Precocious Puberty Research Articles

12212 A Rare Case Of Central Precocious Puberty In A Male Infant With Adrenal Hypoplasia Congenita

Abstract Disclosure: A. Mastoropoulou: None. A.H. Lane: None. Introduction: We describe a male with Adrenal Hypoplasia Congenita (AHC) caused by a novel mutation in NR0B1, who was noted at 9 months of age to have central precocious puberty (CPP). Clinical case: A 3 week-old full-term male presented with hypothermia and lethargy, and a 0.3 kg weight loss since birth. Labs were consistent with adrenal crisis: Na 109 mmol/L, K 10.4 mmol/L, Ca 12.2 mg/dL, glucose 43 mg/dL. He was stabilized with stress dose hydrocortisone (HC), insulin and antibiotics, and was admitted. Subsequent labs revealed cortisol 3.5 ug/dL (4.6-23), ACTH 1,123 pg/mL (7.2-63), 17OHP 181 ng/dL (<199), and a renin of 180 ng/mL/h (1.7-11.2) with aldosterone 14.9 ng/dL (6-179). Abdominal ultrasound was remarkable for non-visualization of the adrenal glands. The patient was discharged with HC, fludrocortisone and sodium supplementation with good tolerance and interval weight gain and normal electrolytes. Genetic testing identified a novel pathogenic c.707G>A [p.Trp236Ter] nonsense variant in the DNA-binding domain of NR0B1 (DAX-1). At 9 months of age, penile enlargement with prepubertal testes was noted. Testosterone was 318 ng/dL (Tanner stage I: 0-18), LH 3.2 mIU/mL (prepubertal: 0-0.3), DHEA-S 2 ug/dL (0-33), FSH 1.1 mIU/mL (prepubertal: 0-1), consistent with hypothalamic pituitary gonadal (HPG) axis activation. Given previous reports of prolonged but self-resolving “mini-puberty of infancy” in AHC, observation was initially chosen. At 12 months of age the bone age was consistent with the Greulich and Pyle 17 months standard. At 13 months of age testosterone remained elevated, therefore based on others’ reported experiences we increased HC from 8 to 15 mg/m2/day and noted a decrease in testosterone to 104 ng/dL within 5 days. HC was further increased to 18 mg/m2/day for 15 days, however testosterone increased to 270 ng/dL, therefore decision was made to treat with Lupron Depot. Conclusions: Historically, hypogonadotropic hypogonadism has been observed in 76% of adolescent patients with AHC who have alterations in NR0B1. CPP has been infrequently described in AHC, and the natural history and management of CPP in this setting is not established. CPP in AHC does not seem to correlate with any particular functional domain of DAX1, because previously reported NR0B1 variants associated with CPP, and this case’s, are dispersed across the gene. The course of our patient suggests multiple underlying mechanisms, including early HPG axis activation as well as the possibility of ACTH dependent precocious puberty. Our observations may contribute to the understanding of factors influencing normal and abnormal puberty in infants. Increased awareness of the possibility of CPP in AHC will aid clinicians in the earlier clinical and laboratory detection of this complication. Presentation: 6/1/2024

8134 Hypothalamic Dlk1 Expression is Not Essential for Preventing Early Maturation of the Reproductive Axis in Female and Male Mice

Abstract Disclosure: D.B. Macedo: None. H. Kim Kyeol: None. A. Abreu: None. A. Latronico: None. R.S. Carroll: None. U.B. Kaiser: None. Background and Objectives: Inactivating mutations in Delta-like homolog 1 (DLK1), a maternally imprinted gene on chromosome 14, have been recognized as a genetic cause of central precocious puberty (CPP) in humans. It is well established that the timing of puberty, especially in females, is highly sensitive to energy stores and metabolic cues, with lower body fat negatively correlated with age at puberty and menarche. Mice lacking Dlk1 have pre- and postnatal growth retardation. Despite their considerably lower body weight (BW), we found that Dlk1 deficient females achieved puberty at the same age as controls (‘relative precocious puberty’). Moreover, Dlk1 deficiency led to activation of the reproductive axis despite lower levels of kisspeptin and leptin, an adipose tissue hormone with a permissive/stimulatory effect on metabolic control of reproduction. Dlk1 is a transmembrane protein that plays an essential role in inhibiting adipocyte differentiation and its biologically active form is soluble. Increased hypothalamic expression postnatally suggests a neuroendocrine function, but the precise mechanism by which DLK1 deficiency leads to CPP is yet to be deciphered. In this study, we aimed to investigate the role of hypothalamic Dlk1 expression on pubertal onset without the interference of diminished BW, using a conditional knockout (cKO) mouse model. Methods and Results: We generated a targeted Dlk1 knockout mouse model by crossing mice expressing Cre recombinase under the control of Nestin gene (Nes Cre), expressed primarily in neuro-epithelial cells, with mice harboring the Dlk1 gene flanked by loxP sites (Dlk1fl). Since Dlk1 is imprinted and expressed only by the paternal allele, we bred heterozygous Dlk1fl/+ male mice with Nes Cre females to obtain Dlk1fl/Cre (Dlk1 cKO), Dlk1+/Cre (Nes Cre) and Dlk1+/+ (wild type - WT). We confirmed by RT-qPCR that Dlk1 mRNA was undetectable in the mediobasal hypothalamus of Dlk1 cKO adult male mice, whereas it was present in Nes Cre and WT mice. We noted a lower BW in Nes Cre compared to WT mice, as previously reported. However, no difference in BW was observed between Dlk1 cKO and Nes Cre mice at the timing of puberty (females: Dlk1 cKO 12.6 ± 0.8 g vs. Nes Cre 13.8 ± 0.2 g, P = 0.18; males: Dlk1 cKO 12.4 ± 0.2 g vs. Nes Cre 13.1 ± 0.5 g, P = 0.26). Puberty onset, assessed by age at vaginal opening (females) and at preputial separation (males), was not affected by hypothalamic Dlk1 deletion (Dlk1 cKO: 35.6 ± 2.7 days vs. Nes Cre 34.6 ± 0.7 days, P = 0.73; Dlk1 cKO 29.0 ± 0.6 vs. Nes Cre: 30.3 ± 0.6 days, P = 0.19). Conclusion: In contrast to global Dlk1 deficiency, targeted deletion of Dlk1 from neuronal and glial cells did not affect body weight or timing of pubertal onset. These findings suggest that Dlk1 originates from peripheral tissues, such as adipocytes, to regulate reproduction and metabolism. Presentation: 6/1/2024

