NBEAL2 mutations and bleeding in patients with gray platelet syndrome

Homozygosity/compound heterozygosity for loss of function mutations in neurobeachin-like 2 (NBEAL2) is causative for Gray platelet syndrome (GPS; MIM #139090), characterized by thrombocytopenia and large platelets lacking α-granules and cargo. Most GPS-associated NBEAL2 mutations generate nonsense codons; frameshifts causing premature translation termination and/or changes in mRNA splicing have also been observed. Data regarding NBEAL2 protein expression in GPS patients is limited. We observed absence of NBEAL2 in platelets from GPS patients with 3 different genotypes, and reduced/truncated platelet NBEAL2 has been reported for others. GPS is commonly associated with mild bleeding, but lifethreatening bleeding has been reported in some cases. A common long-term complication in GPS patients is myelofibrosis; splenomegaly is less common but sometimes of sufficient severity to merit splenectomy. Like GPS patients, mice lacking NBEAL2 expression exhibit macrothrombocytopenia, deficiency of platelet α-granules, splenomegaly, myelofibrosis, impaired platelet function and abnormalities in megakaryocyte development. Animal studies have also reported impaired platelet function in vivo using laser injury and thrombo-inflammation models. NBEAL2 is a large gene with 54 exons, and several putative functional domains have been identified in NBEAL2, including PH (pleckstrin homology) and BEACH (beige and Chediak-Higashi) domains shared with other members of a protein family that includes LYST and LRBA, also expressed by hematopoietic cells. Potential NBEAL2-interacting proteins have recently been identified, and it is expected that current and future efforts will reveal the cellular mechanisms by which NBEAL2 facilitates platelet development and supports hemostatic function.     

Megakaryocytic emperipolesis and platelet function abnormalities in five patients with gray platelet syndrome

The gray platelet syndrome (GPS) is a rare congenital platelet disorder characterized by mild to moderate bleeding diathesis, macrothrombocytopenia and lack of azurophilic α-granules in platelets. Some platelet and megakaryocyte (MK) abnormalities have been described, but confirmative studies of the defects in larger patient cohorts have not been undertaken. We studied platelet function and bone marrow (BM) features in five GPS patients with NBEAL2 autosomal recessive mutations from four unrelated families. In 3/3 patients, we observed a defect in platelet responses to protease-activated receptor (PAR)1-activating peptide as the most consistent finding, either isolated or combined to defective responses to other agonists. A reduction of PAR1 receptors with normal expression of major glycoproteins on the platelet surface was also found. Thrombin-induced fibrinogen binding to platelets was severely impaired in 2/2 patients. In 4/4 patients, the BM biopsy showed fibrosis (grade 2–3) and extensive emperipolesis, with many (36–65%) MKs containing 2–4 leukocytes engulfed within the cytoplasm. Reduced immunolabeling for platelet factor 4 together with normal immunolabeling for CD63 in MKs of two patients demonstrated that GPS MKs display an alpha granule-specific defect. Increased immunolabeling for P-selectin and decreased immunolabeling for PAR1, PAR4 and c-MPL were also observed in MKs of two patients. Marked emperipolesis, specific defect of MK alpha-granule content and defect of PAR1-mediated platelet responses are present in all GPS patients that we could study in detail. These results help to further characterize the disease.     

α-granule biogenesis: from disease to discovery

Platelets are critical to hemostasis and thrombosis. Upon detecting injury, platelets show a range of responses including the release of protein cargo from α-granules. This cargo is synthesized by platelet precursor megakaryocytes or endocytosed by megakaryocytes and/or platelets. Insights into α-granule biogenesis have come from studies of hereditary conditions where these granules are immature, deficient or absent. Studies of Arthrogryposis, Renal dysfunction, and Cholestasis (ARC) syndrome identified the first proteins essential to α-granule biogenesis: VPS33B and VPS16B. VPS33B and VPS16B form a complex, and in the absence of either, platelets lack α-granules and the granule-specific membrane protein P-selectin. Gray Platelet Syndrome (GPS) platelets also lack conventionally recognizable α-granules, although P-selectin containing structures are present. GPS arises from mutations affecting NBEAL2. The GPS phenotype is more benign than ARC syndrome, but it can cause life-threatening bleeding, progressive thrombocytopenia, and myelofibrosis. We review the essential roles of VPS33B, VPS16B, and NBEAL2 in α-granule development. We also examine the existing data on their mechanisms of action, where many details remain poorly understood. VPS33B and VPS16B are ubiquitously expressed and ARC syndrome is a multisystem disorder that causes lethality early in life. Thus, VPS33B and VPS16B are clearly involved in other processes besides α-granule biogenesis. Studies of their involvement in vesicular trafficking and protein interactions are reviewed to gain insights into their roles in α-granule formation. NBEAL2 mutations primarily affect megakaryocytes and platelets, and while little is known about NBEAL2 function some insights can be gained from studies of related proteins, such as LYST.     

