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Practical RetinaIncorporating current trials and technology into clinical practice

Genetic Testing for Retina Specialistsby Edward H. Wood, MD; and Kimberly A. Drenser, MD, PhD

In December 2017, the U.S. Food and Drug Administration approved Lux-terna (voretigene neparvovec-rzyl; Spark Therapeutics, Philadelphia, PA) as a new gene therapy to treat children and adult patients with RPE65 mu-tations resulting in vision loss. The

availability of this landmark treat-ment has brought attention to the need for genetic testing by retina specialists.

For this column, Kimberly Drenser, MD, PhD, and Ed-ward H. Wood, MD, from William Beaumont Hospital

in Royal Oak, MI, provide us with an overview of this important topic. They will begin with a historical perspec-tive, dating back to 1953 during the days of Watson and Crick. They will then review genetic sequencing, pro-vide an overview of the importance of genetic testing in retina, and conclude with American Academy of Ophthal-mology guidelines for genetic testing.

We are grateful to Drs. Drenser and Wood for generously sharing their ex-tensive knowledge on this topic with our community. Given the numerous gene therapies currently in clinical trials, I anticipate that genetic testing will be routinely offered to our pa-tients in our practices. Therefore, this piece will likely be of interest to many of us in the retina community.

When Watson and Crick published their seminal Nature paper1 in 1953 proposing the double-helix, they simultaneously pre-dicted the field of genetic sequencing en-tirely: “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copy-ing mechanism for the genetic material.” Since this time, the scientific progress made within genomics has been remark-able. Sixty-five years later, we are now able to sequence an individual’s entire genome and have begun to treat disease-causing mutations with gene therapy and gene ed-iting tools. But, how do we bridge the gap between genetic testing performed within specialized academic research institutions and biotechnology companies, and genetic testing performed within routine vitreoret-inal clinical practice?

A BRIEF HISTORY OF GENETIC SEQUENCING

The discovery of DNA1 in 1953 and the development of the central dogma2 (DNA makes RNA, RNA makes protein) in 1970 marks a paradigm shift. The discovery of DNA cleav-ing enzymes, type 2 restriction enzymes,2,3 in 1970 followed by the ability to clone DNA via molecules called plasmids4 (1973) allowed for targeted DNA cutting, isolation, and map-ping, thus setting the stage for genetic sequencing. Frederic Sanger’s development of “chain termination” method of se-quencing5 at Oxford (1977) and polymerase chain reaction (PCR) by Kary Mullis (1985)6 revolutionized genetic sequenc-ing. Sanger sequencing was used by the Human Genome Proj-ect7 (1990-2003) and Craig Venter’s group at Celera8 to map the first human genome and remains the gold-standard meth-odology (with error rates less than 1%). However, Sanger sequencing is slow and requires large DNA fragments to be cloned, which contributed to the Human Genome Project costing $2.7 billion.9 These disadvantages inspired scientists to create a second generation of genetic sequencing methods. Next-generation sequencing (NGS) methods allow sequencing of huge numbers of samples at one time, so called “massively parallel sequencing.” This improvement makes it possible to sequence an individual’s entire genome (with the “genome” consisting of 3x109 bases) in “whole genome sequencing”

Edward H. Wood

Kimberly A. Drenser

doi: 10.3928/23258160-20180501-01

Seenu M. Hariprasad Practical Retina

Co-Editor

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(WGS), or the protein-coding regions (“exome” consisting of 3x107 bases accounting for 1% of the entire genome but encompassing roughly 85% of disease-causing mutations) in “whole exome se-quencing,” (WES).9,10 Software analytics compare these findings to reference genomes and identify variants.11

WHY SHOULD RETINA SPECIALISTS CARE ABOUT GENETIC TESTING?

