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Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 118-125
HEMATOPOIESIS
From Puget Sound Blood Center, Seattle, WA; and the Divisions
of Hematology and Medical Genetics, Department of Medicine, University
of Washington, Seattle, WA.
We studied a large kindred with nonsyndromic autosomal dominant
thrombocytopenia to define the phenotype and used genomic linkage
analysis to determine the locus of the abnormal gene. Affected family
members are characterized by lifelong moderate thrombocytopenia
(mean = 42.7 × 109/L) with moderate propensity
toward easy bruising and minor bleeding. Megakaryocytes are present in
bone marrow with reduced frequency, and there are no apparent
abnormalities of myeloid or erythroid cells. This type of inherited
thrombocytopenia has no evident association with hematopoietic
malignancy or progression to aplastic anemia. In the past, members of
this family have failed therapeutic trials of immunosuppression and
splenectomy. In our investigation, we found that affected individuals
had normal platelet size compared with unaffected family members and
modestly increased thrombopoietin levels. Hematopoietic colony assays
from bone marrow and peripheral blood demonstrated that megakaryocyte
precursors (CFU-Mk) were dramatically increased in both number and size
in affected individuals. Bone marrow cells grown in liquid culture with
thrombopoietin failed to develop polyploid cells greater than 8N. Also,
electron microscopy demonstrated that megakaryocytes from an affected
individual had markedly delayed nuclear and cytoplasmic
differentiation. Genome-wide linkage analysis established a single
locus for the disease gene on the short arm of chromosome 10 with a
maximum 2-point lod score of 5.68 (at
Inherited thrombocytopenia is a rare human condition
that is frequently mistaken for an acquired platelet disorder. A
careful medical and family history can generally help categorize
congenital thrombocytopenia based on the duration of symptoms and onset
immediately after birth. A detailed pedigree can often help identify
the inheritance pattern (ie, autosomal recessive, autosomal dominant,
or sex-linked). Furthermore, in some cases, the coexpression of
additional phenotypic anomalies defines clinically recognized syndromes
associated with thrombocytopenia (ie, deafness and nephritis in Alport
syndrome, eczema and immunodeficiency in Wiskott-Aldrich syndrome). The known causes of inherited thrombocytopenia have been recently reviewed.1 It is important to distinguish disorders that
are primarily of platelet number from those that are predominantly of
platelet dysfunction (often with normal numbers). Glanzmann thrombasthenia (GpIIb/IIIa deficiency) and type I von Willebrand disease (vWF deficiency) are examples of such
thrombocytopathies.1,2
In this report, we present new information regarding a family with
autosomal dominant thrombocytopenia that was first described by Bithell
and colleagues in 1965.3 This large family has kept meticulous records, which allow the inheritance pattern to be traced
over 6 generations from a single founder.3 The recent identification of thrombopoietin (TPO) and its receptor, c-Mpl, has
provided the means to study megakaryocyte and platelet development in
vitro.4,5 Studies of rare familial human conditions provide the opportunity to understand normal physiology and identify novel gene
functions in vivo.
