全基因组外显子测序发现Marie Unna遗传性少毛症致病基因EPS8L3

发布时间：2018-06-26 21:58

本文选题：Marie + Unna遗传性少毛症　；参考：《安徽医科大学》2012年博士论文

【摘要】：研究背景Marie Unna遗传性少毛症(Marie Unna hereditary hypotrichosis,MUHH,MIM146550/612841)是一种罕见的呈常染色体显性遗传模式的遗传性毛发疾病。Marie Unna于1925年首次报道1例德国患者。该病发病无男女性别差异,但男性患者病情比女性严重。其临床特点为：患者出生时头发正常或稀少或缺乏,随后开始缓慢生长,但已生长的头发粗糙、不规则扭曲呈金属丝样外观,至青春期头发弥漫性脱落且逐渐加重,严重者可呈全秃。此外,睫毛可缺如,眉毛、腋毛、阴毛和男性胡须稀少,但不伴有其他外胚层结构异常,且体格检查和智力均正常。组织病理学显示毛囊周围有少量炎性细胞浸润或纤维化,但成熟的毛囊数量显著减少、毛囊缩小；光学显微镜镜检显示毛发呈扁平、粗糙和不规则扭曲状；扫描电镜显示可有纵嵴、纵沟、纵裂、不规则横断面、毛小皮广泛的剥脱和毛鞘异常。自1999年至2004年,多个研究小组分别对来自荷兰、英国、德国、比利时、美国和中国等不同种族的MUHH家系进行了连锁和单倍型分析,均将MUHH的致病基因位点定位于8p21。前期许多研究小组先后针对定位区域内人类无毛基因(Human hairless gene,HR)的编码区和剪接位点进行突变分析,但均未检测到HR基因的致病性突变。2009年,张学教授在来自不同种族的19个MUHH家系中发现了HR基因U2HR区域的13种不同突变,建立了MUHH遗传突变谱。随后多个研究小组也在中国、土耳其、德国家系或散发病例中发现了HR基因U2HR区域的致病突变。 2004年Yan等通过微卫星标记研究对1个4代的MUHH家系进行分析,发现该家系的致病基因与8p区域不连锁,提示MUHH具有遗传异质性；2005年Yang等对该家系进行全基因组扫描研究,证实该家系致病基因与8p区域不连锁,并将致病基因定位于1p21.1-1q21.3上D1S248和D1S2345之间的17.5cM区域。随着新一代测序技术的迅猛发展,全基因组外显子测序策略已成功应用于单基因病的致病基因研究,癌症和多基因病易感基因的筛查以及临床上疑难病例的诊断,全基因组外显子测序得到了迅猛的发展。目的结合前期的全基因组定位信息,运用全基因组外显子测序技术搜寻MUHH的致病基因,为将来基因诊断、基因治疗奠定基础。方法(1)从家系1中挑选出2个临床表型典型的患者和1个家系内对照,进行全基因组外显子测序；(2)通过逐步滤过dbSNP数据库和家系内对照中的常见突变,并且结合前期定位信息获得候选基因集；(3)利用Sanger测序,在家系内患者和对照中对候选基因集的突变位点进行测序验证,分析突变点是否与疾病共分离,获得候选基因；(4)在独立的家系2和其他4个散发病例中,对候选基因的编码区外显子和外显子与内含子交界区的序列进行测序,以期在其他病例中发现候选基因的突变；(5)采用PCR反应扩增HR基因U2HR区域,用直接测序方法对候选基因突变筛查阴性的病例对HR基因U2HR区域进行突变检测,并通过PubMed及中国生物医学光盘CBM,对已报道的关于HR基因U2HR区域的突变报道进行总结,以其发现其基因型与表型的关系。结果(1)通过全基因组外显子测序,获得3个全基因组外显子测序样本的SNPs、 indels的数据集合；(2)考虑到引起疾病的变异是罕见的,在家系内对照和公共数据库中(如dbSNP135)中不存在,通过逐步滤过后,满足2个患者共有、dbSNP135和家系内对照不存在的条件的突变点有91个。其中EPS8L3基因上一个错义突变即c.22GA位于定位区域lp21.1-1q21.3,该突变导致了丙氨酸变成苏氨酸,且ANNOVAR和PhastCons软件预测为有害突变,EPS8L3基因作为候选基因作进一步研究；(3)通过Sanger测序对家系内成员(包括全基因组外显子测序的2个患者和1个对照)的该位点进行测序验证,证实8个患者均存在该错义突变,3个对照均无该突变；(4)通过筛查676例正常对照和781例有其他疾病的病例的全基因组外显子测序数据,在676例正常对照和781例其他疾病的病例中均未发现该位点突变,从而说明该突变是一个致病性突变而非多态性变化；(5)通过对家系2的先证者和4例散发病例的EPS8L3基因编码区进行测序,未发现EPS8L3基因潜在性的致病性突变；(6)在家系2的先证者和其母亲中检测到HR基因U2HR区域的1个错义突变：c.73CG(p.pro25ala),而先证者的父亲(正常人)中不存在该突变,该突变曾在荷兰、英国、意大利种族的MUHH患者中报道过；(7)目前已报道HR基因U2HR区域的突变有16种,包括5个起始密码子突变,2个无义突变,7个错义突变,2个终止密码子突变,总结了MUHH临床特点和突变类型,但尚未发现基因型与表型之间的关系。结论(1)结合全基因组连锁分析的定位研究,通过全基因组外显子测序发现了一个中国汉族Maire Unna遗传性少毛症家系的致病基因—EPS8L3基因。证实了全基因组外显子测序结合全基因组定位信息鉴定单基因病致病基因的有效性。(2)通过直接测序的方法在另外1个中国MUHH家系中检测出HR基因U2HR区域错义突变c.73CG(p.pro25ala),丰富了U2HR遗传突变谱,为将来的遗传咨询、产前诊断及基因治疗打下理论基础；同时表明MUHH具有遗传异质性,不同家系致病基因可能不同。
