miRNAs in the spotlight: Making 'silent' mutations speak up.


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miRNAs in the spotlight: Making 'silent' mutations speak up.

  1. 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. 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 (2008). 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.