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Unidade de Endocrinologia do Desenvolvimento e Laboratório de Hormônios e Genética Molecular, LIM/42, Disciplina de Endocrinologia, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo 05403–900, Brazil
Address all correspondence and requests for reprints to: Berenice B. Mendonca, M.D., Hospital das Clínicas, Faculdade de Medicina da Universidade de Saõ Paulo, Disciplina de Endocrinologia, Caixa Postal 3671, São Paulo, 01060-970, Brasil. E-mail: beremen{at}usp.br
Abstract
The currently used cutoff level for ACTH-stimulated 17- hydroxyprogesterone (17OHP) for the diagnosis of the nonclassical (NC) form of 21-hydroxylase deficiency (21OHD), established before molecular studies, is based on the mean + 2 SD of 17OHP levels of obligate heterozygotes. However, carriers of CYP21 mutations present variable ACTH-stimulated 17OHP levels, ranging from normal values up to 30 nmol/liter. The aim of this study was to determine whether ACTH-stimulated 17OHP levels in obligate carriers for 21OHD would be correlated with the impairment of the enzyme activity caused by these mutations, which would affect the 17OHP cutoff level for the diagnosis of the NC form. Fifty-nine parents of patients with the classical and NC forms of 21OHD had their DNA screened for the mutations found in the index case and were divided into three mutation groups according to the impairment of enzyme activity (A = 0%, B = 3%, and C > 20%). All parents carried mutations in one allele (29 of group A, 9 of group B, and 21 of group C). Blood samples were collected at baseline condition and 60 min after ACTH (250 µg iv) to measure 17OHP levels. The levels among groups A, B, and C were compared using the Kruskall Wallis test. ACTH-stimulated 17OHP levels identified 39% of the carriers (9 in group A, 2 in group B and 12 in group C). The mean ± SD basal 17OHP levels in groups A, B, and C were: 2.94 ± 1.89, 1.77 ± 0.81 and 3.90 ± 2.43 nmol/liter, respectively (P > 0.05) and for ACTH-stimulated levels were 12.6 ± 7.2, 13.2 ± 12.9 and 16.8 ± 7.8 nmol/liter, respectively (P > 0.05). Two carriers presented ACTH-stimulated 17OHP levels between 30 and 45 nmol/liter and their entire CYP21 sequencing revealed only one mutation in heterozygous state indicating that the current cutoff level might overestimate the diagnosis of the NC form. We conclude that the variable ACTH-stimulated 17-OHP levels in carriers are not related to CYP21 gene mutations with different impairment of enzyme activity.
CONGENITAL ADRENAL HYPERPLASIA (CAH) due to 21-hydroxylase (21OH) deficiency is the most frequent inborn metabolism error and accounts for 90–95% of CAH cases (1, 2). Molecular defects in the CYP21 gene result in different phenotypes: a severe form (classical) with prenatal virilization of external genitalia in the female fetus and postnatal virilization in both sexes, with or without salt losing, and a milder form [nonclassical (NC)]. In the latter, patients remain asymptomatic or develop symptoms during childhood or at puberty (2). These different clinical forms are caused by mutations with different degrees of enzyme activity impairment.
Mutations that account for 21OH deficiency can be CYP21 deletions, large gene conversions, or point mutations, with the latter present in two-thirds of affected alleles. Speiser et al. (3) divided the patients into three genotype groups, according to the impairment of enzyme activity: group A < 2%, B = 3%, and C > 20% of enzyme activity. They observed that the genotypes A, B, and C correlated with the salt wasting, simple virilizing and NC forms and with decreasing levels of basal and ACTH-stimulated 17-hydroxyprogesterone (17OHP).
Because of the existence of several mutations in the CYP21 gene, most patients are compound heterozygotes, and the clinical form correlates to the allele with higher enzymatic activity (3, 4, 5, 6). In the NC form, the genotype is a combination of mutations that cause moderate impairment (C) in one allele and total (A), severe (B) or moderate impairment of enzymatic activity in the other allele. It was observed that patients with the NC form and genotype A/C had higher ACTH-stimulated 17OHP levels and presented symptoms of androgen excess earlier than patients with genotype C/C, suggesting an influence of the allele A on 17OHP levels and probably on the phenotype (7).
Carriers for 21OH deficiency present variable peak responses of 17OHP levels in the ACTH test, ranging from normal values (<13 nmol/liter) to 30 nmol/liter. This upper value is considered by many investigators as the lower limit for the diagnosis of the NC form (8, 9, 10). However, this value was established before the identification of mutations in the CYP21 gene.
