CYP1B1 variants have low contribution to Pakistani patients with Primary Open Angle Glaucoma Page

Background Glaucoma is a group of complex neurodegenerative ophthalmological disorders and is the second common of cause of irreversible blindness around the globe. Primary open-angle glaucoma (POAG) is the commonest type of all glaucoma and is characterized by elevated intraocular pressure (IOP) leading to optic nerve damage and visual field defects. Inherited mutations in CYP1B1 are a rare cause of POAG. Objective This study was conducted to screen Pakistani population with POAG for CYP1B1 variants. Methods Detailed family history was recorded from all participating families. The disease was confirmed through ophthalmological examinations. Blood samples were collected for genomic DNA extraction. CYP1B1 exons were directly sequenced in one affected individual from each family.

2 Abstract Background Glaucoma is a group of complex neurodegenerative ophthalmological disorders and is the second common of cause of irreversible blindness around the globe. Primary open-angle glaucoma (POAG) is the commonest type of all glaucoma and is characterized by elevated intraocular pressure (IOP) leading to optic nerve damage and visual field defects.
Inherited mutations in CYP1B1 are a rare cause of POAG.

Objective
This study was conducted to screen Pakistani population with POAG for CYP1B1 variants.

Methods
Detailed family history was recorded from all participating families. The disease was confirmed through ophthalmological examinations. Blood samples were collected for genomic DNA extraction. CYP1B1 exons were directly sequenced in one affected individual from each family.

Results
CYP1B1 sequencing revealed a novel heterozygous missense variant, c.649G>A (p.Asp217Asn). All affected individuals having the variant had characteristic glaucomatous changes with mean disease onset at 35 years. The unaffected individuals in the family had no signs of POAG. The prevalence of CYP1B1 associated POAG in study cohort is 4% (1/25).
In silico predictions showed that p.Asp217Asn, substitution affects the structure and function of the protein.

Conclusion
The study suggests that CYP1B1 mutations are considered to have have less contribution in pathogenesis of POAG in Pakistani population studied. Identification of novel dominantly inherited variant shows allelic heterogeneity and may help in early diagnosis for effective 3 management of the disease through genetic counseling. Background Glaucoma, the second most important cause of irreversible blindness in the entire globe, is characterized by raised intraocular pressure (IOP) and damage to retinal ganglionic cells of the retina which together result in cupping of the optic disc and its atrophy with resultant visual field defects. (Wójcik-Gryciuk et al., 2015) Based on anatomy and structure of irido-corneal angle, glaucoma is into Primary open angle glaucoma (POAG) and Primary angle closure glaucoma (PACG). POAG is divided into Adult onset glaucoma which occurs mostly after 40 years of age and Juvenile onset glaucoma which starts between 3 and 40 years of age (Liu and Allingham, 2017).
POAG is the most common variety of glaucoma in Asia and Europe including Pakistan. (Iqbal et al., 2011) It produces more structural damage than other forms of glaucoma due to its asymptomatic progression and late diagnosis in most of the cases (Kwon et al., 2009, Cheng et al., 2012. It is genetically heterogeneous in nature and is usually inherited as an autosomal dominant trait but in the majority of the cases, the pattern of inheritance is complex and multifactorial. Up till now more than 20 loci have been identified for POAG. (Wirtz et al., 2010, Abu-Amero et al., 2012 Myocilin (MYOC) is the most common gene responsible for POAG, but some cases have also been reported with variants in CYP1B1. (Liu andAllingham, 2017, Abu-Amero et al., 2015) CYP1B1 (OMIM 601771) encodes an enzyme, cytochrome P450 B1, which is required for the metabolism of endogenous substrates in ciliary epithelium, and has a role in the production of aqueous humour. (Tang et al., 1996, Stoilov et al., 1998, Civan and Macknight, 2004 The absence or reduced activity of cytochrome P450B1 may result in abnormal flow of aqueous humour and causes increased intraocular pressure and subsequent glaucomatous changes (Micheal et al., 2015). Mutations in CYP1B1 have been 4 associated with anterior segment disorders, including Primary Congenital Glaucoma (PCG), POAG, Juvenile open angle glaucoma (JOAG), Peter's anomaly and Rieger's anomaly. To date, over 200 pathogenic variants have been identified in CYP1B1 and the majority of the mutations are substitutions, followed by insertions and deletions (Stenson et al., 2017). It is noteworthy that CYP1B1 mutations are population specific and have been detected in patients with diverse ethnic backgrounds. (Sheikh et al., 2014, Rauf et al., 2016 The Pakistani population is genetically heterogeneous with combination of several ethnicities. Previous research indicates presence of population specific variants in numerous disease causing genes. Thus, the present study was aimed to investigate the role of CYP1B1 in the pathogenesis of POAG in Pakistani population resides in Sindh province. This may help to study the contribution of CYP1B1 mutation in POAG patients and may also help identify common variants for genetic counseling.

