Inactivating mutations in the MKRN3 gene are cause of familial central precocious puberty

Author(s)

Francisca Delanie Bulcão de Macêdo

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
• Defense date: 2016-04-26

Examining board members

Ana Claudia Latrônico Xavier; Júlio Zaki Abucham Filho; Sonir Roberto Rauber Antonini; Marcello Delano Bronstein

• Advisor: Ana Claudia Latrônico Xavier

Abstract

Most cases of central precocious puberty (CPP) in girls remain idiopathic. The hypothesis of a genetic cause has been strengthened after the discovery of some genes associated with this phenotype, particularly those involved with the kisspeptin system (KISS1 and KISS1R). However, genetic defects in KISS1 and its receptor are rare and have been identified in only a few patients with CPP.over the past years. To date, most genetic studies in CPP was based mainly on a candidate gene approach, including genes selected in animal studies, human models of patients with hypogonadotropic hypogonadism or in genome wide association studies. In the present study, we used whole exome sequencing, a more advanced method of sequencing, to identify variants associated with CPP. Thirty-six patients with the familial form of CPP (19 families) and 213 apparently sporadic cases were initially selected. The familial form was defined by the presence of more than one member affected in the family. Genomic DNA was extracted from peripheral blood leukocytes in all patients. Whole exome sequencing performed by ILLUMINA technique in 40 members of 15 families with CPP, identified inactivating mutations in a single gene, MKRN3, in five out of these families. Analysis of MKRN3 mutations performed by automatic sequencing in two additional families (four patients) identified two novel mutations. MKRN3 is an introless gene located on chromosome 15, in the Prader Willi syndrome critical region, and it is expressed only by the paternal allele due to the maternal imprinting. Following the initial findings, we searched for MKRN3 mutations in 213 patients with apparently sporadic CPP using polymerase chain reaction followed by direct enzymatic purification and automated sequencing (Sanger). Three new mutations and two previously reported, including four frameshifts and one missense variant was identified in six unrelated girls with CPP. All variants were not described in the two databases (1000 Genomes and Exome Variant Server). The familial segregation analysis performed in three out of these girls with apparently sporadic CPP and MKRN3 mutations confirmed the autosomal dominant inheritance with complete penetrance and exclusive transmission through the paternal allele, revealing familial inheritance in apparently sporadic cases. Most of these MKRN3 mutations were frameshifts or nonsense, leading to premature stop codons and truncated proteins, thus demonstrating positive genotype- phenotype correlation. The two missense mutations (p.Arg365Ser and p.Phe417Ile) identified were located within zinc finger motifs, regions predicted to be essential for the protein function. Besides that, all missense mutations were predicted to be pathogenic by in silico analysis. All patients carrying MKRN3 mutations exhibited typical clinical and hormonal features of premature activation of the reproductive axis. The median age of puberty onset was 6.0 years in girls (ranging from 3.0 to 6.5) and 8.0 years in boys (ranging from 5.9 to 8.5). In view of the imprinting phenomenon, methylation analysis was also performed in a subgroup of 52 patients with CPP by MSMLPA technique, but no methylation abnormalities were detected. In conclusion, our work has identified a new gene associated with CPP. Currently, inactivating mutations in MKRN3 represent the most common genetic cause of familial CPP (33%). MKRN3 is the first imprinted gene associated with pubertal disorders in humans. However, its precise mechanism of action in the regulation of GnRH secretion needs further studies (AU)

• FAPESP's process: 13/06391-1 - New perspectives of the genetic study of idiopathic central precocious puberty
• Grantee: Francisca Delanie Bulcão de Macêdo
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)