Next generation genome-wide association tool: design and coverage of a high-throughput European-optimized SNP array

The success of genome-wide association studies has paralleled the development of efficient genotyping technologies. We describe the development of a next-generation microarray based on the new highly-efficient Affymetrix Axiom genotyping technology that we are using to genotype individuals of European ancestry from the Kaiser Permanente Research Program on Genes, Environment and Health (RPGEH). The array contains 674,517 SNPs, and provides excellent genome-wide as well as gene-based and candidate-SNP coverage. Coverage was calculated using an approach based on imputation and cross validation. Preliminary results for the first 80,301 saliva-derived DNA samples from the RPGEH demonstrate very high quality genotypes, with sample success rates above 94% and over 98% of successful samples having SNP call rates exceeding 98%. At steady state, we have produced 462 million genotypes per week for each Axiom system. The new array provides a valuable addition to the repertoire of tools for large scale genome-wide association studies.     

Genotype at the P554L variant of the hexose-6 phosphate dehydrogenase gene is associated with carotid intima-medial thickness

Objective

The combined thickness of the intima and media of the carotid artery (carotid intima-medial thickness, CIMT) is associated with cardiovascular disease and stroke. Previous studies indicate that carotid intima-medial thickness is a significantly heritable phenotype, but the responsible genes are largely unknown. Hexose-6 phosphate dehydrogenase (H6PDH) is a microsomal enzyme whose activity regulates corticosteroid metabolism in the liver and adipose tissue; variability in measures of corticosteroid metabolism within the normal range have been associated with risk factors for cardiovascular disease. We performed a genetic association study in 854 members of 224 families to assess the relationship between polymorphisms in the gene coding for hexose-6 phosphate dehydrogenase (H6PD) and carotid intima-medial thickness.

Methods

Families were ascertained via a hypertensive proband. CIMT was measured using B-mode ultrasound. Single nucleotide polymorphisms (SNPs) tagging common variation in the H6PD gene were genotyped. Association was assessed following adjustment for significant covariates including “classical” cardiovascular risk factors. Functional studies to determine the effect of particular SNPs on H6PDH were performed.

Results

There was evidence of association between the single nucleotide polymorphism rs17368528 in exon five of the H6PD gene, which encodes an amino-acid change from proline to leucine in the H6PDH protein, and mean carotid intima-medial thickness (p = 0.00065). Genotype was associated with a 5% (or 0.04 mm) higher mean carotid intima-medial thickness measurement per allele, and determined 2% of the population variability in the phenotype.

Conclusions

Our results suggest a novel role for the H6PD gene in atherosclerosis susceptibility.     

Workers dominate male production in the neotropical bumblebee Bombus wilmattae (Hymenoptera: Apidae)

Background

Cooperation and conflict in social insects are closely linked to the genetic structure of the colony. Kin selection theory predicts conflict over the production of males between the workers and the queen and between the workers themselves, depending on intra-colonial relatedness but also on other factors like colony efficiency, sex ratios, cost of worker reproduction and worker dominance behaviour. In most bumblebee (Bombus) species the queen wins this conflict and often dominates male production. However, most studies in bumblebees have been conducted with only a few selected, mostly single mated species from temperate climate regions. Here we study the genetic colony composition of the facultative polyandrous neotropical bumblebee Bombus wilmattae, to assess the outcome of the queen-worker conflict over male production and to detect potential worker policing.

Results

A total of 120 males from five colonies were genotyped with up to nine microsatellite markers to infer their parentage. Four of the five colonies were queen right at point of time of male sampling, while one had an uncertain queen status. The workers clearly dominated production of males with an average of 84.9% +/- 14.3% of males being worker sons. In the two doubly mated colonies 62.5% and 96.7% of the male offspring originated from workers and both patrilines participated in male production. Inferring the mother genotypes from the male offspring, between four to eight workers participated in the production of males.

Conclusions

In this study we show that the workers clearly win the queen-worker conflict over male production in B. wilmattae, which sets them apart from the temperate bumblebee species studied so far. Workers clearly dominated male production in the singly as well the doubly mated colonies, with up to eight workers producing male offspring in a single colony. Moreover no monopolization of reproduction by single workers occurred.     

The age-related risk of co-existing meningitis in children with urinary tract infection

Objective

The primary aim of this study was to determine age-stratified rates of co-existing bacterial meningitis in children with urinary tract infection (UTI). The secondary aims of this study were to determine the causative pathogens of UTI, and the clinical features and outcome of children with co-existing meningitis.

Methods

Analysis of data collected over a nine-year period at a tertiary pediatric hospital in Australia. Study population: children below 16 years of age with culture-confirmed UTI and a paired CSF sample.

Results

A total of 748 episodes in 735 cases were included in the final analysis. The commonest pathogens causing UTI were Escherichia coli (67.4%), Enterococcus faecalis (8.4%), Klebsiella oxytoca (3.5%) and Klebsiella pneumoniae (3.5%). Only two (1.2%; 95% CI: 0.15–4.36%) of 163 neonates (between 0 and 28 days of age) with UTI had co-existing meningitis. Both presented with pyrexia, irritability and lethargy, and recovered uneventfully with antibiotic treatment. There were no cases of co-existing meningitis among 499 infants (between 29 days and 12 months of age) with UTI (95% CI: 0.00–0.74%), or any of the 86 children aged 12 months or over (95% CI: 0.00–4.20%).

Conclusions

These findings indicate that clinicians should have a low threshold to perform a lumbar puncture in neonates with UTI, as the risk of co-existing meningitis is not insignificant in this age group. In contrast, beyond the neonatal period, the risk is small and a more selective approach is warranted.     

Molecular characterization of adeno-associated viruses infecting children

Although adeno-associated virus (AAV) infection is common in humans, the biology of natural infection is poorly understood. Since it is likely that many primary AAV infections occur during childhood, we set out to characterize the frequency and complexity of circulating AAV isolates in fresh and archived frozen human pediatric tissues. Total cellular DNA was isolated from 175 tissue samples including freshly collected tonsils (n = 101) and archived frozen samples representing spleen (n = 21), lung (n = 16), muscle (n = 15), liver (n = 19), and heart (n = 3). Samples were screened for the presence of AAV and adenovirus sequences by PCR using degenerate primers. AAV DNA was detected in 7 of 101 (7%) tonsil samples and two of 74 other tissues (one spleen and one lung). Adenovirus sequences were identified in 19 of 101 tonsils (19%), but not in any other tissues. Complete capsid gene sequences were recovered from all nine AAV-positive tissues. Sequence analyses showed that eight of the capsid sequences were AAV2-like (approximately 98% amino acid identity), while the single spleen isolate was intermediate between serotypes 2 and 3. Comparison to the available AAV2 crystal structure revealed that the majority of the amino acid substitutions mapped to surface-exposed hypervariable domains. To further characterize the AAV capsid structure in these samples, we used a novel linear rolling-circle amplification method to amplify episomal AAV DNA and isolate infectious molecular clones from several human tissues. Serotype 2-like viruses were generated from these DNA clones and interestingly, failed to bind to a heparin sulfate column. Inspection of the capsid sequence from these two clones (and the other six AAV2-like isolates) revealed that they lacked arginine residues at positions 585 and 588 of the capsid protein, which are thought to be essential for interaction with the heparin sulfate proteoglycan coreceptor. These data provide a framework with which to explore wild-type AAV persistence in vivo and provide additional tools to further define the biodistribution and form of AAV in human tissues.     

c-Src is involved in regulating signal transmission from PDGFbeta receptor-GPCR(s) complexes in mammalian cells

We have reported that the platelet-derived growth factor receptor-beta (PDGFbeta) forms a novel signaling complex with G protein-coupled receptors (GPCR) (e.g. S1P(1) receptor) that enables more efficient activation of p42/p44 mitogen-activated protein kinase (MAPK) in response to PDGF and sphingosine 1-phosphate (S1P). We now demonstrate that c-Src participates in regulating the endocytosis of PDGFbeta receptor-GPCR complexes in response to PDGF. This leads to association of cytoplasmic p42/p44 MAPK with the receptor complex in endocytic vesicles. c-Src is regulated by G protein betagamma subunits and can interact with beta-arrestin. Indeed, the PDGF-dependent activation of p42/p44 MAPK was reduced by over-expression of the C-terminal domain of GRK2 (sequesters Gbetagamma subunits), the clathrin-binding domain of beta-arrestin and by inhibitors of c-Src and clathrin-mediated endocytosis. Moreover, PDGF and S1P induce the recruitment of c-Src to the PDGFbeta receptor-S1P(1) receptor complex. This leads to a G protein/c-Src-dependent tyrosine phosphorylation of Gab1 and accumulation of dynamin II at the plasma membrane, a step required for endocytosis of the PDGFbeta receptor-GPCR complex. These findings provide important information concerning the molecular organisation of novel receptor tyrosine kinase (RTK)-GPCR signal relays in mammalian cells.     

