Clinical significance of human immunodeficiency virus type 1 replication fitness

The relative fitness of a variant, according to population genetics theory, is that variant's relative contribution to successive generations. Most drug-resistant human immunodeficiency virus type 1 (HIV-1) variants have reduced replication fitness, but at least some of these deficits can be compensated for by the accumulation of second-site mutations. HIV-1 replication fitness also appears to influence the likelihood of a drug-resistant mutant emerging during treatment failure and is postulated to influence clinical outcomes. A variety of assays are available to measure HIV-1 replication fitness in cell culture; however, there is no agreement regarding which assays best correlate with clinical outcomes. A major limitation is that there is no high-throughput assay that incorporates an internal reference strain as a control and utilizes intact virus isolates. Some retrospective studies have demonstrated statistically significant correlations between HIV-1 replication fitness and clinical outcomes in some patient populations. However, different studies disagree as to which clinical outcomes are most closely associated with fitness. This may be in part due to assay design, sample size limitations, and differences in patient populations. In addition, the strength of the correlations between fitness and clinical outcomes is modest, suggesting that, at present, it would be difficult to utilize these assays for clinical management.     

Cytomegalovirus-enhanced induction of chromosome aberrations in human peripheral blood lymphocytes treated with potent genotoxic agents.

Human cytomegalovirus (HCMV) has been shown to increase the frequency of chromosome aberrations, primarily chromatid-type, in human peripheral blood lymphocytes (PBLs). Because HCMV persists in most humans, pathologically activates cells, and may perturb the cell cycle, we investigated the possibility that HCMV-infected cells have a modified sensitivity to chromosome damage induced by genotoxic chemicals. Uninfected PBLs exposed to bleomycin (3 to 100 micrograms/ml) demonstrated a linear increase in the frequency of chromosome aberrations. HCMV infection of PBLs at an intensity that did not cause detectable damage followed by exposure to the same concentrations of bleomycin resulted in a significant enhancement (p less than 0.01) in the frequency of chromosome aberrations relative to the effect of bleomycin alone. A more than additive enhancement of the frequency of chromosome aberrations was also noted in HCMV-infected PBLs exposed to 4-hydroxyaminoquinoline-1-oxide (4-HAQO; 0.1 to 0.3 micrograms/ml) relative to uninfected cells treated with 4-HAQO alone. No increase in the percentage of aberrant cells or the frequency of chromosome aberrations was observed in HCMV-infected cells treated with 4-nitroquinoline-1-oxide (4-NQO) relative to similarly treated uninfected PBLs. These results suggest that HCMV can potentiate the induction of chromosome aberrations in human PBLs caused by potent DNA damaging agents.     

Spontaneous and 4-nitroquinoline 1-oxide-induced G2 chromosome aberrations in lymphoblasts from familial melanoma patients

Lymphoblastoid cell lines established from three unrelated kindreds with familial melanoma (FM) were cytogenetically analyzed for spontaneous and 4-nitroquinoline-1-oxide (4NQO)-induced chromosome aberrations. There were no significant differences between control, FM, or xeroderma pigmentosum (XP) cell lines for spontaneous aberrations. As a group, FM patients, as well as XP patients, had significantly higher 4NQO-induced aberrations than controls when metaphase cells were analyzed 2.5 hours after treatment during the G2 phase of the cell cycle. When selected cell lines were analyzed 18 hours after 4NQO treatment, the frequency of chromosome aberrations in FM cells returned to spontaneous levels, but XP cells retained significantly elevated aberration frequencies. The wide variability for chromosome aberrations within the control. FM relative, and FM patient groups during G2, however, indicated that analysis of total breakage rates alone would not be predictive of susceptibility to FM. Heterogeneity for carcinogen-induced chromosome breakage between some cancer-prone individuals and the possible significance of site-specific chromosome aberrations are discussed.     

Candidate driver genes in focal chromosomal aberrations of stage II colon cancer

Chromosomal instable colorectal cancer is marked by specific large chromosomal copy number aberrations. Recently, focal aberrations of 3 Mb or smaller have been identified as a common phenomenon in cancer. Inherent to their limited size, these aberrations harbour one or few genes. The aim of this study was to identify recurrent focal chromosomal aberrations and their candidate driver genes in a well‐defined series of stage II colon cancers and assess their potential clinical relevance. High‐resolution DNA copy number profiles were obtained from 38 formalin‐fixed, paraffin‐embedded colon cancer samples with matched normal mucosa as a reference using array comparative genomic hybridization. In total, 81 focal chromosomal aberrations were identified that harboured 177 genes. Statistical validation of focal aberrations and identification of candidate driver genes were performed by enrichment analysis and mapping copy number and mutation data of colorectal, breast, and pancreatic cancer and glioblastomas to loci of focal aberrations in stage II colon cancer. This analysis demonstrated a significant overlap with previously identified focal amplifications in colorectal cancer, but not with cancers from other sites. In contrast, focal deletions seemed less tumour type‐specific since they also showed significant overlap with focal deletions of other sites. Focal deletions detected were significantly enriched for cancer genes and genes frequently mutated in colorectal cancer. The mRNA expression of these genes was significantly correlated with DNA copy number status, supporting the relevance of focal aberrations. Loss of 5q34 and gain of 13q22.1 were identified as independent prognostic factors of survival in this series of patients. In conclusion, focal chromosomal copy number aberrations in stage II colon cancer are enriched in cancer genes that contribute to and drive the process of colorectal cancer development. DNA copy number status of these genes correlates with mRNA expression and some are associated with clinical outcome. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Growth, DNA repair, sister chromatid exchange and chromosome studies in fibroblasts fromHuntington's disease patients

Fibroblast cultures from six unrelatedHuntington's Disease (HD) patients and controls and one affected relative of an HD patient were used in studies of cell growth, DNA repair, sister chromatid exchange (SCE) and chromosome aberrations.
There were no significant differences in background levels of SCEs or of chromosome aberrations between HD cultures and controls.
Preliminary results using epidermal growth factor indicated that HD cells may have a lowered relative response to this polypeptide hormone.
Cell growth studies showed no correlation between growth rate and HD.
Increased cell saturation density was recorded in cell lines from four of the HD patients; the remaining three lines from affected individuals (two of them related) were indistinguishable from control cultures. This variation may reflect genetic heterogeneity in HD.
An apparent deficiency in DNA repair capacity following UV irradiation in cultures from three HD patients was subsequently shown to be the result of the increased cell saturation densities in these cultures.     

A comparative cytogenetic study on cases of induced abortions in tcdd-exposed and nonexposed women

In order to evaluate the cytogenetic findings previously obtained on induced abortions in women exposed to 2,3,7,8 tetrachlorodibenzo-p-Dioxim (TCDD) after the Seveso accident, chromosome analyses were carried out on a comparable control sample. The frequencies of aberrant cells, the relative proportions of individual types of chromosome aberration, the average number of lesions per damaged cell, and the frequencies of polyploids do not differ significantly in maternal blood and placenta in the two samples. A highly significant increase in the frequencies of aberrant cells and in the average number of aberrations per damaged cell was found in the fetal tissues in the group of exposed pregnancies. A noticeable feature of the data on the frequencies of cells with aberrations is the marked variability among individuals within tissues and within samples. On the basis of these results, the authors point to the need to continue cytogenetic investigations on interrupted pregnancies in TCDD-exposed and nonexposed women.     

Evolutionary barriers to quinolone resistance in Streptococcus pneumoniae

It is assumed that bacteria always pay a significant physiological price for the acquisition of resistance to antibiotics. To test whether this was the case for a strain of Streptococcus pneumoniae that develops resistance to fluoroquinolone antibiotics, we selected resistance to these agents in a wild-type strain and measured their fitness in comparative growth experiments. The relative growth rate of a mutant strain selected on ciprofloxacin (parC Serine 79 to Tyrosine) was compared with its susceptible isogenic parent and no significant deficit was found (relative fitness 1.15 95% C.I. +/- 0.2.). A double mutant, however, had a relative fitness of 0.81 (parC Serine 79 to Tyrosine gyrA Serine 81 to Tyrosine). Mutant strains selected on gemifloxacin had only a modest increase in minimum inhibitory concentration; thus, second-round mutants were competed with a first-round gyrA Serine 81 to Tyrosine or the susceptible isogenic parent. The growth rate of three double-mutant strains parC Serine 79 to Tyrosine gyrA Serine 81 to Phenylanine, parC Serine 79 to Tyrosine, and Asparagine 83 to Phenylalanine were similar to the isogenic susceptible parent 1.16 (95% C.I. +/- 0.17), 0.99 (95% C.I. +/- 0.05), and 0.95 (95% C.I. +/- 0.05), respectively. These data suggest that mutation in the parC and gyrA genes may, on some occasions, not be associated with a physiological deficit.     

