ORE identifies extreme expression effects enriched for rare variants.

MOTIVATION: Non-coding rare variants (RVs) may contribute to Mendelian disorders but have been challenging to study due to small sample sizes, genetic heterogeneity and uncertainty about relevant non-coding features. Previous studies identified RVs associated with expression outliers, but varying outlier definitions were employed and no comprehensive open-source software was developed. RESULTS: We developed Outlier-RV Enrichment (ORE) to identify biologically-meaningful non-coding RVs. We implemented ORE combining whole-genome sequencing and cardiac RNAseq from congenital heart defect patients from the Pediatric Cardiac Genomics Consortium and deceased adults from Genotype-Tissue Expression. Use of rank-based outliers maximized sensitivity while a most extreme outlier approach maximized specificity. Rarer variants had stronger associations, suggesting they are under negative selective pressure and providing a basis for investigating their contribution to Mendelian disorders. AVAILABILITY AND IMPLEMENTATION: ORE, source code, and documentation are available at https://pypi.python.org/pypi/ore under the MIT license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MRI differential diagnosis of suspected multiple sclerosis.

Diagnosing multiple sclerosis (MS) can be very challenging owing to its variable clinical features and lack of a definitive test. Magnetic resonance imaging (MRI) is a core diagnostic tool in the detection of MS lesions and demonstration of spatial and temporal distribution of disease. Moreover, MRI plays a crucial role in the exclusion of alternative diagnoses of MS. The aim of this review is to describe the typical MRI features of MS and to present a series of common mimics of MS with emphasis on their distinguishing features from MS.

Posterior circulation infarction in patients with traumatic cervical spinal cord injury and its relationship to vertebral artery injury.

STUDY DESIGN: Prospective study. OBJECTIVE: To ascertain the prevalence of posterior circulation stroke in traumatic chronic spinal cord injured (SCI) patients and associated traumatic vertebral artery injuries (VAI). METHODS: All adult patients with cervical SCI and American Spinal Injury Association Impairment Scale (AIS) grade A or B referred for follow-up magnetic resonance imaging of their spinal cord were invited to take part in the study between January 2010 and December 2012 at the National Spinal Injury Centre. Two additional sequences were added to the existing imaging protocol to evaluate the brain and vertebral arteries. RESULTS: Ninety-eight patients were recruited. All imaging were analysed independently by three consultant radiologists. Posterior circulation infarcts were noted in seven (7%) patients. Significant VAI was noted in 13 patients (13%) with 10 occlusions and 3 with high-grade stenosis. However, only one patient had co-existent posterior circulation infarct and significant VAI. CONCLUSION: There is an increased prevalence of posterior circulation infarction in SCI patients. The relationship with associated traumatic VAI requires further investigation.

The popliteal fibular ligament in acute knee trauma: patterns of injury on MR imaging.

OBJECTIVE: To describe the patterns of injury associated with injury to the popliteofibular ligament injury. MATERIALS AND METHODS: A retrospective review was performed of 180 MRI scans undertaken for acute knee trauma. Scans were excluded if the time of injury was over 4 weeks from the time of the scan, or if there was a history of septic arthritis, inflammatory arthropathy, previous knee surgery, or significant artefact. An agreed criterion for assessing the structures of the posterolateral ligamentous complex was defined and in each scan, the popliteofibular ligament (PFL) was scored as normal or injured. The menisci, ligaments, and tendons of each knee were also assessed. RESULTS: The mean age was 25.7 years (range, 9-65 years) and 72.2% (n = 130) patients were male. The PFL was injured in 36 cases (20%). There is a significant association between PFL injury and ACL rupture (p = 0.0001), ITB injury (p = 0.0001), PCL injury (p = 0.0373), in addition to associations with injury to other posterolateral corner structures including the lateral collateral ligament (p = 0.0001), biceps femoris tendon (p = 0.0014), and popliteus tendon (p = 0.0014). Of our series of PFL injuries, nine cases (25%) were associated with further injuries of posterolateral corner structures and in 27 cases (75%) the PFL was the only posterolateral corner structure torn. CONCLUSIONS: PFL injury is not uncommon in acute knee trauma and is associated with significant internal derangement of the knee, especially anterior cruciate ligament rupture, ITB sprain, and injury to other structures within the posterolateral corner.

Validating a threshold of ocular gaze deviation for the prediction of acute ischaemic stroke.

AIM: To determine a threshold at which the degree of ocular gaze deviation (OGD) on axial imaging is highly specific for the prediction of acute ischaemic stroke. MATERIALS AND METHODS: A retrospective analysis of 517 patients who had received MRI with diffusion-weighted imaging (DWI) for suspected acute stroke was performed. The degree of OGD was measured in all patients and the presence and location of infarction determined. The difference in OGD between groups was compared using the independent t-test for normally distributed data and the Mann-Whitney test for non-normal data. The sensitivity and specificity for degrees of OGD in the prediction of acute infarction was calculated using a receiver operating curve (ROC) analysis. RESULTS: The imaging of 448 patients meeting the inclusion criteria was reviewed. Acute infarct was demonstrated in 34.8% (n=156). There was a significant difference in the degree of OGD between patients with an acute infarct and those without evidence of acute ischaemia (p<0.001). ROC curve analysis for OGD demonstrated area under the curve (AUC) = 0.619 with increasing degrees of OGD more specific for acute infarct. OGD >11.95° had a sensitivity of 17% and specificity of 95.9% in predicting acute infarction. CONCLUSION: Significant OGD>11.95° has a high specificity for acute infarct. This threshold may provide a helpful additional sign in the detection of subtle acute infarct, particularly on axial CT brain imaging.

The analysis of costimulatory receptor signaling cascades in normal T lymphocytes using in vitro gene transfer and reporter gene analysis.

Ligation of the antigen receptor and costimulatory receptors on the surface of T lymphocytes initiates intracellular signals that regulate cell-cycle progression and cell differentiation. To effectively manipulate the activation of T cells for immunotherapeutic applications, it will be important to understand how these signaling pathways are integrated to control specific gene transcription events. Here we describe a novel transient transfection procedure that efficiently introduces DNA into non-dividing normal human and murine T lymphocytes while maintaining high cell viability. Using this technique, reporter genes can be introduced to characterize intracellular signaling pathways that regulate specific gene transcription events in normal T-lymphocyte populations. We show that the CD28 receptor can be differentially coupled to downstream signaling pathways in different T-lymphocyte populations. In addition, we demonstrate that a gene encoding a tagged constitutively active mitogen-activated kinase kinase-1 protein can be transfected and rapidly expressed to regulate the expression of Bcl-2 in normal thymocytes.

Ligand-induced desensitization of interleukin 1 receptor-initiated intracellular signaling events in T helper lymphocytes.

Although interleukin 1 (IL-1) receptor signaling events in T helper lymphocytes are incompletely characterized, events associated with translocation of the transcription factor NF-kappa B are receptor-proximal assays of ligand-initiated responses. In this report we demonstrate that the transient nature of IL-1-induced NF-kappa B nuclear translocation occurs as a consequence of ligand-induced receptor desensitization. Other receptor-initiated events including induction of I kappa B alpha phosphorylation, expression of c-jun and junB mRNA, and costimulatory effects on IL-2 synthesis also are altered by IL-1 receptor desensitization. IL-1 receptor desensitization is not initiated by tumor necrosis factor, which also stimulates NF-kappa B translocation, and is not a consequence of alterations in either IL-1 receptor expression or binding affinity. In the absence of IL-1, the effects of desensitization are completely reversed within 18 h. Since IL-1 desensitization is initiated under conditions of low receptor occupancy, it is likely that receptor desensitization results from alterations to a receptor-proximal transducer, rather than from direct modification of the IL-1 receptor. These results suggest that the cyclic nature of the events in the T helper lymphocyte activation program can be controlled, in part, by the reversible desensitization of cell surface IL-1 receptors.

Evidence of eosinophil granule major basic protein in human placenta.

