FRAGILE-X INDUCTION SYSTEMS IN CVS CULTURES - EFFECT ON CYTOGENETIC, PCR, AND GENOMIC SOUTHERN BLOT DNA ANALYSES OF THE FMR-1 GENE

Low fragile X frequencies have been commonly observed in chorionic vil lus sample (CVS) cultures, compared to subsequent analysis in whole bl ood or products of conception (POC). To investigate possible mechanism s 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 l ast 24 hours before harvesting, to FUdR, excess thymidine, and a combi nation of both. For CVS, only those cultures that were exposed to a co mbination of FUdR and excess thymidine showed positive cytogenetic fin dings (1/90 or 1.1%), agreeing with our original positive cytogenetic results (2/86 or 2.3%) for cultures exposed to excess thymidine. Fragi le X frequencies in the POC tissues from this fetus increased to an av erage of 14%. PCR analyses showed full mutations (>200 CGG repeats) in uninduced CVS cultures but induced cultures exhibited apparently smal ler sizes in the range of 120-180 repeats. The results showed variabil ity. In one instance, the banding pattern from one of the uninduced cu ltures was similar to the results where cultures were exposed to a dou ble induction system. When PCR analyses were conducted on induced POC cultures, full mutations were observed in virtually all samples. South ern blot genomic analysis using probe StB12.3 showed an unmethylated f ull mutation in CVS cultures. Southern blot patterns from cultures of muscle revealed size variations of DNA bands in the premu-tation range representing unmethylated DNA as well as methylated full mutations. F inally, variations were also observed in lung and skin cultures, compa red to CVS and muscle. The multiple banding patterns that occurred as a function of methylation appeared to be histiotypic. These results su ggest that fragile X induction in CVS cultures causes a decrease in ap parent CGG) repeat size, an effect not seen or less apparent in most c ultured POC tissues. Alternatively, apparent histiotypic variations in banding patterns observed in PCR and StB12.3 genomic Southern blot an alyses may reflect differences in tissue type mosaicism, methylation, and/or stage of development. Our findings may help explain the relativ ely low cytogenetic frequencies commonly seen in CVS cultures. (C) 199 4 Wiley-Liss, Inc.