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Subhabrata Chakrabarti, meha kabra, Sonika Rathi, Wei Zhang, Anil K Mandal, Sirisha Senthil, Inderjeet Kaur, Hemant Khanna; Digenic Inheritance and Physical Interactions of CYP1B1 and TEK in Primary Congenital Glaucoma. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2122.
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© ARVO (1962-2015); The Authors (2016-present)
Our goal is to understand the molecular underpinnings of autosomal recessive primary congenital glaucoma (PCG) pathogenesis. A recent study implicated heterozygous mutations in TEK (tyrosine kinase, endothelial receptor) in PCG, however, mutations in the second gene were not detected. We undertook this study to assess the prevalence of TEK mutations in a large cohort of PCG cases (n=285) from Southern India, who were devoid of homozygous mutations in the known genes (CYP1B1 and LTBP2).
Screening of the entire coding and untranslated regions of TEK was accomplished through a customized gene panel on a next generation sequencing platform (Ion Proton) using the Ampliseq chemistry. The raw data was analyzed by GATK and imported to the Ion Reporter software (version 5.2) and aligned to the hg19 sequence for further analysis. Each variation had a coverage of 100-500X and was further validated by Sanger sequencing on an automated DNA sequencer (ABI 3130 XL) using the BigDye chemistry. Since, heterozygous TEK mutations were found to co-segregate with heterozygous CYP1B1 variations, we functionally validated the potential genetic interaction between these two genes. To this end, we transiently transfected HEK293 cells with constructs encoding GFP-fused CYP1B1 and HA-tagged TEK, followed by immunoprecipitation and immunoblot analyses.
Eleven novel heterozygous variations in TEK including a nonsense (Y285X) and 10 missense (E103D, R119C, I148T, Y285X, E300G, P304L, H400R, R413W, G743A, G951D, C1044Y) changes were observed in 25/285 cases, with an overall mutation frequency of 8.77% (95%CI, 6.01%-12.63%) in our cohort. These changes were absent in geographically and ethnically matched controls (n=1000). Bioinformatic analysis (SIFT, PolyPhen) indicated these variations to be potentially pathogenic. Interestingly, the heterozygous TEK alleles (E103D, I148T and G743A) co-segregated with heterozygous CYP1B1 alleles (A115P, E229K and R368H) respectively, in PCG-affected children. Furthermore, CYP1B1 and TEK associated with each other in HEK293 cells.
Our study expands the mutation spectrum of TEK as a candidate gene in PCG and suggest that TEK and CYP1B1 genetically and physically interact with each other. Further studies are underway to determine the involvement of CYP1B1 in TEK-mediated angiogenesis and its deregulation in the phenotypic manifestation of PCG.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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