Mutation distributions among patients with congenital adrenal hyperplasia from five regions of Brazil: a systematic review

ABSTRACT Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is an autosomal recessive disorder caused by CYP21A2 gene mutations, and its molecular diagnosis is widely used in clinical practice to confirm the hormonal diagnosis. Hence, considering the miscegenation of the Brazilian population, it is important to determine a mutations panel to optimise the molecular diagnosis. The objective was to review the CYP21A2 mutations' distribution among Brazilian regions.Two reviewers screened Brazilian papers up to February 2020 in five databases. The pair-wise comparison test and Holm method were used in the statistical analysis. Nine studies were selected, comprising 769 patients from all regions. Low proportion of males and salt-wasters was identified in the North and Northeast regions, although without significant difference. Large gene rearrangements also had a low frequency, except in the Center-West and South regions (p < 0.05). The most frequent mutations were p.I172N, IVS2-13A/C>G, p.V281L and p.Q318X, and significant differences in their distributions were found: p.V281L was more frequent in the Southeast and p.Q318X in the Center-West and Northeast regions (p < 0.05). Thirteen new mutations were identified in 3.8%-15.2% of alleles, being more prevalent in the North region, and six mutations presented a founder effect gene. Genotype-phenotype correlation varied from 75.9%-97.3% among regions. The low prevalence of the salt-wasting form, affected males and severe mutations in some regions indicated pitfalls in the clinical diagnosis. The good genotype-phenotype correlation confirms the usefulness of molecular diagnosis; however, the Brazilian population also presents significant prevalence of novel mutations, which should be considered for a molecular panel.

Genetic diagnosis of non-classical 21-hydroxylase deficiency by the new nanopore sequencing detection method / 中华检验医学杂志

Objective:To summarize initial experience of applying nanopore third-generation sequencing detection method (nanopore sequencing) for genetic diagnosis of non-classical 21 hydroxylase deficiency (NC 21-OHD), and to explore its performance and application prospects.Methods:Clinical data of the two NC 21-OHD patients, who were hospitalized at the First Affiliated Hospital of Zhengzhou University in May 2019, were collected. Peripheral venous blood was collected and genome DNA extracted. Genetic variants was detected by nanopore sequencing and underwent bioinformatic analysis. Pathogenetic mutations in CYP21A2 gene were validated with PCR-sanger sequencing in the two patients and their parents.Results:The average reads length and sequence depth in the patient one was 12, 792 bp and 27.19×. The average reads length and sequence depth in the patient two was 13, 123 bp and 21.34×. Compound variants of c.293-13C>G/c.844G>T (p.Val282Leu) and c.332_339delGAGACTAC (p.Gly111Valfs)/c.844G>T (p.Val282Leu) were detected in these two patients, which were consistent with clinical phenotype of NC 21-OHD. Further analysis showed that c.293-13C>G mutation was inherited from her father and c.844G>T (p.Val282Leu) mutation was inherited from her mother for the patient one. The c.844G>T (p.Val282Leu) mutation was inherited from her father and c.332_339delGAGACTAC (p.Gly111Valfs) mutation from her mother.Conclusions:The heterozygous mutations in CYP21A2 gene are the cause of NC 21-OHD in these two patients. Nanopore sequencing technique is a reliable new detection method for patients with NC 21-OHD.

Phenotype and genotype analysis of 6 patients with 21-hydroxylase deficiency / 中华全科医师杂志

The clinical data of 6 patients with 21-hydroxylase deficiency(21-OHD) diagnosed in The People′s Hospital of Xinjiang Uygur Autonomous Region from 2015 to 2020 were retrospectively analyzed. There were 2 male cases manifesting shorter height, high progesterone level and infertility. And 4 cases were females, manifesting primary amenorrhea, heterosexual precocious puberty, fatigue during emergency, decreased physical strength, dark skin, clitoral hypertrophy and vulva fusion. None of the parents had a history of consanguinity. All but one patient received glucocorticoid replacement therapy. The sequencing of exons and introns of 21CYPA2 gene showed tuat 1 case was homozygous mutation and 5 cases were complex heterozygous mutation. In terms of clinical phenotype, 1 case was non-classical (complex heterozygous mutation) and 5 cases were simple virilizing phenotype.

Molecular analysis of the CYP21A2 gene in dried blood spot samples

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder due to a deficiency of enzymes involved in cortisol biosynthesis. In more than 90% of cases, CAH is secondary to deleterious mutations in the CYP21A2 gene leading to 21-hydroxilase deficiency (21OHD). The CYP21A2 gene is located on the short arm of chromosome 6 (6p21·3) and encodes the cytochrome P450C21 enzyme. Neonatal screening programs detect the classic forms of CAH-21OHD quantifying 17OH-progesterone in dried blood spots (DBS). This test is very sensitive, but it has a low specificity, requiring a second sample to confirm the result. In these cases, a second-tier test in the same sample may be useful. Our aim was to evaluate a DNA extraction method from DBS and assess the performance of such DNA in the molecular analysis of the CYP21A2 gene mutations. Twelve individuals, who presumably had CAH based on the initial neonatal screening results, were analyzed using DNA extracted from freshly collected blood on EDTA and DBS. The CYP21A2 gene was analyzed by automated sequencing of all exons and intron boundaries and MLPA analysis in DBS. Molecular analysis results from both extraction methods were compared. In this study, we show that DNA extracted from neonatal screening DBS is a useful tool to define CYP21A2 gene mutations in 21-OHD diagnostic confirmation for the newborn screening program and that its results are comparable to traditional genotyping.

La hiperplasia suprarrenal congénita (HSC) es un desorden autosómico recesivo producido por la deficiencia de alguna de las enzimas involucradas en la biosíntesis de cortisol. Más del 90% se debe a mutaciones en el gen CYP21A2 que genera deficiencia de 21 hidroxilasa (21OHD). Este gen se encuentra en el brazo corto del cromosoma 6 (6p21·3) y codifica para la enzima citocromo P450C21. Los programas de pesquisa neonatal detectan la forma clásica de la HSC-21OHD cuantificando 17OH-progesterona en gota de sangre en papel de filtro (GSPF). Este test es muy sensible, pero tiene baja especificidad , por lo que se utiliza una segunda muestra para confirmar el resultado. En estos casos, una segunda determinación en la misma muestra podría ser de utilidad. Nuestro objetivo fue evaluar el método de extracción de ADN y posterior análisis molecular del gen CYP21A2 en muestras de GSPF. Analizamos doce individuos presumiblemente afectados por HSC en la pesquisa neonatal usando ADN extraído de sangre fresca recolectada sobre EDTA y de GSPF. Realizamos el análisis del gen CYP21A2 mediante secuenciación automática de todos los exones y regiones intrónicas flanqueantes y MLPA en GSPF, y comparamos los resultados con ambos métodos de extracción. En este estudio demostramos que el ADN extraído de GSPF es una herramienta muy útil para analizar las mutaciones del gen CYP21A2 en la confirmación diagnóstica de 21-OHD para los programas de pesquisa neonatal y que los resultados son comparables con la genotipificación tradicional.

Analysis of CYP21A2 gene mutation and phenotype in patients with 21-hydroxylase deficiency / 中华内分泌代谢杂志

Objective To investigate the spectrum of CYP21A2 gene mutation and the correlation between genotype and phenotype in patients with 21-hydroxylase deficiency in Tianjin and surrounding areas.Methods Genomic DNA was extracted from the peripheral blood samples of the proband.Locus-specific PCR,direct sequencing of PCR amplification products,and multiplex ligation-dependent probe amplification were applied to detect pathogenic gene CYP21A2 and the relationship between genotypes and phenotypes was analyzed.Results (1) Of 35 patients with 21-hydroxylase deficiency,25 were classified as salt-wasting phenotype and 10 were simple virilizing phenotype.(2) 69 mutant alleles were detected in a total of 70 alleles in 35 patients.Only one mutant allele was detected in one patient.Two mutant alleles were detected in all other patients,with the mutation detection rate 98.6％.(3) A total of 6 types of mutations were detected,of which c.293-13C/A＞G (I2G) was the most common,accounting for 57.1％ (40/70),followed by 18.6％ (13/70) for large gene deletion or conversion,and 14.3％ (10/70) for p.I173N.In addition,a novel mutation,c.949C＞T (p.R317X),which has not been reported previously,was detected as a pathogenic mutation.(4) Correlation analysis of genotype and phenotype in 35 children showed that the phenotype predicted by genotype was consistent with the actual salt-wasting phenotype in 31 children,and those in three children were inconsistent with the actual clinical phenotype.Conclusion The mutation characteristics of CYP21A2 gene in patients with 21-hydroxylase deficiency in Tianjin and surrounding areas are slightly different from those reported in other regions in China.A mutation c.949C＞T has not been reported,which enriches the mutation spectrum of CYP21A2 gene and provide the foundation for genetic counseling and prenatal diagnosis.