Recruitment of subjects and Clinical Investigations
The study approval was obtained from Ethical Review Committee of Liaquat University of Medical and Health Sciences, Jamshoro. Total 25 families with two or more than two members affected with POAG were recruited for the study. All the families were visited to record detailed medical history and pedigrees were drawn. Informed written consent was obtained from all members of enrolled families for the study. All affected members of the enrolled families were subjected to extensive ophthalmological examination to confirm the diagnosis of POAG. Intra ocular pressure (IOP) was measured by Goldmann applanation tonometry, irido-corneal angles (anterior chamber angles) were evaluated by the Goldmann gonioscope. The fundus was examined using slit lamp biomicroscopy with +90 Diopter lens. Venous blood was taken in EDTA containing tubes from all affected and normal members of enrolled families and genomic DNA was extracted by using standard 5 non-organic protocol (Grimberg et al., 1989).

Mutation Screening and Bioinformatics Analysis:
Both coding exons of CYP1B1 gene were amplified and directly sequenced as described previously (Sheikh et al., 2014) . Chromatograms were analyzed by using Chromas v 1.45 software. The Pathogenic role of the novel variant was assessed by using mutation taster, Sorting Intolerant from Tolerant (SIFT) and HOPE web tools. The Custal Omega was used to find the conservation status of substituted amino acid in seven species. Biochemical properties of wild type and mutated amino acids and their importance in normal structure and function of protein was assessed using HOPE web tool.

Results
Total 25 consanguineous families with two or more than two affected members with POAG were enrolled for this study. Pedigree analysis and medical history showed that all families had dominantly inherited disease with onset between 35 to 55 years.
Ophthalmological examination confirmed the presence of POAG. Both coding exons of CYP1B1 were sequenced in single affected from each family.
The sequencing data revealed a novel heterozygous transition, c.649 G>A, substituting Aspartic acid into Asparagine at codon 217 (p.Asp217Asn) of CYP1B1 in one family,POAG-02, (4%, 1/25) (Fig 1.B). No CYP1B1 variant was found in any other family. Pedigree analysis of POAG-02 revealed an autosomal dominant trait segregating in 8 affected individuals in 2 generations (Fig. 1.A). Mutation analysis showed that all 8 affected were heterozygous carriers of the variant, whereas, phenotypically normal individuals had wildtype genotype. The mean age of affected patients of POAG-02 family was 45 years, ranging from 40 to 88 years. The findings of the clinical examination showed severe characteristic glaucoma features (Table 1). The c.649 G>A, p.Asp217Asn is a novel variant and has not been reported earlier in association with POAG. Bioinformatics studies 6 predicted Aspartic acid at 217 th codon of CYP1B1 as a conserved residue across the six species and its substitution with Asparagine is deleterious with pathogenic effects on normal protein. In addition, this variant was not found in 1000 genome database and Exome Aggregation Consortium (ExAC) database. This variant was also examined in 60 ethnically matched controls and was not detected.

