Sequencing chromosomal abnormalities reveals neurodevelopmental loci that confer risk across diagnostic boundaries.

Balanced chromosomal abnormalities (BCAs) represent a relatively untapped reservoir of single-gene disruptions in neurodevelopmental disorders (NDDs). We sequenced BCAs in patients with autism or related NDDs, revealing disruption of 33 loci in four general categories: (1) genes previously associated with abnormal neurodevelopment (e.g., AUTS2, FOXP1, and CDKL5), (2) single-gene contributors to microdeletion syndromes (MBD5, SATB2, EHMT1, and SNURF-SNRPN), (3) novel risk loci (e.g., CHD8, KIRREL3, and ZNF507), and (4) genes associated with later-onset psychiatric disorders (e.g., TCF4, ZNF804A, PDE10A, GRIN2B, and ANK3). We also discovered among neurodevelopmental cases a profoundly increased burden of copy-number variants from these 33 loci and a significant enrichment of polygenic risk alleles from genome-wide association studies of autism and schizophrenia. Our findings suggest a polygenic risk model of autism and reveal that some neurodevelopmental genes are sensitive to perturbation by multiple mutational mechanisms, leading to variable phenotypic outcomes that manifest at different life stages.

Inherited DOCK2 Deficiency in Patients with Early-Onset Invasive Infections.

Background Combined immunodeficiencies are marked by inborn errors of T-cell immunity in which the T cells that are present are quantitatively or functionally deficient. Impaired humoral immunity is also common. Patients have severe infections, autoimmunity, or both. The specific molecular, cellular, and clinical features of many types of combined immunodeficiencies remain unknown. Methods We performed genetic and cellular immunologic studies involving five unrelated children with early-onset invasive bacterial and viral infections, lymphopenia, and defective T-cell, B-cell, and natural killer (NK)-cell responses. Two patients died early in childhood; after allogeneic hematopoietic stem-cell transplantation, the other three had normalization of T-cell function and clinical improvement. Results We identified biallelic mutations in the dedicator of cytokinesis 2 gene (DOCK2) in these five patients. RAC1 activation was impaired in the T cells. Chemokine-induced migration and actin polymerization were defective in the T cells, B cells, and NK cells. NK-cell degranulation was also affected. Interferon-α and interferon-λ production by peripheral-blood mononuclear cells was diminished after viral infection. Moreover, in DOCK2-deficient fibroblasts, viral replication was increased and virus-induced cell death was enhanced; these conditions were normalized by treatment with interferon alfa-2b or after expression of wild-type DOCK2. Conclusions Autosomal recessive DOCK2 deficiency is a new mendelian disorder with pleiotropic defects of hematopoietic and nonhematopoietic immunity. Children with clinical features of combined immunodeficiencies, especially with early-onset, invasive infections, may have this condition. (Supported by the National Institutes of Health and others.).

Genome-wide identification of polycomb-associated RNAs by RIP-seq.

Polycomb proteins play essential roles in stem cell renewal and human disease. Recent studies of HOX genes and X inactivation have provided evidence for RNA cofactors in Polycomb repressive complex 2 (PRC2). Here we develop a RIP-seq method to capture the PRC2 transcriptome and identify a genome-wide pool of >9000 PRC2-interacting RNAs in embryonic stem cells. The transcriptome includes antisense, intergenic, and promoter-associated transcripts, as well as many unannotated RNAs. A large number of transcripts occur within imprinted regions, oncogene and tumor suppressor loci, and stem cell-related bivalent domains. We provide evidence for direct RNA-protein interactions, most likely via the Ezh2 subunit. We also identify Gtl2 RNA as a PRC2 cofactor that directs PRC2 to the reciprocally imprinted Dlk1 coding gene. Thus, Polycomb proteins interact with a genome-wide family of RNAs, some of which may be used as biomarkers and therapeutic targets for human disease.

Recurrent viral infections associated with a homozygous CORO1A mutation that disrupts oligomerization and cytoskeletal association.

