Correction: Congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency: functional consequences of four CYP11B1 mutations.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Congenital Adrenal Hyperplasias Presenting in the Newborn and Young Infant.

Congenital adrenal hyperplasia includes autosomal recessive conditions that affect the adrenal cortex steroidogenic enzymes (cholesterol side-chain cleavage enzyme; 3ß-hydroxysteroid dehydrogenase; 17α-hydroxylase/17,20 lyase; P450 oxidoreductase; 21-hydroxylase; and 11ß-hydroxylase) and proteins (steroidogenic acute regulatory protein). These are located within the three major pathways of the steroidogenic apparatus involved in the production of mineralocorticoids, glucocorticoids, and androgens. Many countries have introduced newborn screening program (NSP) based on 17-OH-progesterone (17-OHP) immunoassays on dried blood spots, which enable faster diagnosis and treatment of the most severe forms of 21-hydroxylase deficiency (21-OHD). However, in several others, the use of this diagnostic tool has not yet been implemented and clinical diagnosis remains challenging, especially for males. Furthermore, less severe classic forms of 21-OHD and other rarer types of CAHs are not identified by NSP. The aim of this mini review is to highlight both the main clinical characteristics and therapeutic options of these conditions, which may be useful for a differential diagnosis in the neonatal period, while contributing to the biochemical evolution taking place in the steroidogenic field. Currently, chromatographic techniques coupled with tandem mass spectrometry are gaining attention due to an increase in the reliability of the test results of NPS for detecting 21-OHD. Furthermore, the possibility of identifying CAH patients that are not affected by 21-OHD but presenting elevated levels of 17-OHP by NSP and the opportunity to include the recently investigated 11-oxygenated androgens in the steroid profiles are promising tools for a more precise diagnosis and monitoring of some of these conditions.

46,XX DSD due to Androgen Excess in Monogenic Disorders of Steroidogenesis: Genetic, Biochemical, and Clinical Features.

The term 'differences of sex development' (DSD) refers to a group of congenital conditions that are associated with atypical development of chromosomal, gonadal, or anatomical sex. Disorders of steroidogenesis comprise autosomal recessive conditions that affect adrenal and gonadal enzymes and are responsible for some conditions of 46,XX DSD where hyperandrogenism interferes with chromosomal and gonadal sex development. Congenital adrenal hyperplasias (CAHs) are disorders of steroidogenesis that mainly involve the adrenals (21-hydroxylase and 11-hydroxylase deficiencies) and sometimes the gonads (3-beta-hydroxysteroidodehydrogenase and P450-oxidoreductase); in contrast, aromatase deficiency mainly involves the steroidogenetic activity of the gonads. This review describes the main genetic, biochemical, and clinical features that apply to the abovementioned conditions. The activities of the steroidogenetic enzymes are modulated by post-translational modifications and cofactors, particularly electron-donating redox partners. The incidences of the rare forms of CAH vary with ethnicity and geography. The elucidation of the precise roles of these enzymes and cofactors has been significantly facilitated by the identification of the genetic bases of rare disorders of steroidogenesis. Understanding steroidogenesis is important to our comprehension of differences in sexual development and other processes that are related to human reproduction and fertility, particularly those that involve androgen excess as consequence of their impairment.

Novel non-classic CYP21A2 variants, including combined alleles, identified in patients with congenital adrenal hyperplasia.

OBJECTIVE: Congenital adrenal hyperplasia (CAH) is an inborn error of metabolism and a common disorder of sex development where >90% of all cases are due to 21-hydroxylase deficiency. Novel and rare pathogenic variants account for 5% of all clinical cases. Here, we sought to investigate the functional and structural effects of four novel (p.Val358Ile, p.Arg369Gln, p.Asp377Tyr, and p.Leu461Pro) and three combinations of CYP21A2 variants (i.e. one allele containing two variants p.[Ile172Asn;Val358Ile], p.[Val281Leu;Arg369Gln], or p.[Asp377Tyr;Leu461Pro]) identified in patients with CAH. METHODS: All variants were reconstructed by in vitro site-directed mutagenesis, the proteins were transiently expressed in COS-1 cells and enzyme activities directed toward the two natural substrates (17-hydroxyprogesterone and progesterone) were determined. In parallel, in silico prediction of the pathogenicity of the variants based on the human CYP21 X-ray structure was performed. RESULTS: The novel variants, p.Val358Ile, p.Arg369Gln, p.Asp377Tyr, and p.Leu461Pro exhibited residual enzymatic activities within the range of non-classic (NC) CAH variants (40-82%). An additive effect on the reduction of enzymatic activity (1-17%) was observed when two variants were expressed together, as identified in several patients, resulting in either NC or more severe phenotypes. In silico predictions were in line with the in vitro data except for p.Leu461Pro. CONCLUSIONS: Altogether, the combination of clinical data, in silico prediction, and data from in vitro studies are important for establishing a correct genotype and phenotype correlation in patients with CAH.

