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Comparative Evaluation of Diepoxybutane Sensitivity and Cell Cycle
Blockage in the Diagnosis of Fanconi Anemia
By Helga Seyschab, Richard Friedl, Yujie Sun, Detlev Schindler, Holger Hoehn, Sabine Hentze,
and Traute Schroeder-Kurth
Fanconi anemia (FA) is a clinically and genetically heterogenous disease that is usually diagnosed on the basis of chromosomal instability reflecting the hypersensitivity towards
the DNA cross-linking agents diepoxybutane (DEB) andlor
mitomycin C. A lesswell-known cellular feature that characterizes FA patients is an intrinsic cell cycle disturbance consisting of prolonged progression through, and arrest within,
the G2 phase compartment of the cell cycle. In a collaborative blind study, we have evaluated 72-hour lymphocyte cultures from 66 patients with clinicalsuspicionof FA both
for DEB sensitivity and cell cycle disturbance. A concordant
result was obtained in 63 of 66 cases. Each of the 3 discor-
F
ANCON1 ANEMIA (FA)is an autosomal recessive disease that is genetically heterogenous. If untreated, the
course is fatal because of progressive bone marrow failure.
Symptomatic therapy consists of blood cell transfusions and
ccprticoids. Curative therapy consists of bone marrow transplantation. Less than 50% of FA patients exhibit a pattern
of congenital anomalies suggestive of the correct diagnosis
before the onset of aplastic anemia.’ Four complementation
groups have been described.’ The only gene to be cloned to
date specifies complementation group C located on chromosome 9q22.3 Although sequence and molecular structure of
the FACC gene are known: its function remains to be elucidated. The cellular phenotype ofFA consists of chromosomal instability, a cell cycle defect, and increased sensitivity towards certain clastogens as well as
Until the
DNA sequences of all complementation groups are known,
the differential diagnosis of FA depends on the determination
of the cellular phenotype.
When establishing the International Fanconi Anemia Registry (IFAR), Auerbach and Schroeder-Kurth agreed on the
use of diepoxybutane (DEB) as the best discriminating agent
for the diagnosis of FA.’ Because of their increased sensitivity towards DEB, FA cells are by this definition DEB+,
whereas cells of non-FA patients are DEB-. Diagnostic problems arise in a distinct subgroup of patients in whom the
result of the DEB test shows borderline values. A practical
problem with the DEB test is that DEB itself is a powerful
carcinogen. Many laboratories therefore use mitomycin C
(MMC) as a less-dangerous substance for sensitivity testing.
However, results of both agents are not fully concordant?
In contrast to the numerous studies on DEB and MMC testing, the diagnostic potential of the defective cell cycle seen
in FA cells has not beensystematically explored. The present
study therefore has been designed as a comparative study in
which peripheral blood samples of 66 patients with clinical
suspicion of FA were evaluated in parallel for their DEB
sensitivity and cell cycle defect. Our study seeks to answer
the following questions: ( l ) Do DEB and cell cycle testing
identify the same patients as FA or non-FA? (2) If so, what
are the premises under which the cell cycle assay could be
used for the differential diagnosis of FA? (3) If not, what
are the reasons for discordant classification and what are the
limitations of the cell cycle assay?
Blood, Vol 85,No 8 (April 15). 1995:pp 2233-2237
dant, but only 1 of the concordantcases presented with
overt leukemia. Seventeen cases were identified as classical
FA because of their increased DEB sensitivity and G2 phase
blockage. Fivecases showed acellcycle disturbance but
only borderline DEB sensitivity. These casesmight represent
atypical or nonclassical forms of FA. They would have been
missed by cell cycle studies without concomitant DEB testing. Used in conjunction, cytogenetic and flow cytometric
testing provide for the currently optimal diagnosis of FA in
nonleukemic patients.
0 1995 by The American Societyof Hematology.
MATERIALS AND METHODS
Patients. Peripheral blood samples from patients suffering from
hematologic diseases such as anemia, pancytopenia, aplastic anemia,
thrombocytopenia, or combinations of these symptoms and from
patients who presented with a spectrum of congenital phenotypic
anomalies reminiscent of FA were sent to the laboratory at Heidelberg, Germany for DEB testing. With informed consent of the families, an aliquot of the blood sample was transferred in parallel to
the laboratory in Wurzburg, Germany for cell cycle studies. After
3 years of blind, parallel testing the laboratories compared their
results.
