Evaluating the clinical validity of genes related to hemostasis and thrombosis using the Clinical Genome Resource gene curation framework.

BACKGROUND: Inherited bleeding, thrombotic, and platelet disorders (BTPDs) are a heterogeneous set of diseases, many of which are very rare globally. Over the past 5 decades, the genetic basis of some of these disorders has been identified, and recently, high-throughput sequencing has become the primary means of identifying disease-causing genetic variants. OBJECTIVES: Knowledge of the clinical validity of a gene-disease relationship is essential to provide an accurate diagnosis based on results of diagnostic gene panel tests and inform the construction of such panels. The Scientific and Standardization Committee for Genetics in Thrombosis and Hemostasis undertook a curation process for selecting 96 TIER1 genes for BTPDs. The purpose of the process was to evaluate the evidence supporting each gene-disease relationship and provide an expert-reviewed classification for the clinical validity of genes associated with BTPDs. METHODS: The Clinical Genome Resource (ClinGen) Hemostasis/Thrombosis Gene Curation Expert Panel assessed the strength of evidence for TIER1 genes using the semiquantitative ClinGen gene-disease clinical validity framework. ClinGen Lumping and Splitting guidelines were used to determine the appropriate disease entity or entities for each gene, and 101 gene-disease relationships were identified for curation. RESULTS: The final outcome included 68 Definitive (67%), 26 Moderate (26%), and 7 Limited (7%) classifications. The summary of each curation is available on the ClinGen website. CONCLUSION: Expert-reviewed assignment of gene-disease relationships by the ClinGen Hemostasis/Thrombosis Gene Curation Expert Panel facilitates accurate molecular diagnoses of BTPDs by clinicians and diagnostic laboratories. These curation efforts can allow genetic testing to focus on genes with a validated role in disease.

O-linked sialic acid residues on platelet membrane glycoprotein IIb mask the human HPA-9b alloepitope.

Sialic acids occupy the terminal position of glycan chains and have the potential to influence the antigenicity of glycoproteins (GP). The polymorphisms of human platelet alloantigens (HPA)-3 and HPA-9, located near the C-terminus of the extracellular domain of platelet membrane GPIIb, are adjacent to sialyl-core 1 O-glycans emanating from serines 845 and 847. Whether the nearby O-glycans affect the antigenicity of HPA-9b or influence the binding of anti-HPA-9b alloantibodies in clinically significant cases of neonatal alloimmune thrombocytopenia is unknown. To address this issue, we generated a series of O-glycan mutant HPA-9 allele-specific induced pluripotent stem cell lines, differentiated them to megakaryocytes (MKs), and examined their ability to bind HPA-9b-specific alloantibodies. We found that both wild-type MKs treated with neuraminidase and those genetically modified to lack the sialidases ST3GAL1 and ST3GAL2 dramatically increased anti-HPA-9b alloantibody binding, indicating that the HPA-9b epitope is partially masked by terminal sialic acids on nearby O-glycans of GPIIb. Interestingly, mutating the serine residues that carry these glycan chains to alanine actually reduced the binding of anti-HPA-9b alloantibodies, indicating that these 2 O-glycan chains contribute to the presentation of the HPA-9b epitope-perhaps by stabilizing the conformation of the GP in this region. Collectively, our data suggest that detection of anti-HPA-9b alloantibodies may be enhanced through the use of HPA-9b-specific MKs that have been genetically altered to lack nearby terminal sialic acid residues but retain the glycan chains to which they are attached.

ANKRD26-Related Thrombocytopenia and Predisposition to Myeloid Neoplasms.

