s2fieldsofstudy: Biology Medicine

text:
Skin sensitizers differentially regulate signaling pathways in MUTZ-3 cells in relation to their individual potency

Background Due to the recent European legislations posing a ban of animal tests for safety assessment within the cosmetic industry, development of in vitro alternatives for assessment of skin sensitization is highly prioritized. To date, proposed in vitro assays are mainly based on single biomarkers, which so far have not been able to classify and stratify chemicals into subgroups, related to risk or potency. Methods Recently, we presented the Genomic Allergen Rapid Detection (GARD) assay for assessment of chemical sensitizers. In this paper, we show how the genome wide readout of GARD can be expanded and used to identify differentially regulated pathways relating to individual chemical sensitizers. In this study, we investigated the mechanisms of action of a range of skin sensitizers through pathway identification, pathway classification and transcription factor analysis and related this to the reactive mechanisms and potency of the sensitizing agents. Results By transcriptional profiling of chemically stimulated MUTZ-3 cells, 33 canonical pathways intimately involved in sensitization to chemical substances were identified. The results showed that metabolic processes, cell cycling and oxidative stress responses are the key events activated during skin sensitization, and that these functions are engaged differently depending on the reactivity mechanisms of the sensitizing agent. Furthermore, the results indicate that the chemical reactivity groups seem to gradually engage more pathways and more molecules in each pathway with increasing sensitizing potency of the chemical used for stimulation. Also, a switch in gene regulation from up to down regulation, with increasing potency, was seen both in genes involved in metabolic functions and cell cycling. These observed pathway patterns were clearly reflected in the regulatory elements identified to drive these processes, where 33 regulatory elements have been proposed for further analysis. Conclusions This study demonstrates that functional analysis of biomarkers identified from our genomics study of human MUTZ-3 cells can be used to assess sensitizing potency of chemicals in vitro, by the identification of key cellular events, such as metabolic and cell cycling pathways.

Background
Allergic contact dermatitis (ACD) is an inflammatory skin disease characterized by eczema and recurrent episodes of itching [1]. Hundreds of chemicals are known to cause skin sensitization, making ACD the most common immunotoxic condition in humans. ACD affects a significant proportion of the population. In a recent populations-based investigation, it was demonstrated that the prevalence of ACD is increasing, from 7.2% to 12.9% in the study population [2]. According to the REACH (Registration, Evaluation, and Authorization of Chemicals) regulation [3], chemicals within the EU that are produced or imported (>1 ton/year) must be tested for human hazardous effects. Historically, new chemicals have been tested on animals. For cosmetic ingredients, animal testing is now prohibited [4], even if validated alternative assays are unavailable.
The Local Lymph Node Assay (LLNA) [5], which is currently the gold standard in hazard classification for sensitization, has been shown to have an accuracy of prediction of 72% relative to human data, the same as for the Guinea Pig Maximization Test (GPMT) [6]. Taken together, the ethical aspects of animal testing, the need for monitoring of hazardous effects and the discrepancy between animal and human immune mechanisms makes the need for in vitro tests urgent.
Recently, we presented an in vitro test, Genomic Allergen Rapid Detection (GARD), based on MUTZ-3 cells [7,8], that was able to accurately classify compounds as sensitizers or non-sensitizers, using a genomic biomarker signature [9,10]. Furthermore, we reported that sensitizers induced a heterogeneous transcriptional response in MUTZ-3 cells, indicating that different sensitizers affect different pathways. The OECD guidelines on adverse outcome pathways [11] attempts to summarize the biochemical pathways that are associated with skin sensitization, listing the activation of inflammation and cellular stress related pathways as critical events. However, transcriptional data from MUTZ-3 cells indicate that the regulation of biochemical pathways might be more diverse than previously described. This led us to further investigate the signaling pathways in relationship to the chemical reactivity properties of each individual skin sensitizer. Consequently, the aim of the current study was to identify adverse outcome pathways associated with different sensitizers, stratified into various chemical reactivity groups, in order to enhance the understanding of the skin sensitization processes in relation to sensitizer potency.

Transcriptional data set
A panel of 40 chemical compounds, consisting of 20 sensitizers and 20 non-sensitizers were used for cell stimulations of the human myeloid leukemia-derived cell line MUTZ-3 (DSMZ, Braunschweig, Germany), as detailed earlier in [10]. RNA was isolated, processed to cDNA and hybridized to microarrays (Affymetrix®, Santa Clara, CA) [12]. In short, RNA from un-stimulated and chemically stimulated MUTZ-3 cells, from triplicate experiments, were extracted and analyzed. The preparation of labeled sense DNA was performed, according to Affymetrix® GeneChip® Whole Transcript (WT) Sense Target Labeling Assay (100 ng Total RNA Labeling Protocol), using the recommended kits and controls (Affymetrix®). Hybridization, washing and scanning of the Human Gene 1.0 ST Arrays were performed according to the manufacturer's protocol (Affymetrix®). 18 of the 20 sensitizing chemicals [13][14][15][16][17][18] passed QC test after array analysis and were subsequently used for functional analysis (Figure 1). The complete data set consisting of 137 samples has been deposited to ArrayExpress with the accession number [E-MTAB-670].

Microarray data analysis and statistical methods
The microarray data set was normalized and quality checked, using the RMA algorithm in the Affymetrix® Expression Console software (Affymetrix®). Sensitizers were assigned to at least one of the following chemical reactivity mechanisms; addition/elimination to carboxylic acid derivatives (Add/El), coordination bonds (Koor), Michael addition or 1,4-addition to a/b unsaturated carbonyl (MA), nucleophilic aromatic substitution (NAR), Schiff's base reaction (SB), biomolecular nucleophilic substitution (SN2), Prehapten (PreH) and Prohapten (ProH). In the present cohort of sensitizers, 9 different groups of chemical reactivity combinations were identified, as shown in Figure 1. The 9 reactivity groups were statistically tested against a pool of non-sensitizing stimulations and vehicle controls (83 samples in total), using ANOVA. The top 1000 genes from each statistical test were identified, and the significance level at which these top genes were found was recorded. All filtering and statistical tests were performed in Qlucore Omics Explorer 2.3 (QOE; Qlucore AB, Lund, Sweden).

Functional analysis of chemical reactivity groups in IPA®
Each of the 9 gene lists identified with statistical testing was used as input in Ingenuity Pathways Analysis ver. 12710793, as of June 2012 (IPA®; Ingenuity Systems Inc., Redwood City, CA), using fold change and p-values as decision values for significance and enrichment calculations. For each data set the significant canonical pathways, significant transcription regulators and cellular functions identified by IPA® Core Analysis of Human data using default settings were exported and analyzed further in QOE. Hierarchical Cluster Analysis (HCA) was performed using non-normalized data with maximum linkage.

Pathway annotation and grouping for assessment of biological functions
All pathways were named according to the terminology used by IPA®. In addition to pathway names, we also grouped pathways into functional groups, according to the grouping used by IPA® (apoptosis signaling, cell cycle regulation etc.) with all pathways being classified as either signaling or metabolic pathways or both. When a pathway was ambiguously assigned we chose the first listing, according to alphabetical order. Findings were confirmed, using MetaCore™ (Thomson Reuters, NY).

Availability of supporting data
The microarray data set used was deposited at Array Express with the accession number [E-MTAB-670].

MUTZ-3 transcriptional profiling and stratification of sensitizers into chemical reactivity groups
The transcriptome of MUTZ-3 cells stimulated with skin sensitizers and non-sensitizers was analyzed, using DNA microarray technology, with triplicate samples of 40 individual stimulations [10]. Due to insufficient array quality, 2 out of 20 sensitizers were removed from further analysis, leaving 18 triplicates of sensitizers and 20 triplicates of non-sensitizers for analysis in this study. The relationship between the sensitizers' potency, according to the Local Lymph Node Assay, and the designation of these into different chemical reactivity groups are summarized in Figure 1.
A Principal Component Analysis (PCA) analysis of the complete expression data set visualized that the studied chemical reactivities were indeed quite distinct, as shown in Figure 2. Though there is a certain overlap between the groups there are also clear differences, which is a strong indicator that the 9 chemical groups may trigger different biological activities, while the effects of nonsensitizing samples are homogenous. The PCA plot in Figure 2 was based on the 26 most significant genes in the data set from an ANOVA, comparing sensitizers with non-sensitizers. However, the observed pattern is clearly visible also in the complete data set, where no statistical test has guided the PCA. To further ensure that this is a biological finding, randomization and permutation of data was performed, comparing the original data set to randomized and permuted data sets. Statistical significance was only found in the original data set, which shows that the observed patterns are not seen due to random effects in multidimensional data.
The apparent differences in transcriptional profiles among different reactivity groups led us to further explore the differential engagement of signaling and metabolic pathways and the upstream regulators initiating such pathways. To this end, each chemical reactivity group was compared to the set of non-sensitizers in an ANOVA, yielding 9 lists of differentially regulated genes. These 9 gene lists were subsequently imported into IPA® for further analysis.
Using the Core Analysis of the pathway software IPA®, a number of regulated pathways were identified. In total, 387 regulated pathways were detected in the 9 different chemical reactivity groups. By applying a cut-off of three times, the default IPA® significance (−log(p-value) ≤ 3.9), 33 pathways were selected for a comprehensive investigation. The differential engagement patterns of the 33 pathways, related to sensitizer reactivity, are summarized in Figure 3, and detailed in Additional file 1. In addition, Figures 4 and 5 indicate the direction of regulation, as well as the level of significance at which the IPA® input gene lists were identified. When studying the 33 selected pathways, it is evident that increasing potency correlates with enhanced pathway involvement in terms of number of regulated genes, as well as a shift towards down regulation of the pathways components.
Finally, to identify the drivers in relation to potency, analysis of upstream regulators was performed. In Figure 3,  the common regulatory elements for all nine groups are summarized. The significance calculations include a predicted activation or inhibition status, which takes regulation of connected molecules into account. Only upstream regulators predicted to be significantly up or down regulated in four or more chemical groups were reported here. The regulatory elements were studied for their relationship to each other, using the MetaCore™ database. These findings were summarized in Figure 6. A complete list of all regulatory elements predicted is found in the Supplementary materials (Additional file 2).

