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

Published Online First 26 April 2009Background 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 (pcorr = 0.003). The association was male specific (pcorr = 0.018). The COL3A1 association was replicated in an independent adult case control cohort (pcorr = 0.022). Moreover, male specific association to HH (pcorr = 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.B Åsling, J Jirholt, P Hammond, M Knutsson, A Walentinsson, G Davidson, L Agreus, A Lehmann, M Lagerström-Ferme

A combined human in silico and CRISPR/Cas9-mediated in vivo zebrafish based approach to provide phenotypic data for supporting early target validation

This is the final version. Available on open access from Frontiers Media via the DOI in this record. Data availability statement: The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding authors.The clinical heterogeneity of heart failure has challenged our understanding of the underlying genetic mechanisms of this disease. In this respect, large-scale patient DNA sequencing studies have become an invaluable strategy for identifying potential genetic contributing factors. The complex aetiology of heart failure, however, also means that in vivo models are vital to understand the links between genetic perturbations and functional impacts as part of the process for validating potential new drug targets. Traditional approaches (e.g., genetically-modified mice) are optimal for assessing small numbers of genes, but less practical when multiple genes are identified. The zebrafish, in contrast, offers great potential for higher throughput in vivo gene functional assessment to aid target prioritisation, by providing more confidence in target relevance and facilitating gene selection for definitive loss of function studies undertaken in mice. Here we used whole-exome sequencing and bioinformatics on human patient data to identify 3 genes (API5, HSPB7, and LMO2) suggestively associated with heart failure that were also predicted to play a broader role in disease aetiology. The role of these genes in cardiovascular system development and function was then further investigated using in vivo CRISPR/Cas9-mediated gene mutation analysis in zebrafish. We observed multiple impacts in F0 knockout zebrafish embryos (crispants) following effective somatic mutation, including changes in ventricle size, pericardial oedema, and chamber malformation. In the case of lmo2, there was also a significant impact on cardiovascular function as well as an expected reduction in erythropoiesis. The data generated from both the human in silico and zebrafish in vivo assessments undertaken supports further investigation of the potential roles of API5, HSPB7, and LMO2 in human cardiovascular disease. The data presented also supports the use of human in silico genetic variant analysis, in combination with zebrafish crispant phenotyping, as a powerful approach for assessing gene function as part of an integrated multi-level drug target validation strategy.Royal SocietyAstraZenec

Defining the Sister Rat Mammary Tumor Cell Lines HH-16 cl.2/1 and HH-16.cl.4 as an In Vitro Cell Model for Erbb2

Cancer cell lines have been shown to be reliable tools in genetic studies of breast cancer, and the characterization of these lines indicates that they are good models for studying the biological mechanisms underlying this disease. Here, we describe the molecular cytogenetic/genetic characterization of two sister rat mammary tumor cell lines, HH-16 cl.2/1 and HH-16.cl.4, for the first time. Molecular cytogenetic analysis using rat and mouse chromosome paint probes and BAC/PAC clones allowed the characterization of clonal chromosome rearrangements; moreover, this strategy assisted in revealing detected breakpoint regions and complex chromosome rearrangements. This comprehensive cytogenetic analysis revealed an increase in the number of copies of the Mycn and Erbb2 genes in the investigated cell lines. To analyze its possible correlation with expression changes, relative RNA expression was assessed by real-time reverse transcription quantitative PCR and RNA FISH. Erbb2 was found to be overexpressed in HH-16.cl.4, but not in the sister cell line HH-16 cl.2/1, even though these lines share the same initial genetic environment. Moreover, the relative expression of Erbb2 decreased after global genome demethylation in the HH-16.cl.4 cell line. As these cell lines are commercially available and have been used in previous studies, the present detailed characterization improves their value as an in vitro cell model. We believe that the development of appropriate in vitro cell models for breast cancer is of crucial importance for revealing the genetic and cellular pathways underlying this neoplasy and for employing them as experimental tools to assist in the generation of new biotherapies

A Genome-Wide Association Study of Diabetic Kidney Disease in Subjects With Type 2 Diabetes

dentification of sequence variants robustly associated with predisposition to diabetic kidney disease (DKD) has the potential to provide insights into the pathophysiological mechanisms responsible. We conducted a genome-wide association study (GWAS) of DKD in type 2 diabetes (T2D) using eight complementary dichotomous and quantitative DKD phenotypes: the principal dichotomous analysis involved 5,717 T2D subjects, 3,345 with DKD. Promising association signals were evaluated in up to 26,827 subjects with T2D (12,710 with DKD). A combined T1D+T2D GWAS was performed using complementary data available for subjects with T1D, which, with replication samples, involved up to 40,340 subjects with diabetes (18,582 with DKD). Analysis of specific DKD phenotypes identified a novel signal near GABRR1 (rs9942471, P = 4.5 x 10(-8)) associated with microalbuminuria in European T2D case subjects. However, no replication of this signal was observed in Asian subjects with T2D or in the equivalent T1D analysis. There was only limited support, in this substantially enlarged analysis, for association at previously reported DKD signals, except for those at UMOD and PRKAG2, both associated with estimated glomerular filtration rate. We conclude that, despite challenges in addressing phenotypic heterogeneity, access to increased sample sizes will continue to provide more robust inference regarding risk variant discovery for DKD.Peer reviewe

4-aminobutyrate aminotrasferase (ABAT): genetic and pharmacological evidence for an involvement in gastro esophageal reflux disease

Extent: 9p.Gastro-esophageal reflux disease (GERD) is partly caused by genetic factors. The underlying susceptibility genes are currently unknown, with the exception of COL3A1. We used three independent GERD patient cohorts to identify GERD susceptibility genes. Thirty-six families, demonstrating dominant transmission of GERD were subjected to whole genome microsatellite genotyping and linkage analysis. Five linked regions were identified. Two families shared a linked region (LOD 3.9 and 2.0) on chromosome 16. We used two additional independent GERD patient cohorts, one consisting of 219 trios (affected child with parents) and the other an adult GERD case control cohort consisting of 256 cases and 485 controls, to validate individual genes in the linked region through association analysis. Sixty six single nucleotide polymorphism (SNP) markers distributed over the nine genes present in the linked region were genotyped in the independent GERD trio cohort. Transmission disequilibrium test analysis followed by multiple testing adjustments revealed a significant genetic association for one SNP located in an intron of the gene 4-aminobutyrate aminotransferase (ABAT) (Padj = 0.027). This association did not replicate in the adult case-control cohort, possibly due to the differences in ethnicity between the cohorts. Finally, using the selective ABAT inhibitor vigabatrin (c-vinyl GABA) in a dog study, we were able to show a reduction of transient lower esophageal sphincter relaxations (TLESRs) by 57.3611.4 % (p = 0.007) and the reflux events from 3.160.4 to 0.860.4 (p = 0.007). Our results demonstrate the direct involvement of ABAT in pathways affecting lower esophageal sphincter (LES) control and identifies ABAT as a genetic risk factor for GERD.Johan Jirholt, Bengt Åsling, Paul Hammond, Geoffrey Davidson, Mikael Knutsson, Anna Walentinsson, Jörgen M. Jensen, Anders Lehmann, Lars Agreus and Maria Lagerström-Ferme

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.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

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.Comparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Novel genetic marker for dilated end stage oesophagus and oesophageal adenocarcinoma risk? Authors' response

B Åsling, J Jirholt, P Hammond, M Knutsson, A Walentinsson, G Davidson, L Agreus, A Lehmann, M Lagerström-Ferme

Analysis of genetic changes in rat endometrial carcinomas by means of comparative genome 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.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe