【作者】 温文；

【导师】 罗阳；

【作者基本信息】 中国医科大学 ， 细胞生物学， 2010， 博士

【摘要】 论文一一个中国汉族遗传性多发性外生性骨疣家系的致病基因突变研究前言遗传性多发性外生性骨疣(hereditary multiple exostoses, HME)是一种常染色体显性遗传病,以骨骼系统多发性外生性骨疣为特征,常见于长骨干骺端,偶见于长骨骨干。骨疣通常发生在膝关节(70%)、肱骨(50%)、前臂(50%)、肩胛骨(50%)、踝关节(25%)等部位,呈对称性分布。HME发病率在欧洲人群中约1／10万,在华盛顿人群中≥1／5万。HME外显率为95%。约90%的HME患者有受累父／母,约10%有新生基因突变。HME具有遗传异质性,目前研究认为该病至少与三个基因相关：定位在8q24.11-q24.13的EXT1、定位在11p12-p11的EXT2、定位在19p的EXT3,其中EXT1、EXT2已被克隆。此外,HME可能存在修饰EXT1或EXT2突变效果的二次突变,由此与已知EXT基因具有相同羧基端编码产物的EXTL基因家族也有可能是HME的候选致病基因,包括有三个成员：定位在1p36.1的EXTL1、定位在1p11-p12的EXTL2、定位在8p12-p22的EXTL3。迄今为止,尚未在EXT3和EXTL基因家族发现HME致病突变。HME致病突变目前只发现于EXTl和EXT2基因,其中EXT1突变占56%-78%,EXT2突变占21%～44%。本文以1个中国汉族HME家系为研究对象,首先利用单体型分析进行了致病基因定位,然后对候选基因进行DNA传统测序来筛查突变,最后通过限制性酶切多态性分析对突变进行验证。材料与方法1、家系材料中国医科大学医学基因组学教研室收集的1个中国汉族HME家系,症状与HME既往报道相符。在签署书面的知情同意书后,对家系中12名成员采集血样(其中患者5例,男2例、女3例)。2、单体型分析常规酚-氯仿抽提法提取家系成员的外周血DNA。在EXTl和EXT2基因内部分别选取了2个微卫星多态标记,PCR扩增后经8%变性聚丙烯酰胺凝胶电泳和常规银染,读取每个个体的标记基因型。再根据个体基因型和系谱中的亲缘关系,按照孟德尔遗传定律推导出同一条染色体上不同标记构成的单体型。3、候选基因DNA传统测序根据单体型分析确定候选基因后,PCR扩增患者候选基因所有外显子,PCR产物回收纯化后进行DNA传统测序。4、限制性片段长度多态性(restriction fragment length polymorphism, RFLP)分析根据测序发现的突变设计1个错配引物,错配碱基与包括突变碱基在内的邻近碱基构成一个ScaI限制性酶切位点。对家系全体成员及100名无关正常个体使用错配引物进行PCR扩增,产物经ScaI消化,消化产物经8%中性聚丙烯酰胺凝胶电泳和常规银染检测。实验结果1、单体型分析8号染色体的EXT1基因可能为该家系候选致病基因。2、DNA传统测序患者EXT1基因第10外显子存在杂合缺失突变c.1897delC,该突变导致编码的氨基酸从第633位密码子开始移码,在编码了9个氨基酸之后产生终止密码,可能产生一个含有641个氨基酸的截短蛋白(p.Leu633TyrfsX10)。对该家系全体成员进行测序,发现该变异与疾病表型共分离,家系中所有患者都存在该变异,而正常个体都没有。3、RFLP分析ScaI酶切产物电泳可见,家系中患者具有284bp、250bp和34bp三种长度的片段；而家系中正常个体及100名无关正常个体只有284bp片段,即没有ScaI位点,说明家系中正常人及无关正常个体不携带c.1897delC突变,此突变不是SNP。结论首次在HME家系中检测到杂合缺失突变EXT1 c.1897delC (p.Leu633TyrfsX10),此突变是该中国汉族HME家系的致病突变。论文二三个反常性痤疮家系的致病基因突变研究前言反常性痤疮(acne inversa, AI)以往被称为化脓性汗腺炎(hidradenitis suppurativa,HS),以反复发生皮肤脓肿,窦道及瘢痕形成为特征性表现。病情反复发作,此起彼伏,迁延不愈。与寻常性痤疮不同,该病病变主要累及腋前线、乳房下区、外生殖器区、肛周等毛囊皮脂腺单位、顶泌汗腺分布丰富的部位。据国外报道,该病的发病率估计为1：100～600。AI病因和发病机制尚不明确,目前认为是由于毛囊漏斗部的上皮细胞过度角化增生造成毛囊口及皮脂腺的阻塞,皮脂排出不畅,之后继发细菌感染引起。近年来,遗传因素在其发病过程中所起的作用受到越来越广泛的关注,家系研究也表明其遗传方式符合单基因常染色体显性遗传。目前,人类孟德尔遗传(Mendelian Inheritance in Man)数据库将家族性AI (acne inversa, familial)收录为MIM%142690。迄今为止,报道的致病基因座有3个,分别位于1p21.1-1q25.3,6q25.1-25.2以及19号染色体D19S911与D19S1170之间约12Mb的区间,但致病基因尚未明确。基于中国医学科学院基础医学研究所医学遗传学系(以下简称医科院基础所医学遗传学系)的前期工作基础,本文对三个分别来自中国河北省、河南省和英国的AI家系进行了致病基因突变研究。材料与方法1、家系材料医科院基础所医学遗传学系收集到的三个AI家系分别来自中国河北省、河南省和英国,均符合常染色体显性遗传。2、DNA传统测序在中国两个AI家系定位的连锁区域chr19：35,000,000-40,890,000内的212个基因中筛选候选基因,PCR扩增候选基因5′-UTR、编码外显子、3′-UTR,PCR产物进行DNA传统测序。3、高通量测序提取3个AI家系中明确诊断的患者各一位的外周血DNA,送至Roche NimbleGen公司采用Sequence Capture 385K Array捕获序列,继而使用Roche公司454测序仪(RochGS FLX sequencer)测序3个家系联合定位区域chr19：20,916,746-50,538,495的全部外显子组。4、高通量测序结果分析使用IGV1.4.2软件阅读Roche 454AllDiffsReg.primary_target_region.gff3、454HCDiffsReg.primary_target_region.gff3、454AlignmentCoverage.igv文件；使用UltraEdit软件阅读454AllDiffs.txt.454HCDiffs.txt文件,随时比对不同于UCSC Feb.2009 human reference sequence (GRCh37/hg19)的测序结果,筛选可能致病改变。5、高通量测序结果验证使用DNA传统测序技术验证高通量测序结果中的可能致病改变。针对经DNA传统测序证明确实存在的可能致病突变,设计引物,PCR扩增全部家系成员的相应片段,对片段进行变性聚丙烯酰胺凝胶电泳长度分析、或RFLP分析、或高分辨率熔解曲线(High Resolution Melting, HRM)分析,判断该变异是否与疾病表型共分离。实验结果1、DNA传统测序结果完成了25个基因的DNA传统测序,均未发现致病改变,仅发现了100余个已知的SNP,和FLT3LG基因第6外显子中1个尚未报到过的SNP c.502C>G(以起始密码ATG之A为第1位)。2、高通量测序结果在3个患者总计33979个未知改变中筛选出125个可疑改变,其中“HS19”基因第4外显子的缺失改变c.483delC与AI Family 2疾病表型共分离。结论反常性痤疮候选致病基因可能为“HS19”。更多还原

【Abstract】 Paper 1 Mutation Identification in A Han Chinese Kindred with Hereditary Multiple ExostosisIntroductionHereditary multiple exostoses (HME) is an autosomal dominant bone disorder characterized by growth of multiple exostoses-benign cartilage-capped bone tumors that grow outward from the metaphyses of long bones. Exostoses may also occur at other sites, such as the ribs, scapula and pelvis. Prevalence rate is 1/100,000 in Europe, and 1/50,000 in Washington. HME is inherited in an autosomal dominant manner, with a penetrance of 95%. About 90% of individuals with HME have an affected parent; about 10% of patients develop HME as the result of a de novo gene mutation. Three genes are known to be associated with HME:EXT1, EXT2 and EXT3, located on 8q24.11-q24.13, 11p11-p12 and 19p, respectively. However, a second mutational hit may arise in a related gene such as the EXT-like (EXTL) genes or other genes involved in the signaling cascade of chondrocyte proliferation. The EXTL family of genes is related to EXTs by sequence homology. The EXTL family of genes currently consists of three members:EXTL1, EXTL2 and EXTL3, respectively located on 1p36.1, 1p11-p12 and 8p12-p22. To date, no HME disorder has been attributed to mutations in EXT3, EXTL1, EXTL2 and EXTL3.In this study, we recruited one Han Chinese family with typical HME for research. First of all, haplotyping was performed to locate the chromosome position of pathopoiesis gene. Then, we undertook DNA sequencing to identify the mutation. At last, restriction fragment length polymorphism was undertaken to confirm this mutation.Materials and Methods1. SubjectsA four-generation Han Chinese kindred in which HME had affected 8 members (five males and three females) was investigated. Blood samples were collected from the 12 family members by standard procedures with written informed consent.2. Haplotype analysisGenomic DNA was extracted from venous blood samples using proteinase K digestion and standard phenol-chloroform extraction. Using the UCSC Genome Browser on Human 2006 May assembly (http://www.genome.ucsc.edu), two perfect microsatellite repeat sequences each close to the EXT1 gene and the EXT2 gene were selected as genetic markers for haplotyping. The PCR products of the microsatellite markers were separated by electrophoresis on 8% denaturing polyacrylamide gel and allele fragments were detected with routine silver staining, then on the basis of individual’s genotype and kinship, the haplotypes were deduced according to the Mendel’s law of inheritance.3. DNA sequencing of the candidate geneAfter determining the candidate gene by haplotyping, PCR was performed to amplify the exons of the gene, and then the amplicons were sequenced.4. Restriction fragment length polymorphism (RFLP) analysisTo confirm this mutation, we employed a mismatch primer which creates a Scal restriction site in the deletion mutant allele, but not in the normal allele. The PCR products using the mismatch primer were digested with restriction enzyme Scal, separated by 8% neutral polyacrylamide gel electrophoresis and showed by routine silver staining. All the 12 available family members and 100 unrelated Han Chinese normal controls were included in the above-mentioned PCR-RFLP analysis.Results1. Haplotype analysisHaplotype analysis indicated the causative gene of the Han Chinese HME family was possibly the EXT1 gene located on chromosome 8.2. Mutation screeningWe identified a heterozygous deletion, c.1897delC, in exon 10 of the EXT1 gene in the proband. This deletion will result in a frameshift from codon 633 and a premature termination at codon 642, therefore designated as p.Leu633TyrfsX10 at protein level. To confirm this deletion mutation, in all the other available family members, the exon 10 genomic fragment were subjected to automated DNA sequencing after purification. The results showed the cosegregation of mutation c.1897delC with the disease phenotype.3. RFLP analysisBesides the normal 284bp fragment, a 250bp fragment and a 34bp fragment were seen in all affected individuals from the family but were not detected in all unaffected family members or unrelated normal controls. Therefore, this heterozygous mutation could not be a polymorphism and it is the causal mutation for the phenotype of HME in this Chinese family.ConclusionA new heterozygous mutation c.1897de1C (p.Leu633TyrfsX10) in the EXT1 gene was identified as the pathogenic mutation of HME in this Chinese family.Paper 2 Mutation Identification in Three Acne Inversa FamiliesIntroductionAcne inversa (AI), also known as hidradenitis suppurativa (HS), is a chronic relapsing inflammatory disease characterized by recurrent draining sinuses and abscesses. Inflamed lesions are in the apocrine gland-bearing area of the body, most commonly in the axillae, perineum, and inframammary regions. The exact incidence of AI is uncertain, but the literature suggests from 1 in 100 to 1 in 600. AI is now considered a disease of follicular occlusion rather than an inflammatory or infectious process of the apocrine glands. Now, the etiology is predominantly associated with genetic factors. The pattern of transmission in familial cases is consistent with autosomal dominant inheritance (OMIM %142690). At present, three loci for this disorder were reported and localized to chromosome 1,6,19, but no gene is responsible for it.On the basis of the preliminary work of Department of Medical Genetics in Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, we recruited three AI pedigrees for research. Both traditional DNA sequencing and next-generation DNA sequencing were used to identify gene mutations.Materials and Methods1. SubjectsTwo Chinese and one English AI families collected by Department of Medical Genetics in Sciences Chinese Academy of Medical Sciences were recruited.2. Traditional DNA sequencing of the candidate genesWe selected 25 relatively-known genes as candidates. PCR was performed to amplify the exons of these genes, and then the amplicons were sequenced by traditional DNA sequencing.3. Next-generation DNA sequencing of exomesOn the basis of the preliminary haplotyping analysis work of Department of Medical Genetics and one lab in United Kingdom, we used Roche Sequence Capture 385K Array to capture all exomes in chr19:20916746-50538495. Then next-generation DNA sequencing was used to sequence the exomes.4. IGV 1.4.2 was used to read the files of Roche 454AllDiffsReg.primary_target_region.gff3, 454HCDiffsReg.primary_target_region.gff3 and 454AlignmentCoverage.igv. UltraEdit was used to read the files of 454AllDiffs.txt and 454HCDiffs.txt. At the same time, when we found the variants, we did blat on UCSC to find the predicted damaging variants.5. To confirm the next-generation DNA sequencing results, the predicted damaging variants were resequenced by traditional DNA sequencing.8% denaturing polyacrylamide gel electrophoresis or PCR-RFLP or HRM were used to confirm whether the vatients cosegregate with the disease phenotype.Results1. Traditional DNA sequencing of the candidate genes’exonsIn 25 candidategenes, no significant mutation was found, except about 100 known SNP and one new SNP c.502C>G in FLT3LG gene.2. Next-generation DNA sequencing of exomesIn total, there were 33979 unkown varients in these three patients. We found 125 predicted damaging variants. The HRM results showed the cosegregation of a mutation ’HS19’c.483delC with the disease phenotype in AI Family 2.Conclusion’HS19’gene is considered to be associated with AI.更多还原