We investigated the phenotypes and genotypes of a cohort of ‘long-surviving’ individuals with motor neuron disease (MND) to identify potential targets for prognostication.C9orf72 repeat expansions. Data from ancestry-matched Scottish populations (the Lothian Birth Cohorts) were used as controls.Patients were recruited via the Clinical Audit Research and Evaluation for MND (CARE-MND) platform, which hosts the Scottish MND Register. Long survival was defined as > 8 years from diagnosis. 11 phenotypic variables were analysed. Whole genome sequencing (WGS) was performed and variants within 49 MND-associated genes examined. Each individual was screened for SOD1 p.(Ile114Thr) variant. Significant variants in FIG4, hnRNPA2B1, SETX, SQSTM1, TAF15, and VAPB were detected. 2 individuals had a variant in the SPAST gene suggesting phenotypic overlap with hereditary spastic paraplegia (HSP). No long survivors had pathogenic C9orf72 repeat expansions.58 long survivors were identified. Median survival from diagnosis was 15.5 years. Long survivors were significantly younger at onset and diagnosis than incident patients and had a significantly longer diagnostic delay. 42% had the MND subtype of primary lateral sclerosis (PLS). WGS was performed in 46 individuals: 14 (30.4%) had a potentially pathogenic variant. 4 carried the known SOD1 variant p.(Ile114Thr). Our findings confirm that pathogenic expansion of C9orf72 is likely a poor prognostic marker. Genetic screening using targeted MND and/or HSP panels should be considered in those with long survival, or early-onset slowly progressive disease, to improve diagnostic accuracy and aid prognostication.Long survivors are characterised by younger age at onset, increased prevalence of PLS and longer diagnostic delay. Genetic analysis in this cohort has improved our understanding of the phenotypes associated with the The online version contains supplementary material available at 10.1007/s00415-022-11505-0. The Scottish Motor Neuron Disease Register (SMNDR)  platform in 2015) has been collecting data regarding people living with MND  and MND subtypes  in Scotland since 1989 , 4. ReceThere is currently no definition of long survival with MND, with literature ranging from 5 to 10 years –9. PreviSOD1 variants and a specific variant in the UNC13A gene (rs10419420) are thought to impart long survival . , 5. SOD1nt Table  were obsF15 gene . The TAFtenuated .C9orf72 hexanucleotide sequence within the pathogenic range.No patients in this cohort had expansions in the SOD1 p.(Ile114Thr) Scottish founder variant .Two patients had missense mutations in  and PLS . Two pat disease . One indd in ALS , 38. On n ClinVar in a biaSPAST intronic variant; this variant was absent from controls and gnomAD but has been reported in Clinvar associated with autosomal dominantly inherited HSP (SPG4) and had supportive in silico predictors of an effect on splicing [VAPB gene in an individual with PLS. VAPB variants are rare in MND and the gene is small and tolerant to change. However, all splice algorithms predicted a significant reduction in splice efficiency and the variant was absent in controls with a low MAF.Two patients carried a splicing . InteresThe prevalence of long survivors in the Scottish population 8.9%) is comparable with previous European estimates .9% is co, 41. HowOur cohort is enriched for people diagnosed with upper motor neuron predominant MND (PLS). Although not statistically significant compared with incident patients, the male-to-female ratio in the long-surviving group was high (2:1) and this may be due to the contribution of PLS cases which are known to be largely male (ratio 2–4:1) . That PLLong survivors are more likely to start on riluzole than incident patients. This may be related to opportunity in the context of long duration of disease. While riluzole has been trialled predominantly in people with ALS, an appeal was made to the National Institute for Clinical Excellence (NICE) in 2001 to allow its use in other forms of MND . ClearlySOD1 p.(Ile114Thr) variant which is frequently observed in the Scottish population [Only 11.5% of the long surviving cohort had a family history of MND whereas almost a third (30.4%) of the genotyped cohort had a ACMG-AMP likely pathogenic variant. As would be expected, this is higher than when a limited six-gene panel was employed for an unselected cohort of MND patients in Scotland (17%) [FIG4, hnRNPA2B1, SETX, SQSTM1, TAF15, VAPB) in patients with both ALS and PLS without a family history confirms the sporadic nature of variants in these genes in the Scottish population. Although these variants met ACMG-AMP/ACGS classification criteria for being likely pathogenic, it is not possible to determine if they are disease-causing in a rare heterogeneous condition such as MND. We raise the possibility that people with MND carrying mutations in the SPAST gene may have an MND syndrome similar to HSP. In general, HSP will normally present earlier than upper motor neuron predominant ALS/PLS and patients are more likely to have lower limb onset disease and a family history; however, there will be exceptions and overlap [The presence of other rare variants in MND-associated genes  were not available for all patients but future analysis of long survivors might study the temporal relationship of these measures on outcome. Our control population is ancestry but not age or sex-matched and so may not reflect the characteristics of our typically young MND long survivors.We have reported variants as per ACMG-AMP guidelines. MND-associated variants are rare and have variable penetrance and so often struggle to fulfil strict ACMG-AMP criteria. Many likely pathogenic variants were so defined due to their absence in controls and absence in gnomAD alone. However, these guidelines provide a necessary structure for variant assessment and allow us to report variants which might as yet fail to achieve clinical significance but might be of research interest. As this was a research project and results were not relayed to patients or families, frameshift and indel variants were not confirmed by Sanger sequencing and so it is not possible to determine if they are truly disruptive. Our 49-gene panel was comprehensive for MND-spectrum genes but we did not examine the androgen receptor CAG trinucleotide repeat associated with Kennedy disease , nor didC9orf72 pathogenic expansion and presence of the SOD1 p.(Ile114Thr) mutation or other rare mutation might indicate better outcome. Additionally, presence of the SOD1 p.(Ile114Thr) variant might imply long preservation of bulbar function and cognitive function but allow individuals to prepare for NIV requirement. Extended family history, including multisystem disease and neurological syndromes featuring UMN signs/spasticity, might be of particular merit in those with young onset, slowly progressive ALS or PLS; in these cases, MND and HSP genetic testing may be appropriate. Long survivors are more likely to have a likely pathogenic variant than previous estimates of gene carrier status in MND populations; however, this may reflect breadth of our gene panel. The majority of MND patients do not undergo routine genetic testing due to variable variant penetrance, challenges with variant classification and lack of relevant treatment options for people with MND. However, recent evidence suggests that limiting genetic testing to people with presumed prior probability of having a pathogenic mutation (i.e. those with a family history and young-onset disease) may fail to capture a significant proportion of potential actionable or informative genetic mutations [With the benefit of three decades of longitudinal data collection through the Scottish CARE-MND database, we have shown that long surviving people with MND can be characterised by younger age at onset and diagnosis, increased incidence of PLS and, crucially, a longer time to diagnosis . This long period of diagnostic uncertainty for a typically young person is a key target for improvement in care; such patients may otherwise be denied access to designated benefits and specialised support. Early clinical genotyping of such individuals may help to provide reassurance – for example, the absence of a utations . Our stuSupplementary file1 (DOCX 35 KB)Below is the link to the electronic supplementary material.


Mitochondrial dysfunction is a hallmark of cancer. However, it is unclear whether it is a cause of cancer. This two-sample Mendelian randomization (MR) analyses, uses genetic instruments to proxy the exposure of mitochondrial dysfunction and cancer summary statistics as outcomes, allowing for causal inferences.cis) and robustly associated with the mitochondrial molecular alterations were used as instrumental variables, and their causal associations with cancers were examined using summary-data-based MR (SMR) analyses. An additional five MR methods were used as sensitivity analyses to confirm the casual associations. A Bayesian test for colocalization between mitochondrial molecular QTLs and cancer risk loci was performed to provide insights into the potential regulatory mechanisms of risk variants on cancers.Summary statistics from 18 common cancers , gene expression, DNA methylation and protein expression quantitative trait loci  on individuals of European ancestry, were included. Genetic variants located within or close to the 1136 mitochondrial-related genes , NSUN4 expression level with both breast cancer risk  and prostate cancer risk , NSUN4 methylation level with both breast and prostate cancer risk, and VARS2 methylation level with lung cancer risk.We identified potential causal relationships between mitochondrial-related genes and breast, prostate, gastric, lung cancer and melanoma by primary SMR analyses. The sensitivity and the colocalization analyses further refined four genes that have causal effects on three types of cancer. We found strong evidence of positive association of This data-driven MR study demonstrated the causal role of mitochondrial dysfunction in multiple cancers. Furthermore, this study identified candidate genes that can be the targets of potential pharmacological agents for cancer prevention.10.13039/501100009736Allmänna Sjukhusets i Malmö Stiftelse för bekämpande av cancer (20211025).This work was supported by Styrelsen för  Previous studies have shown associations between dysfunctions in mitochondrial DNA (mtDNA), mtDNA copy number or mitochondrial-related nuclear genes and different cancer risks. However, these studies did not investigate causal inferences between mitochondrial dysfunction and cancers. We searched PubMed for studies in any language using the search terms “mitochondrion OR mitochondria OR mitochondrial dysfunction” AND “Mendelian randomization OR Mendelian randomisation” AND “cancer OR cancers”. Of the yielded 4 studies, three studies’ outcomes were COVID-19, dementia and type 2 diabetes, respectively. The other study was a meta-analysis study that presented heterogeneous estimates for the effect of mtDNA copy numbers on different cancer risks and suggested applying Mendelian randomization for unraveling the casual correlation of mtDNA copy number with cancer risk.NSUN4, for both breast and prostate cancer. All associations underscore the importance of mitochondrial dysfunction in the pathogenesis of multiple cancer types.This data-driven study fills the gap by using Mendelian randomization to examine the potential causal relationship between mitochondrial dysfunction characterized by genetic predisposition in all mitochondrial-related genes and common cancer risks. Our findings provide evidence for the potential causal effect of mitochondrial dysfunction on breast, prostate and lung cancer, after sensitivity and colocalization analyses. In addition, we identified a shared putative causal gene, Our data-driven analyses support the increasing values in the application of publicly accessible datasets to inform public health. To date, our European population-based large-scale study and the available evidence, indicate that individuals with mitochondrial dysfunction have a higher risk of a certain type of cancer, and point to the necessity of objective measurement of mitochondrial function in epidemiologic studies. For the identified putative causal genes, it is feasible to be added to the genetic screening project for better cancer prevention. True causal effects of mitochondrial dysfunction on cancers might be more complex and need larger genetic datasets and sophisticated experimental studies to further confirm.,,Mitochondria are the essential organelles that regulate cellular energy production, metabolism, proliferation and apoptosis. An altered mitochondrial function is a well-known hallmark of cancer, which is commonly characterized by abnormal mitochondrial morphology, deficient mitochondrial copy numbers, aberrant energetic metabolism, accumulation of reactive oxygen species (ROS), imbalanced biogenesis and mitophagy.,Mendelian randomization (MR) is a method that uses genetic variants as instrumental variables (IVs) to explore the potential causal association between lifetime exposure and outcome. In MR, the use of the conceptional random allocation of alleles avoids bias from unobserved confounders such as lifestyle and environmental factors and the problem with reverse causality.To our knowledge, there has been no MR study investigating the potential causal relationship between mitochondrial dysfunction and the risk of common types of cancer. Therefore, in this study, we aimed to investigate the causal relationship between mitochondrial dysfunction characterized by genetic predisposition in mitochondrial-related genes and multiple cancer types by the comprehensive two-sample MR analysis.This study was conducted following the reporting guideline of the Strengthening the Reporting of Observational Studies in Epidemiology STROBE, .11cis) that are robustly associated with gene expression were extracted using eQTLs summary statistics obtained from the eQTLGen Consortium (https://www.eqtlgen.org/cis-eqtls.html). The eQTLGen Consortium contains information on 10,317 trait-associated single nucleotide polymorphisms (SNPs) from 31,684 individuals.cis-mQTL instruments for genetic variants robustly associated with mitochondrial gene methylation were extracted using summary data from a meta-analysis of two cohorts (n = 1980).cis-pQTL instruments for genetic variants associated with the expression of mitochondrial-related proteins were selected from five proteome datasets,Psnp-mitodys <5 × 10−8.To generate eQTL instruments for mitochondrial genes, genetic variants located within 1000 kb on either side of the coding sequence  and βSNP-cancer is the estimated effect size of SNP on cancer (the same genetic variant—outcome trait association).