The Bloom's syndrome (BS) gene, BLM, plays an important role in the maintenance of genomic stability in somatic cells. A candidate for BLM was identified by direct selection of a cDNA derived from a 250 kb segment of the genome to which BLM had been assigned by somatic crossover point mapping. In this novel mapping method, cells were used from persons with BS that had undergone intragenic recombination within BLM. cDNA analysis of the candidate gene identified a 4437 bp cDNA that encodes a 1417 amino acid peptide with homology to the RecQ helicases, a subfamily of DExH box-containing DNA and RNA helicases. The presence of chain-terminating mutations in the candidate gene in persons with BS proved that it was BLM.

The Bloom syndrome gene, BLM, encodes a RecQ DNA helicase that when absent from the cell results in genomic instability and cancer predisposition. We show here that BLM is a substrate for small ubiquitin-like modifier (SUMO) modification, with lysines at K317, K331, K334 and K347 being preferred sites of modification. Unlike normal BLM, a double mutant BLM protein with lysine to arginine substitutions at residues 317 and 331 was not modified by SUMO, and it failed to localize efficiently to the PML nuclear bodies. Rather, double mutant BLM protein induced the formation of DNA damage-induced foci (DDI) that contained BRCA1 protein and phosphorylated histone H2AX. Double mutant BLM only partially complemented the genomic instability phenotypes of Bloom syndrome cells as assessed by sister-chromatid exchange and micronuclei formation assays. These results constitute evidence that BLM is a DNA damage sensor that signals the formation of DDI, and they establish SUMO modification as a negative regulator of BLM's signaling function.

Bloom syndrome (BS) is a rare autosomal recessive disorder characterized by growth deficiency, immunodeficiency, genomic instability, and the early development of cancers of many types. BLM, the protein encoded by BLM, the gene mutated in BS, is localized in nuclear foci and absent from BS cells. BLM encodes a DNA helicase, and proteins from three missense alleles lack displacement activity. BLM transfected into BS cells reduces the frequency of sister chromatid exchanges and restores BLM in the nucleus. Missense alleles fail to reduce the sister chromatid exchanges in transfected BS cells or restore the normal nuclear pattern. BLM complements a phenotype of a Saccharomyces cerevisiae sgs1 top3 strain, and the missense alleles do not. This work demonstrates the importance of the enzymatic activity of BLM for its function and nuclear localization pattern. INTRODUCTIONBloom syndrome (BS) is a rare autosomal recessive trait (German, 1993;German and Ellis, 1997). The major clinical manifestations are small stature, sun-sensitive redness of the face, immunodeficiency, male infertility, a predisposition to diabetes, and the development of early cancers of many types. Cells derived from persons with BS exhibit increased numbers of chromatid gaps, breaks, and sister chromatid exchanges (SCEs). Somatic mutations of many types have been documented at multiple loci (reviewed in German, 1993). Biochemical studies have demonstrated a slow replication-fork progression and an abnormal distribution of DNA replication intermediates. Some BS cell lines exhibit increased sensitivity to DNA-damaging agents such as mitomycin C, N-nitroso-N-ethylurea, and ethyl methanesulfonate. Alterations in several enzymes involved in DNA replication and repair have been identified in some but not all BS cell lines (see references in Ellis et al., 1995a).Despite this accumulation of biochemical evidence of disturbances in DNA metabolism, no consistent defect or candidate gene product could be identified by these approaches.The Bloom syndrome gene was cloned using molecular haplotype analysis of affected families and positional cloning methodologies (Ellis et al., 1995a). The mapping of the gene was facilitated by the observation that lymphocytes from affected compound heterozygotes can revert to a normal low SCE frequency phenotype by virtue of recombination within the two copies of the BLM gene itself (Ellis et al., 1995b). These normal circulating cells arise because of a rare somatic recombination event between the maternal and paternal chromosome 15s and generate cells containing a wild-type gene. Molecular haplotype analysis of low-SCE cells and high-SCE cells from several affected individuals narrowed the BLM locus to a 250-kilobase region at 1 5q 26.1. Expressed DNA sequences from this region were selected, and a cDNA clone was found encoding a 1417 amino acid protein with strong amino acid sequence homology with the RecQ family of DNA helicases. DNA sequence analysis of BLM † Corresponding author. E-mail address: nneff@nybc.org.© 1999 by ...

The pseudoautosomal boundaries are the interface between pseudoautosomal and sex chromosome-specific DNA sequences. We have isolated a gene, PBDX, from the human pseudoautosomal boundary region of Xp. The three exons at the 5' end of PBDX are situated in the pseudoautosomal region immediately downstream of MIC2, whereas the other seven exons are in the X-specific region. Hence, PBDX is inherited in two modes: its 5' end is pseudoautosomally inherited and its 3' end is X-linked. The predicted amino acid sequence of the 540 bp coding region is 48% homologous to 12E7, the product of MIC2. By virtue of its position, PBDX becomes an excellent candidate for the XG blood group gene.

