64 Leber Congenital Amaurosis

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L amaurosis (LCA, MIM 204000) represents a group of congenital retinal diseases that lead to blindness, with a worldwide prevalence of three in 100,000.3 Although rare, it accounts for at least 5% of all inherited retinopathies and approximately 20% of children attending schools for the blind.3 We estimate that 180,000 patients are affected worldwide.

In 1869, Leber defined LCA as a congenital form of retinitis pigmentosa, with severe visual loss at or near birth, wandering nystagmus, amaurotic pupils, a pigmentary retinopathy, and autosomal-recessive inheritance.3,54 In 1956, Franceschetti and Dieterli reported a nondetectable electroretinogram (ERG) in the early course of LCA as essential in the diagnosis.25 Dominant inheritance has also been reported, but this is thought to be rare.26,46,74,81,82 LCA is genetically heterogeneous, and since 1996, eight genes12,15,20,29,59,61,67,79,83 (six of which have been cloned) with disparate retinal functions have been implicated. Five of the LCA genes (tables 64.1, 64.2, and 64.3) are expressed in the photoreceptors, namely, retinal guanylate cyclase (GUCY2D), cone-rod homeobox (CRX), Aryl hydrocarbon receptor–interacting protein-like 1 (AIPL-1), retinitis pigmentosa (RP) GTPase interacting protein 1 (RPGRIP-1), and crumbs-like protein 1 (CRB-1), while one is predominantly expressed in the retinal pigment epithelium (RPE), the RPE65 gene.

The study of LCA is proving central to our understanding of normal retinal development and physiology and also improves our understanding of other retinal degenerations. In the near future, LCA may be treatable by pharmacological intervention and/or gene replacement therapy, as both mouse90 and dog1 LCA models showed dramatic short-term improvements in rod and cone physiology and restoration of vision, by ERG, pupillometry, and behavioral testing.1 These future therapies for human LCA will likely be gene-specific, giving major significance to gene identification and geno- type-phenotype studies.

The clinical variability of LCA is striking. We find variability in visual acuities (20/200 to no light perception), visual evolution (stable, deteriorating, or rarely improving),7,30,40,52,75 refractions (from high hyperopia to high myopia), retinal appearance (from near normal to severe pigmentary retinopathy), associated ocular features (keratoconus, cataracts), associated systemic features, and retinal histopathology (from essentially normal43 to extensive degeneration.5,23,51,55,66,74,92,95 Some patients have an essen-

tially normal retinal aspect; others may have yellow flecks, salt-and-pepper changes, a marbled pattern, atrophic changes, nummular pigment clumps, a “macular coloboma,” white dots, or preserved para-arteriolar RPE. Keratoconus and cataracts may develop in the course of the disease in some patients. LCA may rarely be associated with systemic disease, and this adds additional variability to the disease spectrum. Mental retardation, deafness, polycystic kidney disease (also known as Senior Loken disease), skeletal anomalies (also known as Saldino Mainzer disease), or osteopetrosis may be found in addition to the ocular disease.

In approximately 40% of LCA cases it is now possible to identify the causative mutations in one of the seven LCA genes. Several strategies are now available to provide a molecular diagnosis for a child affected with LCA. The most rapid and comprehensive is the new LCA genotyping array (LCA disease chip), which includes all 300 currently documented LCA mutations. In a period of four hours per sample it is possible to determine the genotype in ~35% of the new cases.97 Conventional SSCP, dHPLC combined with automated sequencing also allows identification of mutations but is much more cumbersome. Genotyping LCA patients is extremely helpful for providing 1) a more accurate clinical diagnosis, 2) a more accurate visual prognosis, 3) a molecular classification of disease, 4) a prenatal diagnosis in selected cases, and 5) a way of separating LCA patients who may be treatable in the near future and those that may be treatable later.

