遗传性毛发疾病的致病突变研究

发布时间：2018-05-18 11:32

本文选题：遗传性稀毛症 + 单纯性遗传性稀毛症　；参考：《北京协和医学院》2015年博士论文

【摘要】：第一部分先天性全身毛发增多症中6q22微重复的鉴定先天性全身毛发增多症(congenital generalized hypertrichosis, CGH)以非雄激素依赖的全身性毛发过度生长为主要特征,具有表型异质性及遗传异质性,临床上分型较多,不同亚型之间表型有交叠。已发现的致病改变包括4号、8号、17号、X染色体重排,以及同样位于17号染色体上的ABCA5基因点突变。本研究收集一例CGH家系。先证者多毛表型接近Ambras型,伴先天性视力不良及脊柱侧弯,牙齿自30岁始至完全脱落,其双亲及同胞均表现正常。经知情同意,采集先证者及其母亲、所有四名同胞外周血或口腔上皮细胞,提取基因组DNA。微阵列比较基因组杂交(array comparative genomic hybridization, aCGH)分析发现先证者6q22存在两个微重复(依次称为Dup1与Dup2),经实时荧光定量聚合酶链式反应(quantitive real-time polymerase chain reaction, qPCR)验证在家系中与疾病表型共分离。随后进行一系列qPCR缩小两个微重复边界范围,并尝试多种gap-PCR组合方式最终克隆得到断裂点。将gap-PCR产物进行Sanger测序,结果显示两个微重复边界彼此相连。结合双色荧光原位杂交(fluorescence in situ hybridization, FISH)结果推断重排后染色体结构。重复区内STR提示先证者微重复为父源新生重复。此两个微重复中共包含6个基因,其中GJA1基因为眼齿指发育不良(ODDD)的致病基因,其拷贝数改变可能是患者眼睛与牙齿表型的原因；RSPO3与RNF146基因的功能与调节毛囊周期的Wnt通路相关,其拷贝数改变可能单独或共同导致患者多毛的表型。综上所述,我们首次在CGH患者中发现6号染色体上的染色体重排,确定了重排后的染色体结构,并推测两个微重复为父源新生重复。第二部分Bazex-Dupre-Christol综合征中Xq26.1微重复的鉴定Bazex-Dupre-Christol综合征(Bazex-Dupre-Christol syndrome, BDCS)是一种极罕见的遗传病,其主要临床表现为一个先天性毛发稀少、毛囊性皮肤萎缩与早发性基底细胞癌(basal cell carcinomas, BCCs)的三联征。该病表型轻重不一,且易与其他疾病混淆,因此需进行严格的鉴别诊断。BDCS呈现明显的X连锁显性遗传模式,目前定位至Xq25-q27.1一个11.4Mb的范围内,但该区域内12个在毛囊中对细胞增殖分化起重要作用的候选基因中并未检测到突变。本课题组开展BDCS相关工作初期收集到两个欧洲BDCS家系,通过全基因组拷贝数变异(copy number variation, CNV)分析,在Xq26.1上发现一段两家患者共享的重复区,并经qPCR验证在两个家系中与疾病表型共分离。随后,通过qPCR gap-PCR及Sanger测序确定两家的微重复范围,其中家系2(F2)精确至碱基水平,并推测了各自的重排机制。为了进一步探究该重复区域的致病性,本研究收集到另6个欧洲BDCS家系。本研究前期发现的微重复经qPCR在其余6家患者中均得到验证,并结合gap-PCR及SNP位点扩增测序的方法作为补充,确认各家微重复在家系内所有成员中均与疾病表型共分离。同时,在215个欧洲无关对照人群,共计354条X染色体中未发现类似的微重复。随后,我们确定了新收集6家的微重复范围,其中F4、F7、F9精确到碱基水平,并推测了各自的重排机制。此外,我们还通过单体型分析,证明8家微重复为独立发生。最后,我们构建了Krt15组织特异性过表达的的转基因小鼠模型,以期进行相关的表型观察与功能研究,但并未在预期的位置检测到Igsfl的过表达。综上,我们提供了充分的证据,从遗传学层面说明Xq26.1上的微重复很可能是BDCS的致病突变,但其具体致病机制有待进一步研究。第三部分常染色体显性遗传性稀毛症致病突变鉴定遗传性稀毛症(hereditary hypotrichosis, HH)是一组临床上较少见的疾病,既可单独发生,也可合并其他表型发生。患者通常出生时有头发,几个月后开始脱落,具有很强的临床表型异质性,可为全身受累(generalized)或头皮受累(scalp-limited),伴有或不伴有毛干异常,以及毛囊发育异常等。同时,该病也具很强的遗传异质性,主要为常染色体显性(autosomal dominant, AD)与常染色体隐性(autosomal recessive, AR)。致病基因已知的ADHH按表型可分为三大类,第一类为单纯性遗传性稀毛症(hereditary hypotrichosis simplex, HSS),其中全身受累型的致病基因APCDD1、 RPL21和SNRPE,头皮受累型为CDSN;第二类为Marie Unna型遗传性稀毛症(Marie Unna hereditary hypotrichosis, MUHH),其中1型的致病基因为U2HR,2型为EPS8L3;第三类位羊毛状发(woolly hair, WH),致病基因为KRT74及KRT71。本研究收集三个ADHH家系(F1、F2、F3),分别对其进行基因诊断与相关遗传学研究。在F1中,我们通过等位基因共享分析排除APCDD1、RPL21、CDSN、SNRPE,以及两个KRT基因簇中的基因作为该家系的致病基因,最终在U2HR基因中检测到c.82GC (D28H)杂合突变作为致病突变,可为该家系后续的遗传咨询及产前基因诊断提供基础。在F2中,我们首先确认了患病的父亲携带U2HR基因c.2TC (MIT)杂合突变；随后胎儿经Sanger测序并排除母源污染,确认不携带此突变,不为患者。在F3中,我们先对先证者进行APCDD1和RPL21基因突变筛查,检测到RPL21基因c.95GA (R32Q)杂合突变,并在全部家系成员中验证,确认为致病突变。随后,通过与报道了相同突变的F4和F5共同进行单体型分析,确认三家突变为独立发生,从而进一步确认RPL21为ADHHS的致病基因,同时提示该突变更有可能是gain-of-function突变。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R758.71

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