【作者】 代小华；

【导师】 王擎； 刘静宇；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 遗传性疾病是人的遗传物质在数量、结构或功能上发生改变,干扰了正常的生命活动而引起的疾病,也是一类能够通过生殖细胞传递给后代的疾病。神经系统的遗传病是所有遗传病中最为重要的一类,是神经系统疾病和医学遗传学的重要组成部分。在众多神经系统遗传病中,癫痫(epilepsy)占有重要位置,其发病率很高,Fahr病的致残率很高,智力障碍(mental retardation,MR)的发病率也很高,它们是重要的神经系统疾病。本论文对上述神经系统遗传病进行分子和细胞遗传研究,以期克隆相关致病基因,同时对克隆的致病基因进行功能研究,揭示上述疾病的发病机制,为治疗这类疾病奠定基础。癫痫是一种常见的慢性脑部疾病,由多种病因引起的慢性脑功能障碍综合征,具有发作性、复发性和自然缓解性的特点。据估计,癫痫患者约占世界人口的0.5-1%,极大危害人类健康。根据遗传方式可将其分为四大类:单基因遗传病、多基因病遗传病、染色体畸变遗传病、线粒体遗传病。该病又呈现出高度临床表现异质性和遗传异质性。家族性基底节脑血管钙化又称Fahr病,是一种神经系统锥体外系遗传病,伴随癫痫、运动障碍和智力、意识障碍等发生,大多数家系表现为常染色体显性遗传方式,也有常染色体隐性遗传和性染色体遗传的报道,具有广泛的遗传异致性。智力障碍,也称精神发育迟缓是指18岁以前发育阶段由于遗传因素、环境因素和社会心理因素等各种原因所引起,临床表现为智力明显低下和社会适应能力缺陷为主要特征的一组疾病。许多染色体病伴随智力障碍的发生。(1)在一个来自中国河南的常染色体显性遗传小儿癫痫家系中,通过连锁分析排除所有已知癫痫致病基因和已知位点,对该家系进行全基因组扫描工作,将该家系的致病基因定位在3号染色体长臂上位于D3S3656和D3S1232标记之间大约10.7Mb的区域(3q26.2—q26.33,所获得的两点最大LOD值是4.13)。这是一个新的癫痫致病基因位点。在定位区段内,共有24个已知基因和24个已知的EST。利用突变体检测技术,我们分析了此区域内的18个可能的候选基因,但是没有发现与疾病相关突变。由于我们定位了一个新的癫痫致病位点,该项目的进一步研究有望克隆新的癫痫致病基因。(2)在河南收集并鉴定了一个5代共31名成员的常染色体显性Fahr病大家系,通过连锁分析排除Fahr病已知致病位点14q13.1-q23.1,对该家系进行全基因组扫描工作,将该家系的致病基因定位在8号染色体上两个标记D8S1809和D8S1833之间大约25.0Mb的区域(8p21.2-q11.23),与D8S505紧密连锁,最大两点LOD值是4.10。此位点是世界上第二个Fahr病致病遗传位点。对该区域内的一个在大脑中广泛表达的SNTG1基因进行测序分析,未发现突变。由于目前还没克隆到Fahr病的相关致病基因,所以本研究为进一步克隆到世界上第一个新Fahr病致病基因奠定了基础。(3)对来自河南的伴随智力障碍、发育迟缓和手、面部轻微畸形患者进行染色体核型分析,发现是9p部分三体综合征。家系1内的先证者除表现出典型的9p部分三体综合征外,同时患有癫痫。经染色体G-显带分析发现家系1内的先证者核型为46,XY,-21,+der(21),t(9;21)。其父亲、伯父为46,XY,t(9;21)平衡易位核型,母亲核型正常,可知异常染色体来自父亲。所以家系1的先证者核型为46,XY,-21,+der(21),t(9;21)pat。而家系2的先证者除表现出典型的9p部分三体综合征外,并伴有皮肤湿疹症状。G显带核型为46,XY,+der(5),t(5;9),其母亲为46,XX,t(5;9)平衡易位核型,父亲核型正常,所以家系2的先证者核型为46,XY,+der(5),t(5;9)mat。进一步的临床调查发现家系1内另有癫痫患者,但G-显带分析发现他们的核型是正常的。而家系2中无其他人有智障及皮肤湿疹症状。利用荧光原位杂交分析进一步证明了核型分析结果。染色体断裂点分析表明,家系1内的9p断裂点位于D9S1846与D9S171之间,大约2.9Mb;而家系2内的9p断裂点位于D9S1870和D9S1679之间,大约2.7Mb。两个先征者的精确核型分别是:46,XY,-21,+der(21),t(9;21)(p21.3;q22.3)pat,46,XY,+der(5),t(5;9)(p15.3;p21.3)mat。虽然两个家系内的患者具有相似的9p复制区域24.4/24.7 Mb(9p21.3 to 9pter),但两个家系患者除都具有典型的9p部分三体综合征外,并伴有独特的临床症状。分析发现9p三体与智力障碍相关,9p21.3→9ter这段区域可能存在与智力发育相关的基因。所有这些分子细胞遗传学研究为阐明神经系统遗传病的病理学,以及提供更好的遗传咨询和最终发展有效疾病治疗策略提供了有益的帮助。更多还原

【Abstract】 Genetic disease can be transmitted to offspring by germ cells and caused by instability of genetic materials,including chromosomal aberrations and gene mutations. Genetic disease in the nervous system is not only the most important inherited disorder, but also a very important part of the neurological diseases and medical genetics. Epilepsy and mental retardation are major neurological disorders and have a high incidence.Fahr disease,another neurological disease,has a high disability rate.In this study,a molecular cytogenetic approach was used to study the inherited neurogenetic diseases in order to find the genes or mutations responsible for these diseases. Functional studies on a pathogenic gene will benefit understanding of mechanisms,and could offer a foundation to therapy of these diseases.Epilepsy is a chronic disorder of the brain and characterized by the imbalance of neurotransmitters and the abnormal discharge of the neuron.The main characteristics are paroxysmal,recurrent and natural relief.The incidence of epilepsy is 0.5-1%. Hereditary epilepsies were divided into four groups such as single-gene disorders, polygene disorders,chromosome disease,and mitochondria disease.The disease shows wide phenotypic and genetic heterogeneity.Familial idiopathic basal ganglia calcification (IBGC,Fahr disease) is another inherited neurologic condition characterized by basal ganglia and extra-basal ganglia brain calcifications,parkinsonism,and neuropsychiatric symptoms.The major pattern of inheritance is autosomal dominant and autosomal recessive,but X-linked inheritance was also found,which demonstrates genetic and clinical heterogeneity of familial IBGC.Mental retardation is defined by significant limitations in intellectual functions and adaptive behaviors that occur before the age of 18 years.The etiology for mental retardation is highly heterogeneous,and involves many cellular pathways and processes.Both genetic defects and environmental insults,alone or in combination,are known to cause cognitive impairment in humans.Many chromosomal diseases are associated with mental retardation.