Molecular Vision 2022; 28:203-219 <>
Received 23 August 2022 | Accepted 14 August 2022 | Published 16 August 2022

Identification of numerous novel disease-causing variants in patients with inherited retinal diseases, combining careful clinical-functional phenotyping with systematic, broad NGS panel-based genotyping

Priya R. Gupta,1 Wajiha Kheir,1,2 Bo Peng,3 Jie Duan,3 John P-W. Chiang,3 Alessandro Iannaccone1

1Duke Eye Center, Department of Ophthalmology, Center for Retinal Degenerations and Ophthalmic Genetic Diseases, Duke University School of Medicine, Durham, NC,; 2Current affiliation: Department of Ophthalmology, American University of Beirut, Beirut, Lebanon; 3Molecular Vision Laboratory, Hillsboro, OR

Correspondence to: Alessandro Iannaccone, Duke Eye Center, Ophthalmology, 2351 Erwin Rd, Durham, NC, 27705; Phone: (919) 681-8797; FAX: (919) 684-0433; email:


Purpose: The widespread consensus is that genotyping is essential for patients with inherited retinal disease (IRD). Given the numerous ongoing gene therapy clinical trials for IRDs, identifying the pathogenic mutation in these patients has potential important therapeutic implications. In this study, we demonstrate how we identified with a high degree of confidence numerous novel disease-causing mutations, deletions, and duplications in a large consecutive IRD case series by using a judicious combination of careful, in-depth clinical-functional phenotyping to guide and integrate our genotyping approach.

Methods: We conducted a retrospective analysis of data between November 2016 and March 2018 from the Duke Center for Retinal Degenerations and Ophthalmic Genetic Diseases IRD patient database, which encompassed 378 IRD cases that had not yet been previously genotyped. With the exception of some patients who presented with classical clinical-functional phenotypes that allowed for targeted gene testing, all other subjects systematically underwent next-generation sequencing-based broad, IRD-focused panel testing. Most cases were also tested for parental allele phase. Results were reviewed vis-à-vis the clinical-functional phenotypes for reconciliation and potential addition of supplemental testing such as deletion/duplication microarrays or copy number variant (CNV) analysis. Supplemental testing was driven by an IRD specialist-laboratory consensus, and decisions were clinically or genetically driven or both.

Results: By judiciously using this two-way approach and leveraging to its full potential the benefits of careful, in-depth clinical-functional phenotyping by an experienced IRD specialist, more than 80% of the cases in this series were successfully genotyped. We also identified with a high degree of confidence 52 novel disease-causing mutations, deletions, and duplications.

Conclusions: The combination of meticulous, expert clinical-functional phenotyping studies with systematic next-generation sequencing panel-based genotyping and microarray deletion/duplication testing or CNV analysis as applicable in accordance with the above-mentioned consensus was extremely effective at the diagnostic end, reduced costs, and saved time. IRD specialist-laboratory two-way interactions and case discussions would augment the efficacy of this approach and improve the diagnostic yield in successfully solving and genotyping IRD cases.


Inherited retinal diseases (IRDs) are a group of disorders in which genetic abnormalities cause a wide spectrum of visual disorders. They span from congenital conditions that cause significant visual incapacitation from birth but with limited progression over time, such as achromatopsia, blue cone monochromacy, congenital stationary night blindness, and fundus albipunctatus, to other conditions that have variable ages of onset but are characterized by progressive photoreceptor degeneration that leads to significant progressive loss of vision and often to blindness. IRDs are a vastly heterogenous group of disorders, both in terms of genotype and clinical phenotype (National Organization for Rare Diseases) [1-4]. Its overall incidence is estimated to range from 1 in 3,500 to 1 in 4,000 population worldwide [2,4]. The widespread consensus is that genotyping is essential for patients with IRD [4-6]. Well over 300 causal genes have been discovered thus far (RetNet). Each gene has numerous variants associated with diseases that range from point mutations to large changes (e.g., deletions and duplications). The latter ones are often challenging to detect during initial panel-based sequencing and often require further testing ranging from copy number variant (CNV) determination to DNA microarray analysis.

