miRNA-155 controls mast cell activation by regulating the PI3Kγ pathway and anaphylaxis in a mouse model.

BACKGROUND: Mast cells (MCs) play a central role in allergic and inflammatory disorders by rapid degranulation and release of inflammatory mediators upon antigen-driven engagement of the FcεRI. Receptor-mediated MC responses are controlled by the activation of different isoforms of phosphoinositide-3-kinase (PI3K) and the downstream signaling processes. Recent evidence suggests that miRNAs are important molecular players regulating the PI3K/Akt pathway. METHODS: The role of miR-155 in the regulation of MC functions in vivo was studied in the passive cutaneous anaphylaxis (PCA) MC-dependent model. WT and miR-155(-/-) mice were injected intradermally with anti-DNP-IgE and intravenously with the antigen DNP-HSA. Ear swelling was assessed to evaluate the anaphylactic response. All investigations, to characterize miR-155 specific activities in MCs, were conducted comparing WT and miR-155(-/-) bone marrow-derived MCs (BMMCs). RESULTS: We report that miR-155(-/-) mice display enhanced anaphylaxis reactions. Although miR-155(-/-) BMMCs show normal development, proliferation, and survival, miR-155 deficiency enhances FcεRI-mediated degranulation and release of TNF-α, IL-13, and IL-6. Interestingly, the level of Akt phosphorylation on both of its regulatory residues Thr308 and Ser473 was increased in miR-155(-/-) compared to WT BMMCs. Gene expression profiling showed that miR-155(-/-) BMMCs exhibited significantly increased expression of the adapter PI3Kγ subunits Pik3r5 (p101) and Pik3r6 (p84, p87(PIKAP) ). Furthermore, selective blockade of the PI3Kγ pathway inhibited degranulation in miR-155(-/-) BMMCs. CONCLUSIONS: Thus, we suggest that miR-155 plays a critical role in FcεRI-mediated MC responses by modulating components of the PI3Kγ pathway. This newly identified mechanism of miRNA-controlled MC activation may affect the initiation and maintenance of allergic disorders.

[Clinical and molecular genetic analysis of monozygotic twins displaying stapes gusher syndrome (DFN3)]. / Klinische und molekulargenetische Analyse monozygoter Zwillinge mit Stapes-Gusher-Syndrom (DFN3).

BACKGROUND: DFN3 ( "stapes gusher") is the most frequent form of X-linked hearing impairment. It accounts for up to 0.5% of all cases of severe childhood hearing disorders. PATIENTS AND METHODS: Monozygotic twins with suspected stapes gusher syndrome, their mother, and control individuals were analyzed clinically and genetically. RESULTS: The clinical investigations confirmed a DFN3 phenotype in both brothers who displayed all typical symptoms. A molecular genetic investigation of the POU3F4 gene, which plays an essential role in the development of DFN3, was also performed. No chromosomal aberrations within the coding region of POU3F4were detected. Since several authors have described mutations in the 5' untranslated region of the gene also resulting in a DFN3 phenotype, we screened this area by microsatellite analysis and detected a double deletion localized in the critical interval. This is the first description of a double deletion in the non-coding region of POU3F4 leading to DFN3 phenotype. CONCLUSION: Interestingly, in spite of having an identical genotype, the twins displayed significant phenotypic differences. This underlines the importance of exogenous factors in the development of inherited pathological processes.

Substitutions in the conserved C2C domain of otoferlin cause DFNB9, a form of nonsyndromic autosomal recessive deafness.

DFNB, the nonsyndromic hearing loss with an autosomal recessive mode of inheritance constitutes the majority of severe to profound prelingual forms of hearing impairment, usually leading to inability of speech acquisition. We analyzed a consanguineous family with autosomal recessive deafness which has been shown to segregate within chromosomal region 2p23.1 (DFNB9; MIM 601071). By SSCP analysis and DNA sequencing of the 48 exons of the DFNB9 gene, coding for otoferlin, previously reported mutations in OTOF were excluded. Next to a frequent T > C single nucleotide polymorphism in exon 8, two novel mutations linked in exon 15 of the OTOF long splice form were identified comprising substitutions at positions 490 (Pro > Gln) and 515 (Ile > Thr), both located in the conserved Ca(2+) binding C2C domain of this peptide. Comparisons of homology using human and mice otoferlins and closely related peptides and computer simulation analyses suggest that changes in the mutated segment's secondary structure affect the Ca(2+) binding capacity of the C2C domain in otoferlin.

