Molecular analysis of mutated P450c21 in congenital adrenal hyperplasia

Abstract

Defects in cytochrome P450c21 (21-hydroxylase, 210H) are the most common causes of congenital adrenal hyperplasia (CAH). This recessively inherited disorder demonstrates a wide spectrum of severity as a consequence of impaired production of cortisol and aldosterone and excessive secretion of adrenal androgens. A limited number of mutations accounts for the majority (95%) of mutated alleles and genetic diagnostics based on detection of these common mutations is well established. However, additional rare mutations, usually specific for a single family or population, are responsible for this disorder in certain patients. Severe forms of 210H deficiency result in prenatal virilizing malformations of external genitalia in affected girls. In an attempt to prevent these malformations and avoid reconstructive surgery, prenatal treatment with dexamethasone (DEX) has been employed. Because eukaryotic cytochromes P450 are integral membrane proteins, it has been difficult to purify and crystallize these proteins in order to determine their three dimensional structures. Useful predictions regarding structure-function relationships for P450c21 can be obtained by studying bacterial members of the cytochrome P450 family, in combination with functional analyses of different P450c21 mutants affecting structurally and/or functionally important residues of the protein.

Using the approaches of site-directed mutagenesis and transient expression in COS-I cells, the functional consequences of eight novel missense mutations in CYP21 have been characterized. All of these mutant proteins demonstrated impaired enzyme function and were thus responsible for the disease in the patients in whom they were identified. The degree of functional impairment was generally in agreement with the severity of the symptoms displayed by the patient. By direct sequencing of CYP21, genetic diagnostics was provided for families who do not segregate the common mutations.

The P105L and P453S mutations were found on the same allele in two siblings with late-onset disease. Functional studies of each of these mutant proteins individually revealed an enzymatic activity which was 60-70% of the wild-type, while these two mutations in combination acted in a synergistic manner to reduce the enzymatic activity to 10%. The exact mechanism underlying the functional impairment for these mutant enzymes is not fully understood, since only the Vmax and not the KM value was affected. Loss of proline residues might disrupt the three-dimensional structures of the proteins, resulting in reduced stability. If only one of these mutations is present in a patient with CAH, the phenotype will most likely be subtle, not including prenatal virilization. Two other mutations (R356P and R356Q) were identified at the same position of P450c21. R356P (0.15% of wild-type activity) was found in a boy with the most severe form of the disease and R356Q (0.65% activity) was present in a girl with prenatal virilization but no salt loss. The region where these mutations are located is likely to be involved in interaction with the NADPH-cytochrome P450 reductase. With this addition of R356P and R356Q, there is a clear clustering of missense mutations in arginine residues in this portion of P450c21, supporting the concept that the region is involved in interactions with redox partners, since such interactions apparently involve charged amino acid residues.

Other structurally and/or functionally important portions of P450c21 include the regions where the mutations delE196, G291S and R483P are located. The delE196 and R483P mutant proteins retained some enzymatic activity and were found in patients with more moderate disease manifestations. These mutant proteins were more rapidly degraded in COS-I cells, as assessed by pulse-chase experiments followed by immunoprecipitation. Thus, the decreased catalytic capacity may be explained by altered protein structure and consequently reduced levels of these mutant forms of P450c21. In the bacterial enzymes P450BM-3 and P450cam, the residue corresponding to G291 of P450c21 has been proposed to participate in the electron transfer required for catalysis. Our data on the function of the G291S mutant suggest that this residue is indeed essential for maintenance of enzyme activity. One fundamental difference between prokaryotic and eukaryotic P450s is the membrane localization of the latter. Membrane-anchoring of P450c21 was determined using cell-free in vitro transcription-translation in the presence of microsomal membranes.

Our data indicate that P450c21 binds to the endoplasmic reticulum via only one trans-membrane domain in the most proximal amino-terminal portion of the protein, in agreement with previous reports. We have also demonstrated that the polymorphism (-L10) and the severe P30Q mutation, found in the first and second hydrophobic segments of P450c21 respectively, do not impair the membrane-binding capacity of the protein. Scandinavian experience (44 cases) with prenatal treatment of CAH indicates that such treatment is effective in preventing virilization in affected females. The majority of treated children are born normal in size and they develop and grow normally, compared to untreated controls. However, several adverse events were observed in treated children, such as failure to thrive and late psychomotor development. Women receiving DEX report more side-effects and gain more weight during early pregnancy when treatment is initiated. Therefore, until more experience is gained this treatment must still be considered to be experimental and prospective studies should be designed to exclude possible negative effects on the mother and child.