The N13 component of somatosensory evoked potential (N13 SEP) represents the segmental response of dorsal horn neurons. In this neurophysiological study, we aimed to verify whether N13 SEP might reflect excitability changes of dorsal horn neurons during central sensitization. In 22 healthy participants, we investigated how central sensitization induced by application of topical capsaicin to the ulnar nerve territory of the hand dorsum modulated N13 SEP elicited by ulnar nerve stimulation. Using a double-blind placebo-controlled crossover design, we also tested whether pregabalin, an analgesic drug with proven efficacy on the dorsal horn, influenced capsaicin-induced N13 SEP modulation. Topical application of capsaicin produced an area of secondary mechanical hyperalgesia, a sign of central sensitization, and increased the N13 SEP amplitude but not the peripheral N9 nor the cortical N20-P25 amplitude. This increase in N13 SEP amplitude paralleled the mechanical hyperalgesia and persisted for 120 min. Pregabalin prevented the N13 SEP modulation associated with capsaicin-induced central sensitization, whereas capsaicin application still increased N13 SEP amplitude in the placebo treatment session. Our neurophysiological study showed that capsaicin application specifically modulates N13 SEP and that this modulation is prevented by pregabalin, thus suggesting that N13 SEP may reflect changes in dorsal horn excitability and represent a useful biomarker of central sensitization in human studies. After a tissue injury, chemical mediators generate an area of primary hyperalgesia due to the peripheral sensitization of nociceptive fibres. Inputs into the spinal cord induced by a given stimulus consequently increase3. As a secondary effect, these increased inputs may induce sensitization of second order neurons in the central nervous system5 that manifests with a zone of secondary hyperalgesia, defined as the undamaged area surrounding the injury site with increased sensitivity to mechanical stimulation9.Central sensitization, a key mechanism contributing to several chronic pain conditions, is defined as an increase in the excitability of central nociceptive neurons 10. Peripheral injection and topical application of capsaicin have been found to intensively activate C and Aδ nociceptive nerve fibres, producing burning pain and pronounced flare response spreading beyond the area of primary hyperalgesia11.Different experimental pain models have been devised to investigate the mechanisms underlying central sensitization in humans12, how to reliably quantify central sensitization within the dorsal horn of the human spinal cord is still an issue of controversy. Some studies have shown that the threshold of nociceptive flexion reflex (RIII) is reduced in human models of secondary hyperalgesia13, as well as in patients with chronic pain13. However, in the same studies the RIII amplitude remained unaffected, thus weakening the usefulness of RIII measurements as an objective biomarker of central sensitization15.Although capsaicin and other experimental pain models are commonly used to investigate the mechanisms underlying central sensitization and its association with chronic pain16, probably reflects the segmental postsynaptic response of dorsal horn neurons in the lower cervical grey matter17, presumably neurons in laminae IV–V19. N13 SEPs might therefore constitute a neurophysiological measure sensitive to the excitability changes of dorsal horn neurons during central sensitization. In this neurophysiological study in healthy humans, we investigated if, and how capsaicin-induced central sensitization modulates the dorsal horn N13 SEPs. In addition, we tested whether pregabalin, a first-line treatment for neuropathic pain20 targeting voltage-gated calcium channels expressed in the dorsal horn and the brain21, is able to modulate capsaicin-induced N13 SEP sensitization.The N13 component of somatosensory evoked potentials (N13 SEP) recorded in anterior–posterior direction from the lower neck (Cv6) after upper limb stimulation is mediated by non-nociceptive Aβ fibres. This component, generated at the cervical dorsal hornWe enrolled 10 healthy subjects for each experiment  without symptoms or signs of peripheral or central nervous system disorders or other medical conditions, any drug intake in the past two weeks, known or suspected allergic reactions or hypersensitivity to pregabalin or its components, dermatological disorders or skin lesions affecting the area of capsaicin application, jet lag, irregular working hours, sleep restrictions in the last week, or past drug abuse.Written informed consent was obtained from all participants. This study was approved by the institutional review board of Sapienza, University of Rome (REF.CE 4789-2018) and performed according to the Declaration of Helsinki regarding the use of humans in experimental studies.After informed consent, in a separate pilot session preceding at least one week the first experimental session, all subjects familiarised themselves with the technical procedures, including capsaicin application and electrical stimulation. During this experimental session, we assessed the consistency of secondary hyperalgesia and dynamic mechanical allodynia in the enrolled subjects.21, could prevent central sensitization-induced N13 SEP modulation; in a double-blind placebo-controlled crossover trial, we tested the effect of a single oral dose of pregabalin and placebo on the capsaicin-induced modulation of N13 SEPs.This study consisted of three distinct experiments. Experiment 1 was designed to verify whether central sensitization induced by application of a commercially available capsaicin cream (2.5% capsicum oleoresin corresponding to 0.1% active capsaicin associated with inactive ingredients) to the ulnar nerve territory of the right-hand dorsum affected N13 SEPs generated by ulnar nerve stimulation. In experiment 2, we investigated the time course of capsaicin-induced N13 SEP modulation. Experiment 3 was designed to verify whether the analgesic pregabalin, whose pharmacological target is expressed in the dorsal hornOf 22 enrolled subjects, four participated in all the three experimental procedures. The three experiments were conducted at an interval of at least 3 months thus limiting any carry-over effects. Dorsal horn N13 SEP measures were analysed offline by two investigators who were unaware of the type of session (drug or placebo).2 covering almost the entire ulnar aspect of the hand dorsum . Capsaicin cream was applied for 40 (experiment 1 and 3) and 60 min (experiment 2), before being gently wiped off. We identified the site of primary hyperalgesia corresponding to the area of flare and burning pain, then we used a calibrated 128-mN pinprick probe  to map with six radial pinches the area of secondary pinprick hyperalgesia, defined as the area surrounding the flare zone with increased sensitivity to mechanical pinprick stimulation  were recorded after electrical stimulation of the ulnar nerve at the wrist . The cathode was placed over the distal ulnar nerve, 2 cm proximal to the wrist crease while the anode was placed on the wrist crease . Intensity was set at the threshold for evoking a muscle twitch in the muscles of the hand innervated by the ulnar nerve (10.4 ± 1.2 mA).Three blocks of 500 trials were consecutively collected, superimposed , and averaged. Muscle artefacts were avoided by making the patient as comfortable as possible. Subjects were instructed to lay down on a medical cot in a supine resting position. Automatic artefact rejection was used to eliminate occasional high-amplitude transients (> 100 μV).22. The peripheral (N9) component was recorded with the surface electrode over the Erb point bilaterally, within the angle formed by the posterior border of the clavicular head of the sternocleidomastoid muscle and the clavicle, 2–3 cm above the clavicle. This montage served to monitor the peripheral input into the spinal cord. The dorsal horn N13 component was recorded with a posterior cervical spinal electrode over the sixth cervical spinous process, with an anterior cervical electrode as a reference on the skin surface of the supraglottic region on the midline. In each subject, N20 and P25 components were recorded with a parietal scalp electrode (CP3/CP4) contralateral to the stimulation, placed according to the 10–20 international system with an Fz reference. Impedance was kept below 5000 Ω. Epochs were averaged after automatic artefact rejection. In each subject less than 5% epochs were excluded. Peak amplitude of the different SEP components was manually extracted from the individual waveforms.To record and measure the different SEP components, we followed the International Federation of Clinical Neurophysiology guidelinesWe enrolled 10 healthy participants . Before capsaicin application, using the calibrated 128-mN pinprick probe, we quantified mechanical pain sensitivity in the right-hand dorsum with the 0–100 NRS . After 40 min  we mapped the area of secondary hyperalgesia and asked subjects to quantify mechanical pain sensitivity in the area of secondary hyperalgesia by using the 0–100 NRS.We recorded SEPs in response to electrical stimulation of the ulnar nerve on both arms before and 40 min after capsaicin application to the ulnar nerve territory of the right-hand dorsum .Ten healthy subjects  participated in this experiment. We investigated the time course of capsaicin-induced N13 SEP modulation by recording dorsal horn N13 SEPs after stimulation of the ulnar nerve of the active side (treated with capsaicin) at multiple time intervals, namely before and 20, 40, 90, 120, 140, 160, and 180 min after capsaicin application to the ulnar nerve territory of the right-hand dorsum.Participants were asked to rate the mechanical pain sensitivity in the area surrounding the capsaicin application, at baseline and at each time interval after capsaicin application, by using the 0–100 NRS . In this experiment we did not map the area of secondary hyperalgesia.In this experiment, we enrolled 10 healthy participants . We recorded the N13 SEP after ulnar nerve stimulation of both arms before and 40 and 90 min after capsaicin application on the ulnar nerve territory of the right-hand dorsum. At each time point, participants were asked to rate the mechanical pain sensitivity, in the area surrounding the capsaicin application, by using a numerical rating scale (NRS) ranging from 0 (no pinprick perception) to 100 (maximum pain imaginable). The area of secondary hyperalgesia was mapped 40 min after the capsaicin application, immediately before the SEPs recording, by applying mechanical pinprick stimuli in the area surrounding the capsaicin-induced flare.After the baseline recording, at the time of capsaicin application, subjects were randomised to receive a single oral dose of pregabalin 150 mg or placebo (a vitamin capsule) in a double-blind placebo-controlled crossover design. Placebo and pregabalin capsules were indistinguishable in terms of weight and size. Random numbers were assigned to each treatment condition. A minimum interval of 7 days was required before the subjects returned for their second session to minimise cross-over effects. The effectiveness of blinding was not directly assessed with specific measures.Descriptive summaries for the latencies and amplitudes of each SEP component  for each treatment condition are presented as mean ± standard deviation (SD) .We used the Shapiro–Wilk test, recommended for the analysis of small samples, to assess data distributionIn experiment 1, paired t-test was used to assess NRS score differences before and after capsaicin application. Two-way repeated measures analysis of variances (ANOVA) and post hoc multiple comparison with Sidak’s correction was used to assess the effect of time (before and after capsaicin application) and treatment (capsaicin) and their interaction on latency and amplitude of SEP components.Given that experiment 1 showed that peripheral N9 and cortical N20-P25 did not change, in experiments 2 and 3 the analysis was limited to N13 SEP amplitude.In experiment 2 we performed one-way ANOVA with the Greenhouse–Geisser correction followed by Dunnett's multiple comparisons tests to assess N13 amplitude and NRS score variation across the different time points.In experiment 3, we estimated a sample size of n = 10 subjects needed to detect a difference in N13 amplitude comparable to that observed in experiment 1, based on the effect