Prominent pathways and biological functions
The overall impression is that more genes, associated with various pathways, are down-regulated than upregulated in MUTZ3 cells stimulated with sensitizers. The sensitizers act mainly on cell cycling, cellular stress and injury, and metabolic pathways, in that order. Relevant identified sub functions are apoptosis, cellular growth and development, cellular immune response, intracellular and second messenger signaling, and metabolism and biosynthesis of glycan, lipids, vitamins and cofactors and nucleotides. The general trend is that both metabolic and cell cycle associated pathways are engaged gradually and in correlation with potency. Oxidative stress related pathways are another group of highly engaged pathways. However, these do not correlate with potency in the same manner as the other pathways do. For example, the NRF2-mediated oxidative stress response pathway shows a reversed pattern compared to the other pathways presented in this study. The overall impression of the NRF2 pathway is that phase I and II enzymes, as well as antioxidant proteins, are up-regulated, for an example see Additional file 3. While these trends are quite weak we still think it deserves further investigation. Pathways regulating metabolism and biosynthesis processes are regulated to a lesser extent but still show the same pattern as seen in the cell cycling pathways. Finally, we report 33 potential key regulators of the skin sensitization process found in the analyses. For a complete list of pathways and regulation patterns see Figures 3,4, and 5. One more pathway has a pattern similar to the one observed in NRF2-mediated oxidative stress response pathway, namely Sphingolipid metabolism. The less potent chemical reactivity groups seem to engage this pathway to a larger extent than the more potent ones. A few metabolic pathways are generally regulated. These are the nucleotide and lipid metabolism pathways. Chemicals belonging to the nucleophilic aromatic substitution (NAR) and coordination bonds (Koor) group down regulate nucleotide metabolism the most. As the current chemical selection includes only highly potent sensitizers from the Koor and NAR group, we cannot extrapolate these findings to less potent ones. Also, the NAR group constitutes a somewhat odd case, since this is a small group of highly experimental allergens with limited clinical relevance. The other metabolic pathways are regulated in a more mixed fashion, but again, amino acid metabolism is gradually down-regulated with increasing chemical potency. The Glutathione metabolism pathway is interesting in that it goes from clear up regulation by the less potent chemicals to clear down regulation by the NAR group. While the division of pathways into signaling or metabolic pathways is a somewhat arbitrary matter, we still believe it reflects the major cellular functions quite well. We have followed the KEGG standard [19] at the time, adopted from the IPA® software.
The pathways of cell cycle regulation are also gradually down-regulated with increasing potency. This could indicate that these chemicals induce cell cycle arrest. As discussed later this seems to be connected to activation state of the up-stream regulators identified in the analyses, especially the let-7 microRNAs.
The apoptosis pathway Aryl hydrocarbon receptor (AHR) signaling is also among the frequently regulated. As seen from Figure 3, its usage does increase with potency, but the base level is among the highest observed. Several caspases in the AHR signaling are highly upregulated by many of the chemical groups. Caspases are both involved in cell death processes, like cell shrinkage, membrane blebbing, DNA fragmentation and DNA repair, Figure 3 Hierarchical cluster analysis of pathway and regulatory elements. This hierarchical cluster analysis shows the regulatory elements predicted to have a general activity in the data set, i.e. in at least 4 of the 9 groups. Regulatory elements are colored according to the activation state. Red color indicates activation, while green color indicates inhibition. Dark and pale red/green colors indicate high and low significance of change, respectively. 33 pathways were found to be significantly regulated by the chemicals. These were clustered and colored from white to blue according to the involvement grade of the pathway, as estimated by the number of regulated molecules. The chemical groups were ordered according to their Local Lymph Node Assay potency with the most potent to the left and least potent to the right in the picture. The complete data set is given in Additional file 1.
Cyclins and cell cycle regulation Cellular growth, proliferation and development  and in processes of post-translational modifications, protein degradation and synthesis [20]. There are also connections between apoptosis and cell cycle regulators. Caspase 6, for example, is known to interact directly with the transcription factor Rb [21], which is a key regulator of the Cell Cycle Control of Chromosomal Replication pathway. As described previously, a shift towards down regulation of pathway members is observed with increasing potency. For a detailed list of the molecules in the AHR signaling pathway, see Additional file 4. Finally, lipid metabolism is generally affected by all the chemical groups. For instance, addition/elimination to carboxylic acid derivatives (Add/El) clearly down regulates Sphingolipid metabolism and the remaining chemical groups show a mixed or up-regulated pattern.

Biological activity of specific chemical groups
From the pathway analysis it is clear that various chemical reactivity groups stimulate pathway activation or inhibition differentially. This selective effect is e.g. observed for chemicals of the Michael addition or 1,4-Addition to a/b unsaturated carbonyl (MA) group, which up regulate several of the signaling pathways, as well as chemicals of the addition/ elimination to carboxylic acid derivatives (Add/El). The groups prehapten (Chemical activation outside the skin; PreH) + prohapten (enzymatic activation; ProH) and Add/ El (addition/elimination to carboxylic acid derivatives) both show a very limited activity in most pathways studied.
Considering the activity levels and changes of transcription observed and summarized in Figures 3, 4, and 5, we conclude that there is no single mechanism of action of skin sensitizing chemicals. On the contrary, a range of functions are triggered at different levels by the studied chemical groups. Some chemical groups, notably the most potent ones, down regulate most or all pathways studied. Others, like MA, up regulate relatively many pathways, while e.g. addition/elimination to carboxylic acid derivatives (Add/El) and prohapten +

Add/ El
Amino acid metabolism Valine, Leucine and Isoleucine degradation Glycan Bio-synthesis and Metabolism Glycosaminoglycan degradation

Metabolism of cofactors and vitamins
One carbon pool by folate p-value cut-off for 1000* best genes 8e-9 8e-14 5e-4 3e-3 4e-6 2e-4 2e-4 3e-4 3e-3 q-value cut-off for 1000* best genes 2e-7 2e-12 2e-2 10e-2 1e-4 5e-3 4e-3 8e-3 9e-2 prehapten(ProH + PreH) show a quite limited activity except for a few pathways that seem to always be highly regulated. The method also shows promising results in the detection of pre-and prohapten activation as a grouped class of chemical reactivity. However, it does not distinguish between these two activation routes. In the literature cinnamic alcohol is classified as a prohapten [22,23], but recently it has been shown that cinnamic alcohol autoxidize very rapidly [24], making the older classifications uncertain. Therefore, further investigation into the use of methods for separation of these two classes of chemical reactivity is needed. Especially for prohaptens, it is also crucial to confirm purity levels stated by suppliers as even small amounts of impurities may change the reactivity patterns.
It should be noted that all samples in this study are collected at single concentrations and single timepoints. Most likely, the observed differences between chemical groups depend in some instances on a varying reactivity speed. As an example, investigations of transcription factors, connecting directly to selected cell cycle pathways, show that the activity level of the pathway molecules can depend on temporal factors. Those chemicals that did not show a great deal of activity in the pathway did indeed have a relatively higher activity level among the upstream regulators of these pathways. Thus, there may be pathways that are more consistently regulated than is observed in this study, although such similarities may be masked by varying reactivity of the chemical sensitizers. Figure 6 Network of interactions between transcription regulators observed or predicted in the IPA® analysis of the 9 chemical reactivity groups. Observed expression patterns were analyzed using IPA® in order to predict upstream regulation. The prediction was based on the expression patterns of the chemical groups and only the most significant predictions were included here. The network shows the known interactions between the regulatory elements identified to be up or down regulated in the analysis. Red color indicates a observed/predicted activation and green color indicates a observed/predicted inhibition of the molecule or complex of molecules. All interactions are based on experimental evidence from the scientific literature. Arrow directions show which way the regulation is directed, if known. R: regulation; B: binding; Br: binding regulated by target; RR: bidirectional regulation; Tg: target. The network was extracted from the MetaCore™ database (Thomson Reuters). For details about observation/prediction state, see Additional files 2, 3, 4 and 5.

Upstream regulators
Next, the significantly predicted upstream regulators were investigated. Figure 6 shows the known and experimentally verified interactions in the dataset. The network was manually built, in the IPA® software using molecules predicted to be up or down regulated based on analysis of the expression patterns of the nine chemical reactivity groups studied. The molecular interactions were studied in detail using the MetaCore™ software for pathways analyses focusing on uni-or bidirectional binding and regulation actions of the included molecules. All interactions shown in Figure 6 have been experimentally verified elsewhere and are described in the scientific literature. Several of the transcription regulators included in the network bind to each other, e.g. MYC and ANXA2, RB1 and E2f2 and NFkB and E2f2. Central to the network of Figure 6 is a microRNA group named let-7 (lethal-7). The de-regulation of let-7 miRNAs has previously been described in association to cancer, where the let-7 family members act as tumor suppressors acting e.g. through MYC and E2f2 [25]. Both MYC and E2f2 were predicted to be down regulated by a majority of the chemical reactivity groups in this study. The microRNA group let-7 and specifically the member let-7a-5p was predicted to be significantly activated in six out of the nine groups, with very high activation scores (z-score 2.6 to 6.6) and regulated with borderline detection in the three remaining groups (z-score 1.3 to 1.8 in ProH, ProH + PreH and Add/El). This finding fits well with the known down regulation of let-7 by MYC, through binding of MYC to the transcription start site of the let-7 g pri-miRNA [26]. let-7 is known to be active in cell cycle regulation, DNA replication, cell division, apoptosis and development [27]. In the current study, cell cycle, apoptosis, and nucleotide metabolism pathways were found to be down-regulated. This situation is comparable to many forms of cancer, in which low levels of let-7 have been observed, giving rise to loss of cell cycle control. In our case, down regulation of cell cycle functions is evident for the majority of the chemical reactivity groups studied, while let-7 was predicted to be up regulated. In a review by Hou et al. [28], let-7 RNAs were described as heavily down regulated by environmental chemicals, cigarette smoke and up-regulated by others, such as hexahydro-1,3,5-triazine (RDX). let-7 upregulation has also been demonstrated in skin samples from psoriasis patients compared to healthy controls [29], suggesting these microRNAs to be key players in skin pathogenesis. Two other, specific, microRNAs were also predicted to be significantly regulated namely let-7a-5p and mir-483-3p. We propose that these microRNAs may have similar functions, as described above, in skin, when chemically challenged. For details about molecular interactions found see Additional file 5. let-7 is known to inhibit a number of molecules: ABCC10, BACH1, BLIMP1 (PRDI-BF1), Bax, CCR7, Dicer, EZH2, HMGA2, HMGI/Y, HOXA9, IL-6, NAV1.9, PGHD, SOCS1, UHRF2, and MYC that are all found regulated in the data set. BCL2L1 and DICER1 were found to be up-regulated while all other were down-regulated. Among the transcription regulators MYC and let-7 miRNAs are known to bind and interact bi-directionally. Furthermore, let-7 is regulated by a number of other molecules, several of which were seen regulated in the present analysis, namely PPARA, DROSHA, XPO5, and KDM5B. Finally, let-7 binds to the predicted activated transcription regulator TGFB1.
The NFkB complex was also predicted to be activated in this study. This extensively studied nucleic protein has a wide range of functions. Of interest to this study is mainly its involvement in pathways of Xenobiotics Metabolism, Production of Nitric Oxide and Reactive Oxygen Species in Macrophages and Toll like receptor signaling. NFkB showed a heterogeneous pattern, with up regulation in four groups (MA + Add/El, MA, SN2 and ProH) and down regulation in one (ProH + PreH). This may be a relevant finding given the role this molecule plays in epithelial cells where it on one hand maintains immunological homeostasis and on the other induces inflammatory processes [30]. NFkB has been shown to be able both to activate and inhibit inflammatory pathways in skin [31], a finding that might explain the mixed pattern seen in this study.

