Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2017/02/10/dmd.116.073254.DC1

1521-009X/45/4/375–389$25.00 http://dx.doi.org/10.1124/dmd.116.073254 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 45:375–389, April 2017 Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics Minireview

Alternative Splicing in the Cytochrome P450 Superfamily Expands Protein Diversity to Augment Gene Function and Redirect Human Drug Metabolism s

Andrew J. Annalora, Craig B. Marcus, and Patrick L. Iversen

Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon

Received September 2, 2016; accepted February 6, 2017 Downloaded from ABSTRACT The human genome encodes 57 cytochrome P450 genes, whose to a disease pathology. The expansion of P450 transcript diversity enzyme products metabolize hundreds of drugs, thousands of xeno- involves tissue-specific splicing factors, transformation-sensitive al- biotics, and unknown numbers of endogenous compounds, including ternate splicing, trans-splicing between gene transcripts, single- steroids, retinoids, and eicosanoids. Indeed, P450 genes are the first nucleotide polymorphisms, and epigenetic regulation of alternate line of defense against daily environmental chemical challenges in a splicing. Homeostatic regulation of variant P450 expression is influ- dmd.aspetjournals.org manner that parallels the immune system. Several National Institutes of enced also by nuclear receptor signaling, suppression of nonsense- Health databases, including PubMed, AceView, and Ensembl, were mediated decay or premature termination codons, mitochondrial queried to establish a comprehensive analysis of the full human P450 dysfunction, or host infection. This review focuses on emergent transcriptome. This review describes a remarkable diversification of aspects of the adaptive gene-splicing process, which when viewed the 57 human P450 genes, which may be alternatively processed into through the lens of P450–nuclear receptor gene interactions, resem- nearly 1000 distinct mRNA transcripts to shape an individual’s P450 blesaprimitiveimmune-likesystemthat can rapidly monitor, respond, proteome. Important P450 splice variants from families 1A, 1B, 2C, 2D, and diversify to acclimate to fluctuations in endo-xenobiotic exposure. at ASPET Journals on September 25, 2021 3A, 4F, 19A, and 24A have now been documented, with some displaying Insights gained from this review should aid future drug discovery and alternative subcellular distribution or catalytic function directly linked improve therapeutic management of personalized drug regimens.

Introduction transcripts and polypeptide products will improve our ability to assess the Mapping the human genome sequence was completed in 2001 (Lander functional nature of its role in both physiologic and pathologic conditions et al., 2001), helping to usher in the “postgenomic era” of personalized (La Cognata et al., 2014). The spectrum of alternate splicing mechanisms medicine. Over a decade later, however, the promise of pharmacoge- underlying the expansion of the proteome involves the use of seven main nomics (PGx) has yet to fully materialize, and the composition of the splicing types, as previously described (Blencowe, 2006; Roy et al., human genome remains enigmatic, as numerous questions linger con- 2013). The molecular basis for these mechanisms is complex and beyond cerning the complexity of its content and organization. For example, how the scope of this review; however, a brief description of the phenomenon can a genome with only 22,000 genes produce a proteome with over and its key players is provided in the supplemental materials (see 200,000 distinct proteins? While transcription is a relatively well un- Supplemental Fig. 1). Here we will focus primarily on the spectrum of derstood phenomenon, the mechanisms involved in regulating alternative phenotypic outcomes induced by alternative transcript splicing, in precursor or unprocessed messenger RNA (pre-mRNA) splicing, the order to highlight the array of splice-sensitive features operating in the driving force behind both transcriptome and proteome expansion, remains cytochrome P450 (P450) superfamily, a collection of 57 human genes less appreciated. The ability to link a single gene to its full suite of RNA that coordinate the metabolism of both drugs and endoxenobiotics. This work focuses new attention on the biologic impact of alternative P450 gene splicing, an underappreciated component of phase I drug metabolism that may complicate or disrupt personalized approaches to This research was supported by start-up funds from the Office of Research and medicine. Precision medicine, therefore, through the universal applica- the College of Agricultural Sciences at Oregon State University (Corvallis, OR) awarded to Drs. Marcus and Iversen. tion of genetics, still faces many challenges beyond cost, ethical dx.doi.org/10.1124/dmd.116.073254. considerations, and the need for additional, whole genome sequences. s This article has supplemental material available at dmd.aspetjournals. Multiple resources have now been developed, in addition to the org. Human Genome Project, to address these challenges, including: the

ABBREVIATIONS: BP, benzo(a)pyrene; ER, endoplasmic reticulum; GWAS, genome-wide association studies; NMD, nonsense-mediated decay; NR, nuclear receptor; P450, cytochrome P450; PGx, pharmacogenomics; PPAR, peroxisome proliferator–activated receptors; pre-mRNA, precursor or unprocessed messenger RNA; ROS, reactive oxygen species; SNP, single-nucleotide polymorphism; snRNA, small nuclear RNA; VDR, vitamin D receptor.

375 376 Annalora et al.

Hapmap Project, which enables single-nucleotide polymorphism and intronic-splicing enhancers) with tissue-specific, trans-acting, (SNP) arrays for .100,000 SNPs; the 1000 Genomes Project; the splicing regulatory factors. In this way the same pre-mRNA transcript ENCODE project for noncoding RNAs; and the National Human can be processed into tissue-specific, alternately spliced forms (Wang Genome Research Institute (NHGRI) genome-wide association and Burge, 2008). The cytochrome P450 superfamily of genes is well studies (GWAS) catalog. GWAS have now identified two P450s studied from many perspectives (Guengerich, 2013; Johnson and that represent a biomarker for disease or a drug-response phenotype, Stout, 2013; Pikuleva and Waterman, 2013). As of late 2015, PubMed including: 1) a CYP2C8 association with bisphosphonate-related listed over 23,790 citations referencing human P450 genes (according osteonecrosis of the jaw in multiple myeloma (Sarasquete et al., to GeneCards.org), with much of this work being focused on tissue- 2008); and 2) a CYP2C19 association with clopidogrel interindividual specific expression or catalytic function of individual, reference forms. variation as an antiplatelet drug (Shuldiner et al., 2009). Aside from Alternative splicing of P450s has also been well studied for more than showing strong associations, few pharmacogenetic biomarkers of this two decades; however, the structural and functional diversity of P450 sort have successfully transitioned from discovery to clinical practice splice variants, and their relationship to human health and disease, (Carr et al., 2014). Many GWAS studies have failed to provide remains poorly understood. A summary of known P450 splice variants prognostic value because they are not designed to evaluate how linked to human disease is presented in Table 1. Reports from groups variant gene expression is influenced by both cellular and environ- attempting to synthesize a global view of alternative P450 splicing, mental splicing factors. In this regard, we feel a new appreciation is and its relevance to our understanding of human metabolism in the needed for the complexity of alternative splicing. For not only is the context of PGx or personalized medicine, are exceedingly rare Downloaded from world of both coding and noncoding RNA more diverse and more (Nelson et al., 2004; Turman et al., 2006). complex than we could have imagined even a decade ago, it is teeming Our review has led us to conclude that personalized approaches to with untold amounts of RNA “dark matter” and other relics of the medicine whose sole basis is GWAS may be misleading, in that they fail RNA world, whose structure and function must be fully appreciated to account for variability in compensatory splicing mechanisms, which and classified before a new guiding orthodoxy for “individualized can modulate the expression of some highly penetrant mutations. This medicine” can be safely conceived (St. Laurent et al., 2014; Cowie phenomenon is exemplified by ontogenetic regulatory factors that can et al., 2015). mask the physiologic impact of genetic variations among developmen- dmd.aspetjournals.org tally regulated P450 genes (Hines and McCarver, 2002). To expand on this concept, and explore the array of alternative splicing products that Meta-Analysis of Alternative Gene Splicing in the Cytochrome serve as an improved platform for predicting disease whose basis is the P450 Superfamily functional genome, we annotated all of the known human P450 splice Alternative splicing regulated by tissue-specific factors is a means of variants currently listed in the NCBI’s PubMed, AceView, and Ensembl adapting to physiologic demands. Tissue-specific splice variants may databases (Thierry-Mieg and Thierry-Mieg, 2006; Cunningham et al., arise from tissue-specific promoter elements (Wiemann et al., 2005) or 2015) as of late 2015. From this computational meta-analysis, we found at ASPET Journals on September 25, 2021 tissue-specific–splicing regulatory elements (Black, 2000). Exon and that the 57 human P450 genes produce a P450 “transcriptome cloud” intron definition requires interaction of discrete cis-elements (exonic- composed of at least 965 unique mRNA transcripts (Table 2). This splicing enhancers, exonic-splicing silencers, intronic splicing silencers, number represents all known reference (or wild-type) and variant P450

