Overview of Cytochrome P450 1B1 gene mutations in patients with primary congenital glaucoma

The objective of this study was to investigate the distribution of mutations in the Cytochrome P450 1B1 gene (CYP1B1) in patients with primary congenital glaucoma (PCG) among different populations. All identifiable original studies on CYP1B1 gene mutations of patients with PCG were reviewed. Finally, DNA mutations within the CYP1B1 gene were identified in 542 patients with PCG according to 52 scientific articles and 147 distinct mutations were found. The 3987G>A (G61E) missense mutation is a founder mutation in Middle Eastern population, responsible for 45.52% of CYP1B1 mutations. In Gypsies, missense mutation 7996G>A (E387K) seems to be a founder mutation, accounting for 79.63% of CYP1B1 mutations. It seems that there is no founder mutation in Asian or Caucasian population, but also accumulates in some spots. Mutations 7927G>A (V364M), 7990C>T (L385F) and 8006G>A (R390H) are common in Asian population. In Caucasians, 7940G>A (R368H), 8037dup10, 8006G>A (R390H), 7901del13, 4340delG, 3987G>A (G61E), 7996G>A (E387K), 4490G>A (E229K) and 8005C>T/A (R390C/S) are common mutations. The findings suggest that ethnic differences and the geographical distribution of PCG may be associated with different CYP1B1 mutation patterns. Such information may be useful in developing strategies for reliable clinical genetic testing of patients with PCG and their families.     

湖北地区汉族原发性先天性青光眼患儿CYP1B1基因突变分析

目的研究湖北地区汉族原发性先天性青光眼(primary congenital glaucoma,PCG)患儿CYP1B1基因的突变情况。方法用苯酚-氯仿法从38例原发性先天性青光眼患儿的全血细胞中提取基因组DNA。然后通过聚合酶链反应-单链构象多态性(single-strand conformation polymorphism,PCR-SSCP)银染色法检测CYP1B1基因第2、3外显子的突变情况。结果5例PCG患儿检出CYP1B1基因第3外显子异常DNA片段条带,经测序分析证实为7990C→T,未发现第2外显子存在基因突变。结论CYP1B1基因具有明显的遗传异质性。应用PCR-SSCP技术可初步筛查原发性先天性青光眼患儿CYP1B1基因突变。     

Compound heterozygous mutations in CYP1B1 gene leads to severe primary congenital glaucoma phenotype

AIM: To identify the novel mutation alleles in the CYP1B1 gene of primary congenital glaucoma (PCG) patients at Shandong Province of China, and investigate their correlation with glaucomatous features. METHODS: The DNA from the peripheral blood of 13 congenital glaucoma patients and 50 ethnically matched healthy controls from the affiliated hospital of Qingdao University were extracted. The coding region of the CYP1B1 gene was amplified by PCR and direct DNA sequencing was performed. Disease causing-variants were analyzed by comparing the sequences and the structures of wild type and mutant CYP1B1 proteins by PyMOL software. RESULTS: Two missense mutations, including A330F caused by c.988G>T&c.989C>T, and R390H caused by c.1169G>A, were identified in one of the 13 PCG patients analyzed in our study. A330F mutation was observed to be novel in the Chinese Han population, which dramatically altered the protein structure of CYP1B1 gene, including the changes in the ligand-binding pocket. Furthermore, R390H mutation caused the changes in heme-protein binding site of this gene. In addition, the clinical phenotype displayed by PCG patient with these mutations was more pronounced than other PCG patients without these mutations. Multiple surgeries and combined drug treatment were not effective in reducing the elevated intraocular pressure in this patient. CONCLUSION: A novel A330F mutation is identified in the CYP1B1 gene of Chinese PCG patient. Moreover, in combination with other mutation R390H, this PCG patient shows significant difference in the CYP1B1 protein structure, which may specifically contribute to severe glaucomatous phenotype.     

原发性先天性青光眼患者CYP1B1突变的研究

目的:了解原发性先天性青光眼患者致病基因CYP1B1(Cytochrome P450 family 1 subfamily B polypeptide 1)的变异情况.方法:采用高分辨率熔解(high-resolution melting,HRM)方法,分析20例原发性先天性青光眼患者的CYP1 B1基因热点突变区,同时采用测序的方法验证HRM的检测结果.结果:检出g.6767C＞T(p.D449D)变异2例,g.2527C＞G(p.R48G)变异1例,两种变异共存者1例.结论:在CYP1B1基因突变筛查方法中,HRM具有高度的灵敏性和特异性,可用于筛查原发性先天性青光眼.PCG的原因可能与g.6767C＞T(p.D449D)和g.2527C＞G(p.R48G)的变异有关;两种变异共存者可能导致更严重的PCG.     

Two novel variants in CYP1B1 gene: a major contributor of autosomal recessive primary congenital glaucoma with allelic heterogeneity in Pakistani patients

AIM: To find the CYP1B1 mutations associated with primary congenital glaucoma (PCG) in Pakistani consanguineous pedigrees. METHODS: After getting informed consent, 11 consanguineous pedigrees belonging to different ethnic groups were enrolled. Detailed medical history was recorded and pedigrees were drawn. The standard ophthalmological examination was done to characterize the phenotype. Genomic DNA was extracted from 10 mL whole blood and coding exons and exon intron boundaries of CYP1B1 gene were directly sequenced. Bioinformatics tools were used to model the mutant protein and predict the effect of novel variants on protein structure and function. RESULTS: Sequencing analysis revealed 5 different CYP1B1 variants in 7 families (7/11; 64%), including two novel variants. A common mutation, p.R390H was found in four families, whereas p.P437L was found once in a family. Two novel variants, a homozygous non sense variant p.L13* and a compound heterozygous variant, p.P350T along with p.V364M were segregating with PCG in two families. All the patients had the variable onset and severity of the disease. The success rate of early clinical interventions was observed dependent on mutation types and position. Two different haplotypes were associated with frequently found mutation, p.R390H. CONCLUSION: Identification of novel CYP1B1 variants reassert the genetic heterogeneity of Pakistani PCG patients. The patients with missense mutations show severe phenotypic presentations and poor vision after surgical interventions as compare to patients with null variants. This may help to better understand the role of CYP1B1 mutations in the development of PCG and its course of pathogenicity.     

原发性先天性青光眼CYP1B1基因变异初步研究

目的了解CYP1B1基因变异在中国原发性先天性青光眼(PCG)患者发病中的作用。方法收集来自不同地区的16例PCG患者,对其CYP1B1基因编码外显子进行直接测序,对照组进行单核苷酸多态性分析。结果在1例PCG患者中发现了一种变异,为8006G>A(R390H)。它是位于外显子III的错义突变。还发现了五种单核苷酸多态性,分别为3793T>G,R48G,A119S,A330S,V432L。结论CYP1B1基因是导致中国人PCG患者的致病基因,但也有其他变异可能和PCG变异有关。     

CYP1B1 Mutations are a Major Contributor to Juvenile-Onset Open Angle Glaucoma in Saudi Arabia

To describe the genotype and phenotype in 14 unrelated Saudis with juvenile open angle glaucoma (JOAG). Detailed clinical examination was carried out and we sequenced cytochrome P450, family 1, subfamily B (CYP1B1), Myocilin (MYOC) and latent-transforming growth factor beta-binding protein 2 (LTBP2) genes. Twelve (85.7%) patients had apparent sporadic inheritance and 2 (14.3%) presented with a family history of glaucoma. Overall, 12 patients (85.7%) had CYP1B1 mutation. Nine patients had CYP1B1 mutations in a homozygous status. Eight of these had homozygous p.G61E mutation and one had a silent (no amino acid change) sequence change. Two patients had p.G61E mutation in a compound heterozygous status with another CYP1B1 mutation (p.L432V). Two patients had p.G61E in a heterozygous status with no other mutation, while one patient had no mutation(s). None of the patients had any mutation(s) in the MYOC or LTBP2 genes. JOAG associated with CYP1B1 mutations occurs at a high rate in the Saudi population. A specific genotype-phenotype relationship was not demonstrated.     

Mutations in the CYP1B1 gene may contribute to juvenile-onset open-angle glaucoma

Purpose

Glaucoma is one of the leading causes of blindness in the world. Juvenile-onset open-angle is a subtype of glaucoma. In this context, we investigate the possible mutations in the promoter and coding regions of the CYP1B1 gene among patients suffering juvenile-onset open-angle glaucoma (JOAG).

Methods

The CYP1B1 gene was analysed for mutations in 61 unrelated Taiwanese probands with JOAG and in 100 healthy control subjects. Genomic DNA was extracted from peripheral blood leukocytes and then subjected to PCR. The amplified products were screened for base mutations by autosequence. Next, data from the two groups were compared using the χ² test. Additionally, three-dimensional (3D) modelling of the human wild-type and p.R390H mutation was performed using SWISS-MODEL, an automated homology modelling program. Finally, the figure was prepared for the modelled structures by using the Accelrys ViewerLite 5.0 program.

