WO2011006214A1

WO2011006214A1 - Method of detecting radiation exposure and adverse toxicity thereto

Info

Publication number: WO2011006214A1
Application number: PCT/AU2010/000913
Authority: WO
Inventors: Carl N. Sprung; Michael J. Mckay
Original assignee: Peter Maccallum Cancer Institute
Priority date: 2009-07-16
Filing date: 2010-07-16
Publication date: 2011-01-20

Abstract

The present invention relates generally to an array of nucleic acid molecules, the nucleic acid expression profiles of which are indicative of cellular exposure to radiation, in particular ionizing radiation. In a related aspect, the present invention provides an array of nucleic acid molecules, the nucleic acid expression profiles of which are indicative of susceptibility to adverse radiation toxicity. More particularly, the methods of the present invention are directed to detecting genes, the expression levels or alternative splicing of which are indicators of exposure to radiation and/or susceptibility to adverse radiation toxicity. Accordingly, the present invention provides a valuable means of screening individuals to determine, inter alia, their inadvertent exposure to ionizing radiation or the predisposition of a patient to exhibit susceptibility to adverse radiation toxicity, thereby indicating that an alternative treatment regime should be pursued.

Description

METHOD OF DETECTING RADIATION EXPOSURE AND ADVERSE TOXICITY

THERETO

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Many anti-proliferative agents used to treat conditions such as cancer and infections also have the potential to damage normal cells. Whilst dosage levels are generally selected to

preferentially affect the target (e.g. tumor cells), some patients are particularly susceptible to toxicity, and can suffer undesirable side effects from such treatment.

Radiation therapy, for example, (also referred to as "radiotherapy"), represents one of several treatment strategies used to treat many cancers (e.g., Hodgkin lymphoma, early-stage non- Hodgkin lymphoma, squamous cell cancer of the head and neck, seminoma (a testicular cancer), prostate cancer, early-stage breast cancer, some forms of non-small cell lung cancer, and medulloblastoma (a brain or spinal cord tumor)). The importance of radiation therapy is reflected in its association with positive clinical outcomes. For instance, for early-stage cancers of the windpipe (larynx) and prostate, the rate of cure is essentially the same with radiation therapy as with surgery. In some cases, radiation therapy is combined with other forms of treatment, such as certain kinds of chemotherapy drugs (e.g. cisplatin) which can enhance the effectiveness of radiation therapy.

Radiation therapy is often the treatment of choice (either alone or in combination with other treatment modalities) because malignant cells are particularly vulnerable to radiation toxicity, attributed at least in part to their ability to undergo continual and rapid renewal. Radiotherapy also has several applications in non-malignant conditions, such as the treatment of trigeminal neuralgia, severe thyroid eye disease, pterygium, pigmented villonodular synovitis, prevention of keloid scar growth, and prevention of heterotopic ossification.

Ionizing radiation (IR) is perhaps the most common form of radiation, used to treat about 60% of cancer patients. IR typically works by depositing energy to injure or destroy cells in a target area. In general, cancer cells are selectively damaged because of their high metabolic rate, and normal tissue repairs itself more effectively, resulting in greater net destruction of tumour tissue.

In its most common form, radiation therapy uses an external beam of gamma radiation generated by a linear accelerator. Less commonly, electron or proton beam radiation is used. Proton beam radiation, which can be focused on a very specific area, effectively treats certain cancers in areas in which damage to normal tissue is a particular concern, such as the eye, brain, or spinal cord. All types of external beam radiation are focused on the particular target area of the body that contains the cancer. To avoid overexposing normal tissue, several beam paths are used and surrounding tissues are shielded as much as possible. New technologies of focusing external beam radiation, called intensity modulated radiation therapy (IMRT), help protect surrounding tissues and allow a higher dose of radiation to be delivered to cancer cells.

External beam radiation therapy is typically given as a series of equally divided doses over a prolonged period of time. This method increases the lethal effects of the radiation on cancer cells while decreasing the toxic effects on normal cells. Toxic effects are decreased because normal cells can repair themselves quickly between doses while cancer cells cannot. Typically, a person receives daily doses of radiation over a period of 6 to 8 weeks. To ensure that the same area is treated each time, the person is precisely positioned using foam casts or other devices.

Stereotactic radiation therapy is radiosurgery with precise stereotactic localization of a tumor to deliver a single high dose or multiple, fractionated doses to a small intracranial or other target. Advantages include complete tumor ablation where conventional surgery would not be possible, and minimal adverse effects. Disadvantages include the requirement for surgery and limitations involving the size of the area that can be treated and the potential danger to adjacent tissues because of the high dose of radiation. In addition, it cannot be used in all areas of the body. The patient must also be immobilized and the target area kept completely still.

In other forms of radiation therapy, a radioactive substance may be injected into a vein to travel to the cancer (e.g., radioactive iodine, which is used in treatment of thyroid cancer). Similarly, brachytherapy involves placement of radioactive seeds through CT or

ultrasonographic guidance into the tumor bed itself (e.g, in the prostate or cervix). This technique can achieve higher effective radiation doses over a longer period than could be accomplished by fractionated, external irradiation.

Systemic radioactive isotopes can also be used to direct radiation to cancer in organs that have specific receptors for uptake of the isotope (i.e., radioactive iodine for thyroid cancer) or when using monoclonal antibodies. Isotopes can also accomplish palliation of generalized bony metastases (i.e., radiostrontium for prostate cancer).

Given the invasive nature of many cancers and the proximity of normal cells to the cancerous tissue, there is the ever present risk of adverse toxicity to normal cells by radiation therapy. The potential toxicity to normal cells typically occurs through the same mechanisms by which cancer cells are damaged during radiation therapy. For example, ionizing radiation may damage mRNA, DNA, and proteins directly and/or by generation of highly reactive free radicals. Damage to other cellular components can result in progressive hypoplasia, atrophy, and eventually fibrosis (scarring). There is also the risk of genetic damage from radiation therapy which can result in malignant transformation or a transmissible genetic defect. In fact, - A - the use of radiotherapy for treating non-malignant conditions is generally limited in part by the risk of radiation-induced malignancies.

There are several non-malignant cell types also susceptible to radiation toxicity, including lymphoid cells, gonads, proliferating bone marrow cells, intestinal epithelial cells, epidermis, hepatic cells, epithelium of lung alveoli and biliary passages, kidney epithelial cells, endothelial cells (pleura and peritoneum), nerve cells, bone cells, and muscle and connective tissue cells. Although most patients tolerate radiation therapy, around 1 to 5% of patients suffer from serious debilitating side effects from radiotherapy that can lead to significant morbidity.

Children are generally more susceptible to radiation injury than adults because they have a higher rate of cellular proliferation and a higher number of future cell divisions. Radiation therapy can cause both acute side effects and chronic side effects in the months or years following treatment or after re-treatment (cumulative side effects). For example, in prostate cancer, where the affected prostate gland lies between the bladder and the rectum, radiotherapy can result in late radiation toxicity that affects the rectum, bladder and/or sexual function in 5-10% of patients.

Adverse radiation toxicity has been attributed to the onset of autoimmune disease {e.g., lupus) and genetic mutations. For example, in humans, DNA double-strand break repair proteins such as ataxia telangiectasia mutated (ATM) and DNA ligase IV, when compromised, confer a clinical radiosensitive phenotype. Abrogation of the function of these same proteins also confers radiosensitivity at the cellular level.

Acute radiation toxicity generally presents as damage to epithelial surfaces (e.g., skin, oral, pharyngeal and bowel mucosa, urothelium). If the head and neck area is treated, temporary soreness and ulceration commonly occur in the mouth and throat. If severe, this can affect swallowing, and the patient may need painkillers and nutritional support. The esophagus can also become sore if it is treated directly, or if it receives a dose of collateral radiation during treatment of lung cancer. The lower bowel may be treated directly with radiation (treatment of rectal or anal cancer) or be exposed by radiotherapy to other pelvic structures (prostate, bladder, female genital tract). Typical symptoms are soreness, diarrhoea, and nausea. As part of the general inflammation that occurs, swelling of soft tissues may also cause problems during or following radiotherapy. This is a concern during treatment of brain tumours and brain metastases, especially where there is pre-existing raised intracranial pressure or where the tumour is causing near-total obstruction of a lumen (e.g., trachea or main bronchus).

Radiation toxicity may also lead to infertility, as the gonads (ovaries and testicles) are very sensitive to radiation. In fact, patients undergoing radiotherapy are at risk of being unable to produce gametes following direct exposure to most normal treatment doses of radiation. For this reason, radiation treatment for all body sites is designed to minimize, if not completely exclude dose to the gonads if they are not the primary area of treatment.

Medium and long-term side effects following radiotherapy include fibrosis (scarring), temporary or permanent hair loss, dry mouth (xerostomia), dry eyes (xerophthalmia) and secondary malignancies (cancer). Risk factors for adverse radiation toxicity include concurrent treatment with radiosensitizing drugs and anatomical variations such as congenital malformations, post-surgical adhesions, fat content, and tissue oxygenation.

In many instances, the dose of radiation may be monitored and adjusted so as to manage the risk of adverse toxicity for the benefit of the patient; that is, maximising the level of toxicity towards malignant cells whilst minimising the level of toxicity towards normal tissue.

However, managing the dose of radiation therapy can be problematic, as the final outcome of a dose of radiation can depend on numerous factors, including the nature of the delivered radiation (mode, timing, volume, dose) and the properties of the tumor (cell cycle phase, molecular properties, overall sensitivity to radiation). In any event, the risk of adverse toxicity to normal tissue following radiotherapy must be weighed against the potential gain in treating the malignant cells.

In light of the potential adverse side effects of radiation therapy, several attempts have been made to correlate radiation toxicity with cellular responses to radiation exposure ex vivo. For instance, survival of cultured skin fibroblasts after exposure to IR correlated with acute radiation toxicity in some studies but not others. In another study, lymphocytes from cancer patients with radiation toxicity showed less IR-induced apoptosis than lymphocytes from control patients. Peripheral blood lymphocytes from breast cancer patients with severe skin reactions showed an abnormal increase in chromosome aberrations when the cells were exposed to IR. In these latter two studies, correlations between radiation toxicity and the ex vivo assay suggested the presence of an underlying genetic defect in some radiation sensitive patients. However, there was a large overlap between radiation sensitive patients and controls in these assays, limiting their clinical usefulness. Thus, assays to predict radiation toxicity have yielded mixed results, and the vast majority of adverse reactions remain unexplained.

To date, there is no effective way known to predict whether or not a patient will be susceptible to adverse toxicity following radiation therapy. Thus, there remains a significant need for a method for predicting an adverse clinical response to radiation therapy in a patient, for example, by provide information as to whether the patient is or is not susceptible to adverse radiation toxicity (i.e., whether or not the patient is radiation sensitive).

In work leading up to the present invention, it has been determined that a panel of genes are differentially expressed between individuals who are susceptible to an adverse radiation toxicity response versus individuals who are not. More particularly, this differential expression has been found to take the form of either an up-regulation or down-regulation in the alternatively spliced variant populations of mRNA which are expressed for a given gene. A unique advantage of the screening method which has been developed based on this determination is the fact that specific sequence information in relation to each and every mRNA splice variant form of a given gene is not required. Rather, by merely analysing the occurrence or not of RNA expression from each individual exon of a given gene, a comparison can be made of the range of alternatively spliced mRNA populations as between a patient sample and a control sample. Accordingly, this has provided a simple and routine means of assessing individuals for their susceptibility to adverse radiation toxicity. In a related aspect, it has also been determined that a panel of genes are differentially expressed between cellular populations which have been exposed to ionizing radiation and corresponding populations which have not. When this aspect is considered in terms of harvesting and testing a cellular population from an individual, this provides a means of routinely and simply screening individuals to determine whether they have been exposed to ionizing radiation. The genes which have been identified in accordance with this aspect have been determined to be differentially expressed either at a total RNA level or at the level of changes to the range of alternatively spliced mRNA forms expressed by a given gene. SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

As used herein, the term "derived from" shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source. Further, as used herein the singular forms of "a", "and" and "the" include plural referents unless the context clearly dictates otherwise.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

One aspect of the present invention is directed to screening for susceptibility to adverse radiation toxicity in an individual, said method comprising identifying the mRNA splice variants expressed by one or more genes selected from:

(i) ACTN l AKT3 BHLHB3 ClOorfl l

C14orfl05 Clorflόl CD28 CDCPl

CHLl CRTAP EOMES FGFRl

GPC4 GRPR HNFlB LY9

MERTK MOBKL2B MTTP PARD3

PTPRE RBPMS SERINC2 SGPP2

SHROOM3 SLC2A13 SSFA2 STEAP2

IQGAP2 RAPGEF5 TSPYL5 EPHBl

HNF4G

(ϋ) the genes identified by accession numbers:

NM 001 102 NM 181690 NM 030762 NM 032024

NM 018168 BC128148 NM 006139 NM 022842

NM 006614 NM 006371 NM 005442 NM 0231 10

NM 001448 NM 005314 NM 000458 NM 002348

NM 006343 NM 024761 NM 000253 NM 019619

NM 006504 NM 00100871 1 NM 178865 NM 152386

NM 020859 NM 052885 NM 006751 NM 152999

NM 006633 NM 012294 NM 033512 NM 004441

NM 004133

(iii) CCL20 CDHl CDKL5 CRl EVC2 PTPN 13 ROBOl SLC25A24

ClorGl NETOl

(iv) the genes identified by accession numbers:

NM 004591 NM 004360 NM 001037343 NM 000651

NM 147127 NM^~ ^"080683 NM J33631 NM _213651

NM 030806 NM^~ ^"138966 in a biological sample from said individual wherein the up-regulation of an alternatively spliced mRNA form of the genes of group (i) and/or group (ii) relative to those expressed in a normal sample is indicative of susceptibility to adverse radiation toxicity and/or the down- regulation of an alternatively spliced mRNA form of the genes of group (iii) and/or group (iv) relative to those expressed in a normal sample is indicative of susceptibility to adverse radiation toxicity.

In another aspect there is provided a means for screening for susceptibility to adverse radiation toxicity in an individual, said method comprising identifying the mRNA splice variants expressed by one or more genes selected from:

(i) APBBlIP DPT GPRC5B PSG4

CLU ERAPl PHACTR3 SYNPO; or

CUBN GABBR2 PI 16

(ϋ) the genes identified by accession numbers

NM 019043 NM 001937 NM 016235 NM 002780

NM 001831 NM 001040458 NM 080672 NM_007286; or

NMJ)01081 NM_005458 NM_153370

(iii) CPM INA LPCAT2 PDE4DIP

CPSl ITGA7 LPHN2 PDE5A

DYNClHl ITGA8 LRRC 16A PKD2

FBN2 JAGl NOTCH3 PLCBl

FGD4 KIAA 1622 PARP 14 PNPLA3

FLNA KIF 16B PDE3A QPRT

RPS6KA2; or

(iv) the genes identified by accession numbers

NM 001874 NM 002206 NM 017640 NM 182734

NM 001 122633 NM 003638 NM 000435 NM 025225

NM 001376 NM 000214 NM 017554 NM 014298

NM 001999 NM 058237 NM 000921 NM 021 135

NM 139241 NM 024704 NM 014644

NM 001456 NM 017839 NM 001083 NM 032727 NM 012302 NM 000297

in a fibroblast sample from said individual wherein the up-regulation of an alternatively spliced mRNA form of the genes of group (i) and/or group (ii) relative to those expressed in a normal sample is indicative of susceptibility to adverse radiation toxicity and/or the down- regulation of an alternatively spliced mRNA form of the genes of group (iii) and/or group (iv) relative to those expressed in a normal sample is indicative of susceptibility to adverse radiation toxicity.

In yet another aspect, there is provided a means of screening for susceptibility to adverse radiation toxicity in an individual, said method comprising identifying the mRNA splice variants expressed by one or more genes selected from:

(i) ANKRD29 CRTAP IQGAP2 SCPEPl

ANO5 DCHS2 ITGA4 SGPP2

ATR DGKD KYNU SHROOM3

BHLHB3 DOCK5 MBOAT2 SLC2A13

BHLHB5 DST MCOLN3 SLCO4C1

ClOorfl l EOMES MERTK SSFA2

Clorflόl EPHBl PARD3 STEAP2

CACNB4 GPC4 RAPGEF5 TOMlLl

CHLl HNFlB RBPMS ZFYVE 16

CRIMl INA SCARB2; or

(ϋ) the genes identified by accession numbers:

NM 173505 NM 006371 NM 006633 NM 021626

NM 213599 NM 017639 NM 000885 NM 152386

NM 001 184 NM 152879 NM 003937 NM 020859

NM 030762 NM 024940 NM 138799 NM 052885

NM 152414 NM 183380 NM 018298 NM 180991

NM 032024 NM 005442 NM 006343 NM 006751

BC128148 NM 004441 NM 019619 NM 152999

NM 001005747 NM 001448 NM 012294 NM 005486

NM 006614 NM 000458 NM 00100871 1 NM 001 105251

NM_016441 NM_032727 NM 005506

(iii) FAM49A ROBOl ; or (iv) the genes identified by accession numbers:

NM 030797 NM 133631

in a biological sample from said individual, which biological sample has been exposed to radiation, wherein the up-regulation of an alternatively spliced mRNA form of the genes of group (i) and/or group (ii) relative to those expressed in a normal sample which has been exposed to radiation is indicative of susceptibility to adverse radiation toxicity and/or the down-regulation of an alternatively spliced mRNA form of the genes of group (iii) and/or group (iv) relative to those expressed in a normal sample which has been exposed to radiation is indicative of susceptibility to adverse radiation toxicity.

