Overrepresentation of genetic variation in the AnkyrinG interactome is related to a range of neurodevelopmental disorders

Upon the discovery of numerous genes involved in the pathogenesis of neurodevelopmental disorders, several studies showed that a significant proportion of these genes converge on common pathways and protein networks. Here, we used a reversed approach, by screening the AnkyrinG protein-protein interaction network for genetic variation in a large cohort of 1009 cases with neurodevelopmental disorders. We identified a significant enrichment of de novo potentially disease-causing variants in this network, confirming that this protein network plays an important role in the emergence of several neurodevelopmental disorders.Subject terms: Genetics research, Neurological disorders     

Positional cloning of the gene for X-linked retinitis pigmentosa 3: homology with the guanine-nucleotide-exchange factor RCC1

The gene for retinitis pigmentosa 3 (RP3), the most frequent form of X- linked RP (XLRP), has been mapped previously to a chromosome interval of less than 1000 kbp between the DXS1110 marker and the OTC locus at Xp21.1-p11.4. Employing a novel technique, YAC Representation Hybridization (YRH)', we have recently identified a small XLRP associated microdeletion in this interval, as well as several putative exons including the 3' end of a gene that was truncated by the deletion. cDNA library screening and sequencing of a cosmid centromeric to the deletion has now enabled us to identify numerous additional exons and to detect several point mutations in patients with XLRP. The predicted gene product shows homology to RCC1, the guanine-nucleotide- exchange factor (GEF) of the Ras-like GTPase Ran. Our findings suggest that we have cloned the long-sought RP3 gene, and that it may encode the GEF of a retina-specific GTP-binding protein.      

Glucocorticoids down-regulate dendritic cell function in vitro and in vivo

Exogenous glucocorticoid hormones are widely used as therapeutical agents, whereas endogenous glucocorticoids may act as physiological immunosuppressants involved in the control of immune and inflammatory responses. The optimal activation of T lymphocytes requires two distinct signals: the major histocompatibility complex-restricted presentation of the antigen and an additional co-stimulatory signal provided by the antigen-presenting cells. There is ample evidence that, among the cells able to present the antigen, the dendritic cells (DC) have the unique property to activate antigen-specific, naive T cells in vitro and in vivo, and are therefore required for the induction of primary immune responses. In this work, we tested whether glucocorticoids affected the capacity of DC to sensitize naive T cells. Our data show that, in vitro, the steroid hormone analog dexamethasone (Dex) affects the viability of DC, selectively downregulates the expression of co-stimulatory molecules on viable DC, and strongly reduces their immunostimulatory properties. In vivo, a single injection of Dex results in impaired antigen presenting function, a finding which correlates with reduced numbers of splenic DC. These results show that glucocorticoids regulate DC maturation and immune function in vitro and in vivo and suggest that this mechanism may play a role in preventing overstimulation of the immune system.     

COMPLEXITY OF THE BOVINE MHC CLASS-II SPECIFICITY DW3 AS DEFINED BY ALLOANTISERA

Alloantisera related to the bovine major histocompatibility complex (MHC) class-II specificity Dw3 were investigated by cross-absorption experiments and by application of the monoclonal antibody-specific immobilization of lymphocyte antigen assay (MAILA). The absorption study revealed antibodies specific for an antigenic determinant shared by all Ds03 (Dw3)-positive animals, and several other antibody populations recognizing the locally defined specificites Ds10, Ds11 and Ds15, that are closely associated with Ds03. The results of the MAILA-assay indicate that the Ds03 specificity is probably encoded by DQ, whereas specificities Ds10 and Ds11 are more closely associated with DR molecules. The data presented here provide the first evidence that bovine DR and DQ specificities can be identified separately by serological methods using alloimmune antisera.     

A case of imported paracoccidioidomycosis: an awkward infection in The Netherlands.

