Retinal dystrophies, genomic applications in diagnosis and prospects for therapy

Retinitis pigmentosa (RP)

The most common clinical manifestation of RD is RP. RP is a progressive non-syndromic rod-cone disease and has high levels of clinical and genetic heterogeneity. Variation exists at multiple levels with locus and allelic heterogeneity, incomplete penetrance and variable expression and penetrance all observed (9). The onset of the disease varies with early onset or juvenile RP sufferers affected from as early as the first years of life whereas adult or late onset RP symptoms develop significantly later. Clinical presentations manifest with progressive deterioration of the ability to see in dim light causing night blindness, followed by loss of peripheral vision that slowly encroaches toward the centre of the visual field resulting in tunnel vision (Figure 1). Later stages of the disease can result in complete blindness where the cone photoreceptors are also implicated. Affected photoreceptors undergo apoptosis, which is evident with the thinning of the outer nuclear layer and pigmented deposits or lesions present in the diseased retina (Figure 2). The loss in visual acuity has been shown to be proportionate to the level of deterioration of the fundus. Other clinical manifestations associated with RP include posterior subcapsular cataracts, dust like particles in the vitreous, white dots deep within the retina and Hyaline bodies affecting the optic nerve. The affected region of the retina may be restricted to a specific site, adding further complexity to disease identification (10).

Over 60 disease genes are reported to associate with RP (Table 1) (5,122). Known functions of the encoded proteins can be grouped into five broad categories including: phototransduction; retinal metabolism; RNA splicing; tissue development and maintenance; and cellular structure. Modes of inheritance vary with 15-20% autosomal dominant, 5-20% autosomal recessive, 5-15% X-linked, and simplex or unknown inheritance observed in 40-50% of cases (9). Digenic inheritance has also been observed where heterozygous mutations in two genes ROM1 and PRPH2 have been shown to cause the RP phenotype (77). Due to such complex clinical presentations and genetic factors, even with the latest genetic diagnostic techniques, including NGS strategies, molecular diagnosis is only achieved in approximately 50% of tested RP patients (123). There is also further genetic heterogeneity with some genes implicated in other forms of RD.

Mutations in the gene RHO, encoding rhodopsin which is critical in phototransduction, are a leading cause of RP. Rhodopsin is a 7-transmembrane spanning protein making up approximately 80% of the protein found in the disc membrane of rod outer segments. RHO contains five exons that encode 348 amino acids. The primary function of the protein is to initiate the phototransduction cascade by facilitating the conformational change of 11-cis retinal into 11-trans retinal. Mutations in this gene are seen in ~20-30% of autosomal dominant RP while autosomal recessive inheritance is also observed with certain mutations, but much more rarely. Mutations that lead to recessive forms of inheritance are suggested to still confer a phenotype in the heterozygous state, however it is milder or with onset later in life (124). The severity of the phenotype appears to depend on the location of the mutation in the protein. Patients with mutations leading to abnormal amino acids in the parts of rhodopsin located in the intradiscal space show a less severe phenotype with better visual acuity and improved dark light adaptation, compared with those where mutations affect amino acids in the cytoplasmic space. Patients with mutations occurring within the transmembrane regions of rhodopsin showed an intermediate outcome (125). Intrafamilial variation is also noted amongst patients indicating the likely presence of genetic modifiers and/or environmental factors contributing to the phenotypic effects that are seen (124).

