Genetic variation in EPHA contributes to sensitivity to paclitaxel-induced peripheral neuropathy.

AIMS: Chemotherapy-induced peripheral neuropathy (PN) is a treatment limiting toxicity of paclitaxel. We evaluated if EPHA genetic variation (EPHA4, EPHA5, EPHA6, and EPHA8) is associated with PN sensitivity by accounting for variability in systemic paclitaxel exposure (time above threshold). METHODS: Germline DNA from 60 patients with breast cancer was sequenced. PN was measured using the 8-item sensory subscale (CIPN8) of the patient-reported CIPN20. Associations for 3 genetic models were tested by incorporating genetics into previously published PN prediction models integrating measured paclitaxel exposure and cumulative treatment. Significant associations were then tested for association with PN-related treatment disruption. RESULTS: EPHA5 rs7349683 (minor allele frequency = 0.32) was associated with increased PN sensitivity (ß-coefficient = 0.39, 95% confidence interval 0.11-0.67, p = 0.007). Setting a maximum tolerable threshold of CIPN8 = 30, optimal paclitaxel exposure target is shorter for rs7349683 homozygous (11.6 h) than heterozygous (12.6 h) or wild-type (13.6 h) patients. Total number of missense variants (median = 0, range 0-2) was associated with decreased PN sensitivity (ß-coefficient: -0.42, 95% confidence interval -0.72 to -0.12, P = .006). No association with treatment disruption was detected for the total number of missense variants or rs7349683. CONCLUSION: Isolating toxicity sensitivity by accounting for exposure is a novel approach, and rs7349683 represents a promising marker for PN sensitivity that may be used to individualize paclitaxel treatment.

Scrambler therapy for chemotherapy neuropathy: a randomized phase II pilot trial.

INTRODUCTION: Chemotherapy-induced peripheral neuropathy (CIPN) is a prominent clinical problem, with limited effective therapies. Preliminary non-randomized clinical trial data support that Scrambler Therapy is helpful in this situation. METHODS: Patients were eligible if they had CIPN symptoms for at least 3 months and CIPN-related tingling or pain at least 4/10 in severity during the week prior to registration. They were randomized to receive Scrambler Therapy versus transcutaneous electrical nerve stimulation (TENS) for 2 weeks. Patient-reported outcomes (PROs) were utilized to measure efficacy and toxicity daily for 2 weeks during therapy and then weekly for 8 additional weeks. RESULTS: This study accrued 50 patients, 25 to each of the 2 study arms; 46 patients were evaluable. There were twice as many Scrambler-treated patients who had at least a 50% documented improvement during the 2 treatment weeks, from their baseline pain, tingling, and numbness scores, when compared with the TENS-treated patients (from 36 to 56% compared with 16-28% for each symptom). Global Impression of Change scores for "neuropathy symptoms," pain, and quality of life were similarly improved during the treatment weeks. Patients in the Scrambler group were more likely than those in the TENS group to recommend their treatment to other patients, during both the 2-week treatment period and the 8-week follow-up period (p < 0.0001). Minimal toxicity was observed. CONCLUSIONS: The results from this pilot trial were positive, supporting the conduct of further investigations regarding the use of Scrambler Therapy for treating CIPN.

Biological predictors of chemotherapy-induced peripheral neuropathy (CIPN): MASCC neurological complications working group overview.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common and debilitating condition associated with a number of chemotherapeutic agents. Drugs commonly implicated in the development of CIPN include platinum agents, taxanes, vinca alkaloids, bortezomib, and thalidomide analogues. As a drug response can vary between individuals, it is hypothesized that an individual's specific genetic variants could impact the regulation of genes involved in drug pharmacokinetics, ion channel functioning, neurotoxicity, and DNA repair, which in turn affect CIPN development and severity. Variations of other molecular markers may also affect the incidence and severity of CIPN. Hence, the objective of this review was to summarize the known biological (molecular and genomic) predictors of CIPN and discuss the means to facilitate progress in this field.

Unilateral Epidural Targeting of Resiniferatoxin Induces Bilateral Neurolysis of Spinal Nociceptive Afferents.

OBJECTIVE: This study modeled image-guided epidural drug delivery to test whether intraprocedural distribution of pre-injected contrast reliably predicts the neuroanatomical reach of resiniferatoxin-mediated nociceptive neurolysis. METHODS: Swine (N = 12) received unilateral L4-S2 computed tomography fluoroscopy injections by a blinded neuroradiologist; 0.25 mL of contrast was pre-injected to confirm dorsal periganglionic targeting, followed by a 0.5-mL injection of 5 µg of resiniferatoxin/Tween80 or vehicle control. Epidural contrast distribution was graded according to maximum medial excursion. Spinal cord substance P immunostaining quantified the magnitude and anatomical range of resiniferatoxin activity. RESULTS: Periganglionic injection was well tolerated by all animals without development of neurological deficits or other complications. Swine were a suitable model of human clinical spinal intervention. The transforaminal approach was used at all L4 and 50% of L5 segments; the remaining segments were approached by the interlaminar route. All injections were successful with unilateral contrast distribution for all resiniferatoxin injections (N = 28). Immunohistochemistry showed bilateral ablation of substance P+ fibers entering the spinal cord of all resiniferatoxin-treated segments. The intensity of substance P immunostaining in treated segments fell below the lower 99% confidence interval of controls, defining the knockout phenotype. Substance P knockout occurred over a narrow range and was uncorrelated to the anatomical distribution of pre-injected contrast. CONCLUSIONS: Periganglionic resiniferatoxin/Tween80 induced bilateral ablation of spinal cord substance P despite exclusively unilateral targeting. These data suggest that the location of pre-injected contrast is an imperfect surrogate for the neuroanatomical range of drugs delivered to the dorsal epidural compartment that may fail to predict contralateral drug effects.

Human interleukin-10 delivered intrathecally by self-complementary adeno-associated virus 8 induces xenogeneic transgene immunity without clinical neurotoxicity in swine.

INTRODUCTION: Intrathecal interleukin (IL)-10 delivered by plasmid or viral gene vectors has been proposed for clinical testing because it is effective for chronic pain in rodents, is a potential therapeutic for various human diseases, and was found to be nontoxic in dogs, when the human IL-10 ortholog was tested. However, recent studies in swine testing porcine IL-10 demonstrated fatal neurotoxicity. The present study aimed to deliver vector-encoded human IL-10 in swine, measure expression of the transgene in cerebrospinal fluid and monitor animals for signs of neurotoxicity. RESULTS: Human IL-10 levels peaked 2 weeks after vector administration followed by a rapid decline that occurred concomitant with the emergence of anti-human IL-10 antibodies in the cerebrospinal fluid and serum. Animals remained neurologically healthy throughout the study period. CONCLUSIONS: The findings of the present study suggest that swine are not idiosyncratically sensitive to intrathecal IL-10 because, recapitulating previous reports in dogs, they suffered no clinical neurotoxicity from the human ortholog. These results strongly infer that toxicity of intrathecal IL-10 in large animal models was previously overlooked because of a species mismatch between transgene and host. The present study further suggests that swine were protected from interleukin-10 by a humoral immune response against the xenogeneic cytokine. Future safety studies of IL-10 or related therapeutics may require syngeneic large animal models.

Fatal Meningitis in Swine after Intrathecal Administration of Adeno-associated Virus Expressing Syngeneic Interleukin-10.

Interleukin-10 (IL-10) delivered by intrathecal (i.t.) gene vectors is a candidate investigational new drug (IND) for several chronic neurological disorders such as neuropathic pain. We performed a preclinical safety study of IL-10. A syngeneic large animal model was used delivering porcine IL-10 (pIL-10) to the i.t. space in swine by adeno-associated virus serotype 8 (AAV8), a gene vector that was previously found to be nontoxic in the i.t. space. Unexpectedly, animals became ill, developing ataxia, seizures, and an inability to feed and drink, and required euthanasia. Necropsy demonstrated lymphocytic meningitis without evidence of infection in the presence of normal laboratory findings for body fluids and normal histopathology of peripheral organs. Results were replicated in a second animal cohort by a team of independent experimenters. An extensive infectious disease and neuropathology workup consisting of comprehensive testing of tissues and body fluids in a specialized research veterinary pathology environment did not identify a pathogen. These observations raise the concern that i.t. IL-10 therapy may not be benign, that previously used xenogeneic models testing the human homolog of IL-10 may not have been sensitive enough to detect toxicity, and that additional preclinical studies may be needed before clinical testing of IL-10 can be considered.

A pilot study of minocycline for the prevention of paclitaxel-associated neuropathy: ACCRU study RU221408I.

PURPOSE: Paclitaxel is associated with both an acute pain syndrome (P-APS) and chronic chemotherapy-induced peripheral neuropathy (CIPN). Given that extensive animal data suggest that minocycline may prevent chemotherapy-induced neurotoxicity, the purpose of this pilot study was to investigate the efficacy of minocycline for the prevention of CIPN and the P-APS. METHODS: Patients with breast cancer were enrolled prior to initiating neoadjuvant or adjuvant weekly paclitaxel for 12 weeks and were randomized to receive minocycline 200 mg on day 1 followed by 100 mg twice daily or a matching placebo. Patients completed (1) an acute pain syndrome questionnaire daily during chemotherapy to measure P-APS and (2) the EORTC QLQ-CIPN20 questionnaire at baseline, prior to each dose of paclitaxel, and monthly for 6 months post treatment, to measure CIPN. RESULTS: Forty-seven patients were randomized. There were no remarkable differences noted between the minocycline and placebo groups for the overall sensory neuropathy score of the EORTC QLQ-CIPN20 or its individual components, which evaluate tingling, numbness and shooting/burning pain in hands and feet. However, patients taking minocycline had a significant reduction in the daily average pain score attributed to P-APS (p = 0.02). Not only were no increased toxicities reported with minocycline, but there was a significant reduction in fatigue (p = 0.02). CONCLUSIONS: Results of this pilot study do not support the use of minocycline to prevent CIPN, but suggest that it may reduce P-APS and decrease fatigue; further study of the impact of this agent on those endpoints may be warranted.

Patient-reported (EORTC QLQ-CIPN20) versus physician-reported (CTCAE) quantification of oxaliplatin- and paclitaxel/carboplatin-induced peripheral neuropathy in NCCTG/Alliance clinical trials.

PURPOSE: Clinical practice guidelines on chemotherapy-induced peripheral neuropathy (CIPN) use the NCI Common Terminology Criteria for Adverse Events (CTCAE), while recent clinical trials employ a potentially superior measure, the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-CIPN twenty-item scale (QLQ-CIPN20), a patient-reported outcome (PRO). Practitioners and researchers lack guidance, regarding how QLQ-CIPN20 results relate to the traditional CTCAE during the serial assessment of patients undergoing chemotherapy. METHODS: Two large CIPN clinical trial datasets (538 patients) pairing QLQ-CIPN20 and CTCAE outcomes were analyzed using a multivariable linear mixed model with QLQ-CIPN20 score as the outcome variable, CTCAE grade as the main effect, and patient as random effect (accounting for internal correlation of serial measures). RESULTS: The association between QLQ-CIPN20 scores and CTCAE grades was strong (p < 0.0001), whereby patients with higher CTCAE grade had worse QLQ-CIPN20 scores. Some variation of QLQ-CIPN20 scores was observed based on drug, treatment, and cycle. While there was a marked difference in the mean QLQ-CIPN20 scores between CTCAE grades, the ranges of QLQ-CIPN20 scores within each CTCAE grade were large, leading to large overlap in CIPN20 scores across CTCAE grades. CONCLUSIONS: A strong positive association of QLQ-CIPN20 scores and CTCAE grade provides evidence of convergent validity as well as practical guidance, as to how to quantitatively interpret QLQ-CIPN20 scores at the study level in terms of the traditional CTCAE. The present results also highlight an important clinical caveat, specifically, that conversion of a specific QLQ-CIPN20 score to a specific CTCAE score may not be reliable at the level of an individual patient.

Targeted alignment and end repair elimination increase alignment and methylation measure accuracy for reduced representation bisulfite sequencing data.

BACKGROUND: DNA methylation is an important epigenetic modification involved in many biological processes. Reduced representation bisulfite sequencing (RRBS) is a cost-effective method for studying DNA methylation at single base resolution. Although several tools are available for RRBS data processing and analysis, it is not clear which strategy performs the best and there has not been much attention to the contamination issue from artificial cytosines incorporated during the end repair step of library preparation. To address these issues, we describe a new method, Targeted Alignment and Artificial Cytosine Elimination for RRBS (TRACE-RRBS), which aligns bisulfite sequence reads to MSP1 digitally digested reference and specifically removes the end repair cytosines. We compared this approach on a simulated and a real dataset with 7 other RRBS analysis tools and Illumina 450 K microarray platform. RESULTS: TRACE-RRBS aligns sequence reads to a small fraction of the genome where RRBS protocol targets on and was demonstrated as the fastest, most sensitive and specific tool for the simulated dataset. For the real dataset, TRACE-RRBS took about the same time as RRBSMAP, a third to a sixth of time needed for BISMARK and NOVOALIGN. TRACE-RRBS aligned more reads uniquely than other tools and achieved the highest correlation with 450 k microarray data. The end repair artificial cytosine removal increased correlation between nearby CpGs and accuracy of methylation quantification. CONCLUSIONS: TRACE-RRBS is fast and more accurate tool for RRBS data analysis. It is freely available for academic use at http://bioinformaticstools.mayo.edu/.

National Cancer Institute-supported chemotherapy-induced peripheral neuropathy trials: outcomes and lessons.

Chemotherapy-induced peripheral neuropathy (CIPN) is one of the most common and debilitating complications of cancer treatment. Due to a lack of effective management options for patients with CIPN, the National Cancer Institute (NCI) sponsored a series of trials aimed at both prevention and treatment. A total of 15 such studies were approved, evaluating use of various neuro-modulatory agents which have shown benefit in other neuropathic pain states. Aside from duloxetine, none of the pharmacologic methods demonstrated therapeutic benefit for patients with CIPN. Despite these disappointing results, the series of trials revealed important lessons that have informed subsequent work. Some examples of this include the use of patient-reported symptom metrics, the elimination of traditional--yet unsubstantiated--practice approaches, and the discovery of molecular genetic predictors of neuropathy. Current inquiry is being guided by the results from these large-scale trials, and as such, stands better chance of identifying durable solutions for this treatment-limiting toxicity.

Scrambler Therapy for the management of chronic pain.

PURPOSE: Chronic pain is a widespread and debilitating condition, encountered by physicians in a variety of practice settings. Although many pharmacologic and behavioral strategies exist for the management of this condition, treatment is often unsatisfactory. Scrambler Therapy is a novel, non-invasive pain modifying technique that utilizes trans-cutaneous electrical stimulation of pain fibers with the intent of re-organizing maladaptive signaling pathways. This review was conducted to further evaluate what is known regarding the mechanisms and mechanics of Scrambler Therapy and to investigate the preliminary data pertaining to the efficacy of this treatment modality. METHODS: The PubMed/Medline, SCOPUS, EMBASE, and Google Scholar databases were searched for all articles published on Scrambler Therapy prior to November 2015. All case studies and clinical trials were evaluated and reported in a descriptive manner. RESULTS: To date, 20 reports, of varying scientific quality, have been published regarding this device; all but one small study, published only as an abstract, provided results that appear positive. CONCLUSION: The positive findings from preliminary studies with Scrambler Therapy support that this device provides benefit for patients with refractory pain syndromes. Larger, randomized studies are required to further evaluate the efficacy of this approach.

