RESUMEN
Microdeletions, either subtelomeric or interstitial, are responsible for the mental handicap in approximately 10-20% of all patients. Currently, Multiplex Ligation-dependent Probe Amplification (MLPA) is widely used to detect these small aberrations in a routine fashion. Although cost-effective, the throughput is low and the degree of multiplexing is limited to maximally 40-50 probes. Therefore, we developed an array-based MLPA method, with probes identified by unique tag sequences, allowing the simultaneous analysis of 180 probes in a single experiment thereby covering all known mental retardation loci with at least two probes. We screened 120 patients with idiopathic mental retardation. In this group we detected 6 aberrations giving a detection rate of 5%, consistent with similar studies. In addition we tested 293 patients with mental retardation who were negative for fragile X syndrome and commercially available subtelomeric MLPA. We found seven causative rearrangements in this group (detection rate of 2.4%) thereby illustrating the value of including probes for interstitial microdeletion syndromes and additional probes in the telomeric regions in targeted screening sets for mental retardation. Array-based MLPA may thus be a good candidate to develop probe sets that rapidly detect copy number changes of disease associated loci in the human genome. This method may become a valuable tool in a routine diagnostic setting as it is a fast, user-friendly and relatively low-cost technique providing straightforward results requiring only 125 ng of genomic DNA.
Asunto(s)
Algoritmos , Deleción Cromosómica , Discapacidad Intelectual/genética , Análisis por Micromatrices/métodos , Cartilla de ADN/genética , Sondas de ADN/genética , Humanos , Hibridación Fluorescente in SituAsunto(s)
Infecciones Bacterianas/epidemiología , Genotipo , Trasplante de Hígado , Lectina de Unión a Manosa/genética , Complicaciones Posoperatorias/epidemiología , Donantes de Tejidos , Lectina de Unión a Manosa de la Vía del Complemento/genética , Humanos , Valor Predictivo de las Pruebas , Estudios Retrospectivos , Factores de RiesgoRESUMEN
BACKGROUND: Innate immunity plays a role in controlling adaptive immune responses. METHODS: We investigated the clinical relevance of single nucleotide polymorphisms in 22 genes encoding innate, secreted, and signaling pattern recognition receptors in a total of 520 donor-recipient pairs of postmortem, human leukocyte antigen-DR-compatible kidney transplantations. Associations with rejection incidence were tested in an a priori randomized training set and validation set. RESULTS: Polymorphisms in TLR-3 (rs3775296) in the recipients and in ficolin-2 (rs7851696; Ala258Ser) and C1qR1 (rs7492) in the donors showed the strongest association with severe rejection. In multivariate analysis, presence of the ficolin-2 Ala258Ser variant in the donor predicted lower incidence of severe rejection (odds ratio=0.3; 95% confidence interval, 0.1-0.9; P=0.024) and of graft loss (hazard ratio=0.5; 95% confidence interval, 0.2-1.0; P=0.046) independently of clinical risk factors. Ficolin-2 messenger RNA expression was detected in pretransplantation biopsies from 69 donor grafts. Serum and tissue ficolin-2 levels were unaffected by genotype. Ficolin-2 protein, which bound to dying cells, was detected in donor kidneys in a passenger leukocyte-like pattern. Indeed, monocytes, monocyte-derived macrophages, and peripheral blood mononuclear cells expressed ficolin-2. Donor grafts with the ficolin-2 Ala258Ser variant contained significantly elevated expression of interleukin 6, having ascribed cytoprotective effects. It has been described that Ala258Ser leads to increased binding capacity of ficolin-2 to N-acetylglucosamine. CONCLUSIONS: Presence of the ficolin-2 Ala258Ser polymorphism in the donor independently predicts improved graft outcome. Based on mechanistic data, we propose that this functional polymorphism leads to more efficient handling of injured cells by phagocytozing cells, resulting in decreased intragraft exposure to danger signals and dampened alloimmune responses.
Asunto(s)
Rechazo de Injerto/genética , Supervivencia de Injerto , Inmunidad Innata/genética , Trasplante de Riñón , Lectinas/genética , Polimorfismo de Nucleótido Simple , Donantes de Tejidos , Apoptosis , Biopsia , Exones , Regulación de la Expresión Génica , Genotipo , Rechazo de Injerto/sangre , Rechazo de Injerto/inmunología , Rechazo de Injerto/metabolismo , Humanos , Interleucina-1beta/genética , Interleucina-6/genética , Interleucina-6/metabolismo , Células Jurkat , Estimación de Kaplan-Meier , Trasplante de Riñón/efectos adversos , Trasplante de Riñón/inmunología , Lectinas/sangre , Modelos Logísticos , Análisis Multivariante , Países Bajos , Oportunidad Relativa , Fenotipo , Modelos de Riesgos Proporcionales , Medición de Riesgo , Factores de Riesgo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/patología , Factores de Tiempo , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/genética , FicolinasRESUMEN
We carried out a multicenter performance evaluation of three new DNA-based human leukocyte antigen (HLA) typing assays: INNO-LiPA HLA-A Update, INNO-LiPA HLA-B Update, and INNO-LiPA HLA-DQB1 Update. After optimization, the accuracy rates were all 100%, and the final observed resolutions were 99.4, 92.4, and 85.6%, respectively. These rapid and easy-to-perform assays yielded results fully concordant with other DNA-based tissue typing tests.