CGG-repeat expansion in the DIP2B gene is associated with the fragile site FRA12A on chromosome 12q13.1

A high level of cytogenetic expression of the rare folate-sensitive fragile site FRA12A is significantly associated with mental retardation. Here, we identify an elongated polymorphic CGG repeat as the molecular basis of FRA12A. This repeat is in the 5' untranslated region of the gene DIP2B, which encodes a protein with a DMAP1-binding domain, which suggests a role in DNA methylation machinery. DIP2B mRNA levels were halved in two subjects with FRA12A with mental retardation in whom the repeat expansion was methylated. In two individuals without mental retardation but with an expanded and methylated repeat, DIP2B expression was reduced to approximately two-thirds of the values observed in controls. Interestingly, a carrier of an unmethylated CGG-repeat expansion showed increased levels of DIP2B mRNA, which suggests that the repeat elongation increases gene expression, as previously described for the fragile X-associated tremor/ataxia syndrome. These data suggest that deficiency of DIP2B, a brain-expressed gene, may mediate the neurocognitive problems associated with FRA12A.     

Human fragile site FRA16B DNA excludes nucleosomes in the presence of distamycin

Human fragile sites are weak staining gaps in chromosomes generated by specific culture conditions. The short CGG repeating DNA derived from folate-sensitive fragile sites has been shown to exclude single nucleosomes. To test whether this nucleosome exclusion model provides a general molecular mechanism for the formation of fragile sites, a different class of fragile site, the 33-base pair AT-rich repeating DNAs derived from the rare distamycin-inducible site, FRA16B, was examined for its ability to assemble single nucleosomes and nucleosome arrays using in vitro nucleosome reconstitution methods. The FRA16B DNA fragments strongly exclude nucleosome assembly only in the presence of distamycin, and increasing the number of 33-bp repeats increases the effect of distamycin in the destabilization of the nucleosome formation, suggesting a common mechanism for the formation of fragile sites.     

Dynamic mutations in human genes: A review of trinucleotide repeat diseases

Dynamic mutations in human genes result from unstable trinucleotide repeats embedded within the transcribed region. The changeable nature of these mutations from generation to generation is in contrast to the static inheritance of other single-gene mutational events, e.g. point mutations, deletions, insertions and inversions, typically associated with Mendelian inheritance patterns. Intergenerational instability of dynamic mutations within families has provided an explanation for the genetic anticipation, leading to increasing severity or earlier age of onset in successive generations, associated with certain inherited disorders. While models for genomic instability presume that trinucleotide repeat expansion results from disruption of the DNA replication process, experimental evidence has not yet been obtained in support of this contention. Nevertheless, examples of unstable trinucleotide repeats continue to increase, although not all are associated with a specific phenotype. Five disorders resulting from small-scale expansions of CAG repeats within the protein-coding region are known: spinobulbar muscular atrophy, Huntington’s disease, spinocerebellar ataxia type 1, dentatorubral-pallidoluysian atrophy (DRPLA) and Machado-Joseph disease. A sixth disorder, Haw River syndrome, is allelic to DRPLA. Five folate-sensitive chromosomal fragile sites characterized to date, viz. FRAXA, FRAXE, FRAXF, FRA11B and FRA16A, all have large-scale CGG repeat expansion. Two disorders, fragile X syndrome and FRAXE mental retardation, result from instability of CGG repeats in the 5’ untranslated region ofFMR1 andF M R2 genes respectively. FRA11B lies close to chromosome 1 1q deletion endpoints in many Jacobsen syndrome patients and may be related to the deletion event producing partial aneuploidy for 1lq. Expansion of FRAXF and FRA16A has not been associated with a phenotype. Myotonic dystrophy results from a large-scale CTG expansion in the 3’ untranslated region of the myotonin protein kinase gene while Friedreich’s ataxia has recently been found to have a large-scale GAA repeat in the first intron ofX25. This article reviews the characteristics of trinucleotide repeat disorders and summarizes current understanding of the molecular pathophysiology.     

Human chromosome fragility

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.     

A novel minisatellite repeat expansion identified at FRA16B in a Japanese carrier

Previously, the allelic expansion of a 33-bp AT-rich minisatellite repeat has been reported to cause FRA16B, a distamycin A-inducible fragile site. Here, we identified a novel 35-bp minisatellite repeat at FRA16B in a Japanese carrier. The nucleotide sequence of the 35-bp minisatellite was highly AT-rich and nearly identical to the 33-bp one but with insertion of two nucleotides, thymine and adenine. The copy number of the AT-rich minisatellite was 21 in total in the carrier, while only a few copies of the 33-bp minisatellite were present in a non-carrier Japanese subject. These results suggest that the molecular mechanism involved in the allelic expansion of the minisatellite repeat in FRA16B recognizes both minisatellites, the 33-bp one and the 35-bp one, as an amplicon. These observations were different from the ones at folate-sensitive fragile sites, where the CCG triplet repeat was commonly involved in the allelic expansion. Although a slight reduction in AT content (95% > 90%) in the region of minisatellite expansion in the carrier subject was observed, both AT-content and length of the highly AT-rich region seem to play important roles in the cytogenetic expression of the distamycin A-inducible fragile site. In another normal subject, without fragile site expression, allelic expansion involving the 33-bp minisatellite was observed, and the length of the AT-rich DNA region was increased up to approximately 1000 bp. Since the length of the AT-rich minisatellite region was increased up to approximately 1,100-bp in the carrier subject, the threshold length for the cytogenetic expression of the AT-rich DNA region may be between about 1,000-bp and 1,100-bp.     

