Filaggrin loss-of-function mutations and incident cancer: a population-based study.

BACKGROUND: Loss-of-function mutations in the filaggrin gene (FLG) could have opposing effects on cancer risk, as mutations are associated with both 10% higher serum vitamin D levels, which may protect against cancer, and with impaired skin barrier function, which may lead to higher cancer susceptibility. OBJECTIVES: To investigate the association of the FLG genotype and cancer types in four population-based cohorts. METHODS: A total of 13,376 individuals were genotyped for FLG mutations. Information on cancer was obtained from the Danish Cancer Registry. Persons with a history of cancer at baseline were excluded from prospective analyses. RESULTS: There were 1339 incident cancers (median follow-up 11·4 years). The hazard ratios (HRs) and 95% confidence intervals (CIs) for FLG mutation carriers vs. wild types were: for any cancer (HR 0·95, 95% CI 0·78-1·16), any cancer excluding nonmelanoma skin cancer (NMSC) (HR 1·05, 95% CI 0·84-1·31), head and neck cancer (HR 1·72, 95% CI 0·71-4·15), colorectal cancer (HR 0·82, 95% CI 0·44-1·52), bronchus and lung cancer (HR 1·34, 95% CI 0·77-2·33), breast cancer (HR 0·58, 95% CI 0·30-1·14), uterine cancer (HR 0·42, 95% CI 0·06-3·10), prostate cancer (HR 1·09, 95% CI 0·61-1·94), urinary cancer (HR 1·30, 95% CI 0·51-3·29), malignant melanoma (HR 1·03, 95% CI 0·41-2·58) and NMSC (HR 0·70, 95% CI 0·47-1·05). Among participants aged over 60 years at baseline, we found statistically significant lower risks of all cancers and NMSC among FLG mutation carriers. CONCLUSIONS: The only significant associations between FLG loss-of-function mutations and cancer were in subgroup analyses.

Skin prick test reactivity to aeroallergens by filaggrin mutation status.

BACKGROUND: Studies have shown that filaggrin gene (FLG) mutations are positively associated with sensitization to aero allergens. We hypothesized that FLG mutations would also have an effect on the mean size of positive skin prick test (SPT) reactions as well as the number of positive reactions. OBJECTIVE: To investigate the effect of FLG mutations on the mean size and the number of positive SPT reactions, as well as the association with positive specific IgE. METHODS: A random sample of 3335 adults from the general population in Denmark was genotyped for the R501X and 2282del4 mutations in the FLG. SPT and specific IgE measurements to common aeroallergens were also performed. RESULTS: FLG mutations did not influence the mean size and number of positive SPT reactions. Also, no association was found between FLG mutations and specific IgE measurements. CONCLUSION: Our findings suggest that FLG mutations alone are insufficient to cause secondary sensitization to allergens. The positive association seen in patients must be explained by a combination of further barrier abnormality caused by dermatitis as well as increased allergen exposure.

Muscle velocity recovery cycles in myopathy.

OBJECTIVE: To understand the pathophysiology of myopathies by using muscle velocity recovery cycles (MVRC) and frequency ramp (RAMP) methodologies. METHODS: 42 patients with quantitative electromyography (qEMG) and biopsy or genetic verified myopathy and 42 healthy controls were examined with qEMG, MVRC and RAMP, all recorded from the anterior tibial muscle. RESULTS: There were significant differences in the motor unit potential (MUP) duration, the early and late supernormalities of the MVRC and the RAMP latencies in myopathy patients compared to controls (p < 0.05 apart from muscle relatively refractory period (MRRP)). When dividing into subgroups, the above-mentioned changes in MVRC and RAMP parameters were increased for the patients with non-inflammatory myopathy, while there were no significant changes in the group of patients with inflammatory myopathy. CONCLUSIONS: The MVRC and RAMP parameters can discriminate between healthy controls and myopathy patients, more significantly for non-inflammatory myopathy. MVRC differences with normal MRRP in myopathy differs from other conditions with membrane depolarisation. SIGNIFICANCE: MVCR and RAMP may have a potential in understanding disease pathophysiology in myopathies. The pathogenesis in non-inflammatory myopathy does not seem to be caused by a depolarisation of the resting membrane potential but rather by the change in sodium channels of the muscle membrane.

