Knut Krohn

Alu Elements in ANRIL Non-Coding RNA at Chromosome 9p21 Modulate Atherogenic Cell Functions through Trans -Regulation of Gene Networks

The chromosome 9p21 (Chr9p21) locus of coronary artery disease has been identified in the first surge of genome-wide association and is the strongest genetic factor of atherosclerosis known today. Chr9p21 encodes the long non-coding RNA (ncRNA) antisense non-coding RNA in the INK4 locus (ANRIL). ANRIL expression is associated with the Chr9p21 genotype and correlated with atherosclerosis severity. Here, we report on the molecular mechanisms through which ANRIL regulates target-genes in trans, leading to increased cell proliferation, increased cell adhesion and decreased apoptosis, which are all essential mechanisms of atherogenesis. Importantly, trans-regulation was dependent on Alu motifs, which marked the promoters of ANRIL target genes and were mirrored in ANRIL RNA transcripts. ANRIL bound Polycomb group proteins that were highly enriched in the proximity of Alu motifs across the genome and were recruited to promoters of target genes upon ANRIL over-expression. The functional relevance of Alu motifs in ANRIL was confirmed by deletion and mutagenesis, reversing trans-regulation and atherogenic cell functions. ANRIL-regulated networks were confirmed in 2280 individuals with and without coronary artery disease and functionally validated in primary cells from patients carrying the Chr9p21 risk allele. Our study provides a molecular mechanism for pro-atherogenic effects of ANRIL at Chr9p21 and suggests a novel role for Alu elements in epigenetic gene regulation by long ncRNAs.

Low atmospheric oxygen avoids maturation, senescence and cell death of murine mesencephalic neural precursors

The efficient generation of specific brain cells in vitro may serve as a source of cells for brain repair in several devastating neurological diseases. Production of dopaminergic neurons from precursor cells for transplantation in Parkinsons disease has become a major research goal. We found that murine mesencephalic neurospheres were viable and proliferated, preserved telomerase activity, pluripotency and dopaminergic commitment for many weeks when cultured in 3% O2, whereas exposing these cells to 21% oxygen prohibited long‐term expansion. Microarray data suggest that a variety of genes related to the cell cycle, cell maturation and apoptosis are differentially regulated in midbrain‐derived precursors cultured in 3 versus 21% oxygen after 1–2 months. Taken together, we hypothesize that sustained high oxygen has deleterious effects on the self‐renewal capacity of mesencephalic neural precursors, possibly accelerating maturation and senescence resulting in overall cell loss. Gene regulation governed by low oxygen tension may be relevant to the normal development and survival of midbrain neurons.

Detection of thyroid-stimulating hormone receptor and Gsα mutations: in 75 toxic thyroid nodules by denaturing gradient gel electrophoresis

The actual frequency of constitutively activating thyrotropin receptor or Gsα mutations in toxic thyroid nodules (TTNs) remains controversial as considerable variation in the prevalence of these mutations has been reported. We studied a series of 75 consecutive TTNs and performed mutation screening by the more sensitive method of denaturing gradient gel electrophoresis (DGGE) in addition to direct sequencing. Furthermore, the likelihood of somatic mutations occurring in genes other than that for the thyroid-stimulating hormone receptor (TSHR) and exons 7–9 of the Gsα protein gene was determined by clonality analysis of TTNs, which did not harbor mutations in the investigated genes. In 43 of 75 TTNs (57%) constitutively active TSHR mutations were identified. Six TSHR mutations were detected only by DGGE, underlining the importance of a sensitive screening method. Novel, constitutively activating mutations were identified at positions 425 (Ser→Leu) and 512 (Leu→Glu/Arg). Furthermore, a new base substitution was detected at position Pro639Ala (CCA→GCA). Ten of 20 TSHR or Gsα mutation negative cases (50%) showed nonrandom X-chromosome inactivation, indicating clonal origin. In conclusion, somatic, constitutively activating TSHR mutations appear to be a major cause of TTNs (57%), while mutations in Gsα play a minor role (3%). The mutation negative but clonal cases indicate a probable involvement of somatic mutations other than in the TSH receptor or Gsα genes as the molecular cause of these hot nodules.

