Search Results (123)

Background: Although Short Read Next-Generation Sequencing (SR-NGS) is widely employed in diagnoses, its application in CYP21A2 genotyping remains limited due to its high sequence homology with its pseudogene, CYP21A1P. Herein, we present (a) a complete pipeline for the diagnostic use of SR-NGS in CYP21A2 genotyping following its assessment; (b) two distinct in-house bioinformatics pipelines for variant calling; and (c) the results by implementing this pipeline in diagnoses. Methods: A total of 221 subjects were studied, comprising a pilot group (n = 21), recruited for assessment of the assay, and a study group (n = 200) categorized in three subgroups, referred for CYP21A2 genotyping. Both groups underwent SR-NGS. Two different bioinformatics algorithms for variant calling were applied and variant filtration was performed using VarAFT (v2.17). In the study group, MLPA was additionally employed. Results: The SR-NGS assay, employing GATK HaplotypeCaller, demonstrated 100% sensitivity and specificity when compared to Sanger Sequencing; however, complex CYP21A2 rearrangements cannot be detected. In the study group, pathogenic variants were identified in 52.7%, 100% and 25% of cases in subgroups (a), (b) and (c) respectively, whereas gene duplications accounted for 12.3% (7/57) of subjects tested. Conclusions: This study provides a comprehensive protocol for the use of SR-NGS in a CYP21A2 diagnostic genotyping, integrating complementary bioinformatics pipelines and MLPA for copy number analysis. Full article

Background: Congenital adrenal hyperplasia (CAH) is associated with chronic hormonal dysregulation and prolonged glucocorticoid exposure, which may contribute to alterations in pulmonary physiology. However, small airway function in children with CAH has not been adequately investigated. This study aims to evaluate small airway function in children with congenital adrenal hyperplasia (CAH) using impulse oscillometry system (IOS) and to compare the findings with those of healthy controls. Methods: This cross-sectional case–control study included 27 children with CAH and 27 age- and sex-matched healthy controls. IOS measurements, including resistance at 5 Hz (R5), resistance at 20 Hz (R20), peripheral airway resistance (R5–R20), reactance at 5 Hz (X5) and 20 Hz (X20), reactance area (AX), and resonance frequency (Fres), were obtained in all participants. Demographic, clinical, laboratory, and treatment-related characteristics were also recorded. Results: The median age of the CAH group was 14 years, and 66.7% were female. Compared with controls, children with CAH demonstrated significantly higher R5 values [0.55 vs. 0.38 kPa·s/L, p = 0.031], increased R5–R20 values [0.21 vs. 0.12 kPa·s/L, p = 0.009], and elevated AX values [2.05 vs. 1.02 kPa·s/L, p = 0.001]. In addition, X5 values were significantly more negative in the CAH group [−0.16 vs. −0.10 kPa·s/L, p = 0.001]. No significant differences were observed in R20, X20, and Fres parameters. Based on the z-scores calculated using the reference equations implemented in the device software, abnormal oscillometric findings were most frequently observed for X5 (29.6%), followed by R5–R20 (25.9%) and R5 (22.2%). Conclusions: Children with CAH exhibited IOS abnormalities suggestive of subclinical small airway dysfunction despite the absence of overt respiratory disease. IOS may serve as a sensitive tool for the early detection of small airway involvement in pediatric CAH. Further prospective studies with larger sample sizes are warranted. Full article

The standard-of-car and genetic testing for 21-hydroxylase deficiency congenital adrenal hyperplasia (CAH) is Sanger sequencing of a long-range CYP21A2 amplicon combined with multiplex ligation-dependent probe amplification (MLPA). Many Sanger sequencing protocols capture only selected regions of CYP21A2 and require several sequencing reactions. This retrospective study explores the potential of short-read next-generation sequencing (SRS) to replace Sanger sequencing in CAH genetic testing. A total of 216 participants, including CAH patients, their parents and individuals with no family history of CAH, were included. We assessed the performance of standard SRS analysis and improved upon it with developed computational software AmpliconPipe, which includes copy number calling and caller harmonization for precise variant detection. With these procedures, SRS of the CYP21A2 long-range amplicon achieved 100% sensitivity (CI 95% [94.9, 100]) and 100% specificity (CI 95% [97.4, 100]) for CAH diagnosis, and 100% sensitivity (CI 95% [94.9, 100]) with 98.6% specificity (CI 95% [95.0, 99.6]) for CAH-carrier diagnosis, with better gene coverage outperforming the standard-of-care method, discovering novel variants, and detecting CYP21A2 copy number alterations with high sensitivity. These findings establish SRS as a practical, accurate, and immediately implementable alternative to Sanger sequencing for CAH genetic testing. Full article

