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Cited by 103MicroRNA in disease regulationConnective tissue disorders researchGenomics and Rare Diseases

Gain-of-function mutation of microRNA-140 in human skeletal dysplasia

Open access
Grigelioniene, Giedre, Suzuki, Hiroshi I., Taylan, Fulya, Mirzamohammadi, Fatemeh, Borochowitz, Zvi U., Ayturk, Ugur M., Tzur, Shay, Horemuzova, Eva +12 more·2019·Nature Medicine
DOI 10.1038/s41591-019-0353-2PMC PMC6622181
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Abstract

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression. Heterozygous loss-of-function point mutations of miRNA genes are associated with several human congenital disorders1–5, but neomorphic (gain-of-new-function) mutations in miRNAs due to nucleotide substitutions have not been reported. Here we describe a neomorphic seed region mutation in the chondrocyte-specific, super-enhancer-associated MIR140 gene encoding microRNA-140 (miR-140) in a novel autosomal dominant human skeletal dysplasia. Mice with the corresponding single nucleotide substitution show skeletal abnormalities similar to those of the patients but distinct from those of miR-140-null mice6. This mutant miRNA gene yields abundant mutant miR-140-5p expression without miRNA-processing defects. In chondrocytes, the mutation causes widespread derepression of wild-type miR-140-5p targets and repression of mutant miR-140-5p targets, indicating that the mutation produces both loss-of-function and gain-of-function effects. Furthermore, the mutant miR-140-5p seed competes with the conserved RNA-binding protein Ybx1 for overlapping binding sites. This finding may explain the potent target repression and robust in vivo effect by this mutant miRNA even in the absence of evolutionary selection of miRNA–target RNA interactions, which contributes to the strong regulatory effects of conserved miRNAs7,8. Our study presents the first case of a pathogenic gain-of-function miRNA mutation and provides molecular insight into neomorphic actions of emerging and/or mutant miRNAs. Clinical insights from patients with a rare genetic skeletal disorder led to the discovery of the first case of a pathogenic gain-of-function miRNA mutation.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper reports a heterozygous neomorphic seed-region mutation in MIR140 (miR-140-5p) causing a novel autosomal dominant skeletal dysplasia, with mutant miRNA producing both loss- and gain-of-function effects. Mouse knock-in models recapitulate key skeletal abnormalities distinct from miR-140-null mice, and mutant miR-140-5p competes with Ybx1 at overlapping RNA binding sites.

Key findings

  • The same heterozygous substitution, chr16:g.69967007A>G (MIR140:NR_029681.1:n.24A>G), was found in three affected individuals from two unrelated families; it occurred de novo in P1 and P3, co-segregated in Family 1, and was absent from gnomAD. Clinical features included disproportionate short stature, short limbs, small hands and feet, midface hypoplasia, mild spondylar dysplasia, delayed epiphyseal ossification, and severe brachydactyly with cone-shaped phalangeal epiphyses.
  • CRISPR-Cas9 knock-in mice with the corresponding Mir140 A>G substitution showed delayed secondary ossification, decreased Col10a1 expression, delayed cartilage maturation of larynx/trachea/anterior ribs, severely decreased epiphyseal mineralization, mildly flat vertebral bodies, and a wider basal skull growth plate; these features were distinct from miR-140−/− mice.
  • Small RNA sequencing showed the mutation yielded slightly greater mutant miR-140-5p than wild-type miR-140-5p and decreased two miR-140-3p species, without miRNA-processing defects. RNA-seq showed widespread derepression of wild-type miR-140-5p targets and repression of mutant miR-140-5p targets, including Loxl3, Btg1, and Trps1; the hypoxia pathway was impaired in miR-140G/G but not miR-140−/− chondrocytes, with reduced Hif1a and collagen crosslinking.
  • Ago2 and Ybx1 seCLIP analyses indicated that the mutant miR-140-5p seed competes with Ybx1 for overlapping binding sites, especially in 3′ UTRs; Ybx1 inhibition dampened mutant miRNA-mediated repression. The authors describe this as the first case of a pathogenic gain-of-function miRNA mutation.

Methods

  • Clinical and genetic study of two unrelated families: exome sequencing, whole genome sequencing, Sanger sequencing, and comparative genomic hybridization; the MIR140 variant was identified and confirmed in affected individuals.
  • Mouse model: CRISPR-Cas9 knock-in of the Mir140 A>G substitution; heterozygous (miR-140G/+), homozygous (miR-140G/G), wild-type (miR-140+/+), and miR-140−/− mice were compared using skeletal preparations, histology, micro-CT, BrdU labeling, TUNEL staining, and collagen crosslink analysis.
  • Molecular analyses in primary rib chondrocytes and cell lines: small RNA sequencing, RNA sequencing, qRT-PCR, Western blot, luciferase reporter assays, single-cell dual fluorescence reporter assays, Ago2 and Ybx1 seCLIP, TargetScan prediction, GSEA, and RBP motif analysis.

