Identification of Predictors of Metastatic Potential in Paragangliomas to Develop a Prognostic Score (PSPGL).

Context: Paragangliomas (PGLs) are rare tumors in adrenal and extra-adrenal locations. Metastasis are found in approximately 5% to 35% of PGLs, and there are no reliable predictors of metastatic disease. Objective: This work aimed to develop a prognostic score of metastatic potential in PGLs. Methods: A retrospective analysis was conducted of clinical data from a cohort with PGLs and tumor histological assessment. Patients were divided into metastatic PGL (presence of metastasis) and nonmetastatic PGL (absence of metastasis ≥96 months of follow-up) groups. Univariate and multivariable analysis were performed to identify predictors of metastatic potential. A prognostic score was developed based on coefficients of multivariable analysis. Kaplan-Meier curves were generated to estimate disease-specific survival (DSS). Results: Out of 263 patients, 35 patients had metastatic PGL and 110 patients had nonmetastatic PGL. In multivariable analysis, 4 features were independently related to metastatic disease and composed the Prognostic Score of Paragangliomas (PSPGL): presence of central or confluent necrosis (33 points), more than 3 mitosis/10 high-power field (HPF) (28 points), extension into adipose tissue (20 points), and extra-adrenal location (19 points). A PSPGL of 24 or greater showed similar sensitivity with higher specificity than the Pheochromocytoma of the Adrenal Gland Scaled Score (PASS) and Grading System for Adrenal Pheochromocytoma and Paraganglioma (GAPP). PSPGL less than or equal to 20 was associated with a risk of metastasis of approximately 10%, whereas a PSPGL of 40 or greater was associated with approximately 80%. The presence of metastasis and Ki-67 of 3% or greater were related to lower DSS. Conclusion: The PSPGL, composed of 4 easy-to-assess parameters, demonstrated good performance in predicting metastatic potential and good ability in estimating metastasis risk.

Intraoperative radiofrequency ablation for unresectable abdominal paraganglioma: a case report.

For pheochromocytoma and paraganglioma (PPGL), the efficacy of percutaneous ablative therapies in achieving control of metastatic tumors measuring <3 cm had been demonstrated in only few reports, and intraoperative radiofrequency ablation (RFA) of locally invasive primary PPGLs has not been reported. We presented the case of a 31-year-old man who had a 9-cm functioning unresectable PPGL. He was treated with 13 cycles of cytotoxic chemotherapy without objective tumor response, according to the Response Evaluation Criteria in Solid Tumors (RECIST). Subsequently, magnetic resonance imaging revealed a 9.0 × 8.6 × 6.0-cm retroperitoneal mass that extended to the inferior portion of the inferior vena cava, the inferior mesenteric artery, and the infrarenal aorta. Biochemical evaluation demonstrated high level of plasma normetanephrine (20.2 nmol/L, normal range <0.9 nmol/L). Genetic investigation showed the germline pathogenic variant c.1591delC (p. Ser198Alafs*22) in the SDHB gene. I131-metaiodobenzylguanidine scintigraphy was negative and Ga68-dotatate PET-CT scan showed high tumor uptake without distant metastases. On open laparotomy, tumor debulking was not possible. Therefore, intraoperative RFA was performed by a highly experienced team of interventional radiologists. At 12 months after the RFA, the tumor volume decreased from 208 to 45 mL (78%), plasma normetanephrine decreased from 20.2 to 2.6 nmol/L (87%), and the doxazosin dose was reduced from 16 to 8 mg/day. To our best knowledge, this was the first report on intraoperative RFA that markedly reduced the size of a large primary unresectable PPGL, along with clinical and biochemical responses.

Beckwith-Wiedemann syndrome mimicking the classical form of congenital adrenal hyperplasia in newborn screening.

Beckwith-Wiedemann syndrome (BWS) is a common genetic congenital disease characterized by somatic overgrowth and its broad clinical spectrum includes pre- and post-natal macrosomia, macroglossia, visceromegaly, increased risk of neonatal hypoglycemia, and development of embryonic tumors. BWS occurs due to genetic/epigenetic changes involving growth-regulating genes, located on region 11p15, with an important genotype-phenotype correlation. Congenital adrenal hyperplasia (CAH) comprises a spectrum of autosomal recessive diseases presenting a variety of clinical manifestations due to a deficiency in one of the enzymes involved in cortisol secretion. Early diagnosis based on newborn screening prevents the adrenal crisis and early infant death. However, high 17-hydroxyprogesterone (17-OHP) levels can occur in newborns or premature infants without CAH, in situations of stress due to maternal or neonatal factors. Here, we report new cases of false-positive diagnosis of 21-hydroxylase deficiency during newborn screening - two girls and one boy with BWS. Methylation-specific multiplex ligation-dependent probe amplification revealed a gain of methylation in the H19 differentially methylated region. Notably, all three cases showed a complete normalization of biochemical changes, highlighting the transient nature of these hormonal findings that imitate the classical form of CAH. This report sheds light on a new cause of false-positive 21-hydroxylase deficiency diagnosis during newborn screening: Beckwith-Wiedemann syndrome.

Clinical and Pathological Predictors of Death for Adrenocortical Carcinoma.

Adrenocortical carcinoma (ACC) is a rare and lethal disease with a poor prognosis. This study aims to share our 41-year experience as a referral center, focusing on identifying risk factors associated with ACC mortality. Our retrospective analysis included a cohort of 150 adult patients with ACC in all stage categories, treated between 1981 and 2022. Tumor hormonal hypersecretion was observed in 78.6% of the patients, and the median age of diagnosis was 40 years. The majority presented as European Network for the Study of Adrenal Tumors (ENSAT) III or IV (22.9% and 31.2%, respectively), and the overall mortality rate was 54.6%. Independent predictors of death were elevated secretion of cortisol (HR = 2.0), androstenedione (HR = 2.2), estradiol (HR = 2.8), 17-OH progesterone (HR = 2.0), and 11-deoxycortisol (HR = 5.1), higher Weiss (HR = 4.3), modified Weiss (HR = 4.4), and Helsinki scores (HR = 12.0), advanced ENSAT stage (HR = 27.1), larger tumor size (HR = 2.7), higher Ki-67 percentage (HR = 2.3), and incomplete surgical resection (HR = 2.5). Mitosis greater than 5/50 high-power field (HR = 5.6), atypical mitosis (HR = 2.3), confluent necrosis (HR = 15.4), venous invasion (HR = 2.8), and capsular invasion (HR = 2.4) were also identified as independent predictors of death. Knowing the risk factors for ACC's mortality may help determine the best treatment option.

Renal Function Evolution and Hypoaldosteronism Risk After Unilateral Adrenalectomy for Primary Aldosteronism.

Few studies demonstrated a percentage decrease in the estimated glomerular filtration rate (eGFR) at a single time and the rate of hypoaldosteronism after adrenalectomy for primary aldosteronism (PA). Our aim was to investigate the evolution of renal function and the hypoaldosteronism risk after adrenalectomy for PA. Aldosterone, renin, eGFR, and electrolyte levels were determined before and at 1 week, 1, 3 and 6 months after unilateral adrenalectomy in 94 PA patients (40 men and 54 women). The main outcome was the postoperative eGFR decline using analysis of covariance with the preoperative eGFR as a covariate. eGFR decreased during first postoperative week compared to 3 months before surgery. During the first 6 months, eGFR remained stable at similar levels to the first week after surgery. Age (p=0.001), aldosterone levels (p=0.021) and eGFR 3 months before surgery (p+<+0.0001) had a significant correlation with eGFR during first postoperative week. High aldosterone levels at diagnosis were correlated with decline in renal function in the univariate model (p=0.033). In the multivariate analysis, aldosterone levels at diagnosis had a tendency to be an independent predictor of renal function after surgery (p=0.059). Postoperative biochemical hypoaldosteronism was diagnosed in 48% of the cases after adrenalectomy, but prolonged hyperkalemia occurred in only 4 cases (4.5%). Our findings showed a decrease of eGFR after unilateral adrenalectomy for PA. Additionally, aldosterone levels at diagnosis correlated with postoperative renal function. Postoperative biochemical hypoaldosteronism occurred in almost half of the patients, but prolonged hyperkalemia with fludrocortisone replacement was less frequent.

Rare variants in the MECP2 gene in girls with central precocious puberty: a translational cohort study.