Correlation between platelet phenotype and NBEAL2 genotype in patients with congenital thrombocytopenia and α-granule deficiency

The gray platelet syndrome is a rare inherited bleeding disorder characterized by macrothrombocytopenia and deficiency of alpha (α)-granules in platelets. The genetic defect responsible for gray platelet syndrome was recently identified in biallelic mutations in the NBEAL2 gene. We studied 11 consecutive families with inherited macrothrombocytopenia of unknown origin and α-granule deficiency. All of them underwent NBEAL2 DNA sequencing and evaluation of the platelet phenotype, including a systematic assessment of the α-granule content by immunofluorescence analysis for α-granule secretory proteins. We identified 9 novel mutations hitting the two alleles of NBEAL2 in 4 probands. They included missense, nonsense and frameshift mutations, as well as nucleotide substitutions that altered the splicing mechanisms as determined at the RNA level. All the individuals with NBEAL2 biallelic mutations showed almost complete absence of platelet α-granules. Interestingly, the 13 individuals assumed to be asymptomatic because carriers of a mutated allele had platelet macrocytosis and significant reduction of the α-granule content. However, they were not thrombocytopenic. In the remaining 7 probands, we did not identify any NBEAL2 alterations, suggesting that other genetic defect(s) are responsible for their platelet phenotype. Of note, these patients were characterized by a lower severity of the α-granule deficiency than individuals with two NBEAL2 mutated alleles. Our data extend the spectrum of mutations responsible for gray platelet syndrome and demonstrate that macrothrombocytopenia with α-granule deficiency is a genetic heterogeneous trait. In terms of practical applications, the screening of NBEAL2 is worthwhile only in patients with macrothrombocytopenia and severe reduction of the α-granules. Finally, individuals carrying one NBEAL2 mutated allele have mild laboratory abnormalities, suggesting that even haploinsufficiency has an effect on platelet phenotype.     

Molecular and cellular characterization of a new α-thalassemia mutation (HBA2:c.94A>C) generating an alternative splice site and a premature stop codon

The identification of α-thalassemia (α-thal) due to point mutations has been increasing significantly with the advancement of molecular diagnostic tools. We describe here the molecular and cellular characteristics of the thalassemia mutation HBA2:c.94A>C, a novel point mutation affecting the α2-globin gene, causing a mild α-thal phenotype in a male patient of undisclosed ethnicity, investigated for unexplained microcytosis. The detected mutation is located at the penultimate nucleotide (nt) of the first exon which we postulated might affect pre mRNA splicing. While an in silico analysis did not predict any aberrant splice variants, experimental analysis using our in vitro model for gene expression studies showed utilization of a cryptic splice site at codon 15 that resulted in an aberrant splice variant. As a result, a frameshift in the reading frame of the mature mRNA was produced, leading to the formation of a premature termination codon (PTC) between codons 48 and 49 in exon 2. This in turn leads to nonsense mediated mRNA decay (NMD) and the phenotype of α-thal.     

Molecular and Cellular Characterization of a New α-Thalassemia Mutation (HBA2:c.94A>C) Generating an Alternative Splice Site and a Premature Stop Codon

The identification of α-thalassemia (α-thal) due to point mutations has been increasing significantly with the advancement of molecular diagnostic tools. We describe here the molecular and cellular characteristics of the thalassemia mutation HBA2:c.94A>C, a novel point mutation affecting the α2-globin gene, causing a mild α-thal phenotype in a male patient of undisclosed ethnicity, investigated for unexplained microcytosis. The detected mutation is located at the penultimate nucleotide (nt) of the first exon which we postulated might affect pre mRNA splicing. While an in silico analysis did not predict any aberrant splice variants, experimental analysis using our in vitro model for gene expression studies showed utilization of a cryptic splice site at codon 15 that resulted in an aberrant splice variant. As a result, a frameshift in the reading frame of the mature mRNA was produced, leading to the formation of a premature termination codon (PTC) between codons 48 and 49 in exon 2. This in turn leads to nonsense mediated mRNA decay (NMD) and the phenotype of α-thal.     

Should any genetic defect affecting α‐granules in platelets be classified as gray platelet syndrome?