Linkage analysis using co-phenotypes in the BRIGHT study reveals novel potential susceptibility loci for hypertension

Identification of the genetic influences on human essential hypertension and other complex diseases has proved difficult, partly because of genetic heterogeneity. In many complex-trait resources, additional phenotypic data have been collected, allowing comorbid intermediary phenotypes to be used to characterize more genetically homogeneous subsets. The traditional approach to analyzing covariate-defined subsets has typically depended on researchers' previous expectations for definition of a comorbid subset and leads to smaller data sets, with a concomitant attrition in power. An alternative is to test for dependence between genetic sharing and covariates across the entire data set. This approach offers the advantage of exploiting the full data set and could be widely applied to complex-trait genome scans. However, existing maximum-likelihood methods can be prohibitively computationally expensive, especially since permutation is often required to determine significance. We developed a less computationally intensive score test and applied it to biometric and biochemical covariate data, from 2,044 sibling pairs with severe hypertension, collected by the British Genetics of Hypertension (BRIGHT) study. We found genomewide-significant evidence for linkage with hypertension and several related covariates. The strongest signals were with leaner-body-mass measures on chromosome 20q (maximum LOD = 4.24) and with parameters of renal function on chromosome 5p (maximum LOD = 3.71). After correction for the multiple traits and genetic locations studied, our global genomewide P value was .046. This is the first identity-by-descent regression analysis of hypertension to our knowledge, and it demonstrates the value of this approach for the incorporation of additional phenotypic information in genetic studies of complex traits.     

Selection and the Cell Cycle: Positive Darwinian Selection in a Well-Known DNA Damage Response Pathway

Cancer is a common occurrence in multi-cellular organisms and is not strictly limited to the elderly in a population. It is therefore possible that individuals with genotypes that protect against early onset cancers have a selective advantage. In this study the patterns of mutation in the proteins of a well-studied DNA damage response pathway have been examined for evidence of adaptive evolutionary change. Using a maximum likelihood framework and the mammalian species phylogeny, together with codon models of evolution, selective pressure variation across the interacting network of proteins has been detected. The presence of signatures of adaptive evolution in BRCA1 and BRCA2 has already been documented but the effect on the entire network of interacting proteins in this damage response pathway has, until now, been unknown. Positive selection is evident throughout the network with a total of 11 proteins out of 15 examined displaying patterns of substitution characteristic of positive selection. It is also shown here that modern human populations display evidence of an ongoing selective sweep in 9 of these DNA damage repair proteins. The results presented here provide the community with new residues that may be relevant to cancer susceptibility while also highlighting those proteins where human and mouse have undergone lineage-specific functional shift. An understanding of this damage response pathway from an evolutionary perspective will undoubtedly contribute to future cancer treatment approaches.     

A systematic analysis of human CHMP protein interactions: additional MIT domain-containing proteins bind to multiple components of the human ESCRT III complex

In Saccharomyces cerevisiae 6 closely related proteins (Did2p, Vps2p, Vps24p, Vps32p, Vps60p, Vps20p) form part of the extended ESCRT III complex. This complex is required for the formation of multivesicular bodies and the degradation of internalized transmembrane receptor proteins. In contrast the human genome encodes 10 homologous proteins (CHMP1A (approved gene symbol PCOLN3), 1B, 2A, 2B, 3 (approved gene symbol VPS24), 4A, 4B, 4C, 5, and 6). In this study we have performed a series of protein interaction experiments to generate a more comprehensive picture of the human CHMP protein-interaction network. Our results describe novel interactions between known components of the human ESCRT III complex and identify a range of putative binding partners, which may indicate new ways in which the function of human CHMP proteins may be regulated. In particular, we show that two further MIT domain-containing proteins (AMSH/STAMBP and LOC129531) interact with multiple components of the human ESCRT III complex.     

Characterization of a glutathione metabolic mutant of Mycobacterium tuberculosis and its resistance to glutathione and nitrosoglutathione

Glutathione is a tripeptide and antioxidant, synthesized at high levels by cells during the production of reactive oxygen and nitrogen intermediates. Glutathione also serves as a carrier molecule for nitric oxide in the form of S-nitrosoglutathione. Previous studies from this laboratory have shown that glutathione and S-nitrosoglutathione are directly toxic to mycobacteria. Glutathione is not transported into the cells as a tripeptide. Extracellular glutathione is converted to a dipeptide due to the action of transpeptidase, and the dipeptide is then transported into the bacterial cells. The processing of glutathione and S-nitrosoglutathione is brought about by the action of the enzyme gamma-glutamyl transpeptidase. The function of gamma-glutamyl transpeptidase is to cleave glutathione and S-nitrosoglutathione to the dipeptide (Cys-Gly), which is then transported into the bacterium by the multicomponent ABC transporter dipeptide permease. We have created a mutant strain of Mycobacterium tuberculosis lacking this metabolic enzyme. We investigated the sensitivity of this strain to glutathione and S-nitrosoglutathione compared to that of the wild-type bacteria. In addition, we examined the role of glutathione and/or S-nitrosoglutathione in controlling the growth of intracellular M. tuberculosis inside mouse macrophages.