[Abstract]:Background Marie Unna hereditary hairs (Marie Unna hereditary hypotrichosis, MUHH, MIM146550/612841) is a rare genetic hair disease of autosomal dominant hair disease.Marie Unna in 1925, which was first reported in 1 German patients. The disease has no sex difference between men and women, but the condition of male patients is more serious than that of women. The clinical features are: the hair is normal or scarce or lacking at birth, and then begins to grow slowly, but the growing hair is rough, irregular and twisted in a wire like appearance, and the hair is diffuse and aggravated in puberty. In addition, the eyelashes are absent, the eyebrows, the axillary hair, the pubic hair and the male beard are scarce, but do not accompany Some other ectoderm structures were abnormal, and the physical examination and intelligence were normal. Histopathology showed a small amount of inflammatory cells infiltrating or fibrosis around the hair follicles, but the number of mature follicles decreased significantly and the hair follicles narrowed; the optical microscopy showed that the hair was flat, rough and irregularly twisted; scanning electron microscopy showed a ridge, longitudinal crest, longitudinal crest. Longitudinal crest, longitudinal crest, longitudinal crest, longitudinal crest, longitudinal crest, longitudinal crest, longitudinal crest, longitudinal crest, longitudinal crest, longitudinal crest, longitudinal crest, longitudinal crest, longitudinal crest, longitudinal crest, longitudinal crest, longitudinal crest, longitudinal crest, longitudinal crest, longitudinal crest, longitudinal crest, longitudinal crest, longitudinal ridge, longitudinal From 1999 to 2004, several groups of MUHH families from different races from Holland, Britain, Germany, Belgium, the United States, and China, respectively, conducted a linkage and haplotype analysis of the MUHH's pathogenic gene loci in many studies in the early 8p21.. The group has analyzed the mutation analysis of the coding region and splice site of Human hairless gene (HR) in the locational region, but none of the pathogenic mutations of the HR gene were detected in.2009 years. Professor Zhang found 13 different mutations in the U2HR region of HR gene in 19 MUHH families from different races and established a MUHH genetic mutation. Subsequently, a number of research teams also found mutations in the U2HR region of the HR gene in China, Turkey, German families or sporadic cases.