PURPOSE OF REVIEW: This review describes ANKRD26-related thrombocytopenia (RT) from a molecular, clinical, and laboratory perspective, with a focus on the clinical decision-making that takes place in the diagnosis and management of families with ANKRD26-RT. RECENT FINDINGS: ANKRD26-related thrombocytopenia (ANKRD26-RT) is a non-syndromic autosomal dominant thrombocytopenia with predisposition to hematologic neoplasm. The clinical presentation is variable with moderate thrombocytopenia with normal platelet size and absent to mild bleeding being the hallmark which makes it difficult to distinguish from other inherited thrombocytopenias. The pathophysiology involves overexpression of ANKRD26 through loss of inhibitory control by transcription factors RUNX1 and FLI1. The great majority of disease-causing variants are in the 5' untranslated region. Acute myeloid leukemia, myelodysplastic syndrome, and chronic myelomonocytic leukemia have been reported to occur in the context of germline variants in ANKRD26, with the development of somatic driver mutations in hematopoietic regulators playing an important role in malignant transformation. In the absence of clear risk estimates of development of malignancy, optimal surveillance strategies and interventions to reduce risk of evolution to a myeloid disorder, multidisciplinary evaluation, with a strong genetic counseling framework is essential in the approach to these patients and their families. Gene-specific expertise and a multidisciplinary approach are important in the diagnosis and treatment of patients and families with ANKRD26-RT. These strategies help overcome the challenges faced by clinicians in the evaluation of individuals with a rare, non-syndromic, inherited disorder with predisposition to hematologic malignancy for which large data to guide decision-making is not available.

A Novel PF4-Dependent Platelet Activation Assay Identifies Patients Likely to Have Heparin-Induced Thrombocytopenia/Thrombosis.

BACKGROUND: Almost without exception, patients with heparin-induced thrombocytopenia/thrombosis (HIT) have antibodies that recognize platelet factor 4 (PF4) in a complex with heparin; however, many heparin-treated patients without HIT are also antibody-positive. A platelet activation test, the serotonin release assay (SRA), is useful for identifying a subset of antibodies that are platelet-activating and most likely to cause HIT. However, this "gold standard" assay for HIT diagnosis is technically demanding and is routinely available only through referral laboratories, limiting its availability for timely diagnosis and management. METHODS: We compared the diagnostic performance of the SRA with that of a technically simple platelet activation assay, the PF4-dependent P-selectin expression assay (PEA), which uses platelets pretreated with PF4 as targets for antibody detection. Archived serum samples from 91 patients for whom clinical information (HIT 4Ts [thrombocytopenia, timing of platelet count fall, thrombosis, and other causes of thrombocytopenia] score) was available were used. Patients with an intermediate 4Ts score and a PF4 ELISA (enzyme-linked immunosorbent assay) optical density ≥ 2.0, or a high 4Ts score and a PF4 ELISA optical density ≥ 1.0, were considered HIT positive; others were designated HIT negative. RESULTS: The PEA had higher diagnostic accuracy (area under the curve, 0.92 vs 0.82; P = .02) than the SRA, using this definition of HIT. Eleven of 16 serum samples that were PEA positive and SRA negative were HIT positive. Studies done with identical target platelets and serially diluted samples from patients with HIT showed that the PEA is inherently more sensitive than the SRA for the detection of platelet-activating antibodies. CONCLUSIONS: The PEA is technically less demanding than the SRA and may be more accurate for the diagnosis of HIT.

Two cases of maternal alloimmunization against human neutrophil alloantigen-4b, one causing severe alloimmune neonatal neutropenia.

BACKGROUND: Human neutrophil antigen (HNA)-4a/4bw is encoded by 230G>A in ITGAM, which results in an Arg61His substitution of the αM chain (CD11b) of complement receptor 3 (CR3; CD11b/18 or Mac-1). HNA-4a antibodies have been detected in the sera of female blood donors and in maternal sera that caused alloimmune neonatal neutropenia (ANN), in which maternal immunoglobulin (Ig)G antibodies against a paternally inherited HNA cross the placenta and destroy fetal and neonatal neutrophils. However, to date, antibodies specific for HNA-4b have not been reported. Here, we report the first two examples of HNA-4b antibodies. STUDY DESIGN AND METHODS: The two sera studied were both from previously pregnant females, one a multiparous female blood donor implicated in two separate transfusion reactions and the second a mother whose first pregnancy resulted in the birth of a severely neutropenic (0 × 10(6) neutrophils/L) infant affected with ANN. Serum neutrophil antibody testing was by flow cytometry and CD11b/18 monoclonal antibody immobilization of granulocyte antigens assay, and HNA genotyping was performed by polymerase chain reaction with sequence-specific priming and allele-specific 5' exonuclease assays. RESULTS: Sera from both women contained IgG antibodies reactive only with HNA-4b+ neutrophils and both typed HNA-4a/a. Both were immunized through pregnancy since their husbands and children all typed HNA-4a/b. CONCLUSIONS: The serologic results, together with the genotype results, confirm that these are the first reported cases of neutrophil antibodies specific for HNA-4b.

