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 × 106, 1.08±0.11 × 105, 1.03±0.10 × 104, 1.02±0.09 × 103, 1.04±0.10 × 102 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).
This study investigated possible associations between selected polymorphisms in the dopamine receptor genes DRD1 and DRD3 with the presence of psychotic phenomena or aggressive behaviour in a community based cohort of 134 patients with late onset Alzheimer's disease. An association was found between the presence of psychotic symptoms and aggressive behaviour and the DRD1 polymorphism and between the presence of psychosis, but not aggression, and the DRD3 polymorphism. Specifically, carriers of the DRD1 B2 allele were more likely to be aggressive or experience hallucinations whereas homozygous carriers of the DRD3 1 allele were more likely to experience delusions. (J Neurol Neurosurg Psychiatry 2001;71:777-779) Keywords: Alzheimer's disease; genetics; behaviour Cognitive decline is, by definition, invariably present in Alzheimer's disease. However, various other behavioural and psychological symptoms in dementia (BPSD) are also present, with psychotic symptoms found in about a third of cases and aggressive behaviour in around a half. The mechanism by which BPSD occurs in some patients with Alzheimer's disease is not completely understood but we have previously shown that common genetic polymorphisms in the serotonin receptors 5-HT2A and 5-HT2C are associated with the presence of psychotic phenomena, particularly hallucinations, in Alzheimer's disease.1 More recently, a study of the dopamine DRD1, 2, 3, and 4 receptors 2 has suggested an association between common polymorphisms in the DRD1 and DRD3 receptors and the presence of aggression and psychosis in Alzheimer's disease. This study was designed to examine the role that these polymorphisms might have on the development of specific components of psychotic phenomena-namely, the presence of delusions and hallucinations in patients with late onset Alzheimer's disease. Methods PATIENTSPatients were selected from the Camberwell Dementia Case Register. This is a community based register consisting of patients referred to psychiatric, medical, and social services with the districts of East Lambeth and South Southwark (formerly Camberwell), London, from April 1993 to April 1995. All patients were screened using the minimental state examination (MMSE) and any patients scoring less than 24 points were included for further assessment. Of the 528 patients fulfilling these criteria 374 also fulfilled the The patients and the next of kin or main carer were interviewed in all cases by means of the CAMDEX 5 and the presence of a psychiatric history defined according to these guidelines-that is, an emotional or nervous illness requiring treatment. Presence of psychopathology was assessed by the Manchester and Oxford Universities scale for the psychological assessment of dementia (MOUSE-PAD).6 Due to possible ethnic diVerences in the frequencies of DRD1 and DRD3 receptor polymorphisms only white persons were included in this study. SuYcient genetic material was available for DRD3 genotyping in 129 cases and for DRD1 genotyping in 105 cases.Ethical approval was obtain...
A mouse monoclonal antibody (CT-1) was prepared against the C-terminal peptide sequence of the human insulin receptor beta-subunit (KKNGRILTLPRSNPS). The antibody reacted with native human and rat insulin receptors in solution, whether or not insulin was bound and whether or not the receptor had undergone prior tyrosine autophosphorylation. The antibody also reacted specifically with the receptor beta-subunit on blots of SDS/polyacrylamide gels. Preincubation of soluble receptors with antibody increased the binding of 125I-insulin approx. 2-fold. The antibody did not affect insulin-stimulated autophosphorylation, but increased the basal autophosphorylation rate approx. 2-fold. The amino acid residues contributing to the epitope for CT-1 were defined by construction and screening of an epitope library. Oligonucleotides containing 23 random bases were synthesized and ligated into the vector pCL627, and the corresponding peptide sequences expressed as fusion proteins in Escherichia coli were screened by colony blotting. Reactive peptides were identified by sequencing the oligonucleotide inserts in plasmids purified from positive colonies. Six different positive sequences were found after 900,000 colonies had been screened, and the consensus epitope was identified as GRVLTLPRS. Phosphorylation of the threonine residue within this sequence (corresponding to the known phosphorylation site Thr-1348 in the insulin receptor) decreased the affinity of antibody binding approx. 100-fold, as measured by competition in an e.l.i.s.a. Antibody CT-1 was used for immunoaffinity isolation of insulin receptor from detergent-solubilized human placental or rat liver microsomal membranes. Highly purified receptor was obtained in 60% yield by binding to CT-1-Sepharose immunoadsorbent and specific elution with a solution of peptide corresponding to the known epitope. This approach to purification under very mild conditions may in principle be used with any protein for which an antibody is available and for which a peptide epitope or 'mimotope' can be identified.
Cancer immunotherapy has been revolutionised by the use monoclonal antibodies (mAb) that function through their interaction with Fc gamma receptors (FcγRs). The low-affinity FcγR genes are highly homologous, map to a complex locus at 1p23 and harbour single nucleotide polymorphisms (SNPs) and copy number variation (CNV) that can impact on receptor function and response to therapeutic mAbs. This complexity can hinder accurate characterisation of the locus. We therefore evaluated and optimised a suite of assays for the genomic analysis of the FcγR locus amenable to peripheral blood mononuclear cells and formalin-fixed paraffin-embedded (FFPE) material that can be employed in a high-throughput manner. Assessment of TaqMan genotyping for FCGR2A-131H/R, FCGR3A-158F/V and FCGR2B-232I/T SNPs demonstrated the need for additional methods to discriminate genotypes for the FCGR3A-158F/V and FCGR2B-232I/T SNPs due to sequence homology and CNV in the region. A multiplex ligation-dependent probe amplification assay provided high quality SNP and CNV data in PBMC cases, but there was greater data variability in FFPE material in a manner that was predicted by the BIOMED-2 multiplex PCR protocol. In conclusion, we have evaluated a suite of assays for the genomic analysis of the FcγR locus that are scalable for application in large clinical trials of mAb therapy. These assays will ultimately help establish the importance of FcγR genetics in predicting response to antibody therapeutics.
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