单基因肥胖等单基因病的分子遗传学研究
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摘要
第一部分单基因肥胖的分子遗传学研究
在过去的十几年中,肥胖人口的不断增长已经成为世界范围内公共卫生事业的主要挑战。作为一种复杂疾病,肥胖的产生除了与环境有关外,还与遗传因素关系密切。据估计遗传因素对体重的影响可能达40-70%。由此可见,对肥胖产生分子机制的研究,具有十分重要的意义。鉴于肥胖成因的复杂性,对一些早发、极端、以单基因方式遗传肥胖致病基因的研究成为了人们研究肥胖分子机制的突破口。目前人们已经鉴定出20多种单基因肥胖的致病基因,但只能解释不到5%的极端肥胖,对剩余肥胖分子机制的研究将是人们面临的一个巨大挑战。
人类生长是一个受到多种因素影响的复杂过程,其中遗传因素在身高决定中占主要作用。一直以来,人们对于身材矮小的分子机制的研究较多,而对高大身材,特别是婴幼儿及儿童期身材高大或过度生长的分子机制研究相对较少。儿童期身材高大或者过度生长可能由多种原因导致,其中最常见的原因是肥胖和超重。因此,针对导致儿童期身材高大或过度生长、肥胖或超重的致病基因鉴定对于人们理解儿童生长和机体组成的机制具有十分重要的意义。
本研究中我们收集了1个常染色体显性肥胖伴过度生长家系和4个早发肥胖核心家系。在获得知情同意后,对这5个家系进行了分子遗传学研究。
常染色体显性肥胖伴过度生长家系患者除了肥胖外,两位处于青春期的患者身高要明显高于同龄人,因此我们该家系命名为肥胖伴过度生长家系。我们首先在先证者中筛查了的23个基因,包括已报道的导致非综合征型单基因肥胖的致病基因和一些在转基因小鼠中发现与体重调节相关的基因。在排除这些基因的改变之后,我们使用Affymetrix SNP6.0芯片对部分家系成员分两批进行拷贝数分析和全基因组连锁分析,在12号染色体长臂和短臂各发现一段LOD值为2.4的区域。其中短臂区域位于rs10491968至rs11063753,长度为1,975,647bp；长臂区域位于rs17105259至rs10160961之间,长度2,119,818bp。在得到连锁结果后,我们在连锁区域附近选择微卫星标记进行验证和精细定位,将短臂的连锁区域定位于D12S1050和D12S374之间,长度为2,299,067bp,长臂的连锁区域定位于D12S313与12q/TG之间,长度为1,391,386bp。同时分别在短臂标记D12S1685与长臂标记12q/TTAT,当0=0时,获得了最大LOD值2.68。
在进行全基因组连锁分析的同时,我们对该家系的先证者进行了全外显子组测序。结合全基因组连锁分析和外显子组测序结果,我们在位于12号染色体短臂的EFCAB4B基因中发现了1个在家系患者中共分离的错义突变(p.R466W)。随后我们在319例无关人群中对该变异进行了验证。经验证我们发现两例无关个体(一个超重,一个体重正常)存在与家系内患者相同的改变,因此暂不能确定我们所发现的变异是否是导致家系肥胖表型的分子基础。
针对4个早发肥胖家系,我们根据家系内先证者的临床表现,首先其中两个家系先证者进行MC4R、LEP和LEPR基因突变检测。在家系2先证者中发现LEP基因一个纯合的无义突变(c.163C>T,p.Q55X),该突变为国际首报,其父母为该突变的携带者。为了验证其父母所携带的突变是否来自同一祖先,我们选择LEP基因上下游各2Mb的微卫星标记对该家系进行连锁分析,从而确定了其父母所携带的突变来自于同一祖先。同时,我们在家系3先证者中发现MC4R基因一个纯合无义突变,其父母和子女为该突变的携带者。
对于另外两个家系的先证者,我们则对已报道的导致单基因肥胖的致病基因和一些在转基因小鼠中发现与体重调节相关的23个基因进行了突变检测。结果并没有在这些基因中发现致病改变,但却分别在两个家系先证者中发现LEPR和SIMl基因的几个与体重调节相关的SNP位点。
总之,在本研究中,我们使用全基因组连锁分析和外显子组测序等方法,在一个常染色体显性肥胖伴过度生长家系中发现了一个与患者共分离的罕见变异,尽管该变异的致病性还需要进一步的确定,本文为肥胖这种复杂疾病分子机制的研究提供了新的思路。在两个早发肥胖家系中分别发现了LEP、MC4R基因的纯合无义突变,这是导致这两个家系患者肥胖表型的分子基础。其中LEP基因突变为中国人群中首次报道。
第二部分三种皮肤遗传病的致病突变研究
皮肤病是指皮肤(包括皮肤附属器官毛发、指甲、汗腺等)受到内外因素的影响后,其形态、结构、功能发生的病理改变。皮肤病的主要病因包括感染、过敏和遗传因素影响。因遗传物质改变所引起的皮肤病称为遗传性皮肤病,有约300种单基因遗传性皮肤病。本文对三种单基因遗传性皮肤病(Bazex-Dupre-Christol综合征、X连锁无汗型外胚层发育不良和先天性无痛伴无汗)进行了致病基因突变研究,寻找或鉴定三种遗传性皮肤病的致病基因改变。
Bazex-Dupre-Christol综合征(Bazex-Dupre-Christol syndrome, BDCS)(MIM301845)是一种罕见遗传性皮肤病,1964年由Bazex等人首先报道。BDCS的患者通常表现出广泛的皮肤症状,最为常见的临床表现为少毛、毛囊性皮肤萎缩和早发的基底细胞癌的三联征。除此之外,在一些病人中还会出现粟丘疹、少汗、颅面部色素沉着,毛发上皮瘤,毛干异常等表型。BDCS呈X连锁显性遗传方式遗传,其致病区域在2011年被定位于Xq25-q27.1区域11.4Mb的范围内,但迄今尚未发现致病突变。本研究中我们收集了来自欧洲的两个BDCS家系,采用全外显子组测序技术及Affymetrix SNP6.0技术平台对家系样品进行分析,以求能够寻找到BDCS的致病改变。在得到外显子组测序结果后,我们首先对连锁区域内可疑改变进行Sanger测序验证,但并没有得到与疾病共分离的可疑改变。随后我们对原始bam文件进行分析,对连锁区域内外显子组测序reads覆盖率较低或未覆盖区域,以及在连锁区域中未出现在bam文件中的基因进行补测,结果并没有发现可疑致病改变。通过使用Affymetrix SNP6.0技术平台对家系成员进行拷贝数分析,发现两个家系的患者都在Xq26.1-q26.2之间存在拷贝数增加,但该重复与DGV数据库的已知CNV存在重叠,该拷贝数改变是否致病还需要进一步的分析验证。