7063 Serum Alkaline Phosphatase Level as a Marker for Progressive Central Precocious Puberty

Abstract Disclosure: V.M. Figueredo: None. T. Seeherunvong: None. Background: Precocious puberty is typically associated with a speed up in growth, risk of premature growth plate closure and reduced adult height. Serum alkaline phosphatase (ALP) is a biochemical indicator of bone turnover and studies have shown greater serum ALP levels in pubertal individuals compared to prepubertal controls. We examined serum ALP levels among children with various form of early puberty. We investigated whether serum ALP levels were significantly different between children with premature thelarche or premature adrenarche compared to those with central precocious puberty (CPP). Methods: We conducted a retrospective chart review of patients with early pubertal development attending a pediatric endocrinology clinic between 2017 and 2023. Information on height, weight, serum ALP levels and bone age was obtained. Patients were considered to have CPP based on the results on a GnRH test. Patients with diagnosis of premature adrenarche or premature thelarche were included as controls. This study was approved by Institutional Review Board (IRB). Analysis of data was performed using IBM SPSS Statistics version 29.0.1.0 (171). Results: Among 72 patients included in the study, 22 patients had progressive CPP whereas 50 patients had premature adrenarche or premature thelarche. The prevalence of abnormally elevated serum ALP levels was greater among patients with CPP (22.7%) than among patients with premature adrenarche or premature thelarche (6%). Using logistic binomial regression analysis, serum ALP levels were significantly elevated (p=0.042) in subjects with progressive CPP compared to patients with premature adrenarche or premature thelarche. Both groups had similar BMI values. BMI z-scores and advanced bone age were positively correlated. BMI (p=0.08) and advanced bone age were not significantly different (p=0.12) in the group with CPP compared with the group comprising patients with either premature adrenarche or premature thelarche. Conclusion: These findings suggest that serum ALP levels are helpful as a tool to help distinguish CPP from premature adrenarche or premature thelarche in the evaluation of patients with initial early pubertal development. Presentation: 6/1/2024

8256 Early Life Stress and Pubertal Predictors of Youth Substance Use Initiation: Does Sex Moderate the Relationship Between ELS, Puberty, and Substance Use Initiation?

Abstract Disclosure: A. Donovan: None. S. Assari: None. M. Shaheen: None. C. Grella: None. L. Richter: None. T.C. Friedman: None. Background: Early life stress (ELS) is a construct composed of children’s experiences of abuse, neglect, and conflict. Approximately 20% of adolescents in the United States have experienced ELS at some point, and these experiences influence adolescent and adult health behavior outcomes. Adults with a history of ELS display higher rates of substance use disorders (SUD) and report earlier initiation of substance use. Additionally, ELS has been associated with early puberty, which in turn is linked to earlier substance use initiation. Our study examines the roles of pubertal status, sex hormones (estradiol, testosterone, and DHEA) and ELS as predictors of the initiation of substance use among a nationally representative sample of male and female adolescents in NIDA’s Adolescent Brain Cognitive Development (ABCD) database. Methods: Baseline measures from the ABCD database release 5.0 were used to generate an ELS score (0-60), puberty assessed via parental report on the Pubertal Development Scale (PDS, 1-4), and sex hormones (estradiol, testosterone, and DHEA) were analyzed via salivary samples. The relationship between these scores and time to first use of alcohol, nicotine, and marijuana was determined via survival analysis utilizing a cox regression. Age was tested as a covariate in the interaction of ELS and time to first use of alcohol, nicotine, and marijuana. Data were analyzed overall and for biological boys and girls separately (N=11,866, 52%M 48%F. ages 9-11 at baseline). Results: We observed additive effects of PDS score, estradiol measure, age, and ELS on time to first use of any substance (alcohol, nicotine, or marijuana) and found age (Exp β= 1.029, p<0.001) and ELS (Exp β= 1.017, p<0.05) to be significant predictors of time to first use. After separating by sex, ELS was no longer a significant predictor for males (Exp β= 1.014, p=0.054), but remained a predictor for females (Exp β= 1.019, p<0.05) indicating the overall effect was driven by females. Additionally, salivary estradiol was a significant predictor of first use for females (Exp β= 1.208, p<0.05), while salivary DHEA and testosterone were not predictors for males or females. Conclusions: Our results indicate sex does moderate the relationship between ELS and substance use initiation within our population, with females showing increased risk of initiation with increases in estradiol. Further findings from our study will help to describe sex differences among the factors that increase the risk of substance use initiation among ELS-exposed youth and explore how these vary by substance. ABCD provides an unprecedented opportunity to explore the mechanisms behind complex effects of ELS by sex on risk factors for adolescent substance use initiation. Presentation: 6/1/2024

7640 The first description of an MC4R variant in a patient with Kallmann syndrome and obesity

Abstract Disclosure: A.A. Aslam: None. S. Lim: None. R. Willemsen: None. K. Koysombat: None. M. Young: None. W.S. Dhillo: None. A. Abbara: None. S. Howard: None. E.F. Gevers: None. Introduction: Pathogenic MC4R gene variants result in hyperphagia and early onset obesity but puberty is not usually affected. We describe an MC4R variant in a patient with Kallmann syndrome and obesity. A 16 year old male with repaired Tetralogy of Fallot, anosmia, autism and anxiety, was referred with obesity and delayed puberty. His birth weight was 3360 g (-0.26 SDS). Height was -1.31 SDS, BMI 30.7 kg/m2. He had high arched palate, normal skin and hair colour, mild hypertelorism and upward slanting palpebral fissures. Pubertal staging was A2P1G1 5ml testes bilaterally. Results: LHRH test showed borderline low peaks (LH 5.0 U/L, FSH 2.6 U/L) and inhibin B 108 pg/mL (25-325 pg/mL). MRI brain showed hypoplastic olfactory bulbs, absent olfactory sulci and normal pituitary anatomy. CGH microarray, karyotype and the UK hypogonadism gene panel were normal. He underwent whole genome sequencing in the Genetic Factors Affecting the Timing of Puberty Study; no abnormalities in 52 known genes associated with GnRH deficiency were found but a previously described heterozygous variant was detected, MC4R c.542G>A, p.Gly181Asp, classified pathogenic and absent in control databases. Management: He was commenced on Testosterone but showed progression of testicular size to 10 mL and testosterone was stopped but required restarting. Repeat investigations off treatment, aged 22 years (height 178 cm, BMI 42 kg/m2, testes 15 mL): inhibin B 66 pg/mL, testosterone 2 nmol/L, baseline LH 2.4 U/L, stimulated peak 24.9 U/L, baseline FSH 1.2 U/L, stimulated peak 4.2 U/L. Cortisol and IGF1 were in the normal range. He also developed autoimmune hypothyroidism with raised TPO antibodies (TSH 24.7 mU/L, FT4 5.6 pmol/L). Discussion: Previous in vitro work has shown complete loss of function of MC4R p.Gly181Asp due to reduced cell surface expression and reduced binding to a-MSH. Heterozygous p.Gly181Asp variants have been described in several children/adults with obesity; but hypogonadism in heterozygous carriers has not. Homozygous MC4R p.Gly181Asp was found in a male with obesity and partial HH thought to be due to abnormal GnRH production but with normal olfactory bulbs. We are the first to describe a heterozygous MC4R p.Gly181Asp variant in a patient with partial hypogonadism and anosmia with hypoplastic bulbs in addition to obesity. Interaction between POMC-MC-leptin circuits and Kisspeptin-GnRH circuits is recognised although not completely understood. Previous work has shown that a subset of kisspeptin neurons express MC4R and that KNDy neurones receive synaptic input from POMC neurons. In addition, inhibition of MC4R/MC3R results in delayed onset of puberty in rats. Our results support a role for MC4R in GnRH secretion and potentially olfactory /GnRH neuronal migration. Assessment of spontaneous gonadotrophin production and response of gonadotrophin production to kisspeptin stimulation is currently in progress. Presentation: 6/1/2024

7128 Efficacy and Safety of Leuprolide Acetate 3M Depot in Chinese Children with Central Precocious Puberty: Interim Analysis of Multicenter, Single-Arm, Prospective Study

Abstract Disclosure: X. Luo: Advisory Board Member; Self; Takeda. L. Hou: Advisory Board Member; Self; Takeda. Y. Li: Advisory Board Member; Self; Takeda. Y. Sun: Advisory Board Member; Self; Takeda. X.K. Jin: Employee; Self; Takeda. Background: Central precocious puberty (CPP) due to early activation of the hypothalamic-pituitary-gonadal axis leads to early puberty. CPP has a negative impact on adult height. Leuprolide acetate (LA), a gonadotropin-releasing hormone agonist (GnRHa) is the standard treatment for CPP. As CPP requires prolonged treatment, a 3-month (3M) depot formulation would increase patient compliance and reduce the injection burden. This interim analysis presents the effect of LA 3M depot on the gonadotropic axis and assesses safety in children with CPP. Methods: In this open-label, multicenter, single-arm, and prospective study, 31 children with CPP were included. Key inclusion criteria were the appearance of secondary sexual characteristics below the age of eight years in girls and nine years in boys with a confirmed diagnosis of CPP. Children treated with prior GnRHa and/or having underlying medical conditions were excluded. Eligible children were treated with LA 11.25 mg subcutaneously every 12 weeks. The primary endpoint of this interim analysis was the proportion of children achieving inhibition of peak LH defined as peak LH ≤ 3.0 IU/L in the GnRH stimulation test at weeks 24. The secondary endpoint was safety analysis in the children receiving at least one dose of LA 3M depot. Results: At baseline, 30 girls and one boy were enrolled. The median (95% CI) chronological age (CA) of the girls was 8.0 (7.0, 9.0) years and the boy was 10 years old. Girls had a median height of 136.95 (131.5, 139.7) cm, weight of 30 (27, 34) kg, and BMI of 16.4 (15.1, 17.9) kg/m2. Baseline bone (BA) age was 11 (10, 11) years, and BA/CA was 1.24 (1.2, 1.3). Boy’s characteristics at baseline differed in CA (10 years), height (144 cm), and BMI (17.4 kg/m2) from girls of this cohort. Due to the data collection time points for the interim analysis, 8 out of the 31 enrolled subjects had a visit at week 24, and 30 subjects reached the week 12 visit time point and had a visit at week 12. The percentage of children achieving inhibition of peak LH (≤ 3.0 IU/L) in the GnRH stimulation test at week 12 was 100 (88.4, 100) %. At week 24, all of them achieved inhibition of LH in the GnRH stimulation test 100 (63, 100) %. Decreases in BA/CA from baseline at weeks 12 and 24 are 93 (77.9, 99.2) % and 50 (15.7, 84.3) % respectively. No progression of the Tanner stage was observed in 96.7 (82.8, 99.9) % and 100 (63, 100) % at weeks 12 and 24. At the data cut-off, 4 (13.33%) patients experienced a total of 5 treatment-emergent adverse events (TEAEs) in the safety population of 30 subjects. No grade ≥3 AE or serious AE was reported. The incidence of injection site reaction was 3.33%. Conclusion: LA at 11.25mg/3M depot effectively suppresses the gonadotropic axis at weeks 12 and 24, normalizing growth and reversibly slowing the early onset of puberty in children with CPP. LA in a 3-month injecting frequency is a safe and convenient treatment option to improve patient compliance. Presentation: 6/3/2024

