peerj_json_files/PeerJ_Json_128.json

{
    "v1_col_introduction": "introduction  : Deep brain stimulation (DBS) has emerged as a relatively safe and reversible neurosurgical technique that can be used in the clinical treatment of traditionally treatment resistant psychiatric disorders. DBS enables the adjustable and stable electrical stimulation of targeted brain structures. A recent paper by H\u00f6flich et al1 notes variability in treatment outcomes for DBS patients, which is likely due to variable responses to differences in targeted stimulation regions and in post-operative stimulation parameters. Both sources of variation, the authors note, will effect the stimulation of different brain tissue fibers having different anatomical and functional connections. Furthermore, the authors suggest that not every target will be suitable for every person, as there exists a large degree of inter-individual variability of brain region activation during a reward task in healthy volunteers, and suggest that future work could (and should) focus on developing surgical plans based on individual-specific activations, functional connectivity and/or tractography. This work exemplifies the large degree of clinically relevant biological variability that exists in terms of individual clinical characteristics.\nOngoing clinical trials testing the \u201cEffectiveness of Deep Brain Stimulation for Treating People With Treatment Resistant Obsessive-Compulsive Disorder\u201d2 detail the below exclusion criteria:\n\u2022 current or past psychotic disorder, \u2022 a clinical history of bipolar mood disorder, and/or \u2022 an inability to control suicide attempts, imminent risk of suicide in the investigator's judgment, or\na history of serious suicidal behavior, which is defined using the Columbia-Suicide Severity Rating Scale (C-SSRS) as either: one or more actual suicide attempts in the 3 years before study entry with the lethality rated at 3 or higher, or one or more interrupted suicide attempts with a potential lethality judged to result in serious injury or death.\nThese study criteria exclude the most severe cases of OCD, as many people with severe OCD also have severe depression, usually with passive (and sometimes active) suicidal ideation3-5. Obsessions and compulsions can be quite severe, with very poor insight, sometimes to a delusional or psychotic degree, and there can also be co-occurring psychoses in any individual. Each person is to some degree unique in his or her psychiatric presentation, and a tailored evaluation schema could prove more effective in clinical treatment. Due in part to these above hurdles, there are few detailed descriptions of the efficacy of DBS for OCD, with the number of published case studies on the efficacy of DBS for OCD covering upwards of ~100 people 6-21.\nAn explosive growth in exome and whole genome sequencing (WGS) 22 has occurred in parallel to the emergence of DBS for OCD, led in part by dramatic cost reductions. This in turn has given medical practitioners an efficient and comprehensive means to medically assess coding and non-coding regions of the genome, leading to much promise in terms of assessing and treating human disease. In our own efforts to push forward the field of precision medicine, we report here one effort to integrate the areas of clinical neuropsychiatry, brain machine interfaces and personal genomics in the individualized care of one person. We evaluate and treat an individual with DBS for treatment refractory OCD, gauge the feasibility and usefulness of the medical integration of genetic data stemming from whole genome sequencing, and search for rare variants that might alter the course of medical care for this person. As mentioned above,\n1 2\n3 4 5 6\n7 8 9\n10 11 12 13 14 15 16 17 18 19 20 21\n22 23\n24 25 26 27 28 29 30\n31 32 33 34 35 36 37 38\n39 40 41 42 43 44 45 46 47\nPeerJ reviewing PDF | (v2013:06:578:2:0:NEW 14 Sep 2013)\nR ev ie w in g M an\nus cr ip t\nthere have been relatively few reports on studies detailing the effective application of DBS for OCD; we report here one such study.",
    "v2_col_introduction": "introduction  : There is a substantial body of literature that highlights the breadth of human phenotypic diversity1-12. And yet, despite a body of scientific work demonstrating significant contributions from genetic and environmental heterogeneity to this diversity, relatively broad phenotypic categorizations still dominate traditional medical classifications13-17. Furthermore, over the past 50 years, psychiatry, and medicine in general, has shifted its focus toward providing pre-market proof of the overall efficacy and safety of drugs and other interventions in randomized clinical trials involving hundreds (and sometimes thousands) of people, despite the existence of phenotypic heterogeneity and variable expressivity in nearly every person and every disease over time6,8,9,18. This course of affairs was brought about by a large confluence of societal factors, including safety concerns stemming from numerous biomedical transgressions over the years 19, including the indiscriminate use of lobotomy in the field of psychiatry20,21. However, there is some evidence suggesting that we might be now undergoing a transformation of the medical world22,23, with a return to individual-focused medical care and to the realization that each individual is truly unique, influenced by their own genetic and environmental factors3,5,24-26.\nAlong these lines, deep brain stimulation (DBS) has emerged as a relatively safe and reversible neurosurgical technique that can be used in the clinical treatment of traditionally treatment resistant psychiatric disorders. DBS enables the adjustable and stable electrical stimulation of targeted brain structures. A recent paper by Hoflich et al27 notes variability in treatment outcomes for DBS patients, which is likely due to variable responses to differences in targeted stimulation regions and in postoperative stimulation parameters. Both sources of variation, the authors note, will effect the stimulation of different brain tissue fibers having different anatomical and functional connections. Furthermore, the authors suggest that not every target will be suitable for every person, as there exists a large degree of inter-individual variability of brain region activation during a reward task in healthy volunteers, and suggest that future work could (and should) focus on developing surgical plans based on individualspecific activations, functional connectivity and/or tractography. This work exemplifies the large degree of clinically relevant biological variability that exists in terms of individual clinical characteristics.\nOngoing clinical trials testing the \u201cEffectiveness of Deep Brain Stimulation for Treating People With Treatment Resistant Obsessive-Compulsive Disorder\u201d28 detail the below exclusion criteria:\n\u2022 current or past psychotic disorder, \u2022 a clinical history of bipolar mood disorder, and/or \u2022 an inability to control suicide attempts, imminent risk of suicide in the investigator's judgment, or\na history of serious suicidal behavior, which is defined using the Columbia-Suicide Severity Rating Scale (C-SSRS) as either: one or more actual suicide attempts in the 3 years before study entry with the lethality rated at 3 or higher, or one or more interrupted suicide attempts with a potential lethality judged to result in serious injury or death.\nUnfortunately, these study criteria exclude the most severe cases of OCD, as many people with severe OCD also have severe depression, usually with passive (and sometimes active) suicidal ideation29-31. Obsessions and compulsions can be quite severe, with very poor insight, sometimes to a delusional or psychotic degree, and there can also be co-occurring psychoses in any individual. Each person is to some\n1 2\n3 4 5 6\n7 8\n9 10 11 12 13 14 15 16 17 18 19 20 21 22\n23 24 25 26 27 28 29 30 31 32 33 34\n35 36\n37 38 39 40 41 42 43\n44 45 46 47\nPeerJ reviewing PDF | (v2013:06:578:1:0:NEW 13 Aug 2013)\nR ev ie w in g M an\nus cr ip t\ndegree unique in his or her psychiatric presentation, and a tailored evaluation schema may be more effective in clinical treatment. Indeed, categorical thresholds for clinical trials and/or general psychiatric treatment lack the continuous gradation that would otherwise enable a high degree of treatment precision for any one person. Due in part to these substantial hurdles, there are unfortunately very few detailed descriptions of the efficacy of DBS for OCD, with the number of published case studies on the efficacy of DBS for OCD covering upwards of ~100 people 32-47. This is really quite small, given that there are 6-7 billion people on this planet, with some estimates of the prevalence of OCD ranging from 0.4-1.2% in the community and perhaps more in military veterans 48.\nThere has, in parallel, been an explosive growth in exome and whole genome sequencing (WGS) 25, led in part by dramatic cost reductions. The same is true for genotyping microarrays, which are becoming increasingly denser with various markers while maintaining a relatively stable cost 49. In the medical world, WGS has led to the discovery of the genetic basis of Miller Syndrome 50 and in another instance, it was used to investigate the genetic basis of Charcot-Marie-Tooth neuropathy 51, alongside a discussion of the \u2018return of results\u2019 52. In 2011, the diagnosis of a pair of twins with dopa (3,4-dihydroxyphenylalanine) responsive dystonia (DRD; OMIM #128230) and the discovery through WGS that they carried compound heterozygous mutations in the SPR gene encoding sepiapterin reductase led to supplementation of l-dopa therapy with 5-hydroxytryptophan, a serotonin precursor, resulting in clinical improvements in both twins 53.\nAs the cost of WGS decreases, evidence is emerging that exon capture and sequencing only achieves a high depth of sequencing coverage in about 90% of the exons, whereas WGS does not involve a capture step and thus obtains better coverage on >95% of all exons in the genome. Of course, even the definition of the exome is a moving target, as the research community is constantly annotating and finding new exons not previously discovered 54,55, and therefore WGS is a much more comprehensive way to assess coding and non-coding regions of the genome. Given that WGS can impact clinical care, it is now a matter of economics and feasibility in terms of whether and when WGS will be adopted widely in a clinical setting 25,56.\nIn our own efforts to push forward the field of precision medicine, we are studying individuals and families with a diverse range of illnesses. We report here one effort to integrate the areas of clinical neuropsychiatry, brain machine interfaces and personal genomics in the individualized care of one person. We evaluate and treat an individual with DBS for treatment refractory OCD and also gauge the feasibility and usefulness of the medical integration of genetic data stemming from whole genome sequencing. To date, there have been relatively few reports on studies detailing the effective application of DBS for OCD; we report here one such study.",
    "v3_col_introduction": "introduction  : There is a substantial body of literature spanning hundreds of years that highlights the breadth of human phenotypic diversity1-12. And yet, despite a body of scientific work demonstrating significant contributions from extreme genetic and environmental heterogeneity to this diversity, many have resorted to overly coarse categorizations in psychiatry that bear little resemblance to reality13-17. Over the past 50 years, psychiatry, and medicine in general, has shifted its focus toward providing pre-market proof of the overall efficacy and safety of drugs in randomized clinical trials involving hundreds (and sometimes thousands) of people, despite the existence of extreme phenotypic heterogeneity and variable expressivity in nearly every person and every disease over time6, 8, 9, 18. This course of affairs was brought about by a large confluence of societal factors, including safety concerns stemming from numerous biomedical transgressions over the years 19, including the indiscriminate use of lobotomy in the field of psychiatry20, 21. Unfortunately and perversely, the intense monetary incentives inherent in achieving \u201csuccessful\u201d clinical trials has now led to controversies regarding trial design, data collection, data quality, and reporting22, 23. However, there is some evidence suggesting that we might be undergoing a transformation of the medical world24, 25, with a return to individual-focused medical care and to the realization that each individual is truly unique, influenced by their own genetic and environmental factors3, 5, 26-28. Accordingly, some are beginning to embrace the notion of individualized, participatory medicine29, including the \u201cquantified self movement\u201d30 and the \u201cnetworking of science\u201d model27, 31, 32. We report here our attempt to integrate, in our study of one individual, the areas of clinical neuropsychiatry, personal genomics and brain-machine interfaces.",
    "v1_text": "results : Pertinent clinical symptoms and treatment A 37-year old man and U.S. veteran (here named with pseudonymous initials M.A.) was evaluated by GJL in 2010 for severe, treatment-refractory obsessive compulsive disorder (OCD), which is an illness that can be quite debilitating31. M.A. had a lifelong history of severe obsessions and compulsions, including contamination fears, scrupulosity, and the fear of harming others, with much milder symptoms in childhood that got much worse in his early 20\u2019s. His Yale-Brown Obsessive Compulsive Scale (YBOCS)32,33 ranged from 32-40, indicating extremely severe OCD. Perhaps the worst period of OCD included a 5-day, near continuous, period of tapping on his computer keyboard as a compulsion to prevent harm from occurring to his family members. M.A. had suffered throughout his life from significant periods of depression with suicidal ideation, and he had attempted suicide at least three times. His prior psychiatric history also includes episodes of paranoia relating to anxieties from his OCD, and he continues to be treated with biweekly injections of risperidone. His Global Assessment of Functioning (GAF) typically ranged from 5-15 on a 100 point scale. His treatment history included over 15 years of multiple medication trials, including clomipramine and multiple SSRIs at high doses, including fluoxetine at 80 mg by mouth daily, along with several attempts with outpatient exposure and ritual prevention (ERP) therapy34. M.A. inquired and was evaluated by GJL at the University of Utah and then at two other centers independently offering deep brain stimulation for OCD. One of these centers required (as a condition for eligibility for an ongoing clinical trial) a two-week inpatient hospitalization with intensive ERP, which subsequently occurred and was documented as improving his YBOCS score to 24 at discharge. He maintains that he actually experienced no improvement during that hospitalization, but rather told the therapists what they wanted to hear, as they were \u201ctrying so hard\u201d. See the Supplemental File S11 for other clinical details. The teams at the University of Utah and two other centers declined to perform surgery due to his prior history of severe depression, suicide attempts and possible psychoses with paranoia. Through substantial persistence of M.A. and his family members, a psychiatrist and neurosurgeon at a fourth center decided that he was an appropriate candidate for surgical implantation of the Medtronic Reclaim\u00ae DBS Therapy device for OCD, approved under a Humanitarian Device Exemption (HDE) for people with chronic, severe, treatment-resistant OCD23, and he was implanted in January of 2011 (Fig. 1). The device targets the nucleus accumbens / anterior limb of the internal capsule (ALIC). A detailed account of the surgical procedure can be found in the Supplemental File 11. acknowledgements : We thank the many doctors and other caregivers who have worked with M.A, including Drs. Paul House, Ziad Nahas and Istvan Takacs. GJL thanks Kenyon Fausett (at Medtronic) and Lauren Schrock (at University of Utah) for helping train him to perform DBS programming. M.A. and his family have been extraordinarily cooperative throughout the course of treatment. We also thank Tina Hambuch, Erica Ramos, Dawn Barry and others at Illumina for helping to develop the TruSight Individual Genome Sequencing (IGS) test, a whole-genome sequencing service using Illumina\u2019s short-read sequencing technology in the CLIA-certified, CAP-accredited Illumina Clinical Services Laboratory. They provided fee-for-service whole genome sequencing in the CLIA lab at Illumina, along with generating the clinical report on 344 genes. Julianne O\u2019Daniel graciously provided advice regarding genetic counseling, along with helping interpret findings in the 57 genes that are currently recommended for \u201creturn of results\u201d by the American College of Medical Genetics. References 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 PeerJ reviewing PDF | (v2013:06:578:2:0:NEW 14 Sep 2013) R ev ie w in g M an us cr ip t 1 Hoflich, A., Savli, M., Comasco, E., Moser, U., Novak, K., Kasper, S. & Lanzenberger, R. Neuropsychiatric deep brain stimulation for translational neuroimaging. Neuroimage 79, 30-41, doi:http://dx.doi.org/10.1016/j.neuroimage.2013.04.065 (2013). 2 ClinicalTrials.gov Identifier: NCT00640133: Effectiveness of Deep Brain Stimulation for Treating People With Treatment Resistant Obsessive-Compulsive Disorder, <http://clinicaltrials.gov/show/NCT00640133> (2013). 3 Torres, A. R., Ramos-Cerqueira, A. T., Ferrao, Y. A., Fontenelle, L. F., do Rosario, M. C. & Miguel, E. C. Suicidality in obsessive-compulsive disorder: prevalence and relation to symptom dimensions and comorbid conditions. J Clin Psychiatry 72, 17-26; quiz 119-120, doi:10.4088/JCP.09m05651blu (2011). 4 Alonso, P., Segalas, C., Real, E., Pertusa, A., Labad, J., Jimenez-Murcia, S., Jaurrieta, N., Bueno, B., Vallejo, J. & Menchon, J. M. Suicide in patients treated for obsessive-compulsive disorder: a prospective follow-up study. J Affect Disord 124, 300-308, doi:10.1016/j.jad.2009.12.001 (2010). 5 Balci, V. & Sevincok, L. Suicidal ideation in patients with obsessive-compulsive disorder. Psychiatry Res 175, 104-108, doi:10.1016/j.psychres.2009.03.012 (2010). 6 Roh, D., Chang, W. S., Chang, J. W. & Kim, C. H. Long-term follow-up of deep brain stimulation for refractory obsessivecompulsive disorder. Psychiatry Res 200, 1067-1070, doi:10.1016/j.psychres.2012.06.018 (2012). 7 Goodman, W. K. & Alterman, R. L. Deep brain stimulation for intractable psychiatric disorders. Annu Rev Med 63, 511-524, doi:10.1146/annurev-med-052209-100401 (2012). 8 Blomstedt, P., Sjoberg, R. L., Hansson, M., Bodlund, O. & Hariz, M. I. Deep Brain Stimulation in the Treatment of ObsessiveCompulsive Disorder. World Neurosurg, doi:10.1016/j.wneu.2012.10.006 (2012). 9 Burdick, A. P. & Foote, K. D. Advancing deep brain stimulation for obsessive-compulsive disorder. Expert Rev Neurother 11, 341-344, doi:10.1586/ern.11.20 (2011). 10 Mian, M. K., Campos, M., Sheth, S. A. & Eskandar, E. N. Deep brain stimulation for obsessive-compulsive disorder: past, present, and future. 