Mutations in desmoglein 1 cause diverse inherited palmoplantar keratoderma phenotypes: implications for genetic screening.

The inherited palmoplantar keratodermas (PPKs) are a heterogeneous group of genodermatoses, characterized by thickening of the epidermis of the palms and soles. No classification system satisfactorily unites clinical presentation, pathology and molecular pathogenesis. There are four patterns of hyperkeratosis - striate, focal, diffuse and punctate. Mutations in the desmoglein 1 gene (DSG1), a transmembrane glycoprotein, have been reported primarily in striate, but also in focal and diffuse PPKs. We report seven unrelated pedigrees with dominantly inherited PPK owing to mutations in the DSG1 gene, with marked phenotypic variation. Genomic DNA from each family was isolated, and individual exons amplified by polymerase chain reaction. Sanger sequencing was employed to identify mutations. Mutation analysis identified novel mutations in five families (p.Tyr126Hisfs*2, p.Ser521Tyrfs*2, p.Trp3*, p.Asp591Phefs*9 and p.Met249Ilefs*6) with striate palmar involvement and varying focal or diffuse plantar disease, and the recurrent mutation c.76C>T, p.Arg26*, in two families with variable PPK patterns. We report one recurrent and five novel DSG1 mutations, causing varying patterns of PPK, highlighting the clinical heterogeneity arising from mutations in this gene.

Loss-of-function mutations in the gene encoding filaggrin underlie a Japanese family with food-dependent exercise-induced anaphylaxis.

BACKGROUND: Food-dependent exercise-induced anaphylaxis (FDEIA) is a serious food allergy in which anaphylaxis develops when exercise is performed within several hours after food intake. The precise mechanism underlying allergic sensitization in FDEIA has been an important issue but remains poorly understood. OBJECTIVES: We aimed to elucidate the pathomechanism including the route of allergen sensitization involved in FDEIA. METHODS: A Japanese family with wheat-dependent exercise-induced anaphylaxis (WDEIA), a specific form of FDEIA, were clinically examined. Mutation analysis of the gene encoding filaggrin (FLG) was also performed. RESULTS: Two of the family members were confirmed as WDEIA on the basis of their medical history and positive provocation test results. Notably, the two affected individuals in the family had concomitant ichthyosis vulgaris. Mutation analysis of FLG revealed that they carry one or more loss-of-function mutations that have not been described in the Japanese population. CONCLUSION: These results indicate that FLG mutations might be involved in the pathogenesis of WDEIA in the present case.

Safety and efficacy of multipolar pulmonary vein ablation catheter vs. irrigated radiofrequency ablation for paroxysmal atrial fibrillation: a randomized multicentre trial.

AIMS: The current challenge in atrial fibrillation (AF) treatment is to develop effective, efficient, and safe ablation strategies. This randomized controlled trial assesses the medium-term efficacy of duty-cycled radiofrequency ablation via the circular pulmonary vein ablation catheter (PVAC) vs. conventional electro-anatomically guided wide-area circumferential ablation (WACA). METHODS AND RESULTS: One hundred and eighty-eight patients (mean age 62 ± 12 years, 116 M : 72 F) with paroxysmal AF were prospectively randomized to PVAC or WACA strategies and sequentially followed for 12 months. The primary endpoint was freedom from symptomatic or documented >30 s AF off medications for 7 days at 12 months post-procedure. One hundred and eighty-three patients completed 12 m follow-up. Ninety-four patients underwent PVAC PV isolation with 372 of 376 pulmonary veins (PVs) successfully isolated and all PVs isolated in 92 WACA patients. Three WACA and no PVAC patients developed tamponade. Fifty-six percent of WACA and 60% of PVAC patients were free of AF at 12 months post-procedure (P = ns) with a significant attrition rate from 77 to 78%, respectively, at 6 months. The mean procedure (140 ± 43 vs. 167 ± 42 min, P<0.0001), fluoroscopy (35 ± 16 vs. 42 ± 20 min, P<0.05) times were significantly shorter for PVAC than for WACA. Two patients developed strokes within 72 h of the procedure in the PVAC group, one possibly related directly to PVAC ablation in a high-risk patient and none in the WACA group (P = ns). Two of the 47 patients in the PVAC group who underwent repeat ablation had sub-clinical mild PV stenoses of 25-50% and 1 WACA patient developed delayed severe PV stenosis requiring venoplasty. CONCLUSION: The pulmonary vein ablation catheter is equivalent in efficacy to WACA with reduced procedural and fluoroscopy times. However, there is a risk of thrombo-embolic and pulmonary stenosis complications which needs to be addressed and prospectively monitored. CLINICALTRIALSGOV IDENTIFIER: NCT00678340.