Limitations

  • Stated: seCLIP had low sensitivity for small starting materials such as primary chondrocytes, and intronic seCLIP clusters were considered non-specific; sample sizes were estimated based on prior experience, with examined mouse numbers in Supplementary Table 2; human genome data are protected by Swedish law, with raw Sanger sequencing data available upon request.
  • Evident: the human genetic evidence comes from only three affected individuals in two families, limiting broader genotype–phenotype generalization.
  • Evident: much of the mechanistic evidence relies on mouse chondrocytes and heterologous cell assays, and the paper does not report an independent human cohort validating the proposed Ybx1 competition mechanism.

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

Integrity signals

Registry

Crossref · 10.1038/s41591-019-0353-2

Cites

61 of 61 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

38 of 54 cited sources checked · 15 supported · 0 flagged · accuracy not yet measured

  • Findingcitation

    The substitution is not reported in gnomAD.

    “and is not reported in the gnomAD, a large scale human genome sequence database10”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [10] Lek, M. et al. Analysis of protein-coding genetic variation in 60,706 humans. Nature 536, 285–291 (2016). source

      The Article reSeArcH and ExAC data release sections describe a large human exome-variant database, but identify it as ExAC, not gnomAD. The substitution is not specified in the claim, and no passage in our copy establishes whether it is reported in gnomAD (searched: gnomAD, substitution, and database descriptions). (LLM, conf=0.74)

      Experimental — accuracy not yet measured
  • Premisecitation

    The miR-140-null phenotype included mildly advanced terminal differentiation of growth plate chondrocytes of developing bones.

    “This miR-140-null phenotype included mildly advanced terminal differentiation of growth plate chondrocytes of developing bones6,17.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [6] Nakamura, Y., Inloes, J. B., Katagiri, T. & Kobayashi, T. Chondrocyte-specific microRNA-140 regulates endochondral bone development and targets Dnpep to modulate bone morphogenetic protein signaling. Mol. Cell. Biol. 31, 3019–3028 (2011). source
    • cites [17] Papaioannou, G. et al. MicroRNA-140 provides robustness to the regulation of hypertrophic chondrocyte differentiation by the PTHrP-HDAC4 pathway. J. Bone Miner. Res. 30, 1044–1052 (2015). source
  • Premisecitation

    Loss of miR-140, encoded by Mir140, in mice leads to short stature and craniofacial abnormalities.

    “loss of miR-140, encoded by Mir140, in mice leads to short stature and craniofacial abnormalities6,16”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [6] Nakamura, Y., Inloes, J. B., Katagiri, T. & Kobayashi, T. Chondrocyte-specific microRNA-140 regulates endochondral bone development and targets Dnpep to modulate bone morphogenetic protein signaling. Mol. Cell. Biol. 31, 3019–3028 (2011). source
    • cites [16] Miyaki, S. et al. MicroRNA-140 plays dual roles in both cartilage development and homeostasis. Genes Dev. 24, 1173–1185 (2010). source
  • Findingcitation

    Previously reported YBX1-stabilized transcripts were preferentially repressed in miR-140G/G and miR-140G/+ chondrocytes as compared with miR-140−/− chondrocytes.

    “In addition, previously reported YBX1-stabilized transcripts were preferentially repressed in miR-140G/G and miR-140G/+ chondrocytes as compared with miR-140−/− chondrocytes”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [34] Goodarzi, H. et al. Endogenous tRNA-derived fragments suppress breast cancer progression via ybx1 displacement. Cell 161, 790–802 (2015). source
  • Findingcitation

    An extended search in CNV databases revealed a single individual with heterozygous 1.1Mb deletion with isolated Arnold-Chiari malformation.

    “An extended search in copy number variation (CNV) databases regarding human phenotypes associated with CNVs involving the MIR140 gene revealed a single individual with heterozygous 1.1Mb deletion with isolated Arnold-Chiari malformation41.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [41] Kaminsky, E. B. et al. An evidence-based approach to establish the functional and clinical significance of copy number variants in intellectual and developmental disabilities. Genet. Med. 13, 777–784 (2011). source

      The Results section (CNV Characterization; page unavailable) describes aggregate CNV findings, not an individual with the claimed phenotype. Searches of our copy for MIR140, a 1.1 Mb deletion, and Arnold-Chiari malformation found no passage addressing the claimed case. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
  • Classified as probably not a substantial claim. 36
  • May combine two claims. 32

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    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”
    Quote in the paperNot a substantial claim4 of 5 sources checked
    • cites [1] Mencia, A. et al. Mutations in the seed region of human miR-96 are responsible for nonsyndromic progressive hearing loss. Nat. Genet. 41, 609–613 (2009). source
    • cites [2] Solda, G. et al. A novel mutation within the MIR96 gene causes non-syndromic inherited hearing loss in an Italian family by altering pre-miRNA processing. Hum. Mol. Genet. 21, 577–585 (2012). source

      The Discussion reports a MIR96 point mutation that reduces mature miRNA levels and causes inherited hearing loss. It does not establish an association with several human congenital disorders: the affected family’s hearing loss began between ages 25 and 40, rather than being present at birth. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
    • cites [3] Hughes, A. E. et al. Mutation altering the miR-184 seed region causes familial keratoconus with cataract. Am. J. Hum. Genet. 89, 628–633 (2011). source

      The main text (page unavailable) identifies a heterozygous point mutation in MIR184 associated with familial keratoconus and early-onset cataract, and reports that the mutant loses a tested miR-184 function. This supports an example within the multiply cited statement, but the paper does not establish that the affected individuals’ condition was congenital: it describes the cataract as early-onset. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [4] Iliff, B. W., Riazuddin, S. A. & Gottsch, J. D. A single-base substitution in the seed region of miR-184 causes EDICT syndrome. Invest. Ophthalmol. Vis. Sci. 53, 348–353 (2012). source

      The Results (page unavailable) identify a heterozygous single-base substitution in miR-184 that segregates with EDICT syndrome, which includes congenital cataract. The Discussion (page unavailable) proposes reduced miR-184 expression or activity as possible mechanisms but does not establish loss of function. This paper provides one example, not evidence for several congenital disorders. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [5] Lechner, J. et al. Mutational analysis of MIR184 in sporadic keratoconus and myopia. Invest. Ophthalmol. Vis. Sci. 54, 5266–5272 (2013). source

      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

  • May combine two claims.

    Findingcitation

    Mice with the corresponding single nucleotide substitution show skeletal abnormalities similar to those of the patients but distinct from those of miR-140-null mice.

    “Mice with the corresponding single nucleotide substitution show skeletal abnormalities similar to those of the patients but distinct from those of miR-140-null mice6.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [6] Nakamura, Y., Inloes, J. B., Katagiri, T. & Kobayashi, T. Chondrocyte-specific microRNA-140 regulates endochondral bone development and targets Dnpep to modulate bone morphogenetic protein signaling. Mol. Cell. Biol. 31, 3019–3028 (2011). source

      The section ‘Mir140-null mice show defects in endochondral bone growth’ describes skeletal abnormalities in Mir140-null mice. But the paper describes a Mir140 deletion, not mice with the claimed single-nucleotide substitution; our copy does not report patient abnormalities or compare substitution-bearing mice with null mice. (LLM, conf=0.78)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Super-enhancers commonly drive high-level expression of tissue-specific genes, including miRNA genes, with evolutionarily conserved cell type-specific function.

    “Super-enhancers commonly drive high-level expression of tissue-specific genes, including miRNA genes, with evolutionarily conserved cell type-specific function11–13.”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [11] Suzuki, H. I., Young, R. A. & Sharp, P. A. Super-enhancer-mediated RNA processing revealed by integrative microrna network analysis. Cell 168, 1000–1014 e1015 (2017). source
    • cites [12] Whyte, W. A. et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153, 307–319 (2013). source

      The Results sections on functional attributes and cell identity report higher expression of super-enhancer-associated genes, cell type-specific associations, and an ESC miRNA locus among those genes. The Discussion says these regulatory clusters evolved, but our copy does not establish that their cell type-specific functions are evolutionarily conserved. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [13] Hnisz, D. et al. Super-enhancers in the control of cell identity and disease. Cell 155, 934–947 (2013). source

      The Results section reports that super-enhancers drive high-level expression of associated genes, and the Discussion says those genes tend to have cell type-specific expression and include miRNAs. Our copy does not establish that their cell type-specific function is evolutionarily conserved (pages not provided). (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingcitation

    ceRBP effects are widespread in RNAi and contribute to off-target effects and phenotype modulation.

    “We observed that ceRBP effects are widespread in RNAi and contributes to off-target effects and phenotype modulation40.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [40] Suzuki, H. I., Spengler, R. M., Grigelioniene, G., Kobayashi, T. & Sharp, P. A. Deconvolution of seed and RNA-binding protein crosstalk in RNAi-based functional genomics. Nat. Genet. 50, 657–661 (2018). source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    YBX1 is a multifunctional RBP that stabilizes mRNAs and regulates RNA splicing.

