Chromatin and epigenetic control Capturing the dynamic epigenome R E V I E W
2010. 2010

Henikoff Genome B Deal
Biology
Chromatin and epigenetic control Capturing the dynamic epigenome R E V I E W

Henikoff Genome Biology
112182010. 2010*Correspondence: Full list of author information is available at the end of the article Page 6 of 8 Author details
Traditional methods for epigenomic analysis provide a static picture of chromatin, which is actually a highly dynamic assemblage. Recent approaches have allowed direct measurements of chromatin dynamics, providing deeper insights into processes such as transcription, DNA replication and epigenetic inheritance.

A eukaryotic genome can produce many cell types with widely different morphologies and functions. Given that the diverse cell types of a multicellular organism all con tain the same DNA, there must be information in addition to the DNA sequence itself that controls which genes are expressed in a particular cell type. This extra layer of information was termed 'epigenetic control' by Nanney in 1958 [1]. Epigenetic control in eukaryotes occurs in the context of nucleosome particles, which can occlude or allow access to DNA by the proteins that bind specific sequences and precisely regulate active pro cesses, including transcription and replication. Under standing the molecular basis for epigenetic control is a central goal of chromatin research.

The eukaryotic genome is tightly wrapped by histones to form nucleosomes, which must be densely packed to fit within the confines of the nucleus, overall up to approxi mately 1 millionfold compaction of DNA relative to an extended double helix. Despite these tight con straints, nucleosomes must be able to allow the DNA sequences to be accessible to DNA binding proteins and to the action of 'molecular machines' such as DNA and RNA polymerases, ATP dependent nucleosome remodelers and topoisomerases. Nuclear organization involves multiple levels of chromatin packaging, including compartments, territories and selforganizing nuclear bodies, which might appear to be static at a gross cytological level, but which must be sufficiently dynamic to allow for access of regulatory factors to the DNA (Figure 1). Although the precise nature of chromatin beyond the level of single nucleosomes is unclear [2], some principles are beginning to emerge, such as the fractal globule largescale organization of chromosomes, which allows them to decondense and recondense without becoming entangled [3].

Nucleosome particles consist of around 150 bp of DNA wrapped around an octameric histone core containing two copies of each of the four core histone proteins (H2A, H2B, H3 and H4) [4,5]. The properties of a nucleo some can be altered in various ways, including replace ment of standard histones with specialized variant types, posttranslational modification of histones, movement of the particle relative to the underlying DNA sequence, and partial or complete removal of histones from the DNA. The regulation of chromatin structure to expose or occlude a particular DNA segment is controlled by the dynamic interplay between sequencespecific DNAbinding proteins, histone variants, histonemodifying enzymes, chromatinassociated proteins, histone chaper ones and ATPdependent nucleosome remodelers [6]. Collectively, these factors provide instructions that direct the transcriptional output of the genome, but exactly how this information is imparted and transmitted through cell division is unclear. Approaches to under standing chromatinbased regulation have included the identification of factors involved and mapping of chroma tin proteins and histone modifications across the genome [68]. These approaches have taught us much about the control of transcription in particular and have provided a conceptual framework for further research. However, these methods give only a static snapshot of chromatin, whereas chromatin is actually a dynamic assemblage in which proteins are constantly associating and dissociating [9]. Therefore, understanding chromatinbased regula tion has required the development and application of techniques that can capture these dynamic processes. This review will focus on epigenome dynamics at the level of the nucleosome and will explore how emerging technologies that allow timedependent measurements are yielding deeper insights into the regulation of various genomic processes and the inheritance of geneexpression states.

Defining the epigenome through chromatinmapping studies

Much of our understanding of the epigenome and its influence on regulating gene expression has come from genomewide analyses of steadystate chromatin compo sition combined with genetic and biochemical studies that enable functional interpretation of these maps. To elucidate the primary structure of chromatin, many groups have sought to identify the locations of all nucleosomes across the genome and to understand the factors that dictate their locations. A popular mapping approach is to digest chromatin with micrococcal nuclease, which preferentially cleaves the DNA between nucleosomes, and then to infer nucleosome positions by analyzing the pool of sequences protected by nucleo somes [10]. These studies have collectively shown that certain fundamental rules of nucleosome positioning are common to many eukaryotes. The Saccharomyces cere visiae genome has a large number of wellpositioned nucleosomes covering approximately 80% of the genome, whereas metazoan and plant genomes have a smaller percentage of wellpositioned nucleosomes [1116]. However, all genomes examined show a characteristic distribution of nucleosomes around genes. There are often two wellpositioned nucleosomes that flank the transcription start site (TSS) with a nucleosomedepleted region (NDR) in between [17]. Nucleosomes at the 5' ends of transcribed regions tend to be more precisely localized than those further downstream, and there is often another NDR at the 3' end [14,18]. The overall landscape of nucleosome locations and relative occupancy at a point in time seems to be dictated in part by intrinsic DNA sequence preferences of the nucleo somes themselves, and also by the action of nucleosome remodeling complexes and competition between nucleosomes and sequencespecific DNAbinding proteins such as transcription factors [1921].

Chromatin is further differentiated by variations in the characteristics and composition of nucleosomes. Bio chemical studies of histones have shown that they are heavily modified posttranslationally through the addi tion of acetyl, methyl, phosphoryl and ADPribose groups, as well as peptides such as ubiquitin and SUMO. Mapping of these modified histones has revealed distinct patterns of localization across the genome, and this has led to insights into genomic processes, including trans cription as well as DNA replication and repair. It has emerged that certain histone modifications tend to co occur, and each 'mark' can be broadly categorized as being associated with either actively transcribed genes, silenced genes or transposons [68]. Within these cate gories there are modifications, such as acetylation, that alter the physical properties of nucleosomes directly, and others such as methylation that can create binding sites for other proteins that have specific effects on chromatinbased processes. In terms of function, acetylation of the nucleosomes around TSSs seems to be required to support transcription, presumably by loosening the interaction between histones and DNA, while conversely, the deacetylation of nucleosomes throughout the body of the gene appears to repress spurious antisense trans cription by increasing histone association with the DNA [22,23]. Chromatin modifications that are bound by specific effector proteins can either be involved in the repression of transcription, by mechanisms such as compac tion of nucleosome arrays [24,25], or they can support transcription, by recruiting chromatinremodel ing complexes, modifying enzymes or other complexes involved in elongation or splicing [26,27]. Thus, histone modifications can affect access to DNA directly or indirectly, and also serve as a platform for the coordi nation of successive processes such as transcription and splicing [27].

Nucleosomes are also differentiated by the substitution of canonical histones with the universal variants H2A.Z and H3.3 [28]. These variants are replicationindependent in their assembly, and so must be inserted by disruption of existing nucleosomes. H2A.Z is inserted by the Swr1 ATPdependent nucleosomeremodeling complex into nucleo some cores by partial unwrapping and replacement of an H2A/H2B dimer with an H2A.Z/H2B dimer. To insert H3.3 into the central (H3/H4) 2 tetramer, a nucleosome must be completely unwrapped, a process that amounts to dynamic eviction of the histone core and replacement with two dimeric units of H3.3/H4 [29]. H3.3 replace ment requires a histone chaperone, such as  [33], whereas H3.3 is low in promoter nucleo somes and is enriched in essentially all genebody nucleosomes, with its occupancy positively correlated with the level of transcription [34]. Nucleosomes contain ing H2A.Z but not H3.3 are relatively stable, whereas those that contain both variants may be prone to disassembly in vivo [35] (although not in vitro [36]). Unstable doublevariant nucleosomes are found at TSSs and so may regulate exposure of promoter DNA [37]. Thus, both the replicationindependent replacement of canonical histones with histone variants, and the altered properties of doublevariant nucleosomes that sometimes result, indicate that the nucleosomes that package genes are inherently dynamic. The emerging picture of the epigenome is one in which the composition of chromatin in terms of histone modifications, variants and chromatin associated proteins dictates the intrinsic stability of nucleosomes as well as their propensity to be disrupted or moved by chromatinremodeling enzymes and the transcription machinery. In this way, access to the underlying DNA is regulated [38].

Measuring epigenome dynamics

Given the evidence that many regions of chromatin are in a state of flux, various approaches have been developed to measure chromatin dynamics directly by using tools such as microscopy, mass spectrometry (MS), immuno precipitation of inducible tagged proteins, and metabolic labeling of newly synthesized proteins ( Table 1). The application of these methods has led to unexpected new insights into the regulation of various genomic processes such as transcription, DNA replication and the inheri tance of patterns of gene expression.

Fluorescence recovery after photobleaching

One approach to observing chromatin dynamics in vivo is fluorescence recovery after photobleaching (FRAP) and related cytological methods. In FRAP, a discrete region of a nucleus containing a fluorescently labeled chromatin protein is subjected to laser photobleaching, and the amount of time required for the bleached region to regain fluorescence is measured (Figure 2a). The time required for fluorescence recovery is a measure of the residence time of the protein on chromatin; therefore, this technique can be used to infer the binding kinetics of chromatin proteins [39]. FRAP has the advantage that any protein that can be tagged can be analyzed, and information on the nuclear distribution of each protein can also be obtained. However, in contrast to methods that use genomics tools as a readout, FRAP does not provide information on the specific site to which the factor of interest binds. Furthermore, like all methods that rely on epitopetagged proteins, there is the possibility that the protein will not behave like the native form, producing artifactual results.  In the inducible transgene-based approach, the protein to be assayed is expressed from two different transgenes. One transgene is expressed constitutively and carries an epitope tag while the other is inducible and carries a different epitope tag. Induction of the second transgene allows the measurement of binding kinetics by comparing the relative abundances of the two tags at a given genomic location.

(d) Recombination-induced tag exchange (RITE) can be used to measure the dynamics of chromatin binding and nucleosome assembly by enabling old and new proteins expressed from the same transgene to be distinguished. This method uses a single transgene that encodes two different epitope tags. The first tag is flanked by loxP recombination sites and incorporates a stop codon, whereas the downstream tag is in-frame but comes after the stop codon. Normally, the protein encoded by the transgene has only the first tag, but after induction of Cre recombinase the first tag is removed from the transgene and now the encoded protein contains only the second tag. Dynamics of a RITE-tagged protein can be inferred by comparing the relative abundances of the old and new versions of the protein at a given genomic location. (e) Covalent attachment of tags to capture histones and identify turnover (CATCH-IT) can be used to estimate rates of disassembly and reassembly, or turnover, of native nucleosomes across the genome. In this method, newly synthesized proteins are labeled by treating cells with the methionine analog azidohomoalanine (AHA). Nuclei are isolated from AHA-treated cells and biotin is coupled to AHA-containing nuclear proteins through a reaction of the azide group of AHA with an alkyne linked to biotin. Chromatin is then digested to mononucleosomes, the nucleosomes are extracted, and nucleosomes containing biotinylated histones are purified via streptavidin. Stringent washes are used to remove H2A/H2B dimers and all other DNA-binding proteins from the purified nucleosomes. Microarray analysis or deep sequencing of the purified DNA allows the rates of nucleosome turnover across the genome to be estimated on the basis of the extent of newly synthesized H3/H4 dimer incorporation at each site.  The FRAP method has been used to measure residence times of a wide variety of proteins, including nuclear hormone receptors, transcription factors, chromatin remodel ing enzymes and the histones themselves. The results of these experiments have consistently shown that hormone receptors, transcription factors and remodeling enzymes have residence times on chromatin on the order of seconds [40]. By contrast, photobleaching studies of core histones revealed that these proteins have residence times much longer than most chromatinassociated proteins, on the order of tens of minutes to hours, and that the residence time of H2A and H2B on chromatin is shorter than that of H3 and H4 [41]. These results were confirmed by experiments in which epitopetagged histones were introduced into the slime mold Physarum, and chromatin immunoprecipitation (ChIP) was used to track their incorporation into several active genes over time [42]. Collectively, these results argue strongly that complexes that regulate transcription are unstable, and show that histones also dissociate from DNA over time scales that can be shorter than the cell cycle.

