本文選題：Marie + Unna遺傳性少毛癥　； 參考：《安徽醫(yī)科大學(xué)》2012年博士論文

【摘要】：研究背景Marie Unna遺傳性少毛癥(Marie Unna hereditary hypotrichosis,MUHH,MIM146550/612841)是一種罕見的呈常染色體顯性遺傳模式的遺傳性毛發(fā)疾病。Marie Unna于1925年首次報道1例德國患者。該病發(fā)病無男女性別差異,但男性患者病情比女性嚴(yán)重。其臨床特點為：患者出生時頭發(fā)正常或稀少或缺乏,隨后開始緩慢生長,但已生長的頭發(fā)粗糙、不規(guī)則扭曲呈金屬絲樣外觀,至青春期頭發(fā)彌漫性脫落且逐漸加重,嚴(yán)重者可呈全禿。此外,睫毛可缺如,眉毛、腋毛、陰毛和男性胡須稀少,但不伴有其他外胚層結(jié)構(gòu)異常,且體格檢查和智力均正常。組織病理學(xué)顯示毛囊周圍有少量炎性細(xì)胞浸潤或纖維化,但成熟的毛囊數(shù)量顯著減少、毛囊縮�。还鈱W(xué)顯微鏡鏡檢顯示毛發(fā)呈扁平、粗糙和不規(guī)則扭曲狀；掃描電鏡顯示可有縱嵴、縱溝、縱裂、不規(guī)則橫斷面、毛小皮廣泛的剝脫和毛鞘異常。自1999年至2004年,多個研究小組分別對來自荷蘭、英國、德國、比利時、美國和中國等不同種族的MUHH家系進(jìn)行了連鎖和單倍型分析,均將MUHH的致病基因位點定位于8p21。前期許多研究小組先后針對定位區(qū)域內(nèi)人類無毛基因(Human hairless gene,HR)的編碼區(qū)和剪接位點進(jìn)行突變分析,但均未檢測到HR基因的致病性突變。2009年,張學(xué)教授在來自不同種族的19個MUHH家系中發(fā)現(xiàn)了HR基因U2HR區(qū)域的13種不同突變,建立了MUHH遺傳突變譜。隨后多個研究小組也在中國、土耳其、德國家系或散發(fā)病例中發(fā)現(xiàn)了HR基因U2HR區(qū)域的致病突變。 2004年Yan等通過微衛(wèi)星標(biāo)記研究對1個4代的MUHH家系進(jìn)行分析,發(fā)現(xiàn)該家系的致病基因與8p區(qū)域不連鎖,提示MUHH具有遺傳異質(zhì)性；2005年Yang等對該家系進(jìn)行全基因組掃描研究,證實該家系致病基因與8p區(qū)域不連鎖,并將致病基因定位于1p21.1-1q21.3上D1S248和D1S2345之間的17.5cM區(qū)域。隨著新一代測序技術(shù)的迅猛發(fā)展,全基因組外顯子測序策略已成功應(yīng)用于單基因病的致病基因研究,癌癥和多基因病易感基因的篩查以及臨床上疑難病例的診斷,全基因組外顯子測序得到了迅猛的發(fā)展。 目的結(jié)合前期的全基因組定位信息,運用全基因組外顯子測序技術(shù)搜尋MUHH的致病基因,為將來基因診斷、基因治療奠定基礎(chǔ)。 方法(1)從家系1中挑選出2個臨床表型典型的患者和1個家系內(nèi)對照,進(jìn)行全基因組外顯子測序；(2)通過逐步濾過dbSNP數(shù)據(jù)庫和家系內(nèi)對照中的常見突變,并且結(jié)合前期定位信息獲得候選基因集；(3)利用Sanger測序,在家系內(nèi)患者和對照中對候選基因集的突變位點進(jìn)行測序驗證,分析突變點是否與疾病共分離,獲得候選基因；(4)在獨立的家系2和其他4個散發(fā)病例中,對候選基因的編碼區(qū)外顯子和外顯子與內(nèi)含子交界區(qū)的序列進(jìn)行測序,以期在其他病例中發(fā)現(xiàn)候選基因的突變；(5)采用PCR反應(yīng)擴增HR基因U2HR區(qū)域,用直接測序方法對候選基因突變篩查陰性的病例對HR基因U2HR區(qū)域進(jìn)行突變檢測,并通過PubMed及中國生物醫(yī)學(xué)光盤CBM,對已報道的關(guān)于HR基因U2HR區(qū)域的突變報道進(jìn)行總結(jié),以其發(fā)現(xiàn)其基因型與表型的關(guān)系。 結(jié)果(1)通過全基因組外顯子測序,獲得3個全基因組外顯子測序樣本的SNPs、 indels的數(shù)據(jù)集合；(2)考慮到引起疾病的變異是罕見的,在家系內(nèi)對照和公共數(shù)據(jù)庫中(如dbSNP135)中不存在,通過逐步濾過后,滿足2個患者共有、dbSNP135和家系內(nèi)對照不存在的條件的突變點有91個。其中EPS8L3基因上一個錯義突變即c.22GA位于定位區(qū)域lp21.1-1q21.3,該突變導(dǎo)致了丙氨酸變成蘇氨酸,且ANNOVAR和PhastCons軟件預(yù)測為有害突變,EPS8L3基因作為候選基因作進(jìn)一步研究；(3)通過Sanger測序?qū)蚁祪?nèi)成員(包括全基因組外顯子測序的2個患者和1個對照)的該位點進(jìn)行測序驗證,證實8個患者均存在該錯義突變,3個對照均無該突變；(4)通過篩查676例正常對照和781例有其他疾病的病例的全基因組外顯子測序數(shù)據(jù),在676例正常對照和781例其他疾病的病例中均未發(fā)現(xiàn)該位點突變,從而說明該突變是一個致病性突變而非多態(tài)性變化；(5)通過對家系2的先證者和4例散發(fā)病例的EPS8L3基因編碼區(qū)進(jìn)行測序,未發(fā)現(xiàn)EPS8L3基因潛在性的致病性突變；(6)在家系2的先證者和其母親中檢測到HR基因U2HR區(qū)域的1個錯義突變：c.73CG(p.pro25ala),而先證者的父親(正常人)中不存在該突變,該突變曾在荷蘭、英國、意大利種族的MUHH患者中報道過；(7)目前已報道HR基因U2HR區(qū)域的突變有16種,包括5個起始密碼子突變,2個無義突變,7個錯義突變,2個終止密碼子突變,總結(jié)了MUHH臨床特點和突變類型,但尚未發(fā)現(xiàn)基因型與表型之間的關(guān)系。 結(jié)論(1)結(jié)合全基因組連鎖分析的定位研究,通過全基因組外顯子測序發(fā)現(xiàn)了一個中國漢族Maire Unna遺傳性少毛癥家系的致病基因—EPS8L3基因。證實了全基因組外顯子測序結(jié)合全基因組定位信息鑒定單基因病致病基因的有效性。(2)通過直接測序的方法在另外1個中國MUHH家系中檢測出HR基因U2HR區(qū)域錯義突變c.73CG(p.pro25ala),豐富了U2HR遺傳突變譜,為將來的遺傳咨詢、產(chǎn)前診斷及基因治療打下理論基礎(chǔ)；同時表明MUHH具有遺傳異質(zhì)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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R758.71

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1 張鑫;全基因組外顯子測序發(fā)現(xiàn)Marie Unna遺傳性少毛癥致病基因EPS8L3[D];安徽醫(yī)科大學(xué);2012年

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