Functional studies of novel CYP21A2 mutations detected in Norwegian patients with congenital adrenal hyperplasia

In about 95% of cases, congenital adrenal hyperplasia (CAH) is caused by mutations in CYP21A2 gene encoding steroid 21-hydroxylase (21OH). Recently, we have reported four novel CYP21A2 variants in the Norwegian population of patients with CAH, of which p.L388R and p.E140K were associated with salt wasting (SW), p.P45L with simple virilising (SV) and p.V211M+p.V281L with SV to non-classical (NC) phenotypes. We aimed to characterise the novel variants functionally utilising a newly designed in vitro assay of 21OH enzyme activity and structural simulations and compare the results with clinical phenotypes. CYP21A2 mutations and variants were expressed in vitro. Enzyme activity was assayed by assessing the conversion of 17-hydroxyprogesterone to 11-deoxycortisol by liquid chromatography tandem mass spectroscopy. PyMOL 1.3 was used for structural simulations, and PolyPhen2 and PROVEAN for predicting the severity of the mutants. The CYP21A2 mutants, p.L388R and p.E140K, exhibited 1.1 and 11.3% of wt 21OH enzyme activity, respectively, in vitro. We could not detect any functional deficiency of the p.P45L variant in vitro; although prediction tools suggest p.P45L to be pathogenic. p.V211M displayed enzyme activity equivalent to the wt in vitro, which was supported by in silico analyses. We found good correlations between phenotype and the in vitro enzyme activities of the SW mutants, but not for the SV p.P45L variant. p.V211M might have a synergistic effect together with p.V281L, explaining a phenotype between SV and NC CAH.

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SAT-277 Re-Evaluation of the 17-Hydroxyprogesterone (17-OHP) Screening Threshold for Diagnosing Nonclassic Congenital Adrenal Hyperplasia (NCCAH) in the Era of Liquid Chromatography Tandem-Mass Spectrometry (LC-MS/MS)

Journal of the Endocrine Society ◽

10.1210/js.2019-sat-277 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

Author(s):

Alexander Chesover ◽

Heather Millar ◽

Khosrow Adeli ◽

Mark Palmert ◽

Jill Hamilton

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

Congenital Adrenal Hyperplasia ◽

Tandem Mass ◽

Adrenal Hyperplasia ◽

Chromatography Tandem Mass Spectrometry ◽

Liquid Chromatography Tandem Mass ◽

17 Hydroxyprogesterone

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Prevalence of Nonclassic Congenital Adrenal Hyperplasia in Turkish Children Presenting with Premature Pubarche, Hirsutism, or Oligomenorrhoea

International Journal of Endocrinology ◽

10.1155/2014/768506 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 5

Author(s):

Cigdem Binay ◽

Enver Simsek ◽

Oguz Cilingir ◽

Zafer Yuksel ◽

Ozden Kutlay ◽

...

Keyword(s):

Congenital Adrenal Hyperplasia ◽

Mutational Analysis ◽

Autosomal Recessive Disorder ◽

Adrenal Hyperplasia ◽

Acth Stimulation ◽

Gene Encoding ◽

Turkish Children ◽

17 Hydroxyprogesterone ◽

Ovarian Syndrome ◽

Premature Pubarche

Background. Nonclassic congenital adrenal hyperplasia (NCAH), caused by mutations in the gene encoding 21-hydroxylase, is a common autosomal recessive disorder. In the present work, our aim was to determine the prevalence of NCAH presenting as premature pubarche (PP), hirsutism, or polycystic ovarian syndrome (PCOS) and to evaluate the molecular spectrum ofCYP21A2mutations in NCAH patients.Methods. A total of 126 patients (122 females, 4 males) with PP, hirsutism, or PCOS were included in the present study. All patients underwent an ACTH stimulation test. NCAH was considered to be present when the stimulated 17-hydroxyprogesterone plasma level was >10 ng/mL.Results. Seventy-one of the 126 patients (56%) presented with PP, 29 (23%) with PCOS, and 26 (21%) with hirsutism. Six patients (4,7%) were diagnosed with NCAH based on mutational analysis. Four different mutations (Q318X, P30L, V281L, and P453S) were found in six NCAH patients. One patient with NCAH was a compound heterozygote for this mutation, and five were heterozygous.Conclusion. NCAH should be considered as a differential diagnosis in patients presenting with PP, hirsutism, and PCOS, especially in countries in which consanguineous marriages are prevalent.

