Healthcare Professional

Welcome to the Rare Haematology Resource Centre. This site is intended for healthcare professionals in Australia and New Zealand only. By clicking the link below, you are declaring and confirming that you are a healthcare professional.

You are here

Combined alpha-delta platelet storage pool deficiency is associated with mutations in GFI1B

Molecular Genetics and Metabolism, Volume 120, Issue 3, March 2017, Pages 288 - 294


Combined alpha-delta platelet storage pool deficiency is characterized by the absence or reduction in the number of both alpha granules and dense bodies. This disorder can have variable severity as well as a variable inheritance pattern. We describe two patients from unrelated families with combined alpha-delta storage pool deficiency due to mutations in GFI1B, a zinc finger protein known to act as a transcriptional repressor of various genes. We demonstrate that this disease is associated with either a heterozygous mutation (de novo or familial) abrogating the binding of the zinc fingers with the promoter of its target genes, or by hypomorphic biallelic mutations in GFI1B leading to autosomal recessive inheritance.


  • Combined αδ–storage pool deficiency is characterized by absence or reduction in number of alpha granules and dense bodies.
  • This disorder has variable inheritance and severity.
  • We show that the disease is caused by mutations in GFI1B.
  • Either heterozygous dominant negative mutations (de novo or familial) or hypomorphic biallelic mutations lead to disease.

Keywords: Combined alpha-delta platelet storage pool deficiency, αδ-SPD, GFI1B.

1. Introduction

Normal platelets have 4–8 dense bodies and numerous alpha granules; combined alpha-delta platelet storage pool deficiency (αδ-SPD, OMIM 185050) is defined as the absence or severe deficiency of both alpha and delta granules. In 1979, Weiss et al. first described αδ-SPD in 7 patients [1], followed by a few reports of patients with quite heterogeneous presentations. Indeed, αδ-SPD can be inherited either in an autosomal dominant manner [1] and [2] or appear sporadically [1], [3], and [4]. The severity of granule deficiency also varies [1], along with the surface expression of platelet markers [5]. Since the original description of αδ-SPD, its etiology has remained elusive, partly because of the scarcity of published reports, and partly because of the heterogeneity of the disease.

Isolated deficiency of dense granules characterizes Hermansky-Pudlak Syndrome (HPS), a disorder of lysosome-like organelles presenting with oculocutaneous albinism, a bleeding diathesis and, in 3 of the 10 genetic subtypes of HPS, pulmonary fibrosis. Isolated alpha granule deficiency is called Gray Platelet Syndrome (GPS), based upon the gray appearance of platelets on Wright stain. The genetic basis of GPS was elucidated by three different groups, who demonstrated that biallelic mutations in NBEAL2 cause this disorder [6], [7], and [8]. Recently, a monoallelic mutation in GFI1B was also reported to cause GPS, characterized by macrothrombocytopenia, reduced or absent alpha granules, and dysplastic megakaryocytes [9]. In addition, a GFI1B mutation segregated with an isolated bleeding diathesis of variable severity in a family with mild to moderate macrothrombocytopenia and subtle red cell changes consisting of anisopoikylocytosis and increased red blood cell distribution width [10] and [11], and in a family with thrombocytopenia, enlarged hypogranular platelets and abnormally shaped red blood cells [12]. Indeed, GFI1B is known to participate in both megakaryopoiesis and erythropoiesis [13] and [14].

We now demonstrate that both autosomal dominant and autosomal recessive αδ-SPD are associated with mutations in GFI1B.

2. Methods

2.1. Patients

Patients were enrolled in clinical protocol 76-HG-0238, “Diagnosis and treatment of patients with inborn errors of metabolism and other genetic disorders” (identifier: NCT00369421), approved by the NHGRI Institutional Review Board. Parents gave written, informed consent for their children, in accordance with the Declaration of Helsinki.

2.2. Electron microscopy

Whole mount and thin section electron microscopy was performed as previously described [15] and [16]. Briefly, blood was collected into an ACD tube at room temperature, and the sample was centrifuged to obtain platelet rich plasma (PRP). For the whole mount technique, small drops of the PRP were placed on formvar-coated electron microscopy grids, rinsed for 10–15 s with drops of distilled water, dried from the periphery with filter paper and waved in the air to remove any residual moisture. The grids were then inserted into the electron microscope unfixed and unstained. Whole mount electron microscopy of a typical normal platelet is shown in Fig. 1A. The dense granules are dark opaque round-shaped bodies. Both dense granule calling criteria and the normal range of mean dense granules/platelet is ≥ 1.2 based on a normal donor study of 111 healthy volunteers were previously reported [17].

Fig. 1

Fig. 1

Light and electron microscopy of patients platelets and megakaryocytes.

A: Whole mount electron microscopy of a platelet from a normal control donor. Arrow points to a dense granule. B: Thin section electron microscopy of platelet from a normal donor. Arrow points to an alpha granule. C: Dense bodies were virtually absent on whole mount electron microscopy of platelets from patient 1. D: Small dense bodies (DB) could be found in occasional platelets from patient 1 by thin section electron microscopy. E: Absent alpha granules inside platelets from patient 1; mitochondria have become the predominant organelle, and many are enlarged (arrow). F: Severely deficient–but present–alpha granules in platelets of patient 1. G: Platelets of patient 1 maintain a normal discoid shape, given the normal circumferential coils of microtubules in the equatorial plane. H: Marked increase in the number of granule-sized empty vacuoles (arrow) of some platelets from patient 1. I: Normal transition from rough endoplasmic reticulum to the dense tubular system elements in platelets from patient 1. J: Peripheral smear (Wright-Giemsa stain, 1000 ×) of patient 2 shows a large platelet with cytoplasmic granules and a smaller platelet without any granules. K: The megakaryocytes in the bone marrow biopsy from patient 2 show marked emperipolesis (arrow head) and apparent cytoplasmic pale pink inclusions (arrow, PAS stain, 400 ×). L: Whole mount electron microscopy of a platelet from patient 2 shows no dense granules. M: Thin section electron microscopy shows a platelet of patient 2 with alpha granules (*) and 2 other platelets (arrows) with rare (left) or no (right) alpha granules. N: A platelet of patient 2 with alpha granules (*) and a platelet with rare alpha granules (arrow). O: A high magnification image of two platelets (arrows) of patient 2 without (on the right) and with rare alpha granules (on the left).