We studied 30 members of a single extended family to establish linkage
of inherited thrombocytopenia to a locus of the human genome. This led
to the identification of a 17-centimorgan (cM) region of chromosome 10 as the only site of statistically significant linkage (maximum lod
score = 5.68 by 2-point linkage, Patient recruitment
Preparation of platelet-poor plasma, genomic DNA, and platelets
Flow cytometry Platelets were resuspended in Tris-buffered saline with 10-mmol/L EDTA (TBSE), counted (Coulter platelet analyzer, Santa Ana, CA), and adjusted to approximately 200 × 109/L. Two hundred microliters of platelets were incubated with monoclonal antibodies to glycoproteins (Gp) IIb-IIIa, GpIV, or Gp Ia-IIa (Immunotech, Marseille, France) at a final concentration of 10 µg/mL (30 minutes at 37°C). Platelets were washed twice with TBSE plus 1% human albumin (Alpine Biologics, Blauvelt, NY) by centrifugation at 1500g for 10 minutes. All supernatant was removed, and the pellet was resuspended in 10 µL of fluorescein isothionate (FITC)-conjugated goat antimouse immunoglobulin (Becton Dickenson). The platelets were incubated for 15 minutes in the dark and washed by centrifugation in TBSE plus 1% human albumin. The pellet was resuspended in Hema-Line 2 (ABX Diagnostic, Allentown, PA) and analyzed by flow cytometry.Thrombopoietin ELISA TPO levels were measured from platelet-poor plasma for each study participant using a sandwich ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Samples were measured in duplicate and were repeated in 2 separate experiments to ensure that results were internally consistent; 96-well plates were read using a microplate reader (Bio-Rad 550, Bio-Rad, Hercules, CA), 450- to 570-nm dual wavelength protocol.Preparation of bone marrow and CD34+ peripheral blood One unaffected and 2 affected family members consented to donate bone marrow (5-6 mL) from the iliac crest and a large volume of peripheral blood (50 mL). Bone marrow smears were prepared on cover slips and stained with Wright-Giemsa stain. Next, low-density cells were separated from both bone marrow and peripheral blood cells by overlayering the cells onto Ficoll (1.077-1.08 g/mL; ICN Biomedicals Inc, Costa Mesa, CA) and by centrifugation (400g for 30 minutes). Cells were collected from the interface, washed 2 times in Iscove's modified Dulbecco's medium (IMDM) with 2% fetal calf serum (Stem Cell Technologies, Vancouver, Canada), and counted using a hemocytometer. No further manipulations of bone marrow cells were carried out. Peripheral blood cells were subjected to immunomagnetic enrichment of CD34+ cells using the MidiMACS system (CD34 Select Kit, Miltenyi Biotec, Auburn, CA). Selected cells were washed in IMDM with 2% fetal calf serum (Stem Cell Technologies) and counted.Colony assays Low-density bone marrow cells were mixed with 1% methylcellulose in IMDM containing 30% fetal bovine serum, 1% bovine serum albumin, 0.1-mmol/L -mercaptoethanol, and the following recombinant human
cytokines: 50-ng/mL stem cell factor, 10-ng/mL interleukin-3, 3-U/mL
erythropoietin, and 10-ng/mL granulocyte-monocyte colony-stimulating factor (MethoCult Assay, Stem Cell Technologies). The final
concentration of cells was adjusted to 7.5 × 104
cells/plate (1.1 mL) for each sample. Five replicate plates were made
for each of the affected individuals and the unaffected family member.
After 12 days at 37°C and 5% CO2 in a humidified
incubator, burst-forming units, erythroid (BFU-E), CFU-GM (granulocyte,
monocyte), and mixed-lineage CFU-GEMM (granulocyte, erythroid,
monocyte, megakaryocyte) colonies were counted from each of the plates, and averages were determined for each individual. CFU-Mk colonies were
determined separately using the MegaCult System (Stem Cell Technologies), in which cells were cultured on collagen-based slide
chambers in the presence of recombinant human TPO, interleukin-3, and
interleukin-6. Low-density bone marrow cells were plated at a density
of either 5.0 × 104 or
2.0 × 105 cells per chamber, and CD34-selected
peripheral blood cells were plated at either
4.0 × 103 or 1.2 × 104 cells
per chamber. After 14 days in culture, the chamber slides were
dehydrated and stained for GpIIb/IIIa according to the manufacturer's guidelines. All colony numbers were quantified using an inverted phase
light microscope.
