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Clinical Studies |
Departments of Molecular Medicine (A.N., S.L., A.W., H.L.) and Woman and Child Health (S.L., M.H.), Karolinska Hospital, Stockholm, Sweden
Address all correspondence and requests for reprints to: Anna Wedell, M.D., Ph.D., Department of Molecular Medicine, Karolinska Hospital, S-171 76 Stockholm, Sweden. E-mail: awe{at}gen.ks.se
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Abstract |
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Introduction |
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TopAbstractIntroductionExperimental SubjectsMaterials and MethodsResultsDiscussionReferences |
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The 21-hydroxylase locus has a complicated structure, with an active form of the gene (CYP21) and an inactive pseudogene (CYP21P). The genes are located 3' of each of the genes encoding the fourth component of complement, C4A and C4B. One 21-hydroxylase gene and one C4 gene form a unit, these units are tandemly repeated in the HLA class III gene region on chromosome 6p21.3 (4, 5). There is substantial interindividual variation in the numbers and gross structure of these C4/21-hydroxylase units (6, 7). The majority of the disease-causing mutations in CYP21 have arisen through interaction with CYP21P, and are either deletions of CYP21 resulting from misalignment followed by unequal crossing-over or aberrations that represent deleterious sequences that have been transferred from CYP21P into CYP21 (8, 9, 10). By typing for nine pseudogene-derived mutations by allele-specific PCR, approximately 95% of all mutated CYP21 alleles are detected (11). The remaining rare mutations need to be characterized by sequencing of the CYP21 gene.
We have found a good association between genotype and degree of disease manifestation (12). One mutation (V281L) is responsible for the vast majority of mild CAH cases (13). Genotyping for mild CAH mutations is a valuable diagnostic complement for this form of the disease, which is believed to be largely underdiagnosed. We previously described a rare allele carrying three base substitutions occurring in two siblings with late-onset disease (14). These were a C to T transition 4 bases upstream of translation initiation, a CCG to CTG change at codon 105 (P105L), and a CCC to TCC transition at codon 453 (P453S). A thymine is usually present at position -4 in CYP21P, whereas the two missense mutations are not normally found in the pseudogene. A similar allele, containing the -4 base change and P453S mutation together with yet another missense mutation (R339H) has previously been described in a patient of Turkish origin (15). The P453S mutation is also relatively frequent among mild CAH patients in Texas (16). The functional consequences of R339H and P453S have been analyzed after expression of mutant enzymes carrying each of these two mutations separately (15), whereas the -4 base change and P105L mutation have not been studied functionally. We have now analyzed the degree of functional impairment resulting from the C to T transition at base -4 and the P105L and P453S mutations by in vitro translation and after expression of mutant enzymes in cultured cells. The effects of the two missense mutations were studied both individually and in combination. The results have implications for the identification of subjects with the very mildest forms of CAH.
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Experimental Subjects |
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TopAbstractIntroductionExperimental SubjectsMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionExperimental SubjectsMaterials and MethodsResultsDiscussionReferences |
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The human full-length P450c21 complementary DNA (cDNA) clone phc21, cloned into M13 mp18 (18), was kindly provided by Dr. Bon-chu Chung. A BamHI fragment containing the complete cDNA from the initiation codon together with about 300 bp of 3'-untranslated sequences was inserted into plasmid pGEM3Z. This plasmid was modified to include 10 bp of 5'-untranslated sequences from CYP21, creating pGEM-CYP21. From pGEM-CYP21, a BglII/BamHI fragment containing the CYP21 cDNA together with 10 bp of 5'- and approximately 300 bp of 3'-untranslated sequences was isolated and cloned into the BglII site of the expression vector pCMV4, generating pCMV4-CYP21. A shorter construct, containing only 28 bp of 3'-untranslated sequences, was generated by KpnI cleavage of pCMV4-CYP21.