BACKGROUND: Coronin-1A (CORO1A) is a regulator of actin dynamics important for T-cell homeostasis. CORO1A deficiency causes T(-)B(+) natural killer-positive severe combined immunodeficiency or T-cell lymphopenia with severe viral infections. However, because all known human mutations in CORO1A abrogate protein expression, the role of the protein's functional domains in host immunity is unknown. OBJECTIVE: We sought to identify the cause of the primary immunodeficiency in 2 young adult siblings with a history of disseminated varicella, cutaneous warts, and CD4(+) T-cell lymphopenia. METHODS: We performed immunologic, genetic, and biochemical studies in the patients, family members, and healthy control subjects. RESULTS: Both patients had CD4(+) T-cell lymphopenia and decreased lymphocyte proliferation to mitogens. IgG, IgM, IgA, and specific antibody responses were normal. Whole-genome sequencing identified a homozygous frameshift mutation in CORO1A disrupting the last 2 C-terminal domains by replacing 61 amino acids with a novel 91-amino-acid sequence. The CORO1A(S401fs) mutant was expressed in the patients' lymphocytes at a level comparable with that of wild-type CORO1A in normal lymphocytes but did not oligomerize and had impaired cytoskeletal association. CORO1A(S401fs) was associated with increased filamentous actin accumulation in T cells, severely defective thymic output, and impaired T-cell survival but normal calcium flux and cytotoxicity, demonstrating the importance of CORO1A oligomerization and subcellular localization in T-cell homeostasis. CONCLUSIONS: We describe a truncating mutation in CORO1A that permits protein expression and survival into young adulthood. Our studies demonstrate the importance of intact CORO1A C-terminal domains in thymic egress and T-cell survival, as well as in defense against viral pathogens.

Regulatory T-cell deficiency and immune dysregulation, polyendocrinopathy, enteropathy, X-linked-like disorder caused by loss-of-function mutations in LRBA.

BACKGROUND: A number of heritable immune dysregulatory diseases result from defects affecting regulatory T (Treg) cell development, function, or both. They include immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, which is caused by mutations in forkhead box P3 (FOXP3), and IPEX-like disorders caused by mutations in IL-2 receptor α (IL2RA), signal transducer and activator of transcription 5b (STAT5b), and signal transducer and activator of transcription 1 (STAT1). However, the genetic defects underlying many cases of IPEX-like disorders remain unknown. OBJECTIVE: We sought to identify the genetic abnormalities in patients with idiopathic IPEX-like disorders. METHODS: We performed whole-exome and targeted gene sequencing and phenotypic and functional analyses of Treg cells. RESULTS: A child who presented with an IPEX-like syndrome and severe Treg cell deficiency was found to harbor a nonsense mutation in the gene encoding LPS-responsive beige-like anchor (LRBA), which was previously implicated as a cause of common variable immunodeficiency with autoimmunity. Analysis of subjects with LRBA deficiency revealed marked Treg cell depletion; profoundly decreased expression of canonical Treg cell markers, including FOXP3, CD25, Helios, and cytotoxic T lymphocyte-associated antigen 4; and impaired Treg cell-mediated suppression. There was skewing in favor of memory T cells and intense autoantibody production, with marked expansion of T follicular helper and contraction of T follicular regulatory cells. Whereas the frequency of recent thymic emigrants and the differentiation of induced Treg cells were normal, LRBA-deficient T cells exhibited increased apoptosis and reduced activities of the metabolic sensors mammalian target of rapamycin complexes 1 and 2. CONCLUSION: LRBA deficiency is a novel cause of IPEX-like syndrome and Treg cell deficiency associated with metabolic dysfunction and increased apoptosis of Treg cells.

Spreading of X chromosome inactivation via a hierarchy of defined Polycomb stations.

