ABSTRACT
BACKGROUND: Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by anti-ADAMTS13 autoantibodies inducing a severe deficiency of ADAMTS13. Epitope mapping studies on samples obtained during acute iTTP episodes have shown that the iTTP immune response is polyclonal, with almost all patients having autoantibodies targeting the spacer domain of ADAMTS13. OBJECTIVES: To identify the immunogenic hotspots in the spacer domain of ADAMTS13. PATIENTS/METHODS: A library of 11 full-length ADAMTS13 spacer hybrids was created in which amino acid regions of the spacer domain of ADAMTS13 were exchanged by the corresponding region of the spacer domain of ADAMTS1. Next, the full-length ADAMTS13 spacer hybrids were used in enzyme-linked immunosorbent assay to epitope map anti-spacer autoantibodies in 138 samples from acute and remission iTTP patients. RESULTS: Sixteen different anti-spacer autoantibody profiles were identified with a similar distribution in acute and remission patients. There was no association between the anti-spacer autoantibody profiles and disease severity. Almost all iTTP samples contained anti-spacer autoantibodies against the following three regions: amino acid residues 588-592, 602-610, and 657-666 (hybrids E, G, and M). Between 31% and 57% of the samples had anti-spacer autoantibodies against amino acid regions 572-579, 629-638, 667-676 (hybrids C, J, and N). In contrast, none of the samples had anti-spacer autoantibodies against amino acid regions 556-563, 564-571, 649-656, and 677-685 (hybrids A, B, L, and O). CONCLUSION: We identified three hotspot regions (amino acid regions 588-592, 602-610, and 657-666) in the spacer domain of ADAMTS13 that are targeted by anti-spacer autoantibodies found in a large cohort of iTTP patients.
Subject(s)
ADAMTS13 Protein/immunology , Autoantibodies/immunology , Purpura, Thrombotic Thrombocytopenic , DNA, Intergenic , Epitopes , Humans , Immunoglobulin G , Purpura, Thrombotic Thrombocytopenic/diagnosisABSTRACT
INTRODUCTION: Patients suffering from congenital thrombotic thrombocytopenic purpura (cTTP) have a deficiency in ADAMTS13 due to mutations in their ADAMTS13 gene. OBJECTIVE: The aim of this study was to determine ADAMTS13 parameters (activity, antigen, and mutations), to investigate if the propositus suffered from child-onset cTTP, and to study the in vitro effect of the ADAMTS13 mutations. METHODS: ADAMTS13 activity and antigen were determined using the FRETS VWF73 assay and ELISA and ADAMTS13 mutations via sequencing of the exons. Mutant proteins were expressed in Chinese hamster ovary cells, and their expression was studied using fluorescence microscopy and ELISA. Molecular modeling was used to evaluate the effect of the mutations on ADAMTS13 structure and stability. RESULTS: The propositus was diagnosed with cTTP at the age of 20. ADAMTS13 activity was below 10%, and 2 compound heterozygous mutations, the p.R498C point and the p.G259PfsX133 frameshift mutation, were identified. Expression of ADAMTS13 mutants revealed that the p.R498C and the p.G259PfsX133 mutation cause secretion and translation defects in vitro, respectively. Molecular modeling showed that the R498 intra-domain interactions are lacking in the p.R498C mutant, resulting in protein instability. CONCLUSION: The ADAMTS13 mutations result in a severe ADAMTS13 deficiency explaining the patient's phenotype.
ABSTRACT
Abstract Objective: To analyze if there is an association between the presence of polymorphisms in the LPL gene (rs320, rs285 and rs328) with development of acute ischemic stroke in Colombian population. Methods: In a case control design, 133 acute ischemic stroke patients (clinical diagnosis and x-ray CT) and 269 subjects without stroke as controls were studied. PCR -RFLP technique was used to detect rs320, rs285 and rs328 polymorphisms in the LPL gene. Results: In the present research was not found any association between any of the LPL gene polymorphism and acute ischemic stroke in the population studied; the allele and genotypic frequencies of the studied polymorphisms were similar in cases and controls and followed the Hardy-Weinberg equilibrium. The study was approved by the IRB and each subject signed the informed consent. Conclusion: LPL gene polymorphisms are not genetic markers for the development of stroke in the Colombian sample used.
