Context 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. Objective To screen patients with CPP for MKRN3 and DLK1 mutations and analyze the effects of identified mutations on protein function in vitro. Setting Five academic medical institutions. Participants Eighty-four unrelated children with CPP (79 females, 5 males) and, when available, their first-degree relatives. Design 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. Results Eight heterozygous MKRN3 mutations were identified in 9 unrelated girls with CPP. Five are novel missense mutations, two were previously identified in patients with CPP, and one 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 LH and FSH 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. Conclusions 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.

Context : Central precocious puberty (CPP) results from premature activation of the hypothalamic–pituitary–gonadal axis. Loss-of-function mutations in makorin ring finger 3 ( MKRN3 ), a maternally imprinted gene, have been recognized as the most common genetic cause of CPP. More recently, a complex defect (deletion and duplication) in delta-like 1 homolog ( DLK1 ), another maternally imprinted gene, was associated with CPP and increased body fat in a family with several affected girls. Single nucleotide polymorphisms in or near the MKRN 3 and DLK1 genes have been associated with age of menarche when the variant is inherited from the father. Objectives: To investigate the prevalence of mutations in MKRN3 and DLK1 in a cohort of 50 patients (3 boys) with CPP. Design : The 5’ untranslated region (5’UTR) and coding regions of MKRN3 and DLK1 were amplified and sequenced by Sanger sequencing. Family members of patients with identified MKRN3 or DLK1 variants were included for genetic analysis when DNA was available. Results: We identified five mutations in MKRN3 : four novel missense (p.Tyr117Cys, p.Ile461Phe, p.Met126Val and p.Cys364Phe), and one rare frameshift mutations (p.Ala288Profs*108, gnomADE frequency 8e-06). These mutations were identified in five girls, two of whom had a family history of CPP. The Cys364Phe mutation, located in a key cysteine residue in the E3 ligase RING finger domain of MKRN3, was identified in a proband and her paternal cousin with CPP. Ala288Profs*108 was identified in a proband and in two affected relatives, her sister and a paternal cousin. The patient’s father also harbored the mutation; he did not have known early pubertal development. The fathers of the two girls with Ile461Phe and Met126Val mutations without a known family history of CPP were sequenced and harbored the corresponding mutations. In addition, one homozygous variant was identified in the 5’UTR of DLK1 , c.-217G>T, in an overweight girl without a family history of CPP. This variant has been reported in the heterozygous state at a frequency of 3e-05 in gnomADE. Her father was not available for genetic studies. Interestingly, her mother was homozygous for the wild type allele, suggesting that the mother did not transmit a DLK1 allele to the patient in this region. All patients with mutations had classical features of CPP. Conclusions: In our cohort of CPP, 10% of subjects had MKRN3 mutations. Familial segregation analysis was performed in four cases and in all cases the mutations were paternally inherited. A nucleotide change was identified in the 5’UTR of ...