Differences in hormonal levels between heterozygous CYP21A2 pathogenic variant carriers, non-carriers, and females with non-classic congenital hyperplasia

ABSTRACT

Objective:

CYP21A2 mutation heterozygote carriers seem to have an increased risk of hyperandrogenism. However, the clinical relevance of the heterozygote carrier status and the reliability of hormonal testing in discriminating a carrier from a non-carrier are puzzling questions. We aimed to characterize a population of Portuguese females suspected of having non-classic congenital adrenal hyperplasia (NC-CAH) due to clinical and biochemical criteria and who have undergone CYP21A2 molecular analysis.

Subjects and methods:

Retrospectively, we have analyzed the clinical records of 131 females (32 girls aged 3-9 and 99 adolescents and premenopausal women aged 13-49) who underwent complete CYP21A2 molecular analysis due to suspicion of NC-CAH. We divided included participants into three groups according to the CYP21A2 molecular analysis: NC-CAH females (46), heterozygous carriers (49), and wild type (36). We then compared clinical signs and symptoms as well as biochemical and molecular data between carriers and NC-CAH individuals and between carriers and wild type females. We measured 17OHP by electrochemiluminescence immunoassay.

Results:

Clinical features were similar between groups. Heterozygous carriers presented higher basal and post-cosyntropin 17-hydroxyprogesterone (17OHP) than wild type individuals (p < 0.05) and lower basal and stimulated 17OHP levels than NC-CAH patients (p < 0.05). We discovered a considerable overlap between 17OHP levels among groups. The most common pathogenic variant we identified was p.Val282Leu.

Conclusion:

In this population of hyperandrogenic women and children, heterozygous carriers showed higher basal and stimulated 17OHP than non-carriers although normal basal and stimulated 17OHP responses do not exclude heterozygosity for CYP21A2 pathogenic variants. In this study, only the molecular analysis presented good sensitivity in identifying heterozygotes.

Keywords
Heterozygous carrier; 17-hydroxyprogesterone; precocious pubarche; hyperandrogenism

INTRODUCTION

Congenital adrenal hyperplasia (CAH) entails a group of inborn errors of adrenal steroidogenesis. Deficiency of 21-hydroxylase (21OHD) accounts for 95% of the CAH cases. It results in defective conversion of 17-hydroxyprogesterone (17OHP) to 11-deoxycortisol and leads to hypocortisolism and hyperandrogenism due to the accumulation of precursor steroid hormones. It has a broad spectrum of clinical forms, ranging from classic salt wasting, classic simple virilizing and non-classic (NC). The NC female patients generally present premature pubarche in midchildhood or hirsutism, severe acne, oligomenorrhea, or infertility in adolescence and adulthood.

Non-classic congenital adrenal hyperplasia (NC-CAH) affects around 1 in 100 to 1 in 1,000 individuals ( ¹ ), and the frequency of heterozygote carriers is approximately 1 in 55 in the European population ( ² ).

Pathogenic variants in the CYP21A2 gene cause various degrees of dysfunction in enzymatic activity, and a strong genotype-phenotype correlation emerges, especially in the disease’s most severe forms ( ³ ). Genetically, CAH is an autosomal recessive disorder; therefore, an affected individual must present with at least two biallellic pathogenic variants, and the phenotype is conferred by the less deleterious variant ( ⁴ ).

The clinical relevance of the heterozygote carrier status and the reliability of hormonal testing in discriminating a carrier from a non-carrier are still puzzling questions. A heterozygous carrier’s normal allele should encode a CYP21A2 protein with almost complete activity, which ensures a normal physiological function. However, heterozygous carriers of CYP21A2 pathogenic variants seem to have an increased risk of hyperandrogenism, which may manifest as premature pubarche, accelerated growth, apocrine body odor, and seborrhea in childhood ( ⁵ – ⁸ ) and hirsutism, oligomenorrhoea, acne, and infertility in adolescence and adulthood ( ⁹ – ¹² ).

In cohorts of hyperandrogenic women and children, the frequency of the carrier status varies based on various inclusion criteria and ranges from 8% to 35% ( ⁷ , ¹² – ¹⁵ ). Although some studies have shown a higher prevalence of CYP21A2 carriers in hyperandrogenic individuals than in the general population ( ¹⁶ , ¹⁷ ), others have shown no such difference ( ¹³ , ¹⁸ – ²⁰ ).

We aimed to characterize a population of 131 Portuguese females, aged 3-49, suspected of having NC-CAH due to clinical and biochemical criteria, who have undergone CYP21A2 molecular analysis. In this population of 131 females, we aimed to compare 49 heterozygous carriers of CYP21A2 pathogenic variants with 46 NC-CAH individuals (2 pathogenic variants identified) and 36 subjects without any pathogenic variant on CYP21A2 .

SUBJECTS AND METHODS

Population

We have analyzed a population of 131 females referred to our hospital’s endocrinology and pediatric endocrinology clinics in the past 10 years who underwent CYP21A2 molecular analysis due to suspected NC-CAH. This population included 32 (24%) girls aged 3-9 and referred to our clinics due to precocious pubarche as well as 99 (76%) adolescents and premenopausal women aged 13-49 referred due to hirsutism, oligomenorrhea, severe acne, alopecia, and/or infertility.

We performed CYP21A2 molecular analysis for all these participants. Criteria for the molecular analysis in our center were basal 17 OHP > 2 ng/mL and post-ACTH > 10 ng/mL, familial history of CAH plus symptoms of hyperandrogenism, or severe hyperandrogenic signs or symptoms.

Participants fulfilled clinical and biochemical criteria, and we divided them into three groups according to the CYP21A2 molecular analysis. Group I included 46 females (12 girls, 34 women) with a diagnosis of NC-CAH, which was defined as normal genitalia at birth and clinical signs of hyperandrogenism that appeared later in childhood (pubarche, overgrowth, apocrine body odor) and adolescence (hirsutism, acne, oligomenorrhea, infertility), together with a genetic analysis showing two pathogenic variants in both CYP21A2 alleles (homozygous or compound heterozygous). Group II included 49 heterozygous carriers of the CYP21A2 pathogenic variant (10 girls, 39 women) with clinical features overlapping NC-CAH but having a single disease-causing CAH CYP21A2 pathogenic variant in one allele. Group III included 36 females (10 girls, 26 women) with features overlapping NC-CAH but without any CYP21A2 pathogenic variant identified.

Clinical assessment

An endocrinologist or a pediatrician evaluated all patients enrolled in the study at referral, and clinical signs and symptoms were abstracted from clinical records. Premature pubarche was defined as the development of pubic hair before the age of 8 in girls and 9 in boys ( ²¹ ). Hirsutism was defined as a Ferriman-Gallwey score ≥8 ( ²² ) and oligomenorrhea as menstrual cycles lasting 35-90 days.

Biochemical analysis

We measured dehydroepiandrosterone sulphate (DHEAS), 17OHP, androstenedione, and total testosterone by electrochemiluminescence immunoassay on the Elecsys 2010 Modular Analytics E170 analyzer (Roche Diagnostics). In post-pubertal women, we conducted the analyses in the follicular phase, before 9 am. We performed the stimulation test with administration of 250 µg synthetic cosyntropin (Synachten^®) and measured 17OHP at 0 and at 60 min post-cosyntropin.

Molecular analysis

We selectively amplified functional CYP21A2 gene sequencing by polymerase chain reaction (PCR) into two partially overlapping fragments, avoiding the co-amplification of the pseudogene CYP21A1P ( ²³ , ²⁴ ). Using the complete gene sequencing strategy, on the promoter region, approximately 130bp upstream, we sequenced the start codon by Sanger sequencing. We sequenced all coding sequences and intron-exon boundaries with internal primers that covered the entire CYP21A2 gene. We performed both restriction fragment length polymorphism (RFLP) for large deletion/conversions involving the CYP21A2 promoter region and MLPA for detection of large rearrangements ( ²³ , ²⁴ ). In all cases where it was mandatory, we performed parental segregation studies to determine whether the variants were in cis or in trans.

We determined nomenclature of CYP21A2 variants according to the Ensembl transcript CYP21A2 -002: ENST00000418967.6 (NM_000500.7 from National Center for Biotechnology Information ).

Statistical analysis

We performed statistical analysis using SPSS^® v.20. We applied nonparametric tests to compare independent samples and used chi-square tests to determine the association between categorical variables. We established comparisons between Group II (carriers) and Group I (NC-CAH patients) and between Group II (carriers) and Group III (wild type individuals). We regarded P values below 0.05 as statistically significant.

Ethics approval

We conducted all procedures in this study in accordance with the national committee for data protection and with the 1964 Helsinki declaration or comparable ethical standards. This original study had the approval from the Ethical Committee, Centro Hospitalar Universitário S. João, Porto, Portugal. We obtained informed consent from the participants.

RESULTS

Table 1 summarizes the 131 included females’ ages at referral, clinical phenotypes at presentation, genotypes, and biochemical data.

Table 1
Comparison between age at referral, phenotype at presentation, genotype, and biochemical data of the 131 included females, by age and genotype groups

		All	Group I (+/+)	Group II (+/−)	Group III (−/−)	p
All
Number of individuals, n (%)		131 (100%)	46 (35%)	49 (37%)	36 (28%)
Age at referral, years (median, min-max)		22.6 (3.3-49.6)	23.0 (5.1-49.6)	22.6 (3.3-48.3)	22.5 (3.7-46.0)	NS †
Childhood hyperandrogenism *
Number of individuals (%)		32 (100%)	12 (38%)	10 (31%)	10 (31%)
Age at referral, years (median, min-max)		6.3 (3.3-9.2)	6.9 (5.1-9.6)	5.5 (3.3-7.3)	6.6 (3.7-8.2)	NS †
Phenotype at presentation (%)
	Premature pubarche	97%	92%	100%	100%	NS ▼
	Tall stature	3%	8%
Genotype (number of individuals)
	p.Val282Leu/p.Val282Leu		7 (58%)
	p.Val282Leu/del/conv promotor CYP21A2		2 (17%)
	p.Val282Leu/p.Gly111ValfsTer21		1 (8%)
	p.Val282Leu/p.Pro454Ser		1 (8%)
	p.Val282Leu/p.R444X		1 (8%)
	p.Val282Leu/-			9(90%)
	c.290-13A/C>G/-			1(10%)
Biochemical data
Basal 17OHP, ng/mL (median, min-max)		4.8 (0.4-54.3)	16.7 (1.4-54.3)	3.3 (0.9-9.0)	2.0 (0.4-1.3)	<0.05 †
Pos-ACTH 17OHP, ng/mL (median, min-max)		12.5 (4.4-79.0)	55.1 (17.3-79.0)	11.9 (6.2-13.9)	7.7 (4.4-11.1)	<0.05 †
DHEAS, µg/dL (median, min-max)		86.3 (40.0-178.2)	125.4 (46.0-178.2)	77.5 (46.9-141.8)	74.2 (40.0-150.3)	NS †
Androstenedione, ng/mL (median, min-max)		1.0 (0.0-2.9)	1.5 (0.8-2.9)	0.5 (0.0-1.8)	0.4 (0.03-1.2)	NS †
Total testosterone, ng/mL (median, min-max)		0.1 (0.0-0.3)	0.1 (0.1-0.3)	0.1 (0.1-0.2)	0.0 (0.0-0.2)	NS †
Adolescence or adulthood hyperandrogenism **
Number of individuals (%)		99 (100%)	34 (34%)	39 (39%)	26 (26%)
Age at referral, years (median, min-max)		27.9 (13.2-49.6)	28.2 (13.2-49.6)	27.3 (14.2-48.3)	27.7 (13.5-46.0)	NS †
Phenotype at presentation (%)
	Hirsutism	76%	63%	91%	82%	NS ▼
	Oligomenorrhea	11%	19%	0%	9%
	Severe acne	5%	6%	9%	0%
	Alopecia	5%	6%	0%	9%
	Infertility	3%	6%	0%	0%
Genotype, n (%)
	p.Val282Leu/p.Val282Leu		17(50%)
	p.Val282Leu/del/conv promotor CYP21A2		17(50%)
	p.Val282Leu/-			39(100%)
Biochemical data
Basal 17OHP, ng/mL (median, min-max)		3.4 (0.1-30.6)	7.4 (0.6-30.6)	3.7 (0.6-15.7)	1.7 (0.1-5.7)	<0.05 †
Pos-ACTH 17OHP, ng/mL (median, min-max)		13.0 (2.7-21.9)	16.5 (13.0-21.9)	10.8 (4.2-13.0)	5.5 (2.7-9.5)	<0.05 †
DHEAS, µg/dL (median, min-max)		198.2 (0.7-598.1)	209.3 (0.7-399.4)	138.2 (12.3-598.1)	204.8 (2.1-536.0)	NS †
Androstenedione, ng/mL (median, min-max)		3.2 (0.2-8.8)	3.8 (0.2-8.8)	2.7 (0.2-7.3)	2.2 (0.3-8.4)	NS †
Total testosterone, ng/mL (median, min-max)		0.4 (0.03-134.0)	0.5 (0.0-1.0)	0.6 (0.1-2.1)	0.3 (0.0-134.0)	NS †

• Group I (+/+) included females with a diagnosis of NC-CAH (hyperandrogenism and genetic analysis showing 2 pathogenic variants in the CYP21A2 ). Group II (+/−) included heterozygous carriers of CYP21A2 pathogenic variant. Group III (−/−) included females with features overlapping NC-CAH but without any CYP21A2 pathogenic variant identified.
• ^*
  The “childhood hyperandrogenism” group includes 32 girls aged 3-9 years-old at referral due to precocious pubarche or tall stature.
• ^**
  The “adolescence or adulthood hyperandrogenism” included 99 adolescents and older women aged 13-69 years at referral due to infertility, oligomenorrhea, severe acne, alopecia, and/or hirsutism.
• ^†
  Non-parametric tests (Group II compared to I and III).
• ^▼
  Pearson Chi-square.
• NS: non-significant (p > 0.05); 17OHP: 17-hydroxyprogesterone; DHEAS: dehydroepiandrosterone sulphate.

The participants’ median age at referral was 6.3 years in children and 27.9 years in adolescents and adults. We found no significant differences between groups.

Children presented mostly with premature pubarche (97%), and adolescents and adults presented with hirsutism (76%) and oligomenorrhea (11%). We found no significant differences in presentation between NC-CAH patients (Group I), heterozygous carriers (Group II), and wild type individuals (Group III).

We found statistically significant differences in basal and stimulated 17OHP levels in both the children and the adolescents/adult populations between carriers (Group II) and NC-CAH patients (Group I) and between carriers (Group II) and wild type subjects (Group III).

In children, heterozygous carriers showed higher basal and cosyntropin-stimulated 17OHP than wild type girls (basal 17OHP: median 3.3 vs. 2.0 ng/mL; p < 0.05 stimulated 17OHP: median 11.9 vs. 7.7 ng/mL; p < 0.05) and lower basal and stimulated 17OHP than NC-CAH patients (basal 17OHP: median 3.3 vs. 16.7 ng/mL; p < 0.05; stimulated 17OHP: median 11.9 vs. 55.1 ng/mL; p < 0.05, respectively).

In adolescents and adults, heterozygous carriers showed higher basal and cosyntropin-stimulated 17OHP than wild type women (basal 17OHP: median 3.7 vs. 1.7 ng/mL; p < 0.05; stimulated 17OHP: median 10.8 vs. 5.5 ng/mL; p < 0.05) and lower basal and stimulated 17OHP than NC-CAH patients (basal 17OHP: median 3.7 vs. 7.4 ng/mL; p < 0.05); stimulated 17OHP: median 10.8 vs. 16.5 ng/mL; p < 0.05).

Figure 1 shows basal and post-ACTH 17OHP levels according to groups, for the whole sample. Differences in basal and pos-ACTH 17OHP levels between children and adolescent/adult women were non-significant (medians, respectively, 4.8 vs. 3.4 ng/mL (p = 0.278) and 12.5 vs. 13.0 ng/mL (p = 0.409).

Figure 1
Basal (1A) and pos-ACTH (1B) 17OHP levels (ng/mL) according to groups (median (min-max)) . Group I (+/+) included females with NC-CAH (hyperandrogenism and genetic analysis showing 2 pathogenic variants in the CYP21A2 ). Group II (+/−) included heterozygous carriers. Group III (−/−) included wild type females. *Nonparametric tests were applied to compare Group II (carriers) and Group I (NC-CAH patients), and Group II (carriers) and Group III (wild type individuals) (p < 0.001 for all the comparisons).

We found no statistically significant differences in DHEAS, androstenedione, or total testosterone levels between groups in either children or adolescent and adult women.

