Barteling, Wideke

Hebeda, Konnie M

Henskens, Yvonne M C

Huys, Erik

Jansen, Joop H

Jongmans, Marjolijn C J

Laros-van Gorkom, Britta A P

Preijers, Frank W

Saes, Joline L

Schols, Saskia E M

Simons, Annet

van Bergen, Maaike G J M

van der Reijden, Bert A

Utrecht University Repository

5. Beall CM, Brittenham GM, Strohl KP, et al. Hemoglobin concentrationof high-altitude Tibetans and Bolivian Aymara. Am J Phys Anthropol.1998;106:385-400.6. Looker AC, Dallman PR, Carroll MD, Gunter EW, Johnson CE. Prevalenceof iron deficiency in the United States. JAMA. 1997;277(12):973-976.7. Cho JI, Basnyat B, Jeong C, et al. Ethnically Tibetan women in Nepalwith low hemoglobin concentration have better reproductive out-comes. Evol Med Public Health. 2017;2017(1):82-96.Received: 12 February 2020 Revised: 21 February2020Accepted: 27 February2020DOI: 10.1002/ajh.25770Platelet CD34 expression in apatient with a partial deletionof transcription factorsubunit CBFBTo the Editor:The hematopoietic stem and progenitor cell marker CD34 is impli-cated in cell-cell interactions in the bone marrow niche. Upon hemato-poietic maturation its expression is switched off. Aberrant CD34expression on mature platelets has been associated with monogenicbleeding disorders that are caused by mutations in the transcriptionfactors GFI1B (Growth Factor Independence 1B) and RUNX1 (RUNTrelated transcription factor 1).1,2 These familial disorders are charac-terized by a bleeding diathesis, caused by disturbed megakaryocytedevelopment, which results in the formation of dysfunctional plate-lets. In addition, these disorders are accompanied by abnormal reten-tion of CD34 on platelets, which is not lost during differentiation.In this letter, we report a patient with CD34 expression on plateletsassociated with a partial deletion of the RUNX1 binding partner CBFB(Core Binding Factor B). CBFB is part of a heterodimeric transcriptionfactor complex with either RUNX1, −2 or − 3. CBFB enables thefunction of RUNX1-3 through stabilization of their interaction withDNA. Together with GFI1B and RUNX1, CBFB is now the third hema-topoietic transcriptional regulator that associates with abnormal plate-let CD34 expression.The patient we report here, presented to our outpatient clinic withmultiple bleeding events. A Tosetto bleeding score of 11 wasestablished, consisting of spontaneous epistaxis, prolonged bleedingfrom small wounds, spontaneous bruising and menorrhagia. In addition,she experienced prolonged bleeding after wisdom teeth extraction, andsevere bleeding following hysterectomy requiring relaparotomy andblood transfusion. However, she did not experience postpartum bleed-ing after giving birth to a son, and correction of clavicle hypoplasia as achild was uncomplicated. Both the parents and the patientʼs son werenot affected by a bleeding tendency (Figure 1A). Her medical historyrevealed mild asthma, migraines and a clinical depression for which sheused a selective serotonin reuptake inhibitor (SSRI). Platelet counts, aswell as mean platelet volume, ADP levels, β-thromboglobulin and plate-let factor 4 expression, and the ADP/ATP ratio were within the normalrange (Table S1). The platelets showed no abnormalities in a May-Grünwald Giemsa stained blood film (data not shown). The plateletfunction analyzer measured prolonged closure time after stimulationwith the collagen/epinephrine membrane (>300 seconds). Platelet lighttransmission aggregometry revealed an aggregation defect upon stimu-lation with epinephrine (10 μM), ADP (10 μM) and low concentration ofcollagen (1 μg/mL) (Table S1). Stimulation with high concentrations ofcollagen or ADP resulted in normal aggregation responses, similar to thestrong agonists arachidonic acid, thrombin receptor-activating peptideand ristocetin. The ATP release from δ-granules was measuredfollowing stimulation with a high concentration of collagen (5 μg/mL),epinephrine (5 μM) and the thromboxane analogue U46619 (1 μM)(Table S1). The ATP release was normal after stimulation with a highconcentration of collagen, as expected. No ATP was released uponstimulation with epinephrine and low levels were measured after stimu-lation with U46619. Cessation of the SSRI for 8 weeks did not correctplatelet aggregation defects. Thin section electron microscopy(EM) showed normal platelets, with occasional platelets carrying fewα-granules (Figure 1B). Whole mount EM showed increased numbers ofδ-granules at two independent time points, compared to controls(on average 5.84 δ-granules vs 3.66 per platelet, respectively)(Figure 1C, D).2 Together, these data suggest that the index patient suf-fers from a mild δ-granule secretion defect. Remarkably, the plateletsfrom the son showed defects in the light transmission aggregometrytests upon stimulation with epinephrine and low concentrations of col-lagen and ADP, similar to the index patient (Table S1). The adult son