Fully expanded FMR1 CGG repeats exhibit a length- and differentiation-dependent instability in cell hybrids that is independent of DNA methylation.

The fragile X syndrome is characterized at the molecular level by expansion and methylation of a CGG trinucleotide repeat located within the FMR1 locus. The tissues of most full mutation carriers are mosaic for repeat size, but these mutational patterns tend to be well conserved when comparing multiple tissues within an individual. Moreover, full mutation alleles are stable in cultured fibroblasts. These observations have been used to suggest that fragile X CGG repeat instability normally is limited to a period during early embryogenesis. DNA methylation of the repeat region is also believed to occur during early development, and some experimental evidence indicates that this modification may stabilize the repeats. To study the behavior of full mutation alleles in mitotic cells, we generated human-mouse somatic cell hybrids that carry both methylated and unmethylated full mutation FMR1 alleles. We observed considerable repeat instability and analyzed repeat dynamics in the hybrids as a function of DNA methylation, repeat length and cellular differentiation. Our results indicate that although DNA methylation does correlate with stability in primary human fibroblasts, it does not do so in the cell hybrids. Instead, repeat stability in the hybrids is dependent on repeat length, except in an undifferentiated cellular background where large alleles are maintained with a high degree of stability. This stability is lost when the cells undergo differentiation. These results indicate that the determinants of CGG repeat stability are more complex than generally believed, and suggest an unexpected role for cellular differentiation in this process.

Hypomethylation of an expanded FMR1 allele is not associated with a global DNA methylation defect.

The vast majority of fragile-X full mutations are heavily methylated throughout the expanded CGG repeat and the surrounding CpG island. Hypermethylation initiates and/or stabilizes transcriptional inactivation of the FMR1 gene, which causes the fragile X-syndrome phenotype characterized, primarily, by mental retardation. The relation between repeat expansion and hypermethylation is not well understood nor is it absolute, as demonstrated by the identification of nonretarded males who carry hypomethylated full mutations. To better characterize the methylation pattern in a patient who carries a hypomethylated full mutation of approximately 60-700 repeats, we have evaluated methylation with the McrBC endonuclease, which allows analysis of numerous sites in the FMR1 CpG island, including those located within the CGG repeat. We report that the expanded-repeat region is completely free of methylation in this full-mutation male. Significantly, this lack of methylation appears to be specific to the expanded FMR1 CGG-repeat region, because various linked and unlinked repetitive-element loci are methylated normally. This finding demonstrates that the lack of methylation in the expanded CGG-repeat region is not associated with a global defect in methylation of highly repeated DNA sequences. We also report that de novo methylation of the expanded CGG-repeat region does not occur when it is moved via microcell-mediated chromosome transfer into a de novo methylation-competent mouse embryonal carcinoma cell line.

Clinical outcomes of four patients with microdeletion in the long arm of chromosome 2.

We present clinical outcome, through several years of follow-up, of 4 mentally retarded patients, each with a small interstitial deletion in the long arm of chromosome 2, within a region on which clinical reports are infrequent. Our patient 1 was found to have del(2)(q22.3q23.3); patients 2 and 3, del(2)(q23.3q24.2); and patient 4, del(2) (q24.2q31). By comparison of our cases with each other and with those previously published with comparable interstitial deletion, we attempted to identify characteristic clinical findings. Short neck with excessive cervical skin was seen with monosomy of chromosome 2 bands q22.3-q23.3, while hypertrichosis and a peculiar high pitched cry were seen with monosomy of chromosome 2 bands q23.3-q24.2. As suggested by Moller et al. [1984: Hum Genet 68:77-86], a cleft between the first and second toes was seen with monosomy of chromosome 2 bands q24.2-q31. In addition, seizure disorder was present in patients 1 and 4 (with the more proximal and distal deletions, respectively).

Guidelines for the diagnosis of fragile X syndrome. National Fragile X Foundation.

Direct DNA analysis of the fragile X mutation has become available with the isolation of DNA probes that detect the unstable DNA sequence containing the CGG repeat. We present the various alternatives of combinations of probes and enzymes that can be used for the diagnosis of fragile X syndrome. An overview is given of all the different available probes. A different protocol is presented for postnatal and prenatal diagnosis of fragile X syndrome. This includes Southern blot analysis as well as direct analysis of the CGG repeat by PCR amplification. We discuss the role of constitutional cytogenetic analysis in the diagnosis of mentally retarded subjects and cytogenetic analysis for the diagnosis of fragile X syndrome.

Chromosomal fragile site expression in dogs: I. Breed specific differences.

