Novel CYP1B1 and known PAX6 mutations in anterior segment dysgenesis (ASD)

PURPOSE: The study intended to define the underlying genetic defects for 21 index patients affected with different forms of anterior segment dysgenesis. Sequence analysis for the PAX6, PITX2, FOXC1, and CYP1B1 genes has been implemented for this purpose. METHODS: Ten patients affected with Peters anomaly, 8 with Rieger anomaly, and 3 with aniridia were included in this study. All patients underwent a complete eye examination, including anterior segment evaluation, with slit-lamp microsocopy, fundoscopy, tonography, and gonioscopy. Twenty-one intronic primer pairs were used to amplify the coding exons of the FOXC1, CYP1B1, PITX2, and PAX6 genes for sequence analysis on an automated sequencer (ABI 3730). RESULTS: We were able to detect mutations in 5 of 21 patients with anterior segment malformations. We found mutations in individuals suffering from Rieger anomaly and aniridia, in CYP1B1 and PAX6, respectively. None of the 10 Peters anomaly patients had causative mutations in any of the 4 genes we screened. CONCLUSIONS: Our results suggest primary congenital glaucoma and the anterior segment dysgenesis disorders may share a common molecular pathophysiology in the CYP1B1 pathway.     

Genetic analysis of patients with primary congenital glaucoma

Purpose

To determine the common gene mutation in patients with primary congenital glaucoma (PCG) in the Southeast region of Turkey via genetic analysis and to evaluate whether there were other gene mutations in these patients.

Methods

A total of 25 patients with PCG were included in this study. We performed sequence analysis including all exons of cytochrome p450 1B1 (CYP1B1), myocilin (MYOC), forkhead box C1 (FOXC1), and paired-like homeodomain 2 (PITX2) genes of the obtained samples. Further, we analyzed the results using the Nextgen analysis program.

Results

The CYP1B1 gene mutation was detected in 20 (80%) of 25 patients, and FOXC1 gene mutation was detected in one (4%) patient. The mutation site of nine (45%) of the 20 CYP1B1 genes was found in the second exon. The pathogenic variant (p.Gly61Glu) was observed in 12 (60%) patients (in the first and second exons); the mutation type of six (50%) of these patients was homozygous. The mutation site of one patient with FOXC1 gene mutation was found to be in the first exon; its pathogenic variant was p.Met400lle. The mutation type in this gene was observed to be heterozygous. Lastly, there were no mutations in the MYOC, FOXC1, and PITX2 genes in combination with the CYP1B1 gene mutation.

Conclusion

The most common cause of PCG in our region is the CYP1B1 gene mutation, and the most frequent pathogenic variant is c.182G?>?A (p.Gly61Glu). We also determined that the CYP1B1 gene mutation was alone and did not occur with other gene mutations (MYOC, FOXC1, and PITX2).

     

Clinical and molecular aspects of congenital aniridia – A review of current concepts

Congenital aniridia is a pan ocular disorder characterized by partial or total loss of iris tissue as the defining feature. Classic aniridia, however, has a spectrum of ocular findings, including foveal hypoplasia, optic nerve hypoplasia, nystagmus, late-onset cataract, glaucoma, and keratopathy. The latter three are reasons for further visual compromise in such patients. This entity is often due to mutations in the PAX6 (Paired box protein Pax-6) gene. Recently, aniridia-like phenotypes have been reported due to non-PAX6 mutations as in PITX2, FOXC1, FOXD3, TRIM44, and CYP1B1 as well wherein there is an overlap of aniridia, such as iris defects with congenital glaucoma or anterior segment dysgenesis. In this review, we describe the various clinical features of classic aniridia, the comorbidities and their management, the mutation spectrum of the genes involved, genotype-phenotype correlation of PAX6 and non-PAX6 mutations, and the genetic testing plan. The various systemic associations and their implications in screening and genetic testing have been discussed. Finally, the future course of aniridia treatment in the form of drugs (such as ataluren) and targeted gene therapy has been discussed.     

Molecular Characterization of Newborn Glaucoma Including a Distinct Aniridic Phenotype

Purpose: To characterize the underlying genetic defect in otherwise healthy Saudi newborns with buphthalmos, including those with iris abnormalities.

Methods: Prospective case series of affected Saudi Arabian probands who were referred for genetic counseling over a 4 year period. All had CYP1B1 sequencing. Selected patients with visible iris abnormalities had PAX6, FOXC1, and PITX2 sequencing. CYP1B1-negative patients had LTBP2 sequencing.

