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Little is known about genetic control of the timing of puberty. This study implicates an imprinted gene, MKRN3, in familial central precocious puberty. The data suggest that the encoded protein inhibits puberty. The onset of puberty is first detected as an increase in the amplitude and frequency of pulses of gonadotropin-releasing hormone (GnRH) after a quiescent period during childhood. The reemergence of pulsatile GnRH secretion leads to increases in the secretion of the gonadotropins, luteinizing hormone and follicle-stimulating hormone (FSH), by the pituitary gland and the consequent activation of gonadal function. 1 Early activation of the hypothalamic–pituitary–gonadal axis results in gonadotropin-dependent precocious puberty, also known as central precocious puberty, which is clinically defined by the development of secondary sexual characteristics before the age of 8 years in girls and 9 years in boys. . . .

The identification of loss-of-function mutations in MKRN3 in patients with central precocious puberty in association with the decrease in MKRN3 expression in the medial basal hypothalamus of mice before the initiation of reproductive maturation suggests that MKRN3 is acting as a brake on gonadotropin-releasing hormone (GnRH) secretion during childhood. In the current study, we investigated the mechanism by which MKRN3 prevents premature manifestation of the pubertal process. We showed that, as in mice, MKRN3 expression is high in the hypothalamus of rats and nonhuman primates early in life, decreases as puberty approaches, and is independent of sex steroid hormones. We demonstrated that Mkrn3 is expressed in Kiss1 neurons of the mouse hypothalamic arcuate nucleus and that MKRN3 repressed promoter activity of human KISS1 and TAC3, 2 key stimulators of GnRH secretion. We further showed that MKRN3 has ubiquitinase activity, that this activity is reduced by MKRN3 mutations affecting the RING finger domain, and that these mutations compromised the ability of MKRN3 to repress KISS1 and TAC3 promoter activity. These results indicate that MKRN3 acts to prevent puberty initiation, at least in part, by repressing KISS1 and TAC3 transcription and that this action may involve an MKRN3-directed ubiquitination-mediated mechanism.

Abstract Context Loss-of-function mutations of makorin RING finger protein 3 (MKRN3) are the most common monogenic cause of familial central precocious puberty (CPP). Objective To describe the clinical and hormonal features of a large cohort of patients with CPP due to MKRN3 mutations and compare the characteristics of different types of genetic defects. Methods Multiethnic cohort of 716 patients with familial or idiopathic CPP screened for MKRN3 mutations using Sanger sequencing. A group of 156 Brazilian girls with idiopathic CPP (ICPP) was used as control group. Results Seventy-one patients (45 girls and 26 boys from 36 families) had 18 different loss-of-function MKRN3 mutations. Eight mutations were classified as severe (70% of patients). Among the 71 patients, first pubertal signs occurred at 6.2 ± 1.2 years in girls and 7.1 ± 1.5 years in boys. Girls with MKRN3 mutations had a shorter delay between puberty onset and first evaluation and higher follicle-stimulating hormone levels than ICPP. Patients with severe MKRN3 mutations had a greater bone age advancement than patients with missense mutations (2.3 ± 1.6 vs 1.6 ± 1.4 years, P = .048), and had higher basal luteinizing hormone levels (2.2 ± 1.8 vs 1.1 ± 1.1 UI/L, P = .018) at the time of presentation. Computational protein modeling revealed that 60% of the missense mutations were predicted to cause protein destabilization. Conclusion Inherited premature activation of the reproductive axis caused by loss-of-function mutations of MKRN3 is clinically indistinct from ICPP. However, the type of genetic defect may affect bone age maturation and gonadotropin levels.

Abstract Context Primary aldosteronism (PA) is the most common cause of endocrine hypertension (HT). HT remission (defined as blood pressure <140/90 mm Hg without antihypertensive drugs) has been reported in approximately 50% of patients with unilateral PA after adrenalectomy. HT duration and severity are predictors of blood pressure response, but the prognostic role of somatic KCNJ5 mutations is unclear. Objective To determine clinical and molecular features associated with HT remission after adrenalectomy in patients with unilateral PA. Methods We retrospectively evaluated 100 patients with PA (60 women; median age at diagnosis 48 years with a median follow-up of 26 months). Anatomopathological analysis revealed 90 aldosterone-producing adenomas, 1 carcinoma, and 9 unilateral adrenal hyperplasias. All patients had biochemical cure after unilateral adrenalectomy. KCNJ5 gene was sequenced in 76 cases. Results KCNJ5 mutations were identified in 33 of 76 (43.4%) tumors: p.Gly151Arg (n = 17), p.Leu168Arg (n = 15), and p.Glu145Gln (n = 1). HT remission was reported in 37 of 100 (37%) patients. Among patients with HT remission, 73% were women (P = 0.04), 48.6% used more than three antihypertensive medications (P = 0.0001), and 64.9% had HT duration <10 years (P = 0.0015) compared with those without HT remission. Somatic KCNJ5 mutations were associated with female sex (P = 0.004), larger nodules (P = 0.001), and HT remission (P = 0.0001). In multivariate analysis, only a somatic KCNJ5 mutation was an independent predictor of HT remission after adrenalectomy (P = 0.004). Conclusion The presence of a KCNJ5 somatic mutation is an independent predictor of HT remission after unilateral adrenalectomy in patients with unilateral PA. The impact of KCNJ5 somatic mutations on hypertension remission after adrenalectomy brings new insight into the postoperative follow-up of patients with PA.

Abstract MKRN3 mutations represent the most common genetic cause of central precocious puberty (CPP) but associations between genotype and clinical features have not been extensively explored. This systematic review and meta-analysis investigated genotype-phenotype associations and prevalence of MKRN3 mutations in CPP. The search was conducted in seven electronic databases (Cochrane, EMBASE, LILACS, LIVIVO, PubMed, Scopus, and Web of Science) for articles published until 4 September 2018. Studies evaluating MKRN3 mutations in patients with CPP were considered eligible. A total of 22 studies, studying 880 subjects with CPP, fulfilled the inclusion criteria. Eighty-nine subjects (76 girls) were identified as harboring MKRN3 mutations. Girls, compared with boys, exhibited earlier age at pubertal onset (median, 6.0 years; range, 3.0 to 7.0 vs 8.5 years; range, 5.9 to 9.0; P < 0.001), and higher basal FSH levels (median, 4.3 IU/L; range, 0.7 to 13.94 IU/L vs 2.45 IU/L; range, 0.8 to 13.70 IU/L; P = 0.003), and bone age advancement (ΔBA; median, 2.3 years; range, −0.9 to 5.2 vs 1.2 years; range, 0.0 to 2.3; P = 0.01). Additional dysmorphisms were uncommon. A total of 14 studies evaluating 857 patients were included for quantitative analysis, with a pooled overall mutation prevalence of 9.0% (95% CI, 0.04 to 0.15). Subgroup analysis showed that prevalence estimates were higher in males, familial cases, and in non-Asian countries. In conclusion, MKRN3 mutations are associated with nonsyndromic CPP and manifest in a sex-dimorphic manner, with girls being affected earlier. They represent a common cause of CPP in western countries, especially in boys and familial cases.

Background Recent studies demonstrated that changes in DNA methylation (DNAm) and inactivation of two imprinted genes ( MKRN3 and DLK1 ) alter the onset of female puberty. We aimed to investigate the association of DNAm profiling with the timing of human puberty analyzing the genome-wide DNAm patterns of peripheral blood leukocytes from ten female patients with central precocious puberty (CPP) and 33 healthy girls (15 pre- and 18 post-pubertal). For this purpose, we performed comparisons between the groups: pre- versus post-pubertal, CPP versus pre-pubertal, and CPP versus post-pubertal. Results Analyzing the methylome changes associated with normal puberty, we identified 120 differentially methylated regions (DMRs) when comparing pre- and post-pubertal healthy girls. Most of these DMRs were hypermethylated in the pubertal group (99%) and located on the X chromosome (74%). Only one genomic region, containing the promoter of ZFP57 , was hypomethylated in the pubertal group. ZFP57 is a transcriptional repressor required for both methylation and imprinting of multiple genomic loci. ZFP57 expression in the hypothalamus of female rhesus monkeys increased during peripubertal development, suggesting enhanced repression of downstream ZFP57 target genes. Fourteen other zinc finger ( ZNF ) genes were related to the hypermethylated DMRs at normal puberty. Analyzing the methylome changes associated with CPP, we demonstrated that the patients with CPP exhibited more hypermethylated CpG sites compared to both pre-pubertal (81%) and pubertal (89%) controls. Forty-eight ZNF genes were identified as having hypermethylated CpG sites in CPP. Conclusion Methylome profiling of girls at normal and precocious puberty revealed a widespread pattern of DNA hypermethylation, indicating that the pubertal process in humans is associated with specific changes in epigenetically driven regulatory control. Moreover, changes in methylation of several ZNF genes appear to be a distinct epigenetic modification underlying the initiation of human puberty.