外胚层发育不良(Ectodermal Dysplasia, ED)是一类以牙齿、毛发、指甲等外胚层来源组织发育不良为特征的遗传性综合征的总称。在这些综合征中,最为常见的是X连锁隐性少汗型外胚层发育不良(X-linked hypohidrotic ectodermal dysplasia, XLHED, MIM305100)。XLHED男性患者的临床表现为无汗或少汗毛发稀疏或细黄、少牙或牙齿发育异常的三联征。携带者女性由于X染色体的随机失活会有不同程度的临床表现,另有30%左右的携带者不会表现出任何临床症状。XLHED的致病基因为EDA,定位于Xq12.2-q13.1。到目前为止,HGMD上共记录了大约180种EDA基因的突变。其中98%以上的突变导致XLHED,另有约2%突变会引起另一种X连锁疾病：X连锁显性牙发育不良(X-linked dominant hypodontia, MIM300606)。本研究中我们收集了6个XLHED家系,对家系内患者EDA基因各外显子及外显子-内含子交界处进行PCR扩增后直接测序。我们在6个家系中鉴定出6个致病突变,5个为点突变,1个缺失突变。其中1个点突变(p.Q358L)和缺失突变为首次报道的新突变。采用PCR产物酶切的方法,对p.Q358L进行了验证。为了验证所发现的缺失突变,通过使用定量PCR以检测先证者和其家系成员的相对拷贝数。为了确定先证者的缺失范围,我们在EDA基因第7、8外显子之间以及EDA基因3'UTR末端和其下游2.5kb、5kb、7.5kb范围内分别设计引物进行PCR扩增,从而将缺失范围初步确定为第8外显子上游至EDA基因下游5kb。结合GAP-PCR扩增,最终将家系6先证者X染色体的缺失范围确定为10,297bp,同时发现此缺失突变还导致位于EDA基因下游的AWAT2最后4个外显子的缺失。另外,通过采用PCR直接测序和单体型分析的方法,我们对一个家系中的携带者进行了产前基因诊断。结果显示胎儿不携带致病突变,为正常男性。
先天性无痛伴无汗(Congenial insensitive to pain with anhidrosis, CIPA, MIM256800),是一种很罕见的常染色体隐性遗传病。CIPA患者典型的临床表现为对伤害性刺激无反应,无汗,屡现的高热,自我伤害行为及智力迟缓。CIPA的致病基因为神经营养性酪氨酸激酶受体I型(Human neurotrophic tyrpsine kinase receptor type1, NTRK1),定位于1q21-22,编码含790或796个氨基酸残基的受体酪氨酸激酶TRKA。对于我们收集到的两个CIPA家系,我们对家系内患者NTRK1基因各外显子及外显子-内含子交界处进行PCR扩增后直接测序。在家系1先证者NTRK1基因上发现三个错义突变(p.P397L, p.R692C, p.R771C),均为国际首报。其中P397L和R771C突变来自先证者的母亲,而由于先证者的父亲并不携带R692C突变,我们推测该突变可能是新生突变或者父亲是该突变的嵌合体。通过PCR产物酶切的方法,我们在70名正常对照个体中分别对这
个突变进行了验证。随后我们采用在线预测工具SIFT和Polyphen对三个错义突变的致病性进行了预测,预测结果显示R692C和R771C的致病性要强于P397L。另外,我们在家系2先证者NTRK1基因上发现了一个已知的纯合突变,c.287+2dupT,其父母均为此突变的携带者。我们的发现有助于对这两个家系进行遗传咨询和产前诊断,同时新的突变也扩展了NTRK1的突变谱。
总之,对于这三种遗传性皮肤病,我们采用了不同方法对其进行了致病突变研究。通过对BDCS连锁区域的致病突变筛查,我们基本排除了BDCS是由连锁区域内外显子组区域突变致病的可能。通过对连锁区域内拷贝数分析,‘发现了两个家系患者Xq26.1-q26.2之间存在拷贝数的增加,其致病性需要进一步确定。我们对XLHED和CIPA家系分别进行了致病基因突变检测,鉴定出了每个家系的致病突变,包括5个新突变。我们的检测结果有助于为患者家系提供遗传咨询和产前诊断,同时所发现的新的突变也扩展了EDA、NTRK1基因突变谱。
Part1:Molecular Genetic Studies in Chinese families with Monogenic Obesity
During the past decades, obesity has become the main challenge for public health worldwide. Known as a multifactor disease, obesity is influenced both by genetic and environmental factors. It has been estimated that an inherited component to body weight accounts for40-70%. Therefore, study on the molecular mechanism underlying obesity has great significance. Due to the complexity resulting in obesity, studies on the early-onset, extreme, andmonogenic type of obesity have been chosen as the breakthrough for investigating molecular mechanism of obesity. To date, there are more than20genes, in which mutations result in severe monogenic human obesity, have been identified. However, the monogenic causes of obesity identified so far only account for less than5%of severe obesity. Understanding the molecular mechanism of the other95%of obesity is still a great challenge.
