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Cited by 102Hearing, Cochlea, Tinnitus, GeneticsMicroRNA in disease regulationCongenital Ear and Nasal Anomalies

A novel mutation within the MIR96 gene causes non-syndromic inherited hearing loss in an Italian family by altering pre-miRNA processing

Open access
Soldà, Giulia, Robusto, Michela, Primignani, Paola, Castorina, Pierangela, Benzoni, Elena, Cesarani, Antonio, Ambrosetti, Umberto, Asselta, Rosanna +1 more·2012·Human Molecular Genetics
DOI 10.1093/hmg/ddr493PMC PMC3259013
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Abstract

The miR-96, miR-182 and miR-183 microRNA (miRNA) family is essential for differentiation and function of the vertebrate inner ear. Recently, point mutations within the seed region of miR-96 were reported in two Spanish families with autosomal dominant non-syndromic sensorineural hearing loss (NSHL) and in a mouse model of NSHL. We screened 882 NSHL patients and 836 normal-hearing Italian controls and identified one putative novel mutation within the miR-96 gene in a family with autosomal dominant NSHL. Although located outside the mature miR-96 sequence, the detected variant replaces a highly conserved nucleotide within the companion miR-96*, and is predicted to reduce the stability of the pre-miRNA hairpin. To evaluate the effect of the detected mutation on miR-96/mir-96* biogenesis, we investigated the maturation of miR-96 by transient expression in mammalian cells, followed by real-time reverse-transcription polymerase chain reaction (PCR). We found that both miR-96 and miR-96* levels were significantly reduced in the mutant, whereas the precursor levels were unaffected. Moreover, miR-96 and miR-96* expression levels could be restored by a compensatory mutation that reconstitutes the secondary structure of the pre-miR-96 hairpin, demonstrating that the mutation hinders precursor processing, probably interfering with Dicer cleavage. Finally, even though the mature miR-96 sequence is not altered, we demonstrated that the identified mutation significantly impacts on miR-96 regulation of selected targets. In conclusion, we provide further evidence of the involvement of miR-96 mutations in human deafness and demonstrate that a quantitative defect of this miRNA may contribute to NSHL.

AI snapshot

Written from the paper’s full text.

TL;DR

A novel heterozygous MIR96 +57T>C mutation, located outside mature miR-96 but within the miR-96* seed region, was identified in an Italian family with autosomal dominant progressive non-syndromic hearing loss. Functional assays show the mutation alters pre-miR-96 folding, reduces mature miR-96 and miR-96* without affecting precursor levels, and impairs target regulation, suggesting a quantitative miR-96 defect can contribute to deafness.

Key findings

  • Screening 882 NSHL patients and 836 normal-hearing Italian controls identified miR-96(+57T>C) in one family with autosomal dominant progressive NSHL; the variant was absent in controls and segregated with hearing loss in affected individuals, though it was also present in three normal-hearing children.
  • In HeLa cells, the +57T>C mutant significantly reduced mature miR-96 by 85% (P = 0.0006) and miR-96* by 77% (P = 0.019), while pre-miR-96 levels were unaffected; a compensatory +23A>G mutation restored both mature miRNA levels to values not significantly different from wild-type.
  • The known miR-96(+13G>A) mutation reduced mature miR-96 (P < 0.0001) but did not affect miR-96* levels, and its effect was not rescued by restoring precursor folding, indicating a different pathogenic mechanism from +57T>C.
  • Luciferase assays showed 3 of 8 candidate 3′UTRs (MYRIP, ACVR2B, CACNB4) were regulated by miR-96/miR-96*; the +57T>C mutation reduced silencing by approximately 50%, and MYRIP regulation—dependent uniquely on miR-96—was restored by the double +23A>G+57T>C mutant.

