Challenging Molecular Diagnosis of Congenital Adrenal Hyperplasia (CAH) Due to 21-Hydroxylase Deficiency: Case Series and Novel Variants of CYP21A2 Gene.

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive genetic defects in cortisol synthesis and shows elevated ACTH concentrations, which in turn has downstream effects. The most common variant of CAH, 21-hydroxylase deficiency (21OHD), is the result of pathogenic variants in the CYP21A2 gene and is one of the most common monogenic disorders. However, the genetics of 21OHD is complex and challenging. The CYP21A2 gene is located in the RCCX copy number variation (CNV), a complex, multiallelic, and tandem CNV in the major histocompatibility complex (MHC) class III region on chromosome 6 (band 6p21.3). Here, CYP21A2 and its pseudogene CYP21A1P are located 30 kb apart and share a high nucleotide homology of approximately 98% and 96% in exons and introns, respectively. This high-sequence homology facilitates large structural rearrangements, copy number changes, and gene conversion through intergenic recombination. There is a good genotype-phenotype correlation in 21OHD, and genotyping can be performed to confirm the clinical diagnosis, predict long-term outcomes, and determine genetic counseling. Thus, genotyping in CAH is clinically relevant but the interpretations can be challenging for non-initiated clinicians. Here, there are some concrete examples of how molecular diagnosis can sometimes require the use of multiple molecular strategies.

Genetic Disruption of cyp21a2 Leads to Systemic Glucocorticoid Deficiency and Tissues Hyperplasia in the Teleost Fish Medaka (Oryzias latipes).

cytochrome P-450, 21-hydroxylase (cyp21a2), encodes an enzyme required for cortisol biosynthesis, and its mutations are the major genetic cause of congenital adrenal hyperplasia (CAH) in humans. Here, we have generated a null allele for the medaka cyp21a2 with a nine base-pair insertion which led to a truncated protein. We have observed a delay in hatching and a low survival rate in homozygous mutants. The interrenal gland (adrenal counterpart in teleosts) exhibits hyperplasia and the number of pomca-expressing cells in the pituitary increases in the homozygous mutant. A mass spectrometry-based analysis of whole larvae confirmed a lack of cortisol biosynthesis, while its corresponding precursors were significantly increased, indicating a systemic glucocorticoid deficiency in our mutant model. Furthermore, these phenotypes at the larval stage are rescued by cortisol. In addition, females showed complete sterility with accumulated follicles in the ovary while male homozygous mutants were fully fertile in the adult mutants. These results demonstrate that the mutant medaka recapitulates several aspects of cyp21a2-deficiency observed in humans, making it a valuable model for studying steroidogenesis in CAH.

Targeted long-read sequencing for comprehensive detection of CYP21A2 mutations in patients with 21-hydroxylase deficiency.

BACKGROUND: 21-Hydroxylase deficiency (21-OHD) is caused by pathogenic CYP21A2 variations. CYP21A2 is arranged in tandem with its highly homologous pseudogene CYP21A1P; therefore, it is prone to mismatch and rearrangement, producing different types of complex variations. There were few reports on using only one method to detect different CYP21A2 variants simultaneously. AIMS: Targeted long-read sequencing method was used to detect all types of CYP21A2 variants in a series of patients with 21-OHD. METHODS: A total of 59 patients with 21-OHD were enrolled from Peking Union Medical College Hospital. Long-range locus-specific PCR and long-read sequencing (LRS) were performed to detect the pathogenic variants in CYP21A2. RESULTS: Copy-number variants of CYP21A2 were found in 25.4% of patients, including 5.1% with 3 copies of CYP21A2, 16.9% with 1 copy of CYP21A2, and 3.4% with 0 copy of CYP21A2. The remaining 74.6% of patients had 2 copies of CYP21A2. Pathogenic variants were identified in all 121 alleles of 59 patients. Specifically, single-nucleotide variants and small insertions/deletions (< 50 bp) were detected in 79 alleles, of which conversed from CYP21A1P were detected in 63 alleles, and rare variants were found in the other 16 alleles. Large gene conversions (> 50 bp) from pseudogene were detected in 10 alleles, and different chimeric genes (CYP21A1P/CYP21A2 or TNXA/TNXB) formed by large deletions were detected in 32 alleles. Of all variants, p.I173N was the most common variant (19.0%). CONCLUSIONS: Our study demonstrated that targeted long-read sequencing is a comprehensive method for detecting CYP21A2 variations, which is helpful for genetic diagnosis in 21-OHD patients.

Classic congenital adrenal hyperplasia with unilateral functional adrenal cortical adenoma: case report.

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders related to adrenal steroid biosynthesis, and mainly caused by mutations in the CYP21A2 gene encoding 21-hydroxylase. Adrenal tumors are common in CAH, but functional adrenal tumors are rare. Here, we report a 17-year-old female with virilized external genitalia and primary amenorrhea, accompanied by a right adrenal tumor. Her 17-OHP level was normal, cortisol and androgen levels were significantly elevated, and the tumor pathology showed adrenal cortical adenoma. Gene testing for CYP21A2 showed c.518T > A in exon 4 and c.29313C > G in intron 2. The possibility of untreated classic CAH with 21-OH deficiency causing functional adrenal cortical adenoma should be considered. When clinical diagnosis highly considers CAH and cannot rule out the influence of functional adrenal tumors' secretion function on 17-OHP, gene mutation analysis should be performed.

Genotype-phenotype association in congenital adrenal hyperplasia due to 21-hydroxylase deficiency in children.

OBJECTIVE: The purpose of this study was to investigate the clinical manifestation of various forms of congenital adrenal hyperplasia (CAH) in children of the Republic of Kazakhstan, depending on their genotype. DESIGN: The study analysed 50 patients diagnosed with CAH from 7 regions of Kazakhstan with different ethnic origins: 35 Kazakhs (70.0%), 8 Russians (16.0%), 2 Turks (4.0%), 2 Ukrainians (4.0%), 2 Uzbeks (4%), 1 Uighur (2%). All the children studied were from 0 to 18 years old, and their average age was 5.7 years ±3.9. In addition, all children were divided into groups depending on the form of the disease according to the phenotypic manifestation of the disease: salt-wasting (SW) and simple virile (SV) forms. Most of the patients suffered from SW - 32 (64.0%), and a smaller group had SV - 18 (36.0%), also one boy with SW was diagnosed with TART syndrome. MEASUREMENTS: 50 Kazakh children with the classical form of CAH were analysed. Depending on the severity of the mutations, patients were divided into 4 groups: zero groups (the most severe mutations), A, B, and C. RESULTS: According to the results of the study, the salt-wasting form of CAH turned out to be more common than the simple virile form. A high correlation was observed in groups with mutations of high and moderate severity - 0 and A, while group C showed a strong variability of the phenotype. Thus, the correspondence between genotype and phenotype decreased along with the decrease in the severity of the disease. CONCLUSIONS: The relationship between the genotype and the phenotype of both forms of CAH exists indirectly, through the activity of the 21-hydroxylase enzyme. Mutations in the CYP21A2 gene affect the level of the synthesized enzyme, which, in turn, determines the degree of hormone production in the blood.

Unexpectedly high mutation rate of cyp11b1 compared to cyp21a2 in randomly-selected turkish women: a large screening study.