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.     

Matrix attachment region (MAR) properties and abnormal expansion of AT island minisatellites in FRA16B fragile sites in leukemic CEM cells

AT-rich minisatellites (AT islands) are sites of genomic instability in cancer cells and targets for extremely lethal AT-specific drugs, such as bizelesin. Here we investigated the AT islands in the FRA16B fragile site region for their possible roles in the organization of DNA on the nuclear matrix. The FRA16B AT island nominally spans ~3 kb of mostly >90% A/T DNA. In silico analysis indicates that this domain exhibits characteristics of nuclear matrix attachment regions (MARs): an exceptionally intense computed ‘MAR potential’ and profound duplex destabilization and flexibility. FRA16B repeats specifically bind to isolated nuclear matrices, which indicates their in vitro MAR function. This binding is several-fold greater than that of a known MAR in the c-myc gene. AT islands in fragile sites FRA16B and FRA16D are significantly more abundant in CEM cells that are hypersensitive to bizelesin compared to normal WI-38 cells. FRA16B overabundance in CEM is due to an ~10-fold expansion of FRA16B repeats. The expanded FRA16B minisatellites in CEM cells preferentially localize to the nuclear matrix-associated DNA indicating their in vivo MAR function. The unexpanded repeats in WI-38 cells localize to the loop DNA. The c-myc MAR is also matrix-associated in CEM cells while localizing to loop DNA in WI-38 cells. These results are the first to demonstrate that AT islands in fragile sites can function as MARs both in vitro and in vivo. The ability of FRA16B-mediated MAR sites to rearrange depending on the repeat expansion status could be relevant to both genomic instability of cancer cells and their sensitivity to AT-island targeting drugs.     

FRA2B is distinct from inverted telomere repeat arrays at 2q13

Human chromosome 2 was formed by a telomere-to-telomere fusion of two ancestral ape chromosomes. The fusion point is localized in chromosomal band 2q13, which also contains the rare, folate-sensitive fragile site FRA2B. It has been hypothesized that this fragile site may be related to the presence of interstitial telomeric and subtelomeric sequences, which have come to lie in an inverted repeat arrangement as a result of the fusion event. Fluorescence in situ hybridization of a genomic cosmid c8.1, which spans the fusion point, was carried out on metaphase spreads of an individual who expressed the fragile site at 2q13. We show that the fusion point maps distal to this fragile site. Therefore, we conclude that the inverted arrays of telomeric and subtelomeric sequences found at this fusion point are unlikely to correspond to the rare fragile site at 2q13.     

Chromatin remodeling in the noncoding repeat expansion diseases

Friedreich ataxia, myotonic dystrophy type 1 and 3 forms of intellectual disability, fragile X syndrome, FRAXE mental retardation, and FRA12A mental retardation are repeat expansion diseases caused by expansion of CTG.CAG, GAA.TTC, or CGG.CCG repeat tracts. These repeats are transcribed but not translated. They are located in different parts of different genes and cause symptoms that range from ataxia and hypertrophic cardiomyopathy to muscle wasting, male infertility, and mental retardation, yet recent reports suggest that, despite these differences, the repeats may share a common property, namely the ability to initiate repeat-mediated epigenetic changes that result in heterochromatin formation.     

FRAXE expansion is not a common etiological factor among developmentally delayed males.

Expansion of a (CGG)n trinucleotide repeat unit at FRAXE, a newly defined fragile site distal to FRAXA, at Xq28, is reported to be associated with mild mental retardation. Three hundred developmentally delayed male patients referred for fragile X testing but negative for the FMR-1 gene trinucleotide expansion were screened for the FRAXE expansion. This group of patients had a wide range of intellectual or behavioral problems and included 19 patients who had low-level fragile site expression detected cytogenetically at Xq27-q28. None of the patients tested positive for the FRAXE expansion. These results suggest that FRAXE is not a common etiological factor among this group of patients. The data support the hypothesis that FRAXE is either very rare or a benign fragile site that is not associated with any clinical phenotype, similar to the FRAXF and FRA16A sites.     

