Collagen type III alpha I is a gastro-oesophageal reflux disease susceptibility gene and a male risk factor for hiatus hernia.

BACKGROUND AND OBJECTIVES: Gastro-oesophageal reflux disease (GORD) is a common gastrointestinal disorder with a genetic component. Our aim was to identify genetic factors associated with GORD. PATIENTS AND METHODS: Four separate patient cohorts were analysed using a step-wise approach. (1) Whole genome linkage analysis was performed in 36 families. (2) Candidate genes were tested for GORD association in a trio cohort. (3) Genetic association was replicated in a case-control cohort. We also investigated genetic association to hiatus hernia (HH). (4) Protein expression was analysed in oesophageal biopsies. RESULTS: A region on chromosome 2, containing collagen type III alpha 1 (COL3A1), was identified (LOD = 3.3) in families with dominant transmission of GORD, stratified for hiatus hernia (HH). COL3A1 showed significant association with GORD in an independent paediatric trio cohort (p(corr) = 0.003). The association was male specific (p(corr) = 0.018). The COL3A1 association was replicated in an independent adult case control cohort (p(corr) = 0.022). Moreover, male specific association to HH (p(corr) = 0.019) was found for a SNP not associated to GORD. Collagen type III protein was more abundant in oesophageal biopsies from male patients with GORD (p = 0.03). CONCLUSION: COL3A1 is a disease-associated gene in both paediatric and adult GORD. Furthermore, we show that COL3A1 is genetically associated with HH in adult males. The GORD- and HH-associated alleles are different, indicating two separate mechanisms leading to disease. Our data provides new insight into GORD aetiology, identifying a connective tissue component and indicating a tissue remodelling mechanism in GORD. Our results implicate gender differences in the genetic risk for both for GORD and HH.

A dual-color FISH gene map of the proximal region of rat Chromosome 4 and comparative analysis in human and mouse.

The development and refinement of the rat genome map is a prerequisite for a continued qualified and fruitful use of this model system for the study of complex traits. In two distinct rat cancer models, recurrent amplification affecting the proximal region of rat Chr 4 was detected. To further characterize this region, we turned to the evolutionarily conserved chromosome segments in human Chr 7 and mouse Chrs 5 and 6 to identify functional and positional candidate genes. By means of single- and dual-color FISH on metaphase, prometaphase, and interphase chromatin, 15 genes in rat Chr 4q11-q23 (Cdk5, Hgf, Dmtf1, Abcb1, Cyp51, Cdk6, Tac1, Asns, Cav1, Met, Wnt2, Cftr, Smoh, Braf, Arhgef5) were mapped and aligned. In the course of this work, six cancer-related rat genes were isolated de novo and partly sequenced. Ten loci were also mapped by FISH in the mouse. The map provides the framework for a more detailed genetic characterization of individual tumor amplicons, but may also be valuable for the analysis of this region in other rat models of human complex disease. In addition, our data facilitate the analysis of events in mammalian chromosomal evolution affecting the region. In a comparison with human sequence data, we found that there is considerable conservation in this region both in gene order and in distances between genes. There is a single evolutionary breakpoint between rat and mouse and two between rat and human. Since our analysis shows that the three breaks all occurred in different positions, they must be independent of one another. The data tend to support the notion that the genomic configuration in rat Chr 4 is ancestral compared with that in humans and mice.

Independent amplification of two gene clusters on chromosome 4 in rat endometrial cancer: identification and molecular characterization.

The BDII rat is genetically predisposed to hormone-dependent endometrial adenocarcinoma and was used to model human cancer. Tumors arising spontaneously in strain crosses involving BDII rats were analyzed by means of comparative genome hybridization. The most common aberration was amplification of the proximal region of rat chromosome 4, centered around bands q12-q22. The copy numbers of 15 cancer-related genes from the region were examined in tissue cultures of 11 endometrial carcinomas (10 endometrial adenocarcinomas and 1 endometrial squamous cell carcinoma) and one peritoneal mesothelioma. Amplification in rat chromosome 4 was detected in six tumors (50%), five of which carried two separate amplified regions, situated at 4q12-q13 and 4q21-q22, interrupted by a nonamplified segment at 4q13-q21.1. The genes Cdk6 (cyclin-dependent kinase 6) and Met (hepatocyte growth factor receptor) were located in the core of each amplified region and were amplified most recurrently and at the highest levels among the genes tested. Using fluorescence in situ hybridization on tumor metaphases, it was observed that the amplified Cdk6 and Met sequences were situated on typical homogeneously staining regions (HSRs). In three tumors, both genes were amplified in the same HSRs, whereas in two tumors, the amplified sequences of each gene were situated in separate HSRs. In addition, Cdk6 and Met amplification was consistently associated with a corresponding increase in gene expression, suggesting that the two genes might represent the targets for the amplifications. In the sixth tumor, which carried amplified sequences of Met but not of Cdk6, coexpression of Met and the normally silent hepatocyte growth factor gene (Hgf; the ligand of Met) was observed. This finding suggests that an autocrine signaling circuit might be operating in this particular tumor. Taken together, our findings suggest that up-regulation of Cdk6 and/or Met may contribute to the development of endometrial cancers in the BDII rat.

Between rat and mouse zoo-FISH reveals 49 chromosomal segments that have been conserved in evolution.

Mouse single chromosome paints were applied to rat prophase/prometaphase chromosomes to detect homologous chromosome regions. The analysis revealed 49 rat chromosomal regions ranging in size from whole chromosomes down to small bands near the limit of detection with this method, which was estimated to be 2-3 Mb. When all the painted regions were taken into account, the whole rat genome was covered with mouse-homologous regions, with the exception of small segments near the centromeres and the short arms of Chromosomes (Chrs) 3, 11, 12, and 13. These regions were shown to contain high levels of rat-specific repetitive DNA. The number of conserved segments between rat and mouse detected by our high-resolution zoo-FISH method was significantly higher than that reported in previous studies.

Amplification of Mycn, Ddx1, Rrm2, and Odc1 in rat uterine endometrial carcinomas.

The BDII rat is genetically predisposed to estrogen-dependent endometrial adenocarcinoma and represents a valuable model for this type of tumor. Tumors arising in strain crosses involving the BDII rats had previously been screened for DNA copy number changes using comparative genome hybridization (CGH). It was found that extra copies of the proximal region of rat chromosome (RNO) 6 commonly could be detected in these tumors. Based on RH-mapping data and comparative mapping with mouse and human, seven cancer-related genes were predicted to be situated in RNO6q14-q16. Rat PACs were isolated for the N-myc proto-oncogene (Mycn), apolipoprotein B (Apob), the DEAD box gene 1 (Ddx1), ornithine decarboxylase 1 (Odc1), proopiomelanocortin (Pomc1), ribonucleotide reductase, M2 polypeptide (Rrm2), and syndecan 1 (Sdc1). The localization of the genes to the region was verified by FISH (fluorescence in situ hybridization) mapping, and the detailed order among them was determined by dual-color FISH. By Southern blot analysis, it was found that the Mycn locus was highly amplified in two out of 10 cell cultures derived from the tumors. In one of them (designated RUT30), the amplification level of Mycn was estimated at 140x. Two other genes were coamplified (Ddx1 and Rrm2) at much lower levels. Similarly, in another culture (designated RUT2), Mycn was amplified more than 40x, whereas three of the other genes (Ddx1, Rrm2, and Odc1) were coamplified at lower levels. Using FISH on metaphase chromosomes from the cell cultures analyzed, the amplified sequences were shown to be located in typical HSRs. With competitive RT-PCR, distinct overexpression of Mycn and Ddx1 could be demonstrated in both RUT2 and RUT30. In addition, Mycn was overexpressed in two other tumors not exhibiting Mycn amplification. Taken together, our results suggest that overexpression of Mycn plays an important role in the development of endometrial cancer in the BDII rat. In humans, Mycn amplification has been reported mainly from tumors of neuronal origin. To our knowledge, this is the first report of Mycn amplification and overexpression in hormone-dependent tumors.

Analysis of genetic changes in rat endometrial carcinomas by means of comparative genomic hybridization.

Animals of the BDII inbred rat strain are known to be genetically predisposed to endometrial adenocarcinoma (EAC). Using them as models of human EACs, we studied tumors arising in F1 and F2 progeny from BDII animals crossed to animals from two other inbred strains, in which EACs were quite rare. In order to identify chromosomal regions exhibiting DNA copy number changes, comparative genomic hybridization (CGH) was applied in a series corresponding to 27 different solid tumors, most of which were classified as EACs, from these animals. The main findings from the study were that, although many different chromosomes were involved in copy number variation, some of the changes detected were recurrent and quite specific. Among specific changes found were gains in rat chromosome (RNO) regions 4q12 approximately q22, 6q14 approximately q16, and whole chromosome arms in some of the small metacentric chromosomes (e.g., RNO14, 16, and 18). RNO10 was involved in gain in the terminal and proximal regions. Each of these regions contains previously identified cancer-related genes representing possible candidates to be involved in the development of EAC. Furthermore, it was observed that there were clear differences in the pattern of copy number changes between tumors occurring in the two different crosses, and also between solid tumors and cell cultures. Endometrial cancer is the most common human gynecological cancer, but not much is known about specific genetic changes influencing this disease. Two genetic alterations that have been reported from human endometrial cancer are amplification of the ERBB2 gene and mutations in the 12 codon of the KRAS gene. One case of Erbb2 amplification was found but there were no Kras mutations in the rat material studied. We conclude that molecular genetic analysis of the rat BDII model will provide important new information about EAC in mammals.

Rat-mouse and rat-human comparative maps based on gene homology and high-resolution zoo-FISH.

The laboratory rat, Rattus norvegicus, and the laboratory mouse, Mus musculus, are key animal models in biomedical research. A deeper understanding of the genetic interrelationsships between Homo sapiens and these two rodent species is desirable for extending the usefulness of the animal models. We present comprehensive rat-human and rat-mouse comparative maps, based on 1090 gene homology assignments available for rat genes. Radiation hybrid, FISH, and zoo-FISH mapping data have been integrated to produce comparative maps that are estimated to comprise 83-100% of the conserved regions between rat and mouse and 66-82% of the conserved regions between rat and human. The rat-mouse zoo-FISH analysis, supported by data for individual genes, revealed nine previously undetected conserved regions compared to earlier reports. Since there is almost complete genome coverage in the rat-mouse comparative map, we conclude that it is feasible to make accurate predictions of gene positions in the rat based on gene locations in the mouse.

Ras gene mutations in 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat sarcomas.

Tumor induction in rats by 7,12-dimethylbenz[a]anthracene (DMBA) will generate malignancies that display reproducible chromosomal abnormalities involving rat chromosome (RNO) 2. Thus, it has been reported that rat DMBA erythroleukemias display RNO2 abnormalities, which in this case were closely correlated to mutations in the Nras oncogene located in RNO2q34. Our cytogenetic analysis in a series of 17 DMBA-induced rat sarcomas showed that 11 (65%) tumors had a significant increase in RNO2 copy number. Furthermore, the incidence of point mutations in codons 12, 13 and 61 of Hras, Kras, and Nras was examined in the same set of sarcomas, and mutations were detected in three (18%) tumors, in codon 61 of Kras (CAA-->CAT) (1 of 17) and Nras (CAA-->CTA) (2 of 17). We conclude that the high frequency of RNO2 gain was in accordance with previous studies of DMBA-induced rat neoplasms, supporting the idea of a significant role of RNO2 in DMBA carcinogenesis. However, there was no clear-cut relationship between activated Nras and gain of RNO2 material, implying that mutational activation of Nras is not the causative factor underlying the gain of RNO2 copy number in rat DMBA sarcomas, in contrast to what has been suggested for DMBA-induced erythroleukemias.