In still another aspect, there is provided a means for screening for susceptibility to adverse radiation toxicity in an individual, said method comprising identifying the mRNA splice variants expressed by one or more genes selected from:

(i) APBBlIP CUBN HOXC8 SGCG

CAB39L CYTLl MKX TMEM 155

CDKL2 DPT PI16 VLDLR

CLU GABBR2 PSG4 VSIGl ; or

COL 12Al GPRC5B SEZ6L2

(ϋ) the genes identified by accession numbers

NM 019043 NM 001081 NM 022658 NM 000231

NM 030925 NM 018659 NM 173576 NM 152399

NM 003948 NM 001937 NM 153370 NM 003383

NM 001831 NM 005458 NM 002780 NM_182607

NM_004370 NMJ)16235 NM_012410

(iii) CPM GPRl 77 LPHN2 PDE4DIP

CPSl GSTM3 LRRC33 PLCBl

ERAPl HMGA2 MAP3K5 QPRT

FBN2 INA MCOLN3 RPS6KA2

FGD4 ITGA7 NEDD4L RXFPl

FST ITGA8 NOTCH3; or

(iv) the genes identified by accession numbers

NM 001874 NM 02491 1 NM 012302 NM 014644

NM 001 122633 NM 000849 NM 198565 NM 182734

NM 001040458 NM 003483 NM 005923 NM 014298

NM 001999 NM 032727 NM 018298 NM 021 135

NM 139241 NM 002206 NM 015277 NM 021634

NM 006350 NM 003638 NM 000435

in a fibroblast sample from said individual, which sample has been exposed to radiation, wherein the up-regulation of an alternatively spliced mRNA form of the genes of group (i) and/or group (ii) relative to those expressed in a normal sample which has been exposed to radiation is indicative of susceptibility to adverse radiation toxicity and/or the down-regulation of an alternatively spliced mRNA form of the genes of group (iii) and/or group (iv) relative to those expressed in a normal sample which has been exposed to radiation is indicative of susceptibility to adverse radiation toxicity. In a further aspect, of the present invention is directed to a method of assessing whether an individual has been exposed to radiation, said method comprising measuring the level of expression of one or more genes selected from:

0) BLOC1 S2 C12orf5 Clorfl 83 CDKNlA

EDA2R EI24 FAS FBXO22

GADD45A GDF 15 ISF20L1 MDM2

PHLDA3 PLK2 POLH PPMl D

SESN2 TNFRSFl OB XPC ZNF79; or (ii) the genes identified by accession numbers:

NM 001001342 NM 020375 NM 019099 NM 078467

NM 021783 NM 004879 NM 000043 NM 147188

NM 001924 NM 004864 NM 022767 NM 006882

NM 012396 NM 006622 NM 006502 NM 003620

NM_031459 NM 003842 NM_004628 NM_007135

(iii) ARHGAPI lA ASPM AURKA BUB l

CCNB l CDC20 CENPA CENPE

DEPDCl DLG7 FAM72A GTSEl

INCENP KIF20A KIF23 NEK2

PLKl TACC3 TPX2 UBE2C

H2AX CENPF; or

(iv) the genes identified by accession numbers:

NM 014783 NM 018136 NM 198433 NM 004336

NM 031966 NM 001255 NM 001809 NM 001813

NM 001 1 14120 NM 014750 BC035696 NM 016426

NM 001040694 NM 005733 NM 138555 NM 002497

NM 005030 NM 006342 NM 0121 12 NM 181802

NM 002105 NM 016343

in a biological sample from said individual wherein a higher level of expression of the genes of group (i) and/or group (ii) relative to a normal level is indicative of an individual who has been exposed to radiation and/or a lower level of expression of the genes of group (iii) and/or group (iv) relative to a normal level is indicative of an individual who has been exposed to ionizing radiation.

In another further aspect, the present invention, there is provided a means of assessing whether an individual has been exposed to radiation, said method comprising measuring the level of expression of one or more genes selected from:

(i) CDKNlA PPMlD FTG2 GADD45A

MDM2 SESNl WDR63 RNF 19B

PLK3 SESN2 ZNF79 POLH

PAGl TNFRSFlOB DDB2 EDA2R

PSTPIP2 XPC BLOCl S2 BCL2Ll; or

(ii) the genes identified by accession numbers:

NMJJ78467 NM 003620 NM_006763 NM OO 1924

NMJJ02392 NM_014454 NM_145172 NMJ 53341

NM_004073 NM_031459 NM_007135 NM_006502

NM_018440 NM_003842 NM OOO 107 NM_021783

NM 024430 NM 004628 NM 001001342 NM 138578

(iii) GAS2L3 C13orf34 AURKA FAM83D

SERTAD3 CCNF CKS2 CENPA

HJURP HYLSl CDCA8 CDC25B

KLHL23 SETD8 HlFO KIF 18A

TMEM71 KPNA2 GLIS3 BCOR; or

(iv) the genes identified by accession numbers:

NM_174942 NM_024808 NMJ98433 NM_030919

NM_013368 NM_001761 NM_001827 NM OO 1809

NMJH 8410 NMJ45014 NM_018101 NM_021873

ENST00000392647 NM_020382 NM_005318 NM_031217

NM 144649 NM 002266 NM 001042413 NM 001 123385 in a fibroblast sample from said individual wherein a higher level of expression of the genes of group (i) and/or group (ii) relative to a normal level is indicative of an individual who has been exposed to radiation and/or a lower level of expression of the genes of group (iii) and/or group (iv) relative to a normal level is indicative of an individual who has been exposed to radiation.

In yet another further aspect, the present invention is directed to a method of assessing whether an individual has been exposed to radiation, said method comprising identifying the mRNA splice variants expressed by one or more genes selected from:

0) ASTN2 BBC3 Clorfl 83 CDKNlA

FBXO22 FBXW7 FDXR FHL2

IGFBP4 MDM2 PHLDA3 PLK2

PLK3 PPMlD RGLl SESNl

SESN2 TNC TNFRSFlOD TSGAl O

VWCE XPC GADD45G RRM2B

ASPM AEN; or (ii) the genes identified by access numbers:

NM 198186 NM 001127240 NM 019099 NM 078467

NM 147188 NM 033632 NM 024417 NM 201555

NM 001552 NM 002392 NM O 12396 NM 006622

NM 004073 NM 003620 NM 015149 NM 014454

NM 031459 NM 002160 NM 003840 NM 18291 1

NM 152718 NM 004628 NM_006705 NM_015713

NM Ol 8136 NM_022767

(iii) ANLN AURKA BUBlB CCNBl

CDC25B CDCA2 CENPA CENPE

FAM65B FAM72A FAM83D GTSEl

IL16 INCENP KIF 14 KIF23

NEK2 PLKl PSRCl SGOL2

SH2D3C TROAP UBE2C; or

(iv) the genes identified by accession numbers:

NM O 18685 NMJ 98433 NM_00121 1 NM 031966

NM 021873 NMJ 52562 NMJ)01809 NMJKH813

NM_014722 BC035696 NM_030919 NM O 16426

NMJ 72217 NMJ)01040694 NMJ) 14875 NMJ38555

NM_002497 NM_OO5O3O NMJ)01032290 NM 152524

NM 170600 NM 005480 NM 181802

in a biological sample from said individual wherein the up-regulation of an alternatively spliced mRNA form of the genes of group (i) and/or group (ii) relative to those expressed in a normal sample is indicative of an individual who has been exposed to radiation and/or the down-regulation of an alternatively spliced mRNA form of the genes of group (iii) and/or group (iv) relative to those expressed in a normal sample is indicative of an individual who has been exposed to radiation.

Still another aspect of the present invention, there is provided a means for assessing whether an individual has been exposed to radiation, said method comprising identifying the mRNA splice variants expressed by one or more genes selected from:

(0 FBXW7 PLK3 BTG2 SESNl

SESN2 CDKNl A GDF 15 MDM2

VWCE PPMlD FDXR THSDlP

TP53INP1 LRDD Clorfl83 TRAF4

HISTlHlT IER5 WDR63; or

(ii) the genes identified by accession numbers: NM 033632 NM 004073 NM 006763 NM 014454

NM 031459 NM 078467 NM 004864 NM^~ 002392

NM 152718 NM 003620 NM 024417 NR 002816

NM 033285 NM 018494 NM 019099 NM_. J)04295

NM_005323 NM_016545 NMJ45172

(iii) CCNB l CDC25B FAM83D CCNF

C13orf34 GAS2L3 IER5 TROAP

BUB lB AURKA PLKl HERC4

PSRCl CENPA KIF 18A KIF23

CENPE TPX2 CKAP2 CDC27

ZNF321 ARHGAPI lA

(iv) the genes identified by accession numbers:

NM 031966 NM 021873 NM 030919 NM 001761

NM 024808 NM 174942 NM 016545 NM^" 005480

NM 00121 1 NM 198433 NM 005030 NM 022079

NM 001032290 NM 001809 NM 031217 NM^" 138555

NM 001813 NM 0121 12 NM 018204 NM^" 001 1 14091

NM 203307 NM 014783 in a fibroblast sample from said individual wherein the up-regulation of an alternatively spliced mRNA form of the genes of group (i) and/or group (ii) relative to those expressed in a normal sample is indicative of susceptibility to adverse radiation toxicity and/or the down- regulation of an alternatively spliced mRNA form of the genes of group (iii) and/or group (iv) relative to those expressed in a normal sample is indicative of an individual who has been exposed to radiation.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. IGFBP4 gene expression increases after irradiation. PCR was used to amplify the cDNA derived from the transcriptional products of cell lines that were treated with (10Gy) or without (OGy) ionizing radiation. The amplified products were analysed on a polyacrylamide gel. The relative amounts were calculated using a densitometer and the levels were normalized to PGK expression. Figure 2. PLK2 gene expression increases after irradiation. PCR was used to amplify the cDNA derived from the transcriptional products of cell lines that were treated with (10Gy) or without (OGy) ionizing radiation. The amplified products were analysed on a polyacrylamide gel. The relative amounts were calculated using a densitometer and the levels were normalized to PGK expression.

Figure 3. SESN2 gene expression increases after irradiation. PCR was used to amplify the cDNA derived from the transcriptional products of cell lines that were treated with (10Gy) or without (OGy) ionizing radiation. The amplified products were analysed on a polyacrylamide gel. The relative amounts were calculated using a densitometer and the levels were normalized to PGK expression. Figure 4. Quantitative real-time PCR validation of gene expression modulation. (A) Example of genes (XPC, POLH, CDKNlA and FBXO22) up-regulated 4 hours following 10 Gy IR in lymphoblast cell lines. n=12 cell lines for XPC and POLH, P-value 0 vs 10 Gy=0.0005; n=2 for CDKNlA; n=6 for FBXO22, P-value 0 vs 10 Gy=0.03. (B) Example of genes (CENPE, KIF14, CENPA and ASPM) down-regulated 4 hours after 10 Gy IR in lymphoblast cell lines (LCLs). n=6 for ASPM; n=l for CENPE, KI F 14, CENPA. (C) Example of genes (CDKNlA, POLH and DDB2) up-regulated 4 hours following 10 Gy IR in fibroblasts. n=9 for CDKl A, P- value 0 vs 10 Gy=0.004; n=12 for POLH and DDB2, P-values 0 vs 10 Gy=0.002. (D) Examples of the differential expression within genes (SESNl and MDM2) comparing two probe selection region expression changes after 10 Gy IR in LCLs where there is an induction of one exon. This is a type of validation for intra-gene expression differences which indicates use of alternative transcripts p-value 694 vs 668 at 10 Gy = 0.007; p-value 303 vs 315 at 10 Gy = 0.002. (E) As in D but with PPMl D. P-value 546 vs 538 at 10 Gy = 0.01. (F) An example of differential expression where an exon is down-regulated within a gene. The expression at two probe selection regions in the ASPM gene (ASPM 614 and ASPM 604) are compared after 10 Gy IR in LCLs. P-value 614 vs 604 at 10 Gy = 0.008. (G) As in D but with the FBXW7 gene in fibroblasts. n=12 fibroblast cell lines. P-value 008 vs 971 at 10 Gy = 0.0001. (H) As in D but with MDM2 in fibroblasts. n=12 fibroblast cell lines. P-value 303 vs 315 at 10 Gy = 0.002. P-values are based on Wilcoxon Signed Rank Test. Figure 5. CDKNlA gene shows differential expression of alternative spliced transcripts following 10Gy of IR. Exon expression levels (y-axis-log base 2) of individual probe set regions (x-axis) from twelve different lymphoblast cell lines are shown. No treatment (red) or irradiation with 10Gy (blue) are plotted. Error bars represent standard error of the means. Figure 6. PLK2 gene shows differential expression of alternative spliced transcripts following 10Gy of IR. Exon expression levels (y-axis-log base 2) of individual probe set regions (x-axis) from twelve different lymphoblast cell lines are shown. No treatment (red) or irradiation with 10Gy (blue) are plotted. Error bars represent standard error of the means.

Figure 7. Cell type specific gene expression. ATF3 transcripts were isolated 4 hrs after exposure to 10 Gy of IR. Relative PSR expression levels are shown for the ATF3 gene for untreated (red) or irradiated (blue) in LCLs (A) or fibroblasts (B). AS-ANOVA p-values = (p<0.00001 for A). The fold change between two horizontal bars is 25_.

Figure 8. Gene expression, at the exon level, which show modulated transcription expression products after exposure to IR in lymphoblast cell lines. Examples of up- (A-D) and down- (E- F) regulated gene probe selection regions (PSRs) at the exon level across a gene at 4 hours following 10 Gy IR in LCLs are shown. PSR relative expression level examples are shown for the following genes: EDAR2 (A), ANKRA2 (B), C12orf5 (C), AEN (D), DEPDCl (E), BUBl (F), KIF20A (G) and CENPF (H). Relative expression (y-axis) is plotted for each PSR (points along x-axis). Samples were either not irradiated (red) or irradiated (blue) with 10 Gy of radiation. Genes are orientated 5' to 3' except EDA2R, ANKRA2, DEPDCl and BUBl . Relative expression levels are plotted on a Iog2 scale. Fold change between the two horizontal lines is indicated on the right hand side of the figure. 12 cancer patient samples were used for each point (n=12). Error bars = SEM. Above each graph is a representation of known transcripts.

Figure 9. Gene expression, at the exon level, which show modulated transcription expression products with evident alternative transcripts after exposure to IR in lymphoblast cell lines. Examples of up- (A-D) and down- (E-F) regulated gene probe selection regions (PSRs) at the exon level across a gene at 4 hours following 10 Gy IR in lymphoblast cell lines are shown. PSR relative expression level examples are shown for the following genes: GADD45G (A), XPC (B), ASTN2 (C), Clorfl 83 (D), VWCE (E), BBC3 (F), LRDD (G) and PPMl D (H). Relative expression (y-axis) is plotted for each PSR (points along x-axis). Samples were either not irradiated (red) or irradiated (blue) with 10 Gy of radiation. Genes are orientated 5' to 3' except XPC, ASTN2, Clorfl 83, VWCE, BBC3 and LRDD. Relative expression levels are plotted on a Iog2 scale. Fold change between the two horizontal lines is indicated on the right hand side of the figure. 12 cancer patient samples were used for each point (n=12). Error bars = SEM. Above each graph is a representation of known transcripts.

Figure 10. Gene expression, at the exon level, which show modulated transcription expression products after exposure to IR in fibroblast cell lines. Examples of up- (A-D) and down- (E-F) regulated gene probe selection regions (PSRs) at the exon level across a gene at 4 hours following 10 Gy IR in fibroblasts are shown. PSR relative expression level examples are shown for the following genes: GDF 15 (A), PHLDA3 (B), TSKU (C), TNFRSFl OB (D), GAS2L3 (E), C13orf34 (F), CKS2 (G) and HJURP (H). Relative expression (y-axis) is plotted for each PSR (points along x-axis). Samples were either not irradiated (red) or irradiated (blue) with 10 Gy of radiation. Genes are orientated 5' to 3' except PHLDA3 and TNFRSFl OB. Relative expression levels are plotted on a Iog2 scale. Fold change between the two horizontal lines is indicated on the right hand side of the figure. 12 cancer patient samples were used for each point (n=12). Error bars = SEM. Above each graph is a representation of known transcripts.

