RESUMO
We report on a patient who developed donor-derived cutaneous T-cell lymphoma (CTCL) 4 years after successful treatment of chronic myeloid leukaemia with an allogeneic bone marrow transplant. The patient developed an eczematous rash unresponsive to topical therapy and immunosuppression. When CTCL was diagnosed in the recipient, his sibling donor had been attending his local dermatology unit with a maculosquamous rash, which proved subsequently to be mycosis fungoides. An identical pattern of donor and recipient clonality assessment and T-cell receptor gene sequencing indicated that the CTCL was probably transmitted in the bone marrow harvest. This suggests that CTCL cells circulate in the marrow at an early subclinical stage in this disease. This is the second case of donor-derived CTCL reported to date.
Assuntos
Transplante de Medula Óssea/efeitos adversos , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Micose Fungoide/etiologia , Neoplasias Cutâneas/etiologia , Humanos , Masculino , Pessoa de Meia-Idade , Irmãos , Transplante Homólogo/efeitos adversosRESUMO
Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) and 10.0±1.5 copies/µl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).
Assuntos
Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Plasmídeos/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Calibragem , Clonagem Molecular , DNA , Proteínas de Escherichia coli/genética , Dosagem de Genes , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas Proto-Oncogênicas c-bcr/genética , RNA Mensageiro/metabolismo , Padrões de ReferênciaRESUMO
Hereditary progressive arthro-ophthalmopathy, or "Stickler syndrome," is an autosomal dominant osteochondrodysplasia characterized by a variety of ocular and skeletal anomalies which frequently lead to retinal detachment and precocious osteoarthritis. A variety of mutations in the COL2A1 gene have been identified in "Stickler" families; in most cases studied thus far, the consequence of mutation is the premature generation of a stop codon. We report here the characterization of a COL2A1 gene mutation in the original kindred described by Stickler et al. [1965]. Conformational sensitive gel electrophoresis (CSGE) [Ganguly et al., 1993] was used to screen for mutations in the entire COL2A1 gene in an affected member from the kindred. A prominent heteroduplex species was noted in the polymerase chain reaction (PCR) product from a region of the gene including exons 17 to 20. Direct sequencing of PCR-amplified genomic DNA resulted in the identification of a base substitution at the A-2 position of the 3' splice acceptor site of IVS17. Sequencing of DNA from affected and unaffected family members confirmed that the mutation segregated with the disease phenotype. Reverse transcriptase-PCR analysis of poly A+ RNA demonstrated that the mutant allele utilized a cryptic splice site in exon 18 of the gene, eliminating 16 bp at the start of exon 18. This frameshift eventually results in a premature termination codon. These findings are the first report of a splice site mutation in classical Stickler syndrome and they provide a satisfying historical context in which to view COL2A1 mutations in this dysplasia.
Assuntos
Colágeno/genética , Oftalmopatias/genética , Artropatias/genética , Mutação , Splicing de RNA , Descolamento Retiniano/genética , Adolescente , Idoso , Sequência de Bases , Pré-Escolar , Códon de Terminação , Eletroforese em Gel de Poliacrilamida/métodos , Feminino , Humanos , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Miopia/genética , Linhagem , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , SíndromeAssuntos
Biomarcadores Tumorais/genética , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/genética , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/terapia , Mutação de Sentido Incorreto , Receptores de Fator Estimulador de Colônias/genética , Aloenxertos , Sequência de Bases , DNA de Neoplasias/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Transplante de Células-Tronco , Fatores de TempoAssuntos
Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Sequência de Aminoácidos , Sequência de Bases , Feminino , Humanos , Íntrons , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase em Tempo Real , Deleção de SequênciaRESUMO
Secondary or late graft failure has been defined as the development of inadequate marrow function after initial engraftment has been achieved. We describe a case of profound marrow aplasia occurring 13 years after sibling allogeneic bone marrow transplantation for chronic myeloid leukaemia (CML) in first chronic phase. Although the patient remained a complete donor chimera, thereby suggesting that an unselected infusion of donor peripheral blood stem cells (PBSC) or bone marrow might be indicated, the newly acquired aplasia was thought to be immune in aetiology and some immunosuppression was therefore considered appropriate. Rapid haematological recovery was achieved after the infusion of unselected PBSC from the original donor following conditioning with anti-thymocyte globulin (ATG).