Kinetic methods based on mass spectrometry

Dozens of different histone posttranslational modifica tions have been identified by MS, and, in recent years, MS has also been used to determine the different com bi nations in which they occur [43,44]. To exploit this powerful discriminating tool for measuring nucleosome dynamics, MS has been combined with MeselsonStahl type incorporation of heavy isotopes in protocols designed to distinguish newly synthesized histones from preexisting histones (Figure 2b). By synchronizing cells and releasing them into Sphase with the addition of an amino acid labeled with a heavy isotope, such as 15 N labeled arginine or 13 Clabeled methione, peptides from newly synthesized histones can be distinguished from their counterparts from old histones using MS [45,46]. This allows for changes in modification to be scored and quantified during chromatin assembly. Consistent with classical studies, histone acetylation and deacetylation was found to be highly dynamic. In the case of histone methylation, monomethylation occurred on most H3 residues (lysine (K)4, K9, K27 and K36) soon after syn thesis and incorporation, whereas di and tri methylation occurred more slowly over the course of the cell cycle. An exception is H3K79, which was found to undergo monomethylation continuously on both old and new nucleo somes with very little conversion to the dimethylated form [47]. This strategy for following global histone modification changes should become increasingly power ful as MSbased technologies improve. Together with genomicbased methods described below, MSbased kinetics promises to provide a wealth of information on the dynamics of histone marks and how they might be involved in governing nucleosome dynamics.

Inducible transgene-based methods

In the past few years, the rate of replicationindependent nucleosome disassembly and reassembly, or turnover, has been measured at high resolution across the genome of budding yeast (S. cerevisiae) by using inducible epitope tagged histones as a means of estimating relative nucleo some turnover rates. In this method, cells have two transgenic sources of a particular histone protein: one that is constitutively expressed and has an epitope tag, and another that is inducible and has a different epitope tag. By arresting cells in G1 phase and inducing the second tagged histone, ChIP assays can be performed separately for each tag at multiple time points after induction. Analysis of the resulting DNA by microarrays then allows estimation of nucleosome turnover rates across the genome by comparing the ratio of signals from the two tagged histones (Figure 2c). In addition to measur ing nucleosome turnover kinetics, this approach could also be used to measure the dynamics of other chromatin proteins. One caveat to this approach is that there is a time lag during induction of the transgene and synthesis of the encoded protein, which limits the temporal resolution of kinetic measurements.

Several inducible transgene studies have been carried out using histone H3, and these collectively showed that nucleosome turnover was highest both upstream and downstream of the TSS, whereas turnover in the body of the gene was relatively low regardless of expression level, except at very highly expressed genes [48,49]. Using a similar approach, in which only the inducible epitope tagged histone was used, Jamai et al. [50] found that, in contrast to H3, H2B turns over rapidly within promoters and across gene bodies irrespective of expression level, implying that the turnover of H2A/H2B dimers is a distinct process from the turnover of (H3/H4) 2 tetramers. Results from these studies also indicated that nucleosome turnover is very high at known chromatin boundary elements flanking silenced regions, leading to the sug gest ion that nucleosome turnover might help to prevent the spread of silent chromatin and silencing of nearby genes [48].

Recombination-induced tag exchange

Recombinationinduced tag exchange (RITE) allows one to distinguish between old and new proteins encoded by the same transgene (Figure 2d). In this method, a trans gene encoding the protein of interest is engineered to contain an epitope tag and a stop codon flanked by loxP recombination sites, with a second inframe epitope tag after the stop codon. When Cre recombinase activity is induced, the sequence encoding the first tag is removed, Deal and Henikoff Genome Biology 2010, 11:218 http://genomebiology.com/2010/11/10/218 resulting in all subsequent transcripts encoding a protein with only the second tag. This allows the tracking of proteins produced before and after recombinase induction.

Using this approach, Verzijlbergen et al. [51] tagged H3 in yeast and showed that nucleosome turnover occurs not only in G1 phase but also in G2/M phase, and that the rate is dependent on the expression level of a given gene. The RITE method was also used to examine replace ment of proteasomal subunits in this study, suggesting that it will be useful for studying the dynamics not only of histones but also of other chromatin proteins.

Covalent attachment of tags to capture histones and identify turnover

As an alternative to using transgenes, we developed a general method for estimating nucleosome turnover rates by metabolically labeling newly synthesized histones with an aminoacid analog that could be coupled to an affinity tag. This technique is called covalent attachment of tags to capture histones and identify turnover, or CATCHIT ( Figure 2e). This method has an advantage over pre viously discussed genomic methods in that no transgenes are required and the behavior of the native protein itself is being measured. Furthermore, the temporal resolution is very high as there is no time lag associated with transgene induction and protein synthesis. However, essentially all proteins will be labeled, and therefore CATCHIT analysis is limited to (H3/H4) 2 tetramers as these remain associated with DNA under conditions that remove all other proteins.

Using CATCHIT on Drosophila S2 cells, we found that nucleosome turnover landscapes were very similar to those previously reported for steadystate incor poration of H3.3 [34], and that turnover rates were highest in gene bodies, with the rate being correlated to expression level. By contrast, turnover within promoters was relatively low and seemed to be mostly independent of expression level [52]. Interestingly, nucleosome turn over measurements at sites bound by the origin recog nition complex, which dictates the location and timing of replication origin firing [53,54], showed very high turn over rates compared with those of surrounding regions, indicating that regulated nucleosome turnover might also play an important part in the selection of replication origins and replication timing. This would help to explain the lack of DNA sequence conservation between replica tion origins [55].

Examination of nucleosome turnover at epigenetic regu latory elements bound by either trithorax group (trxG) activators or Polycomb group (PcG) silencer proteins [56] in S2 cells revealed that the rates of turnover were different between these two classes of sites. Turnover rates were higher at trxG sites than at PcG sites, suggesting that differences in nucleosome turnover rates provide the mechanistic basis for the opposing activities of these regulatory proteins on gene expression. We hypothesize that trxG proteins promote turnover to allow greater access of sequencespecific regulators, whereas PcG proteins slow turnover to reduce access. In fact, the PRC1 complex, which is responsible for PcG silencing, causes compaction of the chromatin fiber in vitro [24]. Recent in vivo support for this paradigm of develop mental silencing has come from the finding that the PRC1 complex silences Hox genes by compacting chromatin [25], which presumably results in reduced nucleosome turnover.

Inheritance of chromatin states

A point of contention in the field of epigenetics is the basis of the inheritance of a chromatin state through cell division. One view is that histone modifications are infor mational, by virtue of a 'histone code' analogous with the genetic code [57]. However, it is now clear that nucleo somes are reconstituted from newly synthesized histones many times during a cell cycle, essentially erasing histone modifications and making it unlikely that the modifica tions themselves are capable of transmitting information [52]. An alternative hypothesis is that histone modifica tions and secondary effector proteins that recognize them collectively modulate the intrinsic stability of a given nucleosome as well as its propensity to be re modeled. These characteristics in turn determine how likely a nucleosome is to be disassembled or to change position, and thereby expose the underlying DNA to sequencespecific regulators that control genome output. Rather than constituting an informational code, histone modifications, variants, nucleosome remodelers and other chromatinassociated proteins could be considered as components of a dynamic system that regulates nucleosome turnover and, consequently, DNA exposure to sequencespecific regulators.

The perpetuation of geneexpression states during the cell cycle and through cell divisions may be based on a competition between the binding of sequencespecific regulators and the reassembly of nucleosomes onto a particular DNA segment [58]. For example, an active geneexpression state could be initiated and maintained by binding of an activator that brings along factors that promote nucleosome turnover to favor further activator binding. The active state could then be transmitted through cell division by a process based on the per petu ation of nucleosome turnover, which in some instances would be driven by the continued binding of activators, such as general transcription factors, through mitosis [59]. The silent state would then be inherited by default via silencing complexes that reduce nucleosome turnover through chromatin compaction [24], thereby reducing access of activators to DNA. Thus, nucleosome turnover would simply govern the level of exposure of a gene regulatory site and thereby determine whether the gene will remain active or silent during development [60].

Prospects for the future

New approaches to studying chromatin dynamics have made it clear that chromatin and its associated proteins are in a constant state of flux and are not as stable as once thought. Thus, chromatin dynamics need to be taken into account when interpreting ChIP and other static measures of the epigenome. The combination of static and dynamic mapping of chromatin features along with mechanistic studies has the potential to provide a deep understanding of the epigenome.

Although we are only beginning to understand the mechanisms that maintain the epigenome, it has become clear that chromatin dynamics play a central role in the regulation of genome function. We look forward to the development of new methods for measuring dynamics at each structural level of chromatin from the primary fiber to secondary folding and on to the threedimensional arrangement of the genome in the nucleus. Continuing technological progress is needed to generate a mecha nistic description of the relationship between chromatin dynamics and transcriptional regulation, as well as other genomic processes such as DNA replication, repair, and recombination. This will bring us closer to the holy grail of understanding how the epigenome programs the genome to give the specific patterns of gene expression that define a given cell type and how it allows the stable perpetuation of phenotype.

