A review of keratoconus: Diagnosis, pathophysiology, and genetics

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Major review

A review of keratoconus: Diagnosis, pathophysiology, and genetics Veronica Mas Tur, FEBOa, Cheryl MacGregor, BMa, Rakesh Jayaswal, MBChB, FRCOphth, FRCS (Ed)a, David O’Brart, MD, FRCS, FRCOphthb, Nicholas Maycock, BSc (Hons), MBBS, FRCOphthb,* a b

Eye Department, Queen Alexandra Hospital, Portsmouth, Hants, United Kingdom Department of Ophthalmology, St Thomas’ Hospital, London, United Kingdom

article info

abstract

Article history:

We discuss new approaches to the early detection of keratoconus and recent investigations

Received 8 February 2017

regarding the nature of its pathophysiology. We review the current evidence for its com-

Received in revised form 26 June

plex genetics and evaluate the presently identified genes/loci and potential candidate gene/

2017

loci. In addition, we highlight current research methodologies that may be used to further

Accepted 29 June 2017

elucidate the pathogenesis of keratoconus.

Available online 6 July 2017

ª 2017 Elsevier Inc. All rights reserved.

Keywords: keratoconus genetics review article

1.

Introduction

Keratoconus (KCN) is an asymmetric, progressive ectatic condition that can lead to significant visual impairment.114 Although the disease has high prevalence, the cellular etiology of the disease is not well understood. Studies in varied fields such as genetics, genomics, small biomolecule analyses, and gene expression analysis suggest that the disease may be multifactorial in origin. Furthermore, a variety of genome-wide studies in familial KCN implicate differential loci. Therefore, it is even more evident that the disease may be sporadic and dependent on external factors and stimuli

that lead to the inception and progression of this complex disease. KCN is a bilateral and usually asymmetrical disease in which the ectatic cornea becomes conical in shape. It typically presents in adolescence and progress until the third or fourth decade of life and is one of the commonest reasons for keratoplasty in the developed world,36 although this demand is decreasing with the onset of corneal collagen crosslinking. The etiology of KCN is not fully understood with several different pathways implicateddbiochemical, physical, and geneticdwith the condition being a final common pathway for several different diseases. It can occur as a result of genetic

* Corresponding author: Nicholas Maycock, BSc (Hons), MBBS, FRCOphth, Department of Ophthalmology, St Thomas’ Hospital, Westminster Bridge Road, London SE1 7EH, United Kingdom. E-mail address: [email protected] (N. Maycock). 0039-6257/$ e see front matter ª 2017 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.survophthal.2017.06.009

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predisposition triggered by environmental factors. It may arise as an isolated condition or in association with ocular and systemic disorders such as atopy, vernal disease, Down syndrome, retinitis pigmentosa, Turner syndrome, connective tissue disorders such as Marfan syndrome, Ehlers-Danlos syndrome, osteogenesis imperfecta, and pseudoxanthoma elasticum. KCN has a strong association with eye rubbing, repeated trauma from contact lenses, and allergic eye disease.67,114 Between 8% and 10% of cases have a hereditary component and family history, and an inverse relationship between the severity of the condition and diabetes has been described.45,47,114,117,153 Stromal thinning is thought to be related to a combination of increased activity of proteinase enzymes and decreased proteinase inhibitors with subsequent reduced biomechanical stability.11 KCN affects both genders and all ethnicities. The reported prevalence and incidence is variable. This is probably due to different clinical definitions and diagnostic criteria used between studies and populations. The incidence of KCN in the European white population has been determined to be between 5 and 23, with a mean prevalence of 54, per 100,000.117 There is a higher prevalence in South Asian patients compared with whites.17,108 Based on a 48-year epidemiological study conducted in the United States, KCN was thought to affect approximately 1 person in 2000, with a mean incidence of 2 new cases per 100,000 per year51; however, a recent study by Godefrooij and colleagues has shown both the annual incidence and prevalence to be much higher.45 They conducted an epidemiological study looking at 4.4 million patients in the Netherlands and found the annual incidence was 1:7500 (13.3 cases per 100,000) and the estimated prevalence was 1:375 (265 cases per 100,000). These values are 5 to 10 fold higher than previously reported in population studies, and this is thought to be a result of a combination of earlier and more advanced detection with tomography and comprehensive data collection in the Netherlands. We discuss the diagnosis and pathophysiology of KCN. We review the evidence for the complex genetics of KCN and evaluate the currently identified genes/loci and potential candidate gene/loci. In addition, we highlight current research methodologies that may be used to further elucidate the pathogenesis of KCN.

2.1.

Corneal topography

The following topographic parameters should arouse suspicion and examination for further evidence of the disease: astigmatism >5 diopters (D), and/or keratometry values (K1/ K2) > 48 D70; maximum keratometry (Kmax) reading >49 D; central corneal thickness (CCT) <470 mm; and corneal asphericity >
0.50 mm (see Tables 1 and 2).18 The normal corneal surface is aspherical, ranging from mild oblate to moderate prolate in shape, with most studies suggesting the human cornea Q (asphericity) values range from
0.01 to
0.80 (mean
0.23  0.08) measured in the 4.5-mm optical zone.142 Topography maps with high astigmatism or an asymmetrical bowtie pattern are suggestive of KCN.114 Regular astigmatism will be represented by a bowtie pattern with 2 symmetric segments (see Fig. 1). The symmetrical bowtie is vertical in with-the-rule astigmatism, horizontal if the astigmatism is against-the-rule, and diagonal with oblique astigmatism. Corneal irregularities, or deviations from the symmetrical bowtie pattern, are detected by the curvature map and described in terms of their shape: round, oval, superior steep, inferior steep, irregular, symmetric bowtie with skewed radial axis, inferiorly steep asymmetric bowtie, superiorly steep asymmetric bowtie, or asymmetric bowtie with skewed radial axis (see Fig. 1). These patterns are risk factors for corneal ectatic disorders when accompanied by abnormal tomographic parameters. Within the 5-mm central zone, symmetrically opposite superior and inferior locations are compared. There is a risk of corneal ectasia if the superior value is more than 2.50 D greater than the lower value or the inferior value is more than 1.50 D greater than the upper.114 There is a displacement of the corneal apex with localized areas of steepening. In addition, there is vertical asymmetry in corneal power, skewing of radial axes above and below the horizontal meridian, and focal pachymetric thinning localized to the corneal apex. KCN cones can be classified into (1) nippledthe cone has a diameter 5 mm, round morphology and is located in the central, paracentral, or inferonasal corneal

Table 1 e Amsler-Krumeich Classification for grading keratoconus

2.

Diagnosis

Stage 1

KCN should be suspected in any patient with significant irregular astigmatism, especially if unstable and increasing over time. In the early stages of the disease, there is altered metabolic activity that may lead to biomechanical instability and stretching of the corneal tissues.89 As the disease progresses, there is accompanying tissue loss. In addition, there is a loss of correlation between the anterior and posterior corneal curvature.111 Progressive corneal thinning and distortion causes a conical or cone-shaped protrusion, which may be visible at the slit lamp in advanced cases.46 In early disease, the condition may go undiagnosed unless assessments of the posterior and anterior corneal surfaces are undertaken using corneal tomography.

2

3

4

Findings Eccentric steepening Myopia, induced astigmatism, or both <5.00 D Mean central K readings <48 D Myopia, induced astigmatism, or both from 5.00 to 8.00 D Mean central K readings <53.00 D Absence of scarring Corneal thickness >400 micron Myopia, induced astigmatism, or both from 8.00 to 10.00 D Mean central K readings >53.00 D Absence of scarring Corneal thickness 300e400 micron Refraction not measurable Mean central K readings >55.00 D Central corneal scarring Corneal thickness < 200 micron

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Table 2 e ABCD Grading System for classifying keratoconus ABCD criteria

Stage Stage Stage Stage Stage

0 1 2 3 4

A

B

C

D

ARC (3 mm zone)

PRC (3 mm zone)

Thinnest pachy (mm)

BDVA

>7.25 >7.05 >6.35 >6.15 <6.15

>5.90 mm >5.70 mm >5.15 mm >4.95 mm <4.95 mm

>490 >450 >400 >300 300

20/20 >20/20 <20/40 <20/100 <20/400

mm mm mm mm mm

(<46.5D) (<48.0D) (<53.0D) (<55.0D) (>55.0D)

(<57.25D) (<59.25D) (<65.5D) (<68.5D) (>68.5D)

Scarring



,þ,þþ
,þ,þþ
,þ,þþ
,þ,þþ

ARC, anterior radius of curvature; BDVA, best corrected distance visual acuity; PRC, posterior radius of curvature.

quadrant; (2) ovaldthe cone has a diameter >5 mm and a paracentral/peripheral location, most commonly in the inferotemporal corneal quadrant; (3) keratoglobusdthe cone involves 75% of the cornea.52,109,114

2.2.