Figure 11. Gene expression, at the exon level, which show modulated transcription expression products with evident alternative transcripts after exposure to IR in fibroblast cell lines. Examples of up- (A-D) and down- (E-F) regulated gene probe selection regions (PSRs) at the exon level across a gene at 4 hours following 10 Gy IR in fibroblast cell lines are shown. PSR relative expression level examples are shown for the following genes: SESN2 (A), THSDl P (B), TP53INP1 (C), SESNl (D), AURKA (E), CCNFl (F), FAM83D (G) and KIFl 8A (H). Relative expression (y-axis) is plotted for each PSR (points along x-axis). Samples were either not irradiated (red) or irradiated (blue) with 10 Gy of radiation. Genes are orientated 5' to 3' except THSDlP, TP53INP1 , SESNl , AURKA and KIF 18A. Relative expression levels are plotted on a Iog2 scale. Fold change between the two horizontal lines is indicated on the right hand side of the figure. 12 cancer patient samples were used for each point (n=12). Error bars = SEM. Above each graph is a representation of known transcripts.

Figure 12. Treatment with ionising radiation induces the utilization of an ATSS in RRM2B transcripts in human cell lines. RRM2B transcripts were isolated from LCLs (A-E) or fibroblasts (F-J) 4 hrs after exposure to 10 Gy of IR. The p-value as calculated by an alternative splicing ANOVA specifically designed to select alternative transcripts (Partek Genomics Suite). Relative PSR expression levels are shown for the RRM2B gene for untreated (red) or irradiated (blue) in LCLs (A) or fibroblasts (F). The increase in expression is consistently higher for the irradiated samples for every PSR of the RRM2B transcript except at the 5'end. AS-ANOVA p-values = (p<0.00001). The fold change between two horizontal bars is 2_^5_.. A dose response was run using doses ranging from 1 Gy to 20 Gy using samples isolated 4 hours post-IR (B, G). Validation of exon array dose response expression data at two PSRs (probes 313 and 293-labeled in A) using qPCR is shown (C, H). A time course was run using 2 hr, 4 hr, 8 hr, 24 hr and 48 hr after IR in LCLs (D) and fibroblasts (I). Validation of exon array time course expression data using qPCR is shown (E, J). Error bars = SEM based on three separate cell line data. Figure 13. Gene expression, at the exon level, for genes that show differential transcription products between radiosensitive and non-radiosensitive patient lymphoblast cell lines (LCLs). (A-D) Examples of genes that show a general increased expression across most of the gene in radiosensitive (blue) compared to non-radiosensitive (red) patient cell lines. (E-F) Example of genes that show a general increased expression across most of the gene in non-radiosensitive compared to radiosensitive patient cell lines. The transcript expression at each probe selection region (PSR) for the following genes: RBPMS (A), SGPP2 (B), PARD3 (C), BHLHB3 (D), CRl (E), ROBOl (F), CCL20 (G) and PTPNTI3 (H) is shown. Relative expression (y-axis) is plotted for each PSR (points along x-axis). Genes are orientated 5' to 3' except PARD3, BHLHB3 and ROBOl. Relative expression levels are plotted on a Iog2 scale. Fold change between the two horizontal lines is indicated on the right hand side of the figure. 12 cancer patient samples were used for each point (n=12). Error bars = SEM. Above each graph is a representation of known transcripts.

Figure 14. Gene expression, at the exon level, for genes that show differential transcription products between radiosensitive and non-radiosensitive patient fibroblast cell lines (LCLs). (A- D) Examples of genes that show a general increased expression across most of the gene in radiosensitive (blue) compared to non-radiosensitive (red) patient cell lines. (E-F) Example of genes that show a general increased expression across most of the gene in non-radiosensitive compared to radiosensitive patient cell lines. The transcript expression at each probe selection region (PSR) for the following genes: CLU (A), DPT (B), GABBR2 (C), CUBN (D), LPHN2 (E), 1TGA8 (F), FGD4 (G) and INA (H) is shown. Relative expression (y-axis) is plotted for each PSR (points along x-axis). Genes are orientated 5' to 3' except CLU, DPT, GABBR2, CUBN and ITGA8. Relative expression levels are plotted on a Iog2 scale. Fold change between the two horizontal lines is indicated on the right hand side of the figure. 12 cancer patient samples were used for each point (n=12). Error bars = SEM. Above each graph is a representation of known transcripts. Figure 15. Gene expression, at the exon level, for genes that show differential transcription products between radiosensitive and non-radiosensitive patient lymphoblast cell lines 4 hours after exposure to 10 Gy of radiation. (A-D) Examples of genes that show a general increased expression across most of the gene in radiosensitive (blue) compared to non-radiosensitive (red) patient cell lines. (E-F) Example of genes that show a general increased expression across most of the gene in non-radiosensitive compared to radiosensitive patient cell lines. The transcript expression at each probe selection region (PSR) for the following genes: RBPMS (A), STEAP2 (B), GPC4 (C), MCOLN3 (D), RAPGEF5 (E), ITGA4 (F), FAM49A (G) and ROBOl (H) is shown. Relative expression (y-axis) is plotted for each PSR (points along x- axis). Genes are orientated 5' to 3' except GPC4, MC0LN3, RAPGEF5, FAM49A and ROBOl . Relative expression levels are plotted on a Iog2 scale. Fold change between the two horizontal lines is indicated on the right hand side of the figure. 12 cancer patient samples were used for each point (n=12). Error bars = SEM. Above each graph is a representation of known transcripts. Figure 16. Gene expression, at the exon level, for genes that show differential transcription products between radiosensitive and non-radiosensitive patient fibroblast cell lines 4 hours after exposure to 10 Gy of radiation. (A-D) Examples of genes that show a general increased expression across most of the gene in radiosensitive (blue) compared to non-radiosensitive (red) patient cell lines. (E-F) Example of genes that show a general increased expression across most of the gene in non-radiosensitive compared to radiosensitive patient cell lines. The transcript expression at each probe selection region (PSR) for the following genes: APBBl IP (A), SGCG (B), GABBR2 (C), PSG4 (D), HMGA2 (E), CPM (F), N0TCH3 (G) and MAP3K5 (H) is shown. Relative expression (y-axis) is plotted for each PSR (points along x- axis. Genes are orientated 5' to 3' except GABBR2, PSG4, CPM, NOTCH3 and MAP3K5. Relative expression levels are plotted on a Iog2 scale. Fold change between the two horizontal lines is indicated on the right hand side of the figure. 12 cancer patient samples were used for each point (n=12). Error bars = SEM. Above each graph is a representation of known transcripts. Figure 17. MOBKL2B gene shows differential expression of alternative spliced transcripts of radiosensitive compared to non-radiosensitive samples. Exon expression levels (y-axis-log base 2) of individual probe set regions (x-axis) from six lymphoblast cell lines derived from radiosensitive patients and six derived from non-radiosensitive patients is shown.

Radiosensitive (blue) or non-radiosensitive (red) are plotted. Error bars represent standard error of the means.

Figure 18. Treatment with ionising radiation induces the utilization of an ATSS in MDM2 transcripts in both LCLs and fibroblast cells. An ATSS is predicted in LCLs (A) and primary fibroblasts (C). MDM2 transcripts were isolated 4 hrs after exposure to 2 Gy and 10 Gy (LCL: red: 0 Gy; blue 10 Gy (A); Fibroblasts: red=0 Gy; blue= 2 Gy; green=10 Gy (C)) of ionizing radiation. The p-value as calculated by an ANOVA specifically designed to select alternative splicing events MDM2 transcripts are induced (n=12) 4 hours after radiation as in figure 1. The increase in expression is consistently higher for the irradiated samples for every exon of the MDM2 transcript except at the 5 'end. ANOVA p-values = (pO.OOOOl). (B) qPCR validation of exon array data. A dose response from 1 Gy to 20 Gy is observed. ATSS is validated since probe 315 {yellow) is induced but probe 303 (green) is not induced to the same level in LCLs. qPCR amplicons are from PSRs indicated in panel A. (D) MDM2 reaches a plateau level 4 hours post-IR, but ATSS is evident across all time points. Error bars = SEM based on three separate cell line data.

Figure 19. Irradiation induces the utilization of a putative ATSS in TNFSF9 transcripts with loss of translated coding region. TNFSF9 gene is induced in human LCLs (n=12) 4 hours after exposure to 10 Gy (blue line) of radiation. Two exons are shown in this graph (blue and red bars) that have been interrogated by probes from several PSRs for each exon. The yellow indicates untranslated transcript. TNFSF9 is an example where the radiation-induced transcript would result in loss of translated sequences, thus directly affecting functional elements of the protein. Lost functional elements include the transmembrane domain and known

phoshphorylation sites.

Figure 20. qPCR validation of DNA repair genes that show transcription modulation following 10 Gy IR. Ct values were normalized using PGK. Each bar represents data from 12 different cell lines for both LCL (A) and primary fibroblasts (B) with the following exceptions: 6 samples were used for PCNA and RRM2B in LCLs; 10 samples was used for XPC, RRM2B, REV3L in fibroblasts and 5 samples for, PALB2, EXOl, LlGl and H2AFX in fibroblasts. Gene expression levels were averaged across multiple experiments. Four separate experiments were carried out for POLH, DDB2, APTX, RAD51C, and PALB2 genes; three separate experiments were carried out for PCNA, REV3L, EXOl, and NEIL3 genes; and 2 separate experiments were carried out for XPC, RRM2B, H2AFX, and RAD51 genes. Error bars = SEM (n=12). Each value on an experiment was run in triplicate. All differences were statistically significant p-value >0.05. PSRs used for amplification are: XPC: PSR855; POLH: PSR124; DDB2: PSR663; PCNA: PSR213; RRM2B: PSR293; REV3L: PSR729; APTX: PSR338; H2AFX: PSR185; RAD51C: PSR786; RAD51 : PSRlOO; EXOl : PSR239; PALB2: PSR346; LlGl : PSR905; POLL: PSR904; NEIL3: PSR753.

Figure 21. (A) Irradiation induces FS transcripts. FS gene (FST) is induced in human fibroblasts (n=12) 4 hours following exposure to 10 Gy (blue line) of radiation. The FS level of the corresponding unirradiated cells are shown (red line). Each point along the x-axis represents one potential or known exon. The increase in expression is consistently higher for the irradiated versus the unirradiated samples for every exon in the FS gene. Graph description: All of the known exons of the gene are represented along the x-axis. Data points are derived from 4 probes and are called probe selection regions (PSRs) and more than one PSR may be contained in a known exon if potential splicing sites exist. The y-axis represents relative fluorescence. Fluorescence can vary several fold due to differences in probe hybridization/fluorescence characteristics as well as differences in actual gene expression. The relative expression change between two horizontal bars is 2.5 fold. For interpretation of these graphs the difference between the treatments is the important aspect. For example, FST shows a consistently higher level for the irradiated samples across the gene compared to the unirradiated sample. The p-value as calculated by an ANOVA (Partek Genomics Suite 6.5beta) is statistically significant (p<0.00001). (B) Quantitative real-time PCR (qPCR) validates the exon array data showing an increase in FS transcription after 10 Gy (n=l 1). Error bars = SEM. p-value<0.0005 generated from a paired t-test. (C) Lower levels of FS transcripts are present in radiosensitive patients compared to controls. FS transcripts are induced in human fibroblasts from patients who showed radiosensitivity (blue line: n=6) or not (controls: red-line: n=6). FS transcript levels are lower in the radiosensitive group and are consistent across all exons of the gene. The p-value as calculated by an ANOVA is pO.00001 based on 0, 2 Gy and 10 Gy combined samples. The relative fluorescence difference between horizontal bars is 2.5. (D) qPCR is consistent with exon array data that show radiosensitive (n=5) samples express lower amounts of FS transcripts relative to the controls (n=6) at basal level and after 10 Gy of radiation. Error bars = SEM. p-value = 0.01.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, in part, on the elucidation of gene expression profiles which characterise an individual's susceptibility to adverse radiation toxicity. In a related aspect there have been identified gene expression profiles which are characteristic of cells which have undergone exposure to ionizing radiation. These findings have now facilitated the development of routine means of screening individuals to determine their susceptibility to adverse radiation toxicity and/or their exposure to ionizing radiation. In relation to a first aspect of the present invention, it has been determined that individuals who are susceptible to adverse radiation toxicity exhibit a characteristic cellular gene expression profile, prior to exposure to radiation, which differs to that expressed by individuals who are not susceptible. However, it has been further determined that after exposure to radiation, the exposed cells of a susceptible individual also exhibit a characteristic gene expression profile which is different to that of cells from non-susceptible individuals which have similarly been exposed to ionizing radiation. The gene expression profiles of this aspect of the present invention are characterised by changes to the range of alternatively spliced mRNA forms of a given gene. Accordingly, one aspect of the present invention is directed to screening for susceptibility to adverse radiation toxicity in an individual, said method comprising identifying the mRNA splice variants expressed by one or more genes selected from:

(i) ACTN l AKT3 BHLHB3 ClOorfl l

C14orfl05 Clorflόl CD28 CDCPl

CHLl CRTAP EOMES FGFRl

GPC4 GRPR HNFl B LY9

MERTK MOBKL2B MTTP PARD3

PTPRE RBPMS SERINC2 SGPP2

SHROOM3 SLC2A13 SSFA2 STEAP2

IQGAP2 RAPGEF5 TSPYL5 EPHBl

HNF4G (ii) the genes identified by accession numbers:

NM 001 102 NM 181690 NM 030762 NM 032024

NM 018168 BC128148 NM 006139 NM 022842

NM 006614 NM 006371 NM 005442 NM 0231 10

NM 001448 NM 005314 NM 000458 NM 002348

NM 006343 NM 024761 NM 000253 NM 019619

NM 006504 NM 00100871 1 NM 178865 NM 152386

NM 020859 NM 052885 NM 006751 NM 152999

NM 006633 NM_012294 NM 033512 NM_004441

NM_004133

(iii) CCL20 CDHl CDKL5 CRl

EVC2 PTPN 13 ROBOl SLC25A24

Clorf21 NETOl

(iv) the genes identified by accession numbers:

NM_004591 NM 004360 NM OO 1037343 NM_000651

NMJ47127 NM_080683 NM 133631 NM 213651

NM 030806 NM 138966 in a biological sample from said individual wherein the up-regulation of an alternatively spliced mRNA form of the genes of group (i) and/or group (ii) relative to those expressed in a normal sample is indicative of susceptibility to adverse radiation toxicity and/or the down- regulation of an alternatively spliced mRNA form of the genes of group (iii) and/or group (iv) relative to those expressed in a normal sample is indicative of susceptibility to adverse radiation toxicity.

Although the genes listed in the first aspect of this invention are useful diagnostic markers across a wide range of biological samples, a cohort of genes which are particularly useful in the context of biological samples comprising fibroblasts have also been identified.

Accordingly, in one embodiment, fibroblast gene expression profiles which characterise an individual's susceptibility to adverse radiation toxicity have been identified. These findings are particularly useful since fibroblast populations can be easily and routinely harvested via small skin biopsies, such as punch biopsies. Accordingly, this provides a quick and convenient means for testing individuals to determine whether or not they are susceptible to adverse radiation toxicity.

According to this embodiment, there is provided a means for screening for susceptibility to adverse radiation toxicity in an individual, said method comprising identifying the mRNA splice variants expressed by one or more genes selected from:

(i) APBBlIP DPT GPRC5B PSG4

CLU ERAPl PHACTR3 SYNPO; or

CUBN GABBR2 PI16

(ii) the genes identified by accession numbers:

NM 019043 NM 001937 NM 016235 NM 002780

NM 001831 NM 001040458 NM 080672 NM 007286; or

NMJ)01081 NM_005458 NM_153370

(iii) CPM INA LPCAT2 PDE4DIP

CPSl ITGA7 LPHN2 PDE5A

DYNClHl ITGA8 LRRC 16A PKD2

FBN2 JAGl NOTCH3 PLCBl

FGD4 KIAA 1622 PARP 14 PNPLA3

FLNA KIF 16B PDE3A QPRT

RPS6KA2; or

(iv) the genes identified by accession numbers

NM 001874 NM 002206 NM 017640 NM 182734

NM 001122633 NM 003638 NM 000435 NM 025225

NM 001376 NM 000214 NM 017554 NM 014298

NM 001999 NM 058237 NM 000921 NM 021 135

NM 139241 NM 024704 NM 014644

NM 001456 NM 017839 NM 001083

NM 032727 NM 012302 NM 000297 in a fibroblast sample from said individual wherein the up-regulation of an alternatively spliced mRNA form of the genes of group (i) and/or group (ii) relative to those expressed in a normal sample is indicative of susceptibility to adverse radiation toxicity and/or the down- regulation of an alternatively spliced mRNA form of the genes of group (iii) and/or group (iv) relative to those expressed in a normal sample is indicative of susceptibility to adverse radiation toxicity.

In a related aspect, it has also been determined that in individuals who are susceptible to radiation toxicity there is induced a unique gene expression profile in cells of that individual which have been exposed to ionizing radiation, relative to cells of a non-susceptible individual which have similarly been exposed to ionizing radiation.

