Alterations in chromatin accessibility and DNA methylation in clear cell renal cell carcinoma.

Recent studies have demonstrated that in clear cell renal cell carcinoma (ccRCC) several chromatin remodeling enzymes are genetically inactivated. Although, growing evidence in cancer models has demonstrated the importance of epigenetic changes, currently only changes in DNA methylation can be accurately determined from clinical samples. To address this limitation, we have applied formaldehyde-assisted isolation of regulatory elements (FAIREs) combined with next-generation sequencing (FAIRE-seq) to identify specific changes in chromatin accessibility in clinical samples of ccRCC. We modified the FAIRE procedure to allow us to examine chromatin accessibility for small samples of solid tumors. Our FAIRE results were compared with DNA-methylation analysis and show how chromatin accessibility decreases at many sites where DNA-methylation remains unchanged. In addition, our FAIRE-seq analysis allowed us to identify regulatory elements associated with both normal and tumor tissue. We have identified decreases in chromatin accessibility at key ccRCC-linked genes, including PBRM1, SETD2 and MLL2. Overall, our results demonstrate the power of examining multiple aspects of the epigenome.

Array comparative genomic hybridisation (aCGH) analysis of premenopausal breast cancers from a nuclear fallout area and matched cases from Western New York.

High-resolution array comparative genomic hybridisation (aCGH) analysis of DNA copy number aberrations (CNAs) was performed on breast carcinomas in premenopausal women from Western New York (WNY) and from Gomel, Belarus, an area exposed to fallout from the 1986 Chernobyl nuclear accident. Genomic DNA was isolated from 47 frozen tumour specimens from 42 patients and hybridised to arrays spotted with more than 3000 BAC clones. In all, 20 samples were from WNY and 27 were from Belarus. In total, 34 samples were primary tumours and 13 were lymph node metastases, including five matched pairs from Gomel. The average number of total CNAs per sample was 76 (range 35-134). We identified 152 CNAs (92 gains and 60 losses) occurring in more than 10% of the samples. The most common amplifications included gains at 8q13.2 (49%), at 1p21.1 (36%), and at 8q24.21 (36%). The most common deletions were at 1p36.22 (26%), at 17p13.2 (26%), and at 8p23.3 (23%). Belarussian tumours had more amplifications and fewer deletions than WNY breast cancers. HER2/neu negativity and younger age were also associated with a higher number of gains and fewer losses. In the five paired samples, we observed more discordant than concordant DNA changes. Unsupervised hierarchical cluster analysis revealed two distinct groups of tumours: one comprised predominantly of Belarussian carcinomas and the other largely consisting of WNY cases. In total, 50 CNAs occurred significantly more commonly in one cohort vs the other, and these included some candidate signature amplifications in the breast cancers in women exposed to significant radiation. In conclusion, our high-density aCGH study has revealed a large number of genetic aberrations in individual premenopausal breast cancer specimens, some of which had not been reported before. We identified a distinct CNA profile for carcinomas from a nuclear fallout area, suggesting a possible molecular fingerprint of radiation-associated breast cancer.

Identification and characterisation of constitutional chromosome abnormalities using arrays of bacterial artificial chromosomes.

Constitutional chromosome deletions and duplications frequently predispose to the development of a wide variety of cancers. We have developed a microarray of 6000 bacterial artificial chromosomes for array-based comparative genomic hybridisation, which provides an average resolution of 750 kb across the human genome. Using these arrays, subtle gains and losses of chromosome regions can be detected in constitutional cells, following a single overnight hybridisation. In this report, we demonstrate the efficiency of this procedure in identifying constitutional deletions and duplications associated with predisposition to retinoblastoma, Wilms tumour and Beckwith-Wiedemann syndrome.

Molecular analysis of region t(5;6)(q21;q21) in Wilms tumor.

