Anne E. Kwitek

Progress and prospects in rat genetics: a community view

The rat is an important system for modeling human disease. Four years ago, the rich 150-year history of rat research was transformed by the sequencing of the rat genome, ushering in an era of exceptional opportunity for identifying genes and pathways underlying disease phenotypes. Genome-wide association studies in human populations have recently provided a direct approach for finding robust genetic associations in common diseases, but identifying the precise genes and their mechanisms of action remains problematic. In the context of significant progress in rat genomic resources over the past decade, we outline achievements in rat gene discovery to date, show how these findings have been translated to human disease, and document an increasing pace of discovery of new disease genes, pathways and mechanisms. Finally, we present a set of principles that justify continuing and strengthening genetic studies in the rat model, and further development of genomic infrastructure for rat research.

Rat genetics: attaching physiology and pharmacology to the genome.

During the past five years, the Rat Genome Project has been rapidly gaining momentum, especially since the announcement in August 2000 of plans to sequence the rat genome. Combined with the wealth of physiological and pharmacological data for the rat, the genome sequence should facilitate the discovery of mammalian genes that underlie the physiological pathways that are involved in disease. Most importantly, this combined physiological and genomic information should also lead to the development of better pre-clinical models of human disease, which will aid in the development of new therapeutics.

Rat genetics: attachign physiology and pharmacology to the genome

During the past five years, the Rat Genome Project has been rapidly gaining momentum, especially since the announcement in August 2000 of plans to sequence the rat genome. Combined with the wealth of physiological and pharmacological data for the rat, the genome sequence should facilitate the discovery of mammalian genes that underlie the physiological pathways that are involved in disease. Most importantly, this combined physiological and genomic information should also lead to the development of better pre-clinical models of human disease, which will aid in the development of new therapeutics.

Assessing unmodified 70-mer oligonucleotide probe performance on glass-slide microarrays

BackgroundLong oligonucleotide microarrays are potentially more cost- and management-efficient than cDNA microarrays, but there is little information on the relative performance of these two probe types. The feasibility of using unmodified oligonucleotides to accurately measure changes in gene expression is also unclear.ResultsUnmodified sense and antisense 70-mer oligonucleotides representing 75 known rat genes and 10 Arabidopsis control genes were synthesized, printed and UV cross-linked onto glass slides. Printed alongside were PCR-amplified cDNA clones corresponding to the same genes, enabling us to compare the two probe types simultaneously. Our study was designed to evaluate the mRNA profiles of heart and brain, along with Arabidopsis cRNA spiked into the labeling reaction at different relative copy number. Hybridization signal intensity did not correlate with probe type but depended on the extent of UV irradiation. To determine the effect of oligonucleotide concentration on hybridization signal, 70-mers were serially diluted. No significant change in gene-expression ratio or loss in hybridization signal was detected, even at the lowest concentration tested (6.25 μm). In many instances, signal intensity actually increased with decreasing concentration. The correlation coefficient between oligonucleotide and cDNA probes for identifying differentially expressed genes was 0.80, with an average coefficient of variation of 13.4%. Approximately 8% of the genes showed discordant results with the two probe types, and in each case the cDNA results were more accurate, as determined by real-time PCR.ConclusionsMicroarrays of UV cross-linked unmodified oligonucleotides provided sensitive and specific measurements for most of the genes studied.

The Rat Genome Database, update 2007—Easing the path from disease to data and back again

The Rat Genome Database (RGD, ) is one of the core resources for rat genomics and recent developments have focused on providing support for disease-based research using the rat model. Recognizing the importance of the rat as a disease model we have employed targeted curation strategies to curate genes, QTL and strain data for neurological and cardiovascular disease areas. This work has centered on rat but also includes data for mouse and human to create ‘disease portals’ that provide a unified view of the genes, QTL and strain models for these diseases across the three species. The disease curation efforts combined with normal curation activities have served to greatly increase the content of the database, particularly for biological information, including gene ontology, disease, pathway and phenotype ontology annotations. In addition to improving the features and database content, community outreach has been expanded to demonstrate how investigators can leverage the resources at RGD to facilitate their research and to elicit suggestions and needs for future developments. We have published a number of papers that provide additional information on the ontology annotations and the tools at RGD for data mining and analysis to better enable researchers to fully utilize the database.

Genome Sequencing Reveals Loci under Artificial Selection that Underlie Disease Phenotypes in the Laboratory Rat

Summary Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models. PaperClip

Clinical features and linkage analysis of a family with autosomal dominant juvenile glaucoma

BACKGROUND Juvenile glaucoma is an uncommon form of open-angle glaucoma that is usually recognized during childhood or early adulthood and which often has a strong family history. METHODS The authors clinically characterized a large multigeneration family with autosomal-dominant, juvenile-onset, open-angle glaucoma. Linkage analysis with short tandem repeat polymorphisms was used to evaluate the Riegers syndrome locus as the site of the disease-causing mutation. RESULTS Forty members of a family with a five-generation history of open-angle glaucoma were examined. Clinical data were available from an additional five individuals, three of whom were decreased. Older family members provided limited information about the visual history of five other deceased individuals in the first three generations. Fifty-nine people were at 50% risk of harboring the disease-causing mutation; and of these, 30 were affected with glaucoma by examination or by family history. All affected patients had an affected parent. The average age at diagnosis was 18 years (range, 8-30 years). Affected family members tended to be myopic but lacked other ocular or systemic abnormalities. The intraocular pressures (IOPs) of affected individuals were commonly more than 50 mmHg when they were first examined. Gonioscopy showed the angles to be open, with no abnormal pigmentation, iris processes, or embryonic tissue. Topical medications were initially effective in controlling IOP, but surgery was usually required for long-term pressure control. The Riegers syndrome locus on chromosome 4q25 was excluded as the site of the disease-causing mutation. CONCLUSION Juvenile open-angle glaucoma can occur as an autosomal dominant trait with high penetrance. Genetic linkage analysis of the family reported here has the potential to identify the chromosomal location of a glaucoma-causing gene. This gene is genetically distinct from the chromosome 4 locus that was recently associated with Riegers syndrome.

