【作者】 佃艳；

【导师】 黄尚志；

【作者基本信息】 中国协和医科大学 ， 医学遗传学， 2009， 硕士

【摘要】 随着越来越多的疾病基因或候选基因相继被克隆,基因突变与疾病的关系也得到进一步的阐明。临床上迫切需要简便、快速、敏感度高、特异性好、成本低、自动化、可批量提供的基因突变检测方法,以用于遗传病患者的诊断、携带者的检出及高危胎儿的产前诊断。目前用于基因突变检测的常用方法有多种。本课题建立单基因病新病种的基因诊断研究策略,即从某种单基因遗传病的临床诊断出发,到查阅文献、确定候选基因、设计引物、PCR、测序以确定致病突变,并用于定制的产前诊断。本课题完成了三种单基因病的研究:遗传性对称性色素异常症（DSH）,是一种较少见的常染色体显性遗传性皮肤病,该病的致病基因为RNA特异性腺苷脱氨酶（ADAR）基因。用上述方法对一个两代家系进行突变分析,检测到突变位点为c.1642 delC（p.Pro548GlnfsX15）,并设计针对突变的引物进行等位基因特异性PCR（AS-PCR）,群体验证结果表明该突变为新的致病突变。火棉胶样儿是一种特殊的先天性鱼鳞病,本病遗传异质性很强,涉及的致病基因有多种,分析确定本例最可能是常染色体隐性遗传的板层状鱼鳞病（LI）或非大泡性鱼鳞病样红皮病（NBCIE）,由转谷丙酰胺酶1（TGM1）基因突变所致。通过全基因测序,检测到3种新突变,患儿来自父亲的突变基因为c.694delG（p.Glu232SerfsX98）,而来自母亲的突变基因具有2处突变,c.463C＞T（p.Arg155Trp）和c.578G＞A（p.Trp193X）,并推测其中一处改变系二次突变的结果。湿疹-血小板减少紫癜综合征（WAS）是一种X-连锁隐性遗传性免疫缺陷病,致病基因为WAS蛋白（WASP）基因。患儿的突变为c.107₁08delTT（p.Phe35X）,国外已有报道。受家长的委托,对该家庭进行了定制的产前诊断,胎儿为女性,没有获得突变等位基因。更多还原

【Abstract】 With an increasing number of virulence genes or candidate genes have been cloned, the relation between gene mutations and diseases has been further elaborated. Simple, rapid, highly sensitive and well specific, low cost, automatic, accessible in high through put for gene mutation detection systems are on the urgent need for clinical use, in genetic diagnosis of the patients, carrier detection and prenatal diagnosis for high-risk fetus. There are a variety of methods used for detection of gene mutation currently. The design of this subject was to utilize combinely the most common methods that be used for molecular diagnosis of monogenic disease. That is, from clinical diagnosis of a certain monogenic disease, to literature reviewing, selecting the candidate genes, designing the primers, PCR amplication, sequencing, identifying the pathogenic mutations, and finally, after the pathogenic mutations were identified in the family, custom prenatal diagnosis can be offered to the pregnancy at risk in the family.This topic covers the following three monogenic diseases:1. Dyschromatosis symmetrical hereditaria （DSH）.DSH is a rare autosomal dominant hereditary skin disease characterized by a mixture of hyperpigmented and hypopigmented macules of various sizes on the face and the dorsal aspects of the extremities. The pathogenic gene is the RNA-specific adenosine deaminase （ARAD） gene. We used the above strategy in the analysis of the pedigree and identified a single nucleotide deletion （c.1642 delC, p.Pro548GlnfsX15） in exon3 of ADAR gene in the patients, and the mutation was not detected in the normal family members and in any of the control individuals. This single nucleotide deletion was responsible for the disease in the family and it was a novel pathogenic mutation not reported previously.2. Collodion baby.It is a specific congenital ichthyose. It has strong genetic heterogeneity, and many virulence genes involved. Based on a variety of reasons, we suspected the baby was affected with Lamellar ichthyosis （LI） or nonbullous congenital ichtyosiform erythroderma （NBCIE）, and we selected the transglutaminase 1（TGM1） gene as the candidate gene. We identified three new alternations in TGM1 gene of the proband, a single nucleotide deletion c.694delG （p.Glu232SerfsX98） in exon4, a missense mutation C.463C>T （p.Arg155Trp） in exon3 and a nonsense mutation c.578G>A （p.Trp193X） in exon4. The missense mutation was not detected in his father and in any of the control individuals by AS-PCR. Transmission analysis showed that the father was heterozygous for c.694delG mutation, while the mother carried the two mutations （c.463C>T and c.578G>A）. The c.578G>A （p.Trp193X） was a causal mutation itself and the C.463C>T （p.Arg155Trp） mutation might be causative also. Two causal mutation located on the same allele might caused by repeat mutation, not by conversion.3. Wiskott-Aldrich syndrome （WAS）.WAS is an X-linked recessive hereditary immuno- deficiency disease. It is characterized by eczema, thrombocytopenia and infection. The specific diagnosis is based on the finding of small size of platelets. A double-nucleotide-deletion （c.107₁08delTT, p.Phe35X） was identified in exonl of WASP gene in the proband and his mother. This mutation has been reported. When the mother was in pregnancy again, custom prenatal diagnosis was performed in the request of the family. The fetus was a female and got the normal allele from the mother.更多还原