According to this aspect, there is provided a means of screening for susceptibility to adverse radiation toxicity in an individual, said method comprising identifying the mRNA splice variants expressed by one or more genes selected from:

(1) ANKRD29 CRTAP IQGAP2 SCPEPl

ANO5 DCHS2 ITGA4 SGPP2

ATR DGKD KYNU SHROOM3

BHLHB3 DOCK5 MBOAT2 SLC2A13

BHLHB5 DST MCOLN3 SLCO4C1

ClOorfl l EOMES MERTK SSFA2

Clorflόl EPHBl PARD3 STEAP2

CACNB4 GPC4 RAPGEF5 TOMlLl

CHLl HNFlB RBPMS ZFYVE 16

CRIMl INA SCARB2; or

(ϋ) the genes identified by accession numbers:

NM 173505 NM 006371 NM 006633 NM 021626

NM 213599 NM 017639 NM 000885 NM 152386

NM 001 184 NM 152879 NM 003937 NM 020859

NM 030762 NM 024940 NM 138799 NM 052885

NM 152414 NM 183380 NM 018298 NM 180991

NM 032024 NM 005442 NM 006343 NM 006751

BC128148 NM 004441 NM 019619 NM 152999

NM 001005747 NM 001448 NM 012294 NM 005486

NM 006614 NM 000458 NM 00100871 1 NM 001 105251

NM_016441 NM_032727 NM 005506

(iii) FAM49A ROBOl; or

(iv) the genes identified by accession numbers:

NM_030797 NM_133631 in a biological sample from said individual, which biological sample has been exposed to radiation, wherein the up-regulation of an alternatively spliced mRNA form of the genes of group (i) and/or group (ii) relative to those expressed in a normal sample which has been exposed to radiation is indicative of susceptibility to adverse radiation toxicity and/or the down-regulation of an alternatively spliced mRNA form of the genes of group (iii) and/or group (iv) relative to those expressed in a normal sample which has been exposed to radiation is indicative of susceptibility to adverse radiation toxicity.

In one embodiment of the present invention, it is envisaged that patients would be assessed for susceptibility to adverse radiation toxicity using a protocol where they are screened for alternative splice variant expression before exposure to radiation and are also thereafter screened for alternative splice variant expression after exposure to radiation (for example in the early stages after commencement of radiation therapy). A particularly high level of accuracy is obtained by performing this screening test in this type of two step process.

In another embodiment of this aspect of the invention, characteristic fibroblast gene expression profiles which characterise an individual's susceptibility to adverse radiation toxicity, where that individual has been exposed to radiation have also been identified. As detailed earlier, these findings are particularly useful since fibroblast populations can be easily and routinely harvested via small skin biopsies, such as punch biopsies.

According to this aspect, there is provided a means for screening for susceptibility to adverse radiation toxicity in an individual, said method comprising identifying the mRNA splice variants expressed by one or more genes selected from:

(i) APBBl IP CUBN HOXC8 SGCG

CAB39L CYTLl MKX TMEM 155

CDKL2 DPT PI16 VLDLR

CLU GABBR2 PSG4 VSIGl ; or

COL 12Al GPRC5B SEZ6L2

(ii) the genes identified by accession numbers:

NM 019043 NM 001081 NM 022658 NM 000231

NM 030925 NM 018659 NM 173576 NM 152399

NM 003948 NM 001937 NM 153370 NM 003383

NM 001831 NM 005458 NM 002780 NMJ 82607

NM_004370 NM_016235 NMJ) 12410

(iii) CPM GPRl 77 LPHN2 PDE4DIP

CPSl GSTM3 LRRC33 PLCBl

ERAPl HMGA2 MAP3K5 QPRT

FBN2 INA MCOLN3 RPS6KA2

FGD4 ITGA7 NEDD4L RXFPl

FST ITGA8 NOTCH3; or (iv) the genes identified by accession numbers

NM 001874 NM 02491 1 NM 012302 NM 014644

NM 001 122633 NM 000849 NM 198565 NM 182734

NM 001040458 NM 003483 NM 005923 NM 014298

NM 001999 NM 032727 NM 018298 NM 021 135 NM_139241 NM_002206 NM_015277 NM_021634

NM_006350 NM 003638 NM 000435 in a fibroblast sample from said individual, which sample has been exposed to radiation, wherein the up-regulation of an alternatively spliced mRNA form of the genes of group (i) and/or group (ii) relative to those expressed in a normal sample which has been exposed to radiation is indicative of susceptibility to adverse radiation toxicity and/or the down-regulation of an alternatively spliced mRNA form of the genes of group (iii) and/or group (iv) relative to those expressed in a normal sample which has been exposed to radiation is indicative of susceptibility to adverse radiation toxicity. Reference to "adverse radiation toxicity" should be understood as a reference to the serious side effects which approximately 1-5% of patients suffer subsequently to radiotherapy and which leads to significant morbidity. Accordingly, this phrase is not a reference to the toxicity which is sought to be delivered to a target cell population, such as a malignant tissue. Without limiting the present invention to any one theory or mode of action, radiation therapy can cause both acute side effects and chronic side effects in the months or years following treatment or after re-treatment. It should be understood that both types of side effects are intended to be encompassed within the definition of "adverse radiation toxicity" to the extent that the nature or degree of severity of these side effects falls within the spectrum of those experienced by individuals who fall within the subgroup of patients who experience particularly severe radiotherapy side effects. Accordingly, it would be appreciated that some of the acute or chronic symptoms which fall within the scope of "adverse radiation toxicity" may be the same as those exhibited by individuals who are not regarded as suffering from adverse radiation toxicity. The difference, however, will exist with respect to the severity of the subject side effect, this being more severe or prolonged than that experienced by normal individuals.

Examples of adverse radiation toxicity include, but are not limited to:

- onset of autoimmune disease (e.g. lupus)

genetic mutation

- damage to epithelial surfaces (e.g. skin, oral, pharyngeal, bowel mucosa,

urothelium)

- soreness and ulceration in the mouth and oesophagus

- diarrhoea nausea

- tissue inflammation

- infertility

- fibrosis

- hair loss

- dry mouth

- dry eyes

- secondary malignancies

xerostomia

- xerophthalmia.

Each of the genes detailed in sub-paragraphs (i)-(iv), above, would be well known to the person of skill in the art, as would their encoded protein. The identification of these genes occurred via the use of an exon array rather than classical 3' arrays to determine alternative splicing events in a biological sample. Unlike classical oligonucleotide 3' arrays which have probe sets only at the 3' end of the transcripts, the inventors used an exon array with four probe sets for every known exon which covers every exon of every known gene. Transcripts missing the 3' exon (e.g. through degradation, splicing, or undefined 3' ends) are not detected with conventional 3' assay arrays and transcripts with non-polyadenylated messages or alternative polyadenylation sites are commonly missed.

Reference to "splice variants" should be understood as a reference to the alternative mRNA forms which can result from a single gene during transcription. This is commonly the result of splicing events which occur when the primary RNA transcript is transformed to mRNA.

Accordingly, it is conceivable that more than one form of mRNA may be produced. These splice variants may typically, for example, exhibit differences in the exon array which has been assembled. Without limiting the present invention in any way, six modes of alternative splicing which can occur are: • Exon skipping or cassette exon: In this case, an exon may be spliced out of the primary transcript or retained. This is generally the most common mode in mammalian pre- mRNAs.

• Mutually exclusive exons: One of two exons is retained in mRNAs after splicing, but not both. • Alternative donor site: An alternative 5' splice junction (donor site) is used, changing the 3' boundary of the upstream exon.

• Alternative acceptor site: An alternative 3' splice junction (acceptor site) is used, changing the 5' boundary of the downstream exon.

• Intron retention: A sequence may be spliced out as an intron or simply retained. This is distinguished from exon skipping because the retained sequence is not flanked by introns. If the retained intron is in the coding region, the intron must encode amino acids in frame with the neighbouring exons, or a stop codon or a shift in the reading frame will cause the protein to be non-functional.

• Alternative transcription start sites: There are many genes which are known to have alternative promoters. Use of an alternative promoter can lead to initiation of transcription within a coding exon, which can result in an altered protein product. An alternative promoter may also initiate the transcript in the untranslated region. A third way that alternative promoters can affect function is by use of alternative reading frames, yielding different proteins.

In terms of the first aspect of the present invention, the method of the present invention is predicated on a comparison of the range of mRNA splice variants expressed in a patient test sample with those expressed in a normal sample. The splice variants "expressed in a normal sample" should be understood as a reference to the range of splice variants expressed in a tissue sample from an individual who is not susceptible to adverse radiation toxicity. The results of the normal sample are also herein referred to as the "control sample". In one embodiment, the tissue type of the normal sample corresponds to the tissue type of the test sample. However, without limiting the present invention to any one theory or mode of action, the phenotype which is observed in the context of the present invention is based on a genetic element common to many cell types. Accordingly, in another embodiment, the normal sample is from an individual who is not susceptible to adverse radiation toxicity but is not necessarily a tissue type corresponding to the tissue type of the test sample. The patient test results are likely to be analysed relative to a standard result which reflects individual or collective results obtained from individuals who are not susceptible to adverse radiation toxicity. This form of analysis is in fact the preferred method of analysis since it enables the design of kits which require the collection and analysis of a single biological sample, being a test sample of interest. The standard results which provide the control may be utilised in a variety of ways. For example, a population of normal tissues can be assessed in terms of the range of mRNA splice variant forms which are expressed for a particular gene or genes, thereby providing a standard result against which all future test samples are analysed. Alternatively, a control result can be newly prepared each time a test sample is analysed. It should also be understood that the control sample result may be determined from the subjects of a specific cohort and for use with respect to test samples derived from that cohort.

Accordingly, there may be determined a number of control results which correspond to cohorts which differ in respect of characteristics such as age, gender, ethnicity or health status.

In terms of the second aspect of the present invention, there is provided an alternative means of assessing susceptibility to adverse radiation toxicity, based on exposing to radiation a tissue sample isolated from said individual and thereafter analysing the range of alternatively spliced mRNA forms of the genes hereinbefore defined, either singly or in combination, relative to the corresponding alternatively spliced forms in a tissue sample of a non-susceptible individual who has also been exposed to radiation, preferably a corresponding type of radiation.

Accordingly, reference to splice variants "expressed in a normal sample" in this regard should be understood to have a meaning corresponding to the definition provided earlier but with the exception that the normal sample has been exposed to radiation prior to analysis of its RNA. The tissue type of the normal sample may correspond to the tissue type of the test sample. In another embodiment, the tissue type of the normal sample need not correspond to the tissue type of the test sample.

It would be appreciated by the person of skill in the art that in relation to this second aspect of the present invention, one may choose to harvest a biological sample from a patient prior to the commencement of treatment and to expose that sample to radiation, analogous to that proposed during treatment, in order to determine whether that patient exhibits susceptibility to adverse radiation toxicity. This test may be done instead of or together with the test of the first aspect of this invention. However, it would also be appreciated that where a patient has already commenced a radiation treatment regime, one may isolate a biological sample which, as part of the treatment regime, has been exposed to radiation. This sample may then be tested in accordance with the second aspect of the invention. In this situation, the harvested biological sample already achieves the requirement that it has been exposed to radiation since this has occurred in situ. However, where the biological sample is harvested from a patient who has not commenced radiotherapy or is harvested from a part of the patient's body which has not been exposed to the radiotherapy, then the sample would have to undergo radiation exposure in vitro. It would be appreciated that analysis of the type described in the second aspect of the present invention may be particularly useful where a patient is showing signs, during the treatment regime, of developing adverse radiation toxicity and it is sought to confirm whether the patient is in fact genetically susceptible before treatment proceeds too far.

Reference to "radiation" should be understood as a reference to any form of radiation. In a preferred embodiment, said radiation is ionizing radiation. Reference to "radiation therapy" should be understood as a reference to the use of radiation, such as ionizing radiation, to treat a disease condition to control unwanted cellular proliferation. Radiation therapy, and therefore the applicability of the method of the invention, has use beyond just neoplastic conditions and includes non-neoplastic conditions such as the treatment of trigeminal neuralgia, severe thyroid eye disease, pterygium, pigmented villonodular synovitis, prevention of keloid scar growth and prevention of heterotopic ossification.

Reference to "ionizing radiation" should be understood as a reference to subatomic particles or electromagnetic waves that are sufficiently energetic to detach electrons from atoms or molecules, thereby ionizing them. Without limiting the present invention to any one theory or mode of action, the occurrence of ionization depends on the energy of the impinging individual particles or waves, and not on their number. An intense flood of particles or waves will not cause ionization if these particles or waves do not carry enough energy to be ionizing.

Examples of ionizing particles are energetic alpha particles, beta particles, and neutrons. The ability of electromagnetic waves (photons) to ionize an atom or molecule depends on their wavelength. Radiation on the short wavelength end of the electromagnetic spectrum— ultraviolet, x-rays, and γ rays - is ionizing. Ionizing radiation is generated from radioactive materials, x-ray tubes and particle accelerators, for example. Units of measuring ionizing radiation include:

• The coulomb per kilogram (C/kg). This is the SI unit of ionizing radiation exposure, and measures the amount of radiation required to create 1 coulomb of charge of each polarity in 1 kilogram of matter. • The roentgen (R). This is an older traditional unit that is almost out of use, which represented the amount of radiation required to liberate 1 esu of charge of each polarity in 1 cubic centimeter of dry air. 1 Roentgen = 2.58XlO^"4 C/kg.

The amount of damage done to matter by ionizing radiation is more closely related to the amount of energy deposited rather than the charge. This is termed the absorbed dose.

• The gray (Gy), with units J/kg, is the SI unit of absorbed dose, which represents the amount of radiation required to deposit 1 joule of energy in 1 kilogram of any kind of matter.

• The rad (Roentgen absorbed dose), is the corresponding traditional unit which is 0.01 J deposited per kg. 100 rad = 1 Gy.

Equal doses of different types or energies of radiation cause different amounts of damage to living tissue. For example, 1 Gy of alpha radiation causes about 20 times as much damage as 1 Gy of x-rays. Therefore the equivalent dose was defined to give an approximate measure of the biological effect of radiation. It is calculated by multiplying the absorbed dose by a weighting factor W_R for each type of radiation.

• The sievert (Sv) is the SI unit of equivalent dose. Although it has the same units as grays, J/kg, it is the dose of any type of radiation in Gy that has the same biological effect on a human as 1 Gy of x-rays or gamma radiation.

• The rem (roentgen equivalent man) is the traditional unit of equivalent dose. 1 sievert

= 100 rem. Because the rem is a relatively large unit, typical equivalent dose is measured in millirem (mrem), 10^"3 rem, or in microsievert (μSv), 10^"6 Sv. 1 mrem = 10 μSv.

In a preferred embodiment, the radiation of the first and second aspects of the invention is ionizing radiation.

As detailed hereinbefore, the present invention is predicated, in part, on the determination that individuals who are susceptible to adverse radiation toxicity exhibit a unique gene expression profile, both before exposure to radiation and after such exposure, relative to corresponding individuals who are not susceptible to adverse radiation toxicity. These gene expression profiles are characterised by changes to the range of alternatively spliced mRNA forms of one or more of the genes detailed hereinbefore, when considered either singly or in combination.

Accordingly, reference to the "up-regulation" or "down-regulation" of an "alternatively spliced mRNA form of the genes" should be understood as a reference to up-regulation or down- regulation of an alternatively spliced transcript form or type of a given gene in the patient sample, when considered relative to the range of mRNA splice variant transcript forms (populations) which are found in the control. Without limiting the present invention in any way, the inventor has used probes directed to all the exons of a given gene and has screened the mRNA of a biological sample to determine the expression of each exon. These data enable the skilled person, based only on relative comparisons and without requiring the sequence information for each alternatively spliced mRNA form, to determine the exon characteristics of the mRNA forms which are present in the test sample and the control sample. Accordingly, if one observes the presence of a splice variant transcript form in the test sample which is not present in the control sample, then that splice variant transcript form has been "upregulated". However, if there is the absence of a splice variant transcript form in the test sample, but which splice variant transcript form is present in the control sample, then there has occurred "down- regulation". It should be understood that the subject "up-regulation" or "down-regulation" may occur in relation to one or more splice variant types expressed by a particular gene. The analysis which one performs is therefore a relative analysis which compares the overall range of mRNA splice variants in one sample with the overall range of mRNA splice variants in another sample. There is no need for the skilled person to necessarily obtain the sequence of each splice variant since analysis of the relative expression of the individual exons of a given gene is sufficient. It should also be understood that although one may observe changes to the level of expression of a particular splice variant as between one sample and another, for the purposes of the present invention, in one embodiment, the analysis focuses on the presence or absence of different splice variants and not their individual relative expression levels.

However, where the sequence information or other identifying information of a particular splice variant is known, one may screen for the up-regulation or down-regulation of the level of expression of that particular splice variant in the test sample relative to a normal sample. It should be understood that the "individual" who is the subject of testing may be any human or non-human mammal. Examples of non-human mammals includes primates, livestock animals (e.g. horses, cattle, sheep, pigs, donkeys), laboratory test animals (e.g. mice, rats, rabbits, guinea pigs), companion animals (e.g. dogs, cats) and captive wild animals (e.g. deer, foxes). Preferably the mammal is a human.

In yet another related aspect of the present invention, it has been determined that irrespective of whether or not an individual is susceptible to adverse radiation toxicity, it is possible to screen the individual to determine whether that individual has been exposed to radiation, in particular ionizing radiation. This screening method is also based on the finding that an individual who has been exposed to radiation will exhibit a unique gene expression profile. More specifically, individuals who have been exposed will exhibit both a change in the relative level of expression of a specific set of genes and a change in the range of mRNA splice variants of a specific set of genes.

