Fanconi anemia. (FA) is characterized clinically by a pro- gressive pancytopenia. diverse congenital abnormalities and increased predisposition to malignancy.
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1989 73: 391-396
International Fanconi Anemia Registry: relation of clinical symptoms to diepoxybutane sensitivity AD Auerbach, A Rogatko and TM Schroeder-Kurth
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International
Fanconi
Anemia
Registry:
Diepoxyhutane D. Auerbach,
By Arleen
Andr#{233} Rogatko,
ANCONI ANEMIA (FA) was originally described as an autosomal recessive disorder characterized by a
progressive and increased
pancytopenia, predisposition
diverse congenital to malignancy.”3
it has been recognized that the FA that diagnosis on the basis of clinical difficult and often unreliable.4’5 Although unknown,
the molecular hypersensitivity
crosslinking
agents
genotype.6’7 tify
This
pre-anemic
or
cases
leukemia
who
ing5.8’2 It is useful of FA.’3’4
a unique
marker
characteristic
not
have
for
for prenatal
as well
the
FA
can
be used
to iden-
with
aplastic
anemia
characteristic
physical
as postnatal
find-
diagnosis
Literature reports of FA are undoubtedly biased toward the most severe clinical cases. Since the current concept of the syndrome is based on the originally described patients, who had many congenital malformations, cases that do not conform
to this preconceived
order
to study
spectrum
of diverse
tional The
a large
Fanconi
centralized information clinical
University with
physicians.
FA.
with
Peripheral
with
syndrome,
in 1982.
associated
patients
(IFAR)
the
was
The
for clinical,
on patients features
by their
of the
Registry
repository
will not be diagnosed.
of FA
features
Anemia
Rockefeller
picture
number
full
serves
as a
with
genetic
one
or more
to the IFAR
blood
are
samples
report
here
DEB-induced
blood studied analysis significant
results
We
in
breakage
analysis 328
also
report
the
of 310 cases from clinical differences
Vol 73. No 2 (February),
the
results
of
peripheral
of a discriminant
IFAR between
(DEB) clastogenic
data the groups. effect
AND
METHODS
Blood samples for DEB-testing by the RU Cytogenetwere obtained from patients seen at the RU Outpatient Clinic, or at a referring medical center. In the latter case, samples were sent by overnight express carrier to the RU laboratory. Patients were ascertained on the basis of the presence of congenital malformations known to be associated with FA, hematologic manifestations such as aplastic anemia or leukemia, both malformations and aplastic anemia, or family screening. The primary source of case material for the IFAR was voluntary Patients.
physician
reporting.
base, showing DEB-sensitive We conclude of DEB is a
The
basis
for ascertainment
of the
patients
was
the same as described for the DEB testing. Once a potential case was identified, an IFAR questionnaire form was completed by the referring physician and copies of laboratory reports and other patient records were obtained. Chromosome breakage studies including testing for hypersensitivity to the clastogenic effect of the
From
the Laboratoryfor
Investigative
Dermatology.
The
Rocke-
feller University, New York, the Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center. New York; and Institut fIr Humangenetik und Anthropologie, Universitat FRG.
Submitted
July
Supported
inpart and
1 1. /988; accepted by Public
GM36295
September
Health
(AR.)
Service
from
the
19. 1988. Grants
National
Health, by Basil O’Connor Starter Research Grant the March of Dimes Birth Defects Foundation Clinical
National
from patients considered at risk for FA, Rockefeller University (RU) Cytogenetics
(DEB) and DEB-insensitive that hypersensitivity to the useful discriminator for FA.
Blood,
of a discriminant
chromosomal
specimens by the
Laboratory.
the
M. Schroeder-Kurth
MATERIALS
General
obtained
from each of these individuals for studies of sensitivity to the induction of chromosomal breakage by the DNA crosslinking agent diepoxybutane (DEB). We
to
insensitive (DEB1 cases. Similar methods were applied to the data from the International Fanconi Anemia Registry (IFAR) to determine whether DEB and DEB cases may be considered as distinct clinical entities. and if so. which variables provide the best discrimination between the two groups. We conclude that hypersensitivity to the clastogenic effect of DEB is a useful discriminator for FA. A simplified scoring method for classifying patients on the basis of eight clinical manifestations that are the best predictors for FA is presented. Our data indicate that the clinical diversity in FA is more widespread than previously recognized. 0 1989 by Grune & Stratton. Inc.
