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,
Séverine Clauin,
Tiina Robins,
Patrick Vexiau,
Hélène Blanché,
Christine Bellanne-Chantelot and
Anna Wedell
Department of Molecular Medicine, CMM (L8:02), Karolinska Hospital (S.L., T.R., A.W.), 171 76 Stockholm, Sweden; and Department of Genetics, Fondation Jean Dausset-CEPH (H.B., P.V., S.C., C.B.-C.), 75010 Paris, France
Address all correspondence and requests for reprints to: Svetlana Laji, M.D., Ph.D., Department of Molecular Medicine, CMM (L8:02), Karolinska Hospital, 171 76 Stockholm, Sweden. E-mail: .
Abstract
We studied the functional and structural consequences of two novel missense mutations in CYP21 found in women with hyperandrogenism. The women were predicted to carry mutations by hormonal evaluation, but did not display any of the genotypes commonly associated with congenital adrenal hyperplasia. In one woman the novel mutation V304M was found in homozygous form. After expression in COS-1 cells the mutated enzyme was found to have a residual activity of 46% for conversion of 17-hydroxyprogesterone and 26% for conversion of progesterone compared with the normal enzyme. The V304M variant thus represents the sixth known missense mutation associated with nonclassical disease. A normal degradation pattern for this mutant enzyme indicates that the missense mutation is of functional, rather than structural, importance. The other mutation, G375S, was detected in a young woman with signs of hyperandrogenism, in heterozygous form together with P453S, a mutation known to cause nonclassical congenital adrenal hyperplasia (her genotype was G375S+P453S/wild type). This novel variant almost completely abolished enzyme activity; conversion was 1.6% and 0.7% of normal for 17-hydroxyprogesterone and progesterone, respectively. These results underline the importance of genetic evaluation and counseling in hyperandrogenic women who are predicted to carry congenital adrenal hyperplasia-causing mutations by biochemical tests. It also supports the idea that the heterozygous carrier state for CYP21 mutations can be associated with symptoms of androgen excess in certain susceptible individuals.
DEFECTS IN CORTISOL synthesis form a group of recessive syndromes that all result in increased secretion of ACTH from the pituitary with subsequent enlargement of the adrenals, congenital adrenal hyperplasia (CAH). Mutations in the steroid 21-hydroxylase gene (CYP21) account for more than 90% of all cases of CAH. This enzyme deficiency results in impaired secretion of both cortisol and aldosterone as well as an increased production of adrenal androgens (1, 2, 3, 4). The syndrome has traditionally been divided into three groups according to severity. In the most severe, salt-wasting form, affected children present with salt loss during the neonatal period, and females fetuses will develop virilizing malformations of external genitalia. A slightly less severe form is referred to as simple virilizing CAH. Patients with simple virilizing CAH do not develop life-threatening salt loss, but affected girls are born with virilized genitalia, and boys may develop precocious pseudopuberty during early childhood. The mildest form of the disease is nonclassical (NC) CAH, which presents with various degrees of late-onset symptoms. The most common symptoms in NC CAH are premature pubarche in children, acne, hirsutism, and menstrual irregularities in young women. The severity of hyperandrogenic symptoms varies in these patients; some have minimal symptoms, and others, especially males, are assumed to be asymptomatic. Diagnosis of CAH due to steroid 21-hydroxylase deficiency can be established by serum measurements of the intermediary steroids 17-hydroxyprogesterone (17OHP) and 21-deoxycortisol (21DOF), basally and after an ACTH challenge (5, 6). Heterozygous carriers can be detected by the same approach, although this is less reliable because of overlap with the population of unaffected individuals (6, 7). Due to the variable symptoms and diagnostic difficulties, the contribution of 21-hydroxylase deficiency for hyperandrogenism is not always clear-cut. Genotyping of CYP21 mutations offers a valuable complement to biochemical tests in the diagnostics of CAH.