Regarding genotypes, the majority of the NC-CAH patients (Group I) were homozygous for the p.Val282Leu pathogenic variant (58% of the children and 50% of the adults) or compound heterozygous for the p.Val282Leu/del/conv promotor CYP21A2 pathogenic variants (17% of the children and 50% of the adults). As for the heterozygous carriers (Group II), the majority were simple heterozygous for the p.Val282Leu variant (90% of the children and 100% of the adults).

Clinical features did not significantly differ between patients homozygous for the p.Val282Leu variant and patients with the p.Val282Leu/del/conv genotype. Median basal and stimulated 17OHP were higher in homozygous than in compound heterozygous patients (respectively, 12.3 vs. 6.9 ng/mL (p = 0.367), and 17.8 vs. 19.0 ng/mL (p = 0.141)).

DISCUSSION

We have analyzed a population of 131 females suspected of having NC-CAH due to clinical and biochemical criteria, including 46 NC-CAH individuals, 49 heterozygous carriers, and 36 wild type subjects. We have explored our data in two groups (32 children and 99 adolescents and women), as we realize that young girls with premature pubarche are clinically different from adolescents and women with hirsutism, oligomenorrhea, or infertility. However, we found similar results regardless of the age group, including similar signs and symptoms and various 17OHP levels between groups.

Heterozygous carriers presented higher basal and ACTH-stimulated 17OHP levels than wild type subjects and lower basal and stimulated 17OHP levels than NC-CAH patients. In accordance with our data, some previous studies showed higher basal and/or stimulated 17OHP levels in heterozygous CYP21A2 pathogenic variant carriers than in non-carriers ( ⁵ , ⁹ , ¹⁰ , ¹⁶ , ¹⁷ , ²⁵ – ²⁸ ). Other studies, though, showed no significant differences in hormone levels between heterozygous carriers and the general population ( ¹² , ¹⁴ , ¹⁸ , ¹⁹ ). Conflicting results may have arisen due to differences in the participants’ selection criteria.

In our population, 17OHP concentrations exhibit significant overlap between carriers and the remaining two groups. In heterozygous carriers and wild type individuals, we found post-ACTH 17OHP levels > 3 ng/mL, a cutoff proposed for the identification of carrier status ( ²⁸ , ²⁹ ), and even some > 10 ng/mL, the cutoff usually applied for the NC-CAH diagnosis ( ³⁰ ). Therefore, we state that these groups cannot be distinctly separated according to biochemical criteria and underline that the detection of the carrier status should be based on molecular genotype analysis.

Admoni and cols. distinguished two groups of carriers, a group of symptomatic carriers presenting with premature pubarche, accelerated growth, and hirsutism and a group of asymptomatic family member carriers, and they stated that the symptomatic carriers had higher cosyntropin-stimulated 17OHP levels and a higher rate of the p.Val282Leu pathogenic variant than family member carriers ( ²⁷ ). They suggested that the subjects who carried the mild p.Val282Leu pathogenic variant had higher rates of androgen excess symptoms than the carriers of other severe pathogenic variants and speculated that the impairment of enzymatic activity in the symptomatic carriers results from this mutant allele’s dominant-negative effect on the wild type in which the mutant enzyme may compete or interfere with the wild type for the conversion of 17OHP to 11-deoxycortysol ( ²⁷ , ³¹ ). The monoallelic pathogenic variant affects the normal allele’s function, leading to a slight reduction in the 21OH activity, increased androgens secretion, and subsequent clinical manifestations ( ³¹ ). Other studies have supported this observation and showed higher rates of either PCOS or hirsutism in p.Val282Leu carriers but not in other mutation carriers ( ⁹ , ²⁷ , ³² ). In our population, the large majority of our carriers were simple heterozygous for the p.Val282Leu variant (90% of the children and 100% of the adults), which is the most frequent variant in the Portuguese population ( ³ ). This fact may explain our carriers’ higher 17OHP levels compared to wild type subjects, and we may question whether we would see different results if our heterozygous subjects carried other pathogenic variants.

Possible explanations for the phenotypic heterogeneity among individuals carrying the same pathogenic variant include the activity of other genes encoding proteins with extra-adrenal 21OH activity, the presence of other still unidentified mutations, the activity of other genes encoding proteins with extra-adrenal 21OH activity, and interindividual variation in androgens’ peripheral sensitivity and in the amount of protein produced ( ³³ ).