Abstract Disclosure: S. Beeby: None. C.H. Huzita: None. F.Q. Aratani: None. L.S. Motomia: None. F. Coelho: None. M. Polo: None. C.R. Victor: None. A.O. Hoff: None. J. Chambo: None. V. Srougi: None. F. Ledesma: None. A. Latronico: None. M.Q. Almeida: None. B.B. Mendonca: None. M.C. Fragoso: None. Background: Adrenocortical carcinoma (ACC) is a rare and one of the most aggressive solid tumors, with an incidence of 1-2 cases per million. In adults, hormonal excess is usually manifested by ACTH-independent Cushing's syndrome or mixed endocrine syndrome due to cortisol and androgen production. Isolated aldosterone secretion is an extremely rare condition, with an estimated 2.5% of patients with ACC. We aimed to describe two patients with aldosterone-secreting ACC with poor outcomes. Case 1 - A 34-year-old male who was diagnosed with hypertension at 29 years of age, associated with hypokalemia needing intravenous potassium replacement therapy. Diagnosis of primary hyperaldosteronism was established with aldosterone levels of 72 ng/dL (RR < 23.1 ng/dL) and renin activity of 1.1 ng/mL/h (RR 0.3 to 5.8 ng/mL/h. An abdominal CT scan identified a tumor mass of 9.5 x 8.0 x 4.5 cm. He underwent a left adrenalectomy and histological and immunohistochemistry data suggested an adrenocortical carcinoma (modified Weiss score 5/7, Ki67 40%). Initial staging was ENSAT III and a Helsinki score of 48. There was transient improvement, however recent laboratory studies showed an increase in aldosterone (66,9 ng/dL) accompanied by new episodes of hypokalemia. CT scans on follow-up showed recurrence of disease, with the presence of nodules on the surgical site and abdominal wall. He received 3 lines of systemic chemotherapy and mitotane and underwent 2 other surgical procedures to treat the metastatic lesions. Currently, overall survival is 3 years. Case 2 - A 44-year-old female incidentally found a right adrenal mass (6.6 x 6.4 cm) on MRI during the investigation of elevated liver enzymes and abdominal pain. Laboratory studies showed hypokalemia, with potassium levels of 2 mEq/L (RR 3.5-5 mEq/L), aldosterone levels of 243 ng/dL (RR < 23.1 ng/dL), and renin of 12.2 uUI/mL (RR 4.4 - 46.1 uUI/mL). She underwent right adrenalectomy and histology and immunohistochemistry data showed a modified Weiss 7/7 and Ki67 of 80%. The patient had a germline pathogenic variant of TP53 (p.R337H). Initial staging was ENSAT III and a Helsinki score of 88. She received adjuvant mitotane and systemic chemotherapy. Even so, follow-up imaging showed hepatic metastatic disease, with an elevated aldosterone production surpassing 1000 ng/dL. The patient's overall survival was 2 years and she passed away during surgical procedure for resection of liver metastases. Discussion: ACC is a rare and aggressive tumor, and isolated aldosterone production by such tumors is extremely rare. We presented two cases with poor prognostic features and in which despite adjuvant mitotane and systemic chemotherapy there was disease progression. Due to the rarity of this condition, data on patient outcomes is limited, and further understanding of the molecular signature of these tumors could bring new insights on the tumorigenesis process. Presentation: 6/3/2024

Abstract Disclosure: C.H. Huzita: None. W.M. Lins: None. S.K. Beeby: None. R.I. Lopes: None. F.T. Denes: None. A. Latronico: None. M. Polo: None. C.R. Victor: None. A.A. Hoff: None. B.B. Mendonca: None. M.Q. Almeida: None. M.C. Fragoso: None. Background: Adrenocortical oncocytic tumors (AOT) originate from the adrenal cortex. In the pediatric age group, they are rare and usually non-functional. Less than 20 cases of functional AOT have been reported and only one developed central precocious puberty. We describe the case of a patient with Li Fraumeni syndrome with germline TP53 pathogenic variant (PV) (p.R337H) who developed functional AOT and then central precocious puberty. ClinicalCase: A 2-year and 9-months old girl presented with pubic hair (Tanner stage P3S1) and clitoromegaly (2.6cm). Hormonal study showed elevated total testosterone (70ng/dL,ref. < 48ng/dL), DHEA-S (13490 ng/mL,ref<194 ng/mL) and a positive overnight 1mg dexamethasone suppression test (Cortisol5,8µg/dL). Abdominal ultrasonography showed a right round adrenal mass, which on magnetic resonance imaging (MRI) was well defined (50mm) and there was no evidence of invasion into adjacent organs or vessels. ChestX-ray and liver ultrasound imaging were normal.TheTP53 p.R337HPV was inherited from her father. The patient underwent a right adrenalectomy and oncocytic adrenocortical tumor was identified by histopathology (Biseglia score2/7, Wieneke score1/9 and Weiss score 5). Immunohistochemistry was positive for p53 and Ki67(13%). Following surgical removal of the tumor, the patient subsequently developed hypothalamic-pituitary activation and demonstrated central precocious puberty (Tanner P3S2 and advanced bone age). She was treated with a gonadotropin-releasing hormone agonist. Hormonal and clinical follow up demonstrated delayed pubertal progression. Discussion: AOT rarely presents with excess hormonal production. Herein we describe the case of the youngest child with functional AOT that developed central precocious puberty after the removal of the tumor. The mechanisms that cause hypothalamic-pituitary activation are not well understood, but prolonged exposure to androgen and then its withdrawal might lead to activation of hypothalamic GnRH pulse generated via a feedback system that has matured during the period of exposure to sex steroids.Therefore, clinical follow up of potential secondary effects of excess hormone secretion after tumor removal is important in pediatric patients with virilizing arenocortical tumor. In addition, considering Li Fraumeni syndrome, the screening of other tumors according to the Toronto protocol is required. Presentation: 6/2/2024

Abstract Disclosure: L.S. Santana: None. A.G. Guimaraes: None. F. Freitas-Castro: None. A.W. Maciel: None. G.F. Fagundes: None. M.A. Pereira: None. L.F. Drager: None. L.A. Bortolotto: None. M.C. Fragoso: None. B.B. Mendonca: None. A. Latronico: None. M.Q. Almeida: None. Primary aldosteronism (PA) is the most common cause of endocrine hypertension, with an estimated prevalence of 10-20% in patients referred to tertiary hospitals. The most common causes of PA are bilateral adrenal hyperplasia (BAH) and aldosteronomas. Significant advances in elucidating the pathogenesis of PA have been made over the past decade. Pathogenic/likely pathogenic germline allelic variants in KCNJ5, CACNA1D, CACNA1H, CLCN2, PDE2A, and PDE3B genes have already been documented in individuals diagnosed with PA. However, most of the cases still lack a defined genetic etiology. Genomic investigation of these individuals through whole-exome sequencing (WES) would enable the identification of causal monogenic genotype-phenotype associations in genes not yet linked to bilateral PA. In our study, thirty-one patients were selected, including 29 probands and 2 relatives, with a confirmed diagnosis of bilateral PA by adrenal venous sampling. All probands were previously investigated for KCNJ5 pathogenic/likely pathogenic germline variants. Additionally, all cases also had negative genetic screening for glucocorticoid-remediable PA. WES was performed using BGI Genomics technology, DNA nanoball sequencing, on the DNBSEQ platform. Raw data were processed (bioinformatics) using in-house pipelines. The 31 sequenced samples showed excellent coverage metrics, with coverage ranging from 200-467X and %>10X at 99%. Each patient presented an average of 150,000 variants. After the gene/allelic prioritization workflow, 4 heterozygous SNVs were listed as candidates in 3 out of 29 patients (10.34%). Two of the variants were identified in rare but previously documented germline causes of PA: CACNA1H and CACNA1D (patients 13 and 29, respectively). Patient 4 (hypertension diagnosed at 36 years of age) presented 2 variants in possible new candidate genes, CASZ1 and STRN. The NM_001079843.3(CASZ1):c.1912G>C/p.(Asp638His) variant caught our attention not only for its absence in population genomic databases (gnomAD v.4), but also due to the association between CASZ1 locus and PA. The zinc finger transcription factor CASZ1 has CACNA1D as one of its targets and participates in key biological processes for PA pathogenesis, such as: co-repression of mineralocorticoid receptor transcriptional activity; mediation of tissue response to aldosterone and differential expression in aldosterone-producing cell clusters (APCC) and in zona glomerulosa; its hypoexpression promotes aldosterone-dependent mineralocorticoid receptor transcriptional activity and hyperexpression suppresses aldosterone biosynthesis by adrenal cells. In conclusion, an extremely rare CASZ1 variant was identified in a woman with bilateral PA. The functional characterization of the CASZ1 p.(Asp638His) variant might reinforce its role as a new monogenic cause of PA. Presentation: 6/3/2024

Abstract Disclosure: F. Freitas-Castro: None. L.S. Santana: None. G.F. Fagundes: None. A.F. Afonso: None. E.C. Lobato: None. F.L. Ledesma: None. I.C. Soares: None. B.B. Mendonca: None. M.C. Fragoso: None. A. Latronico: None. C.A. Stratakis: None. M.Q. Almeida: None. Background: Carney-Stratakis syndrome (CSS, OMIM #606864), also reported as “paraganglioma and gastrointestinal stromal tumor syndrome” or Carney-Stratakis dyad, is an autosomal dominant rare syndrome with incomplete penetrance, characterized by the association of paragangliomas (PGL) and/or pheochromocytomas (PHEO) and gastrointestinal stromal tumors (GIST). CSS is mainly caused by germline heterozygous pathogenic variants (PVs) in the succinate dehydrogenase subunit genes (SDHB, SDHC, SDHD), with SDHB and SDHD being the most frequently affected. Aim: To investigate a novel genetic etiology for CSS not associated with germline SDHx defects. Methods: Genetic investigation using SANGER sequencing and multiplex ligation-dependent probe amplification (MLPA) rule out germline defects for SDHx in a 59-year-old woman diagnosed with a 9 cm left PHEO, 4.8 cm PGL and 9.3 cm GIST. Thus, we performed whole exome sequencing of germline DNA (paired with tumor DNA from PHEO, PGL, and GIST) and applied a targeted analysis with the enrichment of 3,485 potential neuroendocrine tumors susceptibility genes associated with cellular response to hypoxia, mitochondrion, Krebs cycle, and tumorigenesis (MAPK, mTOR, ERK, and Wnt signaling). Additionally, we performed RT-qPCR to determine SLC25A11 expression levels in PHEO and PGL samples, stratified into three groups: tumors from the case harboring the SLC25A11 variant, Cluster 1 (n= 4), and Cluster 2 (n= 8). Tumor DNA methylation status was assessed using immunostaining for 5-hydroxymethylcytosine (5hmC). Results: Exome sequencing revealed a new 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). Exome sequencing of the PHEO and PGL did not reveal somatic PVs in genes previously associated with PHEO and PGL. Moreover, somatic c-KIT and PDGFRA PVs were not identified in GIST. Notably, a significant downregulation of SLC25A11 expression was observed in tumor samples harboring the SLC25A11 p.Arg98His variant (0.57 ± 0.13) compared with tumors from cluster 1 (1.39 ± 0.45; p = 0.025) and cluster 2 (1.79 ± 0.71; p < 0.001). Interestingly, immunostaining for 5hmC was negative in all tumors (PHEO, PGL and GIST), indicating tumor hypermethylation. Conclusion: A rare germline deleterious variant in SLC25A11 was identified in a patient with CSS. Moreover, the absence of somatic drivers, hypermethylation status and loss of SLC25A11 expression in the CSS tumors support the involvement of this gene with CSS. 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: 6/1/2024