Coagulation factor XIII: A useful polymorphic genetic marker

Summary The plasmas of two groups of subjects were examined for blood coagulation Factor XIII-A (FXIII-A, F13A) by electrophoresis in agarose using a Tris-EDTA-borate buffer to separate the common variants, F13A^*1, F13A^*2, and F13A^*3. Dimeric subunits were visualized in UV light as monodansyl cadaverine bound to casein at the position of the transglutaminase activity representing F13A. One test group consisted of 307 members of three large Caucasian families. The other consisted of 148 consecutive patients whose plasmas had been sent to the clinical laboratory for determination of prothrombin time. Segregation analysis and father-to-son transmission confirmed that F13A is inherited as an autosomal co-dominant trait. The allelic frequencies in the random sample were F13A^*1=0.82 and F13A^*2=0.18. This sample included both blacks and whites, and the gene frequencies were not significantly different in the two races. The gene frequencies among the unrelated spouses of the three white families were A^*1=0.75, A^*2=0.24, A^*3=0.01. Genetic equilibrium was present in both groups.The degree of polymorphism, the availability of blood, the ease of assessment, the absence of selective pressure, and the uniformity of gene frequencies in two major American ethnic groups make F13A a very useful marker for linkage studies and paternity testing. F13A has been provisionally assigned to chromosome 6. Linkage analysis of our family data did not provide evidence of linkage to two chromosome 6 markers, properdin factor B (BF) and glyoxalase 1 (GLO). The highest lod score (Z) was between F13A and the Kidd (Jk) blood group (Z=0.68 at -0.24).     

Efficiency and robustness of pedigree segregation analysis.

Different pedigree structures and likelihoods are examined to determine their efficiency for parameter estimation under one-locus models. For the cases simulated, family size has little effect; estimates based on unconditional likelihoods are generally more efficient than those based on conditional likelihoods. The proposed method of pedigree analysis under a one-locus model is found to be robust in the analysis of nuclear families: skewness of the data and polygenic inheritance will not lead to the spurious detection of major loci unless they occur simultaneously, and together with a moderate amount of environmental correlation among sibs.     

Improved power by use of a weighted score test for linkage disequilibrium mapping

Association studies offer an exciting approach to finding underlying genetic variants of complex human diseases. However, identification of genetic variants still includes difficult challenges, and it is important to develop powerful new statistical methods. Currently, association methods may depend on single-locus analysis--that is, analysis of the association of one locus, which is typically a single-nucleotide polymorphism (SNP), at a time--or on multilocus analysis, in which multiple SNPs are used to allow extraction of maximum information about linkage disequilibrium (LD). It has been shown that single-locus analysis may have low power because a single SNP often has limited LD information. Multilocus analysis, which is more informative, can be performed on the basis of either haplotypes or genotypes. It may lose power because of the often large number of degrees of freedom involved. The ideal method must make full use of important information from multiple loci but avoid increasing the degrees of freedom. Therefore, we propose a method to capture information from multiple SNPs but with the use of fewer degrees of freedom. When a set of SNPs in a block are correlated because of LD, we might expect that the genotype variation among the different phenotypic groups would extend across all the SNPs, and this information could be compressed into the low-frequency components of a Fourier transform. Therefore, we develop a test based on weighted Fourier transformation coefficients, with more weight given to the low-frequency components. Our simulation results demonstrate the validity and substantially higher power of the proposed method compared with other common methods. This method provides an additional tool to existing methods for identification of causative genetic variants underlying complex diseases.     

