Specific Localization of Missense Mutations in the VHL Gene in Clear Cell Renal Cell Carcinoma.

Missense mutations in the VHL gene during sporadic clear cell renal cell carcinoma were studied to evaluate their localization in relation to functionally important motifs of the VHL protein. Somatic mutations were identified in 124 of 307 samples. All missense mutations in the α-domain were localized in the binding site for elongin C. Substitutions in the ß-domain (77%) were found in the HIF-binding site. Five missense mutations were absent in these sites, which illustrates their role in VHL protein formation or suppressor function of other protein cofactors. Mutation c.392A→T (p.N131I) was identified for the first time. Our results hold much promise to estimate the boundaries of functionally important sites in the VHL suppressor gene and contribute to the interpretation of a pathogenic role of mutations in direct DNA diagnostics.

[Molecular genetic diagnosis of Stargardt disease]. / Molekuliarno-geneticheskaia diagnostika bolezni Shtargardta.

AIM: To comparatively evaluate the efficacy of genetic screening in patients with Stargardt disease (SD) by using an express panel of 5 most common ABCA4 mutations and performing massive parallel sequencing of all coding regions of the ABCA4, ELOVL4, PROM1, and CNGB3 genes. MATERIAL AND METHODS: MLPA analysis for 5 ABCA4 mutations, namely p.G863A, p.L541P, p.A1038V, p.G1961E, and p.P1380L, was done in 54 patients with SD. In 25 patients, massive parallel sequencing of coding regions (exons) and neighboring introns of the ABCA4, ELOVL4, PROM1, and CNGB3 genes was also performed. RESULTS: Gene testing for 5 ABCA4 mutations showed that 50% of patients (27 patients) harbored one mutation and 13% - two mutations. At massive parallel sequencing (25 patients), two pathogenic alleles were found in 21 patients (84%), one mutation - in 23 patients (91.7%). The majority of mutations was accounted for by the ABCA4 gene (83% of all mutation-positive patients). CONCLUSION: Sequencing of exons and neighboring introns of the ABCA4, ELOVL4, PROM1, and CNGB3 genes with the new molecular genetic diagnostic system enabled confirmation of the diagnosis of SD in 84% of patients. High prevalence of p.L541P, p.A1038V, and p.G1961E mutations of the ABCA4 gene has been established.

[Relationship between structural and functional changes in retina in Stargardt disease]. / Vzaimosvyaz' strukturnykh i funktsional'nykh izmenenii setchatki pri bolezni Shtargardta.

AIM: To assess the relationship between structural abnormalities of the junction of the internal and external segments of photoreceptors (IS/OS junction) and functional changes. MATERIAL AND METHODS: The study enrolled 45 patients (90 eyes) with Stargardt disease, of them 22 women and 23 men. Ophthalmic examination included color vision test, static perimetry with a 60° field of view, electrophysiological studies, namely, ganzfeld and multifocal electroretinography (gERG and mfERG), autofluorescence, and optical coherence tomography (OCT). RESULTS: For analysis, the patients were divided into 3 groups depending on whether or not the structure of the IS/OS junction was damaged at OCT. A strong correlation has been found between the level of IS/OS damage and the MD index. There were no differences in foveal, perifoveal, or parafoveal retinal thicknesses. Retinal volumes within the 1-, 3- and 6-mm ring-shaped zones were also similar. All three groups showed a reduction in amplitudes of the major gERG waves and mfERG changes of different severity. The most frequent defect seen in all groups and the ring-shaped zones was a reduction in the amplitude and density of retinal biopotential (P1µv and P1 nv/deg2). CONCLUSION: A direct correlation exists between the extent and severity of structural changes in the IS/OS junction at OCT, visual functions assessed with perimetry, and disturbance of electrogenesis detected by mfERG and gERG.

[Structural and functional analysis of tumor genomes and the development of test systems for early diagnosis, prognosis and cancer therapy optimization].

