Fragile X induction systems in CVS cultures: Effect on cytogenetic, PCR, and genomic southern blot DNA analyses of the FMR‐1 gene

AbstractLow fragile X frequencies have been commonly observed in chorionic villus sample (CVS) cultures, compared to subsequent analysis in whole blood or products of conception (POC). To investigate possible mechanisms for this effect, CVS cultures from a previously identified fragile X positive male, were restudied and compared to subsequent POC cultures from lung, muscle, skin, and thymus. Cultures were exposed, for the last 24 hours before harvesting, to FUdR, excess thymidine, and a combination of both. For CVS, only those cultures that were exposed to a combination of FUdR and excess thymidine showed positive cytogenetic findings (1/90 or 1.1%), agreeing with our original positive cytogenetic results (2/86 or 2.3%) for cultures exposed to excess thymidine. Fragile X frequencies in the POC tissues from this fetus increased to an average of 14%. PCR analyses showed full mutations (>200 CGG repeats) in uninduced CVS cultures but induced cultures exhibited apparently smaller sizes in the range of 120–180 repeats. The results showed variability. In one instance, the banding pattern from one of the uninduced cultures was similar to the results where cultures were exposed to a double induction system. When PCR analyses were conducted on induced POC cultures, full mutations were observed in virtually all samples. Southern blot genomic analysis using probe StB12.3 showed an unmethylated full mutation in CVS cultures. Southern blot patterns from cultures of muscle revealed size variations of DNA bands in the premutation range representing unmethylated DNA as well as methylated full mutations. Finally, variations were also observed in lung and skin cultures, compared to CVS and muscle. The multiple banding patterns that occurred as a function of methylation appeared to be histiotypic.These results suggest that fragile X induction in CVS cultures causes a decrease in apparent CGG repeat size, an effect not seen or less apparent in most cultured POC tissues. Alternatively, apparent histiotypic variations in banding patterns observed in PCR and StB12.3 genomic Southern blot analyses may reflect differences in tissue type mosaicism, methylation, and/or stage of development. Our findings may help explain the relatively low cytogenetic frequencies commonly seen in CVS cultures. © 1994 Wiley‐Liss, Inc.