didnot experience hemostatic challenges and had no clinical history ofbleedings, which may suggest that the bleeding disorder observed inthe index patient is not solely caused by the δ-granule secretion defect.To identify a genetic cause for the bleeding tendency, a panel ofthrombosis and hemostasis associated genes was screened followingwhole exome sequencing.3 No disease-causing variants were called.Exome-wide analysis detected a ~100 Kb heterozygous chromosome16q22.1 deletion that was confirmed by single nucleotide polymor-phism (SNP) array. This deletion covers nine genes, including the lastexon of CBFB (Figure 1F). No disease-causing variants were observed inthe remaining wild type CBFB allele. The mother and son of the indexpatient did not have the 16q deletion. The father was deceased andcould therefore not be tested for the deletion, however, no clinical his-tory of bleedings was reported. The expression of CBFB transcripts wasstudied by 30-Rapid Amplification of cDNA Ends (RACE) and Sangersequencing. Readthrough from exon 5 into intron 5 was observed,predicting the formation of a protein that exceeds 11 amino acids fromthe splice junction. To quantify RNA expression of this transcript in leu-kocytes, a quantitative real-time polymerase chain reaction (RT-qPCR)was designed within intron 5 (Figure 1G). Next to the patient (II.1) andher son (III.1) five additional healthy controls were tested for expressionof this transcript. This showed on average a 10-fold increase of thistranscript in the index case compared to the controls and her son(Figure 1H). Based on findings in the son we concluded that theE136 CORRESPONDENCEThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in anymedium, provided the original work is properly cited and is not used for commercial purposes.δ-granule defect was not caused by the 16q deletion. Yet, the 16q dele-tion in combination with the δ-granule defect might contribute to thebleedings in the index patient. To date, none of the 16q-deleted geneshave been implicated in inherited bleeding or platelet disorders. How-ever, conditional Cbfb knockout in mice resulted in disturbedmegakaryopoiesis and thrombocytopenia.4 Because bleeding disorderscaused by RUNX1 mutations result in platelet CD34 expression andan interaction with CBFB is required for the function of RUNX1, theplatelets were tested for CD34 expression.2 Flow cytometry clearlydetected platelet CD34 expression in the index patient whileF IGURE 1 Legend on next page.CORRESPONDENCE E137platelets from her son and 10 nonrelated healthy controls did not.Thus, the 16q deletion associates with platelet CD34 expression(Figure 1E).Entire heterozygous CBFB deletions have been reported in rarecases with congenital bone abnormalities and mutations in its bindingpartner RUNX2 may cause cleidocranial dysplasia, associated withclavicle hypoplasia.5 The medical record of the patient indicated thatshe had undergone surgery at the age of 13 for a congenital hypoplas-tic clavicle. The mother and son of the index patient had no signs ofclavicle hypoplasia and the father was reported not to have boneabnormalities either. Thus, the 16q deletion associates with plateletCD34 expression, and a congenital clavicle hypoplasia but not withdisturbed platelet δ-granule secretion.To our knowledge, the patient reported here is the first inwhom a deletion of chromosome 16q22.1 partially encompassingCBFB associates with platelet CD34 expression. The deletion isnot causal to the platelet δ-granule abnormalities, because theson who did not inherit the deletion showed similar δ-granuledefects as the mother. Recently, we and others reported CD34expression on platelets from patients with GFI1B variants that didnot necessarily cause bleedings on their own. This suggests thatplatelet CD34 expression may be uncoupled from a bleedingtendency for other transcription factor mutations as well.6,7 Dur-ing endothelial to hematopoietic transition GFI1B is a crucialRUNX1/CBFB target gene, which may explain the similaritiesbetween RUNX1, CBFB and GFI1B mutants.8 As the son had aδ-granule secretion defect, similar to the index patient, but noclinical history of bleedings, the index patientʼs bleeding tendencycannot solely be explained by the δ-granule dysfunction. Thiscase underscores that mutations in various hematopoietic tran-scription factors may result in abnormal platelet developmentassociated with increased CD34 expression, which may aggravatebut are not necessarily the sole cause of the clinically manifestedbleeding tendency.ACKNOWLEDGMENTSThis work was supported by the Landsteiner Foundation for BloodTransfusion Research (project 1531).AUTHOR CONTRIBUTIONSM.G.J.M.v.B., J.L.S., B.A.P.L.-v.G., S.E.M.S., M.C.J.J., J.H.J., and B.A.v.d.R.designed and coordinated research; M.G.J.M.v.B., J.L.S., A.S., K.M.H.,Y.M.C.H., W.B., E.H., and F.W.P. collected and analyzed data; B.A.P.L.