A novel form of human STAT1 deficiency impairing early but not late

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A novel form of human STAT1 deficiency impairing early but not late responses
to interferons
Xiao-Fei Kong,1-4 *Michael Ciancanelli,1 *Sami Al-Hajjar,5,6 Laia Alsina,7 Timothy Zumwalt,7 Jacinta Bustamante,2,3
Jacqueline Feinberg,2,3 Magali Audry,1 Carolina Prando,1 Vanessa Bryant,1 Alexandra Kreins,1,8 Dusan Bogunovic,1
Rabih Halwani,5 Xin-Xin Zhang,4 Laurent Abel,1-3 Damien Chaussabel,7 †Saleh Al-Muhsen,5,6 †Jean-Laurent Casanova,1-5,9
and †Stéphanie Boisson-Dupuis1-3
1St Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY; 2Laboratory of Human Genetics of
Infectious Diseases, Necker Branch, U980, Inserm, Paris, France; 3University Paris Descartes, Necker Medical School, Paris, France; 4French-Chinese
Laboratory of Genetics and Life Science, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, People’s Republic of China; 5Prince Naif
Center for Immunology Research, Department of Pediatrics, College of Medicine, King Saud University, Riyadh, Saudi Arabia; 6Department of Pediatrics, King
Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia; 7Baylor Institute for Immunology Research, Dallas, TX; 8Graduate Program of
Immunology and Microbial Pathogenesis, Weill Cornell Graduate School of Medical Sciences, New York, NY; and 9Pediatric Immunology-Hematology Unit,
Necker Hospital, Paris, France
Autosomal recessive STAT1 deficiency is
associated with impaired cellular responses to interferons and susceptibility
to intracellular bacterial and viral infections. We report here a new form of partial
STAT1 deficiency in 2 siblings presenting
mycobacterial and viral diseases. Both
carried a homozygous missense mutation replacing a lysine with an asparagine
residue at position 201 (K201N) of STAT1.
This mutation causes the abnormal splicing out of exon 8 from most STAT1
mRNAs, thereby decreasing (by ⬃ 70%)
STAT1 protein levels. The mutant STAT1
proteins are not intrinsically deleterious,
in terms of tyrosine phosphorylation, dephosphorylation, homodimerization into
␥-activating factor and heterotrimerization into ISGF-3, binding to specific
DNA elements, and activation of the transcription. Interestingly, the activation of
␥-activating factor and ISGF3 was impaired only at early time points in the
various cells from patient (within 1 hour
of stimulation), whereas sustained impairment occurs in other known forms of
complete and partial recessive STAT1
deficiency. Consequently, delayed responses were normal; however, the early
induction of interferon-stimulated genes
was selectively and severely impaired.
Thus, the early cellular responses to human interferons are critically dependent
on the amount of STAT1 and are essential
for the appropriate control of mycobacterial and viral infections. (Blood. 2010;
Interferons (IFNs) are important mediators of immunity.1 STAT1
plays an important role in mediating the physiologic and therapeutic effects of IFNs in humans.2-4 Human STAT1 deficiency was first
described in patients with Mendelian susceptibility to mycobacterial diseases (MIM209950),5-7 which is characterized by clinical
phenotypes of recurrent and/or disseminated disease caused by
weakly virulent mycobacteria in otherwise healthy patients.8,9 Ten
known patients with partial dominant STAT1 deficiency have been
shown to carry a heterozygous mutation impairing either tyrosine
phosphorylation (L706S; Figure 1A)9 or DNA-binding activity
(E320Q and Q463H, Figure 1A).8 Cells with these heterozygous
alleles displayed an impaired response to IFN-␥ in terms of the
amount of bioactive ␥-activating factor (GAF) produced. The
dominant negative effect of the heterozygous mutations identified
results from the inability of homodimers containing a mutant
STAT1 molecule to enter the nucleus or to bind the consensus GAS
element. By contrast, dominant STAT1 deficiency does not impair
cellular responses to IFN-␣/␤. Its clinical penetrance is incomplete,
as only 6 patients have developed mycobacterial diseases, heterogeneous in their nature, although all patients were cured with
antibiotic treatment.8,9
We also identified 3 patients with complete recessive STAT1
deficiency carrying 2 null alleles, resulting in a complete lack of
STAT1 expression and abolition of the STAT1-dependent responses to both IFN-␥ and IFN-␣/␤ (through GAF and interferon-stimulated gene F3 [ISGF3]; Figure 1A).10,11 The
STAT1-dependent cellular responses to IFN-␭ and interleukin-27
(IL-27) are also abolished in these patients. All patients developed
severe bacillus Calmette-Guérin (BCG) infection after vaccination
and viral infections, and all died between the ages of 11 and
16 months.10,11 Two siblings with partial recessive STAT1 deficiency were recently reported to carry an exonic splicing enhancer
mutation (P696S, Figure 1A), leading to aberrant mRNA splicing,
resulting in the production of only approximately 10% the normal
Submitted April 16, 2010; accepted September 7, 2010. Prepublished online as
Blood First Edition paper, September 14, 2010; DOI 10.1182/blood-2010-04280586.
*M.C. and S.A.-H. contributed equally to this study and are considered
co–second authors.
†S.A.-M., J.-L.C., and S.B.-D. contributed equally to this study and are
considered co–senior authors.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2010 by The American Society of Hematology
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KONG et al
Figure 1. The STAT1 K201N mutation caused susceptibility to mycobacterial and viral infections. (A) The human STAT1 coding region with its known mutations. Coding
exons are numbered with Roman numerals and delineated by vertical bars. The N-terminal domain, coiled-coil domain, DNA-binding domain, linker domain, SH2 domain, tail
segment domain (TS), and transactivation domain (TD) are indicated, together with their amino acid boundaries. Phosphorylation sites, Tyr 701 (pY) and Ser 727 (pS), are
indicated. Mutations in red are recessive mutations associated with complete STAT1 deficiency. Mutations in green are heterozygous mutations associated with partial
dominant STAT1 deficiency. Mutations in blue are recessive mutations associated with partial recessive STAT1 deficiency. The mutation reported here is indicated in italics.
