Fragile Sites and Minisatellite Repeat Instability

Molecular Genetics and Metabolism 70, 99 –105 (2000) doi:10.1006/mgme.2000.2996, available online at http://www.idealibrary.com on

Fragile Sites and Minisatellite Repeat Instability Oliva Handt, 1 Grant R. Sutherland, and Robert I. Richards Centre for Medical Genetics, Department of Cytogenetics and Molecular Genetics, Women’s and Children’s Hospital, North Adelaide, SA 5006, Australia Received March 31, 2000

or dCTP at the time of DNA replication. The distamycin A inducible group contains fragile sites that can be induced by distamycin A, berenil, netropsin, Hoechst 33258, and BrdU (6 – 8). Distamycin A, netropsin, and Hoechst 33258 bind DNA with a high affinity to AT-rich sequences. The BrdU-requiring fragile sites, on the contrary, can only be induced by (BrdU) or bromodeoxycytidine (BrdC). BrdU gets incorporated into the DNA in substitution for thymidine (9) and may also affect the pyrimidine pathways. Both distamycin A inducible and BrdU-requiring fragile sites show spontaneous expression in vitro, the first frequently, the latter rarely (8). The addition of distamycin A and BrdU increases the fragile site expression. The molecular basis for the cytogenetic expression of chromosomal fragile sites is not yet completely understood. The different classes of fragile site have allowed the opportunity to compare the molecular anatomy of chromosomal fragile sites with the aim of identifying features that are common to all classes and that might constitute the essential requirements for fragile site expression. Conversely, elements unique to a particular fragile site class might account for the specific induction chemistry of each class.

Key Words: fragile site; minisatellite; instability; repeat expansion.

CLASSIFICATION OF FRAGILE SITES Fragile sites are specific, inherited chromosome loci that appear as gaps or breaks in chromosomes of cells which are exposed to certain cell culture conditions (1). They were first described in 1965 (2) and have been found on every chromosome with the exception of chromosome 21 (3). On the basis of frequency, fragile sites have been grouped into common and rare fragile sites. The common ones are part of the normal chromosome structure, although the proportion of metaphases showing cytogenetic expression varies between individuals. Common fragile sites are further classified into three groups according to their induction chemistry by either bromodeoxyuridine (BrdU), 5-azacytidine, or aphidicolin, an inhibitor of DNA polymerase ␣. The most prevalent rare fragile site is FRA16B, with an occurrence of 1 in 40 chromosomes in the German population (4); some rare fragile sites have been reported only once (5). Just as for the common fragile sites, the mode of chemical induction provides the basis for classification of the rare fragile sites into three groups: (1) folate sensitive, (2) distamycin A inducible, and (3) BrdU-requiring. Induction of folate sensitive fragile sites is caused by omitting folic acid and thymidine from culture media and hence by lowering the levels of either dTTP

MOLECULAR CHARACTERIZATION OF RARE FOLATE SENSITIVE FRAGILE SITES From a clinical point of view the rare folate sensitive fragile site FRAXA is the most important fragile site. It causes the fragile X syndrome, a familial form of mental retardation. The prevalence of the fragile X syndrome is estimated to be about 1 in

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TABLE 1 Founder Effects in Expanded Repeat Diseases Disease

Reference

Expanded repeat

Spinocerebellar ataxia type 1 Spino-bulbar muscular atrophy Dentatorubral and pallidoluysian atrophy Huntington disease Myotonic dystrophy Fragile X syndrome Friedreich ataxia SCA3/Machado–Joseph disease

16 17

poly CAG poly CAG

18 19 20 21 22 23

poly poly poly poly poly poly

CAG CAG CAG CCG AAG CAG

4000 males and 1 in 6000 females (10) and it is the second most common chromosome abnormality causing mental retardation after trisomy 21. The rare folate sensitive fragile site FRAXE is associated with a mild form of mental retardation (11). No other phenotypic effects have been observed in individuals expressing different fragile sites on one or even both chromosomes (7,12,13). Therefore, it is not surprising that FRAXA was the first fragile site for which the DNA sequence was determined. It was shown that it is caused by a novel mutation mechanism referred to as “dynamic mutation.” A polymorphic CCG trinucleotide repeat, which has up to 55 copies in normal alleles, was expanded beyond 230 copies in fragile site expressing alleles (14). The expanded CCG repeat inactivates the FMR1 gene (15). Other trinucleotide repeat expansions have since then been found in a number of hereditary neurodegenerative disorders which include Huntington’s disease, Friedreich ataxia, and various types of spinocerebellar ataxia (for references see Table 1). Every other rare folate sensitive fragile site that has been positionally cloned also showed expansion of the CCG trinucleotide repeat (24 –28). Most short nonfragile site expressing alleles have imperfect repeat stretches at all these loci which are believed to have a stabilizing effect, whereas fragile site alleles do not usually show interruptions of the expanded repeats. Alleles with long stretches of uninterrupted repeats are at high risk of further repeat expansion. This has not only been shown for the CCG repeat (29) but also for the Friedreich ataxia GAA triplet repeat (30) and for the CAG repeat expanded in spinocerebellar ataxia type I (16) and other neurological disorders.

MINISATELLITE REPEAT EXPANSION AT FRA16B It was hoped that the characterization and comparison of members of all three classes of rare fragile sites on the DNA sequence level would reveal the molecular mechanism causing the gaps and breaks in chromosomes. The first non-folate-sensitive rare fragile site that was characterized by positional cloning was the distamycin A-inducible rare fragile site located at 16q22.1 (31). Analogous to the rare folate sensitive fragile sites, the fragility at FRA16B is caused by repeat expansion. However, in this instance it was found that a 33-bp AT-rich minisatellite repeat was expanded on the fragile chromosomes. Minisatellite or variable number of tandem repeats (VNTR) range from 10 bp to more than 100 bp in length. The finding of an expanded AT-rich minisatellite repeat was in agreement with the knowledge that most chemicals inducing FRA16B bind to AT-rich DNA sequences. The sequence at the fragile site locus was composed of various AT-rich minisatellite repeat motifs of which the 33-bp repeat was shown to be expanded to up to 2000 copies in FRA16B alleles (31). The DNA consensus sequence for the FRA16B repeat is presented in Fig. 1. As the expanded FRA16B alleles were very large (⬃15 to 70 kb) it was not feasible to carry out an amplification across the expanded repeats by polymerase chain reaction (PCR). It was therefore not possible to study features like intergenerational instability, to determine and compare the sequences of different FRA16B alleles, or to check whether long normal alleles are predisposed to expansion. MOLECULAR CHARACTERIZATION OF THE BrdU REQUIRING RARE FRAGILE SITE FRA10B The BrdU requiring rare fragile site FRA10B is located at chromosome band 10q25.2. It can be induced by BrdU or BrdC and is present in about 1 in 40 of the Australian population (12,32,33). The molecular basis for this fragile site was recently determined (34). Sequence analysis revealed a highly ATrich region (91%) in the center, which contained a variety of minisatellite repeats, and nonrepetitive flanking regions of ⬃58% AT content (Fig. 2A). PCR across this region showed that it is very polymorphic in length, with alleles in sizes between ⬃0.8 kb up to more than 5 kb. Individuals heterozygous for FRA10B showed amplification of one fragment less

FRAGILE SITE INSTABILITY

FIG. 1. Comparison of the FRA10B and FRA16B consensus sequences. Identical bases between the two repeats are marked with I, asterisks point to differences that are either due to different nucleotides or to the shorter sequences of the FRA16B repeat. The 11-bp repeat element is presented in capital letters for the forward sequence and in lowercase letters for the reverse sequence.

than 5 kb in size as well as either a large fragment (⬎5 kb) or a null allele (no amplification product). On the basis of their lengths, amplification products could be divided into four groups: small normal (SN), intermediate (INT), large normal (LN), and FRA10B-expressing (EXP). Sequence analysis demonstrated that the length polymorphism was mainly due to differences in repeat copy number. SN, INT, LN, and EXP alleles showed distinct sets of flanking repeat motifs (Fig. 2B). Analysis of these flanking haplotypes revealed that FRA10B-expressing alleles are derived from LN alleles, and that these are derivatives from INT alleles which are in turn formed from SN alleles (Fig. 2B). Such founder effects have also been observed for a number of trinucleotide repeat disorders (Table 1) and therefore seem to be a common property of the dynamic mutation mechanism which gives rise to the expansion of both mini- and microsatellite repeats. At the FRA10B locus the LN and EXP haplotypes were closely related. Surprising was the small size difference between the largest LN alleles which do not express FRA10B (⬃4.5 kb) and the shortest FRA10B-expressing alleles (⬃5 kb). This suggests that there is a threshold in repeat copy number that has to be exceeded for cytogenetic expression of the fragile site. Similar thresholds have also been described at the FRAXA and FRAXE loci (35). Hence this is another feature shared by mini- and microsatellites. The long-range PCR amplification across the FRA10B locus allowed the assessment of mitotic and meiotic instability. One heterozygous FRA10B-expressing individual repeatedly showed one short as well as two large amplification products which were interpreted as proof of somatic (mitotic) instability (34). A number of pedigrees revealed meiotic insta-

