Prevalence of rare F5 variants in general population from Bosnia and Herzegovina.

Human gene F5, encoding coagulation factor V, was previously reported to be highly polymorphic. Apart from FV Leiden, several other rare variants have been detected in clinical practice and associated with thrombotic events, especially in cases when patient's phenotype and FV Leiden genotype were not in agreement. In this study, the prevalence of 17 rare F5 variants has been studied on a sample of 130 healthy adult individuals from the general Bosnian-Herzegovinian population. DNA was isolated from buccal swab samples, while genotyping was performed using MALDI-TOF MS method. The results have shown that Asp2194Gly and Met2120Thr are polymorphic in the study population with minor allele frequencies of 0.077 and 0.073, respectively. Additionally, these two variants were mutually exclusive with FV Leiden and none of them was positively associated with participants' family history of cardiovascular or cerebrovascular diseases. While the obtained results are in agreement with previously reported data for the general Caucasian populations, it is worth noting that only two rare F5 variants were detected in the study population, albeit at considerable frequencies. Still, scientific information on rare F5 variants is rather scarce and further studies aiming to assess functional importance of these variants, as well as their role as prothrombotic factors are necessary.

Tumor cell PD-L1 expression is a strong predictor of unfavorable prognosis in immune checkpoint therapy-naive clear cell renal cell cancer.

BACKGROUND: PD-L1 expression predicts response to immune checkpoint inhibitors in renal cell carcinomas (RCC), but has also been suggested to be linked to poor patient outcome. METHODS: We analyzed PD-L1 in > 1400 RCC in a tissue microarray format by immunohistochemistry. Results were compared with histological tumor type, parameters of cancer aggressiveness, and intratumoral CD8+ cytotoxic cells. RESULT: At a cut-off level of 5% PD-L1 positive tumor cells, PD-L1 positivity was seen in 6.3% of 633 clear cell RCC (ccRCC), 18.2% of 165 papillary RCC, 18.8% of 64 chromophobe RCC, and 41.7% of 103 oncocytomas. In ccRCC, PD-L1 positivity was significantly linked to high ISUP (p < 0.0001), Fuhrman (p < 0.0001), Thoenes grade (p < 0.0001), distant metastasis (p = 0.0042), short recurrence-free (p < 0.0001), and overall survival (p = 0.0002). Intratumoral CD8+ lymphocytes were more frequent in PD-L1 positive (1055 ± 109) than in PD-L1 negative ccRCC (407 ± 28; p < 0.0001). PD-L positive immune cells were seen in 8.2% of all RCC and 13.9% of papillary RCC. In ccRCC, PD-L1 positive immune cells were linked to high numbers of tumor-infiltrating CD8+ cells (p < 0.0001), high ISUP (p < 0.0001), Fuhrman (p = 0.0027), and Thoenes grade (p < 0.0001), and poor tumor-specific survival (p = 0.0280). CONCLUSIONS: These data suggest that PD-L1 expression in highly immunogenic RCCs facilitates immune evasion and contributes to cancer aggressiveness.

Mismatch repair deficiency occurs very rarely in seminomas.

BACKGROUND: Dense tumor-associated lymphocyte infiltration is linked to mismatch repair (MMR) deficiency in colorectal and endometrial cancer. MMR deficiency is of high clinical importance as MMR deficient cancers tend to react favorably to treatment with immune checkpoint inhibitors. Strong lymphocytic infiltration is a morphological hallmark of seminomas. We thus asked whether seminomas may exhibit MMR deficiency at relevant frequency. METHODS: To screen for tumors with MMR deficiency, protein expression of MLH1, PMS2, MSH2, and MSH6 was analyzed by immunohistochemistry (IHC) on a tissue microarray (TMA) containing 574 seminomas. RESULTS: In total, 536 cases were evaluable resulting in 481 seminomas with unequivocally intact MMR protein expression. In 55 cancers, one or several IHC stains were equivocal and lacked detectable MMR protein in both tumor and stromal cells. Large section IHC analysis of all 55 equivocal cases demonstrated substantial staining issues due to improper fixation in 54 cases and identified one tumor with clear-cut MLH1 and PMS2 protein loss. This seminoma showed homogeneous loss of MLH1 and PMS2 in the entire tumor mass whereas minor adjacent foci of associated germ cell neoplasia in situ (GCNIS) were MMR intact. Polymerase chain reaction (PCR) analysis using the 5 microsatellite loci of the "Bethesda Panel" revealed instability in 1 of 4 interpretable loci ("MSI-low") and additional instability of the complex tetra-penta repeat locus MYCL1 in this tumor. CONCLUSIONS: In summary, one single seminoma with MMR deficiency, characterized by protein loss of MLH1 and PMS2, was identified among 536 interpretable seminomas (0.19%). MMR deficiency is not a relevant determinant of lymphocyte influx in seminoma.