Methods

  • Case–control mutational screening: 882 genetically undiagnosed Italian NSHL patients (30% familial, 70% sporadic) and 836 normal-hearing Italian controls; MIR183, MIR96, and MIR182 were prescreened by high-resolution melting followed by direct sequencing.
  • Functional studies: mfold was used to predict pre-miR-96 secondary structure; psiUX expression vectors carrying wild-type, mutant, or double-mutant pre-miR-96 were transiently transfected into HeLa cells, and mature miR-96, miR-96*, and pre-miR-96 levels were measured by real-time RT–PCR using the ΔΔCT method.
  • Target validation: eight candidate target 3′UTRs were cloned into psiCHECK2 and tested by luciferase reporter assays; miRNA expression profiles were also examined in human and mouse tissues, including mouse Organ of Corti.

Limitations

  • Stated: a direct role of miR-96* in NSHL pathogenesis remains unproved; none of the putative miR-96* target sites was validated by luciferase assays; more extensive and detailed experiments are needed to define any role of miR-96* in the inner ear.
  • Stated: the mutation was present in three normal-hearing children, which may reflect age-related onset or incomplete penetrance, so those individuals might still develop hearing loss.
  • Evident from the text: functional assays were performed in HeLa cells rather than inner ear hair cells, and the mutation was characterized in a single Italian family, limiting generalization and leaving the inner-ear-specific mechanism unresolved.

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

Integrity signals

Registry

Crossref · 10.1093/hmg/ddr493

Cites

38 of 38 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

8 of 18 cited sources checked · 1 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    The miR-96, miR-182 and miR-183 miRNA family is essential for differentiation and function of the vertebrate inner ear.

    “Importantly, this highly conserved family of miRNAs shows expression in ciliated neurosensory organs across phyla and has recently been demonstrated to contribute specifically to the differentiation and function of the mechanosensory hair cells in the vertebrate inner ear (10,11).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [10] Soukup, 2009, Residual microRNA expression dictates the extent of inner ear development in conditional Dicer knockout mice, Dev. Biol. source

      The Abstract reports that residual miR-183 expression closely corresponds to the degree of hair-cell differentiation, and the Discussion (‘Hair cell development’; page unavailable) describes a critical role for miRNAs in that process. But this Dicer-knockout study does not establish that the miR-96/182/183 family specifically is essential, or demonstrate its role in hair-cell function. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [11] Li, 2010, MicroRNA-183 family members regulate sensorineural fates in the inner ear, J. Neurosci. source

      The Introduction identifies miR-96, miR-182 and miR-183 as a conserved family expressed in neurosensory organs of vertebrates and invertebrates. The Abstract and Discussion report that altering these miRNAs changes hair-cell numbers and development in zebrafish. The paper does not demonstrate that the entire family is essential for hair-cell function across vertebrates; its hearing-loss evidence concerns miR-96 mutations reported in other studies. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
  • Premisecitation

    Tight regulation of miR-96 levels within the vertebrate inner ear is crucial for the correct function and development of hair cells.

    “Indeed, tight regulation of miR-96 levels within the vertebrate inner ear seems to be crucial for the correct function and development of hair cells, as demonstrated by knockdown experiments in zebrafish (11) and by the phenotypic differences between the heterozygous and the homozygous diminuendo mice (15,16).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [11] Li, 2010, MicroRNA-183 family members regulate sensorineural fates in the inner ear, J. Neurosci. source

      The Results sections on miR-183-family knockdown report that miR-96 knockdown in zebrafish reduced inner-ear hair-cell numbers, supporting a role for miR-96 levels in hair-cell development. The cited paper does not show that the knockdown impaired hair-cell function; the diminuendo-mouse comparison is attributed to other citations in the sentence. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [15] Lewis, 2009, An ENU-induced mutation of miR-96 associated with progressive hearing loss in mice, Nat. Genet. source

      The paper reports different hair-cell defects and hearing phenotypes in heterozygous and homozygous diminuendo mice (passage 8; section labeled Abstract, page unavailable), supporting the part attributed to the mouse study. It studies a mutation in miR-96’s seed region, however, and reports mature miRNAs in both wild-type and mutant hair cells (passage 1); it does not establish that tight regulation of miR-96 levels causes those differences. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [16] Kuhn, 2011, miR-96 regulates the progression of differentiation in mammalian cochlear inner and outer hair cells, Proc. Natl Acad. Sci. USA source
  • Premisecitation

    The diminuendo mouse, which carries a mutant miR-96 seed region, does not have a major effect on miR-96 expression levels.

