11.4.2  BMD Models

There are both rat and canine models for BMD. Curiously, the mouse knockout for mBest-1 had no retinal phenotype and an essentially normal DC ERG with some changes in the luminance response function of the LP [112]. A model for BMD was generated in rats through the delivery to the RPE of BEST-1 mutants (WT, W93C, or R218C) by replication defective adenoviruses [114]. While the expression of the mutant proteins did promote changes in the LP, there was no accumulation of LF or formation of anatomic vitelliform lesions in the rat retinas. A naturally occurring syndrome in canines, canine multifocal retinopathy, has features that are similar to BMD including early elevated retinal lesions associated with the accumulation of turbid fluid, which are associated with LF-like inclusions in the RPE, and later, the atrophy of the outer retina and RPE within these lesions [137].

11.4.3  JXRS Models

A mouse model for retinoschisis was recently developed through the knockout of the intrinsic mouse homologue (Rs1h) [138]. This model simulated much of the phenotype of JXRS in human males with disorganization of the laminated character of the retina with schisis cavity formation at several levels and a negative b-wave in the ERG indicating problems with synaptic transmission. Moreover, recombinant adeno-associated virus delivery of a WT RS1 gene to achieve broad expression in many retinal neurons resulted in a reversal of the negative b-wave to suggest that such a therapy could potentially be beneficial in adult males with JXRS. This model can clearly serve both to delineate the mechanism of the disease and for therapeutic and safety testing.

11.5  Phenotypic Diversity

There is indeed a bounty of genotypic heterogeneity in these and other hereditary retinal and macular degeneration syndromes. However, the genotype alone does little to suggest the range of phenotypes that can and do emerge. The nature of the phenotype that results in

a given disease pattern depends upon the molecular impact of the mutations on the expression, structure, and function of the protein encoded by that gene in the cells in which it is expressed. The phenotype that emerges may also depend on the genotype of the individual such that the modifier genes can affect the impact of other mutant genes on the phenotype (see below). The magnitude of loss of a given protein function, due to the expression of nonsense mutations (premature stop codons) or mutations that impact normal function (hypomorphic alleles) will remove a fraction of normal or WT biological function from the cell. The consequence to the cell will depend upon how much WT protein is lost and the functional insult that the cell ensues as a result, if any. While many WT proteins appear to be expressed in at least some excess (system redundancy), the exact level of WT protein needed to attain a properly differentiated and stable cellular phenotype and function are typically unknown. A given cell requires a certain amount of WT protein in order to carry out and maintain the required function for the cell. Loss of WT protein, for example, through two separate recessive mutations, can create haplotype insufficiency, or the inability of the two mutant alleles to specify the sufficient expression of WT protein, or quasi-WT protein, for adequate function by the cell. In the case of autosomal dominant mutations, where missense mutations are more common, the protein encoded by the mutant gene could sustain a wide variety of structural perturbations that impact protein folding or function. Mutant proteins which misfold may be degraded and hence a fraction of WT protein is subtracted from the cell. Or, mutant proteins may misfold and not be degraded, yet often maintaining sustained structural or functional defects. These mutant proteins may insert properly or improperly into cellular metabolism or structure building, with differing degrees of gain-of-function, to typically promote biochemical or physiological defects. Or, a dominant negative mutant protein may interfere with the normal expression, trafficking, or functioning of the WT protein and impair the amount of WT function that is maintained. For example, a dominant negative mutation could promote protein misfolding and congest the intracellular trafficking steam of the WT protein. Or, if proteins interact, the mutant protein could trap the WT protein in cellular processing or trafficking streams and impair, delay, or obviate the arrival of the WT protein at its cellular zipcode.

Hereditary retinal and macular diseases manifest upon a wide variety of entry level molecular perturbations. The nature of the molecular perturbation at the protein level will dictate the type and level of response of the cell in which it is expressed. Most retinal degenerative diseases emerge in time in that the cells and tissue architecture are fully differentiated and normal appearing after birth. Disease (degeneration) begins at some point in time of the life of the individual and progresses from that point forward. The time until disease begins and the rate at which degeneration ensues are often characteristic of both the gene that is mutated and the specific mutational impact of that mutation on the level of WT protein and the loss or gain-of-functions that are imposed upon the protein gene product. Retinal cells adapt to diverse stresses (e.g., mutant proteins) and the adaptational response is a part of the emergence of the disease process.

It is not in any way surprising that there are such a wide range of phenotypic clinical outcomes for different mutations in a single gene. These are called genotype: phenotype correlations and most commonly arise from allelic heterogeneity. There is strong evidence for a wide phenotypic variability due to the many diverse ABCR, BEST-1, or RS1 mutations (see below). An example of profound phenotypic variability occurs with mutations in the RHO gene, which encodes the rod rhodopsin protein and which was the first gene found to be mutated in a hereditary retinal degeneration [139]. There are over 125 known mutations including missense, truncation, and null varieties. Critically, there is also a crystal structure for rhodopsin which helps to strongly correlate biochemical and cell biological structure functional studies [140]. RHO mutations cause different human retinal degenerations including autosomal dominant and autosomal recessive RP, and autosomal dominant congenital stationary night blindness [141]. Clinical phenotype correlates with the location of the mutation in the protein [142]. Within the autosomal dominant RP mutation set, there is a wide range of phenotypes seen as highly dispersed ages of onset and rates of retinal degeneration. For example, mutation at one of the two cysteines (C187Y) that forms the critical disulfide bond (C110–C187) that is essential to the tertiary structure leads to an early onset (by 6 years of age) and rapidly progressive retinal degeneration [143]. The C187Y mutation prevents the formation of the functional tertiary fold of the protein and is therefore expected to result in a severe

phenotype. Similarly, a mutation at the retinoid binding site (K296E, K296M) also causes a severe early onset retinal degeneration [144, 145]. A mutation in the N-terminus such as P23H causes a more mild disease that can present in the third to fifth decades of life and generally has a much slower rate of progression [146]. The P23H mutation can cause changes in the glycosylation pattern of the folded protein and also impact its trafficking within the rod photoreceptor. In general, mutations in the highly conserved regions of a protein tend to be more deleterious due to their substantial impact on evolved local structures. Indeed, hotspots within the natural mutational landscape of a protein can help to predict highly conserved regions such as the many mutations around R135, which lies within a critical domain for protein activation, and mutations clustered at R135 cause early onset and rapidly degenerating (severe) phenotypes. In the set of dominant congenital stationary night blindness mutations (G90D, T94I, A292E, A295V), the nature of the disease can also be understood from biochemical, biophysical, and structural evaluations of correctly folded proteins that develop the new perturbed functionalities as ground state visual pigments with increased rod photoreceptor dark noise (elevate rod thresholds) [147–149]. RHO mutations demonstrate the rich array of cellular biocomplexity that can result in genotype: phenotype correlations in retinal degenerative diseases.

There is ample evidence for a wide range of phenotypic variability in patients with ABCR, BEST-1, and RS1 mutations. There are only a few ELOVL4 mutations, so a rich landscape of gene-phenotype outcomes is not yet possible. ABCR mutations cause STGD1, FFM, RP19, CORD, and may contribute to AMD. The bulk of mutations are missense, followed by nonsense, small deletions/insertions, and splicing site mutations [36]. Phenotypic intermediates between these clinical anatomic diagnostic categories may also exist [150, 151]. That the spectrum of disease is likely to represent a continuum is suggested by the identification of pedigrees with STGD1 and CORD or RP in different individuals [53]. Truncation mutants of ABCR (hypomorphic mutants) commonly lead to the development of STGD1, missense mutations not involving charged residues commonly promote FFM, and mutations promoting mislocalization of the protein cause RP [151–154], (Wiszniewski et al., 2005). These studies suggest a plethora of functional and structural impairments of the ABCR protein proper that manifest a range of clinical

outcomes at the level of photoreceptor metabolism and survivability. A model for disease severity was proposed and is based upon the amount of active ABCR enzyme activity that results from the two alleles [151]. Single allele mutations in normals and in AMD patients (heterozygotes) are thought to have good enzyme activity. Double allele mutations (most commonly compound heterozygotes) in STGD and CRD patients have less ABCR activity, while severe double mutations leaving essentially no enzyme activity are thought to occur in RP19 (Fig. 11.15). Given the known role of ABCR, and the fact that LF and A2E accumulate in STGD1, it would be compelling to test this hypothesis with fundus autofluorescence measurements in STGD1 disease that occurs on the basis of ABCR mutations of known

functionality [182]. In part, such functionality can come from tissue culture expression systems of mutant and WT ABCR proteins [155]. Clearly, one would expect an earlier onset of measurable A2E and LF accumulation in severe mutations, such as those that cause RP19, and proportionally later age of onset and rate of accumulation in mutations with less functional impact on the protein.

Also, the many mutations in BEST-1 promote a substantial variety of phenotypic outcomes with three different disease states (BMD, AVMD, ARB). In the first two autosomal dominant diseases, the spectrum of severity of mutational impact on the BEST-1 proteins leads to more severe early onset disease (BMD) with severe protein phenotypes or later onset disease

Fig. 11.15  ABCR mutations and the model of decreased enzyme activity. The nature of the mutations in ABCR, or in any other gene, lead to a plethora of possible structural and/or functional deficits at the protein level. A partial or complete loss of protein and function can result from stop mutations or frame-shifting mutations depending upon their location in the molecule. Missense mutations can cause a range of structural or functional defects depending upon which amino acid is mutated, how conserved that particular amino acid is (related to evolved structure/ function), and the biophysical nature of the side chain of the amino acid replacement (e.g., Arg to Glu replaces a positively charged side chain with a negatively charged side chain and would be expected to be deleterious at highly conserved amino

acids whereas Ala to Val conserves the properties of the hydrophobic side chain and would be expected to be less deleterious). Since ABCR is an enzyme, the impact of mutations on enzyme function is expected to play a substantial role in the phenotypic outcomes that become manifest. Generally, premature stop mutations, frame shift mutations, and introns splicing mutations will be more deleterious than missense mutations. As STGD1 is an autosomal recessive disease, the combination of two inherited mutant alleles sets the stage for a wide range of phenotypic diversity. Adapted from van Driel et al. [151], the schematic shows a relationship between the nature of the combined mutations and the severity of the phenotype, measured in terms of a scale of two copies of WT enzyme