The article discusses results of the structural and functional analysis of molecular genetic abnormalities in various malignant tumors. Investigations have discovered more than 20 new markers for sporadic breast cancer. Several of them formed the test system, allowing the diagnosis with a specificity of 100%. Appearance of TMPRSS2/ERG4 chimeric gene is a frequent tumor-specific event, its expression is correlated with more aggressive forms of prostate cancer, may serve as a molecular marker for tumor cells and androgen assessment of tumor response to hormonal therapy. The effective systems for the early diagnosis of cervix and endometrium cancer were developed as well. Mutations in the VHL, deletions of chromosome 3 and methylation of several genes can predict the course and selection of effective therapy of clear cell kidney cancer, a number of molecular markers were identified for early diagnosis and prognosis of recurrence of bladder cancer. For diagnosis, prognosis and treatment of brain tumors we developed an effective complex system of markers. Protocol of molecular genetics investigation reveals the cause of the disease by more than 90% of patients with retinoblastoma. In order to study abnormal methylation in tumor genomes an innovative technology AFLOAT has been developed that allows to efficiently identify new markers with diagnostic value. Test systems of molecular genetic and epigenetic markers for early diagnosis and prognosis as well as for cancer therapy optimization have shown to be effective, have been approved for use in clinical practice and are being introduced into practical healthcare.

[Localization of point mutations in the coding part of the VHL gene in clear cell renal cancer].

VHL gene is often inactivated in sporadic clear cell renal cancer (CCRC) due to somatic mutations, and it's germline mutations cause hereditary CCRC--von Hippel-Lindau syndrome. Localization of mutations in VHL, identification of new mutations and their influence on CCRC progression and sensitivity to targeted therapy are actual problems in modern oncogenetics. We have provided search and characterization of mutations in 248 primary CCRC using SSCP-analysis and sequencing. Somatic mutations were detected in 37.5% of samples, 72% of mutations were identified for the first time. New missense-mutations were analyzed by alignment programs and three-dimensional structure modeling. Mutation frequency was compared in different groups of patients in respect to stage, grade, and metastases. It was demonstrated that 39.1% samples with stage I harbor somatic mutations, however, no association with progression or metastases was found. We also have investigated localization of mutations in the VHL coding part and positions of missense-mutations and inframe deletions/insertions focusing on VHL critical sequences. VHL mutation analysis performed in this study improve the possibilities of laboratory diagnostics of familial and sporadic CCRC.

[Allelic disbalance in 1q32 area and microsatellite instability renal papillary adenocarcinoma].

Papillary adenocarcinoma is an abundant form of renal cell carcinoma. At present any diagnostic and prognostic molecular markers of papillary adenocarcinoma are absent, however some cytogenetic and molecular-genetic features of disease are known. According to literary data, the 1q32 duplication is associated with progressive deterioration of primary tumor. We have done a genetic typing (D1S2142 and D1S3465 locus) of 39 papillary adenocarcinoma cases, used PCR and fragment analyses of the 1q32 area. Frequency of the allelic disbalance was 36.8%; the microsatellite instability was found out in 48.7% of cases. The association of genetic disturbances with clinic-morphological features of papillary adenocarcinoma wasn't revealed. In some cases genetic heterogeneity of tumor-adjacent renal parenchyma and primary tumors was found out at multifocal renal carcinoma. For the first time we ve demonstrated that the allelic disbalance in 1q32 area and the microsatellite instability are frequent molecular-genetic disturbances in sporadic papillary carcinomas at all stages of the disease. Probably, the microsatellite instability is connected with progressive deterioration of primary tumor at renal papillary adenocarcinoma.

[Amplification of intermethylated sites experimental design and results analysis with aims in silico computer software].

