Objectives: To assess the uptake of pegylated interferon (PegIFN) plus ribavirin (RBV)-based regimens in patients with hepatitis C virus (HCV) in a large, single-centre, real-world setting over 10 years.Methods: This was a single centre, retrospective analysis of data from patients who attended their first appointment for treatment of HCV genotype 1–3 between 2003 and 2013. Patients were stratified by HCV genotype. The total number of patients who attended their first appointment, incidence of patients who did not proceed to treatment and associated reasons, and incidence of patients treated were analysed. Sustained virological response (SVR) rates were also reported for all patient populations.Results: Overall, 1,132 patients attended their first appointment; 47.8% were included in the genotype 1 group , 7.7% in the genotype 2 group and 44.5% in the genotype 3 group. A greater proportion of patients received treatment versus those who did not receive treatment . Reasons for declining treatment included: patient declined treatment with PegIFN plus RBV: 35.0%, medical contraindications: 20.3% and mental health-related contraindications: 13.6%. An SVR was achieved in 52.6% of patients who attended their first appointment and 62.3% of patients who received treatment.Conclusions: Approximately half of the patients included in this study achieved an SVR. A noteworthy proportion of patients did not receive treatment due to a reluctance to receive PegIFN plus RBV or contraindications to therapy. Results suggest an ongoing need for improvement in the treatment uptake and overall outcomes – particularly for genotype 2 and 3 patients for whom availability of interferon-free regimens is limited. The introduction of more tolerable direct-acting antiviral regimes may help overcome barriers to uptake demonstrated within this cohort. Furthermore, the global burden of diseases, injuries, and risk factors study showed that in 2010 alone, an estimated 499,000 deaths were related to chronic hepatitis C2. The most recent estimates from the UK suggest that 214,000 people are chronically infected with hepatitis C virus (HCV) nationally; approximately 90% are genotype 1 and genotype 3 infections3.Data from the World Health Organization suggest that 130–150 million people are infected with chronic hepatitis C worldwide, a significant proportion of whom will develop liver cirrhosis or cancer4. Treatment of HCV can be complex as the genotypes do not respond in the same way to some therapies. The armamentarium against HCV now comprises antiviral treatments that can cure approximately 90% of HCV infections, thereby reducing the risk of death from liver cancer and cirrhosis; however, global access to diagnosis and treatment remains poor 1.There are seven known genotypes of HCV, although it is possible for patients to be infected with more than one genotype concurrently7. This dual therapy has been associated with frequent and sometimes serious side effects. These side effects, together with treatment durations of up to 1 year and a number of contraindications to treatment, are often seen as barriers to treatment uptake and adherence for some patients9.Until 2011, the only approved treatment option for patients infected with HCV was a pegylated interferon (PegIFN) plus ribavirin (RBV)-based regimen administered for 48 weeks for genotype 1, and 24 weeks for genotypes 2 and 3. Sustained virological response (SVR) rates reported in the registration studies for the dual therapy, PegIFN plus RBV, were 42–52% for genotype 1 and 76–88% for genotypes 2 and 310; however, the tolerability profiles and contraindications for use of the first-generation triple therapies remain an issue, limiting the number of patients considered suitable for treatment11. Further advances were made in the treatment options for genotype 1 patients with the introduction of IFN-free, direct-acting antiviral regimens in 2013 that have significantly improved treatment uptake, SVR rates and tolerability profiles compared with the previously available dual and triple therapies14. However, the availability of these IFN-free regimens is limited for treatment-naïve, genotype 2 or 3, patients in the UK.In 2011, two first-generation protease inhibitors, telaprevir and boceprevir, were licensed for use alongside PegIFN plus RBV for patients with HCV genotype 1. This triple therapy improved SVR rates for genotype 1 patients from 42–52% to 66–75%Although patients with HCV genotype 1 now have alternative treatment options, patients diagnosed with HCV in the real world who do not qualify for treatment with new direct-acting antivirals often decline treatment with a PegIFN plus RBV-based regimen, as they are unwilling or feel unable to endure the associated side effects. Medical and mental health-related contraindications also pose a barrier to the treatment of a proportion of the HCV-infected cohort.This study was designed to assess the uptake of PegIFN plus RBV-based regimens in patients with chronic hepatitis C in a large, single centre, real-world setting over 10 years of treatment. SVR rates for the intention-to-treat (ITT) and treated-patient populations were compared with those achieved in randomised, controlled trials using similar treatment regimens to determine whether our real-world outcomes for patients with HCV were reflective of those achieved in randomised controlled trials.This study was a single centre, retrospective analysis of data from patients who were referred to, and attended their first appointment at the Liver and Antiviral Unit at St Mary’s Hospital, London , for treatment of HCV genotype 1–3 between 2003 and 2013. All treatments and follow-up appointments were also carried out in the Liver and Antiviral Unit at St Mary’s Hospital. Informed patient consent was not required as no patient identifiable information was collected and data collection was retrospective for service evaluation. The work was originally commissioned as a service evaluation by the Chief of Service for Hepatology, Professor Mark Thursz, who granted permission to use and publish the data. As part of a service evaluation, ethical approval was not required. Procedures followed were in accordance with the ethical standards of clinical treatment and within the Helsinki Declaration of 1975, as revised in 2013. It is the belief of the authors that the results of this evaluation is of interest to the wider medical community.16. The study aimed to assess all patients who were referred to the Antiviral Unit specifically for consideration of treatment by a treating hepatologist or specialist practitioner from the outpatient clinic. Therefore, all patients included in the analysis would have been seen in the outpatient setting by a specialist who intended to treat with PegIFN plus RBV. This, therefore, excluded patients who did not attend or comply with outpatient procedures or who had been deemed unsuitable for treatment by the treating physician. Patients who were referred for treatment but did not attend their first appointment at the Antiviral Unit were not included in this analysis. To ensure that the data analysed only related to patients offered an IFN-based treatment regimen (with or without first-generation protease inhibitors), patients referred for treatment after 2013 were not included in these analyses. All patients referred for treatment were screened for medical and mental health-related contraindications. Patients considered suitable for treatment were offered a PegIFN plus RBV treatment regimen over 24–48 weeks, dependent on genotype and predicted response to treatment. In 2011, when first-generation protease inhibitors became available for use in clinical practice, patients with HCV genotype 1 were offered the opportunity to include boceprevir or telaprevir in their treatment regimen.Referred patients ≥18 years old with virologically confirmed chronic hepatitis C genotype 1–3 were eligible for inclusion in the study. Analysis of the data for patients with genotype 4 HCV have been previously published and so were not reported in this studyData were collected on all referrals to the Liver and Antiviral Unit using information from clinical letters and prospectively collated into a computer-based database during the study period. Database and clinical note analyses were performed to establish the total number of patients referred for treatment who attended their first appointment, the incidence of patients who did not proceed to treatment and reasons thereof, and the incidence of patients treated. The incidence of patients who achieved or failed to achieve an SVR were also reported. Analyses were undertaken on the treated patients in the genotype 1 group to establish the proportion of patients whose treatment regimen included a first-generation protease inhibitor and the SVR rates thereof. Patients were considered to have achieved an SVR if they exhibited undetectable HCV RNA 24 weeks after the completion of their antiviral therapy. All analyses were descriptive and calculations were performed using Microsoft Excel 2016 software.The reasons given for the patients who did not receive treatment were also investigated. Patient notes were used to identify medical and mental health-related contraindications; no retrospective assessments of clinical information were carried out. Therefore, contraindications were only included if they were clearly stated in the notes by the treating medical team. When a clear reason for the patient not receiving treatment was not in the notes the reason was categorised as ‘unknown’. The cirrhotic status of the untreated patients was analysed. A patient was considered to have cirrhosis of the liver in cases where the liver biopsy ISHAK score was 5 or 6 out of 6, or the pathologist reported cirrhosis, or where a Fibroscan score was >12.4 KpA.Analyses were undertaken using the ITT population, which included all patients who were referred for treatment and attended their first appointment at the Liver and Antiviral Unit. Analyses were repeated using the treated-patient population, which included patients who were referred for treatment, attended their first appointment and went on to receive treatment.Patients were stratified by HCV genotype; patients with HCV genotype 1a or 1b were included in their respective subgroups (genotype 1a and genotype 1b). All other genotype 1 patients, including mixed genotype and other subgroups, were included in the ‘genotype 1 other’ subgroup. The genotype 1a, genotype 1b and ‘genotype 1 other’ patient populations collectively made up the overall genotype 1 group. Patients with HCV genotype 2 were included in the genotype 2 group and patients with HCV genotype 3 were included in the genotype 3 group.A total of 1,132 patients with HCV genotypes 1–3 were referred to the Liver and Antiviral Unit for treatment between 2003 and 2013. Of these patients, 47.8% were included in the genotype 1 group , 7.7% were included in the genotype 2 group and 44.5% were included in the genotype 3 group . OverallOf the 1,132 patients who were referred for treatment and attended their first appointment, 15.6% did not receive treatment. The most frequently cited reasons were patient declined treatment with PegIFN plus RBV (35.0%), medical contraindication (20.3%) and mental health-related contraindication (13.6%) . These mOf the patients who did not receive treatment, 17.5% had cirrhosis, 42.9% did not have cirrhosis and 39.6% did not have a cirrhotic status indicated in their notes. In the groups stratified by genotype, cirrhosis was indicated in 18.8% of the patients in the genotype 1 group, no patients in the genotype 2 group and 19.7% of the patients in the genotype 3 group. This was compared with 49.5% of patients in the genotype 1 group, 60.0% of patients in the genotype 2 group and 27.9% of patients in the genotype 3 group who did not have cirrhosis. In the genotype 1 group, 31.7% had no cirrhotic status indicated in their notes, compared with 40.0% of patients in the genotype 2 group and 52.4% of patients in the genotype 3 group .In this real-world study, an SVR was achieved in 52.6% of the patients who were referred for treatment and attended their first appointment. The proportion of patients who achieved an SVR was higher in the genotype 2 and genotype 3 groups compared with the genotype 1 group (63.2% and 60.5% vs 43.4%) .Overall, 955 patients in this study received treatment and were included in the treated-patient population. Of these patients who received treatment 62.3% achieved an SVR. The proportion of patients achieving an SVR in the groups stratified by genotype was higher in the genotype 2 and genotype 3 groups compared with the genotype 1 group  .Protease inhibitors were administered to 19.5% (n=86) of the treated patients in the genotype 1 group. Boceprevir was administered to 6.1% of the treated patients in the genotype 1 group and telaprevir was administered to 13.4% of the treated patients in the genotype 1 group. Overall, 72.1% of the patients who received one of these first-generation protease inhibitors achieved an SVR. Similar SVR rates were achieved with the regimens including boceprevir compared with telaprevir . Further results are presented inData for real world intention-to-treat analysis of a decade's experience of treatment of hepatitis C with interferon-based therapiesUnderlying source data are provided. All data were collated in a computer-based database. Analyses were descriptive and calculations were performed using Microsoft Excel 2016 software. Description of each file can be found in the 'data description' file provided.Click here for additional data file.Copyright: © 2016 Selvapatt N and Brown A2016Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).17. This suggests a failure to treat a large proportion of the HCV-infected population in this region when considering total treatment numbers in this centre of 955 genotype 1–3 patients from 2003–2013 and 118 genotype 4 patients from 2002–201416. Whilst this highlights issues regarding screening and patient identification, it could also reflect sub-optimal treatment uptake rates related to tolerability issues surrounding IFN-based therapies.Results of this real-world, single centre, retrospective analysis of data from a 10-year period show that approximately 85% of patients who attended the Liver and Antiviral Unit for treatment of HCV received treatment. Data from Public Health England’s commissioning template for estimating disease prevalence suggest that the catchment area for the study centre  has an estimated 5,035 hepatitis C-infected individuals Although an SVR was achieved in 62% of the treated patients, only approximately half of the patients who were referred for treatment and attended their first appointment achieved an SVR. When stratified by genotype, as expected, the proportion of patients achieving an SVR was higher in the genotype 2 and genotype 3 groups compared with the genotype 1 group.7. It has been suggested previously that outcomes published for treatment of HCV in clinical studies are often not reflected in real-world clinical practice9. However, this centre is a Central London teaching hospital and regional hepatology referral centre with specialist antiviral clinics and dedicated clinical nurse specialists, consultants, pharmacists and a psychiatry liaison. Therefore, it is possible that the screening and support provided at this centre enabled similar outcomes for the treated-patient population to those seen in a clinical trial-based environment. This level of resource might not be available in other centres. In this study, when the patients who attended the centre for treatment of HCV but did not receive treatment were taken into account, the SVR rates were reduced by approximately 10% across all genotypes. Furthermore, the patients who were referred for treatment and did not attend their first appointment and those with HCV who were not referred for treatment at the Liver and Antiviral Unit were not included in these analyses. The inclusion of these patients would have decreased the proportion of patients achieving an SVR further. We therefore conclude that a sub-optimal number of patients diagnosed with HCV in the UK are currently achieving appropriate treatment outcomes. These findings are in line with previously published findings by other UK-based practitioners9.The proportion of the treated-patient population who achieved an SVR in this study was generally in line with previously reported outcomes in clinical trials25. The key paradigm shift, however, relates to the greater tolerability and acceptability of these treatments compared with IFN-based therapies25. In general these drugs have a narrower side effect profile, are not affected by concomitant opiate substitution and street drug use, and have fewer contraindications14. Taking into consideration the improved tolerability profiles and reduced medical and mental health-related contraindications with these new treatment options, we speculate that in the future a higher proportion of patients who attend the centre for treatment of chronic hepatitis C will proceed to treatment. This will, in turn, increase the overall rate of SVR when considering both a per-protocol and an intention-to-treat perspective. In addition, although a proportion of patients who receive treatment with a PegIFN plus RBV-based regimen after a relapse achieve an SVR26, it has been suggested that deferring treatment until new options are available for these patients might be preferential27.More recently the introduction of direct-acting antivirals have revolutionised chronic hepatitis C treatment with superior outcomes in genotypes 1–3 compared with IFN-based therapies3. The proportion of patients who received treatment was higher in the genotype 2 and 3 groups compared with the genotype 1 group. This could be due to known higher SVR rates and shorter treatment durations for patients with HCV genotype 2 and 3 compared with HCV genotype 17. Pre-2011, patients in the genotype 1 group were offered PegIFN plus RBV for 48 weeks, with a lower probability of achieving an SVR than the genotype 2 and genotype 3 groups. Towards the end of the study period, post-2011, patients in the genotype 1 group were also offered a first-generation protease inhibitor, which improved SVR rates to approximately 70%. One-fifth of the treated patients in the genotype 1 group received first-generation protease inhibitors, which raised the mean SVR rate of the patients in the genotype 1 group slightly. Approximately half of the treated patients in the genotype 1 group and three-quarters of the treated patients in the genotype 2 group achieved an SVR in this study, in line with the PegIFN plus RBV registration trials28. In this study, the proportion of patients in the genotype 3 group who achieved an SVR was slightly lower compared with the rates reported in the registration trials , but were in line with other European studies reporting outcomes of patients with HCV treated with a PegIFN plus RBV-based regimen29. This could be reflective of the real-world baseline demographics of this study population compared with Phase 3 clinical trial cohorts, for characteristics such as co-morbidities, age, fibrosis status, metabolic and IL-28B status.In line with national estimates published by Public Health England, over 90% of the patients in this study had HCV genotype 1 or 3 infections30. Results from a survey of treating physicians in 2010 showed that patient-related barriers, including fear of side effects, concerns regarding treatment duration and concerns regarding treatment effectiveness, were considered the most significant barrier to treatment of HCV in Western Europe31. The side effects associated with PegIFN include autoimmune syndromes, neutropenia, flu-like symptoms and neuropsychiatric disorders; while RBV has been found to induce anaemia30. A recent meta-analysis, including results from nine, Phase 3, clinical trials of sofosbuvir-based regimens, found that the removal of PegIFN and RBV from the treatment regimen led to a substantial improvement in patient-reported health-related quality of life during treatment32. This is in contrast with the substantial decrement in health-related quality of life and productivity reported for patients receiving a PegIFN plus RBV-based regimen36. Medical and mental health-related contraindications made up a further third of the reasons cited for patients not receiving treatment in this study. A US study of 45,690 HCV-infected patients reported that bipolar disorder, anaemia, pregnancy and neutropenia were the most frequently cited contraindications to PegIFN plus RBV-based therapy37. A small proportion of patients were lost to follow-up or moved location and the remaining fifth of patients who did not receive treatment had no clear reason recorded in their patient notes. An economic model from the USA analysing work productivity of patients with HCV genotype 1 compared patients treated with an all oral direct-acting antiviral (ledipasvir/sofosbuvir)-based regimen versus no treatment. Patients with untreated HCV were reported to impose a substantial societal burden due to reduced work productivity; the model predicted that the treatment of patients using a (ledipasvir/sofosbuvir)-based regimen would result in significant cost savings from a societal perspective38.The most commonly recorded reason for patients not receiving treatment (for over a third of patients) was a reluctance to receive a PegIFN plus RBV-based treatment regimen. This is in line with previous studies, reporting that the side effects of PegIFN plus RBV-based regimens are commonly cited as a barrier to initiation or adherence to treatment of HCV39. This indicates a significantly reduced risk of death for HCV-infected patients with cirrhosis who achieve an SVR, although it is important to recognise that patients with cirrhosis have been reported to show a reduced response to antiviral therapy compared to those without cirrhosis40. The recent advent of all oral direct-acting antiviral treatments has increased the capability