For thin sections, the samples were mixed with an equal volume of 0.1% glutaraldehyde buffered in White's saline. The samples were then centrifuged and the supernatant removed and replaced with 3% glutaraldehyde in White saline. This sample was then maintained at 4 °C for 30 min and sedimented to pellet. The supernant was removed and replaced with 1% osmic acid. The samples were then dehydrated with alcohol, embedded in resin, and cut in thin sections using an ultramicrotome. Thin section electron microscopy of a normal platelet is shown in Fig. 1B.

Light microscopy images were obtained using an Olympus BX51 microscope (Olympus Life Science, Center Valley, PA) equipped with an Olympus DP71 camera; image at 1000 × obtained using a UplanSApo 100 × oil immersion objective lens and a UIS2 10 × lens (Olympus). Electron microscopy images were obtained using a JEOL-1400Plus 120 kV transmission electron microscope (JEOL Ltd., Tokyo, Japan), with an Orius model 832 camera and DigitalMicrograph® acquisition software (Gatan Inc., Pleasanton, CA); pictures taken at 15,000–20,000 ×.

2.3. Sequencing

DNA was extracted from leucocytes using standard methods. Exome sequencing was performed in patient 1 as previously described [18]. Briefly, data were generated using an Illumina TruSeq Exome capture kit and the HiSeq2000 sequencing platform (Illumina, Inc., San Diego, CA). Reads were aligned with human reference genome hg19 (NCBI build 37; Feb. 2009) using Novoalign (Novocraft Technologies, Selangor, Malaysia). Variant calling was performed using the Most Probable Genotype (MPG) algorithm [19]. Called variants were annotated using a custom built process, then analyzed using VarSifter [20]. Initial sequence variant analysis was performed using an autosomal recessive genetic model. A second round of analysis employed a new-dominant model, identifying sequence variants that met the following criteria: (1) excellent coverage in all samples from prior Undiagnosed Diseases Program [21] next-generation sequencing projects; (2) variation from the human reference sequence occurring in only the family being studied; and (3) appearance of the candidate mutation only in affected members of the family.

Sanger sequencing was performed in patient 2. All exon and intron-exon boundaries of GFI1B were amplified by polymerase chain reaction (PCR) using genomic DNA as template. PCR was performed in a final volume of 10 μl containing 50 ng of genomic DNA, 1 μM of forward and reverse primers, and 5 μl of AmpliTaq Gold Master Mix (Applied Biosystems, Waltham, MA). The PCR products were purified using ExoSap-IT for 45 min at 37 °C and sequenced in both directions using the same primers and Big Dye terminator kit v3.1 (Applied Biosystems). Reactions were purified over G-50 Sephadex. Linear amplification products were separated in an automated capillary sequencer (ABI PRISM 3130xl Genetic Analyzer, Applied Biosystems). Sequences were analyzed with Sequencher software 4.8 (Gene Codes Corporation, Ann Arbor, MI). Primer sequences are shown in Supplementary file 1.

3. Results

3.1. Cases

Patient 1 is a 13-year-old boy born full term to non-consanguineous parents. Scalp petechiae were noted immediately after birth; evaluation revealed severe thrombocytopenia (platelets as low as 3000/μl), as well as anemia and a granulocytic left shift. A head CT showed enlargement of the cysterna magna and right lateral ventricle. The infant received platelet transfusions at birth with a less than expected recovery of his platelet count. No platelet antibodies were detected. His anemia and left shift corrected spontaneously over the first few months of life, while the thrombocytopenia persisted. Other pertinent findings included subglandular hypospadias and chordee needing urethroplasty, bilateral partial 2–3 toe syndactyly (also present in his sister and maternal aunt), a Factor V Leiden mutation (c.1691G > A, p.Arg506Gln) inherited from his mother, and right-sided periventricular nodular heterotopia with ipsilateral ventriculomegaly. He developed seizures at 5 years of age.

Over the course of his lifetime, patient 1 had easy bruising and petechiae, mainly in the face, neck folds, and extremities, as well as episodes of prolonged epistaxis and bleeding with injuries that did not respond promptly to DDAVP. Platelet counts averaged from 30 to 40,000/μl, but platelet transfusions were required several times for platelet counts under 10,000/μl. A trend of his pertinent blood counts over the first two years of life is provided in Supplementary file 2.

A platelet function assay at 8 months showed a collagen/epinephrine membrane closure time of > 300 s (reference: 80–144) and a collagen/ADP closure time of > 250 s (ref: 45–110). A bone marrow aspirate and biopsy at 2 months showed a hypercellular marrow (98%) with scattered megakaryocytes that were decreased in number, confirmed through immunostaining for von Willebrand factor. The platelets were variable in size; some were quite small, with decreased cytoplasm. Red cell maturation appeared progressive, but the cytoplasm of late normoblasts appeared somewhat ragged. Myeloid maturation was also progressive, but a left shift was appreciated with a relative decrease in mature segmented neutrophils. A repeat bone marrow aspirate and biopsy at 11 months showed a decreased number of small megakaryocytes with one or two large irregular nuclei and abnormally scant cytoplasm showing rare platelet formation. Granulocytic maturation was abnormal, showing hypogranulation. A concurrent peripheral smear showed a circulating megakaryocyte with single abnormal convoluted nucleus and cytoplasm showing numerous small blebs. Platelet electron microscopy at 3 months revealed a total of 21 dense granules within 100 whole mounted platelets–with 83% of platelets having no dense granules and 17% having 1–2 dense granules per platelet–consistent with a severe delta granule storage pool deficiency (Figs. 1C–D). Thin sections of the buffy coat using transmission electron microscopy demonstrated platelets (about 2–5 μm in diameter) with an additional alpha granule deficiency (Figs. 1E–F). Other than granular deficiency, some morphologic abnormalities such as enlarged mitochondria (Fig. 1E) and empty vacuoles (Fig. 1H) were seen. Other platelet components such as the circumferential microtubules (Fig. 1G) and the dense tubular system (Fig. 1I) were unremarkable. Most of the granulocytes present in the buffy coat were also unusual in their morphology and appeared to be in blastic phase or undergoing apoptosis.

