Allele Dependent Silencing of Collagen Type I Using Small Interfering RNAs Targeting 3'UTR Indels - a Novel Therapeutic Approach in Osteogenesis Imperfecta

Osteogenesis imperfecta, also known as “brittle bone disease”, is a heterogeneous disorder of connective tissue generally caused by dominant mutations in the genes COL1A1 and COL1A2, encoding the α1 and α2 chains of type I (pro)collagen. Symptomatic patients are usually prescribed bisphosphonates, but this treatment is neither curative nor sufficient. A promising field is gene silencing through RNA interference. In this study small interfering RNAs (siRNAs) were designed to target each allele of 3''UTR insertion/deletion polymorphisms (indels) in COL1A1 (rs3840870) and COL1A2 (rs3917). For both indels, the frequency of heterozygous individuals was determined to be approximately 50% in Swedish cohorts of healthy controls as well as in patients with osteogenesis imperfecta. Cultures of primary human bone derived cells were transfected with siRNAs through magnet-assisted transfection. cDNA from transfected cells was sequenced in order to measure targeted allele/non-targeted allele ratios and the overall degree of silencing was assessed by quantitative PCR. Successful allele dependent silencing was observed, with promising results for siRNAs complementary to both the insertion and non-insertion harboring alleles. In COL1A1 cDNA the indel allele ratios were shifted from 1 to 0.09 and 0.19 for the insertion and non-insertion allele respectively while the equivalent resulting ratios for COL1A2 were 0.05 and 0.01. Reductions in mRNA abundance were also demonstrated; in cells treated with siRNAs targeting the COL1A1 alleles the average COL1A1 mRNA levels were reduced 65% and 78% compared to negative control levels and in cells treated with COL1A2 siRNAs the average COL1A2 mRNA levels were decreased 26% and 49% of those observed in the corresponding negative controls. In conclusion, allele dependent silencing of collagen type I utilizing 3''UTR indels common in the general population constitutes a promising mutation independent therapeutic approach for osteogenesis imperfecta.     

Purification and characterisation of antigenic gliadins in coeliac disease.

Two gliadins, known to be especially antigenic in coeliac disease, were purified to homogeneity by a series of ion-exchange chromatography steps. Their N-terminal amino acid sequences showed minor differences but clearly classified them as gamma-type gliadins. The purified gliadins were further characterised with respect to amino acid composition, molecular mass and E1(1%)cm at 276 nm. Based on these properties it is suggested that one of them is identical to a gamma-type gliadin, earlier characterised by its nucleotide sequence, whereas the other has not previously been described. The purification procedure may form the basis for the development of a more differentiated analysis of circulating antibodies for diagnosis and makes clinical testing of the toxicity of defined gliadin peptides feasible.     

Chest x ray films from construction workers: International Labour Office (ILO 1980) classification compared with routine readings.

The extent of agreement between International Labour Office (ILO) and clinical readings of chest x ray films from construction workers was studied. From a survey of 5898 workers 258 subjects with a profusion of small opacities of > or = 1/1 and a stratified sample of subjects with profusion < 1/1 were selected. Only 41% of the films classified as ILO profusion category > or = 1/1 were clinically recorded as non-normal for the parenchyma. The proportion of films recorded as pneumoconiotic (or possibly so) was especially low for irregular opacities (22%), but increased with the profusion category (both rounded and irregular) as well as with the size of rounded opacities (p 3/11, q 12/25, r 3/4). Only with the profusion category > or = 2/1 were most of the films recorded as pneumoconiotic. The specificity and sensitivity were highest in the geographical areas where a few clinical readers had assessed many films each. The proportion of false negative clinical reports was low for circumscribed pleural thickening of the chest wall (9%) and diaphragmatic pleural thickening (6%). For calcified pleural changes and for the combination of diffuse pleural thickening and obliteration of the costophrenic angle, false negative reports were absent. The present study shows an unsatisfactory sensitivity for clinical compared with ILO readings as a means for screening the parenchyma of workers with a risk of pneumoconiosis.     

