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Original Studies |
Service d’Endocrinologie et Médecine de la Reproduction, Hopital Necker (C.D., A.D., L.B., F.K.), 75015 Paris; Service de Biochimie Endocrinienne et Moléculaire-INSERM, U-329, Hopital Debrousse (V.T., Y.M.), 69005 Lyon; Laboratoire SESEP, Université de Versailles-Saint Quentin (E.M.), 78000 Versailles; Laboratoire d’Immunologie et d’Histocompatibilité, Hopital St. Louis (D.C.), 75010 Paris, France
Address all correspondence and requests for reprints to: Dr. Frédérique Kuttenn, Service d’Endocrinologie et Médecine de la Reproduction-Hopital Necker, 149 rue de Sèvres 75015 Paris, France.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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As the CYP21 gene encoding the adrenal 21-hydroxylase was first characterized in 1984, the molecular genetic basis of the disease has been extensively investigated (1, 2, 3, 4). To date, according to the Human Gene Mutation Database Cardiff,1 over 50 CYP21 mutations have been reported: 29 point mutations causing CAH and several rearrangements of the CYP21 region, such as large deletions and gene conversions. In vitro activity was determined by site-directed mutagenesis in 15 missense mutations and one splicing mutation (see reviews in Refs. 3, 4, 5). Associations between the three different clinical forms of the disease and specific mutations have emerged, and it appears that most, but not all of, the phenotypic variations among the three forms of 21-hydroxylase deficiency result from allelic variations at the CYP21 locus (6, 7, 8).
The nonclassical form of the disease (NC-CAH) also called late-onset congenital adrenal hyperplasia, occurs in approximately 1 in 1000 of the general population (9) depending on the ethnic group and in up to 6% of hirsute women (10). In women, this form is characterized by a variety of late-onset symptoms, including premature adrenarche, hirsutism, menstrual disturbances, and infertility. Three mutations have been specifically characterized in this form, namely the V281L, P30L, and P453S point mutations (11, 12); these mutations have been associated with about 30–40% of residual enzymatic activity in vitro. However, as the severity of the disease is determined by the activity of the less severely affected of the two alleles, patients presenting with NC-CAH may have 1 of their alleles that is severely affected (conferring 2% or less of normal enzymatic activity). Whether the presence of a severe mutation on 1 allele alters the clinical and/or biological presentation of the disease in NC-CAH patients is not known.
We genotyped a group of 56 NC-CAH women, whose clinical and hormonal status was examined. We sought to determine the prevalence of each type of CYP21 mutation in this group and to analyze the phenotype-genotype correlation in this form of CAH. Indeed, as mentioned, these women may transmit a severe defect of the CYP21 gene, and this fact has important implications for the genetic counseling strategy.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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From 1984–1998, 108 unrelated female patients from various ethnic backgrounds who consulted in the department of Endocrinology at the Hopital Necker (Paris, France) were diagnosed as index cases of NC-CAH. The clinical and hormonal profiles of some of these patients were presented in previous papers (10, 13, 14). Molecular analysis of the CYP21 gene became possible in 1986 and was performed in 56 of these women.
These patients had consulted for hirsutism, menstrual disturbances, and/or infertility. There was no history of virilization at birth. The presence of hirsutism was assessed by the examining physician and defined as a clinical Ferriman score (15) greater than 10. Oligomenorrhea was defined as menstrual cycles longer than 35 days; primary amenorrhea was defined as the absence of menarche at the age of 17 yr; secondary amenorrhea was defined as the cessation of menstruations for more than 3 months.
Patients were diagnosed as having NC-CAH on the basis of late-onset
symptoms and a short-term ACTH-stimulated (0.25 mg, im)
17-hydroxyprogesterone (17OH-P) plasma level higher than 10 ng/mL
(30.25 nmol/L) (16). Basal plasma levels of androgens,
testosterone, and 
4-androstenedione, were
radioimmunoassayed (17). Human leukocyte antigen (HLA)
typing was performed with the lymphotoxicity test (10, 18). A molecular study of the CYP21 gene was performed in 56
women, representing 112 unrelated chromosomes. Both parents were
genotyped for 19 patients.