Given the increasing number of retinal gene therapy clinical trials and other gene or mechanism-driven interventions with promising results [3,5] finding the disease-causing genetic etiologies of IRDs has not only critically important diagnostic, counseling, and prognostic implications for affected patients but also important potential therapeutic implications. However, the best approach for genotyping patients with IRD is still controversial in the ophthalmic community [7-9]. The American College of Medical Genetics (ACMG) has set guidelines and recommendations to standardize the process of identifying novel disease-causing variants [8]. These extremely valuable and helpful guidelines have greatly enhanced the accuracy and reproducibility of molecular genetic diagnostic testing. Among the criteria defined by the ACMG, the phenotype expressed by affected patients is considered no more than a supportive finding in establishing the pathogenicity of novel or otherwise uncertain genetic changes (the so-called variants of uncertain significance [VUS]). Phenotypes are certainly variable, but, at times, they are only partially expressed in affected patients, and their characterization relies heavily on the experience and meticulousness of the examining clinicians. Hence, their value from a strict laboratory perspective is understandably generally limited. For IRDs, however, standardized approaches to phenotyping have long been implemented. Owing to the technological advancements in retinal diagnostics and the relative accessibility of the human eye, a tremendous amount of information is available about IRD-specific features. Thus, by engaging actively in clinician-laboratory two-way interactions, the IRD specialist and ophthalmic geneticist have the unique opportunity to provide especially detailed, standardized, highly reproducible, and accurate ocular and systemic phenotyping information that can significantly augment the ability to reach correct diagnostic conclusions about variants that would otherwise be considered only VUS by pure laboratory criteria and other ambiguous cases.

This approach has led to the efficient identification of the genetic etiology in more than 80% of the cases in our database. Herein, we demonstrate how we identified with a high degree of confidence 52 novel disease-causing mutations, deletions, and duplications in consecutive IRD cases by using this judicious combination of careful, in-depth clinical-functional phenotyping to guide and integrate our genotyping approach and report the results.


We retrospectively reviewed a consecutive case series of 378 patients with previously molecularly uncharacterized IRD who visited the Duke Center for Retinal Degenerations and Ophthalmic Genetic Diseases between November 2016 and March 2018. Institutional review board (IRB) approval was obtained before the initiation of this study, and informed consent was obtained from the patients in accordance with IRB policies. All research procedures were adherent to the tenets of the Declaration of Helsinki. In addition to pedigree collection, phenotyping included an in-depth history collection using a standardized vision and review-of-systems questionnaire, complete eye examinations, imaging studies, various types of visual field and electrophysiological studies, including, when appropriate, full-field flash electroretinography (ffERG), multifocal ERG (mfERG), and electro-oculography (EOG).

Targeted/focused genotyping was undertaken only in highly selected cases where the findings from the aforementioned clinical evaluation by the IRD specialist were highly suggestive of a specific genetic etiology by virtue of clinical presentation, functional findings, family history, inheritance pattern, or a combination thereof (see Appendix 1 for a summary flowchart of the decision-making process). For the remaining majority of patients, next-generation sequencing (NGS)-based panel testing, inclusive of CNV detection approaches once this technology became available, was undertaken at baseline. The NGS-based testing included 942 genes and additional mitochondrial genome and mitochondrial nuclear gene testing. Exons, exon-intron boundary regions, and previously known relevant deep intronic regions were sequenced. Whole gene sequencing was not performed in any of the patients included in this retrospective study. Further details regarding the sequencing methods are available in the Appendix 2 section.

In some cases where the etiology remained elusive, panel-based sequencing was then followed by deletion/duplication (del/dup) microarray testing. One or both parental samples were included for case phase assessment to help determine if variants were present in the cis versus trans configuration whenever possible. In cases where parental samples could not be obtained, possible affected or unaffected children and/or siblings/relatives were tested as surrogate best-available alternatives to parental testing to establish the phase of any detected mutation and to confirm segregation among other affected individuals. All genotyped patients underwent Clinical Laboratory Improvement Amendments (CLIA)-certified testing. Each novel variant was then assessed for pathogenicity using a combination of ClinVar, VarSome, PolyPhen-2 missense variant predictions, and laboratory assessments. This information, combined with phase determination and phenotype data, if all concordant was then used to label a variant as pathogenic or “definitely disease causing.” If there was any missing information or even slight ambiguity in the clinical picture, the variant was labeled as just “likely disease causing.” Additional specifics of the phenotypic criteria used to reach our conclusions are presented in Column M of Appendix 3 (“Phenotype Assessment – Salient Clinical Features”) and 2.


