Damjan Glavač

Germline mutations in the Von Hippel-Lindau disease (VHL) gene in families from North America, Europe, and Japan

Germline mutation analysis was performed in 469 VHL families from North America, Europe, and Japan. Germline mutations were identified in 300/469 (63%) of the families tested; 137 distinct intragenic germline mutations were detected. Most of the germline VHL mutations (124/137) occurred in 1–2 families; a few occured in four or more families. The common germline VHL mutations were: delPhe76, Asn78Ser, Arg161Stop, Arg167Gln, Arg167Trp, and Leu178Pro. In this large series, it was possible to compare the effects of identical germline mutations in different populations. Germline VHL mutations produced similar cancer phenotypes in Caucasian and Japanese VHL families. Germline VHL mutations were identified that produced three distinct cancer phenotypes: (1) renal carcinoma without pheochromocytoma, (2) renal carcinoma with pheochromocytoma, and (3) pheochromocytoma alone. The catalog of VHL germline mutations with phenotype information should be useful for diagnostic and prognostic studies of VHL and for studies of genotype‐phenotype correlations in VHL.

MicroRNAs and cardiac sarcoplasmic reticulum calcium ATPase-2 in human myocardial infarction: expression and bioinformatic analysis.

BackgroundCardiac sarco(endo)plasmic reticulum calcium ATPase-2 (SERCA2) plays one of the central roles in myocardial contractility. Both, SERCA2 mRNA and protein are reduced in myocardial infarction (MI), but the correlation has not been always observed. MicroRNAs (miRNAs) act by targeting 3-UTR mRNA, causing translational repression in physiological and pathological conditions, including cardiovascular diseases. One of the aims of our study was to identify miRNAs that could influence SERCA2 expression in human MI.ResultsThe protein SERCA2 was decreased and 43 miRNAs were deregulated in infarcted myocardium compared to corresponding remote myocardium, analyzed by western blot and microRNA microarrays, respectively. All the samples were stored as FFPE tissue and in RNAlater. miRNAs binding prediction to SERCA2 including four prediction algorithms (TargetScan, PicTar, miRanda and mirTarget2) identified 213 putative miRNAs. TAM and miRNApath annotation of deregulated miRNAs identified 18 functional and 21 diseased states related to heart diseases, and association of the half of the deregulated miRNAs to SERCA2. Free-energy of binding and flanking regions (RNA22, RNAfold) was calculated for 10 up-regulated miRNAs from microarray analysis (miR-122, miR-320a/b/c/d, miR-574-3p/-5p, miR-199a, miR-140, and miR-483), and nine miRNAs deregulated from microarray analysis were used for validation with qPCR (miR-21, miR-122, miR-126, miR-1, miR-133, miR-125a/b, and miR-98). Based on qPCR results, the comparison between FFPE and RNAlater stored tissue samples, between Sybr Green and TaqMan approaches, as well as between different reference genes were also performed.ConclusionCombing all the results, we identified certain miRNAs as potential regulators of SERCA2; however, further functional studies are needed for verification. Using qPCR, we confirmed deregulation of nine miRNAs in human MI, and show that qPCR normalization strategy is important for the outcome of miRNA expression analysis in human MI.

A Novel Polymorphism in the Promoter Region of ERBB4 Is Associated with Breast and Colorectal Cancer Risk

Purpose: The receptor tyrosine kinase ERBB4/HER4 plays a role in cell division, migration, differentiation, as well as apoptosis, and is frequently overexpressed in breast and colorectal tumors. To understand the role of genetic variations in the regulation of ERBB4 expression, we identified new polymorphisms and investigated their functional implication and risk association with breast and colorectal cancer. Experimental Design: We screened colorectal tumors from 92 patients for genetic variants at the ERBB4 ATG −1000 bp 5′-regulatory region by denaturing high-performance liquid chromatography and sequencing. Variants were subjected to DNA-protein interaction analyses (electrophoretic mobility shift assay), reporter gene assays in breast cancer cell lines MDA134 and MDA157, and immunohistochemical analyses of breast tumors. We established genotype frequencies within a breast cancer case-control collection (1,021 cases, 1,015 population-based controls) and a colorectal cancer case-control collection (459 cases, 569 blood donors) using matrix-assisted laser desorption ionization/time of flight mass spectrometry. Adjusted odds ratios (OR) and 95% confidence intervals (CI) were assessed by multivariate logistic regression. Results: We identified five new germ line variants −815 A>T, −782 G>T, −638 insTC, −267 C>G, and −219 del10bp. Two variants showed in vitro functional effects. The −782T allele showed lower protein binding affinity and lower promoter activity compared with the −782G allele, however, the −815T allele showed higher protein binding affinity and higher promoter activity. The −782T variant was identified as a risk allele for breast and colorectal cancer (OR, 1.59; 95% CI, 1.06-2.34 and OR, 2.21; 95% CI, 1.22-3.99, respectively). Conclusion: The ERBB4 −782 G>T polymorphism, by virtue of its in vitro functional implication and incidence, is a risk factor for breast and colorectal cancer.

Long Noncoding RNAs as Biomarkers in Cancer

Long noncoding RNAs (lncRNAs) are a relatively well-characterized class of noncoding RNA (ncRNA) molecules, involved in the regulation of various cell processes, including transcription, intracellular trafficking, and chromosome remodeling. Their deregulation has been associated with the development and progression of various cancer types, the fact which makes them suitable as biomarkers for cancer diagnosis and prognosis. In recent years, detection of cancer-associated lncRNAs in body fluids of cancer patients has proven itself as an especially valuable method to effectively diagnose cancer. Cancer diagnosis and prognosis employing circulating lncRNAs are preferential when compared to classical biopsies of tumor tissues, especially due to their noninvasiveness, and have great potential for routine usage in clinical practice. Thus, this review focuses on summarizing the perspectives of lncRNAs as biomarkers in cancer, based on evaluating their expression profiles determined in body fluids of cancer patients.

Low microsatellite instability and high loss of heterozygosity rates indicate dominant role of the suppressor pathway in squamous cell carcinoma of head and neck and loss of heterozygosity of 11q14.3 correlates with tumor grade

Loss of heterozygosity (LOH) and microsatellite instability (MSI) have been recognized as important events in squamous cell carcinoma of the head and neck (HNSCC), suggesting involvement of both suppressor and mutator pathways. We analyzed 153 HNSCC with 8 Bethesda reference panel markers and 14 microsatellite markers selected from chromosomal regions known to harbor either tumor-suppressor genes or oncogenes. A combination of multiplex fluorescence-based polymerase chain reaction and automatic fragment analysis was performed. LOH was observed in 78% of all tumors. 2% to 17% LOH frequency was observed with Bethesda reference panel markers comparing to higher 8% to 48% LOH in chromosomal areas 3p, 9p, 11q, and 17p. LOH of 11q14.3 correlated with tumor grade. The proportions of high- and low-MSI tumors were 3% and 10%, respectively, but no mutation was identified in MLH1 and MSH2 mismatch repair genes. These results indicate the dominant role of the suppressor in comparison with the mutator pathway in HNSCC carcinogenesis.

Hereditary medullary thyroid cancer in Slovenia : genotype-phenotype correlations