Atopic diseases by filaggrin mutations and birth year.

BACKGROUND: The prevalence of atopic disorders has increased in recent years. The pathogenesis is complex with genetic and environmental risk factors. Filaggrin loss-of-function mutations are common and associated with atopic disorders. We investigated whether the prevalence of filaggrin mutations increased in different birth cohorts in adults from the general population in Denmark. METHODS: Cross-sectional questionnaire and filaggrin gene mutation (R501X and 2282del4) data from 3335 18- to 69-year-old adults were available for analyses. RESULTS: The effect of filaggrin mutations on the prevalence of atopic diseases, albeit not statistically significant, depended mostly on birth year for atopic dermatitis (AD). A nonsignificant increase in the prevalence of filaggrin mutations was noted across birth year groups reporting AD, with 12.9% in adults born in 1936-1949 and 19.0% born in 1976-1988. CONCLUSIONS: If confirmed in other populations, the observed increase suggests that mutation carriers have been more susceptible to environmental changes accentuating the rise in AD prevalence.

Filaggrin loss-of-function mutation R501X and 2282del4 carrier status is associated with fissured skin on the hands: results from a cross-sectional population study.

BACKGROUND: Filaggrin metabolites act as osmolytes and are important for skin hydration. Carriers of filaggrin loss-of-function mutations have a higher prevalence of atopic dermatitis and dry skin. There is also evidence to suggest that filaggrin mutations increase the risk of hand eczema in atopic individuals. In our clinic, we have observed a distinct phenotype of hand eczema in patients with filaggrin mutation carrier status, characterized by fissured dermatitis on the dorsal aspect of the hands and with only sparse involvement of the palms including fine scaling. OBJECTIVES: To investigate whether filaggrin loss-of-function mutations are associated with skin fissures on the hands and/or fingers in the general population. METHODS: Participants in a population-based study were questioned about skin symptoms, genotyped for filaggrin mutation, patch tested for nickel allergy and skin prick tested. RESULTS: In an adjusted logistic regression analysis, filaggrin mutation status was significantly associated with fissured skin on the hands and/or fingers in adults (odds ratio 1·93, 95% confidence interval 1·05-3·55) and showed a nearly significant negative interaction with atopic dermatitis (P=0·055), suggesting that the effect was predominantly in subjects without atopic dermatitis. CONCLUSIONS: Filaggrin loss-of-function mutations seem not only to increase the risk of atopic dermatitis and dry skin but also the risk of fissures on the hands and/or fingers in subjects without atopic dermatitis. Prophylactic emollient therapy should be particularly encouraged in filaggrin loss-of-function mutation carriers.

The filaggrin null genotypes R501X and 2282del4 seem not to be associated with psoriasis: results from general population study and meta-analysis.

BACKGROUND: Psoriasis vulgaris could be associated with the filaggrin null genotype since certain known susceptibility loci for psoriasis are shared with susceptibility loci for atopic dermatitis. Furthermore, filaggrin expression is lowered in psoriatic skin lesions but normally expressed in uninvolved skin. So far five relatively small patient-based case-control studies have rejected a possible association between psoriasis and the two most prevalent filaggrin null mutations, 2282del4 and R501X. OBJECTIVES: To reinvestigate a possible association between psoriasis and filaggrin null mutation status by using cross-sectional general population questionnaire data. Also, to perform a meta-analysis including published studies that investigated the relation between filaggrin gene mutations R501X and 2282del4, respectively, and psoriasis vulgaris. METHODS: Between June 2006 and May 2008, a cross-sectional study was performed in the general population in Copenhagen. A random sample of 7931 subjects aged 18-69 years was invited to participate in a general health examination including a questionnaire and 3471 (43.7%) participated. A total of 3335 (96.1%) individuals were filaggrin genotyped for the 2282del4 and R501X mutations. A meta-analysis was undertaken to investigate the relation between filaggrin gene mutations and psoriasis vulgaris. RESULTS: The prevalence of self-reported psoriasis was 6.7% among the 3240 respondents. The prevalence of the R501X and 2282del4 filaggrin null genotypes was 9.3% in subjects who reported psoriasis and 8.0% in subjects who did not report psoriasis (OR = 1.28; 95% CI = 0.74-1.89; P = 0.78). The meta-analysis found no association between the filaggrin null genotypes R501X and 2282del4 and psoriasis (OR = 1.04; 95% CI = 0.81-1.35). CONCLUSIONS: Psoriasis was not associated with the R501X and 2282del4 filaggrin null genotypes in a general population study and in a meta-analysis on published studies.