Amplification of intermethylated sites (AIMS) is a powerful tool for differential methylation screening of genomes. Its applications have nevertheless been limited until recently for the absence of systemic approach to AIMS experimental design and of appropriate computer software for the analysis of AIMS results. We have developed AIMS in silico computer suggestion tool capable of predicting possible experimental outcomes, which assists in designing AIMS experiments depending on the research aims and available instrumentation, and in analyzing experimental results from the point of view of genomic locations of the DNA fragments under study. With AIMS in silico we have characterized qualitatively and quantitatively AIMS products obtainable under different conditions; to ease experimental design we demonstrate AIMS products hierarchical structure. We discuss examples of designing AIMS experiments and results analysis as well as possible relative to AIMS alternative approaches to differential methylation screening. AIMS in silico computer software is intended to standardize AIMS applications and to turn it into one of the principal approaches towards cancer epigenomes studies as well as towards diagnostics in oncology, including early screening.

[Novel methylation and expression markers associated with breast cancer].

We have developed a modification of methylation sensitive arbitrarily primed PCR, one of the methods of differentially methylated CpG islands in cancer cells genomes screening. Seven genes undergoing abnormal epigenetic regulation in breast cancer, SEMA6B, BIN1, VCPIP1, LAMC3, KCNH2, CACNG4 and PSMF1, have been identified by this method. Methylation and loss of expression frequencies were evaluated for each of the identified genes on 100 paired (cancer/morphologically intact control) breast tissue samples. Significant frequencies of abnormal methylation were detected for SEMA6B, BIN1, and LAMC3 (38%, 18%, and 8% correspondingly). Methylation of the above genes was not characteristic for morphologically intact breast tissues. Downregulation of SEMA6B, BIN1, VCPIP1, LAMC3, KCNH2, CACNG4 and PSMF1 in breast cancer was as frequent as 44-94% by real-time PCR expression assay. The most pronounced functional alterations were demonstrated for SEMA6B and LAMC3 genes, which allows recommending their inclusion into the panels of carcinogenesis diagnostic panels. Fine methylation mapping was performed for the genes most frequently methylated in breast cancer (SEMA6B, BIN1, LAMC3), providing a fundamental basis for the development of effective methylation tests for these genes.

[Inactivation and sensitization of the tumor cells by the gene Bax transfection].

After the transfection of the gene Bax into the cultured tumor cells of human ovary adenocarcinoma SKOV3 and uterus carcinoma HeLa in vitro the high sensitivity of the cells SKOV3 to the protein Bax produced after the gene Bax transfection was found. The sensitivity of the cells HeLa to the gene Bax transfection was much smaller. The hyperexpression of gene Bax and hypersensitivity to doxorubicin were seen in HeLa cells received as a result of the gene Bax transfection and subsequent selection. All cells of the line SKOV3 with the increased expression of the transfected gene Bax died. In the cell line SKOV3 the mutation in a gene Bax was found which has a genotype G7/G9 against a native type of a gene Bax--G8/G8. It was concluded that the found in the exone 3 of the gene Bax mutation G7/G9 in cells SKOV3 results in an inactivation of proapoptotic activity of the protein Bax.

[Analysis of polymorphic variants of gene GIPC1 CGG repeats in healthy individuals and in patients with breast cancer and non-small cell lung cancer].

The GIPC1 gene product promotes clustering of some transmembrane receptors, including those involved in carcinogenesis, and protects them against ubiquitin-dependent degradation. The 5' untranslated region of GIPC1 contains a polymorphic trinucleotide CGG repeat, which has not been characterized earlier. In the present study, we have carried out comparative analysis of the allele and genotype frequencies of this repeat in 129 samples of breast cancer (BC), 58 samples of non-small cell lung cancer (NSCLC), and 215 samples of healthy donors. The CGG repeat in the 5' untranslated GIPC1 gene region was shown to be highly polymorphic and represented by at least eight alleles. Alleles CGG10-13 were major, occurring at frequencies of 22, 41, 27, and 9%, respectively; the total frequency of the remaining alleles was approximately 1%. Heterozygosity of the CGG repeat was 0.70. Allele CGG12 was shown to be associated with high risk of developing NSCLC (alpha = 0.05).