TABLE 1 Pathologies associated with P450 splice variants

P450 Isoform Variant Form Related Disease Reference ΔExon 2 and 6 Ovarian cancer Chinta et al. (2005) CYP1A1 Bauer et al. (2007) Leung et al. (2005) Partial ΔExon 2 Glaucoma Stoilov et al. (1998) CYP1B1 Li et al. (2014) CYP2A6 Deletions Lung cancer Liu et al. (2013b) CYP2B6 ΔExons 4–6 Liver/colon cancer Miles et al. (1989) ΔExons 2 and 9 Essential hypertension Nakayama et al. (2002b) CYP8A1 Nakayama et al. (2002a) CYP11B2 Trans-splicing to CYP11B1 Congenital adrenal hyperplasia Hampf et al. (2001) ΔExons 2, 4, 6, or 8 17-a Hydroxylase deficiency, Yamaguchi et al. (1998) CYP17A1 congenital adrenal hyperplasia Costa-Santos et al. (2004) Hwang et al. (2011) CYP19A1 ΔExon 5 Aromatase deficiency Pepe et al. (2007) CYP24A1 ΔExons 9–11 Breast, colon, and prostate cancer Muindi et al. (2007) ΔExon 2 Atherosclerosis Elmabsout et al. (2012) CYP26B1 Oral cancer Chen et al. (2014) ΔExons 4,6, 7 Cerebrotendinous xanthomatosis Garuti et al. (1996) CYP27A1 Verrips et al. (1997) Chen et al. (1998) ΔExons 4–7 Glioblastoma, leukemia, melanoma, Maas et al. (2001) cervical cancer, ovarian cancer Flanagan et al. (2003) CYP27B1 Fischer et al. (2007) Wu et al. (2007) Fischer et al. (2009b) Intron 2 Alzheimer disease Li et al. (2013a) CYP46A1 Li et al. (2013b) Alternative P450 Splicing Redirects Human Drug Metabolism 377 mRNAs, including some with retained introns, premature termination influence of a point mutation on gene function. Although compu- codons (PTCs), and those subject to nonsense-mediated decay (NMD); tational studies indicate that 85% of all human mRNA comes from a we also included several experimentally derived transcripts described in single major transcript (Gonzàlez-Porta et al., 2013), the P450 PubMed-listed citations (identified by searching CYP* or P450 splice superfamily of genes is highly diverse at the genomic level, making variant or alternative CYP* or P450 splicing) that were not represented it uniquely sensitive to alternate splicing events that alter the biologic in the AceView or Ensembl databases. output of both genetic and epigenetic signaling cascades. Nuclear Our results approximate that the average P450 gene encodes nearly receptors are now recognized for their role in mediating global gene- 20 unique mRNA transcripts, capable of yielding both reference and splicing events (Auboeuf et al., 2005), and an improved framework splice-variant proteins, and an ensemble of noncoding RNA molecules. for how this process functions to regulate drug metabolism appears to Over 53% of these transcripts (515) are predicted to express viable be in order, one that can potentially recontextualize the utility and proteins, on the basis of the most stringent expression criteria used by complexity of the convoluted metabolic crosstalk network that underlies both the AceView and Ensembl databases (Table 2). Subsequent the 48 nuclear receptor (NR) signaling pathways operating in humans analysis of the GeneCards.org website revealed that the 57 human (Meyer, 2007; Tralau and Luch, 2013). P450s are associated with over 48,384 SNPs, approximating to roughly Furthermore, drug metabolizing P450s of families 1–4 have tradi- 850 mutations per P450 gene (Stelzer et al., 2011). Although most P450s tionally been considered more polymorphic than P450s that metabolize fall within this general range of genetic variation, some, including CYPs endogenous, cholesterol-based substrates and are assumed to be more 2C19, 5A1, 19A1, and 39A1, express more than twice as many SNPs sensitive to SNP-based modulation of alternative splicing (Lewiñska Downloaded from as the average, suggesting the accumulation process is nonrandom et al., 2013). On the basis of our analysis, it does appear that that the (Table 2). The total number of diseases associated with each of the majority of steroid-metabolizing P450s, including CYP17A1 (91 SNPs) 57 human genes (1057) was also calculated using the MalaCards Human and CYP21A2 (173 SNPs), are among the least polymorphic P450 Disease Database (Rappaport et al., 2014). P450 families 1 (203) and genes. As described above, mutations in these P450 enzymes are closely 2 (413) account for over half of the total disease associations, whereas linked to congenital defects such as pseudohermaphroditism and adrenal orphan P450s CYP20A1 (1) and CYP39A1 (2) are associated with the hyperplasia (Yamaguchi et al., 1998; Doleschall et al., 2014), and SNPs fewest. may be less well tolerated in these systems owing to their central role in dmd.aspetjournals.org A side-by-side, radar-plots comparison of the total number of the production of sex hormones, progestins, and corticoids. In contrast, alternative P450 splice variants and SNPs for each of the 57 human the 35 drug-metabolizing P450s that compose P450 families 1–4in P450 genes is shown in Fig. 1. Pharmacologically relevant drug targets, humans average over 700 SNPs per isoform, and the 25,659 total such as CYP2C9, CYP2D6, CYP3A5, CYP11A1, and CYP19A1, are polymorphisms in this group represent over 53% of the total P450 among the most highly spliced P450 genes, the majority of which are SNP population. Although CYP2C9 (1422 SNPs), CYP2C18 also subject to trans-splicing events. Although differences in gene (1230 SNPs), and CYP2C19 (2467) are the most polymorphic drug- organization or cell-specific selection pressure may play a role in this metabolizing P450s, they are considerably less polymorphic than at ASPET Journals on September 25, 2021 phenomenon, our observations suggest that drug-dosing guidelines genes like CYP5A1 (5274 SNPs), which metabolizes prostaglan- derived from knowledge of a patient’s individual P450 transcriptome dins, and CYP7B1 (3400 SNPs), which converts cholesterol to bile may be far superior to those resulting from genotyping alone, as SNP acids. CYP5A1 is also known as thromboxane synthase (TBXAS1), a approaches may tend to overstate (or oversimplify) the biologic functionally distinct P450 that catalyzes molecular rearrangements of

TABLE 2 Summary of human P450 splice variants

Variant Disease Ratio of Viable Enzyme Family Transcript Size Exons Amino Acids Mol. Weight SNPs RNAs Links Protein

Gene no. Nucleotides kDa Total no. Total no. Total CYP1 (3) 2683–5988 3–7 512–543 58.2–60.8 49a 1052 203 0.64b (31) CYP2 (16) 1681–5024 5–9 491–596 55.7–67.6 281 11853 413 0.50 (141) CYP3 (4) 1604–2167 13–14 502–534 57.1–61.3 120 3029 93 0.47 (56) CYP4 (12) 1678–3395 5–16 505–638 59.1–71.3 170 9725 37 0.53 (90) CYP5 (1) 2093 13 594 66.9 30 5274 6 0.48 (14) CYP7 (2) 2765–2950 6 504–506 57.7–58.3 4 3688 35 0.80 (3) CYP8 (2) 3974–5632 1–10 501–527 58.1–59.5 9 1752 16 0.77 (7) CYP11 (3) 2311–3558 9 503–521 57.6–60.1 52 1807 74 0.57 (30) CYP17 (1) 2001 8 508 57.4 13 179 31 0.57 (7) CYP19 (1) 4479 10 503 57.9 47 2178 45 0.57 (27) CYP20 (1) 1811 13 470 53.4 21 1308 1 0.54 (11) CYP21 (1) 2153 10 495 56.0 30 91 35 0.52 (16) CYP24 (1) 3266 12 514 58.9 17 711 19 0.46 (8) CYP26 (3) 1569–4536 6–7 497–522 56.2–57.5 23 858 22 0.68 (16) CYP27 (3) 2579–5117 8–9 372–531 42.6–60.2 55 1582 58 0.58 (32) CYP39 (1) 2623 12 469 54.1 15 2179 2 0.50 (8) CYP46 (1) 2186 15 500 56.8 11 952 7 0.57 (6) CYP51 (1) 2817 6 860 98.0 18 388 2 0.65 (12) Total 57 1569–5632 1–16 372–860 42.6–98.0 965 48,384 1097 0.534 (515)

aTotal number of variant RNA transcripts represents the total of all nonredundant transcripts identified for human P450 forms in the AceView, Ensembl, and PubMed databases. Transcripts with the same predicted protein molecular weight (in kDa) with a fewer than four base-pair differences in nucleotide transcript length were considered the same transcript. Data for SNPs was obtained from GeneCards.org, and Disease Links were obtained from the MalaCards.org. bRatio of viable protein represents the fraction of human P450 variant transcripts predicted to code functional proteins using the highest confidence scoring metrics for the AceView (very good) and ENSEMBL (Protein coding/merged Ensembl/Havana). Total number of predicted variant proteins indicated in parenthesis. 378 Annalora et al. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 25, 2021

Fig. 1. Comparative analysis of the complete human cytochrome P450 transcriptome and the total number of single-nucleotide polymorphisms. Radar plot comparison of the total number of (A) human P450 transcript variants and (B) SNPs for each of the 57 human cytochrome P450 genes are shown, on the basis of a meta-analysis of information of the NCBI’s Ensembl, PubMed, and AceView databases and the GeneCards.org website. Total transcript variants for individual P450 genes identified in this study are superimposed (in pink) over the current number listed in the AceView database alone (in purple). The expansion in variant numbers we identified highlights the challenge of predicting interindividual variability in polymorphic P450 gene splicing, predicated on existing transcript databases. Radar plot comparison of the total human SNPs associated (as reported by GeneCards.org) revealed CYP5A1 (or thromboxane synthase 1) as the most polymorphic P450 gene (5274), with CYPs 2C19 (2467), 7B1 (3400), 19A1 (2178), and 39A1 (2129) showing higher rates of polymorphism than the average P450 gene (;850 mutations per gene). (C) Bar graph representation of alternative P450 transcript-to-SNP ratios for each gene is shown at the bottom. CYP2E1, with 24 transcript variants and the lowest number of SNPs (33), displays the highest alternative transcript-to-SNP ratio (0.73) among human P450 genes. CYPs 21A2 (0.33), 27B1 (0.15), and CYP2D6 (0.11) also skew above normal for this ratio (;0.05 alternative transcripts per SNP). Improved understanding of the SNP-based mechanisms that alter P450 gene splicing will facilitate the identification of novel SNP biomarkers that are predictive of drug interactions and human disease. its substrates (Hecker and Ullrich, 1989). CYP5A1-based SNPs represent amounts of polymorphisms compared with all other human P450s, more than 10% of all P450 polymorphisms in humans and are recognized reiterating the possibility that SNP expansion among P450 genes is for altering tissue-specific splicing in blood and lung cells (Wang et al., neither random nor predictable on the basis of the tissue-specific ex- 1994), and for promoting alternative exon 12 inclusion to promote pression or substrate-selectivity of an individual P450. What may be cerebrovascular disease (Kimouli et al., 2009). CYP2E1 displayed the more important with respect to the accumulation of SNPs is their ability lowest number of SNPs (33) and the highest alternative transcript-to-SNP to be silenced or masked via alternative or trans-splicing paradigms that ratio (0.73) among human P450 genes (see Fig. 1). CYPs 21A2 (0.33), render them invisible or inconsequential to the natural selection process. 27B1 (0.15), and CYP2D6 (0.11) also skew above normal P450 The defective nature of a P450-related SNP may depend on its ability to superfamily genes with respect to the average alternative transcript-to- manipulate the P450 transcriptome; therefore, improved understanding SNP ratio of 0.05. Improved understanding of how key SNPs interact of how key mutations alter P450 gene-splicing outcomes may be critical with the environment to alter individual P450 gene-splicing outcomes, to addressing the goal of precision medicine in the post-genomic era. therefore, could be very helpful in improving the predictive power of personalized medicine predicated on genetic screening. Therefore, some SNPs alter gene function by modulating alternative Tissue-Specific Alternative Splicing of Cytochrome P450 Genes: splicing events, but the total number in a population may be irrelevant Historical Perspective from an evolutionary perspective if they do not manifest in alternative During the late 1980s and early 1990s, several groups began to phenotypes. Furthermore, it is intriguing that several steroid hormone- appreciate the possibility that differential RNA splicing might serve as metabolizing P450s, including CYP7B1 (3400 SNPs), CYP19A1 an additional mode for regulating P450 function. In 1987, the first P450 (2178 SNPs), and CYP39A1 (2129 SNPs) display disproportionate splice variant for hepatic P450-1, later renamed CYP2C8, was reported Alternative P450 Splicing Redirects Human Drug Metabolism 379

(Okino et al., 1987). Over the next decade, several examples of alternate splicing were documented for several human P450s, including lung CYP4F2 (Nhamburo et al., 1990) and liver CYP2B6 (Miles et al., 1989; Yamano et al., 1989) and CYP2A7 (Ding et al., 1995). In the latter case, a tissue-specific, alternative splicing effect was documented, in which a 44-kDa CYP2A7-AS splice variant (compared with the 49-kDa wild- type protein) found only in 20% of liver samples was the dominant protein expressed in human skin fibroblasts. Just two years later, a similar pattern of alternative splicing behavior was documented for CYP2C18, which selectively encodes splice variants skipping different combinations of exons 4, 5, 6, and 7 in the epidermis (Zaphiropoulos, 1997). Circular RNA transcripts synthesized from the donor and acceptor sites from the skipped segments of exons 4–7 were also documented and, although the epigenetic function of this class of RNA remains unclear (Salzman et al., 2012), they were easily detected on the basis of their increased stability (Zaphiropoulos, 1997). Important studies that focused on environmentally responsive, alternative P450 Downloaded from splicing among xenobiotic-metabolizing P450s of the rat 2A family were also conducted during this era (Desrochers et al., 1996). By 1999, the CYP2C18 alternative-splicing story became even more complex when it was discovered that CYP2C18 and CYP2C19, which Fig. 2. Tissue-specific and transformation-sensitive alternative splicing of CYP1A1 cluster on chromosome 10q24 with CYP2C9, also participate in tissue- in humans. CYP1A1 is inducible in virtually every tissue of the body; however, a brain-specific CYP1A1 splice variant has been identified that preferentially skips specific, trans-splicing events, among the three genes (Zaphiropoulos, exon 6 (Kommaddi et al., 2007). This shortened form of the enzyme is spectrally 1999). Over the next decade, cell-specific splicing factors responsible for active but does not metabolize benzo(a)pyrene to toxic metabolites in brain. dmd.aspetjournals.org directing the complex, post-transcriptional gene assembly of P450s (and Structural analysis of the CYP1A1 crystal structure (PDB: 4I8V) indicates that removal of exon 6 (in yellow ribbon) would: 1) expand the opening of the pw2b other genes) were identified (Wang and Burge, 2008). However, the substrate access channel, 2) reshape the ligand binding pocket by altering the b1-4 physiologic importance of the more unusual P450 splice variant and sheet, and 3) alter the redox partner binding surface via elimination of the K9 helix. chimeric forms remains unclear, particularly with respect to how their A similar pattern of tissue-specific, alternative exon 2 usage in CYP1A1 has been variable, tissue-specific expression alters interindividual sensitivity to reported in tumor cells (Leung et al., 2005). Ovarian cancer cells skip an 84-bp cryptic intron in exon 2 of CYP1A1 but remain in-frame to produce a catalytically hormones, pharmaceuticals, and xenobiotics. unique splice variant with diminished estradiol metabolism that localizes to the

