Screening of radiation susceptibility genes by single-nucleotide polymorphisms and haplotype analysis

Radiotherapy for breast cancer patients occasionally induces adverse effects. The adverse reactions to radiotherapy are caused by multiple factors, including individual genetic differences. The heterogeneity in normal tissue reactions may result from the combined effect of several different genetic alterations. Single nucleotide polymorphisms (SNPs) and derived haplotypes within multiple genes may be used to detect the genetic alterations related to the heterogeneity in normal tissue reactions. DNA was sampled from 397 Japanese breast cancer patients who qualified for breast-conserving radiotherapy. Using the NCI-CTC scoring system version 2, the patients were grouped according to adverse skin reactions at 3 months of starting radiotherapy (grade = 0, n = 230; grade &#8805; 1, n = 167). Nine hundred and ninety-nine SNPs from 137 candidate genes for radiation susceptibility were genotyped, and haplotype associations between groups were assessed. Global haplotype association analysis (P < 0.05 and FDR < 0.005) indicated that estimated haplotypes in three loci were associated with the risk of adverse skin reactions. Comparison of risk haplotype to the most frequent haplotype in each locus showed haplotype CGATA(OR = 2.04; 95% CI, 1.19-3.52) and TGATG(OR = 5.05; 95% CI, 1.31-19.53) and TGAGG(OR = 12.89; 95% CI, 1.53-108.25) in TGFBI , CT in IL1A (OR = 1.83; 95% CI, 1.17-2.85), and AGT in GSTA4 (OR = 2.77; 95% CI, 1.23-6.22) had significantly greater adverse skin reactions risk.In conclusion, Individual radiosensitivity may be partly determined by combinations of these haplotypes in multiple loci. This would provide an understanding of the mechanisms underlying the genetic variation in radiation sensitivity or resistance among the population, and would show the possibility of prediction of the risk of adverse skin reactions prior to radiation therapy.10th Anniversary of Kazusa Arc International Symposium on Advanced Functional Genomic

Classifying the variation in response to ionizing radiation in human cells by gene expression profiling

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Whole genome-wide screening of cervical lymph node metastasis-associated genetic alterations in oral squamous cell carcinoma of Japanese patients

To precisely identify genetic markers that reflect occurrence of cervical lymph node metastasis (LNM) in Japanese oral squamous cell carcinoma (OSCC) patients, oligonucleotide array-based comparative genomic hybridization (CGH) with individual gene-level resolution and real-time quantitative polymerase chain reaction (QPCR) was conducted using primary tumor materials surgically resected from total of 54 OSCC patients with or without cervical LNM (LNM present, n = 22; LNM absent, n = 32). Frequent gain was observed at 11q13 region exclusively in the patient group with cervical LNM using array-based CGH. This observation was confirmed by real-time QPCR experiments designed for 11 genes (TPCN2, MYEOV, CCND1, ORAOV1, FGF4, TMEM16A, FADD, PPFIA1, CTTN, SHANK2 and DHCR7) in this region. Moreover, we refined a comprehensive physical map of the 11q13 amplification region including 33 kb of DNA sequence covering this region and found a novel breakpoint between MYEOV and CCND1. There were hypothetical two cores in 11q13 region. Combination of copy number amplification at CTTN and/or TPCN2/MYEOV, which were selected from each core separated by novel breakpoint, was most significantly associated with cervical LNM (P = 0.0035). Following detailed assessment of selected loci by real-time QPCR used to be quite adequate strategy for finding out genetic markers in regions with complicated alterations. Further study with larger patient numbers should be conducted to validate this result.The 14th European Cancer Conference, ESTRO26 meeting, European Society for therapeutic radiology and oncolog

Solid-phase multiple displacement amplification for multi-locus genotyping of single chromosome molecules

(1) Background: Despite recent innovations in high throughput shotgun sequencing technologies, complex rearrangements in addition to the original dizygotic existence of homologous chromosomes complicate cancer genome sequencing. Thus there is an urgent need for establishing a convenient method to manipulate long stretches of individual chromosomes for analyzing sequence information.(2) Materials and Methods: A novel methodology has been developed to amplify single chromosomes for genotyping. A key feature of this methodology is a solid-phase multiple displacement amplification, that is an enzymatic reaction of Phi29 DNA polymerase, within a solidified agarose gel. It consists of the following seven steps. (I) Lysis of a limited number of cultured cells within a heated agarose gel solution to release chromosome molecules. (II) Careful aliquoting of small volumes of gel solutions containing limited number of chromosome molecules. (III) Solidification of the gel on ice. (IV) Solid-phase multiple displacement amplification of the gel-immobilized individual chromosome molecules. (V) Recovery of the amplification products by heating. (VI) Screening of target chromosomes by real-time QPCR. (VII) Multi-loci SNP typing using newly developed on-plastic chip allele-specific primer extension method (Michikawa et al., Anal. Sci., 2006, 22, 1537-1545). (3) Results: Utilization of agarose gel as a reaction matrix enabled reliable amplification-ready limited dilution of DNA to the level that homologous chromosomes rarely co-localize. Aggregation of chromosomes during the dilution step was dramatically reduced by incubating the gel solution at alkaline pH and at high temperature. Separation of homologous chromosomes by this method provided reliable determination of multi-loci genotypes on each homologous chromosome. Using this methodology, we have successfully determined haplotype of multiple SNPs in human ATM region that span 200 kilobase pairs. (4) Conclusions: The methodology developed in this study is effective for genotyping individual homologous chromosomes. Since amplified materials are easily recovered in a solution as PCR-ready form, this methodology is not restricted for genotyping. Further applications, such as chromosome-wide sequencing, are considerable.The 14th European Cancer Conference, ESTRO26 meeting, European Society for therapeutic radiology and oncolog