Introduction of mutations
The 5'-end of pGEM-CYP21 was PCR amplified with an upstream primer containing the -4 sequence alteration. The resulting fragment was inserted into pGEM-CYP21, generating pGEM-CYP21(-4). The P105L and P453S mutations were introduced separately into pGEM-CYP21 by site-directed mutagenesis, using two phosphorylated primers (19). One primer overlapped restriction sites for SalI and BglII, but contained a sequence that destroyed the SalI site, and the other primer covered the sequence of each of the two missense mutations. The two primers were annealed to the denatured, double stranded plasmid and directed the synthesis of a new second strand containing the destroyed SalI site and each of the missense mutations separately. The resulting DNA was electroporated into a mismatch repair-defective Escherichia coli strain (BMH 71–18 mutS). Plasmid DNA was purified, SalI treated, and used for electrotransformation of competent E. coli XL-1 blue. DNA from recovered colonies was purified, and SalI-resistant plasmids were screened for the P105L and P453S mutations by PCR, using one primer specific for each mutant sequence. Plasmids pGEM-CYP21(P105L) and pGEM-CYP21(P453S) were cleaved with EcoRI, which cleaves between the two mutations, and KpnI, which cleaves in the 3'-untranslated part of CYP21. The EcoRI/KpnI fragment in pGEM-CYP21(P105L) was replaced by the corresponding fragment from pGEM-CYP21(P453S), generating pGEM-CYP21(P105L+P453S). Finally, BglII/BamHI fragments from the four pGEM-CYP21 constructs were inserted into the BglII site of pCMV4. The 3'-KpnI fragment was removed from all mutant pCMV4-CYP21 constructs, generating pCMV4-CYP21(-4), pCMV4-CYP21(P105L), pCMV4-CYP21(P453S), and pCMV4-CYP21-(P105L+P453S).
In vitro translation
Estimation of translation efficiency was performed using the TNT Coupled Reticulocyte Lysate System (Promega, Madison, WI) according to the manufacturer’s instructions. One microgram of plasmids pGEM-CYP21, pGEM-CYP21(-4), pGEM-CYP21(P105L), pGEM-CYP21(P453S), and pGEM-CYP21(P105L+P453S) was used; incubation times were 15, 30, 45, and 60 min. [35S]Methionine incorporation was measured by scintillation counting after trichloroacetic acid precipitation of translation products. The translation products after 60-min incubation were also subjected to SDS-PAGE to verify the integrity of the 55-kDa 21-hydroxylase protein.
In vitro expression and assay of enzyme activity
Approximately 7 x 106 COS-1 cells were transiently transfected by electroporation (Bio-Rad Gene Pulser, Richmond, CA; 1200 V, 25 µF) with 10 µg of each of the pCMV4-CYP21 constructs together with 2 µg of the ß-galactosidase vector pCH110 (Pharmacia, Sweden), seeded in 6-cm petri dishes, and incubated in DMEM supplemented with 5% calf serum for 24–36 h.
To determine 21-hydroxylase activity in intact cells, 0.2 µCi 3H-labeled substrate (progesterone or 17-OHP) was added to the medium together with 0.5 µmol/L unlabeled steroid. After incubations for 10 min, 240 µL medium were collected in duplicate samples, and steroids were extracted with 300 µL methylene chloride (Sigma Chemical Co., St. Louis, MO), evaporated to dryness, and dissolved in ethanol. The steroids were separated by thin layer chromatography in chloroform-ethyl-acetate (80:20), and radioactivity was measured by liquid scintillation spectrophotometry. Subsequently, the cells were trypsinized, washed three times with PBS, and sonicated. Debris was centrifuged, and protein concentration and ß-galactosidase activity were measured.