Non-classical 21-hydroxylase deficiency of a pedigree and related literature review / 中华内分泌代谢杂志

The clinical data of a patient with non-classical 21-hydroxylase deficiency ( 21-OHD) were retrospectively analyzed. The CYP21A2 gene analysis was performed on the patient and his family members by PCR-DNA direct sequencing. It was found that the proband had a heterozygous mutation [ point mutation:p.Ile173Asn, p. ( Ile237Asn, Val238Glu, Met240Lys ), p. Val282Leu, p. Gln319Ter, p. Arg357Trp, insertion mutation: p.Leu308Phefs?6, deletion/insert mutation: p. Arg484Profs]. Among the members of the family, the patient's eldest sister and three paternal members all carried the p.Val282Leu heterozygous mutation, and the patient's second sister and two maternal members carried the same p. Val282Leu homozygous mutation and other compound heterozygous mutations just as the proband. The proband presented a non-classical phenotype with ultimately normal height and fertility. It is suggested that the potential phenotype of the disease is related to the residual activity of its allele, and there exists a good genotype-phenotype correlation.

Clinical features and genetic characteristics of 33 patients with simple virilizing form of 21-hydroxylase deficiency / 中华内科杂志

Objective To analyze the clinical features and genotypes of adult patients with simple virilizing form of 21-hydroxylase deficiency (SV 21-OHD).Methods This is a retrospective study including 33 patients with SV 21-OHD from January 2015 to March 2018 in the Ninth People's Hospital of Shanghai Jiao Tong University School of Medicine.Results The diagnostic age of the patients was (26.3± 6.5) years old.All patients presented with signs of masculinization,such as short stature (100％),clitoromegaly/microphallus (89.65％,26/29),undeveloped breasts (82.76％,24/29),deep voice (55.17％,16/29) and primary amenorrhea (89.65％,26/29).The serum levels of 17-hydroxyprogesterone (17-OHP),androstenedione (AD) and testosterone were significantly elevated in 90.9％,93.9％ and 91.2％ of the patients,respectively.Thirteen types of mutations were identified in CYP21A2 from these patients.Among them,I173N accounted for 40％ and I2 G accounted for 18.33％.Four patients were found with multiple mutations in CYP21A2.Conclusions Short stature,clitoromegaly/microphallus and primary amenorrhea are the most common clinical features in adult patients with SV 21-OHD.Serum levels of 17-OHP and AD are important indices for the diagnosis and monitoring of the patients.I173N and I2 G are the two most prevalent mutations in patients of the present study.Limitation of clinical recognition and delay in treatment contribute to the short stature of the SV 21-OHD patients.

Compound heterozygosity for a whole gene deletion and p.R124C mutation in CYP21A2 causing nonclassic congenital adrenal hyperplasia

We present a family with 2 members who received long-term steroid treatment for presumed classic congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, until molecular testing revealed nonclassic CAH, not necessarily requiring treatment. A 17-year-old male presented to our clinic on glucocorticoid and mineralocorticoid treatment for classic CAH. He was diagnosed at 4 years of age based on mild-moderate elevations of 17-hydroxyprogesterone (17-OHP) and adrenocorticotropic hormone (ACTH), but without evidence of precocious adrenarche/puberty. Due to his diagnosis, his clinically asymptomatic 3-year-old sister was tested and also found to have elevated ACTH and 17-OHP levels and was started on glucocorticoids for classic CAH. Family history revealed a healthy sibling who had no biochemical evidence of CAH and consanguineous healthy parents. We questioned the diagnosis of classic CAH and performed an ACTH1-24 stimulation test, which showed a level of 17-OHP in the borderline range between classic and nonclassic CAH. Molecular testing, using sequencing and multiplex ligation-dependent probe amplification analysis of CYP21A2, revealed that both affected siblings were compound heterozygotes for a whole-gene deletion and a, likely pathogenic (nonclassical), sequence variant, p.R124C. The asymptomatic father had the same genotype, while the mother showed one deleted copy and 2 active copies, making her an asymptomatic carrier. Our report demonstrates the importance of molecular testing in atypical cases of CAH, as well as the importance of both sequencing and deletion analysis. The results of molecular testing should be interpreted in clinical context, and treatment should be prescribed according to guidelines when available.

A case of 21-hydroxylase deficiency in Turner's syndrome and literature review / 中华内分泌代谢杂志

To improve the recognition and treatment of the combination of classical congenital adrenal hyperplasia (CAH) and Turner's syndrome. A case of 21-hydroxylase deficiency (21-OHD) in 45,X[3] / 46,XX [47] was reported,and the related literatures were reviewed. A 29-year-old woman with 45,X[3] / 46,XX[47] was referred with clitorimegaly and primary amenorrhea. Her height was 150 cm with a weight of 56 kg. Physical examination revealed a Tanner stage Ⅵ for both breast development and pubic hair development. She showed a little signs of Turner' s syndrome, such as cubitus valgus. Lab findings: sex hormones are significantly increased, including progesterone, testosterone, dehydroepiandrosterone, 17 hydroxyprogesterone, dihydrotestosterone, and androstendione. Enhanced CT scan showed bilateral adrenal hyperplasia. Gynecological ultrasound showed that the size of the uterus and ovary were near normal and the endometrium was not clear. By gene mutation screening, two mutation sites were found in CYP21A2 gene, such as IVS2-13C/ A→G and p. Ile173Asn( c. 518T→A). Taken together, the patient was diagnosed as a combination of 21-OHD and Turner syndrome. A total of ten patients associated with CAH in Turner's syndrome have been reported so far. The findings showed that routine karyotyping during investigations of patients presenting with ambiguous genitalia or with a diagnosis of CAH may reveal the concomitant presence of Turner's syndrome. We should make a definite diagnosis and give early treatment as soon as possible.

A case of 21-hydroxylase deficiency in Turner's syndrome and literature review / 中华内分泌代谢杂志

To improve the recognition and treatment of the combination of classical congenital adrenal hyperplasia (CAH) and Turner's syndrome. A case of 21-hydroxylase deficiency (21-OHD) in 45,X[3] / 46,XX [47] was reported,and the related literatures were reviewed. A 29-year-old woman with 45,X[3] / 46,XX[47] was referred with clitorimegaly and primary amenorrhea. Her height was 150 cm with a weight of 56 kg. Physical examination revealed a Tanner stage Ⅵ for both breast development and pubic hair development. She showed a little signs of Turner' s syndrome, such as cubitus valgus. Lab findings: sex hormones are significantly increased, including progesterone, testosterone, dehydroepiandrosterone, 17 hydroxyprogesterone, dihydrotestosterone, and androstendione. Enhanced CT scan showed bilateral adrenal hyperplasia. Gynecological ultrasound showed that the size of the uterus and ovary were near normal and the endometrium was not clear. By gene mutation screening, two mutation sites were found in CYP21A2 gene, such as IVS2-13C/ A→G and p. Ile173Asn( c. 518T→A). Taken together, the patient was diagnosed as a combination of 21-OHD and Turner syndrome. A total of ten patients associated with CAH in Turner's syndrome have been reported so far. The findings showed that routine karyotyping during investigations of patients presenting with ambiguous genitalia or with a diagnosis of CAH may reveal the concomitant presence of Turner's syndrome. We should make a definite diagnosis and give early treatment as soon as possible.