Corneal tomography

Corneal tomography has enabled earlier detection of corneal ectasia as it permits a detailed quantitative examination of both the anterior and posterior corneal surfaces. It is essential to examine the posterior corneal surface to look for early elevation changes, ectasia, and stromal thinning. These findings are often the first clinically detectable structural changes as epithelial remodeling, which may hide an early cone, usually masks early anterior surface changes.116 There is evidence to suggest changes in corneal epithelial thickness patterns can aid in the diagnosis of preclinical disease.18 Reinstein and colleagues showed using the Artemis very high frequency ultrasound scanner that an epithelial doughnut pattern suggests the presence of an underlying stromal cone.116 There is epithelial thinning over the cone surrounded by an annulus of epithelial thickening. Absence of an epithelial doughnut pattern would indicate that abnormal topography was not due to keratoconus, and epithelial

compensation can mask the presence of an underlying cone in early disease. Established disease is characterized by increased steepening of both anterior and posterior corneal surfaces. Areas or zones of increased power may be surrounded by adjacent areas of decreased corneal power,97,151 especially in the adjacent hemi-meridian in cases with noncentral cones. There is a degree of overlap with pellucid marginal degeneration, and the 2 conditions can be misdiagnosed. The latter has a different appearance topographically, with peripheral corneal thinning closer to the limbus.57,66

2.3.

Keratoconus indices

Several indices facilitate the distinction between KCN and the normal cornea, such as differences in the central K value, inferior-superior (I-S) index, KISA index, and keratoconus prediction index87,115; however, detecting keratoconus before there is evidence of deformity has proven difficult. The central K value is calculated by averaging the dioptric power on rings 2e4 of the placido disc. The I-S value is the difference in dioptric power between points on the inferior cornea compared with corresponding points on the superior cornea. The KISA index is derived from 4 indices: central K;

Fig. 1 e Illustration of the curvature map showing different patterns of astigmatism. A: Round. B: Oval. C: Superior steepening. D: Inferior steepening. E: Irregular. F: Symmetric bowtie. G: Symmetric bowtie with skewed radial axis. H: Asymmetric bowtie with inferior steepening. I: Asymmetric bowtie with superior steepening. J: Asymmetric bowtie with skewed radial axis. (Adapted from Rabinowitz et al, 1998)

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I-S index; the astigmatism index (AST), which is a measure of the regular corneal astigmatism (simulated K1
simulated K2); and the skewed radial axis index, an expression of irregular astigmatism occurring in keratoconus.82,87,115 It is calculated as follows: KISA ¼ ðCentral KÞ  ðI
SÞ  ðASTÞ  ðSRAXÞ  100=300

2.4.

The Belin Ambrosio Enhanced Ectasia Display

The aim of the Belin/Ambrosio Enhanced Ectasia Display (BAD) is to enhance early detection of ectatic disease. Used in combination with corneal tomography and a detailed examination of the posterior corneal surface, early diagnosis, treatment, and improved outcomes are possible (see Fig. 2). By combining elevation and pachymetric data, BAD produces a display that is more sensitive at picking up early change by comparing the data to an “enhanced best-fit sphere.” The BAD on the Pentacam (Oculus GmbH) displays the anterior and posterior elevation data relative to the best-fit sphere calculated with a fixed optical zone of 8.0 mm, omitting the 4 mm around the elevated cone.8 It performs regression analysis including standard deviation from the mean on changes in anterior and posterior elevations, corneal thickness at the thinnest point, thinnest point displacement, and

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pachymetric progression. Using these values, it creates a new map at the bottom of the display, applying colors to represent variations from the mean. This “difference map” depicts change in elevation as a series of concentric green, yellow, and red circles. Green represents a change of less than 5 mm on the anterior surface and 12 mm on the posterior surface of the cornea; yellow a change of 5e7 mm for the anterior surface and 12e16 mm for the posterior surface; and red greater than 7 mm anteriorly and greater than 16 posteriorly. The Belin intuitive scale with 61 colors and a 2.5-mm step is the most reliable for elevation maps.19 Yellow indicates a suspicious cornea (at least 1.6 standard deviation from the mean): a posterior elevation value of þ15 at the thinnest point warrants suspicion as it occurs in less than 1% of normal corneas.45 Red indicates an abnormal cornea (at least 2.6 standard deviation from the mean), and green values indicate a normal cornea. In normal eyes, an average elevation value at the thinnest point is 3.6  4.7 mm, with a cutoff for keratoconus at 14 mm.1,64

2.5.

Holladay 6 map display

The Holladay 6 map display was designed by Jack Holladay and Oculus to display all of the necessary information for screening and treating patients in 1 output (see Fig. 3). If there

Fig. 2 e Data output from the Belin/Ambrosio Enhanced Ectasia display showing a patient with early keratoconus: top: baseline maps show presence of cone on both the anterior and posterior surface; middle: exclusion map enhances the visibility of the cone; bottom: change in elevation from baseline to exclusion map shows a significant change to both the posterior surface and anterior surface.

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Fig. 3 e An illustration of the Holladay 6 Map display. The upper row of boxes show general patient data, equivalent K readings, ratio of front to back radii, quality of scan (QS), pupil diameter, and estimated refractive change from refractive surgery. The upper left map shows the axial curvature or power map; the lower left map the front surface tangential curvature (or instantaneous curvature); the central upper map show corneal pachymetry and illustrates that the cornea is a negative meniscus lens (the back surface of curvature is steeper than the front); the central lower map shows relative pachymetry (RP), the values represent the percentage above or below the predicted thickness map; the upper right map shows elevation (microns) above the best-fit toric ellipsoid sphere in the central 8-mm zone; the lower right map shows elevation (microns) of the back surface above the best-fit toric ellipsoid.

is steepening on the tangential map, along with thinning on the relative pachymetry map and elevation on the posterior float >10 mm, then a diagnosis of KCN is evident. The relative pachymetry map shows whether (and by how much) the corneal thickness varies from normal value: thinning represented by warmer colors and thickening by colder colors. The Holladay 6 map display, along with the BAD, is useful for the diagnosis and monitoring of patients with KCN.

2.6.

Corneal pachymetry

It is important to compare the overall corneal thickness, the location of the apex, and thinnest point in each cornea. Values for similar superior and inferior locations in the same cornea and the thinnest point in either eye should not differ by more than 30 mm. In addition, general pachymetry thickness including the thinnest point should not differ by more than 10 mm between left and right eyes. The most important consideration is the displacement of the thinnest corneal location. In the abnormal cornea, the thinnest point (yellow) is displaced inferiorly or inferotemporally, and in the normal

cornea, the central area is of a generally uniform thickness (green). The colors relate to the scale depicted adjacent to the map. Ultrasonic CCT is usually measured at the geometric center or apex, which is not always the thinnest point and may vary from the apex thickness by over 10 mm.7 There is a significant correlation between the thinnest point and the geometric center, and these have been shown to be a significant feature of keratoconus.7 There is a significant variation in the central corneal thickness in the normal population, making a single-point measurement relatively useless. A more sensitive indicator of corneal pathology is the relationship between central and peripheral corneal thickness and how this varies in ectatic disease and edema. It is best displayed using the Pentacam corneal thickness spatial profile and the percentage thickness increase displays. Averaging the pachymetric values along each meridian allows the detection of meridia with the most significant change. Nasal is usually thickest, with temporal thinnest. Pachymetric progression indexes are calculated for each hemi-meridian with each point referenced to a normal database.6 The average thickness on the 1-mm, 2-mm, 3-mm,

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4-mm, and 5-mm concentric rings is represented as numerically as the average progression index (pachymetric progression indexavg). This has statistical significance when comparing normal patients to those with keratoconus (normal pachymetric progression indexavg <1.2 and corneal thickness spatial profile/percentage thickness increase lines within the 95% CI limits).

2.7. Automated detection program for subclinical keratoconus An automated screening program based on artificial intelligence has been developed using the GALILEI Dual-Scheimpflug Analyzer (Ziemer Ophthalmic Systems AG) that permits improved sensitivity of subclinical keratoconus detection. Fifty-six parameters derived from topography, elevation maps, pachymetry, and wavefront were analyzed and used to build an algorithm that could best distinguish subclinical keratoconus from a normal cornea. The posterior asphericity asymmetry index with a cutoff value of 21.5 mm and the corneal volume at 30.8 mm3 were identified as the 2 most discriminant variables among the parameters incorporated in the analysis for differentiating between normal corneas and those with forme fruste keratoconus.87,131 The asphericity asymmetry index was first described by Arce and colleagues in 2010 and is calculated using the best-fit toric and aspheric reference surface that has shown improved detection of subclinical keratoconus compared with the bestfit sphere.12,130 The system is based on an automated decisiontree algorithm that automatically selects variables that best discriminate the study population. This artificial intelligence system has the potential to improve the detection of mild ectatic corneas without requiring preliminary expertise in interpreting corneal imaging. It has been shown to detect forme fruste KCN with 93.7% sensitivity and 97.2% specificity.87,131

2.8.

