Genome-wide analysis of gene expression after one year of venom immunotherapy.

Background Insect venom immunotherapy (VIT) is used to protect patients against Hymenoptera insects' venom allergy (HVA), which can result in severe systemic or even life-threatening conditions. Molecular mechanisms triggered by VIT remain largely unknown. Objective To compare genome-wide gene expression of patients with severe HVA prior to VIT and 12 months after. Methods Whole blood RNA samples were analyzed on an expression array. Results from differential expression obtained on a microarray platform were confirmed by quantitative real -time PCR (qRT-PCR). Subsequently we applied unsupervised clustering. Relative blood cell proportions and gene expression profiles were used as an input to csSAM to compute cell specific differential gene expression. Finally, transcription factor enrichment analysis was performed in MotifLab. Results & conclusions Comparison of genome-wide expression patterns for whole blood and qRT-PCR experiments revealed no significantly up and/or down regulated genes. This has been corroborated by unsupervised clustering. We found a significant upregulation of 26 genes in macrophages, of 15 genes in monocytes and 2 genes in T regulatory cells (Tregs). Analysis of the promoter sequences of these upregulated genes revealed a significant over-representation of binding motifs specific for kruppel-like factor 4, retinoic acid receptor gamma, and vitamin D receptor. Our results indicate that changes of gene expression invoked by VIT in peripheral blood may have a too small effect to be detected by conventional biostatistical approaches. When blood cell composition was taken into account we uncovered numerous changes of cell-specific gene expression. Given the regulatory implications we hypothesize that above-mentioned alterations may contribute to activation of anti-inflammatory signals in the innate branch of the immune system.

A TMC1 (transmembrane channel-like 1) mutation (p.S320R) in a Polish family with hearing impairment.

After excluding frequent mutations in common genes like GJB2, SLC26A4 and MT-RNR1 by straightforward Sanger sequencing in about 20 Polish families with hearing impairment, new and possibly pathogenic mutations were searched for by next-generation sequencing (NGS) screening using a specialised panel including more than 80 genes connected with hearing disorders. Due to high rates of false-positive pathogen predictions for newly discovered single-nucleotide polymorphisms (SNPs), different prediction models were combined to enhance the prediction power. In one family with a record of over four generations, II,3 and II,4 were suspected of hearing impairment without medical records. A male person (III,2) displayed hearing loss of 40 dB hearing level (HL) and his two sons, IV,1 and IV,2, were both affected; one with 90 dB HL and the other with 40 dB HL. Here, one heterozygous, non-synonymous variant was detected, with the SNP causing an amino acid substitution in TMC1 (transmembrane channel-like 1), a gene reported with many mutations in DFNA36 and DFNB7/11 (OMIM #606705 and #600974, respectively). Until now, the substitution p.S320R has not been described in any database. Instead of the significance of this mutation by bioinformatics tools, we confirmed the genotype-phenotype co-segregation in family members. The involvement of TMC1 in hereditary hearing impairment has not been observed in the Polish population so far.

Trefoil factor family member 2 (Tff2) KO mice are protected from high-fat diet-induced obesity.

INTRODUCTION: Trefoil factor family member 2 (Tff2) is a small gut peptide, mainly known for its protective and healing functions. As previously demonstrated, high-fat (HF) feeding can rapidly and specifically modulate Tff2 transcription in key tissues of mice, including the duodenum and mesenteric adipose tissue, therefore suggesting a novel role for this gene in energy balance. DESIGN AND METHODS: To explore whether and how Tff2 can influence feeding behavior and energy metabolism, Tff2 knock-out (KO) mice were challenged with HF diet for 12 weeks, hence food and energy intakes, body composition, as well as energy excretion and serum lipid and hormonal levels were analyzed. Finally, energy efficiency was estimated. RESULTS: Tff2 KO mice showed a greater appetite and higher energy intake compared to wild-type (WT). Consistently, they presented lower levels of serum leptin, and increased transcription of agouti-related protein (Agrp) in the hypothalamus. Though energy and triglyceride fecal excretion were augmented in Tff2 KO mice, digestible energy intake was superior. However, KO mice were finally protected from HF diet-induced obesity, and accumulated less weight and fat depots than WT animals, while keeping a normal lean mass. Energy efficiency was lower in HF-KO mice, while energy expenditure and locomotor activity were globally increased. CONCLUSIONS: The present work demonstrates previously unsuspected roles for Tff2 and suggests it to be a mastermind in the control of energy balance and a promising therapeutic target for obesity.