A protein immunochemically related to the eosinophil granule major basic protein (gMBP) is found in increased concentration in the plasma of pregnant women and has been localized to placental trophoblasts by immunofluorescence. Pregnancy MBP (pMBP) is indistinguishable from gMBP in its reactivity with polyclonal antisera and a panel of 14 mouse mAbs. We report the purification of pMBP from human placenta by: (a) affinity chromatography over mAb immobilized on Sepharose, (b) gel filtration in 6 M guanidine.HCl buffer, and (c) reversed-phase HPLC. Purified pMBP and gMBP are biochemically indistinguishable in that both: (a) bind to DNA, (b) polymerize and bind to carrier proteins via disulfide linkages, (c) have a molecular weight of 14,000, (d) have isoelectric points greater than 10.6, (e) comigrate in two-dimensional gels, (f) coelute during reversed-phase HPLC on C18 columns, (g) have identical peptide maps after three different digestions, and (h) have partial amino acid sequence identity. This physicochemical identity has important implications as to the role of pMBP in human placentation.

Molecular cloning of the human eosinophil peroxidase. Evidence for the existence of a peroxidase multigene family.

Human eosinophil peroxidase (EPO) was purified from eosinophil granules derived from the peripheral blood of patients with eosinophilia. The molecular mass of the H and L subunits was determined by gel filtration to be 57,000 and 11,000 daltons, respectively. The partial amino acid sequences of both subunits were used to construct oligonucleotides for the screening of several cDNA libraries, including one derived from human-induced umbilical cord mononuclear cells. A cDNA clone was isolated corresponding to EPO. The nucleotide sequence revealed an open reading frame of 2,106 bp, corresponding to a prosequence, L chain, and H chain, in this order. Comparison of the EPO nucleotide sequence with other peroxidases, such as myeloperoxidase, suggests the existence of a multigene family.

Direct evidence that a class II molecule and a simple globular protein generate multiple determinants.

We have examined the individual contributions of the I-A kappa alpha chain, the I-A kappa beta chain, and the foreign antigen hen egg-white lysozyme (HEL) in the formation of the determinant being recognized by the T cell receptor. As functional probes we have used (a) a panel of 10 HEL-specific T cell hybridomas, (b) a panel of antigen-presenting cells (APC) possessing mutations in either the I-A kappa alpha or I-A kappa beta chains, and (c) proteolytic fragment of HEL and related synthetic peptides. The ability of the I-A kappa beta and I-A kappa alpha mutant cell lines to present antigen to the 10 T cell hybridomas divided these T cells into six distinct groups. These HEL-specific T cells therefore appear to recognize several distinct domains on the I-A kappa molecule. The 10 T cell hybrids were then shown to recognize at least three distinct determinants on the HEL molecule, with 8 of the 10 hybrids recognizing one of two major determinants HEL(46-61) or HEL(34-45). Combining the response patterns to the panel of I-A kappa mutant APC lines with the antigen specificity revealed that the 10 T cell hybrids recognized at least eight unique determinants formed by the I-A kappa alpha chains, I-A kappa beta chains, and HEL peptides. This analysis provides direct evidence that a large number of different determinants or T cell receptor ligands can be generated from a single Ia molecule and a simple globular protein.

Molecular mechanisms that control expression of the B lymphocyte antigen receptor complex.

The B cell receptor for antigen (BCR) is a complex of membrane immunoglobulin (mIg) and at least two other proteins, Ig alpha (mb-1) and Ig beta (B29). This complex promotes surface expression of the BCR and acts to transduce an activation signal. We have used a system of mu heavy chain constructs transfected into murine B cell lines to probe structure-function relationships in the BCR complex. One mutant mu chain, in which two polar transmembrane residues (Tyr587, Ser588) are replaced with valine, fails to associate with Ig alpha and Ig beta and is incapable of transducing signals as a result of mIg cross-linking. This mutant is expressed on the surface at high levels when transfected into a plasmacytoma line that lacks Ig alpha, whereas wild-type mu is retained in this cell line in the endoplasmic reticulum. Pulse-chase and immunoprecipitation analyses indicate that the mutant is more rapidly released from calnexin than the wild-type mu. Further, transfection of Ig alpha into this Ig alpha-negative cell line allows release of the mu chain from calnexin and surface expression of the BCR. These results identify the transmembrane residues of mu heavy chain that control binding to calnexin and Ig alpha, and suggest that calnexin-dependent intracellular retention is an important control mechanism for expression of the BCR complex.