Testicular adrenal rest tumors (TARTs) are a frequent and clinically relevant complication of congenital adrenal hyperplasia (CAH), particularly its classic forms due to 21-hydroxylase deficiency. Although histologically benign, these masses develop within the rete testis and can progressively compress the seminiferous tubules, leading to fibrosis and, when longstanding, permanent gonadal injury and obstructive azoospermia; they are the foremost cause of impaired fertility in men with classic CAH. Their frequency increases with age, from approximately 25% in childhood to 46% in adulthood. This narrative review summarizes current evidence on the pathogenesis, predisposing factors and diagnostic work-up of TARTs, focusing on two practical questions: the appropriate age to begin monitoring and the imaging tools best suited to it. Sustained ACTH excess is regarded as the dominant growth-promoting signal, while the salt-wasting phenotype, severe CYP21A2 variants and inadequate disease control emerge as the principal predisposing factors. Although current guidelines advise ultrasound monitoring from adolescence, a growing body of pediatric data shows that TARTs can be detected well before puberty in patients at greatest risk. Scrotal ultrasonography remains the first-line technique, complemented by CEUS, elastography and MRI in selected cases. We outline a pragmatic, risk-adapted monitoring scheme and argue that earlier, childhood assessment in the highest-risk patients may help to safeguard future fertility, an approach that still awaits prospective confirmation. Full article

Background and Objectives: Bilateral adrenalectomy (BADx) is a definitive treatment for diverse adrenal disorders, though data across etiologies remain limited. This multicenter study evaluated perioperative risks, long-term survival, and shifts in comorbidities in patients undergoing BADx for Cushing’s syndrome (CS) and non-CS indications. Materials and Methods: We retrospectively analyzed 103 adult patients (CS, n = 52; non-CS, n = 51) from 9 tertiary centers. Primary outcomes included early (≤30 days) and late mortality, complications (adrenal crisis, Nelson’s syndrome), and changes in comorbidity profiles. Results: The cohort (mean age 50.59 ± 13.82 years) had a median follow-up of 96 months. Early mortality occurred exclusively in CS patients (5.8%), while late mortality was comparable between groups. Overall 5-year survival rates were 83.3% for CS and 89.9% for non-CS (p = 0.360), with no significant differences across CS etiologies (p = 0.760). Adrenal crisis was significantly more frequent in CS (23% vs. 7%, p = 0.023). Nelson’s syndrome developed in 28% of Cushing’s disease patients. Postoperatively, both cohorts showed increased reliance on psychiatric and cardiovascular medications. Notably, osteoporosis rates were significantly higher than preoperative levels exclusively in the non-CS group (p = 0.006), highlighting the impact of long-term glucocorticoid replacement in this cohort. Two patients with congenital adrenal hyperplasia demonstrated remarkable long-term survival (8 and 30 years) across different surgical timings. Conclusions: BADx delivers rapid systemic remission and definitive control across a range of adrenal pathologies. However, the primary cause and the timing of surgery significantly impact the clinical course and the risk of complications. The resulting state of irreversible adrenal insufficiency requires comprehensive, lifelong surveillance and tailored hormone replacement. Full article

Background: Polycystic ovarian syndrome (PCOS) and the non-classic form of congenital adrenal hyperplasia (NC-CAH) are hyperandrogenic conditions with overlapping clinical symptoms but different genetic backgrounds. The possible interrelationships between the two conditions remain unclear; thus, the present study aims to investigate the prevalence of CYP21A2 exon 7 genetic variants in patients with PCOS and to explore the possible associations of the polymorphisms with adrenocortical hormonal production. Methods: The CYP21A2 exon 7 region was genotyped in 80 unrelated female patients with PCOS and 12 women with NC-CAH. The associations between genetic variants, clinical characteristics, and adrenocortical hormones were investigated. Results: The pathogenic CYP21A2 NC-CAH variant c.844G>T; p.(Val282Leu) was found in 66.7% (8/12) of patients with NC-CAH but in none of the individuals with PCOS. The benign rs1554305325, rs6465, rs6472, and rs6477 genetic polymorphisms were not related to clinical hyperandrogenism. The rs6472 polymorphic alleles were associated with increased adrenocorticotropic hormone (ACTH) (5.5 vs. 3.4 pmol/L, p = 0.022) and cortisol (460.5 vs. 366.5 nmol/L, p = 0.016) levels. The rs6465 variant alleles were significantly associated with lower pregnenolone (1.43 vs. 3.1 ng/mL, p = 0.031) and ACTH (2.5 vs. 4.5 pmol/L, p = 0.030) levels in the unadjusted model but not after adjustment for potential confounders (p > 0.05). Conclusions: The p.(Val282Leu) variant is very common among Bulgarian patients with NC-CAH but it has not been found in our cohort of women with PCOS. The CYP21A2 exon 7 polymorphisms might be associated with cortisol levels in the patients with PCOS. Further larger studies are needed to confirm or reject the current findings in different ethnic groups. Full article