There is much current interest in the role of the platelet storage pool of α‐granule proteins both in hemostasis and non‐hemostatic events. As well as in the arrest of bleeding, the secreted proteins participate in wound healing, inflammation, and innate immunity while in pathology they may be actors in arterial thrombosis and atherosclerosis as well as cancer and metastasis. For a long time, gray platelet syndrome (GPS) has been regarded as the classic inherited platelet disorder caused by an absence of α‐granules and their contents. While NBEAL2 is the major source of mutations in GPS, other gene variants may give rise to significant α‐granule deficiencies in platelets. These include GATA1, VPS33B, or VIPAS39 in the arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome and now GFI1B. Nevertheless, many phenotypic differences are associated with mutations in these genes. This critical review was aimed to assess genotype/phenotype variability in disorders of platelet α‐granule biogenesis and to urge caution in grouping all genetic defects of α‐granules as GPS. Am. J. Hematol. 91:714–718, 2016. © 2016 Wiley Periodicals, Inc.     

Hb Lynwood [α107(G14) (–T) (α2) HBA2:c.323delT)] in Conjunction with the α3.7 Deletion Produces a Moderately Severe α-Thalassemia Phenotype

We describe a novel frameshift mutation associated with an α-thalassemia (α-thal) phenotype in a patient of Sudanese origin investigated for persistent microcytosis. In addition to the α^3.7 deletion, a novel mutation on the α2 gene was detected: HBA2:c.323delT. This mutation causes a frameshift at codon 107 of the α2 gene. The result is a disturbed amino acid sequence for the following 24 amino acids, and a premature termination codon at position 132.     

Hb Lynwood [α107(G14) (-T) (α2) HBA2:c.323delT)] in conjunction with the α(3.7) deletion produces a moderately severe α-thalassemia phenotype

We describe a novel frameshift mutation associated with an α-thalassemia (α-thal) phenotype in a patient of Sudanese origin investigated for persistent microcytosis. In addition to the α(3.7) deletion, a novel mutation on the α2 gene was detected: HBA2:c.323delT. This mutation causes a frameshift at codon 107 of the α2 gene. The result is a disturbed amino acid sequence for the following 24 amino acids, and a premature termination codon at position 132.     

A Novel β-Thalassemic Allele Due to a Two Nucleotide Deletion: β76 (−GC)

We have identified and characterized a novel β-thalassemic mutation in a North African adult. The molecular defect consists of a two nucleotide (nt) deletion in the β-globin gene at codon 76 [β76 (?GC), c.229–230delGC]. This frameshift mutation generates a TGA stop codon at position 89. The carrier presented with mild microcytic anemia (Hb 12.8 g/dL, MCV 60 fL), no detectable Hb F, an elevated Hb A₂ level (5.5%) with no other mutation in the β-globin gene and none of the more common known deletions in the α-globin cluster. No abnormal hemoglobin (Hb) was present in routine electrophoresis or in high performance liquid chromatography (HPLC) analyses. Pathologic inclusions were absent in both mature red cells and in reticulocytes. This observation reinforces the hypothesis that nonsense and frameshift mutations that result in a premature stop codon in exon 1 or exon 2 inherited in the heterozygous state do not generate dominant β-thalassemia (thal). This is the first example of a premature stop codon at position 89.     

Nonsense mutation in exon V of the factor XI gene does not abolish platelet factor XI expression

Factor XI (FXI) is a plasma glycoprotein produced by the liver that plays an essential role in blood coagulation. Individuals deficient in this protein are prone to bleeding following trauma or surgery. Well-washed platelets possess FXI-like activity and FXI antigen that differs in molecular weight from plasma FXI and is associated with FXI mRNA from platelets in which exon V is absent. Some individuals deficient in plasma FXI produce the platelet form of the protein. The molecular basis for the presence of platelet FXI in plasma FXI-deficient patients is unknown. In the current study, to help determine the mechanism for this differential expression, the FXI genotype was determined for three plasma FXI-deficient individuals who express platelet FXI. All three individuals had a nonsense mutation encoding a stop codon in exon V of the FXI gene that would result in a severely truncated polypeptide. An exon V nonsense mutation in the FXI gene combined with the absence of exon V in platelet FXI mRNA provides a plausible mechanism for the differential expression of platelet FXI in plasma FXI-deficient patients.     

A novel Pro126His beta propeller mutation in integrin alphaIIb causes Glanzmann thrombasthenia by impairing progression of pro-alphaIIbbeta3 from endoplasmic reticulum to Golgi

BackgroundGlanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by lack of platelet aggregation in response to most physiological agonists and caused by either a lack or dysfunction of the platelet integrin αIIbβ3 (glycoprotein IIb/IIIa).PatientsMucocutaneous bleeding manifestations and platelet dysfunction consistent with GT were observed in a 20-year-old proband of a Chinese family.ObjectivesTo determine the molecular basis of GT and characterize the mutation by in vitro expression studies.ResultsAnalysis of the patient's platelets by fluorescence-activated cell sorting demonstrated the presence of trace amounts of β3, exposed on her platelet surface, but a complete absence of αIIbβ3. Sequence analysis revealed a novel C470A transversion in exon 4 of the αIIb gene predicting a Pro126His alteration in the blade 2 of the αIIb β propeller domain. The proband was homozygous for the mutation, the mother and the father were heterozygous, whereas 100 healthy subjects lacked this transversion. Chinese hamster ovary cells cotransfected with cDNAs of mutated αIIb and wild-type β3 failed to express αIIbβ3 on the cell surface as shown by FACS. Western blot analysis of the cell lysates showed no detectable mature αIIb. Immunoprecipitation with antibody against β3 demonstrated pro-αIIb in the cells expressing the mutant αIIbβ3, indicating pro-αIIbβ3 complex formation. Intracellular immunofluorescence studies demonstrated the pro-αIIbβ3 complex that co-localized with an ER marker, but showed minimal co-localization with a Golgi marker.ConclusionsA novel Pro126His mutation in αIIb compromised transport of the pro-αIIbβ3 complex from the endoplasmic reticulum to the Golgi, leading to intracellular retention. The impaired αIIbβ3 transport is responsible for the thrombasthenia in this patient.     

Interaction of heterozygous βo-thalassemia with single functional α-globin gene

In this study, we analyzed the phenotypic manifestations resulting from the interaction of heterozygous β^o-thalassemia(β^o-39 nonsense mutation) with the functional loss of three α-globin structural genes in six subjects, of whom four had the [-αl–] α-globin genotype and two the [–/αThα] α-globin genotype. The β-thalassemia defect was in all cases the nonsense mutation at codon 39. The nondeletion α-thalassemia α^th was the initiation codon mutation (AUG→GUG) of the α-2 gene. In all these subjects hypochromia and microcytosis were more marked than in βα -thalassemia heterozygotes with a full complement of four α-globin genes. All but one had moderate anemia. The α:β globin chain synthesis ratios were consistently decreased. No cases had Hb H on electrophoresis. Subjects with [–/αThα] α-globin genotype had more severe thalassemia-like manifestations than those with [–/-α] α-globin genotype.     

Apolipoprotein A-I deficiency due to a codon 84 nonsense mutation of the apolipoprotein A-I gene.

The molecular genetic defect of a female patient with apolipoprotein A-I (apoA-I) deficiency and premature atherosclerosis was examined. Her parents were first cousins. Her plasma density fraction from 1.063 to 1.21 g/ml contained no apoA-I on SDS/PAGE and no measurable high density lipoprotein cholesterol. Southern blot hybridization showed no gross abnormality to be present in the patient's apoA-I gene and homozygosity for a haplotype of restriction fragment length polymorphisms in the apoA-I gene region. Sequencing after amplification by PCR revealed a codon 84 nonsense mutation (CAG----TAG, Gln----stop) of exon 4 and a codon 67 missense mutation (GCC----ACC, Ala----Thr) of exon 3 in the patient's apoA-I gene. The data from dot-blot hybridization with allele-specific oligonucleotide probes indicated that she was homozygous for the apoA-I gene with regard to the two mutations. The codon 37 missense mutation was also detected in the apoA-I gene of 6 out of 60 controls, who all had normal levels of apoA-I and high density lipoprotein cholesterol, suggesting that the missense mutation is polymorphic and not associated with apoA-I deficiency. These findings indicate that homozygosity for the apoA-I gene with codon 84 nonsense mutation causes the deficiency of apoA-I and of high density lipoprotein cholesterol in the patient.     

α+-Thalassemia Trait Caused by a Nonsense Mutation in the α2-Globin Gene: Codon 54 (CAG>TAG)

We report a new α-thalassemia (α-thal) point mutation detected in a woman with α⁺-thal trait. Sequence analysis identified a nonsense mutation in exon 2 of the α2-globin gene, at amino acid codon 54 (CAG>TAG).     