Patient 2 is an 8-year-old male born to Mexican parents who denied consanguinity. At 3 months of age he had multiple spontaneous petechiae along with isolated thrombocytopenia (platelet count 46,000/μl). At 10 months, a bone marrow biopsy showed increased megakaryocytes suggestive of idiopathic thrombocytopenic purpura. By 3 years of age, he had received three treatments with intravenous immunoglobulins (IVIG) without adequate response. Platelet counts ranged from 30,000 to 50,000/μl at baseline, dropping to 15,000 to 20,000 with acute illnesses. The primary bleeding symptoms included spontaneous bruising, petechiae, and prolonged epistaxis, generally well controlled with anti-fibrinolytic agents, although platelet transfusions were required for significant bleeds and surgical procedures.

Over the past 5 years, peripheral smears showed atypical large platelets and hypogranular platelets (Fig. 1J), and two additional bone marrow aspirates showed megakaryocytic hyperplasia with numerous osteoclast-like forms, occasional small monolobated megakaryocytes, and emperipolesis (Fig. 1K). On whole mount electron microscopy, the platelets contained virtually no dense granules (0.05 dense granules per platelet, 200 platelets counted; Fig. 1L). On thin section electron microscopy, 30–40% of platelets (about 2–5 μm in diameter) had markedly decreased numbers of alpha granules (Fig. 1N–O).

3.2. Genetics

Family pedigrees for patients 1 and 2 are shown in Fig. 2A and B, respectively. For patient 1, prior genetic testing included normal TPO and GATA1 sequencing and a normal chromosome microarray. At the NIH, we performed whole exome sequencing and found a heterozygous nonsense mutation in GFI1B, i.e., NM_004188.5:c.793A > T (NP_004179.3:p.Lys265*; Fig. 2C). The mutation appeared de novo, as it was not seen in either parent, and paternity was confirmed based on the inheritance of polymorphisms using next generation sequencing data. Depth of coverage was 124 × at that position, with 60 reads showing the reference allele and 64 reads showing the minor allele; of the latter, 26 reads were from the plus strand and 38 reads belonged to the minus strand, ruling out strand bias. The mutation was confirmed by Sanger sequencing (Supplementary file 3). This truncating mutation is exceedingly rare, as it was not found in 60,706 individuals in the Exome Aggregation Consortium (ExAC, Cambridge, MA) [22]. The amino acid at that position is well conserved through evolution (Supplementary file 4) [23], and the mutation has a high Phred-scaled CADD score of 43, consistent with a nonsense mutation [24]. Despite the fact that this mutation is truncating, the transcript is predicted to escape nonsense-mediated decay, because the mutation is located only 22 nucleotides upstream of the last exon-exon junction [25].

Fig. 2

Fig. 2

Genetics of αδ-SPD patients.

A: Pedigree for patient 1. Squares denote male individuals and circles female individuals, while triangles represent spontaneous abortions, solid symbols affected family members, open symbols unaffected family members, and arrows indicate proband. Dotted symbols indicate carrier state. All symbols used in accordance with the standardized human pedigree nomenclature [43]. B: Pedigree for patient 2. C: Whole exome short read alignments to the reference sequence for patient 1 (top), his father (middle) and his mother (bottom). Patient 1 has a heterozygous change (A > T) in position 793 arising de novo. D: Sanger sequencing chromatograms for patient 2 (top), his father (middle) and his mother (bottom) showing the heterozygous peak in the parents, and a homozygous mutation in the proband. E: Structure of GFI1B, showing the N-terminal SNAG domain and the six C2H2 zinc finger domains located in the C-terminus; dashed lines indicate exon boundaries. Previously reported mutations are shown above the GFI1B model, while the two novel mutations described here are shown below it. F: Structural consequences of the protein-truncating mutations. Zinc fingers 4 and 5 recognize an AATC core sequence by forming base-specific hydrogen bonds between the side chains of specific amino acid residues (colored in red) and the invariant adenines and cytosine of the core sequence [28]. The side chains of residues colored in green establish hydrophobic contact with sugars or bases of DNA, while residues colored in blue establish hydrophilic electrostatic interactions with the DNA phosphodiester backbone. All contact-establishing amino acids are identical in GFI1B and GFI1 in both humans and rats (the latter used for the study of DNA-GFI1B binding by nuclear magnetic resonance spectroscopy) [28].


Given the finding of a GFI1B mutation in patient 1 with αδ-SPD, Sanger sequencing was performed directly on the GFI1B gene of patient 2. A homozygous mutation was found: NM_004188.5:c.923T > C (NP_004179.3:p.Leu308Pro; Fig. 2D). Each parent had one copy of this mutation (Fig. 2D). The change occurred in a highly conserved amino acid position (Supplementary file 5) [23], and is present in only 1 of 121,194 alleles in the general population and, in particular, in 1 of 11,570 alleles in the Latino population [22]. In silico algorithms predict it to be pathogenic; Polyphen-2 predicts it to be probably damaging [26], SIFT predicts the change to be deleterious [27], and CADD assigns it a Phred-scaled score of 27.5 [24].