Hot microscopic areas of iodine-deficient euthyroid goitres contain constitutively activating TSH receptor mutations

Constitutively activating TSH receptor mutations have been established as the most common molecular basis for the pathogenesis of toxic thyroid nodules. These mutations result in uncontrolled signalling through the TSH receptor that is likely to cause hyperfunction and proliferation. The incidence of toxic multinodular goitres has been demonstrated to be related to iodine deficiency. Moreover, scintigraphically autonomous areas are found in 40% of euthyroid goitres from iodine‐deficient areas. To investigate the molecular cause of these autonomous areas, small autoradiographically hot areas were examined for somatic TSH receptor mutations using archival tissue sections from 14 patients with euthyroid goitres, which had been originally prepared nearly 20 years ago. All patients had received 125I 17 h preoperatively for the autoradiographic investigation of their thyroid. Areas with high and low 125I‐labelling on autoradiography sections were collected separately either from serial paraffin‐embedded tissue sections, or Eukitt‐embedded tissue sections containing the autoradiograph. After genomic DNA extraction, the transmembrane segment of the TSH receptor was PCR‐amplified and directly sequenced. Somatic TSH receptor mutations were identified in areas with high 125I‐labelling in four patients: A623I, L629P, F631L, and T632I. This is the first evidence that TSH receptor mutations occur in microscopic areas with increased 125I‐labelling in euthyroid goiters and it suggests that TSH receptor mutations in these areas confer the potential to develop into toxic thyroid nodules. It is therefore very likely that toxic thyroid nodules originate from small autonomous areas in iodine‐deficient euthyroid goitres that contain a TSH receptor mutation. Copyright

Association of FTO variants with BMI and fat mass in the self-contained population of Sorbs in Germany

The association between common variants in the FTO gene with weight, adiposity and body mass index (BMI) has now been widely replicated. Although the causal variant has yet to be identified, it most likely maps within a 47 kb region of intron 1 of FTO. We performed a genome-wide association study in the Sorbian population and evaluated the relationships between FTO variants and BMI and fat mass in this isolate of Slavonic origin resident in Germany. In a sample of 948 Sorbs, we could replicate the earlier reported associations of intron 1 SNPs with BMI (eg, P-value=0.003, β=0.02 for rs8050136). However, using genome-wide association data, we also detected a second independent signal mapping to a region in intron 2/3 about 40–60 kb away from the originally reported SNPs (eg, for rs17818902 association with BMI P-value=0.0006, β=−0.03 and with fat mass P-value=0.0018, β=−0.079). Both signals remain independently associated in the conditioned analyses. In conclusion, we extend the evidence that FTO variants are associated with BMI by putatively identifying a second susceptibility allele independent of that described earlier. Although further statistical analysis of these findings is hampered by the finite size of the Sorbian isolate, these findings should encourage other groups to seek alternative susceptibility variants within FTO (and other established susceptibility loci) using the opportunities afforded by analyses in populations with divergent mutational and/or demographic histories.

Genetic variation in GPR133 is associated with height: genome wide association study in the self-contained population of Sorbs

Recently, associations of several common genetic variants with height have been reported in different populations. We attempted to identify further variants associated with adult height in a self-contained population (the Sorbs in Eastern Germany) as discovery set. We performed a genome wide association study (GWAS) (approximately 390,000 genetic polymorphisms, Affymetrix gene arrays) on adult height in 929 Sorbian individuals. Subsequently, the best SNPs (P < 0.001) were taken forward to a meta-analysis together with two independent cohorts [Diabetes Genetics Initiative, British 1958 Birth Cohort, (58BC, publicly available)]. Furthermore, we genotyped our best signal for replication in two additional German cohorts (Leipzig, n = 1044 and Berlin, n = 1728). In the primary Sorbian GWAS, we identified 5 loci with a P-value < 10(-5) and 455 SNPs with P-value < 0.001. In the meta-analysis on those 455 SNPs, only two variants in GPR133 (rs1569019 and rs1976930; in LD with each other) retained a P-value at or below 10(-6) and were associated with height in the three cohorts individually. Upon replication, the SNP rs1569019 showed significant effects on height in the Leipzig cohort (P = 0.004, beta = 1.166) and in 577 men of the Berlin cohort (P = 0.049, beta = 1.127) though not in women. The combined analysis of all five cohorts (n = 6,687) resulted in a P-value of 4.7 x 10(-8) (beta = 0.949). In conclusion, our GWAS suggests novel loci influencing height. In view of the robust replication in five different cohorts, we propose GPR133 to be a novel gene associated with adult height.