ZusammenfassungHINTERGRUND: Das medulläre Schilddrüsenkarzinom (MTC) ist ein seltener endokriner Tumor, der sporadisch oder vererbt als Teil von MEN 2A beziehungsweise MEN 2B, oder als familiärer Tumor (FMTC) auftreten kann. Keimbahn-Punkt-Mutationen im RET-Proto-onkogen sind für die Tumorentstehung, die Vererbung und für die große klinische Variabilität verantwortlich. Ziel dieser Studie war es, den Genotyp mit dem Phänotyp (Alter bei Diagnoseerstellung, Geschlecht, TNM Klassifikation und Klinik) von Patienten mit vererbbarem MTC zu korrelieren. PATIENTEN: Von 1997 bis 2003 wurden bei 69 von 98 Patienten mit so genanntem sporadischem MTC Genanalysen durchgeführt. Es wurden 14 (20,2%) Mutationsträger (= Index-Patienten) gefunden. Bei 16 (51,6%) der 31 Verwandten dieser Index-Patienten wurde dieselbe Mutation festgestellt. Ein Patient mit MEN 2B (Mutation im Codon 918) wurde nicht in die Studie aufgenommen. METHODEN: Genomische DNS wurde aus den Leukozyten des peripheren Blutes isoliert. Die Exons 10, 11, 12, 13, 14, 15 und 16 des RET-Protoonkogens wurden in der Polymerase-Ketten-Reaktion (PCR) amplifiziert. Die Punktmutationen im RET-Gen wurden durch Single Strand Conformation Analyse (SSCA) und DNS Sequenzierung entdeckt. Die beobachteten Mutationen wurden durch Restriktions-Enzyme bestätigt. ERGEBNISSE: Mutationen im Codon 634 wurden bei 15 Patienten (50%; Alter: 18–76 Jahre; 6 Familien), im Codon 618 bei 9 Patienten (30%; Alter 12–65; 4 Familien) und im Codon 790 bei 5 Patienten (16,6%; Alter 16–74; 3 Familien) entdeckt. Der Median des Alters bei Diagnosestellung lag bei 31 ± 17,3 (Codon 618); bzw 33 ± 15,9 (Codon 634), bzw 36 ± 23,8 (Codon 790) Jahren. Frauen mit den Mutationen im Codon 618 und im Codon 634 hatten ein medianes Alter bei Diagnoseerstellung von 34,5 ± 15,6 verglichen mit 43,5 ± 22,9 bei den Patienten mit Mutation im Codon 790. Bei Männern war im Gegensatz dazu das Alter bei der Diagnose 26,5 ± 18 im Vergleich zu 16 Jahren bei der Mutation im Codon 790. Der Mann/Frau Quotient war 1:2 bei Patienten mit Veränderungen im Codon 618 und 634 und 1:4 bei Patienten mit Mutation im Codon 790. Ein Phäochromozytom (12/15 Patienten) bzw. ein primärer Hyperparathyreoidismus (6/15 Patienten) wurde nur bei Patienten mit einer Mutation im Codon 634 gefunden. Es wurde ein Patient mit einem FMTC und der Hirschsprungschen Erkrankung in einer Familie mit Mutation im Codon 618 gefunden. SCHLUSSFOLGERUNGEN: Unsere limitierte Studie gibt Hinweise auf eine Korrelation des Genotyps mit der Tumorgröße und dem MTC Tumorstadium vor allem bei Frauen mit einer Mutation im Codon 790. Der Umstand, dass diese Tumore später auftreten und einen weniger aggressiven Verlauf nehmen, sollte bei der Planung einer prophylaktischen Schilddrüsenentfernung Berücksichtigung finden. Das MEN 2A Syndrom wurde ausschließlich bei Patientne mit der Mutation im Codon 634 beobachtet.SummaryBACKGROUND: Medullary thyroid cancer (MTC) is a rare endocrine tumor that may be sporadic or inherited in settings of MEN2A, MEN2B and FMTC. Germline point mutations in the RET proto-oncogene are responsible for tumor occurrence, inheritance and great clinical variability. The aim of this study was to correlate the genotype and phenotype of patients with hereditary MTC (age at diagnosis, sex, TNM classification and clinical features). PATIENTS: Between 1997 and 2003 genetic testing was performed in 69 out of 98 patients with sporadic MTC. Carriage of mutation was found in 14 (20.2%) patients (index patients) and in 16 out of 31 (51.6%) of their relatives. One patient with MEN2B and codon 918 mutation was excluded from further analysis. METHODS: Genomic DNA was isolated from peripheral blood leukocytes. Exons 10, 11, 13, 14, 15 and 16 of the RET proto-oncogene were amplified in polymerase chain reactions. Point mutations of the RET gene were detected with single-strand conformation analysis and DNA sequencing. Detected mutations were confirmed with restriction enzyme analysis. RESULTS: Codon 634 mutations were detected in 15 patients (50%; aged 18–76 years; 6 families), codon 618 in nine patients (30%; aged 12–65 years; 4 families) and codon 790 in five patients (16.6%; aged 16–74 years; 3 families). The median age at diagnosis was 31 ± 17.3, 33 ± 15.9 and 36 ± 23.8 years for patients with codon 618, 634 and 790 mutations. Selected by sex, females with codon mutations 618 and 634 versus 790 had median age at diagnosis of 34.5 ± 15.6 years and 43.5 ± 22.9 years, whereas the inverse result was observed in males (26.5 ± 18.0 versus 16 years). The male/female ratio was 1:2 for patients with codon 618 and 634 mutations and 1:4 for patients with codon 790 mutations. Some of the data suggested correlation between specific genotypes, tumor size, stage of MTC and age at diagnosis. Pheochromocytoma (12 out of 15 patients) and primary hyperparathyroidism (6 out of 15 patients) were diagnosed solely in patients with codon 634 mutations. One patient with FMTC and Hirschprung disease was found in a family with codon 618 mutations. CONCLUSION: Correlation between tumor size, stage of MTC at diagnosis in view of patients age, and specific genotype were indicated in our limited series and were more evident in female patients with codon 790 mutations. Later onset and a probably less aggressive course of MTC in these patients than in patients with other mutations should be considered in planning prophylactic thyroid surgery. MEN2A syndrome was related solely to codon 634 mutations.

A Polymorphism in the TC21 Promoter Associates with an Unfavorable Tamoxifen Treatment Outcome in Breast Cancer