Patients Want Genetic TestingAn interview of 48 patients with retinitis pig-

mentosa (RP) showed that 92% of participants de-sired genetic counseling and 86.5% desired genetic testing.12 Although patients with multifactorial disorders such as age-related macular degeneration (AMD) may also desire genetic testing, the Ameri-can Academy of Ophthalmology (AAO) task force currently recommends against routine genetic test-ing for these conditions:13 “Currently, clinicians should avoid routine genetic testing for genetically complex/multifactorial diseases, such as AMD; instead, genetic testing should be used in Mende-lian (monogenic) disease, such as RP or Stargardt disease.”

Clinical Testing Rarely Predicts Genetic DiagnosisThis is because one retinal phenotype may be

caused by mutations in different genes, or “genetic heterogeneity.” For example, more than 100 dif-

ferent genes are associated with RP, and 23 are as-sociated with LCA.14 Conversely, one gene can be associated with several different phenotypes, or “phenotypic heterogeneity.” For example, CRB1 mutations may cause Leber congenital amaurosis (LCA), RP, and cone-rod dystrophy.15 Many retinal diseases display “allelic heterogeneity” in which many different phenotypes are a result of vari-ous mutations within a single gene. Approximate-ly 250 disease-causing genes have been identified and catalogued in the Retinal Information Network (http://www.sph.uth.tmc.edu/RetNet/). Genetic test-ing serves as a diagnostic tool for many patients with suspected Mendelian disorders, carrying significant impact for patients and their families. Benefits in-clude prognostication, guidance of further testing, re-ferral for syndromes, and management of pathology.

Genetic Interventions Are Only Available to Patients With a Genetic Diagnosis

In essence, gene therapy replaces defective DNA with a normal (wild-type) DNA sequence of a target gene, often using a recombinant viral vector to infect the host cell, express the normal (wild-type) protein, and restore cellular function. Gene therapy is current-ly available for those with RPE65-associated LCA (the U.S. Food and Drug Administration approved voreti-gene neparvovec-rzyl [Luxturna; Spark Therapeutics, Philadelphia, PA] on December 19, 2017). In addition, phase 1/2 gene therapy clinical trials are currently un-derway (Figure 1; https://clinicaltrials.gov/ct2/home),

Figure 1. Enrolling gene therapy clinical trials for patients with inherited retinal disease. Left column: gene involved; middle column: inherited retinal disease; right column: ClinicalTrials.gov identifier.

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and many others are being explored in preclinical ani-mal studies.14 In addition, gene editing with clustered regularly interspaced short palindromic repeats (CRIS-PR) and CRISPR-associated systems (CAS) holds sig-nificant promise for treating genetic diseases by allow-ing for targeted cleavage and insertion of DNA down to a single base pair. Editas Medicine (Cambridge, MA) plans to have three early stage clinical trials and two late-stage clinical trials using CRISPR/CAS underway by 2022, including targeting CEP290-associated LCA. With one gene therapy treatment currently available and many others in the pipeline, the potential signifi-cance of genetic testing for patients with retinal disor-ders has never been higher.

Genetic Testing of Unique Patient Populations Can Generate Novel Discoveries and New Therapeutic Approaches

In our group’s experience, genetic sequencing of a pediatric population with retinal vascular dis-ease has been illuminating and impacted the way we care for patients with Wnt-associated vitreoreti-nopathies (WAVR), such as familial exudative vit-reoretinopathy, Norrie disease, pseudoglioma osteo-porosis syndrome, and retinopathy of prematurity. Genetic testing for individuals with WAVR allows us to properly diagnose and treat the patient as well as counsel family members, which is important in these progressive diseases. Our increased under-standing of the genetic basis for these diseases (cen-tering around wnt/B-catenin signaling pathways16) has inspired the generation of a therapeutic com-pound to target this pathway. One can imagine a similar thread beginning with genetic diagnosis and ending in targeted therapy for other disorders affect-ing unique patient populations.

HOW MAY GENETIC TESTING BE PERFORMED?