The clinical understanding of the diagnostic findings in LCA has been evolving as more patients have molecular diagnoses and it becomes possible to go back and correlate test and phenotype to a confirmed known type of early onset retinal degeneration. Because LCA represents a group of diseases with at least seven and potentially as many as 20 genes, it can be expected that there will be variation in severity at onset and in the severity of the disability. For many years, with the lack of availability of the electroretinogram (ERG) and a reluctance to test infants and young children, many patients did not have an ERG until they were older. By then the patients were frequently blind and had a nondetectable ERG signal. Foxman et al.24 suggested ERG testing in blind infants before the age of 1 year to separate LCA from early onset RP. ERG testing is also crucial to differentiate albinism, complete and incomplete achromatopsia, and complete and incomplete congenital stationary night blindness from LCA.50 Infants can now easily have

standardized ERGs in a visual physiology laboratory, a site which is accustomed to managing infants.

Because the infant ERG is not equivalent to that of older children, caution is needed in interpreting these infantile ERGs. Fulton et al. have published standardized first-year values to assist in the interpretation of waveforms from this age group.30 If it is necessary to perform ERG testing under anesthesia, then great care must be given to interpreting the resulting waveforms. Operating room conditions are seldom standardized, and more importantly some anesthetics may suppress or alter ERG waveforms. We recommend that a child suspected of having LCA or any other retinal dystrophy have an ERG at around age 6 months and then a repeat ERG at 1 year.

Repeat ERGs are important because infantile testing is so difficult—children frequently cry and the electrodes fall out, or the tears may interfere with the signals and the ERG signals still mature in the first year of life.30 It is possible to have a small ERG signal early in the LCA disease process, and this certainly does not preclude the diagnosis of LCA. Also, a small number of children with early visual impairment have developmental delay and with repeat testing may have more robust or normal ERG signals on repeat testing.

The aim of this chapter is to discuss in detail, the seven types of LCA associated with the seven currently known LCA genes. Furthermore, we will summarize the management of the blind infant.

LCA caused by RPE65 defects

LCA caused by mutations in the RPE65 gene result in a block in the retinoid cycle and an inability to restore levels of 11-cis-retinal (table 64.3). RPE65 is abundantly expressed in the retinal pigment epithelium (RPE),37 which plays an important role in the vitamin A cycle. Recently, RPE65 expression has been documented in mammalian cone but not rod photoreceptors.99 The RPE65 gene is located on chromosome 1p22 and was cloned by Hamel et al. in 1993.36 It is highly conserved in all vertebrates. The genomic structure of RPE65 was elucidated by Nicoletti et al.65 and is composed of 14 exons; the protein consists of 533 amino acids. RPE65 mutations have been found both in juvenile RP patients35 and in LCA patients.61 In some populations, RPE65 mutations can account for up to 16% of LCA.64 Other large-scale studies found the relative burden of RPE65 in LCA to be 3%,16 6%,60 and 11.4%.86 The new

LCA disease microarray revealed that 2.4% of LCA patients had RPE65 mutations (N = 205).97

The phenotype of LCA patients resulting from RPE65 defect appears to be distinct, as LCA patients with the RPE65 genotype appear to have measurable visual function, unlike LCA patients with GUCY2D mutations.70 In a longitudinal study of four LCA patients with RPE65 mutations, Lorenz et al.58 found that a typical LCA child with RPE65 mutations would present at 3 or 4 months old with suspected blindness. In bright light, a visual reaction was noted, there was nystagmus, and the pupillary light response was dimished. On retinoscopy, mild hyperopia (+4.00 D) was found, while on fundoscopy at age 3–4 months, mild retinal changes were seen, with increased granularity of the RPE and retinal arteriolar thinning. At age 1 year, measurable visual acuities were present when measured by Teller Acuity Cards, and the ERG showed a measurable cone response and a nondetectable rod response. At age 3 years, the acuity was found to be 20/200. At age 5 years, both cone and rod ERG responses were nondetectable, and the retina revealed optic disk pallor, a bull’s-eye maculopathy, thinned retinal arterioles, and RPE granularity. Acuities remained at 20/200, and the Goldmann visual fields were recordable and showed a well-preserved response.