The novel disease-causing genetic changes that were identified in our case series (shown in bold) are illustrated in Table 1 and Table 2. Table 3 and Table 4 outline the summarized phenotypic data of each solved case. In selected cases, the disease expression proved to be so uniquely typical that it permitted successful targeted testing. This strategy can be especially useful in monogenic disorders and when medical health insurance coverage is limited or not available to affected patients. Some of these cases are illustrated briefly and further underscore the potential importance of meticulous, in-depth phenotyping.

However, the phenotypic presentation of our patients required, in most of cases, a broad, non-targeted approach. Broad NGS-based panels often revealed many novel genetic changes that only met VUS attribution by strict adherence to the ACMG guidelines. In 10 cases (including some homozygous cases), the parental allele phase could not be fully established. However, even in these cases, careful reconciliation of genotypic results with the aforementioned phenotypic data allowed us to molecularly characterize 44 patients harboring 52 novel mutations, deletions, and duplications that we can define as pathogenic with a very high degree of confidence on the basis of the aggregate evaluation of both the genotypic and compelling phenotypic information. In addition, certain atypical and ambiguous phenotypes were clarified only after genotyping. Further details about each of the 44 patients, the novel mutations they harbored, and their detailed phenotypic characteristics that allowed us to conclude that these novel changes were pathogenic are presented in the Appendix 3. Using this approach, we successfully identified the genetic etiology in >80% of the 378 patients with IRD. Only the novel mutations identified in the course of this retrospective investigation are presented herein.

Example cases in which targeted genotyping based on phenotypic presentation proved successful

The following cases demonstrate some of the few instances in which careful phenotyping pointed to either a monogenic disorder or to a condition for which only a few genes were known to be involved in causing the observed phenotype. In these cases, this approach was faster and cheaper than full panel-based sequencing.

Fundus albipunctatus associated with RDH5 mutations

PT33 (Figure 1, Table 2, and Appendix 3) was a 22-year-old white male patient who presented with a history of congenital night blindness and exhibited a typical fundus albipunctatus phenotype based on both clinical and ERG criteria (improved rod response after prolonged dark adaptation and actually normalized in this case). Previous evidence suggested a stationary nature of the condition. However, cases clinically suggestive of fundus albipunctatus could also be precursors to its progressive phenocopy, RLBP1-linked retinitis punctata albescens. Therefore, on the basis of this presentation, targeted testing with a limited panel inclusive of only RDH5 (gene ID: 5959; OMIM 601617) and RLBP1 (gene ID: 6017; OMIM 180090) was undertaken. No mutations were identified in the RLBP1 gene, whereas two novel RDH5 gene mutations were identified (Table 2), segregating correctly in the parents, corroborating the clinical and functional diagnosis of fundus albipunctatus.

Enhanced S-cone syndrome associated with a homozygous NR2E3 mutation

This case was that of a 7-year-old white female patient who presented with congenital night blindness and displayed a typical enhanced S-cone syndrome (ESCS) clinical and ERG phenotype (Figure 2). This prompted targeted sequencing of the NR2E3 (gene ID: 10002; OMIM 604485) gene, which revealed a previously reported homozygous c.119- 2A>C (IVS1–2A>C) mutation. No further testing was necessary, and the diagnosis was rapidly confirmed. This case was recently reported in an NR2E3-focused case series [10].

Dominant retinitis pigmentosa associated with the RHO P23H mutation

This case was that of a 33-year-old male patient of color who was minimally symptomatic and had a strong family history of retinitis pigmentosa (RP) consistent with autosomal dominant inheritance (histories of three generations affected and male-to-male transmission). Despite the fact that the P23H RHO (gene ID: 6010; OMIM 613731) mutation had never been previously reported outside of Caucasian families of Northern European descent, the degeneration showed a strictly altitudinal pattern (Figure 3) [11-13]. In addition, we have reported other black patients with an altitudinal dominant RP phenotype linked to other RHO mutations. Thus, targeted RHO gene testing was undertaken, confirming the phenotypically driven conclusion that the patient's condition was due to a RHO mutation and identifying the well-known disease-causing P23H mutation. It was subsequently established that the patient had mixed African-American and Northern European ancestry.

