Objective: To investigate somatic pathogenic variants (PVs) in the TERT promoter region and variants in the ATRX gene, both related to telomere maintenance in cancer, in a Brazilian cohort of adrenal and extra-adrenal paragangliomas (PPGLs), and to correlate these with metastatic disease as well as with clinical, radiological, and pathological characteristics. Materials and methods: The TERT promoter was analyzed by automated Sanger sequencing or whole-exome somatic sequencing in a cohort of 79 patients with PPGLs (53 non-metastatic and 26 metastatic), encompassing a total of 81 tumors. ATRX was assessed through whole-exome somatic sequencing in 26 patients from this cohort. Results: Germline PVs in Cluster 1A genes were identified in 28 of 79 patients (35.4%), including 20 patients harboring PVs in the Succinate Dehydrogenase Complex Iron Sulfur Subunit B gene (SDHB) (25.3%) and 8 patients (10.1%) with PVs in other Cluster 1A genes. Somatic PVs in the TERT promoter (c.-124C>T/ C228T) were detected in two metastatic PPGLs (2.5% of the total cohort). Three PPGLs (two metastatic), among the 26 patients studied, harbored somatic ATRX variants classified as likely benign (11.5%). Somatic PVs in the TERT promoter were identified in patients with germline SDHB PVs. Among metastatic patients with germline SDHB PVs, the frequency of somatic PVs in the TERT promoter was 16.7%. Conclusion: This study expands the understanding of telomere maintenance mechanisms in PPGLs in a Brazilian cohort enriched for SDHB alterations. Somatic variants in the TERT promoter were associated with aggressive tumor features, such as extra-adrenal location, germline SDHB PVs, and metastatic disease.

CONTEXT:Primary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of ACTH-independent Cushing syndrome (CS), occurring in isolation or as part of Carney complex (CNC); its phenotypic and molecular spectra remain incompletely defined. OBJECTIVE:To characterize phenotypic variability, genotype-phenotype correlations, and molecular findings in PPNAD through integrated clinical, histopathological, and genetic analyses. METHODS:We evaluated 18 index patients with PPNAD (15 with CNC, 3 isolated) and performed cascade genetic screening in relatives (20 variants-positive). Clinical, hormonal, imaging and histopathological data were reviewed; germline and somatic analyses employed targeted next-generation sequencing and copy-number assessment. RESULTS:Overt CS was present in 83% of index cases; 17% had cyclic hypercortisolism. Pathogenic or likely-pathogenic germline PRKAR1A variants were identified in 70.6% probands, including four novel variants; germline PRKACA duplications were detected in two cases. Cascade screening identified eight asymptomatic variant-positive relatives, in whom mild autonomous cortisol secretion (MACS) was observed. Somatic second-hit alterations in tumor tissue included PRKACA duplication, a PRKAR1A frameshift deletion, and 17q24.2 loss of heterozygosity. Histology most often showed classic PPNAD but also nonclassical patterns, expanding the morphological spectrum. Extra-adrenal manifestations were frequent (lentigines 61%, cardiac myxomas 22%). CONCLUSION:In this referral cohort, PPNAD/CNC displays marked phenotypic variability, incomplete penetrance with subclinical cortisol excess, and diverse molecular mechanisms, including novel germline variants and somatic second hits. Some cases lacked identifiable drivers, suggesting additional uncharacterized mechanisms. Systematic genetic screening and recognition of MACS are important for early diagnosis and long-term management.

BACKGROUND:The Brazilian population represents a mosaic of genetic diversity resulting from admixture ancestries. Given reported disparities in primary aldosteronism (PA) genetics across ethnicities, we investigated the genetic spectrum of aldosterone-producing adenomas (APAs) and nodules (APNs) with classical histology in a Brazilian cohort. METHODS:We included 62 lesions (1 case with bilateral APAs) from 61 consecutive patients (median age at PA diagnosis 49 years, 59% women) with PA and classical histology, defined by CYP11B2 immunostaining (HISTALDO consensus). Somatic DNA was extracted from CYP11B2-positive areas of the dominant lesions. Hotspot regions of KCNJ5, ATP1A1, ATP2B3, CACNA1D, and CTNNB1 were initially analyzed by Sanger sequencing. Whole-exome sequencing of paired somatic and germline DNA was subsequently performed in cases without driver variants. RESULTS:Histopathology showed combined APA + aldosterone-producing micronodules as the most frequent subtype (n = 29, 47.54%), followed by isolated APA (n = 20, 32.79%) and APN (n = 5, 8.2%). Somatic pathogenic variants were identified in 82.26% of lesions: KCNJ5 (n = 35, 56.45%), ATP2B3 (n = 7, 11.29%), CACNA1D (n = 5, 8.06%), and ATP1A1 (n = 4, 6.45%). Nine novel variants were identified, including 3 in ATP2B3 (2 exon 8 in-frame deletions and 1 missense), 3 in KCNJ5, 2 in CACNA1D, and 1 in CTNNB1. The frequency of ATP2B3 variants (11.29%) was significantly higher than that reported in other cohorts (4.06%) from different ethnicities (p = .0053). ATP2B3-mutated tumors occurred predominantly in older men and were smaller in size compared with wild-type tumors. Rare germline CACNA1H variants were also detected in 3 patients. CONCLUSION:We confirmed the predominance of known somatic drivers and identified a uniquely high frequency of ATP2B3 variants, refining their clinical phenotype. These findings underscore the influence of population-specific genetic backgrounds and expand the global understanding of PA genetics.