Results

Analysis results indicated two CYP1B1 mutations and five polymorphisms. The prevalence of CYP1B1 gene mutations in this study was 4.92% (3/61). The mutations included a missense mutation (p.Arg390His; 2/3) and a mutation in the 5′-untranslated region (c.1–313A>C; 1/3). Moreover, computer-assisted modelling revealed that this p.R390H mutation affects the intra-molecular interaction in the hydrogen-bonding interaction with Glu387 and Asn428, thus altering significantly the efficiency of the haem-binding and proper folding of the molecule.

Conclusions

As a result, the p.Arg390His mutation might affect the protein structure and, ultimately, the normal function of CYP1B1. Therefore, we suggest that the c.1169G>A (p.Arg390His) mutation of CYP1B1 may be a risk factor for the development of JOAG.     

Novel CYP1B1 mutations and a possible prognostic use for surgical management of congenital glaucoma

AIM: To identify CYP1B1 gene mutations and evaluate their possible role as a prognostic factor for success rates in the surgical management of Egyptian congenital glaucoma patients. METHODS: Totally 42 eyes of 29 primary congenital glaucoma patients were operated on with combined trabeculotomy/trabeculectomy with mitomycin-C and followed up at 1d, 1wk, 1, 6 and 12mo postoperatively. Genomic DNA was extracted from peripheral blood leukocytes. Coding regions of CYP1B1 gene were amplified using 13 pairs of primers, screened for mutations using single-strand conformation polymorphism followed by sequencing of both strands. Efficacy of the operation was graded as either a success [maintaining intraocular pressure (IOP) less than 21 mm Hg with or without anti-glaucoma medication], or a failure (IOP more than 21 mm Hg with topical antiglaucoma medications). RESULTS: Seven novel mutations out of a total of 15 different mutations were found in the CYP1B1 genes of 14 patients (48.2%). The presence of CYP1B1 gene mutations did not correlate with the failure of the surgery (P=0.156, odds ratio=3.611, 95%CI, 0.56 to 22.89); while the positive consanguinity strongly correlated with failure of the initial procedure (P=0.016, odds ratio=11.25, 95%CI, 1.57 to 80.30). However, the Kaplan-Meier survival analysis revealed a significantly lower time of IOP control in the subgroup with mutations in CYP1B1 versus the congenital primary glaucoma group without mutations (log rank test, P=0.015). CONCLUSION: Seven new CYP1B1 mutations are identified in Egyptian patients. Patients harboring confirmed mutations suffered from early failure of the initial surgery. CYP1B1 mutations could be considered as a prognostic factor for surgery in primary congenital glaucoma.     

Molecular Karyotyping of a Dysmorphic Girl from Saudi Arabia with CYP1B1-negative Primary Congenital Glaucoma

Purpose: To report the results of molecular karyotyping for a dysmorphic girl with CYP1B1-negative primary congenital glaucoma from Saudi Arabia, where CYPB1 mutations account for over 90% of cases of primary congenital glaucoma and the remaining cases are idiopathic.

Methods: CYP1B1 sequencing in the affected child; high-resolution array comparative genomic hybridization (array CGH) of the affected child and both unaffected parents (Affymetrix Cytogenetics Whole-Genome 2.7M array; Affymetrix Inc., Santa Clara, CA, USA).

Results: The girl did not harbor CYP1B1 mutation by Sanger sequencing. Array CGH revealed 2 de novo 7p heterozygous duplications (7p21 – 7p14, encompassing 223 genes, and 7p14-7p11.2, encompassing 225 genes) and a 4p homozygous microdeletion (4p14) encompassing one gene only, DTHD1.

Conclusions: The fact that this dysmorphic girl is Saudi Arabian and has CYP1B1-negative primary congenital glaucoma suggests that her glaucoma phenotype is related to her de novo copy number variation. Loss or gain of one or more of the genes encompassed in the identified chromosomal areas may be associated with primary congenital glaucoma and/or other observed phenotypic features.     

Occurrence of MYOC and CYP1B1 variants in juvenile open angle glaucoma Brazilian patients

ABSTRACT

Background: The purpose of this study was to screen juvenile open angle glaucoma (JOAG) patients from Brazil for variants within the MYOC and CYP1B1 genes.

Material and Methods: In this study, we evaluated the coding regions of MYOC and CYP1B1 genes in 100 non-related patients with JOAG and 200 controls through Sanger sequencing. We also tested the most frequent single nucleotide variants of CYP1B1 for association with JOAG.

Results: Sixteen different sequence variants in the MYOC gene were observed in JOAG patients: eight variants were described as neutral and eight were identified in 34 out of 100 patients with JOAG and no controls, thus being considered damaging. In the CYP1B1 gene, nine neutral variants and two damaging alterations were found among JOAG patients. No association between CYP1B1 variants and JOAG was detected.