We have previously described the physical localization of a constitutional t(5;6)(q21;q21) in a patient (tumor cell sample designated as MA214) with bilateral Wilms tumor (WT). We have now physically refined the breakpoints and identified putative gene targets within this region. The translocation breakpoints are contained within a 2.5-Mbp region on 5q21 containing four candidate genes and a 1.3-Mbp region on 6q21 that contains three candidate genes. To explore the role of this region in WT genesis, we have performed loss of heterozygosity (LOH) analysis with markers flanking the translocation breakpoints in tumor from MA214 and a panel of sporadic WT. Alleles were retained for all informative markers used in the MA214 tumor. In sporadic tumors LOH was found in 6 of 63 (9.5%) and 5 of 62 (8%) informative cases for flanking markers D6S301 and D6S1592 on 6q21. LOH was found in 3 of 58 (5.2%) and 2 of 54 (3.6%) for flanking markers D5S495 and D5S409 on 5q21. These preliminary data suggest LOH at the t(5;6)(q21;q21) region is unlikely to be a mechanism for tumor development in MA214, but may be important for a subgroup of sporadic WT.

Intrachromosomal genomic instability in human sporadic colorectal cancer measured by genome-wide allelotyping and inter-(simple sequence repeat) PCR.

We have used genome-wide allelotyping with 348 polymorphic autosomal markers spaced, on average, 10 cM apart to quantitate the extent of intrachromosomal instability in 59 human sporadic colorectal carcinomas. We have compared instability measured by this method with that measured by inter-(simple sequence repeat) PCR and microsatellite instability assays. Instability quantitated by fractional allelic loss rates was found to be independent of that detected by microsatellite instability analyses but was weakly associated with that measured by inter-(simple sequence repeat) PCR. A set of seven loci were identified that were most strongly associated with elevated rates of fractional allelic loss and/or inter-(simple sequence repeat) PCR instability; these seven loci were on chromosomes 3, 8, 11, 13, 14, 18, and 20. A lesser association was seen with two loci flanking p53 on chromosome 17. Coordinate loss patterns for these loci suggest that at least two separate sets of cooperating loci exist for intrachromosomal genomic instability in human colorectal cancer.

The pericentromeric region of human chromosome 11: evidence for a chromosome-specific duplication.

We have identified a chromosome duplication in the pericentromeric region of human chromosome 11 located in 11p11 and 11q14. A detailed physical map of each duplicated region was generated to describe the nature of the duplication, the involvement at the centromere and to resolve the correct maps. All clones were evaluated to ensure they were representative of their genetic origin. The order of clones, based on their marker content, as well as the distance covered was determined by SEGMAP. Each duplication encompasses more than 1 Mb of DNA and appears to be chromosome 11 specific. Ten STS markers were mapped within each duplication. Comparative sequence analysis along the duplication identified 35 nucleotide changes in 2,036 bp between the two copies, suggesting the duplication occurred over 14 million years ago. A suggested organization of the pericentromeric region, including the duplications and alpha-related repetitive sequences, is presented.

Semiautomated clone verification by real-time PCR using molecular beacons.

Conventional, high-throughput PCR analysis of common elements utilizing numerous primer sets and template DNA requires multiple rounds of PCR to ensure optimal conditions. Laborious gel electrophoresis and staining is then necessary to visualize amplification products. We propose novel multicolor molecular beacons, to establish a high-throughput, PCR-based sequence tagged site (STS) detection system that swiftly and accurately confirms marker content in template containing common repeat elements. A simple, one-tube, real-time PCR assay system was developed to specifically detect regions containing CA and GATA repeats. Ninety-six samples can be confirmed for marker content in a closed-tube format in 3 h, eliminating product confirmation on agarose gels and avoiding crossover contamination. Multiple STSs can be detected simultaneously in the same reaction tube by utilizing molecular beacons labeled with multicolor fluorophores. Template DNA from 260 RPCI-11 bacterial artificial chromosome (BAC) clones was examined for the presence of CA and/or GATA repeats using molecular beacon PCR and compared with conventional PCR results of the same clones. Of the 205 clones containing CA and GATA repeats, we were able to identify 129 clones (CA, n = 99; GATA, n = 30) by using molecular beacons and only 121 clones (CA, n = 92; GATA, n = 29) by conventional PCR amplification. As anticipated, 55 clones that contained sequences other than CA or GATA failed molecular beacon detection. Molecular beacon PCR, employing beacons specific for tandem repeat elements, provides a fast, accurate, and sensitive multiplex detection assay that will expedite verification of marker content in a multitude of template containing these repeats.