Protamine-induced immune thrombocytopenia.

BACKGROUND: Protamine is widely used to reverse the anticoagulant effects of heparin. Although mild thrombocytopenia is common in patients given protamine after cardiac procedures, acute severe thrombocytopenia has not been described. We encountered a patient who experienced profound thrombocytopenia and bleeding shortly after administration of protamine and performed studies to characterize the responsible mechanism. STUDY DESIGN AND METHODS: Patient serum was studied for antibodies that recognize protamine, heparin-protamine complexes, and platelets (PLTs) treated with protamine using flow cytometry, enzyme-linked immunosorbent assay, and serotonin release from labeled PLTs. RESULTS: A high-titer immunoglobulin G antibody was detected in patient serum that recognizes protamine in a complex with heparin or PLT surface glycosaminoglycans (GAGs) and activates PLTs treated with protamine at concentrations achieved in vivo after protamine infusion. The antibody is distinctly different from those found in patients with heparin-induced thrombocytopenia on the basis of its failure to recognize heparin in a complex with PLT factor 4 (PF4) and to release serotonin from labeled PLTs in the absence of protamine. CONCLUSIONS: Findings made suggest that the patient's antibody is specific for conformational changes induced in protamine when it reacts with heparin or a PLT surface GAG. Development of severe thrombocytopenia after treatment of this patient with protamine defines a previously undescribed mechanism of drug-induced immune thrombocytopenia. Patients given protamine who produce this type of antibody may be at risk of experiencing thrombocytopenia if given the drug a second time while antibody is still present.

Determination of neutrophil antigen HNA-3a and HNA-3b genotype frequencies in six racial groups by high-throughput 5' exonuclease assay.

BACKGROUND: People with the human neutrophil antigen (HNA)-3b/3b type can make HNA-3a antibodies, which have been reported to cause immune neutropenia disorders and are especially prone to cause severe cases of transfusion-related acute lung injury. However, knowledge of HNA-3 allele frequencies outside Caucasian populations is limited. We developed a high-throughput genotyping assay and determined the HNA-3a/3b genotype frequencies in six different racial and ethnic groups. STUDY DESIGN AND METHODS: Genotyping utilized TaqMan 5' exonuclease chemistry and real-time polymerase chain reaction. A total of 742 DNA samples from six different racial and ethnic groups were genotyped for HNA-3a and HNA-3b. RESULTS: The genotyping assay showed 100% sensitivity and specificity compared to sequencing and phenotyping and had high throughput. A significant percentage of Caucasians (6.5%), Han Chinese (16%), and Asian Indians (6%) typed HNA-3b/3b, but only a small percentage of Hispanics (1%) and no African or Native Americans. CONCLUSIONS: The HNA-3 genotyping assay had high sensitivity, specificity, and sample throughput. HNA-3b/b genotype results determined for 742 individuals representing six different racial and ethnic groups showed that there could be a significant risk of producing anti-HNA-3a in Chinese, as well as in Caucasian and Asian Indian blood donor populations, but a very low risk in Hispanic, African, or Native American populations.

Full-length recombinant choline transporter-like protein 2 containing arginine 154 reconstitutes the epitope recognized by HNA-3a antibodies.

