IV. RESULTADOS Y DISCUSIÓN
4.1. Resultados
4.1.1. Esquematización de los conflictos sociales, hechos mágicos y la
The preliminary dual-FISH experiments were repeated on lymphoblastoid cell lines from affected members of family HUD and on normal controls. Any measurements of signal separation deviating from that predicted from actual genomic distance would be indicative o f rearrangement. Furthermore, if any of the YAC hybridisation signals appeared to be split this would indicate the existence of a rearrangement breakpoint within the YAC.
Pilot experiments on normal nuclei revealed that YAC probes sometimes gave two signals per chromosome rather than the expected single signal. This phenomenon was due to post-replicative cells in G2 o f the cell cycle, with one signal localising to
each chromatid. To eliminate these confusing results patient and normal lymphoblastoid cell lines were starved for fourteen days causing arrest in Gq/Gi phase of the cell cycle and therefore preventing DNA replication from taking place. Pairs of YACs were selected such that a variety o f genomic separations could be assessed across the APC-MCC region. The reporter molecules with which YACs were labelled
and the combinations o f YAC investigated were as indicated in figure 3.26. These YAC combinations were considered most informative for analysis o f the rearrangement in family HUD.
The influence o f sample preparation, hypotonic swelling o f cells, spreading o f cells onto a slide, and the effects of aspects o f the FISH procedure such as dénaturation, on the nature o f chromatin packaging and organisation are unknown. For this reason normal cells were prepared and subjected to FISH in tandem with patient material. These normal cells served as a reference, indicating the results that should be obtained in the absence of a rearrangement, and circumvented the risk that small differences in experimental conditions might affect results. Indeed comparison o f experimental controls and results from the earlier pilot experiment (also conducted on normal material) do display some differences despite using identical methodology. Importantly the linear relationship between signal separation and genomic distance was maintained in both experiments.
Results from the dual-FISH experiment conducted on patient material, alongside results from the relevant normal controls, are presented in table 3.5. Because the patient is heterozygous for the rearrangement the average separations given include interphase measurements taken on both mutant and normal chromosomes. There was no way o f distinguishing between the two chromosomes and thus the inclusion o f the normal chromosome in the average was unavoidable. Average measurements from three out of four YAC combinations were comparable between normal controls and patient nuclei. Only the combination of YACs 19AA9 and 37HG4 on patient nuclei gave measurements significantly different from normal controls. The most obvious difference was seen in the number o f signals scored as overlapping: 40% in patient nuclei compared to only 18.5% in normal nuclei, suggesting that 19AA9 and 37HG4 are situated closer together in this patient. Indeed nuclei in which both pairs of signals were clearly separated were never observed in patient nuclei despite being common in normal controls. The mean separation o f signals in patient nuclei was 0.44 microns compared to 0.58 microns in the normal control. Statistical analysis (standard /-test) revealed this to be a significant difference (P<0.01). This is particularly striking given that 50% of measurements made on patient nuclei were conducted on a normal chromosome. The data suggests that the two YACs had been moved closer by deletion o f the intervening region, or by an
inversion bringing one clone nearer to the other. This latter hypothesis is in accord with the PFGE data.
An inversion shifting the MCC gene (19AA9) closer to APC (37HG4) is also supported by the remaining dual-FISH results which indicate that APC is not moved relative to YAC 9HA11. Thus, it is MCC that is involved in the inversion and not APC. The telomeric break point of the inversion appears to lie in between MCC and YAC 9HA11. That 9HA11 is not involved is indicated by the fact that measurements involving this YAC did not differ significantly between patient and control samples.
Figure 3.30. Double FISH performed on patient nuclei.
a)
b)
c)
a) and b) Combination of YACs 19AA9 (red) and 37HG4 (green) hybridised to a patient lymphoblastoid cell nucleus. Only one pair of signals is well separated.
c) Combination of YACs 19AA9 (red) and 37HG4 (green) hybridised to a normal lymphoblastoid cell nucleus. Both pairs of signals are well separated.
region on natient nuclei and norm al control nuclei.
YAC Physical Total Unresolved Mean Adjusted mean
combination separation of number o f signals (%) separation separation
YACs (kb) signals (microns) (microns)
Control Nuclei 9HA11 + 24ED6 180kb 56 13% 0.45 0.53 19AA9 + 37HG4 300kb 42 17% 0.58 0.66 9HA11 + 37HG4 530kb 50 12% 0.66 0.75 9HA11 + 39GG3 630kb 58 3% 0.69 0.71 Patient Nuclei 9HA11 + 24ED6 180kb 40 25% 0.44 0.59 19AA9 + 37HG4 300kb 48 40% 0.44 0.73 9HA11 + 37HG4 530kb 44 9% 0.76 0.84 9HA11 + 39GG3 630kb 66 6% 0.78 0.84
1- Estimated physical separation of various combinations o f YACs (Kinzler gr uZ., 1991a).
2- Unresolved signals are scored as those in which red and green signals were no longer distinct due to extensive overlap.
3- Mean separation refers to the average distance between signals measured after image capture. Overlapping signals are scored as zero microns separation. 4- The adjusted mean separation is the average measured distance between
Figure 3.31. Hypothetical inversion of a chromosomal fragment encompassing the MCC gene. MCC APC 19AA9 37HG4 MCC APC 19AA9 37HG4
Digoxigenin labelled Biotin labelled 0 1 100 I 200 300I I
Kilo bases
Explanation proposed to explain the fluorescence in situ hybridisation and pulsed field gel electrophoresis data obtained from experiments on family HUD. MCC and APC are moved closer to one another by the inversion o f a fragment containing the MCC gene.