    “YBX1 is a multifunctional RBP that stabilizes mRNAs and regulates RNA splicing33,34.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [33] Lyabin, D. N., Eliseeva, I. A. & Ovchinnikov, L. P. YB-1 protein: functions and regulation. Wiley Interdiscip. Rev. RNA 5, 95–110 (2014). source
    • cites [34] Goodarzi, H. et al. Endogenous tRNA-derived fragments suppress breast cancer progression via ybx1 displacement. Cell 161, 790–802 (2015). source

      The Results section describes YBX1 as a multifunctional RNA-binding protein, and the Introduction and Discussion say it stabilizes target transcripts. Our copy does not say that YBX1 regulates RNA splicing; a search for splicing found no supporting passage. Page numbers were not available in the indexed copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Ybx1 seCLIP analysis identified a binding motif similar to the Ybx1 binding motif reported previously.

    “Next, Ybx1 seCLIP analysis identified a binding motif similar to the Ybx1 binding motif reported previously30–32”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [30] Ray, D. et al. A compendium of RNA-binding motifs for decoding gene regulation. Nature 499, 172–177 (2013). source

      The Large-scale analysis of RBPs section describes how the paper derived RNA-binding motifs, but the passages found do not identify a YBX1 motif. Searches for “YBX1,” “YB-1,” and “Y-box binding protein 1” found no passage establishing the specific motif comparison attributed to this source. (LLM, conf=0.72)

      Experimental — accuracy not yet measured
    • cites [31] Wei, W. J. et al. YB-1 binds to CAUC motifs and stimulates exon inclusion by enhancing the recruitment of U2AF to weak polypyrimidine tracts. Nucleic Acids Res. 40, 8622–8636 (2012). source
    • cites [32] Wu, S. L. et al. Genome-wide analysis of YB-1-RNA interactions reveals a novel role of YB-1 in miRNA processing in glioblastoma multiforme. Nucleic Acids Res. 43, 8516–8528 (2015). source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    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.”
    Quote in the paperNot a substantial claim5 of 6 sources checked
    • cites [1] Mencia, A. et al. Mutations in the seed region of human miR-96 are responsible for nonsyndromic progressive hearing loss. Nat. Genet. 41, 609–613 (2009). source
    • cites [2] Solda, G. et al. A novel mutation within the MIR96 gene causes non-syndromic inherited hearing loss in an Italian family by altering pre-miRNA processing. Hum. Mol. Genet. 21, 577–585 (2012). source

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

      Experimental — accuracy not yet measured
    • cites [3] Hughes, A. E. et al. Mutation altering the miR-184 seed region causes familial keratoconus with cataract. Am. J. Hum. Genet. 89, 628–633 (2011). source
    • cites [4] Iliff, B. W., Riazuddin, S. A. & Gottsch, J. D. A single-base substitution in the seed region of miR-184 causes EDICT syndrome. Invest. Ophthalmol. Vis. Sci. 53, 348–353 (2012). source

      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
    • cites [5] Lechner, J. et al. Mutational analysis of MIR184 in sporadic keratoconus and myopia. Invest. Ophthalmol. Vis. Sci. 54, 5266–5272 (2013). source

      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
    • cites [39] Lewis, M. A. et al. An ENU-induced mutation of miR-96 associated with progressive hearing loss in mice. Nat. Genet. 41, 614–618 (2009). source

      The paper’s discussion reports two single-base changes in the miR-96 seed region in humans with progressive hearing loss, supporting the MIR96 portion of the claim. It does not identify autosomal dominant deafness 50 or address MIR184, EDICT syndrome, or keratoconus. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Gene regulation by conserved miRNAs has a significant impact on 3′ UTR evolution, and evolutionarily maintained target sites mediate actions of miRNAs more effectively than non-conserved target sites.

    “Gene regulation by conserved miRNAs has a significant impact on 3′ UTR evolution, and evolutionarily maintained target sites mediate actions of miRNAs more effectively than non-conserved target sites28.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [28] Grimson, A. et al. MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol. Cell 27, 91–105 (2007). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    miR-140-null mice were previously described.

    “miR-140-null mice were previously described6.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [6] Nakamura, Y., Inloes, J. B., Katagiri, T. & Kobayashi, T. Chondrocyte-specific microRNA-140 regulates endochondral bone development and targets Dnpep to modulate bone morphogenetic protein signaling. Mol. Cell. Biol. 31, 3019–3028 (2011). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Mouse H3K27Ac and Sox9 ChIP-seq profiles were previously described.