Both mutations in our patients are located within C-terminal zinc finger regions. The mutation of patient 1 is located in zinc finger 4 (Fig. 2E), one of the three GFI1B zinc fingers (along with zinc fingers 3 and 5) responsible for DNA binding [28]. The truncated protein lacks amino acid residues responsible for hydrogen, electrostatic and hydrophobic bonds with DNA in zinc fingers 4 and 5 (Fig. 2F). The mutation on patient 2 is located in zinc finger 6 (Fig. 2E).

4. Discussion

Megakaryocytes and erythrocytes derive from the same bipotential progenitor cell, whose proliferation and differentiation are regulated by GFI1B [29]. During megakaryocytic differentiation, the megakaryoblast's DNA replicates without subsequent cell division in a process known as endomitosis, leading to a polyploid cell with multilobulated nuclei known as the promegakaryocyte. The cytoplasm of this cell subsequently expands to give rise to platelets. The conditional absence of Gfi1b in mice leads to arrest at the promegakaryocyte stage, after endomitosis is completed but before the maturation of the cytoplasm [30]. In erythrocytes, the conditional inactivation of Gfi1b leads to impaired differentiation of pro-erythroblasts to mature erythrocytes [31]. A long isoform of GFI1B that contains exon 5 is essential for megakaryopoiesis, while a short isoform lacking exon 5 is essential for its erythropoietic role [14].

GFI1B functions as a transcriptional repressor. Its C-terminal zinc fingers bind to DNA, specifically to the promoter region of target genes. The N-terminal SNAG domain recruits the protein machinery that represses transcription; these proteins include the corepressor CoREST, the histone demethylase LSD1, and the histone deacetylases HDAC1 and HDAC2 [32].

Heterozygous truncating mutations in GFI1B have previously been described in three families with autosomal dominant inheritance of macrothrombocytopenia and reduced alpha granules, as well as anisocytosis and poikilocytosis of red blood cells [33], [10], [11], and [9]. A comparison of findings in the three previously reported families, as well as in the present cases, is provided in Table 1. In the single family reported by Monteferrario et al., there was no mention of the number of delta granules, and in fact the thin section electron microscopy images shown reveal reduced or absent delta granules compared with the control image [9]. In the family reported by Stevenson et al. it is noted that dense granules appeared normal in all family members [11]. However, the same family was previously reported by Ardlie et al. in 1976, where it was mentioned that “there seemed to be paucity of dense bodies” on thin sections [10]. More importantly, there is no mention of whole mount electron microscopy being performed in either family; since dense bodies are inherently electron opaque, it is more accurate to evaluate their number by whole mount electron microscopy than by thin sections [2]. Finally, murine megakaryocytes conditionally deficient in Gfi1b have substantially reduced numbers of dense bodies [34].

Table 1

Comparison of clinical, laboratory and genetic findings in families carrying GFI1B mutations.


Family 1 [9] and [33] Family 2 [10] and [11] Family 3 [12] Patient 1 [current paper] Patient 2 [current paper]
Symptoms Mild-to-severe bleeding Easy bruising, spontaneous epistaxes, large hematomas after injuries Mild bleeding Petechiae; prolonged epistaxis Spontaneous bruising; petechiae; prolonged epistaxis
Platelet count, × 109/L (normal: 150–450) 27–80 61–129 40–115 3–67 15–50
Platelet size Increased Some large platelets Variable degree of enlarged platelets Normal MPV Some large, hypogranular platelets
Alpha granules (normal: numerous) Few Some agranular platelets, some with relatively normal number Variable degree of hypogranular platelets (LM) Decreased Markedly decreased
Dense bodies (mean ≥ 1.2/platelet) N/A Probably decreased [10] or normal [11] (only thin section EM) N/A 0.21 0.05
Bone marrow Dysplastic megakaryocytes, normal [33] to increased [9] in number; emperipolesis Dysplastic megakaryocytes, decreased in number Numerous, occasionally small megakaryocytes; emperipolesis
RBC changes Mild anisopoikilocytosis Anisopoikilocytosis Abnormally shaped Anisocytosis
Associated anomalies PNH; hypospadias Persistent PDA
Mutation c.859C > T (p.Gln287*) c.880_881insC (p.His294fs*307) c.814 + 1G > C (p.G272fs*274) c.793A > T (p.Lys265*) c.923T > C (p.Leu308Pro)
Location Deletion of ZF5 Disruption of ZF5 Deletion of ZF5 Disruption of ZF4 and deletion of ZF5 ZF6
Inheritance AD (inherited) AD (inherited) AD (inherited) AD (de novo) AR

Abbreviations: AD, autosomal dominant; AR, autosomal recessive; EM, electron microscopy; LM, light microscopy; MPV, mean platelet volume; N/A, not assessed; PDA, patent ductus arteriosus; PNH, periventricular nodular heterotopia; ZF, zinc finger.

In all previously reported families, a truncating mutation (nonsense in Monteferrario et al. [9], frameshift in Stevenson et al. [11] and canonical splice site in Kitamura et al. [12]) was identified in or proximal to the fifth zinc finger domain, while in patient 1 in this report a truncating nonsense mutation was identified in the fourth zinc finger domain. The three previously reported truncating mutations have been shown to escape nonsense-mediated decay and to exert a dominant negative effect, inhibiting the transcriptional repression activity of the protein [9], [11], and [12]. It is predicted that, as in the case of the three previously described mutations, the truncating variant found in patient 1 will also escape nonsense-mediated decay, since it is located fewer than 50–55 nucleotides upstream of the last exon-exon junction [25]. The autosomal dominant pattern of inheritance can be explained by a dominant negative mechanism through loss of amino acids participating in DNA binding (Fig. 1F), since the expressed truncated protein will interfere with the wild-type protein by competing for the same DNA binding sites, or by quenching the effects of other cofactors of the transcriptional machinery. Haploinsufficiency of Gfi1b in a mouse model leads to no demonstrable abnormalities, while a homozygous deletion leads to fetal lethality [13]. Thus, completely absent GFI1B repressional activity is not compatible with life, and 50% residual activity is not associated with any clinical consequences. The autosomal recessive pattern of inheritance seen in patient 2 is thus likely explained by hypomorphic loss-of-function mutations. It is unknown how missense variants alter protein function. An altered tertiary structure could either decrease the DNA-binding capacity, or occupy DNA binding sites while lacking the capacity to bind cofactors. Thus, uncertainty remains concerning the precise mechanism leading to the reported phenotype, and whether the severity could be modulated by variants in other, modifying genes.

