title: Mutational analysis of MIR184 in sporadic keratoconus and myopia.
abstract: OBJECTIVE
  A mutation miR-184(+57C>T) in the seed region of miR-184 (encoded by MIR184 [MIM*613146]) results in familial severe keratoconus combined with early-onset anterior polar cataract and endothelial dystrophy, iris hypoplasia, congenital cataract, and stromal thinning (EDICT) syndrome (MIM#614303). In order to investigate the phenotypic spectrum resulting from MIR184 mutation, MIR184 was sequenced in a keratoconus cohort of mixed ethnicity and a Chinese axial myopia cohort.
  
  
  METHODS
  Sequencing of MIR184 was performed in 780 unrelated keratoconus patients and 96 unrelated Han southern Chinese subjects with axial myopia. Effects of identified mutations on RNA secondary structure were predicted computationally using mFold and RNAFold algorithms. MIR184 amplicons from patients harboring mutations were cloned and transfected into human embryonic kidney 293T (HEK293T) cells, and mature mutant miR-184 expression was analyzed by stem-loop real-time quantitative PCR (RT-qPCR).
  
  
  RESULTS
  Two novel heterozygous substitution mutations in MIR184 were identified in the two patients with isolated keratoconus: miR-184(+8C>A) and miR-184(+3A>G). Computational modeling predicted that these mutations would alter the miR-184 stem-loop stability and secondary structure. Ex vivo miR-184 expression analysis demonstrated that miR-184(+8C>A) almost completely repressed the expression of miR-184 (P = 0.022), and miR-184(+3A>G) reduced the expression of miR-184 by approximately 40% (P = 0.002). There was no significant association of rs41280052, which lies within the stem-loop of miR-184, with keratoconus. No MIR184 mutations were detected in the axial myopia cohort.
  
  
  CONCLUSIONS
  Two novel heterozygous substitution mutations in MIR184 were identified in two patients with isolated keratoconus: miR-184(+8C>A) and miR-184(+3A>G). Mutations in MIR184 are a rare cause of keratoconus and were found in 2 of 780 (0.25%) cases.
authors.affiliation: Centre for Vision and Vascular Science, Queen's University Belfast, Belfast, Northern Ireland, United Kingdom.
authors.forename: Judith
authors.initials: J
authors.lastname: Lechner
authors.forename: Ha Ae
authors.initials: HA
authors.lastname: Bae
authors.forename: Jasenka
authors.initials: J
authors.lastname: Guduric-Fuchs
authors.forename: Aine
authors.initials: A
authors.lastname: Rice
authors.forename: Gowthaman
authors.initials: G
authors.lastname: Govindarajan
authors.forename: Salina
authors.initials: S
authors.lastname: Siddiqui
authors.forename: Layal
authors.initials: L
authors.lastname: Abi Farraj
authors.forename: Shea Ping
authors.initials: SP
authors.lastname: Yip
authors.forename: Maurice
authors.initials: M
authors.lastname: Yap
authors.forename: Manoranjan
authors.initials: M
authors.lastname: Das
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Souzeau
authors.forename: Doug
authors.initials: D
authors.lastname: Coster
authors.forename: Richard A
authors.initials: RA
authors.lastname: Mills
authors.forename: Richard
authors.initials: R
authors.lastname: Lindsay
authors.forename: Tony
authors.initials: T
authors.lastname: Phillips
authors.forename: Paul
authors.initials: P
authors.lastname: Mitchell
authors.forename: Manir
authors.initials: M
authors.lastname: Ali
authors.forename: Chris F
authors.initials: CF
authors.lastname: Inglehearn
authors.forename: Periasamy
authors.initials: P
authors.lastname: Sundaresan
authors.forename: Jamie E
authors.initials: JE
authors.lastname: Craig
authors.forename: David A
authors.initials: DA
authors.lastname: Simpson
authors.forename: Kathryn P
authors.initials: KP
authors.lastname: Burdon
authors.forename: Colin E
authors.initials: CE
authors.lastname: Willoughby
mesh_terms: D004247:DNA / Q000032:analysis / Q000235:genetics*; D004252:DNA Mutational Analysis; D005260:Female; D005838:Genotype; D006801:Humans; D007640:Keratoconus / Q000235:genetics* / Q000378:metabolism; D008297:Male; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics*; D008875:Middle Aged; D009154:Mutation; D009216:Myopia / Q000235:genetics* / Q000378:metabolism; D010375:Pedigree; D060888:Real-Time Polymerase Chain Reaction
chemical_list: C534424:MIRN184 microRNA, human; D035683:MicroRNAs; D004247:DNA
keywords: MIR184; corneal dystrophies; hereditary; hsa-miR-184; keratoconus; myopia