(B) The pedigree, phenotype, and genotype of the consanguineous kindred from Saudi Arabia. The proband, P1, II.1 is indicated by an arrow, and presented with disseminated
M avium infection and disseminated varicella. II.3 had disseminated BCG infection and died of septic shock at the age of 3. These 2 patients are referred to as P1 and
P2, respectively. I.1 and I.2 are first cousins. (C) Genomic sequence analysis of exon 8 showing a homozygous G 3 T mutation in P1, leading to the replacement of a lysine
residue by an asparagine residue (K201N/K201N) at position 201 of the protein.
amount of STAT1 protein. However, the mutant STAT1 protein
produced functions normally in terms of phosphorylation, dimerization, and DNA binding. Impaired, but not abolished, responses to
IFN-␣/␤, IFN-␥, IFN-␭, and IL-27 render the patients with this
deficiency susceptible to severe intracellular bacterial and multiple
viral infections.12 Both early and late IFN responses are impaired in
these siblings. We report here the clinical and biologic findings for
2 other siblings from an unrelated consanguineous family with a
new form of partial recessive STAT1 deficiency resulting in severe
impairment of the early, but not late, responses to IFNs.
We investigated 2 siblings (P1 and P2) with mycobacterial diseases from a
consanguineous family originating from and living in Saudi Arabia. P1 did
not have BCG vaccination and developed disseminated mycobacterial
infection at the age of 6 years (positive acid-fast bacillus staining on an
axillary lymph node biopsy). He was then treated with intermittent
isonicotinic acid hydrazide, rifampicin, pyrasinamide, and ethambutol for
12 months with moderate improvement. One year later, he was referred to
King Faisal Specialist Hospital and Research Center with multiple large
cervical lymph nodes more than 1 cm in diameter and a 3-cm ⫻ 3-cm
draining abscess on the scalp. Computed tomography scan showed diffuse
neck nodal diseases, bilateral chest nodular opacities, and multiple calvarial
lytic lesions with subgaleal inflammatory fluid loculation. Mycobacterium
avium was cultured from multiple tissues. The patient’s condition improved
significantly, with resolution of the scalp lesion and cervical lymph nodes
after treatment with cycloserine, ethionamide, ethambutol, and moxifloxacin, according to the results of drug susceptibility tests. At the age of
8 years, P1 developed disseminated varicella, requiring intensive care,
which improved after antiviral treatment. He then suffered from Candida
parapsillosis sepsis resulting from central venous line contamination,
which was resolved by removal of the line and antifungal treatment. At the
age of 9 years, P1 presented persistent vomiting and intermittent seizures
with progressive spasticity. MRI showed multiple vasogenic edemas
involving both cerebral hemispheres and multiple enhancing lesions
throughout the brain parenchyma. M avium was again cultured from brain
tissue, with the same pattern of drug susceptibility. Linezolid treatment was
initiated, together with high-dose IFN-␥. The patient’s condition stabilized
and the seizures stopped, but he became blind. He remains hospitalized,
with severe sequelae. P1 has normal white cell counts with normal CD4⫹
and CD8⫹ populations. His immunoglobulin level is normal, with IgG
antibodies against cytomegalovirus, Epstein-Barr virus (EBV), and Toxoplasma gondii. P2 was the younger sister of P1. She received BCG
vaccination after birth and developed disseminated BCG infection. She died
at the age of 3 years, from septic shock. The patients live and were followed
up in Saudi Arabia, where their informed consent was obtained in
accordance with local regulations, with approval from the Institutional
Review Board. The experiments described here were conducted in the
United States, according to the local regulations and with approval of the
Institutional Review Board of Rockefeller University.
Cells, plasmids, and reagents
EBV-transformed B cells (EBV-B), SV40-transformed fibroblasts
(SV40-fibroblasts), 293T cells, and STAT1-deficient U3C cells were
cultured as previously described.12 Stimulations were performed with the
indicated doses of IFN-␥ (Imukin; Boehringer Ingelheim), IFN-␣
2b (IntronA, Schering Plough), IL-27 (R&D Systems), and IFN-␭ (R&D
Systems). Genomic DNA and cDNA were amplified and sequenced with
STAT1-specific primers. Primer sequences and polymerase chain reaction
(PCR) conditions are available on request. We used 3 different STAT1
expression vectors: pcDNA3.1 STAT1-V5, pcDNA3 STAT1-Flag, and
pcDNA6.0 STAT1-myc. The mutated K201N, E320Q, P696S, L706S,
⌬ex8, and F77A⫹F78A (F77A) STAT1 plasmids were obtained by
site-directed mutagenesis with the Quickchange kit (Stratagene). U3C cells
were transfected with a calcium phosphate–based transfection kit (Invitrogen). Immunoprecipitation and Western blotting were carried out as
previously described,13 with antibodies against Tyr-701-phosphorylated
STAT1 (612132, BD Biosciences Transduction Laboratories), the
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N-terminus of STAT1 (610116, BD Biosciences Transduction Laboratories;
H-95, Santa Cruz Biotechnology), the C-terminus of STAT1 (sc-345, Santa
Cruz Biotechnology), Tyr-690-STAT2 (sc-21689, Santa Cruz Biotechnology), STAT2 (sc-476, Santa Cruz Biotechnology), ␣-tubulin (Santa Cruz
Biotechnology), V5 (Invitrogen), Myc (clone 4A6, Sigma-Aldrich), and
Flag (Sigma-Aldrich). We added 500nM staurosporine (Calbiochem) to
cells after 30 minutes of IFN-␥ stimulation, for pulse-chase experiments
monitoring the kinetics of STAT1 Tyr-701 phosphorylation.