(ADA.)
and
FA are referred
and Traute
Heidelberg,
Internaat
hematologic, Patients
In
the
established
registry
Symptoms
ics Laboratory
phenotype is so variable manifestations alone is
as well as patients
do
abnormalities More recently
basis for the syndrome is to the clastogenic effect of DNA
provides cellular
of Clinical
Sensitivity
Fanconi anemia (FA) is characterized clinically by a progressive pancytopenia. diverse congenital abnormalities and increased predisposition to malignancy. Although a variable phenotype makes accurate diagnosis on the basis of clinical manifestations difficult in some patients. study of cellular sensitivity to the clastogenic effect of DNA crosslinking agents such as diepoxybutane (DEB) has been used to facilitate the diagnosis. Data from DEB-induced chromosomal breakage studies of 328 peripheral blood specimens from patients considered at risk for FA were analyzed using a stepwise multivariate logistic regression. in order to determine which method of representing the data best discriminated between DEB-sensitive (DEBt) and DEB-
F
Relation
tal,
Research
by supportfrom
Investigative schaft Part
Center
ofHealth
Institutes
Grant
and
Trust
by the Deutche
of
No. 5-446 from (ADA.), by a from
University
the
Hospi-
to the Laboratory
for
Forschungsgemein-
(T.M.S.-K.). of
this
work
was
presented
previously
(Blood 70:51a, 1987 fsuppl 1/). Address reprint requests to Arleen tory for Investigative Dermatology, 1230
HL32987
Institutes
RROOJO2
to The Rockefeller
the Pew Memorial
Dermatology.
No.
No.
York
Ave.
The publication
New
York,
NY
article
charge payment. This article “advertisement” in accordance indicate this fact.
must
& Stratton.
form
D. Auerbach, PhD, LaboraThe Rockefeller University,
10021-6399.
costs ofthis
0 1 989 by Grune
in abstract
with
were defrayed
therefore
in part
be hereby
18 U.S. C. section
by page
marked
1 734 solely
to
Inc.
0006-4971/89/7302-0007$3.00/0
1989:
pp 39i-396
391
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AUERBACH,
392
Cultures
were
set up in duplicate
for DEB
studies,
and
the
untreated
preparation
were
analyzed.
To avoid
bias
chromosome
were scored
breakage
frequencies,
and
data
Breaks/cell
Cells with
breaks
Difference Abbreviations:
(%)
cell
Breaks/aberrant
between
Mm,
osomal
FA and non-FA minimum
value;
DEB)
and
cells these
with
purpose of this of representing
analysis was the chromosomal
between
independent the
breaks,
the
variable
predictors
breaks
per
aberrant
that
breaks
(Table
per
1). There
group per
cell,
DEB
analyzed patients
after DEB exposure, appeared to have two
approximately populations
somal
In these individuals, the majority (from 60% to 90%) appeared breakage, while the remainder
each
case
examined
range
exhibited
the
high
calculated
as
breaks
per
aberrant
10% of of lympho-
of DEB-treated cells to have no chromoof cells examined in
number
exchanges typical of FA patients. The chromosomal breakage in these patients 2.56 breaks per cell, while the mean cell
of
breaks
and
mean DEB-induced ranged from 1.06 to breakage frequency was 6. 1 3, similar to
that found in typical FA patients. The DEB-induced chromosomal breakage frequency in the non-FA group was similar to that reported previously for control individuals.8 Analysis of baseline group showed that the differ
from
that
group.
The
range
1.9 breaks
per
chromosomal frequency
found
in normal
of breakage
cell
(mean,
breakage in the in some patients did controls
or in the
in untreated 0.27)
for the
cells FA
non-FA
was
group
0.02 and
they
had
some
Mean
clinical
findings
consistent
with
a diagnosis
Blood Lymphocytes
Median
SE Mean
Mm
Max
104
8.96
8.73
0.448
1.30
23.90
NOn-FA
224
0.06
0.04
0.004
0.00
0.36
FA
104
85.15
1.99
12.60
100.00
Non-FA
224
5.12
4.00
0.28
0.00
22.00
FA
104
10.22
9.65
0.043
3.60
24.90
NOn-FA
224
0.99
1.00
0.04
0.00
6.00
Max,
maximum
value.