The role of HPV RNA transcription, immune response-related gene expression and disruptive TP53 mutations in diagnostic and prognostic profiling of head and neck cancer

Stratification of head and neck squamous cell carcinomas (HNSCC) based on HPV16 DNA and RNA status, gene expression patterns, and mutated candidate genes may facilitate patient treatment decision. We characterize head and neck squamous cell carcinomas (HNSCC) with different HPV16 DNA and RNA (E6*I) status from 290 consecutively recruited patients by gene expression profiling and targeted sequencing of 50 genes. We show that tumors with transcriptionally inactive HPV16 (DNA+ RNA‐) are similar to HPV‐negative (DNA‐) tumors regarding gene expression and frequency of TP53 mutations (47%, 8/17 and 43%, 72/167, respectively). We also find that an immune response‐related gene expression cluster is associated with lymph node metastasis, independent of HPV16 status and that disruptive TP53 mutations are associated with lymph node metastasis in HPV16 DNA‐ tumors. We validate each of these associations in another large data set. Four gene expression clusters which we identify differ moderately but significantly in overall survival. Our findings underscore the importance of measuring the HPV16 RNA (E6*I) and TP53‐mutation status for patient stratification and identify associations of an immune response‐related gene expression cluster and TP53 mutations with lymph node metastasis in HNSCC.

Gene expression analysis reveals evidence for inactivation of the TGF-β signaling cascade in autonomously functioning thyroid nodules

Molecular eventsthat lead to the development of autonomously functioning thyroid nodules (AFTNs) are somatic mutations of the thyrotropin receptor (TSHR) in approximately 60% of the nodules and less frequently, somatic mutations in the Gsα protein. However, AFTNs without known mutations indicate that other causes remain to be identified. Moreover, the impact of constitutively activating TSHR mutations on the signal transduction network of the thyroid epithelial cell is unknown. We therefore investigated gene expression in 15 AFTNs and their surrounding tissue using Affymetrix GeneChips. Most prominently, data analysis revealed a changed pattern of gene expression in the TGF-β signaling cascade and 25 differentially regulated genes in AFTNs, including thyroid peroxidase, type I iodothyronine deiodinase and sialyltransferase (SIAT) 1. Strikingly coexpression of SIAT 1 and TSHR in COS-7 cells increased TSH binding and cell surface expression of the TSHR. Moreover, differences in gene expression patterns for AFTNs with and without TSHR mutations indicate specific alterations of signal transduction in AFTNs without TSHR mutations. These results suggest that AFTNs with TSHR mutations harbor further mechanisms of forward stimulation. Furthermore, they give important leads to elucidate the molecular etiology of AFTNs without TSHR mutations.

Ras mutations are rare in solitary cold and toxic thyroid nodules

OBJECTIVE Activation of ras proto‐oncogenes as a result of point mutations is detectable in a significant percentage of most types of tumour. Similar to neoplasms of other organs, mutations of all three ras genes can be found in thyroid tumours. H‐, K‐ and N‐ras mutations have been detected in up to 20% of follicular adenomas and adenomatous nodules which were not functionally characterized. This raises the question as to whether ras mutations are specific for hypofunctional nodules and TSH receptor mutations for hyperfunctioning nodules.

Perspectives for Improved and More Accurate Classification of Thyroid Epithelial Tumors

CONTEXT Histologic examination of thyroid nodules is the current standard to distinguish benign from malignant thyroid epithelial tumors and to classify histologic subtypes. This review analyzes the problems in histological differential diagnosis as well as contradictions between histology and molecular data and describes possibilities to combine histology with molecular data in an effort to more accurately classify thyroid epithelial tumors. EVIDENCE ACQUISITION Published literature, addressing the current recommendations for thyroid tumor classification, as well as literature on the application of histology and molecular studies on the etiology of thyroid tumors is analyzed. EVIDENCE SYNTHESIS The current histologic criteria to classify thyroid tumors, especially follicular-patterned tumors, are hampered by considerable interobserver variability. The detection of somatic mutations via genotyping and the definition of potentially informative gene expression signatures by microarray analyses, which can distinguish cancer subtypes as well as low- and high-risk cohorts, have recently demonstrated significant diagnostic potential. Moreover, in a routine diagnostic setting, micro-RNA profiling appears most promising due to their relative stability and the high accuracy of their expression profiles. CONCLUSIONS It is very likely that molecular definitions of thyroid tumors mentioned in the current World Health Organization classification will be further developed, leading to future progress in defining thyroid tumor types by an integrated histologic and molecular approach. These integrated classifications need to be evaluated for their specific impact on thyroid tumor diagnosis and prognosis.