Finding causative mutations for retinal disease can be difficult due to the large number of genes responsible, as well as the complexity of the genotype phenotype relationship in these disorders. For example, LCA is caused by eighteen genes [53
], and mutations in these genes can also cause RP, vitreoretinal degeneration, pigmented paravenous chorioretinal atrophy, Senior-Loken syndrome, congenital cone-rod synaptic disease, Joubert syndrome, Meckel syndrome, cone-rod dystrophy, and microphthalmia [73
]. New connections are still being made [72
]. Nonetheless, an accurate molecular diagnosis is essential, not only for gene therapy but also for genetic counseling, pathway specific drug prescription [16
], and prognostics. The two most commonly used clinical mutation identification tools are Sanger sequencing and Allele specific Primer EXtention (APEX) arrays.
Sanger sequencing is considered to be the golden standard for mutation identification due to its accuracy. However, because Sanger sequencing is labor intensive and has high per-base sequencing cost, it can be only effectively used to scan a limited number of exons for mutations, or to confirm mutations found by other methods. Given that 185 retinal disease genes have been identified to date, Sanger sequencing every exon potentially contributing to retinal disease is prohibitively costly and time-consuming. This difficulty drove the development of high throughput, array based genotyping methods for retinal disease.
APEX is an array based SNP genotyping technology [75
] that is used to sequence the base pair following a designed oligonucleotide of choice. Since each spot on the array only sequences a single base pair, only known pathogenic bases are targeted. The current Asper Ophthalmics LCA array includes 780 disease-associated sequence variants identified in 15 LCA and early-onset RP genes [76
]. This method is accurate, but is only able to detect novel mutations if they occur at the same position as a known mutation. Further, only a subset of all known LCA genes and mutations are tested. This process generates a successful diagnosis in ~17%–32% of LCA cases [77
Similarly, three Asper Ophthalmics APEX panels exist for RP, each for a separate form of inheritance. For example, the latest dominant RP panel offered by Asper provides simultaneous detection of 414 known disease-associated variants in 16 genes, while their recessive RP panel screens 594 known mutations in 19 genes [79
]. APEX based diagnostics greatly increase diagnosis efficiency when screening a large number of mutations from multiple genes, allowing for high throughput analysis. However, since current arrays can only detect known mutations in a subset of known RP genes, successful diagnosis is only achieved in 15% of all RP cases [80
]. Further, performing the test requires knowledge about the inheritance pattern of the patient, which is only available for about 50% of patients [82
Molecular diagnosis of patients using current methods is either prohibitively expensive or suffers from very low accuracy. In the following section, we will review molecular diagnosis using NGS based strategies for three common retinal diseases: LCA, RP, and Stargardt disease.
3.1 NGS based molecular diagnosis in Leber’s Congenital Amaurosis
LCA is the most extreme form of inherited retinal disease, as LCA patients typically have severe visual impairment or blindness within the first year of life. Other symptoms include congenital nystagmus, defective pupillary responses, and a reduced signal in electroretinograms (ERG) [83
]. LCA is estimated to affect one in every 30000–80000 individuals and accounts for ~5% of all retinal dystrophies [84
]. It is estimated that about 70% of European LCA cases are explained by known LCA genes [18
], though this number is expected to be lower for different ethnicities [86
Interestingly, mutations in the same gene can cause either syndromic or non syndromic eye disease. For example, mild alleles in CEP290 cause LCA, while complete loss of CEP290 function leads to Joubert syndrome [58
]. Also, the recently identified LCA gene KCNJ13 was previously reported to cause vitreoretinal degeneration [72
], a related but distinct disorder. These findings underscore the genetic heterogeneity and difficulty of genetic diagnosis in LCA.
Compared with microarray based methods, NGS has a lower per base cost and is able to detect novel mutations and novel genes. When preceded by a capture array, which specifically isolates DNA of interest, NGS can efficiently sequence every base pair in a large area of interest. This flexibility allows NGS to frequently identify novel mutations [88
] in inherited human diseases. Indeed, Frauke Coppieters et al. recently diagnosed LCA patients using DNA capture combined with NGS. They were able to confirm previously identified mutations, and found the causal mutations missed by arrays in 3 out 17 patients [90
], an increase in diagnosis rate of over 17%.
Due to the genetic heterogeneity of LCA and low rates of diagnosis, it is evident that a portion of LCA is caused by mutations outside of known eye LCA genes. The high throughput nature of capture-NGS can be used to efficiently query large sets of genes to search for this missing inheritance. Indeed, utilizing a capture chip targeting all known retinal disease genes, recent work in our lab shows that a high percentage of LCA cases are indeed caused by mutations in genes known to cause retinal disease, but previously unlinked with LCA (unpublished data).
In summary, NGS based genetic diagnosis of LCA has several advantages over previous methods, but has the added challenge of properly interpreting NGS data. Also, due to LCA’s genetic heterogeneity, comprehensive screening every base in all known retinal disease genes is desirable. Currently, this can only be efficiently achieved with NGS.
3.2 NGS based molecular diagnosis in RP
Retinitis pigmentosa (RP) is a progressive retinal degeneration, affecting about 1 in 4000 people worldwide [91
]. Initially, rod photoreceptor cells begin to die. Patients at this stage develop early onset night blindness and tunnel vision. As the disease develops, the loss of rod cells begins to damage cone photoreceptors. Their degeneration causes a reduction of central and color vision and may lead to complete blindness. The retinal pigment epithelium (RPE) is the third major type of cell that degenerates over the course of RP, in response to loss of photoreceptor cells. RPE cells release pigment granules as they degenerate, which often accumulate in a bone spicule configuration that further occludes vision.
RP is highly heterogeneous, with autosomal recessive (ar), autosomal dominant (ad), X-linked, digenic, and mitochondrial forms. To date, 52 genes functioning in diverse biological pathways have been linked to RP [92
]. Among them, genes involved in the phototransduction cascade account for a major portion of RP cases. Mutations in phototransduction-related genes RHO and PDE are responsible for 25% adRP cases and 8% of arRP cases, respectively [93
]. Because of its genetic heterogeneity, an accurate molecular diagnosis is challenging. Even with decades of improvement, current diagnostics including Sanger sequencing and APEX still have many limitations.
Recent developments in NGS and DNA capture technology provide a potential new approach to molecular diagnosis in RP. Indeed, it has been shown to have many advantages over current diagnostic methods [19
]. First of all, NGS based molecular diagnosis of RP is the most comprehensive molecular diagnostic method available. By screening of both known and novel mutations in all known RP genes simultaneously, NGS can achieve a significantly higher rate of successful diagnosis. For example, in two recent studies, about 150 RP patients with a variety of inheritance forms were examined by NGS based molecular diagnosis, revealing an overall diagnosis rate of about 50% [19
]. Secondly, some RP cases follow a digenic inheritance model [92
], and approximately 50% of cases have an unknown inheritance model [82
]. NGS can easily be applied to these patients in a single step, making it more applicable than arrays. Finally, the massive sequencing capacity of current NGS machines along with advanced molecular bar-coding technology enables sequencing of multiple samples in parallel [19
]. This significantly improves throughput while reducing time and cost, making NGS an ideal diagnostic platform.
3.3 NGS based molecular diagnosis in Stargardt disease
Stargardt disease is a form of early onset macular degeneration affecting at least 1 in 10000 people [98
], with approximately 31000 affected in the US alone. In Stargardt disease, there is a relatively fast degeneration of the macula caused by the buildup of oily waste deposits called lipofusin (comprised largely of A2E, a vitamin A derivative) in the retinal pigment epithelium (RPE) cell layer. This limits interactions between photoreceptors and the RPE, hampering the ability of photoreceptors to uptake nutrients and perform the visual cycle, leading to their death.
A Stargardt phenotype is only known to be caused by mutations in three genes; ABCA4 [99
], ELOVL4 [100
], and PROM1 [102
]. Of these, ABCA4 is by far the most common cause, and the only known gene for recessive Stargardt disease. ABCA4 functions as a flippase for N-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine, moving these compounds from the luminal to cytoplasmic side of the photoreceptor outer segment discs [103
]. Loss of this flippase function leads to the toxic buildup of lipofusin [104
]. Mutations in ABCA4 also cause a large fraction of RP and cone-rod dystrophy cases, and as a result ABCA4 is a very well studied gene [98
It might seem that Stargardt disease is a poor choice for NGS based diagnosis, as the power of NGS is not as easily justified in a disease that is only typically caused by mutations in one gene. However, even with a huge number of known exonic variants (>600 on the current diagnostic chip), homozygous or compound heterozygous disease causing mutations in ABCA4 are found in only ~30%–40% of patients using APEX arrays [106
]. Using NGS based diagnosis for ABCA4, a group was able to identify mutations in 48% (73/142) of their patients [88
], while a more recent study using NGS was able to solve 33% of cases that remained unsolved after use of an array [89
]. This increase in accuracy is due to the ability of NGS to identify novel, rare, detrimental alleles. Polymorphic loci in ABCA4 are 9–400 times as common as in other retinal disease genes [108
], generating a lot of variety that can only currently be resolved through sequencing. Thus, even in monogenic diseases NGS can offer advantages over array based diagnostic methods.
Regardless of the method used to identify exonic mutations, a molecular diagnosis in Stargardt disease remains elusive in a significant fraction of cases. One explanation for this phenomenon is that intronic, regulatory and/or structural variations, including copy number variations (CNVs) and insertion/deletion mutations, account for a large portion of disease alleles. This hypothesis has been supported by microarray studies, which found ABCA4 haplotypes segregating with disease in families with no identified coding mutations [109
]. The genomic size of the ABCA4 locus is over 128 kb, making it far too large for it to be efficiently Sanger sequenced. However, NGS, through the use of a capture array, could be used to probe the entire genomic region around ABCA4. Understanding the effects of extra-exonic mutations is a topic of current research [110
]. Improvements are also being made in the ability of NGS to detect large deletions and CNVs [111
As our knowledge about other retinal diseases grows, it is likely that they will end up like ABCA4 in that many cases of the disease will not be explainable by protein coding variation alone. Improved molecular diagnosis, and its clinical advantages, will necessitate sequencing of large swaths of the human genome, including the regions containing all known retinal disease genes. By moving toward NGS based diagnosis, it is possible to both improve the efficiency of probing exonic regions and open up whole new areas to directed analysis.