    “Mouse H3K27Ac and Sox9 ChIP-seq profiles were previously described51.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [51] Ohba, S., He, X., Hojo, H. & McMahon, A. P. Distinct transcriptional programs underlie Sox9 regulation of the mammalian chondrocyte. Cell Rep. 12, 229–243 (2015). source

  • May combine two claims.

    Findingown data

    miR-140G/G and miR-140−/− mice were smaller and had shorter noses than wild-type littermates.

    “Although miR-140G/G and miR-140−/− mice were smaller and had shorter noses than wild-type littermates”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Clinical features of the skeletal dysplasia include disproportionate short stature with short limbs, small hands and feet, and midface hypoplasia with small nose.

    “Clinical features of the skeletal dysplasia included disproportionate short stature with short limbs, small hands and feet, and midface hypoplasia with small nose.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Radiological hallmarks were mild spondylar dysplasia, delayed epiphyseal ossification of the hip and knee, and severe brachydactyly with cone shaped phalangeal epiphyses.

    “The radiological hallmarks were mild spondylar dysplasia, delayed epiphyseal ossification of the hip and knee, and severe brachydactyly with cone shaped phalangeal epiphyses”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    The mutant miRNA gene yields abundant mutant miR-140-5p expression without miRNA-processing defects.

    “This mutant miRNA gene yields abundant mutant miR-140-5p expression without miRNA-processing defects.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The basal skull growth plate was wider due to increased length of all layers of chondrocytes in miR-140G/G and miR-140G/+ mice, whereas miR-140−/− mice showed no significant changes.

    “The basal skull growth plate was wider due to the increased length of all layers of chondrocytes in miR-140G/G and miR-140G/+ mice, whereas miR-140−/− mice showed no significant changes”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    P1 and P2 suffered from frequent respiratory infections with prolonged cough and inspiratory stridor.

    “P1 and P2 also suffered from frequent respiratory infections with prolonged cough and inspiratory stridor.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    All three affected individuals had normal intelligence, dentition, hearing, visual acuity, and basic blood tests.

    “All three affected individuals had normal intelligence, dentition, hearing, visual acuity, and basic blood tests.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    P1 had a narrow larynx likely due to floppy cartilage.

    “P1 had a narrow larynx likely due to floppy cartilage”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Target sites of miR-140-5p-G seldom overlapped with those of wild-type miR-140 species and were less conserved than those of wild-type miR-140 species.

    “Target sites of miR-140-5p-G seldom overlapped with those of wild-type miR-140 species (wild-type 5p, 3p.1, and 3p.2) and were less conserved than those of wild-type miR-140 species”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Findingown data

    Out of 61 mouse embryos injected with the guide RNA, repair template and Cas9, 11 mice were positive for the knock-in mutation.

    “Out of 61 mice embryos injected with the guide RNA, repair template and Cas9, 11 mice were positive for the knock-in mutation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Screening P1, P2, and P3 for possible disease causing variants in 378 known skeletal dysplasia genes using WGS data, and screening P2 for gene dose abnormalities, was all negative, excluding involvement of these genes in the skeletal dysplasia phenotype.

    “Screening P1, P2, and P3 for possible disease causing variants in 378 known skeletal dysplasia genes using WGS data, and screening P2 for gene dose abnormalities of the above mentioned skeletal dysplasia genes using custom-designed comparative genome hybridization analysis was all negative (data not shown), excluding involvement of these genes in the skeletal dysplasia phenotype of our patients.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    All selected knock-in mice were compound heterozygous with one allele mutated as desired and the other with various size insertion-deletion mutations.

    “All of these mice were compound heterozygous with one allele mutated as desired and the other with various size insertion-deletion mutations.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The miR-140 mutation found in this study affected the same nucleotide in both families and resulted in gain of a new function as well as loss of the original function, as shown by the transcriptome analysis revealing repression of new target genes and derepression of original targets.

    “In contrast, the miR-140 mutation found in this study affected the same nucleotide in both families and resulted in gain of a new function as well as loss of the original function, as shown by the transcriptome analysis revealing repression of new target genes and derepression of original targets.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The suppressive effect of miR-140-5p-G is particularly strong for 8mer target sites and correlates with the predicted miRNA target efficiency.

    “This suppressive effect of miR-140-5p-G is particularly strong for 8mer target sites (Extended Data Fig. 6b) and correlates with the predicted miRNA target efficiency”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A 3′ UTR reporter assay confirmed that miR-140-5p-G and wild-type miR-140-5p target their respective predicted 8mer target sites with similar strengths, and validated the efficiency of target sites in several genes.

    “A 3′ UTR reporter assay, using minimal target sequences and endogenous 3′ UTR sequences, confirmed that miR-140-5p-G and wild-type miR-140-5p target their respective predicted 8mer target sites with similar strengths, and validated the efficiency of target sites in several genes, respectively”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Genes with the identified 3′ UTR target sites of miR-140-5p-G were downregulated only in miR-140G/G and miR-140G/+ chondrocytes, while genes with wild-type miR-140-5p target sites were upregulated in both miR-140-null and miR-140 mutant chondrocytes.

    “Accordingly, genes with the identified 3′ UTR target sites of miR-140-5p-G were downregulated only in miR-140G/G and miR-140G/+ chondrocytes, while genes with wild-type miR-140-5p target sites were upregulated in both miR-140-null and miR-140 mutant chondrocytes”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Apoptotic cells associated with blood vessels invaded into the epiphysis of wild type mice.

    “White arrow indicates apoptotic cells associated with blood vessels invaded into the epiphysis of wild type mice.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In P1, the right arytenoid cartilage is enlarged with redundant soft tissues and prolapses antero-medially over the larynx during inspiration.

    “The right arytenoid cartilage is enlarged with redundant soft tissues and it prolapses antero-medially over the larynx during inspiration (a).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    miR-140-5p-WT and miR-140-5p-G show similar and higher predicted target site efficiency than miR-140-3p.1 and miR-140-3p.2.

    “miR-140-5p-WT and miR-140-5p-G show similar and higher predicted target site efficiency than miR-140-3p.1 and miR-140-3p.2.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Fiberscopy findings in P1, 44 years of age, are consistent with laryngomalacia.

    “Fiberscopy findings in P1, 44 years of age, consistent with laryngomalacia.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The second nucleotide in miR-140-5p is conserved from human to Australian ghostshark.

    “The second nucleotide in miR-140-5p is conserved from human to Australian ghostshark (indicated by a black arrow).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The pathogenic variant in MIR140 (chr16:g.69967007A>G) was found in whole genome sequencing and exome sequencing data and confirmed by Sanger sequencing in individuals P1, 2, and 3.

    “The pathogenic variant in MIR140 (chr16:g.69967007A>G) (indicated by black arrows) in whole genome sequencing and exome sequencing data (left and right panels), and its Sanger sequencing confirmation (middle panel) in individuals P1, 2, and 3 as in Fig. 1.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingreasoning

    miR-140-5p and miR-140-3p are highly abundant miRNAs in chondrocytes.

    “Note that miR-140-5p and miR-140-3p are highly abundant miRNAs in chondrocytes, reflecting super-enhancer-mediated high transcriptional activity.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    No phenotypic differences were found between males and females.

    “Since we found no phenotypic differences between males and females, males and females were not discriminated for the analysis.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingreasoning

    miR-140-5p is increased and miR-140-3p is decreased in chondrocytes from miR-140G/+ and miR-140G/G mice.

    “consistent with the increase in miR-140-5p and decrease in miR-140-3p in chondrocytes from miR-140G/+ and miR-140G/G mice.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Enrichment of miRNA motifs and Ybx1 motifs was not observed in intronic seCLIP clusters.

    “enrichment of miRNA motifs and Ybx1 motifs was not observed in intronic clusters (Extended Data Fig. 9b and 10b)”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Mutations in approximately 400 different genes are currently known to cause congenital skeletal diseases.

    “Mutations in approximately 400 different genes are currently known to cause congenital skeletal diseases9”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [9] Bonafe, L. et al. Nosology and classification of genetic skeletal disorders: 2015 revision. Am. J. Med. Genet. A 167A, 2869–2892 (2015). source

  • May combine two claims.

    Interpretationown data

    The A>G mutation did not compromise miRNA processing and yielded abundant expression of miR-140-5p-G from primary miR-140 transcripts of which transcription is strongly activated by a super-enhancer in chondrocytes.

    “These data indicate that the A>G mutation did not compromise miRNA processing and yielded abundant expression of miR-140-5p-G from primary miR-140 transcripts of which transcription is strongly activated by a super-enhancer in chondrocytes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    These bone abnormalities are highly consistent with the skeletal dysplasia features of the patients, presenting with delayed secondary ossification, mild platyspondyly, small epiphyses, and scaphocephaly.

    “and are highly consistent with the skeletal dysplasia features of the patients, presenting with delayed secondary ossification, mild platyspondyly, small epiphyses, and scaphocephaly.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The mutation produces both loss-of-function and gain-of-function effects.

    “indicating that the mutation produces both loss-of-function and gain-of-function effects.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The differences in phenotype between miR-140G/G and miR-140−/− mice, and delayed epiphyseal maturation in humans, indicate that this single-base substitution causes a neomorphic, and not just loss-of-function, effect.

