A novel type of RET rearrangement (PTC8) in childhood papillary thyroid carcinomas and characterization of the involved gene (RFG8).

As part of ongoing studies on the RET rearrangement frequency in children with papillary thyroid carcinoma (PTC) after their exposure to radioactive iodine after the Chernobyl reactor accident, new methods for the detection of novel types of RET rearrangements are being developed. In this study, an improved reverse transcription-PCR strategy is used successfully to identify a new type of RET rearrangement. This rearrangement is designated PTC8 and the involved RET-fused gene (RFG) as RFG8. The identification of two reciprocal transcripts coding for the RFG8/RET and RET/RFG8 fusions suggests that the PTC8 rearrangement results from a balanced chromosomal translocation. With a view to clarify its role in tumor induction, we compared the fusion products with those of previously described RET rearrangements. We therefore sequenced and characterized the RFG8 cDNA, which showed no significant similarity to any functional protein described as yet. RFG8 is located on chromosome 18q21-22 and is expressed ubiquitously. Bioinformatic analysis predicts with a high probability that the corresponding rfg8 protein is located in the cytoplasm and is involved putatively in intracellular transport processes. Furthermore, we identified coiled-coil structures upstream of the breakpoint with one of the coiled-coils showing dimerization capability. Thus the rfg8/ret fusion protein exhibits structures for oncogenic activation that are similar to those observed in previously described RET fusions.

Detection of a novel type of RET rearrangement (PTC5) in thyroid carcinomas after Chernobyl and analysis of the involved RET-fused gene RFG5.

A novel type of RET rearrangement, PTC5, was detected in papillary thyroid carcinomas of two patients exposed to radioactive fallout after Chernobyl. Reverse transcription-PCR and rapid amplification of 5'-cDNA ends revealed a fusion of the ret tyrosine kinase (TK) domain with a sequence identical to that described previously as ret-II. Ret-II is a transfection artifact in NIH3T3 cells and has not yet been detected in any human tumor. Overlapping sequences found in the expressed sequence tag databases enabled us to sequence the COOH terminus of the ret-fused gene 5 (RFG5). The combined data made it possible to assemble a full-length rfg5 protein sequence. Computer-assisted analysis of this sequence reveals four putative coiled-coil structures, possibly involved in dimerization, but no membrane-binding sequences. Northern blots show a ubiquitous RFG5 expression in various normal tissues, including the thyroid gland. In addition to the RFG5/RET, we also detected the reciprocal RET/RFG5 transcript in both tumor samples, suggesting that the rearrangement is based on a balanced reciprocal translocation. In agreement with other rearranged TKs, it is concluded that the transforming action of the new fusion protein rfg5/ret in thyroid tumors may be due to an activation of the ret TK by constitutive expression and dimerization potential of the 5'-fused rfg5 protein. Ret immunohistochemistry indicates that the fusion protein is expressed in all cells of PTC5 tumors, suggesting that RFG5/RET rearrangement is an early event in thyroid carcinogenesis.

The transcription coactivator HTIF1 and a related protein are fused to the RET receptor tyrosine kinase in childhood papillary thyroid carcinomas.

Children exposed to radioactive iodine as a consequence of the Chernobyl reactor accident have an increased risk of papillary thyroid carcinomas (PTC). The predominant molecular lesions in these tumors are rearrangements of the RET receptor tyrosine kinase (tk). Here we report on two novel types of RET rearrangement, PTC6 and 7, and describe the fusion products and the ret fused gene (rfg) proteins. Like the other rfg proteins identified so far they are ubiquitously expressed, not membrane-bound and contain coiled coil domains required for constitutive activation of the ret tk domain. In the PTC6 rearrangement the ret tk domain is fused to the aminoterminal part of the human transcription intermediary factor htif 1. In the PTC7 rearrangement the ret tk domain is fused to a novel protein that is strongly related to htif1. Like htif1 it contains a RBCC motif (ring finger, B boxes, coiled coil domain) located in the aminoterminal part and a phd finger and a bromodomain in the carboxyterminal part. Htif1 and related proteins are transcription coactivators for nuclear receptors, thus participating in controlling cellular development, differentiation and homeostasis. This is the first report on their involvement in human thyroid carcinogenesis.

