Blood, 15 June 2001, Vol. 97, No. 12, pp. 4000-4002
CORRESPONDENCE
To the editor:
X chromosome inactivation ratios in female
carriers of X-linked sideroblastic anemia
In heterozygous females, an unbalanced X chromosome
inactivation pattern (skewed lyonization) may cause disease expression of X-linked disorders,1 for example, X-linked
sideroblastic anemia (XLSA).2,3 X chromosome inactivation
analyses such as the polymerase chain reaction (PCR)-based human
androgen receptor assay (HUMARA) can reveal whether a female has a
balanced or a skewed lyonization. Skewing itself can be constitutional
or acquired for many different reasons.1 Moreover, in
female carriers of X-linked disorders, skewed lyonization can be
fortunate or unfortunate.1 The latter means a predominant
inactivation of the X chromosome harboring the wild-type allele. But
the distinction between skewed and balanced lyonization depends on
various arbitrary definitions as well as certain technical variables.
Cazzola et al4 recently reported an Italian family with
females heterozygous for an ALAS2 mutation that may cause
XLSA. The degree of lyonization in these individuals was determined in
leukocytes by the cleavage ratio between alleles from the HUMARA. For
this assay, the methods for the detection and semiquantitative assessment of PCR products have crucial importance. Cazzola et al used
silver-stained nondenaturing polyacrylamide gels and densitometric scanning for determination of cleavage ratios and did not provide methods for their calculation or correction. For semiquantitation, we
recommend the use of an automated laser fluorescence sequencer or a
similar device for enhanced resolution.5
Cazzola et al, like some other authors, attribute "excessive
skewing"4(p4364) to allele ratios higher than
3.0 while allele ratios below 3 are defined as balanced. Elsewhere, a
ratio between 1.85 and 4.0, as found in the 3 female carriers,
has been termed "moderately skewed,"6(p32) and
several other authors considered an "extreme lyonization" or
"monoclonality" only when allele ratios were above
10.7(p62),8(p1581) The allele ratio can also be
translated into the percentage of inactivated X chromosomes harboring
the wild-type allele as follows: [ratio / (ratio + 1)] × 100.9 Thus, for case II-2
in Cazzola et al's report, the ratio of 3.2 would translate into 76%
of cells with an inactive wild-type ALAS2 allele.
But sequence analysis of cDNA derived from her reticulocyte RNA showed
only expression of the wild-type allele. This finding is not discussed
and is difficult to reconcile. Theoretically, a distinct erythroid
lineage-specific X chromosome inactivation pattern (XCIP) due to a
postinactivation selection may provide a possible explanation and could
be resolved by X chromosome inactivation analysis of erythroid precursors.
Unfortunately, the only anemic person in the family reported by
Cazzola et al (the proband; hemoglobin level 5.2 g/dL) was not
informative, but an extremely skewed lyonization, for example, 99% of
cells having an inactive wild-type ALAS2 allele, can be assumed. Using the above formula in cases II-3 and III-2 (with HUMARA cleavage ratios of 4.0), 80% of the cells should have an inactive wild-type ALAS2 allele. It is puzzling how 20%
of cells with an active wild-type ALAS2 allele can
account for the lack of anemia in these individuals.
Finally, the authors postulate familial skewing. But the moderately
skewed XCIP in the 3 females' leukocytes could also be the result of
an age-related stochastic event, as occurs in approximately 35% of
normal females.7 A comparison of leukocyte XCIP with XCIP
from other tissues is needed.
Manuel Aivado, Norbert Gattermann, and Sylvia Bottomley
Correspondence: Manuel Aivado, Department of Hematology,
Oncology, and Clinical Immunology, Heinrich-Heine University,
Moorenstrasse 5, Düsseldorf 40225, Germany
References
1.
Belmont JW.
Genetic control of X inactivation and processes leading to X-inactivation skewing.
Am J Hum Genet.
1996;58:1101-1108[Medline]
[Order article via Infotrieve].
2.
Bottomley SS, Wise PD, Wasson EG, Carpenter NJ.
X- linked sideroblastic anemia in ten female probands due to ALAS2 mutations and skewed X chromosome inactivation [abstract].
Am J Hum Genet.
1998;63:A352.
3.
Aivado M, Rong A, Germing U, Gattermann N.
Skewed X-chromosome inactivation promotes disease manifestation in female members of a family with X-linked sideroblastic anemia [abstract].
Blood.
1999;94:410a.
4.
