Analysis of SIRT1 Gene SNPs and Clinical Characteristics in Medication-Related Osteonecrosis of the Jaw.

Certain genetic factors, including single-nucleotide polymorphisms (SNPs) in the SIRT1 gene, have been linked to medication-related osteonecrosis of the jaw (MRONJ). This study examined four SNPs in the SIRT1 gene and implemented multivariate statistical analysis to analyze genetic and clinical factors in MRONJ patients. Genomic DNA was isolated from peripheral blood samples of 63 patients of European origin treated for MRONJ, and four SNP genotypes in the gene encoding the SIRT-1 protein were determined by Sanger sequencing. The allele frequencies measured in the MRONJ population were compared with allele frequencies measured in the European population in the National Center for Biotechnology Information Allele Frequency Aggregator (NCBI ALFA) database. Genetic and clinical factors were examined with multivariate statistical analysis. A C:A allele distribution ratio of 77.8:22.2 was measured in the rs932658 SNP. In the ALFA project, a C:A allele distribution ratio of 59.9:40.1 was detected in the European population, which was found to be a significant difference (p = 4.5 × 10-5). Multivariate statistical analysis revealed a positive correlation (0.275) between the genotype of SNP rs932658 and the number of stages improved during appropriate MRONJ therapy. It is concluded that allele A in SNP rs932658 in the SIRT1 gene acts as a protective factor in MRONJ.

Genetic Factors Associated with the Development of Neuropathy in Type 2 Diabetes.

Neuropathy is a serious and frequent complication of type 2 diabetes (T2DM). This study was carried out to search for genetic factors associated with the development of diabetic neuropathy by whole exome sequencing. For this study, 24 patients with long-term type 2 diabetes with neuropathy and 24 without underwent detailed neurological assessment and whole exome sequencing. Cardiovascular autonomic function was evaluated by cardiovascular reflex tests. Heart rate variability was measured by the triangle index. Sensory nerve function was estimated by Neurometer and Medoc devices. Neuropathic symptoms were characterized by the neuropathy total symptom score (NTSS). Whole exome sequencing (WES) was performed on a Thermo Ion GeneStudio S5 system determining the coding sequences of approximately 32,000 genes comprising 50 million base pairs. Variants were detected by Ion Reporter software and annotated using ANNOVAR, integrating database information from dbSNP, ClinVar, gnomAD, and OMIM. Integrative genomics viewer (IGV) was used for visualization of the mapped reads. We have identified genetic variants that were significantly associated with increased (22-49-fold) risk of neuropathy (rs2032930 and rs2032931 of recQ-mediated genome instability protein 2 (RMI2) gene), rs604349 of myosin binding protein H like (MYBPHL) gene and with reduced (0.07-0.08-fold) risk (rs917778 of multivesicular body subunit 12B (MVB12B) and rs2234753 of retinoic acid X receptor alpha (RXRA) genes). The rs2032930 showed a significant correlation with current perception thresholds measured at 5 Hz and 250 Hz for n. medianus (p = 0.042 and p = 0.003, respectively) and at 5 Hz for n. peroneus (p = 0.037), as well as the deep breath test (p = 0.022) and the NTSS (p = 0.023). The rs2032931 was associated with current perception thresholds (p = 0.003 and p = 0.037, respectively), deep breath test (p = 0.022), and NTSS (p = 0.023). The rs604349 correlated with values measured at 2000 (p = 0.049), 250 (p = 0.018), and 5 Hz (p = 0.005) for n. medianus, as well as warm perception threshold measured by Medoc device (p = 0.042). The rs2234753 showed correlations with a current perception threshold measured at 2000 Hz for n. medianus (p = 0.020), deep breath test (p = 0.040), and NTSS (p = 0.003). There was a significant relationship between rs91778 and cold perception threshold (p = 0.013). In our study, genetic variants have been identified that may have an impact on the risk of neuropathy developing in type 2 diabetic patients. These results could open up new opportunities for early preventive measures and might provide targets for new drug developments in the future.

