|Rev Esp Endocrinol Pediatr 2017;8(2):17-21 | Doi. 10.3266/RevEspEndocrinolPediatr.pre2017.Oct.432|
|Molecular Advances in the Diagnosis of Central Precocious Puberty|
|Enviado a Revisar: 17 Oct. 2017 | _ACCEPTED: 17 Oct. 2017 | En Publicación: 14 Nov. 2017|
|Ana C. Latronico|
|Unidade de Endocrinologia do Desenvolvimento. Lab. de Hormônios e Genética Molecular/LIM42, Hosp. das Clínicas, Disciplina de Endocrinologia, Faculdade de Medicina da Univ. de São Paulo. São Paulo (Brasil)|
|Correspondencia para Ana C. Latronico, Unidade de Endocrinologia do Desenvolvimento, Lab. de Hormônios e Genética Molecular/LIM42, Hosp. das Clínicas, Disciplina de Endocrinologia, Faculdade de Medicina da Univ. de São Paulo, Av. Dr. Enéas de Carvalho Aguiar, 255, 7° andar, sala 7037, 05403-900, São Paulo, Brasil|
|Table 1 - Monogenic causes of central precocious puberty|
|Figure 1 - Potential strategy for identification of central precocious puberty etiology in girls and boys|
Central precocious puberty (CPP) results from the premature activation of the hypothalamic-pituitary-gonadal axis1, 2. It mimics physiological pubertal development, although at an inappropriate chronological age. CPP is clinically defined by the development of secondary sexual features before 8 years in girls and 9 years in boys2. Several congenital malformations and acquired insults have been associated with CPP1, 2. The range of causes is similar in boys and girls, although idiopathic disease is much more common in girls. Actually, over 90% of the girls and up to 25% of the boys with CPP have an idiopathic form1.
Recent studies have implicated genetic changes in the premature reactivation of GnRH secretion, which was previously termed idiopathic3. In the last 10 years, my research group at Sao Paulo University has searched for potential genetic abnormalities associated with sporadic and familial CPP4-6. Initially, candidate gene approach in combination of Sanger sequencing method was used, and although many negative results were obtained, two important data were achieved involving the kisspeptin function6, 7. More recently, the analysis of familial CPP by next generation sequencing, including exomic and genomic, transformed significantly the current knowledge about the unique pathogenesis of premature sexual development in humans4, 5.
Familial CPP is usually defined by the existence of more than one affected member either in the proband generation or in the pedigree. It was demonstrated that up to 27.5% of CPP cases were familial8. In the majority of these families, the inheritance of the phenotype was consistent with autosomal dominant mode of transmission with incomplete penetrance. Maternal, paternal or both modes of transmission have been described described9. To date, monogenic causes of familial CPP were established, and loss-of-function mutations of MKRN3 are the most prevalent cause of familial CPP (Table 1). Only two mutations, one missense mutation in the gene encoding kisspeptin-1 (KISS1) and one in the gene encoding its receptor (KISS1R), were associated with CPP, despite screening of a large cohort of patients for mutations in these genes, indicating that mutations in KISS1 and KISS1R genes are uncommon cause of CPP.
MKRN3 mutations in familial CPP
The role of MKRN3 in the pathogenesis of CPP was first demonstrated in 2013, when whole exome sequence analysis was performed in 40 members of fifteen families with CPP4. Rigorous criteria were used to filter the variants and identify the mutations likely to be causative of the phenotype for CPP. In this study, 15 individuals (8 girls, and 7 boys) from 5 families with CPP carried MKRN3 mutations predicted to be loss-of-function. Patients 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.
MKRN3, an intronless gene, is located on a complex imprinted locus in chromosome 15q11.2, a Prader-Willi syndrome critical region. All studied families had autosomal dominant inheritance, and they were consistent with the expected transmition of a patternally expressed gene, it means the mutated allele must be transmitted by the father to cause the phenotype. Complete penetrance of the MKRN3 defects was lately illustrated by the description of a girl carrying a MKRN3 mutation detected prior to the onset of puberty10. She was screened at the age of 4, because her older sister had developed CPP at 6 years of age due to the MKRN3 mutation, which was inherited from their asymptomatic father10. During close follow-up, she initially had increased growth velocity at 6, followed by an increased basal LH level and clinical thelarche with rapid progression (Tanner stage 1-3) at the age of 6.7. This prismatic case demonstrated the possibility of very early or even subclinical diagnosis of CPP cases caused by genetic abnormalities.
More recently, the hormonal and genetic features of 20 male patients from 17 families, who had diagnosis of CPP associated with normal MRI of hypothalamic-pituitary region, were evaluated for potential genetic abnormalities.11. After genetic analysis, eight boys from 5 families harbored heterozygous loss-of-function mutations of MKRN3. The frequency of MKRN3 mutations among boys was significantly higher than female data (40% vs. 6.4%, respectively, p < .001). Boys with MKRN3 had later pubertal onset than boys without MKRN3 abnormalities (median age 8.2 vs. 7.0 years, respectively, p = .033). Notably, the determination of age at pubertal onset in boys can be a challenge12. 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.
A high prevalence of mutations in familial cases has been described (achieving up to 46%), while in sporadic forms of CPP the prevalence was variable, from very low in a cohort of patients from Korea to quite high in boys from Brazil13, 14. More than 30 different loss-of-function mutations of MKRN3 have been reported in CPP in the last 5 years11. This high prevalence of MKRN3 mutations in familial cases with CPP can affect the clinical investigation of familial cases, in which genetic studies could precede the brain MRI, that might be postponed (in non mutant cases) or even avoided completely in patients who carry MKRN3 mutations1 (Figure 1). The financial costs of the genetic analysis of MKRN3, an intronless gene, are much lower than the costs of a brain MRI in children, who usually need anesthesia for the scan. Therefore, a reliable family history is a key element for driving the medical decision of whether to do a brain MRI or a genetic test first.
The exact role of MKRN3 in the reproductive axis has been explored after the identification of human mutations. Mkrn3 mRNA levels were high in the arcuate nucleus of prepubertal female and male mice and decreased immediately before puberty, after day15, and remained low after puberty, suggesting a potential inhibitor role on the reproductive axis4. Similar pattern of the circulating MKRN3 levels was demonstrated in humans. Longitudinal studies of healthy Danish girls and boys demonstrated that circulating MKRN3 declined prior to pubertal onset 14-16. A negative correlation between MKRN3 levels and gonadotropin levels was demonstrated in girls. In addition, it was demonstrated lower MKRN3 levels in patients with CPP when compared to pre-pubertal age-matched pairs14.
The precise mechanism by which the inactivation of MKRN3 leads to the early reactivation of pulsatile GnRH secretion remains to be elucidated. It has been suggested that MKNR3 is an E3 ubiquitin ligase, similar to other members of makorin family. Once ubiquitinated, the proteins can be targeted to degradation or directed to other intracellular pathways.
A complex defect of DLK1 gene in a family with CPP
Linkage analysis followed by whole-genome sequencing in an Afro-descendant were performed in a Brazilian family in which 4 girls, including two sisters and two paternal half sisters, had progressive CPP5. Their paternal grandmother reported menarche at 9 years old. The children´s father had normal pubertal timing, suggesting either incomplete penetrance or an imprinted disorder. MKRN3 mutation was excluded in this family with CPP. A complex heterozygous defect of DLK1, another maternally imprinted gene located at chromosome 14q, was identified in the affected members. The DLK1 defect was characterized by a 14-kb deletion and 269-bp duplication and only family members who inherited this defect from their father had central precocious puberty, consistent with the known imprinting of DLK15. DLK1, also known as preadipocyte factor 1, is a transmembrane protein expressed at the cell surface in several embryonic tissues, endocrine glands and a subset of neurons.
It was known that paternal deletions or maternal unipaternal disomy of chromosome 14q locus result in Temple syndrome, a complex disease characterized by prenatal and post natal growth failure, obesity, central precocious puberty, small hands and feet and neurologic abnormalities17. However, the Brazilian patients with a complex defect of DLK1 did not demonstrate additional features of Temple syndrome except for increased fat mass5. Interestingly, serum DLK1 levels were undetectable in all affected individuals, supporting the notion that the genomic deletion leads to complete lack of DLK1 protein in these individuals5. It was known that Dlk1 expression increases in the hypothalamus between birth and adulthood in mice18. In addition, Dauber et al.5 showed that Dlk1 was expressed in kisspeptin neuron-derived cell lines, supporting a potential neuroendocrine function.
The importance of MKRN3 and DLK1 in human puberty initiation was reinforced by large genome-wide studies involving European women19. Genotype data in up to 370,000 women demonstrated that rare variants near of these imprinted genes exhibit large effects when paternally inherited. Additionally, these variants conferred a substantial decrease in the age of menarche, demonstrating parent-of-origin-specific associations concordant with known parental expression patterns19.
The initiation of puberty is thought to result from a decrease in factors that inhibit the release of GnRH combined with an increase in stimulatory factors. Human and animal studies recently indicated that MKRN3 is a strong component of the repressive control of human puberty and represents a new pathway in pubertal regulation. The recent association of a complex genomic defect in DLK1 with nonsyndromic familial CPP, strongly suggests a role of genomic imprinting in regulating the timing of human puberty.
Disclosure statement: The author has nothing to disclose.
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