Regional myocardial perfusion after experimental subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: The pathophysiology of cardiac injury after subarachnoid hemorrhage (SAH) remains controversial. Data from animal models suggest that catecholamine-mediated injury is the most likely cause of cardiac injury after SAH. However, researchers also have proposed myocardial ischemia to be the underlying cause, as a result of coronary artery disease, coronary artery spasm, or hypertension and tachycardia. To test the hypothesis that SAH-induced cardiac injury occurs in the absence of myocardial hypoperfusion, we developed an experimental canine model that reproduces the clinical and pathological cardiac lesions of SAH and defines the epicardial and microvascular coronary circulation. METHODS: Serial ECG, hemodynamic measurements, coronary angiography, regional myocardial blood flow measurements by radiolabeled microspheres, 2D echocardiography, and myocardial contrast echocardiography were performed in 9 dogs with experimental SAH and 5 controls. RESULTS: Regional wall motion abnormalities were identified in 8 of 9 SAH dogs and 1 of 5 controls (Fisher's Exact Test, P=0.02) but no evidence was seen of coronary artery disease or spasm by coronary angiography and of significant myocardial hypoperfusion by either regional myocardial blood flow or myocardial contrast echocardiography. CONCLUSIONS: In this experimental model of SAH, a unique form of regional left ventricular dysfunction occurs in the absence of myocardial hypoperfusion. Future studies are justified to determine the cause of cardiac injury after SAH.

MAGOH interacts with a novel RNA-binding protein.

MAGOH is the human homologue of Drosophila mago nashi, a protein that is required for normal germ plasm development in the Drosophila embryo. Using human MAGOH as a bait protein in a yeast two-hybrid screen, we recovered four independent cDNA clones that encode different lengths of a novel protein containing a conserved RNA-binding region. This gene, designated RBM8, encodes a 173-aa protein that was shown to have an apparent molecular mass of 26 kDa, as demonstrated by in vitro translation assay. The interaction between MAGOH and RBM8 was demonstrated by both yeast two-hybrid and GST fusion protein pull-down assays. Like MAGOH, RBM8 gene is expressed ubiquitously in human tissues; three species of RBM8 mRNA were detected. Also similar to MAGOH, RBM8 expression is serum inducible in quiescent NIH3T3 fibroblast cells.

Genome-wide allelotyping indicates increased loss of heterozygosity on 9p and 14q in early age of onset colorectal cancer.

Colorectal cancer remains a significant public health challenge, despite our increased understanding of the genetic mechanisms involved in the initiation and progression of this disorder. It has become clear that multiple mechanisms lead to the tumorigenic phenotype, with familial predisposition syndromes accounting for less than 15% of all colorectal cancers. A genome-wide scan for loss of heterozygosity (LOH) was carried out with 150 highly polymorphic markers in an effort to identify additional loci involved in colorectal tumorigenesis in DNA samples from 42 colorectal cancer patients. The results confirm earlier observations that tumor DNAs from patients with hereditary nonpolyposis colon cancer (HNPCC) either maintain heterozygosity or exhibit altered or additional alleles. DNAs from patients with early onset colorectal carcinomas (diagnosed prior to age 50) revealed a higher overall degree of LOH than DNAs from patients with sporadic colorectal cancers diagnosed later in life (after age 50). While regions on 1p, 10q and 14q are suggestive, statistical analysis of LOH at these regions failed to reach significance. However, LOH at 9p did reveal a statistically significant increase in the early onset patient group, compared to the greater than age 50 group. LOH on 9p may involve inactivation of p16/CDKN2 through aberrant DNA methylation on the remaining chromosome, resulting in a situation analogous to a homozygous deletion of p16 and providing a selective growth advantage to these cells. This marker may prove to be a useful prognostic indicator for patient stratification in the design of therapy for early onset colorectal cancer patients.

