Setting
In order to investigate the possible role of PROKR2 in the etiopathology of CPP, a prospective, observational multicenter study was set up and carried out over a 2-year period.
4 Italian centres were involved in this study: Department of Woman, Child, General and Specialized Surgery of Università degli Studi della Campania “Luigi Vanvitelli”, Naples, Pediatric Section-Department of Translational Medical Sciences, University of Naples Federico II, Naples, Institute for Maternal and Child Health IRCCS, “Burlo Garofolo”, Trieste, and the Department of Paediatrics, Bologna University, Bologna. Ethics committee of the Università degli Studi della Campania “Luigi Vanvitelli” approved the protocol then subscribed by ethics committee of the other centres. According to the World Medical Association Declaration of Helsinki, written informed consent from parents and oral consent from all participants was collected.
Subjects
We recruited 31 females with diagnosis of CPP that met these following criteria: thelarche occurred before age of 6 years, defined as Tanner stage B ≥ 2 at physical examination, a diagnosis of central HPG activation identified by pubertal basal luteinizing hormone (LH) levels (> 0.3 UI/l) or a positive Gn-RH stimulating test and normal brain MRI.
Exclusion criteria included: congenital defects or abnormalities possibly related to syndromic features, central nervous system pathology (i.e., tumours or nonspecific cerebral anomalies associated with CPP). All the patients involved in this study were Italian.
Protocol
All girls underwent a clinical examination with special regard to auxological parameters and staging of breast development according to Tanner’s classification. Right hand and wrist X-ray was performed for bone age evaluation by TW2 method. Peripheral blood samples were collected for all patients for hormonal dosage and genetic analysis.
Chemiluminescence assay (LIAISON, Diasorin) was used to measure FSH and LH concentrations, with detection limits of 0.06 and 0.05 U/L, respectively, and intra- and inter-assay CV less than 5%. Radioimmunoassay was used to measure serum estradiol (CisBio International). The analytical and functional detection limits for plasma estradiol were 4 and 8 pg/mL, respectively.
GnRH stimulating test was provided in patients in which basal hormone level did not meet diagnostic criteria for CPP. Peak-LH > 5UI/L after administration of 0.1 mg of Relefact LH-RH (Sanofi-Aventis, Frankfurt am Main, Germany) was considered positive.
Thin-section, contrast-enhanced MRI examination of sellar region with T1-weighted and T2-weighted sagittal, coronal sequences, 3DT2 thin section axial sequence and FLAIR and EPI DWI on axial sequence was acquired for all patients.
Genetic analysis
Genomic DNA was extracted from peripheral whole blood using a DNA extraction kit (Promega, Madison WI, USA) following the manufacturer's instructions. Each of the two coding exons and the intron-exon boundaries of the PROKR2 gene was amplified by polymerase chain reaction (PCR) using two couples of primers each and subsequently analysed by direct sequencing (ABI PRISM 3100, Perkin Elmer, USA) under standard conditions. In the attempt to reduce possible bias of selection in the sample due to other mutations, an analogous procedure was used to screen MKRN3 gene sequence to rule out the most frequent genetic cause of CPP nowadays individuated. All genetic analysis was performed at Department of Woman, Child, General and Specialize Surgery of Università degli Studi della Campania “L. Vanvitelli”, Naples, Italy.
Data Analysis
Epidemiological data were expressed as medians (interquartile ranges). The Fisher exact test was used to compare allele frequency of polymorphism found in our screening to Exome Aggregation Consortium (ExAC) dataset. Results reach statistical significance at a p value less than 0.05. All statistical analyses were performed using Stat-Graph Centurion XVII software for Windows.