Improved prenatal detection of fra(X)(q27.3): methods for prevention of false negatives in chorionic villus and amniotic fluid cell cultures.

The reliable detection of fra(X)(q27.3) in prenatal samples is important for providing genetic counseling. We have identified 5 new cases of prenatal fragile X [fra(X)] detection in 3 chorionic villus sample (CVS) and 2 amniotic fluid (AF) cell cultures. In 4 of the 5 cases, either excess thymidine (THY) or a combination of THY and 5-fluorodeoxyuridine (FUdR) was clearly superior to FUdR alone as fra(X) inducers. Amniocytes from one case were cultured only in RPMI-1640 and later exposed to FUdR or THY separately. They showed only 2% fra(X) while parallel cultures initiated in Chang medium and incubated in RPMI for at least 7 days (recovery) before fra(X) induction exhibited strikingly increased fra(X) frequencies. Chang medium alone will not allow fra(X) induction in AF (Jenkins EC, Brown WT [1986]: "Genetic Disorders and the Fetus: Diagnosis, Prevention and Treatment." New York: Plenum Press, pp 185-204). Now, using CVS cells, we report that only 1% and 0% fra(X) were detected using FUdR or THY in cells cultured in RPMI for 4 days after removal from Chang medium. Cells with 7 days "recovery" in RPMI exhibited increases from 2 to 6%. Therefore, we have found that Chang medium is very helpful when the appropriate recovery time in another medium is allowed before fra(X) induction. Some false negative reports can be attributed to: induction in Chang medium alone; lack of sufficient recovery time after initiating cells in Chang before induction; and unavailability of the excess THY fra(X) induction system.(ABSTRACT TRUNCATED AT 250 WORDS)

Progress toward an internal control system for fragile-X induction by 5-fluorodeoxyuridine in whole-blood cultures.

We have been attempting to develop a consistently reliable internal control to assure the effectiveness of the 5-fluorodeoxyuridine (FUdR) fragile-X [fra(X)] induction system. We carried out a systematic study of whole-blood specimens cultured from 56 individuals from two different laboratories. An analysis of nearly 9,000 cells demonstrated: (1) the importance of establishing baseline levels of fragile sites in each laboratory, and (2) that a combination of common fragile sites (different for each laboratory) could serve as a consistently reliable indicator of the effectiveness of the FUdR fra(X) induction system. It was suggested that a non-FUdR culture(s) should be incorporated into a laboratory's fra(X)-screening protocol, so that if there are any doubts about the effectiveness of the FUdR system a comparison to background or spontaneously occurring fragile sites can be made within the laboratory. Repeat cultures are recommended where no increase in common fragile-site frequency is observed in the FUdR induction system, and where fra(X) was strongly suspected but not found. In addition, the necessity of using more than one fra(X) induction system in whole-blood cultures was demonstrated, including the effectiveness of an FUdR/excess thymidine double-induction system. Finally, 2 cases of apparent mosaicism for Klinefelter syndrome in fra(X) individuals were observed.