FA not to 0 to
0.12 breaks per cell (mean, 0.02) for the non-FA group. The difference in the baseline breakage frequencies for these two groups was not statistically significant, indicating that this is not a useful method for discrimination of FA patients. IFAR. Of the 310 patients referred to the IFAR because
ge in DEB-Trea ted Peripheral
for all the parametrizations:
The
absolute
in the
FA
groups
of
sex.
gave
no overlap
or
percent
and
alone
to
(DEB
cell,
cell
was
and
DEB
was
were
or breaks
indicated
cytes.
rearrangements
Breaks
logistic regression at the RU Cytoge-
for the DEB (FA) group compared with the DEB (nonFA) group (Fig 1). There was no significant difference in DEB-induced chromosomal breakage when male and female FA patients were compared. While most FA patients exhibited multiple chromatid breaks and exchanges in most or all
in cell
N
multivariate DEB studies
discriminated
The
results
analysis. A stepwise multivariate logistic regression’6 was applied in two instances: (a) to determine which method of representing the data in the chromosomal breakage studies best discriminated between DEB and DEB cases, and (b) to determine
soup
best
discrimination
as two breaks.
Table 1 . Chrom
The method
which
groups. cells
Discriminant
Parametrizations
Laboratory.
determine
a
A stepwise on data from
performed
netics
consecutive metaphases that appeared intact with sufficiently well defined chromosome morphology were selected for study. Each cell was scored for chromosome number and for the numbers and types of structural abnormalities. Achromatic areas less than a chromatid in width were scored as gaps, while exchange configurations, translocations, and dicentric and ring chromosomes were scored as rearrangements. Gaps were excluded in the calculaof
studies.
DEB
was
selection,
tions
SCHROEDER-KURTH
RESULTS
replicate set of cultures established to serve as untreated controls. DEB (Aldrich Chemical Co. Milwaukee), at a final concentration in the medium of 0. 1 zg/mL, was added to the treated cultures 24 hours after their initiation, thus exposing cells to the chemical for 48 to 72 hours. This concentration was chosen because it induces multiple chromosomal breaks and gaps in FA cells, while having little clastogenic effect on normal cells. The mitotic index was usually not affected by this concentration of DEB. Dilutions were prepared just before addition of DEB to cell cultures. Cultures were harvested after 72 to 96 hours, and chromosome preparations made following standard methods. Since DEB is a suspected carcinogen with unknown risk, appropriate precautions were taken. Cultures with DEB were handled using latex gloves, and all work was done in a vertical laminar flow hood or a chemical fume hood (for the first part of the harvest procedure). Since DEB is rapidly inactivated by concentrated hydrochloric acid (HCI), all disposable culture bottles and pipettes were rinsed with HC1 before being discarded. HC1 was added to spent tissue culture medium before disposal, and was used in case of spillage. Analysis was performed on 50 to 100 Giemsa-stained metaphases from each DEB-treated preparation (25 metaphases in the case of very high breakage). If DEB-induced chromosomal breakage was increased over the normal range, 50 Giemsa-stained metaphases from
AND
whether DEB and DEB cases may be considered as distinct clinical entities, and if so, which variables provide the best discrimination between the two groups. In the second instance, after applying the logistic regression, a method for classifying the patients based on a simplified score was derived.
DNA crosslinking agent DEB were performed at the RU laboratory, Heidelberg, FRG, or elsewhere.8 Patients were classified as FA or non-FA based on sensitivity of cultured peripheral blood lymphocytes to DEB-induced chromosomal breakage. Blood specimens from siblings of FA patients were also tested. DEB test. The method for the DEB test has been previously described in detail.’5 In summary, the culture unit consisted of 0.4 mL of heparinized blood added to 10 mL of RPM! 1640 medium (GIBCO, Grand Island, NY) supplemented with 15% fetal bovine serum (Hazelton Research Products, Denver, PA), 1% 1-glutamine, 1% penicillin-streptomycin solution (GIBCO) and 1% phytohemagglutinin (PHA) (Wellcome Diagnostics, Dartford, England), and incubated for 72 or 96 hours at 37#{176}C in a 5% CO2 atmosphere at high humidity.
ROGATKO.
P
92.00
