WO2008061672A2

WO2008061672A2 - Genetic risk factor for cancer

Info

Publication number: WO2008061672A2
Application number: PCT/EP2007/009909
Authority: WO
Inventors: Karen Miriam Johanna Van Loo; Gerardus Johannes Maria Martens
Original assignee: Synthon B.V.
Priority date: 2006-11-24
Filing date: 2007-11-08
Publication date: 2008-05-29
Also published as: WO2008061672A3

Abstract

The present invention relates to an isolated nucleic acid molecule that has been found to be implied in the susceptibility of an individual to develop cancer. Specifically, the present invention relates to an isolated nucleic acid molecule comprising a variant of the human Aph-1b gene as depicted in Fig. 1 that causes the amino acid residue in position 217 of the encoded Aph-1b to be an aliphatic amino acid, in particular a leucine.

Description

GENETIC RISK FACTOR FOR CANCER

The present invention relates to an isolated nucleic acid molecule that has been found to be implicated in the susceptibility of an individual to develop cancer, in particular colon cancer. The invention also relates to the protein encoded by the nucleic acid and to in vitro methods for diagnosing the susceptibility of an individual to such disorders and complications, to probes, primers and kits for use in this method and to cell lines harboring this nucleic acid molecule and their use in drug screening.

Colorectal cancer is a major cause of death in the western world. Current therapies involve surgical resection combined with chemotherapy (cytotoxic drugs and radiotherapy) . The outcome of the disease is highly dependant on an early diagnosis. It is important that persons that are susceptible to the disease are monitored frequently to diagnose the disease in an early stadium. A method to diagnose the susceptibility of individuals to suffer cancer disease is therefore advantageous.

Notch/Wnt is a signalling pathway involved in self- renewal of the intestinal epithelium. Notch signaling is involved in maintenance of undifferentiated state of APC- mutant neoplastic cells. The Wnt cascade is the driving force behind proliferative adenomas and adenocarcinomas of the intestine .

In most tissues, active Notch signaling is associated with an immature cell type. For terminal differentiation, this signaling has to be down-regulated. In some tissues, the opposite is required and Notch signaling is required for differentiation. In the latter tissues, loss of Notch signaling is associated with blocked differentiation and tumour progression.

Over-expression of various proteins in the Notch signaling cascade has been found in renal cell carcinoma, prostate cancer, multiple myeloma, Hodgkins and anaplastic lymphomas. Accumulating data indicate that deregulated Notch activity is also involved in the genesis of other cancers

(pancreatic, medulloblastoma, mucoepidermoid carcinoma) . Aphl-b is a multipass transmembrane protein that interacts with presenilin and nicastrin as a functional component of the gamma-secretase complex. The gamma-secretase complex is required for the intramembrane proteolysis of a number of membrane proteins, including the amyloid-beta precursor protein and Notch. Goutte et al., (Proc Natl Acad Sci USA. 99(2):775-9

(2002)) concluded that Aphl and presenilins are required for cell surface localization of the Notch component Aph2

(nicastrin) .

Francis et al. (Dev Cell. 3(l):85-97 (2002)) determined that Aphl and Pen2 were required for Glpl/Notch- mediated signaling, both in embryonic patterning and in postembryonic germ line proliferation.

Francis et al. (2002), supra, also observed reduction in gamma-secretase cleavage of beta-APP and Notch substrates and reduction in the levels of processed presenilin. They concluded that APHl and PEN2 are required for Notch pathway signaling .

Nevertheless a direct relation between the Aph-lb protein and cancer has never been established before. In the research that led to the present invention it was surprisingly found that a single-nucleotide polymorphism

(SNP) in the human Aph-lb gene is associated with the susceptibility of an individual to develop cancer. It was more in particular found that the SNP in position 651 of the Aph-lb gene, which changes the amino acid in position 217 in the Aph-lb subunit of gamma-secretase from F to L, is directly linked with the susceptibility of individuals to develop cancer and particularly colon cancer. Table 1 shows the SNP651 genotype and allele distribution in cancer cases and controls of Caucasian origin.

Aph-lb is known as a component of the gamma-secretase complex. Subtle alterations in gamma-secretase subunit composition may lead to a variety of affected developmental signaling pathways and, consequently, aberrations in cell differentiation and growth.

SNP651 (T>G) in exon 6 of the Aph-lb gene showed a highly significant difference in the frequency of its genotype and allele between cancer patients and control groups. The genotype frequency was found to be about 6-7% in the group of colon cancer patients against about 3% in controls (Table Ia) The genotype frequency for throat cancer was found to be 5.3% (Table Ib) .

