CARD15 single nucleotide polymorphisms 8, 12 and 13 are not increased in ethnic Danes with sarcoidosis.

BACKGROUND: Mutations of the caspase-activating recruitment domain 15 (CARD15) gene on chromosome 16 are associated with chronic inflammatory granulomatous bowel disease (Crohn's disease). Sarcoidosis is a systemic granulomatous disease with unknown etiology, which shares histological features with Crohn's disease. OBJECTIVES: To evaluate whether ethnic Danes with sarcoidosis have an increased frequency of CARD15 mutations compared to healthy control subjects. METHODS: Genotyping for CARD15 mutations R702W, G908R, and L1007fsinsC, also designated single nucleotide polymorphism (SNP) SNP8, SNP12 and SNP13, respectively, were performed by capillary electrophoresis single-strand confirmation polymorphism in 53 patients with histologically verified sarcoidosis and in 103 healthy controls. RESULTS: The frequencies of CARD15 mutations in sarcoidosis patients were: SNP8, 4/106 chromosomes (3.8%); SNP12, 2/106 chromosomes (1.9%); SNP13, 2/106 chromosomes (1.9%); SNP8+SNP12+SNP13, 8/106 chromosomes (7.6%). All 8 patients were heterozygous. The frequencies in controls were: SNP8, 9/206 chromosomes (4.4%); SNP12, 2/206 chromosomes (1.0%); SNP13, 4/206 chromosomes (1.9%); SNP8+SNP12+SNP13, 15/206 chromosomes (7.3%). All controls were heterozygous. The differences were not statistically significant (p>0.05). Furthermore, the course of disease was not significantly different in the 8 patients with CARD15 mutations and the 45 patients without mutations. CONCLUSION: The frequency of CARD15 mutations is not increased in ethnic Danish patients with sarcoidosis, and heterozygosity for such mutations apparently has no influence on the course of disease.

Tension-type headache in adolescents and adults: a population based study of 33,764 twins.

The aim of this study was to evaluate the 1-year-period prevalence of tension-type headache in a large population based sample. The study population included 33,764 twins aged 12-41 years old from the population based new Danish Twin Registry. They received a posted headache questionnaire and the response rate was 83.5%. The self-reported 1-year-period prevalence of tension-type headache was 86.0%; 78.9% among men and 92.5% among women. The 1-year-period prevalence of infrequent episodic, frequent episodic and chronic tension-type headache was 63.5, 21.6 and 0.9%, respectively. Frequent episodic and chronic tension-type headache was significantly more frequent in women than men. The prevalence of frequent episodic tension-type headache increased slightly in men until age 39 then it declined, while it increased about 20% point in women from age 12 years to age 20-39 years old and then it declined. Congruently, the prevalence of chronic tension-type headache increased until age 39 and declined thereafter in both sexes. Chronic tension-type headache is rare in persons 12-14 years old. These effects were confirmed by age trends of the different subtypes of tension-type headache using a regression model. The prevalence of migraine varied from 7.0 to 16.8% in men and from 8.2 to 31.0% in women. It increased from age 12 to 34 years and then declined. The risk and frequency of tension-type headache was significantly higher in those with migraine than those who had never had migraine. Future large longitudinal follow-up studies are required.

Cellular mitochondrial heterogeneity in cultured astrocytes as demonstrated by immunogold labeling of alpha-ketoglutarate dehydrogenase.

In brain cells, various metabolites and metabolic pathways, largely of mitochondrial origin, have been shown to be compartmentalized. Attention has therefore been focused on the possible existence of mitochondrial heterogeneity in the brain at the cellular level. To determine whether mitochondria in cultured cortical and cerebellar astrocytes are heterogeneous at the single cell level, immunogold electron microscopy and an antibody against the alpha-ketoglutarate dehydrogenase component of the alpha-ketoglutarate dehydrogenase complex, a marker enzyme for the tricarboxylic acid (TCA) cycle, were employed. The number of gold particles was counted in the mitochondria of 36 and 42 cells from cultured cerebellar and cortical astrocytes, respectively. A test for random distribution (Poisson distribution) of mitochondria according to the number of gold particles was subsequently performed for every one of the 36 and 42 cells as the ratio variance/mean (= index of dispersion). This should be approximately distributed as chi2/degrees of freedom (df) = n - 1, n = number of mitochondria), if the observations obeyed a Poisson distribution. For 26 of the 36 (cerebellar astrocytes) distributions and for 28 of the 42 (cortical astrocytes) distributions a random distribution had to be rejected. These findings therefore strongly indicate that alpha-ketoglutarate dehydrogenase is heterogeneously distributed in mitochondria within individual astrocytes originating either from cerebellum or cerebral cortex. In conclusion, this study underlines the probability that mitochondrial heterogeneity at the single cell level might be extended to involve other metabolic pathways and metabolites.

Frequency of the hemochromatosis HFE mutations C282Y, H63D, and S65C in blood donors in the Faroe Islands.

The aim of the study was to assess the frequencies of the hereditary hemochromatosis HFE mutations C282Y, H63D, and S65C in the population in the Faroe Islands. The series comprised 200 randomly selected blood donors of Faroese heritage. The frequency of the C282Y, H63D, and S65C mutations on the HFE gene was assessed by genotyping using the polymerase chain reaction (PCR) technique and calculated from direct allele counting. We found no C282Y homozygous subjects; 28 (14.0%) subjects were C282Y heterozygous and four subjects were C282Y/H63D compound heterozygous (2.0%). The C282Y allele frequency was 8.0% (95% CI 5.3-10.7%). The series contained three (1.5%) H63D homozygous subjects and 60 (30.0%) H63D heterozygous subjects. The H63D allele frequency was 17.5% (95% CI 13.8-21.2%). There were four (2.0%) S65C heterozygous subjects. The S65C allele frequency was 1.0% (95% CI 0.3-2.5%). The frequency of the C282Y mutation is high in Faroese blood donors, being close to and not significantly different from the frequencies reported in other Scandinavian countries: Denmark 5.7%, Norway 6.6%, Iceland 5.1%, and Sweden 6.1%. The frequency of the H63D mutation in Faroese subjects is significantly higher than the frequency in Denmark 12.8% (p=0.007), Iceland 10.9% (p=0.003), and Sweden 12.4% (p=0.015), but not from the frequency in Norway 11.2% (p=0.063). The frequency of the S65C mutation in Faroese subjects is not significantly different from the frequencies in Denmark 1.5% and Sweden 1.6%. Screening of larger groups of the Faroese population for HFE mutations especially C282Y should be considered in order to establish the penetrance.

Frequency of the C282Y and H63D mutations of the hemochromatosis gene (HFE) in 2501 ethnic Danes.

The aim of the study was to assess the frequency of the C282Y and H63D mutations of the hemochromatosis gene (HFE) in ethnic Danes. The series comprised 2501 subjects (1284 men) of Danish heritage who were drawn at random from the Census Registry in age cohorts of 30, 40, 50, and 60 years. The frequency of the C282Y and H63D mutations was assessed on blood samples by genotyping using a polymerase chain reaction (PCR) technique. The HFE genotype distribution was in Hardy-Weinberg equilibrium (p=0.85). C282Y mutation: 9 subjects (0.36%) were homozygous and 265 subjects (10.6%) were heterozygous. H63D mutation: 40 subjects (1.6%) were homozygous and 584 subjects (23.4%) were heterozygous. C282Y/H63D compound heterozygosity was found in 36 subjects (1.4%). The C282Y allele frequency was 5.7% [95% confidence interval (CI) 5.0-6.3%] and the H63D allele frequency was 13.3% (95% CI 12.3-14.2%). In conclusion, the C282Y frequency is relatively high in the Danes, being close to the frequency in other Scandinavian countries, i.e., Iceland 5.1%, the Faroe Islands 6.6%, and Sweden 5.7%, but significantly lower than in Norway 6.6% (p=0.02). Also, the H63D frequency in Danes is close to and not significantly different from the frequency in Iceland 10.9%, Norway 11.2%, and Sweden 12.4%, but significantly lower than in the Faroe Islands 15.4% (p=0.046).

Antisense downregulation of mutant huntingtin in a cell model.

BACKGROUND: Huntington's disease (HD) is an inherited neurodegenerative disorder which is caused by an expansion of a CAG repeat sequence in the HD gene. The repeat encodes an expanded polyglutamine tract in the protein huntingtin. The still unknown pathological mechanisms leading to death of specific neurons in the brains of HD patients correlate with the expression of mutant huntingtin. Therefore, we have studied whether mutant huntingtin expression can be downregulated by antisense technique. METHODS: NT2 precursor cells and differentiated postmitotic NT2-N neurons, respectively, were transfected with plasmid constructs containing exon 1 of the HD gene with expanded CAG repeats in frame with the reporter protein EGFP. The transfected cell cultures were treated with a phosphorothioated antisense oligonucleotide (PS-ASHD/20+) or a control oligonucleotide either by cotransfection or by addition to the culture medium. RESULTS: Expression of the fusion protein containing the mutant huntingtin fragment resulted in diffuse green fluorescence in the cytoplasm and formation of aggregates in some of the NT2 cells and NT2-N neurons. We obtained antisense sequence-specific inhibition of expression of the fusion protein and/or suppression of the aggregate formation in both cell types. In the NT2 cells the antisense effect was dependent on the way of administration of the oligo. CONCLUSIONS: The PS-antisense oligo is effective in downregulation of mutant huntingtin, and the reduction of aggregate formation is a sensitive biological marker. The findings suggest that antisense knockdown of huntingtin could be a useful strategy for treatment of HD, and could also be suitable for studies of the normal and pathological function of huntingtin in different cellular model systems.

Converging signals synergistically activate the LAMC2 promoter and lead to accumulation of the laminin gamma 2 chain in human colon carcinoma cells.

The trimeric extracellular matrix molecule laminin-5 and its constituent chains (alpha 3, beta 3, gamma 2) are normally not detectable intracellularly in intestinal epithelial cells but the laminin gamma 2 chain can be detected in cancer cells at the invasive front of a subset of colon carcinomas. These cells are subjected to cytokines such as transforming growth factor beta 1 (TGF-beta 1) and hepatocyte growth factor (HGF), produced by the tumour cells or by the surrounding stromal cells. The purpose of the present work was to investigate whether TGF-beta 1 and HGF, known to stimulate the LAMC2 gene encoding the laminin gamma 2 chain, might synergize to activate the LAMC2 promoter, and to identify the promoter elements involved. We find evidence for synergy between TGF-beta and HGF with respect to laminin gamma 2 chain expression and promoter activation and demonstrate that this requires the 5' activator protein-1 (AP-1) element of the promoter and an additional upstream element which is also responsive to co-expression of the Smad3 protein from the TGF-beta signalling pathway. The transcripts encoding the other laminin-5 chains are not synergistically activated by HGF and TGF-beta. Thus the synergistic activation of the LAMC2 gene is mediated via different cis-elements and results in an overproduction of the laminin gamma 2 chain relative to the other laminin-5 constituent chains. This difference may explain why laminin gamma 2 chains accumulate in the cells at the invasive front of colon carcinomas.