A young kidney transplant recipient came to the emergency room with an elevated creatinine. His biopsy showed evidence of T cell–mediated rejection type 3, and the serum tacrolimus level was undetectable. Despite antirejection therapy, his graft function failed to recover, and the patient returned to dialysis.1 The COVID-19 pandemic was especially harsh on transplant recipients receiving chronic immunosuppressive therapy, who experienced a mortality rate of 13%–42%2 compared with the 1%–2% mortality rate observed in the general population. These observations explain the current 10-y non-death-censored renal allograft survival rate after living and deceased donor kidney transplantation remaining around 65% and 50%, respectively, with 15%–25% of recipients dying despite ongoing graft function.3 Faced with these dismal facts, we continue our quest for a more successful alternative—tolerance induction—the Holy Grail of organ transplantation.Obviously, this is a case of noncompliance with immunosuppressive medications. The usual response in this circumstance is frequently harsh. “The patient should not have received a transplant,” “Another transplant is out of the question,” or “Psychiatric evaluation is warranted.” Assigning blame may seem reasonable in this situation, but is it really fair to put it all on the patient? We take medications to improve our health with the good faith expectation that doing so will “make us feel better or cure what ails us.” However, taking immunosuppressive medication is a double-edged sword. Yes, these chronically prescribed toxins can help prevent graft rejection but not without considerable cost, including countless side effects and unintended complications that significantly increase the risk of death from infection, cardiovascular disease, and cancer.Transplantation, Leventhal et al4 report the long-term results of a phase 2 clinical trial for kidney allograft tolerance induction. In this trial, from 3- to 10-y immunosuppression-free kidney allograft survival was achieved in approximately 70% of recipients after persistent chimerism induction accomplished by combining donor hematopoietic stem cell (HSC) transplantation with a conditioning regimen that included the infusion of tolerogenic CD8+/TCR− facilitating cells. Immunosuppression was successfully withdrawn in recipients with persistent high-level chimerism (>95% donor cells) but not in those with transient chimerism. Patient and graft survival are comparable with that of recipients receiving ongoing immunosuppression, whereas the estimated glomerular filtration rate was superior in tolerant versus standard of care recipients. Although no comparison has been made with standard of care, no patients in this trial have experienced recurrence of the original cause of their kidney disease . Encouragingly, chimeric patients were safely vaccinated and developed protective immune responses, and there were no deaths among the 11 recipients with COVID-19 infection.In this issue of 5 Nevertheless, GVHD remains a serious concern in patients with high-level persistent chimerism, and 1 fatal GVHD was also reported in this trial. In more recent phase 3 studies (FREEDOM-1) using the same protocol, a higher incidence of GVHD, including another fatal case, eventually shut down the clinical trial. The more aggressive HSC mobilization strategy in the phase 3 trial may be attributed to the difference in the incidence of GVHD from the phase 2 trial, but safety should be established when using this approach with persistent, high-level donor hematopoietic chimerism.The apparently required persistent presence of chimerism is considered a reliable biomarker of tolerance, but the authors have provided additional urinary cell mRNA profiles associated with tolerance. It is impressive that the incidence of graft-versus-host disease (GVHD) was low compared with regular bone marrow transplant recipients who received a comparable regimen but without facilitating cells.6 They carefully adjusted the dose of CD3+ cells infused with the HSCs to prevent GVHD while promoting HSC engraftment. However, persistent chimerism has been difficult to achieve with low-level T-cell chimerism in HLA-mismatched transplant recipients, and no allograft tolerance has thus far been achieved in the HLA-mismatched cohort using this strategy.Two other chimerism-based strategies have been employed to induce renal allograft tolerance Figure . Using t8 In this strategy, tolerance appeared to be more dependent on regulatory mechanisms.9 Because the chimerism is transient, GVHD is not a concern, but identifying reliable tolerance biomarkers may be critically important because of “presence of chimerism” cannot be used as a biomarker for tolerance.The Massachusetts General Hospital group has achieved long-term (up to 17 y) immunosuppression-free renal allograft survival in HLA-mismatched transplant recipients after induction of transient mixed chimerism.10 published their Nobel-prize-winning report on neonatal tolerance in 1953. As we savor our first taste from the Holy Grail, we do so with the clear understanding that novel immunological interventions will have to be incorporated into the chimerism-based tolerance strategy to make tolerance induction sufficiently easy and safe for universal application.Seven decades have passed since Billingham et al


Saccharomyces cerevisiae carrying different conformational variants of the Rnq1p to obtain Liquid Chromatography-Mass Spectrometry (LC-MS) metabolic profiles and identify key perturbations of prion presence. These studies reveal that prion containing [+RNQ] cells display a significant reduction in amino acid biosynthesis and distinct perturbations in sphingolipid metabolism, with significant downregulation in metabolites within these pathways. Moreover, that native Rnq1p appears to downregulate ubiquinone biosynthesis pathways within cells, suggesting that Rnq1p may play a lipid/mevalonate-based cytoprotective role as a regulator of ubiquinone production. These findings contribute to the understanding of how prion proteins interact in vivo in both their prion and non-prion confirmations and indicate potential targets for the mitigation of these effects. We demonstrate specific sphingolipid centred metabolic disruptions due to prion presence and give insight into a potential cytoprotective role of the native Rnq1 protein. This provides evidence of metabolic similarities between yeast and mammalian cells as a consequence of prion presence and establishes the application of metabolomics as a tool to investigate prion/amyloid-based phenomena.Within the field of amyloid and prion disease there is a need for a more comprehensive understanding of the fundamentals of disease biology. In order to facilitate the progression treatment and underpin comprehension of toxicity, fundamental understanding of the disruption to normal cellular biochemistry and trafficking is needed. Here, by removing the complex biochemistry of the brain, we have utilised known prion forming strains of  Protein misfolding disease can be loosely separated into two groups: the non-transmissible amyloid diseases  and the transmissible amyloid diseases . The amyloid and prion diseases present the same misfolding mechanism and thus prions can be used to recapitulate the biochemical hallmarks of amyloid disease. Prion  (+GAR] and [+SMAUG]) support this Lamarckian inheritance model (The  to [−rnq], as is the atypical prion treatment of GdnHCl curing  can be considerable. Recent literature suggests careful consideration of the data analysis programs used is capable of controlling for these undesirable variables , 33. Res−rnq] and rnq1Δ, [+RNQ] and [−rnq]stress, respectively, as shown in 2 and Q2 values shown in 2 values indicate that residual variation within datasets must exist within the experimental replicates. This variation is visible within the groupings, with PIM results appearing in tighter clusters than NIM supporting the calculated Q2 values. Regardless, apparent clustering trends of groups within the models support the progression to OPLS-DA models shown in 2(X), R2(Y) and Q2 scores giving confidence in the model’s predictive ability, with all models giving statistically significant CV-ANOVA p-values (<0.01).To identify the presence of metabolic changes between the test data sets, standard multivariate analysis was employed. Principal component analysis (PCA) using true features as determined by cross comparison demonstrates clear group separation between [−rnq] and [+RNQ] samples (n = 6) were then uploaded to BioCyc’s omics dashboard. The most significantly altered pathways (as determined by DPSS score via BioCyc) are shown in +RNQ] prion formation, thus highlighting unique features of native Rnq1p. However, ubiquinol-6 biosynthesis appears significantly downregulated in both [−rnq] and [+RNQ] samples suggesting a previously unknown role of Rnq1p as a regulator of ubiquinone-6 biosynthesis. Differences in regulation between loss by deletion of Rnq1p (rnq1Δ) and the prion-form of Rnq1p [+RNQ] were also seen, specifically within the superpathway of geranylgeranyldiphosphate biosynthesis I  and amino acid synthesis, with the general trend revealing downregulation of these pathways within prion containing cells [+RNQ] samples when compared to rnq1Δ.To allow for pathway analysis within BioCyc, all significant punitively identified metabolites  relative abundances were collated (including both PIM and NIM). Standardised relative abundances of the most significantly altered metabolites  from the rnq1Δ, [+RNQ] prion presence, pathways identified as being specific to loss of Rnq1p native function were removed from the analysis between prion-free ([−rnq]), prion-containing ([+RNQ]) and prion-free with mild stress ([−rnq]stress) cells  cells . The sub+RNQ] prion, those perturbed features identified due to loss of Rnq1p function effects and those caused by general stress indicators (as discussed above) were removed from the top 20 most perturbed pathways identified from the prion-free ([−rnq]) vs prion-containing ([+RNQ]) analysis  have created a need for faster, more accurate and reliable computational pre-processing. This has led to the availability of variety of programmes used to construct extracted ion chromatograms (EIC), detect and annotate EIC peaks, align samples, identify and relatively quantify analytes . With a −rnq] with those obtained from the rnq1Δ deletion mutant and loss of function of Rnq1p in its