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Keywords:

  • 21-hydroxylase deficiency;
  • CYP3A7, PXR and CAR allelic variants;
  • external genitalia virilization;
  • fetal androgen metabolism;
  • genotype/phenotype correlation

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The 21-hydroxylase deficiency (21OHD) is caused by CYP21A2 mutations resulting in severe or moderate enzymatic impairments. 21OHD females carrying similar genotypes present different degrees of external genitalia virilization, suggesting the influence of other genetic factors. Single nucleotide variants (SNVs) in the CYP3A7 gene and in its transcription factors, related to fetal 19-carbon steroid metabolism, could modulate the genital phenotype. To evaluate the influence of the 21OHD genotypes and the CYP3A7, PXR and CAR SNVs on the genital phenotype in 21OHD females. Prader scores were evaluated in 183 patients. The CYP3A7, PXR and CAR SNVs were screened and the 21OHD genotypes were classified according to their severity: severe and moderate groups. Patients with severe genotype showed higher degree of genital virilization (Prader median III, IQR III-IV) than those with moderate genotype (III, IQR II-III) (p < 0.001). However, a great overlap was observed between genotype groups. Among all the SNVs tested, only the CAR rs2307424 variant correlated with Prader scores (r2 = 0.253; p = 0.023). The CYP21A2 genotypes influence the severity of genital virilization in 21OHD females. We also suggest that the CAR variant, which results in a poor metabolizer phenotype, could account for a higher degree of external genitalia virilization.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder most commonly caused by 21-hydroxylase deficiency (21OHD). The disease is characterized by the impairment of cortisol synthesis, with or without additional impairment of aldosterone synthesis, leading to excessive ACTH stimulation of the adrenal glands, which ultimately results in adrenal hyperplasia and increased androgen synthesis [1].

CAH presents a spectrum of clinical manifestations as a consequence of different residual 21-hydroxylase activity. These manifestations range from prenatal external genitalia virilization in females, with or without neonatal salt-wasting (SW) crisis (classical forms), to late-onset hyperandrogenic symptoms. The 21OHD is the most common cause of ambiguous genitalia in patients with 46,XX disorder of sex development and the virilization of external genitalia results from increased 19-carbon steroid production by adrenal glands that, peripherally, is converted to dihydrotestosterone [1, 2].

21OHD is caused by mutations of the CYP21A2 gene, and a strong correlation between genotypes and clinical forms is well established. Mutations that cause the total and the near total inhibition of enzymatic activity are associated with the SW and simple virilizing (SV) forms of the disease, respectively. Such genotype/phenotype correlations can be extended to the hormone level; patients carrying mutations that abolish the 21OH's enzymatic activity present higher serum 17OH-progesterone (17OHP) levels than those carrying milder mutations [3-5].

However, in our cohort and in other studies, genotypes do not correlate with the severity of external genitalia virilization among CAH females. In general, quite variable degrees of external genitalia virilization are observed among females carrying similar genotypes. In these patients, Prader scores, which reflect the severity of virilization of the external genitalia, vary ranging from mild clitoral enlargement (score I) to complete fusion of labioscrotal folds with a phallic urethra (score V) [6-9]. These findings suggest an influence of other genetic factors modulating the genital phenotype. Therefore, we hypothesized that the genetic variability related to fetal androgen metabolism could account for these discrepancies.

CYP3A7, a hepatic cytochrome, is the main fetal metabolizer of 19-carbon steroids. Its catalytic activity is detected as early as 7–9 weeks of gestation, which is within the period of external genitalia differentiation [10, 11]. The enzymatic activity of CYP3A7 is highly variable among individuals, and part of this variability is attributed to genetic factors [12-14]. It has been shown that CYP3A7 gene polymorphisms are associated with changes in serum 19-carbon steroids levels during postnatal life [15]. Variable CYP3A7 activity can also be regulated by transcription factors, such as pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), both of which are members of the most important family of CYP3A transcription factors. Furthermore, both transcription factors are expressed in highly variable levels early during fetal life, which correlate with CYP3A7 expression [16]. For these two genes, PXR and CAR, some functional polymorphisms have also been described [17-19].