Evaluation of the effects of Lake Hévíz sulfur thermal water on skin microbiome in plaque psoriasis: An open label, pilot study.

Psoriasis is a chronic inflammatory skin disease. It is associated with changes in skin microbiome. The aim of this study was to evaluate how Lake Hévíz sulfur thermal water influences the composition of microbial communities that colonizes skin in patients with psoriasis. Our secondary objective was to investigate the effects of balneotherapy on disease activity. In this open label study, participants with plaque psoriasis underwent 30-min therapy sessions in Lake Hévíz, at a temperature of 36 °C, five times a week for 3 weeks. The skin microbiome samples were collected by swabbing method from two different areas (lesional skin-psoriatic plaque and non-lesional skin). From 16 patients, 64 samples were processed for a 16S rRNA sequence-based microbiome analysis. Outcome measures were alpha-diversity (Shannon, Simpson, and Chao1 indexes), beta-diversity (Bray-Curtis metric), differences in genus level abundances, and Psoriasis Area and Severity Index (PASI). Skin microbiome samples were collected at baseline, and immediately after treatment. Based on the visual examination of the employed alpha- and beta-diversity measures, no systematic difference based on sampling timepoint or sample location could be revealed in these regards. Balneotherapy in the unaffected area significantly increased the level of Leptolyngbya genus, and significantly decreased the level of Flavobacterium genus. A similar trend was revealed by the results of the psoriasis samples, but the differences were not statistically significant. In patients with mild psoriasis, a significant improvement was observed in PASI scores.

Comparison of surgical strategies in the treatment of low-risk differentiated thyroid cancer.

CONTEXT: Increasing diagnostic sensitivity in the detection of thyroid cancer has led to uncertainties in the optimal surgical approach of the smaller, low risk tumors. Current ATA guidelines consider lobectomy safe between 1 and 4 cm, while ETA advocates for primary total thyroidectomy to avoid reoperation, as final risk stratification is based on the histological results. OBJECTIVE: Our aim was to compare the differences in outcomes that are potentially achievable with adherence to the different guidelines, and also to examine the predictive value of clinical parameters on the incidence of postoperative risk factors. METHODS: We performed a retrospective cohort database analysis to identify the different surgical outcomes (based on postoperative risk factors) using ATA and ETA guidelines; the hypothetical rate of completion thyroidectomy when ATA or ETA recommends lobectomy; the accuracy of our preoperative evaluation; the utility of preoperative findings in predicting the optimal surgical strategy using binary logistic regression. RESULTS: Out of 248 patients, 152 (ATA) and 23 (ETA) cases would have been recommended for initial lobectomy. Following the guidelines, a postoperative risk factor would have been present in 61.8, and 65.2% of the cases, respectively. Except for angioinvasion, tumor size was not a significant predictor for the presence of postoperative risk factors. CONCLUSION: Current pre-operative criteria are inadequate to accurately determine the extent of initial surgery and our postoperative findings verify the frequent need for completion thyroidectomy using both guidelines. As a consequence, in the absence of effective pre-operative set of criteria, we advocate primary total thyroidectomy in most cases.

Co-occurrence of thyroid and breast cancer is associated with an increased oncogenic SNP burden.

BACKGROUND: Epidemiological evidence suggests that synchronous or metachronous presentation of breast and thyroid cancers exceeds that predicted by chance alone. The following potential explanations have been hypothesized: common environmental or hormonal factors, oncogenic effect of the treatment for the first cancer, closer follow-up of cancer survivors, shared underlying genetic risk factors. While some cases were found to be related to monogenic disorders with autosomal inheritance, the genetic background of most cases of co-occurring breast and thyroid cancer is thought to be polygenic. METHODS: In this retrospective case-control study we compared the genetic profile of patients with a history of breast cancer (n = 15) to patients with co-occurring breast and thyroid cancer (n = 19) using next generation sequencing of 112 hereditary cancer risk genes. Identified variants were categorized based on their known association with breast cancer and oncogenesis in general. RESULTS: No difference between patients with breast and double cancers was observed in clinical and pathological characteristics or the number of neutral SNPs. The unweighted and weighted number of SNPs with an established or potential association with breast cancer was significantly lower in the group with breast cancer only (mean difference - 0.58, BCa 95% CI [- 1.09, - 0.06], p = 0.029, and mean difference - 0.36, BCa 95% CI [- 0.70, - 0.02], p = 0.039, respectively). The difference was also significant when we compared the number of SNPs with potential or known association with any malignancy (mean difference - 1.19, BCa 95% CI [- 2.27, - 0.11], p = 0.032 for unweighted, and mean difference - 0.73, BCa 95% CI [- 1.32, - 0.14], p = 0.017 for weighted scores). CONCLUSION: Our findings are compatible with the hypothesis of genetic predisposition in the co-occurrence of breast and thyroid cancer. Further exploration of the underlying genetic mechanisms may help in the identification of patients with an elevated risk for a second cancer at the diagnosis of the first cancer.

Targeted Mutational Profiling and a Powerful Risk Score as Additional Tools for the Diagnosis of Papillary Thyroid Cancer.

Nowadays, the complementary diagnostics based on the suspicious thyroid lesion specific mutational state analysis is indispensable in the clinical practice. We aimed to test and validate our novel 568-mutational hotspot panel (23 cancer-related genes) on papillary thyroid cancers (PTCs) and their tumor-free pairs to find the most powerful mutation pattern related to PTC. The sequencing method was carried on with Ion Torrent PGM on 67 thyroid tissue samples. The most commonly detected mutation was the BRAF c.1799 T > A in all non-classical PTC cases. We utilized a multivariate statistical method (CVA) to determine a discrimination score based on mutational data array and to assess malignancy risk. Based on variants, the BRAF gene has by far the highest indicative power, followed by TSHR and APC. We highlighted novel aspects of the mutational profile and genetic markers of PTC. CVA has correctly assigned most of the samples based on the mutation frequencies and different variables of the selected genes, with high analytical probabilities. The final goal is to set up a new comprehensive rule-in and rule-out test to support the clinical decision making mainly in inconclusive fine-needle aspiration biopsy cases.

[Genetic testing of thyroid nodules using a gene panel developed on a new generation sequencing platform]. / A pajzsmirigygöbök genetikai vizsgálata újgenerációs szekvenáláson alapuló platformon kifejlesztett génpanel segítségével.

Introduction: Twenty-five percent of fine-needle aspiration biopsy samples of thyroid nodules produce indeterminate cytological results. Genetic testing of nodules can contribute to accurate diagnosis. Aim: Developing the first gene panel in Europe utilizing the 23 most relevant thyroid oncogenes with 568 mutations. Method: Examination of the isolated DNA from biopsy samples by Ion Torrent new generation sequencing. Results: The validation of our method was performed on tumor tissue samples, in which 127 genetic variations were identified, yet unknown in thyroid tumors. AXIN1 was the most polymorphic gene, while BRAF c.1799T>A (V600E) was the most frequently identified mutation. We detected 36 clinically relevant variants, 75% of which have not been described in the literature. Six of our 8 cytologically malignant and 8 of our 14 indeterminate as well as 20 of our 28 cytologically benign samples were identified as containing pathologic variants in a driver gene (BRAF c.1799T>A, NRAS c.181C>A). Conclusion: We have developed a validated, reliable new generation sequencing-based method with high positive predictive value (89%) and sensitivity (79%), suitable for the early detection of malignant lesions in the thyroid. Orv Hetil. 2019; 160(36): 1417-1425.

Long-term selective estrogen receptor-beta agonist treatment modulates gene expression in bone and bone marrow of ovariectomized rats.

Gonadal hormones including 17ß-estradiol exert important protective functions in skeletal homeostasis. However, numerous details of ovarian hormone deficiency in the common bone marrow microenvironment have not yet been revealed and little information is available on the tissue-specific acts either, especially those via estrogen receptor beta (ERß). The aim of the present study was therefore to examine the bone-related gene expression changes after ovariectomy (OVX) and long-term ERß agonist diarylpropionitrile (DPN) administration. We found that OVX produced strong and widespread changes of gene expression in both femoral bone and bone marrow. In the bone out of 22 genes, 20 genes were up- and 2 were downregulated after OVX. It is noteworthy that DPN restored mRNA expression of 10 OVX-induced changes (Aldh2, Col1a1, Daam1, Fgf12, Igf1, Il6r, Nfkb1, Notch1, Notch2 and Psen1) suggesting a modulatory role of ERß in bone physiology. In bone marrow, out of 37 categorized genes, transcription of 25 genes were up- and 12 were downregulated indicating that the marrow is highly responsive to gonadal hormones. DPN modestly affected transcription, only expression of two genes (Nfatc1 and Tgfb1) was restored by DPN action. The PI3K/Akt signaling pathway was the most affected gene cluster following the interventions in bone and bone marrow, as demonstrated by canonical variates analysis (CVA). We suggested that our results contribute to a deeper understanding of alterations in gene expression of bone and bone marrow niche elicited by ERß and selective ERß analogs.

[Literature review and presentation of our own research results regarding the effects on bone of tyrosine kinase inhibitors imatinib and nilotinib used in the treatment of oncohematological diseases]. / Az onkohematológiai betegségek kezelésében használt tirozinkináz-gátló imatinib és nilotinib csonthatásainak irodalmi áttekintése és a saját kutatási eredmények bemutatása.

Tyrosine kinase inhibitors are widely used for treatment of certain oncohematological diseases. Several clinical studies have confirmed that specific BCR-ABL tyrosine kinase inhibitors alter the physiological process of bone tissue in a complex and unclearly identified manner. Since these treatments are being given to more and more patients, and the therapy takes decades or lasts even lifelong, it is justifiable to obtain more detailed knowledge of the molecular background of these mechanisms. In this article the authors summarize preliminary research results and human clinical observations on imatinib and nilotinib which are related to bone metabolism, and present the results of their own experiments in in vitro osteoblast cultures. Based on the presented results, the effects of imatinib and nilotinib on bone cells depend on the concentration of imatinib and nilotinib, the maturation stage of the cells and the distribution ratio of receptor tyrosine kinase signaling pathways. In this study the authors firstly prepared a stop-gap, comprehensive review in the Hungarian literature, regarding the effects of tyrosine kinase inhibitors on bone metabolism. In addition they firstly performed whole transcriptome analysis on osteoblasts in order to obtain a better understanding of the cellular molecular mechanisms. Orv. Hetil., 2016, 157(36), 1429-1437.

Effects of imatinib and nilotinib on the whole transcriptome of cultured murine osteoblasts.

Numerous clinical observations have confirmed that breakpoint cluster region-abelson fusion oncoprotein tyrosine kinase inhibitors used in leukemia treatment alter bone physiology in a complex manner. The aim of the present study was to analyze the whole transcriptome of cultured murine osteoblasts and determine the changes following treatment with imatinib and nilotinib using Sequencing by Oligonucleotide Ligation and Detection next generation RNA sequencing. This study also aimed to identify candidate signaling pathways and network regulators by multivariate Ingenuity Pathway Analysis. Based on the right-tailed Fisher's exact test, significantly altered pathways including upstream regulators were defined for each drug. The correlation between these pathways and bone metabolism was also examined. The preliminary results suggest the two drugs have different mechanisms of action on osteoblasts, and imatinib was shown to have a greater effect on gene expression. Data also indicated the potential role of a number of genes and signaling cascades that may contribute to identifying novel targets for the treatment of metabolic bone diseases.

Next-generation sequencing of common osteogenesis imperfecta-related genes in clinical practice.

