GABAA receptor beta subunit mRNA expression in the human alcoholic brain

A competitive RT-PCR assay was used to quantify the expression of the GABA(A) receptor beta(1), beta(2) and beta(3) isoform mRNA transcripts in the superior frontal cortex and motor cortex of 21 control and 22 alcoholic cases. A single set of primers was designed that permitted amplification of all three transcripts and the internal standard simultaneously; differentiation of the individual transcripts was achieved by restriction enzyme digestion. Construction of a standard curve, using the internal standard and a concentration range of beta(2) cRNA-enabled quantitation of mRNA expression levels. No significant difference in mRNA expression was found between the control and alcoholic case groups in either the superior frontal or motor cortex for the beta(2) or beta(3) isoforms. A significant interaction was found between isoform and area, although, the two case groups did not partition on this measure. The interaction was due to a significant difference between superior frontal and motor cortex for the beta(3) isoform; this regional comparison was not significant for beta(2) mRNA. Age at death and post-mortem delay (PMD) had no significant effect on beta mRNA expression in either case group in either region. A beta(1) signal could not be detected in the RT-PCR assay. (C) 2004 Elsevier Ltd. All rights reserved.

Association between chronic fatigue syndrome and the corticosteroid-binding globulin gene ALA SER224 polymorphism

Chronic fatigue syndrome (CFS) is characterized by idiopathic fatigue of greater than 6 months' duration with postexertional exacerbation and many other symptoms. A trend toward relative hypocortisolism is described in CFS. Twin and family studies indicate a substantial genetic etiologic component to CFS. Recently, severe corticosteroid-binding globulin (CBG) gene mutations have been associated with CFS in isolated kindreds. Human leukocyte elastase, an enzyme important in CBG catabolism at inflammatory sites, is reported to be elevated in CFS. We hypothesized that CBG gene polymorphisms may act as a genetic risk factor for CFS. A total of 248 patients with CFS defined by Centers for Disease Control criteria, and 248 controls were recruited. Sequencing and restriction enzyme testing of the CBG gene coding region allowed detection of severe CBG gene mutations and a common exon 3 polymorphism (c.825G --> T, Ala-Ser(224)). Plasma CBG levels were measured in 125 CFS patients and 198 controls by radioimmunoassay. Total and free (calculated and measured) cortisol levels were ascertained in single samples between 8-10 a.m. The age of onset (mid 30s) and gender ratio (2.2:1, female:male) of the patients were similar to those reported in U.S. epidemiologic studies. A trend toward a preponderance of serine(224) homozygosity among the CFS patients was noted, compared with controls (chi(2) = 5.31, P = 0.07). Immunoreactive-CBG (IR-CBG) levels were higher in Serine/Alanine (Ser/Ala) than Ala/Ala subjects and higher again in Ser/Ser subjects, this effect was strongest in controls; Ser/Ser: 46.1 +/- 1.8 (n = 31, P = 0.03) vs. Ser/Ala: 42.4 +/- 1.0 (n = 56, P = 0.05) vs. Ala/Ala: 40.8 +/- 1.7 mug/mL (n = 21). Despite higher CBG levels, there was a nonsignificant trend toward lower total and free plasma cortisol in serine allele positive patients, total cortisol: Ser/Ser: 13.3 +/- 1.4 (n = 34) vs. Ser/Ala: 14.0 +/- 0.7 (n = 66) vs. Ala/Ala: 15.4 +/- 1.0 (n = 23). Homozygosity for the serine allele of the CBG gene may predispose to CFS, perhaps due to an effect on hypothalamic-pituitary-adrenal axis function related to altered CBG-cortisol transport function or immune-cortisol interactions.

Detection and discrimination of herpes simplex viruses, Haemophilus ducreyi, Treponema pallidum, and Calymmatobacterium (Klebsiella) granulomatis from genital ulcers

Background. Genital ulcer disease (GUD) is commonly caused by pathogens for which suitable therapies exist, but clinical and laboratory diagnoses may be problematic. This collaborative project was undertaken to address the need for a rapid, economical, and sensitive approach to the detection and diagnosis of GUD using noninvasive techniques to sample genital ulcers. Methods. The genital ulcer disease multiplex polymerase chain reaction (GUMP) was developed as an inhouse nucleic acid amplification technique targeting serious causes of GUD, namely, herpes simplex viruses (HSVs), Haemophilus ducreyi, Treponema pallidum, and Klebsiella species. In addition, the GUMP assay included an endogenous internal control. Amplification products from GUMP were detected by enzyme linked amplicon hybridization assay (ELAHA). Results. GUMP-ELAHA was sensitive and specific in detecting a target microbe in 34.3% of specimens, including 1 detection of HSV-1, three detections of HSV-2, and 18 detections of T. pallidum. No H. ducreyi has been detected in Australia since 1998, and none was detected here. No Calymmatobacterium ( Klebsiella) granulomatis was detected in the study, but there were 3 detections during ongoing diagnostic use of GUMP-ELAHA in 2004 and 2005. The presence of C. granulomatis was confirmed by restriction enzyme digestion and nucleotide sequencing of the 16S rRNA gene for phylogenetic analysis. Conclusions. GUMP-ELAHA permitted comprehensive detection of common and rare causes of GUD and incorporated noninvasive sampling techniques. Data obtained by using GUMP-ELAHA will aid specific treatment of GUD and better define the prevalence of each microbe among at-risk populations with a view to the eradication of chancroid and donovanosis in Australia.

Teaching principles of genetics through SNP analysis

An understanding of inheritance requires comprehension of genetic processes at all levels, from molecules to populations. Frequently genetics courses are separated into molecular and organismal genetics and students may fail to see the relationships between them. This is particularly true with human genetics, because of the difficulties in designing experimental approaches which are consistent with ethical restrictions, student abilities and background knowledge, and available time and materials. During 2005 we used analysis of single nucleotide polymorphisms (SNPs) in two genetic regions to enhance student learning and provide a practical experience in human genetics. Students scanned databases to discover SNPs in a gene of interest, used software to design PCR primers and a restriction enzyme based assay for the alleles, and carried out an analysis of the SNP on anonymous individual and family DNAs. The project occupied eight to ten hours per week for one semester, with some time spent in the laboratory and some spent in database searching, reading and writing the report. In completing their projects, students acquired a knowledge of Mendel’s first law (through looking at inheritance patterns), Mendel’s second law and the exceptions (the concepts of linkage and linkage disequilibrium), DNA structure (primer design and restriction enzyme analysis) and function (SNPs in coding and non-coding regions), population genetics and the statistical analysis of allele frequencies, genomics, bioinformatics and the ethical issues associated with the use of human samples. They also developed skills in presentation of results by publication and conference participation. Deficiencies in their understanding (for example of inheritance patterns, gene structure, statistical approaches and report writing) were detected and guidance given during the project. SNP analysis was found to be a powerful approach to enhance and integrate student understanding of genetic concepts.