11.2.2  Molecular Genetic Testing

Clinical Laboratory Improvement Amendment (CLIA) testing is available for all of the above genes that underlie juvenile macular degenerations. Clearly, family history is essential to establish a dominant, recessive, or X-linked pattern of the disease. Once accomplished, solid clinical diagnosis is based upon a number of anatomic, psychophysical, physiological, and imaging studies. Historically, before the age of molecular genetics of retinal and allied degenerations, this was the endpoint of the diagnostic and prognostic process. Today, the clinical diagnosis and the family history of genetic patterns is only the beginning of what can emerge as a detailed molecular and genetic diagnosis of the disease. As relational knowledge has accumulated over the last two decades since the identification of the first known human mutation in RP (Dryja et al. 1990), correlates emerged between the classical clinical anatomy of retinal disease (pattern recognition), as supported by ancillary discriminatory or confirmatory testing, and a set of candidate genes within which the mutation must be sought [181].

Molecular genetic testing for retinal degenerations at large first emerged as discrete research-based protocols in molecular genetics labs focused on mutational screening in specific candidate genes. Some of these labs then sought CLIA approval to conduct clinical tests (qualitycontrolled and quality-approved fee-for-service), sometimes using the same toolsets (PCR, sequencing) as were used for research testing. More recently, microarrays have been designed, proven-tested, and manufactured to screen for multiple mutations that often occur in multiple gene sets, all of which underlie a common set of clinical anatomic retinal syndromes [203]. The technology used in these “Gene Chips” is arrayed primer extension (APEX) (Asper Biotech, Tartu, Estonia) in which fluo- rescence-based single nucleotide analog sequencing of mutant, WT, and single nucleotide polymorphism alleles occurs on the basis of programming allele-specific oligonucleotides that are tiled on the chip [50, 51]. We provide a current listing of the types of genetic tests available for the genes that are mutated in this common set of juvenile macular degenerations and the references for site lookup (e.g., Gene Tests) (Table 11.2). Currently, genotyping

chips (gene chips) are available for approximately 400 ABCR mutations that underlie STGD1, CRD3, RP19, and AMD, a set of 11 genes that are known to underlie Leber’s congenital amaurosis (AIPL1, GUCY2D, CRB1, CRX, TULP1, RPGRIP1, RPE65, MERTK, LRAT, RDH12, CEP290), a set of 11 genes that underlie autosomal recessive RP (ABCA4, CNGA1, CRB1, MERTK, PDE6A, PDE6B, RGR, RHO, RLBP1, RPE65, TULP1), and a set of eight genes that underlie Usher syndrome (CDH23, MYO7A, PCDH15, USH1C, USH1G, USH2A, VLGR1, USH3A) [52–55]. Currently, no Gene Chip exists for all of the known genes that underlie juvenile macular degenerations. The ABCR gene chip was reported to identify approximately 70% of known mutations, and successfully diagnosed 71% of new patient cohorts. Also, a high frequency (approximately 10%) of mutational ABCR alleles was identified in the control populations [52, 53]. While genotyping chip technology is exciting, efficient, and cost effective, current technology will detect only some of the known mutations (false negatives), will misdiagnose some mutations (false positives), will identify only some of the new mutations, and thus will leave many patients without a genetic diagnosis despite a clinical diagnosis based upon retinal anatomic appearance and supportive ancillary testing (e.g., ERG, psychophysics). We would strongly recommend that all positive mutational hits on Gene Chips be confirmed by the accepted gold standard of direct amplicon sequencing, and prior to patient and family genetic counseling. Currently, Gene Chip technology is an excellent firstorder high throughput screening tool. As technology and knowledge of new genes/mutations increases, the efficiency and validity of this approach to genotyping are expected to increase substantially over the next 5–10 years. Accurate genotyping is critical to establish the genotype: phenotype correlations that are useful to predict the clinical outcomes for a given patient, the patient’s family, and to establish a rational prognosis. Also, accurate genotyping is essential to emerging and future clinical trials with gene-based therapies [184].

11.2.3  Molecular Biochemistry

and Physiology of Pediatric

Macular Degenerations

To begin to understand the clinical nature of hereditary juvenile macular degeneration phenotypes, it is

important to first understand the molecular biology, molecular biochemistry, and molecular physiology of the disease. This starts with a detailed understanding of the structure and function of the wild type (WT) protein in its native cellular and tissue habitat. The success of human and mammalian vision is critically dependent upon seven cell types in the outer retinal microenvironment: (1) rod PRs, (2) red cone PRs, (3) green cone PRs, (4) blue cone PRs, (5) RPE, (6) bipolar cells, and (7) Müller glial cells. To date, most of the genes that have been found to be mutated in hereditary retinal and macular degenerations are expressed in either the photoreceptors or the RPE in the outer retina. The cellular locations of expression of the genes that are mutated in the described syndromes are indicated (Fig. 11.8).

