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Acquired Aplastic Anaemia (AA) often results from immune destruction of hematopoietic stem and progenitor cells. However, only 60-70% of patients with AA respond to immunosuppressive therapy (IST). There is lack of strong predictive marker for response to IST which can help therapy. Our study sought to pinpoint unique immune markers in AA patients and validate established predictors for response to IST. We enrolled 51 severe AA patients and analyzed 57 immunological parameters via flow cytometry. Additionally, we measured paroxysmal nocturnal hemoglobinuria (PNH) clone, telomere length, and thrombopoietin (TPO) levels prior to IST. After a 6-months follow-up, response was observed. Patients with AA had a distinct immunological signature characterized by absolute lymphopenia, skewed CD4/CD8 ratio with expansion of CD8 T cells with activated and senescent phenotype. Treg counts were reduced, while proportion of Treg A and B was comparable to controls. Treatment response was correlated with elevated Absolute Neutrophil Count (ANC), Absolute Reticulocyte Count (ARC), and reduced CD57+ CD8+ naive cells and B cell % before therapy. However, predictors like TPO, telomere length, and PNH did not emerge as indicators of treatment response. Identifying predictors for treatment response in AA is challenging due to abnormal haematopoiesis, genetic mutations, and treatment variables.
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Living organisms are exposed to exogenous and endogenous agents that affect genomic integrity by creating DNA double strand breaks (DSBs). These breaks are repaired by DNA repair proteins to maintain homeostasis. Defects in DNA repair pathways also affect lymphocyte development and maturation, as DSB sites are critical intermediates for rearrangements required for V(D)J recombination. Recent classifications for inborn errors of immunity (IEIs) have listed DNA repair defect genes in a separate group, which suggests the importance of these genes for adaptive and innate immunity. We report an interesting case of a young female (index P1) with mutations in two different genes, DCLRE1C and FANCA, involved in DNA repair pathways. She presented with clinical manifestations attributed to both defects. With the advent of NGS, more than one defect is increasingly identified in patients with IEIs. Familial segregation studies and appropriate functional assays help ascertain the pathogenicity of these mutations and provide appropriate management and genetic counseling.
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Bioensayo , Anemia de Fanconi , Humanos , Femenino , Genómica , Homeostasis , Inmunidad InnataRESUMEN
Leukocyte adhesion deficiency (LAD) is an immunodeficiency caused by defects in the adhesion of leukocytes (especially neutrophils) to the blood vessel wall. As a result, patients with LAD suffer from severe bacterial infections and impaired wound healing, accompanied by neutrophilia. In LAD-I, characterized directly after birth by delayed separation of the umbilical cord, mutations are found in ITGB2, the gene that encodes the ß subunit (CD18) of the ß2 integrins. In the rare LAD-II disease, the fucosylation of selectin ligands is disturbed, caused by mutations in SLC35C1, the gene that encodes a GDP-fucose transporter of the Golgi system. LAD-II patients lack the H and Lewis Lea and Leb blood group antigens. Finally, in LAD-III, the conformational activation of the hematopoietically expressed ß integrins is disturbed, leading to leukocyte and platelet dysfunction. This last syndrome is caused by mutations in FERMT3, encoding the kindlin-3 protein in all blood cells, involved in the regulation of ß integrin conformation. This article contains an update of the mutations that we consider to be relevant for the various forms of LAD.
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Síndrome de Deficiencia de Adhesión del Leucocito , Humanos , Adhesión Celular/genética , Síndrome de Deficiencia de Adhesión del Leucocito/genética , Antígenos CD18/genética , Antígenos CD18/metabolismo , Leucocitos , MutaciónRESUMEN
DOCK8 deficiency affects various cell subsets belonging to both the innate and adaptive immune systems. Clinical diagnosis is challenging, as many cases present with severe atopic dermatitis as the only initial manifestation. Though flow cytometry helps in the presumptive diagnosis of DOCK8-deficient patients by evaluating their DOCK8 protein expression, it requires subsequent confirmation by molecular genetic analysis. Currently, haematopoietic stem cell transplantation (HSCT) is the only curative treatment option available for these patients. There is a paucity of data from India on the clinical diversity and molecular spectrum of DOCK8 deficiency. In the present study, we report the clinical, immunological and molecular findings of 17 DOCK8-deficient patients from India diagnosed over the last 5 years.