In our population, we found no significant differences in signs and symptoms between NC-CAH patients, heterozygous carriers, and wild type individuals. We only included females in the genetic study for the CYP21A2 gene and selected them based on their hormonal patterns and clinical features; therefore, the similarities between the three groups did not surprise us. Moreover, we obtained our data from clinical records, which we must interpret carefully. Our hyperandrogenic wild type girls probably presented premature adrenarche, a diagnosis of exclusion, while most of our post-menarche woman presented polycystic ovarian syndrome (PCOS). Premature adrenarche and PCOS are difficult to differentiate from NC-CAH and heterozygous carrier status based on clinical findings. We recommend treatment of symptomatic heterozygote carriers for the symptoms of androgen excess with both pharmacological therapies, including glucocorticoids and cosmetic approaches ( ²⁹ ).

It would have been interesting to compare 17OHP levels in individuals showing one hyperandrogenic sign or symptom versus individuals showing three or more hyperandrogenic signs or symptoms, both in the wild type and in the carriers group. However, due to this study’s retrospective nature, we could not conduct this analysis.

Limitations include the small sample size, our study’s retrospective design, and the fact that heterozygous carriers with monoallelic pathogenic variants were not studied for other point sequence variants within regulatory or epigenetic control regions in the CYP21 locus, mainly the analysis of the 3’untranslated region of the CYP21A2 gene (3′UTR region), where it was recently reported that 3’UTR sequence variants in combination with other pathogenic variants may be associated with a mild form of the disease ( ³⁴ ). More accurate biochemical predictors of the heterozygous carrier status, such as the serum 21-deoxycortisol ( ³⁵ , ³⁶ ) or the 17OHP/cortisol ratio ( ³⁷ ), were not available in our population. We did not analyze data regarding 17OHP levels in women without hyperandrogenic manifestations.

In conclusion, we found no significant differences in clinical features between NC-CAH patients, heterozygous carriers, and wild type females. The most common pathogenic variant in the CYP21A2 gene identified among NC-CAH patients and carriers was p.Val282Leu. Heterozygous carriers presented higher basal and post-cosyntropin 17OHP levels than wild type individuals and lower basal and stimulated 17OHP levels than NC-CAH patients. We discovered a considerable overlap between 17OHP levels among groups; therefore, normal basal and stimulated 17OHP responses do not exclude heterozygosity for CYP21A2 pathogenic variants.

• Funding: the authors have no relevant financial or non-financial interests to disclose. The authors have no financial or proprietary interests in any material discussed in this article.