Cytogenetic studies. The buffy coat was suspended in 40 mL of
complete Chromosome Medium 1A (Biotest-Institut, Berlin, Germany) of which 3 X 10 mL was incubated as parallel experimental
lymphocyte cultures for 72 hours at 37.5”C. One culture remained
untreated; one received, after 48 hours, 0.1 &nL MMC (Serva,
Heidelberg, Germany); and the third was treated, after 24 hours,
with 0.1 pglmL DEB (Aldrich, Steinheim, Germany). Colcemid was
added after 70 hours, followed by standard procedures for chromosome preparation and staining with Giemsa solution. One hundred
metaphases were evaluated from each culture, but not all cultures
yielded scorable metaphases. To be included in the study, a cytogenetic result had to be available at least for the DEB-treated culture.
Structural chromosome aberrations were specified as follows: percentage of aberrant metaphases; breaks per cell; aberrations per aberrant cell; and types of aberrations. The cut-off values for the discrimination between FA
and
non-FA patients were
as
described
previously.’
Flow cytometric studies. For cell cycle analysis by means of
5-bromo-2’-deoxyuridine (BrdU)/Hoechst 33258-ethidium bromide
(EB) flow cytometry, mononuclear blood cells were isolated from
Fromthe Department ofHuman Genetics, University of Wurzburg, Wurzburg, Germany; and the Department of Anthropology and
Human Genetics, University of Heidelberg, Heidelberg, Germany.
Submitted August 12, 1994; accepted November 29, 1994.
Supported in part by a DFG grant to SFB I72 (H.H.).
Address reprint requests to Helga Seyschab, PhD, Department of
Experimental Oncology, St Jude Children’s Research Hospital, 332
N Lauderdale, PO Box 318, Memphis, TN 38101-0318.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8508-0006$3.00/0
2233
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SEYSCHAB ET AL
2234
16
32
18
64
80
96
112 12
1281
'4
It
I6
32
40
64
80
96
112 11
BrdU/HOECHST FLUORESCENCE
D
I6
32
heparinized bloodviathe standard Ficoll sedimentation technique
(density. 1.077 glmL: Ficoll-Paque: Pharmacia, Uppsala, Sweden).
A totalof 1 X 10' cellslmL each was placed into 25-cm' culturegrade plastic flasks (Nunc, Wiesbaden, Germany) containing 5 mL
of RPMl 1640 medium (GIBCO, Grand Island, NY), supplemented
with 1% autologous serum. 15% heat-inactivated fetal calf serum
(GIBCO). 1 X IO-' mol/L 2'-deoxyuridine (Sigma, Deisenhofen,
Germany), 2 X IO-' mol/L 1-monothioglycerol (Sigma), and I X
10" mol/L BrdU (Sigma). Cells were stimulated using 1.8 pglmL
phytohemagglutinin (Wellcome Diagnostics, Burgwedel. Germany)
and incubated at37.5"C in 5% COz incubators using atmospheric
oxygen conditions. After 72 hours, the samples were centrifuged
and resuspended in freezing medium containing RPMl 1640. 10%
fetal calf serum, and 10% dimethyl sulphoxide and stored frozen at
-20°C until analysis.
Before bivariate flow cytometric measurements. the frozen aliquots were thawed, pelleted, and resuspended at 4 X IO5 cellslmL
in staining buffer containing 0. I mol/L Tris-HCI. pH 7.4.0.154 moll
L NaCI, 0.5 mmol/L MgCl I mmol/L CaCl?. 0.1% NP40,and
0.2% bovine serum albumin (Sigma). The cells were stained with
1.2 pglmL Hoechst 33258 (Sigma), incubated for 15 minutes at
4°C. then stained with 1.5 pglmL ethidium bromide (Serva). and
incubated for another 15 minutes at 4°C. Dual-parameter flow cytometry was performed with an arc-lamp flow cytometer (ICP-22: Phywe
AG, Goettingen. Germany) interfaced to a personal computer.