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Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency*

Endocrine Reviews ◽

10.1210/edrv.21.3.0398 ◽

2000 ◽

Vol 21 (3) ◽

pp. 245-291 ◽

Cited By ~ 9

Author(s):

Perrin C. White ◽

Phyllis W. Speiser

Keyword(s):

Congenital Adrenal Hyperplasia ◽

External Genitalia ◽

Clinical Severity ◽

Sodium Balance ◽

Adrenal Hyperplasia ◽

Hydroxylase Deficiency ◽

Gene Encoding ◽

Salt Wasting ◽

Direct Mutation ◽

21 Hydroxylase Deficiency

Abstract More than 90% of cases of congenital adrenal hyperplasia (CAH, the inherited inability to synthesize cortisol) are caused by 21-hydroxylase deficiency. Females with severe, classic 21-hydroxylase deficiency are exposed to excess androgens prenatally and are born with virilized external genitalia. Most patients cannot synthesize sufficient aldosterone to maintain sodium balance and may develop potentially fatal “salt wasting” crises if not treated. The disease is caused by mutations in the CYP21 gene encoding the steroid 21-hydroxylase enzyme. More than 90% of these mutations result from intergenic recombinations between CYP21 and the closely linked CYP21P pseudogene. Approximately 20% are gene deletions due to unequal crossing over during meiosis, whereas the remainder are gene conversions—transfers to CYP21 of deleterious mutations normally present in CYP21P. The degree to which each mutation compromises enzymatic activity is strongly correlated with the clinical severity of the disease in patients carrying it. Prenatal diagnosis by direct mutation detection permits prenatal treatment of affected females to minimize genital virilization. Neonatal screening by hormonal methods identifies affected children before salt wasting crises develop, reducing mortality from this condition. Glucocorticoid and mineralocorticoid replacement are the mainstays of treatment, but more rational dosing and additional therapies are being developed.

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Monitoring steroid replacement therapy in children with congenital adrenal hyperplasia

Journal of Pediatric Endocrinology and Metabolism ◽

10.1515/jpem-2016-0203 ◽

2017 ◽

Vol 30 (1) ◽

Cited By ~ 1

Author(s):

Niels H. Birkebaek ◽

David M. Hougaard ◽

Arieh S. Cohen

Keyword(s):

Congenital Adrenal Hyperplasia ◽

Adrenal Hyperplasia ◽

Serum Samples ◽

Monitoring Therapy ◽

Liquid Chromatography Tandem Mass ◽

Radio Immunoassay ◽

Highly Correlated ◽

17 Hydroxyprogesterone ◽

Good Agreement ◽

Time Point

AbstractBackground:The objective of this study was to compare the analysis of 17-hydroxyprogesterone (17-OHP) by radio-immunoassay (RIA) in serum with analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) on dried blood spot samples (DBSS) for monitoring therapy in children with congenital adrenal hyperplasia (CAH), and to investigate differences in 17-OHP values during the day.Methods:Fourteen children (8 females), median age 4.2 (0.3–16.0) years, were studied. Serum samples and DBSS were drawn before hydrocortisone dosing.Results:17-OHP by LC-MS/MS in DBSS were highly correlated to 17-OHP by RIA in serum, r=0.956, p<0.01. A total of 26 three-time-point series were investigated. Using only the afternoon 17-OHP values to determine the hydrocortisone doses would have led to overdosing seven times and underdosing six times.Conclusions:Good agreement was demonstrated between 17-OHP determination by RIA in serum and LC-MS/MS on DBSS. Multiple 17-OHP measurements per day are required to ensure sufficient hydrocortisone dose adjustment.

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Congenital Adrenal Hyperplasia with Salt Wasting Crisis: A Case Report

Journal of Nepal Medical Association ◽

10.31729/jnma.4811 ◽

2020 ◽

Vol 58 (221) ◽

Author(s):

Deependra Mandal ◽

Deepa Khanal ◽

Rajan Phuyal ◽

Uttara Adhikari

Keyword(s):

Congenital Adrenal Hyperplasia ◽

Elevated Level ◽

Autosomal Recessive ◽

Oral Prednisolone ◽

Severe Form ◽

Adrenal Hyperplasia ◽

Autosomal Recessive Disorders ◽

Salt Wasting ◽

17 Hydroxyprogesterone ◽

Recessive Disorders

Congenital Adrenal Hyperplasia is a group of autosomal recessive disorders due to deficienciesof enzymes involved in steroidogenesis. The most common form is a 21-hydroxylase deficiencywhich can be classical or non-classical. The severe form also called Classical Congenital AdrenalHyperplasia is usually detected after birth to infant period. If Congenital Adrenal Hyperplasia is notdiagnosed and treated early, neonates are susceptible to sudden death in the early weeks of life. Wereport a case of thirty-five days male with a salt-wasting variant of congenital adrenal hyperplasia.The diagnosis was based on an elevated level of 17-hydroxyprogesterone. He was managed and lifelong oral Prednisolone and Fludrocortisone were prescribed.

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Reverse circadian glucocorticoid treatment in prepubertal children with congenital adrenal hyperplasia

Journal of Pediatric Endocrinology and Metabolism ◽

10.1515/jpem-2021-0540 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Ilja Dubinski ◽

Susanne Bechtold Dalla-Pozza ◽

Martin Bidlingmaier ◽

Nicole Reisch ◽

Heinrich Schmidt

Keyword(s):

Congenital Adrenal Hyperplasia ◽

Pubertal Development ◽

Adrenal Crisis ◽

Adrenal Hyperplasia ◽

Glucocorticoid Treatment ◽

Steroid Synthesis ◽

Prepubertal Children ◽

Salt Wasting ◽

17 Hydroxyprogesterone

Abstract Objectives Children with salt-wasting congenital adrenal hyperplasia (CAH) have an impaired function of steroid synthesis pathways. They require therapy with glucocorticoid (GC) and mineralocorticoid hormones to avoid salt-wasting crisis and other complications. Most commonly, children receive hydrocortisone thrice daily with the highest dose in the morning, mimicking the regular physiology. However, reverse circadian treatment (RCT) had been suggested previously. In this study, we aimed to determine the efficacy of RCT in prepubertal children with CAH by comparing the salivary 17-hydroxyprogesterone (s17-OHP) levels individually. Methods In this retrospective study, we analyzed the records of children with classical CAH and RCT who were monitored by s17-OHP levels. The study included 23 patients. We identified nine prepubertal children with RCT schemes (three boys and six girls) and compared the s17-OHP levels in the morning, afternoon, and evening. The objective of this study was to demonstrate the non-effectiveness of RCT in terms of lowering the morning s17-OHP concentration. In addition, we compared s17-OHP day profiles in six patients on RCT and non-RCT therapy (intraindividually). Results Eight of nine children with RCT showed higher s17-OHP levels in the morning compared to the evening. In addition, none of the children showed a significant deviation of development. Three children were overweight. No adrenal crisis or pubertal development occurred. Comparison of RCT and non-RCT regimens showed no difference in 17-OHP profiles. Conclusions Our data do not support the use of RCT schemes for GC replacement in children with CAH due to lack of benefits and unknown long-term risks.

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Glucocorticoid Activity of Adrenal Steroid Precursors in Untreated Patients With Congenital Adrenal Hyperplasia

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2019-00547 ◽

2019 ◽

Vol 104 (11) ◽

pp. 5065-5072 ◽

Cited By ~ 5

Author(s):

Manon Engels ◽

Karijn J Pijnenburg-Kleizen ◽

Agustini Utari ◽

Sultana M H Faradz ◽

Sandra Oude-Alink ◽

...

Keyword(s):

Congenital Adrenal Hyperplasia ◽

Clinical Signs ◽

Adrenal Hyperplasia ◽

Blood Concentrations ◽

Adrenal Steroid ◽

17 Hydroxyprogesterone ◽

Cortisol Deficiency ◽

Steroid Precursor

Abstract Context and Objective We describe the clinical features and biochemical characteristics of a unique population of severely affected untreated patients with congenital adrenal hyperplasia (CAH) from an Indonesian population with proven cortisol deficiency but without clinical signs of cortisol deficiency. We evaluated the in vitro glucocorticoid activity of all relevant adrenal steroid precursors occurring in patients with CAH. Design Cross-sectional cohort study and translational research. Intervention/Main Outcome Measures Adrenal steroid precursor concentrations before and 60 minutes after ACTH administration to 24 untreated patients with CAH (3 to 46 years) with proven cortisol deficiency (<500 nmol/L post-ACTH) measured by liquid chromatography–tandem mass spectrometry were compared with six control patients (Mann-Whitney U test). Glucocorticoid receptor (GR) activation was determined by dual-luciferase assays in human embryonic kidney cells transfected with the GR and exposed to increasing amounts of adrenal steroid precursors for 24 hours. Results Blood concentrations of the steroid precursors 11-deoxycortisol (457 nmol/L, P = 0.003), 11-deoxycorticosterone (55 nmol/L, P = 0.003), 17-hydroxyprogesterone (610 nmol/L, P < 0.001), progesterone (29 nmol/L, P < 0.001), and 21-deoxycortisol (73 nmol/L) were strongly elevated compared with control subjects. The GR was activated with comparable potency to cortisol by corticosterone and 21-deoxycortisol or with 4 to 100 times lower potency by 11-hydroxyprogesterone, 11-deoxycortisol, aldosterone, 11-deoxycorticosterone, progesterone, and 17-hydroxyprogesterone. Conclusions We identified strongly elevated adrenal steroid precursor concentrations in blood from untreated patients with CAH and demonstrated glucocorticoid activity of these adrenal precursors in vitro, suggesting a possible role of these precursors in the clinical phenotype of these patients. Further studies are necessary to evaluate the role of these precursors in more detail.

