Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/118—Prognosis of disease development
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/172—Haplotypes

Definitions

• the present invention provides methods and compositions for identifying human subjects with an increased risk of having or developing disease.
• this invention relates to the identification and characterization of polymorphisms in the human chromosome 19q, the region r located approximately 19q 13.2-3 correlated with increased risk of developing disease, in particular cancer and the responsive ⁇ ness of a subject to various treatments for cancer.
• DNA polymorphisms provide an efficient way to study the association of genes and diseases by analysis of linkage and linkage disequilibrum. With the sequencing of the human genome a myriad of hitherto unknown genetic polymorphisms among people have been detected. Most common among these are the single nucleotide polymorphisms, also called SNPs, of which several millions are known. Other ex ⁇ amples are variable number of tandem repeat polymorphisms, insertions, deletions and block modifications. Tandem repeats often have multiple different alleles (vari- ants), whereas the other groups of polymorphisms usually just have two alleles.
• Some of these genetic polymorphisms probably play a direct role in the biology of the individuals, including their risk of developing disease, but the virtue of the major ⁇ ity is that they can serve as markers for the surrounding DNA, and thus serve as leads during as search for a causative gene polymorphism, as substitutes in the evaluation of its role in health and disease, and as substitutes in the evaluation of the genetic constitution of individuals.
• Linkage arises because large parts of chromosomes are passed unchanged from parents to off ⁇ spring, so that minor regions of a chromosome tend to flow unchanged from one generation to the next and also to be similar in different branches of the same fam ⁇ ily. Linkage is gradually eroded by recombination occurring in the cells of the germ- line, but typically operates over multiple generations and distances of a number of million bases in the DNA.
• Linkage disequilibrium deals with whole populations and has its origin in the (distant) forefather in whose DNA a new sequence polymorphism arose.
• the immediate sur ⁇ roundings in the DNA of the forefather will tend to stay with the new allele for many generations.
• Recombination and changes in the composition of the population will again erode the association, but the new allele and the alleles of any other polymor ⁇ phism nearby will often be partly associated among unrelated humans even today.
• a crude estimate suggests that alleles of sequence polymorphisms with distances less than 10000 bases in the DNA will have tended to stay together since modern man arose.
• Linkage disequilibrium is the results of many stochastic events and as such subject to statistical variation occasionally resulting in discontinuities, lack of a monotonic relationship between association and distance and differences between people of different ethnicity. Therefore, it is often advantageous to study more that one se ⁇ quence polymorphism in a given region. This also allows for further definition of the genetic surroundings of the biologically relevant polymorphism by combining the associated alleles of the different markers into a socalled haplotype.
• genotypes i.e. the combined analysis of both chromosomes at a given sequence polymorphism.
• the resulting genotypes of a person, analysed for instance on DNA from peripheral blood leukocytes, are inherently very stable over time. Therefore, this type of analysis can be performed any time in the life of a person and will be applicable to this person for his or her entire life.
• genetic analyses are ideally suited to predict future risks of disease.
• a variety of investigations suggest that many diseases in part are determined by the genetic constitution of the individual.
• One group of genes in particular has been as ⁇ sociated with rare genetic predispositions to cancer.
• DNA repair genes these are the genes involved in maintaining the integrity of a person's DNA, the so-called DNA repair genes.
• DNA repair genes One set of such genes are the XP genes which participate in nucleotide excision repair, and, when mutated, give rise to a 1000 fold increased risk of getting skin cancer.
• XPDe ⁇ one allele of the sequence polymorphism called XPDe ⁇ was associated with a moderately increased risk of getting basal cell carci ⁇ noma, the most common form of skin cancer.
• XPDe ⁇ one allele of the sequence polymorphism called XPDe ⁇ was associated with a moderately increased risk of getting basal cell carci ⁇ noma, the most common form of skin cancer.
• Later other groups have studied the association between sequence polymorphisms in this and other DNA repair genes and various forms of cancer. Some have reported positive results.
• the present invention relates in a first aspect to a group of nucleic acid sequences found to be associated with disease, in particular cancer.
• the invention further re ⁇ lates to transcriptional and translational products of said sequence.
• An allele in the r region can be identified as correlated with an increased risk of developing disease, in particular cancer, the prognosis of developed disease, in particular cancer, and responsiveness to disease treatment, in particular cancer treatment on the basis of statistical analyses of the incidence of a particular allele in individuals diagnosed with disease, in particular cancer.
• the invention relates to a method for estimating the disease risk of an individual comprising
• the estimation of the disease risk of an individual can involve the comparison of the number and/or kind of polymorphic sequences identified with a predetermined dis ⁇ ease risk profile.
• a profile can be based on statistical data obtained for a rele ⁇ vant reference group of individuals.
• the disease is a proliferative dis- ease, such as cancer.
• sequence of the r region is set forth as SEQ ID NO 1 , originating from the clon ⁇ ing of human chromosome 19q published as part of the contig NT_011109 in the database of human sequences established by National Center for Biotechnology Information and located on the internet at http://www.ncbi.nlm.nih.gov/- qenome/quide/human/ .
• the presence of an allele is determined by determining the nucleic acid sequence of all or part of the region according to standard molecular biology protocols well known in the art as described for example in Sambrook et al. (1989) and as set forth in the Examples provided herein or products of the nucleic acid sequences.
• the nucleic acid molecules of the present invention represent in a first aspect nucleic acid sequences forming part of the region r corresponding to position 1522-37752 of SEQ ID NO: 1 , and preferably to certain nucleic acid sequences within the gene referred to herein as RAI.
• RAI nucleic acid sequences forming part of the region r corresponding to position 1522-37752 of SEQ ID NO: 1
• the RAI gene is in particular associated with human cancer diseases.
• the invention relates to a method for estimating the disease prognosis of an individual comprising - in a sample from said individual, assessing in the genetic material a sequence polymorphism
• the estimation of the disease prognosis of an individual can involve the comparison of the number and/or kind of polymorphic sequences identified with a predetermined disease prognosis profile. Such a profile can be based on statistical data obtained for a relevant reference group of individuals.
• a method of identifying a human subject as having an in ⁇ creased likelihood of responding to a treatment comprising a) correlating the pres- ence of an r region allele genotype with an increased likelihood of responding to treatment; and b) determining the r region allele genotype of the subject, whereby a subject having an r region allele genotype correlated with an increased likelihood of responding to treatment is identified as having an increased likelihood of responding to treatment.
• the present invention also relates to method for estimating a treatment re ⁇ sponse of an individual suffering from disease to a disease treatment, comprising
• the estimation of the individual's response to disease treatment can involve the comparison of the number and/or kind of polymorphic sequences identified with a predetermined cancer treatment response profile.
• a predetermined cancer treatment response profile can be based on statistical data obtained for a relevant reference group of individuals.
• the disease is a proliferative disease, such as cancer.
• the invention also comprises primers or probes for use in the invention, as well as kits including these.
• the primers and/or probes are preferably capable of hybridising to SEQ ID NO:1, or a part thereor, in particularly the regions relevant to this inven ⁇ tion, or a part thereof, under stringent conditions, as well as to a sequence comple- mentary thereto.
• the invention also relates to cloning vectors and expression vectors containing the nucleic acid molecules of the invention, as well as hosts which have been transformed with such nucleic acid molecules, including cells genetically engi- neered to contain the nucleic acid molecules of the invention, and/or cells geneti ⁇ cally engineered to express the nucleic acid molecules of the invention.
• the nucleic acids are preferably isolated from the r region and preferably contain one or more sequence polymorphisms as described herein below in more detail.
• hosts also include transgenic non-human animals (or prog- eny thereof).
• the present invention is based on the discovery of the correlation with single nucleotide polymorphisms (SNPs), deletion polymorphisms, insertion poly ⁇ morphisms, dinucleotide polymorphisms and/or tandem repeats in the regions and disease.
• SNPs single nucleotide polymorphisms
• deletion polymorphisms e.g., deletion polymorphisms
• insertion poly ⁇ morphisms e.g., tyl-like polymorphisms
• dinucleotide polymorphisms e.g., tandem repeats in the regions and disease.
• the term human includes both a human having or suspected of having a disease and an a-symptomatic human who may be tested for predisposition or susceptibility to disease. At each position the human may be homozygous for an allele or the hu- man may be a heterozygote.
• Fig. 2 shows a pair-wise linkage disequilibrium between all markers in breast cancer controls.
• Fig. 3 shows an association of single polymorphisms with early breast cancer.
• Fig. 4 shows an association of sets of neighboring SNPs with breast cancer.
• Fig. 5 shows a maximal OR for breast cancer for haplotypes formed by two SNPs.
• Fig. 6 shows a Lazzeroni estimation of the position of the causative variant using young breast cancer.