High prevalence of RET rearrangement in thyroid tumors of children from Belarus after the Chernobyl reactor accident.

RET rearrangement was studied in papillary thyroid carcinomas (PTC) of children exposed to radioactive fallout in Belarus after the Chernobyl accident. To detect RET rearrangement in small tissue samples from thyroidectomy specimen (12 PTC of children; 2 PTC and 1 follicular carcinoma of adults; non-tumorous thyroid tissue of 4 children and 4 adults as controls), a RT-multiplex PCR was developed using primers suited to amplify fragments in different quantities depending on the presence or absence of RET rearrangements in the tissues. The type of rearrangement was determined by RT-PCR and direct sequencing using primers for ret/PTC1, 2 and 3. Two-thirds of the papillary thyroid carcinomas of the children revealed a RET rearrangement, with ret/PTC3 being more frequent by a factor of 3 than ret/PTC1. ret/PTC2 was not detected. All RET rearrangement-positive tumors had lymph node metastasis while half of the tumors with wild-type cRET had not. More than half of the cases with ret/PTC3 expressed not only the ELE/RET transcript as expected, but also the RET/ELE transcript. Intrachromosomal rearrangement involving RET and the adjacent H4 or ELE gene on chromosome no. 10 is a very frequent event in thyroid cancer of children of the Chernobyl-contaminated zone of Belarus.

A new form of RET rearrangement in thyroid carcinomas of children after the Chernobyl reactor accident.

Intrachromosomal rearrangements involving the RET and the adjacent H4 or ELE1 gene are very frequent events in thyroid cancer of children from Belarus after the Chernobyl reactor accident (Klugbauer et al., 1995). The fusion product between ELE1 and RET (RET/ PTC3) seems to be the prevailing type of rearrangement as shown in a recently published study using a novel RT multiplex PCR approach in combination with the identification of the rearrangement type by RT-PCR and direct sequencing. Now we found a new type of RET rearrangement: By the 5'RACE method we demonstrated in cDNA the fusion of the tyrosine kinase domain of RET with a truncated ELE1 gene shorter than the ELE1 in RET/PTC3. Sequencing of genomic DNA revealed a rearrangement breakpoint at position 41 of a new ELE1 intron (522 bp in length). The new oncogene RET/ delta PTC3 is shortened by one ELE1 exon of 144 bp in length. Structural considerations of the ele1 amino terminal of RET/ delta PTC3 suggest that the transforming activity of the fusion protein is apparently not affected by this truncation. The exon lacking in RET/ delta PTC3 was found to code in the reciprocal transcript RET/ delta ELE1 and increased its size by 144 bp. Obviously the new and possibly additional ELE/RET fusion molecules might even increase the high prevalence of ELE1/RET rearrangements in thyroid carcinomas of children after the Chernobyl reactor accident.

Molecular analysis of new subtypes of ELE/RET rearrangements, their reciprocal transcripts and breakpoints in papillary thyroid carcinomas of children after Chernobyl.

A high prevalence of RET rearrangements is found in papillary thyroid carcinomas (PTC) of children from Belarus after the Chernobyl reactor accident. The ELE/RET rearrangement (PTC3) is prevailing. Aberrant types of ELE/RET rearrangement have been found with a truncated ELE1 gene: As compared with the common form (PTC3r1) one aberrant type is shorter by one 144 bp exon (PTC3r2) (three cases); in the second atypic form (PTC3r3) the ELE1 part is 18 bp shorter than in PTC3r1. In agreement with the observation that the oncogenic RET is generated by a paracentric inversion at chromosome 10, we found not only ELE/RET, but also RET/ELE transcripts in these tumors. Sequencing of the breakpoint regions at the genomic DNA level revealed DNA modifications that might be relevant for illegitimate recombination after DNA doublestrand breaks. The high prevalence of ELE/RET rearrangements and various subtypes appears to be typical for radiation-induced thyroid carcinomas of children after the Chernobyl reactor accident.

Pattern of radiation-induced RET and NTRK1 rearrangements in 191 post-chernobyl papillary thyroid carcinomas: biological, phenotypic, and clinical implications.

Molecular genetic aberrations and the related phenotypes were investigated in 191 papillary thyroid carcinomas (PTCs) from patients exposed at young age to radioiodine released from the Chernobyl reactor. A high prevalence of RET gene rearrangements (62.3%) with a significant predominance of ELE1/RET (PTC3) over H4/RET (PTC1) rearrangements was found in PTCs of the first post-Chernobyl decade. NTRK1 rearrangements were rare (3.3%). In 3.3%, we observed novel types of RET rearrangements: GOLGA5/ RET (PTC5), HTIF/RET (PTC6), RFG7/RET (PTC7), and an as yet undefined RFGX/RET.RET rearrangements, preferentially ELE1/RET, are related to rapid tumor development. At longer intervals after exposure to ionizing radiation, the prevalence of RET rearrangements declines with a shift from ELE1/RET to H4/RET, most significantly in female patients. The prevalence of specific types of rearrangements is independent of age at irradiation. A significantly higher prevalence of ELE1/RET was observed in the most heavily contaminated Oblasts, Gomel and Brest, suggesting a preferential formation of this type of rearrangement after high thyroid doses. RET rearrangement is related to aggressive growth: Rearrangement-positive PTCs were in a more advanced pT category and more frequently in the pN1 category at presentation than rearrangement-negative PTCs. ELE1/RET is related to the solid variant of PTC, H4/RET more frequently to typical papillary structures. The genotype/phenotype evaluation of post-Chernobyl PTCs reveals a characteristic spectrum of gene rearrangements that lead to typical phenotypes with important biological and clinical implications.

Fine mapping and single nucleotide polymorphism association results of candidate genes for asthma and related phenotypes.

Several genome-wide screens for asthma and related phenotypes have been published to date but data on fine-mapping are scarce. For higher resolution we performed a fine-mapping study with 2 cM average spacing in often discussed asthma candidate regions (2p, 5q, 6p, 7p, 9q, 11p, and 12q) to narrow down the regions of interest. All participants of a Caucasian family study (97 families with at least two affected sib pairs) were genotyped for 49 supplementary polymorphic dinucleotide markers. Our results indicate increased evidence for linkage on chromosome 6p, 9q, and 12q. These candidate regions were further analyzed with SNP polymorphisms in the endothelin 1 (EDN1), lymphotoxin alpha (LTA), and neuronal nitric oxide synthase (NOS1) genes. In addition, IL4 -590C>T and IL10 -592C>A, localized on chromosomes 5q and 1q, respectively, have been analyzed for SNP association. Of the six SNPs tested, four revealed weak association with the examined phenotypes. These are the IL10 -592C>A SNP in the interleukin 10 gene (p=0.036 for eosinophil cell counts), the 4124T>C SNP in EDN1 (p=0.044 for asthma), the 3391C>T SNP in NOS1 with eosinophil cell counts (p=0.0086), and the 5266C>T polymorphism, also in the NOS1 gene, for high IgE levels (p=0.022). In summary, fine mapping data enable us to confine asthma candidate regions, while variants of EDN1 and NOS1, or nearby genes, may play an important role in this context.

Molecular genetics of childhood papillary thyroid carcinomas after irradiation: high prevalence of RET rearrangement.