Otoferlin couples to clathrin-mediated endocytosis in mature cochlear inner hair cells.

The encoding of auditory information with indefatigable precision requires efficient resupply of vesicles at inner hair cell (IHC) ribbon synapses. Otoferlin, a transmembrane protein responsible for deafness in DFNB9 families, has been postulated to act as a calcium sensor for exocytosis as well as to be involved in rapid vesicle replenishment of IHCs. However, the molecular basis of vesicle recycling in IHCs is largely unknown. In the present study, we used high-resolution liquid chromatography coupled with mass spectrometry to copurify otoferlin interaction partners in the mammalian cochlea. We identified multiple subunits of the adaptor protein complex AP-2 (CLAP), an essential component of clathrin-mediated endocytosis, as binding partners of otoferlin in rats and mice. The interaction between otoferlin and AP-2 was confirmed by coimmunoprecipitation. We also found that AP-2 interacts with myosin VI, another otoferlin binding partner important for clathrin-mediated endocytosis (CME). The expression of AP-2 in IHCs was verified by reverse transcription PCR. Confocal microscopy experiments revealed that the expression of AP-2 and its colocalization with otoferlin is confined to mature IHCs. When CME was inhibited by blocking dynamin action, real-time changes in membrane capacitance showed impaired synaptic vesicle replenishment in mature but not immature IHCs. We suggest that an otoferlin-AP-2 interaction drives Ca(2+)- and stimulus-dependent compensating CME in mature IHCs.

A set of specific miRNAs is connected with murine and human gastric cancer.

MircoRNAs as a new class of regulatory molecules have been investigated in many specific cells and organs in healthy and diseased conditions. Although miRNA signatures can be directly assessed in patients' affected tissues such as tumor sections, recent studies revealed that miRNA profiles can also be obtained indirectly, that is, from the patients' peripheral blood. For better understanding of miRNA's contribution to gastric carcinoma (one of the leading causes of cancer-related mortality worldwide), we screened for deregulated miRNAs in blood collected from human cancer patients and compared the expression patterns with a gastric carcinoma mouse model (Tff1 knock-out). The profiles were assessed using species-specific miRNA microarrays. Among many dozens of deregulated miRNAs (219 in H. sapiens; 75 in M. musculus), a subset of eight miRNAs comparable in sequence from both species was noted. By in silico analysis, their involvement in targeting neoplastic and MAPkinase pathways was demonstrated. We found a high probability of linkage of all noted miRNAs to pathways in cancer with P-values of 0.013 and 0.018 in mice and humans, respectively. Linkage to the MAPK-signaling pathway in mice was observed with a P-value of 0.01. Moreover, when comparing the 219 deregulated miRNAs obtained from blood with deregulated miRNAs derived from gastric cancer (GC) tissues, as published previously, 24 miRNAs were identical. If confirmed in a larger patient pool, these miRNAs could constitute appropriate blood-born biomarkers for GC.

Intermediate- and low-methylation epigenotypes do not correspond to CpG island methylator phenotype (low and -zero) in colorectal cancer.

BACKGROUND: Most recent genome-wide studies on the CpG island methylation in colorectal cancer (CRC) have led to the discovery of at least 3 distinct methylation clusters. However, there remains an uncertainty whether the CRC clusters identified in these studies represent compatible phenotypes. METHODS: We carried out comprehensive genome-scale DNA methylation profiling by Illumina Infinium HumanMethylation27 of 21 DNA pools that represent 84 CRC samples divided according to their high-, intermediate-, and low-methylation epigenotypes (HME, IME, and LME, respectively) and 70 normal-adjacent colonic tissues. We have also examined the relationship among 3 epigenotypes and chromosomal gains and deletions (assessed by Comparative Genomic Hybridization) in a group of 100 CRC samples. RESULTS: The HME subgroup showed features associated with CpG island methylator phenotype - high (CIMP-high) including methylation of specific CpG sites (CpGs) as well as significantly lower mean number of chromosomal imbalances when compared with other epigenotypes. The IME subgroup displayed the lowest number of methylated CpGs (717 vs. 2,399 and 2,679 in HME and LME, respectively) and highest mean number of chromosomal imbalances when compared with HME (P, 0.001) and LME (P, 0.004). A comparison between the methylation profiles of 3 epigenotypes revealed more similarities between the HME and LME (1,669 methylated CpGs overlapped) than HME and IME (673 methylated CpGs overlapped). CONCLUSION: Our results provide evidence that IME and LME CRCs show opposite features to those that have been previously attributed to CIMP-low and CIMP-0 CRCs. IMPACT: These discrepancies should be considered when interpreting the data from a particular epigenotyping method.

Increased susceptibility to Yersinia enterocolitica Infection of Tff2 deficient mice.

TFF2 is one of the members of the trefoil factor family, known for its role in protection of gastrointestinal epithelia upon injury; however, recent studies suggest that TFF2 could also play an important role in the immune system. In the present study Tff2 deficient and wild type mice were infected by Y. enterocolitica which resulted in a lethal outcome in all Tff2 deficient mice, but not in WT animals. Yersinia invaded Peyer's patches more efficiently as shown by high bacterial titers in the KO mice while wild type mice displayed lower titers and a visible bacterial accumulation in the intestine. Bacterial accumulation in Peyer's patches of Tff2 deficient mice was accompanied by increased recruitment of macrophages. While an increased level of MAC-1 positive cells was observed in the spleens of both Tff2 deficient and WT mice at third day post infection, bacterial dissemination to liver, lung and kidneys was observed only in Tff2 knock-out mice. Analysis of the cellular composition of spleen did not reveal any substantial alteration to WT animals, suggesting possible disregulation of hemopoietic cells involved in immune response to Y. enterocolitica. These new data indicate that Tff2 plays an important role in immune response by protecting the organism from consequences of infection and that Tff2 knock-out mice react adversely to bacterial infections, in this case specifically to Y. enterocolitica.

Ergic2, a brain specific interacting partner of Otoferlin.

BACKGROUND: Otoferlin, a postulated calcium sensor of 230 kDa, was proposed to trigger calcium dependent fusion of vesicles with plasma membrane in the ribbon synapses of cochlear IHCs. Otoferlin's interaction with Rab8b and Myo6, proteins involved in the intracellular membrane trafficking, extended the previous hypothesis assigning Otoferlin an additional role in trans-Golgi trafficking. Here, we present another Otoferlin binding partner, Ergic2, a protein with a still unknown function but presenting sequence homology to other proteins involved in ER/Golgi vesicle trafficking. METHODS: Novel binding partners of Otoferlin were searched by yeast two-hybrid screening in a rodent cochlear cDNA library (P3-P15). RT-PCR, western blot, immunohistochemistry staining and co-immunoprecipitation were applied to analyze and confirm an interaction between Ergic2 and Otoferlin. RESULTS: The Y-2-H screening, using baits covering parts of Otoferlin's C2D domain, identified Ergic2 as an interacting protein for Otoferlin. Both are co-expressed (mRNA and protein level) in rodent cochlea and brain before- and after-onset of hearing. By RT-PCR Ergic2 was detected in cochlear IHCs and OHCs and in brain regions where Otoferlin is known to be present. Co-localization studies revealed an overlap of Ergic2 and Otoferlin signals in IHCs and neurons of cerebral cortical layer I making Ergic2 the promising binding candidate. However, while Ergic2 was co-precipitated by an anti-Otoferlin antibody in protein lysates from murine brain, this specific protein interaction was not detected in cochlea. CONCLUSION: Our new data on Otoferlin's interactome suggest that Otoferlin can form different, tissue-specific protein complexes.