BACKGROUND: Recent reports have shown that the HNA-3a leukocyte antigen, a target for antibodies that cause severe transfusion-related acute lung injury, correlates with an arginine 154 (rather than glutamine) polymorphism in choline transporter-like protein 2 (CTL2) but did not show directly that R154 determines HNA-3a. CTL2 peptides containing R154 are recognized by only half of HNA-3a antibodies studied to date. Constructs that react with all HNA-3a antibodies are needed to fully define the HNA-3a epitope. STUDY DESIGN AND METHODS: HEK293 cells were transfected with cDNA encoding full-length CTL2 linked to green fluorescent protein (GFP). Transfectants were selected for GFP expression and tested with antibodies specific for HNA-3a and -3b. RESULTS: Each of 20 HNA-3a antibodies reacted preferentially with HEK293 cells expressing the R154 CTL2 construct. An HNA-3b antibody reacted only with CTL2 (Q154). CONCLUSIONS: These findings provide direct evidence that R154 in the context of full-length CTL2 is both necessary and sufficient to create the HNA-3a epitope but suggest that posttranslational modifications of the protein, for example, S-S bonds or addition of glycans, are necessary for recognition of HNA-3a by many antibodies. This could complicate development of an assay for large-scale screening of blood donors to detect anti-HNA-3a.

HNA-3a-specific antibodies recognize choline transporter-like protein-2 peptides containing arginine, but not glutamine at Position 154.

BACKGROUND: Antibodies specific for the neutrophil antigen HNA-3a cause severe, sometimes fatal transfusion-related acute lung disease (TRALI) when transfused, but it has not been possible to screen blood donors for anti-HNA-3a because using neutrophils as targets was impractical and molecular properties of the antigen were unknown. Recently it was shown that HNA-3a is carried on choline transporter-like protein-2 (CTL2) and that the HNA-3a/b phenotype is closely correlated with an R154Q amino acid polymorphism in CTL2. However, it has not been shown by direct experiment that R154 is essential for the HNA-3a epitope. STUDY DESIGN AND METHODS: Preliminary attempts to express recombinant full-length CTL2 (R154) recognized by anti-HNA-3a were unsuccessful. We therefore tested HNA-3a-specific antibodies from donors implicated in TRALI reactions for reactivity against chemically synthesized linear and cyclic CTL2 peptides containing R154 or Q154. RESULTS: Nine of 20 HNA-3a antibodies recognized the R154, but not the Q154 version of a cyclic 36-residue CTL2 peptide (D131-K166). However, 11 others failed to distinguish between the two versions of this peptide. CONCLUSION: The findings provide direct evidence that R154 in the context of CTL2 D131-K166 is necessary to create the HNA-3a epitope but, in the context of cyclic CTL2 peptide D131-K166, is sufficient to detect only about one-half of the HNA-3a-specific antibodies implicated in TRALI. It is likely that fragments of CTL2 longer than can be made on a large scale with an automated synthesizer will be needed to produce a target capable of detecting all examples of anti-HNA-3a in donated blood.

The neutrophil alloantigen HNA-3a (5b) is located on choline transporter-like protein 2 and appears to be encoded by an R>Q154 amino acid substitution.

The molecular basis of the HNA-3a/b (5b/a) leukocyte antigen system has not yet been defined despite evidence that HNA-3a-specific antibodies are particularly prone to cause severe, often fatal, transfusion-related lung injury. We used genome-wide single nucleotide polymorphism scanning and sequencing of DNA from persons of different HNA-3a/b phenotypes to identify a single single nucleotide polymorphism in exon 7 of the CLT2 gene (SLC44A2) that predicts an amino acid substitution in the first extracellular loop of choline transporter-like protein 2, a member of the choline transporter-like protein family of membrane glycoproteins, and correlates perfectly with HNA-3a/b phenotypes (R154 encodes HNA-3a; Q154 encodes HNA-3b). Mass spectrometric analysis of proteins immunoprecipitated from leukocytes by anti-HNA-3a provided direct evidence that anti-HNA-3a recognizes choline transporter-like protein 2. These findings will enable large-scale genotyping for HNA-3a/b to identify blood donors at risk to have HNA-3a-specific antibodies and should facilitate development of practical methods to detect such antibodies and prevent transfusion-related lung injury.