7269 Interim Analysis of Gonadotropin-Releasing Hormone Agonist (GnRHa) Treatment in Children with Central Precocious Puberty: Focus on FMV Urinary LH Levels and Clinical Outcomes

Abstract Disclosure: X. Luo: Advisory Board Member; Self; Takeda. L. Hou: Advisory Board Member; Self; Takeda. Y. Li: Advisory Board Member; Self; Takeda. Y. Sun: Advisory Board Member; Self; Takeda. X.K. Jin: Employee; Self; Takeda. Background: Central precocious puberty (CPP) refers to early activation of the hypothalamic–pituitary–gonadal (HPG) axis in children. There is an urgent need to understand the treatment pattern and clinical outcomes among children suffering from CPP. Studies have reported that first morning voided (FMV) urinary Luteinizing hormone (LH) and Follicle stimulating hormone (FSH) have substantial association with CPP tested in GnRH stimulation test and can be useful in diagnosis and management of CPP in children. Gonadotropin-releasing hormone agonists (GnRHa) such as Leuprolide acetate (LA) are the recommended treatment for children suffering from CPP. The current study aimed to provide the information on level of LH and FSH in FMV urine when CPP diagnosis at baseline and the present results may also revealed trend of FMV-LH and FMV-FSH change during LA intervention in children with CPP. Here we present interim results from this study. Methods: This is an open-label, multicentre, single arm, prospective study that enrolled 31 subjects with CPP. Individuals were administered LA depot 11.25 mg, subcutaneously every 12 weeks. The study comprises a screening period; a 6-month treatment period: 24 weeks; and a post-treatment follow-up period: 12 weeks. The FMV urinary LH and FSH were diagnosed by Clopper-Pearson method with a 95% confidence interval. Results: Due to the data collection time point for the interim analysis, only 8 female subjects reached the 12-week visit time point and had a 12-week visit for FMV urinary Gn testing. Therefore, for this interim analysis, only FMV Gn decline at week 12 of the 8 subjects who had a 12-week visit will be presented. At baseline, the mean value of FMV urinary LH and FSH is 0.58 IU/L and 2.27 IU/L respectively. The FMV Gn urinary LH was demonstrated a decrease in 87.5% (95% CI: 47.35,99.68) of the children after LA intervention. Additionally, urinary FSH value of FMV urinary Gn was found to decrease in 75.00% of the children (95% CI: 34.91,96.81) compared to baseline FSH value. Conclusion: The present study reveals a decreasing trend in FMV Gn urinary LH and FSH levels in CPP children treated with LA during interim analysis. LA was shown to be safe and effective in ameliorating the level of LH and FSH in children suffering from CPP. From the study, it can be concluded that urinary LH and FSH may act as a potential biomarker for diagnosis and management of CPP. Presentation: 6/3/2024

6669 Association Between Serum Levels of Thyroid Function and Per- and Polyfluoroalkyl Compounds Concentrations in Central Precocious Puberty in Girls

Abstract Disclosure: Y. Li: None. Y. Xing: None. X. Yin: None. J. Yang: None. S. Zang: None. T. Dong: None. Y. Pan: None. J. Dai: None. P. Li: None. Background: Exposure to per- and polyfluoroalkyl substances (PFASs) may interfere with thyroid function in the general population and disturb the timing of puberty onset. Objectives: We investigated the possible relationship between PFASs and thyroid hormone (THs) exposure in girls with central precocious puberty (CPP). Methods: In a prospective study initially established for assessing CPP, 627 serum samples were collected with (n = 226) and without (n = 401) CPP. The concentrations of 25 PFASs, thyroxin (T4), 3,5,3’-triiodothyronine (T3), and 3,3’,5’-triiodothyronine (rT3) were measured. We used logistic regression to examine the association of THs with the odds of having CPP and multiple linear regression to analyze the associations between THs and various PFASs in the CPP and control groups. Results: The levels of T4 in the CPP group were significantly lower than those in the control group, and the levels of T3 and rT3 were significantly higher than those in the control group. After adjusting for confounders, T3 concentrations were associated with 1.526-fold increased odds of having CPP [odds ratio (OR) = 1.526, 95% confidence interval (CI):1.216-1.914]. In the CPP cohort, perfluoroheptanoic acid (PFHpA), perfluoro (2-ethoxyethane) sulphonic acid (PFEESA), and fluorotelomer phosphate diester (6:2/8:2 diPAP) were inversely associated with T3; perfluorobutanoic acid (PFBA) was inversely associated with T4, and PFEESA and 6:2/8:2 diPAP positively associated with T4; PFBA was positively associated with rT3, and perfluorooctane sulfonic acid (PFOS) was inversely associated with rT. In the control cohort, perfluorobutanesulfonic acid (PFBS) was positively associated with T3, and PFEESA and 6:2/8:2 diPAP were inversely associated with T3, and these PFASs had an adverse association with T4. Perfluorohexanoic acid (PFHxA) was positively associated with rT3, and 6:2/8:2 diPAP was negatively associated with rT3. Conclusion: In this study, different measurements of PFASs and THs were observed in the CPP and control groups. Discrepancies in exposure to PFASs were associated with THs in the CPP cohort and healthy girls. Further research is necessary to investigate the mechanism by which PFASs affect the timing of puberty onset and thyroid function. Presentation: 6/1/2024