PURPOSE: Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders resulting from enzyme deficiencies associated with steroidogenesis. The clinical presentation of non-classic CAH (NCAH) in females is often indistinguishable from other hyperandrogenic disorders like polycystic ovary syndrome (PCOS). The data on the prevalence of NCAH in unselected women in the literature is scanty. The research aimed to evaluate the prevalence of NCAH, carrier frequencies, and the correlation between clinical symptoms and genotype in Turkish women. METHODS: The study group comprised two hundred and seventy randomly-selected unrelated asymptomatic women of reproductive age (18-45). Subjects were recruited from female blood donors. All volunteers underwent clinical examination and hormone measurements. The protein-encoding exons and exon-intron boundaries of the CYP21A2, CYP11B1, HSD3ß2 and CYP21A2 promoter were sequenced by direct DNA sequencing. RESULTS: After genotyping, seven (2.2%) individuals were diagnosed with NCAH. The heterozygous carrier frequencies of CYP21A2, CYP21A2 promoter, CYP11B1, and HSD3ß2 genes with 34, 34, 41, and 1 pathologic mutation were determined at 12.6%, 12.6%, 15.2%, and 0.37% of volunteers, respectively. Gene-conversion (GC) frequencies between CYP21A2/CYP21A1P and CYP11B1/CYP11B2 were determined as 10.4% and 14.8%, respectively. CONCLUSION: Despite GC-derived higher mutation frequency determined in the CYP11B1 gene, the reason for the low frequency of NCAH due to 11OHD compared to 21OHD might be that gene-conversion arises with active CYP11B2 rather than an inactive pseudogene. HSD3ß1 exhibits high homology with HSD3ß2 located on the same chromosome; remarkably, it demonstrates low heterozygosity and no GC, most probably the outcome of a tissue-specific expression pattern.

Clinical analysis of 78 patients with nonclassical 21-hydroxylase deficiency.

PURPOSE: Retrospectively analyze the clinical characteristics of patients with nonclassical 21-hydroxylase deficiency (NC21OHD) as well as the relationship between the gene mutations and endocrine hormones. In addition, the relationship between different basal 17-hydroxyprogesterone (17OHP) levels and patients' glucolipid metabolism, hormone levels, pregnancy, and treatment outcomes were examined. METHODS: Clinical data of 78 females with NC21OHD from January 2012 to July 2022 in the Department of Endocrinology and Metabolism of the Third Affiliated Hospital of Guangzhou Medical University were retrospectively analyzed. Diagnosis was based on the 17OHP level combined with clinical manifestations, imaging, and other endocrine hormones and the cytochrome P450 c21, steroid 21-hydroxylase (CYP21A2) gene. RESULTS: The age at diagnosis of the 78 patients was 29.1 ± 4.2 years; 83.3% (65/78) of the patients had menstrual abnormalities, 70 patients were of childbearing age, and 97.1% (68/70) had a history of infertility with a median time of infertility of 3.6 years. Moreover, 71.8% (56/78) of the patients had polycystic ovaries, 26.9% (21/78) had hyperandrogenemia manifestations on physical examination, 66.7% (52/78) had adrenal hyperplasia, 32.1% (25/78) had combined dyslipidemia, and 41.0% (32/78) had combined insulin resistance. Pathogenic mutations were detected in 78.2% (61/78) of the patients with both CYP21A2 alleles; 14.1% (11/78) of the patients had only one allele and 7.7% (6/78) had no pathogenic mutations. The levels of total testosterone (TT), progesterone (P) (0 min, 30 min), and 17-OHP (0 min, 30 min, 60 min) in the adrenocorticotropic hormone (ACTH) stimulation test varied between the groups. Furthermore, patients with NC21OHD were divided into 17OHP < 2 ng/ml, 2 ng/ml < 17OHP < 10 ng/ml, and 17OHP ≥ 10 ng/ml groups according to their different basal 17OHP levels. The 17OHP ≥ 10 ng/ml group had significantly higher TT, FT4, basal and post-stimulation progesterone, and 17OHP, net value added of 17-hydroxyprogesterone (△17OHP), net value added of 17-hydroxyprogesterone/net value added of cortisol ratio (△17OHP/△F), the incidence of adrenal hyperplasia, and number of gene mutations compared to those of the 17OHP < 2 ng/ml group (P < 0.05). NC21OHD infertile patients who received low-dose glucocorticoids showed a significant increase in pregnancy and live birth rates, and a significant decrease in miscarriage rate (all P < 0.05). CONCLUSION: Comprehensive analysis is important as NCCAH diagnoses may be false positive or false negative based on clinical characteristics, hormone levels, and gene detection. Females with NC21OHD showed varying degrees of fertility decline; thus, low doses of glucocorticoid treatment for infertile females with NC21OHD can improve fertility and fertility outcomes.

The discovery of BMS-737 as a potent, CYP17 lyase-selective inhibitor for the treatment of castration-resistant prostate cancer.

