Understanding the nature and timing of leukaemogenic events during the development of childhood acute lymphoblastic leukaemia (ALL) will enable intervention that could prevent ALL in the future. We hypothesised that 9p21 deletion in childhood ALL may unmask predisposing genetic events that would allow us to determine the "nature" of initiating events in childhood ALL; whereas the inclusion, or exclusion, of random �N� nucleotides in normal immunoglobulin gene rearrangements from the developing fetus and the expression of terminal deoxynucleotidyl transferase (TdT) in fetal lymphocytes may allow us to unmask the developmental window during which the first transforming leukaemic event occurs in a pre-leukaemic B cell.
The most frequent genetic abnormality in childhood ALL is deletion of chromosome 9p21, with the minimal region of deletion including the CDKN2-locus, making genes at this locus candidates for a predisposing genetic event in ALL. To determine whether genomic imprinting might be involved in ALL at the 9p21 locus we investigated the imprinting status of the candidate genes CDKN2A, CDKN2B and ARF. No evidence for genomic imprinting of ARF was found in this study. Because no expressed polymorphisms could be identified for CDKN2B, and CDKN2A expression was too low in normal tissues, the imprinting status of these genes could not be evaluated. Furthermore investigations in our laboratory have been unable to find genomic imprinting at any of these genes in mice. However, we have shown variation in allelic expression of ARF, which suggests a role for ARF haploinsufficiency in the onset of childhood ALL.
A key feature of early human fetal lymphoid development is the absence of random �N� nucleotides between the rearranged V[H], D[H] and J[H] gene segments. The addition of �N� nucleotides at these junctions requires the enzyme terminal deoxynucleotidyl transferase (TdT). TdT is reported to not be expressed during early fetal lymphopoiesis but has been observed by the end of the first trimester, but data are sparse.
The reported absence of N nucleotides in the majority of childhood ALLs suggests an early fetal origin. By defining the window-in-time during which TdT-negative B cell development occurs, we will be able to refine the timing of the origin of the B cells that give rise to ALL. Therefore we have sequenced and analysed the V[H]-DJ[H] and D[H]-J[H] junctions from immunoglobulin rearrangements in developing B cells in normal human fetuses aged from 5.1 to 11 weeks gestation.
In this study 73 fetal IgH gene rearrangements were amplified from 21 different fetal liver samples. Only eight of the seventy-three rearrangements (11%) analysed in this study had no �N� nucleotides at the N1 (D[H]-J[H]) junction. This finding contrasts with the 24-28% of fetal rearrangements with no �N� nucleotides that have been reported in the literature. Furthermore, �N� nucleotides were shown to be present in the earliest sample, 5.1 weeks gestation. TdT expression was demonstrated by immunohistochemistry at 7.3 weeks and by RT-PCR at 8.3 weeks. B cell development in the fetal liver was detected as early as 6.5 weeks using flow cytometric analysis.
Then, IgH gene rearrangements from 99 cases of childhood ALL were analysed. In total, 134 clone-specific IgH gene rearrangements were examined in this study. No association was found between the number of �N� nucleotides from complete and incomplete rearrangements at either the N1 (D[H]-J[H]) or N2 (V[H]-DJ[H]) junctions. Nor was any association observed between ALLs from children [less than or equal to] 3 years of age and those >3 years of age at diagnosis. These findings indicate that ALL IgH rearrangements do not have the paucity of �N� nucleotides that has been previously reported.
The findings in this thesis suggest that there is no TdT-negative timepoint during B cell development and that there is no paucity of �N� nucleotides at the N1 junction in either fetal or childhood ALL IgH gene rearrangements.
| Identifer | oai:union.ndltd.org:ADTP/266585 |
| Date | January 2007 |
| Creators | Drake, Kylie Marie, n/a |
| Publisher | University of Otago. Department of Biochemistry |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Kylie Marie Drake |
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