Human growth is a multi-factorial and complex process, while the height is still predominantly under genetic control. Studies on molecular mechanisms resulting in tall stature and'overgrowth'have perhaps occurred in a less systematic manner, comparing with the study of short stature. Tall stature and overgrowth during childhood can be caused by many reasons, while obesity might be the commonest reason for apparent children tall stature and overgrowth. Therefore, identifying genetic causes of tall stature and overgrowth, weight gain can further our knowledge on the important mechanisms, by which childhood growth and body composition are regulated.
In the present study, we have collected one family with autosomal dominant obesity with overgrowth and four nuclear families with early onset obesity. With informed consent of all participating individuals, we performed genetic research in these five families, respectively.
We named the first family Obesity with Overgrowth by the fact that besides obesity, two pubertal patients seemed taller than their peers. Firstly, in the proband of this family, we have screened mutations of23genes associated with monogenic forms of obesity in humans or transgenic mice. After that, Affymetrix SNP6.0array was used to detect the copy number variation and performed whole genome linkage analysis in some of the members of the family. Two regions on the short and long arms of Chromosome12were found and they showed the maximal LOD score of2.4. The short arm region located between rs1049168and rs1106753, with a length of1,975,647bp, while the long arm region located between rs17105259and rs10160961, with a length of2,119,818bp. Based on the results of genome linkage analysis,8polymorphic microsatellite markers were selected,4from the linkage region on the short arm and4from the linkage region on the long arm of Chromosome12, and linkage and haplotype analysis was performed to verify the critical region shown by the results from the SNP array data in family1. Genotyping and haplotype analysis results showed that the short arm linkage region were between D12S1050and D12S374, with a length of2,299,067bp, and a maximal LOD score of2.68was obtained for the maker D12S1685at0=0. The long arm linkage region located between D12S313and12q/TG, with a length of1,3913,86bp, and a maximal LOD score of2.68was obtained for the maker12q/TTAT at θ=0.
We also performed exome sequencing for the proband. Combining the results of exome sequencing with the results of whole genome linkage analysis, we found a missense mutation in the EFCAB4B gene (R466W) that cosegregated with the patients of the family. We genotyped319unrelated controls for the variant using PCR followed by restriction analysis, and found two of them were heterozygous carriers for this variant. One of the carriers is overweight, while the other one is normal, suggesting the variant we found in this family may not be the pathogenic mutation.
Based on the clinical features of the probands of two of the four early onset obese familis, we performed mutation screening of MC4R, LEP and LEPR for both of them. A novel homozygous nonsense mutation in the LEP gene was found in the proband of one family, and both of his parents were carriers of this mutation. To investigate whether the mutations of his parents were inherited from a same ancient, we selected microsatellite markers near the LEP gene and performed haplotype analysis in this family. The results supported our hypothesis. We also found a homozygous nonsense mutation in the MC4R gene in the proband of another family. His parents, his son and daughter were all carriers of this mutation without showing any clinical feature.
We performed mutation screening of23genes associated with monogenic forms of obesity in humans or transgenic mice in the probands of two other families, respectively. No mutation had been found in both of them; however, we did find some coding SNPs known to be associated with body weight in the LEPR and SIM1gene of the patient, respectively.
In conclusion, we used whole genome linkage analysis combining with exome sequcing to perform mutation screening in a family with autosomal dominant obesity with overgrowth, and found a rare variant that cosegregated with the patients of the family. Though the pathgenicity of this variant is to be determined, our study offers a new method for investigating the molecular mechanism of obesity. We also found two homozygous mutations in LEP and MC4R, in patients with extreme early onset obesity, respectively. The mutation in LEP is also the first LEP gene mutation report in China.
Part2:Detection of Pathogenic Mutations of Three Inherited Skin Disorders
Skin disease, or dermatosis, is the disease that involves skin. Dermatosis is defined as the pathological changes of skin (including skin and skin appendages, as hair, nail and sweat glands) resulting from internal or external factors. The main causes of dermatosis are infections, allegies and genetics factors. Dermatosis that is caused by changes of genetic material is named genodermatosis. Most of the genodermatosis, such as vitiligo, psoriasis, et al, are polygenic diseases; however, there are also nearly300monogenic genodermatosis. Herein this thesis, we perform molecular genetics study on three monogenic genodermatosis, Bazex-Dupre-Christol syndrome, X-linked Hypohidrotic Ectodermal Dysplasia, and Congenital insensitivity to pain with anhidrosis, to investigate or detect mutations resulting in these diseases.
Bazex-Dupre-Christol syndrome (BDCS, MIM301845) is a rare genodermatosis that was first recognized by Bazex and colleagues in1964. Affected individuals can manifest a broad range of cutaneous symptoms, while frequently and regularly observed clinical signs comprise the triad of hypotrichosis, follicular atrophoderma, and early onset of basal cell carcinomas (BCCs). Further symptoms include milia, hypohidrosis, hyperpigmentation of the face, trichoepitheliomas and hair shaft abnormalities. BDCS shows an X-linked dominant inheritance mode, the gene for BDCS has been mapped to an11.4Mb interval on chromosome Xq25-q27.1(ChrX:128,242,493-139,631,728) in2011. However, no mutation has been found yet. In the present study, we have collected two BDCS families from Europe. Whole exome sequencing and Affymetrix SNP6.0array was performed to investigate the pathogenic mutation of BDCS. After we received results of exome sequencing, we design primers to validate the variants found in the reported linkage region; however, no potential causative variants were found. Then the raw data of the exome sequencing within the linkage region were reanalyzed. The exons of genes with low or null coverage were picked up and were sequenced; moreover, the genes within the linkage interval that were not shown in the raw data were sequenced, too. Unfortunately, we still did not find any potential causative variants. Using Affymetrix SNP6.0array for copy number variantion detection, we have found duplication on chromosome Xq26.1-q26.2in all of the patients we had. However, the duplication located in a region that had overlap with known CNVs, therefore the pathgenicity of the duplication still need to be further confirmed.
Ectodermal Dysplasia (ED) is not a single disease, but a class of syndromes characterized by abnormal development of two or more of ectodermal-derived structures, such as hair, teeth, and nails and so on. Of all these syndromes, X-linked Hypohidrotic Ectodermal Dysplasia (XLHED, MIM305100) is the most common form. Male patients present the triad of hypohidrosis, hypotrichosis and anodontia. Female carriers, however, show considerably variability in the severity of the disease, and about30%of them do not present obvious manifestations. XLHED is caused by mutations in EDA, which is located at Xq12-13. To date, there are nearly180pathogenic mutations that have been found in the EDA gene. More than98%of these mutations in EDA result in XLHED. However, there are several mutations found to cause another X-linked disease:X-linked dominant incisor hypodontia (MIM300606). To detect mutations of EDA gene that resulted in XLHED in the six families we collected, PCR amplification and automatic sequencing of EDA gene coding region with flanking regions were performed to detect mutations in EDA. We identified six mutations in6Chinese families with XLHED, including two novel mutations, a missense mutation and a deletion. The pathogenicity of novel missense mutation was determined by restriction analysis in family members and in52unrelated female controls. To confirm the deletion, we performed qPCR assays to validate the relative copy number (CNV) of the proband and his mother. Moreover, to explore the extent of the deletion, PCR assays were performed with primer pairs located between exon7and exon8and downstream of exon9. Finally, the deletion size was determined to be10kb by using Gap-PCR. This deletion we found also affected a gene AWAT2, downstream of EDA. Based on the results of mutation analysis, we also performed prenatal diagnosis for one female carrier, who got a male without EDA mutation at the end.
Congenital insensitivity to pain with anhidrosis (CIPA, MIM256800), which is classified as hereditary sensory and autonomic neuropathy type IV (HSAN-IV), is a rare autosomal recessive disorder. CIPA is characterized by absence of reaction to noxious stimuli, anhidrosis (inability to sweat), recurrent episodic fever, self-mutilating behavior, and often mental retardation. Mutations in the human neurotrophic tyrosine kinase receptor type1(NTRK1) are responsible for this disease [3]. NTRK1, which is also designated as TRKA, is located on chromosome1q21-22, and encodes a receptor tyrosine kinase TRKA that contains either790or796amino acids, and is a high-affinity receptor for nerve growth factor (NGF). In two Chinese Han families with CIPA, we found three novel missense mutations (p.P397L, p.R692C, p.R771C) in the proband of family1; his mother carried two of them (p.P397L and p.R771C); while his father didn't carry the third mutation, suggesting p.R692C was de novo or his father was chimera for this mutation. We found homozygous one base pair duplication in the proband of family2. Both of his parents were carriers of this mutation.
In conclusion, we chose several methods to perform pathogenic mutation analysis for these three genodermatosis. Based on the mutation screening within linkage region of BDCS, we have basically ruled out the possibility that BDCS was caused by mutation in the exome of the reported linkage region. We also indentified duplication on chromosome Xq26.1-q26.2in all of the four patients we had, however, the pathgenicity of the duplication still need to be further confirmed. Through mutation detection performed on XLHED and CIPA families, we found pathnogenic mutations in all of the families, including5novel mutations. Our finding should be helpful for genetics counseling and prenatal diagnosis for these families, and the novel mutations also expand the EDA and NTRK1gene mutation spectrum.