We report herein, the discovery of BMS-737 (compound 33) as a potent, non-steroidal, reversible small molecule inhibitor demonstrating 11-fold selectivity for CYP17 lyase over CYP17 hydroxylase, as well as a clean xenobiotic CYP profile for the treatment of castration-resistant prostate cancer (CRPC). Extensive SAR studies on the initial lead 1 at three different regions of the molecule resulted in the identification of BMS-737, which demonstrated a robust 83% lowering of testosterone without any significant perturbation of the mineralocorticoid and glucocorticoid levels in cynomologous monkeys in a 1-day PK/PD study.

Genotypic spectrum of 21-hydroxylase deficiency in an endogamous population.

BACKGROUND: Congenital adrenal hyperplasia (CAH) due to autosomal recessive 21-hydroxylase deficiency (21-OHD) is caused by defects in the CYP21 (CYP21A2) gene. Several mutations have been identified in the CYP21 (CYP21A2) gene of patients with 21-OHD. We aimed at determining the frequency of these mutations among a group of Egyptian patients and studying the genotype-phenotype correlation. METHODS: Forty-seven patients with CAH due to 21-OHD from 42 different families diagnosed by clinical and hormonal evaluation and classified accordingly into salt wasting (SW) and simple virilizing (SV) phenotypes were enrolled. Their ages ranged between 1.78 and 18.99 years. Molecular analysis of the CYP21 (CYP21A2) gene was performed for the detection of eleven common mutations: P30L, I2 splice (I2 G), Del 8 bp E3 (G110del8nt), I172N, cluster E6 (I236N, V237E, M239K), V281L, L307 frameshift (F306 + T), Q318X, R356W, P453S, R483P by polymerase chain reaction (PCR) and reverse hybridization. RESULTS: Disease-causing mutations were identified in 47 patients, 55.31% of them were compound heterozygous. The most frequent mutations were I2 splice (25.43%), followed by cluster E6 (16.66%) and P30L (15.78%). Two point mutations (P453S, R483P) were not identified in any patient. In the SW patients, genotypes were more compatible with their phenotypes. CONCLUSION: Molecular characterization should be considered along with clinical and biochemical diagnosis of CAH since it could confirm the diagnosis, outline the treatment strategy and morbidity, and ensure proper genetic counseling.

First insights into the genetics of 21-hydroxylase deficiency in the Roma population.

BACKGROUND: 21-hydroxylase deficiency (21OHD) is an autosomal recessive disorder with an incidence of 1:10,000-1:20,000 and is the result of various mutations in the CYP21A2 gene. 21OHD has been described in many different populations, but it has not been studied in Roma individuals so far. The aim of the study was to analyse the genotype in Roma patients with 21OHD and the prevalence of the disease in the Roma population of North Macedonia. METHODS: Molecular analysis of the nine most frequent CYP21A2 mutations in all known Roma patients with CAH in North Macedonia, relatives and healthy individuals of Roma ancestry, using the PCR/ACRS method. RESULTS: Ten Roma patients with 21OHD were identified, of which nine had the salt-wasting and one had the simple virilizing form. Calculated incidence of 21OHD in the North Macedonian Roma population was 1:3375. Interestingly, 9/10 patients (90%) were homozygous for the In2G splicing mutation (293-13A/C > G). Standard therapy with hydrocortisone and fludrocortisone had been introduced according to the guidelines. In 16 healthy relatives investigated for CYP21A2 mutations, heterozygosity for the In2G mutation was detected in 13/32 (40.6%) alleles. In 100 healthy Roma individuals, none related to the analysed families, no CYP21A2 mutations were detected. CONCLUSION: The Roma population in North Macedonia had a very high incidence of classic 21OHD. Almost all patients had the severe salt-wasting form and the In2G/In2G genotype.

Reassessment of predictive values of ACTH-stimulated serum 21-deoxycortisol and 17-hydroxyprogesterone to identify CYP21A2 heterozygote carriers and nonclassic subjects.

