135例大前庭导水管耳聋患者SLC26A4基因突变分析

目的研究华东地区非综合征性大前庭导水管耳聋患者SLC26A4基因的突变情况,以进一步了解其突变谱,为阐明EVA的分子机制和指导基因诊断提供依据。方法通过巢式聚合酶链式反应扩增目的片段和Sanger测序对135例明确诊断的散发非综合征性EVA耳聋患者SLC26A4基因的外显子及侧翼序列进行变异检测。结果在135例EVA耳聋患者中,明确为SLC26A4双等位基因突变致病的占74.07%(100/135),单等位基因突变的占7.41%(10/135),18.52%(25/135)的患者未检测到任何SLC26A4基因突变。共检出51种SLC26A4基因突变,其中,c.919-2A>G突变和p.H723R突变最常见,分别占总检出突变的50%(105/210)和10.48%(22/210)。结论与既往报道一致,c.919-2A>G突变为中国人群SLC26A4基因的最常见变异。有相当一部分患者未检出SLC26A4双等位基因突变,提示可能有其他致病因子参与EVA的发生,有待进一步研究阐明。     

95例前庭水管扩大核心家系SLC26A4基因特异突变图谱

目的探讨中国人群中前庭水管扩大（enlarged vestibular aqueduct，EVA）患者SLC26A4基因突变图谱，为相关基因的筛查及临床应用奠定基础。方法采集95例EVA核心家系，募集84名听力正常者及46名内耳结构正常的听力损失患者作为对照。提取基因组DNA，应用聚合酶链反应（PCR）的方法扩增SLC26A4基因的21个外显子，纯化PCR产物后直接测序，使用DNAStar及BioEdit序列比对软件分析SLC26A4基因的突变位点，利用Clustal软件进行同源氨基酸序列比对分析。结果在95例EVA核心家系中，93例发生了SLC26A4基因突变，约占97．9％（93／95）。其中双等位基因突变占88．4％（84／95），单等位基因和未发现突变的家系分别占9．5％和2．1％。93个家系中共发现38种突变，包括23种国际上尚未报道的突变，15种已报突变（5种仅报道于中国家系）。IVS7—2A〉G是所有突变中最常见的突变，其在所有突变体中约占57．63％（102／177），75（75／95）个家系发生了此种突变。结论95例中国EVA核心家系的SLC26A4基因调查中，发现了一个中国特异性的SLC26A4基因突变谱：97．9％的中国EVA患者均可检测到SLC26A4基因的突变，双等位基因突变率约为88．4％；23种新的突变和5种仅报道于中国家系的突变在其他人群未曾检测到；IVS7—2A〉G为中国人群前庭水管扩大患者最常见的突变。     

内耳畸形相关SLC26A4基因的研究

目的:研究内耳畸形相关SLC26A4基因在大前庭水管综合征(LVAS)、Mondini畸形及不伴前庭水管扩大的耳蜗畸形耳聋人群中的突变情况,在分子水平上探讨内耳畸形的发病机制,为基因诊断提供理论基础。方法:收集14例散发LVAS、6例Mondini畸形(伴前庭水管扩大)及7例不伴前庭水管扩大的耳蜗畸形耳聋患者的外周血DNA样本及临床资料,利用PCR扩增目的基因后直接测序的方法对所有患者进行SLC26A4基因全编码序列检测,同时进行GJB2、线粒体12SrRNA 1555/1494位点排除性检测。结果:14例LVAS患者中12例(85.7%)具有SLC26A4双等位基因(纯合或复合杂合)突变,2例(14.3%)有单等位基因突变。6例Mondini畸形患者均具有SLC26A4双等位基因(纯合或复合杂合)突变。7例不伴前庭水管扩大的耳蜗畸形耳聋患者中均未查出SLC26A4基因突变。27例患者均未发现GJB2、线粒体12SrRNA 1555/1494致病突变。结论:LVAS及Mondini畸形与SLC26A4基因突变密切相关,且Mondini畸形比单纯LVAS基因突变率更高,而不伴前庭水管扩大的耳蜗畸形耳聋患者未发现SLC26A4基因突变,尚需对该部分耳聋患者进行深入的分子病因学研究。     

