Analysis of Xq27.3 Fragility Using the Micronucleus-Fluorescence In situ Hybridization Assay

Bjerregaard V.A., Garribba L., McMurray C.T., Hickson I.D., Liu Y.

Significance Dietary folate deficiency is associated with fetal neural tube defects, psychological disorders, and age-associated dementia. However, it remains unclear how folate deficiency could be a causative factor in such a diverse range of disorders. Through analysis of the FRAXA locus, which contains an extensive CGG repeat sequence, we show that folate deprivation triggers extensive mitotic missegregation of the locus. Moreover, the entire chromosome X becomes unstable during a period of long-term folate deprivation. Considering that the human genome contains several loci associated with extensive CGG repeat regions, these findings suggest a mechanism by which folate deficiency contributes to the onset of a wide range of human diseases.

Dockendorff T.C., Labrador M.

The fragile X syndrome (FXS) arises from loss of expression or function of the FMR1 gene and is one of the most common monogenic forms of intellectual disability and autism. During the past two decades of FXS research, the fragile X mental retardation protein (FMRP) has been primarily characterized as a cytoplasmic RNA binding protein that facilitates transport of select RNA substrates through neural projections and regulation of translation within synaptic compartments, with the protein products of such mRNAs then modulating cognitive functions. However, the presence of a small fraction of FMRP in the nucleus has long been recognized. Accordingly, recent studies have uncovered several mechanisms or pathways by which FMRP influences nuclear gene expression and genome function. Some of these pathways appear to be independent of the classical role for FMRP as a regulator of translation and point to novel functions, including the possibility that FMRP directly participates in the DNA damage response and in the maintenance of genome stability. In this review, we highlight these advances and discuss how these new findings could contribute to our understanding of FMRP in brain development and function, the neural pathology of fragile X syndrome, and perhaps impact of future therapeutic considerations.

Bjerregaard V.A., Özer Ö., Hickson I.D., Liu Y.

A fragile site is a chromosomal locus that is prone to form a gap or constriction visible within a condensed metaphase chromosome, particularly following exposure of cells to DNA replication stress. Based on their frequency, fragile sites are classified as either common (CFSs; present in all individuals) or rare (RFSs; present in only a few individuals). Interest in fragile sites has remained high since their discovery in 1965, because of their association with human disease. CFSs are recognized as drivers of oncogene activation and genome instability in cancer cells, while some RFSs are associated with neurodegenerative diseases. This review summaries our current understanding of the nature and causes of fragile site “expression”, including the recently characterized phenomenon of telomere fragility. In particular, we focus on a description of the methodologies and technologies for detection and analysis of chromosome fragile sites.

Fenech M., Bonassi S.

Micronucleus (MN) frequency in cytokinesis-blocked peripheral blood lymphocytes (PBL) has become one of the best-established biomarkers for studying DNA damage occurring in vivo in humans. The application of this method in population biomonitoring studies requires a deep understanding of how lifestyle and common host variables may influence MN frequency in PBL. In this mini-review, an update is provided on results from studies reporting on the impact of age, gender, diet and lifestyle factors (e.g. exercise, alcohol, smoking and recreational drugs) on this biomarker. Evidence from these studies shows that each of these factors, either in isolation or in combination, can significantly influence MN frequency. Proper control for these factors is required to enable better measurement of the impact of other conditions, such as environmental exposure to genotoxins or a susceptible genetic background, on MN frequency in PBL.

Hovhannisyan G.G.

Comet assay and micronucleus (MN) test are widely applied in genotoxicity testing and biomonitoring. While comet assay permits to measure direct DNA-strand breaking capacity of a tested agent MN test allows estimating the induced amount of chromosome and/or genome mutations. The potential of these two methods can be enhanced by the combination with fluorescence in situ hybridization (FISH) techniques. FISH plus comet assay allows the recognition of targets of DNA damage and repairing directly. FISH combined with MN test is able to characterize the occurrence of different chromosomes in MN and to identify potential chromosomal targets of mutagenic substances. Thus, combination of FISH with the comet assay or MN test proved to be promising techniques for evaluation of the distribution of DNA and chromosome damage in the entire genome of individual cells. FISH technique also permits to study comet and MN formation, necessary for correct application of these methods. This paper reviews the relevant literature on advantages and limitations of Comet-FISH and MN-FISH assays application in genetic toxicology.

Lukusa T., Fryns J.P.

