Microarray analysis of human leucocyte subsets: the advantages of positive selection and rapid purification.

BACKGROUND: For expression profiling to have a practical impact in the management of immune-related disease it is essential that it can be applied to peripheral blood cells. Early studies have used total peripheral blood mononuclear cells, and as a consequence the majority of the disease-related signatures identified have simply reflected differences in the relative abundance of individual cell types between patients and controls. To identify cell-specific changes in transcription it would be necessary to profile purified leucocyte subsets. RESULTS: We have used sequential rounds of positive selection to isolate CD4 and CD8 T cells, CD19 B cells, CD14 monocytes and CD16 neutrophils for microarray analysis from a single blood sample. We compared gene expression in cells isolated in parallel using either positive or negative selection and demonstrate that there are no significant consistent changes due to positive selection, and that the far inferior results obtained by negative selection are largely due to reduced purity. Finally, we demonstrate that storing cells prior to separation leads to profound changes in expression, predominantly in cells of the myeloid lineage. CONCLUSION: Leukocyte subsets should be prepared for microarray analysis by rapid positive selection.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

X-inactivation and marker studies in three families with incontinentia pigmenti: implications for counselling and gene localisation

Article first published online: 24 DEC 2001Familial incontinentia pigmenti (IP) is an X-linked dominant disorder with an extremely variable clinical presentation. Ambiguous diagnosis can complicate genetic counselling and attempts to refine the gene location in Xq28. Marked skewing of X-inactivation patterns is a hallmark of IP and provides a means for investigating uncertain cases. We have conducted X-inactivation studies in three families where Xq28 marker studies were at odds with the original clinical assessment. The results indicate that no recombination between the disease locus and Xq28 loci has occurred and suggest that mosaicism is responsible for the discrepancy in one family

Mutations in the DLG3 Gene Cause Nonsyndromic X-Linked Mental Retardation

We have identified truncating mutations in the human DLG3 (neuroendocrine dlg) gene in 4 of 329 families with moderate to severe X-linked mental retardation. DLG3 encodes synapse-associated protein 102 (SAP102), a member of the membrane-associated guanylate kinase protein family. Neuronal SAP102 is expressed during early brain development and is localized to the postsynaptic density of excitatory synapses. It is composed of three amino-terminal PDZ domains, an src homology domain, and a carboxyl-terminal guanylate kinase domain. The PDZ domains interact directly with the NR2 subunits of the NMDA glutamate receptor and with other proteins responsible for NMDA receptor localization, immobilization, and signaling. The mutations identified in this study all introduce premature stop codons within or before the third PDZ domain, and it is likely that this impairs the ability of SAP102 to interact with the NMDA receptor and/or other proteins involved in downstream NMDA receptor signaling pathways. NMDA receptors have been implicated in the induction of certain forms of synaptic plasticity, such as long-term potentiation and long-term depression, and these changes in synaptic efficacy have been proposed as neural mechanisms underlying memory and learning. The disruption of NMDA receptor targeting or signaling, as a result of the loss of SAP102, may lead to altered synaptic plasticity and may explain the intellectual impairment observed in individuals with DLG3 mutations

Mutations of the BRAF gene in human cancer

Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for analysis signalling pathways in which at least one gene is mutated in human cancer. The RAS RAF MEK ERK MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%. Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma