Background: Deficiency of 11-hydroxylase results in the impairment of the last step of cortisol synthesis. In females, the phenotype of this disorder includes different degrees of genital ambiguity and arterial hypertension. Mutations in the CYP11B1 gene are responsible for this disease.

Objective: To screen CYP11B1 gene for mutations in two unrelated Brazilian females with CAH due to 11-hydroxylase deficiency.

Design: The coding and intron-exon junction regions of CYP11B1 were totally sequenced. A putative splice mutation was further investigated by minigene transcription.

Results: We report two novel CYP11B1 mutations in these Brazilian patients. An Arabian Lebanese descendent female was found to be homozygous for a cytosine insertion at the beginning of exon 8 changing the 404 arginine to proline. It alters the open reading frame creating a putative truncated protein at 421 residue which eliminates the domain necessary for the association of heme prosthetic group. A severely virilized female was homozygous for the g.2791G>A transition in the last position of exon 4. This nucleotide is also part of 5' intron 4 donor splice site consensus sequence. Minigene experiments demonstrated that g.2791G>A activated an alternative splice site within exon 4 leading to a 45-bp deletion in the transcript. The putative translation of such modified mRNA indicates a truncated protein at residue 280.

Conclusions: We describe two novel mutations, g.4671 4672insC and g.2791G>A, that drastically affects normal protein structure. These mutations abolish normal enzyme activity leading to a severe phenotype of CAH due to 11-hydroxylase deficiency.