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Síndrome de Job , Humanos , India , Factores de Intercambio de Guanina Nucleótido/genéticaRESUMEN
Background & objectives: Accurate diagnosis of immunodeficiencies requires a critical comparison of values with age-matched controls. In India, the existing reference values for rare lymphocyte subsets are currently not available and we rely on the data originating from other countries for the interpretation of the results. Furthermore, there is limited information on normal variation for these rare-subset parameters in Indian children. So, this study aimed to establish normative values for clinically important lymphocyte subsets in Indian children at different age groups. Methods: 148 children aged ≥16 yr were enrolled in this study. The study population included 61 per cent males and 39 per cent females and was divided into the following groups: cord blood (n=18), 0-6 months (n=9), 6-12 months (n=13), 1-2 yr (n=19), 2-5 yr (n=27), 5-10 yr (n=25) and 10-16 yr (n=37). The absolute and relative percentage of lymphocytes, T, B, natural killer cell, along with activated, naïve and memory subsets, was determined by flow cytometry. Results: Median values and the 10th and 90th percentiles were obtained for 34 lymphocyte sub-populations. The T and B naïve compartments showed a decreasing trend, whereas memory cells showed an increase with age. The activated T cell subset shows an increasing pattern up to one year and then declines gradually. Double negative T cells are relatively stable. TCRgd+T cell percentage increases with age. Interpretation & conclusions: This single-centre pilot study provides preliminary data that justifies the need for future large-scale multi centric studies to generate a reference range for interpreting extended immunophenotyping profiles in the paediatric age group, making it possible for clinicians to assess the immunological status in inborn errors of immunity, infectious and autoimmune diseases.
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Subgrupos Linfocitarios , Subgrupos de Linfocitos T , Masculino , Femenino , Niño , Humanos , Proyectos Piloto , Recuento de Linfocitos , Inmunofenotipificación , Citometría de Flujo , India/epidemiología , Valores de ReferenciaRESUMEN
MOTIVATION: BAli-Phy, a popular Bayesian method that co-estimates multiple sequence alignments and phylogenetic trees, is a rigorous statistical method, but due to its computational requirements, it has generally been limited to relatively small datasets (at most about 100 sequences). Here, we repurpose BAli-Phy as a 'phylogeny-aware' alignment method: we estimate the phylogeny from the input of unaligned sequences, and then use that as a fixed tree within BAli-Phy. RESULTS: We show that this approach achieves high accuracy, greatly superior to Prank, the current most popular phylogeny-aware alignment method, and is even more accurate than MAFFT, one of the top performing alignment methods in common use. Furthermore, this approach can be used to align very large datasets (up to 1000 sequences in this study). AVAILABILITY AND IMPLEMENTATION: See https://doi.org/10.13012/B2IDB-7863273_V1 for datasets used in this study. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Algoritmos , Filogenia , Teorema de Bayes , Indonesia , Alineación de SecuenciaRESUMEN
PURPOSE: Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome mainly caused by uncontrolled activation of antigen presenting cells and CD8 T cells. CD8 T cell exhaustion is a known phenomenon in chronic viral infections and cancer. However, the role of T cell exhaustion is not yet identified in HLH in the background of persistent inflammation. So, currently, we have characterized the CD8 T cells using flow cytometry to understand the phenomenon of exhaustion in these cells in HLH. METHODS: We have comprehensively evaluated lymphocyte subsets and characterized CD8 T cells using immunophenotypic markers like PD1, TIM3, LAG3, Ki67, Granzyme B, etc. in a cohort of 21 HLH patients. Effector cytokine secretion and degranulation by CD8 T cells are also studied. RESULTS: Our findings indicate skewed lymphocyte subsets and aberrantly activated CD8 T cells in HLH. CD8 T cells exhibit significantly increased expression of PD1, TIM3, and LAG3 prominently in primary HLH as compared to controls. PD1 + CD8 T cells express elevated levels of Granzyme B and Ki67. Moreover, CD8 T cells are hypofunctional as evidenced by significantly reduced cytokine secretion and compromised CD107a degranulation. CONCLUSION: The study has revealed that CD8 + cytotoxic T lymphocytes from HLH patients exhibited high expression of exhaustion markers with overall impaired function. To the best of our understanding, this is the first report suggesting functional exhaustion of CD8 T cells in both primary and secondary HLH. Future studies to understand the association of exhaustion with disease outcome are needed for its probable therapeutic implementation.
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Linfocitos T CD8-positivos/inmunología , Linfohistiocitosis Hemofagocítica/inmunología , Adolescente , Adulto , Anciano , Antígenos CD/inmunología , Niño , Preescolar , Citocinas/inmunología , Femenino , Granzimas/inmunología , Receptor 2 Celular del Virus de la Hepatitis A/inmunología , Humanos , Lactante , Antígeno Ki-67/inmunología , Masculino , Persona de Mediana Edad , Fenotipo , Receptor de Muerte Celular Programada 1/inmunología , Adulto Joven , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
World Health Organisation recommends the practice of BCG vaccination at birth in countries which have a high incidence of tuberculosis and/or high leprosy burden. The BCG vaccination is considered safe for a competent immune system. However, in children with weakened immune systems cause of which can be primary or secondary, the vaccine may lead to side effects which can be localized or disseminated. In this study, we report a spectrum of inborn errors of immunity (IEI) commonly referred to as primary immunodeficiency disorders (PIDs) diagnosed in a large cohort of patients presenting with complications to BCG vaccination from India. Retrospective data analysis of patients referred to ICMR- National Institute of Immunohematology (ICMR-NIIH) for IEI workup between 2007 and 2019 was done. IEI was identified in n = 52/90 (57.7%) patients presenting with BCG complications. Of these, n = 13(14.4%) patients were diagnosed with severe combined immune deficiency, n = 15(16.7%) with chronic granulomatous disease, n = 19(21.1%) with Inborn errors of IFN-γ immunity, n = 4(4.4%) with Combined immunodeficiency and n = 1(1.1%) with Leucocyte Adhesion Deficiency type1. Majority of cases with BCGosis (88%) had an underlying IEI. This study strongly highlights the need for evaluation of patients with BCG complications for underlying IEI. While disseminated BCGosis strongly predicts underlying IEI, even localized persistent adenitis may be a warning sign of underlying IEI. It is also strongly recommended to record a family history of previous sibling death prior to administration of this live vaccine and deferring live vaccine till the diagnosis of IEI is ruled out in cases with a positive family history.
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Vacuna BCG/efectos adversos , Enfermedad Granulomatosa Crónica/patología , Inmunodeficiencia Combinada Grave/patología , Tuberculosis Pulmonar/prevención & control , Vacunación/efectos adversos , Vacuna BCG/inmunología , Femenino , Enfermedad Granulomatosa Crónica/diagnóstico , Enfermedad Granulomatosa Crónica/inmunología , Humanos , India , Lactante , Masculino , Mycobacterium tuberculosis/inmunología , Inmunodeficiencia Combinada Grave/diagnóstico , Inmunodeficiencia Combinada Grave/inmunología , Resultado del TratamientoRESUMEN
The mammalian adaptive immune system has evolved over millions of years to become an incredibly effective defense against foreign antigens. The adaptive immune system's humoral response creates plasma B cells and memory B cells, each with their own immunological objectives. The affinity maturation process is widely viewed as a heuristic to solve the global optimization problem of finding B cells with high affinity to the antigen. However, memory B cells appear to be purposely selected earlier in the affinity maturation process and have lower affinity. We propose that this memory B cell selection process may be an approximate solution to two optimization problems: optimizing for affinity to similar antigens in the future despite mutations or other minor differences, and optimizing to warm start the generation of plasma B cells in the future. We use simulations to provide evidence for our hypotheses, taking into account data showing that certain B cell mutations are more likely than others. Our findings are consistent with memory B cells having high-affinity to mutated antigens, but do not provide strong evidence that memory B cells will be more useful than selected naive B cells for seeding the secondary germinal centers.