Chromosome substitution reveals the genetic basis of Dahl salt-sensitive hypertension and renal disease

This study examined the genetic basis of hypertension and renal disease in Dahl SS/Mcwi (Dahl Salt-Sensitive) rats using a complete chromosome substitution panel of consomic rats in which each of the 20 autosomes and the X and Y chromosomes were individually transferred from the Brown Norway (BN) rat onto the Dahl SS/Mcwi genetic background. Male and female rats of each of the two parental and 22 consomic strains (10-12 rats/group) were fed a high-salt (8.0% NaCl) diet for 3 wk. Mean arterial blood pressure rose by 60 mmHg and urinary protein and albumin excretion increased 3- and 20-fold, respectively, in male SS/Mcwi rats compared with BN controls. Substitution of chromosomes 1, 5, 7, 8, 13, or 18 from the BN onto the SS/Mcwi background attenuated the development of hypertension, proteinuria, and albuminuria in male rats. In female rats, substitution of chromosomes 1 and 5 also decreased blood pressure, protein excretion, and albumin excretion. These studies also identified several chromosomes in male (6, 11, Y) and female (4, 6, 11, 19, 20) rats that reduced albuminuria without altering blood pressure. These data indicate that genes contributing to salt-sensitive hypertension are found on multiple chromosomes of the Dahl SS/Mcwi rat. Furthermore, this consomic rat panel provides a stable genetic platform that can facilitate further gene mapping by either linkage studies or the breeding of congenic and subcongenic rats.

The Rat Genome Database (RGD): developments towards a phenome database

The Rat Genome Database (RGD) (http://rgd.mcw.edu) aims to meet the needs of its community by providing genetic and genomic infrastructure while also annotating the strengths of rat research: biochemistry, nutrition, pharmacology and physiology. Here, we report on RGDs development towards creating a phenome database. Recent developments can be categorized into three groups. (i) Improved data collection and integration to match increased volume and biological scope of research. (ii) Knowledge representation augmented by the implementation of a new ontology and annotation system. (iii) The addition of quantitative trait loci data, from rat, mouse and human to our advanced comparative genomics tools, as well as the creation of new, and enhancement of existing, tools to enable users to efficiently browse and survey research data. The emphasis is on helping researchers find genes responsible for disease through the use of rat models. These improvements, combined with the genomic sequence of the rat, have led to a successful year at RGD with over two million page accesses that represent an over 4-fold increase in a year. Future plans call for increased annotation of biological information on the rat elucidated through its use as a model for human pathobiology. The continued development of toolsets will facilitate integration of these data into the context of rat genomic sequence, as well as allow comparisons of biological and genomic data with the human genomic sequence and of an increasing number of organisms.

Evidence of a Functional Role for Mast Cells in the Development of Type 1 Diabetes Mellitus in the BioBreeding Rat

Human type 1 diabetes mellitus (T1DM) arises through autoimmune destruction of pancreatic β cells and is modeled in many respects by the lymphopenic and spontaneously diabetic BioBreeding (BB) DRlyp/lyp rat. Previously, preonset expression profiling of whole DRlyp/lyp pancreatic lymph nodes (PLN) revealed innate immune activity, specifically that of mast cells and eosinophils. Furthermore, we observed that pancreatic islets of DRlyp/lyp rats as well as those of diabetes-inducible BB DR+/+ rats potentially recruit innate cells through eotaxin expression. Here we determine that lifelong eotaxin expression begins before 40 days of life and is localized specifically to β cells. In this report, we find that PLN mast cells are more abundant in DRlyp/lyp compared with related BB DR+/+ rats (2.1 ± 0.9% vs 0.9 ± 0.4% of total cells, p < 0.0001). DRlyp/lyp PLN mast cell gene expression profiling revealed an activated population and included significant overrepresentation of transcripts for mast cell protease 1, cationic trypsinogen, carboxypeptidase A, IL-5, and phospholipase Cγ. In the DR+/+ rat, which develops T1DM upon depletion of T regulator cells, mast cells displayed gene expression consistent with the negative regulation of degranulation, including significant overrepresentation of transcripts encoding tyrosine phosphatase SHP-1, lipid phosphatase SHIP, and E3 ubiquitin ligase c-Cbl. To recapitulate the negative mast cell regulation observed in the DR+/+ rats, we treated DRlyp/lyp rats with the mast cell “stabilizer” cromolyn, which significantly (p < 0.05) delayed T1DM onset. These findings are consistent with a growing body of evidence in human and animal models, where a role for mast cells in the initiation and progression of autoimmune disease is emerging.