As discussed above, a single P450 gene can be translated into nucleus and mitochondria, rather than the ER. Cryptic intron removal in exon at ASPET Journals on September 25, 2021 multiple, variant protein sequences, solely on the basis of tissue-specific 2 eliminates 28-amino-acid residues among helices E, F, and the E–F loop (shown in transparent gold ribbon), which putatively expands the opening of the pw3 substrate differences in the expression of key gene-splicing machinery [e.g., small access channels located above the heme center and helix I. nuclear RNA (snRNAs)]. Several alternately spliced CYP1A1 tran- scripts identified in human brain tissues exemplify this phenomenon, as well as the concept of tissue-specific spliceosome activity (Bauer et al., structure/function and subcellular distribution observed for exon 2007). Inducible CYP1A1 is constitutively expressed in the brain, where 2-modified, CYP1A1 splice variants found both in normal and tumor it is localized to neurons in the cortex, cerebellum, and hippocampus. An tissues remains enigmatic and denotes a higher level of structural 87-bp deletion in exon 6 is observed in the brain-specific CYP1A1 form, plasticity with respect to tertiary structure than might be currently but not in liver (Chinta et al., 2005). Human brain tissues expressing the appreciated or expected from classic structural studies of wild-type Dexon6-CYP1A1 variant do not express wild-type CYP1A1 and they P450 forms (Johnson and Stout, 2013). metabolize known substrates [e.g., benzoxy-resorufin and benzo(a)pyrene Perhaps the most dramatic, and mutually exclusive, example of tissue- (BP)] at different rates than wild-type enzyme (Kommaddi et al., 2007). specific, alternate-exon usage in P450s was documented for CYP4F3, Furthermore, cells expressing wild-type CYP1A1 generate DNA adducts which can be expressed as two distinct splice variant forms (CYP4F3A via the formation of reactive 3-OH-BP products, whereas cells expressing and CYP4F3B). These two CYP4F3 isoforms differ only by their the Dexon6-CYP1A1 variant in brain do not (Kommaddi et al., 2007). variable usage of exons 3 and 4, which encode distinct portions of the active On the basis of the structural analysis of CYP1A1 in Fig. 2 (derived site and substrate access channel, allowing them to fine-tune substrate from PDB: 4I8V; Walsh et al., 2013), tissue-specific, alternative specificity (Christmas et al., 1999, 2001). CYP4F3A is expressed in blood splicing of exon 6 may reshape the size and polarity of the CYP1A1 and bone marrow myeloid cells, selectively expresses cassette exon 4 (and substrate access channel to alter substrate recruitment and recognition not 3), and has low affinity for leukotriene B(4) (LTB4). In contrast, processes and redox-partner binding interactions. Exon 6 of CYP1A1 CYP4F3B, expressed exclusively in the liver and kidney, encodes exon appears to be organized for “plug and play” utilization in the brain, 3 (and not 4) and has high affinity for LTB4, in addition to other where targeted skipping does not compromise the global P450 fold but functional differences. The highly concerted, tissue-specific splicing alters substrate specificity for a spectrum of endogenous substrates, of CYP4F3 exemplifies the functional utility of the modular P450 including testosterone. This tissue-specific exon 6 usage of CYP1A1 gene platform, one that allows cells to sample a continuum of gene is complemented by a similar pattern of alternative exon 2 usage found products as dictated by tissue-specific physiologic stress conditions. in human endothelial cells, leukocytes, and tumor cells (Leung et al., However, it should also be clearly noted that not all P450 genes are 2005, Bauer et al., 2007). As shown in Fig. 2, targeted skipping of an divided into an equal number of coding and noncoding regions, which 84-bp cryptic intron in exon 2 of CYP1A1 produces a catalytically suggests that the accumulation of introns may be an adaptive and ongoing unique splice variant with diminished estradiol metabolism that process. For example, the CYP1B1 gene, which encodes a P450 enzyme localizes to the nucleus and mitochondria rather than the endoplasmic that metabolizes both xenobiotics and endogenous substrates, con- reticulum (ER) (Leung et al., 2005). The added diversity in both tains only two coding exons (Supplemental Fig. 2) and, therefore, is 380 Annalora et al. not subject to the same patterns of alternative splicing as other CYP1 family members 1A1 and 1A2, which have six coding exons (Stoilov et al., 1998). In this regard, the most well studied splice variants of CYP1B1 all represent severe truncations or deletion mutants, not exon skipping (Tanwar et al., 2009), and CYP1B1 retains an unusual, elongated C-terminus, further distinguishing it from other human P450 forms. The CYP1B1 gene is also uniquely located on chromosome 2 at locus 2p21– 22, which in humans is derived from an ancient fusion of chimpanzee chromosomes 2a and 2b (Faiq et al., 2014). It is difficult to speculate why the CYP1B1 gene is organized so differently from closely related CYP1A genes located on chromosome 15. However, observations linking a common set of nuclear proteins to both the alternative splicing process and NMD seem to imply that expansion of gene complexity may be related more to the rate at which a given mRNA transcript is processed by the spliceosome and ribosome, rather than other evolutionary factors (Lejeune and Maquat, 2005; McGlincey and Smith, 2009). Ultimately, the adaptive forces driving the modular organization of Downloaded from genes into variable numbers of introns and exons remain a point of speculation. However, a gene’s intronic complexity clearly influences Fig. 3. Alternative-splicing of the human CYP24A1 gene: tissue- and tumor- specific exons 1, 2, and 10 inclusion. CYP24A1 is a mitochondrial P450 composed its sensitivity to alternate-splicing events and the domain swapping of 11 coding exons that metabolizes the vitamin D hormone to regulate its role in paradigm. The tissue-specific use of alternative promoters and non- calcium homeostasis, cell growth, and immunomodulation. Alternatively spliced AUG translation start-sites also is facilitated by this type of hidden transcript variants have been identified for this gene, including a CYP24A1 splice variant (CYP24sv) specifically expressed in macrophage (Ren et al., 2005), which gene complexity, which allows discrimination among a set of well skips exons 1 and 2. The 372-amino-acid variant protein (;43 kDa) lacks the dmd.aspetjournals.org defined exons and poorly defined pseudoexons. N-terminus, helices A9, A, B, and B9 and portions of the b1 sheet but probably In 2005, a truncated, CYP24A1 splice variant incapable of metabo- retains the ability to bind heme and substrate in some capacity. Ren and coauthors lizing hormonal forms of vitamin D was identified in the human concluded that CYP24sv represents a cytosolic variant with dominant-negative function that quarantines 25-hydroxyvitamin D3 and other hormonal forms of macrophage (Ren et al., 2005). CYP24A1 is the prototypical mito- vitamin D, slowing the rate of their metabolism in the mitochondria or endoplasmic chondrial P450 responsible for the side-chain cleavage of the vitamin reticulum. Structural analysis of CYP24A1 (PDB: 3K9V) suggests that CYP24sv’s D hormone and it possesses a mitochondrial localization sequence in unique N-terminus, derived from intron 2 (shown in green), reshapes the pw2a substrate access channel and provides contacts for sealing the ligand binding pocket the first 30 residues of its N-terminus (Annalora et al., 2004). The via interactions with helices F–G and the b1 and b4 sheets systems. It is notable that CYP24A1 splice variant found in activated macrophage (CYP24A1- exons 1 and 2 exclusion in CYP24A1 is exacerbated in human tumors of the breast at ASPET Journals on September 25, 2021 SV1) lacks 153 N-terminal residues encoded by exons 1 and 2, and and colon, where N-terminally truncated splice variants of approximately 40, 42, and gains eight residues preceding the content from exon 3, via a partial 44 kDa have been identified (Fischer et al., 2009a; Horváth et al., 2010; Scheible et al., 2014). An additional, prostate cancer-related, splice variant of CYP24A1 that insertion of intron 2. CYP24A1-SV1 is thought to function as a soluble cleanly skips exon 10 has also been described (Horvath et al., 2010; Muindi et al., variant that disrupts normal vitamin D hormone metabolism via the 2007). Exon 10 encodes much of the protein’s proximal surface, including the b1–3 sequestration of key intermediates from both wild-type CYP27B1 and sheet, meander region, CYS loop, and portions of the L-helix involved in heme- thiolate bond formation. Variants lacking exon 10 may express defects in heme and CYP24A1 (Ren et al., 2005). In this regard, the CYP24A1-SV1 variant redox partner binding, giving rise to an alternative dominant negative form that may may promote a discrete pattern of vitamin D hormone accumulation in retain membrane-binding features; loss of exon 10 could also refine the alternative the macrophage, and potentially other tissues such as the intestine, where functions of cytosolic splice variants lacking exons 1 and 2. vitamin D hormonal leakage from irritated cells is thought to contribute to the burst of extracellular hormone observed in inflammatory disorders like Crohn’s disease (Abreu et al., 2004; Mangin et al., 2014). A regulation, particularly for variants subject to atypical subcellular structural analysis of the CYP24A1-SV1 variant is shown in Fig. 3 (on trafficking events. the basis of PDB: 3K9V; Annalora et al., 2010), and it highlights how Therefore, although increasingly common in the literature, the the targeted loss of N-terminal residues from exons 1 and 2 could re- functional significance of many tissue-specific P450 variants remains configure the membrane-binding surface and substrate access channel to elusive. CYP2C8, for example, is constitutively expressed in the liver alter the catalytic properties of wild-type CYP24A1. at 5–7% of the total tissue P450, has more than 20 known alternately Interestingly, the orphan P450 CYP27C1, a close relative of spliced variants, and is a breast cancer biomarker owing to its high CYP24A1, encodes a 372-residue wild-type protein that resembles the expression in mammary tumors (Knüpfer et al., 2004). Bimodal CYP24A1-SV1. CYP27C1 lacks a prototypical N-terminus and initiates targeting to both mitochondria and ER have been reported for coding in the middle of canonical helix C, at a position analogous to CYP2C8 and several other P450s, including CYP1A1, 1B1, 2B1, the alternative start site used by CYP24A1-SV1 (Ren et al., 2005). 2E1, and 2D6. Although it is clear that alternative splicing directs this CYP27C1 is expressed in liver, kidney, pancreas, and several other process in some tissues, proteolytic cleavage of N-terminal targeting tissues, and remained an orphan for over a decade (Wu et al., 2006), until sequences can also produce this effect (Bajpai et al., 2014). One 2C8 recently, when it was shown to mediate the conversion of vitamin A1 variant (variant 3; ;44 kDa) with an alternative N-terminus is (all-trans retinol) into vitamin A2 (all-trans 3,4-dehydroretinal) in cell targeted to the mitochondria, where it may contribute to oxidative culture (Kramlinger et al., 2016). It is intriguing that CYP27C1 may stress (Bajpai et al., 2014). The catalytic competency of these represent a codified version of the more soluble P450 isotype re- untethered variants remains a topic of intense debate, as it is often capitulated by CYP24-SV1, and that this transition may mediate a difficult to assign an orphan function to a variant protein that has lost general switch between vitamin A and vitamin D substrate specificity. selectivity to the gene’s prototypical substrates. A review of P450 Improved knowledge of CYP27C1 structure/function could help to splice variants subject to alternate subcellular trafficking is shown in unravel some of the complexity associated with the variant P450 Supplemental Table 1. Computational analysis from the AceView Alternative P450 Splicing Redirects Human Drug Metabolism 381 database (Thierry-Mieg and Thierry-Mieg, 2006) indicate that some intronic SNPs may facilitate the alternate splicing pattern seen for P450 variants may be targeted to the nucleus, peroxisome, plasma CYP24A1 in prostate cancer cells, vitamin D hormone exposure also membrane, or vacuole compartments, in addition to classic targets in putatively alters splicing via discrete interactions among the vitamin D the ER, mitochondria, or cytoplasm. This analysis hints that P450 receptor (VDR) and nuclear-splicing factors like the heterogeneous splice variants, including tumor-specific forms, may be performing nuclear ribonucleoprotein C (hnRNPC; Zhou et al; 2015). Tumor cells an array of unrecognized functions, including diverse roles at the cell overexpressing CYP24A1, therefore, may not retain sufficient hormone surface and beyond, which are rarely considered (Eliasson and to activate the VDR properly, disrupting normal gene-splicing events Kenna, 1996; Stenstedt et al., 2012). needed to properly transduce the hormone’s immunogenic, prodiffer- entiation properties. CYP24A1 splice variants have now been docu- mented among multiple human breast tumor cell lines (e.g., MCF-7 and Transformation-Sensitive Alternative Splicing in the Cytochrome MCF-10) and in both healthy and malignant breast tumor tissues. In P450 Superfamily benign tissue, wild-type CYP24A1 (56 kDa) was the only isoform This transformation-specific, P450 splicing phenomenon was first present. In malignant tissues, three splice variants (40, 42, and 44 kDa) documented in 1996 for the prototype P450, CYP19A1 (or aromatase), were expressed, similar to cultured MCF-7 breast tumor cells that which facilitates estrogen biosynthesis via three successive hydroxyl- express the 42- and 44-kDa variants (Fischer et al., 2009a; Scheible ations of the androgen A ring and displays a unique pattern of exon et al., 2014). A similar pattern of transformation-sensitive splicing is 1 (and cryptic intron) usage in human breast tumor cells and tissues seen in human colon cancer tissues, where the histological grade and the Downloaded from (Zhou et al., 1996). A similar pattern of CYP19A1 exon 1 variant ex- gender of the patient modulates the formation of the three detectable pression also has been documented in sheep, cattle, and rabbit, suggesting CYP24A1 splice variants (Horváth et al., 2010). Peng and coworkers this paradigm is not exclusive to humans or transformed-tissue types (2012) further refined our understanding of this process in the colon by (Vanselow et al., 1999; Bouraïma et al., 2001). More recently, it was demonstrating that cell- or tissue-specific splicing patterns were sup- determined that CYP19A1’s exon 5 also is defined poorly and subject to pressed by parathyroid hormone signaling, but only in the absence of the both splicing mutations and physiologic alternative splicing events (Pepe vitamin D hormone, which may be the master regulator of both et al., 2007). Skipping of exon 5, which encodes complete a-helices D and CYP24A1 gene expression and splicing. dmd.aspetjournals.org E in CYP19A1, eliminates prototypical P450 aromatase activity in both The phenotypic instability exemplified by the CYP24A1 gene in steroidogenic and nonsteroidogenic tissues (Lin et al., 2007; Pepe et al., human tumors complicates the therapeutic landscape from which 2007). These observations suggest a tissue-specific, regulatory mechanism rational drugs for cancer can be conceived (Trump et al., 2006). for aromatization on the basis of alternative exon 5 inclusion in humans. Epigenetic modification of the CYP24A1 gene and its promoter also A structural analysis of this CYP19A1 splice variant is depicted in may contribute to tissue-specific differences in P450 expression Supplemental Fig. 3 (on the basis of PDB: 3S79; Ghosh et al., 2012). (Johnson et al., 2010). Frontiers in this area of research will likely

