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。     

Peters综合征临床特征和基因突变的研究

目的：分析国人Peters综合征的临床特征,并确定致病基因突变,为该病的临床诊断和治疗及发病原因提供依据。方法：选取2012/2015年在常州市第二人民医院眼科就诊的10例先天性角膜混浊的患者,并收集详细的相关临床资料。征得患者及其家系成员的同意后抽血制备基因组DNA,用聚合酶链反应( PCR)对致病基因PITX2的编码区及其临接内含子进行扩增后,直接测序分析该基因。同时检测100位无亲缘关系的正常人外周血标本进行对照验证。结果：患者1例的临床特点包括先天性角膜中央部混浊白斑,伴有相应区域的角膜后部基质变薄和后弹力层缺损,且患者伴有全身系统如心脏和听力损害等改变,符合Peters综合征的临床诊断;该患者PITX2基因突变筛查结果发现了1种新突变,c.788G>A,导致该基因的功能异常,而家属中表型正常者及无亲缘关系的正常对照者均未发现该基因突变。结论：先天性角膜混浊患者10例中检测到1个新PITX2基因突变,符合Peters综合征的临床诊断,这是中国首次报道Peters综合征的PITX2基因突变,结果丰富了PITX2基因突变频谱,并进一步明确了Peters综合征的临床特点,为该病的临床诊断和治疗及发病原因提供了依据。     

PAX6基因与视网膜疾病关系的研究进展

PAX6基因在胚胎发育过程中起重要作用,PAX6基因突变有可能导致先天性无虹膜、视网膜母细胞瘤、黄斑发育不良、Peters异常等眼病。本文主要就PAX6基因的基本背景知识以及PAX6与视网膜疾病的关系进行综述。     

东北地区原发性闭角型青光眼家系PITX2基因和FOXC1基因突变筛查

目的通过分子遗传学研究筛查原发性闭角型青光眼家系PITX2基因和FOXC1基因突变情况。设计实验研究。研究对象东北地区原发性闭角型青光眼4个家系13例患者和24例健康成员。方法应用聚合酶链反应(PCR)扩增PITX2基因和FOXC1基因所有编码外显子,直接测序法筛查致病突变。主要指标PITX2基因、FOXC1基因序列。结果所分析的4个家系13例患者基因组DNA中均未检测到PITX2基因、FOXC1基因突变。结论 PITX2基因和FOXC1基因不是中国东北地区该原发性闭角型青光眼家系的致病病因。     

PAX6基因突变的鉴定与中国家系无虹膜症合并妊娠期糖尿病的产前诊断

目的：探讨1个中国无虹膜症合并妊娠期糖尿病家系的基因缺陷及产前诊断。

方法：收集1个患有无虹膜症合并妊娠期糖尿病的中国家系,从外周血中提取整个家系成员的基因组DNA,通过聚合酶链式反应结合直接测序法,分析人类配对盒基因(PAX6)的编码序列。妊娠18wk时对孕妇进行羊膜穿刺术,并根据突变筛查结果进行遗传学分析。

结果：无虹膜患者在PAX6的第5外显子中存在杂合缺失突变(c.113_129del GGCCGTGCGACATTTCC, p.Arg38ProfsTer12),该患者同时合并妊娠期糖尿病,产前诊断结果提示胎儿具有相同的突变,易患先天性无虹膜症,经产后随访证实。

结论：在中国先天性无虹膜患者中发现了PAX6基因缺失突变,为人类PAX6等位基因变异数据库提供了更多的文献资料,为产前诊断提供了分析依据。     

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.     

Sturge-Weber综合征1例

目的：检测1例中国上皮基底膜角膜营养不良家系TGFBI基因突变类型。

方法：经过详细的病史采集及临床检查后,提取先证者及其家系内其他2例患者和4例有血缘关系的正常家系成员的静脉血白细胞DNA,应用PCR直接测序法对TGFBI的17个外显子进行候选基因的突变检测。

结果：在该家系患者的TGFBI基因4号外显子发现了c.417C>T,导致了杂合突变R124C。家系中正常成员及对照组均未检测到该突变。

结论：本研究首次报道了TGFBI基因的R124C杂合突变导致了上皮基底膜角膜营养不良,拓宽了角膜营养不良的基因型与表现型关系,为进一步的分子遗传学研究奠定了基础。     

先天性无虹膜症家系的基因突变位点研究

目的探讨先天性无虹膜症家系的基因突变位点。方法抽取家系成员的外周血2~5ml,提取DNA;先合成2个多态性微卫星遗传标记(D11S904和D11S935)的引物进行聚合酶链反应(PCR),PCR产物变性后用变性聚丙烯酰胺(PAGE)胶分离,根据带型和家系成员间的关系进行单体型连锁分析,判断家系无虹膜表型是否与PAX6基因相关;PCR扩增PAX6基因的所有外显子,所有PCR产物分别进行单链构象多态性(SSCP)分析,通过患者与正常人带型的差异确定突变发生的外显子,对有差异SSCP带型的PCR产物进行直接DNA测序,找到突变位点。结果该家系先天性无虹膜表型明显与PAX6基因连锁;SSCP分析PAX6基因第9外显子PCR产物,显示患者均有异常带型出现,而家系正常人均无此异常带;测序结果显示突变位点为PAX6基因第9外显子c1080核苷酸C突变为T,使编码精氨酸的密码子突变为终止密码子。结论PAX6基因突变可导致先天性无虹膜。     

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）。     

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。     

Congenital Corneal Opacities: A Review with a Focus on Genetics

Congenital corneal opacities present in approximately 3/100,000 newborns. Many different disorders may result in corneal opacifications of infancy, including Peters' anomaly (PA), congenital hereditary endothelial dystrophy (CHED), congenital hereditary stromal dystrophy (CHSD) and posterior polymorphous dystrophy (PPMD). Current studies have localized defects using genetic testing in PA, CHED, CHSD and PPMD. Identifying mutations for specific disorders may lead to better understanding of the underlying pathogeneses and may help with diagnosis and prognosis. This article will review the clinical presentations, treatments and genetics of Peters' anomaly, congenital hereditary endothelial dystrophy, congenital hereditary stromal dystrophy and posterior polymorphous dystrophy.     

Molecular characterization of Axenfeld-Rieger spectrum and other anterior segment dysgeneses in a sample of Mexican patients

ABSTRACT

Background: Anterior segment dysgenesis (ASD) and Axenfeld-Rieger spectrum (ARS) are mainly due to PITX2 and FOXC1 defects, but it is difficult in some patients to differentiate among PITX2-, FOXC1-, PAX6- and CYP1B1-related disorders. Here, we set out to characterize the pathogenic variants (PV) in PITX2, FOXC1, CYP1B1 and PAX6 in nine unrelated Mexican ARS/ASD patients and in their available affected/unaffected relatives.

Materials and methods: Automated Sanger sequencing of PITX2, FOXC1, PAX6 and CYP1B1 was performed; those patients without a PV were subsequently analyzed by Multiplex Ligation-dependent Probe Amplification (MLPA) for PITX2, FOXC1 and PAX6. Missense variants were evaluated with the MutPred, Provean, PMUT, SIFT, PolyPhen-2, CUPSAT and HOPE programs.

Results: We identified three novel PV in PITX2 (NM_153427.2:c.217G>A, c.233T>C and c.279del) and two in FOXC1 [NM_001453.2:c.274C>T (novel) and c.454T>A] in five ARS patients. The previously reported FOXC1 c.367C>T or p.(Gln123*) variant was identified in a patient with ASD. The ocular phenotype related to FOXC1 included aniridia, corneal opacity and early onset glaucoma, while an asymmetric ocular phenotype and aniridia were associated with PITX2. No gene rearrangements were documented by MLPA analysis, nor were any PV identified in PAX6 or CYP1B1.

Conclusions: Heterozygous PV in the PITX2 and FOXC1 genes accounted for 66% (6/9) of the ARS/ASD cases. The absence of PAX6 or CYP1B1 abnormalities could reflect our small sample size, although their analysis could be justified in ARS/ASD patients that present with congenital glaucoma or aniridia.     

Molecular basis of Peters anomaly in Saudi Arabia

Peters anomaly (PA) and primary congenital glaucoma (PCG) are genetically and phenotypically distinct conditions. Mutations in cytochrome P4501B1 (CYP1B1) are the most common cause of PCG in Saudi Arabia. Recent evidence suggests that there may be common genetic factors to these conditions. To determine the molecular basis of PA, 11 study subjects with PA from 10 Saudi Arabian families were recruited. Experienced ophthalmologists examined all affected subjects and most of their available unaffected relatives. The diagnosis of PA was confirmed by pathological examination of excised corneal buttons in seven subjects. The coding exons of FOXC1, PITX2, and PAX6 were screened and those of CYP1B1 and FOXE3 sequenced. Homozygous CYP1B1 mutations were identified in six individuals in five families. Five individuals were homozygous for G61E and one was homozygous for 143del10. No mutations were identified in FOXC1, PITX2, PAX6, or FOXE3. The clinical or pathologic phenotype of the subjects with CYP1B1 mutations was not different from that of the other PA patients in this study. Two families included at least one individual with homozygous CYP1B1 mutations and no ocular anomalies (nonpenetrant). Mutations in CYP1B1 may be a substantive cause for PA in this population. Thus, PA and PCG may share a common molecular pathophysiology. Indeed, PA and PCG may share the same spectrum of anterior segment dysgenesis. Finally, the occurrence of PA, PCG, and unaffected individuals with identical homozygous CYP1B1 mutations in the same sibship suggests the presence of modifiers that modulate the clinical severity of the phenotypic expression of the same CYP1B1 mutation(s).     

The Spectrum of Associated Ocular and Systemic Malformations

The cases of Peters' anomaly seen over the past decade, at Hospital Ste-Justine in Montreal, are reviewed. Associated ocular anomalies were observed in 50% of cases while 60% of patients presented with associated systemic defects. It is clear, from these patients and those reported in the literature, that Peters' anomaly can be an isolated condition, or part of distinct syndromes: the Krause-Kivlin syndrome or the Peters'-plus syndrome. The authors emphasize the importance for the ophthalmologist to recognize these possibilities if proper management is to be provided.     

