兩個中國遺傳性聾家系的致病基因研究
發(fā)布時間：2018-09-11 19:54
【摘要】：第一部分一個母系遺傳性聾家系的致病基因研究 目的： 研究一個五代母系遺傳性聾大家系的遺傳學病因及發(fā)病機制,闡明該家系成員耳聾的機制,為遺傳性聾的防治提供理論依據(jù)。 方法： 經(jīng)耳聾先證者獲悉一遺傳性聾大家系,經(jīng)家系調(diào)查采集詳細的臨床及聽力學資料,并在簽署知情同意書后采集靜脈血。由靜脈血提取全基因組DNA,對母系成員的線粒體基因組全序列及常見耳聾基因GJB2經(jīng)聚合酶鏈反應(yīng)擴增和測序進行檢測。對檢測到的線粒體DNA耳聾相關(guān)突變應(yīng)用焦磷酸測序進行異質(zhì)性定量,分析突變的異質(zhì)性比例與耳聾程度的相關(guān)性。對突變位點行物種間進化保守性分析。在相同遺傳背景的200名正常聽力對照和806名散發(fā)感音神經(jīng)性聾患者中進行突變篩查。同時建立耳聾患者和正常聽力對照的淋巴細胞系,提取細胞的線粒體總RNA,采用Northern blot檢測基因突變后tRNA水平的變化,并進行線粒體基因的蛋白合成和細胞耗氧量測量。 結(jié)果： 該家系符合母系遺傳規(guī)律,耳聾的發(fā)生與使用氨基糖甙類抗生素無關(guān),表現(xiàn)為雙側(cè)對稱、遲發(fā)性、進行性感音神經(jīng)性聾,以高頻聽力損失為主,逐漸累積全頻,常伴高調(diào)耳鳴�；颊唢D骨高分辨率CT無明顯異常,未伴隨明顯的其他系統(tǒng)疾病,屬于非綜合征型聾。線粒體基因組全序列分析顯示母系成員屬于同一線粒體單倍型Z,共存在43種堿基改變,其中T12201C為異質(zhì)性突變,位于tRNA—His基因二級結(jié)構(gòu)的接受臂,使堿基配對由A-U變?yōu)锳-C,該位點在多物種間高度保守,突變異質(zhì)性比例與耳聾發(fā)病年齡和嚴重程度存在相關(guān)性。其余堿基改變無特殊病理意義。在200名聽力正常對照和806名散發(fā)感音神經(jīng)性聾患者中未篩查到該突變。常見耳聾基因GJB2篩查顯示部分家系成員包括患者和聽力正常者均攜帶235delC雜合突變。成功構(gòu)建患者和正常聽力對照的淋巴細胞系,Northern blot分析顯示突變后tRNA-His水平較正常對照減少75%。線粒體基因的翻譯產(chǎn)物較正常對照減少47%,細胞總耗氧率較正常對照減少36%,差異皆有統(tǒng)計學意義。 結(jié)論： 線粒體基因tRNA His T12201C異質(zhì)性突變?yōu)樵摷蚁党蓡T耳聾的主要分子基礎(chǔ),突變異質(zhì)性比例與耳聾發(fā)病年齡和嚴重程度存在一定相關(guān)性。T12201C突變造成tRNA代謝異常,tRNA His的水平明顯減少,使線粒體蛋白合成和呼吸功能顯著降低,ATP合成減少,活性氧族增加,最終導致耳聾。 第二部分一個Waardenburg綜合征家系的致病基因研究 目的： 研究一個中國Waardenburg綜合征家系的臨床表型特征和遺傳學致病基因,及其基因型和表型間的關(guān)系。 方法： 經(jīng)耳聾先證者獲悉一個綜合征型聾家系,進行家系調(diào)查、聽力學檢測和全身檢查,并在簽署知情同意書后采集靜脈血5ml,提取全基因組DNA。分析整理臨床資料,繪制系譜圖,判斷遺傳方式。聚合酶鏈反應(yīng)擴增候選基因MITF全部9個外顯子及其側(cè)翼序列,PCR產(chǎn)物純化測序后與參考序列比對,尋找突變和多態(tài)改變。對發(fā)現(xiàn)的突變分析氨基酸及編碼蛋白質(zhì)的改變,并進行氨基酸多物種間保守性分析。同時對所有家系成員進行常見耳聾基因GJB2、線粒體DNA 12S rRNA的測序篩查。在100名相同遺傳背景的正常聽力對照中對發(fā)現(xiàn)的突變進行測序篩查。 結(jié)果： 該家系診斷為Waardenburg綜合征Ⅱ型,主要臨床表現(xiàn)為棕褐色雀斑、早白發(fā)、虹膜異色、先天性感音神經(jīng)性聾。根據(jù)系譜圖判斷為常染色體顯性遺傳方式,但家系中不同個體的表型存在明顯差異。MITF基因外顯子全測序在第8外顯子發(fā)現(xiàn)c.[742-743delAAinsT;746-747delCA]雜合突變,12名家系成員攜帶該突變,與臨床表型共分離。突變導致MITF蛋白第248位氨基酸由賴氨酸轉(zhuǎn)變?yōu)榻K止密碼子TAG,正常時419個氨基酸殘基的蛋白截短為247個氨基酸,喪失了蛋白重要的功能結(jié)構(gòu)域bHLH,導致單倍劑量不足。該氨基酸位點在多物種間高度保守。同時發(fā)現(xiàn)先證者不僅攜帶MITF基因的雜合突變,而且攜帶GJB2基因c. [109GA]+[235delC]復(fù)合雜合致病突變。其他家系成員無GJB2基因雙致病突變,所有成員線粒體DNA 12S rRNA均未發(fā)現(xiàn)致病突變。100名聽力正常對照中未發(fā)現(xiàn)MITF基因第8外顯子任何突變或多態(tài)改變,也未發(fā)現(xiàn)GJB2基因c.[109GA]+[235delC]復(fù)合雜合致病突變。 結(jié)論： Waardenburg綜合征Ⅱ型臨床表型多變,該家系成員的主要遺傳學病因為MITF基因c.[742-743delAAinsT; 746-747delCA]雜合突變,這是一個新的WS2復(fù)雜突變。先證者同時存在MITF基因突變和GJB2基因雙致病突變,兩個基因可能在其耳聾的表型中均發(fā)揮作用。研究拓展了WS2基因突變譜,其基因型和表型的關(guān)系需進一步研究。
[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.
【學位授予單位】：復(fù)旦大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R764.43
本文編號：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.
【學位授予單位】：復(fù)旦大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R764.43
【參考文獻】
相關(guān)博士學位論文 前2條
1 楊淑芝;綜合征型耳聾臨床表型特征分析及相關(guān)基因突變研究[D];中國人民解放軍軍醫(yī)進修學院;2006年
2 汪振誠;人線粒體tRNA～（Leu（UUR））基因點突變的研究[D];第二軍醫(yī)大學;2003年
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