New insights into the Tyrolean Iceman's origin and phenotype as inferred by whole-genome sequencing.

The Tyrolean Iceman, a 5,300-year-old Copper age individual, was discovered in 1991 on the Tisenjoch Pass in the Italian part of the Ötztal Alps. Here we report the complete genome sequence of the Iceman and show 100% concordance between the previously reported mitochondrial genome sequence and the consensus sequence generated from our genomic data. We present indications for recent common ancestry between the Iceman and present-day inhabitants of the Tyrrhenian Sea, that the Iceman probably had brown eyes, belonged to blood group O and was lactose intolerant. His genetic predisposition shows an increased risk for coronary heart disease and may have contributed to the development of previously reported vascular calcifications. Sequences corresponding to ~60% of the genome of Borrelia burgdorferi are indicative of the earliest human case of infection with the pathogen for Lyme borreliosis.

Altered miRNA expression patterns in Tff2 knock-out mice correlate with cellular pathways of neoplastic development and caloric metabolism.

The trefoil peptide family, consisting in mammals of three members namely TFF1, 2 and 3, plays a cytoprotective role in epithelial cells of various tissues, mainly in the digestive tract. Tff1, Tff2 or Tff3 knock-out mouse models developed various kinds of gastrointestinal impairment. microRNAs are known to be novel gene regulators. We aimed to investigate the physiological role of such miRNAs in Tff2 knock-out mice. Whole miRNome profiling and in silico analysis were performed for Tff2-KO and WT mice. Our latest data explored the role of miRNAs in the regulatory cascades and molecular processes of Tff2-/- mice. As much as 6% of the Tff2-KO mice miRNome was significantly dys-regulated. Further in silico analysis suggests that the respective dys-regulated part of the miRNome is involved in human pathological processes, including pancreatic, colorectal and basal cell cancer. Additionally, the dys-regulated miRNome targets pathways involved in carbohydrate metabolism and adipocytokine signaling. The latter links deficient caloric maintenance in Tff2 and previous observation in Tff3-KO mice with miRNAs. In summary, our proof-of-concept study indicates that miRNAs may play an important role in the regulatory processes of the trefoil peptide family, especially in the regulation of cancer-related cascades.

Temporal expression pattern of Fkbp8 in rodent cochlea.

BACKGROUND: FKBP8 is a multifunctional protein involved in many distinct processes like formation of central nervous system, viral RNA replication and inhibition of apoptosis. Fkbp8 expression was reported in different tissues, various cell lines and malignancies, in the latter displaying changes during carcinogenesis. Loss of Fkbp8 leads to substantial neurodegenerations during regular mouse development, thus hearing onset in mice could also potentially depend on Fkbp8 expression. Since Fkbp8 is crucial for patterning of neuronal function, we studied its expression during maturation of the rodent auditory function. METHODS: Fkbp8 gene expression in rodent cochlear samples was studied by RT-PCR, qPCR, and western blot. Localization of Fkbp8 transcripts and protein was analyzed by in-situ hybridization and immunohistochemistry. RESULTS: Studies of auditory organ demonstrate that Fkbp8 gene activity is increasing just before hearing onset and gradually decreasing after onset of hearing. Western blot analysis suggests substantial levels of Fkbp8 protein before hearing onset, and slow degradation after onset of hearing. The Fkbp8 mRNA is localized in spiral ganglion of cochlea but its distribution changes over time to the stria vascularis, a finding supported by immunohistochemistry staining. Additionally, in pre-hearing time Fkbp8-specific signal was also observed in the tectorial membrane, whose α- and ß-Tectorin components show similar time-dependent expression of mRNA as Fkbp8. CONCLUSION: These results indicate a temporal shift in expression of Fkbp8 which correlates with cochlear maturation, strongly suggesting a contribution of Fkbp8 to the onset of the rodent hearing processes.