Methylmalonic aciduria combined with congenital adrenal hyperplasia:a case report / 临床儿科杂志

Objective To explore the diagnosis and treatment of a rare case of methylmalonic aciduria combined with congenital adrenal hyperplasia. Methods The clinical and laboratory data of the first case of methylmalonyl CoA mutase deifcient methylmalonic aciduria combined with 21-hydroxylase deifciency in China were analyzed. Results The male patient with age of onset at 3 months presented with feeding dififculty, diarrhea, metabolic acidosis, and psychomotor retardation after polio vaccination or high protein diet. At one year and 8 months of age, methylmalonic aciduria was diagnosed, and the patient was clinically improved after treatment. At 5 years of age, precocious puberty was noticed, and virilizing form of 21-Hydroxylase deifciency was diagnosed. Genetic testing conifrmed 2 known mutations in MUT gene (c.866G?>?C, c.2179C?>?T) and 2 known mutations in CYP21A2 gene (c.188A?>?T, c.518T?>?A). Conclusions The clinical manifestations of inherited metabolic disorders and endocrine diseases are complex and it is rare that multiple disorders occurred simultaneously in one patient. This male patient has two rare diseases, methylmalonic aciduria and 21-hydroxylase deifciency.

Hiperplasia adrenal congénita por déficit de 21 hidroxilasa: un reto diagnóstico y terapéutico / Congenital adrenal hyperplasia due to 21-hydroxylase deficiency: a therapeutic and diagnosis challenge

La hiperplasia adrenal congénita es un conjunto de anomalías con herencia autosómica recesiva por el déficit de una de las cinco enzimas necesarias para la síntesis de cortisol en la corteza adrenal. La causa más frecuente es la deficiencia de 21 hidroxilasa, que explica más del 95% de los casos. La presentación es heterogénea y depende de cuán afectada está la función enzimática y el sexo del paciente. Se clasifica en una variante no clásica y clásica, esta se subclasifica en una forma con pérdidas salinas y virilizante simple. El tratamiento se fundamenta en el uso de glucocorticoides y mineralocorticoides, con un seguimiento estricto para minimizar las reacciones adversas.Objetivo: Revisión descriptiva sobre el estado del arte de la hiperplasia adrenal congénita.Materiales y métodosRevisión no sistemática de la literatura mediante los buscadores Medline, PubMed, LILACS y la herramienta Clinical Key de publicaciones en los últimos diez años. Se usaron las palabas: hiperplasia adrenal congénita, déficit de 21 hidroxilasa y ambigüedad sexual.Discusión y conclusión: Como es una enfermedad de gran variabilidad en la presentación clínica y las características paraclínicas, es necesario que los profesionales de la salud tengan amplio conocimiento en cuanto a su forma de presentación, diagnóstico y manejo en situaciones especiales (crisis adrenal, dosis de estrés, embarazo), además de realizar seguimiento regular e intervenciones tempranas con el fin de mermar las consecuencias deletéreas, derivadas del tratamiento con corticoides en forma crónica.

Congenital adrenal hyperplasia is a group of autosomal recessive anomalies caused by a deficiency of one of the five enzymes required for the synthesis of cortisol in the adrenal cortex. The most common cause is 21-hydroxylase deficiency, which accounts for over 95% of cases. The presentation is heterogeneous and depends on how much the enzymatic function is affected, and sex of the patient. It is classified as a non-classical and classical variant, which is sub-classified into simple virilising and salt loss. The treatment is based on the use of mineralocorticoids and glucocorticoids, with close monitoring to minimise adverse reactions.Objective: To present a descriptive review of the state of art of congenital adrenal hyperplasia.Materials and methods: A non-systematic review of publications in the literature over the past ten years using the Medline, PubMed, LILACS and Clinical Key. The search words used were: congenital adrenal hyperplasia, 21-hydroxylase deficiency, and sexual ambiguity.Discussion and conclusion: Congenital adrenal hyperplasia is a disease of great variability in clinical presentation and para-clinical characteristics. Health professionals should have extensive knowledge in its presentation, diagnosis, and management in special situations (adrenal crisis, stress dose, pregnancy). It also requires regular monitoring and early interventions in order to reduce the deleterious consequences arising from continuous treatment with corticosteroids.

