11.1.2.3  Differential Diagnosis: BMD

AVMD. This is a relatively mild disease compared to BMD and occurs later in life (fourth to fifth decade). There is a decrease in central visual acuity and metamorphopsia with slow progression. The vitelliform lesions of AVMD are smaller (1/4 to 1/3 disk diameter in size) with a central area of hyperpigmentation surrounded by a region of atrophy. There is no secondary retinal detachment that forms. On IVFA, the central aspects of the lesions block and the marginal areas demonstrate window defects. Color vision and ERG are generally normal for the age. Critically, the EOG is also normal. Cone dystrophy. Patients with cone dystrophies are differentiated from those with BMD on the basis of the normal full-field ERG findings in BMD, the abnormal cone ERG findings in cone dystrophies, and the generally normal EOG findings in the latter. Patients with BMD do not have the profound color vision deficits of patients with cone dystrophies and there is typically no photophobia. STGD macular dystrophy with large central flecks could give the mistaken appearance of BMD. The atrophic lesions of late STGD and BMD can appear similar. The EOG can distinguish patients with BMD from those with STGD/ FF. Butterfly Pattern Dystrophy. This macular dystrophy demonstrates foveal pigmentary changes that do not simulate BMD. Nevertheless, like BMD, the ERG is normal and the EOG is reduced. Commonly inherited in autosomal dominant fashion like BMD, the disease can be differentiated by both the clinical anatomic features of the macular lesion and the identification of other family members who express a butterfly pattern dystrophy. Basal laminar drusen. Dense collections of basal laminar drusen in association with soft (exudative) drusen of age-related maculopathy can give the appearance of a vitelliform like lesion and pseudohypopyon and atrophy has occurred in this condition. This is differentiated from BMD by the age and the presence of other drusen in the AMD patient, and a normal EOG. However, some patients with AMD may have late onset BMD and the EOG would be expected to be abnormal. Sorsby macular dystrophy. Pseudoin­ flammatory macular dystrophy is an exudative disciform degeneration of the macula that typically emerges later in life (fourth to fifth decades). There is no vitelliform lesion and the EOG and ERG are normal. North Carolina macular dystrophy. The atrophic macular changes can simulate those found in late BMD. The

abnormal EOG of BMD is a differentiating feature.

Acquired pigment epithelial detachments. These are distinguished by the lack of a vitelliform presentation, the lack of a family history, and the normal EOG findings.

11.1.2.4  Inherited Forms: BMD

BEST1 mutations have been associated with both BMD and AVMD, which may represent a spectrum of phenotypic disease expression. Certain mutations in the peripherin gene (PRPH2) have also been associated with AVMD but not BMD. BEST1 mutations have also been identified in three other conditions that suggest an involvement in ocular development [23]. BEST1 mutations have been identified in a recessive or null form called autosomal recessive bestrophinopathy (ARB), in which high hyperopia and shallow anterior chambers are found in addition to retinal degeneration. They have also been identified in autosomal dominant vitreoretinochoroidopathy and autosomal dominant microcornea, rod–cone dystrophy, early onset cataract, and posterior staphyloma syndrome. Again, there is broad genotypic and phenotypic heterogeneity in BMD that depends upon the gene, the mutation in that gene, and the impact on the cells in which the mutant gene(s) are expressed.

11.1.3  Juvenile X-Linked Retinoschisis

JXRS was first defined by Haas [24]. In male patients with JXRS, a characteristic or pathognomonic starshaped or stellate pattern of retinal cystic spaces surrounds the fovea in all cases (Fig. 11.3). These changes are caused by splitting in the inner retinal region or nerve fiber layer region. There is commonly an associated radial plication of the inner limiting membrane extending from the fovea. There is a variation in the expression and the extent of peripheral retinal schisis cavities.

11.1.3.1  Clinical Features: JXRS

Affected males are commonly identified in the first decade of life (5–10 years) through early vision screening exams. Presenting visual acuity is generally around 20/40 to 20/60 and progressively deteriorates

Fig. 11.3  Fundus appearance in JXRS. Note the radial pattern of parafoveal intraretinal schisis cavities

to generally stabilize by the third decade. All individuals with JXRS have the characteristic foveal cystic changes. With aging and progression of macular changes, older individuals (sixth to seventh decades) rarely have visual acuity better than 20/200. Over time, the foveal microcystic cavities can enlarge and coalesce giving the appearance of a macular hole and foveal atrophy sometimes associated with foveal pigmentary changes. Peripheral schisis cavities are common in the inferotemporal region. As peripheral retinal schisis cavities emerge, it is possible for complications that include vitreous hemorrhage, inner and outer retinal holes, and retinal detachment that emerges due to the latter or due to traction in retinal bands on nonschitic retina, to occur. Leukocoria can occur if the schisis is sufficiently severe. There are nonspecific late pigmentary changes that occur in the regions of schisis cavities indicative of outer retinal changes. Schisis cavities commonly embrace sclerotic retinal vessels that give the cavity a dendritic appearance. The vitreous can show liquefaction, strands, traction bands, and posterior and anterior vitreous detachments. The prevalence of JXRS is estimated to be within the range of 1/7,000 and 1/2,800.

11.1.3.2  Diagnostic Features: JXRS

Color vision disturbances, when identified, can include proton or tritan deficits. Dark adaptation can be normal or extend to the cone threshold plateau. Visual fields show a relative central scotoma mapping the region of foveal schisis and absolute scotomas in the region corresponding to the location of peripheral retinal schisis.

Superonasal scotomas are common given the inferotemporal peripheral schisis cavities. In affected male patients, the ERG is abnormal with a characteristic “negative” ERG to scotopic stimuli. In this negative ERG pattern, the a-wave (photoreceptor driven negative component) is usually normal and the b-wave (bipolar and Muller cell-driven positive component) is substantially attenuated, with a coincident reduction in the b/a wave ratio. Photopic ERG signals are also affected. These finds suggest that primary signal generation by the photoreceptors occurs normally, but secondary signal generation by the bipolar cells is affected in JXRS. ERG signal changes follow disease progression. Carrier females do not have ERG changes. The EOG is initially normal, but may become abnormal as the disease progresses to involve the outer retina and RPE. The IVFA most commonly shows no leakage or staining into the foveal cystic cavities. Late in the disease, the IVFA may show window defects or blocking due to the pigment in the foveal area, or regions of capillary nonperfusion in areas of peripheral retinal schisis associated with pigmentary changes.

11.1.3.3  Differential Diagnosis: JXRS

Goldmann–Favre vitreotapetoretinal syndrome. Patients with Goldmann–Favre syndrome have a combination of retinal and psychophysical findings similar to both JXRS and RP. There is early onset nyctalopia in the first decade with Goldmann–Favre, which is not present in JXRS. The prominent foveal cysts of JXRS do not occur in Goldmann–Favre, where the foveal changes are more microcystic in nature. Peripheral schisis cavities also occur in Goldmann–Favre, as do more extensive vitreal changes. A differentiating feature from JXRS is a diffuse retinal pigmentary degeneration where pigmented spots are common as opposed to the bone spicule changes seen in RP. In Goldmann–Favre syndrome, and unlike the characteristic negative ERG of JXRS, the scotopic and photopic ERGs are markedly attenuated and the remaining responses have a dominant S-cone component. Goldmann–Favre and enhanced S-cone syndrome share this feature, which suggests common molecular genetic origins. During IVFA, there is extensive leak of perifoveal capillaries in Goldmann–Favre which is rare in JXRS. Also, JXRS is an X-linked ­dystrophy whereas Goldmann–Favre is an autosomal ­recessive condition. Wagner vitreoretinal syndrome.

The macula may be involved in Wagner syndrome of myopia, vitreal syneresis, and retinal degeneration. The characteristic negative ERG changes of JXRS are readily distinguished from the normal to subnormal ERG findings in individuals with Wagner syndrome. Also, Wagner has an autosomal dominant mode of inheritance and does not have the foveal cystic changes found in JXRS. Cystoid macular edema. CME is readily differentiated from JXRS because there is commonly no late leakage into the cystic foveal schisis cavities in JXRS, no peripheral schisis cavities in CME, no full-field ERG changes, and no X-linked inheritance in CME.

Degenerative peripheral retinoschisis. This syndrome, which predominantly affects the inferotemporal retina, occurs in older individuals (older than 40) in the absence of foveal schisis changes and has no known genetic causes.

11.1.3.4  Inherited Forms: JXRS

Retinoschisin 1 (RS1) is the only known disease gene underlying JXRS. There is a broad phenotypic heterogeneity in JXRS that depends upon the mutation and its impact on the photoreceptor cells in which the mutant gene is expressed.

11.1.4  Advanced Clinical Assessment

of Juvenile Macular Degenerations

A lack of surrogate markers for assessing the therapeutic efficacy has long hindered research in hereditary macular degenerations [25]. For example, while the full-field ERG is useful for discriminating the more localized pathology of STGD from widespread forms of CRD, it is of little value in the early detection of macular disease and for following patients in longitudinal studies and clinical trials. Visual acuity is critically dependent on the location of degeneration, so that acuity may remain fairly constant in a patient despite extensive extrafoveal atrophy. Traditional static and perimetric fields can be useful for following relative scotomas, but are limited by the need to maintain accurate fixation. Thus, accurate visual field testing typically requires visual acuity of better than 20/100.

Because of the shortcomings of traditional measures in patients with macular degeneration, it has

been important to develop a new set of advanced technologies to address the geographic nature of the disease. The developments have been primarily in three areas; electrophysiology, with the multifocal ERG (mfERG) [26], imaging, with ultrahigh resolution spectral domain optical coherence tomography (SD-OCT), and psychophysical, with fundus-based perimetry. These new modalities can be used for the assessments of efficacy in upcoming treatment trials for hereditary macular degenerations.

The initial electrophysiological technique developed for the objective assessment of the macula was the focal ERG [27, 28]. The focal ERG should be conducted with direct visualization of the fundus to ensure that the response originates from the area of interest [28]. When evaluating acuity loss, the region of interest is typically the fovea. With a test light as small as 4°, the stimulus is typically flickered at a frequency higher than the rod fusion frequency and is concentric with a more intense, steady surround. The sensitivity and utility of this test for documenting the retinal basis of acuity loss has been reviewed previously [29]. It is generally felt that the foveal response drops below the lower normal amplitude limit when the visual acuity is 20/50 or less due to macular degeneration [30]. In STGD, the amplitude may be below the lower limit of normal prior to a substantial loss of acuity, making this an important prognostic test [27, 30, 31].

The mfERG has the advantage of simultaneously measuring retinal function at dozens of locations throughout the macula [26]. With the development of a fundus camera-based stimulus delivery system, it is now possible to monitor fundus position while testing. This is particularly important for patients with STGD, who may use a preferred eccentric locus of fixation. In practice, it is useful to assess the fixation behavior of the patient through a fundus camera prior to mfERG testing. It is then possible to correct for eccentric fixation during testing. Maintaining the position of the optic disk helps assure that the stimulus pattern is centered on the fovea. The fundus of the left eye of a 17-year-old female with STGD is shown (Fig. 11.4a). Characteristic flecks (LF) are scattered throughout the posterior pole, but are not present in the central macula, which has an atrophic appearance (more evident on fluorescein angiography). The mfERG from the same eye is characteristic of responses in a patient with recently-diagnosed STGD. Despite only a modest reduction in acuity, responses from the central 10° are

Fig. 11.4  Phenotypic presentation in STGD. (a) Fundus of a 17-year-old patient with STGD showing lipofuscin accumulation throughout the posterior pole. (b) Humphrey static perimetric fields from central 20° show loss of sensitivity corresponding

to the mfERG regional loss. (c) mfERG shows a selective loss of responses from fovea. (d) Three-dimensional representation of mfERG responses shown in (c)

severely reduced in amplitude, while responses from outside the macula are normal (Figs. 11.4c, d). The pattern of loss in the mfERG corresponds to that seen in the visual field (Fig. 11.4b).

Ultrahigh resolution SD-OCT utilizes Fourier analysis of interference patterns from high speed laser scans. Because of the high acquisition speeds, these new generation devices are particularly well suited for patients with unsteady fixation [32, 33]. Some devices, such as the Heidelberg Spectralis™, have integrated fundus tracking to aid in the registration of scans. In normal subjects, seven distinct retinal layers [34] are readily measurable (Fig. 11.5a). In patients with hereditary macular degeneration, SD-OCT reveals the abnormal ultrastructure of the retina. In a 40-year-old patient with STGD, for example, there are clear undulations in the photoreceptor layer resulting from the yellow flecks throughout the macula (Fig. 11.5b). In more advanced disease, it may be possible to visualize the extent of

photoreceptor-RPE loss and follow progression over time. In a 51-year-old patient with STGD an atrophic region measuring 5.7 mm in diameter is found (Fig. 11.5c). SD-OCT clearly reveals the transition from the photoreceptors to atrophy. Interestingly, this patient retains a patch of photoreceptors in the fovea and still has 20/20 vision in this eye. Foveal sparing is also evident in the mfERG, where the foveal response in the STGD patient (Fig. 11.6a) is comparable to that in the normal subject (Fig. 11.6b), while the surrounding parafoveal region shows substantially reduced responses.