CONTEXT:Bilateral macronodular adrenocortical disease (BMAD) is a rare and often underdiagnosed cause of adrenal Cushing syndrome (CS), with manifestations ranging from mild autonomous cortisol secretion (MACS) to overt CS. ARMC5 and KDM1A are the most frequently implicated genes in familial BMAD; however, long-term follow-up data on affected patients remain limited. OBJECTIVE:This retrospective study assessed the clinical and hormonal variability, genetic profiles, treatment approaches, and outcomes of 17 familial and 9 sporadic BMAD cases over a follow-up period ranging from 8 to 410 months at a Brazilian tertiary center. METHODS:A total of 250 individuals (50 index cases and 200 relatives) were included. Clinical, hormonal, and imaging data, along with histological and genetic analyses of ARMC5 and KDM1A, were evaluated. RESULTS:Among 250 individuals, 104 (26 index and 78 relatives) carried germline pathogenic/likely pathogenic ARMC5 variants. No KDM1A (likely) pathogenic variants were identified in ARMC5-wild-type patients. ARMC5-positive index cases exhibited severe clinical manifestations, evidenced by elevated cortisol levels (urinary, salivary, and post-dexamethasone suppression test) and reduced ACTH and DHEAS levels (P = .005, P = .042, P = .005, P = .041, and P = .007, respectively). Index cases had larger adrenal nodules (P < .0001). Adrenal-sparing surgery achieved 100% remission vs a 40% remission rate for unilateral adrenalectomy. Central nervous system meningiomas were observed in BMAD patients independent of ARMC5 status. Interestingly, malignant neoplasms were notably prevalent among ARMC5-altered individuals. CONCLUSION:The proposed management flowchart highlights the importance of genetic screening and continuous monitoring to mitigate the adrenal insufficiency, MACS recurrence, and tumor risk, while underscoring the need for tailored therapeutic strategies in BMAD, adapted to genetic alterations and ARMC5 status.

The luteinizing hormone/choriogonadotropin receptor (LHCGR) is essential for Leydig cell function, gonadal steroidogenesis, and male sexual differentiation. Loss-of-function variants in LHCGR are a rare cause of 46,XY differences of sex development (DSD). We report 2 46,XY adult sisters born to consanguineous parents, who presented with primary amenorrhea and absent secondary sexual characteristics. Both exhibited tall stature, female external genitalia, hypergonadotropic hypogonadism with very low testosterone levels. Imaging revealed bilateral cryptorchid testes and absence of Müllerian structures. Both sisters underwent bilateral gonadectomy, and histopathological findings provide strong morphological support for Leydig cell hypoplasia. Genetic testing identified a novel homozygous splice-site variant (c.384-2A>G) in LHCGR, predicted to cause aberrant splicing and a loss of receptor function. These findings expand the mutational spectrum of LHCGR-related DSD, a rare cause of 46,XY DSD.

Supplementary Information:Supplementary Figures 1-6 with Figure Legends, Supplementary Table 1 (reference only), Supplementary Table 2, Supplementary Methods, Supplementary References

Cardiac myxomas, though rare, are the most common benign cardiac tumors and may be associated with Carney Complex (CNC). Patients with CNC are at increased risk of developing recurrent myxomas, which can lead to severe complications. We report a case of a 46-year-old woman with CNC and recurrent cardiac myxomas who developed multiple embolic strokes and cerebral aneurysms. Following two hemorrhagic strokes, neuroimaging and biopsy revealed a well-differentiated myxoid neoplasm in the brain parenchyma. Genetic analysis revealed a germline pathogenic PRKAR1A variant, along with loss of heterozygosity (LOH) at chromosome 17q24.2 in the cardiac myxoma, but not in the brain lesion. This case challenges the conventional understanding of cardiac myxomas as strictly benign, suggesting they may exceptionally exhibit distant proliferative behavior, likely through mechanical dissemination and subsequent growth in the brain. Although embolic events are common in cardiac myxomas, the capacity of tumor cells to implant and proliferate in extracardiac sites remains poorly understood. Our findings underscore the importance of maintaining a high index of suspicion for neurological complications in patients with cardiac myxomas, particularly in the setting of CNC. Further investigation is essential to elucidate the mechanisms driving this behavior and to optimize management strategies in similar cases.