Cazzola M, May A, Bergamaschi G, Cerani P, Rosti V, Bishop DF.
Familial-skewed X-chromosome inactivation as a predisposing factor for late-onset X-linked sideroblastic anemia in carrier females.
Blood.
2000;96:4363-4365[Abstract/Free Full Text].
5.
Maes N, De Gheldre Y, De Ryck R, et al.
Rapid and accurate identification of Staphylococcus Species by tRNA intergenic spacer length polymorphism analysis.
J Clin Microbiol.
1997;35:2477-2481[Abstract].
6.
Orstavik KH, Orstavik RE, Eiklid K, Tranebjaerg L.
Inheritance of skewed X chromosome inactivation in a large family with an X-linked recessive deafness syndrome.
Am J Med Genet.
1996;64:31-34[CrossRef][Medline]
[Order article via Infotrieve].
7.
Busque L, Mio R, Mattioli J, et al.
Nonrandom X-inactivation patterns in normal females: lyonization ratios vary with age.
Blood.
1996;88:59-65[Abstract/Free Full Text].
8.
Delabesse E, Aral S, Kamoun P, Varet B, Turhan AG.
Quantitative non-radioactive clonality analysis of human leukemic cells and progenitors using the human androgen receptor (AR) gene.
Leukemia.
1995;9:1578-1582[Medline]
[Order article via Infotrieve].
9.
Monteiro J, Derom C, Vlietinck R, Kohn N, Leeser M, Gregersen PK.
Commitment to X inactivation precedes the twinning event in monochorionic MZ twins.
Am J Hum Genet.
1998;63:339-346[CrossRef][Medline]
[Order article via Infotrieve].
Response:
X chromosome inactivation ratios in female
carriers of X-linked sideroblastic anemia
Aivado et al raise a number of questions concerning our
recent paper on familial-skewed X chromosome inactivation as a
predisposing factor for late-onset X-linked sideroblastic anemia (XLSA)
in carrier females.1 We are pleased to provide them with
technical details that could not be placed in a brief report and also
to have the opportunity of discussing the pathophysiology of
sideroblastic anemia.
Aivado et al correctly state that distinction between skewed and
balanced lyonization depends on various arbitrary definitions as well
as certain technical variables. They claim that we did not provide
methods for calculation of cleavage ratios or their correction but do
not consider that we had just one sentence available for describing
clonal analysis of hematopoiesis. Therefore, we referred the reader to
our previous methodological paper,2 which can provide
technical and methodological details. It is a shame that our colleagues
did not have the chance to read this article.
The decision to use a cleavage ratio equal to 3.0 as the cutoff between
cases with balanced X chromosome inactivation and cases with excessive
skewing was arbitrary by definition. More generally, any cutoff is
arbitrarily established (eg, a hemoglobin level of 12 g/dL for
distinguishing between healthy and anemic women): what counts
is the rationale supporting the arbitrary decision. In the previously
mentioned paper,2 we did perform a detailed analysis of
the literature, which indicated that a value of 3.0 was the best
cutoff. Our German and American colleagues recommend the use of an
automated laser fluorescence sequencer or a similar device for enhanced
resolution: we fully agree and have indeed started to use this
technique in the last few months.
As regards case II-2 in our report, it is true that the ratio of 3.2 would translate into 76% of cells with an inactive wild-type ALAS2 allele [(3.2 × 100) / (1 + 3.2)]. Aivado et
al find it difficult to explain the fact that sequence analysis of cDNA
derived from her reticulocyte RNA showed only expression of the
wild-type allele. They also argue that it is puzzling how 20% to 24%
of cells with an active wild-type ALAS2 allele can account
for the lack of anemia in women II-2, II-3, and III-2. What they do not
account for is the pathophysiology of anemia in XLSA. We are glad to
provide them with the interpretation of these findings that was
included in the first version of our manuscript and eventually had to
be omitted for reasons of space.