(1) A four-generation Chinese family with autosomal dominant febrile seizure and epilepsy was studied by genome-wide linkage analysis after exclusion of known loci and genes for epilepsy.Significant linkage was identified with markers on chromosome 3q26.2-26.33 with a maximum pairwise LOD score of 4.13.Fine mapping defined the new genetic locus within a 10.7 Mb region between markers D3S3656 and D3S1232.Our results identified another novel locus on chromosome 3q26.2-26.33.There are 24 candidate genes and EST in the novel locus region respectively.We analyzed 18 candidate genes in this region,but no mutation was found.A new genetic locus was obtained in our study,which provides a framework for future identification of the specific gene responsible for epilepsy.(2) We characterized a large five-generation Chinese family with Fahr disease,in which 31 family members were used for genome-wide linkage analysis.After exclusion of the unique genetic locus located on chromosome 14q13.1-q23.1,haplotype and two-point LOD analysis had been performed to define the disease gene interval.The Fahr disease locus in the family was mapped to chromosome 8p21.2-q11.23 with the maximum two-point LOD scores of 4.10 for marker D8S505,and the gene was found to be within a 25.0 Mb interval between markers D8S1809 and D8S1833.Mutation analysis of the SNTG1 gene at the locus,which is specifically expressed in the brain,did not reveal a disease causing mutation.To date,the gene responsible for Fahr disease is not found.Our study provides a framework for identification of a first pathogenic gene for Fahr disease.(3) Two Chinese families with metal retardation and delayed development in Henan province were reported.A 9p trisomy was detected by chromosomal analysis.The proband in family 1 was also diagnosed with epilepsy.Conventional GTG-banded chromosome analysis in the proband of family 1 showed 46,XY,-21,+der(21),t(9; 21).His father and uncle’s karyotypes revealed a balanced translocation,46,XY,t(9; 21),and his mother’s karyotype was normal.The abnormal chromosome 21 was a derivative chromosome 21 inherited from his father.Thus,the karyotype of proband in family 1 was 46,XY,-21,+der(21),t(9;21 ) pat.The proband in family 2 had refractoriness eczema except for 9p trisomy symptoms.The karyotype was 46,XY,+der (5),t(5;9).His mother’s karyotype revealed a balanced translocation,46,XY,t(5; 9) and his father’s karyotype was normal.The karyotype of proband in family 2 was 46, XY,+der(5),t(5;9) mat.Further clinical investigation found that there were other individuals with epilepsy disease in family 1.Karyotype analysis found that they all had normal karyotype.In family 2,all individuals had no refractoriness eczema disease except for the proband.To identify the precisely breakpoints,FISH analysis and DNA marker analysis were performed.FISH analysis also showed that the proband in family 1 carried partial 9p trisomy.Molecular genetic analysis defined the precise breakpoint of family 1 on chromosome 9p between markers D9S1846 and D9S171,an interval of about 2.9 Mb on 9p21.3 and the precise breakpoint on chromosome 9p of family 2 between markers D9S1679 and D9S1870,an interval of about 2.7 Mb on 9p21.3.The precise karyotypes of probands in these two families are 46,XY,-21,+der(21),t(9;21)(p21.3;q22.3) pat, and 46,XY,+der(5),t(5;9)(p15.3;p21.3) mat.Although the 9p duplicated region spanned a similar region of 24.4/24.7 Mb(9p21.3 to 9pter),the patients in the two families had a different clinical symptom besides the clinical findings of partial trisomy 9p. These results implicated that trisomy 9p was associated with mental retardation,and that there may be a genomic duplication on chromosome 9pter→9p21.3 responsible for mental retardation and mild facial anomaly.All these results have clinical implications as they provide valuable information for genetic testing and counseling as well as development of future therapeutic strategies for management of inherited neurogenetic disorders.These studies also provide insights into the pathophysiology of congenital neurogenetic diseases.更多还原