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Genetics in medicine : official 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 PeerJ reviewing PDF | (v2013:06:578:2:0:NEW 14 Sep 2013) R ev ie w in g M an us cr ip t journal of the American College of Medical Genetics 13, 255-262, doi:10.1097/GIM.0b013e3182088158 (2011).729 PeerJ reviewing PDF | (v2013:06:578:2:0:NEW 14 Sep 2013) R ev ie w in g M an us cr ip t We show here sagittal and transverse sections taken from CT scans. Imaging was performed before (A) and after (B) M.A. received deep brain stimulation surgery for his treatment refractory OCD. Two deep brain stimulator probes can be seen to be in place from a bifrontal approach (B), with tips of the probes located in the region of the hypothalamus. Leads traverse through the left scalp soft tissues. Streak artifact from the leads somewhat obscures visualization of the adjacent bifrontal and left parietal parenchyma. We did not observe any intracranial hemorrhage, mass effect or midline shift or extra-axial fluid collection. Brain parenchyma was normal in volume and contour. PeerJ reviewing PDF | (v2013:06:578:2:0:NEW 14 Sep 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:06:578:2:0:NEW 14 Sep 2013) R ev ie w in g M an us cr ip t Figure 2 Implementation of the analytic-interpretive split model for the clinical incorporation of a whole genome. We have implemented the analytic-interpretive split model here with M.A., with WGS being performed in a CLIA certified and CAP accredited lab at Illumina as part of the Individual Genome Sequencing test developed by them. The WGS acts as a discrete deliverable clinical unit from which multiple downstream interpretive analyses were performed. We used the ERDS CNV caller, the Golden Helix SVS CNAM for CNV calling, and the Omicial Opal and the AssureRx Health Inc. pipelines for variant annotation and clinical interpretation of genomic variants. By archiving and offering to him the encrypted hard drive containing his \u201craw\u201d sequencing data, any number of people, including the individual and/or his/her health care providers can analyze his genome for years to come. Abbreviations are as follows: CLIA, Clinical Laboratory Improvement Amendments; CAP, College of American methods : discussion : conclusions : One can learn a substantial amount from detailed study of particular individuals (for just a small sampling, see102-109), and we believe that we are entering an era of precision medicine in which we can learn from and collect substantial data on informative individual cases. Incorporating insights from a range of scientific and clinical disciplines into the study and treatment of any one person is therefore beginning to emerge as a tractable, and more holistic, approach, and we document here what we believe to be the first integration of deep brain stimulation and whole genome sequencing for precision medicine in the evaluation, treatment and preventive care for one severely mentally ill individual, M.A. We have shown that DBS has been successful in aiding in the care and beneficial clinical outcome of his treatment refractory OCD, and we have also demonstrated that it is indeed feasible, given current technologies, to incorporate health information from WGS into the clinical care of one person with severe mental illness, including with the return of these health information to him directly. On a comparative level, deep brain stimulation has thus far been a more direct and effective intervention for his mental illness than anything discovered from his whole genome sequencing. Despite this, health information stemming from these WGS data was nevertheless immediately useful in the care of this person, as a variant associated with his ophthalmologic phenotype did indeed inform and enrich his care, and we expect that these data will continue to inform his care as our understandings of human biology and the genetic architecture of disease improves. Of course, the genomic data would have been more helpful if obtained much earlier in his medical course, as it could have provided guidance on which medications to avoid or to provide in increased doses. ethics compliance : Research was carried out in compliance with the Helsinki Declaration. The corresponding author (GJL) conducted all clinical evaluations and he is an adult psychiatry and child/adolescent psychiatry diplomate of the American Board of Psychiatry and Neurology. GJL obtained IRB approval #00038522 at the University of Utah in 2009-2010 to evaluate candidates for surgical implantation of the Medtronic Reclaim\u00ae DBS Therapy for OCD, approved under a Humanitarian Device Exemption (HDE) for people with chronic, severe, treatment-resistant OCD23. The interdisciplinary treatment team consisted of one psychiatrist (GJL), one neurologist and one neurosurgeon. Implantation ultimately occurred on a clinical basis at another site. Written consent was obtained for phenotyping and whole genome sequencing through Protocol #100 at the Utah Foundation for Biomedical Research, approved by the Independent Investigational Review Board, Inc. Informed and written consent was also obtained using the Illumina Clinical Genome Sequencing test consent form, which is a clinical test ordered by the treating physician, clinical results for dbs for treatment-refractory ocd : After healing for one month, the implanted device (equipped with the Kinetra Model 7428 Neurostimulator) was activated on February 14, 2011, with extensive programming by an outpatient psychiatrist, with bilateral stimulation of the ALIC. Final settings were case positive, contact 1 negative on the left side at 2.0 V, frequency 130 Hz, and pulse width 210 usec, and case positive, contact 5 negative on the right side with identical settings. Over the next few months, his voltage was increased monthly in increments of 0.2-0.5 V by an outpatient psychiatrist. He returned to one of the author\u2019s (GJL) for psychiatric treatment in July 2011, at which time his voltage was set at 4.5 V bilaterally. His depression had immediately improved after the surgery, along with many of his most irrational obsessions, but his YBOCS score still remained in the 35-38 range. From July 2011-December 2011, his voltage was increased bilaterally on a monthly basis in increments of 0.2 V, with steady improvement with his OCD until his battery started to lose charge by December 2011. This 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 PeerJ reviewing PDF | (v2013:06:578:2:0:NEW 14 Sep 2013) R ev ie w in g M an us cr ip t caused him considerable anxiety, prompting him to turn off his battery in order to \u201csave battery life\u201d, which unfortunately led to a complete relapse to his baseline state in a 24 hour period, which was reversed when he turned the battery back on. The battery was surgically replaced with a rechargeable Activa RC neurostimulator battery in January 2012, and the voltage has been increased monthly in 0.1-0.2 V increments until the present time (May 2013). At every visit, M.A. has reported improvements, with reductions of his obsessions and compulsions, marked by a steady decline in his YBOCS score (Fig. 3). M.A. has started to participate in many activities that he had never previously been able to engage in. This includes: exercising (losing 50 pounds in two years) and volunteering at the church and other organizations, but not yet able to work in any paid capacity. M.A. also started dating and recently got married, highlighting his improvement in daily functioning, with a GAF score ranging now from 40-50. New issues that M.A. reports are consistent tenesmus, occasional diarrhea (which he can now tolerate despite prior contamination obsessions) and improved vision (going from 20/135 to 20/40 vision, as documented by his optometrist), with him no longer needing to wear glasses. It is unknown whether the DBS implant has contributed to any of these issues. Attempts to add fluoxetine at 80 mg by mouth daily for two months to augment any efficacy from the DBS and ERP were unsuccessful, mainly due to no discernible benefit and prominent sexual side effects. M.A. still receives an injection of 37.5 mg risperidone every two weeks for his past history of psychoses; otherwise, he no longer takes any other medications. There has not been any exacerbation of psychoses in this individual during the two years of treatment with DBS. There are still many challenges in showing how any one mutation can contribute toward a clear phenotype, particularly in the context of genetic background and possible environmental influences 88. Bioinformatics confounders, such as poor data quality 89, sequence inaccuracy, and variation introduced by different methodological approaches90 can further complicate biological and genetic inferences. Although the variants discussed in the results section of our study have been previously associated with mental disease, we caution that the data presented are not sufficient to implicate any particular mutation as being 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 PeerJ reviewing PDF | (v2013:06:578:2:0:NEW 14 Sep 2013) R ev ie w in g M an us cr ip t necessary or sufficient to lead to the described phenotype, particularly given that mental illness results from a complex interaction of any human with their surrounding environment and social support structures. The genetic architecture of most neuropsychiatric illness is still largely undefined and controversial 91-94. We provide our study as a cautionary one: WGS cannot act as a diagnostic and prognostic panacea for neuropsychiatric disorders, but instead could act to elucidate risk factors for psychiatric disease and pharmacogenetic variants that can inform future medication treatments. During our study, we found that M.A. carries at least three alleles that have been associated with neuropsychiatric phenotypes, including variants in BDNF, MTHFR, and ChAT (Table 1). And, although we have discovered informative phamacogenetic variants in this person, these discoveries have not led to the immediate alteration of this person\u2019s medication schema. We have archived these discoveries, as described below, and expect that these variants will be useful over the course of his life-long medical care. We feel that this information is inherently valuable, as one can never predict with certainty what the future might hold, and a more complete medical profile on individual patients will facilitate more informed medical choices. Integrating WGS data into the Electronic Medical Health Record In the context of the incomplete, and sometimes proprietary, nature of human gene mutation databases, it is likely that analyses and medical guidance gleaned from these WGS data will differ from institution to institution. It is therefor important that people be given the opportunity, like with many other traditional medical tests, to obtain \u201csecond opinions\u201d. For this to be possible, one must accurately describe the contents of short-read sequencing data in terms of the existing electronic medical health standards, so that these data can be incorporated into an electronic medical health record. Accurately describing the contents of next generation sequencing (NGS) results is particularly critical for clinical analysis of genomic data. However, genomics and medicine use different, often incompatible terminologies and standards to describe sequence variants and their functional effects. In our efforts to treat this one person with severe mental illness, we have implemented the GVFclin format for the variants that were discovered during the sequencing of his whole genome (see Supplemental File S12). We hope to eventually incorporate his genetic data into his electronic health record, if and when the VistA health information system (HIS) 71-74 is upgraded to allow entry of such data. We did already counsel M.A. regarding several genetic variants that may be clinically relevant to predisposing him to his psychiatric disorder95. evaluation and recruitment for dbs for treatment-refractory ocd : GJL received training regarding DBS for OCD at a meeting hosted by Medtronic in Minneapolis, Minnesota, in September 2009. The same author attended a Tourette Syndrome Association meeting on DBS for Tourette Syndrome, Miami, Florida, in December 2009. Approximately ten candidates were evaluated over a one-year period in 2010. The individual discussed herein received deep brain stimulation surgery at another site, and then returned for follow-up with GJL. Another psychiatrist, author RR, provided ongoing consultation throughout the course of this study. Although other candidates have since returned for follow-up (with GJL), no others have been surgically treated. CLIA WGS and the Management of Results from sequencing data CLIA WGS using the Illumina Individual Genome Sequencing test Whole genome sequencing was ordered on this individual as part of our ongoing effort to implement precision medicine in the diagnosis, treatment, and preventive care for individuals. His genome was sequenced in the Illumina Clinical Services Laboratory (CLIA-certified, CAP-accredited) as part of the TruSight Individual Genome Sequencing (IGS) test, a whole-genome sequencing service using Illumina\u2019s short- read sequencing technology24 (Fig. 2). Although clinical genome sequencing was ordered by GJL on a clinical basis (thus not requiring IRB approval), the clinical phenotyping and collection of blood and saliva for other research purposes was approved by the Institutional Review Board (iIRB) (Plantation, Florida) as part of a study protocol at the Utah Foundation for Biomedical Research (UFBR). Consistent with laboratory-developed tests, WGS has not been cleared or approved by the U.S. Food and Drug Administration25. The entire procedure included barcoded sample tracking of the blood collected by GJL from this person, followed by DNA isolation and sequencing in the Illumina CLIA lab. Data statistics are summarized in Supplemental Fig. S1. WGS data analysis and variant prioritization For the bioinformatics analyses, Illumina utilized the internal assembler and variant caller CASAVA (short for Consensus Assessment of Sequence And VAriation). Reads were mapped to the Genome Reference Consortium assembly GRCh37. Data for sequenced and assembled genomes was provided on one hard drive, formatted with the NTFS file system and encrypted using the open source cross platform TrueCrypt software (www.truecrypt.org) and the Advanced Encryption Standard (AES) algorithm (Federal Information Processing Standards Publication 197). 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 PeerJ reviewing PDF | (v2013:06:578:2:0:NEW 14 Sep 2013) R ev ie w in g M an us cr ip t Genotyping array data was generated in parallel of the CLIA whole genome sequencing, using the Illumina HumanOmni2.5-8 bead chip. The encrypted hard drive contains several files, including a \u201cgenotyping folder\u201d within which there is a genotyping report in a text-based tab-delimited format (see Supplemental File S1). See Supplemental File 11 for more details on the genotyping array data. Insertions, deletions and structural alterations are not validated variant types in the Illumina Clinical Services Laboratory. Insertions and deletions provided in the gVCF file are for investigative or research purposes only. A medical report and the raw genomic data were provided back to the ordering physician (GJL) on an encrypted hard drive as part of the Illumina Understand your Genome Symposium, held in October 2012, which included the clinical evaluation of 344 genes (see Supplemental File S2 and S3)26. To perform more comprehensive downstream analyses using a greater portion of the genomic data, all of the variants that were detected by the Illumina CLIA WGS pipeline were imported and analyzed within the Omicia Opal web-based clinical genome interpretation platform (Fig. 2, Supplemental Fig. S5), version 1.5.027. The Omicia system annotates variants and allows for the identification and prioritization of potentially deleterious alleles. Omicia Scores, which are computationally derived estimates of deleteriousness, were calculated by using a decision-tree based algorithm, which takes as input the Polyphen, SIFT, MutationTaster and PhyloP score(s), and derives an integrative score between 0 and 1. Receiver operating characteristic (ROC) curves are plotted for that score based on annotations from HGMD. For further details on the method and the program see the Supplemental File S11 and www.omicia.com. The AssureRx Health, Inc. annotation and analysis pipeline was used to further annotate variants (see Supplemental File S11 for more detailed methods). We also applied a recently published method, ERDS (Estimation by Read Depth with SNVs) version 1.06.0428, in combination with genotyping array data, to generate a set of CNV calls. ERDS starts from read depth information inferred from BAM files, but also integrates other information including paired end mapping and soft-clip signature, to call CNVs sensitively and accurately. We collected deletions and duplications that were >200 kb in length, with confidence scores of >300. CNVs that were detected by the ERDS method were visually inspected by importing and visualizing the read alignment data in the Golden Helix Genome Browser, version 1.1.1. CNVs were also independently called from Illumina HumanOmni2.5-8v1 genotyping array data. Array intensities were imported and analyzed within the Illumina GenomeStudio software suite, version 1.9.4. LogR values were exported from GenomeStudio and imported into Golden Helix SVS, version 7.7.5. A Copy Number Analysis Method (CNAM) optimal segmentation algorithm was used to generate a list of putative CNVs, which was then restricted to include only CNVs that were >200kb in length with average segment LogR values of > 0.15 and < -0.15 for duplications and deletions, respectively. LogR and covariate values were plotted and visually inspected at all genomic locations where the CNAM method detected a CNV. CNVs that were simultaneously detected by both methods (ERDS and CNAM) were considered to be highly confident CNVs. Highly confident CNVs were, again, visually inspected within Golden Helix Genome Browser to further eliminate any artefactual CNV calls. g : managing sequencing results : There are multiple steps involved in the management of clinical test results, beginning with bar-coded tracking of orders and the return of results to the clinician\u2019s office from the outside CLIA-certified testing facility. The results are transferred to the clinician, who reviews, signs, and interprets the results and incorporates them into the medical health record. The patient is notified, and needed follow-up is arranged. In an ongoing effort to develop ways to incorporate genomic data into clinical EHR, we also collaborated with the Sequence Ontology Group to convert the data into the GVFclin format (see Supplemental File S12). The Genome variant format (GVF), which uses Sequence Ontology to describe genome variation29, has been extended for use in clinical applications. This extended file format, called GVFClin30, adds the 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 PeerJ reviewing PDF | (v2013:06:578:2:0:NEW 14 Sep 2013) R ev ie w in g M an us cr ip t necessary attributes to support Health Level 7 compatible data for clinical variants. The GVF format represents genome annotations for clinical applications using existing EHR standards as defined by the international standards consortium: Health Level 7. Thus, GVFclin can describe the information that defines genetic tests, allowing seamless incorporation of genomic data into pre-existing EHR systems. clia certified wgs results : illumina wgs clinical evaluations : The Illumina WGS clinical evaluation included manual annotation of 344 genes (see Supplemental Fig. S2, Supplemental File S2 and S3), which led to the following conclusion: \u201cNo pathogenic or likely pathogenic variants were found in the 344 genes evaluated that are expected to be clinically significant for the patient. The coverage for these 344 genes is at least 99%. Therefore, significant variants could exist that are not detected with this test.\u201d The clinical evaluation did, however, identify M.A. as a carrier for a variant (c.734G>A ,p.Arg245Gln) in PHYH, which has been associated in the autosomal recessive or compound heterozygote states with Refsum disease, which is an inherited condition that can lead to vision loss, anosmia, and a variety of other signs and symptoms35. In silico prediction programs suggest little impact; however, the variant is rare with a 1000 Genomes frequency of ~0.18%. In this regard, it is worth noting that M.A. has always had poor night vision and enlarged pupils, and, as a result of this genetic finding, we met with M.A.\u2019s treatment team at his Veteran\u2019s Affair\u2019s (V.A.) medical center and learned that he had recently been diagnosed with bilateral cataracts, enlarged pupils, and vision loss. We also learned that M.A.\u2019s mother and maternal grandfather have a history of enlarged pupils with poor vision, and we are currently following up whether this might be related in any way to this particular variant and Refsum disease. disease variant discovery : Further downstream analyses (Fig 2.) identified and prioritized several other potentially clinically relevant variants. Variants that were imported into the Omicia Opal system were filtered to include those that had a high likelihood of being damaging (as defined by an Omicia score > 0.7) and those that have supporting Online Mendelian Inheritance in Man (OMIM; an online database of human genetics and genetic disorders) evidence. We chose to filter based on an Omicia Score of > 0.7 as this value derives a slightly more inclusive list of variants which still cannot be dismissed, but for which we have additional corroborating evidence (i.e., Illumina Genome Network (IGN) validation and annotation). These prioritized variants were further annotated and evaluated by the AssureRx Health, Inc. annotation and analysis pipeline. Prioritized variants are shown in Supplemental File S4 and Supplemental Fig. S3. A 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 PeerJ reviewing PDF | (v2013:06:578:2:0:NEW 14 Sep 2013) R ev ie w in g M an us cr ip t longer list of variants, which were required only to have supporting evidence within the OMIM database, is shown in Supplemental File S5. We highlight here some of the findings: M.A. was found to be a heterozygote for a p.Val66Met change in BDNF, which encodes a protein that is a member of the nerve growth factor (NGF) family. The protein is induced by cortical neurons, and is deemed necessary for the survival of striatal neurons in the brain. In drug na\u00efve patients, BDNF serum levels were found to be significantly decreased in OCD patients when compared to controls (36.90 \u00b1 6.42 ng/ml versus 41.59 \u00b1 7.82 ng/ml; p = 0.043)36, suggesting a link between this protein and OCD. Moreover, a study including 164 proband-parent trios with obsessive-compulsive disorder37 uncovered significant evidence of an association between OCD and all of the BDNF markers that were tested, including the exact variant found here in this person, p.Val66Met. This particular variant has been further studied in a sample of 94 nuclear families38, which included 94 probands with schizophrenia-spectrum disorders and 282 family members. The results of this study suggest that the p.Val66Met polymorphism may play a role in the phenotype of psychosis. Similar anxiety-related behavioral phenotypes have also been observed among mice and humans having the p.Val66Met variant in BDNF39. In humans, the amygdala mediates conditioned fear40, normally inhibited by \u2018executive centers\u2019 in medial prefrontal cortex41. Deep brain stimulation of the pathways between medial prefrontal cortex and the amygdala increased the extinction of conditioned fear in a rat model of OCD42. Studies using functional magnetic resonance imaging (fMRI) demonstrate that humans with the p.Val66Met variant exhibit exaggerated activation of the amygdala in response to an emotional stimulus in comparison to controls lacking the variant43,44. It is thought that this variant may influence anxiety disorders by interfering with the learning of cues that signal safety rather than threat and may also lessen efficacy of treatments that rely on extinction mechanisms, such as exposure therapy39. In this regard, it is interesting to note that this person did indeed obtain very little benefit from exposure therapy prior to surgery. M.A heterozygously carries the p.Glu429Ala allele in MTHFR, encoding a protein that catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate for homocysteine remethylation to methionine, and which has been shown to confer an elevated susceptibility to psychoses. Variants in MTHFR influence susceptibility to occlusive vascular disease, neural tube defects, colon cancer and acute leukemia. Variants in this gene are associated with methylenetetrahydrofolate reductase deficiency. In addition, a meta-analysis comparing 1,211 cases of schizophrenia with 1,729 controls found that the MTHFR p.Glu429Ala allele was associated with susceptibility to schizophrenia45 (odds ratio, 1.19; 95% CI, 1.07- 1.34; p = 0.002). According to the Venice guidelines for the assessment of cumulative evidence in genetic association studies, the MTHFR association exhibited a strong degree of epidemiologic credibility46. Pharmacogenetic studies have found a consistent association between the MTHFR p.Glu429Ala allele and metabolic disorder in adult, adolescent and children taking atypical antipsychotic drugs47,48. M.A. is also heterozygous for the p.Val108Met variant in catechol-O-methyltransferase (COMT), which catalyzes the transfer of a methyl group from S-adenosylmethionine to catecho- lamines, including the neurotransmitters dopamine, epinephrine, and norepinephrine. The minor allele A of this 472G>A variant produces a valine to methionine substitution, resulting in a less thermostable COMT enzyme that exhibits a 3-fold reduction in activity. A substantial body of literature implicates this variant as possibly elevating the risk for various neuropsychiatric disorders in some Caucasian populations but not necessarily in other genetic backgrounds49-55. There is some evidence that MTHFR x COMT genotype interactions might also be occurring in M.A. to influence his neuropsychiatric status56, and the same is true for BDNF x COMT interactions57. pharmacogenetic variants : Pharmacogenetic analyses were performed using the Omicia Opal platform. Pharmacogenetic variants were identified and prioritized by activating the \u201cDrugs and Pharamcology\u201d track within the Opal system and by requiring these variants to have prior evidence within any one of several supporting databases (i.e., OMIM, HGMD, PharmGKB, LSDB and GWAS). Prioritized variants are shown in Supplemental File S6 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 PeerJ reviewing PDF | (v2013:06:578:2:0:NEW 14 Sep 2013) R ev ie w in g M an us cr ip t and Supplemental Fig. S4. A longer, more inclusive list is shown in Supplemental File S7; variants in this file are only required to be detected by the \u201cDrugs and Pharmacology\u201d track in Opal. Variants within Supplemental File S6/S7 were further annotated and analyzed by the AssureRx Health, Inc. pipeline (see Supplemental File S8). Below, we highlight pharmacogenetic variants found to be informative in terms of future medication choices for M.A. M.A. is heterozygous for a c.19G>A p.Asp7Asn allele in ChAT, encoding choline O-acetyltransferase, which synthesizes the neuro-transmitter acetylcholine (Supplemental Fig. S5). This particular variant (rs1880676) is significantly associated with both risk for schizophrenia in Caucasians (P = 0.002), olanzapine response (P = 0.04) and for other psychopathology (P = 0.03)58. Allele A is associated with increased response to olanzapine in people with schizophrenia as compared to allele G. This association was nominally significant (p= 0.04) in the Spanish cohort58. M.A. is homozygous for a p.Ile359Leu change in CYP2C9, and this variant has been linked to a reduction in the enzymatic activity of CYP2C959. CYP2C9 encodes a member of the cytochrome P450 superfamily of enzymes. Cytochrome P450 proteins are mono-oxygenases, which catalyze many reactions associated with drug metabolism as well as reactions associated with the synthesis of cholesterol, steroids and other lipids60. CYP2C9 localizes to the endoplasmic reticulum and its expression is induced by rifampin. CYP2C9 is known to metabolize xenobiotics, including phenytoin, tolbutamide, ibuprofen as well as Swarfarin. Studies identifying individuals who are poor metabolizers of phenytoin and tolbutamide suggest associations between metabolism and polymorphisms found within this gene. CYP2C9 is located within a cluster of cytochrome P450 genes on chromosome 1061. Fluoxetine is commonly used in the treatment of OCD; it has been shown to be as effective as clomipramine and causes less side effects 62,63. CYP2C9 acts to convert fluoxetine to R-norfluoxetine64, and so M.A. may not be able to adequately biotransform fluoxetine65. However, CYP2C9 does not play a rate-limiting role for other SSRIs or clomipramine. In his own treatment experience, M.A. had no response to an 80 mg daily dose of fluoxetine, although he did experience sexual side effects at that dosage. The protein encoded by DPYD is a pyrimidine catabolic enzyme and it acts as the initial and rate-limiting factor in uracil and thymidine catabolism pathways. M.A. was found to be a carrier of two variants in this gene, p.Ile543Val and p.Arg29Cys, for which he is a heterozygote and homozygote, respectively. Variants within DPYD result in dihydropyrimidine dehydrogenase deficiency, an error in pyrimidine metabolism associated with thymine-uraciluria and an increased risk of toxicity in cancer patients receiving 5- fluorouracil chemotherapy. Two transcript variants encoding different isoforms have been described for DPYD66,67. M.A. is heterozygous both for a c.590G>A p.Arg197Gln allele (rs1799930) and a c.803G>A p.Arg268Lys allele (rs1208) in NAT2, encoding an enzyme that functions to both activate and deactivate arylamine and hydrazine drugs and carcinogens 68,69. Genotype AG for rs1799930 is associated with increased risk of toxic liver disease in people with tuberculosis when treated with ethambutol, isoniazid, pyrazinamide and rifampin as compared to genotype GG. Allele G for rs1208 is not associated with risk of hypersensitivity when treated with sulfamethoxazole and trimethoprim in people with infection. copy number variants : ERDS identified 60 putative CNVs, all of which were visually inspected within the Golden Helix Genome Browser. Many of the CNVs detected by the ERDS method were found to be located within chromosomal boundary regions and were determined to be false positives due to highly variable read depth in these regions. The CNAM method detected 35 putative CNVs, which were visually inspected by plotting the LogR and covariate values in Golden Helix SVS. Only six CNVs were simultaneously detected by both the ERDS and CNAM methods, and were visually inspected as further confirmation to be included among the set of highly confident CNVs. High-confidence CNVs are shown in Supplemental File S9. To our knowledge, these CNVs have not been previously associated in any way with M.A.\u2019s disease phenotype, but we are archiving these results for future analysis as knowledge of CNVs and disease associations 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 PeerJ reviewing PDF | (v2013:06:578:2:0:NEW 14 Sep 2013) R ev ie w in g M an us cr ip t expands. return of results : A board-certified genetic counselor was consulted by GJL prior to returning results, and all therapy and counseling was provided by GJL. Although we believe in archiving and managing all genetic results and not just a small subset of genes, we did analyze the 57 genes that are currently recommended for \u201creturn of results\u201d by the American College of Medical Genetics70. These results are shown in Supplemental File S10, and one of us (GJL) met with M.A. to go over the results with him, along with adding some of the findings into his paper-based medical record. Lastly, we did contact the physicians and other officials at the U.S. Veterans Affairs office to offer to incorporate these data into the electronic medical record for M.A. at the VA, but we were informed that the VistA health information system (HIS)71-74 does not currently have the capability to incorporate any genomic variant data. dbs for treatment-refractory ocd : Deep brain stimulation for M.A.\u2019s treatment refractory OCD has provided a quantifiable and significant improvement in the management of his symptoms (Fig. 3). M.A. has regained a quality of life that he had previously not experienced in over 15 years, which is highlighted by him participating in regular exercise, working as a volunteer in his local church, dating, and eventually getting married, all of which act to illustrate a dramatic improvement in his daily functioning since receiving DBS treatment for his OCD. One significant aspect of this study is the rechargeable, and hence depleteable, nature of the Activa RC neurostimulator battery, which serves to illustrate the efficacy of DBS for OCD for this individual. On one such illustrative occasion, M.A. forgot to take the recharging device on a four-day weekend trip. Once his battery was depleted, all of his symptoms gradually returned to their full level over a ~24 hour period, including severe OCD, depression and suicidality. Since that episode, M.A. always takes his recharging device with him on extended trips, but there have been other such instances in which his battery has become depleted for several hours, with the noticeable and intense return of his OCD symptoms and the cessation of his tenesmus. The electrical stimulation is having a demonstrable effect on his OCD, and these data are complementary to other data-sets involving turning DBS devices off for one week at a time19. There are many ethical and regulatory issues relating to deep brain stimulation that have been discussed elsewhere75-81, and we report here our one positive experience, made possible when the US Food and Drug Administration granted a Humanitarian Device Exemption (HDE) to allow clinicians to use this intervention. The rechargeable nature of the new battery has been reassuring to M.A., as he is able to exert self-control over his battery life, whereas he previously had no control with the original \u201csingle-use\u201d battery that must be replaced when the battery depletes (usually at least once annually). We assume that other persons treated with DBS for OCD will likely also start receiving rechargeable batteries. In this regard, it is worth noting that the recent development of an injectable class of cellular-scale optoelectronics paves the way for implanted wireless devices82, and we fully expect that there will be more brain-machine neural interfaces used in humans in the future83-87. returning genetic results : There is considerable controversy in the field of medical genetics concerning the extent of return of genetic results to people, particularly in the context of \u201csecondary\u201d, \u201cunrelated\u201d, \u201cunanticipated\u201d or \u201cincidental\u201d findings stemming from new high-throughput sequencing techniques 96. Some people have concerns regarding the clinical utility of much of the data, and in response have advocated for selectively restricting the returnable medical content. One such set of recommendations has been provided by the American College of Medical Genetics which recently released guidelines in which they recommended the \u201creturn of secondary findings\u201d for 57 genes, without detailed guidance for the rest of the genome 97. These types of recommendations take a more paternalistic approach in returning test results to people, and generally involve a deciding body of people that can range in size from a single medical practitioner to a committee of experts. We believe that anyone should be able to access and manage their own genome data98, just like how anyone should be able to own and manage their medical and radiology test results99, particularly if the testing is performed with suitably appropriate clinical standards in place, i.e. CLIA in America100,101. In this regard, we found by means of WGS that M.A carries a variant in PHYH, this revelation ended up improving his care despite not being related in any known or direct way to his psychiatric disorder, which is the main focus of this study. As stated in our Results section, M.A. has been diagnosed with bilateral cataracts and has been counseled in ways to reduce further damage to and deterioration of his vision. 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 PeerJ reviewing PDF | (v2013:06:578:2:0:NEW 14 Sep 2013) R ev ie w in g M an us cr ip t contributions : GJL conceived of the project, conducted psychiatric evaluations, provided clinical care, analyzed data, supervised other data analyses, and wrote the first draft of the manuscript. JO edited the manuscript extensively and analyzed the data. HF, GH, ESK and MGR analyzed and interpreted the whole genome data. RR leads the Utah Foundation for Biomedical Research and assisted with clinical phenotyping. All authors read, commented on, and approved the final manuscript. conflicts of interest : The corresponding author (GJL) has had informal discussions with representatives from Medtronic, Illumina, and Omicia, Inc., but he has not had any formal consulting role, nor received financial compensation or grants from these or any other for-profit companies performing deep brain stimulation, DNA collection or sequencing. GJL does not hold any patents, and he is unaware of any conflicts of interest on his part. Revenue earned by GJL from providing medical care in Utah is currently donated to the Utah Foundation for Biomedical Research for genetics research. ESK and MGR are co-founders and officers of Omicia, Inc., and GH is an employee of Assure Rx, Inc. All authors read and approved of the content in the manuscript. pathologists; casava, consensus assessment of sequence and variation; erds, : Estimation by Read Depth with SNVs; CNAM, Copy Number Analysis Method; WGS, Whole Genome Sequencing. PeerJ reviewing PDF | (v2013:06:578:2:0:NEW 14 Sep 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:06:578:2:0:NEW 14 Sep 2013) R ev ie w in g M an us cr ip t Figure 3 Yale Brown Obsessive Compulsive Scale (YBOCS) scores were measured for M.A over a three year and seven months period of time. A time series plot (A) shows a steady decline in YBOCS scores over the period of time spanning his DBS surgery (s) and treatment. Incremental adjustments to neurostimulator voltage are plotted over a period of time following DBS surgery. Mean YBOCS scores are plotted for sets of measurements taken before and after Deep Brain Stimulation (DBS) surgery (B). A one-tailed unpaired t test with Welch\u2019s correction results in a p value of 0.0099, demonstrating a significant difference between YBOCS scores measured before and after the time of surgery. PeerJ reviewing PDF | (v2013:06:578:2:0:NEW 14 Sep 2013) R ev ie w in g M an us cr ip t Table 1(on next page) A summary of three clinically relevant alleles found in the sequencing results of M.A. Variations in MTHFR, BDNF, and ChAT were found to be of potential clinical relevance for this person, as they are all implicated in contributing to the susceptibility and development of many neuropsychiatric disorders that resemble those present within M.A. A brief summary of the characteristics of each variation is shown, including the gene name, genomic coordinates, amino acid change, zygosity, variation type, estimated population frequency and putative clinical significance. PeerJ reviewing PDF | (v2013:06:578:2:0:NEW 14 Sep 2013) R ev ie w in g M an us cr ip t Gene name Genomic coordinates Amino acid change Zygosity Variation type Population Frequency Clinical significance MTHFR chr1: 11854476 Glu>Ala heterozygous non-synon T:77% G:23% Susceptibility to psychoses, schizophrenia occlusive vascular disease, neural tube defects, colon cancer, acute leukemia, and methylenetetra-hydrofolate reductase def-iciency BDNF chr11: 27679916 Val>Met heterozygous non-synon C:77% T:23% Susceptibility to OCD, psychosis, and diminished response to exposure therapy CHAT chr10: 50824117 Asp>Asn heterozygous non-synon G:85% A:15% Susceptibility to schizophrenia and other psychopathological disorders. PeerJ reviewing PDF | (v2013:06:578:2:0:NEW 14 Sep 2013) R ev ie w in g M an us cr ip t",
