Doi:10.1016/j.ijrobp.2006.03.006

Int. J. Radiation Oncology Biol. Phys., Vol. 65, No. 3, pp. 646 – 655, 2006 doi:10.1016/j.ijrobp.2006.03.006
GENETIC PREDICTORS OF ADVERSE RADIOTHERAPY EFFECTS: THE
GENE-PARE PROJECT
ALICE Y. HO, M.D.,* DAVID P. ATENCIO, PH.D.,* SHEILA PETERS, B.A.,* RICHARD G. STOCK, M.D.,* SILVIA C. FORMENTI, M.D.,§ JAMIE A. CESARETTI, M.D.,* SHERYL GREEN, M.D.,* BRUCE HAFFTY, M.D.,¶ KAREN DRUMEA, M.D.,ʈ LARISA LEITZIN, M.D.,ʈ ABRAHAM KUTEN, M.D.,ʈ DAVID AZRIA, M.D., PH.D.,# MAHMUT OZSAHIN, M.D., PH.D.,** JENS OVERGAARD, M.D., D.M.SC., F.A.C.R., F.R.C.R.,†† CHRISTIAN N. ANDREASSEN, M.D.,†† CYNTHIA S. TROP, M.D.,‡‡ JANELLE PARK, M.D.,§§ AND BARRY S. ROSENSTEIN, PH.D.*†‡§ Departments of *Radiation Oncology, †Community and Preventive Medicine, and ‡Dermatology, Mount Sinai School of Medicine, New York, NY; §Department of Radiation Oncology, New York University School of Medicine, New York, NY; ¶Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT; ʈDepartment of Oncology, Rambam Medical Center, Haifa, Israel; #Department of Radiation Oncology, CRLC Val d’Aurelle, Montpellier, France; **Department of Radiation Oncology, Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland; ††Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus, Denmark; Departments of ‡‡Urology and §§Radiation Oncology, Bronx VA Purpose: The development of adverse effects resulting from the radiotherapy of cancer limits the use of this treatment
modality. The validation of a test capable of predicting which patients would be most likely to develop adverse
responses to radiation treatment, based on the possession of specific genetic variants, would therefore be of value. The
purpose of the Genetic Predictors of Adverse Radiotherapy Effects (Gene-PARE) project is to help achieve this goal.
Methods and Materials: A continuously expanding biorepository has been created consisting of frozen lympho-
cytes and DNA isolated from patients treated with radiotherapy. In conjunction with this biorepository, a
database is maintained with detailed clinical information pertaining to diagnosis, treatment, and outcome. The
DNA samples are screened using denaturing high performance liquid chromatography (DHPLC) and the
Surveyor nuclease assay for variants in ATM, TGFB1, XRCC1, XRCC3, SOD2, and hHR21. It is anticipated that
additional genes that control the biologic response to radiation will be screened in future work.
Results: Evidence has been obtained that possession of variants in genes, the products of which play a role in
radiation response, is predictive for the development of adverse effects after radiotherapy.
Conclusions: It is anticipated that the Gene-PARE project will yield information that will allow radiation
oncologists to use genetic data to optimize treatment on an individual basis.
2006 Elsevier Inc.
Genetic predictors, Adverse radiotherapy effects, Breast cancer, Prostate cancer.
INTRODUCTION
viewed. In addition, current efforts and techniques used inthe Genetic Predictors of Adverse Radiotherapy Effects The term “adverse radiation effects” can generally be de- (Gene-PARE) project will be discussed as well as future fined as undesirable clinical and physiologic responses sec- directions for developing genetic predictors of radiation- ondary to radiation treatment. In an effort to balance the eradication of clonogenic tumor cells with minimization ofdamage to surrounding normal tissues, the mechanismsunderlying adverse responses to radiation therapy have been GENETIC FACTORS AND RADIOSENSITIVITY
studied by both basic scientists and clinicians In thisarticle, both the historical and current literature examining A variety of patient, tumor, treatment, cellular, and mo- genetic factors in adverse radiation response will be re- lecular factors contribute to the variability in severity of Reprint requests to: Barry S. Rosenstein, Ph.D., Box 1236, Depart- (BSR); New York State Empire Clinical Research Investigator ment of Radiation Oncology, Mount Sinai School of Medicine, One Program Grant; New York State Department of Health Contract Gustave Levy Place, New York, NY 10029. Tel: (212) 241-9408; C017931; Swiss Cancer League grants KFS 539-9-1997 and SKL Fax: (212) 996-8927; E-mail: barry.rosenstein@mssm.edu 778-2-1999; The Danish Cancer Society; and The ESTRO Supported by Department of the Army grants DAMD 17-02-1- 0502, DAMD 17-02-1-0503 and W81XWH-04-0172; American Received Nov 9, 2005, and in revised form Feb 27, 2006.
Cancer Society Research Scholar Grant RSGT-05-200-01-CCE Accepted for publication Mar 1, 2006.
The Gene-PARE project ● A. Y. HO et al. normal tissue reactions exhibited after radiotherapy. Patient characteristics including age, nutritional status, medica- Several studies have attempted to define the relationship tions, body habitus, and coexisting morbidities such as dia- between in vitro radiation response and clinically evident betes or recent surgery all may contribute to radiation tox- effects by correlating fibroblast radiosensitivity with the icity Tumor-related factors such as size, histology, and development of acute and late radiation damage. The un- tumor grade may also affect the reaction to radiotherapy.
derlying hypothesis of these studies is that genetic differ- Variation in treatment-related parameters including treated ences may account for much of the unanticipated severity of volume, field size, anatomic prescription point, total dose, acute and chronic radiation reactions exhibited by some dose per fraction, and use of concomitant chemotherapy radiotherapy patients. Several studies have reported a cor- may also contribute to response heterogeneity. Because of relation between dermal fibroblast radiosensitivity quanti- the steep dose–response relationship for normal tissues, a fied by clonogenic survival assays, measuring the SF (i.e., small difference in dose could produce divergent outcomes the surviving fraction after exposure to 2 Gy of X-rays), andthe severity of both early and late effects In In addition, it has been hypothesized that individual addition, it has been reported that in vitro fibroblast prolif- genetic variations may also influence the development of eration postirradiation may be a useful predictor of wound- adverse radiation responses Evidence in support of healing morbidity for patients with soft tissue sarcoma who this theory was obtained through a study that examined received preoperative radiotherapy However, in con- the incidence and time to development of radiation-induced trast to these positive results, several studies have reported telangiectasia in a cohort of breast cancer patients. A wide a lack of correlation between dermal fibroblast SF with range of values was reported for this patient population either early or late skin reactions Taken together, these despite uniform radiation treatment. Consistent with the studies indicate that skin fibroblast sensitivity correlates results of previous analyses of radiotherapy patients only weakly with assessment of radiation-induced skin it was estimated that approximately 80% to 90% of the variability was attributed to deterministic effects, possibly aris-ing from potential individual genetic differences, whereas only 10% to 20% of the variation resulted from stochastic events For assays of normal tissue radiation response, blood is associated with the random nature of radiation-induced cell considered to be the tissue of choice because of the ease of killing in addition to random variations in dosimetry and collection in a standardized, patient-convenient manner.