Accordingly, a third aspect of the present invention is directed to a method of assessing whether an individual has been exposed to radiation, said method comprising measuring the level of expression of one or more genes selected from:

(i) BLOC1 S2 C12orf5 Clorfl 83 CDKNl A

EDA2R EI24 FAS FBXO22

GADD45A GDF 15 ISF20L1 MDM2

PHLDA3 PLK2 POLH PPMlD

SESN2 TNFRSFlOB XPC ZNF79; or

(ϋ) the genes identified by accession numbers:

NM 001001342 NM 020375 NMJ) 19099 NM 078467

NM 021783 NM 004879 NM 000043 NM 147188

NM 001924 NM 004864 NM 022767 NM 006882

NM 012396 NM 006622 NM 006502 NM 003620

NM_031459 NM_003842 NM_004628 NM_007135

(iii) ARHGAPI lA ASPM AURKA BUB l

CCNBl CDC20 CENPA CENPE

DEPDCl DLG7 FAM72A GTSEl

INCENP KIF20A KIF23 NEK2

PLKl TACC3 TPX2 UBE2C

H2AX CENPF; or

(iv) the genes identified by accession numbers: NM_014783 NM Ol 8136 NM_198433 NM_004336

NM 031966 NMJ)01255 NM OO 1809 NM 001813

NM_001 1 14120 NMJ) 14750 BC035696 NM O 16426

NMJ)01040694 NM_005733 NMJ38555 NM_002497

NM_OO5O3O NM 006342 NM 0121 12 NM 181802

NM 002105 NM 016343 in a biological sample from said individual wherein a higher level of expression of the genes of group (i) and/or group (ii) relative to a normal level is indicative of an individual who has been exposed to radiation and/or a lower level of expression of the genes of group (iii) and/or group (iv) relative to a normal level is indicative of an individual who has been exposed to ionizing radiation.

In one embodiment of this aspect of the present invention, there is provided a means of assessing whether an individual has been exposed to radiation, said method comprising measuring the level of expression of one or more genes selected from:

(i) CDKNlA PPMlD FTG2 GADD45A

MDM2 SESNl WDR63 RNF 19B

PLK3 SESN2 ZNF79 POLH

PAG l TNFRSFlOB DDB2 EDA2R

PSTPIP2 XPC BLOCl S2 BCL2Ll ; or

(ϋ) the genes identified by accession numbers:

NM 078467 NM 003620 NM 006763 NM 001924

NM 002392 NM 014454 NM 145172 NM 153341

NM 004073 NM 031459 NM 007135 NM 006502

NM 018440 NM 003842 NM 000107 NM 021783

NM_024430 NM_004628 NMJ)01001342 NMJ38578

(iii) GAS2L3 C13orf34 AURKA FAM83D

SERTAD3 CCNF CKS2 CENPA

HJURP HYLSl CDCA8 CDC25B

KLHL23 SETD8 HlFO KIF 18A

TMEM71 KPNA2 GLIS3 BCOR; or

(iv) the genes identified by accession numbers:

NM 174942 NM 024808 NM 198433 NM 030919

NM 013368 NM 001761 NM 001827 NM 001809

NM 018410 NM 145014 NM 018101 NM 021873

ENST00000392647 NM 020382 NM 005318 NM 031217

NM 144649 NM 002266 NM 001042413 NM 001123385 in a fibroblast sample from said individual wherein a higher level of expression of the genes of group (i) and/or group (ii) relative to a normal level is indicative of an individual who has been exposed to radiation and/or a lower level of expression of the genes of group (iii) and/or group (iv) relative to a normal level is indicative of an individual who has been exposed to radiation.

In one embodiment, said radiation is ionizing radiation.

In a fourth aspect, the present invention is directed to a method of assessing whether an individual has been exposed to radiation, said method comprising identifying the mRNA splice variants expressed by one or more genes selected from:

(i) ASTN2 BBC3 Clorfl83 CDKNlA

FBXO22 FBXW7 FDXR FHL2

IGFBP4 MDM2 PHLDA3 PLK2

PLK3 PPMlD RGLl SESNl

SESN2 TNC TNFRSFlOD TSGAlO

VWCE XPC GADD45G RRM2B

ASPM AEN; or

(ϋ) the genes identified by access numbers:

NMJ98186 NM 001 127240 NM 019099 NM 078467

NM 147188 NM 033632 NM 024417 NM 201555

NM 001552 NM 002392 NM 012396 NM 006622

NM 004073 NM 003620 NM 015149 NM 014454

NM 031459 NM 002160 NM 003840 NM 18291 1

NMJ 52718 NM 004628 NM_006705 NM_015713

NM_018136 NM_022767

(iii) ANLN AURKA BUBlB CCNBl

CDC25B CDCA2 CENPA CENPE

FAM65B FAM72A FAM83D GTSEl

ILl 6 INCENP KIF 14 KIF23

NEK2 PLKl PSRCl SGOL2

SH2D3C TROAP UBE2C; or

(iv) the genes identified by accession numbers:

NM_018685 NMJ 98433 NMJ)0121 1 NM_031966

NM_021873 NMJ 52562 NM OO 1809 NMJ)Ol 813

NM_014722 BC035696 NM_030919 NMJ) 16426

NMJ 72217 NM_001040694 NM O 14875 NMJ38555

NM_002497 NM_005030 NMJ)01032290 NM 152524

NM 170600 NM 005480 NM 181802

in a biological sample from said individual wherein the up-regulation of an alternatively spliced mRNA form of the genes of group (i) and/or group (ii) relative to those expressed in a normal sample is indicative of an individual who has been exposed to radiation and/or the down-regulation of an alternatively spliced mRNA form of the genes of group (iii) and/or group (iv) relative to those expressed in a normal sample is indicative of an individual who has been exposed to radiation. In one embodiment of this aspect of the present invention, there is provided a means for assessing whether an individual has been exposed to radiation, said method comprising identifying the mRNA splice variants expressed by one or more genes selected from:

(>) FBXW7 PLK3 BTG2 SESNl

SESN2 CDKNlA GDF 15 MDM2

VWCE PPMlD FDXR THSDlP

TP53INP1 LRDD Clorfl 83 TRAF4

HISTlHlT IER5 WDR63; or (ii) the genes identified by accession numbers:

NM 033632 NM 004073 NM 006763 NM 014454

NM 031459 NM 078467 NM 004864 NM 002392

NM 152718 NM 003620 NM 024417 NR 002816

NM 033285 NM 018494 NM 019099 NM_004295

NM_005323 NMJ 16545 NMJ45172

(iii) CCNBl CDC25B FAM83D CCNF

C13orO4 GAS2L3 IER5 TROAP

BUBlB AURKA PLKl HERC4

PSRCl CENPA KlF 18A KIF23

CENPE TPX2 CKAP2 CDC27

ZNF321 ARHGAPl IA; or

(iv) the genes identified by accession numbers:

NM 031966 NM 021873 NM 030919 NM 001761

NM 024808 NM 174942 NM 016545 NM 005480

NM 00121 1 NM 198433 NM 005030 NM 022079

NM 001032290 NM 001809 NM 031217 NM 138555

NM 001813 NM 0121 12 NM 018204 NM 001 1 14091

NM 203307 NM 014783 in a fibroblast sample from said individual wherein the up-regulation of an alternatively spliced mRNA form of the genes of group (i) and/or group (ii) relative to those expressed in a normal sample is indicative of susceptibility to adverse radiation toxicity and/or the down- regulation of an alternatively spliced mRNA form of the genes of group (iii) and/or group (iv) relative to those expressed in a normal sample is indicative of an individual who has been exposed to radiation. In one embodiment, said radiation is ionising radiation.

In relation to the third and fourth aspects of the present invention, reference to "splice variants", "up-regulation", "down-regulation", "alternatively spliced mRNA form of the gene" and "individual" should be understood to have the same meaning as hereinbefore defined.

Reference to "expressed in a normal sample", in the context of the fourth aspect of the present invention, should be understood to have a meaning corresponding to the definition provided with respect to this phrase as defined for the first aspect of the present invention, but with the exception that the normal sample is a sample obtained from any individual who has not been exposed to ionizing radiation. It is irrelevant whether this individual is susceptible to adverse radiation toxicity.

In terms of the third aspect of the present invention, however, reference to "normal level" is directed to the level of the gene expressed by a tissue sample, in one embodiment a corresponding tissue sample, from an individual who has not been exposed to ionizing radiation. It would be appreciated that in the context of this aspect of the present invention, the skilled person is not assessing the existence or not of splice variant populations. Rather, the analysis in relation to the third aspect of the invention is directed to determining whether more or less gene expression product has been produced in the test sample relative to the "normal" sample. Accordingly, the overall level of expression of a gene is analysed and results in relation to levels of expression of individual splice variant forms need not be considered. It should also be understood that whereas the first, second and fourth aspects of the present invention are directed to the analysis of an mRNA expression, this being an appropriate means for assessing mRNA splice variant populations, the third aspect of the present invention is more simply directed to measuring the overall level of expression of a gene. It would be appreciated by the person of skill in the art that this can be achieved by measuring either transcription product or translation product. Reference to "expression product" or "expression of a gene" should therefore be understood as a reference to either a transcription product (such as primary RNA or mRNA) or a translation product such as protein.

Reference to "expression" should therefore be understood as a reference to the transcription and/or translation of a nucleic acid molecule. In this regard, this aspect of the present invention is exemplified with respect to screening for expression products taking the form of RNA transcripts (eg primary RNA or mRNA). Reference to "RNA" should be understood to encompass reference to any form of RNA, such as primary RNA or mRNA. Without limiting the present invention in any way, the modulation of gene transcription leading to increased or decreased RNA synthesis will also correlate with the translation of some of these RNA transcripts (such as mRNA) to produce a protein product. Accordingly, the present invention also extends to detection methodology which is directed to screening for modulated levels or patterns of the protein products. Although one method is to screen for mRNA transcripts and/or the corresponding protein product, it should be understood that the present invention is not limited in this regard and extends to screening for any other form of expression product such as, for example, a primary RNA transcript. It is well within the skill of the person of skill in the art to determine the most appropriate screening target for any given situation.

Reference to "radiation" and "ionizing radiation" in the context of the third and fourth aspects of the invention should be understood to have the same meaning as hereinbefore defined.

The detection method of the present invention can be performed on any suitable biological sample. To this end, reference to a "biological sample" should be understood as a reference to any sample of biological material derived from an animal such as, but not limited to, cellular material (eg. fibroblast), biofluids (eg. blood), faeces, tissue biopsy specimens (eg. skin specifics), surgical specimens or fluid which has been introduced into the body of an animal and subsequently removed (such as, for example, the solution retrieved from an enema wash). The biological sample which is tested according to the method of the present invention may be tested directly or may require some form of treatment prior to testing. For example, a biopsy or surgical sample may require homogenisation prior to testing or it may require sectioning for in situ testing of the expression of individual genes. Alternatively, a cell sample may require permeabilisation prior to testing. Further, to the extent that the biological sample is not in liquid form, (if such form is required for testing) it may require the addition of a reagent, such as a buffer, to mobilise the sample. To the extent that the gene expression product is present in a biological sample, the biological sample may be directly tested or else all or some of the nucleic acid or protein material present in the biological sample may be isolated prior to testing. In yet another example, the sample may be partially purified or otherwise enriched prior to analysis. For example, to the extent that a biological sample comprises a very diverse cell population, it may be desirable to enrich for a sub-population of particular interest. It is within the scope of the present invention for the target cell population or molecules derived therefrom to be treated prior to testing, for example, inactivation of live virus or being run on a gel. It should also be understood that the biological sample may be freshly harvested or it may have been stored (for example by freezing) prior to testing or otherwise treated prior to testing (such as by undergoing culturing).

The choice of what type of sample is most suitable for testing in accordance with the method disclosed herein will be dependent on the nature of the situation. Reference to "nucleic acid molecule" should be understood as a reference to both

deoxyribonucleic acid molecules and ribonucleic acid molecules and fragments thereof. The present invention therefore extends to both directly screening for mRNA in a biological sample or screening for the complementary cDNA which has been reverse-transcribed from an mRNA population of interest. It is well within the skill of the person of skill in the art to design methodology directed to screening for either DNA or RNA. As detailed above, in relation to the third aspect of the present invention, the method of the present invention also extends to screening for the protein product translated from the subject mRNA.

The term "protein" should be understood to encompass peptides, polypeptides and proteins (including protein fragments). The protein may be glycosylated or unglycosylated and/or may contain a range of other molecules fused, linked, bound or otherwise associated to the protein such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.

Reference herein to a "protein" includes a protein comprising a sequence of amino acids as well as a protein associated with other molecules such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.

The proteins encoded by the genes of the present invention may be in multimeric form meaning that two or more molecules are associated together. Where the same protein molecules are associated together, the complex is a homomultimer. An example of a homomultimer is a homodimer. Where at least one marker protein is associated with at least one non-marker protein, then the complex is a heteromultimer such as a heterodimer.

Means of testing for the subject expressed genes in a biological sample can be achieved by any suitable method, which would be well known to the person of skill in the art, such as but not limited to:

(i) In vivo detection. Molecular Imaging may be used following administration of imaging probes or reagents capable of disclosing altered gene expression. Molecular imaging (Moore et al., BBA, 1402:239-249, 1988; Weissleder et al, Nature Medicine 6:351-355, 2000) is the in vivo imaging of molecular expression. (ii) Detection of up-regulation of RNA expression in the cells by Fluorescent In Situ

Hybridization (FISH), or in extracts from the cells by technologies such as Quantitative Reverse Transcriptase Polymerase Chain Reaction (QRTPCR) or Flow cytometric qualification of competitive RT-PCR products (Wedemeyer et al., Clinical Chemistry 48:9 1398-1405, 2002).

(iii) Assessment of expression profiles of RNA, for example by array technologies (Alon et al, Proc. Natl. Acad. ScL USA: 96, 6745-6750, June 1999).

A "microarray" is a linear or multi-dimensional array of preferably discrete regions, each having a defined area, formed on the surface of a solid support. The density of the discrete regions on a microarray is determined by the total numbers of target

polynucleotides to be detected on the surface of a single solid phase support. As used herein, a DNA microarray is an array of oligonucleotide probes placed onto a chip or other surfaces used to detect complementary oligonucleotides from a complex nucleic acid mixture. Since the position of each particular group of probes in the array is known, the identities of the target polynucleotides can be determined based on their binding to a particular position in the microarray.

Recent developments in DNA microarray technology make it possible to conduct a large scale assay of a plurality of target nucleic acid molecules on a single solid phase support.

U.S. Pat. No. 5,837,832 (Chee et al.) and related patent applications describe

immobilizing an array of oligonucleotide probes for hybridization and detection of specific nucleic acid sequences in a sample. Target polynucleotides of interest isolated from a tissue of interest are hybridized to the DNA chip and the specific sequences detected based on the target polynucleotides' preference and degree of hybridization at discrete probe locations. One important use of arrays is in the analysis of differential gene expression, where the profile of expression of genes in different cells or tissues, often a tissue of interest and a control tissue, is compared and any differences in gene expression among the respective tissues are identified. Such information is useful for the identification of the types of genes expressed in a particular tissue type and diagnosis of conditions based on the expression profile.

In one example, RNA from the sample of interest is subjected to reverse transcription to obtain labelled cDNA. See U.S. Pat. No. 6,410,229 (Lockhart et al.) The cDNA is then hybridized to oligonucleotides or cDNAs of known sequence arrayed on a chip or other surface in a known order. In another example, the RNA is isolated from a biological sample and hybridised to a chip on which are anchored cDNA probes. The location of the oligonucleotide to which the labelled cDNA hybridizes provides sequence information on the cDNA, while the amount of labelled hybridized RNA or cDNA provides an estimate of the relative representation of the RNA or cDNA of interest. See

Schena, et al. Science 270:467-470 (1995).

In a preferred embodiment, nucleic acid probes corresponding to the subject nucleic acids are made. The nucleic acid probes attached to the microarray are designed to be substantially complementary to the nucleic acids of the biological sample such that specific hybridization of the target sequence and the probes of the present invention occurs. This complementarity need not be perfect, in that there may be any number of base pair mismatches that will interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention. It is expected that the overall homology of the genes at the nucleotide level probably will be about 40% or greater, probably about 60% or greater, and even more probably about 80% or greater; and in addition that there will be corresponding contiguous sequences of about 8-12 nucleotides or longer. However, if the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence. Thus, by "substantially

complementary" herein is meant that the probes are sufficiently complementary to the target sequences to hybridize under normal reaction conditions, particularly high stringency conditions. A nucleic acid probe is generally single stranded but can be partly single and partly double stranded. The strandedness of the probe is dictated by the structure, composition, and properties of the target sequence. In general, the oligonucleotide probes range from about 6, 8, 10, 12, 15, 20, 30 to about 100 bases long, with from about 10 to about 80 bases being preferred, and from about 15 to about 40 bases being particularly preferred. That is, generally entire genes are rarely used as probes. In some embodiments, much longer nucleic acids can be used, up to hundreds of bases. The probes are sufficiently specific to hybridize to a complementary template sequence under conditions known by those of skill in the art. The number of mismatches between the probe's sequences and their complementary template (target) sequences to which they hybridize during hybridization generally do not exceed 15%, usually do not exceed 10% and preferably do not exceed 5%, as-determined by BLAST (default settings). Oligonucleotide probes can include the naturally-occurring heterocyclic bases normally found in nucleic acids (uracil, cytosine, thymine, adenine and guanine), as well as modified bases and base analogues. Any modified base or base analogue compatible with hybridization of the probe to a target sequence is useful in the practice of the invention. The sugar or glycoside portion of the probe can comprise deoxyribose, ribose, and/or modified forms of these sugars, such as, for example, 2'-O-alkyl ribose. In a preferred embodiment, the sugar moiety is 2'-deoxyribose; however, any sugar moiety that is compatible with the ability of the probe to hybridize to a target sequence can be used. In one embodiment, the nucleoside units of the probe are linked by a phosphodiester backbone, as is well known in the art. In additional embodiments, internucleotide linkages can include any linkage known to one of skill in the art that is compatible with specific hybridization of the probe including, but not limited to phosphorothioate, methylphosphonate, sulfamate (e.g., U.S. Pat. No. 5,470,967) and polyamide (i.e., peptide nucleic acids). Peptide nucleic acids are described in Nielsen et al. (1991)

Science 254: 1497-1500, U.S. Pat. No. 5,714,331 , and Nielsen (1999) Curr. Opin.