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Centro Germinal , Memoria Inmunológica , Inmunidad Adaptativa , Animales , Antígenos , Linfocitos BAsunto(s)
Enfermedades Autoinmunes , Síndrome Linfoproliferativo Autoinmune , Humanos , Síndrome Linfoproliferativo Autoinmune/genética , Enfermedades Autoinmunes/genética , Fenotipo , Mutación , Receptor fas/genética , Apoptosis/genética , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismoRESUMEN
BACKGROUND: Chronic granulomatous disease (CGD) is characterized by mutation in any one of the five genes coding NADPH oxidase components that leads to functional abnormality preventing the killing of phagocytosed microbes by affecting the progression of a respiratory burst. CGD patients have an increased susceptibility to infections by opportunistic and pathogenic organisms. Though initial diagnosis of CGD using a nitroblue tetrazolium (NBT) test or dihydrorhodamine (DHR) test is relatively easy, molecular diagnosis is challenging due to involvement of multiple genes, presence of pseudogenes, large deletions, and GC-rich regions, among other factors. The strategies for molecular diagnosis vary depending on the affected gene and the mutation pattern prevalent in the target population. There is a paucity of molecular data related to CGD for Indian population. METHOD: This report includes data for a large cohort of CGD patients (n = 90) from India, describing the diagnostic approach, mutation spectrum, and novel mutations identified. We have used mosaicism in mothers and the expression pattern of different NADPH components by flow cytometry as a screening tool to identify the underlying affected gene. The techniques like Sanger sequencing, next-generation sequencing (NGS), and Genescan analysis were used for further molecular analysis. RESULT: Of the total molecularly characterized patients (n = 90), 56% of the patients had a mutation in the NCF1 gene, 30% had mutation in the CYBB gene, and 7% each had mutation in the CYBA and NCF2 genes. Among the patients with NCF1 gene mutation, 82% of the patients had 2-bp deletion (DelGT) mutations in the NCF1 gene. In our cohort, 41 different mutations including 9 novel mutations in the CYBB gene and 2 novel mutations each in the NCF2, CYBA, and NCF1 genes were identified. CONCLUSION: Substantial number of the patients lack NCF1 gene on both the alleles. This is often missed by advanced molecular techniques like Sanger sequencing and NGS due to the presence of pseudogenes and requires a simple Genescan method for confirmation. Thus, the diagnostic approach may depend on the prevalence of affected genes in respective population. This study identifies potential gene targets with the help of flow cytometric analysis of NADPH oxidase components to design an algorithm for diagnosis of CGD in India. In Indian population, the Genescan method should be preferred as the primary molecular test to rule out NCF1 gene mutations prior to Sanger sequencing and NGS.
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Enfermedad Granulomatosa Crónica/diagnóstico , Mutación/genética , NADPH Oxidasa 2/genética , NADPH Oxidasas/genética , NADP/metabolismo , Patología Molecular/métodos , Adolescente , Adulto , Niño , Preescolar , Estudios de Cohortes , Análisis Mutacional de ADN , Femenino , Citometría de Flujo , Enfermedad Granulomatosa Crónica/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , India , Lactante , Masculino , Nitroazul de Tetrazolio , Adulto JovenRESUMEN
Prolonged excretion of poliovirus can occur in immunodeficient patients who receive oral polio vaccine, which may lead to propagation of highly divergent vaccine-derived polioviruses (VDPVs), posing a concern for global polio eradication. This study aimed to estimate the proportion of primary immunodeficient children with enterovirus infection and to identify the long-term polio/nonpolio enterovirus excreters in a tertiary care unit in Mumbai, India. During September 2014-April 2017, 151 patients received diagnoses of primary immunodeficiency (PID). We isolated 8 enteroviruses (3 polioviruses and 5 nonpolio enteroviruses) in cell culture of 105 fecal samples collected from 42 patients. Only 1 patient with severe combined immunodeficiency was identified as a long-term VDPV3 excreter (for 2 years after identification of infection). Our results show that the risk of enterovirus excretion among children in India with PID is low; however, systematic screening is necessary to identify long-term poliovirus excreters until the use of oral polio vaccine is stopped.
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Síndromes de Inmunodeficiencia/virología , Poliomielitis/prevención & control , Vacuna Antipolio Oral/administración & dosificación , Poliovirus/inmunología , Esparcimiento de Virus/inmunología , Niño , Preescolar , Enterovirus Humano C/inmunología , Enterovirus Humano C/patogenicidad , Infecciones por Enterovirus/inmunología , Infecciones por Enterovirus/transmisión , Infecciones por Enterovirus/virología , Heces/virología , Femenino , Humanos , Síndromes de Inmunodeficiencia/inmunología , Síndromes de Inmunodeficiencia/patología , India , Lactante , Masculino , Poliomielitis/inmunología , Poliomielitis/transmisión , Poliomielitis/virología , Poliovirus/patogenicidad , RiesgoRESUMEN
Neurological manifestations in familial hemophagocytic lymphohistiocytosis (FHL) are common, seen in up to 73% of patients in their course of disease. However, in majority of the cases central nervous system manifestations are associated with other clinical and laboratory parameters of hemophagocytic lymphohistiocytosis. We report here a case with FHL2 in whom hemophagocytic lymphohistiocytosis was a presenting manifestation which responded to specific therapy, however, there was isolated central nervous system relapse while patient was in remission and off therapy. FHL2 was confirmed on the basis of reduced perforin expression and homozygous mutation in PRF1at codon 637 in exon 3 (c.673C>T p.Arg225Trp).
