Central Precocious Puberty Caused by Mutations in the Imprinted Gene MKRN3

Loss-of-function mutations in the maternally imprinted genes, MKRN3 and DLK1, are associated with central precocious puberty (CPP). Mutations in MKRN3 are the most common known genetic etiology of CPP. This work aimed to screen patients with CPP for MKRN3 and DLK1 mutations and analyze the effects of identified mutations on protein function in vitro. Participants included 84 unrelated children with CPP (79 girls, 5 boys) and, when available, their first-degree relatives. Five academic medical institutions participated. Sanger sequencing of MKRN3 and DLK1 5' upstream flanking and coding regions was performed on DNA extracted from peripheral blood leukocytes. Western blot analysis was performed to assess protein ubiquitination profiles. Eight heterozygous MKRN3 mutations were identified in 9 unrelated girls with CPP. Five are novel missense mutations, 2 were previously identified in patients with CPP, and 1 is a frameshift variant not previously associated with CPP. No pathogenic variants were identified in DLK1. Girls with MKRN3 mutations had an earlier age of initial pubertal signs and higher basal serum luteinizing hormone and follicle-stimulating hormone compared to girls with CPP without MRKN3 mutations. Western blot analysis revealed that compared to wild-type MKRN3, mutations within the RING finger domain reduced ubiquitination whereas the mutations outside this domain increased ubiquitination. MKRN3 mutations were present in 10.7% of our CPP cohort, consistent with previous studies. The novel identified mutations in different domains of MKRN3 revealed different patterns of ubiquitination, suggesting distinct molecular mechanisms by which the loss of MRKN3 results in early pubertal onset.

Background: A correlation between plasma lipids and timing of pubertal development has been hypothesized, though lipid influence remains unclear in central precocious puberty (CPP). Aim: To assess any possible alterations in the lipid profile and triglyceride glucose index (TyG) in children diagnosed with CPP. Patients and Methods: Retrospective single-center study conducted on children (aged 6.3 ± 2.1 years) evaluated for the suspicion of CPP. Results: Based on the results of the gonadotropin releasing hormone (GnRH) test, considering 5 IU/L as cut-off of the luteinizing hormone peak, CPP was confirmed in 43 patients (57.3%). Sixteen (37.2%) had a pathologic body mass index (BMI), with 9 (20.9%) being overweight and 7 (16.27%) obese. High total cholesterol was found in 3 patients with CPP (6.97%), high triglycerides were found in 11 patients with CPP (25.58%), high LDL cholesterol was found in 5 patients with CPP (11.62%), low HDL cholesterol was found in 12/43 patients with CPP (27.9%), a pathologic TyG was found in 13/43 patients with CPP (30.23%). No significant association was observed in the lipid profile for patients with or without CPP, except for HDL cholesterol, which was lower in the CPP group (47.1 ± 10.9; p = 0.033). However, the association between serum HDL cholesterol and CPP was not confirmed at the multivariate logistic regression analysis adjusted for patients' sex and age (p = 0.1; OR: 1.035; 95% CI: 0.993-1.078). Conclusion: The overall lipid profile of our pediatric patients diagnosed with CPP did not differ from patients having idiopathic precocious thelarche or normal variants of puberty development.

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Introduction: Loss-of-function mutation of MKRN3 represents the most frequent genetic cause of familial central precocious puberty (CPP). The outcomes of gonadotropin-releasing hormone analog (GnRHa) treatment in CPP patients with MKRN3 defects are unknown. Objective: To describe the clinical and hormonal features of patients with CPP with or without MKRN3 mutations after GnRHa treatment. Anthropometric, metabolic and reproductive parameters were evaluated. Patients and Methods: Twenty-nine female patients with CPP due to loss-of-function mutations in the MKRN3 and 43 female patients with idiopathic CPP were included. Their medical records were retrospectively evaluated for clinical, laboratory, and imaging study, before, during, and after GnRHa treatment. All patients with idiopathic CPP and 11 patients with CPP due to MKRN3 defects reached final height (FH). Results: At the diagnosis, there were no significant differences between clinical and laboratory features of patients with CPP with or without MKRN3 mutations. A high prevalence of overweight and obesity was observed in patients with CPP with or without MKRN3 mutations (47.3 and 50%, respectively), followed by a significant reduction after GnRHa treatment. No significant differences in the values of mean FH and target height were found between the 2 CPP groups after GnRHa treatment. Menarche occurred at the expected age in patients with or without CPP due to MKRN3 mutations (11.5 ± 1.3 and 12 ± 0.6 years, respectively). The prevalence of polycystic ovarian syndrome was 9.1% in patients with CPP due to MKRN3 mutations and 5.9% in those with idiopathic CPP. Conclusion: Anthropometric, metabolic, and reproductive outcomes after GnRHa treatment were comparable in CPP patients, with or without MKRN3 mutations, suggesting the absence of deleterious effects of MKRN3 defects in young female adults’ life.