OBJECTIVE:In this study, our aim was to search for new genotype-phenotype correlations in patients with Von Hippel-Lindau (VHL) disease. SUBJECTS AND METHODS:We retrospectively studied 53 consecutive patients with VHL disease and confirmed genetic diagnoses from 32 relatives. RESULTS:Most VHL pathogenic or likely pathogenic variants were missense (18 out of 32; 56.25%). The median size of the large carcinoma (RCC) was 3.6 cm (interquartile range, 2.8 to 6.5 cm). Interestingly, the size of the large RCC in patients harboring VHL pathogenic variants (n = 9) was significantly greater than that in patients with VHL likely pathogenic (n = 7) variants (5.4 cm [3.65 to 6.6] vs. 2.9 cm [2.45 to 3.35]; p = 0.008). Moreover, adrenal paraganglioma (PGL) (82.35% vs. 17.65%; p = 0.0001) and pancreatic neuroendocrine tumor (PNET) (81.81% vs. 18.18%; p = 0.007) were associated with missense VHL pathogenic or likely pathogenic variants compared with non-missense defects. In contrast, central nervous system (CNS) hemangioblastomas (HBs) (90.47% vs. 53.12%; p = 0.004), pancreatic cysts (76.19% vs. 28.12%; p = 0.001) and RCCs (57.14% vs. 12.5; p = 0.001) were more common in patients with non-missense VHL variants. CONCLUSION:VHL pathogenic variants were associated with larger RCCs than were VHL likely pathogenic variants.

INTRODUCTION:Worldwide, combined 17-hydroxylase/17,20-lyase deficiency (CYP17D) is a rare form of congenital adrenal hyperplasia, but it is the second most prevalent type in Brazil. An absence of sexual differentiation and hypergonadotropic hypogonadism arise from a reduction in the usual pattern of sex steroid formation in the adrenals and the gonads, and virtually all affected individuals are phenotypically female, regardless of karyotype. The absence of sex steroids precludes bone maturation, allowing an extended growth phase, such that nontreated adult patients usually have a tall eunuchoid appearance. Mineralocorticoid hypertension is an associated feature. OBJECTIVE:To describe the clinical aspects of growth development, bone maturation, and body proportions of a large cohort of Brazilian patients with CYP17D. PATIENTS AND METHODS:The study involved an analysis of the records of 88 patients with CYP17D who were treated at the Federal University of São Paulo Medical School and other Endocrine Reference Centres in Brazil. RESULTS:At diagnosis, the median chronological age and bone age of non-adult patients were 15.8 years (range: 10-20 years; n = 41) and 11 years (7.5-15 years; n = 25), respectively. A delay of ≥ 2 years in bone age was present in 92.5% of cases. In 30 patients, the height and its Z-score were 157 cm (130-171.5 cm) and -0.4 (-3.0 to +1.6), respectively. The span-to-height ratio was high and consistent over time. Final heights were available for 51 patients, of which 77% (25 XY, 14 XX) were in the 50th percentile or higher, and 39% (14 XY, 6 XX) were in the 90th percentile or higher. Only 8% (1 XY, 3 XX) were in the 25th percentile or lower. Of the 42 patients with data available, 11 (26%) had lower Z-scores during childhood and adolescence, and it is plausible that they missed a growth spurt. CONCLUSION:In this large CYP17D cohort, we verified that the prolonged hypoestrogenism that led to delayed or absent puberty was associated with decreased bone age, lower stature in childhood and adolescence, missed growth spurts, an extended growth phase, and greater final heights with frequent eunuchoid appearance.