Biotechnol. 10:71-75.

In certain embodiments, the probe can be a chimeric molecule; i.e., can comprise more than one type of base or sugar subunit, and/or the linkages can be of more than one type within the same primer. The probe can comprise a moiety to facilitate hybridization to its target sequence, as are known in the art, for example, intercalators and/or minor groove binders. Variations of the bases, sugars, and internucleoside backbone, as well as the presence of any pendant group on the probe, will be compatible with the ability of the probe to bind, in a sequence-specific fashion, with its target sequence. A large number of structural modifications, are possible within these bounds. Advantageously, the probes according to the present invention may have structural characteristics such that they allow the signal amplification, such structural characteristics being, for example, branched DNA probes as those described by Urdea et al. {Nucleic Acids Symp.

Ser., 24:197-200 (1991)) or in the European Patent No. EP-0225,807. Moreover, synthetic methods for preparing the various heterocyclic bases, sugars, nucleosides and nucleotides that form the probe, and preparation of oligonucleotides of specific predetermined sequence, are well-developed and known in the art. A preferred method for oligonucleotide synthesis incorporates the teaching of U.S. Pat. No. 5,419,966.

Multiple probes may be designed for a particular target nucleic acid to account for polymorphism and/or secondary structure in the target nucleic acid, redundancy of data and the like. In some embodiments, where more than one probe per sequence is used, either overlapping probes or probes to different sections of a single target gene are used.

That is, two, three, four or more probes, are used to build in a redundancy for a particular target. The probes can be overlapping (i.e. have some sequence in common), or are specific for distinct sequences of a gene. When multiple target polynucleotides are to be detected according to the present invention, each probe or probe group corresponding to a particular target polynucleotide is situated in a discrete area of the microarray.

Probes may be in solution, such as in wells or on the surface of a micro-array, or attached to a solid support. Examples of solid support materials that can be used include a plastic, a ceramic, a metal, a resin, a gel and a membrane. Useful types of solid supports include plates, beads, magnetic material, microbeads, hybridization chips, membranes, crystals, ceramics and self-assembling monolayers. One example comprises a two-dimensional or three-dimensional matrix, such as a gel or hybridization chip with multiple probe binding sites (Pevzner et al., J. Biomol. Struc. & Dyn. 9:399- 410, 1991; Maskos and Southern, Nuc. Acids Res. 20:1679-84, 1992). Hybridization chips can be used to construct very large probe arrays that are subsequently hybridized with a target nucleic acid. Analysis of the hybridization pattern of the chip can assist in the identification of the target nucleotide sequence. Patterns can be manually or computer analyzed, but it is clear that positional sequencing by hybridization lends itself to computer analysis and automation. In another example, one may use an Affymetrix chip on a solid phase structural support in combination with a fluorescent bead based approach. In yet another example, one may utilise a cDNA microarray. As will be appreciated by those in the art, nucleic acids can be attached or immobilized to a solid support in a wide variety of ways. By "immobilized" herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal. The binding can be covalent or non-covalent. By "non-covalent binding" and grammatical equivalents herein is meant one or more of either electrostatic, hydrophilic, and hydrophobic interactions. Included in non-covalent binding is the covalent attachment of a molecule, such as streptavidin, to the support and the non-covalent binding of the biotinylated probe to the streptavidin. By "covalent binding" and grammatical equivalents herein is meant that the two moieties, the solid support and the probe, are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds.

Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules. Immobilization may also involve a combination of covalent and non-covalent interactions.

Nucleic acid probes may be attached to the solid support by covalent binding such as by conjugation with a coupling agent or by covalent or non-covalent binding such as electrostatic interactions, hydrogen bonds or antibody-antigen coupling, or by combinations thereof. Typical coupling agents include biotin/avidin, biotin/streptavidin, Staphylococcus aureus protein A/IgG antibody F_c fragment, and streptavid in/protein A chimeras (T. Sano and C. R. Cantor, Bio/Technology 9:1378-81 (1991)), or derivatives or combinations of these agents. Nucleic acids may be attached to the solid support by a photocleavable bond, an electrostatic bond, a disulfide bond, a peptide bond, a diester bond or a combination of these sorts of bonds. The array may also be attached to the solid support by a selectively releasable bond such as 4,4'-dimethoxytrityl or its derivative. Derivatives which have been found to be useful include 3 or 4 [bis-(4- methoxyphenyl)]-methyl-benzoic acid, N-succinimidyl-3 or 4 [bis-(4-methoxyphenyl)]- methyl-benzoic acid, N-succinimidyl-3 or 4 [bis-(4-methoxyphenyl)]-hydroxymethyl- benzoic acid, N-succinimidyl-3 or 4 [bis-(4-methoxyphenyl)]-chloromethyl-benzoic acid, and salts of these acids.

In general, the probes are attached to the microarray in a wide variety of ways, as will be appreciated by those in the art. As described herein, the nucleic acids can either be synthesized first, with subsequent attachment to the microarray, or can be directly synthesized on the microarray.

The microarray comprises a suitable solid substrate. By "substrate" or "solid support" or other grammatical equivalents herein is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method. The solid phase support of the present invention can be of any solid materials and structures suitable for supporting nucleotide hybridization and synthesis. Preferably, the solid phase support comprises at least one substantially rigid surface on which the primers can be immobilized and the reverse transcriptase reaction performed. The substrates with which the polynucleotide microarray elements are stably associated and may be fabricated from a variety of materials, including plastics, ceramics, metals, acrylamide, cellulose, nitrocellulose, glass, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicates, polycarbonates,

Teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans, and polyamino acids. Substrates may be two-dimensional or three-dimensional in form, such as gels, membranes, thin films, glasses, plates, cylinders, beads, magnetic beads, optical fibers, woven fibers, etc.

In one embodiment, the surface of the microarray and the probe may be derivatized with chemical functional groups for subsequent attachment of the two. Thus, for example, the microarray is derivatized with a chemical functional group including, but not limited to, amino groups, carboxy groups, oxo groups and thiol groups, with amino groups being particularly preferred. Using these functional groups, the probes can be attached using functional groups on the probes. For example, nucleic acids containing amino groups can be attached to surfaces comprising amino groups, for example using linkers as are known in the art; for example, homo-or hetero-bifunctional linkers as are well known. In addition, in some cases, additional linkers, such as alkyl groups (including substituted and heteroalkyl groups) may be used.

In this embodiment, the oligonucleotides are synthesized as is known in the art, and then attached to the surface of the solid support. As will be appreciated by those skilled in the art, either the 5' or 3' terminus may be attached to the solid support, or attachment may be via an internal nucleoside. In an additional embodiment, the immobilization to the solid support may be very strong, yet non-covalent. For example, biotinylated oligonucleotides can be made, which bind to surfaces covalently coated with

streptavidin, resulting in attachment.

The arrays may be produced according to any convenient methodology, such as preforming the polynucleotide microarray elements and then stably associating them with the surface. Alternatively, the oligonucleotides may be synthesized on the surface, as is known in the art. A number of different array configurations and methods for their production are known to those of skill in the art and disclosed in WO 95/251 16 and WO 95/35505 (photolithographic techniques), U.S. Pat. No. 5,445,934 (in situ synthesis by photolithography), U.S. Pat. No. 5,384,261 (in situ synthesis by mechanically directed flow paths); and U.S. Pat. No. 5,700,637 (synthesis by spotting, printing or coupling); the disclosure of which are herein incorporated in their entirety by reference. Another method for coupling DNA to beads uses specific ligands attached to the end of the DNA to link to ligand-binding molecules attached to a bead. Possible ligand-binding partner pairs include biotin-avidin/streptavidin, or various antibody/antigen pairs such as digoxygenin-antidigoxygenin antibody (Smith et al., Science 258:1122-1 126 (1992)). Covalent chemical attachment of DNA to the support can be accomplished by using standard coupling agents to link the 5'-phosphate on the DNA to coated microspheres through a phosphoamidate bond. Methods for immobilization of oligonucleotides to solid-state substrates are well established. See Pease et al., Proc. Natl. Acad. Sci. USA 91(11):5022-5026 (1994). A preferred method of attaching oligonucleotides to solid- state substrates is described by Guo et al., Nucleic Acids Res. 22:5456-5465 (1994). Immobilization can be accomplished either by in situ DNA synthesis (Maskos and Southern, supra) or by covalent attachment of chemically synthesized oligonucleotides (Guo et al., supra) in combination with robotic arraying technologies.

In addition to the solid-phase technology represented by microarray arrays, gene expression can also be quantified using liquid-phase assays. One such system is kinetic polymerase chain reaction (PCR). Kinetic PCR allows for the simultaneous

amplification and quantification of specific nucleic acid sequences. The specificity is derived from synthetic oligonucleotide primers designed to preferentially adhere to single-stranded nucleic acid sequences bracketing the target site. This pair of oligonucleotide primers form specific, non-covalently bound complexes on each strand of the target sequence. These complexes facilitate in vitro transcription of double- stranded DNA in opposite orientations. Temperature cycling of the reaction mixture creates a continuous cycle of primer binding, transcription, and re-melting of the nucleic acid to individual strands. The result is an exponential increase of the target dsDNA product. This product can be quantified in real time either through the use of an intercalating dye or a sequence specific probe. SYBR(r) Green 1, is an example of an intercalating dye, that preferentially binds to dsDNA resulting in a concomitant increase in the fluorescent signal. Sequence specific probes, such as used with TaqMan technology, consist of a fluorochrome and a quenching molecule covalently bound to opposite ends of an oligonucleotide. The probe is designed to selectively bind the target DNA sequence between the two primers. When the DNA strands are synthesized during the PCR reaction, the fluorochrome is cleaved from the probe by the exonuclease activity of the polymerase resulting in signal dequenching. The probe signalling method can be more specific than the intercalating dye method, but in each case, signal strength is proportional to the dsDNA product produced. Each type of quantification method can be used in multi-well liquid phase arrays with each well representing primers and/or probes specific to nucleic acid sequences of interest. When used with messenger RNA preparations of tissues or cell lines, an array of probe/primer reactions can

simultaneously quantify the expression of multiple gene products of interest. See Germer et ai, Genome Res. 10:258-266 (2000); Heid et al, Genome Res. 6:986-994 (1996). (iv) Measurement of altered protein levels in cell extracts, for example by immunoassay.

Testing for protein expression product in a biological sample can be performed by any one of a number of suitable methods which are well known to those skilled in the art.

Examples of suitable methods include, but are not limited to, antibody screening of tissue sections, biopsy specimens or bodily fluid samples.

To the extent that antibody based methods of diagnosis are used, the presence of the marker protein may be determined in a number of ways such as by Western blotting,

ELISA or flow cytometry procedures. These, of course, include both single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.

Sandwich assays are among the most useful and commonly used assays. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample. Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent.

In the typical forward sandwich assay, a first antibody having specificity for the marker or antigenic parts thereof, is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking, covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample.

An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes) and under suitable conditions (e.g. 25⁰C) to allow binding of any subunit present in the antibody.

Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the antigen. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the antigen.

An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody. Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.

By "reporter molecule" as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.

The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and

chemiluminescent molecules. In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of antigen which was present in the sample. "Reporter molecule" also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like. Alternately, fluorescent compounds, such as fluorecein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome- labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope. As in the EIA, the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest.

Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.

(v) Determining altered expression of proteins on the cell surface, for example by

immunohistochemistry.

(vi) Determining altered protein expression based on any suitable functional test, enzymatic test or immunological test in addition to those detailed in points (iv) and (v) above. Without limiting the present invention in any way, and as detailed above, gene expression levels can be measured by a variety of methods known in the art. For example, gene transcription or translation products can be measured. Gene transcription products, i.e., RNA, can be measured, for example, by hybridization assays, run-off assays., Northern blots, or other methods known in the art.

Hybridization assays generally involve the use of oligonucleotide probes that hybridize to the single-stranded RNA transcription products. Thus, the oligonucleotide probes are

complementary to the transcribed RNA expression product. Typically, a sequence-specific probe can be directed to hybridize to RNA or cDNA. A "nucleic acid probe", as used herein, can be a DNA probe or an RNA probe that hybridizes to a complementary sequence. One of skill in the art would know how to design such a probe such that sequence specific

hybridization will occur. One of skill in the art will further know how to quantify the amount of sequence specific hybridization as a measure of the amount of gene expression for the gene was transcribed to produce the specific RNA.

The hybridization sample is maintained under conditions that are sufficient to allow specific hybridization of the nucleic acid probe to a specific gene expression product. "Specific hybridization", as used herein, indicates near exact hybridization (e.g., with few if any mismatches). Specific hybridization can be performed under high stringency conditions or moderate stringency conditions. In one embodiment, the hybridization conditions for specific hybridization are high stringency. For example, certain high stringency conditions can be used to distinguish perfectly complementary nucleic acids from those of less complementarity. "High stringency conditions", "moderate stringency conditions" and "low stringency conditions" for nucleic acid hybridizations are explained on pages 2.10.1-2.10.16 and pages 6.3.1-6.3.6 in Current Protocols in Molecular Biology (Ausubel, F. et ai, "Current Protocols in Molecular Biology", John Wiley & Sons, (1998), the entire teachings of which are

incorporated by reference herein). The exact conditions that determine the stringency of hybridization depend not only on ionic strength (e.g., 0.2.times.SSC, O.l .times.SSC), temperature (e.g., room temperature, 42⁰C, 68⁰C.) and the concentration of destabilizing agents such as formamide or denaturing agents such as SDS, but also on factors such as the length of the nucleic acid sequence, base composition, percent mismatch between hybridizing sequences and the frequency of occurrence of subsets of that sequence within other non- identical sequences. Thus, equivalent conditions can be determined by varying one or more of these parameters while maintaining a similar degree of identity or similarity between the two nucleic acid molecules. Typically, conditions are used such that sequences at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 95% or more identical to each other remain hybridized to one another. By varying hybridization conditions from a level of stringency at which no hybridization occurs to a level at which hybridization is first observed, conditions that will allow a given sequence to hybridize (e.g., selectively) with the most complementary sequences in the sample can be determined. Exemplary conditions that describe the determination of wash conditions for moderate or low stringency conditions are described in Kraus, M. and Aaronson, S., 1991. Methods Enzymol., 200:546-556; and in, Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, (1998). Washing is the step in which conditions are usually set so as to determine a minimum level of complementarity of the hybrids. Generally, starting from the lowest temperature at which only homologous hybridization occurs, each ⁰C. by which the final wash temperature is reduced (holding SSC concentration constant) allows an increase by 1% in the maximum mismatch percentage among the sequences that hybridize. Generally, doubling the concentration of SSC results in an increase in T_n, of about 17⁰C. Using these guidelines, the wash temperature can be determined empirically for high, moderate or low stringency, depending on the level of mismatch sought. For example, a low stringency wash can comprise washing in a solution containing 0.2.times.SSC/0.1% SDS for 10 minutes at room temperature; a moderate stringency wash can comprise washing in a pre-warmed solution (42⁰C) solution containing 0.2.times.SSC/0.1% SDS for 15 minutes at 42⁰C; and a high stringency wash can comprise washing in pre-warmed (68⁰C.) solution containing 0.1. times. SSC/0.1% SDS for 15 minutes at 68⁰C. Furthermore, washes can be performed repeatedly or sequentially to obtain a desired result as known in the art. Equivalent conditions can be determined by varying one or more of the parameters given as an example, as known in the art, while maintaining a similar degree of complementarity between the target nucleic acid molecule and the primer or probe used (e.g., the sequence to be hybridized).