    “In this respect, it should be recalled that the diminuendo mouse, which carries a mutant miR-96 seed region, does not have a major effect on miR-96 expression levels (15).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [15] Lewis, 2009, An ENU-induced mutation of miR-96 associated with progressive hearing loss in mice, Nat. Genet. source

      The passage on the Mirn96 cluster (page unavailable) reports mature miR-96 expression in hair cells of both wild-type and homozygous diminuendo mice, and suggests the mutation does not have a major effect on miRNA processing or export. It does not directly compare miR-96 expression levels, so the claim is more specific than the evidence. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
  • Premisecitation

    Mutations within miR-96 are not a common cause of deafness.

    “Even though mutations within miR-96 are not a common cause of deafness, as evidenced by this and previous works (14,17), the description of novel causative variants might help in elucidating the pathogenic mechanisms underlying the DFNA50-associated phenotype.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [14] Mencía, 2009, Mutations in the seed region of human miR-96 are responsible for nonsyndromic progressive hearing loss, Nat. Genet. source
    • cites [17] Hildebrand, 2010, miRNA mutations are not a common cause of deafness, Am. J. Med. Genet. A source
  • Findingown data

    The identified mutation significantly impacts on miR-96 regulation of selected targets.

    “Finally, even though the mature miR-96 sequence is not altered, we demonstrated that the identified mutation significantly impacts on miR-96 regulation of selected targets.”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 18
  • May combine two claims. 33

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

    Premisecitation

    Point mutations within the seed region of miR-96 cause autosomal dominant non-syndromic sensorineural hearing loss in two Spanish families and in a mouse model of NSHL.

    “Recently, point mutations within the seed region of miR-96 were reported in two Spanish families with autosomal dominant non-syndromic sensorineural hearing loss (NSHL) and in a mouse model of NSHL.”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [14] Mencía, 2009, Mutations in the seed region of human miR-96 are responsible for nonsyndromic progressive hearing loss, Nat. Genet. source
    • cites [15] Lewis, 2009, An ENU-induced mutation of miR-96 associated with progressive hearing loss in mice, Nat. Genet. source

      The paper reports a seed-region miR-96 mutation associated with progressive hearing loss in its diminuendo mouse model. It also reports two different seed-region changes in humans with progressive hearing loss, citing an accompanying study (discussion passage; page unavailable). Our copy does not establish that the human cases were two Spanish families or had autosomal dominant sensorineural hearing loss, and it describes the mouse trait as semi-dominant rather than specifically non-syndromic. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [16] Kuhn, 2011, miR-96 regulates the progression of differentiation in mammalian cochlear inner and outer hair cells, Proc. Natl Acad. Sci. USA source

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

    Premisecitation

    miR-96* has been experimentally detected in humans, mouse, platypus and zebrafish, suggesting that the miRNA* species has been maintained throughout vertebrate evolution, although its sequence is only partially conserved.

    “The miR-96* has been experimentally detected in humans, mouse, platypus and zebrafish (mirBase, http://www.mirbase.org/) (18), suggesting that also the miRNA* species has been maintained throughout vertebrate evolution, although its sequence is only partially conserved.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [18] Soares, 2009, Parallel DNA pyrosequencing unveils new zebrafish microRNAs, BMC Genomics source

      The Results section, “Star sequences of zebrafish microRNAs” (page unavailable), reports detection of zebrafish miRNA* sequences generally. Searches for miR-96*, miR-96, and the named vertebrate species found no passage establishing detection of miR-96* in those species or its partial sequence conservation. (LLM, conf=0.72)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Three (MYRIP, ACVR2B, CACNB4) out of eight luciferase-3'UTR constructs were controlled by human miR-96/miR-96* and the miR-96(+57T>C) mutation led to significantly reduced silencing (~50%) of luciferase expression compared with wild-type pre-miR-96.

    “This reporter system showed that three (MYRIP, ACVR2B, CACNB4) out of eight luciferase-3′UTR constructs were controlled by human miR-96/miR-96* and that the miR-96(+57T>C) mutation led, in all cases, to a significantly reduced silencing (∼50%) of luciferase expression compared with the wild-type pre-miR-96 (Fig. 3A and Supplementary Material, Fig. S5).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The luciferase reporter assays reflect endogenous regulation of miR-96 targets.