Rare fragile sites

Rare folate-sensitive fragile sites are the archetypal trinucleotide repeats. Although the CAG repeat in the androgen receptor, associated with spinobulbar muscular atrophy, was the first to be published in 1991, it was the publication in the same year of the molecular basis of fragile X that focused much attention on trinucleotide repeat expansion as a mutational mechanism. A number of rare fragile sites have had their repeat elements characterised since that time. The so-called "folate-sensitive" fragile sites are likely to be all CCG repeat expansions similar to the fragile X. The folate insensitive fragile sites have more complex longer repeat elements. Only two rare fragile sites (FRAXA and FRAXE) are of unequivocal clinical significance in that they are associated with intellectual disability.     

Instability of the CGG repeat and expression of the FMR1 protein in a male fragile X patient with a lung tumor.

The molecular mechanism of the fragile X syndrome is based on the expansion of an CGG repeat in the 5' UTR of the FMR1 gene in the majority of fragile X patients. This repeat displays instability both between individuals and within an individual. We studied the instability of the CGG repeat and the expression of the FMR1 protein (FMRP) in several different tissues derived from a male fragile X patient. Using Southern blot analysis, only a full mutation is detected in 9 of the 11 tissues tested. The lung tumor contains a methylated premutation of 160 repeats, whereas in the testis, besides the full mutation, a premutation of 60 CGG repeats is detected. Immunohistochemistry of the testis revealed expression of FMR1 in the spermatogonia only, confirming the previous finding that, in the sperm cells of fragile X patients with a full mutation in their blood cells, only a premutation is present. Immunohistochemistry of brain and lung tissue revealed that 1% of the cells are expressing the FMRP. PCR analysis demonstrated the presence of a premutation of 160 repeats in these FMR1-expressing cells. This indicates that the tumor was derived from a lung cell containing a premutation. Remarkably, despite the methylation of the EagI and BssHII sites, FMRP expression is detected in the tumor. Methylation of both restriction sites has thus far resulted in a 100% correlation with the lack of FMR1 expression, but the results found in the tumor suggest that the CpGs in these restriction sites are not essential for regulation of FMR1 expression. This indicates a need for a more accurate study of the exact promoter of FMR1.     

The fragile X syndromes

Fragile X syndrome is a leading cause of mental retardation worldwide, with an incidence of approximately one case in 2000 live births. It is amongst the most common of human genetic diseases, and was the first to be associated with an unstable trinucleotide (CGG) repeat sequence. It is also characterized by a chromosomal fragile site which was the first of (now) four such sites to be identified at the molecular level. Each shows very similar features suggesting that other representatives of this type of fragile site will likely involve similar sequences. As with the other unstable trinucleotide repeats, the sequence at the fragile X locus is found to be remarkably unstable upon genetic transmission, however many features differ from the other repeats. As repeat expansion at the fragile X locus results in loss of expression of the co-resident FMR1 gene, the basis for clinical features is best understood in this disorder. Two additional fragile sites in the vicinity have been identified, and at least one of these is associated with mental retardation.     

RNA-binding proteins hnRNP A2/B1 and CUGBP1 suppress fragile X CGG premutation repeat-induced neurodegeneration in a Drosophila model of FXTAS

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a recently described neurodegenerative disorder of older adult carriers of premutation alleles (60-200 CGG repeats) in the fragile X mental retardation gene (FMR1). It has been proposed that FXTAS is an RNA-mediated neurodegenerative disease caused by the titration of RNA-binding proteins by the CGG repeats. To test this hypothesis, we utilize a transgenic Drosophila model of FXTAS that expresses a premutation-length repeat (90 CGG repeats) from the 5' UTR of the human FMR1 gene and displays neuronal degeneration. Here, we show that overexpression of RNA-binding proteins hnRNP A2/B1 and CUGBP1 suppresses the phenotype of the CGG transgenic fly. Furthermore, we show that hnRNP A2/B1 directly interacts with riboCGG repeats and that the CUGBP1 protein interacts with the riboCGG repeats via hnRNP A2/B1.     

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.     

An origin of DNA replication in the promoter region of the human fragile X mental retardation (FMR1) gene

Fragile X syndrome, the most common form of inherited mental retardation in males, arises when the normally stable 5 to 50 CGG repeats in the 5' untranslated region of the fragile X mental retardation protein 1 (FMR1) gene expand to over 200, leading to DNA methylation and silencing of the FMR1 promoter. Although the events that trigger local CGG expansion remain unknown, the stability of trinucleotide repeat tracts is affected by their position relative to an origin of DNA replication in model systems. Origins of DNA replication in the FMR1 locus have not yet been described. Here, we report an origin of replication adjacent to the FMR1 promoter and CGG repeats that was identified by scanning a 35-kb region. Prereplication proteins Orc3p and Mcm4p bind to chromatin in the FMR1 initiation region in vivo. The position of the FMR1 origin relative to the CGG repeats is consistent with a role in repeat maintenance. The FMR1 origin is active in transformed cell lines, fibroblasts from healthy individuals, fibroblasts from patients with fragile X syndrome, and fetal cells as early as 8 weeks old. The potential role of the FMR1 origin in CGG tract instability is discussed.