Inactivation of Epigenetic Regulators due to Mutations in Solid Tumors.

Main factors involved in carcinogenesis are associated with somatic mutations in oncogenes and tumor suppressor genes representing changes in the DNA nucleotide sequence. Epigenetic changes, such as aberrant DNA methylation, modifications of histone proteins, and chromatin remodeling, are equally important in the development of human neoplasms. From this perspective, mutations in the genes encoding key participants of epigenetic regulation are of particular interest including enzymes that methylate/demethylate DNA, enzymes that covalently attach or remove regulatory signals from histones, components of nucleosome remodeling multiprotein complexes, auxiliary proteins and cofactors of the above-mentioned molecules. This review describes both germline and somatic mutations in the key epigenetic regulators with emphasis on the latter ones in the solid human tumors, as well as considers functional consequences of these mutations on the cellular level. In addition, clinical associations of the somatic mutations in epigenetic regulators are presented, as well as DNA diagnostics of hereditary cancer syndromes due to germline mutations in the SMARC proteins and chemotherapy drugs directly affecting the altered epigenetic mechanisms for treatment of patients with solid neoplasms. The review is intended for a wide range of molecular biologists, geneticists, oncologists, and associated specialists.

[The incidence of AZF deletions, CFTR mutations and long alleles of the ar CAG repeats during the primary laboratory diagnostics in a heterogeneous group of infertily men].

AIM: microdeletions in the AZF region of Y-chromosome, compound heterozygotes of severe and mild CFTR mutations, and long CAG-repeats in the androgen receptor gene (AR) as marker of predisposition are frequently studied as genetic causes of male infertility. A simultaneously testing of the panel including biochemical, immunological, cyto- and molecular genetic markers is often performed during the complex laboratory diagnostics in infertile men. The aim of our work was to identify molecular genetic alterations, which are advisable for simultaneously testing in a man with currently uncertain form of infertility, to increase the informativeness of laboratory diagnostics. MATERIALS AND METHODS: a retrospective study of 885 infertile men was conducted. AZF deletions were determined by multiplex PCR using 10 STS-markers (sY83, sY84, sY86, sY127, sY134, sY143, sY152, sY157, sY254, sY255) and two control loci SRY and AMEL with detection in polyacrylamide gel. Mutations in the CFTR gene (F508del, CFTRdel2.3(21kb), I507del, 1677delTA, 2143delT, 2184insA, 394delTT, W1282X, G542X, N1303K, R334W and 5T) were detected by PCR and SNaPshot. For determination of length of the AR CAG-repeat a fragment analysis of fluorescently labeled PCR products on the 3500xl capillary sequencer was performed. RESULTS: AZF deletions were detected in 8.2% of cases. The largest number of deletions was found in the AZFc subregion (58.9%), while a frequency of deletion in AZFa, AZFb or combined deletions of two and three subregions was 5.5%, 12.3% and 23.3%, respectively. Heterozygous carriage of severe CFTR mutations was detected in 4.7% patients. The most frequent mutation was F508del (83.3%), followed by CFTRdel21kb (7.1%) and W1282X (4.8%). The frequency of the mild splicing 5T mutation was 5.3%, and its incidence was significantly higher than in the previously published control group (p=0.002). AR genotyping revealed that the most prevailing allele was 21 (CAG) (21.5%). Long alleles with 27 or more CAG-trinucleotides were identified in 7.5% of the tested cases. In addition, 7 CAG heterozygotes with Kleinfelter syndrome were found. CONCLUSION: during primary complex laboratory diagnostics in a heterogeneous group of infertile men, it is advisable to detect AZF deletions and the most frequent CFTR mutations, including F508del, CFTRdel21kb, 1677delTA, 2143delT, W1282X and 5T. The more comprehensive analysis of CFTR mutations is justified only in patients with verified obstructive infertility. Sequencing of panels associated with infertility genes using NGS technology is promising.

Structural Alterations in Human Fibroblast Growth Factor Receptors in Carcinogenesis.

