RESUMO
Background: Bruton's tyrosine kinase (BTK) is a cytoplasmic protein involved in the B cell development. X-linked agammaglobulinemia (XLA) is caused by mutation in the BTK gene, which results in very low or absent B cells. Affected males have markedly reduced immunoglobulin levels, which render them susceptible to recurrent and severe bacterial infections. Methods: Patients suspected with X-linked agammaglobulinemia were enrolled during the period of 2010-2018. Clinical summary, and immunological profiles of these patients were recorded. Peripheral blood samples were collected for monocyte BTK protein expression detection and BTK genetic analysis. The medical records between January 2020 and June 2023 were reviewed to investigate COVID-19 in XLA. Results: Twenty-two patients (from 16 unrelated families) were molecularly diagnosed as XLA. Genetic testing revealed fifteen distinct mutations, including four splicing mutations, four missense mutations, three nonsense mutations, three short deletions, and one large indel mutation. These mutations scattered throughout the BTK gene and mostly affected the kinase domain. All mutations including five novel mutations were predicted to be pathogenic or deleterious by in silico prediction tools. Genetic testing confirmed that eleven mothers and seven sisters were carriers for the disease, while three mutations were de novo. Flow cytometric analysis showed that thirteen patients had minimal BTK expression (0-15%) while eight patients had reduced BTK expression (16-64%). One patient was not tested for monocyte BTK expression due to insufficient sample. Pneumonia (n=13) was the most common manifestation, while Pseudomonas aeruginosa was the most frequently isolated pathogen from the patients (n=4). Mild or asymptomatic COVID-19 was reported in four patients. Conclusion: This report provides the first overview of demographic, clinical, immunological and genetic data of XLA in Malaysia. The combination of flow cytometric assessment and BTK genetic analysis provides a definitive diagnosis for XLA patients, especially with atypical clinical presentation. In addition, it may also allow carrier detection and assist in genetic counselling and prenatal diagnosis.
Assuntos
Agamaglobulinemia , COVID-19 , Masculino , Gravidez , Feminino , Humanos , Proteínas Tirosina Quinases/genética , Malásia , COVID-19/genética , Tirosina Quinase da Agamaglobulinemia/genética , Agamaglobulinemia/diagnóstico , Agamaglobulinemia/genéticaRESUMO
OBJECTIVES: To study the incidence of Omicron infections in Malaysia and the exposures that could reduce the hazard of attaining Omicron infection. METHODS: We used a multicenter, prospective cohort to study 482 healthcare workers vaccinated with two and three doses of BNT162b2 for SARS-CoV-2 infection during the Omicron-dominant period in Malaysia. RESULTS: Between January 31 and July 31, 2022, the cumulative incidence was 44.6% (95% CI 40.2-49.1%), and the incidence rate was 3.33 (95% CI 2.91-3.80) per 1000 person-days. Our study found that protection against Omicron infection was significantly higher for persons with previous SARS-CoV-2 infection (hazard ratio [HR] 0.41, 95% CI 0.27-0.62) and persons with a more recent immunity event (<30 days [reference] vs >90 days, HR 3.82, 95%CI 1.34-10.90) from the beginning of the Omicron period. CONCLUSION: Pre-Omicron natural infection and a recent immunity event protect against future Omicron infections.
Assuntos
Vacina BNT162 , COVID-19 , Humanos , Malásia , Estudos Prospectivos , SARS-CoV-2RESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is the most common type of inherited cystic kidney disease. The feasibility of wholeexome sequencing (WES) to obtain molecular diagnosis of ADPKD is still in question as previous studies showed conflicting results. Utilizing WES on a patient with ADPKD, standard bioinformatics pipeline demonstrated no pathogenic variant in the genes of interest. By visualizing read alignments using the Integrative Genomics Viewer, a region with atypical alignment of numerous softclipped reads at exon 45 of polycystin 1, transient receptor potential channel interacting (PKD1) gene was demonstrated. A total of four visual inspection steps were outlined to assess the origin of these softclipped reads as strand bias during capture, poor mapping, sequencing error or DNA template contamination. Following assessment, the atypical alignment at PKD1 was hypothesized to be caused by an insertion/deletion mutation. Sanger sequencing confirmed the presence of a novel 20bp insertion in PKD1 (NM_001009944.3; c.12143_12144insTCCâCCGâCAGâTCTâTCCâCCGâCA; p.Val4048LeufsTer157), which introduced a premature stop codon and was predicted to be pathogenic. The present study demonstrated that WES could be utilized as a molecular diagnostic tool for ADPKD. Furthermore, visual inspection of read alignments was key in identifying the pathogenic variant. The proposed visual inspection steps may be incorporated into a typical WES data analysis workflow to improve the diagnostic yield.