This study is the first to investigate whether single-nucleotide variants (SNVs) related to fetal androgen metabolism could have an influence on the degree of genital virilization in a large cohort of females with 21OHD.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Subjects

This is a retrospective study conducted using a protocol approved by the Ethical Committee of the Hospital das Clínicas, Universidade de São Paulo. Written informed consent was obtained from all patients and/or caretakers.

A total of 183 females with the classical form of 21OHD were selected from the state of São Paulo: 134 patients were followed at the Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo; 45 patients at the Faculdade de Ciências Médicas, Universidade Estadual de Campinas; and 4 at the Irmandade da Santa Casa de Misericórdia, São Paulo.

The SV form was characterized by the presence of prenatal external genitalia virilization. The SW form was characterized by external genital virilization as well as weight loss, vomiting and volume depletion starting from the neonatal period. Basal serum hormone levels are presented as the median value (IQR = interquartile range) as follows: 17OHP, 151 (89–251) ng/ml; testosterone and androstenedione levels in prepubertal patients, 133 (95–297) ng/dl and 8.5 (5–20) ng/ml, respectively. The mean (±SD) age at diagnosis was 24 (±30) days after birth for SW patients and 4.4 (±4.4) years for SV patients.

Degree of external genitalia virilization

The degree of external genitalia virilization before genitoplasty was obtained retrospectively from the medical records by the same physician from each center and were classified according to Prader scores [20]. Prader scores were well documented in 154 medical records and data from the remaining 27 patients were excluded to minimize bias in the classification of Prader III and IV before genitoplasty. Additionally, two other patients, who had been given prenatal dexamethasone treatment, were excluded as well. One patient presented Prader I, 19 with Prader II, 78 with Prader III, 46 with Prader IV and 10 patients with Prader V. A total of 154 cases were considered, 67 of which were the SV form and 87 of which were the SW form.

Hormone assays

Basal serum 17OHP levels were measured by radioimmunoassay, testosterone levels were measured by immunofluorometric assay, and androstenedione levels were measured by chemiluminescence assay. Intra-assay and inter-assay coefficients of variation ranged from 5% to 10%.

Genotype determination

Genomic DNA was extracted from peripheral blood samples using the salting-out or phenol extraction technique.

CYP21A2 gene

The point mutations of CYP21A2 were previously screened by direct sequencing of the entire gene, and large genomic rearrangements were analyzed by either multiplex ligand-dependent probe amplification (MRC Holland, Amsterdam, the Netherlands) or Southern blotting analysis [21-23]. Mutation segregation was determined by genotype analysis of the patients' parents. On the basis of the mutation analysis results, CYP21A2 genotypes were classified into groups A and B [24, 25]. In group A, both alleles carried mutations that cause total or near-total inhibition of 21-hydroxylase enzyme (≤2%), such as: CYP21A2 deletions and large gene conversions, p.W19X, IVS2-13A/C>G (I2 splice), IVS2-2A>G, 8 bp deletion (p.G110Efs), p.Ser170fs (c.511_512insA), exon 6 cluster (p.I236N, p.V237E and p.M239K), p.Leu307fs (c.923_924insT), p.G291S, p.Q318X, p.R356W, p.R408C, p.G424S and p.R444X. Group B was characterized by the p.I172N mutation (3–7% of residual enzymatic activity) with either homozygosity or compound heterozygosity including other severe mutations from group A. The CYP21A2 genotypes were determined in all 154 females with 21OHD.

