The homeobox gene MEIS1 is amplified in IMR-32 and highly expressed in other neuroblastoma cell lines.

Neuroblastoma is a childhood tumour of the sympathetic nervous system that demonstrates striking clinical heterogeneity. In order to determine which genes are abnormally expressed in neuroblastoma, we screened regions of amplification from the short arm of chromosome 2 in the neuroblastoma cell line IMR-32 and found that the homeobox gene, myeloid ecotropic integration site 1 (MEIS1), is highly amplified. MEIS1 normally maps to chromosome band 2p14. High expression of MEIS1 without amplification was also found in other neuroblastoma cell lines, with and without MYCN amplification, and in medulloblastoma and crythroleukaemia cell lines. MEIS1 is highly expressed in cerebellum and ubiquitously expressed in normal immunohaematopoietic tissues and is thought to be important in cell proliferation and differentiation. While several lines of evidence point towards a role for homeobox genes in the development of other malignancies, this is the first report showing the amplification of a homeobox gene in neuroblastoma.

Narrowing of the region of allelic loss in 21q11-21 in squamous non-small cell lung carcinoma and cloning of a novel ubiquitin-specific protease gene from the deleted segment.

We examined 42 fresh non-small cell lung carcinomas for allelic loss using 4 microsatellite markers located in a 4.5 Mb region in 21q11-21, a gene-poor interval recently found by others to be homozygously deleted and exhibiting frequent allelic loss in lung cancer. We found allelic loss across the entire segment in 13/34 informative squamous carcinomas, with 2 cases showing loss in only part of the region. Analysis by fluorescence in situ hybridization of P1-derived artificial chromosomes from the region directly on paraffin sections of the tumor is in concordance with the loss of heterozygosity (LOH) results, and tentatively excludes a 2 Mb segment bearing 2 of the only 3 known genes in the area. Exon trapping in the remaining segment of loss led to identification and cloning of a novel gene spanning 150 kb within the deletion. The full-length gene encodes a protein of 1,055 amino acids with homology to ubiquitin-specific proteases across the eukaryotic evolutionary spectrum. The expressed protein acts as a de-ubiquitinating enzyme as proved by the ability to cleave ubiquitin from a model fusion protein. We found no mutations in the sequence of the functional domains of this gene in any of the LOH-exhibiting tumor DNA samples. It is, however, interesting that genes of the same superfamily have been reported on 3p21, a locus showing the most frequent allelic instability and deletions in lung cancer. Genes Chromosomes Cancer 27:153-161, 2000.

Construction and analysis of a sequence-ready map in 4q25: Rieger syndrome can be caused by haploinsufficiency of RIEG, but also by chromosome breaks approximately 90 kb upstream of this gene.

The autosomal dominant disorder Rieger syndrome (RIEG) shows genetic heterogeneity and has a phenotype characterized by malformations of the anterior segment of the eye, failure of the periumbilical skin to involute, and dental hypoplasia. The main locus for RIEG was mapped to the 4q25-q27 chromosomal segment using a series of cytogenetic abnormalities as well as by genetic linkage to DNA markers. Recently, a bicoid-related homeobox transcription factor gene called RIEG has been cloned, characterized, and proven to cause the 4q25 linked RIEG. Its mode of action in the pathogenesis of RIEG was not conclusively proven, since most etiological mutations detected in the RIEG sequence caused amino acid substitutions or splice changes in the homeodomain. Through FISH analysis of a 460-kb sequence-ready map (PAC contig) around RIEG that we report in this paper, we demonstrate that the 4q25 linked RIEG disorder can arise from the haploid, whole-gene deletion of RIEG, but also from a translocation break 90 kb upstream from the gene. The data provide conclusive evidence that physical or functional haploinsufficiency of RIEG is the pathogenic mechanism for Rieger syndrome. The map also defines restriction fragments bearing sequences with a potential key regulatory role in the control of homeobox gene expression.

Rieger syndrome locus: a new reciprocal translocation t(4;12)(q25;q15) and a deletion del(4)(q25q27) both break between markers D4S2945 and D4S193.

Rieger syndrome (RS) is an autosomal dominant disorder of morphogenesis characterised by malformation of the anterior segment of the eye, dental hypoplasia, and failure of the periumbilical skin to involute. RS has been mapped to the 4q25-q27 chromosomal segment by a series of cytogenetic studies as well as by genetic linkage to DNA markers. It was first localised to chromosome 4q based on an association with a constitutional deletion of 4q23-q27. In this paper we localise the proximal breakpoint of this deletion from the original patient, and we describe a new family with a de novo balanced reciprocal translocation t(4;12)(q25;q15) segregating with full RS in two generations. Using FISH and the P1 artificial chromosomes (PACs) as probes, we have physically localised both the deletion and the translocation breakpoints between genetic markers which are known to be strongly linked to RS. We have mapped both the proximal deletion breakpoint and the translocation breakpoint within a region between two groups of PACs bearing the markers D4S2945 (on the centromeric side) and D4S193 and D4S2940 (on the telomeric side). We believe that these recombinant bacterial clones derived directly from genomic DNA (not subcloned from YACs) will be valuable complementary tools in the efforts to clone the RS gene and to construct a full transcriptional and sequence ready map of this region.

Determination of the organisation of coding sequences within the iduronate sulphate sulphatase (IDS) gene.

The structure of the gene coding for iduronate sulphate sulphatase (IDS) has been determined. We have used exon to exon and vectorette PCR to identify 9 exons within the IDS gene and to characterise the surrounding intron sequences. The results of this study will be useful for the complete analysis of many mutations giving rise to Hunter syndrome. IDS is the first member of the group of lysosomal nonarylsulphatase genes for which the gene structure has been determined. It bears no relationship to the exon organisation of steroid sulphatase, despite the homology between these two proteins. This suggests that the division of the sulphatases into the two subgroups on the basis of substrate specificity is also reflected at the level of gene structure.

Detection of point mutations and a gross deletion in six Hunter syndrome patients.

We have used screening with the polymerase chain reaction and chemical mismatch detection of amplified cDNA to detect and characterize deletions and point mutations in six Hunter Syndrome patients. A high degree of mutational heterogeneity was observed. The first patient is completely deleted for the gene coding for alpha-L-iduronate sulfate sulfatase, while the second has a point mutation that creates a stop codon. The third patient shows a point mutation that creates a novel splice site that is preferentially utilized and results in partial loss of one exon in the RNA. Patients 4, 5, and 6 have point mutations resulting in single amino acid substitutions. Four of the six single-base changes observed in this study were examples of transitions of the highly mutable dinucleotide CpG to TpG. This study has demonstrated a procedure capable of detecting all types of mutation that affect the function of the IDS protein and should enable direct carrier and prenatal diagnosis for Hunter syndrome families.

Evaluation of a reversed passive latex agglutination test for detection of Escherichia coli heat-labile toxin in culture supernatants.

One hundred strains of Escherichia coli were tested for the production of the heat-labile enterotoxin by the Y1 adrenal cell test and a commercially available reversed passive latex agglutination test. The strains were grown in Casamino Acids-yeast extract broth, and filtered culture supernatants were tested for the presence of heat-labile enterotoxin. There was perfect correlation between the Y1 test and the reversed passive latex agglutination test, and the latter was simple to perform and completed within 48 h.