Assuntos
Rim Policístico Autossômico Dominante , Humanos , Rim Policístico Autossômico Dominante/diagnóstico , Rim Policístico Autossômico Dominante/genética , Sequenciamento do Exoma , Códon sem Sentido , Mutação , Canais de Cátion TRPP/genética , DNARESUMO
Background: Inborn errors of immunity (IEIs) are comprised of heterogeneous groups of genetic disorders affecting immune function. In this report, a 17-month-old Malay patient suspected of having Hyper IgM syndrome, a type of IEIs, was described. However, the diagnosis of Hyper IgM syndrome was excluded by the normal functional studies and the mild features of ectodermal dysplasia observed from a further clinical phenotype inspection. Methods: Whole-exome sequencing (WES) was performed to unravel the causative mutation in this patient. Results: The variant analysis demonstrated a novel missense mutation in NFKBIA (NM_020529:c.94A > T,NP_065390:p.Ser32Cys) and was predicted as damaging by in silico prediction tools. The NFKBIA gene encodes for IκBα, a member of nuclear factor kappa B (NF-κB) inhibitors, playing an important role in regulating NF-κB activity. The mutation occurred at the six degrons (Asp31-Ser36) in IκBα which were evolutionarily conserved across several species. Prediction analysis suggested that the substitution of Ser32Cys may cause a loss of the phosphorylation site at residue 32 and a gain of the sumoylation site at residue 38, resulting in the alteration of post-translational modifications of IκBα required for NF-κB activation. Conclusion: Our analysis hints that the post-translational modification in the NFKBIA Ser32Cys mutant would alter the signaling pathway of NF-κB. Our findings support the usefulness of WES in diagnosing IEIs and suggest the role of post-translational modification of IκBα.
Assuntos
Disgamaglobulinemia , Displasia Ectodérmica , Síndrome de Imunodeficiência com Hiper-IgM , Síndromes de Imunodeficiência , Humanos , Inibidor de NF-kappaB alfa/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Mutação de Sentido Incorreto , Displasia Ectodérmica/genética , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismoRESUMO
Blended phenotypes exhibited by a patient may present a challenge to the establishment of diagnosis. In this study, we report a seven-year-old Murut girl with unusual features of Williams-Beuren syndrome (WBS), including recurrent infections and skin abscesses. Considering the possibility of a second genetic disorder, a mutation screening for genes associated with inborn errors of immunity (IEI) was conducted using whole exome sequencing (WES). Analysis of copy number variations (CNVs) from the exome data revealed a 1.53Mb heterozygous deletion on chromosome 7q11.23, corresponding to the known WBS. We also identified a biallelic loss of NCF1, which indicated autosomal recessive chronic granulomatous disease (CGD). Dihydrorhodamine (DHR) flow cytometric assay demonstrated abnormally low neutrophil oxidative burst activity. Coamplification of NCF1 and its pseudogenes identified a GT-deletion (ΔGT) at the start of exon 2 in NCF1 (NM_000265.7: c.75_76delGT: p.Tyr26Hisfs*26). Estimation of NCF1-to-NCF1 pseudogenes ratio using ΔGT and 20-bp gene scans affirmed nil copies of NCF1 in the patient. While the father had a normal ratio of 2:4, the mother had a ratio of 1:5, implicating the carrier of ΔGT-containing NCF1. Discovery of a 7q11.23 deletion involving one NCF1 allele and a ΔGT in the second NCF1 allele explained the coexistence of WBS and CGD in our patient. This study highlights the capability of WES to establish a molecular diagnosis for a case with blended phenotypes, enabling the provision of appropriate prophylactic treatment.