Tamoxifen therapy is a standard in the treatment of estrogen receptor (ER)-positive breast cancer; however, its efficacy varies widely among patients. In addition to interpatient differences in the tamoxifen-metabolizing capacity, there is growing evidence that crosstalk between ER and growth factor signaling contributes to tamoxifen resistance. We focused on TC21, a member of the Ras superfamily, to investigate the influence of the TC21 -582C>T promoter polymorphism on TC21 expression and treatment outcome. Immunohistochemical analyses of breast tumors revealed a higher TC21 expression in ER-negative compared with ER-positive tumors. Expression in ER-positive tumors was higher in carriers of the T allele in an allele dose-dependent manner. Quantitative real-time PCR analyses showed that TC21 mRNA expression is decreased after transfection of ERalpha in ER-negative breast cancer cells MDA-MB-231, UACC893, and BT-20. In MCF7 ER-positive cells, TC21 expression decreased with 17beta-estradiol treatment and increased after treatment with tamoxifen metabolites, 4-OH-tamoxifen, or endoxifen. In patients treated with adjuvant mono tamoxifen, high cytoplasmic TC21 tumor expression or the carriership of the -582T allele conferred increased recurrence rates [n=45: hazard ratio (HR), 3.06; 95% confidence interval (95% CI), 1.16-8.05; n=206: HR, 1.79; 95% CI, 1.08-3.00, respectively]. A combined analysis with the data of the known tamoxifen predictor CYP2D6 showed an improvement of outcome prediction compared with CYP2D6 or TC21 genotype status alone (per mutated gene HR, 2.35; 95% CI, 1.34-4.14). Our functional and patient-based results suggest that the TC21 -582C>T polymorphism improves prediction of tamoxifen treatment outcome in breast cancer.

miRNome in myocardial infarction: Future directions and perspective.

MicroRNAs (miRNAs), which are small and non-coding RNAs, are genome encoded from viruses to humans. They contribute to various developmental, physiological and pathological processes in living organisms. A huge amount of research results revealed that miRNAs regulate these processes also in the heart. miRNAs may have cell-type-specific or tissue-specific expression patterns or may be expressed ubiquitously. Primary studies of miRNA involvement in hypertrophy, heart failure and myocardial infarction analyzed miRNAs that are enriched in or specific for cardiomyocytes; however, growing evidence suggest that other miRNAs, not cardiac or muscle-specific, play a significant role in cardiovascular disease. Abnormal miRNA regulation has been shown to be involved in cardiac diseases, suggesting that miRNAs might affect cardiac structure and function. In this review, we focus on miRNAs that have been found to contribute to the pathogenesis of myocardial infarction (MI) and the response post-MI and characterized as diagnostic, prognostic and therapeutic targets. The majority of these studies were performed using mouse and rat models of MI, with a focus on the identification of basic cellular and molecular pathways involved in MI and in the response post-MI. Much research has also been performed on animal and human plasma samples from MI individuals to identify miRNAs that are possible prognostic and/or diagnostic targets of MI and other MI-related diseases. A large proportion of research is focused on miRNAs as promising therapeutic targets and biomarkers of drug responses and/or stem cell treatment approaches. However, only a few studies have described miRNA expression in human heart tissue following MI.

TMEM25 Is a Candidate Biomarker Methylated and Down-Regulated in Colorectal Cancer

The identification of novel genes involved in colorectal cancerogenesis is of high clinical relevance for early diagnosis, applying new therapeutic strategies and monitoring disease recurrence, in order to reduce disease incidence and mortality. Gene silencing through CpG island hypermethylation is a major epigenetic mechanism involved in cancer development. In our study, we aimed to identify and validate novel genes with a tumour specific DNA methylation profile in colorectal cancer. We performed a whole-genome methylation scan and identified several possible candidate genes that are hypermethylated in tumour in comparison to healthy colon mucosa. Using methylation-specific high-resolution melting analysis in a set consisting of 133 colorectal cancer samples, we were able to confirm an altered CpG site in TMEM25 in 69.2% (92/133) tumours analysed. Furthermore, the expression of TMEM25 was found to be significantly lower in tumour tissue. An inverse correlation between hypermethylation of TMEM25 and TMEM25 down-regulated expression was observed. Our results suggest that epigenetic down-regulation of TMEM25 is cancer-related; we thus suggest that TMEM25 hypermethylation might play a significant role in altering expression of this gene in colorectal cancer.

Identification of novel genes with somatic frameshift mutations within coding mononucleotide repeats in colorectal tumors with high microsatellite instability.

We have systematically retrieved genes with coding mononucleotide repeats from sequence databases and analyzed them for mutations in tumors with high levels of microsatellite instability (MSI‐H). We found somatic frameshift mutations in 7/13 genes previously not analyzed in MSI‐H tumors. According to the frequency of mutations in MSI‐H tumors, these genes could be divided into genes with high coding mononucleotide repeat instability (CMRI‐H) and genes with low coding mononucleotide instability (CMRI‐L). CMR‐H genes were mutated in more than 9/38 and CMRI‐L in less than 4/38 of MSI‐H tumors. Four genes in our study were CMRI‐H and could thus possibly play a role in the development of MSI‐H tumors: TFE3 (9/38), TEF4 (12/38), RGS12 (11/38), and TCF1 (12/38). Our results suggest that systematic identification of genes with CMR in the sequence databases and determination of mutation frequency in MSI‐H tumors might be a powerful tool for identification of new molecular targets in the development of MSI‐H tumors.