Puberty is an essential biological process marking the transition from childhood to adulthood. It can be affected by genetic, nutritional, environmental and socioeconomic factors. Central precocious puberty (CPP) results from the premature activation of the hypothalamic-pituitary-gonadal axis and it mimics physiological pubertal development, although at an inappropriate chronological age, before 8 years in girls and 9 years in boys. Several congenital malformations and acquired insults have been associated with CPP (1). Recent studies have implicated genetic changes in the premature reactivation of GnRH secretion, which was previously termed idiopathic, including chromosomal abnormalities and gene mutations (2-5).

In the last 12 years, my research group at São Paulo University has searched for potential genetic abnormalities associated with sporadic and familial CPP. The analysis of familial CPP by next generation sequencing, including exomic and genomic, changed significantly our current knowledge about the unique pathogenesis of premature sexual development in humans. To date, monogenic causes of familial CPP were established, and loss-of-function mutations of MKRN3 gene are the most prevalent cause of familial CPP (4). The role of this gene in the pathogenesis of CPP was first demonstrated in 2013, when we performed whole exome sequence analysis in 40 members of fifteen families with CPP (2). Thirty-two of them had CPP (including 27 girls and 5 boys). The great majority of the selected families were originated from Brazil. Rigorous criteria were used to filter the variants and identify the mutations likely to be causative of the phenotype for CPP. We were able to identify 15 individuals (8 girls, and 7 boys) from 5 families with CPP who carried MKRN3 mutations. MKRN3 gene is imprinted, with expression only from the paternally inherited allele. All studied families had autosomal dominant inheritance, and they were consistent with the expected inheritance of a paternally expressed gene, it means the mutated allele must be transmitted by the father to cause the phenotype (2-4).

Girls and boys with CPP due to MKRN3 mutations had typical clinical and hormonal features of premature activation of the reproductive axis when compared with those CPP patients without mutations (3-4). There were no identified clinical or hormonal differences between patients with and without MKRN3 mutations.

Recently, clinical, hormonal and genetic features of 20 male patients from 17 families who were initially classified as idiopathic CPP were revised. After genetic analysis, eight boys from 5 families harbored heterozygous loss-of-function mutations of MKRN3 (4). The frequency of MKRN3 mutations among boys was significantly higher than female data (40% vs. 6.4%, respectively, p < 0.001). Boys with MKRN3 defects had later pubertal onset than boys without MKRN3 abnormalities (median age 8.2 vs. 7.0 years, respectively, p = 0.033) (4).  Notably, the determination of age at pubertal onset in boys can be a challenge. Male patients usually remember only late events of puberty, such as the age at initiation of full facial shaving and age of voice change.  The borderline early age at pubertal onset in boys with MKRN3 mutations can compromise the precise identification of puberty by parents and general pediatrics and, therefore, lead to an underestimated incidence of CPP in this group.

Linkage analysis followed by whole-genome sequencing in an Afro-descendant Brazilian family was performed in 4 Brazilian girls, including two sisters and two paternal half-sisters, who had progressive CPP (5). A complex heterozygous defect of DLK1 was identified in this family. The defect was characterized by a ∼14-kb deletion and 269-bp duplication. Only family members who inherited the defect from their father had CPP. DLK1, also known as pre-adipocyte factor 1, is located at chromosome 14q32.2, a region that carries paternally and maternally expressed genes. It was known that maternal uniparental disomy of chromosome 14q region results in Temple syndrome, a complex disease characterized by CPP, prenatal and post-natal growth failure, obesity, small hands and feet and neurologic abnormalities. However, the Brazilian patients with the complex defect of DLK1 did not demonstrate additional features of Temple syndrome, except for increased fat mass. More recently, three novel and rare frameshift mutations of DLK1 (p.Gly199Alafs*11, p.Val271Cysfs*14 and p.Pro160Leufs*50) in five adult women from three families who had menarche at 7- 9 years were described (6). Segregation analysis was consistent with the maternal imprinting of DLK1 in these families.  DLK1 levels were undetectable (ELISA assay) in all affected members with the deletion or frameshift mutations of DLK1, supporting the notion that these defects lead to complete absence of DLK1 protein in these individuals (5-6). Notably, metabolic abnormalities, such as overweight/obesity, hyperlipidemia and early onset glucose intolerance/type 2 diabetes mellitus were more prevalent in the DLK1 mutated adult female patients from the 4 families when compared to the idiopathic treated CPP group (5-6). These metabolic findings in adult women with paternally inherited DLK1 deficiency are very similar to the Dlk1-null mice that gained body weight more rapidly, had major fat depots, enlarged fatty liver, and showed increased circulating levels of triglycerides, cholesterol, and free fatty acids Another intriguing clinical aspect was the diagnosis of polycystic ovary syndrome (PCOS) in the two affected sisters with early menarche. It is known that patients with CPP are prone to developing PCOS, especially if are untreated cases. In the two sisters with a DLK1 pathogenic variant, it is possible that the significant ovarian stimulus was determined by the premature activation of the reproductive axis, and it was worsened by the presence of high insulin levels and metabolic alterations.  A past history of CPP or precocious menarche should be investigated in PCOS women. In addition, loss-of-function variants of DLK1 should be screened in the PCOS women with a past history of CPP or precocious menarche, especially when associated with poor metabolic profile. These recent studies indicate that DLK1 is a novel link between reproduction and metabolism.

Pharmacological and non-pharmacological perturbations of DNA methylation and histone modifications in animal models were able to modulate gene expression, resulting in delayed or advanced puberty, indicating that the initiation of puberty requires repression of target repressor genes. More recently, we have investigated the relationship between DNA methylation pattern and pubertal timing in healthy and central precocious puberty girls. We studied 18 girls with familial CPP and 33 healthy Brazilian girls, including 15 pre- pubertal and 18 post-pubertal (7). We identified 120 differentially methylated regions (DMR) and the majority of them (99%) was hypermethylated in girls at pubertal development. The genomic region with the biggest methylation difference was related to the promoter of a zing finger gene (ZNF57). Furthermore, the majority of differentially methylated CpG sites was hypermethylated in patients with CPP compared to both pre-pubertal (340 out of 419, 81%) and post-pubertal controls (540 out of 606, 89%). Our data suggest the existence of an epigenetic signature underlying the pubertal development that involves a complex network of multiple genes.

1-    Latronico AC, Brito VN, Carel JC. Causes, diagnosis, and treatment of central precocious puberty. Lancet Diabetes Endocrinol. 2016 Mar;4(3):265-274. doi: https://doi.org/10.1016/S2213-8587(15)00380-0.

2-    Abreu AP, Dauber A, Macedo DB, Noel SD, Brito VN, Gill JC, Cukier P, Thompson IR, Navarro VM, Gagliardi PC, Rodrigues T, Kochi C, Longui CA, Beckers D, de Zegher F, Montenegro LR, Mendonca BB, Carroll RS, Hirschhorn JN, Latronico AC, Kaiser UB. Central precocious puberty caused by mutations in the imprinted gene MKRN3. N Engl J Med. 2013 Jun 27;368(26):2467-75. doi: https://doi.org/10.1056/NEJMoa1302160.

3-    Macedo DB, Abreu AP, Reis AC, Montenegro LR, Dauber A, Beneduzzi D, Cukier P, Silveira LF, Teles MG, Carroll RS, Junior GG, Filho GG, Gucev Z, Arnhold IJ, de Castro M, Moreira AC, Martinelli CE Jr, Hirschhorn JN, Mendonca BB, Brito VN, Antonini SR, Kaiser UB, Latronico AC. Central precocious puberty that appears to be sporadic caused by paternally inherited mutations in the imprinted gene makorin ring finger 3. J Clin Endocrinol Metab. 2014 Jun;99(6): E1097-103. doi: https://doi.org/10.1210/jc.2013-3126.

4-    Bessa DS, Macedo DB, Brito VN, França MM, Montenegro LR, Cunha-Silva M, Silveira LG, Hummel T, Bergadá I, Braslavsky D, Abreu AP, Dauber A, Mendonca BB, Kaiser UB, Latronico AC. High Frequency of MKRN3 Mutations in Male Central Precocious Puberty Previously Classified as Idiopathic. Neuroendocrinology. 2017;105(1):17-25.[Pubmed]

5-    Dauber A, Cunha-Silva M, Macedo DB, Brito VN, Abreu AP, Roberts SA, Montenegro LR, Andrew M, Kirby A, Weirauch MT, Labilloy G, Bessa DS, Carroll RS, Jacobs DC, Chappell PE, Mendonca BB, Haig D, Kaiser UB, Latronico AC. Paternally Inherited DLK1 Deletion Associated with Familial Central Precocious Puberty. J Clin Endocrinol Metab. 2017;102(5):1557-1567.[Pubmed]

6-    Gomes LG, Cunha-Silva M, Crespo RP, Ramos CO, Montenegro LR, Canton A, Lees M, Spoudeas H, Dauber A, Macedo DB, Bessa DS, Maciel GA, Baracat EC, Jorge AAL, Mendonca BB, Brito VN, Latronico AC. DLK1 is a novel link between reproduction and metabolism. J Clin Endocrinol Metab. 2018 Nov 19. [Epub ahead of print]. doi: https://doi.org/10.1210/jc.2018-02010.

7-    Bessa DS, Maschietto M, Aylwin CF, Canton APM, Brito VN, Macedo DB, Cunha-Silva M, Palhares HMC, de Resende EAMR, Borges MF, Mendonca BB, Netchine I, Krepischi ACV, Lomniczi A, Ojeda SR, Latronico AC. Methylome profiling of healthy and central precocious puberty girls. Clin Epigenetics. 2018;10(1):146.[Pubmed]