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In recent years, assurance of clonality of the production cell line has been emphasized by health authorities during review of regulatory submissions. When insufficient assurance of clonality is provided, augmented control strategies may be required for a commercial production process. In this study, we conducted a retrospective assessment of clonality of a legacy cell line through analysis of subclones from the master cell bank (MCB). Twenty-four subclones were randomly selected based on a predetermined acceptance sampling plan. All these subclones share a conserved integration junction, thus providing a high level of assurance that the cell population in the MCB was derived from a single progenitor cell. However, Southern blot analysis indicates that at least four subpopulations possibly exist in the MCB. Additional characterization of these four subpopulations demonstrated that the resulting changes in product quality attributes of some subclones are not related to the genetic heterogeneity observed in Southern blot hybridization. Furthermore, process consistency, process comparability, and analytical comparability have been demonstrated in batches produced across varying manufacturing processes, scales, facilities, cell banks, and cell ages. Finally, process and product consistency together with a high level of assurance of clonal origin of the MCB helped clear the hurdle for regulatory approval without requirement of additional control strategies.

PFRED a software application for the design, analysis, and visualization of antisense oligonucleotides and siRNA is described. The software provides an intuitive user-interface for scientists to design a library of siRNA or antisense oligonucleotides that target a specific gene of interest. Moreover, the tool facilitates the incorporation of various design criteria that have been shown to be important for stability and potency. PFRED has been made available as an open-source project so the code can be easily modified to address the future needs of the oligonucleotide research community. A compiled version is available for downloading at https://github.com/pfred/pfred-gui/releases/tag/v1.0 as a java Jar file. The source code and the links for downloading the precompiled version can be found at https://github.com/pfred.

Two intriguing forms of genome structural variation (SV) – dispersed duplications, and de novo rearrangements of complex, multi-allelic loci – have long escaped genomic analysis. We describe a new way to find and characterize such variation by utilizing identity-by-descent (IBD) relationships between siblings together with high-precision measurements of segmental copy number. Analyzing whole-genome sequence data from 706 families, we find hundreds of “IBD-discordant” (IBDD) CNVs: loci at which siblings’ CNV measurements and IBD states are mathematically inconsistent. We found that commonly-IBDD CNVs identify dispersed duplications; we mapped 95 of these common dispersed duplications to their true genomic locations through family-based linkage and population linkage disequilibrium (LD), and found several to be in strong LD with genome-wide association (GWAS) signals for common diseases or gene expression variation at their revealed genomic locations. Other CNVs that were IBDD in a single family appear to involve de novo mutations in complex and multi-allelic loci; we identified 26 de novo structural mutations that had not been previously detected in earlier analyses of the same families by diverse SV analysis methods. These included a de novo mutation of the amylase gene locus and multiple de novo mutations at chromosome 15q14. Combining these complex mutations with more-conventional CNVs, we estimate that segmental mutations larger than 1kb arise in about one per 22 human meioses. These methods are complementary to previous techniques in that they interrogate genomic regions that are home to segmental duplication, high CNV allele frequencies, and multi-allelic CNVs.Author SummaryCopy number variation is an important form of genetic variation in which individuals differ in the number of copies of segments of their genomes. Certain aspects of copy number variation have traditionally been difficult to study using short-read sequencing data. For example, standard analyses often cannot tell whether the duplicated copies of a segment are located near the original copy or are dispersed to other regions of the genome. Another aspect of copy number variation that has been difficult to study is the detection of mutations in the copy number of DNA segments passed down from parents to their children, particularly when the mutations affect genome segments which already display common copy number variation in the population. We develop an analytical approach to solving these problems when sequencing data is available for all members of families with at least two children. This method is based on determining the number of parental haplotypes the two siblings share at each location in their genome, and using that information to determine the possible inheritance patterns that might explain the copy numbers we observe in each family member. We show that dispersed duplications and mutations can be identified by looking for copy number variants that do not follow these expected inheritance patterns. We use this approach to determine the location of 95 common duplications which are dispersed to distant regions of the genome, and demonstrate that these duplications are linked to genetic variants that affect disease risk or gene expression levels. We also identify a set of copy number mutations not detected by previous analyses of sequencing data from a large cohort of families, and show that repetitive and complex regions of the genome undergo frequent mutations in copy number.