The otoferlin interactome in neurosensory hair cells: significance for synaptic vesicle release and trans-Golgi network (Review).

Sound perception in terrestrial vertebrates relies on a structure in the inner ear consisting of the utriculus, sacculus and lagena. In mammals, the lagena has developed into the cochlea where mechanotransduction at ciliated cells leads to ion influx via regulated ion channels. To maintain proper Ca2+ concentration many cellular systems use a variety of functional proteins; the neurosensory systems use calcium-sensors like hippocalcin, visinin or recoverin. In cochlear hair cells the 230 kDa protein otoferlin has been suggested to play this role. While several observations support this hypothesis additional data argue for a more expanded functional profile of otoferlin. Evidence for otoferlin's multiple roles and newer results on otoferlin's interacting partners are presented and the existence of a protein complex as a functional unit ('interactome') in the cochlea and further tissues is suggested.

The intestinal factor Tff3 and a miRNA network regulate murine caloric metabolism.

Genetic impairment of the genes coding for three mammalian trefoil peptides resulted in severe gastrointestinal malfunctions. The trefoil peptides also appear involved in caloric metabolism. Monitoring global miRNA expression of Tff3 deficient mice points to an interplay of Tff3 with a miRNA regulatory network. We identified 21 miRNAs that were deregulated when compared to the wild type strain. In silico evaluation indicated that the majority of the 21 miRNA were connected with the metabolic pathway "glycolysis/gluconeogenesis'' (p=0.032), a signaling pathway including nine target genes Aldh9a1, Aldh2, Pck1, Aldoc, Pgam2, Pck2, Adh4, Adh5, and Fbp1. Association of Tff3 with this metabolic pathway is further supported by the observation that the body mass of adult Tff3 KO mice (five months) showed a clearly reduced weight. Furthermore, the majority of the identified 21 miRNA genes are localized on murine chromosomes 2 and 5 in three clusters (2A1, 2B, 5B3) suggesting a coordinated expression control and function.

Epigenetic alterations by methylation of RASSF1A and DAPK1 promoter sequences in mammary carcinoma detected in extracellular tumor DNA.

Novel strategies for early detection of breast cancer, the most common and second most lethal cancer in women, are urgently needed. Silencing tumor suppressor genes via DNA methylation has established hypermethylation as one of the most frequent molecular alterations that may initiate and drive many types of human neoplasia including breast cancer. Detecting such epigenetic changes in DNA derived not only from tumor tissue, but also from bodily fluids, may be a promising target for the molecular analysis of cancer. In this study we examined serum, a readily accessible bodily fluid known to contain neoplastic DNA, from individuals with breast carcinoma. Using sensitive methylation-specific polymerase chain reaction, we searched for aberrant promoter hypermethylation of two normally nonmethylated genes: RAS association domain family member 1A (RASSF1A) and death-associated protein kinase 1 (DAPK1) in 26 patients with breast cancer, 16 patients with benign breast diseases, and 12 age-matched healthy controls. Hypermethylation of at least one gene was detected in 25/26 (96%) cancer patients, in 7/16 (43%) cases with benign breast diseases, and only 1/12 (8%) control subjects. Furthermore, methylation of both genes was found to be associated with ductal type of breast carcinoma. RASSF1A was hypermethylated in 18/26 cases (69%) and DAPK1 in 23/26 (88%). However, DAPK1 promoter methylation was more pronounced, as 12/23 DAPK1 methylated cases (52%) were strongly methylated (>75%) compared to the weaker methylation of RASSF1A (none of the cases with methylation at the level of >75%). These findings, if confirmed in studies of extended cohorts, may lead to useful clinical application in early diagnosis of breast cancer and better management of the neoplastic disease.

Functional analysis of a novel I71N mutation in the GJB2 gene among Southern Egyptians causing autosomal recessive hearing loss.