11.2.3.1  ABCR

The ABCR protein is expressed to the outer segments of the rod and cone photoreceptors, which are the specialized cellular compartments where phototransduction begins. In the cone photoreceptor, ABCR is localized to the surface plasma membrane at the disk edges as an integral membrane protein in an outsideout orientation (Fig. 11.9). In the rod photoreceptor, due to complete invagination of the outer segment plasma membranes to form topologically closed disks that stack inside an embracing surface plasma membrane, most of the ABCR becomes localized to the edges of internalized disk membranes; a residual fraction of ABCR is expressed to the surface plasma membrane of the outer segment that envelopes the stack of disks. ABCR is known as a retinoid flipase and uses the energy from ATP breakdown to catalyze the transmembrane transfer of ATR-derived molecules in the photoreceptor outer segments [56]. The net vector of retinoid movement is from the outside surface of the visual pigment containing membranes to the inside (cytoplasmic) surfaces. This statement is true regardless of whether the photoreceptor is a rod or cone. While all- trans-retinaldehyde (ATR), resulting from visual pigment bleaching can be carried by ABCR, the preferred substrate is N-retinylidene-phosphatidyl-ethanolamine. The later chemical results from the chemical reaction of ATR with phosphatidyl-ethanolamine (PE) in the disk membranes of the outer segments (Fig. 11.10). The source of ATR is from the hydrolysis of the Schiff

Fig. 11.8  Retinal microenvironment and the location of proteins affected by juvenile macular degenerations. Cellular components of the outer retinal microenvironment are shown schematically with the final localizations of the proteins

ABCR, ELOVL4, BEST-1, and

RS1 which are affected by mutations in the genes causing pediatric macular and retinal degenerations

Photoreceptors

RS1

ELOVL4

ABCR

RPE

BEST1

Bruchs Membrane

Choriocapillaris

base (–C=N–) covalent linkage of all-trans-retinylidene to lysine 296 in both human rod and all human cone (red, green, blue) pigments. This yields ATR and the opsin apoprotein. All-trans-retinylidene chromophore results from the isomerization of 11-cis-retinylidene, the ground state (dark adapted) chromophore for all human and mammalian visual pigments. This isomerization leads to the biochemical activation of the visual

pigments in the rods and cones and initiates the signaling of phototransduction. The sole source of ATR in the eye is the light-dependent bleaching of the rod and cone visual pigments. A substantial to massive amount of ATR is formed each day in the retina. The macular rod photoreceptors are thought to undergo several complete bleaches of all rhodopsin each day [58]. Recent studies have indicated, at least in the rod visual pigment

and rod photoreceptor, that there is a vectorial movement of hydrolyzed ATR from the core of the bleached pigment to the one surface face of the membrane in which rhodopsin is situated as an integral membrane protein. In the rod photoreceptor, most of the visual pigment containing membrane is in the internalized topologically closed disks, with the extracellular surface of the membrane inside the disk, and with the remaining small fraction of rhodopsin in the plasma

membrane (~1.5%). The same process of retinoid flipase must be operative in cone visual pigments as ABCR is localized outside-out in the surface plasma membrane of the cone photoreceptor. For the cone photoreceptors,­ and cone visual pigments, which are entirely localized as integral membrane proteins in the plasma membrane, the same process would deposit ATR on the outside surface of the cone photoreceptors. ATR is a reactive molecule due to its aldehyde moiety

H+

Fig. 11.9  ABCR cellular localization and function. (a) The localization of ABCR protein in the rod and cone photoreceptors is shown at the edges of the disk microenvironments. Peripherin and ROM-1 also localize to this microenvironment and help to specify the shape of the outer disk rim. (b) Molecular schematic of the ABCR protein is shown. ABCR is a retinoid flipase and moves hydrophobic retinoids (ATR, NRPE) from the extracellular surface of the disk (intradiscal environment in a rod) onto the cytoplasmic surface. The retinoid binds to the extracellular domains of the pro-

tein. Energy from ATP breakdown on the cytoplasmic domain of ABCR is used to perform this transport function across the lipid bilayer. Once on the cytoplasmic surface (physically inside the cell), all-trans-retinol dehydrogenase is able to reduce the aldehyde bond of ATR or NRPE to form Vitamin A. Vitamin A is then transported to the RPE through associations with retinol binding proteins in the photoreceptors and IRBP in the subretinal space to the RPE, which absorb it, bind it to other retinoid binding proteins, and esterify it in preparation for the reformation of 11-cis-retinal

cytoplasm H+

and under physiological conditions can rapidly react (seconds to minutes) with primary amines as are commonly found in membrane proteins and in certain membrane lipids (PE). Two molecules of ATR are able to react with a single molecule of membranous PE to form a molecule called N-retinylidene-N-retinyl- ethanolamine (A2E) (Fig. 11.10). A2E not only causes a direct toxicity to RPE cells by several independent mechanisms, but is also is light sensitive and is the key underlying chemical intermediate to phototoxicity after LF accumulation. A2E is a validated chemical target for RPE toxicity, as it occurs in several retinal degenerations including STGD (ABCR and ELOVL4

mediated), FF, BMD, and dAMD. The manner in which A2E is formed is rooted in the visual pigment and retinoid cycle biochemistry, which is sketched out below.

There is a natural process of clearing ATR from the outer segment membranes of both the rod and cone photoreceptors. This is known as the retinoid visual cycle (Fig. 11.11). In both the rod and cone photoreceptors, ATR is converted to all-trans-retinol (Vitamin A (VA)) by a trans-retinol dehydrogenase (tRDH) which is expressed to the outer segment cytoplasm. Hence, in order for ATR, localized after bleaching on the outer leaflet of the plasma membrane in the cones and rods or the internalized leaflet of the disk membrane in rods, to

Fig. 11.9  (continued)

be metabolized to VA, which has minimal chemical reactivity, the ATR must be moved from the extra­ cellular surface of the rod and cone photoreceptor membranes­ onto the cytoplasmic surface. ABCR accomplishes this critical task for the rod and cone photoreceptors by moving ATR from the outside leaflet to

the inside leaflet of visual pigment containing membranes, at the cost of ATP hydrolysis. Once on the intracellular surface tRDH can convert ATR to VA. This enzymatic reduction (aldehyde to alcohol state) reaction does two things for the rod and cone photoreceptors: (1) it regenerates VA, and (2) it removes the

O

Phosphatidyl-ethanolamine (PE)

R1 and R2 are fatty acids (C14-22, ∆=0−6)

O

O

Photoreceptors HO P O Schiff base

O

N

H

O

R1 O

R2 O

O

O

Photoreceptors

Fig. 11.10  Chemical and enzymatic reactions in the formation of A2E. Any delays in the transport of ATR across the photoreceptor outer segment lipid bilayer, or its reduction into Vitamin A create a temporal window allowing the reactive aldehyde group of ATR to covalently bond with primary amines at the membrane plane (proteins, aminolipids). The retinoid is then covalently captured as a Schiff base (–C=N–) to form NHRE which is a relatively stable chemical species. Further reaction with an additional ATR molecule forms A2PE, which is still

attached to the protein or lipid. It is the monomer or dimer forms of NHE or A2PE that are likely to be the primary chemical forms in which retinoid byproducts of photoreceptor bleaching metabolism gain access to the RPE through daily phagocytosis of the shed photoreceptor outer segment disks. Once in the RPE, the lysosome phospholipase D cleaves off the lipid component leaving the cationic bis-retinoid, A2E, as a final toxic chemical species, which apparently cannot be degraded further by known RPE metabolism

toxicity of ATR. VA regenerated in the outer segment cytoplasm of the photoreceptors is then released from the photoreceptors to the subretinal space where it complexes with interstitial retinoid binding protein (IRBP), which transfers hydrophobic VA into the apical surface of the RPE cell, from which it enters the RPE and binds to the cellular retinol binding protein (CRBP). Once in the RPE VA is esterified to intracellular membrane lipids by a protein called lecithin retinyl ester transferase (LRAT), where it is effectively stored in the RPE. Another protein in the RPE is the RPE-65 kD protein (RPE65) which isomerizes­ the all-trans-retinyl ester to 11-cis-retinyl ester and hydrolyzes the molecule to

release 11-cis-retinol (11cRol), which complexes with cellular retinaldehyde binding protein (CRALBP). An 11-cis-retinol dehydrogenase (RHD5) in the RPE oxidizes 11cRol to regenerate 11-cis-retinaldehyde (11cRal), which is essential to regenerate the ground state visual pigments from bleached ­apoproteins. These proteins constitute the major components of the retinoid visual cycle, which occur in the rod dominant retinas in at least two cell types (photoreceptor and RPE) that are critical for the reconversion of the spent ATR back into 11cRal to regenerate visual pigments.

If the ATR is not converted back to 11cRal human vision will eventually stop. The amount of retinoid in

Fig. 11.11  The retinoid visual cycle. The conversion of spent ATR back to 11-cis-retinal requires enzyme conversion reactions which are distributed within both the photoreceptors and RPE. Transfer of retinoids from one membrane surface to another requires binding proteins due to the hydrophobic character of chemicals in this class. tRDH in the photoreceptor cytoplasm reduces ATR to Vitamin A. Vitamin A leaves the photoreceptor and enters into the subretinal space where it is bound by IRBP. IRBP is thought to unload Vitamin A at the RPE apical membrane complex. Vitamin A, once inside the RPE, is bound to CRBP which traffics it to the microsomal membranes where LRAT esterifies Vitamin A onto membrane

lipids. All-trans-retinyl membrane esters result. RPE65, now known to be the retinoid isomerase and possibly the retinyl hydrolase, isomerizes the 11–12 double bond from trans to cis and likely promotes the hydrolysis of the ester group, coupling the energy of hydrolysis into the uphill isomerization reaction. CRALBP then binds 11-cis-retinol and docks with RDH5, the 11-cis-retinol specific dehydrogenase, to regenerate 11-cis-reti- nal. The final product, likely still bound to CRALBP, is trafficked across the apical membrane of the RPE and complexed to IRBP in the subretinal space, from where it is taken up again into the outer segments of the photoreceptors to regenerate the bleached rod or cone visual pigment