A study of FRAXE in mentally retarded individuals referred for fragile X syndrome (FRAXA) testing in the United Kingdom.

The folate-sensitive fragile site FRAXE is located in proximal Xq28 of the human X chromosome and lies approximately 600 kb distal to the fragile X syndrome (FRAXA) fragile site at Xq27.3. The cytogenetic expression of FRAXE is thought to be associated with mental handicap, but this is usually mild compared to that of the more common fragile X syndrome that is associated with the expression of the FRAXA fragile site. The exact incidence of FRAXE mental retardation is uncertain. We describe here the results of a U.K. survey designed to assess the frequency of FRAXE in a population of individuals referred for fragile X syndrome testing and found to be negative for expansion events at the FRAXA locus. No FRAXE expansion events were found in 362 cytogenetically negative males studied, and one expansion event was identified in a sample of 534 males for whom cytogenetic analyses were either unrecorded or not performed. Further FRAXE expansion events were detected in two related females known to be cytogenetically positive for a fragile site in Xq27.3-28. To gain insight into the FRAXE phenotype, the clinical details of the identified FRAXE male plus three other FRAXE individuals identified through previous referrals for fragile X syndrome testing are presented. For the population studied, we conclude that FRAXE mental retardation is a relatively rare but significant form of mental retardation for which genetic diagnosis would be appropriate.     

Genetic mapping of new DNA probes at Xq27 defines a strategy for DNA studies in the fragile X syndrome

The fragile X syndrome is the most common cause of familial mental retardation and is characterized by a fragile site at the end of the long arm of the X chromosome. The unusual genetics and cytogenetics of this X-linked condition make genetic counseling difficult. DNA studies were of limited value in genetic counseling, because the nearest polymorphic DNA loci had recombination fractions of 12% or more with the fragile X mutation, FRAXA. Five polymorphic loci have recently been described in this region of the X chromosome. The positions of these loci in relation to FRAXA were defined in a genetic linkage study of 112 affected families. The five loci--DXS369, DXS297, DXS296, IDS, and DXS304--had recombination fractions of 4% or less with FRAXA. The closest locus, DXS296, was distal to FRAXA and had a recombination fraction of 2%. The polymorphisms at these loci can be detected in DNA enzymatically digested with a limited number of restriction endonucleases. A strategy for DNA studies which is based on three restriction endonucleases and on five probes will detect one or more of these polymorphisms in 94% of women. This strategy greatly increases the utility of DNA studies in providing genetic advice to families with the fragile X syndrome.     

Physical mapping of new DNA probes near the fragile X mutation (FRAXA) by using a panel of cell lines

The fragile X syndrome is a very common disorder, but there has been little progress toward isolating the fragile X mutation (FRAXA). We describe a panel of 14 somatic cell hybrid lines, lymphoblastoid cell lines, and peripheral lymphocytes with X-chromosome translocation or deletion breakpoints near FRAXA. The locations of the breakpoints were defined with 16 established probes between pX45d (DXS100) and St14-1 (DXS52). Seven of the cell lines had breakpoints between the probes RN1 (DXS369) and U6.2 (DXS304), which flank FRAXA at distances of 3-5 centimorgans. The panel of cell lines was used to localize 16 new DNA probes in this region. Six of the probes-VK16, VK18, VK23, VK24, VK37, and VK47--detected loci near FRAXA, and it was possible to order both the X-chromosome breakpoints and the probes in relation to FRAXA. The order of probes and loci near FRAXA is cen-RN1,VK24-VK47-VK23-VK16,FRAXA-++ +VK21A-VK18-IDS-VK37-U6.2-qter. The breakpoints near FRAXA are sufficiently close together that probes localized with this panel can be linked on a large-scale restriction map by pulsed-field gel electrophoresis. This panel of cell lines will be valuable in rapidly localizing other probes near FRAXA.     

A survey of fragile X syndrome in a sample from Spanish Basque country

Fragile X syndrome is the most common inherited form of mental retardation. The syndrome is associated with a CGG repeat expansion in the 5'UTR of the first exon of the FMR1 gene. This gene maps to Xq27.3 and coincides with the cytogenetic fragile site (FRAXA). The present study deals with the prevalence of fragile X syndrome among individuals with mental retardation of unknown cause from institutions and special schools from the Spanish Basque Country. Results of cytogenetic and molecular studies, performed in a group of 134 unrelated individuals (92 males and 42 females) are presented. The cytogenetic marker at Xq27.3 was identified in 12 patients. Other chromosomal abnormalities were found in two cases that this and previous studies confirmed as Angelman and Prader-Willi syndromes. Two males, in whom the cytogenetic marker was identified, were found negative for FRAXA and FRAXE expansion at the molecular level. The present study shows that the frequency of the FRAXA full mutation in individuals of Spanish non-Basque origin is in the range of other Spanish populations. In the sample of Spanish Basque origin we have not found cytogenetic FRAXA site expression, and the CGG repeat size of FMR1 gene is in the normal range. The significance of these results are discussed.     

New polymorphisms at the DXS98 locus and confirmation of its location proximal to FRAXA by in situ hybridization.

The locus DXS98, detected with the 1.5-kb anonymous probe p4D-8, was recently shown to be closely linked and proximal to the locus for the fragile X syndrome, with theta = .05 at lod = 3.406, by utilizing a limited number of meioses informative for a two-allele MspI RFLP. Because DXS98 may be the closest available marker to the fragile X locus (FRAXA), we sought to increase its utility for linkage studies by extending its PIC and confirming its localization to Xq27, proximal to FRAXA. We have isolated 15 kb of genomic DNA (lambda 4D8-3) from the DXS98 locus by using p4D-8 to screen a genomic phage library containing partial Sau3A-digested human DNA. Three additional RFLPs for the enzymes BglII and XmnI were found by using the entire lambda 4D8-3 as probe. Combined heterozygosity for the four RFLPs in 25 unrelated females was 48%, as compared with only 28% when the MspI RFLP alone was used. In situ hybridization of unique sequences from lambda 4D8-3 was performed on metaphase chromosomes of lymphocytes and lymphoblasts from patients with the fragile X syndrome. Grains on the X chromosome were significantly clustered at band Xq27. Following fragile site induction, all nine grains in the q27-28 region were proximal to the fragile site. Confirmation of the location of DXS98 proximal to FRAXA and the new RFLPs at this locus make DXS98 more useful for linkage analysis and physical mapping in the region of the fragile X mutation.     

The role of DNA damage response pathways in chromosome fragility in Fragile X syndrome

FRAXA is one of a number of fragile sites in human chromosomes that are induced by agents like fluorodeoxyuridine (FdU) that affect intracellular thymidylate levels. FRAXA coincides with a >200 CGG•CCG repeat tract in the 5′ UTR of the FMR1 gene, and alleles prone to fragility are associated with Fragile X (FX) syndrome, one of the leading genetic causes of intellectual disability. Using siRNA depletion, we show that ATR is involved in protecting the genome against FdU-induced chromosome fragility. We also show that FdU increases the number of γ-H2AX foci seen in both normal and patient cells and increases the frequency with which the FMR1 gene colocalizes with these foci in patient cells. In the presence of FdU and KU55933, an ATM inhibitor, the incidence of chromosome fragility is reduced, suggesting that ATM contributes to FdU-induced chromosome fragility. Since both ATR and ATM are involved in preventing aphidicolin-sensitive fragile sites, our data suggest that the lesions responsible for aphidicolin-induced and FdU-induced fragile sites differ. FRAXA also displays a second form of chromosome fragility in absence of FdU, which our data suggest is normally prevented by an ATM-dependent process.     

A YAC contig across the fragile X site defines the region of fragility.