    “Taken together, these differences in phenotype between miR-140G/G and miR-140−/− mice, and delayed epiphyseal maturation in humans, indicate that this single-base substitution causes a neomorphic, and not just loss-of-function, effect.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Premisenone given

    These mutations, occurring at multiple different nucleotides of single miRNAs, are thought to primarily cause loss of function.

    “However, these mutations, occurring at multiple different nucleotides of single miRNAs, are thought to primarily cause loss of function”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The disease phenotype may be at least partially explained by potential competition with the RBP Ybx1, and by the super-enhancer-driven high-level miRNA expression.

    “the disease phenotype may be at least partially explained by potential competition with the RBP, Ybx1, and by the super-enhancer-driven high-level miRNA expression”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    A neomorphic mutant miRNA would need to target a biologically important regulatory network in order to produce a disease phenotype.

    “a neomorphic mutant miRNA would need to target a biologically important regulatory network in order to produce a disease phenotype.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Multiple RNA binding proteins (RBPs) are reported to modulate miRNA function.

    “multiple RNA binding proteins (RBPs) are reported to modulate miRNA function29”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [29] van Kouwenhove, M., Kedde, M. & Agami, R. MicroRNA regulation by RNA-binding proteins and its implications for cancer. Nat. Rev. Cancer 11, 644–656 (2011). source

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationnone given

    The present study expands our knowledge on how mutations in miRNAs contribute to human disease development.

    “Overall, the present study expands our knowledge on how mutations in miRNAs contribute to human disease development”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    miR-140-5p exerts greater target repression than miR-140-3p even though the latter is more abundant.

    “suggesting that miR-140-5p exerts greater target repression than miR-140-3p even though the latter is more abundant.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Interpretationcitation

    Given the expansion of miRNA genes in vertebrates, the molecular mechanism revealed in this study has broader implications in understanding how function of new miRNA genes might evolve in humans.

    “In addition, given the expansion of miRNA genes in vertebrates7,8, the molecular mechanism revealed in this present study also has broader implications in understanding how function of new miRNA genes might evolve in humans.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [7] Chen, K. & Rajewsky, N. The evolution of gene regulation by transcription factors and microRNAs. Nat. Rev. Genet. 8, 93–103 (2007). source
    • cites [8] Berezikov, E. Evolution of microRNA diversity and regulation in animals. Nat. Rev. Genet. 12, 846–860 (2011). source

  • May combine two claims.

    Interpretationown data

    P1 had normal bone density for age at 43, which suggests that the heterozygous MIR140 mutation in humans does not lead to bone mass abnormalities.

    “She had normal bone density for the age, which suggests that the heterozygous MIR140 mutation in humans does not lead to bone mass abnormalities.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premiseown data

    The A>G substitution creates a novel Msp I restriction enzyme site (CCGG).

    “This substitution creates a novel restriction enzyme site (Msp I; CCGG).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The sample size for molecular biology experiments was estimated based on prior experience of similar sets of experiments.

    “The sample size was estimated based on our prior experience of similar sets of experiments.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    HEK293T cells are tested for mycoplasma contamination.

    “HEK293T cells are tested for mycoplasma contamination.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Micro-CT analysis was performed in a blinded manner with all mice assigned to coded sample numbers.

    “Micro-CT analysis was performed in a blinded manner with all mice assigned to coded sample numbers.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    miR-140-5p-G follows mechanisms of conventional miRNA targeting as well as other endogenous miRNAs.

    “suggesting that miR-140-5p-G follows mechanisms of conventional miRNA targeting as well as other endogenous miRNAs.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    The high abundance of miR-140-5p and miR-140-3p in chondrocytes reflects super-enhancer-mediated high transcriptional activity.

    “Note that miR-140-5p and miR-140-3p are highly abundant miRNAs in chondrocytes, reflecting super-enhancer-mediated high transcriptional activity.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The sample size was estimated based on prior experience of performing similar sets of experiments.

    “The sample size was estimated based on our prior experience of performing similar sets of experiments and the number of examined mice is shown in Supplemental Table 2.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    The absence of additional mutations in the 2 kb region surrounding the Mir140 gene was confirmed using Sanger sequencing.

    “After confirming the absence of additional mutations in the 2 kb region surrounding the Mir140 gene using Sanger sequencing”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Each single-cell dual fluorescence experiment included non-transfection controls.

    “Each experiment included non-transfection controls.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Selection of a single mature miRNA strand from miRNA duplex is determined by combination of 5′-end nucleotide identity and thermodynamic asymmetry of miRNA duplex termini.

    “Selection of a single mature miRNA strand from miRNA duplex is determined by combination of 5′-end nucleotide identity and thermodynamic asymmetry of miRNA duplex termini (Ref. 20).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Ref. 20

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

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisereasoning

    The PCR amplicon containing the A>G substitution is digested into 98 bp and 119 bp long fragments.

    “The PCR amplicon containing the A>G substitution is digested into 98 bp and 119 bp long fragments.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    The molecular biology experiments were independently repeated at least twice, and all attempts to reproduce the results were successful.

    “We independently repeated the molecular biology experiment at least twice, and all attempts to reproduce the results were successful.”
    • Also stated in: § Statistical analysis · plain
      “In molecular biology experiments, results are representative of more than two independent and reproducible experiments.”
    Quote in the paperNot a substantial claim
61 of 61

  • The paper relies on this without support.

    Neomorphic (gain-of-new-function) mutations in miRNAs due to nucleotide substitutions have not been reported.

    “but neomorphic (gain-of-new-function) mutations in miRNAs due to nucleotide substitutions have not been reported.”

  • The paper relies on this without support.

    Underlying molecular mechanisms are not elucidated for many congenital skeletal diseases.

    “but underlying molecular mechanisms are not elucidated for many of them.”

  • The paper relies on this without support.

    A mutation in the miRNA seed region creates a new repertoire of target genes and the magnitude of such target suppression is typically small.

    “Since a mutation in the miRNA seed region creates a new repertoire of target genes and the magnitude of such target suppression is typically small”
3 of 3

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Authors
Grigelioniene, G., Suzuki, H. I., Taylan, F., Mirzamohammadi, F., Borochowitz, Z. U., Ayturk, U. M., Tzur, S., Horemuzova, E., Lindstrand, A., Weis, M. A., Grigelionis, G., Hammarsjö, A., Marsk, E., Nordgren, A., Nordenskjöld, M., Eyre, D. R., Warman, M. L., Nishimura, G., Sharp, P. A., & Kobayashi, T.
Affiliations
  • Koch Institute for Integrative Cancer Research At MIT (US)
  • Karolinska Institutet (SE)
  • Harvard University (US)
  • Technion – Israel Institute of Technology (IL)
  • Boston Children's Hospital (US)
  • Rambam Health Care Campus (IL)
  • Karolinska University Hospital (SE)
  • University of Washington (US)
  • Saitama Medical University Hospital (JP)
Corresponding author
Kobayashi, Tatsuya
ORCID
Venue
Nature Medicine
Publisher
Springer Science and Business Media LLC
Published
2019-02-25
Volume
25
Issue
4
Pages
583-590
ISSN
  • 1078-8956 (ISSN-L)
  • 1546-170X
DOI
10.1038/s41591-019-0353-2
PMC
PMC6622181
OpenAlex
W2917129046
Semantic Scholar
dffcad30f105f92c7952744d74b10b64d6c8ec7f
Cited by
103
References
65
Citations by year
2026: 5 · 2025: 11 · 2024: 10 · 2023: 18 · 2022: 15 · 2021: 13 · 2020: 22 · 2019: 8 · 2018: 1
FWCI
4.01
Type
journal article
Language
en
Open access
Licence
www.springer.com/tdm (tdm)
Locations
  • Nature Medicine (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (submittedVersion)
  • PubMed Central (submittedVersion)
Funders
  • Eli Lilly and Company
  • Massachusetts Institute of Technology (P30-CA14051)
  • Dana-Farber Cancer Institute
  • Uehara Memorial Foundation (NIH P30)
  • European Society for Paediatric Endocrinology
  • Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology (P30-CA14051)
  • Karolinska Institutet
  • Stockholms Läns Landsting (20150143)
  • Vetenskapsrådet (2018-03046, 2018-)
  • Svenska Läkaresällskapet
  • Stiftelsen Samariten
  • Science for Life Laboratory
  • National Institutes of Health (P30-AR066261, GM034277, AR066261, R01-GM034277)
  • Uppsala Multidisciplinary Center for Advanced Computational Science
  • National Cancer Institute (P01 CA 042063, R01CA133404, CA14051, P30CA014051, P30-CA-14051)
  • National Institute of General Medical Sciences (R01 GM034277)
  • National Institute of Arthritis and Musculoskeletal and Skin Diseases (P30 AR066261, R01AR056645)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Connective tissue disorders research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomics and Rare Diseases
Keywords
microRNA-140, gain-of-function mutations, neomorphic mutations, skeletal dysplasia, chondrocytes, YB-1, post-transcriptional regulation, super-enhancers, mouse model
MeSH terms
Gain of Function Mutation, Animals, Base Sequence, Bone Diseases, Developmental, Female, Homozygote, Humans, Male, Mice, Inbred C57BL, Mice, Mutant Strains, Pedigree, Phenotype, Chondrocytes, MicroRNAs, Transcriptome

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-20.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-20.

Cited sources are checked by gpt-6-sol.

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