• Fig. 7 shows the overall distribution of p-values for sets of markers plotted against the position on the chromosome for basal cell carcinoma and lung cancer.
• posi ⁇ tion on the abscissa we used the median marker position in a given cluster of mark ⁇ ers.
• the ordinate values are the negative logarithms to the overall p-values for a difference between cases and controls associated with a given set of markers.
• Each curve corresponds to a given size of marker sets, i.e. the length of the haplotypes.
• Fig. 8 shows odds ratio for cancer versus control between the two homozygotes of each SNP in relation to location on chromosome 19.
• Fig. 9 shows event free survival for patients who are wild-type carriers of ASE-1 (0) or homozygous or heterozygous carriers of the variant allele (1 ).
• Fig. 11 shows event free survival for women subdivided by ASE-1 genotype.
• 0 homozygous carrier of the wild-type allele of ASE-1
• 1 carrier of the variant allele of ASE-1.
• Fig. 12 shows overall survival for women subdivided by ASE-1 genotype.
• 0 homozygous carrier of the wild-type allele of ASE-1
• 1 carrier of the variant allele of ASE-1
• Fig. 13 shows event free survival for men subdivided by ASE-1 genotype.
• 0 homozygous carrier of the wild-type allele of ASE-1
• 1 carrier of the variant allele of ASE-1.
• Fig. 15 shows Kaplan Meier plot of survival of lung cancer patients in relation to highrisk haplotype
• Fig. 16 shows Kaplan Meier plot of survival in relation to XPD K751Q among those lung cancer patients homozygous for the high risk haplotype.
• Fig. 17 shows Kaplan Meier plot of survival in relation to XPD K751Q among those lung cancer patients not homozygous for the highrisk haplotype.
• the present invention relates to a characterization of a person's present and/or fu ⁇ ture risk of getting certain forms of disease, in particular a proliferative disease, such as cancer.
• the characterization is based on the analysis of sequence polymor ⁇ phisms in a region of chromosome 19q in the person.
• a number of polymorphisms in the chromosomal region 19q 13.2-3 have been identi ⁇ fied and characterised. Surprisingly, the sequence polymorphisms with strongest association to disease appeared to be located outside the gene XPD. More specifi ⁇ cally, the sequences were located in a sub-region harboring the gene RAI and im ⁇ mediately downstream of the RAI gene. The nature of the association between hap- lotype and disease was examined together with the p-values associated with the individual haplotypes. The odds ratios for each haplotype of each set of three neighboring SNPs were also determined. The odds ratio for test of homozygotes of individual markers against cancer status was likewise detemined. The most likely location of a single causative gene variant for individuals of the breast cancer cases was evaluated.
• the region of chromosome 19q is depicted in Figure 1 as it is presently known together with the presently known or suspected genes.
• the arrows indicate the directions of transcription of the genes.
• the absolute chromosome positions shown are from the particular build of NCBPs map of chromsome 19, and will proba- bly change with time. The position of markers used throughout the experiments is indicated.
• the intron-exon structure of the RAI gene is shown together with the posi ⁇ tion of the inter gene region between RAI and XPD genes.
• the region r stretches from the beginning of, but not including the XPD gene, to ap- proximately the end of ERCC1 and includes the genes RAI, LOC162978, and ASE- 1. More specifically r is bounded by and includes the following two sequences: AGAACCCCCG CCCCTCCACC TCGTCTCAAA and TCCCTCCCCA GA- GACTGCAC CAGCGCAGCC, and is defined by SEQ ID NO: 1.
• the region r means SEQ ID NO: 1 and complementary se ⁇ quence as well as transcriptional products and translational products thereof.
• the gene RAI is defined in the claims as including transcribed sequences of the gene plus a 1500 base upstream promoter region. More specifically RAI is bounded by and includes the following sequences: CATAACCACA ATGATGAGCA TGTATTGAGT and ATGTTGTCCA GGCTGGTCTT GAACTCCTGA. In the present context this section of the region relates to SEQ ID NO: 1 bases 7761-22885 and complementary sequence as well as transcriptional products and translational products thereof.
• one preferred section of the region stretches approximately from the the beginning of, but not including the XPD gene, to approximately the end of the RAI gene.
• the region means SEQ ID NO: 1 bases 1 to 25550 and complementary sequence as well as transcriptional products and transla ⁇ tional products thereof.
• one preferred section of the region stretches approximately from the the beginning of, but not including the XPD gene, to approximately within the RAI gene.
• the region means SEQ ID NO: 1 bases 1 to 15698 and complementary sequence as well as transcriptional products and translational products thereof.
• one preferred section of the region r stretches ap ⁇ proximately from outside the XPD gene, to approximately within the RAI gene.
• the region means SEQ ID NO: 1 bases 4528 to 15698 and comple- mentary sequence as well as transcriptional products and translational products thereof.
• one preferred section of the region r stretches approximately from the beginning of, but not including the XPD gene, into the inter gene region between the RAI and XPD gene.
• the region means SEQ ID NO: 1 bases 1 to 1510 and complementary sequence as well as transcriptional products and translational products thereof.
• one preferred section of the region r stretches approxi- mately from the the beginning of, but not including the XPD gene, throughout the inter gene region between the RAI and XPD gene and into the 3' part of the RAI gene.
• the region means SEQ ID NO: 1 bases 1710 to 8685 and complementary sequence as well as transcriptional products and translational products thereof.
• one preferred section of the region r stretches ap ⁇ proximately over the central part of the RAI gene.
• the region means SEQ ID NO: 1 bases 8987 to 12090 and complementary sequence as well as transcriptional products and translational products thereof.
• one preferred section of the region r stretches approximately over the middle and 5' part of the RAI gene.
• the region means SEQ ID NO: 1 bases 15898 to 25550 and complementary sequence as well as tran ⁇ scriptional products and translational products thereof.
• Fragments or parts of the region r as used herein relates to any fragment of at least 5 nucleic acid redues in length, or multiples of 5 nucleic acid residues in length start ⁇ ing from SEQ ID NO: 1 position 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100.
• At least 21 such as at least 22, for example at least 23, such as at least 24, for example at least 26, such as at least 27, for ex- ample at least 28, such as at least 29, for example at least 31 , such as at least 32, for example at least 33, such as at least 34, for example at least 36, such as at least 37, for example at least 38, such as at least 39, for example at least 41 , such as at least 42, for example at least 43, such as at least 44, for example at least 46, such as at least 47, for example at least 48, or at least 100 nucleic acid redues in length, or mutiples of 100 nucleic acid residues in length, starting from SEQ ID NO: 1 posi ⁇ tion 1, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2600, 2700, 2800, 2900
• Multiples are preferably multiples of e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 and 50.
• the length of said fragments will thus be e.g. 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500,
• the length of said fragments will thus be e.g. 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500,
• the length of said fragments will thus be e.g. 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500,
• nucleic acid sequences according to the present invention make it possible to estimate cancer risk in an individual by using sequence polymorphisms originating from a specific region of chromosome 19.
• Lung cancer affects approxi- mately 10-15 percent of smokers and thus approximately 5 percent of the popula ⁇ tion, somewhat varying from country to country.
• Malignant melanoma a sun- induced, often lethal form of skin cancer, affects approximately 700 persons a year in Denmark or about 1 percent of the Danish population.
• sequence polymorphism is understood any single nucleotide, tandem repeat, insertion, deletion or block polymorphism, which varies among humans, whether it is of known biological importance or not.
• one or more single nucleotide polymorphism(s) at a pre ⁇ determined position in the region are identified and used for e.g. cancer risk profiling and/or cancer treatment response profiling.
• Presently preferred polymorphism(s) are listed in Tables 1a, 1b and 1c, more preferably at least two polymorphism(s) are selected, most preferably at least three polymorphism(s) are selected.
• the present invention relates to any polymorphism in the region. Table 1a
• ERCC1-3' C/T rs762562 NT 011109 18180561 57424 37267 rs numbers were derived from the NCBI's database dbSNP.
• TCTTTCTTTCTT rs4572514 AGAACCTGTTCAGGCTGGCGGCTCA[C/T]TTGGATGAAC 28691
• one or more single nucleotide polymorphism(s) at a pre ⁇ determined position in the region are identified and used for e.g. cancer risk profiling and/or cancer treatment response profiling.
• Presently preferred polymorphism(s) are listed in Tables 2a and 2b, more preferably at least two poly- morphism(s) are selected, most preferably at least three polymorphism(s) are se ⁇ lected.
• the present invention relates to any polymorphism in the region.
• RAI intron 1-3 A/C AC092309 25115 39833 19677 rs numbers were derived from the NCBI's database dbSNP.
• one or more single nucleotide polymorphism(s) at a pre ⁇ determined position in the region are identified and used for e.g. cancer risk profiling and/or cancer treatment response profiling.
• Presently preferred polymorphism(s) are listed in Tables 3a, 3b and 3c, more preferably at least two polymorphism(s) are selected, most preferably at least three polymorphism(s) are selected.
• the present invention relates to any polymorphism in the region.
• RAI intron 1-3 A/C AC092309 25115 39833 19677 rs numbers were derived from the NCBI's database dbSNP.
• polymorphism(s) are listed below in tables 3c and 3d, more pref ⁇ erably at least two polymorphism(s) are selected, most preferably at least three polymorphism(s) are selected from SEQ ID NO:1 below Table 3c
• RAI intron 3 A/G rs2017104 NT 011109 18155483 32346 12190 rs numbers were derived from the NCBI's database dbSNP.
• GAGGCTGCAGTGAGCTGT gactgtgcca ctgcactcca rs2097215 TGACAGTAGA CATCCTGTCA T (A/G) ATAAGTCttt ttttttt 1610 rs 11878644 CATCCCCATA CCAAcccacc (c/t) tactgctctg atctcctcct 2790 rs7252567 tttagtagagacatggttccgcca (C/T) gttgcccaggctggtcttgaactc 4428 rs3047560 ACTAAAAATAAAAAATAAAAAAAA(-/AA) ATAGCCGAG- 4797
• polymorphism(s) are those listed below in tables 3e and 3f, more preferably at least two polymorphism(s) are selected, most preferably at least three polymorphism(s) are selected from SEQ ID NO: 1 below:
• RAI intron 3 A/G rs2017104 NT 011109 18155483 32346 12190 rs numbers were derived from the NCBI's database dbSNP.
• polymorphism(s) are those listed in tables 4a and 4b below, more preferably at least two polymorphism(s) are selected, most preferably at least three polymorphism(s) are selected from the polymorphisms shown below:
• RAI-3'3 AAA rs3047560 NT 011109 24055 RAI exon 6 A/T rs6966 NT 011109 28043 RAI intron 3 A/G rs2017104 NT 011109 32346 rs numbers were derived from the NCBI's database dbSNP.
• rs6966 ATTAAGTGCCTTCACACAGC (A/T) CTGGTTTAAT GTTTATAA 8785 rs2017104 gggaggctcg aggcgggc (AJG) gattgcatga gctcaggatt 12190
• one or more single nucleotide polymor ⁇ phism ⁇ ) at a predetermined position in the region are identified and used for e.g. cancer risk profiling and/or cancer treatment response profiling.
• the preferred polymorphism(s) are listed in Tables 5a, more preferably at least two polymorphism(s) are selected, most preferably at least three polymor- phism(s) are selected.
• the present invention relates to any polymorphism in the region.
• RAI intron 1-3 A/C AC092309 25115 39833 19677 rs numbers were derived from the NCBI's database dbSNP.
• polymorphism(s) are those listed in table 5b below, more preferably at least two polymorphism(s) are selected, most preferably at least three polymor ⁇ phism ⁇ ) are selected from the polymorphisms shown below:
• polymorphism(s) are those listed in table 5c below, more preferably at least two polymorphism(s) are selected, most preferably at least three polymorphism(s) are selected from the polymorphisms shown below:
• RAI-i ⁇ -2 A/T rs8112723 NT 011109 18153497 30360 10204 rs numbers were derived from the NCBI's database dbSNP.
• polymorphism(s) are those listed in table 5d below, more preferably at least two polymorphism(s) are selected, most preferably at least three polymorphism(s) are selected from the polymorphisms shown below:
• RAI-3'8 A/G rs8101662 NT 011109 18150911 27774 8516 rs numbers were derived from the NCBI's database dbSNP.
• At least one of the following combinations of polymor ⁇ phisms is included in the methods:
• the method described herein is one in which the tandem repeat is at a position as described in Table 6: Table 6
• the method for diagnosis described herein is preferably one in which the sequence polymorphism is in region r. Testing for the presence of the RAI gene allele is especially preferred because, without wishing to be bound by theoretical considerations, of its association with increased risk of can- cer (as explained herein).
• one or more polymorphism(s) at a predetermined position in the region r are identified and used for e.g. cancer risk profiling and/or cancer treatment response profiling.
• Presently preferred polymorphism(s) are the dinucleotide polymorphism RAI-3'3 in which AA or a deletion is present, the sin ⁇ gle nucleotide polymorphism RAI exon 6 in which A or T is present, and RAI intron 3 in which A or G is present.
• the present invention relates to any polymor ⁇ phism and SNP in the r region.
• the sequence polymorphism of the invention comprises at least one base differ ⁇ ence, such as at least two base differences, such as at least three base differences, such as at least four base differences, such as eighty one base pair differences.
• the sequence polymorphism(s) comprises at least one polymor- phism, such as at least two polymorphisms, such as at least three polymorphisms, such as at least four polymorphisms.
• the sequence polymorphism comprises at least one polymorphism, such as at least two tandem repeat polymorphisms.
• sequence polymorphism may be a combination of single nucleotide poly- morphism and dinucleotide polymorphism, such as one single nucleotide polymor ⁇ phism and one dinucleotide polymorphism.
• the status of the individual may be determined by reference to allelic variation at one, two, three, four or more of the above loci.
• the cell sample used in the present invention may be any suitable cell sample ca ⁇ pable of providing the genetic material for use in the method.
• the cell sample is a blood sample, a tissue sample, a sample of secretion, semen, ovum, a washing of a body surface (e.g. a buccal swap), a clipping of a body surface (hairs, or nails), such as wherein the cell is selected from white blood cells and tumour tissue.
• test sample may equally be a nucleic acid sequence corresponding to the sequence in the test sample, that is to say that all or a part of the region in the sample nucleic acid may firstly be amplified using any convenient technique e.g. PCR, before use in the analysis of variation in the region.
• Detection may be conducted on the sequence of SEQ ID NO: 1 or a complementary sequence as well as on translational (mRNA) and transcriptional products (polypep- tides, proteins) therefrom.
• Muta- tions or polymorphisms within or flanking the r region can be detected by utilizing a number of techniques. Nucleic acid from any nucleated cell can be used as the starting point for such assay techniques, and may be isolated according to standard nucleic acid preparation procedures that are well known to those of skill in the art. In general, the detection of allelic variation requires a mutation discrimination tech ⁇ nique, optionally an amplification reaction and a signal generation system. Table 7 lists a number of mutation detection techniques, some based on the PCR.
• Table 8 illustrates various mutation detection techniques capable of being used for SNP detection.
• Fluorescence Fluorescence: FRET, Fluorescence quenching, Fluorescence polarisation-United Kingdom Patent No. 2228998 (Zeneca Limited)
• Table 9 illustrates examples of further amplification techniques.
• Preferred mutation detection techniques include ARMS, ALEX, COPS, Taqman, Molecular Beacons, RFLP, and restriction site based PCR and FRET techniques.
• Particularly preferred methods include FRET; taqman, ARMS and RFLP based methods.
• mutations or polymorphisms can be detected by using a microassay of nucleic acid sequences immobilized to a substrate or "gene chip” (see, e.g. Cronin, et a!., 1996, Human Mutation 7:244-255).
• Caskey et al. (U.S. Pat. No. 5,364,759) describe a DNA profiling assay for detecting short tri and tetra nucleotide repeat sequences. The process includes ex ⁇ tracting the DNA of interest, such as the RAI gene, amplifying the extracted DNA, and labelling the repeat sequences to form a genotypic map of the individual's DNA.
• the level of RAI gene expression can also be assayed.
• RNA from a cell type or tissue known, or suspected, to express the RAI gene may be isolated and tested utilizing hybridization or PCR techniques such as are described, above.
• the isolated cells can be derived from cell culture or from a patient.
• the analysis of cells taken from culture may be a necessary step in the assessment of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the ef ⁇ fect of compounds on the expression of the RAI gene.
• Such analyses may reveal both quantitative and qualitative aspects of the expression pattern of the RAI gene, including activation or inactivation of RAI gene expression.
• a cDNA molecule is synthesized from an RNA molecule of interest (e.g., by reverse transcription of the RNA mole ⁇ cule into cDNA).
• a sequence within the cDNA is then used as the template for a nucleic acid amplification reaction, such as a PCR amplification reaction, or the like.
• the nucleic acid reagents used as synthesis initiation reagents (e.g., primers) in the reverse transcription and nucleic acid amplification steps of this method are chosen from among the RAI gene nucleic acid reagents described above.
• the preferred lengths of such nucleic acid reagents are at least 9-30 nucleotides.
• the nucleic acid amplification may be performed using radio- actively or non-radioactively labeled nucleotides.
