本文選題：遺傳性稀毛癥 + 單純性遺傳性稀毛癥　； 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：第一部分先天性全身毛發(fā)增多癥中6q22微重復(fù)的鑒定先天性全身毛發(fā)增多癥(congenital generalized hypertrichosis, CGH)以非雄激素依賴的全身性毛發(fā)過度生長為主要特征,具有表型異質(zhì)性及遺傳異質(zhì)性,臨床上分型較多,不同亞型之間表型有交疊。已發(fā)現(xiàn)的致病改變包括4號、8號、17號、X染色體重排,以及同樣位于17號染色體上的ABCA5基因點突變。本研究收集一例CGH家系。先證者多毛表型接近Ambras型,伴先天性視力不良及脊柱側(cè)彎,牙齒自30歲始至完全脫落,其雙親及同胞均表現(xiàn)正常。經(jīng)知情同意,采集先證者及其母親、所有四名同胞外周血或口腔上皮細胞,提取基因組DNA。微陣列比較基因組雜交(array comparative genomic hybridization, aCGH)分析發(fā)現(xiàn)先證者6q22存在兩個微重復(fù)(依次稱為Dup1與Dup2),經(jīng)實時熒光定量聚合酶鏈式反應(yīng)(quantitive real-time polymerase chain reaction, qPCR)驗證在家系中與疾病表型共分離。隨后進行一系列qPCR縮小兩個微重復(fù)邊界范圍,并嘗試多種gap-PCR組合方式最終克隆得到斷裂點。將gap-PCR產(chǎn)物進行Sanger測序,結(jié)果顯示兩個微重復(fù)邊界彼此相連。結(jié)合雙色熒光原位雜交(fluorescence in situ hybridization, FISH)結(jié)果推斷重排后染色體結(jié)構(gòu)。重復(fù)區(qū)內(nèi)STR提示先證者微重復(fù)為父源新生重復(fù)。此兩個微重復(fù)中共包含6個基因,其中GJA1基因為眼齒指發(fā)育不良(ODDD)的致病基因,其拷貝數(shù)改變可能是患者眼睛與牙齒表型的原因；RSPO3與RNF146基因的功能與調(diào)節(jié)毛囊周期的Wnt通路相關(guān),其拷貝數(shù)改變可能單獨或共同導(dǎo)致患者多毛的表型。綜上所述,我們首次在CGH患者中發(fā)現(xiàn)6號染色體上的染色體重排,確定了重排后的染色體結(jié)構(gòu),并推測兩個微重復(fù)為父源新生重復(fù)。第二部分Bazex-Dupre-Christol綜合征中Xq26.1微重復(fù)的鑒定Bazex-Dupre-Christol綜合征(Bazex-Dupre-Christol syndrome, BDCS)是一種極罕見的遺傳病,其主要臨床表現(xiàn)為一個先天性毛發(fā)稀少、毛囊性皮膚萎縮與早發(fā)性基底細胞癌(basal cell carcinomas, BCCs)的三聯(lián)征。該病表型輕重不一,且易與其他疾病混淆,因此需進行嚴格的鑒別診斷。BDCS呈現(xiàn)明顯的X連鎖顯性遺傳模式,目前定位至Xq25-q27.1一個11.4Mb的范圍內(nèi),但該區(qū)域內(nèi)12個在毛囊中對細胞增殖分化起重要作用的候選基因中并未檢測到突變。本課題組開展BDCS相關(guān)工作初期收集到兩個歐洲BDCS家系,通過全基因組拷貝數(shù)變異(copy number variation, CNV)分析,在Xq26.1上發(fā)現(xiàn)一段兩家患者共享的重復(fù)區(qū),并經(jīng)qPCR驗證在兩個家系中與疾病表型共分離。隨后,通過qPCR gap-PCR及Sanger測序確定兩家的微重復(fù)范圍,其中家系2(F2)精確至堿基水平,并推測了各自的重排機制。為了進一步探究該重復(fù)區(qū)域的致病性,本研究收集到另6個歐洲BDCS家系。本研究前期發(fā)現(xiàn)的微重復(fù)經(jīng)qPCR在其余6家患者中均得到驗證,并結(jié)合gap-PCR及SNP位點擴增測序的方法作為補充,確認各家微重復(fù)在家系內(nèi)所有成員中均與疾病表型共分離。同時,在215個歐洲無關(guān)對照人群,共計354條X染色體中未發(fā)現(xiàn)類似的微重復(fù)。隨后,我們確定了新收集6家的微重復(fù)范圍,其中F4、F7、F9精確到堿基水平,并推測了各自的重排機制。此外,我們還通過單體型分析,證明8家微重復(fù)為獨立發(fā)生。最后,我們構(gòu)建了Krt15組織特異性過表達的的轉(zhuǎn)基因小鼠模型,以期進行相關(guān)的表型觀察與功能研究,但并未在預(yù)期的位置檢測到Igsfl的過表達。綜上,我們提供了充分的證據(jù),從遺傳學(xué)層面說明Xq26.1上的微重復(fù)很可能是BDCS的致病突變,但其具體致病機制有待進一步研究。第三部分常染色體顯性遺傳性稀毛癥致病突變鑒定遺傳性稀毛癥(hereditary hypotrichosis, HH)是一組臨床上較少見的疾病,既可單獨發(fā)生,也可合并其他表型發(fā)生�；颊咄ǔ３錾鷷r有頭發(fā),幾個月后開始脫落,具有很強的臨床表型異質(zhì)性,可為全身受累(generalized)或頭皮受累(scalp-limited),伴有或不伴有毛干異常,以及毛囊發(fā)育異常等。同時,該病也具很強的遺傳異質(zhì)性,主要為常染色體顯性(autosomal dominant, AD)與常染色體隱性(autosomal recessive, AR)。致病基因已知的ADHH按表型可分為三大類,第一類為單純性遺傳性稀毛癥(hereditary hypotrichosis simplex, HSS),其中全身受累型的致病基因APCDD1、 RPL21和SNRPE,頭皮受累型為CDSN;第二類為Marie Unna型遺傳性稀毛癥(Marie Unna hereditary hypotrichosis, MUHH),其中1型的致病基因為U2HR,2型為EPS8L3;第三類位羊毛狀發(fā)(woolly hair, WH),致病基因為KRT74及KRT71。本研究收集三個ADHH家系(F1、F2、F3),分別對其進行基因診斷與相關(guān)遺傳學(xué)研究。在F1中,我們通過等位基因共享分析排除APCDD1、RPL21、CDSN、SNRPE,以及兩個KRT基因簇中的基因作為該家系的致病基因,最終在U2HR基因中檢測到c.82GC (D28H)雜合突變作為致病突變,可為該家系后續(xù)的遺傳咨詢及產(chǎn)前基因診斷提供基礎(chǔ)。在F2中,我們首先確認了患病的父親攜帶U2HR基因c.2TC (MIT)雜合突變；隨后胎兒經(jīng)Sanger測序并排除母源污染,確認不攜帶此突變,不為患者。在F3中,我們先對先證者進行APCDD1和RPL21基因突變篩查,檢測到RPL21基因c.95GA (R32Q)雜合突變,并在全部家系成員中驗證,確認為致病突變。隨后,通過與報道了相同突變的F4和F5共同進行單體型分析,確認三家突變?yōu)楠毩l(fā)生,從而進一步確認RPL21為ADHHS的致病基因,同時提示該突變更有可能是gain-of-function突變。