Determination of mRNA levels by quantitative real-time PCR
Total RNA was extracted with Trizol reagent (Invitrogen) from EBV-B cells
or SV40-fibroblasts left unstimulated or stimulated with 103 IU/mL IFN-␣
or IFN-␥ or with 100 ng/mL IL-27 or 20 ng/mL IFN-␭. The RNA was
treated with RNase-free DNase (Roche Diagnostics) and cleaned by
passage through an RNAeasy column (QIAGEN). Reverse transcription
was then carried out directly with random primers, using the TaqMan
Reverse Transcription kit (Applied Biosystems), for the determination of
and IFIT1 mRNA levels, using the TaqMan probes delivered by Applied
Biosystems for these genes. The results were normalized with respect to the
values obtained for the endogenous GUS cDNA. Transcription of the
STAT1 sequence carried by the plasmids used for transfection was
quantified with a pair of primers binding to the 5⬘-untranslated region of
pcDNA3.1 and exon 1 of STAT1, and the results were normalized with
respect to the levels of glyceraldehyde-3-phosphate dehydrogenase mRNA,
with the SYBR Green Master Mix (Applied Biosystems). Representative
results from 2 or 3 independent experiments were compared in unpaired,
2-tailed Student t tests.
Reporter assay and other
We used 300 ng GAS, IFN-stimulated response element (ISRE), IRF1,
IFIT1, IFIT2 firefly luciferase reporter plasmids, separately, to transfect
0.2 million U3C cells. The cells were also transfected with 300 ng wild-type
(WT) or mutant STAT1 expression plasmid and 30 ng of the Renilla
luciferase plasmid. We then stimulated cells, 24 hours after transfection,
with either 103 IU/mL IFN-␣ or 103 IU/mL IFN-␥. The cells were harvested
24 hours after stimulation. Luciferase levels were measured with the Dual
Luciferase assay, according to the manufacturer’s instructions (Promega).
The methods for exon trapping, electrophoretic mobility shift assay, flow
cytometric analysis, and viral assay are described in the supplemental data
(available on the Blood Web site; see the Supplemental Materials link at the
top of the online article).
Identification of the K201N STAT1 allele
We investigated a male proband (P1) presenting both mycobacterial and viral diseases (“Patients”). Sequencing the exons and
flanking intron regions of STAT1, we found a homozygous
nucleotide substitution at position 603 in exon 8 (G 3 T) in
genomic DNA from the patient’s leukocytes (Figure 1A-C). This
substitution replaces a lysine with an asparagine residue (K201N)
in the coiled-coil region of STAT1. No other STAT1 mutations were
found. Genomic DNA sequencing showed that his younger sister
(P2), who had BCG-osis, also carried the homozygous K201N
mutation (data not shown). Both parents were healthy and heterozygous for this mutant allele, and the 2 healthy siblings carried at least
one WT allele, consistent with autosomal recessive segregation.
We excluded the possibility that this mutation was a common or
irrelevant polymorphism by sequencing 200 controls from the
same ethnic group. In addition, alignment of the human STAT1
sequence with those from 23 animal species present in databases
showed the K201 residue to be strictly conserved throughout
evolution. These results suggest that the K201N STAT1 allele
is responsible for the autosomal recessive STAT1 deficiency in
this family.
The K201N mutation causes abnormal STAT1 mRNA splicing
We carried out reverse-transcribed (RT)-PCR to study the STAT1
mRNAs in EBV-B cells and SV40-fibroblasts from a healthy
control person (WT/WT), P1 (K201N/K201N), a healthy heterozygous relative (K201N/WT), and previously described patients with
partial recessive STAT1 deficiency (P696S/P696S)12 or complete
STAT1 deficiency (1928insA/1928insA).10 As expected, 2 different
STAT1 transcripts, STAT1␣ and STAT1␤, were amplified from
healthy control cells, whereas a product of lower molecular weight
(MW) was obtained for STAT1␣ from P696S/P696S cells (Figure
2A). RT-PCR amplified 2 fragments each for STAT1␣ and STAT1␤
from P1 cells: one fragment with the expected MW and another
with a lower MW than the WT mRNA (Figure 2A). Both fragments
were sequenced, and the smaller fragment was found to lack exon
8, whereas the fragment of normal MW corresponded to the
full-length STAT1 gene, including the K201N mutation in exon
8 (Figure 2B). Amplification of the STAT1 mRNA fragment
extending from exon 7 to exon 10 confirmed that exon 8 was
spliced out by an abnormal splicing process, which was also
observed in healthy heterozygous relatives (K201N/WT). Unlike
the previously reported P696S mutation, which leads to exon
23 being spliced out of STAT1␣, but not out of STAT1␤, the K201N
mutation impairs the splicing of both STAT1␣ and STAT1␤
mRNAs. For confirmation of the role of the K201N mutation in the
abnormal splicing of STAT1 mRNA, we transfected HEK293T and
COS-7 cells with the exon trapping pSPL314 mock vector, or with
pSPL3 containing the STAT1 genomic region, including exons
8 and 9 and their surrounding introns, with or without introducing
the K201N mutation (Figure 2C). In both cell types used, the
pattern of splicing differed between the WT and K201N STAT1
alleles. A lower MW product, from which exon 8 had been
eliminated by splicing (Figure 2D-E), was clearly detectable in
cells transfected with the K201N allele and only barely detectable
in cells transfected with the WT allele, whereas the normally
spliced product of the expected MW was barely detectable in
K201N-transfected cells. These results suggest that the K201N
mutation is responsible for the splicing out of STAT1 exon 8 in both
the STAT1␣ and STAT1␤ mRNAs, with residual, leaky splicing in
of exon 8 (resulting in ⬃ 30% the normal amount of full-length
mRNAs, as shown by TA cloning of RT-PCR products, data not
shown). Consistent with this hypothesis, in silico analysis predicted
the creation of a new binding site for ETR-3 (data not shown) by
the K201N mutation, and ETR-3 has been reported to activate exon
STAT1 expression and phosphorylation
The splicing out of exon 8 (92 nucleotides) induces a frameshift
and creates a premature stop codon at position 582, potentially
resulting in the production of a truncated STAT1 protein consisting
of only the first 194 N-terminal amino acids. To investigate whether
such a truncated protein could actually be produced, we generate a
plasmid construct containing the STAT1 with exon 8 deleted
(⌬ex8). We transfected 293T and U3C cells with this construct and
with a WT construct. We analyzed the results, comparing them with
those from P1’s EBV-B cells, by Western blotting with a specific
antibody recognizing the N-terminal part of STAT1 (amino acids
69-169). A truncated protein was produced in 293T cells but was
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KONG et al
Figure 2. Abnormal mRNA splicing resulting from K201N. (A) RT-PCR of a full-length STAT1␣, STAT1␤, STAT1 fragment running from exon 7 to exon 10, and
glyceraldehyde-3-phosphate dehydrogenase from mRNA extracted from the EBV-B cells of a healthy control (WT/WT), P1, and persons with the following genotypes:
K201N/WT, P696S/P696S, and 1928insA/1928insA (⫺/⫺). This result is representative of 3 independent experiments. H2O was used as a negative control for PCR.