Background/Aims: Recently, loss-of-function mutations in the MKRN3 gene have been implicated in the etiology of familial central precocious puberty (CPP) in both sexes. We aimed to analyze the frequency of MKRN3 mutations in boys with CPP and to compare the clinical and hormonal features of boys with and without MKRN3 mutations. Methods: This was a retrospective review of clinical, hormonal and genetic features of 20 male patients with idiopathic CPP evaluated at an academic medical center. The entire coding regions of MKRN3, KISS1 and KISS1R genes were sequenced. Results: We studied 20 boys from 17 families with CPP. All of them had normal brain magnetic resonance imaging. Eight boys from 5 families harbored four distinct heterozygous MKRN3 mutations predicted to be deleterious for protein function, p.Ala162Glyfs*14, p.Arg213Glyfs*73, p.Arg328Cys and p.Arg365Ser. One boy carried a previously described KISS1-activating mutation (p.Pro74Ser). The frequency of MKRN3 mutations among these boys with idiopathic CPP was significantly higher than previously reported female data (40 vs. 6.4%, respectively, p < 0.001). Boys with MKRN3 mutations had typical clinical and hormonal features of CPP. Notably, they had later pubertal onset than boys without MKRN3 abnormalities (median age 8.2 vs. 7.0 years, respectively, p = 0.033). Conclusion: We demonstrated a high frequency of MKRN3 mutations in boys with CPP, previously classified as idiopathic, suggesting the importance of genetic analysis in this group. The boys with CPP due to MKRN3 mutations had classical features of CPP, but with puberty initiation at a borderline age.

Paternally inherited loss-of-function mutations in MKRN3 underlie central precocious puberty (CPP). We describe clinical and genetic features of CPP patients with paternally inherited MKRN3 mutations in two independent families. The single coding exon of MKRN3 was analyzed in three patients with CPP and their family members, followed by segregation analyses. Additionally, we report the patients' responses to GnRH analog treatment. A paternally inherited novel heterozygous c.939C>G, p.(Ile313Met) missense mutation affecting the RING finger domain of MKRN3 was found in a Finnish girl with CPP (age at presentation 6 years). Two Polish siblings (a girl presenting with B2 at the age of 4 years and a boy with adult size testes at the age of 9 years) had inherited a novel heterozygous MKRN3 mutation c.1237_1252delGGAGACACATGCTTTT p.(Gly413Thrfs*63) from their father. The girls were treated with GnRH analogs, which exhibited suppression of the hypothalamic-pituitary-gonadal axis. In contrast, the male patient was not treated, yet he reached his target height. We describe two novel MKRN3 mutations in three CPP patients. The first long-term data on a boy with CPP due to an MKRN3 mutation questions the role of GnRH analog treatment in augmenting adult height in males with this condition. We describe the genetic cause for central precocious puberty (CPP) in two families. This report adds two novel MKRN3 mutations to the existing literature. One of the mutations, p.(Ile313Met) affects the RING finger domain of MKRN3, which has been shown to be important for repressing the promoter activity of KISS1 and TAC3. We describe the first long-term observation of a male patient with CPP due to a paternally inherited MKRN3 loss-of-function mutation. Without GnRH analog treatment, he achieved an adult height that was in accordance with his mid-parental target height.

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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.

The prevalence of central precocious puberty (CPP) in girls has increased worldwide and is often associated with obesity in childhood as well as high fat/high glycemic index diets. Evidence suggests that subjects with obesity present with alterations in appetite-regulating hormones. The arcuate and paraventricular nuclei of the hypothalamus are the centers of action of appetite hormones, as well as the location of gonadotropin-releasing hormone (GnRH) neurons, the activation of which results in the onset of puberty. This anatomical proximity raises the question of possible alterations in appetite-regulating hormones in patients with CPP. Furthermore, diet-induced hypothalamic inflammation constitutes a probable mechanism of the pathophysiology of CPP, as well as alterations in appetite-regulating hormones in young children. In this article, we summarize the evidence investigating whether girls with CPP present with alterations in appetite-regulating hormones. We present evidence that leptin concentrations are elevated in girls with CPP, ghrelin concentrations are lower in girls with CPP, nesfatin-1 and orexin-A concentrations are elevated among girls with premature thelarche, and insulin concentrations are increased in girls with early menarche.

Introduction: Central precocious puberty (CPP), defined as the onset of pubertal signs before the age of 8 in girls and 9 in boys, results from the premature reactivation of the hypothalamic-pituitary-gonadal axis. CPP is familial in as many as 27.5% of the cases, which supports a genetic origin of this disorder. Currently, monogenic defects associated with CPP include inactivating mutations in MKRN3 and rare defects in KISS1, KISS1R and DLK1. In addition, CPP has been associated with complex genetic syndromes resulting from distinct chromosomal abnormalities as Silver-Russel Syndrome and Temple Syndrome. However in many patients its genetic basis remains unknown. Objective: To investigate pathogenic copy number variations (CNVs) in patients with idiopathic CPP associated with dysmorphic features or congenital malformations. Methods: A cohort of 45 patients with idiopathic CPP was initially evaluated for mutations in the coding region of MKRN3. Subsequently, 3 patients screened negative for MKRN3 mutations and with additional dysmorphisms or congenital malformations were selected for chromosome microarray analysis (CMA) using the Affymetrix CytoScan 750k according to manufacturer’s protocols. Results: In patient 1 (P1), a boy presenting CPP at the age of 8 associated with autism spectrum disorder and overweight, a pathogenic previously described heterozygous microdeletion at 16p11.2 (761 kbp), and a microduplication at 18p11.22 (363kbp), a variant of unknown significance (VOUS), were detected. His younger brother, who also has ASD and was recently diagnosed with CPP, also carried the same CNVs. Patient 2 (P2) was a monozygotic twin boy with a past history of hypospadia, an angular deformity of the femur, and pubertal onset at age of 6.5years. His monozygotic twin was equally affected and both developed polycythemia in the beginning of the second decade of life. CMA revealed a heterozygous duplication of 155kbp at 7p15.2, classified as VOUS. The third case (P3), a girl with idiopathic CPP associated with congenital heart disease, exhibited no significant chromosomal rearrangements, except for blocks of homozygosity due to parental consanguinity. Conclusion: Our results underscore the relevance of CMA in the investigation of patients with CPP associated with syndromic features. Parental consanguinity can also be identified by CMA and is associated with an increased risk of congenital anomalies, as was the case of P3. The 16p11.2 microdeletion has been previously associated with ASD, however CPP has not been previously reported in these patients. Further studies are needed to address possible associations between CPP and the CNVs and/or genes presented inside these variant regions identified in P1 and P2. Source of research support: CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico; FAPDF - Fundação de Apoio a Pesquisa do Distrito Federal.

Context: Familial central precocious puberty (CPP) is a prevalent form (about 27.5%) of precocious puberty. Loss-of-function mutations in two maternally imprinted genes, MKRN3 and DLK1, were identified in families with CPP showing dominant autosomal inheritance with paternal transmission. Maternally transmitted CPP was previously demonstrated in up to 60% of families using pedigree analysis, however, no definitive genetic abnormality has been identified so far.ObjectivesTo estimate the prevalence of familial cases in a multiethnic cohort with CPP. To characterize the genetic basis and the mode of inheritance of the affected families. To compare clinical and hormonal features of patients with familial CPP due to different modes of transmission.Patients and MethodsClinical and hormonal data were obtained from medical registries of 495 patients with CPP and no brain MRI alterations. Familial CPP was defined as the presence of one or more close relatives with CPP or precocious menarche (≤9 yr). Sanger sequencing of MKRN3 and DLK1 was performed in 427 index cases. Targeted gene panel sequencing was performed in 79 cases, while whole exome sequencing was performed in 101 cases from 36 familiesResultsAmong 495 index cases, 159 had familial CPP (31%). The mode of transmission of CPP was identified as paternal in 58 (35%), maternal in 59 (38%), indeterminate in 34 (22%), and both maternal and paternal transmission in 8 (5%). Most families with paternal or maternal transmission had 2 generations known to be affected (51 and 77%, respectively). Notably, 67% of index cases with maternally transmitted CPP had their mother affected. In girls with CPP, the median age of thelarche was 6.5 yr in the paternally transmitted group, 6.9 yr in the maternally transmitted group, and 7.3 yr in the indeterminate group (p= 0.547). Median bone age advancement was 2.1, 2.3 and 1.2 yr, respectively (p= 0.013). Basal LH levels were higher in girls with paternally transmitted CPP (p= 0.047). Among those with paternally transmitted CPP, MKRN3 and DLK1 mutations were identified in, respectively, 63.8% and 10.3% of the families.ConclusionsA significant prevalence (31%) of familial CPP was demonstrated in a multiethnic cohort. Maternally transmitted CPP represented the most frequent form of familial CPP (38%). MKRN3 loss-of-function mutations were responsible for most paternally transmitted CPP cases (63.8%), followed by DLK1 loss-of-function mutations (10.3%).Presentation: Monday, June 13, 2022 11:15 a.m. - 11:30 a.m.