CYP3A7, PXR and CAR genes

DNA samples were available for CYP3A7 sequencing in 136 patients and for PXR and CAR gene sequencing in 94 patients. Proximal promoter and coding regions of the CYP3A7 gene, intron 1 of the CYP3AP1 pseudogene and coding regions of the PXR gene were polymerase chain reaction (PCR) amplified as previously described [12, 13, 17, 26, 27]. Exons 1–9 of the CAR gene were PCR amplified with primers designed using Primer 3 program (University of Massachusetts Medical School, USA).

PCR products were purified using the ExoSAP-IT purification Kit (USB Corporation, Cleveland, OH), directly sequenced using the ABI BigDye terminator cycle sequencing kit and subjected to capillary electrophoresis on the ABI PRISM 3100 sequencer (Applied Biosystem, Inc., Foster City, CA). Sequences were analyzed for the presence of variants using the Sequencher TM 4.9 software (Genes Codes Corporation, Ann Arbor, MI). All genotypes resulting in SNVs were confirmed by two independent PCRs followed by sequencing. The access numbers were as follows: CYP3A7-ENSG00000160870, PXR-ENSG00000144852, CAR-ENSG00000143257.

Statistical analysis

SNVs in Hardy–Weinberg equilibrium and with allelic frequency ≥3% were selected for the correlation analysis along with Prader scores. Six SNVs of the CYP3A7 gene, one SNV of the PXR gene and three SNVs of the CAR gene were classified based on the Dominant Genetic Model. To test the role of the 21OHD genotypes and each of the SNVs (as the independent variables), multiple linear regression analyses were performed, using the Prader score as the dependent variable.

Comparisons of numerical variables among groups were performed using Mann–Whitney or Kruskal–Wallis test when appropriate.

Statistical analysis was performed using the SigmaPlot 10.0 software (Sigmaplot Users' Manual, 1993, Jandel Scientific, San Rafael, CA). Statistical significance was established at the level of p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Frequency of CYP21A2 mutations

The two most frequent CYP21A2 point mutations were IVS2–13A/C>G (I2 splice) and p.I172N in 37.6% and 17.8% of all the alleles screened, respectively. Moreover, other severe mutations that lead to the total impairment of enzymatic activity were identified in 28.9% of the alleles screened. Large gene rearrangements, such as CYP21A2 deletions and large conversions, were identified in 11% of all the alleles screened, whereas novel mutations were identified in 2.4% [28-31].

Correlation between 21OHD genotypes, clinical forms, basal hormone levels and Prader scores

Of the 21OHD females who carried a CYP21A2 genotype, 66% were included in group A, 33% corresponded to group B genotype, and 1% carried new mutations, for which the effect on the 21OH's enzymatic activity is unknown. Within group A, 83% of the patients presented the SW form and 17% presented the SV form. Within group B, the percentages of SW and SV patients were 6.5% and 93.5, respectively. CYP21A2 genotypes from groups A and B showed significant association with both the SW and SV forms (p < 0.001).

The medians (IQR) of the basal 17OHP levels of patients in groups A and B were 160 (89–264) ng/ml and 125 (85–208) ng/ml, respectively (p = 0.174). The medians (IQR) of basal androstenedione levels for prepubertal patients in groups A and B were 13.5 (6–30) ng/ml and 8.0 (4–10) ng/ml, respectively (p = 0.003), and basal testosterone levels were 135 (109–345) ng/dl and 92.4 (63–170) ng/dl, respectively (p = 0.013).

The most frequent CYP21A2 mutations and the corresponding Prader scores are shown in Table 1.

Table 1. Degrees of external genitalia virilization (Prader scores) in CAH females bearing the most frequent CYP21A2 genotypes
CYP21A2 mutationNumber of patients (%)Prader I + II (n)Prader III (n)Prader IV (n)Prader V (n)
  1. CAH, congenital adrenal hyperplasia; LR, large gene rearrangements (CYP21A2 deletion and large gene conversion); null, mutations that abolish the enzymatic activity.

LR/LR3 (2)0111
LR/null7 (4)0430
I2 splice/I2 splice33 (18)119112
I2 splice/null47 (25)419204
I2 splice/p.I172N15 (8)21021
p.I172N/null21 (11)71031
p.I172N/p.I172N11 (6)6500

The CAH patients belonging to group A (severe 21OHD genotype) had a significantly higher degree of external genitalia virilization than those belonging to group B (moderate 21OHD genotype) (p < 0.001). However, despite this correlation, the severity of external genitalia virilization had a significant overlap between both groups (Fig. 1), the median Prader score was III for group A (III–IV) and also III (II–III) for group B. For group A patients, there was no difference in the median Prader score between those who were carrying null mutations in both alleles and those who had compound heterozygosis with the I2 splice mutation.

image

Figure 1. Frequencies (%) of Prader scores in each CYP21A2 genotype groups, A and B. A: genotypes predicting total or near total impairment (<2%) of 21-hydroxylase activity; B: genotypes predicting severe impairment of activity (3–7%).

Download figure to PowerPoint

Interestingly, there were no differences in median basal testosterone levels among the Prader score (P) groups, P I and P II, 138 (97–254) ng/dl; P III, 142 (88–351) ng/dl; P IV, 125 (95–229) ng/dl; and P V 129 (108–135) ng/dl (p = 0.921).

Distribution of CYP3A7, PXR and CAR allelic variants

All SNVs identified in the screening of the CYP3A7, PXR and CAR genes were previously described, and their frequencies are depicted in Table 2.

Table 2. Frequency of SNVs in coding and non-coding regions of the CYP3A7, PXR and CAR genes in a Caucasian population and in the CAH cohort under study
SNVGeneNCBIChange of protein or nucleotideCAHCaucasianaEffect on CYP3A activity
  1. =, no change in CYP3A activity; ↑, increase the CYP3A activity; CAH, congenital adrenal hyperplasia; NA, not available; SNV, single-nucleotide variant.

  2. a

    Data obtained from Burk et al. (26), Rodriguez-Antona et al. (12,13), Hustert et al. (17), Zhang et al. (35) and HapMap project http://hapmap.org.

1CYP3A7rs3823647CYP3A7*1B0.41NA
2CYP3A7rs11568825CYP3A7*1C3.33NA
3CYP3A7rs55798860CYP3A7*1D0.41NA
4CYP3A7rs28451617CYP3A7*1E2.24NA
5CYP3A7rs2257401CYP3A7*2258NA
6CYP3A7rs45515892IVS2-159T>C313NA
7CYP3A7rs45446903IVS4-52T>A3.33NA
8CYP3A7rs41279866IVS4-14G>A6.65NA
9CYP3A7rs45600842IVS6+73T>C6.626NA
10PXRrs12721613P27S10=
11PXRrs12721607G36R23=
12PXRrs35761343A370T0.50=
13PXRrs12721616IVS2-29C>T26NA
14PXRrs2276707IVS6-17C>T1915
15PXRrs3732358*UTR15G>A210NA
16CARrs2307424P180P3030NA
17CARrs2502815IVS3-99C>T3023NA
18CARrs2307420IVS5+41A>G1.60NA
19CARrs2307418IVS8+17A>C1213NA
20CARrs35796551*UTRG>T10NA

CYP3A7 gene: in the promoter region, four different alleles were identified: SNV 1, CYP3A7*1B (n = 1 allele); SNV 2, CYP3A7*1C (n = 9 alleles); SNV 3, CYP3A7*1D (n = 1 allele); and SNV 4, CYP3A7*1E (n = 6 alleles), all of which were heterozygous.

In the coding regions, the SNV 5, CYP3A7*2 allele (n = 67 alleles) was identified in linkage disequilibrium with the CYP3A7P1 (CYP3A7_39256-->A) pseudogene allele. On the other hand, SNV 6 (n = 8 alleles), SNV 7 (n = 9 alleles), SNV 8 (n = 18 alleles) and SNV 9 (n = 18 alleles) were identified in the intronic flanking regions (Table 2).

