Central precocious puberty caused by mutations in the imprinted gene MKRN3

Abstract

Background: The onset of puberty is first detected as an increase in pulsatile secretion of gonadotropin-releasing hormone (GnRH). Early activation of the hypothalamic-pituitary-gonadal axis results in central precocious puberty. The timing of pubertal development is driven in part by genetic factors, but only a few, rare molecular defects associated with central precocious puberty have been identified.

Methods: We performed whole-exome sequencing in 40 members of 15 families with central precocious puberty. Candidate variants were confirmed with Sanger sequencing. We also performed quantitative real-time polymerase-chain-reaction assays to determine levels of messenger RNA (mRNA) in the hypothalami of mice at different ages.

Results: We identified four novel heterozygous mutations in MKRN3, the gene encoding makorin RING-finger protein 3, in 5 of the 15 families; both sexes were affected. The mutations included three frameshift mutations, predicted to encode truncated proteins, and one missense mutation, predicted to disrupt protein function. MKRN3 is a paternally expressed, imprinted gene located in the Prader-Willi syndrome critical region (chromosome 15q11-q13). All affected persons inherited the mutations from their fathers, a finding that indicates perfect segregation with the mode of inheritance expected for an imprinted gene. Levels of Mkrn3 mRNA were high in the arcuate nucleus of prepubertal mice, decreased immediately before puberty, and remained low after puberty.

Conclusions: Deficiency of MKRN3 causes central precocious puberty in humans. (Funded by the National Institutes of Health and others.).

Figure 1. Pedigrees of the Families with MKRN3 Mutations

Squares indicate male family members, circles female family members, black symbols clinically affected family members, symbols with black circles asymptomatic carriers, symbols with an X deceased family members, symbols with a question mark family members whose phenotype is unknown, and arrows the proband in each family. The MKRN3 genotype is shown for family members whose DNA was available for genetic studies. A star indicates that the patient was screened by means of Sanger sequencing only. NM denotes nonmutated.

Figure 2. MKRN3 Domains and the Mutations Identified in the Study Families

MKRN3 has four zinc-finger domains: three C3H motifs (blue) and one C3HC4 RING motif (red), which is responsible for ubiquitin ligase activity. The MKRN-specific Cys–His domain (green) is of uncertain function. The numbers correspond to the amino acid positions in the protein. Blue mutation labels and arrows indicate the location of frameshift mutations; the red mutation and arrow indicate the location of a missense mutation.

Figure 3. Mkrn3 Messenger RNA (mRNA) Levels in the Hypothalamic Arcuate Nucleus of Male and Female Mice during Postnatal Development

Total RNA was extracted from the hypothalamic arcuate nucleus of male and female mice at the ages indicated (number of days after birth), and Mkrn3 mRNA was quantified with the use of real-time polymerase-chain-reaction assay. The bar graphs show the relative change in mRNA levels in female mice and male mice, as compared with the level on postnatal day 10 (black bars), normalized to levels of endogenous ribosomal protein L19 mRNA. Mean (±SE) values are shown for three different mice at each age, with each measurement performed in triplicate. Significant differences (P<0.05) were measured by means of a one-way analysis of variance with a post hoc Tukey multiple-comparison test. Asterisks indicate P<0.001.