BACKGROUND: Identification of genetic causes of central precocious puberty have revealed epigenetic mechanisms as regulators of human pubertal timing. MECP2, an X-linked gene, encodes a chromatin-associated protein with a role in gene transcription. MECP2 loss-of-function mutations usually cause Rett syndrome, a severe neurodevelopmental disorder. Early pubertal development has been shown in several patients with Rett syndrome. The aim of this study was to explore whether MECP2 variants are associated with an idiopathic central precocious puberty phenotype. METHODS: In this translational cohort study, participants were recruited from seven tertiary centres from five countries (Brazil, Spain, France, the USA, and the UK). Patients with idiopathic central precocious puberty were investigated for rare potentially damaging variants in the MECP2 gene, to assess whether MECP2 might contribute to the cause of central precocious puberty. Inclusion criteria were the development of progressive pubertal signs (Tanner stage 2) before the age of 8 years in girls and 9 years in boys and basal or GnRH-stimulated LH pubertal concentrations. Exclusion criteria were the diagnosis of peripheral precocious puberty and the presence of any recognised cause of central precocious puberty (CNS lesions, known monogenic causes, genetic syndromes, or early exposure to sex steroids). All patients included were followed up at the outpatient clinics of participating academic centres. We used high-throughput sequencing in 133 patients and Sanger sequencing of MECP2 in an additional 271 patients. Hypothalamic expression of Mecp2 and colocalisation with GnRH neurons were determined in mice to show expression of Mecp2 in key nuclei related to pubertal timing regulation. FINDINGS: Between Jun 15, 2020, and Jun 15, 2022, 404 patients with idiopathic central precocious puberty (383 [95%] girls and 21 [5%] boys; 261 [65%] sporadic cases and 143 [35%] familial cases from 134 unrelated families) were enrolled and assessed. We identified three rare heterozygous likely damaging coding variants in MECP2 in five girls: a de novo missense variant (Arg97Cys) in two monozygotic twin sisters with central precocious puberty and microcephaly; a de novo missense variant (Ser176Arg) in one girl with sporadic central precocious puberty, obesity, and autism; and an insertion (Ala6_Ala8dup) in two unrelated girls with sporadic central precocious puberty. Additionally, we identified one rare heterozygous 3'UTR MECP2 insertion (36_37insT) in two unrelated girls with sporadic central precocious puberty. None of them manifested Rett syndrome. Mecp2 protein colocalised with GnRH expression in hypothalamic nuclei responsible for GnRH regulation in mice. INTERPRETATION: We identified rare MECP2 variants in girls with central precocious puberty, with or without mild neurodevelopmental abnormalities. MECP2 might have a role in the hypothalamic control of human pubertal timing, adding to the evidence of involvement of epigenetic and genetic mechanisms in this crucial biological process. FUNDING: Fundação de Amparo à Pesquisa do Estado de São Paulo, Conselho Nacional de Desenvolvimento Científico e Tecnológico, and the Wellcome Trust.

ß-Catenin-Driven Differentiation Is a Tissue-Specific Epigenetic Vulnerability in Adrenal Cancer.

Adrenocortical carcinoma (ACC) is a rare cancer in which tissue-specific differentiation is paradoxically associated with dismal outcomes. The differentiated ACC subtype CIMP-high is prevalent, incurable, and routinely fatal. CIMP-high ACC possess abnormal DNA methylation and frequent ß-catenin-activating mutations. Here, we demonstrated that ACC differentiation is maintained by a balance between nuclear, tissue-specific ß-catenin-containing complexes, and the epigenome. On chromatin, ß-catenin bound master adrenal transcription factor SF1 and hijacked the adrenocortical super-enhancer landscape to maintain differentiation in CIMP-high ACC; off chromatin, ß-catenin bound histone methyltransferase EZH2. SF1/ß-catenin and EZH2/ß-catenin complexes present in normal adrenals persisted through all phases of ACC evolution. Pharmacologic EZH2 inhibition in CIMP-high ACC expelled SF1/ß-catenin from chromatin and favored EZH2/ß-catenin assembly, erasing differentiation and restraining cancer growth in vitro and in vivo. These studies illustrate how tissue-specific programs shape oncogene selection, surreptitiously encoding targetable therapeutic vulnerabilities. SIGNIFICANCE: Oncogenic ß-catenin can use tissue-specific partners to regulate cellular differentiation programs that can be reversed by epigenetic therapies, identifying epigenetic control of differentiation as a viable target for ß-catenin-driven cancers.