amyloid state ([+RNQ]), reveal an upregulation of those pathways involved in the biosynthesis of many amino acids and key cellular metabolites  compared to those that do contain Rnq1p [−rnq]. Ubiquinol-6 in its reduced and oxidised forms (ubiquinone), plays a pivotal role within the mitochondrial-based electron transport chain, residing within the inner mitochondrial membrane and acting as a lipid-soluble electron carrier and central electron pool, passing electrons on to complex III of the respiratory chain    is obser−rnq] cells of this pathway. However, we also observed that prion containing samples display a widespread downregulation in general biosynthesis, including many pathways that either are or directly affect amino acid regulation. Intriguingly, amino acid restriction in S. cerevisiae is beneficial to the overall health of cells, promoting longevity . These [+RNQ] prion-only differences were found to be within the superpathway of the phenylalanine, tyrosine and tryptophan biosynthetic pathway. Measurement of metabolites within this pathway offer potential for their use as indicative biomarkers for [+RNQ] prion presence by permitting the distinction between the cellular response as a result of oxidative stress to that of misfolded protein presence.The metabolic perturbations observed within prion-free [nditions  were comin vitro tyrosine residues in patients with PD were found to be sequestered into covalently crosslinked homodimers that appear to play a key role in the formation of seed and oligomeric species (Further supporting evidence for the involvement of the tyrosine biosynthetic pathway in protein misfolding and aggregation as identified in this study comes from a separate study on PD (Parkinson’s Disease) were  species . Without+RNQ] prion is the down regulation of sphingolipid recycling and degradation (shown in The most significant perturbation that occurred due to the presence of the  samples simply due to the cells using all their sphingolipid resources available to cope with the number of misfolded proteins. Indeed, within amyloid biology it is well documented that microdomains are formed by the sphingolipids and cholesterols that act as sites for the binding and oligomerisation of amyloidogenic proteins (SC), sphingolipid rafts present in cell plasma membranes have been found to decrease in concentration  trp1-289(UAG) ura3-52 his3-Δ200 leu2-3, 112). Yeast harbouring [+RNQ] and knockout strain Δrnq were kind gifts from the Kent Fungal Group. Yeast were grown at 30°C with shaking at 180 rpm in synthetic complete (SC) media (2% (w/v) glucose, 0.17% Yeast Nitrogen Base , 0.5% ammonium sulphate, the appropriate concentration of yeast synthetic complete supplement mixture or synthetic complete drop-out media supplement). Transient growth on SC media containing 3mM guanidine hydrochloride (GdnHCl) was used as a curing agent in the media of S. cerevisiae cells that required a [−prion] status. Mild oxidative stresses were achieved by the addition of H2O2  to the appropriate culture mediums. Cultures were grown using the filter culture method, . Reversed phase (RP) separation was employed using a 1.7 μm C18 BEH column , heated to 35°C and operated with a 10-minute gradient from 0–50% acetonitrile (0.1% formic acid) at a flow rate of at 500 nL/min. Detection was carried out in both positive and negative mode, respectively. A capillary voltage of 2.5 kV and a cone voltage of 40 V were used. Nitrogen, the desolvation gas, was set to 800 L/hr at a temperature of 400°C. MSE continuum mode was used for data acquisition over the mass range of 50-1200 m/z, with a scan time of 1 second, which was programmed to step between low (10 eV) and elevated ramp collision energies (20–35 eV) with argon was used as the collision gas. The data was collected in Data Independent Acquisition (DIA) MS/MS mode. Leucine-enkephalin (556.2771 m/z) was used as a lock-spray standard injected every 10 seconds for 1 second. Water blanks were also analysed every 50 samples to check for contamination. For quality control, 25 μL of each sample was pooled run ≈ every 10 samples for quality control.et al. (2019). Statistical analysis was performed using SIMCA 14.1 (Umetrics). Punitive compounds and pathways were identified using Metaboanalyst’s Mummichogg function MS Peaks to Pathways (The raw data (.raw) files were automatic peak detected via Masslynx  and then converted to .mzML format using Proteowizards MSConvert. To ensure robustness, the subsequent mzML. files were then analysed using two separate data analysis programmes, MZmine2 and XCMS , and a comparative analysis using overlapping features of the two programmes using VBA programming, adapted from Li Pathways . BioCyc’Pathways . These p


To investigate the diagnostic value of unenhanced CT in mechanical small bowel obstruction (SBO) with small bowel necrosis, and to establish a predictive model.From May 2017 to December 2021, the patients with mechanical SBO admitted to our hospital were retrospectively collected. Taking pathology-confirmed small bowel necrosis as the gold standard, the experimental group was composed of patients with small bowel necrosis confirmed by pathology, and the control group was composed of patients with no intestinal necrosis confirmed by surgery or successful conservative treatment with no recurrence of intestinal obstruction during 1-month followed-up.P = 0.002), diffuse mesenteric haziness (P = 0.010), difference of CT value between mesenteric vessel and aorta (P = 0.025) and U-/C-shaped small bowel loop (P = 0.010) were independent risk factors for the diagnosis of mechanical SBO with small bowel necrosis. Through internal verification, the area under curve (AUC) of the predictive model reached 0.886 (95%CI: 0.824–0.947), and the calibration result was moderate.A total of 182 patients were enrolled in this study, 157 patients underwent surgery, of which 35 patients were accompanied with small bowel necrosis and 122 patients were not (33 patients with ischemic findings at surgery without necrosis). Finally, there were 35 patients in the experimental group and 147 patients in the control group. Multivariable logistic regression showed that increased attenuation of small bowel wall  of unenhanced CT have clinical value in the diagnosis of mechanical SBO with small bowel necrosis. The predictive model based on these four features could achieve satisfactory efficiency.The online version contains supplementary material available at 10.1186/s12880-023-01041-2. Mechanical small bowel obstruction (SBO) was one of the main complications requiring emergency surgery, accounting for 20~50% of emergency surgeries . Among tCurrently, computed tomography (CT) is the first choice for diagnosing intestinal obstruction , and is Therefore, our study aimed to find objective risk factors of mechanical SBO with small bowel necrosis on unenhanced CT imaging, and establish a predictive model.This single-center retrospective study was approved and waived the need for an informed consent by the medical ethics committee of our institution (JJSDYRMYY-YXLL-2021-258).From May 2017 to December 2021, the patients with mechanical SBO admitted to our hospital were retrospectively collected, with “pathology-confirmed small bowel necrosis” as the gold standard. The experimental group consisted of patients with small bowel necrosis confirmed by pathology, while the control group consisted of patients with no small bowel necrosis confirmed by pathology or successful conservative treatment and without recurrence of intestinal obstruction in 1-month followed-up. To be included, patients must meet the following conditions: ⑴ adult patient (≥ 18 years); ⑵CT examination within 12 h after admission; ⑶ unenhanced CT scan was diagnosed as mechanical SBO. Patients who met the following conditions would be excluded: ⑴ non-adult patients; ⑵CT examination for more than 12 h or only enhanced CT, no unenhanced CT; ⑶small bowel obstruction caused by inflammatory bowel disease, abdominal tuberculosis, intestinal tumor , intestinal foreign body, and external abdominal hernia; ⑷ inflammatory obstruction; ⑸the CT imaging only contained part of the abdomen and the data collection could not be completed accurately; ⑹the patients were discharged automatically without treatment. Finally, we screened out 182 patients with mechanical SBO, and 157 patients underwent surgery, of which 35 patients were accompanied by small bowel necrosis and 33 patients were accompanied by small bowel ischemia without necrosis  with 10 years of experience in abdominal CT, who were familiar with the standard of imaging indicators, and blinded to the status of patients , the final value of those imaging indicators were determined by a consensus read. A total of 17 imaging indicators were collected from 182 unenhanced CT data collected by the imaging workstation, which were defined as follows, and the schematic diagram was shown in Fig. The following imaging indicators of unenhanced CT were recorded:(1) Increased attenuation of small bowel wall was subjectively defined as high density of the small bowel wall of a dilated loop compared with the healthy dilated loop , 18.(2) CT value of small bowel wall was defined as the CT value of the axial plane where the intestinal obstruction was most obvious .(3) Small bowel wall annular thickening was defined as annular wall thickness ≥ 2 mm .(4) Small bowel lumen dilation was defined as the maximum diameter of the intestinal lumen at the most obvious dilatation was ≥ 3 cm .(5) Maximum diameter of small bowel lumen dilation was defined as the maximum diameter of the intestinal lumen at the most obvious dilatation .(6) Mesenteric ground glass sign was defined as the adipose tissue density around the blood vessels in the mesentery area was higher than that of the normal mesenteric fat, showing cloudy or ground glass-like changes .(7) CT value of mesenteric vessel was defined as the CT value of the most obvious mesenteric vessels on the cross-section, most of the vessels were vein .(8) Difference CT values between mesenteric vessel and aorta was defined as the CT value of mesenteric vessel minus the CT value of aorta.(9) Mesenteric vasodilation was defined as relative dilatation of the mesenteric vessel around the small bowel obstruction site compared with those distant from the obstruction site .(10) Diffuse mesenteric haziness was defined subjectively as diffuse density increase of mesenteric fat around the small bowel obstruction caused by edema .