Purpose
Many questions concerning Turner syndrome (TS) remain unresolved, such as the long-term complications and, therefore, the optimal care setting for adults. The primary aim of this long-term ...cohort study was to estimate the incidence of comorbid conditions along the life course.
Methods
A total of 160 Italian patients with TS diagnosed from 1967 to 2010 were regularly and structurally monitored from the diagnosis to December 2019 at the University Hospital of Bologna using a structured multidisciplinary monitoring protocol.
Results
The study cohort was followed up for a median of 27 years (IQR 12–42). Autoimmune diseases were the comorbid condition with the highest incidence (61.2%), followed by osteoporosis and hypertension (23.8%), type 2 diabetes (16.2%) and tumours (15.1%). Median age of onset ranged from 22 years for autoimmune diseases to 39 years for type 2 diabetes. Malignant tumours were the most prominent type of neoplasm, with a cumulative incidence of 11.9%. Papillary thyroid carcinoma was the most common form of cancer, followed by skin cancer and cancer of the central nervous system. Only one major cardiovascular event (acute aortic dissection) was observed during follow-up. No cases of ischaemic heart disease, heart failure, stroke or death were recorded.
Conclusions
This cohort study confirms the need for continuous, structured and multidisciplinary lifelong monitoring of TS, thus ensuring the early diagnosis of important comorbid conditions, including cancer, and their appropriate and timely treatment. In addition, these data highlight the need for the increased surveillance of specific types of cancer in TS, including thyroid carcinoma.
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
1–24
ACTH 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 (17OHP
60
) and lower cortisol, whereas LC–MS/MS revealed higher 17OHP (17OHP
LC-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 17OHP
LC-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.
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.
Here, we used an epidemiological approach, which estimates the allelic frequency (q) of an autosomal recessive disorder using the proportion of homozygous patients, the mutational spectrum and the inbreeding coefficient in a sample of affected individuals. We applied this approach to 2 independent Italian cohorts of patients with both clinical and molecular diagnosis of 21OHD‐CAH from mainland Italy (N = 240) and Sardinia (N = 53). We inferred q estimates of 2.87% and 1.83%, corresponding to a prevalence of 1/1214 and 1/2986, respectively. CYP21A2 mutational spectra were quite discrepant between the 2 cohorts, with V281L representing 74% of all the mutations detected in Sardinia vs 37% in mainland Italy.
These findings provide an updated fine‐grained picture of 21OHD‐CAH genetic epidemiology in Italy and suggest the need for a screening approach suitable to the detection of the largest number of clinically significant forms of CAH.
Purpose
11β-Hydroxylase deficiency (11OHD) represents the second most common cause of congenital adrenal hyperplasia. It is caused by mutations in the
CYP11B1
gene localized about 40 kb from the
...CYP11B2
gene with which it shares a homology of 95 %. The asymmetric recombination of these two genes is involved both in 11OHD and in glucocorticoid-remediable aldosteronism (GRA). Our objective was to set up an easy and rapid method to detect these hybrid genes and other kinds of deletions, to improve the molecular diagnosis of 11OHD.
Methods
A set of 8 specific probes for both the
CYP11B1
and the
CYP11B2
genes to be used for multiplex ligation-dependent probe amplification (MLPA) analysis was designed to detect rearrangements of these genes.
Results
The method developed was tested on 15 healthy controls and was proved to be specific and reliable; it led us to identify a novel chimeric
CYP11B2/CYP11B1
gene in one patient that carried the known A306V mutation on the other allele. Specific amplification and sequencing of the hybrid gene confirmed the breakpoint localization in the second intron.
Conclusions
The MLPA kit developed enables the detection of deletions, duplications or chimeric genes and represents an optimal supplement to DNA sequence analysis in patients with 11OHD. In addition, it can also be used to show the presence of the opposite chimaera associated with GRA.
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.
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.
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.
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.
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.
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.
•All novel amino acid changes had mild effects on enzyme function when expressed alone.•In vitro studies confirmed an additive effect between mild variants on the same allele, leading to a more severe phenotype.•In silico predictions were in line with the in vitro data except for the p.Leu461Pro variant.
Mitochondria provide chemical energy for endoergonic reactions in the form of ATP, and their activity must meet cellular energy requirements, but the mechanisms that link organelle performance to ATP ...levels are poorly understood. Here we confirm the existence of a protein complex localized in mitochondria that mediates ATP-dependent potassium currents (that is, mitoK
). We show that-similar to their plasma membrane counterparts-mitoK
channels are composed of pore-forming and ATP-binding subunits, which we term MITOK and MITOSUR, respectively. In vitro reconstitution of MITOK together with MITOSUR recapitulates the main properties of mitoK
. Overexpression of MITOK triggers marked organelle swelling, whereas the genetic ablation of this subunit causes instability in the mitochondrial membrane potential, widening of the intracristal space and decreased oxidative phosphorylation. In a mouse model, the loss of MITOK suppresses the cardioprotection that is elicited by pharmacological preconditioning induced by diazoxide. Our results indicate that mitoK
channels respond to the cellular energetic status by regulating organelle volume and function, and thereby have a key role in mitochondrial physiology and potential effects on several pathological processes.
Mitochondrial morphological and ultrastructural changes occur during apoptosis and autophagy, but whether they are relevant in vivo for tissue response to damage is unclear. Here we investigate the ...role of the optic atrophy 1 (OPA1)-dependent cristae remodeling pathway in vivo and provide evidence that it regulates the response of multiple tissues to apoptotic, necrotic, and atrophic stimuli. Genetic inhibition of the cristae remodeling pathway in vivo does not affect development, but protects mice from denervation-induced muscular atrophy, ischemic heart and brain damage, as well as hepatocellular apoptosis. Mechanistically, OPA1-dependent mitochondrial cristae stabilization increases mitochondrial respiratory efficiency and blunts mitochondrial dysfunction, cytochrome c release, and reactive oxygen species production. Our results indicate that the OPA1-dependent cristae remodeling pathway is a fundamental, targetable determinant of tissue damage in vivo.
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•Mice with controlled Opa1 overexpression are viable and grow normally•Opa1 protects from muscular atrophy, heart and brain ischemia, and liver apoptosis•Opa1 reduces ROS production and cytochrome c release•Opa1-controlled cristae remodeling is a targetable component of tissue damage
Varanita et al. show that a mouse model of controlled overexpression of the master mitochondrial cristae biogenetic factor Opa1 resists skeletal muscle atrophy, heart and brain ischemic damage, and massive liver apoptosis by blunting mitochondrial dysfunction and cytochrome c release. Together with the accompanying paper by Civiletto et al., the authors offer a proof of principle of the potential for therapies targeting cristae remodeling in sporadic and genetic diseases.
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.
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