  • May combine two claims.

    Findingown data

    Both miR-96 and miR-96* levels were significantly increased in the double mutant (+23A>G+57T>C) compared with the +57T>C mutant (P=0.0003 and P=0.02, respectively), restoring mature miRNA expression at levels not significantly different from wild-type.

    “Both miR-96 and miR-96* levels were significantly increased in the double mutant compared with the +57T>C mutant (P= 0.0003 and P= 0.02, respectively) restoring mature miRNA expression at levels not significantly different from the wild-type (Fig. 2C).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The processing of miR-96 in HeLa cells reflects the processing in the human inner ear relevant to hearing loss.

  • May combine two claims.

    Findingown data

    Screening 882 NSHL patients and 836 normal-hearing Italian controls identified one putative novel mutation within the miR-96 gene in a family with autosomal dominant NSHL.

    “We screened 882 NSHL patients and 836 normal-hearing Italian controls and identified one putative novel mutation within the miR-96 gene in a family with autosomal dominant NSHL.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Expression levels of both miR-96 and miR-96* were significantly reduced in the +57T>C mutant compared with wild-type (85% reduction, P=0.0006 for miR-96 and 77% reduction, P=0.019 for miR-96*), whereas precursor levels were unaffected.

    “Interestingly, expression levels of both miR-96 and miR-96* were significantly reduced in the mutant (+57T>C) compared with the wild-type (85% reduction, P= 0.0006 for miR-96 and 77% reduction, P= 0.019, for miR-96*, respectively), whereas the precursor levels were unaffected by the mutation (Fig. 2C).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The processing of miR-96 in HeLa cells reflects the processing in the human inner ear relevant to hearing loss.

  • May combine two claims.

    Findingown data

    The miR-96(+57T>C) variation is located in the stem region of the pre-miRNA and replaces a residue fully conserved throughout vertebrate evolution, from fish to primates.

    “The last variant identified, miR-96(+57T>C) (NR_029512.1:c.57T>C; NT_007933.15:g.67447397A>G) is located in the stem region of the pre-miRNA and replaces a residue that is fully conserved throughout vertebrate evolution, from fish to primates (Fig. 1A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The detected miR-96 variant is located outside the mature miR-96 sequence and replaces a highly conserved nucleotide within the companion miR-96*.

    “Although located outside the mature miR-96 sequence, the detected variant replaces a highly conserved nucleotide within the companion miR-96*, and is predicted to reduce the stability of the pre-miRNA hairpin.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The miR-96(+57T>C) change occurs at position +6, within the miR-96* seed region.

    “In particular, the change occurs at position +6, within the miR-96* seed region (Fig. 1A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The miR-96(+57T>C) substitution introduces a base-pairing mismatch, decreasing the free energy value and creating an enlarged RNA bulge in the pre-miR-96 stem, close to the Dicer cleavage site.

    “The miR-96(+57T>C) substitution introduces a base-pairing mismatch, decreasing the free energy value and creating an enlarged RNA bulge in the pre-miR-96 stem, close to the Dicer cleavage site (Fig. 2A).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The mfold-predicted secondary structure of pre-miR-96 reflects the actual RNA secondary structure.

  • May combine two claims.

    Findingown data

    The miR-96(+57T>C) variant segregates with the phenotype within the family, being present in all affected individuals and absent in the normal hearing individuals III-4 and IV-4.

    “The miR-96(+57T>C) variant segregates with the phenotype within the family, being present in all affected individuals and absent in the normal hearing individuals III-4 and IV-4.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The known miR-96(+13G>A) mutant showed a significant reduction in mature miR-96 (P<0.0001) but miR-96* levels were unaffected.

    “In this case, the mutant showed a significant reduction in mature miR-96 (P< 0.0001) but, unexpectedly, miR-96* levels were unaffected (Fig. 2D), suggesting an at least partially different pathogenic mechanism.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The processing of miR-96 in HeLa cells reflects the processing in the human inner ear relevant to hearing loss.