Paracoccidioidomycosis is an important endemic mycosis in South America. In Europe the disease is very rare and only found as infections in travelers to Latin America. We report here the first case encountered in the Netherlands for which the appropriate diagnosis was not attained for several months. A Dutch 60-year-old man presented with a painful ulceration in the buccal mandibular vestibular mucosa of three months duration. While his medical history was uneventful, he had worked, until 8 years prior to his presentation, as a carpenter for 25 years in the jungles of Peru and Ecuador. An aberrant chest radiograph, CT-scan of the lungs and increased erythrocyte sedimentation rate were suggestive of sarcoidosis or a bronchiolitis obliterans organizing pneumonia. There was no improvement in the patient's symptoms despite the use of budesonide and prednisone medication, as well as tuberculosis prophylaxis with isoniazide and rifampicin, and local use of miconazole. Quite to the contrary, as an irritated, irregular hyperemic mucosa and gingiva with ulceration were noticed during this period of time. These precipitated an incisional biopsy through which a mixed inflammatory cellular infiltrate and large yeast cells were found on histopathologic examination. Based on the patient's travel history and the multiple budding yeastlike cells revealed in the biopsy tissue, the diagnosis of paracoccidioidomycosis was finally made. This was supported by the isolation of Paracoccidioides brasiliensis in culture. Antimycotic oral therapy with itraconazole was started and continued for 15 months. At two and five year follow-ups, the patient was asymptomatic. In Europe, it may be expected that diseases that are endemic in other areas will be seen more frequently in countries where the diseases are not routinely encountered. It is most likely that the use of corticosteroid medication, with its inherent immunosuppressive effect, resulted in the reactivation of an infection acquired many years before in Latin America. The etiologic agent then disseminated from the initial focal point to cause the ensuing oral mucous membrane lesions. The importance of the patient's prolonged residence in Latin America was overlooked. The very long latency of endemic mycoses emphasizes the need for a meticulous history which should include not only recent trips, but also past residence in foreign countries.     

Use of class II tetramers for identification of CD4+ T cells

Multivalent MHC class II molecules containing peptide antigens are useful tools for the detection of antigen specific human CD4+ T cells. Tetramers produced by exogenous peptide loading onto empty class II molecules are comparable to tetramers with peptide tethered to the class II chain covalently, but have many practical advantages. Conditions for optimal peptide loading to generate tetramers are discussed and optimal conditions of using tetramers for staining T cells are examined. As the frequency of antigen specific CD4+ T cells in peripheral blood is low, we demonstrate that an in vitro expansion step is effective in detecting low frequency T cells. Two new applications with tetramers, their uses for mapping T cell epitopes and for the detection of low affinity T cells are described. In a clinical setting, potential applications include using these reagents for monitoring disease progression during clinical intervention.     

Comparison of DNA- and RNA-based methods for detection of truncating BRCA1 mutations

A number of methods are used for mutational analysis of BRCA1, a large multi-exon gene. A comparison was made of five methods to detect mutations generating premature stop codons that are predicted to result in synthesis of a truncated protein in BRCA1. These included four DNA-based methods: two-dimensional gene scanning (TDGS), denaturing high performance liquid chromatography (DHPLC), enzymatic mutation detection (EMD), and single strand conformation polymorphism analysis (SSCP) and an RNA/DNA-based protein truncation test (PTT) with and without complementary 5' sequencing. DNA and RNA samples isolated from 21 coded lymphoblastoid cell line samples were tested. These specimens had previously been analyzed by direct automated DNA sequencing, considered to be the optimum method for mutation detection. The set of 21 cell lines included 14 samples with 13 unique frameshift or nonsense mutations, three samples with two unique splice site mutations, and four samples without deleterious mutations. The present study focused on the detection of protein-truncating mutations, those that have been reported most often to be disease-causing alterations that segregate with cancer in families. PTT with complementary 5' sequencing correctly identified all 15 deleterious mutations. Not surprisingly, the DNA-based techniques did not detect a deletion of exon 22. EMD and DHPLC identified all of the mutations with the exception of the exon 22 deletion. Two mutations were initially missed by TDGS, but could be detected after slight changes in the test design, and five truncating mutations were missed by SSCP. It will continue to be important to use complementary methods for mutational analysis.