βhttps://yanglab.westlake.edu.cn/software/smr/#Overview). Odds ratio (OR) estimates of mitochondrial dysfunction on the risk of cancer were obtained as follows: ORmitodys-cancer = exp (βmitodys-cancer), where OR is the odds ratio estimate per 1-ln increment in mitochondrial genome levels and exp is the base of the natural logarithm.Here, we performed SMR using the Linux version 1.0.3 of SMR software in the command line using default options (www.r-project.org).Sensitivity analyses were conducted after completing the primary SMR analyses with 5 additional MR methods, including MR Egger, weighted median, inverse variance weighting (IVW), simple mode and weighted mode by using the TwoSampleMR R package. Each of these methods calculates the estimates of the causal effect based on slightly different assumptions about the instrument validity and therefore provide robust evidence of our findings . All anahttps://chr1swallace.github.io/coloc/, version 5.1.0) to estimate the posterior probability of shared variants.HEIDI test is one of the colocalization methods that use external reference to estimate the LD. To refine the results, we performed another Bayesian test for the colocalization of two traits using the coloc R package  followed by the HEIDI test (PHEIDI>0.01) implemented in SMR software to investigate if the association was due to a shared causal variant and not pleiotropy. We thus identified 7 association signals across 7 unique genetic loci for breast cancer, 4 association signals across 4 unique genetic loci for prostate cancer and one association signal for gastric cancer. We found no significant genetic correlation for the other cancer types. Sensitivity analysis using additional MR methods relying on similar assumptions was conducted and shown to support our findings  while 1 SD increase of NSUN4 expression was associated with 5% higher risk . When sub-grouping breast cancer according to the intrinsic molecular subtypes, the causal associations showed a similar trend only with luminal A-like breast cancer. Interestingly, we found a robust causal association  between MTX1 expression and luminal A-like breast cancer, specifically. We also found a strong causal association  between COX11 expression and luminal B-like/HER2-negative cancer .For breast cancer, one SD decrease of e cancer . For proNSUN4 increased by rs41293273 is associated with higher risk of both breast cancer and prostate cancer.Most importantly, our results show that the expression level of PSMR < 1.961 × 10−5) and HEIDI test were performed. We identified a total of 15 association signals across 14 unique genetic loci for breast cancer, 11 association signals across 10 unique genetic loci for prostate cancer, one association signal for gastric cancer, 4 association signals across 3 unique genetic loci for lung cancer and 2 association signals across one unique genetic locus for melanoma , and conversely, one SD increase of NSUN4 methylation by rs6681857 was associated with 7% higher risk of breast cancer . Here, in total, we found 6 unique loci that regulated the methylation level of 7 different CpG sites in NUSU4, and were positively associated with the risk of breast cancer   . For lunpG sites . We founNSUN4, which was regulated by rs6682266, rs5013329, rs56063031 and rs6681857, was associated with NSUN4 expression, and VARS2 methylation by rs2596495 was also associated with VARS2 expression  and 3) the absolute value of size effect (β) >0.01 NSUN4 that were causally associated with both breast and prostate cancer were not found to be associated with all available secondary traits  was associated with secondary traits such as metabolic-related blood urea nitrogen and Crohn's disease (NUDT5 methylation associated) was also associated with endocrine-related traits  NUDT5 for prostate cancer; 3) VARS2 for lung cancer and 4) NSUN4 for both breast and prostate cancers. Our results show that genetic determinants of mitochondrial dysfunction were associated with the risk of cancer in a cancer type-specific manner, which provides robust evidence for underlying mechanisms linking the genetic loci, gene expression, and methylation with multiple cancers.In this study, we demonstrate that mitochondrial dysfunction characterized by genetic predisposition has causal effect on cancers, and identified important putative causal mitochondrial-related genes as follows: 1) FDPS has been investigated over decades for its physiological function and was found to be associated with leukemia growth,FDPS has a causal relationship with breast cancer. Studies have shown that knockdown FDPS enhanced apoptosis and ectopic overexpression of FDPS promoted cancer colony growth and proliferation by affecting STAT3, AKT and ERK pathways.https://go.drugbank.com/), more drugs have been investigated such as Ibandronate, Minodronic acid and Incadronic acid that target FDPS.The FDPS is a key enzyme that is involved in the mevalonate pathway to catalyze the biosynthesis of cholesterol and sterol, and to isoprenylate cellular metabolites such as Ras, Rac, Rab and Rho for membrane anchorage and cellular signaling.NSUN4 is an rRNA m5C methyltransferase that can induce the methylation of the 12S rRNA of the small ribosomal subunit joining in mitochondria and promote rRNA rearrangements to form peptidyl transferase center.,NSUN4 is the strongest shared functional candidate at 1p34.NSUN4 methylation was associated with decreased risk of breast cancer only. Importantly, we identified additional 1 genetic locus related to NSUN4 expression and 2 loci related to NSUN4 methylation that were causally associated with both breast and prostate cancer (NSUN4 and both breast and prostate cancers was not caused by horizontal pleiotropy. Together, these results emphasize the potentially important role of NSUN4 in carcinogenesis.e cancer . FurtherNUDT5 is differentially expressed in different types of cancer and positively correlated with aggressive cancer disease phenotype, knockdown of which can suppress the proliferation of cancer cells without inducing DNA oxidative lesion.NUDT5 has a strong causal effect on prostate cancer. Those findings highlight that NUDT5 may represent a promising drug target for cancer prevention and treatment. More studies now focus on the identification of NUDT5 inhibitors from approved drugs and small molecules, and a potent TH5427 was tested and shown to block hormone signaling and disrupt the proliferation of breast cancer cells.VARS2 have been associated with mitochondrial diseases such as complex I defect, early onset of mitochondrial encephalomyopathies and encephalocardiomyopathies,VARS2 as a causal gene only for lung cancer. The mechanisms of VARS2 in lung cancer carcinogenesis need further evaluation by experimental studies.Several mutations in The main strength of the present study is that we performed a comprehensive MR analysis between mitochondrial dysfunction, characterized by genetic predisposition in all known mitochondrial-related genes, and their causal relationship with cancers. The inclusion of all genes related to mitochondria eliminates the selection bias in previous studies and might be able to address mitochondrial dysfunction directly. Secondly, we have included a very large sample size and 18 different cancer outcomes from GWAS summarized statistics, which allowed us to gain sufficient power to elucidate causal relationships and make conclusive estimations for several cancer types. Thirdly, we used SMR as the primary analysis and performed a sensitivity analysis using 5 additional MR approaches and colocalization analysis, which shows the robustness of our findings. Finally, we only included samples of European ancestry, thus, we minimized the biases caused by different genetic backgrounds.This study has several limitations as well. Although we drew on the large available GWAS data sources, no genetic variants were obtained that represent the mitochondrial protein expression and the available eQTL and mQTL datasets did not have information on genetic variants that were associated with gene expression or