两个中国遗传性聋家系的致病基因研究
发布时间：2018-09-11 19:54
【摘要】：第一部分一个母系遗传性聋家系的致病基因研究 目的： 研究一个五代母系遗传性聋大家系的遗传学病因及发病机制,阐明该家系成员耳聋的机制,为遗传性聋的防治提供理论依据。 方法： 经耳聋先证者获悉一遗传性聋大家系,经家系调查采集详细的临床及听力学资料,并在签署知情同意书后采集静脉血。由静脉血提取全基因组DNA,对母系成员的线粒体基因组全序列及常见耳聋基因GJB2经聚合酶链反应扩增和测序进行检测。对检测到的线粒体DNA耳聋相关突变应用焦磷酸测序进行异质性定量,分析突变的异质性比例与耳聋程度的相关性。对突变位点行物种间进化保守性分析。在相同遗传背景的200名正常听力对照和806名散发感音神经性聋患者中进行突变筛查。同时建立耳聋患者和正常听力对照的淋巴细胞系,提取细胞的线粒体总RNA,采用Northern blot检测基因突变后tRNA水平的变化,并进行线粒体基因的蛋白合成和细胞耗氧量测量。 结果： 该家系符合母系遗传规律,耳聋的发生与使用氨基糖甙类抗生素无关,表现为双侧对称、迟发性、进行性感音神经性聋,以高频听力损失为主,逐渐累积全频,常伴高调耳鸣。患者颞骨高分辨率CT无明显异常,未伴随明显的其他系统疾病,属于非综合征型聋。线粒体基因组全序列分析显示母系成员属于同一线粒体单倍型Z,共存在43种碱基改变,其中T12201C为异质性突变,位于tRNA—His基因二级结构的接受臂,使碱基配对由A-U变为A-C,该位点在多物种间高度保守,突变异质性比例与耳聋发病年龄和严重程度存在相关性。其余碱基改变无特殊病理意义。在200名听力正常对照和806名散发感音神经性聋患者中未筛查到该突变。常见耳聋基因GJB2筛查显示部分家系成员包括患者和听力正常者均携带235delC杂合突变。成功构建患者和正常听力对照的淋巴细胞系,Northern blot分析显示突变后tRNA-His水平较正常对照减少75%。线粒体基因的翻译产物较正常对照减少47%,细胞总耗氧率较正常对照减少36%,差异皆有统计学意义。 结论： 线粒体基因tRNA His T12201C异质性突变为该家系成员耳聋的主要分子基础,突变异质性比例与耳聋发病年龄和严重程度存在一定相关性。T12201C突变造成tRNA代谢异常,tRNA His的水平明显减少,使线粒体蛋白合成和呼吸功能显著降低,ATP合成减少,活性氧族增加,最终导致耳聋。 第二部分一个Waardenburg综合征家系的致病基因研究 目的： 研究一个中国Waardenburg综合征家系的临床表型特征和遗传学致病基因,及其基因型和表型间的关系。 方法： 经耳聋先证者获悉一个综合征型聋家系,进行家系调查、听力学检测和全身检查,并在签署知情同意书后采集静脉血5ml,提取全基因组DNA。分析整理临床资料,绘制系谱图,判断遗传方式。聚合酶链反应扩增候选基因MITF全部9个外显子及其侧翼序列,PCR产物纯化测序后与参考序列比对,寻找突变和多态改变。对发现的突变分析氨基酸及编码蛋白质的改变,并进行氨基酸多物种间保守性分析。同时对所有家系成员进行常见耳聋基因GJB2、线粒体DNA 12S rRNA的测序筛查。在100名相同遗传背景的正常听力对照中对发现的突变进行测序筛查。 结果： 该家系诊断为Waardenburg综合征Ⅱ型,主要临床表现为棕褐色雀斑、早白发、虹膜异色、先天性感音神经性聋。根据系谱图判断为常染色体显性遗传方式,但家系中不同个体的表型存在明显差异。MITF基因外显子全测序在第8外显子发现c.[742-743delAAinsT;746-747delCA]杂合突变,12名家系成员携带该突变,与临床表型共分离。突变导致MITF蛋白第248位氨基酸由赖氨酸转变为终止密码子TAG,正常时419个氨基酸残基的蛋白截短为247个氨基酸,丧失了蛋白重要的功能结构域bHLH,导致单倍剂量不足。该氨基酸位点在多物种间高度保守。同时发现先证者不仅携带MITF基因的杂合突变,而且携带GJB2基因c. [109GA]+[235delC]复合杂合致病突变。其他家系成员无GJB2基因双致病突变,所有成员线粒体DNA 12S rRNA均未发现致病突变。100名听力正常对照中未发现MITF基因第8外显子任何突变或多态改变,也未发现GJB2基因c.[109GA]+[235delC]复合杂合致病突变。 结论： Waardenburg综合征Ⅱ型临床表型多变,该家系成员的主要遗传学病因为MITF基因c.[742-743delAAinsT; 746-747delCA]杂合突变,这是一个新的WS2复杂突变。先证者同时存在MITF基因突变和GJB2基因双致病突变,两个基因可能在其耳聋的表型中均发挥作用。研究拓展了WS2基因突变谱,其基因型和表型的关系需进一步研究。
[Abstract]:The first part is a genetic study of a maternal hereditary deafness family.
Objective:
To study the genetic etiology and pathogenesis of maternal hereditary deafness in a large family of five generations, and to elucidate the mechanism of deafness among its members, so as to provide theoretical basis for the prevention and treatment of hereditary deafness.
Method:
A large family with hereditary deafness was investigated and detailed clinical and audiological data were collected. Venous blood was collected after informed consent was signed. Genome-wide DNA was extracted from venous blood. The complete mitochondrial genome sequence of maternal members and the common deafness gene GJB2 were amplified and sequenced by polymerase chain reaction. Pyrophosphate sequencing was used to quantify the heterogeneity of mitochondrial DNA deafness-related mutations and analyze the correlation between the proportion of heterogeneity and the degree of deafness. Evolutionary conservatism among species was analyzed for the mutation sites. The total mitochondrial RNA was extracted from the lymphocyte lines of deaf patients and normal controls. The level of tRNA was detected by Northern blot, and the protein synthesis of mitochondrial gene and the cell oxygen consumption were measured.
Result:
This family is in line with maternal inheritance. The occurrence of deafness has nothing to do with the use of aminoglycoside antibiotics. It is bilateral symmetrical, delayed, progressive sensorineural deafness with high-frequency hearing loss as the main cause, and gradually accumulates full-frequency, often accompanied by high-pitched tinnitus. Mitochondrial genome sequence analysis showed that maternal members belonged to the same mitochondrial haplotype Z, and there were 43 alterations. Among them, T12201C was a heterogeneous mutation located in the receptor arm of the secondary structure of tRNA-His gene, which made the base pairing from A-U to A-C. The site was highly conserved among species and the mutation heterogeneity ratio was high. The mutation was not detected in 200 normal hearing control subjects and 806 sporadic sensorineural deafness patients. The common deafness gene GJB2 screening showed that 235 delC heterozygous mutations were carried in some family members including patients and normal hearing subjects. Northern blot analysis showed that the level of tRNA-His decreased by 75%, the translation products of mitochondrial genes decreased by 47% and the total cell oxygen consumption decreased by 36% compared with normal control.
Conclusion:
The heterogenous mutation of the mitochondrial gene tRNA His T12201C was the main molecular basis of deafness in this family. The proportion of heterogenous mutation was correlated with the age and severity of deafness. The increase in reactive oxygen species leads to deafness.
The second part is a pathogenic gene study of a family with Waardenburg syndrome.
Objective:
To study the clinical phenotypic characteristics and genetic pathogenic genes of a Chinese Waardenburg syndrome family and the relationship between genotype and phenotype.
Method:
The proband of deafness was informed of a family with syndromic deafness. Family investigation, audiological examination and general examination were carried out. After signing the informed consent, 5 ml of venous blood was collected to extract genome-wide DNA. Clinical data were analyzed, pedigree maps were drawn and genetic patterns were judged. Flanking sequences were purified and sequenced by PCR products and compared with reference sequences for mutation and polymorphism. Amino acids and coding proteins were analyzed for mutation, and amino acid polymorphism was analyzed. The common deafness genes GJB2 and mitochondrial DNA 12S rRNA were sequenced and screened for all family members. The mutation was screened in normal hearing control with genetic background.
Result:
The pedigree was diagnosed as Waardenburg syndrome type II. The main clinical manifestations were brown-brown freckles, early white hair, iris heterochromia and congenital sensorineural deafness. 3 delAAinsT; 746-747 delCA] heterozygous mutation, carried by 12 family members and co-segregated with clinical phenotype. The mutation resulted in the conversion of 248 amino acids of MITF protein from lysine to TAG. Normally, the protein of 419 amino acid residues was truncated to 247 amino acids, resulting in the loss of bHLH, an important functional domain of the protein, leading to haploids. The amino acid locus was highly conserved among species. The proband was found to carry not only heterozygous mutations of MITF gene, but also complex heterozygous mutations of GJB2 gene c.109 GA] + [235 delC]. No double pathogenic mutation of GJB2 gene was found in other family members, and no mutation of mitochondrial DNA 12S rRNA was found in all members. No mutation or polymorphism in exon 8 of MITF gene was found in normal controls, and no heterozygous mutation in GJB2 gene C. [109 GA] + [235 delC] was found.
Conclusion:
The main genetic cause of Waardenburg syndrome is the heterozygous mutation of MITF gene C. [742-743 delAAinsT; 746-747 delCA], which is a new complex WS2 mutation. The proband has both MITF gene mutation and GJB2 gene double pathogenic mutation. Both genes may play an important role in the phenotype of deafness. Studies have expanded the mutation spectrum of WS2 gene, and the relationship between genotype and phenotype needs further study.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R764.43
本文编号：2237705
[Abstract]:The first part is a genetic study of a maternal hereditary deafness family.
Objective:
To study the genetic etiology and pathogenesis of maternal hereditary deafness in a large family of five generations, and to elucidate the mechanism of deafness among its members, so as to provide theoretical basis for the prevention and treatment of hereditary deafness.
Method:
A large family with hereditary deafness was investigated and detailed clinical and audiological data were collected. Venous blood was collected after informed consent was signed. Genome-wide DNA was extracted from venous blood. The complete mitochondrial genome sequence of maternal members and the common deafness gene GJB2 were amplified and sequenced by polymerase chain reaction. Pyrophosphate sequencing was used to quantify the heterogeneity of mitochondrial DNA deafness-related mutations and analyze the correlation between the proportion of heterogeneity and the degree of deafness. Evolutionary conservatism among species was analyzed for the mutation sites. The total mitochondrial RNA was extracted from the lymphocyte lines of deaf patients and normal controls. The level of tRNA was detected by Northern blot, and the protein synthesis of mitochondrial gene and the cell oxygen consumption were measured.
Result:
This family is in line with maternal inheritance. The occurrence of deafness has nothing to do with the use of aminoglycoside antibiotics. It is bilateral symmetrical, delayed, progressive sensorineural deafness with high-frequency hearing loss as the main cause, and gradually accumulates full-frequency, often accompanied by high-pitched tinnitus. Mitochondrial genome sequence analysis showed that maternal members belonged to the same mitochondrial haplotype Z, and there were 43 alterations. Among them, T12201C was a heterogeneous mutation located in the receptor arm of the secondary structure of tRNA-His gene, which made the base pairing from A-U to A-C. The site was highly conserved among species and the mutation heterogeneity ratio was high. The mutation was not detected in 200 normal hearing control subjects and 806 sporadic sensorineural deafness patients. The common deafness gene GJB2 screening showed that 235 delC heterozygous mutations were carried in some family members including patients and normal hearing subjects. Northern blot analysis showed that the level of tRNA-His decreased by 75%, the translation products of mitochondrial genes decreased by 47% and the total cell oxygen consumption decreased by 36% compared with normal control.
Conclusion:
The heterogenous mutation of the mitochondrial gene tRNA His T12201C was the main molecular basis of deafness in this family. The proportion of heterogenous mutation was correlated with the age and severity of deafness. The increase in reactive oxygen species leads to deafness.
The second part is a pathogenic gene study of a family with Waardenburg syndrome.
Objective:
To study the clinical phenotypic characteristics and genetic pathogenic genes of a Chinese Waardenburg syndrome family and the relationship between genotype and phenotype.
Method:
The proband of deafness was informed of a family with syndromic deafness. Family investigation, audiological examination and general examination were carried out. After signing the informed consent, 5 ml of venous blood was collected to extract genome-wide DNA. Clinical data were analyzed, pedigree maps were drawn and genetic patterns were judged. Flanking sequences were purified and sequenced by PCR products and compared with reference sequences for mutation and polymorphism. Amino acids and coding proteins were analyzed for mutation, and amino acid polymorphism was analyzed. The common deafness genes GJB2 and mitochondrial DNA 12S rRNA were sequenced and screened for all family members. The mutation was screened in normal hearing control with genetic background.
Result:
The pedigree was diagnosed as Waardenburg syndrome type II. The main clinical manifestations were brown-brown freckles, early white hair, iris heterochromia and congenital sensorineural deafness. 3 delAAinsT; 746-747 delCA] heterozygous mutation, carried by 12 family members and co-segregated with clinical phenotype. The mutation resulted in the conversion of 248 amino acids of MITF protein from lysine to TAG. Normally, the protein of 419 amino acid residues was truncated to 247 amino acids, resulting in the loss of bHLH, an important functional domain of the protein, leading to haploids. The amino acid locus was highly conserved among species. The proband was found to carry not only heterozygous mutations of MITF gene, but also complex heterozygous mutations of GJB2 gene c.109 GA] + [235 delC]. No double pathogenic mutation of GJB2 gene was found in other family members, and no mutation of mitochondrial DNA 12S rRNA was found in all members. No mutation or polymorphism in exon 8 of MITF gene was found in normal controls, and no heterozygous mutation in GJB2 gene C. [109 GA] + [235 delC] was found.
Conclusion:
The main genetic cause of Waardenburg syndrome is the heterozygous mutation of MITF gene C. [742-743 delAAinsT; 746-747 delCA], which is a new complex WS2 mutation. The proband has both MITF gene mutation and GJB2 gene double pathogenic mutation. Both genes may play an important role in the phenotype of deafness. Studies have expanded the mutation spectrum of WS2 gene, and the relationship between genotype and phenotype needs further study.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R764.43
【参考文献】
相关博士学位论文 前2条
1 杨淑芝;综合征型耳聋临床表型特征分析及相关基因突变研究[D];中国人民解放军军医进修学院;2006年
2 汪振诚;人线粒体tRNA～（Leu（UUR））基因点突变的研究[D];第二军医大学;2003年
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