Bestrophin gene mutations in patients with Best vitelliform macular dystrophy.

Best vitelliform macular dystrophy (VMD2) is an autosomal dominant dystrophy with a juvenile age of onset. Mutations in the Bestrophin gene were shown in patients affected with VMD2. In a mutation study, we made three new and interesting observations. First, we identified possible mutation hotspots within the gene, suggesting that particular regions of the protein have greater functional significance than others. Second, we described a 2-bp deletion in a part of the gene where mutations have not previously been reported; this mutation causes a frameshift and subsequent premature termination of the protein. Finally, we have evidence that some mutations are associated with variable expression of the disease, suggesting the involvement of other factors or genes in the disease phenotype.

Mapping, genomic organization and promoter analysis of the human prostate-specific membrane antigen gene.

Prostate-specific membrane antigen (PSMA) is a 100 kDa type II transmembrane protein with folate hydrolase and NAALAdase activity. PSMA is highly expressed in prostate cancer and the vasculature of most solid tumors, and is currently the target of a number of diagnostic and therapeutic strategies. PSMA is also expressed in the brain, and is involved in conversion of the major neurotransmitter NAAG (N-acetyl-aspartyl glutamate) to NAA and free glutamate, the levels of which are disrupted in several neurological disorders including multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease and schizophrenia. To facilitate analysis of the role of PSMA in carcinoma we have determined the structural organization of the gene. The gene consists of 19 exons spanning approximately 60 kb of genomic DNA. A 1244 nt portion of the 5' region of the PSMA gene was able to drive the firefly luciferase reporter gene in prostate but not breast-derived cell lines. We have mapped the gene encoding PSMA to 11p11-p12, however a gene homologous, but not identical, to PSMA exists on chromosome 11q14. Analysis of sequence differences between non-coding regions of the two genes suggests duplication and divergence occurred 22 million years ago.

Transcript mapping of the human chromosome 11q12-q13.1 gene-rich region identifies several newly described conserved genes.

Despite the localization of several human diseases to 11q13, the majority of the genes responsible for these disorders have not yet been cloned. Exon amplification and EST mapping were performed using clones derived from an approximately 1.65-Mb P1 artificial chromosome contig encompassing the region that reportedly harbors the gene mutated in the dominantly inherited eye disorder, Best disease. Fifty-eight exons isolated from the region were sequenced, resulting in 41.3% showing weak or no similarity to database sequences. Four exons had exact matches with human ESTs and 2 exons were highly similar to mouse ESTs. The sequence of 1 of these human ESTs was highly similar to that of the rat Rabin3 and mouse Pat-12 genes, which potentially encode Ras-like GTPase binding proteins. Three exon sequences were similar to those of the inner centromere proteins of Gallus gallus and Xenopus laevis, which are mitotic phosphoproteins, and 1 exon sequence had similarity to the epidermal growth factor-like repeat from several proteins. High-resolution mapping of 34 ESTs binned to the 11q12-q13 region by the Human Transcript Mapping Project identified 5 present in the PAC contig, with 1 of these ESTs identifying a human homologue of the rat synaptotagmin VII gene. Database searches identified two overlapping cDNA clones representing almost the entire open reading frame of this human gene and a sequenced cosmid indicating its partial genomic structure. Further database analyses identified another sequenced cosmid from this region that contained both exon-trap and mapped EST sequences. PowerBLAST and GRAIL analysis of this cosmid sequence identified matches with several other ESTs, the previously described FEN1 gene, and a novel evolutionarily conserved gene. These experiments identify candidate genes for disorders that map to this region and indicate that this is a gene-rich region of the human genome.