Fragile sites are heritable specific chromosome loci that exhibit an increased frequency of gaps, poor staining, constrictions or breaks when chromosomes are exposed to partial DNA replication inhibition. They constitute areas of chromatin that fail to compact during mitosis. They are classified as rare or common depending on their frequency within the population and are further subdivided on the basis of their specific induction chemistry into different groups differentiated as folate sensitive or non-folate sensitive rare fragile sites, and as aphidicolin, bromodeoxyuridine (BrdU) or 5-azacytidine inducible common fragile sites. Most of the known inducers of fragility share in common their potentiality to inhibit the elongation of DNA replication, particularly at fragile site loci. Seven folate sensitive (FRA10A, FRA11B, FRA12A, FRA16A, FRAXA, FRAXE and FRAXF) and two non-folate sensitive (FRA10B and FRA16B) fragile sites have been molecularly characterized. All have been found to represent expanded DNA repeat sequences resulting from a dynamic mutation involving the normally occurring polymorphic CCG/CGG trinucleotide repeats at the folate sensitive and AT-rich minisatellite repeats at the non-folate sensitive fragile sites. These expanded repeats were demonstrated, first, to have the potential, under certain conditions, to form stable secondary non-B DNA structures (intra-strand hairpins, slipped strand DNA or tetrahelical structures) and to present highly flexible repeat sequences, both conditions which are expected to affect the replication dynamics, and second, to decrease the efficiency of nucleosome assembly, resulting in decondensation defects seen as fragile sites. Thirteen aphidicolin inducible common fragile sites (FRA2G, FRA3B, FRA4F, FRA6E, FRA6F, FRA7E, FRA7G, FRA7H, FRA7I, FRA8C, FRA9E, FRA16D and FRAXB) have been characterized at a molecular level and found to represent relatively AT-rich DNA areas, but without any expanded repeat motifs. Analysis of structural characteristics of the DNA at some of these sites (FRA2G, FRA3B, FRA6F, FRA7E, FRA7G, FRA7H, FRA7I, FRA16D and FRAXB) showed that they contained more areas of high DNA torsional flexibility with more highly AT-dinucleotide-rich islands than neighbouring non-fragile regions. These islands were shown to have the potential to form secondary non-B DNA structures and to interfere with higher-order chromatin folding. Therefore, a common fragility mechanism, characterized by high flexibility and the potential to form secondary structures and interfere with nucleosome assembly, is shared by all the cloned classes of fragile sites. From the clinical point of view, the folate sensitive rare fragile site FRAXA is the most important fragile site as it is associated with the fragile X syndrome, the most common form of familial mental retardation, affecting about 1/4000 males and 1/6000 females. Mental retardation in this syndrome is considered as resulting from the abolition of the FMR1 gene expression due to hypermethylation of the gene CpG islands adjacent to the expanded methylated trinucleotide repeat. FRAXE is associated with X-linked non-specific mental retardation, and FRA11B with Jacobsen syndrome. There is also some evidence that fragile sites, especially common fragile sites, are consistently involved in the in vivo chromosomal rearrangements related to cancer, whereas the possible implication of common fragile sites in neuropsychiatric and developmental disorders is still poorly documented.

Durkin S.G., Glover T.W.

Chromosomal fragile sites are specific loci that preferentially exhibit gaps and breaks on metaphase chromosomes following partial inhibition of DNA synthesis. Their discovery has led to novel findings spanning a number of areas of genetics. Rare fragile sites are seen in a small proportion of individuals and are inherited in a Mendelian manner. Some, such as FRAXA in the FMR1 gene, are associated with human genetic disorders, and their study led to the identification of nucleotide-repeat expansion as a frequent mutational mechanism in humans. In contrast, common fragile sites are present in all individuals and represent the largest class of fragile sites. Long considered an intriguing component of chromosome structure, common fragile sites have taken on novel significance as regions of the genome that are particularly sensitive to replication stress and that are frequently rearranged in tumor cells. In recent years, much progress has been made toward understanding the genomic features of common fragile sites and the cellular processes that monitor and influence their stability. Their study has merged with that of cell cycle checkpoints and DNA repair, and common fragile sites have provided insight into understanding the consequences of replication stress on DNA damage and genome instability in cancer cells.

Penagarikano O., Mulle J.G., Warren S.T.

Fragile X syndrome is the most common form of inherited mental retardation. The disorder is mainly caused by the expansion of the trinucleotide sequence CGG located in the 5′ UTR of the FMR1 gene on the X chromosome. The abnormal expansion of this triplet leads to hypermethylation and consequent silencing of the FMR1 gene. Thus, the absence of the encoded protein (FMRP) is the basis for the phenotype. FMRP is a selective RNA-binding protein that associates with polyribosomes and acts as a negative regulator of translation. FMRP appears to play an important role in synaptic plasticity by regulating the synthesis of proteins encoded by certain mRNAs localized in the dendrite. An advancing understanding of the pathophysiology of this disorder has led to promising strategies for pharmacologic interventions.

HAGERMAN R.J., McBOGG P., HAGERMAN P.J.