Figure 1 .
1Dynamic chromatin. Chromatin consists of arrays of nucleosomes (N) with a number of dynamic features such as nucleosome position, histone-variant composition of nucleosomes, post-translational modifications of histones, as well as the binding of transcription factors, chromatin-remodeling complexes, and modification binding proteins. Transcription factors (TFs) and remodeling complexes (R) are in equilibrium between the bound and unbound states, and nucleosomes can slide along DNA, be dislodged from DNA, and be reassembled. In addition, a wide variety of histone modifications (m) can be added and removed enzymatically. The right-angled arrow indicates the transcription start site. DAXX, and various ATPdependent nucleosomeremodel ing complexes, including Chd1 and Atrx [3032]. H2A.Z and H3.3 show partially overlapping distributions: H2A.Z is often enriched at the 1 nucleosome position relative to the TSS and in gene body nucleosomes near the 5' end

Figure 2 .
2Methods for investigating chromatin dynamics. (a)In fluorescence recovery after photobleaching (FRAP), a laser is used to bleach the fluorescence of a chromatin protein in a discrete region of the nucleus. The time required for the fluorescent protein to move back into the bleached region and restore fluorescence is proportional to the residence time of the protein on chromatin. The shorter the residence time of a protein, the faster fluorescence is recovered in the bleached region. (b) Mass spectrometry (MS) can be used to study the dynamics of posttranslational modifications in the total histone pool by briefly labeling newly synthesized histones with a heavy isotope, such as 15 N or 13 C. Histone modifications can then be determined by MS for both the old and new histones, based on the mass difference between these two pools of histones. (c)

The same protein with a di erent tag is induced 3. Rate of incorporation is measured as ratio of two tags 1. Transgene encodes a protein with two epitope tags 2. Induction of recombinase removes one tag, allowing distinction of old and new proteins 1. Newly synthesized proteins are labeled with azidohomoalanine (AHA) 2. Biotin is coupled to AHA-containing proteins 3. Nucleosomes containing newly synthesized histones are puri ed

Table 1 .
1Comparison of methods for measuring chromatin dynamicsMethod
Utility
Benefits
Drawbacks

Fluorescence recovery after
photobleaching (FRAP)

Measurement of chromatin protein
binding kinetics

1. Can be used for nucleosomes as
well as other chromatin binding
proteins
2. Allows observation of protein
location within the nucleus

1. Cannot determine the specific
genomic sites that are bound
2. Requires an epitope-tagged
protein that may not behave exactly
like the native form

MS-based kinetic methods
Measurements of histone
modification kinetics

Can be used for nucleosomes as well
as other chromatin-binding proteins

Cannot determine the kinetics at
specific genomic sites

Inducible transgene-based methods Measurement of nucleosome
turnover kinetics as well as binding of
other chromatin proteins

Can be used for nucleosomes as well
as other chromatin-binding proteins

1. Requires an epitope-tagged
protein
2. Time lag during induction limits
time resolution

Recombination-induced tag
exchange (RITE)

Measurement of nucleosome
turnover kinetics as well as binding of
other chromatin proteins

Can be used for nucleosomes as well
as other chromatin-binding proteins

1. Requires an epitope-tagged
protein that may not behave exactly
like the native form
2. Time lag during recombination
limits time resolution

Covalent attachment of tags to
capture histones and identify
turnover (CATCH-IT)

Measurement of nucleosome
turnover kinetics

1.No transgenes or antibodies are
required
2. Excellent time resolution
3. Can be used on many different
cell types

Only (H3/H4) 2 tetramer incorporation
kinetics can be measured easily

Deal and Henikoff Genome Biology 2010, 11:218
http://genomebiology.com/2010/11/10/218

AcknowledgementsWe thank Melissa Conerly and Paul Talbert for helpful suggestions on improving the manuscript. This work was supported by NIH grant 1R21DA025758 to SH, NIH Postdoctoral Fellowship 1F32GM083449 to RBD, and the Howard Hughes Medical Institute.
Epigenetic control systems. D L Nanney, Proc Natl Acad Sci. 44Nanney DL: Epigenetic control systems. Proc Natl Acad Sci USA 1958, 44:712-717.

Chromatin higher-order structure and dynamics. C L Woodcock, R P Ghosh, Cold Spring Harb Perspect Biol. 2596Woodcock CL, Ghosh RP: Chromatin higher-order structure and dynamics. Cold Spring Harb Perspect Biol 2010, 2:a000596.

Comprehensive mapping of long-range interactions reveals folding principles of the human genome. E Lieberman-Aiden, N L Van Berkum, L Williams, M Imakaev, T Ragoczy, A Telling, I Amit, B R Lajoie, P J Sabo, M O Dorschner, R Sandstrom, B Bernstein, M A Bender, M Groudine, A Gnirke, J Stamatoyannopoulos, L A Mirny, E S Lander, J Dekker, Science. 326Lieberman-Aiden E, van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, Amit I, Lajoie BR, Sabo PJ, Dorschner MO, Sandstrom R, Bernstein B, Bender MA, Groudine M, Gnirke A, Stamatoyannopoulos J, Mirny LA, Lander ES, Dekker J: Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 2009, 326:289-293.

Chromatin structure; oligomers of the histones. R D Kornberg, J O Thomas, Science. 184Kornberg RD, Thomas JO: Chromatin structure; oligomers of the histones. Science 1974, 184:865-868.

Crystal structure of the nucleosome core particle at 2.8 A resolution. K Luger, A W Mader, R K Richmond, D F Sargent, T J Richmond, Nature. 389Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ: Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 1997, 389:251-260.

The role of chromatin during transcription. B Li, M Carey, J L Workman, Cell. 128Li B, Carey M, Workman JL: The role of chromatin during transcription. Cell 2007, 128:707-719.

The complex language of chromatin regulation during transcription. S L Berger, Nature. 447Berger SL: The complex language of chromatin regulation during transcription. Nature 2007, 447:407-412.

Genome-wide views of chromatin structure. O J Rando, H Y Chang, Annu Rev Biochem. 78Rando OJ, Chang HY: Genome-wide views of chromatin structure. Annu Rev Biochem 2009, 78:245-271.

Protein dynamics in the nuclear compartment. G L Hager, C Elbi, M Becker, Curr Opin Genet Dev. 12Hager GL, Elbi C, Becker M: Protein dynamics in the nuclear compartment. Curr Opin Genet Dev 2002, 12:137-141.

Chromatin structure snap-shots: rapid nuclease digestion of chromatin in yeast. N A Kent, J Mellor, Nucleic Acids Res. 23Kent NA, Mellor J: Chromatin structure snap-shots: rapid nuclease digestion of chromatin in yeast. Nucleic Acids Res 1995, 23:3786-3787.

Genome-scale identification of nucleosome positions in S. cerevisiae. G-C Yuan, Y-J Liu, M F Dion, M D Slack, L F Wu, S J Altschuler, O J Rando, Science. 309Yuan G-C, Liu Y-J, Dion MF, Slack MD, Wu LF, Altschuler SJ, Rando OJ: Genome-scale identification of nucleosome positions in S. cerevisiae. Science 2005, 309:626-630.

Flexibility and constraint in the nucleosome core landscape of Caenorhabditis elegans chromatin. S M Johnson, F J Tan, H L Mccullough, D P Riordan, A Z Fire, Genome Res. 16Johnson SM, Tan FJ, McCullough HL, Riordan DP, Fire AZ: Flexibility and constraint in the nucleosome core landscape of Caenorhabditis elegans chromatin. Genome Res 2006, 16:1505-1516.

A genomic code for nucleosome positioning. E Segal, Y Fondufe-Mittendorf, L Chen, A Thastrom, Y Field, I K Moore, J P Wang, J Widom, Nature. 442Segal E, Fondufe-Mittendorf Y, Chen L, Thastrom A, Field Y, Moore IK, Wang JP, Widom J: A genomic code for nucleosome positioning. Nature 2006, 442:772-778.

Nucleosome organization in the Drosophila genome. T N Mavrich, C Jiang, I P Ioshikhes, X Li, B J Venters, S J Zanton, L P Tomsho, J Qi, R L Glaser, S C Schuster, D S Gilmour, I Albert, B F Pugh, Nature. 453Mavrich TN, Jiang C, Ioshikhes IP, Li X, Venters BJ, Zanton SJ, Tomsho LP, Qi J, Glaser RL, Schuster SC, Gilmour DS, Albert I, Pugh BF: Nucleosome organization in the Drosophila genome. Nature 2008, 453:358-362.

Dynamic regulation of nucleosome positioning in the human genome. D E Schones, K Cui, S Cuddapah, T-Y Roh, A Barski, Z Wang, G Wei, K Zhao, Cell. 132Schones DE, Cui K, Cuddapah S, Roh T-Y, Barski A, Wang Z, Wei G, Zhao K: Dynamic regulation of nucleosome positioning in the human genome. Cell 2008, 132:887-898.

R K Chodavarapu, S Feng, Y V Bernatavichute, P Y Chen, H Stroud, Y Yu, J A Hetzel, F Kuo, J Kim, S J Cokus, D Casero, M Bernal, P Huijser, A T Clark, U Krämer, S S Merchant, X Zhang, S E Jacobsen, M Pellegrini, Relationship between nucleosome positioning and DNA methylation. 466Chodavarapu RK, Feng S, Bernatavichute YV, Chen PY, Stroud H, Yu Y, Hetzel JA, Kuo F, Kim J, Cokus SJ, Casero D, Bernal M, Huijser P, Clark AT, Krämer U, Merchant SS, Zhang X, Jacobsen SE, Pellegrini M: Relationship between nucleosome positioning and DNA methylation. Nature 2010, 466:388-392.

Nucleosome positioning: how is it established, and why does it matter?. M Radman-Livaja, O J Rando, Dev Biol. 339Radman-Livaja M, Rando OJ: Nucleosome positioning: how is it established, and why does it matter? Dev Biol 2010, 339:258-266.

Dynamic remodeling of individual nucleosomes across a eukaryotic genome in response to transcriptional perturbation. S Shivaswamy, A Bhinge, Y Zhao, S Jones, M Hirst, V R Iyer, PLoS Biol. 665Shivaswamy S, Bhinge A, Zhao Y, Jones S, Hirst M, Iyer VR: Dynamic remodeling of individual nucleosomes across a eukaryotic genome in response to transcriptional perturbation. PLoS Biol 2008, 6:e65.

The DNA-encoded nucleosome organization of a eukaryotic genome. N Kaplan, I K Moore, Y Fondufe-Mittendorf, A J Gossett, D Tillo, Y Field, E M Leproust, T R Hughes, J D Lieb, J Widom, E Segal, Nature. 458Kaplan N, Moore IK, Fondufe-Mittendorf Y, Gossett AJ, Tillo D, Field Y, LeProust EM, Hughes TR, Lieb JD, Widom J, Segal E: The DNA-encoded nucleosome organization of a eukaryotic genome. Nature 2009, 458:362-366.

Comparison of ABF1 and RAP1 in chromatin opening and transactivator potentiation in the budding yeast Saccharomyces cerevisiae. A Yarragudi, T Miyake, R Li, R H Morse, Mol Cell Biol. 24Yarragudi A, Miyake T, Li R, Morse RH: Comparison of ABF1 and RAP1 in chromatin opening and transactivator potentiation in the budding yeast Saccharomyces cerevisiae. Mol Cell Biol 2004, 24:9152-9164.

Chromatin remodeling complexes: strength in diversity, precision through specialization. B R Cairns, Curr Opin Genet Dev. 15Cairns BR: Chromatin remodeling complexes: strength in diversity, precision through specialization. Curr Opin Genet Dev 2005, 15:185-190.

Histones are first hyperacetylated and then lose contact with the activated PHO5 promoter. H Reinke, W Horz, Mol Cell. 11Reinke H, Horz W: Histones are first hyperacetylated and then lose contact with the activated PHO5 promoter. Mol Cell 2003, 11:1599-1607.

Histone H3 methylation by Set2 directs deacetylation of coding regions by Rpd3S to suppress spurious intragenic transcription. M J Carrozza, B Li, L Florens, T Suganuma, S K Swanson, K K Lee, W J Shia, S Anderson, J Yates, M P Washburn, J L Workman, Cell. 123Carrozza MJ, Li B, Florens L, Suganuma T, Swanson SK, Lee KK, Shia WJ, Anderson S, Yates J, Washburn MP, Workman JL: Histone H3 methylation by Set2 directs deacetylation of coding regions by Rpd3S to suppress spurious intragenic transcription. Cell 2005, 123:581-592.

Chromatin compaction by a polycomb group protein complex. N J Francis, R E Kingston, C L Woodcock, Science. 306Francis NJ, Kingston RE, Woodcock CL: Chromatin compaction by a polycomb group protein complex. Science 2004, 306:1574-1577.

Ring1B compacts chromatin structure and represses gene expression independent of histone ubiquitination. R Eskeland, M Leeb, G R Grimes, C Kress, S Boyle, D Sproul, N Gilbert, Y Fan, A I Skoultchi, A Wutz, W A Bickmore, Mol Cell. 38Eskeland R, Leeb M, Grimes GR, Kress C, Boyle S, Sproul D, Gilbert N, Fan Y, Skoultchi AI, Wutz A, Bickmore WA: Ring1B compacts chromatin structure and represses gene expression independent of histone ubiquitination. Mol Cell 2010, 38:452-464.

Chd1 chromodomain links histone H3 methylation with SAGA-and SLIKdependent acetylation. M G Pray-Grant, J A Daniel, D Schieltz, J R Yates, P A Grant, Nature. 433Pray-Grant MG, Daniel JA, Schieltz D, Yates JR, 3rd, Grant PA: Chd1 chromodomain links histone H3 methylation with SAGA-and SLIK- dependent acetylation. Nature 2005, 433:434-438.

Recognition of trimethylated histone H3 lysine 4 facilitates the recruitment of transcription postinitiation factors and pre-mRNA splicing. R J Sims, S Millhouse, C F Chen, B A Lewis, H Erdjument-Bromage, P Tempst, J L Manley, D Reinberg, Mol Cell. 28Sims RJ, 3rd, Millhouse S, Chen CF, Lewis BA, Erdjument-Bromage H, Tempst P, Manley JL, Reinberg D: Recognition of trimethylated histone H3 lysine 4 facilitates the recruitment of transcription postinitiation factors and pre- mRNA splicing. Mol Cell 2007, 28:665-676.

Assembly of variant histones into chromatin. S Henikoff, K Ahmad, Annu Rev Cell Dev Biol. 21Henikoff S, Ahmad K: Assembly of variant histones into chromatin. Annu Rev Cell Dev Biol 2005, 21:133-153.

Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis. H Tagami, D Ray-Gallet, G Almouzni, Y Nakatani, Cell. 116Tagami H, Ray-Gallet D, Almouzni G, Nakatani Y: Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis. Cell 2004, 116:51-61.

CHD1 motor protein is required for deposition of histone variant H3.3 into chromatin in vivo. A Y Konev, M Tribus, S Y Park, V Podhraski, C Y Lim, A V Emelyanov, E Vershilova, V Pirrotta, J T Kadonaga, A Lusser, D V Fyodorov, Science. 317Konev AY, Tribus M, Park SY, Podhraski V, Lim CY, Emelyanov AV, Vershilova E, Pirrotta V, Kadonaga JT, Lusser A, Fyodorov DV: CHD1 motor protein is required for deposition of histone variant H3.3 into chromatin in vivo. Science 2007, 317:1087-1090.

. A D Goldberg, L A Banaszynski, K M Noh, P W Lewis, S J Elsaesser, S Stadler, Henikoff Genome Deal, Biology, 11Goldberg AD, Banaszynski LA, Noh KM, Lewis PW, Elsaesser SJ, Stadler S, Deal and Henikoff Genome Biology 2010, 11:218 http://genomebiology.com/2010/11/10/218

Distinct factors control histone variant H3.3 localization at specific genomic regions. S Dewell, M Law, X Guo, X Li, D Wen, A Chapgier, R C Dekelver, J C Miller, Y L Lee, E A Boydston, M C Holmes, P D Gregory, J M Greally, S Rafii, C Yang, P J Scambler, D Garrick, R J Gibbons, D R Higgs, I M Cristea, F D Urnov, D Zheng, C D Allis, Cell. 140Dewell S, Law M, Guo X, Li X, Wen D, Chapgier A, DeKelver RC, Miller JC, Lee YL, Boydston EA, Holmes MC, Gregory PD, Greally JM, Rafii S, Yang C, Scambler PJ, Garrick D, Gibbons RJ, Higgs DR, Cristea IM, Urnov FD, Zheng D, Allis CD: Distinct factors control histone variant H3.3 localization at specific genomic regions. Cell 2010, 140:678-691.

The death-associated protein DAXX is a novel histone chaperone involved in the replicationindependent deposition of H3.3. P Drane, K Ouararhni, A Depaux, M Shuaib, A Hamiche, Genes Dev. 24Drane P, Ouararhni K, Depaux A, Shuaib M, Hamiche A: The death-associated protein DAXX is a novel histone chaperone involved in the replication- independent deposition of H3.3. Genes Dev 2010, 24:1253-1265.

Patterning chromatin: form and function for H2A.Z variant nucleosomes. R M Raisner, H D Madhani, Curr Opin Genet Dev. 16Raisner RM, Madhani HD: Patterning chromatin: form and function for H2A.Z variant nucleosomes. Curr Opin Genet Dev 2006, 16:119-124.

Genome-scale profiling of histone H3.3 replacement patterns. Y Mito, J G Henikoff, S Henikoff, Nat Genet. 37Mito Y, Henikoff JG, Henikoff S: Genome-scale profiling of histone H3.3 replacement patterns. Nat Genet 2005, 37:1090-1097.

Nucleosome stability mediated by histone variants H3.3 and H2A. C Jin, G Felsenfeld, Z. Genes Dev. 21Jin C, Felsenfeld G: Nucleosome stability mediated by histone variants H3.3 and H2A.Z. Genes Dev 2007, 21:1519-1529.

A Thakar, P Gupta, T Ishibashi, R Finn, B Silva-Moreno, S Uchiyama, K Fukui, M Tomschik, J Ausio, J Zlatanova, H2A.Z and H3.3 histone variants affect nucleosome structure: biochemical and biophysical studies. 48Thakar A, Gupta P, Ishibashi T, Finn R, Silva-Moreno B, Uchiyama S, Fukui K, Tomschik M, Ausio J, Zlatanova J: H2A.Z and H3.3 histone variants affect nucleosome structure: biochemical and biophysical studies. Biochemistry 2009, 48:10852-10857.

H3.3/H2A.Z double variant-containing nucleosomes mark 'nucleosome-free regions' of active promoters and other regulatory regions. C Jin, C Zang, G Wei, K Cui, W Peng, K Zhao, G Felsenfeld, Nat Genet. 41Jin C, Zang C, Wei G, Cui K, Peng W, Zhao K, Felsenfeld G: H3.3/H2A.Z double variant-containing nucleosomes mark 'nucleosome-free regions' of active promoters and other regulatory regions. Nat Genet 2009, 41:941-945.

Nucleosome destabilization in the epigenetic regulation of gene expression. S Henikoff, Nat Rev Genet. 9Henikoff S: Nucleosome destabilization in the epigenetic regulation of gene expression. Nat Rev Genet 2008, 9:15-26.

Visualizing chromatin dynamics in intact cells. T C Voss, G L Hager, Biochim Biophys Acta. 1783Voss TC, Hager GL: Visualizing chromatin dynamics in intact cells. Biochim Biophys Acta 2008, 1783:2044-2051.

Global nature of dynamic protein-chromatin interactions in vivo: three-dimensional genome scanning and dynamic interaction networks of chromatin proteins. R D Phair, P Scaffidi, C Elbi, J Vecerová, A Dey, K Ozato, D T Brown, G Hager, M Bustin, T Misteli, Mol Cell Biol. 24Phair RD, Scaffidi P, Elbi C, Vecerová J, Dey A, Ozato K, Brown DT, Hager G, Bustin M, Misteli T: Global nature of dynamic protein-chromatin interactions in vivo: three-dimensional genome scanning and dynamic interaction networks of chromatin proteins. Mol Cell Biol 2004, 24:6393-6402.

Kinetics of core histones in living human cells: little exchange of H3 and H4 and some rapid exchange of H2B. H Kimura, P R Cook, J Cell Biol. 153Kimura H, Cook PR: Kinetics of core histones in living human cells: little exchange of H3 and H4 and some rapid exchange of H2B. J Cell Biol 2001, 153:1341-1353.

Replication-independent core histone dynamics at transcriptionally active loci in vivo. C Thiriet, J J Hayes, Genes Dev. 19Thiriet C, Hayes JJ: Replication-independent core histone dynamics at transcriptionally active loci in vivo. Genes Dev 2005, 19:677-682.

Pervasive combinatorial modification of histone H3 in human cells. B A Garcia, J J Pesavento, C A Mizzen, N L Kelleher, Nat Methods. 4Garcia BA, Pesavento JJ, Mizzen CA, Kelleher NL: Pervasive combinatorial modification of histone H3 in human cells. Nat Methods 2007, 4:487-489.

High throughput characterization of combinatorial histone codes. N L Young, P A Dimaggio, M D Plazas-Mayorca, R C Baliban, C A Floudas, B A Garcia, Mol Cell Proteomics. 8Young NL, DiMaggio PA, Plazas-Mayorca MD, Baliban RC, Floudas CA, Garcia BA: High throughput characterization of combinatorial histone codes. Mol Cell Proteomics 2009, 8:2266-2284.

Establishment of histone modifications after chromatin assembly. A N Scharf, T K Barth, A Imhof, Nucleic Acids Res. 37Scharf AN, Barth TK, Imhof A: Establishment of histone modifications after chromatin assembly. Nucleic Acids Res 2009, 37:5032-5040.

In vivo residuespecific histone methylation dynamics. B M Zee, R S Levin, B Xu, G Leroy, N S Wingreen, B A Garcia, J Biol Chem. 285Zee BM, Levin RS, Xu B, LeRoy G, Wingreen NS, Garcia BA: In vivo residue- specific histone methylation dynamics. J Biol Chem 2010, 285:3341-3350.

Kinetics of reestablishing H3 K79 methylation marks in global human chromatin. S M Sweet, M Li, P M Thomas, K R Durbin, N L Kelleher, 10.1074/jbc.M110.145094J Biol Chem. Sweet SM, Li M, Thomas PM, Durbin KR, Kelleher NL: Kinetics of re- establishing H3 K79 methylation marks in global human chromatin. J Biol Chem 2010, doi:10.1074/jbc.M110.145094.

Dynamics of replication-independent histone turnover in budding yeast. M F Dion, T Kaplan, M Kim, S Buratowski, N Friedman, O J Rando, Science. 315Dion MF, Kaplan T, Kim M, Buratowski S, Friedman N, Rando OJ: Dynamics of replication-independent histone turnover in budding yeast. Science 2007, 315:1405-1408.

Genome-wide replication-independent histone H3 exchange occurs predominantly at promoters and implicates H3 K56 acetylation and Asf1. A Rufiange, P-E Jacques, W Bhat, F Robert, A Nourani, Mol Cell. 27Rufiange A, Jacques P-E, Bhat W, Robert F, Nourani A: Genome-wide replication-independent histone H3 exchange occurs predominantly at promoters and implicates H3 K56 acetylation and Asf1. Mol Cell 2007, 27:393-405.

Continuous histone H2B and transcription-dependent histone H3 exchange in yeast cells outside of replication. A Jamai, R M Imoberdorf, M Strubin, Mol Cell. 25Jamai A, Imoberdorf RM, Strubin M: Continuous histone H2B and transcription-dependent histone H3 exchange in yeast cells outside of replication. Mol Cell 2007, 25:345-355.

Recombination-induced tag exchange to track old and new proteins. K F Verzijlbergen, V Menendez-Benito, T Van Welsem, S J Van Deventer, D L Lindstrom, H Ovaa, J Neefjes, D E Gottschling, F Van Leeuwen, Proc Natl Acad Sci. 107Verzijlbergen KF, Menendez-Benito V, van Welsem T, van Deventer SJ, Lindstrom DL, Ovaa H, Neefjes J, Gottschling DE, van Leeuwen F: Recombination-induced tag exchange to track old and new proteins. Proc Natl Acad Sci USA 2010, 107:64-68.

Genome-wide kinetics of nucleosome turnover determined by metabolic labeling of histones. R B Deal, J G Henikoff, S Henikoff, Science. 328Deal RB, Henikoff JG, Henikoff S: Genome-wide kinetics of nucleosome turnover determined by metabolic labeling of histones. Science 2010, 328:1161-1164.

The origin recognition complex protein family. B P Duncker, I N Chesnokov, B J Mcconkey, Genome Biol. 10214Duncker BP, Chesnokov IN, McConkey BJ: The origin recognition complex protein family. Genome Biol 2009, 10:214.

Drosophila ORC localizes to open chromatin and marks sites of cohesin complex loading. H K Macalpine, R Gordân, S K Powell, A J Hartemink, D M Macalpine, Genome Res. 20MacAlpine HK, Gordân R, Powell SK, Hartemink AJ, MacAlpine DM: Drosophila ORC localizes to open chromatin and marks sites of cohesin complex loading. Genome Res 2010, 20:201-211.

In search of the holy replicator. D M Gilbert, Nat Rev Mol Cell Biol. 5Gilbert DM: In search of the holy replicator. Nat Rev Mol Cell Biol 2004, 5:848-855.

Genome regulation by polycomb and trithorax proteins. B Schuettengruber, D Chourrout, M Vervoort, B Leblanc, G Cavalli, Cell. 128Schuettengruber B, Chourrout D, Vervoort M, Leblanc B, Cavalli G: Genome regulation by polycomb and trithorax proteins. Cell 2007, 128:735-745.

Translating the histone code. T Jenuwein, C D Allis, Science. 293Jenuwein T, Allis CD: Translating the histone code. Science 2001, 293:1074-1080.

Histone replacement marks the boundaries of cis-regulatory domains. Y Mito, J G Henikoff, S Henikoff, Science. 315Mito Y, Henikoff JG, Henikoff S: Histone replacement marks the boundaries of cis-regulatory domains. Science 2007, 315:1408-1411.

Mitotic bookmarking of formerly active genes: keeping epigenetic memories from fading. K D Sarge, O K Park-Sarge, Cell Cycle. 8Sarge KD, Park-Sarge OK: Mitotic bookmarking of formerly active genes: keeping epigenetic memories from fading. Cell Cycle 2009, 8:818-823.

Modulation of a transcription factor counteracts heterochromatic gene silencing in Drosophila. K Ahmad, S Henikoff, 10.1186/gb-2010-11-10-218Cell. 104Ahmad K, Henikoff S: Modulation of a transcription factor counteracts heterochromatic gene silencing in Drosophila. Cell 2001, 104:839-847. doi:10.1186/gb-2010-11-10-218

Cite this article as: Deal RB, Henikoff S: Capturing the dynamic epigenome. Cite this article as: Deal RB, Henikoff S: Capturing the dynamic epigenome.

. Genome Biology. 11218Genome Biology 2010, 11:218.


Major Radiodiagnostic Imaging in Pregnancy and the Risk of Childhood Malignancy: A Population-Based Cohort Study in Ontario

Joel G Ray
Department of Medicine
St. Michael's Hospital
TorontoOntarioCanada

Department of Obstetrics and Gynecology
St. Michael's Hospital
TorontoOntarioCanada

Department of Health Policy Management and Evaluation
University of Toronto
TorontoOntarioCanada

Institute for Clinical Evaluative Sciences
University of Toronto
TorontoOntarioCanada

Michael J Schull
Department of Health Policy Management and Evaluation
University of Toronto
TorontoOntarioCanada

Department of Medicine (Division of Emergency Medicine)
Sunnybrook Sciences Centre
Institute for Clinical Evaluative Sciences
University of Toronto
TorontoOntarioCanada

Marcelo L Urquia
Dalla Lana School of Public Health
University of Toronto
TorontoOntarioCanada

John J You
Institute for Clinical Evaluative Sciences
University of Toronto
TorontoOntarioCanada

Department of Medicine
McMaster University
HamiltonOntarioCanada

Department of Clinical Epidemiology & Biostatistics
McMaster University
HamiltonOntarioCanada

Astrid Guttmann
Department of Health Policy Management and Evaluation
University of Toronto
TorontoOntarioCanada

Institute for Clinical Evaluative Sciences
University of Toronto
TorontoOntarioCanada

Department of Paediatrics
The Hospital for Sick Children
TorontoOntarioCanada

Marian J Vermeulen
Institute for Clinical Evaluative Sciences
University of Toronto
TorontoOntarioCanada

Major Radiodiagnostic Imaging in Pregnancy and the Risk of Childhood Malignancy: A Population-Based Cohort Study in Ontario
10.1371/journal.pmed.1000337
Background: The association between fetal exposure to major radiodiagnostic testing in pregnancy-computed tomography (CT) and radionuclide imaging-and the risk of childhood cancer is not established.Methods and Findings:We completed a population-based study of 1.8 million maternal-child pairs in the province of Ontario, from 1991 to 2008. We used Ontario's universal health care-linked administrative databases to identify all term obstetrical deliveries and newborn records, inpatient and outpatient major radiodiagnostic services, as well as all children with a malignancy after birth. There were 5,590 mothers exposed to major radiodiagnostic testing in pregnancy (3.0 per 1,000) and 1,829,927 mothers not exposed. The rate of radiodiagnostic testing increased from 1.1 to 6.3 per 1,000 pregnancies over the study period; about 73% of tests were CT scans. After a median duration of follow-up of 8.9 years, four childhood cancers arose in the exposed group (1.13 per 10,000 person-years) and 2,539 cancers in the unexposed group (1.56 per 10,000 person-years), a crude hazard ratio of 0.69 (95% confidence interval 0.26-1.82). After adjusting for maternal age, income quintile, urban status, and maternal cancer, as well as infant sex, chromosomal or congenital anomalies, and major radiodiagnostic test exposure after birth, the risk was essentially unchanged (hazard ratio 0.68, 95% confidence interval 0.25-1.80).Conclusions: Although major radiodiagnostic testing is now performed in about 1 in 160 pregnancies in Ontario, the absolute annual risk of childhood malignancy following exposure in utero remains about 1 in 10,000. Since the upper confidence limit of the relative risk of malignancy may be as high as 1.8 times that of an unexposed pregnancy, we cannot exclude the possibility that fetal exposure to CT or radionuclide imaging is carcinogenic.

Introduction

Cancer remains the second leading cause of hospitalization and death among children aged 14 y and younger in industrialized nations [1][2][3]. Fewer than 10% of childhood cancers are attributable to a genetic predisposition [4]. With the exception of a few known risk factors, including exposure to ionizing radiation in utero and after birth [5,6], the etiology of most childhood cancer remains unknown.

There has been a substantial increase in the utilization of computed tomography (CT) and radionuclide (nuclear medicine) radiodiagnostic procedures, making them a common source of radiation to patients [7][8][9]. Since these tests are often ordered under emergency circumstances [10], inadvertent exposure in early pregnancy may occur, since half of all pregnancies are unplanned [11].

There may be an association between exposure to ionizing radiation in pregnancy and childhood cancer, yet, the supporting data are somewhat conflicting. For example, studies of cancer incidence following intrauterine exposure to the atomic bomb are inconsistent [12][13][14], as have some [15], but not all [16][17][18] studies examining maternal exposure to plain radiographs in pregnancy. Less is known about the risk related to major radiodiagnostic tests, such as CT and radionuclide imaging, both of which expose the fetus to considerably higher doses of radiation than plain radiographs administered at the same anatomical level [19,20].

We studied women exposed to major radiodiagnostic tests in pregnancy in an effort to provide clinicians and mothers with better estimates of the risk of pediatric malignancy in their offspring.

Methods

Study Design

We completed a retrospective population-based cohort study of women who delivered a liveborn infant in Ontario between April 1, 1992 andMarch 31, 2008. The study was approved by the Ethics Review Board of Sunnybrook Health Sciences Centre.

Participants

We included singleton and multifetal pregnancies, and for the latter we included the first infant birth record to avoid multiple counts per pregnancy. We included maternal-infant pairs comprising term liveborn infants who survived more than 30 d after date of birth. For three reasons, we restricted the sample to term infants $37 wk gestation, and who weighed at least 2,500 g at birth: First, preterm infants are commonly exposed to radiodiagnostic imaging procedures in hospital [21,22], which could potentially contaminate the studied effect of prenatal exposure. Second, since term infants represent 94% of all livebirths [23], our findings would remain applicable to most pregnancies. We defined term infants by actual gestational age from 2002 onward, the time in which it was captured in our dataset; before 2002, term status was based on the absence of any diagnostic codes for prematurity according to the International Classification of Diseases, 9th (ICD-9) and 10th (ICD-10CA) revisions (Table S1). Thus, limiting our cohort to term infants enabled us to better estimate the gestational age of fetal exposure to a major radiodiagnostic test prior to the year 2002.

Exposure and Outcome

The study exposure was CT or radionuclide imaging in an index pregnancy, up to 2 d before the day of delivery (to maximize the likelihood that the fetus was in utero). Prior to the year 2002, gestational age at birth was not recorded in our databases, so the estimated gestational age at exposure was calculated by subtracting 37 wk from the delivery date. This approach has a reported sensitivity of 90% and specificity of 99% [24]. From 2002 onward, we used the recorded gestational age at delivery to determine the timing of exposure to a radiodiagnostic test.

The main study outcome was a confirmed diagnosis of any pediatric malignancy in a child born in the index pregnancy at any time 30 d or more after his/her birth. This 30-d interval was used to exclude infants who died from major morbidity, such as neonatal sepsis or severe anomalies.

Database Sources

We used anonymized databases for the entire province of Ontario, where universal health care, including prenatal care and radiodiagnostic testing, is available to all residents. Databases were linked using unique encrypted identifiers, which enabled us to link radiation exposure in an index pregnancy to a diagnosed malignancy in a child.

Individual obstetrical deliveries were identified in the Canadian Institute for Health Information Discharge Abstract Database (DAD). The DAD contains age and sex, dates of admission, and up to 16 diagnoses coded by ICD-9 up to March 31, 2002, and up to 25 diagnoses coded by ICD-10-CA thereafter. Up to 2002, inpatient admission records for mothers and newborns are probabilistically linked using delivery/birth dates in the same hospital, same residential postal codes, and diagnostic information to create each mother-child pair. This approach leads to successful matching in 95% of cases. Validation studies of similar maternalnewborn linkages have shown that this approach yields high degrees of successful matching, with a sensitivity of about 95% and a specificity of nearly 90% [25][26][27]. In a Canadian study linking newborn birth records and infant death records, over 99% of records were successfully linked [28]. Starting in the fiscal year 2002/2003, a common identifying number between a delivering mother's chart number and her newborn's chart number permitted a deterministic linkage between the two from 2002 and onward. The DAD was also used to define study covariates (Table S1).

