Functional and structural studies on CYP21 mutants in congenital adrenal hyperplasia

Abstract: Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is one of the most common autosomal recessive disorders, affecting around 1/10 000 newborns worldwide. A defect in the gene encoding steroid 21-hydroxylase, CYP21, results in impaired synthesis of cortisol and in most cases also aldosterone. Consequently, the secretion of ACTH by the pituitary gland is increased, resulting in hyperplasia of the adrenal cortex and an excessive secretion of androgens. This steroid hormone imbalance gives rise to a wide spectrum of signs and symptoms ranging from neonatal life-threatening salt-wasting and/or severe prenatal virilization of female external genitalia, to minor signs of hyperandrogenism in adulthood, such as acne, hirsutism and infertility. Nine pseudogene-derived mutations together with a net CYP21 gene deletion are responsible for around 95% of all alleles involved in CAH. However, the remaining 5% of affected alleles harbor rare mutations that are considered to be family- or population-specific. The total number of such reported rare mutations has increased dramatically during the last decade, now accounting for approximately 100 variants. CYP21 is thus the cytochrome P450 for which the largest number of naturally occurring amino acid substitutions (mutations and polymorphisms) has been found. The overall focus of the five studies that form the basis of this thesis was to investigate these rare mutations. The molecular mechanisms underlying the impaired enzyme function have been analyzed by combining experimental mutagenesis and functional enzyme activity assays with structural modeling studies. The functional studies included 11 CYP21 mutations (of which six are novel), the majority representing unique non-pseudogene-derived gene defects specific for certain families or populations detected in patients with hyperandrogenism or CAH. Close relationships between genotype and phenotype were found. Enzyme activities displayed by CYP21 mutants in vitro were reflected in the severity of the clinical signs displayed by the patients, indicating that functional experimental data is useful for classification of mutants according to the different groups of severity seen in CAH. Salt-wasting (SW) CAH is associated with an in vitro activity below 1%, simple virilizing (SV) CAH is associated with activities of around 1 to 15%, and residual activities above 20% of normal are associated with the mildest, non-classical (NC) form of CAH. Functional assays were also useful for discrimination between normal variants and disease-causing mutations, and for evaluations of individual mutations present in multiply mutated alleles. Functional characterization of CYP21 mutants thus provides information that has direct implications for diagnostics, genetic counseling, and clinical management in families who do not segregate the most common CYP21 mutations. The role of CYP21 mutations in hyperandrogenism was also investigated. The results support the concept that heterozygosity for severe CYP21 mutations can be associated with hyperandrogenic symptoms and signs in susceptible individuals. The structural studies included modeling of all known allelic forms of the human CYP21 protein (missense mutants and normal variants, a total of 66 alleles including seven novel mutants), using the crystal structure of rabbit CYP2C5 as a template. Each enzyme variant was evaluated with respect to the following parameters: cumulative frequencies of free energy, surface accessibility, evolutionary conservation, changes in polarity, distances to heme and steroid, and structural information from visual inspection of the model. Relationships between these parameters and associated clinical manifestations seen in CAH patients were found, indicating that this approach provides a new bioinformatic route for the prediction of clinical consequences of disease causing mutations.