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.     

Lens opacity in patients with hypercholesterolemia and ischaemic heart disease

Lens opacity studies were performed using an electronic Lens Opacity Meter (Interzeag Opacity Lensmeter 701) in a population (n = 321) with ischaemic heart disease. These patients are participating in a trial targetting at the reduction of mortality and incidence of myocardial infarction using a cholesterol-lowering drug, simvastatin. A separate study to evaluate the reliability of the method showed good reproducibility. Repeated measurements after a short time-interval (2–10 days) gave statistically lower opacity values either due to a change in lens transparency or perhaps a change in pigment and cell dispersion in the acqueous caused by repeated mydriasis. Lens opacity values showed a highly significant positive correlation to age. Serum cholesterol, systolic blood pressure and smoking habits showed no significant correlations to the levels of lens opacity when adjustments for age were made.Abbreviations HMG-CoA hydroxy-methylglutarylcoenzyme A - 4S Scandinavian Simvastatin Survival Study - LOM lens opacity meter     

IgE-mediated allergy to fungal allergens in Finland with special reference to Alternaria alternata and Cladosporium herbarum.

BACKGROUND: Alternaria alternata and Cladosporium herbarum are common fungi in outdoor environments, but their clinical significance has not been elucidated in Finland. OBJECTIVE: To evaluate the prevalence of IgE-mediated allergy and clinical outcomes caused by sensitization to fungal allergens in patients with suspected allergy. METHODS: Skin prick tests (SPTs) were performed with C. herbarum in 6,376 patients and also with A. alternata in 1,504 of these patients. SPTs were repeated in 40 patients who showed a positive reaction to either allergen using commercial and in-house extracts. The association of SPT with allergen-specific IgE antibodies in serum was evaluated. Seven patients also underwent a conjunctival challenge test with these fungal allergens. RESULTS: The prevalence of positive SPT results to A. alternata and C. herbarum was low (2.8% and 2.7%, respectively). Among the 40 patients, atopic eczema/dermatitis syndrome was found in 58%, asthma in 44%, and rhinitis in 31%. Most of the patients displayed SPT reactions also to several other fungal allergens, and 75% to 80% showed a positive SPT reaction to allergens of pet animals or pollens. Four patients had a positive reaction to A. alternata and 6 to C. herbarum in the conjunctival challenge test. CONCLUSION: In the Finnish population with allergic symptoms, IgE-mediated sensitization to 2 common fungal allergens was rare and of minor clinical importance. SPT reactions to fungi are mostly observed in patients with multiple sensitivity to various allergens.     

Ethylene–Propylene Copolymerisations: Effect of Metallocene Structure on Termination Reactions and Polymer Microstructure

Summary: Ethylene–propylene (EP) copolymerisations were performed with two sterically different metallocenes activated by methylaluminoxane (MAO) in an attempt to better understand the effect of catalyst structure on termination reactions and polymer microstructure. The metallocene precursors under investigation were rac‐dimethylsilylbis(2‐methyl‐4‐phenyl‐1‐indenyl)zirconium dichloride ( 1 ) and a more sterically hindered counterpart rac‐dimethylsilylbis(2‐isopropyl‐4‐[3,5‐dimethylphenyl]indenyl) zirconium dichloride ( 2 ). For both catalyst systems, the most common termination mechanism was chain transfer to aluminium. In addition, for polymer samples polymerised with 1 /MAO, chain growth was terminated by chain transfer to Zr metal in propylene‐rich polymerisations and by chain transfer to ethylene monomer in ethylene‐rich polymerisations. The steric hindrance of 2 was able to suppress the chain transfer to the ethylene monomer, and chain transfer to Zr metal was also found in the ethylene‐rich polymerisations. The greater steric hindrance of 2 also affected the EP copolymer microstructure: regioregularity in the propylene‐rich copolymers was greater and isotacticity less with 2 /MAO than with 1 /MAO.

The catalyst precursors used: rac‐dimethylsilylbis(2‐methyl‐4‐phenyl‐1‐indenyl)zirconium dichloride ( 1 ) and rac‐dimethylsilylbis(2‐isopropyl‐4‐[3,5‐dimethylphenyl]indenyl) zirconium dichloride ( 2 ).     

TK-GFP fusion gene virus vectors as tools for studying the features of HSV-TK/ganciclovir cancer gene therapy in vivo

The fusion gene of herpes simplex virus thymidine kinase and green fluorescent protein (TK-GFP) was shown to be a versatile tool for examining the features of thymidine kinase/ganciclovir gene therapy in vitro. In this study, we used viral vectors carrying the fusion gene to characterize the aspects of this gene therapy form in rodent tumor models. Growth of subcutaneous 9L rat tumors transduced ex vivo with TK-GFP gene was prevented when ganciclovir (GCV) treatment was initiated immediately after tumor inoculation. Established tumors (>100 mm(3)), however, were untreatable despite the initial 55% proportion of TK-GFP positive cells. This was due to a rapid clearance of TK-GFP positive cells, but not GFP positive cells. Propidium iodide staining revealed that TK-GFP lentivirus vector was able to induce apoptosis/necrosis in 9L cells, as opposed to the respective GFP vector. Furthermore, when a subcutaneous nude mouse tumor model was used, the percentage of TK-GFP positive cells in vivo was maintained similarly as in cultured cells, suggesting contribution of a fully functional immune response to the disappearance of fusion gene positive cells. In vivo gene transfer studies: adenovirus TK-GFP vector injections resulted in about 25% gene transfer efficiency to 9L tumors and showed that their growth could be significantly reduced even when the tumor volumes were already >120 mm(3). Part of the effect was shown to be due to cytotoxicity of the vector. In summary, our results demonstrate the utility of TK-GFP fusion gene-carrying viral vectors in animal studies and show that readily detectable therapeutic genes can help us to understand the complicated nature of in vivo cancer gene therapy experiments.     

Phage types and genotypes of Shiga toxin-Producing Escherichia coli O157 in Finland

This study examined Shiga toxin-producing Escherichia coli (STEC) O157, using phage typing, pulsed-field gel electrophoresis, and typing of Shiga toxin variant genes by PCR with restriction fragment length polymorphism in an epidemiological survey of STEC O157 isolated from humans in Finland between 1990 and 1999.     

Training effects of cross-country skiing and running on maximal aerobic cycle performance and on blood lipids

Summary Two experiments were carried out to compare the cardiorespiratory and metabolic effects of cross-country skiing and running training during two successive winters. Forty-year-old men were randomly assigned into skiing (n = 15 in study 1,n = 16 in study 2), running (n = 16 in study 1 andn = 16 in study 2) and control (n = 17 in study 1 andn = 16 in study 2) groups. Three subjects dropped out of the programme. The training lasted 9–10 weeks with 40-min exercise sessions three times each week. The training intensity was controlled at 75%–85% of the maximal oxygen consumption (VO_2max) using portable heart rate metres and the mean heart rate was 156–157 beats·min^–1 in the training groups. In the pooled data of the two studies the mean increase in theVO_2max (in ml·min^–1·kg^–1) on a cycle ergometer was 17% for the skiing group, 13% for the running group and 2% for the control group. The increase inVO_2max was highly significant in the combined exercise group compared to the control group but did not differ significantly between the skiing and running groups. The fasting serum concentrations of lipoproteins and insulin did not change significantly in any of the groups. These results suggested that training by cross-country skiing and running of the same duration and intensity at each session for 9–10 weeks improved equally the cardiorespiratory fitness of untrained middle-aged men.     

The interval of linkage disequilibrium (LD) detected with microsatellite and SNP markers in chromosomes of Finnish populations with different histories

Linkage disequilibrium (LD) has been an efficient tool for fine mapping of monogenic disease genes in population isolates. Its usefulness for identification of predisposing loci for common, polygenic diseases has been challenged on the basis of anticipated allelic and locus heterogeneity. We compared the extent of LD among marker loci in Finnish subpopulations with divergent but well-characterized histories. One study sample represents the early settlement Finnish population, descended from two immigration events 4,000 and 2,000 years ago. The second sample represents the geographically large late settlement region, populated 15 generations ago by several small immigrant groups from the early settlement region. The third is a restricted regional subpopulation in northeastern Finland which was founded 12 generations ago by 39 immigrant families from the late settlement region. We genotyped 243 microsatellite markers and 68 single nucleotide polymorphisms (SNPs) on chromosomes 1q and 5q. The genealogy of the families from the early (n=16) and late settlements (n=54) and the isolated settlement (n=54) was studied in detail back to the 1800s. Microsatellite data revealed greater LD in the young, founder subpopulation than was seen in either of the older populations. Observed linkage disequilibrium correlated not only with physical distance between markers but also with the information content of the markers. Using biallelic SNP markers, significant LD could only be detected up to 0.1 cM. Our results demonstrate the complexity of the concept of 'detectable LD' and emphasize the importance of understanding population history when designing a strategy for disease gene mapping.     

A mouse model for alpha-methylacyl-CoA racemase deficiency: adjustment of bile acid synthesis and intolerance to dietary methyl-branched lipids

alpha-Methylacyl-CoA racemase (Amacr) deficiency in humans leads to sensory motor neuronal and liver abnormalities. The disorder is recessively inherited and caused by mutations in the AMACR gene, which encodes Amacr, an enzyme presumed to be essential for bile acid synthesis and to participate in the degradation of methyl-branched fatty acids. To generate a model to study the pathophysiology in Amacr deficiency we inactivated the mouse Amacr gene. As per human Amacr deficiency, the Amacr(-/-) mice showed accumulation (44-fold) of C27 bile acid precursors and decreased (over 50%) primary (C24) bile acids in bile, serum and liver, however the Amacr(-/-) mice were clinically symptomless. Real-time quantitative PCR analysis showed that, among other responses, the level of mRNA for peroxisomal multifunctional enzyme type 1 (pMFE-1) was increased 3-fold in Amacr(-/-) mice. This enzyme can be placed, together with CYP3A11 and CYP46A1, to make an Amacr-independent pathway for the generation of C24 bile acids. Exposure of Amacr(-/-) mice to a diet supplemented with phytol, a source for branched-chain fatty acids, triggered the development of a disease state with liver manifestations, redefining the physiological significance of Amacr. Amacr is indispensable for the detoxification of dietary methyl-branched lipids and, although it contributes normally to bile acid synthesis from cholesterol, the putative pMFE-1-mediated cholesterol degradation can provide for generation of bile acids, allowing survival without Amacr. Based upon our mouse model, we propose elimination of phytol from the diet of patients suffering from Amacr deficiency.