Results: All 67 probands had corneal enlargement with variable haze/scarring evident to caregivers at birth; 46 had a family history of infantile or early childhood glaucoma. All families were consanguineous except for 6, 2 of which were endogamous. Eight probands had mild ectropion uveae with partial aniridia; 2 probands had thick scarred corneas that precluded careful iris examination. Homozygous or compound heterozygous CYP1B1 mutations were identified in 91% (61/67), including all 8 probands with ectopion uveae and partial aniridia. The common Saudi mutation p.G61E occurred in most cases (38 homozygous, 8 compound heterozygous). Four novel mutations were identified (p.N252K, p.V460E, p.S485F, p.N519D). No mutations were identified in the other screened genes.

Conclusions: Newborn glaucoma on the Arabian Peninsula is typically CYP1B1-related even in the setting of developmental iris abnormality. Mild iris ectropion with partial aniridia in a newborn with glaucoma suggests mutations in CYP1B1 rather than in other genes associated with anterior segment dysgenesis. On the Arabian Peninsula p.G61E mutations are the major cause of newborn glaucoma but novel CYP1B1 mutations continue to be documented. The fact that the 9% of cases that were CYP1B1-negative did not have mutations in LTBP2 suggests that there exists at least 1 additional locus for this condition.     

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.     

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).     

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).     

A novel deletion downstream of the PAX6 gene identified in a Chinese family with congenital aniridia

Purpose: Congenital aniridia, a severe bilateral panocular visual disorder, is an autosomal dominantly inherited eye anomaly. Mutations in the paired box 6 gene (PAX6) have been shown to be responsible for congenital aniridia in most patients. The purpose of the present study was to report clinical features of a Chinese family with congenital aniridia and to screen novel genetic mutations for congenital aniridia.

Methods: All members of a three-generation family underwent comprehensive ophthalmic examination, and 8 of its 25 members were diagnosed with congenital aniridia. The proband was analyzed by exome sequencing and whole genome sequencing, and linkage analysis was performed for the family. The mutation was confirmed by direct DNA sequencing.

Results: Using Illumina’s Human Linkage-12 beadchip microarray (including 6090 SNPs) whole genome scan, the LOD score value showed that the interval on chromosome 11 between rs1389423 to rs910090 exhibited a strong linkage. A novel heterozygous 469 kb deletion mutation within the downstream region of PAX6 (chr11:31189937–31659379) was identified in all affected family members, but not in unaffected family members or 2000 ethnically matched controls.

Conclusion: A novel deletion mutation was identified within the PAX6 downstream region that results in congenital aniridia.     

A novel PAX6 mutation causes congenital aniridia with or without retinal detachment

Background: Aniridia is a rare developmental eye disorder characterized by complete or partial iris hypoplasia often accompanied with other ocular changes that affect the cornea, anterior chamber, lens, retina, and optic nerve. Most cases of aniridia are inherited with an autosomal dominant mode of inheritance caused by PAX6 mutations or deletions. To reveal the underlying genetic defect in a four-generation Iranian family with aniridia, we carried out a genetic screening of PAX6.

Methods: Complete ophthalmic examinations were performed for available affected family members. All PAX6 exons and their flanking regions were sequenced for affected individuals. Candidate variation was screened for segregation in the pedigree by Sanger sequencing. Bioinformatics prediction was done to evaluate the deleterious effects of the mutation on protein product. Real-time PCR was used to investigate the impact of the variant on PAX6 mRNA expression.

Results: All patients were diagnosed with isolated aniridia associated with variable phenotypic features including retinal detachment. A novel heterozygous deletion c.320_348delTGTCCGAGGGGGTCTGTACCAACGATAAC (p.Leu107HisfsX16) on PAX6 gene was detected. Decreased mRNA level of PAX6 in the affected individuals indicated that the mutation caused nonsense-mediated mRNA decay (NMD).

Conclusions: To the best of our knowledge, it is the first report on the genetics of aniridia in Iran. Segregation analysis, bioinformatics prediction and confirmation of NMD, all support the proposition that the novel observed PAX6 mutation is the cause of aniridia in the pedigree. Retinal detachment in some of the affected members, which is a rare reported phenotypic feature of aniridia patients, may be associated with this mutation.     

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异常PITX2及PAX6基因型表型分析

目的：分析中国人Peters异常(PA)患者的临床特征,研究Peters异常患者PITX2及PAX6基因变异情况。

方法：选取2016/2019年在常州市第二人民医院及第三人民医院眼科就诊的15例Peters异常患者,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及PAX6的编码区及其临接内含子进行扩增后,直接测序筛查中国人群Peters异常患者PITX2及PAX6基因变异,异源双链-单链构象多态性分析(HA-SSCP)的方法对突变患者及其家系成员及80例正常对照进行验证; 分析比较国内已报道的Peters异常患者PITX2及PAX6基因突变并研究其相关表型。