Example cases in which broad NGS panel-based testing, and at times subsequent dup/del microarray testing, proved successful

In some cases, broad NGS panel-based testing and, at times, subsequent dup/del microarray testing successfully uncovered the genetic etiology of a patient's IRD. In several situations, phenotyping can be greatly aided and further directed in a “hypothesis testing” manner by genotyping results or at least refined to understand which genetic variants may truly be at play in a given patient or family. In some cases, family history led our investigations and initial interpretations in the incorrect direction and a second round of testing both at the clinical and molecular levels proved essential to resolve ambiguity and establish the genetic etiologies of the conditions exhibited by our patients. In an additional common scenario, initial broad NGS panel-based sequencing, inclusive of CNV detection approaches once this technology became available, did not yield a convincing pathogenic etiology, but subsequent del/dup microarray testing successfully elucidated novel pathogenic variants that escaped detection.

Autosomal recessive bestrophinopathy (ARB) associated with BEST1 changes

Autosomal recessive bestrophinopathy (ARB) associated with a BEST1 (gene ID: 7439; OMIM 607854) mutation and an exon 1–2 deletion in the second allele was found in PT9 (Figure 4, Table 1, and Appendix 3). The patient was a 47-year-old white woman with an atypical clinical presentation and a complex family history, inclusive of a maternal uncle with a form of macular degeneration (MD) with vitelliform features. When first examined at the Duke Center for Retinal Degenerations and Ophthalmic Genetic Diseases in 2014, she exhibited yellowish exudate-like lesions that did not show hyper-autofluorescence (AF) but rather were associated with patches of speckled hypo-AF. Cystoid macular edema (CME) was observed on ocular coherence tomography (OCT). In 2017, the exudate-like features disappeared, and the CME was minimal (not shown). Full-field flash ERG testing revealed a rod>cone (retinitis pigmentosa [RP]-like) pattern of retinal dysfunction of moderate severity.

An initial NGS-based broad panel screening revealed multiple changes. A novel BEST1 c.75C>A, p.Tyr25Ter mutation stood out as very likely to be pathogenic. EOG, which typically exhibits an Arden ratio of <1.5 in Best's disease [14], was performed and supported this possibility in PT9. However, the clinically healthy mother and maternal uncle (i.e., her brother), who was affected with the vitelliform MD, did not exhibit this mutation, and their EOG results were normal. Del/dup microarray testing was therefore performed, revealing a second novel BEST1 exon 1–2 deletion (Table 1). This also shows that while of clear pathogenic potential, the BEST1 exon 1–2 deletion harbored by the mother was phenotypically silent. The father of PT9 was also tested, and the Tyr25Ter BEST1 presumed pathogenic mutation was confirmed to be paternally inherited. Thus, on the basis of these results, it could be convincingly concluded that the patient indeed had BEST1-related disease, as initially suggested by the NGS-based test results, albeit not a dominant one but a form of autosomal recessive bestrophinopathy (ARB) [15].

Consistent with this conclusion, the maternal uncle with the vitelliform MD phenotype was also found to be negative for the BEST1 exon 1–2 deletion, which established that his condition was unrelated to the phenotype expressed by PT9 and was misleading with regard to the initially presumed autosomal dominant inheritance. This information was critically important not only diagnostically but also with regard to the reproductive risk counseling of the proband and other members of her nuclear family. The addition of a diagnostic method that permitted detection of the small deletion (below the typical CNV analysis detection threshold) also revealed a novel disease-causing change.