Evidence for a Founder Effect of SDHB Exon 1 Deletion in Brazilian Patients With Paraganglioma.

CONTEXT: Limited information is available concerning the genetic spectrum of pheochromocytoma and paraganglioma (PPGL) patients in South America. Germline SDHB large deletions are very rare worldwide, but most of the individuals harboring the SDHB exon 1 deletion originated from the Iberian Peninsula. OBJECTIVE: Our aim was to investigate the spectrum of SDHB genetic defects in a large cohort of Brazilian patients with PPGLs. METHODS: Genetic investigation of 155 index PPGL patients was performed by Sanger DNA sequencing, multiplex ligation-dependent probe amplification, and/or target next-generation sequencing panel. Common ancestrality was investigated by microsatellite genotyping with haplotype reconstruction, and analysis of deletion breakpoint. RESULTS: Among 155 index patients, heterozygous germline SDHB pathogenic or likely pathogenic variants were identified in 22 cases (14.2%). The heterozygous SDHB exon 1 complete deletion was the most frequent genetic defect in SDHB, identified in 8 out of 22 (36%) of patients. Haplotype analysis of 5 SDHB flanking microsatellite markers demonstrated a significant difference in haplotype frequencies in a case-control permutation test (P = 0.03). More precisely, 3 closer/informative microsatellites were shared by 6 out of 8 apparently unrelated cases (75%) (SDHB-GATA29A05-D1S2826-D1S2644 | SDHB-186-130-213), which was observed in only 1 chromosome (1/42) without SDHB exon 1 deletion (X2 = 29.43; P < 0.001). Moreover, all cases with SDHB exon 1 deletion had the same gene breakpoint pattern of a 15 678 bp deletion previously described in the Iberian Peninsula, indicating a common origin. CONCLUSION: The germline heterozygous SDHB exon 1 deletion was the most frequent genetic defect in the Brazilian PPGL cohort. Our findings demonstrated a founder effect for the SDHB exon 1 deletion in Brazilian patients with paragangliomas.

The Congenital and Acquired Mechanisms Implicated in the Etiology of Central Precocious Puberty.

The etiology of central precocious puberty (CPP) is multiple and heterogeneous, including congenital and acquired causes that can be associated with structural or functional brain alterations. All causes of CPP culminate in the premature pulsatile secretion of hypothalamic GnRH and, consequently, in the premature reactivation of hypothalamic-pituitary-gonadal axis. The activation of excitatory factors or suppression of inhibitory factors during childhood represent the 2 major mechanisms of CPP, revealing a delicate balance of these opposing neuronal pathways. Hypothalamic hamartoma (HH) is the most well-known congenital cause of CPP with central nervous system abnormalities. Several mechanisms by which hamartoma causes CPP have been proposed, including an anatomical connection to the anterior hypothalamus, autonomous neuroendocrine activity in GnRH neurons, trophic factors secreted by HH, and mechanical pressure applied to the hypothalamus. The importance of genetic and/or epigenetic factors in the underlying mechanisms of CPP has grown significantly in the last decade, as demonstrated by the evidence of genetic abnormalities in hypothalamic structural lesions (eg, hamartomas, gliomas), syndromic disorders associated with CPP (Temple, Prader-Willi, Silver-Russell, and Rett syndromes), and isolated CPP from monogenic defects (MKRN3 and DLK1 loss-of-function mutations). Genetic and epigenetic discoveries involving the etiology of CPP have had influence on the diagnosis and familial counseling providing bases for potential prevention of premature sexual development and new treatment targets in the future. Global preventive actions inducing healthy lifestyle habits and less exposure to endocrine-disrupting chemicals during the lifespan are desirable because they are potentially associated with CPP.

Adult Height of Patients with SHOX Haploinsufficiency with or without GH Therapy: A Real-World Single-Center Study.