结果：Peters异常患者15例PITX2基因突变筛查结果发现了1种新PITX2的突变c.296delG(P.R99fsx56),导致该基因的功能异常,分析突变患者临床特征,该患者右眼诊断为Axenfeld-Rieger综合征(ARS),左眼诊断为Peters异常。而家系成员中该患者父母及无亲缘关系的正常对照者均未发现相同突变,故此突变为新生突变。PAX6基因突变筛查未能发现突变。

结论：PA患者15例中检测到1个新PITX2基因突变,丰富了PITX2基因突变频谱,进一步明确了PA合并ARS眼病的临床特点,为该种少见眼病的临床诊断和发病原因提供了依据。     

PAX6 3′ deletion in a family with aniridia

Background: Aniridia is a congenital panocular malformation defined as iris aplasia or hypoplasia. It can be either isolated or be a part of multiple ocular anomalies such as cataracts, glaucoma, corneal pannus, optic nerve hypoplasia, absence of macular reflex or ectopia lentis. In the majority of cases the disease is caused by mutation in the PAX6 gene.

Material and Methods: A Polish family with aniridia was screened for the presence of genomic rearrangements in PAX6, WT1 and the flanking genes by means of multiplex ligation probe amplification (MLPA). MLPA reaction was performed using the P219-B1 PAX6 commercial kit from MRC-Holland. Additionally, the coding sequence of PAX6 gene was sequenced in the proband. Array comparative genomic hybridization analysis was performed using the NimbleGen CGX-12 format.

Results: MLPA examination revealed a heterozygous deletion of approximately 0.6?Mb, downstream of PAX6 gene on chromosome 11. Four genes lie in the deleted region. Bi-directional sequencing of 14 exons of the PAX6 gene did not reveal any causative alteration. Microarray analysis confirmed the deletion and determined its size which ranged from 598.87–651.76 kb.

Conclusions: A small subset of aniridia cases is caused by rearrangements of PAX6 neighboring regions, and the so-called “position effect” is considered to be the underlying pathogenic mechanism. Molecular testing of aniridia patients should include sequencing of the PAX6 gene, followed by screening for larger structural abnormalities located on chromosome 11p13. MLPA can be a useful method in molecular testing of aniridia patients.     

A Review of the Clinical and Genetic Aspects of Aniridia

Abstract

Aniridia classically presents with a bilateral congenital absence or malformation of the irides, foveal hypoplasia, and nystagmus, and patients tend to develop visually significant pre-senile cataracts and keratopathy. Additionally, they are at high risk for developing glaucoma. Classic aniridia can be genetically defined as the presence of a PAX6 gene deletion or loss-of-function mutation that results in haploinsufficiency. Variants of aniridia, which include a condition previously referred to as autosomal dominant keratitis, are likely due to PAX6 mutations that lead to partial loss of PAX6 function. Aniridia-associated keratopathy (AAK) is a progressive and potentially debilitating problem affecting aniridic patients. The current treatments for AAK are to replace the limbal stem cells through keratolimbal allograft (KLAL) with or without subsequent keratoplasty for visual rehabilitation, or to implant a Boston type 1 keratoprosthesis. Future therapies for AAK may be aimed at the genetic modification of corneal limbal stem cells.     

The Genetics of Intraocular Pressure

Abstract

Glaucoma is a leading cause of irreversible blindness. Intraocular pressure (IOP) is the only modifiable risk factor for glaucoma, yet there is little known about the molecular events that regulate IOP. Genetic and genomic studies have helped identify genes that influence IOP and could lead to the identification of biological pathways that serve as targets for novel pressure-modifying therapies. Genetic linkage studies resulted in the identification of several genes that cause Mendelian (autosomal dominant or autosomal recessive) forms of high-pressure glaucoma, including MYOC. PITX2, FOXC1, and CYP1B1. Classical twin studies suggest that IOP is a heritable trait. More recently, genome-wide association studies (GWAS) have shown that common genetic variants in the GAS7 and TMCO1 genomic regions are associated with elevated IOP. TMCO1 has also been associated with primary open-angle glaucoma in patients with advanced disease. A further study identifying additional genes contributing to IOP will be necessary to fully define the underlying genetic architecture of IOP.     

PAX6 Analysis of Two Unrelated Families from the Arabian Peninsula with Classic Hereditary Aniridia

Introduction: Reports from around the world confirm that heterozygous PAX6 mutation is the major cause of hereditary aniridia (with a classic phenotype of iris hypoplasia, keratopathy, lens opacity, and foveal hypoplasia). However, genotype/phenotype reports are lacking from the Arabian Peninsula, a historically isolated region with a relatively high incidence of recessive disease and thus a potential for phenocopy and pseudodominance. The purpose of this study to assess for PAX6 mutation in two unrelated families with classic hereditary aniridia from the Arabian Peninsula. Methods: Interventional cases series of two unrelated affected Saudi Arabian families. Available family members underwent ophthalmic examination and venous blood sampling for PAX6 sequencing. Results: The pedigrees of both families suggested dominant (or pseudodominant) inheritance of the classic aniridia phenotype. Affected individuals in Family #1 were heterozygous for a novel frameshift PAX6 mutation (c.delA1294). Affected individuals in Family #2 had heterozygosity for a commonly-reported PAX6 nonsense mutation (p.Arg240X). Conclusions: PAX6 haploinsufficiency, the major cause of classic hereditary aniridia worldwide, is also associated with the phenotype in two different families from the Arabian Peninsula. Homozygosity by descent is not expected to affect genotype/phenotype correlation for the classic phenotype.     

