Consistent deletion of a specific chromosome region in a particular tumour type is generally indicative of the presence of a tumour suppressor gene, the loss of function of which is integral to the development or progression of malignancy (Soloman et al, 1991; Weinberg, 1991; Nowell, 1997). In cancers, tumour suppressor genes have been located at sites that show loss of heterozygosity. These are recessive genes where both copies must be inactivated for malignant transformation (Soloman et al, 1991; Weinberg, 1991; Nowell, 1997). In the DS cases investigated in the present study it is unlikely that the interstitial deletions/inversions expose a tumour supressor gene. In these cases only one copy of chromosome 21 is affected whereas all three copies of a tumour suppressor gene must be mutated/inactivated to drive the transformation process. It is possible that the deletions/inversions result in a dominant negative mutation, where the single mutated allele negatively affects the
(Nowell, 1997; Song et al, 1999; Downing et al, 2000; Imai et al, 2000; Preudhomme et al, 2000; Michaud et al, 2002).
4.3.10.1 Disomie homozygosity
In the DS acute leukaemia and DS/non-DS TAM cases there is another mechanism that may be involved in the development and progression of the disorders, namely disomic homozygosity. In the trisomie cells there is an extra paternal or maternal copy of chromosome 21. Where an alteration has occurred on this extra copy of the chromosome, allelic balance would be restored resulting in a restoration of the disomic situation for the region affected. Loss of genetic material from the unique parental chromosome will result in uniparental disomy and a reduction to homozygosity for that particular chromosome region (Abe et al, 1989; Rogan et al, 1995; Lengauer et al, 1998). Loss of allelic variation can unmask a recessive mutant allele which is unopposed by a normal copy of the gene. Abe et al (1989) described disomic homozygosity on 21q in DS TAM cases. Fluorescent banding methods were used to analyse heteromorphisms, inherited variability of sequences in the gene poor regions of chromosomes, to determine the parental origin of chromosome 21 in each case. Parental alleles were designated a, b and c. The trisomie cells of all TAM cases had an ‘aab’ pattern, and two of the three copies of chromosome 21 showed an identical heteromorphism. This study and others revealed disomic homozygosty on 2 1 q ll, leading to speculation that a gene localised to this region was important in the pathology of TAM (Abe et al, 1989; Niikawa et al, 1991; Shen, 1995; Cavani et al, 1998).
4.3.10.2 Inversion of probe order
A small number of metaphase cells in seven cases showed inversions of probe loci (five DS TAM and case 14). This may lead to the inappropriate activation of genes through gene fusion and activation of an oncogene, or may result in the production of a chimaeric protein, similar to the effects caused by translocations. The inversion of probe order may not in fact represent the relocation of sequences after double strand breakage has occurred but may be an artefact of chromatin folding on an already highly condensed chromosome. Early investigations into mapping by FISH indicated that probes must be several megabases apart in order to be mapped with accuracy on metaphase chromosomes (Lawrence et al, 1990; Trask et al, 1991). The commercial
probes analysed in this study were situated at a distance of 3-4Mb. In case 14 there can be no question that an actual inversion has taken place as the regions involved were at least 20Mb apart.
The effects of submicroscopic alterations of genes on chromosome 21 may not be as dramatic as those described above. It is possible that these subtle changes are secondary events occurring during the progression of the disorder. As described previously, DS cases are thought to be susceptible to DNA damage caused by the constitutional trisomy for chromosome 21. This predisposition coupled with further damage perhaps caused by for example rapid proliferation, the build up of toxins or the failure to apoptose during the development/progression of the disorder, may result in DNA breaks that are repaired defectively leading to loss of large tracts of sequences. These deletions may not be random but may be found at specific sites implicated in recombination events such as topoisomerase H, DNA cleavage sites, DNAse I hypersensitive sites or repeat sequences. (Elliot and Jasin, 2002; Zhang et al, 2002). These are sites that have recently been identified at translocation breakpoints in the RUNXl gene in leukaemia patients (Zhang et al, 2002).
4.3.10.3 Amplification of loci
Case 5 was a phenotypically normal infant diagnosed with pre-B ALL. A karyotypic abnormality was detected in the form of a duplication of part of the long arm of one copy of chromosome 21. This case differs from the others investigated here as this was the marker chromosome that allowed the leukaemic cells to be identified. This case was included as a prototype for the examination of abnormalities of chromosome 21. FISH analysis revealed that there was amplification of signals through tandem duplication (four to six times) of the D2IS65-D21S19 region. The amplified region did not extend to the paracentromeric loci and did not include the subtelomeric region. Although not investigated due to lack of material, it is likely that the RUNXl region (situated distal to D21S65 and proximal to D21S55) was also amplified between four to six times. Gene amplification frequently occurs in solid tumours and can be associated with activation of an oncogene, but gene amplification is uncommon in haematological malignancies. Amplification of RUNXl has been described in a number of childhood ALL cases but has not been detected in adult ALL (Niini et al,
2002). Although it is still unknown what role the amplification of RUNXl may have on leukaemogeneisis, mutation of the sequence has so far not been detected in these cases. It is possible that enhanced expression of RUNXl could be an important factor contributing to the pathogenesis and progression of childhood B-lineage ALL. Should this be the case then this may have relevance in the DS AL/TAM cases where there is either congenital or acquired, trisomy for chromosome 21. The altered expression of RUNXl and possibly other chromosome 21 genes may result in a predisposition to developing a pre-leukaemic disorder/leukaemia, but further studies are required to determine whether chromosome 21 genes show enhanced expression and whether there is any effect on protein expression.