The Mutations Research Of CYP1B1 Gene For Primary Congenital Glaucoma In Guangdong Province

Background:Glaucoma is the second largest blinding eye disease, involving the world's six thousand seven million people, 10% of patients with bilateral blindness. Blinding Glaucoma is the main reason for Chinese adults. Glaucoma can be classified according to etiology of primary and secondary glaucoma. According to diffierent etiology, glaucoma can be divided into primary glaucoma and secondary glaucoma. There are three types of primary glaucoma: primary congenital glaucoma, primary open angle glaucoma and primary angle-closure glaucoma.Primary congenital glaucoma (primary congenital glaucoma, PCG) is a good fat in infants and young children inherited cause of blindness, congenital glaucoma is the most common type of male and female incidence rate ratio of about 2:1, more distributed, the treatment less effective, and ultimately lead to the optic nerve damage and atrophy or even blindness, can not see again. More involvement of both eyes simultaneously or successively. Primary congenital glaucoma is characterized by a marked increase of intraocular pressure at birth or early childhood(less than 3 years old), large ocular globes (buphthalmos), corneal edema, and Haab's striae. It is associated with developmental defects in the trabecular meshwork of anterior chamber angle,PCG is the most common childhood glaucoma, which is observed in the neonatal or infantile period, and is an important cause of visual loss in children. The incidence ratio is approximately 1 in 30,000 within live births. IOP is a risk of PCG. it is also called Buphthalmos. The exclusion of other ocular or systemic anomalies, buphthalmos can be one of the basis diagnosis of PCG . When one children occurs with corneal diameter> 12mm or corneal edema, and Haab's striae, intraocular pressure> 2lmmHg or increasing the ratio of cup / disc, and epiphora, photophobia, decreased visual acuity and other clinical manifestations, exclusion of other congenital anomalies , it can be diagnosed for PCG.The incidence of PCG varies among geographic locations and ethnic communities, from 1:10,000–20,000 in western countries, to 1:2,500 and 1:1250 in inbred Slovakian Gypsy and Saudi Arabian populations, respectively. the incidence rate in Israel is 1:1200. So far, three genetic loci have been linked to PCG: GLC3A , GLC3B, and GLC3C. The only gene that has been identified is the cytochrome P4501B1 gene (CYP1B1) linked to GLC3A. CYP1B1 has been implicated in the pathogenesis of PCG. There are only few reports to investigate the genetic characteristics of CYP1B1 gene to primary congenital glaucoma in our country, therefore, it is critical for us to screen CYPlBl gene in patients with PCG in Chinese.Objective:1. Using High-Resolution Melting to detect variations in high-frequency area of CYP1B1 gene (Cytochrome P450, family 1, subfamily B, polypeptide 1);2. The data of whole CYP1B1 gene in normal Han race of Guangdong province can help us to study the variations of CYP1B1 gene in Han race of Guangdong Province. It is critical for us to obtain the sequence of CYP1B1 gene. The obtained sequence will provide not only criterion in Han race of Guangdong province, but also features contrast with PCG group. And laying the foundation for study of the relationship between CYP1B1 variations and PCG;3. Looking for variations outside the high-frequency area involved PCG on the base of datas in Han race of Guangdong province.Methods:1. Preparation of peripheral blood and extract genomic DNA, using polymerase chain reaction (Polymerase chain Reaction, PCR) to amplify fragments in high-frequency area, and then using high-resolution melting (High-Resolution Melting, HRM) to scan and analyse the fragments of CYP1B1. And the fragments will be sequenced directly which can confirm the results of HRM.2. Using the Polymerase chain reaction to amplify the whole CYP1B1 gene of 3 controls. And then sequencing, fragments assembly, homology comparison analysis, variations summary and analysis. providing basic datas of normal CYP1B1 gene for Han race of Guangdong province.3. Using the Polymerase chain reaction to amplify the whole CYP1B1 gene of PCG group. And then sequencing, fragments assembly, homology comparison analysis, variations summary and analysis. Analysing the characteristics of the whole CYP1B1 gene, and finding the pathogenic factors in CYP1B1 gene of PCG in Han race of Guangdong province.Results:1. In 46.2% (6 / 13) of the PCG, we detected 7 CYP1B1 variations, 3 of which were new variations and have never been reported. The others have been reported before. In this study, g.2862G→T(Q159H), g.6549A→G(N377D), g.6598G→A(S393N) was not detected in 3 controls, suggest us: 3 of them maybe new variations, and have correlated with PCG. All the 7 variations are located in the coding region, g.2527C→G (R48G), g.2740G→T (A119S), g.2862G→T (Q159H) located in the second exon, g.6549A→G (N377D), g.6589G→A (R390H), g.6598G→A (S393N) g.6767C→T (D449D) located in the third exon. g.2862G→T (Q159H), g.6549A→G (N377D), g.6589G→A (R390H), g.6598G→A (S393N) have not been detected in controls.2. 24 CYP1B1 variations were detected in controls, and 22 located in the non-coding region, only 2 (g.2740G> T, g.6983T> C) located in the coding region of CYP1B1. g.6983T> C has not been reported in current literatures. g.1274T> C, g.1385C> T located in the flanking sequence, g.2123G> A, g.2373C> T located in the first intron, these four variations have been reported in other ethnic race. g.7162A> G and g.7533G> A located in the third non-coding exon. All the remaining 16 variations of CYP1B1 located in the second intron.3. 134 CYP1B1 gene variations were detected in PCG group. Variations'analysis please see discussion.Conclusion:1.⑴7 CYP1B1 variations were detected in 46.2% (6 / 13) of the PCG. All of them located in the coding region, and 3 variations have not been reported in currently literatures. ⑵g.2527C→G (R48G) has been reported in other ethnic race as a CYP1B1 gene SNPs. We do not rule out the conclusion that this variation has correlation with PCG in Han race of Guangdong province.2.⑴Obtaining basic datas of CYP1B1 gene in Han race of Guangdong province.⑵Sequence analysis shows: 24 CYP1B1 variations were detected in 3 controls, and 17 of them variations have not been reported in currently literatures. All the 24 CYP1B1 variations do not the leading cause of PCG.3.⑴134 CYP1B1 gene variations were detected in PCG group;⑵18 variations which are not the leading cause of CYP1B1 struction or function abnormal were excluded. There are still 116 variations unable to determine whether can cause PCG.