(B) Schematic diagram of the STAT1␣ and STAT1␤ mRNA in the cells of P1. The upper band corresponds to the form with an MW identical to that of the WT mRNA, with normal
splicing and containing the K201N mutation (red line). The lower band was observed for the cells of P1 and corresponds to forms of the STAT1␣ and STAT1␤ mRNAs lacking
exon 8. The exons are numbered with Roman numerals. (C) Schematic diagram of the plasmid used for exon trapping. The genomic STAT1 region from nucleotides 15844 to
16416 (NC_000002) was inserted into the pSPL3 plasmid, between the XhoI and BamHI sites, with or without the K201N mutation (line in red). Exon 8 is shown in the green
box and exon 9 in the blue box. SD6 and SA2 primer positions are indicated. (D) HEK293T and COS-7 cells were transfected with no vector (⫺), pSPL3 mock vector
(M), pSPL3 vector containing the WT STAT1 gene (WT), and pSPL3 vector containing the K201N-mutated STAT1 gene (K201N). RT-PCR was carried out to amplify the
splicing products 24 hours after transfection. (Top panel) PCR with the SD6 and SA2 primers. (Bottom panel) PCR with the glyceraldehyde-3-phosphate dehydrogenase
primers. This result is representative of 2 independent experiments. (E) Schematic diagram of the 3 forms of mRNA splicing products: (i) exon 8 and exon 9 plus vector
sequence; (ii) exon 9 plus vector sequence; and (iii) 263-bp product corresponding to exonic sequence in the vector.
not detectable in U3C cells or in EBV-B cells from P1 (supplemental Figure 1). To assess the expression of full-length STAT1 protein,
we carried out Western blotting on lysates from EBV-B cells and
SV40-fibroblasts from P1 (Figure 3A and data not shown), together
with cells from one healthy control, and from other patients with
partial (P696S/P696S) or complete STAT1 deficiency. Using an
antibody recognizing the C-terminal part of the STAT1␣ isoform,
we showed that STAT1 expression was impaired in the cells of
P1, which nonetheless produced more of this protein (30% of WT
levels) than P696S/P696S cells (10%; Figure 3A). On fluorescenceactivated cell sorter (FACS) analysis with an antibody recognizing
the N-terminus of STAT1, the specific mean fluorescence intensity
of STAT1 was lower in the cells of P1 than in WT/WT, WT/⫺, and
K201N/WT cells but was higher than that in P696S/P696S cells
(Figure 3B). Tyrosine 701-phosphorylated STAT1 levels in response to IFN-␥ and IFN-␣ stimulations in vitro were lower in the
cells of P1, as demonstrated by both Western blotting and FACS
(Figure 3A,C). The different genotypes of cells studied could be
ranked in descending order of Tyr-701-phosphorylated STAT1, as
follows: WT/WT ⬎ K201N/WT ⬎ WT/⫺ ⬎ K201N/K201N ⬎
P696S/P696S ⬎ ⫺/⫺ (Figure 3C). The levels of STAT1 phosphorylation were correlated with the amount of STAT1 protein expressed, suggesting that mutant STAT1 proteins underwent normal
phosphorylation on the Tyr701 residue. As a control, STAT2
phosphorylation levels in response to IFN-␣ stimulation were
found be similar in all the cells studied (Figure 3A). We then
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Figure 3. STAT1 protein expression is impaired in the cells of P1. EBV-B cells from WT/WT, P1, K201N/WT, 1928insA/WT (WT/⫺), P696S/P696S, and 1928insA/1928insA
(⫺/⫺) persons were stimulated with 105 IU/mL IFN-␣ or IFN-␥ or left unstimulated for 30 minutes. (A) Western blotting was carried out with an antibody against
Tyr701-phosphorylated STAT1 (P-STAT1), STAT1␣ (STAT1), Tyr690-phosphorylated STAT2 (P-STAT2), STAT2, and ␣-tubulin as a reference. (B) FACS analysis of intracellular
STAT1 levels with an antibody against the N-terminus of STAT1. Solid area represents isotype control antibody. Solid line indicates STAT1 antibody. (C) FACS analysis of
intracellular Tyr701-phosphorylated STAT1 levels in untreated cells (NS) or in cells treated with 105 IU/mL IFN-␣ or IFN-␥ for 30 minutes. Solid area represents untreated cells.
Thin line indicates 105 IU/mL IFN-␥ stimulation; and thick line, 105 IU/mL IFN-␣ stimulation. Mean fluorescence intensity is indicated. The results are representative of
3 independent experiments.
transfected STAT1-deficient U3C cells with the STAT1 K201N
allele to compare the stability and phosphorylation of the mutant
protein with those of the WT STAT1. The mutant K201N STAT1
was produced in normal amounts and phosphorylated on tyrosine
701 (supplemental Figure 2). The K201N mutation caused the
abnormal splicing out of exon 8 in most mRNAs, resulting in the
impairment, but not total abolition, of full-length STAT1␣ and
STAT1␤ isoform expression. The K201N allele results in lower
levels of STAT1 through abnormal mRNA splicing, as the K201N
missense mutation is not itself intrinsically deleterious for the
expression and phosphorylation of STAT1.
STAT1 dimerization and dephosphorylation
Different mutations in the STAT1 gene cause various functional
defects.17 Certain hydrophilic amino acids in the N-terminal
domain, coiled-coil domain, and DNA-binding domain play an
important role in converting the structure of the molecule from a
parallel dimer to an antiparallel dimer. Mutations affecting these
hydrophilic amino acids may impair the dimerization of unphosphorylated STAT1, resulting in the prolonged phosphorylation and
nuclear retention of STAT1.18-20 We investigated the possible
deleterious effects of K201N on this dimerization by modeling
K201N STAT1 homodimers, based on the known 3-dimensional
structure of WT STAT1 homodimers.20 K201N is not located on the
dimer interface (data not shown). We nevertheless investigated the
dimerization of unphosphorylated and phosphorylated STAT1 by
carrying out coimmunoprecipitation followed by Western blotting
in U3C cells transfected with various combinations of WT and
K201N STAT1 tagged with either Myc or Flag (Figure 4A). The
proteins precipitated with an anti-Flag antibody were subjected to
Western blotting with an anti-Myc antibody. For both phosphorylated and unphosphorylated forms of STAT1, the interaction
between K201N and WT or K201N was normal (Figure 4A; and
data not shown). We then investigated the kinetics of STAT1
activation and dephosphorylation in the cells of P1 and in
STAT1-deficient U3C cells transfected with the mutant K201N
allele. In EBV-B cells and SV40-fibroblasts from P1, STAT1
phosphorylation was observed at 30 minutes, decreasing thereafter
as rapidly as in the control cells, in response to IFN-␥ stimulation
(Figure 4B and data not shown). With staurosporine, which blocks
continuous tyrosine kinase activation, the kinetics of phosphorylation and dephosphorylation were similar in the cells of P1 and in
control cells (Figure 4C). Moreover, in U3C cells transfected with
K201N, STAT1 phosphorylation and dephosphorylation kinetics
were similar to those of the WT protein. This was not the case after
transfection with the F77A STAT1 allele,18 which displays prolonged STAT1 phosphorylation (supplemental Figures 2-3). Thus,
the K201N missense mutation does not affect the phosphorylation,
homodimerization, or dephosphorylation of STAT1.
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KONG et al
Figure 4. K201N mutation does not impair STAT1 dephosphorylation and homodimerization. (A) K201N does not impair homodimerization. U3C cells were transfected
with a combination of mock (M), WT, and K201N-mutated STAT1 plasmids tagged with either Flag or Myc. Proteins were extracted 48 hours after transfection and subjected to
coimmunoprecipitation and Western blotting. (Bottom panel) Western blotting for the detection of Myc or Flag expression in input with a specific antibody. (Top panel)
Immunoprecipitation with an anti-Flag antibody, followed by Western blotting with an anti-Myc or anti-Flag antibody. (B) STAT1 phosphorylation kinetics. EBV-B cells from a
healthy control (WT/WT), P1, a subject heterozygous for the K201N allele (K201N/WT), and a patient with complete STAT1 deficiency (1928insA/1928insA, ⫺/⫺) were
stimulated with 105 IFN-␥ for the time indicated (in minutes). Western blotting was carried out with an antibody against Tyr701-phosphorylated STAT1 (P-STAT1), or STAT1␣
(STAT1), or with an antibody against ␣-tubulin, as a reference. (C) Pulse-chase experiment. EBV-B cells from a healthy control (WT/WT), P1, a subject heterozygous for the
K201N allele (K201N/WT), and a patient with complete STAT1 deficiency (1928insA/1928insA, ⫺/⫺) were stimulated with 105 IU/mL IFN-␥ for 30 minutes, and staurosporine
was added and the mixture incubated for the time indicated. Western blot shows Tyr701-phosphorylated STAT1 and STAT1, with STAT2 as a reference.
Nuclear translocation and DNA-binding activity of the mutant
STAT1 protein
The phosphorylation of STAT1 triggers its translocation into the
nucleus in the form of GAF dimers or ISGF3 trimers, which bind to
consensus promoter sequences and initiate the transcription of
downstream ISGs. Immunofluorescence analyses showed that
STAT1 was constitutively expressed in the cytoplasm of control
fibroblasts and in the fibroblasts of P1 but was not detectable in
fibroblasts from patients with partial (P696S/P696S) or complete
STAT1 deficiency. In response to IFN-␥ or IFN-␣ stimulation,
STAT1 was translocated from the cytoplasm to the nucleus, as
normal, in the fibroblasts of P1 (supplemental Figure 4). We
quantified the DNA-binding activity of mutant STAT1-containing
GAF and ISGF-3 complexes, by carrying out electrophoretic
mobility shift assays with GAS and ISRE probes in cells from P1,
and comparing the results obtained with those for healthy controls
and cells from patients with other forms of STAT1 deficiency.
The cells of P1 displayed levels of GAF binding activity
36% (⫾ 5%) of those from normal controls after 16 minutes
of stimulation with 103 IU/mL or 105 IU/mL IFN-␥. This level
of activity was higher than that of P696S/P696S mutant
(⬃ 8%; Figure 5A) and L706S/WT mutant (impairing phosphorylation and, thereby, DNA binding, ⬃ 20%) cells, but lower than that
of E320Q/WT mutant (impairing DNA binding, ⬃ 40%) cells (data
not shown). After 16 minutes of stimulation with 103 IU/mL or
105 IU/mL IFN-␣, P1 ISGF-3 DNA-binding activity was determined and was shown to be 38% (⫾ 14%) or 62.7% (⫾ 11%) that
of normal controls. These levels were similar to those of the
P696S/P696S mutant after stimulation with 103 IU/mL IFN-␣ but
higher than those observed after stimulation with 105 IU/mL
(Figure 5B). We also evaluated the DNA-binding activity of
K201N STAT1 after its overexpression in U3C cells. Unlike L706S
and E320Q mutant proteins, K201N STAT1 displayed normal
DNA-binding activity, such as P696S STAT1 (supplemental
Figure 5 and data not shown). Furthermore, transcripts lacking
exon 8 had no dominant-negative effect on WT STAT1 GAF-DNA
binding activities in U3C cells in response to IFN-␥ (supplemental
Figure 6).
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Figure 5. Impaired GAF and ISGF-3 DNA binding in the cells of P1 early in stimulation. EBV-B cells from WT/WT, P1, K201N/WT, WT/⫺, P696S/P696S, and ⫺/⫺ subjects
were stimulated with the indicated doses of IFN-␥ (A) or IFN-␣ (B) for 15 minutes. Nuclear proteins were extracted to determine GAF (A,C) and ISGF-3 (B,D) levels, with the
corresponding probe. Kinetics of GAF (C,E-F) or ISGF-3 (D) DNA-binding activity after IFN-␥ and IFN-␣, respectively, as determined with stimulation for the times indicated.
(F) Pulse-chase experiment with the addition of staurosporine after 15 minutes of IFN-␥ stimulation. The results are representative of 3 independent experiments.
(G) Schematic diagram of GAF DNA-binding kinetics in healthy control, P1, P696/P696S, and 1928insA/1928insA cells. The x-axis indicates duration of IFN-␥ stimulation; and
the y-axis, GAF DNA-binding activities.
Kinetics of DNA-binding by STAT1-containing complexes in the
cells of P1
As the patients had low levels of STAT1 in their cells and as some
mutations in the STAT1 coiled-coil region are known to lead to the
accumulation of activated GAF in the nucleus,19 we examined the
kinetics of GAF DNA-binding activity in EBV-B cells and
SV40-fibroblasts from P1 and controls on stimulation with IFN-␣
or IFN-␥, with or without staurosporine. In healthy control cells,
GAF-binding activity was detected after 2 minutes of stimulation,
gradually increasing thereafter to reach a peak at 16 minutes,
and began to decrease again after 30 minutes. The activated GAF
did not disappear until 48 hours of stimulation (Figure 5C,E and
data not shown). With staurosporine, GAF binding rapidly decreased, becoming undetectable at 2 hours (Figure 5F). Com-
parisons with healthy controls showed that the cells of P1 had
defects in GAF-binding activity only at early time points (levels
20%-40% those of controls). However, whereas GAF-binding in
control cells decreased after 30 minutes, GAF levels decreased at
different rates in cells of P1 and in control cells, resulting in similar
levels of GAF-binding in the cells from P1 and control cells after
1 hour of stimulation. In the presence of staurosporine, cells from
P1 displayed lower levels of DNA-binding activity than control
cells at the peak of activation and similar levels of DNA-binding
during deactivation (Figure 5F). The kinetics of ISGF-3 DNAbinding activity after IFN-␣ stimulation followed a pattern similar
to that observed after IFN-␥ stimulation in the cells of P1 (Figure
5D). These results suggest that the K201N mutation impaired the
early response to IFN stimulation in terms of GAF and ISGF-3
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KONG et al
Figure 6. Selective impairment of downstream gene induction in the cells of P1. (A) The K201N allele has normal transcriptional activity. U3C cells were transfected with
M, WT, and K201N mutant alleles, with firefly luciferase under IRF1 or IFIT1 promoters and a Renilla luciferase control, and stimulated with 103 IU/mL IFN-␥ or IFN-␣ for
24 hours. (B) Hotmap of 2 categories of ISGs quantified by quantitative PCR in SV40-fibroblasts from P1, with comparison of the results obtained for WT/WT, L706S/WT,
P696S/P696S, and ⫺/⫺ subjects. The induction of CCL2, CXCL10, and IRF8 was significantly impaired in the cells of P1, as shown by comparison with 5 healthy controls.
These genes are identified as “STAT1 pulse-dependent primary ISGs”; CXCL9, GBP1, CIITA, and IRF1 displayed a similar pattern in the cells of P1 and controls and are
designated as “non-STAT1 pulse-dependent primary ISGs.” (C) After stimulation with 103 IU/mL IFN-␣, CXCL9 induction was significantly impaired in EBV-B cells from P1, at
1-hour and 2-hour time points. However, the level of induction of CXCL9 in the cells of P1 remained higher than those in P696S/P696S and ⫺/⫺ EBV-B cells. The results are
representative of 3 independent experiments. One red asterisk indicates P ⬍ .05; and 3 red asterisks indicate P ⬍ .005.
activation, as the normal peaks of activation were not reached, but
that the late phase of activated GAF and ISGF-3–binding activity
was normal (Figure 5G).
Transcriptional activation and downstream gene induction
We investigated whether the K201N mutation impaired the transcriptional activity of STAT1, by transfecting STAT1-deficient U3C
cells with a WT or mutant STAT1-encoding plasmid, together with
reporter plasmids, with expression of the firefly luciferase under the
control of GAS, ISRE, IRF1, IFIT1, or IFIT2 promoters. Cells
were also cotransfected with a Renilla luciferase reporter as a
reference. After IFN-␥ stimulation, the K201N mutant STAT1
induced the transcription of reporter genes under the control of
GAS, IRF1, or IFIT2 promoters, with the level of induction
attained similar to that for the WT (Figure 6A; and data not shown).
On IFN-␣ stimulation, reporter genes under the control of ISRE,
IFIT1, or IFIT2 promoters were also induced to similar levels by
WT and K201N STAT1 mutant proteins (Figure 6A; and data not
shown). The P696S STAT1 mutant also displayed normal transcriptional activity, compared with the L706S mutant, which was a
loss-of-function mutant in these reporter assays (data not shown).
We then studied the downstream induction of ISGs by quantitative
PCR, in EBV-B cells and SV40-fibroblasts from 5 healthy controls,
K201N/K201N, L706S/WT, P696S/P696S, and 1928insA/1928insA
cells, on stimulation with IFN-␥ or IFN-␣. Comparisons with
healthy controls showed the induction of one group of genes to be
decreased in the K201N/K201N cells after 2 hours of stimulation
with IFN-␥. This group of genes included the CCL2, IRF8, and
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CXCL10 genes. However, another group of genes, including
CXCL9, CIITA, GBP1, and IRF-1, was induced well in cells from
P1 (Figure 6B). The lowest levels of expression of these target
genes were observed in the P696S/P696S and 1928insA/1928insA
fibroblasts. In cells from a patient with dominant STAT1 deficiency
(L706S/WT), induction of the CCL2, CXCL9, and CXCL10 genes
show some decreases. For the IFN-␣–induced genes, we found that
the induction of CXCL9, GBP1, IFIT1, ISG15, and MxA was
STAT1-dependent, whereas the induction of SOCS3 and FOS was
STAT1-independent; CXCL9, GBP1, and IFIT1 were earlyresponse genes for which induction typically peaked 2 hours
after stimulation (Figure 6C; supplemental Figure 7A-B; and data
not shown). In addition, MxA and ISG15 displayed a delayed
mRNA induction peak at approximately 8 hours. EBV-B cells
from P1 contained significantly smaller amounts of CXCL9 and
GBP1 mRNA than control cells after 2 hours of induction, but these
levels remained higher than those in P696S/P696S cells and cells
from a patient with complete STAT1 deficiency (Figure 6C).