Routine genetic testing in the clinic still remains elusive. It is advisable to work in coordination with a geneticist to guide the appropriate testing. Patients with high suspicion for a specific monogenic dis-ease may begin with targeted Sanger sequencing. Syndromic conditions with possible chromosomal alteration may be approached using chromosom-al single nucleotide polymorphism microarrays.17 When a mitochondrial disorder is suspected, mito-chondrial genome sequencing is appropriate. Often querying a panel of genes employing NGS (validated for ophthalmic disease) is chosen.18-20 A variety of companies and organizations provide NGS services for ophthalmic patients (Figure 2). These companies will work with you to provide sample collection kits and often bill directly to insurances. NGS panels for inherited retinal diseases have high diagnostic rates ranging from 50% to 57%.21-23 When NGS panels do not result in a hit, one may choose to proceed onto WES or WGS. This tiered approach, with increasing levels of sequencing minimizes cost, time, and un-necessary data acquisition.

CONCLUSION

Retina specialists have the incredible privilege of being involved in gene therapy and shaping the fu-ture to come. The AAO task force on genetic testing defines a genetic test as the sum of five parts:24 “(1) the clinical determination that a genetic eye disease is likely to be present, (2) the molecular investigation of genomic DNA samples from one or more individu-als, (3) the analysis of the resulting molecular data in the context of relevant published literature and public databases using appropriate statistical meth-ods, (4) the interpretation of the data in the context

Figure 2. Next-generation sequencing resources. Left column: resource type; middle column: company name; right column: company website.

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of the clinical findings, and (5) the counseling of the patient about the interpreted findings and their implications.” When performing genetic testing, the retina specialist should consider all of these com-ponents in order to maximize the positive impact of genetic testing.

REFERENCES

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13. American Academy of Ophthalmology. Recommendations for ge-netic testing of inherited eye diseases – 2014. https://www.aao.org/clinical-statement/recommendations-genetic-testing-of-inherited-eye-d. Published February 2014.

14. Hafler BP. Clinical progress in inherited retinal degenerations: Gene therapy clinical trials and advances in genetic sequencing. Retina. 2017;37(3):417-423.

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17. Lee K, Garg S. Navigating the current landscape of clinical genetic testing for inherited retinal dystrophies. Genet Med. 2015;17(4):245-252.

18. Scott AF, Mohr DW, Kasch LM. Identification of an HMGB3 frame-shift mutation in a family with an X-linked colobomatous microph-thalmia syndrome using whole-genome and X-exome sequencing. JAMA Ophthalmol. 2014;132(10):1215-1220.

19. Liu X, Xiao J, Huang H, et al. Molecular genetic testing in clinical diagnostic assessments that demonstrate correlations in patients with autosomal recessive inherited retinal dystrophy. JAMA Ophthalmol. 2015;133(4):427-436.

20. Taylor RL, Parry NRA, Barton SJ, et al. Panel-based clinical genetic testing in 85 children with inherited retinal disease. Ophthalmology. 2017;124(7):985-991.

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patients with retinal dystrophies using targeted next generation se-quencing. PLoS One. 2016;11(1):e0145951.

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Edward H. Wood, MD, can be reached at Associated Retinal Consultants, William Beaumont Hospital, Neuroscience Center, 3555 W. 13 Mile Road, Suite LL-20, Royal Oak, MI 48073; email: ehuntw@gmail.com.

Seenu M. Hariprasad, MD, can be reached at the Department of Ophthalmology and Visual Science, University of Chicago, 5841 S. Maryland Avenue, MC2114, Chicago, IL 60637; email: retina@uchicago.edu.

Kimberly A. Drenser, MD, can be reached at Associated Retinal Consultants, William Beaumont Hospital, Neuroscience Center, 3555 W. 13 Mile Road, Suite LL-20, Royal Oak, MI 48073; email: kdrenser@arcpc.net.

Disclosures: Drs. Wood and Drenser report no relevant financial disclosures. Dr. Hariprasad is a consultant or on the speakers bureau for Alcon, Allergan, Bayer, OD-OS, Clearside Biomedical, Ocular Therapeutix, Alimera Sciences, Leica, Spark, and Regeneron.