The Genetic And Bioinformatic Analysis Of Chinese CPP Patients With MKRN3 Gene Mutations

Background:Central precocious puberty(CPP)is an endocrine disease which is clinically defined as the development of secondary sexual characteristics before the age of 8 years in girls and 9 years in boys due to the early activation of the hypothalamic-pituitary-gonadal axis.In 2010,a survey on the emergence of secondary sexual signs and the age of menarche in girls in 9 major cities of China showed that more than 10% of girls had breast development before 8 years and approximately 1.65% girls with menarche before 10 years.Studies have indicated that precocious puberty will not only cause short stature in adulthood,social and mental illness during adolescence,but also increase the risk of breast cancer,endometrial cancer,obesity,type 2 diabetes and cardiovascular disease.Therefore,treatment of precocious puberty can help the patients back to the normal physiological process and reduce the risks of suffering subsequent diseases.The timing of puberty is influenced by complex interactions of genetic,environmental,ethnic,nutritional,and socioeconomic factors,among which the genetic factor plays a crucial role.Up to now,disease causing mutations associated with CPP are described in KISS1,KISS1 R,DLK1,MKRN3 genes.Among them,the incidence of MKRN3 mutations caused CPP is relatively high.Since MKRN3 related CPP first published in New England Journal of Medicine in 2013,MKRN3 related had been reported many familial and sporadic cases of CPP.Objective:1.Explore MKRN3 mutations in Chinese girls and boys with CPP,and analyze the genetic characterizations of MKRN3 mutations.2.To observe the clinical phenotypes of patients with MKRN3 mutations,in order to provide the theoretical basis for precise diagnosis and genetic counseling of the Chinese CPP cases.Designs and Methods:1.Inclusion and exclusion criteriaWe enrolled in our study 173 patients(containing 9 probands of familial cases)who were diagnosed with CPP and 43 children had early puberty(3 of whom were described their family history)at endocrinology department in Children's Hospital affiliated to Soochow University of Jiangsu from February 2015 to February 2017.The diagnostic criteria of CPP is in accordance with a consensus published in Chinese Journal of Pediatrics in 2015,excluding to misuse contraceptives,the intracranial lesions,pelvic or adrenal tumors,and other endocrine disorders(such as Mc Cune-Albright syndrome)caused precocious puberty.2.Study designTo collect the clinical data of all research subjects including age at diagnosis,age at puberty onset,bone age,Tanner stage,gonads size,Gn RH stimulation test,and the family history of CPP probands.After the informed consent was obtained from parents of all participants,we collected and stored their blood samples.Genomic DNA was extracted from peripheral blood lymphocytes.The MKRN3 coding region was amplified by polymerase-chain-reaction(PCR).We purified these PCR products and then use the Sanger method to sequence them.We investigated the pathogenicity of MKRN3 variants according to the “Standards and Guidelines for the interpretation of sequence variants” published by American College of Medical Genetics(ACMG)and the use of different silico prediction programs.Correlation between the MKRN3 genotype and clinical phenotype was explored by combining with the Pedigree segregation analysis.3.Methods(1)DNA extraction: It was based on the specification in the commercialized reagent kit(TIANamp Blood DNA kit,TIANGEN biotechnology limited company,Beijing,China).We also made further standardization on their concentrations.(2)MKRN3 gene amplification in PCR: The MKRN3 coding region was amplified with three pairs of primers(5'-CCGGAAGTAGGTAGGAACACAC-3' and 5'-TTCAGCAGCCGAGCCAATCA-3';5'-CTTTCTGGTCGGAAGATGGC-3' and 5'-CACCTGCGGATACACCTAAT-3';5'-TGCAGCGTGGTATGGACAAG-3' and 5'-AGAAGCACTGCCTCAACAGC-3').Then,we purified the PCR products.(3)Sanger sequencing: The PCR product was analysed by ABI3730 xl software,and compared with the corresponding reference sequence(ENST00000314520)from the Ensemble database.If the MKRN3 mutations were found,we would verify the mutation in their parents and grandparents as far as possible.(4)Pathogenicity prediction of new missense mutations: The pathogenicity of the variants was initially predicted by Poly Phen-2,SIFT and Mutation Taster.(5)Correlation analysis between genotype and phenotype: We investigated the population frequency and inheritance pattern of MKRN3 mutations in a large of patients with CPP on basis of the ACMG Guidelines and databases(such as Ex AC Browser,1000 Genomes,Clinvar,HGMD),in order to discuss whether were related to the clinical phenotype of CPP.(6)Bioinformatic analysis of new missense mutations: The 3D structure model of MKRN3 protein was constructed by BLAST/PSI-BLAST,MODELLER V9.19 and VMD1.9.4.Then,we analysed the effect of new missense mutations on protein structure.Result:1.MKRN3 sequencing data: We identified four missense mutations of MKRN3,c.1138G>A;p.Glu380 Lys,c.1420T>A;p.Leu474 Met,c.673C>G;p.Leu225 Val,c.1071C>G;p.Ile357 Met,in two sporadic cases and five familial cases.The first mutation is a de novo mutation,and the others are heterozygous missense mutations inherited from their parents.So far,none of these mutations has been reported and they are all new mutations discovered in our study.2.No significant difference of clinical phenotypes was found between patients with and without mutations.3.According to ACMG standards,we analysed the correlation between genotype and phenotype in MKRN3 mutations,which indicated that two MKRN3 mutations were likely pathogenic,two others were of uncertain significance4.The bioinformatic analysis of the impact of mutations on MKRN3 protein structures:E380K and I357 M both locate at the zinc-binding region(C3HC4 RING finger motif).Since in E380 K,E with negative charges has been changed into K with positive charges,the physicochemical property has totally altered,thereby hydrogen bonds between amino acids could not been formed and the stability of the local structure was impaired,which might affect the binding of zinc iron and thus change the expression of MKRN3 protein.Conclusion:1.MKRN3 mutations are relatively uncommon in Chinese children with CPP,more common in girls,and missense mutations.2.We have observed the MKRN3 mutations both in the familial and sporadic cases.The inheritance pattern of familial cases is autosomal dominant with incomplete sex-dependent penetrance.3.Because of the imprinting inheritance pattern of MKRN3,the detailed family history should be recorded,especially paternal family history.Health children with family history of CPP should seek for early genetic counseling,so as not to miss the best timing of treatment.4.MKRN3 gene analysis should be routinely carried out on patients with familial patients with CPP.5.Up to now,Gn RH analogs are the only reliable treatment for MKRN3 mutant CPP cases.