|Year : 2014 | Volume
| Issue : 7 | Page : 72-79
Phenotypic variability of hyperandrogenemia in females heterozygous for CYP21A2 mutations
Vassos Neocleous1, Christos Shammas1, Alexia AP Phedonos1, Leonidas A Phylactou1, Nicos Skordis2
1 Department of Molecular Genetics, Function and Therapy, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
2 Department of Molecular Genetics, Function and Therapy, The Cyprus Institute of Neurology and Genetics; Division of Pediatric Endocrinology, Paedi Center for Specialized Pediatrics; St. George's University of London Medical School at the University of Nicosia, Nicosia, Cyprus
|Date of Web Publication||20-Nov-2014|
Division of Pediatric Endocrinology, Paedi Center for Specialized Pediatrics. 178 Athalassas Avenue, 2025 Nicosia
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objectives: The objective was to seek evidence on the prevalence and consequences of heterozygous CYP21A2 mutations in girls, adolescent, and adult females with clinical manifestation of androgen excess. Patients and Methods: The study included 64 girls diagnosed with premature adrenarche (PA) in childhood and 141 females with clinical hyperandrogenemia manifested in adolescence or adulthood. Direct DNA sequencing and multiplex ligation-dependent probe amplification analysis were used to identify mutations in the CYP21A2 gene. Results: (1) Thirty-four patients were diagnosed with nonclassical-congenital adrenal hyperplasia (NC-CAH) based on the 17-hydroxyprogesterone (17-OHP) levels and the presence of two mutations in CYP21A2 and therefore were excluded from the study, 66 were found to be heterozygotes and finally 105 had no identifiable mutations. The most frequent mutations among the carriers were the mild p.Val281 Leu and p.Qln318stop. Higher levels of mean stimulated 17-OHP were found in the carriers of the p.Val281 Leu. (2) A notable increased allelic frequency for the known p.Asn493 Ser polymorphism was observed in the pool of females with hyperandrogenemia in whom no mutation was identified. (3) In girls, who presented early with PA, 26.6% were diagnosed with NC-CAH and carried two mutations, 28.7% were identified as heterozygotes 43.7% had no identifiable genetic defect in the translated region of the CYP21A2 gene. On the contrary, in the group of 141 females with late onset hyperandrogenemia, the presence of 2 mutations was detected in 12%, 1 mutation in 33.4% and no mutation in 54.6%. Conclusions: The carrier status for 21-OHD, may be an important factor in the variable phenotype of hyperandrogenism and may be a contributing factor for the early manifestation of the disease.
Keywords: CYP21A2, hyperandrogenemia, polycystic ovary syndrome
|How to cite this article:|
Neocleous V, Shammas C, Phedonos AA, Phylactou LA, Skordis N. Phenotypic variability of hyperandrogenemia in females heterozygous for CYP21A2 mutations. Indian J Endocr Metab 2014;18, Suppl S1:72-9
|How to cite this URL:|
Neocleous V, Shammas C, Phedonos AA, Phylactou LA, Skordis N. Phenotypic variability of hyperandrogenemia in females heterozygous for CYP21A2 mutations. Indian J Endocr Metab [serial online] 2014 [cited 2020 Jul 4];18, Suppl S1:72-9. Available from: http://www.ijem.in/text.asp?2014/18/7/72/145077
| Introduction|| |
The most common form of congenital adrenal hyperplasia (CAH) (95% of all cases) is due to 21-hydroxylase deficiency (21-OHD) resulting from molecular defect in the steroid 21-OH (CYP21A2) gene, with an overall estimated incidence of 1:10,000-1:15,000 for the severe classic form and 1:500-1:100 live births for the nonclassical-CAH (NC-CAH). ,
The incidence of the genetic defects of 21-OHD has been extensively studied, and ethnic specific distribution of mutations has been reported. As expected from the prevalence of CAH and NC-CAH, the frequency of heterozygotes in the population for 21-OHD is quite common and varies considerably, ranging from 1 in 10 to 1 in 60 in certain population and ethnic groups and in some cases it may affect as 1 in 3 of Askenazi Jews. ,,,,,,,,,
Approximately, 95% of the mutated CYP21A2 alleles are the result of recombination events between the homologous CYP21A2P pseudogene and the active CYP21A2 gene, while the remaining 5% represent new mutations.  Several studies in the Mediterranean region, including studies from our group have reported as the most prevalent genetic defects, the mutations c. 655A/C > G (IVS2-13A/C > G), c. 1994C > T (p.Qln318stop), c. 1683G > T (p.Val281 Leu), c. 329_336del (8 bpdelE3). ,,,
Compared to normal female individuals, female carriers for 21-OHD frequently demonstrate an exaggerated secretion of the 21-OH precursors 17-OH progesterone (17-OHP) and P4  and lower levels of 11-deoxycorticosterone  and aldosterone  after adrenocorticotropic hormone (ACTH) administration. In fact, between 50% and 80% of carriers exhibit a 17-OHP level after ACTH stimulation that is above the 95 th percentile of the control value. ,,
The purpose of this study was to seek evidence on the prevalence and consequences of heterozygous CYP21A2 mutations in girls, adolescent, and adult females with clinical signs of androgen excess.
| Patients and Methods|| |
A total of 205 female patients with clinical signs of hyperandrogenemia was evaluated. The inclusion criteria were the clinical manifestation of premature adrenarche (PA) in prepubertal girls and the presence of clinical hyperandrogenemia in adolescent and adult females. Sixty-four presented with PA, which was defined as the development of pubic hair and/or axillary hair prior to 8 years of age. One hundred and forty-one females presented with hyperandrogenemia, which was defined as the presence of hirsutism (Ferriman Gallway score >8)  and/or severe acne according to Androgen Excess Society (AES).  Polycystic ovary syndrome (PCOS) criteria included irregular ovulation, hyperandorgenism, and/or polycystic ovaries as defined by AES.  Basal 17-OHP was measured in all patients. Symptoms of hyperandrogenemia and the elevated 17-OHP levels were used to diagnose the patients with the NC form of CAH. The patients with the NC form had basal 17-OHP values more than 15 nmol/l (often >60 nmol/l probably due to stress during sampling) and/or 17-OHP values, after intravenous administration of 250 μg of ACTH (1-24), >30 nmol/l (normograms for the diagnosis of 21-OHD). Possible heterozygote carriers were considered those patients with baseline 17-OHP > 6 nmol/l and those with baseline 17-OHP <6 nmol/l and ACTH stimulated <30 nmol/l. , Stimulated 17-OHP values >30 nmol/l revealed the possibility of mutations in more than one allele. , Serum 17-OHP concentrations were measured with the commercial Radioimmunoassay method (Beckman Coulter). Three hundred females of Cypriot origin were recruited from the cohort of healthy individuals seeking biochemical evaluation prior to their marriage from the National Unit for Thalassemia at the Makarios Hospital in Nicosia, Cyprus. The numbers were calculated based on the inheritance recessive model of the disease. Informed consent was obtained from all individuals, and the study was approved by the Cyprus National Bioethics Committee.
Amplification of the CYP21A2 gene
The CYP21A2 genes of all patients were analyzed using genomic DNA isolated from peripheral blood samples. Molecular analysis was performed according to a cascade strategy as previously described. ,, The primers P1-P48  were used to amplify the fragment containing the-370 bp CYP21A2 promoter, the 5' untranslated region of the CYP21A2 gene that is mainly located in the first 167 nucleotides upstream the ATG codon and the 3' untranslated region that is 536 nucleotides downstream the TGA stop codon of the CYP21A2 gene.  The polymorphism p.Asn493Ser is found in exon 10 of the CYP21A2 gene, and therefore, was covered with the direct sequencing analysis.
Multiplex ligation-dependent probe amplification analysis
DNA from female patients in this study analyzed by direct sequencing was also examined with the multiplex ligation-dependent probe amplification (MLPA) technique (MRC Holland, Amsterdam, Netherlands). MLPA was employed to investigate any possible large gene deletions and large gene conversions in the CYP21A2 gene.