Structural mutation affecting intracellular transport and cell surface expression of murine class II molecules.

We have selected Ia variants from the Ia+ (H-2d) M12.4.1 B cell lymphoma that are negative on the cell surface for one or both Ia isotypes. The molecular analysis of two such independently selected cell lines, M12.A2 and M12.C3, is reported here. This analysis revealed that the genes encoding Ad beta (M12.A2) and Ed beta (M12.C3) contained identical single-nucleotide transitions that resulted in the substitution of Ser (mutant) for Asn (wild-type) at residue 82/83 of the extracellular NH2-terminal (membrane distal) beta 1 domain. This conservative substitution caused a cytoplasmic accumulation of I-A or I-E molecules in the respective cell line although predicted secondary-structure analysis suggests a minimal effect on protein conformation. Thus, the mutation appears to have either created a negative signal that stops transport or eliminated a positive signal that is required for transport and targeting to the cell surface.

Regulation of murine MHC class II molecule expression. Identification of A beta residues responsible for allele-specific cell surface expression.

A panel of mutant class II genes have been constructed using site-directed mutagenesis and DNA-mediated gene transfer. Using this technique, Ak beta polypeptides have been altered by substituting one or more Ad beta-specific residues at polymorphic positions in the beta 1 domain. Transfection of M12.C3 B lymphoma cells with most mutant Ak beta* genes results in the expression of Ak beta* Ad alpha molecules on the cell surface. However, the substitution of a single d allele residue at position 78 or 86 in the Ak beta polypeptide results in either the complete absence or very low levels, respectively, of cell surface expression of the Ak beta* Ad alpha molecule, but does not alter Ak beta* Ak alpha expression. The T.86 Ak beta* Ad alpha is expressed primarily in an intracellular compartment while the T.78 Ak beta* molecule does not appear to be produced. The core-glycosylated T.78 Ak beta* polypeptide does, however, form a complex intracellularly with the core-glycosylated Ii polypeptide. Substitution of the combination of d allele residues at Ak beta polymorphic positions 9, 12, 13, 14, and 17 results in the absence of Ak beta* Ak alpha cell surface expression but does not alter the expression of this mutant Ak beta* polypeptide with the Ad alpha polypeptide. These allele-specific expression mutants demonstrate that substitution at certain beta 1 domain positions may result in the alteration of Ia cell surface expression and that the transport of Ia molecules from the Golgi apparatus to the cell surface may be regulated by signals that are determined by the interaction of polymorphic residues in both the alpha and beta polypeptides.

DNA sequence analysis of I-Ak beta mutants reveals serologically immunodominant region.

We have produced a series of in vitro serologically selected cell lines that express mutant I-Ak molecules. In this report we describe the DNA sequence analysis of the Ak beta gene of four cell lines that express serologically altered Ak beta polypeptides in association with wild-type Ak alpha polypeptides. Each of the major serologic epitopes on the Ak beta polypeptide has been altered in one or more of the four mutants. In addition, the four mutants exhibit a broad spectrum of functional defects when used to stimulate a panel of T hybridomas of various specificities. The DNA sequence analysis revealed that each mutant had sustained a single nucleotide substitution resulting in a single amino acid substitution. All four independent substitutions occurred within or near the third of the four variable regions defined in the beta 1 domain of the A beta polypeptide by allelic comparisons. These data strongly suggest that the third variable region is the major determinant of alloantigenicity on the Ak beta polypeptide.

Identification of an immunodominant region on the I-A beta chain using site-directed mutagenesis and DNA-mediated gene transfer.

To identify which polymorphic residues determine the allospecific antibody binding sites on A beta polypeptides, mutant Ak beta genes were constructed encoding single or multiple amino acids of the d allele at 14 polymorphic positions in the beta 1 domain. Cell lines expressing these genes were analyzed by quantitative immunofluorescence using 16 mAbs reactive to Ak beta or Ad beta. Substitution of d allele residues at positions 63 and 65-67 in the Ak beta polypeptide resulted in the loss of binding of all Ak beta-reactive antibodies and the gain of binding of most Ad beta-reactive antibodies. Two Ad beta-reactive mAbs bound to the mutant Ak beta polypeptide containing d allele-characteristic residue at position 40. In contrast, substitution of the other polymorphic residues in the NH2-terminal and COOH-terminal regions of the beta 1 domain did not alter antibody binding.

