Common variable immunodeficiency (CVID) is the most common symptomatic primary immunodeficiency (PID). CVID is a heterogenous condition and clinical manifestations may vary from increased susceptibility to infections to autoimmune manifestations, granulomatous disease, polyclonal lymphoproliferation, and increased risk of malignancy. Autoimmune manifestations may, at times, be the first and only clinical presentation of CVID, resulting in diagnostic dilemma for the treating physician.Autoimmune cytopenias (autoimmune haemolytic anaemia and/or thrombocytopenia) are the most common autoimmune complications seen in patients with CVID. Laboratory investigations such as antinuclear antibodies, direct Coomb’s test and anti-platelet antibodies may not be useful in patients with CVID because of lack of specific antibody response. Moreover, presence of autoimmune cytopenias may pose a significant therapeutic challenge as use of immunosuppressive agents can be contentious in these circumstances. It has been suggested that serum immunoglobulins must be checked in all patients presenting with autoimmune cytopenia such as immune thrombocytopenia or autoimmune haemolytic anaemia.nuclear factor kappa B subunit 1 (NF-kB1), Lipopolysaccharide (LPS)-responsive beige-like anchor protein (LRBA), cytotoxic T lymphocyte antigen 4 (CTLA4), Phosphoinositide 3-kinase (PI3K), inducible T-cell costimulatory (ICOS), IKAROS and interferon regulatory factor-2 binding protein 2 (IRF2BP2).It has been observed that patients with CVID and autoimmune cytopenias have a different clinical and immunological profile as compared to patients with CVID who do not have an autoimmune footprint. Monogenic defects have been identified in 10-50% of all patients with CVID depending upon the population studied. Monogenic defects are more likely to be identified in patients with CVID with autoimmune complications. Common genetic defects that may lead to CVID with an autoimmune phenotype include In this review, we update on recent advances in pathophysiology and management of CVID with autoimmune cytopenias. Common variable immunodeficiency (CVID) is the most common symptomatic primary immunodeficiency –3. CVID Of the various autoimmune complications seen in patients with CVID, autoimmune cytopenias  are the most common . LaboratIt has been observed that patients with CVID and autoimmune cytopenias have a different clinical and immunological profile as compared to patients with CVID who do not have an autoimmune footprint . MonogenThis review will elaborate on recent developments in pathophysiology and management of CVID in the context of autoimmune cytopenia.Of the various autoimmune complications in CVID, cytopenia has been reported to be the most common complication . Recent Of the various autoimmune cytopenia in CVID, ITP is the most frequently reported manifestation by several authors , 24–26 IProportion of patients with CVID who develop ITP has been reported to vary from 7.4 to 19% Table 1.At times, ITP may be the first and only symptom of CVID. A French study in 2004 included 21 patients with ITP who were also diagnosed to have CVID . Of thesn = 35): 9 had AIHA, and 11 had Evans syndrome  were observed more commonly in patients with neutropenia . Another colitis .Patients with CVID and AIN have been reported to have poor prognosis. Ghorbani et al. reported a higher frequency of deaths (61.5%) in their cohort of patients with CVID who had AIN . AnotherPernicious anaemia has been reported to be the least common amongst the various autoimmune cytopenia associated with CVID Table 1.12, (4) gastric body mucosal atrophy, and (5) auto-antibodies to intrinsic factor and/or to gastric parietal cells” J recombination. Autoreactive B cells are generally silenced by 3 main mechanisms: deletion, receptor editing, and anergy. Receptor editing and deletion results in central tolerance that omits autoreactive immature B cells. However, a small percentage of autoreactive B cells escape the bone marrow and remain in periphery where anergy renders them unresponsive to antigenic stimuli , CVID and rheumatoid arthritis (RA)  in the serum of patients with CVID lead to inhibition of removal of anergic CD21lo B cells from periphery (lo B cells with low avidity to bind to self-antigens pose a major risk of development of autoimmunity. Murine studies have shown that inactive CD21lo B may overcome the state of anergy during infection where cross-reactive antigenic epitopes present on infectious agents stimulate anergic B cells via innate immune ligands (CD21tis (RA) . Factorseriphery . Presenc ligands .lo B cells in patients with CVID and RA. CD21lo B cells were found to be elevated in these patients. It was further observed that these CD21-/lo B cells are closer to the naïve B cell population  and express germline B-cell receptor (BCR) repertoire that is rich in autoreactive clones. Immunofluorescence assay showed that CD21lo B cells expressed antinuclear antibodies (speckled nuclear and nucleolar pattern) and also expressed autoantibodies against several cytoplasmic structures. CD21lo B cells do not get activated and do no proliferate through BCR and CD40 co-stimulation and showed impaired calcium-mediated signalling. This results in inactivation of a few activation markers on B cells upon BCR triggering. Impaired activation has been linked to impaired proliferation of B cells to antigenic stimuli suggesting their unresponsive stage. The transcriptome analysis of CD21lo B cells revealed up-regulation of several genes implicated in the inhibition of B-cell activation, proliferation, and survival and the downregulation of B-cell activating genes, suggesting an inhibitory gene signature. These results were further confirmed using flow cytometry assays that suggested downregulation of receptors that favour B cell survival and upregulation of receptors that favour B cell inhibition. In addition, the survival potential of CD21lo B cells was compared with that of CD21+ B cells and it was found that CD21lo B cells were prone to die by apoptosis suggesting they have a shorter half-life   .lo B cells and autoimmune cytopenia was reported. Percentage increase in CD21lo B cells was comparable in NI and AI group while it was significantly high in the cy group. This suggests that higher proportion of CD21lo B cells in patients with CVID correlate specifically with an increased risk of autoimmune cytopenia  into non infection (NI), autoimmunity (AI) and autoimmune cytopenia (cy) group. Absolute numbers of B and T cells were low but comparable among the 3 groups. However, a significant association between CD21ytopenia .Role of pro B10 cells  has been reported in a number of immune mediated disorders, such as RA and ITP. Olsen et al. reported that pro B10 cells were increased in patients with CVID who developed autoimmunity and splenomegaly whereas patients with CVID without autoimmunity displayed only a modest increase in these cells . The undRomberg et al. compared the germinal centre (GC) responses of patients with CVID with autoimmune cytopenia (CVID+AIC) and CVID patients with autoimmunity other than cytopenia (CVID-AIC). Irregularly shaped and hyperplastic germinal centres along with increased number of circulating T follicular helper cells were observed in CVID+AIC group while GC structure in CVID-AIC group were found to be small and circular. CVID+AIC cohort had higher CD19hiCD21-/lo B cells compared to CVID-AIC cohort, CD27+IgG+ memory B-cell population and IgA+ B cells were reduced in CVID+AIC group .The study also evaluated somatic hypermutation (SHM) in CD27+IgG+ memory B-cells. Patients with CVID were found to have lower SHM frequencies in heavy chain variable regions (VH) than controls. CVID+AIC patients showed least SHM (7.5 mutations per VH segment) as compared to 15.1 in CVID-AIC group and 18.6 in heathy controls. VH4-34 gene segment was identified in 9.9% of CVID+AIC IgG transcripts while this segment was rarely seen in CVID-AIC group and healthy controls. VH4-34-encoded antibodies have been found to be autoreactive as they bind the conserved I/i carbohydrate self-antigens expressed in red blood cells and other hematopoietic cell lineages .Yu et al. have also reported that patients with autoimmunity with CVID had significantly reduced switched memory B cells .Role of T cell compartment in the development of autoimmune cytopenia in patients with CVID has also been reported by several authors. Disturbed T cell homeostasis underlies the pathogenesis in one third of CVID patients with autoimmune manifestations. These include alterations in number of CD4, CD8 T cells, memory T cells, regulatory T cells, and altered expression of transcripts essential for regulatory T cells functioning.