INTRODUCTION: Heterozygotes (HZs) for 21-hydroxylase deficiency (21OHD) are highly prevalent, ranging from 1:60 to 1:11 for classic and nonclassic (NC) forms, respectively. Detection of HZ and asymptomatic NC by CYP21A2 genotyping is valuable for genetic counselling, but costly, complex and narrowly available. Adrenocorticotropic hormone (ACTH)-stimulated serum 17-hydroxyprogesterone (17P) and 21-deoxycortisol (21DF) discriminate 21OHD phenotypes effectively, notably if measured simultaneously by liquid chromatography-tandem mass spectrometry (LC-MS/MS). OBJECTIVE: This study was performed to reassess former LC-MS/MS-defined post-ACTH 21DF, 17P and cortisol (F) cutoffs in family members at risk for 21OHD. DESIGN AND PATIENTS: Prospective study in which we screened 58 asymptomatic relatives from families with 21OHD patients and compared post-ACTH steroid phenotypes with subsequent genotypes. RESULTS: Post-ACTH 21DF, 17P, F and (21DF + 17P)/F ratio segregate NC, HZ and wild-type (WT) phenotypes (subsequently genotyped) with some overlap. New receiver operating characteristic curve-defined cutoffs for post-ACTH 21DF, 17P and (21DF + 17P)/F ratio are 60 ng/dl, 310 ng/dl and 12 (unitless). Twenty-six of 33 HZ and all 6 NC (82.1%) had post-ACTH 21DF > 60 and 17P > 310 ng/dl, whereas 17/19 WT (89.5%) had values below cutoffs. Post-ACTH 21DF and 17P had a strong positive correlation (r = .9558; p < .001). A (21DF + 17P)/F ratio > 12 correctly identified 36 of 39 HZ plus NC (92.3% sensitivity) with 84.2% specificity (16 of 19 WT). Given the high frequency of 21OHD HZ, the negative prediction of ratio values below 12 excludes heterozygosity in 99.8% and 99.1% for classic and NC mutations, respectively. CONCLUSIONS: Reassessed ACTH-stimulated 21DF and 17P cutoffs by LC-MS/MS (60 and 310 ng/dl, respectively) correctly recognised 82.5% HZ plus NC, but combined precursor-to-product ratio ([21DF + 17P]/F) cutoff of 12 was superior, identifying 92.3% HZ plus NC. Since one WT subject is an outlier (potential HZ), these values would be somewhat better reinforcing their utility for screening asymptomatic relatives at risk for 21OHD.

Exploration of CYP21A2 and CYP17A1 polymorphisms and preeclampsia risk among Chinese Han population: a large-scale case-control study based on 5021 subjects.

BACKGROUND: Several genome-wide association studies have identified single-nucleotide polymorphisms (SNPs), such as rs4409766, rs1004467, and rs3824755 in CYP17A1 and rs2021783 in CYP21A2, as new hypertension susceptibility genetic variants in the Chinese population. This study aimed to look into the relationship between preeclampsia (PE) and these SNPs in Chinese Han women. METHODS: Overall, 5021 unrelated pregnant women were recruited, including 2002 patients with PE and 3019 normal healthy controls. The real-time PCR (TaqMan) method was applied to genotype these four polymorphisms. RESULTS: A statistically obvious difference in the allelic frequencies was observed in CYP21A2 rs2021783 between cases and controls (χ2 = 7.201, Pc = 0.028 by allele), and the T allele was associated with the occurrence and development of PE (OR = 1.151, 95% CI 1.039-1.275). We also found a significant association between rs2021783 and the development of early-onset PE (Pc = 0.008 by genotype, Pc = 0.004 by allele). For rs1004467 and rs3824755, the distribution of allelic frequencies differed markedly between mild PE and control groups (χ2 = 6.843, Pc = 0.036; χ2 = 6.869, Pc = 0.036), and patients with the TT genotype of rs1004467 were less easy to develop mild PE than were those carrying the CT or CC genotype (χ2 = 7.002, Pc = 0.032, OR = 1.306, 95% CI 1.071-1.593). The GG genotype of rs3824755 appeared to a protective effect on the occurrence of mild PE (OR = 0.766, 95% CI 0.629-0.934). CONCLUSIONS: CYP21A2 rs2021783 appears to be closely related to PE susceptibility, and CYP17A1 rs1004467 and rs3824755 seem to be closely associated with mild PE in Han women.

POR polymorphisms are associated with 21 hydroxylase deficiency.

PURPOSE: Genotype-phenotype correlation in congenital 21 hydroxylase deficiency is strong but by no means absolute. Indeed, clinical and hormonal features may vary among patients carrying similar CYP21A2 mutations, suggesting that modifier genes may contribute to the phenotype. Aim of the present study was to evaluate whether polymorphisms in the p450  oxidoreductase (POR) gene may affect clinical features in patients with 21 hydroxylase deficiency METHODS: Sequencing of the POR gene was performed in 96 patients with 21 hydroxylase deficiency (49 classic, 47 non-classic) and 43 control subjects. RESULTS: Prevalence of POR polymorphisms in patients with 21 hydroxylase was comparable to controls and known databases. The rs2228104 polymorphism was more frequently associated with non-classic vs classic 21 hydroxylase deficiency (allelic risk 7.09; 95% C.I. 1.4-29.5, p < 0.05). Classic 21 hydroxylase-deficient carriers of the minor allele in the rs2286822/rs2286823 haplotype presented more frequently the salt-wasting form (allelic risk 1.375; 95% C.I. 1.138-1.137), more severe Prader stage at birth (allelic risk 3.85; 95% C.I. 3.78-3.92), higher ACTH levels, and younger age at diagnosis. CONCLUSIONS: Polymorphisms in the POR gene are associated with clinical features of 21 hydroxylase deficiency both as regards predisposition to classic vs non-classic forms and severity of classic adrenal hyperplasia.

Genetic profiling of CAH Egyptian children: rapid guide to clinical interpretation of common mutations.

OBJECTIVES: The prevalence of CAH in Egypt is reported to be ten times more than that of the worldwide prevalence. The study aimed at genetic screening of children diagnosed with 21-alpha hydroxylase deficiency congenital adrenal hyperplasia (21OHD-CAH). In addition, the study offers a rapid and easy guide for clinical reporting of common mutations for endocrinologists. METHODS: A cohort of 174 unrelated Egyptian children with 21OHD-CAH were screened for 11 common CYP21A2 gene mutations using a strip hybridization assay, and then, bioinformatics analysis was done to report the pathogenicity of the common mutations for clinical classification. RESULTS: The most common mutations were I2 splice and p.Q318X. Deletions/conversions comprised 45.9% of the cohort, whereas 7.4% of the cases were negative for all mutations. The least positively detected point mutations were p.P453S, cluster E6, p.R483P, and p.L307FS, which were detected in fewer than 5% of cases. CONCLUSION: Strip hybridization assay is a rapid screening tool for the diagnosis of CAH. The authors hypothesized an easy and rapid scheme for clinical interpretation of the strip results to gain the highest value of the strip in diagnosis.

Challenges of CYP21A2 genotyping in children with 21-hydroxylase deficiency: determination of genotype-phenotype correlation using next generation sequencing in Southeastern Anatolia.

BACKGROUND/PURPOSE: Although it is known that there is generally a good correlation between genotypes and phenotypes, the number of studies reporting discrepancies has recently increased, exclusively between milder genotypes and their phenotypes due to the complex nature of the CYP21A2 gene and methodological pitfalls. This study aimed to assess CYP21A2 genotyping in children with 21-hydroxylase deficiency (21-OHD) and establish their predictive genotype-phenotype correlation features using a large cohort in Southeastern Anatolia's ethnically diverse population. METHODS: The patients were classified into three groups: salt-wasting (SW), simple virilizing (SV) and non-classical (NC). The genotypes were categorized into six groups due to residual enzyme activity: null-A-B-C-D-E. CYP21A2 genotyping was performed by sequence-specific primer and sequenced with next generation sequencing (NGS), and the expected phenotypes were compared to the observed phenotypes. RESULTS: A total of 118 unrelated children with 21-OHD were included in this study (61% SW, 24.5% SV and 14.5% NC). The pathogenic variants were found in 79.5% of 171 mutated alleles (60.2%, 22.2%, and 17.6% in SW, SV and NC, respectively). Patient distribution based on genotype groups was as follows: null-16.1%, A-41.4%, B-6.0%, C-14.4%, E-22%). In2G was the most common pathogenic variant (33.9% of all alleles) and the most common variant in the three phenotype groups (SW-38.8%, SV-22.2% and NC-23.3%). The total genotype-phenotype correlation was 81.5%. The correlations of the null and A groups were 100% and 76.1%, respectively, while it was lower in group B and poor in group C (71.4% and 23.5%, respectively). CONCLUSION: This study revealed that the concordance rates of the severe genotypes with their phenotypes were good, while those of the milder genotypes were poor. The discrepancies could have resulted from the complex characteristics of 21-OHD genotyping and the limitations of using NGS alone without integrating with other comprehensive methods.

Characterization of Mutations Causing CYP21A2 Deficiency in Brazilian and Portuguese Populations.

Deficiency of 21-hydroxylase enzyme (CYP21A2) represents 90% of cases in congenital adrenal hyperplasia (CAH), an autosomal recessive disease caused by defects in cortisol biosynthesis. Computational prediction and functional studies are often the only way to classify variants to understand the links to disease-causing effects. Here we investigated the pathogenicity of uncharacterized variants in the CYP21A2 gene reported in Brazilian and Portuguese populations. Physicochemical alterations, residue conservation, and effect on protein structure were accessed by computational analysis. The enzymatic performance was obtained by functional assay with the wild-type and mutant CYP21A2 proteins expressed in HEK293 cells. Computational analysis showed that p.W202R, p.E352V, and p.R484L have severely impaired the protein structure, while p.P35L, p.L199P, and p.P433L have moderate effects. The p.W202R, p.E352V, p.P433L, and p.R484L variants showed residual 21OH activity consistent with the simple virilizing phenotype. The p.P35L and p.L199P variants showed partial 21OH efficiency associated with the non-classical phenotype. Additionally, p.W202R, p.E352V, and p.R484L also modified the protein expression level. We have determined how the selected CYP21A2 gene mutations affect the 21OH activity through structural and activity alteration contributing to the future diagnosis and management of CYP21A2 deficiency.

Genetic characterization of a large cohort of Argentine 21-hydroxylase Deficiency.

CONTEXT: 21-hydroxylase deficiency is the most common cause of Congenital Adrenal Hyperplasia. It presents as severe or classical forms-salt wasting and simple virilizing-and a mild or nonclassical (NC). Several studies have reported the frequency of pathogenic variants in different populations, although few of them included a large number of NC patients. OBJECTIVE: To analyse the CYP21A2 gene defects in a large cohort of Argentine patients. DESIGN: Molecular characterization of 628 patients (168 classical, 460 nonclassical, representing 1203 nonrelated alleles), 398 relatives, 126 partners. METHODS: Genetic variants were assessed by allele-specific PCR, PCR-RFLP or direct sequencing. Deletions, duplications and large gene conversions (LGC) were studied by Southern blot/MLPA or long-range PCR. Biological implications of novel variants were analysed by structure-based in silico studies. RESULTS: The most frequent pathogenic variants were p.V282L (58%) in NC alleles and c.293-13C>G (31.8%) and p.I173N (21.1%) in classical. Deletions and LGC were found at low frequency (6.2%), 57 alleles had rare pathogenic variants, and 3 had novel variants: p.(S166F); p.(P189R), p.(R436L). Genotype-phenotype correlation was observed in 98.6% of the cases, 11 asymptomatic first-degree relatives had pathogenic variants in both alleles, and 21/126 partners were carriers. CONCLUSIONS: We conducted a comprehensive genetic characterization of the largest cohort of 21-hydroxylase patients from the region. In particular, we add to the molecular characterization of a large number of NC patients and to the estimation of the disease carrier's frequency in our population.

Identification and circumvention of bottlenecks in CYP21A2-mediated premedrol production using recombinant Escherichia coli.