size of interest . We then performed a two-way repeated measures ANOVA on both arms including the Greenhouse–Geisser (GG) correction involving the effect of time, type of treatment and their interaction. Post hoc analysis of pairwise comparisons was performed using t-tests adjusted for multiple comparisons with Sidak’s correction.We used the Pearson correlation coefficient to assess the correlation between changes  of the N13 SEP amplitude and the mechanical pain sensitivity in the 22 subjects included in this study at the 40-min time interval of the three experiments.p-value All the tests were two-sided and a 2) characterised by increased sensitivity to mechanical pinprick stimulation relative to the baseline assessment .In each subject, capsaicin application to the ulnar nerve territory of the right-hand dorsum induced an area of primary hyperalgesia with flare, with a surrounding area of secondary hyperalgesia  and type of treatment  for the N13 SEP amplitude p < 0.02) , while secondary mechanical hyperalgesia, as quantified by the increase in pain sensitivity relative to baseline, had a longer duration of up to 180 min ,  and time × treatment interaction  . No other adverse events were reported.The correlation analysis in the 22 subjects included in this study, did not show any significant correlation between the N13 SEP amplitude changes and the magnitude changes of mechanical pain sensitivity before and 40 min after capsaicin application .In this neurophysiological study in healthy humans, we show that central sensitization induced by topical capsaicin was associated with an increase in the amplitude of the dorsal horn N13 SEP. Furthermore, we showed that pregabalin, a drug with proven efficacy on dorsal horn neurons, prevented the N13 SEP modulation associated with capsaicin-induced central sensitization. These findings suggest that N13 SEP reflects changes in dorsal horn excitability and might represent a biomarker of central sensitization in humans.12. Further experimental pain models could also be potentially used to investigate N13 SEP changes during central sensitization, such as high-frequency electrical stimulation of the skin, which also elicits robust secondary hyperalgesia, but potentially has a longer and more stable time course than topical capsaicin26.To investigate the effect of central sensitization on the N13 SEP, we used topical capsaicin, a safe and easy-to-use experimental model to induce central sensitization and secondary hyperalgesia, which avoids the often very strong pain induced by other routes of administration such as intradermal injection27.In our experiments, we analysed N13 SEP amplitude changes at multiple time points after capsaicin application which spanned a period of stable secondary hyperalgesia which persisted for up to 180 min. Since all subjects reported robust secondary hyperalgesia between 30 and 60 min after capsaicin application in the experimental session, we used a 40-min time interval in experiments 1 and 3 to assess the capsaicin-induced N13 SEP modulation. In experiment 3, we investigated whether pregabalin also prevented capsaicin-induced N13 SEP modulation at 90 min, given that 60–90 min is the time of maximum observed pregabalin plasma concentration28. N13 SEP characteristics in humans are very similar to those of the N1 spinal potential in animal models, reflecting postsynaptic neuronal responses to inputs conveyed by group I and II peripheral afferent fibres in laminae IV and V of the dorsal horn30. We therefore conjecture that our data indicate that the increase of the N13 SEP after topical capsaicin reflects dorsal horn excitability changes underlying central sensitization.Each experiment in our study (including the placebo treatment session in experiment 3) concordantly demonstrated that topical application of capsaicin cream to the ulnar nerve territory of the hand dorsum produced a mild, though reproducible, increase in the N13 SEP amplitude after electrical stimulation of the ulnar nerve. This increase was associated with an elevated pinprick response in the area of secondary hyperalgesia, which is also a function of central sensitization. The N13 SEP, which is mediated by non-nociceptive Aβ fibres, is generated by segmental postsynaptic responses of dorsal horn neurons at the level of the cervical spinal cord31. Since N13 SEP is elicited by Aß fibre stimulation, its amplitude increase might therefore reflect an excitability change of WDR neurons.Previous studies have shown that capsaicin induces an input amplification in nociceptive specific neurons as well as wide-dynamic range (WDR) neurons with convergent tactile and nociceptive inputs; low-threshold Aß fibre-related neurons with purely tactile inputs are not facilitated22. These findings lend further evidence that the N13 SEP may reflect dorsal horn excitability.Peripheral N9 SEP and cortical N20-P25 SEP did not parallel N13 SEP amplitude modifications across the three time points of the experiment. The cortical SEP components are generated in the primary somatosensory cortex by large myelinated fibres whose collaterals activate dorsal horn neurons generating N13 SEP24. This long-lasting modulation is presumably compatible with post-translational changes associated with the early phase of long-term potentiation underlying central sensitization25. Conversely, secondary hyperalgesia, quantified by the increase in mechanical pain sensitivity in comparison to baseline assessment, had a longer duration. This difference may be compatible with a supra-spinal contribution to this long-lasting increased mechanical pain sensitivity. This interpretation is in line with previous studies showing decay of dorsal horn sensitization after 2 h24, with longer-lasting metabolic changes in supraspinal structures32.The experiment 2 of our study showed that the capsaicin-induced N13 SEP modulation persisted for approximately 2 h, consistent with long-lasting, yet reversible, potentiation of spinal nociceptive pathways34. Notably, we found that pregabalin completely prevented the N13 SEP modulation associated with capsaicin-induced central sensitization, thus the effects on spinal signalling were stronger than those on perception. Pregabalin inhibits voltage-gated calcium channel activity at the presynaptic level, thus reducing the synaptic release of neurotransmitters and activation of postsynaptic neurons21. Although we cannot exclude that pregabalin might affect the N13-SEP through supraspinal pharmacological effects, we consider that the most plausible mechanism is a reduction of the central sensitization induced by capsaicin at the level of the dorsal horn21.In experiment 3, we found that pregabalin reduced the secondary hyperalgesia magnitude in comparison with the placebo. In the pregabalin session the NRS increase was lower than that in the placebo session and this increase had a slight negative tendency at 90 min. The effect of pregabalin we found is consistent with previous studies showing that gabapentinoids reduce hyperalgesic responses associated with capsaicin-induced central sensitizationhttps://www.ema.europa.eu/en/documents/scientific-guideline/guideline-clinical-development-medicinal-products-intended-treatment-pain-first-version_en.pdf). Our data indicate that the dorsal horn mediated-N13 SEP might be used to detect central sensitization in human clinical trials and ultimately demonstrate its modulation by novel analgesics; large group comparison may provide useful data on dorsal horn excitability changes during central sensitization. Since many analgesic drugs have failed when tested in clinical populations35, the use of N13 SEPs in early phase pharmacological trials might hasten the identification and selection of promising drug candidates for chronic pain, thereby helping to increase the likelihood of successful translation from the preclinical to clinical settings and thereby reducing the high costs associated with their development.Our data therefore introduce the possibility of using the N13 SEP as a biomarker to investigate how drugs affect central sensitization in the human spinal cord. European Medicines Agency guidelines on the clinical development of medicinal products intended for the treatment of pain explicitly indicate that objective biomarkers might improve the development of drugs for chronic pain ; furthermore, these two studies do not report whether secondary hyperalgesia developed or not.Our data showing that topical application of capsaicin modulates the N13 SEP amplitude argues against two previous studies showing that motor task and experimental capsaicin-induced pain do not significantly affect N13 SEP15. However, these studies did not consistently report a consensual amplitude increase of the nociceptive flexion reflex15. Furthermore, nociceptive flexion reflex is also affected by ventral horn motoneuron excitability, and, contrary to N13 recordings, needs to employ noxious stimuli which limits patient acceptability. Together, these limitations hamper the usefulness of nociceptive flexion reflex measurements for assessing central sensitization. Previous studies have shown increased amplitude of pinprick-evoked potentials after stimulation of the area of secondary hyperalgesia in healthy humans42. The increased vertex complex of pinprick-evoked potentials may reflect an increased cortical response to A-fibre mechano-nociceptive inputs due to capsaicin-induced central sensitization. Our study, however, suggests that caudal N13 SEPs generated by the dorsal horn may provide a direct measure of increased dorsal horn excitability during central sensitization. The dorsal horn N13 SEP is mediated by non-nociceptive Aβ fibres, which is a crucial advantage in clinical settings since stimulation is set at non-painful levels, hence ensuring subjects’ compliance. A dorsal horn SEP component can be recorded after stimulation of lower limbs (N22 SEP), thus allowing the investigation of central sensitization across multiple sites43.Alternative neurophysiological biomarkers might be used to test central sensitization. Previous studies showed that the nociceptive flexion reflex excitability is increased in patients with chronic pain conditions, presumably associated with central sensitizationWe did not find any significant correlation between the N13 SEP amplitude and the magnitude of secondary hyperalgesia. This finding might be due to a poor amplitude resolution  of N13 SEP recordings, which might prevent the possibility to detect minor excitability changes, still able to produce perceptual changes.44.Admittedly, we cannot exclude that attention might concur in the N13 SEP amplitude changes we found. Previous studies showed that attention modulates dorsal horn mediated nociceptive responses such as the nociceptive flexion reflex12, though this appears to yield lower amplitude SEP components than the more commonly used median nerve stimulation22. The relatively low amplitude of ulnar-nerve-mediated N13 SEPs and the mild amplitude increase after capsaicin application probably prevent the assessment of capsaicin-induced modulation at single subject level. This limitation may hamper the usefulness of our findings in the search for objective evidence of central sensitization in individual patients with neuropathic pain, but at a group level such changes have already been reported for patients with painful cervical radiculopathy45. N13 SEP investigation might still be useful to assess drug effects on central sensitization in pharmacological trials since drug effects are assessed using statistical group comparisons in these studies.In our study, we stimulated the ulnar nerve to elicit N13 SEPs because the topical capsaicin model works best on hairy skinAlthough in our three experiments including 22 participants we found that the capsaicin-induced effect on the N13 SEP modulation is relatively reproducible, further studies, including larger sample of healthy participants, are needed to further support a regular use of N13 SEP as a biomarker of central sensitization.The dorsal horn N13 component of SEPs reflects dorsal horn excitability changes, thus raising the possibility that this spinal SEP component might be used as a biomarker to investigate central sensitization in humans. N13 SEPs may therefore represent a useful tool to detect central sensitization and to test the effects of drugs on pain in pharmacological trials involving humans.Supplementary Figure 1.Supplementary Figure 2.Supplementary Tables.


