Lessons from postgenome-wide association studies: functional analysis of cancer predisposition loci.

In the last few years, genome-wide association studies (GWASs) have identified hundreds of predisposition loci for several types of human cancers. Recent progress has been made in determining the underlying mechanisms through which different single-nucleotide polymorphisms (SNPs) affect predisposition to cancer. Although there has been much debate about the clinical utility of GWASs, less attention has been paid to how GWASs and post-GWASs functional analysis have contributed to understanding the aetiology of cancer. Most common variants associated with cancer risk are localized in nonprotein-coding regions highlighting transcriptional regulation as a common theme in the mechanism of cancer predisposition. Here, we outline strategies to functionally dissect predisposition loci and discuss their limitations as well as challenges for future studies.

A candidate gene approach to searching for low-penetrance breast and prostate cancer genes.

Most cases of breast and prostate cancer are not associated with mutations in known high-penetrance genes, indicating the involvement of multiple low-penetrance risk alleles. Studies that have attempted to identify these genes have met with limited success. The National Cancer Institute Breast and Prostate Cancer Cohort Consortium--a pooled analysis of multiple large cohort studies with a total of more than 5,000 cases of breast cancer and 8,000 cases of prostate cancer--was therefore initiated. The goal of this consortium is to characterize variations in approximately 50 genes that mediate two pathways that are associated with these cancers--the steroid-hormone metabolism pathway and the insulin-like growth factor signalling pathway--and to associate these variations with cancer risk.

Short-term retention of lexical-semantic representations: implications for speech production.

Patients with semantic STM deficits have difficulty comprehending sentences that require the retention of several lexical-semantic representations prior to their integration into higher-level propositions (Martin, 1995: Martin & Romani. 1994). In Experiment 1, patients with a semantic retention deficit had difficulty with the same type of constructions in speech production, namely noun phrases with one or two prenominal adjectives. Their performance improved when they could produce the nouns and adjectives in sentence form, which placed smaller demands on lexical-semantic retention. In Experiment 2 these patients were better able to produce syntactically complex sentences than the prenominal adjective phrases having an equal number of content words, indicating that the findings in Experiment 1 could not be attributed to syntactic complexity. These patients produced more pauses in the sentence constructions in Experiments 1 and 2, suggesting that the timing of such productions is abnormal. In contrast, patient EA, with a phonological retention deficit, performed better than the patients with a semantic retention deficit on the AN phrases despite having a smaller STM span. She showed no significant benefit of producing sentence compared to phrase constructions, and also made fewer and shorter pauses than the other patients. These findings support the multiple capacities view of verbal working memory and suggest that the same semantic retention capacity used in language comprehension is used in speech production.

Dissociable components of short-term memory and their relation to long-term learning.

Short-term memory (STM) includes dissociable phonological and semantic components (R.C. Martin, 1993). Previous findings indicate that phonological STM capacity supports learning of novel phonological forms, such as new vocabulary (e.g., Baddeley, Gathercole, & Papagno, 1998). It was hypothesised that semantic STM capacity would support the learning of novel semantic information. Five aphasic patients were tested who demonstrated deficits in the short-term retention of either phonological or semantic information. Four of the five patients demonstrated learning deficits in a paired associate paradigm that corresponded to their STM deficits. One patient with a severe deficit in phonological STM but a better-preserved ability to retain semantic information showed better learning of new semantic information than new phonological information. Three patients with a greater deficit in semantic than phonological STM showed the reverse. A fifth patient with a severe semantic STM deficit failed to show learning for either type of material. Results suggest that the semantic and phonological components of STM are essential for the long-term learning of corresponding representations in long-term memory.

Increased peroxidation and reduced antioxidant enzyme activity in Alzheimer's disease.

The overall peroxidation activity in brain tissue by region from patients with Alzheimer's disease (AD) and age-matched controls was determined employing the thiobarbituric acid-reactive substances (TBARS) assay, a measure of lipid peroxidation, followed by a determination the activities of the antioxidant enzymes Cu/Zn superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), in the frontal, temporal, and cerebellar cortex of 10 AD and 9 control brains. The level of TBARS was elevated in all regions, with particular statistical significance in the temporal cortex when compared to age-matched controls. SOD activity was significantly decreased in AD frontal and AD temporal cortex, while catalase activity was significantly decreased in AD temporal cortex. There was no significant difference in GSH-Px activity found in any of the regions examined. This study supports the theory that in AD the brain is affected by increased oxidative stress which, when combined with a decrease in SOD activity, produces oxidative alterations, seen most significantly in temporal cortex in AD, where the pathophysiologic changes are most severe.

Quantitative neuronal c-fos and c-jun expression in Alzheimer's disease.

Apoptosis, or programmed cell death, has been proposed as a mechanism of neuropathology in Alzheimer's disease (AD). Activation of immediate early genes (IEG) c-jun and c-fos appears to be required for the initiation of apoptosis. Furthermore, the expression of c-jun is induced in cultured neurons that undergo beta-amyloid-mediated apoptosis suggesting a direct role for c-jun in the apoptosis of AD neurons. Using immunohistochemical methods, we calculated the average number of neuronal profiles per unit area expressing c-Jun and c-Fos within hippocampal regions CA1, CA2/3, and CA4 in postmortem brain samples from AD patients and age-matched non-AD patients. There was an increase in c-Jun-positive and c-Fos-positive neuronal profile density in nearly all AD hippocampal regions examined. In cerebellum there was no evidence of apoptosis as determined by using TUNEL technique, and negligible c-Jun labeling.

Decreased brain glucose metabolism in microvessels from patients with Alzheimer's disease.

We studied brain glucose metabolism in patients with Alzheimer's disease and age-matched controls in vivo by PET and assessed brain glucose utilization and the phosphorylation constant K3 for hexokinase. In addition we determined in vitro the binding of 2DG and measured its phosphorylation to 2DG-phosphate in cerebral microvessels obtained at autopsy from subjects with Alzheimer's disease and age-matched controls. In patients with Alzheimer's disease we found a marked decrease in the kinetic constant K3 for the hexokinase, and a marked decrease in the overall metabolism of glucose in our PET studies; in microvessels there was a marked decrease in the affinity of 2DG and a decrease in hexokinase activity. Alzheimer's disease may be related to a complex alteration in brain glucose metabolism.

Dosimetry and cost of imaging osseointegrated implants with film-based and computed tomography.

Thermoluminescent dosimeters were used to measure radiation doses at craniofacial sites in a tissue-equivalent phantom during film-based multidirectional tomography with the Tomax Ultrascan (Incubation Industries, Ivyland, Pa.) and during computed tomography with the Elscint Excel 2400 (Elscint Corp., Tel Aviv, Israel). Mean absorbed doses for presurgical mandibular and maxillary canine and molar implant assessments were converted to equivalent doses, which were then multiplied by published weighting factors and summed to give effective doses. The computed tomography device consistently delivered higher doses than the Tomax Ultrascan to all anatomic locations; the differences were most pronounced when only one or two implant sites were evaluated. The reasons for the dose disparities are considered both anatomically and procedurally. A survey of examination cost revealed film-based multidirectional tomography to be less expensive than computed tomography.

Niobium filtration of conventional and high-frequency x-ray generator beams for intraoral radiography. Effects on absorbed doses, image density and contrast, and photon spectra.

We have studied the effects of niobium beam filtration on absorbed doses, on image density and contrast, and on photon spectra with conventional and high-frequency dental x-ray generators. Added niobium reduced entry and superficial absorbed doses in periapical radiography by 9% to 40% with film and digital image receptors, decreased the radiation necessary to produce a given image density on E-speed film and reduced image contrast on D- and E-speed films. As shown by increased half-value layers for aluminum, titanium, and copper and by pulse-height analyses of beam spectra, niobium increased average beam energy by 6% to 19%. Despite the benefits of adding niobium on patient dose reduction and on narrowing the beams' energy spectra, the beam can be overhardened. Adding niobium, therefore, strikes the best balance between radiation dose reduction and beam attenuation, with its risks of increased exposure times, motion blur, and diminished image contrast, when it is used at modest thicknesses (30 microns) and at lower kVp (70) settings.

Brain glucose metabolism in Alzheimer's disease.

In vitro determination of brain glucose metabolism in the temporal cortex from patients with Alzheimer's disease showed a marked decrease when compared with nondemented, age-matched control subjects. Additional determinations on normal human temporal cortex obtained at autopsy demonstrated an age-dependent decline in the rate of glucose use. These data provide an in vitro correlate for positron emission tomography studies that suggest decreased brain glucose use in Alzheimer's disease.

Alteration of irradiated shuttle vector processing by exposure of human lymphoblast host cells to single or split gamma-ray doses.

The repair of damaged DNA by mammalian cells exposed to single or split doses of radiation was probed with shuttle vector pZ189. Human lymphoblast hosts who received a single 120 cGy dose 2 h before transfection with 2500 cGy-damaged pZ189 yielded a two-fold higher frequency of progeny plasmids with mutations in their supF-tRNA target genes than did unirradiated host cells. Delaying transfection for 12 h, however, reduced the mutation frequency by half versus unirradiated controls. Plasmid survival was also affected by the time between host cell irradiation and transfection. Splitting doses of 50-500 cGy into two equal fractions separated by 4 h lowered mutation frequency and increased plasmid survival compared with equivalent acute doses; increasing the interval between dose fractions to 8 h, however, lowered plasmid survival compared with acute doses. Sequence analyses of the target gene in mutant plasmids revealed increased multiple-base substitution mutations among progenies recovered from irradiated hosts, indicating enhanced excision repair. These findings support modulation of mammalian cell DNA repair by ionizing radiation, disclose the transient nature of the effect of radiation on DNA repair, and demonstrate a quantitative difference in the effectiveness of single and split doses.

Mutations caused by gamma-radiation-induced double-strand breaks in a shuttle plasmid replicated in human lymphoblasts.

The mutagenicity of open-circular DNA (containing base damage and single-strand breaks) and linear DNA (containing base damage, single-strand breaks, and one double-strand break) produced in vitro by gamma-irradiation of shuttle vector pZ189, was analysed after the plasmid's repair and replication in the human lymphoblast line, GM606. By comparing the survival, mutation frequency, and types of mutations in descendants from the two DNA forms, the effects of the double-strand break were determined. The percentage of viable plasmids from linear DNA was two-fold lower than that from open-circular DNA, 7.8 versus 14.0 (compared with unirradiated, control DNA). The mutation frequency in progenies of the open-circular plasmid was 4.2 +/- 1.7 x 10(-3), compared with 7.8 +/- 0.1 x 10(-3) in progenies of the linear DNA, again, nearly a two-fold difference. Approximately 59% of the mutations from the linear DNA were deletions and 34% were base substitutions. In contrast, only 13% of mutations from open-circular DNA were deletions, but 87% were base substitutions. All recoverable deletions were small, ranging from 1 to 205 base pairs, and the majority contained direct repeats at the deletion junctions, indicating non-homologous recombinations. Thus, mutations found among descendants from the linear and open-circular DNAs were qualitatively similar but quantitatively different. The data suggests that producing one double-strand break in DNA by ionizing radiation causes a two-fold increase in both lethality and mutation frequency.

Mutation spectrum in gamma-irradiated shuttle vector replicated in ataxia-telangiectasia lymphoblasts.

Cells from ataxia-telangiectasia (AT) patients are hypersensitive to the lethal effects of ionizing radiation. To assess radiation mutagenesis in these cells, the SV40-based shuttle vector, pZ189, was used to analyze gamma-ray-induced mutations following the plasmid's replication in AT lymphoblasts. Progenies from the AT line GM2783 exposed to 50 Gy showed a mutation frequency of 7.6 x 10(-3), 63-fold over background; surviving plasmids were 3.4% of control. Both values were essentially the same as those of irradiated plasmids replicated in a normal lymphoblast line, GM606. In addition, pZ189 exposed to 25 Gy of gamma radiation and replicated in another normal lymphoblast line and in cells of two additional AT lymphoblast lines showed similar mutation frequencies and percentages of surviving plasmids. Qualitative comparison of plasmid mutations from AT and normal cells showed no significant differences, indicating that the damaged DNA was repaired with similar fidelity in AT and normal cells. These studies suggest that there is no correlation between the enhanced sensitivity of AT cells to killing by ionizing radiation and gamma-radiation-induced mutagenesis of plasmid DNA processed in these cells.

Dietary aluminum and Alzheimer's disease.

Aluminum accumulation has been implicated in the development of Alzheimer's Disease, a hallmark of which is decreased brain glucose metabolism. Dietary sources of aluminum include that which comes from the contact of food with aluminum cook ware, containers, foil, and utensils. Normal aluminum intake from all sources is estimated as 12-14 mg per day. We have evaluated brain glucose metabolism in the presence of physiologically achievable levels of aluminum, in the range 10(-15) to 10(-5) M. Our results indicate no effect of aluminum in the range tested. Thus the contact of food with aluminum cooking utensils cannot alone raise plasma aluminum concentrations to the millimolar levels required to decrease brain glucose metabolism. Non dietary pathophysiological mechanisms are operating which lead to the accumulation of tissue aluminum, since most of dietary aluminum is excreted by the kidney.

Dependence of the mutation spectrum in a shuttle plasmid replicated in human lymphoblasts on dose of gamma radiation.

The frequencies and types of mutations induced in the target gene, supF-tRNA, of the shuttle vector pZ189 were analysed following the replication of the gamma-irradiated plasmid in the human lymphoblastoid cell line, GM606. The mutation frequency measured in progeny of unirradiated pZ189 was 1.02 x 10(-4), increasing to 17.5 x 10(-4) at 1000 cGy, and to 63.4 x 10(-4) at 5000 cGy, approximately 17- and 62-fold over background levels, respectively. Simultaneously, the number of plasmids capable of replicating in Escherichia coli decreased with increasing radiation dose to 4% of the control value at 5000 cGy. Electrophoresis of the irradiated DNA showed a correlation between increases in mutation frequency and decreases in plasmid survival, and the formation of open-circular and linear DNA. The majority of the spontaneous (69.8%) and induced mutations (85.7%) at 1000 and 79.4% at 5000 cGy) were base substitutions and were generally of similar types among all groups. However, changes at 2500 (12.7%) and 5000 cGy (13.2%) involving A:T base pairs were greater than those in unirradiated controls (3.4%) or those at 1000 cGy (2.0%). This increase in A:T base pair mutations could be a result of reduced repair fidelity when the DNA is extensively damaged by high doses of ionizing radiation.

Cimetidine suppresses chemically induced experimental hepatic porphyria.

The ability of cimetidine to reduce the activity of hepatic aminolevulinic acid synthase (ALA-S) was examined in allylisopropyl acetamide (AIA) treated porphyric adult rats. A dose of 20 mg cimetidine/100 gm body weight resulted in a 50% decrease in the AIA-induced hepatic ALA-S activity compared to rats treated with AIA alone. Heme oxygenase activity was decreased 25% compared to rats treated with AIA alone. The effects of AIA and cimetidine on cytochrome P-450 were not additive, suggesting competition for a common site of interaction. The results suggest that cimetidine may prove to be useful in treating porphyria in humans.