Autologous Bone Marrow Transplantation - Blog Science Connections

Clonal Detection of Remission 733
consistently at some sites and can be monitored there by such restriction
endonucleases.
There are at least sue Hhal sites within the first intron of the HPRT gene
(Fig 1). Hhal cleaves at the sequence CCG:C (G-guanine), but does not cleave
at this sequence when either C is methylated. Hhal site 1 is unmethylated in
active chromosomes. Cleavage at that site changes the mobility of the BamHI
fragment by less than 3%, and this small change in mobility can barely be
detected on agarose gels. Sites H2 to H6 are each methylated in over 95% of
active X chromosomes, but at least one of sites H2 to H6 is unmethylated in
most normal inactive X chromosomes. The use of the second enzyme Hpall
allows interpretation when tumors are resistant to Hhal digestion (25-30% of
cases). Hpall, like Hhal, is sensitive to changes in methylation and cleaves at
the sequence G:CGG except when the C is methylated.
There are at least nine Hpall sites within the HPRT locus (Fig 2). Hpall
sites 1 to 3 are unmethylated in the active chromosome and methylated in the
inactive chromosome. Sites 4 to 9 are methylated in the active chromosome,
and at least one is unmethylated in most inactive X chromosomes.
Applications of this approach have been reported using a single enzyme and
HPRT probe to study clonality of a variety of tumors (8) and to detect a clonal
population of granulocytes in patients with ANLL at presentation, in remission,
and in relapse (2). In the latter group, Fearon et al. documented 3 of
13 clonal remissions by examining the DNA of remission granulocytes. This
suggested that the persistence of a single dominant hematopoietic clone in
patients in clinical remission is not rare.
We have refined the methodology of Vogelstein and colleagues by always
using two enzymes (Hhal and Hpall) and by using a second probe (9-11). The
use of two enzymes allows for interpretation in cases in which the inactive
chromosome is resistant to digestion by one enzyme. Probe I is a 1.4-kb
EcoRLXhoI fragment isolated from a plasmid subclone of the human HPRT
clone Hhl 3, and probe II is a 0.65-kb Hpall fragment derived from probe I. In
situations where the tumor is resistant to both Hpall and Hhal digestion, the
use of the 0.65-kb probe enables recognition of the active chromosome. Hpall
cleaves a 0.65-kb fragment from the active X chromosome. The remainder of
KHobases i
0
1
1
1
2
1
3
1 i
4 5 6
1
7
1
8
T
9
1 1
10
//—i
22
BamHI (BamHI) BamHI
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HQ) I
'
11
1 II I I I "
Hhal sites 1 2 3 4 5 6
Probes £ ^5
Figure 1. Diagram of the 5 '-half of the human H PR T gene. The polymorphic BamHI site
is shown in parentheses, and positions of axons relative to the polymorphic fragments
are indicated. Hhal cleavage sites and hybridization probes referred to in the text are
also
denoted.