To evaluate the effects of long-term tumor necrosis factor (TNF) inhibition on the risk and age at onset of Parkinson disease (PD), we performed a 2-sample Mendelian randomization study using genome-wide association studies (GWAS) summary statistics.TNFRSF1A, the gene encoding TNF receptor 1 (TNFR1), were identified as predictive of pharmacologic blockade of TNFR1 signaling by anti-TNF therapy, based on genetic associations with lower circulating C-reactive protein . The effects of TNF-TNFR1 inhibition were estimated for PD risk  and age at PD onset  using GWAS data from the International Parkinson's Disease Genomics Consortium and 23andMe, Inc. To validate variants as proxies of long-term anti-TNF treatment, we also assessed whether variant associations reflected anticipated effects of TNFR1 inhibition on Crohn disease, ulcerative colitis, and multiple sclerosis risk .Genetic variants in the vicinity of TNF-TNFR1 signaling inhibition was not estimated to affect PD risk  or age at onset . In contrast, genetically indexed TNF-TNFR1 signaling blockade predicted reduced risk of Crohn disease  and ulcerative colitis  and increased multiple sclerosis risk . Findings were consistent across models using different genetic instruments and Mendelian randomization estimators.Our findings do not imply that TNF-TNFR1 signaling inhibition will prevent or delay PD onset.This study provides Class II evidence that TNF-TNFR1 signaling inhibition is not associated with the risk or age at onset of PD. Moreover, results obtained from a sample of patients with IBD may not be generalizable to individuals without IBD.Inflammation in the periphery is hypothesized to contribute to the pathogenesis of Parkinson disease (PD), particularly when present in the gastrointestinal tract.5 In Mendelian randomization studies relating to pharmacologic exposures, genetic variants that perturb the expression or function of a drug's target (often a specific protein) are used to anticipate the effects of modulating this target with therapeutic use.6 Due to Mendelian randomization principles, associations of this genetic variation with diseases are not expected to have arisen from confounding or reverse causation, thus aiding causal inference.7 In this study, we used a Mendelian randomization design to further evaluate the therapeutic potential of targeting TNF signaling for PD prevention or treatment, leveraging genetic variation in or near the gene encoding TNF receptor 1 (TNFR1) that is indicative of the blockade of proinflammatory TNF signaling via this receptor.2Mendelian randomization is a valuable approach to help answer questions in pharmacoepidemiology.8 We focused on single nucleotide polymorphisms (SNPs) in the vicinity of the gene TNFRSF1A, which encodes TNFR1, the principal effector of proinflammatory signaling following TNF agonism.2 We determined the extent to which genetic variation in this gene region is indicative of long-term TNF signaling blockade, based on associations of the regional SNPs with circulating markers of systemic inflammation reported by large genome-wide association studies (GWAS) of C-reactive protein (CRP) and cell count measures.10 We then combined data on selected variants with corresponding association statistics from GWAS of PD traits to test the effects of TNF-TNFR1 signaling inhibition on (1) the risk of PD, measured as self-reported or clinically ascertained disease status; and (2) age at PD onset, measured as self-reported age at motor symptoms manifestation, or age at PD diagnosis when the former was unavailable.12 This study provides Class II evidence that TNF-TNFR1 signaling inhibition is not associated with PD risk or age at onset, according to criteria of the American Academy of Neurology.This study used a 2-sample Mendelian randomization design .8 We foc14 Therefore, researchers have investigated the development of novel therapeutics to selectively antagonize TNFR1 without inhibiting TNFR2-mediated signaling.16 An effective response to anti-TNF treatment leads to lower systemic inflammation, reflected by reductions in circulating inflammatory biomarkers, such as CRP.17 Thus, to validate whether SNPs in the vicinity of TNFRSF1A index the modulation of proinflammatory TNF-TNFR1 signaling, we extracted data on associations of these variants with circulating CRP from a large GWAS meta-analysis  and mean platelet volume (MPV)—from independent GWAS data , whereas TNFR2 signaling contributes to tissue repair and neuron survival.WAS data .10,18TNFRSF1A is located close to genes that encode other proteins with known immune-related roles, such as lymphotoxin β receptor gene LTBR .In total, 23 SNPs were selected solely from within the gene's genomic coordinates and a narrow flank in either direction . The choice of a narrow flanking region was adopted to minimize the possibility that the selected variants might associate with PD traits via pathways other than TNF-TNFR1 signaling, given that 11 This yielded a sample of 37,688 cases and 981,372 controls. Genetic association data for age at PD onset were based on a GWAS comprising 28,568 PD cases from a subset of the cases sampled by the IPDGC and 23andMe, in which the mean age at onset was 61.7 years (range 20–97).12 These samples are described further in Genetic association data for PD risk were derived from a meta-analysis of 16 case–control samples from the International Parkinson's Disease Genomics Consortium (IPDGC) and 23andMe, using the same protocol as adopted in a recent GWAS.19 However, sample overlap is likely to be nominal in this study because the exposure and outcome GWAS were conducted with largely independent samples: samples for CRP and cell count GWAS were derived primarily from population-based cohorts assembled by the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium,9 and PD GWAS samples were derived mainly from independent case–control studies assembled by the IPDGC and the 23andMe user base  to the set of SNPs in the region with CRP association p values under 0.05, to select independent SNPs with the strongest evidence for association with systemic inflammation. Mendelian randomization results based on this selection criterion were then obtained using Wald estimation, given that a single SNP (rs767455) was retained for analyses. Mendelian randomization estimates can be biased when the genetic variants used in analysis are weak instruments for the exposure being indexed.29 Thus, to indicate the strength of this SNP as an instrumental variable, the F statistic for its association with CRP was estimated from the F distribution based on the p value and sample size of SNP–CRP association, with 1 degree of freedom.We conducted Mendelian randomization models based on 3 approaches. In the primary analysis, we applied conservative linkage disequilibrium (LD) clumping (r2 < 0.8). The clumped SNPs were then filtered by requiring false discovery rate–corrected CRP associations with p values under 0.05, and further associations of the variants with both WBC and MPV (using an unadjusted p threshold <0.05), ensuring consistent and robust associations of variants with inflammatory signaling were retained. Next, Mendelian randomization estimates based on the set of retained SNPs were calculated using an extension to the inverse variance weighting (IVW) method with principal components to account for the residual correlations between selected SNPs.30 This method relies on reference data to adjust for estimated correlations between the SNPs, for which we used data on 502 individuals of European ancestry of the 1000 Genomes project, phase 3.31 Principal components explaining over 99% of variance in the weighted correlation matrix were used in the IVW estimator.For secondary analyses, we aimed to retain more variants—which may explain more variance in CRP and therefore potentially improve the power of Mendelian randomization models—by including all regional SNPs after liberal LD clumping .Third, after examining the functional annotation of SNPs in the gene region, we also assessed specific associations of a single SNP (rs1800693) with traits, since this SNP is suggested to affect alternative splicing of the 33 In plots, outputs from logistic regression models were presented as log-odds to scale appropriately alongside βs from linear regression models. Corresponding odds ratios (ORs) and 95% confidence intervals (CIs) for binary outcomes are reported in the Results. Given that we had strong priors for non-null results from positive control analyses, we did not adjust these results for multiple testing correction.All Mendelian randomization outputs were scaled to be expressed per 10% reduction in circulating CRP, indicative of the direction of effect anticipated from TNF inhibition.TwoSampleMR and mendelianRandomization packages.34All analyses were performed in R version 3.6.1 (2019-07-05) using For each GWAS study included in the present work, written informed consent was obtained from all participants and the studies were approved by relevant ethics review boards. Because our study was based on existing summary-level genetic results—i.e., without accessing individual-level genetic data—no additional ethical approval was required.9 Summary statistics from GWAS of WBC and MPV are publicly available at bloodcellgenetics.org/.35 Full PD GWAS data can be obtained via research project applications to 23andMe, Inc. and the IPDGC.The majority of genetic association statistics used in this study are presented in TNFRSF1A were present in the CRP data. One SNP in the region—rs767455—was retained following conservative LD clumping (p = 7.8 × 10−8). It is in high LD with the presumed functional variant (rs1800693) in this region (r2 = 0.84)32 and the variant is also associated with circulating WBC (p = 4.2 × 10−3) and MPV (p = 3.7 × 10−6), with consistent directions for all 3 inflammatory markers. Using this variant in Mendelian randomization models, no effects of TNF-TNFR1 inhibition on PD risk  or age at onset  were predicted .A total of 23 SNPs in the vicinity of clumping . This exredicted . In contp < 0.05. Among these 4 SNPs, one was not associated with either WBC (p = 0.7) or MPV (p = 0.95) and was therefore removed, leaving 3 variants that were associated with all 3 inflammatory markers for secondary Mendelian randomization models . Although we showed no support for the potential of blocking TNF-TNFR1 signaling to lower PD risk, the results should be interpreted carefully in comparison to the observational study for several reasons. First, the 4 TNF inhibitors investigated by Peter et al.4—adalimumab, certolizumab, golimumab, and infliximab—all directly neutralize soluble or transmembrane TNF and disrupt both the TNFR1- and TNFR2-mediated pathways as well as other potential biological effects induced by TNF by any other pathways,36 whereas our results are specific to the selective inhibition of TNF-TNFR1 signaling transduction. However, we note that TNF-TNFR1 signaling is proposed as the major TNF-related neurotoxic pathway to intervene on for PD therapy.14–37 Second, given that IBD is rare, our findings are more relevant for the general population. The models test whether TNF-TNFR1 signaling blockade affects PD onset regardless of whether or not individuals have a background of severe systemic inflammation.38 Consequently, we do not exclude therapeutic benefits in individuals with certain underlying conditions, such as IBD. Third, Mendelian randomization estimates relate to an averaged effect of long-term exposure to the TNF-TNFR1 inhibitors across the life course; therefore, a null result may reflect either a true lack of an overall effect on PD risk at any stage of life or a sum of opposing, period-specific effects that balance out in our models. This scenario might be possible if, for instance, TNF-TNFR1 signaling inhibition is beneficial for PD in