In PXR gene, three missense substitutions were identified: SNV 10 (n = 2 alleles), SNV 11 (n = 4 alleles) and SNV 12 (n = 1 allele). In addition, three other mutations were detected in the intronic regions and the 3′UTR: SNV 13 (n = 4 alleles), SNV 14 (n = 36 alleles) and SNV 15 (n = 4 alleles) (Table 2).

In CAR gene, within the coding regions, the silent SNV 16 was observed in 57 of the alleles, whereas in the intronic regions and the 3′UTR, the following mutations were observed: SNV 17 (n = 57 alleles), SNV 18 (n = 3 alleles), SNV 19 (n = 23 alleles) and SNV 20 (n = 2 alleles) (Table 2).

Correlation between 21OHD genotype, CYP3A7, PXR and CAR allelic variants and Prader score

SNVs 2, 5, 6, 7, 8 and 9 (of CYP3A7 gene), SNV 14 (of PXR gene) and SNVs 17 and 19 (of CAR gene) did not correlate with Prader scores (I–V). Only SNV 16 (rs2307424) and the 21OHD genotype were significantly associated with the degree of external genitalia virilization in a multivariate analysis (r2 = 0.253; p = 0.023), explaining 25% phenotypic genital variability. None of the other SNVs analyzed presented a statistical significance result for correlation (p > 0.005).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

It is well-known that there is a strong correlation between the impairment of enzymatic activity resulting from CYP21A2 mutations and the clinical forms of CAH as well as the basal levels of 17OHP [4, 5, 32]. In the Brazilian patients included in this study, except for the gene founder effect of some mutations [28, 29], the mutation distribution was similar to that of other ethnic groups and we also observed a strong correlation between genotype and phenotype. However, three studies [6, 8, 9] as well as our previous report comprising 85 CAH females from a single center [33] have indicated some controversies in the correlation between genotype and degree of external genitalia virilization.

In the analysis of a recent multicentric study comprising 158 CAH females, a correlation was reported between the severity of 21OHD genotype and the degree of external genitalia virilization [25]. In order to verify these abovementioned discordant results, we increased the size of our series with the completion of a multicentric study involving 154 CAH females. In agreement with the latter European study [25], we also identified a correlation between 21OHD genotype and degree of external genital virilization. Although, our first paper presented a considerable number of patients (n = 85), we cannot rule out the sample size effect [33]. Despite the correlation between 21OHD genotype and genital phenotype in this study, there was great overlap between the degree of external genitalia virilization and 21OHD genotypes, suggesting the involvement of other factors in modulating the genital phenotype.

In the literature, the association of serum androgen levels has also been evaluated with the genital phenotype. In an Indonesian study, that included CAH females with exceptionally late diagnosis, a positive correlation between androgen levels and enlargement of the clitoris during postnatal life was showed [9]. Moreover, Prader scores also varied substantially among different genotypes in that study. The analysis of androgen levels in our cohort indicated a positive correlation between the severity of 21OHD genotypes and either postnatal testosterone or androstenedione levels, but these latter two were not correlated with Prader scores. We suppose that this lack of correlation could be explained by an alternative fetal pathway in the androgen synthesis, which in conditions where 17OHP accumulates, the DHT synthesis bypasses the conventional androstenedione and testosterone precursors and, instead, makes use of androstenediol as a substrate [2, 34].

Although CAH is a monogenic disorder, the interindividual variability of hyperandrogenic manifestations among patients carrying similar 21OHD genotypes could be modulated by a polygenic tract. Therefore, allelic variants in genes related to fetal androgen metabolism could play an important role in the genital phenotype.

