Ewing family tumors (EFTs) are small round blue cell tumors that show features of neuroectodermal differentiation. However, the histogenetic origin of EFTs is still a matter of debate. We used high-density DNA microarrays for the identification of EFT-specific gene expression profiles in comparison with normal tissues of diverse origin. We identified 37 genes that are up-regulated in EFTs compared with normal tissues and validated expression of these genes in EFTs by both conventional and quantitative reverse transcription-polymerase chain reaction. The expression pattern of EFT-associated genes in normal tissues indicated a high similarity between EFTs and fetal and neuronal as well as endothelial tissues and supports the concept that a primitive neural crest-derived progenitor at the transition to mesenchymal and endothelial differentiation is transformed in EFTs. EFT-associated genes could be used for molecular discrimination between EFTs and other small round blue cell tumors and clearly identified a cell line (SK-N-MC) that was initially established as neuroblastoma as being an EFT. Ectopic expression of the EFT-specific EWS-FLI1 fusion protein in human embryonic kidney (HEK293) cells was not sufficient to induce the complete EFT-specific gene expression signature, suggesting that the EFT-specific gene expression profile is not just a consequence of EWS-FLI1 expression but depends on the histogenetic background of the EFT stem cell.

Epigenetic silencing of the FHIT tumor suppressor gene is a novel inactivation mechanism to be considered in the development of intrahepatic cholangiocarcinomas. However, a statistically significant inverse correlation between K-Ras activation and RASSF1A inactivation was not found.

Periocular sebaceous gland carcinomas (SGCs) occur in the eyelids either sporadically or as a phenotypic feature of Muir-Torre syndrome (MTS). In knockout mice mismatch-repair (MMR) defects or inactivation of the fragile histidine triad (FHIT) gene are associated with MTS-like signs, including SGC. To dissect the genetic alterations associated with microsatellite instability (MSI) and inactivation of the FHIT gene, we studied nine periocular SGC specimens from MTS patients. Immunohistochemistry was performed for FHIT, MSH2, MLH1, and MSH6. We assessed MSI as well as loss of heterozygosity (LOH) at the FHIT locus with polymorphic markers and genomic multiplex PCR. Epigenetic silencing was detected by methylation-specific PCR (MSP) and combined bisulfite restriction analysis (COBRA). Our analyses identified two SGCs with FHIT positivity and high-grade MSI, and seven cases with loss of FHIT and microsatellite stability (MSS). MSI correlated with loss of MSH2 and MLH1 immunostaining. Loss-of-function mechanisms affecting the FHIT gene were identified as intragenic deletions eliminating the coding exons 5 and 6 on one hand, and complete biallelic methylation of the FHIT transcription regulatory region on the other hand. Germinal FHIT mutations as a predisposing factor for MTS were excluded in two index patients with cancer in three generations, including an FHIT-negative SGC. Our data suggest that either somatic inactivation of the FHIT gene associated with MSS or inactivation of the MMR system resulting in MSI contribute to the development of periocular SGCs in presumptive MTS.

The CTG18.1 repeat expansion may reduce gene expression of TCF4 and ZEB1, suggesting that a mechanism triggering a loss of function may contribute to FECD. The correlation of CTG18.1 repeat expansion from blood and the cornea may represent the first step toward investigating the potential relevance of testing the blood of cornea donors to minimize the risk of transplanting grafts potentially affected with FECD.

Pluripotency is characterized by specific transcription factors such as OCT4, NANOG, and SOX2, but also by pluripotency-associated microRNAs (miRs). Somatic cells can be reprogrammed by forced expression of these factors leading to induced pluripotent stem cells (iPSCs) with characteristics similar to embryonic stem cells (ESCs). However, current reprogramming strategies are commonly based on viral delivery of the pluripotency-associated factors, which affects the integrity of the genome and impedes the use of such cells in any clinical application. In an effort to establish nonviral, nonintegrating reprogramming strategies, we examined the influence of hypoxia on the expression of pluripotency-associated factors and the ESC-specific miR-302 cluster in primary and immortalized mesenchymal stromal cells (MSCs). The combination of hypoxia and fibroblast growth factor 2 (FGF2) treatments led to the induction of OCT4 and NANOG in an immortalized cell line L87 and primary MSCs, accompanied with increased doubling rates and decreased senescence. Most importantly, the endogenous ECS-specific cluster miR-302 was induced upon hypoxic culture and FGF2 supplementation. Hypoxia also improved reprogramming of MSCs via episomal expression of pluripotency factors. Thus, our data illustrate that hypoxia in combination with FGF2 supplementation efficiently facilitates reprogramming of MSCs.

An expanded TGC allele with more than 50 TGC repeats in intron 2 and the described risk allele G of the polymorphism rs613872 in intron 3 of the TCF4 gene appear as an association to FECD. The chance to be affected by FECD is up to 30 times higher. With molecular genetics also donors with clinically unknown FECD may be detected.

DNA-microarrays allow the analysis of almost the complete gene expression program of tumor samples and normal control samples in a single experiment. This allows the processing of a large number of samples in a reasonable short time. Tumor specific gene expression profiles can be used for molecular tumor classification and as a new diagnostic tool. In addition, the identification of tumor specific genes can help to understand the biology of tumor cells and identified genes can be used for the development of new therapeutic strategies. However, the huge amount of data generated by DNA-microarrays creates new challenges for data analysis. In addition, accuracy and reproducibility of the available techniques require complementary methods for verification of DNA-microarray data.

The purpose of this study was to report on two novel missense mutations of the cornea-specific TGFBI gene in one single patient and in two generations of a family diagnosed with unique corneal dystrophy (CD) phenotypes. Ophthalmologic examination, in several cases ocular coherence tomography of the anterior segment (AS-OCT), was performed in 21 affected patients and in two unaffected members of one affected family. Coding regions of the TGFBI gene were direct sequenced in all 23 individuals. The two novel mutations were verified by RFLP analysis. A novel mutation c.1640T > G (p.Phe574Cys) in exon 12 of the TGFBI gene was detected in one single patient with recurrent granular intrastromal deposits comparable to a type of granular dystrophy. In AS-OCT, the deposit pattern reached up to the Descemet's layer. A further novel mutation c.393G > T(p.Glu131Asp) in exon 4 of the TGFBI gene was detected in all three affected members of one family with superficial cloud- and honeycomb-like opacifications, comparable to a Schnyder corneal dystrophy. Two unaffected members did not carry this alteration. The two identified novel mutations add other two phenotypes in patients suffering from TGFBI-linked CD to those reported so far. In one case, clinical finding indicates a Schnyder corneal dystrophy-like phenotype due to its superficial crystalline shape, and in the second one, granular deposits who reach Descemet's layer indicate a granular CD subtype. Molecular genetic analysis may help to distinguish those subtypes and to decide for specific treatment in time of a wide variation of corneal surgical techniques.