Corneal biomechanics

Laser refractive surgery and corneal diseases that lead to ectasia cause changes in both the optical and mechanical properties of the cornea. Corneal biomechanics is described in terms of corneal hysteresis (CH) and corneal resistance factor (CRF). CH is the difference in pressure between the first and second applanation points and the CRF is related to the elastic properties of the cornea and calculated using a linear equation.103 Two machines are commercially available that can measure corneal biomechanical data in vivodthe Ocular Response Analyzer (Reichert Technologies), a dynamic bidirectional applanation device, and the Corvis ST (Oculus GmbH), a dynamic Scheimpflug analyzer device.86,110 The Ocular Response Analyzer device has 4 components: an infrared light source, a light intensity detector, a solenoid-driven air pump, and a pressure transducer.123 The infrared light shines on the cornea and the detector monitors the intensity of the reflected light. On alignment with the apex of the cornea, the air pump delivers a collimated stream of air, and the cornea begins to flatten (first corneal applanation). The intensity of the reflected light is maximal when the cornea flattens (second corneal applanation) which takes place within approximately

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20 ms. The Corvis ST allows imaging of the cornea’s dynamic deformation to a puff of air. A high-speed Scheimpflug camera records the deformation with full corneal cross-sections, which are then displayed in slow motion on a control panel.53 A precisely metered air pulse causes the cornea to flatten (the first corneal applanation). The cornea continues to move inward until reaching a point of the highest concavity. Because the cornea is viscoelastic, it rebounds from this concavity to another point of applanation (the second applanation) and then to its normal convex curvature. The Corvis ST records throughout the deformation process and therefore gains information concerning the cornea’s viscoelastic properties and stiffness, as well as recording standard tonometry and pachymetry data. In addition, Brillouin microscopy enables 3-dimensional mechanical imaging of the cornea allowing a quantitative assessment of the biomechanical properties of the tissue in high spatial resolution.122,131 Brillouin light scattering involves a spectral shift proportional to the longitudinal modulus of the tissue. The Brillouin frequency and elastic imaging can be recorded via confocal scanning using a 532-nm laser and an ultra-high resolution spectrometer. This technology is still under development but has the unique potential of providing high-resolution maps of corneal elasticity. A decrease in CH and CRF after myopic and hyperopic LASIK surgery has been reported in numerous studies.13,14,19,25,35,55,64,97,107,117,145 The weakening of the corneal structure during surgery is reflected in the reduction of these 2 biomechanical parameters. Eyes with KCN have a significantly lower CCT, CH, and CRF compared with normal eyes.43,103,110,125,126 This may be the consequence of distortion of the lamellar matrix in the stroma that no longer follows an orthogonal pattern, with regions of highly aligned collagen intermixed with regions there is little aligned collagen.37,93 In a retrospective study analyzing a large sample of keratoconic eyes, the corrected distance visual acuity was significantly correlated with the CRF, as well as with mean keratometry, IOP, and spherical equivalence. This correlation may be the consequence of the relationship between CRF and CCT.5 There is no significant difference between mild KCN and normal eyes in terms of CH.43,126 Some studies differentiate forme fruste KCN from normal eyes in terms of CCT: in the low CCT group (<500 mm), CH achieves 91% sensitivity whereas CRF it is only 81% and 87% sensitivity. Specificity was not studied.43

3.

Classification systems

Classifications based on morphology, disease evolution, ocular signs, and index-based systems of keratoconus have been proposed.

3.1.

Amsler-Krumeich

The oldest and most commonly used classification system, based on mean K-readings on the anterior curvature sagittal map, thickness at the thinnest location, and the refractive error of the patient (see Table 1).9,10 It fails to make use of

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current information available and technological advances in corneal imaging.

3.2.

Pathophysiology

5.1.

Histopathology and ultrastructural changes

ABCD

This new classification system looks at the anterior (A) and posterior, or back (B) radius of curvature taken from the 3.0-mm zone centered on the thinnest point, thinnest corneal (C) pachymetry, distance (D) best-corrected vision, and adds a modifier: “e“ for no scarring, “þ” for scarring that does not obscure iris details, and “þþ” for scarring that obscures iris details (see Table 2). It uses the central 3-mm zone centered on the thinnest point as this area represents the ectatic region better than a single-point parameter such as Kmax or maximal elevation.18 This grading system is relatively simple to use and has the advantage of grading each component independently, recognizing subclinical disease, and adding a stage 0 to better reflect an absence of possible disease. The grading system is dependent on tomography to produce both posterior data and thinnest point pachymetry.

4.

5.

Progression of disease

According to the Global Consensus on Keratoconus and Ectatic Diseases (2015), there is no consistent or clear definition of ectasia progression.46 They defined progression by a consistent change in at least 2 of the following parameters: steepening of the anterior or posterior corneal surface and thinning or changes in the pachymetric rate of change. They concluded that specific quantitative data to define progression are lacking and need further investigation. Kmax (maximum anterior curvature) is commonly used to detect progression, but there is a wealth of evidence to highlight its poor suitability for this purpose. It only represents a small area of the anterior curvature and fails to recognize the contribution of the posterior cornea and that progression can occur with no change or even a reduction in Kmax.40,88 Other parameters have been investigated as a means of detecting progression, such as the index of surface variants, the index of height decentration, visual acuity; manifest refraction, and CCT. The index of surface variants and index of height decentration are the most sensitive and specific, with the remainder unreliable and poorly correlated to the severity of disease.84,135 Several other parameters have been shown to document progression (change in posterior elevation maps, change in BCVA, reduction in apical thickness, or an increase in corneal asymmetry), but none of these methods are in the peer-reviewed literature.40 Different approaches have focused on the cone apex as a means of assessing progression. Mahmoud and colleagues developed an index that combines the cone location and magnitude with topographic information to improve the ability to detect KCN progression.88 In addition, the corneal thickness at the thinnest point, anterior and posterior radiuses of curvature taken from the central 3.0-mm optical zone centered on the thinnest point perform well as progression determinants.40

KCN is a multifactorial disease, with several biochemical processes contributing to its development.30,61 KCN is characterized by a central or paracentral stromal thinning, resulting in alteration in the corneal curvature.114 A decrease in keratocyte density, a reduction in the number of lamellae, and a degradation of fibroblasts in the stroma are observed.117,136 In addition, changes in the gross organization of the lamellae and an uneven distribution of collagen fibrillar mass, especially around the apex of the cone, occur in KCN.93 Although stromal thinning in KCN has been attributed to collagen degradation by proteolytic enzymes63,129 or decreased levels of proteinase inhibitors, it has also been proposed that collagen is not lost, but simply redistributed within the cornea by slippage between the lamellae.112 This latter mechanism is supported by the observation of reduced interlamellar adhesion, lamellar interlacing in the apex of KCN corneas, and a reduced number of lamellar insertions into Bowman layer.24,98 Sharply edged defects and interruptions in Bowman layer resulting from collagen bundles separation can be observed in this disease. Breaks in Bowman layer are usually filled with collagen derived from the stroma.114 A positive correlation was reported between the occurrence of breaks in Bowman layer and the extent of such thinning. Additional researchers have reported the reduction of the interfibrillar distance of collagen sheets and the increase of proteoglycans with abnormalities in their configuration as the condition evolves. These changes allow more contact between the collagen sheets and the proteoglycans, thus altering the stroma organization where alterations in interlamellar proteoglycans might contribute to slippage of the lamellae. Meek and colleagues, using synchrotron X-ray scattering patterns, confirmed that a gross rearrangement of vertical and horizontal collagen lamellae occurs in the apical region of advanced keratoconus.93 On the basis of these findings, Meek and colleagues proposed that the loss of structural integrity in the KCN cornea was caused by the presence of abnormal keratocytes and matrix proteins, and upregulated proteolysis triggered an unraveling of lamellae along their length and from their anchors at the limbus, with an opening of the lamellar bifurcations.93 This theory is supported by observations following riboflavin/UVA collagen cross-linking where the proposed cross-linkage of the tissue increases both the resistance of the stroma to enzymatic digestion and the cohesiveness between collagen fibrils and the noncollagenous matrix. Although the reduction in the number of lamellae within the affected region could correspond simply to a redistribution of the collagen within the cornea by slippage between the lamellae, as suggested by Polack112 almost 40 years ago, this explanation by itself does not seem to be enough to account for the stromal thinning, especially in light of more recent studies showing that the collagen lamellae in keratoconus corneas exhibit a significant decrease in number compared

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with controls135 and that KCN diminishes the amount of types I, III, V, and XII collagen. A decrease in the content of proteoglycans, including decorin, lumican, biglycan, and keratocan, was found in KCN. These proteoglycans interact with fibrillar collagens, making them biomechanically strong, refractive, and transparent.135 Furthermore, a decreased concentration of transforming growth factor beta (TGF-b) was observed in KCN. TGF-b can interact with several collagen types and proteoglycans. It is also involved in cell junction, facilitating contact between cells and ECM111,119 as well as lumican and keratocan proteins, as determined using highly sensitive mass spectrometric analysis.