During fetal development, 19-carbon steroids are mainly metabolized by CYP3A7, and 90% of the interindividual variability in the enzymatic activity is genetically determined. Several allelic variants have been described for the CYP3A7 gene. For example, the CYP3A7*1C allele has a stretch of 60 bp substituted by the CYP3A4 promoter. This gene variant leads to its delayed postnatal expression, and it correlates with lower serum DHEAS levels in both healthy female carriers as well as polycystic ovary syndrome patients as compared with wild-type carriers [14, 15, 26]. Another frequent variant, the CYP3A7*2 allele, is associated with higher DHEA hydroxylation [12]. Consequently, we hypothesized that these substitutions could correlate with a less severe degree of genital virilization in CAH. The CYP3A7*1C variant was found in only nine CAH patients, whose Prader scores ranged from II to V. Similarly, the CYP3A7*2 allele, although presenting a higher frequency in our study, was also identified in patients with a wide range of Prader scores. These data suggest that both these CYP3A7 alleles, at least in the heterozygous state, did not influence the CAH genital phenotype.

Recently, it was shown that the expression of CYP3A7 is mainly regulated by the nuclear receptors, PXR and CAR, and that variants of these receptors dramatically impact CYP3A family expression [17, 18, 35]. Considering the functional variants previously described, only SNV 14 was identified in a considerable number of CAH patients without any significant influence on the external genitalia virilization.

The literature has been giving emphasis to SNVs that do not change protein sequence, because they also can modulate an individual's phenotype [36]. We analyzed in this study the influence of 10 such SNVs within the CYP3A7, PXR and CAR genes on Prader scores, and we found that only SNV 16 presented a significant effect on phenotype. Thus, SNV 16 and the 21OH genotype, taken together, explained 25% of all observed phenotypic variability. We speculate that SNV 16 contributes to a poor metabolizer phenotype of 19-carbon steroids, as this variant was found more frequently among highly virilized CAH females. In the literature, there are no functional studies evaluating the effect of this SNV on CAR or CYP3A transcriptional activity. In agreement with our hypothesis that SNV 16 could confer a poor liver metabolizer phenotype, a recent study observed an association of this SNV with early treatment discontinuation in patients presenting with HIV/AIDS due to a higher frequency of side effects [37].