Novel filaggrin mutation but no other loss-of-function variants found in Ethiopian patients with atopic dermatitis.

BACKGROUND: Filaggrin is a key protein involved in maintaining skin barrier function and hydration. Mutations in the filaggrin gene (FLG) cause ichthyosis vulgaris (IV) and are a major predisposing factor for atopic dermatitis (AD) in individuals of European and Asian descent. It has been proposed that FLG mutations are population specific and a difference in the spectra of mutations between different ancestral groups has been described. However, it is unknown whether FLG mutations in the African population are a causative genetic factor for IV and predispose to AD, or whether other mechanisms are more prominent. OBJECTIVES: The present aim was to investigate the role of FLG mutations as predisposing factors for IV or AD among individuals from Ethiopia. METHODS: A case series of Ethiopian patients with AD (n = 103) and IV (n = 7) together with controls (n = 103; subjects without past or present history of AD, dry skin or atopic manifestations) was collected at the outpatient dermatology clinics at ALERT Dermatology Hospital, Tikur Anbessa Hospital and Gondar University Hospital, Ethiopia. AD was diagnosed by a dermatologist using the U.K. Working Party's diagnostic criteria. The IV diagnosis was based on clinical examination and genetic testing of the steroid sulphatase gene to exclude X-linked recessive ichthyosis. Patients were studied with direct sequencing (n = 40) and/or allelic discrimination (n = 110). Immunohistochemical analysis was performed for filaggrin expression in the skin of patients (n = 7) and controls (n = 2). RESULTS: The Ethiopian patients and controls were genotyped for the four previously described common European FLG null mutations (R501X, 2282del4, S3247X, R2447X) and no carriers were found. In one patient with AD a novel heterozygous 2-bp deletion, 632del2, leading to a premature stop codon was revealed by direct sequencing. No additional carrier of this deletion or other mutations was found. In addition, no difference in filaggrin expression was detected in AD or IV skin compared with healthy control skin. CONCLUSIONS: Our results indicate that FLG loss-of-function-variants are less common in patients with IV and AD in the Ethiopian population, suggesting that other factors may be of importance in the pathogenesis in this ethnic group.

Wide spectrum of filaggrin-null mutations in atopic dermatitis highlights differences between Singaporean Chinese and European populations.

BACKGROUND: Null mutations in the filaggrin gene (FLG) cause ichthyosis vulgaris (IV) and predispose to atopic dermatitis (AD). Cohort studies in Europe and Japan have reported an FLG mutation carrier frequency of between 14% and 56%, but the prevalent European FLG mutations are rare or absent in Chinese patients with IV and AD. OBJECTIVES: To investigate further the spectrum of FLG-null mutations in Chinese patients and to compare it with that in other populations. METHODS: We conducted comprehensive FLG genetic analysis in a discovery cohort of 92 Singaporean Chinese individuals with IV and/or moderate-to-severe AD. All detected FLG mutations were then screened in a cohort of 425 patients with AD and 440 normal controls. Results In total, 22 FLG-null mutations, of which 14 are novel, were identified in this study; the combined null FLG genotype of 17 mutations detected in cases and controls showed strong association with AD [Fisher's exact test; P = 5·3 × 10⁻9; odds ratio (OR) 3·3], palmar hyperlinearity (Fisher's exact test; P = 9·0 × 10⁻¹5; OR 5·8), keratosis pilaris (Fisher's exact test; P = 0·001; OR 4·7) and with increased severity of AD (permutation test; P = 0·0063). CONCLUSIONS: This study emphasizes the wider genetic landscape of FLG-null mutations in Asia that is slowly emerging.