To determine 21-hydroxylase activity in cell homogenates, the cells were washed twice with phosphate-buffered saline (Life Technologies, Gaithersburg, MD), and approximately 2 x 106 cells/plate were recovered for homogenization by sonication for 20 s in 500 µL hypotonic buffer [10 mmol/L HEPES (pH 6.2), 10 mmol/L NaCl, and 1.5 mmol/L MgCl2]. The homogenate was centrifuged at 350 x g for 10 min to remove nuclei and whole cells, and the supernatant was collected. To 50 µL supernatant, 4 mmol/L NADPH and 200 mmol/L phosphate buffer, pH 7.2, were added to a final volume of 300 µL, both with and without 20% glycerol. Subsequently, 0.2 µCi 3H-labeled substrate (progesterone or 17-OHP) was added together with 0.5, 2.0, 4.0, or 6.0 µmol/L unlabeled steroid. After incubation at 37 C for 1–2 h, steroids were extracted and analyzed as described above.
Enzyme activities were determined as a percentage of substrate conversion compared to that in cells transfected in parallel by pCMV4-CYP21 encoding wild-type 21-hydroxylase, after correction for total protein content. Assays were performed under initial rate conditions, determined by preliminary time-course experiments. The ratio of ß-galactosidase activity/total protein content was measured in each experiment to verify reproducibility of transformation efficiency. Apparent kinetic constants were calculated after linear regression of the data derived from determinations of enzymatic activity in cell homogenates at each of the four different substrate concentrations, using SigmaPlot and SigmaStat (Jandel Scientific, Erkrath, Germany).
To ascertain the amount of translated P450c21 in transfected cells, proteins from supernatants of homogenized cells were size-separated on a polyacrylamide gel and transferred to a nitrocellulose filter. Immunoblot analyses were performed according to standard procedures, using sera from patients with Addison’s disease containing autoantibodies against P450c21 (20).
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Results |
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TopAbstractIntroductionExperimental SubjectsMaterials and MethodsResultsDiscussionReferences |
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Recombinant P450c21 was transiently expressed in COS-1 cells. The effect of cell culture time after transfection was investigated. Enzyme activity increased with extended cell culture time up to 18 h, after which point it did not increase further. This pattern was the same for the allele carrying the -4 mutation and the wild-type CYP21 gene, indicating that this base substitution does not affect the rate of translation in transfected cells. As any difference in expression levels would affect the apparent maximum velocity of the enzyme, we performed immunoblot analyses of homogenates from cells transfected with the different constructs. Similar amounts of the protein were obtained in each case (data not shown).
Deletion of around 270 bp of the 3'-untranslated sequence of CYP21 in the expression vector pCMV4 resulted in approximately 2-fold higher expression, as determined by enzyme activity, when cells were harvested 8 h after transfection (data not shown). This difference was hardly detectable when the cells were harvested and assayed for enzymatic activity 24 h after transfection. Due to the higher expression early after transfection, constructs with shortened 3'-untranslated sequences were used in all subsequent experiments. Many determinants of messenger ribonucleic acid stability are known to be located in the 3'-untranslated region of genes. Multiple degradative pathways recognize AT-rich motifs (21), and several AT-rich stretches are present in the sequences that were removed.
In vitro translation
To further investigate the effect on translation efficiency of the different sequence alterations, we incubated the four mutant alleles and wild-type CYP21 in a coupled transcription-translation system. None of the mutations, including the base substitution at position -4, affected the rate of translation in reticulocyte extracts (data not shown).
Enzyme activity
To assess the influence of the different base substitutions on
21-hydroxylase activity, we compared the activity of the normal protein
with those of the various mutant forms. All mutant forms of CYP21
(-4, P105L, P453S, and P105L+P453S) were transiently expressed in
COS-1 cells, and enzyme activities toward the two natural substrates
for 21-hydroxylase, 17-OHP and progesterone, were assayed in intact
cells. As illustrated in Fig. 1
, the P105L mutation
reduced enzyme activity to 62% for 17-OHP and 64% for progesterone,
and the P453S mutation reduced activity to 68% and 46%, respectively,
with the wild-type activity being arbitrarily defined as 100%. As the
mutations were found in the same allele in the two patients, the
combined effect of the two missense mutations was analyzed. The
mutations were synergistic, resulting in a reduction of enzyme activity
to 10% of normal for 17-OHP and 7% for progesterone. As expected, the
construct containing the -4 base substitution gave rise to normal
enzyme activity (not shown).