SLC26A4基因热点突变检测对大前庭导水管综合征患儿的诊断作用

目的:利用基因诊断方法检测非综合征性耳聋患者中SLC26A4基因热点突变的发生频率,并通过颞骨CT检查证实基因筛查发现大前庭水管综合征的可行性.方法:研究对象为92例非综合征性耳聋患儿,以序列分析方法检测SLC26A4基因IVS7-2 A>G和H723R突变情况,阳性者进行颞骨CT检查.结果:11例(12.0%)患儿携带有SLC26A4基因的突变,包括双等位基因突变者5例,单等位基因突变者6例.颞骨CT检查发现双等位基因突变者均为大前庭导水管综合征患者.结论:非综合征性耳聋患儿中SLC26A4基因突变发生率较高,耳聋基因检测对大前庭导水管综合征诊断有一定作用.     

国人前庭水管扩大患者SLC26A4基因的特异性突变

目的探讨SLC26A4基因与前庭水管扩大(enlarged vestibular aqueduct,EVA)的关系及其在中国人群EVA患者中突变的频率及分布情况,为相关基因的筛查及临床应用奠定基础。方法采集中国人群中的38例EVA核心家系,提取基因组DNA,应用聚合酶链反应(PCR)的方法扩增SLC26A4基因的21个外显子,纯化PCR产物后直接测序,使用DNAStar及Bioedit序列比对软件分析SLC26A4基因的突变位点。结果35例EVA核心家系的SLC26A4基因发生了突变,所占比例约为92.1%(35/38)。在发现的12种突变类型中,6种为新的突变类型(待发表),其余类型国际上已见报道:分别为IVS7-2A>G、L676Q、H723R、IVS15 5G>A、R409H和M147V。在所有的突变中,IVS7-2A>G突变的发生率最高,约为81.6%(31/38)。结论中国人群的EVA患者中存在SLC26A4基因的多种突变类型,其中IVS7-2A>G突变的发生率最高(81.6%),应视为特异性热点突变,建议在中国人群进行SLC26A4基因的突变热点的普遍筛查,降低EVA患儿的出生率。     

大前庭水管综合征的基因诊断和SLC26A4基因突变分析

目的应用变性高效液相色谱分析和序列分析方法进行大前庭水管综合征患者的SLC26A4（PDS）基因全序列扫描,分析大前庭水管综合征的SLC26A4基因型变化和遗传特征.方法来自35个家庭的38例患者多患中重度感音性耳聋,所有患者颞骨CT均显示前庭水管明显扩大.以高效液相色谱分析结合序列分析进行SLC26A4基因分析.结果共发现32个先证者携带有SLC26A4基因突变,其中纯合突变11例,复合突变9例,单一杂合突变12例,3个先证者未发现突变,在所有受检患者中突变发现率91.4%（32/35）.共发现8种突变类型58个突变,其中IVS7-2 A＞G突变为中国人最常见的SLC26A4突变,共发现其纯合型10例,杂合突变13例,即有71.9%的患者（23/32）携带此种突变,2168A＞G为第二常见突变,共有8名患者携带此杂合突变,另发现1229C＞T,IVS15＋5G＞A,1226 G＞A,1199-1200insT,946 G＞T,916-917 ins G突变形式,后三种突变为国内外尚未报道的新突变.四种复发性突变IVS7-2 A＞G、2168A＞G、1229C＞T、IVS15＋5G＞A在此组病例的30例患者均有出现.三个多发家庭的同胞患者均具有一样的SLC26A4突变.结论大前庭水管综合征是典型的常染色体隐性遗传疾病,SLC26A4基因突变是其明确的致病因素,SLC26A4基因检测是诊断大前庭水管综合征的重要方法之一.     

河南省安阳市特教学校非综合征型聋学生SLC26A4基因突变热点区域的分析

目的利用基因诊断方法调查河南省安阳地区聋哑学生SLC26A4基因突变热点区域的基因突变频率。方法对安阳市特教学校151例聋哑学生采集外周血并提取DNA,以序列分析方法检测SLC26A4基因的突变热点区域（包括外显子7＋8、19、10、17、15）。结果SLC26A4基因突变热点区域序列分析结果显示31.79%（48/151）患者检测到了SLC26A4基因的突变,包括双等位基因突变者26例（17.22%）,单等位基因突变者22例（14.57%）。结论安阳地区耳聋人群的SLC26A4基因突变频率明显高于国内外的报道,提示此地区特有的遗传性致聋病因和此地区人群具有SLC26A4基因热点突变区域的高携带率。     

荧光PCR法在非综合征型遗传性耳聋基因诊断中的应用研究

摘要：目的分析GJB2、SLC26A4和mtDNA12SrRNA基因热点突变在非综合征型遗传性耳聋人群中的突变谱和突变频率。方法采用荧光PCR法,针对本院收集的126例非综合征型耳聋患者进行中国人群常见的3个耳聋致病基因GJB2、SLC26A4和mtDNA 12SrRNA的10个热点突变的筛查,分析总结突变数据。阳性结果进一步采用直接测序法进行验证。结果应用荧光PCR技术在126例非综合征型耳聋（non syndromic hearing loss, NSHL）患者中检测出携带基因突变的患者31例,阳性率为24.6％（31/126）,其中GJB2双等位基因突变、SLC26A4双等位基因突变和12SrRNA的均质突变分别占该人群分子病因的6.35%（8/126）、2.33%（3/126）和3.17%（4/126）。此外,IVS7-2A>G单等位基因突变的检出率高达11.11%（14/126）。GJB2 c.235delC和SLC26A4 IVS7-2A>G是本研究中最为常见的热点突变。进一步采用直接测序法验证阳性位点,其结果与荧光PCR法一致。结论GJB2双等位基因突变是本研究人群最为常见的分子致病因素,其次为SLC26A4双等位基因突变和12SrRNA的均质突变。GJB2 c.235delC和SLC26A4 IVS7-2A>G是本研究中最为常见的热点突变。     

FMCA技术分析大前庭水管综合征家系及产前诊断

目的应用FMCA技术检测双侧前庭水管扩大家系SLC26A4基因,探讨前庭水管扩大与SLC26A4基因的关系及FMCA技术最优条件。方法收集EVA家系临床资料,基于荧光定量PCR熔解曲线平台,运用FMCA技术分析107例正常人,6个EVA家系19位成员的SLC26A4基因IVS7-2A>G(919-2A>G),2168A>G及1229C>T突变位点。并用直接测序技术予以验证。结果 107例正常人中有2例SLC26A4基因杂合突变,突变率为1.87%;EVA家系中有3例SLC26A4基因纯合突变,3例SLC26A4基因复合杂合突变,10例SLC26A4基因杂合突变,3例无SLC26A4基因突变,SLC26A4基因突变率为84.21%。对家系a进行产前诊断,结果为IVS7-2A>G/1229C>T复合杂合突变。所有检测结果与测序结果一致,符合率为100%。结论 FMCA平台能快速检测SLC26A4基因的IVS7-2A>G,2168A>G与1229C>T突变,其操作简单,成本低。且经测序技术验证,结果准确。可作为诊断遗传性疾病及产前诊断的快速有效方法。     