[Abstract]:In the first part of the congenital systemic hairs, 6q22 microsatellosis (congenital generalized hypertrichosis, CGH) is characterized by non androgen dependent systemic hair overgrowth, characterized by phenotypic and genetic heterogeneity, more clinically differentiated and phenotypic among different subtypes. 4, 8, 17, X chromosome rearrangement, and ABCA5 gene point mutation on chromosome 17 were found. A CGH family was collected in this study. This study collected a case of CGH family. The hairy phenotype was close to the Ambras type, with congenital vision and scoliosis, and the tooth from 30 years old to complete shedding. Both parents and siblings were shown. Normal. After informed consent, all four sib peripheral blood or oral epithelial cells were collected and their mother, all four sib peripheral blood or oral epithelial cells, and the genomic DNA. microarray comparative genomic hybridization (array comparative genomic hybridization, aCGH) analysis found that the precursor 6q22 had two microduplication (called Dup1 and Dup2 in turn) by real time fluorescent quantitative polymerase. Quantitive real-time polymerase chain reaction (qPCR) was verified to be co isolated from the disease phenotype in the family. Then a series of qPCR narrowing two micro repetitive boundary ranges, and a variety of gap-PCR combination methods were tried to clone the fracture point. The gap-PCR product was sequenced by Sanger, and the results showed two micro repeat boundaries. In conjunction with the results of fluorescence in situ hybridization (FISH), the structure of the chromosomes after rearrangement was deduced. The STR in the repeat area indicated that the precursor was duplicated as the parent of the parent. The two microsatellites included 6 genes, of which the GJA1 gene was the pathogenetic gene of the eye tooth dysplasia (ODDD) and its copy number. Changes may be the cause of the patient's eye and tooth phenotype; the function of the RSPO3 and RNF146 genes is related to the Wnt pathway that regulates the hair follicle cycle. The change in the number of copies may lead to the patient's hairy phenotype alone or together. In summary, we first found chromosome rearrangements on chromosome 6 in CGH patients and identified the staining after rearrangement. Body structure, and speculate that two microsatellite duplicates. The second part of the Bazex-Dupre-Christol syndrome in the second part of the Bazex-Dupre-Christol syndrome (Bazex-Dupre-Christol syndrome, BDCS) is a rare genetic disease, its main clinical manifestation is a congenital rare hair, hair follicle atrophy and atrophy. The triad of basal cell carcinomas (BCCs) is a triad of early onset basal cell carcinoma (BCCs). The disease is different from other diseases and is easy to be confused with other diseases. Therefore, a strict differential diagnosis of.BDCS is required to present a distinct X linkage dominant genetic model, which is currently located within the range of Xq25-q27.1 a 11.4Mb, but 12 in the region are proliferating in the follicle. Two European BDCS families were collected at the initial stage of BDCS related work, and the whole genome copy number variation (copy number variation, CNV) was analyzed in the group. A segment of duplicated areas shared by two patients was found on Xq26.1, and the disease was verified by qPCR in two families and diseases. Then, the qPCR gap-PCR and Sanger sequencing were used to determine the micro repetition range of the two groups, including the family 2 (F2) to the base level and the respective rearrangement mechanism. In order to further explore the pathogenicity of the