Genomic profiling of circulating tumor DNA derived from cell-free DNA (cfDNA) in blood can provide a noninvasive method for detecting genomic biomarkers to guide clinical decision making for cancer patients. We developed a hybrid capture–based next-generation sequencing assay for genomic profiling of circulating tumor DNA from blood (FoundationACT). High-sequencing coverage and molecular barcode–based error detection enabled accurate detection of genomic alterations, including short variants (base substitutions, short insertions/deletions) and genomic re-arrangements at low allele frequencies (AFs), and copy number amplifications. Analytical validation was performed on 2666 reference alterations. The assay achieved >99% overall sensitivity (95% CI, 99.1%–99.4%) for short variants at AF >0.5%, >95% sensitivity (95% CI, 94.2%–95.7%) for AF 0.25% to 0.5%, and 70% sensitivity (95% CI, 68.2%–71.5%) for AF 0.125% to 0.25%. No false positives were detected in 62 samples from healthy volunteers. Genomic alterations detected by FoundationACT demonstrated high concordance with orthogonal assays run on the same clinical cfDNA samples. In 860 routine clinical FoundationACT cases, genomic alterations were detected in cfDNA at comparable frequencies to tissue; for the subset of cases with temporally matched tissue and blood samples, 75% of genomic alterations and 83% of short variant mutations detected in tissue were also detected in cfDNA. On the basis of analytical validation results, FoundationACT has been approved for use in our Clinical Laboratory Improvement Amendments–certified/College of American Pathologists–accredited/New York State–approved laboratory.

As availability of precision therapies expands, a well-validated circulating cell-free DNA (cfDNA)-based comprehensive genomic profiling assay has the potential to provide considerable value as a complement to tissue-based testing to ensure potentially life-extending therapies are administered to patients most likely to benefit. Additional data supporting the clinical validity of cfDNA-based testing is necessary to inform optimal use of these assays in the clinic. The FoundationOne®Liquid CDx assay is a pan-cancer cfDNA-based comprehensive genomic profiling assay that was recently approved by FDA. Validation studies included >7,500 tests and >30,000 unique variants across >300 genes and >30 cancer types. Clinical validity results across multiple tumor types are presented. Additionally, results demonstrated a 95% limit of detection of 0.40% variant allele fraction for select substitutions and insertions/deletions, 0.37% variant allele fraction for select rearrangements, 21.7% tumor fraction for copy number amplifications, and 30.4% TF for copy number losses. The limit of detection for microsatellite instability and blood tumor mutational burden were also determined. The false positive variant rate was 0.013% (approximately 1 in 8,000). Reproducibility of variant calling was 99.59%. In comparison with an orthogonal method, an overall positive percent agreement of 96.3% and negative percent agreement of >99.9% was observed. These study results demonstrate that FoundationOne Liquid CDx accurately and reproducibly detects the major types of genomic alterations in addition to complex biomarkers such as microsatellite instability, blood tumor mutational burden, and tumor fraction. Critically, clinical validity data is presented across multiple cancer types.

Background: Liquid biopsy offers the ability to non-invasively analyze the genome of a tumor through circulating tumor DNA (ctDNA) to identify targetable and prognostic genomic alterations. Few studies have rigorously analyzed ctDNA results and determined the fidelity with which they recapitulate the genomics of a sequenced tissue sample obtained from the same tumor. The clinical utility study (CUS) for the FoundationACT™ ctDNA assay (Foundation Medicine, Cambridge, MA, USA; NCT02620527) is a multi-center prospective clinical study for multiple solid tumor types to compare genomic profiling of paired tissue and blood samples from the same patient. In this subset of the study, paired specimens from 96 patients with colorectal cancer (CRC) were analyzed with comprehensive genomic profiling (CGP) of the tumor tissue sample (FoundationOne ® ) and blood sample (FoundationACT™). Methods: Both samples underwent CGP using the hybrid capture-based Illumina Hi-Seq technology. Maximum somatic allele frequency (MSAF) was used to estimate the fraction of ctDNA in the sample. The set of genes and targeted regions common to both tumor and liquid were compared for each subject. Results: Among these patients, 61% were male; 74% had clinical stage IV disease, 19% had clinical stage III disease, and 7% had clinical stage II disease. Time between the tissue biopsy and liquid biopsy (range, 0 – 709 days) had a significant impact on the positive percent agreement (PPA) between the two assays. Eighty percent of cases had evidence of ctDNA in the blood (MSAF >0). For all cases with MSAF >0, 171 base substitutions and insertions/deletions (indels) were identified in the tumor, and 79% (PPA) of these identical alterations were also identified in matched ctDNA samples; PPA increased to 87% for cases

Background: F1LCDx is an FDA-approved liquid biopsy test offering pan-tumor profiling of >300 genes with CDx indications for multiple cancer types. One key limitation is that sensitivity for detection of genomic alterations in ctDNA is impaired when the plasma tumor fraction (TF) is low. TF is known to vary in advanced cancer and has been correlated with overall survival (Stover, JCO, 2018). While some ctDNA assays use variant allele fraction (VAF) to quantify tumor content, such methods may be prone to interference from germline variants, copy number alterations and a limited number of mutations. We studied SNP aneuploidy to develop a variant-independent measure of TF which we hypothesized could inform assay sensitivity. Methods: For each liquid sample, coverages from heterozygous SNPs, across the genome, were used to measure allelic deviations from normal via a root-mean-square (RMS) metric. The relationship between RMS and TF was established based on a training data set of clinical samples with known TF that were diluted in silico. In this way, the RMS metric for any sample could be linked to a distribution of TF values. TF is reported when the lower bounds of the predicted TF distribution is >0%. Positive percentage agreement (PPA) of liquid NGS versus tissue genotype was studied in those with a TF reported vs TF not quantified. Results: For a pilot concordance analysis, we studied a real-world cohort of NSCLC patients where NGS was performed on both tissue and on a 70-gene ctDNA assay (FoundationOne®Liquid (F1L), median 314 days between results). Among 251 cases with tissue NGS showing an EGFR L858R or 19del mutation and a F1L result available, the EGFR mutation was detected 90% of the time (57/63) when TF was reported (range 13-74%) but was detected 51% of the time (96/188) when TF was not quantified. Next, we analyzed a cohort of 3 clinical trials in advanced cancers where tissue genotype was known and with F1LCDx results available, including studies of activating PIK3CA mutations in breast cancer, MET exon 14 mutations in NSCLC and BRCA1/2 mutations in prostate cancer. For the subset of patients with TF reported (range 9.6-72%), PPA was 94.7% (breast, n=38), 100% (NSCLC, n=19), and 90.1% (prostate, n=46), versus 66.7% (breast, n=33), 66.7% (NSCLC, n=45) and 89.6% (prostate, n=48) for TF not quantified. Conclusions: We describe a novel method to measure TF in plasma ctDNA based on aneuploidy, which may overcome some limitations of TF measurement based on VAF. When TF confirms tumor content, sensitivity of F1LCDx for short variant detection is excellent (90%-100%), suggesting reduced value from a reflex to tissue genotyping in the absence of a targetable genomic alteration. When TF is not quantified, sensitivity can be lower and the value of reflex to tissue genotyping may be heightened. Citation Format: Meijuan Li, Bernard Fendler, Lei Yang, Russell Madison, Brennan Decker, Ole Gjoerup, Kimberly McGregor, Aparna Aiyer, Jason Hughes, Priti Hegde, Christine Vietz, Geoffrey R. Oxnard. Utility of plasma tumor fraction (TF) to inform sensitivity of FoundationOne Liquid CDx (F1LCDx) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2231.

Lyons et al. show that STAT3 negatively regulates TGF-β signaling via ERBIN and that cell-intrinsic deregulation of TGF-β pathway activation promotes the IL-4/IL-4Rα/GATA3 axis to support atopic phenotypes in humans., Nonimmunological connective tissue phenotypes in humans are common among some congenital and acquired allergic diseases. Several of these congenital disorders have been associated with either increased TGF-β activity or impaired STAT3 activation, suggesting that these pathways might intersect and that their disruption may contribute to atopy. In this study, we show that STAT3 negatively regulates TGF-β signaling via ERBB2-interacting protein (ERBIN), a SMAD anchor for receptor activation and SMAD2/3 binding protein. Individuals with dominant-negative STAT3 mutations (STAT3mut) or a loss-of-function mutation in ERBB2IP (ERBB2IPmut) have evidence of deregulated TGF-β signaling with increased regulatory T cells and total FOXP3 expression. These naturally occurring mutations, recapitulated in vitro, impair STAT3–ERBIN–SMAD2/3 complex formation and fail to constrain nuclear pSMAD2/3 in response to TGF-β. In turn, cell-intrinsic deregulation of TGF-β signaling is associated with increased functional IL-4Rα expression on naive lymphocytes and can induce expression and activation of the IL-4/IL-4Rα/GATA3 axis in vitro. These findings link increased TGF-β pathway activation in ERBB2IPmut and STAT3mut patient lymphocytes with increased T helper type 2 cytokine expression and elevated IgE.

Human MDA5 deficiency features recurrent severe human rhinovirus (HRV) infections. MDA5 serves a protective and nonredundant role in protecting against HRV in the respiratory tract, through its effects on virus sensing and triggering of antiviral IFN signaling., MDA5 is a cytosolic sensor of double-stranded RNA (ds)RNA including viral byproducts and intermediates. We studied a child with life-threatening, recurrent respiratory tract infections, caused by viruses including human rhinovirus (HRV), influenza virus, and respiratory syncytial virus (RSV). We identified in her a homozygous missense mutation in IFIH1 that encodes MDA5. Mutant MDA5 was expressed but did not recognize the synthetic MDA5 agonist/(ds)RNA mimic polyinosinic-polycytidylic acid. When overexpressed, mutant MDA5 failed to drive luciferase activity from the IFNB1 promoter or promoters containing ISRE or NF-κB sequence motifs. In respiratory epithelial cells or fibroblasts, wild-type but not knockdown of MDA5 restricted HRV infection while increasing IFN-stimulated gene expression and IFN-β/λ. However, wild-type MDA5 did not restrict influenza virus or RSV replication. Moreover, nasal epithelial cells from the patient, or fibroblasts gene-edited to express mutant MDA5, showed increased replication of HRV but not influenza or RSV. Thus, human MDA5 deficiency is a novel inborn error of innate and/or intrinsic immunity that causes impaired (ds)RNA sensing, reduced IFN induction, and susceptibility to the common cold virus.