  • May combine two claims.

    Findingown data

    No significant alteration of miR-96, miR-96*, and pre-miR-96 levels was measured for the two known polymorphisms rs73159662 (+42C>T) and rs41274239 (+36T>C), although rs73159662 tends to produce slightly elevated levels of both mature miRNA species.

    “No significant alteration of miR-96, miR-96* and pre-miR-96 levels was measured by real-time RT–PCR, although the rs73159662 mutant tends to produce slightly elevated levels of both mature miRNA species (Supplementary Material, Fig. S3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    miR-96 levels were still markedly reduced in the double mutant (+13G>A+66C>T) compared with wild-type (P<0.0001), and not significantly different from the +13G>A mutant, whereas miR-96* and pre-miRNA levels remained unaffected.

    “Indeed, transfection of a double-mutant vector carrying a compensatory mutation, psiUX-miR-96(+13G>A+66C>T), confirmed this hypothesis: miR-96 levels were still markedly reduced in the double mutant compared with the wild-type (P< 0.0001), and not significantly different from the +13G>A mutant, whereas the miR-96* and pre-miRNA levels remained unaffected (Fig. 2D).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The processing of miR-96 in HeLa cells reflects the processing in the human inner ear relevant to hearing loss.

  • May combine two claims.

    Findingown data

    The miR-96(+57T>C) mutation was found in the heterozygous state in a patient with a family history of autosomal dominant progressive NSHL and was absent in all 839 normal-hearing controls.

    “The miR-96(+57T>C) mutation was found in the heterozygous state in a patient with a family history of autosomal dominant progressive NSHL (with the age of onset ranging from ∼25 to 40) and was absent in all 839 normal-hearing controls.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    The family carrying the +57(T>C) mutation is characterized by a late onset (between 25 and 40 years) and a slow progression of the hearing impairment.

    “In particular, the family carrying the +57(T>C) mutation is characterized by a late onset (between 25 and 40 years) and a slow progression of the hearing impairment.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Neither the proband nor any affected individuals of her family referred visual or olfactory problems; episodes of vertigo were reported in the proband (III-2), her mother (II-2) and her brother (III-3).

    “Neither the proband nor any affected individuals of her family referred visual or olfactory problems; however, episodes of vertigo were reported in the proband (III-2), her mother (II-2) and her brother (III-3).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The proband shows non-syndromic, bilateral, sensorineural deafness that onset as a mild hearing impairment at ∼25 years and slowly progressed first to a severe form at the age of 45 and then to a profound form in the sixth decade.

    “The proband shows non-syndromic, bilateral, sensorineural deafness that onset as a mild hearing impairment at ∼25 years and slowly progressed first to a severe form at the age of 45 and then to a profound form in the sixth decade (Fig. 1D).”
    Quote in the paperNot a substantial claim

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

    Findingnone given

    In the mouse Organ of Corti, miR-96* could be cloned but not reliably quantitated by real-time RT-PCR.

    “In the mouse Organ of Corti, the miR-96* could be cloned, but not reliably quantitated by real-time RT–PCR.”
    Quote in the paperNot a substantial claim

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

    Findingown data

    Two novel MIR183 variants, miR-183(+3G>T) and miR-183(-27C>T), were detected each in one proband; both were located outside the mature miRNA sequence at non-conserved nt residues, were absent in normal-hearing controls, but did not segregate with hearing loss in the corresponding families.

    “Two novel MIR183 variants, miR-183(+3G>T) and miR-183(-27C>T), were detected, each in one proband: both were located outside the mature miRNA sequence at non-conserved nt residues, were absent in normal-hearing controls, but did not segregate with hearing loss in the corresponding families (the variants were absent in at least one affected relative in the corresponding family).”
    Quote in the paperNot a substantial claim

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

    Findingown data

    miR-96* is expressed at very low levels but shows some degree of differential expression across a panel of human and mouse tissues.

    “Profiling of miR-96* expression in a panel of human and mouse tissues evidenced that, although expressed at very low levels, miR-96* shows some degree of differential expression, suggesting that it might be regulated in a tissue-specific manner (Supplementary Material, Fig. S6).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    MYRIP 3'UTR is regulated uniquely by miR-96, and the predicted miR-96* binding site is not functional.