Despite the fact that our proband was not informative for clonal
analysis of hematopoiesis, studies on the erythroid-specific 5-aminolevulinic acid synthase (ALAS2) structure and
expression provided useful information. In fact, although she was
heterozygous for the ALAS2 mutation, only the mutant
ALAS2 mRNA was expressed in her reticulocytes, as happened
with her grandson, who is hemizygous and therefore carries
only the mutant X chromosome. It should be noted that both the
woman and her grandson were under pyridoxine treatment and no
longer anemic at the time they were found to express the mutated
ALAS2 allele. On the other hand, the remaining 3 heterozygous women in this family had normal hemoglobin levels and,
despite unbalanced X chromosome inactivation, expressed the normal
ALAS2 in their reticulocytes. In the proband's daughters, red cell production is essentially sustained by erythroid cells carrying the nonmutant X chromosome as the active one. Even if such
erythroid cells represent only about 20% to 24% of total immature red
cells, they can clearly sustain a normal red cell production.3 Most erythroid precursors expressing the
mutant ALAS2 are ring sideroblasts that die prematurely in
the bone marrow, a mechanism responsible for anemia in hemizygous males
and known as ineffective erythropoiesis. The few mature red cells
deriving from erythroid precursors expressing the mutant gene account
for the slightly increased red blood cell distribution width (RDW) values that are typically observed in heterozygous females. But the RNA
content of reticulocytes expressing the mutant gene is only a small
fraction of total reticulocyte RNA and may or may not be detected using
the cDNA assay employed by us (which is semiquantitative). One
experiment we did not carry out involved administration of pyridoxine
to the nonanemic heterozygous women in order to see its effect on
mutant ALAS2 expression in their reticulocytes. It is
possible that under pyridoxine women II-2 and II-3 would also have
expressed the mutant allele.
Finally, Aivado et al raise doubts about our conclusion that skewing
was familial. They suggest that the moderately skewed X chromosome
inactivation patterns (XCIPs) in the 3 females' leukocytes could also
be the result of an age-related stochastic event. In our previous
paper,2 we studied XCIPs in blood cells from healthy women
belonging to 3 age groups: neonates (umbilical cord blood), women 25 to
32 years old (young women group), and women more than 75 years old
(elderly women). The frequency of skewed X inactivation in
polymorphonuclear cells (PMNs) increased with age: in fact, a cleavage
ratio of at least 3.0 was found in 3 of 36 cord blood samples, 5 of 30 young women, and 14 of 31 elderly women. The inactivation patterns
found in T lymphocytes were significantly related to those observed in
PMNs in both young (P < .001) and elderly women
(P < .01). Based on the above estimates, the probability that the 4 women in our family simply had age-related skewing would be
8 divided by 10 000 [(5 of 30)4], while the probability
that skewing was familial is 9992 divided by 10 000. Consequently our
conclusion had a strong scientific basis.
Aivado et al suggest that a comparison of leukocyte XCIP with XCIP from
other tissues is needed. To define the best control tissue for the
interpretation of X chromosome inactivation patterns in hematopoietic
cells, we previously analyzed X chromosome inactivation patterns in
different peripheral blood cell populations and in hair bulbs from
healthy women belonging to different age groups.2 When
PMNs were compared with hair bulbs,2(Fig3) no relationship
was found with respect to the inactivation ratio (r = .31,
P > .05). There was no difference between young and elderly women in this respect, a cleavage ratio of at least 3.0 in PMNs
being associated with a similar value only in about 50% of hair bulb
DNA from either young or elderly women.
In summary, findings of our study clearly indicate that the most likely
explanation of the above findings is that the proband, despite a
markedly congenitally unbalanced X chromosome inactivation in her
hematopoietic cells, was able to produce normal amounts of red cells
for the first 6 decades of her life, as her daughters and granddaughter
do. In the seventh decade she developed acquired skewing, as do about
one third of elderly women. She unfortunately further inactivated the
parental X chromosome carrying the normal ALAS2 gene, and
when nearly all red cell precursors expressed the mutant gene, she
became severely anemic.
Mario Cazzola and Gaetano Bergamaschi
Correspondence: Maro Cazzola, Division of Hematology, University
of Pavia School of Medicine, IRCCS Policlinico S Matteo, 27100 Pavia,
Italy
References
1.
Cazzola M, May A, Bergamaschi G, Cerani P, Rosti V, Bishop DF.
Familial-skewed X-chromosome inactivation as a predisposing factor for late-onset X-linked sideroblastic anemia in carrier females.
Blood.
2000;96:4363-4365.
2.
Tonon L, Bergamaschi G, Dellavecchia C, et al.
Unbalanced X-chromosome inactivation in haemopoietic cells from normal women.
Br J Haematol.
1998;102:996-1003[CrossRef][Medline]
[Order article via Infotrieve].
3.
Cazzola M, Pootrakul P, Huebers HA, Eng M, Eschbach J, Finch CA.
Erythroid marrow function in anemic patients.
Blood.
1987;69:296-301[Abstract/Free Full Text].