Next generation sequencing (NGS) is a rapidly developing area in genetics. Utilizing this technology in the management of disorders with complex genetic background and not recurrent mutation hot spots can be extremely useful. In this study, we applied NGS, namely semiconductor sequencing to determine the most significant osteogenesis imperfecta-related genetic variants in the clinical practice. We selected genes coding collagen type I alpha-1 and-2 (COL1A1, COL1A2) which are responsible for more than 90% of all cases. CRTAP and LEPRE1/P3H1 genes involved in the background of the recessive forms with relatively high frequency (type VII and VIII) represent less than 10% of the disease. In our six patients (1-41 years), we identified 23 different variants. We found a total of 14 single nucleotide variants (SNV) in COL1A1 and COL1A2, 5 in CRTAP and 4 in LEPRE1. Two novel and two already well-established pathogenic SNVs have been identified. Among the newly recognized mutations, one results in an amino acid change and one of them is a stop codon. We have shown that a new full-scale cost-effective NGS method can be developed and utilized to supplement diagnostic process of osteogenesis imperfecta with molecular genetic data in clinical practice.

PREDICTIVE VALUE OF SOMATIC MUTATIONS FOR THE DEVELOPMENT OF MALIGNANCY IN THYROID NODULES BY CYTOPATHOLOGY.

OBJECTIVE: The purpose of our prospective longitudinal study was to evaluate the predictive efficacy of genetic testing for malignancies in fine-needle aspiration biopsy samples that are cytologically benign at the time of biopsy. METHODS: A total of 779 aspirated cytological samples collected from thyroid nodules of 626 patients were included in a 3-year follow-up study. Consecutive patients with cytologically benign thyroid nodules by the Bethesda System for Reporting Thyroid Cytopathology were enrolled in the study. At enrollment, somatic 1-point nucleotide polymorphisms of BRAF and RAS family genes were tested by melting-point analysis, while RET/PTC and PAX8/PPAR-gamma rearrangements were examined by real-time polymerase chain reaction. The genetic test was considered to be positive if a somatic mutation was found. Malignant cytopathologic diagnoses were confirmed by histopathology. RESULTS: In samples collected from 779 thyroid nodules, there were 39 BRAF, 33 RAS mutations, and 1 RET/PTC rearrangements found at the beginning of the study. No PAX8/PPAR-gamma rearrangement was identified. There were 52 malignant thyroid tumors removed during follow-up, out of which 24 contained a somatic mutation. The specificity of the presence of somatic mutations for malignancies was as high as 93.3%, and sensitivity was 46.2%. The negative predictive value of genetic testing reached 96.0%. CONCLUSION: Our results show that our set of genetic tests can predict the appearance of malignancy in benign thyroid nodules (at the beginning of follow-up) with high specificity and strong negative predictive value. ABBREVIATIONS: BRAF = v-raf murine sarcoma viral oncogene homolog B1 FLUS = follicular lesion of undetermined significance FNAB = fine-needle aspiration biopsy FTC = follicular thyroid carcinoma HRAS = homologous to the oncogene from the Harvey rat sarcoma virus KRAS = homologous to the oncogene from the Kirsten rat sarcoma virus NRAS = first isolated from a human neuroblastoma/neuroblastoma RAS = viral oncogene homolog PAX8 = paired box 8 PCR = polymerase chain reaction PPAR-gamma = peroxisome proliferator-activated receptor gamma PTC = papillary thyroid carcinoma RAS = rat sarcoma RET = rearranged during transfection tyrosine-kinase proto-oncogene SM = somatic mutation SNP = single-nucleotide polymorphism.

Clinical Use of Next-Generation Sequencing in the Diagnosis of Wilson's Disease.

Objective. Wilson's disease is a disorder of copper metabolism which is fatal without treatment. The great number of disease-causing ATP7B gene mutations and the variable clinical presentation of WD may cause a real diagnostic challenge. The emergence of next-generation sequencing provides a time-saving, cost-effective method for full sequencing of the whole ATP7B gene compared to the traditional Sanger sequencing. This is the first report on the clinical use of NGS to examine ATP7B gene. Materials and Methods. We used Ion Torrent Personal Genome Machine in four heterozygous patients for the identification of the other mutations and also in two patients with no known mutation. One patient with acute on chronic liver failure was a candidate for acute liver transplantation. The results were validated by Sanger sequencing. Results. In each case, the diagnosis of Wilson's disease was confirmed by identifying the mutations in both alleles within 48 hours. One novel mutation (p.Ala1270Ile) was found beyond the eight other known ones. The rapid detection of the mutations made possible the prompt diagnosis of WD in a patient with acute liver failure. Conclusions. According to our results we found next-generation sequencing a very useful, reliable, time-saving, and cost-effective method for diagnosing Wilson's disease in selected cases.

Genetic Alterations in Hungarian Patients with Papillary Thyroid Cancer.

The incidence of thyroid cancers is increasing worldwide. Some somatic oncogene mutations (BRAF, NRAS, HRAS, KRAS) as well as gene translocations (RET/PTC, PAX8/PPAR-gamma) have been associated with the development of thyroid cancer. In our study, we analyzed these genetic alterations in 394 thyroid tissue samples (197 papillary carcinomas and 197 healthy). The somatic mutations and translocations were detected by Light Cycler melting method and Real-Time Polymerase Chain Reaction techniques, respectively. In tumorous samples, 86 BRAF (44.2%), 5 NRAS (3.1%), 2 HRAS (1.0%) and 1 KRAS (0.5%) mutations were found, as well as 9 RET/PTC1 (4.6%) and 1 RET/PTC3 (0.5%) translocations. No genetic alteration was seen in the non tumorous control thyroid tissues. No correlation was detected between the genetic variants and the pathological subtypes of papillary cancer as well as the severity of the disease. Our results are only partly concordant with the data found in the literature.

Strong effect of SNP rs4988300 of the LRP5 gene on bone phenotype of Caucasian postmenopausal women.

The purpose of this study was to identify relationships between single nucleotide polymorphisms (SNPs) in the genes of the Wnt pathway and bone mineral density (BMD) of postmenopausal women. We chose this pathway due to its importance in bone metabolism that was underlined in several studies. DNA samples of 932 Hungarian postmenopausal women were studied. First, their BMD values at different sites (spine, total hip) were measured, using a Lunar Prodigy DXA scanner. Thereafter, T-score values and the patients' body mass indices (BMIs) were calculated, while information about the fracture history of the sample population was also collected. We genotyped nine SNPs of the following three genes: LRP5, GPR177, and SP7, using a Sequenom MassARRAY Analyzer 4 instrument. The genomic DNA samples used for genotyping were extracted from the buccal mucosa of the subjects. Statistical analyses were carried out using the SPSS 21 and R package. The results of this analysis showed a significant association between SNP rs4988300 of the LRP5 gene and total hip BMD values. We could not reveal any associations between the markers of GPR177, SP7, and bone phenotypes. We found no effect of these genotypes on fracture risk. We could demonstrate a significant gene-gene interaction between two SNPs of LRP5 (rs4988300 and rs634008, p = 0.009) which was lost after Bonferroni correction. We could firmly demonstrate a significant association between rs4988300 of the LRP5 gene and bone density of the hip on the largest homogeneous postmenopausal study group analyzed to date. Our finding corroborates the relationship between LRP5 genotype and bone phenotype in postmenopausal women, however, the complete mechanism of this relationship requires further investigations.

Rapid and cost effective screening of breast and ovarian cancer genes using novel sequence capture method in clinical samples.

BRCA1 and BRCA2 are two well-known genes in the background of hereditary breast and ovarian cancer. There is also evidence that several other genes play an important role in the pathogenesis of these two malignancies. Latest population-scaled studies showed that certain mutations in different genes could cause similar risk elevation like BRCA2 mutations. In this study we present a new method to analyse the risk assessment of women to breast and ovarian cancer. Using Haloplex, a novel sequence capture method combined with next-generation sequencing we were able to perform rapid and cost-effective screening of 16 genes that could be associated with an increased risk of breast and ovarian cancer. The rapid and cost effective analysis of this 16-gene cohort can reveal the genetic background of approximately 30 % of hereditary and familiar cases of breast and ovarian cancers. Thus, it opens up a new and high-throughput approach with fast turnaround time to the genetic diagnostics of these disorders and may be helpful to investigate other familial genetic disorders as well.

Fast and robust next-generation sequencing technique using ion torrent personal genome machine for the screening of neurofibromatosis type 1 (NF1) gene.

Neurofibromatosis type 1 (NF1) gene exhibits one of the highest spontaneous mutation rates in the human genome. Identification of the NF1 mutation is challenging because the NF1 gene is very large and complex, lacking mutational "hot spots." There is no clustering of mutations, there are several pseudogenes, and a wide spectrum of different types of mutation has been recognized. To date, NF1 mutations or deleted regions have been detected with a number of techniques. With the appearance of next-generation sequencing (NGS) machines, molecular biology is in a new revolutionary phase. Our aim was to work out a method to use the high-throughput NGS machine, Ion Torrent PGM, in diagnostic settings for neurofibromatosis type 1. In our examination, we could reveal 21 distinct variations in NF1 gene in seven patients. This is an absolutely new method for exploring the genetic background of neurofibromatosis type 1 exhibiting the extremely high throughput of NGS in a diagnostic setting.

Marked increase of CYP24A1 mRNA level in hepatocellular carcinoma cell lines following vitamin D administration.

AIM: 1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) inhibits cell growth and induces apoptosis in numerous types of tumors. We aimed to examine the mRNA and protein expression of 1,25(OH)(2)D(3)-inactivating CYP24A1 and mRNA expression of the activating CYP27B1 enzymes, as well as that of vitamin D receptor (VDR), in hepatocellular carcinoma (HCC) cell cultures in response to 1,25(OH)(2)D(3) administration. MATERIALS AND METHODS: Increasing amounts of 1,25(OH)(2)D(3) (0.256-10 nM) were added to cultures of HepG2, Huh-Neo, Hep3B, Huh5-15 human HCC cell lines and cells then incubated for various time periods (30 min-28 h). The mRNA expression was analyzed by real time reverse transcription-polymerase chain reaction (RT-PCR). CYP24A1 protein in HepG2 cells was detected by immuncytochemistry. RESULTS: CYP24A1 mRNA expression significantly (p<0.0001) increased in response to 1,25(OH)(2)D(3) administration in two cell lines: in HepG2 cells, the CYP24A1 mRNA level exhibited 5,300-fold elevation, reaching a maximum value at 8 h; in Huh-Neo cells, the increase was 152-fold that of the baseline value, with the maximum being reached at 14 h. There was no significant change in Hep3B and Huh5-15 cell lines, nor was there any change in CYP27B1 and VDR gene expression in any cell cultures. Immuncytochemistry in HepG2 cells proved that gene activation was followed by CYP24A1 protein synthesis. CONCLUSION: Our novel data indicate that administration of 1,25(OH)(2)D(3) results in a marked increase of CYP24A1 mRNA expression in some, but not all, human HCC lines in vitro. These differences could be dependent upon the origin of the tumor cells.

[Comparative study of somatic oncogene mutations in normal thyroid tissues and thyroid neoplasms]. / Szomatikus onkogén mutációk összehasonlító vizsgálata egészséges és tumoros pajzsmirigy-szövetmintákban.

It is established that numerous somatic oncogene mutation (BRAF, NRAS, HRAS, KRAS) and gene translocations (RET/PTC, PAX8/PPAR-gamma) are associated with the development of thyroid cancer. In this study 22 intraoperative thyroid tissue samples (11 pathologic and 11 normal) were examined. Somatic single nucleotide polymorphisms were analyzed by LigthCycler melting method, while translocations were identified by real-time polymerase chain reaction technique. In tumorous sample 3 BRAF, 2 NRAS and one HRAS mutations were found, as well as one RET/PTC1 translocation. Results confirm international data showing that these oncogene mutations and translocations are linked to thyroid cancer. Cytological examination completed with genetic data may support the diagnosis of thyroid malignancies. In addition, genetic alterations may indicate malignant transformation and may become prognostic factors in future.