title: Variant c.2262A>C in DOCK9 Leads to Exon Skipping in Keratoconus Family.
abstract: OBJECTIVE
  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.affiliation: Department of Genetics and Pharmaceutical Microbiology, Poznan University of Medical Sciences, Poznan, Poland 2Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.
authors.forename: Justyna A
authors.initials: JA
authors.lastname: Karolak
authors.affiliation: Department of Medical Genetics, Medical University of Warsaw, Warsaw, Poland.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Rydzanicz
authors.affiliation: Department of Genetics and Pharmaceutical Microbiology, Poznan University of Medical Sciences, Poznan, Poland 2Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Ginter-Matuszewska
authors.affiliation: Department of Ophthalmology, Hospital Metropolitano, Quito, Ecuador.
authors.forename: Jose A
authors.initials: JA
authors.lastname: Pitarque
authors.affiliation: Department of Ophthalmology, Hospital Metropolitano, Quito, Ecuador.
authors.forename: Andrea
authors.initials: A
authors.lastname: Molinari
authors.affiliation: Elson S. Floyd College of Medicine, Washington State University, Spokane, Washington, United States.
authors.forename: Bassem A
authors.initials: BA
authors.lastname: Bejjani
authors.affiliation: Department of Genetics and Pharmaceutical Microbiology, Poznan University of Medical Sciences, Poznan, Poland 2Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.
authors.forename: Marzena
authors.initials: M
authors.lastname: Gajecka
mesh_terms: D003315:Cornea / Q000473:pathology*; D004484:Ecuador; D005091:Exons; D020869:Gene Expression Profiling; D008040:Genetic Linkage; D020022:Genetic Predisposition to Disease; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007640:Keratoconus / Q000235:genetics* / Q000473:pathology; D020125:Mutation, Missense; D010375:Pedigree; D022821:RNA Splice Sites; D012326:RNA Splicing; D016415:Sequence Alignment
chemical_list: C466321:DOCK9 protein, human; D020662:Guanine Nucleotide Exchange Factors; D022821:RNA Splice Sites