Recent advances in biochemical and molecular analysis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency

The term congenital adrenal hyperplasia (CAH) covers a group of autosomal recessive disorders caused by defects in one of the steroidogenic enzymes involved in the synthesis of cortisol or aldosterone from cholesterol in the adrenal glands. Approximately 95% of all CAH cases are caused by 21-hydroxylase deficiency encoded by the CYP21A2 gene. The disorder is categorized into classical forms, including the salt-wasting and the simple virilizing types, and nonclassical forms based on the severity of the disease. The severity of the clinical features varies according to the level of residual 21-hydroxylase activity. Newborn screening for CAH is performed in many countries to prevent salt-wasting crises in the neonatal period, to prevent male sex assignment in affected females, and to reduce long-term morbidities, such as short stature, gender confusion, and psychosexual disturbances. 17α-hydroxyprogesterone is a marker for 21-hydroxylase deficiency and is measured using a radioimmunoassay, an enzyme-linked immunosorbent assay, or a fluoroimmunoassay. Recently, liquid chromatography linked with tandem mass spectrometry was developed for rapid, highly specific, and sensitive analysis of multiple analytes. Urinary steroid analysis by gas chromatography mass spectrometry also provides qualitative and quantitative data on the excretion of steroid hormone metabolites. Molecular analysis of CYP21A2 is useful for genetic counseling, confirming diagnosis, and predicting prognoses. In conclusion, early detection using neonatal screening tests and treatment can prevent the worst outcomes of 21-hydroxylase deficiency.

CYP21A2 Mutation Analysis in Korean Patients With Congenital Adrenal Hyperplasia Using Complementary Methods: Sequencing After Long-Range PCR and Restriction Fragment Length Polymorphism Analysis With Multiple Ligation-Dependent Probe Amplification Assay

CYP21A2 mutation analysis of congenital adrenal hyperplasia (CAH) is challenging because of the genomic presence of a homologous CYP21A2 pseudogene and the significant incidence of pseudogene conversion and large deletions. The objective of this study was to accurately analyze the CYP21A2 genotype in Korean CAH patients using a combination of complementary methods. Long-range PCR and restriction fragment length polymorphism analyses were performed to confirm valid amplification of CYP21A2 and to detect large gene conversions and deletions before direct sequencing. Multiple ligation-dependent probe amplification (MLPA) analysis was conducted concurrently in 14 CAH-suspected patients and six family members of three patients. We identified 27 CYP21A2 mutant alleles in 14 CAH-suspected patients. The c.293-13A>G (or c.293-13C>G) was the most common mutation, and p.Ile173Asn was the second, identified in 25% and 17.9% of alleles, respectively. A novel frame-shift mutation of c.492delA (p.Glu 164Aspfs*24) was detected. Large deletions were detected by MLPA in 10.7% of the alleles. Mutation studies of the six familial members for three of the patients aided in the identification of haplotypes. In summary, we successfully identified CYP21A2 mutations using both long-range PCR and sequencing and dosage analyses. Our data correspond relatively well with the previously reported mutation spectrum analysis.

Two Novel Complex Mutations of CYP21A2 Gene Causing Simple Virilizing 21-hydroxylase Deficiency / 中国医科大学学报

Objective To investigate the clinical manifestations,laboratory findings and genetic characteristics of two cases with simple virilizing (SV)21-hydroxylase(21OHD). Methods Clinical manifestations and laboratory data were obtained. The promoter and coding areas of CYP21A2 gene were directly sequenced. In silico analysis were used to predict the function of mutations. Results Two patients presented severe virilism. The laboratory examinations showed that plasma ACTH,aldosterone,androstendione and testosterone were significantly increased compared with normal individuals. VCT scan showed hyperplasia of bilateral adrenal. Direct sequencing of CYP21A2 gene showed two complex mutations-H62L/V69L and I2g/10InsL,which were not reported previously. In silico analysis(Polyphen)showed the novel mutation V69L could probably damage the function of CYP21A2 protein. Conclusion The combined mutations,H62L/V69Land I2g/10InsL,could be associated with SV type 21-OHD phenotype.

