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Tim Footz, Adrian Lahola-Chomiak, Ordan J Lehmann, Janey L Wiggs, Michael A Walter; Targeted next generation amplicon sequencing for candidate genes of Pigment Dispersion Syndrome and Pigmentary Glaucoma. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5140. doi: https://doi.org/.
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Pigmentary Glaucoma (PG) is a secondary open-angle glaucoma associated with anterior segment pigment defects typical of Pigment Dispersion Syndrome (PDS). To date, there are no published reports of human genes responsible for PDS or PG, despite strong evidence of a heritable component. We have recently used whole exome sequencing of affected families to identify new candidate genes for PDS/PG, premelanosome protein (PMEL) and cornichon homolog 4 (CNIH4). The melanoma antigen gene melan-A (MLANA) encodes a protein that physically interacts with both PMEL and the ocular albinism 1 protein to regulate melanogenesis and is thus an additional excellent candidate gene. Identification and evaluation of the genetic foundations of PG will foster experiments directed at understanding the root cause of PG and the best treatment and management strategies for patients. Therefore, we critically need fast and inexpensive methods to analyze candidate genes for PG-associated mutations.
Genomic DNA was amplified using standard PCR with primers designed to cover all coding regions of the genes, and included sequences compatible with Illumina next generation sequencing (NGS) technology. Up to 18 amplicons per individual were pooled before a secondary barcoding PCR was performed, and 113 uniquely-indexed individual PDS/PG patients were combined in each NGS run.
High quality sequence was rapidly and cost-effectively obtained using an Illumina MiSeq System. For example, 360Mb of sequence was generated in a single run that targeted 5.1kb of DNA, where 90% of the 1.4 million reads (2x250bp) had a Phred quality score of >Q30, corresponding to an average read-depth of over 100x. This run was estimated to cost about one-fifth the projected amount as commercial Sanger sequencing of the same amplicons. To date we have discovered four PDS-associated PMEL missense mutations in our patients.
The identification of PDS/PG-associated mutations in PMEL, CNIH4 and MLANA in our patient cohort will further clarify the genetic etiology of the disease, establishing a new regime of biochemical reagents (i.e. melanogenesis/pigmentation genes) that can be used to investigate the poorly-understood molecular mechanisms of glaucoma. Improved screening for the genetic causes of glaucoma will lead to earlier diagnosis and treatment to better manage this form of irreversible blindness.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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