WES provides a cost-effective analysis for clinical investigation in families with IRDs as well as research studies aiming to identify novel retinal disease genes. Our study used WES to study 10 families from Alberta, Canada, chosen from a database of ophthalmology patients (IMM) based on the likelihood of identifying novel genetic associations in retinal and ocular disease. Below we discuss these results in context of our genetic and clinical findings. We also describe potential genetic associations in our unsolved cases that provide insights for other investigators of these blinding conditions.
Clinical review of this patient indicated a likely diagnosis of choroideremia, however, molecular testing of CHM/REP1 precluded this diagnosis. Our results identified a RPE65 variant (c.1430?G?>?A: p.(D477G)) previously shown to cause choroidal atrophy [14]. Though RPE65 variants cause recessive Leber congenital amaurosis, this variant has a unique effect and is the only RPE65 variant known to cause a choroideremia-like appearance. Functional studies of the p.(D477G) variant have shown that the protein variant facilitates mono- and di-ubiquitination of the RPE65 protein [15], and another showed that c.1430?G?>?A leads to aberrant RNA splicing [16]. We suggest that cases of choroideremia with negative CHM sequencing should be tested for this RPE65 variant.
Our analysis identified two variants in BBS1 (c.1169?T?>?G: p.(M390R) and c.1040del: p.(M347fs)). The affected males were initially diagnosed as non-syndromic RP but the presence of known BBS1 variants, as well as additional family information (presence of post-axial polydactyly and learning disabilities), altered our diagnosis to Bardet-Biedl Syndrome, a multi-system ciliopathy. The c.1040del variant was previously reported in a large study of BBS1 [17]. In addition, the p.(M390R) variant has been reported to cause a wide spectrum of phenotypes from non-syndromic RP to severe BBS [18] when inherited in a recessive manner. This suggests that genetic modifiers or mutational burden influences phenotypic presentation, a phenomenon that has been documented to M71 impact presentation in ciliopathies such as BBS [19, 20].
We identified a known pathogenic recessive variant in ABCA4 (c.4469?G?>?A: p.(C1490Y)) in the proband of family M53. This prompted a review of clinical data, as well as a search for the second variant, which ultimately identified a deep intronic variant through a collaborator c.4539?+?2028?C?>?T: p.(R1514Lfs*36) [10]. Combined, this data led us to diagnose Stargardt macular dystrophy, an adolescent-onset maculopathy. This case illustrates the importance of identifying heterozygous variants in WES data for recessive conditions, which may act as ‘guides’ for further analysis.
WES analysis of family M73 identified a single variant in PEX6, c.1802G?>?A: p.(R601Q). This variant has been previously associated with a mild peroxisomal biogenesis disorder called Heimler syndrome [12], characterized by macular schisis, SNHL, and dental/nail abnormalities. Although the proband of this family presented with SNHL and macular schisis, no nail or dental abnormalities were identified and normal pipecolic/plasmalogen were noted in blood. These findings indicate that this is either a mild presentation of a peroxisomal disorder, or that our finding is coincidental. cDNA analysis showed no splicing abnormalities; however, this analysis was carried out in EBV transformed lymphoblast cells and may not contain the appropriate PEX6 isoform. It is possible that a second variant is controlling expression of the PEX6 gene through a regulatory sequence, which may be detectable through targeted or NGS.
Drusen are subretinal deposits of lipids and proteins that are a major risk factor for age-related macular degeneration (AMD), though the exact relationship between drusen and photoreceptor death is not clear. We report two pedigrees with early-onset (<40 years of age) bilateral, macular drusen and compound heterozygous variants in LRP1: Family M54 with [c.650?C?>?T];[c.9628?G?>?C] and M70 with [c.2910?G?>?A];[c.11930?C?>?T]. One study showed that the c.650?C?>?T variant may activate a cryptic microRNA binding site, leading to altered expression of LRP1. Although the c.2910?G?>?A:p.(S970?=?) variant is synonymous, in silico splice predictions (SplicePort and Human Splice Finder) for the c.2910?G?>?A variant predict that this likely creates an exonic splice suppressor and alters an exonic splice enhancer near the variation. LRP1 is an intriguing candidate gene in the pathology of drusen formation. First, it has direct interaction with many components of drusen and proteins associated with MD, such as amyloid-beta [21], APOE, complement factors, and components of lipid metabolism. Second, LRP1 is expressed in the retinal pigment epithelium (RPE). The RPE provides nutrients to the neural retina, and the basal RPE is the site of drusen formation. Third, LRP1 provides a fascinating link between the three pathologies M71 involved in age-related MD: lipid metabolism, complement pathway, and extracellular matrix homeostasis (Fig.?3). We hypothesize that dysfunction of LRP1 protein leads to accumulation of extracellular material over time due to altered endocytosis kinetics, resulting in drusen. Due to its multiple roles in MD-related pathways and our interesting genetic results, we hypothesize that LRP1 plays a role in drusen formation. Further studies of this protein and its relationship to drusen formation and MD are necessary.
To the best of our knowledge, Family M72 presents a novel dominantly inherited condition. WES identified two novel variants: c.62?A?>?G: p.(D21G) in STUM, and c.122?A?>?C: p.(E41A) in UBE2U. Of interest, UBE2U has been reported to regulate RNF168 [22], an E2-ubiquitin conjugating enzyme that has been associated with the Radiosensitivity Immunodeficiency Dysmorphic features and Learning difficulties (RIDDLE) syndrome (OMIM 611943). Clinical assessment by a medical geneticist (OC) indicated this family shared some systemic dysmorphisms (short stature, small head circumference, low weight, hypertelorism) and behavioral/learning disabilities, similar to RIDDLE syndrome patients. We predict that the variant leads to an abnormal interaction between variant UBE2U and RNF168 and leads to a RIDDLE syndrome-like phenotype in our patients (Fig.?4). The affected mother of this family also developed breast cancer at age 31. The RNF168 system is involved with the repair of DNA damage and has a physical interaction with BRCA1, the most common cause of genetic breast cancer [23]. The diagnosis of breast cancer in our patient may be unrelated but is an interesting observation in the context of the UBE2U variant.
WES analysis yielded no obvious results to pinpoint the genetic etiology of this retinopathy. Of interest was the heterozygous CNGB3 c.1148del variant, previously associated with achromatopsia, an autosomal recessive cone photoreceptor disease. However, we excluded the likelihood of a second CNGB3 variant as the electrophysiological results make a diagnosis of achromatopsia unlikely; however, the testing revealed a slightly depressed b-wave, indicating a dysfunction between photoreceptors and the interneurons. We identified two heterozygous variants in genes that cause CSNB: GRM6 (c.2092?C?>?G: p.(L698V)) and TRPM (c.3958?G?>?A: p.(E1320K)). Van Genderen et al. (2009) suggested that these two proteins directly interact and that 
TRPM1 is channel-gated by the GRM6 signaling pathway [24]. We postulate that digenic inheritance of these variants is the potential cause of this inherited retinopathy, though further segregation or functional testing is needed to confirm this.
The two affected individuals shared variants in many genes not previously associated with IRD (Table?3), however a direct cause is not apparent. The most interesting candidate gene is CROCC, which encodes rootletin protein, a core component of the ciliary rootlet [25]. Knockout of rootletin leads to loss of the ciliary rootlet and photoreceptor degeneration in mice [26, 27]. Our reported patient variants may underlie the patient phenotype due to a fragile photoreceptor cilium, which could hinder light detection and phototransduction.
Three affected individuals across two generations presented with retinal detachments. WES revealed no variants in known cataract or retinal detachment-associated genes. We observed novel variations in nine potential candidate genes identified by WES (Table?3). Pathogenicity predictor programs and gene expression data further narrowed this to three genes: ELAVL2, WTIP, and ATG2B. Embryonic Lethal Abnormal Vision-Like 2 (ELAVL2) is a neuron-specific RNA binding protein that regulates transcript expression during neuronal development [28, 29]. ELAVL2 is expressed early during retinal development, coincident with the differentiation of retinal neurons [30, 31]. Another notable gene is Wilms Tumour Interacting Protein (WTIP), which is important for cell–cell and cell–extracellular matrix adhesion in the kidney [32]. There is also evidence that WTIP associates with basal bodies of cilia [33]. WTIP expression and function have not been investigated in the eye, but it may play a role in adhesion of the retina to the extracellular matrix and cell–cell adhesion in the lens. Autophagy Related 2B (ATG2B) is a key component of autophagosome biogenesis [34, 35]. The ATG complex is comprised of ATG2A and ATG2B, which are functionally redundant — therefore, single loss-of-function variants are unlikely to produce a phenotype. However, whether they have overlapping expression in the human eye is unknown. The functional consequence of the patient’s missense variant in ATG2B is unknown; however, impaired autophagosome development could result in accumulation of damaged organelles and abnormal proteins, leading to cellular dysfunction and death.