However, initial lymphocyte radiosensitivity studies were disappointing with respect to experimental varia-tion, which confuted the predictive power of this assay.
Because the various lymphocyte cell types display different EFFORTS TO DEVELOP PREDICTIVE ASSAYS
radiation responses, fluctuations in the relative frequency of FOR NORMAL TISSUE RADIOSENSITIVITY
lymphocyte types cause an apparent shift in radiosensitivity The development of an in vitro radiosensitivity assay resulting in large experimental variation However, capable of predicting the extent of normal tissue damage in by taking into account cell-type specific radiosensitivities, ithas been reported that CD4 and CD8 T-lymphocyte radio- radiotherapy patients represents a long-sought goal sensitivity can discriminate differences in radiation-induced Despite limited success, the effort to achieve this objective cytotoxicity between individuals although it is continues because an assay capable of predicting suscepti- premature to use such an approach as a predictive assay.
bility for the development of adverse radiation effectswould allow customization of radiotherapy protocols on an Chromosomal aberrations and micronuclei individual basis. By doing so, it has been estimated that a Additional attempts to find suitable assays include anal- significant improvement in the therapeutic index could be ysis of fibroblast chromosomal aberrations However, achieved This work is also reflective of the new era this technique is time consuming and allows examination of of “individualized” or “personalized” medicine only a limited number of cells. Thus, it is considered im- The goal is therefore to develop a robust, specific assay to practical for cell types that exhibit slow growth and low enable individual dose adjustment based on the response of mitotic indices. Micronucleus induction analysis is another means of detecting chromosomal damage. Although this Numerous assays have been proposed to provide the assay has a well-established role in genetic toxicology clinician with information that predicts the outcome after as a means of biomonitoring human populations and as irradiation and thus guide treatment prescription, but none a biologic indicator of radiation damage efforts to have become established in daily practice. Major difficulties predict radiosensitivity have been inconclusive limiting the success of these assays are lack of sensitivityand specificity, technical burden of the procedures, poor characterization of the assayed cells, and the complexity of Despite multiple and various attempts to develop an assay capable of predicting which patients are susceptible to de- I. J. Radiation Oncology ● Biology ● Physics veloping adverse radiotherapy effects, none of the assays attempting to “pare away” those individuals from the gen- examined to date has proved to be consistently sensitive and eral patient population who are most likely to experience accurate for the prediction of side effects among patients pronounced radiation-induced normal tissue damage. Al- receiving radiation However, new technologies in mo- though these radiosensitive patients may be better suited to lecular biology may promote novel strategies for developing a surgical treatment approach, paradoxically these individ- a predictive assay with clinical applicability. The use of uals could alternatively represent a subset of patients who gene expression arrays that could predict the variation in are actually optimal candidates for radiotherapy, given that normal tissue sensitivity to radiation among individuals their cancers should harbor identical sequence alterations based on the expression patterns of different genes is cur- associated with radiosensitivity. This highlights the poten- rently under investigation. Several studies have demon- tial for radiotherapy dose modification, as radiosensitive strated the predictive power of pretreatment expression pro- tumors theoretically should require lower total treatment filing for human tumors but similar large-scale doses than their genetically nonvariant counterparts. Con- studies on normal tissues to assess the extent of radiation- versely, for the vast majority of patients who do not possess induced toxicity have yet to be reported. In addition, a few genetic variants associated with radiosensitivity, it may be studies have demonstrated meaningful correlations with possible to dose escalate and potentially achieve a larger morbidity by focusing primarily on cytokine responses Another new molecular approach involves analysis of DNAend-binding complexes that form at DNA double strand Inclusion of African-American patients breaks after irradiation. It has been reported that the levels A unique feature of Gene-PARE that distinguishes it of ATM-containing complexes correlated with cellular radi- from its European counterpart, the Genetic Pathways for the osensitivity as measured by the SF Although these Prediction of the Effects of Irradiation (GENEPI) project new molecular approaches appear to be promising, it has not coordinated through the European Society for yet been determined whether any will have clinical appli- Therapeutic Radiology and Oncology (ESTRO), as well as the developing Japanese RadGenomics and the BritishRadiogenomics: Assessment of Polymorphisms for Predict-ing the Effects of Radiotherapy (RAPPER) and Radiation GOAL OF THE GENE-PARE PROJECT
Complications and Epidemiology (RACE) studies is To develop an alternative approach to establish an assay the inclusion of a substantial number of patients of African- predictive of which patients are most likely to experience American ethnicity. Based upon currently funded Gene- radiation-induced complications, a research program has been PARE studies, it is anticipated that at a minimum, approx- initiated to identify the genetic factors associated with clin- imately 500 African-American subjects will be screened for ical radiosensitivity. To achieve this goal, a broad interna- genetic variants associated with clinical radiosensitivity.
tional effort has been organized comprising investigators Screening of these samples may allow identification of from radiation oncology departments in the United States, important genetic predictors specific for this population, as Israel, France, and Switzerland, to create the Gene-PARE genetic alterations that contribute to enhanced radiosensi- project Through the studies currently active in Gene- tivity could differ among ethnicities. Initial results of Gene- PARE, more than 2000 radiotherapy patients will be PARE studies suggest that substantial differences exist be- screened for genetic variants. The primary objective of tween the genetic factors associated with the development Gene-PARE is to establish the genetic alterations, the pres- of adverse radiotherapy effects for African Americans com- ence of which may confer increased susceptibility for de- pared with variants correlated with radiosensitivity in the veloping an adverse response to radiotherapy. Although the general population . This preliminary finding is consistent subjects screened to date are primarily breast and prostate with accumulating pharmacogenomic evidence indicating that cancer patients, the Gene-PARE tissue biorepository is not African Americans have a significantly different spectrum of exclusive to these two types of cancers as it is open to tissue polymorphisms in genes associated with drug metabolism samples from patients diagnosed with any form of cancer compared with those in the general population treated with radiation. For all patients accrued into Gene-PARE studies, a blood sample is obtained for lymphocyte DISTINCTION BETWEEN MUTATIONS, SNPs,
isolation and DNA extraction. In addition, frozen lympho- AND RARE VARIANTS
cytes from patients exhibiting clinical radiosensitivity ornotable genetic characteristics have been used for EBV Several semantic issues deserve mention. Throughout this transformation to create permanent cell lines, which are review, the word “mutation” is generally avoided, as this being used in assays examining the functional significance term is often used to signify a particular DNA sequence variation that exerts a functional impact on the protein By identifying genetic factors associated with radiosen- encoded by the gene. Instead, the term “single nucleotide sitivity, the goal of Gene-PARE is to develop a means to polymorphism” (SNP) is used to indicate a sequence vari- predict which patients are at increased risk for complica- ation in which the less common or minor allele occurs at a tions secondary to radiation treatment. In this sense, we are population frequency Ͼ1% The expression “rare vari- The Gene-PARE project ● A. Y. HO et al. ant” is used to mean a sequence variation for which the atively common in this cohort. Based on a logistic regres- minor allele occurs with a frequency Ͻ1%. Hence, these sion model, a dose–response using the ED terms refer only to the prevalence of a minor allele and do that resulted in a 50% incidence of Grade 3 radiation- not imply whether a particular genetic variant possesses induced fibrosis) was generated for these patients. The find- functional or pathologic significance. The terms “DNA se- ings of this study suggest a correlation between possession quence variation” or “genetic variant/alteration” are used to of the 5557 G¡A variant in ATM and radiosensitivity as the signify SNPs and rare variants. The use of “mutation” is for women who were carriers of this SNP was 52 Gy, limited to avoid any suggestion as to the functional impact on the protein encoded by a gene possessing a particular possess this genetic alteration. These results are consistent with those of Angele et al. who found a significantassociation between homozygote carriers of the G¡A tran-sition at ATM nucleotide 5557 and adverse radiotherapy ROLE OF ATM IN CLINICAL
responses, as well as a separate study that reported a non- RADIOSENSITIVITY
significant overrepresentation of the ATM 5557 A allele During the initial years of the Gene-PARE project, sub- among breast cancer patients with marked alterations in stantial attention was devoted to study of the ATM gene breast appearance after postlumpectomy radiotherapy and its relationship to radiosensitivity, which has pioneered In addition, an association was reported between this SNP the way for examination of other genetic variations as and late morbidity in prostate cancer patients, although it predictors of adverse radiation responses. The ATM protein did not achieve statistical significance because of the small functions as a protein kinase involved in cellular stress responses, cell-cycle checkpoint control and DNA repair Further evidence supporting the relationship between Loss of these functions may subsequently lead to ATM sequence variations and radiosensitivity has been ob- a diminished DNA repair ability and defective cell-cycle tained for prostate cancer patients treated with iodine-125 checkpoint control. The clinical association between pa- (125I) brachytherapy The samples for these patients tients producing nonfunctional ATM protein and the subse- were obtained from the Mount Sinai Prostate Cancer Patient quent devastating responses to ionizing radiotherapy have Tissue Biorepository, which represents a critical resource been described In addition, cells derived from for Gene-PARE. This biorepository maintains DNA and individuals who were heterozygous for a mutation in ATM frozen blood lymphocytes derived from the approximately exhibited a radiosensitivity intermediate between persons 2400 prostate cancer patients treated with radiotherapy and diagnosed with AT and those who were not ATM carriers followed at this medical center over the past 15 years. A pilot study involving ATM screening reported that 10 of the The initial studies examining the role of ATM variants 16 subjects (63%) shown to possess sequence variants ex- in clinical radiosensitivity failed to find a positive correla- hibited at least one form of adverse response (defined as tion between ATM mutation status and the development of erectile dysfunction, late rectal bleeding, or severe urinary enhanced normal tissue damage in breast cancer patients disturbance). In contrast, of the 21 patients who did not . However, all of these studies used a protein trunca- harbor an ATM sequence variation, only 3 (14%) manifested tion test, which only detects genetic alterations that cause radiation-induced adverse responses. Nine of the patients protein truncations. Subsequent to these reports, evidence with sequence alterations specifically possessed missense was obtained that missense mutations, which result in mutations, which encode for amino acid substitutions, and amino acid substitutions rather than protein truncation, are are therefore more likely to possess functional importance.
more prevalent in cancer patients and therefore serve as a In this group, 7 of 9 (78%) exhibited at least one form of more appropriate type of DNA alteration to measure for adverse response. In contrast, among the 28 patients who ascertainment of ATM mutational status did not have a missense alteration, only 6 (21%) displayed In the first Gene-PARE study examining the role of ATM any form of adverse response to the radiotherapy.
mutations in susceptibility to radiotherapy-induced morbid-ity, 46 breast cancer patients were screened for ATM se- ADDITIONAL RADIOSENSITIVITY CANDIDATE
quence variations It was reported that 3 of 3 (100%) GENES UNDER STUDY
of the patients who developed a Grade 3/4 subcutaneousreaction, manifested as either fibrosis or soft-tissue necrosis, Although there is now evidence supporting ATM as a had ATM missense variants. In contrast, only 3 of the 43 gene associated with clinical radiosensitivity, it is nonethe- patients (7%) who did not develop this form of severe less likely that this is not the only gene the alteration of toxicity harbored this type of ATM alteration. In a separate which is responsible for adverse radiotherapy responses.
study, DNA samples isolated from 41 postmastectomy pa- Additional radiosensitivity candidate genes that have been tients who were treated with either a hypofractionated or linked to enhanced radiation responses include TGFB1, standard radiotherapy fractionation protocol were screened XRCC1, XRCC3, SOD2, and hHR21. TGF␤1, the protein Because many of these patients received a hypofrac- encoded by TGFB1, is a key cytokine involved with the tionated treatment, radiation-induced skin fibrosis was rel- regulation of cell growth and immunosuppressive activities.