Fibroblast growth factor (FGF) plays an important role in human embryogenesis, angiogenesis, cell proliferation, and differentiation. Carcinogenesis is accompanied by aberrant constitutive activation of FGF receptors (FGFRs) resulting from missense mutation in the FGFR1-4 genes, generation of chimeric oncogenes, FGFR1-4 gene amplification, alternative splicing shift toward formation of mesenchymal FGFR isoforms, and FGFR overexpression. Altogether, these alterations contribute to auto- and paracrine stimulation of cancer cells and neoangiogenesis. Certain missense mutations are found at a high rate in urinary bladder cancer and can be used for non-invasive cancer recurrence diagnostics by analyzing urine cell pellet DNA. Chimeric FGFR1/3 and amplified FGFR1/2 genes can predict cell response to the targeted therapy in various oncological diseases. In recent years, high-throughput sequencing has been used to analyze exomes of virtually all human tumors, which allowed to construct phylogenetic trees of clonal cancer evolution with special emphasis on driver mutations in FGFR1-4 genes. At present, FGFR blockers, such as multi-kinase inhibitors, specific FGFR inhibitors, and FGF ligand traps are being tested in clinical trials. In this review, we discuss current data on the functioning of the FGFR family proteins in both normal and cancer cells, mutations in the FGFR1-4 genes, and mechanisms underlying their oncogenic potential, which might be interesting to a broad range of scientists searching for specific tumor markers and targeted anti-cancer drugs.

[Genetic and biochemical features of the monogenic hereditary urolithiasis]. / Geneticheskie i biokhimicheskie osobennosti nasledstvennykh monogennykh form mochekamennoi bolezni.

Urolithiasis is a common urological problem. In most cases, this multifactorial pathology develops due to the combination of inherited low-penetrance gene variants and environment factors such as urinary tract infections and unbalanced diet. However, some cases are monogenic. These hereditary forms of urolithiasis manifest in childhood, and are characterized by multiple, bilateral and recurrent kidney stones and progress to chronic renal failure relatively early. Due to widening acceptance of exome and gene panel sequencing, substantially larger percentages of urolithiasis cases are now attributed to hereditary causes, up to 20% among patients of 18 years old or younger. Here we review genetic and biochemical mechanisms of urolithiasis, with an emphasis on its hereditary forms, including fermentopathies (primary hyperoxaluria, adenine phosphorobosyltransferase deficiency, phosphoribosyl-pyrophosphate-synthetase deficiency, xanthinuria, Lesch-Nihan syndrome) and these caused by membrane transport alterations (Dent's disease, familial hypomagnesia with hypercalciuria and nephrocalcinosis, hypophosphatemic urolithiasis, distal tubular acidosis, cystinuria, Bartter's syndrome). We suggest a comprehensive gene panel for NGS diagnostics of the hereditary urolithiasis. It is expected that accurate and timely diagnosis of hereditary forms of urolithiasis would enable the counselling of the carriers in affected families, and ensure personalized management of the patients with these conditions.

[Hormone Resistance and Neuroendocrine Differentiation Due to Accumulation of Genetic Lesions during Clonal Evolution of Prostate Cancer].

Progression of malignant tumors is largely due to clonal evolution of the primary tumor, clones acquiring different sets of molecular genetic lesions. Lesions can confer a selective advantage in proliferation rate or metastasis on the tumor cell population, especially if developing resistance to anticancer therapy. Prostate cancer (PCa) provides an illustrative example of clinically significant clonal evolution. The review considers the genetic alterations that occur in primary PCa and the mechanism whereby hormone-refractory PCa develops on hormone therapy, including mutations and alternative splicing of the androgen receptor gene (AR) and intratumoral androgen synthesis. Certain molecular genetic lesions determine resistance to new generation inhibitors (AR mutations that block the antagonist effect or allow other hormones to activate the receptor) or lead to neuroendocrine differentiation (repression of the AR signaling pathway, TP53 mutations, and amplification of the AURKA or MYCN oncogene). Multistep therapy based on the data about somatic mutations associated with progression and metastasis of the primary tumor can be expected to significantly improve the survival of patients with advanced PCa in the nearest future.