Multivariate analyses of cellular fatty acids and carbohydrates of 1:2:1 and 2:4:2 spirochetes

Delimitation of small-sized spirochetes of the oral cavity can be difficult. The endoflagella pattern has long served as the main criterion for taxonomic distinction. For approved species, e.g. Treponema denticola, several endoflagella patterns are observed, which indicates that this criterion is inadequate. The present study with GC and GC-MS used fatty acids and carbohydrates of whole-cell methanolysates to distinguish 1:2:1 and 2:4:2 subgingival spirochetes. Thirteen fatty acids: C12:0, C13:0, C14:0, Ciso-14:0, C2-OH-14:0, C15:0, Cante-15:0, C16:0, Ciso-16:0, C16:1, C18:0, C18:1, and C18:2, and three carbohydrates: rhamnose, glucose, and glucosamine, were detected. The carbohydrate contents did not differ between the two groups. While 1:2:1 spirochetes contained Ciso-14:0 and Cante-15:0 acid, 2:4:2 spirochetes did not. Also multivariate analyses of quantitative fatty acid data distinguished between these groups.     

Association of total plasma homocysteine with methylenetetrahydrofolate reductase genotypes 677C>T, 1298A>C, and 1793G>A and the corresponding haplotypes in Swedish children and adolescents

We studied 692 Swedish children and adolescents (aged 9-10 or 15-16 years, respectively), in order to evaluate the effect of the methylenetetrahydrofolate reductase (MTHFR) 677C>T, 1298A>C, and 1793G>A polymorphisms on total plasma homocysteine concentrations (tHcy). Genotyping was performed with Pyrosequencing technology. The MTHFR 677C>T polymorphism was associated with increased tHcy concentrations in both the children and the adolescents (P<0.001 for both age groups) in both genders. The effect of MTHFR 1298A>C was studied separately in subjects with the 677CC and 677CT genotypes, and the 1298C allele was found to be associated with higher tHcy levels both when children were stratified according to 677C>T genotypes, and when using haplotype analyses and diplotype reconstructions. The 1793A allele was in complete linkage disequilibrium with the 1298C allele. It was still possible to show that the 1793A allele was associated with lower tHcy levels, statistically significant in the adolescents. In conclusion, a haplotype-based approach was slightly superior in explaining the genetic interaction on tHcy plasma levels in children and adolescents than a simple genotype based approach (R2 adj 0.44 vs. 0.40). The major genetic impact on tHcy concentrations is attributable to the MTHFR 677C>T polymorphism. The common 1298A>C polymorphism had a minor elevating effect on tHcy, whereas the 1793G>A polymorphism had a lowering effect on tHcy.     

Cardiorespiratory fitness relates more strongly than physical activity to cardiovascular disease risk factors in healthy children and adolescents: the European Youth Heart Study.

BACKGROUND: Physical activity and cardiorespiratory fitness are closely related to health variables in adults, especially those considered to be among risk factors for cardiovascular diseases. The possible tracking of cardiovascular disease risk factors from childhood to adulthood makes it important to increase our understanding of the complex relationships between physical activity, cardiorespiratory fitness and cardiovascular risk factors early in life. DESIGN: A cross-sectional, school-based study on healthy children and adolescents, aged 9-10 years (295 girls, 295 boys) and 15-16 years (302 girls, 233 boys) was performed during a school year in Sweden and Estonia, as part of the European Youth Heart Study. METHODS: Total physical activity, and minutes spent in inactivity and activity of moderate or higher intensity were measured by accelerometry. A maximal ergometer bike test was used for estimation of cardiorespiratory fitness. The risk factors included blood pressure and fasting blood levels of insulin, glucose, triglycerides, total cholesterol and high-density lipoprotein cholesterol. RESULTS: Canonical correlations between physical activity and cardiorespiratory fitness versus cardiovascular disease risk factors showed significant associations in both age and sex groups (rc=0.46-0.61, P<0.0001). The cardiorespiratory fitness was found to be the strongest contributor to these relationships. In girls high values of the physical activity variables were also associated with a favourable cardiovascular profile. CONCLUSIONS: Cardiorespiratory fitness relates more strongly to cardiovascular risk factors than components of objectively measured physical activity in children and adolescents. Physical activity becomes more important in the 15-year-old adolescents, indicating that these modifiable lifestyle factors increase in importance with age.     

Ocular motor function in relation to gross motor function in congenital and childhood myotonic dystrophy type 1

Purpose: To assess ocular motor function in congenital and childhood myotonic dystrophy type 1 (DM1) and correlate the results with cytosine‐thymine‐guanine (CTG) repeat size, severity of the disease, myotonia and skeletal muscle function. Methods: A cross‐sectional investigation into strabismus, versions/ductions, saccades, smooth pursuit movements and ptosis was performed on 49 individuals with a confirmed diagnosis of DM1, all diagnosed at <18 years of age and with >40 CTG expansion repeats. The results were correlated with myotonia as well as Hammersmith motor ability scale (HMA). In addition, the ocular results were compared to results from an age and‐ sex‐matched control group. Results: Ocular motor abnormalities were seen in 82%; the most frequent findings were altered conjugate eye movements and ‘pseudoptosis’ while blepharoptosis was rare. Strabismus was most common in the severe congenital subgroup, with a frequency 14 times higher than in the control group. Positive correlations were seen between CTG repeat size and affected eyelids, and between myotonia and affected eyelids; both these findings were most prominent in the mild congenital group. CTG repeat size was also correlated with version/duction defects, and most obviously in the childhood group. Low HMA scores were associated with high occurrence of strabismus and version/duction defects. Conclusion: Abnormalities of ocular motor function are frequently present. CTG repeat size correlates positively with altered versions/ductions and eyelid pathology. Gross motor dysfunction correlates with strabismus and defect versions/ductions, and eyelid pathology indicates involvement of myotonia.     

Adipose tissue volume determination in males by computed tomography and 40K

Seventeen healthy male volunteers with weights ranging from 54 to 145 kg were examined with a Philips Tomoscan 310. The upper attenuation limit of adipose tissue was determined to be -30 HU. The lower attenuation limit was set to -190 HU. Regional and total adipose tissue volumes were calculated from the adipose tissue areas of 22 scans and from the distances between these scans. Three different mathematical formulas were used, which all gave similar results. The adipose tissue area of several trunk scans, as well as the elbow, showed very high correlations (r greater than 0.96) versus the volume determinations based on 22 scans. The visceral adipose tissue area of scan L2-L3 showed a higher correlation (r = 0.986) than any other single scan versus the visceral adipose tissue volume. Total adipose tissue volume determinations with ten selected scans correlated very closely with the results obtained from 22 scans (r = 0.997). The adipose tissue volume of the head and neck region was 1.9 +/- 1.0 per cent of the total volume. Corresponding figures for other regions were: arms 6.8 +/- 1.0 per cent, legs 29.0 +/- 7.3 per cent, subcutaneous part of the trunk 41.4 +/- 7.4 per cent and the visceral region 20.9 +/- 7.0 per cent. With greater total adipose tissue volumes the percentage of the subcutaneous adipose tissue of the trunk increased (r = 0.686; P less than 0.005). There was a very strong negative relationship between the fractional amount of adipose tissue in the legs and in the trunk (r = 0.993, P less than 0.001). The potassium contents of fat-free mass and lean body mass were deduced to be 64.7 and 71.0 mmol/kg, respectively. These calculations were based on adipose tissue volume determinations by computed tomography, on 40K measurements and on the assumption that the volume proportions of fat, water and protein in adipose tissue were 85:13.7:1.3. By using computed tomography (CT) as a standard an optimal weight (W) for height (H) index was constructed by using an iterative correlation technique. The optimal index, i.e. highest correlation and lowest error versus ATCT was found for W/H0.9.     

Visceral fat accumulation in men is positively associated with insulin, glucose, and C-peptide levels, but negatively with testosterone levels

Twenty-three healthy men (age 25 to 50 years), covering a wide range of fatness and body fat distribution, were studied. An oral glucose tolerance test was performed and adipose tissue areas were calculated from computed tomography (CT) scans made at the level of L4/L5. Visceral fat area was associated with elevated concentrations of insulin and C-peptide and with glucose intolerance before and after the oral glucose load. Concentrations of sex-hormone-binding globulin (SHBG), as well as total and free testosterone, were negatively correlated with waist/hip circumference ratio and visceral fat area and also negatively associated with increased glucose, insulin, and C-peptide concentrations. In multiple linear regression, adjusting for age, body mass index, and visceral fat area, serum concentrations of free testosterone were still negatively correlated with glucose, insulin, and C-peptide levels. Without claiming any causality in the observed associations, we conclude that, unlike in women, abdominal fat distribution, insulin, glucose, and C-peptide levels are negatively associated with serum testosterone levels in men.