Levels of filaggrin degradation products are influenced by both filaggrin genotype and atopic dermatitis severity.

BACKGROUND: Filaggrin, coded by FLG, is the main source of several major components of natural moisturizing factor (NMF) in the stratum corneum (SC), including pyrrolidone carboxylic acid (PCA) and urocanic acid (UCA). Loss-offunction mutations in FLG lead to reduced levels of filaggrin degradation products in the SC. It has recently been suggested that expression of filaggrin may additionally be influenced by the atopic inflammatory response. In this study, we investigated the levels of several breakdown products of filaggrin in the SC in healthy controls (CTRL) and patients with atopic dermatitis (AD) in relation to FLG null allele status. We examined the relationship between NMF (defined here as the sum of PCA and UCA) and AD severity. METHODS: The SC levels of filaggrin degradation products including PCA, UCA, histidine (HIS) and tyrosine were determined in 24 CTRL and 96 patients with moderate-to-severe AD. All subjects were screened for 11 FLG mutations relevant for the study population. RESULTS: The levels of PCA, UCA and HIS correlated with FLG genotype. Furthermore, these levels were higher in the CTRL when compared to AD patients with no FLG mutations. Multiple regression analysis showed that NMF levels were independently associated with FLG genotype and severity of disease. CONCLUSION: Decreased NMF is a global feature of moderate-to-severe AD; within AD, FLG genotype is the major determinant of NMF, with disease severity as a secondary modifier. NMF components are reliably determined by a noninvasive and relatively inexpensive tape stripping technique.

FLG mutation p.Lys4021X in the C-terminal imperfect filaggrin repeat in Japanese patients with atopic eczema.

BACKGROUND: Mutations in the gene encoding filaggrin (FLG) have been shown to predispose to atopic eczema (AE). OBJECTIVES: Further to establish population genetics of FLG mutations in the Japanese population and to elucidate effects of FLG mutations to filaggrin biosynthesis in skin of patients with AE. METHODS: We searched for FLG mutations in 19 newly recruited Japanese patients with AE. We then screened 137 Japanese patients with AE and 134 Japanese control individuals for a novel mutation identified in the present study. In addition, we evaluated FLG mRNA expression by real-time reverse transcription-polymerase chain reaction and profilaggrin/filaggrin protein expression by immunohistochemical staining in the epidermis of the patients carrying the novel mutation. RESULTS: We identified a novel FLG nonsense mutation c.12069A>T (p.Lys4021X) in one patient with AE. Upon further screening, p.Lys4021X was identified in four patients with AE (2.9% of all the patients with AE). In total, there are at least eight FLG variants in the Japanese population. Here we show that about 27% of patients in our Japanese AE case series carry one or more of these eight FLG mutations and these variants are also carried by 3.7% of Japanese general control individuals. There is a significant statistical association between the eight FLG mutations and AE (chi(2) P = 6.50 x 10(-8)). Interestingly, the present nonsense mutation is in the C-terminal incomplete filaggrin repeat and is the mutation nearest the C-terminal among previously reported FLG mutations. Immunohistochemical staining for filaggrin revealed that this nonsense mutation leads to remarkable reduction of filaggrin protein expression in the patients' epidermis. CONCLUSIONS: We clearly demonstrated that FLG mutations are significantly associated with AE in the Japanese population. The present results further support the hypothesis that the C-terminal region is essential for proper processing of profilaggrin to filaggrin.

Filaggrin haploinsufficiency is highly penetrant and is associated with increased severity of eczema: further delineation of the skin phenotype in a prospective epidemiological study of 792 school children.