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High aldosterone and cortisol levels in salt wasting congenital adrenal hyperplasia: a clinical conundrum

Journal of Pediatric Endocrinology and Metabolism ◽

10.1515/jpem-2017-0166 ◽

2017 ◽

Vol 30 (12) ◽

Cited By ~ 2

Author(s):

Sirisha Kusuma Boddu ◽

Sheeja Madhavan

Keyword(s):

Congenital Adrenal Hyperplasia ◽

Cross Reactivity ◽

Adrenal Hyperplasia ◽

Salt Wasting ◽

Adrenal Steroid ◽

Wasting Syndrome ◽

Serum Aldosterone ◽

Diagnostic Confusion ◽

Cortisol Levels ◽

17 Hydroxyprogesterone

AbstractBackground:Salt wasting syndrome (hyponatremia, hyperkalemia, dehydration, metabolic acidosis) in early infancy could be caused by either mineralocorticoid deficiency as in congenital adrenal hyperplasia (CAH) and adrenal insufficiency or mineralocorticoid resistance as in pseudohypoaldosteronism (PHA). In salt wasting CAH, serum aldosterone and cortisol levels are expected to be low. Cross reactivity between high levels of adrenal steroid precursors and aldosterone has recently been reported resulting in elevated aldosterone levels in CAH, leading to difficulty in differentiating between CAH and PHA.Case presentation:We report four such cases of salt wasting CAH, where high aldosterone levels and high normal cortisol levels led to initial diagnostic confusion with PHA. Diagnosis of CAH was later established on the basis of significantly elevated adrenocorticotropic hormone (ACTH) stimulated 17-hydroxyprogesterone (17-OHP) values.Conclusions:By reporting these cases we draw attention to the possibility that high levels of adrenal steroid precursors can cross react with aldosterone and cortisol, and underscore the significance of ACTH stimulated 17-OHP values in differentiating CAH and PHA.

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Late-diagnosed salt-wasting congenital adrenal hyperplasia in adult patient

Problems of Endocrinology ◽

10.14341/probl8813 ◽

2018 ◽

Vol 64 (2) ◽

pp. 105-110

Author(s):

Liudmila Ya. Rozhinskaya ◽

Natalia Yu. Kalinchenko ◽

Zhanna E. Belaya ◽

Tatiana S. Zenkova ◽

Alexander S. Lutsenko ◽

...

Keyword(s):

Congenital Adrenal Hyperplasia ◽

Molecular Genetic ◽

Gas Chromatography Mass Spectrometry ◽

Adrenal Hyperplasia ◽

Molecular Genetic Study ◽

Hydroxylase Deficiency ◽

Salt Wasting ◽

Adrenal Cortical Adenoma ◽

17 Hydroxyprogesterone ◽

21 Hydroxylase Deficiency

We describe a unique case of diagnosing salt-wasting congenital adrenal hyperplasia (CAH) caused by 21-hydroxylase deficiency in a 32-year-old male patient. Before establishing the diagnosis, the main patient complaint was infertility. In childhood, the patient suffered from developmental delay, often illness, overconsumption of salt, and often vomiting; he stopped growing at the age of 12 years. In the adolescent period, his physical condition improved. Later, the patient got married, but was infertile. Plasma steroid profiling by gas chromatography-mass spectrometry (GC-MS) revealed markedly increased levels of 17-hydroxyprogesterone and 21-deoxycortisol and a decreased cortisol level, which enabled the diagnosis of CAH and 21-hydroxylase deficiency. The diagnosis was confirmed by a molecular genetic study that detected a CYP21 gene mutation — l2 splice in the homo(hemi)zygotic state, which was characteristic of the salt-wasting form of CAH. Also, the patient had secondary adrenal cortical adenoma caused by prolonged ACTH hyperstimulation and secondary hypogonadism associated with excessive production of adrenal androgens, which led to elevated levels of estrogens inhibiting production of LH and FSH. Treatment of the patient with glucocorticoids and mineralocorticoids and then with androgens improved his clinical condition and hormonal parameters.

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