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miRNAs in the spotlight: Making 'silent' mutations speak up.
- 1. Bet ween Bedside and Bench
miRNAs in the spotlight
Genetic mutations can cause numerous diseases. These alterations affect not only protein-coding genes but also regions that were
until recently thought to be trivial in disease. In ‘Bedside to Bench’, David Salzman and Joanne Weidhaas examine a human study
showing how a silent mutation impairs the binding of miR-196, increasing the risk for Crohn’s disease. Therefore, alterations
of miRNA target sites as pathogenic mechanism begs further investigation. miRNAs themselves can also be the root of disease.
In ‘Bench to Bedside’, Carlo Croce peruses a study in vivo showing evidence of tumor addiction to miR-21 in a mouse model of
cancer, which highlights the role of miRNAs as initiators of disease. Targeting these drivers will help to develop effective drugs.
Since the onset of genomic sequencing, there
has been great interest in identifying genetic
markers to predict an individual’s disease sus-
ceptibility. Although the greatest success in
identifying such genetic alterations has been
in cancer, known mutations only account for
approximately 3–5% of all cancer diagnoses,
reflecting the need to identify and characterize
additional markers.
Genetic mutations have traditionally been
dubbed as deleterious only when they alter
proteinsequence,functionorboth.Butgenome
sequencing has identified a number of single
nucleotide polymorphisms (SNPs) in the open
reading frame, introns and 5′ and 3′ untrans-
lated regions of protein-coding genes. The role
of these variants in disease has not been recog-
nized, and they are referred to as ‘silent’ muta-
tions, as they have no apparent effect on protein
sequence. Consequently, many of them have
been classified as variants of unknown signifi-
cance(VUS)1.This leads tothe crucial question
of whether a silent mutation that does not affect
protein sequence can cause genetic diseases.
In a recent study by Brest et al.2, the authors
show that a silent mutation in the open reading
frameoftheIRGMgeneactsasafunctionalvari-
ant by destroying a miRNA target site, resulting
in the persistence of intracellular bacteria in the
inflamed intestinal epithelium of individuals
with Crohn’s disease. This observation provides
the first evidence that silent mutations may in
factbeclinicallysignificant,especiallyintheset-
ting of disruption of miRNA regulation.
Mounting evidence suggests that some
SNPs occur in cis-regulatory elements and
have a crucial role in regulating the spatial
and temporal timing of gene expression3. But
it is necessary to appreciate that cis-regulatory
(complementary) elements are controlled
by the concomitant expression of a requi-
site trans-acting factor that will probably be
expressed in response to cellular or extracel-
lular signaling events4. miRNAs are a class of
noncoding, 18- to 24-nucleotide, trans-acting
RNAs that act at complementary elements in
the 5′ and 3′ untranslated regions of a target
RNA and negatively regulate gene expression
by inhibiting protein translation5. miRNA-
mediated gene regulation has been implicated
in almost all cellular processes, in particular
cancer, where they control transformation and
tumor cell maintenance6.
The expression of miRNAs seems to be
dynamic and governed by cellular and extra-
cellular stimuli7. There is, therefore, growing
appreciation that miRNAs quickly and effi-
ciently ‘fine tune’ expression to allow cells to
respond to these stimuli8. Because miRNA tar-
geting is based on RNA-RNA complementar-
ity, it has been postulated that single nucleotide
David W. Salzman and Joanne B. Weidhaas
are at the Yale University School of Medicine
Department of Therapeutic Radiology, New Haven,
Connecticut, USA.
