This week I attended the annual meeting of ARVO — the Association for Research in Vision and Opthalmology — which brings together 15,000 clinicians and researchers working in those fields. This is an unusual type of meeting for me, and quite unlike CSHL or Marco Island meetings in that the primary focus is on patient care. Few areas of medicine have benefited from next-generation sequencing more than research into vision disorders. Many groups employing whole-exome or more targeted sequencing presented their work here, and also highlighted the challenges of translating NGS technologies into clinical use.
Inherited Eye Disorders
Vision disorders with a large genetic component range from rare, penetrant Mendelian disorders like retinitis pigmentosa (RP) to common complex diseases (AMD, glaucoma). In many ways, these are well-suited to genetic studies:
- They often manifest phenotypes that can be quantitatively measured and tracked over time.
- For Mendelian disorders, many (if not most) of the common disease-causing genes have been identified.
- Vision disorders are usually non-fatal, meaning that they can be studied over lifetimes and generations.
Next-generation sequencing has become a powerful tool for genetic screening in visual disorders. In autosomal dominant RP, for example, screening a panel of known retinal disease genes uncovers the casual mutation in about 75% of cases. Many groups including ours have employed exome sequencing to search for new causal mutations in the other 25%.
Surprisingly, however, the number of new RP genes identified since exome sequencing became widely available is relatively small. An explanation I can offer from my own experience, and one echoed by many of those presenting at ARVO, is that there are thousands of variants in every individual’s exome and identifying the one that causes disease (especially dominant disease) is hard. By definition, we are looking for a variant in a gene that isn’t yet linked to retinal disease, and that means almost every gene.
Challenges of Mendelian Disorders
Searching for Mendelian disease genes by exome sequencing seems like a straightforward exercise, but I think many of us have come to recognize that it’s not as easy at perhaps it should be. Inherited retinal diseases are a good model to help explain some of the difficulties:
1. Modes of inheritance.
Even with extensive pedigrees and good clinical phenotyping, the inferred mode of inheritance for retinal disorders can be wrong. A study this year by Steve Daiger’s group at the University of Texas- Houston, reported that around 10% of apparently dominant RP pedigrees turn out to be X-linked RP, often with expression of the disease in carrier females. Further complication arises from the fact that 6 of the 23 known genes that cause dominant RP are also linked to recessive RP.
2. Refractory mutations.
By this I mean segmental duplications, large-scale deletions, and other classes of variants that remain difficult to detect by current sequencing technologies. About 5% of disease-causing mutations in dominant RP are large deletions; these remain difficult to detect by capillary or even exome sequencing. Interestingly, many are associated with the gene PRPF31, the only known gene for which haploinsufficiency causes dominant RP.
3. Unexpected causals.
Ed Stone of the University of Iowa presented a fascinating report on Stargardt’s disease, one of the most common forms of inherited eye diseases and caused predominantly by mutations in ABCA4. His group tackled 208 patients with classic (autosomal recessive) Stargardt’s in which only one of the requisite 2 mutations in the gene was observed. It turned out that alternative splicing due to a noncoding or synonymous coding variant explained the biallelic gene loss in 40% of such cases. Many were in regions not captured by current exome kits.
Targeted Panels versus Exomes
Another important debate in retinal genetics surrounds the use of targeted panels versus exome sequencing as the front-line diagnostic tool. Currently, CLIA testing for dominant RP identifies the causal mutation in about 50% of cases. It’s conducted by PCR and Sanger sequencing, with a cost of about $10,000. It seems likely that this routine diagnostic tool will soon be replaced with a next-generation sequencing assay. The question is whether or not that should be a custom panel targeting known retinal disease genes (which seems to explain about 75% of the cases) or exome sequencing.
Both approaches have strengths and weaknesses. Panel testing is cheaper than exome sequencing, can be tweaked to ensure coverage of important genes/regions, and generally yields results that can be confidently reported to the clinician. Exome sequencing provides more information and would likely be performed anyway should a panel test comes back negative. But it comes at a higher cost and might also not sufficiently cover some important targets. It seems likely, too, that an exome approach would often yield reports of “variant of unknown significance” which has limited use to clinicians (and patients for that matter).
It may also turn up variants not relevant for the vision disorder but with important health implications nonetheless; whether and how to report such findings is an important ethical debate.
Clinical Translation and Genetic Counseling
An emphasis on patient counseling and care distinguishes ARVO from the genetics/genomics meetings I more often attend. Hearing from clinicians and genetic counselors provided a new perspective. These individuals are the front line for patients, whereas researchers like myself are often two or three levels removed. I might see sample codes, phenotype information, and demographics like age and ethnicity, but they know the faces and voices and families of their patients.
As a result, we have some philosophical differences in how we conduct the research. I might be comfortable reporting a gene with, say, 90% probability of causing the disease. A clinician or genetic counselor might not.
Ultimately, next-generation sequencing is destined for the clinic, to aid the diagnosis, prognosis, and treatment decisions for patients. Most of us in the field can see this. It must be said that not everyone is convinced, especially those on the clinical side. Some of them may take the initiative on their own, and come to our meetings (ASHG, AGBT, etc.) to learn about NGS and its potential. We will do even better, I think, by taking that message to them at their own society meetings and lecture halls. There is a great deal of common ground between research and clinical care; meetings like ARVO seem like a good place to find it.