A clonal study of hematopoiesis using the M27 beta probe: aberrant band patterns caused by incomplete digestion of a methyl-sensitive enzyme in the the inactive X-chromosome.
The M27 beta probe has been used to determine the clonality of human tumors, based upon X-chromosome inactivation. However, it occasionally gives rise to aberrant results. In this study, the M27 beta probe was used for clonal analysis in Japanese women with clonal stem cell disorders and in those with normal hematopoiesis. Restriction digestion with PstI indicated heterozygosity for the DXS255 locus in 41 out of 50 individuals (82%). Further digestion with HpaII in heterozygous women led to four distinct band patterns: I, both fragments were partially digested; II, either one of the two fragments was completely digested; III, a three-band pattern; and IV, neither fragment was digested. Of 21 hematologically normal females, 17 (81%) and four (19%) had patterns I and III, respectively. In some subjects with pattern I, imbalanced HpaII digestion in the two alleles was seen. Fifteen (65%) of the 23 patients with clonal stem cell disorders had pattern II, while the remainder (35%) had pattern IV. The normal tissues of three acute myeloid leukemia patients with pattern IV all revealed pattern I. It is possible that the aberrant band patterns could be caused by incomplete HpaII digestion in inactive X-chromosomes. In this study, we propose a hypothesis whereby, in normal tissues, aberrant cells, the DXS255 locus of which is not digested with HpaII despite their inactive status, would be mixed with cells demonstrating the usual methylation pattern. In normal tissues, complex of proportion of aberrant cells and skewed Lyonization could produce a variety of band patterns. If a cell with the usual methylation pattern proliferated monoclonally, pattern II would be seen: whereas if an aberrant cell proliferated, pattern IV would be demonstrated.
Jinnai
I
I
First Department of Internal Medicine, Saitama Medical School, Japan.
Yoshida
S
S
Toyoda
A
A
Murohashi
I
I
Bessho
M
M
Hirashima
K
K
Nagai
K
K
Tomonaga
M
M
Boyd
Y
Y
D015139:Blotting, Southern; D015342:DNA Probes; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D005260:Female; D006410:Hematopoiesis; D006579:Heterozygote; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics; D008745:Methylation; D012150:Polymorphism, Restriction Fragment Length; D015533:Transcriptional Activation; D014960:X Chromosome / Q000378:metabolism*
D015342:DNA Probes; D004262:DNA Restriction Enzymes