【作者】 刘璐；

【导师】 陈临琪；

【作者基本信息】 苏州大学 ， 儿科学（专业学位）， 2018， 硕士

【摘要】 [研究背景]中枢性性早熟(central precocious puberty,CPP)是指由于下丘脑-垂体-性腺轴的提前启动,女孩在8岁前,男孩在9岁前出现第二性征发育的儿童内分泌疾病。2010年中国九大城市女童第二性征发育和初潮年龄调查显示:女童在8岁前出现乳房发育者超过10%,而10岁前月经来潮者占1.65%。研究表明,性早熟不仅会造成矮身材,影响儿童的正常发育和社会心理健康,还会导致成年后乳腺癌、子宫内膜癌、肥胖、2型糖尿病和心血管疾病等的发病风险显著增高。因此,儿童性早熟的早期诊断和及时干预,不仅可以使青春发育回归正常的生理过程,还可以减少后期相关疾病发生的风险。青春期启动时间受遗传、环境、种族、营养及社会经济等多方面因素影响,其中遗传因素起到关键性作用。到目前为止,已证实的与CPP相关的致病基因有KISS1、KISS1R、DLK1和MKRN3基因。其中MKRN3基因所致性早熟发病率相对较高,自2013年在新英格兰杂志首次报道以来,目前已有多个家系及散发病例被发现。[研究目的]1.从遗传和基因方向入手,通过检测和分析CPP患儿中MKRN3基因的突变类型和遗传方式,探讨MKRN3基因在中国CPP患儿中的突变规律。2.通过研究不同MKRN3基因突变所对应的临床表型,明确基因型与表型的关系,为CPP患儿的精准诊疗和遗传咨询提供理论依据。[研究设计与方法]1.选取研究对象:选取2015.02-2017.02在江苏省苏州大学附属儿童医院内分泌科就诊及住院的CPP患儿173例(其中9例为家族性CPP),早青春期患儿43例(其中3例有家族史)为研究对象。CPP诊断参照2015年中华医学儿科学分会内分泌遗传代谢学组发表的《中枢性性早熟诊断与治疗共识》,排除误服避孕药,颅内、盆腔、肾上腺等部位的占位性病变和其他内分泌疾病(如Mc Cune-Albright综合征)引起的性早熟。2.研究内容:收集所有患儿的临床资料,包括就诊年龄、发病年龄、骨龄、初诊时第二性征分期、性腺大小、促性腺激素释放激素(Gn RH)激发试验、家族性CPP患儿的家族史等;在征得患儿及其家属同意后,同时采集血样标本。提取血液组织全基因组DNA,用PCR法扩增MKRN3基因片段,纯化PCR产物,用Sanger法进行测序。依据“ACMG基因变异解读指南”,运用多种计算机软件及数据库,对MKRN3基因新突变的致病性进行分析,探索MKRN3基因型与CPP临床表型的相关性。3.研究方法:(1)DNA的提取:采用商品化试剂盒(TIANamp Blood DNA试剂盒,天根生物科技,中国北京),按照说明书方法提取基因组DNA,并对其浓度进行标化。(2)PCR法扩增MKRN3基因:用PCR法扩增MKRN3基因整个编码区,将其扩增为三个重叠的片段(三对引物序列分别为:CCGGAAGTAGGTAGGAACACAC和TTCAGCAGCCGAGCCAATCA;CTTTCTGGTCGGAAGATGGC和CACCTGCGGATACACCTAAT;TGCAGCGTGGTATGGACAAG和AGAAGCACTGCCTCAACAGC)。纯化PCR产物。(3)Sanger法测序:用ABI3730xl系统对PCR产物进行分析,并与参考序列进行比对,参考序列由Ensembl数据库获得(版本序列号ENST00000314520)。家系患儿若发现突变,需在其三代以内直系亲属中进行突变位点验证。(4)新错义突变的致病性预测:用计算机程序(Poly Phen-2,SIFT,Mutation Taster等)对突变的致病性进行初步预测。(5)基因型与表型相关性分析:依据“ACMG基因变异解读指南”,同时运用多种计算机软件及数据库(如Ex AC Browser,1000Genomes,Clinvar,HGMD等),结合家系分离分析,明确基因变异的人群携带率、遗传方式,以及与临床表型的相关性。(6)新错义突变的生物信息学分析:运用计算机软件(BLAST/PSI-BLAST、MODELLER V9.19、VMD1.9.4)构建MKRN3蛋白的三维结构模型,并分析突变所处的空间位置,从而推断其对蛋白功能的影响。[研究结果]1.MKRN3基因测序结果:本研究在2个散发病例(散发共204个)和5个家系病例(家系共12个)中,检测出四种MKRN3基因突变,分别为:c.1138G>A(p.Glu380Lys)、c.1420T>A(p.Leu474Met)、c.673C>G(p.Leu225Val)、c.1071C>G(p.Ile357Met);其中第一个为新发错义突变(父母均无该突变),后三者则为杂合错义突变(父或母有相同突变)。以上4种突变,目前均无文献报道,为本研究发现的新的突变位点。2.MKRN3突变组和非MKRN3突变组的临床特征相比无明显差异。3.综合12个家系病例的基因及临床资料,进行遗传学分析提示:c.1138G>A(p.Glu380Lys)和c.1071C>G(p.Ile357Met)为CPP的可能致病突变;另外两个错义突变的临床意义均不明确。4.MKRN3突变对其蛋白结构及功能影响的生物信息学分析:E380K和I357M均位于锌离子结合区域(C3HC4环状锌指区),而前者由于E(带负电荷)突变为K(带正电荷),使得其物理化学性质完全改变,氨基酸之间的氢键相互作用无法形成,局部结构的稳定性被破坏,并可能导致锌离子结合障碍,故推断其对MKRN3蛋白功能的表达有显著影响。[研究结论]1.本研究中MKRN3突变所致CPP的发生率相对较低,女孩多见,以错义突变为主。2.MKRN3突变在家系及散发CPP病例中均可检测到,其遗传方式为常染色体显性遗传,有性别依赖的不完全外显率。3.基于MKRN3突变的印迹遗传模式,采集病史时不仅要了解母亲一方的家族史,更应详细询问父系家族史。有男性性早熟家族史的健康儿童,应早期进行遗传咨询,以免错过最佳诊疗时机。4.可在家族性CPP的患者中常规进行MKRN3基因检测。5.无论中国还是国外,Gn RH类似物都是MKRN3突变所致CPP的唯一可靠治疗手段。更多还原

【Abstract】 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.更多还原