DNA ploidy evaluation Ficoll-purified bone marrow cells were cultured under serum-free conditions in IMDM with 1% Nutridoma (Sigma Corp, St Louis, MO) and supplemental antibiotics (penicillin and streptomycin, BioWhittaker, Walkersville, MD) at a final concentration of 1 × 106 cells/mL. Recombinant TPO was added at a final concentration of 10 ng/mL (generous gift from Don Foster, Zymogenetics, Seattle, WA), and the cultures were incubated at 37°C in a humidified incubator containing 5% CO2. On day 11, cells were removed from liquid culture and incubated with FITC-labeled CD41a-specific antibody (Pharmingen Corp, San Diego, CA) as recommended (20 µL/106 cells). After gentle washing, the cells were resuspended in buffer containing 50-µg/mL propidium iodide, 0.1% Triton X-100, 0.1% sodium citrate, and 30-µg/mL RNAse (Sigma Corp). Flow cytometry was performed (Becton Dickinson FACScalibers), and ploidy classes for megakaryocytes were determined by plotting the propidium iodide fluorescence of the cells with highest CD41 expression (brightest 10% to 15%) using a semilogarithmic scale.Signal transduction studies Platelets were obtained by centrifugation of platelet-rich plasma (see "Preparation of platelet-poor plasma, genomic DNA, and platelets"). Platelets were prepared by incubation with aspirin, prostaglandin E1, and apyrase as previously described.6 Final cell pellets were resuspended in a modified HEPES-Tyrodes buffer (10-mmol/L HEPES, pH 7.4; 129-mmol/L NaCl; 8.9-mmol/L NaHCO3; 0.8-mmol/L KH2PO4; 0.8-mmol/L MgCl2; 5.6-mmol/L dextrose; 2-U/mL apyrase; 1-mmol/L CaCl2) and were incubated at 37°C in the absence of serum and growth factors for 2 hours. One half of each sample was then stimulated with recombinant human TPO (10 ng/mL) for 10 minutes while the other half remained unstimulated. Protein extracts were prepared using a modified RIPA buffer as previously described.5,7 Samples of unstimulated and stimulated lysates were analyzed by Western blot and probed with a phospho-specific STAT3 (signal transducers and activators of transcription) antibody generously provided by David Frank, Boston, MA (1:3000 in Tris-buffered saline solution plus 0.05% Tween 20 and 3% bovine serum albumin [TBST + BSA]). After extensive washing in TBST, tyrosine-phosphorylated STAT3 protein was visualized using a secondary goat antirabbit immunoglobulin G conjugated to horseradish peroxidase (1:5000 in TBST + BSA; Bio-Rad) and chemiluminescent reagents (New England Nuclear Corp, Boston, MA). The blot was later stripped (50°C for 30 minutes in 62.5-mmol/L Tris, pH 6.8; 2% sodium dodecyl sulfate; 100-mmol/L-mercaptoethanol) and reprobed to
demonstrate equal loading of STAT3 in all lanes (STAT3 polyclonal
antibody K-18, 0.5 µg/mL in TBST + BSA; Santa Cruz Biotechnology,
Santa Cruz, CA).