Epidemiological studies have revealed a connection between thyroid carcinogenesis and a history of radiation. The molecular mechanisms involved are not well understood. It has been claimed that RAS, p53 or GSP mutations and RET or TRK rearrangements might play a role in adult thyroid tumors. In childhood, the thyroid gland is particularly sensitive to ionizing radiation. The reactor accident in Chernobyl provided a unique chance to study molecular genetic aberrations in a cohort of children who developed papillary thyroid carcinomas after a short latency time after exposure to high doses of radioactive iodine isotopes. According to the concepts of molecular genetic epidemiology, exposure to a specific type of irradiation might result in a typical molecular lesion. Childhood papillary thyroid tumors after Chernobyl exhibit a high prevalence of RET rearrangement as almost the only molecular alteration. The majority showed RET/PTC3 (i.e., ELE/RET rearrangements), including several subtypes. Less frequently, RET/PTC1 (i.e., H4/RET rearrangements), and a novel type (RET/PTC5, i.e., RFG5/RET) were observed. Proof of reciprocal transcripts suggests that a balanced intrachromosomal inversion leads to this rearrangement. Breakpoint analyses revealed short homologous nucleotide stretches at the fusion points. In all types of rearrangement, the RET tyrosine kinase domain becomes controlled by 5' fused regulatory sequences of ubiquitously expressed genes that display coiled-coil regions with dimerization potential. Oncogenic activation of RET is apparently due to ligand-independent constitutive ectopic RET tyrosine kinase activity. The analysis of this cohort of children with radiation-induced thyroid tumors after Chernobyl provides insights into typical molecular aberrations in relation to a specific mode of environmental exposure and may serve as a paradigm for molecular genetic epidemiology.

[Radiation-induced thyroid carcinomas in children: high prevalence of RET rearrangement]. / Strahleninduzierte Schilddrüsenkarzinome bei Kindern: Hohe Prävalenz von RET-Rearrangement.

Papillary thyroid carcinomas were observed in children living in the Gomel region of Belarus at the time of the Chernobyl reactor accident in April 1986. Radioactive fallout, iodine-131 in particular, led to thyroid doses of > 10 Gy in some cases. Till now, more than 400 thyroid carcinomas developed. They provide a unique possibility to search for characteristic molecular aberrations. Small fresh frozen thyroid tumor samples from 59 children were available. cDNA after reverse transcription of mRNA was amplified by multiplex PCR and analyzed for the presence of RET rearrangement (PTC1, 2 or 3) by identification-PCR with specific primers and by direct sequencing. A significantly higher prevalence of RET rearrangement was found in the thyroid carcinomas of radiation-exposed children than formerly described for adult thyroid carcinomas. While the prevailing type of RET rearrangement in adult thyroid carcinomas is PTC1 involving RET and the H4 gene, the majority of tumors in radiation-exposed children shows PTC3. In this type of rearrangement the 3'-tyrosin kinase domain of RET becomes dependent on the 5'-regulatory part of the ELE gene. Different breakpoints were found in the ELE gene. Besides ELE/RET transcripts, reciprocal RET/ELE transcripts were expressed indicating a complete inversion of the two genes after double stand break and their functional activity in both rearranged forms. Paracentric inversion on chromosome 10 bringing the functional tyrosine kinase domain of c-RET under the regulatory control of the ubiquitously expressed ELE gene appears to be a typical molecular lesion in thyroid carcinomas of children after radiation. This rearrangement is thought to endow juvenile thyrocytes with a clonal growth advantage and may be a critical initiating event of thyroid carcinogenesis in radiation-exposed children.

Absence of RAS and p53 mutations in thyroid carcinomas of children after Chernobyl in contrast to adult thyroid tumours.

Thyroid carcinomas of an additional series of 34 children exposed to radioactive fall-out after the Chernobyl reactor accident were analysed for mutations in the H-, K- and N-RAS and the p53 gene. Allele-specific oligonucleotide hybridization, single-strand conformation polymorphism (SSCP) and direct sequencing did not disclose mutations in codons 12, 13 and 61 of RAS genes nor mutations in exons 5, 7 and 8 of p53. Considering the recently reported high prevalence of RET rearrangements of the PTC3 type in childhood tumours after Chernobyl (Klugbauer et al, 1995, Oncogene 11: 2459-2467), it follows that RET rearrangements are the most relevant molecular aberration in these radiation-induced tumours. RAS or p53 mutations do not play a role in childhood thyroid carcinogenesis after Chernobyl.

