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Detection of CYP21A2 large gene rearrangements through the Multiplex Ligation-Dependent Probe Amplification methodology and comparison to the Southern blotting results


21-hydroxylase deficiency (21OHD) is a common autosomic recessive disorder characterized by a great phenotypic variability, from prenatal external genitalia virilization in female fetuses to the hirsutism at adulthood, with the possibility the coexistence of a serious aldosterone deficiency that could cause hyponatremic dehydration in the neonatal period. These clinical forms are caused by CYP21A2 gene mutations, which decrease the enzymatic activity at different levels. Due to the presence of a pseudogene, the main origin of mutations is recombination events between the active gene and its pseudogene. These mutations can be CYP21A2 deletions, large gene conversions or microconversions (point mutations). Pseudogene deletions or duplications can also exist, which does not modify the enzyme activity, but can make the analysis of the number of copies of CYP21 units difficult. 21OHD molecular biology is widely known, and molecular diagnosis has been used in the clinical practice. For the screening of point mutations, several methods had been standardized that allow their use on a large scale, as well as in the study of a large number of patients at the same time, like the allele-specific PCR method. However, for the screening of large gene rearrangements, the most common methodology used is Southern blotting, followed by in situ hybridization with specific probes. This technique has some disadvantages like the necessity for large DNA amounts, the use of radioactive material, the carrying out of a larger number of steps, consequently requiring more time to obtain results (±7 days). The Multiplex Ligation-Dependent Probe Amplification (MLPA) technique can also be used to study the number of gene copies, eliminating the aforementioned disadvantages. It is a relatively fast procedure, so that it is possible to get results in two days. However, until now, there is no validation of this method for the detection of large gene rearrangements in 21OHD, and it is unknown whether this technique has the capacity to differentiate all the different types of large gene rearrangements, which can exist in the same patient. Objectives: to compare the sensitivity and the specificity of MLPA methodology for the detection of the CYP21 rearrangements in comparison to previous results from the Southern blotting technique standardized in our laboratory. Casuistic and Methodology: DNA samples from 150 patients with 21OHD will soon be studied considering the number of copies of CYP21 genes by way of MLPA methodology. The kit P050B CAH (MRC-Holland, Amsterdam, The Netherlands) will be used, which contains eight sets of specific probes for CYP21A2 and CYP21A1P genes. The reactions will be carried out in accordance to the manufacturer’s protocol. All of these DNA samples were previously evaluated with Southern blotting, followed by in situ hybridization with CYP21 probe and the results of both methodologies will be compared in each patient. (AU)

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