Miroslaw Andrusiewicz

XbaI and PvuII Polymorphisms of Estrogen Receptor 1 Gene in Females with Idiopathic Scoliosis: No Association with Occurrence or Clinical Form

Introduction XbaI single nucleotide polymorphism (SNP) (A/G rs934099) in estrogen receptor 1 gene (ESR1) was described to be associated with curve severity in Japanese idiopathic scoliosis (IS) patients and in Chinese patients with both curve severity and predisposition to IS. PvuII SNP (C/T rs2234693) of ESR1 was described to be associated with the occurrence of IS in the Chinese population; however, two replication studies did not confirm the findings. The ESR1 SNPs have never been studied in Caucasian IS patients. Methods Case-control study. 287 females with IS underwent clinical, radiological and genetic examinations. The patients were divided into three groups according to curve progression velocity: non-progressive IS, slowly progressive IS (progression <1° per month), and rapidly progressive IS (progression ≥1° per month). The radiological maximum Cobb angle was measured and surgery rate established. A control group consisted of 182 healthy females. Results All results followed Hardy-Weinberg equilibrium. In the case-control study, genotype frequency in the patients did not differ for the XbaI (AA = 33.5%, AG = 49.1%, GG = 17.4%), nor for the PvuII (TT = 26.8%, TC = 50.2%, CC = 23.0%) comparing to controls (AA = 33.5%, AG = 50.5%, GG = 15.9%) and (TT = 23.1%, TC = 51.1%, CC = 25.8%), respectively, p = 0.3685, p = 0.6046. The haplotype frequency for the patients (AT = 47.1%, GC = 39.2%, AC = 8.9%, GT = 2.8%) did not differ from the controls (AT = 44.8%, GC = 37.4%, AC = 14.0%, GT = 3.8%), p = 0.0645. No difference was found either in XbaI (p = 0.8671) or PvuII (p = 0.3601) allele distribution between the patients and the controls. In the case study, there was no significant difference in genotype frequency for the non-progressive, slowly progressive, and rapidly progressive scoliosis. No difference was found in genotype or haplotype distribution for the mean maximum Cobb angle or the surgery rate. Conclusions No association was found between ESR1 XbaI or ESR1 PvuII SNP and idiopathic scoliosis in Caucasian females. None of the previously reported associations could be confirmed, regarding curve severity, progression or operation rate.

Nicotinamide phosphorybosiltransferase overexpression in thyroid malignancies and its correlation with tumor stage and with survivin/survivin DEx3 expression

Nicotinamide phosphorybosiltransferase (NAMPT) plays an important role in the regulation of cellular growth, angiogenesis, and apoptosis in mammalian cells. NAMPT overexpression has been recently found in colorectal, breast, prostatic, gastric, esophageal, pancreatic cancers, and specific NAMPT inhibitors might be adjuvant therapeutic modalities. In this study, we analyzed NAMPT expression in 40 malignant and in 67 benign thyroid tissue samples using qPCR. We also investigated relationships between NAMPT expression and survivin/survivin splicing variants DEx3 and 2B expressions. NAMPT expression was significantly higher in thyroid cancers (P < 0.0001), and it was positively correlated with tumor stage (P = 0.0012; r = 0.493). NAMPT expression was significantly higher in tumors staged pT3 or pT4 (16 cases) than in tumors staged pT1 or pT2 (24 cases) (P = 0.0106). Metastases to the lymph nodes were found in 12 out of 40 cases, and NAMPT expression was higher in the metastatic group (P = 0.0258). Multifocality was not associated with higher NAMPT expression (P = 0.3451). NAMPT expression in thyroid cancers significantly correlated with survivin and with survivin splice variant DEx3 expressions (P < 0.0001; r = 0.624 and P = 0.0239; r = 0.357, respectively). There was no correlation between NAMPT and survivin 2B expressions (P = 0.3508). This is the first study demonstrating NAMPT overexpression in thyroid malignancies using quantitative RT-PCR. Moreover, it shows that NAMPT is upregulated in patients with more advanced tumor stage and metastatic disease which may prove to be clinically relevant. Further studies are needed to explain the role of NAMPT in thyroid cancer biology and the possible use of NAMPT inhibitors in thyroid cancer.