older age due to anti-inflammatory effects, but detrimental earlier in life for other reasons . Mendelian randomization estimates are also averaged across any tissue-specific effects, whereas therapeutic administration of TNF inhibitors may be limited to effects induced in the periphery. Fourth, because the 4 TNF inhibitors examined by Peter et al.4 are large molecules that do not cross the blood–brain barrier (when intact), they are assumed to mitigate peripheral inflammation principally, unless IBD has affected the integrity of the blood–brain barrier to allow TNF inhibitors to act in the cerebral parenchyma. However, the possibilities of opposing period- or tissue-specific effects of TNF inhibition on PD risk are speculative.By comparing patients with IBD with and without anti-TNF treatment in a cohort study, Peter et al.In addition to the risk of PD, we also considered whether TNF-TNFR1 signaling inhibition may affect the age at PD onset for its relevance to the course of disease progression in lieu of large GWAS of longitudinally measured PD progression, which are not currently available. For a progressive disease such as PD, an exposure affecting the age at onset could do so by either delaying the pathogenesis of PD  or modifying the rate of PD progression once the disease process has commenced. In the present study, the long-term blockade of TNF-TNFR1 signaling did not affect the age at PD onset, implying limited potential for TNF inhibitors to affect PD progression (while keeping the aforementioned caveats about interpreting genetic vs pharmacologic results in mind).The strengths of our study include the use of large-scale genetic data in a Mendelian randomization design to facilitate causal inference, robust metrics for the Mendelian randomization analyses , and positive control analyses that indicate the validity of using the chosen genetic variants to predict therapeutic effects. This study's main limitations are that we could not examine whether there is an interaction between inflammatory status and TNF inhibition on PD risk or index the effects of TNF inhibition on direct measures of PD progression.39 Hence, our findings should be considered as part of discourse on the repurposing potential of TNF inhibitors for PD prevention and treatment, particularly if selective inhibitors of TNF-TNFR1 signaling become available. Future Mendelian randomization studies could use large-scale GWAS of PD progression to help evaluate the disease-modifying potential of selective TNFR1 inhibitors for PD treatment.In conclusion, our findings do not support the notion that long-term blockade of TNF-TNFR1 signaling will prevent or delay PD onset. Human genetics provides valuable guidance for therapeutic programs because the chances of successful drug development are substantially improved when genetic evidence links a drug's target to an indication for therapeutic use.


ALCAM, RIPOR2, AFAP1 and MOCOS. In addition, treatment associations overlapped at gene level with baseline clinical and social outcomes. Treatment related DMPs are involved in memory formation—the key agent in trauma focused treatments—and enriched for molecular pathways commonly affected by trauma related disorders. Results were partially replicated in an independent sample of 53 female former child soldiers from Northern Uganda. Our results suggest a molecular impact of psychological treatment in women with war-related childhood trauma.The aftermath of traumatization lives on in the neural and epigenetic traces creating a momentum of affliction in the psychological and social realm. Can psychotherapy reorganise these memories through changes in DNA methylation signatures? Using a randomised controlled parallel group design, we examined methylome-wide changes in saliva samples of 84 female former child soldiers from Eastern DR Congo before and six months after Narrative Exposure Therapy. Treatment predicted differentially methylated positions (DMPs) related to Trial registration: Addressing Heightened Levels of Aggression in Traumatized Offenders With Psychotherapeutic Means . Frequent exposure to severe stressors is decisively detrimental. Such exposure reorganises the functioning of the brain and mind in a lasting, self-perpetuating manner such that simple cues, sometimes arising from imaginative processes alone, may activate a physical response as part of the defence cascade3. Most importantly, the hypothalamic–pituitary–adrenal (HPA) axis may be tilted6, resulting in anything from a long-lasting immune problem9 via deficient metabolic systems12 to the broad trauma related psychopathology maintained by the intrinsic dynamics of traumatic memories.Former child soldiers, particularly female combatants, are at high risk of experiencing multiple, severe and traumatic stressors including violence and repeated rape. These extreme and intense stressors produce lasting changes in mind and body leading to a range of mental disorders. These include posttraumatic stress disorder (PTSD), depression, dissociative symptoms and high levels of aggression against oneself and others14. For example, PTSD and depression have been associated with DNAm changes in blood and buccal cells at genes involved in glucocorticoid functioning, such as the FKBP prolyl isomerase 5 (FKBP5) and the nuclear receptor subfamily 3 group C member 1 (NR3C1)19. Emerging evidence suggests that these associations could even be transferable across generations22, although studies in mammals are still scarce23. Aggression has also been associated with multiple differentially methylated regions (DMRs) in at least 30 gene promoters24. Likewise, antisocial personality disorder has been associated with DNAm changes in the monoamine oxidase A (MAOA) and B (MAOB)25, two genes relevant in the dopaminergic and serotonergic pathways.These processes are realised through epigenetic modifications in neuronal and peripheral tissues, and in particular have been associated with DNA methylation (hereafter DNAm)28. However, so far, there have been no randomised controlled trials using an epigenome-wide association approach to investigate the impact of successful trauma treatment in any sample, let alone in children of war. Given that childhood experiences are important for both aetiology and epigenetic predisposition of mental disorders31, investigating therapeutic impacts on the epigenome in patients with childhood and war-related trauma may support further development of treatment for mental disorders and contribute to our understanding of therapeutic agents.It is yet to be seen whether these epigenetic marks can be modified through treatment such that the clinical symptoms may recede. A few studies with small sample sizes that investigated only a limited number of target genes nonetheless provide early evidence that this may be possible32 using an epigenome-wide approach (EWAS) that investigated associations between differentially methylated positions (DMPs), trauma related mental disorders and social consequences as well as Narrative Exposure Therapy (NET)33 in comparison to a less effective treatment [step 1]. NET is one of the empirically supported psychotherapies for trauma-related disorders34, alongside CPT (Cognitive Processing Therapy), EMDR (Eye Movement Desensitization and Reprocessing) and PE (Prolonged Exposure Therapy). It particularly targets survivors of multiple traumatic stressors. Recent adaptations of NET have successfully treated trauma related suffering, focusing on PTSD, depression and appetitive aggression, and mitigated social problems such as current violent behaviour and perceived social acknowledgment as victim or survivor36. Then, we identified overlap in associated DMPs [step 2] and further investigated outcome and treatment associations using gene-ontology and gene-network enrichments to determine potential functions [step 3]. Finally, we used an independent cohort of 53 female former child soldiers from Northern Uganda who also had been successfully treated using NET to replicate our findings [step 4].We examined a subsample of 84 highly traumatized female former child soldiers who participated in a randomised controlled trialAt baseline, mean Beta per probe ranged from 0.030 to 0.968 with an overall mean value of 0.61 . Most probes  had a methylation status of more than 0.5 (69.7%). Mean DNAm per participant  was 0.61 (SD = 0.008). It did not significantly change over time  nor did it differ between treatment groups , CVB (51 DMPs), AAGS  and SAQ (85 DMPs), but we did not find significant associations with PHQ-9 or AAS. For details on all estimates see supplement Linear models (EWAS) showed a varying number of CpGs that were significantly associated (FDR = 0.05) with clinical and social outcomes at baseline. We found significant DNAm associations with PSS-I , cg12337669 (AFAP1) and cg08739828 , and DNAm increase at cg18803039 (RIPOR2) , CpG sites were found to be associated with more than one outcome, with the highest intersection between PSS-I and PHQ-9 (31 CpGs). Six other CpGs were associated both with treatment and any of the mental health measures  were significantly enriched for multiple networks and functional pathways, after statistical overrepresentation tests in STRING  and cg08739828 (MOCOS) were also not successfully replicated. Regarding the treatment vs. outcome association intersections (131 CpGs related to 62 genes), replication was successful for several loci. Eleven out of 64 (17%) CpGs that were associated with treatment in the discovery sample were successfully replicated, namely cg06125671 (ABTB2) and cg22356726 (KIF20B) with the strongest replications (> 1.5% percentage change), followed by cg12774902 (CHRNA10), cg03259703 (IGSF21), cg16662768 (IRF8), cg09102332 (ITGB8), cg15509069 (SDHAF3), cg04070142 (SLCO1A2), cg06665305 (SOWAHC), cg20401896 (WDPCP), cg26212904 (WSCD1) (> 0.5% percentage change). For details, see supplement For The present study investigated patterns of DNAm associated with trauma related mental problems and psychotherapeutic treatment in female former child soldiers from Eastern DR Congo. DNAm of different CpGs were associated with treatment but were not replicated in an independent sample. Associated genes found in discovery sample play a critical role in memory formation and fear learning/extinction and, in addition, have been associated with processes of the immune system. Moreover, 62 genes were related to both clinical/social outcomes and treatment; replication was successful in 17% of the tested CpGs. Enrichment analyses corroborated that NET might alter gene regulatory processes involved in memory formation  along with alterations of the glucocorticoid and aldosterone system amongst others. Interestingly, genes involved in GnRH signalling were associated with both social acknowledgement and treatment.vs. TAU on DNAm change, we found that treatment significantly predicted DNAm change from baseline to six month follow up in four CpGs . Activated leukocyte cell adhesion molecule (ALCAM) and actin filament associated protein 1 (AFAP1) were replicated, despite the limited power of the replication sample. Products of both genes have previously been associated with the formation of memory. In NET, restructuring of the memory and particularly the establishment of hypersalient/traumatic memories in ‘time and space’ of the autobiographic memory has been postulated as cardinal agent of change for the treatment of PTSD and appetitive aggression37. Substantial evidence corroborates the involvement of actin filaments in neural plasticity, specifically the generation, stabilisation and consolidation of long-term memory38. Actin dynamics interact with several of the identified enriched gene networks, namely glutaminergic signalling, focal adhesion, ECM-receptor interaction (see review by Rudy39) as well as the RAP1 signalling pathway40. ALCAM