Background: Congenital Adrenal Hyperplasia (CAH) is a group of autosomal recessive disorders caused by impaired adrenal steroidogenesis, most frequently due to pathogenic variants in the CYP21A2 gene leading to 21-hydroxylase deficiency (21-OHD). Epidemiology and management vary across the Mediterranean Basin as a result of genetic and healthcare differences. Objective: To provide an overview of the epidemiology, diagnostic approaches and treatment patterns of CAH in Mediterranean countries. Methods: A structured review of the literature was performed using PubMed, using combined disease-related, geographic and methodological terms. Eligible studies reporting on epidemiology, diagnosis, or management of CAH were included. Data on study design, population characteristics, incidence, diagnostics, genetics and treatment availability were extracted. Results: Data were collected from 23 Mediterranean and neighboring regions covering over 8.7 million screened newborns. In countries with established newborn screening (e.g., Spain, Italy, France, Greece), the incidence of classic CAH ranged from 1:10,000 to 1:25,000 live births. Higher rates were reported in parts of North Africa and the Eastern Mediterranean. Diagnostic set-up and access to biochemical and genetic confirmation varied widely. Hydrocortisone remains the primary therapy, while access to mineralocorticoids and modified-release glucocorticoids differed across settings. Conclusions: Overall, considerable heterogeneity in CAH epidemiology and care exists across the Mediterranean region. Genetic factors such as founder effects, consanguinity and healthcare organization contribute to these differences. Expanding newborn screening, improving diagnostics and availability to treatments are critical to reducing disparities in CAH care. Full article

Newborn screening for congenital adrenal hyperplasia (CAH) is effective in identifying patients with severe forms before a potentially lethal crisis, but has a relatively high false-positive rate. The aim of this study was to improve the national neonatal screening program in Sweden and the positive predictive value by implementing LC-MS/MS second-tier testing. A combination of two independent parameters, the steroid hormone ratio (androstenedione+17-hydroxyprogesterone)/cortisol and the concentration of 21-deoxycortisol and adjustment of cut-off levels resulted in an increase in the positive predictive value (PPV) from 14% to 84% for full-term infants. In total, the false-positive screening cases decreased by 88%. CYP21A2 genotyping was used to determine the severity of CAH in identified cases. We report on the stepwise approach that was used to optimize the cut-off levels for full-term and preterm infants in order not to miss any true cases in the process. Full article

21-deoxycortisol is a sensitive and specific blood marker for congenital adrenal hyperplasia (CAH). We postulated that 21-deoxycortisone, the 11β-hydroxysteroid dehydrogenase metabolite of 21-deoxycortisol, may also be an accurate bloodspot marker of CAH. Measurement of 21-deoxycortisone was performed on 42 residual NBS specimens with a true positive result for CAH, 11 with a false negative result, and 439 specimens with a false positive result. For this study, the test was considered positive if 21-deoxycortisone was detected. The sensitivity and specificity of 21-deoxycortisone as a marker for classical CAH was calculated and compared to 21-deoxycortisol data from a previous New Zealand study. The method for 21-deoxycortisone measurement was linear to 1000 nmol/L and precision was 7.3–10.3%. The lower limit of quantification was 2 nmol/L, and recovery was 99%. 21-deoxycortisone was ≥2 nmol/L in all 42 true positive samples and in 10 false negative samples, and was not detected in the false positive group of specimens. The sensitivity of 21-deoxycortisone was 98.1%, and specificity was 100%. In a previous study, the sensitivity of 21-deoxycortisol was 88.7% and specificity was 99.8% in 1910 newborn screening tests carried out between 2018 and 2021. Incorporating 21-deoxycortisone into a second-tier test and adjustment of primary screening protocols could improve the accuracy of newborn screening for CAH. Longer term prospective studies on the performance of 21-deoxycortisone are warranted. Full article

