David Vetrie

The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases.

X-linked agammaglobulinaemia (XLA) is a human immunodeficiency caused by failure of pre-B cells in the bone marrow to develop into circulating mature B cells. A novel gene has been isolated which maps to the XLA locus, is expressed in B cells, and shows mutations in families with the disorder. The gene is a member of the src family of proto-oncogenes which encode protein-tyrosine kinases. This is, to our knowledge, the first evidence that mutations in a src-related gene are involved in human genetic disease.

DNA microarrays for comparative genomic hybridization based on DOP-PCR amplification of BAC and PAC clones

We have designed DOP‐PCR primers specifically for the amplification of large insert clones for use in the construction of DNA microarrays. A bioinformatic approach was used to construct primers that were efficient in the general amplification of human DNA but were poor at amplifying E. coli DNA, a common contaminant of DNA preparations from large insert clones. We chose the three most selective primers for use in printing DNA microarrays. DNA combined from the amplification of large insert clones by use of these three primers and spotted onto glass slides showed more than a sixfold increase in the human to E. coli hybridization ratio when compared to the standard DOP‐PCR primer, 6MW. The microarrays reproducibly delineated previously characterized gains and deletions in a cancer cell line and identified a small gain not detected by use of conventional CGH. We also describe a method for the bulk testing of the hybridization characteristics of chromosome‐specific clones spotted on microarrays by use of DNA amplified from flow‐sorted chromosomes. Finally, we describe a set of clones selected from the publicly available Golden Path of the human genome at 1‐Mb intervals and a view in the Ensembl genome browser from which data required for the use of these clones in array CGH and other experiments can be downloaded across the Internet.

Whole-genome microarrays of fission yeast: characteristics, accuracy, reproducibility, and processing of array data

BackgroundThe genome of the fission yeast Schizosaccharomyces pombe has recently been sequenced, setting the stage for the post-genomic era of this increasingly popular model organism. We have built fission yeast microarrays, optimised protocols to improve array performance, and carried out experiments to assess various characteristics of microarrays.ResultsWe designed PCR primers to amplify specific probes (180–500 bp) for all known and predicted fission yeast genes, which are printed in duplicate onto separate regions of glass slides together with control elements (~13,000 spots/slide). Fluorescence signal intensities depended on the size and intragenic position of the array elements, whereas the signal ratios were largely independent of element properties. Only the coding strand is covalently linked to the slides, and our array elements can discriminate transcriptional direction. The microarrays can distinguish sequences with up to 70% identity, above which cross-hybridisation contributes to the signal intensity. We tested the accuracy of signal ratios and measured the reproducibility of array data caused by biological and technical factors. Because the technical variability is lower, it is best to use samples prepared from independent biological experiments to obtain repeated measurements with swapping of fluorochromes to prevent dye bias. We also developed a script that discards unreliable data and performs a normalization to correct spatial artefacts.ConclusionsThis paper provides data for several microarray properties that are rarely measured. The results define critical parameters for microarray design and experiments and provide a framework to optimise and interpret array data. Our arrays give reproducible and accurate expression ratios with high sensitivity. The scripts for primer design and initial data processing as well as primer sequences and detailed protocols are available from our website.

Exon array CGH: detection of copy-number changes at the resolution of individual exons in the human genome.

The development of high-throughput screening methods such as array-based comparative genome hybridization (array CGH) allows screening of the human genome for copy-number changes. Current array CGH strategies have limits of resolution that make detection of small (less than a few tens of kilobases) gains or losses of genomic DNA difficult to identify. We report here a significant improvement in the resolution of array CGH, with the development of an array platform that utilizes single-stranded DNA array elements to accurately measure copy-number changes of individual exons in the human genome. Using this technology, we screened 31 patient samples across an array containing a total of 162 exons for five disease genes and detected copy-number changes, ranging from whole-gene deletions and duplications to single-exon deletions and duplications, in 100% of the cases. Our data demonstrate that it is possible to screen the human genome for copy-number changes with array CGH at a resolution that is 2 orders of magnitude higher than that previously reported.

Pelizaeus-Merzbacher disease: identification of Xq22 proteolipid-protein duplications and characterization of breakpoints by interphase FISH.

Pelizaeus-Merzbacher disease (PMD) is an X-linked, dysmyelinating disorder of the CNS. Duplications of the proteolipid protein (PLP) gene have been found in a proportion of patients, suggesting that, in addition to coding-region or splice-site mutations, overdosage of the gene can cause PMD. We show that the duplication can be detected by interphase FISH, using a PLP probe in five patients and their four asymptomatic carrier mothers. The extent of the duplication was analyzed in each family by interphase FISH, with probes from a 1. 7-Mb region surrounding the PLP gene between markers DXS83 and DXS94. A large duplication >=500 kb was detected, with breakpoints that differed, between families, at the proximal end. Distinct separation of the duplicated PLP signals could be seen only on metaphase chromosomes in one family, providing further evidence that different duplication events are involved. Quantitative fluorescent multiplex PCR was used to confirm the duplication in patients, by the detection of increased copy number of the PLP gene. Multiallelic markers from the duplicated region were analyzed, since the identification of two alleles in an affected boy would indicate a duplication. The majority of boys were homozygous for all four markers, compared with their mothers, who were heterozygous for one to three of the markers. These results suggest that intrachromosomal rearrangements may be a common mechanism by which duplications arise in PMD. One boy was heterozygous for the PLP marker, indicating a duplication and suggesting that interchromosomal rearrangements of maternal origin also can be involved. Since duplications are a major cause of PMD, we propose that interphase FISH is a reliable method for diagnosis and identification of female carriers.

Gene expression profiling in the myelodysplastic syndromes using cDNA microarray technology

The myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal disorders of the haematopoietic stem cell and primarily involve cells of the myeloid lineage. Using cDNA microarrays comprising 6000 human genes, we studied the gene expression profiles in the neutrophils of 21 MDS patients, seven of which had the 5q‐ syndrome, and two acute myeloid leukaemia (AML) patients when compared with the neutrophils from pooled healthy controls. Data analysis showed a high level of heterogeneity of gene expression between MDS patients, most probably reflecting the underlying karyotypic and genetic heterogeneity. Nevertheless, several genes were commonly up or down‐regulated in MDS. The most up‐regulated genes included RAB20, ARG1, ZNF183 and ACPL. The RAB20 gene is a member of the Ras gene superfamily and ARG1 promotes cellular proliferation. The most down‐regulated genes include COX2, CD18, FOS and IL7R. COX2 is anti‐apoptotic and promotes cell survival. Many genes were identified that are differentially expressed in the different MDS subtypes and AML. A subset of genes was able to discriminate patients with the 5q‐ syndrome from patients with refractory anaemia and a normal karyotype. The microarray expression results for several genes were confirmed by real‐time quantitative polymerase chain reaction. The MDS‐specific expression changes identified are likely to be biologically important in the pathophysiology of this disorder.

Functional diversity for REST (NRSF) is defined by in vivo binding affinity hierarchies at the DNA sequence level

The molecular events that contribute to, and result from, the in vivo binding of transcription factors to their cognate DNA sequence motifs in mammalian genomes are poorly understood. We demonstrate that variations within the DNA sequence motifs that bind the transcriptional repressor REST (NRSF) encode in vivo DNA binding affinity hierarchies that contribute to regulatory function during lineage-specific and developmental programs in fundamental ways. First, canonical sequence motifs for REST facilitate strong REST binding and control functional classes of REST targets that are common to all cell types, whilst atypical motifs participate in weak interactions and control those targets, which are cell- or tissue-specific. Second, variations in REST binding relate directly to variations in expression and chromatin configurations of RESTs target genes. Third, REST clearance from its binding sites is also associated with variations in the RE1 motif. Finally, and most surprisingly, weak REST binding sites reside in DNA sequences that show the highest levels of constraint through evolution, thus facilitating their roles in maintaining tissue-specific functions. These relationships have never been reported in mammalian systems for any transcription factor.

Identification of novel deletion breakpoints bordered by segmental duplications in the NF1 locus using high resolution array-CGH

Background: Segmental duplications flanking the neurofibromatosis type 1 (NF1) gene locus on 17q11 mediate most gene deletions in NF1 patients. However, the large size of the gene and the complexity of the locus architecture pose difficulties in deletion analysis. We report the construction and application of the first NF1 locus specific microarray, covering 2.24 Mb of 17q11, using a non-redundant approach for array design. The average resolution of analysis for the array is ∼12 kb per measurement point with an increased average resolution of 6.4 kb for the NF1 gene. Methods: We performed a comprehensive array-CGH analysis of 161 NF1 derived samples and identified heterozygous deletions of various sizes in 39 cases. The typical deletion was identified in 26 cases, whereas 13 samples showed atypical deletion profiles. Results: The size of the atypical deletions, contained within the segment covered by the array, ranged from 6 kb to 1.6 Mb and their breakpoints could be accurately determined. Moreover, 10 atypical deletions were observed to share a common breakpoint either on the proximal or distal end of the deletion. The deletions identified by array-CGH were independently confirmed using multiplex ligation-dependent probe amplification. Bioinformatic analysis of the entire locus identified 33 segmental duplications. Conclusions: We show that at least one of these segmental duplications, which borders the proximal breakpoint located within the NF1 intron 1 in five atypical deletions, might represent a novel hot spot for deletions. Our array constitutes a novel and reliable tool offering significantly improved diagnostics for this common disorder.

Dual targeting of p53 and c-MYC selectively eliminates leukaemic stem cells.

Summary Chronic myeloid leukaemia (CML) arises following transformation of a haemopoietic stem cell (HSC) by protein-tyrosine kinase BCR-ABL1. Direct inhibition of BCR-ABL1 kinase has revolutionized disease management, but fails to eradicate leukaemic stem cells (LSC), which maintain CML. LSC are independent of BCR-ABL1 for survival, providing a rationale to identify and target kinase-independent pathways. Here we show using proteomics, transcriptomics and network analyses, that in human LSC aberrantly expressed proteins, in both imatinib-responder and non-responder patients are modulated in concert with p53 and c-Myc regulation. Perturbation of both p53 and c-Myc, not BCR-ABL1 itself, leads to synergistic kill, differentiation and near elimination of transplantable human LSC in mice, whilst sparing normal HSC. This unbiased systems approach targeting connected nodes exemplifies a novel precision medicine strategy providing evidence that LSC can be eradicated.

Detection of mutations in COL4A5 in patients with Alport Syndrome

Alport syndrome (AS) can be caused by mutations in COL4A5, one of the six type IV collagen genes. For the purposes of confirming diagnoses, carrier screening and correlating genotype to phenotype, we have screened all 51 exons of this gene by SSCP analysis in 153 families with suspected AS. Mutations were identified in 77 families (of which 20 have previously been reported) and are reported with all available clinical information. All types of mutation were found (missense, nonsense, splicing, small and large deletions and insertions), with the commonest type being those affecting glycine residues in the collagen triple helix. Our 50% detection rate is similar to that of other groups and may imply the presence of mutations outside of the COL4A5 coding region or the existence of a second X‐linked AS gene. Hum Mutat 13:124–132, 1999.