is a member of the immunoglobulin superfamily and important for the growth of midbrain dopamine neurons through trans-heterophilic interactions42. Dopamine neurons in the midbrain are crucial for reinforcement learning . Actin polymerization and midbrain dopamine neurons have both been found to modulate fear extinction in animal studies47. ALCAM was furthermore linked to maintaining the integrity of the blood–brain-barrier and the proliferation of T-cells49. In line with this, NET has previously been found to normalise regulatory T cells50, which have been reported to be reduced in individuals with PTSD51.Using a longitudinal parallel group design to draw causal effects of NET ABTB2 and KIF20B. The former encodes the ankyrin repeat and BTB/POZ domain-containing protein 2, which inhibits the aggregation of alpha-synuclein and exerts a protective effect on dopamine neurons usually killed in Parkinson disease52. The second encodes a kinesin-like protein relevant to cytokinesis during cell division54. Moreover, CHRNA10 encodes neuronal nicotinic acetylcholine receptor (nAChR) complex α9 and α10, and its inhibition has recently been suggested to prevent neuropathic pain55. Deletion of nAChR α9 also resulted in altered bone structure in mice56. Other genes have been related to the immune system , bioenergetics (SDHAF3) and integrins (ITGB8).Further, among the top 0.1% significant CpGs (n = 305), loci on 62 genes were associated both with the effect of treatment and clinical/social outcomes in the discovery sample and 11 (17%) were replicated in the independent sample (percentage change of 0.5% on the same CpG in the same direction). Percentage change in the replication sample was strongest in 57. Increasing evidence suggests that biological, social and environmental factors induce an accelerated epigenetic aging58. Unfortunately, we did not find conclusive results regarding the effect of treatment on epigenetic age, since our groups had differing baseline values and there were no differences in epigenetic age between timepoints. More research is also needed in that direction.Finally, epigenetic age is expected to be related to disease and mental healthcortisol synthesis and secretion have been found associated with treatment in our sample, indicating changes in HPA-regulation, i.e., alterations in the elite force of defence5. Additionally, multiple genes associated with treatment are involved in aldosterone syntheses and secretion. Aldosterone is a mineralocorticoid steroid hormone that is involved in the renin–angiotensin–aldosterone-system (RAAS), crucial for the homeostatic regulation of body fluids and electrolytes. It is also implicated in cardiovascular and metabolic diseases, by mediating immune cell activation59. Elevated aldosterone levels were previously found in patients with depression and PTSD62. Glutaminergic signalling is another pathway related to treatment in our sample. Most cortical synapses in the human brain are glutamatergic (80–90%). Alteration of glutaminergic signalling is essential in the development of trauma related psychiatric disorders, which motivated the investigation into a variety of therapeutics, but with inconsistent outputs63. Further pathways that currently emerged in association with the treatment have traditionally been related to social behaviour. For example, oxytocin signalling has been described to mediate complex social behaviours in animal and human studies64. Moreover, GnRH was related with both social acknowledgement and treatment. GnRH is synthesised and released by GnRH neurons in the hypothalamus, especially during puberty and adolescence.Gene ontology analysis provided further insights into biological mechanisms underlying the changes triggered by NET. Seven out of 63 genes involved in NPFFR2)66, neural functioning (SLC2A4 and KCNG2)68 immune system , chromatic remodelling (UBL4A)69, insulin metabolism (SLC2A4)70, and transcriptional fine tuning in the early post-natal period (DNMT3A)72, amongst others. The multiple DMPs  associated with social acknowledgement indicate a potential sensitivity of DNAm for processes that originate in the social environment.Our analysis revealed a mixture of overlapping epigenetic signatures associated with clinical and social outcomes Fig. . This re73, smoking74 or cancer75. Considering that our study detected small levels of DNAm change after treatment application, our findings should be interpreted with caution. Further studies should also test the hypothesis that therapy treats phenotypic conditions through alternative epigenetic routes rather than by reversing specific methylated positions on the genome.If we assume that complex traits, such as trauma related disorders are in part influenced by epigenetic markers, one might expect that treating mental disorders reverses epigenetic changes in specific loci responsible for the development of symptoms. Our data, however, does not support this hypothesis. It is noteworthy that almost none of the CpGs associated with trauma related disorders were also associated with treatment, despite various intersections at the gene level. This indicates that treatment does not promote a simple reversal of epigenetic alterations on specific CpG sites that correlate with trauma related disorder symptoms. Instead, an adaptation of regulatory routes that are pathological or render survivors socially dysfunctional might contribute to psychological and physiological changes in response to treatment. Additionally, it is still unclear whether and to which degree methylation changes could effectively produce meaningful biological impacts in the context of psychotherapy. Research investigating biomarkers for trauma related disorders are still incipient, compared to substance use76. Secondly, the lack of significant CpG sites associated with PTSD and appetitive aggression might be due to the low variance in these particular phenotypes, since participants were preselected based on presence of a PTSD diagnosis and apparent aggressive behaviour. Thirdly, we should acknowledge that individual measures of DNAm might have been affected by aspects outside of the control of our design, such as menstrual cycle, contact with chemical substances or diet. However, systematic bias should be reduced by the randomized study design. Also, although the estimation and processing of methylation data in our study was done meticulously, we did not confirm it using a validation technique such as pyrosequencing. Further, we did not correct for multiple testing at the level of psychological outcomes, which could reduce the number of significant associations. Also, our replication method supported the association of some DMPs, but the magnitude of change was relatively small, which may mean there was not an effective change in methylation status after 6–9 months of treatment application. As indicated above, methylation change might qualitatively distinguish treatment groups, but the degree of change has still an uncertain meaning. Lastly, we cannot rule out tissue-specificity and genetic population stratification in our study. Genetic restriction should not be mistakenly understood as genetic bias however. Since we applied a paired sample design, baseline versus follow-up, individuals were their own genetic controls.Although our study has several strengths, the results need to be interpreted in light of the following limitations. Firstly, we cannot be sure that the associations found in our study directly reflect gene expression and protein levels in participants, although a growing body of research suggests that DNAm is a reliable epigenetic marker for gene regulationIn conclusion, this study is the first to suggest that evidence based trauma therapy—specifically Narrative Exposure Therapy (NET)—might affect DNAm markers in multiple genes associated with psychiatric conditions such as PTSD, but also with trauma related social problems such as current violent behaviour and social acknowledgement. We found evidence that NET might effectively alter DNAm of loci close to genes of general and specific biological processes and pathways cardinal to trauma related problems and somatic complaints as well as social behaviour and reproduction.32) condition via SPSS by a person who was not involved in interviews or treatment. Four women did not offer saliva samples and were thus excluded from this analysis. The final sample included 84 women (nnet and ntau = 42) with saliva samples collected at baseline (BL) and 6-month follow-ups (FU6). Participants had a mean age of 18 (SD = 1.9) years at baseline. All women had a history of abduction and numerous lifetime traumatic events77. It is very uncommon to smoke tobacco within this micro-culture, and other drugs were not available or affordable for these women, therefore this information was not gathered during interviews. All methods were performed in accordance with the relevant guidelines and regulations.We applied a prospective randomised parallel group design in a sample of 88 female former child soldiers in the Eastern DR Congo. All participants fulfilled the DSM-5 diagnostic criteria for PTSD. Inclusion criteria were female sex, minimum age of 16 years and armed group involvement. Recruited through a local non-profit organisation, patients were invited to meet with psychological interviewers, where the study was outlined in detail and written informed consent was obtained. Simple randomisation was applied to eligible women for allocation to Narrative Exposure Therapy (NET) or treatment-as-usual , depression  and appetitive aggression . Social outcomes were current violent behaviour , feelings of guilt  and perceived recognition of the trauma and support in the women’s social environment . A more detailed description of the sample, study design, questionnaire measures and treatment is provided in Robjant et al.32.Structured psychodiagnostic interviews were conducted by trained local interviewers blind to treatment condition. Baseline assessment included (1) sociodemographic questions, (2) a 44-items checklist for traumatic events and perpetrated acts of violence, and (3) clinical and social outcomes. Clinical outcomes were PTSD . DNA was purified using QIAsymphony DSP DNA Midi Kit  and concentrations were determined using Invitrogen Quant-iT™ PicoGreen™ dsDNA Assay Kit . Bisulfite conversion using 500 ng of genomic DNA was performed using EZ DNA Methylation Gold Kit , and methylation profiling was conducted using the Infinium MethylationEPIC BeadChip array . There were no deviations from the Illumina protocol and all experiments were performed in compliance with GenomeScan Standard Operating Procedures (SOPs). Quality control was carried out using Illumina Technical Controls Plots and using the MethylAid R package85. Less than 16 months elapsed between saliva collection and biochemical analysis. Raw methylation data containing 865,859 probes was processed to remove probes of bad quality , non CpG or SNPs probes , probes at male sex chromosomes (n = 58) and cross-reactive probes . The remaining 698,540 probes were filtered to select differentially methylated positions (DMPs). In total, 305,868 CpGs were selected for statistical analyses. During processing of methylation data, we accounted for batch effects and generated estimates of epigenetic age86, sex , and cell counts87 , which efficiently preserve DNA material at room temperature and above even over long periods of timeDNAm ~ treatment + time + treatment × time + reported age + nr. traumatic events + cell counts, where response was mean Beta across all CpGs. Control variables were set to account for individual differences, whereas participant IDs were set as random effects. Epigenetic age was compared among groups using t-tests.To investigate general differences in overall DNAm between treatment groups, we conducted a generalized