A high-density screen for linkage in multiple sclerosis

To provide a definitive linkage map for multiple sclerosis, we have genotyped the Illumina BeadArray linkage mapping panel (version 4) in a data set of 730 multiplex families of Northern European descent. After the application of stringent quality thresholds, data from 4,506 markers in 2,692 individuals were included in the analysis. Multipoint nonparametric linkage analysis revealed highly significant linkage in the major histocompatibility complex (MHC) on chromosome 6p21 (maximum LOD score [MLS] 11.66) and suggestive linkage on chromosomes 17q23 (MLS 2.45) and 5q33 (MLS 2.18). This set of markers achieved a mean information extraction of 79.3% across the genome, with a Mendelian inconsistency rate of only 0.002%. Stratification based on carriage of the multiple sclerosis–associated DRB1*1501 allele failed to identify any other region of linkage with genomewide significance. However, ordered-subset analysis suggested that there may be an additional locus on chromosome 19p13 that acts independent of the main MHC locus. These data illustrate the substantial increase in power that can be achieved with use of the latest tools emerging from the Human Genome Project and indicate that future attempts to systematically identify susceptibility genes for multiple sclerosis will have to involve large sample sizes and an association-based methodology.     

A germline mutation in BLOC1S3/reduced pigmentation causes a novel variant of Hermansky-Pudlak syndrome (HPS8)

Hermansky-Pudlak syndrome (HPS) is genetically heterogeneous, and mutations in seven genes have been reported to cause HPS. Autozygosity mapping studies were undertaken in a large consanguineous family with HPS. Affected individuals displayed features of incomplete oculocutaneous albinism and platelet dysfunction. Skin biopsy demonstrated abnormal aggregates of melanosomes within basal epidermal keratinocytes. A homozygous germline frameshift mutation in BLOC1S3 (p.Gln150ArgfsX75) was identified in all affected individuals. BLOC1S3 mutations have not been previously described in patients with HPS, but BLOC1S3 encodes a subunit of the biogenesis of lysosome-related organelles complex 1 (BLOC-1). Mutations in other BLOC-1 subunits have been associated with an HPS phenotype in humans and/or mouse, and a nonsense mutation in the murine orthologue of BLOC1S3 causes the reduced pigmentation (rp) model of HPS. Interestingly, eye pigment formation is reported to be normal in rp, but we found visual defects (nystagmus, iris transilluminancy, foveal hypoplasia, reduced visual acuity, and evidence of optic pathway misrouting) in affected individuals. These findings define a novel form of human HPS (HPS8) and extend genotype-phenotype correlations in HPS.     

A simple fluorescent method for detecting mismatched DNAs using a MutS-fluorophore conjugate

A fluorescent method was developed for the detection of unpaired and mismatched DNAs using a MutS-fluorophore conjugate. The fluorophore, 2-(4'-(iodoacetoamido)anilino) naphthalene-6-sulfonic acid (IAANS), was site-specifically attached to the 469 position of Thermus aquaticus (Taq.) MutS mutant (C42A/T469C). The fluorophore labeled residue located at the dimer interface of the protein undergoes a drastic conformational change upon binding with mismatched DNA. The close proximity of the two identical fluorescent molecules presumably causes the self-quenching of the fluorophore, since fluorescence emission of the biosensor decreases with increasing concentrations of mismatched DNA. The order of binding affinity for each unpaired and mismatched DNA obtained by this method was DeltaT (Kd=52 nM)>GT (62 nM)>DeltaC (130 nM)>CT (160 nM)>DeltaG (170 nM)>DeltaA (250 nM)>CC (720 nM)>AT (950 nM). This order is comparable to the previous results of the gel mobility shift assay. Thus, this method can be a simple, useful tool for elucidating the mechanism of DNA mismatch repair as well as a novel probe for detecting of genetic mutation.     

Two C-terminal ankyrin repeats form the minimal stable unit of the ankyrin repeat protein p18INK4c

Ankyrin repeat proteins (ARPs) appear to be abundant in organisms from all phyla, and play critical regulatory roles, mediating specific interactions with target biomolecules and thus ordering the sequence of events in diverse cellular processes. ARPs possess a non-globular scaffold consisting of repeating motifs named ankyrin (ANK) repeats, which stack on each other. The modular architecture of ARPs provides a new paradigm for understanding protein stability and folding mechanisms. In the present study, the stability of various C-terminal fragments of the ARP p18^INK4c was investigated by all-atomic 450 ns molecular dynamics (MD) simulations in explicit water solvent. Only motifs with at least two ANK repeats made stable systems in the available timescale. All smaller fragments were unstable, readily losing their native fold and α-helical content. Since each non-terminal ANK repeat has two hydrophobic sides, we may hypothesize that at least one hydrophobic side must be fully covered and shielded from the water as a necessary, but not sufficient, condition to maintain ANK repeat stability. Consequently, at least two ANK repeats are required to make a stable ARP. Figure Structure of the p18INK4c protein (PDB entry 1IHB, chain B), which is a member of the cyclin-dependent kinase inhibitor (INK) tumor suppressor family with five ankyrin (ANK) repeat modules. The figure was generated by PyMol Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

New teleomorph combinations in the entomopathogenic genus Metacordyceps

The genus Metacordyceps contains arthropod pathogens in Clavicipitaceae (Hypocreales) that formerly were classified in Cordyceps sensu Kobayasi et Mains. Of the current arthropod pathogenic genera of Hypocreales, the genus Metacordyceps remains one of the most poorly understood and contains a number of teleomorphic morphologies convergent with species of Cordyceps s.s. (Cordycipitaceae) and Ophiocordyceps (Ophiocordycipitaceae). Of note, the anamorph genera Metarhizium and Pochonia were found to be associated only with Metacordyceps and demonstrated to be phylogenetically informative for the clade. Several species of Cordyceps considered to have uncertain placements (incertae sedis) in the current taxonomic framework of clavicipitoid fungi were collected during field expeditions mostly in eastern Asia. Species reclassified here in Metacordyceps include Cordyceps atrovirens Kobayasi & Shimizu, Cordyceps indigotica Kobayasi & Shimizu, Cordyceps khaoyaiensis Hywel-Jones, Cordyceps kusanagiensis Kobayasi & Shimizu, Cordyceps martialis Speg., Ophiocordyceps owariensis Kobayasi, Cordyceps pseudoatrovirens Kobayasi & Shimizu and Ophicordyceps owariensis f. viridescens (Uchiy. & Udagawa) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora. Incorporation of these species in a multigene phylogenetic framework of the major clades of clavicipitoid fungi more than doubled the number of species in Metacordyceps and allowed for refinement of morphological concepts for the genus consistent with the phylogenetic structure. Based on these findings we then discuss evolution of this genus, subgeneric relationships, anamorph connections, and suggest additional species that should be confirmed for possible inclusion in Metacordyceps.     

Rapid and Quantitative Assay of Amyloid-Seeding Activity in Human Brains Affected with Prion Diseases

The infectious agents of the transmissible spongiform encephalopathies are composed of amyloidogenic prion protein, PrP^Sc. Real-time quaking-induced conversion can amplify very small amounts of PrP^Sc seeds in tissues/body fluids of patients or animals. Using this in vitro PrP-amyloid amplification assay, we quantitated the seeding activity of affected human brains. End-point assay using serially diluted brain homogenates of sporadic Creutzfeldt–Jakob disease patients demonstrated that 50% seeding dose (SD₅₀) is reached approximately 10¹⁰/g brain (values varies 10^8.79–10.63/g). A genetic case (GSS-P102L) yielded a similar level of seeding activity in an autopsy brain sample. The range of PrP^Sc concentrations in the samples, determined by dot-blot assay, was 0.6–5.4 μg/g brain; therefore, we estimated that 1 SD₅₀ unit was equivalent to 0.06–0.27 fg of PrP^Sc. The SD₅₀ values of the affected brains dropped more than three orders of magnitude after autoclaving at 121°C. This new method for quantitation of human prion activity provides a new way to reduce the risk of iatrogenic prion transmission.     

Use of a bait impact index to assess effects of bait application against populations of Formosan subterranean termite (Isoptera: Rhinotermitidae) in a large area

The effects of bait applications on the overall activity of the Formosan subterranean termite, Coptotermes formosanus Shiraki, in a town of 1.2 km2 were assessed between 2001 and 2003 by using a bait impact index. The index incorporated the amount of baits applied and the distance between bait application loci and the monitoring stations from which termite activity was measured. Even with the collaboration of only 35% of the residents and incomplete treatment records, the bait impact index demonstrated that more baits applied in proximity to monitoring loci significantly caused the decline of C. formosanus activity.     

Transmyocardial revascularization aggravates myocardial ischemia around the channels in the immediate phase

We examined whether transmyocardial revascularization (TMR) relieves myocardial ischemia by increasing regional perfusion via the transmural channels in acute canine experiments. Regional blood flow during transient coronary ligation (2 min) was compared before and 30 min after TMR, and at the third transient ischemia the mid-left ventricle (LV) was cut and immediately frozen along the short axis for the analysis of NADH fluorescence in the regions around the TMR channels. In low-resolution analysis (2-4 g tissue or 2-3 cm(2) area), regional perfusion was not significantly altered after TMR, and NADH fluorescence was observed throughout the ischemic region without significant spatial variation. High-resolution analysis (2.8 mg, 1 mm x 1 mm) revealed that the flow after TMR was lower, and NADH fluorescence was higher in the regions close to the channels (1-2 mm) than in the regions 3-4 mm away from them. Creating TMR channels did not improve the regional perfusion and rather aggravated the local ischemia in the vicinity of the channels in the immediate phase.     

Examining the Polymorphisms in the Hypoxia Pathway Genes in Relation to Outcome in Colorectal Cancer

Introduction

Colorectal cancer is a common malignancy. Identification of genetic prognostic markers may help prognostic estimations in colorectal cancer. Genes that regulate response to hypoxia and other genes that are regulated under the hypoxic conditions have been shown to play roles in cancer progression. In this study, we hypothesized that genetic variations in the hypoxia pathway genes were associated with the risk of outcome in colorectal cancer patients.

Methods

This study was performed in two phases. In the first phase, 49 SNPs from six hypoxia pathway genes (HIF1A, HIF1B, HIF2A, LOX, MIF and CXCL12) in 272 colorectal cancer patients were analyzed. In the second phase, 77 SNPs from seven hypoxia pathway genes (HIF1A, HIF1B, HIF2A, HIF2B, HIF3A, LOX and CXCL12) were analyzed in an additional cohort of 535 patients. Kaplan Meier, Cox univariate and multivariable regression analyses were performed to analyze the relationship between the SNPs and overall survival (OS), disease free survival (DFS) or disease specific survival (DSS). Since this was a hypothesis-generating study, no correction for multiple testing was applied.

Results

In phase I, one SNP (HIF2A rs11125070) was found to be associated with DFS in multivariable analysis; yet association of a proxy polymorphism (HIF2A rs4953342) was not detected in the phase II patient cohort. In phase II, associations of two SNPs (HIF2A rs4953352 and HIF2B rs12593988) were significant in both OS and DFS multivariable analyses. However, association of HIF2A rs4953352 was not replicated in the phase I cohort using a proxy SNP (HIF2A rs6706003).

Conclusion

Overall, our study did not find a convincing evidence of association of the investigated polymorphisms with the disease outcomes in colorectal cancer.