BACKGROUND: Null mutations within the filaggrin gene (FLG) cause ichthyosis vulgaris and are associated with atopic eczema. However, the dermatological features of filaggrin haploinsufficiency have not been clearly defined. OBJECTIVES: This study investigated the genotype-phenotype association between detailed skin phenotype and FLG genotype data in a population-based cohort of children. METHODS: Children (n = 792) aged 7-9 years were examined by a dermatologist. Features of ichthyosis vulgaris, atopic eczema and xerosis were recorded and eczema severity graded using the Three Item Severity score. Each child was genotyped for the six most prevalent FLG null mutations (R501X, 2282del4, R2447X, S3247X, 3702delG, 3673delC). Fisher's exact test was used to compare genotype frequencies in phenotype groups; logistic regression analysis was used to estimate odds ratios and penetrance of the FLG null genotype and a permutation test performed to investigate eczema severity in different genotype groups. RESULTS: Ten children in this cohort had ichthyosis vulgaris, of whom five had mild-moderate eczema. The penetrance of FLG null mutations with respect to flexural eczema was 55.6% in individuals with two mutations, 16.3% in individuals with one mutation and 14.2% in wild-type individuals. Summating skin features known to be associated with FLG null mutations (ichthyosis, keratosis pilaris, palmar hyperlinearity and flexural eczema) showed a penetrance of 100% in children with two FLG mutations, 87.8% in children with one FLG mutation and 46.5% in wild-type individuals (P < 0.0001, Fisher exact test). FLG null mutations were associated with more severe eczema (P = 0.0042) but the mean difference was only 1-2 points in severity score. Three distinct patterns of palmar hyperlinearity were observed and these are reported for the first time. CONCLUSIONS: Filaggrin haploinsufficiency appears to be highly penetrant when all relevant skin features are included in the analysis. FLG null mutations are associated with more severe eczema, but the effect size is small in a population setting.

Analysis of Taiwanese ichthyosis vulgaris families further demonstrates differences in FLG mutations between European and Asian populations.

BACKGROUND: Mutations in the gene encoding filaggrin (FLG) were identified to underlie ichthyosis vulgaris (IV) and also shown to predispose to atopic eczema. Until now, no FLG mutations have been described in the Taiwanese population. OBJECTIVES: To elucidate filaggrin mutations in the Taiwanese population and further to clarify the population genetics of filaggrin gene mutations in the Asian populations. METHODS: In the present study, 12 individuals from four unrelated Taiwanese IV families were examined for FLG mutations. We carried out comprehensive sequencing of the entire FLG coding region using an overlapping polymerase chain reaction strategy. RESULTS: We identified three FLG mutations in the Taiwanese IV families. One mutation E1795X was a previously unidentified FLG mutation, which might be specific to the Taiwanese. Interestingly, another FLG mutation 3321delA is prevalent in the Japanese population and the other mutation Q2417X was found in the Singaporean Chinese population. No FLG mutation identified in the white European population was found in the Taiwanese population. CONCLUSIONS: The present findings suggest that the Taiwanese population, as an East Asian group, share FLG mutations with both the Japanese and the Singaporean Chinese population. In addition, these results exemplify differences in the population genetics of filaggrin between Europe and Asia.

Ichthyosis vulgaris: novel FLG mutations in the German population and high presence of CD1a+ cells in the epidermis of the atopic subgroup.

BACKGROUND: Ichthyosis vulgaris (IV) is a genetic disorder with a prevalence of 1:250-1000 caused by filaggrin (FLG) mutations, which also predispose to atopic diseases. OBJECTIVES: To study the genotype/phenotype relationship in IV and to analyse whether the suggested skin barrier defect is associated with differences of epidermal dendritic cells. PATIENTS/METHODS: We evaluated a cohort of 26 German patients with IV, established an IV severity score and analysed epidermal ultrastructure, histology, filaggrin and CD1a antigens. Mutations were screened by restriction enzyme analysis. Particular sequencing techniques allowed the complete FLG analysis to reveal novel mutations. RESULTS: The combined null allele frequency of R501X and 2282del4 was 67.3%. Patients also showed the mutations S3247X and R2447X as well as five novel FLG mutations: 424del17 and 621del4 (profilaggrin S100 domain), 2974delGA (repeat 2), R3766X (repeat 10(1)) and E4265X (repeat 10(2)). Their combined allele frequency in controls was <0.7%. No mutation was found in one IV patient, all in all approximately 27% were heterozygous, and the majority (approximately 69%) showed two null alleles. The IV severity score and ultrastructure showed a significant correlation with genotypes. Interestingly, CD1a cell counts showed a significant difference between nonatopic and atopic IV patients both with eczema and without eczema. CONCLUSIONS: We confirm that the mutations R501X and 2282del4 represent the most frequent genetic cause in German IV patients. The novel mutations are probably population and family specific. The observed differences of CD1a cells support the hypothesis that there is a barrier defect that predisposes to atopic manifestations, possibly independent of atopic eczema.