    “While the construct lacking the miR-96*-binding site was still significantly responsive to miR-96 repression (P< 0.01), the mutant deprived of miR-96-binding site was not (Fig. 3A), indicating that MYRIP 3′UTR is regulated uniquely by miR-96, and that the predicted miR-96* binding site is not functional.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The luciferase reporter assays reflect endogenous regulation of miR-96 targets.

  • May combine two claims.

    Interpretationown data
    § A novel mutation within the MIR96 gene causes non-syndromic inherited hearing loss in an Italian family by altering pre-miRNA processing

    A novel mutation within the MIR96 gene causes non-syndromic inherited hearing loss in an Italian family by altering pre-miRNA processing.

    “A novel mutation within the MIR96 gene causes non-syndromic inherited hearing loss in an Italian family by altering pre-miRNA processing”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The processing of miR-96 in HeLa cells reflects the processing in the human inner ear relevant to hearing loss.
    • Rests on an assumption: The presence of the miR-96(+57T>C) variant in three normal-hearing children is due to incomplete penetrance or age-related onset of hearing loss.

  • May combine two claims.

    Interpretationown data

    The miR-96(+57T>C) mutation causes a reduction in both mature miRNAs but does not alter the expression level of the miRNA precursor, suggesting a defect in Dicer-mediated cleavage.

    “The miR-96(+57T>C) mutation causes a reduction in both mature miRNAs but does not alter the expression level of the miRNA precursor, thus suggesting a defect in the Dicer-mediated cleavage.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The mfold-predicted secondary structure of pre-miR-96 reflects the actual RNA secondary structure.

  • May combine two claims.

    Interpretationown data

    The mutation hinders precursor processing, probably interfering with Dicer cleavage.

    “Moreover, miR-96 and miR-96* expression levels could be restored by a compensatory mutation that reconstitutes the secondary structure of the pre-miR-96 hairpin, demonstrating that the mutation hinders precursor processing, probably interfering with Dicer cleavage.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The processing of miR-96 in HeLa cells reflects the processing in the human inner ear relevant to hearing loss.
    • Rests on an assumption: The mfold-predicted secondary structure of pre-miR-96 reflects the actual RNA secondary structure.

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Standard curves were generated to verify that the RT-PCR assays had comparable amplification efficiencies.

    “Standard curves were generated to verify that the RT–PCR assays had comparable amplification efficiencies (see Materials and Methods and Supplementary Material, Fig. S2).”
    Quote in the paperSubstantial claim

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

    Interpretationown data

    A direct role of miR-96* in NSHL pathogenesis remains unproved.

    “In the case of the miR-96(+57T>C) mutation, however, a direct role of miR-96* in NSHL pathogenesis has still to be proved.”
    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 study was approved by the local Ethical Committee of the University of Milan and performed according to the Declaration of Helsinki and Italian legislation on sensitive data recording.

    “This study was approved by the local Ethical Committee of the University of Milan and was performed according to the Declaration of Helsinki and to the Italian legislation on sensible data recording.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    Real-time RT-PCR conditions were set up to favor amplification of mature miR-96 and miR-96* forms, and melting curve analysis confirmed a single amplification product.

    “Although in principle the primers for the amplification of mature miR-96 and miR-96* can also amplify the pre-miRNA, real-time RT–PCR conditions were set up to favor the amplification of the mature forms and the analysis of melting curves confirmed the presence of a single amplification product (data not shown).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The phenotypic differences with previously reported families might be partly explained by molecular findings: a quantitative defect of miR-96 expression may lead to delayed or reduced penetrance of hearing impairment, whereas mutations directly impairing miR-96 target recognition would probably result in an earlier defect of hearing.

    “The phenotypic differences with the previously reported families might be partly explained by molecular findings: a quantitative defect of miR-96 expression may lead to a delayed or reduced penetrance of the hearing impairment, whereas mutations directly impairing miR-96 target recognition would probably result in an earlier defect of hearing.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    The psiUX constructs used for miRNA expression in luciferase assays contained only the miR-96 precursor, avoiding confounding by the related miR-183.