The kit detects mutations for exons 1, 3, 4, 6, and 8; among these are c. 329_336del (8 bpdelE3), c. 999T > A (p.Ile172Asn), c. 1380T > A; c. 1383T > A; c. 1389T > A (Cluster E6) and c. 1994C > T (p.Qln318stop) mutations. Furthermore, this kit contains three CYP21A1P-specific probes, three TNXB probes, one C4A probe, one C4B probe, and one probe for the CREBL1 gene which is located q-telomeric of TNXB. In addition, two other probes located on chromosome 6p21.3, 1 Y-chromosome specific gene (UTY) and 16 reference probes are included. Briefly, 50-200 ng DNA was denatured and hybridized overnight at 60°C with the SALSA probe mix. Samples were then treated with Ligase-65 enzyme for 15 min at 54°C, the reactions were stopped by incubation at 98°C for 5 min. Finally, polymerase chain reaction amplification was carried out using BigDye terminator v1.1, cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). Amplification products were run on an automated Applied Biosystems 3130 xl Genetic Analyzer. The raw data were analyzed by using Coffalyzer 9.4 Software (MRC Holland, Amsterdam, Netherlands). The size of migration of exon-specific peaks was identified according to their migration relative to Gene Scan 600 LIZ size standard (Applied Biosystems, Foster City, CA, USA).
Comparisons of 17-OHP levels were performed using parametric methods (t-test procedures). A two-tailed alpha level of 0.05 was used to establish statistical significance for the p.Asn493Ser polymorphism. All statistics analyses were performed using Statistical Analysis System (SAS) software Version 9.2, SAS Institute Inc., Cary, NC, USA.
| Results|| |
CYP21A2 genotypic analysis
Thirty-four patients were diagnosed with NC-CAH based on the 17-OHP levels and genotypic analysis (i.e. identified to carry 2 mutations) and were excluded from the study, 66 were identified as being heterozygotes for mutations in CYP21A2 [Table 1] and 105 had no mutation in the CYP21A2 [Table 2], [Table 3], [Table 4] and [Table 5].
|Table 1: Mutation frequency of affected alleles from 66 unrelated hyperandrogenic female patients|
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|Table 2: Genotypic analysis, basal, and ACTH stimulated 17‑OHP levels in 19 CYP21A2 heterozygote girls with premature adrenarche. The polymorphism p.Asn493Ser was identified in heterozygosity in patients 1, 2 and 18|
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|Table 3: Clinical features, genotypic analysis, basal, and ACTH stimulated 17‑OHP levels in 17 CYP21A2 heterozygote adolescent girls with hyperandrogenemia. The polymorphism p.Asn493Ser was identified in heterozygosity in patients 3-5, 13 and 16|
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|Table 4: Clinical features, genotypic analysis, basal, and ACTH stimulated 17‑OHP levels in 30 CYP21A2 heterozygote adult females with hyperandrogenemia. The polymorphism p.Asn493Ser was identified in heterozygosity in patients 4, 10, 23, and 27|
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|Table 5: Allelic frequency of p.N493S polymorphism in 300 controls, girls with PA with and without CYP21A2 mutations, and adult females with and without CYP21A2 mutations|
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The frequency of the molecular defects detected in the 66 heterozygote females (19 girls with PA and 47 adolescents and adults with hyperandrogenemia) are shown in [Table 1]. In the total pool of the 66 unrelated heterozygote alleles, the most frequent mutation was c. 1683G > T (p.Val281 Leu) (53.0%), followed by c. 1994C > T (p.Qln318stop) (18.2%), c. 2665C > T (p.Pro482Ser) (10.6%), c. 1752G > A (p.Val304Met) (6.1%), c. 2578C > T (p.Pro453Ser) (6.1%), c. 2296G > A (p.Ala391Thr) (1.5%), large deletion/conversion exons 1-4 (1.5%), large deletion/conversion exons 6-8 and c. 707_714delGAGACTAC (8 bpdelE3) (1.5%). The rare c. 1752G > A (p.Val304Met) was first found in a 7-year-old girl with PA and two adolescent females with PCOS. The large deletion/conversion of exons 1-4 and large deletion/conversion of exons 6-8 were, respectively, found in two young girls at the age of 7 and 9 years, both presented with PA.
The rare c. 1752G > A (p.Val304Met) was first found in a 7-year-old girl with PA and two adolescent females with PCOS. The large deletion/conversion of exons 1-4 and large deletion/conversion of exons 6-8 were, respectively, found in two young girls at the age of 7 and 9 years, both diagnosed with mild NC-CAH and PA.
In 32, out of the 47 (68.1%), heterozygote adolescents and adults with hyperandrogenemia, who fulfilled the criteria of PCOS the most frequent mutation was p.Val281 Leu (73.9%).
Baseline and stimulated 17-OHP levels were compared according to mutation found in the 66 carriers. There was no statistically significant difference in the baseline mean 17-OHP levels. Stimulated 17-OHP, however, was higher in carriers of the c. 1683G > T (p.Val281 Leu) mutation compared to the mean 17-OHP found in carriers of the other mutations (21.1 nmol/L vs. 16.6 nmol/L, P = 0.02).
A notable increased allelic frequency of 54.3% (57/105) for the known p.Asn493Ser polymorphism was observed in the pool of females with hyperandrogenemia and no mutation when compared with age-matched controls of Cypriot origin (37% [111/300], P = 0.0001) [Table 5]. The allelic frequency of p.Asn493Ser was 19.7% (13/66) in the group of the 66 CYP21A2 heterozygotes ([P = 0.01] vs. controls and [P = 0.0001] vs. no mutation). In the subgroup of girls with PA, the known polymorphism p.Asn493Ser was detected in 18/28 (64.3%) patients, who concurrently had no identified mutations in the CYP21A2: (P = 0.008) versus controls and (P = 0.02) versus 26.3% in the group of 19 heterozygotes [Table 5].
In the total group of 64 girls, who presented early with PA, 17 (26.6%) were diagnosed with NC-CAH and carried 2 mutations, 19 (28.7%) were identified as heterozygotes, and 28 (43.7%) had no identifiable genetic defect in the translated region of the CYP21A2 gene. On the contrary, in the group of 141 females with late onset hyperandrogenemia, the presence of 2 mutations was detected in 17 (12%), 1 mutation in 47 (33.4%) and no mutation in 77 (54.6%) [Figure 1].
|Figure 1: Girls with premature adrenarche (n = 64) and females (n = 141) who exhibited 2, 1, and no mutations in the CYP21A2 gene|
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| Discussion|| |
The present study was designed to seek evidence on the prevalence and consequences of heterozygous CYP21A2 mutations in females with clinical hyperandrogenemia. Most of the females with clinical signs of hyperandrogenism exhibited a significant elevation of stimulated 17-OHP and had identifiable heterozygous mutations in the CYP21A2 gene. This finding was indicative of carrier status for 21-OHD and was further confirmed with direct DNA sequencing and MLPA analysis for mutations in the CYP21A2 gene.
Several investigators have shown a high rate of heterozygosity for CYP21A2 mutations in girls, female adolescents and women with clinical symptoms of androgen excess. ,,,[27.28] The presence of heterozygous CYP21A2 mutations is associated with an increased risk of clinical and biochemical hyperandrogenism. ,,, The clinical expression of CYP21A2 heterozygosity prepubertally is consistent with the study by Dacou-Voutetakis and Dracopoulou,  who reported a high incidence of molecular defects of the CYP21A2 gene in girls with PA, although in a significant number of cases the genotype could not be predicted by the age of onset of PA. A high prevalence of heterozygous mutations was also reported in a cohort of French Mediterranean girls with isolated premature pubarche.  In a similar manner as in the case of the 19 heterozygous girls of the present study, the French girls with PA were found to exhibit high 17-OHP levels.  In addition, a number of other studies have described both males and females with the milder form of NC-CAH carrying only 1 mutation in their CYP21A2 gene. 
The most frequent mutation among these identified female carriers was the mild c. 1683G > T (p.Val281 Leu) with a frequency of 53.0%. Back in 2006 Admoni et al.  proposed a dominant-negative effect of the mutant c. 1683G > T (p.Val281 Leu) allele on the wild type by drastically reducing its activity. The mutant enzyme may interfere or compete with the wild type for the conversion of 17-OHP to 11-deoxycortisol. The c. 1683G > T (p.Val281 Leu) mutation is quite common in our population and a high rate seen in the symptomatic carriers group of the present study can be attributed to the high rate observed. ,, In a random screening of healthy individuals, often used as controls in our laboratory, c. 1683G > T (p.Val281 Leu) was determined in 26 out of 600 alleles or 1 in 23. 
The question of the contribution of the CYP21A2 heterozygosity to the pathogenesis of PCOS has been recently studied by Settas et al. who reported no significant input as the frequency of the CYP21A2 heterozygous mutations in PCOS women and controls did not differ. This discrepancy in the literature could be partly explained by the different methodologies used and the diversity of the clinical phenotype among the patients.
The higher ACTH stimulated 17-OHP mean values exhibited in the group of carriers with the c. 1683G > T (p.Val281 Leu) mutation compared with carriers of other mutations suggest increased exposures to androgens and point to more severe impairment of 21-OHD in the symptomatic c. 1683G > T (p.Val281 Leu) carriers. This finding supports the already identified notion that carriers of the mild c. 1683G > T (p.Val281 Leu) missense mutation exhibit higher ACTH-stimulated 17-OHP values and higher rates of either PCOS  irregular menses or hirsutism.  Patients that exhibit stimulated 17-OHP levels >60.5 nmol/l are consistent with either the homozygous or the compound heterozygous state.
In the present study, the rare missense c. 1752G > A (p.Val304Met) mutation and the less frequent severe large deletions del1-4 and del6-8 were identified. We highlight that c. 1752G > A (p.Val304Met) is a mild and rare mutation and after expression in COS-1 cells the mutated enzyme was found to have residual activity of 46% for conversion of 17-OHP and 26% for conversion of progesterone compared with the normal enzyme.  To our knowledge, c. 1752G > A (p.Val304Met) missense mutation was reported only by our group in Cypriot patients with the NC-CAH form  and in a 24-year-old female patient of Asian origin who presented with hirsutism, acne, and alopecia.  The c. 1752G > A (p.Val304Met) missense mutation is located at a region suggested to be involved in substrate interaction in a model of the protein. 
In the females with clinical hyperandrogenemia in whom the direct sequencing of the coding region and the proximal promoter region of CYP21A2 gene did not identify any putative new mutation, the polymorphism p.Asn493Ser was identified to be the leading sequence alteration. Among these 57 patients, six were homozygotes with the rest 51 being heterozygotes for the p.Asn493Ser substitution. Most of the patients with p.Asn493Ser presented with elevated baseline or ACTH-stimulated 17-OHP levels and clinical signs of androgen excess. Taking into account the finding from a previous study by our group that the allelic frequency of p.Asn493Ser in the general population of Cyprus is quite high 37.0%  and also that in the CYP21A2 heterozygotes (n = 66) also tested in the present study to be significantly lower to 19.7%, it may be suggested that a possible implication of this alteration could contribute in the pathogenesis of hyperandrogenemia.
Various studies demonstrated this substitution as a naturally occurring polymorphism  and some as a disease-causing mutation  but its influence on residual enzyme activity has never been tested in vitro. A recent study on a molecular model of human CYP21A2 demonstrated unchanged the electrostatic charge of the non-conserved amino acid alteration p.Asn493Ser.  Such a study identified in Mexican CAH patients and healthy controls a higher proportion of homozygosity for the p.Asn493Ser substitution.  The presence of elevated basal and ACTH-stimulated 17-OHP, PA, hyperandrogenism, and PCOS in the female patients of the present study identified in heterozygosity or homozygosity only with p.Asn493Ser and with no other pathogenic mutation (s) is suggestive of a direct role of this substitution in the disease manifestation of NC-CAH.
Although no association could be found between the presenting symptom and the type of the molecular defect, it is clear that heterozygote females have a tendency to manifest androgen excess in early life. Heterozygotes for mutations in the CYP21A2 gene may have mild biochemical abnormalities and in general they do not display a serious endocrine disorder.  Regardless of these general correlations, the 21-OHD phenotype does not always correlate precisely with the genotype,  suggesting that other genes influence the clinical manifestations.
The novelty provided by the present study is the tendency of heterozygote females to manifest androgen excess and PA in early life. In addition, the high carrier rate of the p.Asn493Ser in female patients with androgen excess with no other pathogenic mutation (s) is suggestive of a possible role of this substitution in the disease manifestation of NC-CAH.
| Conclusion|| |
The carrier status for 21-OHD, may be an important factor in the variable phenotype of hyperandrogenism and may be a contributing factor for the early manifestation of the disease. Although the clinical expression of NC-CAH is not solely depended on the genotype, discrimination between mild and severe alleles should be made. The clinical relevance of the early detected molecular defect is emphasized as these girls have a higher probability of having earlier onset of puberty, clinical hyperandrogenemia at puberty, menstrual disturbances, and fertility problems in adulthood and moreover compromised final height. The detection of patients with NC-CAH phenotypes tested negative for mutations in the CYP21A2 gene underlines the importance to screen for other genes responsible for NC-CAH.
| Acknowledgments|| |
The Project UGEIA/ΔUGEIA/0609/(BIE)/27 is co-financed by the European Development Fund and the Republic of Cyprus through the Research Promotion Foundation.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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