INTRODUCTION: Isolated SHOX haploinsufficiency is a common monogenic cause of short stature. Few studies compare untreated and rhGH-treated patients up to adult height (AH). Our study highlights a growth pattern from childhood to AH in patients with SHOX haploinsufficiency and analyzes the real-world effectiveness of rhGH alone or plus GnRH analog (GnRHa). METHODS: Forty-seven patients (18 untreated and 29 rhGH-treated) with SHOX haploinsufficiency were included in a longitudinal retrospective study. Adult height was attained in 13 untreated and 18 rhGH-treated (rhGH alone [n = 8] or plus GnRHa [n = 10]) patients. RESULTS: The untreated group decreased height SDS from baseline to AH (-0.8 [-1.1; -0.4]), with an increase in the prevalence of short stature from 31% to 77%. Conversely, the rhGH-treated group had an improvement in height SDS from baseline to AH (0.6 [0.2; 0.6]; p < 0.001), with a reduction in the prevalence of short stature (from 61% to 28%). AH in the rhGH-treated patients was 1 SD (6.3 cm) taller than in untreated ones. Regarding the use of GnRHa, the subgroups (rhGH alone or plus GnRHa) attained similar AH, despite the higher prevalence of pubertal patients and worse AH prediction at the start of rhGH treatment in patients who used combined therapy. CONCLUSION: The use of rhGH treatment improves AH in patients with SHOX haploinsufficiency, preventing the loss of height potential during puberty. In peripubertal patients, the addition of GnRHa to rhGH allows AH attainment similar to the AH of patients who start rhGH alone in the prepubertal age.

WT1 Pathogenic Variants are Associated with a Broad Spectrum of Differences in Sex Development Phenotypes and Heterogeneous Progression of Renal Disease.

Wilms' tumor suppressor gene 1 (WT1) plays an essential role in urogenital and kidney development. Heterozygous germline pathogenic allelic variants of WT1 have been classically associated with Denys-Drash syndrome (DDS) and Frasier syndrome (FS). Usually, exonic pathogenic missense variants in the zinc finger region are the cause of DDS, whereas pathogenic variants affecting the canonic donor lysine-threonine-serine splice site in intron 9 cause FS. Phenotypic overlap between WT1 disorders has been frequently observed. New WT1 variant-associated phenotypes, such as 46,XX testicular/ovarian-testicular disorders of sex development (DSD) and primary ovarian insufficiency, have been reported. In this report, we describe the phenotypes and genotypes of 7 Brazilian patients with pathogenic WT1 variants. The molecular study involved Sanger sequencing and massively parallel targeted sequencing using a DSD-associated gene panel. Six patients (5 with a 46,XY karyotype and 1 with a 46,XX karyotype) were initially evaluated for atypical genitalia, and a 46,XY patient with normal female genitalia sought medical attention for primary amenorrhea. Germ cell tumors were identified in 2 patients, both with variants affecting alternative splicing of WT1 between exons 9 and 10. Two pathogenic missense WT1 variants were identified in two 46,XY individuals with Wilms' tumors; both patients were <1 year of age at the time of diagnosis. A novel WT1 variant, c.1453_1456 (p.Arg485Glyfs*14), was identified in a 46,XX patient with testicular DSD. Nephrotic proteinuria was diagnosed in all patients, including 3 who underwent renal transplantation after progressing to end-stage kidney disease. The expanding phenotypic spectrum associated with WT1 variants in XY and XX individuals confirms their pivotal role in gonadal and renal development as well as in tumorigenesis, emphasizing the clinical implications of these variants in genetic diagnosis.

Targeted massively parallel sequencing panel to diagnose genetic endocrine disorders in a tertiary hospital

Abstract Objectives To analyze the efficiency of a multigenic targeted massively parallel sequencing panel related to endocrine disorders for molecular diagnosis of patients assisted in a tertiary hospital involved in the training of medical faculty. Material and methods Retrospective analysis of the clinical diagnosis and genotype obtained from 272 patients in the Endocrine unit of a tertiary hospital was performed using a custom panel designed with 653 genes, most of them already associated with the phenotype (OMIM) and some candidate genes that englobes developmental, metabolic and adrenal diseases. The enriched DNA libraries were sequenced in NextSeq 500. Variants found were then classified according to ACMG/AMP criteria, with Varsome and InterVar. Results Three runs were performed; the mean coverage depth of the targeted regions in panel sequencing data was 249×, with at least 96.3% of the sequenced bases being covered more than 20-fold. The authors identified 66 LP/P variants (24%) and 27 VUS (10%). Considering the solved cases, 49 have developmental diseases, 12 have metabolic and 5 have adrenal diseases. Conclusion The application of a multigenic panel aids the training of medical faculty in an academic hospital by showing the picture of the molecular pathways behind each disorder. This may be particularly helpful in developmental disease cases. A precise genetic etiology provides an improvement in understanding the disease, guides decisions about prevention or treatment, and allows genetic counseling.