linear mixed model using the formula EWAS: an epigenome wide association study (EWAS) approach to find DNAm associations with outcomes and treatment by fitting robust generalized linear models using the formula Y ~ X + reported age + nr. of lifetime traumatic events + cell counts. We chose a robust model to account for potential violation of statistical assumptions. For outcomes, Y was the M-value  at baseline for each probe, and X was one of the outcomes. We fitted these model structures for each outcome separately to avoid multicollinearity. For treatment, Y was the Beta value difference between FU6 and BL, X was treatment condition, and cell counts were the mean cell count values between BL and FU6. Reported age, number of lifetime traumatic events (perpetrated and experienced) and cell counts were treated as covariates to control for individual differences and past experiences. P-values were adjusted for multiple testing after Benjamini-Hochberg88 and used to rank probes by significance (cut-off: FDR = 0.05) per outcome. QQ and MD plots were used to evaluate model fits. Statistical models were implemented in lme489 and limma90, using R 3.5.191 in RStudio92. [2] Probe annotation, CpG/gene intersections: first, metadata for probes were gathered using minfi93 and complemented with the updated Illumina EPIC array annotation release 03.13.201794. CpGs that did not correspond to a specific gene were manually investigated using the UCSC genome browser (GRCh37/hg19 assembly), and the closest protein-coding gene was selected. All gene symbols were double-checked against the most recent HUGO (Human Genome Organization) nomenclature. Second, to find biomarkers in our sample that were both associated with clinical/social outcomes and with treatment, we intersected CpGs and genes associated with those variables. For this, we selected the top 0.1% CpGs (305 probes) ordered by adjusted p-value for each variable. Contrary to a conservative FDR cut-off, this allowed us to inspect a more comparable number of sets per association. [3] Gene network analysis: to investigate gene function and important biological pathways, we conducted a gene-network and gene-ontology analysis using STRING95. [4] Replication: statistical analyses were replicated in a subsample of female former child soldiers abducted by the Lord’s Resistance Army in Northern Uganda of whom all received NET (N = 53). These participants had a mean age of 34 (SD = 9.1) years and a history of trauma and perpetration comparable to the discovery sample. Phenotype and saliva samples were collected at baseline, 4-month (FU4) and 10-month (FU10) follow-up interviews. Phenotypic data and DNAm processing are described elsewhere97. Baseline biomarker correlates were investigated for clinical outcomes only: PTSD , depression (HSCL99) and appetitive aggression (AAS). To also control for trauma load, we used a trauma checklist specific for the Ugandan context, where information about violence exposure and perpetration was present100. Linear models were calculated for those CpG sites found in associations within the discovery sample by using the formula and procedure mentioned above. Replication was considered successful for the effect of treatment when the direction of DNAm change was identical with a minimum percentage change of 0.5% in both discovery and replication samples. Average percentage change in CpG methylation across all CpG sites for the entire discovery sample was 0.26% (SD = 0.89).To investigate the effect of treatment on DNAm, we performed an analysis in four steps: [1] All participants have given written informed consent. Concerning the discovery sample, the study protocol was approved by the Ethical Commission of the University of Konstanz and the Social Funds of the DR Congo. Concerning the replication sample, the study protocol was approved by the Ethical Commission of the University of Konstanz, the Gulu University Research and Ethics Committee (Uganda), the Lacor Hospital Institutional Research Ethics Committee (Uganda), and the Ugandan National Council of Science and Technology.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.


In 2011, through a multipartner Integrated Family Health Initiative (IFHI), CARE started supporting maternal and neonatal health (MNH) improvement goals in 8 of 38 districts in Bihar, India. The programme included a frontline health worker (FHW) component offering health advice through household visits and benefited from CARE’s direct engagement during IFHI, which then evolved into statewide Technical Support Unit (TSU) to the Government of Bihar in 2014.Using eight rounds of state-representative household surveys with mothers of infants aged 0–2 months (N=73 093) linked with two facility assessments conducted during 2012–2017, we assessed changes in FHW visit coverage, intensity and quality between IFHI and TSU phases. Using logistic regression models, we ascertained associations between FHW outputs and three MNH core practices: ≥3 antenatal care check-ups (ANC3+), institutional delivery and early breastfeeding initiation.Women’s receipt of 1+ FHW visits declined from 60.2% (IFHI phase) to 46.3% (TSU phase) in the eight IFHI districts, being below 40% statewide during the TSU phase. Despite a parallel decline in FHW visit quality measured as the number of health advice received, all three outcomes improved during the TSU versus IFHI phase in IFHI districts. We found significant positive associations between all three outcomes and receipt of 1+ FHW visits and programme phase (TSU vs IFHI) in the eight IFHI districts. During the TSU phase, receipt of 2+ FHW visits in the third trimester increased the odds of women receiving ANC3+ (adjusted OR (aOR)=1.21; 95% CI: 1.13 to 1.31), delivering in a facility  and initiating breast feeding early . Independent of the number and timing of FHW visits, we also found positive associations between women reporting higher than lower quality of FHW interactions and receiving outcome-specific advice and all three MNH outcomes.Implementation of large community-based interventions under the technical support model should be continuously and strategically evaluated and adapted. Programmes with community-based components can accelerate improvements in maternal and neonatal health (MNH) in low-resource countries.In recent years, frontline health worker (FHW) programmes provided household visits and provision of health advice to pregnant and postpartum women and infants in Bihar with support from development partners, which included CARE.The initial, eight-district phase  that started in 2011 and benefited from CARE’s direct engagement evolved into a Technical Support Unit (TSU) phase in 2014.The coverage, intensity and quality of FHW services were greater during the IFHI than the TSU phase.During the TSU phase, receipt of 2+ FHW visits in the third trimester increased the odds of women receiving ≥3 antenatal care check-ups, delivering in a facility and initiating breast feeding early.Independent of the number and timing of FHW visits, we also found positive associations between women reporting higher than lower quality of FHW interactions and receiving outcome-specific advice and all three MNH outcomes.Given continuous improvements in MNH outcomes in Bihar, implementation of large community-based interventions under the TSU model should be continuously and strategically evaluated and adapted.Community-based interventions have long been considered important components of comprehensive approaches to accelerate improvements in reproductive, maternal, newborn and child health and nutrition (RMNCHN) outcomes in low/middle-income countries (LMICs). They promote appropriate care seeking and improved home practices mainly through home-based counselling or participatory women’s groups.et alet alet alet al,Walt With a population of over 1.3 billion people, India accounts for 12% of maternal deaths22CARE India is a non-profit organisation and a lead partner in a multiorganisational platform aiming to improve RMNCHN in Bihar.23During 2014, in close coordination with GoB, the programme evolved into a Technical Support Unit (TSU) and was rolled out in all 38 districts of Bihar covering over 104 million people. This phase was characterised by a considerable reduction in direct engagement in programme activities by CARE, yet deeper focus on capacity building, strengthening the supervisory system, synergising and focusing efforts on select interventions ,This analysis focuses on the FHW component of the programme in Bihar during 2011–2017. We aimed to: (1) assess and compare the coverage, intensity, and quality of FHWs’ interactions with pregnant women during household visits during IFHI (eight districts) and TSU (statewide) phases, and (2) ascertain and compare associations between FHW programme outputs and the uptake of three core maternal and neonatal health (MNH) programme practices: ≥3 ANC check-ups (ANC3+), institutional delivery and early (<1 hour of birth) breastfeeding initiation. Of note, early initiation of breast feeding was a key programme target in Bihar during both IFHI and TSU phases; ANC and institutional delivery became specific programme targets only in 2015 and 2016, respectively, after the programme started to benefit from a multidimensional quality improvement initiative implemented in all public health facilities in the state between 2014 and 2017.24Women and their families were not directly involved in setting the research questions or the outcome measures for the programme or this specific analysis of programme data. However, these were developed in consultation with and guidance from community leaders, and women and their families were intimately involved in the design and implementation of the programme in Bihar. Through community sensitisation meetings, they received information about the programme and its goals, which in turn helped motivate their participation in household surveys. We intend to disseminate the main results of this analysis to the community in Bihar through CARE India colleagues who continue to be engaged in the programme to serve the community.We used eight rounds of household surveys conducted by CARE with mothers of infants 0–2 months old in Bihar. Four survey rounds (P1–P4) were conducted about 6 months apart during the IFHI phase (2012–2013) in all 137 blocks in eight districts using lot quality assurance sampling methodology; the total P1–P4 sample was 10 408 mothers . Systema10.1136/bmjgh-2020-004389.supp1Supplementary dataSurvey questionnaires collected information on women’s sociodemographic characteristics, obstetric history, JSY programme participation, interactions with FHWs and the types of health advice received from them during and after pregnancy, receipt of ANC and delivery care as well as neonatal care practices. While some variation between survey questionnaires existed for P1–P4 vs S1–S4 surveys, for this analysis, we selected questions that were consistently asked across survey rounds to derive FHW and outcome indicators of interest.We generated FHW programme output variables: a binary (yes/no) indicator to identify women who received 1+ FHW visits during pregnancy; a four-category variable derived from three survey questions  ever come to your home to talk to you about your or your expected baby’s health?’, ‘Was it a health-related visit?’, and ‘How many visits did you receive from FHWs during the third trimester of pregnancy?’) and categorised as: received no FHW visits during pregnancy, received 1+ FHW visit(s) but not in the last trimester of pregnancy, received only 1 FHW visit during the last trimester of pregnancy, and received 2+ FHW visits during the last trimester of pregnancy; indicators capturing specific types of FHW advice ; and an index of the quality of interactions with FHWs derived from questions regarding advice received across five domains: birth preparedness for institutional delivery, birth preparedness for home delivery, recognition and care seeking for maternal complications, essential newborn care practices and postpartum family planning. This quality index accounts for the number of domains of advice received (0–5 range). For ease of interpretation, in the analysis, we used a binary FHW interaction quality variable categorised as ‘high quality’ if the index is ≥4 (90th percentile of index score) and ‘average or lower quality’ if the index is <4.We explored trends in several outcomes: ANC ; institutional delivery; presence of skilled birth attendant for home deliveries; skin-to-skin practice right after birth; early initiation of breast feeding, defined as breast feeding initiated <1 hour after birth and estimated only among women with a vaginal delivery; and newborn weighing. For the analysis of FHW visits’ effects, we selected three outcomes based on their relevance in the MNH field and the availability of strong evidence around community-based interventions’ success on improving these outcomesWe identified potential confounders of the associations of interest. Sociodemographic factors included maternal age, parity, religion, caste, mother’s and husband’s education and literacy, tertiles of a household wealth index generated as time-varying price-weighted sum of 25 household items (P1–P4) and 27 items (S1–S4) assessed in each survey round. Contextual factors included: JSY programme participation (yes/no) available in all survey rounds, and SHG membership (yes/no) only available in S1–S4. Facility-level factors in block-level facilities where the woman resided were obtained from two rounds of comprehensive facility assessments conducted in 2015 (linked with P1–P4 and S1 data) and 2016 (linked with S2–S4 data). They included the level of block-level facility (lowest level considered if more than one facility per block as most likely first source of care), and the ratio of clinical staff  filled to that approved in the block-level facility.For our first study objective, we first examined trends in FHW programme outputs by survey round and inclusion in the IFHI phase, and trends in outcomes by survey round and place of delivery  when applicable. Multivariable logistic regression models were fitted for each of the outcomes using only the data from the eight IFHI districts comparing the TSU with the IFHI phase. Models were adjusted for women’s receipt of 1+ FHW visits, an interaction term between programme phase and receipt of 1+ FHW visits, and the potential confounders listed above; the model fitted for early breastfeeding initiation was also adjusted for whether the woman delivered in a facility or not. Models were estimated using generalised estimating equations to account for clustering of facility-level covariate data. In regression analyses, we controlled for all potential confounders noted above.For the second study objective, we fitted a second set of four multivariable logistic regression models using only the TSU phase (S1–S4) data. Models were adjusted for the FHW household visit frequency, the quality of interactions with FHWs, the provision of outcome-specific advice as well as the potential confounders listed above. Compared with the first set of regression models, these models were adjusted for SHG membership and IFHI district status. Models were estimated using generalised estimating equations to account for clustering of facility-level covariate data. Using this second set of regression models, we estimated the predicted marginal probabilities of the outcomes under a variety of hypothetical scenarios to illustrate the relative impact of FHW visit frequency, quality of interactions and provision of outcome-specific advice compared with the impact of changes in other factors known to influence MNH outcomes: female literacy, SHG membership and delivery in a health facility.All analyses were conducted in Stata V.15 and accounted for the complex survey design using Taylor’s linearisation method. Data can be made available upon request and under a data use agreement with CARE, India.About three in four women in our samples were 20–29 years old and had two or three children . The majIn the eight IFHI districts, the coverage of pregnant women with 1+ FHW visits during pregnancy peaked at 65.0% in round P4 at the end of the IFHI phase but declined during the TSU phase to about 40% (p<0.001); this level of coverage was above that in the 30 non-IFHI districts, which was stagnant at <40% until the end of 2017 . CoveragDespite declines in FHW outputs, we found a significant increase in ANC3+ between the time of the S1 survey and the period captured by S2–S4 surveys, more so among the non-IFHI than IFHI districts p<0.001; . InstituWe compared FHW programme outputs, MNH outcomes, and contextual measures between the IFHI and the TSU phase in the eight IFHI districts, and between the 8 IFHI vs 30 non-IFHI districts during the TSU phase. While, on average, 53.4% of pregnant women in the eight IFHI districts received 2+ FHW visits during the third trimester of pregnancy during the IFHI phase, the proportion declined to 31.2% during the TSU phase (p<0.001); only 26.6% of pregnant women received such visits in the 30 non-IFHI districts during the TSU phase . Of womeAfter adjustment for potential confounders, we found significant positive associations between the three MNH outcomes  and receipt of 1+ FHW visits and programme phase (TSU vs IFHI) in IFHI districts . DeliverDuring the TSU phase, compared with women in the 30 non-IFHI districts, those in the eight IFHI districts had higher odds of delivering in a health facility , yet lower odds of having ANC3+ check-ups  to 6% (S3 data)—the trend in institutional deliveries remained flat during this time as the programme in Bihar also prioritised improving quality of care in public facilities rather than reaching a higher institutional delivery target . Followi34 38 41 42Visits by FHWs have plausibly driven the increasing proportion of women initiating breast feeding early—they appear to, both directly and indirectly through institutional delivery, influence early breastfeeding practices. Only 18.0% (statewide TSU phase average) of pregnant women received advice from FHWs to start breast feeding early, a level that is comparable with that reported in the evaluation of a similar programme in Uttar Pradesh.This study has several limitations. We rely on cross-sectional household survey data and cannot infer causality in observed associations. The sample frame for these surveys is limited to areas served by AWCs, thus, possibly, excluding some limited areas in the state. Women’s self-reported information in surveys is subject to social desirability and recall bias—the former would bias our associations upwards by causing women with more exposure to FHW advice to over-report positive behaviours; the latter would only affect our findings if differential, making associations of interest appear stronger. As the case with most studies, there is risk for residual confounding. Programme components that may have played a role in the observed associations, such as mass-media education campaigns, interactions with FHWs and other health providers during immunisation campaigns and VHSND, were not measured in household surveys in Bihar. Of note, our ‘falsification test’ worked well and gives confidence in our results—the only significant association between our control outcome  and FHW exposure relates to the quality of interactions with FHWs, which can be explained by women being receptive to various channels of information. Importantly, given the tautological nature of the relationship between JSY programme participation and institutional deliveries in India, it is difficult to assess the true contribution of the FHW programme to changes in institutional deliveries during the period of study. A sensitivity analysis that adjusted our facility delivery models for JSY participation showed a significant effect of this programme on women delivering in a facility .Implementation of FHW interventions should be continuously and strategically evaluated and adapted in Bihar. Attention should be given to addressing potential barriers to obtaining sustainable impact at scale, including weak leadership and supervision, limited implementation management capacity and variable engagement, competition between programme components, variation in implementation fidelity and FHW motivation. The relative costs and benefits of supporting FHW programmes alone or in tandem with other strategies to improve MNH outcomes should be considered. Adoption of a stepwise approach to FHW programme scale-up and integration into the health system and a thorough preparedness prior to transitioning to a technical support model for implementing and maintaining programmes at scale appear to be key to achieving positive and sustainable results.10 44


Mycobacterium tuberculosis (MTB) would be advantageous in reducing the amplification of additional drug resistance and disease transmission.With the advancement of next generation sequencing technologies (NGS), whole-genome sequencing (WGS) has been deployed to a wide range of clinical scenarios. Rapid and accurate classification of drug-resistant n = 10) and INH resistant clones (n = 42) isolated from clinical specimens. Non-synonymous variants within the katG or inhA gene associated with INH resistance was identified using Nanopore sequencing coupled with a corresponding analytical workflow.In this study, a long-read sequencing approach was subjected to the whole-genome sequencing of clinical MTB clones with susceptibility test profiles, including isoniazid (INH) susceptible clones  or Thr579Asn (AUC = 0.875).In total, 54 nucleotide variants within the Taken together, we curated lists of coding variants associated with differential INH resistance using Nanopore sequencing, which may constitute an emerging platform for rapid and accurate identification of drug-resistant MTB clones. Mycobacterium tuberculosis (MTB) is still considered a health threat with about 10 million incident cases of tuberculosis (TB) and 1 million deaths every year [katG gene converts INH to its active form, which in turn acts by blocking the biosynthesis of mycolic acid which is required for cell wall synthesis [Infection with ynthesis . Active ynthesis . INH resynthesis .Drug resistance in MTB is routinely evaluated using phenotypic methods, such as susceptibility tests or genotypic approaches ; howeverWe conducted WGS to identify non-synonymous variants within the katG and inhA genes in three groups of MTB clones, including (1) susceptible isolates, (2) low INH-resistant isolates, and (3) high INH-resistant isolates.Enrollment of clinical isolates was reviewed and approved by the Institutional Review Board of Taipei Medical University . In total, 50 MTB isolates were collected from clinical specimens at Taipei Municipal Wan Fang Hospital.