Resumen Objetivo: Determinar la asociación entre los polimorfismos en el gen LPL (rs320, rs285 y rs328), y la enfermedad cerebrovascular isquémica aguda en una muestra de población colombiana. Métodos: A partir de un diseño de casos y controles, se estudiaron 133 casos con enfermedad cerebrovascular isquémica aguda (diagnóstico clínico y TAC), y 269 controles sin enfermedad cerebrovascular. Se examinó los polimorfismos rs320, rs285 y rs328 en el gen LPL con la técnica PCR-RFLP. Resultados: En el presente estudio no se encontró asociación entre rs320, rs285 y rs328 con la enfermedad cerebrovascular isquémica aguda en la muestra analizada; siendo las frecuencias alélicas y genotípicas de los polimorfismos similares entre casos y controles, y se encontró en equilibrio de Hardy-Weinberg. El estudio fue avalado por el comité de ética de las instituciones vinculadas y todos los pacientes dieron consentimiento informado. Conclusión: Los polimorfismos en el gen de la LPL no tienen utilidad como marcadores genéticos asociados con la presentación de la enfermedad cerebrovascular isquémica aguda en la muestra analizada.
Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Genetic Markers , Brain Ischemia/genetics , Stroke/genetics , Lipoprotein Lipase/genetics , Polymorphism, Genetic , Polymorphism, Restriction Fragment Length , Case-Control Studies , Polymerase Chain Reaction , Colombia , Alleles , GenotypeABSTRACT
OBJECTIVE: To analyze if there is an association between the presence of polymorphisms in the LPL gene (rs320, rs285 and rs328) with development of acute ischemic stroke in Colombian population. METHODS: In a case control design, 133 acute ischemic stroke patients (clinical diagnosis and x-ray CT) and 269 subjects without stroke as controls were studied. PCR -RFLP technique was used to detect rs320, rs285 and rs328 polymorphisms in the LPL gene. RESULTS: In the present research was not found any association between any of the LPL gene polymorphism and acute ischemic stroke in the population studied; the allele and genotypic frequencies of the studied polymorphisms were similar in cases and controls and followed the Hardy-Weinberg equilibrium. The study was approved by the IRB and each subject signed the informed consent. CONCLUSION: LPL gene polymorphisms are not genetic markers for the development of stroke in the Colombian sample used.
OBJETIVO: Determinar la asociación entre los polimorfismos en el gen LPL (rs320, rs285 y rs328), y la enfermedad cerebrovascular isquémica aguda en una muestra de población colombiana. MÉTODOS: A partir de un diseño de casos y controles, se estudiaron 133 casos con enfermedad cerebrovascular isquémica aguda (diagnóstico clínico y TAC), y 269 controles sin enfermedad cerebrovascular. Se examinó los polimorfismos rs320, rs285 y rs328 en el gen LPL con la técnica PCR-RFLP. RESULTADOS: En el presente estudio no se encontró asociación entre rs320, rs285 y rs328 con la enfermedad cerebrovascular isquémica aguda en la muestra analizada; siendo las frecuencias alélicas y genotípicas de los polimorfismos similares entre casos y controles, y se encontró en equilibrio de Hardy-Weinberg. El estudio fue avalado por el comité de ética de las instituciones vinculadas y todos los pacientes dieron consentimiento informado. CONCLUSIÓN: Los polimorfismos en el gen de la LPL no tienen utilidad como marcadores genéticos asociados con la presentación de la enfermedad cerebrovascular isquémica aguda en la muestra analizada.