X chromosome inactivation (XCI) achieves dosage balance in mammals by repressing one of two X chromosomes in females. During XCI, the long noncoding Xist RNA and Polycomb proteins spread along the inactive X (Xi) to initiate chromosome-wide silencing. Although inactivation is known to commence at the X-inactivation center (Xic), how it propagates remains unknown. Here, we examine allele-specific binding of Polycomb repressive complex 2 (PRC2) and chromatin composition during XCI and generate a chromosome-wide profile of Xi and Xa (active X) at nucleosome-resolution. Initially, Polycomb proteins are localized to ∼150 strong sites along the X and concentrated predominantly within bivalent domains coinciding with CpG islands ("canonical sites"). As XCI proceeds, ∼4000 noncanonical sites are recruited, most of which are intergenic, nonbivalent, and lack CpG islands. Polycomb sites are depleted of LINE repeats but enriched for SINEs and simple repeats. Noncanonical sites cluster around the ∼150 strong sites, and their H3K27me3 levels reflect a graded concentration originating from strong sites. This suggests that PRC2 and H3K27 methylation spread along a gradient unique to XCI. We propose that XCI is governed by a hierarchy of defined Polycomb stations that spread H3K27 methylation in cis.

Next-generation sequencing strategies enable routine detection of balanced chromosome rearrangements for clinical diagnostics and genetic research.

The contribution of balanced chromosomal rearrangements to complex disorders remains unclear because they are not detected routinely by genome-wide microarrays and clinical localization is imprecise. Failure to consider these events bypasses a potentially powerful complement to single nucleotide polymorphism and copy-number association approaches to complex disorders, where much of the heritability remains unexplained. To capitalize on this genetic resource, we have applied optimized sequencing and analysis strategies to test whether these potentially high-impact variants can be mapped at reasonable cost and throughput. By using a whole-genome multiplexing strategy, rearrangement breakpoints could be delineated at a fraction of the cost of standard sequencing. For rearrangements already mapped regionally by karyotyping and fluorescence in situ hybridization, a targeted approach enabled capture and sequencing of multiple breakpoints simultaneously. Importantly, this strategy permitted capture and unique alignment of up to 97% of repeat-masked sequences in the targeted regions. Genome-wide analyses estimate that only 3.7% of bases should be routinely omitted from genomic DNA capture experiments. Illustrating the power of these approaches, the rearrangement breakpoints were rapidly defined to base pair resolution and revealed unexpected sequence complexity, such as co-occurrence of inversion and translocation as an underlying feature of karyotypically balanced alterations. These findings have implications ranging from genome annotation to de novo assemblies and could enable sequencing screens for structural variations at a cost comparable to that of microarrays in standard clinical practice.

Loss of the DNA Repair Gene RNase H2 Identifies a Unique Subset of DDR-Deficient Leiomyosarcomas.

Targeting the DNA damage response (DDR) pathway is an emerging therapeutic approach for leiomyosarcoma (LMS), and loss of RNase H2, a DDR pathway member, is a potentially actionable alteration for DDR-targeted treatments. Therefore, we designed a protein- and genomic-based RNase H2 screening assay to determine its prevalence and prognostic significance. Using a selective RNase H2 antibody on a pan-tumor microarray (TMA), RNase H2 loss was more common in LMS (11.5%, 9/78) than across all tumors (3.8%, 32/843). In a separate LMS cohort, RNase H2 deficiency was confirmed in uterine LMS (U-LMS, 21%, 23/108) and soft-tissue LMS (ST-LMS; 30%, 39/102). In the TCGA database, RNASEH2B homozygous deletions (HomDels) were found in 6% (5/80) of LMS cases, with a higher proportion in U-LMS (15%; 4/27) compared with ST-LMS (2%; 1/53). Using the SNiPDx targeted-NGS sequencing assay to detect biallelic loss of function in select DDR-related genes, we found RNASEH2B HomDels in 54% (19/35) of U-LMS cases with RNase H2 loss by IHC, and 7% (3/43) HomDels in RNase H2 intact cases. No RNASEH2B HomDels were detected in ST-LMS. In U-LMS patient cohort (n = 109), no significant overall survival difference was seen in patients with RNase H2 loss versus intact, or RNASEH2B HomDel (n = 12) versus Non-HomDel (n = 37). The overall diagnostic accuracy, sensitivity, and specificity of RNase H2 IHC for detecting RNA-SEH2B HomDels in U-LMS was 76%, 93%, and 71%, respectively, and it is being developed for future predictive biomarker driven clinical trials targeting DDR in U-LMS.