methylation level in the X chromosome, Y chromosome and mitochondrial genome; the mitochondrial genome-wide associated genetic variants in this study mainly laid on the mitochondrial-related nuclear genome rather than the mitochondrial genome itself because a mitochondrial genome-specific QTL dataset has not yet been developed. Moreover, GWAS dataset that directly reflected on mitochondrial dysfunction is not available, hence we cannot assess the direction of causal relationship by using bi-directional MR based on current software resources. In this study, we showed that causal effects of mtDNA copy number variations and cancers were bi-directional in a cancer-specific manner. However, GWAS summary statistics of mtDNA copy number variation are potentially underpowered to detect the direction of causal association between mitochondrial dysfunction and cancers. Furthermore, univariable MR estimates the total effect of exposure on the outcome. As an extension, multivariable MR simultaneously estimates several potentially related exposures with a shared set of SNPs on the outcome using GWAS summary statistics, allowing for the assessment of the direct causal effect of a single exposure on the outcome. In this study, the exposure was mitochondrial dysfunction characterized by predisposition in the mitochondrial-related gene, which only can be retrieved from the QTL datasets other than the GWAS dataset. Thus, we are unable to perform multivariable MR to estimate the direct causal effects of mitochondrial dysfunction on cancers. Further studies should be conducted on the question of whether mitochondrial dysfunction is causally associated with cancer when GWAS or more advanced methods are available.This study leverages MR to examine the potential causal relationship between mitochondrial dysfunction characterized by genetic predisposition in mitochondrial-related genes and cancer, and demonstrates the importance of mitochondrial dysfunction in the pathogenesis of multiple cancer types. The identified putative genes can function as potential pharmacological targets for cancer treatment and prevention, further research could explore details of the underlying biological mechanisms.YL designed the study and performed the statistical analysis; YL, KS, NZ, XW and AAM participated in data interpretation; YL and AAM wrote the first draft, and KS, NZ, XW and JS revised the article. YL and NZ had direct access and responsibility for verifying all data reported in the manuscript. All authors read and approved the submitted version of the manuscript.Data used in this study are available from the referenced peer-reviewed studies and listed in The authors declare that there is no conflict of interest associated with this manuscript.


By studying the interplay among genetic variants, gene expression, and cis-regulatory domains, we ascertain that changes in coordinated regulatory activity tag alterations in gene expression levels , unveil case-specific QTL effects, and identify regulatory machinery changes for genes affecting synaptic function and dendritic spine morphology in schizophrenia. Altogether, we show that accounting for coordinated regulatory activity provides a novel mechanistic approach to reduce the search space for unveiling genetically perturbed regulation of gene expression in schizophrenia.Schizophrenia is a polygenic psychiatric disorder with limited understanding about the mechanistic changes in gene expression regulation. To elucidate on this, we integrate interindividual variability of regulatory activity (ChIP-sequencing for H3K27ac histone mark) with gene expression and genotype data captured from the prefrontal cortex of 272 cases and controls. By measuring interindividual correlation among proximal chromatin peaks, we show that regulatory element activity is structured into 10,936 and 10,376 cis-regulatory domains in cases and controls, respectively. The schizophrenia-specific cis-regulatory domains are enriched for fetal-specific ( Schizophrenia (SCZ) is a severe mental illness that oftentimes leads to a lifetime of chronic disability. While several lines of evidence converge on the neurodevelopmental origin for the disorder and augment the impact of both genetic and environmental factors in disease aetiology, the pathophysiology of SCZ remains incompletely understood with little progress in novel treatment development , 2.The last decades of extensive research have yielded valuable insights into the genomic and molecular underpinnings of SCZ. Large-scale genomic analyses triggered by prior studies of strong heritability estimates for SCZ 60–80%) , 4 have 0% , 4 haGene transcription profiles are defined by the activity of regulatory elements (REs) that overlap with open chromatin. Since the activity of REs are modulated by genetic variation, changes in chromatin accessibility result in gene expression variability and hence constitute as an intermediate phenotype for profiling eQTL effects on gene expression –20. SystMolecular and phenotype data for HBCC was accessed through dbGaP . All patients met DSM-IV criteria for a lifetime Axis I diagnosis of psychiatric disorders including schizophrenia or schizoaffective disorder. Controls had no history of psychiatric diagnoses or addictions. For genotype data, we determined the intersect of single nucleotide variant (SNV) content across three Illumina genotyping arrays  after filtering the SNVs using standard procedure with PLINK v2.0  and impuFor CRD calling, we used the pipeline developed in Delaneau et al. . First, qvalue package [To assess the features of SCZ-specific CRDs, we considered only CRDs in SCZ cases composed of peaks not regrouping into any CRD in controls. For peak activity estimation, we used ChIP-sequencing peak quantifications uncorrected for covariates and applied Mann-Whitney U test per peak activity between SCZ cases and controls. Significant differences between SCZ cases and controls were determined at false discovery rate (FDR) 5% using R/ package . For est/clusterProfiler package [Differential CRD activity analysis and differential gene expression analysis were carried out using DESeq2 . Signifi package  was usedBoth MAGMA v1.10  and partQTL mapping was carried out using the standard procedure implemented in the QTLtools software package . SpecifiWe used QTLtools cis permuation pass  to identTo quantify gene-CRD pairs identified as significant at FDR 5% across SCZ cases and controls, we used PC analysis-based dimensionality reduction. For each gene-CRD pair, we aggregated gene expression with CRD activity and used the coordinates on PC1 as new pseudo-phenotypes for QTL mapping in a cis window. This effectively gave us eCRDQTL-CRD-gene triplets consisting of a genetic variant, a CRD, and a gene, all associated with each other.We applied a Bayesian Network approach to infer the most likely causal relationship for eCRDQTL-CRD-gene triplets common to SCZ cases and controls and conducted the analyses separately in SCZ cases and in controls. This approach allowed to estimate the most likely network from which the observed data originates by calculating the posterior probabilities for the three possible causal models:  (i) caus/clusterProfiler package [To provide confidence for retrieved probabilities, we carried out 100 bootstrapping runs for each tested triplet separately for SCZ cases and controls using sampling with replacement. We estimated how many times the most probable model across bootstrapping runs for each triplet was the same as in the original Bayesian Network results and filtered out all triplets that fell below a confidence threshold of 55%: this corresponds to the lower quartile value in SCZ cases. R package  was usedExtended methods are provided in the Supplementary.n = 40,819), 31.4%  and 38.4%  of the peaks, respectively , regrouping 28.9%  1.52) and significantly depleted of those captured in adults [p = 3.04 × 10−50, OR = 0.57; Fig. p = 0.008; Supplementary Fig. POU3F1 , KIF5A nsporter ), NECAB1 neurons ), and PDr ligand ) , whereas down-regulated DACs were not  . We set out to determine the differences in regulatory activity between SCZ cases and controls and to investigate their relation to genes that were differentially expressed (DEGs) between the two groups. At FDR 5%, we identified 1141 CRDs  and 1363 genes  to be differentially active and expressed in SCZ cases, respectively Table . The det65) Fig. , or baseCorrelating gene expression and CRD activity  in cis using linear regression revealed 95, 634, and 1197 CRD-gene associations in SCZ cases, in controls, and in the combined set, respectively, at FDR 5% Table . Most gen = 42) and 16% of eQTLs (n = 158) showed SCZ-specificity, i.e., these affected CRD activity or gene expression only in SCZ cases or displayed significant change in effect size compared to controls , and 987 and 6,716 functionally independent eQTLs in SCZ cases and controls, respectively Table . The strls Table . The SCZls Table . Colocalls Table . Of H3K2ls Table .Fig. 3SCTo assess common genetic regulation of coordinated regulatory activity and gene expression, we investigated the association of aCRD-QTL and eQTL effects on the other molecular phenotype. Specifically, we correlated aCRD-QTLs with gene expression and eQTLs with CRD activity over gene-CRD associations detected across all samples at nominal significance. We identified that up to 47% of the eQTL and aCRD-QTL variants had an effect on CRD activity and gene expression, respectively  and controls (n = 105)  that affected both molecular phenotypes and by that determined eCRDQTL-CRD-gene triplets for causal inference. Using Bayesian Networks (BN), we tested three relationship patterns: (i) causal model in which the genetic variant affects first the activity of the CRD which then regulates the gene expression, (ii) reactive model in which the genetic variant affects the expression of the gene which then modulates the CRD activity, and (iii) independent model in which the genetic variant affects the gene and the CRD independently; and studied these relationships separately in SCZ cases  than in controls  . THBS2 is an extracellular matrix protein of the central nervous system that is secreted by astrocytes to control excitatory synaptogenesis. Importantly, small GTPase proteins have been shown to play a key component in the synaptogenic signalling cascade downstream of the protein and its neuronal calcium channel subunit receptor [THBS2 expression did not translate via the same regulatory mechanism in both states. Interestingly, this association was identified only in individuals with African American ancestry (Supplementary Fig. THBS2 expression in SCZ cases was mediated by the regrouping of the identified regulatory regions and represents a dysregulated step within an abnormal molecular cascade affecting synapse function in SCZ.A perturbation in the regulatory mechanism of gene expression in SCZ is exemplified by a triplet consisting of an eCRD-QTL chr6:169646282:A:T associated to gene receptor –52. BaseDeciphering regulatory mechanisms of gene expression that reflect molecular perturbation in SCZ are under extensive scrutiny yet are hindered by the complexity of the SCZ phenotype and scarcity of relevant molecular data. Studying regulatory activity that tracks changes in gene expression requires a higher order analysis approach for signal discernment due to narrow variability range in regulatory activity and extensive multiple testing burden , 22. Her+ nuclei samples overlap with those detected in bulk DLPFC [(Supplementary Fig. First, our results support the neurodevelopmental hypothesis for schizophrenia. Multiple studies have highlighted the concordance of SCZ heritability enrichment for open chromatin regions in fetal and in SCZ DLPFC samples and the stability of methylation and expression feature deviations in fetal brain development that persist into adulthood for those affected by the disorder , 22, 55.Altogether, we have outlined that leveraging higher-order structural resolution of regulatory activity allows to reduce the search space for unveiling genetically perturbed regulation of gene expression specific to SCZ. We anticipate that cell-type specific gene expression and open chromatin exposure profiles in larger sample sets would allow better delimitation of CRD chromatin peak content, facilitate the identification of trans-regulatory hubs across different chromosomes as well as enhance more robust detection of origin effect for gene expression deviations, thereby increasing our understanding of perturbed functional pathways underlying schizophrenia and for prioritizing targets for experimental investigation and novel treatment development.Supplementary InformationSupplementary tables


Measurement of T cell receptor (TCR) or B cell receptor (BCR) gene utilization may be valuable in monitoring the dynamic changes in donor-reactive clonal populations following transplantation and enabling adjustment in therapy to avoid the consequences of excess immune suppression or to prevent rejection with contingent graft damage and to indicate the development of tolerance.We performed a review of current literature to examine research in immune repertoire sequencing in organ transplantation and to assess the feasibility of this technology for clinical application in immune monitoring.We searched MEDLINE and PubMed Central for English-language studies published between 2010 and 2021 that examined T cell/B cell repertoire dynamics upon immune activation. Manual filtering of the search results was performed based on relevancy and predefined inclusion criteria. Data were extracted based on study and methodology characteristics.Our initial search yielded 1933 articles of which 37 met the inclusion criteria; 16 of these were kidney transplant studies (43%) and 21 were other or general transplantation studies (57%). The predominant method for repertoire characterization was sequencing the CDR3 region of the TCR β chain. Repertoires of transplant recipients were found to have decreased diversity in both rejectors and non-rejectors when compared to healthy controls. Rejectors and those with opportunistic infections were more likely to have clonal expansion in T or B cell populations. Mixed lymphocyte culture followed by TCR sequencing was used in 6 studies to define an alloreactive repertoire and in specialized transplant settings to track tolerance.Methodological approaches to immune repertoire sequencing are becoming established and offer considerable potential as a novel clinical tool