Table Ia

Aph-lb SNP651 t>g genotype and allele distribution in patients and controls

SNP = Single-Nucleotide Polymorphism NBS = Nijmegen Biomedical Study

Table Ib

SNP = Single-Nucleotide Polymorphism NBS = Nijmegen Biomedical Study

This T>G substitution leads to a change in amino acid residue 217 from phenylalanine to leucine (Phe/Leu) . Amino acid residue 217 is conserved among all known Aph-lb sequences and also among all sequences of its paralogue Aph- Ia from man to worm (either Phe or Tyr) .

In view of the observed magnitude of the association between SNP651 and the susceptibility to develop cancer, the presence of this genotype in an individual is a significant genetic risk factor that together with other genetic or environmental risk factors increases the likelihood of becoming diseased. In view of the above, the present invention thus provides the use of an isolated nucleic acid molecule comprising a variant of the human Aph-lb gene as depicted in Fig. 1 (SEQ ID NO:1) that causes the amino acid residue in position 217 of the encoded Aph-lb to be an aliphatic amino acid, in particular a leucine, in the diagnosis of the susceptibility of an individual to develop cancer. In a specific embodiment, the variant Aph-lb gene has the nucleotide sequence as depicted in any one of the Figs. 2A-F (SEQ ID NOS:3-8), or a fragment thereof that comprises the codon encoding amino acid residue 217 of the encoded component Aph-lb. The isolated nucleic acid of the invention is in a further embodiment a nucleic acid encoding a polypeptide having the amino acid sequence as depicted in Fig. 4 (SEQ ID NO: 9). Due to the degeneracy of the genetic code one amino acid sequence can be encoded by various nucleic acid sequences.

The isolated nucleic acid molecule which hybridizes under high stringency conditions to a nucleotide sequence selected from the group consisting of the sequences as shown in Fig. 2A-F (SEQ ID NOS: 2-7) and the complement of the sequence as shown in Fig. 2A-F (SEQ ID NOS:2-7), with the proviso that the nucleic acid has a codon selected from TTA, TTG, CTT, CTC, CTA, CTG in the position encoding the amino acid residue in position 217 of the encoded Aph-lb. Preferably, the codon in the position encoding the amino acid residue in position 217 of the encoded Aph-lb is TTG.

The skilled person knows how to define "high stringency" conditions for the different hybridisation techniques that are generally available. In general stringency is determined by various factors, among which the temperature, the solvent (i.e. aqueous or with formamide) , the volume of the hybridisation solution and length of hybridisation and the salt concentration. High stringency is usually a temperature above 50⁰C and a salt concentration of at least 2x SSC.

The invention further relates to the use of an isolated nucleic acid molecule comprising the complement of a sequence described above in the diagnosis of the susceptibility of an individual to develop cancer.

According to a further aspect thereof the invention provides the use of an isolated polypeptide encoded by the nucleic acid molecule, or a fragment of said polypeptide, which polypeptide or fragment comprises an aliphatic amino acid, in particular a leucine, in the position that corresponds with position 217 in human Aph-lb as depicted in Fig. 3 (SEQ ID NO: 8) in the diagnosis of the susceptibility of an individual to develop cancer. Suitably, the isolated polypeptide has the amino acid sequence of Fig. 4 (SEQ ID NO: 9) or is an isolated polypeptide comprising an amino acid sequence which is more than about 90 percent identical to the amino acid sequence of Fig. 3 (SEQ ID NO: 8) and which has an aliphatic amino acid, in particular a leucine, in the position that corresponds with position 217 in human Aph-lb.

The invention also relates to the use of a fusion protein comprising the isolated polypeptide and to an antibody, or an antigen-binding fragment thereof, which selectively binds to the polypeptide, in particular to a part that comprises the amino acid in position 217 of Aph-lb in the diagnosis of the susceptibility of an individual to develop cancer.

The variant gene and the encoded polypeptide and products derived therefrom can thus suitably be used in the diagnosis of the susceptibility of an individual to develop cancer. For this, primers and probes can be derived from the variant gene. "Probes" or "primers" are oligonucleotides that hybridize in a base-specific manner to a complementary strand of nucleic acid molecules. The term "primer" in particular refers to a single-stranded oligonucleotide which acts as a point of initiation of template-directed nucleic acid synthesis using well-known methods (e.g., PCR, LCR, NASBA, etc.) including, but not limited to those described herein. Typically, a probe or primer comprises a region of nucleotide sequence that hybridizes to at least about 15, typically about 20-25, and more typically about 40-75, like 40, 50 or 75, consecutive nucleotides of a nucleic acid molecule comprising a contiguous nucleotide sequence selected from the sequences shown in Figs. 2A-P (SEQ ID NOS: 2-7), the complement of the sequences shown in Figs. 2A-F (SEQ ID NOS: 2-7), or a sequence encoding the amino acid sequence as shown in Fig. 4 (SEQ ID NO: 9) . In preferred embodiments, a probe or primer comprises 100 or fewer nucleotides, preferably from 6 to 50 nucleotides, preferably from 12 to 30 nucleotides. In other embodiments, the probe or primer is at least 70% identical to the above nucleotide sequence or to the complement of the above nucleotide sequence, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95% identical, or even capable of selectively hybridizing to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence. Often, the probe or primer further comprises a label, e.g., radioisotope, fluorescent compound, enzyme, or enzyme co-factor. The probe or primer of the invention should contain a nucleotide that is the complement of position 651 of either the known or the variant Aph-lb gene. Representative oligonucleotides useful as probes or primers are given in Table 2.

Table 2 primer nucleotide sequence name

T-primer forward primer: 5¹- AATAAACCTGGCGTCAGCATTT -3¹ reverse primer: 5¹- AGTCGGCTTTACACTGTCCCA -3¹

G-primer forward primer: 5'- AATAAACCTGGCGTCAGCATTG -3' reverse primer: 5¹- AGTCGGCTTTACACTGTCCCA -3'

The nucleic acid molecules that can be used in the invention such as those described above can be identified and isolated using standard molecular biology techniques and the sequence information provided in Figs. 1 and 2A-P (SEQ ID NOS: 1-7) . For example, nucleic acid molecules can be amplified and isolated by the polymerase chain reaction using synthetic oligonucleotide primers designed based on one or more of the sequences provided in Figs. 1 and 2A-F (SEQ ID NOS: 1-7) and/or the complement thereof, or designed based on sequences encoding the amino acid sequence provided in Fig. 4 (SEQ ID NO: 9). See generally PCR Technology: Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY, N. Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al . , Nucleic Acids Res., 19:4967 (1991); Eckert et al., PCR Methods and Applications, 1:17 (1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. No. 4,683,202. The nucleic acid molecules can be amplified using cDNA, mRNA or genomic DNA as a template, cloned into an appropriate vector and characterized by DNA sequence analysis.

Other suitable amplification methods include the ligase chain reaction (LCR) (see Wu and Wallace, Genomics, 4:560 (1989), Landegren et al . , Science, 241:1077 (1988), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989)), and self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87:1874 (1990)) and nucleic acid based sequence amplification (NASBA) . The latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively. The amplified DNA can be radiolabelled or provided with another suitable label, such as a fluorescent label, and used as a probe. Such probe can be used for detecting another amplified nucleic acid molecule or for screening.

Antisense nucleic acid molecules for use according to the invention can be designed using the nucleotide sequences of Figs. 1 and 2A-F (SEQ ID NOS: 1-7) and/or the complement and/or a portion thereof, and/or a sequence encoding the amino acid sequence of Fig. 4 (SEQ ID NO: 9), or encoding a portion thereof, and constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid molecule (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.

Alternatively, the antisense nucleic acid molecule can be produced biologically using an expression vector into which a nucleic acid molecule has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid molecule will be of an antisense orientation to a target nucleic acid of interest) . A further approach can include an RNA interference approach by expressing double stranded RNA or si-RNA.

The invention further relates to an in vitro method of diagnosing a susceptibility to develop cancer in an individual, comprising screening for the presence of SNP651 (SEQ ID NO: 10) in the gene encoding the human Aph-lb, which SNP651 is more frequently present in a population of individuals susceptible to develop cancer than in the general population, and wherein the presence of SNP651 is indicative of the existence of cancer or the susceptibility to develop cancer. The SNP651 is more frequently present when there is a significant increase in its occurrence as compared to the normal population. In a specific embodiment the normal population and the patient are Caucasian.

The subjects may have been previously diagnosed as having cancer or the screening may be used in conjunction with diagnostic efforts.

The method comprises determining the presence of SNP651 in the genotype of the subject. The genotype that is detected indicates that the subject is likely to have the phenotypic response associated with that genotype.

SNP651 is a single nucleotide polymorphism in whichthe T in position 651 of the coding part of the gene (wherein the start codon ATG represent positions 1-3) is changed into

G.

The in vitro method of diagnosis according to the invention is suitable for all types of cancer, and in particular for colon cancer or throat cancer.

Diagnosis of a susceptibility to develop cancer is thus made by detecting the SNP651 polymorphism in the Aph-lb gene. The polymorphism is the change of one nucleotide, resulting in a change in the encoded amino acid. In a first method of diagnosing a susceptibility to develop cancer, hybridization methods, such as Southern analysis, Northern analysis, or in situ hybridizations are used. These techniques are well known in the art and are for example disclosed in Current Protocols in Molecular Biology, Ausubel, F. et al . , eds . , John Wiley & Sons, including all supplements .

For example, a biological sample from a test subject (a "test sample") of genomic DNA, RNA, or cDNA, is obtained from an individual suspected of having, being susceptible to or predisposed for cancer (the "test individual"). The individual can be an adult, child, or fetus. The test sample can be from any source which contains genomic DNA, such as a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract a or other organs. A test sample of DNA from fetal cells or tissue can be obtained by appropriate methods, such as by amniocentesis or chorionic villus sampling.

The DNA, RNA, or cDNA sample is then examined to determine whether the polymorphism in Aph-lb is present. The presence of the polymorphism can be indicated by hybridization of the gene in the genomic DNA, RNA, or cDNA to a nucleic acid probe. A "nucleic acid probe", as used herein, can be a DNA probe or an RNA probe; the nucleic acid probe contains the SNP651 polymorphism in Aph-lb. The probe can be any of the nucleic acid molecules described above (e.g., the gene, a fragment, a vector comprising the gene, a probe or primer, etc. )

To diagnose a susceptibility to develop cancer, a hybridization sample is formed by contacting the test sample containing the Aph-lb gene, with at least one nucleic acid probe. A preferred probe for detecting mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA sequences described herein. The nucleic acid probe can be, for example, a full-length nucleic acid molecule, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to appropriate mRNA or genomic DNA. For example, the nucleic acid probe can be all or a portion of the sequence shown in Figs. 2A-F (SEQ ID NOS: 2-7), or the complement thereof, or a portion thereof, or can be a nucleic acid encoding all or a portion of the amino acid sequence shown in Fig. 4 (SEQ ID NO: 9). Suitable probes for use in the diagnostic assays of the invention comprise a nucleotide that is complementary to position 651 of the Aph-lb gene or variant thereof.

The hybridization sample is maintained under conditions, which are sufficient to allow specific hybridization of the nucleic acid probe to the Aph-lb gene. "Specific hybridization", as used herein, indicates exact hybridization (e.g., with no mismatches). Specific hybridization can be performed under high stringency conditions or moderate stringency conditions. In a particularly preferred embodiment, the hybridization conditions for specific hybridization are high stringency. The specific hybridization can take place over the complete length of the probe but should in any case take place over the region that contains the SNP651.

Specific hybridization, if present, is then detected using standard methods. If specific hybridization occurs between the nucleic acid probe and the Aph-lb gene in the test sample, then the Aph-lb gene has the polymorphism that is present in the nucleic acid probe. Specific hybridization of the nucleic acid probe is indicative of a polymorphism in the Aph-lb gene, and is therefore diagnostic for a susceptibility to develop cancer.

In Northern analysis (see for example Current Protocols in Molecular Biology, Ausubel, F. et al, eds., John Wiley & Sons, supra) , the hybridization methods described above are used to identify the presence of the SNP651 polymorphism, associated with a susceptibility to develop cancer. For Northern analysis, a test sample of RNA is obtained from the individual by appropriate means. Specific hybridization of a nucleic acid probe, as described above, to RNA from the individual is indicative of the SNP651 polymorphism in the Aph-lb gene, and is therefore diagnostic for a susceptibility to develop cancer. Alternatively, a peptide nucleic acid (PNA) probe can be used instead of a nucleic acid probe in the hybridization methods described above. PNA is a DNA mimic having a peptide- like, inorganic backbone, such as N- (2-aminoethyl) glycine units, with an organic base (A, G, C, T or U) attached to the glycine nitrogen via a methylene carbonyl linker (see, for example, Nielsen, P. E. et al., Bioconjugate Chemistry, 1994, 5, American Chemical Society, p. 1 (1994) . The PNA probe can be designed to specifically hybridize to the Aph-lb gene having the SNP651 polymorphism associated with a susceptibility to develop cancer. Hybridization of such PNA probe to the Aph-lb gene is diagnostic for a susceptibility to develop cancer.

Sequence analysis can also be used to detect the SNP651 polymorphism in the Aph-lb gene. A test sample of DNA or RNA is obtained from the test individual. PCR or other appropriate methods can be used to amplify the gene, and/or its flanking sequences, if desired. The sequence of the Aph- lb gene, or a fragment of the gene, or cDNA, or fragment of the cDNA, or mRNA, or fragment of the mRNA, is determined, using standard methods. The sequence of the gene, gene fragment, cDNA, cDNA fragment, mRNA, or mRNA fragment is compared with the known nucleic acid sequence of the gene, cDNA (e.g., Fig. 1 (SEQ ID NO: 1), or a nucleic acid sequence encoding the amino acid sequence of Fig. 4 (SEQ ID NO: 9), or a fragment thereof) or mRNA, as appropriate. The presence of a polymorphism at 651 in the coding sequence of the Aph-lb gene or an amino acid substitution at position 217 of the encoded Aph-lb protein indicates that the individual has a susceptibility to develop cancer.

Allele-specific oligonucleotides can also be used to detect the presence of the SNP651 polymorphism in Aph-lb, through the use of dot-blot hybridization of amplified oligonucleotides with allele-specific oligonucleotide (ASO) probes (see, for example, Saiki, R. et al., (1986), Nature (London) 324:163-166). An "allele-specific oligonucleotide" (also referred to herein as an "allele-specific oligonucleotide probe") is an oligonucleotide of approximately 10-50 base pairs, preferably approximately 15- 30 base pairs, that specifically hybridizes to Aph-lb, and that contains a polymorphism associated with a susceptibility to develop cancer. An ^'allele-specific oligonucleotide probe that is specific for the SNP651 polymorphism in Aph-lb can be prepared, using standard methods (see Current Protocols in Molecular Biology, supra) .

To identify the SNP651 polymorphism in the Aph-lb gene that is associated with a susceptibility to develop cancer, a test sample of DNA is obtained from the individual. PCR can be used to amplify all or a fragment of Aph-lb, and its flanking sequences. The DNA containing the amplified Aph- lb (or fragment of the gene) is dot-blotted, using standard methods (see Current Protocols in Molecular Biology, supra) , and the blot is contacted with the oligonucleotide probe. The presence of specific hybridization of the probe to the amplified Aph-lb is then detected. Specific hybridization of an allele-specific oligonucleotide probe to DNA from the individual is indicative of the SNP651 polymorphism in Aph- lb, and is therefore indicative of a susceptibility to develop cancer. In another embodiment, arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual, can be used to identify the SNP651 polymorphism in Aph-lb. For example, in one embodiment, an oligonucleotide array can be used. Oligonucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. One such oligonucleotide probe could be used to detect the presence of SNP651 in the target nucleic acid sequence segments from an individual. These oligonucleotide arrays, also described as "Genechips™, " have been generally described in the art, for example, U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and 92/10092. These arrays can generally be produced using mechanical synthesis methods or light directed synthesis methods which incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods. See Fodor et al., Science, 251:767-777

(1991), Pirrung et al . , U.S. Pat. No. 5,143,854 (see also PCT Application No. WO 90/15070) and Fodor et al . , PCT Publication No. WO 92/10092 and U.S. Pat. No. 5,424,186, the entire teachings of each of which are incorporated by reference herein. Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. No. 5,384,261, the entire teachings of which are incorporated by reference herein.

Once an oligonucleotide array is prepared, a nucleic acid of interest is hybridized with the array and scanned for polymorphisms. Hybridization and scanning are generally carried out by methods described in, e.g. Published PCT Application Nos. WO 92/10092 and WO 95/11995, and U.S. Pat. No. 5,424,186, the entire teachings of which are incorporated by reference herein. In brief, a target nucleic acid sequence, which includes one or more previously identified polymorphic markers is amplified by well known amplification techniques, e.g., PCR. Typically, this involves the use of primer sequences that are complementary to the two strands of the target sequence both upstream and downstream from the polymorphism. Asymmetric PCR techniques may also be used. Amplified target, generally incorporating a label, is then hybridized with the array under appropriate conditions. Upon completion of hybridization and washing of the array, the array is scanned to determine the position on the array to which the target sequence hybridizes. The hybridization data obtained from the scan is typically in the form of fluorescence intensities as a function of location on the array .

Other methods of nucleic acid analysis can be used to detect the SNP651 polymorphism .in Aph-lb. Representative methods . include direct manual sequencing (Church and Gilbert,

(1988), Proc. Natl. Acad. Sci. USA 81:19911995; Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. 74:5463-5467; Beavis et al.

U.S. Pat. No. 5,288,644); automated fluorescent sequencing; single-stranded conformation polymorphism assays (SSCP) ; clamped denaturing gel electrophoresis (CDGE) ; denaturing gradient gel electrophoresis (DGGE) (Sheffield, V. C. et al .

(19891) Proc. Natl. Acad. Sci. USA 86:232-236), mobility shift analysis (Orita, M. et al. (1989) Proc. Natl. Acad.

Sci. USA 86:2766-2770), restriction enzyme analysis (Flavell et al. (1978) Cell 15:25; Geever, et al . (1981) Proc. Natl.

Acad. Sci. USA 78:5081); heteroduplex analysis; chemical mismatch cleavage (CMC) (Cotton et al. (1985) Proc. Natl.

Acad. Sci. USA 85:4397-4401); RNase protection assays (Myers,

R. M. et al. (1985) Science 230:1242); use of polypeptides which recognize nucleotide mismatches, such as E. coli mutS protein; allele-specific PCR, for example.