I. J. Radiation Oncology ● Biology ● Physics It is also associated with the deposition of extracellular sion, whereas amino acid substitutions resulting from vari- matrix proteins and plays a central role in radiation-induced ants present in exons may alter protein function. Even SNPs fibrosis The primary function of the XRCC1 protein is present within noncoding regions could be of significance to coordinate the activities of the enzymes that perform base through their affect upon RNA stability or splicing mecha- excision repair of radiation-induced damage. Cells lacking a functional XRCC1 protein have demonstrated a hypersen- The “allelic architecture” of complex traits has received sitivity to radiation XRCC3 is involved in recom- significant attention Susceptibility to adverse binational repair of radiation-induced DNA double strand radiotherapy responses can be conceptualized through the breaks SOD2 encodes the manganese superoxide dis- two competing theories for the genetic basis of complex mutatse that represents an important line of cellular antiox- traits The first theory, the so-called “common dis- idant defense against the reactive oxygen species induced ease/ common variant hypothesis,” suggests that the inher- by irradiation hHR21 is the human homolog of the ited basis of complex traits is most likely the result of yeast rad21 the encoded protein of which is involved genetic variants characterized by relatively high allelic fre- with repair of DNA double stand breaks sister chro- quencies According to this theory, common SNPs in a limited number of genes are responsible for the inheri- To summarize this work, a correlation between radiosen- tance of complex traits. However, this approach to identify sitivity and the presence of a Pro/Pro at codon 10 and the genes associated with complex traits has achieved only T/T genotype in position Ϫ509 of TGFB1 has been reported modest success. Therefore, the alternative “rare variant” A relationship has also been demonstrated between hypothesis has been proposed, which suggests that a large the SOD2 codon 16 Val/Ala, XRCC3 codon 241 Thr/Thr pool of alleles is accountable for the development of com- and XRCC1 codon 399 Arg/Arg genotypes and an increased plex traits The most realistic model for complex risk of radiation-induced fibrosis Another study screened genetic traits likely incorporates aspects of both theories, three SNPs in XRCC1 and detected an association with with predisposing alleles of varying population frequencies radiosensitivity for patients possessing either the codon 194 present in the same and different genes. The Gene-PARE Arg/Trp alone or in combination with the codon 399 Arg/ project will not be limited by either of these theories, as the Gln genotype Finally, a T¡C transition at position approach being used in the studies that constitute this 1440 of the open reading frame of hHR21 has been found in project routinely involves screening the entire coding por- 6 of 19 radiation-sensitive cancer patients In aggregate, these studies support the general hypothesis A question also arises as to the types of mutations that that genetic factors play a significant role as predictors of may be associated with clinical radiosensitivity. The studies adverse radiotherapy responses. It is also important to note reporting the results of ATM screening lend support to an that the postmastectomy radiotherapy breast cancer patients association between minor sequence alterations, such as who were screened through Gene-PARE for ATM variants SNPs and rare variants, with susceptibility to adverse effects have also been examined for SNPs in the additional genes of radiotherapy In contrast, evidence has been cited above From the results obtained, it appears that susceptibility to the development of radiation-induced fi- of pathogenic truncating mutations, which are typically the brosis depends critically upon the total number of genetic type of mutation found in individuals with AT appear variants possessed rather than on any single genetic alter- not to have been radiosensitive. It is possible that the ation or gene affected These findings suggest that presence of a null mutation in one copy of the ATM gene clinical normal tissue radiosensitivity should be regarded as does not confer clinical radiosensitivity, whereas possession a complex genetic trait that is dependent on the effect of of a functional but altered ATM protein may result in an increased risk for the development of an adverse response toradiation treatment.
Cellular radiosensitivity and possession ofgenetic variants Radiosensitivity and tolerance dose The Human Genome Project is a well-publicized example The question may also be raised as to whether a small of the increasing effort to unravel the genetic variation difference in cellular survival associated with possession of underlying complex diseases and traits by illustrating the genetic variants that confers a relatively small increase in genetic differences existing between individuals The cellular radiosensitivity could account for an increased se- role of SNPs and rare variants, which constitute approxi- verity in radiation response. In fact, the performance of a mately 90% of naturally occurring sequence variations, is of simple calculation demonstrates that this is a likely out- particular importance SNPs and rare variants are come. For example, an SF for cells from an individual not known to potentially affect phenotype, although they have possessing variants associated with radiosensitivity may be often been regarded as genetic changes without functional 0.5, whereas for a person possessing genetic variants caus- significance. However, these sequence alterations may in ing mild radiosensitivity, the SF could be 0.3. Considering fact have an important biologic impact as genetic variants a protocol involving the use of 25 2-Gy fractions, at the located within regulatory regions could affect gene expres- completion of treatment, cellular survival would be approx- The Gene-PARE project ● A. Y. HO et al. imately 3 ϫ 10Ϫ8 for normal patients whereas it would be8 ϫ 10Ϫ14 for patients possessing radiosensitivity alleles.
This effectively represents the biologic impact of an 88 Gy total treatment dose for radiosensitive patients compared with 50 Gy for the patients not harboring such genetic alterations. This large biologically effective dose could cer- tainly account for adverse effects from the radiation treat- ment. In fact, when taking into account the relatively steep increase in the complication curves for normal tissue re- sponses and the practice of treating to normal tissue toler-ance, only a small increase in radiosensitivity could result in a large increase in the probability of normal tissue radiation- It is also important to note that this small increase in radiosensitivity may be difficult to detect through routine cellular radiosensitivity studies, considering the limitations in accuracy and precision of in vitro assays. Thus, when taking into account the steep slope of the normal tissuedose– complication curves, it is likely that a relatively mod- est, and possibly undetectable effect upon protein function, resulting in mild cellular radiosensitivity, could still substan- tially increase the probability for an adverse clinical response.
Thus it may prove difficult or impossible to detect through functional assays the impact of a genetic variant that causes Denaturing high-performance liquid chromatography andthe Surveyor nuclease assay The principal screening techniques for identification of genetic variants in the Gene-PARE project are denaturing high-performance liquid chromatography (DHPLC) and the Surveyor nuclease assay. These are both robust techniques that can be used to screen any gene in a large population for single nucleotide substitutions as well as small deletions andinsertions The main advantage of DHPLC lies in its rapid and accurate identification of polymorphisms and rare genetic variants in an automated fashion with a high level of sensitivity and specificity The samples obtained through Gene-PARE are also being screened using a complementary methodology that uses Surveyor nuclease (Trangenomic, Inc., Omaha, NE), which is a mismatch- specific DNA endonuclease. It is a member of the CEL nuclease family of plant DNA endonucleases. Surveyor nuclease cleaves with high specificity at the 3= side of any mismatch site in both DNA strands, including all base substitutions and insertion/deletions up to at least 12 nucle- otides. When mutant and wild-type alleles are mixed,heated, and then cooled to form heteroduplexes, Surveyor nuclease cleaves the heteroduplex fragments. The cleavage products are then analyzed using the same HPLC platform used for DHPLC but performed under nondenaturing con- ditions. This assay is performed under high sensitivity con- ditions in which the DNA is stained with a fluorescent probeand detected using a fluorescence detector. Hence the use of this approach permits the recognition of certain variants that are difficult to identify using DHPLC, which may require samples to be run at multiple denaturing temperatures to be I. J. Radiation Oncology ● Biology ● Physics detected. A further advantage in the use of the Surveyor will enable radiation oncologists to take greater advantage nuclease assay is that it provides information not only as to of the increasingly powerful and inexpensive methodologies the presence of a genetic alteration, but also its relative to sequence DNA in anticipation of the day when patients position in the DNA fragment being analyzed diagnosed with cancer arrive at their initial radiation oncol- Although genotyping assays designed to detect common ogy consultation armed with their full genome sequenced SNPs may be less costly to perform, these assays are limited By identifying genetic predictors of radiosensi- to detection of already known SNPs and are not designed to tivity, Gene-PARE may help cancer patients avoid serious discover new sequence variants. Of greatest importance, complications that lead to severe morbidity, or even mor- DHPLC and the Surveyor assay are capable of detecting tality, arising from organ damage secondary to radiother- virtually all variants in a gene, rather than just specific apy. In addition, it could be discovered through this work that there exists a small radiosensitive portion of the popu-lation and that standard treatment doses are effectivelybeing limited by their radiation tolerance. If these individ- CONCLUSION
uals can be identified through genetic screening, it may then The goal of the Gene-PARE project is to identify the be revealed that the vast majority of people are more resis- genetic sequence variants that are predictive for the devel- tant to radiation than generally assumed. This finding might opment of adverse effects resulting from radiotherapy. To permit radiation oncologists to be more aggressive and to accomplish this objective, a clinical database and bioreposi- dose escalate, which could translate not only into improved tory of frozen lymphocytes derived from cancer patients clinical outcomes for radiotherapy patients but also to more treated with radiation have been established. DNA isolated frequently provide safe treatment of relatively radioresis- from each tissue sample is being screened for variants in tant cancers. Thus, the results of the research conducted genes associated with radiation responses. It is expected that under Gene-PARE will help in the development of a the results of Gene-PARE will enable the greater use of data predictive test that will provide individuals faced with a generated as part of the Human Genome Project and the diagnosis of cancer, and to their doctors, critical information emerging field of radiogenomics. In addition, Gene-PARE that is necessary to reach optimal treatment decisions.
REFERENCES
1. Anscher MS, Vujaskovic Z. Mechanisms and potential tar- 13. Jones IM, Thomas CB, Xi T, et al. The genetic basis for gets for prevention and treatment of normal tissue injury after variation in radiation sensitivity in the general population.
radiation therapy. Semin Oncol 2005;32:S86 –S91.
Radiat Res 2005;163:700 –701.
2. McBride WH, Chiang CS, Olson JL, et al. A sense of danger 14. Bourguignon MH, Gisone PA, Perez MR, et al. Genetic and from radiation. Radiat Res 2004;162:1–19.
epigenetic features in radiation sensitivity. Part II: implica- 3. Stone HB, Coleman CN, Anscher MS, et al. Effects of tions for clinical practice and radiation protection. Eur J Nucl radiation on normal tissue: Consequences and mechanisms.
Med Mol Imaging 2005;32:351–368.
Lancet Oncol 2003;4:529 –536.
15. Safwat A, Bentzen SM, Turesson I, et al. Deterministic 4. Denham JW, Hauer-Jensen M. The radiotherapeutic inju- rather than stochastic factors explain most of the variation in ry—a complex ‘wound.’ Radiother Oncol 2002;63:129 –145.
the expression of skin telangiectasia after radiotherapy. Int J 5. Hall EJ. Do no harm—normal tissue effects. Acta Oncol Radiat Oncol Biol Phys 2002;52:198 –204.
16. Tucker SL, Geara FB, Peters LJ, et al. How much could the 6. Bentzen SM, Overgaard J. Patient-to-patient variability in the radiotherapy dose be altered for individual patients based on expression of radiation-induced normal tissue injury. Semin a predictive assay of normal-tissue radiosensitivity? Ra- Radiat Oncol 1994;4:68 – 80.
diother Oncol 1996;38:103–113.
7. Jackson A, Kutcher GJ, Yorke ED. Probability of radiation- 17. Turesson I, Joiner MC. Clinical evidence of hypersensitivity induced complications for normal tissues with parallel archi- to low doses in radiotherapy. Radiother Oncol 1996;40:1–3.
tecture subject to non-uniform irradiation. Med Phys 1993; 18. Fletcher GH. Regaud lecture perspectives on the history of radiotherapy. Radiother Oncol 1988;12:iii–v, 253–271.
8. Tucker SL, Turesson I, Thames HD. Evidence for individual 19. Mackay RI, Hendry JH. The modelled benefits of individu- differences in the radiosensitivity of human skin. Eur J alizing radiotherapy patients’ dose using cellular radiosensi- tivity assays with inherent variability. Radiother Oncol 1999; 9. Fernet M, Hall J. Genetic biomarkers of therapeutic radiation sensitivity. DNA Repair (Amst) 2004;3:1237–1243.
20. Evans WE, Relling MV. Moving towards individualized 10. Baumann M, Holscher T, Begg AC. Towards genetic pre- medicine with pharmacogenomics. Nature 2004;429:464 – diction of radiation responses: ESTRO’s GENEPI project.
Radiother Oncol 2003;69:121–125.
21. Fierz W. Challenge of personalized health care: To what 11. Andreassen CN, Alsner J, Overgaard J. Does variability in extent is medicine already individualized and what are the normal tissue reactions after radiotherapy have a genetic future trends? Med Sci Monit 2004;10:RA111–RA123.
basis—where and how to look for it? Radiother Oncol 2002; 22. Gurwitz D, Livshits G. Personalized Medicine Europe: Health, Genes and Society: Tel-Aviv University, Tel-Aviv, 12. Andreassen CN. Can risk of radiotherapy-induced normal Israel, June 19 –21, 2005. Eur J Hum Genet 2006;14:376 – tissue complications be predicted from genetic profiles? Acta 23. Agren A, Brahme A, Turesson I. Optimization of uncompli- The Gene-PARE project ● A. Y. HO et al. cated control for head and neck tumors. Int J Radiat Oncol biological indicator of radiation damage. Mutat Res Biol Phys 1990;19:1077–1085.