−2 and 10−4 dilutions of the standard inoculum was spread on 7H10 agar with or without an anti-TB drugs, including Isoniazid (INH), Rifampin (RIF), Ethambutol (EM), Streptomycin (S) and concentrations in microgram/milliliter. Drug resistance was defined as more than 1% colony growth in the presence of the drug compared to colony growth in the absence of the drug.All isolates in this study were examined at the Department of Laboratory Medicine, Taipei Municipal Wan Fang Hospital using an agar proportion assay in accordance to the Clinical and Laboratory Standards Institute (CLSI) methods. In brief, bacterial suspensions with a turbidity equivalent to 1.0 McFarland standard were prepared from fresh MTB colonies cultured on Lowenstein-Jensen medium. After examination of the turbidity using a nephelometer, the suspension was applied as the inoculum for all dilutions. One hundred microliters of 10MTB isolates were cultured on Lowenstein-Jensen media in the absence of an anti-MTB drug. Multiple colonies from a single isolate were emulsified and inactivated in 200 μL nuclease-free water at 95 °C for 15 min. Genomic (g)DNA was extracted using the Presto™ Mini gDNA Bacteria Kit  according to the manufacturer’s instructions. The DNA concentration was measured using a fluorometric kit  and a Qubit fluorometer .https://epi2me.nanoporetech.com). The coverage rate of sequenced reads to the MTB reference was estimated by applying FastQ custom alignment (ONT). Analytical results of variant classification were aligned to the MTB reference  using Bacterial Small Variant Calling workflow composed of the Medaka variant calling pipeline with long-read datasets via the EPI2ME Labs Launcher (ONT). The sequencing quality, reading depth, and variant calling with long-read datasets were repeatedly assessed using the CLC genomics workbench .WGS of MTB gDNA was conducted using a third-generation long read-sequencing approach. In brief, 500 ng of extracted gDNA was first homogenized with 8000 bps using a g-TUBE device  according to the manufacturer’s instructions. The fractionated gDNA was subjected to library construction using a Ligation Sequencing Kit , Oxford, UK) coupled with a Native Barcoding Expansion kit  according the manufacturer’s protocol. The barcoded library was captured, washed, and eluted from magnetic beads . Seven hundred nanograms of the pooled library was loaded and sequenced on MinION flow cells . The sequence read number of each sample was 160,000 ~ 200,000 to meet a reading depth of 200. For analysis of sequencing results, the MinION-sequenced reads were uploaded through the EPI2ME desktop agent (ONT), and the quality and quantity of sequencing results were assessed using the EPI2ME website algorithm  according to the manufacturer’s instructions. In brief, an MTB isolate was subcultured on 7H10 agar . Fresh colonies were resuspended with glass beads in a saline-Tween solution, and the turbidity was adjusted to a McFarland standard of 0.5. After 15 min, 100 µl of the resuspension was poured and mixed with 11 ml 7H9 broth containing oleic acid albumin-dextrose-catalase . Diluent (100 µl) was inoculated into each well of the MYCOTB plate that was covered with permanent plastic seals and incubated at 37 °C in 5% COp values of < 0.05 . The utility of identified variants for predicting INH resistance was evaluated using the receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) ratio as implemented in R programming.Experimental results were statistically analyzed using a one- or two-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison post-hoc test. Analytical results are presented as the mean ± standard error of the mean (SEM) and considered significant at Drug-resistance patterns determined by the agar proportion method are displayed in Table The HMW gDNA extracted from the clinical isolates was subjected to sequencing using a long-read sequencer . Numbers with an average of sequenced and aligned reads per sample were filtered and analyzed using the CLC Genomics Workbench  and EPI2ME desktop agent algorithm (ONT). As shown in Table katG gene, while inhA mutations were identified in a small number of INH resistant strains [katG or inhA gene  found in all isolates enrolled in this study, five non-synonymous variants within the ly Table , lower. ly Table , lower [inhA gene in susceptible isolates  according to the results of DST assay. The minimal inhibitory concentration (MIC) test was subsequently conducted to evaluate the impact of individual genotyping variant on the resistance signature of recruited MTB isolates toward INH. MIC results showed significant INH-resistance (2 μg/mL) with sole presence of the Ser315Thr variant within the katG gene (Table Correlation of the Ser315Thr variant within the orldwide . In totaOverall agreement between the phenotypic and genotypic approaches for INH-resistance of MTB isolates was estimated in this study. Results of WGS and MIC tests showed better agreement (Fig. katG or inhA gene were identified using the ONT platform in this study. The putative impacts of these variants on the INH-resistance signature should be further demonstrated.The turnaround time of DSTs for MTB is a major challenge relevant to the efficacy of precise treatment . PhenotySer315Thr has been widely demonstrated to be a high-confidence and frequent polymorphism that confers an INH-resistance signature of MTB , which wAgreement between the results of the phenotypic test and genotyping analysis is considered a critical issue when evaluating the utility of WGS results on determining the drug-resistance signature of MTB infection . PhenotyIn our study, the long read-sequencing platform was subjected to SNP calling within the entire MTB genome. In addition to the high-confidence and frequent Ser315Thr mutations, the emerging Thr579Asn variant relevant to high INH-resistance was characterized using MIC and WGS analyses. Moreover, Cohen's kappa value indicated better agreement between the MIC and WGS analyses of differentiating high INH-resistance of clinical MTB. These results suggested the practicable adoption of the ONT sequencing approach as an alternative to the current diagnostic method for DSTs of MTB.


The soft tissue reconstruction around proximal or total humeral megaprostheses following limb-sparing resection of a primary malignant bone tumor can be a challenge. A surgical technique to overcome these challenges is the Bateman-type soft tissue reconstruction, which is performed as a lateral acromion and trapezoid transfer aiming to improve soft tissue coverage and postoperative function. However, the functional outcome and the endoprosthetic complications of this procedure have hardly been evaluated. Our study shows that the Bateman-type reconstruction is a feasible treatment option, but the postoperative functional outcome is overall limited although good to excellent functional results can be achieved in individual patients. The risk for revision surgery is high within the first year, but remains low thereafter.We aimed to evaluate the functional outcome and endoprosthetic complications following the Bateman-type soft tissue reconstruction around proximal or total humeral replacements in patients undergoing resection of a primary malignant bone tumor. Between September 2001 and December 2018, a total of 102 patients underwent resection of a primary malignant bone tumor and subsequent reconstruction with a modular humeral megaprosthesis in our department. Fifteen (15%) of these patients underwent a Bateman-type soft tissue reconstruction and were included in this retrospective study. The median Musculoskeletal Tumor Society (MSTS) score was 21, the median Toronto Extremity Salvage Score (TESS) was 70, and the median American Shoulder and Elbow Surgeons (ASES) score was 72. Fifty-three percent (8/15) of all patients required a revision surgery after a median time of 6 months. There were 2 soft tissue failures, 3 infections and 3 tumor recurrences. The revision-free implant survivorship amounted to 53%  28–81) after 1 year and 47% (95% CI 22–73) at last follow-up. The Bateman-type reconstruction is a feasible option for soft tissue reconstruction but functional outcome is overall limited and the risk for revision surgery within the first postoperative year is high. The humerus is a common site for primary malignant bone tumors . While tSurgeons have attempted several surgical techniques using muscle transfers and en-bloc flaps to address these challenges ,15,16,17However, despite the need for improved techniques in soft tissue reconstruction around proximal and total humeral megaprostheses, this procedure has only been investigated by a single case report .Thus, the aim of our study was to evaluate the functional outcome and endoprosthetic complications of a Bateman-type reconstruction performed as a lateral acromion and trapezoid transfer in patients with primary malignant bone tumor who underwent resection and megaprosthetic proximal or total humerus replacement.Between September 2001 and December 2018, a total of 580 patients underwent resection of a primary malignant tumor of the long bones and subsequent reconstruction with a modular megaprosthesis in our department. In 102 of these patients (18%), location of the tumor was the proximal humerus. 15 of these patients (15%) underwent the Bateman-type reconstruction and were included in this retrospective study , Table 2Patients’ demographics, tumour characteristics, surgical and oncological treatment as well as endoprosthetic failure and its subsequent treatment were retrospectively retrieved from patients’ electronic medical records. Functional outcome was determined in surviving patients at last follow-up and included evaluation of the shoulder range of motion (ROM) and determination of 3 standardized scoring systems: the Musculoskeletal Tumor Society (MSTS) score, the Toronto Extremity Salvage Score (TESS) and the American Shoulder and Elbow Surgeons Score (ASES) ,21,22.Fifteen patients  with a median BMI of 25 (Interquartile range (IQR) 22–27) and a median age at the time of surgery of 19 (IQR 15–34) years were available for analysis after a median follow-up of 49 (IQR 25–100) months. Patients who died of their disease or underwent revision surgery were included irrespective of the follow-up period; for all other patients the minimum follow-up was 24 months. Seven patients died of their disease after a median time of 25 (IQR 20–49) months. In the 8 surviving patients, the median follow-up amounted to 80 (IQR 47–113) months. Functional outcome data were available for 6 of these patients as 1 patient lived overseas and 1 patient was not contactable. The overall patient survival probability was 80% (95% CI 60–100) after 2 years and 50% (95% CI 24–76) after 5 years. Demographic, oncological and surgical details are presented in ®  . ThLimb-survivorship following excision of bone tumors and subsequent megaprosthetic reconstruction is overall good to excellent as Jeys et al. reported a long-term amputation rate of only 8.9% (112 of 1261 patients) in these patients with local recurrence  and infection  being the “single biggest risk factor” for amputation ,31. SimiWe acknowledge several limitations of our study. (1) The study follows a retrospective design and the reported endoprosthetic complications must be considered as low-end estimates as patients could have been treated elsewhere. (2) The number of patients in this study is small. While this can be attributed to the rarity of this approach, we are able—to the best of our knowledge—to report the largest cohort of patients undergoing a Bateman-type procedure in musculoskeletal oncology treated with a single design modular implant at a single tertiary university hospital. (3) Functional results were only available in 6 of 8 surviving patients as 1 patient lived overseas and 1 patient was not contactable. However, we repeatedly tried to contact the remaining patient but were unable to schedule a follow-up examination to determine shoulder ROM and determine the functional outcome scores. (4) Furthermore, there was no control group of patients that were treated with a different muscle transfer of free flaps for this indication.The Bateman-type reconstruction is a feasible option for soft tissue reconstruction around megaprosthetic proximal or total humeral replacements in patients undergoing limb-sparing resection of a primary malignant bone tumor of the humerus. While the functional outcome is overall limited, good to excellent functional results can be achieved in individual patients. The risk for revision surgery is high within the first postoperative year but remains low thereafter. Limb-survivorship after the first and fifth postoperative year is overall acceptable.