Association between COMT (Val158Met) functional polymorphism and early onset in patients with major depressive disorder in a European multicenter genetic association study.

The available data from preclinical and pharmacological studies on the role of the C-O-methyl transferase (COMT) support the hypothesis that abnormal catecholamine transmission has been implicated in the pathogenesis of mood disorders (MD). We examined the relationship of a common functional polymorphism (Val108/158Met) in the COMT gene, which accounts for four-fold variation in enzyme activity, with 'early-onset' (EO) forms (less than or equal to 25 years) of MD, including patients with major depressive disorder (EO-MDD) and bipolar patients (EO-BPD), in a European multicenter case-control sample. Our sample includes 378 MDD (120 EO-MDD), 506 BPD (222 EO-BPD) and 628 controls. An association was found between the high-activity COMT Val allele, particularly the COMT Val/Val genotype and EO-MDD. These findings suggest that the COMT Val/Val genotype may be involved in EO-MDD or may be in linkage disequilibrium with a different causative polymorphism in the vicinity. The COMT gene may have complex and pleiotropic effects on susceptibility and symptomatology of neuropsychiatric disorders.

Recent advances in the diagnosis of malignant hyperthermia susceptibility: how confident can we be of genetic testing?

Malignant hyperthermia (MH) is a condition that manifests in susceptible individuals only on exposure to certain anaesthetic agents. Although genetically heterogeneous, mutations in the RYR1 gene (19q13.1) are associated with the majority of reported MH cases. Guidelines for the genetic diagnosis for MH susceptibility have recently been introduced by the European MH Group (EMHG). These are designed to supplement the muscle biopsy testing procedure, the in vitro contracture test (IVCT), which has been the only means of patient screening for the last 30 years and which remains the method for definitive diagnosis in suspected probands. Discordance observed in some families between IVCT phenotype and susceptibility locus genotype could limit the confidence in genetic diagnosis. We have therefore assessed the prevalence of 15 RYR1 mutations currently used in the genetic diagnosis of MH in a sample of over 500 unrelated European MH susceptible individuals and have recorded the frequency of RYR1 genotype/IVCT phenotype discordance. RYR1 mutations were detected in up to approximately 30% of families investigated. Phenotype/genotype discordance in a single individual was observed in 10 out of 196 mutation-positive families. In five families a mutation-positive/IVCT-negative individual was observed, and in the other five families a mutation-negative/IVCT-positive individual was observed. These data represent the most comprehensive assessment of RYR1 mutation prevalence and genotype/phenotype correlation analysis and highlight the possible limitations of MH screening methods. The implications for genetic diagnosis are discussed.

A novel system for convenient detection of low-affinity receptor-ligand interactions: chelator-lipid liposomes engrafted with recombinant CD4 bind to cells expressing MHC class II.

The interactions of cell surface receptors with their ligands, crucial for initiating many immunological responses, are often stabilized by receptor dimerization/oligomerization, and by multimeric interactions between receptors on one cell with their ligands or cognate receptors on the apposing cell. Current techniques for studying receptor-ligand interactions, however, do not always allow receptors to move laterally to enable dimerization/ oligomerization, or to interact multimerically with ligands on cell surfaces. For these reasons detection of low- affinity receptor-ligand interactions has been difficult. Utilizing a novel chelator-lipid, nitrilotriacetic acid di-tetradecylamine (NTA-DTDA), we have developed a convenient liposome system for directly detecting low-affinity receptor-ligand interactions. Our studies using recombinant soluble forms of murine CD40 and B7.1, and murine and human CD4, each possessing a hexhistidine tag, showed that these proteins can be anchored or 'engrafted' directly onto fluorescently labelled liposomes via a metal-chelating linkage with NTA-DTDA, permitting them to undergo dimerization/oligomerization and multimeric binding with ligands on cells. Fluorescence- activated cell sorter (FACS) analyses demonstrated that while there is little if any binding of soluble forms of murine CD40 and B7.1, and murine and human CD4 to cells, engrafted liposomes bind specifically to cells expressing the appropriate cognate receptor, often giving a fluorescence 4-6-fold above control cells. Such liposomes could detect directly the low-affinity interaction of murine CD40 and B7.1 with CD154- and CD28-expressing cells, respectively, and the interaction of CD4 with MHC Class II, which has hitherto defied direct detection except through mutational analysis and mAb blocking studies.

Engrafting costimulator molecules onto tumor cell surfaces with chelator lipids: a potentially convenient approach in cancer vaccine development.

The genetic modification of cells to develop cell-based vaccines and to modulate immune responses in vivo can be risky and inconvenient to perform in clinical situations. A novel chelator lipid, nitrilotriacetic acid di-tetradecylamine (NTA-DTDA) that, via the NTA group has high affinity for 6His peptide, was used to directly anchor recombinant forms of T cell costimulatory molecules containing a C-terminal 6-His sequence onto tumor cell surfaces. Initial experiments using murine P815 tumor cells established the optimum conditions for incorporating NTA-DTDA onto the membranes of cells. P815 cells with incorporated NTA-DTDAbound hexahistidine-(6His)-tagged forms of the extracellular domains of murine B7.1 and CD40 (B7.1-6H and CD40-6H) at very high levels (fluorescence 200-300-fold above background), and both proteins could be anchored onto the cells simultaneously. Significant loss of the anchored or "engrafted" protein occurred through membrane internalization following culture of the cells under physiological conditions, but P815 cells with engrafted B7.1-6H and/or CD40-6H stimulated the proliferation of allogenic and syngeneic splenic T cells in vitro, and generated cytotoxic T cells when used as vaccines in syngeneic animals. Furthermore, the immunization of syngeneic mice with P815 cells engrafted with B7.1-6H or with B7. 1-6H and CD40-6H induced protection against challenge with the native P815 tumor. The results indicate that the use of chelator lipids like NTD-DTDA to engraft costimulatory and/or other molecules onto cell membranes could provide a convenient alternative to transfection in the development of cell-based vaccines and for modulation of immune function.