However, the induction of MxA, ISG15, and IFIT1 was not impaired in
cells from P1 (supplemental Figure 7A-B and data not shown).
We then studied the late response in cells from P1. HLA-DR
displayed a classic delayed response to IFN-␥ stimulation in
fibroblasts.13 SV40-fibroblasts from P1 showed normal HLA-DR
induction with either 10 IU/mL or 103 IU/mL IFN-␥, whereas
HLA-DR could not be up-regulated in the fibroblasts from a patient
with complete STAT1 deficiency and only residual levels of
induction were observed in P696S/P696S fibroblasts (Figure 7A).
We then assessed the delayed response by determining the protection against viral challenge conferred by IFN-␣ in the fibroblasts.
Prior treatment with IFN-␣ for 18 hours decreased vesicular
stomatitis virus (VSV) load by a factor of 106 with respect to
untreated cells, demonstrating the effective protection of the cells
of P1 by IFN-␣ (Figure 7B). Prior treatment with IFN-␣ also
protected the cells of P1 against VSV-induced cell death, as
observed in the healthy control (Figure 7C). Thus, impairment of
the early response is not sufficient to abolish the protection
mediated by IFN-␣.
P1’s response to IL-27 and IFN-␭
It has been shown that IL-27 activates STAT1, leading to GAF
production. The EBV-B cells of P1 produced smaller amounts of
GAF than those of healthy controls (Figure 7D). We then studied
the transcriptional activation of downstream genes, by quantitative
PCR. The transcriptional activation of CXCL9 and CXCL10 was
significantly impaired in the cells of P1 after only 1 or 2 hours of
stimulation with IL-27, but this activation was similar to that of
controls after 8 hours of stimulation (Figure 7E; supplemental
Figure 8). By contrast, the induction of IRF1 and GBP1 was not
impaired even at early time points in P1 cells, consistent with the
selective impairment of induction of early ISGs in P1 cells, but not
in P696S/P696S cells or in complete STAT1-deficient cells (data
now shown). Moreover, on IFN-␭ stimulation, the cells of P1
displayed normal induction of IFIT1 and ISG15 at various time
points (Figure 7F; supplemental Figure 9), whereas P696S/P696S
cells and complete STAT1-deficient cells displayed an impaired
response. The reason for these differences in cellular phenotype
between P1 and P696S/P696S cells may be the lower level of
IFN-␭ receptor expression in EBV-B cells, which could not
phosphorylate large amounts of STAT1. In conclusion, the cells of
P1 displayed defects in their response to IL-27 stimulation, but not
in their response to IFN-␭.
We report here 2 siblings with a new form of partial recessive
STAT1 deficiency. They are homozygous for a nucleotide substitution leading to both an amino acid substitution (K201N) and a
frameshift splice defect of exon 8. Their cells contain approximately 30% the normal amount of STAT1 protein, exclusively in
the full-length K201N STAT1␣ and ␤ forms. Partial STAT1
deficiency was first described in a kindred with the P696S
mutation.12 These 2 different exonic missense mutations, K201N
and P696S, probably lead to abnormal RNA splicing by introducing exonic enhancer effects through different mechanisms. Despite
the location of K201N substitution in the coiled-coil domain,
STAT1 dephosphorylation and dimerization were normal, consistent with the presence of the K201N residue on the outside of the
homodimer interface.18-20 Mutations of some of the lysine residues
of STAT1 mimicking the acetylated form impair the nuclear
translocation of STAT1.21,22 However, K201N-mutated STAT1 was
translocated from the cytoplasm to the nucleus. The K201N STAT1
protein, like the P696S STAT1 allele, was intrinsically fully
functional in terms of phosphorylation, dimerization, translocation,
dephosphorylation, DNA binding, and transcriptional activation.
However, K201N, like the P696S allele, caused an aberrant mRNA
splicing, decreasing the amounts of STAT1 protein in the cells.
Cells from patients with the P696S/P696S genotype, however,
contain smaller amount of STAT1 (⬃ 10%) than cells from patients
with the K201N/K201N genotype (⬃ 30%), resulting in patients
with the K201N mutation having a less severe cellular phenotype
on IFN stimulation.
However, the most interesting observation revealed by this
experiment of Nature is that the disease-causing K201N allele
impairs early, but not late cellular responses to IFNs,23,24 unlike the
P696S allele, which impairs both early and late responses to IFNs.
The correct duration and magnitude of IFN-induced transcriptional
activation are essential to ensure optimal IFN activity, so the
regulation of STAT1 activation has been investigated at multiple
levels.25 On IFN stimulation, STAT1 undergoes rapid and transient
tyrosine phosphorylation, peaking after approximately 16 minutes
and gradually decreasing thereafter. In the positive regulation of
STAT1, IFN production,26,27 receptor densities,28 kinase activities,29,30 and nuclear factors are tightly regulated to control the
physiologic response to IFN stimulation. For STAT1 deactivation,
tyrosine phosphatase activity,31,32 the export of STAT1 from the
nucleus,33 the SOCS and PIAS protein families,34 and the methylation and acetylation of STAT121,22,35 have been associated with
control of the parabolic response to IFN stimulation. Two phases of
the cellular response to IFNs can be defined in terms of STAT1
activation: an early response characterized by peak levels of
activated STAT1 in the nucleus and a delayed response, in which
activated STAT1 is present at low levels in the nucleus. However,
little is known about the physiologic importance of these 2 separate
phases. We demonstrate here the importance of the early peak
response. We provide here the first evidence that impairment of the
early human IFN response may cause clinical susceptibility to
mycobacterial and viral infections.