Assuntos
Doenças da Medula Óssea/etiologia , Transplante de Medula Óssea/efeitos adversos , Leucemia Mielogênica Crônica BCR-ABL Positiva/complicações , Soro Antilinfocitário/uso terapêutico , Doenças da Medula Óssea/tratamento farmacológico , Doenças da Medula Óssea/patologia , Transplante de Medula Óssea/métodos , Feminino , Teste de Histocompatibilidade , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Pessoa de Meia-Idade , Transplante de Células-Tronco de Sangue Periférico , Irmãos , Transplante Homólogo , Resultado do TratamentoRESUMO
1. The involvement of nitric oxide (NO) in enteric neural pathways underlying reflex responses of the longitudinal muscle (LM) and circular muscle (CM) layers activated by mucosal stimulation was examined in the isolated guinea-pig distal colon. 2. A segment of colon spanned two partitions (10 mm apart), which divided the organ bath into three chambers: a recording chamber where LM and CM tension was measured; a stimulation chamber where mucosal stimulation was applied; and a middle chamber separating them. 3. Brushing the mucosa anal and oral to the recording site evoked simultaneous oral contraction and anal relaxation of both the LM and CM. 4. N omega-nitro-L-argininel-NA; 100 microM) or N omega-nitro-L-arginine methyl ester (L-NAME; 100 microM) applied to the middle chamber or stimulation chamber decreased the oral contractile response of the LM and CM (by about 30-40 %), but increased the anal relaxation (> 600 %) and exposed an anal contraction (> 1000 % increase) of both muscles. The addition of L-NA to the recording chamber reduced the anal relaxation of the LM and CM and the anal contraction of the LM, but slightly increased the anal contraction of the CM. 5. S-Nitroso-N-acetylpenicillamine (SNAP; 10 microM), an NO donor, reversed the effects of L-NA in the middle or stimulation chambers. 6. 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ; 10 microM), a soluble guanylate cyclase inhibitor, mimicked the effects of L-NAin the middle chamber or stimulation chamber, but these effects were not reversed by SNAP. 7. The oral contractile responses, and the anal relaxation and contractile responses of the LM and CM produced by L-NA in the stimulation or middle chambers, were blocked by hexamethonium (300 microM) in any chamber. Atropine (1 microM) in the recording chamber reduced the contractile responses of the LM and CM. 8. In conclusion, endogenous NO facilitates and depresses release of acetylcholine from interneurons in ascending and descending nervous pathways, respectively. These NO effects are mediated through soluble guanylate cyclase in cholinergic interneurons
Assuntos
Colo/fisiologia , Músculo Liso/fisiologia , Óxido Nítrico/fisiologia , Sistema Nervoso Parassimpático/fisiologia , Reflexo/fisiologia , Animais , Vias Autônomas/fisiologia , Colo/inervação , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Cobaias , Técnicas In Vitro , Mucosa Intestinal/inervação , Mucosa Intestinal/fisiologia , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Relaxamento Muscular/efeitos dos fármacos , Relaxamento Muscular/fisiologia , Músculo Liso/inervação , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I , Penicilamina/análogos & derivados , Penicilamina/farmacologia , S-Nitroso-N-AcetilpenicilaminaRESUMO
1. We investigated the effects of sympathetic nerve stimulation within ascending and descending reflex pathways underlying the peristaltic reflex in the guinea-pig distal colon. 2. A three-chambered partitioned bath was used to divide a segment of distal colon into stimulation, recording and intermediate regions. The effects of lumbar colonic nerves (LCN) could be localized to the intermediate region by surgical lesions of the mesentery and by application of guanethidine (3 microM) to the stimulation and recording chambers. 3. Brush stroking the mucosa in the anal and oral stimulation chambers elicited a synchronous contraction of the longitudinal muscle (LM) and circular muscle (CM) oral to, and transient relaxation of the LM and CM anal to, the stimulus, respectively. 4. After N omega-nitro-L-arginine (L-NA; 100 microM) in the oral and intermediate chambers, mucosal stimulation in the oral chamber elicited a prolonged descending inhibitory and excitatory complex in both the LM and CM in the anal recording chamber. This was blocked by hexamethonium (300 microM), which did not affect the transient relaxation response recorded in control conditions. 5. Stimulation of the LCN (1200 pulses, 20 Hz), delivered to the intermediate region, abolished the oral contraction and the L-NA-induced anal complex in both the LM and CM, but was without effect on the transient hexamethonium-resistant anal relaxation. These effects of LCN stimulation were reversed by phentolamine (3 microM) or yohimbine (100 nM), but not propranolol (10 microM), when added to the intermediate chamber. 6. LCN stimuli (2-20 Hz, 600 micros pulses) directed to the recording chamber elicited synchronous relaxations in the LM and CM that were unaffected by hexamethonium (300 microM), but were reduced by yohimbine and usually blocked by the further addition of propranolol (10 microM). 7. In conclusion, sympathetic nerve stimulation inhibits orally and anally projecting cholinergic interneurones underlying the peristaltic reflex in the distal colon. In addition, the LM and CM relax synchronously following release of sympathetic neurotransmitter, over a range of stimulus frequencies.
Assuntos
Colo/inervação , Colo/fisiologia , Interneurônios/fisiologia , Peristaltismo/fisiologia , Sistema Nervoso Simpático/fisiologia , Agonistas alfa-Adrenérgicos/farmacologia , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Vias Autônomas/efeitos dos fármacos , Vias Autônomas/fisiologia , Colo/efeitos dos fármacos , Estimulação Elétrica , Cobaias , Técnicas In Vitro , Interneurônios/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Agonistas Nicotínicos/farmacologia , Sistema Nervoso Parassimpático/efeitos dos fármacos , Sistema Nervoso Parassimpático/fisiologia , Peristaltismo/efeitos dos fármacos , Reflexo/efeitos dos fármacos , Reflexo/fisiologia , Sistema Nervoso Simpático/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
An eight-year-old boy was referred for dental assessment of dentinogenesis imperfecta, a full clinical examination also revealed joint hypermobility and some features of mild osteogenesis imperfecta although he had suffered few fractures. Analysis of the collagens produced by both gingival and skin fibroblast cultures showed the synthesis and intracellular retention of an abnormal alpha 2(I) chain that migrated faster than normal on SDS-PAGE. Cyanogen bromide peptide mapping of this intracellular protein indicated a probable deletion in the N-terminal peptide alpha 2CB4. The denaturation temperature of the mutant protein was only 36 degrees C, some 6 degrees C below normal. At 37 degrees C secretion of abnormal protein was not detectable but a lower temperature (30 degrees C) some was secreted into the medium. RT-PCR amplification of mRNA coding for alpha 2CB4 revealed a heterozygous deletion of the 108 bp exon 21 of COL1A2. Sequencing of PCR amplified genomic DNA identified a G --> A transition in the moderately conserved + 5 position of the IVS 21 5' consensus splice site causing the skipping of exon 21. Hybridization with allele-specific oligonucleotides showed no other family member had this base change. Since the cDNA deletion was associated with the (-) allele of a Pvu II polymorphism in exon 25 of COL1A2 we could demonstrate that the mutant pre-mRNA was alternatively spliced yielding both full length and deleted transcripts. Family genotype analysis indicated the mutation had originated in the paternal alpha 2(I) gene.