+ T cells in CVID associated autoimmune cytopenia. This reduction was accompanied by activated phenotype with an increased expression of HLA-DR and CD95 markers on CD4+ T cells. Patients with other autoimmune manifestations did not show this T and B cell phenotype  and CD4+ T (41.7%) cells. There was inversion of CD4/CD8 ratio (70.8%). Patients with decreased circulating B and CD4+ T cells were significantly more likely to have auto-immune cytopenias and lymphoproliferative disease .It has also been reported that patients with CVID with autoimmune manifestations have significantly reduced proportion of CD8+ T cells .Herrera et al. compared the absolute numbers of T, B and NK cells among patients with CVID. Lymphocyte profiles were compared between patients with CVID with autoimmunity and patients with CVID without autoimmunity and healthy controls. CD4+ T cell numbers in patients with CVID without AI were significantly lower compared with the control group. Patients with CVID with AI had increased CD4+CD45RO+ memory T cell populations compared with healthy controls ..51).Olsen et al. reported altered proportions of regulatory lymphocytes in patients with CVID. Patients with autoimmunity had reduced levels of resting Tregs and activated Tregs predominantly seen in patients with autoimmunity and splenomegaly. The impaired functioning of activated Tregs was further indicated by reduced expression of cytotoxic T lymphocyte antigen 4 (CTLA-4) on its surface .+CD25HIGHFOXP3+ Tregs in patients with CVID with autoimmunity as compared to patients with CVID without autoimmunity. Forkhead box P3 protein (FOXP3) mRNA (messenger ribonucleic acid) expression was also found to be reduced in patients with CVID compared to healthy controls and the reduction was more pronounced in patients who had autoimmune cytopenia . The key proteins involved in functioning of Treg, including FoxP3, Granzyme A, XCL1 (lymphotactin), pSTAT5 , and GITR (glucocorticoid induced tumor necrosis factor receptor related protein) were found to be significantly reduced in patients with CVID with autoimmunity. Results suggest that these proteins may be involved in Treg-mediated autoimmunity in patients with CVID .Although defects in innate immunity have been reported in patients CVID, their correlation with autoimmunity has not been investigated in detail. Taraldsrud et al. and Sharifi et al. studied the role of Toll-like receptors (TLR) in the pathogenesis of CVID , 55. It Rezaei et al. measured various cytokines, especially type I interferons (IFN), in patients with CVID. It has been postulated that increased IFN-α/β may result in dysregulation of peripheral tolerance by activating immature dendritic cells . They maAutoimmune cytopenia may also be seen in several other disorders such as inborn errors of immunity , acquired causes such as . The pathophysiology of autoimmune cytopenia in several of these disorders especially those associated with inborn errors of immunity is more complex and similar to the mechanisms associated with CVID. On the other hand, the pathophysiology of autoimmune cytopenia in acquired disorders such as SLE or drug induced cytopenia is primarily associated with generation of auto-antibodies  , 59.Autoimmune cytopenia in patients with autoimmune lymphoproliferative syndrome is primarily due to defective apoptosis of lymphocytes mediated through the Fas/Fas ligand pathway . On the Monogenic defects have been identified in a small proportion of patients with CVID. These monogenic defects may have an important pathophysiological link with autoimmune cytopenia.TNFESF13B gene encodes for TACI , a member of tumour necrosis factor receptor superfamily expressed on B cells. TACI has been found to play important role in the B cell development. Monoallelic heterozygous and biallelic (compound heterozygous and homozygous) defects in the gene encoding for TACI have been reported to cause CVID. However, there are speculations that TACI defects are diseases modifying rather than disease causing because several healthy individuals have been reported to have same defect but do not develop any clinical manifestations. A recent study from Greece reported that monoallelic defects in TACI may act as susceptibility or disease modifying factor in the pathogenesis of CVID. It was, however, observed that patients with CVID with TACI defects had significantly higher risk of autoimmune cytopenia as compared to patients with CVID without any TACI defect (I defect . In addiI defect . It is iI defect .Tumor necrosis factor receptor superfamily member 13C (TNFRSF13C) encodes for BAFF-R (B-cell activating factor receptor) that functions as a pro-survival factor for B cells. Variants in BAFF-R lead to arrest of developing B cells at immature/transitional B cells stage. Similar to the TACI defect, the variants in BAFF-R may possibly be disease modifying rather than disease causing. A few patients with BAFF-R deficiency have been reported to develop autoimmune manifestations . The exaInducible T cell costimulator (ICOS), a member of CD28/CTLA-4 family, plays important role in regulating T cell responses. ICOS deficiency was the first identified genetic defect in patients with CVID. ICOS ligand is expressed in monocytes, dendritic cells and B cells. In addition to hypogammaglobulinemia and recurrent infections, these patients have also been reported to develop autoimmune manifestations especially autoimmune neutropenia , 73 74)74). The LRBA gene, is a critical protein involved in the expression and intracellular trafficking of CTLA4 protein. Costimulatory signal between T cells and antigen presenting cells (APCs) using CD28 (on T cells) and CD80/86 on APCs is crucial in the activation of T cells. CTLA-4 has higher affinity for CD80/86 and outcompetes CD28 in binding to CD80/86, CTLA-4, therefore, constitute an important immune check-point by preventing overactivation of T cells. CTLA-4 is an important mechanism by which Tregs exert their inhibitory effect on activated T cells. Patients with homozygous or compound heterozygous variants in LRBA gene and heterozygous variants in CTLA-4 gene fail to express CTLA-4 protein on surface and have been reported to develop CVID phenotype with autoimmunity, lymphoproliferation and inflammation (Lipopolysaccharide-responsive and beige-like anchor (LRBA) protein encoded by the ammation . LRBA deammation .rd of all cases)  , 73. LRBrd of all patients with Evans syndrome (autoimmune haemolytic anaemia and thrombocytopenia) especially defects in LRBA and CTLA-4 gene. Other genetic defects reported in patients with Evans syndrome include heterozygous loss of function mutation in TNFRSF6 gene, CBL gene and ADAR1 gene; heterozygous gain of function mutations in STAT3 gene and PIK3CD gene; and compound heterozygous mutations in RAG1 gene. In addition, somatic mutations in TNFRSF6 and KRAS genes and possibly pathogenic variants in several other genes were also reported. Patients with Evans syndrome who had a monogenic defect were more likely to have hypogammaglobulinemia and lymphoproliferation as compared to the patients with Evans syndrome who had no monogenic defects  of PI3Kδ [APDS 1] or loss of function mutations in PIK3R1 gene that encodes for regulatory subunit (p85α) of PI3Kδ [APDS2] (Patients with activated Phosphoinositide 3-kinase (PI3) δ syndrome (APDS) present with a CVID or hyper IgM phenotype with predominant clinical manifestation of autoimmunity  and lymphoproliferation , 81. APD [APDS2] . The end [APDS2] .NFKB2 lead to a CVID phenotype along with a distinct pattern of autoimmune manifestations. Unlike most patients with CVID wherein autoimmune cytopenia is the most common autoimmune manifestations, this is not the most common autoimmune manifestation in patients with NFKB2 gene mutation . As a result of haploinsufficiency of NFKB2, there is loss of central tolerance mechanism leading to accumulation of auto-reactive T cells.Heterozygous pathogenic variants in the mutation . AutoimmIKZF1 gene. The somatic mutation in IKZF1 gene predispose to development of malignancy while more recently patients with germline mutations have been reported to develop immunodeficiency that commonly presents as CVID. Patients with CVID with germ line mutations in IKZF1 also develop autoimmune cytopenia. IKAROS as a transcription factor controls development of autoimmunity by promoting the B cell anergy and by regulating the TLR pathway signalling , monogenic defects may possibly be identified more commonly in this subset of patients with CVID. In 2 studies that have reported monogenic defects in children with Evans syndrome (autoimmune haemolytic anaemia and thrombocytopenia), more than 2/3d CTLA-4 , 78.TNFRSF13; 1 LRBA; 1 NF-κB2; and 1 caspase recruitment domain 11 (CARD11)}. Authors concluded that patients who had recurrent and/or refractory autoimmune cytopenia; propensity to develop recurrent infections and family history of autoimmunity/immunodeficiency need evaluation for underlying monogenic defects  to identify pathogenic variants in their cohort of children with refractory ITP and it was observed that 9.1% children had pathogenic variants related to CVID {5  defects .As alluded to previously, autoimmune cytopenia may be the first and only symptom of CVID. This may lead to a diagnostic conundrum as patients with CVID may not produce an adequate autoantibody response and some of the diagnostic laboratory investigations such as direct Coombs’ test, anti-platelet antibodies or anti-neutrophil antibodies may give normal results. Thus, it might be prudent to check serum immunoglobulin levels in all patients with unexplained cytopenia .Glucocorticoids have remained the standard of care in autoimmune cytopenia in CVID . A revieIn a retrospective multicentre study on 33 patients with CVID-associated refractory immune cytopenias, rituximab showed an initial response rate of 80% and a sustained response rate of 50% at a mean follow-up of 39 months .There are conflicting reports on role of splenectomy in the management of cytopenia in CVID. Initial reports showed an increased rate of mortality following splenectomy . HoweverCurrent guidelines on chronic ITP and aplastic anaemia recommend the usage of thrombopoietin-receptor agonists (TPO-A) as a second or third-line agent in refractory cases . In CVIDMonogenic forms of CVID are often associated with autoimmune cytopenia . AlthougThe mTOR pathway has been reported to be activated inn patients with APDS and mTOR inhibitor, sirolimus has been recommended for management of cytopenia and lymphoproliferation . Howeverth of cases where it was carried out. It was also suggested that immunoglobulin replacement therapy does not prevent or ameliorate disease related complications in patients with CTLA-4 haploinsufficiency. Hematopoietic stem cell transplantation (HSCT) is an effective option for refractory cytopenia. Autoimmune cytopenia in patients with LRBA deficiency have also been managed on similar lines as in patients with CTLA-4 haploinsufficiency. However, rituximab has been used less commonly while abatacept has been used more commonly in the former group  are more likely to have an underlying monogenic defect. The treatment of choice for autoimmune cytopenia in CVID remains corticosteroids. However, biologic drugs and several targeted treatments are now being explored.SC- Preparation of the first draft of the manuscript; editing of manuscript; literature review. PB- Preparation of the first draft of the manuscript; editing of manuscript; literature review. RT-Preparation of the first draft of the manuscript; editing of manuscript; literature review. AJ- Preparation of the first draft of the manuscript; editing of manuscript; literature review; critical review and final approval. SSh/AR- Editing of manuscript, review of literature. SSi- Critical revision of manuscript, review of literature and final approval. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Evidence supporting the benefits of locomotor training (LT) to improve walking ability following stroke are inconclusive and could likely be improved with a better understanding of the effects of individual parameters i.e., body weight support (BWS), speed, and therapist assistance and their interactions with walking ability and specific impairments. We evaluated changes in muscle activity of thirty-seven individuals with chronic stroke (> 6 months), in response to a single session of LT at their self-selected or fastest-comfortable speed (FS) with three levels of BWS , and at FS with 30% BWS and seven different combinations of therapist assistance at the paretic foot, non-paretic foot, and trunk. Altered Muscle Activation Pattern (AMAP), a previously developed tool in our lab was used to evaluate the effects of LT parameter variation on eight lower-extremity muscle patterns in individuals with stroke. Repeated-measures mixed-model ANOVA was used to determine the effects of speed, BWS, and their interaction on AMAP scores. The Wilcoxon-signed rank test was used to determine the effects of therapist-assisted conditions on AMAP scores. Increased BWS mostly improved lower-extremity muscle activity patterns, but increased speed resulted in worse plantar flexor activity. Abnormal early plantar flexor activity during stance decreased with assistance at trunk and both feet, exaggerated plantar flexor activity during late swing decreased with assistance to the non-paretic foot or trunk, and diminished gluteus medius activity during stance increased with assistance to paretic foot and/or trunk. Therefore, different sets of training parameters have different immediate effects on activation patterns of each muscle and gait subphases.The online version contains supplementary material available at 10.1007/s00221-023-06551-7. Stroke survivors frequently present with difficulty in walking. Thirty percent of community dwelling stroke survivors cannot walk independently  influence different gait deficits . Data from three stroke survivors were not included in the analysis due to incomplete data sets; of these two were unable to complete all the LTSS parameters due to fatigue, and ground reaction force (GRF) data were missing for one stroke survivors. Individuals with stroke were included if they had a unilateral lesion; the ability to walk independently for at least 10 m at a self-selected speed ≤ 1.0 m/s; revealed no severe perceptual or cognitive deficits, no significant lower limb contractures, no severe osteoarthritis or prior pathological fracture, no significant cardiovascular impairments contraindicative to walking; were free of significant lower-extremity joint pain, range of motion limitations, and profound sensory deficits (including proprioception). Individuals who did not meet these criteria were excluded. Demographic characteristics are reported in Table Forty individuals with chronic stroke (> 6 months) and 20 similarly aged controls with no neurological or orthopedic impairments, were studied during a single session of locomotor training  in nine conditions presented in randomized order. Manipulation variables included three speeds  and three levels of BWS . Individuals with stroke walked on the treadmill at their self-selected and fastest-comfortable walking speed at three levels of BWS . Since stroke survivors with a self-selected speed ≤ 1.0 m/s were included in the study, we used pre-defined speeds i.e., at 0.3, 0.6, 0.9 m/s for our healthy participants to match their walking speeds with those of stroke survivors. A safety harness attached to the Constant Force Body Weight Support system mounted to the laboratory ceiling was worn by participants to provide BWS for the experimental conditions and also protect them in the event of loss of balance. Thus, they were not allowed to use the handrails for additional support. Individuals with stroke were allowed to adjust their self-selected and fast walking speeds at 15% and 30% BWS if it changed due to the increased BWS. Additionally, all stroke survivors completed 7 conditions at their fastest speed with 30% BWS and therapist assistance presented in randomized order. Conditions with therapist assistance consisted of paretic foot (P), non-paretic foot (N), trunk (T), paretic foot with trunk (PT), non-paretic foot with trunk (NT), both feet (PN), and both feet with trunk (PNT). One 30-s trial per condition was conducted for stroke survivors and controls. All participants were allowed as much rest as needed between trials, but not less than 2 min. Therapist assistance for the paretic and non-paretic foot was designed to promote step length symmetry with therapists’ hands placed at the ankle. Therapists assisted with trunk movement in the mediolateral direction to promote weight-shift symmetry. All LTElectromyographic (EMG) data were collected from 8 muscles in both legs  including: tibialis anterior (TA), soleus (SO), medial gastrocnemius (MG), vastus medialis (VM), rectus femoris (RF), lateral hamstring (LH), medial hamstring (MH), and gluteus medius (GM). Here we present EMG data for the paretic leg for stroke survivors and bilaterally for controls. GRF were captured using two force plates embedded in the instrumented split-belt treadmill . EMG and ground reaction force data were sampled at 2000 Hz except for one stroke survivor where data were accidentally sampled at 1000 Hz.K-means cluster analysis was used to identify the “on” and “off” periods of EMG activity , first (SS1) and second halves of single-leg stance (SS2), second double support (DS2), and first (SW1) and second halves of swing (SW2). Within each of these regions, AMAP scores were computed for timing and amplitude components of the EMG activity. The timing component was equal to the “on” time within each region expressed as a percentage of the total duration of that region:The amplitude component was equal to the integrated EMG i.e., net area under the curve (AUC) of the “on” time for each region expressed as a percentage of the total integrated EMG of all “on” periods of the gait cycle:z-scores. A z-score is a measure of the number of standard deviations an element falls above or below the population mean. In the current study means and standard deviations of healthy controls within each speed and BWS condition for both the amplitude and timing components of each region were used as normal patterns (Eqs. SS condition.Equations  and 2)  2) were STROKE represent the scores of each stroke survivor for a given LTSS condition; Amplitude and Timing component MeanHEALTHY represents the group mean of healthy individuals walking at speeds and BWS similar to individuals with stroke; Amplitude and Timing component SDHEALTHY represents group SD of healthy individuals walking at speeds and BWS similar to individuals with stroke. Calculation of AMAP scores for stroke survivors requires comparison to mean and SD of healthy individuals at similar speed and BWS (MeanHEALTHY and SDHEALTHY). Thus, based