More Clinical Overlap between 22q11.2 Deletion Syndrome and CHARGE Syndrome than Often Anticipated.

CHARGE (coloboma, heart defects, atresia of choanae, retardation of growth and development, genital hypoplasia, and ear abnormalities) and 22q11.2 deletion syndromes are variable, congenital malformation syndromes that show considerable phenotypic overlap. We further explored this clinical overlap and proposed recommendations for the genetic diagnosis of both syndromes. We described 2 patients clinically diagnosed with CHARGE syndrome, who were found to carry a 22q11.2 deletion, and searched the literature for more cases. In addition, we screened our cohort of CHD7 mutation carriers (n = 802) for typical 22q11.2 deletion features and studied CHD7 in 20 patients with phenotypically 22q11.2 deletion syndrome but without haploinsufficiency of TBX1. In total, we identified 5 patients with a clinical diagnosis of CHARGE syndrome and a proven 22q11.2 deletion. Typical 22q11.2 deletion features were found in 30 patients (30/802, 3.7%) of our CHD7 mutation-positive cohort. We found truncating CHD7 mutations in 5/20 patients with phenotypically 22q11.2 deletion syndrome. Differentiating between CHARGE and 22q11.2 deletion syndromes can be challenging. CHD7 and TBX1 probably share a molecular pathway or have common target genes in affected organs. We strongly recommend performing CHD7 analysis in patients with a 22q11.2 deletion phenotype without TBX1 haploinsufficiency and conversely, performing a genome-wide array in CHARGE syndrome patients without a CHD7 mutation.

Derivatization of the tricarboxylic acid cycle intermediates and analysis by online solid-phase extraction-liquid chromatography-mass spectrometry with positive-ion electrospray ionization.

The analysis of cellular metabolic processes is of fundamental biological interest. Cellular metabolites, such as the intermediates of the tricarboxylic acid (TCA) cycle, provide essential information about the metabolic state of the cell. Not only is the TCA cycle a key factor in the energy regulation within aerobic cells, it possibly also plays a role in cell signaling. This paper describes a novel derivatization strategy, using the empirically selected N-methyl-2-phenylethanamine as derivatization reagent with a carbodiimide as co-reagent, for the selective derivatization of carboxylic acids, such as the di- and tri-carboxylic acids of the TCA cycle. Optimization of the derivatization protocol is described. This procedure enables analysis of the derivatives using on-line solid-phase extraction and reversed-phase liquid chromatography in combination with sensitive positive-ion electrospray ionization mass spectrometry. The complete procedure, involving the use of core-shell silica column material, allows the rapid analysis of TCA cycle intermediates in sample matrices, here shown for pig heart tissue extracts, with a good linearity over 3-4 orders of magnitude. Detection limits range from 12 to 1000 nM, depending on the analyte.

Venous thromboembolism in ANCA-associated vasculitis--incidence and risk factors.

OBJECTIVES: In patients with ANCA-associated vasculitis (AAV), an increased incidence of venous thromboembolism (VTE), mainly during active disease, has been described. In a large cohort of AAV patients, we assessed the incidence of VTE and its relation with disease activity and classic risk factors for VTE. METHODS: Patients newly diagnosed with AAV between 1990 and 2005 and treated with cyclophosphamide and corticosteroids were included. Data were retrospectively retrieved from charts and by questionnaire. The incidence of VTE associated with and following a diagnosis of AAV was calculated (VTE/100 person-years) and related to periods with active disease. RESULTS: One hundred and ninety-eight patients with AAV were followed for 6.1 (0.2-17.6) yrs. In 23 patients (12%), 25 VTEs (17 deep venous thromboses, 3 pulmonary emboli, 5 both) occurred in association with AAV, of which 52% occurred during active disease, defined as 3 months before and after diagnosis or relapse of AAV. Overall, VTE incidence was 1.8/100 person-years, increasing to 6.7/100 during active disease. VTEs occurred significantly less frequently in patients with WG than in patients with microscopic polyangiitis and renal limited vasculitis. Classic risk factors were present in most patients at some moment during follow-up. There were no significant differences in classic risk factors between patients with and without AAV-associated VTE. CONCLUSIONS: Patients with AAV have an increased risk of developing VTEs, especially when AAV is active. This finding could not be explained by classic risk factors, but is probably related to endothelial changes and hypercoagulability induced by AAV and its therapy.

On-line continuous-flow, multi-protein biochemical assays for the characterization of bioaffinity compounds using electrospray quadrupole time-of-flight mass spectrometry.

The applicability of a homogeneous on-line continuous-flow, multi-protein biochemical assay was demonstrated for the interaction between fluorescein-biotin and streptavidin and for digoxin and anti-digoxigenin using electrospray quadrupole time-of-flight mass spectrometry (Q-TOF MS). In the on-line continuous-flow biochemical MS-based system several receptors (e.g., streptavidin and anti-digoxigenin, respectively) were allowed to react with corresponding reporter ligands (e.g.,fluorescein-biotin and digoxin, respectively). The methodology presented allows the simultaneous measurement of affinity and molecular mass of an active compound. By using automated MS and MS-MS switching functions of the Q-TOF, structure information is obtained allowing the characterization of bioactive compounds. No cross-reactivities were observed between the two model systems fluorescein-biotin/streptavidin and digoxin/anti-digoxigenin.

Continuous-flow, on-line monitoring of biospecific interactions using electrospray mass spectrometry.

A continuous-flow analytical screening system is presented using electrospray mass spectrometry to measure the interaction of biologically active compounds with soluble affinity proteins. The biochemical detection system is based on a solution-phase, homogeneous assay. In a first step, compounds to be screened (e.g., biotinylated compounds, concentration range 10-1,000 nmol/L) are injected into a continuous-flow reaction system and allowed to react with the affinity protein (e.g., streptavidin, concentration range 3-48 nmol/L). Subsequently, a reporter ligand (fluorescein-labeled biotin 96 nmol/L) is added to saturate the remaining free binding sites of the affinity protein and the concentration of unbound reporter ligand is measured using electrospray MS in the selectedion monitoring mode. The presence of active compounds in the sample results in an increase of the concentration of unbound reporter ligands. The feasibility of a homogeneous MS-based biochemical assay is demonstrated using streptavidin/biotin and anti-digoxigenin/digoxin as model systems. Compared to radioactive or fluorescence-based biochemical assays, the present assay format does not require the synthesis and purification of labels. Various analytical conditions were investigated to determine the ability of MS to measure the biochemical interactions. The availability of a single ligand that can be detected at 10-50 nmol/L concentrations by electrospray MS is sufficient to set up the biochemical assay. For the biospecific interactions studies, detection limits of 10-100 nmol/L were obtained.

Regeneration and tolerance factor is expressed during T-lymphocyte activation and plays a role in apoptosis.

Regeneration and tolerance factor (RTF) is a protein cloned from the thymus and expressed on B lymphocytes in normal pregnancy, B lymphocytic leukemia lines, and T and B lymphocytes in individuals with HIV infection. Findings, using the Jurkat T-cell model, revealed that RTF is upregulated after activation and anti-RTF antibody-induced apoptosis. In this article anti-RTF antibody-induced apoptosis of both unstimulated and activated T lymphocytes. RTF expression was examined in human PBMC or purified T lymphocytes after their in vitro activation. Kinetic studies indicated maximal RTF cell surface expression on activated T lymphocytes occurred between expression of the early activation antigen CD69 and the IL-2alpha receptor (CD25) by multiparameter flow cytometry. RTF receptor expression correlated with Fas (CD95) and CD25 receptor expression (r2 = 0.6 and 0.5, respectively). RTF surface expression was dependent on the stimuli used to activate T lymphocytes. T lymphocytes obtained maximal RTF expression when activated through the TCR signal complex using anti-CD3epsilon antibody alone when compared with T lymphocytes activated with costimulation provided by anti-CD28 antibody alone or with anti-CD28 and anti-CD3epsilon antibody. RTF is expressed under conditions of both activation and anergy. The RTFs increased concentration on the surface of anergic T cells may protect these cells from apoptosis because increased RTF concentrations inhibited anti-RTF induced apoptosis. These data further characterize the expression of RTF on activated T lymphocytes and the role of anti-RTF antibody in T-lymphocyte apoptosis.