Adrenal crisis and mortality rate in adrenal insufficiency and congenital adrenal hyperplasia

ABSTRACT Primary adrenal insufficiency (PAI) is characterized by the inability of the adrenal cortex to produce sufficient amounts of glucocorticoids and/or mineralocorticoids. Addison's disease (AD) and congenital adrenal hyperplasia (CAH) are the most frequent disorders in adults and children, respectively. Despite the diagnostic advances and the availability of glucocorticoid and mineralocorticoid replacements, adrenal crisis (AC) is still a potentially lethal condition contributing to the increased mortality, not only during the first year of life, but also throughout life. Failure in increasing glucocorticoid doses during acute stress, when greater amounts of glucocorticoids are required, can lead to AC and an increase morbimortality rate of PAI. Considering a mortality rate of 0.5 per 100 patient years, up to 1,500 deaths from AC are expected in Brazil in the coming decade, which represents an alarming situation. The major clinical features are hypotension and volume depletion. Nonspecific symptoms such as fatigue, lack of energy, anorexia, nausea, vomiting, and abdominal pain are common. The main precipitating factors are gastrointestinal diseases, other infectious disease, stressful events (e.g., major pain, surgery, strenuous physical activity, heat, and pregnancy), and withdrawal of glucocorticoid therapy. Suspected AC requires immediate therapeutic action with intravenous (iv) hydrocortisone, fluid infusion, monitoring support, and antibiotics if necessary. AC is best prevented through patient education, precocious identification and by adjusting the glucocorticoid dosage in stressor situations. The emergency card, warning about acute glucocorticoid replacement, has high value in reducing the morbidity and mortality of AC.

High-throughput splicing assays identify missense and silent splice-disruptive POU1F1 variants underlying pituitary hormone deficiency.

Pituitary hormone deficiency occurs in ∼1:4,000 live births. Approximately 3% of the cases are due to mutations in the alpha isoform of POU1F1, a pituitary-specific transcriptional activator. We found four separate heterozygous missense variants in unrelated individuals with hypopituitarism that were predicted to affect a minor isoform, POU1F1 beta, which can act as a transcriptional repressor. These variants retain repressor activity, but they shift splicing to favor the expression of the beta isoform, resulting in dominant-negative loss of function. Using a high-throughput splicing reporter assay, we tested 1,070 single-nucleotide variants in POU1F1. We identified 96 splice-disruptive variants, including 14 synonymous variants. In separate cohorts, we found two additional synonymous variants nominated by this screen that co-segregate with hypopituitarism. This study underlines the importance of evaluating the impact of variants on splicing and provides a catalog for interpretation of variants of unknown significance in POU1F1.

Adrenal crisis and mortality rate in adrenal insufficiency and congenital adrenal hyperplasia.

Primary adrenal insufficiency (PAI) is characterized by the inability of the adrenal cortex to produce sufficient amounts of glucocorticoids and/or mineralocorticoids. Addison's disease (AD) and congenital adrenal hyperplasia (CAH) are the most frequent disorders in adults and children, respectively. Despite the diagnostic advances and the availability of glucocorticoid and mineralocorticoid replacements, adrenal crisis (AC) is still a potentially lethal condition contributing to the increased mortality, not only during the first year of life, but also throughout life. Failure in increasing glucocorticoid doses during acute stress, when greater amounts of glucocorticoids are required, can lead to AC and an increase morbimortality rate of PAI. Considering a mortality rate of 0.5 per 100 patient years, up to 1,500 deaths from AC are expected in Brazil in the coming decade, which represents an alarming situation. The major clinical features are hypotension and volume depletion. Nonspecific symptoms such as fatigue, lack of energy, anorexia, nausea, vomiting, and abdominal pain are common. The main precipitating factors are gastrointestinal diseases, other infectious disease, stressful events (e.g., major pain, surgery, strenuous physical activity, heat, and pregnancy), and withdrawal of glucocorticoid therapy. Suspected AC requires immediate therapeutic action with intravenous (iv) hydrocortisone, fluid infusion, monitoring support, and antibiotics if necessary. AC is best prevented through patient education, precocious identification and by adjusting the glucocorticoid dosage in stressor situations. The emergency card, warning about acute glucocorticoid replacement, has high value in reducing the morbidity and mortality of AC.