Divergently transcribed overlapping genes expressed in liver and kidney and located in the 11p15.5 imprinted domain.

Human chromosomal band 11p15.5 has been shown to contain genes involved in the development of several pediatric and adult tumors and in Beckwith-Wiedemann syndrome (BWS). Overlapping P1 artificial chromosome clones from this region have been used as templates for genomic sequencing in an effort to identify candidate genes for these disorders. PowerBLAST identified several matches with expressed sequence tags (ESTs) from fetal brain and liver cDNA libraries. Northern blot analysis indicated that two of the genes identified by these ESTs encode transcripts of 1-1.5 kb with predominant expression in fetal and adult liver and kidney. With RT-PCR and RACE, full-length transcripts were isolated for these two genes, with the largest open reading frames encoding putative proteins of 253 and 424 amino acids. Database comparison of the predicted amino acid sequence of the larger transcript indicated homology to integral membrane organic cation transporters; hence, we designate this gene ORCTL2 (organic cation transporter-like 2). An expressed sequence polymorphism provided evidence that the ORCTL2 gene exhibits "leaky" imprinting in both human fetal kidney and human fetal liver. The mouse orthologue (Orctl2) was identified, and a similar polymorphism was used to demonstrate maternal-specific expression of this gene in fetal liver from interspecific F1 mice. The predicted protein of the smaller gene showed no significant similarity in the database. Northern and RACE analyses suggest that this gene may have multiple transcription start sites. Determination of the genomic structure in humans indicated that the 5'-end of this transcript overlaps in divergent orientation with the first two exons of ORCTL2, suggesting a possible role for antisense regulation of one gene by the other. We, therefore, provisionally name this second transcript ORCTL2S (ORCTL2-antisense). The expression patterns of these genes and the imprinted expression of ORCTL2 are suggestive of a possible role in the development of Wilms tumor (WT) and hepatoblastoma. Although SSCP analysis of 62 WT samples and 10 BWS patients did not result in the identification of any mutations in ORCTL2 or ORCTL2S, it will be important to examine their expression pattern in tumors and BWS patients, since epigenetic alteration at these loci may play a role in the etiology of these diseases.

Nibrin, a novel DNA double-strand break repair protein, is mutated in Nijmegen breakage syndrome.

Nijmegen breakage syndrome (NBS) is an autosomal recessive chromosomal instability syndrome characterized by microcephaly, growth retardation, immunodeficiency, and cancer predisposition. Cells from NBS patients are hypersensitive to ionizing radiation with cytogenetic features indistinguishable from ataxia telangiectasia. We describe the positional cloning of a gene encoding a novel protein, nibrin. It contains two modules found in cell cycle checkpoint proteins, a forkhead-associated domain adjacent to a breast cancer carboxy-terminal domain. A truncating 5 bp deletion was identified in the majority of NBS patients, carrying a conserved marker haplotype. Five further truncating mutations were identified in patients with other distinct haplotypes. The domains found in nibrin and the NBS phenotype suggest that this disorder is caused by defective responses to DNA double-strand breaks.

Linkage-disequilibrium mapping without genotyping.

Genomic mismatch scanning (GMS) is a technique that enriches for regions of identity by descent (IBD) between two individuals without the need for genotyping or sequencing. Regions of IBD selected by GMS are mapped by hybridization to a microarray containing ordered clones of genomic DNA from chromosomes of interest. Here we demonstrate the feasibility and efficacy of this form of linkage-mapping, using congenital hyperinsulinism (HI), an autosomal recessive disease, whose relatively high frequency in Ashkenazi Jews suggests a founder effect. The gene responsible (SUR1) encodes the sulfonylurea receptor, which maps to chromosome 11p15.1. We show that the combination of GMS and hybridization of IBD products to a chromosome-11 microarray correctly maps the HI gene to a 2-Mb region, thereby demonstrating linkage-disequilibrium mapping without genotyping.