Other RP disease genes implicated in the phototransduction process have an expected frequency of less than 2-5% amongst affected individuals. Autosomal dominant inheritance is observed due to mutations in RDH12, encoding retinol dehydrogenase-12, which is responsible for metabolising all-trans and -cis retinols (14). GUCA1B encodes guanylate cyclase-activating protein 2, which is responsible for activating photoreceptor guanylate cyclases for the conversion of cGMP to cGTP involved with the hyperpolarisation response to light (13). Mutations in this gene lead to autosomal dominant RP. Autosomal recessive inheritance is seen associated with mutations in PDE6A and PDE6B, which encode phosphodiesterase 6A & 6B responsible for the α and β subunits respectively of a key enzyme that maintains cytoplasmic cGMP concentration crucial for rod cell phototransduction (16,18). Mutations in CNGA1 and CNGB1 also follow an autosomal recessive inheritance pattern and these genes encode the α and β subunits of cyclic nucleotide gated ion channels responsible for opening of calcium channels after binding of the cGMP/cGTP ligand (21,23). The SAG gene encodes the arrestin protein responsible for the inactivation of the phototransduction cascade, specifically acting on the activated rhodopsin molecule. While mutations in SAG are implicated in a form of CSNB, discussed below, a homozygous 1-bp deletion (c.1147delA) has been observed in three unrelated individuals with RP (20).

Mutations in RP disease-causing genes that encode proteins associated with retinal metabolism generally follow an autosomal recessive inheritance pattern. These include ABCA4, which encodes the ATP-binding cassette subfamily A member-4, a transmembrane protein that facilitates the removal of all-trans retinaldehyde from the photoreceptor (3); the LRAT gene which encodes lecithin retinol acyltransferase, an enzyme located in the RPE that initiates the reactions where all-cis retinal is derived from all-trans retinol (vitamin A) (28); the RBP3 gene which encodes retinal binding protein 3 that is secreted from rod photoreceptors and responsible for the transportation of retinoids from the photoreceptor to the RPE and for the binding of fatty acids in the interphotoreceptor matrix (30); the RGR gene which encodes the G protein-coupled receptor retinal that is located in the RPE and preferentially binds all-trans retinal facilitating its conversion into 11-cis retinal (32); and the RLBP1 gene which encodes retinaldehyde-binding protein 1 found in the RPE (33). RPE65 that encodes retinal specific protein 65 kD is responsible for the conversion of all-trans retinyl ester to 11-cis retinol in the RPE. Mutations in this gene are most usually associated with a severe autosomal recessive form of RD called LCA (26). Interestingly, mutations in RPE65 have also been observed segregating in an autosomal dominant pattern in RP, highlighting further the variation of outcomes from mutations within the same gene (27).

Genes encoding splicing factors have also been implicated in the expression of an RP phenotype and all follow an autosomal dominant inheritance pattern. These genes include PRPF31, PRPF8, PRPF3, PAP1, SNRNP200 and PRPF6 (38,41,42,45,46,48). It is interesting that despite deficiencies in splicing having an effect on processes of the entire cell, mutations in the above-mentioned genes only confer an RP phenotype, and there are various reasons considered for this. These include that the splicing factor affected acts specifically on genes which are expressed in the retina, such as RHO (126), or that the splicing of genes outside the retina may be affected, but the phenotype is only evident in the retina due to its specific rapid turnover requirements (127). Some genes may be important in spliceosome assembly and maturation, and studies are ongoing to understand the mechanisms causing the splicing abnormalities (128,129).

Retinal tissue development, differentiation and maintenance is critical for proper photoreceptor function, and mutations in genes encoding factors critical in these processes can cause RP including: RP1, NR2E3, CRX, NRL, SEMA4A, FAM161A, TULP1, RP2, CRB1 and IMPDH1 (2,52,58,60,62,63,65,67,69,130). A subgroup of these genes, namely CRX, NRL and NR2E3 are known to interact during retinal neurogenesis. CRX is a transcription factor that regulates retinal cellular differentiation and also plays a role in the maintenance of neural bipolar cells in the adult retina (54). Interestingly a p.Arg41Gln missense mutation has been shown to cause RP in one study while being associated with cone-rod dystrophy (CORD) in another (55,131). NRL encodes a neural retina leucine zipper transcription factor and NR2E3 encodes a ligand-activated transcription factor. These are exclusively expressed in rod photoreceptors and are required for their differentiation during retinal development (52,60).

The functions of the photoreceptors can only take place through specialised cellular structures and development and maintenance of these are critical for normal retinal function. Mutations in genes encoding proteins in this group that have been associated with autosomal recessive RP include: PROM1, MAK, IMPG2, DHDDS, CLRN1, and USH2A (73,79,81,83,88,89) while genes associated with autosomal dominant RP include: FSCN2, ROM1 and PRPH2 (75,77,85,86). PROM1 encodes Prominin-1, which is a highly conserved protein across the animal kingdom that plays a crucial role in disc membrane morphogenesis in rod photoreceptors (83). MAK encodes the male germ cell-associated kinase involved in regulation of cilium length that connects the outer segments to the cell bodies within the photoreceptor (81). The interphotoreceptor matrix proteoglycan is encoded by the gene IMPG2, and is a part of the extracellular matrix which connects the photoreceptor outer segments to the RPE (79). DHDDS encodes a dehydrodolichyl diphosphate synthase, which localises to the inner segments of the photoreceptor and plays an important role in the glycosylation of rhodopsin (89). CLRN1 and USH2A encode clarin-1 and usherin, respectively, and are both implicated in autosomal recessive forms of syndromic and non-syndromic RP. The clarin-1 protein contains five transmembrane domains that secure the protein into the plasma membrane and is thought to include roles in both hair cell and photoreceptor synapses (88). Usherin is a plasma membrane bound protein with a large extracellular domain, which performs structural and signalling functions through interactions with the cilium of photoreceptors and hair cells (73). Usherin knockout mouse models reveal progressive degeneration of the retina indicating usherin is involved in photoreceptor maintenance (132). Mutations in CLRN1 and USH2A are predominantly associated with Usher syndrome, a disorder which presents with both retinal degeneration and sensorineural hearing loss (132,133).

BEST1 mutations are found in a group of RDs termed ‘Bestrophinopathies’ inherited in both autosomal dominant and recessive patterns (96,134). The BEST1 protein is predominately expressed in the basolateral membrane of the RPE and has been proposed to be an ion channel regulator interacting with calcium activated chloride channels (CaCC). The majority of missense mutations are found in the conserved N-terminal of the protein which contains the membrane spanning and calcium interaction domains (135).

Congenital stationary night blindness (CSNB)

CSNB is a non-progressive form of night blindness, also known as nyctalopia, where patients find it difficult to see in relatively low light intensities. Presentation onset is from an early age and can also include decreased visual acuity, myopia, nystagmus and strabismus (136). Photoreceptor function as measured by electroretinogram (ERG) may indicate absence of rod pathway function, or there may be incomplete rod and cone dysfunction. Varying abnormalities may be observed on fundus examination. Complete CSNB results from defects in bipolar cell signalling pathways, resulting in only one intact alternate pathway (137). There are currently 11 genes where mutations have been identified in patients with CSNB (Table 2). Known functions of these genes include roles in calcium channel function with respect to CACNA1F, CACNA2D4 and TRPM1 (137,141,148), calcium-binding in CABP4 (142), glutamate receptor functions in GRM6 (143) and involvement in the phototransduction cascade in GNAT1, GRK1, PDE6B, SAG and RHO (138-140,146,147).

The incomplete form of X-linked CSNB (CSNB2) is caused by disruption of the CACNA1F gene, which encodes the α1F subunit of calcium channels located at the synaptic connection between the rod photoreceptor and the bipolar cell (148,149). The calcium channel regulates the release of glutamate into the synaptic cleft depending on the membrane potential of the bipolar cell or photoreceptor. It has been shown that loss of function mutations of the gene disrupt the calcium ion flow at this synapse resulting in a non-functional channel and therefore loss of function of the photoreceptor (151). The CSNB2 phenotype that is observed includes a diminished scotopic b-wave on ERG, indicating a diminution in the signalling between rod photoreceptors and bipolar cells (149). There have been more than 50 reported mutations in this gene that result in various forms of protein truncation and calcium channel loss of function (136,152).

The complete form of X-linked CSNB (CSNB1) is caused by mutations in the gene NYX, which is predominantly expressed in the retina. Although the exact mechanism is yet to be known, the encoded protein nyctalopin is a small leucine-rich proteoglycan that when mutated is believed to disrupt bipolar amacrine and ganglion cell signalling (150). Truncated proteins are typically non-functional and would be expected to cause the phenotype observed. The CSNB1 phenotype is distinguished from CSNB2 with ERG as both rod and cone photoreceptor function is affected (150). Deletions within the gene have also been observed in mice which have resulted in the no b-wave (“nob”) phenotype (153).

Autosomal recessive forms of CSNB can be broken up into 3 clinical sub-types including incomplete forms, complete forms and Oguchi disease. Oguchi disease is clinically distinct from the other autosomal recessive CSNBs due to characteristic ERG abnormalities including an absent rod response with normal cone responses. Fundal changes known as the Mizuo-Nakamura phenomenon are also observed whereby during light exposure and adaption the retina has a metallic, golden brown appearance. This appearance disappears however after the retina is returned to a complete dark adaptation (154). There are now two forms of Oguchi disease known, type 1 and type 2, which are caused by mutations in SAG and the G-protein dependent receptor kinase 1 gene GRK1 respectively. Investigations into a cohort of Japanese patients indicated that arrestin and rhodopsin kinase genes are possible candidates for the phenotype due to their association with the inactivation of rhodopsin in the recovery phase of phototransduction (146). A SAG c.1147delA mutation was observed more frequently in affected unrelated Japanese individuals, which reflects a founder effect (147). Mutations in SAG have also been found in patients with an RP phenotype, suggesting variable expression associated with mutations within the gene (20). GRK1 mutations are mainly reported to cause an Oguchi phenotype, although there have also been reports of an RP phenotype (155).

Autosomal dominant forms of CSNB are caused by mutations in GNAT1, PDE6B, and RHO, all of which are involved in aspects of the phototransduction cascade (138,139,146). GNAT1 encodes Guanine nucleotide-binding protein α, α-transducing activity polypeptide 1 which assembles the α subunit in the rod transducing protein that stimulates the coupling of rhodopsin and GMP during the photoreceptor visual response. PDE6B codes for the β subunit of a membrane bound enzyme, phosphodiesterase 6B, that is responsible for the hyperpolarization of the rod photoreceptor. Mutations in RHO are more usually seen in patients with autosomal dominant RP but have also been identified in CSNB depending on the amino acids that are affected (156). One study identified the RHO c.884C>T:p.Ala295Val missense mutation in a family with autosomal dominant CSNB, where patients had a diminished sensitivity and response to light at lower intensities without any additional retinal degeneration (157).

Progressive cone and cone-rod dystrophies

The onset of progressive cone (COD) and CORD is usually during early childhood or adolescence. Affected individuals usually present with only cone photoreceptor involvement (COD) or cone followed by rod degeneration (CORD). Differences between the two dystrophies are apparent with additional rod involvement leading to an increase in severity with most sufferers reaching legal blindness by the age of 40 (158). On fundus examination, appearance of the macula varies with some cases presenting with an atrophic appearance (Figure 3), or retinal pigment deposits. Currently there are over 30 genes described with reported disease-causing mutations (Table 3), with various roles in similar functional groupings as those described for the rod dystrophies, but with specificity of function to the cone photoreceptors. As shown in previous review of the literature, molecular causes are able to be identified in approximately 20% and 74% of autosomal dominant and X-linked COD/CORD respectively, while 23-25% of autosomal recessive pedigrees can be resolved (200). This indicates that many underlying disease genes remain to be identified especially in the autosomal dominant and recessive forms of these conditions.

Subgroups of genes where mutations lead to COD and CORD are also associated with other forms of RD, presumably based on their relatively ubiquitous functions across the photoreceptors and/or retina. As an example, ABCA4 mutations are further implicated in RP, and a form of juvenile macular degeneration also known as Stargardts disease (3,201). ABCA4 is the most frequently identified disease gene in COD and CORD of autosomal inheritance with studies reporting its frequency in 9% and 26% of cases respectively (158). Furthermore, truncating mutations involving ABCA4 are more evident in the CORD cohort compared to the COD cohort, reportedly seen in 76% and 63% respectively (158,200).

Achromatopsia

The stationary forms of cone dystrophy can exist in two forms whereby complete or incomplete achromatopsia results in the loss of all colour perception, or the perception of only a specific colour respectively. Tritanopia, or defective blue vision is an autosomal dominant phenotype that is caused by mutations in the gene OPN1SW, which encodes the short wave sensitive opsin that detects blue light (202). Genes currently known to be associated with autosomal recessive complete achromatopsia include CNGA3, CNGB3, GNAT2, PDE6C and PDE6H. CNGB3 alone is responsible for up to 50% of complete achromatopsia in affected individuals (203). CNGA3 and CNGB3 encode the α and β subunits of cGMP-gated channels located in the cone photoreceptor, which are involved in key steps of phototransduction (204,205). GNAT2 encodes the cone-specific α subunit of transducin, which is the cone visual pigment that induces one of the first steps of the phototransduction cascade (206). PDE6C and PDE6H encode the cone specific α and gamma subunits of a cGMP phosphodiesterase respectively, which is an enzyme responsible for the conversion of cGMP to 5’-GMP during light exposure (163,164).

The genetic overlap across the non-syndromic RDs including RP, COD/CORD and the stationary forms is quite complex and is illustrated in Figure 4. Genetic overlap is expected to a certain degree due to fundamental similarities in photoreceptor structures and cellular processes despite independence of their scotopic and photopic roles.

Leber congenital amaurosis (LCA)

The most common non-syndromic generalised RD is LCA. The onset of LCA is early with affected individuals developing symptoms within the first year of life. Clinical features include poor vision, nystagmus, and no measurable light response on ophthalmic examination with ERG (207). A characteristic finding is Franschetti’s oculo-digital sign where patients repeatedly rub and poke their eyes. The physical appearance of the retina varies in early stages, however retinal pigmentary changes can be observed with progression of disease (207). Interestingly, there have been suggestions of genotype-phenotype patterns of retinal appearance including a translucent RPE appearance with white dots with RPE65 gene mutations and progressive macular atrophy prominent in NMNAT1, but which is also seen in patients with AIPL1 and CRB1 mutations (208). To date there are over 20 genes associated with LCA with nearly all following an autosomal recessive inheritance pattern (Table 4). Despite this, the underlying genetic causes of LCA are not fully known (231), with genetic overlap with other forms of RD present (Figure 4).

Choroideremia

The only X-linked form of non-syndromic generalised RD is choroideremia, which is caused by mutations in the gene CHM (232). Patients present during the second decade of life with night blindness and progressive degeneration of photoreceptors, the RPE and the choroid. Characteristically, affected males have an appearance of chorioretinal scalloped atrophy in the midperipheral fundus. CHM encodes REP-1, a subunit of the intracellular trafficking protein rab protein 1 which is responsible for the intracellular transport of proteins and organelles (233). Multiple non-synonymous mutations along with insertions and deletions have been reported to be associated with the disease (234). Heterogeneity is evident among choroideremia patients with some atypical presentations being first identified as RP on clinical examination. Studies using genomic tools to provide molecular diagnosis are proving useful in refining clinical diagnosis (235).

This review of clinical features and underlying genetic causes in the non-syndromic degenerative and stationary RDs including RP, COD/CORD, LCA, CSNB and achromatopsia indicates that while there are specific groupings, there is also a degree of clinical overlap and genetic causes in these conditions (Figure 4). Knowledge of these overlapping clinical features and genetic causes is important in design of molecular diagnostic approaches, molecular genetic result interpretation and work towards therapies in these conditions.