In conclusion, we observed in this multicentric study that the impairment of enzymatic activity predicted by 21OHD genotypes influences the degree of external genitalia virilization in CAH females. In addition, we observed that the CAR variant SNV 16, which is related to fetal 19-carbon steroids metabolism, had a modulatory effect on the genital phenotype. We speculate that, although CAH is a monogenic disorder, the hyperandrogenic phenotype could be modulated by a polygenic pathway, involving either genes related to the fetal androgen synthesis and/or metabolism.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study was supported by FAPESP # 2008/57616-5, L. C. K. by FAPESP # 2008/55546-0, T. A. B and B. B. M by CNPq # 305117/2009-2 and # 305743/2011-2, respectively.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Speiser PW, White PC. Congenital adrenal hyperplasia. N Engl J Med 2003: 349: 776788.
  • 2
    Auchus RJ, Miller WL. Congenital adrenal hyperplasia – more dogma bites the dust. J Clin Endocrinol Metab 2012: 97: 772775.
  • 3
    Bachega TA, Billerbeck AE, Madureira G et al. Molecular genotyping in Brazilian patients with the classical and nonclassical forms of 21-hydroxylase deficiency. J Clin Endocrinol Metab 1998: 83: 44164419.
  • 4
    Wilson RC, Nimkarn S, Dumic M et al. Ethnic-specific distribution of mutations in 716 patients with congenital adrenal hyperplasia owing to 21-hydroxylase deficiency. Mol Genet Metab 2007: 90: 414421.
  • 5
    Finkielstain GP, Chen W, Mehta SP et al. Comprehensive genetic analysis of 182 unrelated families with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Endocrinol Metab 2011: 96: E161E172.
  • 6
    Giwercman YL, Nordenskjöld A, Ritzén EM et al. An androgen receptor gene mutation (E653K) in a family with congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency as well as in partial androgen insensitivity. J Clin Endocrinol Metab 2002: 87: 26232628.
  • 7
    Bachega TA, Billerbeck AE, Parente EB et al. Multicentric study of Brazilian patients with 21-hydroxylase deficiency: a genotype-phenotype correlation. Arq Bras Endocrinol Metabol 2004: 48: 697704.
  • 8
    Sugiyama Y, Mizuno H, Hayashi Y et al. Severity of virilization of external genitalia in Japanese patients with salt-wasting 21-hydroxylase deficiency. Tohoku J Exp Med 2008: 215: 341348.
  • 9
    Goossens K, Juniarto AZ, Timmerman MA et al. Lack of correlation between phenotype and genotype in untreated 21-hydroxylase-deficient Indonesian patients. Clin Endocrinol (Oxf) 2009: 71: 628635.
  • 10
    Yang HY, Lee QP, Rettie AE, Juchau MR. Functional cytochrome P4503A isoforms in human embryonic tissues: expression during organogenesis. Mol Pharmacol 1994: 46: 922928.
  • 11
    Stevens JC, Hines RN, Gu C et al. Developmental expression of the major human hepatic CYP3A enzymes. J Pharmacol Exp Ther 2003: 307: 573582.
  • 12
    Rodriguez-Antona C, Jande M, Rane A, Ingelman-Sundberg M. Identification and phenotype characterization of two CYP3A haplotypes causing different enzymatic capacity in fetal livers. Clin Pharmacol Ther 2005: 77: 259270.
  • 13
    Rodriguez-Antona C, Axelson M, Otter C, Rane A, Ingelman-Sundberg M. A novel polymorphic cytochrome P450 formed by splicing of CYP3A7 and the pseudogene CYP3AP1. J Biol Chem 2005: 280: 2832428331.
  • 14
    Smit P, van Schaik RH, van der Werf M et al. A common polymorphism in the CYP3A7 gene is associated with a nearly 50% reduction in serum dehydroepiandrosterone sulfate levels. J Clin Endocrinol Metab 2005: 90: 53135316.
  • 15
    Goodarzi MO, Xu N, Azziz R. Association of CYP3A7*1C and serum dehydroepiandrosterone sulfate levels in women with polycystic ovary syndrome. J Clin Endocrinol Metab 2008: 93: 29092912.
  • 16
    Vyhlidal CA, Gaedigk R, Leeder JS. Nuclear receptor expression in fetal and pediatric liver: correlation with CYP3A expression. Drug Metab Dispos 2006: 34: 131137.
  • 17
    Hustert E, Zibat A, Presecan-Siedel E et al. Natural protein variants of pregnane X receptor with altered transactivation activity toward CYP3A4. Drug Metab Dispos 2001: 29: 14541459.
  • 18
    Ikeda S, Kurose K, Jinno H et al. Functional analysis of four naturally occurring variants of human constitutive androstane receptor. Mol Genet Metab 2005: 86: 314319.
  • 19
    Wang XD, Li JL, Su QB et al. Impact of the haplotypes of the human pregnane X receptor gene on the basal and St John's wort-induced activity of cytochrome P450 3A4 enzyme. Br J Clin Pharmacol 2009: 67: 255261.
  • 20
    Prader A. Genital findings in the female pseudo-hermaphroditism of the congenital adrenogenital syndrome; morphology, frequency, development and heredity of the different genital forms. Helv Paediatr Acta 1954: 9: 231248.
  • 21
    Bachega TA, Billerbeck AE, Madureira G et al. Low frequency of CYP2B deletions in Brazilian patients with congenital adrenal hyperplasia due to 21-hydroxylas deficiency. Hum Hered 1999: 49: 914.
  • 22
    Paulino LC, Araujo M, Guerra G Jr, Marini SH, De Mello MP. Mutation distribution and CYP21/C4 locus variability in Brazilian families with the classical form of the 21-hydroxylase deficiency. Acta Paediatr 1999: 88: 275283.
  • 23
    Costa-Barbosa FA, Tonetto-Fernandes VF, Carvalho VM et al. Superior discriminating value of ACTH-stimulated serum 21-deoxycortisol in identifying heterozygote carriers for 21-hydroxylase deficiency. Clin Endocrinol (Oxf) 2010: 73: 700706.
  • 24
    Speiser PW, Dupont J, Zhu D et al. Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Invest 1992: 90: 584595.
  • 25
    Welzel M, Schwarz HP, Hedderich J et al. No correlation between androgen receptor CAG and GGN repeat length and the degree of genital virilization in females with 21-hydroxylase deficiency. J Clin Endocrinol Metab 2010: 95: 24432450.
  • 26
    Burk O, Tegude H, Koch I et al. Molecular mechanisms of polymorphic CYP3A7 expression in adult human liver and intestine. J Biol Chem 2002: 277: 2428024288.
  • 27
    Du J, Shi Y, Zhang A et al. Screening for SNPs and haplotypes in the CYP3A7 gene in Chinese populations. Pharmacogenomics 2007: 8: 559566.
  • 28
    Billerbeck AE, Bachega TA, Frazatto ET et al. A novel missense mutation, GLY424SER, in Brazilian patients with 21-hydroxylase deficiency. J Clin Endocrinol Metab 1999: 84: 28702872.
  • 29
    Billerbeck AE, Mendonca BB, Pinto EM, Madureira G, Arnhold IJ, Bachega TA. Three novel mutations in CYP21 gene in Brazilian patients with the classical form of 21-hydroxylase deficiency due to a founder effect. J Clin Endocrinol Metab 2002: 87: 43144317.
  • 30
    Lau IF, Soardi FC, Lemos-Marini SH, Guerra G Jr, Jr BMT, De Mello MP. H28 + C insertion in the CYP21 gene: a novel frameshift mutation in a Brazilian patient with the classical form of 21-hydroxylase deficiency. J Clin Endocrinol Metab 2001: 86: 58775880.
  • 31
    Coeli FB, Soardi FC, Bernardi RD et al. Novel deletion alleles carrying CYP21A1P/A2 chimeric genes in Brazilian patients with 21-hydroxylase deficiency. BMC Med Genet 2010: 11: 104.
  • 32
    Stikkelbroeck NM, Hoefsloot LH, de Wijs IJ, Otten BJ, Hermus AR, Sistermans EA. CYP21 gene mutation analysis in 198 patients with 21-hydroxylase deficiency in The Netherlands: six novel mutations and a specific cluster of four mutations. J Clin Endocrinol Metab 2003: 88: 38523859.
  • 33
    Rocha RO, Billerbeck AE, Pinto EM et al. The degree of external genitalia virilization in girls with 21-hydroxylase deficiency appears to be influenced by the CAG repeats in the androgen receptor gene. Clin Endocrinol (Oxf) 2008: 68: 226232.
  • 34
    Kamrath C, Hochberg Z, Hartmann MF, Remer T, Wudy SA. Increased activation of the alternative “backdoor” pathway in patients with 21-hydroxylase deficiency: evidence from urinary steroid hormone analysis. J Clin Endocrinol Metab 2012: 97: 367375.
  • 35
    Zhang B, Xie W, Krasowski MD. PXR: a xenobiotic receptor of diverse function implicated in pharmacogenetics. Pharmacogenomics 2008: 9: 16951709(Review).
  • 36
    Goode DL, Cooper GM, Schmutz J et al. Evolutionary constraint facilitates interpretation of genetic variation in resequenced human genomes. Genome Res 2010: 20: 301310.
  • 37
    Wyen C, Hendra H, Siccardi M et al. German Competence Network for HIV/AIDS Coordinators. Cytochrome P450 2B6 (CYP2B6) and constitutive androstane receptor (CAR) polymorphisms are associated with early discontinuation of efavirenz-containing regimens. J Antimicrob Chemother 2011: 66: 20922098.