• enough amplified product may be made such that the product may be visualized by standard ethidium bromide staining or by utilizing any other suitable nucleic acid staining method.
• RAI gene expression assays "in situ", i.e., directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary.
• Nucleic acid reagents such as those described above may be used as probes and/or prim ⁇ ers for such in situ procedures (see, for example, Nuovo, G. J., 1992, “PCR In Situ Hybridization: Protocols And Applications", Raven Press, NY).
• stan ⁇ dard Northern analysis can be performed to determine the level of mRNA expres ⁇ sion of the RAI gene.
• Another method for detecting sequence polymorphism is by analysing the activity of gene products resulting from the sequences. Accordingly, in one embodiment the detection uses the activity of the RAI gene product as compared to a reference in the method. In particular if the activity of the genes are decreased or increased by at least or about 50 %, such as at least or about 40%, for example at least or about 30%, such as at least or about 20%, for example at least or about 10%, such as at least or about 10%, for example at least or about 5%, such as at least or about 2%, it indicates a sequence polymorphism in the gene.
• the present invention may combine the result of sequence polymorphism within the region r with sequence polymorphism outside the region in order to increase the probability of the correlation.
• the primer nucleotide sequences of the invention further include: (a) any nucleotide sequence that hybridizes to a nucleic acid molecule of the region r or its comple ⁇ mentary sequence or RNA products under stringent conditions, e.g., hybridization to filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about 45°C followed by one or more washes in 0.2x SSC/0.1% SDS at about 50-65 0 C, or (b) under highly stringent conditions, e.g., hybridization to filter-bound nucleic acid in 6x SSC at about 45°C followed by one or more washes in 0.1 x SSC/0.2% SDS at about 68°C, or under other hybridization conditions which are apparent to those of skill in the art (see, for example, Ausubel F.M.
• stringent conditions e.g., hybridization to filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about 45°C
• nucleic acid molecule that hybrid- izes to the nucleotide sequence of (a) and (b), above is one that comprises the complement of a nucleic acid molecule of the region s or r or a complementary se ⁇ quence or RNA product thereof.
• nucleic acid molecules comprising the nucleotide sequences of (a) and (b) comprises nucleic acid mole ⁇ cule of RAI or a complementary sequence or RNA product thereof.
• oli- gos deoxyoligonucleotides
• TM melting temperature
• Exemplary highly stringent conditions may refer, e.g., to washing in 6x SSC/0.05% sodium pyrophosphate at 37 0 C (for about 14-base oligos), 48°C (for about 17-base oligos), 55°C (for about 20-base oligos), and 6O 0 C (for about 23-base oligos).
• the invention further provides nucleotide primers or probes which de- tect the r region polymorphisms of the invention.
• the assessment may be conducted by means of at least one nucleic acid primer or probe, such as a primer or probe of DNA, RNA or a nucleic acid analogue such as peptide nucleic acid (PNA) or locked nucleic acid (LNA).
• the nucleotide primer or probe is preferably capable of hybridis ⁇ ing to a subsequence of the region corresponding to SEQ ID NO: 1, or a part thereof, or a region complementary to SEQ ID NO: 1.
• an allele-specific oligonucleotide probe capable of detecting a r region polymorphism at one or more of positions in the r region as defined by the positions in SEQ ID NO: 1.
• the allele-specific oligonucleotide probe is preferably 5-50 nucleotides, more pref ⁇ erably about 5-35 nucleotides, more preferably about 5-30 nucleotides, more pref ⁇ erably at least 9 nucleotides.
• Such probes will be apparent to the molecular biologist of ordinary skill.
• Such probes are of any convenient length such as up to 50 bases, up to 40 bases, more conveniently up to 30 bases in length, such as for example 8-25 or 8- 15 bases in length.
• such probes will comprise base sequences entirely complementary to the corresponding wild type or variant locus in the region. How- ever, if required one or more mismatches may be introduced, provided that the dis ⁇ criminatory power of the oligonucleotide probe is not unduly affected.
• the probes of the invention may carry one or more labels to facilitate detection.
• the primers and/or probes are capable of hybridizing to and/or amplifying a subsequence hybridizing to a single nucleotide polymorphism contain ⁇ ing the sequence shown herein selected from the group of subsequences below or a sequence complementary thereto, wherein the polymorphism is denoted as for ex ⁇ ample T/C:
• gctgcagtga gctgt (-/ACACCTGTGGTCCCAGCTACTCTGG AAGCTGAGGTGGGAGGATCGCTTGAGCCCAAGAGGTGGAGGCTG-
• CAGTGAGCTGT gactgtgcca ctgcactcca
• the primers and/or probes are capable of hybridizing to and/or amplifying a subsequence hybridizing to a single nucleotide polymorphism containing the sequence shown herein selected from the group of subsequences below or a sequence complementary thereto, wherein the polymorphism is denoted as for example T/C:
• the primers and/or probes are capable of hybridizing to and/or amplifying a subsequence hybridizing to a single nucleotide polymorphism containing the sequence shown herein selected from the group of subsequences below or a sequence complementary thereto, wherein the polymorphism is denoted as for example TYC:
• gctgcagtga gctgt (-/ACACCTGTGGTCCCAGCTACTCTGG AAGCTGAGGTGGGAGGATCGCTTGAGCCCAAGAGGTGGAGGCTG-
• CAGTGAGCTGT gactgtgcca ctgcactcca
• the primers and/or probes are capable of hybridizing to a subsequence selected from the group of subsequences below:
• the primers and/or probes are capable of hybridizing to a subsequence selected from the group of subsequences below:
• TGTTGTCCAA GCTGGCAGAG (A/G) TTTTTGTTTG TTTGTTTGAG 5.
• CTCGGGAGGCTGAGGCAGGAGAATC (A/G) CTTGAACTCAGGCA-
• primers and/or probes capable of hybridizing to a subse ⁇ quence selected from the group of subsequences below:
• gggaggctcg aggcgggc (A/G) gattgcatga gctcaggatt are the primers and/or probes capable of hybridizing to a subsequence selected from the group of subsequences below:
• the primers and/or probes are capable of hybridizing to a subsequence selected from the group of subsequences below:
• primers and/or probes are capable of hybridizing to a subsequence selected from the group of subsequences below:
• primers and/or probes are capable of hybridizing to a subsequence selected from the group of subsequences below:
• CTTGCTACAGAATTACAGGCA GCGCCACCGCTCCGGGCTAA 2.
• CTAAAGACTACA VA
• tgc ctt cac aca get ctg gtt taa tg 20.
• egg get aca ggg tta cct gag 40.
• age tgc cag age tgc ctg ggc One or more primers able to detect subsequences by hybridisation as described may in a particularly preferred embodiment of the method of the invention be se ⁇ lected from
• a diagnostic nucleic acid primer capable of detecting a r region polymorphism at one or more of positions in the r region as defined by the in SEQ ID NO: 1.
• the primer or probe may be a diagnostic nucleic acid primer defined as an allele specific primer, used, generally together with a constant primer, in an amplification reaction such as a PCR reaction, which provides the discrimination between alleles through selective amplification of one allele at a particular sequence position.
• the diagnostic primer is preferably 5-50 nucleotides, more preferably about 5-35 nucleo ⁇ tides, more preferably about 5-30 nucleotides, more preferably at least 9 nucleo ⁇ tides.
• diagnostic primers compris- ing the sequences set out below as well as derivatives thereof wherein about 6-8 of the nucleotides at the 3' terminus are identical to the sequences given below and wherein up to 10, such as up to 8, 6, 4, 2, or 1 of the remaining nucleotides may be varied without significantly affecting the properties of the diagnostic primer.
• up to 10 such as up to 8, 6, 4, 2, or 1 of the remaining nucleotides may be varied without significantly affecting the properties of the diagnostic primer.
• At least two sets of primer(s) and/or probe(s), such as at least three sets of primer(s) and/or probe(s) may be combined in the method thereby increasing the correlation probability.
• This second or other set of primer(s) and/or probe(s) may be a nucleotide or nucleotide analogues hybridising to a region within the region r or to a sequence different from the region r. Said sequence differ- ent from the region r is preferably a region in chromosome 19, preferably in chromo ⁇ some 19q.
• such second or other primer or probe may be selected from one or more of the sequences below, or the complementary strands:
• the primers and probes may be manufactured using any convenient method of syn ⁇ thesis. Examples of such methods may be found in standard textbooks, for example "Protocols for Oligonucleotides and Analogues; Synthesis and Properties," Methods in Molecular Biology Series; Volume 20; Ed. Sudhir Agrawal, Humana ISBN: 0- 89603-247-7; 1993; Lsup.st Edition. If required the primer(s) and probe(s) may be labelled to facilitate detection.
• a diagnostic kit comprising at least one diagnostic primer of the invention and/or at least one al- lele-specific oligonucleotide primer of the invention.