to treat patients with cirrhosis compared to IFN-based regimens, although it is not clear yet how this may alter the natural history of disease42.Approximately a fifth of patients with HCV genotypes 1 or 3 who did not receive treatment had cirrhosis. A recent study of HCV-infected patients showed that patients with cirrhosis who achieved an SVR had a 5-year mortality rate of 5%, rising to over 15% for patients who did not achieve an SVR. After adjustment for potential confounding factors, achieving an SVR was found to be associated with an approximately 74% decreased risk of all-cause mortality in the cirrhotic cohortA number of limitations should be considered for this study including the retrospective nature of the analyses and the lack of comprehensive baseline demographics. The overall treatment population included patients who attended their first appointment at the Liver and Antiviral Unit; therefore, patients who were considered eligible and referred for treatment but did not attend their first appointment, for whatever reason, were not included in these analyses. This study is likely to have significantly under-reported the number of patients overall who were not included in the intention-to-treat analysis. Given that the analyses included only those patients who were referred directly for treatment, it is likely to exclude a multitude of patients who were either not referred or self-elected not to embark on antiviral therapies before an opportunity to be referred for treatment was offered. The concern is that this group of patients may have since developed complications of the virus or remain unlinked to care and have ongoing potential to develop HCV-related complications. More tolerable and acceptable treatments, initiated at an earlier stage with less need for specialist involvement, would conceivably increase treatment uptake rates and as a result, reduce long-term disease burden. The treatment options available for patients with HCV genotype 1 changed during the study period, with the introduction of first-generation protease inhibitors in 2011; the genotype 1 data should be interpreted with this in mind.Results from this 10-year retrospective analysis of real-world data suggest that half of the patient population who attended the Liver and Antiviral Unit for IFN-based treatment of HCV went on to achieve an SVR. Furthermore, over half of the patients with HCV genotype 1 and one-third of the patients with HCV genotypes 2 and 3 failed to achieve an SVR. A noteworthy proportion of patients did not receive treatment due to a reluctance to receive a PegIFN plus RBV-based regimen or contraindications to therapy which may not be relevant to current direct-acting antiviral treatments. Whilst interferon therapies offer reasonable treatment outcomes for carefully selected patients at a population level, issues pertaining to patient perceptions and contraindications are a barrier for upscaling of treatments. Despite these major advances in the therapeutic options available for treatment of HCV, there remains an ongoing need for improvement in the treatment uptake and overall outcomes for the HCV-infected genotype 2 and 3 patients in our UK-based centre.The data referenced by this article are under copyright with the following copyright statement: Copyright: © 2016 Selvapatt N and Brown AData associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).10.5256/f1000research.9114.d13355943F1000Research: Dataset 1. Data for real world intention-to-treat analysis of a decade's experience of treatment of hepatitis C with interferon-based therapies, Many thanks for asking me to review this article. Though no longer topical (as the treatments have progressed) this paper remains of interest as it provides some real-world insights, which are unlikely to be repeated, from an era of HCV care. I believe it is suitable for indexing, but have some minor recommendations. I wonder if it is overly long in its present form, with some minor repetition in introduction & discussion, and with topics discussed that are not directly relevant to the questions posed. However this will ultimately depend on the editorial flexibility and approach of the journal.a greater proportion of patients received treatment compared with those who did not receive treatment ’ do not provide any useful information . After reading the abstract I was especially interested in the data on those that did not receive treatment (which is largely missing from the literature), but on reading the article itself it is apparent that this data is not of such utility unfortunately. This is because there are confounders in that other specialist clinicians have already effectively excluded many individuals by not referring on for treatment. There is also the issue of not considering those that did not attend. Though unavoidable issues with the design of this research, it does relate to the issue of using ITT analyses (as the denominators used are not translatable to any other settings). It also means that comments such as ‘In line with national estimates published by Public Health England, over 90% of the patients in this study had HCV genotype 1 or 3 infections’ should be avoided as you are not comparing like with like. The study examined only G1-3 and excluded G4-6. It is not clear as to why this was the case and also it means that statements such as ‘ The authors comment that their SVR rates for G1 and G2 are comparable to the trial data, potentially due to the clinical and patient support infrastructure they have in place – but that infrastructure was presumably also there for the G3 patients (who had a lower than expected SVR rate) and therefore I think such conclusions may be too speculative. Other comments:It is more generally accepted that there are 6 distinct genotypes of HCV – not 7 as stated in paragraph 2 of the introduction.The graphing in figures 4 and 5 does not need both columns as the outcome for each is dichotomous (either SVR or failure) – therefore just the SVR column would be better for clarity.I could not link on my computer to the datasets and so could not assess them.  I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The article stands as a final word on a closed era of HCV treatment. I wonder if in the rapidly changing world of HCV treatments the introduction should be updated a little, especially the second to last paragraph that implies that in the real world some patients with G1 might still be treated with PEG RIBA. I also question the relavence of the presenting of the data separating referrals to the anti viral unit  and those actually treated . This is a little artificial as we don't know how many people were seen in other hepatology clinics and never referred because they were either deemed unsuitable or uninterested. Other than these few tweaks I approve.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


In this mini-review, we summarize the main genetic findings discovered in Hispanics and discuss the limitations and challenges of performing genetic studies in these populations. Finally, we present possible next steps to make studies in Latino populations more valuable in providing a deeper understanding of T2D and anticipate their future application to the development of predictive and preventive medicine and personalized therapies.Type 2 diabetes (T2D) affects 415 million people worldwide, and has a much higher prevalence in Hispanics (16.9%), compared to non-Hispanic whites (10.2%). Genome-wide association studies and whole-genome and whole-exome sequencing studies have discovered more than 100 genetic regions associated with modified risk for T2D. However, the identified genetic factors explain a very small fraction of the estimated heritability. Until recently, little attention has been put in studying other non European populations that suffer from a higher burden of T2D, such as Hispanics/Latinos. In the past few years, genetic studies in Hispanic populations have started to provide new insights into the genetic architecture of T2D in this ancestry group. Of note, several genetic variants that are absent or very rare in non-Hispanic populations but more common in Hispanics have shown from moderate to strong association with T2D and have provided new insights into the biology of T2D, which may be ultimately useful for developing novel therapeutic strategies applicable to all populations. Studying diverse populations can also improve the ability to find the causal variants in known T2D  Type 2 diabetes (T2D) affects more than 415 million people worldwide and is predicted to be the 7th leading cause of death in 2030 (Genome-wide association studies (GWAS) have been able to identify more than 100 loci associated with T2D. However, until very recently, most GWAS have been performed in populations of European ancestry . Even inGenetic studies in diverse populations are essential for several reasons. First, finding a population-specific variant associated with T2D can help identify subjects at high risk for T2D in that particular population, who could be selected for lifestyle or therapeutic preventive intervention. Second, the discovery of causal genes in these populations can expand our understanding of T2D or lead to a potential therapeutic target that could be valuable even in populations where the genetic variant that prompted the discovery is not present.During the past few years, several studies conducted in Latino populations have revealed novel associations that have improved our knowledge of the biology of T2D, and also proposed novel therapeutic targets or personalized strategies. In this review, we will describe such studies and illustrate how they might lead to potential therapeutic targets for T2D. Finally, we will suggest future research to improve the performance and interpretation of GWAS in non-European populations.loci were identified at genome-wide statistical significance , the authors replicated several loci previously found in European populations, indicating that the majority of common genetic risk factors are transferrable to Latin American populations.The first GWAS for T2D in Hispanic populations was performed in the Mexican American population of Starr County in 2011 , 9. AlthAs part of the SIGMA, the SIGMA T2D Consortium has shed new light on the genetic architecture of T2D in Mexicans, and resulted in several discoveries that may result in future therapeutic strategies  and solute carrier family 16 member 11 (SLC16A13) genes and represented one of the strongest signals identified to date , in a haplotype present at ~30% frequency in Mexico, ~10% in East Asian, and rare in Europeans and African Americans. Simultaneously, a genome-wide significant signal was identified in East Asians near SLC16A13, in a variant which is in tight linkage disequilibrium with the top variant identified in Mexicans (r2 = 0.97), and, therefore, likely representing and replicating the same signal  that was strongly associated with T2D , conferring one of the highest effect sizes identified at that time . Functional exploration of transcriptional activity suggests that this variant confers an intermediate molecular phenotype between MODY and common T2D, although carriers of this variant are clinically indistinguishable from non-carriers with T2D, highlighting the need to use genetic methods to identify them. Patients with MODY who carry complete loss-of-function mutations in HNF1A have improved sensitivity to sulfonylureas, compared to insulin and metformin treatment  homeobox A gene  (IGF2), isoform 2. We demonstrated that this variant prevents the expression of this isoform in vitro, and that carriers of this variant show reduced expression of isoform 2 IGF2 in liver and adipose tissue ex vivo. Interestingly, higher expression of isoform 2 of IGF2 in adipose tissue was associated with T2D and with increased plasma glycated hemoglobin (HbA1c) in individuals without diabetes in vivo. These results suggest that reducing the levels of this isoform could be a therapeutic strategy for preventing or treating T2D. The identification of loss-of-function variants associated with reduced risk of disease is of particular interest, as their protective genetic effect can be potentially recapitulated by pharmacological inhibition . However, to assess the viability of such a strategy, it is crucial to exclude the possibility that carriers of these T2D-protective variants might be at increased risk for other conditions, so as to reduce the chance of potential adverse effects when instituting a therapy that targets this mechanism  . This vaechanism . To mitiContemporary Hispanic/Latino individuals may descend from diverse ancestries, each of which may be associated differently with T2D . The genEach of these differences in ancestries, as well as the degree of admixture, needs to be carefully taken into account to avoid artifactual associations driven by confounders (population stratification). Several methods that address the particularities of Hispanic and Latino populations have been developed and shown to overcome this type of inflation .KCNQ1, which was further replicated in the Meta-analysis of type 2 DIabetes in African Americans (MEDIA) consortium  . The uninsortium . The autMTNR1B (rs10830963) with acute insulin response in a locus that was previously reported to be associated with fasting glucose levels in Europeans  could be properly imputed if it was present in Europeans, this variant cannot currently be imputed in Latinos due to the lack of sufficiently large Latino-specific samples in current reference panels. To address this block, the ongoing NHLBI-funded initiative, the Trans-Omics for Precision Medicine , will release high coverage whole-genome sequencing data of 62,000 participants, of whom 10% are of Hispanic ancestry. This sequence data will eventually be very valuable as a highly precise reference panel for genotype imputation in Latin Americans.One major limitation is the reduced resolution of GWAS analyses compared to which can be achieved in European populations. A key step in modern GWAS is the imputation of genotypes, for which a population-specific reference panel is required . The abiThe majority of genetic associations with complex diseases, including those in T2D, fall within non-coding regions: only in a few cases can a coding variant that drives the association signal for a non-coding proxy be identified and presumed to be causal to enable the design of functional experiments. Non-coding variation is less well characterized for functional impact, which makes a mechanistic interpretation challenging. Currently, the interrogation of expression quantitative trait loci (eQTLs) is a powerful tool to assess if a non-coding variant has a regulatory effect, and additionally identify the effector genes and tissue of action for a given association. The GTEx Consortium represents one of the largest multi-tissue eQTL dataset, as it consists of genotype and gene expression data for 544 individuals and 53 tissues . HoweverTherefore, significant investment is needed to assemble the first eQTL datasets including Latin American samples, perhaps starting with relevant tissues for T2D such as pancreatic islets, in order to improve the interpretation of the GWAS results in these populations.Once a genetic association with a disease such as T2D is discovered, it can be very useful to analyze whether this association is specific for T2D or it is also associated with other traits or diseases. This can be specifically relevant as follow-up of loss-of-function protective variants for T2D. Before considering an association as a potential therapeutic target, phenome-wide association analysis can allow discarding the possibility that the protective variant might be associated with increased risk of other diseases or impair fertility. Large-scale biobanks with genetic data, such as the UK Biobank , 36, theThe study of genetics of T2D in Latin Americans is still in its infancy, but has already provided exciting and promising results. The sample size of the largest studies  currently analyzed in Latin American populations is comparable to those published in 2008 for Europeans, but has already provided several novel findings of potential therapeutic relevance that would also be applicable beyond the population where they were discovered. This should serve as a motivation to pursue larger-scale association analyses to find additional relevant therapeutic targets and to better understand the pathophysiology of T2D. While increasing the sample size or extending the allele frequency spectrum in European populations can provide additional novel associations for T2D, the expected population impact of the novel associations will be smaller, as the associations would have been identified earlier otherwise. However, studying different populations may provide novel associations with larger effect sizes that might be more amenable to functional studies.In parallel to focusing on increasing the sample sizes of GWAS in Latin American populations, genomic resources that enable the functional interpretation of genetic results and building infrastructures to establish large-scale biobanks with access to genetic information will enhance the success of the discovery of potential therapeutic targets in these populations.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chronic social isolation renders 5-HT neurons insensitive to SK2 blockade, however inhibition of the upregulated SK3 channels restores normal excitability. In vivo, we demonstrate that inhibiting SK channels normalizes chronic social isolation-induced anxiety/depressive-like behaviors. Our experiments reveal a causal link for the first time between SK channel dysregulation and 5-HT neuron activity in a lifelong stress paradigm, suggesting these channels as targets for the development of novel therapies for mood disorders.The activity of serotonin (5-HT) neurons is critical for mood regulation. In a mouse model of chronic social isolation, a known risk factor for depressive illness, we show that 5-HT neurons in the dorsal raphe nucleus are less responsive to stimulation. Probing the responsible cellular mechanisms pinpoints a disturbance in the expression and function of small-conductance CaDOI:http://dx.doi.org/10.7554/eLife.21416.001 Major depressive disorder is a common and debilitating disease that interferes with the afflicted person’s everyday life. While some patients do benefit from antidepressant treatments, these medications need to be taken for several weeks before they become effective. Still, a large proportion of patients do not recover fully and some do not respond at all to the existing treatments. As a result, there is a need to find new and more effective treatments for depression.The most widely used antidepressant drugs target the chemical messenger or neurotransmitter called serotonin. The majority of nerve cells that produce serotonin are located in a region deep in the brain known as the dorsal raphe nucleus. When active, these nerve cells release serotonin; this in turn controls the cells’ own activity as well as the activity of a large number of connected nerve cells located throughout the brain. Any disruption in this system will have a widespread impact and can potentially increase the risk of disturbed moods. However, it was not exactly clear what alters the activity of serotonin-producing nerve cells in depression.Now, Sargin et al. have identified a previously unknown mechanism that underlies changes to the activity of serotonin-producing nerve cells. Keeping mice isolated for a prolonged period elicits the symptoms of depression. Sargin et al. found that serotonin-producing nerve cells were dramatically less active in isolated mice and that a specific type of ion channel protein (the SK3 channel) was more abundant in these nerve cells. A higher amount of this channel inhibits the activity of nerve cells. Blocking these inhibitory SK3 channels (using a drug that can be obtained from bee venom) restored normal activity in the serotonin-producing cells. Moreover, this treatment alleviated the depressive symptoms of the isolated mice.The findings of Sargin et al. suggest a new way to treat the symptoms of depression. Yet to translate them into an accessible treatment for patients, future work will be required to develop drugs that can specifically and potently target the affected channel.DOI:http://dx.doi.org/10.7554/eLife.21416.002 Major depression is a prevalent and debilitating disease for which standard treatments remain ineffective. Social isolation has long been implicated as a risk factor for depression in humans  channels. Furthermore, inhibition of SK channels normalizes the activity of 5-HT neurons and restores the behavioral deficits observed after chronic social isolation.Here, we show that chronic social isolation leads to a reduction in the excitability of DRN 5-HT neurons. Their firing activity to optical, electrophysiological, and neuromodulatory stimulation are all reduced after social isolation. Specifically, we have identified that the reduction in the firing activity of 5-HT neurons results from alterations in the function and expression of small-conductance CaTph2 promoter . Firing frequency of 5-HT neurons from single-housed mice in response to light was significantly reduced compared to those from group-housed littermates . In contDRN 5-HT neurons receive numerous inputs  that relBrown et al., 2001A key characteristic of 5-HT neurons is their long-duration action potential followed by a medium duration AHP . Since A2+) (2+ channels (VGCC) would modify the firing activity of 5-HT neurons. Using the VGCC blocker CdCl2, the difference in firing frequency of 5-HT neurons from group- and single-housed mice was abolished (2+ levels relative to baseline (dF/F) in response to a set number of action potentials were similar between 5-HT neurons of group- and single-housed mice  , we exambolished . 