Background: Secondary pseudohypoaldosteronism (PHA) is a rare, transient condition caused by renal tubular resistance to aldosterone, most commonly associated with urinary tract infection (UTI) and/or congenital anomalies of the kidney and urinary tract (CAKUT). It mimics primary adrenal disorders, presenting with life-threatening electrolyte disturbances in early infancy. Case Presentation: We report a male infant admitted twice within the first four months of life with severe dehydration, hyponatremia, hyperkalemia, metabolic acidosis, and acute kidney injury (AKI). Urine cultures grew Klebsiella pneumoniae and later Escherichia coli. Imaging studies demonstrated obstructive CAKUT, including posterior urethral valves, bilateral megaureters, hydronephrosis, and bladder diverticulosis. Congenital adrenal hyperplasia was excluded. Further evaluation showed markedly elevated plasma renin and aldosterone levels, confirming secondary PHA. The patient was successfully treated with intravenous fluids, electrolyte correction, and antibiotic therapy. Subsequently, oral sodium chloride and bicarbonate supplementation were added. Stepwise surgical correction of the urinary tract anomalies was initiated. Conclusions: Secondary PHA should be considered in infants presenting with failure to thrive, dehydration, hyponatremia, and hyperkalemia, particularly in the presence of UTI or CAKUT. Early recognition and differentiation from primary adrenal disorders are essential to prevent life-threatening complications. Prompt correction of electrolyte imbalance and management of the underlying urinary tract pathology are crucial for favorable outcomes. Full article

Background/Objectives: Non-classical congenital adrenal hyperplasia (NCCAH) due to 21-hydroxylase deficiency represents the mildest form of congenital adrenal hyperplasia and is frequently diagnosed only after the onset of clinical signs in childhood. Newborn screening programs for CAH are primarily designed to detect classical forms and show limited sensitivity for NCCAH. The clinical significance of neonatal 17-hydroxyprogesterone (17-OHP) values below recall thresholds remains incompletely defined. Methods: We retrospectively analyzed clinical, auxological, hormonal, and genetic data from pediatric patients diagnosed with NCCAH between 2018 and 2023 at a tertiary referral center. Neonatal screening 17-OHP concentrations, basal and ACTH-stimulated 17-OHP levels at diagnosis, bone age advancement, pubertal status, and hydrocortisone treatment were evaluated. Correlations between hormonal parameters, age at onset, and treatment dose were assessed. Results: Thirty-five patients (30 females) were included, with a mean age at clinical onset of 7.52 ± 0.36 years for females and 6.25 ± 0.29 years for males. Premature pubarche was the most frequent presenting sign (94.3%), and central precocious puberty was diagnosed in 31.4% of cases. The mean neonatal screening 17-OHP level was 4.53 ± 0.7 ng/mL; only two patients exceeded the screening recall cut-off. At diagnosis, mean basal and ACTH-stimulated 17-OHP levels were 15.1 ± 3.35 and 55.2 ± 11.3 ng/mL, respectively. Age at clinical onset was inversely correlated with both basal and stimulated 17-OHP levels, while hydrocortisone dose correlated positively with biochemical severity. Bone age advancement was observed in all patients. Conclusions: Most children with NCCAH display mildly elevated neonatal 17-OHP values that do not trigger screening recall. Higher biochemical severity is associated with earlier clinical presentation and higher glucocorticoid requirements. Neonatal 17-OHP concentrations, even when below cut-off values, may represent an early indicator of disease severity and warrant further investigation. Full article