Population study of thrombophilic markers and pharmacogenetic markers of warfarin prevalence in Bosnia and Herzegovina.

AIM: To investigate the prevalence of common genetic variants that can serve as markers of thrombophilia and warfarin pharmacogenetics in Bosnia and Herzegovina. METHODS: The study was performed between August and October 2017 on 130 healthy unrelated adult volunteers from Bosnian-Herzegovinian population sample. The prevalence of the following genetic variants was determined: F5 c.1601G>A (factor V Leiden), F2 c.*97G>A (factor II or prothrombin mutation), F13A1 (factor XIII) c.103G>T, MTHFR (methylenetetrahydrofolate reductase) c.665C>T and c.1286A>C, as well as PAI-1 (plasminogen activator inhibitor 1) c.-816A>G and c.-844G>A as markers of thrombophilia risk, and *2 and *3 alleles of CYP2C9 (cytochrome P450 2C9) and five variants of VKORC1 (vitamin K epoxide reductase complex subunit 1) as markers of warfarin pharmacogenetics. DNA was isolated from buccal swabs using salting out method, while genotyping was performed using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. RESULTS: Minor allele frequencies for two main thrombophilia risk factors, F5 c.1601G>A and F2 c.*97G>A were 0.023 and 0.008, respectively. Combined data for the markers of warfarin pharmacogenetics imply that 57.4% study participants can be expected to metabolize warfarin at an extensive, 40.3% at intermediate, and 2.3% at a poor rate. CONCLUSION: This study reports the first extensive population genetic data for thrombophilia and warfarin pharmacogenetic markers in Bosnia and Herzegovina. Allele frequencies of genetic variants are within the general average for European populations, and their presence implies the necessity of introduction of personalized medicine in warfarin-mediated antithrombotic therapy.

Cystic fibrosis presentation in del. F508 and p. Tyr109Glyfs compound heterozygote CFTR state: a case report.

The diagnosis of cystic fibrosis (CF) is commonly confirmed by molecular genetics with the presence of specific mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene. We report a case of cystic fibrosis (CF) in a 15-year-old female patient who is a compound heterozygote for CFTR gene, with delta F508 and Tyr109Glyfs mutations detected. This is the first detailed description of such a case in the medical literature. The primary CF presentation occurred at the age of 9 in the form of gastrointestinal symptoms including greasy, bulky, and foul-smelling stool. The patient exhibited delayed growth, with her height and weight being below the 5th centile for age according to the World Health Organization growth curves. Pancreatic enzyme supplement treatment was started immediately, alongside high-fat and high-calorie diet, resulting in patient's recovery and development. DNA analysis of CFTR gene demonstrated the presence of del. F508 mutation and a rare combining deletion and insertion mutation p. Tyr109Glyfs. The combination of the two mutations is very rare in CF patients and is therefore valuable to document this case in order to provide information on disease progression, therapy options, and outcomes. With standard treatment and early diagnosis, the patient is currently doing well and is not restricted by the disease in her daily and sports activities.

Evaluating Guideline Adherence for T1 Bladder Cancer Treatment and Surveillance: A Retrospective German Multicenter Observation.

INTRODUCTION: To mirror guideline-adherence for pT1 bladder cancer treatment in Northern Germany. MATERIALS AND METHODS: Overall, 111 patients with pT1 diagnosis were treated at 4 institutions. Guideline-adherence was defined as repeat resection, instillation, and quarterly cystoscopy. Patient characteristics and pathological parameters were assessed. We summarized patients using descriptive analyses and evaluated guideline-adherence within selected subgroups. We created a multivariable model to identify predictors of guideline-adherence. RESULTS: Median age was 75 years (range 39-94 years), multifocal tumors were found in 44.1%, early instillation was performed in 33.3%, and repeat resection was performed in 77.5%. Of 62.2% who underwent instillation, 59.4% received BCG, while 40.6% received Mitomycin C or other agents. Cystoscopic follow-up was performed in 81.8%. Guideline-adherence was met in 56.8%. Patients aged below the median met adherence metrics more often compared to those above the median (66.7 vs. 46.3%; p = 0.030). Men more frequently met adherence metrics compared to women (62.1 vs. 37.5%; p = 0.038). More patients with multifocal tumors met all 3 adherence metrics (69.4 vs. 48.0%; p = 0.050), as compared to those with unifocal lesions. In multivariable analyses, age-adjusted comorbidity (OR 0.75; 95% CI 0.59-0.94; p = 0.011) and multifocality (OR 2.62; 95% CI 1.09-6.27; p = 0.031) were predictors of guideline-adherence. CONCLUSIONS: We found non-adherence in more than one-third of patients and disparities among patients of different age and according to tumor focality. Larger samples and prospective studies are needed to delineate and eradicate treatment disadvantages in these high-risk patients.

Impact of the Ki-67 labeling index and p53 expression status on disease-free survival in pT1 urothelial carcinoma of the bladder.

BACKGROUND: The identification of protein biomarkers to guide treatment decisions regarding adjuvant therapies for high-risk non-muscle-invasive bladder cancer (NMIBC) has been of increasing interest. Evidence of the impact of tumor suppressor gene product p53 and cell proliferation marker Ki-67 on oncologic outcomes in bladder cancer patients at highest risk of recurrence and progression is partially contradictory. We sought to mirror contemporary expression patterns of p53 and Ki-67 in a select cohort of patients with pT1 bladder cancer. METHODS: Patients from four Northern German institutions with a primary diagnosis of pT1 bladder cancer between 2009 and 2016 and complete data regarding p53 or Ki-67 expression status were included for final analyses. Baseline patient characteristics (age, gender, age-adjusted Charlson comorbidity index) and tumor characteristics [diagnostic sequence, tumor focality, concomitant carcinoma in situ, 1973 World Health Organization (WHO) grading, lymphovascular invasion, adjuvant instillation therapy] were abstracted by retrospective chart review. Immunohistochemistry for detection of p53 and Ki-67 expression was performed according to standardized protocols. Microscopic analyses were performed by central pathologic review. First, we compared patients with positive vs. negative p53 expression and Ki-67 labeling index [>40% vs. ≤40%; cutoffs based on best discriminative ability in univariable Cox regression analysis with disease-free survival (DFS) as endpoint] with regard to baseline and tumor characteristics. Second, we evaluated the effect of biomarker positivity on DFS by plotting univariable Kaplan-Meier curves and performing uni- and multivariable Cox regression analyses. RESULTS: Of 102 patients with complete information on p53 status, 44 (43.1%) were p53 positive, and they more often harbored concomitant carcinoma in situ (50.0% vs. 27.6%; P=0.032) and 1973 WHO grade 3 (97.7% vs. 69.0%; P=0.001) compared to their p53 negative counterparts. Of 79 patients with complete information on Ki-67 expression status, 30 (38.0%) had a labeling index >40%. Mean Ki-67 labeling index was higher in WHO grade 3 vs. grade 2 tumors (45.8 vs. 29.7; P=0.004). At a median follow-up of 51.0 months, 31/91 patients with complete follow-up information (34.1%) suffered from disease recurrence or progression. In univariable Kaplan-Meier analyses, no difference regarding DFS was found in p53 positive vs. negative (P=0.8) or Ki-67 labeling index >40% vs. ≤40% (P=0.078) patients. In multivariable analyses, Ki-67 labeling index >40% remained an independent predictor of DFS [hazard ratio (HR), 2.66; 95% confidence interval (CI), 1.02-6.95; P=0.046], after adjusting for p53 expression and lymphovascular invasion. However, p53 status was not associated with our endpoint (P=0.8). CONCLUSIONS: While we found an association of a Ki-67 labeling index >40% and shorter DFS in pT1 bladder cancer patients, this did not hold true for p53 positivity. Future research is needed to identify additional microscopic and molecular risk factors and biomarker panels to improve risk stratification and guide adjuvant therapies in those patients.

MALDI imaging-based identification of prognostically relevant signals in bladder cancer using large-scale tissue microarrays.

OBJECTIVE: Although most patients with urinary bladder cancer present with noninvasive and low-malignant stages of the disease, about 20% eventually develop life-threatening metastatic tumors. This study was designed to evaluate the potential of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to identify molecular markers predicting the clinical course of bladder cancer. MATERIALS AND METHODS: We employed MALDI-MSI to a bladder cancer tissue microarray including paraffin-embedded tissue samples from 697 patients with clinical follow-up data to search for prognostically relevant associations. RESULTS: Analysis of our MALDI imaging data revealed 40 signals in the mass spectra (m/z signals) associated with epithelial structures. The presence of numerous m/z signals was statistically related to one or several phenotypical findings including tumor aggressiveness (stage, grade, or nodal status; 30 signals), solid (5 signals) or papillary (3 signals) growth patterns, and increased (6 signals) or decreased (12 signals) cell proliferation, as determined by Ki-67 immunohistochemistry. Two signals were linked with tumor recurrence in noninvasive (pTa category) tumors, of which one was also related to progression from pTa-category to pT1-category disease. The absence of one m/z signal was linked with decreased survival in the subset of 102 muscle-invasive cancers. CONCLUSION: Our data demonstrate the suitability of combining MSI and large-scale tissue microarrays to simultaneously identify and validate clinically useful molecular markers in urinary bladder cancer.

Reduced membranous MET expression is linked to bladder cancer progression.

The MET protein is involved in the malignant progression of different tumors. This study aimed to analyze the relationship of MET expression with tumor phenotype and clinical outcome in bladder cancer and the role of gene amplification for MET overexpression. A bladder cancer tissue microarray containing 686 bladder cancers was analyzed by immunohistochemistry and by fluorescence in situ hybridization. MET immunostaining was seen in normal urothelium and was recorded in 459 of 560 analyzable urothelial carcinomas (82.0%). Low MET staining was associated with a more unfavorable tumor phenotype. MET staining was seen in 89.8% of 266 pTa, 81.1% of 132 pT1, and 69.4% of 160 pT2-4 cancers (P < 0.0001). MET staining was detectable in 92.4% of 66 grade 1, 85.6% of 257 grade 2, and 75.1% of 237 grade 3 cancers (P = 0.001). MET expression status was not associated with overall or tumor-specific survival in muscle-invasive cancers (pT2-4), tumor progression in pT1 cancers, or recurrences in pTa tumors. Only four of the analyzed tumors (0.8%) showed amplification of the MET gene. We conclude that MET is not overexpressed in urothelial cancer but rather downregulated in a fraction of cancers. Accordingly, rare amplification of the genomic area including the MET gene was not associated with MET protein overexpression.

Effects on transcriptional regulation and lipid droplet characteristics in the liver of female juvenile pigs after early postnatal feed restriction and refeeding are dependent on birth weight.

Epidemiological and experimental data indicate that caloric restriction in early postnatal life may improve liver lipid metabolism in low birth weight individuals. The present study investigated transcriptional and metabolic responses to low (U) and normal (N) birth weight (d 75, T1) and postnatal feed restriction (R, 60% of controls, d 98, T2) followed by subsequent refeeding until d 131 of age (T3). Liver tissue studies were performed with a total of 42 female pigs which were born by multiparous German landrace sows. Overall, 194 genes were differentially expressed in the liver of U vs. N (T1) animals with roles in lipid metabolism. The total mean area and number of lipid droplets (LD) was about 4.6- and 3.7 times higher in U compared to N. In U, the mean LD size (µm(2)) was 24.9% higher. 3-week feed restriction reduced total mean area of LDs by 58.3 and 72.7% in U and N, respectively. A functional role of the affected genes in amino acid metabolism was additionally indicated. This was reflected by a 17.0% higher arginine concentration in the liver of UR animals (vs. NR). To evaluate persistency of effects, analyses were also done after refeeding period at T3. Overall, 4 and 22 genes show persistent regulation in U and N animals after 5 weeks of refeeding, respectively. These genes are involved in e.g. processes of lipid and protein metabolism and glucose homeostasis. Moreover, the recovery of total mean LD area in U and N animals back to the previous T1 level was observed. However, when compared to controls, the mean LD size was still reduced by 23.3% in UR, whereas it was increased in NR (+24.7%). The present results suggest that short-term postnatal feed restriction period programmed juvenile U animals for an increased rate of hepatic lipolysis in later life.

PTEN deletions are related to disease progression and unfavourable prognosis in early bladder cancer.

AIMS: This study aimed to determine the prevalence and clinical significance of deletions of the tumour suppressor gene PTEN in bladder cancer. METHODS AND RESULTS: A tissue microarray with 686 bladder cancers was analysed for PTEN deletions by fluorescence in-situ hybridization. PTEN mutations were analysed in nine tumours with heterozygous PTEN deletion. Heterozygous PTEN deletions were present in 16.5% of tumours and were associated with grade (P = 0.0024) and p53 status (P = 0.0141), but not linked to stage (P = 0.0965). PTEN deletions were seen in 5.8% of pTaG1, 10.9% of pTaG2, 29.0% of pTaG3, 16.7% of pT1G2, 22.2% of pT1G3, 17.7% of pT2-4G2 and 20.9% of pT2-4G3 tumours (P = 0.0235). PTEN deletions were associated significantly with recurrences in pTa tumours (P = 0.0173), progression in pT1 tumours (P = 0.0016), but not with overall or cancer-specific survival in pT2 tumours. Multivariate analyses including grade and PTEN deletions revealed that PTEN deletions but not grade were associated independently with recurrence in pTa tumours (P = 0.0377) and progression in pT1 tumours (P = 0.0030). No inactivating PTEN mutations were found. CONCLUSIONS: PTEN is linked to aggressive tumour phenotype and to unfavourable outcome in early bladder cancer. Heterozygous PTEN loss, i.e. reduced PTEN gene dosage, might be sufficient to cause aggressive tumour behaviour in bladder cancer cells.

Molecular epidemiology of multiple endocrine neoplasia 2: implications for RET screening in the new millenium.

OBJECTIVE: Twenty years ago, the groundbreaking discovery that rearranged during transfection (RET) mutations underlie multiple endocrine neoplasia 2 (MEN2) and familial medullary thyroid cancer (FMTC) ushered in the era of personalized medicine. MEN2-associated signs, taking time to manifest, can be subtle. This study sought to clarify to what extent conventional estimates of 1:200 000-500 000 underestimate the incidence of RET mutations in the population. DESIGN: Included in this retrospective investigation were 333 RET carriers born between 1951 and 2000 and operated on at the largest German surgical referral center (286 carriers) or elsewhere (47 carriers). METHODS: To estimate the incidence of RET mutations, the number of RET carriers born in Germany in five decades (1951-1960, 1961-1970, 1971-1980, 1981-1990, and 1991-2000) was divided by the corresponding number of German live births. RESULTS: Owing to improved diagnosis and capture of FMTC and MEN2 patients, minimum incidence estimates increased over time: overall from 5.0 (1951-1960) to 9.9 (1991-2000) per million live births and year (P=0.008), and by American Thyroid Association/ATA class from 1.7 to 3.7 for ATA class C (P=0.008); from 1.8 to 2.7 for ATA class A (P=0.017); from 1.5 to 2.2 for ATA class B (P=0.20); and from 0 to 1.4 for ATA class D mutations per million live births and year (P=0.008). Based on 1991-2000 incidence estimates the prevalence in Germany is ∼1:80 000 inhabitants. CONCLUSIONS: The molecular minimum incidence estimate of ≈1:100 000 was two- to fivefold greater than conventional estimates of 1:200 000-500 000.

Reduced CD151 expression is related to advanced tumour stage in urothelial bladder cancer.

AIMS: CD151 belongs to the group of tetraspanins and is aberrantly expressed in different tumours and differential expression has been associated with prognosis. The aim of this study was to clarify the relationship of CD151 expression with tumour phenotype and clinical outcome in bladder cancer. METHODS: A bladder cancer tissue microarray containing samples from 686 urothelial bladder cancers was analysed by immunohistochemistry. RESULTS: Membranous CD151 immunostaining was recorded in 409 (66.0%) of 620 analysable cases. High CD151 expression was seen in normal urothelium and in most non-invasive tumours. Low CD151 expression levels were associated with a more unfavourable tumour phenotype. CD151 staining was seen in 71.5% of 284 pTa, 62.1% of 145 pT1 and 60.4% of 187 pT2-4 cancers (p = 0.0033). CD151 staining was detectable in 77.3% of 75 grade 1, 71.1% of 273 grade 2 and 57.7% of 272 grade 3 cancers (p < 0.0001). CD151 expression status was not associated with overall or tumour-specific survival in muscle-invasive cancers (pT2-4), tumour progression in pT1 and recurrences in pTa tumours. CONCLUSION: On the basis of our data we conclude that loss of CD151 may contribute to bladder cancer progression through attenuation of cell adhesion. In clinically defined subgroups CD151 expression does not provide additional prognostic information.