Integrating circulating T follicular memory cells and autoantibody repertoires for characterization of autoimmune disorders.

Introduction: Autoimmune diseases are heterogeneous and often lack specific or sensitive diagnostic tests. Increased percentages of CD4+CXCR5+PD1+ circulating T follicular helper (cTfh) cells and skewed distributions of cTfh subtypes have been associated with autoimmunity. However, cTfh cell percentages can normalize with immunomodulatory treatment despite persistent disease activity, indicating the need for identifying additional cellular and/or serologic features correlating with autoimmunity. Methods: The cohort included 50 controls and 56 patients with autoimmune cytopenias, gastrointestinal, pulmonary, and/or neurologic autoimmune disease. Flow cytometry was used to measure CD4+CXCR5+ T cell subsets expressing the chemokine receptors CXCR3 and/or CCR6: CXCR3+CCR6- Type 1, CXCR3-CCR6- Type 2, CXCR3+CCR6+ Type 1/17, and CXCR3- CCR6+ Type 17 T cells. IgG and IgA autoantibodies were quantified using a microarray featuring 1616 full-length, conformationally intact protein antigens. The 97.5th percentile in the control cohort defined normal limits for T cell subset percentages and total number (burden) of autoantibodies. Results: This study focused on CD4+CXCR5+ T cells because CXCR5 upregulation occurs after cognate T-B cell interactions characteristic of autoimmune diseases. We refer to these cells as circulating T follicular memory (cTfm) cells to acknowledge the dynamic nature of antigen-experienced CXCR5+ T cells, which encompass progenitors of cTfh or Tfh cells as well as early effector memory T cells that have not yet lost CXCR5. Compared to controls, 57.1% of patients had increased CXCR5+CXCR3+CCR6+ cTfm1/17 and 25% had increased CXCR5+CXCR3-CCR6+ cTfm17 cell percentages. Patients had significantly more diverse IgG and IgA autoantibodies than controls and 44.6% had an increased burden of autoantibodies of either isotype. Unsupervised autoantibody clustering identified three clusters of patients with IgG autoantibody profiles distinct from those of controls, enriched for patients with active autoimmunity and monogenic diseases. An increased percentage of cTfm17 cells was most closely associated with an increased burden of high-titer IgG and IgA autoantibodies. A composite measure integrating increased cTfm1/17, cTfm17, and high-titer IgG and/or IgA autoantibodies had 91.1% sensitivity and 90.9% specificity for identifying patients with autoimmunity. Percentages of cTfm1/17 and cTfm17 percentages and numbers of high-titer autoantibodies in patients receiving immunomodulatory treatment did not differ from those in untreated patients, thus suggesting that measurements of cTfm can complement measurements of other cellular markers affected by treatment. Conclusions: This study highlights two new approaches for assessing autoimmunity: measuring CD4+CXCR5+ cTfm subsets as well as total burden of autoantibodies. Our findings suggest that these approaches are particularly relevant to patients with rare autoimmune disorders for whom target antigens and prognosis are often unknown.

MolBioLib: a C++11 framework for rapid development and deployment of bioinformatics tasks.