In aromatase, exon 5 skipping naturally suppresses androgen metab- address the coordinated role that nuclear hormone receptors and at ASPET Journals on September 25, 2021 olism by redefining the determinants of substrate recognition. While epigenetic splicing factors (including noncoding forms of RNA) play transfection studies suggest CYP19A1 variant proteins are encoded in managing both the transcriptome and the epigenome. and not subject to NMD, their ability to alter the safety and efficacy of It should be noted that the CYP27B1 gene, which is highly related to several CYP19A1 (aromatase) inhibitor drugs [e.g., Arimidex (anastro- CYP24A1, was also recognized early as being sensitive to transformation- zole), Aromasin (exemestane), Femara (letrozole), and Teslac (testolac- specific alternative splicing in malignant human glioma cells (Maas et al., tone)] remains unclear (Hadfield and Newman, 2012) and an area of 2001). CYP27B1 variant expression has now been documented in tumors active investigation (Liu et al., 2013a; Liu et al., 2016). of the breast, skin, cervix, kidney, and ovaries (Diesel et al., 2004; Fischer The tissue-specific, alternative start-site usage of CYP24A1, dis- et al., 2007, 2009b; Wu et al., 2007). Tissue-specific CYP27B1 splicing cussed above, appears to be exacerbated in several human transcript also is common in healthy skin, where a 59-kDa variant with a 3-kDa variants in which an array of truncated splice variants of approximately insertion between exons 2 and 3 and exclusive to skin was observed 40, 42, and 44 kDa have been identified with similar N-terminal and linked to the activity of cAMP response element–binding elements modifications (Fischer et al., 2009a; Horváth et al., 2010; Scheible et al., (Flanagan et al., 2003). Fluctuations in skin cell density, calcium con- 2014). An additional, cancer-related, splice variant of CYP24A1 that centrations, and UV-B light exposure were also shown to affect the cleanly skips exon 10 has also been documented (Muindi et al., 2007; splicing process (Seifert et al., 2009). Collectively, these findings help to Horvath et al., 2010). In CYP24A1, exon 10 encodes much of the validate our core hypothesis that alternative gene splicing, dictated by protein’s proximal surface, including the CYS loop, and a portion of both cellular and environmental factors such as UV light exposure and helix L involved in heme-thiolate bond formation and adrenodoxin nutritional status, can alter a patient’s metabolic profile in tissue-, age-, recognition (shown in Fig. 3). Although the function of this cancer- sex-, exposure-, and disease-specific ways that are difficult to predict. specific variant remains unknown, it may have reduced ability to bind Research focused on CYPs 19A1, 24A1, and 27B1 is among the most heme and redox partners compared with truncated variants (skipping comprehensive on transformation-sensitive alternative splicing in human exons 1 and 2), and thus may represent an alternative, dominant-negative P450s, and the literature is replete with anecdotal reports of alternative variant also capable of modulating vitamin D hormone metabolism and P450 splicing among various transformed cell types and tissues. For metabolite trafficking at the plasma membrane level. As discussed example, CYP2E1 transcripts that selectively skip exons (2, 2–3, and previously, the cellular mechanisms regulating CYP24A1 alternative 2,4,5–59,6) were identified in lung carcinoma cell lines but not in spicing in the macrophage remains unclear, although cell-specific corresponding lung tumor tissue or liver extracts, where splice variants variations in splicing factors (e.g., heterogeneous ribonuclear protein were found to be noninducible by ethanol exposure (Bauer et al., 2005). A1) are thought to be responsible (Ren et al., 2005). A more complex The authors noted at the time that none of these splice variants were pattern of “transformation-sensitive” alternative CYP24A1 gene splic- accounted for in the National Institutes of Health (NIH) AceView ing also has been documented in human tumors of the prostate (Muindi database, highlighting the longstanding challenge associated with et al., 2007), breast (Fischer et al., 2009a; Scheible et al., 2014), and cataloging splice variant forms derived from cell-specific abnormalities colon (Horváth et al., 2010; Peng et al., 2012). It is thought that whereas versus those generated from normal, tissue-specific splicing paradigms. 382 Annalora et al.

Furthermore, several orphan P450s, including CYP1B1, CYP2S1, and The CYP2D gene family also is complex, clustered, and subject to CYP2W1 are highly associated with specific human tumor types, making trans-splicing events; it is composed of CYP2D6 and 4 pseudogenes them important prodrug targets for chemoprevention strategies (Stenstedt (CYP2D7P1 and 2 and CYP2D8P1 and 2). Alternative splicing of the et al., 2012). Unfortunately, as discussed above, the alternative splicing CYP2D family pre-mRNAs has been detected in human liver, breast, behavior of orphan P450s is often as poorly understood as are their and lung tissue (Huang et al., 1996, 1997), including a highly expressed cryptic functions. splice variant that completely skips exon 6. As shown in Supplemental Interestingly, orphan CYP2W1, which is expressed in human tumor Fig. 4B, analysis of the CYP2D6 crystal structure (PDB: 4WNU: Wang types of the colon, is associated almost exclusively with aberrant P450 et al., 2015) reveals that exon 6 encodes the complete coding sequence behavior, including an inverted orientation in the ER membrane, and for helix I, and that skipping this important region would dramatically glycosylation-sensitive trafficking to the plasma membrane (Gomez alter protein structure/function by removing key catalytic residues (e.g., et al., 2010; Stenstedt et al., 2012). The increased expression of Thr-309) and remodeling the distal pocket of the active site. Alternate CYP2W1 in tumors is associated with demethylation of a CpG island CYP2D6 exon usage is tissue-selective, and variant forms skipping exon in the exon 1-intron 1 junction of the gene. How this mechanism relates 3 and portions of exon 4 have been documented that alter enzyme to the paucity of normal CYP2W1 expression in healthy tissues remains function and subcellular localization (Sangar et al., 2010). CYP2D6 unclear, but it implies that the expression of some specialized P450s may polymorphisms are linked to modified enzyme activity and may only be invoked under extreme cellular stress, when an alternative P450 serve as biomarkers for poorly metabolizing phenotypes sensitive to activity may be critical for balancing cellular homeostasis. The abnormal dose-dependent drug toxicity. Recently it was reported that intronic Downloaded from expression and trafficking of CYP2W1 to the plasma membrane could polymorphisms in CYP2D6*41 individuals (considered intermediate promote an autoimmune response as part of its mechanism. A similar metabolizers) could dramatically increase the expression levels of autoimmune reaction is associated with aberrant CYP2E1 trafficking in CYP2D6 variants lacking exon 6 (Toscano et al., 2006). These animal tissues treated with halothane (Eliasson and Kenna, 1996). The findings, coupled with the complex organization of the CYP2D gene NIH’s AceView and Ensembl (Cunningham et al., 2015) databases cluster, indicate that both alternative and trans-splicing mechanisms indicate that CYP2W1 encodes at least 11 transcript variants, three of may underlie the complex genotype-phenotype relationships asso- which lack one or more exons and are associated with hepatocellular ciated with variable CYP2D6 metabolism in humans, and that dmd.aspetjournals.org carcinomas, adenocarcinomas, and neuroblastomas. Nearly 200 of the CYP2D6 is well organized structurally to exploit both tissue- and 965 alternatively spliced P450 transcripts we identified in public database transformation-selective splicing mechanisms targeting helices C searches were associated with a well-characterized human tumor type. and D (exon 3) and helix I (exon 6). Improved understanding of their tumor-specific cellular roles and the The CYP3A gene family (comprising CYPs 3A4, 3A5, 3A7, and mechanisms regulating their induction should aid in the identification of 3A43) is also highly complex in humans, with each gene consisting of improved disease biomarkers and P450 variant-specific therapeutic agents. 13 exons (with ;71–88% amino acid identity) that cluster on chromo-