• kits may comprise appropriate packaging and instructions for use in the methods of the invention.
• Such kits may further comprise appropriate buffer(s) and polymerase(s) such as thermostable polymerases, for example taq polymerase.
• kits can comprise means for amplifying the relevant sequence such as primers, polymerase, deoxynucleotides, buffer, metal ions; and/or means for dis ⁇ criminating the polymorphism, such as one or a set of probes hybridising to the poly ⁇ morphic site, a sequence reaction covering the polymorphic site, an enzyme or an antibody; and/or a secondary amplification system, such as enzyme-conjugated antibodies, or fluorescent antibodies.
• the kit-of-parts preferably also comprises a detection system, such as a fluorometer, a film, an enzyme reagent or another highly sensitive detection device.
• kits for detect ⁇ ing the presence of a polypeptide or nucleic acid of the invention in a biological sample i.e., a test sample.
• a biological sample i.e., a test sample.
• kits for detect ⁇ ing the presence of a polypeptide or nucleic acid of the invention in a biological sample i.e., a test sample.
• kits for detect ⁇ ing the presence of a polypeptide or nucleic acid of the invention in a biological sample i.e., a test sample.
• kits can be used, e.g., to determine if a subject is suffering from or is at increased risk of developing a disorder associated with a dis ⁇ order-causing allele, or aberrant expression or activity of a polypeptide of the inven- tion.
• the kit can comprise a labeled compound or agent capable of detecting the polypeptide or mRNA or DNA or RAI gene sequences, e.g., encoding the polypeptide in a biological sample.
• the kit can further comprise a means for de ⁇ termining the amount of the polypeptide or mRNA in the sample (e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide).
• Kits can also include instructions for observing that the tested subject is suffering from or is at risk of developing a disorder associ ⁇ ated with aberrant expression of the polypeptide if the amount of the polypeptide or mRNA encoding the polypeptide is above or below a normal level, or if the DNA correlates with presence of an RAI allele that causes a disorder.
• the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide of the invention; and, op ⁇ tionally, (2) a second, different antibody which binds to either the polypeptide or to the first antibody and is conjugated to a detectable agent.
• a first antibody e.g., attached to a solid support
• a second, different antibody which binds to either the polypeptide or to the first antibody and is conjugated to a detectable agent.
• An allele in the r region can be identified as correlated with an increased risk of de ⁇ veloping cancer on the basis of statistical analyses of the incidence of a particular allele in two groups of individuals with and without cancer, respectively, according to the ⁇ 2 test, which is well known in the art. Furthermore, an allele in the region can be identified as an allele correlated with prognosis of cancer on the basis of statistical analyses of the incidence of a particular allele in individuals demonstrating different prognostic characteristics. Identification of humans having increased likelihood of responding to treat ⁇ ment
• the present invention provides a method for identifying a human subject as having an increased likelihood of responding positively to a cancer treatment, comprising determining the presence in the subject of a s or r re ⁇ gion allele genotype correlated with an increased likelihood of positive response to treatment, whereby the presence of the genotype identifies the subject as having an increased likelihood of responding to cancer treatment.
• the treatment mentioned herein may be any cancer treatment, such as conventional cancer treatment, for example X-ray, chemotherapeutics, surgical excision or com ⁇ binations thereof.
• Gene products of the region r or peptide fragments thereof can be prepared for a variety of uses.
• such gene products, or peptide fragments thereof can be used for the generation of antibodies, in diagnostic assays.
• the gene products of the invention include, but are not limited to, human RAI gene products. In the following the invention is described in relation to RAI gene product.
• Gene product sometimes referred to herein as an "protein” or “polypeptide”, in- eludes those gene products encoded by the RAI gene sequences shown as position 7760-22885 in SEQ ID NO: 1.
• gene product variants are gene products comprising amino acid residues encoded by the polymorphisms.
• Such gene product variants also include a variant of the RAI gene product.
• RAI gene products may include proteins that represent functionally equi ⁇ valent gene products.
• functionally equivalent RAI gene products are naturally occurring gene products.
• Functionally equivalent RAI gene products also include gene products that retain at least one of the biological activities of the RAI gene products described above, and/or which are recognized by and bind to antibodies (polyclonal or monoclonal) directed against RAI gene prod ⁇ ucts.
• the terms "spe- cifically bind” and “specifically recognize” refer to antibodies that bind to RAI gene product epitopes at a higher affinity than they bind to non-RAI (e.g., random) epi ⁇ topes.
• Such antibodies may include, but are not limited to, polyclonal antibodies, mono- clonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above, including the polyclonal and monoclonal antibodies described below.
• mAbs mono- clonal antibodies
• Such antibod ⁇ ies may be used, for example, in the detection of a gene product in a biological sample and may, therefore, be utilized as part of a diagnostic or prognostic tech ⁇ nique whereby patients may be tested for abnormal levels of gene products, and/or for the presence of abnormal forms of such gene products.
• Such antibodies may also be utilized in conjunction with, for example, compound screening schemes, as described, below, for the evaluation of the effect of test compounds on gene product levels and/or activity.
• various host animals may be immunized by injection with a RAI gene product, or a portion thereof.
• Such host animals may include, but are not limited to rabbits, mice, and rats, to name but a few.
• Various adjuvants may be used to increase the immunological response, de ⁇ pending on the host species, including but not limited to Freund's (complete and in ⁇ complete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
• BCG Bacille Calmette-Guerin
• Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as a gene product, or an antigenic functional derivative thereof.
• an antigen such as a gene product, or an antigenic functional derivative thereof.
• host animals such as those described above, may be immunized by injection with gene product supplemented with adjuvants as also described above.
• Monoclonal antibodies which are homogeneous populations of antibodies to a par ⁇ ticular antigen, may be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein (1975, Nature 256:495- 497; and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. U.S.A.
• Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
• the hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.
• chimeric antibodies In addition, techniques developed for the production of "chimeric antibodies" (Morri ⁇ son, et al., 1984, Proc. Natl. Acad. Sci., 81 :6851-6855; Neuberger, et al., 1984, Na ⁇ ture 312:604-608; Takeda, et al., 1985, Nature, 314:452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used.
• a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a mur ⁇ ine mAb and a human immunoglobulin constant region.
• An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, referred to as complementarity determining regions (CDRs).
• CDRs complementarity determining regions
• the extent of the framework region and CDRs have been precisely defined (see, "Sequences of Proteins of Im ⁇ munological Interest", Kabat, E. et al., U.S. Department of Health and Human Ser ⁇ vices (1983) ).
• humanized antibodies are antibody molecules from non- human species having one or more CDRs from the non-human species and a framework region from a human immunoglobulin molecule.
• Antibody fragments that recognize specific epitopes may be generated by known techniques.
• such fragments include but are not limited to: the F(ab') 2 fragments, which can be produced by pepsin digestion of the antibody molecule and the Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
• Fab expression libraries may be constructed (Huse, et al., 1989, Science 246:1275-1281) to allow rapid and easy identification of mono ⁇ clonal Fab fragments with the desired specificity.
• Immunoassays for gene products, conserved variants, or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a tissue ex- tract, freshly harvested cells, or lysates of cells in the presence of a detectably la ⁇ beled antibody capable of identifying gene product, conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of tech ⁇ niques well-known in the art.
• the biological sample may be brought in contact with and immobilized onto a solid phase support or carrier, such as nitrocellulose, that is capable of immobilizing cells, cell particles or soluble proteins.
• a solid phase support or carrier such as nitrocellulose, that is capable of immobilizing cells, cell particles or soluble proteins.
• the support may then be washed with suitable buffers followed by treatment with the detectably labeled gene product specific anti ⁇ body.
• the solid phase support may then be washed with the buffer a second time to remove unbound antibody.
• the amount of bound label on the solid support may then be detected by conventional means.
• solid phase support or carrier any support capable of binding an antigen or an antibody.
• supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified cellu ⁇ loses, polyacrylamides, gabbros, and magnetite.
• the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
• the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
• the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
• the surface may be flat such as a sheet, test strip, etc.
• Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
• RAI gene product-specific antibody can be detectably labeled is by linking the same to an enzyme, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, ⁇ -glycero- phosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, ⁇ -galactosidase, ribonucle- ase, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
• the detection can be accomplished by colorimetric methods that employ a chromogenic substrate for the enzyme. Detection may also be ac- complished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
• Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, by Ia- beling the antibody with a fluorescent compound.
• fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phyco- erythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
• the antibody can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series or by coupling it to a chemilumines- cent compound.
• Described herein are various applications of gene sequences, gene products, in ⁇ cluding peptide fragments and fusion proteins thereof, and of antibodies directed against gene products and peptide fragments thereof.