Because of poor fixation, traditional static and kinetic perimetry is often unreliable in patients with hereditary macular degeneration. With the advent of confocal scanning laser ophthalmoscopes, fundusrelated microperimetry incorporating automatic eye trackers circumvent the limitations of traditional perimetry. A commercially available instrument, the MP1 Microperimeter™ (Nidek Technologies, Padova, Italy)

Fig. 11.5  Spectral domain optical coherence tomography. (a) SD-OCT from normal subject. Seven distinct retinal layers can be readily visualized in this horizontal scan through the fovea.

(b) Horizontal scan through the fovea of the patient with STGD. Note the disruption of the photoreceptor and RPE layers due to deposits. (c) Horizontal scan through the fovea of the patient with STGD and large atrophic region. Note the preservation of the patch of photoreceptors in the fovea underlying 20/20 visual acuity

a

b

c

allows for a detailed examination of the macular function and exact, point-by-point correlation of the fundus appearance and retinal sensitivity [35]. For example, a circumscribed region of reduced sensitivity in a 37-year-old patient with 20/100 visual acuity is shown (Fig. 11.7a). On the other hand, results from a patient with advanced-stage disease show an absolute scotoma (>20 dB loss) in the atrophic region and reduced sensitivity throughout the posterior pole (Fig. 11.7b).

A key attribute of the mfERG, SD-OCT, and fundus microperimetry is that they can all be obtained with direct visualization of the fundus. Furthermore, results from these tests can be precisely registered to provide an integrated description of retinal function and

structure in hereditary macular degeneration. In the future, these tests will facilitate a more rapid assessment of efficacy in the treatment trials for these diseases.

11.2  Molecular and Cellular

Pathophysiology of Pediatric

Macular Degenerations

and Potential Therapeutics

The basic knowledge base is well established on macular degenerations that occur most commonly in children. STGD, BMD, CRD, and JXRS now have clear

Fig. 11.6  mfERG of a STGD Patient. mfERG responses (left) and three-dimensional representation (right) from an STGD patient whose SD-OCT is shown in Fig. 11.5c. Responses from

the STGD patient are present in the fovea, but lower than normal throughout the parafovea

molecular and cellular underpinnings. However, while we understand the genetic origin of such diseases, and the impact of mutations on the protein structure and related function, the molecular pathways in which the disease target proteins operate, and how the diseases emerge in time in photoreceptors and RPE cells all remain to be better understood. Here, we will detail the known genes that are mutated in these diseases, the cellular environments in which they are expressed, and the biochemical and physiological pathways in which those encoded proteins operate. We also present to the current state of knowledge of how the molecular failures of the mutant proteins are thought to operate in cellular systems in which they are expressed to manifest cellular and retinal disease, and how gene therapeutics might be used to intervene in these diseases.

11.2.1  Molecular Genetics of Pediatric

Macular Degenerations

Information on the genes and proteins for pediatric macular degenerations is provided (Table 11.1). STGD occurs in both autosomal recessive and autosomal dominant patterns and is associated with macular flecks at the level of the RPE that are associated with RPE changes and atrophy. It occurs with a frequency of about 1/10,000 [36]. To date, ABCR (ABCA4), ELOVL4, and PROM1 have been the sole genes identified as mutated in autosomal recessive and dominant STGD. Mutations in the ABCR gene are the cause of most forms of autosomal recessive STGD (STGD1). The large ABCR gene has been mapped to the short arm of human chromosome 1 (1p13-p21) (Mendelian Inheritance in Man (MIM) ID: 601691), has 50 exons