Electron microscopy After 10 days in liquid culture (10-ng/mL TPO, serum-free), a portion of the marrow culture was recovered by low-speed centrifugation (200g for 5 minutes) and was enriched for megakaryocytes by discontinuous BSA density gradient sedimentation as previously described.5,7 The fractions with highest megakaryocyte numbers were pooled and collected by low-speed centrifugation. The cell pellet was preserved by gently adding fixative containing 1% glutaraldehyde and 4% formaldehyde. The pellet was minced to provide fragments about 1 mm in maximum diameter. These fragments were postfixed in 2% osmium tetroxide and processed into Eponate 12. Each plastic block was cut to provide sections 0.5 µm thick, which were stained (methylene blue-azure II dye) and examined by light microscopy to select the block with maximum cell density. This block was cut into thin sections (60-90 nm), stained with lead acetate and uranyl acetate, and examined by electron microscopy.Genome-wide linkage analysis Genomic localization was performed using the Weber-9 human linkage set, consisting of 388 microsatellite markers with an average distance of 10 cM between markers. Polymerase chain reaction of genomic DNA and allelic assignments for the initial 16 participants (Figure 1) were done by Research Genetics Corp (Birmingham, AL). This service reports a genotyping error rate of less than 1%.8 Using that data and allelic frequencies obtained from the web sites of CEPH (Centre d'Etude du Polymorphisme Humain; www.cephb.fr/cgi-bin/wdb/ceph/systeme/form) and CHLC (Cooperative Human Linkage Center; http://cgap.nci.nih.gov/CHLC), 2-point linkage analysis was performed for each individual marker using the LINKAGE analysis software.9,10 An autosomal dominant, fully penetrant gene with an allele frequency of 10 4 and
no phenocopies was modeled (see "Autosomal dominant inheritance pattern"). The initial genomic scan had a maximum
potential lod score of 3.31. A lod score is the logarithm of ratio of
the likelihood of the data given linkage at a certain recombination
fraction divided by the likelihood of the data assuming no linkage. A
lod score of 3 or more is generally considered significant evidence of
genetic linkage (ie, cosegregation of the marker and disease). A
chromosome 10 region that achieved a significant lod score was studied
further by adding additional markers from the PE
Biosystems high-density microsatellite set (Foster City,
CA) and accrual of additional family members for genotyping. After the
initial genomic scan, all subsequent genotyping was performed in our
laboratory using an ABI 310 genetic analyzer. Linkage analysis was then
used to calculate lod scores for the extended pedigree, delimit the range of linkage, and identify recombination events.
Autosomal dominant inheritance pattern We obtained peripheral venous blood from 28 members of a single pedigree and classified them as thrombocytopenic (less than 125 × 109/L) or normal (150 × 109/L to 400 × 109/L). We were able to assign affectation status to all individuals without ambiguity. There were no instances of transmission of thrombocytopenia from an asymptomatic individual (ie, carrier) that might suggest incomplete penetrance. Males and females were equally likely to transmit the thrombocytopenic trait and to inherit it, suggesting that there is no sex-linked component. There was no consanguinity within the family, and accurate records have been maintained for 6 generations. These data were completely consistent with an autosomal dominant inheritance of thrombocytopenia in this family (Figure 1).Medical history Individual members of this family have come to medical attention at various times over the past 30 years and have been extensively evaluated to determine the nature of their thrombocytopenia. At least 3 individuals were treated for presumed immune-mediated thrombocytopenia with immunosuppression followed by splenectomy without any improvement in platelet levels. There were no major bleeding diatheses even with minor surgery and childbirth, suggesting that the residual platelets have normal hemostatic function. None of the affected individuals developed additional hematopoietic problems, such as myelodysplasia and leukemia, which have been described as occurring in another familial thrombocytopenia syndrome.11 Autologous platelet survival studies done elsewhere in 3 individuals showed moderately decreased survival likely
insufficient to account for the degree of thrombocytopenia.
Thrombocytopenia associated with increased white blood cells and polymorphonuclear granulocytes We analyzed peripheral blood to determine the platelet count, mean platelet volume (MPV), hematocrit, mean corpuscular volume (MCV), total white blood cells, and complete differential using an automated blood analyzer (Table 1). Most notably, the affected individuals were thrombocytopenic, with a mean platelet count of 42.7 × 109/L (range, 18 × 109/L to 106 × 109/L) compared with a mean of 241.8 × 109/L for unaffected family members (range, 162 × 109/L to 342 × 109/L). Morphologically, the platelets of affected individuals appeared normal without clumping or variation in size (Figure 2). Moreover, the MPV was equivalent between the affected and unaffected family members, distinguishing this form of inherited thrombocytopenia from others that are associated with either large platelets (ie, macrothrombocytes, observed in Alport syndrome, May-Hegglin anomaly, and grey platelet syndrome) or small platelets (ie, microthrombocytes, seen in Wiskott-Aldrich syndrome). The total white blood cell count was significantly higher in individuals with thrombocytopenia than without thrombocytopenia (10.8 × 109/L vs 6.2 × 109/L, respectively, P = .002). This was primarily due to an increased number of neutrophils for thrombocytopenic individuals. The lymphocyte and mononuclear cell numbers were not statistically different for these populations. Average hematocrits were similar in the thrombocytopenic and unaffected groups (47.5% vs 47.1%, respectively), but the MCV was significantly lower in affected family members than in unaffected individuals (P = .02). Because the percentage of women was higher among thrombocytopenic (11 of 14; 79%) than among unaffected (6 of 16; 38%) individuals, we stratified our data by sex. Although the number of observations is relatively small, a lower MCV was observed in affected males and females than in unaffected males and females (data not shown). Mean corpuscular hemoglobin concentration and red cell distribution width showed no statistically significant differences between affected and unaffected family members.