• Such applications include, for example, prognostic and diagnostic evaluation of a disease, such as cancer, and the identification of subjects with a predisposition to such disorders, as described above.
• the method according to the invention may be used in relation to any cancer form, such as, but not limited to, skin carcinoma including malignant melanoma, breast cancer, lung cancer, colon cancer and other cancers in the gastro-intestinal tract, prostate cancer, lymphoma, leukemia, multiple myeloma, pancreas cancer, head and neck cancer, ovary cancer and other gynecological cancers.
• the method is relevant for skin cancer, lung cancer, colon cancer, multiple myeloma, and breast cancer, such as skin cancer, breast cancer, multiple myeloma, and lung cancer, such as skin cancer, breast cancer and lung cancer, such as skin cancer and breast cancer, preferably wherein the skin cancer is basal cell carcinoma, such as lung cancer.
• the cancer is multiple myeloma
• the cancer is breast cancer.
• the cancer may also be skin cancer, preferably basal cell carcinoma, for example early age basal cell carcinoma.
• the method is relevant for both early age cancer and later age cancer, such as early age breast cancer, and such as later age breast cancer.
• the method is of particular relevance for lung cancer, such as in patients with XPD exon 23 M .
• the method is also in particular relevant for early age skin cancer, such as early age basal cell carcinoma.
• Gene nucleic acid sequences can be utilized for transferring re ⁇ combinant nucleic acid sequences to cells and expressing said sequences in recipi- ent cells. Such techniques can be used, for example, in marking cells or for the treatment of cancer. Such treatment can be in the form of gene replacement ther ⁇ apy. Specifically, one or more copies of a normal RAI gene or a portion of the RAI gene that directs the production of an RAI gene product exhibiting normal RAI gene function, may be inserted into the appropriate cells within a patient, using vectors that include, but are not limited to, adenovirus, adeno-associated virus, and retrovi ⁇ rus vectors, in addition to other particles that introduce DNA into cells, such as lipo ⁇ somes.
• the invention may be used in relation to inflammatory dis- eases, such as, but not limited thereto, rheumatoid arthritis, colitis ulcerosa, Crohn's disease, thyroiditis, neural inflammation as in Alzheimer's disease, and Guillain- Barre syndrome.
• inflammatory dis- eases such as, but not limited thereto, rheumatoid arthritis, colitis ulcerosa, Crohn's disease, thyroiditis, neural inflammation as in Alzheimer's disease, and Guillain- Barre syndrome.
• the primary effectors i.e. the mutations causing cancer according to the present invention may be found within the region including and flanked by the marker RAI- 37 and the polymorphism having the sequence
• the region and markers herein represents the region around position 24000 which is shown to be important in relation to can ⁇ cer according to the present invention.
• the primary effector may be selected from the group consisting of
• the primary effectors i.e. the mutations causing cancer according to the present invention may be be selected from the group consisting of
• the primary effectors i.e. the mutations causing cancer according to the present invention may be be selected from the group consisting of
• the primary effectors i.e. the mutations causing cancer ac ⁇ cording to the present invention may further be be selected from the group consist ⁇ ing of
• the primary effectors i.e. the mutations causing cancer according to the present invention may further be be selected from the group con ⁇ sisting of
• the primary effectors i.e. the mutations causing cancer ac ⁇ cording to the present invention may further be be selected from the group consist ⁇ ing of
• the primary effectors i.e. the mutations causing cancer according to the present invention may further be be selected from the group consisting of
• the primary effectors i.e. the mutations causing cancer according to the present invention may further be be selected from the group consisting of
• the primary effectors i.e. the mutations causing cancer according to the present invention may further be be selected from the group consisting of
• the primary effector may be the mutation corresponding to the sequence TTTTAG- TAGAGACATGGTTCCGCCA[C/ ⁇ GTTGCCCAGGCTGGTCTTGAACTCC posi ⁇ tioned at 18146823 of Contig nt_011109.
• the primary effec ⁇ tor may be the mutation corresponding to the sequence ctggggaggctgaggcagga- gaatc[A/G]cttgaaaccgggaggcggaggttgt positioned at 18147126 of Contig nt_011109.
• the primary effector may be the mutation corresponding to the sequence GATTGTCATGT[G/T]ACATCAGCCAATACT posi ⁇ tioned at 18146233 of Contig nt_011109.
• the primary effector may be the mutation corresponding to the sequence caggcggatca- caaggtcaggagtt[C/T]gagaccagcctggccaacacagtga positioned at 18148193 of Contig nt_011109.
• the primary effector may be the mutation corre ⁇ sponding to the sequence CACAGTGAAAC[C/T]CCATCTCTACTAAA positioned at 18149120 of Contig nt_011109.
• the primary effec- tor may be the mutation corresponding to the sequence AGCCTGGCCAACATG[CZG]TGAAACCCCGTCTCT positioned at 18150815 of Contig nt_011109.
• the primary effector may be the muta ⁇ tion corresponding to the sequence ctcgggaggctgaggcagga- gaatc[A/G]cttgaactcaggaggcagaggttgc positioned at 18150911 of Contig nt_011109.
• the primary effector may be the mutation corresponding to the sequence AAGTTTCTCTATT[GZT]TGTTTATAAACA positioned at 18151158 of Contig nt_011109.
• the primary effector may be the mutation corresponding to the sequence CCCTATGTTGTCCAAGCTGGCAGAG[AZG]TTTTT-GTTTGTTTGTTTGAGAGGGA positioned at 18150199 of Contig nt_011109.
• the primary effector may be the mutation corresponding to the sequence ac- taaaataaaaaataaaaaaaaaaaa[-ZAA]atagccgagcatggtggtgggtgcc positioned at 18147192 of Contig nt_011109.
• the primary effector may be the muta- tion corresponding to the sequence AAAAAACTAAAGTGGGGTTTGCGGG[GZT]- AGTGGGAGGGCCCTTCCTGCTAGGT positioned at 18147886 of Contig nt_011109 .
• the primary effector may be the mutation cor ⁇ responding to the sequence AAAATTAGCCGG[AZG]CGCCATGGCGGGAG posi ⁇ tioned at 18149154 of Contig nt_011109.
• the primary effector may be the mutation corresponding to the sequence GGTTTAT[ATTTT]Ntgagatggatttt positioned at 18147012 of Contig nt_011109.
• the primary effectors may however be any combination of the polymorphisms.
• polymorphisms employed herein for providing a method andZor composi- tions for identifying human subjects with an increased risk of having or developing disease
• a number of polymorphisms are novel and have been identified by the pre ⁇ sent inventors.
• the position according to Contig nt_011109 and the nucleotide sequence of the identified polymorphism are provided, whereas the novel polymorphisms cannot be assigned trivial names or identification numbers according to the dbSNP database.
• the primary effectors i.e. the mutations causing cancer according to the present invention may be found within the region around position 39000 which is shown to be important in relation to cancer according to the present invention.
• the region includes and is flanked by the marker Rai intron 1 and the polymorphism having the sequence TCCAGCCTGGGCAAGAA[C/G]AGTGAAACTCCAGCTT corresponding to position 18165052 of contig nt_011109.
• the primary effector may be selected from the group consisting of
• the primary effectors i.e. the mutations caus ⁇ ing cancer according to the present invention may be be selected from the group consisting of
• the primary effectors i.e. the mutations causing cancer according to the present invention may be be selected from the group consisting of
• the primary effectors i.e. the mutations causing cancer according to the present invention may be be selected from the group consisting of
• the primary effectors i.e. the mutations caus ⁇ ing cancer according to the present invention may be be selected from the group consisting of
• the primary effectors i.e. the mutations causing cancer according to the present invention may be be selected from the group consisting of
• the primary effector may be selected from the group con ⁇ sisting of
• the primary effector may be the muta ⁇ tion corresponding to the sequence ggagcttgcagtgagctga- gatcgc[A/G]ccactgcactccagcctgggcgaca positioned at 18159091 of Contig nt_011109.
• the primary effector may be the mutation corre ⁇ sponding to the sequence TTCTCCTGACCTC[A/G]TGATCCGCCCACCTCGG posi ⁇ tioned at 18159263 of Contig nt_011109.
• the primary effector may be the mutation corresponding to the sequence GGGATTACAGG- CATGC[A/G]CCACCAGGCCCAGCTAATTTTTGT positioned at 18160363 of Contig nt_011109.
• the primary effector may be the mutation corre ⁇ sponding to the sequence TCCAATGGTGACA[A/C]CAGTAAGAGCAGTTAACAG positioned at 18160936 of Contig nt_011109.
• the primary effector may be the mutation corresponding to the sequence TCCAATGGTGACAA[C/G]AGTAAGAGCAGTTAACAG positioned at 18160937 of Contig nt_011109.
