Regulation of B-cell development and function by microRNAs

Immunological Reviews

Volumn 253, Issue 1, 2013, Pages 25-39

  de Yébenes, Virginia G ^a   Bartolomé Izquierdo, Nahikari ^a   Ramiro, Almudena R ^a  

^a CENTRO NACIONAL DE INVESTIGACIONES CARDIOVASCULARES CNIC   (Spain)

Author keywords

Bone marrow; Dicer; Germinal center; Lymphomagenesis; MicroRNA

Indexed keywords

MICRORNA; MICRORNA 155;

B LYMPHOCYTE; B LYMPHOCYTE DIFFERENTIATION; BIOGENESIS; BONE MARROW; CELL MATURATION; GENE TARGETING; GROWTH REGULATION; HUMAN; HUMORAL IMMUNITY; LYMPHOCYTE FUNCTION; ONCOGENE; PRIORITY JOURNAL; REVIEW; TUMOR SUPPRESSOR GENE;

ANIMALS; B-LYMPHOCYTES; CELL DIFFERENTIATION; CELL LINEAGE; CELL PROLIFERATION; GENE EXPRESSION REGULATION, DEVELOPMENTAL; HUMANS; MICRORNAS; ORGAN SPECIFICITY;

EID: 84875760707     PISSN: 01052896     EISSN: 1600065X     Source Type: Journal    
DOI: 10.1111/imr.12046     Document Type: Review

References (146)

1. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993;75:843-854.
2. Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 1993;75:855-862.
3. Reinhart BJ, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 2000;403:901-906.
4. Pasquinelli AE, et al. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature 2000;408:86-89.
5. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed RNAs. Science 2001;294:853-858.
6. Lau NC, Lim LP, Weinstein EG, Bartel DP. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 2001;294:858-862.
7. Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science 2001;294:862-864.
8. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009;136:215-233.
9. Liu Q, Paroo Z. Biochemical principles of small RNA pathways. Annu Rev Biochem 2010;79:295-319.
10. Ebert MS, Sharp PA. Roles for microRNAs in conferring robustness to biological processes. Cell 2012;149:515-524.
11. Krol J, Loedige I, Filipowicz W. The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet 2010;11:597-610.
12. Yang JS, Lai EC. Alternative miRNA biogenesis pathways and the interpretation of core miRNA pathway mutants. Mol Cell 2011;43:892-903.
13. van Kouwenhove M, Kedde M, Agami R. MicroRNA regulation by RNA-binding proteins and its implications for cancer. Nat Rev Cancer 2011;11:644-656.
14. Voinnet O. Origin, biogenesis, and activity of plant microRNAs. Cell 2009;136:669-687.
15. Pasquinelli AE. MicroRNAs and their targets: recognition, regulation and an emerging reciprocal relationship. Nat Rev Genet 2012;13:271-282.
16. Ozsolak F, et al. Chromatin structure analyses identify miRNA promoters. Genes Dev 2008;22:3172-3183.
17. Corcoran DL, Pandit KV, Gordon B, Bhattacharjee A, Kaminski N, Benos PV. Features of mammalian microRNA promoters emerge from polymerase II chromatin immunoprecipitation data. PLoS ONE 2009;4:e5279.
18. O'Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT. c-Myc-regulated microRNAs modulate E2F1 expression. Nature 2005;435:839-843.
19. He L, et al. A microRNA component of the p53 tumour suppressor network. Nature 2007;447:1130-1134.
20. Chang TC, et al. Widespread microRNA repression by Myc contributes to tumorigenesis. Nat Genet 2008;40:43-50.
21. Ma L, et al. miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis. Nat Cell Biol 2010;12:247-256.
22. Melnyk CW, Molnar A, Baulcombe DC. Intercellular and systemic movement of RNA silencing signals. EMBO J 2011;30:3553-3563.
23. Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 2007;9:654-659.
24. Mittelbrunn M, et al. Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells. Nat Commun 2011;2:282.
25. Mittelbrunn M, Sanchez-Madrid F. Intercellular communication: diverse structures for exchange of genetic information. Nat Rev Mol Cell Biol 2012;13:328-335.
26. Bernstein E, et al. Dicer is essential for mouse development. Nat Genet 2003;35:215-217.
27. Xiao C, Rajewsky K. MicroRNA control in the immune system: basic principles. Cell 2009;136:26-36.
28. Belver L, Papavasiliou FN, Ramiro AR. MicroRNA control of lymphocyte differentiation and function. Curr Opin Immunol 2011;23:368-373.
29. Cheloufi S, Dos Santos CO, Chong MM, Hannon GJ. A dicer-independent miRNA biogenesis pathway that requires Ago catalysis. Nature 2010;465:584-589.
30. Cifuentes D, et al. A novel miRNA processing pathway independent of Dicer requires Argonaute2 catalytic activity. Science 2010;328:1694-1698.
31. Yang JS, et al. Conserved vertebrate mir-451 provides a platform for Dicer-independent, Ago2-mediated microRNA biogenesis. Proc Natl Acad Sci U S A 2010;107:15163-15168.
32. Pritchard CC, Cheng HH, Tewari M. MicroRNA profiling: approaches and considerations. Nat Rev Genet 2012;13:358-369.
33. Mullokandov G, et al. High-throughput assessment of microRNA activity and function using microRNA sensor and decoy libraries. Nat Methods 2012;9:840-846.
34. de Yebenes VG, Ramiro AR. MicroRNA activity in B lymphocytes. Methods Mol Biol 2010;667:177-192.
35. de Yebenes VG, et al. miR-181b negatively regulates activation-induced cytidine deaminase in B cells. J Exp Med 2008;205:2199-2206.
36. Casola S. Mouse models for miRNA expression: the ROSA26 locus. Methods Mol Biol 2010;667:145-163.
37. O'Connell RM, Rao DS, Chaudhuri AA, Baltimore D. Physiological and pathological roles for microRNAs in the immune system. Nat Rev Immunol 2010;10:111-122.
38. Gentner B, et al. Stable knockdown of microRNA in vivo by lentiviral vectors. Nat Methods 2009;6:63-66.
39. Chaudhuri AA, et al. Oncomir miR-125b regulates hematopoiesis by targeting the gene Lin28A. Proc Natl Acad Sci USA 2012;109:4233-4238.
40. Rao DS, O'Connell RM, Chaudhuri AA, Garcia-Flores Y, Geiger TL, Baltimore D. MicroRNA-34a perturbs B lymphocyte development by repressing the forkhead box transcription factor Foxp1. Immunity 2010;33:48-59.