Similar platelet-specific proteins found on the surface platelets from thrombocytopenic and nonthrombocytopenic individuals Flow cytometry of affected versus unaffected family members showed no major differences in expression of GpIIb/IIIa, GpIV, and GpIa/IIa (data not shown). These assays were difficult to compare because they were run as samples became available, and the number of platelets in thrombocytopenic samples was low. Nonetheless, these data demonstrate that there are no major differences in cell surface expression for several well-characterized platelet proteins.Examination of the bone marrow We examined bone marrow from 2 affected patients and 1 unaffected family member. Megakaryocytes were evident in all 3 samples, although the number of mature, multilobulated megakaryocytes was reduced in the 2 thrombocytopenic individuals and the size of the average megakaryocyte was decreased (Figure 3). Erythroid and myeloid maturation appeared to be unaffected.
Intact tyrosine phosphorylation of STAT3 in platelets from thrombocytopenic individuals Fifty milliliters of blood was obtained from 1 unaffected and 2 affected individuals, and platelets were isolated for signaling studies. Platelets were divided into 2 aliquots, and half were stimulated with TPO (10 ng/mL) for 10 minutes before lysis. Whole-cell extracts from each patient were then analyzed by Western blot and probed with an antibody specific for tyrosine-phosphorylated STAT3 (Figure 4). We found that both the thrombocytopenic and unaffected platelets were capable of mediating TPO-induced STAT phosphorylation. This demonstrates that early signaling events in response to TPO stimulation are similar in affected and unaffected platelets. This finding is in marked contrast to patients with the TAR syndrome (thrombocytopenia with absent radii syndrome), in which a failure of TPO-induced signaling has been demonstrated.12
TPO levels are elevated in affected versus unaffected individuals Thrombocytopenic individuals had an average TPO value of 94.1 ± 63.9 pg/mL (range, 26.6-224.7 pg/mL) compared with 48.7 ± 42.4 pg/mL (range, 8.1-155 pg/mL) for unaffected family members (P = .04, 2-tailed Student t test). TPO levels in plasma are primarily regulated by platelets and megakaryocytes, which internalize and degrade TPO bound to Mpl receptors.13,14Therefore, the minimal elevation in TPO levels, despite marked reduction in circulating platelets, suggests that a compensatory increase in TPO uptake and degradation is occurring in the marrow.Megakaryocytes lack full polyploidization Low-density cells from bone marrow were cultured for 11 days under serum-free conditions in the presence of recombinant human TPO (10 ng/mL). Then, 2 × 106 to 3 × 106 cells were double-stained with FITC-conjugated CD41 antibody and propidium iodide. Cells were analyzed by flow cytometry to determine ploidy classes for the CD41+ cells (Figure 5A). We found that megakaryocytes from an unaffected family member contained megakaryocytes with ploidy classes of 2N, 4N, 8N, 16N, and 32N. In contrast, the 2 affected thrombocytopenic individuals had predominantly 2N and 4N megakaryocytes with a few 8N cells but no higher ploidy classes. The CD41 staining was similar in all 3 samples, and the highest 10% to 15% of all cells were analyzed. Representative megakaryocytes from an unaffected and a thrombocytopenic family member are shown in Figure 5B (grown in vitro with 10-ng/mL TPO for 7 days).