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shows the kinetic results from a
representative experiment, and the corresponding apparent kinetic
constants are given in Table 1. The experiments were
performed both with and without the addition of 20% glycerol, with
essentially the same results. As shown, Km was in the same
range among the various P450c21s, whereas the maximum velocity was
decreased for the mutant enzymes.
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The presence of the P105L and P453S mutations in the pseudogene was investigated in 34 healthy parents of CAH patients from families in which C4/21-hydroxylase haplotypes have previously been determined (17, 22). These subjects represent 74 unrelated CYP21P alleles. Genotyping was performed by allele-specific PCR, essentially as previously described (11). Neither of the 2 mutations was present in any of the CYP21P alleles analyzed.
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Discussion |
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TopAbstractIntroductionExperimental SubjectsMaterials and MethodsResultsDiscussionReferences |
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The known missense mutations in CYP21 that have been shown to
result in partial enzyme inactivation are listed in Table 2. I172N, V281L, and P30L all derive from the pseudogene
and are relatively common among CAH patients, accounting for roughly
20%, 6%, and 2% of all affected CYP21 alleles in Scandinavia (12)
(Wedell, A., unpublished observations). The I172N mutation is
associated with prenatal virilization of females, whereas the V281L
mutation is associated with mild CAH without virilizing malformations
such as clitoromegaly. It is practical to relate to these two mutations
because they are common, whereas for the rare alleles, large numbers of
patients are not available for phenotypic evaluation. Based on the
synergistic effect of the two mutations in vitro, it is
reasonable to expect that P105L and P453S both would result in the
mildest form of CAH if they were present separately in a CYP21 allele,
i.e. the form of CAH that is believed to be largely
underdiagnosed. It is interesting that the only other allele containing
P453S that was completely sequenced also carried an additional
mutation, R339H (15). This allele was found in a markedly hirsute woman
with clitoromegaly. In this case, the combined effect of the two
mutations was not studied in cultured cells.
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The C to T transition at position -4 did not affect CYP21 gene function, as expected. It is known that as long as a purine is present in position -3 before the initiation codon, translation is usually not impaired (36). As a thymine is usually present at position -4 in the pseudogene, it can be assumed that this base substitution has arisen by the same mechanism as the most common disease-causing CYP21 mutations, i.e. by transfer of a CYP21P sequence into CYP21. We could not detect the P105L or P453S mutations among Scandinavian CYP21P alleles. However, P453S has been reported to occur in the pseudogene in the Texan population, where this mutation is quite frequent (16). It is likely that there exists a rare, distantly related CYP21P allele carrying the P453S mutation in the Scandinavian population, and that P453S can be expected to occur in additional CAH patients also in this ethnic group.
We have analyzed approximately 350 Scandinavian CAH alleles and only found the P105L and P453S mutations in a single family of Scandinavian ancestry. The siblings of this family had a slightly more severe phenotype than the mildest forms of CAH; the girl had clitoromegaly, which is generally not seen in association with the mild V281L mutation. The functional studies of mutant enzymes show that the mutations would produce a more subtle phenotype when found alone than when combined, as was the case in this family. This together with the fact that the global frequency of P453S is not negligible motivates us to include these mutations when using genotyping to ascertain mild, undiagnosed cases of CAH.
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Footnotes |
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Received January 29, 1996.
Revised August 21, 1996.
Accepted September 10, 1996.
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References |
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TopAbstractIntroductionExperimental SubjectsMaterials and MethodsResultsDiscussionReferences |
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Asn or Arg356Trp causes steroid 21-hydroxylase
deficiency. J Biol Chem. 265:3549–3552.
-helices in mammalian cytochromes
P450. Biochemistry. 28:3762–3770.[CrossRef][Medline]
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, Normal
P450c21; 
, P105L; 
, P453S; 
, P105L+P453S.