The CYP21 gene is located adjacent to a highly homologous pseudogene (CYP21P) on chromosome 6p21.3 (8, 9, 10). This genomic structure predisposes to misalignment during meiosis, which after recombination or gene conversion can result in either CYP21 deletion or in the transfer of deleterious CYP21P sequences to CYP21. Nine such pseudogene-derived mutations account for about 95% of all affected CYP21 alleles in different ethnic groups. With few exceptions, there are good relationships between genotype and clinical disease presentation (phenotype) (11, 12, 13). Due to the complicated structure of the locus, with transfer of sequences between CYP21P and CYP21, genetic diagnostics of 21- hydroxylase deficiency is not always entirely straightforward. One pitfall regarding the presence of alleles harboring more than one disease-causing mutation. Multiply mutated alleles account for about 5% of all CAH alleles, and all combinations of mutations can occur (14). The correct linkage phase, i.e. whether mutations are present in the same or in different alleles, can be determined by segregating the mutations from the parents. If parents are not available, this can instead be performed by allele-specific PCR (15).
By sequencing the complete CYP21 gene in individuals presenting with hyperandrogenism and slightly elevated 17OHP and 21DOF levels after ACTH stimulation, we identified two novel missense mutations, V304M and G375S. V304M was found in both alleles of one woman, whereas G375S was found in a doubly mutated allele (G375S+P453S) in heterozygous form. To investigate whether the mutations could be responsible for the phenotypes of the patients, the mutations were reconstructed by in vitro site-directed mutagenesis, and the functional properties of the mutant enzymes were analyzed and compared with the normal protein. Activities toward the two natural substrates, 17OHP and progesterone, were determined. In addition, apparent kinetic constants were calculated for one of the mutants. To assess whether the observed impaired function was due to reduced enzyme stability, the half-lives of mutant proteins were determined by following their degradation patterns in intact COS-1 cells.
Subjects and Methods
Subjects
The novel mutations (V304M and G375S) were detected in two females (DOH94 and DOH14) who consulted the Department of Endocrinology and Diabetology, Hôpital Saint-Louis (Paris, France), with symptoms of hyperandrogenism. Patient DOH94 (born in 1968) presented with hirsutism, acne, and alopecia at the age of 24 yr. She was of Asian origin, and consanguinity could not be ruled out in this family. After ACTH stimulation, the levels of 17OHP and 21DOF were elevated in this patient (Table 1
), and she was considered to have mild (nonclassic) congenital adrenal hyperplasia. Sequencing of the entire coding region of CYP21 revealed that she was homozygous for a novel missense mutation (V304M). The other female, DOH14 (born in 1969), also suffered from acne and alopecia but did not have any problems with hirsutism at the age of 17 yr. Her hormonal levels were only slightly elevated upon ACTH stimulation (Table 1). Sequencing of CYP21 detected another novel mutation (G375S) in combination with a mutation causing mild CAH (P453S); both mutations were present in heterozygous form. Allele-specific PCR (15) showed that the mutations were present in the same allele and that an allele with normal sequence at both positions also was present. Her genotype was thus G375S+P453S/wild type (WT).
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Construction of the pCMV4 expression vector containing the CYP21 cDNA, pCMV4-CYP21, has previously been described (16) as well as introduction of mutations by site-directed mutagenesis using the pALTER-1 system (17). PmlI/KpnI fragments of pALTER-CYP21(V304) and pALTER-CYP21(G375S) were transferred to pCMV4-CYP21, thereby generating pCMV4-CYP21(V304M) and pCMV4-CYP21(G375S). The construct of pCMV4-CYP21(G375S+P453S) was generated by transferring a BglII/SauI fragment from pCMV4-CYP21(G375S) to pCMV4-CYP21(P453S). The complete CYP21 cDNA was sequenced to verify the correct incorporation of mutations and to exclude additional sequence aberrations.