[Spectrum and frequencies of RB1 gene structural defects in retinoblastoma]. / Spektr i chastota strukturnoi patologii gena RB1 pri retinoblastome.

The spectrum and frequencies of RB1 structural defects were studied in tumors and peripheral blood lymphocytes of patients with various forms of retinoblastoma. Single strand conformation polymorphism (SSCP) and heteroduplex (HA) analyses, along with direct sequencing, revealed 47 mutations, including 24 new ones. Of these, 42.5% were nonsense mutations, 15% were missense mutations, 15% affected splicing sites, and 27.5% were frameshifts resulting from microdeletions or microinsertions. Six polymorphisms were found, including three new ones located in the coding region. Microsatellite analysis with markers Rbint2, Rbint20, D13S262, and D13S284 revealed a loss of heterozygosity for at least one marker in 71% tumors.

[RB1 and CDKN2A functional defects resulting in retinoblastoma]. / Funktsional'naia patologoia genov RB1 i CDKN2A, privodiashchaia k razvitiiu retinoblastomy.

Multiplex methylation-sensitive PCR was employed in studying the methylation of the RB1 and CDKN2A/p16 promoter regions in 52 retinoblastomas. Aberrant methylation inactivating RB1 was detected in 14 (27%) tumors. Methylation of p16 was for the first time observed in retinoblastoma (9 tumors, 17%). Both promoters proved to be methylated in two tumors. In four tumors, aberrant methylation was combined with structural defects of both RB1 alleles. Aberrant methylation of the p16 promoter was the second mutation event in two tumors and was not accompanied by RB1 defects in one tumor. Complex testing for RB1 mutations, loss of heterozygosity, and functional inactivation of the two genes revealed a molecular defect in at least one allele in 51 (98%) tumors.

[Profile of methylation of certain tumor growth suppressing genes in non-small cell lung cancer]. / Profil' metilirovaniia nekotorykh genov-supressorov opukholevogo rosta pri nemelkokletochnom rake legkogo.

Multiplex methylation-sensitive PCR was employed in studying the methylation of CpG islands in the RB1, p16/CDKN2A, p15/CDKN2B, p14/ARF, CDH1, HIC1, and N33 5' regions in non-small cell lung cancer (51 tumors). Methylation was observed for the two suppressor genes involved in controlling the cell cycle through the Cdk-Rb-E2F signaling pathway, RB1 (10/51, 19%) and p16 (20/51, 39%). The highest methylation frequencies were established for CDH1 (72%) and HIC1 (82%). The CpG islands of p14 and p15 proved to be nonmethylated. At least one gene was methylated in 90% (46/51) tumors and no gene, in 10% (5/51) tumors. In addition, the genes were tested for methylation in peripheral blood lymphocytes of healthy subjects. Methylation frequency significantly differed between tumors and normal cells in the case of RB1, p16, CDH1, HIC1, and N33. Gene methylation frequency was tested for association with histological type of the tumor and stage of tumor progression. Methylation index of a panel of tumor suppressor genes was established for groups of tumors varying in clinical and morphological parameters.

[Diagnostics for epigenetic pathology in hereditary and oncologic diseases]. / Diagnostika épigeneticheskoi patologii pri nasledstvennykh i onkologicheskikh zabolevaniiakh.

The review considers the epigenetic defects and their diagnostics in several hereditary disorders and tumors. Aberrant methylation of the promoter or regulatory region of a gene results in its functional inactivation, which is phenotypically similar to structural deletion. Screening tests were developed for Prader-Willi, Angelman, Wiedemann-Beckwith, and Martin-Bell syndromes and mental retardation FRAXE. The tests are based on allele methylation analysis by methylation-specific or methylation-sensitive PCR. Carcinogenesis-associated genes (RB1, CDKN2A, ARF14, HIC1, CDI, etc.) are often methylated in tumors. Tumors differ in methylation frequencies, allowing differential diagnostics. Aberrant methylation of tumor suppressor genes occurs in early carcinogenesis, and its detection may be employed in presymptomatic diagnostics of tumors.

[Abnormal methylation of several tumor suppressor genes in sporadic breast cancer]. / Anomal'noe metilirovanie nekotorykh genov-supressorov pri sporadicheskom rake molochnoi zhelezy.

Multiplex methylation-sensitive PCR was employed in studying the methylation of CpG islands in the RB1, p16/CDKN2A, p15/CDKN2B, p14/ARF, CDH1, MGMT, HIC1, and N33 promoter regions in breast carcinoma (105 tumors). Methylation was often observed for the two major suppressor genes involved in cell-cycle control through the Cdk-Rb-E2F signaling pathway, RB1 (18/105, 17%) and p16 (59/105, 56%); both genes were methylated in 13 tumors. Methylation involved p15 in two (2%) tumors; CDH1, in 83 (79%) tumors; MGMT, in eight (8%) tumors, and N33, in nine (9%) tumors. The p14 promoter was not methylated in the tumors examined.

[Abnormal methylation of p16/CDKN2A AND p14/ARF genes GpG Islands in non-small cell lung cancer and in acute lymphoblastic leukemia]. / Sravnitel'nyi analiz anomal'nogo metilirovaniia CpG-ostrovkov, raspolozhennykh v promotornykh oblastiakh genov p16/CDKN2A i p14/ARF, pri nemelkokletochnom rake legkogo i ostrom limfoblastnom leikoze.

Multiplex methylation-sensitive PCR and methylation-specific PCR were employed in studying the methylation of CpG islands in the p16/CDKN2A and p14/ARF promoter and the first exon regions in non-small cell lung cancer (54 samples) and acute B-cell lymphoblastic leukemia (61 samples). Differences in methylation were detected between types of neoplasia as well as between CpG islands studied within the same types of tumors. High level of the p16/CDKN2A first exon CpC island methylation was revealed in non-small cell lung cancer (68%) and in acute B-cell lymphoblastic leukemia (55%) and the CpG island of p14/ARF first exon was nonmethylated in these types of tumors. The methylation of CpG-rich fragments of genes p16/CDKN2A and p14/ARF promoters was analysed. As was found out, CpG islands located in 5' areas of one and the same gene can differ in methylation frequencies. The comparison of sensitivity between methylation-specific PCR and methylation-sensitive PCR used in the methylations studies was carried out.

[Analysis of the allelic polymorphism of the five X-linked (CA)n dinucleotide repeats in Russia]. / Analiz allel'nogo polimorfizma piati X-stseplennykh dinukleotidnykh povtorov (CA)n v Rossii.

A PCR-based survey of allelic polymorphism of three microsatellite markers, DXS998, DXS548, and FRAXAC1, mapped to chromosome region Xp27.3, and two microsatellite markers, DXS8091 and DXS1691 located on Xq28 was carried out using a series of DNA samples obtained from 98 unrelated individuals from Russia. The number of alleles detected on electrophregrams for each marker tested was 4, 6, 4, 5, and 3, respectively. The values of heterozygosity index for the markers examined were 0.65, 0.27, 0.38, 0.70, and 0.29, respectively. The observed distribution of the allelic frequencies for each microsatellite marker examined fitted Hardy--Weinberg expectations. The values of individualization potential determined for each marker were 0.24, 0.53, 0.43, 0.12, and 0.52, respectively. In the sample tested the genotype distribution with regard to above loci was determined. The perspectives of using the analyzed allelic polymorphisms for indirect DNA diagnostics of the monogenic diseases located in this chromosome region (X-linked mental retardations, FRAXA and FRAXE) as well as for human population genetics and personal identification is discussed.