5-HT nebolished , we nextbolished , the firbolished . The intsed mice , indicatCa2.2) and SK3 (KCa2.3), exist in the rodent DRN  in a group of single-housed mice 30 min before each test.Single-housed mice showed significantly longer latencies to start feeding in the novelty suppressed feeding test compared to the group-housed mice , indicatIn the tail suspension test, chronic social isolation resulted in an increase in depressive-like behavior. Single-housed mice showed significantly increased cumulative immobility . This imIn addition to exploring the behavioral data for each individual test we also combined the z-scores across all behavioral tests to produce an integrated emotionality score for each mouse . IntegraOur results demonstrate that chronic social isolation results in a reduction in the excitability of 5-HT neurons and renders them less responsive to stimulation. We demonstrate that inhibiting SK channels, critical regulators of AHP in these cells, can reverse the reduced excitability. This work provides the first direct link between a chronic social isolation paradigm and functional alterations in 5-HT neurons themselves. We also reveal that SK2 contributes to normal regulation of 5-HT neuronal excitability but ceases to modulate these neurons significantly after chronic stress. Behavioral analysis showed that chronic social isolation increases anxiety/depression-like behaviors, which can be normalized upon inhibition of SK channels by acute systemic apamin.In humans, polymorphisms on the gene encoding the SK3 channel have been associated with neuropsychiatric disorders characterized by emotional dysregulation including schizophrenia, bipolar disorder, and anorexia nervosa . AdminisIMSR_JAX:014555) that express channelrhodopsin EYFP fusion protein driven by the Tph2 promoter (ad libitum access to both food and water.All experiments were performed in Tg(Tph2-COP4*H134R/EYFP)5Gfng mice  was included during the recovery period to maintain 5-HT synthesis (2/5% CO2 at 31–33ºC flowing at a rate of 3–4 ml/min. Patch pipettes (2–4 MΩ) contained the following composition: 120 mM potassium gluconate, 5 mM KCl, 2 mM MgCl2, 4 mM K2-ATP, 0.4 mM Na2-GTP, 10 mM Na2-phosphocreatine, and 10 mM HEPES buffer (adjusted to pH 7.3 with KOH). Neurons in DRN were visualized with a fixed-staged microscope (Olympus BX50WI) and 5-HT neurons were targeted based on the expression of EYFP. Recordings were focused on the dorsomedial subregion of dorsal raphe, where most of the EYFP positive neurons were visible. Whole-cell recordings were made in voltage-clamp or current-clamp mode with a Multiclamp 700B amplifier (Molecular Devices). Voltage-clamp recordings were performed at −75 mV. Optogenetic stimulation  was performed using LED light . For electrophysiological stimulation, action potentials were elicited by applying brief depolarizing current pulses  at 20 Hz from a membrane potential of −70 mV. All data were acquired at 20 kHz and low-pass filtered at 3 kHz using pClamp10.2 and Digidata1440 software.Coronal brainstem slices (400 µm) comprising the dorsal raphe nucleus were obtained using a Dosaka Pro-7 Linear Slicer (SciMedia) in ice-cold oxygenated sucrose-substituted artificial cerebrospinal fluid (ACSF). The slices were then recovered for a minimum of 2 hr in ACSF solution containing: 128 mM NaCl, 10 mM D-glucose, 26 mM NaHCOynthesis . Recordi1A receptor currents. WAY100635  was applied in ACSF to block 5-HT1A receptor responses. OrexinB  and R-(-) Phenylephrine hydrochloride  were applied in bath to measure responses of 5-HT neurons to excitatory neuromodulation. Picrotoxin  and CGP52432  were included together in bath solution to block GABAA and GABAB receptor responses, respectively. Cadmium chloride  was used to block voltage gated calcium channels. Lei-Dab7  was included in ACSF as a blocker of SK2 channels. Apamin  was used as a blocker of SK2 and SK3 channels.5-CT  was bath applied to probe 5-HTThe calcium dye Fluo5F  was included in the pipette, along with Alexa Fluor-594 hydrazide (20 μM), for visualization of the neuron. Multiphoton imaging was performed using a titanium:sapphire laser (Newport) tuned to wavelength 800 nm and an Olympus Fluoview FV1000 microscope equipped with a 60X objective . Images were acquired at a rate of ∼10 frames/s. Emissions were recorded in the green channel  and the red channel . Analysis was performed using Fluoview software by selecting a pan-somatic area of interest and measuring the increases in green fluorescence relative to baseline fluorescence (dF/F).oC with primary antibodies . Immunoreactive bands were visualized using HRP-coupled secondary antibodies by ECL detection methods (Bio-Rad). Band intensities were analyzed using Bio-Rad Image Laboratory software and calculated relative to β-actin as the loading control.For Western blot analysis, brainstem slices were prepared in the same way as for electrophysiological experiments. DRN was then dissected and immediately frozen on dry ice. The tissue was lysed in lysis buffer (Biobasic) supplemented with protease and phosphatase inhibitors. Protein concentration was measured using Bradford assay (Sigma). For immunoblotting, 10 μg protein was loaded per lane onto 4–20% Mini-Protean precast polyacrylamide gels (Bio-Rad), separated by electrophoresis and transferred onto a nitrocellulose membrane (Bio-Rad). Membranes were then blocked with 5% non-fat milk in Tris-buffered saline and incubated overnight at 4To assess anxiety- and depression-like behaviors, the novelty suppressed feeding, tail suspension, and sucrose preference tests were performed. Each test was separated by a minimum of 3 days. Mice were habituated to the testing room for at least 30 min. Apamin  or vehicle (0.9% NaCl) was injected intraperitoneally (i.p.) 30 min before each test .Following food deprivation for 18 hr, mice were provided with a single food pellet placed in the middle of a dimly lit plastic (45 x 45x 20 cm) box. The floor of the box was covered with bedding 2 cm deep. The latency to start feeding in a 5 min assay was measured by an observer blinded to the groups/treatment. Following the test, each mouse was returned to its homecage and the amount of food consumed within 5 min was measured.Mice were individually suspended by their tails within a three-walled plastic box (45 x 25 x 25 cm) using adhesive tape securely attached to the tip of the tail. The test lasted for 6 min and the behavior of each mouse was videotaped. The amount of time spent immobile per minute was measured by an observer blinded to the groups and treatment and expressed as the cumulative immobility.Mice were habituated to drinking 2% sucrose from two bottles prior to the test. In order to assess the drinking amount, group-housed mice were housed in pairs or singles for a period of 3 days. Mice were given a choice of 2% sucrose or water in their homecage for 2 days. Water and sucrose intake were measured daily and the position of the bottles were changed after the first day. Drugs were administered on both days of the test. Data were expressed as the ratio of sucrose to water consumed and the average of both days were taken.Z-scoring methodology was used as previously described  to assest-test, two-way ANOVA or repeated-measures two-way ANOVA. Newman-Keuls post-hoc test was performed when appropriate. Data were expressed as mean ± S.E.M. and evaluated at a significance level of 0.05.Electrophysiological responses were calculated as changes in membrane potential or holding current resulting from manipulations such as 473 nm light exposure or the bath application of drugs and analyzed by Clampfit (Molecular Devices) software. Spike frequencies for input-output curves were measured by counting the number of spikes in response to 500 ms depolarizing current steps. Spike frequencies for optogenetic and electrophysiological stimulations were determined by counting the number of spikes over a 2 s stimulation period. For illustrative purposes, averaged traces showing current responses and AHPs were compiled using Axograph software. Quantification of AHP potentials was performed by detecting action potentials automatically with a derivative threshold of 20 mV/ms by Axograph software. AHP area (mVxs) is measured as the area under the AHP between the midpoint of each spike (taken as time=0) and the 100 ms time point corresponding to the medium AHP . The max In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by two peer reviewers, one of whom, Sacha B Nelson, is a member of our Board of Reviewing Editor and the evaluation has been overseen by Richard Aldrich as the Senior Editor.Thank you for submitting your work entitled "Chronic social isolation reduces 5-HT neuronal activity via upregulated SK3 calcium-activated potassium channels" for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in Although the topic of the experiments is interesting and the results presented are convincing, the reader is left unsure of the behavioral impact of the changes in excitability observed and as to whether this is indeed as suggested a "promising therapeutic target for disorders of emotional disturbance such as depression and anxiety." It remains possible that SK channels are too broad a target to be useful, and even that these changes in excitability are consequences of, but not causally related to, the effects of social deprivation. For example, one reviewer noted (during the reviewer discussion) that "a more recent paper used a somewhat similar model in mice :2928-37. doi: 10.1038/npp.2014.162) and doesn't seem to see much on the anxiety tests; they did voltammetry measures of 5-HT release in different conditions, and rather see a trend for increased rather than decreased 5-HT release (see their table1). " While it would be feasible with significant additional work to shore up the present results with additional behavioral studies , this would likely take several months to complete and it is the journal policy to reject manuscripts which cannot be accepted without work that could be completed in 1-2 months at most. In this case, however, we would be willing to consider a new manuscript on the same topic, provided it addressed the issue of behavioral relevance. But the reviewer and reviewing editor felt that in its present state the manuscript would be more appropriate for a more specialized journal.Reviewer #1:The authors show that social isolation results in reduced excitability of serotonergic neurons in the mouse dorsal raphe, perhaps explaining the impact of social isolation on depression related symptoms. The experiments demonstrate concisely and convincingly that the decreased excitability is due to an increased afterhyperpolarization (AHP) associated with increased expression of the SK3 subtype of small conductance Ca-activated potassium (SK) channels. Interestingly, polymorphisms of the SK3 gene are known to be risk factors for psychiatric disease.The paper is short and to the point, and the experiments are well done and clearly illustrated.I had only minor suggestions for clarifying the physiological results presented in the manuscript. However, after discussion with the other reviewer, I am persuaded that