and 49 introns, and covers a span of approximately 130 kbp (kilo base pairs) (pre-mRNA is 128,313 nt; processed mRNA is 7,326 nt; open reading frame is 6,819 nt) [14]. ABCR mutations (autosomal recessive) are also found in a variant of STGD called FF (MIM ID: 248200) in which fleck lesions occur throughout the retina proper and also in the macula, but there are less notable RPE changes. By definition FF emerges later in life as an adult form of STGD. If vision loss begins within the first two decades of life, STGD is the preferred designation and if it occurs later and has a more progressive course, then FF is preferred (Weleber 1994). STGD is often associated with central vision loss most commonly due to paracentral scotomas, while FF typically is not associated with central vision loss. ABCR mutations are also found in a form of recessive CRD (CORD3) (MIM: 604116) and even in RP (RP19) (MIM: 601718) (Martinez-Mir et al. 1997; [15, 16, 193]). ABCR mutations may provide modifier genes with the risk of development of age-related macular degeneration (AMD) (ARMD2) (MIM: 153800) [37]. Finally, ABCR mutations may contribute to bull’s-eye maculopathy [38]. Human ABCR encodes a 2,273 amino acid ATP-binding cassette transporter, retina-specific protein (220 kD) which is expressed in both rod and cone photoreceptors. Previously known as the photoreceptor rim protein (RmP), ABCR is a large integral membrane glycoprotein localized at the edges of the disks in both rods and cones. While initially thought to be expressed only in rod disk membranes, recent studies showed that primate foveal and peripheral cones also express ABCR to their surface membranes [39, 40]. Curiously, no dominant mutations have been found in the ABCR gene. Thus, as already well established for retinal and macular degenerations that occur in adults, different mutations in a single gene cause different clinical syndromes. Or, as is also well established­ for ABCR mutations in STGMD disease, different mutations in a single gene can promote a wide range of phenotypic variability. The primary variables in phenotypic diversity are the age of onset and the rate of retinal degeneration or demise. A database for ABCR mutations is found on the Retina International Mutations Database (http://www.retina-international.com/sci-news/muta- tion.htm), and at RetNet (http://www.sph.uth.tmc.edu/ Retnet/).

The ELOVL4 gene, which is mutated in autosomal dominant STGD, maps to the long arm of chromosome 6 (6q14) (STGD3) (MIM ID: 600110) and has six exons and five introns and covers a span of approximately 40 kbp (kilo base pairs) (pre-mRNA is 32,694 nt; processed mRNA is 3,085 nt) [6, 7, 17, 41]. For STGD3 cases that have arisen in North America, there appears to be a founder effect mutation for ELOVL4 that originates from an individual in the Great Scottish-Irish wave of immigration during the mid 1700s [41, 183]. ELOVL4 encodes an enzyme of 314 amino acids (37 kD) that is involved in the elongation of very long-chain fatty acids. Like the recessive STGD gene, ABCR, ELOVL4 is expressed in the rod and cone photoreceptors of the mammalian retina. ELOVL4 is expressed in the inner segments of both the rod and cone photoreceptors and is an integral membrane protein that resides in the endoplasmic reticulum, where fatty acid synthesis occurs. The car- boxyl-terminus of the protein contains an ER-retention signal (KXKXX, where K is lysine and X is any amino acid). Photoreceptor outer segments provide a unique membranous environment for the phototransduction cascade or apparatus that is highly dependent upon lipid composition. In order to support efficient phototransduction and recovery, proteins must diffuse quickly within these outer segment membranes. The role of ELOVL4 in long-chain fatty acid synthesis is not yet completely worked out, but there is evidence to suggest a prime role in very long-chain polyunsaturated fatty acid synthesis. Such lipids are found in high concentration in the photoreceptor outer segments and play a role in the high levels of membrane fluidity characteristic of this microenvironment and critical to its functional role (see below). To date, only three mutations in ELOVL4 have been identified as STGD3 is a rare syndrome. A database for ELOVL4 mutations is found on the Retina International Mutations Database (http://www.retina-international.com/sci- news/mutation.htm) and at RetNet (http://www.sph. uth.tmc.edu/Retnet/).

An additional autosomal dominant STGD dystrophy gene maps to the short arm of chromosome 4 (4p15.32) (STGD4) (MIM ID: 603786) [42]. Recently, a disease gene (PROM1 R373C missense mutation) mapping to this locus was identified for STGD4 and for an autosomal dominant macular dystrophy