The examples shown in Fig I illustrate the principle of qualitative
and quantitative cell cycle differences that can be assessed by the
BrdUlHoechst cell cycle assay. Figure IA depicts the 72-hour cell
',
40
M
89
96
112
BrdU/HOECHST FLUORESCENCE
l
Fig l . Bivariate cytograms of
peripheral blood lymphocytes
stimulated with phytohemagglutinin and cultured for 72
hours without treatment. Each
cytogram represents the BrdUl
Hoechst andthe EB fluorescence
by increasing channel numbers.
The quantitative analysis
is
given in Table 1. (A) Healthy donor. The respectivecellcycle
phasesoffourcellcyclesdisplayed are denoted asfollows:
GO-G1 and G 2 are the respective
compartments of the first cycle;
Gl', Gl", and G l " ' stand forthe
second, third, and fourth cell cycle G1-phases, respectively; G2'
and 62" are the respective G 2
compartments of the second
and third cellcycle. (B) FA patient. The elevated G 2 phases of
the first and second cell cycle
are
marked by arrows. IC and D) Cell
cycle analysis over four consecutive cell cycles (CC) of the cytograms shown in the respective
upper panels.
cycle distribution of lectin-activated mononuclear blood cells from
a 6-year-old healthy donor. Most of his cells have progressed into
the second and third cell cycle. There are very few cells left in the
G2 phase compartment ofthefirstcell
cycle. In contrast, the 72hour cell cycle distribution of a 5-year-old DEB' patient with the
clinical diagnosis of FA (Fig IB) exhibits striking accumulations of
second cell
cells in the G2 phase compartments of thefirstand
cycle (arrowheads). Exposure toMMC or DEB accentuates these
accumulations in FA cultures, but, in addition, causes other cell
cycle changes that are not found in untreated cultures such as used
for this study (data not shown). The quantitative evaluation of these
cytograms (shown in thebottom panels ofFig 1) uses electronic
framing ofthe respective cell cycles and computerized cell cycle
fitting as described by Rabinovitch et al."' An important prerequisite
for the applicability of the BrdU-Hoechst technique in thehuman
peripheral blood cell system is that incorporation of the base analogue does not by itself cause a cell cycle disturbance. This fact has
been established in previous studies withhealthyblood donors of
various ages."
RESULTS
Cytogenetic testing. Table 1 summarizes the results of
the cytogenetic testing. Usingthe criteria established previously by Schroeder-Kurth et al," the lymphocytes of 42 of
the 66 patients tested were not sensitive towards DEB, because their blood cultures showedlessthan 20% aberrant
cells after DEB exposure. The breakage rate in this group
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2235
DIAGNOSIS OF FANCONIANEMIA
Table 2. Resutts of Cell Cvcle Testing
Table 1. Results of Cytogenetic Testing
Sum of G1
Sum of S
Sum of G2
% Aberrant Cellst
Breaks/Cells
Aberrant Cell
Category
N
PhasesIGF
PhasedGF
PhasedGF
1-20
1
4-42
0.03-0.28
0.0-0.1
0.04-1.28
1.90-8.55
0.05-0.85
1.60-6.00
0.65-2.30
1.0-1.7
1.0-1.7
1.0-2.5
1.2-8.15
1 .O-1.8
1.2-6.2
1.8-2.8
Controls
G2G2'
30
43
23
0.43 f 0.06
0.45 ? 0.09
0.27 f 0.07+
0.40 2 0.06
0.40 f 0.07
0.31 ? 0.06*
0.17 ? 0.03
0.15 f 0.05
0.42 f 0.10*
Aberrationd
Category
N
42
0-1
42
Spont
MMC
38
DEB'S 54-1 19
17
Spont
MMC 56-1 13
DEB*§
5
DEB-
00
5-40
00
29-68
Seventy-two-hour lymphocytecultures were treated after 24 hours
with 0.1 pg/mL DEB. Standard chromosome preparations were scored
for structural chromosome aberrations. The results are ranges of values. Not all patient cultures yielded scorable metaphases in the last
two categories.
Abbreviations: DEB-, lack of DEB sensitivity; DEB+. DEB sensitive;
DEB*, borderline sensitive; MMC, MMC-induced breakage; Spont,
spontaneous chromosome breakage.
t Ranges of aberrations observed.
Includes 1 case with exceptional sensitivity (see Table 4, case no.
6).
5 Results of spontaneous and MMC-induced breakage are given in
Table 4.
*
was less than 0.28 breakdcell, and there were no cells with
more than 1.7 breaks. This group will be referred to as DEB-.
The cells of 19 patients proved sensitive towards DEB, as
evidenced by their elevated chromosomal breakage rates. In
these DEB-sensitive patients, an average of 88.6% aberrant
cells and an average of more than 5.63 breakskell were
found, including single metaphases with more than 10
breaks. This group will be referred to as DEB+ (Table 1).
The cytogenetic classification of 5 of the 66 patients proved
difficult. These patients were classified as FA-like, because
their cells showed a significant increase of chromosomal
instability after exposure to DEB. However, their values
remained well below the level of 80% to 100% aberrant cells
typically found in FA cultures. Moreover, not a single cell
with more than 10 aberrations was found in this group. These
patients will be referred to as DEB'. More details on these
patients will be given in Table 4 (see below). A cytogenetic
result was not obtained in all untreated (spontaneous) or
MMC-treated cultures. However, the data in Table 1 show
that DEB+ cases did not overlap with DEB- cases in their
percentages of aberrant cells after MMC exposure. This
finding also held for the category breaks per cell, but was not
true for the untreated cultures. The evaluation of spontaneous
breakage obviously does in no way suffice for the distinction
between FA and non-FA patients.
Cellcycletesting.
The results of cell cycle testing are
summarized in Table 2. The cell cycle distributions of patients and controls are presented as cumulative proportions
of cells within each of three cell cycle compartments (GI,
S, and G2) of four consecutive cell cycles. Because the
fraction of noncycling cells (GO) varies greatly among individuals (eg, as a function of donor age), cumulative cell
cycle distributions were calculated relative to the growth
fraction. The growth fraction (GF) was defined as the sum
of all proliferating cells except the nonproliferating GO phase
Cell cycle analysis was performed on72-hour lymphocyte cultures.
Proliferation-independent parameters were calculated as a ratio of
the sum of the respective cell cycle compartments versus the growth
fraction. The values are mean f SD.
P < .0001 (Student's t-test).
cells. Table 2 shows that the cell cycle parameters of 43 of
the 66 patients did not deviate significantly from those of
30 healthy controls. Specifically, there was no evidence for
G2 phase blockage (sum of G2 phases/GF). This group of
patients will be referred to as G2-. The lymphocyte cultures
of each of the remaining 23 patients showed a marked shift
of their cell cycle distributions. In addition to deviations
affecting the G1- and S-phase compartments, there were
highly significant accumulations of cells in the G2-phase
compartments. Such G2-phase accumulations were described previously for patients with Fanconi
The
group of 23 patients exhibiting a disturbed cell cycle distribution will be denoted as G2+.
Correlationbetween DEB and cell cycletesting.
Because the primary emphasis of this study was onthe comparison between DEB and cell cycle testing, only those results
are shown in Table 3. DEB- patients were, as a rule, G2(1 exception), whereas DEB+ patients were G2+ (2 exceptions). All 5 DEB' cases showed G2-phase blockage. In the
DEB+ group, the degree of DEB sensitivity (expressed as
breaks per cell) was weakly correlated to the extent of G2
phase blockage ( r = .36). However, this weak correlation
was caused by a single patient whose lymphocytes were both
highly DEB sensitive and G2 arrested. When this patient's
data were omitted from the regression analysis, chromosome
breakage rates and the extent of G2-phase blockage were
unrelated ( r = M).
Exceptional cases. Table 4 lists clinical, cytogenetic,
andflow cytometric details of exceptional cases. None of
these patients displayed radial ray defects, hyperpigmentation, or other dysmorphic features, except microcephaly (patients no. l and 2). The group of DEB' (borderline-positive)
patients consisted of 2 patients with the clinical diagnosis
Table 3. Correlation Between DEB and Cell Cycle Testing in 66
Patients With Clinical Suspicion of FA
G2-
DEBDEB+
DEB*$
41
2t
-
G2+
It
17
5
Sixty-six patients with clinicalsuspicion of FA were tested in parallel
for DEB sensitivity and cell cycle disturbance. The numbers of patients
in each category is listed.
t Patients no. 6 through 8, Table 4.