7381 Overexpression of Makorin Ring Finger Protein 3 in the Arcuate Nucleus of Postpubertal Female Mice Impacts Neurokinin B and Kisspeptin Co-Expressing Neurons

Abstract Disclosure: S.A. Roberts: None. A. Fontes: None. S. Blau: None. M. Magnuson: None. K.M. McKnight: None. M. Sena-Esteves: None. R.S. Carroll: None. U.B. Kaiser: None. Background: Makorin ring finger protein 3 (MKRN3), an upstream inhibitor of GnRH release, is an important regulator of the age of menarche. Loss-of-function mutations in MKRN3 are the most common genetic etiology of central precocious puberty. However, its potential role in delayed puberty or hypogonadism is not well defined. In mice, expression of Mkrn3 in the hypothalamic arcuate nucleus (ARC), where kisspeptin, neurokinin B and dynorphin (KNDy) co-expressing neurons are located, is high early in life and declines before pubertal onset. Neonatal hypothalamic overexpression of Mkrn3 leads to delayed puberty in female mice and decreased neurokinin B (NKB) and kisspeptin protein levels in the ARC peripubertally. Conversely, Mkrn3-deficient peripubertal mice exhibit increased NKB protein levels in the ARC. We hypothesized that Mkrn3 overexpression in the ARC of postpubertal female mice would result in suppression of the HPG axis, manifested as hypogonadotropic hypogonadism. Methods and Results: Twelve week old adult wild type female mice were injected stereotaxically into the ARC with a novel adeno-associated virus overexpressing Mkrn3 (AAV-Mkrn3-IRES-EGFP; denoted AAV-Mkrn3) or a control virus (AAV-EGFP). Mean body weight did not differ between groups post-injection. AAV-Mkrn3 injected mice spent significantly more time (62.7%) in diestrus three to six weeks post-injection compared to controls (41.3%; P<0.01). Eight weeks post-injection, gonad-intact females were injected with an NKB receptor (NK3R) agonist, senktide (5 nmol or 10 nmol ip). AAV-Mkrn3 mice had a decreased peak LH response twenty minutes post-injection, compared to controls (P<0.001) at both doses. Peak LH levels were also reduced in response to senktide (5 nmol ip) in ovariectomized and estradiol capsule replaced (OVX+E2) AAV-Mkrn3 injected females, compared to controls (P< 0.001). In contrast, in both gonad-intact and OVX+E2 female mice, kisspeptin-10 (1 nmol ip) resulted in similar peak serum LH levels in AAV-Mkrn3 injected mice compared to controls. Immunohistochemistry in the ARC of intact and OVX+E2 AAV-Mkrn3 injected females showed significantly decreased NKB protein expression in AAV-Mkrn3 mice compared to controls. In response to OVX, serum LH levels increased similarly between AAV-Mkrn3 and control-injected mice. LH pulsatility in OVX females, assessed over 3 hours, showed that mean basal LH levels, number of pulses, pulse amplitude and area under the curve did not differ significantly between groups. Conclusions: These findings indicate that MKRN3 can exert inhibitory effects on the reproductive axis in female mice beyond the juvenile period, which has implications as a potential therapeutic target. The blunted response to senktide, and decreased NKB protein levels in the setting of Mkrn3 overexpression, further supports the impact of Mkrn3 on the neuropeptides and/or receptors of KNDy neurons. Presentation: 6/2/2024

12427 MKRN3 Methylation Abnormalities In Patients With Pubertal Disorders

Abstract Disclosure: N.C. Grosek: None. T. Silva: None. J.C. Magnotto: None. A. Latronico: None. A.P. Canton: None. Y. Chan: None. R.S. Carroll: None. U.B. Kaiser: None. Background and Objectives: Central Precocious Puberty (CPP) is caused by early reactivation of pulsatile GnRH secretion before age 8 years in girls and 9 years in boys. Delayed puberty (DP) is defined by the absence or incomplete development of sexual characteristics by age 13 years in girls and 14 years in boys. Environmental cues affect the HPG axis and timing of puberty via epigenetic mechanisms such as DNA methylation (DNAm). Inactivating mutations in the maternally imprinted Makorin Ring Finger 3 (MKRN3) gene are the most common genetic defect associated with CPP, representing ∼9% of patients with sporadic and familial CPP. Genomic imprinting occurs through differential methylation of CpG islands at a gene promoter or enhancer region. Differentially methylated regions (DMRs) regulate genetic imprinting. We hypothesized that transcription factor binding sites (TFBS) for ZFP57 (CpG57) andNRF1 (CpGs54+55) within the islet 3.7kb upstream of MKRN3 (MRKN3 islet) may impact expression. This study aimed to analyze if aberrant MKRN3 islet DNAm changes are associated with disorders of pubertal timing. Methods and Results: Germline DNA from 2 control patients was used to analyze the MKRN3 promoter region and islet DNAm profile. Bisulfite-treated DNA was amplified by PCR with primers targeting MKRN3 promoter and islet and submitted to Sanger sequencing. All CpG islands in the promoter region were 100% methylated, but those in the MKRN3 islet were differentially methylated with ∼50-60% methylated. DNA from our CPP cohort (n=71) underwent Next-Generation Sequencing analysis of bisulfite-treated DNA of these regions to identify the DMR potentially associated with MKRN3 imprinting. The MKRN3 islet covered CpG59-CpG49. Next, samples from our CPP cohort and patients with DP (n=44) and normal pubertal timing (n=33; 45% prepubertal, 55% pubertal) underwent pyrosequencing analyses of MKRN3islet. To compare DNAm levels between controls and our cohorts at each CpG island, T-tests were used: reported as mean±SEM. In the CPP cohort, 4/11 CpGs were significantly hypomethylated compared to controls (CpGs 54, 53, 51, 50). Specifically, the NRF1 TFBS was hypomethylated compared to controls (37.8 vs 41.6 ± 1.1, p<0.001) and DP (41.4 vs 43.8 ± 0.83, p=0.044). In the DP group, we found no significant difference in DNAm compared to controls. But they had hypermethylation of 4/11 CpGs compared to the CPP group, including the NRF1 TFBS (CpGs 55, 53, 51,50). Neither the CPP nor DP cohorts had significant DNAm differences at the ZFP57 TFBS compared to controls. Conclusion: Our study showed hypomethylation of 4 out of 11 CpGs in MKRN3 islet, including the NRF1 TFBS, in CPP compared to control and DP groups. On the other hand, NRF1 TFBS was hypermethylated in DPP compared to CPP. Further studies are needed to determine if these alternations in methylation are associated with MKRN3 expression levels and pubertal initiation. Presentation: 6/1/2024

