Comparing G: multivariate analysis of genetic variation in multiple populations.

The additive genetic variance-covariance matrix (G) summarizes the multivariate genetic relationships among a set of traits. The geometry of G describes the distribution of multivariate genetic variance, and generates genetic constraints that bias the direction of evolution. Determining if and how the multivariate genetic variance evolves has been limited by a number of analytical challenges in comparing G-matrices. Current methods for the comparison of G typically share several drawbacks: metrics that lack a direct relationship to evolutionary theory, the inability to be applied in conjunction with complex experimental designs, difficulties with determining statistical confidence in inferred differences and an inherently pair-wise focus. Here, we present a cohesive and general analytical framework for the comparative analysis of G that addresses these issues, and that incorporates and extends current methods with a strong geometrical basis. We describe the application of random skewers, common subspace analysis, the 4th-order genetic covariance tensor and the decomposition of the multivariate breeders equation, all within a Bayesian framework. We illustrate these methods using data from an artificial selection experiment on eight traits in Drosophila serrata, where a multi-generational pedigree was available to estimate G in each of six populations. One method, the tensor, elegantly captures all of the variation in genetic variance among populations, and allows the identification of the trait combinations that differ most in genetic variance. The tensor approach is likely to be the most generally applicable method to the comparison of G-matrices from any sampling or experimental design.

Effects of SCA1, MJD, and DPRLA triplet repeat polymorphisms on cognitive phenotypes in a normal population of adolescent twins.

The expansion of unstable trinucleotide CAG repeat polymorphisms of a number of genes causes several neurodegenerative disorders with decreased cognitive function, the severity of the disorder being related to allele length at the triplet repeat locus. While the effects of repeat length have been well studied in clinical samples, there has been little investigation of the effects of triplet repeat variation in the normal range for these genes. We have, therefore, examined linkage and association for three CAG triplet repeat markers (Spinocerebellar Ataxia Type 1, SCA1; Machado-Joseph Disease, MJD; Dentatorubro-pallidoluysian Atrophy, DRPLA) to assess their contribution to variation in cognitive ability (IQ, reading ability, processing speed) in a normal, unselected sample of adolescent twins (248 dizygotic (DZ) sibling pairs, aged 16 years). Association tests, performed in Mx and QTDT, showed a consistent positive association of SCA1 with Arithmetic (P = 0.04). While association was supported between SCA1 and Cambridge reading scores and between DRPLA and inspection time, results were inconsistent across software packages. Given the number of statistical tests performed, it is unlikely that trinucleotide repeat variation in the normal range for these genes influences variation in normal cognition.

Searching for new markers of endogenous steroid administration in athletes: "looking outside the metabolic box".

A simple means of detecting the abuse of steroids that also occur naturally is a problem facing doping control laboratories. Specific markers are required to allow the detection of the administration of these steroids. These markers are commonly measured using a set of data obtained from the screening of samples by gas chromatography-mass spectrometry (GC-MS). Doping control laboratories further need to confirm identified abuse using techniques such as gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). An interesting urinary species was found while following the pharmacokinetics and changes to the steroid profile from single and multiple oral doses of the International Olympic Committee/World Anti Doping Agency (IOC/WADA) prohibited substance, dehydroepiandrosterone (DHEA). The urine samples collected from the administration studies were subject to GC-MS and GC-C-IRMS steroid analysis following cleanup by solid phase extraction techniques. A useful urinary product of DHEA administration was detected in the urine samples from each of the administration studies and was identified by GC-MS experiments to be 3alpha,5-cyclo-5alpha-androstan-6beta-ol-17-one (3alpha,5-cyclo). This compound occurs naturally but the concentrations of 3alpha,5-cyclo were elevated following both the single DHEA administration (up to 385 ng/mL) and multiple DHEA administrations (up to 1240 ng/mL), in relation to those observed prior to these administrations (70 and 80 ng/mL, respectively). A reference distribution of urine samples collected from elite athletes (n = 632) enabled the natural concentration range of 3alpha,5-cyclo to be established (0-280 ng/mL), with a mean concentration of 22 ng/mL. Based on this an upper 3alpha,5-cyclo concentration limit of 140 ng/mL is proposed as a GC-MS screening marker of DHEA abuse in athletes. GC-C-IRMS analysis revealed significant 13C depletion of 3alpha,5-cyclo following DHEA administration. In the single administration study, the delta13C value of 3alpha,5-cyclo changed from -24.3 per thousand to a minimum value of -31.1 per thousand at 9 h post-administration, before returning to its original value after 48 h. The multiple administration study had a minimum delta13C 3alpha,5-cyclo of -33.9 per thousand during the administration phase in contrast to the initial value of -24.2 per thousand. Preliminary studies have shown 3alpha,5-cyclo to most likely be produced from DHEA sulfate found at high levels in urine. The complementary use of GC-MS and GC-C-IRMS to identify new markers of steroid abuse and the application of screening criteria incorporating such markers could also be adapted by doping control laboratories to detect metabolites of androstenedione, testosterone and dihydrotestosterone abuse.

Detection of mRNA from the immediate early gene of human cytomegalovirus in infected cells by in vitro amplification.

It would be desirable to have a facile means of detecting the presence of viral DNA or mRNA in any given biological sample, especially in view of the growing immunocompromised population in which the diagnosis of viral disease is a common problem. In vitro amplification of DNA using methods such as the polymerase chain reaction, offers a sensitive means of detecting both DNA and mRNA. We have used the polymerase chain reaction to detect DNA and mRNA from human cytomegalovirus infected human fibroblasts. Viral mRNA was differentiated from DNA using primers which flank a splice junction, resulting in a smaller product for the mRNA template. A cDNA was prepared from total RNA using a primer specific for the gene of interest, in this case the major immediate early transcript of human cytomegalovirus. The cDNA was then amplified using a modified polymerase chain reaction protocol. mRNA from the major immediate early gene was detected in cultured fibroblasts as early as 6 h after infection, and continued to be expressed for at least 96 h post infection. Sensitive and facile detection of viral mRNA should facilitate diagnostic and basic studies of viral pathogenesis.

Analysis of DNA in fresh and fixed tissue by the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify viral or oncogene sequences from frozen or formalin-fixed, paraffin-embedded tissue sections. Methods for preparing fixed, embedded colonic tissue for PCR amplification of c-K-ras sequences from genomic DNA and for amplification of viral DNA from other tissues, including brain, lung, and liver, were evaluated. The effect of formalin fixation on the efficiency of amplification was also determined. While there seemed to be only a modest variation in the efficiency of the PCR for amplification of single-copy human genes, regardless of the methods used for tissue preparation, amplification of viral DNA sequences against a human genomic DNA background was more efficient when the DNA was purified to some degree before amplification of the tissue. We used the PCR to examine frozen and fixed embedded tissue sections for the presence of Epstein-Barr virus (EBV) and cytomegalovirus (CMV) DNA. One patient with a heart-lung transplant succumbed to a lymphoproliferative disorder, and EBV genome was present in tissues with abnormal lymphoid infiltrates. CMV was also present in bronchial lavages from the same patient, where cytologic diagnosis was not apparent. Another patient with a liver transplant showed CMV genome in multiple liver biopsies, with negative histologic results for CMV. In vitro DNA amplification with the PCR demonstrated sensitivity superior to that of histology in detecting CMV and EBV in the cases examined.

New semisolid agar for the detection of motile salmonellae.

A semisolid selective motility agar based on selenite broth and designated semisolid selenite fecal (SSF) agar was developed for recovery of motile salmonellae from large numbers of rodent fecal specimens. The medium is easily prepared and inoculated; results are readily interpreted. When combined with selenite or gram-negative broth enrichment, SSF agar yielded significantly better Salmonella isolation from experimentally infected rodents than a standard battery of media. Only 14 non-Salmonella isolates from 1,002 Salmonella-free rodent fecal specimens migrated on SSF agar.