Survivin Delta Ex3 Overexpression in Thyroid Malignancies

Context Thyroid cancer incidence has increased significantly during the past decades and is the most common type of endocrine malignancy. Many factors in thyroid cancers were studied as independent predictors of a poor prognosis. Objective The objective of the study was to evaluate survivin expression – BIRC5 and its splice variants: survivin delta Ex3 and survivin 2B in benign and malignant thyroid nodules. Design Thyroid tissues samples from a group of 50 patients consisting of: 29 patients with thyroid cancers (including medullary, papillary, follicular and undifferentiated types), as well as from 21 patients with non-cancerous thyroid tissues (including: 11 benign thyroid lesions and 10 healthy thyroid samples). Main Outcome Measures The analysis of the survivin gene expression and evaluation of the level of splice variants were performed using quantitative RT-PCR. Results A statistically significant higher level of expression of survivin gene – BIRC5 was detected in thyroid malignant nodules, when compared with benign lesions and healthy thyroid samples. Moreover, the comparison of survivin relative expression in different staged tumors (pT1, pT3, and pT4) revealed a much higher amount of BIRC5 transcripts in tumor tissues of pT3/pT4. The comparison of survivin expression between benign thyroid nodules and healthy thyroid did not reveal significant differences. Importantly, high expression rate of the survivin delta Ex3 splice variant characterized thyroid carcinomas. Conclusion The results suggest that survivin, especially survivin delta Ex3 splice variant being overexpress, is a characteristic feature of thyroid malignancy.

The association between irisin and muscle metabolism in different thyroid disorders

Irisin is a new adipo‐myokine, encoded by the FNDC5 gene. Currently, there is a discussion regarding the relation between thyroid function and irisin concentration. This prospective study assesses the influence of thyrometabolic changes on serum irisin concentration in association with altered muscle metabolism. This is performed on a large cohort of patients affected by severe hypo‐ or hyperthyroidism, as well as by the expression of the FNDC5 gene in thyroid tissue affected by different pathologies.

Selection of reliable reference genes in eutopic and ectopic endometrium for quantitative expression studies.

PURPOSE Physiological changes during menstrual cycle cause the endometrium and endometriosis to develop specific kind of tissues, especially in regard to the gene expression profiles, which may include also housekeeping genes, commonly used as reference genes (RGs) in quantitative studies. Reverse transcription, followed by quantitative polymerase chain reaction (RT-qPCR) is the most precise and commonly used method in gene expression studies. In order to reduce effects of technical approaches and biological variability of genes expression level, the studies often employ RGs in experimental data normalization. However, the expression of RGs is not always stable and depends on several variables. Thus, the selection of appropriate RG is one of the most significant steps to obtain reliable results in RT-qPCR-based methods. MATERIAL AND METHODS With the usage of RT-qPCR, we researched the expression of seven genes (ACTB, B2M, G6PD, GAPD, GUSB, HPRT and PPIA) as reliable reference genes in eutopic and ectopic endometrial tissue specimens obtained during standard surgery of women of reproductive age. Stability of expression level was analyzed by the most universal MS Excel plug-ins including: geNorm, NormFinder and BestKeeper. The descriptive statistics were evaluated using Statistica software. RESULTS The distribution of threshold (Ct) values was not equal. We identified genes with higher expression level (referring to Ct values) such as ACTB and B2M, medium e.g., GAPD and low expression level, e.g., G6PD and HPRT. We demonstrated that the stability of the analyzed reference genes was not homogenous, and different algorithms pointed to PPIA, GAPD and B2M as the most stable ones in eutopic and ectopic endometrium. On the contrary to these, GUSB and G6PD were the most unstable ones. CONCLUSIONS In RT-qPCR-based analyses of gene expression level in eutopic and ectopic endometrium, we strongly recommend that a minimum of two reference genes are to be used and we determined that the most suitable seem to be PPIA and GAPD.

ESR1 and ESR2 genotypes and the age at menarche in idiopathic scoliosis

Background Environmental and genetic factors have influence on the age at menarche (AAM). Disturbance of the AAM in patients with idiopathic scoliosis (IS) were postulated [1]. Estrogen receptor genes 1 and 2 (ESR1, ESR2) single nucleotide polymorphisms (SNP) in IS were suggested to have some association with predisposition to IS [2]. ESR SNPs were reported to have association with AAM in healthily females [3].

Replication study of estrogen receptor 1 XbaI polymorphism in adolescent idiopathic scoliosis (AIS) Caucasian population

Background It has been suggested that XbaI single nucleotide polymorphism (SNP) (A/G rs934099) in estrogen receptor 1 (ESR1) may be associated with curve severity in Japanese AIS patients [1] and with both curve severity and predisposition for AIS in Chinese patients [2]. However, replication studies have not confirmed these findings [3]. The role of the XbaI ESR1 polymorphism rs9340799 in AIS has never been described in Caucasian AIS patients.

Trichrome Mallory's stain may indicate differential rates of RNA synthesis in eutopic and ectopic endometrium.

Mallorys triple staining is a histochemical technique used mainly for analysing connective tissues and glands and other tissues. We have described the differences in the nuclear staining between eutopic and ectopic endometrium as well as endometrial hyperplasia and adenocarcinoma using the Mallorys method. The ultrastructural differences between eutopic and ectopic endometrium have been detected. In normal and hyperplastic endometrium the presence of stromal cell nuclei with an increased affinity to aniline blue has been observed. The affinity has disappeared after digestion of tissues with RNase. In cases of endometriosis, independently of cell types, the nuclei have shown affinity to orange G. Similar effects in adenocarcinoma have been noted. The ultrastructural studies have shown that in normal endometrium the stroma contained cells with euchromatic and low electron density cell nuclei. In endometriosis heterochromatic cell nuclei present both in the stroma and within glands have been detected. The results indicate that the Mallorys technique may be a useful tool for recognizing the differences between eutopic and ectopic endometrium. The affinity for aniline blue in normal and hyperplastic endometrium occurs most likely due to increased RNA synthesis. Based on Mallorys staining a similarity between hyperplasia and unchanged endometrium in contrast to similar results of the staining obtained in cases of adenocarcinoma and endometriosis may be suggested.

Increased expression of PELP1 in human sperm is correlated with decreased semen quality

Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is a scaffolding protein involved in both genomic and nongenomic estrogen signal transduction pathways. To date, the role of PELP1 protein has yet to be characterized in human sperm and has not been associated with sperm parameters. To confirm the presence of PELP1 in human sperm, fresh semen samples were obtained from 178 donors. The study was designed to establish both mRNA and protein presence, and protein cellular localization. Additionally, the number of PELP1-positive spermatozoa was analyzed in men with normal and abnormal semen parameters. Sperm parameters were assessed according to the World Health Organization (WHO) 2010 standards. The presence of PELP1 in spermatozoa was investigated using four precise, independent techniques. The qualitative presence of transcripts and protein was assessed using reverse transcription-polymerase chain reaction (RT-PCR) and western blot protocols, respectively. The cellular localization of PELP1 was investigated by immunocytochemistry. Quantitative analysis of PELP1-positive cells was done by flow cytometry. PELP1 mRNA and protein was confirmed in spermatozoa. Immunocytochemical analysis identified the presence of PELP1 in the midpieces of human sperm irrespective of sperm parameters. Becton Dickinson fluorescence-activated cell sorting (FACSCalibur™) analysis revealed a significantly lower number of PELP1-positive cells in males with normal semen parameters versus abnormal samples (42.78% ± 11.77% vs 61.05% ± 21.70%, respectively; P = 0.014). The assessment of PELP1 may be a time-saving method used to obtain information about sperm quality. The results of our study suggest that PEPL1 may be utilized as an indicator of sperm quality; thereby, PELP1 may be an additional biomarker useful in the evaluation of male infertility.

Survivin DEx3 as a biomarker of thyroid cancers: A study at the mRNA and protein level

Survivin and its splice variants DEx3 and 2B are involved in pathogenesis of numerous types of cancer. Proliferating cell nuclear antigen (PCNA) level correlates with cellular proliferation. The present study aimed to analyze the potential utility of survivin and its splice variants DEx3 and 2B as biomarkers for thyroid cancer. PCNA, survivin and its splice variants DEx3 and 2B expressions were analyzed in 22 tissue samples (15 thyroid cancers and 7 benign lesions) by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry (IHC). There was significantly higher staining for survivin (P=0.019), survivin DEx3 (P=0.001), survivin 2B (P=0.0149) and PCNA (P=0.0237) in thyroid malignant tumors when compared with benign lesions. The receiver operating characteristics curve analysis has shown that the cut-off points of survivin IHC expression >2 [sensitivity 46.7%; specificity 100%; area under curve (AUC) 0.810; P=0.0005] and survivin DEx3 IHC expression >0 (sensitivity 86.7%; specificity 100%; AUC 0.933; P<0.0001) were the best predictors of thyroid malignancy. Additionally, PCNA staining >1 (sensitivity 93.3%; specificity 71.4%; AUC 0.790; P=0.0243) and survivin 2B >2 (sensitivity 46.7%; specificity 100%; AUC 0.824; P=0.0002) were the best predictors of thyroid cancer. In conclusion, the present study exhibited that survivin DEx3 expression has high specificity and sensitivity for discrimination between benign thyroid lesions and cancers. Survivin DEx3 may be considered a biological marker of thyroid malignancy and therefore applied in clinical practice.