Divergent Roles of CAAX Motif-signaled Posttranslational Modifications in the Regulation and Subcellular Localization of Ral GTPases

The Ras-like small GTPases RalA and RalB are well validated effectors of RAS oncogene-driven human cancer growth, and pharmacologic inhibitors of Ral function may provide an effective anti-Ras therapeutic strategy. Intriguingly, although RalA and RalB share strong overall amino acid sequence identity, exhibit essentially identical structural and biochemical properties, and can utilize the same downstream effectors, they also exhibit divergent and sometimes opposing roles in the tumorigenic and metastatic growth of different cancer types. These distinct biological functions have been attributed largely to sequence divergence in their carboxyl-terminal hypervariable regions. However, the role of posttranslational modifications signaled by the hypervariable region carboxyl-terminal tetrapeptide CAAX motif (C = cysteine, A = aliphatic amino acid, X = terminal residue) in Ral isoform-selective functions has not been addressed. We determined that these modifications have distinct roles and consequences. Both RalA and RalB require Ras converting CAAX endopeptidase 1 (RCE1) for association with the plasma membrane, albeit not with endomembranes, and loss of RCE1 caused mislocalization as well as sustained activation of both RalA and RalB. In contrast, isoprenylcysteine carboxylmethyltransferase (ICMT) deficiency disrupted plasma membrane localization only of RalB, whereas RalA depended on ICMT for efficient endosomal localization. Furthermore, the absence of ICMT increased stability of RalB but not RalA protein. Finally, palmitoylation was critical for subcellular localization of RalB but not RalA. In summary, we have identified striking isoform-specific consequences of distinct CAAX-signaled posttranslational modifications that contribute to the divergent subcellular localization and activity of RalA and RalB.     

Evidence for a major gene influence on tumor necrosis factor-alpha expression in tuberculosis: path and segregation analysis

OBJECTIVE: Tuberculosis (TB) is a growing global public health problem. Several studies suggest a role for host genetics in disease susceptibility, but studies to date have been inconsistent and a comprehensive genetic model has not emerged. A limitation of previous genetic studies is that they only analyzed the binary trait TB, which does not reflect disease heterogeneity. Furthermore, these studies have not accounted for the influence of shared environment within households on TB risk, which may spuriously inflate estimates of heritability. METHODS: We conducted a household contact study in a TB-endemic community in Uganda. Antigen-induced tumor necrosis factor-alpha (TNFalpha) expression, a key component of the underlying immune response to TB, was used as an endophenotype for TB. RESULTS: Path analysis, conducted to assess the effect of shared environment, suggested that TNFalpha is heritable (narrow sense heritability = 34-66%); the effect of shared environment is minimal (1-14%), but gene-environment interaction may be involved. Segregation analysis of TNFalpha suggested a major gene model that explained one-third of the phenotypic variance, and provided putative evidence of natural selection acting on this phenotype. CONCLUSION: Our data further support TNFalpha as an endophenotype for TB, as it may increase power to detect disease-predisposing loci.     

A biosensor generated via high-throughput screening quantifies cell edge Src dynamics

Fluorescent biosensors for living cells currently require laborious optimization and a unique design for each target. They are limited by the availability of naturally occurring ligands with appropriate target specificity. Here we describe a biosensor based on an engineered fibronectin monobody scaffold that can be tailored to bind different targets via high-throughput screening. We made this Src-family kinase (SFK) biosensor by derivatizing a monobody specific for activated SFKs with a bright dye whose fluorescence increases upon target binding. We identified sites for dye attachment and changes to eliminate vesiculation in living cells, providing a generalizable scaffold for biosensor production. This approach minimizes cell perturbation because it senses endogenous, unmodified target, and because sensitivity is enhanced by direct dye excitation. Automated correlation of cell velocities and SFK activity revealed that SFKs are activated specifically during protrusion. Activity correlates with velocity, and peaks 1-2 μm from the leading edge.     