As can be seen in Table 1, a common finding in bone marrow biopsy seems to be that of dysplastic megakaryocytes and emperipolesis. It is more difficult, however, to generalize regarding the number of megakaryocytes, as they have been variously described as decreased (patient 1), normal to slightly increased [33], or increased in number (patient 2 and [9]). It is likely that these differences stem from the fact that the number of megakaryocytes in the bone marrow has been assessed subjectively.

Patient 1 showed a left shift and a relative decrease in the number of mature bone marrow granulocytes early in life. GFI1B is not expressed in granulocyte-monocyte progenitor cells; however, it is then re-expressed in circulating monocytes and granulocytes, pointing towards a role in mobilizing these cells from the bone marrow to the periphery [35]. In fact, immature myeloid cells and monocytes, as well as a reduced number of granulocytes, have previously been described in the bone marrow of Gfi11b/1b knock-in mice [36]. The reason for the spontaneous resolution of anemia seen in patient 1–and the lack of a more prominent red blood cell phenotype in general in patients with GFI1B deficiency–is puzzling, especially considering that GFI1B is still needed for adult erythropoiesis, just as it is for adult megakaryopoiesis [30].

Mutations in another transcription factor, GATA1, lead to dyserythropoietic anemia, macrothrombocytopenia, and deficiency of alpha granules [37]. In addition, some mutations in this X-linked gene cause absence of both alpha and delta granules [38]. The similar phenotypes caused by mutations in these two transcription factors is not surprising, especially in light of the fact that GFI1B is known to interact with GATA1 [39], [40], and [41].

Both patients described here had congenital anomalies, including hypospadias, periventricular nodular heterotopia and syndactyly in patient 1, and persistent patent ductus arteriosus in patient 2. The syndactyly in patient 1 is likely unrelated to the GFI1B mutation, as it segregated independently in the family. It is unknown, however, if the other anomalies are related to the GFI1B variants, or to variants in other genes. Even assuming that variants in other genes were to be found, these would be unlikely to explain the paucity of delta granules, since the fact that the two unrelated patients described here both had decreased number of delta granules and rare variants in GFI1B likely means that there is an association between these findings. In addition, as previously explained, experimental animal data support the role of GFI1B deficiency in dense body reduction [34].

In conclusion, we demonstrate that αδ-SPD is associated with mutations in GFI1B. Our finding of the underlying etiology of αδ-SPD not only expands the known spectrum of pathology associated with mutations in GFI1B, but should open new avenues of investigation into novel therapeutic options for this rare disorder. As an example, it is known that valproic acid, a histone deacetylase inhibitor, induces expression of GFI1B [42]. Further investigations carry the potential to develop interventions that could change the course of αδ-SPD and related disorders.

The following are the supplementary data related to this article.

Download file

Supplementary file 1

Primers used for Sanger sequencing in patient 2.


Download file

Supplementary file 2

Pertinent blood counts during patient's 1 first two years of life.


Supplementary file 3

Supplementary file 3

Mutation confirmation by Sanger sequencing for patient 1, using Mutation Surveyor® DNA variant analysis software (SoftGenetics, State College, PA). The three upper tracks represent data for the forward primer, while the three lower tracks represent data for the reverse primer. Tracks 1 and 6 correspond to the reference peak profile, tracks 2 and 5 correspond to the sample peak profile, and tracks 3 and 4 correspond to the “mutation electropherogram”, with taller peaks indicating greater discordance between the reference and the sample peaks.


Download file

Supplementary file 4

Conservation through 100 vertebrate species for the amino acid position mutated in patient 1 (p.Lys265); hg19, The mutated amino acid position is highlighted in light blue, and depicted tracks include bar graphs for the PhyloP and PhastCons scores, as well as a Multiz alignment of 100 vertebrate species.


Download file

Supplementary file 5

Conservation through 100 vertebrate species for the amino acid position mutated in patient 2 (p.Leu308); hg19, The mutated amino acid position is highlighted in light blue, and depicted tracks include bar graphs for the PhyloP and PhastCons scores, as well as a Multiz alignment of 100 vertebrate species.



Contributions: CRF wrote the initial draft of the manuscript. DC provided expert platelet pathology interpretation. KLS and MCM performed genetic sequencing. DA and MGA provided management and clinical information for patient 1, while SMA provided clinical management for patient 2. TCM analyzed whole exome sequencing data in patient 1. WAG assisted with manuscript preparation and oversaw the study. All authors read and approved the final manuscript.

Disclosure of Conflicts of Interest

The authors declare that they have no competing financial interests.


This study was supported by the Intramural Research Program of the National Human Genome Research Institute, National Institutes of Health. The authors thank the patients and their families for their kind support and assistance. We dedicate this work to the late Dr. James G. White, who contributed to the evaluation of patient 1.