Pseudo-vitelliform occult macular dystrophy associated with an RP1L1 mutation

The pseudo-vitelliform occult macular dystrophy presentation associated with an RP1L1 (gene ID: 94137; OMIM 608581) mutation in PT35 is perhaps the most challenging and least definitive of the cases in our series. The patient was a 26-year-old black man who presented with unexplained bilateral subretinal fluid and vitelliform changes on OCT with markedly depressed mfERG response amplitude (Figure 5) and normal EOG results (with an Arden ratio well above 1.5) [16]. The left eye had atypical hyporeflective subretinal pseudo-vitelliform lesions on OCT. Thus, this presentation was inconsistent with a BEST1-related disorder and, instead, suggested a possible variant of occult macular dystrophy (OMD), as this presentation was previously reported to occur in patients with OMD [17]. Consistent with this impression, a broad NGS-based panel failed to identify any BEST1 gene changes but revealed a novel RP1L1 change instead (Table 2 and Appendix 3). The clinical diagnosis of OMD could also be confirmed molecularly, at least in part, in this case [17]. The patient was found to also carry 3 VUS, all predicted to be benign using the PolyPhen-2 software and positive in the patient's asymptomatic mother. Two of the VUS were in the ABCA4 (gene ID: 24; OMIM 601691) gene. The first was the c.618C>G, p.Ser206Arg change. Current evidence in the literature is conflicting regarding whether this variant is benign or pathogenic (ClinVar). At times, ABCA4 variants have been reported to be associated with dominant inheritance. Thus, we examined the mother of our patient, who carried this variant, and found that she was asymptomatic and disease-free, providing no evidence that this variant may act dominantly. The other ABCA4 VUS was the c.2546T>C, p.Val849Ala change, which was predicted to be completely benign using the Polyphen-2 software, with a near 0 pathogenicity score. It was also considered a benign polymorphism after evaluation using MutationTaster. The mother's proband did not carry this variant. Even if we postulate that the latter ABCA4 variant could somehow have a pathogenic effect and that, coupled with the former variant, could be responsible for the patient's phenotype, the observed pseudo-vitelliform presentation is entirely inconsistent with an ABCA4-related phenotype. Thus, is this context, in silico and in vivo predictions were consistent with one another and supported a lack of association between the macular dystrophy and these ABCA4 changes. Lastly, the patient also exhibited a PROM1 (gene ID: 8842; OMIM 604365) c.1928C>G, p.Ala643Gly change. Our patient's asymptomatic and disease-free mother possessed this PROM1 variant with no visual consequences. Both Polyphen-2 and MutationTaster predicted this VUS to be completely benign, with a near 0 pathogenicity score by the former and a benign polymorphism prediction by the latter. Thus, we feel fairly confident in predicting that only the RP1L1 mutation was responsible for the phenotype found in this patient. To the patient's knowledge, his father did not have ocular symptoms, however his father was available for neither phase assessment nor eye examinations. Therefore we could not distinguish between the following scenarios: our patient exhibited a de novo mutation not carried by either parent, the father possessed the novel RP1L1 variant but demonstrated no clinical features on ocular exam, or the father possessed the novel RP1L1 variant with mild clinical phenotype detectable on ocular examination but was asymptomatic. Each of these scenarios has been previously reported in OMD families [18]. Thus, while this case remains especially challenging and with the aforementioned caveats, genetic testing revealed a plausible molecular explanation for the observed phenotype with potentially important implications due to the dominant nature of OMD.

Severe cone-rod dystrophy associated with ABCA4 mutations

The patient was a 30-year-old white man who presented with severe diffuse retinopathy, a clinical phenotype of profound loss of vision, minimal residual peripheral fields, and essentially nonrecordable ffERG responses in the presence of disseminated atrophic and pigmentary nummular lesions resembling a disseminated generalized multifocal chorioretinitis (Figure 6). However, he had a history of childhood-onset Stargardt disease. Consistent with this, a broad NGS panel revealed two ABCA4 mutations, a deep intronic mutation (IVS38–10T>C) and a pathogenic synonymous change (p.Val2114Val), segregating correctly in the parents. Both mutations have been documented in the literature, with the synonymous change shown to introduce a premature splice leading to a large deletion in exon 46 [19]. After this discovery, wide-field fundus AF revealed a pattern of peripapillary AF sparing [20] (green arrow) and residual hyper-AF flecks in the far periphery (white arrow), entirely consistent with an ABCA4-linked phenotype but far more serious and advanced than the original diagnosis of Stargardt disease. Thus, despite the disease features that initially suggested more likely sequelae of a primary inflammatory disorder, genetic testing established an autosomal recessive condition instead. Anti-retinal autoantibody (AR-AAbs) testing and retinal immunohistochemistry were also performed for this patient. The results were positive for multiple AR-AAbs and retinal staining, strongly suggesting that a secondary autoimmune component (with supportive evidence of leakage on fluorescein angiography and improved vision by intravitreal steroids and a systemic immunomodulatory regimen, not shown) likely accounted, at least in part, for the disseminated chorioretinal lesions that, to our knowledge, are otherwise not outright typical for ABCA4-associated disease.

Usher syndrome type 2A associated with an exon 27 deletion in the second USH2A allele

PT43 (Figure 7, Table 2, and Appendix 3) was a 42 year-old white male patient with a history of congenital sloping high-frequency sensorineural hearing loss and RP that suggested Usher syndrome type 2. However, it could have been also a case of pseudo-Usher syndrome due to an RPGR (gene ID: 6103; OMIM 312610) mutation [1,21]. The clinical phenotype of typical RP exhibited by this patient was associated with disseminated peripheral nummular cobblestone-like atrophic spots, overall good central AF integrity with a perifoveal “bull's eye” pattern, and peripheral hypo-AF spots, but a good ellipsoid zone foveal residue on OCT (Figure 7). Despite the mild CME, the central acuity (20/25 in the left eye) was good. A close correspondence was observed between the area of best-preserved AF and the size of the III4e target (red) on Goldmann-style semi-automated kinetic Octopus perimetry (Figure 7). An initial NGS screening revealed multiple changes in various genes, including a known disease-causing, paternally inherited mutation in USH2A (gene ID: 7399; OMIM 608400; c.1256G>T, p.Cys419Phe), but no second mutation in this gene was detected. Subsequent del/dup microarray testing revealed that the other, hidden pathogenic allele was a novel large exon-27 USH2A deletion, which was maternally inherited, confirming the initially suspected diagnosis of Usher syndrome type 2A.


As gene therapy opportunities for IRDs continue to emerge and progress, and other gene-specific treatments are also being developed, far better therapeutic options for these previously incurable retinal disorders are becoming a reality [3,9]. Therefore, identifying the pathogenic genetic etiologies of IRDs is no longer medically necessary only for diagnostic, counseling, and reproductive risk assessment purposes but also for compelling therapeutic implications and is a top priority in the field of IRDs [4,7,22].

The ACMG guidelines emphasize the importance of genotype data with far less relevance placed on clinical phenotyping during the search for novel variants. This is an excellent general advice due to the lack of standardization of clinical data in many medical fields and the heterogeneity and often incompleteness of the clinical workups performed by many referring medical providers. Indeed, it has been our experience that the clinical diagnosis of suspected IRD in patients referred to our clinic is incorrect in approximately 50% of the cases.

Thus, from a strict laboratory perspective, the low level of relevance assigned by the ACMG guidelines to phenotypic information is completely understandable. When a novel genetic change that may be disease causing or is an outright VUS is found in a patient with IRD, it would be ideal to also perform a detailed in vitro or otherwise in vivo (e.g., in zebrafish, cultured inducible pluripotent stem cells, or retinal organoids) characterization of the various uncertain genetic changes. However, this is far beyond the scope of clinical diagnostic testing and would be time-consuming and expensive to implement on a vast scale. IRDs are relatively unique compared with most other genetic disorders owing to the high accessibility of the human eye for detailed examination and testing. IRD specialists have already come together to define optimal approaches and protocols for IRD phenotyping [7,22] that are being broadly followed by IRD specialists worldwide. This affords IRD specialists an opportunity to maximize the throughput of high-quality, high-fidelity clinical and functional phenotype data. In turn, this information can then guide far more refined genotyping interpretations. Likewise, when genotyping information reveals paths to disease etiologies that had not been previously fully suspected, such as in the cases illustrated in Figure 4 and Figure 6, the current clinical and functional phenotyping capabilities afforded to IRD specialists permit the recognition, at times in retrospect, of disease features that had not been initially captured (e.g., the peripapillary sparing and peripheral flecks in the ABCA4-positive patient illustrated in Figure 6), which help confirm the molecular diagnosis. In PT9 (Figure 4), the initial and correct impression that the first BEST1 mutation was predicted to be pathogenic in view of a vertical history of vitelliform disease was in fact misleading as to the true inheritance pattern and actual final diagnosis. It was not until the EOG results in the proband's parents were found to be normal that the pursuit of a possible second CNV in BEST1 became evidently necessary. Once a del/dup microarray test confirmed that the atypical vitelliform phenotype of PT9 was recessive in nature (and thus an ARB) and not dominant, both the phenotype in the proband and the lack thereof in the mother, who carried the novel BEST1 exon 1–2 deletion, which was phenotypically silent in the carrier state, were clarified. Thus, in the field of IRDs, a two-way interaction between IRD specialists and molecular genetic laboratories offers the unique opportunity to leverage the power of in-depth phenotyping. For both IRD specialists and laboratories, this allows for a greatly increased level of certainty in identifying novel disease-causing genetic variants without requiring complex, time-consuming, and expensive experiments on a systematic scale.

Taking advantage of this systematic approach, which is summarized schematically in the flowchart in Appendix 3, we identified 52 novel pathogenic variants in 27 IRD genes. The associated phenotypes are summarized in Appendix 4. The identified variants ranged from point mutations, splice site mutations, deletions, and duplications. We have illustrated cases in which our methodical approach to clinical phenotyping allowed for the identification of just a few genes (sometimes even just a single gene) that would be sufficient to sequence and identify the causal variant in selected patients. The use of this approach led to significant savings in terms of both cost and time. We acknowledge that as sequencing techniques have become more streamlined and less expensive, many cases could have been reasonably identified by starting off with IRD-focused NGS-based panel testing, which is now widely available and far cheaper than even just a few years ago. These panels also often allow the identification of mutations and variants that may not be disease causing but may have relevance as phenotype modifiers and, at times, may be important for family-wide counseling. For these reasons, we presently advocate the utilization of CLIA-certified broad NGS-based panel testing as the first-line testing option.

For most other patients, phenotyping was followed by IRD-focused broad NGS-based panel testing. CNV determination became available as part of the initial panel sequencing partway through our study period. Whereas CNV analysis did not always lead to diagnosis in our cohort because it best detects genomic changes larger than 3 exons in size (i.e., larger than any novel variants found in our patients), it has been shown to be of conceptual utility in other studies and is emerging as an essential part of the genotyping approach. In many cases, careful reconciliation of clinical data with NGS panel-based sequencing was sufficient in diagnosing the genetic etiology. In certain cases, follow-up del/dup microarray testing was critical in establishing the diagnosis and in finding novel variants, and can remain essential in identifying a second allele in autosomal recessive patients. The common thread in all of our cases was the initial step of in-depth clinical and functional phenotyping, the benefit of which cannot be sufficiently emphasized.

In summary, in the hands of IRD specialists, judicious use of meticulous, standardized, expert clinical-functional phenotyping combined with systematic, broad NGS panel-based genotyping was extremely effective for providing critical genetic diagnoses to patients with IRD and for the discovery of a large number of novel genetic IRD variants that could be characterized as causal with a high degree of confidence. Increasing these two-way interactions between IRD investigators and CLIA-certified molecular diagnostic laboratories warrants the continuous improvement in the yield of molecular genetic diagnostic testing in our field. This will ultimately facilitate the common goal of achieving genotypic characterization of all patients with IRD as we continue to remain steadily on the path to an increasing number of gene- and mutation-specific interventions, as well as treatments aimed at tackling gene- or mutation-specific driven mechanisms (e.g., translational readthrough-inducing drugs for stop-codon mutations or drugs interfering with vitamin A metabolism or recycling in ABCA4, LRAT, or RPE65-related diseases) [4,7,9,22].

Appendix 1. Flowsheet.

Appendix 2. Supplemental Methods.

Appendix 3. Novel IRD variants.

Appendix 4. Phenotype number case series.


This research was funded by an unrestricted institutional grant to the Duke Eye Center by Research to Prevent Blindness, Inc., New York, NY and by private donations to the Duke Eye Center Retinal Degenerations Research Fund (AI) and the Retina Genetics Fund (AI), respectively. Portions of these data were accepted and presented at the ARVO 2018 meeting.


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