Individuals who are homozygous for the 2282del4 and R501X filaggrin null mutations do not always develop dermatitis and complete long-term remission is possible.

BACKGROUND: About 8-10% of the general population in Europe carry a null mutation in the filaggrin gene which is associated with early onset of atopic dermatitis as well as persistence into adulthood. No studies have investigated whether individuals with the homozygous filaggrin null genotype always develop dermatitis. OBJECTIVES: The aim of this study was to describe the natural history of individuals with no filaggrin expression. MATERIALS: Three study populations were included: (i) a random sample of 3335 subjects aged 18-69 years from the general population in Copenhagen who underwent general health examination; (ii) a total of 499 patients seen in our dermatitis clinic since 2009 and who were filaggrin genotyped as a part of the routine diagnostic work up; and (iii) a prospective, longitudinal, birth cohort study of 411 children born to mothers with a history of asthma. Filaggrin genotyping was performed for the 2282del4 and R501X mutations. RESULTS: Filaggrin homozygous/compound heterozygous individuals accounted for 0.3% of adults, 3% of dermatitis patients and 0.7% of children. Respectively, one of nine adults and one of three children never experienced dermatitis until now. All hospital patients had atopic dermatitis with onset during (early) childhood. Year-long complete remission was observed in half of patients. CONCLUSIONS: The natural history of individuals with the filaggrin null genotype is fairly good in the sense that they may not develop dermatitis at all, and if they do, they may experience complete remission.

Association between filaggrin null mutations and concomitant atopic dermatitis and contact allergy.

BACKGROUND: The phenotypic traits of people with the filaggrin mutation (FLG) genotype and atopic dermatitis (AD) are still under elucidation, and the association with concomitant AD and contact allergy (CA) has not previously been examined. AIM: To assess FLG status in a subset of patients with AD and a minimum of one positive patch-test reaction. METHODS: In total, 430 people from a hospital population and 3335 people from the general population were tested for FLG mutations by DNA hybridization to paramagnetic polystyrene beads and analysis on a multiplex analysis system. All of the individuals in the hospital population had a minimum of one CA. AD was diagnosed according to the UK Working Party Criteria, (questions-only version). Individuals from the hospital population who had both AD and CA were considered as cases, and comparison of mutation carrier frequency was estimated (χ(2) test) against individuals without AD but with CA from the hospital population, individuals from the general population, and individuals with AD from the general population. RESULTS: The mutation frequency in patients with AD and CA in the hospital population was significantly less than that of people with AD from the general population (OR = 0.54; 95% CI 0.30-0.98). No difference in mutation frequency was found between individuals with and without AD in the hospital population (OR = 1.40; 95% CI 0.70-2.79), or between individuals with AD and CA in the hospital population and in the overall general population (OR = 1.29; 95% CI 0.76-2.20). CONCLUSIONS: The spectrum of observable traits characteristic for the FLG mutation genotype in patients with AD is at present not defined. Our results indicate that the subset of patients with both AD and CA represent a phenotype of AD that is not associated with FLG mutations.

Early detection of evolving critical illness myopathy with muscle velocity recovery cycles.