there is still an open question as to the behavioral relevance, and hence the general interest, of the results. Although it is clear that social isolation is a risk factor for depressive symptoms and anxiety, it is not clear whether the observed change in DRN excitability plays a causal role in this.Reviewer #2:The work nicely demonstrates that rearing mice in social isolation lowers the excitability of serotonin raphe neurons, by modifying the afterhyperpolarization (AHP) responses of the 5-HT neurons, a biophysical property that is controlled by SK channels; they find that SK3 channel expression is increased in the raphe of single-reared mice and that pharmacological blockade of SK3 normalizes AHP and raphe neuron excitability. The paper is well done, but without addition of behavioral data, it remains limited in its conclusions and does not address the possible behavioral correlates of SK3 changes (nor the possible upstream mechanisms that induce SK3 up regulation). Thus, although presenting an interesting novel observation in the field, the results seemed somewhat too preliminary to conclude on the potential relevance of SK3 up regulation for the effects of stress on mood disorders.Specific remarks1) Antagonist rescues hypoactivity of the 5-HT neurons. What about behaviour? There are no behavioural data to show that the protocol of social isolation really changes behaviour, nor how SK3 antagonism could modify them. This is difficult to predict from the published literature since SK3 overexpression was found to cause learning deficits but no anxiety phenotypes , while SK3-KO appear to have increased anxiety .2) Increased of SK3 is shown with western blots, however this shows an unequal signal among cases; thus dot plots would be preferable to histograms in 3) In the Results section the authors claim that difference in excitability did not result from changes in GABA tone mentioning data not shown. It would be important to show these data since there is literature showing that stress affects excitability of GABA neurons but not 5-HT neurons .4) When measuring AHP amplitudes and areas, at which frequency were the cells firing ? The aut5) Because of the heterogeneity of the dorsal raphe neurons, the authors need to specify more precisely the localisation of their recordings.[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your article has been reviewed by two peer reviewers: Sacha B Nelson (Reviewer #1), who is a member of our Board of Reviewing Editors, and Patricia Gaspar (Reviewer #2). The evaluation has been overseen by Richard Aldrich as the Senior Editor.Thank you for submitting your article "Chronic social isolation reduces 5-HT neuronal activity via upregulated SK3 calcium-activated potassium channels" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:2+-activated potassium (SK) channels. They go on to show that isolation also impacts behaviors associated with anxiety and depression in rodents and that these changes are reversed by inhibiting SK channels.The authors nicely demonstrate that rearing mice in social isolation lowers the excitability of serotonin raphe neurons, by modifying their afterhyperpolarizations (AHPs), a biophysical property that is controlled by small-conductance CaEssential revisions:The requested revisions are minor and this should not need to go back for another round of review. The full text of the reviews is given below.Reviewer #1:In this revised manuscript, the authors have now added three behavioral tests to support the idea that the changes in DRN SK3 activity they see following social isolation has effects on feeding and the anxiety/depression related behavior of immobility following tail suspension. The results and some other minor fixes add confidence that the observed changes in the excitability of serotonergic neurons are more likely to have a behavioral impact. There is some concern of course that changes elsewhere (e.g. in the gut) could have affected the feeding behaviors, but this concern is difficult to address.Reviewer #2:The authors have addressed my previous concerns concerning the validation of the social isolation model in mice, by adding behavioral experiments that demonstrate the effectiveness of their protocol to induce anxiety-like phenotypes. Moreover they indicate a causal link between SK3 upregulation and these phenotypes by showing a partial rescue of the behavioral phenotype with Apamin. Finally methodological precisions and complementary explanations were added to the text as requested.Minor comments:1) In their rebuttal, the authors explain the difference of their results with the Dankovski 2014 paper as a difference in protocol. However all the other supporting papers they cite have been done in rats (and they do not cite the Dankovski paper). If this is the first time the social separation model could be valuably transferred to mice, this would be worth mentioning more explicitly for future studies in mice.2) In this regard, information about the mouse strain/gender of mice used in the different experiments is important; I could not find this info in the new version of the manuscript.3) Results, subsection “Social isolation reduces excitability of 5-HT neurons”, first paragraph: explain what picrotoxin and CGP52432 are doing (as in the Methods).4) Results, subsection “Systemic apamin treatment normalizes anxiety- and depressive-like 160 behaviors in socially isolated mice”, last paragraph: to see how the z-score was calculated, I went to "Lin and Sibille 2015"; this was a waste of time as the Methods only refers to Guilloux 2011. Suggestion is to keep only the useful reference.5) Conclusion - It is interesting that SK channels are a druggable compound for depression. Since this is not entirely new, as it had already been put forth and tested - to some extent - by Crespi in 2010, it may be useful to add a sentence as to why no one has really followed this up. Are there issues of toxicity? A need for more selective agents? [Editors’ note: the author responses to the first round of peer review follow.][…] Although the topic of the experiments is interesting and the results presented are convincing, the reader is left unsure of the behavioral impact of the changes in excitability observed and as to whether this is indeed as suggested a "promising therapeutic target for disorders of emotional disturbance such as depression and anxiety." It remains possible that SK channels are too broad a target to be useful, and even that these changes in excitability are consequences of, but not causally related to, the effects of social deprivation. For example, one reviewer noted (during the reviewer discussion) that "a more recent paper used a somewhat similar model in mice :2928-37. doi: 10.1038/npp.2014.162) and doesn't seem to see much on the anxiety tests; they did voltammetry measures of 5-HT release in different conditions, and rather see a trend for increased rather than decreased 5-HT release (see their table1). " While it would be feasible with significant additional work to shore up the present results with additional behavioral studies , this would likely take several months to complete and it is the journal policy to reject manuscripts which cannot be accepted without work that could be completed in 1-2 months at most. In this case, however, we would be willing to consider a new manuscript on the same topic, provided it addressed the issue of behavioral relevance. But the reviewer and reviewing editor felt that in its present state the manuscript would be more appropriate for a more specialized journal.We appreciate that the editors and reviewers brought a recent paper into discussion that uses a different model of social isolation and employed voltammetry to address questions with respect to serotonin release. This gives us an opportunity to talk about our model of social isolation in a more detailed manner. We isolate mice at the age of postnatal (P) day 21 upon weaning and this social isolation continues for more than 7 weeks. Our behavioral work in the new Indeed, it has been shown in various studies that there is a critical developmental window for isolation rearing experiments in rodents and that single-housing after this critical developmental window does not lead to the same robust behavioral effects .In the above-mentioned paper by Dankoski et al. 2016, mice were socially isolated later, starting at adolescence (P35-42), for a relatively brief period of 3 weeks. In this model, single-housed mice did not show robust behavioral changes compared to the paired controls. Although single- housing mice in adolescence may certainly have stressful effects , the Dankoski et al. model likely represents a milder stress than ours. Still interestingly, citalopram-induced facilitation of serotonin release was blocked in this model of social isolation suggesting that even milder stressful conditions may have an impact on serotonin release under challenge.Our electrophysiological findings that serotonin neurons become less responsive to stimulation upon chronic social isolation beginning at P21 are consistent with previous reports showing decreased release of 5-HT in postsynaptic areas after single-housing rodents starting at this developmentally-sensitive time .Reviewer #1:[…] I had only minor suggestions for clarifying the physiological results presented in the manuscript. However, after discussion with the other reviewer, I am persuaded that there is still an open question as to the behavioral relevance, and hence the general interest, of the results. Although it is clear that social isolation is a risk factor for depressive symptoms and anxiety, it is not clear whether the observed change in DRN excitability plays a causal role in this.We thank the reviewer for raising the point with respect to the behavioral relevance of our findings. We have now performed the behavioral experiments showing that this chronic social isolation paradigm elicits significant differences on anxiety- and depressive-like behaviors.Furthermore, we demonstrate that this group difference can be rescued with SK channel blockade by apamin used at a dose shown to enhance the excitability of raphe neurons in vivo .Reviewer #2:[…] Specific remarks1) Antagonist rescues hypoactivity of the 5-HT neurons. What about behaviour? There are no behavioural data to show that the protocol of social isolation really changes behaviour, nor how SK3 antagonism could modify them. This is difficult to predict from the published literature since SK3 overexpression was found to cause learning deficits but no anxiety phenotypes , while SK3-KO appear to have increased anxiety .We appreciate the valuable feedback with respect to the requirement of behavioral experiments. We used male mice for our experiments. Jacobsen et al., 2011 reported an antidepressant-like phenotype in SK3 deficient male mice (reduced immobility in tail suspension and forced swim tests), which supports our findings in this study.With the suggestion of the reviewer, we now conducted the necessary behavioral experiments and added them into the manuscript. A new Interestingly, Jacobsen et al., 2011 reported an anxiety-like effect in one of the tests (zero maze) in SK3 deficient female mice. Whether sex differences exist in our model is an interesting point and will be important to follow up in a future study.2) Increased of SK3 is shown with western blots, however this shows an unequal signal among cases; thus dot plots would be preferable to histograms in We now included the dot plots instead of the histograms for the western blot data in 3) In the Results section the authors claim that difference in excitability did not result from changes in GABA tone mentioning data not shown. It would be important to show these data since there is literature showing that stress affects excitability of GABA neurons but not 5-HT neurons .We thank the reviewer for raising this important point. We have now included these data in 4) When measuring AHP amplitudes and areas, at which frequency were the cells firing ? The autTo measure AHP amplitudes and areas, we selected the minimal depolarizing current step required to elicit action potentials in these cells. We calculated the AHPs from the first spike generated in response to the rheobase current. The frequency of firing at this point is similar between the groups . Of note, we found the frequency differences between the groups in response to stronger depolarizing current steps (>250pA).5) Because of the heterogeneity of the dorsal raphe neurons, the authors need to specify more precisely the localisation of their recordings.We now included this in the Materials and methods section.