for pre- and post-transplant immune monitoring. But by virtue of its invasive nature, biopsy is normally employed only when clinical data raise diagnostic suspicion. Donor-derived cell-free DNA (dd-cfDNA) has shown potential as a biomarker for early detection of immune activation and serial surveillance . Novel sGenomic analysis of the T and B cell receptor  repertoires promises to further our understanding of immune rejection and allows us to develop clinical biomarkers of graft injury for predicting short- and long-term graft outcomes , 3 T andvia B cell receptor (BCR)-mediated recognition of an HLA-peptide complex presented by helper T cells. Once activated, the B cells mature and differentiate into antibody-secreting plasma cells or memory B cells. A single activated B cell may generate 5000 plasma cells in a week and up to 1012 antibodies per day  recognition of a cognate antigenic peptide presented on a class I or class II major histocompatibility complex (MHC). This interaction is stimulatory, and its strength depends on the density and affinity of the TCR-MHC interaction . AntigenOnce antigen specific recognition occurs, the second signal involves engagement of immune checkpoints which are receptor-ligand pairs belonging to either the TNF or immunoglobulin superfamily. These act to either co-stimulate or co-inhibit the T cell response upon TCR engagement thereby directing and tightly regulating the subsequent T cell response Figure 1TCR engagement and co-stimulation together lead to downstream activation of the calcineurin/NFAT, MAP kinase, and NF-kB pathways which in turn lead to the transcription of cytokines which provide the third signal for T cell proliferation and differentiation . BecauseLast, the third signal which occurs upon antigen specific TCR engagement and co-stimulation, is the evolution of cytokines to direct T cell proliferation and differentiation. The prototypical signal responsible for T cell proliferation is IL2 which binds the IL2 receptor (IL2R) on T cells. Downstream activation of the mechanistic target of rapamycin (MTOR) through IL2R favours the proliferation and differentiation of conventional T cells whereas IL2R engagement in the presence of MTOR inhibition favours the proliferation and differentiation of T regulatory cells through STAT5 signaling . The IL23B cell signaling and activation is a fundamental step in the humoral immune response and in AMR. First, immature B cells released from the bone marrow circulate in the secondary lymphoid organs (spleen and lymph nodes) until an antigen is encountered . RecogniB cells have also been targeted for immunosuppression in transplant. Rituximab is directed against CD20 on the B cell surface and has been used extensively in treatment of AMR as well as desensitization therapies . B cell 46 to 108 unique T cells as identified by their TCR sequence. Such repertoire diversity underlies the breadth of the adaptive immune response against an array of immune targets. BCR diversity is generated via a similar V(D)J gene rearrangement process , diversity (D), and joining (J) while α gene recombination involves V and J segments only. TCR β gene recombination occurs in a stepwise fashion in which D-to-J rearrangement is followed by V-to-DJ rearrangement Figure 2 process .Considering this profound diversity and specificity, the identification of unique TCR and BCR sequences using lymphocyte receptor sequencing provides an important opportunity to characterize and monitor the dynamic changes in the immune repertoire in response to an antigenic stimulus, particularly HLA molecules, in organ transplantation. This review describes a systematic synthesis of current knowledge and methods for using lymphocyte receptor repertoire sequencing in the setting of solid organ transplantation. First, we summarize different sequencing approaches and compare their relative advantages in a clinical setting. Next, we review current data pertaining to alloreactivity and tolerance in renal transplant and the more limited data in other solid organ transplants. Finally, we discuss promising areas for future study in this space.5Two authors (P.W. and D.P.C) performed the searches in MEDLINE and PubMed Central, SCOPUS and EMBASE library databases. Commencing with T-cell and B-cell receptor sequencing, five refined key search terms were used in combinations for a total of six queries using the Boolean operator “AND”, as this allowed for only studies mentioning key search terms to be included Table 1.Articles were then evaluated for inclusion in the study according to the workflow process summarized in Study characteristics were categorized and summarized. Details included publication reference , receptor type (eg. TCR or BCR), organ transplant, sequencing region (eg. TCRβ sequencing), PCR method (eg. multiplex), starting material (eg. RNA), and whether a mixed lymphocyte reaction was conducted to identify clonal targets. For applicable studies, the subtype of immune cells investigated was noted. Other notable study characteristics were documented at the discretion of the reviewer.66.1vs 35%), which could be due to the role of T cells in multiple mechanisms of graft injury, but perhaps also because there are established B cell methods including DSA testing. Not only are T cells involved directly in CMR, but they also play a role in the evolution of antibody-producing B cells that underly AMR. This points to the potential clinical utility of TCR sequencing in monitoring early phases of the alloimmune response with the goal of early intervention to prevent irreversible graft. BCR sequencing may also have clinical potential as it could provide further insight into the dynamics of AMR which is generally a more destructive form of graft injury.A summary of the studies included in this review and their characteristics are outlined in 6 cells/mL, with a 5-fold increase in frequency from seeding to harvesting defined as donor-reactive clones. Harvested cells were then fluorescence activated cell sorted (FACS) to isolate CFSElow proliferating cells as well as CD4+, CD8+, and Tregs. Taken together, MLC approaches have been similar although with slight variations in culturing protocols.For purposes of identifying alloreactive clones, generation of donor-specific T or B cells could be a valuable resource. Mixed lymphocyte culture (MLC) coupled with receptor sequencing was a common method used amongst studies. Pre-transplant culturing of irradiated donor PBMCs with proliferation dye carboxyfluorescein diacetate succinimidyl ester (CFSE) stained recipient PBMCs over 5-7 days , 83, 91 The TCRβ chain was the genomic region sequenced in all 24 TCR sequencing studies, with 4 studies targeting both TCRα and TCRβ chains. Since each T cell can potentially express more than one α chain, but only one β chain, the β chain is considered a precise candidate to uniquely identify a given T cell clone . All stuDiffering approaches were used to generate sequencing reads. Of the 25 studies that detailed PCR methods, 20 (80%) used a multiplex PCR method. This versatile approach accepts either gDNA or RNA input, but the mix of primers targeting only known V gene alleles means that novel alleles cannot be detected. It is also prone to amplification biases which can be