Apolipoprotein E genotype affects tissue metallothionein levels: studies in targeted gene replacement mice.

The apolipoprotein E (APOE) genotype is an important risk factor for ageing and age-related diseases. The APOE4 genotype (in contrast to APOE3) has been shown to be associated with oxidative stress and chronic inflammation. Metallothioneins (MT) exhibit antioxidant and anti-inflammatory activity, and MT overexpression has been shown to increase lifespan in mice. Interactions between APOE and MT, however, are largely unknown. Hence, we determined the effect of the APOE4 versus APOE3 genotype on MT levels in targeted gene replacement mice. APOE4 versus APOE3 mice exhibited significantly lower hepatic MT1 and MT2 mRNA as well as lower MT protein levels. The decrease in hepatic MT protein levels in APOE4 as compared to APOE3 mice was accompanied by lower nuclear Nrf1, a protein partly controlling MT gene expression. Cell culture experiments using hepatocytes identified allyl-isothiocyanate (AITC) as a potent MT inductor in vitro. Therefore, we supplemented APOE3 and APOE4 mice with AITC. However, AITC (15 mg/kg b.w.) could only partly correct for decreased MT1 and MT2 gene expression in APOE4 mice in vivo. Furthermore, cholesterol significantly decreased both Nrf1 and MT mRNA levels in Huh7 cells indicating that differences in MT gene expression between the two genotypes could be related to differences in hepatic cholesterol concentrations. Overall, present data suggest that the APOE genotype is an important determinant of tissue MT levels in mice and that MT gene expression may be impaired by the APOE4 genotype.

Nrf2-dependent gene expression is affected by the proatherogenic apoE4 genotype-studies in targeted gene replacement mice.

An apoE4 genotype is an important risk factor for cardiovascular and other chronic diseases. The higher cardiovascular disease risk of apoE4 carriers as compared to the apoE3 genotype has been mainly attributed to the differences in blood lipids between the two genotype subgroups. Recently, a potential protective role of the transcription factor Nrf2 in cardiovascular disease prevention has been suggested. In this study, we show that Nrf2-dependent gene expression is affected by the apoE genotype. ApoE4 vs. apoE3 mice exhibited lower hepatic Nrf2 nuclear protein levels. Furthermore, mRNA and protein levels of Nrf2 target genes including glutathione-S-transferase, heme oxygenase-1 and NAD(P)H dehydrogenase, quinone 1 were significantly lower in apoE4 as compared to apoE3 mice. Lower hepatic mRNA levels of phase II enzymes, as observed in apoE4 vs. apoE3 mice, were accompanied by higher mRNA levels of phase I enzymes including Cyp26a1 and Cyp3a16. Furthermore, miRNA-144, miRNA-125b, and miRNA-29a involved in Nrf2 signaling, inflammation, and regulation of phase I enzyme gene expression were affected by the apoE genotype. We provide first evidence that Nrf2 is differentially regulated in response to the apoE genotype.

Sequence analysis of CYP21A1P in a German population to aid in the molecular biological diagnosis of congenital adrenal hyperplasia.

BACKGROUND: The high homology between the CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide 2) and CYP21A1P (cytochrome P450, family 21, subfamily A, polypeptide 1 pseudogene) genes is the major obstacle to risk-free genetic diagnosis of congenital adrenal hyperplasia, especially regarding the quantification of gene dosage. Because of the lack of a comprehensive study providing useful information about the detailed genetic structure of CYP21A1P, we used a large data set to analyze and characterize this pseudogene. METHODS: We amplified and directly sequenced the CYP21A1P and CYP21A2 genes of 200 unrelated individuals. The resulting sequence data were aligned against the manually curated transcript ENST0000448314 from Havana/Vega matching to the genebuild ENSG00000198457; all differences were documented. Copy number was measured by multiplex ligation-dependent probe amplification when necessary. RESULTS: We found that 40 potentially variable positions in CYP21A2 were conserved in CYP21A1P in all study participants. In addition, we detected 14 CYP21A1P variants that were not previously reported in either CYP21A2 or CYP21A1P. Unlike CYP21A2, CYP21A1P possessed certain mutation haplotypes. CONCLUSIONS: The genetic structure of CYP21A1P and the potential risks of false conclusions it may introduce are essential considerations in designing a PCR-based diagnosis procedure for congenital adrenal hyperplasia.