Homozygosity mapping with SNP arrays confirms 3p21 as a recessive locus for gray platelet syndrome and narrows the interval significantly

Gray platelet syndrome (GPS) is an inherited bleeding disorder characterized by thrombocytopenia and the absence of α-granules in platelets. Patients with GPS present with mild to moderate bleeding and many develop myelofibrosis. The genetic cause of GPS is unknown. We present 2 Native American families with a total of 5 affected persons and a single affected patient of Pakistani origin in which GPS appears to be inherited in an autosomal recessive manner. Homozygosity mapping using the Affymetrix 6.0 chips demonstrates that all 6 GPS-affected persons studied are homozygous for a 1.7-Mb region in 3p21. Linkage analysis confirmed the region with a logarithm of the odds score of 2.7. Data from our families enabled us to significantly decrease the size of the critical region for GPS from the previously reported 9.4-Mb region at 3p21.     

X-linked gray platelet syndrome due to a GATA1 Arg216Gln mutation

We identified a family with gray platelet syndrome (GPS) segregating as a sex-linked trait. Affected males had a mild bleeding disorder, thrombocytopenia, and large agranular platelets characteristic of GPS, while obligate carrier females were asymptomatic but had dimorphic platelets on peripheral smear. Associated findings included mild erythrocyte abnormalities in affected males. Linkage analysis revealed a 63 cM region on the X chromosome between markers G10578 and DXS6797, which segregated with the platelet phenotype and included the GATA1 gene. Sequencing of GATA1 revealed a G-to-A mutation at position 759 corresponding to amino acid change Arg216Gln. This mutation was previously described as a cause of X-linked thrombocytopenia with thalassemia (XLTT) but not of gray platelet syndrome. Our findings suggest that XLTT is within a spectrum of disorders constituting the gray platelet syndrome, and we propose that GATA1 is an upstream regulator of the genes required for platelet alpha-granule biogenesis.     

Novel nonsense mutation in the platelet glycoprotein Ibbeta gene associated with Bernard-Soulier syndrome

Bernard-Soulier syndrome (BSS) is an autosomal recessive bleeding disorder caused by quantitative or qualitative abnormalities in the glycoprotein (GP) Ib/IX/V complex, the platelet receptor for von Willebrand factor. This complex is composed of four subunits, GPIbalpha, GPIbbeta, GPIX, and GPV, and the coordinated assembly of GPIbalpha, GPIbbeta, and GPIX is required for the efficient surface expression of a functional complex. We report here a novel nonsense mutation of the GPIbbeta gene associated with BSS. Flow cytometric analysis of the patient's platelets showed markedly reduced GPIbalpha and absent GPIX surface expression. Immunoblot analysis of solubilized platelets showed that a small amount of GPIbalpha was detected; however, GPIbbeta and GPIX were undetectable. DNA sequencing analysis revealed a novel nonsense mutation of the GPIbbeta gene that converts Trp (TGG) to a stop codon (TAG) at residue 123. Transient transfection studies revealed that the mutant GPIbbeta polypeptide was not detected in the transfected 293T cells, suggesting that null expression of the mutant GPIbbeta impairs expression of the GPIbalpha and GPIX subunits and results in a BSS phenotype in the patient.     

A novel mutation that leads to a congenital factor XI deficiency in a Japanese family

We have identified a novel mutation leading to a congenital deficiency of the coagulation factor XI (FXI) in a Japanese family. A propositus was a 42-year-old female patient without bleeding tendency. Coagulant activity and the antigen level of FXI in her plasma were below the detectable range. The nucleotide sequences of the FXI gene of this patient were determined by a direct sequence method established in this study. A novel nonsense mutation (CAA; Gly263 --> TAA; stop) was identified in exon 8 of the FXI gene. Her parents are first cousins, and a polymerase chain reaction-restriction-fragment length polymorphism analysis revealed that her parents were heterozygous at this nucleotide position. This patient inherited mutant alleles from her parents and is homozygous at this nucleotide position. The nonsense mutation in the FXI gene is responsible for her deficiency of FXI.     

Human alpha2-globin nonsense-mediated mRNA decay induced by a novel alpha-thalassaemia frameshift mutation at codon 22

We describe a novel alpha-thalassaemia determinant in a 3-year-old girl presenting a mild microcytic and hypochromic anaemia, and normal haemoglobin A2 level. Molecular studies revealed heterozygosity for a novel microdeletion (-C) at codon 22 of the alpha2-globin gene. As the frameshift mutation generates a premature translation termination codon at position 48/49, we investigated the effect of the nonsense codon on the alpha2-globin gene expression. Although it does not affect RNA splicing, the premature nonsense codon induces accelerated mRNA degradation. To our knowledge, this is the first time the nonsense-mediated mRNA decay has been reported to occur in human alpha-globin mRNA.