In a particularly practical embodiment of a method for diagnosing the susceptibility to develop cancer, the screening for the presence of SNP651 (SEQ ID NO: 10) in the gene encoding the human Aph-lb comprises the steps of: a) provision of nucleic acid from the individual to be tested; b) amplification of part of the nucleic acid of the individual to be tested with a primer comprising a contiguous nucleotide sequence, which is at least partially complementary to a part of the nucleotide sequence of said Aph-lb nucleic acid and which is capable of acting as a primer for said Aph-lb nucleic acid when maintained under conditions for primer extension; c) determining whether the amplification product is formed. This embodiment is particularly advantageous when the primer is complementary to a stretch of nucleic acid located immediately upstream from the position of SNP651, and ends with an A or a C. When the genomic DNA of the individual contains a T at position 651, the primer with the 3' A (A- primer) will result in primer extension, whereas the primer with the C at the end (C-primer) will not be extended. When both primers are used, the PCR reaction on the nucleic acid sample of an individual having T/T at position 651 will not produce an amplification product with the C-primer but will result in an amplification product with the A-primer. An individual having the SNP651 mutation at both alleles will produce a product with the C-primer but not with the A- primer. The sample of a heterozygous individual will show a product with both primers. Although, two PCR' s with the two primers has the additional advantage of obtaining information about the homozygosity or heterozygosity of the individual, one PCR with one primer will also give the indication whether or not the individual has the genetic risk factor. Instead of PCR any other amplification technique, such as NASBA or LCR, can be used.

The nucleic acids, probes, primers, polypeptides and antibodies described herein can be used in methods of diagnosis of a susceptibility to develop cancer, as well as in kits useful for diagnosis of such susceptibility. The invention also relates to a reagent and a kit for diagnosing an individual for susceptibility to develop cancer .

A reagent of the invention is for assaying a sample for the presence of a variant Aph-lb nucleic acid, said reagent comprising a nucleic acid comprising a contiguous nucleotide sequence which is at least partially identical to the complement of a part of the nucleotide sequence of said variant Aph-lb nucleic acid and comprises the codon encoding amino acid 217 in the Aph-lb protein. The reagent is suitably a probe or primer.

Suitably the probe or primer comprises a contiguous nucleotide sequence which is completely identical to the complement of a part of the nucleotide sequence of said Aph- Ib nucleic acid.

A reagent kit of the invention is for assaying a sample for the presence of a Aph-lb nucleic acid, comprising, in particular in separate containers: a) one or more labeled nucleic acids comprising a contiguous nucleotide sequence which is at least partially identical to the complement of a part of the nucleotide sequence of said Aph-lb nucleic acid and comprises the codon encoding amino acid 217 in the Aph-lb protein, and b) reagents for detection of said label. Suitably the labeled nucleic acid comprises a contiguous nucleotide sequences which is completely identical to the complement of a part of the nucleotide sequence of said Aph-lb nucleic acid.

The present invention further relates to a cell line harboring the SNP651 polymorphism in the Aph-lb gene. This cell ^"line produces an Aph-lb protein in which the amino acid in position 217 is a leucine instead of a phenylalanine. The cell line is either a transgenic cell line or a cell line derived from an individual that has the polymorphism.

The presence of SNP651 in an individual is indicative for an increased susceptibility to develop cancer. The presence of SNP651 defines a novel subgroup in the group of patients susceptible to cancer and thereby, as a logical consequence, at least one causative factor responsible for development of the disease in this subgroup. Specific targeting of this factor offers the possibility to provide to this subgroup of patients an improved, possibly additional, treatment protocol especially designed to target the causative factor and/or its consequence. Therefore, the present invention also relate to the use of the presence of SNP651 in a patient suffering from cancer, in the treatment of the patient.

In the present application the term "cancer" is used to indicate any form of cancer. However, when the general term is used herein also the more specific colon cancer and throat cancer are intended. Every disclosure herein that mentions cancer in general is intended to be also a specific disclosure in relation to colon cancer and throat cancer.

The present invention will be further illustrated in the Examples that follow and that are not intended to limit the invention in any way. Figure 1 shows the 905 bp cDNA sequence of the known human Aph-lb gene (accession AL136671 from GenBank) encoding the variant component Aph-lb of the human gamma-secretase gene as identified according to the invention. SNP651 is the nucleotide at position 651 of the coding sequence in which the ATG corresponds to positions 1-3.

Figures 2A-F show examples of variant Aph-lb genes of the invention. Figure 3 shows the known amino acid sequence of human component Aph-lb of the human gamma-secretase gene as found in the UniProtKB/Swiss-Prot at entry Q8WW43. The length is 257 amino acids, the molecular weight is 28460 Da. In this application the amino acid in position 217 in this figure will always be designated as "the amino acid residue in position 217" thus referring to the protein that naturally occurs in humans, even when the actual position of that amino acid in an amino acid sequence is not position 217. Figure 4 shows the amino acid sequence of an example of a variant Aph-lb protein.

Figure 5 shows the nucleotide sequence of the genomic region around SNP651 of the human Aph-lb gene. Exon 6 is shown in bold, primers are indicated with rectangles. The last nucleotide of the forward primer is specific for the T or G allele of SNP651.

Figure 6 shows the results of the PCR genotyping of SNP651 of the human Aph-lb gene. TT means the presence of a phenylalanine at position 217. TG: individual heterozygous for SNP651, meaning 50% phenylalanine and 50% leucine at position 217. GG: individual homozygous for a leucine at position 217.

EXiAMPLE Materials and Methods

Genomic DNA Isolation from Tissues and Blood

Patients from the Nijmegen area, The Netherlands, with colon cancer and throat cancer were screened. Diagnosis was made according to standardized protocols. Controls were obtained from the Nijmegen Biomedical Study.

Genomic DNA was isolated from whole blood using FlexiGene DNA kit (Qiagen) . Analysis of SNP651 of the human Aph-lb gene

Genotyping for variation in SNP651 of the human Aph- lb gene was performed via Polymerase Chain Reaction (PCR) . For each DNA sample (control and patient) , two PCRs were performed: the first reaction with a specific primer for the T-allele (TTT, encoding a phenylalanine at amino acid 217), and the second reaction specific for the G-allele (TTG, encoding for a leucine) . Briefly, the PCR mixture contained -10 ng gDNA, 10 mM Tris-HCl (pH 8.3), 2.5 mM MgCl₂, 50 mM KCl, 0.1% gelatin per ml, 200 mM each of the four deoxynucleotide triphosphates, 0.6 μM of each oligonucleotide primer (forward primer: 5'- AATAAACCTGGCGTCAGCATTT -3' = T- primer, or forward primer 5'- AATAAACCTGGCGTCAGCATTG -3' = G- primer, both in combination with reversed primer: 5¹- AGTCGGCTTTACACTGTCCCA -3') (Fig. 5) and 0.6 units of Taq polymerase (Fermentas) in a total volume of 20 ml. To distinguish between T- and G-alleles, the PCR conditions were optimised. Reactions were amplified for 30 cycles at 94°C for 1 min, 65°C for 30 sec, 72°C for 1 min, followed by a final extension step at 72°C for 10 minutes. PCR products were analysed on 1% agarose gels.

A PCR product of 370 bp observed with the forward T- primer means the presence of an Aph-lb protein with a phenylalanine at position 217, whereas a product of 370 bp obtained with the forward G-primer stands for an Aph-lb protein with a leucine. Via screening with the two specific forward primers, in two separate reactions, three combinations can be obtained: 1) only a product with the T- primer: homozygous for the T-allele (TT), 2) a product with both primers: heterozygous (TG) and 3) only a band with the G-primer: homozygous for the G-allele (GG) (fig. 12) . Results

In an attempt to link the SNP651 in Aph-lb to the susceptibility to cancer, the sequence variation at SNP651 (amino acid position 217) of the human Aph-lb gene was studied in a large control group and patient subpopulations . This study resulted in a significantly increased correlation between the prevalence of coloncancer and the SNP. (Table Ib) .

List of Sequence ID numbers

SEQ ID NOS

Fig. 1 SEQ ID NO:1 known Aph~lb gene

Fig. 2A SEQ ID NO:2 novel Aph-lb variant Fig. 2B SEQ ID NO:3 novel Aph-lb variant

Fig. 2C SEQ ID NO: 4 novel Aph-lb variant

Fig. 2D SEQ ID NO:5 novel Aph-lb variant

Fig. 2E SEQ ID NO: 6 novel Aph-lb variant

Fig. 2F SEQ ID NO:7 novel Aph-lb variant Fig. 3 SEQ ID N0:8 known Aph-lb protein

Fig. 4 SEQ ID NO:9 novel Aph-lb protein

Fig. 5 SEQ ID NO: 10 SNP651

Claims

1. Use of an isolated nucleic acid molecule comprising a variant of the human Aph-lb gene as depicted in Fig. 1 (SEQ ID N0:l) that causes the amino acid residue in position 217 of the encoded Aph-lb to be an aliphatic amino acid, in particular a leucine, for diagnosing the susceptibility of an individual to cancer.

2. Use as claimed in claim 1, wherein the variant Aph-lb gene has the nucleotide sequence as depicted in any one of the Figs. 3A-F (SEQ ID NOS:3-8), or a fragment thereof that comprises the codon encoding amino acid residue 217 of the encoded Aph-lb.

3. Use of an isolated nucleic acid encoding a polypeptide having the amino acid sequence as depicted in Fig. 4 (SEQ ID NO: 9) for diagnosing the susceptibility of an individual to cancer.

4. Use of an isolated nucleic acid molecule which hybridizes under high stringency conditions to a nucleotide sequence selected from the group consisting of the sequences as shown in any one of the Figs. 3A-F (SEQ ID NOS: 2-7) and the complement of the sequence as shown in any one of the Figs. 3A-F (SEQ ID NOS:2-7), for diagnosing the susceptibility of an individual to cancer, with the proviso that the nucleic acid has a codon selected from TTA, TTG, CTT, CTC, CTA, CTG in the position encoding the amino acid residue in position 217 of the encoded Aph-lb.