Comparison of oxaliplatin and paclitaxel-induced neuropathy (Alliance A151505).

PURPOSE: Oxaliplatin and paclitaxel are commonly used chemotherapies associated with acute and chronic neuropathies. There is a need to better understand the similarities and differences of these clinical syndromes. METHODS: Neuropathy data were pooled from patients receiving adjuvant oxaliplatin and weekly paclitaxel or every 3 weeks of paclitaxel. Patients completed daily questionnaires after each chemotherapy dose and the European Organization for Research and Treatment of Cancer quality-of-life questionnaire for patients with chemotherapy-induced peripheral neuropathy before each chemotherapy cycle and for 12 months post-treatment. RESULTS: Acute neuropathy symptoms from both drugs peaked around day 3. Acute symptoms experienced in cycle 1 predicted occurrence in subsequent cycles. Paclitaxel-induced acute symptoms were similar in intensity in each cycle and largely resolved between cycles. Oxaliplatin-induced acute symptoms were about half as severe in the first cycle as in later cycles and did not resolve completely between cycles. Both drugs caused a predominantly sensory chronic neuropathy (with numbness and tingling being more common than pain). Oxaliplatin-induced neuropathy worsened after the completion of treatment and began to improve 3 months post-treatment. In contrast, paclitaxel-induced neuropathy began improving immediately after chemotherapy cessation. During treatment, the incidence of paclitaxel sensory symptoms was similar in the hands and feet; with oxaliplatin, the hands were affected more than the feet. Both paclitaxel- and oxaliplatin-induced acute neurotoxicity appeared to predict the severity of chronic neuropathy, more prominently with oxaliplatin. CONCLUSIONS: Knowledge of the similarities and differences between neuropathy syndromes may provide insight into their underlying pathophysiology and inform future research to identify preventative treatment approaches.

Sequencing of Charcot-Marie-Tooth disease genes in a toxic polyneuropathy.

OBJECTIVE: Mutations in Charcot-Marie-Tooth disease (CMT) genes are the cause of rare familial forms of polyneuropathy. Whether allelic variability in CMT genes is also associated with common forms of polyneuropathy-considered "acquired" in medical parlance-is unknown. Chemotherapy-induced peripheral neuropathy (CIPN) occurs commonly in cancer patients and is individually unpredictable. We used CIPN as a clinical model to investigate the association of non-CMT polyneuropathy with CMT genes. METHODS: A total of 269 neurologically asymptomatic cancer patients were enrolled in the clinical trial Alliance N08C1 to receive the neurotoxic drug paclitaxel, while undergoing prospective assessments for polyneuropathy. Forty-nine CMT genes were analyzed by targeted massively parallel sequencing of genomic DNA from patient blood. RESULTS: A total of 119 (of 269) patients were identified from the 2 ends of the polyneuropathy phenotype distribution: patients that were most and least susceptible to paclitaxel polyneuropathy. The CMT gene PRX was found to be deleteriously mutated in patients who were susceptible to CIPN but not in controls (p = 8 × 10(-3)). Genetic variation in another CMT gene, ARHGEF10, was highly significantly associated with CIPN (p = 5 × 10(-4)). Three nonsynonymous recurrent single nucleotide variants contributed to the ARHGEF10 signal: rs9657362, rs2294039, and rs17683288. Of these, rs9657362 had the strongest effect (odds ratio = 4.8, p = 4 × 10(-4)). INTERPRETATION: The results reveal an association of CMT gene allelic variability with susceptibility to CIPN. The findings raise the possibility that other acquired polyneuropathies may also be codetermined by genetic etiological factors, of which some may be related to genes already known to cause the phenotypically related Mendelian disorders of CMT.

Calcium and Magnesium Use for Oxaliplatin-Induced Neuropathy: A Case Study to Assess How Quickly Evidence Translates Into Practice.

Substantial research efforts have focused on methods of treating and preventing oxaliplatin-associated neuropathy, the dose-limiting toxicity associated with this drug. Administration of intravenous calcium and magnesium (CaMg) before and after oxaliplatin has been the most studied approach to preventing oxaliplatin-induced neuropathy. Although early reports demonstrated potential benefit, subsequent larger trials failed to confirm the efficacy of CaMg in preventing this adverse effect. This article explores how accumulating evidence for and against the use of CaMg for preventing oxaliplatin-induced neuropathy has impacted clinical practice.

Intraneural convection enhanced delivery of AAVrh20 for targeting primary sensory neurons.

Gene therapy using adeno-associated virus (AAV) is an attractive strategy to treat disorders of the peripheral nervous system (PNS), such as chronic pain or peripheral neuropathies. Although intrathecal (IT) administration of AAV has been the standard in the field for targeting the PNS, it lacks anatomical specificity and results in wide rostro-caudal distribution of the vector. An alternative approach is to deliver AAV directly to the peripheral nerve axon. The present study employed convection-enhanced delivery (CED) of a novel AAV serotype, AAVrh20, expressing enhanced green fluorescent protein (EGFP) into rat sciatic nerve investigating its efficacy, anatomical selectivity, and safety, compared to the IT route. Intraneural CED resulted in transduction confined to the ipsilateral L4 and L5 DRG while IT administration led to promiscuous DRG transduction encompassing the entire lumbar region bilaterally. The transduction rate for intraneural AAV administration was similar to IT delivery (24% for L4 and 31.5% for L5 DRG versus 50% for L4 and 19.5% for L5 DRG). The use of hyperosmotic diluent did not further improve the transduction efficiency. AAVrh20 was superior to reference serotypes previously described to be most active for each route. Intraneural CED of AAV was associated with transient allodynia that resolved spontaneously. These findings establish intraneural CED as an alternative to IT administration for AAV mediated gene transfer to the PNS and, based on a reference rodent model, suggest AAVrh20 as a superior serotype for targeting the PNS.

Preclinical toxicity evaluation of AAV for pain: evidence from human AAV studies and from the pharmacology of analgesic drugs.

Gene therapy with adeno-associated virus (AAV) has advanced in the last few years from promising results in animal models to >100 clinical trials (reported or under way). While vector availability was a substantial hurdle a decade ago, innovative new production methods now routinely match the scale of AAV doses required for clinical testing. These advances may become relevant to translational research in the chronic pain field. AAV for pain targeting the peripheral nervous system was proven to be efficacious in rodent models several years ago, but has not yet been tested in humans. The present review addresses the steps needed for translation of AAV for pain from the bench to the bedside focusing on pre-clinical toxicology. We break the potential toxicities into three conceptual categories of risk: First, risks related to the delivery procedure used to administer the vector. Second, risks related to AAV biology, i.e., effects of the vector itself that may occur independently of the transgene. Third, risks related to the effects of the therapeutic transgene. To identify potential toxicities, we consulted the existing evidence from AAV gene therapy for other nervous system disorders (animal toxicology and human studies) and from the clinical pharmacology of conventional analgesic drugs. Thereby, we identified required preclinical studies and charted a hypothetical path towards a future phase I/II clinical trial in the oncology-palliative care setting.

Advances in small bowel neuroendocrine neoplasia.

PURPOSE OF REVIEW: This review aims at summarizing progress in clinical trials and basic science redefining the diagnosis and treatment of well differentiated small intestine neuroendocrine tumors (SI-NET). RECENT FINDINGS: Two clinical trials demonstrated antitumor activity of the long-acting somatostatin analogues octreotide long-acting release and lanreotide for advanced SI-NET. The mammalian target of rapamycin (mTOR) inhibitor everolimus is another treatment option for patients with SI-NET, but awaits definitive proof of benefit in the ongoing RAD001 In Advanced Neuroendocrine Tumors study (RADIANT-4). Two whole exome/genome-sequencing studies reported in the past year provided the first genome-wide analysis of large sets of SI-NET at nucleotide resolution. Candidate therapeutically relevant alterations were found to affect SRC, SMAD genes, aurora kinase A, epidermal growth factor receptor, heat shock protein 90, and platelet-derived growth factor receptor as well as mutually exclusive amplification of RAC-alpha serine/threonine-protein kinase (AKT1) or AKT2 and other alterations of PI3K/Akt/mTOR signaling genes. The gene CDKN1B is inactivated by small insertions/deletions in 8% of patients with SI-NET suggesting cell cycle inhibitors as new candidate drugs for SI-NET. Circulating tumor cells and tumor-derived RNA in the blood are promising clinical tests for SI-NET. SUMMARY: Clinical and genomic research may merge in the near future to re-shape clinical trials and to define the 'personalized' treatment options for patients with SI-NET.

mRNA-seq with agnostic splice site discovery for nervous system transcriptomics tested in chronic pain.

mRNA-seq is a paradigm-shifting technology because of its superior sensitivity and dynamic range and its potential to capture transcriptomes in an agnostic fashion, i.e., independently of existing genome annotations. Implementation of the agnostic approach, however, has not yet been fully achieved. In particular, agnostic mapping of pre-mRNA splice sites has not been demonstrated. The present study pursued dual goals: (1) to advance mRNA-seq bioinformatics toward unbiased transcriptome capture and (2) to demonstrate its potential for discovery in neuroscience by applying the approach to an in vivo model of neurological disease. We have performed mRNA-seq on the L4 dorsal root ganglion (DRG) of rats with chronic neuropathic pain induced by spinal nerve ligation (SNL) of the neighboring (L5) spinal nerve. We found that 12.4% of known genes were induced and 7% were suppressed in the dysfunctional (but anatomically intact) L4 DRG 2 wk after SNL. These alterations persisted chronically (2 mo). Using a read cluster classifier with strong test characteristics (ROC area 97%), we discovered 10,464 novel exons. A new algorithm for agnostic mapping of pre-mRNA splice junctions (SJs) achieved a precision of 97%. Integration of information from all mRNA-seq read classes including SJs led to genome reannotations specifically relevant for the species used (rat), the anatomical site studied (DRG), and the neurological disease considered (pain); for example, a 64-exon coreceptor for the nociceptive transmitter substance P was identified, and 21.9% of newly discovered exons were shown to be dysregulated. Thus, mRNA-seq with agnostic analysis methods appears to provide a highly productive approach for in vivo transcriptomics in the nervous system.

SAAP-RRBS: streamlined analysis and annotation pipeline for reduced representation bisulfite sequencing.

UNLABELLED: Reduced representation bisulfite sequencing (RRBS) is a cost-effective approach for genome-wide methylation pattern profiling. Analyzing RRBS sequencing data is challenging and specialized alignment/mapping programs are needed. Although such programs have been developed, a comprehensive solution that provides researchers with good quality and analyzable data is still lacking. To address this need, we have developed a Streamlined Analysis and Annotation Pipeline for RRBS data (SAAP-RRBS) that integrates read quality assessment/clean-up, alignment, methylation data extraction, annotation, reporting and visualization. This package facilitates a rapid transition from sequencing reads to a fully annotated CpG methylation report to biological interpretation. AVAILABILITY AND IMPLEMENTATION: SAAP-RRBS is freely available to non-commercial users at the web site http://ndc.mayo.edu/mayo/research/biostat/stand-alone-packages.cfm.