Postoperative risk stratification based on expression of PDE4D variants selected according to TMPRSS2-ERG fusion status and postoperative clinical variables

Abstract

The present invention relates to a method for postoperative risk stratification of prostate cancer subjects, comprising determining the transmembrane protease serine 2-ETS-related gene (TMPRSS2-ERG) fusion status in a biological sample obtained from the subject, determining the gene expression profile of each of one or more phosphodiesterase 4D variants in the biological sample obtained from the subject, determining the expression-based risk score of the subject based on the gene expression profile of the selected phosphodiesterase 4D variant, and determining the postoperative prognostic risk score based on the expression-based risk score and the postoperative clinical variables of the subject, wherein the selected phosphodiesterase 4D variant is selected based on the TMPRSS2-ERG fusion status. This can improve the stratification of subjects in the postoperative setting and can obtain better postoperative secondary treatment decisions.

Description

Field of the Invention

[0001] The present invention relates to a method for postoperative risk stratification of prostate cancer subjects. In addition, the present invention relates to a diagnostic kit, to the use of the kit in the method for postoperative risk stratification of prostate cancer subjects, to the use of the gene expression profile of phosphodiesterase 4D variants in postoperative risk stratification of prostate cancer subjects, and to a corresponding computer program product. Background of the Invention

[0003] Cancer is a class of diseases in which a group of cells present uncontrolled growth, invasion and sometimes metastasis. These three malignant properties of cancer distinguish it from benign tumors, which are self-limiting and do not invade or metastasize. Prostate cancer (PCa) is the most common non-skin malignancy in men. It presents a heterogeneous disease with various potentials that gradually develop into fatal forms of the disease. In Europe, it is estimated that among 417,000 new cases per year, approximately 92,000 will die from their disease (see Ferlay J.et al., GLOBOCAN 2012v1.0, Cancer Incidence and MortalityWorldwide:IARC CancerBase No.11[Internet], Lyon, France, International Agencyfor Research on Cancer, 2013).

[0004] Secondary treatment (secondary treatment) decision after primary intervention should follow the risk classification after surgery. In the past, multiple algorithms for risk assessment after treatment have been developed (see Lughezani G.et al., "Predictive and prognostic models in radical prostatectomy candidates: A critical analysis of the literature ", European Urology, Vol.58, No.5, pages 687-700, 2010). These risk stratification models are based on clinical variables after treatment, such as pathological Gleason score or TNM malignant tumor classification. Based on the expected disease recurrence and the risk of developing into a wider form of disease (i.e. metastatic disease), clinicians can decide to perform many auxiliary secondary treatments, such as radiotherapy (such as external particle beam radiotherapy (EBRT)), hormone therapy (such as anti-androgen therapy), chemotherapy (such as docetaxel or cabazitaxel), immunotherapy or any combination thereof. However, because this treatment has obvious side effects, it should only be used for patients who really need this invasive treatment (aggressive therapy) to prevent disease progression and ultimately prevent death related to prostate cancer.

[0005] WO 2016 / 193110A1 discloses a method for diagnosing, monitoring or prognosing prostate cancer or the state of prostate cancer progression, wherein the state of prostate cancer is determined based on the expression level of phosphodiesterase 4D (PDE4D) variants. SUMMARY OF THE INVENTION

[0007] One object of the present invention is to provide a method for postoperative risk stratification of prostate cancer subjects, which can allow for better postoperative secondary treatment decisions. Another object of the present invention is to provide a diagnostic kit, the use of the kit in a method for postoperative risk stratification of prostate cancer subjects, the use of a gene expression profile of phosphodiesterase 4D variants in postoperative risk stratification of prostate cancer subjects, and corresponding computer program products.

[0008] In a first aspect of the present invention, a method for risk stratification of a prostate cancer subject after surgery is provided, comprising:

[0009] - determining the transmembrane protease serine 2-ETS related gene (TMPRSS2-ERG) fusion status in a biological sample obtained from the subject,

[0010] - determining a gene expression profile for each of the one or more phosphodiesterase 4D variants in a biological sample obtained from the subject,

[0011] - determining an expression-based risk score for the subject based on the gene expression profile of the selected phosphodiesterase 4D variant, and

[0012] - determining a post-operative prognostic risk score for the subject based on the subject's expression-based risk score and post-operative clinical variables,

[0013] The selected phosphodiesterase 4D variants were selected based on their TMPRSS2-ERG fusion status.

[0014] Wherein, if the TMPRSS2-ERG fusion status is positive, the selected phosphodiesterase 4D variant is selected to be the first phosphodiesterase 4D variant, and if the TMPRSS2-ERG fusion status is negative, the selected phosphodiesterase 4D variant is selected to be the second phosphodiesterase 4D variant.

[0015] It is known that the cAMP signaling pathway plays an important role in the occurrence and progression of prostate cancer (see Merkle D. and Hoffmann R., "Roles of cAMP and cAMP-dependent protein kinase in the progression of prostate cancer: Cross-talk with the androgen receptor", Cellular Signalling, Vol. 23, No. 3, pages 507-515, 2011). Although the adenylate cyclase family is responsible for the synthesis of cAMP, cyclic nucleotide phosphodiesterase (PDE) seems to represent the only cellular mechanism for its destruction. PDE provides both signal termination function and, more importantly, compartmentalization of cAMP signaling within the cell 3D matrix. This is achieved by spatially discrete disruption of cAMP by subsets of different PDE isoforms sequestered by locally located ankyrin / signalosomes (see, e.g., Conti M. and Beavo J., "Biochemistry and physiology of cyclic nucleotidephosphodiesterases: essential components in cyclic nucleotide signaling", Annual Review of Biochemistry, Vol. 76, pages 481-511, 2007). Therefore, changes in the expression and / or activity of different PDE isoforms can alter downstream signaling pathways during disease development and progression, providing potential targets for novel biomarkers and targeted therapeutic interventions. In fact, changes in the expression of PDE4 family members that degrade cAMP appear to be associated with many different diseases, including stroke, acrodysplasia, schizophrenia, and COPD. Recently, studies have shown that downregulation of a specific PDE4 isoform (PDE4D7) may have an impact on prostate cancer (see, e.g., R.et al., "Humanphosphodiesterase 4D7 (PDE4D7) expression is increased in TMPRSS2-ERG positive primary prostate cancer and independently adds to a reduced risk of post-surgical disease progression", Britsch Journal of Cancer, Vol.113, No.10, pages1502-1511, 2015). The PDE4D7 isoform is a so-called long isoform because it contains both the UCR1 and UCR2 regulatory domains. UCR1 is present in long, but not short, PDE4 isoforms and can be regulated by various protein kinases including PKA and MK2, and also determines the functional consequences of phosphorylation of the ERK catalytic unit. Functionally, it provides a partial cellular desensitization system for cAMP, and, for example, causes crosstalk between signal transduction pathways that lead to activation of ERK and AMPK.

[0016] By determining the expression-based risk score of prostate cancer objects based on the gene expression profile of phosphodiesterase 4D variants, other molecular information representing disease biology is obtained. The prognostic ability of phosphodiesterase 4D variants can be used for postoperative patient risk assessment by determining a postoperative prognostic risk score, which is not only based on the postoperative clinical variables of the object, but also based on the risk score based on expression. This can allow for improved stratification of objects in postoperative settings, which can lead to better secondary treatment decisions. For example, a postoperative prognostic risk score can provide better suggestions for whether certain subgroups of prostate cancer patients choose specific postoperative secondary treatment. In addition, the inventors found that the prognostic ability of different phosphodiesterase 4D variants depends on the molecular subtype of prostate cancer, particularly on whether the TMPRSS2-ERG fusion state of prostate cancer is positive or negative. Therefore, by selecting a phosphodiesterase 4D variant for postoperative prognostic risk score according to the TMPRSS2-ERG fusion state, the reliability and expressiveness of the postoperative prognostic risk score can be improved. In this regard, we note that the "selection" of the phosphodiesterase 4D variant is associated with the utilization of the phosphodiesterase 4D variant in the post-operative prognostic risk score according to the TMPRSS2-ERG fusion status, wherein if the TMPRSS2-ERG fusion status is positive, the selected phosphodiesterase 4D variant is selected to be the first phosphodiesterase 4D variant, and if the TMPRSS2-ERG fusion status is negative, the selected phosphodiesterase 4D variant is selected to be the second phosphodiesterase 4D variant. The gene expression profile of the used phosphodiesterase 4D variant can be determined in a biological sample obtained from the subject as one of many gene expression profiles for different phosphodiesterase 4D variants without knowing the TMPRSS2-ERG fusion status in the biological sample.

[0017] The term "biological sample" or "sample obtained from a subject" refers to any biological material obtained from a subject, such as a prostate cancer patient, by a suitable method known to those skilled in the art. The biological sample used can be collected in a clinically acceptable manner, such as in a manner that preserves nucleic acids (particularly RNA) or proteins.

[0018] Biological samples can include body tissues and / or body fluids, such as, but not limited to, blood, sweat, and urine. In addition, biological samples can contain cell extracts derived from epithelial cells or cell populations comprising epithelial cells, such as cancerous epithelial cells or epithelial cells derived from tissues suspected of being cancerous. The biological sample can include a cell population derived from glandular tissue, such as the sample can be derived from the prostate of a male subject. In addition, if necessary, cells can be purified from the obtained body tissues and body fluids and then used as biological samples. In some embodiments, the sample can be a tissue sample, a urine sample, a urine sediment sample, a blood sample, a saliva sample, a semen sample, a sample comprising circulating tumor cells, an extracellular vesicle, a sample comprising exosomes secreted by the prostate, or a cell line or a cancer cell line.

[0019] In a particular embodiment, a biopsy sample or a surgical resection sample may be obtained and / or used. Such a sample may include cells or cell lysates.

[0020] It is also conceivable that the content of the biological sample is subjected to an enrichment step. For example, the sample can be contacted with a part that is specific to the cell membrane or organelle of some cell types, such as prostate cells, with, for example, magnetic particle functionalization. The material concentrated by the magnetic particles can be used for detection and analysis steps as described above or below subsequently.

[0021] Furthermore, cells (eg, tumor cells) can be enriched by filtration processes of body fluids or liquid samples (eg, blood, urine, etc.). Such filtration processes can also be combined with enrichment steps based on ligand-specific interactions as described above.

[0022] The term "prostate cancer" refers to a cancer of the prostate gland in the male reproductive system, which occurs when prostate cells mutate and begin to multiply uncontrollably. Typically, prostate cancer is associated with elevated levels of prostate-specific antigen (PSA). In one embodiment of the invention, the term "prostate cancer" relates to a cancer with a PSA level greater than 4.0. In another embodiment, the term relates to a cancer showing a PSA level greater than 2.0. The term "PSA level" refers to the concentration of PSA in the blood in ng / ml.

[0023] The term "non-progressive prostate cancer status" means that the individual sample does not show parameter values ​​indicative of "biochemical recurrence" and / or "clinical recurrence".

[0024] The term "progressive prostate cancer status" refers to an individual sample showing parameter values ​​indicative of "biochemical recurrence" and / or "clinical recurrence".

[0025] The term "biochemical recurrence" generally refers to the biological value of recurrence, where PSA is elevated, indicating the presence of prostate cancer cells in the sample. However, other markers useful for detecting the presence or suspicion of their presence may also be used.

[0026] The term "clinical recurrence" refers to the presence of clinical signs indicating the presence of tumor cells, for example as measured using in vivo imaging.

[0027] As used herein, the term "prognosis for prostate cancer" refers to, for example, a prediction of the course or outcome of a prostate cancer diagnosed or detected within a certain time period, during treatment, or after treatment. The term also refers to determining the chance of survival or recovery from the disease, and predicting the expected survival time of an object. Specifically, prognosis can be related to determining the possibility of survival of an object in a future period of time (for example, 6 months, 1 year, 2 years, 3 years, 5 years, 10 years or any other time period).

[0028] Preferably, if the TMPRSS2-ERG fusion status is positive, the selected phosphodiesterase 4D variant is selected to be phosphodiesterase 4D variant 7 (PDE4D7), and if the TMPRSS2-ERG fusion status is negative, the selected phosphodiesterase 4D variant is selected to be phosphodiesterase 4D variant 5 (PDE4D5) or phosphodiesterase 4D variant 9 (PDE4D9).

[0029] As described above, the inventors have found that the prognostic power of different phosphodiesterase 4D variants depends on the molecular subtype of prostate cancer, in particular, on whether the TMPRSS2-ERG fusion status of the prostate cancer is positive or negative. In prostate cancer with a positive TMPRSS2-ERG fusion status, PDE4D7 was found to provide good prognostic power, while this was not the case in prostate cancer with a negative TMPRSS2-ERG fusion status. In contrast, in prostate cancer with a negative TMPRSS2-ERG fusion status, PDE4D5 and PDE4D9 were found to have suitable prognostic power, while the same prognostic power was not found in prostate cancer with a negative TMPRSS2-ERG fusion status.

[0030] The term "phosphodiesterase 4D7" or "PDE4D7" refers to the splice variant 7 of human phosphodiesterase PDE4D, i.e., the human phosphodiesterase PDE4D7 gene, such as the sequence defined by the NCBI reference sequence: NM_001165899.1, in particular the nucleotide sequence shown in SEQ ID NO: 6, which corresponds to the sequence of the NCBI reference sequence of the PDE4D7 transcript, and also relates to the corresponding amino acid sequence, such as that shown in SEQ ID NO: 7, which corresponds to the protein sequence encoding the PDE4D7 polypeptide defined in the NCBI protein accession reference sequence NP_001159371.1. The term "phosphodiesterase 4D7" or "PDE4D7" also relates to an amplicon that can be generated by the primer pair PDE4D7_forward primer (SEQ ID NO: 8) and PDE4D7_reverse primer (SEQ ID NO: 9) and can be detected by the probe SEQ ID NO: 10.

[0031] The PDE4D7 polypeptide can also be detected using the primer pair PDE4D7-2_Forward Primer (SEQ ID NO:11) and PDE4D7_Reverse Primer (SEQ ID NO:12) and can be detected by the probe SEQ ID NO:13.

[0032] The term "phosphodiesterase 4D5" or "PDE4D5" refers to the splice variant 5 of human phosphodiesterase PDE4D, i.e., the human phosphodiesterase PDE4D5 gene, such as the sequence defined by the NCBI reference sequence: NM_001197218.1, in particular the nucleotide sequence shown in SEQ ID NO: 1, which corresponds to the sequence of the NCBI reference sequence of the above-mentioned PDE4D5 transcript, and also relates to the corresponding amino acid sequence shown in SEQ ID NO: 2, which corresponds to the protein sequence encoding the PDE4D5 polypeptide defined by the NCBI protein accession reference sequence NP_001184147.1. The term "phosphodiesterase 4D5" or "PDE4D5" also relates to an amplicon that can be generated by the primer pair PDE4D5_forward primer (SEQ ID NO: 3) and PDE4D5_reverse primer (SEQ ID NO: 4) and can be detected by the probe SEQ ID NO: 5.

[0033] The term "phosphodiesterase 4D9" or "PDE4D9" relates to the splice variant 9 of human phosphodiesterase PDE4D, i.e. the human phosphodiesterase PDE4D9 gene, such as the sequence defined by the NCBI reference sequence: NM_001197220.1, in particular the nucleotide sequence shown in SEQ ID NO: 14, which corresponds to the sequence of the NCBI reference sequence of the PDE4D9 transcript described above, and also to the corresponding amino acid sequence shown in SEQ ID NO: 15, which corresponds to the protein sequence encoding the PDE4D9 polypeptide defined by the NCBI protein accession reference sequence NP_001184149.1. The term "phosphodiesterase 4D9" or "PDE4D9" also relates to an amplicon that can be generated by the primer pair PDE4D9_forward primer (SEQ ID NO: 16) and PDE4D9_reverse primer (SEQ ID NO: 17) and can be detected by the probe SEQ ID NO: 18.

[0034] The terms "PDE4D5", "PDE4D7" and "PDE4D9" also include nucleotide sequences that are highly homologous to PDE4D5, PDE4D7 and PDE4D9, respectively, such as nucleic acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences shown in SEQ ID NO: 1, 6 or 14, respectively, or amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 2, 7 or 15, respectively, or encoding amino acid sequences that are at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 99% identical to SEQ ID NO: 2, 7 or 15, respectively. NO:2, 7 or 15 has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in NO:2, 7 or 15, or an amino acid sequence encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:1, 6 or 14, respectively.

[0035] Preferably, the postoperative clinical variables include one or more of the following: (i) prostate specific antigen (PSA) level; (ii) pathological Gleason score (pGS); (iii) surgical margin (SM); (iv) extracapsular extension (ECE); (v) seminal vesicle invasion (SVI); and (vi) lymph node infiltration (LNI).

[0036] As mentioned above, a variety of algorithms for post-treatment risk assessment have been developed in the past (see Lughezani G. et al., "Predictive and prognostic models in radical prostatectomy candidates: A critical analysis of the literature", European Urology, Vol. 58, No. 5, pages 687-700, 2010). The postoperative CAPRA-S score is one of the most widely validated clinical risk algorithms for postoperative decision support (see Cooperberg MR et al., "The CAPRA-S score: A straightforward tool for improved prediction of outcomes after radical prostatectomy", Cancer, Vol. 117, No. 22, pages 5039-5046, 2011). The score is a combination of clinically available information, i.e., preoperative PSA, pathological Gleason score (pGS), surgical margin (SM), extracapsular extension (ECE), seminal vesicle invasion (SVI) and lymph node invasion (LNI). The score was initially published in 2011 and has since been validated in several studies (see, e.g., Punnen S. et al., "Multi-institutional validation of the CAPRA-S score to predict disease recurrence and mortality after radical prostatectomy", European Urology, Vol. 65, No. 6, pages 1171-1177, 2014). By combining the molecular information provided by the expression-based risk score with information from this extensively validated postoperative clinical variables, a postoperative prognostic risk score with improved prognostic power can be obtained.

[0037] It is also preferred that the method further comprises:

[0038] - Determine the subject's Cancer of the Prostate Risk Assessment after Surgery (CAPRA-S) score,

[0039] The postoperative prognostic risk score was determined by combining the expression-based risk score and the CAPRA-S score.

[0040] As mentioned above, the CAPRA-S score is one of the most widely validated clinical risk algorithms for decision support after prostate cancer surgery. It provides a categorical score between 1-12, divided into three categories: low risk (CAPRA-S score 0-2), medium risk (CAPRA-S score 3-5) and high risk (CAPRA-S score 6-12). In view of its current level of validation as a prognostic algorithm in prostate cancer and its easy-to-interpret single score output, the CAPRA-S score can be advantageously combined with an expression-based risk score as a postoperative prognostic risk score, and the combined score can be easily determined in clinical practice compared to using only the postoperative CAPRA-S algorithm, and can further improve postoperative prognosis.

[0041] It is further preferred that the CAPRA-S score is categorized, wherein a number of points are added to the post-operative prognostic risk score according to the category, preferably in the range of 1-3.

[0042] According to the categories of the CAPRA-S score, by adding multiple points to the postoperative prognostic risk score, the corresponding categories (e.g., high risk) help to make the postoperative prognostic risk score easy to understand and intuitively reflect the categories. In particular, if the number of points is in the range of 1-3, a higher value corresponds to a higher risk, while a lower value corresponds to a lower risk (as shown in the CAPRA-S score).

[0043] It is further preferred that the expression-based risk score is a value within a predetermined range, wherein a number of points are added to the post-operative prognostic risk score according to said value, preferably within the range of 0-3.

[0044] This has the advantage that the expression-based risk score is treated in substantially the same manner as the CAPRA-S score when determining the postoperative prognostic risk score. In particular, if the points are in the range of 0-3, the expression-based risk score and the CAPRA-S score have substantially the same impact on the expression-based risk score, resulting in a good balance of the prognostic information provided by the two scores.

[0045] In a preferred embodiment, the expression-based risk score is a value in the range of 1-5, and if the value is in the range of 1 to <2, 3 points are added to the postoperative prognostic risk score, if the value is in the range of 2 to <3, 2 points are added, if the value is in the range of 3 to <4, 1 point is added, and if the value is in the range of 4 to <5, no points are added.

[0046] Preferably, the method further comprises:

[0047] - Based on the postoperative prognostic risk score, recommend secondary treatment after surgery for the subject,

[0048] Wherein, the secondary treatment after surgery is selected from: radiotherapy, hormone therapy, chemotherapy, immunotherapy or any combination thereof.

[0049] The radiation therapy may be, for example, external beam radiation therapy (EBRT), the hormone therapy may be, for example, anti-androgen therapy, and the chemotherapy may be based on, for example, docetaxel or cabazitaxel.

[0050] Further preferably, the method comprises:

[0051] - normalizing the gene expression profile of the selected phosphodiesterase 4D variants relative to one or more reference genes selected from the group consisting of: Homo sapiens hypoxanthine phosphoribosyltransferase 1 (HPRT1), tubulin-alpha-1b (TUBA1B), Homo sapiens pumilio RNA-binding family member (PUM1) and Homo sapiens TATA box binding protein (TBP), wherein the expression-based risk score is determined based on the normalized gene expression profile.

[0052] By normalizing the gene expression profile relative to one or more reference genes and determining an expression-based risk score based on the normalized gene expression profile, the variability in determining the expression-based risk score can be reduced. This makes it possible to distinguish between actual changes in the gene expression profile and changes due to the measurement method. In this regard, it has been found that HPRT1, TUBA1B, PUM1 and TBP are particularly suitable for use as reference genes for normalizing the phosphodiesterase 4D variant gene expression profile.

[0053] Gene expression profile can be determined by detecting mRNA expression using one or more and / or one or more sets of primers and / or probes. In addition, gene expression profile can be determined by amplification-based methods and / or microarray analysis and / or RNA sequencing. Determining gene expression profile can include performing real-time quantitative polymerase chain reaction (RT-qPCR) on RNA extracted from biological samples. In other embodiments, gene expression profile is determined by RNA sequencing, conventional PCR (using endpoint analysis such as by gel electrophoresis) or multiplex PCR. In the case of RT-qPCR, determination of gene expression profile can include determining the threshold cycle (Ct) value of each gene of phosphodiesterase 4D variants and one or more reference genes. PCR can be performed using at least one primer and / or probe for measuring a reference gene selected from HPRT1, TUBA1B, PUM1 and TBP.

[0054] Preferably, the one or more reference genes include at least two or at least three of HPRT1, TUBA1B, PUM1 and TBP. In a particularly preferred embodiment, the one or more reference genes include all of HPRT1, TUBA1B, PUM1 and TBP.

[0055] Other reference genes that may be used in addition or instead for normalizing the phosphodiesterase 4D variant gene expression profile include: Homo sapiens actin beta mRNA (ACTB); Homo sapiens 60S acidic ribosomal phosphoprotein P0 mRNA (RPLP0); polymerase (RNA) II (DNA-directed) polypeptide A, 220 kDa (POLR2A); beta-2-microglobulin (B2M); and aminolevulinate-delta synthase (ALAS-1).

[0056] Further preferably, the expression-based risk score is determined based on the gene expression profile of the selected phosphodiesterase 4D variant using a scoring function derived from the gene expression profile of a biological sample of a prostate cancer subject.

[0057] At this, particularly preferably described scoring function is based on standardized gene expression spectrum, for example, with respect to the standardized gene expression spectrum of all HPRT1, TUBA1B, PUM1 and TBP, described scoring function is derived from the corresponding standardized gene expression spectrum of prostate cancer object biological sample.In a preferred embodiment, scoring function is linear transformation, and it converts the standardized gene expression spectrum into the value of predetermined range, for example the scope of above-mentioned 1-5.Can be by considering the frequency distribution of the standardized gene expression spectrum value of phosphodiesterase 4D variant of the biological sample of prostate cancer object colony, and by determining the conversion that converts the frequency distribution into the required range to determine this conversion.By utilizing this scoring function, the risk score based on expression can be represented in the user-intuitive mode, for example in the range of small positive values.This is similar to other classifications used in clinical routine, for example histopathological grading (Gleason) or multi-parameter MRI radiation score (PIRADS).

[0058] In a particular embodiment, the expression-based risk score is determined as follows:

[0059] EBRS=(((PDE4D_norm+A)*B)+1), 1)

[0060] where "EBRS" is the expression-based risk score, "PDE4D_norm" is the normalized phosphodiesterase 4D variant gene expression profile value, and A and B are variables.

[0061] In one example, when the phosphodiesterase 4D variant is selected to be PDE4D7, A can be about 6-8, such as 6.7167499999999, B can be 0.4-0.45, such as 0.420780231744713, and the risk score based on expression can be a value ranging from 1 to 5 (as described above). In another example, when the phosphodiesterase 4D variant is selected to be PDE4D5, A can be about 4-6, such as 4.59820000000001, B can be 0.5-0.6, such as 0.556517867701789. In another example, when the phosphodiesterase 4D variant is selected to be PDE4D9, A can be about 3-5, such as 3.909299999999999, and B can be 0.5-0.6, such as 0.548770240189875.

[0062] The expression-based risk score can also be classified or categorized into one of at least two risk groups depending on the value of the expression-based risk score. For example, there are two risk groups, or three risk groups, or four risk groups, or more than four predefined risk groups. Each risk group covers a respective (non-overlapping) range of expression-based risk scores. For example, one risk group can include all expression-based risk scores from 1 to <2, another risk group includes from 2 to <3, another risk group includes from 3 to <4 and another risk group includes from 4 to <5.

[0063] Particularly preferably, determining the gene expression profile of the selected phosphodiesterase 4D variant comprises performing RT-qPCR on RNA extracted from a biological sample, wherein a Cq value is determined for each of the selected phosphodiesterase 4D variant and one or more reference genes, and wherein determining the expression-based risk score comprises normalizing the Cq value of the selected phosphodiesterase 4D variant using the Cq value of each gene in the one or more reference genes, and calculating the expression-based risk score as a linear function of the normalized Cq value. For example, the normalized Cq value of the selected phosphodiesterase 4D variant can be generated by applying the following formula:

[0064] N(Cq PDE4D )=Mean(Cq ref_genes )–(Cq PDE4D ), 2)

[0065] Where N(Cq PDE4D ) is the normalized gene expression profile value (quantization cycle, Cq) of the selected phosphodiesterase 4D variants, Mean (Cq ref_genes ) is the arithmetic mean of the PCR Cq values ​​of one or more reference genes, Cq PDE4D are the PCR Cq values ​​of selected phosphodiesterase 4D variants.

[0066] In another aspect of the present invention, a diagnostic kit is provided, comprising:

[0067] at least one primer and / or probe for determining the fusion status of transmembrane protease serine 2-ETS related gene (TMPRSS2-ERG) in a biological sample obtained from a subject with prostate cancer,

[0068] at least one primer and / or probe for determining a gene expression profile of each of the one or more phosphodiesterase 4D variants obtained from a biological sample of the subject; and

[0069] Optionally comprising at least one primer and / or probe for determining the gene expression profile of one or more reference genes selected from the group consisting of: Homo sapiens hypoxanthine phosphoribosyltransferase 1 (HPRT1), tubulin alpha-1b (TUBA1B), Homo sapiens pumilio RNA-binding family member (PUM1), and Homo sapiens TATA box binding protein (TBP); and

[0070] Optionally comprising at least one substance for determining the level of prostate specific antigen (PSA) in a biological sample obtained from said subject; and

[0071] Instructions for calculating a post-operative prognostic risk score based on a gene expression profile of a selected phosphodiesterase 4D variant of the subject and post-operative clinical variables, the instructions being stored on a computer program product that, when executed by a computer, perform a method comprising:

[0072] - Determine TMPRSS2-ERG fusion status,

[0073] - determining an expression-based risk score for said subject based on the gene expression profile of the selected phosphodiesterase 4D variant, and

[0074] - determining a post-operative prognostic risk score for said subject based on said expression-based risk score and post-operative clinical variables of said subject,

[0075] The selected phosphodiesterase 4D variants were selected based on their TMPRSS2-ERG fusion status.

[0076] wherein if the TMPRSS2-ERG fusion status is positive, the selected phosphodiesterase 4D variant is selected to be the first phosphodiesterase 4D variant, and if the TMPRSS2-ERG fusion status is negative, the selected phosphodiesterase 4D variant is selected to be the second phosphodiesterase 4D variant,

[0077] Optionally, wherein the method comprises:

[0078] - normalizing the gene expression profile of the selected phosphodiesterase 4D variant relative to one or more reference genes, wherein the expression-based risk score is determined based on the normalized gene expression profile of the selected phosphodiesterase 4D variant,

[0079] Optionally, the at least one substance used to determine the prostate specific antigen (PSA) level may be, for example, a PSA-specific antibody or the like.

[0080] In another aspect of the present invention, there is provided a use of the kit according to claim 13 in a method for postoperative risk stratification of prostate cancer subjects.

[0081] In another aspect of the present invention, there is provided a method for using a gene expression profile of a selected phosphodiesterase 4D variant in postoperative risk stratification of a prostate cancer subject, comprising:

[0082] - determining the transmembrane protease serine 2-ETS related gene (TMPRSS2-ERG) fusion status in a biological sample obtained from said subject,

[0083] - determining a gene expression profile for each of one or more phosphodiesterase 4D variants in a biological sample obtained from said subject,

[0084] - determining an expression-based risk score for said subject based on the gene expression profile of the selected phosphodiesterase 4D variant, and

[0085] - determining a post-operative prognostic risk score for said subject based on said expression-based risk score and post-operative clinical variables of said subject,

[0086] The selected phosphodiesterase 4D variants were selected based on their TMPRSS2-ERG fusion status.

[0087] Wherein, if the TMPRSS2-ERG fusion status is positive, the selected phosphodiesterase 4D variant is selected to be the first phosphodiesterase 4D variant, and if the TMPRSS2-ERG fusion status is negative, the selected phosphodiesterase 4D variant is selected to be the second phosphodiesterase 4D variant.

[0088] In another aspect of the present invention, there is provided a computer program product comprising instructions, which, when executed by a computer, causes the computer to perform a method comprising:

[0089] - determining the transmembrane protease serine 2-ETS related gene (TMPRSS2-ERG) fusion status in a biological sample obtained from a subject with prostate cancer,

[0090] - determining a gene expression profile for each of one or more phosphodiesterase 4D variants in a biological sample obtained from said subject,

[0091] - determining an expression-based risk score for said subject based on the gene expression profile of the selected phosphodiesterase 4D variant, and

[0092] - determining a post-operative prognostic risk score for said subject based on said expression-based risk score and post-operative clinical variables of said subject,

[0093] The selected phosphodiesterase 4D variants were selected based on their TMPRSS2-ERG fusion status.

[0094] Wherein, if the TMPRSS2-ERG fusion status is positive, the selected phosphodiesterase 4D variant is selected to be the first phosphodiesterase 4D variant, and if the TMPRSS2-ERG fusion status is negative, the selected phosphodiesterase 4D variant is selected to be the second phosphodiesterase 4D variant.

[0095] It should be understood that the method of claim 1, the diagnostic kit of claim 12, the use of the diagnostic kit of claim 13, the use of the gene expression profile of claim 14 and the computer program of claim 15 have similar and / or or identical preferred embodiments, in particular as defined in the dependent claims.

[0096] It shall be understood that a preferred embodiment of the invention can also be any combination of the dependent claims or the above embodiments with the respective independent claim.