The aim of our study was to determine whether the variable responses of 17OHP levels after ACTH test in genotyped carriers for 21OH deficiency was dependent on the impairment of enzyme activity caused by the mutation (alleles A, B, or C) and consequently, in an attempt to establish suitable cutoffs for the diagnosis of heterozygotes for the classical and NC forms.
Patients and Methods
This study was approved by the Ethics Committee, and all subjects gave their informed consent.
We studied 59 parents (29 males and 30 females) of patients with different clinical forms of 21OH deficiency. All women were premenopausal and were not receiving any steroid medication including oral contraceptives, for at least 3 months before the study.
Molecular studies
DNA samples were obtained from peripheral blood leukocytes by standard procedures. The parents had their DNA screened for the mutations found in the index case through the following methodologies: Southern blotting studies to determine large rearrangements (11); allele-specific PCR (12, 13) for the determination of point mutations (P30L, I2 splice, Del 8 nt, I172N, Cluster, V281L, Q318x, R356W, Ins T, P453S); and BanI restriction of PCR products (14) for the G424S mutation found in Brazilian patients. Direct sequencing of the entire CYP21 gene (exons, introns, and 250 bp of the promoter region) was performed in two cases with ACTH-stimulated 17OHP levels higher than 30 nmol/liter, to rule out the presence of CYP21 mutations in both alleles.
The parents were classified into three different genotype groups (A, B, and C) according to the previously described impairment of enzyme activity (3, 15, 16, 17).
An acute ACTH stimulation test was performed between 0700 and 0900 h, in the follicular phase of the menstrual cycle in females (d 2–8 of the cycle). An iv catheter was placed in the forearm, and the subject was allowed to rest for 30 min. Blood samples were obtained before and 60 min after 250 µg of ACTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) iv injection to measure 17OHP and cortisol levels. 17OHP was measured by tritiated RIA after hexane/ethylacetate extraction without chromatography after demonstration that the antibody was adequately specific, by Abraham’s method (18), slightly modificated (19). The intra and interassay coefficients of variation were respectively less than 10% and 18%. All samples were measured in two assays. Cortisol was measured by precoated iodide RIA, a 125I competitive protein binding assay from INCSTAR Corp. (Stillwater, MN).
Statistical analysis
We compared basal and ACTH-stimulated 17OHP and cortisol values among groups A, B, and C. The differences were evaluated using the Kruskal Wallis test.
Results
Mutations were detected in one allele in all parents (Table 1
). The CYP21 deletions and large gene conversions were found in 11.9% of the subjects. The most frequent point mutations were V281L (32.2%), followed by I2 splice (18.6%), Q318x (11.9%), I172N (10.1%), R356W and G424S (5.1% each), Ins T (3.4%), P30L, Del 8 nt, Cluster and P453S (1.7% each). Twenty-nine parents belonged to group A, 9 to group B, and 21 to group C.
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. All individuals presented normal ACTH-stimulated cortisol levels (>497 nmol/liter). Nine subjects from group A, two from group B, and twelve from group C presented elevated response of 17OHP after ACTH test (>13 nmol/liter). ACTH-stimulated 17OHP levels failed to discriminate 61% of the obligate heterozygotes, who had a response within the normal range. The lowest value of ACTH-stimulated 17OHP in the obligate heterozygotes was 2.1 nmol/liter, and the highest value was 45 nmol/liter. Two carriers (cases 9 and 31) who had the highest ACTH-stimulated 17OHP levels, 33 and 45 nmol/liter, respectively, had the entire CYP21 gene sequenced. Only one mutation in heterozygous state was found in each subject, one from group A and the other from group B.
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, Fig. 1). We also analyzed the effect of the most representative mutations from groups A (large rearrangements and I2 splice), B (I172N), and C (V281L) on basal and ACTH-stimulated 17OHP levels and found no statistically difference either (P > 0.05). Elevated ACTH-stimulated 17OHP levels were found in none of 7 carriers of large mutations, in 5 of 11 carriers of the I2 splice, in one of 6 carriers of the I172N, and in 11 of 19 carriers of V281L mutation.
