Late Diagnosis of NBEAL2-related Gray Platelet Syndrome in Finnish Siblings with Lifelong Thrombocytopenia

Abstract

In the case of familial thrombocytopenia, a congenital defect should be considered; however, in particular older patients who have had thrombocytopenia for a long time have often not yet been genetically analyzed using modern sequencing methods. Remarkably, sometimes they are still suspected of suffering from chronic immune thrombocytopenia until genetic testing reveals a congenital defect. We report on elderly Finnish siblings (both older than 60 years) with lifelong thrombocytopenia. The lifelong bleeding tendency in both the siblings was usually treated with tranexamic acid and platelet transfusions when necessary. In 2022, the older brother presented at the University Hospital in Helsinki because he had recently been suffering from gastrointestinal bleeding and also had mild pancytopenia. Because his sister lived abroad, the Finnish colleagues recommended that the sister should present to the University Hospital in Freiburg. Independent genetic testing of both the siblings using NGS identified the diagnosis of NBEAL2-associated gray platelet syndrome. The disease comprises macrothrombocytopenia and a reduction of α-granules in platelets, resulting in a grayish appearance of platelets on the blood smear. Patients usually suffer from a mild to moderate bleeding diathesis. Interestingly, during the last years a more syndromic character of the disease has been described: besides the platelet phenotype, the immune system can also be affected. In the course of the disease patients may develop pancytopenia, splenomegaly, and bone marrow fibrosis. Comprehensive diagnostics including molecular genetic analyses are particularly important to provide these patients with adequate care and treatment.

Keywords

platelet disorder - congenital thrombocytopenia - gray platelet syndrome - NBEAL2 - α storage pool disease

Introduction

Congenital thrombocytopenia is a heterozygous group of disorders, mainly affecting platelet production in the bone marrow.[1] Increased turnover of platelets in spleen or liver may also arise due to inherited altered glycosylation profile of the platelets.[2] Gray platelet syndrome (GPS, MIM # 139090) is a rare bleeding disorder of mild to moderate severity characterized by moderate macrothrombocytopenia and grayish appearance of the patient's platelets in the peripheral blood smear due to a severe shortage (or even absence) of α-granules.[3] [4] In 2011 biallelic pathogenic alterations in NBEAL2, which contains 54 exons, have been identified to cause the disease.[5] [6] [7] The encoded large scaffolding protein, neurobeachin-like protein 2, is composed of 2,754 amino acids and contains the BEACH domain making it a member of the BEACH domain-containing family.[8] The protein is involved in granule trafficking and the development of α-granules.[9] [10] Alpha-granules are the most abundant platelet granules and the main storage granule in platelets. They store proteins that promote platelet adhesiveness (e.g., VWF, thrombospondin, fibrinogen), blood coagulation (e.g., factors V, XI, and XIII), inflammation, and wound healing (e.g., cytokines, pro- and anti-inflammatory factors) when secreted during platelet activation.[11] [12] Some α-granule proteins are synthesized in megakaryocytes and differentially sorted and packed into these granules, whereas others are either passively or actively taken up from the plasma by receptor-mediated endocytosis.[13] Small or differentially packed α-granules can still be seen in GPS platelets. Also, a consistent reduction of α-granule cargo has been shown.[14] [15] NBEAL2 is expressed in hematopoietic cells, including megakaryocytes, platelets, monocytes, neutrophils, and NK (natural killer) cells. Nbeal2 is important for normal granule function in megakaryocytes, platelets, and in a variety of myeloid and lymphoid cells in mice.[16] [17] This has also been shown for a larger cohort of GPS patients, in which multiple types of blood cells are deficient in granule proteins, and the plasma proteome has a proinflammatory profile. Immune dysregulation is overrepresented in GPS patients, comprising autoimmune diseases, positive autoantibodies, and reduced leukocyte counts.[18] Interestingly, NBEAL2 deficiency leads to low CTLA-4 expression in patient-derived effector T-cells, while their regulatory T-cells appear unaffected.[19] In humans, autosomal-dominant, inherited, disease-causing variants in CTLA4 are associated with autoimmunity, immunodeficiency, and lymphoproliferation (MIM # 123890). These findings give evidence that GPS is a syndromic disease affecting not only platelets, but also immune cells. Over time the patients can develop pancytopenia, splenomegaly, and bone marrow fibrosis.[20] Besides NBEAL2, alterations in other genes (e.g., GFI1B, GATA1, VPS33B, and VIPAS39) may also be associated with pale platelets due to a significant reduction in α-granules.[21] [22] However, phenotypes of these diseases may vary.