In summary, the clinical phenotype of LCA patients with RPE65 mutations may consist of severe visual impairment in infancy, with gradual visual improvement in the first few years of life, unlike the visual performance that is usually seen in LCA. Gradually, visual function may then decline and is associated with nondetectable ERG responses, and retinal findings indicating a diffuse retinal degeneration. This suggests that RPE65 type LCA represents a rod-cone dystrophy. We also found that RPE65 type LCA patients have measurable visual acuities (20/200), Goldmann visual fields, and small ERG amplitudes followed by slow deterioration of their visual function when measured over 20 years.15

In an important study by Porto et al.,72 the retinal phenotype of a fetus with LCA and mutations in the RPE65 gene was assessed by histopathological and immunocytochemical analysis. They obtained retinal tissue from a voluntarily aborted 33-week-old fetus with a homozygous C330Y RPE65 mutation and studied the histopathological changes compared to an age-matched fetus. They found 55% fewer cell nuclei in the outer nuclear layer (ONL) in the central retina, while the midperiphery and far periphery counts were similar to those of the control. The cell counts of the inner nuclear layer (INL) (i.e., bipolar, horizontal, and amacrine cells) were also less in the RPE65 fetus, while the ganglion cell layer (GCL) was normal. Specific immunolabeling revealed both rod and cone photoreceptor outer segment abnormalities. No apoptosis or gliosis was detected, and there was no neurite sprouting or Muller cell activation,

as is seen in some RP retinal specimens. The RPE showed abnormal inclusions, while the Bruch’s membrane was thickened, and the choriocapillaris was engorged.

These are the first histopathological retinal changes documented in a human LCA baby with a known gene defect, in this case RPE65, and therefore are very important. The results are also unexpected, as the animal models and their responses to treatments indicate essentially normal retinal architecture and viable photoreceptors. Not all human LCA retinas with RPE65 mutations are necessarily abnormal, however; in Van Hooser et al.,90 a relatively normal retinal architecture was noted by optical coherence tomography in vivo in an LCA patient with RPE65 defects. Also, the question remains whether the changes found in the Porto et al.72 study represent retinal degeneration or a failure to develop normal retinal cell numbers. Finally, it remains to be determined whether abnormal retinal histology can still support gene therapy.

The clinical phenotype and ERG phenotype of carriers (parents with LCA offsprings) of heterozygous mutations of LCA genes may have distinct features, which may give insight into the pathophysiology of the causative gene and point to the defective gene in the offspring.47 The phenotype of carrier parents who harbor heterozygous RPE65 mutations has not yet been extensively studied but is predicted to reveal a rod-cone-type dysfunction. Felius et al.22 found extensive dark-adapted visual field defects (1–2 log units above normal) and delayed 30-Hz flicker ERG in one carrier parent of a RPE65 mutation (but not in the other) of a child with juvenile RP.

In the RPE65 mouse model of LCA66 (knock-out of the RPE65 gene, RPE65-/-), rods and cones are present at birth and appear normal with intact outer segments until 15 weeks, when the outer nuclear layer declines from 10–11 layers to 8–9 layers and then down to 7 layers at 28 weeks. The rod ERG is absent from the beginning, but the cone ERG is intact. Furthermore, although opsin levels are normal, the rhodopsin molecule is absent. In conclusion, the RPE-/- mouse model likely represents a rod-cone degeneration and therefore provides a model of human LCA.

The RPE65 dog model4 of LCA is found in the Swedish Briard dog and represents a natural knock-out of the RPE65 gene.91 A 4-bp deletion is present in the RPE65 gene in homozygous state in exon 5 and results in a frameshift and a premature stop codon, with two thirds of the polypeptide missing, making this likely a null allele.91 The dogs develop an autosomal-recessive early-onset, slowly progressive retinal dystrophy. Histopathology of the dog shows a relatively wellpreserved retina, with relative minor photoreceptor changes but with prominent inclusions in the RPE layer and slowly progressive degeneration.3 The rod and cone ERG are essentially nondetectable.