[Editors' note: the author responses to the re-review follow.][…] Essential revisions:The requested revisions are minor and this should not need to go back for another round of review. The full text of the reviews is given below.[…] Reviewer #2:1) In their rebuttal, the authors explain the difference of their results with the Dankovski 2014 paper as a difference in protocol. However all the other supporting papers they cite have been done in rats (and they do not cite the Dankovski paper). If this is the first time the social separation model could be valuably transferred to mice, this would be worth mentioning more explicitly for future studies in mice.We thank the reviewer for noticing the requirement of additional references for mouse studies on social isolation. We now included additional references in the Introduction.2) In this regard, information about the mouse strain/gender of mice used in the different experiments is important; I could not find this info in the new version of the manuscript.We used only male mice in our study. The gender and the background of our mice are included in the Methods section.3) Results, subsection “Social isolation reduces excitability of 5-HT neurons”, first paragraph: explain what picrotoxin and CGP52432 are doing (as in the Methods).We added this information to this subsection.4) Results, subsection “Systemic apamin treatment normalizes anxiety- and depressive-like 160 behaviors in socially isolated mice”, last paragraph: to see how the z-score was calculated, I went to "Lin and Sibille 2015"; this was a waste of time as the Methods only refers to Guilloux 2011. Suggestion is to keep only the useful reference.The Lin and Sibille 2015 reference is no longer included.5) Conclusion - It is interesting that SK channels are a druggable compound for depression. Since this is not entirely new, as it had already been put forth and tested - to some extent - by Crespi in 2010, it may be useful to add a sentence as to why no one has really followed this up. Are there issues of toxicity? A need for more selective agents?We thank the reviewer for raising this important point. We now included in the Discussion what additional steps/approaches are required for targeting SK channels for treatment of depression. Including these points also gave us a chance to explain the translational aspect of our study with the goal of finding better treatment strategies for depression and anxiety.


Plasmodium parasites, is thought to be one of the strongest selective forces that has shaped the genome of modern humans and was endemic in Europe until recent times. Due to its eradication around mid-twentieth century, the potential selective history of malaria in European populations is largely unknown. Here, we screen 224 ancient European genomes from the Upper Palaeolithic to the post-Roman period for 22 malaria-resistant alleles in twelve genes described in the literature. None of the most specific mutations for malaria resistance, like those at G6PD, HBB or Duffy blood group, have been detected among the available samples, while many other malaria-resistant alleles existed well before the advent of agriculture. We detected statistically significant differences between ancient and modern populations for the ATP2B4, FCGR2B and ABO genes and we found evidence of selection at IL-10 and ATP2B4 genes. However it is unclear whether malaria is the causative agent, because these genes are also involved in other immunological challenges. These results suggest that the selective force represented by malaria was relatively weak in Europe, a fact that could be associated to a recent historical introduction of the severe malaria pathogen.Malaria, caused by  Plasmodium falciparum is one of the main causes of child mortality worldwide, although other species from the same genus, P. vivax, P. malariae and P. ovale are also causative agents of the disease. Therefore, it is not surprising that malaria is one of the strongest known selective pressures that have recently shaped the human genome. Malaria is the evolutionary driving force behind sickle-cell disease (HbS), thalassemia, glucose-6-phosphatase deficiency (G6PD) and other erythrocyte defects that together comprise the most common Mendelian diseases of humankind. Remarkably, populations from different geographical areas have developed different genetic mechanisms adapted to malaria resistance; for instance, the HbS allele at the HBB gene is common in Africa but rare in Southeast Asia, whereas the HbE alelle shows a reversed pattern. Resistance to malaria includes primarily genes involved in immunological response, but also some involved in inflammation and cell adhesion and even genes related to metabolic pathways. The fact that different malaria-resistance alleles have arisen in different places suggests that this adaptation occurred relatively recent in human history, at least well after the Out of Africa migration1.The malaria parasite P. falciparum may have rapidly spread from its African tropical origins to other tropical and subtropical regions of the world only within the last 6,000 years2. The recent origin of the worldwide P. falciparum populations, which are the most malignant of human malarial parasites, may account for its virulence3. Thus, haplotype analysis at the G6PD locus suggests that the African resistance allele to P. falciparum originated within the last 10,000 years4 while a similar analysis at the HbE alleles (variants of the HBB gene) in Southeast Asia yielded an even more recent date, around 5,000 years ago5. These and other estimates associate the resistance to P. falciparum with the onset of agriculture and the emergence of favourable environments for mosquito proliferation after the clearance of woodlands for farming. Falciparum-like illness (I.e. miasmas) appears to be very recent in Europe. It seems to have spread from India to the West, appearing in ancient Greece only by the 4th century BCE, where it was implicated in the decline of many city-state populations2. The retrieval of the eradicated European P. falciparum from 70-year-old slides from a malaria-endemic region in Spain confirms a recent, phylogenetic link with present-day Indian haplotypes6. Therefore, it seems unlikely that P. falciparum would have had a significant impact on prehistoric European genomes.P. vivax  is likely an ancient parasite that evolved in Africa and spread to the rest of the world with the Out of Africa migration, around 60,000 years ago2. Phylogenetic analyses suggest that P. vivax could have an African origin but has largely disappeared from this continent after the spread of Duffy negative resistance7. The biology of P. vivax that makes it highly adapted to maintain itself in small mobile groups of hosts and its mild effect also supports the idea that it is an ancient human parasite. The limited data available does show that P. vivax and P. falciparum may have had at least partially different effects on the human genome1.On the other hand, 2. However, while there is information of some malaria-resistance alleles from Europe, notably from the Mediterranean area (such as the HBB and the G6PD variants), little is known about the magnitude of selection due to malaria in the continent because of the eradication of the parasite around 1950. The recent retrieval of more than 250 ancient genomes from different moments of the prehistory of Europe11 and the publication of several Genome wide association studies (GWAS) on malaria13 now allows for a genome-wide screening of malaria-resistance alleles along space and time. Using this approach, we aim to understand the role of this disease as a selective force in the shaping of the gene pool of modern Europeans.Malaria was endemic in Europe until very recently. Historical data describe malaria as affecting people as north as England, Scandinavia or RussiaG6PD, HBB, ACKRQ, FCGR2B, TIRAP, ATP2B4, GRK5, IL-10, MARVELD3, CD36, CD40LG and ABO blood group system  were already present prior to the arrival of farming, in Upper Palaeolithic and/or Mesolithic individuals. For other loci, the derived alleles are only seen in very recent periods. For instance, only one of the Iron Age individuals carries the T allele (rs2230345) at GRK5 gene, one individual carries the T allele (rs8176746) at ABO gene and only two individuals (one Bronze Age Russian and one Post-Roman British) carry the C allele (rs1050501) at the FCGR2B gene.Even with such data limitations, we were able to detect a substantial number of carriers of the proposed malaria resistance alleles in our ancient genome dataset Table . It is nFCGR2B) , rs4951074 (ATP2B4)  and rs8176746 (ABO) . The three genes show lower frequencies than expected of the derived alleles in ancient populations as compared to present-day Europeans. Samples with data from these three statistically significant ancient vs modern variants are not geographically clustered, as can be seen in Fig. For three genetic variants, we observed significant frequency differences between ancient and modern European populations from the 1,000 Genomes project, after adjusting for multiple tests: rs1050501 FCGRB  was 34%, statistically significant higher than the 16% of the derived alleles in North-European populations  . This trend cannot be discerned in the ancient samples  .Although the problems of assessing population structure with a dataset of low-coverage, geographically and temporally