Expression of P450c21 in mammalian COS-1 cells and assay of enzyme activity
Transient transfection of COS-1 cells was initially performed by electroporation (Gene Pulser, Bio-Rad Laboratories, Inc., Richmond, CA; 1200 V, 25 µF). Approximately 1 x 106 COS-1 cells were transfected with 2 µg of each of the pCMV4-CYP21 constructs together with 0.25 µg ß-galactosidase vector pCH110 (Pharmacia Biotech, Uppsala, Sweden), seeded in 3.5-cm petri dishes, and incubated in DMEM supplemented with 10% calf serum for 24 h.
To determine 21-hydroxylase activity in intact COS-1 cells, 0.1 µCi 3H-labeled substrate (17OHP or progesterone) was added to the medium together with 2 µmol/liter unlabeled steroid and 4 mmol/liter NADPH. After incubations at 37 C for 15 min, 200 µl medium were collected in duplicate samples, and steroids were extracted with 300 µl methylene chloride (Sigma, St. Louis, MO), evaporated to dryness, and dissolved in ethanol. The steroids were separated by TLC in chloroform-ethylacetate (80:20), and radioactivity was measured by liquid scintillation spectrophotometry. Subsequently, the cells were trypsinized and sonicated (once, 20 sec) and subjected to measurements of protein content and ß-galactosidase activity.
To determine apparent kinetic constants, intact cells were incubated as described above together with 0.5, 1.0, 2.0, 3.0, 4.0, or 7.0 µmol/liter unlabeled steroid. After incubation at 37 C for 15 min, steroids were extracted and analyzed as described above.
Enzyme activities were expressed as a percentage of substrate conversion, taking the activity of cells transfected in parallel by pCMV4-CYP21 encoding WT P450c21 as 100%, after correction for total protein content. The ratio of ß-galactosidase activity to total protein content was measured in each experiment to verify the reproducibility of transfection efficiency. Apparent kinetic constants were calculated after linear regression of the data derived from determinations of enzymatic activity in intact cells at each of the six different substrate concentrations.
Another transfection method was tested and used for approximately half of the experiments analyzed. By using transient transfection of COS-1 cells with lipofectin (FuGene, Amersham Pharmacia Biotech, Arlington Heights, IL) the number of cells needed for each experiment could be reduced, and the cell survival upon transfection increased. Approximately 2 x 105 cells were transfected using 3 µl FuGene with 1 µg of each of the pCMV4-CYP21 constructs and 0.5 µg ß-galactosidase vector pCH110 (Pharmacia Biotech), seeded in 3.5-cm petri dishes, and incubated in DMEM supplemented with 10% calf serum for 48 h. Apparent kinetic constants were thereafter determined as described above.
To ascertain the amount of translated P450c21 in transfected cells, proteins from supernatants of homogenized cells were size-separated in polyacrylamide gel and transferred to a nitrocellulose membrane. Immunoblot analyses were performed according to standard procedures, using polyclonal antibodies against human P450c21.
Assay of enzyme stability
COS-1 cells were transiently transfected with each of the pCMV4-CYP21 constructs using lipofectin as described above. After 48 h of incubation, cells received fresh medium (DMEM without serum) supplemented with cycloheximide (10 µg/µl). The cells were then harvested in PBS (pH 7.2) for 0, 4, 8, 12, and 24 h upon cycloheximide addition. The cells were thereafter sonicated, and the homogenates achieved were analyzed for P450c21 content by Western blot analysis. At each time point, 15 µg total protein were separated on a 10% polyacrylamide gel, and a polyclonal antibody was used to detect P450c21.
To ensure that the amount of loaded protein was equal for each time point, the blots were stained with Ponceau staining, and the intensity of the staining was quantified with a luminescent image analyzer (LAS-1000, Fujifilm, Fuji Corp., Tokyo, Japan). The degradation patterns of each of the mutant enzymes as well as of the WT protein were analyzed and quantified using the same analyzer (LAS-1000, Fujifilm), and half-lives were calculated.