    "v2_text": "results : Pertinent clinical symptoms and treatment A 37-year old man and U.S. veteran (here named with pseudonymous initials M.A.) was evaluated by GJL in 2010 for severe, treatment-refractory obsessive compulsive disorder (OCD), which is an illness that can be quite debilitating63. M.A. had a lifelong history of severe obsessions and compulsions, including contamination fears, scrupulosity, and the fear of harming others, with much milder symptoms in childhood that got much worse in his early 20\u2019s. His Yale-Brown Obsessive Compulsive Scale (YBOCS)64,65 ranged from 32-40, indicating extremely severe OCD. Perhaps the worst period of OCD included a 5-day, near continuous, period of tapping on his computer keyboard as a compulsion to prevent harm from occurring to his family members. M.A. had suffered throughout his life from significant periods of depression with suicidal ideation, and he had attempted suicide at least three times. His prior psychiatric history also includes episodes of paranoia relating to anxieties from his OCD, and he continues to be treated with biweekly injections of risperidone. His treatment history included over 15 years of multiple medication trials, including clomipramine and multiple SSRIs at high doses, including fluoxetine at 80 mg by mouth daily, along with several attempts with outpatient exposure and ritual prevention (ERP) therapy66. M.A. inquired and was evaluated by GJL at the University of Utah and then at two other centers independently offering deep brain stimulation for OCD. One of these centers required (as a condition for eligibility for an ongoing clinical trial) a two-week inpatient hospitalization with intensive ERP, which was documented as improving his YBOCS score to 24 at discharge. He maintains that he actually experienced no improvement during that hospitalization, but rather told the therapists what they wanted to hear, as they were \u201ctrying so hard\u201d. See the Supplemental File S11 for other clinical details. The teams at the University of Utah and two other centers declined to perform surgery due to his prior history of severe depression, suicide attempts and possible psychoses with paranoia. Through substantial persistence of M.A. and his family members, a psychiatrist and neurosurgeon at a fourth center decided that he was an appropriate candidate for surgical implantation of the Medtronic Reclaim\u00ae DBS Therapy device for OCD, approved under a Humanitarian Device Exemption (HDE) for people with chronic, severe, treatment-resistant OCD57, and he was implanted in January of 2011 (Fig. 1). The device targets the nucleus accumbens / anterior limb of the internal capsule (ALIC). A detailed account of the surgical procedure can be found in the Supplemental File 11. 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 PeerJ reviewing PDF | (v2013:06:578:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t acknowledgements : We thank the many doctors and other caregivers who have worked with M.A, including Drs. Paul House, Ziad Nahas and Istvan Takacs. GJL thanks Kenyon Fausett (at Medtronic) and Lauren Schrock (at University of Utah) for helping train him to perform DBS programming. M.A. and his family have been extraordinarily cooperative throughout the course of treatment. We also thank Tina Hambuch, Erica Ramos, Dawn Barry and others at Illumina for helping to develop the TruSight Individual Genome Sequencing (IGS) test, a whole-genome sequencing service using Illumina\u2019s short-read sequencing technology in the CLIA-certified, CAP-accredited Illumina Clinical Services Laboratory. They provided fee-for-service whole genome sequencing in the CLIA lab at Illumina, along with generating the clinical report on 344 genes. Julianne O\u2019Daniel graciously provided advice regarding genetic counseling, along with helping interpret findings in the 57 genes that are currently recommended for \u201creturn of results\u201d by the American College of Medical Genetics. discussion : DBS for treatment-refractory OCDDeep brain stimulation for M.A.\u2019s treatment refractory OCD has provided a quantifiable and significant improvement in the management of his symptoms (Fig. 3). M.A. has regained a quality of life that he had previously not experienced in over a decade, which is highlighted by him participating in regular exercise, working as a volunteer in his local church and becoming engaged to be married, all of which act to illustrate a dramatic improvement in his daily functioning since receiving DBS treatment for his OCD. One significant aspect of this study is the rechargeable, and hence depleteable, nature of the Activa RC neurostimulator battery, which serves to illustrate the efficacy of DBS for OCD for this individual. On one such illustrative occasion, M.A. forgot to take the recharging device on a four-day weekend trip. Once his battery was depleted, all of his symptoms gradually returned to their full level over a ~24 hour period, including severe OCD, depression and suicidality. Since that episode, M.A. always takes his recharging device with him on extended trips, but there have been other such instances in which his battery has become depleted for several hours, with the noticeable and intense return of his OCD symptoms and the cessation of his tenesmus. The electrical stimulation is having a demonstrable effect on his OCD, and these data are complementary to other data-sets involving turning DBS devices off for one week at a time45. There are many ethical and regulatory issues relating to deep brain stimulation that have been discussed elsewhere107-113, and we report here our one positive experience, made possible when the US Food and Drug Administration granted a Humanitarian Device Exemption (HDE) to allow clinicians to use this intervention. The rechargeable nature of the new battery has been reassuring to M.A., as he is able to exert self-control over his battery life, whereas he previously had no control with the original \u201csingle-use\u201d battery that must be replaced when the battery depletes (usually at least once annually). We assume that other persons treated with DBS for OCD will likely also start receiving rechargeable batteries. In this regard, it is worth noting that the recent development of an injectable class of cellular-scale 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 PeerJ reviewing PDF | (v2013:06:578:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t optoelectronics paves the way for implanted wireless devices114, and we fully expect that there will be more brain-machine neural interfaces used in humans in the future115-119. methods : conclusions : One can learn a substantial amount from detailed study of particular individuals (for just a small sampling, see155-162), and we believe that we are entering an era of precision medicine in which we can learn from and collect substantial data on informative individual cases. Incorporating insights from a range of scientific and clinical disciplines into the study and treatment of any one person is therefore beginning to emerge as a tractable, and more holistic, approach, and we document here what we believe to be the first integration of deep brain stimulation and whole genome sequencing for precision medicine in the evaluation, treatment and preventive care for one severely mentally ill individual, M.A. We have shown that DBS has been successful in aiding in the care and beneficial clinical outcome of his treatment refractory OCD, and we have also demonstrated that it is indeed feasible, given current technologies, to incorporate health information from WGS into the clinical care of one person with severe mental illness, including with the return of these health information to him directly. On a comparative level, deep brain stimulation has thus far been a more direct and effective intervention for his mental illness than anything discovered from his whole genome sequencing, although the detection and preventive care for his bilateral cataracts was brought about by the WGS. Of course, the genomic data would have been more helpful if obtained much earlier in his medical course, as it could have provided guidance on which medications to avoid or to provide in increased doses, such as fluoxetine. There are still only sparse data on the effectiveness of DBS for treatment refractory OCD, and current trials and treatment criterion make difficult the implementation and application of this technology for people with severe and treatment refractory forms of OCD, despite clinical promise in this realm (as demonstrated here in our own study). There is currently an intense drive toward individualized data-driven medical care, with the field of cancer medicine being the canonical example, as it is no longer enough to say that a person has cancer, as this distinction is uninformative due to the fact that there are many different well defined molecular etiologies of cancer163. This allows for more precise and targeted therapy, and we fully expect this to occur in the field of psychiatry as hundreds to thousands of psychiatric illnesses become better defined by more precise, molecular, means. This is of particular interest for brain implantable devices that allow for adjustable treatment, such as DBS, as each person could be individually treated (and perhaps even self-tuned) in a precise way to maximize efficacy. We have also found that there are still many challenges in incorporating high-throughput genomics data into the medical health record of any individual, given that many electronic medical record systems are not yet fully compatible with these data. There are also other more fundamental difficulties in the application of genomics guided medicine, as the causal influence of any one, or set, of genetic variant is not at all clear in most cases. Many have proposed using WGS or other genomics data in terms of informing health risk profiles at the individual level164,165, and still others claim that these data lead to a diagnosis in up to 27% of some rare disease cases in which they are used166. We find that health information stemming from WGS cannot currently act as a diagnostic and prognostic panacea, particularly in this case of severe mental illness where the genetic architecture of this class of diseases is unknown. We did find, however, that health information stemming from these data were nevertheless immediately useful in the care of this person, as a variant associated with his ophthalmologic phenotype did indeed inform and enrich his care, and we expect that these data will continue to inform his care as our 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 PeerJ reviewing PDF | (v2013:06:578:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t understandings of human biology and the genetic architecture of disease improves. clinical results for dbs for treatment-refractory ocd : After healing for one month, the implanted device (equipped with the Kinetra Model 7428 Neurostimulator) was activated on February 14, 2011, with extensive programming by an outpatient psychiatrist, with bilateral stimulation of the ALIC. Final settings were case positive, contact 1 negative on the left side at 2.0 V, frequency 130 Hz, and pulse width 210 usec, and case positive, contact 5 negative on the right side with identical settings. Over the next few months, his voltage was increased monthly in increments of 0.2-0.5 V by an outpatient psychiatrist. He returned to one of the author\u2019s (GJL) for psychiatric treatment in July 2011, at which time his voltage was set at 4.5 V bilaterally. His depression had immediately improved after the surgery, along with many of his most irrational obsessions, but his YBOCS score still remained in the 35-38 range. From July 2011-December 2011, his voltage was increased bilaterally on a monthly basis in increments of 0.2 V, with steady improvement with his OCD until his battery started to lose charge by December 2011. This caused him considerable anxiety, prompting him to turn off his battery in order to \u201csave battery life\u201d, which unfortunately led to a complete relapse to his baseline state in a 24 hour period, which was reversed when he turned the battery back on. The battery was surgically replaced with a rechargeable Activa RC neurostimulator battery in January 2012, and the voltage has been increased monthly in 0.1-0.2 V increments until the present time (May 2013). At every visit, M.A. has reported improvements, with reductions of his obsessions and compulsions, marked by a steady decline in his YBOCS score (Fig. 3). M.A. has started to participate in many activities that he had never previously been able to engage in. This includes: exercising (losing 50 pounds in two years) and volunteering at the church and other organizations. In fact, M.A. started dating and recently became engaged to be married, highlighting his improvement in daily functioning. New issues that M.A. reports are consistent tenesmus, occasional diarrhea (which he can now tolerate despite prior contamination obsessions) and improved vision (going from 20/135 to 20/40 vision, as documented by his optometrist), with him no longer needing to wear glasses. It is unknown whether the DBS implant has contributed to any of these issues. Attempts to add fluoxetine at 80 mg by mouth daily for two months to augment any efficacy from the DBS and ERP were unsuccessful, mainly due to no discernible benefit and prominent sexual side effects. M.A. still receives an injection of 37.5 mg risperidone every two weeks for his past history of psychoses; otherwise, he no longer takes any other medications. There has not been any exacerbation of psychoses in this individual during the two years of treatment with DBS. CLIA certified Whole Genome Sequencing results The Illumina WGS clinical evaluation included manual annotation of 344 genes (see Supplemental Fig. S2, Supplemental File S2 and S3), which led to the following conclusion: \u201cNo pathogenic or likely pathogenic variants were found in the 344 genes evaluated that are expected to be clinically significant for the patient. The coverage for these 344 genes is at least 99%. Therefore, significant variants could exist that are not detected with this test.\u201d The clinical evaluation did, however, identify M.A. as a carrier for a variant (c.734G>A ,p.Arg245Gln) in PHYH, which has been associated in the autosomal recessive or compound heterozygote states with Refsum disease, which is an inherited condition that can lead to vision loss, anosmia, and a variety of other signs and symptoms67. In silico prediction programs suggest little impact; however, the variant is rare with a 1000 Genomes frequency of ~0.18%. In this regard, it is worth noting that M.A. has always had poor night vision and enlarged pupils, and, as a result of this genetic finding, we met with M.A.\u2019s treatment team at his Veteran\u2019s Affair\u2019s (V.A.) medical center and learned that he had recently been diagnosed with bilateral cataracts, enlarged pupils, and vision loss. We also learned that M.A.\u2019s mother and maternal grandfather have a history of enlarged pupils with poor vision, and we are currently following up whether this might be related in any way to this particular variant and Refsum disease. This finding is one example of why it is important to archive and re-interpret his genome going forward, as any 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 PeerJ reviewing PDF | (v2013:06:578:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t number of variants could influence his medical care over the course of his life. To achieve this, one of us (GJL) has previously argued in favor of an analytic-interpretive split in the area of clinical genomics, in which WGS is discrete deliverable clinical unit, allowing for multiple downstream interpretive analyses, by any number of people, including the individual and/or his/her health care providers59. We have implemented that model here with M.A. by archiving and offering to him and his health care providers the encrypted hard drive containing his \u201craw\u201d sequencing data, along with analyzing the data with several downstream pipelines (Fig. 2). Further downstream analyses identified and prioritized several other potentially clinically relevant variants. Variants that were imported into the Omicia Opal system were filtered to include those that had a high likelihood of being damaging (as defined by an Omicia score > 0.7) and those that have supporting Online Mendelian Inheritance in Man (OMIM; an online database of human genetics and genetic disorders) evidence. We chose to filter based on an Omicia Score of > 0.7 as this value derives a slightly more inclusive list of variants which still cannot be dismissed, but for which we have additional corroborating evidence (i.e., Illumina Genome Network (IGN) validation and annotation). These prioritized variants were further annotated and evaluated by the AssureRx Health, Inc. annotation and analysis pipeline. Prioritized variants are shown in Supplemental File S4 and Supplemental Fig. S3. A longer list of variants, which were required only to have supporting evidence within the OMIM database, is shown in Supplemental File S5. We highlight here some of the findings: M.A. was found to be a heterozygote for a p.Val66Met change in BDNF, which encodes a protein that is a member of the nerve growth factor (NGF) family. The protein is induced by cortical neurons, and is deemed necessary for the survival of striatal neurons in the brain. In drug na\u00efve patients, BDNF serum levels were found to be significantly decreased in OCD patients when compared to controls (36.90 \u00b1 6.42 ng/ml versus 41.59 \u00b1 7.82 ng/ml; p = 0.043)68, suggesting a link between this protein and OCD. Moreover, a study including 164 proband-parent trios with obsessive-compulsive disorder69 uncovered significant evidence of an association between OCD and all of the BDNF markers that were tested, including the exact variant found here in this person, p.Val66Met. This particular variant has been further studied in a sample of 94 nuclear families70, which included 94 probands with schizophrenia-spectrum disorders and 282 family members. The results of this study suggest that the p.Val66Met polymorphism may play a role in the phenotype of psychosis. Similar anxiety-related behavioral phenotypes have also been observed among mice and humans having the p.Val66Met variant in BDNF71. In humans, the amygdala mediates conditioned fear72, normally inhibited by \u2018executive centers\u2019 in medial prefrontal cortex73. Deep brain stimulation of the pathways between medial prefrontal cortex and the amygdala increased the extinction of conditioned fear in a rat model of OCD74. Studies using functional magnetic resonance imaging (fMRI) demonstrate that humans with the p.Val66Met variant exhibit exaggerated activation of the amygdala in response to an emotional stimulus in comparison to controls lacking the variant75,76. It is thought that this variant may influence anxiety disorders by interfering with the learning of cues that signal safety rather than threat and may also lessen efficacy of treatments that rely on extinction mechanisms, such as exposure therapy71. In this regard, it is interesting to note that this person did indeed obtain very little benefit from exposure therapy prior to surgery. M.A heterozygously carries the p.Glu429Ala allele in MTHFR, encoding a protein that catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate for homocysteine remethylation to methionine, and which has been shown to confer an elevated susceptibility to psychoses. Variants in MTHFR influence susceptibility to occlusive vascular disease, neural tube defects, colon cancer and acute leukemia. Variants in this gene are associated with methylenetetrahydrofolate reductase deficiency. In addition, a meta-analysis comparing 1,211 cases of schizophrenia with 1,729 controls found that the MTHFR p.Glu429Ala allele was associated with susceptibility to schizophrenia77 (odds ratio, 1.19; 95% CI, 1.07- 1.34; p = 0.002). According to the Venice guidelines for the assessment of cumulative evidence in genetic association studies, the MTHFR association exhibited a strong degree of epidemiologic credibility78. Pharmacogenetic studies have found a consistent association between the MTHFR p.Glu429Ala allele and metabolic disorder in adult, adolescent and children taking 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 PeerJ reviewing PDF | (v2013:06:578:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t atypical antipsychotic drugs79,80. M.A. is heterozygous for a c.19G>A p.Asp7Asn allele in ChAT, encoding choline O-acetyltransferase, which synthesizes the neuro-transmitter acetylcholine (Supplemental Fig. S5). This particular variant (rs1880676) is significantly