Possible influence of multiple SNP markers to urological morbidity induced by radiotherapy with carbon-ions among 133 prostate cancer patients

Purpose: To develop efficient predictive method for risk of dysuria after radiotherapy (RT). Patients and Methods: A total of 197 prostate cancer patients who underwent C-ion RT at a total dose of 64.4 +/- 2.7 GyE and evaluated for urinary morbidity (dysuria) according to the Late Effects of Normal Tissue/Subjective, Objective, Management, and Analytic scoring system (LENTSOMA). Three hundred seventy-three SNPs in 109 candidate genes were genotyped by MassARRAY system. Patients were categorized into control (grade 0) and case (grade >1) groups. First, association between the genotype at each SNP site and dysuria were assessed using the Fisher exact test (P ) had 3 or more risk genotypes. Conclusions: Although more patients are required to validate the results, this study supports the assumptions that radiosensitivity is caused by multigenetic factors and that the number of high-risk genotypes on SNPs might predict radiosensitivity.The 13th International Congress of Radiation Researc

Risk estimation of radiation-induced normal tissue injury on cancer patients by convenient analysis of marker SNPs

It has been increasingly recognized that cancer patient\u27s genetic information significantly influences therapeutic outcomes. In this study, a novel experimental system was developed for estimating risk of radiation-induced normal tissue injury on individual cancer patients based on analysis of marker SNP genotypes, which had been screened by a large association study on genes involved in DNA damage repair, cell cycle control, redox regulation, inflammation and so on. These marker SNPs were analyzed simultaneously using patient\u27s blood-derived genomic DNA by a novel DNA chip method that provided optical detection of typing results. The resulting spot images on the DNA chips were captured by a digital camera and processed by computerized tool to call genotype of SNPs. The genotype information was then referred a database made by the foregoing large association study to statistically calculate risk of radiation-induced normal tissue injury. This system does not require specific instruments and can be easily operated in a short time. It is suitable to be used at laboratory of clinical hospitals.Cancer Models & Mechanism

On-chip optical detection system for allele-specific extension of 3\u27-LNA modified oligonucleotides

Single nucleotide polymorphisms (SNPs) are useful as genetic association markers for various human diseases as well as for prediction of individual responses to therapeutic treatment such as drugs and ionizing radiation. For routine molecular biology research and bedside clinical diagnosis, readily available technologies are required to genotype limited numbers of SNPs that were selected in previous large scale association studies. To this end, easy and rapid protocols with inexpensive instruments running at reasonable cost are required for the technology to be widely adopted.\n In the present work, a novel optical detection system for on-chip allele-specific primer extension has been developed to conveniently genotype multiple SNPs. Optimization of the procedure was achieved by i) locked nucleic acid (LNA) modification of the 3\u27-end of immobilized oligonucleotide primer, ii) titration of magnesium concentration of the reaction mixture iii) utilization of optimum reaction temperature. Efficient primer extension without an annealing step using double-stranded template DNAs was demonstrated for LNA-modified oligonucleotides immobilized on an S-Bio PrimeSurface plastic base. This property provided simplification of experimental procedures and reduction of reaction time to as short as 10 minutes at a constant temperature of 65 oC. Incorporation of biotin-dUTP during primer extension, followed by binding of alkaline phosphatase-conjugated streptavidin, allowed optical detection of the typing results through precipitation of colored alkaline phosphatase substrate onto the surface of the plastic base. Oligonucleotide primer sets were designed to genotype three SNPs in the genes APEX1, TGFB1 and SOD2, previously investigated for association with radiation sensitivity. The simultaneous evaluation of these SNPs in 25 individuals has produced considerably reliable results. \n The experimental system developed in this study is not oriented towards high throughput analysis. Rather, limited numbers of SNPs are easily analyzed within a couple of hours. Dividing the surface of the plastic base into multiple areas can increase the number of individuals analyzed per chip without affecting experimental processes. In conclusion, all the benefits described above make this system applicable to routine molecular biology research and bedside clinical diagnosis.HUGO\u27s 11th Human Genome Meeting （HGM2006

Reliable and Fast Allele-Specific Extension of 3\u27-LNA Modified Oligonucleotides Covalently Immobilized on a Plastic Base, Combined with Biotin-dUTP Mediated Optical Detection

In the present work, a convenient microarray SNP typing system has been developed using a plastic base that covalently immobilizes amino-modified oligonucleotides. Reliable SNP allele discrimination was achieved by using allelic specificity-enhanced enzymatic extension of immobilized oligonucleotide primer, with a locked nucleic acid (LNA) modification at the SNP-discriminating 3\u27-end nucleotide. Incorporation of multiple biotin-dUTP molecules during primer extension, followed by binding of alkaline phosphatase-conjugated streptavidin, allowed optical detection of the genotyping results through precipitation of colored alkaline phosphatase substrates onto the surface of the plastic base. Notably, rapid primer extension was demonstrated without a preliminary annealing step of double-stranded template DNA, allowing overall processes to be performed within a couple of hours. Simultaneous evaluation of three SNPs in the genes TGFB1, SOD2 and APEX1, previously investigated for association with radiation sensitivity, in 25 individuals has shown perfect assignment with data obtained by another established technique (Massarray system)

Prediction of human mammary tumor cell radiosensitivity: Radiosensitivity predicted by the comet assay and expression pattern of genes induced by irradiation using cDNA microarray

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