8428 Rare Variants in the MECP2 Gene in Two Boys with Central Precocious Puberty

Abstract Disclosure: A.P. Canton: None. J.B. Mebarak: None. M. Magnuson: None. S. Roberts: None. N. Mauras: None. M. Benson: None. S. Witchel: None. R.S. Carroll: None. A. Latronico: None. U.B. Kaiser: None. A. Abreu: None. Introduction: Central precocious puberty (CPP) occurs more frequently in girls and is usually labelled as idiopathic; however, in boys organic causes are more frequent. Identification of imprinted genes causing CPP have revealed epigenetic mechanisms underlying puberty. MECP2, an X-linked gene, encodes a methylated DNA reader protein with a role in gene transcription. MECP2 loss-of-function mutations usually cause Rett syndrome, a severe neurodevelopmental disorder that may be associated with early pubertal development. Recently, rare variants in MECP2 have been recognized in girls with sporadic CPP with or without mild neurodevelopmental disorders. In our cohort of 78 patients with idiopathic CPP, no MECP2 mutations were identified in 73 girls. In this study, five boys with CPP were evaluated for potential MECP2 sequence variants. Methods and Results: Five boys with CPP were screened for MECP2 sequence variants using Sanger sequencing. At the time of CPP diagnosis, they had median (interquartile range) chronological age 9.2 yr (4.3), bone age advancement 2.1 yr (3.5), height SDS 2.3 (1.5), basal LH levels 1.6 IU/L (0.9), and testosterone levels 370 ng/dL (570). Organic causes of CPP were excluded. No MKRN3 or DLK1 mutations were identified. Familial segregation analysis was performed when appropriate. We identified a hemizygous MECP2 variant in a boy (Patient 1) who presented at age 2.7 yr with sporadic CPP. In addition, he had speech delay and behavioral changes, defined as autism spectrum disorder by neuropsychological assessment. He harbored an extremely rare (gnomAD AF=0.000004956) missense variant (p.Val312Ile) in exon 3 of MECP2, encoding the transcriptional repression domain, critical for protein function. The p.Val312Ile mutation was classified as likely pathogenic (ACMG criteria) with a potential association with the phenotype. Another hemizygous MECP2 variant was identified in a boy (Patient 2) who presented at age 8.0 yr with sporadic CPP; he had no neurodevelopmental conditions. He harbored a rare (gnomAD AF=0.00008) missense variant (p.Arg366Cys) in exon 3, encoding the C-terminal domain, classified as a variant of uncertain significance (ACMG criteria). Familial segregation analysis revealed that both boys inherited the MECP2 variants from their unaffected mothers, consistent with a pattern of clinical variability described in women with defects in X-linked genes.Conclusions: We identified rare MECP2 variants in two boys with sporadic CPP without classic features of Rett syndrome, expanding the phenotype of patients with MECP2 mutations, as described in girls. The MECP2 p.Val312Ile variant was likely pathogenic, whereas the functional significance of the p.Arg366Cys variant in still indeterminate. These findings provide additional evidence for a role of MECP2, an epigenetic factor, in the hypothalamic control of pubertal timing. Presentation: 6/1/2024