Anthropometric, metabolic, and reproductive outcomes of patients with central precocious puberty treated with leuprorelin acetate 3-month depot (11.25 mg).

OBJECTIVES: Longer-acting gonadotropin-releasing hormone analogs (GnRHa) have been widely used for central precocious puberty (CPP) treatment. However, the follow-up of patients after this treatment are still scarce. Our aim was to describe anthropometric, metabolic, and reproductive follow-up of CPP patients after treatment with leuprorelin acetate 3-month depot (11.25 mg). METHODS: Twenty-two female patients with idiopathic CPP were treated with leuprorelin acetate 3-month depot (11.25 mg). Their medical records were retrospectively evaluated regarding clinical, hormonal, and imaging aspects before, during, and after GnRHa treatment until adult height (AH). RESULTS: At the diagnosis of CPP, the mean chronological age (CA) was 8.2 ± 1.13 year, and mean bone age (BA) was 10.4 ± 1.4 year. Mean height SDS at the start and the end of GnRHa treatment was 1.6 ± 0.8 and 1.3 ± 0.9, respectively. The mean duration of GnRHa treatment was 2.8 ± 0.8 year. Mean predicted adult heights (PAH) at the start and the end of GnRH treatment was 153.2 ± 8.6 and 164.4 ± 7.3 cm, respectively (p<0.05). The mean AH was 163.2 ± 6.2 cm (mean SDS: 0.1 ± 1). All patients were within their target height (TH) range. There was a decrease in the percentage of overweight and obesity from the diagnosis until AH (39-19% p>0.05). At the AH, the insulin resistance and high LDL levels were identified in 3/17 patients (17.6%) and 2/21 patients (9.5%), respectively. The mean CA of menarche was 12.2 ± 0.5 years. At the AH, PCOS was diagnosed in one patient (4.8%). CONCLUSIONS: Long-term anthropometric, metabolic, and reproductive follow-up of patients with CPP treated with longer-acting GnRHa revealed effectivity, safety, and favorable outcomes.

The phenotypic spectrum associated with OTX2 mutations in humans.

Objective: The transcription factor OTX2 is implicated in ocular, craniofacial, and pituitary development. Design: We aimed to establish the contribution of OTX2 mutations in congenital hypopituitarism patients with/without eye abnormalities, study functional consequences, and establish OTX2 expression in the human brain, with a view to investigate the mechanism of action. Methods: We screened patients from the UK (n = 103), international centres (n = 24), and Brazil (n = 282); 145 were within the septo-optic dysplasia spectrum, and 264 had no eye phenotype. Transactivation ability of OTX2 variants was analysed in murine hypothalamic GT1-7 neurons. In situ hybridization was performed on human embryonic brain sections. Genetically engineered mice were generated with a series of C-terminal OTX2 variants. Results: Two chromosomal deletions and six haploinsufficient mutations were identified in individuals with eye abnormalities; an affected relative of one patient harboured the same mutation without an ocular phenotype. OTX2 truncations led to significant transactivation reduction. A missense variant was identified in another patient without eye abnormalities; however, studies revealed it was most likely not causative. In the mouse, truncations proximal to aa219 caused anophthalmia, while distal truncations and the missense variant were tolerated. During human embryogenesis, OTX2 was expressed in the posterior pituitary, retina, ear, thalamus, choroid plexus, and partially in the hypothalamus, but not in the anterior pituitary. Conclusions: OTX2 mutations are rarely associated with hypopituitarism in isolation without eye abnormalities, and may be variably penetrant, even within the same pedigree. Our data suggest that the endocrine phenotypes in patients with OTX2 mutations are of hypothalamic origin.

Genetic and clinical aspects of paediatric pheochromocytomas and paragangliomas.