SUMMARY: We developed MolBioLib to address the need for adaptable next-generation sequencing analysis tools. The result is a compact, portable and extensively tested C++11 software framework and set of applications tailored to the demands of next-generation sequencing data and applicable to many other applications. MolBioLib is designed to work with common file formats and data types used both in genomic analysis and general data analysis. A central relational-database-like Table class is a flexible and powerful object to intuitively represent and work with a wide variety of tabular datasets, ranging from alignment data to annotations. MolBioLib has been used to identify causative single-nucleotide polymorphisms in whole genome sequencing, detect balanced chromosomal rearrangements and compute enrichment of messenger RNAs (mRNAs) on microtubules, typically requiring applications of under 200 lines of code. MolBioLib includes programs to perform a wide variety of analysis tasks, such as computing read coverage, annotating genomic intervals and novel peak calling with a wavelet algorithm. Although MolBioLib was designed primarily for bioinformatics purposes, much of its functionality is applicable to a wide range of problems. Complete documentation and an extensive automated test suite are provided. AVAILABILITY: MolBioLib is available for download at: http://sourceforge.net/projects/molbiolib CONTACT: ohsumit@molbio.mgh.harvard.edu.

Transposon activation mutagenesis as a screening tool for identifying resistance to cancer therapeutics.

BACKGROUND: The development of resistance to chemotherapies represents a significant barrier to successful cancer treatment. Resistance mechanisms are complex, can involve diverse and often unexpected cellular processes, and can vary with both the underlying genetic lesion and the origin or type of tumor. For these reasons developing experimental strategies that could be used to understand, identify and predict mechanisms of resistance in different malignant cells would be a major advance. METHODS: Here we describe a gain-of-function forward genetic approach for identifying mechanisms of resistance. This approach uses a modified piggyBac transposon to generate libraries of mutagenized cells, each containing transposon insertions that randomly activate nearby gene expression. Genes of interest are identified using next-gen high-throughput sequencing and barcode multiplexing is used to reduce experimental cost. RESULTS: Using this approach we successfully identify genes involved in paclitaxel resistance in a variety of cancer cell lines, including the multidrug transporter ABCB1, a previously identified major paclitaxel resistance gene. Analysis of co-occurring transposons integration sites in single cell clone allows for the identification of genes that might act cooperatively to produce drug resistance a level of information not accessible using RNAi or ORF expression screening approaches. CONCLUSION: We have developed a powerful pipeline to systematically discover drug resistance in mammalian cells in vitro. This cost-effective approach can be readily applied to different cell lines, to identify canonical or context specific resistance mechanisms. Its ability to probe complex genetic context and non-coding genomic elements as well as cooperative resistance events makes it a good complement to RNAi or ORF expression based screens.

Rethinking Immunological Risk: A Retrospective Cohort Study of Severe SARS-Cov-2 Infections in Individuals With Congenital Immunodeficiencies.

BACKGROUND: Debates on the allocation of medical resources during the coronavirus disease 2019 (COVID-19) pandemic revealed the need for a better understanding of immunological risk. Studies highlighted variable clinical outcomes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in individuals with defects in both adaptive and innate immunity, suggesting additional contributions from other factors. Notably, none of these studies controlled for variables linked with social determinants of health. OBJECTIVE: To determine the contributions of determinants of health to risk of hospitalization for SARS-CoV-2 infection among individuals with inborn errors of immunodeficiencies. METHODS: This is a retrospective, single-center cohort study of 166 individuals with inborn errors of immunity, aged 2 months through 69 years, who developed SARS-CoV-2 infections from March 1, 2020, through March 31, 2022. Risks of hospitalization were assessed using a multivariable logistic regression analysis. RESULTS: The risk of SARS-CoV-2-related hospitalization was associated with underrepresented racial and ethnic populations (odds ratio [OR] 4.50; 95% confidence interval [95% CI] 1.57-13.4), a diagnosis of any genetically defined immunodeficiency (OR 3.32; 95% CI 1.24-9.43), obesity (OR 4.24; 95% CI 1.38-13.3), and neurological disease (OR 4.47; 95% CI 1.44-14.3). The COVID-19 vaccination was associated with reduced hospitalization risk (OR 0.52; 95% CI 0.31-0.81). Defects in T cell and innate immune function, immune-mediated organ dysfunction, and social vulnerability were not associated with increased risk of hospitalization after controlling for covariates. CONCLUSIONS: The associations between race, ethnicity, and obesity with increased risk of hospitalization for SARS-CoV-2 infection indicate the importance of variables linked with social determinants of health as immunological risk factors for individuals with inborn errors of immunity.

