Search

Carp pituitary proteins and glycoproteins were separated by the combination of immobilized metal affinity chromatography (IMAC) and affinity chromatography on Con A-Sepharose. The protein fractions were analysed by SDS-PAGE. Luteinizing hormone (in the α1β and α2β forms), growth hormone, free α1 subunit and β subunit of thyroid-stimulating hormone were identified by N-terminal amino acid sequencing. N-linked oligosaccharide chains of thyroid-stimulating hormone (β TSH) were separated after fluorescent labelling on a GlycoSep N column and treated by exoglycosidases. Among the saccharide components, complex and hybrid structures terminating SO4-GalNAc-GlcNAc-Man-, and high mannose structures, with 1 to 8 mannose units attached to the oligosaccharide core (GlcNAc)2(Man)3, were found.

Germline DNA testing using the next-gene-ration sequencing (NGS) technology has become the analytical standard for the diagnostics of hereditary diseases, including cancer. Its increasing use places high demands on correct sample identification, independent confirmation of prioritized variants, and their functional and clinical interpretation. To streamline these processes, we introduced parallel DNA and RNA capture-based NGS using identical capture panel CZECANCA, which is routinely used for DNA analysis of hereditary cancer predisposition. Here, we present the analytical workflow for RNA sample processing and its analytical and diagnostic performance. Parallel DNA/RNA analysis allowed credible sample identification by calculating the kinship coefficient. The RNA capture-based approach enriched transcriptional targets for the majority of clinically relevant cancer predisposition genes to a degree that allowed analysis of the effect of identified DNA variants on mRNA processing. By comparing the panel and whole-exome RNA enrichment, we demonstrated that the tissue-specific gene expression pattern is independent of the capture panel. Moreover, technical replicates confirmed high reproducibility of the tested RNA analysis. We concluded that parallel DNA/RNA NGS using the identical gene panel is a robust and cost-effective diagnostic strategy. In our setting, it allows routine analysis of 48 DNA/RNA pairs using NextSeq 500/550 Mid Output Kit v2.5 (150 cycles) in a single run with sufficient coverage to analyse 226 cancer predisposition and candidate ge-nes. This approach can replace laborious Sanger confirmatory sequencing, increase testing turnaround, reduce analysis costs, and improve interpretation of the impact of variants by analysing their effect on mRNA processing.

The Guidelines for Clinical Practice for carriers of pathogenic variants in clinically relevant cancer predisposition genes define the steps of primary and secondary prevention that should be provided to these individuals at high risk of developing hereditary cancer in the Czech Republic. The drafting of the guidelines was organized by the Oncogenetics Working Group of the Society for Medical Genetics and Genomics of J. E. Purkyně Czech Medical Society (SLG ČLS JEP) in cooperation with the representatives of oncology and oncogynecology. The guidelines are based on the current recommendations of the National Comprehensive Cancer Network (NCCN), European Society of Medical Oncology (ESMO) and take into account the capacity of the Czech healthcare system.

Male breast cancer (mBC) is a rare cancer diagnosis that constitutes less than 1 % of all breast cancer cases globally. Genetic factors play an important role in the mBC risk. Germline pathogenic variants (PVs) in cancer predisposition genes could be identified in about 15 % of cases. We performed germline genetic testing in 248 Czech mBC patients and 3,626 non-cancer male controls using next-generation sequencing by the CZECANCA panel (226 genes). We identified 46/248 (18.5 %) carriers of PVs in the established mBC predisposition genes, primarily in BRCA2 (N = 34), less frequently in BRCA1 (N = 7) and PALB2 (N = 5). The presence of a PV in these genes significantly increased the mBC risk (OR 44.04; 5.82; and 8.26, respectively). Additionally, we identified 16 carriers of PVs in candidate mBC genes, but only PVs in CHEK2 were significantly associated with increased mBC risk (OR = 4.98). The significance of 26 germline alterations in 23/192 additionally analysed genes remained uncertain. The carriers of PVs in BRCA1 and CHEK2 were significantly younger (55.8 and 52.6 years, respectively) than non-carriers (64.8 years), and all carriers of PVs in the established genes had more frequently grade G3 tumours and positive family cancer history. Our study underscores the critical role of BRCA2 in mBC predisposition while also highlighting the potential contributions of additional genes that warrant further investigation. Moreover, it supports and justifies universal genetic testing for all mBC patients to generally improve early cancer detection and tailored treatment.

The guidelines for clinical practice for carriers of pathogenic variants in clinically relevant genes predisposing to Lynch syndrome and colorectal cancer define the steps of primary and secondary prevention that should be provided to the individuals at high risk of developing hereditary cancer in the Czech Republic. The drafting of the guidelines was organized by the Oncogenetics Working Group of the Society for Medical Genetics and Genomics of J. E. Purkyně Czech Medical Society, in cooperation with representatives of oncology, oncogynecology, and gastroenterology. The guidelines are based on the current recommendations of the National Comprehensive Cancer Network (NCCN), European Society of Medical Oncology (ESMO) and take into account the capacity of the Czech healthcare system.

Background: Hereditary cancer syndromes are an important subset of malignant cancers caused by pathogenic variants in one of many known cancer predisposition genes. Diagnosis of cancer predisposition is based on genetic testing using next-generation sequencing. This allows many genes to be analysed at once, increasing the number of variants identified. The correct classification of the variants found is essential for the clinical interpretation of genetic test results., Purpose: The aim of this study is to summarise the rules for classifying identified variants within individual laboratories and to present the process for creating a common classification. In the Czech Republic, the sharing of identified genetic variants and the development of their consensus classification among national laboratory diagnostic communities is carried out within the Czech Cancer Panel for Clinical Application (CZECANCA) consortium of scientific and diagnostic oncogenetic laboratories. Consensus for variant classification follows a defined protocol. Sharing the results and consensus classification accelerates and refines the release of genetic test results, harmonises results between laboratories and thus contributes to improving the care of individuals at high risk of cancer and their relatives.

Background: Breast cancer is a complex, multifactorial disease influenced by many genetic factors. Besides the relatively rare pathogenic variants in high or moderate penetrant cancer predisposition genes, breast cancer risk is modified by numerous low risk alleles considered to be polygenic genetic factors. While the risks associated with individual polygenic loci are negligible, its cumulative effect can reach clinically significant values and it can be expressed as a polygenic risk score (PRS). PRS is recently considered to be a possible tool improving assessment of absolute and cumulative risks at the individual level., Purpose: Several single nucleotide polymorphism sets for PRS assessment have recently been developed and prepared for their implementation into clinical practice. The following text aims to explain the fundamental principles of the PRS assessment and its interpretation as a candidate prediction tool. The use of the PRS should always depend on genetic analysis of pathogenic variants in cancer predisposition genes including its current limitations.

Balance control is a critical task of daily life, the ability to maintain upright posture becomes of particular concern during aging when the sensory and motor system becomes deteriorated. Falls contribute to the most deaths caused by injury within the aged population, and the mortality rate following a fall is drastically elevated. Longitudinal and reliable assessment of balance control abilities is a critical point in the prediction of increased risk of falling in an elderly population. The primary aim of the study was to evaluate the efficiency of the Homebalance test in the identification of persons being at higher risk of falling. 135 subjects (82 women and 53 men) with geriatric syndrome have been recruited and the Homebalance and the Tinetti Balance test were performed. Results of both tests strongly correlated proving the good performance of the Homebalance test. Standing balance declines with increasing body mass index in both genders. Analysis of fluctuations of the center of pressure (COP) revealed higher frequency and magnitude in mediolateral direction COP movements when compared women to men. A strong negative correlation has been found between Tinetti static balance score and the total length of the COP trajectory during the examination on Homebalance (r = -0.6, p<0.001). Although both methods revealed good performance in detecting balance impairment, Homebalance test possesses higher precision due to the continuous nature of COP-derived parameters. In conclusion, our data proved that the Homebalance test is capable to identify persons with impaired balance control and thus are at higher risk of falling.

Ovarian cancer is one of the most common gynecologic cancers with the highest mortality rate over a long period. Genetic predisposition to ovarian cancer is unusually high. In the Czech Republic, causal mutation in any ovarian cancer predisposition gene is identified in approximately 30% of the ovarian cancer patients. Therefore, according to the current guidelines, all ovarian cancer patients should be provided with genetic testing. The BRCA1 and BRCA2 are the two major ovarian cancer predisposition genes. Nevertheless, mutations in other predisposition genes, including RAD51C and RAD51D, are associated with high ovarian cancer risk. Mutations in RAD51C and RAD51D are found in 1% of ovarian cancer patients in each respective gene. Currently, identification of germline mutation in RAD51C and RAD51D is primarily of preventive importance but it potentially could make a prognostic difference. The aim of this review is to summarize the recent RAD51C and RAD51D knowledge, including the biological function, cancer risks associated with germline mutations, and recommendations for mutation carriers.

Background: Ovarian cancer is a disease with high mortality. Approximately 1,000 women are diagnosed with ovarian cancer in the Czech Republic annually. Women harboring a mutation in cancer-predisposing genes face an increased risk of tumor development. Mutations in BRCA1, BRCA2, BRIP1, and Lynch syndrome genes (RAD51C, RAD51D, and STK11) are associated with a high risk of ovarian cancer, and mutations in ATM, CHEK2, NBN, PALB2, and BARD1 appear to increase the risk. Our aim was to examine the frequency of mutations in cancer-predisposing genes in the Czech Republic., Materials and Methods: We analyzed 1,057 individuals including ovarian cancer patients and 617 non-cancer controls using CZECANCA panel next-generation sequencing on the Illumina platform. Pathogenic mutations in high-risk genes, including CNVs, were detected in 30.6% of patients. The mutation frequency reached 25.0% and 18.2% in subgroups of unselected ovarian cancer patients and patients with a negative family cancer history, respectively. The most frequently mutated genes were BRCA1 and BRCA2. The overall frequency of mutations in non-BRCA genes was comparable to that in BRCA2. The mutation frequency in ovarian cancer patients aged &gt;70 years was three times higher than that in patients diagnosed before the age of 30., Conclusion: Ovarian cancer is a heterogeneous disease with a high proportion of hereditary cases. The lack of efficient screening for early diagnosis emphasizes the importance of identifying carriers of mutations in ovarian cancer-predisposing genes; this is because proper follow-up and prevention strategies can reduce overall ovarian cancer-related mortality. This work was supported by grants AZV 15-27695A, SVV2019/260367, PROGRES Q28/LF1. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 7. 3. 2019 Accepted: 24. 4. 2019.

Background: Hereditary mutations in the CHEK2 gene (which encodes CHK2 kinase) contribute to a moderately increased risk of breast cancer (BC) and other cancers. Large variations in the frequency of CHEK2 mutations and the occurrence of variants of unknown clinical significance (VUS) complicate estimation of cancer risk in carriers of germline CHEK2 mutations., Patients and Methods: We performed mutation analysis of 1,526 high-risk Czech BC patients and 3,360 Czech controls. Functional analysis was performed for identified VUS using a model system based on a human RPE1-CHEK2-KO cell line harboring biallelic inactivation of endogenous CHEK2., Results: The frequency of ten truncating CHEK2 variants differed markedly between BC patients (2.26%) and controls (0.11%; p = 4.1 × 1012). We also found 23 different missense variants in 4.5% patients and in 4.0% of controls. The most common was p.I157T, which was found in patients and controls with the same frequency. Functional analysis identified nine functionally deleterious VUS, another nine functionally neutral VUS, and four intermediate VUS (including p.I157T). We found that carriers of truncating CHEK2 mutations had a high BC risk (OR 8.19; 95% CI 4.11-17.75), and that carriers of functionally deleterious missense variants had a moderate risk (OR 4.06; 95% CI, 1.37-13.39). Carriers of these mutations developed BC at 44.4 and 50.7 years, respectively. Functionally neutral and functionally intermediate missense variants did not increase the BC risk. BC in CHEK2 mutation carriers was frequently ER-positive and of higher grade. Notably, carriers of CHEK2 mutations developed second cancers more frequently than BRCA1/BRCA2/PALB2/p53 or mutation non-carriers., Conclusion: Hereditary CHEK2 mutations contribute to the development of hereditary BC. The associated cancer risk in mutation carriers increases with the number of affected individuals in a family. Annual follow-up with breast ultrasound, mammography, or magnetic resonance imaging is recommended for asymptomatic mutation carriers from the age of 40. Surgical prevention and specific follow-up of other tumors should be considered based on family cancer history. The work was supported by grants from the Czech Health Research Council of the Ministry of Health of the Czech Republic NR 15-28830A, 16-29959A, NV19-03-00279, projects of the PROGRES Q28/LF1, GAUK 762216, SVV2019 / 260367, PRIMUS/17/MED/9, UNCE/MED/016, Progress Q26, LQ1604 NPU II and project AV&#268;R Qualitas. The analysis of a set of unselected controls was made possible by the existence and support of the scientific infrastructure of the National Center for Medical Genomics (LM2015091) and its project aimed at creating a reference database of genetic variants of the Czech Republic (CZ.02.1.01/0.0/0.0/16&#95;013/0001634). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 2. 4. 2019 Accepted: 14. 5. 2019.

Individuals with hereditary cancer syndromes form a minor but clinically important subgroup of oncology patients, comprising several thousand cases in the Czech Republic annually. In these patients, the identification of pathogenic mutations in cancer susceptibility genes has an important predictive and, in some cases, prognostic value. It also enables rational preventive strategies in asymptomatic carriers from affected families. More than 150 cancer susceptibility genes have been described so far; however, mutations in most of them are very rare, occurring with substantial population variability, and hence their clinical interpretation is very complicated. Diagnostics of mutations in cancer susceptibility genes have benefited from the broad availability of next-generation sequencing analyses using targeted gene panels. In order to rationalize the diagnostics of hereditary cancer syndromes in the Czech Republic, we have prepared the sequence capture panel "CZECANCA", targeting 219 cancer susceptibility genes. Besides more than 50 clinically important high- and moderate-penetrance susceptibility genes, the panel also targets less common candidate genes with uncertain clinical relevance. Alongside the panel design, we have optimized the analytical and bioinformatics pipeline, which will facilitate establishing a collective nationwide database of genotypes and clinical data from the analyzed individuals. The key objective of this project is to provide diagnostic laboratories in the Czech Republic with a reliable procedure and collective database improving the clinical utility of next-generation sequencing analyses in high-risk patients, which would help improve the interpretation of rare or population-specific variants in cancer susceptibility genes.

Background: The PALB2 (FANCN) gene was identified as a component of endogenous BRCA2 complex that encodes a DNA repair protein participating along with BRCA1 and BRCA 2 proteins in DNA double-strand break repair. Hereditary PALB2 mutations are associated with an increased risk of breast and pancreatic cancers in heterozygotes. Breast cancer risk for PALB2 mutation carriers has recently been estimated at 33-58% depending on family history of breast cancer; pancreatic cancer risk in carriers of PALB2 mutations has not been precisely quantified, yet., Materials and Results: Results of a study identifying PALB2 mutations in high-risk, BRCA1/2-negative, breast and/or ovarian cancer patients in the Czech Republic indicate that the frequency of hereditary PALB2 mutations in our population is quite high. Interestingly, almost 20% of all recognized mutations comprised large genomic rearrangements. The highest proportion of PALB2 mutations (comparable with the number of mutations reported for BRCA2) was found in a subgroup of hereditary breast cancer patients (5.5%). Frequency of mutations in an independent group of Czech unselected pancreatic cancer patients was approximately 1.3%., Conclusion: Considering the frequency of pathogenic, hereditary PALB2 mutations in our population, their phenotypic similarity to BRCA2, and expected risk of breast cancer associated with PALB2 mutations, its screen-ing (including large genomic rearrangements) should be encouraged in patients from hereditary breast cancer families. The follow-up of pathogenic PALB2 mutation carriers should be similar to that in BRCA2 mutation carriers, enabling early diagnosis, prevention, and possible targeted therapy. Preventive surgical interventions for the carriers could be considered in case of strong family cancer history and evident segregation of a pathogenic mutation with a tumor phenotype. Additional analysis of various cancer patient populations and further meta-analyses will be necessary for accurate assessment of PALB2 gene penetrance and its significance for the risk of pancreatic and other cancers.