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bility: e.g., a decrease or increase in repeat copy number from parent to offspring which resulted in a shift in the size of PCR products between related individuals. Mitotic and meiotic instability have also been described at trinucleotide repeats (reviewed in 36) and are hence a third property shared between mini- and microsatellite repeats. COMPARISON OF DISTAMYCIN A INDUCIBLE AND BrdU REQUIRING RARE FRAGILE SITES One question arising from the fact that FRA16B is sensitive to distamycin A and BrdU, whereas FRA10B can only be induced by BrdU, is whether FRA16B and FRA10B share structural features. Both fragile site loci revealed expansion of AT-rich minisatellite repeats; their close relationship becomes obvious in Fig. 1. The FRA10B and FRA16B

FIG. 2. Representation of the AT-rich minisatellite region at the FRA10B locus. (A) Illustration of one FRA10B allele demonstrating the differences in percentages of AT/GC-values across FRA10B. The central region (91% AT) contains numerous minisatellite repeats that are depicted in different patterns. The flanking regions consist of nonrepetitive sequences that still have quite a high AT content (58%). (B) Simplified illustration of the FRA10B minisatellite repeat region. Presented are three groups of repeat motifs that were termed proximal (triangle), expanded (diamond), and SnaBI (hexagon). Variation in the sequences is shown as different shading or patterns. Stars point out identical repeat motifs between short normal (SN), intermediate (INT), long normal (LN), or FRA10B-expressing alleles. Only SN alleles were sequenced over the full length; question marks represent gaps in the sequences and dashes depict absence of a repeat motif. The repeats are flanked on both sides by nonrepetitive sequences (NR).

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consistency and the fact that members of all classes of rare fragile sites have now been characterized at the molecular level, the relation between sequence composition and induction chemistry of fragile sites is still unclear. COMMON FRAGILE SITES

FIG. 3. Examples for the ability of AT-rich minisatellite repeats to form hairpins. Similar secondary structures have been described for Apo B VNTR dimeric repeats (36).

consensus sequences share— depending on the sequence alignment— up to 31 identical bases (Fig. 1). The 5⬘-ends of the repeats reveal the highest homology with 12 identical base pairs. This includes an 11-bp sequence that is present in its inverse form further 3⬘ of the repeat motif. The FRA10B and FRA16B repeats both contain three copies each of these 11-bp sequence elements (Fig. 1). The inverted repeats are able to form hairpin structures (Fig. 3). Similar AT-rich repeats are, for example, present at the ApoB locus (37), the COL2A1 minisatellite region (38), and in the human minisatellite MSY1 (39). This widespread occurrence suggests that AT-rich minisatellite repeats might be of functional significance. A possible biological role for AT-rich minisatellite repeats is that they might function as matrix attachment regions (MAR), which are DNA sequences that are attached to the nuclear matrix (reviewed in 40). They are on average 500 bp long, occur about every 30 kb, and may play a role in DNA replication and transcription. The AT-rich class of MARs are often enriched in inverted repeats which is also the case for the FRA10B and FRA16B minisatellite repeats. The chemicals that induce the cytogenetic expression of FRA16B are known to preferentially bind to AT-rich DNA sequences. This is in agreement with the ascertainment of an expanded AT-rich minisatellite repeat at the fragile site. But despite this

It has been hypothesized that fragile sites predispose to chromosome breakage at the fragile site loci and that they may lead to cancer (41). A number of common fragile sites have been positionally cloned in recent years (42– 46) but although there is evidence for tumor suppressor genes at these loci, the results are as yet not conclusive. Common fragile sites seem to represent regions of fragility, rather than specific loci. No repeat expansion has been found at any of the common fragile sites studied. Nevertheless, they seem to be regions of the genome that attract interspersed repeats and are targets for viral integration. Future research will show whether the fragility at common fragile sites is in fact associated with cancer and whether it is caused by the formation of unusual secondary structures or a clustering of high flexibility regions as proposed by Mishmar et al. (45,47). MINISATELLITES AND DISEASE Minisatellites have repeatedly been correlated with human disease. In some cases the VNTR domains have been found to act as transcriptional regulators. Examples for this are first, that genetic susceptibility to insulin dependent diabetes mellitus type 2 (IDDM2) is encoded by the number of minisatellite repeats located 600 bp upstream of the insulin gene (48,49) and second, that the majority of patients with progressive myoclonus epilepsy of the Unverricht–Lundborg type (EPM1) have expansions to more than 40 copies of a dodecamer repeat which is located upstream of the 5⬘-transcription start site of the cystatin B gene, as compared to 2–3 repeat copies in normal alleles (50). There is more and more evidence that minisatellite repeats could be involved in disease epidemiology. For example, the minisatellite repeat at the 3⬘ end of the human apolipoprotein B gene is likely to play a role in coronary disorders (51). Ogilvie et al. (52) proposed that a polymorphism in the serotonin transporter gene is associated with susceptibility to depression in more than 10% of affected individuals. Another illustration for the influence of VNTR elements on the sus-

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ceptibility of disease was recently given by Deckert et al. (53): a repeat polymorphism in the promoter of the monoamine oxidase A gene was shown to be associated with panic disorder. It is likely that other VNTR polymorphisms afford susceptibility to additional diseases. These findings give only a glimpse into this exciting research area and reveal that the copy number of minisatellite repeats can not only determine the genetic stability of a chromosome locus, as in the case of fragile sites, but it can also influence the level of gene expression if the repeat is located within or close to a gene. This underlines the importance of understanding the molecular mechanism causing minisatellite instability. MECHANISMS GENERATING INSTABILITY AT REPEAT LOCI Whereas microsatellites, which comprise trinucleotide repeats, seem to mutate primarily by strand slippage during DNA replication (54), instability present at minisatellites is thought to be due to recombinational processes. Bois and Jeffreys (55) differentiate between mutation processes at GC-rich and AT-rich minisatellite repeats. Although instability at GC-rich minisatellite repeats is well studied, the processes causing it are still poorly understood. It is likely that meiotic instability of GC-rich minisatellites involves gene conversion-like events, whereas mitotic instability is caused by unequal sister chromatid exchange and intramolecular recombination, although replication slippage can not be excluded (reviewed in 55). Even less is known about mechanisms causing instability at AT-rich minisatellite repeats. Only the minisatellite present at the 3⬘ end of the human apolipoprotein B gene has been studied in some detail. It consists of a dimeric AT-rich core repeat of 30 bp (56,57). Linkage disequilibrium between flanking polymorphisms and Apo B minisatellite alleles was observed (37), which is in agreement with intrallelic mutation events like replication slippage and/or unequal sister chromatid exchange. Ellsworth et al. (37) proposed that instability of repeat arrays depends on the DNA sequence and their ability to form secondary structures. This accords with the fact that AT-rich minisatellite repeats like the ones detected at the FRA10B and FRA16B loci consist of shorter inverted repeats and are able to form hairpin structures (Fig. 3). Debrauwere et al. stated in a recent review that no general rule can explain the instability at micro- and minisatellites because it is controlled by their sequence

environment and biological activities (58). Understanding of mutation mechanisms causing the instability of minisatellite repeat arrays might therefore involve the detailed analysis of each single VNTR locus—a task that is not feasible with current technology. EXPECTATIONS FOR THE FUTURE With increasing DNA sequencing information becoming publicly available, it emerges that finishing the sequencing of the human genome is on no account the end of all activities but only the closure of the first chapter in the book of understanding human molecular genetics. Many more chapters are to follow and one of them will certainly be dealing with the biological roles that repeat sequences play—not only in the formation of fragile sites on chromosomes but also in the regulation of gene expression and possibly as matrix attachment regions. Sequencing of repeat arrays has proven to be cumbersome and time consuming. Advances in technology will hopefully open up faster ways of determining the exact composition of repeat tracts which will consequently enable us to gain a better understanding of not only the mutation mechanisms generating instability at fragile site loci but also of how the repeat number influences gene expression. ACKNOWLEDGMENTS The authors are supported by funds from the National Health and Medical Research Council of Australia, the WCH Research Foundation, and the Anti-Cancer Foundation of South Australia.

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