MKNR3 is a paternally expressed gene whose mutations are the main cause of central precocious puberty (CPP). Protein circulating levels can be easily measured, as demonstrated in idiopathic CPP and healthy controls. No data are available for patients harboring an MKRN3 mutation. Our aim was to perform MKRN3 mutation screening and to investigate if circulating protein levels could be a screening tool to identify MKRN3 mutation in CPP patients. We enrolled 140 CPP girls and performed MKRN3 mutation analysis. Patients were stratified into two groups: idiopathic CPP (iCPP) and MKRN3 mutation-related CPP (MKRN3-CPP). Clinical characteristics were collected. Serum MKRN3 values were measured by a commercially available ELISA assay kit in MKRN3-CPP and a subgroup of 15 iCPP patients. We identified 5 patients with MKRN3 mutations: one was a novel mutation (p.Gln352Arg) while the others were previously reported (p.Arg328Cys, p.Arg345Cys, p.Pro160Cysfs*14, p.Cys410Ter). There was a significant difference in circulating MKRN3 values in MKRN3-CPP compared to iCPP (p < 0.001). In MKRN3-CPP, the subject harboring Pro160Cysfs*14 presented undetectable levels. Subjects carrying the missense mutations p.Arg328Cys and p.Gln352Arg showed divergent circulating protein levels, respectively 40.56pg/mL and undetectable. The patient with the non-sense mutation reported low but measurable MKRN3 levels (12.72pg/mL). MKRN3 defect in patients with CPP cannot be predicted by MKRN3 circulating levels, although those patients presented lower protein levels than iCPP. Due to the great inter-individual variability of the assay and the lack of reference values, no precise cut-off can be identified to suspect MKRN3 defect.

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

BackgroundMakorin ring finger protein 3 gene (MKRN3) gene mutation is the most common genetic cause of central precocious puberty (CPP) in children. Due to the lack of ideal MKRN3‐modified animal model (MKRN3‐modified mice enter puberty only 4–5 days earlier than normal mice), the related research is limited.MethodsTherefore, the MKRN3‐modified rabbit was developed using CRISPR (clustered regularly interspaced short palindromic repeats) gene editing technology. The genotype identification and phenotype evaluation of MKRN3‐modified rabbits were carried out.ResultsThe first estrus of MKRN3‐modified female rabbits was observed ~27 days earlier than that of wild‐type female rabbits, with a typical CPP phenotype. This study found increased gonadotropin releasing hormone (GnRH) and decreased gonadotropin inhibiting hormone (GnIH) in the hypothalamus of the CPP rabbit model with MKRN3 gene mutation. Although this study failed to fully clarify the pathogenesis of CPP caused by MKRN3 mutation, it found some differentially expressed genes and potential pathways through transcriptome sequencing.ConclusionsThis study established a novel CPP model: paternal MKRN3 gene‐modified rabbit. It is hoped that the establishment of this model will help researchers better understand, treat, and prevent CPP in the future.

Purpose: Central precocious puberty (CPP), which is characterized by premature activation of the hypothalamus-pituitary-gonadal axis, is more common in girls. Although genetic factors, such as mutations in MKRN3, have been identified, their role in CPP remains a subject of investigation. This study aimed to evaluate the role of MKRN3 in CPP development. Materials and Methods: This retrospective study included 70 patients with CPP. Demographic, anthropometric, and laboratory data were collected. Genetic analysis of MKRN3 pathogenic variants was performed in 22 patients using Sanger sequencing, following DNA isolation. Results: Seventy patients were diagnosed with CPP, of which 62(88.5%) were female and 8(11.5%) were male. The mean age of symptom onset was 7.1 years for girls and 5.7 years for boys, with the mean age at diagnosis being 7.8 years and 5.9 years, respectively. The mean age of the patients in whom the mutations were investigated was 7.8 years. Genetic analysis revealed no pathogenic MKRN3 variants. The rs2239669 variant, classified as benign, was detected in 11 heterozygous and in one patient in homozygous form. Conclusion: The detection of only a benign variant in MKRN3, with no pathogenic variants identified, may be attributed to the absence of a positive family history in all patients and the relatively higher mean age of the cohort. A limitation of this study is the small number of patients analyzed genetically and the inability to assess other genes related to central precocious puberty.