24. MacKay RI, Niemierko A, Goitein M, et al. Potential clinical 42. Geard CR, Chen CY. Micronuclei and clonogenicity follow- impact of normal-tissue intrinsic radiosensitivity testing. Ra- ing low- and high-dose-rate gamma irradiation of normal diother Oncol 1998;46:215–216.
human fibroblasts. Radiat Res 1990;124:S56 –S61.
25. Dubray B, Pavy JJ, Giraud P, et al. [Predictive tests of 43. Shibamoto Y, Streffer C, Fuhrmann C, et al. Tumor radio- response to radiotherapy. Assessment and perspectives in sensitivity prediction by the cytokinesis-block micronucleus 1997] (in French). Cancer Radiother 1997;1:473– 483.
assay. Radiat Res 1991;128:293–300.
26. Loeffler JS, Harris JR, Dahlberg WK, et al. In vitro radio- 44. Champion AR, Hanson JA, Venables SE, et al. Determina- sensitivity of human diploid fibroblasts derived from women tion of radiosensitivity in established and primary squamous with unusually sensitive clinical responses to definitive radi- cell carcinoma cultures using the micronucleus assay. Eur J ation therapy for breast cancer. Radiat Res 1990;121:227– 45. Twardella D, Chang-Claude J. Studies on radiosensitivity 27. Oppitz U, Baier K, Wulf J, et al. The in vitro colony assay: from an epidemiological point of view– overview of methods A predictor of clinical outcome. Int J Radiat Biol 2001;77: and results. Radiother Oncol 2002;62:249 –260.
46. Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of 28. Akudugu JM, Bell RS, Catton C, et al. Wound healing diffuse large B-cell lymphoma identified by gene expression morbidity in STS patients treated with preoperative radio- profiling. Nature 2000;403:503–511.
therapy in relation to in vitro skin fibroblast radiosensitivity, 47. Pomeroy SL, Tamayo P, Gaasenbeek M, et al. Prediction of proliferative capacity and TGF-beta activity. Radiother On- central nervous system embryonal tumour outcome based on gene expression. Nature 2002;415:436 – 442.
29. Begg AC, Russell NS, Knaken H, et al. Lack of correlation 48. van ’t Veer LJ, Dai H, van de Vijver MJ, et al. Gene of human fibroblast radiosensitivity in vitro with early skin expression profiling predicts clinical outcome of breast can- reactions in patients undergoing radiotherapy. Int J Radiat cer. Nature 2002;415:530 –536.
49. Torres-Roca JF, Eschrich S, Zhao H, et al. Prediction of 30. Stewart CC, Stevenson AP, Habbersett RC. The effect of radiation sensitivity using a gene expression classifier. Can- low-dose irradiation on unstimulated and PHA-stimulated cer Res 2005;65:7169 –7176.
human lymphocyte subsets. Int J Radiat Biol Relat Stud Phys 50. Sotiriou C, Lothaire P, Dequanter D, et al. Molecular pro- filing of head and neck tumors. Curr Opin Oncol 2004;16: 31. Crompton NE, Ozsahin M. A versatile and rapid assay of radiosensitivity of peripheral blood leukocytes based on 51. Lonning PE, Sorlie T, Borresen-Dale AL. Genomics in DNA and surface-marker assessment of cytotoxicity. Radiat breast cancer-therapeutic implications. Nat Clin Pract Oncol 32. Crompton NE, Miralbell R, Rutz HP, et al. Altered apoptotic 52. Quarmby S, West C, Magee B, et al. Differential expression profiles in irradiated patients with increased toxicity. Int J of cytokine genes in fibroblasts derived from skin biopsies of Radiat Oncol Biol Phys 1999;45:707–714.
patients who developed minimal or severe normal tissue 33. Crompton NE, Shi YQ, Emery GC, et al. Sources of varia- damage after radiotherapy. Radiat Res 2002;157:243–248.
tion in patient response to radiation treatment. Int J Radiat 53. Ismail SM, Buchholz TA, Story M, et al. Radiosensitivity is Oncol Biol Phys 2001;49:547–554.
predicted by DNA end-binding complex density, but not by 34. Ozsahin M, Ozsahin H, Shi Y, et al. Rapid assay of intrinsic nuclear levels of band components. Radiother Oncol 2004; radiosensitivity based on apoptosis in human CD4 and CD8 T-lymphocytes. Int J Radiat Oncol Biol Phys 1997;38:429 – 54. West CM, McKay MJ, Holscher T, et al. Molecular markers predicting radiotherapy response: Report and recommenda- 35. Ozsahin M, Crompton NE, Gourgou S, et al. CD4 and CD8 tions from an International Atomic Energy Agency technical T-lymphocyte apoptosis can predict radiation-induced late meeting. Int J Radiat Oncol Biol Phys 2005;62:1264 –1273.
toxicity: A prospective study in 399 patients. Clin Cancer 55. Iwakawa M, Imai T, Harada Y, et al. [RadGenomics project] (in Japanese). Nippon Igaku Hoshasen Gakkai Zasshi 2002; 36. Azria D, Gourgou S, Sozzi WJ, et al. Concomitant use of tamoxifen with radiotherapy enhances subcutaneous breast 56. Rosenstein BS. ATM Mutations and the Development of fibrosis in hypersensitive patients. Br J Cancer 2004;91: Severe Radiation-Induced Morbidity Following Radiother- apy for Breast Cancer. The Fourth Era of Hope Meeting for 37. Rigaud O, Guedeney G, Duranton I, et al. Genotoxic effects the Department of Defense (DOD) Breast Cancer Research of radiotherapy and chemotherapy on the circulating lym- phocytes of breast cancer patients. II. Alteration of DNA 57. Holden C. Race and medicine. Science 2003;302:594 –596.
repair and chromosome radiosensitivity. Mutat Res 1990; 58. Brookes AJ. The essence of SNPs. Gene 1999;234:177–186.
59. Shiloh Y. ATM and related protein kinases: Safeguarding 38. Miller B, Potter-Locher F, Seelbach A, et al. Evaluation of genome integrity. Nat Rev Cancer 2003;3:155–168.
the in vitro micronucleus test as an alternative to the in vitro 60. Savitsky K, Bar-Shira A, Gilad S, et al. A single ataxia chromosomal aberration assay: position of the GUM Work- telangiectasia gene with a product similar to PI-3 kinase.
ing Group on the in vitro micronucleus test. Gesellschaft fr¨ Science 1995;268:1749 –1753.
Umwelt-Mutations-forschung. Mutat Res 1998;410:81–116.
