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A. Ramirez-Miranda, B. Buentello-Volante, J. C. Zenteno; Multiplex Ligation-Dependent Probe Amplification (mlpa) Analisys in Mexican Individuals With Congenital Aniridia and No Pax6 Mutations Detected by Direct Automated Sequencing. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3735.
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PAX6 is a phylogenetically conserved transcription factor almost universally employed to direct eye development that regulates the tissue-specific expression of diverse molecules, including transcription factors, cell adhesion molecules, hormones, and structural proteins. Its mutations, frequently point mutations that generate the introduction of a premature stop codon, lead to a variety of hereditary ocular malformations being the most common congenital aniridia (AN2; OMIM 106210). However, not all aniridia cases can be explained by mutations in the PAX6 gene, but gross deletion can be detected by MLPA. In this study we perform the first MLPA analisys in mexican individuals with congenital aniridia.
An affected family underwent full ophthalmologic examination including visual acuity, slit lamp inspection, intraocular pressure. Blood samples were drawn by venipuncture, and genomic DNA was extracted, PCR amplification of the complete coding region of the PAX6 gene (14 exons). Direct automated sequencing of PAX6, samples were analyzed in an ABI Prism 310 Genetic Analyzer . Wild-type and mutated sequences were compared manually. The samples also were studied by means of multiplex ligation-dependent probe amplification (SALSA MLPA Kit P219) from MRC Holland after treating the samples with ligase,PCR amplification was performed with the specific SALSA FAM PCR primers. Electrophoresis of PCR products was performed using an ABI PRISM 310 . Data analysis was performed by exporting the peak areas to a Microsoft Excel file.
Sequencing analysis of the coding region and exon/intron junctions of PAX6 in the affected members failed to detect any deleterious mutation. MLPA analisys showed no gaining or losing in DNA material in the affected members, even in the remote 3' regulatory elements required for initiation of PAX6 expression.
Although the entire PAX6 sequence is analyzed in congenital aniridia patients in some cases no mutations are detected. We speculate that the probable source for aniridia phenotype are mutations in remote 3' regulatory elements required for initiation of PAX6 expression, or a deletion of several contiguous exons, deletion of a single exon, or deletion outside the coding region of PAX6. For these type of genetic alterations MLPA provides a great tool for expending the universe of the PAX6 genotype-phenotype correlations, but a more specific test is required for the rest of the individuals that show no alterations of the PAX6 gene, or to study a new locus for this small percentage of aniridia patients.
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