41. Chi SW, Zang JB, Mele A, Darnell RB. Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 2009;460:479-486.
42. Hafner M, et al. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 2010;141:129-141.
43. Dorsett Y, et al. MicroRNA-155 suppresses activation-induced cytidine deaminase-mediated Myc-Igh translocation. Immunity 2008;28:630-638.
44. Teng G, et al. MicroRNA-155 is a negative regulator of activation-induced cytidine deaminase. Immunity 2008;28:621-629.
45. Lim LP, et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 2005;433:769-773.
46. Baek D, Villen J, Shin C, Camargo FD, Gygi SP, Bartel DP. The impact of microRNAs on protein output. Nature 2008;455:64-71.
47. Selbach M, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N. Widespread changes in protein synthesis induced by microRNAs. Nature 2008;455:58-63.
48. Rajewsky K. Clonal selection and learning in the antibody system. Nature 1996;381:751-758.
49. Chen CZ, Li L, Lodish HF, Bartel DP. MicroRNAs modulate hematopoietic lineage differentiation. Science 2004;303:83-86.
50. Li QJ, et al. miR-181a is an intrinsic modulator of T cell sensitivity and selection. Cell 2007;129:147-161.
51. Ebert PJ, Jiang S, Xie J, Li QJ, Davis MM. An endogenous positively selecting peptide enhances mature T cell responses and becomes an autoantigen in the absence of microRNA miR-181a. Nat Immunol 2009;10:1162-1169.
52. Fragoso R, et al. Modulating the strength and threshold of NOTCH oncogenic signals by mir-181a-1/b-1. PLoS Genet 2012;8:e1002855.
53. Koralov SB, et al. Dicer ablation affects antibody diversity and cell survival in the B lymphocyte lineage. Cell 2008;132:860-874.
54. He L, et al. A microRNA polycistron as a potential human oncogene. Nature 2005;435:828-833.
55. Ventura A, et al. Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters. Cell 2008;132:875-886.
56. Chang TC, et al. Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis. Mol Cell 2007;26:745-752.
57. Raver-Shapira N, et al. Transcriptional activation of miR-34a contributes to p53-mediated apoptosis. Mol Cell 2007;26:731-743.
58. Hermeking H. The miR-34 family in cancer and apoptosis. Cell Death Differ 2010;17:193-199.
59. Concepcion CP, et al. Intact p53-dependent responses in miR-34-deficient mice. PLoS Genet 2012;8:e1002797.
60. Hu H, et al. Foxp1 is an essential transcriptional regulator of B cell development. Nat Immunol 2006;7:819-826.
61. Martin F, Kearney JF. B1 cells: similarities and differences with other B cell subsets. Current Opin Immunol 2001;13:195-201.
62. Montecino-Rodriguez E, Dorshkind K. B-1 B cell development in the fetus and adult. Immunity 2012;36:13-21.
63. Thomas MD, Kremer CS, Ravichandran KS, Rajewsky K, Bender TP. c-Myb is critical for B cell development and maintenance of follicular B cells. Immunity 2005;23:275-286.
64. Monticelli S, et al. MicroRNA profiling of the murine hematopoietic system. Genome Biol 2005;6:R71.
65. Zhou B, Wang S, Mayr C, Bartel DP, Lodish HF. miR-150, a microRNA expressed in mature B and T cells, blocks early B cell development when expressed prematurely. Proc Natl Acad Sci USA 2007;104:7080-7085.
66. Xiao C, et al. MiR-150 controls B cell differentiation by targeting the transcription factor c-Myb. Cell 2007;131:146-159.
67. Mondol V, Pasquinelli AE. Let's make it happen: the role of let-7 microRNA in development. Curr Topics Cell Dev Biol 2012;99:1-30.
68. Thornton JE, Gregory RI. How does Lin28 let-7 control development and disease? Trends Cell Biol 2012;22:474-482.
69. Viswanathan SR, Daley GQ. Lin28: a microRNA regulator with a macro role. Cell 2010;140:445-449.
70. Buganim Y, et al. Single-cell expression analyses during cellular peprogramming reveal an early stochastic and a late hierarchic phase. Cell 2012;150:1209-1222.
71. Yuan J, Nguyen CK, Liu X, Kanellopoulou C, Muljo SA. Lin28b reprograms adult bone marrow hematopoietic progenitors to mediate fetal-like lymphopoiesis. Science 2012;335:1195-1200.
72. Busslinger M. Transcriptional control of early B cell development. Annu Rev Immunol 2004;22:55-79.
73. Allman D, Pillai S. Peripheral B cell subsets. Curr Opin Immunol 2008;20:149-157.
74. Carsetti R, Rosado MM, Wardmann H. Peripheral development of B cells in mouse and man. Immunol Rev 2004;197:179-191.
75. Wardemann H, Yurasov S, Schaefer A, Young JW, Meffre E, Nussenzweig MC. Predominant autoantibody production by early human B cell precursors. Science 2003;301:1374-1377.
76. Pillai S, Cariappa A. The follicular versus marginal zone B lymphocyte cell fate decision. Nat Rev Immunol 2009;9:767-777.
77. Li Y, Li H, Weigert M. Autoreactive B cells in the marginal zone that express dual receptors. J Exp Med 2002;195:181-188.