Borderline positive; patients no. 1 through 5, Table 4.
*
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SEYSCHAB ET AL
2236
Table 4. Exce@ional Cams: Clinical, Cytogenetic, andFlow Cytometric Details
Case
No.
Clinical Information
Culture
Transient thrombocytopenia.
microcephaly, A A ?
Anemia, thrombocytopenia,
microcephaly
Anemia, thrombocytopenia;
BMT+
Mild anemia,
thrombocytopenia; twin
brother to no. 5
AA (deceased); twin brother
to no. 4
6
AA, ALL (deceased)
7
MDS,
AML?
8
(deceased)
AA, ALL; BMT'
Lymphocyte
Test
% Aberrant
Cells
Spont 1
Spont
0.09 2
MMC 1
MMC 2
DEB 1
DEB
0.90 2
Spont
0.56
MMC
DEB
Spont
MMC
DEB
Spont 1
Spont 2
MMC
DEB 1
DEB 2
Spont 1
Spont 2
MMC
DEB 1
DEB 2
Spont
MMC
DE B
Spont 1
Spont 2
MMC
DEB
Spont
DEB
5
6
48
14
33
32
38
44
50
3
28
29
12
5
54
12
64
11
11
52
10
68
85
100
0.92
1.40
0.03
0.56
0.65
0.13
0.05
1.90
0.16
1.98
0.13
0.12
1.60
0.40
2.30
2.85
10.0
+++
+++
64
100
5
10
BreaksKell
Aberrant
0.05
1.20
0.22
1.10
2.20
7.20
0.05
0.14
Aberrations1
Cell
1.o
1.3
1.8
1.3
2.3
2. l
1.5
2.0
2.8
1.o
1.8
1.8
1.1
1.o
3.2
1.3
2.2
1.2
1.1
2.3
1.1
2.6
35.0
10.0
++c
2.8
5.8
1.o
1.2
GF
Flow Cytometric
Classification
DEB*
0.478
G2'
DEB*
0.400
G2'
DEB*
0.347
G2'
DEB*
0.397
0.373
G2'
DEB*
0.516
0.435
G2
DEB"'
0.097
G2-
DEB-
0.305
0.212
G2'
G2-
DEB-
0.319
G2'
Cytogenetic
Classification
X21
Detailed clinical, cytogenetic and flow cytometric data from the exceptional cases found in this study.
Abbreviations: AA, aplastic anemia; AML, acute myeloid leukemia; BMT', successful bone marrowtransplantation; DEBX, borderline sensitivity
to DEB, atypical Fanconi anemia (FA-like syndrome); DEB+, sensitive to DEB (classical FA); DEB-, not sensitive to DEB (non-FA); DEB"',
extremely sensitive to DEB (no quantitative evaluation possible); G2'. G2-phase blockage (typical for FA); G2-, no GP-phase blockage (non-FA);
ZG2/GF, sum of G2 phases over growth fraction; MDS, Myelodysplastic syndrome; Spont, spontaneous chromosome breakage.
of anemia and thrombocytopenia (patients no. 2 and 3) and
3 patients with the diagnosis of incipient aplastic anemia
(patients no. 1,4,and 5). In contrast to their borderline DEB
and MMC sensitivities on repeated testing, each of these
patients displayed unequivocal evidence for GZphase
blockage. Table 4 also includes data on the 3 patients in this
studyin whom DEB and cell cycle testing were clearly
discordant (see Table 3). The clinical parameter common to
these three discordant individuals (patients no. 6 through 8)
was the presence of hematoproliferative disease, although
the presence of acute lymphoblastic leukemia (ALL) is not
typical for FA.'4 Whereas patients no. 6 and 7 experienced
a rapid progression of their disease precluding any further
cytogenetic or cell genetic testing, patient no. 8 is alive and
well after a successful bone marrow transplant.