repeat area, this study collected 6 European BDCS families. The remaining 6 patients were tested and combined with gap-PCR and SNP amplification sequencing methods to confirm that all the families in each family were separated from the disease phenotype in all members of the family. At the same time, no similar micro repetition was found in 354 X chromosomes in 215 European unrelated populations. Then, we identified a new collection of 6 families. The micro repetition range, in which F4, F7, F9 are accurate to the base level, and speculates the respective rearrangement mechanism. In addition, we have also demonstrated that 8 micro duplication is independent by haplotype analysis. Finally, we constructed a transgenic mouse model of Krt15 specific overexpression in order to conduct related phenotypic observation and functional research, but not The overexpression of Igsfl was detected in the expected position. To sum up, we provided sufficient evidence that the microsatellite on Xq26.1 might be the pathogenetic mutation of BDCS from the genetic level, but its specific pathogenesis remains to be further studied. Third the hereditary dilute hairs of the autosomal dominant hereditary dilute hairs are identified (Heredita Ry hypotrichosis, HH) is a rare group of diseases that can occur either alone or in combination with other phenotypes. The patients usually have hair when they are born and begin to fall off a few months later, with a strong clinical phenotypic heterogeneity, for generalized or scalp involvement (scalp-limited) with or without abnormality of hair. At the same time, the disease also has very strong genetic heterogeneity, mainly autosomal dominant (autosomal dominant, AD) and autosomal recessive (autosomal recessive, AR). The known ADHH can be divided into three major categories according to the phenotype, the first category is pure hereditary dilute hair disease (hereditary hypotrichosis simplex, HSS). The pathogenic genes APCDD1, RPL21, SNRPE, and the scalp affected type were CDSN; the second type was Marie Unna hereditary dilute hair disease (Marie Unna hereditary hypotrichosis, MUHH), of which the pathogenic genes of type 1 were U2HR, type 2, and third types of hair like hair. Three ADHH families (F1, F2, F3) were set up for genetic diagnosis and related genetics. In F1, we eliminated APCDD1, RPL21, CDSN, SNRPE, and two KRT gene clusters by allelic sharing analysis, and finally detected the heterozygous mutation as a pathogenic process in the U2HR gene. In F2, we first confirmed that the sick father carried the U2HR gene c.2TC (MIT) heterozygous mutation; then the fetus was sequenced by Sanger and excluded from mother source pollution, confirming that the mutation was not carried, not the patient. In F3, we first performed APCDD1 and RPL21 to the precursor to the precursor. Gene mutation screening, detection of the RPL21 gene c.95GA (R32Q) heterozygous mutation, and confirmed in all family members, confirmed as the pathogenic mutation. Subsequently, by using the same mutation of F4 and F5 to carry out the haplotype analysis, we confirmed that three mutations were independent, and then confirmed the RPL21 as the pathogenicity gene of ADHHS, and suggested the process. The change may be a gain-of-function mutation.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R758.71

【共引文獻】

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