The fragile X (marker X) syndrome is a relatively common form of X-linked mental retardation. The karyotypic hallmark of the syndrome consists of a pronounced constriction near the terminus of the long arm of the X chromosome (fragile site), expressed in vitro only under conditions where thymidylate production is blocked (reduced folate levels and/or addition of methotrexate or 5-fluorodeoxyuridine). Clinical features associated with the syndrome include macroorchidism, large or prominent ears, and significant emotional dysfunction. In the present review, historical, diagnostic, biochemical, and clinical aspects of this syndrome are presented. Recent anecdotal reports of clinical improvement following high dose folic acid treatment will be discussed.

Fenech M.

The cytokinesis-block micronucleus cytome assay is a comprehensive system for measuring DNA damage, cytostasis and cytotoxicity. DNA damage events are scored specifically in once-divided binucleated (BN) cells and include (a) micronuclei (MNi), a biomarker of chromosome breakage and/or whole chromosome loss, (b) nucleoplasmic bridges (NPBs), a biomarker of DNA misrepair and/or telomere end-fusions, and (c) nuclear buds (NBUDs), a biomarker of elimination of amplified DNA and/or DNA repair complexes. Cytostatic effects are measured via the proportion of mono-, bi- and multinucleated cells and cytotoxicity via necrotic and/or apoptotic cell ratios. Further information regarding mechanisms leading to MNi, NPBs and NBUDs formation is obtained using centromere and/or telomere probes. The assay is being applied successfully for biomonitoring of in vivo genotoxin exposure, in vitro genotoxicity testing and in diverse research fields such as nutrigenomics and pharmacogenomics as well as a predictor of normal tissue and tumor radiation sensitivity and cancer risk. The procedure can take up to 5 days to complete.

Lindberg H.K., Wang X., Järventaus H., Falck G.C., Norppa H., Fenech M.

Micronuclei are formed from chromosomes and chromosomal fragments that lag behind in anaphase and are left outside daughter nuclei in telophase. They may also be derived from broken anaphase bridges. Nuclear buds, micronucleus-like bodies attached to the nucleus by a thin nucleoplasmic connection, have been proposed to be generated similarly to micronuclei during nuclear division or in S-phase as a stage in the extrusion of extra DNA, possibly giving rise to micronuclei. To better understand these phenomena, we have characterized the contents of 894 nuclear buds and 1392 micronuclei in normal and folate-deprived 9-day cultures of human lymphocytes using fluorescence in situ hybridization with pancentromeric and pantelomeric DNA probes. Such information has not earlier been available for human primary cells. Surprisingly, there appears to be no previous data on the occurrence of telomeres in micronuclei (or buds) of normal human cells in general. Our results suggest that nuclear buds and micronuclei have partly different mechanistic origin. Interstitial DNA without centromere or telomere label was clearly more prevalent in nuclear buds (43%) than in micronuclei (13%). DNA with only telomere label or with both centromere and telomere label was more frequent in micronuclei (62% and 22%, respectively) than in nuclear buds (44% and 10%, respectively). Folate deprivation especially increased the frequency of nuclear buds and micronuclei harboring telomeric DNA and nuclear buds harboring interstitial DNA but also buds and micronuclei with both centromeric and telomeric DNA. According to the model we propose, that micronuclei in binucleate lymphocytes primarily derive from lagging chromosomes and terminal acentric fragments during mitosis. Most nuclear buds, however, are suggested to originate from interstitial or terminal acentric fragments, possibly representing nuclear membrane entrapment of DNA that has been left in cytoplasm after nuclear division or excess DNA that is being extruded from the nucleus.

Wojda A., Zietkiewicz E., Witt M.

Studies have shown a significant increase in chromosome aneuploidy with age. The aim of this study was to elucidate whether the age-related changes in the level of hypoploidy correlate with the occurrence of micronuclei (MN) and chromosome nondisjunction (ND) in men and women. We analyzed cytokinesis-blocked (binucleated) lymphocytes treated with cytochalasin B, from 127 donors varying in gender and age including 53 centenarians. Fluorescent in situ hybridization with probes specific for several autosomes (1, 4, 6, 8, 20) and for the sex chromosomes was applied to analyze the chromosomal content of MN and to analyze the frequency of reciprocal loss and gain due to ND in binucleated interphase cells. The general level of MN in Giemsa-stained preparations was higher in women and in both genders increased with age until approximately 70 years and ranged, depending on age group, from 0.5 to 1.4% in men and from 0.9 to 1.8% in women. Gender-related differences were mostly observed in the younger age groups (< or =50 years), with an almost two-fold difference between men and women (P < 0.005). Frequencies of autosome-positive MN in both genders and of sex chromosome-positive MN in men were comparable and remained unchanged in older groups. The frequency of X-positive MN in women was higher than the average frequency of autosome-positive MN and continued to increase until the oldest age. The frequency of NDs involving the analyzed chromosomes was on average two-fold higher in women than in men. In both genders, the frequency of NDs increased with age and was, on average, an order of magnitude higher than that of cells with MN, consistent with the previous reports that the efficiency of elimination of micronucleated cells is higher than of the cells presenting chromosome ND.

Leopardi P.

Folic acid plays a key role in the maintenance of genomic stability, providing methyl groups for the conversion of uracil to thymine and for DNA methylation. Besides dietary habits, folic acid metabolism is influenced by genetic polymorphism. The C677T polymorphism of the methylene-tetrahydrofolate reductase (MTHFR) gene is associated with a reduction of catalytic activity and is suggested to modify cancer risk differently depending on folate status. In this work the effect of folic acid deficiency on genome stability and radiosensitivity has been investigated in cultured lymphocytes of 12 subjects with different MTHFR genotype (four for each genotype). Cells were grown for 9 days with 12, 24 and 120 nM folic acid and analyzed in a comprehensive micronucleus test coupled with centromere characterization by CREST immunostaining. In other experiments, cells were grown with various folic acid concentrations, irradiated with 0.5 Gy of gamma rays and analyzed in the micronucleus test. The results obtained indicate that folic acid deficiency induces to a comparable extent chromosome loss and breakage, irrespective of the MTHFR genotype. The effect of folic acid was highly significant (P < 0.001) and explained >50% of variance of both types of micronuclei. Also nucleoplasmic bridges and buds were significantly increased under low folate supply; the increase in bridges was mainly observed in TT cells, highlighting a significant effect of the MTHFR genotype (P = 0.006) on this biomarker. Folic acid concentration significantly affected radiation-induced micronuclei (P < 0.001): the increased incidence of radiation-induced micronuclei with low folic acid was mainly accounted for by carriers of the variant MTHFR allele (both homozygotes and heterozygotes), but the overall effect of genotype did not attain statistical significance. Treatment with ionizing radiations also increased the frequency of nucleoplasmic bridges. The effect of folic acid level on this end-point was modulated by the MTHFR genotype (P for interaction = 0.02), with TT cells grown at low folic acid concentration apparently resistant to the induction of radiation-induced bridges. Finally, the effect of in vitro folate deprivation on global DNA methylation was evaluated in lymphocytes of six homozygous subjects (three CC and three TT). The results obtained suggest that, under the conditions of this work, folic acid deprivation is associated with global DNA hypermethylation.

Bardoni B., Davidovic L., Bensaid M., Khandjian E.W.

Fragile X syndrome (FXS) – the leading cause of inherited mental retardation – is an X-linked disease caused by loss of expression of the FMR1 (fragile X mental retardation 1) gene. In addition to impairment of higher-cognitive functions, FXS patients show a variety of physical and other mental abnormalities. FMRP, the protein encoded by the FMR1 gene, is thought to play a key role in translation, trafficking and targeting of mRNA in neurons. To better understand FMRP's functions, the protein partners and mRNA targets that interact with FMRP have been sought. These and functional studies have revealed links with processes such as cytoskeleton remodelling via the RhoGTPase pathway and mRNA processing via the RNA interference pathway. In this review, we focus on recent insights into the function of FMRP and speculate on how the absence of FMRP might cause the clinical phenotypes seen in FXS patients. Finally, we explore potential therapies for FXS.

Schwartz M., Zlotorynski E., Kerem B.

Fragile sites are specific loci that form gaps and constrictions on chromosomes exposed to partial replication stress. Fragile sites are classified as rare or common, depending on their induction and frequency within the population. These loci are known to be involved in chromosomal rearrangements in tumors and are associated with human diseases. Therefore, the understanding of the molecular basis of fragile sites is of high significance. Here we discuss the works performed in recent years that investigated the characteristics of fragile sites which underlie their inherent instability.

Tabolacci E., Nobile V., Pucci C., Chiurazzi P.

A dynamic mutation in exon 1 of the FMR1 gene causes Fragile X-related Disorders (FXDs), due to the expansion of an unstable CGG repeat sequence. Based on the CGG sequence size, two types of FMR1 alleles are possible: “premutation” (PM, with 56-200 CGGs) and “full mutation” (FM, with >200 triplets). Premutated females are at risk of transmitting a FM allele that, when methylated, epigenetically silences FMR1 and causes Fragile X syndrome (FXS), a very common form of inherited intellectual disability (ID). Expansions events of the CGG sequence are predominant over contractions and are responsible for meiotic and mitotic instability. The CGG repeat usually includes one or more AGG interspersed triplets that influence allele stability and the risk of transmitting FM to children through maternal meiosis. A unique mechanism responsible for repeat instability has not been identified, but several processes are under investigations using cellular and animal models. The formation of unusual secondary DNA structures at the expanded repeats are likely to occur and contribute to the CGG expansion. This review will focus on the current knowledge about CGG repeat instability addressing the CGG sequence expands.