A form of familial sex-linked mental retardation has been associated with the expression of a fragile site near the terminal end of the long arm of the X chromosome. Previous reports on the fragile X chromosome showed expression of the fragile site to be limited to chromosome preparations from peripheral blood lymphocytes of mentally retarded males and their female relatives in families in which the disorder was segregating. Fragile site expression has also been shown to be a function of the medium employed in cell culture. The fragile X chromosome could only be demonstrated in lymphocytes cultured in medium 199 or media deprived of folic acid. This study was undertaken to develop a method for demonstrating the fragile X chromosome in cultured skin fibroblasts. Fibroblast cell lines from five patients (two mentally retarded males, two obligate carrier females, and a potential carrier female) from a family in which familial sex-linked mental retardation was known to be segregating were established and routinely maintained in a complete culture medium. Forty-three hours prior to chromosome harvest, cells from each patient were transferred to media deficient in folic acid. Under conditions of folic acid deprivation, it was possible to elicit expression of the fragile X chromosome in skin fibroblasts from all five patients studied. No fragile X chromosomes were detected in fibroblasts from three normal control subjects. In a preliminary assessment of the reliability of the fibroblast method, three patients (two mentally retarded males and a potential carrier female) from a second unrelated family in which the disorder is known to be segregating were studied with this method. The fragile X chromosome could be demonstrated in fibroblasts from both of the retarded male patients but could not be. demonstrated in fibroblast chromosome preparations from the potential carrier female. Lymphocytes for all patients studied were grown under similar folate deprived conditions for the purpose of comparing the effectiveness of fibroblast culture with lymphocyte culture in demonstrating the expression of the fragile X chromosome. Neither tissue was shown to consistently provide a higher frequency of expression of the fragile X chromosome. In addition to folate deprivation, it was shown that two other features of the fibroblast method influenced the frequency of expression of the fragile X chromosome. The fragile site was expressed at a significantly higher frequency in chromosome preparations in which the chromosomes were not severely contracted. The frequency of expression in fibroblasts was also shown to be significantly higher with a hypotonic treatment at chromosome harvest using 1% NaCitrate rather than 0.075M KC1. Because fragile site expression was shown to be a function of the degree of chromosome condensation, two agents, 5-BrdU and actinomycin-D, were studied to examine their decondensation effects on the frequency of expression. Neither BrdU nor actinomycin D proved effective in accentuating the frequency of expression. Since fibroblasts behave much like amniocytes in terms of cell culture and chromosome harvest, the development of a method for demonstrating the fragile X chromosome in cultured skin fibroblasts is a step toward the prospect of reliable antenatal diagnosis of familial sex-linked mental retardation associated with a fragile X chromosome. / Medicine, Faculty of / Medical Genetics, Department of / Graduate