HDAC8 Loss of Function and SHOX Haploinsufficiency: Two Independent Genetic Defects Responsible for a Complex Phenotype.

We report a patient with developmental delay, brachydactyly type E, short stature, and tetralogy of Fallot. Brachydactyly-mental retardation syndrome (BDMR) was suspected based on the phenotype; however, array CGH excluded a 2q37 deletion, but identified a deletion encompassing the SHOX gene. BDMR is characterized by cognitive impairment, skeletal abnormalities involving hands and feet, short stature, and overweight. Most affected individuals carry relatively large 2q37 deletions encompassing HDAC4. This gene encodes a histone deacetylase involved in epigenetic regulation of cell growth and differentiation, specifically during endochondral bone formation in chondrocyte hypertrophy. Since SHOX haploinsufficiency can cause skeletal defects and short stature but would not fully explain the clinical picture of this patient, exome sequencing was performed, and a heterozygous HDAC8 frameshift mutation was identified. HDAC8 is a distinct histone deacetylase involved in cohesin recycling and is responsible for an X-linked dominant Cornelia de Lange-like phenotype. A new blended clinical phenotype may be explained by the result of a dual molecular diagnosis, which represents a combination of 2 independent genetic defects, with relevant implications for genetic counseling, clinical management, and prognosis.

Mutational and functional studies on NR5A1 gene in 46,XY disorders of sex development: identification of six novel loss of function mutations.

OBJECTIVE: To study the functional properties of six novel missense mutations of the NR5A1 gene encoding the steroidogenic factor 1 (SF-1) identified in six patients with 46,XY disorders of sex development (DSD) and to describe their relative phenotype-genotype relationship. DESIGN: Genetic and functional studies. SETTING: University department. PATIENT(S): Six 46,XY DSD patients. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sanger sequencing and multiplex ligation-dependent probe amplification analysis to identify the mutations or deletions/duplications of the NR5A1 gene. Functional studies by transactivation assays to predict the impact of mutations on molecular function. RESULT(S): NR5A1 exons sequencing identified in six 46,XY DSD patients six novel mutations: p.T40R, p.T47C, p.G328W, p.A351E, p.R427W, and p.Q460R. Five missense variants were heterozygous, and one was homozygous (p.R427W). Functional analysis revealed a significant loss of DNA-binding and transactivation ability for all variants, except for p.Q460R, which showed a modest reduced activity compared with that of the wild-type protein. Phenotypes associated with these mutations varied from males with spontaneous puberty, substantial T production, and possible fertility, to females with and without müllerian structures and primary amenorrhea. CONCLUSION(S): We describe six novel mutations in NR5A1 gene and showed that they might affect protein structure, therefore compromising seriously the SF-1 role in regulating gonadal development. Clinically, we suggest that NR5A1 analysis should be performed whenever atypical sex organs are evidenced or there is an abnormal sexual development, to have proper diagnosis and better management of patients.

Two Moroccan Sisters Presenting with a Severe Salt-Wasting Form of Congenital Adrenal Hyperplasia but Normal Female Genitalia.

We report the case of 2 sisters (46,XX) born from consanguineous Moroccan parents. Both sisters had normal female genitalia, but within 2 weeks after birth, they presented with a severe salt-wasting crisis. Hormonal investigations suggested the diagnosis of congenital adrenal hyperplasia, which was confirmed by subsequent molecular analysis to be caused by 3ß-hydroxysteroid dehydrogenase type 2 deficiency. Here, we discuss the main features like onset, possible complications, genetics, and replacement therapy of this rare disease.

Congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency: functional consequences of four CYP11B1 mutations.

Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited endocrine disease. Steroid 11ß-hydroxylase deficiency (11ß-OHD) is the second most common form of CAH. The aim of the study was to study the functional consequences of three novel and one previously described CYP11B1 gene mutations (p.(Arg143Trp), p.(Ala306Val), p.(Glu310Lys) and p.(Arg332Gln)) detected in patients suffering from classical and non-classical 11ß-OHD. Functional analyses were performed by using a HEK293 cell in vitro expression system comparing wild type (WT) with mutant 11ß-hydroxylase activity. Mutant proteins were examined in silico to study their effect on the three-dimensional structure of the protein. Two mutations (p.(Ala306Val) and p.(Glu310Lys)) detected in patients with classical 11ß-OHD showed a nearly complete loss of 11ß-hydroxylase activity. The mutations p.(Arg143Trp) and p.(Arg332Gln) detected in patients with non-classical 11ß-OHD showed a partial functional impairment with approximately 8% and 6% of WT activity, respectively. Functional mutation analysis allows the classification of novel CYP11B1 mutations as causes of classical and non-classical 11ß-OHD. The detection of patients with non-classical phenotypes underscores the importance to screen patients with a phenotype comparable to non-classical 21-hydroxylase deficiency for mutations in the CYP11B1 gene in case of a negative analysis of the CYP21A2 gene. As CYP11B1 mutations are most often individual for a family, the in vitro analysis of novel mutations is essential for clinical and genetic counselling.

Thyrotropin-stimulating hormone receptor gene analysis in pediatric patients with non-autoimmune subclinical hypothyroidism.

CONTEXT: Mutations in TSH receptor (TSHR) are notoriously associated with congenital hypothyroidism as well as with non-autoimmune subclinical hypothyroidism, a mild form of TSH resistance that is not as well characterized by diagnostic procedures. OBJECTIVE: The genetic analysis of the TSHR gene was performed to determine the prevalence of TSHR gene mutations in non-autoimmune subclinical hypothyroidism during the pediatric age. The new mutations were studied for genotypic-phenotypic correlation. PATIENTS: Thirty-eight children (ages 0.5-18.0 yr) affected by non-autoimmune subclinical hypothyroidism diagnosed in our center (follow-up from 1 to 11.5 yr) and normal at neonatal screening were enrolled in the genetic study. In 11 cases, the relatives were included in the genetic analysis. RESULTS: Eleven different mutations of the TSHR gene were identified in 11 of the 38 patients. Two are new: the nonsense mutation C31X and the missense mutation P68S, which shows a decrease in TSH binding capacity but not in biological activity. In all cases the carrier parent was identified. CONCLUSIONS: To date, this study demonstrates the highest prevalence (29%) of TSHR gene mutations in children and adolescents with non-autoimmune subclinical hypothyroidism not selected by neonatal screening. Functional studies show that some mutations cause a slight inactivation of the TSHR. This reveals a possible limit of the in vitro study or of the knowledge of mechanisms involving TSHR, or that other candidate genes must be considered.

Three novel AMH gene mutations in a patient with persistent mullerian duct syndrome and normal AMH serum dosage.

BACKGROUND: Persistentmullerian duct syndrome (PMDS) is characterized by the presence of uterus, fallopian tubes and the upper part of the vagina in 46,XY patients with perfectly virilized external genitalia. It is mostly caused by mutations of the AMH or AMH type 2 receptor (AMHR2) gene. The AMH serum level is very often low or undetectable in the AMH gene defect and normal in the AMHR2 gene defect. AIM: We investigate an Italian patient, genotypically and phenotypically male, observed at 1 month of age for a right inguinal hernia that at surgery showed the presence of both testes in the same hernial sac and uterus and fallopian tubes in the abdomen. RESULTS: Genetic tests first showed the absence of the common 27-bp deletion in the AMHR2 gene, then the presence of three new sequence variations in the AMH geneleading to the following variants: the p.A405P carried by the paternal allele; the p.G326V plus the p.V508A carried by the maternal allele. CONCLUSIONS: The determination of serum AMH, performed after the genetic analysis, showed a normal level for age, suggesting that these mutations may affect the function and the bioactivity of the hormone and not the secretion rate.

Functional characterization of naturally occurring NR3C2 gene mutations in Italian patients suffering from pseudohypoaldosteronism type 1.

OBJECTIVE: The renal form of pseudohypoaldosteronism type 1 (PHA1) is a rare disease caused by mutations in the human mineralocorticoid receptor gene (NR3C2). DESIGN: Aim of the study was to analyze the NR3C2 gene in three Italian patients with clinical signs of renal PHA1 and to evaluate the distribution of the -2G > C, c.538A > G, and c.722C > T single nucleotide polymorphism (SNP) pattern in the PHA1 patients and in 90 controls of the same ethnic origin. METHODS: Analysis of the NR3C2 gene sequence and of the polymorphic SNP markers. Functional characterization of the detected novel NR3C2 mutations utilizing aldosterone-binding assays and reporter gene transactivation assays. RESULTS: One novel nonsense (Y134X) and one novel frameshift (2125delA) mutation were detected. They exhibited no aldosterone binding and no transactivation abilities. No mutation was detected in the third patient. Haploinsufficiency of NR3C2 was ruled out by microsatellite analysis in this patient. The c.722T SNP was detected in 97% of alleles in the Italian population which is significantly different from the general German or US population. CONCLUSIONS: Molecular analysis of the NR3C2 gene in PHA1 patients is warranted to detect novel mutations in order to clarify the underlying genetic cause, which may extend the insight into relevant functional regions of the hMR protein. The effect the different distribution of the c.722T SNP is not clear to date. Further studies are necessary to provide evidence as to a possible advantage of a less sensitive hMR in southern countries.