X nondisjunction & inactivation patterns in childhood ALL

X chromosome inactivation is a gene dosage compensation mechanism. De novo methylation of one X during early female embryonic development shuts down the expression of a large number of genes. Thus, in the descendant somatic cells only either one of the two X homologues remains active. Primary or acquired deviations of a random inactivation pattern can occur with increasing age, but may also indicate pre-existent X-encoded heterozygous genetic defects or the clonal expansions of genetically altered benign or neoplastic cell clones. In childhood acute lymphoblastic leukemia (ALL), acquired X chromosome duplications are the most abundant numerical abnormality. We therefore want to investigate the X nondisjunction and inactivation mechanisms in such male and female ALL cases. Our interest derives from the fact that in females either the active or inactive X may be duplicated in a random or nonrandom fashion, whereas in male patients the only active homologue can be duplicated. Such duplication should therefore concur with an equivalent dosage increase of the respective active genes. This, in turn, may influence and deregulate the expression of many other genes, even on the autosomes, and by that contribute to the neoplastic transformation process. Alternatively, the inactive X in females may be duplicated in a nonrandom fashion or a duplicated active X, either in a female or male patient, may become de novo inactivated. The presence and extent of X inactivation skewing is best assessed by the simultaneous quantification of allele-specific methylation patterns of polymorphic gene regions. We will apply methylation- sensitive PCR (MS-PCR) to analyze polymorphisms in the HUMARA and FMR1 genes and to quantify the allele- specific methylation profile of the XIST and SYBL1 genes. XIST is responsible for X-inactivation and methylated on the active, but nonmethylated on the inactive X. SYBL1 is a unique gene that maps to the pseudoautosomal region and is nonmethylated on the active X, but methylated on the inactive X and Y chromosome. Irrespective of the outcome of our investigations, the results will be of eminent interest, because they will provide important clues not only about the role of X-encoded genes in the pathogenesis of the respective leukemias, but also about the chromosomal selection and inactivation process itself.

The aim of our project was to investigate the mechanism that leads to of the gain of one or more extra X chromosomes in hyperdiploid cases of childhood acute lymphoblastic leukemia (ALL) and its potential biological consequences. This acquired abnormality is not only the most common numerical aberration in childhood ALL, but also in non-Hodgkin lymphoma (NHL) and occurs in both male and female patients. Our particular interest derives from the fact that females have two and male only one X chromosome in normal cells. To compensate for an otherwise double gene dosage in females, one of the two X chromosomes becomes inactivated in a random manner in each cell during early fetal development. Thus, in female leukemia patients either an active or inactive X might be acquired in a random or non-random fashion, whereas in male patients only the sole active homologue can be duplicated. However, a supernumerary active X could subsequently also become de novo inactivated in either sex. A random involvement of either the active or inactive X might provided an explanation for the difference in the treatment response and outcome (boys fare worse). As planned, we started to investigate this issue with methylation-specific PCR (MS PCR), which enables a semi-quantitative estimation of the distribution of active and inactive X chromosomes through analysis of associated polymorphic differentially methylated sequences. We spend a lot of time to test and evaluate different published variations of MS-PCR with the aim to improve the sensitivity of the quantification process. Having had analyzed already a considerable number of cases with this technique, we realized the limitations of MS PCR for this particular purpose, particularly in cases with low blast cells numbers. To overcome these problems, we developed a novel dual-color DNA/RNA Fluorescence in situ hybridisation (FISH) assay that finally enabled us to simultaneously enumerate the active and inactive X chromosomes on a single cell level. Following the successful evaluation of the assay on control samples from healthy individuals and cases with various constitutional X chromosome aneuploidies, we analyzed as planned 54 hyperdiploid cases of childhood ALL, but also 29 cases of NHL. The results of our successful investigations revealed that the acquisition of one or more X chromosomes occurs in a non-random manner that only depends on the number of affected chromosomes, but not on the sex of the patient. This finding does therefore not explain the sex-related differences in treatment outcome. However, it corroborates previously established evidence that the maldistribution of chromosomes occurs in a single step rather than consecutively during several cell divisions. Therefore, our findings provide important clues about the still rather enigmatic biological cause and consequences of simple numerical chromosome changes that lead to the development of these hematologic diseases.