Hgfr/Met oncogene acts as target for gene amplification in DMBA-induced rat sarcomas: free chromatin fluorescence in situ hybridization analysis of amplicon arrays in homogeneously staining regions.

Analysis of chromosome rearrangements in tumors is an important means for revealing genetic pathways underlying tumorigenesis and tumor progression. In five of 17 DMBA-induced rat sarcomas, cytogenetic analysis had disclosed homogeneously staining regions (hsrs), which are generally accepted to be cytogenetic signs of gene amplification. Using comparative genomic hybridization (CGH), regional increases in DNA copy number of the proximal part of rat chromosome (RNO) 4 were detected in four of the tumors harboring hsrs. Amplification of the Hgfr/Met oncogene, located at RNO4q21.2, was detected by fluorescence in situ hybridization (FISH) in five tumors. In four of them, a number of flanking genes located in the close vicinity of Hgfr/Met, including Cav1 (q21.1), Wnt2 (q21.2-q21.3), and Cftr (q21.3), also were amplified, though amplification was seen in a lower fraction of the cells than was Hgfr/Met amplification. In the fifth tumor (BL150T), Hgfr/Met was amplified in all cells and was the sole amplified gene of those tested. In addition, the Hgfr/Met FISH signals in BL150T were tightly clustered and formed compact and intense spots compared with the signals seen in the other four tumors. Application of the free chromatin FISH technique to BL150T showed that the genomic Hgfr/Met probe stained the extended chromatin fibers of up to 1.5 Mb with an almost uninterrupted signal, indicating that the BL150T amplicon was build up solely of Hgfr/Met gene sequences. Our results suggest that the Hgfr/Met oncogene is the primary target for amplification in a subset of rat DMBA sarcomas.

Genomewide assessment of genetic alterations in DMBA-induced rat sarcomas: cytogenetic, CGH, and allelotype analyses reveal recurrent DNA copy number changes in rat chromosomes 1, 2, 4, and 7.

Rat sarcomas, induced by subcutaneous injections of 7,12-dimethylbenz[a]anthracene (DMBA), were studied with the objective of identifying critical chromosome regions associated with tumorigenesis. We employed three genomewide screening techniques-cytogenetics, CGH, and allelotyping-in 19 DMBA-induced sarcomas in F1 (BN/Han x LE/Mol) rats. The most conspicuous finding in the cytogenetic analysis was a high incidence of trisomy for rat chromosome 2 (RNO2). Signs of gene amplification (hsr) were also seen in several tumors. The CGH analysis revealed that gains in copy number were much more common than losses. The gains mostly affected RNO2 (10/19), RNO12q (7/19), and RNO19q (5/19), as well as the proximal part of RNO4 (8/19) and the distal part of RNO7 (7/19). Reduction in copy number was seen in RNO17 (2/19). For the allelotyping, we used 318 polymorphic microsatellite marker loci covering the entire genome. We identified regions of allelic imbalance affecting most of the rat chromosomes. The highest incidences of recurrent allelic imbalance were observed at loci in certain regions in RNO1, 2, 4, and 7 and at lower incidences in parts of RNO12, 16, 18, and 19. The combined results suggested that genetic alterations detected in RNO2 and RNO12 usually corresponded to complete or partial trisomy, whereas those in RNO1 and RNO7 seemed to involve regional deletions and/or gains. Furthermore, we could confirm that copy number gains occur proximally in RNO4, where a previous study showed amplification of the Met oncogene in a subset of these tumors.