    “In this case, the psiUX constructs used for miRNA expression contained only the miR-96 precursor, thus avoiding the confounding effect of cloning the related miR-183.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    miR-183 was used as an internal control to normalize for transfection efficiency.

    “In all experiments, miR-183 was used as internal control to normalize for transfection efficiency.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    A SNP within miR-146a that confers increased risk of thyroid cancer hinders pre-miRNA processing and affects the seed region of the companion miR-146a*.

    “Interestingly, one SNP within the miR-146a, which confers an increased risk to develop thyroid cancer, not only hindered the pre-miRNA processing, but also affected the seed region of the companion miR-146a*, similarly to the miR-96(+57T>C) mutation.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [26] Jazdzewski, 2009, Polymorphic mature microRNAs from passenger strand of pre-miR-146a contribute to thyroid cancer, Proc. Natl Acad. Sci. USA source

  • May combine two claims.

    Study design and conductnone given

    Clinical history ruled out environmental factors as cause of the hearing loss and physical examination did not reveal any evidence of syndromic features.

    “Clinical history ruled out environmental factors as cause of the hearing loss and physical examination did not reveal any evidence of syndromic features.”
    Quote in the paperSubstantial claim

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

    Interpretationown data

    miR-96* might be regulated in a tissue-specific manner.

    “Profiling of miR-96* expression in a panel of human and mouse tissues evidenced that, although expressed at very low levels, miR-96* shows some degree of differential expression, suggesting that it might be regulated in a tissue-specific manner (Supplementary Material, Fig. S6).”
    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

    Signed informed consent was obtained from all participants and from parents of subjects younger than 18 years.

    “Signed informed consent was obtained from all participants and from parents of subjects younger than 18 years.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    The MIR96 mutation reported here is the third mutation described in this gene in humans and the first in a population different from that of the original report.

    “This is the third mutation described in this gene in humans, and the first in a different population from that of the original report (14).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [14] Mencía, 2009, Mutations in the seed region of human miR-96 are responsible for nonsyndromic progressive hearing loss, Nat. Genet. source

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Standard curves verified that the RT-PCR assays for wild-type and mutant miR-96, miR-96*, and pre-miR-96 had comparable amplification efficiencies.

    “Conversely, standard curves were generated to verify that the RT–PCR assays to detect: (i) wild-type miR-96, miR-96(+13G>A) or miR-96(+23A>G); (ii) wild-type miR-96* and miR-96*(+66T>C); (iii) wild-type pre-miR-96, pre-miR-96(+42C>T) and pre-miR-96(+36T>C) had comparable amplification efficiencies (Supplementary Material, Fig. S2).”
    Quote in the paperSubstantial claim

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

    Premisecitation

    miR-146a* is expressed in both normal and tumor thyroid tissues, and altered regulation of miRNA* targets in heterozygotes compared with homozygotes was suggested as a possible mechanism underlying tumor predisposition.

    “The miR-146a* was expressed in both normal and tumor thyroid tissues and the altered regulation of the miRNA* targets in heterozygotes compared with homozygotes was suggested as the possible mechanism underlying tumor predisposition (26).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [26] Jazdzewski, 2009, Polymorphic mature microRNAs from passenger strand of pre-miR-146a contribute to thyroid cancer, Proc. Natl Acad. Sci. USA source

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

    Premisecitation

    Common SNPs within miRNA genes can lead to impaired miRNA processing and might be associated with increased susceptibility to human diseases.

    “Other studies have reported that common SNPs within miRNA genes can lead to impaired miRNA processing, and might be associated with increased susceptibility to human diseases (23–25).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [23] Duan, 2007, Single nucleotide polymorphism associated with mature miR-125a alters the processing of pri-miRNA, Hum. Mol. Genet. source
    • cites [24] Sun, 2009, SNPs in human miRNA genes affect biogenesis and function, RNA source
    • cites [25] Jazdzewski, 2008, Common SNP in pre-miR-146a decreases mature miR expression and predisposes to papillary thyroid carcinoma, Proc. Natl Acad. Sci. USA source

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

    Interpretationreasoning

    The pathogenic mechanisms underlying each family's phenotype might be at least partially different.

