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Cited by 78Corneal surgery and disordersMicroRNA in disease regulationRetinal Development and Disorders

Mutational Analysis of MIR184 in Sporadic Keratoconus and Myopia

Open access
Lechner, Judith, Bae, Ha Ae, Guduric-Fuchs, Jasenka, Rice, Aine, Govindarajan, Gowthaman, Siddiqui, Salina, Abi Farraj, Layal, Yip, Shea Ping +15 more·2013·Investigative Opthalmology & Visual Science
DOI 10.1167/iovs.13-12035
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Abstract

PURPOSE: 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.

AI snapshot

Written from the paper’s full text.

TL;DR

Sequencing MIR184 in 780 keratoconus patients and 96 Han Chinese axial myopia subjects identified two novel heterozygous MIR184 substitutions in isolated keratoconus (2/780, 0.25%) and none in myopia. Both mutations reduced mature miR-184 expression in vitro, while rs41280052 was not associated with keratoconus.

Key findings

  • Two novel heterozygous MIR184 mutations, miR-184(+8C>A) and miR-184(+3A>G), were found in two Caucasian patients with isolated keratoconus; no MIR184 mutations were detected in the 96 axial myopia subjects. Mutations in MIR184 were a rare cause of keratoconus, found in 2/780 (0.25%) cases.
  • Computational modelling predicted altered stem-loop stability: WT miR-184 ΔG = -35.50 kcal/mol, miR-184(+3A>G) ΔG = -34.70 kcal/mol, and miR-184(+8C>A) ΔG = -33.40 kcal/mol, corresponding to changes of +0.8 and +2.1 kcal/mol.
  • Ex vivo expression analysis showed miR-184(+8C>A) almost completely repressed mature miR-184 expression (P = 0.022), while miR-184(+3A>G) reduced expression by approximately 40% (P = 0.002); WT transfection significantly increased miR-184 expression (P < 0.001).
  • SNP rs41280052 showed no significant association with keratoconus: minor allele frequency was 1.3% in cases (692 patients) versus 1.7% in controls (1865 individuals), χ² = 0.86, 1 d.f., p = 0.3537.

Methods

  • Sanger sequencing of MIR184 was performed in 780 unrelated keratoconus patients (667 European Caucasian, 123 South Indian) and 96 unrelated Han southern Chinese subjects with axial myopia plus 96 controls; rs41280052 was genotyped in 692 Caucasian keratoconus cases and 1865 unaffected controls from the Blue Mountains Eye Study.
  • Effects of mutations on RNA secondary structure were predicted computationally using mFold and RNAFold, with nucleotide conservation assessed via the UCSC genome browser.
  • MIR184 amplicons from patients with mutations were cloned and transfected into HEK293T cells; mature mutant miR-184 expression was analysed by stem-loop RT-qPCR relative to miR-103 and miR-218, with at least three independent biological replicates and REST analysis.

Limitations

  • Stated: penetrance of miR-184(+3A>G) is not complete, segregation of miR-184(+8C>A) could not be verified, and an elderly carrier parent could not undergo clinical examination, so keratoconus or forme fruste could not be excluded.
  • Stated: miRNA target mRNAs can be predicted in silico but require functional characterisation; the study did not functionally characterise downstream targets.
  • Evident: only two mutation carriers were identified, limiting genotype–phenotype inference; myopia screening was limited to 96 Han Chinese subjects, so the absence of MIR184 mutations in myopia is not definitive.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1167/iovs.13-12035

Cites

43 of 43 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 8 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Sequencing of the MIR184 gene in the parents revealed that the father carries the same mutation (+3A>G).

    “Sequencing of the MIR184 gene in the parents revealed that the father carries the same mutation (+3A>G).”
    Quote in the paperNot a substantial claim
  • Findingown data

    Two novel heterozygous substitution mutations in MIR184, miR-184(+8C>A) and miR-184(+3A>G), were identified in two patients with isolated keratoconus.

    “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).”
    • Also stated in: § MiR-184 sequencing · plain
      “Two novel heterozygous substitution mutations in MIR184 were identified in two patients from the keratoconus cohort: miR-184(+3A>G) and miR-184(+8C>A) (Fig. 1).”
    Quote in the paperSubstantial claim
  • Findingown data

    miR-184(+8C>A) almost completely repressed the expression of miR-184 (P = 0.022).

    “Ex vivo miR-184 expression analysis demonstrated that miR-184(+8C>A) almost completely repressed the expression of miR-184 (P = 0.022)”
    • Also stated in: § Ex vivo miR-184 expression analysis · plain
      “While miR-184(+8C>A) almost completely repressed the expression of miR-184, miR-184(+3A>G) reduced the expression of miR-184 by approximately 40%.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The HEK293T cell model is a valid system for assessing the effect of MIR184 mutations on miR-184 processing in human corneal tissue.
  • Findingnone given

    The miR-184(+3A>G) mutation was predicted to reduce the base pair probability at position 3 of the miRNA stem loop structure in the junction region between single-stranded RNA and double-stranded RNA.

    “The miR-184(+3A>G) mutation was predicted to reduce the base pair probability at position 3 of the miRNA stem loop structure in the junction region between single-stranded RNA and double-stranded RNA.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The computational predictions of RNA secondary structure (mFold/RNAFold) reflect the actual secondary structure and processing of miR-184 in vivo.
  • Findingown data

    Computational modelling predicted that the identified MIR184 mutations alter the miR-184 stem loop stability and secondary structure.

    “Computational modelling predicted that these mutations alter the miR-184 stem loop stability and secondary structure.”
    • Also stated in: § MiR-184 modelling · plain
      “Both mutations, miR-184(+3A>G) and miR-184(+8C>A) alter the stem loop secondary structure of miR-184.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [30]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • Rests on an assumption: The computational predictions of RNA secondary structure (mFold/RNAFold) reflect the actual secondary structure and processing of miR-184 in vivo.
  • May combine two claims. 8
  • Classified as probably not a substantial claim. 2

  • May combine two claims.

    Findingown data

    The UK patient carrying miR-184(+8C>A) had definite keratoconus on slit-lamp biomicroscopy and Orbscan II topography, with no evidence of anterior segment dysgenesis, no iris or lenticular abnormalities, and normal fundi.

    “On slit-lamp biomicroscopy there was definite keratoconus and Orbscan II (Bausch & Lomb, Salt Lake City, UT, USA) topography confirmed keratoconus (Fig. 1).”
    • Also stated in: § Clinical details · plain
      “There was no evidence of anterior segment dysgenesis, no iris or lenticular abnormalities, and the fundi were normal.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both variants were absent from ethnically matched controls without ocular disease (96 UK and 96 Australian controls) and the 1000 Genomes Project data, release 12 -May 2012.

    “Both variants were absent from ethnically matched controls without ocular disease (96 UK and 96 Australian controls) and the 1000 Genomes Project data, release 12 -May 2012 (http://browser.1000genomes.org/index.html;accessed July 2012).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The Australian patient carrying miR-184(+3A>G) had bilateral keratoconus with anterior stromal scarring on the apex of the cone, prominent corneal nerves, and unique cortical granular cataract, with no iris abnormalities or anterior segment dysgenesis, and normal fundi.

    “On slit-lamp biomicroscopy there was definite keratoconus with anterior stromal scarring on the apex of the cone, prominent corneal nerves and unique cortical granular cataract.”
    • Also stated in: § Clinical details · plain
      “There were no iris abnormalities or anterior segment dysgenesis, and the fundi were normal.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Findingown data

    The cytosine at position 8 of pre-miR-184 is highly conserved across all species and the adenine at position 3 is well conserved although a guanine is present in some rodents.

    “The cytosine at position 8 of pre-miR-184 is highly conserved across all species and the adenine at position 3 is well conserved although a guanine is present in some rodents (Figure 2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The minimum free energy prediction (ΔG) for WT miR-184 was ΔG = -35.50kcal/mol compared to miR-184(+3A>G) with ΔG = -34.70kcal/mol and miR-184(+8C>A) with ΔG = -33.40kcal/mol.

    “The minimum free energy prediction (ΔG) for WT miR-184 was ΔG = -35.50kcal/mol compared to miR-184(+3A>G) with ΔG = -34.70kcal/mol and miR-184(+8C>A) with ΔG = -33.40kcal/mol (Figure 2).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The computational predictions of RNA secondary structure (mFold/RNAFold) reflect the actual secondary structure and processing of miR-184 in vivo.

  • May combine two claims.

    Findingown data

    The mutation miR-184(+8C>A) was identified in the NHLBI GO Exome Sequencing Project dataset with a minor allele frequency of 0.01% while miR-184(+3A>G) was not present in the ESP data.

    “The mutation miR-184(+8C>A) was identified in the NHLBI GO Exome Sequencing Project dataset (Exome Variant Server, NHLBI Exome Sequencing Project (ESP), Seattle, WA (http://evs.gs.washington.edu/EVS/;accessed July 2012) with a minor allele frequency of 0.01% while miR-184(+3A>G) was not present in the ESP data.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Cloning and transfection of mutant MIR184 transcripts into HEK293T cells resulted in a decrease in mature miR-184 expression levels compared to transfected miR-184 WT transcripts, indicating altered miRNA processing.

    “Cloning and transfection of mutant MIR184 transcripts into HEK293T cells resulted in a decrease in mature miR-184 expression levels compared to transfected miR-184 WT transcripts indicating altered miRNA processing.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The HEK293T cell model is a valid system for assessing the effect of MIR184 mutations on miR-184 processing in human corneal tissue.

  • May combine two claims.

    Interpretationown data

    The impact of the miR-184(+3A>G) mutation on the level of miR-184 expression was less than that observed for the miR-184(+8C>A), consistent with the incomplete penetrance of this mutation in the Australian family.

    “The impact of the miR-184(+3A>G) mutation on the level of miR-184 expression was less than that observed for the miR-184(+8C>A) (Figure 2), consistent with the incomplete penetrance of this mutation in the Australian family.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The HEK293T cell model is a valid system for assessing the effect of MIR184 mutations on miR-184 processing in human corneal tissue.

  • Classified as probably not a substantial claim.

    Premisecitation

    The structures of miRNA precursors are crucial for recognition and cleavage by Drosha and Dicer proteins during miRNA processing.

    “The structures of miRNA precursors are crucial for recognition and cleavage by Drosha and Dicer proteins during miRNA processing 37 .”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [37]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

9 of 9

  • The paper relies on this without support.

    The HEK293T cell model is a valid system for assessing the effect of MIR184 mutations on miR-184 processing in human corneal tissue.

    “The resulting recombinant plasmids containing inserts with mutations in MIR184 and WT controls were transfected into Human Embryonic Kidney 293T (HEK293T) cells from the American Type Culture Collection (ATCC).”
    “Cloning and transfection of mutant MIR184 transcripts into HEK293T cells resulted in a decrease in mature miR-184 expression levels compared to transfected miR-184 WT transcripts indicating altered miRNA processing.”

  • The paper relies on this without support.

    The computational predictions of RNA secondary structure (mFold/RNAFold) reflect the actual secondary structure and processing of miR-184 in vivo.

    “Secondary structural effects of identified mutations were assessed computationally using mFold (http://mfold.rna.albany.edu/?q=mfold) 30 and RNAFold algorithms (http://www.tbi.univie.ac.at/RNA/index.html).”
    “Computational modelling predicted that these mutations alter the miR-184 stem loop stability and secondary structure.”

  • The paper relies on this without support.

    The absence of MIR184 mutations in 96 Han Chinese axial myopia patients is sufficient to conclude that MIR184 does not play a major role in myopia pathogenesis in this population.

    “Screening of MIR184 in a cohort of 96 myopia patients from Hong Kong did not reveal any mutations, indicating that in this patient population MIR184 does not play a major role in myopia pathogenesis.”

  • The paper relies on this without support.

    The keratoconus cohort of 780 unrelated patients is representative of sporadic keratoconus patients of mixed ethnicity, such that the observed mutation frequency (2/780) estimates the population frequency.

    “A total of 780 unrelated keratoconus patients were recruited; 667 of European Caucasian and 123 of South Indian origin.”
    “Mutations in MIR184 are a rare cause of keratoconus and were found in 2/780 (0.25%) cases.”

  • The paper relies on this without support.

    The controls used for the rs41280052 association analysis are appropriate and comparable to the keratoconus cases.

    “Unaffected individuals (n=1865) were obtained from the Blue Mountains Eye Study.All were Caucasian aged over 49 years of age and had an ophthalmological examination; the recruitment of this cohort has been described previously 32 .”

  • The paper relies on this without support.

    Structural changes induced by mutations interfere with miRNA processing, altering miRNA expression or downstream processes.

    “Structural changes induced by mutations are likely to interfere with the processing of miRNAs, altering miRNA expression or modifying downstream processes and pathways.”
6 of 6

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Heterozygous loss-of-function point mutations of miRNA genes are associated with several human congenital disorders.

    “Heterozygous loss-of-function point mutations of miRNA genes are associated with several human congenital disorders1–5”

    From Gain-of-function mutation of microRNA-140 in human skeletal dysplasia (2019) · cites this paper as [5]

    The Abstract (page unavailable) reports two heterozygous MIR184 substitutions in patients with keratoconus and shows that both reduced mature miR-184 expression; it also links a MIR184 mutation to EDICT syndrome, which includes congenital cataract. The Discussion (page unavailable) mentions MIR96 mutations associated with progressive hearing loss, but does not establish that heterozygous loss-of-function mutations across miRNA genes are associated with several congenital disorders. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

  • Besides gene dosage abnormalities, single nucleotide substitutions in two miRNA genes have been identified in human diseases: MIR96 mutations in autosomal dominant deafness 50 and MIR184 mutations in EDICT syndrome and familial and sporadic cases of keratoconus.

    “Besides gene dosage abnormalities37,38, single nucleotide substitutions in two miRNA genes have been identified in human diseases: MIR96 mutations in autosomal dominant deafness 50 (#613704) and MIR184 mutations in EDICT syndrome (#614303) and familial and sporadic cases of keratoconus1–5,39.”

    From Gain-of-function mutation of microRNA-140 in human skeletal dysplasia (2019) · cites this paper as [5]

    Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Transfected mutation is named in opposite directions

    Minor

    The methods list the transfected variant as +3G>A, whereas the figure caption identifies the corresponding six-replicate variant as +3A>G.

1 of 1

Cites

Flagged first, then by citations

1 of the 44 references couldn’t be matched to a known paper.

Research works printed but not linked: 1

  1. Rabinowitz YS. Keratoconus. Surv Ophthalmol 1998;42:297-319.

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Related

Authors
Lechner, J., Bae, H. A., Guduric-Fuchs, J., Rice, A., Govindarajan, G., Siddiqui, S., Abi Farraj, L., Yip, S. P., Yap, M., Das, M., Souzeau, E., Coster, D., Mills, R. A., Lindsay, R., Phillips, T., Mitchell, P., Ali, M., Inglehearn, C. F., Sundaresan, P., Craig, J. E., Simpson, D. A., Burdon, K. P., & Willoughby, C. E.
Affiliations
  • Queen's University Belfast (GB)
  • Flinders University (AU)
  • Centre for Vision and Vascular Science, Queen's University Belfast, Belfast, Northern Ireland, United Kingdom
  • University of Leeds (GB)
  • Aravind Eye Hospital (IN)
  • Leeds Institute of Molecular Medicine, University of Leeds, Leeds, United Kingdom
  • Hong Kong Polytechnic University (HK)
  • Richard Lindsay and Associates, East Melbourne, Victoria, Australia
  • The University of Sydney (AU)
Corresponding author
Willoughby, Colin E.
ORCID
Venue
Investigative Opthalmology & Visual Science
Publisher
Association for Research in Vision and Ophthalmology (ARVO)
Published
2013-08-05
Volume
54
Issue
8
Pages
5266
ISSN
  • 0146-0404 (ISSN-L)
  • 1552-5783
DOI
10.1167/iovs.13-12035
OpenAlex
W1996952783
Semantic Scholar
08b6f0e441dd1e07b0ac1dd06e0c9561b8dd444d
Cited by
78
References
44
Citations by year
2026: 3 · 2025: 4 · 2024: 4 · 2023: 4 · 2022: 8 · 2021: 5 · 2020: 7 · 2019: 10 · 2018: 4 · 2017: 9 · 2016: 8 · 2015: 9 · 2014: 1 · 2013: 2
FWCI
4.75
Type
journal article
Language
en
Open access
Licence
other-oa
Locations
  • Investigative Ophthalmology & Visual Science (publishedVersion, public-domain)
  • PubMed (publishedVersion)
  • Research Portal (Queen's University Belfast) (submittedVersion, other-oa)
  • eCite Digital Repository (University of Tasmania) (submittedVersion)
  • Flinders Academic Commons (Flinders University) (submittedVersion)
  • Figshare (submittedVersion)
  • PolyU Institutional Research Archive (Hong Kong Polytechnic University) (submittedVersion)
Funders
  • Queen's University Belfast
  • Public Health Agency (RRG/3240/05)
  • Queen's University
  • Directorate for Biological Sciences
  • Medical Research Council
  • Biotechnology and Biological Sciences Research Council (BB/H005498/1)
  • National Health and Medical Research Council
Topics
  • Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Corneal surgery and disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Retinal Development and Disorders
Keywords
miR-184, keratoconus, axial myopia, novel mutations, miRNA seed region, miRNA secondary structure, Chinese patients, genotype-phenotype correlation, iris hypoplasia
MeSH terms
DNA, DNA Mutational Analysis, Female, Genotype, Humans, Keratoconus, Male, Middle Aged, Mutation, Myopia, Pedigree, MicroRNAs, Real-Time Polymerase Chain Reaction

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