61. Lavin MF, Birrell G, Chen P, et al. ATM signaling and 39. Fenech M. The cytokinesis-block micronucleus technique genomic stability in response to DNA damage. Mutat Res and its application to genotoxicity studies in human popula- tions. Environ Health Perspect 1993;101(Suppl 3):101–107.
62. McKinnon PJ. ATM and ataxia telangiectasia. EMBO Rep 40. Muller WU, Streffer C, Wuttke K. Micronucleus determina- tion as a means to assess radiation exposure. Stem Cells 63. Gotoff SP, Amirmokri E, Liebner EJ. Ataxia telangiectasia.
Neoplasia, untoward response to x-irradiation, and tuberous 41. Muller WU, Nusse M, Miller BM, et al. Micronuclei: A sclerosis. Am J Dis Child 1967;114:617– 625.
I. J. Radiation Oncology ● Biology ● Physics 64. Morgan JL, Holcomb TM, Morrissey RW. Radiation reac- tients with severe late responses to radiation therapy. Cancer tion in ataxia telangiectasia. Am J Dis Child 1968;116:557– 84. Cesaretti JA, Stock RG, Lehrer S, et al. ATM sequence 65. Pandita TK, Hittelman WN. Increased initial levels of chro- variants are predictive of adverse radiotherapy response mosome damage and heterogeneous chromosome repair in among patients treated for prostate cancer. Int J Radiat Oncol ataxia telangiectasia heterozygote cells. Mutat Res 1994;310: Biol Phys 2005;61:196 –202.
85. Martin M, Lefaix J, Delanian S. TGF-beta1 and radiation 66. Parshad R, Sanford KK, Jones GM, et al. G2 chromosomal fibrosis: A master switch and a specific therapeutic target? Int radiosensitivity of ataxia-telangiectasia heterozygotes. Can- J Radiat Oncol Biol Phys 2000;47:277–290.
cer Genet Cytogenet 1985;14:163–168.
86. Marsin S, Vidal AE, Sossou M, et al. Role of XRCC1 in the 67. Shiloh Y, Parshad R, Sanford KK, et al. Carrier detection in coordination and stimulation of oxidative DNA damage re- ataxia-telangiectasia. Lancet 1986;1:689 – 690.
pair initiated by the DNA glycosylase hOGG1. J Biol Chem 68. Sanford KK, Parshad R, Price FM, et al. Enhanced chromatid damage in blood lymphocytes after G2 phase x irradiation, a 87. Thompson LH, West MG. XRCC1 keeps DNA from getting marker of the ataxia-telangiectasia gene. J Natl Cancer Inst stranded. Mutat Res 2000;459:1–18.
88. Liu Y, Masson JY, Shah R, et al. RAD51C is required for 69. Paterson MC, MacFarlane SJ, Gentner NE, et al. Cellular Holliday junction processing in mammalian cells. Science hypersensitivity to chronic gamma-radiation in cultured fi- broblasts from ataxia-telangiectasia heterozygotes. Kroc 89. Zelko IN, Mariani TJ, Folz RJ. Superoxide dismutase mul- tigene family: A comparison of the CuZn-SOD (SOD1), 70. Weeks DE, Paterson MC, Lange K, et al. Assessment of Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, chronic gamma radiosensitivity as an in vitro assay for het- evolution, and expression. Free Radic Biol Med 2002;33: erozygote identification of ataxia-telangiectasia. Radiat Res 90. McKay MJ, Troelstra C, van der Spek P, et al. Sequence 71. Appleby JM, Barber JB, Levine E, et al. Absence of muta- conservation of the rad21 Schizosaccharomyces pombe DNA tions in the ATM gene in breast cancer patients with severe double-strand break repair gene in human and mouse.
responses to radiotherapy. Br J Cancer 1997;76:1546 –1549.
72. Ramsay J, Birrell G, Lavin M. Testing for mutations of the 91. Birkenbihl RP, Subramani S. Cloning and characterization of ataxia telangiectasia gene in radiosensitive breast cancer rad21 an essential gene of Schizosaccharomyces pombe in- patients. Radiother Oncol 1998;47:125–128.
volved in DNA double-strand-break repair. Nucleic Acids 73. Clarke RA, Goozee GR, Birrell G, et al. Absence of ATM truncations in patients with severe acute radiation reactions.
92. Pati D, Zhang N, Plon SE. Linking sister chromatid cohesion Int J Radiat Oncol Biol Phys 1998;41:1021–1027.
and apoptosis: Role of Rad21. Mol Cell Biol 2002;22:8267– 74. Oppitz U, Bernthaler U, Schindler D, et al. Sequence anal- ysis of the ATM gene in 20 patients with RTOG grade 3 or 93. Quarmby S, Fakhoury H, Levine E, et al. Association of 4 acute and/or late tissue radiation side effects. Int J Radiat transforming growth factor beta-1 single nucleotide polymor- Oncol Biol Phys 1999;44:981–988.
phisms with radiation-induced damage to normal tissues in 75. Weissberg JB, Huang DD, Swift M. Radiosensitivity of breast cancer patients. Int J Radiat Biol 2003;79:137–143.
normal tissues in ataxia-telangiectasia heterozygotes. Int J 94. Andreassen CN, Alsner J, Overgaard M, et al. Prediction of Radiat Oncol Biol Phys 1998;42:1133–1136.
normal tissue radiosensitivity from polymorphisms in candi- 76. Concannon P, Gatti RA. Diversity of ATM gene mutations date genes. Radiother Oncol 2003;69:127–135.
detected in patients with ataxia-telangiectasia. Hum Mutat 95. Moullan N, Cox DG, Angele S, et al. Polymorphisms in the DNA repair gene XRCC1, breast cancer risk, and response to 77. Gilad S, Khosravi R, Shkedy D, et al. Predominance of null radiotherapy. Cancer Epidemiol Biomarkers Prev 2003;12: mutations in ataxia-telangiectasia. Hum Mol Genet 1996;5: 96. Severin DM, Leong T, Cassidy B, et al. Novel DNA se- 78. Telatar M, Teraoka S, Wang Z, et al. Ataxia-telangiectasia: quence variants in the hHR21 DNA repair gene in radiosen- identification and detection of founder-effect mutations in the sitive cancer patients. Int J Radiat Oncol Biol Phys 2001;50: ATM gene in ethnic populations. Am J Hum Genet 1998;62: 97. Cavalli-Sforza LL. The Human Genome Diversity Project: 79. Iannuzzi CM, Atencio DP, Green S, et al. ATM mutations in Past, present and future. Nat Rev Genet 2005;6:333–340.
female breast cancer patients predict for an increase in radi- 98. Lee JE, Choi JH, Lee JH, et al. Gene SNPs and mutations in ation-induced late effects. Int J Radiat Oncol Biol Phys clinical genetic testing: Haplotype-based testing and analy- sis. Mutat Res 2005;573:195–204.
