Genetic newborn screening and digital technologies: A project protocol based on a dual approach to shorten the rare diseases diagnostic path in Europe.

Since 72% of rare diseases are genetic in origin and mostly paediatrics, genetic newborn screening represents a diagnostic "window of opportunity". Therefore, many gNBS initiatives started in different European countries. Screen4Care is a research project, which resulted of a joint effort between the European Union Commission and the European Federation of Pharmaceutical Industries and Associations. It focuses on genetic newborn screening and artificial intelligence-based tools which will be applied to a large European population of about 25.000 infants. The neonatal screening strategy will be based on targeted sequencing, while whole genome sequencing will be offered to all enrolled infants who may show early symptoms but have resulted negative at the targeted sequencing-based newborn screening. We will leverage artificial intelligence-based algorithms to identify patients using Electronic Health Records (EHR) and to build a repository "symptom checkers" for patients and healthcare providers. S4C will design an equitable, ethical, and sustainable framework for genetic newborn screening and new digital tools, corroborated by a large workout where legal, ethical, and social complexities will be addressed with the intent of making the framework highly and flexibly translatable into the diverse European health systems.

'Single-subject studies'-derived analyses unveil altered biomechanisms between very small cohorts: implications for rare diseases.

MOTIVATION: Identifying altered transcripts between very small human cohorts is particularly challenging and is compounded by the low accrual rate of human subjects in rare diseases or sub-stratified common disorders. Yet, single-subject studies (S3) can compare paired transcriptome samples drawn from the same patient under two conditions (e.g. treated versus pre-treatment) and suggest patient-specific responsive biomechanisms based on the overrepresentation of functionally defined gene sets. These improve statistical power by: (i) reducing the total features tested and (ii) relaxing the requirement of within-cohort uniformity at the transcript level. We propose Inter-N-of-1, a novel method, to identify meaningful differences between very small cohorts by using the effect size of 'single-subject-study'-derived responsive biological mechanisms. RESULTS: In each subject, Inter-N-of-1 requires applying previously published S3-type N-of-1-pathways MixEnrich to two paired samples (e.g. diseased versus unaffected tissues) for determining patient-specific enriched genes sets: Odds Ratios (S3-OR) and S3-variance using Gene Ontology Biological Processes. To evaluate small cohorts, we calculated the precision and recall of Inter-N-of-1 and that of a control method (GLM+EGS) when comparing two cohorts of decreasing sizes (from 20 versus 20 to 2 versus 2) in a comprehensive six-parameter simulation and in a proof-of-concept clinical dataset. In simulations, the Inter-N-of-1 median precision and recall are > 90% and >75% in cohorts of 3 versus 3 distinct subjects (regardless of the parameter values), whereas conventional methods outperform Inter-N-of-1 at sample sizes 9 versus 9 and larger. Similar results were obtained in the clinical proof-of-concept dataset. AVAILABILITY AND IMPLEMENTATION: R software is available at Lussierlab.net/BSSD.

Correction to: binomialRF: interpretable combinatoric efficiency of random forests to identify biomarker interactions.

An amendment to this paper has been published and can be accessed via the original article.

binomialRF: interpretable combinatoric efficiency of random forests to identify biomarker interactions.

BACKGROUND: In this era of data science-driven bioinformatics, machine learning research has focused on feature selection as users want more interpretation and post-hoc analyses for biomarker detection. However, when there are more features (i.e., transcripts) than samples (i.e., mice or human samples) in a study, it poses major statistical challenges in biomarker detection tasks as traditional statistical techniques are underpowered in high dimension. Second and third order interactions of these features pose a substantial combinatoric dimensional challenge. In computational biology, random forest (RF) classifiers are widely used due to their flexibility, powerful performance, their ability to rank features, and their robustness to the "P > > N" high-dimensional limitation that many matrix regression algorithms face. We propose binomialRF, a feature selection technique in RFs that provides an alternative interpretation for features using a correlated binomial distribution and scales efficiently to analyze multiway interactions. RESULTS: In both simulations and validation studies using datasets from the TCGA and UCI repositories, binomialRF showed computational gains (up to 5 to 300 times faster) while maintaining competitive variable precision and recall in identifying biomarkers' main effects and interactions. In two clinical studies, the binomialRF algorithm prioritizes previously-published relevant pathological molecular mechanisms (features) with high classification precision and recall using features alone, as well as with their statistical interactions alone. CONCLUSION: binomialRF extends upon previous methods for identifying interpretable features in RFs and brings them together under a correlated binomial distribution to create an efficient hypothesis testing algorithm that identifies biomarkers' main effects and interactions. Preliminary results in simulations demonstrate computational gains while retaining competitive model selection and classification accuracies. Future work will extend this framework to incorporate ontologies that provide pathway-level feature selection from gene expression input data.

