17q21.31 duplication causes prominent tau-related dementia with increased MAPT expression.

To assess the role of rare copy number variations in Alzheimer's disease (AD), we conducted a case-control study using whole-exome sequencing data from 522 early-onset cases and 584 controls. The most recurrent rearrangement was a 17q21.31 microduplication, overlapping the CRHR1, MAPT, STH and KANSL1 genes that was found in four cases, including one de novo rearrangement, and was absent in controls. The increased MAPT gene dosage led to a 1.6-1.9-fold expression of the MAPT messenger RNA. Clinical signs, neuroimaging and cerebrospinal fluid biomarker profiles were consistent with an AD diagnosis in MAPT duplication carriers. However, amyloid positon emission tomography (PET) imaging, performed in three patients, was negative. Analysis of an additional case with neuropathological examination confirmed that the MAPT duplication causes a complex tauopathy, including prominent neurofibrillary tangle pathology in the medial temporal lobe without amyloid-ß deposits. 17q21.31 duplication is the genetic basis of a novel entity marked by prominent tauopathy, leading to early-onset dementia with an AD clinical phenotype. This entity could account for a proportion of probable AD cases with negative amyloid PET imaging recently identified in large clinical series.

Meta-analysis of 74,046 individuals identifies 11 new susceptibility loci for Alzheimer's disease.

Eleven susceptibility loci for late-onset Alzheimer's disease (LOAD) were identified by previous studies; however, a large portion of the genetic risk for this disease remains unexplained. We conducted a large, two-stage meta-analysis of genome-wide association studies (GWAS) in individuals of European ancestry. In stage 1, we used genotyped and imputed data (7,055,881 SNPs) to perform meta-analysis on 4 previously published GWAS data sets consisting of 17,008 Alzheimer's disease cases and 37,154 controls. In stage 2, 11,632 SNPs were genotyped and tested for association in an independent set of 8,572 Alzheimer's disease cases and 11,312 controls. In addition to the APOE locus (encoding apolipoprotein E), 19 loci reached genome-wide significance (P < 5 × 10(-8)) in the combined stage 1 and stage 2 analysis, of which 11 are newly associated with Alzheimer's disease.

Subtle metabolic and liver gene transcriptional changes underlie diet-induced fatty liver susceptibility in insulin-resistant mice.

AIMS/HYPOTHESIS: Complex changes in gene expression are associated with insulin resistance and non-alcoholic fatty liver disease (NAFLD) promoted by feeding a high-fat diet (HFD). We used functional genomic technologies to document molecular mechanisms associated with diet-induced NAFLD. MATERIALS AND METHODS: Male 129S6 mice were fed a diet containing 40% fat (high-fat diet, HFD) for 15 weeks. Glucose tolerance, in vivo insulin secretion, plasma lipid profile and adiposity were determined. Plasma metabonomics and liver transcriptomics were used to identify changes in gene expression associated with HFD-induced NAFLD. RESULTS: In HFD-fed mice, NAFLD and impaired glucose and lipid homeostasis were associated with increased hepatic transcription of genes involved in fatty acid uptake, intracellular transport, modification and elongation, whilst genes involved in beta-oxidation and lipoprotein secretion were, paradoxically, also upregulated. NAFLD developed despite strong and sustained downregulation of transcription of the gene encoding stearoyl-coenzyme A desaturase 1 (Scd1) and uncoordinated regulation of transcription of Scd1 and the gene encoding sterol regulatory element binding factor 1c (Srebf1c) transcription. Inflammatory mechanisms appeared to be stimulated by HFD. CONCLUSIONS/INTERPRETATION: Our results provide an accurate representation of subtle changes in metabolic and gene expression regulation underlying disease-promoting and compensatory mechanisms, collectively contributing to diet-induced insulin resistance and NAFLD. They suggest that proposed models of NAFLD pathogenesis can be enriched with novel diet-reactive genes and disease mechanisms.

No effect of APOE and PVRL2 on the clinical outcome of multiple sclerosis.

