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782 Part VII Hematologic Malignancies

Reactivation of Random X Polyclonal
paternal X inactivation proliferation

Oocyte

Sperm Female Morula Blastocyst
zygote

Monoclonal
proliferation
Fig. 56.7 X-CHROMOSOME–LINKED ENZYME GLUCOSE-6-PHOSPHATE DEHYDROGENASE
(G6PD) AS A MARKER TO INVESTIGATE CLONAL DEVELOPMENT OF HUMAN HEMATOPOI-
ETIC DISORDERS. Early in embryogenesis, regions of all but one X chromosome are inactivated in each
cell containing two or more X chromosomes. The choice of maternal versus paternal X chromosome for
inactivation is random. Once the inactivation occurs, it is fixed and is stably transmitted to daughter cells
during mitosis (Lyon hypothesis). Females who are heterozygous for the common B type and the less frequent
A type, G6PD (localized on Xq27), are mosaic. This cellular mosaicism is used to study monoclonal versus
polyclonal cell proliferation and development of malignant hematopoietic diseases. (Courtesy Dr. W Raskind,
University of Washington, Seattle.)

is not restricted to the cell lineages. Individuals with Turner or G6PD type, but these clonal cells were Ph-negative; thus leukemic
Klinefelter syndrome are mosaic for XX or XY and monosomy X cells transformation predates development of the chromosomal abnormal-
or XXY and XY cells, respectively. The mosaicism created by ity. This observation provided evidence that CML has a multistep
X-chromosome inactivation in females is much more widely appli- pathogenesis. Application of G6PD studies to hematologic malignan-
cable and has provided fundamental insights into the pathogenesis of cies demonstrated the clonal and stem cell origin for AML, ALL,
hematologic malignancies. Original studies with X-linked glucose-6- Ph-negative myeloproliferative neoplasm (MPN), myelodysplastic
phospate dehydrogenase (G6PD) as a marker of clonality were based syndrome (MDS), and CLL. G6PD studies were particularly useful
on the Lyon hypothesis, which asserts that early in embryogenesis, in the investigation of red blood cells and platelets in hematologic
one X chromosome in females is inactivated in somatic cells and the malignancies because the absence of nuclei in these cells means they
activation status is stably transmitted to daughter cells during mitosis cannot be studied by cytogenetics or DNA analysis. Although it is
(Fig. 56.7). The choice of maternal versus paternal X-chromosome now considered common knowledge that hematologic malignancies
inactivation is random; however, once it occurs, it is maintained in are characterized by clonal development, this understanding is greatly
all daughter cells. Random X inactivation occurs by embryonic day owing to what is now known as classic Fialkow’s work, whose profound
6.5 around the start of gastrulation and results in a mosaic pattern insight contributed much to current concepts and understanding.
that characterizes adult females. Therefore an adult female is a mosaic Despite the importance of the G6PD approach, it is limited by
for two-cell populations, one expressing genes from an active X the rarity of females who are heterozygous for the G6PD isoenzyme.
chromosome and the other expressing genes from the inactive X An alternative and more extensive DNA-based X-chromosome clonal
chromosome. Incidentally, mammalian X-chromosome inactivation assay uses common polymorphic markers that are caused by changes
is a mechanism that equalizes the dosage of X-linked genes between in DNA methylation patterns that accompany inactivation of the X
sexes. Although the exact mechanism of X-chromosome inactivation chromosome. These X-linked loci, such as phosphoglycerine kinase,
remains to be elucidated, the process of X inactivation starts with hypoxanthine phosphoribosyltransferase, DXS25 (M27β), and
methylation of CpG islands. The inactivation process is believed to human androgen receptor (HUMARA), have been subsequently
occur before differentiation of the embryonic stem cell into various extensively used in assessment of clonality, and now it is possible to
cell lineages. Hematopoietic cells do not originate from a single identify clonal cell populations in virtually all females. DNA-based
embryonic stem cell but from several progenitors, thereby allowing marker systems rely on a sequence polymorphism that has adjacent
for mosaic expression from both X chromosomes. differences in methylation on the active and inactive X chromosomes.
The observation that human females are heterozygous for the The inactive X chromosome is more highly methylated than its active
−
G6PD variant A and A and that two mosaic cell populations may be homologue, but this is only true for certain regions of genes as 10%
distinguishable by electrophoretic mobility was reported in the 1960s. to 20% of X-linked genes escape inactivation and can be found both
The X-inactivation G6PD mosaic system was then applied to the in clusters and in isolation. The most widely used HUMARA assay
study of clonality in human tumors (uterine leiomyomas) in 1964 by appears to maintain the stringent methylation differences. The
Gartler and Linden. In females who are heterozygous for the G6PD number of CAG tandem repeats differentiates the maternal from the
polymorphism and have malignant hematologic disorders such as paternal X chromosome.
CML, the finding of a single G6PD type in marrow or blood cells The DNA-based X-chromosome clonal assay is limited to females
and both the A and B type G6PD in tissues not involved by the younger than 60 years because they usually have 1 : 1 distribution of
malignant process demonstrated that CML was of clonal origin and two-mosaic–cell population. A ratio greater than 3 : 1 is found in
provided evidence that the malignant transformation occurred at the women older than 60 years, probably as a result of stem cell kinetics
level of a stem cell common to most hematopoietic cell lineages. influenced by X-linked genetic factors. When the ratio of two cell
Additional studies with heterozygous G6PD females who had CML populations is greater than 3 : 1, this phenomenon is called a skewed
demonstrated that some CML-derived B lymphocytes had a single X-inactivation pattern. With the HUMARA assay, acquired unequal