Another major finding of this study is the dissociation of
DNA-binding activity and the induction of target genes after
stimulation with IFNs. The impairment of early transcriptional
factor activation does not cause a decrease in mRNA induction for
all downstream ISGs. Careful investigation of ISG induction, by
quantitative PCR, identified 2 groups of downstream genes in
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KONG et al
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Figure 7. Delayed interferon responses, IL-27 and IFN-␭ responses in the cells of P1. (A) HLA-DR induction in SV-40 fibroblasts of WT/WT, P1, P696S/P696S, and ⫺/⫺
subjects after 48 hours of stimulation with the indicated dose of IFN-␥. HLA-DR was quantified by FACS analysis. Solid area represents no stimulation; thin solid line, 10 IU/mL
IFN-␥; and thick solid line, 103 IU/mL IFN-␥. (B) VSV viral titer after VSV challenge. The SV40-fibroblasts of P1 controlled VSV replication after prior treatment of IFN-␣ for
18 hours. Bold line indicates without IFN-␣; and dashed line, with 105 IU/mL IFN-␣. (C) Cell viability after VSV challenge. The viability of SV40-fibroblasts from P1 was similar to
that of healthy control cells after prior treatment with 105 IU/mL IFN-␣ for 18 hours. The results are representative of 2 independent experiments. (D) GAF DNA-binding activity
was impaired in the EBV-B cells of P1, as shown by comparison with the cells of healthy controls after stimulation with 100 ng/mL IL-27 for 15 minutes. (E) On stimulation with
100 ng/mL IL-27, CXCL9 induction was significantly impaired in the EBV-B cells of P1 at the 1-hour and 2-hour time points, as shown by comparison with healthy controls.
However, CXCL9 induction was nonetheless stronger in the cells of P1 than in P696S/P696S and ⫺/⫺ EBV-B cells. (F) After stimulation with 20 ng/mL IFN-␭, IFIT1 induction
was normal in the EBV-B cells of P1. This level of induction was greater than that observed in P696S/P696S and ⫺/⫺ EBV-B cells. The results are representative of
3 independent experiments. One red asterisk indicates P ⬍ .05; and 3 red asterisks indicate P ⬍ .005.
K201N/K201N cells. One group of genes was found to display
impaired induction in K201N/K201N cells, consistent with a
dependence on the early peak of GAF activation, whereas the genes
of the second group were induced normally. The mechanisms of
inducible mRNA transcription in response to stimulation have been
widely studied recently and seem to involve several sequential
processes, from chromatin remodeling and histone modification to
the binding of transcription factors to DNA and RNA polymerase
II recruitment.36 There may be various reasons for the existence of
the 2 groups of induced genes identified in K201N/K201N cells.
First, the promoter structures may account for the differences in
induction pattern. These effects may result from the presence of
cis-elements, CpG islands, and differences in G plus C content.37-39
Second, the 2 categories of inducible ISGs in K201N/K201N cells
may differ in terms of the roles played by several cotranscriptional
factors40-42 and histone regulation proteins, such as HDAC1, CBP,
and BRG1.43-45 Third, the threshold level of STAT1, which binds to
the inducible gene loci, required to induce efficient transcription
may differ for different genes.46,47 Further careful investigation is
required to resolve this point. In any case, the genes not induced in
K201N/K201N cells, such as IRF8 in response to IFN-␥48,49 and
CXCL9 in response to IFN-␣,50 are the best candidate genes for
patients with unexplained mycobacterial and viral diseases,
The authors thank David Levy, Stephane Smale, Bertrand Boisson,
Sun Xiao-Jian, Minji Byun, and Melina Herman for helpful
discussions and critical reading; David Levy (pIFIT2), John Hiscott
(pIFIT1), and Georg Kochs (pIRF1) for providing luciferase
reporter plasmids; Ron Liebman, Tatiana Kochetkov, Erin Kirk,
Yelena Nemirovskaya, Lucile Jannière, Martine Courat, and Tony
Leclerc for technical and secretarial assistance; and all members of
the Laboratory of Human Genetics of Infectious Diseases for
helpful discussions.
X.-F.K. is supported by a Choh-Hao Li Memorial Fund Scholar
award and the Shanghai Educational Development Foundation.
The Laboratory of Human Genetics of Infectious Diseases is
supported by grants from the Rockefeller University Center for
Clinical and Translational Science (grant 5UL1RR024143), the
Rockefeller University, the Bill and Melinda Gates Foundation, the
St Giles Foundation, the Jeffrey Modell Foundation and Talecris
Biotherapeutics, National Institute of Allergy and Infectious Diseases (grant 1R01AI089970), the Schlumberger Foundation, the
BNP-Paribas Foundation, the Institut Universitaire de France, and
the European Union (grant QLK2-CT-2002-0046).
Contribution: X.-F.K., S.A.-M., J.-L.C., and S.B.-D. were responsible for the conception and design of the experiments; X.-F.K. and
M.C. performed the experiments; X.-F.K., M.C., M.A., J.-L.C., and
S.B.-D. analyzed the data; S.A.-H., R.H., and S.A.-M. treated the
patients; L. Alsina, T.Z., J.B., J.F., M.A., C.P., V.B., A.K., D.B.,
X.-X.Z., L. Abel, and D.C. contributed reagents/materials/analysis
tools and edited the paper; X.-F.K., J.-L.C., and S.B.-D. wrote the
paper; and J.-L.C. supervised all work.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Jean-Laurent Casanova, St Giles Laboratory
of Human Genetics of Infectious Diseases, Rockefeller Branch,
Rockefeller University, 1230 York Ave, New York, NY 10065;
e-mail: [email protected]
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2010 116: 5895-5906
doi:10.1182/blood-2010-04-280586 originally published
online September 14, 2010
A novel form of human STAT1 deficiency impairing early but not late
responses to interferons
Xiao-Fei Kong, Michael Ciancanelli, Sami Al-Hajjar, Laia Alsina, Timothy Zumwalt, Jacinta
Bustamante, Jacqueline Feinberg, Magali Audry, Carolina Prando, Vanessa Bryant, Alexandra
Kreins, Dusan Bogunovic, Rabih Halwani, Xin-Xin Zhang, Laurent Abel, Damien Chaussabel, Saleh
Al-Muhsen, Jean-Laurent Casanova and Stéphanie Boisson-Dupuis
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of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.