Inhibition of CYP21A2 Enzyme Activity Caused by Novel Missense Mutations Identified in Brazilian and Scandinavian Patients

Abstract

Background: Most patients with 21-hydroxylase deficiency carry CYP21A1P-derived mutations, but an increasing number of novel and rare mutations have been reported in disease-causing alleles.

Objective: Functional effects of three novel (p.G56R, p.L107R, p.L142P) and one recurrent (p.R408C) CYP21A2 mutations were investigated. The degree of enzyme impairment caused by p.H62L alone or combined to p.P453S was also analyzed.

Design: The study included 10 Brazilian and two Scandinavian patients. To determine the deleterious role of each mutant protein, in vitro assays were performed in transiently transfected COS-1 cells. For a correct genotype-phenotype correlation, the enzymatic activities were evaluated toward the two natural substrates, 17-hydroxyprogesterone and progesterone.

Results: Low levels of residual activities obtained for p.G56R, p.L107R, p.L142P, and p.R408C mutants classified them as classical congenital adrenal hyperplasia mutations, whereas the p.H62L showed an activity within the range of nonclassical mutations. Apparent kinetic constants for p.H62L confirmed the nonclassical classification as the substrate binding capacity was within the same magnitude for mutant and normal enzymes. A synergistic effect was observed for the allele bearing the p.H62L+p.P453S combination because it caused a significant reduction in the enzymatic activity.

Conclusions: We describe the functional analysis of five rare missense mutations identified in Brazilian and Scandinavian patients. The p.G56R, p.L107R, and p.L142P are reported for the first time. Most probably these novel mutations are closer to null than the p.I172N, but for the p.G56R, that might not be the case, and the p.H62L is definitely a nonclassical mutation.

More than 90% of cases of congenital adrenal hyperplasia (CAH) are due to 21-hydroxylase deficiency (21OHD), which results from mutations in the 21-hydroxylase gene (CYP21A2) (1). The disorder may be classified into clinical forms that vary from a mild/late-onset nonclassical (NC) to a severe/early-onset classical form that can manifest with or without salt wasting (SW or SV), depending on whether the enzymatic deficit affects both the glucocorticoid and mineralocorticoid pathways or whether the latter is preserved. Additionally, due to androgen overproduction, females are born with virilized external genitalia and males develop precocious puberty (1, 2).

The CYP21A2 gene maps to the short arm of chromosome 6 (6p21.3) (3). CYP21A2 and the CYP21A1P, a nonfunctional pseudogene, are genomically organized in tandem to the 3′-end of C4B and C4A, respectively (4). The majority of disease-causing mutations in CYP21A2 alleles are CYP21A1P-derived sequences transferred to the active gene by deletions or macro- or microconversions (5). In addition, an increasing number of novel and rare mutations have been reported (www.imm.ki.se/CYPalleles/cyp21.htm).

In general, there is a good genotype-phenotype correlation. Furthermore, there is also a good correlation between in vitro studies with mutated enzymes and in vivo disease severity. Therefore, in vitro analysis is proposed as a good complement to evaluate the residual enzymatic activity caused by novel mutations to establish a genotype-phenotype correlation and enable improved genetic counseling (6, 7).

In this report, we describe the functional analyses of three novel (p.G56R, p.L107R, p.L142P) and one recurrent (p.R408C) mutation (8) identified in Brazilian patients. The rare p.H62L was also analyzed because it was found in alleles bearing two different combinations, p.P34L+p.H62L as part of a chimeric gene in an allele carrying a 30-kb deletion in a Brazilian patient and p.H62L+p.P453S found in two Scandinavian patients.

Patients and Methods

Patients

This study was approved by the Ethics Committee of the Universidade Estadual de Campinas (São Paulo, Brazil), and an informed consent was obtained from patients and relatives.

Clinical and molecular data of the 12 patients are summarized in Table 1.

TABLE 1.