Trafficking from bedside to bench Typically in translational research, a discovery in cell or molecular biology is later exploited to improve patient care. Occasionally, information flows in the opposite direction. Lo et al. found that patients with an autoimmune disorder caused by deficiency of a protein called LRBA responded dramatically to the drug abatacept (see the Perspective by Sansom). Abatacept contains a segment of a potent inhibitory immune receptor, CTLA4. Experiments prompted by this observation revealed the relationship between the two proteins: LRBA controls the intracellular trafficking and degradation of CTLA4. This information may further improve patient care, because other clinically approved drugs have the desired mechanism of action with potentially fewer side effects. Science , this issue p. 436 ; see also p. 377

Knowledge of biological relatedness between samples is important for many genetic studies. In large-scale human genetic association studies, the estimated kinship is used to remove cryptic relatedness, control for family structure, and estimate trait heritability. However, estimation of kinship is challenging for sparse sequencing data, such as those from off-target regions in target sequencing studies, where genotypes are largely uncertain or missing. Existing methods often assume accurate genotypes at a large number of markers across the genome. We show that these methods, without accounting for the genotype uncertainty in sparse sequencing data, can yield a strong downward bias in kinship estimation. We develop a computationally efficient method called SEEKIN to estimate kinship for both homogeneous samples and heterogeneous samples with population structure and admixture. Our method models genotype uncertainty and leverages linkage disequilibrium through imputation. We test SEEKIN on a whole exome sequencing dataset (WES) of Singapore Chinese and Malays, which involves substantial population structure and admixture. We show that SEEKIN can accurately estimate kinship coefficient and classify genetic relatedness using off-target sequencing data down sampled to ~0.15X depth. In application to the full WES dataset without down sampling, SEEKIN also outperforms existing methods by properly analyzing shallow off-target data (~0.75X). Using both simulated and real phenotypes, we further illustrate how our method improves estimation of trait heritability for WES studies., Author summary Inference of genetic relatedness from molecular markers has broad applications in many areas, including quantitative genetics, forensics, evolution and ecology. Classic estimators, however, are not suitable for low-coverage sequencing data, which have high levels of genotype uncertainty and missing data. We evaluate existing methods and describe a new method for kinship estimation using sparse sequencing data. Our method leverages correlations between neighboring markers and models genotype uncertainty in kinship estimators for both homogeneous populations and admixed populations. We show that our method can accurately estimate kinship coefficient even when the sequencing depth is as low as ~0.15X, while existing methods have strong downward bias. Our method can be applied to estimate kinship using sparse off-target data and thus enables control of family structure and estimation of heritability in target sequencing studies, in which the deeply sequenced target regions are often too small to infer genetic relatedness. Even for whole exome sequencing, we show that our method can improve kinship and heritability estimation by including off-target data, compared to conventional analyses solely based on the target regions.

The transcriptional programs that guide lymphocyte differentiation depend on the precise expression and timing of transcription factors (TFs). The TF BACH2 is essential for T and B lymphocytes and is associated with an archetypal super-enhancer (SE). Single-nucleotide variants in the BACH2 locus are associated with several autoimmune diseases, but BACH2 mutations that cause Mendelian monogenic primary immunodeficiency have not previously been identified. Here we describe a syndrome of BACH2-related immunodeficiency and autoimmunity (BRIDA) that results from BACH2 haploinsufficiency. Affected subjects had lymphocyte-maturation defects that caused immunoglobulin deficiency and intestinal inflammation. The mutations disrupted protein stability by interfering with homodimerization or by causing aggregation. We observed analogous lymphocyte defects in Bach2-heterozygous mice. More generally, we observed that genes that cause monogenic haploinsufficient diseases were substantially enriched for TFs and SE architecture. These findings reveal a previously unrecognized feature of SE architecture in Mendelian diseases of immunity: heterozygous mutations in SE-regulated genes identified by whole-exome/genome sequencing may have greater significance than previously recognized.

Lucas et al. identify humans with a gain-of-function mutation in PIK3R1, encoding the p85α subunit of PI3K. The splice site mutation causes in-frame skipping of exon 11, resulting in altered p85α association with p110δ that stabilizes the catalytic subunit but fails to properly inhibit catalytic activity. The patients have immunodeficiency and lymphoproliferation with skewing of CD8+ T cells toward terminally differentiated and senescent effector cells that have shortened telomeres., Class IA phosphatidylinositol 3-kinases (PI3K), which generate PIP3 as a signal for cell growth and proliferation, exist as an intracellular complex of a catalytic subunit bound to a regulatory subunit. We and others have previously reported that heterozygous mutations in PIK3CD encoding the p110δ catalytic PI3K subunit cause a unique disorder termed p110δ-activating mutations causing senescent T cells, lymphadenopathy, and immunodeficiency (PASLI) disease. We report four patients from three families with a similar disease who harbor a recently reported heterozygous splice site mutation in PIK3R1, which encodes the p85α, p55α, and p50α regulatory PI3K subunits. These patients suffer from recurrent sinopulmonary infections and lymphoproliferation, exhibit hyperactive PI3K signaling, and have prominent expansion and skewing of peripheral blood CD8+ T cells toward terminally differentiated senescent effector cells with short telomeres. The PIK3R1 splice site mutation causes skipping of an exon, corresponding to loss of amino acid residues 434–475 in the inter-SH2 domain. The mutant p85α protein is expressed at low levels in patient cells and activates PI3K signaling when overexpressed in T cells from healthy subjects due to qualitative and quantitative binding changes in the p85α–p110δ complex and failure of the C-terminal region to properly inhibit p110δ catalytic activity.

Beware of T cells that don't know how to stop During an infection, T cells divide extensively and secrete proteins that can severely damage tissues. But T cells know when to stop—they express proteins on their surface such as CTLA4, which put on the brakes. Kuehn et al. now report genetic evidence of the importance of CTLA4 in humans (see the Perspective by Rieux-Laucat and Casanova). They identified six patients with mutations in one copy of CTLA4 . Patients presented with symptoms of an overzealous immune response, with immune cells infiltrating their organs. The findings support the idea that CTLA4 tells the immune system when enough is enough. Science , this issue p. 1623 ; see also p. 1560

BACKGROUND The study of autoinflammatory diseases has uncovered mechanisms underlying cytokine dysregulation and inflammation. METHODS We analyzed the DNA of an index patient with early-onset systemic inflammation, cutaneous vasculopathy, and pulmonary inflammation. We sequenced a candidate gene, TMEM173, encoding the stimulator of interferon genes (STING), in this patient and in five unrelated children with similar clinical phenotypes. Four children were evaluated clinically and immunologically. With the STING ligand cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), we stimulated peripheral-blood mononuclear cells and fibroblasts from patients and controls, as well as commercially obtained endothelial cells, and then assayed transcription of IFNB1, the gene encoding interferon-β, in the stimulated cells. We analyzed IFNB1 reporter levels in HEK293T cells cotransfected with mutant or nonmutant STING constructs. Mutant STING leads to increased phosphorylation of signal transducer and activator of transcription 1 (STAT1), so we tested the effect of Janus kinase (JAK) inhibitors on STAT1 phosphorylation in lymphocytes from the affected children and controls. RESULTS We identified three mutations in exon 5 of TMEM173 in the six patients. Elevated transcription of IFNB1 and other gene targets of STING in peripheral-blood mononuclear cells from the patients indicated constitutive activation of the pathway that cannot be further up-regulated with stimulation. On stimulation with cGAMP, fibroblasts from the patients showed increased transcription of IFNB1 but not of the genes encoding interleukin-1 (IL1), interleukin-6 (IL6), or tumor necrosis factor (TNF). HEK293T cells transfected with mutant constructs show elevated IFNB1 reporter levels. STING is expressed in endothelial cells, and exposure of these cells to cGAMP resulted in endothelial activation and apoptosis. Constitutive up-regulation of phosphorylated STAT1 in patients’ lymphocytes was reduced by JAK inhibitors. CONCLUSIONS STING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory disease caused by gain-of-function mutations in TMEM173. (Funded by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases; ClinicalTrials.gov number, NCT00059748.)

504 Background: Microsatellite instability (MSI) testing has become critically important in clinical cancer care of patients with cancer given the recent pan-tumor FDA approval of pembrolizumab for use in patients with MSI-High (MSI-H) tumors. We previously demonstrated the robustness of a novel proprietary algorithm for determination of MSI status via NGS from solid tumor biopsy specimens (J Clin Oncol 34, 2016 (suppl; abstr 1523)). Traditional MSI tests such as PCR or IHC are impractical for pan-tumor adoption, as MSI-H prevalence outside of gastrointestinal and endometrial cancers is usually < 1%. NGS-based ctDNA profiling provides an opportunity for both MSI and actionable alteration testing in patients in whom tissue-based biopsy is not available. Methods: Genomic DNA (gDNA) from five previously characterized MSI-H cell lines: (DLD1, 22Rv1, LNCap, RL952, CL188), and one MSS cell line (SCC9) was enzymatically-fragmented to simulate ctDNA and titrated to various dilution levels with DNA from a healthy hapmap subject (NA12878). Samples were screened with a 70-gene panel, FoundationOne Liquid, that includes 180 mononucleotide repeat sequences (8-26bp long in the human reference genome). Length variability in the 180 repeat loci was utilized to generate an overall MSI score via principal components analysis. The NGS based MSI algorithm was applied to all the samples. Results: Assessment of these six cell lines, targeting five dilution levels confirmed by SNP mixing ratios, show that our NGS based MSI test for liquid biopsies has 96% sensitivity at > 2% tumor fraction with 100% PPV. The regression intercept of the MSI-H dilution samples with the pre-established MSI-H calling threshold shows our method has a LOD of 1.03% tumor fraction. MSI-H prevalence data from liquid biopsies of gastrointestinal tumors obtained during clinical care will also be presented. Conclusions: These data demonstrate the feasibility of using NGS-based liquid biopsy assays for MSI testing. This ctDNA-based approach will allow for increased access to checkpoint inhibitors in a pan-tumor setting, which would be especially relevant for cancers where routine MSI testing is impractical or when tissue is not available.

Elevated basal serum tryptase levels are present in 4–6% of the general population, but the cause and relevance of such increases are unknown1, 2. Previously, we described subjects with dominantly inherited elevated basal serum tryptase levels associated with multisystem complaints including cutaneous flushing and pruritus, dysautonomia, functional gastrointestinal symptoms, chronic pain, and connective tissue abnormalities, including joint hypermobility. Here we report the identification of germline duplications and triplications in the TPSAB1 gene encoding α-tryptase that segregate with inherited increases in basal serum tryptase levels in 35 families presenting with associated multisystem complaints. Individuals harboring alleles encoding three copies of α-tryptase had higher basal serum levels of tryptase and were more symptomatic than those with alleles encoding two copies, suggesting a gene-dose effect. Further, we found in two additional cohorts (172 individuals) that elevated basal serum tryptase levels were exclusively associated with duplication of α-tryptase–encoding sequence in TPSAB1, and affected individuals reported symptom complexes seen in our initial familial cohort. Thus, our findings link duplications in TPSAB1 with irritable bowel syndrome, cutaneous complaints, connective tissue abnormalities, and dysautonomia.

Relationships between physicochemical drug properties and toxicity were inferred from a data set consisting of animal in vivo toleration (IVT) studies on 245 preclinical Pfizer compounds; an increased likelihood of toxic events was found for less polar, more lipophilic compounds. This trend held across a wide range of types of toxicity and across a broad swath of chemical space.

Chinese hamster ovary (CHO) cells have been used widely in the pharmaceutical industry for production of biological therapeutics including monoclonal antibodies (mAb). The integrity of the gene of interest and the accuracy of the relay of genetic information impact product quality and patient safety. Here we employed next-generation sequencing, particularly RNA-seq, and developed a method to systematically analyze the mutation rate of the mRNA of CHO cell lines producing a mAb. The effect of an extended culturing period to mimic the scale of cell expansion in a manufacturing process and varying selection pressure in the cell culture were also closely examined.

Mutations in the LRBA gene (encoding the lipopolysaccharide-responsive and beige-like anchor protein) cause a syndrome of autoimmunity, lymphoproliferation, and humoral immune deficiency. The biological role of LRBA in immunologic disease is unknown. We found that patients with LRBA deficiency manifested a dramatic and sustained improvement in response to abatacept, a CTLA4 (cytotoxic T lymphocyte antigen-4)-immunoglobulin fusion drug. Clinical responses and homology of LRBA to proteins controlling intracellular trafficking led us to hypothesize that it regulates CTLA4, a potent inhibitory immune receptor. We found that LRBA colocalized with CTLA4 in endosomal vesicles and that LRBA deficiency or knockdown increased CTLA4 turnover, which resulted in reduced levels of CTLA4 protein in FoxP3(+) regulatory and activated conventional T cells. In LRBA-deficient cells, inhibition of lysosome degradation with chloroquine prevented CTLA4 loss. These findings elucidate a mechanism for CTLA4 trafficking and control of immune responses and suggest therapies for diseases involving the CTLA4 pathway.

Protein interfaces are thought to be distinguishable from the rest of the protein surface by their greater degree of residue conservation. We test the validity of this approach on an expanded set of 64 protein-protein interfaces using conservation scores derived from two multiple sequence alignment types, one of close homologs/orthologs and one of diverse homologs/paralogs. Overall, we find that the interface is slightly more conserved than the rest of the protein surface when using either alignment type, with alignments of diverse homologs showing marginally better discrimination. However, using a novel surface-patch definition, we find that the interface is rarely significantly more conserved than other surface patches when using either alignment type. When an interface is among the most conserved surface patches, it tends to be part of an enzyme active site. The most conserved surface patch overlaps with 39% (+/- 28%) and 36% (+/- 28%) of the actual interface for diverse and close homologs, respectively. Contrary to results obtained from smaller data sets, this work indicates that residue conservation is rarely sufficient for complete and accurate prediction of protein interfaces. Finally, we find that obligate interfaces differ from transient interfaces in that the former have significantly fewer alignment gaps at the interface than the rest of the protein surface, as well as having buried interface residues that are more conserved than partially buried interface residues.