Genotype-phenotype correlation in 27 pediatric patients in congenital adrenal hyperplasia due to 21-hydroxylase deficiency in a single center

PURPOSE: The purpose of the study was to evaluate endocrine patterns of patients with congenital adrenal hyperplasia and each gene mutation and to analyze the correlation between each phenotype and genotype. METHODS: This was a retrospective study of the patients with congenital adrenal hyperplasia in the pediatric outpatient clinic at the Samsung Medical Center from November 1994 to December 2012. We analyzed the medical records of 27 patients (male, 19; female, 8) with congenital adrenal hyperplasia who had been diagnosed by genetic testing to have 21-hydroxylase deficiency. RESULTS: In genetic analysis of 54 alleles from 27 patients, 13 types of mutations were identified. The distribution of 21-hydroxylase deficiency gene mutations revealed that intron 2 splice site (c.293-13A/C>G) mutations and large deletions were the most common, at 31.5% and 22.2% respectively, followed by p.I173N, p.R356W, and p.I172N mutations at 11.1%, 9.3%, and 9.3%, respectively. Other mutations were observed at 1.9-3.7%. No novel mutations were detected. CONCLUSION: The analysis of 54 alleles revealed 13 types of mutation. The salt wasting form showed a good correlation between genotype and phenotype, but the simple virilizing and nonclassic forms showed inconsistencies between genotype and phenotype. The distribution of CYP21A2 mutations was evaluated for 21-hydroxylase deficiency patients from a single center. This study provides limited data on mutation spectrum and genotype-phenotype correlation of 21-hydroxylase deficiency in Korea.

Mechanism of varied responsiveness to ACTH in patients with polycystic ovary syndrome / 中华内分泌代谢杂志

Objective To explore the potential mechanism of adrenal androgen excess in patients with polycystic ovary syndrome (PCOS), ACTH stimulation test was conducted and the polymorphisms in the promoter region of CYP21 A2 gene were screened to verify the variations related to the responsiveness to ACTH stimulation. Methods 30 healthy women and 101 PCOS patients, matched for age, were recruited from Ruijin hospital. Blood biochemical examinations were taken and sex hormone profiles obtained at baseline. 17 hydroxyprogesterone( 17OHP)was measured at 0 and 60 min in an ACTH stimulation test. The -710 bp -1 bp of the promoter region of CYP21A2 was sequenced in 87 PCOS patients and 30 control subjects. Results According to the post-stimulation 17 OHP levels obtained from 30 healthy women,PCOS patients were allocated into one group with high responsiveness to ACTH ( HR-PCOS, n = 21) and the other with normal responsiveness to ACTH ( NR-PCOS, n = 80). Compared with NR-PCOS subjects, HR-PCOS patients had higher testosterone( P<0.05), basal and post-stimulation 17OHP (both P<0.01)and dehydroepiandrosterone sulfate levels (P<0.05 or P<0.01) .whereas serum cortisol and androstenedione levels were not significantly different before and after ACTH stimulation test. The genotypes of locus -535 were well correlated with post-stimulation 17OHP levels (r = 0. 20,P = 0. 03) in PCOS patients and the control subjects. The genotype T/T or allele T was significantly more frequent in subjects with a higher fertile of post-stimulation 17OHP (P<0.05 or P<0. 01). The odds ratio(OR)for higher responsiveness to ACTH in women with allele T at -535 was 3. 69 (95% CI 1. 69-8. 06,P = 0. 000 7). Conclusions The PCOS patients with higher responsiveness to ACTH are characterized by severe hyperandrogenemia and adrenal androgenexcess,suggesting that adrenal androgen excess in some PCOS patients may be due to higher responsiveness to ACTH. The polymorphism of -535C>T in the promoter region of CYP21 A2 may play a role in regulating 21 hydroxylase gene expression and further influencing 17OHP responsiveness to ACTH.

Heterozygosis for CYP21A2 mutation considered as 21-hydroxylase deficiency in neonatal screening / Heterozigose para mutação no gene CYP21A2 considerada como deficiência de 21-hidroxilase na triagem neonatal

Steroid 21-hydroxylase deficiency (21-OHD) accounts for more than 90 percent of congenital adrenal hyperplasia. CAH newborn screening, in general, is based on 17-hydroxyprogesterone dosage (17-OHP), however it is complicated by the fact that healthy preterm infants have high levels of 17-OHP resulting in false positive cases. We report on molecular features of a boy born pre-term (GA = 30 weeks; weight = 1,390 g) with elevated levels of 17-OHP (91.2 nmol/L, normal < 40) upon neonatal screening who was treated as having CAH up to the age of 8 months. He was brought to us for molecular diagnosis. Medication was gradually suspended and serum 17-OHP dosages mantained normal. The p.V281L mutation was found in compound heterozygous status with a group of nucleotide alterations located at the 3' end intron 4 and 5' end exon 5 corresponding to the splice site acceptor region. Molecular studies continued in order to exclude the possibility of a nonclassical 21-OHD form. The group of three nucleotide changes was demonstrated to be a normal variant since they failed to interfere with the normal splicing process upon minigene studies.

A deficiência de 21-hidroxilase (21-OHD) é uma doença autossômica recessiva que contribui com mais de 90 por cento dos casos de hiperplasia congênita da adrenal. O teste de dosagem de 17-hidroxiprogesterona (17-OHP) por radioimunoensaio em amostras de sangue colhidas em papel de filtro tem sido o método mais usado nos programas de triagem neonatal. No entanto, essa triagem pode apresentar alto número de falso-positivos pelo fato de os recém-nascidos prematuros apresentarem dosagens mais elevadas deste esteróide. Apresentamos aqui os estudos moleculares de uma criança, sexo masculino, nascida pré-termo (IG = 30 sem; peso = 1.390 g) que apresentava valores elevados de 17-OHP sérica (91,2 nmol/L, normal < 40) na triagem neonatal e que foi tratada como portadora da forma clássica da 21-OHD até a idade de 8 meses quando nos foi encaminhada para diagnóstico molecular. A terapia foi, então, gradativamente descontinuada, sendo que as concentrações séricas de 17-OHP se mantiveram normais. A mutação p.V281L foi encontrada em heterozigose composta com um grupo de alterações no terminal 3' do íntron 4 e no terminal 5' do éxon 5 correspondendo à região do sítio aceptor de splicing. A análise do gene CYP21A2 prosseguiu para se excluir a possibilidade de a criança ser afetada com a forma não-clássica de 21-OHD. Pela análise de minigene ficou demonstrado que o grupo de três trocas nucleotídicas não afeta o processo normal de transcrição. Concluindo, a criança é apenas heterozigota da mutação p.V281L sem necessidade de tratamento.

Utilidad del estudio molecular de CYP21A2 en el manejo prenatal de hiperplasia suprarrenal congénita: detección de dos nuevas mutaciones en Chile / Molecular study of CYP21A2 gene for prenatal diagnosis of congenital adrenal hyperplasia: Report of a family

Prenatal treatment of pregnancies at risk of congenital adrenal hyperplasia (CAH) may prevent ambiguous genitalia in female fetuses. We present the prenatal treatment performed in an extended family with two mutations. The proband, a boy with CAH-salt losing form, and his relatives were studied. The proband's paternal uncles/aunts were married to the maternal aunts/uncles, respectively. The relatives had normal basal and stimulated 170HProgesterone levels, which did not clarify their carrier status. The CYP21A2 gene was sequenced. The proband and the paternal relatives harbored a Q318X, R483W mutation in one alíele. The maternal relatives and the proband exhibited an R483 frameshift mutation. Early dexametasone treatment was given during two pregnancies and stopped when male gender was confirmed by early ultrasonography Both newborns were healthy and had normal 170HProgesterone levels. This family had three mutations which abolish the 21-hydroxilase activity. Two mutations were detected in codon 483 of CYP21A2 gene, exon 10, which have not been reported previously in Latin-America. The molecular study performed in this family allowed us to give an appropriate genetic counseling and prenatal treatment.