Mutations in the extracellular domain of the 55-kD tumor-necrosis factor (TNF) receptor (TNFRSF1A), a key regulator of inflammation, define a periodic-fever syndrome, TRAPS (TNF receptor-associated periodic syndrome [MIM 142680]), which is characterized by attacks of fever, sterile peritonitis, arthralgia, myalgia, skin rash, and/or conjunctivitis; some patients also develop systemic amyloidosis. Elsewhere we have described six disease-associated TNFRSF1A mutations, five of which disrupt extracellular cysteines involved in disulfide bonds; four other mutations have subsequently been reported. Among 150 additional patients with unexplained periodic fevers, we have identified four novel TNFRSF1A mutations (H22Y, C33G, S86P, and c.193-14 G-->A), one mutation (C30S) described by another group, and two substitutions (P46L and R92Q) present in approximately 1% of control chromosomes. The increased frequency of P46L and R92Q among patients with periodic fever, as well as functional studies of TNFRSF1A, argue that these are low-penetrance mutations rather than benign polymorphisms. The c.193-14 G-->A mutation creates a splice-acceptor site upstream of exon 3, resulting in a transcript encoding four additional extracellular amino acids. T50M and c.193-14 G-->A occur at CpG hotspots, and haplotype analysis is consistent with recurrent mutations at these sites. In contrast, although R92Q also arises at a CpG motif, we identified a common founder chromosome in unrelated individuals with this substitution. Genotype-phenotype studies identified, as carriers of cysteine mutations, 13 of 14 patients with TRAPS and amyloidosis and indicated a lower penetrance of TRAPS symptoms in individuals with noncysteine mutations. In two families with dominantly inherited disease and in 90 sporadic cases that presented with a compatible clinical history, we have not identified any TNFRSF1A mutation, despite comprehensive genomic sequencing of all of the exons, therefore suggesting further genetic heterogeneity of the periodic-fever syndromes.

Familial amyloid polyneuropathy type I is an autosomal dominant disorder caused by mutations in the transthyretin (TTR) gene; however, carriers of the same mutation exhibit variability in penetrance and clinical expression. We analyzed alleles of candidate genes encoding non-fibrillar components of TTR amyloid deposits and a molecule metabolically interacting with TTR [retinol-binding protein (RBP)], for possible associations with age of disease onset and/or susceptibility in a Portuguese population sample with the TTR V30M mutation and unrelated controls. We show that the V30M carriers represent a distinct subset of the Portuguese population. Estimates of genetic distance indicated that the controls and the classical-onset group were furthest apart, whereas the late-onset group appeared to differ from both. Importantly, the data also indicate that genetic interactions among the multiple loci evaluated, rather than single-locus effects, are more likely to determine differences in the age of disease onset. Multifactor dimensionality reduction indicated that the best genetic model for classical onset group versus controls involved the APCS gene, whereas for late-onset cases, one APCS variant (APCSv1) and two RBP variants (RBPv1 and RBPv2) are involved. Thus, although the TTR V30M mutation is required for the disease in Portuguese patients, different genetic factors may govern the age of onset, as well as the occurrence of anticipation.

Protein kinase C (PKC) isozymes play major roles in human diseases, including cancer. Yet, the poor understanding of isozymes-specific functions and the limited availability of selective pharmacological modulators of PKC isozymes have limited the clinical translation of PKC-targeting agents. Here, we report the first small-molecule PKCδ-selective activator, the 7α-acetoxy-6β-benzoyloxy-12-O-benzoylroyleanone (Roy-Bz), which binds to the PKCδ-C1-domain. Roy-Bz potently inhibited the proliferation of colon cancer cells by inducing a PKCδ-dependent mitochondrial apoptotic pathway involving caspase-3 activation. In HCT116 colon cancer cells, Roy-Bz specifically triggered the translocation of PKCδ but not other phorbol ester responsive PKCs. Roy-Bz caused a marked inhibition in migration of HCT116 cells in a PKCδ-dependent manner. Additionally, the impairment of colonosphere growth and formation, associated with depletion of stemness markers, indicate that Roy-Bz also targets drug-resistant cancer stem cells, preventing tumor dissemination and recurrence. Notably, in xenograft mouse models, Roy-Bz showed a PKCδ-dependent antitumor effect, through anti-proliferative, pro-apoptotic, and anti-angiogenic activities. Besides, Roy-Bz was non-genotoxic, and in vivo it had no apparent toxic side effects. Collectively, our findings reveal a novel promising anticancer drug candidate. Most importantly, Roy-Bz opens the way to a new era on PKC biology and pharmacology, contributing to the potential redefinition of the structural requirements of isozyme-selective agents, and to the re-establishment of PKC isozymes as feasible therapeutic targets in human diseases.