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Enfermedades Desmielinizantes/etiología , Linfohistiocitosis Hemofagocítica/genética , Trastornos del Movimiento/etiología , Debilidad Muscular/etiología , Mutación Missense , Perforina/genética , Adulto , Tronco Encefálico/diagnóstico por imagen , Preescolar , Codón/genética , Ciclosporina/uso terapéutico , Enfermedades Desmielinizantes/diagnóstico por imagen , Dexametasona/uso terapéutico , Exones/genética , Femenino , Ferritinas/sangre , Lóbulo Frontal/diagnóstico por imagen , Humanos , Inmunosupresores/uso terapéutico , Linfohistiocitosis Hemofagocítica/sangre , Linfohistiocitosis Hemofagocítica/diagnóstico por imagen , Linfohistiocitosis Hemofagocítica/tratamiento farmacológico , Imagen por Resonancia Magnética , Masculino , Metotrexato/uso terapéutico , Perforina/deficiencia , Recurrencia , Infecciones del Sistema Respiratorio/etiologíaRESUMEN
Chronic granulomatous disease (CGD) is a group of inherited disorder of phagocytes, resulting from mutations in the components of the NADPH oxidase complex. Reduced or absent oxygen radical synthesis seen in these patients leads to impaired killing of intracellular bacteria and fungi. CGD clinically presents with recurrent and life-threatening infections as well as granulomatous inflammatory responses. p47phox encoded by the NCF1 gene is the most common autosomal recessive form of CGD which is often clinically milder. Here, we are presenting the data on clinical and immunological findings in 21 Indian patients with Del GT mutation in the NCF1 gene. Diagnosis of these patients was based on detailed clinical evaluation, measurement of respiratory burst activity by nitro blue tetrazolium and dihydrorhodamine-1,2,3 assay, expression of p47phox by flow cytometry, and molecular confirmation by GeneScan method. Seventeen male and four female patients with median age of onset of 1 year ranging from 1.5 months to 6 years were included in the study. Sixty-two percent (13 out of 21) of patients belonged to a consanguineous marriage with only one family having a history of a previous sibling death. Significant variability in clinical presentation was observed in spite of identical genetic defect ranging from asymptomatic to very severe presentation leading to early death or requiring transplantation. However, none of these patients showed difference in immunological parameters to account for this variability. Thus, this study highlights the phenotypic heterogeneity seen in these patients with Del GT mutation in the NCF1 gene and its implication in management of these patients.