Newborn screening (NBS) is a well-established public health program that enables early detection and treatment of rare disorders in newborns, preventing severe complications or death. Despite its recognized importance, the scope and implementation of NBS programs vary across Southeastern (SE) and Central Europe. This study aimed to evaluate the current status of NBS in 16 countries of SE and Central Europe and assess progress since the previous survey in 2021. A structured questionnaire was distributed to national experts between April and December 2025, collecting data on program organization, coverage, diseases included, laboratory methods, confirmatory testing, consent practices, and future expansion plans. All countries reported universal screening for congenital hypothyroidism, except Kosovo, where a national NBS is in the process of being established. Expanded NBS using tandem mass spectrometry was available in Austria, Bulgaria, Croatia, Cyprus, Greece, Hungary, North Macedonia, Romania, and Slovenia. Spinal muscular atrophy screening became universal in Austria, Croatia, Hungary, Serbia, and Slovenia. Most countries reported plans for further expansion, with congenital adrenal hyperplasia, severe combined immunodeficiency, spinal muscular atrophy, and cystic fibrosis being the most frequently targeted conditions. Although notable infrastructural progress has been achieved, financial constraints, lack of staff, and organizational barriers remain key challenges. The study’s assessment of program effectiveness was further limited by the absence of region-wide systems for capturing end-to-end performance indicators, such as the age of the infant at treatment initiation or missed cases. Regional collaboration and adoption of best practices are therefore vital to ensure equitable access and continuous advancement of NBS programs. Full article

Serum and urinary steroid profiles are altered in hormone-producing adrenal adenomas, Cushing’s or Conn’s syndrome, or adrenocortical carcinoma. Definitive diagnosis of inherited congenital adrenal hyperplasia is usually accomplished by measuring the blood levels of adrenal hormones and precursor steroids. Neonatal diagnosis of congenital adrenal hyperplasia is complicated. Alternative methods such as gas chromatography/mass spectrometry or liquid chromatography/mass spectrometry have been used for the diagnosis of congenital adrenal hyperplasia. This review covers the current application of gas chromatography/mass spectrometry or liquid chromatography/mass spectrometry in the interpretation of steroid profiles in different clinical and diagnostic settings. In the future, mass spectrometry may provide more information to assist in the choice of routine DNA analysis. Full article

Background and Clinical Significance: This report presents the case of a 33-year-old female with recurrent miscarriage, investigated for an adrenal cortical adenoma characterized by autonomous secretion of 17-hydroxyprogesterone (17-OHP). The findings challenge the established diagnostic paradigm, which predominantly attributes elevated serum 17-OHP to congenital adrenal hyperplasia (CAH) or non-classical CAH (NCCAH). Case Presentation: The patient was found to have elevated serum 17-OHP and a 2 cm left adrenal mass. Normal testosterone and precursor levels, along with whole-exome sequencing (WES), argued against a diagnosis of non-classical 21-hydroxylase deficiency (NC-21OHD). An ACTH stimulation test elicited a mild-to-moderate rise in 17-OHP, while adrenal venous sampling (AVS) confirmed marked lateralization of 17-OHP hypersecretion to the left side. Postoperative normalization of 17-OHP levels further supported the diagnosis of a 17-OHP-secreting tumor. Histopathological analysis identified tumor regions with non-uniformly high expression of CYP17A1 and CYP21A2. Preliminary transcriptomic profiling revealed that differentially expressed genes (DEGs) were enriched in microRNA-related and PI3K-Akt signaling pathways. Conclusions: This paradigm-shifting case indicates that, in addition to 21OHD, a 17-OHP-hypersecreting adrenal adenoma should be considered in the differential diagnosis of elevated 17-OHP. The integration of multimodal diagnostic techniques, particularly AVS, is valuable for localizing hormonally active tumors. Preliminary mechanistic insights suggest a potential role for epigenetic dysregulation in the pathogenesis of this tumor type. Full article

This study was designed to assess the effectiveness of neonatal congenital adrenal hyperplasia (CAH) screening in Guangzhou, China. A total of 818,417 newborns were screened for CAH by measuring 17-hydroxyprogesterone (17-OHP) concentrations. Cut-off values were stratified based on gestational age (GA) and the timing of sample collection. Neonates with initial positive results (17-OHP ≥ cut-off value) were recalled for a second dried blood spot sample to reassess 17-OHP levels. Confirmatory testing involved biochemical analyses, Sanger sequencing, and multiplex ligation-dependent probe amplification of the CYP21A2 gene. From 2018 to 2024, a total of 40 patients with classical 21-hydroxylase deficiency were identified, including 28 cases (70%) of the salt-wasting form and 12 cases (30%) of the simple virilizing form. The overall incidence of CAH was 1 in 20,653 (95% confidence interval: 1:34,928, 1:14,661). No statistically significant differences in prevalence were observed between sexes or between preterm and full-term infants (p > 0.05). 17-OHP concentrations are influenced by GA and the timing of sample collection. The screening efficiency for CAH could be improved by adopting a multitiered cut-off value system adjusted for GA and collection time. Full article