DNA analysis
Peripheral blood samples were obtained from the patients and
both parents (if available) after informed consent for this study had
been given, according to our institutional guidelines. Genomic DNAs
were prepared as previously described (19). Twenty
patients were genotyped by Mornet’s group; 13 of them were previously
described (20, 21). For the other patients, molecular
genetic defects of the CYP21 gene were studied according to a cascade
strategy. Selective amplification of the CYP21 gene was performed by
PCR in several fragments using the oligonucleotides shown in Table 1
. For each pair of primers, at least 1
oligonucleotide was specific for the CYP21 gene. Firstly, DNA was
screened for 6 frequent CYP21 mutations. A fragment was amplified by
PCR methods and digested with appropriate restriction enzyme as
follows: P30L (AC fragment digested by AciI), IVS2-13
C/A
G termed in the text as intron 2 mutation (BC fragment digested
by AluI), I172N mutation in exon 4 (DI fragment digested by
BsrI), cluster of 3 mutations (I236N, V237E, and M239K) in
exon 6 designated CL6 (DG fragment digested by MboI), the
V281L (DG fragment digested by HgiAI or
BsiHKA-I), and Q318X (DG fragment digested by
PstI). Digested fragments were separated on 4% agarose
(P30L, V281L, CL6, and Q318X) or 10% polyacrylamide gels
(IVS2–13A/C G and I172N). Mutations are designated according to the
convention of Beaudet and Tsui (22) as modified by
Antonarakis (23). Nucleotide numbering is based on the
gene sequence reported by Higashi et al. (24),
where 1 is the first nucleotide of the initiating ATG codon. Secondly,
if no mutation was detected on at least 1 allele, direct sequencing of
PCR products (DH, then FC, and finally BE) was performed with a 373A
model automatic sequencer (Applied Biosystems, Inc., Foster City, CA)
as previously described (25). Finally, if a large lesion
(deletion or gene conversion) was suggested by either PCR or sequencing
or if both parents were not available for genotyping, a Southern blot
analysis was performed as follows. Human leukocyte DNA was digested
with Taq1 restriction enzyme, and Southern blotting studies
were performed as previously described (19). Blots were
probed with a mixture of two fragments: a 500-bp
BamHI-KpnI fragment of the C4 complementary DNA
and a 3.1-kb genomic EcoRI-BamHI fragment of the
5.5-kb BglII-BamHI fragment encompassing the
entire CYP21 gene cloned in the BamHI site of
Bluescript SK+ plasmid (2).
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Student’s t test was used for comparing quantitative
variables. 
2 test was used for comparing
nonquantitative variables. Data are presented as the mean ±
SD.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In our group of 56 women, the mean age at diagnosis was 22.5 ± 7.5 yr and ranged from 12–41. Hirsutism was present in almost all patients (96%). Menstrual cycles were considered normal in 29% of the women; 47% had oligomenorrhea; 11% had secondary amenorrhea; 13% had primary amenorrhea. The mean age at menarche was 12.6 ± 1.5 yr (after exclusion of the women with primary amenorrhea).
The mean 17OH-P level was 12.5 ± 13.6 ng/mL (37.8 ± 41.1
nmol/L) basally and 45 ± 22.4 ng/mL (136.1 ± 67.8 nmol/L)
after ACTH. The stimulated 17OH-P level was greater than 20 ng/mL (60.5
nmol/L) in most patients. Seven women had stimulated 17OH-P levels
between 10 and 20 ng/mL (30.2 and 60.5 nmol/L), a range of possible
overlapping values between NC-CAH patients and heterozygous subjects.
The mean plasma testosterone level was 0.84 ± 0.38 ng/mL (2.9
±1.3 nmol/L). The mean plasma
4-androstenedione level was 4.04 ± 1.74
ng/mL (14 ± 6 nmol/L). Seventy-five percent of the women had at
least one HLA B14 haplotype, 18.75% were B14/B14, and 33.3% were
A33-B14. The clinical and hormonal profiles as well as the proportion
of HLA B14 haplotype of some of these patients have been reported
previously (10, 13, 14). They remain similar throughout
the on-going progression of the study of our NC-CAH population
(10, 13, 14).
Molecular evaluation
Mutations. The distribution of the mutations detected in the
CYP21 gene on the 112 chromosomes examined is shown in Table 2. According to previous in
vitro expression studies, these mutations are grouped by the
degree of predicted enzymatic activity. The mutations V281L, P30L, and
P453S, that lead to moderate alteration of enzymatic function (with
30–40% of remaining activity) were detected on 60% of the
chromosomes examined. The overall predominant mutation was V281L, which
was found on 51% of alleles and in 80% of women.
