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Eileen Hwang, Denise Morgan, Leah Owen, John H Fingert, Paul S Bernstein, Margaret M DeAngelis; Long-range polymerase chain reaction validates copy number variation in autosomal dominant optic pit. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5402. doi: https://doi.org/.
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A Utah family with optic pit maculopathy was found to have a copy number variation upstream of the MMP19 gene on chromosome 12 that segregated with disease. However, initial assessment of copy number indicated a possible duplication, which differed from a previously published report of a family with a triplication in the same region (Hazlewood, 2015). Studies were performed to further investigate the number of copies.
Four of 16 individuals in the three-generation pedigree were affected by optic pit maculopathy. Leukocyte DNA from all individuals were tested using two different real-time polymerase chain reaction (PCR) copy number variation assays designed to target the previously reported region upstream of MMP19. A long-range PCR design was created to span the breakpoint that would produce a single 500-bp product in the case of a duplication, and both a 500-bp and an 8.5 kb product in the case of a triplication.
Results from the real-time PCR copy number assays showed that affected individuals had 2.8-4.1 copies compared to 1.5-2.3 copies in unaffected individuals. Real-time PCR copy number assay results were found to be affected by sample-specific error. Long range PCR of DNA from affected individuals demonstrated 8.5 kb and 500 bp products, providing independent evidence for a tandem triplication on the affected allele, i.e. four total copies. Long range PCR of DNA from unaffected individuals resulted in no product, as expected. These tests permitted early diagnosis of five members of the youngest generation (ages 10 to 17) who did not yet manifest clinical disease. These minors likely require closer follow up and could benefit from earlier intervention.
Fine distinctions of copy number with TaqMan qPCR assays may be limited. Long-range PCR can be utilized to clarify copy number variation and most importantly validate methodology when repetitive elements such as Alus underlie disease pathobiology. High quality DNA extractions are required for long regions, and PCR parameters must be optimized. The Utah family with autosomal dominant optic pit could be related to the Iowa family in which this copy number variation was first described.Reference: Hazlewood et al. (2015) Heterozygous Triplication of Upstream Regulatory Sequences Leads to Dysregulation of Matrix Metalloproteinase 19 in Patients with Cavitary Optic Disc Anomaly. Human Mutation. 36:369-378.
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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