Abstract 18

Mutations of the AR gene in 46.XY subjects cause a spectrum of AIS, ranging from normal female external genitalia (CAIS) to ambiguous genitalia or infertile males and boys with reduced virilization at puberty (PAIS). The AR gene is composed of eight exons. Exon 1 encodes the 5′-untranslated region and N-terminal modulatory region. Exons 2 and 3 encode the DNA binding domain and the first point of the bipartite nuclear localization signal. Exons 4-7 and a small part of exon 8 encode the androgen binding domain (ABD). McPhaul et al defined the ABD as beginning at residue 692, and pointed out that 19 of 21 substitution mutations in the ABD clustered in two regions ("hot spots") that occupy only 35% of the ABD: residues 728 to 774 (exon 5) and residues 826 to 866 (exon 7). We have investigated 34 families with one or more 46,XY affected members with pseudohermaphroditism for AR gene mutations. Eight patients had classical clinical and laboratory features of the AIS, 3 CAIS and 5 PAIS and 26 subjects had pseudohermafroditism of undetermined cause. Genomic DNA was obtained from peripheral blood leukocytes from all subjects. The entire exon 5, which encodes part of the androgen binding domain of the AR, was amplified by polymerase chain reaction (PCR) and screened for sequence-dependent differences in its melting characteristics by denaturing gradient gel electrophoresis (DGGE). A mutant control (Y763C) and a normal control were used in the DGGE. PCR fragments with altered mobility in DGGE analysis were directly sequenced by dideoxy nucleotide chain-termination method. Two different single nucleotide base substitutions were found. In two affected sisters with PAIS amino acid residue 742 was changed from methionine (ATG) to valine (GTG). In one patient with CAIS, arginine (CGA) at residue 752 was changed to a stop codon (TGA). Both mutations were located in the androgen binding domain and each has been previously reported in one family with AIS. We conclude that 2/8 families with AIS and 0/26 with MPH of undetermined origin had mutations in exon 5 of the AR. Further molecular analysis of other exons of the AR gene may provide basis for MPH in the remaining Brazilian patients.