Clinical and molecular data of CAH patients

Case	Sex	Age at diagnosis (months)	Clinical data	Basal 17OHP (nmol/liter)	Na+/K+ (mmol/liter)	Phenotype	Genotype (paternal allele/maternal allele)	Nationality
1	F	17	Preterm (GW 36) and small for gestational age (weight z-score −3.1 and height z-score −4.2); clitoromegaly and partial fusion of the labioscrotal folds at birth; Prader II	> 6 ndma	143/4.7	SV	p.G56R/IVS2–13A/C>G	Brazilian
2b	F	3	Ambiguous genitália at birth; Prader III	> 6 ndm	nt	SV	p.L107R/p.I172N	Brazilian
3b	M	37	Precocious puberty	> 6 ndm	nt	SV	p.L107R/p.I172N	Brazilian
4	F	0.8	Failure to thrive (weight z-score −0.92); clitoromegaly at birth; Prader I-IIc	> 6 ndm	126/5.5	SW	p.L142P/IVS2–13A/C>G	Brazilian
5d	F	0.9	Ambiguous genitália at birth; Prader III	49	135/7.2	SW	p.R408C/IVS2–13A/C>G	Brazilian
6d	F	1	Ambiguous genitália at birth; Prader IV	> 6 ndm	127/7.1	SW	p.R408C/IVS2–13A/C>G	Brazilian
7d	M	0.5	Failure to thrive	> 6 ndm	130/6.6	SW	p.R408C/IVS2–13A/C>G	Brazilian
8	F	2	High basal level of 17OHP in the neonatal screening program; ambiguous genitália at birth; Prader V	50	nt	SV	p.R408C/p.I172N	Brazilian
9	F	0.27	Ambiguous genitalia prenatally diagnosed on a routine ultrasound with 16 GW; Prader IV	35	127/7.1	SW	p.R408C/p.R356W	Brazilian
10	F	0.5	Ambiguous genitália; Prader IV	> 6 ndm	119/5.3	SW	c.920_921insT/p.P34 L+p.H62L+IVS2–13A/C>G+ c.329_336delGAGACTACe	Brazilian
11	F	72	Pseudoprecocious puberty; advanced bone age	nt	nt	SV	p.H62L+p.P453S/IVS2–13A/C>Gf	Norwegian
12	M	132	Precocious puberty with 4 yr; accentuated pubertal development at the age of 10 yr resulted in short stature (160 cm)	151g	nt	SV	p.H62L+p.P453S/c.920_921insT+ p.Q318Xf	Swedish

^a

Reference value for ndm is 5.97 nmol/liter.

^b

Siblings.

^c

For follow-up, malnourishment was discarded; treatment with hydrocortisone and fludrocortisone (0.05 mg/d) was introduced after the first 24 d of life; at the age of 5 months, she presented with hyponatremia and hyperkalemia; therefore, the fludrocortisone dosage was raised to 0.1 mg/d, resulting in electrolyte equilibrium (Na⁺ = 134 mmol/liter and K⁺ = 4.8 mmol/liter); at the age of 11 yr, due to bad compliance to the treatment, the steroid 17OHP levels raised to 78 nmol/liter in combination with elevated levels androstenedione (4 mg/ml) and high plasma renin activity [17.4 μg/liter·h (normal values between 0.15 and 2.33 μg/liter·h; radioimmunoassay, Incstar, Stillwater, MN; solid phase)].

^d

Siblings.

^e

Mutations within a chimeric gene in a 30-kb deleted allele.

^f

Parental DNA could not be obtained for segregation analysis, but all mutations were found in heterozygous form, and the most likely genotype is shown here.

^g

Reference value is 1.6–7.6 nmol/liter.

F, Female; M, male; ndm, nondiluted measurements; nt, not transmitted; GW, gestational weeks.