Differences of sex development (DSD) represent a group of congenital conditions that affect human sex development and maturation owing to discrepancies of chromosomal, gonadal and phenotypic sex. The Chicago consensus classifies DSD as sex chromosome DSD, 46,XY DSD and 46,XX DSD, with subclassifications according to gonadal determination into testes and ovaries and hormone-dependent differentiation of Müllerian and Wolffian embryonic structures into female-typical or male-typical internal and external sex organs. DSD may occur as an isolated condition or as part of a complex syndrome. Diagnosis is based on clinical characteristics, imaging studies, hormonal measurements and genetic investigations. Management includes lifelong psychosocial support, hormonal treatments and surgical interventions that require personalization for each case as DSD encompasses a wide variety of aetiologies and presentations. This personalization must also consider individual values and preferences to ensure that clinical care is tailored to meet the unique needs and circumstances of each person, ideally provided by a care team with diverse specialities. This care involves psycho-educational counselling on the condition and its consequences, considering family and cultural norms. Additional efforts are needed to bridge gaps in knowledge related to diagnosis, management and long-term outcomes. Enhancing our understanding of the distinctions between sex and gender in societies is essential as greater awareness will inform and enrich public debates. Differences of sex development (DSD) encompass a group of rare congenital conditions characterized by atypical sex development. In this Primer, Flück and colleagues provide insights into our current understanding of rare DSD pathophysiology, its prevalence and diagnosis as well as challenges and controversies related to clinical management.

Background:Carney-Stratakis syndrome (CSS), a rare condition characterized by paragangliomas and/or pheochromocytomas and gastrointestinal stromal tumors (GIST), is caused by germline heterozygous pathogenic variants in the succinate dehydrogenase subunit genes (SDHB, SDHC, SDHD). Methods:Histological, genetic, and functional analyses were conducted in a 59-year-old female with CSS (9 cm left pheochromocytoma, 4.8 cm paraganglioma, and 9.3 cm GIST). Whole-exome sequencing (WES) of germline DNA paired with tumor DNA was performed. Results:WES identified a rare heterozygous germline variant (c.293G>A/p.Arg98His) in the mitochondrial 2-oxoglutarate/malate carrier gene (SLC25A11). This variant, located in a highly conserved residue of the SLC25A11 mitochondrial carrier domain, is predicted to be deleterious in silico (REVEL score = 0.81). WES of pheochromocytoma, paraganglioma, and GIST did not reveal somatic pathogenic variants in genes previously associated with these tumors. A significant reduction in SLC25A11 expression was observed in the tumors of this patient with the SLC25A11 c.293G>A variant (0.69 ± 0.003) compared to tumors from cluster 1 (1.39 ± 0.45; P = 0.0229) and cluster 2 (1.79 ± 0.71; P = .0154). Consistent with the mRNA findings, SLC25A11 protein levels were markedly reduced in the pheochromocytoma and paraganglioma compared to other tumors. Negative staining for 5-hydroxymethylcytosine in all 3 tumors suggests a DNA hypermethylation profile characteristic of cluster 1A, despite normal SDHB expression levels. However, genome-wide copy number variation analysis did not reveal any loss of heterozygosity at the SLC25A11 locus. Conclusion:The loss of SLC25A11 expression in tumors, the absence of somatic drivers, and the hypermethylation status strongly support the role of SLC25A11 in CSS pathogenesis.

BACKGROUND:46,XY gonadal dysgenesis is classified as complete (CGD) or partial (PGD) subtypes. The phenotype of PGD and the long-term outcome is not clearly defined. OBJECTIVE:To evaluate clinical features and pubertal outcome of PGD in a large cohort, using CGD as a comparator for diagnostic clarity. METHODS:Patients with 46,XY GD were identified from the I-DSD Registry and data on phenotype, genetics, biochemistry, gonadal histology, and pubertal development were collated in 3 categories; CGD (n = 100), PGD assigned female (PGDf, n = 107), and male (PGDm, n = 103) at birth. RESULTS:Most individuals with PGD presented with atypical genitalia in infancy, though, 18% of PGDf presented with delayed puberty and 8% with virilization. A genetic etiology was identified in 42% of the cohort, with common gene defects in SRY and WT1 in CGD and NR5A1 in PGD. Gonadal pre-/malignancy was found in 33.8% in CGD, 19.7% in PGDf, and 8.8% in PGDm. Among the PGDm (>13 years) with at least 1 gonad, 80% had spontaneous pubertal onset and 59% achieved Tanner G5 without hormone treatment. Labioscrotal gonads at presentation and testosterone response to human chorionic gonadotropin predicted onset of spontaneous puberty. In PGDf with gonads, 42% developed spontaneous virilization at puberty. Sex was reassigned in 16.1% and 5.3% of individuals with PGDf and PGDm, respectively. CONCLUSION:This study highlights the heterogeneous phenotype of PGD and the consequent diagnostic challenge. Many PGD patients with preserved gonads have the potential to develop puberty spontaneously, though further study is needed to determine the risk of developing gonadal tumors.

Disclosure: F. Freitas-Castro: None. G.F. Fagundes: None. L.S. Santana: None. A.F. Afonso: None. F.L. Ledesma: None. I.C. Soares: None. B.B. Mendonca: None. A. Latronico: None. M.Q. Almeida: None. Background: Pheochromocytomas and paragangliomas (PPGLs) are associated with germline genetic defects in 30-50% of cases and are categorized into three transcriptional clusters. Cluster 1, subdivided into Clusters 1A and 1B, involves genes associated with cellular pseudohypoxia. Cluster 1A encompasses mutations in genes encoding Krebs cycle enzymes (e.g., succinate dehydrogenase complex subunits SDHA, SDHB, SDHC, and SDHD), leading to the accumulation of oncometabolites such as succinate and pyruvate, which increase the stabilization of hypoxia-inducible factor 2-alpha (HIF-2α). Aim: To investigate novel genetic etiologies in patients with PPGLs. Methods: Whole-exome sequencing (WES) of paired germline and tumor DNA was performed on 50 patients with PPGLs who lacked germline defects in previously known susceptibility genes. A targeted analysis enriched for 3,485 genes involved in cellular responses to hypoxia, mitochondrial function, the Krebs cycle, and tumorigenesis was conducted. Results: A germline c.280A>T (p.Lys94*) variant in the Pyruvate Dehydrogenase Phosphatase Catalytic Subunit 2 (PDP2) gene was identified in a 43-year-old female diagnosed with a 10.5 cm abdominal PGL. This stop-codon variant is absent from population databases and has been classified as a variant of uncertain significance. Tumor WES revealed no oncogenic variants. Additionally, immunohistochemistry for SDHB was positive in the tumor. The patient had no family history of cancer. Among her six siblings, one was tested for the variant, with a negative result. She has no children, and her parents are deceased. PDP2 gene encodes an enzyme responsible for dephosphorylating and reactivating the E1 alpha subunit of the pyruvate dehydrogenase complex (PDC), a critical regulator of mitochondrial metabolism. While the PDC has been implicated in glucose metabolism and tumor aggression in other cancers, such as neuroblastoma, it has not been previously associated with PPGLs. No specific phenotype has been associated with this gene. Conclusion: We report, for the first time, a rare loss-of-function PDP2 variant in a patient with abdominal PGL, which may contribute to HIF-2α stabilization and the pathogenesis of PPGL. Ongoing functional studies aim to further investigate this hypothesis. Support: Sao Paulo Research Foundation (FAPESP) grant 2019/15873-6 (to M.Q.A.), and FAPESP post-doctoral fellowship 2021/11240-9 (to F.F-C.). Presentation: Sunday, July 13, 2025

Objective Germline and somatic drivers are identified in 30% and 40% of pheochromocytomas and paragangliomas (PPGLs), respectively. In this study, we investigated the genetic landscape of PPGLs in a Brazilian cohort. Methods We studied 182 index patients with PPGLs (116 females and 66 males), comprising 118 pheochromocytoma and 70 paraganglioma cases. Our optimized sequencing strategy included SANGER sequencing, targeted next-generation sequencing panel, and whole-exome sequencing. Results Germline and somatic pathogenic or likely pathogenic variants in susceptibility genes were identified in 88 (48.4%) and 18 (10.4%) cases, respectively. SDHB was the most frequently affected gene, identified in 30 patients (16.5%), with a germline SDHB exon 1 deletion present in 46.7% of these cases. The Brazilian cohort exhibited a higher rate of germline diagnoses when compared to the European (31%), American (27%), and Chinese (21%) cohorts (P < .001). Five germline variants in new susceptibility genes were identified: (1) Three CHEK2 likely pathogenic or pathogenic variants (c.475T > C/p.Tyr159His; c.362G > A/p.Cys121Tyr; c.319 + 2T > A); and (2) Two BRCA2 pathogenic variants (c.3680_3681delTG/p.Leu1227fs and c.7806-2A > C). These variants are unreported in the Brazilian genomic variant repository. CHEK2 immunostaining was negative in the three tumors, with one case exhibiting CHEK2 loss of heterozygosity. Moreover, the prevalence of CHEK2 or BRCA2 pathogenic or likely pathogenic variants in our cohort was significantly higher compared to global population databases (P < .0001 and P = .0004, respectively). Conclusion Our cohort of PPGLs demonstrated a high frequency of germline diagnoses. Additionally, our findings suggest CHEK2 and BRCA2 as potential susceptibility genes for PPGLs.