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Congenital adrenal hyperplasia (CAH) is a common genetic disorder in endocrinology, especially its milder clinical presentation, often caused by a partial or total deficiency of the 21-hydroxylase enzyme located in the adrenal cortex. CAH is characterized by the overproduction of androgen, along with variable degrees of cortisol and aldosterone deficiency. The age at diagnosis can provide some information about underlying mutations, with those diagnosed at birth/early infancy more likely to have severe enzymatic defects, which may include adrenal insufficiency, sexual development disorders, short stature in adulthood, hirsutism, and a higher risk for metabolic syndrome and infertility. Non-classic CAH, a milder form of CAH, is usually manifested later in life and is a common differential diagnosis of Polycystic Ovary Syndrome and should be actively evaluated during initial studies of clinical or biochemical hyperandrogenism. The main goals of CAH treatment are hormone supplementation for severe cases, controlling adrenal androgen overproduction to minimize long-term side effects, managing fertility and genetic counseling, and optimizing patients' quality of life.

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CYP21A2 deficiency represents 95% of congenital adrenal hyperplasia (CAH) cases, a group of genetic disorders that affect steroid biosynthesis. The genetic and functional analysis provide critical tools to elucidate complex CAH cases. One of the most accessible tools to infer the pathogenicity of new variants is in silico prediction. Here, we analyzed the performance of in silico prediction tools to categorize missense single nucleotide variants (SNVs) of CYP21A2. SNVs of CYP21A2 characterized in vitro by functional assays were selected to assess the performance of online single and meta predictors. SNVs were tested separately or in combination with the related phenotype (severe or mild CAH form). In total, 103 SNVs of CYP21A2 (90 pathogenic and 13 neutral) were used to test the performance of 13 single-predictors and four meta-predictors. All SNVs associated with the severe phenotypes were well categorized by all tools, with an accuracy of between 0.69 (PredictSNP2) and 0.97 (CADD), and Matthews' correlation coefficient (MCC) between 0.49 (PoredicSNP2) and 0.90 (CADD). However, SNVs related to the mild phenotype had more variation, with the accuracy between 0.47 (S3Ds&GO and MAPP) and 0.88 (CADD), and MCC between 0.18 (MAPP) and 0.71 (CADD). From our analysis, we identified four predictors of CYP21A2 variant pathogenicity with good performance, CADD, ConSurf, DANN, and PolyPhen2. These results can be used for future analysis to infer the impact of uncharacterized SNVs in CYP21A2.

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Congenital adrenal hyperplasia (CAH) secondary to 21-hydroxylase deficiency is an autosomal recessive disorder. The 21-hydroxylase enzyme P450c21 is encoded by the CYP21A2 gene located on chromosome 6p21.33 within the HLA major histocompatibility complex. This locus also contains the CYP21A1P, a non-functional pseudogene, that is highly homologous to the CYP21A2 gene. Other duplicated genes are C4A and C4B, that encode two isoforms of complement factor C4, the RP1 gene that encodes a serine/threonine protein kinase, and the TNXB gene that, encodes the extracellular matrix glycoprotein tenascin-X (TNX). TNX plays a role in collagen deposition by dermal fibroblasts and is expressed in the dermis of the skin and the connective tissue of the heart and skeletal muscle. During meiosis, misalignment may occur producing large gene deletions or gene conversion events resulting in chimeric genes. Chimeric recombination may occur between TNXB and TNXA. Three TNXA/TNXB chimeras have been described that differ in the junction site (CH1 to CH3) and result in a contiguous CYP21A2 and TNXB gene deletion, causing CAH-X syndrome. TNXB deficiency is associated with Ehlers Danlos syndrome (EDS). EDS comprises a clinically and genetically heterogeneous group of connective tissue disorders. As molecular analysis of the TNXB gene is challenging, the TNX-deficient type EDS is probably underdiagnosed. In this minireview, we will address the different strategies of molecular analysis of the TNXB-gene, as well as copy number variations and genetic status of TNXB in different cohorts. Furthermore, clinical features of EDS and clinical recommendations for long-term follow-up are discussed.

