13 novel mutations in NLRP7 were identified. We confirm that mutations in NLRP7 affect female but not male reproduction, and provide evidence that transcript variant 2 of NLRP7 is the important isoform in this condition. The mutation clustering seen confirms that the LRR is critical for normal functioning of NLRP7.

Methylation of the cytosine residues within the CpG dinucleotides plays an important role in the fundamental cellular processes, human diseases and even cancer. The DNA methylation represents a very stable sign and therefore may be used as a valuable marker for cancer screening. Epigenetic cancer biomarkers are independent of classical morphology and thus show extensive potential to overcome the limitations of cytology. Several epigenetic cancer markers have been reported to be detectable in body fluids such as bronchial aspirate, sputum, plasma and serum.Short stature homeobox gene 2 (SHOX2) encodes a homeo-domain transcription factor, which has been identified as a close homologue of the SHOX gene and both genes are involved in skeletogenesis and heart development. Methylation of SHOX2 gene has been shown to be present at high prevalence in carcinomas of lung, however may also be used to identify other tumour entities.In the presented study, we have compared suitability of two types of material associated with lung cancer for the detection of SHOX2 methylation. We have confirmed that methylation of SHOX2 gene represents reliable marker of lung malignancies. The parallel tests in the blood plasma revealed that it may represent a good alternative material for testing of the SHOX2 methylation, making the test available to patients who are unable to undergo bronchoscopy.

Lung carcinoma is the most frequently occurring cancer worldwide and the Non-small Cell Lung Cancer (NSCLC) subtype represents 80% of all diagnosed cases. Epidermal growth factor receptor (EGFR) has an important dual role in NSCLC patients. On one hand, EGFR is frequently mutated in many types of tumors, which leads to deregulation of important downstream pathways including those affecting cell proliferation, differentiation and migration. On the other hand, presence of certain activating mutation leads to increased sensitivity of EGFR to tyrosine kinase inhibitors (TKIs) treatment. Detection of these mutations is essential for identification of NSCLC patients who would profit from such therapy. However, due to the nature of available tumor material and the relatively high number of mutation hot spots, such DNA analysis may be challenging and time consuming. Here we present an approach combining direct sequencing and SNaPshot assay for identification of EGFR mutations in FFPE tissues as well as in rarely analyzed cytological smears. Using this strategy on the set of 450 tested NSCLC samples; we have identified 29 activating mutations and 14 variants, which might be interesting in predicting the efficiency of TKI therapy.Key words: non-small cell lung cancer, SNaPshot analysis, sequencing, EGFR mutations Lung carcinoma is the most frequently occurring cancer worldwide and is responsible for one third of the deaths resulting from malignant diseases [1]. In Slovakia, the lung cancer is the second most common cancer (following the colon cancer). There are 21 new cases per 100 000 inhabitants diagnosed each year (11 in men and 10 in women population) [2]. Primary lung cancer may be divided into two different histological groups: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The latter group represents almost 80% of all cases. NSCLC can be further divided into several other subtypes: squamous (epidermoid), adeno, large cell, neuroendocrine carcinoma and special types of carcinomas with pleiomorphic, sarcomatoid or sarcomatous elements [3].Lung cancer treatment depends on the tumor stage and type. Therapy of lung carcinomas is based on the combination of different approaches, especially surgery, chemotherapy and radiotherapy. In SCLC patients, chemotherapy using the anti-tumor cytotoxic drugs, e.g. platinum derivatives, taxol or cyclophosphamides is the most commonly used treatment. In advanced stages of NSCLC, a personalized biological treatment with TKIs or monoclonal antibodies may be used [4].Application of the EGFR monoclonal antibodies (cetuximab, panitumumab) leads to efficient blocking of several signaling pathways, e.g. MAPK cascade [5]. TKI treatment (erlotinib, gefitinib, afatinib), is frequently used in patients with NSCLC and detected EGFR activating mutations [6]. TK inhibitors are small molecules that bind to the activation loop of cytoplasmic domain of EGFR receptor and suppress its activity by competing with binding of ATP. Gefitinib, a reversible competitive inhibitor [7], has...

The presence of BCR-ABL oncogene mutations in patients with chronic myeloid leukemia (CML) may be responsible for the failure of tyrosine kinase inhibitor (TKI) treatment. The aim of the study was to evaluate the frequency of BCR-ABL gene mutations in patients with CML treated with tyrosine kinase inhibitors. Our lab received 64 samples (34 women, 30 men) from patients with CML who failed or had suboptimal response to TKI treatment. The mutation analysis was performed in 61 patients with CML, 3 patients could not be tested because of inadequate RNA quality. An 866 base pair fragment containing the ABL kinase domain was amplified in a seminested RT (reverse transcriptase)-PCR and then sequenced using Applied Biosystems BigDye Terminator chemistry with two pairs of primers. We analyzed 61 patients with CML, 11 mutations were detected in 13 (21%) patients and SNP (single nucleotide polymorphism) in 6 patients (10%). In addition to 9 point mutations (G250E / F317L, F359V, L387M, Y253H, M388L, M244V, T315I, D276G), 35 bp insertion between exons 8 and 9 and deletion exon 7 were detected. Our results demonstrate that direct sequencing is suitable for routine clinical monitoring patients with CML and may be useful for optimizing therapy.

Abstract:Introduction: It has been confi rmed, that there is a causal relationship between persistent infection of high risk human papillomavirus (HR-HPV) and the development of cervical cancer. In population of women older than 30 years HPV infection becomes a signifi cant etiological factor of precancerous lesion of the cervix, but HPV infection may spontaneously regress in the majority of the cases. Patients and methods:The analysed study group consisted of 397 samples with cytological diagnosis of atypical squamous cells of unknown signifi cance (ASCUS). All cases underwent HPV DNA testing using the Hybrid Capture 2 (HC2) assay. We analysed prevalence of HR-HPV and a viral load expressed as relative light units/ cut off ratio (RLU/CO) in different age groups with cytological diagnoses of ASCUS. Results: The prevalence of HR-HPV with cytological diagnoses of ASCUS was detected in 44 %. The prevalence of HR-HPV between patients aged 17-29 and between patients aged 30-40 was 55 % and 48 % respectively and we detected signifi cant reduction of prevalence (28 %) in patients older than 41 years. Conclusion: Based on the results of presented study we assumed that age the 40 and over is crucial for the development of serious precancerous lesions in Slovakia, thus this age group is the most suitable for HPV triage of ASCUS. As a refi nement of that type of ASCUS triage we recommend to add to the algorithm quantitative measurement of viral load in the specimens in the form of RLU/CO ratio (Fig. 3, Ref. 27). Text in PDF www.elis.sk.