Multiple sclerosis (MS) is a common inflammatory disease of the central nervous system unsurpassed for its variability in disease outcome. Apolipoprotein E (APOE) is involved in neuronal remodelling and several studies have attempted to examine the effect of APOE on MS disease severity, but its function in modifying the course of MS is controversial. It has been suggested recently that PVRL2, not APOE, is the locus on chromosome 19 which influences clinical outcome of MS. A cohort of sporadic MS cases, taken from opposite extremes of the putative distribution of long-term outcome using the most stringent clinical criteria to date, was used to determine the role of APOE and PVRL2 on MS disease severity. The MS cases selected represent the prognostic best 5% (benign MS) and worst 5% (malignant MS) of cases in terms of clinical outcome assessed by the EDSS. Genotyping the two sets of MS patients (112 benign and 51 malignant) and a replication cohort from Sardinia provided no evidence to suggest that APOE or PVRL2 have any outcome modifying activity. We conclude that APOE and PVRL2 have little or no effect on the clinical outcome of MS.

Genetic control of plasma lipid levels in a cross derived from normoglycaemic Brown Norway and spontaneously diabetic Goto-Kakizaki rats.

AIMS/HYPOTHESIS: Dyslipidaemia is a main component of the insulin resistance syndrome. The inbred Goto-Kakizaki (GK) rat is a model of spontaneous type 2 diabetes and insulin resistance, which has been used to identify diabetes-related susceptibility loci in genetic crosses. The objective of our study was to test the genetic control of lipid metabolism in the GK rat and investigate a possible relationship with known genetic loci regulating glucose homeostasis in this strain. MATERIALS AND METHODS: Plasma concentration of triglycerides, phospholipids, total cholesterol, HDL, LDL and VLDL cholesterol were determined in a cohort of 151 hybrids of an F2 cross derived from GK and non-diabetic Brown Norway (BN) rats. Data from the genome-wide scan of the F2 hybrids were used to test for evidence of genetic linkage to the lipid quantitative traits. RESULTS: We identified statistically significant quantitative trait loci (QTLs) that control the level of plasma phospholipids and triglycerides (chromosome 1), LDL cholesterol (chromosome 3) and total and HDL cholesterol (chromosomes 1 and 5). These QTLs do not coincide with previously identified diabetes susceptibility loci in a similar cross. The significance of lipid QTLs mapped to chromosomes 1 and 5 is strongly influenced by sex. CONCLUSION/INTERPRETATION: We established that several genetic loci control the quantitative variations of plasma lipid variables in a GKxBN cross. They appear to be distinct from known GK diabetes QTLs, indicating that lipid metabolism and traits directly relevant to glucose and insulin regulation are controlled by different gene variants in this strain combination.

Enhanced insulin secretion and cholesterol metabolism in congenic strains of the spontaneously diabetic (Type 2) Goto Kakizaki rat are controlled by independent genetic loci in rat chromosome 8.

AIMS/HYPOTHESIS: Genetic investigations in the spontaneously diabetic (Type 2) Goto Kakizaki (GK) rat have identified quantitative trait loci (QTL) for diabetes-related phenotypes. The aims of this study were to refine the chromosomal mapping of a QTL ( Nidd/gk5) identified in chromosome 8 of the GK rat and to define a pathophysiological profile of GK gene variants underlying the QTL effects in congenics. METHODS: Genetic linkage analysis was carried out with chromosome 8 markers genotyped in a GKxBN F2 intercross previously used to map diabetes QTL. Two congenic strains were designed to contain GK haplotypes in the region of Nidd/gk5 transferred onto a Brown Norway (BN) genetic background, and a broad spectrum of diabetes phenotypes were characterised in the animals. RESULTS: Results from QTL mapping suggest that variations in glucose-stimulated insulin secretion in vivo, and in body weight are controlled by different chromosome 8 loci (LOD3.53; p=0.0004 and LOD4.19; p=0.00007, respectively). Extensive physiological screening in male and female congenics at 12 and 24 weeks revealed the existence of GK variants at the locus Nidd/gk5, independently responsible for significantly enhanced insulin secretion and increased levels of plasma triglycerides, phospholipids and HDL, LDL and total cholesterol. Sequence polymorphisms detected between the BN and GK strains in genes encoding ApoAI, AIV, CIII and Lipc do not account for these effects. CONCLUSIONS/INTERPRETATION: We refined the localisation of the QTL Nidd/gk5 and its pathophysiological characteristics in congenic strains derived for the locus. These congenic strains provide novel models for testing the contribution of a subset of GK alleles on diabetes phenotypes and for identifying diabetes susceptibility genes.