The gene BLM, mutated in Bloom syndrome (BS), encodes the nuclear protein BLM, which when absent, as it is from most BS cells, results in genomic instability. A manifestation of this instability is an excessive rate of sister-chromatid exchange (SCE). Here we describe the effects on this abnormal cellular phenotype of stable transfection of normal BLM cDNAs into two types of BS cells, SV40-transformed fibroblasts and Epstein-Barr virus (EBV)-transformed lymphoblastoid cells. Clones of BLM-transfected fibroblasts produced normal amounts of BLM by western blot analysis and displayed a normal nuclear localization of the protein by immunofluorescence microscopy. They had a mean of 24 SCEs/46 chromosomes, in contrast to the mean of 69 SCEs in controls transfected only with the vector. BLM-transfected fibroblast clones that expressed highest levels of the BLM protein had lowest levels of SCE. The lymphoblastoid cells transfected with BLM had SCE frequencies of 22 and 42 in two separate experiments in which two different selectable markers were used, in contrast to 57 and 58 in vector-transfected cells; in this type cell, however, the BLM protein was below the level detectable by western blot analysis. These experiments prove that BLM cDNA encodes a functional protein capable of restoring to or toward normal the uniquely characteristic high-SCE phenotype of BS cells.

We studied the feasibility of a novel approach to localize breast cancer susceptibility genes, using a low-density genomewide panel of single-nucleotide polymorphisms and taking advantage of large regions of linkage disequilibrium (LD) flanking Jewish disease genes in high-risk cases. With Affymetrix GeneChip arrays, we genotyped 8,576 polymorphisms in three sets of Ashkenazi Jewish breast cancer cases: a ''validation'' set of 27 breast cancer cases, all of whom carried the BRCA2*6174delT founder mutation; a ''field'' set of 19 breast cancer cases from male breast cancer kindreds, which simulated conditions for finding new genes; and a ''test'' set of 57 probands from breast cancer kindreds (4 or more cases/ kindred), in which mutations in BRCA1 and BRCA2 had been excluded. To identify associations, we compared the frequency of genotypes and haplotypes in cases vs. controls by the Fisher's exact test and a maximum likelihood ratio test. In the ''validation'' set, we demonstrated the presence of a region of linkage disequilibrium on BRCA2*6174delT chromosomes that spanned over 5 million bases. In the ''field'' set, we showed that this large region of linkage disequilibrium flanking BRCA2 was detectable despite the presence of heterogeneity in the sample set. Finally, in the ''test'' set, at least three regions of interest emerged that could contain novel breast cancer genes, one of which had been identified previously by linkage analysis. While these results demonstrate the feasibility of genome-wide association strategies, further application of this approach will critically depend on optimizing the density and distribution of SNPs and the size and type of study design. Genet. Epidemiol. 30:48-61, 2006. r 2005 Wiley-Liss, Inc.

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With the large numbers of single nucleotide polymorphisms (SNPs) available and new technologies that permit high throughput genotyping, we have investigated the possibility of the localization of disease genes with genome-wide panels of SNP markers and taking advantage of the linkagedisequilibrium (LD) between the disease gene and closely linked markers. For this purpose, we selected cases from the Ashkenazi Jewish population, in which the mutant alleles are expected to be identical by descent from a common founder and the regions of LD encompassing these mutant alleles are large. As a validation of this approach for localization, we performed two trials: one in autosomal recessive Bloom syndrome, in which a unique mutation of the BLM gene is present at elevated frequencies in cases, and the other in autosomal dominant hereditary nonpolyposis colorectal cancer (HNPCC), in which a unique mutation of MSH2 is present at elevated frequencies. In the Bloom syndrome trial, we genotyped 3,258 SNPs in 10 Jewish Bloom syndrome cases and 31 non-Bloom syndrome Jewish persons as a comparison group. In the HNPCC trial, we genotyped 8,549 SNPS in 13 Jewish HNPCC cases whose colon cancers exhibited microsatellite instability and in 63 healthy Jews as a comparison group. To identify significant associations, we performed (a) Fisher's exact test comparing genotypes at each locus in cases versus controls and (b) a haplotype analysis by estimating the frequency of haplotypes with the expectation-maximization algorithm and comparing haplotype frequencies in cases versus controls by logistic regression and a maximum likelihood ratio method. In the Bloom syndrome trial, by Fisher's exact test, statistically significant association was detected at a single locus, TSC0754862, which is a locus 1.7 million bp from BLM. Two-locus, three-locus, and four-locus haplotypes that included TSC0754862 and flanked BLM were also statistically more frequent in cases versus controls. In the HNPCC trial, although a significant P value was not obtained by the single SNP genotype analysis, significant associations were detected for several multilocus haplotypes in an 11-million-bp region that contained the MSH2 gene. This work demonstrates the power of the LD mapping approach in an isolated population and its general applicability to the identification of novel cancer-causing genes.