-v.G., and M.C.J.J. arranged for obtaining patient informed consent; andM.G.J.M.v.B. and B.A.v.d.R. wrote the manuscript that was criticallyrevised by all authors.Maaike G.J.M. van Bergen1 , Joline L. Saes2,3, Annet Simons4,Konnie M. Hebeda5, Yvonne M.C. Henskens1,6, Wideke Barteling1,Erik Huys1, Britta A.P. Laros-van Gorkom2,3, Saskia E.M. Schols2,3,Frank W. Preijers1, Marjolijn C.J. Jongmans4,7,8, Joop H. Jansen1,Bert A. van der Reijden11Department of Laboratory Medicine, Laboratory of Hematology,Radboudumc and Radboud Institute for Molecular Life Sciences,Nijmegen, The Netherlands2Department of Hematology, Radboudumc, Nijmegen, The Netherlands3Hemophilia Treatment Center Nijmegen – Eindhoven – Maastricht,Nijmegen, The Netherlands4Department of Human Genetics, Radboudumc, Nijmegen,The Netherlands5Department of Pathology, Radboudumc, Nijmegen, The Netherlands6Central Diagnostic Laboratory, Unit for Hemostasis and Transfusion,Maastricht University Medical Center, Maastricht, The Netherlands7Department of Medical Genetics, University Medical Center Utrecht,Utrecht, The Netherlands8Princess Máxima Center for Pediatric Oncology, Utrecht,The NetherlandsF IGURE 1 Family history, platelet phenotyping and genetic analyses in a patient with a partial deletion of CBFB. A, Family pedigree with oneaffected family member with a bleeding disorder as well as a heterozygous deletion of chromosome 16q22.1. Besides patient II.1 none of theother family members had a clinical presentation of a bleeding tendency. The father of the index patient (I1) was deceased and unavailable forfurther testing. No clinical abnormalities were reported. □ represents male,  represents females. B, Transmission electron microscopy imagesshow platelets with few α-granules (as depicted with an arrowhead) as well as platelets with normal amounts of α-granules. (JEM 1400 Flash, Jeol,Tokyo, Japan) C, D, Whole mount electron microscopy image revealed on average increased numbers of δ-granules (5.84 granules/platelet, blackdots depicted with arrowheads) in the platelets from index II.1 compared to healthy controls (3.66 granules/platelet). (JEM 1400 Flash, Jeol,Tokyo, Japan) Whole mount EM was performed twice at different time points, showing similar results. E, CD34 expression was measured onCD42b positive platelets. CD34 was expressed on platelets derived from the index patient II.1 (n = 2), whereas the platelets from the son (III.1) aswell as healthy controls (n = 10) showed no CD34 expression. F, The last exon of CBFB together with eight other genes were deleted asobserved in WES (not shown) and SNP array. The proximal breakpoint is situated within intron 5 of CBFB. The father (I1) of index patient II.1 wasunavailable for genotyping. The mother (I2) did not carry the deletion nor did the son (III.1). G, 30-RACE using an oligo-dT primer was performed,followed by amplification of the cDNA from exon5 onwards and subsequently a semi-nested PCR was performed. Sanger sequencing identifieda transcript that predicts the coding for 11 amino acids prior to encountering a stop codon into CBFB intron 5. H, High expression of intron5 sequences was observed in blood mononuclear cells from the index patient II.1, whereas low expression was observed for five healthycontrols and the index case her son (III.1). Expression was measured with RT-qPCR with primers indicated with the black boxes in panel G.Expression was normalized against the housekeeping gene Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primer sequences are given inTable S2E138 CORRESPONDENCECorrespondenceBert A. van der Reijden, Geert Grooteplein zuid 10,Nijmegen 6525 GA, the Netherlands.Email: bert.vanderreijden@radboudumc.nlDOI 10.1002/ajh.25770ORCIDMaaike G.J.M. van Bergen https://orcid.org/0000-0001-9846-1777REFERENCES1. Monteferrario D, Bolar NA, Marneth AE, et al. A dominant-negative GFI1Bmutation in the gray platelet syndrome.NEngl JMed. 2014;370(3):245-253.2. Marneth AE, Van Heerde WL, Hebeda KM, et al. Platelet CD34 expres-sion and α/δ-granule abnormalities in GFI1B-and RUNX1-related famil-ial bleeding disorders. Blood. 2017;129(12):1733-1736.3. Saes JL, Simons A, de Munnik SA, et al. Whole exome sequencing inthe diagnostic workup of patients with a bleeding diathesis.Haemophilia. 2019;25(1):127-135.4. Wang CQ, Chin DW, Chooi JY, et al. Cbfb deficiency results in differ-entiation blocks and stem/progenitor cell expansion in hematopoiesis.Leukemia. 2015;29(3):753-757.5. Khan A, Hyde RK, Dutra A, Mohide P, Liu P. Core binding factor beta(CBFB) haploinsufficiency due to an interstitial deletion at 16q21q22resulting in delayed cranial ossification, cleft palate, congenital heartanomalies, and feeding difficulties but favorable outcome. Am J MedGenet A. 2006;140(21):2349-2354.6. van Oorschot R, Marneth AE, Bergevoet SM, et al. Inherited missensevariants that affect GFI1B function do not necessarily cause bleedingdiatheses. Haematologica. 