• the primary effector may be the mutation corresponding to the sequence tacaggcgcccgccac- cacccccag[A/C]taatttttgtatttttagtagagac positioned at 18161433 of Contig nt_011109.
• the primary effector may be the mutation corre ⁇ sponding to the sequence TTGCCTCAGCCTCCTGA[G/T]TAGCTGGGATTGGAATGAGA positioned at 18161694 of Contig nt_011109.
• the primary effec- tor may be the mutation corresponding to the sequence TACGA- TAAATAGCTAGA[C/GACCTTGGCGCCACCATCT ⁇ positioned at 18161841 of Contig nt_011109.
• the primary effector may be the mutation corresponding to the sequence AAAATAATAATAATAATAT- TAA[C/T]CCTGACCTTGGCGCCACCATCT positioned at 18161896 of Contig nt_011109.
• the primary effector may be the muta ⁇ tion corresponding to the sequence tcgtcctgctacagaatta- caggca[C/T]gcgccaccgctccgggctaattttt positioned at 18162206 of Contig nt_011109.
• the primary effector may be the mutation corresponding to the sequence CCTCATGAGCCACCCAC[CZT]TCGGCCTCCCAAAGTGCT posi- tioned at 18162309 of Contig nt_011109.
• the primary effector may be the mutation corresponding to the sequence TGAGCCACCGCGCCC[A/G]GCCGAGACTCACTATTT positioned at 18162356 of Contig nt_011109.
• the primary effector may be the mu ⁇ tation corresponding to the sequence taaagcgggag- gatggcttgaacct[A ⁇ 3]ggaggcggaggttgcagtgagccga positioned at 18162599 of Contig nt_011109. In a further embodiment.
• the primary effec ⁇ tor may be the mutation corresponding to the sequence GGAGAGAAGGAGCAGA- GAAC[A/C]TCTCTATGTGGCCA positioned at 18162903 of Contig nt_011109.
• the primary effector may be the mutation corre- sponding to the sequence ATCCTAAAGACTAC[A/C]TTTCCCAGCATCCCA posi ⁇ tioned at 18162970 of Contig nt_011109.
• the primary effector may be the mutation corresponding to the sequence TTTCCCAG- CATCCCA[C/T]TGCAATGAGGCTCCTGGCC positioned at 18162986 of Contig nt_011109.
• the primary effector may be the mutation corresponding to the sequence TCCTGACTCCAGTG[A/C]GGTGCCTACAGTCCTG positioned at 18163200 of Contig nt_011109. In yet a preferred embodiment the primary effector may be the mutation corresponding to the sequence TTCCAGCCTGGGCAAGAA[C/G]AGTGAAACTCCAGCTT positioned at 18165052 of Contig nt_011109. In a further preferred embodiment the primary effectors may however be any combination of the polymorphisms. RAI function
• the primary effector influences activity or expression of RAI or XPD.
• the effector could modify the sequence of RAI protein, through amino acid substitution, splicing or termination.
• one likely position of the effector is in the 3' portion of the gene and that the effector modifies mRNA expression and stability (22).
• the effector was a modification of an enhancer situated between RAI and XPD, again modulating the expression of RAI, and possibly modulating other nearby genes as well.
• the primary effector may be located in the promoter or first intron of RAI and influence the transcriptional activity of the gene.
• RAI protein is an inhibitor of ReIA, a subunit of the transcription factor NF- ⁇ B (23, 24). NF- ⁇ B has long been implicated in both cell proliferation and apoptosis. Modulation of NF- ⁇ B may well be part of a "crunch-time scenario", in ⁇ voked when the cell has to muster its forces and make life-and-death decisions. A recent scientific paper suggests that the choice between the cell survival and death is regulated by the relative activity of the two subunits encoded by ReIA and c-rel, respectively (25). By neutralising the ReIA product, RAI protein would presumably shift this balance towards apoptosis. RAI protein may also influence p53 activity (26).
• the present invention relates to method of estimating the disease risk of an individ ⁇ ual comprising further a predictor of RAI action.
• the length of the RAI transcript may be used as a predictor.
• the quantity of RAI transcripts may be used as a predictor.
• the present invention comprises the combined RAI transcript characteristics described above as a predictor of RAI action and the estimation of the disease risk of an individual. Examples
• HRT hormone replacement therapy
• menopausal status were also recorded.
• blood, urine, fat tissue and other biological material was sam ⁇ pled and stored in a biobank at -15O 0 C.
• Cohort members were identified by a unique identification number, which is allocated to every Danish citizen by the Central Population Registry. Cohort members were linked to The Central Population Regis ⁇ try for information on vital status and immigration. Information on cancer occurrence among cohort members was obtained through record linkage to the Danish Cancer Registry, which collects information on all inhabitants in Denmark who develop can ⁇ cer (7). Linkage was performed by use of the personal identification number.
• Basal cell carcinoma BCC basal cell carcinoma
• the groups of Caucasian Americans with and without BCC have been described previously (Athas et al, Cancer Res. 51 :5786-5793, 1991; Wei et al, Proc. Natl. Acad. Sci USA, 90: 1614-8, 1994). Briefly, the study was a clinic based case control study at the Johns Hopkins Hospital, which serves multiple participating dermatologists in Maryland. Cases were histo-pathologically confirmed primary BCCs and were diagnosed be ⁇ tween 1987 and 1990. The controls were patients from the same physician practices and had a diagnosis of mild skin disorders. All participants were Caucasians living near Baltimore and were between 20 and 60 years of age.
• the controls were fre- quency matched to the cases by age and sex. Cases and controls with any other forms of cancer were excluded.
• the study subjects were asked if they had any blood relatives with skin cancer, and were asked to specify the type of cancer. Study subjects with relatives with basal cell carcinoma and squamous cell carcinoma and 'skin cancer' were included in the group of subjects with a family of skin cancer. Subjects with relatives with melanoma were not included.
• the subjects gave informed consent, were examined by dermatologists, com ⁇ pleted a structured questionnaire and provided blood. Available frozen lymphocytes were genotyped. Initially, 71 cases and 118 controls were included in this study. However, the number of persons varied between analyses, as the supply of DNAs gradually was depleted. In case of the SNP RAI intron 1 only 133 persons could be genotyped reliably.
• the examples relate to prediction from sequence polymorphisms in and around the region r to cancer, see Fig. 1 for an overview of a subregion of chromosome 19q.
• 27 markers were investigated within this 69 kb stretch for associa ⁇ tion with breast, lung, skin cancer, and multiple myeloma, using linkage disequilib ⁇ rium mapping based on single markers, as well as, on haplotypes combining several neighboring markers.
• haplotypes with maximal association to all three cancers are centered around the gene RAI.
• Table 10 lists the polymorphisms used in this study, their nature, the numbers in the NCBI database dbSNP, the sequence defining the SNPs, and their position therein, the currently estimated chromsome positions and their relative position within the region of interest (which can readily be calculated based on the information provided in the table).
• Table 11 lists the primers used for the PCR reactions.
• Table 12 lists the probes used for detection and typing.
• Table 13 lists the PCR regimen for the poly ⁇ morphisms using Lightcycler, Taqman and ABI 2720.
• rs numbers were derived from the NCBI's database dbSNP; Nucleotide sequence of polymorphisms identified in the present invention with no trivial name as yet:
• Haplotype trend regression is basically a two-stage procedure. First, genotype results in combination with population assumptions such as Hardy-Weinberg equilibrium are used to con ⁇ struct all haplotype probabilities corresponding to a given set of markers for each individual. Secondly, the disease state (1 for cases, 0 for controls) is regressed on the haplotype probabilities of all individuals, resulting in a p-value for the overall as- sociation of the set of markers with disease, and parameters for association for each specific haplotype with disease.
• population assumptions such as Hardy-Weinberg equilibrium are used to con ⁇ struct all haplotype probabilities corresponding to a given set of markers for each individual.
• the disease state (1 for cases, 0 for controls) is regressed on the haplotype probabilities of all individuals, resulting in a p-value for the overall as- sociation of the set of markers with disease, and parameters for association for each specific haplotype with disease.
• the HelixTree program will use sets of markers of defined size, typically 2 to 4 neighboring markers at a time, to scan the entire region. It can then be used to derive frequencies of the individual haplotypes, to calculate an overall p-value for the distribution of haplotypes covering a given set of markers among cases and controls, and to calculate p-values for the distribution of each hap ⁇ lotype derived from a given set of markers.
• the program RASCAL for performing gene localization according to Lazzeroni (16) was implemented in Delphi (B.A. Nex ⁇ , unpublished). We used a bootstrap set of 10000 sets of haplotypes selected from the original set with replacement, and to avoid a Q-form with negative values we used a relaxation value of 0.25. A 95% con- fidence interval for the location of the causative gene variant was derived from the Q-form. Places, where it took on a value less that 3.85 above the minimum, were considered inside the confidence interval.
• primers and probes can be designed based on the information provided herein, in order to detect polymorphism by other means than sequencing, for example by PCR ampliation methods as also used herein.
• Primers were purchased from DNA Technology (Aarhus, Denmark), probes for the lightcycler were obtained from MolBiol (Tvhofer Weg 11-12, Berlin) and the probes for Taqman were supplied by Applied Biosystem.
• 5'-cta gag get cag tgt taa tct gtt cct ERCC1-3' 5' gga cag atg gca atg atg g g
• 5'-cta gag get cag tgt taa tct gtt cct ERCC1-3' 5' gga cag atg gca atg atg g g
• R is Roche buffer
• H homebrew buffer
• M mastermix
• T denotes Tagman technique
• L Lightcycler
• A ABI2720 se ⁇ quencing, respectively.
• R is Roche buffer: 10 pmole primers
• H is Homebrew: 10 pmole primers 1 pmole probes
• Polymorphisms by real-time PCR using Taqman probes. Polymor ⁇ phisms analyzed on Taqman were scored on the basis of the relative reaction of the 2 probes. Some polymorphisms were analysed using the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, Ca, USA). PCR Primers and Taqman probes were designed using Primer Express v 1.0 (Applied Biosystems).
• the reactions were performed in MicroAmp optical tubes sealed with MicroAmp op ⁇ tical caps (Applied Biosystems) containing a 10 ⁇ l reaction volume (Mastermix): 1x Taqman buffer A, 2.5mM MgCI 2 , 200 ⁇ M each of dATP dCTP, dGTP, 400 ⁇ M dUTP, 80OnM each primer, 200nm each probe, 0,01 U/ ⁇ L AmpErase UNG, 0,025 U/ ⁇ L AmpliTaq Gold Polymerase. Tubes were incubated at 50 0 C for 2 min followed by 10 min at 95 0 C. The incubation was succeeded by 45 cycles with thermal cycler condi ⁇ tions as shown in Table 13.
• XPD exon 10 PCR was performed by rapid-cycling in a reaction vol ⁇ ume of 20 ⁇ l with 0.5 ⁇ M of each primer, 0.045 ⁇ M of anchor and sensor probe, 3.5 mM MgCI 2, approximately 7 - 25 ng genomic DNA, and 2 ⁇ l LightCycler DNA Master Hybridization probe buffer (Roche Molecular Biochemicals, Cat. No 2158 825).
• This buffer contains Taq DNA polymerase, dNTP mix, and 10 mM MgC ⁇ and 5% DMSO.
• the temperature cycling consisted of denaturation at 95 0 C for 2 sec, followed by 46 cycles consisting of 10 sec at 95 0 C, 15 sec at 53°C, and 30 sec at 72°C, see table 4a for PCR regimen for the polymorphisms using LightCyclerTM.
• the last annealing pe- riod at 72°C was extended to 120 sec followed by denaturation for 10 sec at 95°C.
• the melting profile was determined by a temperature ramp from 50 0 C to 95°C with a rate of 0.1 degree/sec.
• the region consists of 2 major haplotype blocks with a few interspersed markers in between.
• One haplotype block spans roughly 16 markers and 26 kb from XPD exon6 to RAI intron1-3, while the other haplotype block spans 6 markers and 9 kb from RAI-5' to ERCC1-3'3.
• Fig. 9 depicts the relative risks and the lower confidence limits of the relative risks for postmenopausal breast cancer below age 55 as a function of the location of the markers. The values were initially calculated with the wild-type allele as reference, however, if the calculation gave a RR values less than 1 we used the reciprocal value of the RR and the reciprocal value of the high confidence limit in- stead.
• haplotype trend regression is basically a two-stage procedure. First, genotype results in combination with population as- sumptions such as Hardy-Weinberg equilibrium are used to construct all haplotype probabilities corresponding to a given set of markers for each individual.
• the disease state (1 for cases, 0 for controls) is regressed on the haplotype prob ⁇ abilities of all individuals, resulting in a p-value for the overall association of the set of markers with disease, and parameters for association for each specific haplotype with disease.
• the HelixTree program will use sets of markers of defined size, typi ⁇ cally 2 to 4 neighboring markers at a time, to scan the entire region. It can then be used to derive frequencies of the individual haplotypes, to calculate an overall p- value for the distribution of haplotypes covering a given set of markers among cases and controls, and to calculate p-values for the distribution of each haplotype derived from a given set of markers.
• Fig. 4 shows the overall distribution of p-values for sets of markers plotted against the position on the chromosome for breast cancer.
• position on the abscissa we used the median marker position in a given set of markers.
• the ordinate values are the negative logarithms to the overall p-values for a difference between cases and controls associated with a given set of markers.
• Each curve corresponds to a given size of marker sets, i.e. the number of markers in the haplotypes.
• Fig. 4 The results in Fig. 4 suggest that the association with breast cancer has two peaks. One was located at roughly 24 kb and one at 39 kb. When the cases were broken down into early and late breast cancer the peak at 24 kb was present in both groups, while the peak at 39 kb was only present in the older group. The peaks were clearly present in curves for haplotypes of 2, 3 and 4 neighboring markers. Larger sets were less informative, presumably due to increased degrees of freedom, but they essentially corroborated the results (results not shown).
• this association at 24 kb was present for breast cancer both in young and in older female cases. This is the first time an association to RAI has been re ⁇ ported for cancers in older populations.
• the position of the optimal set of markers corresponds to the 3' part of the gene RAI and the inter-gene region between RAI and XPD and was identical for young and older cancer cases.
• the peak at 39 kb which is only present in the older breast cancers correspond to the 5' part of RAI.
• haplotypes for the set of 2 markers, located at 39 kb. Again the deviation of young and old breast cancer cases from the controls had similarity (RAI intron1-2 9 RAI intron1-3 a up; RAI intron1-2 g RAI intron1-3° down). The same pattern was present in the lung cancer data. This is remarkable, as neither the young breast cancers nor the lung cancers showed a peak in this position, and sug- gests an underlying similarity of haplotypes in the different groups.
• DNA was isolated from lymphocytes from humans from the American cohort of pa ⁇ tients with basal cell carcinoma and controls, described in Materials and Methods, was typed with respect to a number of sequence polymorphisms located in and around the claimed region r. The salient features of the Americant cohort are shown in Table 16.
• Figure 7 shows the same kind of result for early basal cell carcinoma. Again we found a strong association with the region around RAI, with the curve for sets of 3 markers possibly shifted slightly to the right, and the curve for 4 markers showing suggestions of an extra association at higher positions.
• DNA from humans from the Danish cohort of patients with lung cancer and controls was typed with respect to a number of sequence polymorphisms located in and around the claimed region r.
• Fig. 7 shows that also this disease is associated with sets of markers span ⁇ ning the distal end of RAI and the inter-gene region in the population with the XPD exon23 AA genotype. This is the group of patients free of influence of the XPD gene. No association was evident in the two other groups (results not shown).
• the ASE-1 genotype influences relapse-free survival and survival in Multiple Mye ⁇ loma 391 patients diagnosed with multiple myeloma were found eligible for transplantation in Denmark in the period 1993-2004.
• the multiple myeloma patients were treated with high dose alkylating chemotherapy followed by autologous bone marrow transplantation.
• the overall survival was sig- nificantly shorter for patients who experienced relapse than for patients who did not.
• ASE-1 genotype was found to strongly influence the event-free survival (EFS) and overall survival in patients with multiple myeloma who were auto-transplanted.
• EFS event-free survival
• variant allele carries had 1.5 year longer event-free survival, see Fig. 9.
• the EFS was simi ⁇ lar for men and women, although the difference was only statistically significant for women (Table 18). This means that the ASE-1 genotype can predict who will benefit most from the present treatment regimen.
• Fig. 11 illustrates, female carriers of the variant allele of ASE-1 polymorphism had a longer relapse-free period than homozygous carriers of the wild-type allele (median 1479 days vs. 714 days), while Figure 12 illustrates that they lived longer (median 3015 vs. 1897). Similar analyses for the men revealed the same tenden ⁇ cies but no statistically significant differences (median 963 vs 736 days for the re ⁇ lapse-free period; mean 2747 vs. 2030 days for the survival), Fig. 13 and Fig. 14, respectively.
• High-risk haplotype carriers (defined as ERCC1 exon 4 M , ASE1 exon1 GG , RAI In- tron 1 M ) had a mean survival of 359 days compared to a mean survival of 244 for non-carriers (Table 19 and Fig. 15).
• the genotype XPD K751Q was also deter ⁇ mined.
• the p-value is for a cox regression.

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