Assuntos
Sequenciamento do Exoma , Doença Granulomatosa Crônica/genética , NADPH Oxidases/genética , Síndrome de Williams/genética , Criança , Deleção Cromossômica , Variações do Número de Cópias de DNA , Feminino , Dosagem de Genes , Predisposição Genética para Doença , Doença Granulomatosa Crônica/diagnóstico , Doença Granulomatosa Crônica/imunologia , Doença Granulomatosa Crônica/terapia , Humanos , Mutação , Fenótipo , Valor Preditivo dos Testes , Síndrome de Williams/diagnóstico , Síndrome de Williams/imunologia , Síndrome de Williams/terapiaRESUMO
Primary immunodeficiency diseases refer to inborn errors of immunity (IEI) that affect the normal development and function of the immune system. The phenotypical and genetic heterogeneity of IEI have made their diagnosis challenging. Hence, whole-exome sequencing (WES) was employed in this pilot study to identify the genetic etiology of 30 pediatric patients clinically diagnosed with IEI. The potential causative variants identified by WES were validated using Sanger sequencing. Genetic diagnosis was attained in 46.7% (14 of 30) of the patients and categorized into autoinflammatory disorders (n = 3), diseases of immune dysregulation (n = 3), defects in intrinsic and innate immunity (n = 3), predominantly antibody deficiencies (n = 2), combined immunodeficiencies with associated and syndromic features (n = 2) and immunodeficiencies affecting cellular and humoral immunity (n = 1). Of the 15 genetic variants identified, two were novel variants. Genetic findings differed from the provisional clinical diagnoses in seven cases (50.0%). This study showed that WES enhances the capacity to diagnose IEI, allowing more patients to receive appropriate therapy and disease management.
Assuntos
Sequenciamento do Exoma , Doenças Genéticas Inatas/genética , Imunidade Celular/genética , Imunidade Inata/genética , Síndromes de Imunodeficiência/genética , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Malásia , Masculino , Projetos PilotoRESUMO
BACKGROUND: A retrospective review of clinical manifestations and demographic pattern of patients diagnosed as chronic granulomatous disease (CGD) from 7 hospitals in Malaysia. An analysis of the available database would establish clinical characteristics, diagnoses and outcome including microbiologic pattern. Studying the demography allows us to document the occurrence of CGD amongst multiethnic groups and its geographical distribution for Malaysia. METHODS: Data from the Malaysia Primary Immunodeficiency Network (MyPIN) with cases of CGD diagnosed from 1991 until 2016 were collated and analysed. RESULTS: Twenty patients were diagnosed as CGD. Males (N = 13, 65%) outnumber females (N = 7, 35%). CGD is commonest amongst the Malays (65%) followed by the Chinese (15.0%), Indians (10.0%) and natives of Borneo (10.0%), reflecting the ethnic composition of the country. The mean age of diagnosis was 3.7 years. There was a positive family history in 40% of the cases. Abscess was the main presenting feature in 16 patients (80%) with one involving the brain. Pneumonia occurred in 10 (50%) and one with complicated bronchiectasis. Catalase-positive bacteria were the most commonly isolated pathogen with Chromobacterium violaceum predominating (N = 5, 25%) with consequent high mortality (N = 4, 80%). All CGD patients with C. violaceum infection displayed CD4 + (T helper cells) lymphopenia. CONCLUSION: This study has shown CGD occurs in the major ethnic groups of Malaysia. To the best of our knowledge, this is the first and the largest series of chronic granulomatous disease in South East Asia which may be reflective of similar clinical pattern in the region. C. violaceum infection is associated with a higher mortality in CGD patients in Malaysia. All the CGD patients with C. violaceum infection in this patient series displayed CD4 + (T helper) lymphopenia. We recorded rare clinical manifestation of CGD viz. brain abscess and bronchiectasis.