some 7q21–22.1, where the CYP3A43 gene is in a head-to-head at ASPET Journals on September 25, 2021 orientation with the other three genes (Finta and Zaphiropoulos, 2000). Trans -Splicing of Cytochrome P450 Gene Chimeras Several chimeric forms of CYP3A mRNAs have been described, in It is well understood that both transformation-sensitive cellular changes which CYP3A43 exon 1 is joined at CYP3A4 and CYP3A5 canonical and P450 polymorphisms can alter protein expression in multiple ways splice sites, implying a trans-splicing mechanism that bypasses classic that are often difficult to predict from primary sequence analysis alone. transcriptional control paradigms. In Supplemental Fig. 5A, a structural This challenge is exacerbated in situations where chimeric P450 genes analysis of CYP3A4 (on the basis of PDB: 4K9T; Sevrioukova and are created via trans-splicing events among extended (or multiple) Poulos, 2013) reveals the highly complex segmentation of the CYP3A4 pre-mRNA transcripts. Therefore, although mutations in a gene like gene into 13 exonic domains, which, as described above, may potentiate CYP2C9 are linked to poor metabolism of several drugs, including rapid translation upon induction (Lejeune and Maquat, 2005; McGlincy phenytoin and tolbutamide, the role that alternative or trans-gene and Smith, 2008). Primary amino acid sequence alignments of key splicing plays in manifesting a deleterious CYP2C9-related metabolic CYP3A genes suggest exon 1 may encode distinct transmembrane anchor phenotype are rarely considered. For example, several CYP2C9 splice helices and membrane-binding architecture (Supplemental Fig. 5B), variants (CYP2C9sv) have been identified (Ohgiya et al., 1992), hinting that CYP3A chimeras may encode a spectrum of phenotypes with including a liver-specific form that skips exon 2 (Ariyoshi et al., unique tissue-specific distribution and functionalities. A CYP3A4 trans- 2007). The internally truncated CYP2C9 variant is spectrally active splicing variant containing CYP3A43 exon 1, followed by CYP3A4 but does not metabolize prototype CYP2C9 substrates. A structural exons 4–13, has been described; it retains detectable testosterone 6-b- analysis of the CYP2C9 crystal structure (PDB: 4NZ2; Brändén et al., hydroxylase activity despite losing membrane-binding features encoded 2014; shown in Supplemental Fig. 4A) hint that removal of exon 2 by exons 2 and 3, comprising helices A9 and A, and portions of the b1-1 would reshape portions of the reference protein relative to its membrane- sheet (Supplemental Fig. 5C). A CYP3A4 trans-splicing variant binding surface, substrate access channel, active site, and redox partner containing CYP3A43 exon 1, followed by CYP3A4 exons 7–13, has binding surface. CYP2C9 is located within a cluster of P450 genes on been studied also, and it displays minimal 6-b-hydroxylase activity chromosome 10q24 and is subject to nonrandom, trans-splicing events (Supplemental Fig. 5D). This story is further complicated by the presence with neighboring CYPs 2C8, 2C18, and 2C19 in liver and skin, where of additional CYP3A pseudogenes (CYP3AP1 and CYP3AP2) in the CYP2C18exon1–like sequences were found spliced into different intergenic regions between CYP3A4, 3A7, and 3A5, where discrete combinations of introns and exons from the CYP2C9 genes (Warner exons from the pseudogenes also have been found trans-spliced into et al., 2001). Common 2C9-related SNPs linked to various metabolic CYP3A7 transcripts (Finta and Zaphiropoulos, 2002). Ultimately, disorders, therefore, may only function to modulate the innate splicing CYP3A4 and CYP3A5 splicing complexity may help to explain how process indirectly. This is particularly true if their coding regions are intronic SNPs in CYP3A4*22 patients alter tacrolimus metabolism, spliced out of chimeric 2C forms being expressed on an interindivid- without introducing a nonsynonymous mutation to an exon (Elens ual basis, highlighting another major flaw in GWAS targeting of the et al., 2011). The predictive power of GWAS may therefore need to be components of gene clusters. reconsidered, particularly with respect to the most complex P450 gene Alternative P450 Splicing Redirects Human Drug Metabolism 383 families defined by the presence of pseudogenes (e.g., CYP 2C, 2D, proteins) may account for interindividual variability in recognizing 3A, 21A, and 51A families) and trans-splicing mechanisms. the CYP3A5*3 SNP, there are currently no models to explain how this complex, salt-sensitive splicing phenomenon is being regulated in the kidney. The alternative splicing of CYP3A5 variant exem- SNP-Sensitive Alternative Splicing in the Cytochrome P450 plifies why SNP-based approaches to personalized medicine are so Superfamily challenging to implement, when the physiologic impact of a given As discussed above, tissue-specific, alternative and trans-splicing SNP may be the opposite of what is expected. Computational approaches behaviors have now been documented for several important P450 gene that consider a patient’s age, genotype, nutrition, and disease status may families; however, SNPs can destabilize this process in complex ways ultimately be needed to make good predictions concerning the impact of that are often difficult to predict. SNPs that target cryptic intronic a given SNP on an individual’s transcriptome. recognition elements or discrete intron/exon splice junction boundaries The challenge of addressing SNPs for personalized medicine is further can alter translation start-and-stop site usage, and the expression ratios complicated by observations that some xenobiotics, including amino- among wild-type and variant P450 transcripts. Some P450 SNPs are glycosides and cyclohexamides (Busi and Cresteil, 2005), can alter the exceedingly rare, occurring in less than 1% of the population. These rare proofreading capabilities of the ribosome, allowing inhibition of pre- mutations are linked to congenital defects, including glaucoma (CYP1B1; mature stop codons and translational read-thru of alternate transcripts that Stoilov et al., 1998; Tanwar et al., 2009; Sheikh et al., 2014), 17-a may or may not remain in-frame with respect to the reference transcript. hydroxylase deficiency (CYP17A1; Yamaguchi et al., 1998; Costa- Improved knowledge of a patient’s chemical exposure history and Downloaded from Santos et al., 2004; Hwang et al., 2011; Qiao et al., 2011), congenital epigenetic status, therefore, may become increasingly important when adrenal hyperplasia (CYP21A2; Robins et al., 2006; Lee, 2013; Szabó making predictions about food and drug safety. Furthermore, personal- et al., 2013; Sharaf et al., 2015), spina bifida (CYP26A1; Rat et al., ized approaches to medicine solely on the basis of SNPs identified via 2006), focal facial dermal dysplasia (CYP26C1; Slavotinek et al., 2013), cohort studies or GWAS may be fundamentally flawed and potentially and cerebrotendinous xanthomatosis (CYP27A1; Garuti et al., 1996; misleading, in that they fail to account for the milieu of compensatory Verrips et al., 1997; Chen et al., 1998; Tian and Zhang, 2011). Other cellular mechanisms that are adept at masking the most deleterious effects P450 polymorphisms that alter splicing are associated with neurologic of even the most highly penetrant mutations. dmd.aspetjournals.org and metabolic diseases, including Parkinson’s disease (CYP2D6, Denson et al., 2005; CYP2J2, Searles Nielsen et al., 2013), hypertension (CYP4A11, Zhang et al., 2013; CYP17A1, Wang et al., 2011), breast Addressing Nontraditional P450 Function and Trafficking Events cancer (CYP2D6, Huang et al., 1996; CYP19A1, Kristensen et al., 2000;) Our meta-analysis and review of splice variability within the colon cancer (CYP2W1,Stenstedt et al., 2012), and lung cancer cytochrome P450 superfamily has revealed an under-appreciated (CYP2D6, Huang et al., 1997; CYP2F1, Tournel et al., 2007). However, level of alternative transcript expansion and variant protein structural the complex etiology of these disorders can make it difficult to interpret complexity that is poorly addressed by conventional P450 structure/ at ASPET Journals on September 25, 2021 the exact role that a P450 SNP might play in the actual onset or function paradigms. Well studied splice variants from the CYP1A progression of a disease. Multiple studies of various intergenic, intronic, family reveal a sophisticated domain-swapping mechanism that and exonic polymorphisms that alter human drug and xenobiotic allows for tissue-specific sampling of alternative protein conforma- metabolism have also been reported (CYP1A1, Allorge et al., 2003; tional space. Discrete arrays of tissue-specific snRNAs (U1–U9) and CYP2C19, de Morais et al., 1994; Ibeanu et al., 1999; Satyanarayana ancillary splicing factors can alter the biologic syntax of a gene, thus et al., 2009; Sun et al., 2015; CYP2D6, Toscano et al., 2006; Lu et al., providing a granular mechanism for this natural phenomenon that is 2013; Wang et al., 2014; CYP3A4, Elens et al., 2011; and CYP3A5, highly responsive to environmental factors, including diet and chemical Kuehl et al., 2001; Busi and Cresteil, 2005; Lee et al., 2007; García- exposure. In human brain, the alternative splicing of CYP1A1 appears Roca et al., 2012). In many cases, the most pharmacologically relevant linked to a tissue-specific sensitivity to a reactive P450-product (e.g., SNPs are those affecting the innate splicing behavior of a given gene. 3-hydroxybenzo[a]pyrene; Kommaddi et al., 2007). How this gene- This observation highlights why knowledge of a patient’s discrete specific sensitivity is transduced and imprinted at the genetic level P450 transcriptome is so important when selecting a personalized remains enigmatic, but the cellular microenvironment clearly dictates medicine regimen. what hidden structural complexity of a P450 gene may be exploited. It is now clear that numerous P450-related SNPs are found between This observation helps explain why some P450 variants, like CYP24A1- genes and exons, or in the 59 or 39 untranslated regions of RNA SV1, which are normally expressed in the immune system, can become transcripts. Polymorphisms in this class have now been identified for major players in tumor cells, where they may elaborate a common virtually every human P450 isoform, with each having the potential emergency function, such as the suppression of vitamin D hormone to alter splicing in unique ways in different individuals. For example, metabolism. This cellular phenomenon corresponds well with related the CYP3A5*3 SNP (rs776746, also known as 6986A.G) intro- vitamin D hormone–scavenging strategies, such as the 3-epimerization duces a mutation in intron 3 that alters normal splicing of exon 4, pathway, which can be induced to modify the vitamin D hormone’s producing a nonfunctional, truncated CYP3A5 protein that predom- chemical ring structure to limit its efficient catabolism via CYP24A1 inates in many Caucasians (Kuehl et al., 2001). Expression of the (Rhieu et al., 2013). To date the enzyme responsible for the 3-epimerization wild-type CYP3A5*1 protein is highly variable among populations of the vitamin D hormone remains unknown (Bailey et al., 2013). andislinkedtoanincreasedsensitivitytosalt-inducedhypertension. New appreciation for the increasingly diverse and complex roles that Interestingly, some individuals who are homozygous CYP3A5*3/*3 P450 splice variants play in normal biology should help reinvigorate can express both the truncated variant and properly spliced, wild-type discussions of P450 gene structure and evolution, particularly with CYP3A5 (Lin et al., 2002). Because the CYP3A5*3 mutation occurs respect to the selective forces that regulate P450 gene duplication and in intron 3, over 100 base pairs upstream from the splice donor site of mutation events. Although the origin and expansion of spliceosomal exon 4, it is unclear