OBJECTIVE: To investigate the sensitivity of muscle velocity recovery cycles (MVRCs) for detecting altered membrane properties in critically ill patients, and to compare this to conventional nerve conduction studies (NCS) and quantitative electromyography (qEMG). METHODS: Twenty-four patients with intensive care unit acquired weakness (ICUAW) and 34 healthy subjects were prospectively recruited. In addition to NCS (median, ulnar, peroneal, tibial and sural nerves) and qEMG (biceps brachii, vastus medialis and anterior tibial muscles), MVRCs with frequency ramp were recorded from anterior tibial muscle. RESULTS: MVRC and frequency ramp parameters showed abnormal muscle fiber membrane properties with up to 100% sensitivity and specificity. qEMG showed myopathy in 15 patients (63%) while polyneuropathy was seen in 3 (13%). Decreased compound muscle action potential (CMAP) amplitude (up to 58%) and absent F-waves (up to 75%) were frequent, but long duration CMAPs were only seen in one patient with severe myopathy. CONCLUSIONS: Altered muscle fiber membrane properties can be detected in patients with ICUAW not yet fulfilling diagnostic criteria for critical illness myopathy (CIM). MVRCs may therefore serve as a tool for early detection of evolving CIM. SIGNIFICANCE: CIM is often under-recognized by intensivists, and large-scale longitudinal studies are needed to determine its incidence and pathogenesis.

Filaggrin null mutations increase the risk and persistence of hand eczema in subjects with atopic dermatitis: results from a general population study.

BACKGROUND: Hand eczema is prevalent in the general population. It remains unclear whether or not filaggrin gene (FLG) null mutations increase the overall risk of hand eczema or only increase the risk of hand eczema in subjects with atopic dermatitis. OBJECTIVES: To investigate the association between FLG null mutations and hand eczema. METHODS: A random sample of 3335 adults from the general population in Denmark was patch tested, FLG genotyped for R501X and 2282del4 null mutations and questioned about hand eczema. RESULTS: Participants with combined presence of atopic dermatitis and FLG null mutation status had a significantly higher prevalence of hand eczema, an earlier onset of hand eczema and a higher persistence of hand eczema compared with subjects with normal FLG status and absence of atopic dermatitis. Logistic regression analyses revealed positive associations between hand eczema within the past 12 months and FLG null mutation status in participants with a history of atopic dermatitis [odds ratio (OR) 2.98; 95% confidence interval (CI) 1.27-7.01], but not in subjects without atopic dermatitis (OR 0.82; 95% CI 0.41-1.67). CONCLUSIONS: FLG null mutations were significantly associated with hand eczema (< 12 months) in subjects with atopic dermatitis. Combined atopic dermatitis and filaggrin null mutation status was strongly associated with early onset of hand eczema and hand eczema persistence.

The association between null mutations in the filaggrin gene and contact sensitization to nickel and other chemicals in the general population.

BACKGROUND: It was recently shown that filaggrin gene (FLG) null mutations are positively associated with nickel sensitization. We have hypothesized that histidine-rich filaggrin proteins in the epidermis chelate nickel ions and prevent their skin penetration and exposure to Langerhans cells. Furthermore, we have proposed that the low degree of genetic predisposition to nickel sensitization found by a Danish twin study was explained by a high prevalence of ear piercing among participants resulting in 'bypassing' of the filaggrin proteins. OBJECTIVES: To investigate the association between FLG null mutations and (nickel) contact sensitization. METHODS: A random sample of 3335 adults from the general population in Denmark was patch tested and genotyped for R501X and 2282del4 in the FLG gene. RESULTS: The combined carrier frequency of FLG null mutations was 8·1%. Nickel, fragrance and contact sensitization to at least one allergen were not associated with FLG null mutations. A crude analysis on women who did not have ear piercings revealed a positive association between FLG null mutations and nickel sensitization [8·3% vs. 2·4%; odds ratio (OR) 3·71, 95% confidence interval (CI) 0·73-18·96] as well as between FLG null mutations and allergic nickel dermatitis (8·3% vs. 1·3%; OR 6·75, 95% CI 1·17-38·91). FLG mutation status and atopic dermatitis were positively associated with neomycin or ethylenediamine sensitization. CONCLUSIONS: This study suggests that FLG null mutations may be a risk factor for the development of nickel sensitization. However, ear piercing was a much stronger risk factor in our general population and we could therefore identify a positive association only in women without ear piercings. Contact sensitization to specific chemicals is related to treatment exposure.

Nuclear translocation of endonuclease G in degenerating neurons after permanent middle cerebral artery occlusion in mice.

Endonuclease G (EndoG) is a mitochondrial enzyme, known to be involved in caspase-independent cell death following translocation to the cellular nucleus. Nuclear translocation of EndoG has been observed in the ischemic area following transient occlusion of the middle cerebral artery (MCA) in mice, but not after permanent MCA occlusion. In this study we investigated the cellular and temporal expression of EndoG in infarcted cortex during the first 24 h after permanent MCA occlusion in mice, using immunohistochemistry, quantitative rt-PCR and cell specific immunoflourescence markers. EndoG translocated from the cytoplasm to the nucleus as early as 4 h and with a significant increase in the number of EndoG positive nuclei at 12 and 24 h after MCA occlusion. Nuclear translocation of EndoG was observed in degenerating NeuN positive neurons that were evenly distributed throughout the developing infarct. Translocation of EndoG was supported by unaltered EndoG mRNA levels. EndoG was neither expressed in GFAP positive astrocytes nor in CD11b positive microglia/macrophages. In contrast, CD11b positive microglia, but not infiltrating CD11b positive bone marrow-derived macrophages, were shown to express activated caspase-3. The translocation of EndoG to the nucleus of neurons in the infarct implicates EndoG in ischemic neuronal degeneration after permanent MCA occlusion in mice. Increased knowledge about EndoG involvement in ischemic neuronal cell death in mice might offer a promise to control processes involved in neuronal cell death pathways in stroke.

Microglia and macrophages express tumor necrosis factor receptor p75 following middle cerebral artery occlusion in mice.

The proinflammatory and potential neurotoxic cytokine tumor necrosis factor (TNF) is produced by activated CNS resident microglia and infiltrating blood-borne macrophages in infarct and peri-infarct areas following induction of focal cerebral ischemia. Here, we investigated the expression of the TNF receptors, TNF-p55R and TNF-p75R, from 1 to 10 days following permanent occlusion of the middle cerebral artery in mice. Using quantitative polymerase chain reaction (PCR), we observed that the relative level of TNF-p55R mRNA was significantly increased at 1-2 days and TNF-p75R mRNA was significantly increased at 1-10 days following arterial occlusion, reaching peak values at 5 days, when microglial-macrophage CD11b mRNA expression was also increased. In comparison, the relative level of TNF mRNA was significantly increased from 1 to 5 days, with peak levels 1 day after arterial occlusion. In situ hybridization revealed mRNA expression of both receptors in predominantly microglial- and macrophage-like cells in the peri-infarct and subsequently in the infarct, and being most marked from 1 to 5 days. Using green fluorescent protein-bone marrow chimeric mice, we confirmed that TNF-p75R was expressed in resident microglia and blood-borne macrophages located in the peri-infarct and infarct 1 and 5 days after arterial occlusion, which was supported by Western blotting. The data show that increased expression of the TNF-p75 receptor following induction of focal cerebral ischemia in mice can be attributed to expression in activated microglial cells and blood-borne macrophages.

Improved hybridisation potential of oligonucleotides comprising O-methylated anhydrohexitol nucleoside congeners.

The hybridising potential of anhydrohexitol nucleoside analogues (HNAs) is well documented, but tedious synthesis of the monomers hampers their development. In a search for better analogues, the synthesis of two new methylated anhydrohexitol congeners 1 and 2 was accomplished and the physico-chemical properties of their respective oligomers were evaluated. Generally, oligonucleotides (ONs) containing the 3'-O-methyl derivative 1 showed a small increase in thermal stability towards complementary sequences as compared to HNA. Compared to the altritol modification, 3'-O-methylation seems to cause a small decrease in thermal stability of duplexes, especially when targeting RNA. These results suggest the possibility of derivatisation of the 3'-hydroxyl group of altritol-containing congeners without significantly affecting the thermal stability of the duplexes. The methyl glycosidic analogues 2 likewise increased the affinity for RNA in comparison with well-known HNA, while at the same time being economically more favorable monomers. However, homopolymers of 2 displayed self-pairing, but not so homopolymers of 1. Upon incorporation of the hexitols within RNA sequences in an effort to induce a beneficial pre-organised structure, the positive effect of the 3'-O-methyl derivative 1 proved larger than that of 2.

