle glaucoma in Caucasians from the USA. Hum Mol Genet 2011; 20: 4707–13. Kuehn MH, Wang K, Roos B et al. Chromosome 7q31 POAG locus: ocular expression of caveolins and lack of association with POAG in a US cohort. Mol Vis 2011; 8: 430–5.

55. Malik SR, Sing G, Gupta AK. Posterior embryotoxon. J All India Ophthalmol Soc 1969; 17: 115–6. 56. Liu Y, Allingham RR. Molecular genetics in glaucoma. Exp Eye Res 2011; 93: 331–9. 57. Hindorff LA, Sethupathy P, Junkins HA et al. Potential etiologic and functional implications of genome-wide association loci for human diseases and traits. Proc Natl Acad Sci U S A 2009; 106: 9362–7. 58. Parton RG, Simons K. The multiple faces of caveolae. Nat Rev Mol Cell Biol 2007; 8: 185–94. 59. Ward LD, Kellis M. Interpreting noncoding genetic variation in complex traits and human disease. Nat Biotechnol 2012; 30: 1095–106. 60. Wang K, Li M, Hakonarson H. Analysing biological pathways in genome-wide association studies. Nat Rev Genet 2010; 11: 843–54. 61. Teslovich TM, Musunuru K, Smith AV et al. Biological, clinical and population relevance of 95 loci for blood lipids. Nature 2010; 466: 707–13. 62. Sandhu MS, Waterworth DM, Debenham SL et al. LDL-cholesterol concentrations: a genome-wide association study. Lancet 2008; 371: 483–91. 63. Barabasi AL, Gulbahce N, Loscalzo J. Network medicine: a network-based approach to human disease. Nat Rev Genet 2011; 12: 56–68. 64. Goh KI, Cusick ME, Valle D, Childs B, Vidal M, Barabasi AL. The human disease network. Proc Natl Acad Sci U S A 2007; 104: 8685–90. 65. Oti M, Snel B, Huynen MA, Brunner HG. Predicting disease genes using protein-protein interactions. J Med Genet 2006; 43: 691–8. 66. Acharya M, Huang L, Fleisch VC, Allison WT, Walter MA. A complex regulatory network of transcription factors critical for ocular development and disease. Hum Mol Genet 2011; 20: 1610–24. 67. Berry FB, Lines MA, Oas JM et al. Functional interactions between FOXC1 and PITX2 underlie the sensitivity to FOXC1 gene dose in Axenfeld-Rieger syndrome and anterior segment dysgenesis. Hum Mol Genet 2006; 15: 905–19. 68. Manolio TA, Collins FS, Cox NJ et al. Finding the missing heritability of complex diseases. Nature 2009; 461: 747–53.

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