associated with both risk for schizophrenia in Caucasians (P = 0.002), olanzapine response (P = 0.02) and for other psychopathology (P = 0.03)81. M.A. is also heterozygous for the p.Val108Met variant in catechol-O-methyltransferase(COMT), which catalyzes the transfer of a methyl group from S-adenosylmethionine to catecho- lamines, including the neurotransmitters dopamine, epinephrine, and norepinephrine. The minor allele A of this 472G>A variant produces a valine to methionine substitution, resulting in a less thermostable COMT enzyme that exhibits a 3-fold reduction in activity. A substantial body of literature implicates this variant as possibly elevating the risk for various neuropsychiatric disorders in some Caucasian populations but not necessarily in other genetic backgrounds82-88. There is some evidence that MTHFR x COMT genotype interactions might also be occurring in M.A. to influence his neuropsychiatric status89, and the same is true for BDNF x COMT interactions90. Pharmacogenetic analyses were performed using the Omicia Opal platform. Pharmacogenetic variants were identified and prioritized by activating the \u201cDrugs and Pharamcology\u201d track within the Opal system and by requiring these variants to have prior evidence within any one of several supporting databases (i.e., OMIM, HGMD, PharmGKB, LSDB and GWAS). Prioritized variants are shown in Supplemental File S6 and Supplemental Fig. S4. A longer, more inclusive list is shown in Supplemental File S7; variants in this file are only required to be detected by the \u201cDrugs and Pharmacology\u201d track in Opal. Variants within Supplemental File S6/S7 were further annotated and analyzed by the AssureRx Health, Inc. pipeline (see Supplemental File S8). M.A. is homozygous for a p.Ile359Leu change in CYP2C9, and this variant has been linked to a reduction in the enzymatic activity of CYP2C991. CYP2C9 encodes a member of the cytochrome P450 superfamily of enzymes. Cytochrome P450 proteins are mono-oxygenases, which catalyze many reactions associated with drug metabolism as well as reactions associated with the synthesis of cholesterol, steroids and other lipids92. CYP2C9 localizes to the endoplasmic reticulum and its expression is induced by rifampin. CYP2C9 is known to metabolize xenobiotics, including phenytoin, tolbutamide, ibuprofen as well as Swarfarin. Studies identifying individuals who are poor metabolizers of phenytoin and tolbutamide suggest associations between metabolism and polymorphisms found within this gene. CYP2C9 is located within a cluster of cytochrome P450 genes on chromosome 1093. Fluoxetine is commonly used in the treatment of OCD; it has been shown to be as effective as clomipramine and causes less side effects 94,95. CYP2C9 acts to convert fluoxetine to R-norfluoxetine96, and so M.A. may not be able to adequately biotransform fluoxetine97. However, CYP2C9 does not play a rate-limiting role for other SSRIs or clomipramine. In his own treatment experience, M.A. had no response to an 80 mg daily dose of fluoxetine, although he did experience sexual side effects at that dosage. The protein encoded by DPYD is a pyrimidine catabolic enzyme and it acts as the initial and rate-limiting factor in uracil and thymidine catabolism pathways. M.A. was found to be a carrier of two variants in this gene, p.Ile543Val and p.Arg29Cys, for which he is a heterozygote and homozygote, respectively. Variants within DPYD result in dihydropyrimidine dehydrogenase deficiency, an error in pyrimidine metabolism associated with thymine-uraciluria and an increased risk of toxicity in cancer patients receiving 5- fluorouracil chemotherapy. Two transcript variants encoding different isoforms have been described for DPYD98,99. A copy number variant (CNV) analysis was performed using the estimation by read depth with singlenucleotide variants (ERDS) method62 in combination with the Golden Helix Copy Number Analysis Method (CNAM) optimal segmentation algorithm applied to Illumina HumanOmni2.5-8v1 genotyping array data. ERDS identified 60 putative CNVs, all of which were visually inspected within the Golden 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 PeerJ reviewing PDF | (v2013:06:578:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t Helix Genome Browser. Many of the CNVs detected by the ERDS method were found to be located within chromosomal boundary regions and were determined to be false positives due to highly variable read depth in these regions. The CNAM method detected 35 putative CNVs, which were visually inspected by plotting the LogR and covariate values in Golden Helix SVS. Only six CNVs were simultaneously detected by both the ERDS and CNAM methods, and were visually inspected as further confirmation to be included among the set of highly confident CNVs. High-confidence CNVs are shown in Supplemental File S9. To our knowledge, these CNVs have not been previously associated in any way with M.A.\u2019s disease phenotype. Although we believe in archiving and managing all genetic results and not just a small subset of genes, we did analyze the 57 genes that are currently recommended for \u201creturn of results\u201d by the American College of Medical Genetics100. These results are shown in Supplemental File S10. Lastly, in an ongoing effort to develop ways to incorporate genomic data into clinical electronic health records, we collaborated with the team of Karen Eilbeck to convert the data into the GVFclin format (see Supplemental File S12). The Genome variant format (GVF), which uses Sequence Ontology to describe genome variation101, has been extended for use in clinical applications. This extended file format, called GVFClin102, adds the necessary attributes to support Health Level 7 compatible data for clinical variants. The GVF format represents genome annotations for clinical applications using existing EHR standards as defined by the international standards consortium: Health Level 7. Thus, GVFclin can describe the information that defines genetic tests, allowing seamless incorporation of genomic data into pre-existing EHR systems. We did contact the physicians and other officials at the U.S. Veterans Affairs office to offer to incorporate these data into the electronic medical record for M.A., but we were informed that the VistA health information system (HIS)103-106 does not currently have the capability to incorporate any genomic variant data. During our study, we found that M.A. carries at least three alleles that have been associated with neuropsychiatric phenotypes, including variants in BDNF, MTHFR, and ChAT (Table 1). There are, however, still many challenges in showing how any one mutation can contribute toward a clear phenotype, particularly in the context of genetic background and possible environmental influences 120. Bioinformatics confounders, such as poor data quality 121, sequence inaccuracy, and variation introduced by different methodological approaches122 can further complicate biological and genetic inferences. Although the variants discussed in the results section of our study have been previously associated with mental disease, we caution that the data presented are not sufficient to implicate any particular mutation as being necessary or sufficient to lead to the described phenotype, particularly given that mental illness results from a complex interaction of any human with their surrounding environment and social support structures. The genetic architecture of most neuropsychiatric illness is still largely undefined and controversial 123-126, and our data also does not allow us to exclude the possibility of polygenic and epistatic modes of inheritance 127-134. We provide our study as a cautionary one: WGS cannot act as a diagnostic and prognostic panacea, but instead could act to elucidate potential risk factors for some illnesses. There are certainly other variants and/or environmental interactions that have influenced or will influence the clinical course of M.A., and there will likely be many more gene-gene and geneenvironment interactions occurring and impacting various phenotypes developing over the course of his life135-147. In the context of the incomplete, and sometimes proprietary, nature of human gene mutation databases, it is likely that analyses and medical guidance gleaned from these WGS data will differ from institution to institution. It is therefor important that people be given the opportunity, like with many other traditional medical tests, to obtain \u201csecond opinions\u201d. For this to be possible, one must accurately describe the contents of short-read sequencing data in terms of the existing electronic medical health standards, so that these data can be incorporated into an electronic medical health record. Accurately describing the contents of next generation sequencing (NGS) results is particularly critical for clinical analysis of genomic data. However, genomics and medicine use different, often incompatible terminologies and standards to describe sequence variants and their functional effects. In our efforts to treat this one person with severe mental illness, we have implemented the GVFclin format for the variants that were discovered during the sequencing of his whole genome (see Supplemental File S12). We hope to eventually incorporate his genetic data into his electronic health record, if and when the VistA health information system (HIS)103-106 is upgraded to allow entry of such data. We did already counsel M.A. regarding several genetic variants that may be clinically relevant to predisposing him to his psychiatric disorder148. There is, however, considerable controversy in the field of medical genetics concerning the return of genetic results to people, particularly in the context of \u201csecondary\u201d, \u201cunrelated\u201d, \u201cunanticipated\u201d or \u201cincidental\u201d findings stemming from new high-throughput sequencing techniques. Some people worry about returning the results of such tests, due to their concerns regarding clinical utility, and in response have advocated for selectively restricting the returnable medical content. One such set of recommendations has been provided by the American College of Medical Genetics which recently released guidelines in which they recommended the \u201creturn of secondary findings\u201d for 57 genes, without detailed guidance for the rest of the genome 149. These types of recommendations can take a more paternalistic approach in returning test results to people, and generally involve a deciding body of people that can range in size from a single medical practitioner to a committee of experts. We believe that anyone should be able to access and manage their own genome data150, just like how anyone should be able to own and manage their medical and radiology test results151, particularly if the testing is performed with suitably appropriate clinical standards in place, i.e. CLIA in America56,152. 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 PeerJ reviewing PDF | (v2013:06:578:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t We tend to think that whole genome sequencing will eventually become like many other laboratory tests, without the risks inherent to many surgical procedures and other medical interventions. There is currently an ongoing project in America to collect phenotype and genetic data on one million U.S. veterans 153. We have readily demonstrated herein that it is possible to sequence the whole genome of a veteran in a CLIAcertified laboratory, so that these results can be offered to this veteran, and we are working now to determine if we can incorporate any of these results into his electronic medical record at the VA. We also note that there are efforts underway to create \u201ca national resource with linked genealogy and phenotypic data: the Veterans Genealogy Project\u201d, and the authors of that paper note the potential of linking this with the genetic information obtained via the Million Veteran Program154. ethics compliance : Research was carried out in compliance with the Helsinki Declaration. The corresponding author (GJL) conducted all clinical evaluations and he is an adult psychiatry and child/adolescent psychiatry diplomate of the American Board of Psychiatry and Neurology. GJL obtained IRB approval #00038522 at the University of Utah in 2009-2010 to evaluate candidates for surgical implantation of the Medtronic Reclaim\u00ae DBS Therapy for OCD, approved under a Humanitarian Device Exemption (HDE) for people with chronic, severe, treatment-resistant OCD57. The interdisciplinary treatment team consisted of one psychiatrist (GJL), one neurologist and one neurosurgeon. Implantation ultimately occurred on a clinical basis at another site. Written consent was obtained for phenotyping and whole genome sequencing through Protocol #100 at the Utah Foundation for Biomedical Research, approved by the Independent Investigational Review Board, Inc. Informed and written consent was also obtained using the Illumina Clinical Genome Sequencing test consent form, which is a clinical test ordered by the treating physician, G.J.L. 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 PeerJ reviewing PDF | (v2013:06:578:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t evaluation and recruitment for dbs for treatment-refractory ocd : GJL received training regarding DBS for OCD at a meeting hosted by Medtronic in Minneapolis, Minnesota, in September 2009. The same author attended a Tourette Syndrome Association meeting on DBS for Tourette Syndrome, Miami, Florida, in December 2009. Approximately ten candidates were evaluated over a one-year period in 2010. The individual discussed herein received deep brain stimulation surgery at another site, and then returned for follow-up with GJL. Another psychiatrist, author RR, provided ongoing consultation throughout the course of this study. Although other candidates have since returned for follow-up (with GJL), no others have been surgically treated. clia whole genome sequencing and the management of results from : sequencing data Whole genome sequencing was ordered on this individual as part of our ongoing effort to implement precision medicine in the diagnosis, treatment, and preventive care for individuals. His genome was sequenced in the Illumina Clinical Services Laboratory (CLIA-certified, CAP-accredited) as part of the TruSight Individual Genome Sequencing (IGS) test, a whole-genome sequencing service using Illumina\u2019s short- read sequencing technology58. Although clinical genome sequencing was ordered by GJL on a clinical basis (thus not requiring IRB approval), the clinical phenotyping and collection of blood and saliva for other research purposes was approved by the Institutional Review Board (iIRB) (Plantation, Florida) as part of a study protocol at the Utah Foundation for Biomedical Research (UFBR). Consistent with laboratory-developed tests, WGS has not been cleared or approved by the U.S. Food and Drug Administration59. The entire procedure included barcoded sample tracking of the blood collected by GJL from this person, followed by DNA isolation and sequencing in the Illumina CLIA lab. Data statistics are summarized in Supplemental Fig. S1. For the bioinformatics analyses, Illumina utilized the internal assembler and variant caller CASAVA (short for Consensus Assessment of Sequence And VAriation). Reads were mapped to the Genome Reference Consortium assembly GRCh37. Data for sequenced and assembled genomes was provided on one hard drive, formatted with the NTFS file system and encrypted using the open source cross platform TrueCrypt software (www.truecrypt.org) and the Advanced Encryption Standard (AES) algorithm (Federal Information Processing Standards Publication 197). Genotyping array data was generated in parallel of the CLIA whole genome sequencing, using the Illumina HumanOmni2.5-8 bead chip. The encrypted hard drive contains several files, including a \u201cgenotyping folder\u201d within which there is a genotyping report in a text-based tab-delimited format (see Supplemental File S1). See Supplemental File 11 for more details on the genotyping array data. Insertions, deletions and structural alterations are not validated variant types in the Illumina Clinical Services Laboratory. Insertions and deletions provided in the gVCF file are for investigative or research purposes only. A medical report and the raw genomic data were provided back to the ordering physician (GJL) on an encrypted hard drive as part of the Illumina Understand your Genome Symposium, held in October 2012, which included the clinical evaluation of 344 genes (see Supplemental File S2 and S3)60. To perform more comprehensive downstream analyses using a greater portion of the genomic data, all of the variants that were detected by the Illumina CLIA WGS pipeline were imported and analyzed within the Omicia Opal web-based clinical genome interpretation platform (Supplemental Fig. S5), version 1.5.061. The Omicia system annotates variants and allows for the identification and prioritization of potentially deleterious alleles. Omicia Scores, which are computationally derived estimates of deleteriousness, were calculated by using a decision-tree based algorithm, which takes as input the Polyphen, SIFT, MutationTaster and PhyloP score(s), and derives an integrative score between 0 and 1. Receiver operating characteristic (ROC) curves are plotted for that score based on annotations from HGMD. For further details on the method and the program see the Supplemental File S11 and www.omicia.com. The AssureRx Health, Inc. annotation and analysis pipeline was used to further annotate variants (see Supplemental File S11 for more detailed methods). We also applied a recently published method, ERDS (Estimation by Read Depth with SNVs) version 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 PeerJ reviewing PDF | (v2013:06:578:1:0:NEW 13 Aug 2013) R ev ie w in g M an us cr ip t 1.06.0462, in combination with genotyping array data, to generate a set of CNV calls. ERDS starts from read depth information inferred from BAM files, but also integrates other information including paired end mapping and soft-clip signature, to call CNVs sensitively and accurately. We collected deletions and duplications that were >200 kb in length, with confidence scores of >300. CNVs that were detected by the ERDS method were visually inspected by importing and visualizing the read alignment data in the Golden Helix Genome Browser, version 1.1.1. CNVs were also independently called from Illumina HumanOmni2.5-8v1 genotyping array data. Array intensities were imported and analyzed within the Illumina GenomeStudio software suite, version 1.9.4. LogR values were exported from GenomeStudio and imported into Golden Helix SVS, version 7.7.5. A Copy Number Analysis Method (CNAM) optimal segmentation algorithm was used to generate a list of putative CNVs, which was then restricted to include only CNVs that were >200kb in length with average segment LogR values of > 0.15 and < -0.15 for duplications and deletions, respectively. LogR and covariate values were plotted and visually inspected at all genomic locations where the CNAM method detected a CNV. CNVs that were simultaneously detected by both methods (ERDS and CNAM) were considered to be highly confident CNVs. Highly confident CNVs were, again, visually inspected within Golden Helix Genome Browser to further eliminate any artefactual CNV calls. A board-certified genetic counselor was consulted by GJL prior to returning results, and all therapy and counseling was provided by GJL. contributions : GJL conceived of the project, conducted psychiatric evaluations, provided clinical care, analyzed data, supervised other data analyses, and wrote the manuscript. JO edited the manuscript extensively and analyzed the data. HF, GH, ESK and MGR analyzed and interpreted the whole genome data. RR leads the Utah Foundation for Biomedical Research. All authors read, commented on, and approved the final manuscript. conflicts of interest : The corresponding author (GJL) has had informal discussions with representatives from Medtronic, Illumina, and Omicia, Inc., but he has not had any formal consulting role, nor received financial compensation or grants from these or any other for-profit companies performing deep brain stimulation, DNA collection or sequencing. GJL does not hold any patents, and he is unaware of any conflicts of interest on his part. Revenue earned by GJL from providing medical care in Utah is currently donated to the Utah Foundation for Biomedical Research for genetics research. ESK and MGR are co-founders and officers of Omicia, Inc., and GH is an employee of Assure Rx, Inc. All authors read and approved of the content in the manuscript.",