(11) Small bowel fecal gas sign was defined as the presence of fecal material containing air bubbles in a single segment in the small bowel lumen proximal to the obstructive zone .(12) U-/C-shaped small bowel loop was defined as the obstruction points at both ends of a dilated intestine, forming a “U”-shaped or “C”-shaped dilated intestinal loop, and the mesentery in the intestinal loop is stretched and unified gather between two obstruction points .(13) Ascites was defined as intraperitoneal fluid visible to the naked eye on the cross-Sect.  (14) Peritoneal thickening was subjectively defined as the thickening of peritoneum around intestinal obstruction, paracolic sulcus or pelvis compared with normal peritoneum .(15) Pneumatosis intestinalis was defined as the presence of gas in the intestinal wall .(16) Portal venous gas was defined as the presence of gas in the portal vein .(17) Whirlpool sign was defined as a soft tissue mass with internal structures of swirling mesenteric vessels and fat attenuation reflecting mesenteric torsion .Categorical data are presented as number and percentage and were compared by Pearson’s chi-square test or Fisher’s exact test. When continuous variables conformed to normal distribution, variables were expressed as mean ± standard deviation and compared using t test; when they conformed to non-normal distribution, variables were expressed as median sum (range) and compared using Wilcoxon test.Inter observer variability assessment was used Cohen kappa test for categorical variables and intraclass correlation coefficient (ICC) for continuous variables . The 95%P<0.05 was determined to be statistically significant. We developed the predictive nomogram by R 4.2.1 (http://www.r-project.org) with the rms package. C-statistic was adopted to validate the nomogram internally. To reduce overfit bias, we validated the nomogram internally by bootstraps with 1000 resamples.All data were analyzed using SPSS 23 Statistics software , and The general characteristics of the study population were presented in Table The results of the consensus reading and inter observer agreements were presented in Table P = 0.001), increased attenuation of small bowel wall , small bowel wall annular thickening , mesenteric ground glass sign , diffuse mesenteric haziness , U/C-shaped small bowel loop , ascites  and peritoneal thickening  between the experimental and control group , difference CT values between mesenteric vessel and aorta , diffuse mesenteric haziness , and U-/C-shaped small bowel loop  were independent risk factors for the diagnosis of mechanical SBO with small bowel necrosis , which could be reflected on CT imaging .The exudation in different tissues presented different characteristics on CT imaging. (1) The exudation in intestinal wall was usually presented as a thickened, double-layered ring sign or target sign. Sometimes it could also cause oozing blood in the intestinal wall, which could be easily identified as a mixed high-density shadow on unenhanced CT imaging . UnfortuIn the process of metamorphism, according to the different stages of pathological changes, the intestinal wall shows successively thickening, weakening or disappearance of reinforcement, or thin paper-like shape . In our The CT findings of intestinal hyperplasia stage are thickening of intestinal wall and lead-like ankyloses, which usually indicates severe or intestinal ischemia . U-/C-shOur study also found that mechanical SBO patients without history of abdominal surgery were more likely to be complicated with small bowel necrosis. This may be the deviation caused by our small case. Theoretically, the history of abdominal surgery is related to intestinal adhesion, and adhesion bands and internal hernias caused by intestinal adhesion are more likely to cause closed-loop intestinal obstruction, leading to small bowel necrosis , 22, 23 Darras et al.  suggesteThe limitations of this study were as follows: Firstly, the small sample size and high proportion of surgical patients may lead to potential bias. Secondly, the predictive model was only validated internally instead of externally. Thirdly, this study did not distinguish simple and complicated mechanical SBO. According to the classification of mechanical SBO, it would be better to distinguish simple and complicated mechanical SBO. However, it is always difficult to accurately distinguish them in clinical practice .Multiple features  of unenhanced CT have clinical value in the diagnosis of mechanical SBO complicated with small bowel necrosis. The predictive model for mechanical SBO achieved satisfactory efficiency.Below is the link to the electronic supplementary material.Supplementary Material 1


RPGR-targeted therapy for XLRP. Data presented concerned RPGR structure and mutation types known to cause XLRP, RPGR mutation–associated retinal phenotype diversity, patterns in genotype/phenotype relationships, disease onset and progression from natural history studies, and the various functional and structural tests used to monitor disease progression. Panel recommendations include considerations, such as genetic screening and other factors that can impact clinical trial inclusion criteria, the influence of age on defining and stratifying participant cohorts, the importance of conducting natural history studies early in clinical development programs, and the merits and drawbacks of available tests for measuring treatment outcomes. We recognize the need to work with regulators to adopt clinically meaningful end points that would best determine the efficacy of a trial. Given the promise of RPGR-targeted gene therapy for XLRP and the difficulties encountered in phase III clinical trials to date, we hope these recommendations will help speed progress to finding a cure.X-linked retinitis pigmentosa (XLRP) is a rare inherited retinal disease manifesting as impaired night vision and peripheral vision loss that progresses to legal blindness. Although several trials of ocular gene therapy for XLRP have been conducted or are in progress, there is currently no approved treatment. In July 2022, the Foundation Fighting Blindness convened an expert panel to examine relevant research and make recommendations for overcoming the challenges and capitalizing on the opportunities in conducting clinical trials of RPGR-associated XLRP.Examination of relevant data and recommendations for the successful clinical development of gene therapies for  Key topics were:The Expert Panel's goal was to formulate recommendations for designing XLRP gene therapy clinical studies to provide the best chance of successful treatment development. This article summarizes the meeting's presentations and discussions and the panel's recommendations.–RPGR was first identified in 1996 as the gene responsible for the most common form of XLRP.,RPGR is found in a variety of tissues throughout the body, including in the eyes, brain, lungs, testes, and kidneys.,,,,,RPGR mutations have been associated with XLRP and represent a wide variety of types, including missense, nonsense, frameshift, splice, and insertion/deletion.–RPGR occur, they are not common and often share a common ancestor rather than arising independently.,Located on Xp11.4,RPGR variants, as with other genes, is spread across multiple individual databases, some of which are proprietary.–RPGR-targeted therapy for XLRP in part because discussions with the US Food and Drug Administration (FDA) have suggested that a genetic therapy must target a gene with variants that have been validated for pathogenicity . The FDA has recognized ClinGen as a valid source of human genetic mutation interpretation data, and work on the X-linked retinal disease domain is on-going.,Limits on data accessibility, as well as lack of information on phenotypic and functional effects associated with mutations, hamper the clinical use of genetic variant information. The information on RPGR mutations fall within the broad category of progressive diffuse photoreceptor diseases. Traditionally, they have been classified into the subgroups “rod dystrophy,” rod-cone dystrophy , cone-rod dystrophy (CRD), and cone dystrophy (CD) based on the pattern and extent of rod versus cone photoreceptor degeneration from full-field flash electroretinography (ffERG).,,,Clinical phenotypes associated with pathogenic RPGR-targeted gene therapy trials, it is necessary to differentiate between rod- and cone-predominant dystrophy phenotypes due to their differing clinical appearance and progression patterns. RP, which includes simple/nonsyndromic, syndromic, and systemic disease, is the most common retinal dystrophy, with a worldwide prevalence ranging from approximately 1:3700 to 1:8300, reflecting regional differences.,,,,,,,,,,In the context of defining target populations for ,RPGR mutations are the cause in the majority of them.,,,–,,The cone dystrophies—CRD, in which cone dysfunction precedes rod dysfunction, and CD, in which rod photoreceptors remain unaffected—are less common than RP, with a global prevalence of approximately 1:30,000 to 1:40,000.RPGR mutations is comparatively later than that for RPGR-associated XLRP. Male patients with XLRP typically are diagnosed in childhood or early adolescence, whereas X-linked CD and CRD symptom onset typically occurs in adulthood.,,RPGR-associated retinal dystrophies in the Netherlands by Talib et al. found the median age of onset for those with XLRP (n = 52) was 5 years compared with 23 years for those with CD/CRD (n = 22).Age of onset for X-linked CD and CRD arising from RPGR mutations (approximately 70%) present as the XLRP phenotype, with a much smaller proportion leading to X-linked CRD (up to 23%) and an even smaller proportion resulting in CD (approximately 7%).,,,,,,COD1, which maps to a region harboring RPGR, and identified 2 mutations in RPGR ORF15 (ORF+1343_1344delGG and ORF+694_708del15) that were present in affected individuals but not in control subjects.RPGR ORF15 3′ region that resulted in premature protein truncation in an analysis of 2 families from the Netherlands with X-linked CD.RPGR mutations resulting in the XLRP phenotype may be concentrated in exons 1 to 14 and the proximal part ORF15, with a “watershed” zone of mutations in ORF15 occurring at about region 949 to 1047 that may be associated with either XLRP or X-linked cone dystrophies.Most ,,,P < 0.05), and all had elevated thresholds on subsequent annual visits. The remaining 5 participants with normal