Evaluating single-subject study methods for personal transcriptomic interpretations to advance precision medicine.

BACKGROUND: Gene expression profiling has benefited medicine by providing clinically relevant insights at the molecular candidate and systems levels. However, to adopt a more 'precision' approach that integrates individual variability including 'omics data into risk assessments, diagnoses, and therapeutic decision making, whole transcriptome expression needs to be interpreted meaningfully for single subjects. We propose an "all-against-one" framework that uses biological replicates in isogenic conditions for testing differentially expressed genes (DEGs) in a single subject (ss) in the absence of an appropriate external reference standard or replicates. To evaluate our proposed "all-against-one" framework, we construct reference standards (RSs) with five conventional replicate-anchored analyses (NOISeq, DEGseq, edgeR, DESeq, DESeq2) and the remainder were treated separately as single-subject sample pairs for ss analyses (without replicates). RESULTS: Eight ss methods (NOISeq, DEGseq, edgeR, mixture model, DESeq, DESeq2, iDEG, and ensemble) for identifying genes with differential expression were compared in Yeast (parental line versus snf2 deletion mutant; n = 42/condition) and a MCF7 breast-cancer cell line (baseline versus stimulated with estradiol; n = 7/condition). Receiver-operator characteristic (ROC) and precision-recall plots were determined for eight ss methods against each of the five RSs in both datasets. Consistent with prior analyses of these data, ~ 50% and ~ 15% DEGs were obtained in Yeast and MCF7 datasets respectively, regardless of the RSs method. NOISeq, edgeR, and DESeq were the most concordant for creating a RS. Single-subject versions of NOISeq, DEGseq, and an ensemble learner achieved the best median ROC-area-under-the-curve to compare two transcriptomes without replicates regardless of the RS method and dataset (> 90% in Yeast, > 0.75 in MCF7). Further, distinct specific single-subject methods perform better according to different proportions of DEGs. CONCLUSIONS: The "all-against-one" framework provides a honest evaluation framework for single-subject DEG studies since these methods are evaluated, by design, against reference standards produced by unrelated DEG methods. The ss-ensemble method was the only one to reliably produce higher accuracies in all conditions tested in this conservative evaluation framework. However, single-subject methods for identifying DEGs from paired samples need improvement, as no method performed with precision> 90% and obtained moderate levels of recall. http://www.lussiergroup.org/publications/EnsembleBiomarker.

Precision drug repurposing via convergent eQTL-based molecules and pathway targeting independent disease-associated polymorphisms.

Repurposing existing drugs for new therapeutic indications can improve success rates and streamline development. Use of large-scale biomedical data repositories, including eQTL regulatory relationships and genome-wide disease risk associations, offers opportunities to propose novel indications for drugs targeting common or convergent molecular candidates associated to two or more diseases. This proposed novel computational approach scales across 262 complex diseases, building a multi-partite hierarchical network integrating (i) GWAS-derived SNP-to-disease associations, (ii) eQTL-derived SNP-to-eGene associations incorporating both cis- and trans-relationships from 19 tissues, (iii) protein target-to-drug, and (iv) drug-to-disease indications with (iv) Gene Ontology-based information theoretic semantic (ITS) similarity calculated between protein target functions. Our hypothesis is that if two diseases are associated to a common or functionally similar eGene - and a drug targeting that eGene/protein in one disease exists - the second disease becomes a potential repurposing indication. To explore this, all possible pairs of independently segregating GWAS-derived SNPs were generated, and a statistical network of similarity within each SNP-SNP pair was calculated according to scale-free overrepresentation of convergent biological processes activity in regulated eGenes (ITSeGENE-eGENE) and scale-free overrepresentation of common eGene targets between the two SNPs (ITSSNP-SNP). Significance of ITSSNP-SNP was conservatively estimated using empirical scale-free permutation resampling keeping the node-degree constant for each molecule in each permutation. We identified 26 new drug repurposing indication candidates spanning 89 GWAS diseases, including a potential repurposing of the calcium-channel blocker Verapamil from coronary disease to gout. Predictions from our approach are compared to known drug indications using DrugBank as a gold standard (odds ratio=13.1, p-value=2.49x10-8). Because of specific disease-SNPs associations to candidate drug targets, the proposed method provides evidence for future precision drug repositioning to a patient's specific polymorphisms.

Workshop during the Pacific Symposium of Biocomputing, Jan 3-7, 2019: Reading between the genes: interpreting non-coding DNA in high-throughput.

Identifying functional elements and predicting mechanistic insight from non-coding DNA and noncoding variation remains a challenge. Advances in genome-scale, high-throughput technology, however, have brought these answers closer within reach than ever, though there is still a need for new computational approaches to analysis and integration. This workshop aims to explore these resources and new computational methods applied to regulatory elements, chromatin interactions, non-protein-coding genes, and other non-coding DNA.

Developing a 'personalome' for precision medicine: emerging methods that compute interpretable effect sizes from single-subject transcriptomes.

The development of computational methods capable of analyzing -omics data at the individual level is critical for the success of precision medicine. Although unprecedented opportunities now exist to gather data on an individual's -omics profile ('personalome'), interpreting and extracting meaningful information from single-subject -omics remain underdeveloped, particularly for quantitative non-sequence measurements, including complete transcriptome or proteome expression and metabolite abundance. Conventional bioinformatics approaches have largely been designed for making population-level inferences about 'average' disease processes; thus, they may not adequately capture and describe individual variability. Novel approaches intended to exploit a variety of -omics data are required for identifying individualized signals for meaningful interpretation. In this review-intended for biomedical researchers, computational biologists and bioinformaticians-we survey emerging computational and translational informatics methods capable of constructing a single subject's 'personalome' for predicting clinical outcomes or therapeutic responses, with an emphasis on methods that provide interpretable readouts. Key points: (i) the single-subject analytics of the transcriptome shows the greatest development to date and, (ii) the methods were all validated in simulations, cross-validations or independent retrospective data sets. This survey uncovers a growing field that offers numerous opportunities for the development of novel validation methods and opens the door for future studies focusing on the interpretation of comprehensive 'personalomes' through the integration of multiple -omics, providing valuable insights into individual patient outcomes and treatments.

Interpretation of 'Omics dynamics in a single subject using local estimates of dispersion between two transcriptomes.

Calculating Differentially Expressed Genes (DEGs) from RNA-sequencing requires replicates to estimate gene-wise variability, a requirement that is at times financially or physiologically infeasible in clinics. By imposing restrictive transcriptome-wide assumptions limiting inferential opportunities of conventional methods (edgeR, NOISeq-sim, DESeq, DEGseq), comparing two conditions without replicates (TCWR) has been proposed, but not evaluated. Under TCWR conditions (e.g., unaffected tissue vs. tumor), differences of transformed expression of the proposed individualized DEG (iDEG) method follow a distribution calculated across a local partition of related transcripts at baseline expression; thereafter the probability of each DEG is estimated by empirical Bayes with local false discovery rate control using a two-group mixture model. In extensive simulation studies of TCWR methods, iDEG and NOISeq are more accurate at 5%90%, recall>75%, false_positive_rate<1%) and 30%

Novel disease syndromes unveiled by integrative multiscale network analysis of diseases sharing molecular effectors and comorbidities.

BACKGROUND: Forty-two percent of patients experience disease comorbidity, contributing substantially to mortality rates and increased healthcare costs. Yet, the possibility of underlying shared mechanisms for diseases remains not well established, and few studies have confirmed their molecular predictions with clinical datasets. METHODS: In this work, we integrated genome-wide association study (GWAS) associating diseases and single nucleotide polymorphisms (SNPs) with transcript regulatory activity from expression quantitative trait loci (eQTL). This allowed novel mechanistic insights for noncoding and intergenic regions. We then analyzed pairs of SNPs across diseases to identify shared molecular effectors robust to multiple test correction (False Discovery Rate FDReRNA < 0.05). We hypothesized that disease pairs found to be molecularly convergent would also be significantly overrepresented among comorbidities in clinical datasets. To assess our hypothesis, we used clinical claims datasets from the Healthcare Cost and Utilization Project (HCUP) and calculated significant disease comorbidities (FDRcomorbidity < 0.05). We finally verified if disease pairs resulting molecularly convergent were also statistically comorbid more than by chance using the Fisher's Exact Test. RESULTS: Our approach integrates: (i) 6175 SNPs associated with 238 diseases from ~ 1000 GWAS, (ii) eQTL associations from 19 tissues, and (iii) claims data for 35 million patients from HCUP. Logistic regression (controlled for age, gender, and race) identified comorbidities in HCUP, while enrichment analyses identified cis- and trans-eQTL downstream effectors of GWAS-identified variants. Among ~ 16,000 combinations of diseases, 398 disease-pairs were prioritized by both convergent eQTL-genetics (RNA overlap enrichment, FDReRNA < 0.05) and clinical comorbidities (OR > 1.5, FDRcomorbidity < 0.05). Case studies of comorbidities illustrate specific convergent noncoding regulatory elements. An intergenic architecture of disease comorbidity was unveiled due to GWAS and eQTL-derived convergent mechanisms between distinct diseases being overrepresented among observed comorbidities in clinical datasets (OR = 8.6, p-value = 6.4 × 10- 5 FET). CONCLUSIONS: These comorbid diseases with convergent eQTL genetic mechanisms suggest clinical syndromes. While it took over a decade to confirm the genetic underpinning of the metabolic syndrome, this study is likely highlighting hundreds of new ones. Further, this knowledge may improve the clinical management of comorbidities with precision and shed light on novel approaches of drug repositioning or SNP-guided precision molecular therapy inclusive of intergenic risks.

Single subject transcriptome analysis to identify functionally signed gene set or pathway activity.

Analysis of single-subject transcriptome response data is an unmet need of precision medicine, made challenging by the high dimension, dynamic nature and difficulty in extracting meaningful signals from biological or stochastic noise. We have proposed a method for single subject analysis that uses a mixture model for transcript fold-change clustering from isogenically paired samples, followed by integration of these distributions with Gene Ontology Biological Processes (GO-BP) to reduce dimension and identify functional attributes. We then extended these methods to develop functional signing metrics for gene set process regulation by incorporating biological repressor relationships encoded in GO-BP as negatively_regulates edges. Results revealed reproducible and biologically meaningful signals from analysis of a single subject's response, opening the door to future transcriptomic studies where subject and resource availability are currently limiting. We used inbred mouse strains fed different diets to provide isogenic biological replicates, permitting rigorous validation of our method. We compared significant genotype-specific GO-BP term results for overlap and rank order across three replicate pairs per genotype, and cross-methods to reference standards (limma+FET, SAM+FET, and GSEA). All single-subject analytics findings were robust and highly reproducible (median area under the ROC curve=0.96, n=24 genotypes × 3 replicates), providing confidence and validation of this approach for analyses in single subjects. R code is available online at http://www.lussiergroup.org/publications/PathwayActivity.

Reading Between the Genes: Computational Models to Discover Function from Noncoding DNA.

Noncoding DNA - once called "junk" has revealed itself to be full of function. Technology development has allowed researchers to gather genome-scale data pointing towards complex regulatory regions, expression and function of noncoding RNA genes, and conserved elements. Variation in these regions has been tied to variation in biological function and human disease. This PSB session tackles the problem of handling, analyzing and interpreting the data relating to variation in and interactions between noncoding regions through computational biology. We feature an invited speaker to how variation in transcription factor coding sequences impacts on sequence preference, along with submitted papers that span graph based methods, integrative analyses, machine learning, and dimension reduction to explore questions of basic biology, cancer, diabetes, and clinical relevance.

A genome-by-environment interaction classifier for precision medicine: personal transcriptome response to rhinovirus identifies children prone to asthma exacerbations.

OBJECTIVE: To introduce a disease prognosis framework enabled by a robust classification scheme derived from patient-specific transcriptomic response to stimulation. MATERIALS AND METHODS: Within an illustrative case study to predict asthma exacerbation, we designed a stimulation assay that reveals individualized transcriptomic response to human rhinovirus. Gene expression from peripheral blood mononuclear cells was quantified from 23 pediatric asthmatic patients and stimulated in vitro with human rhinovirus. Responses were obtained via the single-subject gene set testing methodology "N-of-1-pathways." The classifier was trained on a related independent training dataset (n = 19). Novel visualizations of personal transcriptomic responses are provided. RESULTS: Of the 23 pediatric asthmatic patients, 12 experienced recurrent exacerbations. Our classifier, using individualized responses and trained on an independent dataset, obtained 74% accuracy (area under the receiver operating curve of 71%; 2-sided P = .039). Conventional classifiers using messenger RNA (mRNA) expression within the viral-exposed samples were unsuccessful (all patients predicted to have recurrent exacerbations; accuracy of 52%). DISCUSSION: Prognosis based on single time point, static mRNA expression alone neglects the importance of dynamic genome-by-environment interplay in phenotypic presentation. Individualized transcriptomic response quantified at the pathway (gene sets) level reveals interpretable signals related to clinical outcomes. CONCLUSION: The proposed framework provides an innovative approach to precision medicine. We show that quantifying personal pathway-level transcriptomic response to a disease-relevant environmental challenge predicts disease progression. This genome-by-environment interaction assay offers a noninvasive opportunity to translate omics data to clinical practice by improving the ability to predict disease exacerbation and increasing the potential to produce more effective treatment decisions.