The functional significance of genetic variation within the beta-adrenoceptor.

The beta-1 adrenoceptor is an archetypal G-coupled protein receptor that controls sympathetic responses in the heart, kidney and adipocytes. It has been widely exploited as a drug target with the development of antagonists to treat cardiovascular diseases such as hypertension, angina and heart failure. Signalling through the receptor is modulated by desensitization and beta1- adrenoceptor down-regulation. It is also affected by in vitro substitution of specific amino acid residues within the beta-1 adrenoceptor. Amino acid substitutions also occur naturally due to polymorphic variation within the human beta-1 adrenoceptor gene itself. Since these variants are common (typically being present in > 5% of the population), the pharmacogenetic implications are enormous. A number of these variants have been identified, although two have been the particular focus of recent publications: a serine to glycine substitution at position 49 (49S > G) and an arginine to glycine at position 389 (389R > G). The data on the in vitro behaviour of these two receptor variants is reviewed here, along with the evidence that they may affect both the risk of cardiovascular disease and the therapeutic response to beta-1 adrenoceptor antagonists.

Greater inotropic and cyclic AMP responses evoked by noradrenaline through Arg389 beta 1-adrenoceptors versus Gly389 beta 1-adrenoceptors in isolated human atrial myocardium.

1. We studied the biochemical and contractile responses of isolated human myocardial tissue expressing native receptor variants of the 389G>R beta(1)-adrenoceptor polymorphism. 2. Right atrial appendage was obtained from homozygous RR patients (n=37) and homozygous GG patients (n=17) undergoing elective cardiac surgery. The positive inotropic effect of noradrenaline in these tissues, mediated through beta(1)-adrenoceptors, was studied using electrically stimulated (1 Hz) atrial strips, as well as the effects of noradrenaline on cyclic AMP levels and cyclic AMP-dependent protein kinase. 3. Tissue from RR homozygotes (n=14) showed significantly increased inotropic potency to noradrenaline (-log EC(50), M=6.92+/-0.12) compared to GG homozygotes (n=8, -log EC(50), M=6.36+/-0.11, P<0.005). This difference was not dependent on tissue basal force. 4. Tissue cyclic AMP levels (pmol mg(-1)) were also greater in RR homozygotes (basal 34.8+/-3.7 n=12, 300 nM noradrenaline 41.4+/-7.6 n=9, 30 micro M noradrenaline 45.2+/-3.2 n=22, 0.2 mM isoprenaline 48.3+/-4.2 n=16) compared to GG homozygotes (basal 30.7+/-4.4 n=5, 300 nM noradrenaline 32.6+/-6.92 n=5, 30 micro M noradrenaline 38.1+/-3.1 n=8, 0.2 mM isoprenaline 42.6+/-5.2 n=6, P=0.007). There were no differences between the variants in terms of cyclic AMP-dependent protein kinase activity. 5. These data provide the first evidence that enhanced G-protein coupling of the R389 beta(1)-adrenoceptor variant reported in rodent fibroblast expression systems is also present in native human receptors. The functional consequence of this is to significantly alter the inotropic potency of beta(1)-adrenoceptor activation depending on its genotype at the 389 position.

Up-regulation of novel intermediate filament proteins in primary fiber cells: an indicator of all vertebrate lens fiber differentiation?