Genetic dissection of region around the Sa gene on rat chromosome 1: evidence for multiple loci affecting blood pressure.

A region with a major effect on blood pressure (BP) is located on rat chromosome 1 in the vicinity of the Sa gene, a candidate gene for BP regulation. Previously, we observed a single linkage peak for BP in this region in second filial generation rats derived from a cross of the spontaneously hypertensive rat (SHR) with the Wistar-Kyoto rat (WKY), and we have reported the isolation of the region containing the BP effect in reciprocal congenic strains (WKY.SHR-Sa) and (SHR.WKY-Sa) derived from these animals. Here, we report the further genetic dissection of this region. Two congenic substrains each were derived from WKY.SHR-Sa (WISA1 and WISA2) and SHR.WKY-Sa (SISA1 and SISA2) by backcrossing to WKY and SHR, respectively. Although there was some overlap of the introgressed regions retained in the various substrains, the segments in WISA1 and SISA1 did not overlap. Furthermore, although the Sa allele in WISA1, WISA2, and SISA2 remained donor in origin, recombination in SISA1 reverted it back to the recipient (SHR) allele. Surprisingly, all 4 substrains demonstrated a highly significant BP difference compared with that of their respective parental strain, which was of a magnitude similar to those seen in the original congenic strains. The findings strongly indicate that there are at least 2 quantitative trait loci (QTLs) affecting BP in this region of rat chromosome 1. Furthermore, the BP effect seen in SISA1 indicates that at least a proportion of the BP effect of this region of rat chromosome 1 cannot be due to the Sa gene. SISA1 contains an introgressed segment of <3 cM, and this will facilitate the physical mapping of the BP QTL(s) located within it and the identification of the susceptibility-conferring genes. Our observations serve to illustrate the complexity of QTL dissection and the care needed to interpret findings from congenic studies.

A high-resolution consensus linkage map of the rat, integrating radiation hybrid and genetic maps.

We have constructed a high-resolution consensus genetic map of the rat in a single large intercross, which integrates 747 framework markers and 687 positions of our whole-genome radiation hybrid (RH) map of the rat. We selected 136 new gene markers from the GenBank database and assigned them either genetically or physically to rat chromosomes to evaluate the accuracy of the integrated linkage-RH maps in the localization of new markers and to enrich existing comparative mapping data. These markers and 631 D-Got- markers, which are physically mapped but still uncharacterized for evidence of polymorphism, were tested for allele variations in a panel of 16 rat strains commonly used in genetic studies. The consensus linkage map constructed in the GK x BN cross now comprises 1620 markers of various origins, defining 840 resolved genetic positions with an average spacing of 2.2 cM between adjacent loci, and includes 407 gene markers. This whole-genome genetic map will contribute to the advancement of genetic studies in the rat by incorporating gene/EST maps, physical mapping information, and sequence data generated in rat and other mammalian species into genetic intervals harboring disease susceptibility loci identified in rat models of human genetic disorders.

Homozygous deletion in the coding sequence of the c-mer gene in RCS rats unravels general mechanisms of physiological cell adhesion and apoptosis.

The RCS rat presents an autosomal recessive retinal pigment epithelium dystrophy characterized by the outer segments of photoreceptors being phagocytosis-deficient. A systematic genetic study allowed us to restrict the interval containing the rdy locus to that between the markers D3Mit13 and D3Rat256. We report the chromosomal localization of the rat c-mer gene in the cytogenetic bands 3q35-36, based on genetic analysis and radiation hybrid mapping. Using a systematic biocomputing analysis, we identified two strong related candidate genes encoding protein tyrosine kinase receptors of the AXL subfamily. The comparison of their expression patterns in human and mice tissues suggested that the c-mer gene was the best gene to screen for mutations. RCS rdy- and RCS rdy+ cDNAs were sequenced. The RCS rdy- cDNAs carried a significant deletion in the 5' part of the coding sequence of the c-mer gene resulting in a shortened aberrant transcript encoding a 20 amino acid peptide. The c-mer gene contains characteristic motifs of neural cell adhesion. A ligand of the c-mer receptor, Gas6, exhibits antiapoptotic properties.

