Abstract

CUKIER, Priscilla; BACHEGA, Tânia A. S. S.; MENDONCA, Berenice B.  and  BILLERBECK, Ana Elisa C.. Use of nonradioactive labeling to detect large gene rearrangements in 21-hydroxylase deficiency. Rev. Hosp. Clin. [online]. 2004, vol.59, n.6, pp. 369-374. ISSN 0041-8781.  http://dx.doi.org/10.1590/S0041-87812004000600010.

PURPOSE: To establish the Southern blotting technique using hybridization with a nonradioactive probe to detect large rearrangements of CYP21A2 in a Brazilian cohort with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH-21OH). METHOD: We studied 42 patients, 2 of them related, comprising 80 non-related alleles. DNA samples were obtained from peripheral blood, digested by restriction enzyme Taq I, submitted to Southern blotting and hybridized with biotin-labeled probes. RESULTS: This method was shown to be reliable with results similar to the radioactive-labeling method. We found CYP21A2 deletion (2.5%), large gene conversion (8.8%), CYP21AP deletion (3.8%), and CYP21A1P duplication (6.3%). These frequencies were similar to those found in our previous study in which a large number of cases were studied. Good hybridization patterns were achieved with a smaller amount of DNA (5 mg), and fragment signs were observed after 5 minutes to 1 hour of exposure. CONCLUSIONS: We established a non-radioactive (biotin) Southern blot/hybridization methodology for CYP21A2 large rearrangements with good results. Despite being more arduous, this technique is faster, requires a smaller amount of DNA, and most importantly, avoids problems with the use of radioactivity.

Keywords : Biotin labeling; Large rearrangements; Southern blotting; CAH-21OH.