5.2.

Postkeratoplasty keratoconus

There have been several case reports of recurrence of keratoconus-like pathology in the donor corneal button post keratoplasty.33,56 Findings described include increased astigmatism, subepithelial and anterior stromal scarring, corneal thinning, Vogt striae, and Munson sign.23,68,141 The incidence of recurrence of KCN is rare occurring at a rate of 5.4e11.7% with a latency of 17.9e21.00 years.106,113 There are 3 reported mechanisms:

5.2.1.

The donor route

The transmission of undiagnosed keratoconus in the donor cornea has been reported in several case reports.61,69,146 Longterm follow-up analysis is needed to further investigate this mechanism of transmission.

5.2.3.

KCN. It was first reported in 2002 that VSX1 mutations cause keratoconus and posterior polymorphous dystrophy. Two mutations in VSX1 (R166W and L159M) were reported to be associated with KCN. VSX1 is a member of the paired-like homeodomain transcription factors. This gene encodes a pair-like homeodomain protein that binds to the core of the locus control region of the red and green visual pigment gene cluster and may regulate expression of the cone opsin genes during embryonic development.31,102 It is expressed in several ocular tissues, including the nuclear layer of the retina, and embryonic craniofacial tissue.48,50,124 The expression of VSX1 in human or mouse cornea is still up for debate because many studies did not confirm the expression in cornea.31,50,102,149 Mouse models with the loss of VSX1 function did not reveal cornea-related phenotypes.124 Since the initial report in 2002, mutations in VSX1 have been demonstrated to be associated with and other corneal dystrophies21,34,38,41,95,96,104,121; however, many studies did not identify any potential VSX1 mutations in keratoconic patients.4,44,54,71,83,133,137 It remains unclear whether and how mutations in VSX1 contribute to the pathogenesis of KCN.4,22 It is suggested that mutations within VSX1 only affect a small number of those with KCN. This is consistent with the concept of genetic heterogeneity in keratoconus.

The host route

It is thought that remaining host keratocytes migrate into and repopulate the donor graft button producing abnormal collagen.2,100 Another possible route is residual basal epithelial cells secrete enzymes that lead to a loss of collagen.29,42 The host route seems to be the most accepted mechanism given the slow onset of pathological changes.

5.2.2.

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Mechanical trauma

Mechanical trauma such as contact lens wear and eye rubbing can lead to graft destabilization and recurrence of pathology.92 It is difficult to determine whether mechanical trauma is a major cause of recurrence as there are several case reports of recurrence where there has been no mechanical trauma or contact lens wear. Although each route can explain the recurrence in certain situations, further investigation and studies are required to understand the process in more detail.

6.2.

The dedicator of cytokinesis 9 (DOCK9) is a possible candidate gene that encodes a member of the DOCK protein family that possesses guanosine triphosphate/guanosine diphosphate exchange factor activity and specifically activates G-protein CDC42 involved in intracellular signaling networks (see Tables 3 and 4). The expression patterns were observed in keratoconic and nonkeratoconic corneas as well as in lymphoblastoid cell lines. Recently, mutation Gln754His was reported through sequencing candidate genes in a previously identified linkage locus, 13q32.140 This locus was first identified by Gajecka and colleagues in a large Ecuadorian family and was reported to segregate under an autosomal-dominant model.32,44 A mutation screening of 8 candidate genes within the 13q32 locus identified 3 different sequence variants in the DOCK9 gene. This locus contains additional genes, IPO5 (importin 5) and STK24 (serine/threonine kinase 24). All 3 genes are expressed in the human cornea, but detailed expression analyses are required to determine their role in KCN pathogenesis.138

6.3.

6.

Genetics

6.1.

Visual system homeobox 1 gene

Visual system homeobox 1, a protein that in humans is encoded by the Visual system homeobox 1 (VSX1) gene, plays a role in craniofacial and ocular development. VSX1 (OMIM 605020) is located on chromosome 20p11eq11,48,143 a linkage locus known for a corneal dystrophy called posterior polymorphous dystrophy (PPCD).50 PPCD has been associated with

Dedicator of cytokinesis 9

Transforming growth factor beta-induced gene

Another gene called transforming growth factor beta-induced (TGFbI ) gene, a cytokine, is responsible for many dominant corneal dystrophies.80 It is a potent regulator of the extracellular matrix formation, during tissue injury and repair. Recently, a novel nonsense mutation of TGFbI (G535X) was observed in a Chinese patient with KCN. Although mouse embryos that lack TGFb1 have normal signs of ocular development, TGFb1 is well known to be involved in corneal fibrosis and scar formation.143 An increase in TGFb pathway markers was seen in severe KCN cases.124

778

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Table 3 e Some of the genes reported in keratoconus using different genomic approaches

Table 4 e Candidate genes with mutations identified in patients with keratoconus

Genes

Reference

Genes VSX1 SOD1

Method

Population

Candidate gene Candidate gene Candidate gene

European European European

49,137,139

European European Korean, Chinese Korean, Japanese Northern Irish Ecuadorian Americans

39

SOD1 HGF

Candidate gene Candidate gene Candidate gene Candidate gene Linkage Linkage Linkage/candidate gene Candidate gene GWAS

144,145

RAB3GAP1

GWAS

LOX MPDZ-NFIB

Linkage/GWAS GWAS/candidate gene

BANP-ZNF469

GWAS/candidate gene

Americans Australian, Americans Australian, Americans Americans European, Asian, and Australian European, Asian, and Australian Americans European Korean

VSX1 TGFB1 COL4A3/ COL4AF FLG ZEB1 IL1A IL1B MIR184 DOCK9 CAST

COL5A1 Linkage/GWAS KRT72 Gene expression TIMP1, TIMP3, Gene expression CFL1, and BMP4

59 59,132

TGFBI

72 148

MIR184

94 55 32 79

COL4A3/ COL4A4 FLG

26,152

Craniofacial and ocular development. Major cytoplasmic antioxidant enzyme. Metabolizes superoxide radicals. Defense against oxidative stress. Cytokine interacting with an extracellular matrix protein playing a role in tissue injury and repair. Expressed in the cornea and lens, 30 untranslated region of 2 target genes, inositol polyphosphate phosphatase-like 1 and integrin beta 4. Involved in corneal healing after injury. Corneal collagen structure, function, and/or development during embryology. Apoptosis-related, genetic risk factor for atopic dermatitis.

78

28 3,85

3,85

77 99 73

6.4. Candidate genes associated with keratoconus superoxide dismutase 1 Several reports have suggested the potential contribution of superoxide dismutase 1 (SOD1) in KCN.144 SOD1 (OMIM 147450) maps to the 20p11.2 and encodes a major cytoplasmic antioxidant enzyme that metabolizes superoxide radicals and provides a defense against oxygen toxicity.101 Mutations in the SOD1 gene have been implicated in familial amyotrophic lateral sclerosis.101,118 No KCN phenotype is known in amyotrophic lateral sclerosis patients. To date, it is widely accepted that oxidative stress plays a critical role in the progression of KCN.118,133 Numerous reports have shown an accumulation of cytotoxic byproducts, mitochondrial DNA damage, and high levels of oxidative stress in keratoconus corneas.13,14,25,108 SOD1 was selected as a candidate gene and was examined in many studies; however, no mutations were found in KCN patients.38,44,121,133,144,145 It remains unclear whether SOD1 plays a role in the pathogenesis of keratoconus.

6.5.

Physiological role

miR-184

miR-184 is a microRNA. microRNAs (miRNAs) are small regulatory strands of RNA with 19e25 nucleotides in length. miRNAs bind to complementary sequences mostly in the 30 untranslated region of mRNA of target genes and lead to mRNA degradation or translational suppression. Recently, a mutation altering the miR-184 seed region was reported in a family with KCN and early-onset anterior polar cataract.55

This genomic region chr15q22-q25 was previously mapped as a KCN linkage locus.35,55 This 5 Mb linkage region was enriched in affected and unaffected family members using a custom sequence capture array from NimbleGen. The enriched DNA was sequenced using second-generation DNA sequencing (Genome Analyzer II from Illumina), identifying a mutation (r.57c>u) within miR-184.55 miR-184 is abundantly expressed in the cornea and lens.58,120 The authors hypothesized that miR-184 with the r.57c>u mutation fails to compete with another miRNAemiR-205 for overlapping target sites on the 30 untranslated region of 2 target genes, inositol polyphosphate phosphatase-like 1 (INPPL1) and integrin beta 4 (ITGB4) while these 2 target genes are involved in corneal healing after injury as the principal component of corneal basal epithelial hemidesmosomes.55 The finding of mutations in the seed region of miR-184 suggests that regulatory variants may directly impact transcriptional activity of key genes in corneal development and maintenance. It will be necessary to replicate this original finding in other studies of keratoconus.

6.6.

