Loss of function,missense, and intronic variants in NOTCH1 confer different risks for left ventricular outflow tract obstructive heart defects in two European cohorts

Loss of function variants in NOTCH1 cause left ventricular outflow tract obstructive defects (LVOTO). However, the risk conferred by rare and noncoding variants in NOTCH1 for LVOTO remains largely uncharacterized. In a cohort of 49 families affected by hypoplastic left heart syndrome, a severe form of LVOTO, we discovered predicted loss of function NOTCH1 variants in 6% of individuals. Rare or low-frequency missense variants were found in 16% of families. To make a quantitative estimate of the genetic risk posed by variants in NOTCH1 for LVOTO, we studied associations of 400 coding and noncoding variants in NOTCH1 in 1,085 cases and 332,788 controls from the UK Biobank. Two rare intronic variants in strong linkage disequilibrium displayed significant association with risk for LVOTO amongst European-ancestry individuals. This result was replicated in an independent analysis of 210 cases and 68,762 controls of non-European and mixed ancestry. In conclusion, carrying rare predicted loss of function variants in NOTCH1 confer significant risk for LVOTO. In addition, the two intronic variants seem to be associated with an increased risk for these defects. Our approach demonstrates the utility of population-based data sets in quantifying the specific risk of individual variants for disease-related phenotypes.     

Experience with a metered-dose inhaler with a spacer in the pediatric emergency department

Studies of adults suggest that metered-dose inhalers with spacers are as effective as hand-held nebulizers for bronchodilator delivery. We studied 13 children with acute asthma. They received two puffs every 2 minutes from metered-dose inhalers with spacers (range, 4 to 14 puffs) titrated until improvement stopped. Peak expiratory flow increased 34% for metered-dose inhalers with spacers after the first 2 puffs and increased 87% for metered-dose inhalers with spacers after dose titration. After titration, respiratory rate decreased by 12%, heart rate increased by 2%, and breath sounds improved in 92% of the patients. We concluded that the metered-dose inhalers with spacers are an effective device for the treatment of asthma in the pediatric emergency department and that the use of metered-dose inhalers with spacers with titration can achieve significant bronchodilation in the treatment of patients with acute asthma.     

A study comparing biopharmaceutic characteristics of four once daily controlled release diltiazem preparations

Summary— In the present study we have compared the steady state biopharmaceutic characteristics of four diltiazem once daily controlled release capsules: Mono-Tildiem LP 300® (300 mg), Adizem® XL (300 mg)¹, Cardizem® (300 mg) and Dilacor® (240 mg). Sixteen healthy male volunteers (aged 22.9 ± 3.3 years, range 19–31 years) completed an open label, multiple oral dose, randomized, four-period crossover study without a washout period in between. The volunteers received each diltiazem formulation once daily for four days. Trough diltiazem and metabolites plasma concentrations were determined on days 3 and 4. The 24-h plasma concentration-time profiles were assessed after the dose on day 4 of each period. The following steady state pharmacokinetic parameters for diltiazem were calculated: the minimum plasma concentration (c_min), the maximum plasma concentration (c_max), the time to reach that concentration (t_max), the time interval during which the plasma concentration exceeds 50% of c_max (t₅₀), the area under the plasma concentration-time curve (AUC_72–96) and the peak-to-trough fluctuation (PTF). For the metabolites of diltiazem, N-mono-desmethyl-diltiazem (NDM) and desacetyldiltiazem (DAD), AUC_72–96 (AUC_NDM and AUC_DAD) and the ratio metabolite/parent compound were calculated. Steady state was achieved on day 3. Except one, all controlled release formulations have satisfactory controlled release properties allowing once daily administration. However, significant (P < 0.05) differences were found between the pharmacokinetic characteristics which do not allow exchange of the various formulations. Concentrations well below 50 ng·mL^-1 in the morning hours were observed for Dilacor® (240 mg) and Adizem® XL (300 mg), which could be a disadvantage of these formulations as it is well-known that ischaemic events occur at a higher rate during that part of the day. The plasma concentration profiles of NDM and DAD, the major circulating metabolites, parallel the plasma concentration profiles for the parent compound. From a clinical point of view, all treatments were well tolerated.     

Segmental intestinal muscular thinning: a possible cause of intestinal obstruction in the newborn

Intestinal obstruction proximal to a transition zone without an interposed physical barrier usually indicates Hirschsprung disease. The authors report one case of focal small bowel muscular thinning just distal to a transition zone that produced clinical and radiographic findings that simulated long-segment Hirschsprung disease in a 2-day-old infant.     