[0097] These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter. Sequence Listing <110> Royal Philips Ltd. <120> Postoperative risk stratification based on expression of PDE4D variants selected according to TMPRSS2-ERG fusion status and postoperative clinical variables <130> 2017PF02537 <160> 65 <170> PatentIn version 3.5 <210> 1 <211> 7979 <212> DNA <213> Homo sapiens <400> 1 cagcagcagg ctcagacctg cttccctgga catttccggg accgtgagcg agggaaccac 60 gttgccctgg attcttgcca gctgtacaaa gttgaccagg aaaatggctc agcagacaag 120 cccggacact ttaacagtac ctgaagtgga taatccgcat tgtccaaacc cgtggctgaa 180 cgaagacctt gtgaaatcct tgcgagaaaa cctgttgcag catgagaagt ccaagacagc 240 gaggaaatcg gtttctccca agctctctcc agtgatctct ccgagaaatt cccccaggct 300 tctgcgcaga atgcttctca gcagcaacat ccccaaacag cggcgtttca cggtggcaca 360 tacatgtttt gatgtggaca atggcacatc tgcgggacgg agtcccttgg atcccatgac 420 cagcccagga tccgggctaa ttctccaagc aaattttgtc cacagtcaac gacgggagtc 480 cttcctgtat cgatccgaca gcgattatga cctctctcca aagtctatgt cccggaactc 540 ctccattgcc agtgatatac acggagatga cttgattgtg actccatttg ctcaggtctt 600 ggccagtctg cgaactgtac gaaacaactt tgctgcatta actaatttgc aagatcgagc 660 acctagcaaa agatcaccca tgtgcaacca accatccatc aacaaagcca ccataacaga 720 ggaggcctac cagaaactgg ccagcgagac cctggaggag ctggactggt gtctggacca 780 gctagagacc ctacagacca ggcactccgt cagtgagatg gcctccaaca agtttaaag 840 gatgcttaat cgggagctca cccatctctc tgaatgagt cggtctggaa atcaagtgtc 900 agagtttata tcaacat tcttagataa gcaacatgaa gtggaaattc cttctccaac 960 tcagaaggaaaggaaaaagaaagacc aatgtctcag atcagtggag tcaagaatt 1020 gatgcacagc tctagtctga ctattcaag tatcccagg tttggagtta aaactgaaca 1080 agaagagatgtc cttgccaagg aactagaaga tgtgacaaa tggggtctc atgttttcag 1140 atagcagag ttgtctgta accggccctt gactgttatc atgcacca ttttcagga 1200 acgggattta ttaaaaacat ttaaaattcc agtagatact ttatacat atcttatgac 1260 tctcgaagac cattaccatg ctgatgtggc ctacacac atatccatg ctgcagatgt 1320 tgtccagtct actcatgtgc tattatac acctgctttg gaggctgtgt ttacagattt 1380 ggagattctt gcagcaattt ttgccagtgc atacatgat gtagatcatc ctggtgtgtc 1440 caatcaattt ctgatcaata caaactctga acttgccttg atgtacaatg attcctcagt 1500 cttagagaac catcatttgg ctgtgggctt taaattgctt caggaagaaa actgtgacat 1560 tttccagaat ttgaccaaaa aacaaagaca atctttaagg aaaatggtca ttgacatcgt 1620 acttgcaaca gatatgtcaa aacacatgaa tctactggct gatttgaaga ctatggttga 1680 aactaagaaa gtgacaagct ctggagttct tcttcttgat aattattccg ataggattca 1740 ggttcttcag aatatggtgc actgtgcaga tctgagcaac ccaacaaagc ctctccagct 1800 gtaccgccag tggacggacc ggataatgga ggagttcttc cgccaaggag accgagagag 1860 ggaacgtggc atggagataa gccccatgtg tgacaagcac aatgcttccg tggaaaaatc 1920 acaggtgggc ttcatagact atattgttca tcccctctgg gagacatggg cagacctcgt 1980 ccaccctgac gcccaggata ttttggacac tttggaggac aatcgtgaat ggtaccagag 2040 cacaatccct cagagcccct ctcctgcacc tgatgaccca gaggagggcc ggcagggtca 2100 aactgagaaa ttccagtttg aactaacttt agaggaagat ggtgagtcag acacggaaaa 2160 ggacagtggc agtcaagtgg aagaagacac tagctgcagt gactccaaga ctctttgtac 2220 tcaagactca gagtctactg aaattcccct tgatgaacag gttgaagagg aggcagtagg 2280 ggaagaagag gaaagccagc ctgaagcctg tgtcatagat gatcgttctc ctgacacgta 2340 acagtgcaaa aactttcatg cctttttttt ttttaagtag aaaaattgtt tccaaagtgc 2400 atgtcacatg ccacaaccac ggtcacacct cactgtcatc tgccaggacg tttgttgaac 2460 aaaactgacc ttgactactc agtccagcgc tcaggaatat cgtaaccagt ttttcacct 2520 ccatgtcatc cgagcaaggt ggacatcttc acgaacagcg ttttaacaa gatttcagct 2580 tggtagagct gacaaagcag ataaaatcta ctccaaatta ttttcaagag agtgtgactc 2640 atcaggcagc ccaaaagttt attggacttg gggtttctat tcctttttat ttgtttgcaa 2700 tattttcaga agaaaggcat tgcacagagt gaacttaatg gacgaagcaa caaatatgtc 2760 aagaacagga catagcacga atctgttacc agtaggagga ggatgagcca cagaaattgc 2820 ataatttct aatttcaagt cttcctgata catgactgaa tagtgtggtt cagtgagctg 2880 cactgacctc tacattttgt atgatatgta aaacagattt tttgtagac ttacttttat 2940 tattaaatgt attgaggtat tatatttaaa aaaaactatg ttcagaactt catctgccac 3000 tggttatttt tttctaagga gtaacttgca agttttcagt acaaatctgt gctacactgg 3060 ataaaaatct aatttatgaa ttttacttgc accttatagt tcatagcaat taactgattt 3120 gtagtgattc attgtttgtt ttatatacca atgacttcca tattttaaaa gagaaaaaca 3180 actttatgtt gcaggaaacc ctttttgtaa gtctttatta tttactttgc atttgtttc 3240 actctttcca gataagcaga gttgctcttc accagtgttt ttcttcatgt gcaaagtgac 3300 tatttgttct ataatacttt tatgtgtgtt atatcaaatg tgtcttaagc ttcatgcaaa 3360 ctcagtcatc agttcgtgtt gtctgaagca agtgggagat atataaatac ccagtagcta 3420 aaatggtcag tcttttttag atgttttcct acttagtatc tcctaataac gttttgctgt 3480 gtcactagat gttcatttca caagtgcatg tctttctaat aatccacaca tttcatgctc 3540 tataatcca cacatttcat gctcattttt attgttttta cagccagtta tagtaagaaa 3600 aaggtttttc cccttgtgct gctttataat ttagcgtgtg tctgaacctt atccatgttt 3660 gctagatgag gtcttgtcaa atatatcact accattgtca ccggtgaaaa gaaacaggta gttaagttag ggttaacatt catttcaacc acgaggttgt atatcatgac tagcttttac tcttggttta cagagaaag ttaaacagcc aactaggcag tttttaaga tattaacaat attachment attachment actatcct atttggtttt aatgatttca ccatgggatt aagaactata tcaggaacat ccctgagaaa cggttttaag tgtagcaact actcttcctt aatggacagc cacataacgt gtaggaagtc ctttatcact tatcctcgat ccataagcat atcttgcaga ggggaactac ttctttaaac acatggaggg aaagagatg atgccactgg caccagaggg ttagtactgt gatgcatcct aaaatatttta ttatattggt aaaaattctg gttaaataaa aaattagaga tcactcttgg ctgatttcag crack tgtattacag ttttagat taattcctag tgtttacctg attatagcag ttggcatcat ggggcattta attctgactt tatccccacg tcagccttaa taagtcttc tttaccttct ctatgaagac tttaaagcccc aaataatcat ttttcacatt gatattcaag aattgagata gatagaagcc 4380 aaagtgggta tctgacaagt ggaaatca acgtttaaga agaattacaa ctctgaaag 4440 catttatatg tggaacttct caggagcct cctggggact ggaagtaag tcatcagcca 4500 ggcaaatgac tcatgctgaa gagagtcccc atttcagtcc cctgagatct agctgatgct 4560 tagatcctttt gaataaaaa ttatgcttt attackactctga tctttacat aaagcagaag 4620 aggaatcaac tagttaattg caagttct actctgttc ctctgtaaag atcagatggt 4680 aatctttca atagaaaaaaaagacg tatgtttgac caagtagtttt cacagaata 4740 tttgggaact tgttctttt aattttatt gtccctgagt gaagtctga aagaaaggta 4800 aagagtctag agtttattcc tctttccaa acatctcat tcctctcctc cctacactta 4860 gtatttcccc cacagagtgc ctagaatcttt aaatgaat aaaaaaa gcagcaat 4920 gtcattaaca atccagacc tgaagggta aagggtttat aactgcacta ataagagag 4980 gctcttttt ttcttccag ttgttggtt tttaatgta ccgtgttgta aagataccca 5040 ctaatggaca atcaaattgc agaaaaggct caatatccaa gagacaggga ctaatgcact 5100 gtacaatctg cttatccttg cccttctctc ttgccaaagt gtgcttcaga aatatatact 5160 gctttaaaaa agaataaaag aatatccttt tacaagtggc tttacatttc ctaaaatgcc 5220 ataagaaaat gcaatatctg ggtactgtat ggggaaaaaa atgtccaagt ttgtgtaaaa 5280 ccagtgcatt tcagcttgca agttactgaa cacaataatg ctgttttaat tttgttttat 5340 atcagttaaa attcacaata atgtagatag aacaaattac agacaaggaa agaaaaaact 5400 tgaatgaaat ggattttaca gaaagcttta tgataatttt tgaatgcatt atttattttt 5460 tgtgccatgc attttttttc tcaccaaatg accttacctg taatacagtc ttgtttgtct 5520 gtttacaacc atgtatttat tgcaatgtac atactgtaat gttaattgta aattatctgt 5580 tcttattaaa acatcatccc atgatgggat ggtgttgata tatttggaaa ctcttggtga 5640 gagaatgaat ggtgtgtata catactctgt acatttttct tttctcctgt aatatagtct 5700 tgtcacctta gagcttgttt atggaagatt caagaaaact ataaaatact taaagatata 5760 taaatttaaa aaaacatagc tgcaggtctt tggtcccagg gctgtgcctt aactttaacc 5820 aatattttct tctgttttgc tgcatttgaa aggtaacagt ggagctaggg ctgggcattt 5880 tacatccagg cttttaattg attagaattc tgccaatagg tggattttac aaaaccacag 5940 acaacctctg aaagattctg agacccttt gagacagaag ctcttaagta cttcttgcca 6000 gggagcagca ctgcatgtgt gatggttgtt tgccatctgt tgatcaggaa ctacttcagc 6060 tacttgcatt tgattatttc ctttttttt ttttttaact cggaaacaca actggggaaa 6120 tatattcttt cccagtgatt ataaacaatc ttttcttttt ttttaagtcc ttttggcttc 6180 tagagctcat aggaaaatgg acttgatttg aaattggagc cagagtttac tcgtgttggt 6240 tatctattca tcagcttcct gacatgttaa gagaatacat taaagagaaa atactgtttt 6300 ttaatcctaa aatttttctt ccactaagat aaaccaaatg tccttacata tatgtaaacc 6360 catctattta aacgcaaagg tgggttgatg tcagtttaca tagcagaaag cattcactat 6420 cctctaagat ttgtttctgc aaaactttca ttgctttaga atttaaaat ttcaccttgt 6480 acaatggcca gcccctaaag caggaaacat ttataatgga ttatatggaa acatcctccc 6540 agtacttgcc cagcccttga atcatgtggc ttttcagtga aaggaaaagat tcttttcta 6600 ggaaaaatga gcctatttta ttttatttta ttttattttt tgacacaaac tgtagatttt 6660 agcagccctg gcccaaagga atttgattac ttttgttttta aacagtacaa aggggacact 6720 ataattacaa aaacatcctt aactgatttg agttgtttt atttctttgg atatattttc 6780 agagtggtaa attgtgtgtg agaattacaa atgattattc ttttagtggt ttcttagcct 6840 ctcttacagc ccacggggat agtactgtac atcaatacct tcatatgaaa tttttatatg 6900 caatgaaaat aaaagcatgg gttgattctg cctatttatg actcaatctt ttacaataa 6960 aagattattc attttaaatt atagttcaat cagcatgtct cttaggatac tgaacgtggt 7020 7080 acttgagtaa ttttgctaca ttaaaaatta ccaaaattac ttaagatggcc tataagatta 7140 agcatggtgt tttctaagca agctttgaaa ggggccttcc atacttactt aattgaatat 7200 tctgggatat tgaaaattat tcagatactt gatacttt ttggttacc tactccgcaa 7260 actacaaagt tttaggact acacataag ttaatgac acagtgtttg ctttcatgga 7320 gcttacagtc tggagggac aaaggcttaa acaatactca tataattata tatgtgatca 7380 gtacaatgaa ggagctcagt gggtaaata agcaggaacc tgaacttgat ctgttccgga 7440 gggccacaga aggctcctt gaggccttga gaagtgat tgcatctgag tctgaagga 7500 ttgtaagagg taactaggga aaagttgac aggagagga aggggatcca ghaagaaac 7560 atttgcaag atcttgaggc ataatgagc tgagacatc tggaaact gaggaaaagt 7620 gagagagtag gcaggggcctg gagccgcaga gccattgcta accatcctgt gtgagatatc 7680 ccccattctg tagctttatt ctcataaccc tgctcaattt tcttataac acttctcaca 7740 gatttatata cgtgttgtt ttgttatct gtctctccca ccagaccaca gctccatgag 7800 agcaaggtct ttgcttacca atatatcact agcacttaa actatgcctg gtacacagta 7860 ggttcttaat atgtgttgaa tatagccatc aaattgatat tggatataat tcaatctgat 7920 aagatatttt gagatattaa agagttttta acttgatacc ataaaaaaa aaaaaaaaa 7979 <210> 2 <211> 745 <212> PRT <213> Homo sapiens <400> 2 Put Ala Gln Gln Thr Ser Pro Asp Thr Leu Thr Val Pro Glu Val Asp 1 5 10 15 Asn Pro His Cys Pro Asn Pro Trp Leu Asn Glu Asp Leu Val Lys Ser 20 25 30 Leu Arg Glu Asn Leu Leu Gln His Glu Lys Ser Lys Thr Ala Arg Lys 35 40 45 Ser Val Ser Pro Lys Leu Ser Pro Val Ile Ser Pro Arg Asn Ser Pro 50 55 60 Arg Leu Leu Arg Arg Met Leu Leu Ser Ser Asn Ile Pro Lys Gln Arg 65 70 75 80 Arg Phe Thr Val Ala His Thr Cys Phe Asp Val Asp Asn Gly Thr Ser 85 90 95 Ala Gly Arg Ser Pro Leu Asp Pro Met Thr Ser Pro Gly Ser Gly Leu 100 105 110 Ile Leu Gln Ala Asn Phe Val His Ser Gln Arg Arg Glu Ser Phe Leu 115 120 125 Tyr Arg Ser Asp Ser Asp Tyr Asp Leu Ser Pro Lys Ser Met Ser Arg 130 135 140 Asn Ser Ser Ile Ala Ser Asp Ile His Gly Asp Asp Leu Ile Val Thr 145 150 155 160 Pro Phe Ala Gln Val Leu Ala Ser Leu Arg Thr Val Arg Asn Asn Phe 165 170 175 Ala Ala Leu Thr Asn Leu Gln Asp Arg Ala Pro Ser Lys Arg Ser Pro 180 185 190 Met Cys Asn Gln Pro Ser Ile Asn Lys Ala Thr Ile Thr Glu Glu Ala 195 200 205 Tyr Gln Lys Leu Ala Ser Glu Thr Leu Glu Glu Leu Asp Trp Cys Leu 210 215 220 Asp Gln Leu Glu Thr Leu Gln Thr Arg His Ser Val Ser Glu Met Ala 225 230 235 240 Ser Asn Lys Phe Lys Arg Met Leu Asn Arg Glu Leu Thr His Leu Ser 245 250 255 Glu Met Ser Arg Ser Gly Asn Gln Val Ser Glu Phe Ile Ser Asn Thr 260 265 270 Phe Leu Asp Lys Gln His Glu Val Glu Ile Pro Ser Pro Thr Gln Lys 275 280 285 Glu Lys Glu Lys Lys Lys Arg Pro Met Ser Gln Ile Ser Gly Val Lys 290 295 300 Lys Leu Met His Ser Ser Ser Leu Thr Asn Ser Ser Ile Pro Arg Phe 305 310 315 320 Gly Val Lys Thr Glu Gln Glu Asp Val Leu Ala Lys Glu Leu Glu Asp 325 330 335 Val Asn Lys Trp Gly Leu His Val Phe Arg Ile Ala Glu Leu Ser Gly 340 345 350 Asn Arg Pro Leu Thr Val Ile Met His Thr Ile Phe Gln Glu Arg Asp 355 360 365 Leu Leu Lys Thr Phe Lys Ile Pro Val Asp Thr Leu Ile Thr Tyr Leu 370 375 380 Met Thr Leu Glu Asp His Tyr His Ala Asp Val Ala Tyr His Asn Asn 385 390 395 400 Ile His Ala Ala Asp Val Val Gln Ser Thr His Val Leu Leu Ser Thr 405 410 415 Pro Ala Leu Glu Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Ile 420 425 430 Phe Ala Ser Ala Ile His Asp Val Asp His Pro Gly Val Ser Asn Gln 435 440 445 Phe Leu Ile Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Ser 450 455 460 Ser Val Leu Glu Asn His His Leu Ala Val Gly Phe Lys Leu Leu Gln 465 470 475 480 Glu Glu Asn Cys Asp Ile Phe Gln Asn Leu Thr Lys Lys Gln Arg Gln 485 490 495 Ser Leu Arg Lys Met Val Ile Asp Ile Val Leu Ala Thr Asp Met Ser 500 505 510 Lys His Met Asn Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys 515 520 525 Lys Val Thr Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr Ser Asp Arg 530 535 540 Ile Gln Val Leu Gln Asn Met Val His Cys Ala Asp Leu Ser Asn Pro 545 550 555 560 Thr Lys Pro Leu Gln Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met Glu 565 570 575 Glu Phe Phe Arg Gln Gly Asp Arg Glu Arg Glu Arg Gly Met Glu Ile 580 585 590 Ser Pro Met Cys Asp Lys His Asn Ala Ser Val Glu Lys Ser Gln Val 595 600 605 Gly Phe Ile Asp Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp 610 615 620 Leu Val His Pro Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp Asn 625 630 635 640 Arg Glu Trp Tyr Gln Ser Thr Ile Pro Gln Ser Pro Ser Pro Ala Pro 645 650 655 Asp Asp Pro Glu Glu Gly Arg Gln Gly Gln Thr Glu Lys Phe Gln Phe 660 665 670 Glu Leu Thr Leu Glu Glu Asp Gly Glu Ser Asp Thr Glu Lys Asp Ser 675 680 685 Gly Ser Gln Val Glu Glu Asp Thr Ser Cys Ser Asp Ser Lys Thr Leu 690 695 700 Cys Thr Gln Asp Ser Glu Ser Thr Glu Ile Pro Leu Asp Glu Gln Val 705 710 715 720 Glu Glu Glu Ala Val Gly Glu Glu Glu Glu Ser Gln Pro Glu Ala Cys 725 730 735 Val Ile Asp Asp Arg Ser Pro Asp Thr 740 745 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PDE4D5_forward primer <400> 3 gcttctcagc agcaacatc 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primary PDE4D5_reverse <400> 4 tgccattgtc cacatcaaaa 20 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PDE4D5 probe <400> 5 acagcggcgt ttcacggtgg caca 24 <210> 6 <211> 8130 <212> DNA <213> Homo sapiens <400> 6 agattatagc ccagcgtacg agaagcacga gtcctatagt tggcgtaccc tgaggcctgc 60 cagttcctgc cttaatgcat atgtagtcgt aattgagttc tgacacggcc ttggatgttt 120 ctgtcctaaa tagctgacat tgcatcttca agactgtcat tccagttggc ttttgagtgg 180 atacgtgcag tgagatcatt gacactggaa acactagttc ccattttaat tacttaaaac 240 accacgatga aaagaaatac ctgtgatttg ctttctcgga gcaaaagtgc ctctgaggaa 300 acactacatt ccagtaatga agaggaagac cctttccgcg gaatggaacc ctatcttgtc 360 cggagacttt catgtcgcaa tattcagctt ccccctctcg ccttcagaca gttggaacaa 420 gctgacttga aaagtgaatc agagaacatt caacgaccaa ccagcctccc cctgaagatt 480 ctgccgctga ttgctatcac ttctgcagaa tccagtggtt ttgatgtgga caatggcaca 540 tctgcgggac ggagtccctt ggatcccatg accagcccag gatccgggct aattctccaa 600 gcaaattttg tccacagtca acgacgggag tccttcctgt atcgatccga cagcgattat 660 gacctctctc caaagtctat gtcccggaac tcctccattg ccagtgatat acacggagat 720 gacttgattg tgactccatt tgctcaggtc ttggccagtc tgcgaactgt acgaaacaac 780 tttgctgcat taactaattt gcaagatcga gcacctagca aaagatcacc catgtgcaac 840 caaccatcca tcaacaaagc caccataaca gaggaggcct accagaaact ggccagcgag 900 accctggagg agctggactg gtgtctggac cagctagaga ccctacagac caggcactcc 960 gtcagtgaga tggcctccaa caagtttaaa aggatgctta atcgggagct cacccatctc 1020 tctgaaatga gtcggtctgg aaatcaagtg tcagagttta tatcaacac attcttagat 1080 aagcaacatg aagtggaat tccttctcca actcagaagg aaaggagaa aaagaaaga 1140 ccaatgtctc agatcagtgg agtcaagaa ttgatgcaca gctctagtct gactattca 1200 agtatcccaa ggtttggagt taaactgaa ggaagatg tccttgccaa ggaactagaa 1260 gatgtgaaca aatggggtct tcatgttttc agaatagcag agttgtctgg taaccggccc 1320 ttgactgtta tcatgcacac cattttcag gaacgggatt tattaaaaac atttaaaatt 1380 ccagtagata ctttaattac atatcttatg actctcgaag accattacca tgctgatgtg 1440 gcctatcaca acaatatcca tgctgcagat gttgtccagt ctactcatgt gctattatct 1500 acacctgctt height height shoes height height shoes height 1560 gcaatacatg atgtagatca tcctggtgtg tccaatcaat ttctgatcaa tacaactct 1620 gaacttgcct tgatgtacaa tgattcctca gtcttagaga accatcattt ggctgtgggc 1680 tttaaattgc ttcaggaga aactgtgac attttccaga atttgaccaa aaaacaaaga 1740 caatctttaa ggaaaatggt cattgacatc gtacttgcaa cagatgtc aaaacacatg 1800 aatctactgg ctgatttgaa gactatggtt gaactaaga aagtgacaag ctctggagtt 1860 cttctcttg atattattc cgataggatt caggttctc agaatatggt gcactgtgca 1920 gatctgagca acccaaaa gcctctccag ctgtaccgcc agtggacgga ccggataatg 1980 gaggagttct tccgccaagg agaccgagag agggacgtg gcatggagat aagccccatg 2040 tgtgacaagc acatgctc cgtggaaaaa tcacaggtgg gcttcataga ctatattgtt 2100 catcccctct gggagacatg ggcagacctc gtccaccctg acggcccagga tattttggac 2160 actttggagg acaatcgtga atggtaccag agcacaatcc ctcagagccc ctctcctgca 2220 cctgatgacc cagaggagggg ccggcaggggt caactgaga aattccagtt tgaactact 2280 ttagaggaag atggtgagtc agacacggaa aaggacagtg gcagtcaagt ggaagagac 2340 actagctgca gtgactccaa gactcttgt actcagact cagagctac tgaaattccc 2400 cttgatgaac aggttgaaga ggaggcagta ggggaagaag aggaagcca gcctgaagcc 2460 tgtgtcatag atgatcgttc tcctgacacg taacagtgca aaaactttca tgccttttt 2520 ttttttaagt agaaaaattg tttccaagt gcatgtcaca tgccacaacc acggtcacac 2580 ctcactgtca tctgccagga cgtttgttga acaaactga ccttgactac tcagtccagc 2640 gctcaggaat atcgtaacca gttttcac ctccatgtca tccgagcaag gtggacatct 2700 tcacgacacag cgttttaac aagatttcag cttggtagag ctgacaagc agataaaatc 2760 tactccaaat tatttcaag agagtgtgac tcatcaggca gcccaaagt tattggact 2820 tggggttctt atttccttttttgtttgc atattttca gagaaaggc attgcacaga 2880 gtgaacttaa tggacgaagc aaaaatg tcagacag gatagcac gatctgtta 2940 ccagtaggag gaggatgagc cacagaaatt gcatatttt ctaatttca gtcttcctga 3000 tacatgactg atagtgtgg ttcagtgagc tgcactgacc tctacatttt gtatgatatg 3060 taaaacagat ttttgtaga gcttacttt attattaat gtattgaggt attattatta 3120 aaaaaaacta tgttcagaac ttcatctgcc actggttatt ttttctaag gagtaacttg 3180 caagttttca gtacaaatct gtgctacact ggataaaaat ctaatttatg aattttactt 3240 gcaccttata gttcatagca attaactgat ttgtagtgat tcattgtttg ttttatatac 3300 caatgacttc catattttaa aagagaaaaa caactttatg ttgcaggaaa cccttttgtgt 3360 aagtcttat tatttacttt gcattttgtt tcactctttc cagataagca gagttgctct 3420 tcaccagtgt ttttcttcat gtgcaaagtg actatttgtt ctataatact tttatgtgtg 3480 ttatatcaaa tgtgtcttaa gcttcatgca aactcagtca tcagttcgtg ttgtctgaag 3540 caagtgggag atatataaat acccagtagc taaaatggtc agtcttttt agatgttttc 3600 ctacttagta tctcctaata acgttttgct gtgtcactag atgttcattt cacaagtgca 3660 tgtctttcta ataatccaca catttcatgc tctaataatc cacacatttc atgctcattt 3720 ttattgtttt tacagccagt tatagtaaga aaaaggtttt tccccttgtg ctgctttata 3780 atttagcgtg tgtctgaacc ttatccatgt ttgctagatg aggtcttgtc aaatatatca 3840 ctaccattgt caccggtgaa aagaaacagg tagttaagtt agggttaaca ttcatttcaa 3900 ccacgaggtt gtatatcatg actagctttt actcttggtt tacagagaaa agttaaacag 3960 ccaactaggc agttttaag aatattaaca atatattaac aaacaccaat acaactaatc 4020 ctatttggtt ttaatgattt caccatggga ttaagaacta tatcaggaac atccctgaga 4080 aacggtttta agtgtagcaa ctactcttcc ttaatggaca gccacataac gtgtaggaag 4140 tcctttatca cttatcctcg atccataagc atactctgca gaggggaact acttctttaa 4200 acacatggag ggaaagaaga tgatgccact ggcaccagag ggttagtact gtgatgcatc 4260 ctaaaatatt tattatattg gtaaaaattc tggttaaata aaaaattaga gatcactctt 4320 ggctgatttc agcaccagga actgtattac agttttagag attaattcct agtgtttacc 4380 tgattatagc agttggcatc atggggcatt taattctgac tttatcccca cgtcagcctt 4440 aataaagtct tctttacctt ctctatgaag actttaaagc ccaaataatc atttttcaca 4500 ttgatattca agaattgaga tagatagaag ccaaagtggg tatctgacaa gtggaaaatc 4560 aaacgtttaa gagaattac aactctgaaa agcattatta tgtggaactt ctcaggagc 4620 ctcctgggga ctggaagta agtcatcagc caggcaatg actcatgctg aagagagtcc 4680 ccatttcagt cccctgagat ctagctgatg cttagatcct ttgaataaa aattatgtct 4740 ttataactct gatctttac attaagcaga agaggaatca actaagttaat tgcaggtttt 4800 ctactctgtt tcctctgtaa agatcagatg gtaatctttc aaataagaaaaataaga 4860 cgtatgtttg accaagtagt ttcacaagaa tatttgggaa cttgttttctt ttaattttat 4920 ttgtccctga gtgaagtcta gaagaaagg taaagagtct agagtttt cctctttcca 4980 aaacattctc attcctcc tccctacact tagtatttcc cccacagagt gcctagaatc 5040 ttaataatga aaaataa aaagcagcaat atgtcattaa aaatccaga cctgaaaggg 5100 taaagggttt atactgcac taataagag aggcttttt ttttctttcc agttttgttgg 5160 ttttaatgg taccgtgttg taagatacc cactaatgga caatcaatt gcagaaaagg 5220 ctcaatcc agagacagg gactaatgca ctgtacaatc tgcttacct tgccctctc 5280 tcttgccaaa gtgtgcttca gaaatatata ctgctttaaa aaagataaa agatatcct 5340 tttacaagtg gctttacatt tcctaaaatg ccataagaaa atgcaatc tggtactgt 5400 atggggaaaa aaatgtccaa gtttgtgtaa aaccagtgca ttcagcttg caagttactg 5460 aacacataa tgctgtttta atttgtttt atatcagtta aattcacaa taatgtagat 5520 agaaaaattt acagaaagg aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagct 5580 tatgataatt ttgaatgca ttatttttt ttgtgccat gcatttttt tctcaccaaa 5640 tgaccttacc tgtaatacag tcttgtttgt ctgtttacaa ccatgtattt attgcaatgt 5700 acatactgta atgttaattg taaattatct gttcttatta aaacatcatc ccatgatggg 5760 atggtgttga tatatttgga aactctttggt gagagaatga atggtgtgta tacatactct 5820 gtacatttt cttttctcct gtaatatagt cttgtcacct tagagcttt ttatggaaga 5880 ttcaagaaaa ctataaaata cttaaagata tataaattta aaaaaacata gctgcaggtc 5940 tttggtccca gggctgtgcc ttactttaa ccaatatttt cttctgtttt gctgcatttg 6000 aaaggtaaca gtggagctag ggctggggcat tttacatcca ggcttttaat tgattagaat 6060 tctgccaata ggtggatttt acaaaaccac agaaccctc tgaaagattc tgagaccctt 6120 ttgagacaga agctcttaag tacttcttgc cagggagcag cactgcatgt gtgatggttg 6180 tttgccatct gttgatcagg aactacttca gctacttgca tttgattatt tccttttttt 6240 ttttttttaa ctcggaaaca caactggga aatatattct ttcccagtga ttataaacaa 6300 tctttttctt ttttttaagt ccttttggct tctagagctc ataggaaaat ggacttgatt 6360 tgaaattgga gccagagttt actcgtgttg gttatctatt catcagcttc ctgacatgtt 6420 aagagaatac attaagaga aaatactgtt ttttaatcct aaaatttttc ttccactaag 6480 ataaaccaaa tgtccttaca tatatgtaaa cccatctatt taaacgcaaa ggtgggttga 6540 tgtcagttta catagcagaa agcattcact atcctctaag atttgtttct gcaaaacttt 6600 cattgcttta gaattttaaa atttcacctt gtacaatggc cagcccctaa agcaggaaac 6660 atttataatg gattatatgg aaacatcctc ccagtacttg cccagccctt gaatcatgtg 6720 gcttttcagt gaaggaaag attcttttc taggaaaaat gagcctattt tattttattt 6780 tattttattt tttgacacaa actgtagatt ttagcagccc tggcccaaag gaatttgatt 6840 acttttgttt taaacagtac aaaggggaca ctataattac aaaaacatcc ttaactgatt 6900 tgagttgttt ttattcttt ggatatattt tcagagtggt aaattgtgtg tgagaattac 6960 aaatgattat tcttttagtg gtttcttagc ctctcttaca gcccacgggg atagtactgt 7020 acatcaatac cttcatatga aatttttata tgcaatgaaa ataaagcat gggttgattc 7080 tgcctatta tgactcaatc ttttacaaat aaaagattat tcattttaaa ttatagttca 7140 atcagcatgt ctcttaggat actgaacgtg gttgaatga areatagtg acatcataag 7200 7260 taccaaaatt acttagatgg cctataagat taagcatggt gttttctaag caagctttga 7320 aaggggcctt ccatacttac ttaattgaat attctgggat attgaaaatt attcagatac 7380 ttgacaatta tttttggtta cctactccgc aaactacaaa gttttaaagga ctcaacaata 7440 agttaatgag acacagtgtt tgctttcatg gagcttacag tctggagggg acaaaggctt 7500 aaacaatact catataatta tatatgtgat cagtacaatg aaggagctca gtggggtaaa 7560 taagcaggaa cctgaacttg atctgttccg gagggccaca gaaggcttcc ttgaggcctt 7620 gagaaagtga tttgcatctg agttctgaag gattgtaaga ggtaactagg gaaaaagttg 7680 acaggaagag gaaggggatc cagacaagaa acatttgcaa agatcttgag gcataaatga 7740 gcttgagaca tctggagaaa ctgaggaaaa gtgagagagt aggcagggcc tggagccgca 7800 gagccattgc taaccatcct gtgtgagata tcccccattc tgtagcttta ttctcataac 7860 cctgctcaat tttctttata acacttctca cagatttata tacgtgtttg ttttgttat 7920 ctgtctctcc caccagacca cagctccatg agagcaaggt ctttgcttac caatatatca 7980 ctagcactta aaactatgcc