CFU-Mk increased in affected individuals Colony assays were performed to determine the number of erythroid, myeloid, and megakaryocytic progenitors in this form of hereditary thrombocytopenia. An equal number of low-density bone marrow cells or peripheral blood cells were plated in semisolid methylcellulose medium in the presence of multiple growth factors as described in the "Materials and methods." After 12 days, BFU-E, CFU-GM, and CFU-GEMM were counted. As shown in Table 2 and Table 3, thrombocytopenic patients had moderately increased BFU-E and normal CFU-GM and CFU-GEMM in the bone marrow. In contrast, peripheral blood from an affected individual had markedly higher BFU-E, CFU-GM, and CFU-GEMM, indicating a greater number of progenitors in circulation. Qualitatively, the erythroid colonies from thrombocytopenic patients were less strongly hemaglobinized and had less discrete borders than those from the unaffected individual. Also, there were significantly more scattered cells and diffuse colonies of unclear lineage in the cultures from thrombocytopenic patients.
Electron microscopy evaluation of megakaryocytes in hereditary
thrombocytopenia
Genomic linkage analysis
In this paper, we describe a single extended family with autosomal
dominant thrombocytopenia that results in a phenotype of incomplete
maturation of megakaryocytes. This syndrome is characterized by mild to
moderate bruising throughout life, normal erythroid and myeloid cells,
moderately elevated plasma TPO levels, expanded progenitors across all
hematopoietic lineages, and dramatic expansion of immature
megakaryocytes (CFU-Mk). Through a genomic scan with follow-up linkage
analysis, we have established a locus of linkage encompassing a maximal
region of below 20 cM on human chromosome 10. We propose that a
mutation of 1 allele in this region inhibits terminal differentiation
of megakaryocytes, either through a loss of function or
dominant-negative mechanism of action.
We wish to acknowledge Nancy Lin for assistance with the
hematopoietic colony assays, Jennifer Luthi for CD34+
enrichment of peripheral blood cells, Gayle Teramura for flow cytometry
of platelets, Don Foster for purified human TPO, David Frank for
phospho-STAT3 antibody, David Thorning for electron microscopy, and
Sarah Serafimidis for administrative assistance and editing. Most
importantly, we wish to thank the family members who participated in
this study. Without their enthusiastic support and assistance, this
work would not have been possible.
Submitted November 29, 1999; accepted February 18, 2000.
Supported by the Doris Duke Charitable Foundation, Clinical Scientist
Award (J.G.D.).
G.P.J. is an American Heart Association clinician-scientist awardee.
Reprints: Jonathan Drachman, Puget Sound Blood Center, 921 Terry Ave, Seattle, WA 98104; e-mail: drachman{at}u.washington.edu.
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 U.S.C.
section 1734.
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| Copyright © 2000 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
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Abstract
Introduction
= 0). By recruiting
additional family members, the genomic region was narrowed to 17 centimorgans. We conclude that a gene in this locus plays an important
role in megakaryocyte endomitosis and terminal maturation.
(Blood. 2000;96:118-125)
) or
stimulated (+) with recombinant human TPO for 10 minutes, 10 ng/mL.
Cellular extracts were analyzed by Western blot (7.5% acrylamide gel)
and probed with a phospho-STAT3-specific antibody. Below, the blot was
stripped and reprobed with a STAT3 antibody to confirm equal protein in
each lane. At left, extracts from parental Ba/F3 and Ba/F3-mMpl cells
(a cell line engineered to express the murine Mpl receptor) were used
as negative and positive control, respectively, for TPO-dependent STAT3
phosphorylation.