78. Martin F, Kearney JF. Positive selection from newly formed to marginal zone B cells depends on the rate of clonal production, CD19, and btk. Immunity 2000;12:39-49.
79. Stavnezer J, Guikema JE, Schrader CE. Mechanism and regulation of class switch recombination. Annu Rev Immunol 2008;26:261-292.
80. Di Noia JM, Neuberger MS. Molecular mechanisms of antibody somatic hypermutation. Annu Rev Biochem 2007;76:1-22.
81. Peled JU, et al. The biochemistry of somatic hypermutation. Annu Rev Immunol 2008;26:481-511.
82. de Yebenes VG, Ramiro AR. Activation-induced deaminase: light and dark sides. Trends Mol Med 2006;12:432-439.
83. Liu M, et al. Two levels of protection for the B cell genome during somatic hypermutation. Nature 2008;451:841-845.
84. Pasqualucci L, et al. Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas. Nature 2001;412:341-346.
85. Ramiro AR, et al. AID is required for c-myc/IgH chromosome translocations in vivo. Cell 2004;118:431-438.
86. Ramiro AR, et al. Role of genomic instability and p53 in AID-induced c-myc-Igh translocations. Nature 2006;440:105-109.
87. Robbiani DF, et al. AID is required for the chromosomal breaks in c-myc that lead to c-myc/IgH translocations. Cell 2008;135:1028-1038.
88. Landgraf P, et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 2007;129:1401-1414.
89. Basso K, et al. Identification of the human mature B cell miRNome. Immunity 2009;30:744-752.
90. Kuchen S, et al. Regulation of microRNA expression and abundance during lymphopoiesis. Immunity 2010;32:828-839.
91. Belver L, de Yebenes VG, Ramiro AR. MicroRNAs prevent the generation of autoreactive antibodies. Immunity 2010;33:713-722.
92. Hobeika E, et al. Testing gene function early in the B cell lineage in mb1-cre mice. Proc Natl Acad Sci USA 2006;103:13789-13794.
93. Kil LP, et al. Btk levels set the threshold for B-cell activation and negative selection of autoreactive B cells in mice. Blood 2012;119:3744-3756.
94. Honigberg LA, et al. The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy. Proc Natl Acad Sci USA 2010;107:13075-13080.
95. Xu S, Guo K, Zeng Q, Huo J, Lam KP. The RNase III enzyme Dicer is essential for germinal center B-cell formation. Blood 2012;119:767-776.
96. Rodriguez A, et al. Requirement of bic/microRNA-155 for normal immune function. Science 2007;316:608-611.
97. Thai TH, et al. Regulation of the germinal center response by microRNA-155. Science 2007;316:604-608.
98. Trotta R, et al. miR-155 regulates IFN-gamma production in natural killer cells. Blood 2012;119:3478-3485.
99. O'Connell RM, et al. Sustained expression of microRNA-155 in hematopoietic stem cells causes a myeloproliferative disorder. J Exp Med 2008;205:585-594.
100. O'Connell RM, Taganov KD, Boldin MP, Cheng G, Baltimore D. MicroRNA-155 is induced during the macrophage inflammatory response. Proc Natl Acad Sci USA 2007;104:1604-1609.
101. O'Connell RM, et al. MicroRNA-155 promotes autoimmune inflammation by enhancing inflammatory T cell development. Immunity 2010;33:607-619.
102. Eis PS, et al. Accumulation of miR-155 and BIC RNA in human B cell lymphomas. Proc Natl Acad Sci USA 2005;102:3627-3632.
103. Tili E, Croce CM, Michaille JJ. miR-155: on the crosstalk between inflammation and cancer. Int Rev Immunol 2009;28:264-284.
104. Costinean S, et al. Pre-B cell proliferation and lymphoblastic leukemia/high-grade lymphoma in E(mu)-miR155 transgenic mice. Proc Natl Acad Sci USA 2006;103:7024-7029.
105. Tam W. Identification and characterization of human BIC, a gene on chromosome 21 that encodes a noncoding RNA. Gene 2001;274:157-167.
106. Vigorito E, et al. microRNA-155 regulates the generation of immunoglobulin class-switched plasma cells. Immunity 2007;27:847-859.
107. Garrett-Sinha LA, et al. PU.1 and Spi-B are required for normal B cell receptor-mediated signal transduction. Immunity 1999;10:399-408.
108. Basso K, et al. BCL6 positively regulates AID and germinal center gene expression via repression of miR-155. J Exp Med 2012;209:2455-2465.
109. Victora GD, Nussenzweig MC. Germinal centers. Annu Rev Immunol 2012;30:429-457.
110. Sernandez IV, de Yebenes VG, Dorsett Y, Ramiro AR. Haploinsufficiency of activation-induced deaminase for antibody diversification and chromosome translocations both in vitro and in vivo. PLoS ONE 2008;3:e3927.
111. Takizawa M, et al. AID expression levels determine the extent of cMyc oncogenic translocations and the incidence of B cell tumor development. J Exp Med 2008;205:1949-1957.
112. O'Connell RM, Chaudhuri AA, Rao DS, Gibson WS, Balazs AB, Baltimore D. MicroRNAs enriched in hematopoietic stem cells differentially regulate long-term hematopoietic output. Proc Natl Acad Sci USA 2010;107:14235-14240.
113. Kim SW, Ramasamy K, Bouamar H, Lin AP, Jiang D, Aguiar RC. MicroRNAs miR-125a and miR-125b constitutively activate the NF-kappaB pathway by targeting the tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20). Proc Natl Acad Sci USA 2012;109:7865-7870.
114. Gururajan M, et al. MicroRNA 125b inhibition of B cell differentiation in germinal centers. Int Immunol 2010;22:583-592.
115. Malumbres R, et al. Differentiation stage-specific expression of microRNAs in B lymphocytes and diffuse large B-cell lymphomas. Blood 2009;113:3754-3764.
116. Chaudhuri AA, et al. MicroRNA-125b potentiates macrophage activation. J Immunol 2011;187:5062-5068.
117. Lu J, et al. MicroRNA expression profiles classify human cancers. Nature 2005;435:834-838.
118. Esquela-Kerscher A, Slack FJ. Oncomirs - microRNAs with a role in cancer. Nat Rev Cancer 2006;6:259-269.
119. Volinia S, et al. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci USA 2006;103:2257-2261.
120. Calin GA, Croce CM. MicroRNA signatures in human cancers. Nat Rev Cancer 2006;6:857-866.
121. Garzon R, Calin GA, Croce CM. MicroRNAs in Cancer. Annu Rev Med 2009;60:167-179.
122. Xiao C, et al. Lymphoproliferative disease and autoimmunity in mice with increased miR-17-92 expression in lymphocytes. Nat Immunol 2008;9:405-414.
123. Mu P, et al. Genetic dissection of the miR-17~92 cluster of microRNAs in Myc-induced B-cell lymphomas. Genes Dev 2009;23:2806-2811.
124. Olive V, et al. miR-19 is a key oncogenic component of mir-17-92. Genes Dev 2009;23:2839-2849.
125. Garzon R, Marcucci G, Croce CM. Targeting microRNAs in cancer: rationale, strategies and challenges. Nat Rev Drug Disc 2010;9:775-789.
126. Costinean S, et al. Src homology 2 domain-containing inositol-5-phosphatase and CCAAT enhancer-binding protein beta are targeted by miR-155 in B cells of Emicro-MiR-155 transgenic mice. Blood 2009;114:1374-1382.
127. Pedersen IM, et al. Onco-miR-155 targets SHIP1 to promote TNFalpha-dependent growth of B cell lymphomas. EMBO Mol Med 2009;1:288-295.
128. Dagan LN, Jiang X, Bhatt S, Cubedo E, Rajewsky K, Lossos IS. miR-155 regulates HGAL expression and increases lymphoma cell motility. Blood 2012;119:513-520.
129. Rai D, Kim SW, McKeller MR, Dahia PL, Aguiar RC. Targeting of SMAD5 links microRNA-155 to the TGF-beta pathway and lymphomagenesis. Proc Natl Acad Sci USA 2010;107:3111-3116.
130. Valeri N, et al. Modulation of mismatch repair and genomic stability by miR-155. Proc Natl Acad Sci USA 2010;107:6982-6987.
131. Tili E, et al. Mutator activity induced by microRNA-155 (miR-155) links inflammation and cancer. Proc Natl Acad Sci USA 2011;108:4908-4913.
132. Medina PP, Nolde M, Slack FJ. OncomiR addiction in an in vivo model of microRNA-21-induced pre-B-cell lymphoma. Nature 2010;467:86-90.
133. Fulci V, et al. Quantitative technologies establish a novel microRNA profile of chronic lymphocytic leukemia. Blood 2007;109:4944-4951.
134. Lawrie CH, et al. MicroRNA expression distinguishes between germinal center B cell-like and activated B cell-like subtypes of diffuse large B cell lymphoma. Int J Cancer 2007;121:1156-1161.
135. Jongen-Lavrencic M, Sun SM, Dijkstra MK, Valk PJ, Lowenberg B. MicroRNA expression profiling in relation to the genetic heterogeneity of acute myeloid leukemia. Blood 2008;111:5078-5085.
136. Santanam U, et al. Chronic lymphocytic leukemia modeled in mouse by targeted miR-29 expression. Proc Natl Acad Sci USA 2010;107:12210-12215.
137. Enomoto Y, et al. Emu/miR-125b transgenic mice develop lethal B-cell malignancies. Leukemia 2011;25:1849-1856.
138. Li C, et al. Copy number abnormalities, MYC activity, and the genetic fingerprint of normal B cells mechanistically define the microRNA profile of diffuse large B-cell lymphoma. Blood 2009;113:6681-6690.
139. Calin GA, et al. Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 2002;99:15524-15529.
140. Calin GA, et al. MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proc Natl Acad Sci USA 2004;101:11755-11760.
141. Calin GA, et al. A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. N Engl J Med 2005;353:1793-1801.
142. Klein U, et al. The DLEU2/miR-15a/16-1 cluster controls B cell proliferation and its deletion leads to chronic lymphocytic leukemia. Cancer Cell 2010;17:28-40.
143. Pichiorri F, et al. Downregulation of p53-inducible microRNAs 192, 194, and 215 impairs the p53/MDM2 autoregulatory loop in multiple myeloma development. Cancer Cell 2010;18:367-381.
144. Zhao JL, Rao DS, Boldin MP, Taganov KD, O'Connell RM, Baltimore D. NF-kappaB dysregulation in microRNA-146a-deficient mice drives the development of myeloid malignancies. Proc Natl Acad Sci USA 2011;108:9184-9189.
145. Visone R, et al. Karyotype-specific microRNA signature in chronic lymphocytic leukemia. Blood 2009;114:3872-3879.
146. Labbaye C, Testa U. The emerging role of MIR-146A in the control of hematopoiesis, immune function and cancer. J Hematol Oncol 2012;5:13.