The early embryonic development and expression patterns of the eye lens specific cytoskeletal proteins, CP49 and CP95, were determined for the chick and were found to be similar in both human and mouse. These proteins, as well as their homologs in other species, are obligate polymerization partners which form unique filamentous structures termed "beaded filaments." CP49 and CP95 appeared as protein products after 3 days of embryonic development in the chick during the elongation of primary fiber cells. Although limited data were obtained for human embryos at these early developmental timepoints, they were consistent with the interpretation that the up-regulation of these lens specific proteins began only after the initiation of lens vesicle closure. In situ hybridization with the mouse lens confirmed that message levels for beaded filament proteins were greatly elevated in differentiating primary fiber cells. Nuclease protection assays established that mRNA levels for CP49 remained relatively constant while CP95 mRNA levels increased once the process of secondary fiber formation was under way. Although present in relatively low abundance, the mRNA for a unique splice variant of CP49, CP49(INS), was also detected early in embryonic development and into adulthood. Peptide-specific antibodies directed against unique predicted sequences were able to confirm the protein expression of CP49(INS) in both embryonic and adult chick lens cells. These data present the first detailed study of the expression of CP49 and CP95 during early lens development. They suggest that the up-regulated expression of CP49 and CP95 could serve as pan-specific markers for all vertebrate lens fiber development.

The eye lens cytoskeleton.

During lens cell differentiation there are a number of very characteristic morphological changes that occur. These include a 50- to 100-fold increase in cell length as the equatorial lens epithelial cells differentiate into fibre cells and the loss of the cellular organelles such as mitochondria, nuclei, Golgi apparatus and endoplasmic reticulum. Coincident with these changes are dramatic alterations in the organisation of the lens fibre cell cytoskeleton and in particular the lens-specific intermediate filament network comprising CP49 and filensin. Cell shape and cell polarisation as well as tissue integrity are all processes that depend upon the cytoskeleton and are therefore important to the lens. The unique aspects of the lenticular cytoskeleton are the subject of this review.

Three murine cataract mutants (Cat2) are defective in different gamma-crystallin genes.

A number of murine cataract mutations have been localized to chromosome 1 close to the gamma-crystallin gene cluster (Cryg) (Everett et al., 1994, Genomics 20: 429-434; Löster et al., 1994, Genomics 23: 240-242). Based on the size of the mapping or allelism tests they have not been shown to be genetically distinct and have been assigned to locus symbol Cat2. Here we assign three mutations to the respective gamma-crystallin gene. Using a systematic candidate gene approach to analyze the entire Cryg cluster, an A-->G transition was found in exon 2 of Cryga for the ENU-436 mutation and is designated Cryga1Neu. The mutant allele Crygbnop (formerly Cat2(nop)) is caused by a replacement of 11 bp by 4 bp in the third exon of Crygb, while a C-->G transversion in exon 3 of Cryge has been found for the Cryget (formerly Cat2(t)) mutation. For the mutation Cryga1Neu, an Asp-->Gly exchange is deduced, whereas the mutations Crygbnop and Cryget lead to the formation of in-frame stop codons and give rise to truncated proteins of 144 and 143 amino acids, respectively. The effects of the mutations upon gamma-crystallin structure are likely to be quite different. The Cryga1Neu mutation is expected to affect the link between Greek-key motifs 2 and 3, whereas both Crygbnop and Cryget mutations are supposed to truncate the fourth Greek-key motif. All three mutations are predicted to alter protein folding of the gamma-crystallins and result in lens cataract, but the phenotype for each is quite distinctive.

The beaded filament of the eye lens: an unexpected key to intermediate filament structure and function.

In 1959, an unusual filamentous polymer, now called the beaded filament, was described in the lens of the eye. The constituent proteins, assembly properties and functions of the beaded filament have been elusive. The recent publication of the sequences for two major lens filament proteins (CP49 and filensin) and the reconstitution in vitro of structures closely resembling beaded filaments, suggests that the beaded filament is related structurally to intermediate filaments (IFs). The association of the lenticular chaperones, the alpha-crystallins, with the filament contributes to the characteristic beaded morphology, as well as giving important clues to the function of this unusual filament in the lens. These recent results have several implications for IF function and assembly.