RESUMO
Pigmentary glaucoma has recently been associated with missense mutations in PMEL that are dominantly inherited and enriched in the protein's fascinating repeat domain. PMEL pathobiology is intriguing because PMEL forms functional amyloid in healthy eyes, and this PMEL amyloid acts to scaffold melanin deposition. This is an informative contradistinction to prominent neurodegenerative diseases where amyloid formation is neurotoxic and mutations cause a toxic gain of function called "amyloidosis". Preclinical animal models have failed to model this PMEL "dysamyloidosis" pathomechanism and instead cause recessively inherited ocular pigment defects via PMEL loss of function; they have not addressed the consequences of disrupting PMEL's repetitive region. Here, we use CRISPR to engineer a small in-frame mutation in the zebrafish homolog of PMEL that is predicted to subtly disrupt the protein's repetitive region. Homozygous mutant larvae displayed pigmentation phenotypes and altered eye morphogenesis similar to presumptive null larvae. Heterozygous mutants had disrupted eye morphogenesis and disrupted pigment deposition in their retinal melanosomes. The deficits in the pigment deposition of these young adult fish were not accompanied by any detectable glaucomatous changes in intraocular pressure or retinal morphology. Overall, the data provide important in vivo validation that subtle PMEL mutations can cause a dominantly inherited pigment pathology that aligns with the inheritance of pigmentary glaucoma patient pedigrees. These in vivo observations help to resolve controversy regarding the necessity of PMEL's repeat domain in pigmentation. The data foster an ongoing interest in an antithetical dysamyloidosis mechanism that, akin to the amyloidosis of devastating dementias, manifests as a slow progressive neurodegenerative disease.
Assuntos
Glaucoma de Ângulo Aberto , Doenças Neurodegenerativas , Animais , Humanos , Adulto Jovem , Amiloide/metabolismo , Olho/metabolismo , Glaucoma de Ângulo Aberto/metabolismo , Antígeno gp100 de Melanoma/genética , Melanossomas/genética , Melanossomas/metabolismo , Doenças Neurodegenerativas/metabolismo , Peixe-ZebraRESUMO
Current diagnostic standards for lymphoproliferative disorders include multiple tests for detection of clonal immunoglobulin (IG) and/or T-cell receptor (TCR) rearrangements, translocations, copy-number alterations (CNAs), and somatic mutations. The EuroClonality-NGS DNA Capture (EuroClonality-NDC) assay was designed as an integrated tool to characterize these alterations by capturing IGH switch regions along with variable, diversity, and joining genes of all IG and TCR loci in addition to clinically relevant genes for CNA and mutation analysis. Diagnostic performance against standard-of-care clinical testing was assessed in a cohort of 280 B- and T-cell malignancies from 10 European laboratories, including 88 formalin-fixed paraffin-embedded samples and 21 reactive lesions. DNA samples were subjected to the EuroClonality-NDC protocol in 7 EuroClonality-NGS laboratories and analyzed using a bespoke bioinformatic pipeline. The EuroClonality-NDC assay detected B-cell clonality in 191 (97%) of 197 B-cell malignancies and T-cell clonality in 71 (97%) of 73 T-cell malignancies. Limit of detection (LOD) for IG/TCR rearrangements was established at 5% using cell line blends. Chromosomal translocations were detected in 145 (95%) of 152 cases known to be positive. CNAs were validated for immunogenetic and oncogenetic regions, highlighting their novel role in confirming clonality in somatically hypermutated cases. Single-nucleotide variant LOD was determined as 4% allele frequency, and an orthogonal validation using 32 samples resulted in 98% concordance. The EuroClonality-NDC assay is a robust tool providing a single end-to-end workflow for simultaneous detection of B- and T-cell clonality, translocations, CNAs, and sequence variants.