Therapeutic studies

Acland et al.1 studied the effects of RPE65 gene replacement in the Briard dog. Subretinal injections in one eye of three dogs containing the AAV virus with cDNA of dog RPE65, with a CMV promotor, B-actin enhancer, and internal ribosome entry sequence were performed at age 4 months. Rodmediated and cone-mediated ERGs, visual evoked potential, pupillometry, and behavioral testing all showed dramatic improvements in visual function at about 8 months of age. In eyes that were treated with subretinal AAV-RPE65, a rod and cone ERG response was found that represents approximately 16% of normal, which was significantly different from untreated or intravitreally injected eyes. These results were obtained in three eyes, which were injected subretinally in only one of the four retinal quadrants. Genomic PCR and RT PCR demonstrated expression of the wild-type message in the retina and RPE, while immunoblots showed persistant RPE65 protein in RPE cells. This is the first study to demonstrate restoration of visual function in a large-eyed animal model with LCA due to an RPE gene defect. It is currently not known whether the retinas of human LCA patients with RPE65 defects are intact and whether the photoreceptor layer is present. The time window before the retina undergoes cell death is also not known. Also, whether LCA photoreceptor gene replacements have similar dramatic effects will now have to be evaluated. A human clinical trial involving well-characterized LCA gene defects in babies with LCA is likely not far off.

Van Hooser et al.,90 using recent knowledge that RPE65 mutations in mice lead to an inability to form 11-cis-retinal (which binds to rod opsin to form the light-sensitive photopigment rhodopsin), supplemented the mouse diet with the oral vitamin A derivative 9-cis-retinal, and consequently showed short-term improvements in rod photopigment production and rod physiology. The long-term consequences of this intervention are not yet known.

LCA caused by GUCY2D defects

LCA caused by mutations in the retinal guanylate cyclase gene, GUCY2D, is the result of a block in the recovery of the phototransduction cascade (table 64.3). The phototransduction gene retinal guanylate cyclase, GUCY2D, was cloned by Shyjan et al. in 1992 and mapped to 17p13.1.77 Camuzat et al.8 mapped a LCA gene by homozygosity mapping to the same 17p13.1 interval. In 1996, Perrault et al.67 reported mutations in GUCY2D in four unrelated LCA probands. The human gene contains 20 exons (figure 64.1) and has thus far been implicated in autosomal-recessive (AR) LCA67 and auto- somal-dominant (AD) cone-rod dystrophy45,69 (also known as CORD6). GUCY2D is expressed in the photoreceptor outer segments but at higher levels in cones than in rods.18,56

F 64.1 Cone-mediated 30-Hz flicker (top) and white-flash ERG (bottom) of the right eye (OD) of a normal 45-year-old; the father (middle); and mother (right) of a LCA patient with compound heterozygous GUCY2D mutations. The father carries a 1-bp deletion (bp 2843 del G) in exon 15 of GUCY2D, and the mother carries a L954P mutation in GUCY2D, also in exon 15. Both the 30-Hz flicker and white-flash cone mediated amplitudes are clearly decreased in comparison to normal.

Functional studies have revealed that mutations in the kinase homology domain of the retinal guanylate cyclase severely compromise the ability to produce cGMP.21 We found LCA mutations in the extracellular, kinase homology, and catalytic domains and performed expression experiments to compare their impact on enzyme activity. We found that LCA mutations from the extracellular domain (C105Y and L325P) cause a mild decrease in the catalytic ability of the enzyme,48 while catalytic domain mutations (L954P and P858S) severely compromise the ability of GCAP to stimulate cGMP production and cause dominant negative effects on the wild-type allele.49 As we added more of the mutant allele, we noted a progressive decrease in the ability of the enzyme to produce cGMP.49 Dominant negative effects are seen with dominant mutations, we were therefore surprised to find dominant negative behavior with autosomal-recessive mutations.88 Classical teaching about recessive mutations dictates that in the heterozygous state, the wild-type allele provides enough protein product to have a normal phenotype. Membrane guanylyl cyclases are thought to exist in a dimeric state,10,87 and our results showing a significant decrease in wild-type RetGCf-1 activity when L954P or P858S are coexpressed with wild-type suggest that any heterodimers that are formed are inactive or poorly active.88

