Genotype and phenotype correlation in 103 individuals with 2q37 deletion syndrome reveals incomplete penetrance and supports HDAC4 as the primary genetic contributor

The 2q37 deletion syndrome, also described in the literature as brachydactyly‐mental retardation syndrome (MIM 600430), is caused by deletion or haploinsufficiency of the HDAC4 gene, which encodes the histone deacetylase 4 protein. Although the most commonly described hallmark features of the 2q37 deletion syndrome include brachydactyly type E, developmental delay, obesity, autistic features, and craniofacial or skeletal dysmorphism, a literature review of 101 published cases plus two newly reported individuals indicates that there is a high degree of variability in the presence of some of the features that are considered the most characteristic of the syndrome: overweight and obesity (34%), cognitive‐behavioral issues (79%), dysmorphic craniofacial features (86%), and type E brachydactyly (48%). These features overlap with other neurodevelopmental conditions, including Smith‐Magenis syndrome (SMS), and may be incompletely penetrant or demonstrate variable expressivity, depending on the specific chromosomal anomaly. With the advent of fluorescence in situ hybridization (FISH), array‐based comparative genomic hybridization, and next‐generation DNA sequencing, more detailed molecular diagnoses are possible than in years past, enabling refined characterization of the genotype–phenotype correlation for subjects with 2q37 deletions. In addition, investigations into molecular and gene expression networks are expanding in neurodevelopmental conditions, and we surveyed HDAC4 downstream gene expression by quantitative real‐time polymerase chain reaction, further implicating HDAC4 in its role in the regulation of RAI1. Correlation of clinical data defining the impact on downstream gene expression and the potential clinical associations across neurodevelopment will improve our understanding of these complex conditions and potentially lead to common therapeutic approaches.     

An observational study of variation in GPs' out-of-hours emergency referrals.

Out-of-hours organisations are responsible for the care of patients 70% of the time, and their GPs act as gatekeepers to secondary care services. This observational study identifies the variations in GPs' out-of-hours referral rates to secondary care and factors that could explain these variations. One hundred and forty-nine GPs who worked in one UK general practice out-of-hours cooperative which served 19 practices with 167 000 registered patients. Data on patients who accessed the out-of-hours service over 3 years (2001-2004) were examined. Factors thought to be predictors of variation in referral rates were investigated using logistic regression analysis. There was a fivefold difference in referral rates between the lowest and highest referring quartiles of GPs (OR [odds ratio] = 4.56, CI [confidence interval] = 3.86 to 5.38). The sex (female) of the clinician, the time of the consultation (11 pm to 7 am), and the place of the consultation (home visit) accounted for some, but not all, of the increased referral rates. A doctor working out-of-hours disproportionately influences the fate of the patient, the number of hospital admissions, and extra costs to the health service. There is a need for follow-up studies to investigate the factors associated with referral behaviour, and how the variation relates to patient factors and the resources available. These findings could be used when planning the staffing of out-of-hours services to optimise appropriate care and minimise patients' exposure to unnecessary intrusive and expensive hospital care.     

Development of a quantitative monoclonal antibody‐based immunoassay for humicola ianuginosa on rice grains and comparison with conventional assays

A sensitive, specific, quantitative enzyme‐linked immunosorbent assay (ELISA) has been developed that can be used to determine the extent of mycelial growth of a sporulating thermophilic fungus, Humicola lanuginosa on the surface of rice grains. The assay employs a monoclonal antibody EC6, developed in a previous study, which does not recognize spores of the fungus. Using antigen‐coated wells, a direct linear relationship was established between dilutions of extracts from freeze‐dried mycelium (0.5 to 3 μg/ml) and absorbance values but to eliminate day‐to‐day variations it was found to be necessary to run a dilution series, prepared from stock freeze‐dried mycelium, with every test sample. The ELISA method was compared with conventional quantitative methods. Estimates of total mycelial length in freeze‐dried material by ELISA were found to be in the same order of magnitude as those determined by ergosterol and a theoretical calculation. The ELISA method also compared favourably with direct linear measurements (by photomicrography) of live mycelium present in aliquots from homogenates of a 1 cm² plug taken from a plate but estimates of the latter by the dilution plate count method were much lower. In assays with inoculated rice grains, the quantitative ELISA method proved more sensitive than either the ergosterol method or direct plating of surface‐sterilized grains. The ELISA method also has the advantage of being highly specific and quick to conduct.     

Mode of delivery and risk of developing allergic disease

The aim of this study was to quantify the relationship between mode of delivery and subsequent incidence of allergic disease. The analysis is based on data derived from a birth cohort of 24,690 children who contributed data to the West Midlands General Practice Research Database. We found no convincing evidence to suggest that babies born by caesarean, forceps, or breech delivery had an increased risk of developing allergic disease.     

Guidelines to avoid personnel contamination by infective agents in research laboratories that use human tissues

Summary The personnel in laboratories that utilize tissue and fluids from humans and other primates are at risk for infection with agents, including the viruses causing hepatitis, AIDS, and other infective agents such as mycobacteria tuberculosis. To minimize the chance of infection of laboratory personnel, carefully organized policies and procedures to minimize exposure to infective agents must be established in research laboratories. We outline some of the approaches of hospital clinical laboratories which have proved most effective in minimizing transmission of infections from samples to laboratory personnel. Also, we discuss simple considerations important in the use and in the selection of safety equipment. These guidelines and references to other safety information are provided to aid research laboratories in establishing safety procedures that will minimize chances of personnel contamination with infective agents from research samples.     

Quantification of Human Immunodeficiency Virus Type 1 RNA Levels in Plasma by Using Small-Volume-Format Branched-DNA Assays

We have developed small-volume (50 or 250 μl)-format branched-DNA assays for human immunodeficiency virus type 1 (HIV-1) RNA for use with specimens in which the volume is limited and/or a high viral load is anticipated. These formats exhibited good correlation with the standard 1-ml format; high specificity, reproducibility, and linearity; and no significant difference in the quantification of HIV-1 subtypes.     

Development of a simple method for rapid isolation of polymorphonuclear leukocytes from human blood

Polymorphonuclear leukocytes (PMNs; commonly known as neutrophils) play essential roles in innate immunity and inflammation. Although there are standardized methods for the isolation of human neutrophils, they are time consuming and demand considerable technical expertise, making them unfeasible for many clinical applications. Here, we describe a simple and time-efficient technique for the isolation of human neutrophils, which adapts a readily available commercial cell preparation tube (CPT) currently in use for isolation of peripheral blood mononuclear cells (PBMC) and plasma and is now adapted to also yield neutrophils. The total time required for neutrophil isolation was less than 1 hr. Neutrophils isolated by this method were highly purified (> or =97%) as assessed by surface expression of the neutrophil specific marker, CD66b. Neutrophils isolated by this method were functional as demonstrated by their ability to secrete interleukin-1 receptor antagonist (IL-1RA). Neutrophils isolated using this new technique secreted significant amounts of soluble IL-1RA (929.3+/-197 pg/10(6)cells/mL) in response to lipopolysaccharide (LPS). Use of this adapted CPT method allows simultaneous isolation of functional human neutrophils as well as PBMC and plasma. Adoption of this new method will allow the conduct of different neutrophil assays at any clinical site without requiring trained laboratory personnel or a large staff time commitment.     

Expression of VE-cadherin in zebrafish embryos: a new tool to evaluate vascular development.

We have identified the zebrafish homologue of VE-cadherin and documented its expression in the developing vascular system. The zebrafish VE-cadherin gene is specifically expressed in the vascular endothelial cell lineage beginning with the differentiation and migration of angioblasts and persists throughout vasculogenesis, angiogenesis, and endocardium development. Staining zebrafish embryos by whole-mount in situ hybridization with the VE-cadherin probe provides a method to screen embryos for vascular defects. To illustrate this utility, we used VE-cadherin expression to demonstrate a conservation of vascular endothelial growth factor-A (VEGF-A) function. The morpholino antisense oligonucleotide knockdown of VEGF-A function in zebrafish embryos results in a loss of angiogenic blood vessels, as indicated by the lack of VE-cadherin expression in the intersegmental vasculature. This loss can be restored in embryos supplemented with either zebrafish or human VEGF-A, the latter indicating that genes crucial to angiogenesis have highly conserved functional activities in vertebrates.