N-of-1-pathways MixEnrich: advancing precision medicine via single-subject analysis in discovering dynamic changes of transcriptomes.

BACKGROUND: Transcriptome analytic tools are commonly used across patient cohorts to develop drugs and predict clinical outcomes. However, as precision medicine pursues more accurate and individualized treatment decisions, these methods are not designed to address single-patient transcriptome analyses. We previously developed and validated the N-of-1-pathways framework using two methods, Wilcoxon and Mahalanobis Distance (MD), for personal transcriptome analysis derived from a pair of samples of a single patient. Although, both methods uncover concordantly dysregulated pathways, they are not designed to detect dysregulated pathways with up- and down-regulated genes (bidirectional dysregulation) that are ubiquitous in biological systems. RESULTS: We developed N-of-1-pathways MixEnrich, a mixture model followed by a gene set enrichment test, to uncover bidirectional and concordantly dysregulated pathways one patient at a time. We assess its accuracy in a comprehensive simulation study and in a RNA-Seq data analysis of head and neck squamous cell carcinomas (HNSCCs). In presence of bidirectionally dysregulated genes in the pathway or in presence of high background noise, MixEnrich substantially outperforms previous single-subject transcriptome analysis methods, both in the simulation study and the HNSCCs data analysis (ROC Curves; higher true positive rates; lower false positive rates). Bidirectional and concordant dysregulated pathways uncovered by MixEnrich in each patient largely overlapped with the quasi-gold standard compared to other single-subject and cohort-based transcriptome analyses. CONCLUSION: The greater performance of MixEnrich presents an advantage over previous methods to meet the promise of providing accurate personal transcriptome analysis to support precision medicine at point of care.

kMEn: Analyzing noisy and bidirectional transcriptional pathway responses in single subjects.

MOTIVATION: Understanding dynamic, patient-level transcriptomic response to therapy is an important step forward for precision medicine. However, conventional transcriptome analysis aims to discover cohort-level change, lacking the capacity to unveil patient-specific response to therapy. To address this gap, we previously developed two N-of-1-pathways methods, Wilcoxon and Mahalanobis distance, to detect unidirectionally responsive transcripts within a pathway using a pair of samples from a single subject. Yet, these methods cannot recognize bidirectionally (up and down) responsive pathways. Further, our previous approaches have not been assessed in presence of background noise and are not designed to identify differentially expressed mRNAs between two samples of a patient taken in different contexts (e.g. cancer vs non cancer), which we termed responsive transcripts (RTs). METHODS: We propose a new N-of-1-pathways method, k-Means Enrichment (kMEn), that detects bidirectionally responsive pathways, despite background noise, using a pair of transcriptomes from a single patient. kMEn identifies transcripts responsive to the stimulus through k-means clustering and then tests for an over-representation of the responsive genes within each pathway. The pathways identified by kMEn are mechanistically interpretable pathways significantly responding to a stimulus. RESULTS: In ∼9000 simulations varying six parameters, superior performance of kMEn over previous single-subject methods is evident by: (i) improved precision-recall at various levels of bidirectional response and (ii) lower rates of false positives (1-specificity) when more than 10% of genes in the genome are differentially expressed (background noise). In a clinical proof-of-concept, personal treatment-specific pathways identified by kMEn correlate with therapeutic response (p-value<0.01). CONCLUSION: Through improved single-subject transcriptome dynamics of bidirectionally-regulated signals, kMEn provides a novel approach to identify mechanism-level biomarkers.

USP15 regulates type I interferon response and is required for pathogenesis of neuroinflammation.

Genes and pathways in which inactivation dampens tissue inflammation present new opportunities for understanding the pathogenesis of common human inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis and multiple sclerosis. We identified a mutation in the gene encoding the deubiquitination enzyme USP15 (Usp15L749R) that protected mice against both experimental cerebral malaria (ECM) induced by Plasmodium berghei and experimental autoimmune encephalomyelitis (EAE). Combining immunophenotyping and RNA sequencing in brain (ECM) and spinal cord (EAE) revealed that Usp15L749R-associated resistance to neuroinflammation was linked to dampened type I interferon responses in situ. In hematopoietic cells and in resident brain cells, USP15 was coexpressed with, and functionally acted together with the E3 ubiquitin ligase TRIM25 to positively regulate type I interferon responses and to promote pathogenesis during neuroinflammation. The USP15-TRIM25 dyad might be a potential target for intervention in acute or chronic states of neuroinflammation.

Integrative genomics analyses unveil downstream biological effectors of disease-specific polymorphisms buried in intergenic regions.

Functionally altered biological mechanisms arising from disease-associated polymorphisms, remain difficult to characterize when those variants are intergenic, or, fall between genes. We sought to identify shared downstream mechanisms by which inter- and intragenic single nucleotide polymorphisms (SNPs) contribute to a specific physiopathology. Using computational modeling of 2 million pairs of disease-associated SNPs drawn from genome wide association studies (GWAS), integrated with expression Quantitative Trait Loci (eQTL) and Gene Ontology functional annotations, we predicted 3,870 inter-intra and inter-intra SNP pairs with convergent biological mechanisms (FDR<0.05). These prioritized SNP pairs with overlapping mRNA targets or similar functional annotations were more likely to be associated with the same disease than unrelated pathologies (OR>12). We additionally confirmed synergistic and antagonistic genetic interactions for a subset of prioritized SNP pairs in independent studies of Alzheimer's disease (entropy p=0.046), bladder cancer (entropy p=0.039), and rheumatoid arthritis (PheWAS case-control p<10-4). Using ENCODE datasets, we further statistically validated that the biological mechanisms shared within prioritized SNP pairs are frequently governed by matching transcription factor binding sites and long-range chromatin interactions. These results provide a "roadmap" of disease mechanisms emerging from GWAS and further identify candidate therapeutic targets among downstream effectors of intergenic SNPs.

Analysis of protein-coding genetic variation in 60,706 humans.

Large-scale reference data sets of human genetic variation are critical for the medical and functional interpretation of DNA sequence changes. Here we describe the aggregation and analysis of high-quality exome (protein-coding region) DNA sequence data for 60,706 individuals of diverse ancestries generated as part of the Exome Aggregation Consortium (ExAC). This catalogue of human genetic diversity contains an average of one variant every eight bases of the exome, and provides direct evidence for the presence of widespread mutational recurrence. We have used this catalogue to calculate objective metrics of pathogenicity for sequence variants, and to identify genes subject to strong selection against various classes of mutation; identifying 3,230 genes with near-complete depletion of predicted protein-truncating variants, with 72% of these genes having no currently established human disease phenotype. Finally, we demonstrate that these data can be used for the efficient filtering of candidate disease-causing variants, and for the discovery of human 'knockout' variants in protein-coding genes.

THEMIS is required for pathogenesis of cerebral malaria and protection against pulmonary tuberculosis.

We identify an N-ethyl-N-nitrosourea (ENU)-induced I23N mutation in the THEMIS protein that causes protection against experimental cerebral malaria (ECM) caused by infection with Plasmodium berghei ANKA. Themis(I23N) homozygous mice show reduced CD4(+) and CD8(+) T lymphocyte numbers. ECM resistance in P. berghei ANKA-infected Themis(I23N) mice is associated with decreased cerebral cellular infiltration, retention of blood-brain barrier integrity, and reduced proinflammatory cytokine production. THEMIS(I23N) protein expression is absent from mutant mice, concurrent with the decreased THEMIS(I23N) stability observed in vitro. Biochemical studies in vitro and functional complementation in vivo in Themis(I23N/+):Lck(-/+) doubly heterozygous mice demonstrate that functional coupling of THEMIS to LCK tyrosine kinase is required for ECM pathogenesis. Damping of proinflammatory responses in Themis(I23N) mice causes susceptibility to pulmonary tuberculosis. Thus, THEMIS is required for the development and ultimately the function of proinflammatory T cells. Themis(I23N) mice can be used to study the newly discovered association of THEMIS (6p22.33) with inflammatory bowel disease and multiple sclerosis.