在过去的十几年中,肥胖人口的不断增长已经成为世界范围内公共卫生事业的主要挑战。作为一种复杂疾病,肥胖的产生除了与环境有关外,还与遗传因素关系密切。据估计遗传因素对体重的影响可能达40-70%。由此可见,对肥胖产生分子机制的研究,具有十分重要的意义。鉴于肥胖成因的复杂性,对一些早发、极端、以单基因方式遗传肥胖致病基因的研究成为了人们研究肥胖分子机制的突破口。目前人们已经鉴定出20多种单基因肥胖的致病基因,但只能解释不到5%的极端肥胖,对剩余肥胖分子机制的研究将是人们面临的一个巨大挑战。
人类生长是一个受到多种因素影响的复杂过程,其中遗传因素在身高决定中占主要作用。一直以来,人们对于身材矮小的分子机制的研究较多,而对高大身材,特别是婴幼儿及儿童期身材高大或过度生长的分子机制研究相对较少。儿童期身材高大或者过度生长可能由多种原因导致,其中最常见的原因是肥胖和超重。因此,针对导致儿童期身材高大或过度生长、肥胖或超重的致病基因鉴定对于人们理解儿童生长和机体组成的机制具有十分重要的意义。
本研究中我们收集了1个常染色体显性肥胖伴过度生长家系和4个早发肥胖核心家系。在获得知情同意后,对这5个家系进行了分子遗传学研究。
常染色体显性肥胖伴过度生长家系患者除了肥胖外,两位处于青春期的患者身高要明显高于同龄人,因此我们该家系命名为肥胖伴过度生长家系。我们首先在先证者中筛查了的23个基因,包括已报道的导致非综合征型单基因肥胖的致病基因和一些在转基因小鼠中发现与体重调节相关的基因。在排除这些基因的改变之后,我们使用Affymetrix SNP6.0芯片对部分家系成员分两批进行拷贝数分析和全基因组连锁分析,在12号染色体长臂和短臂各发现一段LOD值为2.4的区域。其中短臂区域位于rs10491968至rs11063753,长度为1,975,647bp；长臂区域位于rs17105259至rs10160961之间,长度2,119,818bp。在得到连锁结果后,我们在连锁区域附近选择微卫星标记进行验证和精细定位,将短臂的连锁区域定位于D12S1050和D12S374之间,长度为2,299,067bp,长臂的连锁区域定位于D12S313与12q/TG之间,长度为1,391,386bp。同时分别在短臂标记D12S1685与长臂标记12q/TTAT,当0=0时,获得了最大LOD值2.68。
在进行全基因组连锁分析的同时,我们对该家系的先证者进行了全外显子组测序。结合全基因组连锁分析和外显子组测序结果,我们在位于12号染色体短臂的EFCAB4B基因中发现了1个在家系患者中共分离的错义突变(p.R466W)。随后我们在319例无关人群中对该变异进行了验证。经验证我们发现两例无关个体(一个超重,一个体重正常)存在与家系内患者相同的改变,因此暂不能确定我们所发现的变异是否是导致家系肥胖表型的分子基础。
针对4个早发肥胖家系,我们根据家系内先证者的临床表现,首先其中两个家系先证者进行MC4R、LEP和LEPR基因突变检测。在家系2先证者中发现LEP基因一个纯合的无义突变(c.163C>T,p.Q55X),该突变为国际首报,其父母为该突变的携带者。为了验证其父母所携带的突变是否来自同一祖先,我们选择LEP基因上下游各2Mb的微卫星标记对该家系进行连锁分析,从而确定了其父母所携带的突变来自于同一祖先。同时,我们在家系3先证者中发现MC4R基因一个纯合无义突变,其父母和子女为该突变的携带者。
对于另外两个家系的先证者,我们则对已报道的导致单基因肥胖的致病基因和一些在转基因小鼠中发现与体重调节相关的23个基因进行了突变检测。结果并没有在这些基因中发现致病改变,但却分别在两个家系先证者中发现LEPR和SIMl基因的几个与体重调节相关的SNP位点。
总之,在本研究中,我们使用全基因组连锁分析和外显子组测序等方法,在一个常染色体显性肥胖伴过度生长家系中发现了一个与患者共分离的罕见变异,尽管该变异的致病性还需要进一步的确定,本文为肥胖这种复杂疾病分子机制的研究提供了新的思路。在两个早发肥胖家系中分别发现了LEP、MC4R基因的纯合无义突变,这是导致这两个家系患者肥胖表型的分子基础。其中LEP基因突变为中国人群中首次报道。
第二部分三种皮肤遗传病的致病突变研究
皮肤病是指皮肤(包括皮肤附属器官毛发、指甲、汗腺等)受到内外因素的影响后,其形态、结构、功能发生的病理改变。皮肤病的主要病因包括感染、过敏和遗传因素影响。因遗传物质改变所引起的皮肤病称为遗传性皮肤病,有约300种单基因遗传性皮肤病。本文对三种单基因遗传性皮肤病(Bazex-Dupre-Christol综合征、X连锁无汗型外胚层发育不良和先天性无痛伴无汗)进行了致病基因突变研究,寻找或鉴定三种遗传性皮肤病的致病基因改变。
Bazex-Dupre-Christol综合征(Bazex-Dupre-Christol syndrome, BDCS)(MIM301845)是一种罕见遗传性皮肤病,1964年由Bazex等人首先报道。BDCS的患者通常表现出广泛的皮肤症状,最为常见的临床表现为少毛、毛囊性皮肤萎缩和早发的基底细胞癌的三联征。除此之外,在一些病人中还会出现粟丘疹、少汗、颅面部色素沉着,毛发上皮瘤,毛干异常等表型。BDCS呈X连锁显性遗传方式遗传,其致病区域在2011年被定位于Xq25-q27.1区域11.4Mb的范围内,但迄今尚未发现致病突变。本研究中我们收集了来自欧洲的两个BDCS家系,采用全外显子组测序技术及Affymetrix SNP6.0技术平台对家系样品进行分析,以求能够寻找到BDCS的致病改变。在得到外显子组测序结果后,我们首先对连锁区域内可疑改变进行Sanger测序验证,但并没有得到与疾病共分离的可疑改变。随后我们对原始bam文件进行分析,对连锁区域内外显子组测序reads覆盖率较低或未覆盖区域,以及在连锁区域中未出现在bam文件中的基因进行补测,结果并没有发现可疑致病改变。通过使用Affymetrix SNP6.0技术平台对家系成员进行拷贝数分析,发现两个家系的患者都在Xq26.1-q26.2之间存在拷贝数增加,但该重复与DGV数据库的已知CNV存在重叠,该拷贝数改变是否致病还需要进一步的分析验证。
外胚层发育不良(Ectodermal Dysplasia, ED)是一类以牙齿、毛发、指甲等外胚层来源组织发育不良为特征的遗传性综合征的总称。在这些综合征中,最为常见的是X连锁隐性少汗型外胚层发育不良(X-linked hypohidrotic ectodermal dysplasia, XLHED, MIM305100)。XLHED男性患者的临床表现为无汗或少汗毛发稀疏或细黄、少牙或牙齿发育异常的三联征。携带者女性由于X染色体的随机失活会有不同程度的临床表现,另有30%左右的携带者不会表现出任何临床症状。XLHED的致病基因为EDA,定位于Xq12.2-q13.1。到目前为止,HGMD上共记录了大约180种EDA基因的突变。其中98%以上的突变导致XLHED,另有约2%突变会引起另一种X连锁疾病：X连锁显性牙发育不良(X-linked dominant hypodontia, MIM300606)。本研究中我们收集了6个XLHED家系,对家系内患者EDA基因各外显子及外显子-内含子交界处进行PCR扩增后直接测序。我们在6个家系中鉴定出6个致病突变,5个为点突变,1个缺失突变。其中1个点突变(p.Q358L)和缺失突变为首次报道的新突变。采用PCR产物酶切的方法,对p.Q358L进行了验证。为了验证所发现的缺失突变,通过使用定量PCR以检测先证者和其家系成员的相对拷贝数。为了确定先证者的缺失范围,我们在EDA基因第7、8外显子之间以及EDA基因3'UTR末端和其下游2.5kb、5kb、7.5kb范围内分别设计引物进行PCR扩增,从而将缺失范围初步确定为第8外显子上游至EDA基因下游5kb。结合GAP-PCR扩增,最终将家系6先证者X染色体的缺失范围确定为10,297bp,同时发现此缺失突变还导致位于EDA基因下游的AWAT2最后4个外显子的缺失。另外,通过采用PCR直接测序和单体型分析的方法,我们对一个家系中的携带者进行了产前基因诊断。结果显示胎儿不携带致病突变,为正常男性。
先天性无痛伴无汗(Congenial insensitive to pain with anhidrosis, CIPA, MIM256800),是一种很罕见的常染色体隐性遗传病。CIPA患者典型的临床表现为对伤害性刺激无反应,无汗,屡现的高热,自我伤害行为及智力迟缓。CIPA的致病基因为神经营养性酪氨酸激酶受体I型(Human neurotrophic tyrpsine kinase receptor type1, NTRK1),定位于1q21-22,编码含790或796个氨基酸残基的受体酪氨酸激酶TRKA。对于我们收集到的两个CIPA家系,我们对家系内患者NTRK1基因各外显子及外显子-内含子交界处进行PCR扩增后直接测序。在家系1先证者NTRK1基因上发现三个错义突变(p.P397L, p.R692C, p.R771C),均为国际首报。其中P397L和R771C突变来自先证者的母亲,而由于先证者的父亲并不携带R692C突变,我们推测该突变可能是新生突变或者父亲是该突变的嵌合体。通过PCR产物酶切的方法,我们在70名正常对照个体中分别对这
个突变进行了验证。随后我们采用在线预测工具SIFT和Polyphen对三个错义突变的致病性进行了预测,预测结果显示R692C和R771C的致病性要强于P397L。另外,我们在家系2先证者NTRK1基因上发现了一个已知的纯合突变,c.287+2dupT,其父母均为此突变的携带者。我们的发现有助于对这两个家系进行遗传咨询和产前诊断,同时新的突变也扩展了NTRK1的突变谱。
总之,对于这三种遗传性皮肤病,我们采用了不同方法对其进行了致病突变研究。通过对BDCS连锁区域的致病突变筛查,我们基本排除了BDCS是由连锁区域内外显子组区域突变致病的可能。通过对连锁区域内拷贝数分析,‘发现了两个家系患者Xq26.1-q26.2之间存在拷贝数的增加,其致病性需要进一步确定。我们对XLHED和CIPA家系分别进行了致病基因突变检测,鉴定出了每个家系的致病突变,包括5个新突变。我们的检测结果有助于为患者家系提供遗传咨询和产前诊断,同时所发现的新的突变也扩展了EDA、NTRK1基因突变谱。
Part1:Molecular Genetic Studies in Chinese families with Monogenic Obesity
During the past decades, obesity has become the main challenge for public health worldwide. Known as a multifactor disease, obesity is influenced both by genetic and environmental factors. It has been estimated that an inherited component to body weight accounts for40-70%. Therefore, study on the molecular mechanism underlying obesity has great significance. Due to the complexity resulting in obesity, studies on the early-onset, extreme, andmonogenic type of obesity have been chosen as the breakthrough for investigating molecular mechanism of obesity. To date, there are more than20genes, in which mutations result in severe monogenic human obesity, have been identified. However, the monogenic causes of obesity identified so far only account for less than5%of severe obesity. Understanding the molecular mechanism of the other95%of obesity is still a great challenge.
Human growth is a multi-factorial and complex process, while the height is still predominantly under genetic control. Studies on molecular mechanisms resulting in tall stature and'overgrowth'have perhaps occurred in a less systematic manner, comparing with the study of short stature. Tall stature and overgrowth during childhood can be caused by many reasons, while obesity might be the commonest reason for apparent children tall stature and overgrowth. Therefore, identifying genetic causes of tall stature and overgrowth, weight gain can further our knowledge on the important mechanisms, by which childhood growth and body composition are regulated.
In the present study, we have collected one family with autosomal dominant obesity with overgrowth and four nuclear families with early onset obesity. With informed consent of all participating individuals, we performed genetic research in these five families, respectively.
We named the first family Obesity with Overgrowth by the fact that besides obesity, two pubertal patients seemed taller than their peers. Firstly, in the proband of this family, we have screened mutations of23genes associated with monogenic forms of obesity in humans or transgenic mice. After that, Affymetrix SNP6.0array was used to detect the copy number variation and performed whole genome linkage analysis in some of the members of the family. Two regions on the short and long arms of Chromosome12were found and they showed the maximal LOD score of2.4. The short arm region located between rs1049168and rs1106753, with a length of1,975,647bp, while the long arm region located between rs17105259and rs10160961, with a length of2,119,818bp. Based on the results of genome linkage analysis,8polymorphic microsatellite markers were selected,4from the linkage region on the short arm and4from the linkage region on the long arm of Chromosome12, and linkage and haplotype analysis was performed to verify the critical region shown by the results from the SNP array data in family1. Genotyping and haplotype analysis results showed that the short arm linkage region were between D12S1050and D12S374, with a length of2,299,067bp, and a maximal LOD score of2.68was obtained for the maker D12S1685at0=0. The long arm linkage region located between D12S313and12q/TG, with a length of1,3913,86bp, and a maximal LOD score of2.68was obtained for the maker12q/TTAT at θ=0.
We also performed exome sequencing for the proband. Combining the results of exome sequencing with the results of whole genome linkage analysis, we found a missense mutation in the EFCAB4B gene (R466W) that cosegregated with the patients of the family. We genotyped319unrelated controls for the variant using PCR followed by restriction analysis, and found two of them were heterozygous carriers for this variant. One of the carriers is overweight, while the other one is normal, suggesting the variant we found in this family may not be the pathogenic mutation.
Based on the clinical features of the probands of two of the four early onset obese familis, we performed mutation screening of MC4R, LEP and LEPR for both of them. A novel homozygous nonsense mutation in the LEP gene was found in the proband of one family, and both of his parents were carriers of this mutation. To investigate whether the mutations of his parents were inherited from a same ancient, we selected microsatellite markers near the LEP gene and performed haplotype analysis in this family. The results supported our hypothesis. We also found a homozygous nonsense mutation in the MC4R gene in the proband of another family. His parents, his son and daughter were all carriers of this mutation without showing any clinical feature.
We performed mutation screening of23genes associated with monogenic forms of obesity in humans or transgenic mice in the probands of two other families, respectively. No mutation had been found in both of them; however, we did find some coding SNPs known to be associated with body weight in the LEPR and SIM1gene of the patient, respectively.
In conclusion, we used whole genome linkage analysis combining with exome sequcing to perform mutation screening in a family with autosomal dominant obesity with overgrowth, and found a rare variant that cosegregated with the patients of the family. Though the pathgenicity of this variant is to be determined, our study offers a new method for investigating the molecular mechanism of obesity. We also found two homozygous mutations in LEP and MC4R, in patients with extreme early onset obesity, respectively. The mutation in LEP is also the first LEP gene mutation report in China.
Part2:Detection of Pathogenic Mutations of Three Inherited Skin Disorders
Skin disease, or dermatosis, is the disease that involves skin. Dermatosis is defined as the pathological changes of skin (including skin and skin appendages, as hair, nail and sweat glands) resulting from internal or external factors. The main causes of dermatosis are infections, allegies and genetics factors. Dermatosis that is caused by changes of genetic material is named genodermatosis. Most of the genodermatosis, such as vitiligo, psoriasis, et al, are polygenic diseases; however, there are also nearly300monogenic genodermatosis. Herein this thesis, we perform molecular genetics study on three monogenic genodermatosis, Bazex-Dupre-Christol syndrome, X-linked Hypohidrotic Ectodermal Dysplasia, and Congenital insensitivity to pain with anhidrosis, to investigate or detect mutations resulting in these diseases.
Bazex-Dupre-Christol syndrome (BDCS, MIM301845) is a rare genodermatosis that was first recognized by Bazex and colleagues in1964. Affected individuals can manifest a broad range of cutaneous symptoms, while frequently and regularly observed clinical signs comprise the triad of hypotrichosis, follicular atrophoderma, and early onset of basal cell carcinomas (BCCs). Further symptoms include milia, hypohidrosis, hyperpigmentation of the face, trichoepitheliomas and hair shaft abnormalities. BDCS shows an X-linked dominant inheritance mode, the gene for BDCS has been mapped to an11.4Mb interval on chromosome Xq25-q27.1(ChrX:128,242,493-139,631,728) in2011. However, no mutation has been found yet. In the present study, we have collected two BDCS families from Europe. Whole exome sequencing and Affymetrix SNP6.0array was performed to investigate the pathogenic mutation of BDCS. After we received results of exome sequencing, we design primers to validate the variants found in the reported linkage region; however, no potential causative variants were found. Then the raw data of the exome sequencing within the linkage region were reanalyzed. The exons of genes with low or null coverage were picked up and were sequenced; moreover, the genes within the linkage interval that were not shown in the raw data were sequenced, too. Unfortunately, we still did not find any potential causative variants. Using Affymetrix SNP6.0array for copy number variantion detection, we have found duplication on chromosome Xq26.1-q26.2in all of the patients we had. However, the duplication located in a region that had overlap with known CNVs, therefore the pathgenicity of the duplication still need to be further confirmed.
Ectodermal Dysplasia (ED) is not a single disease, but a class of syndromes characterized by abnormal development of two or more of ectodermal-derived structures, such as hair, teeth, and nails and so on. Of all these syndromes, X-linked Hypohidrotic Ectodermal Dysplasia (XLHED, MIM305100) is the most common form. Male patients present the triad of hypohidrosis, hypotrichosis and anodontia. Female carriers, however, show considerably variability in the severity of the disease, and about30%of them do not present obvious manifestations. XLHED is caused by mutations in EDA, which is located at Xq12-13. To date, there are nearly180pathogenic mutations that have been found in the EDA gene. More than98%of these mutations in EDA result in XLHED. However, there are several mutations found to cause another X-linked disease:X-linked dominant incisor hypodontia (MIM300606). To detect mutations of EDA gene that resulted in XLHED in the six families we collected, PCR amplification and automatic sequencing of EDA gene coding region with flanking regions were performed to detect mutations in EDA. We identified six mutations in6Chinese families with XLHED, including two novel mutations, a missense mutation and a deletion. The pathogenicity of novel missense mutation was determined by restriction analysis in family members and in52unrelated female controls. To confirm the deletion, we performed qPCR assays to validate the relative copy number (CNV) of the proband and his mother. Moreover, to explore the extent of the deletion, PCR assays were performed with primer pairs located between exon7and exon8and downstream of exon9. Finally, the deletion size was determined to be10kb by using Gap-PCR. This deletion we found also affected a gene AWAT2, downstream of EDA. Based on the results of mutation analysis, we also performed prenatal diagnosis for one female carrier, who got a male without EDA mutation at the end.
Congenital insensitivity to pain with anhidrosis (CIPA, MIM256800), which is classified as hereditary sensory and autonomic neuropathy type IV (HSAN-IV), is a rare autosomal recessive disorder. CIPA is characterized by absence of reaction to noxious stimuli, anhidrosis (inability to sweat), recurrent episodic fever, self-mutilating behavior, and often mental retardation. Mutations in the human neurotrophic tyrosine kinase receptor type1(NTRK1) are responsible for this disease [3]. NTRK1, which is also designated as TRKA, is located on chromosome1q21-22, and encodes a receptor tyrosine kinase TRKA that contains either790or796amino acids, and is a high-affinity receptor for nerve growth factor (NGF). In two Chinese Han families with CIPA, we found three novel missense mutations (p.P397L, p.R692C, p.R771C) in the proband of family1; his mother carried two of them (p.P397L and p.R771C); while his father didn't carry the third mutation, suggesting p.R692C was de novo or his father was chimera for this mutation. We found homozygous one base pair duplication in the proband of family2. Both of his parents were carriers of this mutation.
In conclusion, we chose several methods to perform pathogenic mutation analysis for these three genodermatosis. Based on the mutation screening within linkage region of BDCS, we have basically ruled out the possibility that BDCS was caused by mutation in the exome of the reported linkage region. We also indentified duplication on chromosome Xq26.1-q26.2in all of the four patients we had, however, the pathgenicity of the duplication still need to be further confirmed. Through mutation detection performed on XLHED and CIPA families, we found pathnogenic mutations in all of the families, including5novel mutations. Our finding should be helpful for genetics counseling and prenatal diagnosis for these families, and the novel mutations also expand the EDA and NTRK1gene mutation spectrum.
引文
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3. Wu, Y., Overweight and obesity in China. BMJ,2006.333(7564):p.362-3.
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