e-mail: joanne.weidhaas@yale.edu
■ BeDSIDe To BeNCh
Making ‘silent’ mutations speak up
David W Salzman & Joanne B Weidhaas
Lumen
Inflammation
Autophagy
Bacterial
lysis
miR-196
miR-196
IRGM
AAAAAATTG
Silent mutation
(risk variant)
Mucosal epithelial cell
Extracellular
bacteria
IRGM
Figure 1 Regulation of Crohn’s disease by miRNA-196. miR-196 is upregulated by inflammation in the
epithelial lining of the mucosa and targets the IRGM protective variant mRNA in healthy individuals,
decreasing IRGM amounts and allowing for proper clearing of intracellular Crohn’s disease–associated
bacteria. But miR-196 does not target the IRGM risk allele mRNA harboring a silent mutation, which
results in increased IRGM and impaired clearing of intracellular bacteria. Persistence of intracellular
bacteria associates with different miR-196–mediated regulation of IGRM in Crohn’s disease phenotypes.
MaryLouQuillen
934 volume 17 | number 8 | august 2011 nature medicine
©2011NatureAmerica,Inc.Allrightsreserved.
- 2. B e t w e e n B e d s i d e an d B e n c h
alterations, such as SNPs, in cis-regulatory ele-
ments can alter target gene regulation by either
creating or destroying miRNA target sites9.
Indeed, SNPs occurring at cis-regulatory ele-
ments have been shown to either enhance or
inhibit the recognition of a miRNA, causing
aberrant gene expression and disease pheno-
types10,11. However, the interaction of specific
miRNAs with cis-acting sequence variants
and the environmental cues required for their
expression are just coming to light.
Crohn’s disease is an inflammatory bowel
disease caused by the persistence of bacteria in
the epithelial lining of the gastrointestinal tract.
There seems to be an inherent genetic compo-
nent to Crohn’s disease, as an individual’s risk
is directly associated with the amount of first-
and second-degree relatives with the disease.
Genetic mapping in people with this disorder
has uncovered numerous mutations—in par-
ticular, a silent mutation in IRGM (encoding
immunity-related GTPase family, M), a gene
required for autophagy of intracellular bac-
teria that is highly prevalent in individuals of
European descent12. Brest et al.2 found that
the protective IRGM variant, but not the risk-
associated allele, is a target site for the miR-196
family, which normally results in a decrease in
cellular IRGM protein amounts in inflamed
epithelial cells. miR-196 was also overexpressed
in inflamed epithelial mucosa of the gut in
people with Crohn’s disease, but this did not
result in decreased expression of cellular IRGM
in the presence of the risk allele. miR-196 was
first found to be crucial in development, and
subsequently in cancer progression, through
regulation of HOX genes13.
ThepathophysiologicalrelevanceofmiR-196–
mediated suppression of IRGM protective vari-
ant is that decreased amounts of IRGM were
necessary to clear Crohn’s disease–associated
adherent invasive E. coli from the gut through
autophagy (Fig. 1). Thus, individuals with
Crohn’s disease with the risk allele were unable
to clear these bacteria, resulting in an accumu-
lation of intracellular bacteria and disease pro-
gression.
The observation from the study of Brest et
al.2 provides direct evidence that a silent muta-
tion can, in fact, have a function in disease, and
it can do so by creating an aberrant cis-regu-
latory element for a miRNA. In the future, the
risk of a particular silent mutation should be
assessed by its ability to functionally regulate
gene expression under different environmental
conditions. The finding that miR-196 is specifi-
cally expressed in the inflamed mucosa to tar-
get IRGM and that, in this scenario, the silent
mutation in IRGM becomes crucial in disease
causation2 is the perfect proof of this concept.
The identification of such functional variants
may therefore not be possible by comparing
their altered frequency in control and disease
populations without understanding concurrent
physiology of the patients.
Althoughthisworksubstantiallyadvancesour
understanding of Crohn’s disease, it has much
broader implications for the clinical assessment
of silent mutations and other VUS in disease.
