Using Genomic Data to Find Disease-Modifying Loci in Huntington's Disease (HD).

In this chapter, genetic modifiers are defined, and the rationale for investigating them in HD explained. Issues involved in modeling the phenotype are discussed, using age at motor onset as an example. The statistical methods for analyzing genetic data (linkage and association) are discussed, along with the advantages and disadvantages of each. In particular, the advantage of a genome-wide approach over one based on candidate genes is stressed. Genome-wide association studies (GWAS) are current method of choice to detect genetic modifiers. The power of GWAS is discussed, along with sources of error, and how these might be detected and corrected. Extensions to GWAS, such as gene- and pathway-wide analyses, are discussed, and also how GWAS may be used to estimate genetic risks and trait heritability. Since GWAS are most effective to detect common genetic variants, methods for analyzing rare variation are also discussed. The uses of other types of genomic data (notably, expression) are discussed, and how they might be integrated with genetic data to find causal genes and variants. The chapter ends with a short overview of future prospects for detecting genetic modifiers of HD.

Last Glacial Maximum ages for robust humans at Kow Swamp, southern Australia.

The Kow Swamp people are a fossil population of robust modern humans. We report optically stimulated luminescence (OSL) ages on sediments from Kow Swamp that are at odds with radiocarbon ages obtained previously for the site. The calibrated 14C ages place the Kow Swamp people in the period 15-9 ka. Our single aliquot OSL ages suggest that they lived around the time of the Last Glacial Maximum (LGM) between 22 and 19 ka. An LGM age for the Kow Swamp people is supported by palaeoenvironmental reconstruction. The shoreline silt, in which most of them were interred, was deposited by high lake levels between 26 and 19 ka. Few robust people were left after 19 ka when a sand lunette formed. Climate change may explain the demise of this unusual genetic population.

Evaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyes.

Two-dimensional difference gel electrophoresis (2-D DIGE) enables an increased confidence in detection of protein differences. However, due to the nature of the minimal labelling where only approximately 5% of a given protein is labelled, spots cannot be directly excised for mass spectrometry (MS) analysis and detection sensitivity could be further enhanced. Amersham Biosciences have developed a second set of CyDye DIGE Cy 3 and Cy5 dyes, which aim to overcome these limitations through saturation-labelling of cysteine residues. The dyes were evaluated in relation to their sensitivity and dynamic range, their useability as multiplexing reagents and the possibility of direct spot picking from saturation-labelled gels for MS analysis. The saturation-labelling dyes were superior in sensitivity to their minimal-labelling counterparts, silver stain and Sypro Ruby, however, the resulting 2-D spot pattern was significantly altered from that of unlabelled or minimal-labelled protein. The dyes were found to be useful as multiplexing reagents although preferential labelling of proteins with one dye over another was observed but was controlled for through experimental design. Protein identities were successfully obtained from material directly excised from saturation-labelled gels eliminating the need for post-stained preparative gels.

Fluorescence two-dimensional difference gel electrophoresis and mass spectrometry based proteomic analysis of Escherichia coli.

Separation and relative quantitation of complex protein mixtures remain two of the most challenging aspects of proteomics. Here an advanced technique called fluorescence difference 2-D gel electrophoresis technology (2D-DIGE) has been applied to a model system study of the Escherichia coli proteome after benzoic acid treatment. The molecular weight and charge matched cyanine dyes enable pre-electrophoretic labelling of control and treated samples which are then mixed and run in the same gel. Pooled control and treated samples labelled with Cy trade mark 3 were used as an internal standard for both Cy5 labelled control and treated E. coli samples. Together with DeCyder trade mark imaging analysis software, more accurate quantitative analysis than conventional two-dimensional polyacrylamide gel electrophoresis was achieved. Using matrix-assisted laser desorption/ionization-time of flight and quadrupole-time of flight mass spectrometry a total of 179 differentially expressed protein spots were identified. These included enzymes, stress related and substrate (e.g. amino acids, maltose, ribose and TRP repressor) binding proteins. Of the spots analysed, 77% contained only one protein species per spot, hence the change in protein expression measured was solely attributed to the identified protein. Many membrane proteins and protein isoforms were identified indicating both adequate solubilization of E. coli samples and potential post-translational modification. The results indicate that the regulatory mechanisms following benzoic acid treatment of E. coli are far more complicated than hitherto expected.