Download Supplementary Materials and Methods. Patient description. A 6

Supplementary Materials and Methods.
Patient description. A 6-month-old male was hospitalized with vomiting, a cough,
fever, and hepatosplenomegaly. Fever (38-40oC), severe pallor of the skin and
mucosa, tachycardia, systolic breath, distended abdomen, splenomegaly (8 cm), and
hepatomegaly (10 cm) were noted at physical examination. Blood parameters were:
white blood cells (WBC) 35.3 x 109/L (neutrophils: 23%, lymphocytes: 37%,
monocytes: 2%, basophils: 38%), hemoglobin (Hb) 5.4 g/dL, platelets (PLT) 168 x
109/L. Myeloblastic morphology was assessed. The bone marrow aspirate was
hypercellular, with 90% blasts. AML-M5 was diagnosed. Metaphase spreads from
bone marrow cells were obtained: 30 metaphases were analyzed and a
t(X;6)(p11.2;q23) was observed in all of them as the sole abnormality.
Treatment with AraC, VP16 and daunorubicin was started, but the patient showed
serious side effects associated with the chemotherapy. A second cycle with
prednisone, tioguanine, vincristine and adriamicine was started, with no response to
the treatment. Several complications (pneumonia, anemia, and thrombocytopenia)
arose; the patient died after 2 months, because of cardiorespiratory failure, secondary
to pneumonia.
Rearrangement mapping and cloning. Fluorescence in situ hybridization (FISH)
experiments were performed on metaphase spreads from the patient’s bone marrow
aspirate, prepared according to standard procedures (2). Genomic clones that mapped
to
the
Xp11.2
and
6q23
(http://www.chori.org/bacpac/)
regions
library.
were
Mapping
selected
results
from
are
the
RPCI
summarized
in
Supplementary Figure 1i and ii. The result of a representative FISH experiment is
pictured in Supplementary Figure 1iii (see below). More specifically:
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Clones’ names are as follows: 1. RP11-149B9; 2. RP11-119E20; 3. RP11167P23; 4. RP11-465E19; 5. RP5-1158B12; 6. RP11-349J5; 7. RP1-32B1; 8.
RP11-12A2; 9. RP11-331O9.
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RACE-PCR and RT-PCR experiments were performed with the SMARTTM
RACE cDNA AMPLIFICATION KIT (Clontech, Palo Alto, CA), according
to manufacturer’s directions (data not shown). All RACE and RT-PCR
products were cloned using the Topo-TA cloning kit (Invitrogen, Carlsbad,
CA) and sequenced.
-
Mapping details:

Supplementary Figure 1i depicts a portion of the Xp11.2 region, from
28 to 58Mb, which is schematically represented by a black horizontal
line; numbers indicate position along the chromosome (Mb);

Supplementary Figure 1ii shows a portion of the 6q23 region, from
135 to 154Mb, which is schematically represented by a black
horizontal line; numbers indicate position along the chromosome
(Mb);