For the study exposure-a major radiodiagnostic test performed on the mother up to one day before her delivery date-we used the Ontario Health Insurance Plan (OHIP) database, which captures all billing information for physician services, including inpatient and outpatient major radiodiagnostic services [29,30]. In an audit of a representative sample of 11,824 outpatient CT scans and 11,867 outpatient magnetic resonance imaging (MRI) scans identified from 29 randomly selected hospitals across Ontario, 95% of the procedures studied in the chart audit had a corresponding claim in the OHIP database, indicating that these administrative databases have high accuracy in identifying imaging procedures [30]. In the current study, major radiodiagnostic testing included all forms of CT and radionuclide imaging (Table S2). Because the OHIP database does not capture all billings for inpatient radionuclide imaging before 2006, the DAD was used to identify inpatient radionuclide tests performed throughout the study period (Table S2).

For the study outcome-pediatric malignancy-each child record was linked to the Ontario Cancer Registry (OCR), a computerized database of information on all Ontario residents who have been newly diagnosed with cancer [31]. All new cases of cancer, except for nonmelanoma skin cancer, are registered, and about 80% have the tissue pathological diagnosis recorded within the OCR. Validation studies have shown the OCR to be effective in ascertaining cancer cases in the province, with a sensitivity of 98% [32]. Close to 400,000 records are submitted to the OCR annually, and coded using ICD-9 diagnostic codes (Table S1). Incident cases of pediatric malignancy were captured according to a date of diagnosis between May 1, 1991 (a minimum of 30 d after the earliest birth in the study cohort) and March 31, 2009 (a maximum of 18 y after the earliest birth in the cohort).

Mortality data were retrieved from the Registered Persons Database (RPDB), which contains demographic information for all OHIP-eligible individuals. Income quintile and rurality were defined using Statistics Canada census data. The OHIP database was also used to identify outpatient claims for prenatal ultrasonography at any point in pregnancy (Table S2) [33].

Statistical Analysis

We measured the absolute number of radiodiagnostic tests performed in each livebirth pregnancy in a given year and calculated the annual test rate per 1,000 livebirths, from 1992 to 2008. Changes over time in the rates for radionuclide tests, CT, and both were analyzed using a Cochran-Armitage test for trend.

Time-to-event analyses were conducted using Cox proportional hazards regression models, to derive a hazard ratio (HR) and 95% confidence interval (CI) for pediatric malignancy among children born to mothers exposed versus not exposed to major radiodiagnostic imaging in pregnancy.

The period of observation for each child started 31 d after birth, to ensure that he or she survived long enough to develop, or to be diagnosed with, a malignancy. A child was censored (i.e., determined to have not had a study outcome event) at the point in time in which he or she either reached the end of the period of March 31, 2009 without having experienced a study outcome event or died during the period of study. While emigration from the province could not be ascertained, the annual rate is less than 1% (http://www.statcan.gc.ca/pub/75-001-x/2008110/t/ 10711/5800473-eng.htm); such persons were classified as being event-free up to March 31, 2009.

The HR was adjusted for maternal age at delivery (in years), income quintile, urban status, and maternal cancer diagnosed in the index pregnancy or up to 6 mo thereafter, as well as infant sex, a chromosomal or congenital anomaly documented at the time of birth or up to 365 d thereafter (present or absent), and exposure to CT or radionuclide imaging (as present or absent) starting 31 d after birth and up to 365 d before either the date that he/she experienced a study outcome event or the date that he/she was censored. The latter considered postnatal exposure to the same ionizing radiation sources as in pregnancy. Information about maternal parity, prior pregnancy loss, smoking, and medication use were not available.

All p-values were two-sided, at a significance level of 0.05. All statistical analyses were performed using SAS for UNIX, Version 9.1 (SAS Institute).

Results

There were 2,018,924 maternal-child pairs initially generated, of which 183,442 (9.1%) were excluded for the following reasons: newborn weight ,2,500 g or .6,000 g (n = 113,718); preterm delivery ,37 wk gestation (n = 50,881); multiple births subsequent to the first birth (n = 10,116); maternal age ,16 y or .50 y (n = 4,091); nonresident of Ontario (n = 2,310); infant death #30 d after birth (n = 1,514); stillbirth (n = 780); extreme post-term birth .43 wk gestation (n = 29); and incorrect coding of birth date (n = 3). Among those excluded, the rate of stillbirth was not significantly different among mothers exposed (0.65%) and not exposed (0.84%) to major radiodiagnostic testing in pregnancy. Among all mothers exposed to major radiodiagnostic testing in pregnancy, the characteristics of those who were included and excluded are listed in Table S3.

The final cohort comprised 1,835,517 maternal-child pairs. The demographic characteristics of the 5,590 mothers exposed to major radiodiagnostic testing in pregnancy were generally similar to the unexposed cohort of 1,829,927 women (Table 1). However, the rate of diagnosed cancer during or soon after pregnancy was higher in the exposed mothers (0.63% versus 0.050%), as was the rate of early prenatal ultrasonography (33.0% versus 19.0%).

Among the liveborn infants of mothers exposed and not exposed to a major radiodiagnostic test, just under half were female, and the mean (standard deviation [SD]) gestational ages at birth were 39.1 (1.2) and 39.3 (1.1) wk, respectively ( Table 1). The rates of chromosomal (0.13% versus 0.11%) and congenital (3.9% versus 3.7%) anomalies were about the same in both groups, as were the rates of CT or radionuclide imaging after birth (4.4% versus 4.4%).

The overall rate of exposure to major radiodiagnostic testing in pregnancy was 3.0 per 1,000, which increased significantly over time, peaking at 6.3 per 1,000 livebirth pregnancies in 2008 (trend p,0.001) (Figure 1). Exposure to major radiodiagnostic testing in pregnancy occurred at an estimated mean (SD) gestational age of 15.7 (12.8) wk, and about half were completed before 14 wk gestation ( Table 1). 15% of women had two or more tests performed in pregnancy. About 73% of all major radiodiagnostic tests were CT scans, of which 68% were of an extremity or head, nearly 10% of the thorax, and 22.6% of the abdomen, spine, or pelvis (Table 1).

There were 35,487 and 16,326,410 persons-years of follow-up in the exposed and unexposed groups, respectively. Among the entire cohort of 1,835,517 children, the median (interquartile range [IQR]) duration of follow-up was 8.91 (4.83-13.00) y. A total of 1,015,789 (55.3%) children were followed to age 8 y, 797,940 (43.5%) to age 10 y, 573,445 (31.2%) to age 12 y, and 341,432 (18.6%) to age 14 y.

A total of four childhood cancers occurred in the exposed group (1.13 per 10,000 person-years) and 2,539 cancers in the unexposed group (1.56 per 10,000 person-years), corresponding to a crude HR of 0.69 (95% CI 0.26-1.82) (Figure 2; Table 2). After adjusting for potential confounders, the risk was not significantly higher among exposed versus unexposed maternal-child pairs (HR 0.68, 95% CI 0.25-1.80). Upon adding into the sample previously excluded infants at extremes of gestational age and birthweight, the crude (0.71, 95% CI 0.29-1.70) and adjusted (0.69, 95% CI 0.29-1.66) HR remained unchanged. We could not specify the types of cancers arising in the exposed group because of a strict privacy policy at our institution about the reporting of specific information for cell sizes under the number 5.

Discussion

About 1 in 160 term pregnancies now appear to be exposed to a major radiodiagnostic test. The offspring of women exposed to major radiodiagnostic testing in pregnancy do not appear to be at higher risk of childhood malignancy than the children of unexposed mothers. Moreover, the overall prevalence of pediatric malignancy following exposure to CT or radionuclide imaging in pregnancy was under 0.07%, an incidence rate of 1.13 per 10,000 person-years.

Study Strengths and Limitations

Among those exposed to major radiodiagnostic testing, totaling nearly 5,600 pregnancies and 35,000 persons-years of follow-up, just four cases of childhood malignancy were identified. The incidence rate of 1.56 cancers per 10,000 person-years, based on Table 1. Characteristics of mothers and their infants who were and who were not exposed to a major radiodiagnostic testing in pregnancy.   2,539 events arising among 1,829,995 unexposed pregnancies, is highly concordant with population studies from Canada, the United States, Ireland, and Europe [1][2][3]. Post hoc, considering the number of participants enrolled in the current study, at a conventional p-value of 0.05, we had just under 20% statistical power to detect the observed difference of 0.40 per 10,000 personyears in the incidence rate of childhood malignancy between exposed and unexposed mothers. To achieve a power of 80%, approximately 60 and 19,613 cancers would be needed in the exposed and unexposed groups, respectively-about eight times higher than the current number of events. That so few cases of childhood malignancy were identified among the exposed is a testament to the difficulty of conducting research in this area, and why previous data may have been lacking. Nonetheless, the wide confidence limits of our relative risk estimates do not exclude the possibility that fetal exposure to CT or radionuclide imaging in pregnancy is carcinogenic, ranging from as low as one-quarter, to as high as 1.8 times, that of an unexposed pregnancy. We did not include women who had a radiodiagnostic test in a pregnancy that ended in a spontaneous or therapeutic termination before 20-wk gestation. However, this study's focus was on childhood malignancy after 30 d of life, rather than on pregnancy loss, and it is unlikely that including that group of women would have systematically biased the association between the study exposure and outcome. Moreover, the rates of stillbirth after 20 wk among excluded women exposed (0.65%) and not exposed (0.84%) to major radiodiagnostic testing in pregnancy did not differ significantly. By limiting our sample to term deliveries between 37 and 43 wk gestation and restricting the period of exposure up to 2 d before a woman's date of delivery, we improved the accuracy of our determination of the timing of exposure to radiodiagnostic testing. At the same time, our findings may not apply to preterm infants, who both have higher rates of congenital and chromosomal anomalies, as well as major radiodiagnostic testing soon after birth (Table S3) [21,22].

We did not have on record whether a woman's pregnancy was known at the time of exposure, or if she received lead apron shielding of the abdominal-pelvic area. Assuming that shielding occurred in most cases, as is routine practice, then this may partly explain the low rate of childhood malignancy in the exposed group. Moreover, two-thirds of CT scans were of an extremity or head, with the remaining sites in closer proximity to the fetus, but only 23% were of the abdomen, spine, or pelvis. Hence, much of our CT data reflects ''low-risk'' scans, in terms of fetal radiation exposure.

We also do not possess information about the generation(s) of CT scans that were used, or the measured dose of ionizing radiation that a woman and her fetus were exposed to. The cited average effective maternal radiation dose of CT of the head or neck is about 2 millisievert (mSv); for a CT of the chest, spine, abdomen, or pelvis it ranges from 6 to 8 mSv [9,34]; and for a ventilation-perfusion lung scan it is about 2.5 mSv [34]. While these doses are between 10 to 100 times more than that of an equivalent single plain X-ray [34], the effective radiation dose to the fetus remains lower than that to the mother [18,35]. The rate of major radiodiagnostic testing in pregnancy in our study is certainly lower than when plain X-rays were performed in pregnancy for maternal pelvimetry [36]. In the era of the current study, pelvimetry was not routine, but we would have missed plain X-rays performed for other reasons.

Mothers, as well as clinicians of all types-internists, surgeons, radiologists, obstetricians and midwives, pediatricians, and family and emergency medicine physicians-need data to better estimate the risk of exposure to radiodiagnostic testing in pregnancy. Our findings, though statistically underpowered, inform this process in a manner that has been lacking to date, especially using population-based data over a long period of follow-up, and within a health care system that provides universal coverage of radiological testing and health care.

Clinical Implications

Our findings can help inform clinicians and mothers about the risk of childhood malignancy following major radiodiagnostic testing in pregnancy. First, the absolute risk appears to be low (about 0.07% or 1 per 10,000 person-years), while the relative risk is not statistically significantly higher than unexposed controls, notwithstanding the wide confidence limits. However potentially small that risk may be, beta hCG testing should continue to be done in all potentially pregnant women before undergoing major radiodiagnostic testing, and lead apron shielding used in all women of reproductive age, whether or not pregnant [6,37]. Furthermore, nonradiation-emitting imaging (e.g., MRI and ultrasonography) should be considered first, when appropriate.

Some pregnant women will nevertheless be faced with the decision to undergo CT or nuclear imaging because the test is clinically warranted. Unfortunately, some health care providers may be unwilling to counsel a woman about the related fetal risks or may provide misinformation [37,38]. This issue seems more pressing than ever, given that we and others [9] have noted that CT testing is on the rise, and has become the mainstay for the investigation of pulmonary embolism [39], stroke [40], appendicitis [41], and other common conditions encountered in emergency situations [10]. Both physiological and anatomical changes in pregnancy may obscure the clinical diagnosis of pulmonary embolism [42], for example, such that CT or radionuclide testing may be especially warranted. Delaying the diagnosis of such conditions may postpone therapy, in turn, jeopardizing both mother and child [43]. Table 2. Risk of childhood malignancy in the offspring of women exposed to major radiodiagnostic testing in pregnancy compared to unexposed women. When indicated, major radiodiagnostic testing in pregnancy should be carried out, along with brief counseling [37]. The latter will hopefully lessen the level of anxiety experienced by an expectant mother (and her family), not only at the time of illness, but after her child is born.  Table S3 Characteristics of mothers and their infants who were exposed to a major radiodiagnostic testing in pregnancy and who were included or excluded from the study.

Supporting Information

Editors' Summary

Background In industrialized countries, childhood cancer (any form of cancer in a child aged 14 years or under) remains a major cause of death. With the exception of a few known risk factors, such as acquired genetic predisposition to cancer, which accounts for about 10% of all childhood cancers, the etiology of most childhood cancer remains unknown. There is thought to be an association between exposure to ionizing radiation in pregnancy and the subsequent risk of development of cancer in the exposed mother's child, but the evidence base to support this association is conflicting. For example, studies examining maternal exposure to plain radiographs in pregnancy and subsequent childhood cancer are inconsistent. Furthermore, although their use has dramatically increased over the past two decades, little is known about the cancer risk related to certain types of radiodiagnostic tests, such as CT and radionuclide imaging, both of which expose the fetus to considerably higher doses of radiation than plain radiographs administered at the same anatomical level.

Why Was This Study Done? Many women could be exposed to major radiodiagnostic tests, such as those used in emergency situations, before they are aware that they are pregnant, as almost 50% of pregnancies are unplanned. This situation means that it is important to determine the subsequent cancer risk to any child exposed to maternal radiodiagnostic tests before birth.

What Did the Researchers Do and Find? The researchers conducted a retrospective population-based cohort study of women who delivered a live infant in Ontario, Canada between April 1, 1992 and March 31, 2008. The basis of the research was an anonymized database for the whole province of Ontario, where universal health care, including prenatal care and radiodiagnostic testing, is available to all residents. Database characteristics allowed the researchers to link maternal radiation exposure (a major radiodiagnostic test performed on the mother up to one day before her delivery date) in a specific (index) pregnancy to a subsequent malignancy in the child. After birth, maternal-infant pairs were only followed up if the infant was delivered at term, weighed 2,500 g or more, and survived for at least 30 days. The researchers were able to follow up 1,835,517 maternalchild pairs. The overall rate of exposure to major radiodiagnostic testing in pregnancy was 3.0 per 1,000 and occurred at an estimated mean gestational age of 15.7 weeks. A total of four childhood cancers occurred in the exposed group and 2,539 cancers in the unexposed group corresponding to a crude hazard ratio of 0.69, which did not significantly change after adjustments were made for potential confounding factors, such as maternal age, sex, and the presence of any chromosomal or congenital anomalies in the infant. The overall prevalence of childhood cancer following exposure to CT or radionuclide imaging in pregnancy is under 0.07%, giving an incidence rate of 1.13 per 10,000 person-years.

What Do These Findings Mean? These findings can help inform clinicians and mothers about the risk of childhood malignancy following major radiodiagnostic testing in pregnancy. The absolute risk appears to be low, while the relative risk is not materially higher than that of unexposed controls. However, as the upper confidence limit of the relative risk of malignancy may be a maximum of 1.8 times that of an unexposed pregnancy, the possibility that fetal exposure to CT or radionuclide imaging is carcinogenic cannot be excluded. Because this finding means that a very slight risk may exist, beta hCG testing should continue to be done in all potentially pregnant women before undergoing major radiodiagnostic testing, and lead apron shielding used in all women of reproductive age, whether or not known to be pregnant. In addition, nonradiation-emitting imaging, such as MRI and ultrasonography, should be considered first, when clinically appropriate. However, some pregnant women will still be faced with the decision to undergo CT or nuclear imaging because the test is clinically warranted. The findings of this study suggest that when clinically indicated, major radiodiagnostic testing in pregnancy should be performed, along with brief counseling to help lessen the anxiety experienced by an expectant mother before and after the birth of her child.

Figure 1 .
1Annual rate of major radiodiagnostic testing in pregnancy in Ontario over time. Data are presented for radionuclide testing (lower solid line), CT scan (upper dashed line), and both (solid bars with total number per year). doi:10.1371/journal.pmed.1000337.g001

Figure 2 .
2Risk of childhood cancer in the offspring of women exposed (lower solid) and not exposed (upper dashed) to a major radiodiagnostic test in pregnancy. doi:10.1371/journal.pmed.1000337.g002

Additional
Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10. 1371/journal.pmed.1000337. N For information for patients and caregivers on radiodiagnostic testing, see The Royal College of Radiologists N The National Cancer Institute provides information about childhood cancer N CureSearch for Children's Cancer provides additional information about research into childhood cancer

Table S1
S1Diagnostic and procedural codes used to identify the cohort, comorbidity, and outcome features.Found at: doi:10.1371/journal.pmed.1000337.s001 (0.03 MB
DOC)

Table S2 Codes used to identify inpatient or outpatient major
radiodiagnostic imaging (for the study exposure) as well as
outpatient prenatal ultrasonography.
Found at: doi:10.1371/journal.pmed.1000337.s002 (0.04 MB
DOC)

Found at: doi:10.1371/journal.pmed.1000337.s003 (0.04 MB DOC)
PLoS Medicine | www.plosmedicine.org
September 2010 | Volume 7 | Issue 9 | e1000337
AcknowledgmentsChaim Bell and Richard Glazer provided some helpful suggestions about the manuscript.
Leading Causes of Death and Hospitalization in Canada. Ottawa: Public Health Agency of Canada. Leading Causes of Death and Hospitalization in Canada. Ottawa: Public Health Agency of Canada, Available: http://www.phac-aspc.gc.ca/publicat/lcd- pcd97/pdf/lcd-pcd-t1-eng.pdf. Accessed 10 July 2010.

Trends in childhood cancer incidence in the. A M Linabery, J A Ross, Cancer. 112Linabery AM, Ross JA (2008) Trends in childhood cancer incidence in the U.S. (1992-2004). Cancer 112: 416-432.

Childhood cancer in Ireland: a population-based study. M Stack, P M Walsh, H Comber, C A Ryan, O 'lorcain, P , Arch Dis Child. 92Stack M, Walsh PM, Comber H, Ryan CA, O'Lorcain P (2007) Childhood cancer in Ireland: a population-based study. Arch Dis Child 92: 890-897.

Inherited susceptibility for pediatric cancer. S E Plon, K Nathanson, Cancer J. 11Plon SE, Nathanson K (2005) Inherited susceptibility for pediatric cancer. Cancer J 11: 255-267.

Risk of childhood cancer from fetal irradiation. R Doll, R Wakeford, Br J Radiol. 70Doll R, Wakeford R (1997) Risk of childhood cancer from fetal irradiation. Br J Radiol 70: 130-139.

Imaging the pregnant patient for nonobstetric conditions: algorithms and radiation dose considerations. S J Patel, D L Reede, D S Katz, R Subramaniam, J K Amorosa, Radiographics. 27Patel SJ, Reede DL, Katz DS, Subramaniam R, Amorosa JK (2007) Imaging the pregnant patient for nonobstetric conditions: algorithms and radiation dose considerations. Radiographics 27: 1705-1722.

Recent trends in utilization rates of abdominal imaging: the relative roles of radiologists and nonradiologist physicians. D C Levin, V M Rao, L Parker, A J Frangos, J H Sunshine, J Am Coll Radiol. 5Levin DC, Rao VM, Parker L, Frangos AJ, Sunshine JH (2008) Recent trends in utilization rates of abdominal imaging: the relative roles of radiologists and nonradiologist physicians. J Am Coll Radiol 5: 744-747.

Trends in the utilization of medical procedures that use ionizing radiation. M Bhargavan, Health Phys. 95Bhargavan M (2008) Trends in the utilization of medical procedures that use ionizing radiation. Health Phys 95: 612-627.

Exposure to low-dose ionizing radiation from medical imaging procedures. R Fazel, H M Krumholz, Y Wang, J S Ross, J Chen, N Engl J Med. 361Fazel R, Krumholz HM, Wang Y, Ross JS, Chen J, et al. (2009) Exposure to low-dose ionizing radiation from medical imaging procedures. N Engl J Med 361: 849-857.

Increasing utilization of computed tomography in the adult emergency department. J Broder, D M Warshauer, Emerg Radiol. 13Broder J, Warshauer DM (2006) Increasing utilization of computed tomography in the adult emergency department, 2000-2005. Emerg Radiol 13: 25-30.

Evidence for suboptimal use of periconceptional folic acid supplements globally. J G Ray, G Singh, R F Burrows, BJOG. 111Ray JG, Singh G, Burrows RF (2004) Evidence for suboptimal use of periconceptional folic acid supplements globally. BJOG 111: 399-408.

In-utero exposed atomic bomb survivors: cancer risk update. Y Yoshimoto, R Delongchamp, K Mabuchi, Lancet. 344Yoshimoto Y, Delongchamp R, Mabuchi K (1994) In-utero exposed atomic bomb survivors: cancer risk update. Lancet 344: 345-346.

Cancer after intrauterine exposure to the atomic bomb. R W Miller, J D Boice, Jr , Radiat Res. 147Miller RW, Boice JD, Jr. (1997) Cancer after intrauterine exposure to the atomic bomb. Radiat Res 147: 396-397.

Solid cancer incidence in atomic bomb survivors exposed in utero or as young children. D L Preston, H Cullings, A Suyama, S Funamoto, N Nishi, J Natl Cancer Inst. 100Preston DL, Cullings H, Suyama A, Funamoto S, Nishi N, et al. (2008) Solid cancer incidence in atomic bomb survivors exposed in utero or as young children. J Natl Cancer Inst 100: 428-436.