A beta polymorphic residues responsible for class II molecule recognition by alloreactive T cells.

In an effort to characterize the ligand that is bound by T helper lymphocyte antigen receptors, we have begun to identify class II polymorphic residues that comprise part of the allospecific TCR binding sites. Site-directed mutagenesis was used to construct mutant Ak beta (Ak beta*) genes that encode polypeptides into which single or multiple residues of the Ad beta polypeptide have been substituted in the beta 1 domain. A panel of cloned cell lines expressing the mutant Ak beta* Ak alpha or Ak beta* Ad alpha molecules was analyzed for the ability to stimulate Ak or Ad alloreactive T cell hybridomas. Substitution of d for k residues at specific positions in the beta 1 domain resulted not only in the loss of epitopes recognized by Ak-reactive T cells but, more importantly, in the gain of epitopes recognized by Ad-reactive T cells. Some of the polymorphic residues identified as contributing to the T cell epitopes are the same residues that contribute to the serologically immunodominant epitope. Other T cell epitopes map to positions predicted to be located either in an alpha-helix forming one side, or in a beta-pleated sheet forming the bottom of the putative antigen binding site. Thus, unlike serologic epitopes, TCR epitopes can be determined by A beta polymorphic residues in many different regions of the beta 1 domain and frequently depend upon contributions of A alpha polymorphic residues.

The role of polymorphic I-Ak beta chain residues in presentation of a peptide from myelin basic protein.

Proteins encoded by genes in the MHC are highly polymorphic. For class II proteins the highest level of polymorphism is found in distinct regions of variability, notably in the membrane-distal domains. To investigate the role of such residues in antigen presentation, we have tested cells transfected with wild-type or mutant I-Ak beta chains for their ability to present the NH2-terminal peptide of myelin basic protein to a panel of T cell clones. We were unable to detect a gross effect on peptide binding, in that all of the mutant cell lines presented antigen to at least one of the cloned T cells. However, the results imply that the more NH2-terminal residues, particularly 12 and 14, are involved in peptide interactions. Mutations at these residues presented antigen only at high antigen concentrations. Furthermore, residues of the more COOH-terminal regions appear to determine TCR interactions. Mutations in the predicted alpha-helical regions of the beta chain affected antigen presentation without abolishing peptide binding.

Major histocompatibility complex-restricted antigen presentation to antigen-reactive T cells by B lymphocyte tumor cells.

Previous reports have demonstrated that accessory cells function to present soluble protein antigens in association with gene products encoded within the I region of the major histocompatibility complex (MHC) to antigen-reactive T helper cells. The biochemical events that occur during antigen presentation are, however, not well-documented primarily because of the difficulties involved in purifying sufficient numbers of homogeneous antigen-presenting cells. In this paper, a number of Ia-positive B lymphocyte tumor lines are shown to be capable of presenting soluble protein antigens to antigen-reactive continuous T cell lines in an MHC-restricted fashion. The characterization of the antigen presentation function of these tumor cells indicates that the tumor cells have many of the functional antigen-presenting characteristics previously thought to be limited to macrophages. These tumor cells should provide a useful model system for determining the biochemical events that occur in antigen uptake and processing as well as for determining the potential interactions between processed antigen and Ia molecules on the plasma membrane of these antigen-presenting cells.

Immunoglobulin structure: amino terminal sequences of kappa chains from genetically similar mice (BALB/c).

The amino terminal portion of 20 kappa chains from the highly inbred BALB/c mouse has been examined on an automatic protein sequencer. These proteins can be divided into at least nine groups (subgroups) based on sequence patterns which are so distinct that each subgroup is probably encoded by at least one germ-line gene. The subgroups of mouse kappa chains are generally quite different from those of human kappa chains.