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INTRODUCTION: Congenital adrenal hyperplasia (CAH), a genetic disease characterized by defective cortisol synthesis and excessive levels of sex hormones, can cause precocious puberty in both sexes in untreated individuals and virilization in female patients with a 46, XX karyotype. The female paraurethral (Skene's) gland has been reported as prostate analogous. Growth of prostate tissue is associated with androgen production; therefore, prostate-specific antigen (PSA) levels may represent a marker of virilization in 46, XX patients with CAH. OBJECTIVES: To describe PSA levels in 46, XX patients and evaluate whether higher PSA levels are associated with androgenization and the severity of the disease. STUDY DESIGN: Sixty-six patients with CAH and a 46, XX karyotype were included, irrespective of age. Serum PSA, testosterone, 17-hydroxyprogesterone (17-OHP) and androstenedione levels were measured. Patients' age, age at diagnosis, forms of the disease, Prader classification, bone age assessment, sex of rearing, surgery, and the presence of clinical complications were obtained from their medical records. RESULTS: The mean age of patients was 11.45 ± 10.74 years. Forty-three patients (65%) were diagnosed neonatally at a median of 0.08 years (mean 1.47 ± 2.34 years), with registers of 17-OHP measurements (Guthrie test) being available in 51%. Testosterone, 17-OHP and androstenedione were significantly high. PSA was detectable in 25% of cases (levels >0.01 ng/ml), with a mean of 0.03 ± 0.09 ng/ml, and only in patients over five years of age. A correlation was found between PSA and age (p < 0.001), age at diagnosis (p = 0.002), testosterone (p = 0.001) and androstenedione (p = 0.023). There was no correlation between PSA and the forms of CAH or Prader classification. A sub-analysis of the patients over five years of age in whom PSA was detectable also showed that there was a correlation between PSA (p < 0.05) and age at analysis, age at diagnosis, testosterone and androstenedione levels. DISCUSSION: Limitations of this study include the small sample size due to the rareness of the disease, its retrospective nature, the absence of a control group, the fact that the sample was selected at two referral centers, which could have resulted in a selection bias, and the use of different reference values in the different laboratories conducting the PSA tests. CONCLUSIONS: PSA is detectable in 25% of 46, XX patients with CAH, only after five years of age. PSA level increases significantly with age, age at diagnosis, and testosterone and androstenedione levels, confirming a correlation between PSA levels and elevated androgen levels.

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CONTEXT: The syndrome CAH-X is due to a contiguous gene deletion of CYP21A2 and TNXB resulting in TNXA/TNXB chimeras. OBJECTIVE: To analyze TNXB gene status and to clinically evaluate the Ehlers-Danlos syndrome phenotype in a large cohort of Argentine congenital adrenal hyperplasia (CAH) patients to assess the prevalence of this condition in our population. METHODS: TNXB gene analysis was performed in 66 nonrelated CAH patients that were carriers of the CYP21A2 gene deletion. A molecular strategy based on multiplex ligation-dependent probe amplification and Sanger sequencing analysis was developed allowing for the detection of different, previously described TNXA/TNXB chimeras, named CH1, CH2, and CH3. The main outcome measures were TNXB status of CAH patients that were carriers of the CYP21A2 deletion in the homozygous or heterozygous state. RESULTS: TNXA/TNXB CH1 was found in 41%, CH2 in 29%, and CH3 in 1% of nonrelated alleles carrying the CYP21A2 deletion. Thus, overall 71% of alleles were found to carry a contiguous gene deletion. Sixty-seven percent of patients analyzed had a monoallelic form and 6% a biallelic form. All patients with the biallelic form had severe skin hyperextensibility and generalized joint hypermobility. CONCLUSION: Based on the high frequency of TNXB alterations found in CYP21A2 deletion carrier alleles, we recommend evaluating TNXB status in these patients, and assessing connective tissue dysplasia, including cardiologic alterations in positive cases. The number of patients undergoing cardiological evaluation should be expanded to determine the incidence of structural and functional abnormalities in this cohort.