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Finally, three novel CYP21 mutations were found. The first was a missense mutation, R435C, located at codon 435 in exon 10 and changing an arginine (CGC) to a cysteine (TGC). The second was a missense mutation L317M located at codon 317 in exon 8 and changing a leucine (CTG) to a methionine (ATG), two nonpolar amino acids. Alignment of P450c21 protein in five species (human, bovine, mouse, swine, and sheep) showed that the arginine in position 435 and the leucine in position 317 are highly conserved. The last novel lesion was a gene conversion replacing the 5'-end of CYP21 by the corresponding sequence of the CYP21P pseudogene. This gene conversion differs from the classic one by having the junction site upstream of exon 3 and located in intron 2 between nucleotides 9 and 45, where four nucleotides TGTT were deleted in the functional gene.
Genotypes and phenotype-genotype correlation
We classified our patients into three groups, A, B, and C,
according to the CYP21 allelic combinations, as shown in Table 3. Group A included 18 women (32% of all
patients) with 2 mild mutations. Twelve of these women were homozygous
for the V281L mutation, which accounts for 67% of the patients in that
group and for 21% of all 56 patients, making it the most common
genotype in the study population. Group A women showed a large range of
hormone levels, with no actual phenotype specificity according to the
mutations involved. Indeed, in the genetically homogeneous V281L/V281L
group, the ranges of hormone levels were as large as in the other
associations, V281L/P30L and V281L/P453S, of this group A (Table 3).
The P30L mutation was not associated with a more severe presentation,
as had previously been suggested (4). However, the small
number of women with this mutation in our group does not allow us to
reach a definitive conclusion.
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4-androstenedione [3.2 ± 1.37
vs. 4.6 ± 1.94 ng/mL (11.1 ± 4.7 vs.
15.9 ± 6.7 nmol/L); P = 0.02] levels tended to
be lower in the presence of the intron 2 mutation despite an important
overlap in individual values. In contrast, in our sample, patients who
carry the I172N mutation on 1 allele and any mild mutation on the other
allele did not show a milder phenotype than patients with a nonsense
mutation on 1 allele and any mild mutation on the other allele, as
shown in Table 3
for stimulated 17OH-P levels.
We compared the clinical and biological phenotypes between genotypic
groups A and B to determine whether the presence of a severe mutation
in NC-CAH women was associated with more severe manifestations. The
clinical characteristics of the patients are presented in Table 4. No significant difference was found
between the two groups for age at diagnosis, prevalence of hirsutism,
or adult height. Although the proportions of women with irregular or
regular cycles did not differ between the two groups (about two thirds
of women had menstrual disturbances), a higher prevalence of primary
amenorrhea was observed in group B. The mean basal plasma 17OH-P level
was significantly higher (P = 0.05, unilateral
Student’s t test) in group B patients [14.8 ± 13;
range, 1.6–54 ng/mL (44.7 ± 39.3; range, 4.8–163.3 nmol/L)]
compared with group A patients [6.2 ±5.2; range, 1.9–23 ng/mL
(18.7 ± 15.7; range, 5.7–69.5 nmol/L); Fig. 1]. A significant difference
(P < 0.001, by unilateral Student’s t
test) was also observed between groups B and A when ACTH-stimulated
levels were compared [51.9 ±21.7; range, 19–107 and 32.3 ±14.3;
range, 16.9–59 ng/mL (156.9 ± 65.6; range, 57.4–323.6 and
97.7 ± 43.2; range, 51.1–178.4 nmol/L), respectively; Fig. 1].
No significant difference was observed between the mutation groups for
mean basal plasma testosterone and
4-androstenedione levels (Fig. 2).
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4-androstenedione level was 3.1 ng/mL (10.7
nmol/L).
One patient is considered separately. She was homozygous for a newly
described 5'-end gene conversion. This woman, a native of Iraq,
reported a high degree of consanguinity in her family. She had been
diagnosed at the age of 42 yr because of a severe hirsutism. She
presented with a particularly pronounced biological phenotype; indeed,
her testosterone plasma level was 2.3 ng/mL (7.9 nmol/L), her
4-androstenedione level was 8.3 ng/mL (28.8
nmol/L), and her basal 17-OHP level was 67 ng/mL (202.7 nmol/L). These
were the highest values recorded for any of these three parameters in
our series.