Here, we present the case of two siblings (older brother P1 and his sister P2) in their 60s with a lifelong thrombocytopenia and a late genetic diagnosis of GPS. The case illustrates the importance of genetic analysis for accurate diagnosis and personalized treatment.

Case Presentation

All procedures were conducted in accordance with the declaration of Helsinki. P2 was included in the study “biochemical and molecular genetic characterization of inherited platelet disorders” approved by Albert-Ludwigs-University Freiburg’s institutional review board (EK584/17).

Patient 1 (Brother)

We received a referral concerning Patient 1 (P1), a 66-year-old male, a few years ago. He had initially been diagnosed with familial thrombocytopenia not otherwise specified at the age of 8. His platelet counts have ranged from 30 to 50 G/L resulting in easy bruising tendency throughout his life. No major bleedings occurred until 2022. Yet, tranexamic acid and single platelet transfusions had been given as preventive measures occasionally. He had had two minor surgical procedures for nasal cartilage and mandibular cyst in 1995 and 2005, respectively.

The patient was referred to us after being diagnosed with stenotic bicuspid aortic valve needing an operation in 2023. His blood picture then showed mild normocytic anemia, leukocytopenia with moderate lymphocytopenia, mild neutropenia, and thrombocytopenia (range 26 to 50 G/L). His spleen size was mildly enlarged measuring 13.5 cm vertically by ultrasound. His parents had had normal platelet counts; however, one sister was known to be thrombocytopenic. By the time of referral, P1 had also recently presented with repetitive gastrointestinal bleeding episodes due to Helicobacter pylori negative hemorrhagic gastritis in a local hospital. The bleedings had led to significant anemia (Hb nadir 81 g/L) and iron deficiency that had been corrected by intravenous iron supplementation and occasional red blood cell and platelet transfusions. The exact bleeding site remained unclear after several gastro- and colonoscopies and capsule endoscopy for the small intestine. However, some angiodysplasia patches in the sigmoid colon were noticed and small polyps removed.

Due to three-lineage cytopenia in the patient's CBC, we performed peripheral blood and bone marrow (BM) morphological analyses and examined his BM histology, karyotype, and a myeloid NGS panel. The patient's platelets had a shadow appearance; however, no pathology was observed in the BM analyses. His plasma thrombin time, activated thromboplastin time, and prothrombin time were within normal limits. Since then, no anemia has reoccurred. Mild leukocytopenia, resulting mainly from moderate lymphopenia, has been stable.

A transcatheter aortic valve implantation (TAVI) procedure was performed successfully in February 2024. Prior to the procedure, 2 units of platelets were administered, and an additional 2 units were given during the procedure. The procedure was completed without immediate complications. A 29-mm VLV Evolut FX Medtronic bioprosthetic valve was implanted. No bleeding complications occurred. Platelet count increased from 32 to 95 following the platelet transfusions. ECG showed no conduction disturbances or bundle branch block. The patient was discharged on the first postoperative day. Aspirin was not initiated due to the patient's significant bleeding risk and a likely decreased risk of thrombosis. The cardiologist recommended avoiding tranexamic acid for 1 to 3 months after the procedure.

Patient 2 (Sister)

Before the genetic analysis was performed for P1, the Finnish hematologists recommended that his sister should contact the pediatric coagulation outpatient clinic at the University Hospital Freiburg in Germany. Therefore, in July 2023, the 64-year-old woman (P2) presented in the pediatric coagulation outpatient clinic in Freiburg for the first time. Thrombocytopenia had been diagnosed in 1989; since 1993 an α-storage pool disease had been suspected; however, at that time the genetic correlate of this disease was still unknown. The bleeding score according to ISTH-BAT of 13 points (normal range for women up to 5 points) results from very heavy menstrual bleeding before menopause, which led to a transfusion when she was a teenager. In addition, P2 experienced bleeding after a tonsillectomy and tooth extractions. Nosebleeds are a minor problem. The patient described that her platelets are usually between 40 and 50 G/L, with a minimum of 30 G/L and a maximum of 70 G/L. She always coped well with tranexamic acid therapy during menstruation. Before the gallbladder was removed, she had received platelet concentrate. Cataract surgery in 2024 went without any complications using tranexamic acid. On the date of the visit in Freiburg, her platelet count was 48 G/L (Ref. 176–391 G/L) and MPV 12.1 fL (Ref.[7] [8] [9] [10] [11] [12] fL). Red and white blood cells were all within normal ranges. Peripheral blood smear for P2 showed partly non-granulated, pale platelets ([Fig. 1A]). We performed platelet flow cytometry as previously described,[23] which surprisingly revealed a combined secretion defect. CD62-P (α-granule secretion marker) and CD63 (lysosomal and delta-granule marker) exposure after thrombin activation was severely reduced ([Fig. 1B, 1C]). Normal values were achieved for VWF binding after ristocetin stimulation, fibrinogen binding after ADP stimulation, platelet surface expression of GPIb/IX (measured with CD42a), GPIb (CD42b), and GPIIb/IIIa (CD41). Slightly elevated anti-cardiolipin antibodies (IgG 43 U/mL, norm <40 U/mL; measured 2022 in Switzerland) and slightly elevated anti-phospholipid antibodies (β2-GP-IgG 19, norm <14; measured 2023 in Freiburg) may refer to an additional immunological disease. The other parameters of the blood count, the liver and kidney values, and the serum electrolytes were unremarkable.

Fig. 1 Blood smear and platelet flow cytometry for P2. (A) Large, partly non-granulated, pale platelets (indicated by black arrow) in the peripheral blood smear of P2 (Giemsa stain, magnification 60x). Platelet flow cytometry for P2 compared with healthy controls (day control and 20 independent measurements from 10 controls as mean ± standard error of the mean). Platelet surface exposure of (B) CD62 (P-selectin) and (C) CD63 stimulated with thrombin (concentrations: 0, 0.05, 0.1, 0.2, 0.5, 1 U/mL) are severely reduced.

Genetic Analysis Independently Performed Revealed a Homozygous 5-base Pair Duplication in NBEAL2 Present in Both Siblings

In-house NGS gene panels were used for genetic analysis of peripheral blood in Helsinki (297-gene germline testing for hematology diseases, customized core exome kit, Twist Bioscience, USA) and in Freiburg (95-gene panel, custom designed, Illumina, USA) independently for the two affected siblings P1 and P2. A list of genes (included in the hematology diseases customized core exome kit) is available on request. The list of all genes included in the Freiburg panel has been previously described.[24] High throughput sequencing was performed on a NovaSeq 6000 platform and a MiSeq (Illumina, USA), respectively. We identified a homozygous 5-base pair duplication (NM_015175.3(NBEAL2):c.4371_4375dup, rs1233482159) in exon 28 of the NBEAL2 gene ([Fig. 2A, B]) to be present in both the siblings. The duplication leads to a change in the reading frame and a premature stop codon (p.Glu1459Alafs*43) resulting in a non-functional protein ([Fig. 2C]). The parents of the two siblings are already deceased and therefore genetic analysis is not possible. However, we performed CNV analysis in the siblings without detecting any evidence of a monoallelic exon or gene deletion masking homozygosity. This duplication is listed in gnomAD Exomes (v4.1) with an allele frequency of 0.000616% globally, and 0.00942% in Finns (9 alleles in total, 5 of them in the European Finnish population, only heterozygotes reported in gnomAD). In ClinVar the duplication is listed as likely pathogenic for GPS (Accession: VCV001705844.1, Submitter: ISTH-SSC Genomics in Thrombosis and Hemostasis, September 17, 2022). The duplication has already been described compound heterozygous with a splice-site mutation in one patient.[5]

Fig. 2 Genetic analyses for the siblings identifying biallelic NBEAL2 (NM_015175.3):c.4371_4375dup. (A) NGS for P1 (genome analysis toolkit [GATK]). (B) Confirmation of the homozygous 5-base pair duplication identified via panel NGS using direct sequencing of NBEAL2 exon 28 for P2 (Chromatograms loaded in SEQUENCE Pilot®; JSI medical systems, Ettenheim, Germany). (C) Adapted NBEAL2 protein with the pleckstrin homology domain in BEACH domain containing proteins (PH_BEACH in orange), the Beige and Chediak-Higashi domain (BEACH in red), and the WD40 repeats. NBEAL2 frameshift variants listed in ClinVar are visualized using the Protein Paint web application (https://pecan.stjude.cloude/home). Framed in blue the p.Glu1459Alafs*43 loss of function variant identified in the siblings P1 and P2.

Discussion

“Loss of function” variants in NBEAL2 lead to GPS. We showed cosegregation with disease independently from the working group in two family members in a gene known to cause the disease. Therefore, we classify the duplication as pathogenic (ACMG criteria: PVS1, PM2, PP1, PP4, PP5). NGS finally revealed the genetic background of the familiar primary thrombocytopenia. In recent years, there has been a debate in the literature as to whether all cases of pale platelets should be referred to as GPS and GFI1B-related pale platelets should be designated as autosomal dominant GPS.[25] [26] [27] However, because of phenotypical differences between the different diseases affecting α-granule biogenesis or function, the term gray platelet syndrome should be used for NBEAL2-related GPS like listed in OMIM. Ultimately, molecular genetic analyses is the only way to make a specific diagnosis for these overlapping phenotypes.

Before the NBEAL2 defect had been identified, bone marrow biopsy, which did not show signs of bone marrow fibrosis or myelodysplastic syndrome, had been performed for P1 because of three-lineage cytopenia. The additionally performed cytogenetic analysis was normal, and a somatic gene panel test remained without myeloid mutations. For the treatment of P1 the use of thrombopoietin agonist medication (or any other platelet-increasing medical treatment) was not recommended because of risk for bone marrow fibrosis associated with GPS. His sister P2 presented with normal red and white blood cell counts. Regular follow-up examinations should be performed to detect any bone marrow fibrosis that may develop. P2's bleeding phenotype decreased with menopause; P1 was only mildly affected until he developed intestinal bleeding in 2022. Tranexamic acid worked well for both the siblings to prevent/treat bleeding during minor surgeries. Nevertheless both the siblings needed platelet transfusions at least once in their life.

Alpha-granule deficiency is a constant feature of GPS. Platelets characteristically show absent or markedly reduced α-granules and frequently display prominent vacuolization of the cytoplasm, whereas the content of dense granules and lysosomes seems to be preserved.[28] [29] Flow cytometry analysis performed for P2 showed reduced exposure of CD62 after ADP-induced platelet activation as expected. However, in addition, CD63 exposure (lysosomal and delta-granule secretory marker) was also reduced.

Mayer et al showed that NBEAL2 interacts with the guanine nucleotide exchange factor DOCK7 probably via its PH-BEACH domain and the endoplasmic reticulum export factor SEC16A to regulate actin reorganization, cytoskeletal rearrangements, and vesicular transport. DOCK7 is nearly depleted from platelets in GPS patients and in Nbeal2^−/− mice.[10] The reduced CD63 exposure after activation observed in P2's platelets may hint to a functional delta-granule secretion defect due to an altered cytoskeleton arrangement.

NGS is an important tool to identify individual diagnosis and provide appropriate therapy, although NGS may not always be able to identify the genetic alteration causing the inherited thrombocytopenia. In conclusion, NGS should be performed in cases of familiar or lifelong thrombocytopenia. Patients with NBEAL2-related GPS present not only with thrombocytopenia, but also with an α-granule defect and in some cases with an additional delta-granule defect. Furthermore, patients with NBEAL2-related GPS can develop myelofibrosis and/or immunological alterations. Because of the risk of developing bone marrow fibrosis platelet-increasing medical treatment should be avoided.

Conflicts of Interest

The authors declare that they have no conflict of interest.

Acknowledgments

Barbara Zieger, Terhi Friman, and Ulla Wartiovaara-Kautto are members of the European Reference Network on Rare Haematological Diseases (ERN-EuroBloodNet) - Project ID N° 101085717.

• References

• 1 Nurden AT, Nurden P. Inherited thrombocytopenias: history, advances and perspectives. Haematologica 2020; 105 (08) 2004-2019
• 2 Zieger B, Boeckelmann D, Anani W. et al. Novel GNE gene variants associated with severe congenital thrombocytopenia and platelet sialylation defect. Thromb Haemost 2022; 122 (07) 1139-1146
• 3 White JG, Brunning RD. Neutrophils in the gray platelet syndrome. Platelets 2004; 15 (05) 333-340
• 4 Raccuglia G. Gray platelet syndrome. A variety of qualitative platelet disorder. Am J Med 1971; 51 (06) 818-828
• 5 Kahr WH, Hinckley J, Li L. et al. Mutations in NBEAL2, encoding a BEACH protein, cause gray platelet syndrome. Nat Genet 2011; 43 (08) 738-740
• 6 Gunay-Aygun M, Falik-Zaccai TC, Vilboux T. et al. NBEAL2 is mutated in gray platelet syndrome and is required for biogenesis of platelet α-granules. Nat Genet 2011; 43 (08) 732-734
• 7 Albers CA, Cvejic A, Favier R. et al. Exome sequencing identifies NBEAL2 as the causative gene for gray platelet syndrome. Nat Genet 2011; 43 (08) 735-737
• 8 Cullinane AR, Schäffer AA, Huizing M. The BEACH is hot: a LYST of emerging roles for BEACH-domain containing proteins in human disease. Traffic 2013; 14 (07) 749-766
• 9 Lo RW, Li L, Leung R, Pluthero FG, Kahr WHA. NBEAL2 (neurobeachin-like 2) is required for retention of cargo proteins by α-granules during their production by megakaryocytes. Arterioscler Thromb Vasc Biol 2018; 38 (10) 2435-2447
• 10 Mayer L, Jasztal M, Pardo M. et al. Nbeal2 interacts with Dock7, Sec16a, and Vac14. Blood 2018; 131 (09) 1000-1011
• 11 Golebiewska EM, Poole AW. Platelet secretion: from haemostasis to wound healing and beyond. Blood Rev 2015; 29 (03) 153-162
• 12 Chen CH, Lo RW, Urban D, Pluthero FG, Kahr WH. α-granule biogenesis: from disease to discovery. Platelets 2017; 28 (02) 147-154
• 13 Battinelli EM, Thon JN, Okazaki R. et al. Megakaryocytes package contents into separate α-granules that are differentially distributed in platelets. Blood Adv 2019; 3 (20) 3092-3098
• 14 Bottega R, Nicchia E, Alfano C. et al. Gray platelet syndrome: novel mutations of the NBEAL2 gene. Am J Hematol 2017; 92 (02) E20-E22
• 15 Nurden AT, Nurden P. The gray platelet syndrome: clinical spectrum of the disease. Blood Rev 2007; 21 (01) 21-36
• 16 Claushuis TAM, de Stoppelaar SF, de Vos AF. et al. Nbeal2 deficiency increases organ damage but does not affect host defense during gram-negative pneumonia-derived sepsis. Arterioscler Thromb Vasc Biol 2018; 38 (08) 1772-1784
• 17 Drube S, Grimlowski R, Deppermann C. et al. The neurobeachin-like 2 protein regulates mast cell homeostasis. J Immunol 2017; 199 (08) 2948-2957
• 18 Sims MC, Mayer L, Collins JH. et al; NIHR BioResource. Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome. Blood 2020; 136 (17) 1956-1967
• 19 Delage L, Carbone F, Riller Q. et al. NBEAL2 deficiency in humans leads to low CTLA-4 expression in activated conventional T cells. Nat Commun 2023; 14 (01) 3728
• 20 Tariq H, Perez Botero J, Higgins RA, Medina EA. Gray platelet syndrome presenting with pancytopenia, splenomegaly, and bone marrow fibrosis. Am J Clin Pathol 2021; 156 (02) 253-258
• 21 Monteferrario D, Bolar NA, Marneth AE. et al. A dominant-negative GFI1B mutation in the gray platelet syndrome. N Engl J Med 2014; 370 (03) 245-253
• 22 Tubman VN, Levine JE, Campagna DR. et al. X-linked gray platelet syndrome due to a GATA1 Arg216Gln mutation. Blood 2007; 109 (08) 3297-3299
• 23 Neubauer K, Boeckelmann D, Koehler U. et al. Hereditary neuralgic amyotrophy in childhood caused by duplication within the SEPT9 gene: a family study. Cytoskeleton (Hoboken) 2019; 76 (01) 131-136
• 24 Boeckelmann D, Wolter M, Neubauer K. et al. Hermansky-Pudlak syndrome: identification of novel variants in the genes HPS3, HPS5, and DTNBP1 (HPS-7). Front Pharmacol 2022; 12: 786937
• 25 Nurden AT, Nurden P. Should any genetic defect affecting α-granules in platelets be classified as gray platelet syndrome?. Am J Hematol 2016; 91 (07) 714-718
• 26 Palma-Barqueros V, Revilla N, Sánchez A. et al. Inherited platelet disorders: an updated overview. Int J Mol Sci 2021; 22 (09) 4521
• 27 Boeckelmann D, Glonnegger H, Sandrock-Lang K, Zieger B. Pathogenic aspects of inherited platelet disorders. Hamostaseologie 2021; 41 (06) 460-468
• 28 White JG. Ultrastructural studies of the gray platelet syndrome. Am J Pathol 1979; 95 (02) 445-462
• 29 Bottega R, Pecci A, De Candia E. et al. Correlation between platelet phenotype and NBEAL2 genotype in patients with congenital thrombocytopenia and α-granule deficiency. Haematologica 2013; 98 (06) 868-874

Publication History

Received: 15 May 2025

Accepted: 27 August 2025

Article published online:
15 October 2025