Supplementary Figure 1iii represents the result of FISH experiments
with the genomic clone RP11-32B1 on bone marrow metaphase
spreads from our patient [because this clone spans the breakpoint, three
fluorescent signals can be seen on metaphases carrying the
rearrangement, recognizing the normal chromosome 6 (indicated with
1, in Supplementary Figure 1iii) as well as the derivative chromosomes
6 (2) and X (3)].
Involved transcripts. Figure 1i reports the structure of the MYB (blue), GATA1 (red),
and MYB-GATA1 transcripts. MYB: Blue boxes represent the 15 exons. The black
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arrow in exon 1 indicates the transcription start site. The light blue box and the gridpattern box represent the 5’ and 3’-UTRs, respectively. Functional domains, along
with the corresponding coding exons, are indicated as follows: R1-3 = three
regulatory DNA-binding domains; TAD = transactivating domain; NRD = negative
regulatory domain. GATA1: Red boxes represent the 6 exons. The black arrow in exon
2 indicates the transcription start site. The light red box and the grid-pattern box
represent the 5’ and 3’-UTRs, respectively. The 2 zinc-finger DNA-binding domains
of the gene along with the corresponding coding exons are indicated. MYB-GATA1:
the fusion transcript is generated by MYB exons 1-8 and GATA1 exons 5-6, the
retained functional domains of the original genes are indicated.
Quantitative PCR (Taqman assay). Figure 1ii reports the results of the quantitative
PCR experiments. Samples were normalized on 18S and GADPH genes. Assay
sequences are available upon request. Total RNA from normal bone marrow (BM) of
an adult healthy donor was used as calibrator. Probe GATAe1-2 suggests the presence
of a truncated GATA1 (exons 1-4), lacking the second zinc-finger domain, while
complete loss of the wt GATA1 allele is indicated by the GATAe4-5. Probe MYBe8-9
detects wt MYB only, which is over-expressed. Both probes MYBe2-3 and GATAe56 detect the MYB-GATA1 transcript, which has levels higher than the typical MYB and
GATA1 levels in normal bone marrow.
The reactions were performed on the ABI/Prism 7900 HT Sequence Detector System
(Applied Biosystems), using a pre-PCR step of 10 min at 95°C, followed by 40 cycles
of 15 sec at 95°C and 60 sec at 60°C. cDNA (5ng) was amplified (in triplicate) in a
reaction volume of 15 µL containing 7.5µl of TaqMan PCR Mastermix 2x No UNG
(Applied Biosystems, Foster City, CA) and 0.75 µl of TaqMan Gene expression assay
20x (both Applied Biosystems, Foster City, CA).
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Transduction and transplantation of lin- cells. The MYB-GATA1 fusion transcript
was cloned into the PINCO retroviral vector (PINCO-MYB-GATA1). An empty
vector was used as a negative control in all experiments (PINCO-ev). Lin– cells from
wild-type (WT), Tp53 null (p53–/–), or GATA1low mice were prepared and infected:
transduction efficiency was 50% for PINCO-MYB-GATA1 and 80% for PINCO-ev.
Infected cells were then selected for GFP as previously described (2). WT, syngeneic
recipient mice (C57BL/6) were lethally irradiated with 9 Gy and reinoculated
intravenously with 300,000 WT, p53–/– or GATA1low donor lin– cells, infected with
either one of the two constructs. 500,000 spleen cells obtained from an untreated WT
mouse (C57/BL6, as the WT recipients) were concomitantly inoculated into the
recipient irradiated mice. Transduced mice are indicated as follows: WT-PINCO-ev
and WT-PINCO-MYB-GATA1; p53-/-PINCO-ev and p53-/-PINCO-MYB-GATA1;
GATA1low-PINCO-ev and GATA1low-PINCO-MYB-GATA1. Animals were checked
periodically for clinical signs of disease. Peripheral blood smears were stained with
May-Grünwald-Giemsa (MGG), and blood counts were performed on a COULTER
AC.T 5diff hematology cell counter (Beckman Coulter). All mouse work was
performed in accordance with national guidelines and was approved by the
Institutional Review Board of the European Institute of Oncology.
Blood smears analysis. Analyses were performed at successive time points (6, 11, 13,
and 15 months) following injection of lin- cells (Figure 2i). A normal distribution of
hematopoietic lineages through time is evident in mPev mice (top part). Progression
towards overt acute leukemia, instead, is present in the mMG1 mouse, with MDS-like
features at 11 months (presence of dysplastic granulocytes) and clear leukemic
phenotypes at later time points (accumulation of myeloid blasts, with morphologically
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normal granulocytes at 13 months, as well as blast accumulation in the absence of
myeloid differentiation at 15 months).
Immunophenotypic characterization. Flow cytometry was performed on peripheral
blood and bone marrow cells. Staining was conducted with: CD38FITC/Sca-1PE/7AAD/Gr1PeCy7/CD8APC/CD45APC-Cy7;
AAD/Gr1PeCy7/CD11bAPC/CD45APC-Cy7;
CD41FITC/TER-119PE/7CD4FITC/CD117PE/7-
AAD/CD19PeCy7/CD8APC/CD45APC-Cy7; CD38FITC/Sca-1PE/7-AAD/CD34APC/CD45APCCy7
; antibodies (all monoclonal) were purchased from BD Biosciences (Mountain
View, CA), and used at the recommended concentration. Controls included
substitution of the relevant monoclonal antibody by IgG of the same isotype.
Following NH4Cl red cell lysis, acquisition was performed using a six-colour flow
cytometer (Facs-CANTO, BD-Bioscences).
Serial transplantation. 15-20x106 spleen cells from leukemic mice were injected in
the tail vein of C57BL/6 recipient mice at each passage (4 mice at the 1 st passage, 8 at
the 2nd, 4 at the 3rd). Mice were monitored for clinical signs of the disease.
MYB-GATA1 RT-PCR analysis. MYB-GATA1-specific RT-PCR reactions were
performed in the GATA1low-PINCO-MYB-GATA1 leukemic mouse (mMG1). RNA
was extracted from spleen cells using the Illustra RNA-spin isolation KIT (GE, Little
Chalfont, UK), according to manufacturer’s instructions. cDNA was prepared with
the SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA), following
manufacturer’s instructions. Primers sequences used for the amplification of the
fusion transcript are as follows: MYB: 5’-AGCCCACTGTTAACAACGACT-3’ and
GATA: 5’-CATTTCTCCGCCACAGTGTC-3’. Following the PCR reaction, PCR
products have been cloned into a TOPO-TA vector (Invitrogen, Carlsbad, CA, USA).
Transformation was performed, according to the instruction given by the
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manufacturer, into competent cells provided by the TOPO-TA kit (Invitrogen,
Carlsbad, CA, USA). Colonies were grown, colony PCR was performed using T7 and
m13rev primers (provided by the kit), and the PCR products were sequenced. 6/24
products corresponded to the fusion transcript, the other clones representing products
derived from unspecific annealing of the primers.
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