2019;104(6):e260-e264.7. Rabbolini DJ, Morel-Kopp MC, Chen Q, et al. Thrombocytopenia andCD34 expression is decoupled from alpha-granule deficiency withmutation of the first growth factor-independent 1B zinc finger.J Thromb Haemost. 2017;15(11):2245-2258.8. Thambyrajah R, Patel R, Mazan M, et al. New insights into the regula-tion by RUNX1 and GFI1(s) proteins of the endothelial to hematopoi-etic transition generating primordial hematopoietic cells. Cell Cycle.2016;15(16):2108-2114.SUPPORTING INFORMATIONAdditional supporting information may be found online in theSupporting Information section at the end of this article.Received: 15 February 2020 Revised: 3 March2020Accepted: 9 March2020DOI: 10.1002/ajh.25817Source plasma collection inthe United States: Toward amore personalized approachTo the Editor:Donor plasmapheresis is one of the most frequent medical proce-dures performed in healthy individuals in the United States (U.S.).In 2017 alone, approximately 30 million liters of sourceplasma were collected in approximately 40 million procedures inthe U.S.Donated plasma is integral to satisfying the growing need forplasma-based medicines. This increasing need is driven by demo-graphic changes, epidemiology, improvements in diagnostic rates, andnew and expanding therapeutic uses. Key indications include inheritedand acquired immune deficiency disorders and autoimmune disorders.The majority of the global source plasma supply is collected in theU.S., where donors are compensated and federal regulations permit morefrequent and higher volume donations than in Europe. The U.S. is self-sufficient for source plasma, a goal that many European countries stillaim to achieve. However, there currently is a critical shortage of plasma-derived medicines, particularly of intravenous immunoglobulin (IVIG)1 inthe U.S. and abroad that is impacting patients.Concerns for donor safety focus on well-understood short-termeffects, most importantly, vasovagal hypotensive events and, less fre-quently, citrate reactions. Longer-term potential complications, includ-ing iron or protein depletion and osteoporosis, are less well-characterized. Previous studies have demonstrated a temporary reduc-tion in serum protein levels but have also shown a rebound effect, evenwith intensive donation schedules.2 Regular monitoring of proteinlevels in serial plasma donors has been incorporated into federal regula-tions as a safety measure (21CFR630.15 and 21CFR640.65). Thehypothesized risks of osteoporosis due to citrate effects and iron deple-tion have not been confirmed and long-term observational studies haveestablished the safety of regular donations.3A nomogram regulating the volume of source plasma that can beextracted per donation serves as a key instrument to ensure donorsafety and, in particular, to reduce the likelihood of hypotensiveevents. The U.S. Food and Drug Administration (FDA) issued its cur-rent plasmapheresis nomogram in 1992.4 To maximize ease of useand to minimize operator error, the nomogram consists simply ofthree allowable collection volume categories based on donor weight.For nearly 30 years, this nomogram has proven to be effective,resulting in a strong safety record for donor plasmapheresis. Reactionrates are very low, with fewer than 0.03% severe reactions.5 However,while the 1992 nomogram fulfilled the objective of simplicity, it has limi-tations. It does not account for height or body mass index (BMI), nor forhematocrit levels. These factors are known to influence the total plasmavolume (TPV) of a donor and would be valuable components of a morepersonalized approach to calculate target collection volumes.6 Moreover,the current weight-based step-wise approach leads to abrupt targetchanges of up to 20% between groups. In summary, while the safetyprofile has been good across the donor population, there could be sub-groups of donors at increased, yet currently unquantified risk.Here, we report a systematic retrospective analysis of a largereal-world data set of source plasma collections following currentU.S. standards to better understand the implications and potentialopportunities for improvement. De-identified data from 111 916 rou-tine plasma collections (all-comers) performed in February 2019 at86 nationwide U.S. plasma donation centers (Octapharma Plasma,Charlotte, North Carolina) were obtained. For each donation, donorweight, height and hematocrit level, as well as the plasma volumeCORRESPONDENCE E139This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,provided the original work is properly cited.

Platelet CD34 expression in a patient with a partial deletion of transcription factor subunit CBFB

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Platelet CD34 expression in a patient with a partial deletion of transcription factor subunit CBFB

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