mitigated with molecular barcoding . Five st6.2Lymphocyte receptor sequencing has been applied most extensively in the context of renal transplantation to identify and track the specific alloreactive T cell repertoire, as well as characterize general trends in the peripheral and graft infiltrating T cell repertoire.To define an ‘alloreactive T cell fingerprint’ Morris et al. developed a novel method using a MLC of recipient PBMCs labelled with CFSE and irradiated donor PBMCs labeled with Cell Tracer Violet for 6 days. The cultured cells were then FACS sorted for CD4+ and CD8+ CFSE-low cells and then their TCR β-chain CDR3 regions were sequenced. Putative donor-reactive clones are then defined as those that expanded at least 5-fold relative to their abundance in the baseline pre-MLC recipient repertoire .in vitro alloreactive response, in terms of diversity and expansion of clones, correlated well with the magnitude of the alloreactive response post-transplant. CKBMT patients who developed tolerance had a significant reduction in both number and diversity of circulating donor reactive T cells while those who experience rejection after IS withdrawal did not. This supports a role for clonal deletion as a mechanism of tolerance  was withdrawn, and 2 conventional living donor renal transplant patients. In the two conventional renal transplant patients CD4+ but not CD8+ donor reactive T cells increased in the post-transplant peripheral repertoire over time. In these patients, the magnitude of the olerance .Savage et al. then leveraged TCR sequencing to study the role of T regulatory cells in maintaining tolerance in the same CKBMT patient population which included 3 tolerant and 1 non-tolerant subject after 10 months of IS withdrawal. They used FACS sorting for CD4+CD25+CD127- Treg lineage markers and TCR sequencing to define Treg repertoires under 3 different conditions: unstimulated, after stimulation with activated donor B-cells, or after stimulation with MLC in the presence of CTLA4Ig. The ‘activated donor B-cell method’ identified the most biologically relevant repertoire of Tregs that correlated best with tolerance by clonal fraction and cumulative frequency at 6 months. Tregs identified by this method were not expanded in the non-tolerant subject suggesting that tolerance despite lack of persistent mixed chimerism in these CKBMT patients may be in part explained by T regulatory cells .These studies not only highlighted how TCR sequencing can be used to elucidate the biology of tolerance in specialized renal transplant cases, but also provide a basis for further studies using TCR sequencing of the alloreactive repertoire as a biomarker in conventional renal transplant. Aschauer et al. leverage a similar method in a single-center prospective cohort study designed to evaluate the expansion or deletion of alloreactive T cells in the post-transplant repertoire of patients with or without acute TCMR. Secondary outcomes included local T cell expansion in the graft during a rejection episode and changes in clonal diversity over time in rejecting versus non-rejecting patients . Using aIn a separate study aimed at understanding the impact of different induction therapies on the alloreactive repertoire in 5 patients with pre-formed DSA induced with rabbit anti-thymocyte globulin (rATG) plus immunoadsorption and 5 without DSA given Basiliximab. MLC and TCR sequencing was performed at baseline and the alloreactive repertoire tracked for a year. They showed that over 1 year the percentage of peripheral CD4+ but not CD8+ donor-reactive T cells increased at similar frequencies across both groups . While tThe remaining studies in this space aim to measure general features of the T and B cell repertoire in patients prior to and after transplant. The conclusions that can be drawn from these studies as to the clinical utility of TCR sequencing in transplant are limited by a lack of systematic approaches and standardized metrics.Two studies examined trends in the T cell repertoire of patients both pre- and post- renal transplant who do experience rejection. Lai et al. characterized the peripheral T cell repertoire in 10 transplant recipients preoperatively, then on postoperative day (POD) 1 and POD 7. These were then compared to a cohort of 10 healthy controls at a single time point. Repertoire overlap between patients, also known as the public repertoire, was minimal. High-abundance clones in individual patients were present at all time points suggesting that abundant clones can be reliably tracked in the peripheral circulation of an individual over time . No diffThe presumption that graft-infiltrating clones occur in peripheral blood underlies the use of TCR sequencing as a monitoring tool in renal allograft rejection. Weinberger et al. attempted to quantify this using nephrectomy samples from 10 patients of whom one was a transplant patient suffering from acute T cell mediated rejection. They showed poor clonal correlation between the B and T cell receptor profiles of tissue and peripheral samples, but that the correlation was greatest among the top 20 most abundant T cell clones . There wWhile these studies that quantify general TCR repertoire metrics in renal transplantation suffer from heterogenous study designs, low patient numbers, and highly variable approaches to data analysis, they offer initial guidance to the feasibility and potential of this technology in a clinical setting and several key technical insights. First, TCR repertoire diversity, CDR3 length, and gene usage are all commonly used metrics in this setting. Second, highly abundant clones in the repertoire can be more reliably tracked over time for a given patient. Third, the graft infiltrating profile correlates best with the peripheral repertoire for highly abundant clones but that this correlation is not quantitative. Future studies will have to better define the nature of highly abundant clones and delineate their cognate targets to better understand their role as a clinical tool in renal transplant.IGHV3-23 gene was used significantly more in patients who develop rejection across all time-points  sequencing. Pineda et al. performed longitudinal BCR sequencing in renal transplant patients, dividing them into three cohorts consisting of: patients who did not reject and did not sustain chronic graft damage, patients who did not reject but did sustain chronic graft damage, and patients who did reject and sustain chronic graft damage . This ste-points , in linee-points . Moreovee-points . While t6.3TCR sequencing has also been evaluated in other solid organ transplant studies including liver, intestinal, and heart transplant.Two studies examined the role of TCR sequencing in liver transplant. Yang et al. investigated general differences in TCR repertoire dynamics between liver recipients and healthy controls . TCR seqIntestinal allografts are rich in lymphoid tissue, which over time engage in a bidirectional exchange with the recipient in which various degrees of bone marrow and peripheral blood chimerism occur or the allograft can become repopulated with recipient lymphocytes. Two detailed studies from the Sykes group leverage TCR sequencing to understand the biology of this unique case study in SOT. First, using MLC and TCR