Implications of the results
Activation of MUTZ-3 cells with skin sensitizers resulted in regulation of a number of previously known, as well as novel, canonical pathways. The 33 most significantly regulated pathways were studied in detail. After stratification of sensitizing chemicals into chemical reactivity groups, it was evident that pathway engagement and the number of regulated molecules within such pathways were linked to the sensitizing potency of the stimulatory agent. For instance, cell cycle associated pathways were engaged gradually in correlation with the sensitizing potency of the chemicals used for cell stimulations. These observations can be utilized when designing tools for assessment of sensitizing potency of chemicals in predictive assays. Currently, the primary prediction call obtained from GARD is binary, i.e. compounds are classified as sensitizers or nonsensitizers. However, this readout can now for the first time be complemented with a potency call based on mechanistic information obtained through pathway analysis. While implementation of such classifications are not straight-forward, similar attempts have been done within cancer research, where pathway activity has successfully been associated with subtypes of cancer and progression of disease [32][33][34].
Oxidative stress related pathways are also in the group of highly engaged pathways. These do not correlate with potency to the same extent that cell cycle pathways do. For example, the NRF2-mediated oxidative stress response pathway shows a higher engagement by less potent chemical groups compared to more potent ones. This may be a consideration when studying more clinically relevant allergens, as these are rarely in the strong to extreme but rather in the week to medium range of potency.
Pathways regulating metabolism and biosynthesis processes constitute the third large group of canonical pathways identified to be regulated in skin sensitization. These pathways are regulated to a lesser extent but still show the same pattern as seen in the cell cycling pathways.
No canonical pathway was unanimously regulated within each data set. The importance of this fact becomes apparent when considering the design of various proposed tests for animal-free assessment of sensitization, which today are mainly based on analysis of single biomarkers. It is evident that excessively simplistic tests lack the power to accurately assess compounds as sensitizers or non-sensitizers, if the target chemical domain is wide. Recognition of the specific subtype of chemicals to be tested based on reactive mechanism or otherwise, will be crucial in order to train and configure predictive models accurately.

Conclusions
In the present transcriptional profiling study, we have shown that skin sensitizers trigger a large number of signaling and metabolic pathways in MUTZ-3 cells. Among these, we found examples of well-known pathways, as well as pathways previously not described in relation to chemical sensitization. Importantly, a majority of the pathways described show a clear correlation with both chemical reactivity and sensitizing potency.
The strongest correlation between pathway usage and potency is found in the cell cycle, nucleotide metabolism and cancer pathways. As these groupings are obviously rather arbitrary it is probably more correct to say that functions involving cell cycle regulation and cell cycle control both in healthy and diseased conditions correlate very well with chemical potency. Nucleotide metabolism is tightly connected to the cell cycle and consequently a down regulation of nucleotide metabolism in parallel to a down regulation of cell cycle functions is expected. The apoptosis pathway Aryl Hydrocarbon Receptor signaling is a novel finding with regard to potency prediction. This pathway is among the highly regulated ones but still shows a decrease in activity with decrease in potency.
Finally, transcription regulation of the sensitization process was investigated, by prediction of activation and inhibition of upstream regulators. A network of regulatory elements that seemed to be generally involved was identified. These seemed to be centered on the transcription regulator MYC and the miRNA let-7.
The presented results have implications for the design of predictive in vitro test for assessment of sensitization potency. Few chemicals appear to induce identical responses in vitro, indicating that excessively simplistic assays will fail to correctly classify unknown samples. Subdivisions among sensitizers, by reactive domain or otherwise, should be considered with caution and predictive models should be trained accordingly. In brief, we demonstrate for the first time that genomic approaches contain valuable mechanistic information associated with potency assessment of chemicals, which is in line with the current demand of evidence based toxicology.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Chemistry

text:
Structural Features of the Interaction between Human 8-Oxoguanine DNA Glycosylase hOGG1 and DNA.

The purpose of the present review is to summarize the data related with the structural features of interaction between the human repair enzyme 8-oxoguanine DNA glycosylase (hOGG1) and DNA. The review covers the questions concerning the role of individual amino acids of hOGG1 in the specific recognition of the oxidized DNA bases, formation of the enzyme–substrate complex, and excision of the lesion bases from DNA. Attention is also focused upon conformational changes in the enzyme active site and disruption of enzyme activity as a result of amino acid mutations. The mechanism of damaged bases release from DNA induced by hOGG1 is discussed in the context of structural dynamics.

mispair with adenine in the Hoogsteen mode, which results in a c to A substitution in the first replication cycle (a 8-oxoG/A mismatched pair is formed), followed by the incorporation of Т opposite A in the second round of replication, thereby producing a G/c → t/A transversion [15,16].
to prevent 8-oxoG accumulation in DnA, there is a pathway to protect cells against the mutagenic effect (the GO-system) [17,18]. this pathway has been described in details for Escherichia coli. It consists of three enzymes: Fpg (MutM), a specific n-glycosylase/ AP-lyase that catalyzes the excision of 8-oxoG; MutY, a specific n-glycosylase that removes adenine opposite 8-oxoG; and Mutt, a nucleotide hydrolase involved in the cleavage of the pyrophosphate bond in 8-oxo-dGtP. eukaryotic cells have structural or functional analogs of these bacterial enzymes [19,20]. the excision of 8-oxoguanine from DnA in eukaryotes is carried out by the 8-oxoguanine glycosylase (OGG1) [21]. each human cell has been shown to contain approximately 50 thousand copies of OGG1 that protect the genomic DnA from the accumulation of oxidized purine nucleotides [22].
tHe catalytic mecHaniSm oF tHe ogg1 Protein the hOGG1 enzyme acts both as a DnA-glycosylase and a β-lyase, hydrolysing the n-glycosidic bond of the damaged base to release 8-oxoG, followed by catalytic cleavage of the 3′-phosphodiester bond. the chemical mechanism of action was first suggested by Wallace et al. for endonuclease III of E. coli [29]. the main ideas proposed in that study were experimentally proved by Lloyd et al. [30] using the analysis of cross-links formed between the enzyme and DnA molecule. According to the proposed mechanism, upon incubation of DnA and the enzyme, followed by sodium borohydride treatment, a covalent bond was formed between the two molecules evidencing the occurrence of Schiff base intermediate.
the catalytic action of hOGG1 involves a mechanism by which the ε-amino group of Lys249 participates in the removal of the 8-oxoG base from the c1' of the ribose moiety and promotes the elimination of the 3'-phosphodiester bond through the formation of a Schiff base intermediate with c1' of the deoxyribose moiety ( Fig. 1) [31][32][33]. Verdine et al. earlier showed that K249Q mutant hOGG1 exhibited no catalytic activity but retained the ability for the recognition of oxidatively damaged DnA [21]. the second chemical event is the scission of phosphodiester bond at the c3 of the 2′-deoxyribose moiety via β-elimination (AP-lyase activity).
Based on numerous structural and biochemical studies, Verdine et al. [34] proposed a role of 8-oxoG being displaced in acid/base interactions with the nε, (Lys249 amino group), c2', and O4' atoms. the structure of the intermediate reduced by sodium borohydride, produced through the reaction of hOGG1 with 8-oxoG-containing DnA, has been characterized. the concerted pathway REVIEWS 8-oxoG base to be removed from the DnA strand is not released but retained in the active site and acts as a cofactor at the β-elimination stage. the n9 of the enzyme in the 8-oxoG base is positioned near nε and O4', which permits the transfer of a proton between these two atoms. Figure 1 illustrates the enzymatic mechanism of hOGG1 as described [34]. the initial attack of the sidechain amino group of Lys249 on the c1' of the deoxyribose sugar results in cleavage of the glycosidic bond to produce an 8-oxoGanion. the anion deprotonates the ε-nH 2 of Lys249 to form the aminal intermediate 3. the protonated 8-oxoGН transfers the proton to the О4' of the ribose sugar, with the aminal intermediate 3 rearranging to a Schiff base 4 with a sugar ring opening. the Schiff base 4 with a proton on the nε of Lys249 donates it back to 8-oxoGto yield 8-oxoGН and an uncharged Schiff base 5 promoting the loss of the 3' phosphate via β-elimination. DnA with a 5' phosphate group and a positively charged intermediate 6 arise. the intermediate carries an α,β-unsaturated Schiff base at the 3' end with a positive charge. upon hydrolysis, the intermediate 6 releases the enzyme and a DnA molecule with 4-hydroxy-2,3-pentenal-1 at the 3'end.
the Protein Data Bank currently holds information on 27 structures of hOGG1. the crystal structures of native hOGG1 [35] and DnA-bound hOGG1 have been solved: catalytically inactive K249Q mutant hOGG1 with 8-оxoG-containing DnA [35,36], n149c with 8-оxoG-containing DnA and intact DnA [37], D268n with 8-оxoG-containing DnA and DnA with a tetrahydrofuran residue (F-ligand) -a "stop" substrate for hOGG1 [33,38]; and the Wt hOGG1 complex with the F-ligand [33]. In addition, the irreversibly linked adduct of hOGG1 with AP substrate has been described [34], formed through a borohydride-trapped Schiff bases for hOGG1 variant forms (H270A, Q315A, Q315F, G42A) containing mutation of amino acids that bind 7,8-dihydro-8-oxoguanine in the native structure [32]. the structural analysis has also been conducted for a late-stage intermediate wherein 8-oxoG is almost completely inserted in the active site; however, catalytically active conformation has not been achieved yet [32].
Structural cHaracteriSticS oF K249Q hogg1 lacKing enzymatic actiVity the first structure of complex of hOGG1 with 8-oxoG-containing substrate was obtained for hOGG1 with a К149Q mutation [36]. It was shown earlier [21] that the mutant form, in which Lys249 is replaced with Glu, lacks catalytic activity but retains substrate recognition. Since the authors [36] failed to yield high-quality crystals for full-length hOGG1 K249Q complexed with the DnA duplex containing 8-oxoG/c, limited diges-tion by trypsin allowed to remove unstructured aminoand carboxyl-termini as well as amino acids at positions 80-82, facilitating crystallization and the analysis of the hOGG1 core domain comprising residues 12-325 (Fig. 2).
It has been demonstrated that hOGG1 shares a common fold with the members of the superfamily of DnA repair enzymes involved in base excision repair (Ber), such as endonuclease III and alkyl-DnA-glycosylase AlkA from E. coli [39]. the repair proteins of this family occur in numerous organisms, from bacteria to mammals, repairing DnA bases damaged by oxidation, alkylation, and deamination. these enzymes possess a unique structural motif "helix-hairpin-helix" (HhH) [40], followed by a Gly/Pro-rich loop and conserved residues of Gly, Pro and Asp (HhH-GPD). the hOGG1 structure also contains two α-helix domains shared by all members of the HhH-GPD superfamily and antiparallel β-sheet present in the alkyl-DnA-glycosylase AlkA only.
the protein has high affinity to 8-oxoG-containing DnA (Fig. 2). the 8-oxoG base flips out of the DnA helix and fits into the pocket of the enzyme's active site, which is in agreement with similar structures in other members of the HhH-GPD superfamily [31,40,41]. In the case of the 8-oxo-dG base, the heterocyclic compound exists in the syn-conformation in relation to the glycosidic bond; although, upon binding to the active site of hOGG1, it takes the anti-conformation; i.e., as is the case for a normal duplex DnA. the flipped-out conformation of the glycosilic moiety and DnA backbone leads to the extrusion of 8-oxoG out of the DnA helix and insertion deeply into the active site of hOGG1.
the interaction of hOGG1 with the phosphate groups of 8-oxoG-containing DnA, 8-oxoG itself, and the complementary cytosine contribute to a contact surface of 2.268 Å 2 [36]. though in most DnA-binding proteins the contact region contains many lysine and arginine residues, for the interaction with the phosphates groups of hOGG1, the DnA backbone is bound to a nearly uncharged groove lined by a single basic His270 residue. the resulting complex is unique in a way that hOGG1 contains many α-helices with the n-termini oriented towards the DnA (Fig. 2). this disposition of α-helices enhances the helix-dipole interaction, promoting dipole electrostatic contacts rather than salt bridges while binding DnA substrates. Only one of the helices, αL, is involved in direct contact with the DnA backbone. the αL helix with a loop and the αK helix form the conserved motif HhH. In addition to the interaction through the αL helix (Val250 and Gln249) and the phosphate group p -2 , the highly conserved glycine residue (Gly245), located within the loop, forms a hydrogen bond with the phosphate p -3 (Fig. 3). the structural motif HhH is brought into con-tact with a DnA substrate from the 3'-side of the oxidized base; at this site, the structure of duplex DnA is very similar to the B-form. consequently, the HhH motif is mainly responsible for positioning the mutated base in the DnA duplex towards the active site pocket.
the phosphate groups p -1 , p 0 and p 1 play an important role in the stabilization of the uncommon DnA backbone conformation at the mutagenic site. the rotation required for flipping 8-oxoG out of the helix causes inward rotation of oxygen atoms of p -1 and imposes extra strain on the ribose phosphate DnA backbone. to decrease electrostatic repulsion between the closely positioned p-1 and p 1 , a partially hydrated ca 2+ ion, present in the crystal microenvironment, is brought in between and secured through a direct bond with p 1 and the water bridge with p -1 (Fig. 3B). Although ca 2+ could be displaced by Mg 2+ under physiological conditions, it does not come in direct contact with the protein, but its ligand, the water molecule, forms a hydrogen bond with DnA, thereby stabilizing its flipped-out and bent conformation.
the complementary cytosine locates inside the helix; however, it hardly stacks with neighboring nucleobases from the 5' side due to a kink in the chain that orients the duplex away from the enzyme. Beyond the active site, the DnA conformation is similar to that of canonical B-DnA form (Fig. 2).
the void remaining in the duplex after extrusion of 8-oxoG, is filled in by the amino acid residue of the conserved nnn-element (a motif of three Asn residues); namely, Asn149 that forms a hydrogen bond via its side-chain amide carbonyl with the exocyclic nH 2 of the estranged cytosine (Fig. 3B). In addition, hOGG1 plunges the indole ring of tyr203 into the space between c 0 (estranged cytosine) and the base on the 5'-side (Fig. 3B), thus unstacking the two bases and creating a sharp kink in the DnA molecule, which significantly improves access to the Watson-crick edge of base from the minor-groove side. c 0 gets unstacked from the base on its 3'-side ( Fig. 3B Т 1 base). the residues Arg154 and Arg204 of hOGG1 move together toward c 0 from the minor groove; one arginine above REVIEWS and the other below the plane of the pyrimidine ring, simultaneously creating hydrogen bonds with the acceptor n3 and O2 atoms of the estranged c 0 . these bonds seem to be exceptionally strong and occur in the presence of adjacent acceptor atoms, which are unique to cytosine among the other DnA bases. Along with the interaction of Asn149 and c° amide carbonyl of the enzyme and the estranged cytosine, respectively, up to five hydrogen bonds could be created.
the role of Asn149 has been elucidated in the work [35] that demonstrated that Asn149 formed a hydrogen bond with n4 of the exocyclic nH 2 of the cytosine pairing with 8-oxoG. the hydrogen bonds made by the guanidine group of Arg204 with the n1 and O2 form a unique recognition site for the estranged cytosine and appear to play an essential role in the specific recognition of the estranged cytosine typical of hOGG1.
recognition of 8-oxoG at the active site is accomplished by specific contacts between the lesion base and the amino acids. the enzyme recognizes the urea fragment in 8-oxoG containing the С8-carbonyl group, the n7 and n9 atoms, with n7 forming a hydrogen bond with the carbonyl of Gly42. Of all contacts associated with 8-oxoG, only the one made by Gly42 that would be different with oxoG versus guanine. therefore, the authors of [36] conclude that 8-oxoG is discriminated from G by a single hydrogen bond. Significantly, the essential amino acid residue Gly42 is the only residue that the β-sheet domain contributes to the hOGG1-DnA interface.
In addition to the aforementioned residues responsible for 8-oxoG recognition, other residues of the hOGG1 active site also contribute to recognition. Phe319 and cys253 stack toward opposite π-faces of the 8-oxoG, sandwiching the base in the active site ( Fig. 4). the Gln315 amide nH 2 -group, in cooperation with a tightly bound water molecule, interacts with О 6 of 8-oxoG, and the Gln315 side-chain carbonyl forms two hydrogen bonds with n1 and n 2 H of 8-oxoG. Another tightly bound water molecule is hydrogen bonded to O 6 . Gln315 and Gly42, including the water molecules trapped in the active site, are not chemically able to form hydrogen bonds with A, c, and t through donor/acceptor interactions.
the structural analysis in [36] provides insight into the role of the catalytically important residues Lys249 and Asp268. Lys249 is located ~2.5 Å off the c1' of 8-oxoG close to the space in the active site into which the lesion base is extruded, with the Asp268 is suitably disposed to assist the protonation/deprotonation of Lys249. the intermediate, formed by an attack of the deoxyribose sugar by Lys249, rearranges to a Schiff base (Fig. 1). this rearrangement requires deprotonation of the Lys249 side-chain amino group, presuma-bly by Asp268, and O1' protonation, for which His270 is suitably positioned. this role of His270 sheds light on why this residue is invariant in members of the HhH-GPD superfamily, since it is involved in the catalyzing of the formation of Schiff base intermediates.
catalytically actiVe Hogg1 comPleX WitH tHe "StoP" SubStrate the structure of hOGG1 complex with DnA containing 2-oxomethyl-2-oxo-tetrahydrofuran (F) instead of 8-oxoG has been reported in [33]. this DnA substrate acts as a stop substrate for hOGG1. It is shown that in the produced structure (PDB ID: 1Fn7) Asp268 is positioned rather far from Lys249 to initiate deprotonation of its side-chain amino group; the n-O distance is 3.7 Å. In addition, Asp268 does not contact with the His270 that is likely needed for hydrogen bonding to O1' of the ribose sugar as it was found in K249Q hOGG1 structure [36]. taken together, the absence of the lesion base being flipped out of the helix and into the active site pocket causes conformational changes in the enzyme.
Based on the findings of [33], it was concluded that, firstly, the 8-oxoG-recognizing pocket in hOGG1 well fits the structure of the lesion, positioning Phe319, His270, and Asp268 in the appropriate arrangement. Secondly, the role of Asp268 in the deprotonating of Lys249 has been neither confirmed nor excluded. A hypothesis is proposed that this residue creates an electrostatic field that stabilizes the positive charge developing in the transition state, especially at O1' and c1' of the 8-oxoG deoxyribose. hogg1 conFormational cHangeS uPon dna binding the crystal structures of native hOGG1 and that bound to 8-oxoG-containing DnA were obtained and described by Bjørås et al. [35] at a resolution of 2.15 Å. It was found that the hOGG1 structure significantly differs from those of the free form and the one com-plexed with 8-oxoG-containing DnA (Fig. 5). the Phe319, cys253, Gly42, Gln43, Phe45, and Gln315 residues are responsible for recognition, which is consistent with other findings [36]. Phe319 and cys253 sandwich 8-oxoG, whereas Gly42, Gln43, and Phe45 interact with the major-groove edge, recognizing the protonated n7 of 8-oxoG. the Gln315 amide oxygen is hydrogen-bonded to the n1-imino-and n2-amino groups of the ring involved in base pairing. the Phe319 residue takes different conformations in the free form of hOGG1 and that bound to DnA (Fig. 5A). When bound to DnA, the aromatic ring of Phe319 is oriented almost perpendicular to its position in the free form. the sidechain of Gln315 in the free enzyme is positioned under the aromatic ring of Phe319 (Fig. 5A). In complex with DnA, the nitrogen atom of the amide moiety of Gln315 and the carbonyl oxygen of Pro266 located on the opposite side of the binding site are involved in hydrogen bonding. this causes a significant conformational change in the enzyme, thereby creating a tight pocket for binding the damaged base.
In comparison with [36] a slightly different point of view for the role of His270 has been proposed [35]. It was demonstrated that conformational changes in the lesion-recognition pocket are accompanied by a change in the orientation of His270, which forms two hydrogen bonds when bound to DnA: one between the nε2 of the imidazole ring and the 5'-phosphate of 8-oxoG, the other between nδ1 and the carboxyl group of Asp322 (Fig. 5A). In the free enzyme, one hydrogen bond with Asp322 is retained, although the imidazole ring of His270 rotates by more than 90° as compared to the DnA-bound conformation; packs against the phenyl ring of Phe319 and forms two layers of a sandwich completed by Gln315. the His270 rotamer is not compatible with the Phe319 and Gln315 conformations required for specific binding of the 8-oxoG in DnA. the side-chain conformations of Phe319 and Gln315 depend on the His270 conformation, which is itself governed in the 8-oxoG-DnA complex by interaction with the 5'-phosphate of 8-oxoG nucleotide. Overall, binding of the ribose-phosphate backbone to DnA influences the conformation of His270, which in turn causes a conformational alteration to Phe319 and Gln315, thus allowing entry of the aberrant base into the pocket.
Hence, the side-chains in Phe319, Gln315, and His270 behave as an entity, flipping between the closed and open states when binding the 8-oxoG-base.
In the free form, the amino acid region of 146 to 151 adopts a conformation different from that in a DnAbound state wherein the atoms could shift away from their positions by up to 4-9 Å. Most pronounced changes occur within the center of the motif, with the sidechain of Asn149 in the free form extending back to-  [36]) using the software package PyMOL [43] REVIEWS ward the enzyme and hydrogen bonding to the amide oxygen and e-amino group of the catalytic residue Lys249 (Fig. 5B). However, during complex formation with 8-oxoG-DnA, this oxygen atom retracts by ≈ 9 Å to pack against the estranged cytosine. the remaining residues of Asn in the nnn triplet, 150 and 151, whose hydrogen bonds in a DnA-bound structure stabilize the cytosine-recognizing protein site, are exposed to a solvent in the free form. the conformation of this inter-helical peptide seen in the free enzyme is unable to bind to 8-oxoG in its the extrahelical conformation. Flipping of this enzyme segment into target DnA, as well as flipping of the scissile base into the enzyme pocket, requires conformational alterations of the protein to allow the chemistry to take place.
An important sequel of His270 re-orientation in the hOGG1-8-oxoG-DnA complex is an incremental shift of the hinge and strand between Pro266 and trp272, which moves the side-chain of Asp268 by over 1.5 Å as compared to its position in the free enzyme (Fig. 5C).
In the DnA-bound hOGG1 complex with F-ligand [33], the Lys249 residue is located away from the carboxyl group of Asp268, which challenges its role in the deprotonating of the nucleophile, thus indicating an alternative function for this residue in the transition state charge stabilization of the deoxyribose ring. In addition, the carboxyl group of Asp268 and the imidazole group of His270 in the DnA-bound structure are sterically close for weak hydrogen bonding which would favor proton abstraction by Asp268, promoting its indirect catalytic contribution. On the other hand, the side-chains of Asp268 and His270 in the free hOGG1 are positioned >4 Å apart, which essentialy prevents their hydrogen bonding (Fig. 5C). By contrast, the side-chains of Lys249 and Asp268 sterically allow a hydrogen bond formation between the ε-aminoand carboxyl groups, respectively, as well as another hydrogen bond between the ε-amino group of Lys249 and the side-chain oxygen of Asn149 (Fig. 5C). the side-chain configuration of Lys249 in the active site corresponds to the protonated ε-aminogroup stabilized by the neutral hydrogen bond with the amide oxygen atom of Asn149 and the hydrogen-bonded deprotonated side-chain carboxyl of Asp268.
According to the catalytic mechanism for OGG1 [29][30][31], Lys249 provides a nucleophile attack on the c1' of the deoxyribose moiety, displacing 8-oxoG and generating Schiff's base covalent intermediate [39]. the protonated side-chain of Lys249 in the free enzyme lacks this function that would require a lone pair of electrons as in the neutral ε-nH 2 . therefore, the Lys249 residue should be deprotonated for the reaction to proceed. this would only be possible if the interaction with Asp268 stabilizing the protonated Lys249 is cleared upon binding to the substrate. It was shown in [35] that binding of 8-oxo-dG promotes a change in the position of Asp268 (through movements in Phe319 and Phe270), which breaks its ion-pair/hydrogen bond to Lys249. At the same time, to promote the interaction between the enzyme and the estranged cytosine, the inter-helical hinge is extruded, accompanied by a loss of hydrogen bonding between Lys249 and the side-chain carbonyl group of Asn149. Following the removal of these neutralizing interactions, the protonated ε-amino group of Lys249 should be disfavored with regard to the neutral state generated by proton abstraction from the carboxyl group of Asp268, in parallel to its movement and loss of the hydrogen bond with Lys249.
Overall, the findings suggest [35] that the hydrogen bonds established by Asp268 and Asn149 with a protonated nitrogen of the ε-amino group of Lys249 act as trigger locks in the free enzyme. One hydrogen bond is involved in 8-oxoG recognition; the other, in the recognition of cytosine. Both should be removed for the enzyme to trigger a nucleophilic attack on the c1' of the deoxyribose.