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Classical 3ß-HSD deficiency due to mutations in the HSD3B2 gene is responsible for a rare form of congenital adrenal hyperplasia (CAH) and is identified by varying degrees of salt wasting. Preimplantation genetic diagnosis (PGD) was performed in a couple carrying mutation c.690 G>A in the HSD3B2 gene. Four polymorphic short tandem repeat markers closely linked to the HSD3B2 gene (D1S185, D1S453, D1S514, D1S540) for linkage analysis in conjunction with the direct mutation analysis were used in embryo genotyping. Two CODIS STRs (VWA and THO1) were also used to confirm embryo zygosity and rule out possible contaminations. Finally, SRY and AMYLOGENIN markers were used for embryo sex determination. PGD was performed by fluorescent multiplex seminested polymerase chain reaction and sequencing. Six embryos were tested and one male carrier embryo was transferred, resulting in the birth of a healthy boy.

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The capability of plant growth promoting microbes to survive under abiotic stresses has important significance for improving plant growth and productivity. Among the various plant growth promoting biomolecules produced by microbes, exopolysaccharide (EPS) help microbes to survive in inhospitable environments and endure environmental stressful conditions. In the present study, a yeast strain CAH2 was isolated from Beta vulgaris rhizosphere soil and identified as Rhodotorula sp., based on the partial 18S rRNA gene sequence analysis. Rhodotorula sp. strain CAH2 was found to tolerate higher concentrations of Al (6 mM), NaCl (150 mM) and PEG-6000 (15%, w/v). The strain CAH2 was shown to produce 7.5 g L-1 of EPS in the production medium with sucrose and yeast extract as a carbon and nitrogen sources, respectively. The EPS yield was increased constantly with increasing concentrations of Al, NaCl and PEG-6000. The structural feature of EPS studied through FT-IR and NMR spectral analysis confirmed the presence of glucose, mannose and galactose. The yeast strain CAH2 was produced multiple plant growth promoting traits in the presence and absence of abiotic stresses. Finally, these results indicate that the production of EPS could be safeguard the plant growth promoting Rhodotorula sp. strain CAH2 from unfavourable environmental conditions.

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3ßHSD2 enzyme is crucial for adrenal and gonad steroid biosynthesis. In enzyme deficiency states, due to recessive loss-of-function HSD3B2 mutations, steroid flux is altered and clinical manifestations result. Deficiency of 3ßHSD2 activity in the adrenals precludes normal aldosterone and cortisol synthesis and the alternative backdoor and 11-oxygenated C19 steroid pathways and the flooding of cortisol precursors along the Δ5 pathway with a marked rise in DHEA and DHEAS production. In gonads, it precludes normal T and estrogen synthesis. Here, we review androgen-dependent male differentiation of the external genitalia in humans and link this to female development and steroidogenesis in the developing adrenal cortex. The molecular mechanisms governing postnatal adrenal cortex zonation and ZR development were also revised. This chapter will review relevant clinical, hormonal, and genetic aspects of 3ßHSD2 deficiency with emphasis on the significance of alternate fates encountered by steroid hormone precursors in the adrenal gland and gonads. Our current knowledge of the process of steroidogenesis and steroid action is derived from pathological conditions. In humans the 3ßHSD2 deficiency represents a model of nature that reinforces our knowledge about the role of the steroidogenic alternative pathway in sex differentiation in both sexes. However, the physiological role of the high serum DHEAS levels in fetal life as well as after adrenarche remains to be elucidated.

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We present a case of a woman with a history of Congenital Adrenal Hyperplasia (CAH) diagnosed at the age of 12, who was referred to our unit for surgical treatment. Despite the initial diagnosis was an indirect inguinal hernia, it was a misdiagnosis. Once in our service, this was corrected into clitoromegaly secondary to CAH. Physical examination and imaging test discarded other abnormalities, such as secondary effects androgenization. Regarding surgical treatment, the techniques used were Spencer and Allen combined with Kumar, which are the most used for clitoroplasty but also less used in Peru.

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Until 2005, questions regarding medical treatment and diagnostic information on Disorders of Sex Development (DSD) were not systematically discussed with both the patients and their families; however, the way these patients are currently treated have been changing with time. Interventional changes in the clinical-psychotherapeutic-surgical areas of DSD determine not only different medical recommendations but also help to place the patient and the family into the decisional process of therapy. We must consider two paradigmatic periods that have influenced and transformed the clinical management framework of patients with DSD: a) The "Money era" (1955), which emphasized the role of the gonads as the diagnostic criterion, having the environment as determinant of the sex identity; and b) The Chicago Consensus (2005) phase, in which the role of genetics and molecular biology was critical for an early identification, as well as in building a proper sex identity, emphasizing ethical questions and the "stigma culture". In addition, recent data have focused on the importance of interdisciplinarity and statements on questions concerning Human Rights as key factors in treatment decision making. Despite each of these management models being able to determine specific directions and recommendations regarding the clinical handling of these patients, we verify that a composite of these several models is the clinical routine nowadays. In the present paper, we discuss these several paradigms, and pinpoint clinical differences and their unfolding regarding management of DSD patients and their families.

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Congenital adrenal hyperplasia, one of the most frequent autosome recessive disorders, is caused by defects in steroidogenic enzymes involved in the cortisol biosynthesis. Approximately 95% of the cases are caused by abnormal function of the 21-hydroxylase enzyme. This deficiency leads to androgen excess, consequently, to virilization and rapid somatic growth with accelerated skeletal maturation. Mutations in CYP21A2 are responsible for different forms of 21-hydroxylase deficiency. Mild impairment in the enzymatic activity causes the non-classic or late-onset congenital adrenal hyperplasia that is observed with a prevalence of 1 in 1000 subjects in different populations. The present paper describes a de novo mutation that occurred in the paternal meiosis. The child, who was conceived by in vitro fertilization, presented with precocious puberty and diagnosed with non-classical 21-hydroxylase deficiency. DNA sequencing showed the compound heterozygosis for a de novo CYP21A1P/A2 chimeric gene and the p.Val281Leu mutation inherited from her mother, who was heterozygous for the mutation. The chimeric gene showed pseudogene-derived sequence from 5'-end to intron 3 and CYP21A2 sequences from intron 3 to 3'-end of the gene. Sequencing analysis of the father did not show any mutation. The multiplex ligation-dependent probe amplification (MLPA) assay did not indicate loss of DNA discarding gene deletion but confirmed the chimeric gene. In addition, supernumerary copies of CYP21A1P were observed for both parents and for the affect child. Since paternity has been confirmed, those results suggest that a de novo large gene conversion in the paternal meiosis could have occurred by misalignment of alleles bearing different copy numbers of genes in CYP21 locus.

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OBJETIVO: O objetivo deste estudo foi avaliar pacientes com HAC clássica antes e após tratamento com glicocorticoides e/ou mineralocorticoides e comparar o perfil metabólico entre o grupo bem controlado (BC) e mal controlado (MC). SUJEITOS E MÉTODOS: Foram selecionados pacientes recém-diagnosticados e pacientes em acompanhamento por HAC, forma clássica, em uso regular ou não de glicocorticoides/mineralocorticoides do Serviço de Genética do Hupes-UFBA, atendidos de março/2004 a maio/2006. Todos os pacientes foram submetidos a avaliação clínica detalhada e exames laboratoriais (glicemia, sódio e potássio, colesterol total, HDL, LDL, triglicerídeos, ácido úrico, leptina, 17-hidroxiprogesterona, testosterona total, peptídeo C e insulina). Os pacientes com valores normais de andrógenos foram classificados como bem controlados (BC) e os com valores elevados de andrógenos em uso ou não de glicocorticoides/mineralocorticoides foram classificados como mal controlados (MC). RESULTADOS: Foram estudados 41 pacientes com HAC: 11 no grupo BC e 30 no grupo MC. Leptina e LDL colesterol estavam mais elevados no grupo BC que no MC (p < 0,05). Valores de ácido úrico eram menores no grupo BC quando comparados com MC (p < 0,05). CONCLUSÃO: O controle adequado da HAC com glicocorticoides parece seguro, pois está associado a alterações discretas no perfil lipídico e da leptina. Não observamos outras alterações metabólicas associadas ao uso de glicocorticoides. O motivo para o menor valor de ácido úrico encontrado nos pacientes com HAC bem controlada não é conhecido e deve ser mais bem estudado.

OBJECTIVE: The objective of this study was to evaluate patients with classic CAH before and after treatment with glucocorticoids/mineralocorticoid and compare the metabolic profile of the well controlled (WC) and poorly controlled (PC) group. SUBJECTS AND METHODS: We selected newly diagnosed patients and patients monitored for CAH, classical form, regularly using or not glucocorticoids/mineralocorticoid in the Genetics Service Hupes-UFBA, seen from March/2004 to May/2006. All patients underwent detailed clinical evaluation and laboratory tests (glucose, sodium and potassium; total cholesterol, HDL, LDL, triglycerides and uric acid; leptin, 17-hydroxyprogesterone, total testosterone, C peptide, and insulin). Patients with normal androgens were classified as well controlled (WC), and those with high levels of androgens either using or not glucocorticoids/mineralocorticoids were classified as poorly controlled (PC). RESULTS: We studied 41 patients with CAH: 11 in the WC group and 30 in PC group. Leptin and LDL cholesterol levels were higher in WC than in the PC group (p < 0.05). Uric acid values ​​were lower in WC compared with the PC group (p < 0.05). CONCLUSION: Adequate control of CAH with steroids seems safe, as it is associated with only mild changes in lipid profile and leptin values. No other metabolic abnormality was associated with glucocorticoid use. The reason for lower uric acid levels found in WC CAH patients is unknown and should be further studied.

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CONTEXT: Molecular diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) has not been straightforward. OBJECTIVE: To conduct a comprehensive genetic analysis by Multiplex Ligation dependent Probe Amplification (MLPA) and evaluate its reliability for the molecular CAH-21OHD diagnosis. PATIENTS AND METHODS: We studied 99 patients from 90 families with salt-wasting (SW; n=32), simple-virilizing (SV; n=29), and non-classical (NC; n=29) CAH-21OHD. Molecular analysis was sequentially performed by detecting the most frequent point mutations by allele-specific oligonucleotide polymerase chain reaction (ASO-PCR), large rearrangements by MLPA, and rare mutations by direct sequencing. Parental segregation was evaluated. RESULTS: ASO-PCR detected microconversions in 164 alleles (91.1%). MLPA identified CYP21A1P large conversions to CYP21A2 in 7 of the remaining 16 (43.7%), 30-kb deletions including the 3'-end of CYP21A1P, C4B, and the 5'-end of CYP21A2 in 3 of the 16 (18.7%), and a complete CYP21A2 deletion in one (6.3%). Five alleles (2.7%) required direct sequencing; three mutations located in the CYP21A2 gene and two derived from CYP21A1P were found. No parental segregation was observed in patients with the c.329_336del and/or the CL6 cluster mutations. These cases were not diagnosed by ASO-PCR, but MLPA detected deletions in the promoter region of the CYP21A2 gene, explaining the genotype/phenotype dissociation. CONCLUSION: Using the proposed algorithm, all alleles were elucidated. False-positive results in MLPA occurred when mutations or polymorphisms were located close to the probe-binding regions. These difficulties were overcome by the association of MLPA with ASO-PCR and paternal segregation. Using these approaches, we can successfully use MLPA in a cost-effective laboratory routine for the molecular diagnosis of CAH-21OHD.

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