RESUMO
X-linked agammaglobulinemia is a rare primary immunodeficiency due to a BTK mutation. The patients are characteristically deficient in peripheral B cells and serum immunoglobulins. While they are susceptible to infections caused by bacteria, enteroviruses, and parasites, fungal infections are uncommon in XLA patients. Here, we report a boy of Malay ethnicity who suffered from recurrent upper respiratory tract infections and severe progressive necrotizing fasciitis caused by Saksenaea erythrospora. Immunological tests showed a B cell deficiency and hypogammaglobulinemia. Whole-exome sequencing identified a dinucleotide deletion (c.1580_1581del) in BTK, confirmed by Sanger sequencing and predicted to be disease causing by in silico functional prediction tools (Varsome and MutationTaster2) but was absent in the gnomAD database. This mutation resulted in a frameshift and premature termination (p.C527fs), which disrupted the protein structure. The mother was heterozygous at the mutation site, confirming her carrier status. Flow cytometric analysis of monocyte BTK expression showed it to be absent in the patient and bimodal in the mother. This study describes a novel BTK mutation in a defined hotspot and an atypical fungal phenotype in XLA. Further studies are required to understand the pathogenesis of fungal infection in XLA.
Assuntos
Agamaglobulinemia/genética , Fasciite Necrosante/genética , Tirosina Quinase da Agamaglobulinemia/genética , Linfócitos B/metabolismo , Pré-Escolar , Humanos , Masculino , Mutação/genéticaRESUMO
Autoinflammatory disorders are characterized by dysregulated innate immune response, resulting in recurrent uncontrolled systemic inflammation and fever. Gain-of-function mutations in NLRC4 have been described to cause a range of autoinflammatory disorders. We report a twelve-year-old Malay girl with recurrent fever, skin erythema, and inflammatory arthritis. Whole exome sequencing and subsequent bidirectional Sanger sequencing identified a heterozygous missense mutation in NLRC4 (NM_001199138: c.1970A > T). This variant was predicted to be damaging in silico, was absent in public and local databases and occurred in a highly conserved residue in the leucine-rich repeat (LRR) domain. Cytokine analysis showed extremely high serum IL-18 and IL-18/CXCL9 ratio, consistent with other NLRC4-MAS patients. In summary, we identified the first patient with a novel de novo heterozygous NLRC4 gene mutation contributing to autoinflammatory disease in Malaysia. Our findings reinforce the likely pathogenicity of specific LRR domain mutations in NLRC4 and expand the clinical spectrum of NLRC4 mutations.
Assuntos
Doenças Autoimunes/genética , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas de Ligação ao Cálcio/genética , Artrite/genética , Quimiocina CXCL9/sangue , Criança , Eritema/sangue , Feminino , Febre/genética , Humanos , Interleucina-18/sangue , Mutação , Domínios Proteicos , SíndromeRESUMO
Dysregulation of matrix metalloproteinases (MMPs) activity is known in many pathological conditions with which most of the conditions are related to elevate MMPs activities. Ficus deltoidea (FD) is a plant known for its therapeutic properties. In order to evaluate the therapeutic potential of FD leaf extract, we study the enzymatic inhibition properties of FD leaf extract and its major bioactive compounds (vitexin and isovitexin) on a panel of MMPs (MMP-2, MMP-8 and MMP-9) using experimental and computational approaches. FD leaf extract and its major bioactive compounds showed pronounced inhibition activity towards the MMPs tested. Computational docking analysis revealed that vitexin and isovitexin bind to the active site of the three tested MMPs. We also evaluated the cytotoxicity and cell migration inhibition activity of FD leaf extract in the endothelial EA.hy 926 cell line. Conclusively, this study provided additional information on the potential of FD leaf extract for therapeutical application.
Assuntos
Apigenina/farmacologia , Ficus/química , Inibidores de Metaloproteinases de Matriz/farmacologia , Extratos Vegetais/farmacologia , Apigenina/química , Apigenina/metabolismo , Domínio Catalítico , Linhagem Celular , Movimento Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Metaloproteinase 2 da Matriz/química , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 8 da Matriz/química , Metaloproteinase 8 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/química , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Extratos Vegetais/química , Folhas de Planta/químicaRESUMO
High-depth next generation sequencing data provide valuable insights into the number and distribution of RNA editing events. Here, we report the RNA editing events at cellular level of human primary monocyte using high-depth whole genomic and transcriptomic sequencing data. We identified over a ten thousand putative RNA editing sites and 69% of the sites were A-to-I editing sites. The sites enriched in repetitive sequences and intronic regions. High-depth sequencing datasets revealed that 90% of the canonical sites were edited at lower frequencies (<0.7). Single and multiple human monocytes and brain tissues samples were analyzed through genome sequence independent approach. The later approach was observed to identify more editing sites. Monocytes was observed to contain more C-to-U editing sites compared to brain tissues. Our results establish comparable pipeline that can address current limitations as well as demonstrate the potential for highly sensitive detection of RNA editing events in single cell type.