tHe role oF asp268 in tHe catalytic action oF hogg1
Asp268 is catalytically important for both hOGG1 and other members of the structural family to which it belongs [26,44]. Substitution of residue 268 for Ala or Asn abrogates the glycosylase and AP-lyase activities; however, substrate recognition is retained.
Initially, it was suggested that Asp268 could be important for oxidized base excision through deprotonation of Lys249, thereby converting the catalytically inactive cation into a nucleophilic neutral amine [36]. However, this is inconsistent with the fact that Asp located at the end of the α-protein helix needs to be less basic by several orders of magnitude. the fixed position should prevent the rotation of Asp268 around c α -c β to interact with Lys249. the X-ray crystal structure of the native enzyme bound to AP-DnA [33] showed that Asp268 and Lys249 have no contact. Indeed, only the free form of the protein permits hydrogen bonding between the two, with Asp268 preserving its position in the α-helix, whereas Lys249 swivels about to enable contact [35]. Importantly, even though in the case of weakly hydrogen-bonded Glu268 and Lys249 (in mutant D268e) and a longer side-chain, the side-chain of Glu268 lacks contact with Lys249. It is likely that if the pK a of Lys249 declines slightly due to the sequence context to produce a certain amount of neutral amine, this could be sufficient to break the glycosidic bond and excise the aberrant base.
to elucidate the role of Asp268 in the hOGG1-dependent catalysis, this residue was substituted for as-paragine (D268n), glutamate (D268e), and glutamine (D268Q) [38]. It was demonstrated that Asp268, located at the n-terminus of the α-helix (Fig. 6), plays a dual role in the catalysis of base excision and DnA strand scission. the mutation of this residue to asparagine led to a significant decline in the enzyme activity of D268n hOGG1. the D268n crystal structure first revealed the contribution of this nucleophilic Lys residue (Lys249) in the recognition of an aberrant guanine (8-oxoG or F).
Analysis of this structure allowed to suggest that 8-oxoG excision was performed through dissociative mechanism consisting of cleavage of the glycosidic bond, deprotonation of Lys249 (perhaps, by 8-oxoG anion) followed by subsequent linkage of Lys249, forming a Schiff base between the ε-nH 2 -group and С1' of the deoxyribose sugar. On the other hand, a change from aspartic acid to glutamine (D268Q hOGG1) and glutamate (D268e hOGG1), although it led to a protein fold disruption but did not abrogate catalytic activity.
It is shown [38] that complexes of Asp268 mutated hOGG1 with DnA are structurally similar to that of K249Q mutant hOGG1 [36]. the root mean square deviation of the protein backbone coordinates between the D268n and K249Q hOGG1 structures is 0.32 Å, which confidently lends credence to the identity of the structures. comparison between the structure of wildtype and D268e mutant hOGG1 bound to F-analog yields root mean square deviations of 0.45 Å and 0.59 Å for Wt hOGG1/F and hOGG1 D268Q/F structures, REVIEWS respectively. this is indicative of the fact that no global conformational changes occur following mutations of active site amino acid. In all three structures, considerable alterations were only observed in the active site, especially in the first three residues of helix α-M (residues 269, 270 and 271). In all three structures, the nucleophilic ε-nH 2 -group of Lys249 has a relatively fixed position, even though it is located at the end of a long alkyl chain. In this conformation, the ε-nH 2 -group of Lys249 is located near the c1' of the deoxyribose moiety (3.4 Å), but in an orientation unfavorable to a straightforward nucleophilic attack on the glycosidic bond (Fig. 6). the ε-nH 2 -group of Lys249 seems to be hydrogen-bonded to the sulfhydryl of cys253, which is, by contrast, attached to the π-system of the 8-oxoG through a van der Waals interaction. Since cys253 is located on the top face of the ribose moiety, Lys249 cannot form contact with cys253 when the sugar is attacked from the underside. In the wild-type enzyme, the carbonyl group of Asn268 is hydrogen-bonded to the His270 side-chain, whereas in the D268n mutant, the nH 2 -amide group of Asn268 is positioned too far off His270 for hydrogen bonding to occur. the absence of bonding clarifies the relatively weak binding of DnA to the D268n mutant variant, since His270 is in direct contact with the phosphate backbone on the 5'-side of the lesion. the remaining part of the active site and recognition pocket in D268n and K249Q mutant hOGG1 bound to 8-oxoG-DnA are structurally identical. An alternative view of the role of Asp268 is the electrostatic stabilization of the positive charge on the O4' of the deoxyribose sugar in the transition state during base excision [38]. this is in agreement with the fact that in all hOGG1 structures with Asp at position 268, the carboxyl oxygen is positioned near the O4' of the sugar phosphate backbone DnA, at a distance of 3.2 Å. Due to the strong interaction with its α-helix, the orientation of the Asn268 side-chain in D268n is nearly the same as that of Asp268 in the wild-type enzyme. consequently, a change of Asp to Asn at position 268 leads to the substitution of a negatively charged oxygen for a neutral nH 2 group, maintaining the position in the active site. the imide nitrogen of Asn268 and O4' of the deoxyribose moiety (3.4 Å) are positioned at a longer distance than would be expected the hydrogen bonding distance between these atoms; however, this is sufficient for O4' to experience the partially positive electrostatic field of the amide protons on Asn268. It is concluded [38] that the charge change from Asp (δ -) to Asn (δ + ) at position 268 increases the transition state energy for base excision due to the enchanced positive charge on O4', which results in a significant decline in reaction rates. taken together, it was obtained [38] that hOGG1 containing Asn in place of Asp268 (D268n) exhibits no catalytic activity. the amino acid mutations D268Q or D268e, even though they confer a sterically disfavored conformation, do not diminish the catalytic activity. these findings argue against the role of Asp268 as an acid/basic catalyst in hOGG1 [36]; however, they support its involvement in the charge appearance on the О4' of the deoxyribose sugar in 8-oxoG. recognition oF 8-oxog 8-oxoG and the G base structurally differ by two positions: С8 carries О or Н; and n7 contains Н or a lone pair of electrons, respectively. For this reason, the Н atom adjacent to n7 in 8-oxoG is capable of hydrogen bonding with the carboxyl of the Gly42 main chain, whereas G is not. to clarify the structural features of hOGG1 in complex with 8-oxoG and intact G, n149c mutant hOGG1, lacking catalytic activity, was probed tested [37]. cys149 was connected with a linker to the С4 of cytosine complementary to 8-oxoG through a disulfide bond, thereby preventing the dissociation of the enzyme-DnA complex. the X-ray structures for n149c hOGG1 complexed with 8-oxoG-, G-, and 7-deaza-G-containing DnA duplexes have been solved.
the global structures of hOGG1 enzyme complexed with 8-oxoG-and G-containing DnA reveal a drastic helical axis kinks of ~70 and ~80 о , respectively, at the plane of the extruded base. the 8-oxoG base is located deeply in the active site pocket, whereas G lies against the enzyme surface at an exo-site positioned about 5 Å away from the pocket (Fig. 7A,B). the G base interacts with two active site residues Phe319 and His270 but these contacts differ from those made by 8-oxoG. In the 8-oxoG-containing complex, His270 is not in contact with the damaged base but forms hydrogen bond with its 5'-phosphate. In a G-containing DnA, His270 interacts with the π-system of the base, without hydrogen bonding to its phosphate.
Free energy calculations made for the interactions of 8-oxoG and G with the hOGG1 active site (ΔA 1 and ΔA 2 respectively) using quantum mechanics/molecular dynamics simulation techniques [37] showed that the value for free energy discrimination ΔΔA = ΔA 1 -ΔA 2 is -6.8 kcal/mol, which indicates a 10 5 -fold preference for binding 8-oxoG in relation to G into the active site.
the hydrogen bond between the carbonyl oxygen of Gly42 and the proton at n7 in 8-oxoG strongly stabilizes the complex conformation. In the case of G, this will be replaced by coulomb repulsion between the carbonyl oxygen of Gly42 and the lone pair of electrons at n7, if G and Gly42 are positioned similarly as in the 8-oxoG complex.
the study [37] provides insight into the recognition mechanism for the lesion base. It is likely that base extrusion involves more than one step and cannot occur as a single process but rather progresses through multiple discrete states. this conclusion is supported by the fact that G-bound hOGG1 could mimic the intermediate produced upon binding to 8-oxoG, immediately before flipping out into the enzyme pocket. the multi-stage recognition mechanism for an aberrant base by hOGG1 is also corroborated in the study whereby 2-aminopurine and tryptophan fluorescence emissions were used for recording the dynamics of conformation changes [45,46].
When compared, the structures of G-and 8-oxoG-DnA bound to the enzyme reveal events taking place at the final extrusion step. At the 3'-side (Fig.  7, left flank) of the damaged base the structures are quite similar. they retain the hydrogen bond contact with the HhH, containing Gly245, Gln249, and Val250, as well as electrostatic interaction with the divalent metal ion (Fig. 7A). the only difference is the 3'-phosphate of the extra-helical nucleoside hydrogen bonded to Lys249 in the G-containing complex; whereas in the 8-oxoG-bound structure, Lys249 is unable to approach the 3'-phosphate and must rotate to face the active site to promote catalysis. It has been suggested [37] that the contacts on the 3'-side of the lesion, even more likely that of the 3' phosphate of the DnA molecule with Lys249, are made before the base extrusion is complete. From the 5'-side of the extra-helical nucleoside, the helix conformation for the G-and 8-ox-oG-structure varies. consequently, when bound to G-DnA, the helix has a more pronounced bend (~80° versus ~70°); the duplex from the 5'-terminus is also over-rotated by ~20° about the vertical axis (Fig. 7B). this discrepancy is due to the loss of hydrogen bonds established between the 3'-and 5'-phosphates and the main chain nH-group of Asn150 and the side-chain nH-group of His270 in the complex with 8-oxoG-containing DnA. A divalent cation ca 2+ that coordinates the 3'-phosphate and stabilizes the bend by inner-and outer-sphere contacts to the bases flanking the extra-helical nucleoside is also missing in the G complex (Fig. 7A). these contacts in the 8-oxoG complex are formed only after the target base has been inserted into the lesion recognition pocket. Despite the apparent advances in understanding the structural features underlying the high specificity towards damaged base, it still remains unclear how hOGG1 recognizes 8-oxoG inside the DnA helix. According to [47] this issue could be addressed by identifying hOGG1 variants that recognize the intrahelical lesion base but with a diminished capacity for binding the lesion base outside the helix.