Pattern of Sequence Variation Across 213 Environmental Response Genes

To promote the clinical and epidemiological studies that improve our understanding of human genetic susceptibility to environmental exposure, the Environmental Genome Project (EGP) has scanned 213 environmental response genes involved in DNA repair, cell cycle regulation, apoptosis, and metabolism for single nucleotide polymorphisms (SNPs). Many of these genes have been implicated by loss-of-function mutations associated with severe diseases attributable to decreased protection of genomic integrity. Therefore, the hypothesis for these studies is that individuals with functionally significant polymorphisms within these genes may be particularly susceptible to genotoxic environmental agents. On average, 20.4 kb of baseline genomic sequence or 86% of each gene, including a substantial amount of introns, all exons, and 1.3 kb upstream and downstream, were scanned for variations in the 90 samples of the Polymorphism Discovery Resource panel. The average nucleotide diversity across the 4.2 MB of these 213 genes is 6.7 × 10^-4, or one SNP every 1500 bp, when two random chromosomes are compared. The average candidate environmental response gene contains 26 PHASE inferred haplotypes, 34 common SNPs, 6.2 coding SNPs (cSNPs), and 2.5 nonsynonymous cSNPs. SIFT and Polyphen analysis of 541 nonsynonymous cSNPs identified 57 potentially deleterious SNPs. An additional eight polymorphisms predict altered protein translation. Because these genes represent 1% of all known human genes, extrapolation from these data predicts the total genomic set of cSNPs, nonsynonymous cSNPs, and potentially deleterious nonsynonymous cSNPs. The implications for the use of these data in direct and indirect association studies of environmentally induced diseases are discussed.     

Patterns of aneuploidy in stage IV clear cell renal cell carcinoma revealed by comparative genomic hybridization and spectral karyotyping

We report the use of spectral karyotyping (SKY) and comparative genomic hybridization (CGH) to describe the numerous genomic imbalances characteristic of stage IV clear cell renal cell carcinoma (CCRCC). SKY and CGH were performed on 10 cell lines established from nephrectomy specimens, and CGH on uncultured material from five of the primary renal tumors. The mutational status of VHL (3p25) and MET (7q31), genes implicated in renal carcinogenesis, were determined for each case. Each case showed marked aneuploidy, with an average number of copy alterations of 14.6 (+/-2.7) in the primary tumors and 19.3 (+/-4.6) in the cell lines. Both whole-chromosome and chromosome-segment imbalances were noted by CGH: consistent losses or gains included +5q23-->ter (100%), -3p14-->ter (80%), and +7 (70%). All VHL mutations and 83% of the genomic imbalances found in the primary tumors were also found in the cell lines derived from them. SKY showed many complex structural rearrangements that were undetected by conventional banding analysis in these solid tumors. All cases with VHL inactivation had 3p loss and 5q gain related primarily to unbalanced translocations between 3p and 5q. In contrast, gains of chromosome 7 resulted primarily from whole-chromosome gains and were not associated with mutations of MET. SKY and CGH demonstrated that genomic imbalances in advanced RCC were the result of either segregation errors [i.e., whole chromosomal gains and losses (7.8/case)] or chromosomal rearrangements (10.7/case), of which the majority were unbalanced translocations.     

A549 lung epithelial cells grown as three-dimensional aggregates: alternative tissue culture model for Pseudomonas aeruginosa pathogenesis

A three-dimensional (3-D) lung aggregate model was developed from A549 human lung epithelial cells by using a rotating-wall vessel bioreactor to study the interactions between Pseudomonas aeruginosa and lung epithelial cells. The suitability of the 3-D aggregates as an infection model was examined by immunohistochemistry, adherence and invasion assays, scanning electron microscopy, and cytokine and mucoglycoprotein production. Immunohistochemical characterization of the 3-D A549 aggregates showed increased expression of epithelial cell-specific markers and decreased expression of cancer-specific markers compared to their monolayer counterparts. Immunohistochemistry of junctional markers on A549 3-D cells revealed that these cells formed tight junctions and polarity, in contrast to the cells grown as monolayers. Additionally, the 3-D aggregates stained positively for the production of mucoglycoprotein while the monolayers showed no indication of staining. Moreover, mucin-specific antibodies to MUC1 and MUC5A bound with greater affinity to 3-D aggregates than to the monolayers. P. aeruginosa attached to and penetrated A549 monolayers significantly more than the same cells grown as 3-D aggregates. Scanning electron microscopy of A549 cells grown as monolayers and 3-D aggregates infected with P. aeruginosa showed that monolayers detached from the surface of the culture plate postinfection, in contrast to the 3-D aggregates, which remained attached to the microcarrier beads. In response to infection, proinflammatory cytokine levels were elevated for the 3-D A549 aggregates compared to monolayer controls. These findings suggest that A549 lung cells grown as 3-D aggregates may represent a more physiologically relevant model to examine the interactions between P. aeruginosa and the lung epithelium during infection.     

Development of hatching blastocysts from immature human oocytes following in-vitro maturation and fertilization using a co-culture system

Recently, in-vitro maturation (IVM) of immature human oocytes recovered from non-stimulated follicles has been applied in the treatment of infertility. However, in previous reports, very few embryos cultured in conventional medium have reached the expanded blastocyst stage following in-vitro maturation and fertilization (IVM/IVF). The objective of this study was to investigate whether the developmental competence of human embryos following IVM/IVF could be enhanced by the use of a human ampullary cell co-culture system. Immature human oocytes were aspirated from small follicles at Caesarean section and then cultured in medium containing human menopausal gonadotrophin for 36 to 48 h, followed by insemination. Zygotes were randomly cultured either in conventional culture medium alone or in the co-culture system. Of 48 embryos cultured in conventional medium alone, all arrested at the 2-16- cell stage on day 3 after insemination. Of 46 embryos cultured in the co-culture system, 26 embryos (56.5%) arrested at the 2-16-cell stage. Six embryos (13%) developed to the morula stage. Fourteen embryos (30.4%) developed to expanded blastocysts and two blastocysts were hatching on day 7 after insemination. We conclude that co-culture significantly enhances the development of blastocysts in embryos resulting from IVM/IVF.