Production of polyclonal antibodies
Polyclonal antibodies for the human P450c21 were produced in rabbit (Innovagen AB, Lund, Sweden). A five-amino acid-long peptide representing the carboxy-terminal end of P450c21 (amino acids 491–495, PGQSQ) was used for immunization. After the third booster dose, serum was collected from the rabbit and subjected to purification by affinity column chromatography. The polyclonal antibody was specific for human P450c21 in immunoblot experiments.
Genotyping of the novel mutations in the general population
The frequency of the novel mutations in the general French population was investigated using PCR-restriction fragment length polymorphism in 130 unrelated control subjects. The V304M and G375S mutations generate restriction sites for NcoI and AluI, respectively.
Results
Enzyme activity of mutants
To assess the influence of the missense mutations on 21-hydroxylase activity, the mutations were reconstructed by site-directed mutagenesis. All mutant forms as well as the WT protein of CYP21 were transiently expressed in COS-1 cells, and enzyme activities toward the two natural substrates, 17OHP and progesterone, were assayed in intact cells. The activity of the normal protein was arbitrarily defined as 100%.
As illustrated in Fig. 1, the V304M mutation reduced enzyme activity to 46% for 17OHP and 26% for progesterone, whereas the G375S mutation reduced activity to 1.6% and 0.7%, respectively. When the G375S mutation was expressed in combination with the more common P453S mutation, as found in the patient, the activity of the enzyme was totally abolished (Fig. 1). As previously reported, the P453S mutant protein has a good capacity for substrate conversion (62% and 64% for 17OHP and progesterone, respectively) (16), and the fatal reduction in enzymatic capacity of the particular allele found in patient DOH14 must thus be assigned to the novel G375S mutation.
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, the apparent kinetic constants are listed, and as shown, the Km values for the normal and mutant (V304M) proteins are of the same range for both substrates, whereas the maximum velocity is decreased for the mutant enzyme (by ANOVA, P < 0.05).
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To determine the half-lives of the WT and mutant proteins, the degradation pattern of the enzymes was followed in mammalian cells. All mutant forms of P450c21 were transiently expressed in COS-1 cells, and the amount of P450c21 protein was analyzed at different time points (0, 4, 8, 12, and 24 h) preceded by blocking of translation using cycloheximide. The apparent half-lives were not greatly different for the WT (13.0 h; minimum, 7.8 h; maximum, 23.6 h) and mutant proteins (V304M: 9.7 h; minimum, 7.5 h; maximum, 12.9 h; G375S: 10.7 h; minimum, 8.3 h; maximum, 14.1 h; G375S+P453S: 11.3 h), and thus the novel mutations did not have any drastic effect on the stability of the protein.
To verify that all variants of the enzyme were expressed, immunoblotting was performed from homogenates of cells transfected with the different constructs. All forms of P450c21 were produced in comparable amounts, as visualized on Western blots (data not shown).
Screening for the novel mutations
To assess the frequency of the novel mutations in the general population, 130 unrelated French control subjects were genotyped by PCR-restriction fragment length polymorphism. Among the 130 subjects screened, no individuals were found to be carriers of the novel mutations.
Discussion
The prevalence of NC CAH is estimated to range from 0.1–3.7% depending on ethnic group (18). The incidence of NC CAH among hyperandrogenic women varies among reports and ranges from 1.2% in a Californian study (19) to nearly 14% in a population from New York (20). It is likely that differences in ethnicity as well as in diagnostic criteria contribute to the varying proportions of mild CAH cases detected among hyperandrogenic patients. The diagnosis of NC CAH can be established by determination of basal or stimulated plasma 17OHP and 21DOF values (5). This approach can also be used to identify heterozygous carriers, although this is less reliable due to overlap with the normal, unaffected population (6, 7). Genotyping of CYP21 mutations is therefore a valuable complement in the diagnostics of 21-hydroxylase deficiency.
