遲發(fā)性耳聾家系臨床表型及相關(guān)基因突變研究
發(fā)布時間：2018-08-17 09:05
【摘要】：目的：耳聾是導(dǎo)致人類交流障礙最常見的疾病，，其病因中最主要的因素為遺傳因素，而聽力正常人群中耳聾基因的攜帶率并不低，這也是耳聾發(fā)病率之所以居高不下的重要原因之一。雖然目前國內(nèi)外學(xué)者對遺傳性耳聾的研究很多，但仍有一部分耳聾病人無法確定其致病原因。本課題組收集了5個遲發(fā)性耳聾家系，擬通過分析和總結(jié)其臨床表型的特征與基因型的相關(guān)性，探討常見遲發(fā)性耳聾家系的分子基因，為這些家庭提供遺傳咨詢和婚育指導(dǎo)，力爭豐富遺傳性耳聾基因突變譜。 方法：收集病例，采集家系信息，分別編號為：HBZ-01、HBZ-02、HBX、HBD、HBM。收集家系成員血液樣本，提取外周靜脈血基因組DNA，采取PCR擴(kuò)增后直接測序，首先篩查國人常見突變基因，包括GJB2、SLC26A4以及線粒體基因（mtDNA12S rRNA）。篩查陽性者，分析其臨床表型與基因型的相關(guān)性，對耳聾患者及家庭成員提供遺傳咨詢；篩查陰性者，依據(jù)表型相似性原則，在目前已定位的DFNA中篩選可疑致病基因，陽性者，同前；陰性者，進(jìn)行耳聾基因組高通量測序，分析可疑的致病基因位點，陽性者對該突變點進(jìn)行PCR擴(kuò)增，擴(kuò)增產(chǎn)物進(jìn)行Sanger測序驗證，依舊為陰性者下一步將考慮進(jìn)行全外顯子組測序。 結(jié)果: 1在1個遺傳方式不明的耳聾小家系（HBZ-01家系）中檢測出GJB2基因的復(fù)合雜合突變c.35insG/c.232GA，家系耳聾成員中相同的突變類型卻出現(xiàn)不同的臨床表型，患者發(fā)病年齡為0~20歲，均為感音神經(jīng)性耳聾，語后聾患者早期表現(xiàn)為高頻聽力下降，后逐漸累及中、低頻。c.35insG/c.232GA復(fù)合雜合突變在HBZ-01家系內(nèi)與耳聾表型共分離。初步結(jié)果顯示，在耳聾人群中GJB2基因的這種復(fù)合雜合突變較少。 2在1個表現(xiàn)為常染色體顯性遺傳非綜合征型耳聾的河北家系（HBZ-02家系）中，發(fā)現(xiàn)該家系的表型中除了聽力下降外，部分患者還伴有前庭功能障礙。我們將可能的致病基因鎖定在已報道的COCH基因上，篩查后在其第4和11外顯子上發(fā)現(xiàn)了新的突變位點，其中第4外顯子上為致病位點，c.226GA (p.76AT)，該突變與臨床表型共分離，在多個物種間亦為保守性序列。第11外顯子上為c.1055GA (p.352TS)，后經(jīng)驗證發(fā)現(xiàn)該位點為多態(tài)性改變。COCH基因是為數(shù)不多的耳蝸和前庭共相關(guān)基因，對其分子機(jī)制的全面認(rèn)識還需深入的研究。HBZ-02家系的臨床表型與以往報道的DFNA9家系相似，這也進(jìn)一步證實了表型相似性原則，準(zhǔn)確判斷特征性的遺傳性耳聾表型對指導(dǎo)基因型分析具有重要意義。 3在1個常染色體顯性非綜合征型耳聾家系（HBX家系）中首次發(fā)現(xiàn)了中國人群中報道較少見的EYA4基因突變，c.544_545insA，該位點突變位于EYA4基因第8外顯子，導(dǎo)致氨基酸的移碼突變（p.E182X），c.544_545insA突變在HBX家系家系內(nèi)與耳聾表型共分離。與以往國外報道的DFNA10家系不同的是，該家系聽力曲線圖不是典型的“谷型”圖而是平坦型或陡降型，發(fā)病年齡跨度較大，分布在20~40歲之間，部分成員伴有耳鳴。該突變也是首次發(fā)現(xiàn)的新突變。 4其他2個家系(HBM家系、HBD家系)，利用基于下一代測序技術(shù)和外顯子捕獲技術(shù)的耳聾基因組高通量測序（含已知的130個耳聾基因），未發(fā)現(xiàn)明確的致病基因，提示這兩個家系可能由一些少見的或未知的耳聾基因缺陷致聾，下一步可考慮進(jìn)行全外顯子組測序。 結(jié)論： 1由于遺傳性耳聾遺傳的異質(zhì)性，不同基因的突變可以導(dǎo)致同一種臨床表型；同一基因不同位點的突變可以導(dǎo)致不同的臨床表型，甚至是同一基因同一突變位點在同一家系的不同個體之間表型也可能不同。 2準(zhǔn)確識別特征性的耳聾臨床表型可以指導(dǎo)基因型的診斷。 3目前仍有許多耳聾患者的致病基因不明，遺傳性耳聾基因數(shù)據(jù)庫仍待完善。
[Abstract]:Objective: Deafness is the most common cause of communication disorders in humans. Genetic factors are the main cause of deafness, and the carrying rate of deafness genes is not low in normal hearing population. This is also one of the important reasons for the high incidence of deafness. We collected 5 families with delayed hearing loss to analyze and summarize the correlation between their clinical phenotype and genotype, and to explore the molecular genes of common families with delayed hearing loss, so as to provide genetic counseling and guidance in marriage and childbearing for these families, so as to enrich the genetic hearing loss. Gene mutation spectrum.
Methods: Collect the cases and collect the family information, which are HBZ-01, HBZ-02, HBX, HBD, HBM. Collect the blood samples of family members, extract the genomic DNA of peripheral venous blood, and then sequence it directly after PCR amplification. First, screen the common mutant genes in China, including GJB2, SLC26A4 and mitochondrial gene (mtDNA 12S rRNA). Correlation between clinical phenotype and genotype provides genetic counseling for deaf patients and family members; screening negative patients, according to the principle of phenotypic similarity, screening suspicious pathogenic genes in the currently located DFNA, positive, same; negative people, high-throughput sequencing of the deaf genome, analysis of suspicious pathogenic gene loci, positive pairs The mutation point was amplified by PCR, and the amplified product was verified by Sanger sequencing. Whole exome sequencing would be considered in the next step for those still negative.
Result:
1. Complex heterozygous mutation c.35insG/c.232GA of GJB2 gene was detected in a small family with unknown hereditary pattern of deafness (HBZ-01). The same mutation type in the deaf members of the family showed different clinical phenotypes. The onset age of the patients ranged from 0 to 20 years old, and they were all sensorineural deafness. After gradual involvement, low-frequency. c.35insG/c.232GA complex heterozygous mutation was co-segregated with the deaf phenotype in HBZ-01 family. Preliminary results showed that there were fewer such complex heterozygous mutations in the GJB2 gene in deaf people.
2 In a Hebei pedigree with autosomal dominant nonsyndromic hearing loss (HBZ-02), besides hearing loss, some patients were found to have vestibular dysfunction. The mutation site in exon 4 was the pathogenic site, C. 226GA (p. 76AT), which was co-segregated with clinical phenotype and conserved in many species. The clinical phenotype of HBZ-02 family is similar to that of the previously reported DFNA9 family, which further confirms the principle of phenotypic similarity. It is important to accurately determine the characteristic phenotype of hereditary deafness for guiding genotypic analysis.
3. A rare EYA4 gene mutation, c.544_545insA, was found in an autosomal dominant nonsyndromic deafness family (HBX family) for the first time in China. The mutation was found in exon 8 of the EYA4 gene, resulting in amino acid frameshift mutation (p.E182X), c.544_545insA mutation was separated from the phenotype of deafness in HBX family. Different from the previously reported DFNA 10 family, the audiogram of this family is not a typical "trough" pattern but a flat or steep descending pattern. The onset age span is large, ranging from 20 to 40 years old, and some members have tinnitus.
4 In the other two families (HBM family, HBD family), high-throughput sequencing of the deaf genome (including 130 known deafness genes) based on next-generation sequencing and exon capture techniques was used. No definite pathogenic genes were found, suggesting that these two families may be deaf from some rare or unknown deafness gene defects. The next step may be considered. Whole exon group sequencing.
Conclusion:
Because of the genetic heterogeneity of hereditary deafness, mutations in different genes can lead to the same clinical phenotype; mutations in different loci of the same gene can lead to different clinical phenotypes, and even the same gene at the same mutation locus may lead to different phenotypes among different individuals in the same family.
2 accurately identifying the clinical phenotype of characteristic deafness can guide the diagnosis of genotypes.
3 there are still many unknown genes of deafness patients. The genetic deafness gene database needs to be improved.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R764.43
[Abstract]:Objective: Deafness is the most common cause of communication disorders in humans. Genetic factors are the main cause of deafness, and the carrying rate of deafness genes is not low in normal hearing population. This is also one of the important reasons for the high incidence of deafness. We collected 5 families with delayed hearing loss to analyze and summarize the correlation between their clinical phenotype and genotype, and to explore the molecular genes of common families with delayed hearing loss, so as to provide genetic counseling and guidance in marriage and childbearing for these families, so as to enrich the genetic hearing loss. Gene mutation spectrum.
Methods: Collect the cases and collect the family information, which are HBZ-01, HBZ-02, HBX, HBD, HBM. Collect the blood samples of family members, extract the genomic DNA of peripheral venous blood, and then sequence it directly after PCR amplification. First, screen the common mutant genes in China, including GJB2, SLC26A4 and mitochondrial gene (mtDNA 12S rRNA). Correlation between clinical phenotype and genotype provides genetic counseling for deaf patients and family members; screening negative patients, according to the principle of phenotypic similarity, screening suspicious pathogenic genes in the currently located DFNA, positive, same; negative people, high-throughput sequencing of the deaf genome, analysis of suspicious pathogenic gene loci, positive pairs The mutation point was amplified by PCR, and the amplified product was verified by Sanger sequencing. Whole exome sequencing would be considered in the next step for those still negative.
Result:
1. Complex heterozygous mutation c.35insG/c.232GA of GJB2 gene was detected in a small family with unknown hereditary pattern of deafness (HBZ-01). The same mutation type in the deaf members of the family showed different clinical phenotypes. The onset age of the patients ranged from 0 to 20 years old, and they were all sensorineural deafness. After gradual involvement, low-frequency. c.35insG/c.232GA complex heterozygous mutation was co-segregated with the deaf phenotype in HBZ-01 family. Preliminary results showed that there were fewer such complex heterozygous mutations in the GJB2 gene in deaf people.
2 In a Hebei pedigree with autosomal dominant nonsyndromic hearing loss (HBZ-02), besides hearing loss, some patients were found to have vestibular dysfunction. The mutation site in exon 4 was the pathogenic site, C. 226GA (p. 76AT), which was co-segregated with clinical phenotype and conserved in many species. The clinical phenotype of HBZ-02 family is similar to that of the previously reported DFNA9 family, which further confirms the principle of phenotypic similarity. It is important to accurately determine the characteristic phenotype of hereditary deafness for guiding genotypic analysis.
3. A rare EYA4 gene mutation, c.544_545insA, was found in an autosomal dominant nonsyndromic deafness family (HBX family) for the first time in China. The mutation was found in exon 8 of the EYA4 gene, resulting in amino acid frameshift mutation (p.E182X), c.544_545insA mutation was separated from the phenotype of deafness in HBX family. Different from the previously reported DFNA 10 family, the audiogram of this family is not a typical "trough" pattern but a flat or steep descending pattern. The onset age span is large, ranging from 20 to 40 years old, and some members have tinnitus.
4 In the other two families (HBM family, HBD family), high-throughput sequencing of the deaf genome (including 130 known deafness genes) based on next-generation sequencing and exon capture techniques was used. No definite pathogenic genes were found, suggesting that these two families may be deaf from some rare or unknown deafness gene defects. The next step may be considered. Whole exon group sequencing.
Conclusion:
Because of the genetic heterogeneity of hereditary deafness, mutations in different genes can lead to the same clinical phenotype; mutations in different loci of the same gene can lead to different clinical phenotypes, and even the same gene at the same mutation locus may lead to different phenotypes among different individuals in the same family.
2 accurately identifying the clinical phenotype of characteristic deafness can guide the diagnosis of genotypes.
3 there are still many unknown genes of deafness patients. The genetic deafness gene database needs to be improved.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R764.43
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