Mutation analysis of CYP11B1 and CYP11B2 in patients with increased 18-hydroxycortisol production

BACKGROUND: In patients with glucocorticoid remediable aldosteronism (GRA), a rare hypertensive disorder caused by the presence of a chimeric aldosterone synthase (CYP11B2) and 11beta-hydroxylase (CYP11B1) gene, high level of urinary 18-hydroxycortisol (18OHF) excretion are observed. In some patients with hypertension, increased urinary 18OHF secretion is also found in the absence of the hybrid CYP11B1/CYP11B2 gene. We hypothesised that gene variants of CYP11B1 or CYP11B2 may be linked to this abnormal glucocorticoid production. METHODS: The urinary steroid profile was analysed by gas chromatography/mass spectrometry in 429 hypertensive patients and 98 (23%) thereof tested positive for increased 18OHF excretion. After correction for total cortisol excretion, 12 subjects showed an abnormally high 18OHF excretion. For genotyping DNA was obtained from six of these patients. All were tested negative for the hybrid CYP11B1/CYP11B2 gene and were further analysed for mutations in all exons and promoter regions of both CYP11B1 and CYP11B2 by single strand conformation polymorphism (SSCP) and sequencing when appropriate. RESULTS: The genetic analysis of the two genes revealed the presence of nine molecular variants in CYP11B2 and three in CYP11B1. In addition to published polymorphic sites, we identified two new variants in CYP11B2 but no new variants in CYP11B1. The newly identified CYP11B2 mutations are a C/T single nucleotide exchange located in the first intron and a double nucleotide exchange at the 3'-splice site of exon 8. The mutated sequence corresponds to the sequence of CYP11B1 indicating a gene conversion. This suggests that the mutant is not likely to affect splicing. Thus, none of the genetic variants identified explains the high urinary excretion of 18OHF. CONCLUSIONS: We present here a complete method for the genetic analysis of the CYP11B1 and CYP11B2 genes. By this method we could not identify genetic variants responsible for a GRA-like phenotype. The presence of high levels of 18OHF should not be used alone as a diagnosis tool for GRA.