Assuntos
Rearranjo Gênico , Transtornos Linfoproliferativos , DNA , Genômica , Humanos , Imunoglobulinas , Transtornos Linfoproliferativos/diagnóstico , Transtornos Linfoproliferativos/genéticaRESUMO
The ancient paralogs premelanosome protein (PMEL) and glycoprotein nonmetastatic melanoma protein B (GPNMB) have independently emerged as intriguing disease loci in recent years. Both proteins possess common functional domains and variants that cause a shared spectrum of overlapping phenotypes and disease associations: melanin-based pigmentation, cancer, neurodegenerative disease and glaucoma. Surprisingly, these proteins have yet to be shown to physically or genetically interact within the same cellular pathway. This juxtaposition inspired us to compare and contrast this family across a breadth of species to better understand the divergent evolutionary trajectories of two related, but distinct, genes. In this study, we investigated the evolutionary history of PMEL and GPNMB in clade-representative species and identified TMEM130 as the most ancient paralog of the family. By curating the functional domains in each paralog, we identified many commonalities dating back to the emergence of the gene family in basal metazoans. PMEL and GPNMB have gained functional domains since their divergence from TMEM130, including the core amyloid fragment (CAF) that is critical for the amyloid potential of PMEL. Additionally, the PMEL gene has acquired the enigmatic repeat domain (RPT), composed of a variable number of imperfect tandem repeats; this domain acts in an accessory role to control amyloid formation. Our analyses revealed the vast variability in sequence, length and repeat number in homologous RPT domains between craniates, even within the same taxonomic class. We hope that these analyses inspire further investigation into a gene family that is remarkable from the evolutionary, pathological and cell biology perspectives.
Assuntos
Melanócitos/metabolismo , Glicoproteínas de Membrana/metabolismo , Mutação , Doenças Neurodegenerativas/patologia , Antígeno gp100 de Melanoma/metabolismo , Sequência de Aminoácidos , Proteínas Amiloidogênicas/metabolismo , Animais , Biologia Computacional/métodos , Humanos , Glicoproteínas de Membrana/genética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Filogenia , Pigmentação , Domínios Proteicos , Homologia de Sequência , Antígeno gp100 de Melanoma/genéticaRESUMO
Clonality analysis of immunoglobulin (IG) or T-cell receptor (TR) gene rearrangements is routine practice to assist diagnosis of lymphoid malignancies. Participation in external quality assessment (EQA) aids laboratories in identifying systematic shortcomings. The aim of this study was to evaluate laboratories' improvement in IG/TR analysis and interpretation during five EQA rounds between 2014 and 2018. Each year, participants received a total of five cases for IG and five cases for TR testing. Paper-based cases were included for analysis of the final molecular conclusion that should be interpreted based on the integration of the individual PCR results. Wet cases were distributed for analysis of their routine protocol as well as evaluation of the final molecular conclusion. In total, 94.9% (506/533) of wet tests and 97.9% (829/847) of paper tests were correctly analyzed for IG, and 96.8% (507/524) wet tests and 93.2% (765/821) paper tests were correctly analyzed for TR. Analysis scores significantly improved when laboratories participated to more EQA rounds (p=0.001). Overall performance was significantly lower (p=0.008) for non-EuroClonality laboratories (95% for IG and 93% for TR) compared to EuroClonality laboratories (99% for IG and 97% for TR). The difference was not related to the EQA scheme year, anatomic origin of the sample, or final clinical diagnosis. This evaluation showed that repeated EQA participation helps to reduce performance differences between laboratories (EuroClonality versus non-EuroClonality) and between sample types (paper versus wet). The difficulties in interpreting oligoclonal cases highlighted the need for continued education by meetings and EQA schemes.
Assuntos
Rearranjo Gênico , Genes de Imunoglobulinas , Genes Codificadores dos Receptores de Linfócitos T , Transtornos Linfoproliferativos/genética , Técnicas de Diagnóstico Molecular , Reação em Cadeia da Polimerase , Humanos , Ensaio de Proficiência Laboratorial , Transtornos Linfoproliferativos/imunologia , Transtornos Linfoproliferativos/patologia , Variações Dependentes do Observador , Valor Preditivo dos Testes , Garantia da Qualidade dos Cuidados de Saúde , Controle de Qualidade , Reprodutibilidade dos TestesAssuntos
Neoplasias Hepáticas , Linfoma de Células T , Neoplasias Esplênicas , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Linfoma de Células T/metabolismo , Linfoma de Células T/patologia , Masculino , Pessoa de Meia-Idade , Neoplasias Esplênicas/metabolismo , Neoplasias Esplênicas/patologiaRESUMO
A scoping literature review found evidence supporting the hypothesis that a population's pollution status could help refine classification of emerging infectious disease (EID) hotspots. Systematic literature reviews and studies designed to specifically test the predictive value of pollutant status on EID risk are recommended.
Y a-t-il un lien entre l'exposition aux polluants et les maladies infectieuses émergentes? Une recension extensive de la littérature a permis de trouver des données probantes appuyant l'hypothèse que l'état de pollution de la population pourrait aider à raffiner la classification des points chauds des maladies infectieuses émergentes (MIE). Des examens systématiques de la littérature et des études conçues spécifiquement pour tester la valeur prédictive de l'état des polluants sur le risque des MIE sont recommandés.(Traduit par Isabelle Vallières).
Assuntos
Doenças Transmissíveis Emergentes/etiologia , Poluentes Ambientais/toxicidade , Animais , Suscetibilidade a Doenças , HumanosRESUMO
ATM kinase modulates pathways implicated in premature ageing and ATM genotype predicts survival, yet immunodeficiency in ataxia telangiectasia is regarded as mild and unrelated to age. We address this paradox in a molecularly characterised sequential adult cohort with classical and mild variant ataxia telangiectasia. Immunodeficiency has the characteristics of premature ageing across multiple cellular and molecular immune parameters. This immune ageing occurs without previous CMV infection. Age predicts immunodeficiency in genetically homogeneous ataxia telangiectasia, and in comparison with controls, calendar age is exceeded by immunological age defined by thymic naïve CD4+ T cell levels. Applying ataxia telangiectasia as a model of immune ageing, pneumococcal vaccine responses, characteristically deficient in physiological ageing, are predicted by thymic naïve CD4+ T cell levels. These data suggest inherited defects of DNA repair may provide valuable insight into physiological ageing. Thymic naïve CD4+ T cells may provide a biomarker for vaccine responsiveness in elderly cohorts.
Assuntos
Envelhecimento/imunologia , Ataxia Telangiectasia/imunologia , Linfócitos T CD4-Positivos/imunologia , Adulto , Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia , Contagem de Células , Proteínas de Ciclo Celular/genética , Separação Celular , Proteínas de Ligação a DNA/genética , Feminino , Citometria de Fluxo , Humanos , Masculino , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Recent research has suggested that negative stereotypes about aging may have a detrimental influence on older adults' memory performance. This study sought to determine whether stereotype-based influences were moderated by age, education, and concerns about being stigmatized. Possible mechanisms underlying these influences on memory performance were also explored. The memory performance of adults aged 60 to 70 years and 71 to 82 years was examined under conditions designed to induce or eliminate stereotype threat. Threat was found to have a greater impact on performance in the young-old than in the old-old group, whereas the opposite was observed for the effects of stigma consciousness. In both cases, the effects were strongest for those with higher levels of education. Further analyses found little evidence in support of the mediating roles of affective responses or working memory. The only evidence of mediation was found with respect to recall predictions, suggesting a motivational basis of threat effects on performance. These findings highlight the specificity of stereotype threat effects in later adulthood as well as possible mechanisms underlying such effects.
Assuntos
Envelhecimento/psicologia , Memória , Estereotipagem , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Molecular characterization of T-cell receptor junctional region sequences in cutaneous T-cell lymphoma had not been previously reported. We have examined in detail the features of the T-cell receptor beta (TCRB) gene rearrangements in 20 individuals with well-defined stages of cutaneous T-cell lymphoma (CTCL) comprising 10 cases with early-stage mycosis fungoides (MF) and 10 cases with late-stage MF or Sezary syndrome. Using BIOMED-2 PCR primers, we detected a high frequency of clonally rearranged TCR gamma and TCRB genes (17/20 and 15/20 cases, respectively). We carried out sequencing analysis of each complete clonal variable (V)beta-diversity (D)beta-joining(J)beta fingerprint generated by PCR amplification, and determined the primary structure of the Vbeta-Dbeta-Jbeta junctional regions. We observed considerable diversity in the T-cell receptor Vbeta gene usage and complementarity-determining region 3 loops. Although we found that TCRB gene usage in CTCL and normal individuals share common features, our analysis also revealed preferential usage of Jbeta1 genes in all cases with advanced stages of disease.
Assuntos
Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/genética , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/genética , Linfoma de Células T/genética , Neoplasias Cutâneas/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Linfoma de Células T/imunologia , Micose Fungoide/genética , Micose Fungoide/imunologia , Análise de Sequência de DNA , Síndrome de Sézary/genética , Síndrome de Sézary/imunologia , Neoplasias Cutâneas/imunologiaRESUMO
Malignant and reactive lymphoproliferations in most cases can be distinguished by histology and immunohistology alone; however, in T-cell lymphoproliferations and lymphoproliferations of unknown origin, histology often is inconclusive, and there is no reliable protein marker of malignancy. At the genomic level T-cell neoplasms clonally rearrange T-cell receptor (TCR) genes that can serve as clonal markers. In comparison with Southern blot analysis, polymerase chain reaction (PCR) techniques increasingly are being used to detect these rearrangements because PCRs are rapid, easy to perform, and can be used to amplify poor-quality deoxyribonucleic acid (DNA) from paraffin-embedded formalin-fixed biopsies. Nevertheless there are a number of problems associated with the detection of gene rearrangements using PCR. The foremost of these is improper primer annealing that will lead to false-negative or false-positive PCR results that may arise from poor primer design, especially for TCRB genes with an extensive variable (V), diversity (D), and joined (J) gene repertoire. There also can be difficulty in discriminating between clonal and polyclonal PCR products unless specific methods such as heteroduplex analysis or gene scanning are used. In this chapter, we describe methods, derived from a recent European collaborative BIOMED-2 program, for the detection of TCRG, B, and D rearrangements. TCRB VJ and DJ gene rearrangements are detected using 23 VB primers, 13 JB primers, and 2 DB primers in 3 multiplex tubes. TCRG VJ gene rearrangements are detected with four VG and two JG primers in two multiplex tubes, and TCRD VJ, VD, DJ, and DD rearrangements are detected with six VD primers, four JD primers, and two DD primers in one multiplex tube. Gaussian distributions of polyclonal PCR products are seen at specific size ranges for both TCRB and TCRG. Interpretation of TCRD rearrangements is more complex because of the wide range in PCR product size and often low numbers of TCRGD cells in target tissue. For all loci, some indication of gene usage can be ascertained by labeling the primers with different fluorochromes and gene scan analysis of PCR products. Furthermore the complementarity of the TCR loci affords an unprecedented high clonal detection rate. In addition, we also describe a set of control gene primers designed to amplify amplicons of 100, 200, 300, 400, and 600 bp for the assessment of the integrity and amplifiability of DNA.
Assuntos
Rearranjo Gênico do Linfócito T/genética , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/genética , Genes Codificadores da Cadeia delta de Receptores de Linfócitos T/genética , Genes Codificadores da Cadeia gama de Receptores de Linfócitos T/genética , Linfoma de Células T/diagnóstico , Reação em Cadeia da Polimerase/métodos , Primers do DNA , Humanos , Linfoma de Células T/genéticaRESUMO
Omenn syndrome (OS) is characterised by hepatosplenomegaly, lymphadenopathy, erythema, eosinophilia, elevated IgE, oligoclonal T cell expansions and recombinase activating gene (RAG) mutations. We investigated 9 cases of OS to correlate genotype with immunophenotype using a two-color flow cytometry with monoclonal antibodies against CD3 and TCRVB families to map TCRVB usage. T and B clonal cell populations were examined in peripheral blood lymphocytes by PCR and sequencing of TCRB/TCRG T cell and IGH FR2/FR3 B cell products. RAG and Artemis genes were sequenced from genomic DNA. All patients demonstrated absent TCRVB families; six had predominant TCRVB families, six oligoclonal TCR gene rearrangements including TCRGD rearrangements. One demonstrated functional IGH rearrangement, an observation not previously reported. In this clinically homogeneous population, with similar immunological phenotype, RAG mutations were identified in only 2/9 patients. OS is a genetically heterogeneous condition, and patients with similar immunophenotypes may have as yet unidentified gene defects.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Homeodomínio/genética , Mutação , Imunodeficiência Combinada Severa/genética , Sequência de Bases , DNA/genética , Endonucleases , Feminino , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Rearranjo Gênico da Cadeia gama dos Receptores de Antígenos dos Linfócitos T , Genótipo , Humanos , Imunofenotipagem , Lactente , Recém-Nascido , Masculino , Proteínas Nucleares/genética , Imunodeficiência Combinada Severa/imunologia , SíndromeRESUMO
Anaplastic large cell lymphoma (ALCL), CD30+, is a subtype of T-non-Hodgkin's lymphoma (NHL). Its most common form is a classical systemic type that involves multiple nodal and extranodal sites. In this study, morphologic, immunohistologic, and genetic studies were performed on ALCL cases in Pakistani patients. The median age of the patients in this study was 45 years (age range: 5-70 years), with a male to female ratio of 3.4:1. Thirty-seven (37) patients were diagnosed to have Ki-1 (CD30+) ALCL, which constituted 2% of all NHLs and 12.6% of all T-NHLs, over a period of 11 years (January 01, 1992-December 31, 2002). The tumors were of either T- or null-cell type with constant (100%) expression of CD30 (Ki-1). The majority of the cases (89.2%) expressed EMA, whereas 40.5% of the cases expressed either CD45 (LCA), CD45RO (UCHL1), or ALK. The mean age of ALCL patients with null-cell phenotype was 33.8 years as compared to those with T-cell phenotype having a mean age of 36.3 years. Out of the 37 cases diagnosed as ALCL, amplifiable DNA was isolated from 28 cases, which were further assessed for T-cell clonality for T-cell receptor (TCR)-beta, gamma, and immunoglobulin heavy chain (IgH) for the FR2 and FR3 regions. The polymerase chain reaction (PCR) technique demonstrated clonal rearrangement of the TCR beta, gamma, and IgH regions in 15 (53.6%), 11 (39.3%), and 2 (7.1%) ALCL cases, respectively, out of 28 cases. Association of Epstein-Barr virus (EBV) was noted in seven out of 28 cases (25%) of ALCL by PCR, whereas ISH for EBV-encoded nuclear RNA-1 (EBER-1) detected the presence of EBV in two (16.7%) out of 12 cases, where one was T-cell ALCL and the other null-cell ALCL. Immunostaining for LMP-1 could not be performed, because tissue material was not available. In conclusion, our study demonstrated that the prevalence of ALCL in Pakistan is comparable to that reported for some of the Asian communities and by the International Lymphoma Study Group and that EBV could be partly responsible for the pathogenesis of ALCL.
Assuntos
Infecções por Vírus Epstein-Barr/epidemiologia , Infecções por Vírus Epstein-Barr/virologia , Linfoma Difuso de Grandes Células B/epidemiologia , Linfoma Difuso de Grandes Células B/virologia , Infecções Tumorais por Vírus/epidemiologia , Infecções Tumorais por Vírus/virologia , Adolescente , Adulto , Idoso , Quinase do Linfoma Anaplásico , Antígenos CD20/metabolismo , Criança , Pré-Escolar , Infecções por Vírus Epstein-Barr/patologia , Feminino , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Rearranjo Gênico da Cadeia gama dos Receptores de Antígenos dos Linfócitos T , Herpesvirus Humano 4 , Humanos , Cadeias Pesadas de Imunoglobulinas , Imuno-Histoquímica , Hibridização In Situ , Antígeno Ki-1/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pessoa de Meia-Idade , Paquistão , Reação em Cadeia da Polimerase , Prevalência , Proteínas Tirosina Quinases/metabolismo , RNA Viral/metabolismo , Receptores Proteína Tirosina Quinases , Infecções Tumorais por Vírus/patologiaRESUMO
The authors describe two pediatric cases of large granular lymphocytosis presenting early in the second decade of life with neutropenia and sepsis. They are among the youngest described in the literature. This report focuses on the advantages of detailed immunophenotypic and molecular analysis and highlights some of the controversies and uncertainties in the management of these patients, particularly the choice of immunosuppressive therapy. Immunosuppressive therapy in the two children described in this report resulted in improvement of neutropenia and clinical status, but this was not accompanied by the disappearance of the clonal population.