Synthetic glucocorticoids such as methylprednisolone are compounds of fundamental interest to the pharmaceutical industry as their modifications within the sterane scaffold lead to higher inflammatory potency and reduced side effects compared with their parent compound cortisol. In methylprednisolone production, the complex chemical hydroxylation of its precursor medrane in position C21 exhibits poor stereo- and regioselectivity making the process unprofitable and unsustainable. By contrast, the use of a recombinant E. coli system has recently shown high suitability and efficiency. In this study, we aim to overcome limitations in this biotechnological medrane conversion yielding the essential methylprednisolone-precursor premedrol by optimizing the CYP21A2-based whole-cell system on a laboratory scale. We successfully improved the whole-cell process in terms of premedrol production by (a) improving the electron supply to CYP21A2; here we use the N-terminally truncated version of the bovine NADPH-dependent cytochrome P450 reductase (bCPR-27 ) and coexpression of microsomal cytochrome b5 ; (b) enhancing substrate access to the heme by modification of the CYP21A2 substrate access channel; and (c) circumventing substrate inhibition which is presumed to be the main limiting factor of the presented system by developing an improved fed-batch protocol. By overcoming the presented limitations in whole-cell biotransformation, we were able to achieve a more than 100% improvement over the next best system under equal conditions resulting in 691 mg·L-1 ·d-1 premedrol.

A rare CYP21A2 haplotype clarifies the phenotype-genotype discrepancy in an Italian patient with Non Classical Congenital Adrenal Hyperplasia (NC-CAH).

RCCX haplotypes with two copies of the CYP21A2 gene and one copy of the CYP21A1P pseudogene have been widely described in different populations. In most cases, the CYP21A2-like gene downstream of the TNXA gene showed a wild-type sequence or the c.293-13A/C > G variant while the CYP21A2 gene next to TNXB carried the p.(Gln319Ter) variant. Here is the discovery of a novel rare CYP21A2 haplotypes detected in an Italian patient with Non Classical Congenital Adrenal Hyperplasia (NC-CAH). The molecular family study was performed clarifying the previously found phenotype-genotype discrepancy.

Steroid biomarkers for identifying non-classic adrenal hyperplasia due to 21-hydroxylase deficiency in a population of PCOS with suspicious levels of 17OH-progesterone.

OBJECTIVE: We aimed at defining the most effective routine immunoassay- or liquid chromatography-tandem mass spectrometry (LC-MS/MS)-determined steroid biomarkers for identifying non-classic adrenal hyperplasia due to 21-hydroxylase deficiency (21-NCAH) in a PCOS-like population before genotyping. METHODS: Seventy PCOS-like patients in reproductive age with immunoassay-determined follicular 17OH-progesterone (17OHP) ≥ 2.00 ng/mL underwent CYP21A2 gene analysis and 1-24ACTH test. Serum steroids were measured by immunoassays at baseline and 60 min after ACTH stimulation; basal steroid profile was measured by LC-MS/MS. RESULTS: Genotyping revealed 23 21-NCAH, 15 single allele heterozygous CYP21A2 mutations (21-HTZ) and 32 PCOS patients displaying similar clinical and metabolic features. Immunoassays revealed higher baseline 17OHP and testosterone, and after ACTH stimulation, higher 17OHP (17OHP60) and lower cortisol, whereas LC-MS/MS revealed higher 17OHP (17OHPLC-MS/MS), progesterone and 21-deoxycortisol and lower corticosterone in 21-NCAH compared with both 21-HTZ and PCOS patients. Steroid thresholds best discriminating 21-NCAH from 21-HTZ and PCOS were estimated, and their diagnostic accuracy in identifying 21-NCAH from PCOS was established by ROC analysis. The highest accuracy was observed for 21-deoxycortisol ≥ 0.087 ng/mL, showing 100% sensitivity, while the combination of 17OHPLC-MS/MS ≥ 1.79 ng/mL and corticosterone ≤ 8.76 ng/mL, as well as the combination of ACTH-stimulated 17OHP ≥ 6.77 ng/mL and cortisol ≤ 240 ng/mL by immunoassay, showed 100% specificity. CONCLUSIONS: LC-MS/MS measurement of basal follicular 21-deoxycortisol, 17OHP and corticosterone seems the most convenient method for diagnosing 21-NCAH in a population of PCOS with a positive first level screening, providing high accuracy and reducing the need for ACTH stimulation test.