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N.W. Keiser, S.B. Schwartz, I.M. MacDonald, S.G. Jacobson, J. Bennett; The Identification of Mutations in the REP–1 Promoter in a Patient with Choroideremia. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2451.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Choroideremia is an X–linked retinal degeneration caused by mutations in the REP–1 gene. These mutations result in stop codons that lead to truncation of the REP–1 protein, ablating its function. We identified a patient with the diagnosis of choroideremia, but there were no mutations in the coding region of the REP–1 gene. To identify the molecular basis of disease in this individual, we undertook an analysis of his REP–1 promoter and analyzed the expression of the mRNA transcript and protein. Methods:Lymphoblast cell lines were generated from the patient and other individuals with and without choroideremia, and genomic DNA, mRNA, and protein was prepared from these cells. Regions upstream of the REP–1 gene were amplified by PCR from genomic DNA. The PCR products were cloned and sequenced, and the sequences were compiled and aligned to screen for polymorphisms. Various fragments of the mutant and normal REP–1 promoter were cloned into reporter vectors, allowing for the activity of each promoter region to be measured in vitro. Presence of REP–1 transcript was assessed by RT–PCR, and REP–1 protein was detected by Western blot. Results: Sequence analysis of the REP–1 promoter revealed many nucleotide changes in the patient as compared to controls. Identical changes were also detected in the patient’s mother, who is a carrier for choroideremia. Transitions and transversions were found throughout the entire length of the promoter, and insertions and deletions were also detected, and ranged from one to several nucleotides in length. Several of these mutations were located in putative transcription factor binding sites. However, in vitro analysis of the mutated promoter showed that these mutations did not decrease the level of transcription. Furthermore, RT–PCR and Western blot analysis showed that lymphoblasts from the patient express REP–1 mRNA and have a normal level of REP–1 protein. Conclusions: A patient with choroideremia was found to have no coding region mutations consistent with the disease, but harbors numerous mutations in the promoter of the REP–1 gene. These mutations do not lead to a decrease in gene expression in vitro or in cells isolated from the patient. This suggests that mutations in the promoter may affect elements that regulate REP–1 expression specifically in the eye, leading to choroideremia. This is the first evidence that mutations in the upstream region of REP–1 can be associated with choroideremia.
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