Structural assessment of PITX2, FOXC1, CYP1B1, and GJA1 genes in patients with Axenfeld-Rieger syndrome with developmental glaucoma

PURPOSE: Axenfeld-Rieger (AR) is an autosomal dominant disorder with phenotypic heterogeneity characterized by anterior segment dysgenesis, facial bone defects, and redundant periumbilical skin. The PITX2 gene, on chromosome 4q25, and the FOXC1 gene, on chromosome 6p25, have been implicated in the different phenotypes of the syndrome through mutational events. Recently, the CYP1B1 gene was found to be associated with Peters' anomaly, and the gene associated with oculodentodigital dysplasia syndrome, which presents some similarities with AR, was identified (connexin 43--GJA1 gene). The purpose of this study was to evaluate PITX2, FOXC1, CYP1B1, and GJA1 gene mutations in Brazilian families with AR. METHODS: Eight unrelated patients affected by AR (all eight with glaucoma and three with systemic manifestations) and their families were ophthalmologically evaluated and their blood was collected for DNA extraction purposes. The coding regions of PITX2, FOXC1, CYP1B1, and GJA1 genes were completely evaluated through direct sequencing. RESULTS: The frequency of mutations in the FOXC1, GJA1, PITX2, and CYP1B1 genes in this study were 25%, 12.5%, 0% and 0%, respectively. In the FOXC1 gene, two GGC triplet insertions (GGC375ins and GGC447ins) defined as a polymorphism, and two new mutations--a deletion (718 to 719delCT) and a nonsense mutation (Trp152STOP)--were identified. One polymorphism (Ala253Val) was identified in the GJA1 gene in the same family presenting the Trp152STOP mutation in the FOXC1 gene. In this family harboring both structural alterations, two patients who carried the GJA1 (Ala253Val) and FOXC1 (Trp152STOP) mutations developed less severe glaucoma compared with family members presenting the FOXC1 (Trp152STOP) mutation alone. CONCLUSIONS: Two new structural alterations in the FOXC1 gene and a polymorphism in the GJA1 gene were first described in Brazilian patients with AR and developmental glaucoma. A polymorphism in the GJA1 gene (Ala253Val), for the first time identified in association with AR, raises the possibility of its participation as a modifier gene.     

A Novel CYP1B1 Mutation with Congenital Glaucoma and Total Aniridia

Purpose: Primary congenital glaucoma is a common disorder in the Middle East mainly caused by mutations in the the CYP1Bl gene. We report a family with three siblings that presented with recalcitrant childhood glaucoma, aniridia in two siblings with a novel CYP1B1 gene mutation.

Materials and methods: Review of pedigree, clinical history and clinical course of the family. Genetic testing in the affected family members.

Results: Three sisters presented with clinical findings of severe congenital glaucoma and a positive family history. Clinical examination of two of sisters revealed corneal scarring, bilateral aniridia with severe glaucoma that required multiple surgical procedures to control intraocular pressure. The third sibling presented with garden-variety primary congenital glaucoma. Genetic analysis revealed a novel CYP1B1 gene mutation (g.8291 C?>?T; p.S485F).

Conclusion: CYP1B1 mutation related congenital glaucoma can present with an extreme form of anterior segment dysgenesis that includes recalcitrant glaucoma, corneal opacification and aniridia.     

Genomics and anterior segment dysgenesis: a review

Anterior segment dysgenesis refers to a spectrum of disorders affecting structures in the anterior segment of the eye including the iris, cornea and trabecular meshwork. Approximately 50% of patients with anterior segment dysgenesis develop glaucoma. Traditional genetic methods using linkage analysis and family‐based studies have identified numerous disease‐causing genes such as PAX6, FOXC1 and PITX2. Despite these advances, phenotypic and genotypic heterogeneity pose continuing challenges to understand the mechanisms underlying the complexity of anterior segment dysgenesis disorders. Genomic methods, such as genome‐wide association studies, are potentially an effective tool to understand anterior segment dysgenesis and the individual susceptibility to the development of glaucoma. In this review, we provide the rationale, as well as the challenges, to utilizing genomic methods to examine anterior segment dysgenesis disorders.     

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。