Genetic mapping of blood pressure quantitative trait loci in Milan hypertensive rats.

In a previous study, by using a candidate gene approach, we detected in both Milan hypertensive rats and humans a polymorphism in the alpha-adducin gene (ADD1) that was associated with blood pressure and renal sodium handling. In the present study, a genomewide search with 264 informative markers was undertaken in 251 (Milan hypertensive strain x Milan normotensive strain) F2 rats to further investigate the contribution of the adducin gene family (Add1, Add2, and Add3) and to identify novel quantitative trait loci (QTLs) that affect blood pressure. The influence of 2 different methods of blood pressure measurement, the intracarotid catheter and the tail-cuff method, was also evaluated. We found evidence that QTLs affected systolic blood pressure (SBP) measured at the carotid (direct SBP) on rat chromosome 1 with a logarithm of the odds (LOD) score peak of 3.3 on D1Rat121 and on rat chromosome 14 on Add1 locus (LOD=3.2). A QTL for SBP measured at the tail (indirect SBP) was found on rat chromosome 10 around D10Rat33 (LOD=5.0). All of these QTLs identified chromosomal regions not detected in other rat studies and harbor genes (Na(+)/H(+) exchanger A3; alpha-adducin; alpha(1B)-adrenergic receptor) that may be involved in blood pressure regulation. Therefore, these findings may be relevant to human hypertension, also in consideration of the biochemical and pathophysiological similarities between MHS and a subgroup of patients of primary hypertension, which led to the identification of alpha-adducin as a candidate gene in both species.

High-throughput scanning of the rat genome using interspersed repetitive sequence-PCR markers.

We report the establishment of a hybridization-based marker system for the rat genome based on the PCR amplification of interspersed repetitive sequences (IRS). Overall, 351 IRS markers were mapped within the rat genome. The IRS marker panel consists of 210 nonpolymorphic and 141 polymorphic markers that were screened for presence/absence polymorphism patterns in 38 different rat strains and substrains that are commonly used in biomedical research. The IRS marker panel was demonstrated to be useful for rapid genome screening in experimental rat crosses and high-throughput characterization of large-insert genomic library clones. Information on corresponding YAC clones is made available for this IRS marker set distributed over the whole rat genome. The two existing rat radiation hybrid maps were integrated by placing the IRS markers in both maps. The genetic and physical mapping data presented provide substantial information for ongoing positional cloning projects in the rat.

Characterization of newly developed SSLP markers for the rat.

We have isolated more than 12,000 clones containing microsatellite sequences, mainly consisting of (CA)n dinucleotide repeats, using genomic DNA from the BN strain of laboratory rat. Data trimming yielded 9636 non-redundant microsatellite sequences, and we designed oligonucleotide primer pairs to amplify 8189 of these. PCR amplification of genomic DNA from five different rat strains yielded clean amplification products for 7040 of these simple-sequence-length-polymorphism (SSLP) markers; 3019 markers had been mapped previously by radiation hybrid (RH) mapping methods (Nat Genet 22, 27-36, 1998). Here we report the characterization of these newly developed microsatellite markers as well as the release of previously unpublished microsatellite marker information. In addition, we have constructed a genome-wide linkage map of 515 markers, 204 of which are derived from our new collection, by genotyping 48 F2 progeny of (OLETFxBN)F2 crosses. This map spans 1830.9 cM, with an average spacing of 3.56 cM. Together with our ongoing project of preparing a whole-genome radiation hybrid map for the rat, this dense linkage map should provide a valuable resource for genetic studies in this model species.

Detailed comparative gene map of rat chromosome 1 with mouse and human genomes and physical mapping of an evolutionary chromosomal breakpoint.

We report the localization of 92 new gene-based markers assigned to rat chromosome 1 by linkage or radiation hybrid mapping. The markers were chosen to enrich gene mapping data in a region of the rat chromosome known to contain several of the principal quantitative trait loci in rodent models of human multifactorial disease. The composite map reported here provides map information on a total of 139 known genes, including 80 that have been localized in mouse and 109 that have been localized in human, and integrates the gene-based markers with anonymous microsatellites. The evolutionary breakpoints identifying 16 segments that are homologous regions in the human genome are defined. These data will facilitate genetic and comparative mapping studies and identification of novel candidate genes for the quantitative trait loci that have been localized to the region.

A gene map of the rat derived from linkage analysis and related regions in the mouse and human genomes.

We report the localization by linkage analysis in the rat genome of 148 new markers derived from 128 distinct known gene sequences, ESTs, and anonymous sequences selected in GenBank database on the basis of the presence of a repeated element. The composite linkage map of the rat contributed by our group integrates mapping information on a total of 370 different known genes, ESTs, and anonymous mouse or human sequences, and provides a valuable tool for comparative genome analysis. 206 and 254 homologous loci were identified in the mouse and human genomes respectively. Our linkage map, which combines both anonymous markers and gene markers, should facilitate the advancement of genetic studies for a wide variety of rat models characterized for complete phenotypes. The comparative genome mapping should define genetic regions in human likely to be homologous to susceptibility loci identified in rat and provide useful information for the identification of new potential candidates for genetic disorders.

A radiation hybrid map of the rat genome containing 5,255 markers.

A whole-genome radiation hybrid (RH) panel was used to construct a high-resolution map of the rat genome based on microsatellite and gene markers. These include 3,019 new microsatellite markers described here for the first time and 1,714 microsatellite markers with known genetic locations, allowing comparison and integration of maps from different sources. A robust RH framework map containing 1,030 positions ordered with odds of at least 1,000:1 has been defined as a tool for mapping these markers, and for future RH mapping in the rat. More than 500 genes which have been mapped in mouse and/or human were localized with respect to the rat RH framework, allowing the construction of detailed rat-mouse and rat-human comparative maps and illustrating the power of the RH approach for comparative mapping.

Complete genome searches for quantitative trait loci controlling blood pressure and related traits in four segregating populations derived from Dahl hypertensive rats.

The Dahl salt-sensitive rat is one of the principal animal models of hereditary hypertension. Genome-wide searches were undertaken to detect quantitative trait loci (QTLs) that influence blood pressure, cardiac mass, and body weight in four F2 populations derived from Dahl salt-sensitive rats and different inbred normotensive control strains of rat. We detected three QTLs associated with one or more of the phenotypes, using a stringent statistical criterion for linkage (p < 0.00003). These included a novel QTL linked to blood pressure on rat Chromosome (Chr) 12, and another QTL on rat Chr 3 linked to body weight. A QTL on rat Chr 10 for which linkage to blood pressure has been described in other crosses was found to be a principal determinant of blood pressure and cardiac mass in some but not all of the crosses examined here. Three other regions showed evidence of linkage to these phenotypes with a less stringent statistical criterion of linkage at QTLs previously reported in other studies. As part of our study, microsatellite markers have been developed for three candidate genes for investigation in hypertension, and the genes have been localized by linkage mapping. These are: the rat Gs alpha subunit (Gnas) gene, the alpha-1B adrenergic receptor (Adra1b), and the Na+, K+-ATPase beta2 subunit (Atp1b2) gene.

An improved linkage map of rat chromosome 3 with three mapping panels.

Our purposes were to develop an improved linkage map for rat Chromosome 3 and to develop new markers polymorphic between Dahl salt-sensitive (S) and Dahl salt-resistant (R) rats. The linkage mapping panel consisted of three F2 populations totaling 359 rats. Twenty-five new markers were developed and placed on the linkage map. About half of these markers (13) were polymorphic between S and R rats. The final map spans 124.7 centiMorgans (cM) and includes 64 markers. The average distance between adjacent markers is 1.9 cM, and the largest separation is 10.5 cM.