For example, there are many silent mutations in
BRCA1andBRCA2classifiedas‘uninformative’
or ‘not clinically significant’ by the US National
Institutes of Health Breast Cancer Information
Core database and Myriad Genetics, as they are
not predicted to alter BRCA protein sequence
or function1. Yet many of the individuals with
these VUS still develop breast and/or ovarian
cancer. In light of the observations by Brest et
al.2, clinicians and genetic counselors may need
toreconsidertheirassessmentof BRCAVUS,as
well as SNPs disrupting miRNA binding sites
outside of protein coding sequences.
As it is possible that many of these silent
mutations or noncoding region SNPs alter
miRNA target sites and that their function will
depend on miRNA expression alterations in
response to physiologic conditions—similar to
that of the IRGM risk-associated allele—it will
be crucial to bring the study of such variants
back to the bench to understand their impor-
tance in human disease.
COMPETING FINANCIAL INTERESTS
The authors declare competing financial interests:
details accompany the full-text HTML version of the
paper at http://www.nature.com/naturemedicine/.
1. Easton, D.F. et al. Am. J. Hum. Genet. 81, 873–883
(2007).
2. Brest, P. et al. Nat. Genet. 43, 242–245 (2011).
3. Pastinen, T. & Hudson, T.J. Science 306, 647–650
(2004).
4. Pastinen, T. et al. Hum. Mol. Genet. 15 Spec No 1,
R9–R16 (2006).
5. Bartel, D.P. Cell 116, 281–297 (2004).
6. Esquela-Kerscher, A. & Slack, F.J. Nat. Rev. Cancer 6,
259–269 (2006).
7. van Rooij, E. et al. Science 316, 575–579 (2007).
8. Bartel, D.P. & Chen, C.Z. Nat. Rev. Genet. 5, 396–400
(2004).
9. Saunders, M.A. et al. Proc. Natl. Acad. Sci. USA 104,
3300–3305 (2007).
10. Abelson, J.F. et al. Science 310, 317–320 (2005).
11. Chin, L.J. et al. Cancer Res. 68, 8535–8540
(2008).
12. McCarroll, S.A. et al. Nat. Genet. 40, 1107–1112
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13. Chen, C. et al. J. Cell. Mol. Med. 15, 14–23 (2011).
■ BeNCh To BeDSIDe
Understanding cancer gene dependency
Carlo M Croce
Carlo M. Croce is in the Department of Molecular
Virology, Immunology and Medical Genetics,
The Ohio State University School of Medicine,
Columbus, Ohio, USA.
e-mail: carlo.croce@osumc.edu
Most human cancers are driven by somatic
genetic alterations, which often involve onco-
genes and tumor suppressor genes1. For exam-
ple, chromosome translocations involving the
MYC oncogene on chromosome 8 and one
of three immunoglobulin loci dysregulate the
MYC oncogene, leading to the development
of Burkitt’s lymphoma1. The BCR-ABL fusion
gene resulting from the Philadelphia chromo-
some causes elevation of the activity of the Abl
tyrosine kinase and is responsible for chronic
myelogenous leukemia (CML)1. Because such
driver gene alterations are essential for tumor
cellgrowth,survivalorboth,theirreversioncan
lead to selective death of cancer cells1.
Specific drugs, such as imatinib in the case
of CML, can target the consequences of such
genetic alterations2. Treatment with imatinib
leads to complete remission in more than 95%
of patients with CML in the chronic phase with
few side effects2, indicating that targeting the
activated Abl tyrosine kinase can selectively
result in the death of cancer cells with consid-
erable benefit for the patients. Given that the
malignancy is caused by the expression of the
BCR-ABL fusion gene, the results suggest that
reverting cancer ‘drivers’ can lead to tumor
regression and possibly cure.
The expression of additional cancer genes
can be altered during tumor progression and
can contribute to tumor growth and spreading;
therefore, targeting these additional alterations
may have some effect. But such alterations may
not be present in every cell within a tumor.
Another possibility is that cooperation of can-
cer genes may be required for or may speed up
nature medicine volume 17 | number 8 | august 2011 935
©2011NatureAmerica,Inc.Allrightsreserved.