5. Use as claimed in claim 4, wherein the codon in the position encoding the amino acid residue in position 217 of the encoded Aph-lb is TTG.

6. Use as claimed in any one of the claims 1-5, wherein the isolated nucleic acid molecule comprises the complement of a sequence as defined in any one of the claims

1-5.

7. Use of an isolated polypeptide encoded by a nucleic acid molecule as defined in any one of the claims 1- 6, or a fragment of said polypeptide, which polypeptide or fragment comprises an aliphatic amino acid, in particular a leucine in the position that corresponds with position 217 in human Aph-lb as depicted in Fig. 3 (SEQ ID NO: 8) in the diagnosis of the susceptibility of an individual to cancer.

8. Use as claimed in claim 1, wherein the isolated polypeptide has the amino acid sequence of Fig. 4 (SEQ ID N0:9) .

9. Use as claimed in claim 7, wherein the isolated polypeptide comprises an amino acid sequence which is more than about 90 percent identical to the amino acid sequence of Fig. 3 (SEQ ID NO: 8) and which has an aliphatic amino acid, in particular a leucine, in the position that corresponds with position 217 in human component Aph-lb.

10. Use of a fusion protein comprising an isolated polypeptide defined in claim 8 or 9 in the diagnosis of the susceptibility of an individual to cancer.

11. Use of an antibody, or an antigen-binding fragment thereof, which selectively binds to a polypeptide as defined in any one of the claims 7-10 in the diagnosis of the susceptibility of an individual to cancer.

12. Use of a reagent, comprising a probe or primer for assaying a sample for the presence of a variant Aph-lb nucleic acid in a method for diagnosing an individual for susceptibility to develop cancer.

13. Use as claimed in claim 12, wherein said reagent comprises a nucleic acid comprising a contiguous nucleotide sequence which is at least partially identical to the complement of a part of the nucleotide sequence of said variant Aph-lb nucleic acid and comprises the codon encoding amino acid 217 in the Aph-lb protein.

14. Use as claimed in claim 13, wherein the reagent is suitably a probe or primer.

15. Use as claimed in claim 14, wherein the probe or primer comprises a contiguous nucleotide sequence which is completely identical to the complement of a part of the nucleotide sequence of said Aph-lb nucleic acid.

16. Use of a reagent kit, comprising one or more labeled nucleic acids and reagents for detection of said label, for assaying a sample for the presence of a Aph-lb nucleic acid in the diagnosis of an individual for susceptibility to develop cancer.

17. Use as claimed in claim 16, wherein the kit comprises in separate containers: a) one or more labeled nucleic acids comprising a contiguous nucleotide sequence which is at least partially identical to the complement of a part of the nucleotide sequence of said Aph-lb nucleic acid and comprises the codon encoding amino acid 217 in the Aph-lb protein, and b) reagents for detection of said label.

18. Use as claimed in claim 17, wherein the labeled nucleic acid comprises a contiguous nucleotide sequences which is completely identical to the complement of a part of the nucleotide sequence of said Aph-lb nucleic acid.

19. An in vitro method of diagnosing the susceptibility to develop cancer in an individual, comprising screening for the presence of SNP651 (SEQ ID NO: 10) in the gene encoding the human Aph-lb, which SNP651 is more frequently present in an individual susceptible to developing cancer than in a healthy individual, and wherein the presence of SNP651 is indicative of the existence of cancer or susceptibility to develop cancer.

20. The method of claim 19, wherein the cancer is selected from colon cancer, throat cancer.

21. Method as claimed in claim 19 or 20, wherein the individual to be tested is Caucasian.

22. Method as claimed in any one of the claims 19-21, wherein the screening for the presence of SNP651 (SEQ ID NO: 10) in the gene encoding the human Aph-lb comprises the steps of: a) provision of nucleic acid from the individual to be tested; b) amplification of part of the nucleic acid of the individual to be tested with a primer which is at least partially complementary to a part of the nucleotide sequence of said Aph-lb nucleic acid and which is capable of acting as a primer for said Aph-lb nucleic acid when maintained under conditions for primer extension; c) determining whether the amplification product is formed.

23. Method as claimed in claim 22, wherein the primer is complementary to a part of the Aph-lb gene located immediately upstream from the position of SNP651 and ends with an A or a C at the position of SNP651.

24. Method for determining genetic predisposition to susceptibility to develop cancer, which comprises: (i) obtaining a sample of DNA from a test subject; and (ii) assaying said sample for the presence of SNP651 in the Aph-1 gene.

25. Use of a cell line harboring the SNP651 polymorphism in the Aph-lb gene in the diagnosis of the susceptibility of an individual to cancer.

26. Use of the presence of SNP651 in a patient suffering from cancer in the treatment of the patient.