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C. J. Spellicy, S. J. Bowne, L. S. Sullivan, D. Hughbanks-Wheaton, D. G. Birch, J. R. Heckenlively, S. P. Daiger; Using Peripheral Blood Leukocytes to Detect PRPF31 Haploinsufficiency. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1302. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To test a novel method of screening for PRPF31 mutations in patients with retinitis pigmentosa using peripheral blood leukocytes as a model system. Mutations in pre-mRNA splicing factor 31 (PRPF31) cause autosomal dominant retinitis pigmentosa (adRP). PRPF31 accounts for 8% adRP. The disease mechanism by which PRPF31 mutations cause disease is thought to be haploinsufficiency.
Blood samples were collected from unaffected control individuals and from patients with known PRPF31 mutations. Leukocytes were isolated using the LeukoLOCKTM system from Ambion. RNA was processed from the leukocyte population. Additional blood samples were sent to the Coriell Institute for Medical Research where they were immortalized with Epstein Barr virus. PRPF31 levels were measured using real time quantitative PCR (RT-qPCR). Comparisons in PRPF31 mRNA levels were made between unaffected and affected individuals in both the lymphoblast and leukocyte mRNAs.
Initial data shows that PRPF31 mRNA levels can be measured in both leukocytes and lymphoblast cell lines using RT-qPCR. Experiments are underway to confirm that reduced levels of PRPF31 mRNA can be detected in patients carrying known PRPF31 mutations as compared to control individuals.
Lymphoblast cell lines from affected individuals with PRPF31 mutations have roughly half the amount of PRPF31 mRNA as control individuals. If experiments using leukocytes validate the ability to detect half levels of PRPF31 mRNA in patients with mutations, measuring PRPF31 mRNA levels in leukocytes may represent a novel and efficient way of identifying PRPF31 as a target for mutation screening. Furthermore, adRP patients with low levels of PRPF31 mRNA in leukocytes, but without detectable mutations in the PRPF31 coding sequence, become candidates for additional analysis to identify mutations in promoter and/or regulatory regions.
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