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Enfermedad Granulomatosa Crónica/genética , Enfermedad Granulomatosa Crónica/inmunología , NADPH Oxidasas/genética , Niño , Preescolar , Consanguinidad , Femenino , Humanos , India , Lactante , Masculino , Mutación/genética , NADPH Oxidasas/inmunología , Fagocitos/inmunología , Estallido Respiratorio/genética , Estallido Respiratorio/inmunologíaAsunto(s)
Antígenos HLA-DR/genética , Inmunidad Celular/inmunología , Inmunidad Humoral/inmunología , Inmunodeficiencia Combinada Grave/inmunología , Inmunodeficiencia Combinada Grave/terapia , Agammaglobulinemia/genética , Agammaglobulinemia/inmunología , Recuento de Linfocito CD4 , Relación CD4-CD8 , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Preescolar , Proteínas de Unión al ADN/genética , Antígenos HLA-DR/inmunología , Humanos , Masculino , Proteínas Nucleares/genética , Factores de Transcripción del Factor Regulador X/genética , Inmunodeficiencia Combinada Grave/genética , Transactivadores/genética , Factores de Transcripción/genéticaRESUMEN
Pyoderma gangrenosum (PG) is an uncommon noninfectious neutrophilic dermatosis characterized by recurrent, sterile, necrotic skin ulcers. It is commonly associated with underlying systemic disease like inflammatory bowel disease, rheumatoid arthritis and hematological malignancies. Pathogenesis of PG remains unclear though aberrant immune responses have been implicated. The diagnosis of PG is of exclusion and management is empirical with local or systemic immunosuppressive therapy. LAD-I is a rare form of autosomal recessive disorders caused by mutations of the gene ITGB2, clinically characterized by recurrent severe bacterial infection, impaired pus formation, poor wound healing and persistent neutrophilia. Though skin ulcerations are common, predominant clinical presentation as PG is unusual in LAD-I. Here we present four Indian patients with LAD-I from three unrelated families initially diagnosed as PG due to chronic recurrent skin ulcerations requiring steroids and antibiotics for healing, associated with atrophic scar formation. All these four patients had persistent neutrophilia without history of delayed cord separation and showed moderate expression of CD18 (19 to 68%) on neutrophils. Sequencing of the entire coding region and intronic splice sites of the ITGB2 gene from the genomic DNA of these patients revealed a novel common mutation IVS10+4A>G. LAD-I should be kept in mind while evaluating patients with PG especially those with persistent neutrophila in the absence of other rheumatological disorders. Diagnosis of LAD-I in these cases is extremely important for management as treating these patients without adequate antibiotic cover may prove fatal and these patients often require hematopoietic stem cell transplantation for permanent cure.
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Antígenos CD18/genética , Intrones , Síndrome de Deficiencia de Adhesión del Leucocito/diagnóstico , Síndrome de Deficiencia de Adhesión del Leucocito/genética , Mutación , Piodermia Gangrenosa/diagnóstico , Adolescente , Niño , Preescolar , Consanguinidad , Análisis Mutacional de ADN , Diagnóstico Diferencial , Femenino , Humanos , Inmunofenotipificación , Recuento de Leucocitos , Masculino , Neutrófilos/metabolismo , Fenotipo , Úlcera Cutánea/patologíaRESUMEN
PURPOSE: Leukocyte adhesion deficiency type-I (LAD-I) is caused by mutations in the ITGB2 gene, encoding the ß2-subunit of ß2-integrin (CD18) which leads to markedly reduced expression of CD18 on leukocytes resulting into recurrent life threatening infections. Here we aim to identify the molecular defects underlying LAD-I in Indian patients and correlate with the clinical presentation. METHODS: Blood was collected from 30 patients and their parents for absolute neutrophil count, expression of CD18 and CD11 by flow cytometry and DNA extraction. PCR and DNA sequencing of the ITGB2 gene was done for mutation characterization. RESULTS: Phenotypically, 22 patients were LAD-I(0), 1 was LAD-I(-) and 7 were LAD-I(+) showing no expression and reduced expression of CD18 respectively. Nine novel mutations in 15 patients and 11 known mutations in 16 patients were detected. Prenatal diagnosis was performed for 5 families. CONCLUSION: In this study 30 patients were phenotypically and genotypically evaluated for a less known disease LAD-I. Unavailability of curative options to majority of the patients and high cost of supportive care emphasize the need to increase awareness about a suspicious case so that timely management can be given to the patient and prenatal diagnosis can be offered to their families.
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Antígenos CD18/genética , Síndrome de Deficiencia de Adhesión del Leucocito/genética , Mutación , Análisis Mutacional de ADN , Femenino , Humanos , India , Lactante , Recién Nacido , Síndrome de Deficiencia de Adhesión del Leucocito/diagnóstico , Leucocitos/metabolismo , Leucocitos/patología , Masculino , Diagnóstico Prenatal , Población Blanca/genéticaRESUMEN
OBJECTIVES: Primary Immunodeficiency diseases (PID) are a heterogeneous group of inherited disorders of immune system. Immunophenotypic evaluation of PIDs using flowcytometry provides important clues for diagnosis of these disorders, though confirmation requires identification of underlying molecular defects. Prenatal diagnosis (PND) forms an important component of management in families affected with severe PID. However, molecular diagnostic facilities for each of these diseases are not available and may not be possible to perform in all cases. In such scenario we opted for phenotypic prenatal diagnosis by cordocentesis for families with index case having immunophenotypically well characterized PID. METHODS: Normal reference ranges of lymphocyte subsets, CD 18/CD11 integrins on leukocytes, MHC class II expression and oxidative burst activity of fetal neutrophils at 18 weeks of gestation were previously established on 30 cord blood samples. PND was performed in 13 families with PIDs. Maternal contamination was ruled out by VNTR analysis. RESULTS: Out of 13 fetuses, nine were found to be unaffected (three cases with leukocyte adhesion deficiency (LAD-I), four cases with severe combined immunodeficiency diseases (SCID), one with X-linked agammaglobulinemia (XLA), and one with chronic granulomatous disease (CGD)] and three were found to be affected (one with T-B+NK-SCID, one with MHC class II deficiency and one with LAD-I). Diagnosis was confirmed by testing the cord blood samples after delivery and further follow-up of the children. In one family diagnosis could not be offered due to maternal contamination. No procedure related complications were observed. CONCLUSION: Flowcytometry offers rapid and sensitive method for prenatal diagnosis and genetic counseling for selected phenotypically well characterized PID in cases where molecular diagnostic facilities are not available.
