Predicting stroke through genetic risk functions: the CHARGE Risk Score Project.

BACKGROUND AND PURPOSE: Beyond the Framingham Stroke Risk Score, prediction of future stroke may improve with a genetic risk score (GRS) based on single-nucleotide polymorphisms associated with stroke and its risk factors. METHODS: The study includes 4 population-based cohorts with 2047 first incident strokes from 22,720 initially stroke-free European origin participants aged ≥55 years, who were followed for up to 20 years. GRSs were constructed with 324 single-nucleotide polymorphisms implicated in stroke and 9 risk factors. The association of the GRS to first incident stroke was tested using Cox regression; the GRS predictive properties were assessed with area under the curve statistics comparing the GRS with age and sex, Framingham Stroke Risk Score models, and reclassification statistics. These analyses were performed per cohort and in a meta-analysis of pooled data. Replication was sought in a case-control study of ischemic stroke. RESULTS: In the meta-analysis, adding the GRS to the Framingham Stroke Risk Score, age and sex model resulted in a significant improvement in discrimination (all stroke: Δjoint area under the curve=0.016, P=2.3×10(-6); ischemic stroke: Δjoint area under the curve=0.021, P=3.7×10(-7)), although the overall area under the curve remained low. In all the studies, there was a highly significantly improved net reclassification index (P<10(-4)). CONCLUSIONS: The single-nucleotide polymorphisms associated with stroke and its risk factors result only in a small improvement in prediction of future stroke compared with the classical epidemiological risk factors for stroke.

Simultaneous identification of multiple mammalian species from mixed forensic samples based on mtDNA control region length polymorphism

Molecular species identification in mixed or contaminated biological material has always been problematic. We developed a simple and accurate method for mammal DNA identification in mixtures, based on interspecific mitochondrial DNA control region length polymorphism. Contrary to other published methods dealing with species mixtures, our protocol requires a single universal primer pair and amplification step, and is not based on a pre-defined panel of species. This protocol has been routinely employed by our laboratory for species identification in dozens of human and animal forensic caseworks. Six representative forensic caseworks involving the specific identification of mixed animal samples are reported in this paper, in order to demonstrate the applicability and usefulness of the method.

Dramatic influence of V beta gene polymorphism on an antigen-specific CD8+ T cell response in vivo.

According to recent crystallographic studies, the TCR-alpha beta contacts MHC class I-bound antigenic peptides via the polymorphic V gene-encoded complementarity-determining region 1 beta (CDR1 beta) and the hypervariable (D)J-encoded CDR3 beta and CDR3 alpha domains. To evaluate directly the relative importance of CDR1 beta polymorphism on the fine specificity of T cell responses in vivo, we have taken advantage of congenic V beta a and V beta b mouse strains that differ by a CDR1 polymorphism in the V beta 10 gene segment. The V beta 10-restricted CD8+ T cell response to a defined immunodominant epitope was dramatically reduced in V beta a compared with V beta b mice, as measured either by the expansion of V beta 10+ cells or by the binding of MHC-peptide tetramers. These data indicate that V beta polymorphism has an important impact on TCR-ligand binding in vivo, presumably by modifying the affinity of CDR1 beta-peptide interactions.

Single Subject Finger Somatotopy Mapping at 7T.

Introduction: The primary somatosensory cortex (SI) contains Brodmann areas (BA) 1, 2, 3a, and 3b. Research in non-human primates showed that BAs 3b, 1, and 2 each contain one full representation of the hand with separate representations for each finger. This research also showed that the finger representation in BA3b has larger and clearer finger somatotopy than BA1 and 2. Although several efforts to map finger somatotopy in SI by fMRI have been made at 1.5 and 3T these studies have yielded variable results and were not able to detect single subject finger somatotopy, probably due to the limited spatial extent of the cortical areas representing a digit (close to the resolution in most fMRI experiments), complications due to acquisition of consistent maps for individual subjects (Schweizer et al 2008), or inter-individual variability in sulcal anatomy impeding group studies. Here, we used 7T fMRI to investigate finger somatotopy in SI, some of its functional characteristics, and its reproducibility. Methods: Eight right-handed male subjects were scanned on a 7T scanner (Siemens Medical, Germany) with an 8-channel Tx/Rx rf-coil (Rapid Biomedical, Germany). 1.3x1.3x1.3mm3 resolution fMRI data were acquired using a sinusoidal readout EPI sequence (Speck et al, 2008) and FOV=210mm, TE/TR=27ms/2.5s, GRAPPA=2. Each volume contained 28 transverse slices covering SI. A single EPI volume with 64 slices was acquired to aid coregistration. 1x1x1mm3 anatomical data were acquire using the MP2RAGE sequence (Marques et al, 2009; TE/TR/TI1,2/TRmprage=2.63ms/7.2ms/0.9,3.2s/5s). Subjects were positioned supine in the scanner with their right arm comfortably against the magnet bore. An experimenter was positioned at the entrance of the bore where he could easily reach and stroke successively the two distal phalanxes of each digit. The order of stroked digit was D1 (thumb)-D3-D5-D2-D4, with 20s ON, 10s OFF alternated. This sequence was repeated four times per run and two functional runs were acquired per subject. Realignment, smoothing (FWHM 2 mm), coregistration of the anatomical to the fMRI data and calculation of t-statistics were done using SPM8. An SI mask was obtained via an F-contrast (p<0.001) over all digits. Within the mask, voxels were labeled with the number of the digit demonstrating the highest t-value for that particular voxel. Results: For all subjects, areas corresponding to the five digits were identified in contralateral SI. BA3b showed the most consistent somatotopic finger representation (see an example in Fig.1). The five digits were localized in a consecutive order in the cortex, with D1 most anterior, inferior and distal and D5, most posterior, superior and medial (mean distance between centres of mass of digit representations ±stderr: 4.2±0.7mm; see Fig. 2). The analysis of average beta values within each finger representation region revealed the specificity of the somatotopic region to the tactile input for each tested finger (except digit 4 and 5). Five of these subjects also presented an orderly and consecutive representation of the five digits in BA1 and 2. Conclusions: Our data reveal that the increased BOLD sensitivity at 7T and the high spatial resolution used in this study allow consistent somatotopic mapping using human touch as a stimulus and that human SI contains at least three separate regions that contain five separate representations of all single contralateral fingers. Moreover, adjacent fingers were represented at adjacent cortical regions across the three SI regions. The spatial organization of SI as reflected in individual subject topography corresponds well with previous electrophysiological data in non-human primates. The small distance between digit representations highlights the need for the high spatial resolution available at 7T.

Evidence against close linkage of the loci for fraXq of Martin-Bell syndrome and for factor IX.

Linkage between the loci for fraXq of Martin-Bell syndrome and factor IX was studied in nine families exhibiting this syndrome by means of a restriction fragment length polymorphism at the factor IX locus. Computer analysis of the data indicates there to be no evidence for close linkage between the syndrome and the factor IX locus.

Deviant trajectories of cortical maturation in 22q11.2 deletion syndrome (22q11DS): a cross-sectional and longitudinal study.

22q11.2 deletion syndrome (22q11DS) is associated with an increased susceptibility to develop schizophrenia. Despite a large body of literature documenting abnormal brain structure in 22q11DS, cerebral changes associated with brain maturation in 22q11DS remained largely unexplored. To map cortical maturation from childhood to adulthood in 22q11.2 deletion syndrome, we used cerebral MRI from 59 patients with 22q11DS, aged 6 to 40, and 80 typically developing controls; three year follow-up assessments were also available for 32 patients and 31 matched controls. Cross-sectional cortical thickness trajectories during childhood and adolescence were approximated in age bins. Repeated-measures were also conducted with the longitudinal data. Within the group of patients with 22q11DS, exploratory measures of cortical thickness differences related to COMT polymorphism, IQ, and schizophrenia were also conducted. We observed deviant trajectories of cortical thickness changes with age in patients with 22q11DS. In affected preadolescents, larger prefrontal thickness was observed compared to age-matched controls. Afterward, we observed greater cortical loss in 22q11DS with a convergence of cortical thickness values by the end of adolescence. No compelling evidence for an effect of COMT polymorphism on cortical maturation was observed. Within 22q11DS, significant differences in cortical thickness were related to cognitive level in children and adolescents, and to schizophrenia in adults. Deviant trajectories of cortical thickness from childhood to adulthood provide strong in vivo cues for a defect in the programmed synaptic elimination, which in turn may explain the susceptibility of patients with 22q11DS to develop psychosis.

Mutation hot spots in 5q31-linked corneal dystrophies.

Mutations in the BIGH3 gene on chromosome 5q31 cause four distinct autosomal dominant diseases of the human cornea: granular (Groenouw type I), Reis-Bücklers, lattice type I, and Avellino corneal dystrophies. All four diseases are characterized by both progressive accumulation of corneal deposits and eventual loss of vision. We have identified a specific recurrent missense mutation for each type of dystrophy, in 10 independently ascertained families. Genotype analysis with microsatellite markers surrounding the BIGH3 locus was performed in these 10 families and in 5 families reported previously. The affected haplotype could be determined in 10 of the 15 families and was different in each family. These data indicate that R555W, R124C, and R124H mutations occurred independently in several ethnic groups and that these mutations do not reflect a putative founder effect. Furthermore, this study confirms the specific importance of the R124 and R555 amino acids in the pathogenesis of autosomal dominant corneal dystrophies linked to 5q.

Extra-pair paternity, testes size and testosterone level in relation to colour polymorphism in the barn owl Tyto alba

In many bird populations, individuals display one of several genetically inherited colour morphs. Colour polymorphism can be maintained by several mechanisms one of which being frequency-dependent selection with colour morphs signalling alternative mating strategies. One morph may be dominant and territorial, and another one adopt a sneaky behaviour to gain access to fertile females. We tested this hypothesis in the barn owl Tyto alba in which coloration varies from reddish-brown to white. This trait is heritable and neither sensitive to the environment in which individuals live nor to body condition. In Switzerland, reddish-brown males were observed to feed their brood at a higher rate and to produce more offspring than white males. This observation lead us to hypothesize that white males may equalise fitness by investing more effort in extra-pair copulations. This hypothesis predicts that lighter Coloured males produce more extra-pair young, have larger testes and higher levels of circulating testosterone. However, our results are not consistent with these three predictions. First, paternity analyses of 54 broods with a total of 211 offspring revealed that only one young was not sired by the male that was feeding it. Second, testes size was not correlated with male plumage coloration suggesting that white males are not sexually more active. Finally, in nestlings at the time of feather growth testosterone level was not related to plumage coloration suggesting that this androgen is not required for the expression of this plumage trait. Our study therefore indicates that in the barn owl colour polymorphism plays no role in the probability of producing extra-pair young.

Common variants in the ATP2B1 gene are associated with susceptibility to hypertension: the Japanese Millennium Genome Project.

Hypertension is one of the most common complex genetic disorders. We have described previously 38 single nucleotide polymorphisms (SNPs) with suggestive association with hypertension in Japanese individuals. In this study we extend our previous findings by analyzing a large sample of Japanese individuals (n=14 105) for the most associated SNPs. We also conducted replication analyses in Japanese of susceptibility loci for hypertension identified recently from genome-wide association studies of European ancestries. Association analysis revealed significant association of the ATP2B1 rs2070759 polymorphism with hypertension (P=5.3×10(-5); allelic odds ratio: 1.17 [95% CI: 1.09 to 1.26]). Additional SNPs in ATP2B1 were subsequently genotyped, and the most significant association was with rs11105378 (odds ratio: 1.31 [95% CI: 1.21 to 1.42]; P=4.1×10(-11)). Association of rs11105378 with hypertension was cross-validated by replication analysis with the Global Blood Pressure Genetics consortium data set (odds ratio: 1.13 [95% CI: 1.05 to 1.21]; P=5.9×10(-4)). Mean adjusted systolic blood pressure was highly significantly associated with the same SNP in a meta-analysis with individuals of European descent (P=1.4×10(-18)). ATP2B1 mRNA expression levels in umbilical artery smooth muscle cells were found to be significantly different among rs11105378 genotypes. Seven SNPs discovered in published genome-wide association studies were also genotyped in the Japanese population. In the combined analysis with replicated 3 genes, FGF5 rs1458038, CYP17A1, rs1004467, and CSK rs1378942, odds ratio of the highest risk group was 2.27 (95% CI: 1.65 to 3.12; P=4.6×10(-7)) compared with the lower risk group. In summary, this study confirmed common genetic variation in ATP2B1, as well as FGF5, CYP17A1, and CSK, to be associated with blood pressure levels and risk of hypertension.

Interactome mapping of the phosphatidylinositol 3-kinase-mammalian target of rapamycin pathway identifies deformed epidermal autoregulatory factor-1 as a new glycogen synthase kinase-3 interactor.

The phosphatidylinositol 3-kinase-mammalian target of rapamycin (PI3K-mTOR) pathway plays pivotal roles in cell survival, growth, and proliferation downstream of growth factors. Its perturbations are associated with cancer progression, type 2 diabetes, and neurological disorders. To better understand the mechanisms of action and regulation of this pathway, we initiated a large scale yeast two-hybrid screen for 33 components of the PI3K-mTOR pathway. Identification of 67 new interactions was followed by validation by co-affinity purification and exhaustive literature curation of existing information. We provide a nearly complete, functionally annotated interactome of 802 interactions for the PI3K-mTOR pathway. Our screen revealed a predominant place for glycogen synthase kinase-3 (GSK3) A and B and the AMP-activated protein kinase. In particular, we identified the deformed epidermal autoregulatory factor-1 (DEAF1) transcription factor as an interactor and in vitro substrate of GSK3A and GSK3B. Moreover, GSK3 inhibitors increased DEAF1 transcriptional activity on the 5-HT1A serotonin receptor promoter. We propose that DEAF1 may represent a therapeutic target of lithium and other GSK3 inhibitors used in bipolar disease and depression.

Respiratory nitric oxide and pulmonary artery pressure in children of aymara and European ancestry at high altitude.

Invasive studies suggest that healthy children living at high altitude display pulmonary hypertension, but the data to support this assumption are sparse. Nitric oxide (NO) synthesized by the respiratory epithelium regulates pulmonary artery pressure, and its synthesis was reported to be increased in Aymara high-altitude dwellers. We hypothesized that pulmonary artery pressure will be lower in Aymara children than in children of European ancestry at high altitude, and that this will be related to increased respiratory NO. We therefore compared pulmonary artery pressure and exhaled NO (a marker of respiratory epithelial NO synthesis) between large groups of healthy children of Aymara (n = 200; mean +/- SD age, 9.5 +/- 3.6 years) and European ancestry (n = 77) living at high altitude (3,600 to 4,000 m). We also studied a group of European children (n = 29) living at low altitude. The systolic right ventricular to right atrial pressure gradient in the Aymara children was normal, even though significantly higher than the gradient measured in European children at low altitude (22.5 +/- 6.1 mm Hg vs 17.7 +/- 3.1 mm Hg, p < 0.001). In children of European ancestry studied at high altitude, the pressure gradient was 33% higher than in the Aymara children (30.0 +/- 5.3 mm Hg vs 22.5 +/- 6.1 mm Hg, p < 0.0001). In contrast to what was expected, exhaled NO tended to be lower in Aymara children than in European children living at the same altitude (12.4 +/- 8.8 parts per billion [ppb] vs 16.1 +/- 11.1 ppb, p = 0.06) and was not related to pulmonary artery pressure in either group. Aymara children are protected from hypoxic pulmonary hypertension at high altitude. This protection does not appear to be related to increased respiratory NO synthesis.

In vitro whole-genome analysis identifies a susceptibility locus for HIV-1.

Advances in large-scale analysis of human genomic variability provide unprecedented opportunities to study the genetic basis of susceptibility to infectious agents. We report here the use of an in vitro system for the identification of a locus on HSA8q24.3 associated with cellular susceptibility to HIV-1. This locus was mapped through quantitative linkage analysis using cell lines from multigeneration families, validated in vitro, and followed up by two independent association studies in HIV-positive individuals. Single nucleotide polymorphism rs2572886, which is associated with cellular susceptibility to HIV-1 in lymphoblastoid B cells and in primary T cells, was also associated with accelerated disease progression in one of two cohorts of HIV-1-infected patients. Biological analysis suggests a role of the rs2572886 region in the regulation of the LY6 family of glycosyl-phosphatidyl-inositol (GPI)-anchored proteins. Genetic analysis of in vitro cellular phenotypes provides an attractive approach for the discovery of susceptibility loci to infectious agents.