SLC26A4基因突变致聋患者的临床表现与康复措施的选择

目的分析非综合征性耳聋患儿的致聋基因和携带SLC26A4基因突变者的临床表现、康复设备使用及康复教育机构的选择情况。方法对195例非综合征性耳聋患儿进行耳聋基因芯片筛查。对34例携带SLC26A4基因突变的耳聋患儿进行了进一步的测序分析,并对这些聋儿进行病史和听力学检查回顾性分析以及康复设备使用和康复教育机构选择的随访。结果①在195例耳聋患者中,基因芯片检测出的遗传性耳聋比率为43．59％,其中GJB2、SLC26A4致病突变的携带率分别为24．10％、17．44％。②在34例携带SLC26A4基因突变的耳聋患者中,芯片检测出纯合或者复合杂合突变者13例。通过随后的测序方法,在15例SLC26A4基因单杂合突变患者中检出第二个致病突变位点,诊断为复合杂合突变患者。这34名患儿中,中度耳聋3人,重度耳聋12人,极重度耳聋19人;听力波动者10人,没有明显波动者24人127人选择了助听器,7人进行了人工耳蜗手术。28例SLC26A4基因纯合或者复合杂合突变患者中,前庭水管扩大者20人,1人正常,7人没有接受CT检查。③34名携带SLC26A4基因突变的聋儿中,3～6岁者共19人,其中13人选择了康复中心,3人选择了普通幼儿园,3人选择了家庭康复。6岁以上者15人,13人选择了普通小学,2人选择了聋校。结论①结果表明,遗传因素是儿童耳聋的重要致病原因,GJB2和SLC26A4是两个主要的致病基因。②测序和芯片联合的方法可以增加SLC26A4突变致聋的诊断率。③在3～10岁年龄段,91．18％的SLC26A4基因突变患儿为重度一极重度耳聋,听力无明显波动者多于听力波动者,影像学多显示为前庭水管扩大。④康复中心和普通小学是携带SLC26A4基因突变的耳聋儿童最常选择的康复教育机构。     

The influence of mutations in the SLC26A4 gene on the temporal bone in a population with enlarged vestibular aqueduct

OBJECTIVE: To correlate genetic and audiometric findings with a detailed radiologic analysis of the temporal bone in patients with enlarged vestibular aqueduct (EVA) to ascertain the contribution of SLC26A4 gene mutations to this phenotype. DESIGN: A retrospective review of patients with EVA identified in a database of pediatric hearing-impaired patients. SETTING: A tertiary care pediatric referral center. PATIENTS: Seventy-one children with EVA and screening results for SLC26A4 mutations. MAIN OUTCOME MEASURES: Genetic screening results, audiometric thresholds, and radiographic temporal bone measurements. RESULTS: Seventy-one children with EVA were screened for SLC26A4 mutations. Mutations were found in 27% of children overall, while only 8% had biallelic mutations. The mean initial pure-tone average (PTA) was 59 dB; the mean final PTA was 67 dB. A bilateral EVA was found in 48 (67%) of the children; a unilateral EVA was found in 23 (33%). Progressive hearing loss (in at least 1 ear) was seen in 29 (41%) of the patients. The strongest genotype-phenotype interaction was seen in children with a bilateral EVA. Among children with SLC26A4 mutations, there was a significantly wider vestibular aqueduct at the midpoint and a wider vestibule width (P < .05) than in children without the mutation. Among patients with a bilateral EVA, children with any SLC26A4 mutation were more likely to have a more severe final PTA (64 dB vs 32 dB), larger midpoint measurement (2.1 vs 1.1 mm), and larger operculum measurement (3.0 vs 2.0 mm) than those without the mutation in their better-hearing ear (P < .05). CONCLUSIONS: In a population of pediatric patients with an EVA and hearing loss, SLC26A4 mutations are a contributor to the phenotype. Our data suggest that other genetic factors also have important contributions to this phenotype. The presence of an abnormal SLC26A4 allele, even in the heterozygous state, was associated with greater enlargement of the vestibular aqueduct, abnormal development of the vestibule, and possibly a stable hearing outcome.     

广东省59例大前庭水管综合征患者SLC26 A4基因突变及表型分析

目的：探讨广东省大前庭水管综合征（enlargement of vestibular aqueduct syndrome,EVAS）患者SLC26 A4基因位点突变及相关听力表型,为研究 EVAS 发病机制提供参考。方法采用基因芯片法对59例EVAS患儿进行 SLC26 A4基因 IVS7－2 A＞G：2168 A＞G 位点检测,并行颞骨 CT 影像学检查。结果59例EVAS患者中21例（35．59％）为SLC26 A4双等位基因（纯合或复合杂合）突变,其中16例为IVS7－2 A＞G纯合突变,2例为2168A＞G纯合突变,3例为IVS7－2A＞G、2168A＞G复合杂合突变,这21例CT均显示为双侧前庭水管扩大或其他内耳畸形;38例为 SLC26 A4单等位基因突变,其中31例为 IVS7－2 A＞G 杂合突变,7例为2168A＞G杂合突变,这38例中4例为前庭水管扩大伴Mondini畸形,2例表型正常,其余均为双侧前庭水管扩大。59例患儿均表现为重度－极重度聋。结论本组EVAS患者中SLC26 A4基因IVS7－2 A＞G位点的突变发生率最高,其次为2168A＞G;均表现为双耳重度或极重度感音神经性听力损失。     

SLC26A4 mutations are associated with a specific inner ear malformation

BACKGROUND AND AIM: Inner ear anomalies have been reported in approximately 30% of children with early onset deafness. Identification of causative genetic factors in a large proportion of these patients was not successful. Mutations in the SLC26A4 gene have been detected in individuals with enlarged vestibular aqueduct (EVA) or Mondini dysplasia. We aimed to characterize the inner ear anomalies associated with SLC26A4 mutations. METHODS: The SLC26A4 gene has been screened for mutations in 16 subjects from 14 unrelated Turkish families with a variety of inner ear anomalies ranging from Michel aplasia to incomplete partition-II and EVA. None of the patients was diagnosed to have a recognizable genetic syndrome. Additional four patients with Pendred syndrome from three families were included. RESULTS: Only one patient with EVA was found to have a heterozygous mutation (c.1586delT) in SLC26A4. All patients with Pendred syndrome had homozygous mutations and were noted to have either EVA or EVA associated with incomplete partition-II on the computed tomography of the temporal bone. CONCLUSION: SLC26A4 mutations are not associated with a large spectrum of inner ear anomalies. They, instead, result in a specific morphological appearance consistent with EVA or incomplete partition-II.     

Molecular analysis of the GJB2, GJB6 and SLC26A4 genes in Korean deafness patients

OBJECTIVES: Mutations in the GJB2, GJB6 and SLC26A4 genes are a frequent cause of hearing loss in a number of populations. However, little is known about the genetic causes of hearing loss in the Korean population. METHODS: We sequenced the GJB2 and GJB6 genes to examine the role of mutations in these genes in 22 hearing loss patients. We also sequenced the SLC26A4 gene in seven patients with inner ear malformations, including enlarged vestibular aqueduct (EVA) revealed by computer tomography. RESULTS: Coding sequence mutations in GJB2 were identified in 13.6% of the patients screened. Two different mutations, 235delC and T86R were found in three unrelated patients. The 235delC was the most prevalent mutation with an allele frequency of 6.9% in our patient group. No mutations, including 342-kb deletion, were found in GJB6 gene. Three different variants of SLC26A4 were identified in the EVA patients, including one novel mutation. Four EVA patients carried two mutant alleles of SLC26A4, and at least one allele in all patients was the H723R mutation, which accounted for 75% of all mutant alleles. CONCLUSIONS: Our results suggest that GJB2 and SLC26A4 mutations together make up a major cause of congenital hearing loss in the Korean population. Further studies may be able to identify other common variants that account for a significant fraction of hearing loss in the Korean population.     

Novel compound heterozygous mutations in SLC26A4 gene in a Chinese Han family with enlarged vestibular aqueduct

ObjectiveTo identify the disease-related SLC26A4 mutants in a Chinese Han pedigree associated with Enlarged vestibular aqueduct (EVA).MethodsEVA diagnosis was based on the family history, clinical examinations, systematically audiometric evaluations, high-resolution computed tomography (HRCT) of the temporal bone, and magnetic resonance imaging (MRI) of inner ear. Sanger sequencing and mutation analysis of the SLC26A4 gene were performed in all members of this family to identify the disease-related SLC26A4 mutants. Mutations in the SLC26A4 gene were compared with 200 ethnically matched control persons to exclude common polymorphism.ResultsAll members in this family were negative for systemic and thyroid diseases. There were three subjects (I-2, II-2 and II-3) with bilateral sensorineural deafness since childhood. Temporal bone HRCT scans and inner ear MRI showed bilateral enlarged vestibular aqueduct with Mondini malformation in II-2 and II-3. A novel SLC26A4 splice-site mutation c.1001 + 5G > C was identified in compound heterozygosity with the mutation c.919-2A > G in the proband and in II-2. This novel compound heterozygote of two splice site mutations was not found in 200 normal hearing Chinese Han controls.ConclusionsA novel splice site mutation of c.1001 + 5G > C was identified, and the novel compound heterozygote of two splice site mutations, c.1001 + 5G > C and c.919-2A > G, in the SLC26A4 gene has been linked to hearing impairment in EVA patients.     

SLC26A4 p.Thr410Met homozygous mutation in a patient with a cystic cochlea and an enlarged vestibular aqueduct showing characteristic features of incomplete partition type I and II

Mutations of SLC26A4 are associated with incomplete partition type II (IP-II) and isolated enlargement of the vestibular aqueduct (EVA). We experienced a congenitally deaf 6-year-old boy with a rare p.Thr410Met homozygous mutation in SLC26A4 who underwent bilateral cochlear implantation. He had bilateral inner ear malformation, in which the dilated vestibule and EVA were identical to those in IP-II, but the cochlea lacking a bony modiolus resembled that in incomplete partition type I. These results suggest that homozygous mutations in SLC26A4 are always associated with EVA, while the severity of cochlear malformation may vary depending on the type of SLC26A4 mutation.     

先天性内耳畸形与SLC26A4基因相关性的研究

目的分析婴幼儿、儿童先天性感音神经性聋(sensorineural hearing loss,SNHL)中先天性内耳畸形患儿的SLC26A4基因突变发生的概率,突变类型以及基因突变与各种内耳畸形之间的关系。方法回顾性分析125例(225耳)先天性内耳畸形的SNHL患儿的影像学及听力学资料,收集其中77例先天性内耳畸形患儿及正常对照组100例的外周血。提取基因组DNA,聚合酶链反应扩增SLC26A4,直接测序分析突变。结果 77例样本中79.2%(42/53)的前庭导水管扩大组、90%(9/10)的Mondini畸形组、7.1%(1/14)其他内耳畸形组中的患者发现有SLC26A4基因突变。共发现SLC26A4基因突变类型16种,其中4种为新发现的类型。69.8%(44/63)前庭导水管扩大(enlarged vestibular aqueduct,EVA)和Mondini畸形组中发现c.919-2A>G(IVS7-2A>G)突变,等位基因突变频率50%(63/126),是SLC26A4基因最常见的突变类型。EVA组和Mondini组分别与正常对照组等位基因突变频率比较差异有显著性(P=0.000,P<0.01)。结论 SLC26A4基因突变是EVA和Mondini畸形的主要原因之一,c.919-2A>G(IVS7-2A>G)是最常见的突变类型。     

SIX1 mutation associated with enlargement of the vestibular aqueduct in a patient with branchio-oto syndrome

OBJECTIVES:: The objectives of this study were to identify SIX1 gene mutations in a patient with branchio-oto syndrome (BO) and to clarify the relationship between SIX1 mutation and enlargement of the vestibular aqueduct (EVA). METHODS:: A genetic study and retrospective chart review for a patient in whom EYA1 mutation had already been excluded was conducted. We studied a Japanese patient who had autosomal-dominant mixed hearing loss, a unilateral ear pit and unilateral EVA, and who was previously diagnosed as having BO. We searched for SIX1 and SLC26A4 mutations using polymerase chain reaction and direct gene sequencing. RESULTS:: The patient carried a heterozygous A-->G mutation at nucleotide 386 within exon 1 of SIX1 that resulted in substitution of a cysteine for a tyrosine at codon 129 (Y129C) of the gene product. Y129C is a previously identified SIX1 mutation and was not detected in any of our 164 control chromosomes. No SLC26A4 mutations were identified. CONCLUSION:: Y129C mutation in SIX1 may cause EVA as well as BO.