how this SNP alters normal recognition of the introns remains highly debated (Rogozin et al., 2012; Qu et al., 2014), intron 3-exon 4 junction. Although variations in the expression of the intron-exon boundaries of 17 of 18 mammalian P450 families appear ubiquitous splicing factors (e.g., U1 small nucleolar ribonuclear to be well conserved across almost 420 million years of evolution, as 384 Annalora et al. evidenced by the fugu (pufferfish) genome (Nelson, 2003); only the splicing regulatory-factor proteins) and alter RNA polymerase (Pol) II CYP39 gene is missing from fugu and all known fish. The bulk of P450 processivity. Furthermore, because some introns are defined by AU-AC structural diversity encoded by the human genome, therefore, likely junctions rather than GU-AG junctions, the splicing of these introns may predates the divergence of tetrapods from ray-finned fish over 420 million be less sensitive to ROS-mediated events and, therefore, may be subject years ago. In this regard, brain-selective expression of CYP1A1 variants to a different suite of regulatory-splicing mechanisms. For example, the lacking exon 6 in humans may be no more evolved than tissue-specific induction of environmentally responsive genes like adenosine-deaminase expression of CYP19A1 splice variants found in fugu. In each case, the could alter the recognition of a prototypical AU-AC junction in a induction of P450 splice variants appears to provide a novel mechanism similar way, via adenosine-to-inosine (A-to-I) RNA editing (Solomon for modulating steroid hormone pleiotropism among a diverse set of et al., 2013). Even mitochondrial genes lacking spliceosomal introns target tissues. could be affected by this phenomenon, as the discrete dinucleotide The selective pressures that created the modular framework of the sequences that coordinate their autocatalytic, self-splicing events P450 gene, therefore, likely predate the Cambrian explosion 540 million could also be potentially altered by P450-mediated ROS modulation years ago, and lurk somewhere in the history of deuterostome evolution (Zimmerly and Semper, 2015). (Nelson, 2003). However, contemporary analyses of P450 splicing mechanisms are needed to help elaborate the relationship between alternative subcellular trafficking and alternative P450 variant function Shaping an Improved Roadmap toward Precision Medicine in the mitochondria, nucleus, peroxisome, cytoplasm, or plasma Because changes in mitochondrial function can alter global alternative Downloaded from membrane (Supplemental Table 2). Because alternative splicing and splicing events, it is not surprising that both phenomenon are linked to alternative start-site usage can generate even greater P450 structural the induction of cellular heterogeneity and human disease pathology diversity than those predicted to arise from post-translational modi- progression (Raj and van Oudenaarden, 2008; Hanahan and Weinberg, fications of intracellular targeting motifs, the mechanisms underlying 2011; Pagliarini et al., 2015). Despite a growing appreciation for the role their emergence and regulation deserve greater attention. For example, that alternative splicing plays in promoting phenotypic variability, the if subtle differences in the membrane-binding features of CYP1A1 and identification of genetic polymorphisms linked to disease, which may or CYP1A2 can modulate redox partner recruitment and protein interactions may not alter gene-splicing events, remains a core focus of pharmaco- dmd.aspetjournals.org within ordered and disordered regions of the lipid bilayer (Park et al.; genomics. Mutations in single genes have now been identified for over 2015), metamorphic structural changes associated with some P450 4000 human diseases, of which 5–15% are the result of SNPs resulting in variants may underlie an even greater array of uncharacterized (and nonsense mutations (Mort et al., 2008). However, SNPs occur in coding potentially moonlighting) P450 functions (Zhao and Waterman 2011; regions of genes with a frequency of approximately 1.5%, and of those, Lamb and Waterman, 2013). only one-third are expected to result in nonsynonymous mutations and a As the landscape of alternate P450 gene functions continues to much smaller number alter pre-mRNA processing, leading to a frequency expand, the cellular basis for alternatively targeting microsomal in change of phenotype of only ;0.5%. When one considers that humans at ASPET Journals on September 25, 2021 P450 forms to the mitochondria (e.g., CYP1A1, CYP1A2, CYP1B1 express almost 50,000 SNPs across the 57 human P450 genes, fewer than CYP2B1, CYP2B2, CYP2E1, CYP3A1, and CYP3A2) is still being 250 may be capable of significantly altering a patient’s metabolic investigated (Anandatheerthavarada et al., 1997). However, a putative phenotype, an insight we contend may help streamline PGx approaches role in modulating destructive, subcellular reactive oxygen species to personalized medicine (Zhou et al., 2009; Zanger and Schwab, 2013). (ROS) production has recently been proposed (Dong et al., 2013). For example, although CYP2D6 genotyping is no longer recommended This theory emerges from evidence that the alternative trafficking of in the clinical setting for tamoxifen treatments (Abraham et al., 2010; the related NADPH:quinone oxidase-1 (NQO1) gene, which limits Wu, 2011; Lum et al., 2013), the FDA still considers CYP2D6 clinically ROS by preventing semiquinone redox cycling, can protect the actionable from a PGx perspective for codeine and other drugs (Crews mitochondria from stress-induced oxidative damage. P450 variants et al., 2014). Unfortunately, over 74 allelic variants of CYP2D6 have may potentially play a similar role by redirecting the metabolism of already been described, which greatly complicates genetic testing and the reactive subcellular compounds. However, because P450s can also clinician’s ability to select the appropriate pharmacotherapy and dose become uncoupled and directly produce ROS via the release of (Zhou, 2009). Fortunately, organizations such as the Clinical Pharma- reactive products (i.e., via uncoupled Fenton-type reactions), further cogenetics Implementation Consortium (CPIC) and the Pharmacoge- attention to the role of alternative P450 splicing in the modulation of nomics Knowledgebase (PharmGKB) are working to standardize mitochondrial stress and dysfunction seem warranted. In this regard, it methodologies for PGx data analysis and clinical guidance for specific is intriguing that many P450 splice variants that undergo alternative gene-drug pairings (Caudle et al., 2016). However, CPIC concedes trafficking often lack a significant portion of the membrane binding that some rare P450 variants may not be included in genetic tests, and domain and/or substrate-access architecture (typically encoded by that patients may be assigned “wild-type” genetics by default (Crews exons 1–3), which may expand heme accessibility or the rate of et al., 2014). When paired with additional uncertainties regarding uncoupled ROS production. When paired with observations that P450-specific genotype/phenotype associations and the untold num- alternative splicing is a primary mechanism by which mitochondria bers of unclassified SNPs, it becomes clear why accurate genetic alter cellular phenotypes to mitigate environmental stress (Guantes screening remains challenging, and only one aspect of the therapeutic et al., 2015), the recognition of new, nonclassic roles for P450 variants decision-making process. linked to disease seems probable. Ultimately, the falling cost and broader availability of pyrosequencing In this regard, it is interesting that the consensus sequence (GU-AG) technologies support our call for improved RNA sequence strategies for of most RNA splice junctions contains a minimum of two guanine personalized medicine, as accurate identification of the patient’s functional nucleotides, which are highly sensitive to ROS-mediated oxidation. In genome is a crucial component of precision medicine. Although tran- the presence of radical oxygen, guanine is rapidly converted to 8-oxo- scriptomic analysis offers superior guidance in the design of personalized guanine and related metabolites that disrupt normal base pairing and local therapeutic options, its broad implementation will require technical secondary structure. Formation of 8-oxo-guanine at splice junctions can improvements to sample collection and processing that are also problem- impede target site recognition by splicing factors (e.g., snRNAs and atic for genomic testing. In this regard, complementary metabolomics Alternative P450 Splicing Redirects Human Drug Metabolism 385 approaches directed at variant-specific metabolism may provide more P450s like CYP3A4 (Arora and Iversen, 2001), whose metabolism of feasible, short-term improvements to PGx screening and precision-based drugs like tamoxifen is linked to genotoxicity (Mahadevan et al., 2006). As approaches to medicine (Beger et al., 2016). Innovative, gene-directed our appreciation for transcriptome expansion and the mechanisms of therapeutic technologies such as splice-altering antisense oligonucleotides alternative P450 gene-splicing evolve, new therapeutic gene-editing and CRISPR/Cas9 genome-editing systems may also become feasible options will probably emerge that could scarcely be predicted using tools for manipulating a patient’s transcriptome to optimize therapeutic genetic testing alone. outcomes. Key examples of splice-switching technology already being In summary, the human cytochrome P450 family transcriptome investigated to treat human disease are listed in Supplemental Table 2. In contains over 965 different variant forms (Table 2), many with common this regard, our group has participated in the development of eteplirsen, the structural features sensitive to alternative splicing events that expand new antisense oligonucleotide drug that received accelerated approval P450 protein diversity. The transcription and processing of P450 gene from the FDA for the treatment of Duchenne muscular dystrophy (DMD; transcripts is complex and coordinately regulated within the nucleus by Syed, 2016; Niks and Aartsma-Rus, 2017). Eteplirsen’s development multiple factors, including NR signaling via environmental sensors like evolved from early studies of exon skipping in the murine dystrophin the peroxisome proliferator–activated receptors (PPARs) PPARg and model (Fall et al., 2006; Fletcher et al., 2007; Adams et al., 2007; Mitrpant PPARa, which interact with the PGC-1a transcriptional coactivator to et al., 2009), a canine model of DMD (McClorey et al., 2006b), and in regulate oxidative metabolism and mitochondrial biogenesis (Wu et al., human muscle explants (McClorey et al., 2006a). Our group has also 1999; Monsalve et al., 2000). Multiple steroids, including products of employed exon-skipping oligomers to refine the immune response– CYPs 1A, 1B, 2A, 2B, 2C, 2D, 3A, 7A, 17A, 19A, 24A1, and 51A Downloaded from mediated gene expression of CD45 protein-tyrosine phosphatase in a metabolism, bind NRs and lead to interactions with NR coregulators murine anthrax model (Panchal et al., 2009; Mourich and Iversen, 2009), through LxxLL or FxxLF motifs that modulate the assembly of the of interleukin-10 in an Ebola virus lethal-challenge mouse model (Panchal spliceosome complex and pre-mRNA splicing (Auboeuf et al., 2005). et al., 2014) and of CTLA-4 in a murine model of autoimmune diabetes The androgen receptor (AR), which binds multiple metabolites of CYPs (Mourich et al., 2014). We hypothesize that similar splice-altering 2A, 2C, 2D, 3A, 17A, 19A, and 21A, can also directly interact with technology may be useful in redirecting the function of drug metabolizing nucleolar splicing factors (e.g., U5 small nucleolar ribonuclear protein), dmd.aspetjournals.org at ASPET Journals on September 25, 2021