on the training speed of a stroke survivor  we used means (MeanHEALTHY) and standard deviations (SDHEALTHY) of controls walking without therapist assistance at speeds of 0.3 m/s , 0.6 m/s  and 0.9 m/s  at corresponding BWS (15% or 30%) to compute AMAP scores in Eqs. . Therefore, our analysis produced an amplitude and a timing score of each stroke survivor for each of the 8 muscles and walking conditions for each of the six regions.Amplitude and Timing component AMAP Score in Eqs.  requirinSS could result in either a worse score, if it shifts further from zero and becomes less similar to normal outside the bounds of normal variability for that muscle within that region, or an improved score, if it shifts closer to zero and becomes more similar to normal. Therefore, the effect of increases and decreases in EMG activity depends on how that change compares to the change in activity observed in the similar healthy condition. AMAP scores of stroke survivors  for each LTSS condition were compared with their baseline AMAP scores to evaluate the response to each training parameter.The AMAP scores for timing and amplitude quantify EMG patterns of stroke survivors relative to those of similarly aged control participants and their range of typically observed variability. The AMAP scores for each condition was computed by comparing EMG patterns of individuals with stroke to that of healthy individuals during similar walking conditions. An AMAP score closer to zero would suggest that EMG activity is closer to that of healthy individuals’ walking at similar speed and BWS and vice versa. Note that an increase in the EMG activity of individuals with stroke following LTp ≤ 0.05, with Bonferroni-correction used to correct for multiple comparisons. Mean and SD values of untransformed AMAP scores are reported in Table 1 of supplementary material. The Wilcoxon-signed rank test was used to compare AMAP scores between each of therapist-assisted conditions and baseline self-selected walking at 0% BWS. For each of the Wilcoxon-signed rank tests, statistical significance adjusted for multiple comparisons was set at p < 0.007. Mean and SD values of AMAP scores for each condition are reported in Table 2 of supplementary material. All statistics were performed in SPSS version 25  and SAS version 9.4 .A repeated-measures mixed-model ANOVA was used to determine the effects of speed, BWS, and their interaction, on AMAP scores during walking with 0%, 15% or 30% BWS at self-selected or fast walking speeds without therapist assistance. To meet normality assumptions required for mixed-model comparisons, if the AMAP scores were not normally distributed they were first log transformed and if normality assumptions were still not met, were square root transformed across all conditions to improve model fit for each muscle’s amplitude and/or timing component. Statistical significance was set a priori at Baseline Patterns : We derived AMAP scores for EMG amplitude and timing of individuals with stroke at self-selected walking speed relative to controls’ EMG patterns at similar speeds. At this baseline, AMAP scores of individuals with stroke revealed exaggerated activity during DS1 and SW2 and diminished activity during SS2 for SO and MG. We also observed exaggerated activity for TA during DS2 and for GM during DS2, SW2 and diminished activity for GM during SS1. In most stroke survivors, AMAP patterns for MH, LH, RF, and VM were not dissimilar from controls, meaning they fell within the range of typically observed variability. The data used for baseline scores have been reported in our previous work defining the AMAP  = 5.35, p = 0.0224], DS2 , and for SO timing scores during DS1 , SW2 . Significant main effects of speed were also observed for MG amplitude scores  and timing scores  during DS1 and for amplitude scores only during SS2 . Specifically, relative to baseline AMAP scores, increased speed revealed worse SO amplitude scores  during SS1  and DS2 . Increased speed also led to worse SO timing scores during DS1 and SW2 . Similarly, increased speed led to worse MG amplitude scores during DS1 and SS2 , and worse timing scores during DS1 . At baseline, both SO and MG amplitude are diminished in individuals with stroke during stance phase, but here an increase in amplitude with speed results in worse AMAP scores because it exceeded the level of normal activity seen in healthy controls. There were no significant effects of speed on MH activity patterns. For the remainder of the significant changes observed in each muscle pattern, the AMAP scores were within the normal range at baseline and remained within the normal range  following LTSS conditions involving speed change.BWS effect: Increasing the amount of BWS  mostly tended to improve muscle activation. Significant main effects of weight were seen for SO AMAP scores during DS1 , SS1 , and SW2 ; for MG AMAP scores during SW2 , and DS1 . Significant main effects of weight were also seen for TA AMAP scores during DS2 , for GM AMAP scores during SS1 , DS2 , SS2  and SW1 . Specifically, the AMAP scores for SO improved with increased BWS relative to baseline scores during DS1 and SW2  and during SS1 . With increased BWS, AMAP scores for MG amplitude during DS1 were worse . MG amplitude and timing scores improved during SW2 . AMAP scores improved significantly with increased BWS for the TA timing component during DS2 . The scores improved during SS1 and SS2 for GM amplitude and during SS1 for timing . There was also an improvement in scores for GM amplitude during DS2 and SW1 , and for timing during DS2 . For the remainder of the significant changes observed in each muscle pattern the AMAP scores were within the normal range at baseline and remained within the normal range .Interaction of speed and BWS: There were no significant interaction effects.Effects of therapist assistance at 30% BWS and fast walking speeds: Significant changes with therapist assistance were seen mostly for timing and amplitude scores in SO, MG, and GM activity. SO amplitude AMAP scores improved significantly relative to baseline  during SS1 and SS2 for all LTSS parameters and during DS1 following trunk and both feet assistance. SO amplitude AMAP scores improved significantly during SW2  following non-paretic foot assistance  to identify the combination of LTns Figs. , 3, whicns Figs. . We founIncreased speed resulted in worse amplitude and timing AMAP scores seen as increased activity  for SO and MG. A previous study evaluating the effects of BWS training at different speeds demonstrated that plantarflexor activity at faster speeds was greater than at slower speeds  during stance with increased BWS . Typically, one would expect diminished plantarflexor activity with increased BWS during stance phase  during early stance and late swing, and improved AMAP scores  during mid-stance. Individuals with stroke often have an early activation of plantar flexors during stance along with decreased amplitude during mid and late stance  of plantarflexor activity with other stance phase muscle activity  during stance and increased BWS without therapist assistance improved AMAP score  during swing, but therapist assistance to trunk and both feet led to worse AMAP scores  during swing. Diminished baseline GM activity seen in stroke gait relative to healthy controls during stance improved with increased BWS and therapist assistance to paretic foot, trunk, and trunk with paretic foot. We believe that therapist assistance to the trunk and paretic leg may promote appropriate weight bearing during stance phase facilitating increased GM activity during stance. At baseline, during swing we observed exaggerated GM activity in individuals with stroke. Consistent with previous literature , they became worse  with assistance to non-paretic foot or trunk, and no changes were observed for the timing patterns with therapist assistance. Therefore, typically-used LTSS parameters may not be ideal to improve altered TA activity in individuals with stroke. Activation patterns of MH, LH, RF, and VM in stroke survivors were similar to healthy controls at baseline and likely did not require large modifications to improve their patterns. Although this limits our ability to evaluate the effects of LTSS parameters for these muscles, we have discussed in our previous work that SO, MG, and GM are typically more associated with functional walking ability of individuals with stroke . Plantarflexor activity during swing improves with non-paretic foot assistance aimed at facilitating spatiotemporal gait symmetry. Additionally, LTSS with increased BWS and not speed may result in a plantarflexor pattern more similar to healthy individuals suggesting that use of faster speeds for LTSS should be considered in this light given the goals of the therapist. Based on the current results, we believe that optimally configured LT has the potential to improve muscle activity patterns which could lead to an improved walking ability in individuals with stroke.In conclusion, our results demonstrate that a specific set of LTSupplementary file1 (DOCX 44 kb)Supplementary file2 (DOCX 51 kb)Supplementary file3 (DOCX 46 kb)Below is the link to the electronic supplementary material.