Two novel mutations of the PAX6 gene causing different phenotype in a cohort of Chinese patients

Purpose

Aniridia (AN) is a rare congenital panocular disorder caused by the mutations of the paired box homeotic gene 6(PAX6) gene. The PAX6gene is also involved in other anterior segment malformations including Peters anomaly. We studied the PAX6gene mutations in a cohort of affected individuals with different clinical phenotype including AN, coloboma of iris and choroid, or anterior segment malformations.

Patients and methods

Six unrelated families and 10 sporadic patients were examined clinically. After informed consent was obtained, genomic DNA was extracted from the venous blood of all participants. Mutation screening of all exons of the PAX6gene was performed by direct sequencing of PCR-amplified DNA fragments. Multiplex ligation-dependent probe amplification (MLPA) was performed to detect large deletions.

Results

By clinical examination, the patients and the pedigrees were divided into the following three groups: AN, coloboma of iris and choroids, and the anterior segment malformations including peters anomaly. Sequencing of the PAX6gene, three intragenic mutations including a novel heterozygous splicing-site mutations c.357-3C>G (p.Ser119fsX) were identified in the patients of the AN group. A novel missense mutation c.643T>C (p.S216P) was detected in the anterior segment malformation group. The mutation p.S216P located in the homeodomain region of the PAX6 caused the phenotype of Peters anomaly in family A6 with different expressing. Through MLPA analysis, a large deletion including the whole PAX6gene and DKFZ p686k1684gene was detected in one sporadic patient from the AN group. Neither intragenic mutation nor large deletion was identified in the group with coloboma of iris and choroid.

Conclusion

Our findings further confirmed that different kind of mutations might cause different ocular phenotype, and clearly clinical phenotype classification might increase the mutation detection rate of the PAX6gene.     

Subject index

Peters anomaly (PA) and primary congenital glaucoma (PCG) are genetically and phenotypically distinct conditions. Mutations in cytochrome P4501B1 (CYP1B1) are the most common cause of PCG in Saudi Arabia. Recent evidence suggests that there may be common genetic factors to these conditions. To determine the molecular basis of PA, 11 study subjects with PA from 10 Saudi Arabian families were recruited. Experienced ophthalmologists examined all affected subjects and most of their available unaffected relatives. The diagnosis of PA was confirmed by pathological examination of excised corneal buttons in seven subjects. The coding exons of FOXC1, PITX2, and PAX6 were screened and those of CYP1B1 and FOXE3 sequenced. Homozygous CYP1B1 mutations were identified in six individuals in five families. Five individuals were homozygous for G61E and one was homozygous for 143del10. No mutations were identified in FOXC1, PITX2, PAX6, or FOXE3. The clinical or pathologic phenotype of the subjects with CYP1B1 mutations was not different from that of the other PA patients in this study. Two families included at least one individual with homozygous CYP1B1 mutations and no ocular anomalies (nonpenetrant). Mutations in CYP1B1 may be a substantive cause for PA in this population. Thus, PA and PCG may share a common molecular pathophysiology. Indeed, PA and PCG may share the same spectrum of anterior segment dysgenesis. Finally, the occurrence of PA, PCG, and unaffected individuals with identical homozygous CYP1B1 mutations in the same sibship suggests the presence of modifiers that modulate the clinical severity of the phenotypic expression of the same CYP1B1 mutation(s).     

A new autosomal dominant Peters’ anomaly phenotype expanding the anterior segment dysgenesis spectrum

Purpose: To test the association of genes involved in anterior segment development in a family with autosomal dominantly inherited Peters’ anomaly (PA) with a unique ocular phenotype. Methods: Six members of a five‐generation family with PA were extensively phenotyped and linkage analysis of candidate genes, namely, PAX6, PITX2, FOXC1, CYP1B1 and MAF, was performed. Results: The complete pedigree consisted of 38 members, 19 of whom were affected. The six probands examined had bilateral microcornea, corneal opacity, iridocorneal adhesions, nystagmus and strabismus, but cataract, keratolenticular adhesions, glaucoma and posterior embryotoxon were absent. PAX6 gene mutations had been previously excluded in one of the affected members. DNA markers for candidate genes CYP1B1 on 2p22, PITX2 on 4q25, PAX6 on 11p13, MAF on 16q23 and FOXC1 on 6p25 were genotyped. Highly negative lod scores were obtained for all markers. Conclusions: The exclusion of these genes as likely candidates supports the hypothesis that the ocular phenotype associated with PA segregating in this family is a distinct, new, autosomal dominant entity in the anterior segment dysgenesis spectrum.     

Corneoscleral transplantation in congenital corneal staphyloma and Peters' anomaly

An infant born with bilateral corneal clouding diagnosed clinically as congenital anterior staphyloma and Peters' anomaly was confirmed histopathologically. This case report demonstrates one clinical spectrum of Peters' anomaly. The clinical course and histopathologic findings are detailed as is a unique surgical approach of corneoscleral graft used to perserve the right globe.