A related aspect of the present invention provides a molecular array, which array comprises a plurality of:

(i) nucleic acid molecules comprising a nucleotide sequence corresponding to any one or more of the genes hereinbefore described or a sequence exhibiting at least 80% identity thereto or a functional derivative, fragment, variant or homologue of said nucleic acid molecule; or (ii) nucleic acid molecules comprising a nucleotide sequence capable of hybridising to any one or more of the sequences of (i) under medium stringency conditions or a functional derivative, fragment, variant or homologue of said nucleic acid molecule; or

(iii) nucleic acid probes or oligonucleotides comprising a nucleotide sequence capable of hybridising to any one or more of the sequences of (i) under medium stringency conditions or a functional derivative, fragment, variant or homologue of said nucleic acid molecule; or

(iv) probes capable of binding to any one or more of the proteins encoded by the nucleic acid molecules of (i) or a derivative, fragment or, homologue thereof.

Preferably, said percent identity is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Low stringency includes and encompasses from at least about 1% v/v to at least about 15% v/v formamide and from at least about IM to at least about 2M salt for hybridisation, and at least about IM to at least about 2M salt for washing conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.0 IM to at least about 0.15M salt for hybridisation, and at least about 0.0 IM to at least about 0.15M salt for washing conditions. In general, washing is carried out at T_m = 69.3 + 0.41 (G + C) % [19] = -12°C. However, the T_m of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatched based pairs (Bonner et al (1973) J. MoI. Biol. 81 :123).

Preferably, the subject probes are designed to bind to the nucleic acid or protein to which they are directed with a level of specificity which minimises the incidence of non-specific reactivity. However, it would be appreciated that it may not be possible to eliminate all potential cross-reactivity or non-specific reactivity, this being an inherent limitation of any probe based system.

In terms of the probes which are used to detect the subject proteins, they may take any suitable form including antibodies and aptamers.

A library or array of nucleic acid or protein probes provides rich and highly valuable information. Further, two or more arrays or profiles (information obtained from use of an array) of such sequences are useful tools for comparing a test set of results with a reference, such as another sample or stored calibrator. In using an array, individual probes typically are immobilized at separate locations and allowed to react for binding reactions. Primers associated with assembled sets of markers are useful for either preparing libraries of sequences or directly detecting markers from other biological samples. A library (or array, when referring to physically separated nucleic acids corresponding to at least some sequences in a library) of gene markers exhibits highly desirable properties. These properties are associated with specific conditions, and may be characterized as regulatory profiles. A profile, as termed here refers to a set of members that provides diagnostic information of the tissue from which the markers were originally derived. A profile in many instances comprises a series of spots on an array made from deposited sequences.

A characteristic patient profile is generally prepared by use of an array. An array profile may be compared with one or more other array profiles or other reference profiles. The comparative results can provide rich information pertaining to disease states, developmental state, receptiveness to therapy and other information about the patient.

Another aspect of the present invention provides a diagnostic kit for assaying biological samples comprising an agent for detecting one or more genes as hereinbefore defined and reagents useful for facilitating the detection by said agent. Further means may also be included, for example, to receive a biological sample. The agent may be any suitable detecting molecule.

The present invention is further described by the following non-limiting examples: EXAMPLE 1 Materials and Methods Cell lines and culture

LCLs were derived from patient blood as described (Severin et al., 2001). The radiosensitivity selection was exclusively based on Radiation Therapy Oncology Group (RTOG) grades of 3 or above. Six 'late' reactors and 6 controls (those individuals that show normal radiosensitivity) were analysed (Leong et al., 2000; Severin et al., 2001). LCLs were grown in RPMI media supplemented with 10% FBS and 20ug/ml gentamicin and incubated in a 5% CO₂ humidified incubator. All patients have given written informed consent and studies have been approved by the Peter MacCallum Cancer Centre Ethics Committee and Monash University Ethics

Committee.

RNA Isolation

Cells were grown to a density of 1 x 10⁷, pelleted, resuspended in 3 ml PBS and an equal volume of Trizol (Invitrogen, Carlsbad, CA, USA) was added, mixed and the aqueous layer was mixed with and equal volume of 70 percent ethanol and added onto a RNeasy column (Qiagen, Venlo, The Netherlands). The RNA extraction was continued by using the RNAeasy method as per manufactures recommendation except starting with the addition of the sample of Buffer RWl . RNA concentration and integrity was determined by analysing on a bioanalyzer 6000 Nano Labchip as per the manufacturer's recommendations (Agilent, Santa Clara, CA, USA). RNA was determined to be of high enough quality if a minimum RIN of 8.5 was obtained.

Exon arrays GeneChip Human Exon 1.0 ST Array analysis was performed as per the 'GeneChip Whole Transcript (WT) Sense Target labelling assay Manual' (Affymetrix, Santa Clara, CA, USA). The rRNA from lug of total RNA was reduced using a RiboMinus Human/Mouse

Transcriptome Isolation Kit (Invitrogen, Carlsbad, CA, USA). Exon array analysis

For this investigation the 'core set' that is defined by over 200,000 probe set regions

(Affymetrix.com) was analysed. Assessment of array quality was determined using Expression Console (Affymetrix.com). Gene expression was assessed using R, normalized using RMA and analysed using Significance analysis of microarrays (SAM; (Tusher et al., 2001).

Alternative splicing was determined using Alternative Splicing ANOVA from Partek analysis package (Partek, St Louis, MO, USA). Transcriptional validation

Primers were designed to candidate exons or genes using 'Primer 3' program (Co). Normal PCR amplification was carried out using 1.25 Units Go Taq polymerase (Promega, UK), 200 nM primers, 500 ng cDNA, with a cycling protocol of 95⁰C: 2'; ((95⁰C: 45 sec; 6O⁰C: 60 sec; 72⁰C: 45 sec) x 30); 72⁰C: 5 min. Products were run on a 4 percent agarose gel or a 7 percent polyacrylamide gel to determine amplification of the proper sized product. Real-time PCR was performed using these primers under the following conditions. Sybr Green Master Mix (Applied Biosystems, Wisconsin, USA) was mixed with 100 ng of total RNA. The cycling steps were as follows. 95⁰C: 2'; ((95⁰C: 45 sec; 6O⁰C: 60 sec; 72⁰C: 45 sec) x 30); 72⁰C: 5 min with a melting curve step following. Quantitation was performed using PGK and /or GAPDH as normalization controls.

Results Radiation Response

Processed RNA from six different lymphoblast cell lines that were exposed to either 10 Gy of radiation or no treatment were run on Affymetrix exon arrays to examine the transcriptional profile in response to radiation. The exon array platform has enabled the comprehensive characterisation of alternative transcripts following IR. 1 ,674 genes have been identified that have an alternative splicing signature in response to IR showing statistical significance with a p-value of less than 0.1 using RMA normalization and Partek's ANOVA alternative splicing algorithm. All known exons are covered by probes on these arrays, thus enabling the determination of an alternative splicing profile on a whole genome level. Genes previously identified to be responsive to IR as obtained from the literature were confirmed in the lymphoblast cell lines.

Validation of individual gene radiation response

The expression levels obtained from the exon array data were validated for many of the candidate genes. PCR was performed on the cDNA derived from the transcriptional products and the amplified products were analysed on a polyacrylamide gel. The relative amounts were calculated using a densitometer with normalization to expression of the housekeeping gene, PGK. Examples of results were plotted for IGFBP4, PLK2 and SESN2 (Figures 1 -3).

Furthermore, real-time quantitative PCR was also used to validate transcript level changes for XPC, POLH, CDKNlA, FBXO22, CENPE, KIF 14, CENPA and ASPM in lymphoblast cell lines (Figure 4A and 4B) and for genes CDKNlA, POLH and DDB2 in fibroblasts (Figure 4C). This validation effort using PCR indicated that the exon array platform was yielding very consistent and reliable results.

Individual gene radiation AS response

Alternative splicing can be determined from probe sets that have a discordance in the amount of expression modulation following IR treatment compared to the majority of the probe sets (Partek Genomic Suite). This can be graphed as a function of probe set region average expression levels. CDKNlA, a p53 responsive gene was induced greater than two fold four hours after 10 Gy of IR and appeared to have at least two splice products indicating a lesser amount of the full length RNA isoform (Figure 5). Examples of other genes also found to show expression patterns suggestive of alternative splicing included PLK2 and ATF3 (Figures 6 and 7). Furthermore, additional examples of alternative transcript modulation following 10 Gy IR was observed in genes in lymphoblast cell lines (Figures 8 and 9) and fibroblast cell lines (Figures 10 and 1 1). Note that a large amount of the expression variation can be attributed to probe-specific effects, thus, of higher importance to determine alternative splicing, is to track the difference between treatments rather than between probe sets, although these can be important in some cases.

Validation of individual gene radiation AS response Quantitative real time PCR and conventional PCR was carried out to validate the levels of transcriptional expression in genes predicted to have alternative splice (AS) products as indicated from the exon array data. A number of genes were validated with varied inductions between exonic regions indicating the induction of AS after irradiation in lymphoblast cell lines (Figures 4D, 4E and 12H) and fibroblast cell lines (Figure 4F, 4G, 4H and 12J).

A survey of the number of AS predictions that involve the 5' and 3' ends were determined. It was found that there was a strong bias towards AS occurring at the terminus of a gene, typically in the untranslated regions which potentially could be regulatory. There may be a global mechanism to co-ordinately regulate the formation of AS products, perhaps at the polymerase level as a previous study has found using UV radiation (Munoz et al. 2009 Cell 137 p708-720), a splicesome component, or other global regulator protein or RNA.

Radiosensitivity Response Whole genome gene expression was determined in lymphoblast cell lines derived from radiosensitive individuals using whole transcript exon array analysis. The transcriptional response to radiation in these cell lines from radiosensitive individuals was compared to controls including a comprehensive survey of alternative splicing. Patterns of alternative splicing were found that suggested genes that would enable the differentiation of clinically RS individuals from controls. These same cell lines were also irradiated with 10 Gy of IR and found a differential in the transcriptional signal between the radiosensitive and the control cell lines. Lists of genes using the alternative splice ANOVA (Partek Genomics Suite) were generated showing the top candidate genes with p-values less than 0.1 for both basal and 10Gy treatment. 936 genes were found to have a difference in alternative splicing between radiosensitive and non-radiosensitive cell lines at basal levels and have a p-value of less than 0.1. 4,569 were found genes to have a difference in alternative splicing between radiosensitive and non-radiosensitive cell lines after treatment with 10 Gy of ionizing radiation and have a p- value of less than 0.1. The top candidate genes show very clear expression profiles which can separate the radiosensitive samples from the non-radiosensitive samples (Figures 13-16). These results suggest that radiosensitivity can be predicted prior to or following irradiation of cell lines derived from radiosensitive patients.

Individual gene radiosensitivity AS response Alternative splicing can be determined from probe sets that have a discordance in the amount of expression modulation following IR treatment compared to the majority of the probe sets and this can be graphed as a function of probe set region average expression levels. Adverse Radiation Toxicity

1. Patients who are susceptible to adverse radiation toxicity exhibit a characteristic

cellular gene expression profile, prior to exposure to radiation, which differs to that expressed by individuals who are not susceptible

(i) alternatively spliced genes which are up-regulated in patients who are susceptible to adverse radiation toxicity

Tables IA and IB: Alternatively spliced genes which are up or down regulated in lymphoblast cell lines derived from radiosensitive patients (12 cell lines) verses control patients (12 cell lines) as indicated (RS v CL). The gene names are the HUGO gene nomenclature approved symbols and the gene identification is the RefSeq ID number. The p-value is based on AS ANOVA (Partek Genomic Suite statistical package).

Table IA

(ϋ) alternatively spliced genes which are down-regulated in patients who are susceptible to adverse radiation toxicity

Table IB

(iii) alternatively spliced genes which are up-regulated in the fibroblasts of patients who are susceptible to adverse radiation toxicity

Tables 2A and 2B: Alternatively spliced genes which are up or down regulated in fibroblasts from radiosensitive patients (12 cell lines) verses control patients (12 cell lines) as indicated (RS v CL). The gene names are the HUGO gene nomenclature approved symbols and the gene identification is the RefSeq ID number. The p-value is based on AS ANOVA (Partek Genomic Suite statistical package).

Table 2A

(iv) alternatively spliced genes which are down-refiulated in the fibroblasts of patients who are susceptible to adverse radiation toxicity

Table 2B

2. Patients who are susceptible to adverse radiation toxicity exhibit a characteristic

cellular gene expression profile, after exposure to ionizing radiation, which differs to that expressed by individuals who are not susceptible.

Tables 3A and 3B: Alternatively spliced genes which are up or down regulated in lymphoblast cell lines derived from radiosensitive patients (12 cell lines) verses control patients (12 cell lines) 4 hours after irradiation with 10 Gy as indicated (RS v CL). The gene names are the HUGO gene nomenclature approved symbols and the gene identification is the RefSeq ID number. The p-value is based on AS ANOVA (Partek Genomic Suite statistical package).

Table 3A

Table 3B

(iii) alternatively spliced genes which are up-regulated in the fibroblasts of patients who are susceptible to adverse radiation toxicity Tables 4A and 4B: Alternatively spliced genes which are up or down regulated in fibroblasts from radiosensitive patients verses (12 cell lines) control patients (12 cell lines) 4 hours after irradiation with 10 Gy as indicated (RS v CL). The gene names are the HUGO gene nomenclature approved symbols and the gene identification is the RefSeq ID number. The p-value is based on AS ANOVA (Partek Genomic Suite statistical package).

Table 4A

(iv) alternatively spliced genes which are down-regulated in the fibroblasts of patients who are susceptible to adverse radiation toxicity

Table 4B

Radiation Exposure

3. Patients who have been exposed to radiation exhibit a unique gene expression profile which differs to that expressed by individuals who have not been exposed to radiation.

(i) genes which are up-regulated in terms of their level of expression in patients who have been exposed to radiation

Tables 5A and 5B: Genes which are up or down regulated in lymphoblast cell lines (24 cell lines) 4 hours after irradiation with 10 Gy as indicated (10 v 0 Gy). The gene names are the HUGO gene nomenclature approved symbols and the gene identification is the RefSeq ID number. The p-value is based on ANOVA (Partek Genomic Suite statistical package).

Table 5A

(ii) genes which are down-regulated in terms of their level of expression in patients who have been exposed to radiation

Table 5B

(iii) alternatively spliced genes which are up-regulated in patients who have been exposed to radiation

Tables 6A and 6B: Alternatively spliced genes which are up or down regulated in lymphoblast cell lines (24 cell lines) 4 hours after irradiation with 10 Gy as indicated (post-IR). The gene names are the HUGO gene nomenclature approved symbols and the gene identification is the RefSeq ID number. The p-value is based on AS ANOVA (Partek Genomic Suite statistical package).

Table 6A

(iv) alternatively spliced genes which are down-regulated in patients who have been

exposed to radiation

Table 6B

(v) genes which are increased in terms of their level of expression in fibroblasts in patients who have been exposed to radiation

Tables 7A and 7B: Genes which are up or down regulated in fibroblasts (24 cell lines) 4 hours after irradiation with 10 Gy as indicated (10 v 0 Gy). The gene names are the HUGO gene nomenclature approved symbols and the gene identification is the RefSeq ID number. The p-value is based on ANOVA (Partek Genomic Suite statistical package).

Table 7A

(vi) genes which are decreased in terms of their level of expression in fibroblasts in patients who have been exposed to radiation

Table 7B

(vii) alternatively spliced genes which are up-regulated in fibroblasts in patients who have been exposed to radiation

Tables 8A and 8B: Alternatively spliced genes which are up or down regulated in fibroblasts (24 cell lines) 4 hours after irradiation with 10 Gy as indicated (10 v 0 Gy). The gene names are the HUGO gene nomenclature approved symbols and the gene identification is the RefSeq ID number. The p-value is based on AS ANOVA (Partek Genomic Suite statistical package).

Table 8A

(viii) alternatively spliced genes which are down-reRulated in patients who have been exposed to radiation

Table 8B

EXAMPLE 2

IDENTIFICATION OF A NOVEL GLOBAL RADIATION RESPONSE INVOLVING USE OF ALTERNATIVE TRANSCRIPTION START SITES

A fundamental global transcriptional radiation response has been identified from the examination of transcripts generated in response to ionizing radiation at the exon level across the whole genome. Gene expression has been comprehensively tracked with a high degree of sensitivity, interrogating all known and predicted exons. 58% and 54% of the top 100 genes for lymphoblast cell lines (LCLs) and fibroblasts, respectively, suggested the utilization of an alternative transcription start site (ATSS) or be protected yielding different transcripts in response to ionizing radiation. Genes that are alternatively spliced following irradiation have been identified. An alternative splicing ANOVA algorithm developed by Partek Inc has been applied to determine alternatively spliced exons across the whole genome. ATSSs are also detected with the exon array format. Twelve lymphoblast cell lines (LCL) and twelve primary fibroblast cell lines were irradiated and the gene expression exon arrays were run. A high number of genes that showed ATSS usage at the 5' end of the transcript after treatment with 10 Gy of radiation were found. This indicates that the use of an ATSS is a common response and certainly is important in the regulation of gene expression in response to ionizing radiation. Specifically, over 50% of genes on the lists (from both LCLs (58%) and fibroblasts (54%) of the top 100 alternatively spliced genes based on ANOVA p-values), showed unusual responses at the 5' exon(s) and about half of these clearly showed alternative transcriptional start site usage, with the other half showing a protection of a short section of the transcript, relative to the rest of the transcript, again, this regulation predominantly in the 5' region of the transcripts. Examples of induction of transcripts that use ATSSs are shown in Figures 8-19. Furthermore, the change in expression levels and use of alternative transcriptional start sites for a number of genes using quantitative real-time PCR (qPCR; Figures 2, 12 and 18) has been validated. Pilot studies have been performed using additional exon arrays to examine the effect of dose on the use of ATSSs. A direct correlation was found of increased alternative spliced products and dose (Figures 12 and 18). Additionally, the effect of time on the use of alternative transcriptional start sites was examined and it was determined some genes showed increases in the alternative splice product while others showed different kinetics over time (Figures 12 and 18). Genes that show different response kinetics in different cell types were also found (Figure 12). There has been testing of all possible sequence motifs down to 5bp within lOObp upstream of the TSS of PI s for 20 ATSS genes in LCLs. Fisher's Exact Test was used to compute statistically significant differences of motifs presence at each location between the upregulated transcripts and the reference set of all human genes. A p-value of < 0.001 was considered significant in this pilot study. Several consensus motifs near the Pl promoters were identified, including one 5 base sequence that shows 95% presence in ATSS genes (n=20) and only 47% in all genes (Fisher's p-value=0.00001).

These multiple sets of data support the hypothesis that radiation induces a large set of genes that use a common mechanism of ATSS. This is a new paradigm in molecular radiation biology and has far reaching consequences for radiobiology especially in the medical and occupational radiation areas. This global radiation stress response is relevant to wide variety of stress responses. Different radiation type/modalities such as UV and microbeam radiotherapy respond similarly in that they use ATSSs. Use of ATSS as a principal mechanism for the global response to ionizing radiation. qPCR validation of genes identified by exon array analysis to utilize alternative transcriptional start sites in response to radiation qPCR is used to validate the use of ATSSs. The kinetics of the alternative transcriptional start site genes are validated by using qPCR at the same time points (0, 4hr, 8hr, 24hr and 48hr) and doses (0 Gy, 1 Gy, 2 Gy, 5 Gy, 10 Gy, 20 Gy). Examination of lower dose response is relevant to some types of medical, background and occupational exposures. Therefore, additional doses (0.1 Gy and 0.01 Gy) and time points (30', lhr, 2hr, 2day, 4day 7day) are investigated on selected genes using qPCR to obtain a more refined view of the radiation response. Expansion of time points to include both shorter and longer time points provides further insight about the kinetics of the radiation response for the ATSS genes. qPCR is routinely run in 384 well formats. Robotics is used to distribute samples accurately into the plate which enables performance of multiple plates per day if necessary and therefore it is possible to perform sufficient numbers of qPCR assays to test the many conditions.

Protein analysis in genes that have altered translational products in response to radiation. Radiation-induced ATSS can produce a truncated protein. Examples of this have been found in radiation induced transcripts, for example, the TNFSF9 gene ATSS is within the translated region (Figure 19). In this case functional domains are not present which can alter protein function. Sites for phoshphorylation and other protein modifications are also lost in TNFSF9 which could also have consequences for protein action. Protein assays (eg. western blots) will be run to verify the different isoforms for those proteins. Cell lines are transfected with plasmids that over-express either the long or short form of the protein. Radiosensitivity is measured after over-expression with clonogenic and DNA damage and repair assays

Specific radiation response as a function of cell type.

Cell type specificity is also an important factor in the spectrum of ATSS radio-responsive genes (Figures 7 and 12). Cell type specific responses to radiation are addressed by examining the radiation response using exon arrays in keratinocytes to provide the spectrum of ATSS genes in the cell line including those that differ from LCLs and fibroblasts. A variety of cell types including myocytes, endothelial cells and tumour cells are analysed for a selection of genes using qPCR to determine how and to what degree ATSS genes are modulated in these cell types.

DNA repair genes

Understanding the molecular mechanisms of DNA repair genes in response to DNA damaging agents such as ionizing radiation is important for radiotherapy. DNA repair genes are likely candidates for radiosensitivity. Therefore, we have investigated the DNA repair gene response to radiation to further understand the complex nature of the radiation response. We have comprehensively assessed DNA repair gene expression using the Affymetrix exon array platform where each gene is extensively covered with an average of 40 probes. Here, we have identified the extent of transcriptional regulation in the known DNA repair genes in response to IR exposure. We have found a radiation response in a high number of DNA repair genes (Figure 20). Altered response in any of these genes may predict radiosensitivity. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

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Germer et al, Genome Res. 10:258-266 (2000)

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Claims

CLAIMS:

1. A method of screening for susceptibility to adverse radiation toxicity in an individual, said method comprising identifying the mRNA splice variants expressed by one or more genes selected from:

(') ACTNl AKT3 BHLHB3 ClOorfl l

C14orfl05 Clorflόl CD28 CDCPl

CHLl CRTAP EOMES FGFRl

GPC4 GRPR HNFlB LY9

MERTK MOBKL2B MTTP PARD3

PTPRE RBPMS SERINC2 SGPP2

SHROOM3 SLC2A13 SSFA2 STEAP2

IQGAP2 RAPGEF5 TSPYL5 EPHBl

HNF4G

(ϋ) the genes identified by accession numbers:

NM 001 102 NM 181690 NM 030762 NM 032024

NM 018168 BC128148 NM 006139 NM 022842

NM 006614 NM 006371 NM 005442 NM 0231 10

NM 001448 NM 005314 NM 000458 NM 002348

NM 006343 NM 024761 NM 000253 NM 019619

NM 006504 NM 001008711 NM 178865 NM 152386

NM 020859 NM 052885 NM 006751 NM 152999

NM 006633 NM_012294 NM_033512 NM_004441

NM_004133

(iii) CCL20 CDHl CDKL5 CRl

EVC2 PTPN 13 ROBOl SLC25A24

Clorf21 NETOl

(iv) the genes identified by accession numbers:

NM_004591 NM 004360 NMJ)Ol 037343 NM_000651

NMJ47127 NM 080683 NM 133631 NM 213651

2. A method of screening for susceptibility to adverse radiation toxicity in an individual, said method comprising identifying the mRNA splice variants expressed by one or more genes selected from:

(1) ANKRD29 CRTAP IQGAP2 SCPEPl

ANO5 DCHS2 ITGA4 SGPP2

ATR DGKD KYNU SHROOM3

BHLHB3 DOCK5 MBOAT2 SLC2A13

BHLHB5 DST MCOLN3 SLCO4C1

ClOorfl l EOMES MERTK SSFA2

Clorflόl EPHBl PARD3 STEAP2

CACNB4 GPC4 RAPGEF5 TOMl Ll

CHLl HNFlB RBPMS ZFYVE 16

CRIMl INA SCARB2; or

(ϋ) the genes identified by accession numbers:

NM 173505 NM 006371 NM 006633 NM 021626

NM 213599 NM 017639 NM 000885 NM 152386

NM 001 184 NM 152879 NM 003937 NM 020859

NM 030762 NM 024940 NM 138799 NM 052885

NM 152414 NM 183380 NM 018298 NM 180991

NM 032024 NM 005442 NM 006343 NM 006751

BC128148 NM 004441 NMJ) 19619 NM 152999

NM 001005747 NM 001448 NM 012294 NM 005486

NM 006614 NM 000458 NM 00100871 1 NM 001 105251

NM_016441 NM_032727 NM 005506

(iii) FAM49A ROBOl ; or

(iv) the genes identified by accession numbers:

NM_030797 NMJ 33631

3. The method according to claim 1 or 2 wherein said method comprises both screening a patient prior to exposure to radiation and thereafter screening said patient subsequently to exposure to said radiation.

4. A method of screening for susceptibility to adverse radiation toxicity in an individual, said method comprising identifying the mRNA splice variants expressed by one or more genes selected from:

(i) APBBl IP DPT GPRC5B PSG4

CLU ERAPl PHACTR3 SYNPO; or

CUBN GABBR2 PI16

(ϋ) the genes identified by accession numbers

NM 019043 NM 001937 NM 016235 NM 002780

NM 001831 NM 001040458 NM 080672 NM 007286; or

NMJ)01081 NM 005458 NMJ53370

(iii) CPM INA LPCAT2 PDE4DIP

CPSl ITGA7 LPHN2 PDE5A

DYNClHl ITGA8 LRRC 16A PKD2

FBN2 JAGl NOTCH3 PLCBl

FGD4 KIAA 1622 PARP 14 PNPLA3

FLNA KIF 16B PDE3A QPRT

RPS6KA2; or

(iv) the genes identified by accession numbers

NM 001874 NM 002206 NM 017640 NM 182734

NM 001122633 NM 003638 NM 000435 NM 025225

NM 001376 NM 000214 NM 017554 NM 014298

NM 001999 NM 058237 NM 000921 NM 021 135

NM 139241 NM 024704 NM 014644

NM 001456 NM 017839 NM 001083

NM 032727 NM 012302 NM 000297

5. A method of screening for susceptibility to adverse radiation toxicity in an individual, said method comprising identifying the mRNA splice variants expressed by one or more genes selected from: 0) APBBl IP CUBN HOXC8 SGCG

CAB39L CYTLl MKX TMEM 155

CDKL2 DPT PI16 VLDLR

CLU GABBR2 PSG4 VSIGl ; or

COLl 2Al GPRC5B SEZ6L2

Oi) the genes identified by accession numbers:

NM 019043 NM 001081 NM 022658 NM 000231

NM 030925 NM 018659 NM 173576 NM 152399

NM 003948 NM 001937 NM 153370 NM 003383

NM 001831 NM 005458 NM 002780 NM_182607

NM_004370 NM_016235 NM_012410

(iii) CPM GPRl 77 LPHN2 PDE4DIP

CPS l GSTM3 LRRC33 PLCBl

ERAPl HMGA2 MAP3K5 QPRT

FBN2 INA MCOLN3 RPS6KA2

FGD4 ITGA7 NEDD4L RXFPl

FST ITGA8 NOTCH3; or

(iv) the genes identified by accession numbers:

NM 001874 NM 02491 1 NM 012302 NM 014644

NM 001 122633 NM 000849 NM 198565 NM 182734

NM 001040458 NM 003483 NM 005923 NM 014298

NM 001999 NM 032727 NM 018298 NM 021 135

NM 139241 NM 002206 NM 015277 NM 021634

NM 006350 NM 003638 NM 000435

in a fibroblast sample from said individual, which sample has been exposed to radiation, wherein the up-regulation of an alternatively spliced mRNA form of the genes of group (i) and/or group (ii) relative to those expressed in a normal sample which has been exposed to radiation is indicative of susceptibility to adverse radiation toxicity and/or the down-regulation of an alternatively spliced mRNA form of the genes of group (iii) and/or group (iv) relative to those expressed in a normal sample which has been exposed to radiation is indicative of susceptibility to adverse radiation toxicity.

6. The method according to claim 4 or 5 wherein said method comprises both screening a patient prior to exposure to radiation and thereafter screening said patient subsequently to exposure to said radiation.

7. A method of assessing whether an individual has been exposed to radiation, said method comprising identifying the mRNA splice variants expressed by one or more genes selected from:

0) ASTN2 BBC3 Clorfl 83 CDKNlA

FBXO22 FBXW7 FDXR FHL2

IGFBP4 MDM2 PHLDA3 PLK2

PLK3 PPMlD RGLl SESNl

SESN2 TNC TNFRSFlOD TSGAlO

VWCE XPC GADD45G RRM2B

ASPM AEN; or

(ϋ) the genes identified by access numbers:

NMJ98186 NM 001127240 NM 019099 NM 078467

NM 147188 NM 033632 NM 024417 NM 201555

NM 001552 NM 002392 NM 012396 NM 006622

NM 004073 NM 003620 NMJ) 15149 NM 014454

NM 031459 NM 002160 NM 003840 NM 18291 1

NM 152718 NM 004628 NM 006705 NMJ) 15613

NMJ)18136 NM_022767

(iii) ANLN AURKA BUB l B CCNBl

CDC25B CDCA2 CENPA CENPE

FAM65B FAM72A FAM83D GTSEl

ILl 6 INCENP KIF14 KIF23

NEK2 PLKl PSRCl SGOL2

SH2D3C TROAP UBE2C; or

(iv) the genes identified by accession numbers:

NMJ) 18685 NMJ98433 NM OOl 21 1 NMJB l 966

NM_021873 NMJ 52562 NM OO 1809 NMJ)01813

NMJ) 14722 BC035696 NM_030919 NMJ 16426

NMJ 72217 N M_001040694 NM O 14875 NMJ38555

NM_002497 NM_005030 NMJ)01032290 NM 152524

NM 170600 NM 005480 NM 181802

8. A method of assessing whether an individual has been exposed to radiation, said method comprising identifying the mRNA splice variants expressed by one or more genes selected from:

(i) FBXW7 PLK3 BTG2 SESNl

SESN2 CDKNlA GDF 15 MDM2

VWCE PPMlD FDXR THSDlP

TP53INP1 LRDD Clorfl83 TRAF4

HISTlHlT IER5 WDR63; or

(ϋ) the genes identified by accession numbers:

NM 033632 NM 004073 NM 006763 NM 014454

NM 031459 NM 078467 NM 004864 NM 002392

NM 152718 NM 003620 NM 024417 NR 002816

NM 033285 NM 018494 NM 019099 NM _004295

NM_005323 NM_016545 NM_145172

(iii) CCNBl CDC25B FAM83D CCNF

C13orf34 GAS2L3 IER5 TROAP

BUBlB AURXA PLKl HERC4

PSRCl CENPA KIF 18A KIF23

CENPE TPX2 CKAP2 CDC27

ZNF321 ARHGAPI lA

(iv) the genes identified by accession numbers:

NM 031966 NM 021873 NM 030919 NM 001761

NM 024808 NM 174942 NM 016545 NM 005480

NM 00121 1 NM 198433 NM 005030 NM 022079

NM 001032290 NM 001809 NM 031217 NM 138555

NM 001813 NM 0121 12 NM 018204 NM 0011 14091

9. A method of assessing whether an individual has been exposed to radiation, said method comprising measuring the level of expression of one or more genes selected from:

(i) BLOC1S2 C12orf5 Clorfl 83 CDKNlA

EDA2R EI24 FAS FBXO22 GADD45A GDF 15 ISF20L1 MDM2

PHLDA3 PLK2 POLH PPMlD

SESN2 TNFRSFlOB XPC ZNF79; or

(ϋ) the genes identified by accession numbers:

NM 001001342 NM 020375 NM 019099 NM 078467

NM 021783 NM 004879 NM 000043 NM 147188

NM 001924 NM 004864 NM 022767 NM 006882

NM 012396 NM 006622 NM 006502 NM 003620

NM 031459 NM_003842 NM_004628 NM_007135

(iii) ARHGAPI lA ASPM AURKA BUBl

CCNBl CDC20 CENPA CENPE

DEPDCl DLG7 FAM72A GTSEl

INCENP KIF20A KIF23 NEK2

PLKl TACC3 TPX2 UBE2C

H2AX CENPF; or

(iv) the genes identified by accession numbers:

NM 014783 NM 018136 NM 198433 NM 004336

NM 031966 NM 001255 NM 001809 NM 001813

NM 001 1 14120 NM 014750 BC035696 NM 016426

NM 001040694 NM 005733 NM 138555 NM 002497

NM 005030 NM 006342 NM 0121 12 NM 181802

NM 002105 NM 016343

10. A method of assessing whether an individual has been exposed to radiation, said method comprising measuring the level of expression of one or more genes selected from:

0) CDKNlA PPMlD FTG2 GADD45A

MDM2 SESNl WDR63 RNF 19B

PLK3 SESN2 ZNF79 POLH

PAGl TNFRSFlOB DDB2 EDA2R

PSTPIP2 XPC BLOC1S2 BCL2Ll ; or

(ii) the genes identified by accession numbers:

NM_078467 NM_003620 NM_006763 NM_001924

NM_002392 NM_014454 NMJ 45172 NMJ 53341

NM_004073 NMJB l 459 NM_007135 NM_006502

NM 018440 NM 003842 NM 000107 NM 021783 NM_024430 NM 004628 NM OO 1001342 NM 138578

(iii) GAS2L3 C13orf34 AURKA FAM83D

SERTAD3 CCNF CKS2 CENPA

HJURP HYLSl CDCA8 CDC25B

KLHL23 SETD8 HlFO KIF 18A

TMEM71 KPNA2 GLIS3 BCOR; or

(iv) the genes identified by accession numbers:

NM 174942 NM 024808 NM 198433 NM 030919

NM 013368 NM 001761 NM 001827 NM 001809

NM 018410 NM 145014 NM 018101 NM 021873

ENST00000392647 NM 020382 NM 005318 NM 031217

1 1. The method according to any one of claims 1-lOwherein said radiation is ionizing radiation.

12. The method according to any one of claims 1, 2, 3, 7 or 9 wherein said biological sample is a blood sample.

13. The method according to any one of claims 4, 5, 6, 8 or 10 wherein said fibroblast sample is a skin sample.

14. The method according to claim 9 or 10 wherein said level of expression is RNA levels.

15. The method according to claim 14 wherein said RNA is mRNA.

16. The method according to any one of claims 1 -13 wherein said level of expression is assessed by analysing protein levels.