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). Discussion
The currently used cutoff level for ACTH-stimulated 17OHP for the diagnosis of NC form of 21OH deficiency is >30 nmol/liter. This value corresponds to the upper limit of obligate heterozygotes for 21OH deficiency in the normogram, established before molecular studies became available, by New et al. (8). However, in this present study, there were two fully genotyped carriers (one male and one female) with ACTH-stimulated 17OHP values above the current cutoff levels (33 and 45 nmol/liter). Our findings corroborate the considerable debate regarding the minimum ACTH-stimulated 17OHP value used to diagnose the NC form of 21OH deficiency. Some investigators have proposed that the diagnosis of the NC form of 21OH deficiency should be based on ACTH-stimulated 17OHP levels >45 or >60 nmol/liter, corresponding to the lowest 17OHP levels found in some patients with the NC form (8, 10, 20).
The NC form has a higher frequency of unidentified alleles compared with the classical form in several population studies (4, 6, 21, 22, 23), suggesting incorrect diagnosis of this form. Considered together, the presence of carriers with ACTH-stimulated 17OHP levels >30 nmol/liter and the higher number of unidentified alleles in the NC form, indicate that the current cutoff for diagnosis of NC 21OH deficiency needs to be reevaluated.
Heterozygotes for CYP21 mutations have been found in a high frequency in the general population, and many attempts have been made to detect heterozygosity for 21OH deficiency using steroid concentration analysis. ACTH-stimulated 17OHP level is the most frequently used analysis, but there is a considerable overlap between the heterozygotes and the control population, making the prediction of carriers difficult (24, 25, 26, 27). In our sample, ACTH-stimulated 17OHP levels identified only 39% of the genotyped carriers. Therefore, a normal 17OHP response to ACTH-stimulation (<13 nmol/liter) does not exclude heterozygosity for 21OH deficiency, making molecular studies necessary to discriminate between normal and heterozygote subjects, which is important in genetic counseling.
Mutations in the CYP21 gene impair enzyme activity at different degrees, which are responsible for the different clinical forms. In a previous study, we observed that patients with the NC A/C genotype presented higher ACTH-stimulated 17OHP levels and earlier symptom onset than those with the NC C/C genotype (7). These data suggest an influence of the allele carrying the severe mutation (allele A) on the phenotype. Therefore, we hypothesized whether the variable ACTH-stimulated 17OHP levels in the heterozygotes correlated to the severity of enzyme activity impairment determined by the mutation. However, our data did not confirm this hypothesis because basal and ACTH-stimulated 17OHP levels in carriers of alleles A or B were not different from those carriers of allele C. Similarly, Witchel and Lee (26), studying 25 carriers of allele A found that 52% of them presented elevated ACTH-stimulated 17OHP levels.
There have been only two studies in the literature analyzing 17OHP levels according to the mutation group in hyperandrogenic women (27, 28). The joint analysis of these two casuistics, including 16 hyperandrogenic women who were heterozygotes for CYP21 mutations, disclosed elevated ACTH-stimulated 17OHP levels in 28% (2/7) of the carriers with the allele A and in 89% (8/9) of the carriers with the V281L mutation. They found higher ACTH-stimulated 17OHP levels in those bearing mutations from group C. The latter authors (28) suggested a dominant negative effect of the V281L mutation to explain the increased 17OHP levels conferred by this specific mutation with moderate impairment of enzyme activity. However, the number of cases does not permit statistically significant results. We analyzed the effect of the V281L mutation and of the most frequent mutations from each group (large rearrangements, I2 splice and I172N) in our series and did not find statistically significant differences in basal and ACTH-stimulated 17OHP levels among these specific mutations.
We conclude that the degree of enzyme activity conferred by the mutant allele is not the only factor responsible for the variable responses of ACTH-stimulated 17OHP in heterozygotes for 21-hydroxylase deficiency. Therefore, other factors besides the degree of enzyme impairment activity must influence 17OHP levels, such as individual variations on metabolic clearance. The presence of one complete genotyped carrier with ACTH-stimulated 17OHP levels of 45 nmol/liter, indicates that the CYP21 genotyping is advisable for the differential diagnosis between heterozygous state and NC form in patients with ACTH-stimulated 17OHP levels between 10 and 45 nmol/liter.
Acknowledgments
We thank the staff of Laboratório de Hormônios e Genética Molecular LIM/42 for technical assistance and Sonia Strong for English revision.
Footnotes
This work was partially supported by FAPESP Grant 95/8325-6, and T.A.S.S.B. was supported by Fundacaõ de Amparo a Pesquisa do Estado de Saõ Paulo, Grant 98/00243-9.
Abbreviations: CAH, Congenital adrenal hyperplasia; 17OHP, 17-hydroxyprogesterone; NC, nonclassical; 21OHD, 21-hydroxylase deficiency.
Received March 15, 2001.
Accepted November 5, 2001.
References
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