REVIEWS
A structural analysis has been carried out [47] for hOGG1 variants with mutated amino acids that are in contact with 8-oxoG. As in similar studies [37], the authors employed the disulfide cross-linking strategy for irreversibly linking cysteine in hOGG1 with the С4 of cytosine complementary to the oxidized guanine. Mutations at position His270, which interacts with the 5'-phosphate (H270A mutant hOGG1), and at position Gln315 contacting with the outer face of the DnA molecule (Q315A mutant hOGG1), do not affect the structure but eliminate its functionality.
On the other hand, an Ala substitution at position Gly42 (G42A mutant hOGG1), removing a specific contact with 8-oxoG [36], disfavors binding of hOGG1 to DnA. As mentioned above, Gly42 is the only residue in hOGG1 that directly distinguishes between G and 8-oxoG: n7-H of 8-oxoG forms hydrogen bond with the carbonyl oxygen of Gly42 [37]. A substitution of the hydrogen atom at c α of Gly42 for a bulkier methyl group of Ala sterically impedes the binding of 8-oxoG in the active site pocket (Fig. 8A), followed by a conformational rearrangement in hOGG1.
A mutation to Q315F, which sterically disfavored the entry of 8-oxoG into the active site pocket, was studied for two variants: Q315F*149 and Q315F*292, wherein the cytosine of complementary strand was covalently linked to cys149 or cys292, respectively. the Q315F*149 hOGG1 variant actually failed to insert the 8-oxoG base into the recognition pocket but only in the exo-site (Fig. 8B). However, Q315F*292 allowed a nearly complete insertion of 8-oxoG, thus promoting hydrogen bond formation between Gly42 and n7-H of 8-oxoG (Fig. 8C). the authors suggest that it is the covalent bond to the remote cys292 residue that stabilizes the weak interaction of 8-oxoG with the recognition pocket. notwithstanding that 8-oxoG is inserted into the pocket, no cleavage takes place. Importantly, a Gln to Phe mutation in Q315F mutant hOGG1 abrogated the specificity for both intact and 8-oxoG-DnA.
to clarify the role of certain amino acid residues in the hOGG1 active site, participating in 8-oxoG binding, a photocleavable analog of 8-oxoG carrying the С6 o-nitrophenylisopropyl group (Pc) was synthesized [32]. the use of this analog in a DnA substrate coupled with flash-freezing shed light on the structure of a very late-stage intermediate in the base excision.
the structure of the Pc-bound enzyme does not differ from that of the G-bound complex; i.e., the modified Pc-base was positioned in the exo-site of the hOGG1 enzyme [37]. upon irradiation of the crystal with 373nm laser light for 30 s at 4°c, the Pc group was cleaved, unmasking a 8-oxoG/С base pair (Fc-complex). Subsequent cryotrapping in liquid nitrogen and analysis of the captured structure demonstrated that the 8-oxoG localizes in the active site pocket in the same position as in the hOGG1/8-oxoG complex [36,47]. notably, the hydrogen bond contact , recognizing the 8-oxoG base and formed by the n7-H of 8-oxoG and the carbonyl oxygen of Gly42 is retained, although the bond is longer than that in the 8-oxoG-bound substrate (Lrc-complex, 3.4 versus 2.8 Å) (Fig. 9).
no other active site residues are in contact with 8-oxoG. the three key amino acids in hOGG1, known to establish contacts with 8-oxoG, in particular Phe319, cys253, and Gln315, were shifted away from their positions in the Fc-complex, as observed in earlier described structures [36,47] (Fig. 4, Fig. 9A). In addition, the contact between His270 and the 5′-phosphate of 8-oxoG found in the Lrc-complex is not observed in the Fc-complex. this is replaced by His270 stacked with Phe319, whereas the hydrogen bond between His270 and Asp322 existing in the Fc-complex is not seen in the hOGG1/8-oxoG lesion recognition complex (Lrc). the catalytically important nucleophilic side-chain of Lys249 in the Fc-complex is disordered and is not involved in the salt bridge with cys253 (Lys249(nH 3 + )/cys253(S -)) predicted to play a role in 8-oxoG recognition [37]. the difference between the Fc-and Lrc-complexes is not limited with side-chain motions. the α-O helix, harboring the active site residues Gln315, Phe319, and Asp322, is retracted from the active site in the Fc (Fig. 9B).
Overall, the structure studied in [32] is the very late-stage intermediate discovered to date in the DnA glycosylase reaction, wherein the 8-oxoG base has achieved nearly complete insertion, with the active site not yet accommodated for the base excision to occur (see also [46]). It is also evidenced [32] that the transition of the target base from the exo-site to the pocket proceeds much faster than the subsequent conformational rearrangements for the active site to achieve catalytic competence. the strategy of disulfide cross-linking [37,47] enabled the generation of DnA-enzyme adducts in crystal form for an X-ray analysis. the hOGG1 structure, in which the intact G was extruded out of the double-stranded DnA and inserted into the pocket, was obtained and analyzed [48]. no cleavage of the n-glycosidic bond was observed. the failure to break this bond was not due to the disulfide crosslink in the enzyme-DnA adduct, since the G to 8-oxoG replacement in this complex did promote base excision. these findings indicate a mechanism by which the G base is rejected late in the base excision pathway after it has been mistakenly inserted into the hOGG1 active site. this mechanism is triggered on those rare occasions when G overcomes the transition energy barrier from the exo-site to the active site pocket. the mechanism of the rejection of G remains unknown. It was earlier shown that the n-glycosidic bond in G nucleoside is more labile to hydrolysis at neutral pH than in 8-oxoG [49]. For this reason, the discrimination between the n-glycosidic bonds of G and 8-oxoG cannot be explained by a variation in bond stability.
there is also evidence in support of the existence of a mechanism by which G is rejected once it has been presented to the recognition pocket [46]. using stopped-flow kinetics, the catalytic effects of hOGG1 with specific and non-specific DnA substrates were studied. the combination of tryptophan and 2-aminopurine fluorescence was used to follow the conformational dynamics of DnA, as well as the conformational transitions in the enzyme. the duplex DnA molecules used contained either 8-oxoG or a non-damaged G base. When binding to hOGG1, duplex DnA exhibited double helix disruption (judged by an increase in the 2-aminopurine fluorescence) at ~10 and 20 ms, respectively. this is due to DnA bending and flipping G or 8-oxoG out of the helix [46]. At > 20 ms in the case of 8-oxoG, a decline in the 2-aminopurine fluorescence was recorded, which was likely to correspond to the entry of amino acids into the DnA gap and complex stabilization. no similar changes in the 2-aminopurine fluorescence were observed with the G-base. this means that binding of hOGG1 to a non-specific DnA substrate could lead to DnA bending and eversion of the G base into the exo-site; however, G is rejected and the enzyme fails to achieve a catalytically competent state.
Verdine et al. [48] proposed that the energy barrier of the transition state for breaking the n-glycosidic bond of G could be higher than that for 8-oxoG because of the conformational changes in the active site or deprivation of the transition state stabilization (enzyme-G) ü through hydrogen bonding with Gly42. In addition, the G base in the active site is in a slightly different position relative to 8-oxoG as described recently [37]. this REVIEWS could prevent the enzyme from adopting the optimal conformational state required for an attack on the С1' of the deoxyribose sugar by the Lys249 side-chain amino group. this is in good agreement with the finding indicating that introduction of active site mutations, that even slightly disturb 8-oxoG disposition, namely D268n [38] and Q315F [47], dramatically (but not completely) decreases the hOGG1 catalytic activity.
Lukina et al. [50] engineered c253L and c253I mutant hOGG1 forms with an occluded active site pocket by replacement of a cys253 to a bulky leucine or isoleucine. Despite the perturbed active site geometry afforded by this mutation and dramatic decline in catalytic activity, the enzyme was still catalytically active. these findings argue for the concept of active site plasticity that postulates that the hOGG1 active site is flexible enough to mitigate the steric hindrance brought about by mutations [50]. the engineered complex [48] is structurally very similar to that produced with an 8-oxoG lesion. the differences in the interaction with G and 8-oxoG could be attributed to conformational adjustments to the lesion recognition pocket. It is suggested that while scanning for the target base, hOGG1 can occasionally flip the non-damaged G base into its active site [48]. the obtained data provide a basis to conclude that such erroneous insertions of G into the hOGG1 active site do not result in n-glycosidic bond cleavage due to the discrimination capacity between 8-oxoG and G at the catalytic stage.
there is a hypothesis that hOGG1 captures altered dynamic characteristics in the 8-oxoG·c base pair relative to the G·c base pair. this was tested by assessing the spontaneous opening of complementary pairs in double-stranded DnA using nMr spectroscopy and proton exchange [51]. It was demonstrated that the rate of spontaneous opening of 8-oxoguanine and the lifetime of the base in the extra-helical state are the same as those of a canonical guanine-cytosine base pair. this finding does not support the role of the opening dynamics of 8-oxoguanine in the recognition of the lesion by DnA glycosylases.