COL4A3 and COL4A4

Another hypothetical explanation for KCN pathogenesis could be related to underlying changes in the corneal collagen structure, function, and/or during embryonic development. COL4A3 and COL4A4 mutation analysis, however, revealed no pathogenic variants in 107 patients with KCN. Interestingly, significant allele frequency (genetic variants) was found in KCN cases that are D326Y variant in COL4A3 and M1237V and F1644F in COL4A4.39,134 Another mutation study on 15 Ecuadorian families with KCN identified missense mutations but none of them segregated in with family members. In parallel, 50 patients were investigated for COL8A1 and COL8A2, but yet again, no pathogenic mutation was detected.59 Thus, the role of collagen mutations remains debatable.4 Recent investigations have shown that the keratocyte apoptosis observed in keratoconic cornea emphasized the role of apoptotic processes in the pathogenesis. The apoptosis-related genetic risk factor for atopic dermatitis is filaggrin (FLG) mutations, expressed in the corneal epithelium.65 Loss of function of FLG alleles (R501X and 2282del4) was found in 5 KCN cases, suggesting the role of FLG in pathogenesis (see Tables 3 and 4).39

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6.7.

Genome-wide association studies and KCN

Multiple approaches have been used to identify common genetic factors that influence health and complex diseases. These include whole-genome sequencing, whole-exome sequencing, targeted resequencing, and functional studies in transcriptome level. The genetic etiology of many complex diseases, including Fuchs corneal dystrophy (FECD) and central corneal thickness, genome-wide association studies (GWASs) are useful tools to identify single nucleotide polymorphisms.16 The allele frequency differs significantly between cases and controls, which is taken into account in identifying the associated risk or protective nature of the genetic factors. Recent GWAS reveals few candidate genes identified including IL1B, CDH11, NUB1, COL27A1, and hepatocyte growth factor (HGF) RAB3GAP1 and LOX which are associated with risk factor for KCN.81,90,133 Interleukin 1 (IL1) released and triggered by the corneal epithelial cell during keratocyte apoptosis has been reported in 60% of keratoconic corneas.34,38,105,121 The guanosine triphosphatase activating protein subunit 1 (RAB3GAP1) gene mutations have been previously reported to be associated with Warburg Micro Syndrome with ocular disorders.121 HGF expression in corneal keratinocytes is upregulated in response to corneal injury, which has binding site for proinflammatory cytokine IL-6, which is elevated in KCN patients.4 The association of HGF with KCN suggests the potential involvement of inflammatory pathway. Global gene expression analysis was studied by Nielsen and colleagues, using microarrays for epithelial RNA from KCN patients and healthy controls.65 They observed differential expression of 471 genes of which 47 had significantly increased expression and 9 had reduced. Of these, 2 genes, lysyl oxidase (LOX ) and TIMP3 have been reported by numerous groups. A number of studies also found tissue inhibitor of metalloproteinase 1 (TIMP1) to be significantly reduced in KC corneas compared with normal.62,90,91 The lysyl oxidase group of enzymes has been shown to have lower expression and lesser activity in KC corneas. Katoh and colleagues found human angiopoietin isoform (ANGPTL7) messenger ribonucleic acid (mRNA) to be upregulated in keratoconic corneas indicating the involvement of the WNT/ beta-catenin signaling pathway.60 This is particularly interesting because a more recent study specifically on the Wnt signaling pathway demonstrates that Secreted frizzledrelated protein 1 (SFRP1) protein is highly expressed in KC epithelia at both the RNA and protein levels compared to normal. Apart from this, recent literature suggests that inflammatory molecules and abnormal levels of enzymes are present in subjects with KCN.74,75,134 Lema and colleagues have demonstrated that tears from KC patients have higher levels of interleukin 6 (IL6), tumor necrosis factor alpha (TNF-a), and MMP9 compared to healthy controls.74e76 A second GWAS in keratoconus was reported by Burdon and colleagues in Australia using pooled DNA in 97 keratoconus patients and 216 controls.26 Although no variants reached genome-wide significance, the most significant association was with SNP rs1014091, located upstream of the hepatocyte growth factor (HGF ) gene. Further genotyping additional tagging SNPs for the HGF gene identified another

779

SNP rs3735520 with significant association (P-value 9.9  10
7). This SNP was also found to be associated with serum HGF level in normal individuals (P value 0.036). Interestingly, the HGF gene has been associated with refractive error in several populations including Han Chinese and whites.147,153 The association of HGF with keratoconus suggests the potential involvement of inflammatory pathway.26

7.

Future directions

So far, genetic studies have suggested that KCN has clinical variability and may be linked to multiple chromosomal regions, consistent with polygenic mode of inheritance. Despite several genomic loci, mutations were reported for disease susceptibility, but lack of validation in larger numbers suggests genetic heterogeneity in KCN.150 The whole-exome or genome sequencing and GWAS are significantly useful techniques to explore novel genes and their functions in cellular pathways, which will provide the exact pathology of KCN, thereby aiding in designing better treatment modules. For example, LOX polymorphisms are associated with the treatment of collagen cross-linking to ensure that only “genotypically suitable” patients will undergo the gene-specific treatment, thus fulfilling the promise of personalized genomic medicine.28 Increasing our knowledge of genome sequence functionality will take us a step further in personalized medicine.

8.

Summary

KCN is a complex disorder and involves multiple genes and various mechanisms that contribute to the clinical disease etiology. As such, devising a gene therapy strategy for this disease is fraught with risk and requires a better molecular understanding of the disease; however, certain genes such as VSX1, DOCK9, or TGFB1 may have an essential, albeit sufficient role in the disease.27 Such a gene (or set of genes) delivered to the cornea via viral vectors or nanoparticles under the control of a corneaspecific promoter could hold promise for treatment.127,128 Recent genome technology development has enabled novel and high-throughput genetic approaches to study both Mendelian and complex disorders. Among these approaches, whole-exome or genome sequencing will allow identification of the causal mutations in multiplex families with keratoconus.15,20,140 The existing linkage data on these families will be tremendously useful in the interpretation of exome sequencing data. We expect to see more publications using this approach to study keratoconus in the near future. Another approach is to perform genome-wide association studies in a large number of keratoconus cases and controls using high-density SNP arrays. This approach has been shown to be very promising in keratoconus.26,78 Because GWAS studies need thousands of cases and controls, different laboratories will need to collaborate and combine each available dataset to identify genome-wide significant associations, which will help identify new genes involved in keratoconus pathogenesis. The available genome-wide genotype data will make it possible to study any potential gene-environment interactions.

780

9.

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Conclusions

Keratoconus is an asymmetrical bilateral corneal ectasia that causes significant visual morbidity. Early detection is now a feasible proposition with improved corneal tomography and methods of diagnosis and surveillance; however, current methodologies to measure corneal biomechanics are limited and require further investigation. A number of genes and candidate loci have been identified, and as we elucidate a greater understanding of the pathophysiology behind the condition, earlier diagnosis, new treatments, and improved outcomes will be possible.

10.

Methods of literature search

A literature search was conducted using the following search engines: MyAthens, OVID Medline, EMBASE, Cochrane database, Proquest, ScienceDirect, Springerlink, Oxford Journals, Google, and personal knowledge of the literature.

11.

Disclosures

No author has any conflict of interest in this paper, financial or otherwise.

references

1. Abad JC, Rubinfeld RS, Del Valle M, et al. Vertical D: a novel topographic pattern in some keratoconus suspects. Ophthalmology. 2007;114(5):1020e6 2. Abelson MB, Collin HS, Gillette JE. Recurrent keratoconus after keratoplasty. Am J Ophthalmol. 1980;90:672e8 3. Abu A, Frydman M, Marek D, et al. Deleterious mutations in the Zinc-Finger 469 gene cause brittle cornea syndrome. Am J Hum Genet. 2008;82(5):1217e22 4. Aldave AJ, Yellore VS, Salem AK, et al. No VSX1 gene mutations associated with keratoconus. Invest Ophthalmol Vis Sci. 2006;47:2820e2 5. Alio JL, Pinero DP, Aleson A, et al. Keratoconus-integrated characterization considering anterior corneal aberrations, internal astigmatism, and corneal biomechanics. J Cataract Refract Surg. 2011;37:552e68 6. Ambrosio R Jr, Alonson RS, Luz A, et al. Corneal-thickness spatial profile and corneal volume distribution: tomographic indices to detect keratoconus. J Cataract Refract Surg. 2006;32(11):1851e9 7. Ambrosio R Jr, Belin MW. Imaging of the cornea: topography vs tomography. J Refract Surg. 2010;26:847e9 8. Ambrosio R Jr, Nogueira LP, Caldas DL, et al. Evaluation of corneal shape and biomechanics before LASIK. Int Ophthalmol Clin. 2011;51(2):11e38 9. Amsler M. Le keratocone fruste au javal. Ophthalmologica. 1938;96:77e83 10. Amsler M. Keratocone classique et keratocone fruste, arguments unitaire. Ophthalmologica. 1946;111:96e101