Microsatellite and chromosome instability in squamous cell laryngeal carcinoma.

Head-and neck squamous cell carcinoma (HNSCC) represents almost 5% of all malignancies in Europe. The aetiology of HNSCC is complex, with both genetic and mutagenic factors involved. The aim of the present study was to investigate the loss of heterozygosity (LOH), mainly at tumour suppressor loci (using markers D1S2883, D2S123, D3S1611, D5S346, D7S501, D8S254, TP53, NM23), microsatellite instability (BAT25, 26, 40) and (bleomycin test) in patients with squamous cell larynx cancer. In a group of 20 patients LOH was observed mainly at the loci 3p (64.7%), 8q (71.4%), 17q (M1-30.8%, M2-25%, M3-38.5%). Despite chromosomal instability detected by bleomycin no microsatellite instability was observed.

[Mutational analysis of the connexin26 gene in sporadic cases of moderate to profound deafness]. / Mutationsanalyse des Connexin26-Gens bei sporadischen Fällen mittel-bis hochgradiger Schwerhörigkeit.

Non-syndromic neurosensory recessive deafness (NSRD) is one of the most common human sensory disorders. Mutations in the connexin 26 gene have been established as a major cause of inherited and sporadic non-syndromic deafness in different populations. The CX26 gene encodes the gap junction protein connexin 26 (beta-2, GJB2), whose expression was shown in several tissues and in the cochlea. The 30delG mutation is the most frequent mutation in the CX26 gene. It represents a deletion of guanosine (G) in a sequence of six Gs extending from position 30 to 35 of the CX26 cDNA. The deletion creates a frameshift resulting in a premature stop codon and a non-functional intracellular domain in the protein. The 30delG mutation can be detected at the molecular level using PCR followed by BsiYI digestion. We screened 164 mainly German patients with non-syndromic sporadic deafness for this mutation to determine its distribution in the German population. The frequency of the mutation in our analyzed patients was lower than in other studies and therefore indicates its dependency on geographically distinct populations.

Frequent D-loop polymorphism in mtDNA enables genotyping of 1400-year-old human remains from Merowingian graves.

Improvements of DNA extraction and amplification techniques presently enable DNA analysis of ancient DNA (aDNA) from samples which range from several hundred years of age up to possibly 5000 years. Taking advantage of the abundance of mitochondrial DNA and its polymorphic D-loop sequence, ten individuals from multiple burial sites of the Merowingian culture (South Germany), estimated to be about 1400 years old, were genotyped to determine possible kinship. Moreover, gonosomal DNA markers from the X- and Y-chromosome were applied for sex determination of the remains. In all individuals investigated, deviations from the Anderson mtDNA consensus sequence were observed, all representing substitutions (7 transitions and 3 transversions). Although such mutations have been reported from recent populations, our study constitutes the first description of these mtDNA mutations from numerous aDNA samples recovered from multiple burial sites. The results obtained by molecular anthropology can aid in describing kinship relations and burial customs of ancient remains.

Assessing genetic heterogeneity of renal cell tumors.

Renal cell tumors display a highly variable morphology which is also reflected at the genomic level. Such heterogeneity was at first monitored by cytogenetic means (numerical and structural chromosomal aberrations); in the meantime, more refined molecular techniques allow the assessment of DNA losses or gains in metaphase chromosomes or tissue sections. Moreover, genomic instability can be monitored using microsatellite probes. All these methods document specific characteristics of certain renal cell tumor types, e.g. telomeric associations in chromophobe carcinomas or oncocytomas, typical losses in 3p in clear cell carcinomas or trisomy 7 in renal cell adenomas and carcinomas. Next to examples demonstrating these alterations the Heidelberg classification of renal cell tumors that is based on genomic observations is discussed.