Mutations in GJB2, a gene encoding the gap junction protein connexin 26 (Cx26), are a major cause for inherited and sporadic non-syndromic hearing loss, albeit with highly variable clinical effects. To determine new mutations and their frequencies in a Southern Egyptian population restriction fragment length polymorphism, gene sequencing, and single strand conformational polymorphism revealed only 2 mutations for GJB2: c.35delG and p.I71N. The allelic frequency of the c.35delG mutation was 8.7% (found in 27 out of 310 investigated alleles) resulting in a relatively low carrier frequency (1.6%) in Upper Egypt. The new mutation, a substitution of isoleucin (I) (a non-polar amino acid) by the polar amino acid asparagin (N), was localized within the conserved Cx26 structure. The functional significance of p.I71N was tested by injection of cRNA into Xenopus laevis oocytes. Cx26 hemi-channel activity was measured by depolarization activated conductance in non-coupled oocytes. As a result, the p.I71N mutated channel was non-functional. The study discloses a novel, functionally relevant GJB2 mutation and defines the contribution of Cx26 alterations to the hearing loss in the Southern Egyptian population.

Otoferlin interacts with myosin VI: implications for maintenance of the basolateral synaptic structure of the inner hair cell.

Otoferlin has been proposed to be the Ca(2+) sensor in hair cell exocytosis, compensating for the classical synaptic fusion proteins synaptotagmin-1 and synaptotagmin-2. In the present study, yeast two-hybrid assays reveal myosin VI as a novel otoferlin binding partner. Co-immunoprecipitation assay and co-expression suggest an interaction of both proteins within the basolateral part of inner hair cells (IHCs). Comparison of otoferlin mutants and myosin VI mutant mice indicates non-complementary and complementary roles of myosin VI and otoferlin for synaptic maturation: (i) IHCs from otoferlin mutant mice exhibited a decoupling of CtBP2/RIBEYE and Ca(V)1.3 and severe reduction of exocytosis. (ii) Myosin VI mutant IHCs failed to transport BK channels to the membrane of the apical cell regions, and the exocytotic Ca(2+) efficiency did not mature. (iii) Otoferlin and myosin VI mutant IHCs showed a reduced basolateral synaptic surface area and altered active zone topography. Membrane infoldings in otoferlin mutant IHCs indicated disturbed transport of endocytotic membranes and link the above morphological changes to a complementary role of otoferlin and myosin VI in transport of intracellular compartments to the basolateral IHC membrane.

Intestinal expression of TFF and related genes during postnatal development in a piglet probiotic trial.

Trefoil factor family (TFF) peptides provide protective and reparative effects by enhancing epithelial integrity and promoting mucosal restitution. TFF peptide expression is induced after mucosal damage. These processes are of central physiological relevance during the postnatal intestinal development and are strongly influenced during the weaning period. In piglets, weaning at early maturation stages frequently causes mucosal inflammation. The aim of this study was to evaluate postnatal intestinal TFF expression in a piglet probiotic trial. Low intestinal TFF2 expression was measured at early maturation stages. Weaning, however, was associated with a distinct response of increased TFF2 expression, indicating an important role in enhancing mucosal integrity. In the distal jejunum and ileum weaning could as well be associated with increased TFF3 mRNA levels. Differential TFF1 expression was not detected. Furthermore, TFF2 localization studies in different intestinal loci were performed by means of immunohistochemistry. Expression of selected genes (TGFA, EGFR, Cox-2) known to promote TFF signaling showed differential expression pattern as well, thereby providing further functional background. Furthermore, the expression patterns of EGFR observed in this study contribute to an advanced view of previous findings of EGFR regulation mainly obtained in rodents. An upregulated EGFR expression during early postnatal development suggests a local relevance to porcine intestinal maturation. However, a feed supplementation with the probiotic strain Enterococcus faecium did not influence TFF expression.

Rab8b GTPase, a protein transport regulator, is an interacting partner of otoferlin, defective in a human autosomal recessive deafness form.

Mutations within OTOF encoding otoferlin lead to a recessive disorder called DFNB9. Several studies have indicated otoferlin's association with ribbon synapses of cochlear sensory hair cells, as well as data showing the protein's presence in neurons, nerve fibers and hair cells, suggesting a more ubiquitous function. Otoferlin's co-localization not only with ribbon synaptic proteins, but also with additional endosomal (EEA1) or Golgi proteins (GM130) were motivation for a search for further binding partners of otoferlin by a yeast two-hybrid screen in a rodent cochlear cDNA library (P3-P15). This screen identified Rab8b GTPase as a novel interacting partner, substantiated by transient co-expression and co-localization in HEK 293 cells and co-immunoprecipitation of the complex using tagged proteins in vitro and native proteins from cochlea. This finding implies that otoferlin could be a part of components contributing to trans-Golgi trafficking.

Lack of Tff3 peptide results in hearing impairment and accelerated presbyacusis.

Tff peptides are secreted mainly by the gastrointestinal epithelial cells and their primary role is maintaining normal structure and function of mucous epithelia. Ongoing studies on their expression pattern have disclosed other sites of their synthesis thus revealing additional physiological functions in the organism. Here we present new data about Tff3 expression in the cochlea of the rodent inner ear. On the basis of RT-PCR we describe the presence of Tff3 transcripts in both, a mouse cDNA library isolated from whole cochleae from postnatal days 3-15 (P3-P15), and also in cochlear tissue. By using a riboprobe for the fragment containing exon 1, 2 and 3 of Tff3, in situ hybridization, localized Tff3 signals in neurons of spiral ganglion and vestibular organ. We did not observe any abnormalities in the middle ear of Tff3 knock-out mice, neither did histological examination of the inner ear indicate any gross morphological changes in the cochlea. However, ABR (auditory evoked brain stem responses) audiograms revealed that the Tff3 knock-out animals show an accelerated presbyacusis and a hearing loss of about 15 dB at low frequencies increasing to 25 dB loss at higher frequencies. These findings suggest that Tff3 could play a role in neurosensory signaling. Further studies are needed to clarify this new function in the auditory system.

The CpG island methylator phenotype correlates with long-range epigenetic silencing in colorectal cancer.

The CpG island methylator phenotype (CIMP), characterized by an exceptionally high frequency of methylation of discrete CpG islands, is observed in 18% to 25% of sporadic colorectal cancers. Another hypermethylation pattern found in colorectal cancers, termed long-range epigenetic silencing, is associated with DNA/histone methylation in three distinct gene clusters at chromosome 2q14.2, showing that DNA hypermethylation can span larger chromosomal domains and lead to the silencing of flanking, unmethylated genes. We investigated whether these two phenotypes are interrelated in colorectal cancers. The CIMP status of 148 sporadic colorectal cancers was determined by methylation-specific PCR. We determined the BRAF V600E mutation by mutant allele-specific PCR amplification. The methylation status of the MLH1 gene and of three CpG islands (EN1, SCTR, and INHBB), corresponding to three distinct clusters along 2q14.2, was determined by methylation-specific PCR. The average number of sites showing methylation in CIMP+ tumors was 2.21, compared with 1.22 for CIMP- individuals, and this difference was highly significant (P = 3.6 x 10(-8), Mann-Whitney test). Moreover, all CIMP+ tumors showed hypermethylation of at least one of these loci, in contrast to CIMP- tumors, where 18 (16%) samples remained unmethylated. The mean number of simultaneously hypermethylated CpG islands at 2q14.2 differs significantly between CIMP- and CIMP+ tumors, suggesting varying effects of domain silencing in this region. Given that the number of hypermethylated loci at 2q14.2 likely affects the range of silenced flanking genes, high frequency of simultaneous hypermethylation of three CpG islands (EN1, SCTR, and INHBB) may have potential influence on specific characteristics of CIMP+ colorectal cancers.