In vitro functional studies of rare CYP21A2 mutations and establishment of an activity gradient for nonclassic mutations improve phenotype predictions in congenital adrenal hyperplasia.

Background: A detailed genotype-phenotype evaluation is presented by studying the enzyme activities of five rare amino acid substitutions (Arg233Gly, Ala265Ser, Arg341Trp, Arg366Cys and Met473Ile) identified in the CYP21A2 gene in patients investigated for Congenital adrenal hyperplasia (CAH).

Objective: To investigate whether the mutations identified in the CYP21A2 gene are disease causing and to establish a gradient for the degree of enzyme impairment to improve prediction of patient phenotype.

Design And Patients: The CYP21A2 genes of seven patients investigated for CAH were sequenced and five mutations were identified. The mutant proteins were expressed in vitro in COS-1 cells, and the enzyme activities towards the two natural substrates were determined to verify the disease-causing state of the mutations. The in vitro activities of these rare mutations were also compared with the activities of four mutations known to cause nonclassic CAH (Pro30Leu, Val281Leu, Pro453Ser and Pro482Ser) in addition to an in silico structural evaluation of the novel mutants.

Main Outcome Measure: To verify the disease-causing state of novel mutations.

Results: Five CYP21A2 mutations were identified (Arg233Gly, Ala265Ser, Arg341Trp, Arg366Cys and Met473Ile). All mutant proteins exhibited enzyme activities above 5%, and four mutations were classified as nonclassic and one as a normal variant. By comparing the investigated protein changes with four common mutations causing nonclassic CAH, a gradient for the degree of enzyme impairment could be established. Studying rare mutations in CAH increases our knowledge regarding the molecular mechanisms that render a mutation pathogenic. It also improves phenotype predictions and genetic counselling for future generations.