Assuntos
Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Edição de RNA , Análise de Sequência de RNA/métodos , Transcriptoma , Humanos , Monócitos/metabolismo , Motivos de NucleotídeosRESUMO
X-linked agammaglobulinemia (XLA) is a rare genetic disorder, caused by mutations in BTK (Bruton's Tyrosine Kinase) gene. Deep high-throughput RNA sequencing (RNA-Seq) approach was utilized to explore the possible differences in transcriptome profiles of primary monocytes in XLA patients compared with healthy subjects. Our analysis revealed the differences in expression of 1,827 protein-coding genes, 95 annotated long non-coding RNAs (lncRNAs) and 20 novel lincRNAs between XLA patients and healthy subjects. GO and KEGG pathway analysis of differentially expressed (DE) protein-coding genes showed downregulation of several innate immune-related genes and upregulation of oxidative phosphorylation and apoptosis-related genes in XLA patients compared to the healthy subjects. Moreover, the functional prediction analysis of DE lncRNAs revealed their potential role in regulating the monocytes cell cycle and apoptosis in XLA patients. Our results suggested that BTK mutations may contribute to the dysregulation of innate immune system and increase susceptibility to apoptosis in monocytes of XLA patients. This study provides significant finding on the regulation of BTK gene in monocytes and the potential for development of innovative biomarkers and therapeutic monitoring strategies to increase the quality of life in XLA patients.
Assuntos
Agamaglobulinemia/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Imunidade Inata , Transcriptoma , Adulto , Tirosina Quinase da Agamaglobulinemia/deficiência , Tirosina Quinase da Agamaglobulinemia/genética , Agamaglobulinemia/imunologia , Apoptose , Estudos de Casos e Controles , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Humanos , Masculino , Monócitos/metabolismo , Fosforilação OxidativaRESUMO
Differential gene and transcript expression pattern of human primary monocytes from healthy young subjects were profiled under different sequencing depths (50M, 100M, and 200M reads). The raw data consisted of 1.3 billion reads generated from RNA sequencing (RNA-Seq) experiments. A total of 17,657 genes and 75,392 transcripts were obtained at sequencing depth of 200M. Total splice junction reads showed an even more significant increase. Comparative analysis of the expression patterns of immune-related genes revealed a total of 217 differentially expressed (DE) protein-coding genes and 50 DE novel transcripts, in which 40 DE protein-coding genes were related to the immune system. At higher sequencing depth, more genes, known and novel transcripts were identified and larger proportion of reads were allowed to map across splice junctions. The results also showed that increase in sequencing depth has no effect on the sequence alignment.
Assuntos
Perfilação da Expressão Gênica/métodos , Leucócitos Mononucleares/química , Análise de Sequência de RNA/métodos , Adulto , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Imunidade , Masculino , Mapas de Interação de ProteínasRESUMO
Transcriptome analyses based on high-throughput RNA sequencing (RNA-Seq) provide powerful and quantitative characterization of cell types and in-depth understanding of biological systems in health and disease. In this study, we present a comprehensive transcriptome profile of human primary monocytes, a crucial component of the innate immune system. We performed deep RNA-Seq of monocytes from six healthy subjects and integrated our data with 10 other publicly available RNA-Seq datasets of human monocytes. A total of 1.9 billion reads were generated, which allowed us to capture most of the genes transcribed in human monocytes, including 11,994 protein-coding genes, 5558 noncoding genes (including long noncoding RNAs, precursor miRNAs, and others), 2819 pseudogenes, and 7034 putative novel transcripts. In addition, we profiled the expression pattern of 1155 transcription factors (TFs) in human monocytes, which are the main molecules in controlling the gene transcription. An interaction network was constructed among the top expressed TFs and their targeted genes, which revealed the potential key regulatory genes in biological function of human monocytes. The gene catalog of human primary monocytes provided in this study offers significant promise and future potential clinical applications in the fields of precision medicine, systems diagnostics, immunogenomics, and the development of innovative biomarkers and therapeutic monitoring strategies.
Assuntos
Perfilação da Expressão Gênica , Monócitos/metabolismo , Fatores de Transcrição/genética , Transcriptoma , Adulto , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Dados de Sequência Molecular , Cultura Primária de Células , Proteínas/genética , Pseudogenes , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Análise de Sequência de RNARESUMO
Crystal structures of holo vitamin D receptor (VDR) revealed a canonical conformation in which the ligand is entrapped in a hydrophobic cavity buried in the ligand-binding domain (LBD). The mousetrap model postulates that helix 12 is positioned away from the domain to expose the interior cavity. However, the extended form of helix 12 is likely due to artifacts during crystallization. In this study, we set out to investigate conformational dynamics of apo VDR using molecular dynamics simulation on microsecond timescale. Here we show the neighboring backbones of helix 2-helix 3n and beta strand 2-helix 6 of LBD, instead of the helix 12, undergo large-scale motion, possibly gating the entrance of ligand to the ligand binding domain. Docking analysis to the simulated open structure of VDR with the estimated free energy of -37.0 kJ/mol, would emphasise the role of H2-H3n and S2-H6 in facilitating the entrance of calcitriol to the LBD of VDR.
Assuntos
Receptores de Calcitriol/química , Apoproteínas/química , Apoproteínas/metabolismo , Sítios de Ligação , Humanos , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Análise de Componente Principal , Conformação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Receptores de Calcitriol/metabolismoRESUMO
Long non-coding RNAs (lncRNAs) have been shown to possess a wide range of functions in both cellular and developmental processes including cancers. Although some of the lncRNAs have been implicated in the regulation of the immune response, the exact function of the large majority of lncRNAs still remains unknown. In this study, we characterized the lncRNAs in human primary monocytes, an essential component of the innate immune system. We performed RNA sequencing of monocytes from four individuals and combined our data with eleven other publicly available datasets. Our analysis led to identification of ~8000 lncRNAs of which >1000 have not been previously reported in monocytes. PCR-based validation of a subset of the identified novel long intergenic noncoding RNAs (lincRNAs) revealed distinct expression patterns. Our study provides a landscape of lncRNAs in monocytes, which could facilitate future experimental studies to characterize the functions of these molecules in the innate immune system.
Assuntos
Perfilação da Expressão Gênica/métodos , Monócitos/metabolismo , RNA Longo não Codificante/genética , Análise de Sequência de RNA/métodos , Transcriptoma , Linfócitos B/metabolismo , Sequência de Bases , Mapeamento Cromossômico , Genoma Humano/genética , Humanos , Modelos Genéticos , RNA Longo não Codificante/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
Bruton's tyrosine kinase (BTK), encoded by the BTK gene, is a cytoplasmic protein critical in B cell development. Mutations in the BTK gene cause X-linked agammaglobulinemia (XLA), a primary immunodeficiency with characteristically low or absent B cells and antibodies. This report describes a five year-old boy who presented with otitis externa, arthritis, reduced immunoglobulins and no B cells. Flow cytometry showed undetectable monocyte BTK expression. Sequencing revealed a novel mutation at exon 13 of the BTK gene which created a de novo splice site with a proximal 5 nucleotide loss resulting in a truncated BTK protein. The patient still suffered from ear infection despite intravenous immunoglobulin replacement therapy. In this study, mosaicism was seen only in the mother's genomic DNA. These results suggest that a combination of flow cytometry and BTK gene analysis is important for XLA diagnosis and carrier screening.