Allyl-, butyl- and phenylethyl-isothiocyanate activate Nrf2 in cultured fibroblasts.

The isothiocyanate sulforaphane (SFN) has been shown to induce phase 2 and antioxidant enzymes in cultured cells and in vivo via a Nrf2 dependent signal transduction pathway. However, little is known regarding the effect of structurally related compounds such as allyl isothiocyanate (AITC), butyl isothiocyanate (BITC) and phenylethyl isothiocyanate (PEITC) on Nrf2 target gene expression. In this study AITC, BITC and PEITC significantly increased phosphorylation of ERK1/2, an upstream target of Nrf2 in NIH3T3 fibroblasts. EKR1/2 phosphorylation was accompanied by an increased nuclear translocation and transactivation of Nrf2. AITC, BITC and PEITC significantly enhanced mRNA and protein levels of the Nrf2 targets γ-glutamyl cysteine synthetase (γGCS), heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase (NQO1). HO-1 and γGCS both contain CpG islands within their promoter region. However, analysis of DNA methylation status in NIH3T3 cells indicated that expression of these genes may not be dependant on promoter methylation. Current data indicate that not only SFN but also other aliphatic and aromatic isothiocyanates such as AITC, BITC and PEITC induce phase 2 and antioxidant enzymes in cultured fibroblasts.

Y chromosome loss is a frequent early event in urothelial bladder cancer.

AIMS: Y chromosome losses have been described in 10-40% of bladder cancers and were suggested to be age-related. The clinical significance of chromosome Y losses is largely unknown, since only small sets of male bladder cancer patients have been evaluated in previous studies. The aim of this study was to further clarify the potential relevance of Y chromosome losses in bladder cancer with respect to clinical outcome and patient age. METHODS: A pre-existing bladder cancer tissue microarray (TMA) with clinical follow-up data including 516 urothelial bladder cancers from male patients was utilised in this study. Y chromosome losses were analysed by multicolour fluorescence in situ hybridisation (FISH) using a centromere Y probe and a centromere X probe. p53 immunostaining data were available for all patients from a previous study. RESULTS: Y chromosome losses were seen in 23% of 477 interpretable cancers from male patients. There was no significant difference in patient age in tumours with (67.4 +/- 4.3 years) or without (67.3 +/- 2.3 years) Y chromosome losses (p = 0.9068). Y chromosome losses were equally frequent in tumours of all grades (p = 0.7831) and stages (p = 0.6140). There was also no association with p53 immunostaining (p = 0.4092). Y chromosome losses were not associated with survival in 224 invasive urothelial cancers (pT2-4; p = 0.2324), an increased risk for recurrences in 197 pTa tumours (p = 0.7649) or increased progression risk in 76 pT1 tumours (p = 0.4582). CONCLUSION: The data of this study show that Y chromosome losses are frequent in urothelial bladder cancer of all grades and stages, which could imply that loss of the Y chromosome is an early event in bladder cancer development. p53 mediated genomic instability is evidently not required for the development of Y chromosome losses. Since there was no correlation between Y chromosome losses and clinical outcome, detection of Y losses has no clinical relevance in urothelial bladder cancer.

Association analysis of serotonin receptor 1B (HTR1B) and brain-derived neurotrophic factor gene polymorphisms in Borderline personality disorder.

The purpose of this study was to test for association between Borderline personality disorder (BPD) and variants of the HTR1B and the brain-derived neurotrophic factor (BDNF) gene. We genotyped four HTR1B and the functional BDNF G196A marker in 161 Caucasian BPD patients and 156 healthy controls. There were no significant differences between groups in genotype or haplotype distribution of HTR1B markers or in genotype distribution of the BDNF marker. Logistic regression analyses revealed an over-representation of the BDNF 196A allele in HTR1B A-161 allele carrying BPD patients.