OBJECTIVE: Few and conflicting reports have characterized the genetics of paediatric pheochromocytomas and paragangliomas (PPGLs). This study aimed to investigate the clinical and genetic features of Brazilian children with PPGL. PATIENTS AND METHODS: This study included 25 children (52% girls) with PPGL. The median age at diagnosis was 15 years (4-19). The median time of follow-up was 145 months. The genetic investigation was performed by Sanger DNA sequencing, multiplex ligation-dependent probe amplification and/or target next-generation sequencing panel. RESULTS: Of the 25 children with PPGL, 11 (44%), 4 (16%), 2 (8%), 1 (4%) and 7 (28%) had germline VHL pathogenic variants, SDHB, SDHD, RET and negative genetic investigation, respectively. Children with germline VHL missense pathogenic variants were younger than those with SDHB or SDHD genetic defects [median (range), 12 (4-16) vs. 15.5 (14-19) years; P = .027]. Moreover, 10 of 11 cases with VHL pathogenic variants had bilateral pheochromocytoma (six asynchronous and four synchronous). All children with germline SDHB pathogenic variants presented with abdominal paraganglioma (one of them malignant). The two cases with SDHD pathogenic variants presented with head and neck paraganglioma. Among the cases without a genetic diagnosis, 6 and 2 had pheochromocytoma and paraganglioma, respectively. Furthermore, metastatic PPGL was diagnosed in four (16%) of 25 PPGL. CONCLUSIONS: Most of the paediatric PPGL were hereditary and multifocal. The majority of the affected genes belong to pseudohypoxic cluster 1, with VHL being the most frequently mutated. Therefore, our findings impact surgical management and surveillance of children with PPGL.

Performance of mutation pathogenicity prediction tools on missense variants associated with 46,XY differences of sex development.

OBJECTIVES: Single nucleotide variants (SNVs) are the most common type of genetic variation among humans. High-throughput sequencing methods have recently characterized millions of SNVs in several thousand individuals from various populations, most of which are benign polymorphisms. Identifying rare disease-causing SNVs remains challenging, and often requires functional in vitro studies. Prioritizing the most likely pathogenic SNVs is of utmost importance, and several computational methods have been developed for this purpose. However, these methods are based on different assumptions, and often produce discordant results. The aim of the present study was to evaluate the performance of 11 widely used pathogenicity prediction tools, which are freely available for identifying known pathogenic SNVs: Fathmn, Mutation Assessor, Protein Analysis Through Evolutionary Relationships (Phanter), Sorting Intolerant From Tolerant (SIFT), Mutation Taster, Polymorphism Phenotyping v2 (Polyphen-2), Align Grantham Variation Grantham Deviation (Align-GVGD), CAAD, Provean, SNPs&GO, and MutPred. METHODS: We analyzed 40 functionally proven pathogenic SNVs in four different genes associated with differences in sex development (DSD): 17ß-hydroxysteroid dehydrogenase 3 (HSD17B3), steroidogenic factor 1 (NR5A1), androgen receptor (AR), and luteinizing hormone/chorionic gonadotropin receptor (LHCGR). To evaluate the false discovery rate of each tool, we analyzed 36 frequent (MAF>0.01) benign SNVs found in the same four DSD genes. The quality of the predictions was analyzed using six parameters: accuracy, precision, negative predictive value (NPV), sensitivity, specificity, and Matthews correlation coefficient (MCC). Overall performance was assessed using a receiver operating characteristic (ROC) curve. RESULTS: Our study found that none of the tools were 100% precise in identifying pathogenic SNVs. The highest specificity, precision, and accuracy were observed for Mutation Assessor, MutPred, SNP, and GO. They also presented the best statistical results based on the ROC curve statistical analysis. Of the 11 tools evaluated, 6 (Mutation Assessor, Phanter, SIFT, Mutation Taster, Polyphen-2, and CAAD) exhibited sensitivity >0.90, but they exhibited lower specificity (0.42-0.67). Performance, based on MCC, ranged from poor (Fathmn=0.04) to reasonably good (MutPred=0.66). CONCLUSION: Computational algorithms are important tools for SNV analysis, but their correlation with functional studies not consistent. In the present analysis, the best performing tools (based on accuracy, precision, and specificity) were Mutation Assessor, MutPred, and SNPs&GO, which presented the best concordance with functional studies.