ESPE Abstracts

Background: MKNR3 is a paternally expressed gene whose loss-of-function mutations cause Central precocious puberty (CPP). The precise molecular disruption produced by MKRN3 mutations remains unclear albeit protein structure suggests MKRN3 could play a role in proteasome ubiquitination. Circulating MKRN3 levels has been negatively associated to LH peak, estradiol and kisspeptin in idiopathic CPP and healthy controls. However, no literature data is available for plasma MKRN3 levels in CPP harbouring a MKRN3 mutation. Our aim was to investigate for the first time a possible correlation between circulating levels of MKRN3 and different types of MKRN3 mutations.

Material & Methods: we selected five girls with familial CPP carrying different mutations in MKRN3 identified by direct sequencing of the coding region. CPP diagnosis was based on telarche onset before 8 years of age and LH-peak response to GnRH test >5mUI/mL. Circulating MKRN3 levels were measured by Biocompare ELISA assay kit (sensitivity 4.688 pg/ml and inferior detection limit of 7.8 pg/mL). Additionally, in two CPP patients we performed allelic expression analysis by expressed single-nucleotide polymorphism (SNP)-based approach. RNA was extracted from blood white cells samples of patients and their fathers, PCR analysis on RNA retro-transcripts was performed.

Results: Median age of our cohort at diagnosis was 7.6 years old. Two new mutations p.Gln352Arg and p.Ser165Cys, were identified previously described in literature. Subject 1 harbours a frameshift mutation (Pro160Cysfs*14) and an undetectable plasma MKRN3 level. Subjects 2, 3 and 4 carrying missense mutations, namely p.Ser165Cys, p.Arg328Cys and p.Gln352Arg, show divergent circulating plasma protein levels, respectively 27.32pg/ml, 40.56pg/ml and undetectable. Interestingly, patient with a nonsense mutation p.Cys410Ter reported low but measurable circulating levels (12.72 pg/mL). Allelic expression analysis was performed in patients 4, 5 and in their parents confirmed patients expressed MKRN3 paternal allele. Same analysis for other patients is ongoing.

Discussion and conclusions: No correlation between circulating MKRN3 and type of mutation has been identified, remarkably circulating MKRN3 protein levels were detectable even in presence of a non-sense mutation, while missense mutation shown an unpredictable behaviour. Allelic expression analysis excluded that detected protein levels were due to reactivation of the maternal allele, consistent with the expression of wild-type protein in humans and mice. Unlike what has been proved for DLK1 mutations, these preliminary data demonstrate circulating MKRN3 could not be used as a potential screening tool for the diagnosis of familial CPP due to MKRN3 mutations.