Single nucleotide polymorphism screening and association analysis--exclusion of integrin beta 7 and vitamin D receptor (chromosome 12q) as candidate genes for asthma.

BACKGROUND: The human genes coding for integrin beta 7 (ITGB7) and vitamin D receptor (VDR) are two of the several candidate genes for asthma and related phenotypes found in a promising candidate region on chromosome 12q that has been identified in multiple genomewide screens and candidate gene approaches. METHODS: All exons, including parts of the neighbouring introns, and the predicted promoter region of the ITGB7 gene were screened for common polymorphisms in 32 independent asthmatic and healthy probands, resulting in the detection of two single nucleotide polymorphisms (SNPs) unknown so far. In addition to these SNPs, five already described SNPs of the ITGB7 and one in the human VDR gene were analysed in a Caucasian sib pair study of 176 families with at least two affected children, using matrix assisted laser desorption/ionization time of flight mass spectrometry. All confirmed SNPs were tested for linkage/association with asthma and related traits (total serum IgE level, eosinophil cell count and slope of the dose-response curve after bronchial challenge). RESULTS: Two new variations in the ITGB7 gene were identified. The coding SNP in exon 4 causes a substitution of the amino acid GLU by VAL, whereas the other variation is non-coding (intron 3). None of the eight analysed SNPs, of either the ITGB7 or the VDR genes, showed significant linkage/association with asthma or related phenotypes in the family study. CONCLUSIONS: These findings indicate that neither the human ITGB7 nor the VDR gene seem to be associated with the pathogenesis of asthma or the expression of related allergic phenotypes such as eosinophilia and changes in total IgE level.

NTRK1 re-arrangement in papillary thyroid carcinomas of children after the Chernobyl reactor accident.

The prevalence of NTRK1 re-arrangement was determined in papillary thyroid carcinomas (PTCs) of children from Belarus who had been exposed to radioactive iodine after the Chernobyl reactor accident; 81 tumors were included, all of which were devoid of RET re-arrangement as analyzed in a current study on genomic alterations in PTC. Oncogenic fusion of the NTRK1 tyrosine kinase domain with the amino-terminal part of the tropomyosin gene (TPM3/NTRK1, trk) was observed in 5 tumors. A single tumor exhibited a TPR/NTRK1 fusion (TRK-T2). Reciprocal NTRK1/TPM3 transcripts were found in 4 of 5 tumors with TPM3/NTRK1 re-arrangement, indicating an intra-chromosomal balanced reciprocal inversion. No phenotypic differences from other post-Chernobyl childhood PTCs were detected. As compared with the high prevalence of RET re-arrangements reported for thyroid carcinomas of children after the Chernobyl reactor accident, NTRK1 re-arrangements appear rare. Our results confirm that activation of receptor tyrosine kinase genes plays the predominant role in post-Chernobyl childhood thyroid carcinogenesis.

RET rearrangements in radiation-induced papillary thyroid carcinomas: high prevalence of topoisomerase I sites at breakpoints and microhomology-mediated end joining in ELE1 and RET chimeric genes.