pupil thresholds also subsequently had normal ffERGs after age 5 years and exhibited no XLRP symptoms.RPGR-related forms are among the most severe forms of RP. As noted above, XLRP symptom onset typically occurs in childhood or early adolescence.RPGR-associated XLRP based on genotype and other factors.,,P < 0.001).RPGR-associated RP, CD, or CRD, BCVA also declined more quickly for individuals with mutations in ORF15 (0.022 logMAR/year) compared with those with mutations in exons 1 to 14 (0.015 logMAR/year). However, the difference was not statistically significant.RPGR mutations. In addition, the investigators genotyped individuals for coding single nucleotide polymorphisms (SNPs) in 4 modifier genes with products that interact with RPGR protein: RPGRIP1, RPGRIP1L, CEP290, and IQCB1. Individuals were stratified based on ffERG and Humphrey visual field (VF) testing into clinical phenotypes of grade 1 (mild), 2 (moderate), or 3 (severe). The findings showed a range of disease severity between and within families.RPGR exons 1 to 14 resulted in a more severe phenotype than those in ORF15: individuals with mutations in this area were more likely to have moderate or severe disease (P = 0.016), whereas the disease severity distribution for individuals with mutations in ORF15 was roughly equal across all 3 phenotype grades. This study also found that individuals with null alleles were more likely to have moderate or severe disease (P = 0.0038), whereas the disease severity distribution for individuals with mutations resulting in variant protein was roughly equal across all three phenotype grades. Two coding SNPs in RPGRIP1L (R744Q) and IQCB1 (I393N) interacting with RPGR also were significantly associated with more severe disease (P < 0.05).RPGR.Several studies have investigated disease severity and progression rate in individuals with ,P = 0.05). This study also illustrated the utility of GVF area in determining RP disease progression. For the entire cohort, annual GVF area loss rates varied significantly based on target size, with a faster deterioration rate seen for the smaller targets III4e (10.7%) and I4e (12.5%) than for the larger V4e target .XLRP is associated with progressive visual function loss revealed by testing visual acuity and VF. However, because visual acuity can be preserved until later stages of RP progression,–n = 10) individuals with RPGR-associated RP showed good repeatability.RPGR-associated retinopathy also generally showed good interocular symmetry, with variations between individuals and based on the measurement used. In this study, based on Octopus mean sensitivity values, the median annual rate of VF decline was 7.62%, and the overall annual rate of exponential decline was 4.67%.Static automated perimetry's (SAP's) usefulness to evaluate VF in XLRP may vary based on the instrument used. Several studies have found that Humphrey static perimetry test-retest variability in RP can be high, especially in regions with low sensitivity of 8 to 12 dB.,RPGR-associated retinopathy. Mean follow-up was 2.8 years, and most participants had XLRP. The method resulted in a high test-retest reliability and good overall interocular symmetry in mean sensitivity (MS) and volumetric indices. Mean annual progression rates of MS, total volume, and central 3-degree field volume  were comparable with rates recorded in previous studies of full-field static perimetry measured with the Octopus 900 perimeter.,RPGR) showed retinal sensitivity improvement on MAIA microperimetry in the treated eye from 0.5 dB to 6.6 dB after 3 months.Fundus-driven perimetry, or microperimetry, is a solution for quantifying the central VF and monitoring progression in individuals with inherited retinal disease (IRD) who have relatively unstable fixation, offering test-retest repeatability and correlation to retinal pathology.n = 10) exhibited a significantly higher annual rate of rod ERG threshold elevation (0.22 log/year) than those with autosomal dominant RP .P = 0.027).Several studies have investigated visual function loss in RP and XLRP using ffERG. Birch et al. showed that, over a 4-year interval in a cohort of 67 individuals with RP and measurable rod ERG at baseline, 64% and 60%, respectively, showed rod and cone ERG amplitude declines, with a larger annual change in rod ERG threshold versus cone ERG threshold.,Full-field stimulus testing (FST) has been used in natural history studies and clinical trials of gene therapy for rare IRDs, such as Usher syndrome type 2A and Leber congenital amaurosis.,P < 0.001), which is similar to the annual GVF decrease observed over 4 years by Xu et al. (7.2%).n = 20) who underwent SD-OCT twice on the same day.RPGR-associated RP found a positive correlation between EZ dimension and disease progression, with an overall annual exponential EZ width decline rate of 8.2%.en face OCT imaging to quantify the entire EZ area as proof-of-concept to support this measure's utility to monitor disease progression.–2 to 2.40 mm2 over 1 year.,Structural tests also can be used to determine XLRP disease progression and may have advantages over VF as clinical trial outcomes measures because of the shorter duration needed to document change.RPGR-targeted gene therapy clinical studies in XLRP are summarized below. These recommendations are based on careful consideration of the available literature, as well as the panel's experience in XLRP and RPGR research, including clinical trials. The panel hopes these recommendations can help guide discussions among investigators, industry, and regulators working toward the ultimate goal of curing affected individuals and preventing blindness.The Expert Panel's recommendations for designing  •RPGR, might impact phenotype and confound analysis.Expert genetic review boards for clinical trials are essential to help determine inclusion and exclusion criteria based on genetic mutations. For example, a board may make a recommendation that only pathogenic variants in RPGR be considered initially for inclusion in a clinical trial. Pathogenic variants in genes other than •RP2 mutation pathology, identify and document the pathogenic RPGR mutation, and define the break points and DNA level change of structural variants.○A pathogenic CHM mutation associated with X-linked choroideremia, should be excluded.At minimum, it is important to confirm that the mode of inheritance is X-linked, exclude an •Because cone-rod versus rod-cone phenotypes typically require different measures of progression, they should not be mixed in one arm of a trial.•○However, trials should collect as much information as possible about the pathogenic mutations as part of standard genetic work-up in anticipation that technology will be available in the near future to allow investigators to better correlate XLRP genotype and phenotype.It is too early in our understanding of genotype/phenotype relationships and of modifier gene effects in XLRP to use specific mutations or mutation types to balance treatment groups or stratify trial participants.•Because of the wide phenotypic variation among female carriers, clinical trials should include (or should focus on) only male participants and/or have separate trials or cohorts for affected female individuals.•○Participants must have residual microperimetry or static perimetry responses or residual EZ on OCT to allow for an outcome to be measured.○Children, adolescents, or young adults with XLRP may be the most likely age group to benefit.Given the rapid progression of vision loss, age <35 years is a reasonable inclusion parameter for an XLRP RPGR gene therapy trial because:•Because of the relationship between age and severity, it may be desirable to stratify by age; however, stratification beyond age 35 years is probably not meaningful.•○Consideration should be given to increasing the number of patients in early clinical studies, because the volume of phase I/II data needed for this type of analysis is more than is typically collected in a clinical development program.Gathering preliminary efficacy data from phase I/II clinical trials, although not their primary purpose, may help inform inclusion criteria, population stratification factors, and outcomes measures for phase III trials, allowing investigators to enrich later trials with the types of participants in which there was the largest efficacy signal.•Although a 2-year phase III clinical trial may be sufficient, a longer duration (3–4 years) may be required to demonstrate treatment efficacy for an RPGR-targeted therapy for XLRP, particularly when using VF as an outcome measure.•○Having more than one therapy dose arm in a phase III trial dilutes the efficacy, safety, and tolerability outcomes data.○Determining the maximal tolerable dose is done early in clinical development  and changes in structure (e.g. OCT) could be a useful compound end point in phase III trials because of its potential to show the benefit of therapy even if the primary end point is not met.•RPGR-targeted gene therapy clinical development program for XLRP, because the data they provide can help:○Determine cohort size needed to demonstrate efficacy.○Validate age as a disease progression biomarker for stratification.○Determine additional biomarkers that indicate fast versus slow progression, which could be used to select and stratify participants.○Determine the VF cutoff point for inclusion, which must allow for treatment efficacy measurement.○Validate efficacy end points selection, such as EZ changes or changes on microperimetry or static perimetry.○Determine expected disease progression over a certain amount of time to provide a benchmark against which to demonstrate lack of disease progression with treatment, which may be a reasonable measure of efficacy in XLRP (versus the higher bar of vision improvement).Natural history studies are a critical component of an •○However, because of the rapid disease progression with XLRP, a shorter natural history study of <2 years could be of value if the cohort size was large (approximately 200 participants) and the outcome measures focused on changes that could be detected in this time frame, such as EZ changes.Ideally, natural history studies for XLRP should be conducted prior to conducting clinical trials and, given the typical small cohort size for a rare disease like XLRP, should be of 2 to 3 years’ duration.•It would be useful for the field if such data is made accessible in an open clinical trial data repository (there are a few) or via a publication.