80. Andreassen CN, Overgaard J, Alsner J, et al. ATM sequence 99. Mehrian-Shai R, Reichardt JK. A renaissance of “biochem- variants and risk of radiation-induced subcutaneous fibrosis ical genetics”? SNPs, haplotypes, function, and complex after post-mastectomy radiotherapy. Int J Radiat Oncol Biol diseases. Mol Genet Metab 2004;83:47–50.
100. Erichsen HC, Chanock SJ. SNPs in cancer research and 81. Angele S, Romestaing P, Moullan N, et al. ATM haplotypes treatment. Br J Cancer 2004;90:747–751.
and cellular response to DNA damage: Association with 101. Lawrence RW, Evans DM, Cardon LR. Prospects and pitfalls breast cancer risk and clinical radiosensitivity. Cancer Res in whole genome association studies. Philos Trans R Soc Lond B Biol Sci 2005;360:1589 –1595.
82. Andreassen CN, Alsner J, Overgaard J, et al. TGFB1 poly- 102. Newton-Cheh C, Hirschhorn JN. Genetic association studies morphisms are associated with risk of late normal tissue of complex traits: Design and analysis issues. Mutat Res complications in the breast after radiotherapy for early breast cancer. Radiother Oncol 2005;75:18 –21.
103. Hirschhorn JN. Genetic approaches to studying common 83. Hall EJ, Schiff PB, Hanks GE, et al. A preliminary report: diseases and complex traits. Pediatr Res 2005;57:74R–77R.
Frequency of A-T heterozygotes among prostate cancer pa- 104. Hirschhorn JN, Daly MJ. Genome-wide association studies The Gene-PARE project ● A. Y. HO et al. for common diseases and complex traits. Nat Rev Genet ysis in hereditary breast and ovarian cancers. Hum Mutat 105. Halushka MK, Fan JB, Bentley K, et al. Patterns of single- 117. Choy YS, Dabora SL, Hall F, et al. Superiority of denaturing nucleotide polymorphisms in candidate genes for blood- high performance liquid chromatography over single- pressure homeostasis. Nat Genet 1999;22:239 –247.
stranded conformation and conformation-sensitive gel elec- 106. Doris PA. Hypertension genetics, single nucleotide polymor- trophoresis for mutation detection in TSC2. Ann Hum Genet phisms, and the common disease:common variant hypothe- sis. Hypertension 2002;39:323–331.
118. Jones AC, Austin J, Hansen N, et al. Optimal temperature 107. Pritchard JK. Are rare variants responsible for susceptibility selection for mutation detection by denaturing HPLC and to complex diseases? Am J Hum Genet 2001;69:124 –137.
comparison to single-stranded conformation polymorphism 108. Su Y, Swift M. Outcomes of adjuvant radiation therapy for and heteroduplex analysis. Clin Chem 1999;45:1133–1140.
breast cancer in women with ataxia-telangiectasia mutations.
119. Wagner T, Stoppa-Lyonnet D, Fleischmann E, et al. Dena- J Am Med Assoc 2001;286:2233–2234.
turing high-performance liquid chromatography detects reli- 109. Bremer M, Klopper K, Yamini P, et al. Clinical radiosensi- ably BRCA1 and BRCA2 mutations. Genomics 1999;62: tivity in breast cancer patients carrying pathogenic ATM gene mutations: No observation of increased radiation-in- 120. Gross E, Arnold N, Pfeifer K, et al. Identification of specific duced acute or late effects. Radiother Oncol 2003;69:155– BRCA1 and BRCA2 variants by DHPLC. Hum Mutat 2000; 121. Nickerson ML, Weirich G, Zbar B, et al. Signature-based 110. Gatti RA, Tward A, Concannon P. Cancer risk in ATM analysis of MET proto-oncogene mutations using DHPLC.
heterozygotes: A model of phenotypic and mechanistic dif- ferences between missense and truncating mutations. Mol 122. Bernstein JL, Teraoka S, Haile RW, et al. Designing and Genet Metab 1999;68:419 – 423.
implementing quality control for multi-center screening of 111. Huber CG, Oefner PJ, Bonn GK. High-resolution liquid mutations in the ATM gene among women with breast can- chromatography of oligonucleotides on nonporous alkylated cer. Hum Mutat 2003;21:542–550.
123. Qiu P, Shandilya H, D’Alessio JM, et al. Mutation detection using Surveyor nuclease. Biotechniques 2004;36:702–707.
124. Bannwarth S, Procaccio V, Paquis-Flucklinger V. Surveyor 112. Kuklin A, Munson K, Gjerde D, et al. Detection of single- Nuclease: A new strategy for a rapid identification of het- nucleotide polymorphisms with the WAVE DNA fragment eroplasmic mitochondrial DNA mutations in patients with analysis system. Genet Test 1997;1:201–206.
respiratory chain defects. Hum Mutat 2005;25:575–582.
113. Varghese S, Schmidt-Ullrich RK, Dritschilo A, et al. En- 125. Shi R, Otomo K, Yamada H. Temperature-mediated hetero- hanced radiation late effects and cellular radiation sensitivity duplex analysis for the detection of drug-resistant gene mu- in an ATM heterozygous breast cancer patient. Radiat Oncol tations in clinical isolates of Mycobacterium tuberculosis by denaturing HPLC, SURVEYOR nuclease. Microbes Infect 114. O’Donovan MC, Oefner PJ, Roberts SC, et al. Blind analysis of denaturing high-performance liquid chromatography as a 126. Janne PA, Borras AM, Kuang Y, et al. A rapid and sensitive tool for mutation detection. Genomics 1998;52:44 – 49.
enzymatic method for epidermal growth factor receptor mu- 115. Liu W, Smith DI, Rechtzigel KJ, et al. Denaturing high tation screening. Clin Cancer Res 2006;12:751–758.
performance liquid chromatography (DHPLC) used in the 127. Shendure J, Porreca GJ, Reppas NB, et al. Accurate multi- detection of germline and somatic mutations. Nucleic Acids plex polony sequencing of an evolved bacterial genome.
116. Arnold N, Gross E, Schwarz-Boeger U, et al. A highly 128. Pennisi E. Biochemistry. Cut-rate genomes on the horizon? sensitive, fast, and economical technique for mutation anal-

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