Abnormal Methylation of Several Tumor Suppressor Genes in Sporadic Breast Cancer

Multiplex methylation-sensitive PCR was employed in studying the methylation of CpG islands in the R1, p16/CDKN2, p15/CDKN2, p14/ARF, DH1, MGMT, HIC1, and N33 promoter regions in breast cancer (105 tumors). Methylation was often observed for the two major suppressor genes involved in controlling the cell cycle through the Cdk–Rb–E2F signaling pathway, R1 (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.     

Reduced representation bisulfite sequencing design for assessing the methylation of human CpG islands in large samples

The reduced representation bisulfite sequencing (RRBS) method has been developed for the high-throughput analysis of DNA methylation based on the sequencing of genomic libraries treated with sodium bisulfite by next-generation approaches. In contrast to whole-genome sequencing, the RRBS approach elaborates specific endonucleases to prepare libraries in order to produce pools of CpG-rich DNA fragments. The original RRBS technology based on the use of the MspI libraries allows one to increase the relative number of CpG islands in the pools of genomic fragments compared to whole-genome bisulfite sequencing. Nevertheless, this technology is rarely used due to the high cost compared with bisulfite methylation analysis with hybridization microarrays and significant residual amount of data represented by the sequences of genomic repeats that complicates the alignment and is not of particular interest for developing DNA methylation markers, which is often the main goal of biomedical research. We have developed an algorithm for estimating the likelihood that recognition sites of restriction endonucleases will be represented in CpG islands and present a method of reducing the effective size of the RRBS library without a significant loss of the CpG islands based on the use of the XmaI endonuclease for library preparation. In silico analysis demonstrates that the optimum range of the XmaI-RRBS fragment lengths is 110–200 base pairs. The sequencing of this library allows one to assess the methylation status of over 125000 CpG dinucleotides, of which over 90000 belong to CpG islands.

     

Analysis of Polymorphic Variants of Gene <Emphasis Type="Italic">GIPC1</Emphasis> 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 CGG_10–13 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 CGG ₁₂ was shown to be associated with high risk of developing NSCLC (α = 0.05).     

Molecular Analysis of Structural Abnormalities in Papillary Thyroid Carcinoma Genome

Rearrangements of the protooncogene RET (RET/PTC) and somatic mutations of the gene BRAF are the most common events in the etiopathogenesis of papillary thyroid carcinoma (PTC). The rates of RET/PTC rearrangements and BRAF mutations in different nodular formations of the thyroid gland (TG) have been estimated. Comparative expression analysis of the extracellular (EC) and tyrosine kinase (TK) domains of RET has shown that 14% (12 out of 85) of PTC cases are RET/PTC-positive, including one RFP/RET-, two RET/PTC3-, and seven RET/PTC1-positive tumors, as well as two unidentified chimeric RET/PTC oncogenes. The standard T1796A transversion in the gene BRAF has been found in 60% (55 out of 91) PTC cases with the use of amplification refractory mutation system–polymerase chain reaction (ARMS–PCR). Somatic mutation G1753A and deletion del1800_1811 have been identified in PTC for the first time. The absence of the BRAF mutations in RET/PTC-positive tumors allows these two genetic defects to be regarded as alternative mechanisms of the RAS–RAF–MEK–ERK mitogen-activated protein (MAP) kinase cascade activation in PTCs. In none of the three follicular thyroid carcinomas (FTCs), 11 follicular thyroid adenomas (FTAs), and 13 nodular goiters have either BRAF mutations or RET/PTC rearrangements been found. Thus, the RET/PTC chimeric oncogenes and BRAF somatic mutations are specific markers of PTC and can be used for the preoperative diagnosis of these tumors.     

VHL inactivation in sporadic clear cell renal carcinoma

Clear cell renal carcinoma (CCRC) accounts for 75% of all renal cancer cases. The majority of CCRCs displays inactivation of the VHL suppressor gene as a result of mutations, allelic deletions, and/or methylation. Data on the effect of VHL inactivation on the prognosis in CCRC are discrepant. Comprehensive molecular genetic analysis of VHL was performed for 64 CCRCs: mutations were identified by single strand conformation polymorphism analysis and subsequent sequencing, a loss of heterozygosity was studied with two STR markers, and methylation was assessed by methylation-sensitive PCR. In total, 17 somatic mutations, including 12 new ones, were found in VHL. Allelic deletions of VHL were detected in 31.6% of cases; methylation was observed in 7.8% of cases. In total, VHL was inactivated in 46.9% of CCRC cases and in 51.7% of patients with CCRC stage I. The frequencies of mutations, loss of heterozygosity, and methylation did not correlate with clinical features of CCRC or pathological characteristics of the tumor. Studies of the molecular genetics alterations of VHL are thought to facilitate the identification of diagnostic and prognostic markers of renal cancer, e.g., the selection of an optimal panel of methylated suppressor genes.     

DNA methylation in the promoter regions of the laminin family genes in normal and breast carcinoma tissues

Molecular Biology - Extracellular glycoproteins of the laminin family are essential components of basement membranes involved in a number of biological processes, including tissue differentiation,...     

Novel markers of gene methylation and expression in breast cancer

An optimized methylation-sensitive restriction fingerprinting technique was used to search for differentially methylated CpG islands in the tumor genome and detected seven genes subject to abnormal epigenetic regulation in breast cancer: SEMA6B, BIN1, VCPIP1, LAMC3, KCNH2, CACNG4, and PSMF1. For each gene, the rate of promoter methylation and changes in expression were estimated in tumor and morphologically intact paired specimens of breast tissue (N = 100). Significant methylation rates of 38, 18, and 8% were found for SEMA6B, BIN1, and LAMC3, respectively. The genes were not methylated in morphologically intact breast tissue. The expression of SEMA6B, BIN1, VCPIP1, LAMC3, KCNH2, CACNG4, and PSMF1 was decreased in 44–94% of tumor specimens by the real-time RT-PCR assay. The most profound changes in SEMA6B and LAMC3 suggest that these genes can be included in biomarker panels for breast cancer diagnosis. Fine methylation mapping of the most frequently methylated CpG islands (SEMA6B, BIN1, and LAMC3) provides a fundamental basis for developing efficient methylation tests for these genes.     

Amplification of intermethylated sites experimental design and results analysis with <Emphasis Type="Italic">AIMS in silico</Emphasis> 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 of 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.