Fig. 4. Endoxenobiotic crosstalk among cytochrome P450 and nuclear receptor genes coordinate alternative splicing and resemble a primitive immune system. Human tissues are subject to exposure from over 400 FDA-approved drugs, .10,000 xenobiotics, and untold numbers of endogenous substrates and their metabolites (x . 100,000). Cytochrome P450 genes participate in phase I detoxification of many of these compounds, including model substrates benzo[a]pyrene (via CYP3A4) and calcifediol (via CYP24A1). P450 genes are classically induced to silence endoxenobiotic signaling through cognate nuclear receptors, which modulate global gene expression and splicing events by “coloring” or modulating the composition of coregulatory factors that comprise both the transcription complex and the spliceosome complex, which ultimately alter the nature of both ribosome assembly and gene expression (Auboeuf et al., 2005). Model substrates are subject to metabolism by a finite population of P450s in a given tissue, however, and because each gene is sensitive to an infinite number of environmentally sensitive, alternative splicing events, each individual may express a unique, tissue-specific “P450 gene cloud” comprising both wild-type (WT) and splice-altered variant forms (e.g., SV1, SV2, etc.). P450 splice variants can: 1) display reduced ability to metabolize model substrates, 2) function as dominant negatives to sequester compounds from metabolism or potentiate basal NR-mediated signaling, or 3) function as a conformationally distinct protein with alternative metabolic function or cellular role. When coupled with existing paradigms of alternate P450 trafficking and membrane- associated cooperativity, an integrated network of crosstalk among 57 P450 and 48 NR genes begins to emerge, as novel P450 metabolites may engage NR signaling pathways in unique ways that reprogram gene splicing and expression to promote cellular homeostasis in the face of endocrine disruption. NR signaling cascades can alter both the transcriptome and epigenome of an individual, providing an elegant feedback mechanisms for adaptation to cellular stress created by unique personal history and disease status. 386 Annalora et al. indicating a receptor-mediated role in transcription that is coupled to pre- References mRNA splicing mechanisms (Zhao et al., 2002). Vitamin D receptor Abraham JE, Maranian MJ, Driver KE, Platte R, Kalmyrzaev B, Baynes C, Luccarini C, Shah M, activation mediated by metabolites of CYPs 2R, 2J, 3A, 11A, 27A, 24A, Ingle S, Greenberg D, et al. 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Arch Biochem Biophys 425:133–146. protein kinase delta (PKCd) among other pre-mRNAs (Apostolatos et al., Annalora AJ, Goodin DB, Hong WX, Zhang Q, Johnson EF, and Stout CD (2010) Crystal structure of CYP24A1, a mitochondrial cytochrome P450 involved in vitamin D metabolism. JMolBiol Downloaded from 2010). Collectively, these data reveal a novel P450-based mechanism for 396:441–451. adaptive transcriptome remodeling, whereby xenobiotics and endogenous Apostolatos H, Apostolatos A, Vickers T, Watson JE, Song S, Vale F, Cooper DR, Sanchez-Ramos “ J, and Patel NA (2010) Vitamin A metabolite, all-trans-retinoic acid, mediates alternative substrates, monitored by one of several tissue- or disease-specific P450 splicing of protein kinase C deltaVIII (PKCdeltaVIII) isoform via splicing factor SC35. J Biol clouds,” are metabolized in a coordinated fashion that harmonize NR Chem 285:25987–25995. 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Clin Endocrinol Metab 89:43–48. Performed data analysis: Annalora, Iversen. Cowie P, Hay EA, and MacKenzie A (2015) The noncoding human genome and the future of personalised medicine. Expert Rev Mol Med 17:e4. Wrote or contributed to the writing of the manuscript: Annalora, Iversen, Cunningham F, Amode MR, Barrell D, Beal K, Billis K, Brent S, Carvalho-Silva D, Clapham P, Marcus. Coates G, Fitzgerald S, et al. (2015) Ensembl 2015. Nucleic Acids Res 43:D662–D669. Alternative P450 Splicing Redirects Human Drug Metabolism 387

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