Finally, in two patients, only one mutation was detected even after complete sequencing of the CYP21 gene. These two women were therefore considered to be heterozygous patients. Their levels of stimulated 17OH-P [12 and 14 ng/mL (36.3 and 42.3 nmol/L)] were compatible with this status.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The distribution of the genetic alterations in the 56 patients confirms the clear predominance, in NC-CAH, of the V281L mutation in exon 7. This result is in agreement with those obtained in smaller series and probably reflects a founder effect. Furthermore, the close association between this V281L mutation and HLA group B14 (10) is confirmed; indeed, 90% of the chromosomes carrying this mutation were characterized as B14 in the HLA system, and when the HLA status was B14, the V281L mutation was found in all but 1 case. However, we did not observe the previously reported high frequency for the other mutations associated with a mild enzymatic defect, P30L and P453S. In particular, the P453S mutation was detected in 12% of our patients, whereas Owerbach et al. (26) found it in 46% of patients in a group of 13 American women with NC-CAH. These differences may reflect the different origins of the populations.
Severe mutations of the CYP21 gene were detected on 35% of the chromosomes and were present on at least one allele in 63% of our patients. The most frequent anomaly was the intron 2 splicing mutation, which accounts for half of the severely mutated alleles. In this group of NC-CAH women, the intron 2 mutation tended to be associated with a less severe phenotype than did the other severe mutations, as has been described in the classical form of the disease. Indeed, although this mutation has been usually associated with the salt-wasting form of the disease (6, 7, 27), the possibility of milder clinical symptoms has been reported (28, 29) and a low, but measurable, enzymatic activity after transfection in vitro has been shown (30). The high prevalence of this mutation found in 27% of our 56 patients may constitute a source of phenotypic variability in NC-CAH women. Deletion of the CYP21 gene was detected in 9% of women and was the second most frequent severe mutation in our group. This result emphasizes the importance of familial molecular study including analysis of haplotype segregation or, if the family is not available, of the need for a specific genotypic analysis strategy to distinguish between hemizygous and homozygous subjects for a mild mutation.
A major result of our work is the high proportion of NC-CAH women carrying a severe mutation of the CYP 21 gene. An abstract reporting a large number of patients (12) and previous smaller studies had already suggested similarly high proportions (31, 32). From a practical point of view, this result means that about two thirds of the women with NC-CAH may give birth to children with a classical form of the disease if the father is heterozygous for the classical disease. As the prevalence of heterozygous subjects for the classical form of 21-hydroxylase deficiency has been estimated to be 1 in 50 in the general population (33), the probability for such an event may be estimated at 1 in 200. This underlines the need for molecular characterization of the CYP-21 gene in women with NC-CAH and, if a severe mutation is detected, for biological and molecular study of the partner to evaluate the risk of the classical disease in their descendants, in accordance with our previously reported strategy (5).
Seven of the 56 women had a peak 17OH-P level after ACTH of between 10–20 ng/mL. In 2 patients, only 1 mutation was found despite complete sequencing of the entire gene. These women should be considered simple heterozygotes and not NC-CAH, even if we could not rule out a second mutation in a regulatory sequence. In the other 5 patients, two mutations were identified; 4 were homozygous for mild mutations, and 1 was compound heterozygous for a mild and a severe mutation. In these 5 NC-CAH women, peak 17OH-P levels after ACTH were between 16.9–19 ng/mL (51.1 and 57.4 nmol/L), higher than in the two simple heterozygous women. This emphasizes the need for complete sequencing of the entire CYP21 gene in women with this biological phenotype. Additional studies would be useful to determine the frequency of NC-CAH as opposed to simple heterozygotes in this population.
We report three genetic lesions never previously described. Although
these mutations have not been analyzed in vitro, their
association with impaired enzymatic activity is highly probable. The
two missense mutations, L317M and R435C, were associated with a severe
mutation, IVS2–113A/CG and Q318X, respectively, on the other allele
and with typical symptomatic late-onset adrenal hyperplasia
[postpubertal hirsutism, oligomenorrhea, and post-ACTH 17OH-P levels
of 63 and 53 ng/mL (190.6 and 160.3 nmol/L), respectively]. As the
severity of the 21-hydroxylase deficiency is generally determined by
the less severely affected of the two alleles, the two proteins
containing their respective mutation, L317M or R435C, must have a
residual 21-hydroxylase activity similar to that found with other mild
mutations. Therefore, the conservative neutral nonpolar amino
substitution, L317M, may be considered a mild mutation. In contrast,
the R435C mutation, which changes a basic amino acid (arginine) to a
neutral polar amino acid (cysteine) and affects an amino acid located
near Cys428, which is considered to be the heme
ligand (34), should be more severe. Although the phenotype
of the patient carrying this mutation suggested that R435C was a mild
mutation, further in vitro expression analysis is necessary
to confirm this hypothesis. The last novel genetic lesion is a gene
conversion of the 5'-end of the CYP21 gene, found in a woman homozygous
for this mutation, who presented with an especially pronounced NC-CAH
phenotype. Moreover, as the resulting gene differs from the functional
gene by the presence of two deleterious mild mutations, one
corresponding to the promoter region mutation carried by the pseudogene
(which has only 20% of the transcriptional activity of the CYP21
promoter) (35), and the other corresponding to the P30L
mutation in exon 1 (which results in an enzyme with 30–40% of
residual activity) (36), we hypothesize that this mutation
may be responsible for a remaining 21-hydroxylase activity below 20%
and may therefore constitute an intermediate defect between known
severe and mild mutations.
