The Detection of numerical chromosome abnormalities by FISH for prenatal diagnosis

100 SUMMARY Chromosomal mosaicism is the presence of two or more karyotypically different cell lines in an individual. It may take the form of generalised mosaicism or may be confined either to the placenta or the embryo. Confined placental mosaicism (CPM) may be associated with intrauterine growth retardation (IUGR), fetal loss or poor perinatal outcome. The adverse influence of CPM depends on its level and the chromosomes which are involved. In the first part of this study, the relation of IUGR and placental mosaicism was studied using conventional cytogenetic analysis through tissue culture techniques and interphase- FISH technique. The results obtained with these techniques were compared to evaluate the usefulness of interphase-FISH to detect chromosomal aneuploidies. In the second part, trisomies 18 and 21 were detected by dual-colour interphase-FISH on uncultured chorionic villus samples (CVS). The aim of this part of the study was to show that trisomies 18 and 21 can be rapidly detected by dual-colour FISH using uncultured CVS for prenatal diagnosis. In the third part of this project, the presence of some commonly reported chromosome trisomies such as 7, 16, 18 and 21 were studied in IUGR placental tissues, using dual-colour interphase-FISH. Six placental samples from cases having undergone pre-natal diagnosis (3 of these cases having been previously identified as having chromosome abnormalities), and 23 placental samples from pregnancies complicated by unexplained IUGR (total 29 samples) were used in the first part of the study. Conventional cytogenetic analysis obtained by tissue culture using amnion, chorion and six sites of the placental tissue showed that seventeen cases were chromosomaUy normal, while the remaining 12 cases had some numerical abnormalities. Eight of twenty-three IUGR cases showed different mosaicism in the first101 part of study (this excludes the one case of proven MCC). This means that the rate of placental mosaicism was 34.8% (8/23) in this group (although if the two cases of tetraploidy are excluded this would give a rate of 26% (6/23). Out of these twelve samples, three cases of trisomy 21 were detected, two of which were complete trisomy 21 with 47,XY,+21 karyotypes and the other was mosaic 47,XX,+21/46,XX. Two cases were found to be mosaic trisomy 16. One case showed trisomy 15. Mosaic trisomy 5 was also found in one sample. Another sample showed a mosaicism of trisomy 20. Monosomy for a sex chromosome was found in one sample with a 45,X/46,XX mosaic karyotype. Three of the 12 samples were polyploidies. One of the polyploidies was a pure triploidy (69,XXX), two cases showed low level mosaicism with tetraploidy, having 92,XXXX/46,XX (19% of tetraploid and 81% of diploid cell line) and 92,XXYY/46,XY (21% of tetraploid and 79% of diploid cell line) karyotypes. The level of mosaicism (obtained by conventional cytogenetic analysis) between different regions of the placenta, amnion and chorion in each sample was analysed using the %2 test and some statistically significant differences were found, but no consistent pattern was evident. On identification of a mosaicism using conventional cytogenetics, the same regions of placenta, amnion and chorion were used to detect abnormalities by interphase- FISH using the relevant probes. Interphase-FISH and conventional cytogenetic results were compared by using the %2 test in order to show the usefulness of one as opposed to the other. The results showed that there was no significant difference between conventional cytogenetic results and interphase-FISH results, therefore, interphase-FISH technique can be used for the detection of numerical chromosome abnormalities instead of conventional cytogenetic analysis when rapid results are required, and without the necessity for conventional culturing.102 94 chorionic villus samples (CVS) were used to detect trisomy 18 and trisomy 21 by dual-colour interphase-FISH using the probes LI. 84 (specific for chromosome 18) and YAC 831B9 (specific for chromosome 21) in the second part of the study. One case showed a trisomy 18 and another three cases showed trisomy 21. The FISH results were later compared with conventional cytogenetic results obtained by the tissue culture techniques, and no discrepancies were found In the third part of the study, 34 placental samples with IUGR were used to detect the most common autosomal trisomies 7, 16, 18 and 21. Six different regions from each placental sample were used to prepare the interphase-FISH slides following the development of the touch preparation protocol. Dual-colour interphase-FISH experiments were accomplished using the probes digoxigenin-labelled D7Z1 with biotin- labelled D16Z2 and digoxigenin-labelled LI. 84 with biotin-labelled YAC 831B9. A total of 600 interphase cells from each sample (100 cells from each region) were analysed. Six of these 34 samples showed chromosome abnormalities, one complete trisomy 21, 3 trisomy 18 mosaics and 2 trisomy 21 mosaics. No chromosomal aneuploidy for chromosomes 7 and 16 was found. Placental mosaicism was found in 5/34 (14.7%) of the IUGR samples for detection of trisomoies 7, 16 18 and 21 in the third part of this study.