tggtacacag taggttctta atatgtgttg aatatagcca 8040 tcaaattgat attggatata attcaatctg ataagatatt ttgagatatt aaagagtttt 8100 taacttgata ccataaaaaa aaaaaaaaa 8130 <210> 7 <211> 748 <212> PRT <213> Homo sapiens <400> 7 Met Lys Arg Asn Thr Cys Asp Leu Leu Ser Arg Ser Lys Ser Ala Ser 1 5 10 15 Glu Glu Thr Leu His Ser Ser Asn Glu Glu Glu Asp Pro Phe Arg Gly 20 25 30 Met Glu Pro Tyr Leu Val Arg Arg Leu Ser Cys Arg Asn Ile Gln Leu 35 40 45 Pro Pro Leu Ala Phe Arg Gln Leu Glu Gln Ala Asp Leu Lys Ser Glu 50 55 60 Ser Glu Asn Ile Gln Arg Pro Thr Ser Leu Pro Leu Lys Ile Leu Pro 65 70 75 80 Leu Ile Ala Ile Thr Ser Ala Glu Ser Ser Gly Phe Asp Val Asp Asn 85 90 95 Gly Thr Ser Ala Gly Arg Ser Pro Leu Asp Pro Met Thr Ser Pro Gly 100 105 110 Ser Gly Leu Ile Leu Gln Ala Asn Phe Val His Ser Gln Arg Arg Glu 115 120 125 Ser Phe Leu Tyr Arg Ser Asp Ser Asp Tyr Asp Leu Ser Pro Lys Ser 130 135 140 Met Ser Arg Asn Ser Ser Ile Ala Ser Asp Ile His Gly Asp Asp Leu 145 150 155 160 Ile Val Thr Pro Phe Ala Gln Val Leu Ala Ser Leu Arg Thr Val Arg 165 170 175 Asn Asn Phe Ala Ala Leu Thr Asn Leu Gln Asp Arg Ala Pro Ser Lys 180 185 190 Arg Ser Pro Met Cys Asn Gln Pro Ser Ile Asn Lys Ala Thr Ile Thr 195 200 205 Glu Glu Ala Tyr Gln Lys Leu Ala Ser Glu Thr Leu Glu Glu Leu Asp 210 215 220 Trp Cys Leu Asp Gln Leu Glu Thr Leu Gln Thr Arg His Ser Val Ser 225 230 235 240 Glu Met Ala Ser Asn Lys Phe Lys Arg Met Leu Asn Arg Glu Leu Thr 245 250 255 His Leu Ser Glu Met Ser Arg Ser Gly Asn Gln Val Ser Glu Phe Ile 260 265 270 Ser Asn Thr Phe Leu Asp Lys Gln His Glu Val Glu Ile Pro Ser Pro 275 280 285 Thr Gln Lys Glu Lys Glu Lys Lys Lys Arg Pro Met Ser Gln Ile Ser 290 295 300 Gly Val Lys Lys Leu Met His Ser Ser Ser Leu Thr Asn Ser Ser Ile 305 310 315 320 Pro Arg Phe Gly Val Lys Thr Glu Gln Glu Asp Val Leu Ala Lys Glu 325 330 335 Leu Glu Asp Val Asn Lys Trp Gly Leu His Val Phe Arg Ile Ala Glu 340 345 350 Leu Ser Gly Asn Arg Pro Leu Thr Val Ile Met His Thr Ile Phe Gln 355 360 365 Glu Arg Asp Leu Leu Lys Thr Phe Lys Ile Pro Val Asp Thr Leu Ile 370 375 380 Thr Tyr Leu Met Thr Leu Glu Asp His Tyr His Ala Asp Val Ala Tyr 385 390 395 400 His Asn Asn Ile His Ala Ala Asp Val Val Gln Ser Thr His Val Leu 405 410 415 Leu Ser Thr Pro Ala Leu Glu Ala Val Phe Thr Asp Leu Glu Ile Leu 420 425 430 Ala Ala Ile Phe Ala Ser Ala Ile His Asp Val Asp His Pro Gly Val 435 440 445 Ser Asn Gln Phe Leu Ile Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr 450 455 460 Asn Asp Ser Ser Val Leu Glu Asn His His Leu Ala Val Gly Phe Lys 465 470 475 480 Leu Leu Gln Glu Glu Asn Cys Asp Ile Phe Gln Asn Leu Thr Lys Lys 485 490 495 Gln Arg Gln Ser Leu Arg Lys Met Val Ile Asp Ile Val Leu Ala Thr 500 505 510 Asp Met Ser Lys His Met Asn Leu Leu Ala Asp Leu Lys Thr Met Val 515 520 525 Glu Thr Lys Lys Val Thr Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr 530 535 540 Ser Asp Arg Ile Gln Val Leu Gln Asn Met Val His Cys Ala Asp Leu 545 550 555 560 Ser Asn Pro Thr Lys Pro Leu Gln Leu Tyr Arg Gln Trp Thr Asp Arg 565 570 575 Ile Met Glu Glu Phe Phe Arg Gln Gly Asp Arg Glu Arg Glu Arg Gly 580 585 590 Met Glu Ile Ser Pro Met Cys Asp Lys His Asn Ala Ser Val Glu Lys 595 600 605 Ser Gln Val Gly Phe Ile Asp Tyr Ile Val His Pro Leu Trp Glu Thr 610 615 620 Trp Ala Asp Leu Val His Pro Asp Ala Gln Asp Ile Leu Asp Thr Leu 625 630 635 640 Glu Asp Asn Arg Glu Trp Tyr Gln Ser Thr Ile Pro Gln Ser Pro Ser 645 650 655 Pro Ala Pro Asp Asp Pro Glu Glu Gly Arg Gln Gly Gln Thr Glu Lys 660 665 670 Phe Gln Phe Glu Leu Thr Leu Glu Glu Asp Gly Glu Ser Asp Thr Glu 675 680 685 Lys Asp Ser Gly Ser Gln Val Glu Glu Asp Thr Ser Cys Ser Asp Ser 690 695 700 Lys Thr Leu Cys Thr Gln Asp Ser Glu Ser Thr Glu Ile Pro Leu Asp 705 710 715 720 Glu Gln Val Glu Glu Glu Ala Val Gly Glu Glu Glu Glu Ser Gln Pro 725 730 735 Glu Ala Cys Val Ile Asp Asp Arg Ser Pro Asp Thr 740 745 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PDE4D7_forward primer <400> 8 gaacattcaa cgaccaacca 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PDE4D7_reverse primer <400> 9 tgccattgtc cacatcaaaa 20 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PDE4D7 probe <400> 10 ctgccgctga ttgctatcac ttctgca 27 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PDE4D7 Forward Primer 2 <400> 11 cgctgattgc tatcacttct gc 22 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PDE4D7 Reverse primer <400> 12 gtcgttgact gtggacaaaa tttg 24 <210> 13 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> PDE4D7 Probe 2 <400> 13 ttcccttgga tcccatgacc agcccataag ggaa 34 <210> 14 <211> 8395 <212> DNA <213> Homo sapiens <400> 14 ttctcactgc cctgcggtgt tttgaactgc cttcttacag acgtcataca gcccttgagg 60 aatagtttct gcctggtgag attgaatgat agttctcatt cacaaaaccc tggattctaa 120 gcagggacac acagaaatta ctttcgcagg taaatcagcc cacccagcca aagtgtggag 180 agatttgttc cttggctgac ttctttgctc cacggagagg agtgttttcc tgtgcttgcc 240 ctgaaatgga acttccttga cagctctccc gtgttacagt acctcccggt cattttcttt 300 ttctctctct ctacctgcgc tcttcgagtg tcagaaacct ttaaagctgt tactatggaa 360 ttgcaaaaaa gagatcaagt gactctttca ctatgctggt ttcccttgtg acccagatga 420 agaatcaatt cagaattcag ttcctccctt ggcattgcaa gacacagaag aaactgtcac 480 540 tagaaccttg cccatgagct ggaccgagga caggagatgg actccaggaa aattggattt 600 cttcaagcag cctcccttgg aaatggata tctttaaaat cttctttgca gaaagacagt 660 tagaatgtat taatcagaat agttgaagac ttattttcct ttttattttt tttcaaaatg 720 agcattatta tgaagccaag atcccgatct acaagttccc taaggactgc agaggcagtt 780 tgttttgatg tggacaatgg cacatctgcg ggacggagtc ccttggatcc catgaccagc 840 ccaggatccg ggctaattct ccaagcaaat tttgtccaca gtcaacgacg ggagtccttc 900 ctgtatcgat ccgacagcga ttatgacctc tctccaaagt ctatgtcccg gaactctccc 960 attgccagtg atatacacgg agatgacttg attgtgactc catttgctca ggtcttggcc 1020 agtctgcgaa ctgtacgaaa caactttgct gcatacta atttgcaaga tcgagcacct 1080 agcaaaagat cacccatgtg caaccaacca tccatcaaca aagccaccat aacagaggag 1140 gcctaccaga aactggccag cgagaccctg gaggagctgg actggtgtct ggaccagcta 1200 gagaccctac agaccaggca ctccgtcagt gagatggcct ccaacaagtt taaaaggatg 1260 cttaatcggg agctcaccca tctctctgaa atgagtcggt ctggaaatca agtgtcagag 1320 tttatatcaa acacattctt agataagcaa catgaagtgg aaattccttc tccaactcag 1380 aaggaaaagg agaaaaagaa aagaccaatg tctcagatca gtggagtcaa gaaattgatg 1440 cacagctcta gtctgactaa ttcaagtatc ccaaggtttg gagttaaaac tgaacaagaa 1500 gatgtccttg ccaaggaact agaagatgtg aacaaatggg gtcttcatgt tttcagaata 1560 gcagagttgt ctggtaaccg gcccttgact gttatcatgc acaccatttt tcaggaacgg 1620 gatttattaa aaacatttaa aattccagta gatactttaa ttacatatct tatgactctc 1680 gaagaccatt accatgctga tgtggcctat cacaacaata tccatgctgc agatgttgtc 1740 cagtctactc atgtgctatt atctacacct gctttggagg ctgtgtttac agatttggag 1800 attcttgcag caatttttgc cagtgcaata catgatgtag atcatcctgg tgtgtccaat 1860 caatttctga tcaatacaaa ctctgaactt gccttgatgt acaatgattc ctcagtctta 1920 gagaaccatc atttggctgt gggctttaaa ttgctcagg aagaaactg tgacattttc 1980 cagaatttga ccaaaaaaca aagacaatct ttaggaaaa tggtcattga catcgtactt 2040 gcacagata tgtcaaaca catgaatcta ctggctgatt tgagactat ggttgaaact 2100 aagaaagtga caagctctgg agttctctt cttgataatt attccgatag gattcaggtt 2160 cttcagaata tggtgcactg tgcagatctg agcacccca caaagcctct ccagctgtac 2220 cgccagtgga cggaccggat atggaggag ttctccgcc aaggaccg agagaggga 2280 cgtggcatgg agataagccc catgtgtgac aagcacaatg cttccgtgga aaatcacag 2340 gtgggcttca tagactatat tgttcatccc ctctgggaga catgggcaga cctcgtccac 2400 cctgacgccc aggatattt ggacacttg gaggacaatc gtgaatggta ccagagcaca 2460 atccctcaga gcccctctcc tgcacctgat gacccagagg agggccggca gggtcaact 2520 gagaaatttcc agttgaact aactttagag gagatggtg agtcagacac ggaaaggac 2580 agtggcagtc aagtgaaga agacactagc tgcagtgact ccagactct ttgtactca 2640 gactcagagt ctactgaaat tccccttgat gaacaggttg aagaggaggc agtaggggaa 2700 gaagaggaaa gccagcctga agcctgtgtc atagatgatc gttctcctga cacgtaacag 2760 tgcaaaaact ttcatgcctt tttttttt aagtagaaaa attgtttcca aagtgcatgt 2820 cacatgccac aaccacggtc acacctcact gtcatctgcc aggacgtttg ttgaacaaaa 2880 ctgaccttga ctactcagtc cagcgctcag gaatatcgta accagttttt tcacctccat 2940 gtcatccgag caaggtggac atcttcacga acagcgtttt taacaagatt tcagcttggt 3000 agagctgaca aagcagataa aatctactcc aaattatttt caagagagtg tgactcatca 3060 ggcagcccaa aagtttattg gacttggggt ttctattcct ttttattgtgt ttgcaatatt 3120 ttcagaagaa aggcattgca cagagtgaac ttaatggacg aagcaacaaa tatgtcaaga 3180 acaggacata gcacgaatct gttaccagta ggaggaggat gagccacaga aattgcataa 3240 ttttctaatt tcaagtctttc ctgatacatg actgaatagt gtggttcagt gagctgcact 3300 gacctctaca ttttgtatga tatgtaaaac agattttttg tagagcttac ttttattatt 3360 aaatgtattg aggtattata tttaaaaaaa actatgttca gaacttcatc tgccactggt 3420 tatttttttc tagggagtaa cttgcaagtt ttcagtacaa atctgtgcta cactggataa 3480 aaatctaatt tatgaatttt acttgcacct tatagttcat agcaattaac tgatttgtag 3540 tgattcattg tttgttttat ataccaatga cttccatatt ttaaaagaga aaaacaactt 3600 tatgttgcag gaaacccttt ttgtaagtct ttattattta ctttgcattt tgtttcactc 3660 tttccagata agcagagttg ctcttcacca gtgttttct tcatgtgcaa agtgactatt 3720 tgttctataa tacttttatg tgtgttatat caaatgtgtc ttaagcttca tgcaaactca 3780 gtcatcagtt cgtgttgtct gaagcaagtg ggagatatat aaatacccag tagctaaaat 3840 ggtcagtctt ttttagatgt tttcctactt agtatctcct aataacgttt tgctgtgtca 3900 ctagatgttc atttcacaag tgcatgtctt tctaataatc cacacatttc atgctctaat 3960 aatccacaca tttcatgctc atttttattg tttttacagc cagttatagt aagaaaaagg 4020 tttttcccct tgtgctgctt tataatttag cgtgtgtctg aaccttatcc atgtttgcta 4080 gatgaggtct tgtcaatat atcactacca ttgtcaccgg tgaaaagaaa caggtagtta 4140 agttagggtt aacattcatt tcaccacga ggttgtatat catgactagc ttttactt 4200 ggtttacaga gaaagttaa acagccaact aggcagtttt windowatt aacaatat 4260 taacaacac caatacact aatcctattt gttttaatg atttcaccat gggattaga 4320 actatatcag gaacatccct gagaacggt tttaagtgta gcaactactc ttccttaatg 4380 gabagccaca taacgtgtag gaagtcctttt atcacttatc ctcgatccat aagcatatct 4440 tgcagagggg aactacttct ttaaacacat ggagggaag aagatgatgc cactggcacc 4500 agagggttag tactgtgatg catcctaaaa tattttat attggtaaaa attctggtta 4560 aaaaaaat tagagatcac tctggctga ttcagcacc aggactgta tcagtttt 4620 agagattaat tcctagtgtt tacctgatta tagcagttgg catcatgggg catttaattc 4680 tgactttatc cccacgtcag ccttataaa gtctcttta ccttcttat gagacttta 4740 aagcccaaat atcattttt cacattgata ttcagaatt gagatagata gaagccaag 4800 tgggtatctg acaagtggaa aatcaaacgt ttaagagaa ttacaactct gaaaagcatt 4860 tatatgtgga acttctcaag gagcctcctg gggactggaa agtaagtcat cagccaggca 4920 aatgactcat gctgaagaga gtccccattt cagtcccctg agatctagct gatgcttaga 4980 5040 atcaactagt taattgcaag gtttctactc tgtttcctct gtaaagatca gatggtaatc 5100 5160 ggaacttgtt tcttttaatt ttattgtcc ctgagtgaag tctagaaaga aaggtaaaga 5220 gtctagagtt tattcctctt tccaaaacat tctcattcct ctcctcccta cacttagtat 5280 ttcccccaca gagtgcctag aatcttaata atgaataaaa taaaaagcag caatatgtca 5340 ttaacaaatc cagacctgaa agggtaagg gtttataact gcaccataataa agaggctc 5400 tttttttttc ttccagtttg ttggttttta atggtaccgt gttgttaaaga tacccactaa 5460 tggacaatca aattgcagaa aaggctcaat atccaagaga caggactaa tgcactgtac 5520 aatctgctta tccttgccct tctctctgc caagtgtgc ttcagaata tatactgctt 5580 taaaaaagaa taaaagaata tccttttaca agtggcttta cattcctaa atgccataa 5640 gaaaatgcaa tatctgggta ctgtatgggg aaaaaatgt ccaagtttgt gtaaaaccag 5700 tgcatttcag cttgcaagtt actgaacaca ataatgctgt ttatatttg ttatatca 5760 gttaaaatttc acaataatgt agatagaca attackacagac aaggaaa aaacttgaa 5820 tgaaatggat tttacagaa gctttatgat aattttgaa tgcattattt attttgtg 5880 ccatgcatttt tttctcac caaatgacct tacctgtaat acagcttgt tgtctgttt 5940 acaaccatgt atttattgca atgtacatac tgtaatgtta attgtaaatt atctgttctt 6000 attaaaacat catcccatga tgggatggtg ttgatatatt tggaactct tggtgagaga 6060 atgaatggtg tgtatacata ctctgtacat tttctttc tcctgtaata tagtcttgtc 6120 accttagagc ttgtttatgg aagattcaag aaactataa atacttaa gatatataa 6180 tttaaaaaaa catagctgca gtctttggt cccaggggctg tgccttaact ttaccata 6240 ttttctctg tttgctgca ttgaaggt aacagtggag ctagggctgg gcattttaca 6300 tccaggcttt taattgatta gattctgcc ataggtgga ttttacaaaa ccacagacaa 6360 cctctgaaag attctgagac ccttttgaga cagaagctct taagtacttc ttgccaggga 6420 gcagcactgc atgtgtgatg gttgtttgcc atctgttgat caggaactac ttcagctact 6480 tgcatttgat tattccttt tttttttt tattactcgga aacacaactg gggaaatata 6540 ttctttccca gtgattataa acaatctttttttttttt aagtcctttt ggcttctaga 6600 gctcatagga aaatggactt gatttgaat tggagccaga gtttactcgt gttggttatc 6660 tattcatcag cttcctgaca tgttaagaga atacattaaa gagaaaatac tgttttttaa 6720 tcctaaaatt ttctctccac taagataaac caatagtcct tacatatatg taaacccatc 6780 tatttaaacg caaggtggg ttgatgtcag ttcatagc agaagcatt cactaccctc 6840 tagatttgt ttctgcaaaa ctttcattgc tttagaattt taaaatttca ccttgtacaa 6900 tggccagccc ctaaagcagg aaacatttat atggattat atggaacat cctcccagta 6960 cttgcccagc ccttgaatca tgtggcttt cagtgaaagg aaagattctt tttctaggaa 7020 aaatgagcct attttttt attttttt attttttgac acaaactgta gattttagca 7080 gccctggccc aaaggaattt gattactttt gttttaaaca gtacaaaggg gacactataa 7140 ttacaaaaac atccttaact gatttgagtt gtttttattt ctttggatat attttcagag 7200 tggtaaattg tgtgtgagaa ttacaaatga ttatctttt agtggtttct tagcctctct 7260 tacagcccac ggggatagta ctgtacatca ataccttcat atgaaatttt tatatgcaat 7320 gaaataaaaa gcatgggttg attctgccta tttatgactc aatcttttac aaataaaaga 7380 ttattcattt taaattatag ttcaatcagc atgtctctta ggatactgaa cgtggttgaa 7440 atgaaaggat agtgacatca taagttagta ctgatattca taaccaaata aagccaactt 7500 gagtaatttt gctacattaa aaattaccaa aattacttag atggcctata agattaagca 7560 tggtgttttc taagcaagct ttgaaagggg ccttccatac ttacttaatt gaatattctg 7620 ggatattgaa aattattcag atacttgaca attatttttg gttacctact ccgcaaacta 7680 caaagtttta aggactcac ataagttaa tgagacacag tgttgcttt catggagctt 7740 acagtctgga ggggacaaag gcttaaacaa tactcatata attatatg tgatcagtac 7800 aatgaaggag ctcagtgggg taaatagca ggaacctgaa cttgatctgt tccggaggc 7860 cacagaaggc ttccttgagg ccttgagaaa gtgatttgca tctgagttct gaaggattgt 7920 aagaggtaac tagggaaaa gttgacagga agaggaaggg gatccagaca agaaacattt 7980 gcaaagtct tgaggcataa atgagcttga gatactgga gaactgagg aaagtgaga 8040 gagtaggcag ggcctggagc cgcagagcca ttgctaacca tcctgtgtga gatatccccc 8100 attctgtagc tttattctca taaccctgct caatttctt tataacactt ctcacagatt 8160 tatatacgtg ttgttttg tatatctgtct ctcccaccag accacagctc catgagagca 8220 aggtctttgc ttaccaatat atcactagca cttaaaacta tgcctgtac acagtaggtt 8280 cttaatatgt gttgaata gccatcaaat tgatattgga tataattca tctgataga 8340 tattttgaga tattaagag tttttaactt gataccataa aaaaaaaaaaaaaaaaaa 8395 <210> 15 <211> 679 <212> PRT <213> Homo sapiens <400> 15 Met Ser Ile Ile Met Lys Pro Arg Ser Arg Ser Thr Ser Ser Leu Arg 1 5 10 15 Thr Ala Glu Ala Val Cys Phe Asp Val Asp Asn Gly Thr Ser Ala Gly 20 25 30 Arg Ser Pro Leu Asp Pro Met Thr Ser Pro Gly Ser Gly Leu Ile Leu 35 40 45 Gln Ala Asn Phe Val His Ser Gln Arg Arg Glu Ser Phe Leu Tyr Arg 50 55 60 Ser Asp Ser Asp Tyr Asp Leu Ser Pro Lys Ser Met Ser Arg Asn Ser 65 70 75 80 Ser Ile Ala Ser Asp Ile His Gly Asp Asp Leu Ile Val Thr Pro Phe 85 90 95 Ala Gln Val Leu Ala Ser Leu Arg Thr Val Arg Asn Asn Phe Ala Ala 100 105 110 Leu Thr Asn Leu Gln Asp Arg Ala Pro Ser Lys Arg Ser Pro Met Cys 115 120 125 Asn Gln Pro Ser Ile Asn Lys Ala Thr Ile Thr Glu Glu Ala Tyr Gln 130 135 140 Lys Leu Ala Ser Glu Thr Leu Glu Glu Leu Asp Trp Cys Leu Asp Gln 145 150 155 160 Leu Glu Thr Leu Gln Thr Arg His Ser Val Ser Glu Met Ala Ser Asn 165 170 175 Lys Phe Lys Arg Met Leu Asn Arg Glu Leu Thr His Leu Ser Glu Met 180 185 190 Ser Arg Ser Gly Asn Gln Val Ser Glu Phe Ile Ser Asn Thr Phe Leu 195 200 205 Asp Lys Gln His Glu Val Glu Ile Pro Ser Pro Thr Gln Lys Glu Lys 210 215 220 Glu Lys Lys Lys Arg Pro Met Ser Gln Ile Ser Gly Val Lys Lys Leu 225 230 235 240 Met His Ser Ser Ser Leu Thr Asn Ser Ser Ile Pro Arg Phe Gly Val 245 250 255 Lys Thr Glu Gln Glu Asp Val Leu Ala Lys Glu Leu Glu Asp Val Asn 260 265 270 Lys Trp Gly Leu His Val Phe Arg Ile Ala Glu Leu Ser Gly Asn Arg 275 280 285 Pro Leu Thr Val Ile Met His Thr Ile Phe Gln Glu Arg Asp Leu Leu 290 295 300 Lys Thr Phe Lys Ile Pro Val Asp Thr Leu Ile Thr Tyr Leu Met Thr 305 310 315 320 Leo Glu Asp His Tyr His Ala Asp Val Ala Tyr His Asn Asn Ile His 325 330 335 Ala Ala Asp Val Val Gln Ser Thr His Val Leu Leu Ser Thr Pro Ala 340 345 350 Leu Glu Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Ile Phe Ala 355 360 365 Ser Ala Ile His Asp Val Asp His Pro Gly Val Ser Asn Gln Phe Leu 370 375 380 Ile Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Ser Ser Val 385 390 395 400 Leu Glu Asn His His Leu Ala Val Gly Phe Lys Leu Leu Gln Glu Glu 405 410 415 Asn Cys Asp Ile Phe Gln Asn Leu Thr Lys Lys Gln Arg Gln Ser Leu 420 425 430 Arg Lys Met Val Ile Asp Ile Val Leu Ala Thr Asp Met Ser Lys His 435 440 445 Met Asn Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys Lys Val 450 455 460 Thr Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr Ser Asp Arg Ile Gln 465 470 475 480 Val Leu Gln Asn Met Val His Cys Ala Asp Leu Ser Asn Pro Thr Lys 485 490 495 Pro Leu Gln Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met Glu Glu Phe 500 505 510 Phe Arg Gln Gly Asp Arg Glu Arg Glu Arg Gly Met Glu Ile Ser Pro 515 520 525 Met Cys Asp Lys His Asn Ala Ser Val Glu Lys Ser Gln Val Gly Phe 530 535 540 Ile Asp Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp Leu Val 545 550 555 560 His Pro Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp Asn Arg Glu 565 570 575 Trp Tyr Gln Ser Thr Ile Pro Gln Ser Pro Ser Pro Ala Pro Asp Asp 580 585 590 Pro Glu Glu Gly Arg Gln Gly Gln Thr Glu Lys Phe Gln Phe Glu Leu 595 600 605 Thr Leu Glu Glu Asp Gly Glu Ser Asp Thr Glu Lys Asp Ser Gly Ser 610 615 620 Gln Val Glu Glu Asp Thr Ser Cys Ser Asp Ser Lys Thr Leu Cys Thr 625 630 635 640 Gln Asp Ser Glu Ser Thr Glu Ile Pro Leu Asp Glu Gln Val Glu Glu 645 650 655 Glu Ala Val Gly Glu Glu Glu Glu Ser Gln Pro Glu Ala Cys Val Ile 660 665 670 Asp Asp Arg Ser Pro Asp Thr 675 <210> 16 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PDE4D9_forward primer <400> 16 atgagcatta ttatgaagcc aagatc 26 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PDE4D9_reverse primer <400> 17 gtgccattgt ccacatcaaa ac 22 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PDE4D9 probe <400> 18 ctacaagttc cctaaggact gcagagg 27 <210> 19 <211> 1435 <212> DNA <213> Homo sapiens <400> 19 ggcggggcct gcttctctc agcttcaggc ggctgcgacg agccctcagg cgaacctctc 60 ggctttcccg cgcggcgccg cctctgctg cgcctccgc tcctctctg ctcgccacc 120 ggcttcctcc tctgagcag tcagcccgcg cgccggccgg ctccgttatg gcgacccgca 180 gccctggcgt cgtgattagt gatgatgaac caggttatga ccttgattta ttttgcatac 240 ctaatcatta tgctgaggat ttggaaaggg tgtttattcc tcatggacta attatggaca 300 ggactgaacg tcttgctcga gatgtgatga aggagatggg aggccatcac attgtagccc 360 tctgtgtgct caaggggggc tataaattct ttgctgacct gctggattac atcaaagcac 420 tgaatagaaa tagtgataga tccattccta tgactgtaga tttatcaga ctgaagagct 480 attgtaatga ccagtcaaca ggggacataa aagtaattgg tggagatgat ctctcaactt 540 taactggaaa gaatgtcttg attgtggaag atataattga cactggcaaa acaatgcaga 600 ctttgctttc cttggtcagg foottaatc caaagatggt caaggtcgca agcttgctgg 660 tgaaaggac cccacgaagt gttggatata agccagactt tgttggattt gaaattccag 720 acaagtttgt tgtaggatat gcccttgact ataatgaata cttcagggat ttgaatcatg 780 tttgtgtcat tagtgaaact ggaaaagcaa aataacaaagc ctaagatgag agttcaagtt 840 gagtttggaa acatctggag tcctattgac atcgccagta aaattatcaa tgttctagtt 900 ctgtggccat ctgcttagta gagctttttg catgtatctt ctaagaattt tatctgtttt 960 gtactttaga aatgtcagtt gctgcattcc taaactgttt atttgcacta tgagcctata 1020 gactatcagt tccctttggg cggattgttg tttaacttgt aaatgaaaaa attctcttaa 1080 1140 attagtttt taattggtat tttaattttt atatatgcag gaagaatag aagtgattga 1200 atattgttaa ttataccacc gtgtgttaga aaagtaagaa gcagtcaatt ttcacatcaa 1260 agacagcatc taagaagttt tgttctgtcc tggaattatt ttagtagtgt ttcagtaatg 1320 ttgactgtat ttccaactt gttcaatta ttaccagtga atctttgtca gcagttccct 1380 tttaaatgca atcataaa ttcccaaaa tttaaaaaaaaaaaaaaaaaaaaaaaaa 1435 <210> 20 <211> 218 <212> PRT <213> Homo sapiens <400> 20 Met Ala Thr Arg Ser Pro Gly Val Val Ile Ser Asp Asp Glu Pro Gly 1 5 10 15 Tyr Asp Leu Asp Leu Phe Cys Ile Pro Asn His Tyr Ala Glu Asp Leu 20 25 30 Glu Arg Val Phe Ile Pro His Gly Leu Ile Met Asp Arg Thr Glu Arg 35 40 45 Leu Ala Arg Asp Val Met Lys Glu Met Gly Gly His His Ile Val Ala 50 55 60 Leu Cys Val Leu Lys Gly Gly Tyr Lys Phe Phe Ala Asp Leu Leu Asp 65 70 75 80 Tyr Ile Lys Ala Leu Asn Arg Asn Ser Asp Arg Ser Ile Pro Met Thr 85 90 95 Val Asp Phe Ile Arg Leu Lys Ser Tyr Cys Asn Gln Ser Thr Gly 100 105 110 Asp Ile Lys Val Ile Gly Gly Asp Asp Leu Ser Thr Leu Thr Gly Lys 115 120 125 Asn Val Leu Ile Val Glu Asp Ile Ile Asp Thr Gly Lys Thr Met Gln 130 135 140 Thr Leu Leu Ser Leu Val Arg Gln Tyr Asn Pro Lys Met Val Lys Val 145 150 155 160 Ala Ser Leu Leu Val Lys Arg Thr Pro Arg Ser Val Gly Tyr Lys Pro 165 170 175 Asp Phe Val Gly Phe Glu Ile Pro Asp Lys Phe Val Val Gly Tyr Ala 180 185 190 Leu Asp Tyr Asn Glu Tyr Phe Arg Asp Leu Asn His Val Cys Val Ile 195 200 205 Ser Glu Thr Gly Lys Ala Lys Tyr Lys Ala 210 215 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> HPRT1_forward primer <400> 21 gaggatttgg aaagggtgtt <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer HPRT1_reverse <400> 22 acagagggct acaatgtgat g 21 <210> 23 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> HPRT1 probe <400> 23 acgtcttgct cgagatgtga tgaagg 26 <210> 24 <211> 1771 <212> DNA <213> Homo sapiens <400> 24 ggcggccagg ccgggcgcgg agtgggcgcg cggggccgga ggaggggcca gcgaccgcgg 60 caccgcctgt gcccgcccc ccctccgcag ccgctactta agaggctcca gcgccggccc 120 cgccctagtg cgttacttac ctcgactctt agcttgtcgg ggacggtaac cgggacccgg 180 tgtctgctcc tgtcgccttc gcctcctaat ccctagccac tatgcgtgag tgcatctcca 240 tccacgtgg ccaggctggt gtccagattg gcaatgcctg ctgggagctc tactgcctgg 300 aacacggcat ccagcccgat ggccagatgc caagtgacaa gaccattggg ggaggagatg 360 actccttcaa caccttcttc agtgagacgg gcgctggcaa gcacgtgccc cgggctgtgt 420 ttgtagactt ggaacccaca gtcattgatg aagttcgcac tggcacctac cgccagctct 480 tccaccctga gcagctcatc acaggcaagg aagatgctgc caataactat gcccgagggc 540 actacaccat tggcaaggag atcattgacc ttgtgttgga ccgaattcgc aagctggctg 600 accagtgcac cggtcttcag ggcttcttgg ttttccacag ctttggtggg ggaactggtt 660 ctgggttcac ctccctgctc atggaacgtc tctcagttga ttatggcaag aagtccaagc 720 tggagttctc catttaccca gcaccccagg tttccacagc tgtagttgag ccctacaact 780 ccatcctcac cacccacacc accctggagc actctgattg tgccttcatg gtagacaatg 840 aggccatcta tgacatctgt cgtagaaacc tcgatatcga gcgcccaacc tacactaacc 900 ttaaccgcct tattagccag attgtgtcct ccatcactgc ttccctgaga tttgatggag 960 ccctgaatgt tgacctgaca gaattccaga ccaacctggt gccctacccc cgcatccact 1020 tccctctggc cacatatgcc cctgtcatct ctgctgagaa agcctacat gaacagcttt 1080 ctgtagcaga gatcaccaat gcttgctttg agccagccaa ccagatggtg aaatgtgacc 1140 ctcgccatgg taaatacatg gcttgctgcc tgttgtaccg tggtgacgtg gttcccaaag 1200 atgtcaatgc tgccattgcc accatcaaaa ccaagcgcag catccagttt gtggattggt 1260 gccccactgg cttcaaggtt ggcatcaact accagcctcc cactgtggtg cctggtggag 1320 acctggccaa ggtacagaga gctgtgtgca tgctgagcaa caccacagcc attgctgagg 1380 cctgggctcg ctggaccac aagtttgacc tgatgtatgc caagcgtgcc tttgttcact 1440 ggtacgtggg tgaggggatg gaggaaggcg agttttcaga ggcccgtgaa gatatggctg 1500 cccttgagaa ggattatgag gaggttggtg tggattctgt tgaaggagag ggtgaggaag 1560 aaggagagga atactaatta tccattcctt ttggccctgc agcatgtcat gctcccagaa 1620 tttcagcttc agcttaactg acagacgtta aagctttctg gttagattgt tttcacttgg 1680 tgatcatgtc ttttccatgt gtacctgtaa tatttttcca tcatatctca aagtaaagtc 1740 attacked in 1771 <210> 25 <211> 451 <212> PRT <213> Homo sapiens <400> 25 Met Arg Glu Cys Ile Ser Ile His Val Gly Gln Ala Gly Val Gln Ile 1 5 10 15 Gly Asn Ala Cys Trp Glu Leu Tyr Cys Leu Glu His Gly Ile Gln Pro 20 25 30 Asp Gly Gln Met Pro Ser Asp Lys Thr Ile Gly Gly Gly Asp Asp Ser 35 40 45 Phe Asn Thr Phe Phe Ser Glu Thr Gly Ala Gly Lys His Val Pro Arg 50 55 60 Ala Val Phe Val Asp Leu Glu Pro Thr Val Ile Asp Glu Val Arg Thr 65 70 75 80 Gly Thr Tyr Arg Gln Leu Phe His Pro Glu Gln Leu Ile Thr Gly Lys 85 90 95 Glu Asp Ala Ala Asn Asn Tyr Ala Arg Gly His Tyr Thr Ile Gly Lys 100 105 110 Glu Ile Ile Asp Leu Val Leu Asp Arg Ile Arg Lys Leu Ala Asp Gln 115 120 125 Cys Thr Gly Leu Gln Gly Phe Leu Val Phe His Ser Phe Gly Gly Gly 130 135 140 Thr Gly Ser Gly Phe Thr Ser Leu Leu Met Glu Arg Leu Ser Val Asp 145 150 155 160 Tyr Gly Lys Lys Ser Lys Leu Glu Phe Ser Ile Tyr Pro Ala Pro Gln 165 170 175 Val Ser Thr Ala Val Val Glu Pro Tyr Asn Ser Ile Leu Thr Thr His 180 185 190 Thr Thr Leu Glu His Ser Asp Cys Ala Phe Met Val Asp Asn Glu Ala 195 200 205 Ile Tyr Asp Ile Cys Arg Arg Asn Leu Asp Ile Glu Arg Pro Thr Tyr 210 215 220 Thr Asn Leu Asn Arg Leu Ile Ser Gln Ile Val Ser Ser Ile Thr Ala 225 230 235 240 Ser Leu Arg Phe Asp Gly Ala Leu Asn Val Asp Leu Thr Glu Phe Gln 245 250 255 Thr Asn Leu Val Pro Tyr Pro Arg Ile His Phe Pro Leu Ala Thr Tyr 260 265 270 Ala Pro Val Ile Ser Ala Glu Lys Ala Tyr His Glu Gln Leu Ser Val 275 280 285 Ala Glu Ile Thr Asn Ala Cys Phe Glu Pro Ala Asn Gln Met Val Lys 290 295 300 Cys Asp Pro Arg His Gly Lys Tyr Met Ala Cys Cys Leu Leu Tyr Arg 305 310 315 320 Gly Asp Val Val Pro Lys Asp Val Asn Ala Ala Ile Ala Thr Ile Lys 325 330 335 Thr Lys Arg Ser Ile Gln Phe Val Asp Trp Cys Pro Thr Gly Phe Lys 340 345 350 Val Gly Ile Asn Tyr Gln Pro Pro Thr Val Val Pro Gly Gly Asp Leu 355 360 365 Ala Lys Val Gln Arg Ala Val Cys Met Leu Ser Asn Thr Thr Ala Ile 370 375 380 Ala Glu Ala Trp Ala Arg Leu Asp His Lys Phe Asp Leu Met Tyr Ala 385 390 395 400 Lys Arg Ala Phe Val His Trp Tyr Val Gly Glu Gly Met Glu Glu Gly 405 410 415 Glu Phe Ser Glu Ala Arg Glu Asp Met Ala Ala Leu Glu Lys Asp Tyr 420 425 430 Glu Glu Val Gly Val Asp Ser Val Glu Gly Glu Gly Glu Glu Glu Gly 435 440 445 Glu Glu Tyr 450 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> K-ALPHA-1_forward primer <400> 26 tgactccttc aacaccttct tc 22 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> K-ALPHA-1_reverse primer <400> 27 tgccagtgcg aacttcat 18 <210> 28 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> K-ALPHA-1 probe <400> 28 ccgggctgtg tttgtagact tgga 24 <210> 29 <211> 5416 <212> DNA <213> Homo sapiens <400> 29 agtgggccgc catgttgtcg gagtgaaagg taagggggag cgagagcgcc agagagagaa 60 gatcgggggg ctgaaatcca tcttcatcct accgctccgc ccgtgttggt ggaatgagcg 120 ttgcatgtgt cttgaagaga aaagcagtgc tttggcagga ctctttcagc ccccacctga 180 aacatcaccc tcaagaacca gctaatccca acatgcctgt tgttttgaca tctggaacag 240 ggtcgcaagc gcagccacaa ccagctgcaa atcaggctct tgcagctggg actcactcca 300 gccctgtccc aggatctata ggagttgcag gccgttccca ggacgacgct atggtggact 360 acttctttca gaggcagcat ggtgagcagc ttgggggagg aggaagtgga ggaggcggct 420 ataataatag caaacatcga tggcctactg gggataacat tcatgcagaa catcaggtgc 480 gttccatgga tgaactgaat catgattttc aagcacttgc tctggaggga agagcgatgg 540 gagagcagct cttgccaggt aaaaagtttt gggaaacaga tgaatccagc aaagatggac 600 caaaaggaat attcctgggt gatcaatggc gagacagtgc ctggggaaca tcagatcatt 660 cagtttccca gccaatcatg gtgcagagaa gacctggtca gagtttccat gtgaacagtg 720 aggtcaattc tgtactgtcc ccacgatcgg agagtggggg actaggcgtt agcatggtgg 780 agtatgtgtt gagctcatcc ccgggcgatt cctgtctaag aaaaggagga tttggcccaa 840 gggatgcaga cagtgatgaa aacgacaaag gtgaaaagaa gaacaagggt acgtttgatg 900 gagataagct aggagatttg aaggaggagg gtgatgtgat ggacaagacc aatggtttac cagtgcagaa tgggattgat gcagacgtca aagattttag ccgtacccct ggtaattgcc agaactctgc taatgaagtg gatcttctgg gtccaaacca gatggttct gagggcttag cccagctgac cagcaccaat ggtgccaagc ctgtggagga tttctccaac atggagtccc aggtgtccc cttggacccc atggaacatg tgggcatgga gcctcttcag tttgattatt caggcacgca ggtacctgtg caggcagcag cagcaactgt gggacttttt cactacaatt 1320. ctcaacaaca gctgttccaa agacctaatg cgcttgctgt ccagcagttg acagctgctc agcagcagca gtatgcactg gcagctgctc atcagccgca catcggttta gctcccgctg cgtttgtccc caatccatac atcatcagcg ctgctcccc agggacggac ccctacacag ctggattggc tgcagcagcg acactaggcc cagctgtggt ccctcaccag tattatggag ttactccctg gggagtctac cctgccagtc ttttccagca gcaagctgcc gctgccgctg cagcaacta ttcagctaat cacagacca ccccacaggc tcagcaagga cagcagcagg ttctccgtgg aggagccagc caacgtcctt tgaccccaaa ccagaaccag cagggacagc 1680 aaacggatcc ccttgtggca gctgcagcag tgaattctgc ccttgcattt ggacaaggtc 1740 tggcagcagg catgccaggt tatccggtgt tggctcctgc tgcttactat gaccaaactg 1800 gtgcccttgt agtgaatgca ggcgcgagaa atggtcttgg agctcctgtt cgacttgtag 1860 ctcctgcccc agtcatcatt agttcctcag ctgcacaagc agctgttgca gcagccgcag 1920 cttcagcaaa tggagcagct ggtggtcttg ctggaacaac aaatggacca tttcgccctt 1980 taggaacaca gcagcctcag ccccagcccc agcagcagcc caataacaac ctggcatcca 2040 gttctttcta cggcaacaac tctctgaaca gcaattcaca gagcagctcc ctcttctccc 2100 agggctctgc ccagcctgcc aacacatcct tgggattcgg aagtagcagt tctctcggcg 2160 ccaccctggg atccgccctt ggagggtttg gaacagcagt tgcaaactcc aacactggca 2220 gtggctcccg ccgtgactcc ctgactggca gcagtgacct ttataagagg acatcgagca 2280 gcttgacccc cattggacac agtttttata acggccttag cttttcctcc tctcctggac 2340 ccgtgggcat gcctctccct agtcagggac caggacattc acagacacca cctccttccc 2400 tctcttcaca tggatcctct tcaagcttaa acctgggagg actcacgaat ggcagtggaa 2460 gatacatctc tgctgctcca ggcgctgaag ccaagtaccg cagtgcaagc agcgcctcca 2520 gcctcttcag cccgagcagc actcttttct cttcctctcg tttgcgatat ggaatgtctg 2580 atgtcatgcc ttctggcagg agcaggcttt tggaagattt tcgaaacaac cggtacccca 2640 atttacaact gcgggagatt gctggacata taatggaatt ttcccaagac cagcatgggt 2700 ccagattcat tcagctgaaa ctggagcgtg ccacaccagc tgagcgccag cttgtcttca 2760 atgaaatcct ccaggctgcc taccaactca tggtggatgt gtttggtaat tacgtcattc 2820 agaagttctt tgaatttggc agtcttgaac agaagctggc tttggcagaa cggattcgag 2880 gccacgtcct gtcattggca ctacagatgt atggctgccg tgttatccag aaagctcttg 2940 agtttattcc ttcagaccag caggtaatta atgagatggt tcgggaacta gatggccatg 3000 tcttgaagtg tgtgaaagat cagaatggca atcacgtggt tcagaaatgc attgaatgtg 3060 3120. tacagcccca gtctttgcaa tttatcatcg atgcgtttaa gggacaggta tttgccttat ccacacatcc ttatggctgc cgagtgattc agagaatcct ggagcactgt ctccctgacc 3180. agcactccc tattttagag gagcttcacc agcacacaga gcagcttgta caggatcaat atggaaatta tgtaatccaa catgtactgg agcacggtcg tcctgaggat aaaagcaaaa ttgtagcaga aatccgaggc aatgtacttg tattgagtca gcacaaattt gcaagcaatg ttgtggagaa gtgtgttact cacgcctcac gtacggagcg cgctgtgctc atcgatgagg 3420 tgtgcaccat gaacgacggt ccccacagtg ccttatacac catgatgaag gaccagtatg ccaactacgt ggtccagaag atgattgacg tggcggagcc aggccagcgg aagatcgtca 3540. tgcataagat ccggccccac atcgcaactc ttcgtaagta cacctatggc aagcacattc tggccaagct ggagaagtac tacatgaaga acggtgttga cttagggccc atctgtggcc cccctaatgg tatcatctga ggcagtgtca cccgctgttc cctcattccc gctgacctca 3720 ctggcccact ggcaaatcca accagcaacc agaaatgttc tagtagtag tctgagacgg 3780. gcaagtggtt gctccaggat tactccctcc tccaaaaag gatcaatc cacgagtgga 3840 aaagcctttg taatttaat ttttacac atacatgta ctatttttt taattgacta 3900 attgccctgc tgttttactg gtgtatagga tacttgtaca taggtacca atgtacatgg 3960 gaggccacat atttgttca ctgttgtatc tatattcac atgtggaaac ttcaggggtg 4020 gttggtttaa aaaaaaaaaaaaaaaaagctttttag 4080 ctcatttgcc tggccggcaa gttttgcaa tagcttcc ccacctcctc attttagtaa 4140 aaaaaaaaaaaaaaaaaacctgagaa gtttgaattg tgttaatg acccaaact 4200 ggcatttaac actgtttata aaaaatatat attatatatta attatatatta atgaaaag 4260 gttcagagt tgctaaagct tcagtttgtg acataagtt tatgaaattc taaaaaatgc 4320 cttttttgga gactatatta tgctgagaa ggctgttcgt gaggagga tgcgagcacc 4380 cagaacgtct ttgaggctg ggcggtgtg attgtttact gcctactgga tttttcta 4440 ttaacattga aaggtaaat ctgattattt agcatgagaa aaaaaatcc aactctgctt 4500 ttggtcttgc ttctaaat fathers tacttggtgt agactttgca fathers tttgtagtat tttcttgttt tgatgtctaa tctgtatcta taatgtaccc tagtagtcga acatactttt gattgtacaa ttgtacattt gtatacctgt aatgtaaatg tggagaagtt tgaatcaaca taacacgtt ttttggtaag aaaagagaat tagccagccc tgtgcattca gtgtatattc tcacctttta tggtcgtagc atatagtgtt gtatattgta aattgtaatt tcaaccagaa gtaaattttt ttcttttgaa ggaataaatg ttctttatac agcctagtta atgtttaaaa agaaaaaaat agcttggttt tatttgtcat ctagtctcaa gtatagcgag attctttcta aatgttattc aagattgagt tctcactagt gtttttttaa tcctaaaaaa gtaatgtttt gattttgtga cagtcaaaag gacgtgcaaa agtctagcct tgcccgagct ttccttacaa tcagagcccc tctcaccttg taaagtgtga atcgcccttc ccttttgtac family ctgtattttg cattttgtct acttgtaagt cattgtaaca cattgtcaat tttccttgtt tgaataga attgtaacac tacacggtgt acatttccag agccttgtgt atatttccaa tgaacttttt tgcaagcaca cttgtaacca tatgtgtata attaacaaac 5280 ctgtgtatgc ttatgcctgg gcaactattt tttgtaactc ttgtgtagat tgtctctaaa 5340 caatgtgtga tctttatttt gaaaaataca gaactttgga atctgaaaaa aaaaaaaaa 5400 aaaaaaaaaaaaaaa 5416 <210> 30 <211> 5410 <212> DNA <213> Homo sapiens <400> 30 agtgggccgc catgttgtcg gagtgaaagg taagggggag cgagagcgcc agagagagaa 60 gatcgggggg ctgaaatcca tcttcatcct accgctccgc ccgtgttggt ggaatgagcg 120 ttgcatgtgt cttgaagaga aaagcagtgc tttggcagga ctctttcagc ccccacctga 180 aacatcaccc tcaagaacca gctaatccca acatgcctgt tgttttgaca tctggaacag 240 ggtcgcaagc gcagccacaa ccagctgcaa atcaggctct tgcagctggg actcactcca 300 gccctgtccc aggatctata ggagttgcag gccgttccca ggacgacgct atggtggact 360 acttctttca gaggcagcat ggtgagcagc ttgggggagg aggaagtgga ggaggcggct 420 ataatatag siaacatcga tggcctactg gggaataacat tcatgcagaa catcaggtgc 480 gttccatgga tgactgaat catgatttc aagcacttgc tctggaggga agagcgatgg 540 gagagcagct cttgccaggt aaaagtttt gggaaacaga tgaatccagc aagatggac 600 caaaaggaat attcctgggt gatcaatggc gagacagtgc ctggggaaca tcagatcatt 660 cagtttccca gccaatcatg gtgcagagaa gacctggtca gagttttccat gtgaacagtg 720 aggtcaatc tgtactgtcc ccacgatcgg agagtgggg actaggcgtt agcatggtgg 780 agtatgtgtt gagctcatcc ccgggcgatt cctgtctaag aaaggagga tttggcccaa 840 gggatgcaga cagtgatgaa acgacaaag gtgaaaagaa gaacaagggt acgttttgatg 900 gagataagct aggatttg aaggaggagg gtgatgtgat ggacaagacc aatggtttac 960 cagtgcagaa tgggattgat gcagacgtca aagattttag ccgtacccct ggtaattgcc 1020 agaactctgc taatgaagtg gatctctctg gtccaacca gatggttct gaggggcttg 1080 cccagctgac cagcaccaat gtgccaagc ctgtggagga tttctccaac atggagtccc 1140 agagtgtccc cttggacccc atggaacatg tgggcatgga gcctcttcag tttgattatt 1200 caggcacgca ggtacctgtg gactcagcag cagcaactgt gggacttttt gactacaatt 1260 ctcaacaaca gctgttccaa agacctaatg cgcttgctgt ccagcagttg acagctgctc 1320 agcagcagca gtatgcactg gcagctgctc atcagccgca catcggttta gctcccgctg 1380 cgtttgtccc caatccatac atcatcagcg ctgctccccc agggacggac ccctacacag 1440 ctggattggc tgcagcagcg acactaggcc cagctgtggt ccctcaccag tattatggag 1500 ttactccctg gggagtctac cctgccagtc ttttccagca gcaagctgcc gctgccgctg 1560 cagcaactaa ttcagctaat caacagacca ccccacaggc tcagcaagga cagcagcagg 1620 ttctccgtgg aggagccagc caacgtcctt tgaccccaaa ccagaaccag cagggacagc 1680 aaacggatcc ccttgtggca gctgcagcag tgaattctgc ccttgcattt ggacaaggtc 1740 tggcagcagg catgccaggt tatccggtgt tggctcctgc tgcttactat gaccaaactg 1800 gtgcccttgt agtgaatgca ggcgcgagaa atggtcttgg agctcctgtt cgacttgtag 1860 ctcctgcccc agtcatcatt agttcctcag ctgcacaagc agctgttgca gcagccgcag 1920 cttcagcaaa tggagcagct ggtggtcttg ctggaacaac aaatggacca tttcgccctt 1980 taggaacaca gcagcctcag ccccagcccc agcagcagcc caataacaac ctggcatcca 2040 gttctttcta cggcaacaac tctctgaaca gcaattcaca gagcagctcc ctcttctccc 2100 agggctctgc ccagcctgcc aacacatcct tgggattcgg aagtagcagt tctctcggcg 2160 ccaccctggg atccgccctt ggagggtttg gaacagcagt tgcaaactcc aacactggca 2220 gtggctcccg ccgtgactcc ctgactggca gcagtgacct ttataagagg acatcgagca 2280 gcttgacccc cattggacac agtttttata acggccttag cttttcctcc tctcctggac 2340 ccgtgggcat gcctctccct agtcagggac caggacattc acagacacca cctccttccc 2400 tctcttcaca tggatcctct tcaagcttaa acctgggagg actcacgaat ggcagtggaa 2460 gatacatctc tgctgctcca ggcgctgaag ccaagtaccg cagtgcaagc agcgcctcca 2520 gcctcttcag cccgagcagc actcttttct cttcctctcg tttgcgatat ggaatgtctg 2580 atgtcatgcc ttctggcagg agcaggcttt tggaagattt tcgaaacaac cggtacccca 2640 atttacaact gcgggagatt gctggacata taatggaatt ttcccaagac cagcatgggt 2700 ccagattcat tcagctgaaa ctggagcgtg ccacaccagc tgagcgccag cttgtcttca 2760 atgaaatcct ccaggctgcc taccaactca tggtggatgt gtttggtaat tacgtcattc 2820 agaagttctt tgaatttggc agtcttgaac agaagctggc tttggcagaa cggattcgag 2880 gccacgtcct gtcattggca ctacagatgt atggctgccg tgttatccag aaagctcttg 2940 agtttattcc ttcagaccag cagaatgaga tggttcggga actagatggc catgtcttga 3000 agtgtgtgaa agatcagaat ggcaatcacg tggttcagaa atgcattgaa tgtgtacagc 3060 cccagtcttt gcaatttatc atcgatgcgt ttaagggaca ggtatttgcc ttatccacac 3120 atccttatgg ctgccgagtg attcagagaa tcctggagca ctgtctccct gaccagacac 3180 tccctatttt agaggagctt caccagcaca cagagcagct tgtacaggat caatatggaa 3240 attatgtaat ccaacatgta ctggagcacg gtcgtcctga ggataaaagc aaaattgtag 3300 cagaaatccg aggcaatgta cttgtattga gtcagcacaa atttgcaagc aatgttgtgg agaagtgtgt tactcacgcc tcacgtacgg agcgcgctgt gctcatcgat gaggtgtgca 3420. 3480. cggtccccac agtgccttat acaccatgat gaaggaccag tatgccaact acgtggtcca gagatgatt gacgtggcgg agccaggcca gcggagatc gtcatgcata 3540 agatccggcc ccacatcgca actcttcgta actctcact tggcaagcac attctggcca 3600. agctggagaa gtactacatg aagaacggtg ttgacttagg gcccatctgt ggccccccta atggtatcat ctgaggcagt gtcacccgct gttccctcat tcccgctgac ctcactggcc 3720. cactggcaaa tccaaccagc aaccagaat gttctagtgt aggtctgag acgggcaagt ggttgctcca ggattactcc ctcctccaaa aaaggaatca aatccacgag tggaaaagcc tttgtaatt tattttatt acacataaca tgtactattt tttttaattg actaattgcc ctgctgtttt actggtgtat aggatacttg tacataggta accaatgtac atgggaggcc acatattttg ttcactgttg tatctatatt tcacatgtgg aaactttcag ggtggttggt ttaaaaaaaaaaaagctt taaaaaaaaaaaaaaag gaaaggtttt ttagctcatt 4080 tgcctgccg gcaagttttg caatagctc ttcccacct cctcatttta gtaaaaaaca 4140 aaaaaaaaaaaaacctg agaagtttga attgtagtta atgacccca aactggcatt 4200 taacactgtt taaaaaaat attattattattattattattattaatga aaggttttca 4260 gagttgctaa agcttcagtt tgtgacatta agtttagaa attctaaaaa atgccttttt 4320 tggagactat attatgctga agaagctgt tcgtgaggag gagatgcgag cacccagaac 4380 gtcttttgag gctgggcggg tgtgattgtt tactgcctac tggatttt tctattaaca 4440 ttgaaaggta aaatctgatt atttagcatg agaaaaaaaa atccactct gctttggtc 4500 ttgcttctat aaatatatag tgtatacttg gtgtagactt tgcatatata caaatttgta 4560 gtattttct gttttgatgt ctaatctgta tctataatgt accctagtag tcgaacatac 4620 ttttgattgt acaattgtac atttgtatac ctgtaatgta aatgtggaga agttttgaatc 4680 aacataaaca cgttttttgg taagaaaga gattagcca gccctgtgca ttcagtgtat 4740 attctcacct tttatggtcg tagcatatag tgttgtatat tgtaaattgt aatttcaacc 4800 agaagtaaat ttttttcttt tgaaggaata aatgttcttt atacagccta gttaatgttt 4860 aaaaagaaaa aatagcttg gttttattg tcatctagtc tcaagtatag cgagattctt 4920 tctaaatgtt attcaagatt gagttctcac tagtgttttt ttaatcctaa aaaagtaatg 4980 ttttgattt gtgacagtca aaaggacgtg caaaagtcta gccttgcccg agctttcctt 5040 acaatcagag cccctctcac cttgtaaagt gtgaatcgcc cttcccttt gtacagaaga 5100 tgaactgtat tttgcatttt gtctacttgt aagtgaatgt aacatactgt caattttcct 5160 tgtttgaata tagaattgta acactacacg gtgtacattt ccagagcctt gtgtatattt 5220 ccaatgaact ttttgcaag cacacttgta accatatgtg tataattaac aaacctgtgt 5280 atgcttatgc ctgggcaact atttttgta actcttgtgt agattgtctc taaacaatgt 5340 gtgatcttta ttttgaaaaa tacagaactt tggaatctga aaaaaaaaa aaaaaaaaa 5400 aaaaaaaaaa 5410 <210> 31 <211> 1188 <212> PRT <213> Homo sapiens <400> 31 Met Ser Val Ala Cys Val Leu Lys Arg Lys Ala Val Leu Trp Gln Asp 1 5 10 15 Ser Phe Ser Pro His Leu Lys His His Pro Gln Glu Pro Ala Asn Pro 20 25 30 Asn Met Pro Val Val Leu Thr Ser Gly Thr Gly Ser Gln Ala Gln Pro 35 40 45 Gln Pro Ala Ala Asn Gln Ala Leu Ala Ala Gly Thr His Ser Ser Pro 50 55 60 Val Pro Gly Ser Ile Gly Val Ala Gly Arg Ser Gln Asp Asp Ala Met 65 70 75 80 Val Asp Tyr Phe Phe Gln Arg Gln His Gly Glu Gln Leu Gly Gly Gly 85 90 95 Gly Ser Gly Gly Gly Gly Tyr Asn Asn Ser Lys His Arg Trp Pro Thr 100 105 110 Gly Asp Asn Ile His Ala Glu His Gln Val Arg Ser Met Asp Glu Leu 115 120 125 Asn His Asp Phe Gln Ala Leu Ala Leu Glu Gly Arg Ala Met Gly Glu 130 135 140 Gln Leu Leu Pro Gly Lys Lys Phe Trp Glu Thr Asp Glu Ser Ser Lys 145 150 155 160 Asp Gly Pro Lys Gly Ile Phe Leu Gly Asp Gln Trp Arg Asp Ser Ala 165 170 175 Trp Gly Thr Ser Asp His Ser Val Ser Gln Pro Ile Met Val Gln Arg 180 185 190 Arg Pro Gly Gln Ser Phe His Val Asn Ser Glu Val Asn Ser Val Leu 195 200 205 Ser Pro Arg Ser Glu Ser Gly Gly Leu Gly Val Ser Met Val Glu Tyr 210 215 220 Val Leu Ser Ser Ser Pro Gly Asp Ser Cys Leu Arg Lys Gly Gly Phe 225 230 235 240 Gly Pro Arg Asp Ala Asp Ser Asp Glu Asn Asp Lys Gly Glu Lys Lys 245 250 255 Asn Lys Gly Thr Phe Asp Gly Asp Lys Leu Gly Asp Leu Lys Glu Glu 260 265 270 Gly Asp Val Met Asp Lys Thr Asn Gly Leu Pro Val Gln Asn Gly Ile 275 280 285 Asp Ala Asp Val Lys Asp Phe Ser Arg Thr Pro Gly Asn Cys Gln Asn 290 295 300 Ser Ala Asn Glu Val Asp Leu Leu Gly Pro Asn Gln Asn Gly Ser Glu 305 310 315 320 Gly Leu Ala Gln Leu Thr Ser Thr Asn Gly Ala Lys Pro Val Glu Asp 325 330 335 Phe Ser Asn Met Glu Ser Gln Ser Val Pro Leu Asp Pro Met Glu His 340 345 350 Val Gly Met Glu Pro Leu Gln Phe Asp Tyr Ser Gly Thr Gln Val Pro 355 360 365 Val Asp Ser Ala Ala Ala Thr Val Gly Leu Phe Asp Tyr Asn Ser Gln 370 375 380 Gln Gln Leu Phe Gln Arg Pro Asn Ala Leu Ala Val Gln Gln Leu Thr 385 390 395 400 Ala Ala Gln Gln Gln Gln Tyr Ala Leu Ala Ala Ala His Gln Pro His 405 410 415 Ile Gly Leu Ala Pro Ala Ala Phe Val Pro Asn Pro Tyr Ile Ile Ser 420 425 430 Ala Ala Pro Pro Gly Thr Asp Pro Tyr Thr Ala Gly Leu Ala Ala Ala 435 440 445 Ala Thr Leu Gly Pro Ala Val Val Pro His Gln Tyr Tyr Gly Val Thr 450 455 460 Pro Trp Gly Val Tyr Pro Ala Ser Leu Phe Gln Gln Gln Ala Ala Ala 465 470 475 480 Ala Ala Ala Ala Thr Asn Ser Ala Asn Gln Gln Thr Thr Pro Gln Ala 485 490 495 Gln Gln Gly Gln Gln Gln Val Leu Arg Gly Gly Ala Ser Gln Arg Pro 500 505 510 Leu Thr Pro Asn Gln Asn Gln Gln Gly Gln Gln Thr Asp Pro Leu Val 515 520 525 Ala Ala Ala Ala Val Asn Ser Ala Leu Ala Phe Gly Gln Gly Leu Ala 530 535 540 Path Gly Met Pro Gly Tyr Pro Val Leu Path Pro Path Path Tyr Tyr Asp 545 550 555 560 Gln Thr Gly Ala Leu Val Val Asn Ala Gly Ala Arg Asn Gly Leu Gly 565 570 575 Path Pro Val Arg Leu Val Path Pro Path Pro Val Ile Ile Ser Ser Ser 580 585 590 Ala Ala Gln Ala Ala Val Ala Ala Ala Ala Ser Ala Asn Gly Ala 595 600 605 Ala Gly Gly Leu Ala Gly Thr Thr Asn Gly Pro Phe Arg Pro Leu Gly 610 615 620 Thr Gln Gln Pro Gln Pro Gln Pro Gln Gln Gln Pro Asn Asn Asn Leu 625 630 635 640 Ala Ser Ser Ser Phe Tyr Gly Asn Asn Ser Leu Asn Ser Asn Ser Gln 645 650 655 Ser Ser Ser Leu Phe Ser Gln Gly Ser Ala Gln Pro Ala Asn Thr Ser 660 665 670 Leu Gly Phe Gly Be Be Be Be Leu Gly Ala Thr Leu Gly Be Ala 675 680 685 Leu Gly Gly Phe Gly Thr Ala Val Ala Asn Ser Asn Thr Gly Ser Gly 690 695 700 Ser Arg Arg Asp Ser Leu Thr Gly Ser Ser Asp Leu Tyr Lys Arg Thr 705 710 715 720 To Be To Be Leu Thr Pro Ile Gly His To Be Phe Tyr Asn Gly To Be Leu 725 730 735 Phe Ser Ser Ser Pro Gly Pro Val Gly Met Pro Leu Pro Ser Gln Gly 740 745 750 Pro Gly His Ser Gln Thr Pro Pro Pro Ser Leu Ser Ser His Gly Ser 755 760 765 Ser Ser Ser Leu Asn Leu Gly Gly Leu Thr Asn Gly Ser Gly Arg Tyr 770 775 780 Ile Ser Ala Ala Pro Gly Ala Glu Ala Lys Tyr Arg Ser Ala Ser Ser 785 790 795 800 Ala Ser Ser Leu Phe Ser Pro Ser Ser Thr Leu Phe Ser Ser Ser Arg 805 810 815 Leu Arg Tyr Gly Met Ser Asp Val Met Pro Ser Gly Arg Ser Arg Leu 820 825 830 Leu Glu Asp Phe Arg Asn Asn Arg Tyr Pro Asn Leu Gln Leu Arg Glu 835 840 845 Ile Ala Gly His Ile Met Glu Phe Ser Gln Asp Gln His Gly Ser Arg 850 855 860 Phe Ile Gln Leu Lys Leu Glu Arg Ala Thr Pro Ala Glu Arg Gln Leu 865 870 875 880 Val Phe Asn Glu Ile Leu Gln Ala Ala Tyr Gln Leu Met Val Asp Val 885 890 895 Phe Gly Asn Tyr Val Ile Gln Lys Phe Phe Glu Phe Gly Ser Leu Glu 900 905 910 Gln Lys Leu Ala Leu Ala Glu Arg Ile Arg Gly His Val Leu Ser Leu 915 920 925 Ala Leu Gln Met Tyr Gly Cys Arg Val Ile Gln Lys Ala Leu Glu Phe 930 935 940 Ile Pro Ser Asp Gln Gln Val Ile Asn Glu Met Val Arg Glu Leu Asp 945 950 955 960 Gly His Val Leu Lys Cys Val Lys Asp Gln Asn Gly Asn His Val Val 965 970 975 Gln Lys Cys Ile Glu Cys Val Gln Pro Gln Ser Leu Gln Phe Ile Ile 980 985 990 Asp Ala Phe Lys Gly Gln Val Phe Ala Leu Ser Thr His Pro Tyr Gly 995 1000 1005 Cys Arg Val Ile Gln Arg Ile Leu Glu His Cys Leu Pro Asp Gln 1010 1015 1020 Thr Leu Pro Ile Leu Glu Glu Leu His Gln His Thr Glu Gln Leu 1025 1030 1035 Val Gln Asp Gln Tyr Gly Asn Tyr Val Ile Gln His Val Leu Glu 1040 1045 1050 His Gly Arg Pro Glu Asp Lys Ser Lys Ile Val Ala Glu Ile Arg 1055 1060 1065 Gly Asn Val Leu Val Leu Ser Gln His Lys Phe Ala Ser Asn Val 1070 1075 1080 Val Glu Lys Cys Val Thr His Ala Ser Arg Thr Glu Arg Ala Val 1085 1090 1095 Leu Ile Asp Glu Val Cys Thr Met Asn Asp Gly Pro His Ser Ala 1100 1105 1110 Leu Tyr Thr Met Met Lys Asp Gln Tyr Ala Asn Tyr Val Val Gln 1115 1120 1125 Lys Met Ile Asp Val Ala Glu Pro Gly Gln Arg Lys Ile Val Met 1130 1135 1140 His Lys Ile Arg Pro His Ile Ala Thr Leu Arg Lys Tyr Thr Tyr 1145 1150 1155 Gly Lys His Ile Leu Ala Lys Leu Glu Lys Tyr Tyr Met Lys Asn 1160 1165 1170 Gly Val Asp Leu Gly Pro Ile Cys Gly Pro Pro Asn Gly Ile Ile 1175 1180 1185 <210> 32 <211> 1186 <212> PRT <213> Homo sapiens <400> 32 Met Ser Val Ala Cys Val Leu Lys Arg Lys Ala Val Leu Trp Gln Asp 1 5 10 15 Ser Phe Ser Pro His Leu Lys His His Pro Gln Glu Pro Ala Asn Pro 20 25 30 Asn Met Pro Val Val Leu Thr Ser Gly Thr Gly Ser Gln Ala Gln Pro 35 40 45 Gln Pro Ala Ala Asn Gln Ala Leu Ala Ala Gly Thr His Ser Ser Pro 50 55 60 Val Pro Gly Ser Ile Gly Val Ala Gly Arg Ser Gln Asp Asp Ala Met 65 70 75 80 Val Asp Tyr Phe Phe Gln Arg Gln His Gly Glu Gln Leu Gly Gly Gly 85 90 95 Gly Ser Gly Gly Gly Gly Tyr Asn Asn Ser Lys His Arg Trp Pro Thr 100 105 110 Gly Asp Asn Ile His Ala Glu His Gln Val Arg Ser Met Asp Glu Leu 115 120 125 Asn His Asp Phe Gln Ala Leu Ala Leu Glu Gly Arg Ala Met Gly Glu 130 135 140 Gln Leu Leu Pro Gly Lys Lys Phe Trp Glu Thr Asp Glu Ser Ser Lys 145 150 155 160 Asp Gly Pro Lys Gly Ile Phe Leu Gly Asp Gln Trp Arg Asp Ser Ala 165 170 175 Trp Gly Thr Ser Asp His Ser Val Ser Gln Pro Ile Met Val Gln Arg 180 185 190 Arg Pro Gly Gln Ser Phe His Val Asn Ser Glu Val Asn Ser Val Leu 195 200 205 Ser Pro Arg Ser Glu Ser Gly Gly Leu Gly Val Ser Met Val Glu Tyr 210 215 220 Val Leu Ser Ser Ser Pro Gly Asp Ser Cys Leu Arg Lys Gly Gly Phe 225 230 235 240 Gly Pro Arg Asp Ala Asp Ser Asp Glu Asn Asp Lys Gly Glu Lys Lys 245 250 255 Asn Lys Gly Thr Phe Asp Gly Asp Lys Leu Gly Asp Leu Lys Glu Glu 260 265 270 Gly Asp Val Met Asp Lys Thr Asn Gly Leu Pro Val Gln Asn Gly Ile 275 280 285 Asp Ala Asp Val Lys Asp Phe Ser Arg Thr Pro Gly Asn Cys Gln Asn 290 295 300 Ser Ala Asn Glu Val Asp Leu Leu Gly Pro Asn Gln Asn Gly Ser Glu 305 310 315 320 Gly Leu Ala Gln Leu Thr Ser Thr Asn Gly Ala Lys Pro Val Glu Asp 325 330 335 Phe Ser Asn Met Glu Ser Gln Ser Val Pro Leu Asp Pro Met Glu His 340 345 350 Val Gly Met Glu Pro Leu Gln Phe Asp Tyr Ser Gly Thr Gln Val Pro 355 360 365 Val Asp Ser Ala Ala Ala Thr Val Gly Leu Phe Asp Tyr Asn Ser Gln 370 375 380 Gln Gln Leu Phe