In 2004, Yan and other MUHH families of 1 4 generations were analyzed by microsatellite markers. It was found that the pathogenic gene of the family was not linked to the 8p region, suggesting that MUHH had genetic heterogeneity. In 2005, Yang and so on carried out complete genome scanning to confirm that the pathogenic gene of the family was not linked to the 8p region, and the pathogenic gene was located in 1p21.. The 17.5cM region between 1-1q21.3 and D1S2345. With the rapid development of a new generation sequencing technology, the whole genome exons sequencing strategy has been successfully applied to the study of the pathogenic genes of monogenetic disease, the screening of cancer and polygenic disease susceptible genes and the diagnosis of clinically difficult cases. The whole genome exons sequencing has been rapidly developed. Development.
Objective to search for the whole genome location information in the early stage, the whole genome exon sequencing technology is used to search the pathogeny genes of MUHH, so as to lay the foundation for the future gene diagnosis and gene therapy.
Methods (1) 2 patients with typical clinical phenotypes and 1 family controls were selected from family 1 to carry out complete genome exon sequencing; (2) by gradually filtering the common mutations in the dbSNP database and the family control, and using the pre location information to obtain the candidate gene sets; (3) Sanger sequencing was used in the patients and the controls in the family. The mutation sites of the candidate gene set were sequenced to verify whether the mutant points were separated from the disease and obtained the candidate genes. (4) the sequences of the exons and exons and introns of the candidate genes were sequenced in an independent family line 2 and 4 other sporadic cases, in order to find the candidate genes in other cases. (5) amplification of the U2HR region of the HR gene by PCR reaction, the mutation detection of the U2HR region of the HR gene by direct sequencing of the candidate gene mutation screening, and the report on the reported mutation of the U2HR region of the HR gene by PubMed and the Chinese Biomedical disc CBM, in order to find its genotype and its genotype. The relationship of phenotypes.
Results (1) the total genomic exons were sequenced, and 3 total genomic exon sequencing samples were obtained for SNPs, indels data collection; (2) it was rare to take into account the mutation of the disease, and did not exist in the family control and the public database (such as dbSNP135). After gradual filtration, 2 patients were met, dbSNP135 and family members were used. There are 91 mutation points for the conditions that do not exist, of which a missense mutation in the EPS8L3 gene is that c.22GA is located in the location area lp21.1-1q21.3, which causes alanine to become threonine, and ANNOVAR and PhastCons software are predicted to be harmful mutations, and the EPS8L3 gene is used as a candidate gene for further study; (3) Sanger sequencing is used in the family by Sanger sequencing. The loci were sequenced by members (including 2 patients with total exon sequencing and 1 controls), which confirmed that 8 patients had this missense mutation and 3 controls had no mutation; (4) the exon sequencing data of the whole base group of 676 normal controls and 781 cases of other diseases were screened in 676 normal controls and 781. The mutation was not found in the cases of other diseases, indicating that the mutation was a pathogenic mutation, not a polymorphic change. (5) the EPS8L3 gene coding region of the family 2 and 4 sporadic cases was sequenced, and the potential mutation of the EPS8L3 gene was not found; (6) the precursor of the family 2 and its mother. 1 missense mutations in the U2HR region of the HR gene were detected, c.73CG (p.pro25ala), and the mutation was not found in the father's father (normal person), which had been reported in the MUHH patients in Holland, the United Kingdom, and the Italy race; (7) there were 16 mutations in the U2HR region of the HR gene, including 5 initial codon mutations and 2 non sense mutations. 7 missense mutations and 2 stop codon mutations were summarized. The clinical characteristics and mutation types of MUHH were summarized, but the relationship between genotype and phenotype was not found.
Conclusion (1) combined with the localization of whole genome linkage analysis, a whole genome exon sequencing was used to detect the pathogenicity gene EPS8L3 gene of a Chinese Han Maire Unna hereditary less hairy family, which confirmed the effectiveness of the whole genome exon sequencing combined with the whole genome location information identification gene disease gene. (2) Direct sequencing has detected the missense mutation c.73CG (p.pro25ala) of the U2HR region of the HR gene in 1 other Chinese MUHH families, enriching the genetic mutation spectrum of U2HR, providing a theoretical basis for future genetic counseling, prenatal diagnosis and gene therapy, and indicating that MUHH has genetic heterogeneity, and the pathogenicity genes of different families may be different.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R758.71

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