  • [1] H.J. Weiss, L.D. Witte, K.L. Kaplan, B.A. Lages, A. Chernoff, H.L. Nossel, D.S. Goodman, H.R. Baumgartner. Heterogeneity in storage pool deficiency: studies on granule-bound substances in 18 patients including variants deficient in alpha-granules, platelet factor 4, beta-thromboglobulin, and platelet-derived growth factor. Blood. 1979;54:1296-1319
  • [2] J.G. White, S. Keel, M. Reyes, S.M. Burris. Alpha-delta platelet storage pool deficiency in three generations. Platelets. 2007;18:1-10 10.1080/09537100600800172 Crossref
  • [3] A. Kosch, B. Kehrel, U. Nowak-Göttl, J. Häberle, H. Jürgens. Thrombocytic alpha-delta-storage-pool-disease: shortening of bleeding time after infusion of 1-desamino-8-D-arginine vasopressin. Klin. Pädiatr.. 1999;211:198-200 10.1055/s-2008-1043787 Crossref
  • [4] D.A. Biddle, T.G. Neto, A.N. Nguyen. Platelet storage pool deficiency of alpha and delta granules. Arch. Pathol. Lab. Med.. 2001;125:1125-1126 10.1043/0003-9985(2001)125<1125:PSPDOA>2.0.CO;2
  • [5] B. Lages, S.J. Shattil, D.F. Bainton, H.J. Weiss. Decreased content and surface expression of alpha-granule membrane protein GMP-140 in one of two types of platelet alpha delta storage pool deficiency. J. Clin. Invest.. 1991;87:919-929 10.1172/JCI115099 Crossref
  • [6] M. Gunay-Aygun, T.C. Falik-Zaccai, T. Vilboux, Y. Zivony-Elboum, F. Gumruk, M. Cetin, M. Khayat, C.F. Boerkoel, N. Kfir, Y. Huang, D. Maynard, H. Dorward, K. Berger, R. Kleta, Y. Anikster, M. Arat, A.S. Freiberg, B.E. Kehrel, K. Jurk, P. Cruz, J.C. Mullikin, J.G. White, M. Huizing, W.A. Gahl. NBEAL2 is mutated in gray platelet syndrome and is required for biogenesis of platelet α-granules. Nat. Genet.. 2011;43:732-734 10.1038/ng.883 Crossref
  • [7] C.A. Albers, A. Cvejic, R. Favier, E.E. Bouwmans, M.-C. Alessi, P. Bertone, G. Jordan, R.N.W. Kettleborough, G. Kiddle, M. Kostadima, R.J. Read, B. Sipos, S. Sivapalaratnam, P.A. Smethurst, J. Stephens, K. Voss, A. Nurden, A. Rendon, P. Nurden, W.H. Ouwehand. Exome sequencing identifies NBEAL2 as the causative gene for gray platelet syndrome. Nat. Genet.. 2011;43:735-737 10.1038/ng.885 Crossref
  • [8] W.H.A. Kahr, J. Hinckley, L. Li, H. Schwertz, H. Christensen, J.W. Rowley, F.G. Pluthero, D. Urban, S. Fabbro, B. Nixon, R. Gadzinski, M. Storck, K. Wang, G.-Y. Ryu, S.M. Jobe, B.C. Schutte, J. Moseley, N.B. Loughran, J. Parkinson, A.S. Weyrich, J. Di Paola. Mutations in NBEAL2, encoding a BEACH protein, cause gray platelet syndrome. Nat. Genet.. 2011;43:738-740 10.1038/ng.884 Crossref
  • [9] D. Monteferrario, N.A. Bolar, A.E. Marneth, K.M. Hebeda, S.M. Bergevoet, H. Veenstra, B.A.P. Laros-van Gorkom, M.A. MacKenzie, C. Khandanpour, L. Botezatu, E. Fransen, G. Van Camp, A.L. Duijnhouwer, S. Salemink, B. Willemsen, G. Huls, F. Preijers, W. Van Heerde, J.H. Jansen, M.J.E. Kempers, B.L. Loeys, L. Van Laer, B.A. Van der Reijden. A dominant-negative GFI1B mutation in the gray platelet syndrome. N. Engl. J. Med.. 2014;370:245-253 10.1056/NEJMoa1308130 Crossref
  • [10] N.G. Ardlie, W.W. Coupland, G.I. Schoefl. Hereditary thrombocytopathy: a familial bleeding disorder due to impaired platelet coagulant activity. Aust. NZ J. Med.. 1976;6:37-45 Crossref
  • [11] W.S. Stevenson, M.-C. Morel-Kopp, Q. Chen, H.P. Liang, C.J. Bromhead, S. Wright, R. Turakulov, A.P. Ng, A.W. Roberts, M. Bahlo, C.M. Ward. GFI1B mutation causes a bleeding disorder with abnormal platelet function. J. Thromb. Haemost.. 2013;11:2039-2047 10.1111/jth.12368 Crossref
  • [12] K. Kitamura, Y. Okuno, K. Yoshida, M. Sanada, Y. Shiraishi, H. Muramatsu, R. Kobayashi, K. Furukawa, S. Miyano, S. Kojima, S. Ogawa, S. Kunishima. Functional characterization of a novel GFI1B mutation causing congenital macrothrombocytopenia. J. Thromb. Haemost.. 2016;14:1462-1469 10.1111/jth.13350 Crossref
  • [13] S. Saleque, S. Cameron, S.H. Orkin. The zinc-finger proto-oncogene Gfi-1b is essential for development of the erythroid and megakaryocytic lineages. Genes Dev.. 2002;16:301-306 10.1101/gad.959102 Crossref
  • [14] L.M. Polfus, R.K. Khajuria, U.M. Schick, N. Pankratz, R. Pazoki, J.A. Brody, M.-H. Chen, P.L. Auer, J.S. Floyd, J. Huang, L. Lange, F.J.A. van Rooij, R.A. Gibbs, G. Metcalf, D. Muzny, N. Veeraraghavan, K. Walter, L. Chen, L. Yanek, L.C. Becker, G.M. Peloso, A. Wakabayashi, M. Kals, A. Metspalu, T. Esko, K. Fox, R. Wallace, N. Franceshini, N. Matijevic, K.M. Rice, T.M. Bartz, L.-P. Lyytikäinen, M. Kähönen, T. Lehtimäki, O.T. Raitakari, R. Li-Gao, D.O. Mook-Kanamori, G. Lettre, C.M. van Duijn, O.H. Franco, S.S. Rich, F. Rivadeneira, A. Hofman, A.G. Uitterlinden, J.G. Wilson, B.M. Psaty, N. Soranzo, A. Dehghan, E. Boerwinkle, X. Zhang, A.D. Johnson, C.J. O'Donnell, J.M. Johnsen, A.P. Reiner, S.K. Ganesh, V.G. Sankaran. Whole-exome sequencing identifies loci associated with blood cell traits and reveals a role for alternative GFI1B splice variants in human hematopoiesis. Am. J. Hum. Genet.. 2016;99:481-488 10.1016/j.ajhg.2016.06.016 Crossref