Mutation analysis

Genomic DNA was obtained from peripheral blood by phenol/chloroform extraction (9). CYP21A2 was specifically amplified in two or three fragments, depending on the presence or absence of the intron 2 variant IVS2–13C (10). Each fragment was sequenced using internal primers as described previously (10). For nonpseudogene-derived mutations, restriction enzyme digestion or allele-specific oligonucleotide PCR was used to follow the segregation in the family and analyze 50 nonrelated normal individuals. Nucleotide and amino acid numbering followed the published sequence (11) (National Center for Biotechnology Information, no. M12792, Bethesda, MD).

Construction of plasmids and mutagenesis

The constructions of pALTER-CYP21 plasmids for site-directed mutagenesis and pCMV4-CYP21 plasmids for expression analyses have been described previously (12, 13). Mutations were introduced into the pALTER-CYP21 with the Stratagene QuickChange site-directed mutagenesis kit (AH Diagnostics, Stockholm, Sweden) following the manufacturer’s instructions except that XL1-Blue MRF1′ Kan supercompetent cells (Stratagene, Stockholm, Sweden) were used.

Expression of CYP21A2 and enzyme activity assays

After sequencing each cloned CYP21A2 cDNA carrying the mutations, in vitro expression experiments were performed. Transfections in COS-1 cells with wild-type and mutant CYP21A2 proteins and enzyme activity assays were conducted as described before (14) using 12- instead of 6-well plates.

Detailed description of the in vitro CYP21A2 expression assay has recently been covered (14). The experiments were repeated five times for each mutant CYP21A2 using ³H-labeled substrates, either 17-hydroxyprogesterone (17OHP) or progesterone (Amersham Biosciences, Sweden). After thin-layer chromatography, substrates and products were quantified by liquid scintillography. The cells were harvested by trypsination and the ratio of β-galactosidase activity to total protein content was measured to verify the transfection efficiency. Enzyme activities were expressed as a percentage of conversion, taking the apparent specific activity of the wild-type CYP21A2 as 100%.

To determine the apparent kinetic constants, transiently transfected COS-1 cells were incubated with six different steroid concentrations: 0.5, 1.0, 2.0, 3.0, 4.0, and 7.0 μmol/liter. After incubation, steroids were extracted and analyzed as described above. The experiments were performed three times for each substrate.

Western blotting

Western blot analyses were performed using polyclonal antibodies raised in rabbit against human CYP21 as primary antibody and antirabbit IgG (Santa Cruz Biotechnology, Scandinavian Diagnostic Services, Falkenberg, Sweden) as secondary antibody (14).

Results

The p.G56R mutation (c.166G>A) was identified in the paternal allele of a patient with SV-21OHD. She is compound heterozygous with the IVS2–13A/C>G mutation (Table 1). The novel mutation p.G56R almost abolished the enzyme activity toward 17OHP but retained 1.4% of the activity with progesterone as substrate (Fig. 1A). The p.G56R was not found in other disease-related (n = 135) or nondisease-related alleles (n = 85), including nonrelated controls and normal alleles in the obligatory heterozygous carriers.

Fig. 1.

A, Enzymatic activities of normal and mutant P450c21 expressed intact COS-1 cells. The activities of the mutant enzymes are represented as percentage of the wild-type (WT) activity, which is arbitrarily defined as 100%. The values shown in the figure are for the conversion of 17OHP to 11-deoxycortisol and for the conversion of progesterone to 11-deoxycorticosterone at a substrate concentration of 2 μm. The bars represent the mean ± 1 sd of five independent experiments. B, Apparent kinetic constants for the wild-type (P450c21) and the p.H62L mutant proteins; values are shown as the mean ± 1 sd for three (n = 3) independent experiments. C, Western blot of CYP21A2 proteins expressed in COS-1 cells. The proteins were collected directly from the culture plate wells after treating the cells with lysis buffer followed by protein separation in SDS-PAGE. Normal and mutant CYP21A2 proteins were revealed in immunoblottings by using CYP21 rabbit polyclonal antibodies. V_max, Maximum velocity; K_m, Michaelis-Menten constant.

The p.L107R mutation (c.320T>G) was identified in the maternal allele in two siblings. Both the girl and her brother have the SV-21OHD and carry the p.I172N mutation on the other allele (Table 1). The p.L142P (c.425T>C) was detected in the maternal allele of a SW patient who also carries the p.I172N on the other allele (Table 1). Both the p.L107R and the p.L142P reduced the enzyme activity to the level of SW mutations (Fig. 1A). Neither p.L107R nor p.L142P was found in other disease-related (n = 72) or nondisease-related alleles tested (n = 76).

The p.R408C mutation (c.1222C>T) was identified in five Brazilian patients from three nonrelated families (Table 1). The in vitro assay resulted in an almost abolished enzyme activity toward progesterone (Fig. 1A).

The p.H62L (c.185A>T) was associated with other mutations in both Brazilian and Scandinavian patients. The maternally inherited allele in the Brazilian patient (Table 1) harbors a chimeric gene present in a 30-kb deletion allele. This gene carries p.P34L (c.101C>T) and p.H62L mutations, in addition to the pseudogene-derived IVS2–13A/C>G and c.329_336delGAGACTAC. The p.P34L and p.H62L were always associated with chimeric genes in 30-kb deleted alleles in Brazilian patients. Such alleles (∼20% of deleted alleles; FB Coeli, Lemos-Marini SHV, Araújo M, Paulino LC, Lau IF, Bernardi RD, Petroli RJ, Soardi FC, Guerra-Junior G, and De Mello MP, unpublished data) do not bear the p.P30L mutation, which is usually present in chimeric genes. In the Scandinavian patients (Table 1), p.H62L is associated with the nonclassical p.P453S mutation (12). The p.H62L reduced the enzyme activity to 44 and 21% for 17OHP and progesterone, respectively (Fig. 1A). Kinetic parameters were investigated for this mutation. Data indicate a reduction in the maximum velocity when compared with wild type (t test, P < 0.05), whereas the Michaelis-Menten constant values were in the same range of magnitude for both substrates (Fig. 1B). For comparison purposes, p.P453S activity was evaluated under the same experimental conditions as for the p.H62L. Individually, both mutations have similar effects on enzyme activity (Fig. 1A). However, when the p.H62L mutation was expressed in combination with the p.P453S mutation, the activity of the enzyme was reduced to 4.1 and 2.3% toward 17OHP and progesterone, respectively, indicating a synergistic effect.

Western blotting (Fig. 1C) confirmed expression of comparable amounts of wild-type and all mutant CYP21A2 proteins.

Discussion

Transient expression of the p.G56R, p.L107R, p.L142P, and p.R408C mutants in COS-1 showed CYP21A2 enzymatic activity values within the range of classical CAH mutations. The result of enzyme activity obtained for p.G56R indicates that it might be a SV-related mutation, whereas p.I172N, usually associated with a SV phenotype, was not used as an internal control. This mutation should be included in the future functional in vitro studies of new CYP21A2 mutants. Evidences for the important role of p.G56 residue are its highly conserved position among mammalian CYP21 proteins and its location between two membrane contact regions. The p.G56R changes a neutral residue to a basic hydrophilic in the surface region; this probably disturbs membrane interactions (15).

The p.L142P demonstrated a reduction in the enzyme activity to the level of SW mutations. The importance of p.L142 residue for the enzyme activity is highlighted by its conserved position located within D-helix of the human CYP21A2 (15). The substitution for a proline might lead to an altered structure causing function impairment as proline is known to potentially break α-helix structures (16). Considering the genotype, case 4, who carries p.L142P, would be expected to have SV-21OHD because she is compound heterozygous with p.I172N. However, the biochemical findings at diagnosis indicated the severe form of the disease (Table 1). Furthermore, her clinical and laboratorial follow-up confirmed the SW-21OHD. The p.I172N mutation is reported to present high variability in genotype-phenotype correlations (17). Although this mutation was not analyzed in this study, its variable expression and/or the patient’s genetic background may explain the case 4 phenotype.

The p.L107R demonstrated to be a SW mutation. The p.L107 corresponding amino acid in other mammalian CYP21A2 is either a leucine or an isoleucine dictating the conservation of a hydrophobic residue. This position is within an important region for substrate access and product release (18). The substitution for a charged residue (R) is likely to disrupt the enzyme function, as confirmed by the nearly null activity observed in the functional studies (Fig. 1A).

The p.R408C is a rare nonpseudogene-derived mutation described before in Brazilians (9) and was associated with SW-21OHD. The identification of additional patients with this mutation indicates that it is diffusing in this population; therefore, it should be considered in CYP21A2 screening programs. Enzyme activity data described here establish a good genotype-phenotype correlation as cases 5–7 and 9 that are compound heterozygous with null mutations and have a SW phenotype. The p.R408 is the third residue in the ERR-triad (E351/R354/R408), which acts in the meander as an important stabilizer of the three-dimensional structure that allow covalent binding of the heme group (16).

The p.H62L is also a rare mutation first described in French patients (19). In two Scandinavian patients, p.H62L mutation was identified in association with p.P453S. Both patients also presented a phenotype that was clearly much more severe than the NC-21OHD normally associated with p.P453S. In functional assays the p.H62L mutant protein showed an activity compatible with a NC mutation. Determination of apparent kinetic constants revealed that the substrate binding capacity was within the same magnitude for mutant and normal enzyme (Fig. 1B). In vitro activity data revealed a synergistic effect of p.H62L+p.P453, which may explain the mild SV phenotype of the Scandinavian patients. Similar results on these mutations were published during revision of this paper (20).

In summary, we describe five rare, including three novel CYP21A2 mutations causing 21OHD. The novel mutations presented activities closer to null than the p.I172N, except p.G56R that might be SV related. The p.H62L is definitely a NC mutation, but the combination with p.P453S brings the allele closer to mild SV characteristics. Interestingly, all three novel mutations were associated with the IVS2–13C variant, which can be dropped out on CYP21A2 gene PCR selection. We have described before the novel c.82_83insC mutation (10) also present in such alleles. Therefore, the data presented here highlight once again the importance of considering this effect when analyzing IVS2–13A/C or IVS2–13C/G heterozygous individuals. Furthermore, this paper reinforces that biochemical studies on new mutations are important tools to be used when establishing a correct genotype-phenotype correlation, thus improving the clinical management of patients as well as the genetic counseling for the affected families (7, 8, 16).

Acknowledgments

We thank Maria Madalena Vasconcelos Rosa and Ann-Christin Thelander for technical support; Myriano Henriques de Oliveira Junior for assisting with final art; and Marina de Melo Higdon for reading the manuscript.

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo Grant 05/00981-5 and PhD Grant 03/01785-0 (to F.C.S.); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Grant 1919-5; Conselho Nacional de Desenvolvimento Científico e Tecnológico (Brazil); Karolinska Institutet; Sällskapet Barnavård; Stiftelsen Frimurare Barnhuset; Söderberg Foundation (Centrum för Molekylär Medicin); the Swedish Research Council (Grant 12198); the Novo Nordisk Foundation; The Centre of Gender Related Medicine; Karolinska Institutet; and the Stockholm County Council.

Disclosure statement: All authors have nothing to disclose.

Abbreviations

 
• CAH,

  Congenital adrenal hyperplasia;

 
• NC,

  nonclassical;

 
• 21OHD,

  21-hydroxylase deficiency;

 
• 17OHP,

  17-hydroxyprogesterone;

 
• SV,

  simple virilizing;

 
• SW,

  salt wasting.