The extraembryonic ectoderm (ExE) of the mouse conceptus is known to play a role in embryo patterning by signaling to the underlying epiblast and surrounding visceral endoderm. Bmp4 is one of the key ExE signaling molecules and has been recently implicated to participate in regulating development and migration of the anterior visceral endoderm (AVE). However, it remains unclear when exactly BMP4 signaling starts to regulate AVE positioning. To examine this, we have chosen to affect BMP4 function at two different time points, at embryonic day 5.25 (E5.25), thus before AVE migration, and E5.75, just after AVE migration. To this end, an RNAi technique was used, which consisted of the injection of Bmp4 dsRNA into the proamniotic cavity of the egg cylinder followed by its targeted electroporation into the ExE. This resulted in specific knockdown of Bmp4. It was found that Bmp4 RNAi at E5.25, but not at E5.75, led to an abnormal pattern of expression of the AVE marker Cerberus-like. Thus, BMP4 signaling appears to affect the expression of Cer1 at a specific time window. This RNAi approach provides a convenient means to study spatial and temporal function of genes shortly after embryo implantation.

Familial amyloid polyneuropathy (FAP) is a lethal autosomal dominant disorder in which fibrils derived from mutant forms of transthyretin (TTR), the normal plasma carrier of thyroxine (T(4)) and retinol-binding protein, are deposited in tissues. Over 80 TTR sequence variants are associated with FAP, but the amino-acid substitutions alone do not completely explain the variability in disease penetrance, pathology and clinical course. To analyze the factors possibly contributing to this phenotypic variability, we characterized the variations within the wild-type and mutant (Val30Met) TTR genes and their flanking sequences by performing extended microsatellite haplotype analyses, sequencing and single-nucleotide polymorphism haplotyping of genomic DNA from Portuguese and Swedish carriers of V30M. We identified 10 new polymorphisms in the TTR untranslated regions, eight resulting from single-base substitutions and two arising from insertion/deletions in dinucleotide repeat sequences. The data suggest that the onset of symptoms of FAP V30M may be modulated by an interval downstream of TTR on the accompanying noncarrier chromosome (defined by microsatellites D18S457 and D18S456), but not by the immediately 5'- and 3'-flanking sequences of TTR. During the course of these studies, we also encountered the first instance in which the previously described intragenic haplotype III may be associated with V30M FAP in the Portuguese population.

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Detection and quantification of microRNAs (miRNAs) in exhaled breath condensate (EBC) has been poorly explored. Therefore we aimed to assess miRNAs in EBC as potential biomarkers to diagnose and endotype asthma in school aged children. In a cross sectional, nested case control study, all the asthmatic children (n = 71) and a random sample of controls (n = 115), aged 7 to 12 years, attending 71 classrooms from 20 local schools were selected and arbitrarily allocated to the development or validation set. Participants underwent skin-prick testing, spirometry with bronchodilation, had exhaled level of nitric oxide determined and EBC collected. Based on previous studies eleven miRNAs were chosen and analyzed in EBC by reverse transcription-quantitative real-time PCR. Principal component analysis was applied to identify miRNAs profiles and associations were estimated using regression models. In the development set (n = 89) two clusters of miRNAs were identified. After adjustments, cluster 1 and three of its clustered miRNAs, miR-126-3p, miR-133a-3p and miR-145-5p were positively associated with asthma. Moreover miR-21-5p was negatively associated with symptomatic asthma and positively associated with positive bronchodilation without symptoms. An association was also found between miR-126-3p, cluster 2 and one of its clustered miRNA, miR-146-5p, with higher FEF25-75 reversibility. These findings were confirmed in the validation set (n = 97) where two identical clusters of miRNAs were identified. Additional significant associations were observed between miR-155-5p with symptomatic asthma, negative bronchodilation with symptoms and positive bronchodilation without symptoms. We showed that microRNAs can be measured in EBC of children and may be used as potential biomarkers of asthma, assisting asthma endotype establishment.

Background: Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNAmediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages.