The fragile X(q27) form of X‐linked mental retardation: FUdR as an inducing agent for fra(X)(q27) expression in lymphocytes, fibroblasts, and amniocytes

Abstract

The effect of FUdR on the expression of fra(X)(q27) was examined in lymphocytes and/or fibroblasts from 16 affected males and 5 carriers from 10 families; six different culture media were used: F10, 5% serum, pH 7.3(37 degrees C); medium 199, 5% serum, pH 7.6(37 degrees C); folate-free 199, 5% serum, pH 7.6(37 degrees C), and these three media with FUdR (0.05 micron). In lymphocytes there was no significant difference in the percentage of fra(X) expressing cells between any of the FUdR-containing media. The highest percentage of expressing cells seen in lymphocytes with FUdR was 56%. The average enhancement in males with FUdR in the 199 and folate-free 199 media was 30%. This relative enhancement with FUdR was very much higher in a few blood specimens delayed in transit and FUdR may prevent some of the false-negative results obtained from mailed specimens. FUdR did not induce the marker in four obligate carriers with previously negative results. The fibroblasts from affected males were grown in the six specific media for the last 48 hr. Two of the six media yielded reproducibly positive results. These were 199-FUdR and folate-free 199-FUdR with mean percentages of expressing cells of 12.8 +/- 7.1% and 11.3 +/- 6.1%, respectively. F10-FUdR, which contains thymidine, did not permit expression of the marker in fibroblasts and there was no difference in the percentage of fra(X) expression in 199-FUdR media with or without folate. It was concluded that FUdR shows promise as an agent to permit prenatal diagnosis of the condition and to enhance the detection of the marker in lymphocyte cultures.

Fragile X syndrome in Calcutta, India

“…Cytogenetic analysis About 5 mL of venous blood was drawn from patients and 0´5 mL of leucocytes was inoculated in folate-de®cient medium 199 to induce the fragile sites, following the method of Brookwell et al 12 Metaphase chromosomes were prepared and processed for solid staining using the protocol described by Rooney and Czepulkowski. 13 About 100 metaphases were scored per patient and those with fragile sites were photographed.…”

Improved fragile site detection with trimethoprim

“…The fragile site frequency (33%) found in fragile X-affected males was comparable with that (30%) reported by Lejeune (1982), and we found a remarkably similar frequency for the fragile sites on chromosome 8 (31%), chromosome 10 (32%), and chromosome 16 (32%). Similar frequencies, however, were obtained by Brookwell et al (1982) for affected fragile X males with FUDR added to medium 199. This contrasts with a lower value of 21% in our FUDR cultures, but this may reflect our lower baseline in medium 199 (13%) compared with the 25% reported by these workers, possibly a result of differences in culture variables.…”

“…The presence of folic acid or thymidine in the medium significantly depresses the expression of fragile sites (Sutherland 1977). Since then, folic acid inhibitors such as methotrexate and fluorodeoxyuridine (FUDR) in combination with various culture media have been widely used to enhance fragile site expression (Sutherland 1979;Fonatsch 1981;Glover 1981;Mattei et al 1981;Brookwell et al 1982). Excess thymidine or specially prepared media that are folic acid free or folatereduced have also been used (Sutherland 1979;Brookwell et al 1982;Sutherland et al 1985).…”

Differential expression of fragile site Xq27 in cultured fibroblasts from hemizygotes and heterozygotes and its implications for prenatal diagnosis

“…In fact, only a few laboratories have reported consistent success in demonstrating fra(XI(q27) in cultured fibroblasts (Jacky and Dill, 1980;Fonatsch, 1981;Mattei et al, 1981a, b;Tommerup et al, 1981;Glover, 1981;Brookwell et al, 1982;Gardner et al, 1982;Schmidt et al, 1982;Bryant, 1983;Barbie et al, 1983;Schmidt, 1985).…”