How common are common fragile sites in humans: interindividual variation in the distribution of aphidicolin-induced fragile sites

To obtain an estimate of the variation in common fragile sites (CFSs) among individuals, aphidicolin (APC)-induced chromosomal breakage data were analyzed for 20 karyotypically normal adult humans. As it is specifically designed to meet the analytical requirements for considering fragile sites as presence/absence characters in single individuals, the FSM methodology (B?hm et al., 1995) was used to statistically distinguish fragile from nonfragile sites. These analyses indicated that the APC-induced fragile sites are not ubiquitous but vary extensively among individuals; the per-individual number of fragile sites ranged from as few as seven to as many as 20. Of the 45 different sites identified as fragile, 19 (42%) occurred in more than half of the individuals, but only two sites (3p14 and 16q23) were fragile in all of the individuals; 12 (27% of the total) were fragile in single individuals only. Although these analyses provide statistical confirmation (and initial estimates of population variation) for 43 of the 88 APC-inducible fragile sites currently recognized as occurring among humans, they are consistent with the hypothesis that many of the currently recognized human CFSs have been erroneously identified. These results indicate the need for per-individual statistical identification of CFSs for larger samples of individuals and that studies of particular fragile sites should be conducted on individuals documented to be fragile at the loci under consideration.     

Minimum sample sizes for identifying chromosomal fragile sites from individuals: Monte Carlo estimation

A Monte Carlo simulation procedure was used to estimate the exact level of the standardized X ² test statistic (X _s ²) for randomness in the FSM methodology for the identification of fragile sites from chromosomal breakage data for single individuals. A random-number generator was used to simulate 10 000 chromosomal breakage data sets, each corresponding to the null hypothesis of no fragile sites for numbers of chromosomal breaks (n) from 1 to 2000 and at three levels of chromosomal band resolution (k). The reliability of the test was assessed by comparisons of the empirical and nominal α levels for each of the corresponding values of n and k. These analyses indicate that the sparse and discrete nature of chromosomal breakage data results in large and unpredictable discrepancies between the empirical and nominal α levels when fragile site identifications are based on small numbers of breaks (n < 0.5 k). With n≥ 0.5 k, the distribution of X _s ² appears to be stable and non-significant differences in the empirical and nominal α levels are generally obtained. These results are inherent to the nature of the data and are, therefore, relevant to any statistical model for the identification of fragile sites from chromosomal breakage data. For FSM identification of fragile sites at α = 0.05, we suggest that n≥ 0.5 k is the minimum reliable number of mapped chromosomal breaks per individual. Received: 28 April 1997 / Accepted: 1 July 1997     

How common are common fragile sites: variation of aphidicolin-induced chromosomal fragile sites in a population of the deer mouse (Peromyscus maniculatus)

Aphidicolin (APC)-induced chromosomal gaps and breaks were analyzed for ten deer mice (Peromyscus maniculatus) from a natural population. The FSM statistical methodology was used to identify fragile sites as chromosomal loci exhibiting significantly non-random numbers of gaps/breaks in each individual and enabled an assessment of variation in fragile sites among the individuals. The individual deer mice exhibited as few as 7 to as many as 19 of the populational total of 34 sites. Two sites were fragile in all individuals and 13 sites were fragile in single individuals only. Defined by populational frequencies of greater than 50%, high-frequency fragile sites constituted 26% of the populational total. Approximately 35% of the total fragile sites were fragile in 20–40% of the population (low-frequency fragile sites) and about 38% were fragile in single individuals only. Analysis of the data pooled over all individuals identified significantly non-random breakage at 80 sites, 47 of which were not identified as fragile in any single individual. It appears, therefore, that fragile site identifications from pooled data have fostered an inflated estimate of the numbers and frequencies of common fragile sites. Comparison of the fragile site and spontaneous breakage (control) data suggest that APC-induced fragile sites represent regions of chromosomes that experience elevated levels of somatic mutation. Additionally, the occurrence of APC-induced fragile sites at or near the interstitial breakpoints of two pericentric-inversion polymorphisms in this population supports the hypothesis that fragile sites experience an increased rate of meiotic chromosomal mutation and are predisposed to undergo phylogenetic rearrangement. Received: 22 January 1997 / Accepted: 24 February 1997     

Identifying chromosomal fragile sites from individuals: a multinomial statistical model

The inability to identify fragile sites from data for single individuals remains the major obstacle to determining whether these chromosomal loci are predisposed to cancer-causing and evolutionary rearrangements. We describe a novel statistical model that is amenable to data from single individuals and that establishes site-specific chromosomal breakage as nonrandom with respect to the distribution of total breakage. Our method tests incrementally smaller subsets of the data for homogeneity under a multinomial model that assigns equal probabilites to a maximal set of nonfragile sites and unrestricted probabilities to the remaining fragile sites with significantly higher numbers of breaks. We show how standardized Pearson's chi-square (X ²) and likelihood-ratio (G ²) statistics can be appropriately used to measure goodness-of-fit for sparse contingency (individual-based) data in this model. A sample application of this approach indicates extensive variation in fragile sites among individuals and marked differences in fragile-site inferences from pooled as opposed to per-individual data.     

Testing the nonrandomness of chromosomal breakpoints using highest observed breakages

To determine whether a chromosomal band is a fragile site rather than a spontaneous breakpoint, an essential step is to test the nonrandomness of breakage at the region. In this paper, the nonapplicability of the testing procedure introduced by Bohm et al. is discussed, and a new detection procedure is proposed. This new procedure considers the relations of one site with the others, and can be applied to tests of the nonrandomness of breakpoints under either the proportional probability model, or the equiprobability model. A data set for Chinese patients with colorectal carcinoma is analyzed as an illustration of the proposed method. Received: 27 August 1998 / Accepted: 18 December 1998     

Lack of an Additive Effect between the Deletions of Klf5 and Nkx3-1 in Mouse Prostatic Tumorigenesis

Prostate cancer is one of the most common malignancies.The development and progression of prostate cancer are driven by a series of genetic and epigenetic events including gene amplification that activates oncogenes and chromosomal deletion that inactivates tumor suppressor genes.Whereas gene amplification occurs in human prostate cancer,gene deletion is more common,and a large number of chromosomal regions have been identified to have frequent deletion in prostate cancer,suggesting that tumor suppressor inactivation is more common than oncogene activation in prostatic carcinogenesis (Knuutila et al.,1998,1999;Dong,2001).Among the most frequently deleted chromosomal regions in prostate cancer,target genes such as NKX3-1 from 8p21,PTENfrom 10q23 andATBF1 from 16q22 have been identified by different approaches (He et al.,1997;Li et al.,1997;Sun et al.,2005),and deletion of these genes in mouse prostates has been demonstrated to induce and/or promote prostatic carcinogenesis.For example,knockout of Nkx3-1 in mice induces hyperplasia and dysplasia (Bhatia-Gaur et al.,1999;Abdulkadir et al.,2002) and promotes prostatic tumorigenesis (Abate-Shen et al.,2003),while knockout of Pten alone causes prostatic neoplasia (Wang et al.,2003).Therefore,gene deletion plays a causal role in prostatic carcinogenesis (Dong,2001).     

The neurobeachin gene spans the common fragile site FRA13A

Common fragile sites are normal constituents of chromosomal structure prone to chromosomal breakage. In humans, the cytogenetic locations of more than 80 common fragile sites are known. The DNA at 11 of them has been defined and characterized at the molecular level. According to the Genome Database, the common fragile site FRA13A maps to chromosome band 13q13.2. Here, we identify the precise genomic position of FRA13A, and characterize the genetic complexity of the fragile DNA sequence. We show that FRA13A breaks are limited to a 650 kb region within the neurobeachin (NBEA) gene, which genomically spans approximately 730 kb. NBEA encodes a neuron-specific multidomain protein implicated in membrane trafficking that is predominantly expressed in the brain and during development.     

Circadian control of neurogenesis

The life-long addition of new neurons has been documented in many regions of the vertebrate and invertebrate brain, including the hippocampus of mammals (Altman and Das, 1965; Eriksson et al., 1998; Jacobs et al., 2000), song control nuclei of birds (Alvarez-Buylla et al., 1990), and olfactory pathway of rodents (Lois and Alvarez-Buylla, 1994), insects (Cayre et al., 1996) and crustaceans (Harzsch and Dawirs, 1996; Sandeman et al., 1998; Harzsch et al., 1999; Schmidt, 2001). The possibility of persistent neurogenesis in the neocortex of primates is also being widely discussed (Gould et al., 1999; Kornack and Rakic, 2001). In these systems, an effort is underway to understand the regulatory mechanisms that control the timing and rate of neurogenesis. Hormonal cycles (Rasika et al., 1994; Harrison et al., 2001), serotonin (Gould, 1999; Brezun and Daszuta, 2000; Beltz et al., 2001), physical activity (Van Praag et al., 1999) and living conditions (Kemperman and Gage, 1999; Sandeman and Sandeman, 2000) influence the rate of neuronal proliferation and survival in a variety of organisms, suggesting that mechanisms controlling life-long neurogenesis are conserved across a range of vertebrate and invertebrate species. The present article extends these findings by demonstrating circadian control of neurogenesis. Data show a diurnal rhythm of neurogenesis among the olfactory projection neurons in the crustacean brain, with peak proliferation during the hours surrounding dusk, the most active period for lobsters. These data raise the possibility that light-controlled rhythms are a primary regulator of neuronal proliferation, and that previously-demonstrated hormonal and activity-driven influences over neurogenesis may be secondary events in a complex circadian control pathway.     

Location of zinc and 65Zn in spinal ganglia of the rat

Following the works of Velazquez et al. (1999), Jo-Seung et al. (2000), Wang et al. (2001), Danscher et al. (2001) and the criteria of Zinc-containing neurons established by Frederickson et al.(2000), we have found the presence and localisation of Zinc in the neurons of the dorsal root ganglia of Wistar rat, by using Timm's thecnique and by studying the autoradiographic uptake of 65Zn. The agreement between the results of both techniques allows us to classify these spinal ganglion neurons as Zinc-containing neurons and also, to confirm some of the results of Velazquez et al. (1999).     

A Space–Time Scan Statistic for Detecting Emerging Outbreaks

Summary As a major analytical method for outbreak detection, Kulldorff's space–time scan statistic (2001, Journal of the Royal Statistical Society, Series A 164, 61–72) has been implemented in many syndromic surveillance systems. Since, however, it is based on circular windows in space, it has difficulty correctly detecting actual noncircular clusters. Takahashi et al. (2008, International Journal of Health Geographics 7 , 14) proposed a flexible space–time scan statistic with the capability of detecting noncircular areas. It seems to us, however, that the detection of the most likely cluster defined in these space–time scan statistics is not the same as the detection of localized emerging disease outbreaks because the former compares the observed number of cases with the conditional expected number of cases. In this article, we propose a new space–time scan statistic which compares the observed number of cases with the unconditional expected number of cases, takes a time‐to‐time variation of Poisson mean into account, and implements an outbreak model to capture localized emerging disease outbreaks more timely and correctly. The proposed models are illustrated with data from weekly surveillance of the number of absentees in primary schools in Kitakyushu‐shi, Japan, 2006.     

Hypothesis regarding the nature of the fragile X mutation

Summary Pembrey et al. (1985) proposed a hypothesis regarding the nature of the fragile X [fra(X)] mutation. Recently they analyzed DNA linkage data (Winter and Pembrey 1986) that we and others have published on fra(X) pedigrees, found significant linkage heterogeneity, and modified their hypothesis to explain the observations. We would like to point out that their modified hypothesis is not supported by the data available.     

A comparative review of statistical methods for discovering differentially expressed genes in replicated microarray experiments

MOTIVATION: A common task in analyzing microarray data is to determine which genes are differentially expressed across two kinds of tissue samples or samples obtained under two experimental conditions. Recently several statistical methods have been proposed to accomplish this goal when there are replicated samples under each condition. However, it may not be clear how these methods compare with each other. Our main goal here is to compare three methods, the t-test, a regression modeling approach (Thomas et al., Genome Res., 11, 1227-1236, 2001) and a mixture model approach (Pan et al., http://www.biostat.umn.edu/cgi-bin/rrs?print+2001,2001a,b) with particular attention to their different modeling assumptions. RESULTS: It is pointed out that all the three methods are based on using the two-sample t-statistic or its minor variation, but they differ in how to associate a statistical significance level to the corresponding statistic, leading to possibly large difference in the resulting significance levels and the numbers of genes detected. In particular, we give an explicit formula for the test statistic used in the regression approach. Using the leukemia data of Golub et al. (Science, 285, 531-537, 1999), we illustrate these points. We also briefly compare the results with those of several other methods, including the empirical Bayesian method of Efron et al. (J. Am. Stat. Assoc., to appear, 2001) and the Significance Analysis of Microarray (SAM) method of Tusher et al. (PROC: Natl Acad. Sci. USA, 98, 5116-5121, 2001).     

Micronucleus formation in lymphocytes of patients with fraXq27 syndrome

Human chromosome carry different fragile sites, but the most interesting one is the fragile X chromosome, especially in relation to mental retardation. In this report we present micronucleus formation in 5 individuals with the fragile X syndrome and 11 control subjects whose lymphocytes were cultured under 1) hydroxyurea treatment condition suitable for the increasing expression of the fraXq27, 2) control conditions. It has been found that hydroxyurea (HU) added to the final concentration 1.3 X 10(-5)M increases the frequency of the fraXq and micronucleus formation in comparison with culture without HU. The same concentration of HU has induced neither "nonspecific" "spontaneous" chromosomal breakage nor micronucleus formation in the control subjects.     

Fitting genetic mapping functions based on sperm typing: results for three chromosomal segments in cattle

Genetic mapping functions translate the observed recombination rate between two loci into the corresponding map distance in Morgan units. Different mapping functions give different weights to multiple crossing overs and therefore lead to different results. This points out that not every function is best suited to fit a data set. The data used in this study originated from 2214 sperm from 37 Norwegian bulls, which were genotyped for 11 markers. The optimal functions for the chromosomes 6, 23 and the sex chromosome of cattle were derived using the maximum likelihood method, the likelihood ratio test and empirical discriminant analysis. It became apparent that for each chromosome a different function fitted the data best. These were the function of Rao et al. (Human Heredity 1977, 27, 99–104) with p = 0·63 for chromosome 6, the function of Goldgar & Fain (American Journal of Human Genetics 1988, 43, 38–45) with c₀ = 0·42, c₁ = 0·47, c₂ = 0·07 and c₃ = 0·04 for chromosome 23 and the function of Felsenstein (Genetics 1979, 91, 769–75) with K = 0·23 for the sex chromosome. The well known functions of Haldane (Journal of Genetics 1919, 8, 299–309) and Kosambi (Annals of Eugenics 1944, 12, 172–5) were shown to be suboptimal in most cases. A function is said to be multilocus feasible if the evaluation of the probability of all possible recombination events does not lead to negative values. The optimal function for chromosome 23 turned out to be multilocus feasible, whereas the functions for chromosome 6 and the sex chromosome were not. The choice of the correct mapping function is shown to have a considerable impact in mapping studies, when double recombinations have to be taken into account. Since there is no unique best mapping function, it is argued that it might be useful to use a simple parametric mapping function (like the one of Felsenstein 1979) and to estimate the respective parameter specifically for a given data set.     

Unbiased and Locally Efficient Estimation of Genetic Effect on Quantitative Trait in the Presence of Population Admixture

Summary Population admixture can be a confounding factor in genetic association studies. Family‐based methods ( Rabinowitz and Larid, 2000 , Human Heredity 50, 211–223) have been proposed in both testing and estimation settings to adjust for this confounding, especially in case‐only association studies. The family‐based methods rely on conditioning on the observed parental genotypes or on the minimal sufficient statistic for the genetic model under the null hypothesis. In some cases, these methods do not capture all the available information due to the conditioning strategy being too stringent. General efficient methods to adjust for population admixture that use all the available information have been proposed ( Rabinowitz, 2002 , Journal of the American Statistical Association 92, 742–758). However these approaches may not be easy to implement in some situations. A previously developed easy‐to‐compute approach adjusts for admixture by adding supplemental covariates to linear models ( Yang et al., 2000 , Human Heredity 50, 227–233). Here is shown that this augmenting linear model with appropriate covariates strategy can be combined with the general efficient methods in Rabinowitz (2002) to provide computationally tractable and locally efficient adjustment. After deriving the optimal covariates, the adjusted analysis can be carried out using standard statistical software packages such as SAS or R . The proposed methods enjoy a local efficiency in a neighborhood of the true model. The simulation studies show that nontrivial efficiency gains can be obtained by using information not accessible to the methods that rely on conditioning on the minimal sufficient statistics. The approaches are illustrated through an analysis of the influence of apolipoprotein E (APOE) genotype on plasma low‐density lipoprotein (LDL) concentration in children.     

129 The molecular mechanism of breakage at fragile site FRA16D

Replication stress induces physical breakage at discrete loci in chromosomes, which can be visualized on a metaphase chromosome spread. These common fragile sites (CFS) are conserved across species and are hotspots for sister chromatid recombination, viral integration, rearrangements, translocations, and deletions (Glover et al 2005). Despite multiple theories, the molecular mechanisms of CFS expression and genomic instability are still not well understood. The fragile site FRA16D is of special interest because it is the second most highly expressed fragile site and is located within the WWOX tumor suppressor gene. Previous data identified a polymorphic AT repeat within a FRA16D subregion called F1 that causes chromosome fragility and replication fork stalling in a yeast model (Zhang and Freudenreich 2007). Recently, we have found that breakage increases in an AT repeat length-dependent manner. Our results suggest that the AT repeat in the context of F1 forms a secondary structure, making the region more vulnerable to breakage.     

Aphidicolin-Induced FRA3B Breakpoints Cluster in Two Distinct Regions

The common fragile site at chromosomal band 3p14.2 (FRA3B) is the most sensitive single site in the human genome to induced chromosomal lesions. This fragile site may predispose chromosome 3p to breakage that is commonly observed in lung, renal, and many other cancers. We previously used aphidicolin induction of FRA3B expression in a chromosome 3-only somatic cell hybrid to generate a series of hybrids with breakpoints in the 3p14.2 region. These breakpoints were localized to two distinct clusters, separated by 200 kb, that lie on either side of a region of frequent breakage within FRA3B as observed by FISH analysis. Seven proximal aphidicolin-induced breakpoints were localized at or near the end of a THE element. The THE-1 element is flanked by LINE andAlurepetitive elements. The eight distal aphidicolin-induced breakpoints clustered in a region capable of forming multiple hairpin-like structures. Thus repetitive elements and hairpin-like structures may be responsible for chromosome fragility in this region.     

Susceptibility of heterochromatin to aphidicolin-induced chromosomal breakage

The distribution of aphidicolin-induced chromosomal lesions was analyzed to determine the relative breakage susceptibility of euchromatin and heterochromatin in the cactus mouse, Peromyscus eremicus. The observed breakage was tested against expected distributions corresponding to the karyotypic proportions of autosomal euchromatin, autosomal heterochromatin, X-chromosomal euchromatin, and X-chromosomal heterochromatin. The distribution of induced breakage was independent of sex but dependent on the individual. In all individuals, there was a highly significant (P0.0001) deficiency in the number of breaks observed as compared to expected in autosomal heterochromatin. Sparse observations in the X chromosome and the absence of breaks in the Y chromosome precluded valid statistical tests of the sex-chromosomal distribution of induced breakage. These data indicate that the autosomal heterochromatin of Peromyscus is resistant to aphidicolin-induced chromosomal breakage and argue against a simple relationship between late replication and a general mechanism for chromosomal fragility.     

DNA instability at chromosomal fragile sites in cancer

Human chromosomal fragile sites are specific genomic regions which exhibit gaps or breaks on metaphase chromosomes following conditions of partial replication stress. Fragile sites often coincide with genes that are frequently rearranged or deleted in human cancers, with over half of cancer-specific translocations containing breakpoints within fragile sites. But until recently, little direct evidence existed linking fragile site breakage to the formation of cancer-causing chromosomal aberrations. Studies have revealed that DNA breakage at fragile sites can induce formation of RET/PTC rearrangements, and deletions within the FHIT gene, resembling those observed in human tumors. These findings demonstrate the important role of fragile sites in cancer development, suggesting that a better understanding of the molecular basis of fragile site instability is crucial to insights in carcinogenesis. It is hypothesized that under conditions of replication stress, stable secondary structures form at fragile sites and stall replication fork progress, ultimately resulting in DNA breaks. A recent study examining an FRA16B fragment confirmed the formation of secondary structure and DNA polymerase stalling within this sequence in vitro, as well as reduced replication efficiency and increased instability in human cells. Polymerase stalling during synthesis of FRA16D has also been demonstrated. The ATR DNA damage checkpoint pathway plays a critical role in maintaining stability at fragile sites. Recent findings have confirmed binding of the ATR protein to three regions of FRA3B under conditions of mild replication stress. This review will discuss recent advances made in understanding the role and mechanism of fragile sites in cancer development.