Use of nonradioactive labeling to detect large gene rearrangements in 21-hydroxylase deficiency.
OBJECTIVE
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).


METHODS
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 mug), 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.
Cukier
Priscilla
P
Discipline od Endocrinology, Hospital das Clínicas, Faculty of Medicine, University of São Paulo, São Paulo, SP, Brazil.
Bachega
Tânia A S S
TA
Mendonça
Berenice B
BB
Billerbeck
Ana Elisa C
AE
D000312:Adrenal Hyperplasia, Congenital / Q000235:genetics*; D000483:Alleles; D001710:Biotin / Q000031:analogs & derivatives*; D015139:Blotting, Southern / Q000379:methods*; D004247:DNA / Q000032:analysis*; D003845:Deoxycytosine Nucleotides; D005260:Female; D017353:Gene Deletion; D020440:Gene Duplication; D015321:Gene Rearrangement / Q000235:genetics*; D006801:Humans; D008297:Male; D009693:Nucleic Acid Hybridization
D003845:Deoxycytosine Nucleotides; C083589:biotin-14-dCTP; D001710:Biotin; D004247:DNA