11. Andreassen T, Simonsen AH, Oxlund H. Biomechanical properties of keratoconus and normal corneas. Exp Eye Res. 1980;31(4):435e41 12. Arce C. Qualitative and quantitative analysis of aspheric symmetry and asymmetry on corneal surfaces. In: Poster presented at: the ASCRS Symposium and Congress;. Boston. 13. Arnal E, Peris-Martinez C, Menezo JL, et al. Oxidative stress in keratoconus? Invest Ophthalmol Vis Sci. 2011;52:8592e7 14. Atilano SR, Coskun P, Chwa M, et al. Accumulation of mitochondrial DNA damage in keratoconus corneas. Invest Ophthalmol Vis Sci. 2005;46:1256e63 15. Bamshad MJ, Ng SB, Bigham AW, et al. sequencing as a tool for Mendelian disease gene discovery. Nat Rev Genet. 2011;12:745e55 16. Baratz KH, Tosakulwong N, Ryu E, et al. E2-2 protein and Fuchs’s corneal dystrophy. N Engl J Med. 2010;11: 1016e24 17. Barsam A, Petrushkin H, Brennan N, et al. Acute corneal hydrops in keratoconus: a national prospective study of incidence and management. Eye. 2015;29:469e74 18. Belin MW, Duncan J, Ambrosio R Jr, et al. A new tomographic method of grading keratoconus: the ABCD Grading system. Int J Kerat Ect Cor Dis. 2015;4(3):85e93 19. Belin MW, Khachikian SS. An introduction to understanding elevation-based topography: how elevation data are displayed e a review. Clin Exp Ophthalmol. 2009;37:14e29 20. Bick D, Dimmock D. Whole exome and whole genome sequencing. Curr Opin Pediatr. 2011;23:594e600 21. Bisceglia L, Ciaschetti M, De Bonis P, et al. VSX1 mutational analysis in a series of Italian patients affected by keratoconus: detection of a novel mutation. Invest Ophthalmol Vis Sci. 2005;46:39e45 22. Bisceglia L, De Bonis P, Pizzicoli C, et al. Linkage analysis in keratoconus: replication of locus 5q21.2 and identification of other suggestive Loci. Invest Ophthalmol Vis Sci. 2009;50:1081e6 23. Bourges JL, Savoldelli M, Dighiero P, et al. Recurrence of keratoconus characteristics: a clinical and histological follow-up analysis of donor grafts. Ophthalmology. 2003;110:1920e5 24. Bron AJ. Keratoconus. Cornea. 1988;7(3):163e9 25. Buddi R, Lin B, Atilano SR, et al. Evidence of oxidative stress in human corneal diseases. J Histochem Cytochem. 2002;50:341e51 26. Burdon KP, Macgregor S, Bykhovskaya Y, et al. Association of polymorphisms in the hepatocyte growth factor gene promoter with keratoconus. Invest Ophthalmol Vis Sci. 2011;52:8514e9 27. Burdon KP, Vincent A. Insights into keratoconus from a genetic perspective. Clin Exp Optom. 2013;2:146e54 28. Bykhovskaya Y, Li X, Epifantseva I, et al. Variation in the lysyl oxidase (LOX) gene is associated with keratoconus in family-based and case-control studies. Invest Ophthalmol Vis Sci. 2012;7:4152e7 29. Cannon DJ, Foster CS. Collagen cross linking in keratoconus. Invest Ophthalmol Vis Sci. 1978;17:63e5 30. Chaerkady R, Shao R, Scott SG, et al. The keratoconus corneal proteome: loss of epithelial integrity and stromal degeneration. J Proteomics. 2013;87:122e31 31. Chow RL, Volgyi B, Szilard RK, et al. Control of late off-center cone bipolar cell differentiation and visual signaling by the homeobox gene Vsx1. Proc Natl Acad Sci U S A. 2004;101:1754e9 32. Czugala M, Karolak JA, Nowak DM, et al. Novel mutation and three other sequence variants segregating with phenotype at keratoconus 13q32 susceptibility locus. Eur J Hum Genet. 2012;20:389e97

s u r v e y o f o p h t h a l m o l o g y 6 2 ( 2 0 1 7 ) 7 7 0 e7 8 3

33. Damaske E. Value of therapeutic soft contact lenses in the treatment of recurrent corneal erosions. Ophthalmologica. 1979;178(5):289e96 34. Dash DP, George S, O’Prey D, et al. Mutational screening of VSX1 in keratoconus patients from the European population. Eye (Lond). 2010;24:1085e92 35. Dash DP, Silvestri G, Hughes AE. Fine mapping of the keratoconus with cataract locus on chromosome 15q and candidate gene analysis. Mol Vis. 2006;12:499e505 36. Davidson AE, Hayes S, Hardcastle AJ, et al. The pathogenesis of keratoconus. Eye. 2014;28:189e95 37. Daxer A, Fratzl P. Collagen fibril orientation in the human corneal stroma and its implication in keratoconus. Invest Ophthalmol Vis Sci. 1997;38:121e9 38. De Bonis P, Laborante A, Pizzicoli C, et al. Mutational screening of VSX1, SPARC, SOD1, LOX, and TIMP3 in keratoconus. Mol Vis. 2011;17:2482e94 39. Droitcourt C, Touboul D, Ged C, et al. A prospective study of filaggrin null mutations in keratoconus patients with or without atopic disorders. Dermatology. 2011;4:336e41 40. Duncan JK, Belin MW, Borgstrom M. Assessing progression of keratoconus: novel tomographic determinants. Eye Vis (Lond). 2016;11:3e6 41. Eran P, Almogit A, David Z, et al. The D144E substitution in the VSX1 gene: a non-pathogenic variant or a disease causing mutation? Ophthalmic Genet. 2008;29:53e9 42. Feizi S, Javadi M, Kanavi M. Recurrent keratoconus in corneal graft after DALK procedure. J Ophthalmic Vis Res. 2012;7(4):328e31 43. Fontes BM, Ambrosio R Jr, Jardim D, et al. Corneal biomechanical metrics and anterior segment parameters in mild keratoconus. Ophthalmology. 2010;117:673e9 44. Gajecka M, Radhakrishna U, Winters D, et al. Localization of a gene for keratoconus to a 5.6-Mb interval on 13q32. Invest Ophthalmol Vis Sci. 2009;50:1531e9 45. Godefrooij DA, Ardine de Wit G, Uiterwaal CS, et al. Agespecific incidence and prevalence of keratoconus: a nationwide registration study. Am J Ophthalmol. 2017;175:169e72 46. Gomes JA, Tan D, Rapuano CJ, et al. Global consensus on keratoconus and ectatic disease. Cornea. 2015;34: 359e69 47. Hammerstein W. Zur genetic des keratoconus. Albrecht Von Graefes Arch Klin Exp Ophthalmol. 1974;190:293e308 48. Hayashi T, Huang J, Deeb SS. RINX(VSX1), a novel homeobox gene expressed in the inner nuclear layer of the adult retina. Genomics. 2000;67:128e39 49. Heon E, Greenberg A, Kopp KK, et al. VSX1: a gene for posterior polymorphous dystrophy and keratoconus. Hum Mol Genet. 2002;11(9):1029e36 50. Heon E, Mathers WD, Alward WL, et al. Linkage of posterior polymorphous corneal dystrophy to 20q11. Hum Mol Genet. 1995;4:485e8 51. Hofstetter HW. A keratoscopic survey of 13,395 eyes. Am J Optom Arch Am Acad Optom. 1959;36:3e11 52. Hom M, Bruce AS. Manual of contact lens prescribing and fitting. London, Butterworth-Heineman; 2006, pp 503e44 53. Hong J, Xu J, Wei A, et al. A new tonometerdthe Corvis ST tonometer: clinical comparison with non-contact and Goldmann applanation tonometers. Invest Ophthalmol Vis Sci. 2013;54(1):659e65 54. Hosseini SM, Herd S, Vincent AL, et al. Genetic analysis of chromosome 20-related posterior polymorphous corneal dystrophy: genetic heterogeneity and exclusion of three candidate genes. Mol Vis. 2008;14:71e80 55. Hughes AE, Bradley DT, Campbell M, et al. Mutation altering the miR-184 seed region causes familial keratoconus with cataract. Am J Hum Genet. 2011;89:628e33

