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OBJECTIVE: We aimed to develop accurate and user-friendly genetic assays to identify the inherited neutrophil antigen-2 (HNA-2) deficiency in humans. BACKGROUND: HNA-2 is one of the most important neutrophil antigens implicated in a number of human disorders. HNA-2 deficiency or HNA-2 null is a common phenotype observed in 3%-5% Americans. HNA-2 null individuals are at risk to produce isoantibodies (or alloantibodies) that play important roles in transfusion-related acute lung injury, immune neutropenia, and bone marrow graft failure. We previously demonstrated that the CD177 coding SNP 787A > T (c.787A > T) is the most important genetic determinant for HNA-2 deficiency. However, reliable genetic assays are not available for routine clinical laboratory application up to now. STUDY DESIGN AND METHODS: A novel polymerase chain reaction (PCR) strategy was used to determine genotypes of the CD177 SNP c.787A > T. In the simplified PCR assay, all allele specific primers and internal control primers were included in the same reaction, which ensures reliability of the assay. In addition, a novel high-throughput nested TaqMan assay was developed to determine genotypes of c.787A > T for large population genetic analysis of HNA-2 deficiency. RESULTS: CD177 SNP c787A > T genotypes of 396 subjects were 100% concordant among the single PCR reaction method, the nested TaqMan assay, and Sanger Sequencing analysis. Out of 396 subjects, all 18 donors with the CD177 STP homozygous genotype were HNA-2 null. CONCLUSION: The novel PCR-based genotyping assay is accurate to identify HNA-2 deficient individuals and is suitable for clinical laboratories. In addition, the innovative high-throughput nested TaqMan assay will be useful for large-scale population screens and genetic studies of HNA-2 deficiency.
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Isoantígenos , Neutrófilos , Humanos , Reproducibilidad de los Resultados , Isoantígenos/genética , Genotipo , HomocigotoRESUMEN
BACKGROUND: Human neutrophil antigen-2 (HNA-2) is exclusively expressed on neutrophils. HNA-2-deficient individuals (HNA-2 null) are susceptible to produce isoantibodies. The nonsense CD177 coding single-nucleotide polymorphism (SNP) c.787A>T has been demonstrated as the primary genetic mechanism for HNA-2 deficiency. We hypothesized that the other genetic variants also contribute to HNA-2 expression variation and deficiency. STUDY DESIGN AND METHODS: The deficiency, density, and percentage of HNA-2 antigen on neutrophils from 292 healthy blood donors were determined in flow cytometry. CD177 genotypes were determined by genomic DNA sequence analyses. The full-length CD177 cDNAs were amplified and sequenced. Additionally, the whole CD177 genomic sequence in eight HNA-2-null immunized women and four HNA-2-positive donors were analyzed with next-generation sequencing. The associations of CD177 SNP genotypes with HNA-2 expression variation were statistically analyzed. RESULTS: A functional CD177 SNP c.1291G>A was identified in the current study. Atypical (trimodal) HNA-2 expression phenotype was consistently observed in donors carrying the heterozygous c.1291G/A genotype. Phenotype-genotype analyses of SNP c.787A>T and SNP c.1291G>A revealed that all homozygous 787T-1291G (TG/TG) genotype donors were HNA-2 null in healthy blood donors. On the other hand, five of eight HNA-2-immunized females were homozygous for the 787T-1291G (TG/TG) genotype while the other three HNA-2-immunized females had the 787T-1291G/787A-1291A (TG/AA) genotype and the lowest HNA-2 expression was observed in healthy subjects with the 787T-1291G/787A-1291A (TG/AA) and 787A-1291A/787A-1291A (AA/AA) genotype. CONCLUSION: The CD177 SNP c.1291G>A is a genetic determinant for the atypical and low HNA-2 expression, which also contributes to HNA-2 deficiency phenotype.
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Isoantígenos/genética , Neutrófilos/metabolismo , Polimorfismo de Nucleótido Simple/genética , Receptores de Superficie Celular/genética , Secuencia de Bases , ADN Complementario/genética , ADN Complementario/metabolismo , Femenino , Citometría de Flujo , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Genotipo , Humanos , Isoantígenos/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
Human neutrophil antigen 2 (HNA-2) deficiency is a common phenotype as 3-5% humans do not express HNA-2. HNA-2 is coded by CD177 gene that associates with human myeloproliferative disorders. HNA-2 deficient individuals are prone to produce HNA-2 alloantibodies that cause a number of disorders including transfusion-related acute lung injury and immune neutropenia. In addition, the percentages of HNA-2 positive neutrophils vary significantly among individuals and HNA-2 expression variations play a role in human diseases such as myelodysplastic syndrome, chronic myelogenous leukemia, and gastric cancer. The underlying genetic mechanism of HNA-2 deficiency and expression variations has remained a mystery. In this study, we identified a novel CD177 nonsense single nucleotide polymorphism (SNP 829A>T) that creates a stop codon within the CD177 coding region. We found that all 829TT homozygous individuals were HNA-2 deficient. In addition, the SNP 829A>T genotypes were significantly associated with the percentage of HNA-2 positive neutrophils. Transfection experiments confirmed that HNA-2 expression was absent on cells expressing the CD177 SNP 829T allele. Our data clearly demonstrate that the CD177 SNP 829A>T is the primary genetic determinant for HNA-2 deficiency and expression variations. The mechanistic delineation of HNA-2 genetics will enable the development of genetic tests for diagnosis and prognosis of HNA-2-related human diseases.