DISCUSSION
There is an impressive body of evidence that substantiates
the validity of the DEB test for the diagnosis of Fanconi
anernia.I5 Indeed, the DEB test undoubtedly represents the
gold standard against which any new test must be measured.
Despite this excellent record, the DEB test isnot trivial
because it requires a high degree of cytogenetic expertise
and meticulous attention to cell culture and safety conditions.
These limitations have motivated several groups to look for
alternatives to cytogenetic testing. One such alternative is
the cell cycle test, which is based on the observation that
FA cells experience difficulties in traversing the S and G2
compartments of the cell cycle.6~"~'6
A number of reports
describe the diagnostic potential of the cell cycle test for
Fanconi anemia, 13.17-20but this is the first study that blindly
compares cell cycle and DEB testing.
Our comparative study shows a remarkable concordance
between the results of parallel DEB and cell cycle testing in
patients with clinical suspicion of FA. A concordant result
was obtained in 63 of 66 cases. All 3 discordant cases, but
only 1 of the 63 concordant cases had clinical evidence for
overt leukemia. It is conceivable that leukemic cells lack the
cell cycle disturbance that is characteristic of nonneoplastic
FA cells. The selective growth advantage of leukemic cells
would be difficult to reconcile with a severe inhibition of
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DIAGNOSIS OF FANCONIANEMIA
cell cycle progression. We conclude from these results that
both increased DEB sensitivity and a cell cycle disturbance
should be considered as consistent manifestations of the FA
gene defect(s) in nonleukemic peripheral blood mononuclear
cells.
A similar high degree of concordance between cell cycle
and cytogenetic results was reported by Berger et al, 2o who
used nitrogen mustard in both cytogenetic and cell cycle
testing. More importantly, these investigators also failed to
obtain concordance in 3 patients with myelodysplasia or
overt leukemia. The presence of hematoproliferative disease
therefore imposes a definitive limitation on the cell cycle
test.
Given the likely extent of genetic heterogeneity in FA,
the high rate of concordance between DEB+ and G2+-positive cases is rather impressive. In our cohort of 66 patients,
the DEB test proved more sensitive than the cell cycle assay
with regard to uncovering possible genetic heterogeneity,
because 5 of the 24 DEB-positive patients showed unusually
low levels of DEB sensitivity. These patients may represent
a special complementation group or a subgroup denoting an
FA-like entity. The course of their disease differs somewhat
from classical FA in that their hematologic symptoms were
less severe and progressed very slowly, if at all. Because a
G2-phase cell cycle disturbance was observed in all of these
patients, DEB testing seems to single out subgroups of patients with atypical FA or FA-like disease that would have
been missed by the cell cycle studies alone. On the other
hand, the cell cycle disturbance may precede the onset of
severe aplastic anemia, as has been observed previously in
a 2-year-old child.”
In summary, our study confirms that both analytical methods can reliably differentiate between FA and non-FA cells.
Cultured lymphocytes with elevated sensitivity towards DEB
will display a G2-phase cell cycle defect and vice versa.
However, this holds only for nonleukemic cells. Despite this
limitation, we still consider the cell cycle test a valuable
adjunct to the differential diagnosis of childhood anemias.
Owing to its speed and simplicity, the cell cycle test permits
a far more liberal screening of patients withany type of
dysmorphia or hematologic disturbance that could herald
Fanconi anemia. In particular, the many patients carrying the
clinical diagnosis of aplastic anemia could be prescreened
by cell cycle testing. Confirmatory DEB studies would be
required only in cases with evidence for G2-phase arrest.
Used in such a sequence, cell cycle and DEB testing offer
a fast and economical approach towards the definitive diagnosis of a disease whose clinical presentation is quite variable.’ The unambiguous classification at the level of the
cellular phenotype provides the only solid basis for current
efforts to identify complementation groups and to characterize their underlying genetic defect(s) at the molecular level.
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1995 85: 2233-2237
Comparative evaluation of diepoxybutane sensitivity and cell cycle
blockage in the diagnosis of Fanconi anemia
H Seyschab, R Friedl, Y Sun, D Schindler, H Hoehn, S Hentze and T Schroeder-Kurth
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