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Citometría de Flujo , Síndromes de Inmunodeficiencia/diagnóstico , Diagnóstico Prenatal , Femenino , Sangre Fetal/citología , Humanos , Inmunofenotipificación , Subgrupos Linfocitarios/metabolismo , EmbarazoRESUMEN
BACKGROUND: X-linked hyper-IgM (XHIM) is a primary immunodeficiency disorder characterized by recurrent infections, low serum IgG and IgA and normal or elevated IgM. It results from mutations in the CD40 ligand (CD40L) gene. Confirmation of diagnosis with identification of underlying molecular defect is important for the initiation of appropriate therapeutic interventions, including immunoglobulin replacement, antibiotics and bone marrow transplantation. METHODS: To investigate the molecular basis of XHIM, we evaluated 7 patients with suspected XHIM and abnormal CD40L expression on activated CD4(+) T lymphocytes. The entire coding region and intronic splice sites of the CD40L gene were sequenced from the genomic DNA of the patients. RESULTS: 7 mutations; 3 nonsense (c.172delA, c.A229T, c.C478T), 1 missense (c.A506G) and 3 splice sites [c.346+2(TâC), c.289-1(GâC), c.346+1(GâT)] were identified, out of which 5 were novel. CONCLUSION: A wide heterogeneity in the nature of mutations has been observed in Indian XHIM patients in the present study. Identification of mutations in this rare disorder will help in genetic diagnosis in affected families which could be further useful in prenatal diagnosis.
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Síndrome de Inmunodeficiencia con Hiper-IgM Tipo 1/diagnóstico , Síndrome de Inmunodeficiencia con Hiper-IgM Tipo 1/etiología , Ligando de CD40/genética , Ligando de CD40/metabolismo , Estudios de Casos y Controles , Niño , Preescolar , Análisis Mutacional de ADN , Expresión Génica , Humanos , Inmunofenotipificación , India , Lactante , Masculino , Mutación , Fenotipo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Assessment of natural killer cells (NK-cell) cytotoxicity is used not only in research settings but is also important in diagnosis of various diseases. NK-cell cytotoxicity assays are based on measurement of target cells killed by cytotoxic cells analyzed either by chromium (51Cr) release assay or flow cytometry. Both these methods use peripheral blood mononuclear cells (PBMC) or pure NK-cell population and hence require large volume of blood sample which is difficult to obtain in pediatric patients and patients with cytopenia. Hence, a flow cytometric assay was designed to determine NK cell activity using whole blood, eliminating the need for isolation of PBMCs or pure NK cells. This assay is based on a dual fluorescent staining of target cells (K562 cell line). The DIOC18 dye labeled K562 cells are incubated with whole blood and then counterstained with 7-AAD enabling the measurement of dead target cell and then percent cytotoxicity is calculated. This study compared the NK cell cytotoxicity using PBMC and whole blood in clinically relevant samples. There was no significant difference between two assays in the measurement of lytic activity or in-reproducibility in the repeated samplings of healthy individuals. The whole blood assay required less volume of blood and also less processing time as compared to PBMC assay. It was also validated by testing patients diagnosed with familial hemophagocytic lymphohistiocytosis expected to have low NK-cell activity. This assay is rapid, sensitive and reproducible and requires significantly less volume of blood which is important for clinical evaluation of NK-cell function.