The fragile X syndrome is a common cause of mental retardation and is associated with a fragile site at Xq27.3 (FRAXA). Recently, evidence has been presented for the role of methylation and genomic imprinting in the expression of the disease. We have identified a site of methylation in patients by long range restriction mapping of the region. In this paper we present a YAC contig of this area, localise the CpG sequences which are methylated, and show by in situ hybridisation that the site of fragility lies within this region.     

A common fragile site at Xq27: theoretical and practical implications.

The fragile site at Xq27 (FRAXA) is associated with a common form of X-linked mental retardation (Martin-Bell syndrome). It is induced in culture by conditions of thymidylate stress and is generally considered a rare fragile site found only in association with an X-linked form of mental retardation. Using a somatic cell hybrid system, we previously demonstrated that fragile-X expression can be induced by thymidylate stress in normal X chromosomes at low levels (4%-5%). In the present report, significantly higher levels of fragile-X expression (6%-28%) have been induced in lymphocytes or lymphoblasts of all seven control males using high doses of aphidicolin (1.5 microM). Similar high levels of expression (10%-12%) were observed in both of two normal male chimpanzees (Pan troglodytes). These data demonstrate that Xq27 contains a common fragile site (FRAXD) that is ancestral to the divergence of man and the chimpanzee. Presence of a common and a rare fragile site in the same metaphase chromosome band does not prove that they are identical and may, in fact, represent two unrelated fragile sites. However, the possibility exists that the common fragile site at Xq27 may be the substrate for unequal recombination events that produces the rare fragile site associated with Martin-Bell syndrome. In addition, presence of a common fragile site at Xq27 may explain the occasional observation of low-frequency fragile-X expression in normal control individuals. Caution is therefore warranted in the interpretation of low-level fragile-X expression in diagnostic and prenatal diagnostic settings.     

Linkage homogeneity near the fragile X locus in normal and fragile X families

The fragile X syndrome locus, FRAXA, is located at Xq27. Until recently, few polymorphic loci had been genetically mapped close to FRAXA. This has been attributed to an increased frequency of recombination at Xq27, possibly associated with the fragile X mutation. In addition, the frequency of recombination around FRAXA has been reported to vary among fragile X families. These observations suggested that the genetic map at Xq27 in normal populations was different from that in fragile X populations and that the genetic map also varied within the fragile X population. Such variability would reduce the reliability of carrier risk estimates based on DNA studies in fragile X families. Five polymorphic loci have now been mapped to within 4 cM of FRAXA--DXS369, DXS297, DXS296, IDS, and DXS304. The frequency of recombination at Xq26-q28 was evaluated using data at these loci and at more distant loci from 112 families with the fragile X syndrome. Two-point and multipoint linkage analyses failed to detect any difference in the recombination fractions in fragile X versus normal families. Two-point and multipoint tests of linkage homogeneity failed to detect any evidence of linkage heterogeneity in the fragile X families. On the basis of this analysis, genetic maps derived from large samples of normal families and those derived from fragile X families are equally valid as the basis for calculating carrier risk estimates in a particular family.     

Triplet repeat expansion at the FRAXE locus and X-linked mild mental handicap.

We have recently shown that the expression of the FRAXE fragile site in Xq28 is associated with the expansion of a GCC trinucleotide repeat. In the families studied, FRAXE expression is also associated with mild mental handicap. Here we present data on families that previously had been diagnosed as having the fragile X syndrome but that later were found to be negative for trinucleotide repeat expansion at the FRAXA locus. In these families we demonstrate the presence of a GCC trinucleotide repeat expansion at the FRAXE locus. Studies of the FRAXE locus of normal individuals show that they have 6-25 copies of the repeat, whereas affected individuals have > 200 copies. As in the fragile X syndrome, the amplified CpG residues are methylated in affected males.     

Absence of expression of the FMR-1 gene in fragile X syndrome

We previously reported the isolation of a gene (FMR-1) expressed in brain at the fragile X locus. One exon of this gene lies within an EcoRI fragment that exhibits length variation in fragile X patients. This exon also contains the CGG repeat within the CpG island hypermethylated in fragile X patients. To study the involvement of the FMR-1 gene in the fragile X syndrome, its expression was studied in lymphoblastoid cell lines and leukocytes derived from patients and normal controls. FMR-1 mRNA was absent in the majority of male fragile X patients, suggesting a close involvement of this gene in development of the syndrome. Normal individuals and carriers all show expression. The methylation status of the BssHII site at the CpG island was also studied by Southern blot analysis of DNA from patients, carriers, and controls. The minority of fragile X affected males that show expression of FMR-1 demonstrated an associated incomplete methylation of the BssHII site.     

The gene encoding L1, a neural adhesion molecule of the immunoglobulin family, is located on the X chromosome in mouse and man

The murine and human genes for the L1 neural adhesion molecule were shown to lie on conserved regions of the X chromosome to which genes responsible for several neuromuscular diseases have been mapped and which are adjacent to the fragile site (FRAXA) associated with mental retardation. By pulsed-field gel mapping we have demonstrated physical linkage between the L1 gene and other genes located in Xq28: L1 lies between the eye pigment RCP, GCP locus and the glucose-6-phosphate dehydrogenase (G6PD) gene. This location is compatible with the implication of the L1 molecule in one of the X-linked neuromuscular diseases mapped to this region.     

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.     

Review: The fragile X syndrome: Isolation of the FMR-1 gene and characterization of the fragile X mutation

Fragile X syndrome, associated with the fragile X chromosome, is the most common cause of familial mental retardation. A breakthrough has been made in molecular biological research into the fragile X site. In this review we describe the molecular investigations that have led to the isolation of the FMR-1 gene. The nature of the fragile X mutation as well as the implications of the DNA test for the mutation are discussed.     

CGG repeats associated with DNA instability and chromosome fragility form structures that block DNA synthesis in vitro.

A large increase in the length of a CGG tandem array is associated with a number of triplet expansion diseases, including fragile X syndrome, the most common cause of heritable mental retardation in humans. Expansion results in the appearance of a fragile site on the X chromosome in the region of the CGG array. We show here that CGG repeats readily form a series of barriers to DNA synthesis in vitro. There barriers form only when the (CGG)n strand is used as the template, are K(+)-dependent, template concentration-independent, and involve hydrogen bonding between guanines. Chemical modification experiments suggest these blocks to DNA synthesis result from the formation of a series of intrastrand tetraplexes. A number of lines of evidence suggest that both triplet expansion and chromosome fragility are the result of replication defects. Our data are discussed in the light of such evidence.     

Fragile (X) expression: Relationship to the cell cycle

Summary The fragile (X) chromosome demonstrable in individuals with one type of X-linked mental retardation is seldom, if ever, seen in more than 50% of cells of affected individuals. We have devised a model to explain this apparent 50% maximum, one essential feature of which is that the fragile (X) will not be seen in cells in their first division in thymidine-depleted media. The validity of our model was tested on lymphoblastoid cell lines from affected males by treating the cells with fluorodeoxyuridine (FUdR) to induce the marker and/or bromodeoxyuridine (BrdU) to determine the cell cycle. We have evidence that the fragile (X) is present in cells in the first and subsequent these observations our model is not valid and the 50% expression of the fragile site at Xq(28) and other unusual properties of this region of the X chromosome remain unexplained.This work was supported by Grant HD 07879 from the National Institutes of Health     

Thymidylate synthase-deficient Chinese hamster cells: a selection system for human chromosome 18 and experimental system for the study of thymidylate synthase regulation and fragile X expression.

Chinese hamster lung (CHL) V79 cells already deficient in hypoxanthine phosphoribosyltransferase were exposed to uv light and selected for mutations causing deficiency of thymidylate synthase (TS) by their resistance to aminopterin in the presence of thymidine and limiting amounts of methyl tetrahydrofolate. Three of seven colonies chosen for initial study were shown to be thymidylate synthase deficient (TS-) by enzyme assay, thymidine auxotrophy, and their inability to incorporate labeled deoxyuridine into their DNA in vivo. Complementation analysis of human X TS- hamster hybrids revealed that TS activity segregated with human chromosome 18. Southern analysis of a panel of 14 human X hamster hybrids probed with complementary DNA from mouse TS confirmed the chromosome assignment of TS to human chromosome 18; quantitative Southern blotting using unbalanced human cell lines further localized the gene to 18q21.31----qter. Another hybrid was generated that contained a human X chromosome with the Xq28 folate-dependent fragile site as its only human chromosome in a hamster TS- background. The fragile site could be easily and reproducibly expressed in this hybrid without the use of antimetabolites simply by removing exogenous thymidine from the medium. These TS-deficient cells are useful for: somatic cell genetics as a unique selectable marker for human chromosome 18, studies on regulation of the TS gene, and analysis of the fragile (X) chromosome and other folate-dependent fragile sites.     

Evaluation of the fragile X (FRAXA) syndrome with methylation-sensitive PCR

The fragile X (FRAXA) syndrome is the most common form of inherited mental retardation in males. Its peculiar pattern of inheritance results from the parent of origin-specific expansion of a CGG-repeat within the FMR1 gene on the X chromosome. In patients, gene function is abolished by hypermethylation of the promoter and the massively expanded repeat. We have developed a methylation-sensitive polymerase chain reaction (MS-PCR) strategy that combines repeat-length and methylation analysis of the CGG-repeat and the promoters of the FMR1 and XIST genes. The allelic methylation of the latter opposes that of the FMR promoter and serves as an internal control and standard for semiquantitative analyses. This system enables the delineation of 11 distinct patterns encountered in nonaffected, carrier, and affected males and females. We have evaluated our system on well-defined samples with different FMR1 mutations and have used it for the diagnostic evaluation of 253 male and 80 female probands. In the male group, we have identified five full mutations, and three gray-zone and premutation alleles with 54, 55, and 62 repeats, respectively. The female group consists of 33 normal homozygote and 41 heterozygote individuals, two of whom harbor a gray-zone allele with 47 repeats, none with a premutation, and six with a full mutation. Our MS-PCR approach allows the currently most comprehensive diagnostic evaluation of the FRAXA syndrome in a cost- and time-efficient fashion. In addition, it is a valuable tool for the analysis of clonality and skewing phenomena in females.     

Molecular genetic analysis of the FMR-1 gene in a large collection of autistic patients

A genetic etiology in autism is now strongly supported by family and twin studies. A 3:1 ratio of affected males to females suggests the involvement of at least one X-linked locus in the disease. Several reports have indicated an association of the fragile X chromosomal anomaly at Xq27.3 (FRAXA) with autism, whereas others have not supported this finding. We have so far collected blood from 105 simplex and 18 multiplex families and have assessed 141 patients by using the Autism Diagnostic Interview-Revised (ADI-R), the Autism Diagnostic Observation Scale, and psychometric tests. All four ADI-R algorithm criteria were met by 131 patients (93%), whereas 10 patients (7%) showed a broader phenotype of autism. Southern blot analysis was performed with three different enzymes, and filters were hybridized to an FMR-1-specific probe to detect amplification of the CCG repeat at FRAXA, to the complete FMR-1 cDNA probe, and to additional probes from the neighborhood of the gene. No significant changes were found in 139 patients (99%) from 122 families, other than the normal variations in the population. In the case of one multiplex familiy with three children showing no dysmorphic features of the fragile X syndrome (one male meeting 3 out of 4 ADI-algorithm criteria, one normal male with slight learning disability but negative ADI-R testing, and one fully autistic female), the FRAXA full-mutation-specific CCG-repeat expansion in the genotype was not correlated with the autism phenotype. Further analysis revealed a mosaic pattern of methylation at the FMR-1 gene locus in the two sons of the family, indicating at least a partly functional gene. Therefore, we conclude that the association of autism with fragile X at Xq27.3 is non-existent and exclude this location as a candidate gene region for autism. Received: 25 October 1996 / Accepted: 10 March 1997