中国一先天性无虹膜家系PAX6基因新致病突变位点的筛查及验证

背景 先天性无虹膜是临床上罕见的先天性遗传性眼病.研究显示,配对盒转录因子6基因(PAX6)与先天性无虹膜症密切相关,但不同患者中PAX6基因的突变位点不同. 目的 对中国一常染色体显性遗传先天性无虹膜家系进行PAX6基因突变位点分析. 方法 于2014年8月在郑州大学第一附属医院收集一汉族先天性无虹膜家系,采集该家系9名成员及同期100名健康体检者的外周静脉血10 ml,采用标准酚-氯仿提取法提取基因组DNA,对PCR扩增产物进行测序、对比及突变分析.采用实时荧光定量PCR法验证和比较该家系中患病者与该家系表型正常者和健康对照者淋巴细胞中PAX6 mRNA的相对含量. 结果 该家系共3代9名成员,遗传方式符合常染色体显性遗传.家系中共5例患病者,成年患病者均表现为虹膜缺失和白内障,儿童患病者表现为无虹膜;其他4名家系成员表型正常.测序结果显示,家系患病者均存在11号染色体PAX6基因10号外显子的移码突变,第796位核糖核苷酸G缺失(c.796 del G),产生提前终止密码子,而家系正常成员及100名对照者均无此突变.实时荧光定量PCR结果显示,家系中患病者淋巴细胞中PAX6 mRNA表达水平比家系中正常成员约低50％,差异有统计学意义(Z=-2.449,P=0.016). 结论 PAX6基因c.796 del G为此先天性无虹膜家系的致病突变位点,扩增了PAX6基因突变谱.     

PAX6基因在一先天性无虹膜家系中的突变筛查

目的研究PAX6基因突变是否是导致一先天性无虹膜家系致病的原因。方法收集一先天性无虹膜家系,制备外周血基因组DNA,PCR扩增PAX6基因的外显子及其邻近的内含子,应用单链构象多态性（SSCP）法检测,如果发现变异条带,将相应的扩增产物回收并纯化后进行PAX6基因测序。测序结果与GenBank公布的PAX6基因正常序列比对,寻找有无突变。结果本家系43名成员中有8例患病,遗传方式符合常染色体显性遗传特点,40岁以上的4例患者眼压高于35mmHg。所有患者中未发现全身并发症。在家系所有患者中均未发现异常条带。结论 PAX6基因与该先天性无虹膜家系无关。该家系的致病基因有待进一步通过全基因组扫描的方法来确定。     

先天性无虹膜症一家系PAX6基因Q310X突变的致病研究

目的 探讨先天性无虹膜症一家系的致病基因突变情况与发病机制.方法 采用病例对照研究方法.对该家系所有成员21人进行全面的眼科检查,同时进行家系调查并采集外周血样本,分离单个核细胞;用基因组DNA纯化试剂盒提取基因组DNA,以先证者DNA为模板聚合酶链反应法扩增转录因子PAX6基因全部14个外显子,用双脱氧末端终止法进行测序分析.结果 测序结果发现先证者Ⅲ2的PAX6基因在第11外显子上有Q310X(c.1378C>T)无义突变.它导致了第301位氨基酸密码子由CAA改变为TAX(Q301X),编码的谷氨酰胺突变为强终止密码子.对该家系中所有21名成员PAX6基因测序,结果发现所有10例患者都携带这一突变,而11名正常人则未检测到这一改变.结论 PAX6基因Q310X的无义突变所致PAX6蛋白翻译提前终止是此先天性无虹膜症家系的致病原因.     

土家族中一个先天性无虹膜家系的临床特点和PAX6基因突变位点分析

目的：确认土家族中一个先天性无虹膜家系的PAX6基因致病突变并分析其临床特点。方法：实验研究。详细询问家族病史并对该家系中所有7 例成员（4 例患者,3 例正常人）进行详细的眼部检查,采集家系成员及100例（50例土家族人和50例汉族人）正常对照者的外周静脉血,提取DNA;对先证者PAX6基因的全部外显子进行PCR扩增及测序;对家系中所有成员和正常对照者进行PAX6基因突变位点的验证检测。结果：该家系中患者主要以虹膜缺损、白内障、眼球震颤、黄斑中心凹发育不良和角膜病变为主要临床表现,虹膜缺损轻重不一,角膜病变和白内障情况随年龄增加而加重。该家系的4 例患者均在第3 外显子与内含子3 交界处出现一个杂合突变（c.357+1G > A）,正常家系成员及正常对照者均无此突变。结论：该先天性无虹膜家系患者虹膜缺损程度不一。PAX6是该家系的致病基因,该家系患者PAX6基因的突变位点是杂合突变(c.357+1G > A）。     

先天性无虹膜家系中发现PAX6基因新的致病突变

目的对中国一先天性无虹膜家系进行PAX6基因突变检测,以确定其突变位点。方法实验研究。收集一先天性无虹膜家系,采集该家系患者及健康成员的外周静脉血,收集100名健康人外周血作为正常对照,采用Sanger测序的方法对PAX6基因的11个外显子（外显子4-14）以及外显子-内含子连接区域进行测序,随后进行家系共分离分析以及正常样本的对照分析。结果该家系8名成员经全面眼科检查,3名确诊为先天性无虹膜,且合并有复杂的眼部表型,包括不同程度的角膜病变、不同类型的白内障、黄斑发育不良、轻度上睑下垂和轻度眼球水平震颤等。在该家系患者中发现一个新杂合突变[c.569_570delinsACGG（p.Ile190Asnfs*18）],该突变可致PAX6基因编码的蛋白截短,该突变符合共分离且在100名正常对照者中未检测到。结论PAX6基因第8外显子上一个新的杂合突变[c.569_570delinsACGG（p.Ile190Asnfs*18）]为本研究中先天性无虹膜家系的致病突变,该突变与先天性无虹膜有关,本研究扩大了PAX6基因的突变频谱。     

三个先天性无虹膜症家系中PAX6基因的突变分析

背景 人类配对盒基因(PAX6)编码一个转录调节子,对眼和大脑形态的形成起关键作用.PAX6突变可导致许多先天性眼部发育异常,如先天性无虹膜症,通常为常染色体显性遗传方式. 目的 对三个先天性无虹膜症家系成员进行PAX6基因分析,探索这些家系发病的遗传基础. 方法 收集三个先天性无虹膜症家系的5例患病者和正常成员13名的外周血标本提取DNA,根据PAX6基因的序列设计4～ 13外显子序列,通过聚合酶链反应(PCR)扩增引物并测序,将目标序列与已发表的PAX6基因序列进行对比分析.结果 三个家系中共有5例患病者,在家系A中2例患者发现一个杂合突变(c.718 C＞T),导致第240位氨基酸由精氨酸突变为终止密码子(p.A rg240X),而其他正常表型者未发现此突变.家系B中的患病者和正常成员均未检测到PAX6基因的突变.家系C中1例患病者发现c.331 delG(p.Val111SerfsX13)的缺失突变,此单个碱基的缺失造成了移码突变,使PAX6蛋白羧基端的299个氨基酸缺失,而此家系的其他正常表型成员未发现此突变. 结论 家系A和家系C先天性无虹膜症的致病与PAX6基因的突变有关.     

中国一先天性无虹膜家系的遗传分析及PAX6基因突变位点的检测

背景 先天性无虹膜是双眼发病的遗传性疾病,目前的研究表明先天性无虹膜患者配对盒转录因子6(PAX6)基因突变位点具有多样性. 目的 通过目标序列捕获测序结合一代测序验证技术对1个中国先天性无虹膜家系进行基因突变位点的筛查和遗传分析.方法 采用横断面研究方法,本研究组于2016年3月纳入在郑州大学第一附属医院确诊的1个中国汉族先天性无虹膜家系,并对该家系成员进行致病突变基因检测.该家系全体成员均接受神经系统、口服葡萄糖耐量试验等全身体格检查以及眼科相关检查.采集现存家系所有成员前臂静脉血10 ml以提取基因组DNA,以先证者基因组DNA为模板行前房角发育异常致病基因的目标基因定点捕获测序分析,经与各基因库比对筛选出候选致病基因位点,采用PCR法对该家系成员行致病基因位点DNA片段扩增,采用Sanger测序技术在该家系除先证者以外的2例患病者和表型正常成员中进行候选致病基因验证. 结果 该家系共3代9名成员,Ⅰ1去世,现存8位成员,包括患病者3例(Ⅱ2及其子代Ⅲ1、Ⅲ2)和表型正常者5人,符合常染色体显性遗传模式.所有家系成员未发现神经系统异常,口服葡萄糖耐量试验结果均呈阴性.3例患病者视力均明显下降且不能矫正,眼压平均值为21 mmHg(1 mmHg=0.133 kPa),患者均存在虹膜完全缺如、角膜基质层混浊、眼球水平震颤、黄斑中心凹发育不良症状.此外,Ⅱ2患者存在左眼上睑下垂、右眼先天性白内障表现,Ⅲ2同时存在双眼先天性白内障、双侧晶状体不全脱位.先证者目标序列捕获测序分析及数据库比对显示,所有患病者PAX6基因第6号外显子上碱基替换c.183C＞A,经Sanger测序验证后证实突变基因与表型共分离. 结论 PAX6基因c.183C＞A突变是该先天性无虹膜家系的致病突变位点.     

先天性无虹膜合并先天性白内障家系致病基因突变分析

目的：对先天性无虹膜合并先天性白内障家系进行PAX6基因突变位点筛查,丰富该致病基因的突变谱。

方法：选取就诊于北京同仁医院眼科门诊的1个先天性无虹膜合并先天性白内障家系和100名健康志愿者,采集外周静脉血,提取基因组DNA,采用直接测序法进行PAX6基因突变位点的筛查。

结果：该家系中先证者和其他患者均表现为无虹膜合并白内障,PAX6基因测序结果显示,该致病基因第11外显子无义突变c.991 C>T,造成PAX6基因编码的蛋白截短(R331X),从而使该蛋白失去功能,且该突变在家系内与疾病表型共分离,不存在于家系内及家系外健康样本的基因中。

结论：PAX6 R331X突变与先天性无虹膜合并先天性白内障的发生有关。     

一先天性无虹膜家系PAX6基因遗传筛查及临床表型

目的探讨我国常染色体显性遗传先天性无虹膜一家系患者的致病基因突变位点及其临床表型。方法实验研究。于南京医科大学第一附属医院眼科收集一先天性无虹膜家系,共8名家庭成员,其中3名患者,2名正常同胞,3名配偶。完善该家系内所有参与者的眼科检查,采集该家系成员的外周静脉血,提取基因组DNA,扩增PAX6基因的全部编码区及外显子-内含子交界区剪切位点附近的序列,直接测序法确定该家系的致病突变。结果遗传学筛查结果证实该家系的致病突变为位于PAX6基因第7号外显子与第7号内含子交接处的杂合突变（c.357+5G>A）。生物信息学分析结果表明该突变可导致正常剪切位点的缺失,产生移码突变,形成截短蛋白p.Ser121Asnfs*30。该家系中3例患者均表现出典型的先天性无虹膜症的临床表型,表现为虹膜发育不全,与此同时,该家系内患者还具有上睑下垂、白内障、眼球震颤、青光眼及玻璃体混浊等眼部异常。结论PAX6 c.357+5G>A杂合突变为该家系的致病突变,是该家系发生先天性无虹膜及上睑下垂、白内障、眼球震颤、青光眼及玻璃体混浊等一系列临床表型的主要致病原因。     

视网膜色素变性患者视紫红质E341ter基因突变及临床表型分析

目的 探讨常染色体显性遗传视网膜色素变性患者视紫红质基因突变及其与临床表型的关系。方法 应用聚合酶链反应（polymerase chain reaction,PCR)和单链构象多态性（single strand conformation polymorphism,SSCP)技术,对13个常染色体显性遗传视网膜色素变性家系的27例成员,进行视紫红质基因整个编码区的突变筛选,对SSCP检测有变异带的外显子PCR产物进行测序;同时应用裂隙灯、眼底镜、动静态视地和视网膜电流图（ERG）对患者进行临床检测。随机收集30例正常人进行对照检测。结果 发现1个家系患者有视紫红质E341ter突变,呈杂合子,密码子341第一个碱基由G变成T。该家系临床表现为青年期出现夜盲,视力和视野损害较重,ERG检查杆体和锥体无反应或仅有较小的锥体反应。结论 视紫红质基因突变家系的视网膜色素变性病史开始于杆体功能的丢失,进而累及锥体系统,并最终导致视功能严重丧失。视紫红质E341ter突变被认为是该家系的病因。     

Two nonsense mutations of PAX6 in two Japanese aniridia families: case report and review of the literature

PURPOSE: To identify PAX6 mutations in patients from four Japanese families with aniridia. METHODS: Polymerase chain reaction (PCR)-single stand conformational polymorphism (SSCP) analysis (SSCA) was performed in probands of the families, and restriction analysis using MaeIII or AvaI was carried out in other affected family members. RESULTS: PCR-SSCA demonstrated in the proband from one family an extra-band in the PCR product for PAX6 exon 8. Base sequence analysis revealed that the patient is a heterozygote for a C to T transition mutation at codon 203. DNAs from the patient and another affected member in the same family were cut with MaeIII into two fragments, while non-affected members in the family showed only one MaeIII fragment, the result confirmed the mutation. In another family, PCR-SSCA revealed an extra-band in the PCR product for exon 9. Sequencing detected a C-->T substitution at codon 240 in the patient, the mutation resulted in loss of an AvaI site. AvaI cleavage analysis confirmed the mutation in the patient. The two transition mutations observed in the two families also predict the conversion of arginine to a stop codon (R203X and R240X, respectively) around the homeodomain (HD), leading to the truncation of the PAX6 protein within its glycine-rich region. No abnormal SSCP bands or abnormal restriction fragments were detected in patients from the other two families. CONCLUSIONS: The two mutations sites identified in the two families, one at codon 203 and the other at codon 240, are those most frequently observed among 118 previously reported PAX6 mutations. This indicates that the two mutations are two hot-spots in the gene.     

PAX6 gene mutations and genotype-phenotype correlations in sporadic cases of aniridia from India

In order to understand the underlying molecular genetic defect causing aniridia in India, eight probands from sporadic cases were screened for all 14 exons of the PAX6 gene using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Direct sequencing of the SSCP variants revealed a nonsense mutation (R317X) in the eleventh exon leading to a premature termination of the PAX6 protein in the proline-serine-threonine (PST)-rich domain in two probands. Another proband exhibited an intronic polymorphism (IVS 9-12 C-T). The mutation resulted in loss of function of the PAX6 protein along with variable phenotypic manifestations in the probands. This is the first report describing a PAX6 gene mutation in aniridia cases from India and highlights the variable expressivity in phenotypes due to haploinsufficiency.     

Genotype-phenotype correlation in X-linked retinitis pigmentosa 2 (RP2)

In order to understand the underlying molecular genetic defect causing aniridia in India, eight probands from sporadic cases were screened for all 14 exons of the PAX6 gene using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Direct sequencing of the SSCP variants revealed a nonsense mutation (R317X) in the eleventh exon leading to a premature termination of the PAX6 protein in the proline-serine-threonine (PST)-rich domain in two probands. Another proband exhibited an intronic polymorphism (IVS 9–12 C-T). The mutation resulted in loss of function of the PAX6 protein along with variable phenotypic manifestations in the probands. This is the first report describing a PAX6 gene mutation in aniridia cases from India and highlights the variable expressivity in phenotypes due to haploinsufficiency.     

A Novel PAX6 Mutation in Chinese Patients with Severe Congenital Aniridia

Purpose: We identified a novel mutation in Paired Box gene 6 (PAX6) and characterized its associated clinical features of severe ocular malformation in a Chinese family with congenital aniridia. Methods: We studied two patients with aniridia from a Chinese family. All patients and noncarriers in this family underwent full ophthalmologic, general and urinary examinations. Total genomic DNA was isolated from peripheral blood of two aniridia patients. PAX6 levels were determined by PCR and its mutational status was determined by sequencing. Direct sequencing detected variations in PAX6. Results: Patients had bilateral congenital nystagmus, anterior polar cataract, absence of iris tissue, and foveal hypoplasia with severely reduced visual acuity. A novel heterozygous PAX6 mutation in exon 6 c.662G>A (p.W100X) was identified which created a premature termination codon. This observed sequence alteration was not found in 100 normal controls and has not been previously reported. Conclusions: We identified a novel PAX6 mutation in a family with severe ocular malformation. Our study expands the mutational spectrum of PAX6 and enriches our knowledge of the relationship between genotype and phenotype due to these mutations.