Surprisingly, two women in our series carried a potentially severe mutation on both alleles. The first woman was homozygous for the I172N mutation, which is usually associated with the classical simple virilizing form of the disease. Such a genotype in women with a NC-CAH phenotype and no neonatal virilization had been previously reported in a girl diagnosed at the age of 1 yr because of secondary virilization (8). Another study described a NC-CAH woman with the I172N/Q318X genotype but without any virilization (7). Finally, two patients with a homo/hemizygous I172N mutation were reported to exhibit obvious NC-CAH in a Hungarian population (37). Our observation confirms that the I172N mutation in combination with another severe defect of the CYP21 gene may be associated with a nonclassical phenotype. However, although virilization was absent at birth in our patient, her clinical and biological phenotype was particularly marked. It may be considered an intermediate presentation between the simple virilizing and NC-CAH forms that illustrates the concept of a phenotypic continuum among the three forms of 21-hydroxylase deficiency. The mild clinical and biological NC-CAH phenotype of the other woman homozygous for the intron 2 splice mutation is more surprising. The possibility of PCR artifacts has been eliminated. Although this mutation usually seems associated with severe alteration of the 21-hydroxylase function, our description suggests that partially preserved enzymatic activity, as encountered in NC-CAH, remains possible. A sufficient amount of normal splicing should occur in vivo in adrenals, resulting in enough residual activity to explain this NC-CAH phenotype. These two observations indicate that the phenotype cannot always be predicted from the genotype and may be even milder than would be expected. This failure of strict correspondence of phenotype and genotype must be borne in mind when performing a prenatal fetal DNA analysis. Predicting phenotype must remain cautious.
No study of phenotype-genotype correlation has ever reported any
difference between NC-CAH patients on the basis of genotype. However,
the patient samples were always too small to allow for any
classification. Our series of 56 women offers adequate material for
this type of analysis. It would be interesting to determine whether
particular combinations of mutations could yield a more predictable
degree of 21-hydroxylase dysfunction than can be predicted based on
each allele’s in vitro activity alone. When we consider the
hormone levels within the group of genetically patients homozygous for
the V281L mutation, it appears quite heterogeneous. In addition, there
is no significant difference between the hormone profiles of the V281L
homozygous patients and the other patients of group A with a V281L
mutation combined with a P453S or a P30L mutation. Although no
definitive conclusion can be drawn from this small group of patients,
these results strengthen the fact that the genotype cannot be
completely predictive of phenotype. Despite the lack of obvious
distinctive phenotypic criteria, the clinical phenotype among the
series of 56 women tended to be more marked when a severe mutation was
found, as suggested by the higher proportion of women with primary
amenorrhea in group B. However, women with the mildest symptoms were
observed in both genotype subgroups. Although mean basal and stimulated
17OH-P levels were significantly higher in women carrying 1 severe
mutation (group B), there was a considerable overlap between groups A
and B. This overlap was also observed for testosterone and
4-androstenedione plasma levels. Therefore, it
appears that in any given NC-CAH subject, the genotype, more
specifically the presence of a severe mutation, cannot be inferred from
the phenotype. This result together with the observation of symptomatic
and asymptomatic forms of NC-CAH in the same family confirm that
variability in phenotypic expression is conditioned by mechanisms other
than genetic heterogeneity at the CYP 21 locus. Modifying genes may be
involved as well as other factors, such as individual differences in
17OH-P adrenal and extraadrenal metabolic pathways (38),
cortisol requirement, and lastly individual peripheral sensitivity to
androgen oversecretion (10).
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Footnotes |
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Received March 24, 2000.
Revised July 18, 2000.
Accepted September 7, 2000.
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References |
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