These in vitro findings prompted us to test the hypothesis of in vivo dominant negative effects, by ERG of parents of LCA children with GUCY2D mutations.47 We found normal rod ERGs (see figure 64.1) but significant (see figure 64.1) and repeatable cone ERG abnormalities in parents of LCA

patients.47 Our findings are most consistent with a mild cone dysfunction in the carrier state, and this correlates well with the expression profile of GUCY2D, which is much higher in cones than in rods,18,56 and with the GUCY2D knock-out mouse, which develops a cone dystrophy.96 We then studied a second LCA family with a GUCY2D mutation, and we found very similar results47 (not shown). These findings have prompted us to make the following two hypotheses:

1.Heterozygous parents of LCA patients have an ERG phenotype and that this ERG phenotype is LCA genespecific, and these changes may point to the causal gene or pathway. A simple ERG of the parents at the time of the initial visit of the LCA child may therefore direct the subsequent genotyping strategy.

2.Because the ERG of the LCA child is nondetectable by definition, it has no information content in terms of pathophysiology. We propose that the ERG phenotypes of carriers of LCA genes give insights into the pathophysiology of LCA itself.

In some populations, GUCY2D may be the most commonly mutated LCA gene, with 20% of LCA patients carrying GUCY2D mutations.68 Other studies found the relative burden to be 6%.16,60 The phenotype of GUCY2D type LCA patients may be distinct, as most authors have noted a severe phenotype (with vision in the light perception or count finger range) with high hyperopia and photoaversion,70 while others found a severe but stable phenotype over a period of 20 years, with an essentially normal retinal appearance.16

In the GUCY2D mouse model of LCA96 (knock-out of the GUCY2D gene, GUCY2D-/-), rods and cones are present at birth with normal OS. By 5 weeks, there is a dramatic decrease in the number of cones only. By age 1 month, the cone ERG was not detectable, and despite their normal number and morphology, the rod ERG was markedly decreased. This model represents a severe cone-rod degeneration with much more cone than rod disease and therefore again only partially mimics human LCA.

In the GUCY2D chicken model of LCA76 (naturally occurring deletions in both copies of the GUCY2D gene, GUCY2D-/-), a different pathological response occurs. At hatching, photoreceptors are normal in number and morphology, but on day 7, their numbers start to decline from the center to the periphery, and by 6–8 months, the entire photoreceptor layer is gone. The RPE layer then undergoes atrophy.76 While the number of photoreceptors is still normal, it is of interest that the ERG is nondetectable. Semple-Rowland et al.76 propose the following set of events to explain the LCA type disease in this rd chicken: A deletion and insertion of the GUCY2D gene representing a null allele results in an absent cGMP and permanent closure of the cGMP-gated cation channels. This would lead to chronic hyperpolarization of the cells, as in constant light exposure.

Glutamate levels would be chronically elevated, and phototransduction would not be able to take place, which explains the nondetectable ERG. Evidently, these circumstances do not impair normal rod and cone development but do cause a rapid cone rod degeneration. This excellent model of human LCA may represent an early biochemical dysfunction with a superimposed cone rod degeneration.

LCA caused by CRX defects

LCA caused by mutations in the CRX gene result in a block in photoreceptor outer segment development and inability to express several key retinal genes (table 64.3). The conerod homeobox gene CRX was cloned by Freund et al. by screening a human retinal cDNA library with a fragment of a homologous gene.27 CRX resides on chromosome 19q13.3, has three exons, and encodes a protein with 299 amino acids. CRX is highly conserved in the animal kingdom, is specifically expressed in developing photoreceptors and the pineal gland, and plays an important role in regulating important photoreceptor genes, including rhodopsin, and is necessary for the formation of rod and cone outer segments.31,32 CRX is implicated in a variety of severe retinal diseases, including autosomal-dominant LCA,29 autosomal-recessive LCA,85 autosomal-dominant cone rod dystrophy (CORD2),28 and autosomal-dominant RP.81 CRX mutations are probably a rare cause of LCA. We estimate that CRX mutations are responsible for approximately 2–3% of LCA,16 which is in agreement with others.60 Three regions of the predicted CRX protein are highly conserved and include the homeobox, the WSP domain, and the OTX tail domain. According to Rivolta et al.,74 CRX mutations fall into two groups. Group 1 are missense mutations and one short in-frame deletion that preferentially affect the homeobox domain. In the second group are nine frameshift mutations, which are all found in the terminal exon 3. Most frameshift mutations are assumed to create null alleles because they lead to premature termination of translation. This is not the case for the exon 3 CRX mutations, according to Rivolta et al.,74 because of new emerging information about mRNA decay mechanisms that lead to rapid mRNA degradation and no translated protein when the mutations occur upstream of an intron. As the CRX frameshift mutations occur in a terminal exon, they escape detection, and the mRNA molecule is predicted to be stable and translated. Therefore, CRX mutations are dominant, fully penetrant, and likely act through a dominant negative mechanism to cause the disease phenotype.74

Most authors report a severe phenotype for patients with mutations in CRX,16,79 while we have reported one LCA patient with a heterozygous CRX mutation (A177 1 bp del) and a marked improvement in acuity, visual field, and cone ERG when measured over a period of 11 years.46 We found a dramatic improvement in Snellen visual acuity from

20/900 at age 6 to 20/150 at age 11, with a rapid change at age 10, at the same time that we found measurable cone ERG activity and visual fields. Furukawa et al.32 found that both rod and cone photoreceptor outer segments were not developed in the CRX knock-out mouse (CRX -/-) and also found that in the heterozygous knock-out mouse (CRX +/-), the cone photoreceptor outer segments were initially shorter than the wild-type mouse. They noted the absence of a cone ERG in their heterozygous (CRX +/-) four-week old mice, a time when the wild-type cone ERG is already present. At two months, the cone ERG of the heterozygotes (CRX +/-) increased but was still slightly decreased in comparison to wild-type. After a delay of six months, the heterozygous mice (CRX +/-) developed normal cone ERGs and normal-length cone outer segments. The delay in cone ERG and cone photoreceptor outer segment formation in the heterozygous knock-out mouse (CRX +/-) is initially similar to the delay in the cone ERG development of our LCA patient with the A177 1bp del CRX mutation and may correspond to delayed expression of cone opsin genes and/or cone outer segment formation in our patient.

We have not yet examined the ERGs of a carrier parent with a CRX mutation, but Swaroop et al.85 found mild rod and cone ERG abnormalities in both parents of a LCA child with a homozygous CRX mutation.

LCA caused by AIPL-1 defects

LCA caused by AIPL-1 mutations potentially result in a defect in the biosynthesis of the phototransduction enzyme phosphodiesterase (table 64.3). The gene for aryl hydrocarbon receptor–interacting protein like-1, AIPL-1, was discovered and cloned by Sohocki et al. in 1999,79 by screening a human cDNA library containing retinal and pineal gland sequences. It is located on chromosome 17p13.1 and contains eight exons. It is expressed in the retina and pineal gland, but its exact function is not yet known. AIPL-1 interacts with NUB-1, which is thought to control cell cycle progression.2 Immunocytochemistry studies with an AIPL-1 antibody revealed that AIPL-1 is exclusively expressed in rod and not cone photoreceptors.89

The original pedigree for linkage analysis was of Pakistani origin, and many LCA patients developed keratoconus. We also found AIPL-1 mutations in four Pakistani pedigrees with LCA from remote mountain villages who exhibited a severe phenotype, with count finger to light perception vision, severe retinal degeneration, a maculopathy, and keratoconus.11 We estimate that mutations in AIPL-1 are found in up to 7% of patients with LCA.80

ERGs of carrier parents of AIPL-1 mutations may provide insight into the pathophysiology of LCA caused by AIPL-1. We found significant rod isolated ERG abnormalities and normal cone mediated ERGs in an AIPL-1 carrier parents

with a single copy of W88X (figure 64.2), suggesting that AIPL-1 type LCA represents a rod-cone disease (Dharmaraj et al., 2003, personal communication).

LCA caused by CRB-1 defects

LCA caused by mutations in the crumbs gene, CRB-1 result in a defect in the molecular scaffold that controls zonula adherens assembly and in elongation of the photoreceptor outer segment (table 64.3). The crumbs homolog 1 gene, CRB1, was cloned by den Hollander et al.,13,14 and mutations were found in 10 of 15 patients with RP12,13 a specific form of ARRP with preservation of the para-arteriolar RPE (PPRPE type of RP). The gene resides on 1q31, has 12 exons, and encodes a protein of 1376 amino acids. The CRB1 protein shows conspicuous structural similarity to drosophila crumbs, a protein essential in establishing and maintaining epithelial cell polarity of ectodermally derived cells. Because of the severity of the PPRPE type RP, CRB1 was postulated also to cause LCA. Den Hollander et al.12 and Lotery et al.59 recently found that 13% of cases with LCA can be explained by mutations in the CRB1 gene, making it a common and important gene for LCA. In addition, CRB1 mutations were identified in five of nine patients with RP and Coats’-like exudative vasculopathy, a severe complication of RP.12 LCA patients with CRB1 mutations in three of seven cases also show the PPRPE picture. Although the number of patients with LCA and CRB1 mutations is still relatively small, it is noted that three of seven LCA patients and at most one of 15 patients with RP12 or ARRP carry two CRB1 protein truncating mutations, suggesting that combinations of severe CRB1 mutations cause LCA and that combinations of severe and moderately severe mutations cause RP12 or ARRP. Further details of the CRB1 phenotype of LCA and the carrier phenotype are still lacking.

LCA caused by RPGRIP-1 defects

LCA caused by mutations in the gene RPGRIP-1, result in defects in the connecting cilium, which connects the inner and outer segment of photoreceptors (table 64.3) and disk morphogenesis.98 The RPGRIP-1 defects may result in problems with vesicular trafficking of proteins. The RPGRIP-1 gene, encoding the retinitis pigmentosa GTPase regulator interacting protein-1, was discovered by Boyle and Wright6 by performing protein-protein interaction studies using RPGR as bait in yeast-two hybrid screening of retinal cDNA libraries. Mutations in the RPGR gene are the major cause of X-linked RP3. RPGRIP1 resides on 14q11 and has 25 exons. It is expressed in rods and cones and localizes to the connecting cilium, which connects the inner to the outer segment.42 The RPGRIP1 protein may be a structural component of the ciliary axoneme.42 Dryja et al.20 found that 6%

F 64.2 Scotopic rod-mediated ERGs, maximal ERGs, 30Hz flicker, and photopic ERGs of normal (bottom), two LCA sibs (middle, ages 30 and 27 years), and carrier mother of the sibs (top, age 47 years). The sibs are both homozygous for the W88X mutation in AIPL-1, while the mother is a heterozygous carrier of this

of LCA harbor mutations in this gene. The LCA phenotype has not yet been delineated; nor has the carrier phenotype.

LCA caused by RDH12 defects

The relatively new LCA gene RDH12 maps to 14q23.3 consists of 7 exons and encodes a retinol dehydrogenase expressed in photoreceptors, which participates in the vitamin A cycle, as does RPE65.44 The retinol dehydrogenase encoded by RDH12 is involved in the conversion of all trans retinal to all trans retinol. The exact biochemical consequences of RDH12 defects are not yet known. Clinically, the LCA and/or juvenile RP patients harboring RDH12 mutations thus far were found to have a severe and progressive rod-cone dystrophy with severe macular atrophy but no or mild hyperopia.44,71

mutation. The ERGs of the affected LCA sibs are nondetectable, consistent with LCA. The mother’s ERG clearly shows an abnormality in the rod-mediated signals, where the rod amplitudes are markedly decreased compared to normal. Cone-mediated signals appear normal.

Management of the blind infant

The role of the primary ophthalmologist making the initial diagnosis of LCA is difficult, complex, and consists of at least six aspects.