Conclusion: While MYOC damaging alterations were highly prevalent (34%), CYP1B1 damaging variants were less frequent (2%) in this cohort of Brazilian JOAG patients.     

原发性先天性青光眼CYP1B1基因新变异

目的 探讨CYP1B1基因变异在湖南地区原发性先天性青光眼患者中的分布.方法 病例对照研究.收集来自湖南地区的13例原发性先天性青光眼患者的临床资料进行分析,对13例患者的CYP1B1基因编码外显子进行直接测序和聚合酶链反应-限制性内切酶技术检测.结果 13例原发性先天性青光眼患者中,有1例发现一种基因新突变(c.C319G,L107V),是位于外显子2的错义突变.100例正常人中未见L107V突变.同时发现已报道的4种单核苷酸多态位点,分别为R48G、A119S、V432L、D449D.结论 CYP1B1基因L107V突变可能是导致湖南地区原发性先天性青光眼患者的致病原因之一.     

CYP1B1基因缺失小鼠在高糖皮质激素环境下对青光眼的易感性

目的观察高浓度糖皮质激素对CYP1B1^-/-小鼠青光眼易感性的影响。方法采用成年CYP1B1^-/-小鼠作为实验模型,以同龄C57BL／6J小鼠作为对照。每3天结膜下注射0．04mL倍他米松,用Tonopen眼压（IOP）笔每周定期测定小鼠IOP,于第一次给药前,给药后4、8、12周分别摘除眼球,制备石蜡切片,光学显微镜下观察小鼠视网膜形态和厚度,采用TUNEL法检测视网膜神经节细胞（RGCs）的凋亡。结果与给药前相比,2组小鼠给药后4、8、12周的IOP均较前升高,差异有统计学意义（P〈0．05）;CYP1B1^-/-小鼠的IOP在给药后8周、12周较C57BL／6J小鼠显著升高,差异有统计学意义（P〈0．05）。随着倍他米松注射时间的推移,2组小鼠视网膜纤维层均变薄,各时间组的总体差异有统计学意义（P〈0．05）;且CYP1B1^-/-小鼠视网膜纤维层厚度在给药后8周、12周较C57BL／6J小鼠显著变薄,差异有统计学意义（P〈0．05）。2组小鼠RGCs凋亡的速度与给药前相比均显著增加,差异有统计学意义（P〈0．05）;且CYP1B1^-/-小鼠与C57BL／6J小鼠相比结果更为显著（P〈0．05）。结论在高浓度糖皮质激素的诱导下,CYP1B1^-/-小鼠对青光眼的易感性增加。     

Molecular analysis of the CYP1B1 gene: identification of novel truncating mutations in patients with primary congenital glaucoma

BACKGROUND: Mutations and polymorphisms have been identified in the CYP1B1 gene; while mutations that affect the conserved core structures of cytochrome P4501B1 result in primary congenital glaucoma (PCG), mutations in other regions hold the potential to define differences in estrogen metabolism. In the present study, we analyzed the CYP1B1 gene in Mexican patients with PCG and described four novel mutations. MATERIALS AND METHODS: The sample included 12 nonrelated cases with PCG. Analysis of coding regions of the CYP1B1 gene was performed through PCR and DNA sequencing analysis from genomic DNA. RESULTS AND DISCUSSION: Molecular analysis of the CYP1B1 gene showed the following molecular defects: (1) a novel single-base pair deletion within codon 370 (1454delC) that produces a substitution of leucine instead of proline and a premature stop codon 57 amino acids after the last original amino acid; this family also harbored a novel polymorphic variant of the cytochrome P4501B1 with six single-nucleotide polymorphisms (142C-->G; 355G-->T; 729G-->C; 4326C-->G; 4360C-->G and 4379C-->T); (2) a novel single-base pair deletion within codon 277 (1176delT) that results in a premature stop codon; (3) a novel single-base pair deletion within codon 179 (880delG) that produces a substitution of arginine instead of alanine and a premature stop codon 17 amino acids downstream from the last original amino acid, and (4) a duplication (or insertion) of ten base pairs within codon 404 (1556dupATGCCACCAC) that results in a premature stop codon 26 amino acids after the last original amino acid. We also observed in 2 nonrelated patients a deletion of 13 bp (1410_1422delGAGTGCAGGCAGA) previously reported for other populations. CONCLUSION: We reported four novel mutations and a novel polymorphic variant in the CYP1B1 gene in PCG in the Mexican population; it has important implications in diagnosis and genetic counseling.     

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.