Discussion
Glaucoma is a chronic and heterogeneous group of ocular disorders characterized by raised intraocular pressure (IOP) causing degeneration and atrophy of optic nerve fibers and manifesting as visual field defects and irreversible blindness if remain untreated.
Many studies have suggested the role of elevated IOP in the pathogenesis of POAG indicating its close association in its development but the etiology of glaucoma is still unclear. Genetic and molecular studies have indicated an important role of genes in the development of POAG like many other ocular disorders. Identification of disease causing genes may provide insight into the pathogenesis of inherited disorders. (Fingert, 2011) This study was aimed to evaluate the role of CYP1B1 variants in families affected with dominantly inherited POAG in Sindh Province. Twenty five families affected with POAG were screened for variants in CYP1B1; a heterozygous CYP1B1 variant, segregating with the disease phenotype was found in all 8 affected individuals of family POAG2. The prevalence of the CYP1B1 was 4% (1/25) in our group of POAG patients; whereas, the previous study on 40 Pakistani families affected with POAG described CYP1B1 disease causing variants in 10% (4/40) of the patients. In addition, the prevalence of CYP1B1 in non-familial cases (3%, 6/190) was not significantly different from our findings (Micheal, Ayub et al. 2015). The variation of affected individuals in familial cases may be due to different ethnic backgrounds of the patients within Pakistani population. Moreover, results of this study in familial POAG are comparable with the findings of an Indian study, where CYP1B1 variants were found in 4% of the POAG patients (4/200) (Melki et al., 2004).
The patients included in this study had the onset of POAG from 38-50 years (average= 43.3 years). All patients had normal or near-normal IOP as they underwent early trabeculectomy soon after diagnosis with POAG except one (patient IV: 12). There was complete cupping in the patient (IV:12) who had not undergone trabeculectomy, whereas all other affected individuals exhibited significant cupping corresponding to respective reduced visual acuity.
c.649 G>A, p.Asp217Asn is predicted to be pathogenic by mutation taster as disease causing and damaging. Whereas SIFT web tool indicted it as pathogenic. According to HOPE tool, the mutated amino acid (Asparagine) is neutral whereas the wild-type amino acid (Aspartic Acid) has a negative charge. The wild type amino acid forms linkages with Arginine at 122 nd and 183 rd positions of CYP1B1 protein; however incorporation of mutated amino acid causes alterations in the polarity and affects ionic interactions of the protein (Fig 2 A). In addition, the mutated amino acid is located in a highly important domain that has the vital role in interactions between different protein domains and it also affects binding capabilities of the protein with other molecules (Fig 2 A). The mutated amino acid thus affects the overall activity of the protein thus results in the manifestation of glaucomatous symptoms. Aspartic acid at 217 th position is not fully conserved in all seven species studied (Fig 2 B), but it is still important for normal function of the protein in humans.
Previously, only one study was conducted on the role of CYP1B1 in Pakistani patients with family history of POAG and revealed one novel heterozygous mutation (p.D316V) and a previously reported p.E229K mutation. Moreover, three novel heterozygous variants 8 (p.T234K, p.A287P & p.Q362*) and three previously reported heterozygous variants (p.G61E, p.E229K & p.R368H) were detected in 190 sporadic POAG patients (Michael S, Ayub 2015). It is significant that, heterozygous CYP1B1 variants cause POAG, whereas in case of familial PCG, heterozygous are phenotypically normal (Sheikh et al., 2014).
CYP1B1 mutations have shown interfamilial clinical variations; some patients may have congenital glaucoma, whereas other carrying same mutation may have POAG (Melki et al., 2004). In our study, all the affected have onset from 35 to 50 years and family history did not reveal incidence of congenital glaucoma cases. Interestingly, homozygous CYP1B1 mutations have also been associated with POAG, where heterozygous individuals with CYP1B1 variants were phenotypically normal (Acharya et al., 2006). Similarly, in Iranian patients with POAG; homozygous CYP1B1 variants are associated with POAG and JOAG (Suri et al., 2008). It is hypothesized that there are other genetic or epigenetic factors which influence the expression and pathogenicity of CYP1B1 variants. Mutation in CYP1B1 gene may be an important risk factor for early-onset POAG (Vincent et al., 2002).

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