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M. N. Preising, M. Willer, B. Lorenz; Evaluation of Lyonization in Cultured Lymphocytes From Carriers of Choroideremia. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2306.
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Cassels et al. have reported that a REP1 polymorphism partially escapes from Lyonization in unaffected female carriers. The present study investigated the transcription levels of benign variants and pathogenic REP1 mutations in lymphocyte cultures from females.
cDNA fragments covering four types of mutations in the REP1 gene (one polymorphism D601S (c.1833C/A), one deletion c.581del14bp, one insertion c.1168ins50bp, one nonsense R270X (c.838C>T)) were amplified from 10 single cells each. The PCR products were analyzed for the frequencies of transcripts from the inactivated allele using the ABI PRISM SNaPshot Multiplex Kit and a capillary sequencer for fragment analysis.
The benign variant was evaluated in two females, and showed predominant transcription of the mutant allele. Transcription of the inactivated allele was seen in 1% to 5% of transcripts. The pathogenic mutations were evaluated in a single carrier for each mutation type. Ninety percent of cells carrying the insertion predominantly expressed the wildtype allele with a mean escape from lyonization of about 4% (range 20% to 1.6%). Cells carrying the deletion transcribed the minor allele at mean of 25% (range 0% to 50%). Fifty percent of the cells showed predominant transcription of the mutant allele. We could not amplify the expected PCR product from the cells carrying the nonsense mutation.
Cultured lymphocyte cells from females carrying mutant alleles of REP1 show escape from lyonization, ranging from a few to 50% of transcripts affecting the mutant as well as the wild type allele. We saw differences among the mutations as to the predominantly transcribed allele as well as to the amount of the minor allele. Whether this is due to the situation in cultured cells or to functional consequences of the type of mutation has yet to be evaluated.
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