sequencing they define a graft-versus-host (GvH) and a host-versus-graft (HvG) alloreactive T cell profile . InstancBCR sequencing has been used to monitor the response to immunosuppression in a cohort of 12 heart transplant recipients of whom 6 participants had moderate-to-severe acute rejection . This st6.4Opportunistic infections are not only a serious complication of immunosuppression in solid organ transplant but also predispose patients to immune alloreactivity. Common infectious pathogens include Cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK polyoma virus, Varicella zoster virus (VZV), Herpes simplex virus (HSV), and Pneumocystis jirovecii. Lymphocyte receptor sequencing has the potential of offering insight not only into the risk and dynamics of opportunistic infections in solid organ transplant, but also the mechanistic underpinnings of how viral infections predispose patients to immune rejection.CMV is a common viral infection that is associated with allograft rejection, although the mechanistic underpinning of this phenomenon is poorly understood. TCR sequencing offers a unique lens through which to interrogate the hypothesis that cross-reactivity of T cells directed at alloantigen and CMV underlies this phenomenon. Using an observational cohort study design, Stranavova et al. showed a significant association between CMV immediate-early protein 1 (IE-1) specific T cell activity by IFNγ ELISPOT and acute renal allograft rejection and function . These dEBV is a common virus that seldom causes pathology in healthy immunocompetent adults. CD8 T cells provide important protection against EBV. GLCTLVAML (GLC) is a highly conserved immunogenic EBV peptide targeted by a restricted T cell repertoire that is highly conserved between individuals. Nguyen et al. leveraged TCR sequencing to quantify clonotypic diversity and abundance of T cells targeting GLC in healthy individuals, then tracked the dynamics of CD8+ GLC-specific clones over time in a cohort of lung transplant recipients before and after EBV exposure. They showed that, on average, the GLC-specific CD8+ TCRαβ repertoire consisted of 10 TCRαβ clonotypes per individual, and that this repertoire was stable before and after immunosuppression in lung-transplant patients without EBV reactivation. In the setting of clinical EBV reactivation, GLC CD8+ T cells expanded in the transplant recipients. There was marked overlap between repertoire at different time points, although the dominant clonotype changed with the onset of profound viremia . AnotherReactivation of BK polyoma virus, and BK nephropathy, in the setting of immunosuppression is another important infectious cause of graft loss which is currently best diagnosed with histology. Histological features of BK nephropathy can however mimic those of acute cellular rejection. This is further complicated by the possible co-existence of these conditions. Treatment of acute rejection involves increasing immunosuppression transiently which would worsen BK viremia and BK nephropathy as T cell mediated cellular immunity is the key to controlling BK infection. Indeed, TCR sequencing and flow cytometry with cell-sorting was used to show that BK viral clearance in transplant patients correlates with the repertoire diversity of BK virus specific T cells and markers of exhaustion in BK specific CD4+ T cells . This meVZV, HSV, and Pneumocystis jirovecii are less studied in SOT through immune receptor sequencing methods. VZV complications are uncommon as most recipients are seropositive , but her7In this article, we present a first, formal review of lymphocyte receptor sequencing in SOT to monitor alloreactivity, to describe how the technique is currently being used to monitor renal, liver, intestinal, and heart transplants, and to explore its potential in furthering our understanding of opportunistic infections and immune rejection.From a methodologic standpoint, sequencing of the CDR3 region of the TCRβ locus was the preferred method for quantifying the T cell repertoire. In both renal and liver transplant recipients, broad alterations are observed in the T cell repertoire with decreased repertoire diversity relative to controls, minimal public repertoire overlap between patients, and reproducible overlap in private repertoire over time , 95. FurBCR sequencing was less commonly studied and only applied to describe trends in the repertoire before and after transplant. These studies showed an association between specific IGHV gene sequences and class-switched or highly mutated IgM sequences and rejection , 84. SomPracticality and financial considerations cannot be understated when considering a clinical assay. A TCR/BCR sequencing kit from private biotech companies can cost upwards of several thousand dollars, and sequencing services can cost even more. Meanwhile, Enzyme Linked Immunosorbent Spot (ELISPOT) kits can be more cost-effective (Abcam). In terms of assay time required, ELISPOT can take up to several days for optimal cytokine secretion. Although the initial MLC to establish the alloreactive repertoire can take several days, subsequent receptor sequencing procedures for routine monitoring would only require 2 days. Using nanopore sequencing, this time can be further decreased. While the cost-effectiveness of TCR/BCR sequencing assays may be improved upon as the technology matures, it shows promise as an efficient yet precise monitoring technique for SOT.An immediate question this research poses is whether tools are available to predict antigen specificity based on TCR or BCR sequences. Studies on antigen prediction are limited, but two studies provide a glimpse for its potential. Grouping of lymphocyte interactions by paratope hotspots (GLIPH) is an algorithm developed by Glanville et al. that defines TCR specificity groups based on a dataset of MHC tetramer-sorted cells with structural data . It is aDespite the scope and depth of this review, several limitations remain regarding translation to clinical diagnostic practice. First, the conclusions we can draw are limited by the quality of the evidence. While we have performed a comprehensive review of the literature, the studies on this topic are small observational cohort studies and case reports. These studies by their very nature are hypothesis generating. Second, prior to the advent of TCR sequencing, ELISPOT has been used to quantitate the allo-specific memory T cell response. While these data are beyond the scope of this paper, ELISPOT has shown promise in some clinical trials , 114, anTaken together, the findings outlined above point to the promise of lymphocyte receptor sequencing in detecting and monitoring the early phases of allorecognition and rejection in a clinically relevant setting. While the data is promising, larger prospective studies in a more generalizable SOT population are needed.KRS and PAK conceived the study idea and PAK obtained research funding. PW developed a search protocol and conducted the review. PW and DPC extracted the data. KRS and PAK supervised the study progress and provided regular feedback. PW and DPC wrote the draft of the manuscript. RS-P and CP were contributing members of the TCR research planning team. All authors contributed to the article and approved the submitted version.