HPSE2, whereas other rare families carry variants in LRIG2.LRIG2 is immunodetected in pelvic ganglia sending autonomic axons into the bladder. Moreover, Lrig2 mutant mice have abnormal urination and abnormally patterned bladder nerves. We hypothesized that peripheral neurogenic defects underlie LRIG2-associated bladder dysfunction.Urofacial, or Ochoa, syndrome (UFS) is an autosomal recessive disease featuring a dyssynergic bladder with detrusor smooth muscle contracting against an undilated outflow tract. It also features an abnormal grimace. Half of individuals with UFS carry biallelic variants in LRIG2-associated UFS and studied Lrig2 homozygous mutant mice with ex vivo physiological analyses.We describe a new family with HPSE2 sequencing was normal, he carried a homozygous, predicted pathogenic, LRIG2 stop variant . Lrig2 mutant mice had enlarged bladders. Ex vivo physiology experiments showed neurogenic smooth muscle relaxation defects in the outflow tract, containing the urethra adjoining the bladder, and in detrusor contractility. Moreover, there were nuanced differences in physiological outflow tract defects between the sexes.The index case presented antenatally with urinary tract (UT) dilatation, and postnatally had urosepsis and functional bladder outlet obstruction. He had the grimace that, together with UT disease, characterizes UFS. Although LRIG2 variants, the full UFS phenotype occurs with biallelic stop or frameshift variants, but missense variants lead to bladder-limited disease. Our murine observations support the hypothesis that UFS is a genetic autonomic neuropathy of the bladder affecting outflow tract and bladder body function.Putting this family in the context of all reported UT disease-associated   Congenital UT malformations (UTMs) occur in 4 per 1000 human births,,UFS is an autosomal recessive condition causing lifelong ill health with potentially fatal complications.,HPSE2 (OMIM UFS2 #236730), which encodes heparanase-2, a protein that binds heparan sulfate and also inhibits the endoglycosidase enzymatic activity of a related protein called heparanase.,As reviewed,HPSE2 variants. A small number of individuals with UFS have been reported to carry biallelic variants of LRIG2 (OMIM USF2 #615112).,LRIG2 codes for leucine rich repeats and immunoglobulin-like domains 2, 1 of 3 mammalian LRIG proteins.Lrig2 or Hpse2 mutant mice manifest aberrant urinary voiding,,Not all individuals with UFS have LRIG2 variant, and place this in the context of all other UT disease-associated LRIG2 variants reported to date. We hypothesized that peripheral neurogenic defects underlie LRIG2-associated bladder dysfunction. Accordingly, we studied Lrig2 mutant mice with a battery of ex vivo physiology analyses. Our murine observations support the hypothesis that UFS is a genetic, autonomic neuropathy of the bladder with functional defects in both the outflow tract and detrusor smooth muscle in the bladder body.We report here a new family with UFS, associated with a homozygous The family described below was assessed at the Department of Clinical and Molecular Genetics and the Department of Pediatric Surgery, Pediatric Urology and Renal Transplant Unit, Hospital Vall D´Hebron, Barcelona, Spain. The parents, legal guardians, gave written informed consent, as approved by Vall d'Hebron Hospital Ethics Committee, for molecular genetic testing. The family also gave permission for their clinical story to be told, and informed consent for their child’s face to be shown here.Lrig2 mice carrying a targeted deletion of exon 12,Lrig2 (Mut) mice were generated by mating heterozygous parents. Lrig2 protein is present in normal mouse bladders but is not immunodetected in Mut bladders.Lrig2 Mut mice were culled by cervical dislocation in accordance with Schedule 1 of the Animals (Scientific Procedures) Act 1986 (ASPA) and with local Animal Welfare Ethical Review Body (AWERB) approval (University of Manchester). Experiments were performed on bladder preparations from WT and Mut mice as described previously for Hpse2 mutant mice.Lrig2 Mut outflow tracts to relax in response to electrical field stimulation  or the directly acting nitric oxide (NO) donor, sodium nitroprusside (SNP), were compared with WT littermates. Relaxation was measured against a background of preconstriction, which was evoked in males by the α-1 receptor agonist, phenylephrine . Female outflow tracts contracted poorly to PE, confirming an earlier report.2+ influx through voltage-gated Ca2+ channels to elicit contraction. Bladder bodies were subjected to EFS  to induce frequency-dependent contractions mediated by acetylcholine-releasing parasympathetic nerves. Contraction was also evoked directly by the muscarinic agonist, carbachol (10 nM – 50 μM) and compared between WT and Mut mice.WT and . Histologic evaluation of mouse bladder bodies was performed as previously described.,Detailed protocols are provided in the Supplementary MethodsDetails of data and statistical analysis are given in the HPSE2 and LRIG2 were sequenced . No variants were found in HPSE2 but a homozygous variant (NM_014813.2): c.1939C>T (p.Arg647∗), classified as pathogenic, according to American College of Medical Genetics guidelines,LRIG2. The stop codon was in exon 14 coding for an immunoglobulin-like domain , the percentage area positive for collagen in detrusor was similar between the genotypes , coherence was similar in sex-matched mutants and wild-types . The current ex vivo physiology experiments complement our initial published study,Lrig2 mutant mice have abnormal urination patterns (like people with UFS), as well as abnormally patterned bladder nerves (as assessed by whole-mount peripherin immunostaining). The novel mouse studies we report here, which point to mainly neurogenic functional defects, support the overall idea that biallelic LRIG2 variants lead to human bladder disease through autonomic neural defects that perturb bladder function.This study advances our understanding of the genetics and pathophysiology of early onset bladder disease, combining insights from human disease and a mouse model. We place the current LRIG2 in a hitherto unreported family with UFS. The variant in exon 14 results in a stop codon in the second immunoglobulin-like domain of the extracellular part of the protein. Putting this new family in the context of all reported UT-disease associated LRIG2 variants, as depicted in ,,LRIG2 and HPSE2 variants as a cause of congenital bladder obstruction whether a UFS grimace is present or not. The patient we describe in the current report was exposed to gestational diabetes mellitus. Human clinical studies as well as animal experiments suggest that maternal diabetes mellitus can perturb UT development.We describe a variant in ,LRIG2 and HPSE2 in healthy bladders, and how these processes go wrong in the presence of germline variants to generate the clinical phenotypes. LRIG2 and heparanase-2 proteins have been detected in nerves within the late first trimester in human fetal bladder,,,Lrig2 or Hpse2 mutant mice both have abnormally patterned bladder nerves.,,LRIG2-associated bladder dysfunction.Individuals with UFS usually present with dysfunctional urinary voiding and urosepsis in early childhood,Lrig2 mutant mice, we observed reduced nitrergic-nerve mediated outflow relaxation. The outflow maintained its responsiveness to SNP, implying that the loss of nitrergic relaxation in the mutant outflow was due to less NO reaching the muscle rather than an altered NO action. This accords with a report that Lrig2 mutant mouse outflow tracts appear to contain fewer nitrergic nerves than WT controls.Lrig2 mutant outflows were otherwise “physiologically normal.” Contractions induced by KCl or the α1-receptor agonist PE were preserved in mutant outflow tracts, again indicating that nonneurogenic contraction of the outflow smooth muscle is normal in Lrig2 Mut mice. Cautiously extrapolating to humans, male UFS patients might be predicted to have a functional bladder outflow obstruction with an important neurogenic component, specifically with diminished nitrergic-nerve function. Interestingly, when adjusted for KCl, male outflow tract contractile responses to PE were greater in mutants than controls, suggesting that postsynaptic functions in the smooth muscle of the bladder outflow tract may also be altered. Given that LRIG2 is normally detected in bladder nerves rather than SM,For optimal voiding to occur, the outflow tract must relax when the bladder body contracts. NO released from nitrergic nerves is considered the primary mechanism of outflow tract relaxation during micturition in mammals.Lrig2 mutant mouse outflow tracts differed in detail from the male mutants, showing a more dramatic loss of nitrergic relaxation, yet the functional effect would be the same, that is, bladder outflow obstruction. Indeed, reviewing the collective but limited literature  mutant mice compared with control littermates.Lrig2 mutation on muscle contraction evoked directly by membrane depolarization or receptor stimulation also diverged between the sexes. The male Lrig2 mutant bladder generated a normal level of contraction in response to depolarization with KCl but was hyper-responsive to muscarinic receptor-mediated stimulation. In female mice, contractile responses to both KCl and muscarinic receptor stimulation were suppressed in the Lrig2 mutant bladder body. The muscarinic M3 receptor is the predominant mediator of muscarinic stimulation in mouse and human bladder bodiesOur investigation of the bladder body of ex vivo physiological defects in female Lrig2 mutant bladder bodies, compared with males, correlates with the more marked change in bladder weight found in female mutants. Although in both sexes the bladder-to-whole body weight ratios were increased compared to WT mice, only the female mutants had an absolute increase in bladder weight compared with the WT controls. This difference may be a primary feature of the disease or, perhaps more likely, it reflects a more marked growth response to outflow obstruction in females than was present in males. In general, differences between males and females in their physiological responses could be a direct consequence of the mutation in Lrig2, or they could occur secondary to outflow obstruction. The current study could not address this question, but we note that there was no evidence of a fibrotic or inflammatory process in the detrusor smooth muscle of either sex on histology. The only difference found between mutants and controls was an increased collagen coherence in the detrusor layer of male mutants compared with male controls. We speculate that this may reflect higher hydrostatic pressures within these bladders, by analogy the relation between intraluminal pressure and collagen coherence in (nonmutant) arteries.Hpse2 mutant miceex vivo physiology studies in Lrig2 mutant mice could be complemented by performing in vivo experiments measuring bladder-urethra reflexesThe apparently more severe Lrig2 mutant animals have broadly similar ex vivo physiological defects in the bladder outflow and body as seen in juveniles. This allows us to make the important conclusions that the defects in juveniles cannot be simply explained away as delayed maturation but instead represent more fundamental neural functional aberrations that do not correct themselves with age. Future ex vivo physiological experiments on neonatal and even late fetal mutant mice will be needed to establish the exact onset of these defects. In fact, we have previously reported that neonatal Lrig2 mutant bladder display deregulated levels of transcripts, as assessed by RNA sequencing, implicated in neural differentiation.We undertook another set of experiments with the focus on adult (8–12 weeks old) male mice. We show that these older in vivo cystometry with combined bladder and outflow monitoring.Lrig2 mutant mice will address endogenous defects in the bladder body and outflow interact to cause a functional dyssynergia. As pointed out earlier in this study, people with UFS not only have urination defects but are at risk of ascending bacterial urosepsis that can damage the kidneys. Our previous studyLrig2 mutant mice, so we did not repeat this investigation in the current study. In our animal facility, the Lrig2 mutant mice are bred and maintained in a clean environment, and they do not appear to suffer from spontaneous urosepsis in this setting.An important avenue of future study will be to investigate the dyssynergia between bladder and outflow using LRIG2 variants can cause UFS. It also emphasizes that UT disease in UFS begins before birth. Putting this family in the context of all reported UT disease-associated LRIG2 variants reported, full-blown UFS occurs with loss of function variants, but missense variants lead to bladder-limited disease without the grimace. For the clinician, it is important to consider LRIG2 and HPSE2 variants as a cause of congenital bladder obstruction whether a UFS grimace is present or not. Moreover, our murine observations support the idea that UFS is a genetic autonomic neuropathy of the bladder affecting functionality of the outflow tract and the bladder body. The nuanced sex differences in abnormal ex vivo physiology in Lrig2 mutant mice may suggest that drug treatments could be tailored for males or females with UFS and should prompt further studies of possible sex differences in bladder outflow and body physiology in humans.This study highlights several notable clinical, genetic, and pathobiological points. The current case constitutes further evidence that biallelic All the authors declared no competing interests.


We identified over 50 CSF proteins dysregulated in DLB, enriched in myelination processes among others. The dopamine biosynthesis enzyme DDC was the strongest dysregulated protein, and could efficiently discriminate DLB from controls and AD (AUC:0.91 and 0.81 respectively). Classification modeling unveiled a 7-CSF biomarker panel that better discriminate DLB from AD (AUC:0.93). A custom multiplex panel for six of these markers  was developed and validated in independent cohorts, including an AD and DLB autopsy cohort. This DLB CSF proteome study identifies DLB-specific protein changes and translates these findings to a practicable biomarker panel that accurately identifies DLB patients, providing promising diagnostic and clinical trial testing opportunities.Diagnosis of dementia with Lewy bodies (DLB) is challenging and specific biofluid biomarkers are highly needed. We employed proximity extension-based assays to measure 665 proteins in the cerebrospinal fluid (CSF) from patients with DLB ( This study characterizes the CSF proteome changes underlying Dementia with Lewy Bodies (DLB) and identifies pathophysiological and diagnostic leads associated to this cause of dementia. Findings have been translated into a biomarker panel that could identify DLB patients with high accuracy across different cohorts. DLB is pathologically characterized by the intraneuronal accumulation of α-synuclein (α-syn) in Lewy bodies in the neocortex2. The clinical and pathological presentation strongly overlap with AD, challenging differential diagnosis and leading to a large proportion of miss- or undiagnosed DLB patients5.Dementia with Lewy Bodies (DLB) is one of the most common forms of dementia in the aged population after Alzheimer’s disease (AD)8, recent developments using seed amplification assays allow to detect α-syn brain proteinopathy in CSF and skin12. These assays can discriminate DLB from control or AD patients with high accuracy12. However, α-syn pathology is not unique for DLB patients and more than 40% of AD cases can present with this comorbid pathology13. Similarly, previous studies have shown that the core CSF biomarkers used to support AD diagnosis (amyloid β peptide (Aβ1-42), total tau (tTau) and phosphorylated tau (pTau))14 provide limited diagnostic accuracy for discriminating DLB from AD since they are also abnormal in almost 25–40% of DLB patients due to the presence of comorbid AD pathology18. Additional markers reflecting different, specific, and unique aspects of DLB pathophysiology are needed, which could be useful for different contexts of use in both clinical settings  and trials .Limited number of biomarkers have been widely analyzed to date in DLB. Despite previous studies on α-syn in cerebrospinal fluid (CSF) showing conflicting results21. The few DLB proteomic studies performed to date did not yield many biomarker candidates, which could be due to the limited sample size (30–40 samples per group) relative to DLB heterogeneity25. We here employed a high-throughput proteomics method (immune-based proximity extension assay (PEA)) that allows analysis of large cohorts, with the additional advantage that custom multiplex immunoassays including the markers of interest can be smoothly developed for large-scale validation26. We have applied this workflow to (i) define CSF proteomic changes underlying DLB pathogenesis and (ii) to identify, develop and validate multiplex biomarker assays that could aid in the specific diagnosis of DLB.CSF proteome profiling allows to identify changes covering a wide range of biological processes in vivo. As observed within the AD field, such analysis can help to define the molecular mechanisms involved in disease pathogenesis and reveal promising biomarker candidates21. Custom multiplex panels were developed and validated in two independent clinical cohorts  and one AD/DLB autopsy-confirmed cohort (n = 76). The demographic characteristics and AD CSF biomarkers are described in Table 28 score was higher in DLB compared to controls. The total NPI score was also higher in DLB compared to AD in the discovery and clinical validation cohort 1, but not in the clinical validation cohort 2. The Unified Parkinson’s Disease Rating Scale (UPDRS-III)29 was similar across the clinical validation cohorts.An overview of the study design is presented in Fig. q < 0,05). Six of these proteins were upregulated in DLB  and eight proteins were downregulated  are involved in the biosynthesis of dopamine and serotonin , growth control (GH), corticotropin release from pituitary gland (CRH), immune function (FCER2) and extracellular remodeling (MMP1). DDC showed the strongest effects  followed by GH, MMP1, FCER2, and CRH  were reflecting different biological processes including myelination regulation, tooth development, Notch signaling, steroid metabolism, muscle structure development or ovulation cycle  were analyzed separately, indicating that AD pathology comorbidities did not influence the performance of the model , we observed that the performance of the CSF panel could better discriminate DLB from controls and showed similar AUCs for the discrimination of DLB from AD .DDC, the strongest dysregulated marker that is increased in DLB, could discriminate DLB from controls with high accuracy , immune function (FCER-2), intra and extracellular remodeling , regulation of the hypothalamic–pituitary–adrenal axis (CRH) and neuropeptide degradation (THOP1).r > 0.70, Fig. DDC DLBvs.CON: 0.81; AUCDDC DLBvs.AD: 0.59; AUCcustom panel DLBvs.CN: 0.90; AUCcustom panel DLBvs.AD: 0.68; Fig. DDC aDLBvs.CON: 0.95; AUCDDC DLBvs.AD: 0.86; AUCcustom panel aDLBvs.CN: 1.00; AUCcustom panel aDLBvs.aAD: 0.90; Fig. To validate the performance of our discovery findings, we developed custom multiplex PEA panels measuring six out of the seven proteins from the DLB diagnostic panel, including DDC. Custom assays showed low coefficients of variation (mean intra- and inter-assay CVs of 5% and 9% respectively) and >90% detectability (Supplementary Table 30 and DLB stage31). We observed that CSF DDC positively correlated with parkinsonism severity as measured by UPDRS-III in the clinical validation cohort 1 , but not in the clinical validation cohort 2  with different measures of DLB pathophysiology when available , which was validated in independent external cohorts, including one with neuropathology confirmation. The DLB-related markers were also dysregulated in the prodromal and symptomatic phase of patients with PD, further supporting that the proteins identified reflect known biological processes associated with DLB pathophysiology such as the biosynthesis of dopamine or α-synuclein proteopathy.33. To this end, in this large CSF proteome study, we analyzed more than 100 CSF samples from DLB and cognitively unimpaired controls, but also samples from patients with AD dementia. We detected up to 90 proteins dysregulated in the CSF of DLB cases, but only 14 survived correction for multiple testing. Larger sample sizes might still be required to detect additional biomarker candidates due to clinical heterogeneity in DLB. Thanks to the inclusion of the AD dementia group, we could show that up to 55% of these 90 proteins were dysregulated in DLB specifically . We identified an additional subset of CSF proteins (27%) that were differentially regulated in both DLB and AD patients compared to controls, but with different protein abundance also between DLB and AD. While some of these were decreased in DLB and increased in AD , some were especially dysregulated in AD as previously reported 21, and some were more prominently dysregulated in DLB . These last shared but different protein profiles might be explained by the clinicopathological overlap across these two dementias34. Most of the proteins identified were downregulated in DLB compared to controls, a pattern observed in a previous proteome study35 but also in recent DLB transcriptomic studies39. It has been suggested that such a strong downregulated pattern in DLB could be due to demyelination processes38. In line with those findings, we observed that the proteins dysregulated in DLB were especially enriched in processes associated with negative regulation of myelination. Furthermore, previous research indicates that α-syn can induce myelin loss in neurons and oligodendroglia (precursor) cells41. Importantly, α-syn-induced myelination deficits are involved in the development of multiple system atrophy41. It would be thus of interest to investigate how the identified proteins relate to potential magnetic resonance imaging abnormalities and the importance of myelination processes in the pathophysiology of DLB. Of note, the three top proteins dysregulated in DLB  can regulate the hypothalamic-pituitary-adrenal (HPA) axis43, suggesting that one of the major systems associated with stress response and behavioral dysfunction might be involved in DLB pathophysiology. This is in line with previous studies showing hypothalamic dysfunction in DLB45.Biofluid-based biomarkers specifically associated with DLB are strongly needed not only to improve timely diagnosis and diagnostic accuracy, but also to monitor the different biological mechanisms involved in DLB pathophysiology and as surrogate markers for clinical trials46. This marker was not identified in previous transcriptomics studies. However, these analyses were performed in the anterior cingulate cortex38, an area with limited DDC expression47. It should also be noticed that genetic expression is not per se a proxy reflecting protein abundance48, which might be affected by other post-translational factors . The use of different technologies  together with the limited sample size may also explain why DDC was not detected in the few CSF proteomics studies performed so far24. Of note, we have also developed a DDC immunoassay that have shown similar changes in the clinical validation cohort 1, supporting the robustness of the findings . Previous studies have shown that serum DDC enzyme activity is elevated in patients using levodopa with peripheral decarboxylase inhibitors (PDI), underpinning the effect of dopaminergic treatment on DDC activity49. It is important to note that the increase of CSF DDC levels detected in this study are likely not driven by PDI treatment as i) PDIs do not cross the blood-brain barrier and can thus not influence DDC activity/levels in the brain and ii) we obtained similar results when only DLB patients that did not have any parkinsonism medication were included in the analysis (n = 60). Dysfunction of the dopaminergic system due to nigrostriatal degeneration is a well-established pathophysiological feature of DLB50. Previous studies have shown that nigrostriatal degeneration as well as antagonist of dopamine receptors increase DDC mRNA and activity in different models46. These data do not only align with our findings but also suggest that the increased DDC levels might be a response to the nigrostriatal degeneration and could thus be a very relevant biomarker for DLB diagnosis and disease monitoring. We could validate the high discriminatory accuracy for DLB vs controls (AUC: 0.91) in three independent cohorts, including a neuropathological one (AUCs 0.81–0.95). Considering that current DLB diagnostic guidelines include supportive imaging biomarkers as proxy of nigrostriatal degeneration4, it would be of interest to specifically analyze whether CSF DDC measurements could be an alternative or complementary diagnostic test to classical imaging scans. DDC measurements have the additional advantage of being quantitative over the binary classification with α-syn seed amplification assays, meaning that, DDC measurements might be relevant to track disease staging and for monitoring treatment responses.The strongest dysregulated CSF protein in the DLB group was DDC, an enzyme involved in the biosynthesis of dopamine, which lends biological support to our biomarker discovery design56, likely explaining the higher performance to discriminate these two dementias compared to DDC alone. To validate these findings in independent cohorts, we successfully developed custom multiplex assays for six out of the seven selected markers. The protein effect sizes obtained with these custom assays in the three validation cohorts correlated well with those obtained in the discovery cohort (r coefficients ranging between 0.70 and 0.99), and the high discriminative values were mostly validated (AUCs ≥ 0.80), supporting the relevance and robustness of our findings. In the clinical validation cohort 2 we observed, however, lower accuracy when discriminating DLB vs. AD (AUC 0.68), which was not dependent on whether cases were DAT positive or negative. Sensitivity analysis using only RBD-positive DLB cases in the clinical validation cohort 2 showed a similar performance to that observed in the discovery cohort. We also observed that the total NPI score, in contrast to the different scores observed for the discovery or the clinical validation cohort 1, was similar between DLB and AD cases in clinical validation cohort 2. Overall, these data suggest that the heterogeneity of the clinical diagnosis of DLB based on different supportive features  may impact biomarker data; and that the panel is likely most sensitive and specific in typical DLB. Understanding which factors may influence CSF DDC levels is of paramount importance for the potential future implementation of this marker and the corresponding DLB diagnostic panel.To translate the CSF proteome findings into practical biomarker tools for routine diagnostics and clinical trials, we applied classification analysis and identified a panel of seven CSF proteins to discriminate DLB from controls and AD dementia with high accuracy (AUC of 0.95 and 0.93 respectively). This panel combined proteins associated to DLB  but also proteins specifically related to AD 8. We however explored the association between the CSF DLB markers identified in this study and specific DLB pathophysiological features. Even though these specific results should be interpreted with caution considering the limited sample size, the positive association of CSF DDC with UPDRS-III, α-syn load in different brain areas, α-syn Braak stage and DLB stages supports the potential of this marker to track disease progression already from very early stages. This is further supported by the data obtained from the PD cohorts in which CSF DDC was already increased in the prodromal stage of the disease. The other biomarkers within the DLB panel  also associated with some DLB pathophysiological features, but results were not consistent across cohorts. This calls for a systematic analysis of all these CSF markers in cohorts with large associated neuropathological characterization. These markers were also dysregulated in prodromal PD and showed nominal significance at the clinical stage. The fact that the DLB markers identified in this study are also dysregulated in PD is not surprising considering both disorders are characterized by α-syn pathology and dopaminergic cell loss in the substantia nigra57. Thus, despite the fact that the identified markers are specific for DLB in the context of neurodegenerative dementias, they might be dysregulated in other α-synucleopathies or disorders characterized by dopamine deficiency. Overall, all these data support that CSF DDC (and maybe the panel) could be a useful quantitative tool not only to track nigrostriatal degeneration and disease stage, but also to select prodromal cases or monitor treatment effects in clinical trials. Specific follow-up studies including different Parkinsonian disorders and Lewy bodies dementias in the same data set are needed to understand the role of these markers across the different disorders and define their potential context of use.We could not investigate the association between CSF DDC or the other DLB markers  and CSF α-syn levels, as this information was not available in our data sets. Still, the added value of such information might be limited considering the variable results on CSF α-syn observed across studiesn = 13), such data highlights the importance of including treatment information in biomarker studies. Additional analyses are needed to confirm the relevance of treatment effect on CSF FCER2 abundance. Importantly, medication did not drive the main differences observed between groups for the other DLB-related proteins . Considering the clinicopathological overlap with AD34, we cannot exclude that the potential misdiagnosis of DLB patients may have influenced our discovery results. However, samples were collected in well-characterized biobanks from specialized memory units, and DLB diagnosis was either autopsy confirmed or supported by DAT scans in more than one third of the patients. Moreover, our sensitivity analysis showed similar diagnostic accuracies in DLB cases with positive AD CSF biomarker profile, and we further validated the biomarker panel in a CSF AD/DLB autopsy confirmed cohort. The similar differences observed for CSF DDC, FCER2, CRH, and MMP3 also in the PD data sets call for additional studies that evaluate the performance of these markers in other α-synucleinopathies , other dementia types with motor dysfunctions  or other conditions with dopamine deficiency . This DLB study covers already three phases of the biomarker development workflow58 including the retrospective analysis of four cohorts coming from different memory clinics with their own intrinsic methodologies and clinical characteristics, which ultimately supports the reproducibility and robustness of the findings. Still, future studies are needed to define the clinicopathological correlations and trajectories between the biomarker panel and different measures associated with DLB pathophysiology, including other relevant markers, such as α-syn seed amplification assays59, in longitudinal and prospective studies using independent large and highly phenotyped cohorts. This will help to define their potential context of use within different settings in clinical practice and trials . We envision that CSF DDC and the panel developed within this study could be relevant complementary and quantitative diagnostic tests reflecting different biological aspects associated with DLB pathophysiology in the context of dementias. This quality may make them suitable to improve diagnosis and staging along the DLB continuum but also to monitor treatment response in clinical trials targeting different mechanisms61.Despite the large number of samples and proteins analyzed in this DLB-specific study, there are still relevant limitations. We observed that Parkinsonian medication moderately influenced the levels of some CSF proteins. Despite the number of cases that underwent Parkinsonian medication was limited . Informed consent was obtained from all subjects or their authorized representatives.n = 534) included CSF samples from patients diagnosed with DLB (n = 109), AD (n = 235) and 190 cognitively unimpaired controls 65. Three additional independent CSF cohorts were used for validation of the customized panels (see methods below): clinical validation cohort 1 63, clinical validation cohort 2 (from the Sant Pau Initiative on Neurodegeneration (SPIN) cohort: 55 DLB, 55 AD, and 55 controls)66 and an AD/DLB autopsy confirmed cohort (from BIODEM and the neurobiobank of the Institute Born-Bunge (IIB)/UAntwerp: 17 autopsied confirmed DLB (aDLB) and 30 autopsied confirmed AD (aAD))68. An additional 29 cognitively unimpaired controls from BIODEM-UAntwerp were included in this cohort but these were not autopsy confirmed68. CSF was collected by lumbar puncture and processed and stored at all sites in agreement with the JPND-BIOMARKAPD guidelines, thereby minimizing the influence of potential pre-analytical factors69.An overview of the study design is presented in Fig.  