[Polymorphisms of KITLG, SPRY4, and BAK1 genes in patients with testicular germ cell tumors and individuals with infertility associated with AZFc deletion of the Y chromosome].

Testicular cancer is the most common form of solid cancer in young men. Testicular cancer is represented by testicular germ cell tumors (TGCTs) derived from embryonic stem cells with different degrees of differentiation in about 95% of cases. The development of these tumors is related to the formation of a pool of male germ cells and gametogenesis. Clinical factors that are predisposed to the development of germ-cell tumors include cryptorchidism and testicular microlithiasis, as well as infertility associated with the gr/gr deletion within the AZFс locus. KITLG, SPRY4, and BAK1 genes affect the development of the testes and gametogenesis; mutations and polymorphisms of these genes lead to a significant increase in the risk of the TGCT development. To determine the relationship between gene polymorphisms and the development of TGCTs, we developed a system for detection and studied the allele and genotype frequencies of the KITLG (rs995030, rs1508595), SPRY4 (rs4624820, rs6897876), and BAK1 (rs210138) genes in fertile men, patients with TGCTs, and patients with infertility that have the AZFс deletion. A significant association of rs995030 of the KITLG gene with the development of TGCTs (p = 0.029 for the allele G, p = 0.0124 for the genotype GG) was revealed. Significant differences in the frequencies of the studied polymorphisms in patients with the AZFc deletion and the control group of fertile men were not found. We showed significant differences in the frequencies for the combination of all high-risk polymorphisms in the control group, patients with the AZFc deletion and patients with TGCTs (p (TGCTs-AZF-control) = 0.0207). A fivefold increase in the frequency of the combination of all genotypes in the TGCT group (p = 0.0116; OR = 5.25 [1.44-19.15]) and 3.7-fold increase was identified in patients with the AZFc deletion (p = 0.045; OR = 3.69 [1.11-12.29]) were revealed. The genotyping of patients with infertility caused by the AZFc deletion can be used to identify individuals with an increased risk of TGCTs.

[PCA3 AND TMPRSS2:ERG GENES EXPRESSION IN BIOPSIES OF BENIGN PROSTATE HYPERPLASIA, INTRAEPITHELIAL NEOPLASIA, AND PROSTATE CANCER].

Morphological analysis of the biopsies for prostate cancer (PCa) often is a difficult task due to heterogeneity and multifocality of tumors. At the same time, a lot of data exist about the potential molecular genetic markers of PCa. The aim of our study is to determine of PCA3 and TMPRSS2:ERG genes expression in benign hyperplasia (BPH), low and high grade intraepithelial neoplasia (PIN), PCa for revealing of diagnostic value of those genes expression in benign and precancerous changes in prostate. Total RNA was isolated from 53 biopsies, reverse transcription was performed, gene expression was determined by real time PCR (RT-PCR) then deltaCt index was determined as Ct(PCA3)--Ct(KLK3). Average deltaCt and its SD in BPH were 8.28 ± 3.13, low PIN--8.56 ± 2.64, high PIN--8.98 ±1.69, PCa--1.08 ± 2.36. We have demonstarted that deltaCt did not differ in patients with BPH, low and high grade PIN, whereas significantly increased in PCa relative to any of the three groups listed above (p < 0.0001). Expression of TMPRSS2:ERG was absent in BPH, PIN, but it was detected in 40% (4/10) of PCa cases. ROC-analysis showed that the AUC (area under ROC-curve with 95% CI, p < 0.0001) was 0.98 ± 0.02 in the analysis of a combination of overexpression of PCA3 and TMPRSS2:ERG. Thus, the expression analysis of the PCA3 and chimeric oncogene TMPRSS2:ERG in biopsy cannot be used for differential diagnosis of BPH, low and high grade PIN. However, overexpression of PCA3 and expression of TMPRSS2:ERG are characteristic in PCa. Expression analysis of these genes by the proposed RT-PCR modification at the threshold level deltaCt 3,22 has diagnostic accuracy 90% to detect PCa in biopsy specimens.