  • [15] J.G. White. Use of the electron microscope for diagnosis of platelet disorders. Semin. Thromb. Hemost.. 1998;24:163-168 10.1055/s-2007-995836 Crossref
  • [16] J.G. White. Electron-dense chains and clusters in platelets from patients with storage pool-deficiency disorders. J. Thromb. Haemost.. 2003;1:74-79 Crossref
  • [17] C.B. Uhl, R.L. Barness, M.C. Olson, S.C. Gossman, S. Erdogan, S.I. Gamb, J.E. Charlesworth, R. Lingineni, S.C. Bryant, R.S. Miller, J.L. Salisbury, W.L. Nichols, J.G. White. Platelet storage pool deficiency: establishment of reference ranges for platelet dense granule count by transmission electron microscopy. Blood. 2013;122:3549
  • [18] D.R. Adams, M. Sincan, K. Fuentes Fajardo, J.C. Mullikin, T.M. Pierson, C. Toro, C.F. Boerkoel, C.J. Tifft, W.A. Gahl, T.C. Markello. Analysis of DNA sequence variants detected by high-throughput sequencing. Hum. Mutat.. 2012;33:599-608 10.1002/humu.22035 Crossref
  • [19] J.K. Teer, L.L. Bonnycastle, P.S. Chines, N.F. Hansen, N. Aoyama, A.J. Swift, H.O. Abaan, T.J. Albert, NISC Comparative Sequencing Program, E.H. Margulies, E.D. Green, F.S. Collins, J.C. Mullikin, L.G. Biesecker. Systematic comparison of three genomic enrichment methods for massively parallel DNA sequencing. Genome Res.. 2010;20:1420-1431 10.1101/gr.106716.110 Crossref
  • [20] J.K. Teer, E.D. Green, J.C. Mullikin, L.G. Biesecker. VarSifter: visualizing and analyzing exome-scale sequence variation data on a desktop computer. Bioinforma. Oxf. Engl.. 2012;28:599-600 10.1093/bioinformatics/btr711 Crossref
  • [21] W.A. Gahl, J.J. Mulvihill, C. Toro, T.C. Markello, A.L. Wise, R.B. Ramoni, D.A. Adams, C.J. Tifft. The NIH Undiagnosed Diseases Program and network: applications to modern medicine. Mol. Genet. Metab.. 2016;117:393-400 Crossref
  • [22] M. Lek, K. Karczewski, E. Minikel, K. Samocha, E. Banks, T. Fennell, A. O'Donnell-Luria, J. Ware, A. Hill, B. Cummings, T. Tukiainen, D. Birnbaum, J. Kosmicki, L. Duncan, K. Estrada, F. Zhao, J. Zou, E. Pierce-Hoffman, D. Cooper, M. DePristo, R. Do, J. Flannick, M. Fromer, L. Gauthier, J. Goldstein, N. Gupta, D. Howrigan, A. Kiezun, M. Kurki, A. Levy Moonshine, P. Natarajan, L. Orozco, G. Peloso, R. Poplin, M. Rivas, V. Ruano-Rubio, D. Ruderfer, K. Shakir, P. Stenson, C. Stevens, B. Thomas, G. Tiao, M. Tusie-Luna, B. Weisburd, H.-H. Won, D. Yu, D. Altshuler, D. Ardissino, M. Boehnke, J. Danesh, E. Roberto, J. Florez, S. Gabriel, G. Getz, C. Hultman, S. Kathiresan, M. Laakso, S. McCarroll, M. McCarthy, D. McGovern, R. McPherson, B. Neale, A. Palotie, S. Purcell, D. Saleheen, J. Scharf, P. Sklar, S. Patrick, J. Tuomilehto, H. Watkins, J. Wilson, M. Daly, D. MacArthur. Analysis of Protein-coding Genetic Variation in 60,706 Humans. (bioRxiv, 2015) 10.1101/030338
  • [23] W.J. Kent, C.W. Sugnet, T.S. Furey, K.M. Roskin, T.H. Pringle, A.M. Zahler, D. Haussler. The human genome browser at UCSC. Genome Res.. 2002;12:996-1006 10.1101/gr.229102 (Article published online before print in May 2002)
  • [24] M. Kircher, D.M. Witten, P. Jain, B.J. O'Roak, G.M. Cooper, J. Shendure. A general framework for estimating the relative pathogenicity of human genetic variants. Nat. Genet.. 2014;46:310-315 10.1038/ng.2892
  • [25] E. Nagy, L.E. Maquat. A rule for termination-codon position within intron-containing genes: when nonsense affects RNA abundance. Trends Biochem. Sci.. 1998;23:198-199 Crossref
  • [26] I.A. Adzhubei, S. Schmidt, L. Peshkin, V.E. Ramensky, A. Gerasimova, P. Bork, A.S. Kondrashov, S.R. Sunyaev. A method and server for predicting damaging missense mutations. Nat. Methods. 2010;7:248-249 10.1038/nmeth0410-248
  • [27] P. Kumar, S. Henikoff, P.C. Ng. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat. Protoc.. 2009;4:1073-1081 10.1038/nprot.2009.86 Crossref
  • [28] S. Lee, K. Doddapaneni, A. Hogue, L. McGhee, S. Meyers, Z. Wu. Solution structure of Gfi-1 zinc domain bound to consensus DNA. J. Mol. Biol.. 2010;397:1055-1066 10.1016/j.jmb.2010.02.006
  • [29] V. Randrianarison-Huetz, B. Laurent, V. Bardet, G.C. Blobe, F. Huetz, D. Duménil. Gfi-1B controls human erythroid and megakaryocytic differentiation by regulating TGF-beta signaling at the bipotent erythro-megakaryocytic progenitor stage. Blood. 2010;115:2784-2795 10.1182/blood-2009-09-241752 Crossref
  • [30] A. Foudi, D.J. Kramer, J. Qin, D. Ye, A.-S. Behlich, S. Mordecai, F.I. Preffer, A. Amzallag, S. Ramaswamy, K. Hochedlinger, S.H. Orkin, H. Hock. Distinct, strict requirements for Gfi-1b in adult bone marrow red cell and platelet generation. J. Exp. Med.. 2014;211:909-927 10.1084/jem.20131065 Crossref
  • [31] L. Vassen, H. Beauchemin, W. Lemsaddek, J. Krongold, M. Trudel, T. Möröy. Growth factor independence 1b (gfi1b) is important for the maturation of erythroid cells and the regulation of embryonic globin expression. PLoS One. 2014;9 e96636 10.1371/journal.pone.0096636
  • [32] S. Saleque, J. Kim, H.M. Rooke, S.H. Orkin. Epigenetic regulation of hematopoietic differentiation by Gfi-1 and Gfi-1b is mediated by the cofactors CoREST and LSD1. Mol. Cell. 2007;27:562-572 10.1016/j.molcel.2007.06.039 Crossref
  • [33] R. Kurstjens, C. Bolt, M. Vossen, C. Haanen. Familial thrombopathic thrombocytopenia. Br. J. Haematol.. 1968;15:305-317
  • [34] L. Vassen, T. Moroy. Growth factor independence 1b (Gfi1b) is an essential regulator of late stage megakaryocyte maturation and platelet production. Blood. 2011;118:2358
  • [35] L. Vassen, T. Okayama, T. Möröy. Gfi1b: green fluorescent protein knock-in mice reveal a dynamic expression pattern of Gfi1b during hematopoiesis that is largely complementary to Gfi1. Blood. 2007;109:2356-2364 10.1182/blood-2006-06-030031 Crossref
  • [36] K. Fiolka, R. Hertzano, L. Vassen, H. Zeng, O. Hermesh, K.B. Avraham, U. Dührsen, T. Möröy. Gfi1 and Gfi1b act equivalently in haematopoiesis, but have distinct, non-overlapping functions in inner ear development. EMBO Rep.. 2006;7:326-333 10.1038/sj.embor.7400618 Crossref
  • [37] J.G. White, W.L. Nichols, D.P. Steensma. Platelet pathology in sex-linked GATA-1 dyserythropoietic macrothrombocytopenia I ultrastructure. Platelets. 2007;18:273-283 10.1080/09537100601065825 Crossref
  • [38] J.G. White, A. Thomas. Platelet structural pathology in a patient with the X-linked GATA-1, R216Q mutation. Platelets. 2009;20:41-49 10.1080/09537100802406661 Crossref
  • [39] D.-Y. Huang, Y.-Y. Kuo, J.-S. Lai, Y. Suzuki, S. Sugano, Z.-F. Chang. GATA-1 and NF-Y cooperate to mediate erythroid-specific transcription of Gfi-1B gene. Nucleic Acids Res.. 2004;32:3935-3946 10.1093/nar/gkh719 Crossref
  • [40] P. Rodriguez, E. Bonte, J. Krijgsveld, K.E. Kolodziej, B. Guyot, A.J.R. Heck, P. Vyas, E. de Boer, F. Grosveld, J. Strouboulis. GATA-1 forms distinct activating and repressive complexes in erythroid cells. EMBO J.. 2005;24:2354-2366 10.1038/sj.emboj.7600702 Crossref
  • [41] E. Anguita, A. Villegas, F. Iborra, A. Hernández. GFI1B controls its own expression binding to multiple sites. Haematologica. 2010;95:36-46 10.3324/haematol.2009.012351 Crossref
  • [42] R. Zini, R. Norfo, F. Ferrari, E. Bianchi, S. Salati, V. Pennucci, G. Sacchi, C. Carboni, G.B. Ceccherelli, E. Tagliafico, S. Ferrari, R. Manfredini, A.G.I.M.M. Investigators. Valproic acid triggers erythro/megakaryocyte lineage decision through induction of GFI1B and MLLT3 expression. Exp. Hematol.. 2012;40:1043-1054.e6 10.1016/j.exphem.2012.08.003 Crossref
  • [43] R.L. Bennett, K.S. French, R.G. Resta, D.L. Doyle. Standardized human pedigree nomenclature: update and assessment of the recommendations of the National Society of Genetic Counselors. J. Genet. Couns.. 2008;17:424-433 10.1007/s10897-008-9169-9 Crossref


a Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, United States

b Division of Genetics and Metabolism, Children's National Health System, Washington, DC, United States

c Special Coagulation Laboratory, Division of Hematopathology, Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN, United States

d Division of Hematology and Oncology, Department of Pediatrics, University of New Mexico, Albuquerque, NM, United States

e NIH Undiagnosed Diseases Program, NIH Common Fund, National Institutes of Health, Bethesda, MD, United States

f Office of the Clinical Director, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, United States

g Johns Hopkins University School of Medicine, Department of Pediatrics, McKusick-Nathans Institute of Genetic Medicine, Baltimore, MD, United States

Corresponding author at: 10 Center Drive, MSC 1851, Building 10, Room 10C-103, Bethesda, MD 20892-1851, United States.