First and foremost it is essential that the proper diagnosis is made, and both overlapping ocular and systemic diseases must be ruled out. The main ocular diseases are albinism, achromatopsia, congenital stationary night blindness, optic nerve hypoplasia, delayed visual maturation, and cortical visual impairment. The main systemic diseases are all peroxisomal disorders, neuronal lipoid fuscinosis, abetalipoproteinemia, Bardet-Biedl syndrome, Alstrom syndrome, Senior Loken syndrome, Joubert syndrome, and Saldino-Mainzer syndrome.50

Second, the recessive inheritance must be communicated and counseled. If not possible, a genetic counselor must be

consulted. Third, the visual prognosis must be given. Most patients with LCA will have significant lifelong visual handicaps and need extra help at home, school, and work. Most LCA patients have stable visual function, while a subgroup may decline, and rare improvements have been documented. Fourth, a molecular diagnosis is now a must as it is relatively easy to do and provides essential information to the family (carrier status, prenatal screening), the eye-care giver, and the scientist (prognosis, definitive diagnosis, treatment trials). Fifth, it is essential to provide the family with information such as family support groups, websites, and other information (www.FFB.ca; www.FFB.org; www.cafamily.org.uk; American Council of the Blind www.acb.org). Finally, it is imperative that the family is put in touch with a local blindness institute for social, psychological, and low-vision support. A good example is the Los Angeles Blind Children’s Center (see Toni Marcy in Heckenlively 198839). Many issues relating to a mother’s and father’s feelings of guilt and the affected child’s issues of selfstimulation (mannerisms of “blindisms”) if he or she is not stimulated to explore the outside world are addressed at this type of institution. Their program consists of six goals, including 1) acceptance of the blindness, 2) promotion of parent-child attachment, 3) furthering gross motor development (motility training), 4) stimulating object handling, 5) stimulating language development, and 6) prevention of deviant behavior (autism and self-stimulatory behavior).

Summary

In summary, there are currently seven genes responsible for LCA, and mutations in these genes can explain approximately 40% of the cases. Genotype-phenotype correlations of both the LCA and the carrier phenotype suggest that although highly variable, there are clinical features that are specific for the gene defect. These studies are still in progress, but initial results show that GUCY2D defects can lead to a severe cone-rod type LCA, with poor but stable visual function, an essentially normal retinal appearance, and a cone dysfunction in the obligate carriers. RPE65 defects lead to a different phenotype, with an initially milder form of LCA than GUCY2D, with signs of retinal degeneration and slowly progressive visual loss later in life. AIPL-1 defects lead to a very severe form of LCA, often with cataracts, keratoconus and a maculopathy, while some carriers have a striking rod dysfunction on ERG. CRX defects lead to a variable LCA phenotype, with most LCA patients showing a severe retinal degeneration and visual loss, but may rarely be associated with an improving phenotype. The carrier signs seem to be a rod and cone dysfunction on ERG. CRB-1 defects can lead to a unique LCA phenotype, with preservation of the paraarteriolar RPE. The complete phenotype and the carrier phenotype of CRB-1 are still unknown. The RPGRIP-1

phenotype has not yet been fully delineated but appears to consist of a severe pigmentary retinopathy. Similarly, the RDH12 phenotype has not been fully delineated but appears to consist of a severe rod-cone degeneration with a prominent maculopathy.

At least seven retinal and/or RPE genes are responsible for LCA, but five of the seven genes (GUCY2D, RPE65, CRX, AIPL-1, and CRB-1) are also associated with adult or later onset retinal diseases such as cone-rod or rod-cone degenerations. Future therapies for LCA may be gene specific, making genotype-phenotype correlations very useful in reclassifying LCA at the molecular level.

Support for the author’s LCA studies comes from both the Foundation Fighting Blindness Canada and the FRSQ. The author is much indebted to colleagues Drs. Rando Allikmets, Frans Cremers, Gerry Fishman, Pierre Lachapelle, Irma Lopez, Irene Maumenee, and Melanie Sohocki for their thoughtful discussions.

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