A gene map of the Best's vitelliform macular dystrophy region in chromosome 11q12-q13.1.

Best's vitelliform macular dystrophy is an autosomal dominant disorder of unknown causes. To identify the underlying gene defect the disease locus has been mapped to an approximately 1.4-Mb region on chromosome 11q12-q13.1. As a prerequisite for its positional cloning we have assembled a high coverage PAC contig of the candidate region. Here, we report the construction of a primary transcript map that places a total of 19 genes within the Best's disease region. This includes 14 transcripts of as yet unknown function obtained by EST mapping and/or cDNA selection and five genes mapped previously to the interval (CD5, PGA, DDB1, FEN1, and FTH1). Northern blot analyses were performed to determine the expression profiles in various human tissues. At least three genes appear to be good candidates for Best's disease based on their abundant expression in retina or retinal pigment epithelium. Additional information on the functional properties of these genes, as well as mutation analyses in Best's disease patients, have to await their further characterization. [The GenBank/EMBL accession numbers and details of the isolation, localization, and characterization of ESTs and selected cDNAs are available as online supplements in Online Tables 1-3 at http://www.genome.org.]

Contig maps and genomic sequencing identify candidate genes in the usher 1C locus.

Usher syndrome 1C (USH1C) is a congenital condition manifesting profound hearing loss, the absence of vestibular function, and eventual retinal degeneration. The USH1C locus has been mapped genetically to a 2- to 3-cM interval in 11p14-15.1 between D11S899 and D11S861. In an effort to identify the USH1C disease gene we have isolated the region between these markers in yeast artificial chromosomes (YACs) using a combination of STS content mapping and Alu-PCR hybridization. The YAC contig is approximately 3.5 Mb and has located several other loci within this interval, resulting in the order CEN-LDHA-SAA1-TPH-D11S1310-(D11S1888/KCNC1 )-MYOD1-D11S902D11S921-D11S 1890-TEL. Subsequent haplotyping and homozygosity analysis refined the location of the disease gene to a 400-kb interval between D11S902 and D11S1890 with all affected individuals being homozygous for the internal marker D11S921. To facilitate gene identification, the critical region has been converted into P1 artificial chromosome (PAC) clones using sequence-tagged sites (STSs) mapped to the YAC contig, Alu-PCR products generated from the YACs, and PAC end probes. A contig of >50 PAC clones has been assembled between D11S1310 and D11S1890, confirming the order of markers used in haplotyping. Three PAC clones representing nearly two-thirds of the USH1C critical region have been sequenced. PowerBLAST analysis identified six clusters of expressed sequence tags (ESTs), two known genes (BIR, SUR1) mapped previously to this region, and a previously characterized but unmapped gene NEFA (DNA binding/EF hand/acidic amino-acid-rich). GRAIL analysis identified 11 CpG islands and 73 exons of excellent quality. These data allowed the construction of a transcription map for the USH1C critical region, consisting of three known genes and six or more novel transcripts. Based on their map location, these loci represent candidate disease loci for USH1C. The NEFA gene was assessed as the USH1C locus by the sequencing of an amplified NEFA cDNA from an USH1C patient; however, no mutations were detected.

The mammalian homologue of mago nashi encodes a serum-inducible protein.

The products of at least 11 maternal effect genes have been shown to be essential for proper germ plasm assembly in Drosophila melanogaster embryos. Here we report the isolation and characterization of the mammalian counterpart for one of these genes (named MAGOH for mago nashi homologue). The predicted amino acid sequence of mouse and human MAGOH are completely identical; MAGOH homologues from the nematode Caenorhabditis elegans and rice grain Oryza sativa also show a remarkable degree of amino acid conservation. MAGOH was mapped to chromosome 1p33-p34 in the human and a syntenic region of chromosome 4 in the mouse. Of note, MAGOH mRNA expression is not limited to germ plasm, but is expressed ubiquitously in adult tissues and can be induced by serum stimulation of quiescent fibroblasts.