Peripheral blood lymphocytes from clinically normal Doberman pinscher and boxer dogs were cultured for folate-sensitive and, in preliminary studies, aphidicolin-inducible fragile site expression. Both autosomal and X chromosomal fragile sites were observed in canine cells cultured under folate/thymidine depletion and in cells cultured in medium containing aphidicolin. Results from the three dogs evaluated for both folate-sensitive and aphidicolin-inducible fragile site expression showed that the frequency of fragile site expression was significantly (P less than 0.05) greater in cells cultured in medium containing aphidicolin than in cells cultured in folate/thymidine-depleted medium. Cells from the boxer dog expressed a high percentage (66.67%) of aphidicolin-inducible fragile sites in contrast to the Doberman pinscher dog in which only 21.10% of the lymphocytes expressed aphidicolin-inducible fragile sites. The frequencies of spontaneous and folate-sensitive fragile site expression did not vary significantly by breed of dog. Age of dog was significantly and positively correlated with frequency of folate-sensitive fragile site expression in dogs of the boxer breed, but not in dogs of the Doberman pinscher breed. The dog X chromosome expressed three folate-sensitive and aphidicolin-inducible fragile sites. The G-band location of these three fragile sites showed homology with three recognized constitutive common fragile sites on the human X chromosome: Xp22, Xq21, and Xq27.2. Two specific autosomal fragile sites were identified, one on the distal end of the long arm of chromosome 1 and one on the distal end of the long arm of chromosome 8. Other autosomal fragile sites were also apparent but could not be assigned reliably to specific chromosomes.

Chromosomal fragile site expression in dogs: II. Expression in boxer dogs with mast cell tumors.

Peripheral blood lymphocytes from boxer dogs with a history of cutaneous mast cell tumors were cultured for fragile site expression. As in a control group of dogs, cells from these dogs expressed folate-sensitive autosomal and X chromosome fragile sites. Cells from boxer dogs with mast cell tumors expressed the same three common fragile sites on the X chromosome as cells from control dogs. Three folate-sensitive autosomal fragile sites not observed in cells from the control dogs were identified in cells from boxers with mast cell tumors. These included fragile sites near the telomeres of the arms of chromosomes 3 and 4 and a fragile site on the distal half of chromosome 15. Cells from boxers with mast cell tumors showed a greater frequency of fragile site expression than did cells from control dogs, but this observation was attributed to an unintended selection bias for younger boxer dogs without mast cell tumors and older boxer dogs with mast cell tumors and an increased frequency of fragile site expression with increasing age in dogs of the boxer breed.

A novel Robertsonian translocation in a family of Walker hounds.

A 5-year-old female Walker hound was presented to the Washington State University Veterinary Teaching Hospital as a result of a narrowing of the vulva, which prevented natural breeding. All other physical and clinical findings were normal. Cytogenetic analysis disclosed a chromosome number of 77, with three metacentric chromosomes comprised of two X chromosomes and a Robertsonian translocation of two acrocentric autosomes, chromosomes 21 and 33. Cytogenetic analysis of two full-sister siblings with histories of absence of estrus disclosed one with the same translocation and one with a normal female chromosome constitution. The propositus was artificially inseminated with semen from a karyotypically normal male Walker hound and gave birth to nine live grossly normal pups, six females and three males. Another female pup was stillborn but was grossly normal. Cytogenetic analysis of the live pups disclosed that four (three males and one female) of the nine had the same translocation in all lymphocytes. The remaining five pups (five females) had normal female chromosome constitutions. The litter size was average for this breed. This is a previously unreported Robertsonian translocation in dogs.

The giemsa banding pattern of canine chromosomes, using a cell synchronization technique.

Cytogenetic investigations of the domestic dog, Canis familiaris, were performed on the Doberman pinscher and two Boxer dogs. Conventional homogeneously stained and G-banded metaphases from peripheral blood lymphocyte cultures synchronized with amethopterin and bromodeoxyuridine were studied. These procedures permitted the unequivocal identification of all canine chromosomes. A canine chromosome idiogram was constructed on the basis of the G-banding pattern at the haploid 327-band resolution level. The secondary constrictions and tapering of the telomeric regions characteristic of several canine chromosomes are described. Q-, C-, and NOR-banding were also performed and the salient features are described. This karyotype should enhance the value of the canine species in cytogenetic investigations.

Cytogenetic evaluation of four canine mast cell tumors.

We evaluated four canine cutaneous mast cell tumors cytogenetically. All four tumors contained both hypodiploid and hyperdiploid cells, an increase in the number of metacentric chromosomes, exchange configurations, and cells showing loss of an X chromosome. All tumors contained metaphases with chromosome gaps and breaks at frequencies greater than observed spontaneous chromosome breaks in normal cultured canine peripheral blood lymphocytes. Three of the four tumors had a normal modal chromosome number of 78. The fourth tumor had a modal chromosome number of 93, which represented 15% of the cells evaluated from this tumor.

Guidelines for the preparation and analysis of the fragile X chromosome in lymphocytes.

The following guidelines were adopted by an Ad Hoc Committee convened at the Fourth International Workshop on the Fragile X Syndrome and X-Linked Mental Retardation to establish minimum cytogenetic standards for the preparation and analysis of the fragile X chromosome. The intention of the committee was to develop and provide practical standards for the routine cytogenetic detection of the fragile X. The guidelines describe reasonable criteria for effective tissue culture methods for eliciting the Xq27.3 fragile site in vitro and for the analysis of such chromosome preparations.

FMC-HU-1-B: a lymphoma B-cell line with unusual characteristics.

A cell line (FMC-Hu-1-B) was established from a biopsy of an abdominal mass of a child with non-Burkitt's lymphoma. The establishment of the cell line initially required the presence of normal bone marrow stromal cells and phytohaemagglutinin stimulated leucocyte conditioned medium. The cell line lacked Epstein-Barr virus nuclear antigen and exhibited numerous chromosomal abnormalities. Cell-surface marker analysis using a panel of monoclonal antibodies revealed only markers of the B lineage. Within the B-cell lineage FMC-Hu-1-B seemed to occupy a level of maturation equivalent to normal mature B-cells (surface membrane IgM, secretion of immunoglobulin and FMC-1 positive). However, the cells also weakly expressed the common acute lymphoblastic leukaemia antigen, normally found on early precursors of B-cells. The cells appear to secrete auto-stimulatory growth factor(s).

Heritable fragile sites on human chromosomes. XI. Factors affecting expression of fragile sites at 10q25, 16q22, and 17p12.

The fragile sites at 10q25, 16q22, and 17p12 can all be induced in lymphocyte culture by BrdU or BrdC added 6-12 hrs prior to harvest. Without induction, fra(10)(q25) is rarely expressed spontaneously, whereas fra(16)(q22) is frequently expressed spontaneously. Fra(17)(p12) is frequently expressed spontaneously but is probably expressed only after induction in some individuals. Distamycin A, netropsin, and Hoechst 33258 induced high levels of expression of fra(16)(q22) and fra(17)(p12) but did not enhance expression of fra(10)(q25). The mechanisms of induction of fra(16)(q22) by BrdU and distamycin A appear to be different, since the time of induction by BrdU reaches a maximum about 12 hrs prior to harvest whereas induction by distamycin A requires much longer exposure. The fragile sites at 10q25 and 16q22 were both induced in fibroblast culture by BrdU. Fra(17)(p12) is accepted as a fragile site because preliminary studies show that it behaves similarly in lymphocyte culture to fra(16)(q22); however, there is only limited evidence for fragility at 17p12.

Thymidylate synthetase inhibitors and fragile site expression in lymphocytes.

The ability of three thymidylate synthetase inhibitors, fluorodeoxyuridine, fluorodeoxycytidine, and trifluorothymidine, to induce the expression of eight different folate-sensitive fragile sites has been investigated in 22 patients and compared with the efficacy of simple folate deprivation for inducing fragile site expression. Fluorodeoxyuridine and fluorodeoxycytidine were equal in their ability to elicit fragile site expression but fluorodeoxycytidine proved less cytotoxic under comparable culture conditions. Both fluorodeoxyuridine and fluorodeoxycytidine were found to be more efficient than trifluorothymidine at comparable concentrations but less efficient than simple folate deprivation in eliciting fragile site expression in lymphocytes. Since the three inhibitors induced expression of eight different folate-sensitive fragile sites, it is likely that all folate-sensitive fragile sites have a common underlying mechanism of expression. The practical application of thymidylate synthetase inhibitors in the routine detection of heritable fragile sites is discussed.

Heritable fragile sites on human chromosomes. X. New folate-sensitive fragile sites: 6p23, 9p21, 9q32, and 11q23.

Four new folate-sensitive fragile sites are documented at 6p23, 9p21, 9q32, and 11q23. These have all been shown to be heritable except for the one at 9p21, which has been seen only in a single individual. As with the other autosomal fragile sites, these appear to be innocuous in heterozygotes.

Fragile sites in chromosomes: possible model for the study of spontaneous chromosome breakage.

The tissue culture condition that is required for the type of chromosome breakage seen at most fragile sites, namely, the absence of folic acid and thymidine in the medium, greatly enhanced micronucleus formation in proliferating lymphocyte cultures from normal individuals. This suggests that chromosome breakage at fragile sites and the apparently spontaneous damage that gives rise to micronuclei are controlled by the same mechanism.

Fragile X chromosome and chromosome condensation.

The effect of chromosome condensation on the frequency of expression of the fragile X chromosome was examined. Chromosome decondensation substances were tested for their ability to elicit expression or improve frequencies of expression of the fragile X chromosome in five patients. The substances tested included the AT specific DNA ligands ethidium bromide, Hoechst 33258, and netropsin, and the GC specific substances actinomycin D and olivomycin. Under culture conditions appropriate for eliciting fragile X expression none of the decondensation compounds studied significantly altered frequencies of expression, nor did any of the substances elicit fragile X expression under conditions that normally suppress fragile X expression. The fragile X was found to be more frequently evident in less condensed chromosome preparations from fibroblasts. The implications of these findings with respect to the nature of fragile sites are discussed.

Expression in fibroblast culture of the satellited-X chromosome associated with familial sex-linked mental retardation.

The satellited-X chromosome previously shown in lymphocyte culture to be associated with certain types of sex-linked mental retardation has, for the first time, been demonstrated in cultured skin fibroblasts and lymphocytes from two affected males and an obligate carrier female. These findings provide a basis for reliable diagnosis of female carriers and for the development of prenatal diagnosis.