concluSionS
In this review, we have focused on the structures of the DnA repair enzyme human 8-oxoguanine-DnA-glycosylase in free form and in complexes with DnA substrates. the currently available literature and data on the 3D structures of hOGG1 deposited in the Protein Data Bank (PDB) have been summarized. Lys249 and Asp268 are shown to be the key amino acids responsible for catalysis. Gly42, Asn149, cys253, His270, Gln315, and Phe319 are the amino acids important for discrimination between 8-oxoG and G and for binding the target base in the active site pocket. the hOGG1-mediated mechanism of oxidized base excision is reviewed in terms of structural dynamics. It obvious that the eversion of damaged base from the DnA helix into active site of enzyme cannot occur as a concerted one-step process but, it seems to proceed through multiple discrete states. It is reasonable to hypothesize that base-specific cleavage by hOGG1 is controlled throughout the interaction: lesion recognition, base extrusion, binding of 8-oxoG into the active site pocket, and the catalytic hydrolysis of the n-glycosidic bond.


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Genome-wide analysis of SRSF10-regulated alternative splicing by deep sequencing of chicken transcriptome

Splicing factor SRSF10 is known to function as a sequence-specific splicing activator that is capable of regulating alternative splicing both in vitro and in vivo. We recently used an RNA-seq approach coupled with bioinformatics analysis to identify the extensive splicing network regulated by SRSF10 in chicken cells. We found that SRSF10 promoted both exon inclusion and exclusion. Functionally, many of the SRSF10-verified alternative exons are linked to pathways of response to external stimulus. Here we describe in detail the experimental design, bioinformatics analysis and GO/pathway enrichment analysis of SRSF10-regulated genes to correspond with our data in the Gene Expression Omnibus with accession number GSE53354. Our data thus provide a resource for studying regulation of alternative splicing in vivo that underlines biological functions of splicing regulatory proteins in cells.

Sample preparation and RNA-seq analysis
It is known that phosphorylated SRSF10 functions as a sequencespecific splicing activator that is capable of regulating alternative splicing both in vitro and in vivo [1,2]. To understand the global profile of the alternative splicing network regulated by SRSF10 in vivo, we carried out an RNA-seq approach to analyze the effects of SRSF10 depletion on splicing regulation in chicken DT40 cells. The general strategy for sample preparation for RNA-seq was provided in Fig. 1A and the detailed experimental procedures were as follows. Wild type (WT) and SRSF10-knockout (KO) DT40 cells were cultured in a standard medium to 90% confluence. Total RNA was isolated from these cells, and cDNA libraries were prepared individually by performing a series of experiments, including poly(A) enrichment, RNA fragmentation, random hexamer-primed cDNA synthesis, linker ligation, size selection and PCR amplification. After selecting approximately 200-bp fragments by agarose gel electrophoresis, Illumina paired-end sequencing adapters were ligated to the DNA fragments for PCR amplification and sequencing with an Illumina HiSeq™ 2000 system.
The bioinformatics analysis pipeline for RNA-seq data was provided in Fig. 1B. Briefly, images generated by sequencers were converted by base calling into nucleotide sequences, which were called raw reads. Then raw reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence to get clean reads with high quality. The clean reads were then aligned to the reference chicken genome (release: galGal4) and the corresponding recently annotated genes (galGal4.0) using the TopHat program (v2.0.6) [3]. In the alignment, preliminary experiments were performed to optimize the alignment parameters to provide the most information on the alternative splicing events. Cufflinks [4] was used to construct KO and WT DT40 transcriptomes and Cuffmerge merges the transcriptomes with the reference gene annotations (galGal4.0) to generate a new comprehensive transcriptome. Finally, a Java program called ASD (alternative splicing detector, available at http://www.novelbio.com/asd/ASD.html) was developed to detect and assess alternative splicing events between the two samples [5].

Contents lists available at ScienceDirect
Genomics Data

SRSF10 both activates and represses exon inclusion
There are five major modes of alternative splicing (AS) events described in metazoan organisms, including cassette exon, alternative 3′ splice site, alternative 5′ splice site, mutually exclusive exon and retained intron ( Fig. 2A). With a p value cutoff of b 0.05, we were able to identify 167 events belonging to the five major modes of AS from a total number of 10,652 splicing events that changed significantly between the WT and the KO samples by ASD. Specifically, the majority of the affected splicing events (130) belonged to the cassette exon Cufflinks and Cuffmerge were then used to construct and merge DT40 transcriptomes. The merged transcriptome and the read counts in exonic reads and junction reads were then further analyzed using our Java program ASD to identify differential alternative splicing events between WT and KO samples. category. Strikingly, analytical results by ASD indicated that SRSF10 could both activate and repress exon inclusion (Fig. 2B). RT-PCR validation of SRSF10-affected splicing events further demonstrated this prediction. In addition, the selection of alternative 3′ and 5′ splice sites was similarly affected by SRSF10. Taken together, these results provide strong evidence that SRSF10 can promote both exon inclusion and exclusion in vivo.

Function enrichment analysis of SRSF10-affected genes
To understand how loss of SRSF10 affects cellular biological processes, we next performed gene ontology (GO) and pathway analysis of SRSF10-affected genes by using ClueGO [6]. Function enrichment analysis indicated that these affected spliced genes are involved in multiple processes, including histone methylation, response to external stimulus, glucose metabolic processes and mitochondrion distribution (Fig. 3). These results illustrated that SRSF10 might play an important role in a wide range of biological processes by controlling its downstream splicing targets.

Discussion
We present here a unique dataset of RNA-seq results in which we globally analyzed SRSF10-regulated AS events based on deep sequencing of the chicken transcriptome. We found that SRSF10 is involved in all of the common modes of AS, with cassette exons being the most frequent targets for SRSF10 regulation. We also found that SRSF10 could both activate and repress exon inclusion in vivo. Finally, SRSF10affected splicing genes were involved in multiple processes, indicating a role of SRSF10 in these processes. This dataset is of high quality and will facilitate future study of SRSF10 function in vivo and will also improve understanding of AS regulation in vivo.


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Very small deletions within the NESP55 gene in pseudohypoparathyroidism type 1b

Pseudohypoparathyroidism (PHP) is caused by reduced expression of genes within the GNAS cluster, resulting in parathormone resistance. The cluster contains multiple imprinted transcripts, including the stimulatory G protein α subunit (Gs-α) and NESP55 transcript preferentially expressed from the maternal allele, and the paternally expressed XLas, A/B and antisense transcripts. PHP1b can be caused by loss of imprinting affecting GNAS A/B alone (associated with STX16 deletion), or the entire GNAS cluster (associated with deletions of NESP55 in a minority of cases). We performed targeted genomic next-generation sequencing (NGS) of the GNAS cluster to seek variants and indels underlying PHP1b. Seven patients were sequenced by hybridisation-based capture and fourteen more by long-range PCR and transposon-mediated insertion and sequencing. A bioinformatic pipeline was developed for variant and indel detection. In one family with two affected siblings, and in a second family with a single affected individual, we detected maternally inherited deletions of 40 and 33 bp, respectively, within the deletion previously reported in rare families with PHP1b. All three affected individuals presented with atypically severe PHP1b; interestingly, the unaffected mother in one family had the detected deletion on her maternally inherited allele. Targeted NGS can reveal sequence changes undetectable by current diagnostic methods. Identification of genetic mutations underlying epigenetic changes can facilitate accurate diagnosis and counselling, and potentially highlight genetic elements critical for normal imprint setting.

INTRODUCTION
Pseudohypoparathyroidism (PHP) is caused by insensitivity to parathormone (PTH), and the majority of cases are associated with insufficiency of the G protein stimulatory alpha subunit (Gs-a). 1 In PHP type 1a (OMIM #103580), additional features may include obesity, short stature, brachydactyly, ectopic ossification and cognitive impairment. PHP1b (OMIM #603233) is classically defined as sharing the biochemical but not the skeletal features of PHP1a, though this distinction is not absolute. 2 PHP1b is not associated with coding mutations of GNAS, but with epigenetic errors altering its expression. The GNAS gene cluster (OMIM þ 139320) on human chromosome 20 is under complex imprinted regulation, with transcripts variably expressed from the maternal allele, the paternal allele, or biallelically, in a tissue-dependent fashion. 3 The clinical suspicion of PHP1b is confirmed by detecting abnormal DNA methylation at imprinting control regions within the GNAS cluster. [4][5][6] Some patients show alterations at multiple imprinting control regions (ICRs) within the cluster, but the most consistent aberration is hypomethylation of the A/B differentially methylated region directly upstream of the major coding isoform of GNAS. Isolated hypomethylation of this region is the commonest alteration in familial PHP1b; in all cases so far described, it has been associated with deletion of a presumed regulatory element within the STX16 gene. 7,8 Much rarer are deletions affecting NESP55; [9][10][11] these are accompanied by epigenetic disruption throughout the locus, and evidence from murine models suggests they disrupt transcription and thereby primary imprint setting throughout the GNAS cluster. 12 However, apart from these and some cases of paternal uniparental disomy (UPD20pat), 13 the majority of PHP1b cases seem to be caused by a primary epimutation without an underlying genetic aberration.
We hypothesised that additional genetic variants may result in epimutations of the GNAS differentially methylated regions and screened PHP1b patients, using genomic next-generation sequencing through the GNAS cluster.

Patient recruitment: ethics
Clinical diagnosis of PHP1b was made locally by referring clinicians, and patients were consented into the research study 'Imprinting disorders-finding out why' (IDFOW: Southampton and South West Hampshire Research Ethics approval 07/H0502/85) through the UK Comprehensive Local Research network (www.southampton.ac.uk/geneticimprinting/informationpatients/ imprintingfindingoutwhy.page, accessed September 2013). Diagnosis was confirmed by methylation-specific PCR and microsatellite analysis as described. 14 Twenty patients had PHP1b with methylation anomalies affecting the GNAS AB, XLAS, NESP-AS and NESP differentially methylated regions (DMRs), but no evidence of UPD20 or NESP deletion; 9 STX16 deletions were also excluded; and there was no evidence of hypomethylation at any other imprinted locus. Two control PHP1b patients were included: one with UPD20pat and one with a deletion of STX16.
Clinical findings in the two families with genetic variants are presented in Table 1. Briefly: Family 1: a non-dysmorphic sister and brother presented with exerciseinduced muscle cramping in childhood and hypocalcemia. Skeletal survey was unremarkable. The mother was clinically unaffected.
Family 2: a male presented at 2 years 3 months with faltering growth, increasing weight and global developmental delay, a consistently raised serum phosphate and subsequently a raised serum PTH. Skeletal survey was unremarkable. Family history was non-contributory ( Figure 1h).

Bioinformatic analysis
We developed a bioinformatic pipeline for identifying both single-nucleotide variants (SNVs) and copy number variants (CNVs  16 ) to avoid misalignment that might cause mismatches and compromise base quality recalibration. Base quality recalibration was done using GATK ' Table  Recalibration' function, which analysed several covariates to remove systematic biases and improve accuracy of quality scores. 17 All produced aligned files were further used to calculate average read depth in 1000 bps non-overlapping windows within the captured region (Supplementary Table 2). The BAM files were also used to normalise read depth between samples, which was used to compare samples between different pools (Supplementary Table 2). These realigned and recalibrated BAM files were then used to determine SNVs and CNVs. Variant (SNVs eg SNPs and indel alleles) predictions and genotyping were performed using the GATK 'Unified Genotype' function for each sample by computing allele frequency distribution, 18 and raw variant calls were outputted in VCF (variant call format). An individual variant filtration (such as Quality by Deptho2.0, Fisher Strand Bias460.0 etc) was performed for both SNPs and indels using GATK Variant Filtration method to remove low quality and potentially false positive sites. Variant data were further annotated by Annovar to provide functional annotation of variants. 19 Pindel (version 0.2.4 18 ), a structural variant detection tool that can detect breakpoints of large deletions and medium-sized insertions from paired-end short reads, was used to perform CNV predictions. These CNVs were further filtered based on variant size, alternate allele frequency, CA/GT homopolymer repeats and known SNPs. These filters increased sensitivity and specificity of the predictions, which reduced false negatives and false positives.

RESULTS
In seven PHP1b patients, the GNAS locus (chr20.GRCh37: g.57200800_57627000) was enriched by targeted capture, and analysed by next-generation sequencing. On average, more than 98% of the captured regions had read depth greater than 10, showing that the sequence data were of high quality in our region of interest (Supplementary Table 3). Case 1 of family 1, one of two affected siblings, harboured a 40-bp deletion spanning the genomic co-ordinates chr20.GRCh37:g.57416653_57416693del (family 1, Figure 1c). This deletion was intronic to both NESP55 and NESP-AS. It was not present in dbSNP137, Repeat Masker (http:// genome.ucsc.edu) or in 168 control samples. The deletion was detected in the affected sibling and also in the siblings' mother who showed no clinical signs of PHP1b. To determine the parental origin of the deletion in the mother, her DNA was amplified after digestion with methylation-specific restriction enzymes BstUI and McrBc (which digest only unmethylated and methylated DNA, respectively). The deletion was detectable only in DNA restricted by McrBc, demonstrating that it was present on her unmethylated, maternally inherited allele (Figure 1d and g). The maternal grandmother of the affected sibs also harboured the deletion, though methylation-specific restriction analysis in her case showed it to be on the paternally inherited allele (Figure 1g).
Building on this finding, the genomic region chr20.GRCh37: g.57413337_57419096 was amplified by long-range PCR and sequenced by NGS in samples from an additional 14 PHP1b patients with no known underlying genetic aberration. A 33-bp intronic deletion was identified (chr20.GRCh37:g.57418256_57418290del) in one further patient (case 9, Figure 1, Table 1). Targeted sequencing showed the same deletion to be present in his unaffected mother. The deletion was too distant from the DMR to use methylation-specific restriction to determine its origin in the mother. The deletion was not present in the maternal grandmother of patient 2 (data not shown); the maternal grandfather was deceased and no DNA was available for analysis, but he was not known to have any clinical features of PHP1b. The variant in family 2 represented a deletion of two of three copies of an 18-bp repeat element; deletion of one copy was represented within dbSNP137 as rs36230182 (minor allele frequency unknown); however, there was no evidence of any deletion in 168 normal controls. In the remaining 13 patients, CNVs were called by Pindel and SNPs were called by GATK, but no novel variants were identified (data not shown).
DNA methylation analysis showed the affected sibs in family 1 had essentially complete paternalisation of the GNAS cluster, while hypomethylation in proband 2 was incomplete, except at the GNAS A/B DMR (Supplementary Table 1).

DISCUSSION
Like other imprinting disorders, PHP1b is clinically and molecularly heterogeneous. Sporadic PHP1b is associated with UPD20pat and epimutation of DMRs throughout the GNAS cluster; most heritable PHP1b results from epimutation of the GNAS A/B DMR-associated with maternal STX16 deletions-and rarely from deletions of NESP55 of maternal origin. We used NGS of the GNAS locus to seek genetic lesions underlying epimutations in PHP1b patients. In two affected siblings, a variant was found in the first intron of NESP55, within the 3-kb deletion described by Bastepe et al. 9 Further localised sequencing within this region revealed another deletion in a third, unrelated individual. Both variants were maternally transmitted, but the mothers showed no clinical or epigenetic evidence of PHP1b. In family 1, the clinically unaffected mother inherited the deletion from her own mother, showing that maternal inheritance of this deletion does not always cause PHP1b.
Large NESP55 deletions are believed to abrogate germline transcription from the NESP promoter, resulting in aberrant methylation setting throughout the whole imprinted cluster. 9,12 Notably, the variants reported here are close to the transcriptional start site of NESP55, overlapping those described by Bastepe, Chillambhi and Richard et al. [9][10][11] Such deletions potentially delineate critical sequences for imprint setting of this locus.
We detected variants in only two of 20 PHP1b index cases (albeit with different extents of sequencing). NESP deletions 41 kb were not detected in any individual. By contrast, STX16 deletions are found in essentially all individuals with GNAS A/B hypomethylation. It remains unclear whether genetic NESP mutations are uncommon in PHP1b. It may be that in the patients we studied, some variants remained undetected because of discontinuities in sequence coverage; CNVs (like the STX16 deletion) may arise between repetitive sequences, but these sequences cannot be unambiguously captured, results in reduced or absent coverage. Further variants may lie outside the region we targeted, in more distant chromatin-regulating domains. Alternatively, it may be that the majority of PHP1b is indeed sporadic, caused by epimutations without direct genetic cause. Genetic mutations underlying epimutations have been reported very infrequently in other imprinting disorders, including Russell-Silver Syndrome, transient neonatal diabetes mellitus and Beckwith-Wiedemann syndrome associated with KCNQ1OT1 (imprinting control region 2) hypomethylation. However, a significant minority of BWS patients with H19 (ICR1) hypermethylation have maternally inherited deletions or point mutations of ICR1. 20 Genomic analysis of other patient groups could potentially identify mutations that would help to uncover further determinants of epigenetic function.
It is striking that the individuals with NESP55 deletions were not identical, either in phenotype or in DNA methylation status. Clinical features in the affected individuals were somewhat atypical of PHP1b, which is associated chiefly with biochemical features of parathormone resistance in the absence of dysmorphism. 3 The probands of family 1 have mild learning difficulties, and one has calcifications in brain tissue; the proband in family 2 has educational difficulties warranting educational support, and has short stature, which is more typical of PHP1a. It is recognised that a minority of individuals with PHP1b epimutations have clinical features overlapping with PHP1a. [21][22][23][24] In the three individuals with PHP1b, the deletions resulted in epimutations throughout the GNAS cluster; but while the siblings in family 1 had almost complete hypomethylation, the proband in family 2 had less marked hypomethylation, except at the GNAS A/B DMR. In family 1, a deletion was transmitted maternally through three generations, but only the two-third-generation cases 1 and 2 presented with PHP1b. This may be because the deletion is coincidental to PHP-though given the presence of deletions in two of 20 pedigrees, and their absence from controls and databases, this seems unlikely. It may be that these small deletions are not fully penetrant, and do not always cause PHP1b. Alternatively, the severity of the deletion's effect may increase with successive generations, causing apparent genetic anticipation-this phenomenon has been observed in maternally transmitted BWS associated with hypermethylation of H19. 15 Investigation of further cases will shed light on these unresolved issues. In summary, genomic sequencing is a powerful tool with potential to reveal non-coding genetic variants that may underlie epigenetic disorders.