Justyna A Karolak1, Malgorzata Rydzanicz2, Barbara Ginter-Matuszewska1, Jose A Pitarque3, Andrea Molinari3, Bassem A Bejjani4, Marzena Gajecka1. 1. Department of Genetics and Pharmaceutical Microbiology, Poznan University of Medical Sciences, Poznan, Poland 2Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland. 2. Department of Medical Genetics, Medical University of Warsaw, Warsaw, Poland. 3. Department of Ophthalmology, Hospital Metropolitano, Quito, Ecuador. 4. Elson S. Floyd College of Medicine, Washington State University, Spokane, Washington, United States.
Abstract
PURPOSE: Keratoconus (KTCN) is a degenerative disorder of the eye that is characterized by a conical shape and thinning of the cornea, resulting in impaired visual function. Previously, we identified heterozygous single base-pair substitutions in DOCK9, IPO5, and STK24, showing concurrent 100% segregation with the affected phenotype in an Ecuadorian family. As the pathogenic consequences of these variants were not obvious, we performed in vitro splicing analyses to determine their functional significance. METHODS: We generated expression constructs using patient DNA as a template corresponding to the wild-type and mutant alleles of DOCK9, IPO5, and STK24. After transfecting HeLa cells with each construct, total RNA samples were extracted, reverse transcribed, and amplified using specific primers. RESULTS: In vitro splicing analysis revealed that only c.2262A>C in exon 20 of DOCK9 led to aberrant splicing, resulting in the changed ratio between two protein isoforms: a normal transcript and a transcript with exon skipping. The exon skipping causes a premature stop codon, disrupting the functional domains of DOCK9 protein, which may alter the biological role of DOCK9 as a Cdc42 activator. CONCLUSIONS: Based on in vitro results, we demonstrated that c.2262A>C substitution in DOCK9, previously identified in KTCN-affected members of an Ecuadorian family, leads to a splicing aberration. However, because the mutation effect was observed in vitro, a definitive relationship between DOCK9 and KTCN phenotype could not be established. Our results indicate that further elucidation of the causes of KTCN is needed.
PURPOSE: Keratoconus (KTCN) is a degenerative disorder of the eye that is characterized by a conical shape and thinning of the cornea, resulting in impaired visual function. Previously, we identified heterozygous single base-pair substitutions in DOCK9, IPO5, and STK24, showing concurrent 100% segregation with the affected phenotype in an Ecuadorian family. As the pathogenic consequences of these variants were not obvious, we performed in vitro splicing analyses to determine their functional significance. METHODS: We generated expression constructs using patient DNA as a template corresponding to the wild-type and mutant alleles of DOCK9, IPO5, and STK24. After transfecting HeLa cells with each construct, total RNA samples were extracted, reverse transcribed, and amplified using specific primers. RESULTS: In vitro splicing analysis revealed that only c.2262A>C in exon 20 of DOCK9 led to aberrant splicing, resulting in the changed ratio between two protein isoforms: a normal transcript and a transcript with exon skipping. The exon skipping causes a premature stop codon, disrupting the functional domains of DOCK9 protein, which may alter the biological role of DOCK9 as a Cdc42 activator. CONCLUSIONS: Based on in vitro results, we demonstrated that c.2262A>C substitution in DOCK9, previously identified in KTCN-affected members of an Ecuadorian family, leads to a splicing aberration. However, because the mutation effect was observed in vitro, a definitive relationship between DOCK9 and KTCN phenotype could not be established. Our results indicate that further elucidation of the causes of KTCN is needed.
Authors: Michal Kabza; Justyna A Karolak; Malgorzata Rydzanicz; Michał W Szcześniak; Dorota M Nowak; Barbara Ginter-Matuszewska; Piotr Polakowski; Rafal Ploski; Jacek P Szaflik; Marzena Gajecka Journal: Eur J Hum Genet Date: 2017-02-01 Impact factor: 4.246
Authors: Justyna A Karolak; Tomasz Gambin; Malgorzata Rydzanicz; Piotr Polakowski; Rafal Ploski; Jacek P Szaflik; Marzena Gajecka Journal: PeerJ Date: 2020-04-14 Impact factor: 2.984