Assuntos
Processamento Alternativo/genética , Colágeno/genética , Dentinogênese Imperfeita/genética , Osteogênese Imperfeita/genética , Deleção de Sequência , Sequência de Aminoácidos , Sequência de Bases , Células Cultivadas , Criança , Colágeno/química , Eletroforese em Gel de Poliacrilamida , Éxons , Gengiva , Humanos , Masculino , Dados de Sequência Molecular , Mutação , Linhagem , Mapeamento de Peptídeos , Reação em Cadeia da Polimerase , Pele , TemperaturaRESUMO
Platelet adhesion is a critical, early event in hemostasis. It is a complex process which involves platelet sticking, activation, granule release, and a series of morphologic changes which appear to enhance contact. The membrane glycoprotein (GP) Ib, which is thought to play an important role in adhesion, is missing in the Bernard Soulier Syndrome (BSS) and platelets in this syndrome have a structural defect which results in giant size on peripheral smear. Correlative SEM/TEM studies on adherent BSS platelets were used to address two questions: 1) Does the absence of GP Ib in BSS platelets result in abnormalities in the morphologic changes which ordinarily accompany adhesion to formvar? and 2) Is the giantism seen in BSS platelets related to or reflected in abnormalities in organization of the cytoskeletons of adherent platelets?
Assuntos
Transtornos Plaquetários/sangue , Plaquetas/citologia , Agregação Plaquetária , Transtornos Plaquetários/genética , Citoesqueleto/ultraestrutura , Heterozigoto , Humanos , Microscopia Eletrônica , Microscopia Eletrônica de VarreduraRESUMO
The Ehlers-Danlos syndrome (EDS) is a heterogeneous group of inherited connective tissue disorders characterised by skin hyperextensibility, joint hypermobility, easy bruising, and cutaneous fragility. Nine discrete clinical subtypes have been classified. We have investigated the molecular defect in a patient with clinical features of Ehlers-Danlos syndromes types I/II and VII. Electron microscopy of skin tissue indicated abnormal collagen fibrillogenesis with longitudinal sections showing a marked disruption of fibril packing giving very irregular outlines to transverse sections. Analysis of the collagens produced by cultured fibroblasts showed that the type V collagen had a population of alpha 1 (V) chains shorter than normal. Peptide mapping suggested a deletion within the triple helical domain. RTPCR amplification of mRNA covering the whole of this domain of COL5A1 showed a deletion of 54 bp. Although six Gly-X-Y triplets were lost, the essential triplet amino acid sequence and C-propeptide structure were maintained allowing mutant protein chains to be incorporated into triple helices. Genomic DNA analysis identified a de novo G+3-->T transversion in a 5' splice site of one COL5A1 allele. This mutation is analogous to mutations causing exon skipping in the major collagen genes, COL1A1, COL1A2, and COL3A1, identified in several cases of osteogenesis imperfecta and EDS type IV. These observations support the hypothesis that type V, although quantitatively a minor collagen, has a critical role in the formation of the fibrillar collagen matrix.
Assuntos
Colágeno/genética , Síndrome de Ehlers-Danlos/genética , Mutação , Pele/patologia , Adulto , Estatura , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Éxons , Feminino , Humanos , Microscopia Eletrônica , Gravidez , Proteínas/química , Escoliose , Pele/químicaRESUMO
Blau syndrome (MIM 186580), first described in a large, three-generation kindred, is an autosomal, dominantly inherited disease characterized by multiorgan, tissue-specific inflammation. Its clinical phenotype includes granulomatous arthritis, skin rash, and uveitis and probably represents a subtype of a group of clinical entities referred to as "familial granulomatosis." It is the sole human model with recognizably Mendelian inheritance for a variety of multisystem inflammatory diseases affecting a significant percentage of the population. A genomewide search for the Blau susceptibility locus was undertaken after karyotypic analysis revealed no abnormalities. Sixty-two of the 74-member pedigree were genotyped with dinucleotide-repeat markers. Linkage analysis was performed under a dominant model of inheritance with reduced penetrance. The marker D16S298 gave a maximum LOD score of 3.75 at theta = .04, with two-point analysis. LOD scores for flanking markers were consistent and placed the Blau susceptibility locus within the 16p12-q21 interval.