To evaluate the newly developed next generation sequencing assay by analyzing patients with isolated eye abnormalities collected in earlier studies including patients enrolled through eyeGENE program: This project began several years ago in the OGFVB/NEI. We developed a screening tool by the resequencing CHIP on 93 genes provided an opportunity for these samples from patients enrolled in NEI clinical research for years. Our manuscript was published in the IVOS later in 2011. Later on, we adapted the next generation sequencing (NGS) technology. We developed a new panel composed of 184 genes related to retinal function and development. By NGS and microdroplet PCR technology, we have been more actively analyzing patient with variety categories of retinal dystrophies in a more efficient and accurate approach. We hired a company, RainDance Technologies, Inc., in designing a primer library for Retinal Dystrophy Panel (RD panel). Using the developed primer library, RainDance Technologies, Inc. we have performed NGS sequencing for more than 275 samples with a variety of ophthalmic diseases. These samples are 250 samples for retinal dystrophies from beginning, 7 samples for a pilot project related to AMD patients, 24 samples related to corneal dystrophies. Dr. Fanns group from ITBP/NINDS assisted with data processing and bioinformatics analysis. We have identified more than 100 candidate mutations. With the efforts of lab technicians, summer interns and special volunteers, we have been working on verification of the identified targets by Sanger sequencing. Molecular mechanisms in many patients with sporadic retinal diseases or differential diagnostic difficulties have been revealed by this approach. Several cases went through the clinical verification by an outside CLIA lab and have been reported back to clinicians/patients. We are summarizing the data and preparing for manuscript. We are continuing the procedures to analyze more patients with RP. Those samples have been sequenced in our newly purchased MiSeq next generation Sequencer from Illumina. The data is now in processing and bioinformatics analysis at ITBP/NINDS. To analyze the PAX2 gene structure in patients collected in earlier studies: This is a continuation of my clinical ophthalmic research from Minnesota and collaboration. We developed a clinical DNA testing of PAX2 gene for Renal-coloboma Syndrome and have been collecting tested samples for genetic heterogeneity analysis. After I joined NEI, we are extending this study. We have collaborated with Dr. Brian Brooks to study more patients &their PAX2 genes. We are extending the study in order to validate the protocol as a clinical test for patients. To evaluate retinal related genes for mutation in patients with Occult Macular Syndrome for genetic modification: Occult Macular Dystrophy is a very unique macular dystrophy. Clinical diagnosis is usually difficult and fully based on multifocal ERG. Recently, this disorder was found related to mutations in a gene called RP1L1 in Japanese patients and verified in other ethnic populations. NEI ophthalmologists have been clinically evaluating patients with OMD and we have been developing a research procedure to screening the entire coding region of RP1L1 gene. With several students efforts, we found the reported mutations in 3 of 6 index patients. However, published patient analysis indicated a reduced penetrance of OMD in individual carrying the mutation. We identified a large pedigree with multiple occurrences of OMD and positive with the common mutation. We have analyzed the family members for segregation of the mutation and disease. A reduced penetrance of clinical presentation of OMD in the mutation carriers in this family has been revealed. We have analyzed the potential genetic modifiers using our NGS platform: Although, we did not find potential genetic modifiers in this family, we are planning to publish the study. To develop induced pluripotent stem cell protocol to understand the differential gene expression. In the past year, we attempted to adapt the iPS cells reprogramming procedure for the purpose of understanding differential gene expression. Our goal is to explore the timing and level of gene expression among the retinal related genes. These procedures would enable us to explore the functional genomics, especially the profile and regulation of retinal genes. We have successfully reprogrammed and induced iPS cells from peripheral blood cells. We are evaluating the timing and profile of retinal genes at different stages of iPS cells reprogramming and maintenance. We have been also collaborating with Dr. Guokai Chen, from the iPSC and Genome Engineering Core Facility of National Heart, Lung, and Blood Institute to profile the gene expression during the induced differentiation in the early neural stages and neural crest stages. To develop RNA sequencing based assay to generate transcriptome profiling in different cell types including the cell obtained through procedures modified from iPS protocols: By our NGS project, we have screening patients for mutations in retinal related genes. We have found known mutations in the known genes for the clinical conditions;we have found novel variations predicted as or likely mutations in the known genes for the clinical conditions;and we found known mutations not previously correlated to the clinical conditions. For those known mutations or predicted mutations in known genes, we can interpret by the evidences for the predicted functional consequences. However, we frequently found variations with uncertainty for their functional consequences in those known genes and we occasionally found known mutations in irrelevant genes for other retinal conditions in patients with retinal dystrophies. To clarify the correlation or exclude the functional involvement, we need to study the functional consequences of candidate variations at an alternative level such as but not limited to transcription and/or translation. As many retinal genes do not express in PBMC, we could not study gene expression directly in PBMC cells. Therefore, we are considering the evaluation of newly identified variation in a personal model system, such as skin biopsy or induced pluripotent stem cells. We have been collecting samples from different stages of iPS cells. We have RNA-sequenced several stages of cells through an outside service provider. We have asked Agilent to design a target transcripts enrichment custom specific kit for our study of 240 target transcripts. We are working on optimization of the procedure and have generated preliminary data and data analysis is in progress. To identify the breakpoints of RS1 gene partial deletions in two retinoschisis patients: We are providing clinical genetic testing to NEI clinics for X-lined Juvenile Retinoschisis: Where some patients had a partial gene deletion. It was difficult to report back to patient each time without knowing the breakpoint of deletion and unable to carry out carrier analysis in mother and sisters. We had a couple of cases recently. We decided to analyze these cases for their genomic breakpoints. With the help of a summer student, we have successfully determined the breakpoints and defined the deleted sequences. We wrote a manuscript for these results that was published in 2013.
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