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.

A major quantitative trait locus influences hyperactivity in the WKHA rat.

The syndrome of hyperactivity describes behavioural disorders existing mainly in children and characterized by increased levels of motor activity, inattention and impulsivity. Overall the aetiology is poorly understood due to the heterogeneity of the pathology although psychological, biological and social factors acting singly or in concert are generally thought to be involved. In animal studies the observed hyperactivity phenotype results from relative participation of exploration, emotionality and general activity. Studies using brain lesions, neuropharmacology and gene knock-out strategies have shown that specific elements of the brain dopaminergic system can subserve hyperactivity. Evidence of a genetic contribution comes from family and twin studies but also from the ability to select divergent animal lines on the basis of their differential activity. The Wistar-Kyoto (WKY) and Wistar-Kyoto hyperactive (WKHA) rats are such strains--distinct for their low and high activity scores in a novel environment, respectively. Here, we report the detection of a major hyperactivity-related QTL on chromosome 8, explaining 29% of the variance of an intercross between these strains. This study represents the first behavioural QTL analysis in rat and provides a new starting point for biologically categorizing different forms of hyper-activity.

Chromosomal mapping of genetic loci associated with non-insulin dependent diabetes in the GK rat.

Goto-Kakizaki (GK) rats are a well characterized model for non-insulin dependent diabetes mellitus (NIDDM). We have used a combination of physiological and genetic studies to identify quantitative trait loci (QTLs) responsible for the control of glucose homeostasis and insulin secretion in a F2 cohort bred from spontaneously diabetic GK rats. The genetic dissection of NIDDM allowed us to map up to six independently segregating loci predisposing to hyperglycaemia, glucose intolerance or altered insulin secretion, and a seventh locus implicated in body weight. QTLs implicated in glucose tolerance and adiposity map to the same region of rat chromosome 1, and may indicate the influence of a single locus. Our study demonstrates that distinct combinations of genetic loci are responsible for different physiological characteristics associated with the diabetic phenotype in the GK rat, and it constitutes an important step for directing the search for the genetic factors involved in human NIDDM.

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.

Genetic mapping of two blood pressure quantitative trait loci on rat chromosome 1.

A genetic map for rat chromosome 1 was constructed using 66 microsatellite markers typed on either or both of two populations derived from inbred Dahl salt-sensitive (S) rats: F2(LEW x S) n = 151, and F2(WKY x S) n = 159. These populations had been raised on a high salt (8% NaCl) diet. Systolic blood pressure and heart weight were found to be genetically linked to two separate regions on rat chromosome 1 in the F2(LEW x S) population. One region was centered around the anonymous SA locus and accounted for 24 mmHg of blood pressure. The other region was 55 cM from the SA locus centered around a cluster of cytochromes P450 loci, and accounted for 30 mmHg of blood pressure. Since blood pressure and heart weight were highly correlated these same regions were also linked to heart weight. These results were cross-specific as linkage of these chromosome 1 regions to blood pressure and heart weight was not observed in several other F2 populations derived by crossing S and other normotensive control strains. This is presumably due to different alleles and/or different genetic backgrounds in the various populations. The SA region of chromosome 1 was found to influence body weight in F2(LEW x S) rats. Combining the present data with our previously published data on the F2(LEW x S) population showed that four separate quantitative trait loci with additive effects accounted for 106 mmHg and 38% of the total variance of blood pressure and for 506 mg and 34% of the total variance of heart wt.

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.

Allosteric properties of haemoglobin beta 41 (C7) Phe-->Tyr: a stable, low-oxygen-affinity variant synthesized in Escherichia coli.

In human deoxy haemoglobin, the alpha 42(C7)Tyr-residue is hydrogen-bonded to beta 99(G1)Asp which stabilizes the low-oxygen-affinity deoxy conformation. We engineered a haemoglobin with Tyr for Phe at the homologous C7 position in beta-chains. The oxygen affinity of the variant is decreased about two-fold relative to Hb A while keeping similar KR and KT values. This mutant may be a candidate for the development of an artificial oxygen carrier, as it would not require an external effector for significant oxygen unloading in vivo.

Impaired glucose homeostasis in adult rats from hyperglycemic mothers.

The purpose of our study was to investigate whether nondiabetic gestational hyperglycemia during fetal life could have additional effects on glucose homeostasis and insulin secretion in the adult rat. Hyperglycemia without the main other metabolic disorders and vascular injuries associated with diabetes was produced in unrestrained pregnant rats by continuous glucose infusion during the last week of pregnancy. Control rats were infused with distilled water. Compared with controls, the newborns from hyperglycemic rats were hyperglycemic and hyperinsulinemic. When studied longitudinally up to 3 mo, they showed slightly but significantly increased basal plasma glucose levels and normal basal insulin concentrations compared with controls. Glucose tolerance and insulin secretion in response to a glucose load (0.5 mg/kg, i.v.) were altered: Plasma glucose values were more increased at 5 min and remained higher 90 min after glucose injection; incremental plasma insulin values and the insulinogenic indexes (delta IRI/delta G) were always lower in rats from hyperglycemic mothers than in controls. These alterations were more and more marked with advancing age (1-3 mo). These data show that gestational hyperglycemia may lead to persistent impairment of glucose homeostasis and insulin secretion in the adult rat.

Insulin secretion in adult rats after intrauterine exposure to mild hyperglycemia during late gestation.

We investigated the effects of intrauterine mild hyperglycemia during late fetal life on glucose regulation and insulin secretion in adult rats. Unrestrained pregnant rats were continuously infused with glucose during the last week of pregnancy to induce mild hyperglycemia (6.5-8 mM). Control rats were infused with a glucose-free solution. The offspring were studied, as adults, from 1 to 20 mo by performing glucose tolerance and insulin secretion tests. Young-adult rats from hyperglycemic dams showed mild glucose intolerance and impairment of glucose-induced insulin secretion. This situation gradually evolved to basal hyperglycemia and severe impairment of glucose tolerance and insulin secretion. Insulin secretion was also studied in vitro in 20-mo-old rats with the isolated perfused-pancreas technique. Insulin release in response to glucose stimulation from pancreases of hyperglycemic dams was similar to that of controls, and the response to arginine was increased but not significantly. The possible involvement of enhanced sympathetic nervous system activity in the impairment of insulin secretion in adult rats from hyperglycemic mothers was then investigated by performing glucose tolerance and insulin secretion tests in the presence of the alpha 2-blocker idazoxan in 8-mo-old rats. Under these conditions, rats from hyperglycemic dams recovered almost normal glucose tolerance, and glucose-induced insulin secretion was greatly improved. These data show that mild hyperglycemia induced in the fetus during late pregnancy leads to persistent impairment of glucose regulation and insulin secretion. They suggest that the impairment of insulin secretion in vivo results from a perturbation of the neuroregulation of insulin secretion rather than an intrinsic pancreatic beta-cell defect.

Inheritance of diabetes mellitus as consequence of gestational hyperglycemia in rats.

Our study investigated whether a deterioration of glucose homeostasis and insulin secretion in adult female rats from hyperglycemic dams could be transmitted to the next generation independent of genetic interferences. Dams (F0) were rendered hyperglycemic by continuous glucose infusion during the last week of pregnancy. Females born of these rats (F1) exhibited glucose intolerance and impaired insulin secretion in vivo at adulthood. When they were 3 mo old, they were matched with males born of control dams. During pregnancy, their glucose tolerance remained impaired compared with that of controls. Consequently, F2 newborns of F1 hyperglycemic dams showed the main features of newborns from diabetic mothers: they were hyperglycemic, hyperinsulinemic, and macrosomic. As adults, they displayed basal hyperglycemia and defective glucose tolerance and insulin secretion. This indicates that the long-range deteriorating effects on glucose homeostasis of gestational hyperglycemia in the F1 generation are transmitted to the F2 generation and suggests that a perturbed fetal metabolic environment contributes to the inheritance of diabetes mellitus.

Steric and hydrophobic determinants of the solubilities of recombinant sickle cell hemoglobins.

Models for the structure of the fibers of deoxy sickle cell hemoglobin (Hb Hb S, beta 6 Glu-->Val) have been obtained from X-ray and electron microscopic studies. Recent molecular dynamics calculations of polymer formation give new insights on the various specific interactions between monomers. Site-directed mutagenesis with expression of the Hb S beta subunits in Escherichia coli provides the experimental tools to test these models. For Hb S, the beta 6 Val residue is intimately involved in a specific lateral contact, at the donor site, that interacts with the acceptor site of an adjacent molecule composed predominantly of the hydrophobic residues Phe 85 and Leu 88. Comparing natural and artificial mutants indicates that the solubility of deoxyHb decreases in relation to the surface hydrophobicity of the residue at the beta 6 position with Ile > Val > Ala. We also tested the role of the stereospecific adjustment between the donor and acceptor sites by substituting Trp for Glu at the beta 6 location. Among these hydrophobic substitutions and under our experimental conditions, only Val and Ile were observed to induce polymer formation. The interactions for the Ala mutant are too weak whereas a Trp residue inhibits aggregation through steric hindrance at the acceptor site of the lateral contact. Increasing the hydrophobicity at the axial contact between tetramers of the same strand also contributes to the stability of the double strand. This is demonstrated by associating the beta 23 Val-->Ile mutation at the axial contact with either the beta 6 Glu-->Val or beta 6 Glu-->Ile substitution in the same beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional consequences of mutations at the allosteric interface in hetero- and homo-hemoglobin tetramers.

A seminal difference exists between the two types of chains that constitute the tetrameric hemoglobin in vertebrates. While alpha chains associate weakly into dimers, beta chains self-associate into tightly assembled tetramers. While heterotetramers bind ligands cooperatively with moderate affinity, homotetramers bind ligands with high affinity and without cooperativity. These characteristics lead to the conclusion that the beta 4 tetramer is frozen in a quaternary R-state resembling that of liganded HbA. X-ray diffraction studies of the liganded beta 4 tetramers and molecular modeling calculations revealed several differences relative to the native heterotetramer at the "allosteric" interface (alpha 1 beta 2 in HbA) and possibly at the origin of a large instability of the hypothetical deoxy T-state of the beta 4 tetramer. We have studied natural and artificial Hb mutants at different sites in the beta chains responsible for the T-state conformation in deoxy HbA with the view of restoring a low ligand affinity with heme-heme interaction in homotetramers. Functional studies have been performed for oxygen equilibrium binding and kinetics after flash photolysis of CO for both hetero- and homotetramers. Our conclusion is that the "allosteric" interface is so precisely tailored for maintaining the assembly between alpha beta dimers that any change in the side chains of beta 40 (C6), beta 99 (G1), and beta 101 (G3) involved in the interface results in increased R-state behavior. In the homotetramer, the mutations at these sites lead to the destabilization of the beta 4 hemoglobin and the formation of lower affinity noncooperative monomers.

Enhanced insulin secretory response to acetylcholine by perifused pancreas of 5-day-old preobese Zucker rats.

The insulin secretion rate in response to different secretagogues and neurotransmittors was studied in perifused pancreas of 5-day-old lean (Fa/Fa) and preobese (fa/fa) Zucker rats. Glucose (16.6 mM) alone or in combination with 20 mM arginine or 5 mM theophylline induced a net stimulation of insulin secretion. This effect was similar in the two groups. By contrast, the stimulatory effect of acetylcholine on glucose-induced insulin secretion was significantly higher in preobese pups than in lean rats. There was also a tendency toward a higher inhibitory effect of norepinephrine on insulin secretion in preobese than in lean rats, but this difference did not reach statistical significance. Together these results demonstrate a normal insulin secretion in response to nutrient secretagogues in preobese fa/fa rats but an enhanced effect of acetylcholine. This latter effect may be related to the changes in the autonomic nervous system activity, which is usually described in obese fa/fa rats.

Analysis of quantitative trait loci for blood pressure on rat chromosomes 2 and 13. Age-related differences in effect.

Previous studies have suggested the presence of quantitative trait loci (QTLs) influencing blood pressure on rat chromosomes 2 and 13. In this study, we mapped the QTLs in F2 rats derived from a cross of the spontaneously hypertensive rat and the Wistar-Kyoto rat and analyzed the effect of the QTLs on blood pressures measured longitudinally between 12 and 25 weeks of age. We analyzed 16 polymorphic markers spanning 147.3 cM on chromosome 2 and 13 markers spanning 91.6 cM on chromosome 13. Both chromosomes contained QTLs with highly significant effects on blood pressure (peak logarithm of the odds [LOD] scores, 5.64 and 5.75, respectively). On chromosome 2, the peak was localized to a position at anonymous marker D2Wox7, 2.9 cM away from the gene for the sodium-potassium ATPase alpha 1-subunit. On chromosome 13, the major peak coincided with the marker D13Mit2, 21.7 cM away from the renin gene, but there was a suggestion of multiple peaks. The effect of the QTL on chromosome 2 was seen throughout from 12 to 25 weeks of age, whereas interestingly, the effect for the QTL on chromosome 13 was maximal at 20 weeks of age but disappeared at 25 weeks of age, presumably because of the effect of either epistatic factors or environmental influences. The findings provide important information on QTLs influencing blood pressure on rat chromosomes 2 and 13 that will be useful in localizing and identifying the causative genes and emphasize the importance of age being taken into account when the effects of individual QTLs on a trait that shows significant age-related changes are being analyzed.

Quantitative trait loci in genetically hypertensive rats. Possible sex specificity.

We performed a total genome screen in an F2 cross derived from the stroke-prone spontaneously hypertensive rat and the normotensive Wistar-Kyoto rat. Blood pressure at baseline and after 1% NaCl was measured by radiotelemetry; other phenotypes included heart rate, motor activity, left ventricle weight to body weight ratio, and vascular smooth muscle cell polyploidy, a measure of vascular hypertrophy. Quantitative trait loci affecting a given phenotype were mapped relative to microsatellite markers by using the MAPMAKER/QTL 1.1 computer package. We identified three blood pressure quantitative trait loci, two on rat chromosome 2 and one on rat chromosome 3. The quantitative trait loci close to genetic markers D2Mgh12 ("suggestive" linkage, with a maximal logarithm of the odds [LOD] score of 3.1) and D3Mgh16 (significant linkage, with a maximal LOD score of 5.6) showed possible sex specificity in the male F2 cohort only. This was confirmed by the likelihood ratio test for the difference in locus effects between the sexes. We also identified a new quantitative trait locus for LV hypertrophy on rat chromosome 14 ("suggestive" linkage, with a maximal LOD score of 3.1). The sex specificity of blood pressure quantitative trait loci will be important in designing congenic strains and substrains for fine genetic mapping and for identifying genes that regulate blood pressure.

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.

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.