    “Interestingly, also the three affected families (the two Spanish and the here reported Italian one) show some degree of phenotypic variation, suggesting that the pathogenic mechanisms underlying each family's phenotype might be at least partially different.”
    Quote in the paperNot a substantial claim
41 of 41

  • The paper relies on this without support.

    The processing of miR-96 in HeLa cells reflects the processing in the human inner ear relevant to hearing loss.

    “To this aim, expression vectors (psiUX) containing genomic DNA fragments spanning both miR-183 and miR-96 precursors, and corresponding to either the miR-96 wild-type or the miR-96(+57T>C) sequences, were generated and used to transiently transfect HeLa cells.”

  • The paper relies on this without support.

    The mfold-predicted secondary structure of pre-miR-96 reflects the actual RNA secondary structure.

    “we used the mfold program (19) to examine how the newly identified nt variation could alter the predicted RNA secondary structure of pre-miR-96.”

  • The paper relies on this without support.

    The luciferase reporter assays reflect endogenous regulation of miR-96 targets.

    “we co-transfected a vector expressing either the wild-type or the mutant pre-miR-96 hairpin into HeLa cells, together with a construct containing the luciferase reporter cDNA coupled to the 3′UTR of each target gene (psiCHECK2-3′UTR).”

  • The paper relies on this without support.

    The presence of the miR-96(+57T>C) variant in three normal-hearing children is due to incomplete penetrance or age-related onset of hearing loss.

    “this may be due either to the age-relatedness of hearing loss in the family (all non-penetrants are below the average age of onset of the disease among affected relatives) or to incomplete penetrance.”
4 of 4

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 [2]

    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

  • 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 [2]

    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

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.

  • Conflicting numbers of normal-hearing controls

    Minor

    The paper reports screening 836 normal-hearing controls, but later says the +57T>C mutation was absent in all 839 controls.

  • Opposite substitutions reported at position +66

    Minor

    The compensatory mutation at position +66 is consistently identified as C>T elsewhere, but the RT-PCR assay description calls it T>C.

2 of 2

Cites

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1 of the 39 references couldn’t be matched to a known paper.

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Related

Authors
Soldà, G., Robusto, M., Primignani, P., Castorina, P., Benzoni, E., Cesarani, A., Ambrosetti, U., Asselta, R., & Duga, S.
Affiliations
  • University of Milan (IT)
  • Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico (IT)
Corresponding author
Soldà, Giulia
ORCID
Venue
Human Molecular Genetics
Publisher
Oxford University Press (OUP)
Published
2011-10-28
Volume
21
Issue
3
Pages
577-585
ISSN
  • 0964-6906 (ISSN-L)
  • 1460-2083
DOI
10.1093/hmg/ddr493
PMC
PMC3259013
OpenAlex
W2109723356
Cited by
102
References
39
Citations by year
2026: 1 · 2025: 5 · 2024: 10 · 2023: 6 · 2022: 6 · 2021: 2 · 2020: 6 · 2019: 9 · 2018: 7 · 2017: 9 · 2016: 5 · 2015: 13 · 2014: 7 · 2013: 13 · 2012: 3
FWCI
3.61
Type
journal article
Language
en
Open access
Licence
cc-by-nc
Locations
  • Human Molecular Genetics (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (submittedVersion, cc-by-nc)
  • PubMed Central (submittedVersion, cc-by-nc)
Topics
  • Life Sciences › Neuroscience › Sensory Systems › Hearing, Cochlea, Tinnitus, Genetics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Health Sciences › Medicine › Genetics › Congenital Ear and Nasal Anomalies
Keywords
miR-96, non-syndromic sensorineural hearing loss, pre-miRNA processing, compensatory mutations, microRNA biogenesis, inner ear development, Italian families
MeSH terms
Gene Expression Regulation, Hearing Loss, Sensorineural, HeLa Cells, Humans, Italy, Mutation, Nucleic Acid Conformation, RNA Precursors, RNA Processing, Post-Transcriptional, MicroRNAs

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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.

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