disparate ancient genomes cannot be overlooked we sought to explore the genetic structure among ancient and modern European with an Fe) Table . The FSTgs Table . A G-tesgs Table . This su15. The “Selection” software estimates a posterior probability of selection coefficients for heterozygous haplotypes  and for homozygous derived haplotypes  for each SNP and also the allelic age. The mean values of the selection coefficients and the likelihood values are shown in Fig. To further explore the possibility that changes in our time series data could be attributed to selection acting on the derived alleles, we used a Bayesian method that estimates the frequency trajectory of the alleles based on observed frequencies and sampling datesIL-10 gene with a favoured derived-homozygous genotype  and rs10900585 SNP at ATP2B4 gene, with a favoured heterozygous genotype ; p is the probability of the selection coefficient to be <0.Two genetic variants showed statistically significant evidence of positive selection on the derived allele: rs1800890 SNP at ABO blood group gene. The age estimates of all alleles , Ovalocytosis (in South East Asian groups) and G6PD deficiency19. The first two have little if any effect on P. falciparum while G6PD seems to have more effect on P. vivax than on P. falciparum. So far, none of the ancient genomes available displays any mutation at the crucial G6PD or Duffy loci. In addition, we do not find a single carrier of the HBB mutation. We don’t know if this is attributable to the rather limited sample size or if their absence reflects a relatively recent origin of these mutations among European populations. The frequencies of these mutations are very low in current populations . However, the sample size for some of the SNPs, such as the rs372091 at the HBB locus, is not that small and currently includes 155 ancient genomes.20. At present, only one complete genome 21 and several Neolithic individuals from the oriental Mediterranean peninsulas (Greece and Anatolia)23 derive from the thermo-Mediterranean zone21. However, a significant number of samples derive from regions that are close to the Mediterranean shores  represents a limitation of our survey. This is partially attributable to the warm environmental conditions in the area, which are unfavourable to DNA preservationTIRAP gene (rs8177374)  Table  that shoFCGR2B, ATP2B4 and ABO) show differences in the allelic frequency between ancient and modern Europeans. This could in principle be the signal of recent events of positive selection acting upon the associated SNPs.Three genes (FCGR2B gene (an ILE > THR change in the 232 residue of the protein) has been proposed to have a strong protective effect against severe malaria  in African populations due to the enhancement of phagocythosis of P. falciparum infected-human erythrocytes25. This could explain the high frequency of this mutation in malaria endemic areas, although the same mutation has also been described as causative of Systemic Lupus Erythematosus26. The only two individuals that carry this mutation are from periods and regions where malaria would be endemic in historical times: one is a Bronze Age individual from southern Russia and the other one an Anglo-Saxon from southern England. However, some older specimens , we consulted a genome browser with information about such signaturesATP2B4 and rs1800890 at IL-10 genes. This suggests that selection acted on these genes, an evidence that should be further explored from a functional point of view. The fact that no previous evidence of selective sweeps on these genes has been reported could be explained if the timing of the selective event was very recent or if the effect was regionally restricted. Nevertheless, it is reasonable to discard large-scale and dramatic selective events such as those seen for the lactase gene9.We have modelled all the SNPs with an approach that rejects neutrality across a time series and found evidence for selection in SNPs rs10900585 at P. vivax or to an undetectable adaptive response related to a very recent arrival to the European continent . We have shown however that, despite the large geographic distribution, malaria was likely not an important selective pressure in ancient European populations and not nearly as important as it is in Africa today. Additionally we demonstrate that the study of the selective effect of malaria on ancient genomes is a feasible approach that will likely provide more information in the future with increasing sample sizes, especially from the Mediterranean area. With additional genomic data, and also the retrieval of DNA from the pathogen directly from ancient bones31, it would be possible to check if local selective events took place and to understand the role of malaria in the shaping of modern European genomes.In conclusion, it seems that most of the screened alleles were equally prevalent in past than in present time; in those alleles were differences are statistically significant, we cannot discard the effect of drift or other selective forces acting upon these immunity genes. This could indicate a weak adaptation to malaria, due to either the benignity of symptoms caused by sensu lato- genomes with a mean sequence coverage >1x published so far39. We have also included those genomes for which only targeted capture data is available42. Our final dataset comprises genome-wide-data from 224 individuals  and Mediterranean G6PD (rs5030868) forms. Additional G6PD mutations associated to malaria resistance have also been included 43.HBB) determines the sequence of two types of polypeptidic chains in adult haemoglobin, HbA. Variants in the HBB gene such as HbC and HbS change the structure of the erythrocytes and confer resistance to P. falciparum in Asian and African populations44. The mutations that cause both HbS (rs334) and HbC (rs33930165) were screened45. Recent studies based on GWAS have found an additional mutation in the HBB gene related to malaria resistance (rs372091)12. HbE caused by rs33950507 mutation) is a mainly Asiatic variant of HBB gene that also confers resistance to the infection of P. falciparum46.Haemoglobin beta locus (ACKR1 gene) confers resistance to P. vivax and P. knowlesi through the mutation rs2814778 that changes the structure of the protein, which is a receptor for Plasmodium in the erythrocytes47. This phenotype is especially present in West African populations were P. vivax originated13.Duffy blood group system, Fy (a-b-) (MARVELD3 (rs2334880), ATP2B4 (rs4951074 and rs10900585) and ABO (rs81767199)12. The MARVELD3 gene encodes for a tight junction-associated transmembrane protein of endothelial cells, a key point in the pathology of malaria infection. This mutation is supposed to confer resistance to P. falciparum malaria although it is not confirmed12. The ABO gene encodes for a glycosyltransferase enzyme. Mutations in this enzyme determine the ABO blood group. The individuals who are homozygous for a single nucleotide deletion (rs8176719) are classified as blood group O48. O blood group is linked with a reduced risk of severe malaria49. However, we note that, besides malaria, the O blood group has been associated to susceptibility to other pathogens such as Helicobacter pylori50 and severe cholera51. We have also analyzed the rs8176746 SNP. This mutation, which is in LD with the rs8176719 variant, determines the production of B antigens48. The presence of the derived allele in this locus is associated with an increased risk of severe malaria28. Evidence of malaria protection has been also described at CD40LG gene in Gambian populations: homozygotes for the derived rs3092945 allele show a reduced risk of suffering severe malaria. However, a significant increase of severe malaria risk was found for the same allele in Kenyan populations28. In the same study, a severe malaria protective effect of the SNP rs201346212 heterozygous genotype (CD36 gene) was described. Other possible evidences on P. falciparum malaria resistance include additional genes such as GRK5, a G protein receptor (mutation rs2230345)52, IL-10 (rs1800890), FCGR2B (rs105050) and TIRAP (rs8177374).Additional mutations have been detected by GWAS analysis at 14 and the Tuscans (TSI) population of the 1,000 Genomes project53 as a reference panel.Due to the low coverage in many ancient samples and gaps associated to the targeted capture data, we performed imputations of SNPs not covered by any DNA read using Beagle-4 softwarepost-mortem DNA degradation54. To control for this confounding factor we have screened the genetic background in which the SNP is located, following the same approach as in ref. Some of the alleles screened involve C to T or G to A changes that could be affected by The genotypes obtained from genome sequence data and validated with Beagle-4 software were used to generate genotype frequencies. The allelic frequencies were estimated using both validated genome sequence data and targeted capture data. Because of the impossibility to infer genotypes in the case of targeted capture data only one random allele per position was taken into account.The allelic frequencies obtained with validated genotypes and targeted capture data were stratified by geographic position of the samples  and period .To explore potential differences in the genotypic composition between modern South (Iberians and Tuscans) and North Europeans  populations, we tested two different genetic models (codominant and logistic additive models) for each SNP, with a case-control approach. A likelihood ratio test was estimated for each SNP and both models using a Bonferroni correction. A Fisher’s exact test with a case-control approach was used for comparing allele frequencies in ancient versus modern European populations in those SNPs with more than one detected case of derived allele. Due to the assumption that ancient frequencies are directly comparable to the modern ones, the Fisher’s exact test is anti-conservative here; however we have included the p-value in the cases were we found an>1 OR, just as an additional, supporting evidence.55 was calculated. The fixation index was determined performing all the possible pairwise calculations comparing North-South present-day Europeans, the five European populations of 1000 genomes, North-South ancient Europeans and Period-stratified ancient Europeans . Nine SNPs that exhibited variability . The likelihood ratio G-statistic was performed on a contingency table of alleles at one locus x sampling unit; the individual genotypes were randomly permutated among samples and the G-statistic obtained from the original dataset is compared to the G-statistics obtained from the permuted datasets, yielding a P-value of the test.Additionally the G-test was calculated as it is described in Goudet https://github.com/Schraiber/selection) as described in ref. http://cran.r-project.org) software.To test for the presence of selection driving the genotypic frequencies in past-populations and reject neutrality we used a specific software (Supplementary Dataset 1Supplementary Figures