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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.

Impact of Molecular Genetics on Congenital Adrenal Hyperplasia Management

Congenital adrenal hyperplasia (CAH) is a family of autosomal recessive disorders caused by mutations in genes encoding the enzymes involved in one of the 5 steps of adrenal steroid synthesis or the electron donor P450 oxidoreductase (POR) enzyme. Steroid 21-hydroxylase deficiency (21-OHD), the principal focus of this review, accounts for about 90–95% of all CAH cases, and its biochemical and clinical severity depends on the underlying CYP21A2 gene disruption. Molecular genetic advancements have been achieved in recent years, and the aim of this review is to attempt to highlight its contribution to the comprehension and management of the disease. When possible, we will try to achieve this goal also by providing some results from our personal experience regarding: some aspects of CYP21A2 gene analysis, with basic genotype/phenotype relationships; its crucial role in both genetic counselling and in prenatal diagnosis and treatment in families at risk for 21-OHD; its help in the comprehension of the severity of the disease in patients diagnosed by neonatal screening and possibly treated before an evident salt-loss crisis or before performing adequate blood sampling; its usefulness in the definition of post ACTH 17-hydroxyprogesterone values, discriminating between non-classic, heterozygote and normal subjects; and finally the contribution of genes other than CYP21A2 whose function or dysfunction could influence 21-hydroxylase activity and modify the presentation or management of the 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 11β-hydroxylase deficiency (11β-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 11β-OHD. Functional analyses were performed by using a HEK293 cell in vitro expression system comparing wild type (WT) with mutant 11β-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 11β-OHD showed a nearly complete loss of 11β-hydroxylase activity. The mutations p.(Arg143Trp) and p.(Arg332Gln) detected in patients with non-classical 11β-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 11β-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.

A sequence variation in 3’UTR of CYP21A2 gene correlates with a mild form of congenital adrenal hyperplasia

Background: Congenital adrenal hyperplasia (CAH) is mainly caused by the deficiency of the 21-hydroxylase enzyme coded by the CYP21A2 gene. However, some alleles in the non-classical form (NC-CAH) remain without identified mutations, suggesting the involvement of regulatory regions. Aim: Our objective was to study an allele carrying the variant *13 G>A in the 3’UTR of the CYP21A2 gene identified in some patients with a mild form of NC-CAH in order to verify the possible implication of this variation with the phenotype observed. Subjects and methods: Among all the subjects in whom the CYP21A2 gene was analyzed, 14 patients and 7 relatives heterozygous or homozygous for the *13 G>A substitution in 3’UTR were selected. Sequencing of DNA, genotyping, multiplex ligation-dependent probe amplification (MLPA), in vitro studies and bioinformatic analysis were performed. Results: The haplotype of the *13 G>A allele was identical in all the subjects with a monomodular structure composed by one C4A gene and one CYP21A2 gene without a second module with the CYP21A1P pseudogene. No other concomitant mutations were found in the region extending from 3 kb in the promoter and encompassing the polyadenylation signal. Both bioinformatic analysis and in vitro studies predicted an alteration of the RNA folding and expression, but no miRNA target sequences were found in this region. Conclusions: The identification of a substitution in the 3’UTR of the gene associated with a mild form of NC-CAH suggests the importance of analyzing the CYP21A2 untranslated regions to better characterize and treat this subgroup of patients.

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

Background:Persistentmüllerian 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.

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 3β-hydroxysteroid dehydrogenase type 2 deficiency. Here, we discuss the main features like onset, possible complications, genetics, and replacement therapy of this rare disease.

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.

A genetic epidemiology study of congenital adrenal hyperplasia in Italy

Congenital adrenal hyperplasia due to 21‐hydroxylase deficiency (21OHD‐CAH) is an autosomal recessive disorder affecting steroidogenesis, due to mutations in CYP21A2 (6p21.3). 21OHD‐CAH neonatal screening is based on 17‐hydroxyprogesterone (17OHP) serum levels, showing high type I error rate and low sensitivity to mild CAH forms.