12672 MKRN3 Methylation Abnormalities In Patients With Pubertal Disorders

Abstract Disclosure: N.C. Grosek: None. T. Silva: None. J.C. Magnotto: None. A. Latronico: None. A.P. Canton: None. Y. Chan: None. R.S. Carroll: None. U.B. Kaiser: None. Background and Objectives: Central Precocious Puberty (CPP) is caused by early reactivation of pulsatile GnRH secretion before age 8 years in girls and 9 years in boys. Delayed puberty (DP) is defined by the absence or incomplete development of sexual characteristics by age 13 years in girls and 14 years in boys. Environmental cues affect the HPG axis and timing of puberty via epigenetic mechanisms such as DNA methylation (DNAm). Inactivating mutations in the maternally imprinted Makorin Ring Finger 3 (MKRN3) gene are the most common genetic defect associated with CPP, representing ∼9% of patients with sporadic and familial CPP. Genomic imprinting occurs through differential methylation of CpG islands at a gene promoter or enhancer region. Differentially methylated regions (DMRs) regulate genetic imprinting. We hypothesized that transcription factor binding sites (TFBS) for ZFP57 (CpG57) andNRF1 (CpGs54+55) within the islet 3.7kb upstream of MKRN3 (MRKN3 islet) may impact expression. This study aimed to analyze if aberrant MKRN3 islet DNAm changes are associated with disorders of pubertal timing. Methods and Results: Germline DNA from 2 control patients was used to analyze the MKRN3 promoter region and islet DNAm profile. Bisulfite-treated DNA was amplified by PCR with primers targeting MKRN3 promoter and islet and submitted to Sanger sequencing. All CpG islands in the promoter region were 100% methylated, but those in the MKRN3 islet were differentially methylated with ∼50-60% methylated. DNA from our CPP cohort (n=71) underwent Next-Generation Sequencing analysis of bisulfite-treated DNA of these regions to identify the DMR potentially associated with MKRN3 imprinting. The MKRN3 islet covered CpG59-CpG49. Next, samples from our CPP cohort and patients with DP (n=44) and normal pubertal timing (n=33; 45% prepubertal, 55% pubertal) underwent pyrosequencing analyses of MKRN3islet. To compare DNAm levels between controls and our cohorts at each CpG island, T-tests were used: reported as mean±SEM. In the CPP cohort, 4/11 CpGs were significantly hypomethylated compared to controls (CpGs 54, 53, 51, 50). Specifically, the NRF1 TFBS was hypomethylated compared to controls (37.8 vs 41.6 ± 1.1, p<0.001) and DP (41.4 vs 43.8 ± 0.83, p=0.044). In the DP group, we found no significant difference in DNAm compared to controls. But they had hypermethylation of 4/11 CpGs compared to the CPP group, including the NRF1 TFBS (CpGs 55, 53, 51,50). Neither the CPP nor DP cohorts had significant DNAm differences at the ZFP57 TFBS compared to controls. Conclusion: Our study showed hypomethylation of 4 out of 11 CpGs in MKRN3 islet, including the NRF1 TFBS, in CPP compared to control and DP groups. On the other hand, NRF1 TFBS was hypermethylated in DPP compared to CPP. Further studies are needed to determine if these alternations in methylation are associated with MKRN3 expression levels and pubertal initiation. Presentation: 6/1/2024

6825 α-Klotho Levels in Girls with Central Precocious Puberty

Abstract Disclosure: I. Hwang: None. E. Kim: None. Background: The exact mechanisms that trigger the onset of puberty are not fully understood. While it is known that elevated estradiol during puberty induces an increase in IGF-1, which subsequently promotes a rise in α-klotho levels, the relevance of these elevated α-klotho levels in children with Central Precocious Puberty (CPP) remains to be established. Given the potential significance of α-klotho in pubertal development, we initiated this study to determine whether increased α-klotho levels in CPP children are meaningfully pronounced and if these levels could serve as a reliable marker for CPP diagnosis and monitoring. Methods: A total of 139 girls were enrolled in the study, comprised of 82 diagnosed with CPP and 57 who were healthy prepubertal controls. From March 2020 to May 2023, we assessed both α-klotho levels and clinical parameters. α-klotho concentrations were measured using an α-klotho ELISA kit. For the girls with CPP, we additionally analyzed samples taken 6 months after GnRH agonist treatment. Result: α-Klotho levels were higher in the CPP group compared with the controls (CPP group: 2529 ± 999 ng/mL; control group: 1802 ± 675 pg/mL) (P < 0.001). α-Klotho significantly decreased after 6 months on GnRH agonist treatment.(2147± 789 pg/mL) (P < 0.001). α-Klotho levels were positively correlated with height-SDS, weight-SDS, bone age, ALP, IGF-1 SDS, baseline FSH and LH. α-Klotho relationship with IGF-1 SDS, baseline LH studied by partial correlation with adjustment for age, height and weight SDS (r= 0.275, p= 0.003; r= -0.229, p= 0.014). A ROC curve analysis identified an α-klotho cut-off differentiating CPP from controls, with a cut-off of 1914 pg/mL distinguishing CPP girls from controls with a sensitivity of 69.5% and specificity of 70.2%; the area under the curve was 0.723. Conclusion: Our study indicates a notable association between elevated α-klotho levels and CPP, especially as these levels adjust following GnRH agonist treatment. Based on our findings, considering α-klotho concentrations might offer a way to distinguish CPP from controls. Further studies could elucidate the potential of α-klotho as a biomarker for CPP diagnosis and monitoring. Presentation: 6/1/2024

7341 Establishing a Multidisciplinary Care Program for patients with Klinefelter Syndrome and other Aneuploidy X and Y Variations