Nonimmunological connective tissue phenotypes in humans are common among some congenital and acquired allergic diseases. Several of these congenital disorders have been associated with either increased TGF-β activity or impaired STAT3 activation, suggesting that these pathways might intersect and that their disruption may contribute to atopy. In this study, we show that STAT3 negatively regulates TGF-β signaling via ERBB2-interacting protein (ERBIN), a SMAD anchor for receptor activation and SMAD2/3 binding protein. Individuals with dominant-negative

Germline loss-of-function mutations in the transcription factor signal transducer and activator of transcription 3 ( STAT3 ) cause immunodeficiency, whereas somatic gain-of-function mutations in STAT3 are associated with large granular lymphocytic leukemic, myelodysplastic syndrome, and aplastic anemia. Recently, germline mutations in STAT3 have also been associated with autoimmune disease. Here, we report on 13 individuals from 10 families with lymphoproliferation and early-onset solid-organ autoimmunity associated with 9 different germline heterozygous mutations in STAT3 . Patients exhibited a variety of clinical features, with most having lymphadenopathy, autoimmune cytopenias, multiorgan autoimmunity (lung, gastrointestinal, hepatic, and/or endocrine dysfunction), infections, and short stature. Functional analyses demonstrate that these mutations confer a gain-of-function in STAT3 leading to secondary defects in STAT5 and STAT1 phosphorylation and the regulatory T-cell compartment. Treatment targeting a cytokine pathway that signals through STAT3 led to clinical improvement in 1 patient, suggesting a potential therapeutic option for such patients. These results suggest that there is a broad range of autoimmunity caused by germline STAT3 gain-of-function mutations, and that hematologic autoimmunity is a major component of this newly described disorder. Some patients for this study were enrolled in a trial registered at www.clinicaltrials.gov as #NCT00001350.

Regulatory motifs can be found by local multiple alignment of upstream regions from coregulated sets of genes, or regulons. We searched for regulatory motifs using the program AlignACE together with a set of filters that helped us choose the motifs most likely to be biologically relevant in 17 complete microbial genomes. We searched the upstream regions of potentially coregulated genes grouped by three methods: (1) genes that make up functional pathways; (2) genes homologous to regulons from a well-studied species (Escherichia coli); and (3) groups of genes derived from conserved operons. This last group is based on the observation that genes making up homologous regulons in different species are often assorted into coregulated operons in different combinations. This allows partial reconstruction of regulons by looking at operon structure across several species. Unlike other methods for predicting regulons, this method does not depend on the availability of experimental data other than the genome sequence and the locations of genes. New, statistically significant motifs were found in the genome sequence of each organism using each grouping method. The most significant new motif was found upstream of genes in the methane-metabolism functional group inMethanobacterium thermoautotrophicum. We found that at least 27% of the known E. coli DNA-regulatory motifs are conserved in one or more distantly related eubacteria. We also observed significant motifs that differed from the E. coli motif in other organisms upstream of sets of genes homologous to known E. coli regulons, including Crp, LexA, and ArcA in Bacillus subtilis; four anaerobic regulons in Archaeoglobus fulgidus (NarL, NarP, Fnr, and ModE); and the PhoB, PurR, RpoH, and FhlA regulons in other archaebacterial species. We also used motif conservation to aid in finding new motifs by grouping upstream regions from closely related bacteria, thus increasing the number of instances of the motif in the sequence to be aligned. For example, by grouping upstream sequences from three archaebacterial species, we found a conserved motif that may regulate ferrous ion transport that was not found in individual genomes. Discovery of conserved motifs becomes easier as the number of closely related genome sequences increases.

AlignACE is a Gibbs sampling algorithm for identifying motifs that are over-represented in a set of DNA sequences. When used to search upstream of apparently coregulated genes, AlignACE finds motifs that often correspond to the DNA binding preferences of transcription factors. We previously used AlignACE to analyze whole genome mRNA expression data. Here, we present a more detailed study of its effectiveness as applied to a variety of groups of genes in the Saccharomyces cerevisiae genome. Published functional catalogs of genes and sets of genes grouped by common name provided 248 groups, resulting in 3311 motifs. In conjunction with this analysis, we present measures for gauging the tendency of a motif to target a given set of genes relative to all other genes in the genome and for gauging the degree to which a motif is preferentially located in a certain distance range upstream of translational start sites. We demonstrate improved methods for comparing and clustering sequence motifs. Many previously identified cis-regulatory elements were found. We also describe previously unidentified motifs, one of which has been verified by experiments in our laboratory. An extensive set of AlignACE runs on randomly selected sets of genes and on sets of genes whose upstream regions contain known transcription factor binding sites serve as controls.