    "v3_text": "methods : results : CLIA certified Whole Genome Sequencing results The Illumina WGS clinical evaluation included manual annotation of 344 genes (see Fig. 1, Supplemental File S2 and S3), which led to the following conclusion: \u201cNo pathogenic or likely pathogenic variants were found in the 344 genes evaluated that are expected to be clinically significant for the patient. The coverage for these 344 genes is at least 99%. Therefore, significant variants could exist that are not detected with this test.\u201d The clinical evaluation did, however, identify M.A. as a carrier for a mutation (c.734G>A ,p.Arg245Gln) in PHYH, which has been associated in the autosomal recessive or compound heterozygote states with Refsum disease, which is an inherited condition that can lead to vision loss, anosmia, and a variety of other signs and symptoms43. In silico prediction programs suggest little impact; however, the variant is rare with a 1000 Genomes frequency of ~0.18%. The frequency with which the variant was seen in cases compared to both controls and the 1000 Genomes population frequency is suggestive that it might contribute to disease development. In this regard, it is worth noting that M.A. has always had poor night vision and enlarged pupils. As a result of this genetic finding, we met with M.A.\u2019s treatment team at his Veteran\u2019s Affair\u2019s (V.A.) medical center, and we learned that he had recently been diagnosed with bilateral cataracts, affecting his vision. We also learned that M.A.\u2019s mother and maternal grandfather have a history of enlarged pupils with poor vision, and we are currently following up whether this might be related in any way to this particular mutation and Refsum disease. Further downstream analyses identified and prioritized a number of potentially clinically relevant variants. Variants that were imported into the Omicia Opal system were filtered to include those that had a high likelihood of being damaging (as defined by an Omicia score > 0.7) and those that have supporting Online Mendelian Inheritance in Man (OMIM; an online database of human genetics and genetic disorders) evidence. We chose to filter based on an Omicia Score of > 0.7 as this value derives a slightly more inclusive list of variants which still cannot be dismissed, but for which we have additional corroborating evidence (i.e., Illumina Genome Network (IGN) validation and annotation). These prioritized variants were further annotated and evaluated by the AssureRx Health, Inc. annotation and analysis pipeline. Prioritized variants are shown in Supplemental File S4 and Supplemental Fig. S2. A longer list of variants, which were required only to have supporting evidence within the OMIM database, is shown in Supplemental File S5. We highlight here some of the findings: M.A. was found to be a heterozygote for a p.Val66Met change in BDNF, which encodes a protein that is a member of the nerve growth factor (NGF) family. The protein is induced by cortical neurons, and is deemed necessary for the survival of striatal neurons in the brain. In drug na\u00efve patients, BDNF serum levels were found to be significantly decreased in OCD patients when compared to controls (36.90 \u00b1 6.42 ng/ml versus 41.59 \u00b1 7.82 ng/ml; p = 0.043)44, suggesting a link between this protein and OCD. Moreover, a study including 164 proband-parent trios with obsessive-compulsive disorder45 uncovered significant evidence of an association between OCD and all of the BDNF markers that were tested, including the exact variant found here in this person, p.Val66Met. This particular variant has been further studied in a sample of 94 nuclear families46, which included 94 probands with schizophrenia-spectrum disorders and 282 family members. The results of this study suggest that the p.Val66Met polymorphism may play a role in the phenotype of psychosis. Similar anxiety-related behavioral phenotypes have also been observed among mice and humans having the p.Val66Met variant in BDNF47. In humans, the amygdala mediates conditioned fear48, normally inhibited by \u2018executive centers\u2019 in medial prefrontal cortex49. Deep brain stimulation of the pathways between medial prefrontal cortex and the amygdala increased the extinction of conditioned fear in a rat model of OCD50. Studies using functional magnetic 5 PeerJ reviewing PDF | (v2013:06:578:0:1:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t Lyon et al. 2013 resonance imaging (fMRI) demonstrate that humans with the p.Val66Met variant exhibit exaggerated activation of the amygdala in response to an emotional stimulus in comparison to controls lacking the variant51, 52. It is thought that this variant may influence anxiety disorders by interfering with the learning of cues that signal safety rather than threat and may also lessen efficacy of treatments that rely on extinction mechanisms, such as exposure therapy47. In this regard, it is interesting to note that this person did indeed obtain very little benefit from exposure therapy prior to surgery. M.A heterozygously carries the p.Glu429Ala allele in MTHFR, encoding a protein that catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate for homocysteine remethylation to methionine, and which has been shown to confer an elevated susceptibility to psychoses. Variants in MTHFR influence susceptibility to occlusive vascular disease, neural tube defects, colon cancer and acute leukemia. Mutations in this gene are associated with methylenetetra-hydrofolate reductase deficiency. In addition, a meta-analysis comparing 1,211 cases of schizophrenia with 1,729 controls found that the MTHFR p.Glu429Ala allele was associated with susceptibility to schizophrenia53 (odds ratio, 1.19; 95% CI, 1.07- 1.34; p = 0.002). According to the Venice guidelines for the assessment of cumulative evidence in genetic association studies, the MTHFR association exhibited a strong degree of epidemiologic credibility54. Pharmacogenetic studies have found a consistent association between the MTHFR p.Glu429Ala allele and metabolic disorder in adult, adolescent and children taking atypical antipsychotic drugs55, 56. M.A. is heterozygous for a c.19G>A p.Asp7Asn allele in ChAT, encoding choline O-acetyltransferase, which synthesizes the neuro-transmitter acetylcholine (see Supplemental Fig. S3). This particular variant (rs1880676) is significantly associated with both risk for schizophrenia in Caucasians (P = 0.002), olanzapine response (P = 0.02) and for other psychopathology (P = 0.03)57. M.A. is also heterozygous for the p.Val108Met variation in catechol-O-methyltransferase(COMT), which catalyzes the transfer of a methyl group from S-adenosylmethionine to catecho- lamines, including the neurotransmitters dopamine, epinephrine, and norepinephrine. The minor allele A of this 472G>A variant produces a valine to methionine substitution, resulting in a less thermostable COMT enzyme that exhibits a 3-fold reduction in activity. A substantial body of literature implicates this variant as possibly elevating the risk for various neuropsychiatric disorders in some Caucasian populations but not necessarily in other genetic backgrounds58-64. There is some evidence that MTHFR x COMT genotype interactions might also be occurring in M.A. to influence his neuropsychiatric status65, and the same is true for BDNF x COMT interactions66. Pharmacogenetic analyses were performed using the Omicia Opal platform. Pharmacogenetic variants were identified and prioritized by activating the \u201cDrugs and Pharamcology\u201d track within the Opal system and by requiring these variants to have prior evidence within a number of supporting databases (i.e., OMIM, HGMD, PharmGKB, LSDB and GWAS). Prioritized variants are shown in Supplemental File S6 and Supplemental Fig. S4. A longer, more inclusive list is shown in Supplemental File S7; variants in this file are only required to be detected by the \u201cDrugs and Pharmacology\u201d track in Opal. Variants within Supplemental File S6/S7 were further annotated and analyzed by the AssureRx Health, Inc. pipeline (see Supplemental File S8). M.A. is homozygous for a p.Ile359Leu change in CYP2C9, and this mutation has been linked to a reduction in the enzymatic activity of CYP2C967. CYP2C9 encodes a member of the cytochrome P450 superfamily of enzymes. Cytochrome P450 proteins are mono-oxygenases, which catalyze many reactions associated with drug metabolism as well as reactions associated with the synthesis of cholesterol, steroids and other lipids68. CYP2C9 localizes to the endoplasmic reticulum and its expression is induced by rifampin. CYP2C9 is known to metabolize xenobiotics, including phenytoin, tolbutamide, ibuprofen as well as S-warfarin. Studies identifying individuals who are poor metabolizers of phenytoin and tolbutamide suggest associations between metabolism and polymorphisms found within this gene. CYP2C9 is located within a cluster of cytochrome P450 genes on chromosome 1069. Fluoxetine is commonly used in the treatment of OCD; it has been shown to be as effective as clomipramine and causes 6 PeerJ reviewing PDF | (v2013:06:578:0:1:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t Lyon et al. 2013 less side effects70, 71. CYP2C9 acts to convert fluoxetine to R-norfluoxetine72, and so M.A. may not be able to adequately biotransform fluoxetine73. However, CYP2C9 does not play a rate-limiting role for other SSRIs or clomipramine. In his own treatment experience, M.A. had no response to an 80 mg daily dose of fluoxetine, although he did experience sexual side effects at that dosage. The protein encoded by DPYD is a pyrimidine catabolic enzyme and it acts as the initial and rate-limiting factor in uracil and thymidine catabolism pathways. M.A. was found to be a carrier of two variations in this gene, p.Ile543Val and p.Arg29Cys, for which he is a heterozygote and homozygote, respectively. Mutations within DPYD result in dihydropyrimidine dehydrogenase deficiency, an error in pyrimidine metabolism associated with thymine-uraciluria and an increased risk of toxicity in cancer patients receiving 5-fluorouracil chemotherapy. Two transcript variants encoding different isoforms have been described for DPYD74, 75. A copy number variant (CNV) analysis was performed using the estimation by read depth with single-nucleotide variants (ERDS) method42 in combination with the Golden Helix Copy Number Analysis Method (CNAM) optimal segmentation algorithm applied to Illumina HumanOmni2.5-8v1 genotyping array data. ERDS identified 60 putative CNVs, all of which were visually inspected within the Golden Helix Genome Browser. Many of the CNVs detected by the ERDS method were found to be located within chromosomal boundary regions and were determined to be false positives due to highly variable read depth in these regions. The CNAM method detected 35 putative CNVs, which were visually inspected by plotting the LogR and covariate values in Golden Helix SVS. Only six CNVs were simultaneously detected by both the ERDS and CNAM methods, and were visually inspected as further confirmation to be included among the set of highly confident CNVs. High-confidence CNVs are shown in Supplemental File S9. To our knowledge, these CNVs have not been previously associated in any way with M.A.\u2019s disease phenotype. Although we believe in archiving and managing all genetic results and not just a small subset of genes, we did analyze the 57 genes that are currently recommended for \u201creturn of results\u201d by the American College of Medical Genetics76. These results are shown in Supplemental File S10. discussion : There is a rich literature of case studies that have been incredibly illustrative over the years (for just a small sampling, see96-103). We anticipate that others will be able to capture and document many more such cases going forward, as we are entering an era of individualized medicine, made possible mainly by the introduction of the internet and the \u201cnetworking of science\u201d revolution that is currently underway27, 32, 104-109. Incorporating insights from a range of scientific and clinical disciplines into the study and treatment of any one person is beginning to emerge as a tractable, and more holistic, approach. We document here what we believe to be the first integration of whole genome sequencing and deep brain stimulation in the evaluation and treatment of one severely mentally ill individual, M.A. We found that M.A. carries at least three alleles that have been associated with neuropsychiatric phenotypes, including variants in BDNF, MTHFR, and ChAT (Table 1). There are certainly other mutations and/or environmental interactions that have influenced or will influence his clinical course, and we subscribe to the model that there will be numerous gene-gene and gene-environment interactions occurring over the lifetime of M.A. impacting various phenotypes 110-122. This includes the mutation in PHYH, which has led to the discovery of bilateral cataracts, enlarged pupils, and vision loss in M.A., which may or may not be associated with Refsum disease. That is why it is so important to archive and re-interpret his genome going forward, as longitudinal phenotyping data from M.A. and other members of his family emerges. We have previously argued in favor of an analytic-interpretive split in the area of clinical genomics, in which WGS can be a discrete deliverable clinical unit, allowing for multiple downstream interpretive analyses, by any number of people, including the individual and/or his/her health care providers39. We 8 PeerJ reviewing PDF | (v2013:06:578:0:1:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t Lyon et al. 2013 have implemented that model here with M.A. by archiving and offering to him the encrypted hard drive containing his \u201craw\u201d sequencing data, along with analyzing the data with several downstream pipelines. Due to the incomplete and sometimes proprietary nature of databases containing information pertaining to human genetic variation, it is likely that analyses and medical guidance gleaned from these data will differ from institution to institution. For this reason, we stress the need to collate and offer these raw data to individuals so that they can, like with many other traditional medical tests, obtain \u201csecond opinions\u201d. We hope to eventually incorporate his genetic data into his electronic health record, which is made easier by the fact that he is a U.S. veteran, meaning that we can attempt to incorporate his WGS data information into the highly successful VistA health information system (HIS)123-126, perhaps using the GVFclin format127. We did already counsel M.A. regarding several genetic variants that may be clinically relevant to predisposing him to his psychiatric disorder128. We believe that anyone should be able to access and manage their own genome data129, just like how anyone should be able to own and manage their medical and radiology test results130, particularly if the testing is performed with suitably appropriate clinical standards in place, i.e. CLIA in America131, 132. Others have described the extraordinary amount of hype and genetic exceptionalism in the field of genomics133, alongside the rise of genetic determinism134. We tend to think that whole genome sequencing will eventually become like many other laboratory tests, and it certainly won\u2019t be as dangerous as the many thousands of surgical procedures and other interventions that occur every day in medicine. There is currently an ongoing project in America to collect phenotype and genetic data on one million U.S. veterans135. We sincerely hope that the Million Veteran Program will reconsider their current position of not returning any genetic results, as they currently claim in their current consent form (accessed April 27, 2013)136. We have readily demonstrated herein that it is possible to sequence the whole genome in a CLIA-certified laboratory, so that these results can be offered to this veteran (and other veterans), and we are working now to determine if we can incorporate any of these results into his electronic medical record at the VA. We also note that there are efforts underway to create \u201ca national resource with linked genealogy and phenotypic data: the Veterans Genealogy Project\u201d, and the authors of that paper note the potential of linking this with the genetic information obtained via the Million Veteran Program137. There has also been a substantial reluctance among most members of the neurologic and neurosurgical community (in America at least) to work with psychiatrists to implement any further neurosurgical procedures in the field of psychiatry. This is primarily the legacy of an era of psychosurgery with the completely indiscriminate and imprecise use of lobotomy in the field of psychiatry20, 21. It is also a byproduct of the artificial separation of the disciplines of psychiatry, neurology and neurosurgery, which has created departmental siloes and turf battles over which diseases \u201cbelong\u201d to which discipline, despite the fact that these diseases all involve mainly one organ: the brain138. There is an ongoing clinical trial testing the \u201cEffectiveness of Deep Brain Stimulation for Treating People With Treatment Resistant Obsessive-Compulsive Disorder\u201d76. However, some of the extensive exclusion criteria in that trial include: \u201ccurrent or past psychotic disorder\u201d, \u201ca clinical history of bipolar mood disorder\u201d, and/or \u201can inability to control suicide attempts, imminent risk of suicide in the investigator's judgment, or a history of serious suicidal behavior, which is defined using the Columbia-Suicide Severity Rating Scale (C-SSRS) as either (1) one or more actual suicide attempts in the 3 years before study entry with the lethality rated at 3 or higher, or (2) one or more interrupted suicide attempts with a potential lethality judged to result in serious injury or death\u201d. It is therefore sad and ironic that some people in America with the most severe cases of OCD are being excluded from the ongoing clinical trial for DBS due to the above exclusion criteria76. We are publishing our initial two-year experience with this one individual, so that others in the world can learn about this result. This is not an instance of selective reporting139, as the clinician and corresponding author on this paper has only been intimately involved with this single case, although he is aware of two other successful unpublished outcomes obtained by others. There are a considerable number of ethical and regulatory issues relating to deep brain stimulation that have been discussed elsewhere140-146, and we simply wish to report our one very positive experience, only made possible when the US Food and Drug Administration granted a Humanitarian Device Exemption (HDE) to allow clinicians to use this 9 PeerJ reviewing PDF | (v2013:06:578:0:1:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t Lyon et al. 2013 intervention on American soil. The rechargeable nature of the new battery has also been very reassuring to M.A., as he is able to exert self-control over his battery life, whereas he previously had no control with the original \u201csingle-use\u201d battery that must be replaced when the battery depletes (usually at least once annually). We assume that other persons treated with DBS for OCD will likely also start receiving rechargeable batteries. Unfortunately, the artificial split between neurology, psychiatry and neurosurgery has made it incredibly difficult to develop teams that can easily push forward the re-emerging field of neuromodulation, mainly due to the fact that most neurosurgeons and neurologists do not obtain sufficient training or expertise with these severe psychiatric phenotypes, and conversely most psychiatrists get very little training or exposure to neurosurgery and even in some cases neurology. It is also worth noting that the recent development of an injectable class of cellular-scale optoelectronics paves the way for implanted wireless devices147, and we fully expect that there will be more brain-machine neural interfaces used in humans in the future148-152. We hope very much that the recently announced BRAIN initiative 148, 149 will incorporate feedback and advice from clinical investigators in the fields of psychiatry, neurology, neurosurgery and neuropathology. We close with an excerpt of a quote from M.A. upon turning on his device, after leaving it off for one day: \u201cI turned back on the device Saturday afternoon after I became suicidal, and my mood immediately improved in a surprising and dramatic fashion. One second the battery was off and I was not just wishing I were dead, but realizing I needed to kill myself to spare myself future misery, and the next second I came to the realization that my battery wasn't working. I turned on the battery and a second later I was no longer wishing I were dead and was smiling because the internal pain was gone.