DLB n = 09, AD  with additionally negative AD CSF biomarkers in all cases75. Mini-Mental State Examination (MMSE) was used as a measure of global cognition. Motor parkinsonism was assessed using section III of the Unified Parkinson’s Disease Rating Scale (UPDRS)29. Core and supportive clinical features31 were recorded locally in each memory unit66. Evaluation of REM Sleep Behavior Disorder (RBD) includes an interview with a sleep specialist, a full nocturnal video-polysomnography, the Mayo Sleep questionnaire (cutoff ≥1) or caregivers reporting that a patient would ‘act out’ their dreams and moves extensively during their sleep as previously described76. Supportive neuropsychiatry symptoms  were compiled according to the Neuropsychiatry Inventory and summarized in one single score 28. Levels of CSF Aβ42, tTau and pTau(181) (‘AD CSF biomarkers’) were used to support AD diagnoses. These markers were analyzed locally as part of the diagnostic procedure using commercially available kits (VUmc and UAntwerp: ELISA INNOTEST Aβ(1-42), hTAUAg, phospho-Tau or VUmc: Aβ(1-42), t-TAUAg, phospho-Tau181 Elecsys biomarker assays (Roche Diagnostics GmbH); Penn: Luminex xMAP INNO-BIA AlzBio3; Luminex Corp, Austin, TX; SPIN: Lumipulse G600, Fujirebio)78. Positive CSF AD biomarker profile was defined locally as increased tTau/Aβ42 ratio in the cohorts from ADC (>0.46) and Pennsylvania (>0.30); and low Aβ42/40 ratio (<0.062) and high total tau (>456 pg/ml) or p-tau (>63 pg/ml) in the SPIN cohort80.All participants of every cohort underwent standard neurological and cognitive assessments and diagnosis was assigned according to international consensus criteria for DLB31. Comorbid AD pathology, which is common in DLB patients18, was present in up to 34 DLB patients (32%) as measured by AD CSF biomarker profile. From all the DLB patients, only 13 DLB patients had medication for parkinsonian symptoms, 60 did not have such a treatment and this information was not available for 36 cases.In the discovery cohort, DLB neuropathology was confirmed in 14 DLB patients 13%) by autopsy .In the clinical validation cohorts 1 and 2, DAT scans supported DLB diagnoses in 18 and 21 patients respectively  or cerebrovascular lesions (n = 3) were reported in 10 cases. Coexisting cerebrovascular lesions (n = 8), TDP pathology (n = 1) or cerebral amyloid angiopathy (n = 1) were reported within the aAD neuropathological group. The control group of this cohort was not autopsy confirmed. It consisted of volunteers, mainly spouses of patients who visited the memory clinic. The inclusion criteria for these volunteers were: (1) no neurological or psychiatric antecedents; (2) no organic disease involving the central nervous system following extensive clinical examination; and (3) normal neuropsychological exam. Exclusion criteria consisted of brain tumors, large cerebral infarction/bleeding, strategic infarctions, other neurodegenerative diseases, severe head trauma, epilepsy, brain infections, severe depression, unregulated diabetes mellitus, untreated thyroid disorders, or any severe somatic comorbidity that interferes with study participation81.The AD/DLB autopsy validation cohort included cases with a definite diagnosis according to international neuropathological examination guidelines for DLBPatient demographics and clinical and biochemical values from all cohorts used in this study are listed in Table 21, CSF proteins (979) were quantified using the 11 specific and validated multiplex antibody-based protein panels based on the proximity extension assay (PEA) that were available at the time in which the analysis was performed as previously described  were calculated using the 4 CSF QC samples. No cross-reactivity between assays for specific proteins was detected. Assay parameters including LOD, detectability and CVs are included in Supplementary Table Custom-designed multiplex-PEA assays were developed by the manufacturer following standardized protocols32 (http://www.ppmi-info.org/data) on March 28th, 2023. The PPMI study is registered with ClinicalTrials.gov (number NCT01141023). The study protocols and the manuals related to different procedures for clinical assessment, biochemical phenotyping as well as sample handling, collection and storage is described online (http://www.ppmi-info.org/study-design). Data from samples collected at baseline were used for the analysis. Two different sets were available with PEA CSF proteome  from the Parkinson´s Progressive Markers Initiative PPMI82. For each pairwise comparison, multiplicity was taken into account by controlling the False Discovery Rate (FDR)83 at q ≤ 0.05 based on the number of features analyzed. We next evaluated which CSF protein combination (CSF panels) could best discriminate the groups of interest while keeping the number of markers to the minimum, so that they can be ultimately translated into small, practical custom panel21. For this purpose, binary classification signatures (DLB vs. CON and DLB vs. AD) were constructed by way of penalized generalized linear modeling (GLM) with an elastic net penalty  in the discovery CSF cohort using the glmnet package and including age and sex as covariates84. This penalty enables estimation in settings where the feature-to-sample ratio is too high for standard generalized linear regression. Moreover, it performs automatic feature decorrelation as well as feature selection. For each classification exercise, we compare multiple models which reflect (a) a grid of values for the elastic-net mixing parameter, reflecting strong decorrelation to a pure logistic lasso regression and (b) a grid of values reflecting the maximum number of proteins that may be selected under each model (21 markers maximum). The former grid (a) considers that we have little information on the collinearity burden in the data. The latter grid (b) considers that we want to keep the number of selected proteins relatively low for the future development of customized panels. The optimal penalty parameters in the penalized models were determined based on  10-fold cross-validation of the model likelihood82. The cross-validation was performed with balanced folds, by which each fold has an outcome group ratio close to the corresponding ratio in the full data set, also referred to as stratified cross-validation. The predictive performance of all models was assessed by way of (the comparison of) Receiver Operating Characteristic (ROC) curves and Area Under the ROC Curves (AUCs). The model with the highest AUC and lowest number of markers for each classification signature was selected. The fold-based selection proportions for each marker were assessed to identify and select the most promising markers within each model . To reflect the manual selection pressure for these final marker sets, each final logistic signature was subjected to a ridge-regularization with a penalty parameter of 0.1. The performance (AUC) was evaluated by internal validation: repeated 5-fold cross-validation with 1000 repeats. The 95% confidence interval around the resulting AUCs was based on resampling quantiles (percentile method). External validations assessed the performance of the final models with the markers of interest in the validation cohorts using ROC analysis.All data preprocessing and analyses were conducted using R version 3.5.3 and SPSS version 25. Between-group analyses for the demographic variables were performed using two-sided one-way analysis of variance in normally distributed continuous data or Pearson’s chi-square test for categorical variables. Analysis of covariance was performed when an association between classical AD CSF biomarker and age and/or sex were detected. Adjustment for multiple testing was performed using Bonferroni method. Non-Gaussian distributed data were analyzed using Kruskal-Wallis Test. For the CSF proteome data, differences in protein abundance between pairs of clinical groups were evaluated using nested linear models as previously described, in which for each individual protein feature, we assessed if its addition to a base model containing age and gender contributed to model fit85. UPENN values had lower means for Aβ42 on the Innotest, which were first linearly transformed to normalize to the same mean. Passing-Bablock transformation formulas were calculated based on individuals with both Luminex and Innotest values for Aβ42 (n = 32), tTau (n = 32) and pTau (n = 27) and used the formulas to estimate the equivalent Innotest values for those samples measured with Luminex platform only (transformed_ Aβ42 = (Luminex_ Aβ42*4.65) − 36.23; transformed_tTau = (Luminex_tTau*5.28) − 2.03; transformed_ptau = (Luminex_pTau*1.88) + 27.36). The association between the markers of interest with DLB pathophysiological features  was analyzed by either correlation analysis or ANOVA. Stratification or post-hoc analysis with DLB pathophysiological features could not be performed due to the limited sample size of some subgroups.Non-parametric correlation analysis was performed to understand the associations between the proteins within the CSF panels and the classical AD CSF biomarkers or cognitive function (MMSE score) using the complete discovery cohort without stratifying per diagnostic category and conditioning on age and sex as covariates. Innotest values generated for the Amsterdam Dementia Cohort were adjusted for drift over time as previously described86 selecting GO Biological Processes as ontology source. All the CSF proteins optimally analyzed with Olink arrays (n = 645 protein gene products) were included as the enrichment background. Default parameters were used for the analysis in which terms with a p-value < 0.01, a minimum count of 3, and an enrichment factor > 1.5 were collected and grouped into clusters based on their membership similarities.Functional enrichment analysis was performed using MetascapeFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary