Genetic Evidence Implicates the Immune System and Cholesterol Metabolism in the Aetiology of Alzheimer's Disease

Background: Late Onset Alzheimer's disease (LOAD) is the leading cause of dementia. Recent large genome-wide association studies (GWAS) identified the first strongly supported LOAD susceptibility genes since the discovery of the involvement of APOE in the early 1990s. We have now exploited these GWAS datasets to uncover key LOAD pathophysiological processes. Methodology: We applied a recently developed tool for mining GWAS data for biologically meaningful information to a LOAD GWAS dataset. The principal findings were then tested in an independent GWAS dataset.

The Structural and Molecular Biology of Type I Galactosemia: Enzymology of Galactose 1-phosphate Uridylyltransferase

Reduced galactose 1-phosphate uridylyltransferase (GAIT) activity is associated with the genetic disease type 1 galactosemia. This results in an increase in the cellular concentration of galactose 1-phosphate. The accumulation of this toxic metabolite, combined with aberrant glycoprotein and glycolipid biosynthesis, is likely to be the major factor in molecular pathology. The mechanism of GAIT was established through classical enzymological methods to be a substituted enzyme in which the reaction with UDP-glucose results in the formation of a covalent, UMP-histidine adduct in the active site. The uridylated enzyme can then react with galactose 1-phosphate to form UDP-galactose. The structure of the enzyme from Escherichia coli reveals a homodimer containing one zinc (II) and one iron (11) ion per subunit. This enzymological and structural knowledge provides the basis for understanding the biochemistry of this critical step in the Leloir pathway. However, a high-resolution crystal structure of human GAIT is required to assist greater understanding of the effects of disease-associated mutations. (C) 2011 IUBMB IUBMB Life, 63(9): 694-700, 2011

Genetic susceptibility to invasive meningococcal disease: MBL2 structural polymorphisms revisited in a large case–control study and a systematic review

Invasive infection caused by Neisseria meningitidis is a worldwide public health problem. Previous reports have indicated that carriage of common ‘defective’ structural polymorphisms of the host mannose-binding lectin gene (MBL2) greatly increases an individual’s risk of developing the disease. We report the largest case–control study so far to investigate the effect of these polymorphisms in meningococcal disease (296 PCR-positive cases and 5196 population controls, all of European ancestry) and demonstrate that no change in risk is associated with the polymorphisms overall or in any age-defined subgroup. This finding contrasts with two smaller studies that reported an increase in risk. A systematic review of all studies of MBL2 polymorphisms in people of European ancestry published since 1999, including 24 693 individuals, revealed a population frequency of the combined ‘defective’MBL2 allele of 0.230 (95% confidence limits: 0.226–0.234). The past reported associations of increased risk of meningococcal disease were because of low ‘defective’ allele frequencies in their study control populations (0.13 and 0.04) that indicate systematic problems with the studies. The data from our study and all other available evidence indicate that MBL2 structural polymorphisms do not predispose children or adults to invasive meningococcal disease.

Genetic susceptibility to invasive meningococcal disease: MBL2 structural polymorphisms revisited in a large case-control study and a systematic review.

AbstractInvasive infection caused by Neisseria meningitidis is a worldwide public health problem. Previous reports have indicated that carriage of common 'defective' structural polymorphisms of the host mannose-binding lectin gene (MBL2) greatly increases an individual's risk of developing the disease. We report the largest case-control study so far to investigate the effect of these polymorphisms in meningococcal disease (296 PCR-positive cases and 5196 population controls, all of European ancestry) and demonstrate that no change in risk is associated with the polymorphisms overall or in any age-defined subgroup. This finding contrasts with two smaller studies that reported an increase in risk. A systematic review of all studies of MBL2 polymorphisms in people of European ancestry published since 1999, including 24 693 individuals, revealed a population frequency of the combined 'defective'MBL2 allele of 0.230 (95% confidence limits: 0.226-0.234). The past reported associations of increased risk of meningococcal disease were because of low 'defective' allele frequencies in their study control populations (0.13 and 0.04) that indicate systematic problems with the studies. The data from our study and all other available evidence indicate that MBL2 structural polymorphisms do not predispose children or adults to invasive meningococcal disease.

Non-genetic mechanisms communicating antibiotic resistance:rethinking strategies for antimicrobial drug design

Introduction: Infections by multidrug-resistant bacteria are of great concern worldwide. In many cases, resistance is not due to the presence of specific antibiotic-modifying enzymes, but rather associated with a general impermeability of the bacterial cell envelope. The molecular bases of this intrinsic resistance are not completely understood. Moreover, horizontal gene transfers cannot solely explain the spread of intrinsic resistance among bacterial strains. Areas covered: This review focuses on the increased intrinsic antibiotic resistance mediated by small molecules. These small molecules can either be secreted from bacterial cells of the same or different species (e.g., indole, polyamines, ammonia, and the Pseudomonas quinolone signal) or be present in the bacterial cell milieu, whether in the environment, such as indole acetic acid and other plant hormones, or in human tissues and body fluids, such as polyamines. These molecules are metabolic byproducts that act as infochemicals and modulate bacterial responses toward antibiotics leading to increasing or decreasing resistance levels. Expert opinion: The non-genetic mechanisms of antibiotic response modulation and communication discussed in this review should reorient our thinking of the mechanisms of intrinsic resistance to antibiotics and its spread across bacterial cell populations. The identification of chemical signals mediating increased intrinsic antibiotic resistance will expose novel critical targets for the development of new antimicrobial strategies.

Genetic analysis of the O7-polysaccharide biosynthesis region from the Escherichia coli O7:K1 strain VW187

We recently cloned biosynthesis genes for the O7-lipopolysaccharide (O7-LPS) side chain from the Escherichia coli K-1 strain VW187 (M. A. Valvano, and J. H. Crosa, Infect. Immun. 57:937-943, 1989). To characterize the O7-LPS region, the recombinant cosmids pJHCV31 and pJHCV32 were mutagenized by transposon mutagenesis with Tn3HoHo1, which carries a promoterless lac operon and can therefore generate lacZ transcriptional fusions with target DNA sequences. Cells containing mutated plasmids were examined for their ability to react by coagglutination with O7 antiserum. The LPS pattern profiles of the insertion mutants were also investigated by electrophoresis of cell envelope fractions, followed by silver staining and immunoblotting analysis. These experiments identified three phenotypic classes of mutants and defined a region in the cloned DNA of about 14 kilobase pairs that is essential for O7-LPS expression. Analysis of beta-galactosidase production by cells carrying plasmids with transposon insertions indicated that transcription occurs in only one direction along the O7-LPS region. In vitro transcription-translation experiments revealed that the O7-LPS region encodes at least 16 polypeptides with molecular masses ranging from 20 to 48 kilodaltons. Also, the O7-LPS region in VW187 was mutagenized by homologous recombination with subsets of the cloned O7-LPS genes subcloned into a suicide plasmid vector. O7-LPS-deficient mutants of VW187 were complemented with pJHCV31 and pJHCV32, confirming that these cosmids contain genetic information that is essential for the expression of the O7 polysaccharide.

Genetic variation in MME in relation to neprilysin protein and enzyme activity, Aβ levels, and Alzheimer's disease risk

Neprilysin (NEP), also known as membrane metalloendopeptidase (MME), is considered amongst the most important ß-amyloid (Aß)-degrading enzymes with regard to prevention of Alzheimer's disease (AD) pathology. Variation in the NEP gene (MME) has been suggested as a risk factor for AD. We conducted a genetic association study of 7MME SNPs - rs1836914, rs989692, rs9827586, rs6797911, rs61760379, rs3736187, rs701109 - with respect to AD risk in a cohort of 1057 probable and confirmed AD cases and 424 age-matched non-demented controls from the United Kingdom, Italy and Sweden. We also examined the association of these MME SNPs with NEP protein level and enzyme activity, and on biochemical measures of Aß accumulation in frontal cortex - levels of total soluble Aß, oligomeric Aß(1-42), and guanidine-extractable (insoluble) Aß - in a sub-group of AD and control cases with post-mortem brain tissue. On multivariate logistic regression analysis one of the MME variants (rs6797911) was associated with AD risk (P = 0.00052, Odds Ratio (O.R. = 1.40, 95% confidence interval (1.16-1.70)). None of the SNPs had any association with Aß levels; however, rs9827586 was significantly associated with NEP protein level (p=0.014) and enzyme activity (p=0.006). Association was also found between rs701109 and NEP protein level (p=0.026) and a marginally non-significant association was found for rs989692 (p=0.055). These data suggest that MME variation may be associated with AD risk but we have not found evidence that this is mediated through modification of NEP protein level or activity.

Genetic characterization of tigecycline resistance in clinical isolates of Enterobacter cloacae and Enterobacter aerogenes

OBJECTIVES:
The intrinsically encoded ramA gene has been linked to tigecycline resistance through the up-regulation of efflux pump AcrAB in Enterobacter cloacae. The molecular basis for increased ramA expression in E. cloacae and Enterobacter aerogenes, as well as the role of AraC regulator rarA, has not yet been shown. To ascertain the intrinsic molecular mechanism(s) involved in tigecycline resistance in Enterobacter spp., we analysed the expression levels of ramA and rarA and corresponding efflux pump genes acrAB and oqxAB in Enterobacter spp. clinical isolates.

METHODS:
The expression levels of ramA, rarA, oqxA and acrA were tested by quantitative real-time RT-PCR. The ramR open reading frames of the ramA-overexpressing strains were sequenced; strains harbouring mutations were transformed with wild-type ramR to study altered ramA expression and tigecycline susceptibility.

RESULTS:
Tigecycline resistance was mediated primarily by increased ramA expression in E. cloacae and E. aerogenes. Only the ramA-overexpressing E. cloacae isolates showed increased rarA and oqxA expression. Upon complementation with wild-type ramR, all Enterobacter spp. containing ramR mutations exhibited decreased ramA and acrA expression and increased tigecycline susceptibility. Exceptions were one E. cloacae strain and one E. aerogenes strain, where a decrease in ramA levels was not accompanied by lower acrA expression.

CONCLUSIONS:
Increased ramA expression due to ramR deregulation is the primary mediator of tigecycline resistance in clinical isolates of E. cloacae and E. aerogenes. However, some ramA-overexpressing isolates do not show changes in ramR, suggesting alternate pathways of ramA regulation; the rarA regulator and the oqxAB efflux pump may also play a role in tigecycline resistance in E. cloacae.

Nontypable Haemophilus influenzae displays a prevalent surface structure molecular pattern in clinical isolates

Non-typable Haemophilus influenzae (NTHi) is a gram negative pathogen that causes acute respiratory infections and is associated with the progression of chronic respiratory diseases. Previous studies have established the existence of a remarkable genetic variability among NTHi strains. In this study we show that, in spite of a high level of genetic heterogeneity, NTHi clinical isolates display a prevalent molecular feature, which could confer fitness during infectious processes. A total of 111 non-isogenic NTHi strains from an identical number of patients, isolated in two distinct geographical locations in the same period of time, were used to analyse nine genes encoding bacterial surface molecules, and revealed the existence of one highly prevalent molecular pattern (lgtF+, lic2A+, lic1D+, lic3A+, lic3B+, siaA-, lic2C+, ompP5+, oapA+) displayed by 94.6% of isolates. Such a genetic profile was associated with a higher bacterial resistance to serum mediated killing and enhanced adherence to human respiratory epithelial cells.

Genetic and morphological characterization of Cladobotryum species causing cobweb disease of mushrooms

Cladobotryum dendroides (= Dactylium dendroides) has hitherto been regarded as the major causal agent of cobweb disease of the cultivated mushroom, Agaricus bisporus. Nucleotide sequence data for the internal transcribed spacer (ITS) regions of four Cladobotryum/Hypomyces species reported to be associated with cobweb disease, however, indicate that the most common pathogen is now C. mycophilum. This cobweb pathogen varies somewhat in conidial septation from published descriptions of C. mycophilum and lacks the distinctive colony odor. ITS sequencing revealed minor nucleotide variation which split isolates of the pathogen into three subgroups, two comprising isolates that were sensitive to methylbenzimidazole carbamate (MBC) fungicides and one comprising MBC-resistant isolates. The MBC-resistant isolates, which were only obtained from Ireland and Great Britain, clustered together strongly in randomly amplified polymorphic DNA (RAPD) PCR analysis, suggesting that they may be clonal. The MBC-sensitive isolates were more diverse. A RAPD fragment of 800 to 900 bp, containing a microsatellite and found in the MBC-resistant isolates, also indicated their clonal nature; the microsatellites of these isolates contained the same number of GA repeats. Smaller, polymorphic microsatellites, similarly comprising GA repeats, in the MBC-sensitive isolates in general correlated with their geographic origin.

Molecular comparison of Scytalidium thermophilum isolates using RAPD and ITS nucleotide sequence analyses

Scytalidium thermophilum plays an important role in determining selectivity of compost produced for growing Agaricus bisporus. The objective of this study was to characterise S. thermophilum isolates by random amplified polymorphic DNA (RAPD) analysis and sequence analysis of internally transcribed spacer (ITS) regions of the rDNA, to assess the genetic variation exhibited by this species complex and to compare this with existing morphological and thermogravimetric data. RAPD analysis of 34 isolates from various parts of the world revealed two distinct groups, which could be separated on the basis of the differences in the banding patterns produced with five random primers. Nucleotide sequence analysis of the ITS region, which was ca 536 bp in length, revealed only very minor variation among S. thermophilum isolates examined. Several nucleotide base changes within this region demonstrated variation. Genetic distance values among type 1 and 2 S. thermophilum isolates, as determined by ITS sequence analysis, varied by a value of 0.005 %. Molecular analyses carried out in the present study would suggest that isolates within this species complex exhibit genetic differences which correlate well with morphological variation and thermogravimetric data previously determined.

Molecular Validation of the Schizophrenia Spectrum

Background: Early descriptive work and controlled family and adoption studies support the hypothesis that a range of personality and nonschizophrenic psychotic disorders aggregate in families of schizophrenic probands. Can we validate, using molecular polygene scores from genome-wide association studies (GWAS), this schizophrenia spectrum? Methods: The predictive value of polygenic findings reported by the Psychiatric GWAS Consortium (PGC) was applied to 4 groups of relatives from the Irish Study of High-Density Schizophrenia Families (ISHDSF; N = s) differing on their assignment within the schizophrenia spectrum. Genome-wide single nucleotide polymorphism data for affected and unaffected relatives were used to construct per-individual polygene risk scores based on the PGC stage-I results. We compared mean polygene scores in the ISHDSF with mean scores in ethnically matched population controls (N = 929). Results: The schizophrenia polygene score differed significantly across diagnostic categories and was highest in those with narrow schizophrenia spectrum, lowest in those with no psychiatric illness, and in-between in those classified in the intermediate, broad, and very broad schizophrenia spectrum. Relatives of all of these groups of affected subjects, including those with no diagnosis, had schizophrenia polygene scores significantly higher than the control sample. Conclusions: In the relatives of high-density families, the observed pattern of enrichment of molecular indices of schizophrenia risk suggests an underlying, continuous liability distribution and validates, using aggregate common risk alleles, a genetic basis for the schizophrenia spectrum disorders. In addition, as predicted by genetic theory, nonpsychotic members of multiply-affected schizophrenia families are significantly enriched for replicated, polygenic risk variants compared with the general population.

Genetic basis of Congenital Erythrocytosis:mutation update and online databases

Congenital Erythrocytosis (CE), or congenital polycythemia, represents a rare and heterogeneous clinical entity. It is caused by deregulated red blood cell production where erythrocyte overproduction results in elevated hemoglobin and hematocrit levels. Primary congenital familial erythrocytosis is associated with low erythropoietin (Epo) levels and results from mutations in the Epo receptor gene (EPOR). Secondary congenital erythrocytosis arises from conditions causing tissue hypoxia and results in increased Epo production. These include hemoglobin variants with increased affinity for oxygen (HBB, HBA mutations), decreased production of 2,3-bisphosphoglycerate due to BPGM mutations, or mutations in the genes involved in the hypoxia sensing pathway (VHL, EPAS1 and EGLN1). Depending on the affected gene, CE can be inherited either in an autosomal dominant or recessive mode, with sporadic cases arising de novo. Despite recent important discoveries in the molecular pathogenesis of CE, the molecular causes remain to be identified in about 70% of the patients. With the objective of collecting all the published and unpublished cases of CE the COST action MPN&MPNr-Euronet developed a comprehensive internet-based database focusing on the registration of clinical history, hematological, biochemical and molecular data (http://www.erythrocytosis.org/). In addition, unreported mutations are also curated in the corresponding Leiden Open Variation Database (LOVD). This article is protected by copyright. All rights reserved.