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Estudios de Asociación Genética , Enfermedades Genéticas Congénitas , Isoantígenos/genética , Neutrófilos/metabolismo , Receptores de Superficie Celular/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Proteínas Ligadas a GPI/biosíntesis , Proteínas Ligadas a GPI/deficiencia , Proteínas Ligadas a GPI/genética , Regulación de la Expresión Génica , Genotipo , Voluntarios Sanos , Heterocigoto , Humanos , Isoantígenos/biosíntesis , Masculino , Persona de Mediana Edad , Neutrófilos/patología , Polimorfismo de Nucleótido Simple , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/deficiencia , Eliminación de SecuenciaRESUMEN
BACKGROUND: Possible transfusion-related acute lung injury (pTRALI) cases by definition have a clear temporal relationship to an alternative recipient risk factor for acute respiratory distress syndrome (ARDS). We questioned whether transfusion factors are important for the development of pTRALI. STUDY DESIGN AND METHODS: In this nested case-control study, we prospectively identified 145 consecutive patients with pTRALI and randomly selected 163 transfused controls over a 4-year period at the University of California at San Francisco and the Mayo Clinic (Rochester, Minnesota). RESULTS: For pTRALI, we found evidence against transfusion being important: receipt of plasma from female donors (odds ratio [OR], 0.82; 95% confidence interval [CI], 0.29-2.3; p = 0.70), total number of units transfused (OR, 0.99; 95% CI, 0.89-1.10; p = 0.86), and number of red blood cell and whole blood units transfused (OR, 0.78; 95% CI, 0.59-1.03; p = 0.079). In contrast, we found that risk for pTRALI was associated with additional recipient factors: chronic alcohol abuse (OR, 12.5; 95% CI, 2.8-55; p < 0.001), current smoker (OR, 4.2; 95% CI, 1.67-10.8; p = 0.0024), shock before transfusion (OR, 4.6; 95% CI, 2.0-10.7; p < 0.001), and positive fluid balance before transfusion (OR, 1.32/L; 95% CI, 1.20-1.44; p < 0.001). CONCLUSION: Recipient risk factors for ARDS rather than transfusion risk factors predominate in pTRALI.
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Lesión Pulmonar Aguda/etiología , Reacción a la Transfusión , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Análisis Multivariante , Estudios Prospectivos , Factores de Riesgo , Adulto JovenRESUMEN
OBJECTIVE: Transfusion-related acute lung injury is the leading cause of transfusion-related mortality. A prospective study using electronic surveillance was conducted at two academic medical centers in the United States with the objective to define the clinical course and outcomes in transfusion-related acute lung injury cases. DESIGN: Prospective case study with controls. SETTING: University of California, San Francisco and Mayo Clinic, Rochester. PATIENTS: We prospectively enrolled 89 patients with transfusion-related acute lung injury, 164 transfused controls, and 145 patients with possible transfusion-related acute lung injury. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Patients with transfusion-related acute lung injury had fever, tachycardia, tachypnea, hypotension, and prolonged hypoxemia compared with controls. Of the patients with transfusion-related acute lung injury, 29 of 37 patients (78%) required initiation of mechanical ventilation and 13 of 53 (25%) required initiation of vasopressors. Patients with transfusion-related acute lung injury and possible transfusion-related acute lung injury had an increased duration of mechanical ventilation and increased days in the ICU and hospital compared with controls. There were 15 of 89 patients with transfusion-related acute lung injury (17%) who died, whereas 61 of 145 patients with possible transfusion-related acute lung injury (42%) died and 7 of 164 of controls (4%) died. Patients with transfusion-related acute lung injury had evidence of more systemic inflammation with increases in circulating neutrophils and a decrease in platelets compared with controls. Patients with transfusion-related acute lung injury and possible transfusion-related acute lung injury also had a statistically significant increase in plasma interleukin-8, interleukin-10, and interleukin-1 receptor antagonist posttransfusion compared with controls. CONCLUSIONS: In conclusion, transfusion-related acute lung injury produced a condition resembling the systemic inflammatory response syndrome and was associated with substantial in-hospital morbidity and mortality in patients with transfusion-related acute lung injury compared with transfused controls. Patients with possible transfusion-related acute lung injury had even higher in-hospital morbidity and mortality, suggesting that clinical outcomes in this group are mainly influenced by the underlying acute lung injury risk factor(s).
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Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/fisiopatología , Unidades de Cuidados Intensivos/estadística & datos numéricos , Reacción a la Transfusión , Lesión Pulmonar Aguda/inmunología , Adolescente , Adulto , Anciano , Citocinas/metabolismo , Femenino , Humanos , Tiempo de Internación/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Alta del Paciente/estadística & datos numéricos , Estudios Prospectivos , Respiración Artificial , Factores de RiesgoRESUMEN
Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related mortality. Antibodies to HNA-3a are commonly implicated in TRALI. We hypothesized that HNA-3a antibodies prime neutrophils (PMNs) and cause PMN-mediated cytotoxicity through a two-event pathogenesis. Isolated HNA-3a+ or HNA-3a- PMNs were incubated with plasma containing HNA-3a antibodies implicated in TRALI, and their ability to prime the oxidase was measured. Human pulmonary microvascular endothelial cells (HMVECs) were activated with endotoxin or buffer, HNA-3a+ or HNA-3a- PMNs were added, and the coculture was incubated with plasma+/-antibodies to HNA-3a. PMN-mediated damage was measured by counting viable HMVECs/mm2. Plasma containing HNA-3a antibodies primed the fMLP-activated respiratory burst of HNA-3a+, but not HNA-3a-, PMNs and elicited PMN-mediated damage of LPS-activated HMVECs when HNA-3a+, but not HNA-3a-, PMNs were used. Thus, antibodies to HNA-3a primed PMNs and caused PMN-mediated HMVEC cytotoxicity in a two-event model identical to biologic response modifiers implicated in TRALI.