Abstract Disclosure: C.J. Romero: None. N.R. Malhotra: None. A. Norris-Brilliant: None. C. Mintz: None. Objectives: Klinefelter syndrome (KS) is one of the most common genetic aneuploidies denoted by a XXY karyotype and affects up to 1 in 600 patients. Most patients are not diagnosed until adolescent years or adulthood. KS is one of the leading causes of male infertility and testosterone insufficiency. KS is also associated with developmental delays, particularly speech and language delay. Advances in genetics screenings has led to early identification of KS and other aneuploidy X and Y variations, access to neurodevelopmental screening and fertility preservation resources during adolescence and young adulthood. We believe a multidisciplinary program for KS can provide the necessary resources for patients and families at any age from infancy to young adulthood. Methods: A group of faculty from the divisions of pediatric endocrinology, genetics, urology and psychiatry assembled in 2023 to establish a multidisciplinary clinic (MDC) to provide comprehensive care to patients/parents; this ranged from prenatal diagnosis to young adulthood. Two major challenges of care are access to neuro-psychological evaluations and fertility preservation options. Testosterone supplementation requires appropriate referral to endocrinology and timing of treatment remains controversial. Our goals include: expertise guidance and planning after prenatal diagnosis or later in age; access to cost effective neuro-psychological testing during early childhood years; urological consultation beginning in early puberty to educate and prepare patients for choices of surgical and non-surgical options of fertility preservation. Results: Our current center cares for 12 patents ranging from ages 3 months to 21 years. Of this cohort, 3 (25%) of the patients had initial consultations with the parents during pregnancy. 7 (58%) of the patients were of pubertal age that one would consider testosterone replacement, but only 3 (43%) were taking testosterone. Only 1 patient of school age reported neuro-psychological evaluation that was completed at a different institution. No patients received testosterone supplementation during year one of life. Conclusions: A multidisciplinary team can offer simultaneous assessment, anticipatory guidance and management for KS patients and their families. It is a comprehensive resource to our community. Prenatal genetic screenings bring awareness to this diagnosis and therefore timely education toward understanding the lifelong management of KS. Identifying developmental delays early in childhood will help with scholastic success. Monitoring of pubertal development will ensure appropriate pubertal progression in addition to education and management of fertility preservation. Undoubtedly future urological advances will assist patients more effectively. We believe our model provides the access to resources that allow our patients and family to successfully thrive. Presentation: 6/2/2024

Alternate-day fasting delays pubertal development in normal-weight mice but prevents high-fat diet-induced obesity and precocious puberty

Background/ObjectivesChildhood obesity, particularly in girls, is linked to early puberty onset, heightening risks for adult-onset diseases. Addressing childhood obesity and precocious puberty is vital to mitigate societal burdens. Despite existing costly and invasive medical interventions, introducing lifestyle-based alternatives is essential. Our study investigates alternate-day fasting’s (ADF) impact on pubertal development in normal-weight and high-fat diet (HFD)-induced obese female mice.MethodsFour groups of female mice were utilized, with dams initially fed control chow during and before pregnancy. Post-parturition, two groups continued on control chow, while two switched to an HFD. Offspring diets mirrored maternal exposure. One control and one HFD group were subjected to ADF. Morphometry and hormone analyses at various time points were performed.ResultsOur findings demonstrate that ADF in normal-weight mice led to reduced body length, weight, uterine, and ovarian weights, accompanied by delayed puberty and lower levels of sex hormones and growth hormone (GH). Remarkably, GH treatment effectively prevented ADF-induced growth reduction but did not prevent delayed puberty. Conversely, an HFD increased body length, induced obesity and precocious puberty, and altered sex hormones and leptin levels, which were counteracted by ADF regimen. Our data indicate ADF’s potential in managing childhood obesity and precocious puberty.ConclusionsADF reduced GH and sex hormone levels, contributing to reduced growth and delayed puberty, respectively. Therefore, parents of normal-weight children should be cautious about prolonged overnight fasting. ADF prevented HFD-induced obesity and precocious puberty, offering an alternative to medical approaches; nevertheless, further studies are needed for translation into clinical practice.

A systematic review on the impact of SARS-CoV-2 viral infection on precocious puberty

Precocious puberty is defined as the onset of sexual characteristics before age 8 in girls and 9 in boys, with serious physical and psychological implications, including advanced bone maturation, early growth cessation, and increased behavioral issues. As per the studies done by Gupta et al precocious puberty cases have risen during the pandemic, prompting inquiries into the potential link between SARS-CoV-2 and early pubertal onset. PubMed, Scopus Web of Science, Google Scholar, and other electronic databases were used for the literature search. The search was restricted to studies published between January 2020 and July 2024. The study has shown a concerning increase in precocious puberty cases during the pandemic, with reports of a significant rise ranging from 9.8% to 93%. The increase clearly shows the concern of having a massive impact on the puberty timing in girls. The data reveal a consistent increase in the incidence of precocious puberty during the pandemic, with relative increases ranging from 0.2 average incidence per month pre-pandemic to 5.9 average per month pandemic incidence. These findings underscore the need for localized public health strategies and further research to fully understand the long-term implications of early puberty in the context of the COVID-19 pandemic. This review emphasizes the importance of continued monitoring and research to mitigate the potential long-term health risks associated with precocious puberty, including metabolic, cardiovascular, and psychological disorders.

7267 Study of the effect of GnRHa on BMI in girls with central precocious puberty

7267 Study of the effect of GnRHa on BMI in girls with central precocious puberty

Screening of MKRN3, DLK1, KISS1, KISS1R, and PROKR2 genes sequences in related girls with precocious puberty for a personalized management

Screening of MKRN3, DLK1, KISS1, KISS1R, and PROKR2 genes sequences in related girls with precocious puberty for a personalized management

Personalized dietary strategies for managing precocious puberty: Insights from nutrigenetic interactions

Personalized dietary strategies for managing precocious puberty: Insights from nutrigenetic interactions

Genetic Polymorphisms of Prokineticins and Prokineticin Receptors Associated with Human Disease

Prokineticins (PKs) are low molecular weight proteins that exert their effects by binding to two seven-transmembrane G-protein-coupled receptors (prokineticin receptors, PKRs). The prokineticin system is an important player in the development of various diseases. Several polymorphisms that are associated with infertility, neuroendocrine disorders, Hirschsprung’s syndrome (HSCR), idiopathic central precocious puberty (CPP) and congenital disorders such as Kallmann syndrome (KS) have been described for both the PKs and PKR genes. The aim of this study is to summarize and describe the impact of PK/PKR polymorphisms on the pathogenesis and outcome of the above diseases, highlighting the PK system as a therapeutic target and diagnostic biomarker in pathological conditions.