Background: Analysis of the major breast cancer (BC) predisposition genes BRCA1 and BRCA2 enables identification of high-risk individuals. Specialized programs enrolling the carriers of BRCA1/2 mutations facilitate an improvement in prevention and early diagnostics in asymptomatic individuals and rationalize the selection of individualized treatment in case of a BC onset. However, the carriers of mutations in the major predisposition genes represent only approximately 25% of cases among high-risk BC patients. Numerous candidate predisposing genes for breast and other cancers have recently been identified. The risk of cancer development associated with alterations in these genes is lower, and there is a considerable population variability in different regions worldwide., Aim: We have performed mutation analyses of moderate-risk cancer susceptibility genes to evaluate their clinical importance for genetic counseling in high-risk patients suffering from breast and other cancers in the Czech population., Results: Czech oncological patients were analysed for mutation in ATM, CHEK2, NBS1 (NBN) and PALB2 genes. The majority of analyzed individuals represent the population of high-risk BRCA1/2-negative BC patients., Conclusions: Based on results of this study, we recommend an analysis of recurrent truncating mutations in the CHEK2 gene (the c.1100delC mutation and a large deletion affecting exons 9-10) in BRCA1/2-negative patients from high-risk BC families. A clinical assessment of missense variants in CHEK2 is not suitable. A routine mutation analysis of the ATM and NBS1 (NBN) genes is not recommended in BC patients due to the low frequency of alterations in these genes in the Czech Republic. An identification of truncating mutations in the PALB2 gene is important in BRCA1/2-negative BC patients from families with a strong history of BC (HBC families). The frequency of PALB2 mutations may be comparable to the frequency of mutations in the BRCA2 gene in Czech HBC families.

Heterozygous carriers of germ-line mutations in the BRCA1 gene are at high risk for the development of breast and ovarian cancer. Inactivating mutations have been identified in the whole coding region of the gene, however, repeatedly occuring mutations can explain a large proportion of gene alterations detected in certain ethnic groups. In Czech patients, the 5382insC and 185delAG mutations may account for approximately 50% of all BRCA1 abnormalities (unpublished data). In the present study, a rapid and simple method to identify these short insertions and deletions that alter the size of the polymerase chain reaction (PCR) product is described. The analysis involves the separation of fragments amplified with primers that flank altered sites in the BRCA1 gene on non-denaturing polyacrylamide gels containing Spreadex Polymer NAB. The increased resolving power of Spreadex gels enables full separation of two DNA fragments that differ by 1-bp on gels that are 5 cm long. The method gave interpretable results with the genetic material obtained from all tested mutation carriers and control persons. Defective alleles were also detected in DNA samples from carriers of the 1135delA mutation in BRCA1 and the 4206ins4 mutation in BRCA2. These results suggest that electrophoresis on Spreadex gels can be used universally for detection of the most frequent frameshift mutations in BRCA genes. The method is suitable even for rapid screening of frequent germ-line mutations in BRCA genes in breast and ovarian cancer patients not selected for family history of cancer or age at diagnosis.