REFERENCES

• ¹
  Miller WL. Clinical review 54: Genetics, diagnosis, and management of 21-hydroxylase deficiency. J Clin Endocrinol Metab. 1994;78(2):241-6.
• ²
  Baumgartner-Parzer SM, Nowotny P, Heinze G, Waldhausl W, Vierhapper H. Carrier frequency of congenital adrenal hyperplasia (21-hydroxylase deficiency) in a middle European population. J Clin Endocrinol Metab. 2005;90(2):775-8.
• ³
  Santos-Silva R, Cardoso R, Lopes L, Fonseca M, Espada F, Sampaio L, et al. CYP21A2 Gene Pathogenic Variants: A Multicenter Study on Genotype-Phenotype Correlation from a Portuguese Pediatric Cohort. Horm Res Paediatr. 2019;91(1):33-45.
• ⁴
  Speiser PW, Arlt W, Auchus RJ, Baskin LS, Conway GS, Merke DP, et al. Congenital Adrenal Hyperplasia Due to Steroid 21-Hydroxylase Deficiency: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2018;103(11):4043-88.
• ⁵
  Dacou-Voutetakis C, Dracopoulou M. High incidence of molecular defects of the CYP21 gene in patients with premature adrenarche. J Clin Endocrinol Metab. 1999;84(5):1570-4.
• ⁶
  Knorr D, Bidlingmaier F, Höller W, Kuhnle U, Meiler B, Nachmann A. Is heterozygosity for the steroid 21-hydroxylase deficiency responsible for hirsutism, premature pubarche, early puberty, and precocious puberty in children? Acta Endocrinol Suppl (Copenh). 1986;279:284-9.
• ⁷
  Paris F, Tardy V, Chalançon A, Picot MC, Morel Y, Sultan C. Premature pubarche in Mediterranean girls: high prevalence of heterozygous CYP21 mutation carriers. Gynecol Endocrinol. 2010;26(5):319-24.
• ⁸
  Binay C, Simsek E, Cilingir O, Yuksel Z, Kutlay O, Artan S. Prevalence of nonclassic congenital adrenal hyperplasia in Turkish children presenting with premature pubarche, hirsutism, or oligomenorrhoea. Int J Endocrinol. 2014;768506.
• ⁹
  Witchel SF, Aston CE. The role of heterozygosity for CYP21 in the polycystic ovary syndrome. J Pediatr Endocrinol Metab. 2000;13 Suppl 5:1315-7.
• ¹⁰
  Azziz R, Owerbach D. Molecular abnormalities of the 21-hydroxylase gene in hyperandrogenic women with an exaggerated 17-hydroxyprogesterone response to short-term adrenal stimulation. Am J Obstet Gynecol. 1995;172(3):914-8.
• ¹¹
  Neocleous V, Shammas C, Phedonos A, Phylactou L, Skordis N. Phenotypic variability of hyperandrogenemia in females heterozygous for CYP21A2 mutations. Indian J Endocrinol Metab. 2014;18:S72-9.
• ¹²
  Gao Y, Yu B, Mao J, Wang X, Nie M, Wu X. The prevalence of heterozygous CYP21A2 deficiency in patients with idiopathic acne, hirsutism, or both. Endocrine. 2020;67(3):665-72.
• ¹³
  Glintborg D, Hermann AP, Brusgaard K, Hangaard J, Hagen C, Andersen M. Significantly higher adrenocorticotropin-stimulated cortisol and 17-hydroxyprogesterone levels in 337 consecutive, premenopausal, caucasian, hirsute patients compared with healthy controls. J Clin Endocrinol Metab. 2005;90(3):1347-53.
• ¹⁴
  Kelestimur F, Everest H, Dundar M, Tanriverdi F, White C, Witchel SF. The frequency of CYP 21 gene mutations in Turkish women with hyperandrogenism. Exp Clin Endocrinol Diabetes. 2009;117(5):205-8.
• ¹⁵
  Witchel SF, Smith R, Tomboc M, Aston CE. Candidate gene analysis in premature pubarche and adolescent hyperandrogenism. Fertil Steril. 2001;75(4):724-30.
• ¹⁶
  Blanché H, Vexiau P, Clauin S, Le Gall I, Fiet J, Mornet E, et al. Exhaustive screening of the 21-hydroxylase gene in a population of hyperandrogenic women. Hum Genet. 1997;101(1):56-60.
• ¹⁷
  Witchel SF, Lee PA, Suda-Hartman M, Hoffman EP. Hyperandrogenism and manifesting heterozygotes for 21-hydroxylase deficiency. Biochem Mol Med. 1997;62(2):151-8.
• ¹⁸
  Knochenhauer ES, Cortet-Rudelli C, Cunnigham RD, Conway-Myers BA, Dewailly D, Azziz R. Carriers of 21-hydroxylase deficiency are not at increased risk for hyperandrogenism. J Clin Endocrinol Metab. 1997;82(2):479-85.
• ¹⁹
  Escobar-Morreale HF, San Millan JL, Smith RR, Sancho J, Witchel SF. The presence of the 21-hydroxylase deficiency carrier status in hirsute women: phenotype-genotype correlations. Fertil Steril. 1999;72(4):629-38.
• ²⁰
  Potau N, Rique S, Eduardo I, Marcos V, Ibanez L. Molecular defects of the CYP21 gene in Spanish girls with isolated precocious pubarche. Eur J Endocrinol. 2002;147(4):485-8.