Technologies to measure whole-genome mRNA abundances1,2,3 and methods to organize and display such data4,5,6,7,8,9,10 are emerging as valuable tools for systems-level exploration of transcriptional regulatory networks. For instance, it has been shown that mRNA data from 118 genes, measured at several time points in the developing hindbrain of mice, can be hierarchically clustered into various patterns (or 'waves') whose members tend to participate in common processes5. We have previously shown that hierarchical clustering can group together genes whose cis-regulatory elements are bound by the same proteins in vivo6. Hierarchical clustering has also been used to organize genes into hierarchical dendograms on the basis of their expression across multiple growth conditions7. The application of Fourier analysis to synchronized yeast mRNA expression data has identified cell-cycle periodic genes, many of which have expected cis-regulatory elements8. Here we apply a systematic set of statistical algorithms, based on whole-genome mRNA data, partitional clustering and motif discovery, to identify transcriptional regulatory sub-networks in yeast—without any a priori knowledge of their structure or any assumptions about their dynamics. This approach uncovered new regulons (sets of co-regulated genes) and their putative cis-regulatory elements. We used statistical characterization of known regulons and motifs to derive criteria by which we infer the biological significance of newly discovered regulons and motifs. Our approach holds promise for the rapid elucidation of genetic network architecture in sequenced organisms in which little biology is known.

Background Identifying genetic syndromes that lead to significant atopic disease can open new pathways for investigation and intervention in allergy. Objective We sought to define a genetic syndrome of severe atopy, increased serum IgE levels, immune deficiency, autoimmunity, and motor and neurocognitive impairment. Methods Eight patients from 2 families with similar syndromic features were studied. Thorough clinical evaluations, including brain magnetic resonance imaging and sensory evoked potentials, were performed. Peripheral lymphocyte flow cytometry, antibody responses, and T-cell cytokine production were measured. Whole-exome sequencing was performed to identify disease-causing mutations. Immunoblotting, quantitative RT-PCR, enzymatic assays, nucleotide sugar, and sugar phosphate analyses, along with matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry of glycans, were used to determine the molecular consequences of the mutations. Results Marked atopy and autoimmunity were associated with increased T H 2 and T H 17 cytokine production by CD4 + T cells. Bacterial and viral infection susceptibility were noted along with T-cell lymphopenia, particularly of CD8 + T cells, and reduced memory B-cell numbers. Apparent brain hypomyelination resulted in markedly delayed evoked potentials and likely contributed to neurologic abnormalities. Disease segregated with novel autosomal recessive mutations in a single gene, phosphoglucomutase 3 ( PGM3 ). Although PGM3 protein expression was variably diminished, impaired function was demonstrated by decreased enzyme activity and reduced uridine diphosphate–N-acetyl-D-glucosamine, along with decreased O- and N-linked protein glycosylation in patients' cells. These results define a new congenital disorder of glycosylation. Conclusions Autosomal recessive hypomorphic PGM3 mutations underlie a disorder of severe atopy, immune deficiency, autoimmunity, intellectual disability, and hypomyelination.

Chromosome correlation maps display correlations between the expression patterns of genes on the same chromosome. Using these maps, we show here that adjacent pairs of genes, as well as nearby non-adjacent pairs of genes, show correlated expression independent of their orientation. We present specific examples of adjacent pairs with highly correlated expression patterns, in which the promoter of only one of the two genes contains an upstream activating sequence (UAS) known to be associated with that expression pattern. Finally, we show that genes with similar functions tend to occur in adjacent positions along the chromosomes. Our results suggest that, in certain chromosomal expression domains, an UAS can affect the transcription of genes that are not immediately downstream from it.

Whole-genome mRNA quantitation can be used to identify the genes that are most responsive to environmental or genotypic change. By searching for mutually similar DNA elements among the upstream non-coding DNA sequences of these genes, we can identify candidate regulatory motifs and corresponding candidate sets of coregulated genes. We have tested this strategy by applying it to three extensively studied regulatory systems in the yeast Saccharomyces cerevisiae: galactose response, heat shock, and mating type. Galactose-response data yielded the known binding site of Gal4, and six of nine genes known to be induced by galactose. Heat shock data yielded the cell-cycle activation motif, which is known to mediate cell-cycle dependent activation, and a set of genes coding for all four nucleosomal proteins. Mating type alpha and a data yielded all of the four relevant DNA motifs and most of the known a- and alpha-specific genes.