Point of care ultrasonography (POCUS) is increasingly being used as an adjunct to physical examination in internal medicine and subspecialties. In nephrology practice, it is particularly valuable in the objective assessment of fluid volume status. In addition, POCUS enables the physicians to better appreciate the anatomic and pathophysiologic changes that occur during the course of patient management. The intent of this case file is to demonstrate the improvement in sonographic markers of congestive state during ultrafiltration therapy in a patient with heart failure.A 74-year-old gentleman with a history of hypertension, diabetes mellitus type 2, coronary artery disease, congestive heart failure with reduced left ventricular ejection fraction (<20%), and chronic kidney disease stage 3 was admitted to the hospital for acute exacerbation of heart failure. He presented with a serum creatinine of 2.7 mg/dL, which slowly trended down to 2.3 mg/dL with intravenous loop diuretic therapy over the next 5 days and started to trend up again prompting nephrology consultation. As the serum creatinine continued to rise (4.1 mg/dL) with oliguria and hypotension (systolic blood pressure 85-90 mmHg), continuous renal replacement therapy was initiated. He was also started on dobutamine drip by the cardiology team. A POCUS examination was performed by the nephrologist including a focused echocardiogram, lung ultrasound, and a venous excess Doppler ultrasound (VExUS). Echo was significant for a plethoric inferior vena cava (IVC) measuring approximately 2.5 cm in maximal diameter (online Video S1A) with almost no respiratory variation consistent with very high right atrial pressure. There was significantly decreased left ventricular ejection fraction. Parasternal short axis view demonstrated a ‘D’-shaped left ventricle with interventricular septal flattening throughout the cardiac cycle suggestive of right ventricular pressure and volume overload (online Video S1B). It was likely that the compression of the left ventricle was leading to a reduction in stroke volume, contributing to hypotension. Lung ultrasound demonstrated B-line pattern bilaterally, consistent with pulmonary edema/interstitial syndrome (online Video S1C). VExUS scan demonstrated D-only pattern on hepatic vein Doppler, which means there is flow towards the heart only during diastole . Similarly, portal vein Doppler was suggestive of severe congestion with 100% pulsatility and possible flow reversal during systole . Based on these findings, the rate of ultrafiltration was increased despite hypotension and the patient was intubated soon after this scan was performed. A repeat POCUS examination was performed 4 days later after approximately 10 liters net ultrafiltration. The patient remained intubated at that time though the systolic blood pressure improved to ~110 mmHg. Inferior vena cava was still plethoric  but the venous Doppler showed significant improvement in hepatic and portal vein waveforms. Both S (systolic) and D (diastolic) waves were below the baseline on the hepatic vein tracing indicating near-normal venous return  and the portal vein pulsatility has substantially improved to ~30% . Interestingly, the left ventricle appeared round on the parasternal short axis view suggesting improvement in right ventricular volume and pressure overload and thus improved left ventricular outflow (online Video S2B). As expected, lung ultrasound showed improvement with predominantly A-line pattern though there were B-lines at the bases (online Video S2C). In addition, the urine output also picked up though the patient was never completely anuric.In summary, it is important for the nephrologists to be aware that not all hypotension is due to volume depletion and cases of severe congestion with dilated right ventricle compromising the left ventricular outflow as demonstrated here will respond to volume removal The author declares that no conflict of interest exists.Informed consent has been obtained from the patient for the publication of this case study. Video S1 Video S2


Lithium (Li) metal batteries (LMBs) have received extensive research attention in recent years because of their high energy density. However, uncontrollable Li dendrite growth deteriorates the battery life and brings about severe safety hazards. The rational design of battery separators is an effective approach to regulate uniform Li metal deposition towards boosted cycle life and safety of LMBs. Herein, we review the recent research progress concerning this issue, including mechanically strengthened separator fabrication, functional separator construction towards regulated Li ion deposition, and flame-retardant separator design. Moreover, the key issues and prospects of optimal design of separators are clarified for future development. This minireview is expected to bring new insight into developing advanced separators for long-life and safe LMBs. Nevermaterial ,7. Howevmaterial ,9,10.Various strategies have been proposed to address the above challenges in LMBs, including constructing functional artificial SEI ,12,13,14The designing and optimization of separators, including modifying separators with functional polymers ,31, carbStrengthening separators has been deemed an effective strategy to block the growth of Li dendrites and prevent the short-circuit of LMBs. However, the Li dendrite is sharp and has a high Young’s modulus to pierce commercial separators such as polypropylene (PP) or polyethylene (PE) films . Natural2 nanoparticles with 500 nm diameter and commercial PP film  separator, in which compact graphene and Al2O3 powder were coated on each side of the separator  and polar chemical absorption layer (TiO2), which demonstrate a very high reversible specific capacity beyond 1000 mA h g−1 over 300 cycles at 0.5 C [Since Li ions prefer to be absorbed by lithiophilic sites via electrostatic interactions, inorganic materials or heteroatom-doped carbon materials are also efficient in increasing the rate and homogeneity of Li ion flux. Zhang et al.  utilizedat 0.5 C .−2 with a deposition capacity of 2 mA h cm−2 3 are the most common inorganic flame retardants, and efficiently reduce the surface temperature of the combustible materials and inhibit the combustible gas by releasing lots of water at elevated temperature and absorbing plenty of heat when combined with water vapor. Organic materials are mostly halogen- or phosphorus-based molecules that exhibit flame retardation by suppressing chain reactions and flame propagation through radical scavenging processes or physical isolation mechanisms [3 and Al-doped Li6.75La3Zr1.75Ta0.25O12(LLZTO) (3 and LLZTO to poly(vinylidenefluoride-hexafluoropropylene) (PVDF-HFP) by abundant hydrogen bonds and van der Waals forces of the bilayer separators. LLZTO shows excellent Li-ion conductivity, and MoO3 exhibits remarkable flame-retardant capabilities by lowering the generation of volatile hydrocarbon species and flammable benzene species. A Li|LiFePO4 cell with such bilayer separator also exhibited lower charge-transfer resistance and faster reaction kinetics compared to those with Celgard 2325 and PVDF-HFP  a. CombusPVDF-HFP b. The di in LMBs c.Because some flame retardants can increase electrolyte viscosity and thus reduce the ionic conductivity of electrolytes, thermal-sensitive polymers have been adopted as a protective layer to prevent the exposure of flame retardants to the electrolyte and sustain the ionic conductivity . Cui et (1)Most of the reported functional separators have been developed with top-down strategies such as the tap-casting method, which leads to a lack of control over the thickness and the homogeneous distribution of different components. More elaborate bottom-up strategies should be designed to develop uniform and homogenous separators.(2)To minimize sacrifice in both volumetric and gravitation energy density, the thickness of the modification layer should be controlled at <1 µm  and its density should be reduced as low as possible. In this regard, novel nanostructured nanomaterials, especially 2D graphene and boron nitride, are good options.(3)To avoid peeling of the modification layer from the separator during Li plating/stripping cycles, molecular and structural designs to improve the flexibility and elasticity of the modification layer, as well as its adhesion on the separator, are highly desirable.(4)To help eliminate battery thermal runaway caused by Li dendrites and improve battery safety, the thermal conductivity of the separator should be further improved for better battery heat dissipation; few studies have focused on this.(5)+, or even react with electrolyte or Li metal electrodes, especially under complex electrochemical conditions. These “activations” (absorption and reaction) together with the influence on battery performance should be carefully studied.The “activation” of functional separators should be understood in depth. Some of the designed separators are not electrochemically inert, and they may absorb Li(6)−2 and 3 mAh cm−2, respectively, because the area capacity of commercial energy-type LMBs is >3 mAh cm−2 and the discharging/charging current is >3 mA cm−2 based on 1 C rate. The electrolyte amount should be controlled at <10 µL mAh−1 (requirement of lean electrolyte), and the capacity ratio of Li metal anode and cathode should be <5 to maintain high volumetric energy density.The performance of the designed separators should be evaluated under practical conditions. The areal current density and capacity for Li|Li symmetric cells and Li metal full cells should be higher than 3 mA cmThe latest developments on functional separators for long-life and safe Li metal batteries have been summarized and discussed in this minireview, including mechanically strengthened separator fabrication, functional separator construction towards regulated Li ion deposition, and flame-retardant separator design. The basic mechanism for mechanically strengthened separator fabrication is attributable to the mechanical barrier effect with higher puncture strength, strong enough to bend the tip of the Li dendrite. Functional separator construction towards regulated Li ion deposition can construct even lithiophilic sites for uniform nucleating and growth. Flame-retardant separator design aims at controlling thermal runaway by forming dense separators to interrupt battery reactions or releasing flame retardants to prevent burning. Although various strategies have been applied to improve the functional separators for practical applications of LIBs, there are still some challenges that need to be addressed.


TMCC2, SFMBT2, AKAP6 and PHYHIP. Differentially methylated probes were independent of known PD genetic risk alleles. Meta-analysis provided suggestive evidence for a differentially methylated locus within the chromosomal region affected by the PD-associated 22q11.2 deletion. Our findings elucidate novel disease pathways in PD and DLB and generate hypotheses for future molecular studies of Lewy body pathology.Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) are closely related progressive disorders with no available disease-modifying therapy, neuropathologically characterized by intraneuronal aggregates of misfolded α-synuclein. To explore the role of DNA methylation changes in PD and DLB pathogenesis, we performed an epigenome-wide association study (EWAS) of 322 postmortem frontal cortex samples and replicated results in an independent set of 200 donors. We report novel differentially methylated replicating loci associated with Braak Lewy body stage near  Parkinson’s disease and dementia with Lewy bodies are closely related neurodegenerative disorders, although the epigenetic similarities are not well known. Here, the authors study Lewy pathology and DNA methylation in postmortem human frontal cortex, identifying differentially methylated genomic loci. Current treatment for PD and DLB is merely symptomatic, and improved understanding of the molecular disease mechanisms is urgently needed in order to facilitate the development of targeted disease-modifying therapy. Over the last decade, large-scale genome-wide association studies (GWAS) have identified an increasing number of common genetic risk loci for PD3, and more recently also for DLB5, providing novel insights into pathogenesis. In contrast, previous studies have only explored to a limited degree how disease susceptibility and progression may be shaped by epigenetics. Epigenetic processes contribute to variability in complex traits such as aging and disease7 and involve mechanisms of gene regulation that are partially dynamic over time, cell-type specific and influenced by both genetic and environmental factors.Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) are progressive and debilitating neurodegenerative disorders with complex aetiology and overlapping clinical and neuropathological features. These disorders make up the second most common cause of neurodegeneration and dementia after Alzheimer’s disease (AD), and as their prevalence increases with longer life expectancy, the current estimate of 6 million PD patients worldwide is anticipated to more than double by 204011. In contrast, similar efforts in PD or DLB have thus far only been reported for very limited sample sizes14, although larger EWAS in PD have been performed on whole blood17. A recent study investigated frontal cortex neurons isolated by a flow cytometry-based approach and detected widespread differences in DNA methylation between 57 PD patients and 48 controls, with replication in a smaller dataset, highlighting dysregulation of TET2 in particular18.DNA methylation at CpG dinucleotides is the most frequently studied epigenetic mechanism in complex disease. Previous EWAS analyses of AD-associated neuropathology have identified replicable and consistent patterns of differentially methylated CpGs across the genome in postmortem human brain tissue, contributing important insights to disease mechanisms19. According to the staging system proposed by Braak20, Lewy pathology in PD spreads in a predictable pattern over the course of the disease from the olfactory bulb and brainstem (Braak Lewy body stages 1–3) in a caudal-to-rostral manner, next to limbic regions (Braak Lewy body stage 4), reaching the neocortex in the last stages (Braak Lewy body stages 5 and 6). Only the retrospective information about clinical symptom progression distinguishes late-stage PD from DLB, where cortical Lewy pathology and associated cognitive symptoms are already present early in the disease course21. Of note, cellular and proteomic studies have demonstrated that neuronal stress and dysfunction are evident in the cortex also at the very early stages of PD-related pathology, long before Lewy bodies appear in the same brain region22. Previous transcriptome studies on post-mortem human brain tissue across Braak Lewy body stages have also highlighted early changes23. We hypothesized that epigenetic dysregulation contributes to the common molecular pathogenesis of Lewy body disorders, and that an increasing degree of disease-related changes should be detectable in frontal cortex as the disease progresses into more advanced stages.Lewy bodies and Lewy neurites are intraneuronal aggregates composed mainly of misfolded α-synuclein protein and constitute the neuropathological hallmark lesions of both PD and DLBTo dissect the role of DNA methylation changes in PD and DLB, we performed a methylome-wide association study of 322 postmortem frontal cortex samples with Braak Lewy body stages 0–6 and replicated our findings in an independent dataset from 200 donors. The size and direction of effects for the most associated probes were highly correlated across the two datasets, providing a strong indication of consistent results. We report 4 replicating CpGs significantly associated with Braak stage, explore these loci in relation to known genetic risk factors and publicly available data and provide a benchmark for further epigenetic studies of post-mortem human brain tissue in Lewy body disorders.n = 73), donors without clinical neurological symptoms but with incidental Lewy body disease at autopsy (iLBD) (n = 29), clinically diagnosed and pathologically confirmed PD patients (n = 139) and DLB patients (n = 81) , data on 583,192 CpG probes in 322 individuals were included in statistical analyses. We used a published algorithm and reference data to estimate the proportion of NeuN positive neurons in each tissue sample and included this variable as a covariate (see Methods). We observed a non-significant trend towards lower estimated proportion of neurons in samples with higher Braak Lewy body stage .For the discovery stage of our analysis, we used superior frontal gyrus grey matter tissue samples from the Netherlands Brain Bank (NBB) and Normal Aging Brain Collection, Amsterdam (NABCA) with available data on Lewy body Braak stage. We included samples from controls without any records of neurological or psychiatric disease during life , 24 CpG probes were associated with Braak α-synuclein stage at FDR < 0.05 in the discovery stage    , cg09985192 (annotated to AKAP6) and cg04011470 (annotated to PHYHIP)  emerged with the fifth strongest p-value in the meta-analysis, despite not being FDR-significant in the discovery stage alone. In meta-analysis, the three most significant probes also passed the more conservative Bonferroni-corrected threshold . Quantile-quantile and Manhattan plots are shown in Supplementary Figures In addition to the two-stage discovery and replication approach, we also meta-analysed the 564,387 probes passing QC in both datasets. In a fixed-effect inverse variance meta-analysis, 35 probes were significant at FDR < 0.05 and 14 of these were associated at CYP2E1   , where the random effect of total genome-wide DNA methylation captures the correlation structure between probes and directly controls for the genomic inflation28. Comparing results obtained using MOA as implemented in the OSCA software (see Methods) to our linear regression model, we found that effect estimates were strongly correlated (Pearson r2 0.91) and effect directions all concordant  with consistent direction as the primary analysis, indicating that the strongest association signals are likely driven by differences in Lewy body stage partly across and partly within diagnostic groups  in the discovery dataset was 0.81 . Including the first three surrogate variables yielded a similar estimate .We used the omics restricted likelihood method (OREML) implemented in the OSCA software package to estimate the phenotypic variance for Braak Lewy body attributable to all probes in the dataset29. In this previous work, active promoters and enhancers were identified by assay for transposase-accessible chromatin sequencing (ATAC-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) for histone modifications H3K27ac and H3K4me3. Table Sites profiled by the MethylationEPIC array primarily map to promoters and enhancers in the human genome where variability in CpG methylation is likely to affect the functional regulation of nearby genes. To potentially nominate genes and cell-types implicated by our Braak Lewy body stage-associated CpGs, we assessed the overlap between probe positions and genomic annotations from a recent publication characterizing the noncoding regulatory regions and enhancer-promoter interactome of major cell types in the human cortexTMCC2 on chromosome 1, overlapping with a predicted enhancer in both neurons, oligodendrocytes, and astrocytes, and only 164 base pairs upstream of the transcription start site of a 4 exon isoform (ENST00000481950.2) of the TMCC2 gene.The strongest associated replicating probe, cg07107199 maps to an intron of SFMBT2 on chromosome 8, where there is evidence of enhancer activity in microglia and astrocytes, but most pronounced in microglia  falls within the ~3 Mb region typically affected in the 22q11.2 deletion syndrome, which has been implicated in a number of clinical phenotypes, including idiopathic PD32. We found no evidence for the presence of any 22q11.2 deletion in the NBB dataset based on analysis of runs of homozygosity in genotype data (see Methods).The DNA methylation site nominated primarily from meta-analysis, cg03318382, is located ~13 kb upstream of BIN3, a genome-wide significant PD risk locus3. Comparing DNA methylation and genotype data in our discovery sample, we found no association between cg04011470 and rs2280104, nor with any other single-nucleotide polymorphism (SNP) within a 1 Mb window around the probe position. Similarly, we found no significant methylation-quantitative loci (mQTL) for cg04011470 among 277 cis-SNPs in publicly available frontal cortex data summary statistics from the Brain xQTL server (n = 543)33 nor data from a 2018 brain mQTL study (estimated effective n = 1160)34.Of note, one of the significant CpG probes (cg04011470) is located less than 500 kb from rs2280104 near SNCA promoter region in PD has been reported in a number of previous studies, both in brain and blood37. We therefore assessed CpG probes in the SNCA locus specifically, but found no significant CpGs when correcting for multiple testing of 109 probes within 500 kb of significant SNPs .Turning to other known risk loci for PD, we extracted results from all 26,743 CpG probes located within 500 kb of any significant GWAS SNP reported by Nalls et al., but cg04011470 remained the only association significant when correcting for multiple testing. Hypomethylation of the Next, we assessed both NBB genotypes and publicly available data for cis-mQTLs within 500 kb of our remaining 3 Braak stage-associated CpGs, but found no SNPs significantly associated with methylation levels for any of the probes. These results suggest that the DNA methylation differences associated with Lewy body pathology do not appear to be mediated by underlying genetic variability.26. Our study design mirrors a series of successful bulk cortex EWAS studies of AD neuropathology, where the number of significant CpG associations has increased from a handful in the first published studies9 to several hundred in recent meta-analyses11, providing novel insights into disease pathways and mechanisms. We demonstrate here that the same approach is feasible also for Lewy body pathology, the hallmark of PD and DLB.We performed a methylome-wide association study across Braak Lewy body stages in postmortem human frontal cortex, and followed up our findings in an independent dataset, identifying 4 novel differentially methylated loci that replicated across sample series. Including a total of 522 participants, we analysed a larger sample than the few previously published EWAS studies of human brain tissue in Lewy body disordersTMCC2, encoding transmembrane and coiled-coil domains protein 2, which is primarily expressed in brain and blood and localizes predominantly to the endoplasmatic reticulum38. The protein has been shown to interact with apolipoprotein E (APOE) in an allele-specific manner, with evidence indicating that interaction between TCMM2 and the AD-associated APOE ε4 allele may alter amyloid β production39. Our findings suggest the possibility that TMCC2 may also have a role in α-synuclein pathology.Differentially methylated sites are located in promoters or enhancers, and interpretations linking association signals to specific genes should be made with caution in the absence of functional evidence. The strongest associated replicating probe is located in a predicted enhancer within an intronic region of SFMBT2 in microglia may be linked to Lewy body pathology. SFMBT2 encodes Scm-like with four malignant brain tumour domains 2. Apart from being reported as a microglial signature gene30 and differentially expressed in PD31, its function in the human brain is largely unexplored. AKAP6 upstream of cg09985192 encodes A-kinase anchoring protein 6, which is abundantly expressed in various brain regions and striated muscle. Genetic variability in the AKAP6 locus has been associated with general cognitive function in a large GWAS meta-analysis, but the implicated molecular mechanism is unknown40.Based on genomic annotations from human cortex overlapping with the location of cg14511218, we raise the hypothesis that dysregulation of PHYHIP was located less than 500 kb from the top associated SNP identified in a recent PD GWAS analysis. Studies of the association between common SNPs, gene expression, histone modifications and CpG methylation in the human brain have revealed that the different types of QTLs show a certain degree of overlap and are all enriched among disease-associated GWAS SNPs33, raising the possibility that our finding could be linked to the same mechanism as the adjacent GWAS signal. However, in the same study, SNP effects on RNA expression were only mediated fully by epigenetic variation in 9% of loci, so simple one-to-one relationships remain the exception. Consequently, we are not surprised that we did not identify significant mQTLs for cg04011470 or any of the other differentially methylated CpGs, which is also in line with a recent large meta-analysis of AD neuropathology EWAS11. The lack of mQTLs is still compatible with a hypothesis that genetic and epigenetic association signals from the same genomic region may represent independent disease mechanisms converging on the same gene.We noted with interest that the differentially methylated probe cg04011470 in p < 0.05 in both the NBB and the BDR datasets. This approach is less conservative than the standard two-stage discovery and replication method and should therefore be interpreted with caution. The strongest additional signal emerging in our meta-analysis was an association between Lewy pathology and hypermethylation of cg03318382, located within the ~3 Mb region typically affected in the 22q11.2 deletion syndrome. This deletion occurs in at least 1 in 4000 births and has been linked to a number of clinical phenotypes including DiGeorge syndrome, velocardiofacial syndrome and psychotic illness41. Early-onset parkinsonism reported as a clinical feature in known carriers of the 22q11.2 deletion sydrome42 prompted further investigations confirming its role as a risk factor for PD, also predisposing to early onset43. It is not clear which genes within the region are important for PD pathogenesis, although COMT, encoding catechol-o-methyltransferase has been highlighted as an interesting candidate32. We found CpG hypermethylation upstream of SEPTIN5, which encodes septin 5. Septins are guanosine-5'-triphosphate (GTP)-binding proteins that contribute to the regulation of synaptic vesicle trafficking and neurotransmitter release, and septin 5 has been shown to interact with parkin, a protein implicated in early-onset autosomal recessive PD44. Our findings highlight the possibility that epigenetic changes in the 22q11.2 region may be associated with disease risk in individuals with normal copy number due to dysregulation of one or more of the same genes that contribute to the 22q11.2 deletion syndrome.In meta-analysis, we identified 14 CpGs associated with Braak Lewy body stage with consistent direction of effect and SNCA has been reported in postmorten human brain. One study showed significant differences in both substantia nigra and cortex35, whereas another found significant change in substantia nigra only36, with similar methylation levels in patients and controls in putamen and the anterior cingulate. Furthermore, SNCA hypomethylation has been demonstrated in whole blood and cultured mononuclear cells, also with an mQTL association to susceptibility SNPs for PD and DLB in brain and blood45, but the methodologies used have been different from the present study. A number of factors, including choice of brain region, array design and adjustment for cell composition may have contributed to our inability to replicate this signal.Hypomethylation at the promoter in intron 1 of CYP2E1, yet there was no similar signal in the larger NBB data. The association is not FDR-significant in BDR data, but the region stands out from the rest of the results, as is apparent in the Manhattan plot .Our primary analysis model assumed Braak Lewy body stage as a quantitative variable and did not take diagnosis into account, following the approach of previous major EWAS of AD neuropathologyp < 0.05 in the BDR data as evidence of replication, yet we acknowledge that a significance threshold correcting for multiple testing also in the replication stage would have been more robust.Despite being larger than previous brain EWAS in PD and DLB, the sample size of our study was still modest, limiting our statistical power for hypothesis-free association analysis. We note that although the overall pattern of association of the top probes was highly similar, discovery phase results did not reach FDR adjusted significance in the alternative analysis approach based on the more conservative MOA method. It is biologically plausible, however, that Lewy body pathology has widespread effects on methylation causing some degree of inflation that does not result from confounding, which is also compatible with the high observed estimate of the proportion of variance attributable to all probes (0.81). Furthermore, we considered it justified to interpret a two-sided As the total number of samples have passed one thousand for meta-analyses of EWAS in AD neuropathology, a larger set of robustly associated CpGs can be utilized for enrichment analyses, linking the results collectively to molecular mechanisms and pathways. We hope to see a similar development in Lewy neuropathology EWAS through collaborative efforts in the years to come. Furthermore, cross-trait analyses are warranted to assess the degree of epigenetic overlap versus disease-specific methylation patterns across neurodegenerative disorders. Understanding causality and the temporal sequence of disease-linked processes is also of major importance and could potentially be addressed through studies in model systems as well as larger collections of postmortem samples in the very early stages of neuropathological change. Finally, more studies are needed to identify the genetic and environmental determinants driving disease-associated methylation changes in neurodegenerative disorders.In summary, we performed the first well-powered two-stage EWAS of Lewy body neuropathology in postmortem human frontal cortex, providing evidence for significant methylation differences associated with Braak Lewy body stages and linking novel genomic loci to the pathology underlying PD and DLB. These findings generate hypotheses for further molecular studies and hold promise that future meta-analyses in larger sample sets will be as fruitful in Lewy neuropathology as in recent studies of AD-related changes. Complementing other genomic approaches such as GWAS and gene expression studies, investigations of DNA methylation may provide important contributions to our basic understanding of disease mechanisms and ultimately facilitate the development of disease-modifying therapy for PD and DLB.www.brainbank.nl) and Normal Aging Brain Collection, Amsterdam (NABCA)51. Written informed consent for the use of tissue samples and clinical information for research purposes was collected from the donors or their next of kin. The Medical Ethics Committee of the VU University Medical Centre, Amsterdam, approved all procedures of NBB and NABCA. The EWAS study was approved by the Regional Committee for Health and Medical Research Ethics, Norway. Standardized brain autopsies and neuropathological examinations were performed by experienced neuropathologist or neuroanatomist (AR and WB), including assessment of Lewy body-related α-synuclein pathology according to BrainNet Europe guidelines52. Clinical information was extracted from medical records provided by the NBB. PD diagnosis was based on the combination of clinical parkinsonism according to UK Parkinson’s Disease Society Brain Bank53 or Movement Disorders Society54 criteria and moderate to severe loss of neuromelanin-containing neurons in the substantia nigra in association with Lewy pathology in at least the brainstem, with or without limbic and cortical brain regions55. Criteria for DLB were a clinical diagnosis of probable DLB according to the consensus criteria of the DLB Consortium21 combined with presence of limbic-transitional or diffuse-neocortical Lewy pathology upon autopsy. Dementia was diagnosed clinically during life by a neurologist or geriatrician, or retrospectively based on neuropsychological test results showing disturbances in at least two core cognitive domains or Mini-Mental State Examination (MMSE) score <20. The distinction between DLB and PD with dementia (PDD) (74 out of 139 PD patients) was made based on the “1-year rule”, classifying dementia presenting before or within 1 year of parkinsonism onset as DLB21. Tissue slices of 50–100 mg macroscopically spanning all cortical layers, grey matter only, were cut from frozen frontal cortex tissue blocks, collected at autopsy and stored at −80 °C until further processing, in a cryostat, and DNA was extracted using the Qiagen AllPrep DNA/RNA/miRNA Universal Kit according to the manufacturer’s instructions.Samples in the discovery dataset were obtained from the Netherlands Brain Bank  and five brain banks (the Cardiff brain donations were banked in London). BDR is approved as a Research Tissue Bank by the National Research Ethics Service. All participants have given informed consent. Details on recruitment and cohort setup have been thoroughly described in a previous publication56. Post-mortem brains underwent full neuropathological dissection, sampling, and characterization by experienced neuropathologists in each of the five network brain banks using a standardized BDR protocol which was based on the BrainNet Europe initiative52. Additional information on the BDR DNA methylation dataset can be found in Shireby et al.25.A subset of donors from the BDR DNA methylation dataset24. Quality control was carried out in PLINK 1.957. Quality control included filtering of variants and individuals based on call rate (<0.95), Hardy-Weinberg equilibrium (p < 0.000001), relatedness (pi-hat > 0.125) and excess heterozygosity (>4 SD from mean) as well as sex-check and ancestry assessment based on principal component plots. Samples failing genotyping QC were excluded from all analyses. Genotypes were imputed using the Michigan Imputation Server58 using default settings and reference data from the Haplotype Reference Consortium59, and SNPs with an imputation r2 < 0.3 were filtered out. The NeuroChip array has also been designed to allow for screening for known pathogenic mutations in relevant Mendelian neurodegenerative genes. We identified no carriers of definitely or probably pathogenic variants in SNCA, LRRK2, and VPS35 in our discovery sample set. We used the “homozyg” function in plink 1.9 with window size 2.5 Mb and otherwise default parameters to screen for runs of homozygosity on chromosome 22, which could indicate the presence of a 22q11 microdeletion.All NBB samples were genotyped using the NeuroChip Consortium Array 60 package. For rigorous quality control, we applied a series of checks and filtering steps taking advantage of the minfi and wateRmelon v.1.26.061 R packages. CpG sites with a beadcount <3 in 5% of samples or detection p-value > 0.05 in 1% of samples were filtered out using the pfilter function in the wateRmelon package. No samples had detection p-value > 5% in 5% of sites. Two samples in the NBB data and 19 samples in the BDR data were removed due to low median signal intensities as flagged by the minfi getQC function. No outliers were detected by the wateRmelon outlyx function or inspection of bisulfite conversion or other control probe metrics. Sex chromosome CpGs were used to estimate sample sex and two samples failing sex-check were removed. We estimated the proportion of NeuN positive cells in each sample using reference data from flow sorted frontal cortex cell populations62 as implemented in the minfi package. Two outlier samples with low NeuN proportions were removed from the NBB dataset. We evaluated different normalization methods, ultimately selecting the wateRmelon dasen method61, which generated the minimum mean difference in beta value across technical replicate pairs in NBB data and has been successfully employed in previous AD EWAS with similar study design. The normalized MethylSet data object was mapped to the genome and probes on sex-chromosomes, probe sequences containing SNPs of any minor allele frequency in the MethylationEPIC annotation and previously reported cross-reactive probes63 were filtered out. It has been shown that a considerable proportion of methylation array CpGs have large measurement errors, making them unsuitable for statistical association testing in complex disorders. Taking advantage of the technical replicates in the NBB dataset we used the CpGFilter v1.1 package to compute the intra-class correlation coefficient (ICC), which characterizes the relative contribution of the biological variability to the total variability for each probe64. The authors of CpGFilter recommend discarding all probes with ICC below the median. We chose, however, to only filter out the lowest quartile. Finally, the MethylSet data object was converted to beta values and the wateRmelon pwod function was used to filter out values lying more than four times the interquartile range from the mean, outliers assumed to result from rare SNP artifacts.500 ng DNA from each sample was bisulfite treated and assessed using the Illumina Infinium MethylationEPIC BeadChip, assigning samples randomly to arrays. The NBB experiment included technical replicates for 30 samples, which were used for quality checks only and removed before statistical hypothesis testing. For both datasets, all data processing was performed using R 4.0.3. Raw signal intensity data were imported into R using the minfi v1.36.065. To further minimize the risk of unknown batch effects affecting the results, we estimated surrogate variables (SVs) using the sva v3.38.0 package66. In line with previous work, we evaluated models with different numbers of SVs and observed the effects on the test statistic distribution11. We found that including 3 SVs led the inflation measure λ to fall below 1.2 in the NBB analysis, without further improvement when additional SVs were added. Linear regression was performed using the limma package67 with the following model in both discovery and replication:There is no general consensus on whether to use beta or M-values for statistical analyses of methylation array data. We used beta values for our main analyses because this method has been favoured in previously published studies on AD neuropathology that inspired our work. However, we also repeated the statistical analyses with M-values, confirming that the main results obtained were highly similar  and defining Braak stage as a binary variable (0–2 versus 3–6).We also explored alternative linear models including neuropathological diagnosis as a categorical variable  tool34 to assess mQTLs in publicly available data.In the main analysis we adjusted for multiple testing using the Benjamini-Hochberg false discovery rate method, which is recommended as default for microarray studies in the limma package. Despite being somewhat less conservative than the Bonferroni method, we considered this to be justified given our two-stage design with independent replication. Fixed-effect meta-analysis was performed using the meta v5.2–0 package69. We nevertheless attempted to estimate smoking scores in the NBB data using the EpiSmokEr v0.1.0 package70 with default parameters. All donors were classified as either former or current smokers, and quantitative scores were not associated with Braak Lewy body stage or neuropathological diagnosis. We therefore chose not to include any smoking estimate in the model.Smoking exposure during life would have been a relevant variable to include as a covariate but was not available in our brain bank data. Tools have been developed to estimate smoking exposure based on reference data from association studies of smoking and DNA methylation in whole blood, yet this kind of method has been shown to have limited value when applied to human postmortem brain data in an analysis using tissue from the nucleus accumbens, and the same study found no evidence of shared differentially methylated CpGs across blood and brain28. Probes with mean methylation beta values below 0.025 or above 0.975 where removed and probe values were adjusted for sex, age at death, cell composition, experiment plate and postmorten interval, but not surrogate variables. Mixed linear model-based omic association (MOA) was then applied to analyse association between Braak Lewy body stage and adjusted probes.We converted the final matrices of NBB and BDR methylation beta values from the DNA methylation data processing detailed above and converted to BOD-format for further analyses using the OSCA v0.46 softwareThe same covariates were used when estimating the omics relationship matrix (ORM) using the OSCA software, followed by restriction maximum likelihood analysis (OREML) to estimate the phenotypic variance attributable to all probes.Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1-6Reporting Summary