Three missense mutations in CYP21 are known to be common causes of NC CAH when present in homozygous form or as part of a compound genotype, including another CYP21 mutation. These are V281L (21), P453S (22, 23), and P30L (24). These mutations all result in a residual enzyme activity of 20–60% in vitro (16, 24, 25, 26). Two other rare missense mutations have been associated with mild enzyme impairment: P105L (16) and R339H (26).
It has been discussed whether the heterozygous carrier state for CYP21 mutations contributes to hyperandrogenism. Although mothers of CAH children, who are obligate heterozygote carriers of disease-causing CYP21 mutations, generally do not show symptoms or signs of androgen excess (27), heterozygotes for CYP21 mutations have been found among hyperandrogenic patients more frequently than in the general population (28, 29). In addition, almost one third of patients with acne have been found to be carriers of CYP21 mutations (30), and as many as 38% of a group of children with premature adrenarche in the Greek population were found to be heterozygous for one of nine common mutations in the 21-hydroxylase gene (31). The frequency of alleles associated with nonclassical CAH (V281L, P30L, P453S) was 33% among these children, whereas only 4% of the general population were found to carry one of these three mutations. In the same cohort, 8% of the children were diagnosed with nonclassical CAH. In addition, children with premature pubarche and adolescent girls with hyperandrogenism were found to be heterozygous carriers in a similar proportion (35%) compared with 6% of control subjects (29). However, these studies are small, and the concept of manifesting heterozygotes remains controversial.
The exhaustive screening of CYP21 mutations in a population of hyperandrogenic patients, who were predicted to be homo- or heterozygotes for CYP21 mutations on the basis of 17OHP and 21DOF responses to adrenal stimulation, was previously reported (28). Two mutant alleles were found in 15 of 16 patients (94%) predicted to be homozygous (1 patient was heterozygous), whereas 1 mutant allele was identified in 39 of 53 predicted heterozygotes (74%). Mutations associated with severe forms of CAH were detected on 21% of the altered alleles. These results thus confirm that homozygotes are readily detected by biochemical screening, whereas heterozygotes are more difficult to discriminate from the normal population, underlining the importance of genetic evaluation and counseling in this group of patients. In addition to screening for known mutations, the complete CYP21 gene was sequenced in these patients. We hereby detected two novel missense mutations, the functional relevance of which have now been investigated.
One of the women studied was homozygous for the novel missense mutation (V304M) at the age of 24 yr. The biochemical evaluation of stimulated 17OHP and 21DOF levels indicated that she was homozygous for 21-hydroxylase deficiency. The V304M mutant was partially active (46% and 26% of WT for 17OHP and progesterone, respectively), and determination of apparent kinetic constants revealed that the substrate binding capacity (Km) was of the same magnitude for mutant and normal enzymes. We can thus confirm the diagnosis of NC CAH in this patient. The other novel mutation (G375S) was detected in a girl with even milder symptoms who presented with acne and alopecia, but lacked hirsutism. This variant was found in one of her alleles in combination with P453S, a mutation associated with nonclassical CAH. In vitro expression analysis revealed that G375S almost completely abolished enzymatic capacity (1.6% and 0.7% of WT activity for 17OHP and progesterone, respectively), whereas the combined allele (G375S+P453S) resulted in an enzyme with no residual activity. Her other allele was normal, although the possibility remains that a mutation exists in a regulatory region of the gene that was not analyzed.