Intrauterine exposure to diagnostic X rays and risk of childhood leukemia subtypes. E Naumburg, R Bellocco, S Cnattingius, P Hall, J D Boice, Jr , Radiat Res. 156Naumburg E, Bellocco R, Cnattingius S, Hall P, Boice JD, Jr., et al. (2001) Intrauterine exposure to diagnostic X rays and risk of childhood leukemia subtypes. Radiat Res 156: 718-723.

Radiation dose effects in relation to obstetric xrays and childhood cancers. A Stewart, G W Kneale, Lancet i. Stewart A, Kneale GW (1970) Radiation dose effects in relation to obstetric x- rays and childhood cancers. Lancet i: 1185-1188.

Epidemiological studies of children irradiated in utero. J F Bithell, Baverstock KF, Stather JWTaylor and FrancisLondonLow dose radiation: biological bases of risk assessmentBithell JF (1989) Epidemiological studies of children irradiated in utero. Baverstock KF, Stather JW, eds. Low dose radiation: biological bases of risk assessment. London: Taylor and Francis. pp 77-87.

Children's exposure to diagnostic medical radiation and cancer risk: epidemiologic and dosimetric considerations. M S Linet, K P Kim, P Rajaraman, Pediatr Radiol. 39SupplLinet MS, Kim KP, Rajaraman P (2009) Children's exposure to diagnostic medical radiation and cancer risk: epidemiologic and dosimetric considerations. Pediatr Radiol 39 Suppl 1: S4-S26.

Guidelines for computed tomography and magnetic resonance imaging use during pregnancy and lactation. M M Chen, F V Coakley, A Kaimal, R K Laros, Jr , Obstet Gynecol. 112Chen MM, Coakley FV, Kaimal A, Laros RK, Jr. (2008) Guidelines for computed tomography and magnetic resonance imaging use during pregnancy and lactation. Obstet Gynecol 112: 333-340.

Childhood leukaemia following medical diagnostic exposure to ionizing radiation in utero or after birth. R Wakeford, Radiat Prot Dosimetry. 132Wakeford R (2008) Childhood leukaemia following medical diagnostic exposure to ionizing radiation in utero or after birth. Radiat Prot Dosimetry 132: 166-174.

Patient dose in neonatal units. K Smans, L Struelens, M Smet, H Bosmans, F Vanhavere, Radiat Prot Dosimetry. 131Smans K, Struelens L, Smet M, Bosmans H, Vanhavere F (2008) Patient dose in neonatal units. Radiat Prot Dosimetry 131: 143-147.

Dose and image quality optimization in neonatal radiography. E D Dougeni, H B Delis, A A Karatza, C P Kalogeropoulou, S G Skiadopoulos, Br J Radiol. 80Dougeni ED, Delis HB, Karatza AA, Kalogeropoulou CP, Skiadopoulos SG, et al. (2007) Dose and image quality optimization in neonatal radiography. Br J Radiol 80: 807-815.

Determinants of preterm birth rates in Canada from 1981 through 1983 and from 1992 through 1994. K S Joseph, M S Kramer, S Marcoux, A Ohlsson, S W Wen, N Engl J Med. 339Joseph KS, Kramer MS, Marcoux S, Ohlsson A, Wen SW, et al. (1998) Determinants of preterm birth rates in Canada from 1981 through 1983 and from 1992 through 1994. N Engl J Med 339: 1434-1439.

Sensitivity and specificity of computerized algorithms to classify gestational periods in the absence of information on date of conception. S Toh, A A Mitchell, M M Werler, S Hernandez-Diaz, Am J Epidemiol. 167Toh S, Mitchell AA, Werler MM, Hernandez-Diaz S (2008) Sensitivity and specificity of computerized algorithms to classify gestational periods in the absence of information on date of conception. Am J Epidemiol 167: 633-640.

Using discharge abstracts to evaluate a regional perinatal network: assessment of the linkage procedure of anonymous data. C Quantin, B Gouyon, P Avillach, C Ferdynus, P Sagot, Int J Telemed Appl. 181842Quantin C, Gouyon B, Avillach P, Ferdynus C, Sagot P, et al. (2009) Using discharge abstracts to evaluate a regional perinatal network: assessment of the linkage procedure of anonymous data. Int J Telemed Appl 2009: 181842.

Probabilistic record linkage is a valid and transparent tool to combine databases without a patient identification number. N Méray, J B Reitsma, A C Ravelli, G J Bonsel, J Clin Epidemiol. 60Méray N, Reitsma JB, Ravelli AC, Bonsel GJ (2007) Probabilistic record linkage is a valid and transparent tool to combine databases without a patient identification number. J Clin Epidemiol 60: 883-891.

Data linkage methods used in maternally-linked birth and infant death surveillance data sets from the United States. A A Herman, B J Mccarthy, J M Bakewell, R H Ward, B A Mueller, Paediatr Perinat Epidemiol. 11SupplHerman AA, McCarthy BJ, Bakewell JM, Ward RH, Mueller BA, et al. (1997) Data linkage methods used in maternally-linked birth and infant death surveillance data sets from the United States (Georgia, Missouri, Utah and Washington State), Israel, Norway, Scotland and Western Australia. Paediatr Perinat Epidemiol 11 Suppl 1: 5-22.

An assessment of the validity of a computer system for probabilistic record linkage of birth and infant death records in Canada. The Fetal and Infant Health Study Group. M Fair, M Cyr, A C Allen, S W Wen, G Guyon, Chronic Dis Can. 21Fair M, Cyr M, Allen AC, Wen SW, Guyon G, et al. (2000) An assessment of the validity of a computer system for probabilistic record linkage of birth and infant death records in Canada. The Fetal and Infant Health Study Group. Chronic Dis Can 21: 8-13.

Diagnostic services in Ontario: descriptive analysis and jurisdictional review. Toronto: Institute for Clinical Evaluative Sciences. J J You, D A Alter, K Iron, P M Slaughter, A Kopp, You JJ, Alter DA, Iron K, Slaughter PM, Kopp A, et al. (2007) Diagnostic services in Ontario: descriptive analysis and jurisdictional review. Toronto: Institute for Clinical Evaluative Sciences, Available: http://www.ices.on.ca/file/ diagnostic_services_ontario_oct16.pdf. Accessed 10 July 2010.

Non-adherence to recommendations for further testing after outpatient CT and MRI. J J You, A Laupacis, A Newman, C M Bell, Am J Med. 123e8You JJ, Laupacis A, Newman A, Bell CM (2010) Non-adherence to recommendations for further testing after outpatient CT and MRI. Am J Med 123: 557.e1-557.e8.

Epidemiologic research using the Ontario Cancer Registry. L D Marrett, E A Clarke, J Hatcher, H K Weir, Can J Public Health. 77SupplMarrett LD, Clarke EA, Hatcher J, Weir HK (1986) Epidemiologic research using the Ontario Cancer Registry. Can J Public Health 77 Suppl 1: 79-85.

Cancer incidence registration and trends in Ontario. J R Mclaughlin, N Kreiger, L D Marrett, E J Holowaty, Eur J Cancer. 27McLaughlin JR, Kreiger N, Marrett LD, Holowaty EJ (1991) Cancer incidence registration and trends in Ontario. Eur J Cancer 27: 1520-1524.

Proliferation of prenatal ultrasonography. J J You, D A Alter, T A Stukel, S D Mcdonald, A Laupacis, CMAJ. 182You JJ, Alter DA, Stukel TA, McDonald SD, Laupacis A (2010) Proliferation of prenatal ultrasonography. CMAJ 182: 143-151.

Effective doses in radiology and diagnostic nuclear medicine: a catalog. F A MettlerJr, W Huda, T T Yoshizumi, M Mahesh, Radiology. 248Mettler FA, Jr., Huda W, Yoshizumi TT, Mahesh M (2008) Effective doses in radiology and diagnostic nuclear medicine: a catalog. Radiology 248: 254-263.

Radiation dose to the fetus for pregnant patients undergoing multidetector CT imaging: Monte Carlo simulations estimating fetal dose for a range of gestational age and patient size. E Angel, C V Wellnitz, M M Goodsitt, N Yaghmai, J J Demarco, Radiology. 249Angel E, Wellnitz CV, Goodsitt MM, Yaghmai N, DeMarco JJ, et al. (2008) Radiation dose to the fetus for pregnant patients undergoing multidetector CT imaging: Monte Carlo simulations estimating fetal dose for a range of gestational age and patient size. Radiology 249: 220-227.

Childhood cancer after prenatal exposure to diagnostic X-ray examinations in Britain. R H Mole, Br J Cancer. 62Mole RH (1990) Childhood cancer after prenatal exposure to diagnostic X-ray examinations in Britain. Br J Cancer 62: 152-168.

Saving lives and changing family histories: appropriate counseling of pregnant women and men and women of reproductive age, concerning the risk of diagnostic radiation exposures during and before pregnancy. R L Brent, Am J Obstet Gynecol. 200Brent RL (2009) Saving lives and changing family histories: appropriate counseling of pregnant women and men and women of reproductive age, concerning the risk of diagnostic radiation exposures during and before pregnancy. Am J Obstet Gynecol 200: 4-24.

Physicians' perceptions of teratogenic risk associated with radiography and CT during early pregnancy. S Ratnapalan, N Bona, K Chandra, G Koren, AJR. 182Ratnapalan S, Bona N, Chandra K, Koren G (2004) Physicians' perceptions of teratogenic risk associated with radiography and CT during early pregnancy. AJR 182: 1107-1109.

CT pulmonary angiography: a comparative analysis of the utilization patterns in emergency department and hospitalized patients between. J D Prologo, R C Gilkeson, M Diaz, J Asaad, AJR. 183Prologo JD, Gilkeson RC, Diaz M, Asaad J (2004) CT pulmonary angiography: a comparative analysis of the utilization patterns in emergency department and hospitalized patients between 1998 and 2003. AJR 183: 1093-1096.

Imaging of the brain and cerebral vasculature in patients with suspected stroke: advantages and disadvantages of CT and MRI. C S Kidwell, A W Hsia, Curr Neurol Neurosci Rep. 6Kidwell CS, Hsia AW (2006) Imaging of the brain and cerebral vasculature in patients with suspected stroke: advantages and disadvantages of CT and MRI. Curr Neurol Neurosci Rep 6: 9-16.

The impact of helical computed tomography on the negative appendectomy rate: a multi-center comparison. K Kim, C C Lee, K J Song, W Kim, G Suh, J Emerg Med. 34Kim K, Lee CC, Song KJ, Kim W, Suh G, et al. (2008) The impact of helical computed tomography on the negative appendectomy rate: a multi-center comparison. J Emerg Med 34: 3-6.

Clinical practice. The evaluation of suspected pulmonary embolism. P F Fedullo, V F Tapson, N Engl J Med. 349Fedullo PF, Tapson VF (2003) Clinical practice. The evaluation of suspected pulmonary embolism. N Engl J Med 349: 1247-1256.

Medical problems during pregnancy. E Mason, K Rosene-Montella, R Powrie, Med Clin North Am. 82Mason E, Rosene-Montella K, Powrie R (1998) Medical problems during pregnancy. Med Clin North Am 82: 249-269.