781

56. Jahne M. Recurrence of keratoconus after keratoplasty. Z Artzl Fortbild (Jena). 1974;68(9):434e6 57. Karabatsas CH, Cook SD. Topographic analysis in pellucid marginal degeneration and keratoglobus. Eye (Lond). 1996;10:451e5 58. Karali M, Peluso I, Gennarino VA, et al. miRNeye: a microRNA expression atlas of the mouse eye. BMC Genomics. 2010;11:715 59. Karolak JA, Kulinska K, Nowak DM, et al. Sequence variants in COL4A1 and COL4A2 genes in Ecuadorian families with keratoconus. Mol Vis. 2011;17:827e43 60. Katoh Y, Katoh M. Comparative integromics on angiopoietin family members. Int J Mol Med. 2006;17:1145e9 61. Kenney CM, Brown DJ. The cascade hypothesis of keratoconus. Cont Lens Anterior Eye. 2003;26:139e46 62. Kenney MC, Chwa M, Atilano SR, et al. Increased levels of catalase and cathepsin V/L2 but decreased TIMP-1 in keratoconus corneas: Evidence that oxidative stress plays a role in this disorder. Invest Ophthalmol Vis Sci. 2005;46:823e32 63. Kenney M, Chwa M, Opbroek AJ, et al. Increased gelatinolytic activity in keratoconus cultures. A correlation to an altered matrix metalloproteinase-2/tissue inhibitor of metalloproteinase ratio. Cornea. 1994;13(2):114e24 64. Khachikian SS, Belin MW. Posterior elevation in keratoconus. Ophthalmology. 2009;116(4). 816, 816.e1; author reply: 16e17. 65. Kim WJ, Rabinowitz YS, Meisler DM, et al. Keratocyte apoptosis associated with keratoconus. Exp Eye Res. 1999;5:475e81 66. Krachmer JH. Pellucid marginal corneal degeneration. Arch Ophthalmol. 1978;96:1217e21 67. Krachmer JH, Feder RS, Belin MW. Keratoconus and related non-inflammatory corneal thinning disorders. Surv Ophthalmol. 1984;28:293e322 68. Kremer I, Eagle RC, Rapuano CJ, et al. Histological evidence of recurrent keratoconus seven years after keratoplasty. Am J Ophthalmol. 1995;119(4):511e2 69. Krivey D, McCormick S, Zaidman GW, et al. Postkeratoplasty keratoconus in a non-keratoconic patient. Am J Ophthalmol. 2001;131(5):653e4 70. Krumeich JH, Kezirian GM. Circular keratotomy to reduce astigmatism and improve vision in stage I and II keratoconus. J Refract Surg. 2009;25(4):357e65 71. Lam HY, Wiggs JL, Jurkunas UV. Unusual presentation of presumed posterior polymorphous dystrophy associated with iris heterochromia, band keratopathy, and keratoconus. Cornea. 2010;29:1180e5 72. Lechner J, Dash DP, Muszynska D, et al. Mutational spectrum of the ZEB1 gene in corneal dystrophies supports a genotypephenotype correlation. Invest Ophthalmol Vis Sci. 2013;54(5):3215e23 73. Lee J-E, Oum BS, Choi HY, et al. Evaluation of differentially expressed genes identified in keratoconus. Mol Vis. 2009;15:2480e7 74. Lema I, Duran JA. Inflammatory molecules in the tears of patients with keratoconus. Ophthalmology. 2005;112:654e9 75. Lema I, Duran JA, Ruiz C, et al. Inflammatory response to contact lenses in patients with keratoconus compared with myopic subjects. Cornea. 2008;27:758e63 76. Lema I, Sobrino T, Duran JA, et al. Subclinical keratoconus and inflammatory molecules from tears. Br J Ophthalmol. 2009;93:820e4 77. Li X, Bykhovskaya Y, Canedo ALC, et al. Genetic association of COL5A1 variants in keratoconus patients suggests a complex connection between corneal thinning and keratoconus. Invest Ophthalmol Vis Sci. 2013;54(4): 2696e704

782

s u r v e y o f o p h t h a l m o l o g y 6 2 ( 2 0 1 7 ) 7 7 0 e7 8 3

78. Li X, Bykhovskaya Y, Haritunians T, et al. A genome-wide association study identifies a potential novel gene locus for keratoconus, one of the commonest causes for corneal transplantation in developed countries. Hum Mol Genet. 2012;21:421e9 79. Li X, Bykhovskaya Y, Tang YG, et al. An association between the calpastatin (CAST) gene and keratoconus. Cornea. 2013;32(5):696e701 80. Li X, Rabinowitz YS, Tang YG, et al. Two-stage genome-wide linkage scan in keratoconus sib pair families. Invest Ophthalmol Vis Sci. 2006;47:3791e5 81. Li X, Yang H, Rabinowitz YS. Keratoconus: Classification scheme based on videokeratography and clinical signs. J Cataract Refract Surg. 2009;35(9):1597e603 82. Liskova P, Ebenezer ND, Hysi PG, et al. Molecular analysis of the VSX1 gene in familial keratoconus. Mol Vis. 2007;13:1887e91 83. Lively GD, Koehn D, Hedberg-Buenz A, et al. Quantitative trait loci associated with murine central corneal thickness. Physiol Genomics. 2010;2:281e6 84. Lopes BT, Ramos IC, Faria-Correia T, et al. Correlation of topometric and topographic indices with visual acuity in patients with KCN. J Kerat Ect Cor Dis. 2012;1(3):167e72 85. Lu Y, Vitart V, Burdon KP, et al. Genome-wide association analyses identify multiple loci associated with central corneal thickness and keratoconus. Nat Genet. 2013;45(2):155e63 86. Luce DA. Determining in vivo biomechanical properties of the cornea with an ocular response analyzer. J Cataract Refract Surg. 2005;31(1):156e62 87. Maeda N, Klyce SD, Smolek MK, et al. Automated keratoconus screening with corneal topography analysis. Invest Ophthalmol Vis Sci. 1994;35(6):2749e57 88. Mahmoud AM, Nunez MX, Blanco C, et al. Expanding the cone location and magnitude index to include corneal thickness and posterior surface information for the detection of KCN. Am J Ophthalmol. 2013;156(6):1102e11 89. Mannion LS, Tromans C, O’Donnell C. Reduction in corneal volume with severity of keratoconus. Curr Eye Res. 2011;36(6):522e7 90. Manolio TA. Genome-wide association studies and assessment of the risk of disease. N Engl J Med. 2010;2:166e76 91. Matthews FJ, Cook SD, Majid MA, et al. Changes in the balance of the tissue inhibitor of matrix metalloproteinases (TIMPs)-1 and -3 may promote keratocyte apoptosis in keratoconus. Exp Eye Res. 2007;84:1125e34 92. McGhee CNJ. 150 years of practical observations in the conical cornea e what have we learned? Clin Exp Ophthalmol. 2009;37(2):160e76 93. Meek KM, Tuft SJ, Huang Y, et al. Changes in collagen orientation and distribution in keratoconus corneas. Invest Ophthalmol Vis Sci. 2005;46:1948e56 94. Mikami T, Meguro A, Teshigawara T, et al. Interleukin 1 beta promoter polymorphism is associated with keratoconus in a Japanese population. Mol Vis. 2013;19:845e51 95. Mintz-Hittner HA, Semina EV, Frishman LJ, et al. VSX1 (RINX) mutation with craniofacial anomalies, empty sella, corneal endothelial changes, and abnormal retinal and auditory bipolar cells. Ophthalmology. 2004;111:828e36 96. Mok JW, Baek SJ, Joo CK. VSX1 gene variants are associated with keratoconus in unrelated Korean patients. J Hum Genet. 2008;53:842e9 97. Montalban P, Alio JL, Javaloy J, et al. Correlation of anterior and posterior corneal shape in keratoconus. Cornea. 2013;32(7):916e21