\u201d acknowledgements : We thank the many doctors and other caregivers who have worked with M.A, including Drs. Paul House, Ziad Nahas and Istvan Takacs. GJL thanks Kenyon Fausett (at Medtronic) and Lauren Schrock (at University of Utah) for helping train him to perform DBS programming. M.A. and his family have been extraordinarily cooperative throughout the course of treatment. We also thank Tina Hambuch, Erica Ramos, Dawn Barry and others at Illumina for helping to develop the TruSight Individual Genome Sequencing (IGS) test, a whole-genome sequencing service using Illumina\u2019s next-generation sequencing (NGS) technology in the CLIA-certified, CAP-accredited Illumina Clinical Services Laboratory. They provided fee-for-service whole genome sequencing in the CLIA lab at Illumina, along with generating the 10 PeerJ reviewing PDF | (v2013:06:578:0:1:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t Lyon et al. 2013 clinical report on 344 genes. Julianne O\u2019Daniel graciously provided advice regarding genetic counseling, along with helping interpret findings in the 57 genes that are currently recommended for \u201creturn of results\u201d by the American College of Medical Genetics. Han Fang provided statistical and other advice. ethics compliance : Research was carried out in compliance with the Helsinki Declaration. The corresponding author (GJL) conducted all clinical evaluations and he is an adult psychiatry and child/adolescent psychiatry diplomate of the American Board of Psychiatry and Neurology. GJL obtained IRB approval #00038522 at the University of Utah in 2009-2010 to evaluate candidates for surgical implantation of the Medtronic Reclaim\u00ae DBS Therapy for OCD, approved under a Humanitarian Device Exemption (HDE) for people with chronic, severe, treatment-resistant OCD33. The interdisciplinary treatment team consisted of one psychiatrist (GJL), one neurologist and one neurosurgeon. Implantation ultimately occurred on a clinical basis at another site. Written consent was obtained for phenotyping and whole genome sequencing through Protocol #100 at the Utah Foundation for Biomedical Research, approved by the Independent Investigational Review Board, Inc. Informed and written consent was also obtained using the Illumina Clinical Genome Sequencing test consent form, which is a clinical test ordered by the treating physician, G.J.L. evaluation and recruitment for dbs for treatment-refractory ocd : GJL received training regarding DBS for OCD at a meeting hosted by Medtronic in Minneapolis, Minnesota, in September 2009. The same author attended a Tourette Syndrome Association meeting on DBS for Tourette Syndrome, Miami, Florida, in December 2009. Approximately ten candidates were evaluated over a one-year period in 2010. The individual discussed herein received deep brain stimulation surgery at another site, and then returned for follow-up with GJL. Another psychiatrist, author RR, provided ongoing consultation throughout the course of this study. Although other candidates have since returned for follow-up (with GJL), no others have been surgically treated. A 37-year old man and U.S. veteran (here named with pseudonymous initials M.A.) was evaluated by GJL in 2010 for severe, treatment-refractory obsessive compulsive disorder (OCD), which is an illness 2 PeerJ reviewing PDF | (v2013:06:578:0:1:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t Lyon et al. 2013 that can be quite debilitating34. M.A. had a lifelong history of severe obsessions and compulsions, including contamination fears, scrupulosity, and the fear of harming others, with much milder symptoms in childhood that got much worse in his early 20\u2019s. His Yale-Brown Obsessive Compulsive Scale (YBOCS)35, 36 ranged from 32-40, indicating extremely severe OCD. Perhaps the worst period of OCD included a 5-day, near continuous, period of tapping on his computer keyboard as a compulsion to prevent harm from occurring to his family members. M.A. had suffered throughout his life from significant periods of depression with suicidal ideation, and he had attempted suicide at least three times. His prior psychiatric history also includes episodes of paranoia relating to anxieties from his OCD, and he continues to be treated with biweekly injections of risperidone. His treatment history included over 15 years of multiple medication trials, including clomipramine and multiple SSRIs at high doses, including fluoxetine at 80 mg by mouth daily, along with several attempts with outpatient exposure and ritual prevention (ERP) therapy37. M.A. inquired and was evaluated by GJL at the University of Utah and then at two other centers independently offering deep brain stimulation for OCD. One of these centers required (as a condition for eligibility for an ongoing clinical trial) a two-week inpatient hospitalization with intensive ERP, which was documented as improving his YBOCS score to 24 at discharge. He maintains that he actually experienced no improvement during that hospitalization, but rather told the therapists what they wanted to hear, as they were \u201ctrying so hard\u201d. See the Supplemental File S11 for other clinical details. The teams at the University of Utah and two other centers declined to perform surgery due to his prior history of severe depression, suicide attempts and possible psychoses with paranoia. Through substantial persistence of M.A. and his family members, a psychiatrist and neurosurgeon at a fourth center decided that he was an appropriate candidate for surgical implantation of the Medtronic Reclaim\u00ae DBS Therapy device for OCD, approved under a Humanitarian Device Exemption (HDE) for people with chronic, severe, treatment-resistant OCD33, and he was implanted in January of 2011. The device targets the nucleus accumbens / anterior limb of the internal capsule (ALIC). clia whole genome sequencing and the management of results from : Sequencing data Whole genome sequencing was ordered on this individual in order to glean clinically relevant genetic information that might shed light on the etiology of his psychiatric condition. His genome was sequenced in the Illumina Clinical Services Laboratory (CLIA-certified, CAP-accredited) as part of the TruSight Individual Genome Sequencing (IGS) test, a whole-genome sequencing service using Illumina\u2019s next-generation sequencing (NGS) technology38. Although clinical genome sequencing was ordered by GJL on a clinical basis (thus not requiring IRB approval), the clinical phenotyping and collection of blood and saliva for other research purposes was approved by the Institutional Review Board (iIRB) (Plantation, Florida) as part of a study protocol at the Utah Foundation for Biomedical Research (UFBR). Consistent with laboratory-developed tests, WGS has not been cleared or approved by the U.S. Food and Drug Administration39. The entire procedure included barcoded sample tracking of the blood collected by GJL from this person, followed by DNA isolation and sequencing in the Illumina CLIA lab. Data statistics are summarized in Supplemental Fig. S1. For the bioinformatics analyses, Illumina utilized the internal assembler and variant caller CASAVA (short for Consensus Assessment of Sequence And VAriation). Reads were mapped to the Genome Reference Consortium assembly GRCh37. Data for sequenced and assembled genomes was provided on one hard drive, formatted with the NTFS file system and encrypted using the open source cross platform TrueCrypt software (http://www.truecrypt.org) and the Advanced Encryption Standard (AES) algorithm (Federal Information Processing Standards Publication 197). The encrypted hard drive contains several files, including a \u201cgenotyping folder\u201d within which there is a genotyping report (see Supplemental File S1) in a text-based tab-delimited format and including a header with a minimum of the following columns: 3 PeerJ reviewing PDF | (v2013:06:578:0:1:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t Lyon et al. 2013 SNP Name: The snp identifier. An rsID for dbSNP content, Illumina labels otherwise. GC Score: This score is a quality metric that generally indicates reliability of the genotypes called. The GenCall score is a value with a maximum of 1 assigned to every genotype called. GenCall scores are calculated using information from the clustering of the samples. Each SNP is evaluated based on the angle of the clusters, dispersion of the clusters, overlap between clusters, and intensity. Genotypes with lower GenCall scores are located furthest from the center of a cluster and have a lower reliability. Allele A \u2013 Forward: A allele call relative to the Forward sequence as defined by the SNP source database (dbSNP, 1000Genomes, etc.) Allele B \u2013 Forward: B allele call relative to the Forward sequence as defined by the SNP source database (dbSNP, 1000Genomes, etc.) Allele A \u2013 Design: A allele call relative to the probe used. Allele B \u2013 Design: B allele call relative to the probe used. Insertions, deletions and structural alterations are not validated variant types in the Illumina Clinical Services Laboratory. Insertions and deletions provided in the gVCF file are for investigative or research purposes only. A medical report and the raw genomic data were provided back to the ordering physician (GJL) on an encyrypted hard drive as part of the Illumina Understand your Genome Symposium, held in October 2012, which included the clinical evaluation of 344 genes (see Supplemental File S2 and S3)40. To perform more comprehensive downstream analyses using a greater portion of the genomic data, all of the variants that were detected by the Illumina CLIA WGS pipeline were imported and analyzed within the Omicia Opal web-based clinical genome interpretation platform, version 1.5.041. The Omicia system annotates variants and allows for the identification and prioritization of potentially deleterious alleles. Omicia Scores, which are a computationally derived measure of deleteriousness, were calculated by using a decision-tree based algorithm, which takes as input the Polyphen, SIFT, MutationTaster and PhyloP score(s), and derives an integrative score between 0 and 1. Receiver operating characteristic (ROC) curves are plotted for that score based on annotations from HGMD. For an Omicia Score of 0.85, this analyses results in a 1% false positive rate. For further details on the method and the program see the Supplemental File S11 and www.omicia.com. The AssureRx Health, Inc. annotation and analysis pipeline was used to further annotate variants (see Supplemental File S11 for more detailed methods). We also applied a recently published method, ERDS (Estimation by Read Depth with SNVs) version 1.06.0442, in combination with genotyping array data, to generate a set of CNV calls. ERDS starts from read depth information inferred from BAM files, but also integrates other information including paired end mapping and soft-clip signature, to call CNVs sensitively and accurately. We collected deletions and duplications that were >200 kb in length, with confidence scores of >300. CNVs that were detected by the ERDS method were visually inspected by importing and visualizing the read alignment data in the Golden Helix Genome Browser, version 1.1.1. CNVs were also independently called from Illumina HumanOmni2.5-8v1 genotyping array data. Array intensities were imported and analyzed within the Illumina GenomeStudio software suite, version 1.9.4. LogR values were exported from GenomeStudio and imported into Golden Helix SVS, version 7.7.5. A Copy Number Analysis Method (CNAM) optimal segmentation algorithm was used to generate a list of putative CNVs, which was then restricted to include only CNVs that were >200kb in length with average segment LogR values of > 0.15 and < -0.15 for duplications and deletions, respectively. LogR and covariate values were plotted and visually inspected at all genomic locations where the CNAM method detected a CNV. CNVs that were simultaneously detected by both methods (ERDS and CNAM) were considered to be highly confident CNVs. Highly confident CNVs were, again, visually inspected within Golden Helix Genome Browser to further eliminate any artefactual CNV calls. A board-certified genetic counselor was consulted by GJL prior to returning results, and all therapy and counseling was provided by GJL. 4 PeerJ reviewing PDF | (v2013:06:578:0:1:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t Lyon et al. 2013 clinical results for dbs for treatment-refractory ocd : After healing for one month, the implanted device (equipped with the Kinetra Model 7428 Neurostimulator) was activated on February 14, 2011, with extensive programming by an outpatient psychiatrist, with bilateral stimulation of the ALIC. Final settings were case positive, contact 1 negative on the left side at 2.0 V, frequency 130 Hz, and pulse width 210 usec, and case positive, contact 5 negative on the right side with identical settings. Over the next few months, his voltage was increased monthly in increments of 0.2-0.5 V by an outpatient psychiatrist. He returned to one of the author\u2019s (GJL) for psychiatric treatment in July 2011, at which time his voltage was set at 4.5 V bilaterally. His depression had immediately improved after the surgery, along with many of his most irrational obsessions, but his YBOCS score still remained in the 35-38 range. From July 2011-December 2011, his voltage was increased bilaterally on a monthly basis in increments of 0.2 V, with steady improvement with his OCD until his battery started to lose charge by December 2011. This caused him considerable anxiety, prompting him to turn off his battery in order to \u201csave battery life\u201d, which unfortunately led to a complete relapse to his baseline state in a 24 hour period, which was reversed when he turned the battery back on. The battery was surgically replaced with a rechargeable Activa RC neurostimulator battery in January 2012, and the voltage has been increased monthly in 0.1-0.2 V increments until the present time (May 2013). At every visit, M.A. has reported improvements, with reductions of his obsessions and compulsions, marked by a steady decline in his YBOCS score (Fig. 2). M.A. has started to participate in many activities that he had never previously been able to engage in. This includes: exercising (losing 50 pounds in two years) and volunteering at the church and other organizations. In fact, M.A. started dating and recently became engaged to be married, highlighting his 7 PeerJ reviewing PDF | (v2013:06:578:0:1:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t Lyon et al. 2013 improvement in daily functioning. New issues that M.A. reports are consistent tenesmus, occasional diarrhea (which he can now tolerate despite prior contamination obsessions) and improved vision (going from 20/135 to 20/40 vision, as documented by his optometrist), with him no longer needing to wear glasses. It is unknown whether the DBS implant has contributed to any of these issues. Attempts to add fluoxetine at 80 mg by mouth daily for two months to augment any efficacy from the DBS and ERP were unsuccessful, mainly due to no discernible benefit and prominent sexual side effects. M.A. still receives an injection of 37.5 mg risperidone every two weeks for his past history of psychoses; otherwise, he no longer takes any other medications. The most significant aspect of this presentation is the rechargeable, and hence depletable, nature of the Activa RC neurostimulator battery, which serves to illustrate the efficacy of DBS for OCD for this individual. On one such occasion, M.A. forgot to take the recharging device on a four-day weekend trip. Once his battery was depleted, all of his symptoms gradually returned to their full level over a ~24 hour period, including severe OCD, depression and suicidality. Since that episode, M.A. always takes his recharging device with him on extended trips, but there have been other such instances in which his battery has become depleted for several hours, with the noticeable and intense return of his OCD symptoms and the cessation of his tenesmus. The electrical stimulation is clearly having an effect on his OCD, and these data are complementary to other data-sets involving turning DBS devices off for one week at a time77. The efficacy is also in agreement with a burgeoning literature related to the mechanism and efficacy of deep brain stimulation for OCD in some people77-92. It is worth noting that M.A. was declined for DBS at three centers due to his prior history of suicidality, suicide attempts, and possible psychoses. Although there is a considerable degree of caution and outright skepticism in the medical community regarding the use of DBS for OCD, it is a fact that many people with severe OCD also have severe depression, usually with passive (and sometimes active) suicidal ideation93-95. In addition, the obsessions and compulsions can be quite severe, sometimes to a delusional or psychotic degree, and there can also be co-occurring psychoses in any individual. In this regard, there has not been any exacerbation of psychoses in this individual during the prior two years of treatment, and the benefits of DBS have clearly outweighed the risks related to his prior history of psychoses. contributions : GJL conceived of the project, conducted psychiatric evaluations, provided clinical care, analyzed data, supervised other data analyses, and wrote the manuscript. JO, GH, ESK and MGR analyzed and interpreted the whole genome data. RR leads the Utah Foundation for Biomedical Research. All authors read, commented on, and approved the final manuscript. conflicts of interest : The corresponding author (GJL) has had informal discussions with representatives from Medtronic, Illumina, and Omicia, Inc., but he has not had any formal consulting role, nor received financial compensation or grants from these or any other for-profit companies performing deep brain stimulation, DNA collection or sequencing. GJL does not hold any patents, and he is unaware of any conflicts of interest on his part. Revenue earned by GJL from providing medical care in Utah is currently donated to the Utah Foundation for Biomedical Research for genetics research. ESK and MGR are co-founders and officers of Omicia, Inc., and GH is an employee of Assure Rx, Inc. All authors read and approved of the content in the manuscript.",
    "url": "https://peerj.com/articles/178/reviews/",
    "review_1": "Kevin Black \u00b7 Sep 16, 2013 \u00b7 Academic Editor\nACCEPT\nThank you for addressing the concerns raised by the reviewers. I agree that your changes have improved the quality of the manuscript.\n\nIn the revision you perhaps inadvertently removed from the discussion your previous mention of placebo effects. I believe it would further improve the manuscript to reinstate it, especially since the control condition is a wait list rather than a placebo treatment. I suggest one possible solution here:\n\n\"The most surprising findings were the non-significant interaction effects on IPAQ. [INSERT] As noted above, sample size limited power to detect this interaction. A placebo effect in the active treatment group may also explain this result. Various additional explanations can be entertained. [END] Firstly, a large number ...\"\n\nPlease notify the production staff of your decision on this suggested change.\n\nNevertheless, since this is a small change I chose \"accept\" rather than \"minor revisions\".",
    "review_2": "Kevin Black \u00b7 Jul 31, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nI agree with nearly all the comments of the two reviewers. New data are not required, so the revisions are minor in scope, but revisions to the text are of major importance before the manuscript can be accepted for publication.\n\nIt is OK to keep the statement \u201cPhysical activity as a treatment for depression can be delivered in the form of Internet-based self-help.\u201d Similarly, you can keep \"This study has introduced a new, potentially effective Internet-based treatment for depression based on a physical activity intervention.\" However, all claims that this study shows that physical activity per se is effective at treating major depression must be eliminated. For instance, the claim \"physical activity is effective for depressive symptoms for people with major depression\" is not supported by the data.\n\nAdd a short statement to the Discussion about potential concerns with Internet-delivered therapy (e.g. missing physical signs of depression such as agitation or retardation; difficulty identifying clients adequately for follow-up of increasingly suicidal patients, including involuntary treatment if required).\n\nPlease add the rationale for including anxiety and quality of life as outcomes (don't eliminate them from the report).\n\nPlease check, but the between-group effect size of 0.61 in the abstract appears to be an error. Also, the last two confidence intervals in the 2nd paragraph of Discussion are identical, also probably an error.\n\nPlease change \"amount of participants\" to \"number of participants\". Also, \"free of change\" should read \"free of charge.\"",
    "review_3": "Reviewer 1 \u00b7 Jul 31, 2013\nBasic reporting\nThe study addresses and interesting topic which is of relevance. The manuscript seems to adhere to the all PeerJ policies, is written well, conforms to the PeerJ templates, includes appropriate tables and figures, and is self-contained. I would suggest, however, that the authors could improve their introduction to better demonstrate how their work fits into the broader field of knowledge. Specifically, the introduction could be improved by including a better description of what mechanics or theories of behaviour change they utilized in an effort to change physical activity in their intervention (and why they chose those ones), and how/why it was anticipated that physical activity would change depression.\nExperimental design\nThe manuscript presents an original primary research study within the Journal scope. The research question was clearly defined and the research was conducted in conformity with prevailing ethical standards of the field. Suggestions to improve the research question and the clarity of the methods section follows: (1) The objective listed in the abstract needs to state the objective of the study, rather than summarize the background literature. (2) It was unclear why anxiety and quality of life were included as outcomes. Please either add rationale for these outcomes in the introduction or exclude them for analyses. (3) Please include the information about the study drop-out during treatment (described on page 6) in the Figure 1. (4) It was unclear from this description of the study drop-out how many of the study participants completed some, but not all, of the modules and why they were excluded from the analyses. (5) More information is needed about the procedures applied in delivering the intervention (how did it all work), and more info is needed about intervention content and modules as well. Perhaps the authors could provide examples of some excerpts of the text? At present if would be very hard to replicate this study on the basis of the information provided.\nValidity of the findings\nThe data and analyses seemed reasonably sound and controlled, but suggestions for bettering the conclusions follow: (1) The authors should give a rationale for not included this group in the 6 mo. follow-up analyses. (2) A few times, the authors make conclusions beyond the scope of the results of their data. For example, in the abstract they state, \u201cPhysical activity as a treatment for depression can be delivered in the form of Internet-based self-help.\u201d Be it that physical activity was not found to differ between the control and treatment group, it seems unjust to make such claims. The authors should take consideration to adjust conclusions throughout the manuscript. (3) In the conclusion section, they refer to physical activity as a secondary outcome measure. This seems odd based on the study rationale that physical activity is the mechanism of depression change. (4) More should be included in the conclusions about what specifically about their study may have lead to a lack of sig. Between-group effect of physical activity behaviour change. As is, they list general rationales that may apply to most interventions. (5) The authors suggest that a physical activity ceiling effect may explain the lack of sig. between-group change, but physically active people were screened out prior to data collection. This rationale and a more specific description of the screening process is needed.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"Internet-delivered therapist-guided physical activity for mild to moderate depression: a randomized controlled trial (v0.1)\". PeerJ https://doi.org/10.7287/peerj.178v0.1/reviews/1",
    "review_4": "Reviewer 2 \u00b7 Jul 23, 2013\nBasic reporting\nPlease include the basic inclusion criteria for the study in addition to the exclusion criteria.\nExperimental design\nAs written, the intervention is not described in enough detail to be reproduced. An overview of the specific content covered in each of the modules is warranted as is a description of what the participants were encouraged/expected to do each week. Concrete examples of the \u201cweekly planning of exercise\u201d and \u201chome assignments\u201d and therapist-participant interactions would be helpful.\n\nThe first paragraph of the Discussion states that a correlation was expected between changes in depressive symptoms and changes in physical activity. This hypothesis was not stated earlier in the manuscript, nor was it tested statistically. This statement should either be removed from the Discussion or, preferably, addressed using available data.\n\nRelated to the above point, the provision of a pedometer is mentioned in the Intervention section, but there is no information regarding its purpose or use. If the pedometer isn\u2019t relevant to the study, it should be removed from the report. If it is an important component of the intervention, its purpose should be described in the Methods section. Moreover, if pedometer data are available, it seems like they would be useful in addressing a number of issues (particularly in light of the problems with the IPAQ), such as whether the intervention affected physical activity levels or whether changes in physical activity were associated with changes in depressive symptoms.\n\nPlease report the results of the sample size calculations for the primary outcomes in the Methods section. From the Discussion, it appears that these analyses were conducted for the secondary outcome measures, so I assume they were also done for the primary measures. This information should be reported.\nValidity of the findings\nThis statement in the discussion is untrue and contradictory: \u201cIn summary, the findings in this study indicate that physical activity is effective for depressive symptoms for people with major depression, but there is no evidence of effectiveness in raising levels of physical activity.\u201d The study did NOT indicate anything about the effects of physical activity on depressive symptoms because there were no changes in physical activity and because there was no demonstration of a relationship between physical activity and depressive symptoms. Instead, the study indicated that participating in the internet-based self-help intervention improved depressive symptoms; however, as the authors note, the mechanism for this effect is unknown and appears NOT to be related to physical activity (at least as measured by IPAQ).\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"Internet-delivered therapist-guided physical activity for mild to moderate depression: a randomized controlled trial (v0.1)\". PeerJ https://doi.org/10.7287/peerj.178v0.1/reviews/2",
    "pdf_1": "https://peerj.com/articles/178v0.2/submission",
    "pdf_2": "https://peerj.com/articles/178v0.1/submission",
    "all_reviews": "Review 1: Kevin Black \u00b7 Sep 16, 2013 \u00b7 Academic Editor\nACCEPT\nThank you for addressing the concerns raised by the reviewers. I agree that your changes have improved the quality of the manuscript.\n\nIn the revision you perhaps inadvertently removed from the discussion your previous mention of placebo effects. I believe it would further improve the manuscript to reinstate it, especially since the control condition is a wait list rather than a placebo treatment. I suggest one possible solution here:\n\n\"The most surprising findings were the non-significant interaction effects on IPAQ. [INSERT] As noted above, sample size limited power to detect this interaction. A placebo effect in the active treatment group may also explain this result. Various additional explanations can be entertained. [END] Firstly, a large number ...\"\n\nPlease notify the production staff of your decision on this suggested change.\n\nNevertheless, since this is a small change I chose \"accept\" rather than \"minor revisions\".\nReview 2: Kevin Black \u00b7 Jul 31, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nI agree with nearly all the comments of the two reviewers. New data are not required, so the revisions are minor in scope, but revisions to the text are of major importance before the manuscript can be accepted for publication.\n\nIt is OK to keep the statement \u201cPhysical activity as a treatment for depression can be delivered in the form of Internet-based self-help.\u201d Similarly, you can keep \"This study has introduced a new, potentially effective Internet-based treatment for depression based on a physical activity intervention.\" However, all claims that this study shows that physical activity per se is effective at treating major depression must be eliminated. For instance, the claim \"physical activity is effective for depressive symptoms for people with major depression\" is not supported by the data.\n\nAdd a short statement to the Discussion about potential concerns with Internet-delivered therapy (e.g. missing physical signs of depression such as agitation or retardation; difficulty identifying clients adequately for follow-up of increasingly suicidal patients, including involuntary treatment if required).\n\nPlease add the rationale for including anxiety and quality of life as outcomes (don't eliminate them from the report).\n\nPlease check, but the between-group effect size of 0.61 in the abstract appears to be an error. Also, the last two confidence intervals in the 2nd paragraph of Discussion are identical, also probably an error.\n\nPlease change \"amount of participants\" to \"number of participants\". Also, \"free of change\" should read \"free of charge.\"\nReview 3: Reviewer 1 \u00b7 Jul 31, 2013\nBasic reporting\nThe study addresses and interesting topic which is of relevance. The manuscript seems to adhere to the all PeerJ policies, is written well, conforms to the PeerJ templates, includes appropriate tables and figures, and is self-contained. I would suggest, however, that the authors could improve their introduction to better demonstrate how their work fits into the broader field of knowledge. Specifically, the introduction could be improved by including a better description of what mechanics or theories of behaviour change they utilized in an effort to change physical activity in their intervention (and why they chose those ones), and how/why it was anticipated that physical activity would change depression.\nExperimental design\nThe manuscript presents an original primary research study within the Journal scope. The research question was clearly defined and the research was conducted in conformity with prevailing ethical standards of the field. Suggestions to improve the research question and the clarity of the methods section follows: (1) The objective listed in the abstract needs to state the objective of the study, rather than summarize the background literature. (2) It was unclear why anxiety and quality of life were included as outcomes. Please either add rationale for these outcomes in the introduction or exclude them for analyses. (3) Please include the information about the study drop-out during treatment (described on page 6) in the Figure 1. (4) It was unclear from this description of the study drop-out how many of the study participants completed some, but not all, of the modules and why they were excluded from the analyses. (5) More information is needed about the procedures applied in delivering the intervention (how did it all work), and more info is needed about intervention content and modules as well. Perhaps the authors could provide examples of some excerpts of the text? At present if would be very hard to replicate this study on the basis of the information provided.\nValidity of the findings\nThe data and analyses seemed reasonably sound and controlled, but suggestions for bettering the conclusions follow: (1) The authors should give a rationale for not included this group in the 6 mo. follow-up analyses. (2) A few times, the authors make conclusions beyond the scope of the results of their data. For example, in the abstract they state, \u201cPhysical activity as a treatment for depression can be delivered in the form of Internet-based self-help.\u201d Be it that physical activity was not found to differ between the control and treatment group, it seems unjust to make such claims. The authors should take consideration to adjust conclusions throughout the manuscript. (3) In the conclusion section, they refer to physical activity as a secondary outcome measure. This seems odd based on the study rationale that physical activity is the mechanism of depression change. (4) More should be included in the conclusions about what specifically about their study may have lead to a lack of sig. Between-group effect of physical activity behaviour change. As is, they list general rationales that may apply to most interventions. (5) The authors suggest that a physical activity ceiling effect may explain the lack of sig. between-group change, but physically active people were screened out prior to data collection. This rationale and a more specific description of the screening process is needed.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"Internet-delivered therapist-guided physical activity for mild to moderate depression: a randomized controlled trial (v0.1)\". PeerJ https://doi.org/10.7287/peerj.178v0.1/reviews/1\nReview 4: Reviewer 2 \u00b7 Jul 23, 2013\nBasic reporting\nPlease include the basic inclusion criteria for the study in addition to the exclusion criteria.\nExperimental design\nAs written, the intervention is not described in enough detail to be reproduced. An overview of the specific content covered in each of the modules is warranted as is a description of what the participants were encouraged/expected to do each week. Concrete examples of the \u201cweekly planning of exercise\u201d and \u201chome assignments\u201d and therapist-participant interactions would be helpful.\n\nThe first paragraph of the Discussion states that a correlation was expected between changes in depressive symptoms and changes in physical activity. This hypothesis was not stated earlier in the manuscript, nor was it tested statistically. This statement should either be removed from the Discussion or, preferably, addressed using available data.\n\nRelated to the above point, the provision of a pedometer is mentioned in the Intervention section, but there is no information regarding its purpose or use. If the pedometer isn\u2019t relevant to the study, it should be removed from the report. If it is an important component of the intervention, its purpose should be described in the Methods section. Moreover, if pedometer data are available, it seems like they would be useful in addressing a number of issues (particularly in light of the problems with the IPAQ), such as whether the intervention affected physical activity levels or whether changes in physical activity were associated with changes in depressive symptoms.\n\nPlease report the results of the sample size calculations for the primary outcomes in the Methods section. From the Discussion, it appears that these analyses were conducted for the secondary outcome measures, so I assume they were also done for the primary measures. This information should be reported.\nValidity of the findings\nThis statement in the discussion is untrue and contradictory: \u201cIn summary, the findings in this study indicate that physical activity is effective for depressive symptoms for people with major depression, but there is no evidence of effectiveness in raising levels of physical activity.\u201d The study did NOT indicate anything about the effects of physical activity on depressive symptoms because there were no changes in physical activity and because there was no demonstration of a relationship between physical activity and depressive symptoms. Instead, the study indicated that participating in the internet-based self-help intervention improved depressive symptoms; however, as the authors note, the mechanism for this effect is unknown and appears NOT to be related to physical activity (at least as measured by IPAQ).\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"Internet-delivered therapist-guided physical activity for mild to moderate depression: a randomized controlled trial (v0.1)\". PeerJ https://doi.org/10.7287/peerj.178v0.1/reviews/2\nReview 5: nan\nReview 6: \n",
    "LLM_extracted_review": "[\"Review 1: The manuscript's quality has improved, but the previous mention of placebo effects should be reinstated in the discussion, especially since the control condition is a wait list. The non-significant interaction effects on IPAQ may be explained by a placebo effect in the active treatment group. \\n\\nReview 2: Minor revisions are needed, particularly to eliminate unsupported claims that physical activity is effective for treating major depression. A rationale for including anxiety and quality of life as outcomes should be added, and potential concerns with Internet-delivered therapy should be discussed. There are also errors in the effect size and confidence intervals that need correction.\\n\\nReview 3: The introduction could better demonstrate how the work fits into the broader field of knowledge, particularly regarding behavior change theories. The objective in the abstract should state the study's objective rather than summarize background literature. More information is needed about study drop-out, intervention procedures, and content to allow for replication. Conclusions should be adjusted to avoid overstating the findings, and specific reasons for the lack of significant between-group change should be included.\\n\\nReview 4: Basic inclusion criteria should be added. The intervention description lacks detail for reproducibility, and examples of weekly planning and assignments should be included. The hypothesis regarding the correlation between depressive symptoms and physical activity was not stated earlier and should be addressed. The purpose of the pedometer should be clarified, and sample size calculations for primary outcomes should be reported. The discussion contains contradictory statements regarding the effectiveness of physical activity on depressive symptoms.\"]"
}