A quantitative in situ hybridization and polymerase chain reaction study of microglial-macrophage expression of interleukin-1beta mRNA following permanent middle cerebral artery occlusion in mice.

Interleukin-1beta (IL-1beta) is known to play a central role in ischemia-induced brain damage in rodents. In comparison to the rat, however, the available data on the cellular synthesis of IL-1beta mRNA and protein in the mouse are very limited. Here, we report on the time profile, the topography and the quantitative, cellular expression of IL-1beta mRNA in mice subjected to permanent occlusion of the distal middle cerebral artery (MCA). The in situ hybridization analysis showed that IL-1beta mRNA was expressed during the first post-surgical hour in a small number of high-expressing macrophage-like cells, located in cortical layers I and II of the future infarct. At 2 h, a significant number of faintly labeled IL-1beta mRNA-expressing cells had appeared in the developing peri-infarct, and the number remained constant at 4 h and 6 h, when the hybridization signal began to distribute to the cellular processes. Quantitative PCR performed on whole hemispheres showed a significant 20-fold increase in the relative level of IL-1beta mRNA at 12 h and a highly significant 42-fold increase at 24 h, at which time single IL-1beta mRNA-expressing cells were supplemented by aggregates and perivascular infiltrates of intensely labeled IL-1beta mRNA-expressing cells. Immunohistochemistry and double immunohistochemical stainings in addition to combined in situ hybridization, confirmed that the intensely labeled IL-1beta mRNA-expressing and IL-1beta protein synthesizing cells predominantly were glial fibrillary acidic protein-immunonegative, macrophage associated antigen-1-immunopositive microglia-macrophages. By day 5 there was a dramatic decline in the relative level of IL-1beta mRNA in the ischemic hemisphere. In summary, the data provide evidence that permanent occlusion of the distal MCA in mice results in expression of IL-1beta mRNA and IL-1beta synthesis in spatially and temporally segregated subpopulations of microglia and macrophages.

Polymorphism of exon 3 of the HLA-G gene.

HLA-G is a non-classical MHC class I gene with a limited tissue distribution. The most pronounced expression is detected in the cytotrophoblast of first trimester placenta. It is possible to detect mRNA for HLA-G in preimplantation blastocysts where expression is correlated with a high cleavage rate of embryos. HLA-G seems to play an important role in the feto-maternal relationship. The polymorphism of the HLA-G locus is not fully clarified. One study has shown extensive nucleotide sequence variation in the exon 3 (alpha-2 domain) in healthy African Americans. A few studies in other populations have only revealed a limited polymorphism. We investigated the polymorphism of the exon 3 of HLA-G by means of Polymerase Chain Reaction (PCR)-Single Strand Conformation Polymorphism (SSCP)- and DNA sequencing analysis in a Danish population. We detected four single-base substitutions in exon 3 compared to the sequence of HLA-6.0 (G*01011); one of these has not been reported before. We also found a deletion of the first base of codon 130 or the third of codon 129 in a heterozygous individual. This study, together with previous results, suggests that the polymorphism of exon 3 of the HLA-G gene in Caucasians is limited, in contrast to that observed in Americans originating from Africa. Implications of this discrepancy and the detected deletion in relation to certain disorders of pregnancy are discussed.

Increased RNA affinity of HNA analogues by introducing alkoxy substituents at the C-1 or C-3 position.

1,5-Anhydrohexitol nucleoside congeners with alkoxy substituents, were prepared, resulting in a further improvement of their RNA affinity and antisense potential.

Non-invasive method for sampling and extraction of mouse DNA for PCR.

We adapted a non-invasive, fast, reliable and inexpensive procedure for the sampling and extraction of deoxyribonucleic acid (DNA) for genetic testing of mice. The procedure is based on a simple DNA extraction procedure used in the forensic genetic testing of humans. It involves mouth swabbing of the inner cheek using a cotton stick, followed by alkaline lysis of the harvested buccal epithelial cells. This procedure allows for repeated sampling and genetic testing of the individual mouse, and it is faster, simpler and, in our hands, more reliable than the currently used routine procedures for the sampling and extraction of mouse DNA. Current procedures all involve biopsy of a piece of the tail, ear or toe, followed by lengthy procedures to release and isolate the DNA.