• ²¹
  Voutilainen R, Jaaskelainen J. Premature adrenarche: etiology, clinical findings, and consequences. J Steroid Biochem Mol Biol. 2015;145:226-36.
• ²²
  Hatch R, Rosenfield RL, Kim MH, Tredway D. Hirsutism: implications, etiology, and management. Am J Obstet Gynecol. 1981;140(7):815-30.
• ²³
  Loidi L, Quinteiro C, Parajes S, Barreiro J, Leston DG, Cabezas-Agricola JM, et al. High variability in CYP21A2 mutated alleles in Spanish 21-hydroxylase deficiency patients, six novel mutations and a founder effect. Clin Endocrinol (Oxf). 2006;64(3):330-6.
• ²⁴
  Carvalho B, Pereira M, Marques CJ, Carvalho D, Leao M, Oliveira JP, et al. Comprehensive genetic analysis and structural characterization of CYP21A2 mutations in CAH patients. Exp Clin Endocrinol Diabetes. 2012;120(9):535-9.
• ²⁵
  Ostlere LS, Rumsby G, Holownia P, Jacobs HS, Rustin MH, Honour JW. Carrier status for steroid 21-hydroxylase deficiency is only one factor in the variable phenotype of acne. Clin Endocrinol (Oxf). 1998;48(2):209-15.
• ²⁶
  Kulle AE, Riepe FG, Hedderich J, Sippell WG, Schmitz J, Niermeyer L, et al. LC-MS/MS based determination of basal- and ACTH-stimulated plasma concentrations of 11 steroid hormones: implications for detecting heterozygote CYP21A2 mutation carriers. Eur J Endocrinol. 2015;173(4):517-24.
• ²⁷
  Admoni O, Israel S, Lavi I, Gur M, Tenenbaum-Rakover Y. Hyperandrogenism in carriers of CYP21 mutations: the role of genotype. Clin Endocrinol (Oxf). 2006;64(6):645-51.
• ²⁸
  Napolitano E, Manieri C, Restivo F, Composto E, Lanfranco F, Repici M, et al. Correlation between genotype and hormonal levels in heterozygous mutation carriers and non-carriers of 21-hydroxylase deficiency. J Endocrinol Invest. 2011;34(7):498-501.
• ²⁹
  Ahmadi S, Alvi S, Urban RJ. Nonclassic congenital adrenal hyperplasia and the heterozygote carrier. Expert Rev Endocrinol Metab. 2013;8(3):239-46.
• ³⁰
  Azziz R, Hincapie LA, Knochenhauer ES, Dewailly D, Fox L, Boots LR. Screening for 21-hydroxylase-deficient nonclassic adrenal hyperplasia among hyperandrogenic women: a prospective study. Fertil Steril. 1999;72(5):915-25.
• ³¹
  Félix-López X, Riba L, Ordóñez-Sánchez ML, Ramírez-Jiménez S, Ventura-Gallegos JL, Zentella-Dehesa A, et al. Steroid 21-hydroxylase (P450c21) naturally occurring mutants I172N, V281L and I236n/V237E/M239K exert a dominant negative effect on enzymatic activity when co-expressed with the wild-type protein. J Pediatr Endocrinol Metab. 2003;16(7):1017-24.
• ³²
  Bachega, TA, Brenlha EM, Billerbeck AE, Marcondes JA, Madureira G, Arnhold IJ, et al. Variable ACTH-stimulated 17-hydroxyprogesterone values in 21-hydroxylase deficiency carriers are not related to the different CYP21 gene mutations. J Clin Endocrinol Metab. 2002;87(2):786-90.
• ³³
  Witchel SF, Bhamidipati DK, Hoffman EP, Cohen JB. Phenotypic heterogeneity associated with the splicing mutation in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Endocrinol Metab 1996;81(11):4081-8.
• ³⁴
  Neocleous V, Fanis P, Toumba M, Phedonos AAP, Picolos M, Andreou E, et al. Variations in the 3’UTR of the CYP21A2 Gene in Heterozygous Females with Hyperandrogenaemia. Int J Endocrinol. 2017;8984365-8984365.
• ³⁵
  Cristoni S, Cuccato D, Sciannamblo M, Bernardi LR, Biunno I, Gerthoux P, et al. Analysis of 21-deoxycortisol, a marker of congenital adrenal hyperplasia, in blood by atmospheric pressure chemical ionization and electrospray ionization using multiple reaction monitoring. Rapid Commun Mass Spectrom. 2004;18(1):77-82.
• ³⁶
  Miller WL. Congenital Adrenal Hyperplasia: Time to Replace 17OHP with 21Deoxycortisol. Horm Res Paediatr. 2019;26:1-5.
• ³⁷
  Guarnotta V, Niceta M, Bono M, Marchese S, Fabiano C, Indelicato S, et al. Clinical and hormonal characteristics in heterozygote carriers of congenital hyperplasia. J Steroid Biochem Mol Biol. 2020;198:105554.

Publication Dates

• Publication in this collection
  23 Mar 2022
• Date of issue
  Mar-Apr 2022

History

• Received
  24 Apr 2021
• Accepted
  03 Nov 2021