To determine whether the reduced activity of the mutants was due to reduced stability of the mutated protein, the half-lives of the WT and mutated enzymes were determined based on the degradation pattern of normal and mutant proteins in mammalian COS-1 cells. These data showed that neither variant affected the stability of the mutated protein to any great extent. The findings suggest that it is the function rather than the overall structure of the mutated proteins that is impaired. Based on the structure of the prokaryotic cytochrome P450 monooxygenase P450BM-3 (32) and on the computerized three-dimensional model of P450c21 (33, 34) the novel V304M mutation is located in a region of the protein (helix I) that is assumed to form a part of the substrate binding pocket. According to the same models the G375S mutation is located in a ß-strand (ß2-2) between helixes K and K', a region of the protein that has not been proposed to have a specific function in BM-3. However, this area of the protein (BM-3) is located proximal to a structure called the meander, which forms the inner portion of the redox docking site. A conserved E-R-R motif is also present in the meander and assists in stabilizing the three-dimensional structure of this region, locking the position of the Cys pocket.
Recently, the structure of the first mammalian microsomal cytochrome P450 enzyme was determined (35). The overall pattern of mammalian and bacterial P450 enzymes seems to be conserved, but, as expected, the substrate-binding sites diverge among the different enzymes. The V304 position is conserved among different prokaryotic P450s, and a change from one nonpolar residue to another (valine to methionine) will not change the helix formation drastically, which is also presented in the fairly preserved function of the V304M mutant enzyme. The mutant enzyme did not show any change in substrate recognition, since the Km was not affected. This indicates that V304 does not directly contact the substrate, but, rather, could be one of many residues forming the boundaries of the substrate binding pocket. No mutation in CYP21 has to date been determined to affect the Km of the enzyme, and all mutations associated with nonclassical CAH show Km values that are of the same magnitude as for the normal protein (16, 24, 25).
The microsomal P450s are integral membrane proteins, and the N-terminus provides the only transmembrane domain. However, when the N-terminus is removed from P4502C5, the protein still binds to membranes, but can be dissociated in high salt buffers, suggesting that there are other membrane-binding interactions present along the protein surface (35). One of these hydrophobic areas of P4502C5 (residues 376–379) corresponds to the ß2-2 strand in BM-3, the same region where our novel G375S mutant is located. The membrane-binding properties of this mutant enzyme, however, have not been tested in this study, although changing the nonpolar residue glycine to the polar residue serine would deteriorate the hydrophobicity of this region and thereby the association with the membranes and the orientation of the protein. Altered orientation could disrupt the entrance to the substrate access channel that, as in BM-3, is buried in the hydrophobic core of the membrane.
We have characterized two novel missense mutations in CYP21 that cause impaired enzyme function. One of the mutations (V304M) represents the sixth known alteration, causing a mild degree of enzyme dysfunction associated with NC CAH. The other allele is a severe mutation that would be expected to result in the most severe form of CAH if combined with another null mutation. Both patients described have residual enzyme function of about 25–50% of normal. We conclude that genetic investigation is a valuable diagnostic complement in cases of hyperandrogenism, when biochemical testing has indicated a defective 21-hydoxylase function. Although most obligate carriers of CYP21 mutations are completely normal with regard to endocrine status, these findings together with the results of previous studies suggest that the heterozygous state for CYP21 mutations may be associated with symptoms of androgen excess in certain susceptible individuals. Which factors influence this interindividual variation in sensitivity to a slightly reduced residual enzyme activity is unknown, just as it is unknown why some individuals with NC CAH show clear symptoms of androgen excess whereas others have minor symptoms or are asymptomatic.
Acknowledgments
Footnotes
This work was supported by the Swedish Medical Research Council (Project 12198); the Novo Nordisk, Emil and Vera Cornell, Fredrik and Ingrid Thuring Foundations; the Stockholm County Council; the Frimurare Barnhuset; the Society for Child Care; the Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche; and the Conseil Régional d’Ile de France.
Abbreviations: 
4A, 4-Androstenedione; CAH, congenital adrenal hyperplasia; CYP21, CYP21P; 21DOF, 21-deoxycortisol; 17OHP, 17-hydroxyprogesterone; NC, nonclassic; WT, wild type.
Received September 20, 2001.
Accepted January 4, 2002.
References
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