Nickel potentiates the genotoxic effect of benzo[a]pyrene in Chinese hamster lung V79 cells

The modulating effect of acute exposure to NiCl2 on the induction of chromosome aberrations by a model carcinogen, benzo[a]pyrene (B[a]P), was examined in Chinese hamster V79 lung cells. At concentrations up to 20 microg/ml (84.2 microM), NiCl2 did not significantly increase the frequency of chromosome aberrations in V79 cells when the cells were exposed concomitantly to 0.5 microg/ml B[a]P. Addition of the S15 liver microsomal fraction together with the B[a]P did not alter the results. Addition of NiCl2 2 hr before treatment of cells with 0.5 microg/ml B[a]P also did not result in a significant elevation of the frequency of chromosome aberrations, even at NiCl2 concentrations as high as 20 microg/ml. Contrasting sharply with these findings, when V79 cells were treated with NiCl2 immediately after B[a]P exposure, a significant increase in the frequency of chromosome damage was observed at NiCl2 concentrations as low as 5 microg/ml (21.1 microM). NiCl2-mediated enhancement of chromosome damage was also observed when V79 cells were exposed to the reactive B[a]P intermediate, benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (BPDE). In the BPDE-treated cells, the level of NiCl2-mediated enhancement was similar to that observed with the tumor promoter 12-o-tetradecanoylphorbol-13-acetate (TPA, 100 ng/ml). These results are consistent with the view that the effect of nickel (II) on B[a]P-induced genetic damage is dependent on the relative times of exposure to Ni2+ and B[a]P. NiCl2 did not enhance the frequency of chromosome aberrations induced by Chromium (VI), regardless of the order of addition of the chemicals to the V79 cells. These results suggest that nickel may act as a promoter of chemically-induced genetic damage through induction of error-prone repair.     

Analysis by FISH of the spectrum of chromosome aberrations induced by X-rays in G0 human lymphocytes and their fate through mitotic divisions in culture

The induction, distribution, and persistence of chromosome aberrations in human lymphocytes exposed to X-rays in G0 were analyzed in 48-, 70-, and 94-hr cultures by conventional metaphase analysis and painting of chromosomes 1, 2, and 4 by FISH. All cells that had been scored by FISH were relocated to determine by differential staining of chromatids whether they had passed through 1, 2, or > or =3 divisions. FISH revealed a dose-dependent induction of stable and unstable aberrations, while chromatid labeling showed mitotic lag caused by irradiation in G0. Relative to their DNA contents, there was a small but significant overrepresentation of chromosome 4 and underrepresentation of chromosome 2 among the aberrations involving chromosomes 1, 2, and 4. FISH slightly underestimated the genomic frequency of unstable aberrations measured by conventional metaphase analysis. There was a slight excess of translocations relative to dicentrics, but the data are compatible with the 1:1 ratio expected from cytogenetic theory. Many of the translocations were apparently incomplete (i.e., nonreciprocal). Incomplete translocations were more frequent at higher X-ray dose and in first division, suggesting that they may be associated with complex damage and are more apt to be lost in mitosis than complete translocations. Among the incomplete translocations, t(Ab) outnumbered t(Ba) -- a difference ascribable to the FISH technique. Aberration frequencies declined as the cells divided in culture. The overall decline in the frequency of aberrant cells (approximately 29% per cell generation) reflects a rapid decline in dicentrics and fragments (approximately 60% per cell generation) and the relative stability of translocations. The frequency of translocation-bearing cells underwent a modest decline in culture (approximately 13% per cell generation).     

Chromosomal abnormalities subdivide ependymal tumors into clinically relevant groups

Ependymoma occurs most frequently within the central nervous system of children and young adults. We determined relative chromosomal copy-number aberrations in 44 ependymomas using comparative genomic hybridization. The study included 24 intracranial and 20 spinal cord tumors from pediatric and adult patients. Frequent chromosomal aberrations in intracranial tumors were gain of 1q and losses on 6q, 9, and 13. Gain of 1q and loss on 9 were preferentially associated with histological grade 3 tumors. On the other hand, gain on chromosome 7 was recognized almost exclusively in spinal cord tumors, and was associated with various other chromosomal aberrations including frequent loss of 22q. We conclude that cytogenetic analysis of ependymomas may help to classify these tumors and provide leads concerning their initiation and progression. The relationship of these aberrations to patient outcome needs to be addressed.     

Analysis of bleomycin-induced chromosomal aberrations in Chinese hamster primary embryonic cells by FISH using arm-specific painting probes.

Chinese hamster primary embryonic cells (at G1 phase) were treated with 1.0 or 3.0 microg/ml bleomycin and chromosomal aberrations in first division metaphases were analysed by fluorescence in situ hybridization (FISH) using arm-specific painting probes for chromosomes 3, 4, 8 and 9. We observed that bleomycin induced all classes of chromosome-type aberrations very efficiently. The interesting findings were: (i) the frequency of induced interstitial translocations (i.e. insertions) was approximately equal to that of reciprocal translocations; (ii) the frequency of induced pericentric inversions was higher than that of centric rings. In our earlier studies, we found that X-rays induced a low frequency of interstitial translocations in comparison with reciprocal translocations and equal frequencies of centric rings and pericentric inversions. These data suggest that bleomycin differs from X-rays with respect to the induction of some specific types of aberrations. The results of a chi2 test examining the hypothesis that formed aberrations among the chromosomes or chromosome arms are randomly distributed on the basis of their relative lengths revealed a differential involvement of these chromosomes in the aberrations following exposure to bleomycin. In general, chromosome 8 was found to be more involved in induced aberrations than expected, chromosome 4 was randomly involved, whereas chromosomes 3 and 9 were less involved. This study demonstrates the utility of arm-specific painting probes for efficient detection of a large variety of chromosomal aberrations induced by bleomycin.     

Genome wide array comparative genomic hybridisation analysis of premalignant lesions of the stomach.

BACKGROUND: Gastric cancer is one of the most frequent malignancies in the world, ranking fifth in the Netherlands as a cause of cancer death. Surgery is the only curative treatment for advanced cases, but results of gastrectomy largely depend on the stage of the disease. A better understanding of the mechanisms of progression from a preneoplastic condition through intraepithelial neoplasia to invasive cancer may provide information relevant to designing focused prevention strategies. METHODS: Because the pattern of chromosomal aberrations in precursors of gastric cancer is unclear, 11 gastric polyps with intraepithelial neoplasia (three hyperplastic polyps and eight adenomas) were analysed by microarray comparative genomic hybridisation to study chromosomal instability in precursors of gastric cancer. RESULTS: Chromosomal aberrations were detected in all specimens. Adenomas showed no more chromosomal aberrations than did the hyperplastic polyps. The most frequent aberrations were gain of 7q36 and 20q12, and loss of 5q14-q21 in the adenomas, and loss of 15q11-14, 1p21-31, and 21q11-21.2 in the hyperplastic polyps. The most frequent chromosomal aberration in common to both types was loss of 9p21.3. CONCLUSION: Hyperplastic polyps showed many chromosomal aberrations, confirming that neoplastic transformation can occur in these lesions. These observations are consistent with the existence of two morphologically and genetically distinct pathways to gastric cancer-the hyperplastic polyp pathway and the (intestinal type) adenoma pathway. The relative contribution of each to gastric carcinogenesis in general, and how they compare to patterns of chromosomal aberrations in the more prevalent flat foci of intraepithelial neoplasia remain to be determined.     

Small fitness effect of mutations in highly conserved non-coding regions

Comparison of human and mouse genomes has revealed that many non-coding regions have levels of sequence conservation similar to protein-coding genes. These regions have attracted a lot of attention as potentially functional genomic sequences. However, little is known about the effect mutations in these conserved non-coding regions have on fitness and how many of them are present in the human genome as deleterious polymorphisms. To gain insight into the selective constraints imposed on conserved non-coding and protein-coding regions, we compared substitution rates in primate and rodent lineages and analyzed the density and allele frequencies of human polymorphism. Genomic regions conserved between primate and rodent groups show higher relative conservation within rodents than within primates. Thus, our analysis indicates a genome-wide relaxation of selective constraint in the primate lineage, which most likely resulted from a smaller effective population size. We found that this relaxation is much more profound in conserved non-coding regions than in protein-coding regions, and that mutations at a large proportion of sites in conserved non-coding regions are associated with very small fitness effect. Data on human polymorphism are also consistent with very weak selection in conserved non-coding regions. This staggering enrichment in sites at the borderline of neutrality can be explained by assuming an important role for synergistic epistasis in the evolution of non-coding regions. Our results suggest that most individual mutations in conserved non-coding regions are only slightly deleterious but are numerous and may have a significant cumulative impact on fitness.     

Subtelomeric chromosome aberrations: still a lot to learn

Subtelomeric chromosome aberrations: still a lot to learn.Cryptic subtelomeric chromosome aberrations are a significant cause of mental retardation (MR). More than 4000 patients have been investigated, and the mean overall prevalence of subtelomeric rearrangements has been found to be 5.2%. In order to contribute to knowledge on the clinical presentation of subtelomeric rearrangements, we retrospectively studied patients with unexplained MR who had been evaluated for subtelomeric abnormalities by different fluorescence in situ hybridization (FISH) techniques. Hundred and two patients had an unexplained combination of MR with dysmorphism, congenital anomalies, and/or a positive family history and were investigated by total subtelomeric (TS) FISH (89/102), or by total painting (TP) in an obligate carrier in the case of familial MR (13/102). In 59 additional patients, a sequence-specific FISH was performed on clinical indication. In the 102 patients studied by TS or TP, six pathogenic aberrations (5.9%) were found in addition to one polymorphism. In total, eight clinically significant subtelomeric aberrations were found in the 161 index patients; four of these eight aberrations were familial. We report on the clinical presentation of all patients with an aberration and review the relevant literature. Factors complicating the interpretation of subtelomeric rearrangements are discussed, such as the occurrence of variants, clinical variability, and limited knowledge of the phenotype.     

A simple method for calculating risks before DNA analysis.

Calculation of carrier risk of an X linked disease may be performed on a small computer after DNA analysis, but a method for rapid hand estimation of the risk is still useful for a quick check of the results and weighing the relative importance of each element of information, such as the determination of a haplotype. Each risk estimation is a function of a prior risk and the product of likelihood ratios and these terms are derived themselves from parameters such as fitness or the relative mutation rate in male and female gametes. Even if it is often difficult to have strong experimental estimation of these variables, the existence of a normal father or grandfather must be considered whenever male fitness is not null. The likelihood ratio for a woman for not being a carrier, when her father is not affected and her mother has herself a likelihood R for not having the mutated gene, may be expressed as the ratio 2R/(CmR + 1), with Cm being a function of male fitness and relative mutation rate. Cm represents the odds ratio for the mother of a carrier not to be a carrier, given that the father of the known carrier is not affected. This formula can be used recurrently and reduces to 2R/(R + 1) in lethal X linked disease. When likelihood ratios are expressed as an algebraic function, maximum values are easily determined, hence fixing the limits of DNA analysis.     

Strength characteristics of a healthy urban adult population

Summary A random sample of 778 subjects representing ages 25, 35, 45 and 55 years were studied for the amount of habitual physical activity, their anthropometric structure, vertical jumping height, trunk extension and flexion torques and dynamic endurance fitness of trunk extension and flexion. The proportion of subjects who were habitually physically active did not change systematically with age. The highest number of physically inactive subjects was found in men and women in the 35-year age group. The results in all the tests used to assess the strength characteristics were statistically significant when related to sex (P<0.001) and in all, except the relative maximal isometric torque of trunk extension, when related to age (P<0.001). Everyday physical activity was related to the variation in vertical jumping height (P<0.001), as well to the dynamic endurance fitness (P<0.001) of trunk extension and flexion. The decline in vertical jumping height and dynamic endurance fitness of trunk extension and flexion fitness was found to start at carlier ages than that of relative maximal isometric trunk extension and flexion torques. Dynamic endurance fitness of trunk flexion showed a more pronounced decline with age than trunk extension fitness.     

Self‐reported health,self‐reported fitness,and all‐cause mortality: Prospective cohort study

Objectives. Self‐reported health has a well established relationship to later mortality, although the reasons are not entirely understood. This study examined the association of a similar self‐reported measure of fitness with mortality and compared it to that of self‐reported health. Design. The study had a prospective cohort design with multiple sampling points. Methods. Participants were 858 men and women from Glasgow and the surrounding area of Scotland, aged 59 when self‐reported health and fitness data were first collected in 1991/92. They were re‐interviewed at age 64 and 69 and mortality was tracked for 16.5 years in total. Hazard ratios for all cause mortality were estimated for those that reported poor health or poor fitness relative to others their age, taking into account a range of covariates, some of which were also time varying. Results. In both unadjusted and covariate adjusted models, self‐reported fitness was at least as good a predictor of mortality as self‐reported health. In a mutually adjusted model, both again emerged as significant predictors. Poor subjective health with poor subjective fitness appeared to be a particularly lethal combination. Conclusion. Both self‐reported health and self‐reported fitness were independent predictors of mortality. Where the objective assessment of aerobic fitness is not feasible, a simple measure of subjective fitness could prove a useful alternative.     

Generation,long-term persistence,and neuronal differentiation of cells with nuclear aberrations in the adult zebrafish brain

Zebrafish, like other teleosts, continuously produce new cells in numerous regions of the adult brain. Immunolabeling employing antisera against phosphorylated histone-H3 and 5-bromo-2′-deoxyuridine revealed that approximately 6%–7% of such cells exhibited nuclear aberrations. These aberrations, presumably the result of mitotic segregation defects, included single and multiple laggards (both during metaphase and anaphase) and anaphase bridges. Cells with such aberrations persisted long-term and comprised, when examined 7.5 months after their generation, approximately 2.5% of the total population of adult-born cells. The drop in relative frequency of aberrations in the course of further development appears to be caused by elimination of cells with nuclear aberrations, presumably by apoptotic cell death. The cells with nuclear aberrations that persisted long-term were capable of neuronal differentiation, as demonstrated by combining anti-5-bromo-2′-deoxyuridine immunohistochemistry with immunostaining against the neuronal marker protein Hu or the enzyme tyrosine hydroxylase, a marker of catecholaminergic neurons. We hypothesize that the alterations in chromosome number and/or chromosome structure caused by nuclear aberrations do not necessarily result in loss of vital functions or in tumorigenesis. Instead, cells with such aberrations are able to undergo what appears to be normal development.     

Genetic effects of methylmercury in human chromosomes: I. A cytogenetic study of people exposed through eating contaminated fish

An analysis of chromosomal aberrations (structural and numerical) and sister chromatid exchange (SCE) was carried out on 16 people exposed to methylmercury through eating fish caught in Cartagena Bay (Columbia), an area of known methylmercury contamination. Fourteen people whose diet consisted mainly of fish caught in another, noncontaminated area of the Atlantic acted as controls. The results showed a significant difference between the experimental and control groups in methylmercury (MM) concentrations measured in hair and peripheral blood. Subsequently, significant differences between levels of organic mercury in blood and hair were found when all the individuals studied were classified in two groups according to their blood mercury levels. When achromatic lesions were included, the frequency of structural chromosome aberrations differed significantly between the exposed and control groups. Furthermore, a significant correlation (p less than 0.05) was found between structural chromosome aberrations and groups (exposed and control). When achromatic lesions were excluded from the analyses, these differences were not found. There was a significant correlation between SCE and age. This is the first report of a study on the frequency of SCE in a population exposed to methylmercury.