Children exposed to radioactive iodine after the Chernobyl reactor accident frequently developed papillary thyroid carcinomas (PTC). The predominant molecular lesions in these tumors are rearrangements of the RET receptor tyrosine kinase gene. Various types of RET rearrangements have been described. More than 90% of PTC with RET rearrangement exhibit a PTC1 or PTC3 type of rearrangement with an inversion of the H4 or ELE1 gene, respectively, on chromosome 10. To obtain closer insight into the mechanisms underlying PTC3 inversions, we analyzed the genomic breakpoints of 22 reciprocal and 4 nonreciprocal ELE1 and RET rearrangements in 26 post-Chernobyl tumor samples. In contrast to previous assumptions, an accumulation of breakpoints at the two Alu elements in the ELE1 sequence was not observed. Instead, breakpoints are distributed in the affected introns of both genes without significant clustering. When compared to the corresponding wildtype sequences, the majority of breakpoints (92%) do not contain larger deletions or insertions. Most remarkably, at least one topoisomerase I site was found exactly at or in close vicinity to all breakpoints, indicating a potential role for this enzyme in the formation of DNA strand breaks and/or ELE1 and RET inversions. The presence of short regions of sequence homology (microhomologies) and short direct and inverted repeats at the majority of breakpoints furthermore indicates a nonhomologous DNA end-joining mechanism in the formation of chimeric ELE1/Ret and Ret/ELE1 genes.

Bacterial phylogeny based on comparative sequence analysis.

Comparative sequence analysis of small subunit rRNA is currently one of the most important methods for the elucidation of bacterial phylogeny as well as bacterial identification. Phylogenetic investigations targeting alternative phylogenetic markers such as large subunit rRNA, elongation factors, and ATPases have shown that 16S rRNA-based trees reflect the history of the corresponding organisms globally. However, in comparison with three to four billion years of evolution the phylogenetic information content of these markers is limited. Consequently, the limited resolution power of the marker molecules allows only a spot check of the evolutionary history of microorganisms. This is often indicated by locally different topologies of trees based on different markers, data sets or the application of different treeing approaches. Sequence peculiarities as well as methods and parameters for data analysis were studied with respect to their effects on the results of phylogenetic investigations. It is shown that only careful data analysis starting with a proper alignment, followed by the analysis of positional variability, rates and character of change, testing various data selections, applying alternative treeing methods and, finally, performing confidence tests, allows reasonable utilization of the limited phylogenetic information.

Proteome analysis of rat hepatomas: carcinogen-dependent tumor-associated protein variants.

Proteome analysis led to the identification and characterization of tumor-associated protein variants by two-dimensional electrophoresis and mass spectrometry. We focused on comparing the influence of genotoxic nitroso compounds N-methyl-N-nitrosourea, diethylnitrosamine and N-nitrosomorpholine and the nongenotoxic peroxisome proliferator Nafenopin as tumor-inducing agents on the protein pattern of rat hepatomas. We found several tumor-associated variants that represent members of the aldo-keto reductase superfamily. Their induction and/or inhibition was specifically related to the carcinogen used for tumor induction. The most prominent tumor-associated protein, rat aldose reductase-like protein-1 (rARLP-1) (69% sequence identity to lens aldose reductase) and three additional types of rARLP-1 were detected in nitroso compound-induced rat hepatomas, while rat aldo-keto reductase protein-c (Rak-c), a novel tumor-associated variant (65% sequence identity with 3alpha-hydroxysteroid dehydrogenase) was discovered in N-methyl-N-nitrosourea-induced hepatomas only. 3Alpha-hydroxysteroid dehydrogenase and delta4-3-ketosteroid-5beta-reductase, both liver-specific enzymes, were reduced in amount in all hepatomas investigated, independent of their mode of induction. We conclude, that detoxification enzymes like 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) and delta4-3-ketosteroid-5beta-reductase (5beta-Red) might be replaced in hepatomas by tumor-associated proteins that are often present in the embryonal state, like the rARLPs or the Rak-c protein. Their induction appears to reflect an altered constitutive pattern of detoxification enzymes, detoxifying toxic aldehydes being induced by nitroso compounds. In contrast, members of the aldo-keto reductase superfamily have not been found in Nafenopin-induced hepatomas. The pattern of tumor-associated protein variants is apparently characteristic for a given group of initiating carcinogens. The hypothesis is proposed that carcinogens leave specific fingerprints at the proteome level of manifest liver tumors.