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Several studies have shown heterogeneity in frequencies of 6q deletions depending on several factors. First, the approach used. Cytogenetic measurements show frequencies varying from 4% to 13% in ALL (Hayashi Y. et a l, 1990) (Raimondi S. C., 1993) or sometimes as high as 23% in adults T-cell leukaemia/lymphoma (Kamada N. et a l,

1992). FISH detects a frequency between 6% and 44% in B-CLL and ALL (Merup M.

et a l, 1998; Stilgenbauer S. et a l, 1999; Zhang Y. et a l, 1997) while frequency based on LOH also varies greatly between studies: 13% (Takeuchi S. et a l, 1995b), 2% (Cave H. et a l, 1996), 6.7% (Gerard B. et a l, 1997), 15% (Takeuchi S. et a l, 1998) and 34% (Merup M. e ta l, 1998).

Second, different clinical cohorts of patients (from ALL to NHL to CLL) show differences in incidence.

Third, frequencies vary in different age groups (adults and children). The majority of studies focused on childhood patients and even within this group incidence varied extensively from 34% (Merup M. et a l, 1998) to 6.4% (Gerard B. et a l, 1997) to 15% (Takeuchi S. et a l, 1998). Little information has been available on 6q deletions in adult ALL.

region may affect final results, as different studies have used different markers.

Finally, frequencies also vary between different ALL subgroups depending on immunophenotypes (B or T cell lineages) with T-ALL probably showing the highest incidence, from 23% by cytogenetic analysis (Theile M. et a l, 1996) to 41% by LOH studies (Hatta Y. et a l, 1999; Hatta Y. et a l, 1998).

Discrepancies, in incidence between LOH and cytogenetic might be attributed to the use of PCR as a basis for analysis in the LOH studies which overcomes the common problems associated with cytogenetic analysis of leukaemia and lymphoma samples, mainly the poor yield o f the mitoses and fuzzy appearance o f the chromosomes. Furthermore, detection o f the small interstitial deletions may be beyond the resolution of the cytogenetic analysis whereas LOH is a powerful technique for detection o f such small deletions, as mentioned above.

These discrepancies are not restricted to chromosome 6. LOH analysis o f chromosome 9p and 12p in childhood ALL showed that deletions o f those two arms by LOH were much more frequent than those reported from the cytogenetics studies (Takeuchi S. et a l, 1996; Takeuchi S. et a l, 1995a; Takeuchi S. et a l, 1995b; Takeuchi S. et a l,

1997a).

At the time the investigation presented in this thesis started, FISH studies were being conducted to investigate 6q deletion by a colleague (Mr Paul Sinclair) while I was assigned the task to investigate 6q deletions by using LOH. This approach has the advantage of being easily applicable to large cohort of patients, multiple sites can be analysed simultaneously and small deletions undetected by other techniques can be detected easily.

In addition, at the time this study commenced some information was already available on the region o f minimal deletion while less was known about the extent of the region, in term o f size and sequence. This was mainly due to the lack o f a finalised genomic map o f the area of interest because of gaps in the region o f 6q 16-21.

FISH analysis o f 6 cases o f ALL had defined a common region of deletion between M6P1 locus at 6q 14-15 and the FYN locus at 6q21 (Menasce L. P. et a l, 1994b) and this region covered several MB in size. This RMD had been narrowed down by another FISH study by the same group, which defined the RMD between markers D6S447 and

D6S246 (Sherratt T. et al., 1997) and estimated the distance between the two markers to be approximately 2 MB.

This represented the starting point of our investigation through collaboration with Dr Harrison (the principal Investigator of Sherrat’s study, at the time based in Manchester, UK). In 1998 the scientists in our laboratory began FISH investigation using VAC clones and redefined a RMD telomeric to D6S447 and centromeric to marker D6S268 (Jackson A. et a l, 2000) (see Chapter 3, Figure 3.4) and redefined the region of deletion to approximately 1.8MB. At the time, the physical distance between markers D6S447 and D6246 had been estimated to be larger than 3.5MB, using Y AC clones identified in the process (Jackson A. et a l, 2000).

However, because of a gap between markers D6S447 and D6S268 the correct estimate of the size and gene in the region were yet uncertain. I have described in Chapter 3 the effort made to identify clones from within this region.

Several clones were isolated in the process (Chapter 3) and most recently two new Y AC clones from the ICI and ICRF libraries have allowed bridging the gap between D6447 and D6S268 (A. Mungall and colleagues, Sanger Centre, Personal communication). The distance between D6S447 (in clone dJ359N14) and D6S268 (in clone dJ67A8) has now been estimated to be 1,848,070 bp (1.8MB). The specific co-ordinates of this region are listed below (courtesy of A. Mungall at the Sanger Centre, Hixton, Cambridge, UK). This is smaller than we previously estimated. However, it is apparent from other studies resulting from accurate mapping and sequencing of the human genome that overall the size of the genome is in fact “shrinking” from a postulated 3.9GB to 3.2GB according to recent estimations (Dr M Ross, Sanger Centre, personal communication) and therefore this finding is very much in line with similar observations on other chromosomes.

Subsequence "dJ359N14" 2863749 3000047 contains D6S447 Subsequence "bA437I16" 2999948 3063753 Subsequence "bAl 14NT' 3061754 3180774 Subsequence "bA183D15" 3178775 3252682 Subsequence y R 3 9 E C l I " 3250683 33 796 75 Subsequence "dJ101M23" 3393688 3377676 Subsequence "dJ134E15" 3393589 3570751

Subsequence "dJ474G24" 3570652 3594201 Subsequence "dJ354M18" 3628873 3594106 Subsequence "bA404H14" 3658736 3628774 Subsequence "dJ335El" 3802441 3658637 Subsequence "dJ448Kl" 3856246 3802342 Subsequence "bA294Hll" 3994152 3856147 Subsequence ”dJ60O19" 4155407 3994053 Subsequence "yX2SFI0" 415340S 4165164 Subsequence *'bA59I9” 4163165 4348475 Subsequence "bA300Dll" 4346476 4430756 Subsequence "bA41204" 4428756 4558901

Subsequence **dJ67A8** 4711819 4556902 contains D6S268

In addition, another gap between a region containing markers D6S283 and D6S301 on the centromeric side and D6S447 on the telomeric side had also been recently completely mapped and found to encompass 2.3MB region (http://www.ensembl.org/Homo sapiens/mapview?chr=61

For both regions, however, the correct estimate will become available in the next few months as soon as the sequence o f chromosome 6 is completed (A Mungall, personal communication). Analysis of candidate genes within these regions will be presented below, in the following paragraphs.