Assuntos
Agamaglobulinemia/diagnóstico , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Proteínas Tirosina Quinases/genética , Tirosina Quinase da Agamaglobulinemia , Agamaglobulinemia/genética , Pré-Escolar , Análise Mutacional de DNA , Doenças Genéticas Ligadas ao Cromossomo X/genética , Humanos , Masculino , Mutação Puntual , Sítios de Splice de RNARESUMO
BACKGROUND: Chronic granulomatous disease (CGD) is a rare primary immunodeficiency (PID) caused by a dysfunctional respiratory burst enzyme NADPH-oxidase. The concurrence of Klinefelter's Syndrome (KS) and CGD would be extremely rare. OBJECTIVE: We describe the study of a family where the youngest male child had X-linked CGD (X-CGD) while his older brother was both an X-CGD carrier and a Klinefelter. METHODS: Flow cytometry was used to study respiratory burst and gp91-phox expression, while genetic investigation was done by RT-PCR, PCR and X-chromosome short tandem repeat (X-STR) analysis. RESULTS: The Dihydrorhodamine (DHR) assay showed the patient's neutrophils failed to produce a respiratory burst, while both the mother and an older brother showed a bimodal response. gp91-phox expression was absent in the patient's neutrophils, and bimodal in the mother's and brother's neutrophils. The patient's cDNA showed a C>T change at nucleotide 676 of the CYBB gene. The same change was seen in the patient's gDNA, while the brother and mother were heterozygous, with C and T, in this position. The c.676C>T is a nonsense mutation that leads to premature termination of the gp91-phox protein. The brother karyotyped as 47, XXY and X chromosome analysis showed that he had inherited both his mother's X chromosomes. CONCLUSIONS: This study showed that the patient had gp91-phox deficient CGD while his older brother was a CGD carrier and a Klinefelter, who had inherited both his mother's X chromosomes. This is the first report of such a concurrence in an individual, and argues for family members to be included in PID studies.
Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/genética , Doença Granulomatosa Crônica/genética , Síndrome de Klinefelter/genética , Linhagem , Adulto , Feminino , Regulação Enzimológica da Expressão Gênica/genética , Doenças Genéticas Ligadas ao Cromossomo X/enzimologia , Doença Granulomatosa Crônica/enzimologia , Humanos , Lactente , Síndrome de Klinefelter/enzimologia , Masculino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Repetições de Microssatélites , Mães , NADPH Oxidase 2 , NADPH Oxidases/biossíntese , NADPH Oxidases/genética , Explosão Respiratória/genética , IrmãosRESUMO
X-linked agammaglobulinemia (XLA) is a rare genetic disorder caused by mutations in the Bruton's tyrosine kinase (BTK) gene. These mutations cause defects in early B cell development. A patient with no circulating B cells and low serum immunoglobulin isotypes was studied as were his mother and sister. Monocyte BTK protein expression was evaluated by flow cytometry. The mutation was determined using PCR and followed by sequencing. Flow cytometry showed the patient lacked BTK protein expression in his monocytes while the mother and sister had 62% and 40% of the monocytes showing BTK protein expressions respectively. The patient had a novel base substitution in the first nucleotide of intron 9 in the BTK gene, and the mutation was IVS9+1GAssuntos
Agamaglobulinemia/genética
, Doenças Genéticas Ligadas ao Cromossomo X/genética
, Mutação
, Proteínas Tirosina Quinases/genética
, Tirosina Quinase da Agamaglobulinemia
, Sequência de Bases
, Criança
, Feminino
, Citometria de Fluxo
, Heterozigoto
, Humanos
, Masculino
, Dados de Sequência Molecular
, Linhagem
, Reação em Cadeia da Polimerase Via Transcriptase Reversa
RESUMO
Proteasomes are responsible for generating peptides presented by class I MHC molecules of the immune system. ß5t, a recently identified proteasome component, is specifically expressed in thymic cortical epithelial cells (cTECs) and plays a pivotal role in generating an immunocompetent repertoire of class I MHC-restricted CD8(+) T cells. Here, we report that ß5t is detectable in the thymus as early as E12.5 mouse embryos. We also found that ß5t expression in cTECs was detectable in mice deficient for RelB or Rag2, indicating that ß5t in cTECs is expressed in the absence of thymic medulla formation or thymocyte development beyond the CD4(-) CD8(-) stage. ß5t expression in the embryonic thymus was not detectable in Foxn1-deficient nude mice, although its expression was not reduced in mice deficient for both CCR7 and CCR9, in which fetal thymus colonization by leukocytes is defective. These results indicate that ß5t expression in cTECs is dependent on Foxn1 but independent of thymocyte crosstalk or thymic medulla formation.