Fragile sites, chromosomal lesions, tandem repeats, and

IntroductionThe terms “fragility” and “fragile site,” coined in 1969–70, refer to unusual secondary constrictions in chromosomes, that are distinct from the primary constrictions of the centromeres (Schmid and Vischer, 1969; Magenis et al., 1970). Under specific conditions of replicative stress, they can also manifest as chromatin gaps, breaks, or failed chromatin compaction on metaphase chromosomes. Fragile sites are found across the genome, such as in the heterochromatic regions harboring classical satellite repeats on chromosomes 1, 9, 15, 16, and Y, as well as the common and rare fragile sites (Figure 1). Fragile sites can also arise at telomeres, at telomere fusions, and at other specific genetic loci. Due to their genome-wide prevalence, fragile sites have been found to be associated with genetic and genomic instability, and are extensively linked to many disease phenotypes, including neurological disorders (sections 2.1, 2.2), immunodeficiency–centromeric instability–facial anomalies (ICF) syndrome (section 2.3), and cancer progression.FIGURE 1. Repeat Tracts, fragile sites, and disease. (A) Categories of fragile site constrictions observable on human chromosomes. (B) Fragile sites can occur at the telomere or centromere, observed on chromosomes 1, 2, 9, 10, 15, 16, Y, and X. Telomere ends display “fragile-site like” appearances that are TRF1-dependant and APH inducible (Sfeir et al., 2009). The centromere of every chromosome is the “primary constriction,” composed of repeats. (C) Secondary constrictions are present on chromosomes 1, 9, 16, 15, and Y. Cytogeneticists were aware of these gaps prior to FRAXA and modeled it after the secondary constrictions (Lubs, 1969). These are composed of the classical satellite repeat DNAs, where four types (I-IV) of satellite DNA are located in the heterochromatic regions of chromosomes 1, 9, 15, 16, and Y, the total amount on these chromosomes and the proportion of the types being different (Vogt, 1990). Satellite regions of chromosomes 9 and Y, whose composition is the most complicated, and chromosome 15 is less complex, but like 9 and Y, it comprises all four types of satellite DNA. Chromosome 1 has type II satellite DNA, with the proportion of the remaining types being less. The C segment of chromosome 16 comprises only type II. The size/length of the secondary constrictions is highly-polymorphic amongst individuals, and segregates as a heritable state (Chromosomes: Guttenbach and Schmid, 1994). (D) In Immunodeficiency, Centromeric instability & Facial anomalies (ICF) syndrome, satellites I, II, and III of chromosomes 1, 9, and 16 are hypomethylated and show secondary constrictions within these regions. ICF syndrome was recently shown to be caused by four mutations in four genes: ICF1/DNMT3B, ICF2/ZBTB24, ICF3/CDCA7, and ICF4/HELLS (van den Boogaard et al., 2017). Satellite-containing regions on chromosomes 1, 9, and 16 are hypomethylated in individuals affected by ICF syndrome, and these show a variety of aberrant chromosomes: secondary constrictions, multibranched chromosome arms, whole arm deletions, duplications, isochromosomes, and centromeric fragility. As with fragile sites, these involved double-strand DNA breaks (Sawyer et al., 1995; Tuck-Muller et al., 2000). (Chromosomes: Tuck-Muller et al., 2000). (E) Many non-folate sensitive fragile sites have been mapped at repetitive regions. The interstitial telomeric repeat on chromosome 2, the AT-repeats of FRA10B and FRA16B, and the GAA repeat of FXN on chromosome 9 has “fragile-like” characteristics. Fragile site at 2q13-14 at an interstitial inverted head-to-head array of the telomeric repeat (TAGAGGG)54-(CCCTAA)104, a result of an ancient telomeric fusion, not “telomere healing” event (IJdo et al., 1991; Bosco and de Lange, 2012). Notably, there are other interstitial telomeric sequences (Wells et al., 1990). A “fragile-like” site has been reported at 9q21.1 in the expanded (GAA)N repeat in FXN, which causes Friedreich’s ataxia (Kumari et al., 2015). FRA10B at 10q25.2 is induced by BrdU and mapped to an expanded ∼42 bp AT-rich minisatellite repeat (Sutherland et al., 1980; Hewett et al., 1998) (Chromosomes: Bosco and de Lange, 2012; Sutherland et al., 1980; Felbor et al., 2003). (F) Various presentations of the FRAXA site in CGG-expanded FXS patient cells (Crippa et al., 1984; Fitchett and Seabright, 1984; Savage and Fitchett, 1988): chromatid breaks/gaps, isochromatid breaks, isolated double-minutes, deleted X’s, secondary duplications (double satellite). Satellite association and variations in length of the nucleolar constriction of normal and variant human G chromosomes. (Chromosomes: Lubs, 1969). (G) Ribosomal DNAs can vary the length of the chromosome by varying lengths of the secondary constrictions (stalks) of the acrocentrics (Chr 13, 14, 15, 21, and 22) on which they reside (Orye, 1974; Cheung et al., 1989; Heliot et al., 1997).The first fragile site was observed in 1965 (Dekaban, 1965), followed by the discovery of the first disease-associated fragile site at the fragile X locus (Lubs, 1969), later demonstrated to be Martin-Bell syndrome (Richards et al., 1981). This initial discovery remained largely ignored until it was serendipitously induced in specific folate-deficient culture conditions, leading to the renaming of the disease to fragile X syndrome (FXS) (Sutherland, 1977) (reviewed in (Hecht and Kaiser-McCaw, 1979). Since then, discovery of these sites at specific loci has broadened.Fragile site classificationsThe current classifications of fragile sites fall into two categories largely based on frequency of expression and induction method: common fragile sites (CFSs) and rare fragile sites (RFSs). The Human Genome Database documents ∼90 CFSs and ∼30 RFSs that have been cytogenetically observed and documented in previous studies (reviewed by (Feng and Chakraborty, 2017).CFSs are present in a large proportion of the population, and are induced by aphidicolin, 5-azacytidine, and bromodeoxyuridine (BrdU) (Glover et al., 1984; Yunis and Soreng, 1984; Sutherland et al., 1985b). RFSs are observed to a maximal frequency of 5% in the population (Schmid et al., 1986) and can be induced by folate deficiency/thymidylate stress, distamycin A, and BrdU (Sutherland, 1983; Hecht and Sutherland, 1984; Sutherland et al., 1985a). Detailed protocols for the detection and analysis of both CFSs and RFSs have been published recently (Bjerregaard et al., 2018). As CFSs are linked to regions of chromosomal rearrangements in cancer, this group of fragile sites has been far more extensively studied than RFSs (reviewed in (Dillon et al., 2010; Ozeri-Galai et al., 2012; Sarni and Kerem, 2016; Glover et al., 2017; Irony-Tur Sinai and Kerem, 2019; Kaushal and Freudenreich, 2019). Harnessing knowledge about CFSs could empower the field of RFSs and provide important clues as to how fragility contributes to other disease phenotypes and genetic abnormalities (i.e., repeat instability).The current distinction between common and rare fragile sites is problematic, being based both on the conditions that induce their expression, and the frequency with which they are present in the population (Hecht, 1986; Mrasek et al., 2010). There is no clear numerical delineation between the frequency of “common” and “rare” fragile sites. Some CFSs are rare in their manifestation, suggesting they are not ubiquitously present in all individuals or might be observed at lower levels (e.g., FRA2D, FRA18B, and FRA9D are expressed in
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