Comparison of microsatellites, single-nucleotide polymorphisms (SNPs) and composite markers derived from SNPs in linkage analysis

There is growing evidence that a map of dense single-nucleotide polymorphisms (SNPs) can outperform a map of sparse microsatellites for linkage analysis. There is also argument as to whether a clustered SNP map can outperform an evenly spaced SNP map. Using Genetic Analysis Workshop 14 simulated data, we compared for linkage analysis microsatellites, SNPs, and composite markers derived from SNPs. We encoded the composite markers in a two-step approach, in which the maximum identity length contrast method was employed to allow for recombination between loci. A SNP map 2.3 times as dense as a microsatellite map (approximately 2.9 cM compared to approximately 6.7 cM apart) provided slightly less information content (approximately 0.83 compared to approximately 0.89). Most inheritance information could be extracted when the SNPs were spaced < 1 cM apart. Comparing the linkage results on using SNPs or composite markers derived from them based on both 3 cM and 0.3 cM resolution maps, we showed that the inter-SNP distance should be kept small (< 1 cM), and that for multipoint linkage analysis the original markers and the derived composite markers had similar power; but for single point linkage analysis the resulting composite markers lead to more power. Considering all factors, such as information content, flexibility of analysis method, map errors, and genotyping errors, a map of clustered SNPs can be an efficient design for a genome-wide linkage scan.     

Impacts of climate change on national biodiversity population trends

Climate change has had well‐documented impacts on the distribution and phenology of species across many taxa, but impacts on species’ abundance, which relates closely to extinction risk and ecosystem function, have not been assessed across taxa. In the most comprehensive multi‐taxa comparison to date, we modelled variation in national population indices of 501 mammal, bird, aphid, butterfly and moth species as a function of annual variation in weather variables, which through time allowed us to identify a component of species’ population growth that can be associated with post‐1970s climate trends. We found evidence that these climate trends have significantly affected population trends of 15.8% of species, including eight with extreme (> 30% decline per decade) negative trends consistent with detrimental impacts of climate change. The modelled effect of climate change could explain 48% of the significant across‐species population decline in moths and 63% of the population increase in winged aphids. The other taxa did not have significant across‐species population trends or consistent climate change responses. Population declines in species of conservation concern were linked to both climatic and non‐climatic factors respectively accounting for 42 and 58% of the decline. Evident differential impacts of climate change between trophic levels may signal the potential for future ecosystem disruption. Climate change has therefore already driven large‐scale population changes of some species, had significant impacts on the overall abundance of some key invertebrate groups and may already have altered biological communities and ecosystems in Great Britain.     

Use of novel nest boxes by carmine bee‐eaters (Merops nubicus) in captivity

Carmine bee‐eaters make attractive additions to zoo aviaries but breeding programs have had challenges and limited success. The objectives of this study were to document nesting behavior of Carmine bee‐eaters in a captive setting and compare reproductive success between a novel nest box (plastic, 17 × 30 × 22 cm) and a PVC pipe model used previously (30 cm long, 8 cm in diameter). Three bee‐eater pairs were given access to seven nest chambers (six novel boxes, one PVC model). Behavioral observations occurred during a 15‐min period in the morning or afternoon before egg production and continued until chicks fledged for a total of 87 observation periods (21.75 hr). All occurrences by an individual bird entering or exiting a nest tunnel, food provision, and the time (min) spent inside a nest cavity were documented. Additionally, daily temperature within each nest chamber was recorded. Before eggs were produced the average daily temperature (23.02°C) within the nest chambers did not differ, suggesting that nest cavity choice was not influenced by temperature. No differences were detected among pairs in percent of observed time spent inside their nest cavities or number of times a nest tunnel was entered during the incubation or fledging periods. During incubation females spent a greater percent of observed time inside the nest cavity than males (P=0.02). During the fledging period food provision did not differ between the pairs, however males entered their nest tunnels more often per hour than females (P=0.03), and males tended to provide food more often than females (P=0.053). Two pairs nested in novel nest boxes and successfully fledged one chick each. The pair that nested in the PVC model did not fledge a chick. A nest box that aids in keeping eggs intact is essential for breeding bee‐eaters in captivity, and maintaining captive populations will provide opportunities for zoo visitors to enjoy these birds and will reduce the need to remove birds from the wild. Zoo Biol 0:1–13, 2007. © 2007 Wiley‐Liss, Inc.