98. Morshige N, Wahlert A, Kenney M, et al. Second harmonic imaging microscopy of normal and keratoconus cornea. Invest Ophthalmol Vis Sci. 2007;48(3):1087e94 99. Nielsen K, Birkenkamp-Demtro¨der K, Ehlers N, et al. Identification of differentially expressed genes in keratoconus epithelium analyzed on microarrays. Invest Ophthalmol Vis Sci. 2003;44(6):2466e76. 100. Nirankari VS, Karseh J, Bastion F, et al. Recurrence if keratoconus in a donor cornea 22yrs after successful keratoplasty. Br J Ophthalmol. 1983;67(1):23e8 101. Noor R, Mittal S, Iqbal J. Superoxide dismutaseeapplications and relevance to human diseases. Med Sci Monit. 2002;8:210e5 102. Ohtoshi A, Wang SW, Maeda H, et al. Regulation of retinal cone bipolar cell differentiation and photopic vision by the CVC homeobox gene Vsx1. Curr Biol. 2004;14:530e6 103. Ortiz D, Pinero D, Shabayek MH, et al. Corneal biomechanical properties in normal, post-laser in situ keratomileusis, and keratoconic eyes. J Cataract Refract Surg. 2007;33:1371e5 104. Paliwal P, Singh A, Tandon R, et al. A novel VSX1 mutation identified in an individual with keratoconus in India. Mol Vis. 2009;15:2475e9 105. Paliwal P, Tandon R, Dube D, et al. Familial segregation of a VSX1 mutation adds a new dimension to its role in the causation of keratoconus. Mol Vis. 2011;17:481e5 106. Patel SV, Malta JB, Banitt MR, et al. Recurrent ectasia in corneal grafts and outcomes if repeat keratoplasty for keratoconus. Br J Ophthalmol. 2009;93(2):191e7 107. Pathak D, Nayak B, Singh M, et al. Mitochondrial complex 1 gene analysis in keratoconus. Mol Vis. 2011;17: 1514e25 108. Pearson AR, Soneji B, Sarvananthan N, et al. Does ethnic influence the incidence or severity of keratoconus. Eye. 2000;14(4):625e8 109. Perry HD, Buxton JN, Fine BS. Round and oval cones in keratoconus. Ophthalmology. 1980;87:905e9 110. Pinero DP, Alcon N. In vivo characterization of corneal biomechanics. J Cataract Refract Surg. 2014;40:870e87 111. Pinero DP, Alio´ JL, Aleso´n A, Vergara ME, Miranda M. Corneal volume, pachymetry and correlation of anterior and posterior corneal shape in subclinical and different stages of clinical KCN. J Cataract Refract Surg. 2010;36(5):814e25 112. Polack FM. Contributions of electron microscopy to the study of corneal pathology. Surv Ophthalmol. 1976;20:375e414 113. Pramanik S, Musch DC, Sutphin JE. Extended long-term outcomes of penetrating keratoplasty for keratoconus. Ophthalmology. 2006;113(9):1633e8 114. Rabinowitz YS. Keratoconus. Surv Ophthalmol. 1998;42:297e319 115. Rabinowitz YA, Rasheed K. KISA% index: a quantitive videokeratography algorithm embodying minimal topographic criteria for diagnosis keratoconus. J Cataract Refract Surg. 1999;25(10):1327e35 116. Reinstein DZ, Archer TJ, Gobbe M. Corneal epithelial thickness profile in the diagnosis of keratoconus. J Refract Surg. 2009;25(7):604e10 117. Romero-Jimenez M, Santodomingo-Rubido J, Wolffsohn JS. Keratoconus: a review. Cont Lens Anterior Eye. 2010;33:157e66 118. Rosen DR, Siddique T, Patterson D, et al. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature. 1993;362: 59e62 119. Runager K, Basaiawmoit RV, Deva T, et al. Human phenotypically distinct TGFBI corneal dystrophies are linked

s u r v e y o f o p h t h a l m o l o g y 6 2 ( 2 0 1 7 ) 7 7 0 e7 8 3

120.

121.

122.

123.

124.

125.

126.

127.

128.

129.

130.

131.

132.

133.

134.

to the stability of the fourth FAS1 domain of TGFBIp. J Biol Chem. 2011;286:4951e8 Ryan DG, Oliveira-Fernandes M, Lavker RM. MicroRNAs of the mammalian eye display distinct and overlapping tissue specificity. Mol Vis. 2006;12:1175e84 Saee-Rad S, Hashemi H, Miraftab M, et al. Mutation analysis of VSX1 and SOD1 in Iranian patients with keratoconus. Mol Vis. 2011;17:3128e36 Scarcelli G, Pineda R, Yun SH. Brillouin optical microscopy for corneal biomechanics. Invest Ophthalmol Vis Sci. 2012;53(1):185e90 Schweitzer C, Roberts CJ, Mahmoud AM, et al. Screening of forme fruste keratoconus with the ocular response analyzer. Invest Ophthalmol Vis Sci. 2010;51:2403e10 Semina EV, Mintz-Hittner HA, Murray JC. Isolation and characterization of a novel human paired- like homeodomain-containing transcription factor gene, VSX1, expressed in ocular tissues. Genomics. 2000;63:289e93 Shah S, Laiquzzaman M. Comparison of corneal biomechanics in pre and post refractive surgery and keratoconic eyes by the Ocular Response Analyzer. Cont Lens Anterior Eye. 2009;32:129e32 Shah S, Laiquzzaman M, Bhoiwani R, et al. Assessment of the biomechanical properties of the cornea with the ocular response analyzer in normal and keratoconic eyes. Invest Ophthalmol Vis Sci. 2007;48:3026e31 Sharma A, Rodier JT, Tandon A, et al. Attenuation of corneal myofibroblast development through nanoparticlemediated soluble transforming growth factor-beta type II receptor (sTGFbetaRII) gene transfer. Mol Vis. 2012;18:2598e607 Sharma A, Tovey JC, Ghosh A, et al. AAV serotype influences gene transfer in corneal stroma in vivo. Exp Eye Res. 2010;3:440e8 Sherwin T, Brookes NH, Loh IP, et al. Cellular incursion into Bowman’s membrane in the peripheral cone of the keratoconic cornea. Exp Eye Res. 2002;74(4):473e82 Smadja D, Santhiago MR, Mello GR, et al. Influence of the reference surface shape for discriminating between normal corneas, subclinical keratoconus and keratoconus. J Refract Surg. 2013;29(4):274e81 Smadja D, Touboul D, Cohen A, et al. Detection of subclinical keratoconus using an automated decision tree classification. Am J Ophthalmol. 2013;156(2):237e46.e1 Stabuc-Silih M, Ravnik-Glavac M, Glavac D, et al. Polymorphisms in COL4A3 and COL4A4 genes associated with keratoconus. Mol Vis. 2009;15:2848e60 Stabuc-Silih M, Strazisar M, Hawlina M, et al. Absence of pathogenic mutations in VSX1 and SOD1 genes in patients with keratoconus. Cornea. 2010;29:172e6 Sutton G, Madigan M, Roufas A, et al. Secreted frizzledrelated protein 1 (SFRP1) is highly upregulated in keratoconus epithelium: A novel finding highlighting a new potential focus for keratoconus research and treatment. Clin Exp Ophthalmol. 2010;38:43e8

783

135. Suzuki M, Amano S, Honda N, et al. Longitudinal changes in corneal irregular astigmatism and visual acuity in eyes with KCN. Jpn J Ophthalmol. 2007;51(4):265e9 136. Takahashi A, Nakayasu K, Okisaka S, et al. Quantitative analysis of collagen fiberin keratoconus. Nihon Ganka Gakkai Zasshi. 1990;94:1068e73 137. Tang YG, Picornell Y, Su X, et al. Three VSX1 gene mutations, L159M, R166W, and H244R, are not associated with keratoconus. Cornea. 2008;27:189e92 138. Tang YG, Rabinowitz YS, Taylor KD, et al. Genomewide linkage scan in a multigeneration Caucasian pedigree identifies a novel locus for keratoconus on chromosome 5q14.3-q21.1. Genet Med. 2005;7:397e405 139. Tanwar M, Kumar M, Nayak B, et al. VSX1 gene analysis in keratoconus. Mol Vis. 2010;16:2395e401 140. Teer JK, Mullikin JC. Exome sequencing: the sweet spot before whole genomes. Hum Mol Genet. 2010;19:R145e51 141. Thalasselis A, Etcheparaborda J. Recurrent keratoconus 40 years after keratoplasty. Ophthalmic Physiol Opt. 2002;22(4):330e2 142. Tourquetti L, Ferrara P. Corneal asphericity changes after implantation of intrastromal corneal ring segments in Keratoconus. J Emmetropia. 2010;1:178e81 143. Tyynismaa H, Sistonen P, Tuupanen S, et al. A locus for autosomal dominant keratoconus: linkage to 16q22.3-q23.1 in Finnish families. Invest Ophthalmol Vis Sci. 2002;43:3160e4 144. Udar N, Atilano SR, Brown DJ, et al. SOD1: a candidate gene for keratoconus. Invest Ophthalmol Vis Sci. 2006;47:3345e51 145. Udar N, Atilano SR, Small K, et al. SOD1 haplotypes in familial keratoconus. Cornea. 2009;28:902e7 146. Unal M, Yucel L, Akar Y, et al. Recurrence of keratoconus in 2 corneal grafts after penetrating keratoplasty. Cornea. 2007;26(3):322e4 147. Veerappan S, Pertile KK, Islam AF, et al. Role of the hepatocyte growth factor gene in refractive error. Ophthalmology. 2010;117:239, 245.e1ee2. 148. Wang Y, Jin T, Zhang X, et al. Common single nucleotide polymorphisms and keratoconus in the Han Chinese population. Ophthalmic Genet. 2013;34(3):160e6 149. Watson TC, Chow RL. Absence of Vsx1 expression in the normal and damaged mouse cornea. Mol Vis. 2011;17:737e44 150. Wheeler J, Hauser M, Afshari NA, Allingham RR, Liu Y. The genetics of keratoconus: A review. Reprod Syst Sex Disord 2012 151. Wilson SE, Lin DT, Klyce SD. Corneal topography of keratoconus. Cornea. 1991;10(1):2e8 152. Yanovitch T, Li YJ, Metlapally R, et al. Hepatocyte growth factor and myopia: genetic association analyses in a Caucasian population. Mol Vis. 2009;15:1028e35 153. Zhou L, Sawaguchi S, Twining SS, et al. Expression of degradative enzymes and protease inhibitors in corneas with keratoconus. Invest Ophthalmol Vis Sci. 1998;39:1117e24