Rethinking immunologic risk: a retrospective cohort study of severe SARS-CoV-2 infections in individuals with congenital immunodeficiencies.

Background: Debates on the allocation of medical resources during the COVID-19 pandemic revealed the need for a better understanding of immunologic risk. Studies highlighted variable clinical outcomes of SARS-CoV-2 infections in individuals with defects in both adaptive and innate immunity, suggesting additional contributions from other factors. Notably, none of these studies controlled for variables linked with social determinants of health. Objective: To determine the contributions of determinants of health to risk of hospitalization for SARS-CoV-2 infection among individuals with inborn errors of immunodeficiencies. Methods: This is a retrospective, single-center cohort study of 166 individuals with inborn errors of immunity, aged two months through 69 years, who developed SARS-CoV-2 infections from March 1, 2020 through March 31, 2022. Risks of hospitalization was assessed using a multivariable logistic regression analysis. Results: The risk of SARS-CoV-2-related hospitalization was associated with underrepresented racial and ethnic populations (odds ratio [OR] 5.29; confidence interval [CI], 1.76-17.0), a diagnosis of any genetically-defined immunodeficiency (OR 4.62; CI, 1.60-14.8), use of B cell depleting therapy within one year of infection (OR 6.1; CI, 1.05-38.5), obesity (OR 3.74; CI, 1.17-12.5), and neurologic disease (OR 5.38; CI, 1.61-17.8). COVID-19 vaccination was associated with reduced hospitalization risk (OR 0.52; CI, 0.31-0.81). Defective T cell function, immune-mediated organ dysfunction, and social vulnerability were not associated with increased risk of hospitalization after controlling for covariates. Conclusions: The associations between race, ethnicity, and obesity with increased risk of hospitalization for SARS-CoV-2 infection indicate the importance of variables linked with social determinants of health as immunologic risk factors for individuals with inborn errors of immunity. Highlights: What is already known about this topic? Outcomes of SARS-CoV-2 infections in individuals with inborn errors of immunity (IEI) are highly variable. Prior studies of patients with IEI have not controlled for race or social vulnerability. What does this article add to our knowledge ? For individuals with IEI, hospitalizations for SARS-CoV-2 were associated with race, ethnicity, obesity, and neurologic disease. Specific types of immunodeficiency, organ dysfunction, and social vulnerability were not associated with increased risk of hospitalization. How does this study impact current management guidelines? Current guidelines for the management of IEIs focus on risk conferred by genetic and cellular mechanisms. This study highlights the importance of considering variables linked with social determinants of health and common comorbidities as immunologic risk factors.

Sensitive, specific polymorphism discovery in bacteria using massively parallel sequencing.

Our variant ascertainment algorithm, VAAL, uses massively parallel DNA sequence data to identify differences between bacterial genomes with high sensitivity and specificity. VAAL detected approximately 98% of differences (including large insertion-deletions) between pairs of strains from three species while calling no false positives. VAAL also pinpointed a single mutation between Vibrio cholerae genomes, identifying an antibiotic's site of action by identifying sequence differences between drug-sensitive strains and drug-resistant derivatives.

Analysis of combinatorial CRISPR screens with the Orthrus scoring pipeline.

The continued improvement of combinatorial CRISPR screening platforms necessitates the development of new computational pipelines for scoring combinatorial screening data. Unlike for single-guide RNA (sgRNA) pooled screening platforms, combinatorial scoring for multiplexed systems is confounded by guide design parameters such as the number of gRNAs per construct, the position of gRNAs along constructs, and additional features that may impact gRNA expression, processing or capture. In this protocol we describe Orthrus, an R package for processing, scoring and analyzing combinatorial CRISPR screening data that addresses these challenges. This protocol walks through the application of Orthrus to previously published combinatorial screening data from the CHyMErA experimental system, a platform we recently developed that pairs Cas9 with Cas12a gRNAs and enables programmed targeting of multiple genomic sites. We demonstrate Orthrus' features for screen quality assessment and two distinct scoring modes for dual guide RNAs (dgRNAs) that target the same gene twice or dgRNAs that target two different genes. Running Orthrus requires basic R programming experience, ~5-10 min of computational time and 15-60 min total.

The CIP2A-TOPBP1 axis safeguards chromosome stability and is a synthetic lethal target for BRCA-mutated cancer.

BRCA1/2-mutated cancer cells adapt to the genome instability caused by their deficiency in homologous recombination (HR). Identification of these adaptive mechanisms may provide therapeutic strategies to target tumors caused by the loss of these genes. In the present study, we report genome-scale CRISPR-Cas9 synthetic lethality screens in isogenic pairs of BRCA1- and BRCA2-deficient cells and identify CIP2A as an essential gene in BRCA1- and BRCA2-mutated cells. CIP2A is cytoplasmic in interphase but, in mitosis, accumulates at DNA lesions as part of a complex with TOPBP1, a multifunctional genome stability factor. Unlike PARP inhibition, CIP2A deficiency does not cause accumulation of replication-associated DNA lesions that require HR for their repair. In BRCA-deficient cells, the CIP2A-TOPBP1 complex prevents lethal mis-segregation of acentric chromosomes that arises from impaired DNA synthesis. Finally, physical disruption of the CIP2A-TOPBP1 complex is highly deleterious in BRCA-deficient tumors, indicating that CIP2A represents an attractive synthetic lethal therapeutic target for BRCA1- and BRCA2-mutated cancers.

Three-dimensional simulation of anisotropic cell-driven collagen gel compaction.

Tissue equivalents (TEs), formed by entrapping cells in a collagen gel, are an important model system for studying cell behavior. We have previously (Barocas and Tranquillo in J Biomech Eng 117:161-170, 1997a) developed an anisotropic biphasic theory of TE mechanics, which comprises five coupled partial differential equations describing interaction among cells and collagen fibers in the TE. The model equations, previously solved in one or two dimensions, were solved in three dimensions using an adaptive finite-element platform. The model was applied to three systems: a rectangular isometric cell traction assay, an otherwise- acellular gel containing two islands of cells, and an idealized tissue-engineered cardiac valve leaflet. In the first two cases, published experimental data were available for comparison, and the model results were consistent with the experimental observations. Fibers and cells aligned in the fixed direction in the isometric assay, and a region of strong fiber alignment arose between the two cell islands. For the valve problem, the alignment predicted by the model was generally similar to that observed experimentally, but an asymmetry in the experiment was not captured by the model.

Human RELA haploinsufficiency results in autosomal-dominant chronic mucocutaneous ulceration.

The treatment of chronic mucocutaneous ulceration is challenging, and only some patients respond selectively to inhibitors of tumor necrosis factor-α (TNF). TNF activates opposing pathways leading to caspase-8-mediated apoptosis as well as nuclear factor κB (NF-κB)-dependent cell survival. We investigated the etiology of autosomal-dominant, mucocutaneous ulceration in a family whose proband was dependent on anti-TNF therapy for sustained remission. A heterozygous mutation in RELA, encoding the NF-κB subunit RelA, segregated with the disease phenotype and resulted in RelA haploinsufficiency. The patients' fibroblasts exhibited increased apoptosis in response to TNF, impaired NF-κB activation, and defective expression of NF-κB-dependent antiapoptotic genes. Rela+/- mice have similarly impaired NF-κB activation, develop cutaneous ulceration from TNF exposure, and exhibit severe dextran sodium sulfate-induced colitis, ameliorated by TNF inhibition. These findings demonstrate an essential contribution of biallelic RELA expression in protecting stromal cells from TNF-mediated cell death, thus delineating the mechanisms driving the effectiveness of TNF inhibition in this disease.