Gln Arg Pro Asn Ala Leu Ala Val Gln Gln Leu Thr 385 390 395 400 Ala Ala Gln Gln Gln Gln Tyr Ala Leu Ala Ala Ala His Gln Pro His 405 410 415 Ile Gly Leu Ala Pro Ala Ala Phe Val Pro Asn Pro Tyr Ile Ile Ser 420 425 430 Ala Ala Pro Pro Gly Thr Asp Pro Tyr Thr Ala Gly Leu Ala Ala Ala 435 440 445 Ala Thr Leu Gly Pro Ala Val Val Pro His Gln Tyr Tyr Gly Val Thr 450 455 460 Pro Trp Gly Val Tyr Pro Ala Ser Leu Phe Gln Gln Gln Ala Ala Ala 465 470 475 480 Ala Ala Ala Ala Thr Asn Ser Ala Asn Gln Gln Thr Thr Pro Gln Ala 485 490 495 Gln Gln Gly Gln Gln Gln Val Leu Arg Gly Gly Ala Ser Gln Arg Pro 500 505 510 Leu Thr Pro Asn Gln Asn Gln Gln Gly Gln Gln Thr Asp Pro Leu Val 515 520 525 Ala Ala Ala Ala Val Asn Ser Ala Leu Ala Phe Gly Gln Gly Leu Ala 530 535 540 Ala Gly Met Pro Gly Tyr Pro Val Leu Ala Pro Ala Ala Tyr Tyr Asp 545 550 555 560 Gln Thr Gly Ala Leu Val Val Asn Ala Gly Ala Arg Asn Gly Leu Gly 565 570 575 Ala Pro Val Arg Leu Val Ala Pro Ala Pro Val Ile Ile Ser Ser Ser 580 585 590 Ala Ala Gln Ala Ala Val Ala Ala Ala Ala Ala Ser Ala Asn Gly Ala 595 600 605 Ala Gly Gly Leu Ala Gly Thr Thr Asn Gly Pro Phe Arg Pro Leu Gly 610 615 620 Thr Gln Gln Pro Gln Pro Gln Pro Gln Gln Gln Pro Asn Asn Asn Leu 625 630 635 640 Ala Ser Ser Ser Phe Tyr Gly Asn Asn Ser Leu Asn Ser Asn Ser Gln 645 650 655 Ser Ser Ser Leu Phe Ser Gln Gly Ser Ala Gln Pro Ala Asn Thr Ser 660 665 670 Leu Gly Phe Gly Be Be Be Be Leu Gly Ala Thr Leu Gly Be Ala 675 680 685 Leu Gly Gly Phe Gly Thr Ala Val Ala Asn Ser Asn Thr Gly Ser Gly 690 695 700 Ser Arg Arg Asp Ser Leu Thr Gly Ser Ser Asp Leu Tyr Lys Arg Thr 705 710 715 720 To Be To Be Leu Thr Pro Ile Gly His To Be Phe Tyr Asn Gly To Be Leu 725 730 735 Phe Ser Ser Ser Pro Gly Pro Val Gly Met Pro Leu Pro Ser Gln Gly 740 745 750 Pro Gly His Ser Gln Thr Pro Pro Pro Ser Leu Ser Ser His Gly Ser 755 760 765 To Be To Be Leu Asn Leu Gly Gly Leu Thr Asn Gly Ser Gly Arg Tyr 770 775 780 Ile Ser Ala Ala Pro Gly Ala Glu Ala Lys Tyr Arg Ser Ala Ser Ser 785 790 795 800 Ala Ser Ser Leu Phe Ser Pro Ser Ser Thr Leu Phe Ser Ser Ser Arg 805 810 815 Leu Arg Tyr Gly Met Ser Asp Val Met Pro Ser Gly Arg Ser Arg Leu 820 825 830 Leu Glu Asp Phe Arg Asn Asn Arg Tyr Pro Asn Leu Gln Leu Arg Glu 835 840 845 Ile Ala Gly His Ile Met Glu Phe Ser Gln Asp Gln His Gly Ser Arg 850 855 860 Phe Ile Gln Leu Lys Leu Glu Arg Ala Thr Pro Ala Glu Arg Gln Leu 865 870 875 880 Val Phe Asn Glu Ile Leu Gln Ala Ala Tyr Gln Leu Met Val Asp Val 885 890 895 Phe Gly Asn Tyr Val Ile Gln Lys Phe Phe Glu Phe Gly Ser Leu Glu 900 905 910 Gln Lys Leu Ala Leu Ala Glu Arg Ile Arg Gly His Val Leu Ser Leu 915 920 925 Ala Leu Gln Met Tyr Gly Cys Arg Val Ile Gln Lys Ala Leu Glu Phe 930 935 940 Ile Pro Ser Asp Gln Gln Asn Glu Met Val Arg Glu Leu Asp Gly His 945 950 955 960 Val Leu Lys Cys Val Lys Asp Gln Asn Gly Asn His Val Val Gln Lys 965 970 975 Cys Ile Glu Cys Val Gln Pro Gln Ser Leu Gln Phe Ile Ile Asp Ala 980 985 990 Phe Lys Gly Gln Val Phe Ala Leu Ser Thr His Pro Tyr Gly Cys Arg 995 1000 1005 Val Ile Gln Arg Ile Leu Glu His Cys Leu Pro Asp Gln Thr Leu 1010 1015 1020 Pro Ile Leu Glu Glu Leu His Gln His Thr Glu Gln Leu Val Gln 1025 1030 1035 Asp Gln Tyr Gly Asn Tyr Val Ile Gln His Val Leu Glu His Gly 1040 1045 1050 Arg Pro Glu Asp Lys Ser Lys Ile Val Ala Glu Ile Arg Gly Asn 1055 1060 1065 Val Leu Val Leu Ser Gln His Lys Phe Ala Ser Asn Val Val Glu 1070 1075 1080 Lys Cys Val Thr His Ala Ser Arg Thr Glu Arg Ala Val Leu Ile 1085 1090 1095 Asp Glu Val Cys Thr Met Asn Asp Gly Pro His Ser Ala Leu Tyr 1100 1105 1110 Thr Met Met Lys Asp Gln Tyr Ala Asn Tyr Val Val Gln Lys Met 1115 1120 1125 Ile Asp Val Ala Glu Pro Gly Gln Arg Lys Ile Val Met His Lys 1130 1135 1140 Ile Arg Pro His Ile Ala Thr Leu Arg Lys Tyr Thr Tyr Gly Lys 1145 1150 1155 His Ile Leu Ala Lys Leu Glu Lys Tyr Tyr Met Lys Asn Gly Val 1160 1165 1170 Asp Leu Gly Pro Ile Cys Gly Pro Pro Asn Gly Ile Ile 1175 1180 1185 <210> 33 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PUM1_forward primer <400> 33 gccagcttgt cttcaatgaa at 22 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PUM1_reverse primer <400> 34 caaagccagc ttctgttcaa g 21 <210> 35 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PUM1 probe <400> 35 atccaccatg agttggtagg cagc 24 <210> 36 <211> 1921 <212> DNA <213> Homo sapiens <400> 36 ggcggaagtg acattatcaa cgcgcgccag gggttcagtg aggtcgggca ggttcgctgt 60 ggcgggcgcc tgggccgccg gctgtttaac ttcgcttccg ctggcccata gtgatctttg 120 cagtgaccca gcatcactgt ttcttggcgt gtgaagataa cccaaggaat tgaggaagtt 180 gctgagaaga gtgtgctgga gatgctctag gaaaaaattg aatagtgaga cgagttccag 240 cgcaagggtt tctggttgc caagaagaaa gtgaacatca tggatcagaa caacagcctg 300 ccaccttacg ctcagggctt ggcctcccct cagggtgcca tgactcccgg aatccctatc 360 tttagtccaa tgatgccta tggcactgga ctgaccccac agcctattca gaacaccaat 420 agtctgtcta ttttggaaga gcaacaaagg cagcagcagc aacaacaaca gcagcagcag 480 square square square square square 540 cagcagcagc agcagcagca acaggcagtg gcagctgcag ccgttcagca gtcaacgtcc 600 cagcaggcaa cagggaac ctcaggccag gcaccacagc tcttccactc agagactctc 660 acaactgcac ccttgccggg caccactcca ctgtatccct cccccatgac tcccatgacc 720 cccatcactc ctgccacgcc agcttcggag agttctggga ttgtaccgca gctccaaaat 780 attgtatcca cagtgaatct tggttgtaaa cttgacctaa agaccattgc acttcgtgcc 840 cgaaacgccg aatataatcc caagcggttt gctgcggtaa tcatgaggat aagagagcca 900 cgaaccacgg cactgatttt cagttctggg aaaatggtgt gcacaggagc caagagtgaa 960 gaacagtcca gactggcagc aagaaaatat gctagagttg tacagaagtt gggtttcca 1020 gctaagttct tggacttcaa gattcagaat atggtgggga gctgtgatgt gaagtttcct 1080 ataaggttag aaggccttgt gctcacccac caacaattta gtagttatga gccagagtta 1140 tttcctggtt taatctacag aatgatcaaa cccagaattg ttctccttat ttttgtttct 1200 ggaaaagttg tattaacagg tgctaaagtc agagcaagaa tttatgaagc atttgaaaac 1260 atctacccta ttctaaaggg attcaggaag acgacgtaat ggctctcatg tacccttgcc 1320 tcccccacccc ccttcttttt ttttttttaa acaaatcagt ttgttttggt acctttaaat 1380 ggtggtgttg tgagaagatg gatgttgagt tgcagggtgt ggcaccaggt gatgcccttc 1440 tgtaagtgcc caccgcggga tgccgggaag gggcattatt tgtgcactga gaacaccgcg 1500 cagcgtgact gtgagttgct cataccgtgc tgctatctgg gcagcgctgc ccatttattt 1560 atatgtagat tttaaacact gctgttgaca agttggtttg agggagaaaa ctttaagtgt 1620 taaagccacc tctataattg attggacttt ttaattttaa tgtttttccc catgaaccac 1680 agtttttata tttctaccag aaaagtaaaa atctttttta aaagtgttgt ttttctaatt 1740 tataactcct aggggttatt tctgtgccag acacattcca cctctccagt attgcaggac 1800 agaatatatg tgttaatgaa aatgaatggc tgtacatatt ttttctttc ttcagagtac 1860 tctgtacaat aaatgcagtt tataaaagtg ttagattgtt gttaaaaaaa aaaaaaaaa 1920 in 1921 <210> 37 <211> 339 <212> PRT <213> Homo sapiens <400> 37 Met Asp Gln Asn Asn Ser Leu Pro Pro Tyr Ala Gln Gly Leu Ala Ser 1 5 10 15 Pro Gln Gly Ala Met Thr Pro Gly Ile Pro Ile Phe Ser Pro Met Met 20 25 30 Pro Tyr Gly Thr Gly Leu Thr Pro Gln Pro Ile Gln Asn Thr Asn Ser 35 40 45 Leu Ser Ile Leu Glu Glu Gln Gln Arg Gln Gln Gln Gln Gln Gln Gln 50 55 60 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 65 70 75 80 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Ala 85 90 95 Val Ala Ala Ala Ala Val Gln Gln Ser Thr Ser Gln Gln Ala Thr Gln 100 105 110 Gly Thr Ser Gly Gln Ala Pro Gln Leu Phe His Ser Gln Thr Leu Thr 115 120 125 Thr Ala Pro Leu Pro Gly Thr Thr Pro Leu Tyr Pro Ser Pro Met Thr 130 135 140 Pro Met Thr Pro Ile Thr Pro Ala Thr Pro Ala Ser Glu Ser Ser Gly 145 150 155 160 There Will Be Pro Gln Leo Gln Donkey There Will Be Thr There Will Be Leo Gly Cys 165 170 175 Lys from Asp to Lys Thr Ile to Arg to Arg Asn to Glu Tyr 180 185 190 Asn Pro Lys Arg Phe Ala Ala Val Ile Met Arg Ile Arg Glu Pro Arg 195 200 205 Thr Thr Ala Leu Ile Phe Ser Ser Gly Lys Met Val Cys Thr Gly Ala 210 215 220 Lys Ser Glu Glu Gln Ser Arg Leu Ala Ala Arg Lys Tyr Ala Arg Val 225 230 235 240 Val Gln Lys Leu Gly Phe Pro Ala Lys Phe Leu Asp Phe Lys Ile Gln 245 250 255 Asn Met Val Gly Ser Cys Asp Val Lys Phe Pro Ile Arg Leu Glu Gly 260 265 270 Leu Val Leu Thr His Gln Gln Phe Ser Ser Glu Pro Glu Leu Phe 275 280 285 Pro Gly Leopard Tyr Arg Met Leopard Lys Pro Arg Leopard Val Leo Leopard 290,295,300 Phe Val Ser Gly Lys Val Val Leu Thr Gly Ala Lys Val Arg Ala Glu 305 310 315 320 Ile Tyr Glu Ala Phe Glu Asn Ile Tyr Pro Ile Leu Lys Gly Phe Arg 325 330 335 Lys Thr Thr <210> 38 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> TBP_forward primer <400> 38 gccaagaaga aagtgaacat cat 23 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TBP_reverse primer <400> 39 atagggattc cgggagtcat 20 <210> 40 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TBP probe <400> 40 tcagaacaac agcctgccac ctta 24 <210> 41 <211> 1852 <212> DNA <213> Homo sapiens <400> 41 accgccgaga ccgcgtccgc cccgcgagca cagagcctcg cctttgccga tccgccgccc 60 gtccacaccc gccgccagct caccatggat gatgatatcg ccgcgctcgt cgtcgacaac 120 ggctccggca tgtgcaaggc cggcttcgcg ggcgacgatg ccccccgggc cgtcttcccc 180 tccatcgtgg ggcgccccag gcaccagggc gtgatggtgg gcatgggtca gaaggattcc 240 tatgtgggcg acgaggccca gagcaagaga ggcatcctca ccctgaagta ccccatcgag 300 cacggcatcg tcaccaactg ggacgacatg gagaaaatct ggcaccacac cttctacaat 360 gagctgcgtg tggctcccga ggagcacccc gtgctgctga ccgaggcccc cctgaacccc 420 aaggccaacc gcgagaagat gacccagatc atgtttgaga ccttcaacac cccagccatg 480 tacgttgcta tccaggctgt gctatccctg tacgcctctg gccgtaccac tggcatcgtg 540 atggactccg gtgacggggt cacccacact gtgcccatct acgaggggta tgccctcccc 600 catgccatcc tgcgtctgga cctggctggc cgggacctga ctgactacct catgaagatc 660 ctcaccgagc gcggctacag cttcaccacc acggccgagc gggaaatcgt gcgtgacatt 720 aaggagaagc tgtgctacgt cgccctggac ttcgagcaag agatggccac ggctgcttcc 780 agctcctccc tggagaagag ctacgagctg cctgacggcc aggtcatcac cattggcaat 840 gagcggttcc gctgccctga ggcactcttc cagccttcct tcctgggcat ggagtcctgt 900 ggcatccacg aaactacctt caactccatc atgaagtgtg acgtggacat ccgcaaagac ctgtacgcca acacagtgct gtctggcggc accaccatgt accctggcat tgccgacagg 1020 atgcagaagg agatcactgc cctggcaccc agcacaatga agatcaagc cattgctcct 1080. cctgagcgca agtactccgt gtggatcggc ggctccatcc tggcctcgct gtccaccttc 1140 cagcagatgt ggatcagcaa gcaggagtat gacgagtccg gccctccat cgtccaccgc aaatgcttct aggcggacta tgacttagtt gcgttacacc ctttcttgac aaaacctaac ttgcgcagaa aacaagatga gattggcatg gctttatttg ttttttttgt tttgttttgg tttttttttt ttttttggct tgactcagga tttaaaaact ggacggtga aggtgacagc agtcggttgg agcgagcatc ccccaaagtt cacaatgtgg ccgaggactt tgattgcaca ttgttgtttt tttaatagtc attccaaata tgagatgcgt tgttacagga agtcccttgc catcctaaaa gccaccccac ttctctctaa ggagaatggc ccagtcctct cccaagtcca 1560 cacaggggag gtgatagcat tgctttcgtg taaattatgt aatgcaaaat ttttttaatc 1620 ttcgccttaa tactttttta ttttgtttta ttttgaatga tgagccttcg tgccccccct 1680 tcccccttt ttgtccccca acttgagatg tatgaaggct tttggtctcc ctgggagtgg 1740 gtggaggcag ccagggctta cctgtacact gacttgagac cagttgaata aaagtgcaca 1800 cttaaaaat gaaaaaaaaa aaaaaaaaa aaaaaaaaaa aaaaaaaaa aa 1852 <210> 42 <211> 375 <212> PRT <213> Homo sapiens <400> 42 Met Asp Asp Asp Ile Ala Ala Leu Val Val Asp Asn Gly Sister Gly Met 1 5 10 15 Cys Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg Ala Val Phe Pro 20 25 30 Ser Ile Val Gly Arg Pro Arg His Gln Gly Val Met Val Gly Met Gly 35 40 45 Gln Lys Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys Arg Gly Ile 50 55 60 Leu Thr Leu Lys Tyr Pro Ile Glu His Gly Ile Val Thr Asn Trp Asp 65 70 75 80 Asp Met Glu Lys Ile Trp His His Thr Phe Tyr Asn Glu Leu Arg Val 85 90 95 Ala Pro Glu Glu His Pro Val Leu Leu Thr Glu Ala Pro Leu Asn Pro 100 105 110 Lys Ala Asn Arg Glu Lys Met Thr Gln Ile Met Phe Glu Thr Phe Asn 115 120 125 Thr Pro Ala Met Tyr Val Ala Ile Gln Ala Val Leu Ser Leu Tyr Ala 130 135 140 Ser Gly Arg Thr Thr Gly Ile Val Met Asp Ser Gly Asp Gly Val Thr 145 150 155 160 His Thr Val Pro Ile Tyr Glu Gly Tyr Ala Leu Pro His Ala Ile Leu 165 170 175 Arg Leu Asp Leu Ala Gly Arg Asp Leu Thr Asp Tyr Leu Met Lys Ile 180 185 190 Leu Thr Glu Arg Gly Tyr Ser Phe Thr Thr Thr Ala Glu Arg Glu Ile 195 200 205 Val Arg Asp Ile Lys Glu Lys Leu Cys Tyr Val Ala Leu Asp Phe Glu 210 215 220 Gln Glu Met Ala Thr Ala Ala Ser Ser Ser Ser Leu Glu Lys Ser Tyr 225 230 235 240 Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg Phe Arg 245 250 255 Cys Pro Glu Ala Leu Phe Gln Pro Ser Phe Leu Gly Met Glu Ser Cys 260 265 270 Gly Ile His Glu Thr Thr Phe Asn Ser Ile Met Lys Cys Asp Val Asp 275 280 285 Ile Arg Lys Asp Leu Tyr Ala Asn Thr Val Leu Ser Gly Gly Thr Thr 290 295 300 Met Tyr Pro Gly Ile Ala Asp Arg Met Gln Lys Glu Ile Thr Ala Leu 305 310 315 320 Ala Pro Ser Thr Met Lys Ile Lys Ile Ile Ala Pro Pro Glu Arg Lys 325 330 335 Tyr Ser Val Trp Ile Gly Gly Ser Ile Leu Ala Ser Leu Ser Thr Phe 340 345 350 Gln Gln Met Trp Ile Ser Lys Gln Glu Tyr Asp Glu Ser Gly Pro Ser 355 360 365 Ile Val His Arg Lys Cys Phe 370 375 <210> 43 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> ACTB_forward primer <400> 43 ccaaccgcga gaagatga 18 <210> 44 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ACTB_reverse primer <400> 44 ccagaggcgt acagggatag 20 <210> 45 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ACTB probe <400> 45 ccatgtacgt tgctatccag gct 23 <210> 46 <211> 1229 <212> DNA <213> Homo sapiens <400> 46 gtctgacggg cgatggcgca gccaatagac aggagcgcta tccgcggttt ctgattggct 60 actttgttcg cattataaaa ggcacgcgcg ggcgcgaggc ccttctctcg ccaggcgtcc 120 tcgtggaagt gacatcgtct ttaaaccctg cgtggcaatc cctgacgcac cgccgtgatg 180 240. cccagggaag acagggcgac ctggaagtcc aactacttcc ttaagatcat ccaactattg gatgattatc cgaaatgttt cattgtggga gcagacaatg tgggctccaa gcagatgcag cagatccgca tgtcccttcg cgggaaggct gtggtgctga tgggcaagaa caccatgatg 420. cgcaaggcca tccgagggca cctggaaaac aacccagctc tggagaaact gctgcctcat atccggggga atgtgggctt tgtgttcacc aaggaggacc tcactgagat cagggacatg 480 ttgctggcca ataaggtgcc agctgctgcc cgtgctggtg ccattgcccc atgtgaagtc 540 actgtgccag cccagacac tggtctcggg cccgagaga cctccttttt ccaggcttta ggtatcacca ctaaaatctc caggggcacc attgaaatcc tgagtgatgt gcagctgatc aagactggag acaaagtggg agccagcgaa gccacgctgc tgaacatgct caacatctcc cccttctcct ttgggctggt catccagcag gtgttcgaca atggcagcat ctacaaccct 780 gaagtgcttg atatcacaga ggaaactctg cattctcgct tcctggaggg tgtccgcaat gttgccagtg tctgtctgca gattggctac ccaactgttg catcagtacc ccattctatc <h2 style=";text-align:left;direction:ltr">atcaacgggt acaaacgagt cctggccttg tctgtggaga cggattacac cttccccactt 960<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gctgaaaagg tcaaggcctt cttggctgat ccatctgcct ttgtggctgc tgcccctgtg 1020<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gctgctgcca ccacagctgc tcctgctgct gctgcagccc cagctaaggt tgaagccaag 1080<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gaagagtcgg aggagtcgga cgaggatatg ggatttggtc tctttgacta atcaccaaaa 1140<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agcaaccaac ttagccagtt ttatttgcaa aacaaggaaa taaaggctta cttctttaaa 1200<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aagtaaaaaaaaaaaaaaaaaaaaaaaaaa 1229<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 47<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 317<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> PRT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Homo sapiens<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 47<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Met Pro Arg Glu Asp Arg Ala Thr Trp Lys Ser Asn Tyr Phe Leu Lys<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1 5 10 15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ile Ile Gln Leu Leu Asp Asp Tyr Pro Lys Cys Phe Ile Val Gly Ala<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 20 25 30<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Asp Asn Val Gly Ser Lys Gln Met Gln Gln Ile Arg Met Ser Leu Arg<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 35 40 45<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Gly Lys Ala Val Val Leu Met Gly Lys Asn Thr Met Met Arg Lys Ala<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 50 55 60<h2 style=";text-align:left;direction:ltr"> Ile Arg Gly His Leu Glu Asn Asn Pro Ala Leu Glu Lys Leu Leu Pro 65 70 75 80 His Ile Arg Gly Asn Val Gly Phe Val Phe Thr Lys Glu Asp Leu Thr 85 90 95 Glu Ile Arg Asp Met Leu Leu Ala Asn Lys Val Pro Ala Ala Ala Arg 100 105 110 Ala Gly Ala Ile Ala Pro Cys Glu Val Thr Val Pro Ala Gln Asn Thr 115 120 125 Gly Leu Gly Pro Glu Lys Thr Ser Phe Phe Gln Ala Leu Gly Ile Thr 130 135 140 Thr Lys Ile Ser Arg Gly Thr Ile Glu Ile Leu Ser Asp Val Gln Leu 145 150 155 160 Ile Lys Thr Gly Asp Lys Val Gly Ala Ser Glu Ala Thr Leu Leu Asn 165 170 175 Met Leu Asn Ile Ser Pro Phe Ser Phe Gly Leu Val Ile Gln Gln Val 180 185 190 Phe Asp Asn Gly Ser Ile Tyr Asn Pro Glu Val Leu Asp Ile Thr Glu 195 200 205 Glu Thr Leu His Ser Arg Phe Leu Glu Gly Val Arg Asn Val Ala Ser 210 215 220 Val Cys Leu Gln Ile Gly Tyr Pro Thr Val Ala Ser Val Pro His Ser 225 230 235 240 Island Island Asn Gly Tyr Lys Arg Val Leu Ala Leu Ser Val Glu Thr Asp 245 250 255 Tyr Thr Phe Pro Leu Ala Glu Lys Val Lys Ala Phe Leu Ala Asp Pro 260 265 270 Ser Ala Phe Val Ala Ala Ala Pro Val Ala Ala Ala Thr Thr Ala Ala 275 280 285 Pro Ala Ala Ala Ala Ala Pro Ala Lys Val Glu Ala Lys Glu Glu Ser 290 295 300 Glu Glu Ser Asp Glu Asp Met Gly Phe Gly Leu Phe Asp 305 310 315 <210> 48 <211> 317 <212> PRT <213> Homo sapiens <400> 48 Met Pro Arg Glu Asp Arg Ala Thr Trp Lys Ser Asn Tyr Phe Leu Lys 1 5 10 15 Island Island Gln Leu Leu Asp Asp Tyr Pro Lys Cys Phe Island Val Gly Ala 20 25 30 Asp Asn Val Gly Ser Lys Gln Met Gln Gln Ile Arg Met Ser Leu Arg 35 40 45 Gly Lys Ala Val Val Leu Met Gly Lys Asn Thr Met Met Arg Lys Ala 50 55 60 Ile Arg Gly His Leu Glu Asn Asn Pro Ala Leu Glu Lys Leu Leu Pro 65 70 75 80 His Ile Arg Gly Asn Val Gly Phe Val Phe Thr Lys Glu Asp Leu Thr 85 90 95 Glu Ile Arg Asp Met Leu Leu Ala Asn Lys Val Pro Ala Ala Ala Arg 100 105 110 Ala Gly Ala Ile Ala Pro Cys Glu Val Thr Val Pro Ala Gln Asn Thr 115 120 125 Gly Leu Gly Pro Glu Lys Thr Ser Phe Phe Gln Ala Leu Gly Ile Thr 130 135 140 Thr Lys Ile Ser Arg Gly Thr Ile Glu Ile Leu Ser Asp Val Gln Leu 145 150 155 160 Ile Lys Thr Gly Asp Lys Val Gly Ala Ser Glu Ala Thr Leu Leu Asn 165 170 175 Met Leu Asn Ile Ser Pro Phe Ser Phe Gly Leu Val Ile Gln Gln Val 180 185 190 Phe Asp Asn Gly Ser Ile Tyr Asn Pro Glu Val Leu Asp Ile Thr Glu 195 200 205 Glu Thr Leu His Ser Arg Phe Leu Glu Gly Val Arg Asn Val Ala Ser 210 215 220 Val Cys Leu Gln Ile Gly Tyr Pro Thr Val Ala Ser Val Pro His Ser 225 230 235 240 Ile Ile Asn Gly Tyr Lys Arg Val Leu Ala Leu Ser Val Glu Thr Asp 245 250 255 Tyr Thr Phe Pro Leu Ala Glu Lys Val Lys Ala Phe Leu Ala Asp Pro 260 265 270 Ser Ala Phe Val Ala Ala Ala Pro Val Ala Ala Ala Thr Thr Ala Ala 275 280 285 Pro Ala Ala Ala Ala Ala Pro Ala Lys Val Glu Ala Lys Glu Glu Ser 290 295 300 Glu Glu Ser Asp Glu Asp Met Gly Phe Gly Leu Phe Asp 305 310 315 <210> 49 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> RPLP0_forward primer <400> 49 taaaccctgc gtggcaat 18 <210> 50 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> RPLP0_reverse primary <400> 50 acatttcgga taatcatcca atagttg 27 <210> 51 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> RPLP0 probe <400> 51 aagtagttgg acttccaggt cgcc 24 <210> 52 <211> 2458 <212> DNA <213> Homo sapiens <400> 52 cagaagaagg cagcgcccaa ggcgcatgcg cagcggtcac tcccgctgta tattaaggcg 60 ccggcgatcg cggcctgagg ctgctcccgg acaagggcaa cgagcgtttc gtttggactt 120 ctcgacttga gtgcccgcct cttcgccgc cgcctctgca gtcctcagcg cagttatgcc 180 cagttcttcc cgctgtgggg acacgaccac ggaggaatcc ttgcttcagg gactcgggac 240 cctgctggac cccttcctcg ggtttagggg atgtggggac caggagaaag tcaggatccc 300 taagagtctt ccctgcctgg atggatgagt ggcttcttct ccacctagat tctttccaca 360 ggagccagca tacttcctga acatggagag tgttgttcgc cgctgcccat tcttatcccg 420 agtcccccag gcctttctgc agaaagcagg caaatctctg ttgttctatg cccaaaactg 480 ccccaagatg atggaagttg gggccaagcc agcccctcgg gcattgtcca ctgcagcagt 540 acactaccaa cagatcaaag aaacccctcc ggccagtgag aaagacaaaa ctgctaaggc 600 caaggtccaa cagactcctg atggatccca gcagagtcca gatggcacac agcttccgtc 660 tggacacccc ttgcctgcca caagccaggg cactgcaagc aaatgccctt tcctggcagc 720 acagatgaat cagagaggca gcagtgtctt ctgcaaagcc agtcttgagc ttcaggagga 780 tgtgcaggaa atgaatgccg tgaggaaaga ggttgctgaa acctcagcag gccccagtgt 840 ggttagtgtg aaaaccgatg gaggggatcc cagtggactg ctgaagaact tccaggacat 900 catgcaaaag caaagaccag aaagagtgtc tcatcttctt caagataact tgccaaaatc 960 tgtttccact tttcagtatg atcgtttctt tgagaaaaaa attgatgaga aaaagaatga 1020 ccacacctat cgagttttta aaactgtgaa ccggcgagca cacatcttcc ccatggcaga 1080 tgactattca gactccctca tcaccaaaaa gcaagtgtca gtctggtgca gtaatgacta 1140 cctaggaatg agtcgccacc cacgggtgtg tggggcagtt atggacactt tgaaacaaca 1200 tggtgctggg gcaggtggta ctagaaatat ttctggaact agtaaattcc atgtggactt 1260 agagcgggag ctggcagacc tccatgggaa agatgccgca ctcttgtttt cctcgtgctt 1320 tgtggccaat gactcaaccc tcttcaccct ggctaagatg atgccaggct gtgagattta 1380 ctctgattct gggaaccatg cctccatgat ccaagggatt cgaaacagcc gagtgccaaa 1440 gtacatcttc cgccacaatg atgtcagcca cctcagagaa ctgctgcaaa gatctgaccc 1500 ctcagtcccc aagattgtgg catttgaaac tgtccattca atggatgggg cggtgtgccc 1560 actggaagag ctgtgtgatg tggcccatga gtttggagca atcaccttcg tggatgaggt 1620 ccacgcagtg gggctttatg gggctcgagg cggagggatt ggggatcggg atggagtcat 1680 gccaaaaatg gacatcattt ctggaacact tggcaaagcc tttggttgtg ttggagggta 1740 catcgccagc acgagttctc tgattgacac cgtacggtcc tatgctgctg gcttcatctt 1800 caccacctct ctgccaccca tgctgctggc tggagccctg gagtctgtgc ggatcctgaa 1860 gagcgctgag ggacgggtgc ttcgccgcca gcaccagcgc aacgtcaaac tcatgagaca 1920 gatgctaatg gatgccggcc tccctgttgt ccactgcccc agccacatca tccctgtgcg 1980 ggttgcagat gctgctaaaa acacagaagt ctgtgatgaa ctaatgagca gacataacat 2040 ctacgtgcaa gcaatcaatt accctacggt gccccgggga gaagagctcc tacggattgc 2100 ccccacccct caccacacac cccagatgat gaactacttc cttgagaatc tgctagtcac 2160 atggaagcaa gtggggctgg aactgaagcc tcattcctca gctgagtgca acttctgcag 2220 gaggccactg cattttgaag tgatgagtga aagagagaag tcctatttct caggcttgag 2280 caagttggta tctgctcagg cctgagcatg acctcaatta tttcacttaa ccccaggcca 2340 ttatcatatc cagatggtct tcagagttgt cttatatgt gaattaagtt atattaaatt 2400 ttaatctata gtaaaaacat agtcctggaa ataaattctt gcttaaatgg tgaaaaaa 2458 <210> 53 <211> 2281 <212> DNA <213> Homo sapiens <400> 53 cagaagaagg cagcgcccaa ggcgcatgcg cagcggtcac tcccgctgta tattaaggcg 60 ccggcgatcg cggcctgagg ctgctcccgg acaagggcaa cgagcgtttc gtttggactt 120 ctcgacttga gtgcccgcct ccttcgccgc cgcctctgca gtcctcagcg cagtctttcc 180 acaggagcca gcatacttcc tgaacatgga gagtgttgtt cgccgctgcc cattcttatc 240 ccgagtcccc caggcctttc tgcagaaagc aggcaaatct ctgttgttct atgcccaaaa 300 ctgccccaag atgatggaag ttggggccaa gccagcccct cgggcattgt ccactgcagc 360 agtacactac caacagatca aagaaacccc tccggccagt gagaaagaca aaactgctaa 420 ggccaaggtc caacagactc ctgatggatc ccagcagagt ccagatggca cacagcttcc 480 gtctggacac cccttgcctg ccacaagcca gggcactgca agcaaatgcc ctttcctggc 540 agcacagatg aatcagagag gcagcagtgt cttctgcaaa gccagtcttg agcttcagga 600 ggatgtgcag gaaatgaatg ccgtgaggaa agaggttgct gaaacctcag caggccccag 660 tgtggttagt gtgaaaaccg atggagggga tcccagtgga ctgctgaaga acttccagga 720 catcatgcaa aagcaaagac cagaaagagt gtctcatctt cttcaagata acttgccaaa 780 atctgtttcc acttttcagt atgatcgttt ctttgagaaa aaaattgatg agaaaaagaa 840 tgaccacacc tatcgagttt ttaaaactgt gaaccggcga gcacacatct tccccatggc 900 agatgactat tcagactccc tcatcaccaa aaagcaagtg tcagtctggt gcagtaatga 960 ctacctagga atgagtcgcc acccacgggt gtgtggggca gttatggaca ctttgaaaca 1020 acatggtgct ggggcaggtg gtactagaaa tatttctgga actagtaaat tccatgtgga 1080 cttagagcgg gagctggcag acctccatgg gaaagatgcc gcactcttgt tttcctcgtg 1140 ctttgtggcc aatgactcaa ccctcttcac cctggctaag atgatgccag gctgtgagat 1200 ttactctgat tctgggaacc atgcctccat gatccaaggg attcgaaaca gccgagtgcc 1260 aaagtacatc ttccgccaca atgatgtcag ccacctcaga gaactgctgc aaagatctga 1320 cccctcagtc cccaagattg tggcatttga aactgtccat tcaatggatg gggcggtgtg 1380 cccactggaa gagctgtgtg atgtggccca tgagtttgga gcaatcacct tcgtggatga 1440 ggtccacgca gtggggcttt atggggctcg aggcggaggg attggggatc gggatggagt 1500 catgccaaaa atggacatca tttctggaac acttggcaaa gcctttggtt gtgttggagg 1560 gtacatcgcc agcacgagtt ctctgattga caccgtacgg tcctatgctg ctggcttcat 1620 cttcaccacc tctctgccac ccatgctgct ggctggagcc ctggagtctg tgcggatcct 1680 gaagagcgct gagggacggg tgcttcgccg ccagcaccag cgcaacgtca aactcatgag 1740 acagatgcta atggatgccg gcctccctgt tgtccactgc cccagccaca tcatccctgt 1800 gcgggttgca gatgctgcta aaaacacaga agtctgtgat gaactaatga gcagacataa 1860 catctacgtg caagcaatca attaccctac ggtgccccgg ggagaagagc tcctacggat 1920 tgcccccacc cctcaccaca caccccagat gatgaactac ttccttgaga atctgctagt 1980 cacatggaag caagtggggc tggaactgaa gcctcattcc tcagctgagt gcaacttctg 2040 caggaggcca ctgcattttg aagtgatgag tgaaagagag aagtcctatt tctcaggctt 2100 gagcaagttg gtatctgctc aggcctgagc atgacctcaa ttatttcact taaccccagg 2160 ccattatcat atccagatgg tcttcagagt tgtctttata tgtgaattaa gttatattaa 2220 atttaatct atagtaaaa catagtcctg gaaataaatt cttgcttaaa tggtgaaaaa 2280 at 2281 <210> 54 <211> 640 <212> PRT <213> Homo sapiens <400> 54 Met Glu Ser Val Val Arg Arg Cys Pro Phe Leu Ser Arg Val Pro Gln 1 5 10 15 Ala Phe Leu Gln Lys Ala Gly Lys Ser Leu Leu Phe Tyr Ala Gln Asn 20 25 30 Cys Pro Lys Met Met Glu Val Gly Ala Lys Pro Ala Pro Arg Ala Leu 35 40 45 Ser Thr Ala Ala Val His Tyr Gln Gln Ile Lys Glu Thr Pro Pro Ala 50 55 60 Ser Glu Lys Asp Lys Thr Ala Lys Ala Lys Val Gln Gln Thr Pro Asp 65 70 75 80 Gly Ser Gln Gln Ser Pro Asp Gly Thr Gln Leu Pro Ser Gly His Pro 85 90 95 Leu Pro Ala Thr Ser Gln Gly Thr Ala Ser Lys Cys Pro Phe Leu Ala 100 105 110 Ala Gln Met Asn Gln Arg Gly Ser Ser Val Phe Cys Lys Ala Ser Leu 115 120 125 Glu Leu Gln Glu Asp Val Gln Glu Met Asn Ala Val Arg Lys Glu Val 130 135 140 Ala Glu Thr Ser Ala Gly Pro Ser Val Val Ser Val Lys Thr Asp Gly 145 150 155 160 Gly Asp Pro Ser Gly Leu Leu Lys Asn Phe Gln Asp Ile Met Gln Lys 165 170 175 Gln Arg Pro Glu Arg Val Ser His Leu Leu Gln Asp Asn Leu Pro Lys 180 185 190 Ser Val Ser Thr Phe Gln Tyr Asp Arg Phe Phe Glu Lys Lys Ile Asp 195 200 205 Glu Lys Lys Asn Asp His Thr Tyr Arg Val Phe Lys Thr Val Asn Arg 210 215 220 Arg Ala His Ile Phe Pro Met Ala Asp Asp Tyr Ser Asp Ser Leu Ile 225 230 235 240 Thr Lys Lys Gln Val Ser Val Trp Cys Ser Asn Asp Tyr Leu Gly Met 245 250 255 Ser Arg His Pro Arg Val Cys Gly Ala Val Met Asp Thr Leu Lys Gln 260 265 270 His Gly Ala Gly Ala Gly Gly Thr Arg Asn Ile Ser Gly Thr Ser Lys 275 280 285 Phe His Val Asp Leu Glu Arg Glu Leu Ala Asp Leu His Gly Lys Asp 290 295 300 Ala Ala Leu Leu Phe Ser Ser Cys Phe Val Ala Asn Asp Ser Thr Leu 305 310 315 320 Phe Thr Leu Ala Lys Met Met Pro Gly Cys Glu Ile Tyr Ser Asp Ser 325 330 335 Gly Asn His Ala Ser Met Ile Gln Gly Ile Arg Asn Ser Arg Val Pro 340 345 350 Lys Tyr Ile Phe Arg His Asn Asp Val Ser His Leu Arg Glu Leu Leu 355 360 365 Gln Arg Ser Asp Pro Ser Val Pro Lys Ile Val Ala Phe Glu Thr Val 370 375 380 His Ser Met Asp Gly Ala Val Cys Pro Leu Glu Glu Leu Cys Asp Val 385 390 395 400 Ala His Glu Phe Gly Ala Ile Thr Phe Val Asp Glu Val His Ala Val 405 410 415 Gly Leu Tyr Gly Ala Arg Gly Gly Gly Ile Gly Asp Arg Asp Gly Val 420 425 430 Met Pro Lys Met Asp Ile Ile Ser Gly Thr Leu Gly Lys Ala Phe Gly 435 440 445 Cys Val Gly Gly Tyr Ile Ala Ser Thr Ser Ser Leu Ile Asp Thr Val 450 455 460 Arg Ser Tyr Ala Ala Gly Phe Ile Phe Thr Thr Ser Leu Pro Pro Met 465 470 475 480 Leu Leu Ala Gly Ala Leu Glu Ser Val Arg Ile Leu Lys Ser Ala Glu 485 490 495 Gly Arg Val Leu Arg Arg Gln His Gln Arg Asn Val Lys Leu Met Arg 500 505 510 Gln Met Leu Met Asp Ala Gly Leu Pro Val Val His Cys Pro Ser His 515 520 525 Ile Ile Pro Val Arg Val Ala Asp Ala Ala Lys Asn Thr Glu Val Cys 530 535 540 Asp Glu Leu Met Ser Arg His Asn Ile Tyr Val Gln Ala Ile Asn Tyr 545 550 555 560 Pro Thr Val Pro Arg Gly Glu Glu Leu Leu Arg Ile Ala Pro Thr Pro 565,570,575 His His Thr Pro Gln Met Met Asn Tyr Phe Leu Glu Asn Leu Leu Val 580,585,590 Thr Trp Lys Gln Val Gly Leu Glu Leu Lys Pro His Ser Ser Ala Glu 595,600,605 Cys Asn Phe Cys Arg Arg Pro Leu His Phe Glu Val Met Ser Glu Arg 610 615 620 Glu Lys Ser Tyr Phe Ser Gly Leu Ser Lys Leu Val Ser Ala Gln Ala 625,630,635,640 <210> 55 <211> 640 <212> PRT <213> Homo sapiens <400> 55 Met Glu Ser Val Val Arg Arg Cys Pro Phe Leu Ser Arg Val Pro Gln 1 5 10 15 Ala Phe Leu Gln Lys Ala Gly Lys Ser Leu Leu Phe Tyr Ala Gln Asn 20 25 30 Cys Pro Lys Met Met Glu Val Gly Ala Lys Pro Ala Pro Arg Ala Leu 35 40 45 Ser Thr Ala Ala Val His Tyr Gln Gln Ile Lys Glu Thr Pro Pro Ala 50 55 60 Ser Glu Lys Asp Lys Thr Ala Lys Ala Lys Val Gln Gln Thr Pro Asp 65 70 75 80 Gly Ser Gln Gln Ser Pro Asp Gly Thr Gln Leu Pro Ser Gly His Pro 85 90 95 Leu Pro Ala Thr Ser Gln Gly Thr Ala Ser Lys Cys Pro Phe Leu Ala 100 105 110 Ala Gln Met Asn Gln Arg Gly Ser Ser Val Phe Cys Lys Ala Ser Leu 115 120 125 Glu Leu Gln Glu Asp Val Gln Glu Met Asn Ala Val Arg Lys Glu Val 130 135 140 Ala Glu Thr Ser Ala Gly Pro Ser Val Val Ser Val Lys Thr Asp Gly 145 150 155 160 Gly Asp Pro Ser Gly Leu Leu Lys Asn Phe Gln Asp Ile Met Gln Lys 165 170 175 Gln Arg Pro Glu Arg Val Ser His Leu Leu Gln Asp Asn Leu Pro Lys 180 185 190 Ser Val Ser Thr Phe Gln Tyr Asp Arg Phe Phe Glu Lys Lys Ile Asp 195 200 205 Glu Lys Lys Asn Asp His Thr Tyr Arg Val Phe Lys Thr Val Asn Arg 210 215 220 Arg Ala His Ile Phe Pro Met Ala Asp Asp Tyr Ser Asp Ser Leu Ile 225 230 235 240 Thr Lys Lys Gln Val Ser Val Trp Cys Ser Asn Asp Tyr Leu Gly Met 245 250 255 Ser Arg His Pro Arg Val Cys Gly Ala Val Met Asp Thr Leu Lys Gln 260 265 270 His Gly Ala Gly Ala Gly Gly Thr Arg Asn Ile Ser Gly Thr Ser Lys 275 280 285 Phe His Val Asp Leu Glu Arg Glu Leu Ala Asp Leu His Gly Lys Asp 290 295 300 Ala Ala Leu Leu Phe Ser Ser Cys Phe Val Ala Asn Asp Ser Thr Leu 305 310 315 320 Phe Thr Leu Ala Lys Met Met Pro Gly Cys Glu Ile Tyr Ser Asp Ser 325 330 335 Gly Asn His Ala Ser Met Ile Gln Gly Ile Arg Asn Ser Arg Val Pro 340 345 350 Lys Tyr Ile Phe Arg His Asn Asp Val Ser His Leu Arg Glu Leu Leu 355 360 365 Gln Arg Ser Asp Pro Ser Val Pro Lys Ile Val Ala Phe Glu Thr Val 370 375 380 His Ser Met Asp Gly Ala Val Cys Pro Leu Glu Glu Leu Cys Asp Val 385 390 395 400 Ala His Glu Phe Gly Ala Ile Thr Phe Val Asp Glu Val His Ala Val 405 410 415 Gly Leu Tyr Gly Ala Arg Gly Gly Gly Ile Gly Asp Arg Asp Gly Val 420 425 430 Met Pro Lys Met Asp Ile Ile Ser Gly Thr Leu Gly Lys Ala Phe Gly 435 440 445 Cys Val Gly Gly Tyr Ile Ala Ser Thr Ser Ser Leu Ile Asp Thr Val 450 455 460 Arg Ser Tyr Ala Ala Gly Phe Ile Phe Thr Thr Ser Leu Pro Pro Met 465 470 475 480 Leu Leu Ala Gly Ala Leu Glu Ser Val Arg Ile Leu Lys Ser Ala Glu 485 490 495 Gly Arg Val Leu Arg Arg Gln His Gln Arg Asn Val Lys Leu Met Arg 500 505 510 Gln Met Leu Met Asp Ala Gly Leu Pro Val Val His Cys Pro Ser His 515 520 525 Ile Ile Pro Val Arg Val Ala Asp Ala Ala Lys Asn Thr Glu Val Cys 530 535 540 Asp Glu Leu Met Ser Arg His Asn Ile Tyr Val Gln Ala Ile Asn Tyr 545 550 555 560 Pro Thr Val Pro Arg Gly Glu Glu Leu Leu Arg Ile Ala Pro Thr Pro 565 570 575 His His Thr Pro Gln Met Met Asn Tyr Phe Leu Glu Asn Leu Leu Val 580 585 590 Thr Trp Lys Gln Val Gly Leu Glu Leu Lys Pro His Ser Ser Ala Glu 595 600 605 Cys Asn Phe Cys Arg Arg Pro Leu His Phe Glu Val Met Ser Glu Arg 610 615 620 Glu Lys Ser Tyr Phe Ser Gly Leu Ser Lys Leu Val Ser Ala Gln Ala 625 630 635 640 <210> 56 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> ALAS-1_forward primer <400> 56 agccacatca tccctgt 17 <210> 57 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ALAS-1_reverse primer <400> 57 cgtagatgtt atgtctgctc at 22 <210> 58 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ALAS-1 probe <400> 58 tttagcagca tctgcaaccc gc 22 <210> 59 <211> 3212 <212> DNA <213> Homo sapiens <400> 59 gagtaggcgc gagctaagca ggaggcggag gcggaggcgg agggcgaggg gcggggagcg 60 ccgcctggag cgcggcaggt catattgaac attccagata cctatcatta ctcgatgctg 120 ttgataacag caagatggct ttgaactcag ggtcaccacc agctattgga ccttactatg 180 aaaaccatgg ataccaaccg gaaaacccct atcccgcaca gcccactgtg gtccccactg 240 tctacgaggt gcatccggct cagtactacc cgtcccccgt gccccagtac gccccgaggg 300 tcctgacgca ggcttccaac cccgtcgtct gcacgcagcc caaatcccca tccgggacag 360 tgtgcacctc aaagactaag aaagcactgt gcatcacctt gaccctgggg accttcctcg 420 tgggagctgc gctggccgct ggcctactct ggaagttcat gggcagcaag tgctccaact 480 ctgggataga gtgcgactcc tcaggtacct gcatcaaccc ctctaactgg tgtgatggcg 540 tgtcacactg ccccggcggg gaggacgaga atcggtgtgt tcgcctctac ggaccaaact 600 tcatccttca ggtgtactca tctcagagga agtcctggca ccctgtgtgc caagacgact 660 ggaacgagaa ctacgggcgg gcggcctgca gggacatggg ctataagaat aatttttact 720 ctagccaagg aatagtggat gacagcggat ccaccagctt tatgaaactg aacacaagtg 780 ccggcaatgt cgatatctat aaaaaactgt accacagtga tgcctgttct tcaaaagcag 840 tggtttcttt acgctgtata gcctgcgggg tcaacttgaa ctcaagccgc cagagcagga 900 ttgtgggcgg cgagagcgcg ctcccggggg cctggccctg gcaggtcagc ctgcacgtcc 960 agaacgtcca cgtgtgcgga ggctccatca tcacccccga gtggatcgtg acagccgccc 1020 actgcgtgga aaaacctctt aacaatccat ggcattggac ggcatttgcg gggattttga 1080 gacaatcttt catgttctat ggagccggat accaagtaga aaaagtgatt tctcatccaa 1140 attatgactc caagaccaag aacaatgaca ttgcgctgat gaagctgcag aagcctctga 1200 ctttcaacga cctagtgaaa ccagtgtgtc tgcccaaccc aggcatgatg ctgcagccag 1260 aacagctctg ctggatttcc gggtgggggg ccaccgagga gaaagggaag acctcagaag 1320 tgctgaacgc tgccaaggtg cttctcattg agacacagag atgcaacagc agatatgtct 1380 atgacaacct gatcacacca gccatgatct gtgccggctt cctgcagggg aacgtcgatt 1440 cttgccaggg tgacagtgga gggcctctgg tcacttcgaa gaacaatatc tggtggctga 1500 taggggatac aagctggggt tctggctgtg ccaaagctta cagaccagga gtgtacggga 1560 atgtgatggt attcacggac tggatttatc gacaaatgag ggcagacggc taatccacat 1620 ggtcttcgtc cttgacgtcg ttttacaaga aaacaatggg gctggttttg cttccccgtg 1680 catgatttac tcttagagat gattcagagg tcacttcatt tttattaaac agtgaacttg 1740 tctggctttg gcactctctg ccattctgtg caggctgcag tggctcccct gcccagcctg 1800 ctctccctaa ccccttgtcc gcaaggggtg atggccggct ggttgtgggc actggcggtc 1860 aagtgtggag gagaggggtg gaggctgccc cattgagatc ttcctgctga gtcctttcca 1920 ggggccaatt ttggatgagc atggagctgt cacctctcag ctgctggatg acttgagatg 1980 aaaaaggaga gacatggaaa gggagacagc caggtggcac ctgcagcggc tgccctctgg 2040 ggccacttgg tagtgtcccc agcctacctc tccacaaggg gattttgctg atgggttctt 2100 agagccttag cagccctgga tggtggccag aaataaaggg accagccctt catgggtggt 2160 gacgtggtag tcacttgtaa ggggaacaga aacatttttg ttcttatggg gtgagaatat 2220 agacagtgcc cttggtgcga gggaagcaat tgaaaaggaa cttgccctga gcactcctgg 2280 tgcaggtctc cacctgcaca ttgggtgggg ctcctgggag ggagactcag ccttcctcct 2340 catcctccct gaccctgctc ctagcaccct ggagagtgca catgcccctt ggtcctggca 2400 gggcgccaag tctggcacca tgttggcctc ttcaggcctg ctagtcactg gaaattgagg 2460 tccatggggg aaatcaagga tgctcagttt aaggtacact gtttccatgt tatgtttcta 2520 cacattgcta cctcagtgct cctggaaact tagcttttga tgtctccaag tagtccacct 2580 tcatttaact ctttgaaact gtatcatctt tgccaagtaa gagtggtggc ctatttcagc 2640 tgctttgaca aaatgactgg ctcctgactt aacgttctat aaatgaatgt gctgaagcaa 2700 agtgcccatg gtggcggcga agaagagaaa gatgtgtttt gttttggact ctctgtggtc 2760 ccttccaatg ctgtgggttt ccaaccaggg gaagggtccc ttttgcattg ccaagtgcca 2820 taaccatgag cactactcta ccatggttct gcctcctggc caagcaggct ggtttgcaag 2880 aatgaaatga atgattctac agctaggact taaccttgaa atggaaagtc atgcaatccc 2940 atttgcagga tctgtctgtg cacatgcctc tgtagagagc agcattccca gggaccttgg 3000 aaacagttgg cactgtaagg tgcttgctcc ccaagacaca tcctaaaagg tgttgtaatg 3060 gtgaaaacgt cttccttctt tattgcccct tcttatttat gtgaacaact gtttgtcttt 3120 ttttgtatct ttttaaact gtaaagttca attgtgaaaa tgaatatcat gcaaataaat 3180 tatgcaattt ttttttcaaa gtaaaaaaaa aa 3212 <210> 60 <211> 4945 <212> DNA <213> Homo sapiens <400> 60 ttcatttccc agacttagca caatctcatc cgctctaaac aacctcatca aaactacttt 60 ctggtcagag agaagcaata attattatta acatttatta acgatcaata aacttgatcg 120 cattatggcc agcactatta aggaagcctt atcagttgtg agtgaggacc agtcgttgtt 180 tgagtgtgcc tacggaacgc cacacctggc taagacagag atgaccgcgt cctcctccag 240 cgactatgga cagacttcca agatgagccc acgcgtccct cagcaggatt ggctgtctca 300 acccccagcc agggtcacca tcaaaatgga atgtaaccct agccaggtga atggctcaag 360 gaactctcct gatgaatgca gtgtggccaa aggcgggaag atggtgggca gcccagacac 420 cgttgggatg aactacggca gctacatgga ggagaagcac atgccacccc caaacatgac 480 cacgaacgag cgcagagtta tcgtgccagc agatcctacg ctatggagta cagaccatgt 540 gcggcagtgg ctggagtggg cggtgaaaga atatggcctt ccagacgtca acatcttgtt 600 attccagaac atcgatggga aggaactgtg caagatgacc aaggacgact tccagaggct 660 caccccagc tacaacgccg acatccttct ctcacatctc cactacctca gagagactcc 720 tcttccacat ttgacttcag atgatgttga taaagcctta caaaactctc cacggttaat 780 gcatgctaga aacacagggg gtgcagcttt tattttccca aatacttcag tatatcctga 840 agctacgcaa agaattacaa ctaggccaga tttaccatat gagcccccca ggagatcagc 900 ctggaccggt cacggccacc ccacgcccca gtcgaaagct gctcaaccat ctccttccac 960 agtgcccaaa actgaagacc agcgtcctca gttagatcct tatcagattc ttggaccaac 1020 aagtagccgc cttgcaaatc caggcagtgg ccagatccag ctttggcagt tcctcctgga 1080 gctcctgtcg gacagctcca actccagctg catcacctgg gaaggcacca acggggagtt 1140 caagatgacg gatcccgacg aggtggcccg gcgctgggga gagcggaaga gcaaacccaa 1200 catgaactac gataagctca gccgcgccct ccgttactac tatgacaaga acatcatgac 1260 caaggtccat gggaagcgct acgcctacaa gttcgacttc cacgggatcg cccaggccct 1320 ccagccccac cccccggagt catctctgta caagtacccc tcagacctcc cgtacatggg 1380 ctcctatcac gcccacccac agaagatgaa ctttgtggcg ccccaccctc cagccctccc 1440 cgtgacatct tccagttttt ttgctgcccc aaacccatac tggaattcac caactgggg 1500 tatatacccc aacactaggc tccccaccag ccatatgcct tctcatctgg gcacttacta 1560 ctaaagacct ggcggaggct tttcccatca gcgtgcattc accagcccat cgccacaaac 1620 tctatcggag aacatgaatc aaaagtgcct caagaggaat gaaaaaagct ttactggggc 1680 tgggggaagga agccgggggaa gagatccaaa gactcttggg aggagttac tgaagtctta 1740 1800 accttgtaaa agacagtgta tgtagaagca tgaagtctta agcaaagt gccaaagaaa 1860 gtggtcttaa gaaatgtata aactttagag tagagtttgg aatcccacta atgcaaactg 1920 ggatgaaact aaagcaatag aaacaacaca gttttgacct aacataccgt ttataatgcc 1980 attttaagga aaactacctg tatttaaaaa tagaaacata tcaaaaacaa gagaaaagac 2040 acgagagaga ctgtggccca tcaacagacg ttgatatgca actgcatggc atgtgctggt 2100 ttggttgaaa tcaaatacat tccgtttgat ggacagctgt cagctttctc aaactgtgaa 2160 gatgacccaa agtttccaac tcctttacag tattaccgg actatgact aaaaggtggg 2220 actgaggatg tgtatagagt gagcgtgtga ttgtagacag aggggtgaag aaggaggagg 2280 aagaggcaga gaaggaggag accaggggctg ggaagaaac ttctcaagca atgagactg 2340 gactcaggac atttggggac tgtgtacaat gagttatgga gactcgaggg ttcatgcagt 2400 cagtgttata ccaacccag tgttaggaga aaggacacag cgtaatggag aagggaag 2460 tagtagaatt cagaaaaaaaatgcgcatc tctttctttg ttgtcaat gaaaatttta 2520 actggaattg tctgatattt agagaaaca ttcaggacct catcattatg tggggttt 2580 gttctccaca gggtcaggta agagatggcc ttctggctg ccacaatcag aaatcacgca 2640 ggcattttgg gtaggcggcc tccagttttc ctttgagtcg cgaacgctgt gcgtttgtca 2700 gatgaagta tacaagtca tgttttcccc ccttttatta taatattat ataacttatg 2760 catttataca ctacgagttg atctcggcca gccaagaca cacgacaaaa gagacaatcg 2820 atataatgtg gccttgaatt ttactctgt atgcttaatg tttacaat gaagttatta 2880 2940 ttatacagga gtctgcattt gcactttttt tagtgactaa agttgcttaa tgaaaacatg 3000 tgctgaatgt tgtggatttt gtgttataat ttactttgtc caggaacttg tgcaagggag 3060 agccaaggaa ataggatgtt tggcacccaa atggcgtcag cctctccagg tccttcttgc 3120 ctcccctcct gtcttttatt tctagcccct tttggaacag areaccccg ggtttcacat 3180 tggagcctcc atatttatgc ctggaatgga aagaggccta tgaagctggg gttgtcattg 3240 agaaattcta gttcagcacc tggtcacaaa tcacccttaa ttcctgctat gattaaaata 3300 catttgttga acagtgaaca agctaccact cgttaaggcaa actgtattat tactggcaaa 3360 taagcgtca tggatagctg caatttctca ctttacagaa aaagggata acgtctagat 3420 ttgctgcggg gtttctcttt caggagctct cactaggtag acagctttag tcctgctaca 3480 tcagagttac ctgggcactg tggcttggga ttcactagcc ctgagcctga tgttgctggc 3540 tatcccttga agaaatgtt tatttccata atctagagtc agtttccctg ggcatctttt 3600 ctttgaatca caaatgctgc caaccttggt ccaggtgaag gcaactcaaa aggtgaaaat 3660 acaaggtgac cgtgcgaagg cgctagccga aacatcttag ctgaataggt ttctgaactg 3720 gcccttttca tagctgtttc agggcctgtt tttttcacgt tgcagtcctt ttgctatgat 3780 tatgtgaagt tgccaaacct ctgtgctgtg gatgttttgg cagtgggctt tgaagtcggc 3840 aggacacgat taccaatgct cctgacaccc cgtgtcattt ggattagacg gagcccaacc 3900 atccatcatt ttgcagcagc ctgggaaggc ccacaaagtg cccgtatctc cttagggaaa 3960 ataaataaat acaatcatga aagctggcag ttaggctgac ccaaactgtg ctaatggaaa 4020 agatcagtca tttttatttt ggaatgcaaa gtcaagacac acctacattc ttcatagaaa 4080 tacacattta cttggataat cactcagttc tctcttcaag actgtctcat gagcaagatc 4140 ataaaaacaa gacatgatta tcatattcaa ttttaacaga tgttttccat tagatccctc 4200 aaccctccac ccccagtcca ggttattagc aagtcttatg agcaactggg ataattttgg 4260 ataacatgat aatactgagt tccttcaaat acataattct taaattgttt caaaatggca 4320 tattactct gttactgttg taatctatt ccaagcccc ctccaggtca tattcataat 4380 tgcatgaacc tttctct gtttgtccct gtctcttggc tgccctt gtatactcag 4440 actcctgtac aatcttactc ctgctggcaa gagatttgtc ttctttctt gtcttcatt 4500 ggctttcgggg ccttgtatgt ggtaaaatca ccaaatcaca gtcaagactg tgttttgtt 4560 cctagtttga tgccttatg tcccggaggg gttcacaag tgcttgtca ggactgctgc 4620 agttagaagg ctcactgctt ctcctaagcc ttctgcacag atgtgcacc tgcacccag 4680 gagcaggagc cggaggagct gccctctgac agcaggtgca gcagagatgg ctacagctca 4740 ggagctggga aggtgatggg gcacagggaa agcacagatg ttctgcagcg cccaagtg 4800 acccattgcc tggaaga gagaaaata tttttaaaa agctagttta tttagcttct 4860 cattaattca ttcaataa gtcgtgaggt gactaattag agataaaaa ttactttgga 4920 ctactcaaa atacaccaaaaaaa 4945 <210> 61 <211> 492 <212> PRT <213> Homo sapiens <400> 61 Met Ala Leu Asn Ser Gly Ser Pro Pro Ala Ile Gly Pro Tyr Tyr Glu 1 5 10 15 Asn His Gly Tyr Gln Pro Glu Asn Pro Tyr Pro Ala Gln Pro Thr Val 20 25 30 Val Pro Thr Val Tyr Glu Val His Pro Ala Gln Tyr Tyr Pro Ser Pro 35 40 45 Val Pro Gln Tyr Ala Pro Arg Val Leu Thr Gln Ala Ser Asn Pro Val 50 55 60 Val Cys Thr Gln Pro Lys Ser Pro Ser Gly Thr Val Cys Thr Ser Lys 65 70 75 80 Thr Lys Lys Ala Leu Cys Ile Thr Leu Thr Leu Gly Thr Phe Leu Val 85 90 95 Gly Ala Ala Leu Ala Ala Gly Leu Leu Trp Lys Phe Met Gly Ser Lys 100 105 110 Cys Ser Asn Ser Gly Ile Glu Cys Asp Ser Ser Gly Thr Cys Ile Asn 115 120 125 Pro Ser Asn Trp Cys Asp Gly Val Ser His Cys Pro Gly Gly Glu Asp 130 135 140 Glu Asn Arg Cys Val Arg Leu Tyr Gly Pro Asn Phe Ile Leu Gln Val 145 150 155 160 Tyr Ser Ser Gln Arg Lys Ser Trp His Pro Val Cys Gln Asp Asp Trp 165 170 175 Asn Glu Asn Tyr Gly Arg Ala Ala Cys Arg Asp Met Gly Tyr Lys Asn 180 185 190 Asn Phe Tyr Ser Ser Gln Gly Ile Val Asp Asp Ser Gly Ser Thr Ser 195 200 205 Phe Met Lys Leu Asn Thr Ser Ala Gly Asn Val Asp Ile Tyr Lys Lys 210 215 220 Leu Tyr His Ser Asp Ala Cys Ser Ser Lys Ala Val Val Ser Leu Arg 225 230 235 240 Cys Ile Ala Cys Gly Val Asn Leu Asn Ser Ser Arg Gln Ser Arg Ile 245 250 255 Val Gly Gly Glu Ser Ala Leu Pro Gly Ala Trp Pro Trp Gln Val Ser 260 265 270 Leu His Val Gln Asn Val His Val Cys Gly Gly Ser Ile Ile Thr Pro 275 280 285 Glu Trp Ile Val Thr Ala Ala His Cys Val Glu Lys Pro Leu Asn Asn 290 295 300 Pro Trp His Trp Thr Ala Phe Ala Gly Ile Leu Arg Gln Ser Phe Met 305 310 315 320 Phe Tyr Gly Ala Gly Tyr Gln Val Glu Lys Val Ile Ser His Pro Asn 325 330 335 Tyr Asp Ser Lys Thr Lys Asn Asn Asp Ile Ala Leu Met Lys Leu Gln 340 345 350 Lys Pro Leu Thr Phe Asn Asp Leu Val Lys Pro Val Cys Leu Pro Asn 355 360 365 Pro Gly Met Met Leu Gln Pro Glu Gln Leu Cys Trp Ile Ser Gly Trp 370 375 380 Gly Ala Thr Glu Glu Lys Gly Lys Thr Ser Glu Val Leu Asn Ala Ala 385 390 395 400 Lys Val Leu Leu Ile Glu Thr Gln Arg Cys Asn Ser Arg Tyr Val Tyr 405 410 415 Asp Asn Leu Ile Thr Pro Ala Met Ile Cys Ala Gly Phe Leu Gln Gly 420 425 430 Asn Val Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Thr Ser 435 440 445 Lys Asn Asn Ile Trp Trp Leu Ile Gly Asp Thr Ser Trp Gly Ser Gly 450 455 460 Cys Ala Lys Ala Tyr Arg Pro Gly Val Tyr Gly Asn Val Met Val Phe 465 470 475 480 Thr Asp Trp Ile Tyr Arg Gln Met Arg Ala Asp Gly 485 490 <210> 62 <211> 479 <212> PRT <213> Homo sapiens <400> 62 Met Ala Ser Thr Ile Lys Glu Ala Leu Ser Val Val Ser Glu Asp Gln 1 5 10 15 Ser Leu Phe Glu Cys Ala Tyr Gly Thr Pro His Leu Ala Lys Thr Glu 20 25 30 Met Thr Ala Ser Ser Ser Ser Asp Tyr Gly Gln Thr Ser Lys Met Ser 35 40 45 Pro Arg Val Pro Gln Gln Asp Trp Leu Ser Gln Pro Pro Ala Arg Val 50 55 60 Thr Ile Lys Met Glu Cys Asn Pro Ser Gln Val Asn Gly Ser Arg Asn 65 70 75 80 Ser Pro Asp Glu Cys Ser Val Ala Lys Gly Gly Lys Met Val Gly Ser 85 90 95 Pro Asp Thr Val Gly Met Asn Tyr Gly Ser Tyr Met Glu Glu Lys His 100 105 110 Met Pro Pro Pro Asn Met Thr Thr Asn Glu Arg Arg Val Ile Val Pro 115 120 125 Ala Asp Pro Thr Leu Trp Ser Thr Asp His Val Arg Gln Trp Leu Glu 130 135 140 Trp Ala Val Lys Glu Tyr Gly Leu Pro Asp Val Asn Ile Leu Leu Phe 145 150 155 160 Gln Asn Ile Asp Gly Lys Glu Leu Cys Lys Met Thr Lys Asp Asp Phe 165 170 175 Gln Arg Leu Thr Pro Ser Tyr Asn Ala Asp Ile Leu Leu Ser His Leu 180 185 190 His Tyr Leu Arg Glu Thr Pro Leu Pro His Leu Thr Ser Asp Asp Val 195 200 205 Asp Lys Ala Leu Gln Asn Ser Pro Arg Leu Met His Ala Arg Asn Thr 210 215 220 Gly Gly Ala Ala Phe Ile Phe Pro Asn Thr Ser Val Tyr Pro Glu Ala 225 230 235 240 Thr Gln Arg Ile Thr Thr Arg Pro Asp Leu Pro Tyr Glu Pro Pro Arg 245 250 255 Arg Ser Ala Trp Thr Gly His Gly His Pro Thr Pro Gln Ser Lys Ala 260 265 270 Ala Gln Pro Ser Pro Ser Thr Val Pro Lys Thr Glu Asp Gln Arg Pro 275 280 285 Gln Leu Asp Pro Tyr Gln Ile Leu Gly Pro Thr Ser Ser Arg Leu Ala 290 295 300 Asn Pro Gly Ser Gly Gln Ile Gln Leu Trp Gln Phe Leu Leu Glu Leu 305 310 315 320 Leu Ser Asp Ser Ser Asn Ser Ser Cys Ile Thr Trp Glu Gly Thr Asn 325 330 335 Gly Glu Phe Lys Met Thr Asp Pro Asp Glu Val Ala Arg Arg Trp Gly 340 345 350 Glu Arg Lys Ser Lys Pro Asn Met Asn Tyr Asp Lys Leu Ser Arg Ala 355 360 365 Leu Arg Tyr Tyr Tyr Asp Lys Asn Ile Met Thr Lys Val His Gly Lys 370 375 380 Arg Tyr Ala Tyr Lys Phe Asp Phe His Gly Ile Ala Gln Ala Leu Gln 385 390 395 400 Pro His Pro Pro Glu Ser Ser Leu Tyr Lys Tyr Pro Ser Asp Leu Pro 405 410 415 Tyr Met Gly Ser Tyr His Ala His Pro Gln Lys Met Asn Phe Val Ala 420 425 430 Pro His Pro Pro Ala Leu Pro Val Thr Ser Ser Ser Phe Phe Ala Ala 435 440 445 Pro Asn Pro Tyr Trp Asn Ser Pro Thr Gly Gly Ile Tyr Pro Asn Thr 450 455 460 Arg Leu Pro Thr Ser His Met Pro Ser His Leu Gly Thr Tyr Tyr 465 470 475 <210> 63 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> TMPRSS2-ERG_forward primer <400> 63 ctggagcgcg gcaggaa 17 <210> 64 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> TMPRSS2-ERG_reverse primer <400> 64 ccgtaggcac actcaaacaa cga 23 <210> 65 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> TMPRSS2-ERG probe <400> 65 ttatcagttg tgagtgagga c 21 BRIEF DESCRIPTION OF THE DRAWINGS

[0099] In the following figures:

[0100] Figure 1A flow chart schematically and exemplarily illustrating an embodiment of a method for risk stratification of prostate cancer subjects after surgery.

[0101] Figure 2 Shown is a Kaplan Meier curve analysis of the PDE4D7-based expression-based risk score measured in a TMDERS2-ERG fusion-negative (TMRPRSS2-ER−) prostate cancer patient cohort (n=258 patients; 43.8% events).

[0102] Figure 3 Shown is a Kaplan Meier curve analysis of the PDE4D7-based expression-based risk score measured in a TMPRRS2-ERG fusion-positive (TMRPRSS2-ERG+) prostate cancer patient cohort (n=281 patients; 38.1% events).

[0103] Figure 4 Shown is a Kaplan Meier curve analysis of the PDE4D5-based expression-based risk score measured in a TMDERS2-ERG fusion-negative (TMRPRSS2-ER−) prostate cancer patient cohort (n=261 patients; 44.1% events).

[0104] Figure 5 Shown is a Kaplan Meier curve analysis of the PDE4D5-based expression-based risk score measured in a TMPRRS2-ERG fusion-positive (TMRPRSS2-ERG+) prostate cancer patient cohort (n=282 patients; 38.3% events).

[0105] Figure 6 Shown is a Kaplan Meier curve analysis of the PDE4D9-based expression-based risk score measured in a TMPRRS2-ERG fusion-negative (TMRPRSS2-ER−) prostate cancer patient cohort (n=261 patients; 43.9% events).

[0106] Figure 7 Shown is a Kaplan Meier curve analysis of the PDE4D9-based expression-based risk score measured in a TMPRRS2-ERG fusion-positive (TMRPRSS2-ERG+) prostate cancer patient cohort (n=282 patients; 38.3% events).

[0107] Figure 8ROC curve analysis showing the postoperative prognostic risk score of the TMPRSS2-ERG fusion status-dependent CAPRA-S score combined with the expression-based risk score based on PDE4D7 or PDE4D9 ("CAPRA-S&PDE4D7&9 score") relative to the CAPRA-S classification in predicting biochemical recurrence (BCR) 5 years after surgery, performed in the RP cohort with complete 5-year follow-up (radical prostatectomy; n=482 patients; 35.5% BCR events within 5 years after treatment).

[0108] Fig. 9 ROC curve analysis of the postoperative prognostic risk score based on the TMPRSS2-ERG fusion status-dependent CAPRA-S score combined with the expression-based risk score based on PDE4D7 or PDE4D9 ("CAPRA-S&PDE4D7&9 score") relative to the CAPRA-S classification in predicting clinical (i.e., metastatic) recurrence (CR) 10 years after surgery is shown in the RP cohort with complete 10-year follow-up (radical prostatectomy; n=337 patients; 18.1% BCR events within 10 years after treatment).

[0109] Fig.10 ROC curve analysis of the postoperative prognostic risk score based on the TMPRSS2-ERG fusion status-dependent CAPRA-S score combined with the expression-based risk score based on PDE4D7 or PDE4D9 ("CAPRA-S&PDE4D7&9 score") relative to the CAPRA-S classification in predicting prostate cancer-specific survival (PCSS) 10 years after surgery was shown in the RP cohort with complete follow-up of 10 years (radical prostatectomy; n=304 patients; 8.2% of prostate cancer deaths within 10 years after treatment).

[0110] Fig.11 ROC curve analysis of the postoperative prognostic risk score based on the TMPRSS2-ERG fusion status-dependent CAPRA-S score combined with the expression-based risk score based on PDE4D7 or PDE4D9 ("CAPRA-S&PDE4D7&9 score") relative to the CAPRA-S classification in predicting clinical (i.e., metastatic) recurrence (CR) 10 years after surgery is shown in the RP cohort with complete 10-year follow-up (radical prostatectomy; pGleason>6; n=231 patients; 26.4% BCR events within 10 years after treatment).

[0111] Fig.12ROC curve analysis of the postoperative prognostic risk score based on the TMPRSS2-ERG fusion status-dependent CAPRA-S score combined with the expression-based risk score based on PDE4D7 or PDE4D9 ("CAPRA-S&PDE4D7&9 score") relative to the CAPRA-S classification in predicting 10-year prostate cancer-specific survival (PCSS) after surgery in the RP cohort with complete 10-year follow-up (radical prostatectomy; pGleason>6; n=198 patients; 12.6% of prostate cancer mortality events within 10 years after treatment).

[0112] Fig.13 ROC curve analysis of the postoperative prognostic risk score based on the TMPRSS2-ERG fusion status-dependent CAPRA-S score combined with the expression-based risk score based on PDE4D7 or PDE4D9 ("CAPRA-S&PDE4D7&9 score") relative to the CAPRA-S classification in predicting prostate cancer-specific survival (PCSS) 10 years after surgery in the RP cohort with complete follow-up of 10 years (radical prostatectomy; SRT=salvage radiotherapy; n=93 patients; 23.7% of prostate cancer deaths within 10 years after treatment).

[0113] Fig.14 ROC curve analysis of the postoperative prognostic risk score showing the TMPRSS2-ERG fusion status-dependent CAPRA-S score combined with the expression-based risk score based on PDE4D7 or PDE4D9 ("CAPRA-S & PDE4D7 & 9 score") relative to the CAPRA-S classification in predicting prostate cancer-specific survival (PCSS) 10 years after surgery in the RP cohort with complete 10-year follow-up (radical prostatectomy; SADT = salvage androgen deprivation therapy; n = 68 patients; 30.9% of prostate cancer deaths within 10 years after treatment).

[0114] Fig.15 Shown is a Kaplan Meier curve analysis of the CAPRA-S score to predict biochemical recurrence (BCR) in the prostate cancer surgery cohort (n=536 patients; 40.9% events).

[0115] Fig.16 Kaplan Meier curve analysis of the postoperative prognostic risk score based on the TMPRSS2-ERG fusion status-dependent CAPRA-S score combined with the expression-based risk score based on PDE4D7 or PDE4D9 ("CAPRA-S & PDE4D7 & 9 score") is shown to predict biochemical recurrence (BCR) in the prostate cancer surgery cohort (n = 536 patients; 40.9% events).

[0116] Fig.17 Shown is a Kaplan Meier curve analysis of the CAPRA-S score to predict clinical (ie, metastatic) recurrence in the prostate cancer surgery cohort (n=536 patients; 13.6% events).

[0117] Fig.18 Kaplan Meier curve analysis of the postoperative prognostic risk score based on the TMPRSS2-ERG fusion status-dependent CAPRA-S score combined with an expression-based risk score based on PDE4D7 or PDE4D9 ("CAPRA-S & PDE4D7 & 9 score") is shown to predict clinical (i.e., metastatic) recurrence in the prostate cancer surgery cohort (n = 536 patients; 13.6% events).

[0118] Fig.19 Shown is a Kaplan Meier curve analysis of the CAPRA-S score to predict prostate cancer-specific death in the prostate cancer surgery cohort (n=536 patients; 5.2% events).

[0119] Fig. 20 Kaplan Meier curve analysis of the postoperative prognostic risk score based on the TMPRSS2-ERG fusion status-dependent CAPRA-S score combined with an expression-based risk score based on PDE4D7 or PDE4D9 ("CAPRA-S & PDE4D7 & 9 score") is shown to predict prostate cancer-specific death in the prostate cancer surgery cohort (n = 536 patients; 5.2% events).

[0120] Fig.21 Shown is a Kaplan Meier curve analysis of the CAPRA-S score to predict prostate cancer-specific death in the prostate cancer surgery cohort following salvage radiation therapy (SRT; n=173 patients; 14.5% events).

[0121] Fig. 22 Kaplan Meier curve analysis of the postoperative prognostic risk score based on the TMPRSS2-ERG fusion status-dependent CAPRA-S score combined with an expression-based risk score based on PDE4D7 or PDE4D9 ("CAPRA-S & PDE4D7 & 9 score") is shown to predict prostate cancer-specific death in the prostate cancer surgery cohort after salvage radiation therapy (SRT; n = 173 patients; 14.5% events).

[0122] Fig.23Shown is a Kaplan Meier curve analysis of the CAPRA-S score to predict prostate cancer-specific death in the prostate cancer surgery cohort after salvage androgen deprivation therapy (SADT; n=118 patients; 20.3% events).

[0123] Fig.24 Kaplan Meier curve analysis of the postoperative prognostic risk score based on the TMPRSS2-ERG fusion status-dependent CAPRA-S score combined with an expression-based risk score based on PDE4D7 or PDE4D9 ("CAPRA-S & PDE4D7 & 9 score") is shown to predict prostate cancer-specific death in the prostate cancer surgery cohort after salvage androgen deprivation therapy (SADT; n = 118 patients; 20.3% events).

[0124] Implementation Detail

[0125] A review of postoperative risk stratification

[0126] Figure 1 A flow chart schematically and exemplarily illustrates an embodiment of a method for post-operative risk stratification of prostate cancer subjects.

[0127] The method starts at step S100 .

[0128] In step S102, a biological sample is obtained from each patient of the first group of patients (objects) diagnosed with prostate cancer. Preferably, these prostate cancer patients are monitored for prostate cancer within a period of time after obtaining the biological sample, for example, at least one year, or at least two years, or about five years.

[0129] In step S104, a gene expression profile of PDE4D5, PDE4D7 and PDE4D9 is obtained for each biological sample obtained from the first group of patients, for example, by performing RT-qPCR (real-time quantitative PCR) on RNA extracted from each biological sample. The exemplary gene expression profile includes expression levels (e.g., values) of PDE4D5, PDE4D7 and PDE4D9, which can be standardized using a set of reference genes such as HPRT1, TUBA1B, PUM1 and / or TBP. In one embodiment, the gene expression profile values ​​of PDE4D5, PDE4D7 and PDE4D9 are standardized relative to one or more reference genes selected from HPRT1, TUBA1B, PUM1 and TBP, for example, at least one, or at least two, or at least three or preferably all of these reference genes. Further in step 104, the TMPRSS2-ERG fusion state is obtained for each biological sample obtained from the first group of patients, for example, by performing RT-qPCR.

[0130] In step S106, a scoring function for specifying an expression-based risk score is determined based on the gene expression profiles of PDE4D5, PDE4D7 and PDE4D9 obtained from at least some of the biological samples from the first group of patients, respectively, and the respective results obtained from the monitoring. In particular, to determine the scoring function based on the gene expression profile of PDE4D7, only the gene expression profiles from biological samples in which the TMPRSS2-ERG fusion state is positive are used. Conversely, to determine the scoring function based on the gene expression profiles of PDE4D5 and PDE4D9, respectively, only the gene expression profiles from biological samples in which the TMPRSS2-ERG fusion state is negative are used. In a preferred embodiment, the scoring function is a linear transformation that converts the standardized gene expression profile into a predetermined range of values, such as the above-mentioned range of 1-5. As described above, determining such a conversion can be performed by considering the frequency distribution of the standardized gene expression profile values ​​of PDE4D5, PDE4D7 and PDE4D9 of a group of biological samples of prostate cancer subjects (here, a TMPRSS2-ERG fusion status positive subset and a TMPRSS2-ERG fusion status negative subset of the first group of patients) and by determining a conversion that converts the frequency distribution to a desired range. In a specific embodiment, the expression-based risk score is determined as shown in equation (1) above.

[0131] In step S108, a biological sample is obtained from a patient (subject or individual). The patient may be a new patient or one of the first group of patients.

[0132] In step S110, the gene expression profiles of PDE4D5, PDE4D7 and PDE4D9 are obtained, respectively, for example by performing PCR on the biological sample. In one embodiment, the gene expression profile values ​​of PDE4D5, PDE4D7 and PDE4D9 are respectively normalized relative to one or more reference genes selected from HPRT1, TUBA1B, PUM1 and TBP, for example at least one, or at least two, or at least three, or preferably all of these reference genes. This is substantially the same as step S104. In addition, the TMPRSS2-ERG fusion state of the biological sample obtained from the patient is obtained, for example by performing RT-qPCR, as in step S104.

[0133] Other reference genes that may be used additionally or alternatively in steps S104 and S110 include: Homo sapiens actin β mRNA (ACTB); Homo sapiens 60S acidic ribosomal phosphoprotein P0 mRNA (RPLP0); polymerase (RNA) II (DNA-directed) polypeptide A, 220 kDa (POLR2A); beta-2-microglobulin (B2M); and aminolevulinate-δ synthase (ALAS-1).

[0134] In step S112, an expression-based risk score for the patient is determined. If the TMPRSS2-ERG fusion status of the biological sample obtained from the patient is positive, an expression-based risk score is determined based on the gene expression profile of PDE4D7 using a derived scoring function for PDE4D7. Conversely, if the TMPRSS2-ERG fusion status of the biological sample obtained from the patient is negative, an expression-based risk score is determined based on the gene expression profile of PDE4D5 or PDE4D9 using a derived scoring function for PDE4D5 or PDE4D9.

[0135] In step S114, a post-operative prognostic risk score of the patient is determined based on the patient's expression-based risk score and post-operative clinical variables. This is explained in more detail in the following description.

[0136] At S116, based on the postoperative prognostic risk score, a treatment recommendation can be provided, for example, to the patient or his guardian, a doctor, or another medical staff. To this end, the postoperative prognostic risk score can be classified into one of the predetermined risk groups based on the value of the postoperative prognostic risk score. Providing treatment recommendations may include one or more of the following: a) proposing a treatment plan for the patient based on the specified risk group, wherein at least two risk groups are associated with different treatments; b) calculating a postoperative disease recurrence or progression risk prediction for a patient after prostate surgery; and c) calculating a treatment response prediction for a patient after prostate surgery. Example therapies include at least radiotherapy, hormone therapy, chemotherapy, immunotherapy, or any combination thereof.

[0137] The method ends at S118.

[0138] Each risk group may be relevant to the therapy proposed separately because its aggressiveness is different.The therapy of every kind of proposal can be based on the result of the first group of patients assigned as risk group, and is predicted to provide the therapy of minimal aggressiveness, and it does not exceed the threshold clinical risk of prostate cancer development.In some cases, compared with the situation of other risk prediction methods such as postoperative CAPRA-S score, this makes new patient be assigned to the risk group relevant to the less aggressive proposed therapy.

[0139] In one embodiment, in steps S104 and S110, the gene expression profile is determined by detecting mRNA expression using one or more and / or one or more sets of primers and / or probes.

[0140] Table 1 shows a detailed description of PDE4D5, PDE4D7, PDE4D9 and one or more reference genes, including their transcript IDs (NCBI RefSeq) and the corresponding amino acid sequences of primer pairs and probes. The table also shows the sense primers and antisense primers for each gene and the probe sequences that specifically bind to the amplicon. Table 1 also includes the corresponding information for TMPRSS2-ERG.

[0141] Table 1: Exemplary primer and probe nucleic acid sequences

[0142]

[0143]

[0144] To investigate the prognostic power of PDE4D5, PDE4D7, and PDE4D9 in postoperative patient risk assessment in dependence on molecular subtypes of prostate cancer, we investigated the association of postoperative risk variables and algorithms such as postoperative CAPRA-S score with disease recurrence and progression. A combined model of expression-based risk score with postoperative variables was developed in the surgical cohort. The results suggest that the reliability and expressivity of the postoperative prognostic risk score can be improved by selecting the phosphodiesterase 4D variants used for the postoperative prognostic risk score according to TMPRSS2-ERG fusion status. Example

[0145] Patient cohorts and samples

[0146] A radical prostatectomy (RP) patient cohort was used, and its demographic data are shown in Table 2 . A small clamp biopsy sample (approximately 1 mm × 2 mm) of a representative tumor area was collected from the resected prostate from 656 consecutive patients who underwent surgery at a large clinical center in Germany between 2000 and 2004. Of the 656 patients, 600 were selected for RNA next-generation sequencing based on the RNA quality and concentration in the samples after nucleic acid extraction. After quality control of the RNAseq data, 575 patient samples were found to meet the requirements for statistical analysis.

[0147] Table 2: Demographics of the Radical Prostatectomy (RP) Patient Cohort

[0148]

[0149] Patient age, preoperative PSA, tumor percentage in biopsy, prostate volume and PSA density, minimum and maximum values ​​in the cohort, and median values ​​are shown in brackets in the table. For the CAPRA-S risk category, the number and percentage of patients in each risk group are shown in the table. In postoperative pathology, pathological Gleason score (pGS) and Gleason grade group, pathological stage, surgical margin status after prostatectomy, and tumor infiltration status of seminal vesicles and pelvic lymph nodes are shown (shown in the number and percentage of patients). In this regard, it should be noted that extracapsular extension is not provided as the main parameter, but is derived from the pathological stage pT3a. Follow-up shows the average and median follow-up period of all patients several months after surgery. The outcome category exemplifies the cumulative 5-year and 10-year biochemical recurrence (BCR) and clinical recurrence to metastasis (CR) after surgical primary treatment. The treatment category lists the cumulative 5-year and 10-year start of salvage radiotherapy (SRT) or salvage androgen deprivation therapy (SADT) after surgery. Mortality is shown as prostate cancer specific survival (PCSS) and overall survival (OS). For all outcomes, the number of men experiencing the outcome / total number of men at each 5 or 10 years of follow-up is shown, with the percentage of events given in parentheses.

[0150] Laboratory methods

[0151] All laboratory methods used, including oligonucleotide primers and probes for RT-qPCR (quantitative real-time PCR), RNA extraction, quality control, and procedures for including / discarding samples from statistical analyses, were described previously. Table 1 also lists the primers and probes used for RT-qPCR to measure the gene of interest and the reference gene.

[0152] result

[0153] Postoperative prognostic risk score based on expression-based risk score and postoperative clinical variables

[0154] Figure 2Kaplan Meier curve analysis of expression-based risk scores based on PDE4D7 measured for a cohort of TMDERS2-ERG fusion-negative ("TMRPRSS2-ER-") prostate cancer patients (n=258 patients; 43.8% events) is shown. The PDE4D7 score is divided into four groups: score (1-2): all PDE4D7 score values ​​are between 1 and <2; score (2-3): all PDE4D7 score values ​​are between 2 and <3; score (3-4): all PDE4D7 score values ​​are between 3 and <4; score (4-5): all PDE4D7 scores are between 4-5. The score categories in Kaplan Meier were associated with the patient's BCR (biochemical recurrence) progression-free survival time after surgery, expressed in months. The logrank test p value was determined to be p=0.1. This indicates that there was no significant difference in BCR progression-free survival among the four PDE4D7 score categories. The following supplementary table shows the number of patients at risk for each PDE4D7 score category, i.e., shows the patients at risk for each PDE4D7 category at any time interval +20 months after surgery: Score (1-2): 11, 7, 7, 6, 5, 5, 4, 1, 0, 0; Score (2-3): 110, 78, 64, 54, 49, 35, 25, 11, 3, 0; Score (3-4): 130, 101, 83, 76, 71, 62, 53, 25, 4, 0; Score (4-5): 7, 6, 4, 2, 2, 0, 0, 0, 0, 0.

[0155] Figure 3Kaplan Meier curve analysis of the expression-based risk score based on PDE4D7 measured in a cohort of TMPRRS2-ERG fusion-positive ("TMRPRSS2-ERG+") prostate cancer patients (n=281 patients; 38.1% events) is shown. The PDE4D7 score values ​​are divided into four groups: score (1-2): all PDE4D7 score values ​​are between 1 and <2; score (2-3): all PDE4D7 score values ​​are between 2 and <3; score (3-4): all PDE4D7 score values ​​are between 3 and <4; score (4-5): all PDE4D7 score values ​​are between 4-5. In Kaplan Meier, the score categories were associated with the patient's BCR (biochemical recurrence) progression-free survival time after surgery, expressed in months. The logrank test p value was determined to be p<0.0001. This indicates that there are significant differences in BCR progression-free survival across the four PDE4D7 score categories. The lower the PDE4D7 risk score, the higher the associated risk of experiencing disease recurrence after primary treatment. The following supplementary table shows the number of patients at risk for each PDE4D7 score category, i.e., shows the patients at risk for each PDE4D7 category at any time interval after surgery +20 months: score (1-2): 2 0, 0, 0, 0, 0, 0, 0, 0, 0; score (2-3): 37, 25, 15, 12, 10, 7, 7, 1, 0, 0; score (3-4): 187, 158, 130, 120, 108, 92, 73, 36, 6, 0; score (4-5): 55, 46, 44, 40, 36, 31, 28, 17, 4, 0.

[0156] Figure 4Kaplan Meier curve analysis of expression-based risk score based on PDE4D5 for a cohort of TMDERS2-ERG fusion-negative ("TMRPRSS2-ER-") prostate cancer patients (n=261 patients; 44.1% events) is shown. The PDE4D5 score is divided into four groups: score (1-2): all PDE4D5 score values ​​are between 1 and <2; score (2-3): all PDE4D5 score values ​​are between 2 and <3; score value (3-4): all PDE4D5 scores are between 3 and <4; score (4-5): all PDE4D5 scores are between 4-5. In Kaplan Meier, the score categories were associated with the patient's BCR (biochemical recurrence) progression-free survival time after surgery, expressed in months. The logrank test p value was determined to be p<0.0001. This indicates that there are significant differences in BCR progression-free survival rates among the four PDE4D5 score categories. The lower the PDE4D5 risk score, the higher the associated risk of experiencing disease recurrence after primary treatment. The following supplementary table shows the number of patients at risk for each PDE4D5 score category, i.e., shows patients at risk for each PDE4D5 category at any time interval after surgery +20 months: score (1-2): 9, 3, 3, 2, 1, 1, 1, 1, 0, 0; score (2-3): 51, 29, 23, 19, 17, 14, 11, 3, 0, 0; score (3-4): 167, 131, 108, 94, 89, 73, 61, 29, 6, 0; score (4-5): 34, 31, 26, 25, 21, 15, 10, 5, 1, 0.

[0157] Figure 5Kaplan Meier curve analysis of the expression-based risk score based on PDE4D5 measured in a cohort of TMPRRS2-ERG fusion-positive ("TMRPRSS2-ERG+") prostate cancer patients (n=282 patients; 38.3% events) is shown. The PDE4D5 score is divided into four groups: score (1-2): all PDE4D5 scores have values ​​between 1 and <2; score (2-3): all PDE4D5 scores have values ​​between 2 and <3; score (3-4): all PDE4D5 scores have values ​​between 3 and <4; score (4-5): all PDE4D5 scores are between 4-5. In Kaplan Meier, the score categories were associated with the patient's BCR (biochemical recurrence) progression-free survival time after surgery, expressed in months. The logrank test p value was determined to be p<0.39. This indicates that there was no significant difference in BCR progression-free survival across the four PDE4D5 score categories. The following supplementary table shows the number of patients at risk in each PDE4D5 score category, that is, the number of patients at risk in each PDE4D5 category at any time interval +20 months after surgery: Score (1-2): 5, 4, 2, 2, 2, 2, 1, 1, 0, 0; Score (2-3): 92, 71, 56, 52, 46, 38, 33, 17, 3, 0; Score (3-4): 171, 141, 121, 108, 96, 80, 66, 31, 6, 0; Score (4-5): 14, 13, 10, 10, 10, 10, 8, 5, 1, 1, 0.

[0158] Figure 6Kaplan Meier curve analysis of expression-based risk scores based on PDE4D9 measured in a cohort of TMDERS2-ERG fusion-negative ("TMRPRSS2-ER-") prostate cancer patients (n=262 patients; 43.9% events) is shown. The PDE4D9 score is divided into four groups: score (1-2): all PDE4D9 scores are between 1 and <2; score (2-3): all PDE4D9 scores are between 2 and <3; score (3-4): all PDE4D9 scores are between 3 and <4; score (4-5): all PDE4D9 scores are between 4-5. The score categories in Kaplan Meier were associated with the patient's BCR (biochemical recurrence) progression-free survival time after surgery, expressed in months. The logrank test p value was determined to be p<0.0001. This indicates that there are significant differences in BCR progression-free survival rates for the four PDE4D9 score categories. The lower the PDE4D9 risk score, the higher the associated risk of experiencing disease recurrence after primary treatment. The following supplementary table shows the number of patients at risk for each PDE4D9 score category, that is, the number of patients at risk for each PDE4D9 category at any time interval +20 months after surgery: Score (1-2): 6, 0, 0, 0, 0, 0, 0, 0, 0; Score (2-3): 59, 37, 33, 31, 29, 24, 19, 9, 1, 0; Score (3-4): 194, 145, 117, 101, 91, 73, 62, 26, 6, 0; Score (4-5): 13, 12, 10, 8, 8, 6, 2, 2, 0, 0.

[0159] Figure 7Kaplan Meier curve analysis of expression-based risk scores based on PDE4D9 measured in a cohort of TMPRRS2-ERG fusion-positive ("TMRPRSS2-ERG+") prostate cancer patients (n=282 patients; 38.3% events) is shown. The PDE4D9 score is divided into four groups: score (1-2): all PDE4D9 scores are between 1 and <2; score (2-3): all PDE4D9 scores are between 2 and <3; score (3-4): all PDE4D9 scores are between 3 and <4; score (4-5): all PDE4D9 scores are between 4-5. The score categories in Kaplan Meier were associated with the patient's BCR (biochemical recurrence) progression-free survival time after surgery, expressed in months. The logrank test p value was determined to be p<0.01. This shows limited significant differences in BCR progression-free survival across the four PDE4D9 score categories. The following supplementary table shows the number of patients at risk for each PDE4D9 score category, i.e., the number of patients at risk for each PDE4D9 category at any time interval +20 months after surgery: Score (1-2): 3, 1, 0, 0, 0, 0, 0, 0, 0; Score (2-3): 119, 88, 70, 63, 56, 44, 37, 21, 2, 0; Score (3-4): 151, 131, 111, 102, 91, 80, 66, 29, 8, 0; Score (4-5): 9, 9, 8, 7, 7, 6, 5, 4, 0, 0.

[0160] Figure 2-Figure 7 The Kaplan Meier curve analysis shown in clearly shows that the prognostic power of different phosphodiesterase 4D variants depends on the molecular subtype of prostate cancer, in particular, on whether the TMPRSS2-ERG fusion status of the prostate cancer is positive or negative. In TMPRSS2-ERG fusion status-positive prostate cancer, PDE4D7 was found to provide good prognostic power ( Figure 3 ), but not in TMPRSS2-ERG fusion-negative prostate cancer ( Figure 2 In contrast, in TMPRSS2-ERG fusion-negative prostate cancer, both PDE4D5 and PDE4D9 were found to have suitable prognostic abilities ( Figure 4 and 6 ), but the same prognostic ability was not found in prostate cancer with negative TMPRSS2-ERG fusion status ( Figure 5 and 7 ).

[0161] Based on these findings, a combination of the CAPRA-S score and the PDE4D7 and PDE4D9 scores that depend on the TMPRSS2-ERG fusion status was developed, as shown in Table 3, to generate a total TMPRSS2-ERG_CAPRA-S&PDE4D7&PDE4D9 postoperative prognostic score (hereinafter also referred to as "CAPRA-S&PDE4D7&9 score" or "total score" for brevity). The CAPRA-S score (see Cooperberg MR et al., column 1 in Table 3) is divided into three risk groups (low risk, intermediate risk and high risk; column 2 in Table 3). Each risk group is associated with an increased score (low risk = 1 point; intermediate risk = 2 points; high risk = 3 points). The PDE4D7 score is divided into four groups (PDE4D7 score category 1 = PDE4D7 score 1 to <2; PDE4D7 score category 2 = PDE4D7 score 2 to <3; PDE4D7 score category 3 = PDE4D7 score 3 to <4; PDE4D7 score category 4 = PDE4D7 score 4-5). Since PDE4D7 is inversely proportional to the risk of progression, each PDE4D7 score category is associated with a reduction in score (PDE4D7 score category 1: 3 points; PDE4D7 score category 2: 2 points; PDE4D7 score category 3: 1 point; PDE4D7 score category 4: 0 points; Table 3, column 3). The combined score CAPRA-S & PDE4D7 (Table 3, column 4) generated for samples with positive TMPRSS2-ERG fusion events is the sum of the points given in columns 2 (CAPRA-S category) and 3 (PDE4D7 score category) of Table 3. To generate the CAPRA-S&PDE4D9 combined score, the same steps as for PDE4D7 were performed, except that this score was calculated based on PDE4D9 for samples that were negative for TMPRSS2-ERG fusion events (Table 3 columns 5 and 6). An overall TMPRSS2-ERG_CAPRA-S&PDE4D7&PDE4D9 score for all samples was constructed by adding the CAPRA-S&PDE4D7 and CAPRA-S&PDE4D9 scores into a single score, whether positive or negative for TMPRSS2-ERG fusion, where each sample was associated with a score between 1-6 (Table 3 column 7).

[0162] Table 3: Combination of CAPRA-S scores with PDE4D7 and PDE4D9 scores depending on TMPRSS2-ERG fusion status (SC = scoring category).

[0163]

[0164]

[0165] Figure 8ROC curve analysis of the postoperative prognostic risk score based on the combination of the TMPRSS2-ERG fusion status-dependent CAPRA-S score and the expression-based risk score based on PDE4D7 or PDE4D9 ("CAPRA-S & PDE4D7 & 9 score") relative to the CAPRA-S category is shown to predict biochemical recurrence (BCR) 5 years after surgery in the RP cohort with a complete follow-up of 5 years (radical prostatectomy; n = 482 patients; 35.5% BCR events within 5 years after treatment). The respective AUCs (areas under the curve) and p-values ​​for statistical differences between the ROC curves were determined to be 0.79 and 0.78 and p = 0.64, respectively.

[0166] Fig. 9 ROC curve analysis of the postoperative prognostic risk score based on the TMPRSS2-ERG fusion status-dependent CAPRA-S score and the expression-based risk score combination based on PDE4D7 or PDE4D9 ("CAPRA-S & PDE4D7 & 9 score") relative to the CAPRA-S category is shown to predict clinical (i.e., metastatic) recurrence (CR) 10 years after surgery in RP with a complete 10-year follow-up (radical prostatectomy; n = 337 patients; 18.1% BCR events within 10 years after treatment). The respective AUCs (areas under the curve) and p-values ​​for statistical differences between the ROC curves were determined to be 0.85 and 0.83 and p = 0.13, respectively.

[0167] Fig.10 ROC curve analysis of the postoperative prognostic risk score based on the TMPRSS2-ERG fusion status-dependent CAPRA-S score and the expression-based risk score combination based on PDE4D7 or PDE4D9 ("CAPRA-S & PDE4D7 & 9 score") relative to the CAPRA-S category is shown to predict 10-year postoperative prostate cancer-specific survival (PCSS) in the RP cohort with 10 years of complete follow-up (radical prostatectomy; n = 304 patients; 8.2% prostate cancer death events within 10 years after treatment). The respective AUCs (areas under the curve) and p-values ​​for statistical differences between the ROC curves were determined to be 0.91 and 0.85 and p = 0.014, respectively.

[0168] Fig.11ROC curve analysis of the postoperative prognostic risk score based on the TMPRSS2-ERG fusion status-dependent CAPRA-S score and the expression-based risk score combination based on PDE4D7 or PDE4D9 ("CAPRA-S & PDE4D7 & 9 score") relative to the CAPRA-S category is shown to predict clinical (i.e., metastatic) recurrence (CR) 10 years after surgery in the RP subcohort with 10 years of complete follow-up (radical prostatectomy; pGleason>6; n = 231 patients; 26.4% BCR events within 10 years after treatment). The respective AUCs (areas under the curve) and p values ​​for the statistical differences between the ROC curves were determined to be 0.8 and 0.76 and p = 0.06, respectively.

[0169] Fig.12 ROC curve analysis of the postoperative prognostic risk score based on the TMPRSS2-ERG fusion status-dependent CAPRA-S score and the expression-based risk score combination based on PDE4D7 or PDE4D9 ("CAPRA-S & PDE4D7 & 9 score") relative to the CAPRA-S category is shown to predict 10-year prostate cancer-specific survival (PCSS) after surgery in the RP subcohort with 10-year complete follow-up (radical prostatectomy; pGleason>6; n = 198 patients; 12.6% prostate cancer death events within 10 years after treatment). The respective AUCs (areas under the curve) and p-values ​​for statistical differences between the ROC curves were determined to be 0.87 and 0.79 and p = 0.013, respectively.

[0170] Fig.13 ROC curve analysis of the postoperative prognostic risk score based on the TMPRSS2-ERG fusion status-dependent CAPRA-S score and the expression-based risk score combination based on PDE4D7 or PDE4D9 ("CAPRA-S & PDE4D7 & 9 score") relative to the CAPRA-S category is shown to predict 10-year postoperative prostate cancer-specific survival (PCSS) in the RP subcohort with 10-year complete follow-up (radical prostatectomy; SRT = salvage radiation therapy; n = 93 patients; 23.7% prostate cancer death events within 10 years after treatment). The respective AUCs (areas under the curve) and p-values ​​for the statistical differences between the ROC curves were determined to be 0.81 and 0.72 and p = 0.03, respectively.

[0171] Fig.14ROC curve analysis of the postoperative prognostic risk score based on the TMPRSS2-ERG fusion status-dependent CAPRA-S score and the expression-based risk score combination based on PDE4D7 or PDE4D9 ("CAPRA-S & PDE4D7 & 9 score") relative to the CAPRA-S category is shown to predict 10-year postoperative prostate cancer-specific survival (PCSS) in the RP subcohort with 10-year complete follow-up (radical prostatectomy; SADT = salvage androgen deprivation therapy; n = 68 patients; 30.9% prostate cancer death events within 10 years after treatment). The respective AUCs (areas under the curve) and p-values ​​for statistical differences between the ROC curves were determined to be 0.81 and 0.72 and p = 0.05, respectively.

[0172] Figure 8-14 The ROC curve analysis shown shows that for all analyzed cases, the AUC of the postoperative prognostic risk score based on the combination of the TMPRSS2-ERG fusion status-dependent CAPRA-S score and the expression-based risk score based on PDE4D7 or PDE4D9 (CAPRA-S&PDE4D7&9 score) is greater than the CAPRA-S category. In certain cases, for example, predicting 10-year prostate cancer-specific survival (PCSS) after surgery ( Fig.10 ), predicting prostate cancer-specific survival (PCSS) 10 years after surgery ( Fig.12 ), predicting prostate cancer-specific survival (PCSS) 10 years after surgery ( Fig.13 ) and predicting 10-year prostate cancer-specific survival (PCSS) after surgery ( Fig.14 ), this difference was statistically significant (significance was defined as alpha < 0.05).

[0173] Fig.15Kaplan Meier curve analysis of CAPRA-S score is shown to predict biochemical recurrence (BCR) in the prostate cancer surgery cohort (n = 536 patients; 40.9% events). The CAPRA-S score is divided into two groups: low risk and intermediate risk (CAPRA-S score category (1-2)) and high risk (CAPRA-S score category (3)). CAPRA-S score category (1-2) and score category (3) are associated with the patient's BCR (biochemical recurrence) progression-free survival time after surgery in Kaplan Meier, expressed in months. The logrank test p value was determined to be p < 0.0001, and the hazard ratio was determined to be HR = 5.0. The following supplementary list shows the number of patients at risk for the analyzed CAPRA-S score categories, i.e., shows patients at risk at any time interval +20 months after surgery: CAPRA-S score category (1-2): 461, 385, 327, 293, 224, 182, 86, 17, 0; CAPRA-S score category (3): 75, 34, 19, 16, 11, 7, 7, 4, 0, 0.

[0174] Fig.16 Kaplan Meier curve analysis of the postoperative prognostic risk score based on the combination of the TMPRSS2-ERG fusion status-dependent CAPRA-S score and the expression-based risk score based on PDE4D7 or PDE4D9 ("CAPRA-S & PDE4D7 & 9 score") is shown to predict biochemical recurrence (BCR) in the prostate cancer surgery cohort (n = 536 patients; 40.9% events). The CAPRA-S & PDE4D7 & 9 score is divided into two groups: score (1-3) and score (4-6). The CAPRA-S & PDE4D7 & 9 score category (1-3) and the score category (4-6) are associated with the patient's BCR (biochemical recurrence) progression-free survival time after surgery in Kaplan Meier, expressed in months. The logrank test p value was determined to be p < 0.0001, and the hazard ratio was determined to be HR = 4.8. The following supplementary list shows the number of patients at risk for the analyzed CAPRA-S&PDE4D7&9 score categories, i.e., shows patients at risk at any time interval +20 months after surgery: Score category (1-3): 422, 363, 313, 280, 256, 214, 173, 84, 17, 0; Score category (4-6): 114, 56, 33, 29, 24, 17, 16, 6, 0, 0.

[0175] Fig.17Kaplan Meier curve analysis of CAPRA-S score is shown to predict clinical (i.e., metastatic) recurrence in a prostate cancer surgery cohort (n=536 patients; 13.6% events). The CAPRA-S score is divided into two groups: low risk and intermediate risk (CAPRA-S score category (1-2)) versus high risk (CAPRA-S score category (3)). CAPRA-S score category (1-2) versus score category (3) were associated with the patient's CR (clinical recurrence) progression-free survival time after surgery in Kaplan Meier, expressed in months. The logrank test p value was determined to be p<0.0001, and the hazard ratio was determined to be HR=6.9. The following supplementary list shows the number of patients at risk for the analyzed CAPRA-S score categories, i.e., shows patients at risk at any time interval + 20 months after surgery: CAPRA-S score category (1-2): 460, 419, 401, 380, 352, 299, 248, 123, 19, 0; CAPRA-S score category (3): 74, 61, 50, 45, 37, 27, 25, 9, 1, 0.

[0176] Fig.18 Kaplan Meier curve analysis of the postoperative prognostic risk score based on the combination of the TMPRSS2-ERG fusion status-dependent CAPRA-S score and the expression-based risk score based on PDE4D7 or PDE4D9 ("CAPRA-S & PDE4D7 & 9 score") is shown to predict clinical (i.e., metastatic) recurrence in the prostate cancer surgery cohort (n = 536 patients; 13.6% events). The CAPRA-S & PDE4D7 & 9 score was divided into two groups: score (1-3) and score (4-6). The CAPRA-S & PDE4D7 & 9 score category (1-3) was associated with the patient's CR (clinical recurrence) progression-free survival time after surgery in the Kaplan Meier compared with the score category (4-6), expressed in months. The logrank test p value was determined to be p < 0.0001, and the hazard ratio was determined to be HR = 9.5. The following supplementary list shows the number of patients at risk for the CAPRA-S&PDE4D7&9 score categories, i.e., shows patients at risk at any time interval +20 months after surgery: Score categories (1-3): 422, 393, 375, 355, 331, 285, 234, 116, 18, 0; Score categories (4-6): 112, 87, 76, 70, 58, 41, 39, 16, 2, 0.

[0177] Fig.19Kaplan Meier curve analysis of CAPRA-S score is shown to predict prostate cancer specific death in the prostate cancer surgery cohort (n = 536 patients; 5.2% events). The CAPRA-S score is divided into two groups: low risk and intermediate risk (CAPRA-S score category (1-2)) and high risk (CAPRA-S score category (3)). CAPRA-S score category (1-2) and score category (3) are associated with the patient's PCSS (prostate cancer specific survival) progression-free survival time after surgery in Kaplan Meier, expressed in months. The logrank test p value was determined to be p < 0.0001, and the hazard ratio was determined to be HR = 11.8. The following supplementary list shows the number of patients at risk for the analyzed CAPRA-S score categories, i.e., shows patients at risk at any time interval +20 months after surgery: CAPRA-S score category (1-2): 461, 429, 408, 386, 357, 303, 251, 130, 22, 4; CAPRA-S score category (3): 75, 71, 61, 53, 42, 34, 25, 11, 1, 0.

[0178] Fig. 20 Kaplan Meier curve analysis of the postoperative prognostic risk score based on the TMPRSS2-ERG fusion status-dependent CAPRA-S score and the expression-based risk score combination based on PDE4D7 or PDE4D9 ("CAPRA-S & PDE4D7 & 9 score") is shown to predict prostate cancer-specific death in the prostate cancer surgery cohort (n = 536 patients; 5.2% events). The CAPRA-S & PDE4D7 & 9 score was divided into two groups: score (1-3) and score (4-6). The CAPRA-S & PDE4D7 & 9 score category (1-3) was associated with the patient's PCSS (prostate cancer-specific survival) progression-free survival time after surgery in Kaplan Meier, expressed in months. The logrank test p value was determined to be p < 0.0001, and the hazard ratio was determined to be HR = 22.0. The following supplementary list shows the number of patients at risk for the analyzed CAPRA-S&PDE4D7&9 score categories, i.e., shows patients at risk at any time interval +20 months after surgery: Score category (1-3): 422, 395, 378, 359, 334, 288, 237, 123, 20, 3; Score category (4-6): 114, 105, 91, 80, 65, 49, 39, 18, 3, 1.

[0179] Fig.21Kaplan Meier curve analysis of CAPRA-S score is shown to predict prostate cancer specific death in the prostate cancer surgery cohort after salvage radiation therapy (SRT; n = 173 patients; 14.5% events). The CAPRA-S score is divided into two groups: low risk and intermediate risk (CAPRA-S score category (1-2)) vs. high risk (CAPRA-S score category (3)). CAPRA-S score category (1-2) vs. score category (3) were associated with the PCSS (prostate cancer specific survival) progression-free survival time of patients after SRT in Kaplan Meier, expressed in months. The logrank test p value was determined to be p < 0.0001, and the hazard ratio was determined to be HR = 3.8. The following supplementary list shows the number of patients at risk for the analyzed CAPRA-S score categories, i.e., shows patients at risk at any time interval +20 months after surgery: CAPRA-S score category (1-2): 115, 112, 106, 96, 81, 61, 53, 29, 6, 4; CAPRA-S score category (3): 58, 54, 47, 37, 27, 18, 15, 6, 1, 0.

[0180] Fig. 22 Kaplan Meier curve analysis of the postoperative prognostic risk score based on the TMPRSS2-ERG fusion status-dependent CAPRA-S score and the expression-based risk score combination based on PDE4D7 or PDE4D9 ("CAPRA-S & PDE4D7 & 9 score") is shown to predict prostate cancer-specific death in the prostate cancer surgery cohort after salvage radiotherapy (SRT; n = 173 patients; 14.5% events). The CAPRA-S & PDE4D7 & 9 score was divided into two groups: score (1-3) and score (4-6). The CAPRA-S & PDE4D7 & 9 score category (1-3) was associated with the patient's PCSS (prostate cancer specific survival) progression-free survival time after surgery in Kaplan Meier compared with the score category (4-6), expressed in months. The logrank test p value was determined to be p < 0.0001, and the hazard ratio was determined to be HR = 6.4. The following supplementary list shows the number of patients at risk for the analyzed CAPRA-S&PDE4D7&9 score categories, that is, the number of patients at risk in any time interval +20 months after surgery: Score category (1-3): 93, 93, 89, 82, 70, 56, 49, 25, 4, 3; Score category (4-6): 80, 73, 64, 51, 38, 23, 19, 10, 3, 1.

[0181] Fig.23Kaplan Meier curve analysis of CAPRA-S score is shown to predict prostate cancer specific death in the prostate cancer surgery cohort after salvage androgen deprivation therapy (SADT; n = 118 patients; 20.3% events). The CAPRA-S score is divided into two groups: low risk and intermediate risk (CAPRA-S score category (1-2)) vs. high risk (CAPRA-S score category (3)). CAPRA-S score category (1-2) vs. score category (3) were associated with the patient's PCSS (prostate cancer specific survival) progression-free survival time after SADT in Kaplan Meier, expressed in months. The logrank test p value was determined to be p = 0.03, and the hazard ratio was determined to be HR = 2.4. The following supplementary list shows the number of patients at risk for the analyzed CAPRA-S score categories, i.e., shows patients at risk at any time interval +20 months after surgery: CAPRA-S score category (1-2): 68, 65, 62, 56, 49, 37, 32, 24, 5, 4; CAPRA-S score category (3): 50, 46, 40, 31, 21, 13, 11, 6, 1, 0.

[0182] Fig.24 Kaplan Meier curve analysis of the postoperative prognostic risk score based on the combination of the TMPRSS2-ERG fusion status-dependent CAPRA-S score and the expression-based risk score based on PDE4D7 or PDE4D9 ("CAPRA-S & PDE4D7 & 9 score") is shown to predict prostate cancer-specific death (SADT; n = 118 patients; 20.3% events) in the prostate cancer surgery cohort after salvage androgen deprivation therapy. The CAPRA-S & PDE4D7 & 9 score was divided into two groups: score (1-3) and score (4-6). The CAPRA-S & PDE4D7 & 9 score category (1-3) was associated with the patient's PCSS (prostate cancer-specific survival) progression-free survival time after SADT in Kaplan Meier compared with the score category (4-6), expressed in months. The logrank test p value was determined to be p = 0.0007, and the hazard ratio was determined to be HR = 5.2. The following supplementary list shows the number of patients at risk for the analyzed CAPRA-S&PDE4D7&9 score categories, i.e., shows patients at risk at any time interval +20 months after surgery: Score category (1-3): 51, 51, 49, 45, 40, 32, 28, 20, 4, 3; Score category (4-6): 67, 60, 53, 42, 30, 18, 15, 10, 2, 1.

[0183] Figure 15-20It was shown that when the CAPRA-S score categories were compared with the CAPRA-S&PDE4D7&9 score categories, the hazard ratios were increased in the groups of patients with lower and higher risks of progression to the various tested clinical endpoints (BCR, metastasis, prostate cancer death). Figure 21-24 Show that the increased hazard ratio is equivalent to Fig.19 and 20 , but now for men receiving salvage radiation therapy (SRT) and / or salvage androgen deprivation therapy (SADT).

[0184] Although on Figure 8-24 The analysis shown provides an example in which the post-surgical prognostic risk score is determined based on a combination of a TMPRSS2-ERG fusion status-dependent CAPRA-S score and an expression-based risk score based on PDE4D7 or PDE4D9 ("CAPRA-S & PDE4D7 & 9 score"), but comparable results can be obtained if the post-surgical prognostic risk score is determined based on a combination of a TMPRSS2-ERG fusion status-dependent CAPRA-S score and an expression-based risk score based on PDE4D7 or PDE4D5 ("CAPRA-S & PDE4D7 & 5 score"), i.e., if in Table 3, if the TMPRSS2-ERG fusion status is negative, the CAPRA-S & PDE4D5 combined score based on the CAPRA-S score and PDE4D5 is used instead of the CAPRA-S & PDE4D9 combined score.

[0185] While TMPRSS2-ERG fusion status can be determined in a biological sample obtained from a subject, for example by performing PCR on the biological sample, this information can also be obtained from sequencing data, for example using algorithms such as RNA-Seq.

[0186] Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims.

[0187] In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality.

[0188] Figure 1One or more steps of the method shown may be implemented in a computer program product that can be executed on a computer. The computer program product may include a non-transitory computer-readable recording medium, such as a disk, a hard disk drive, etc., on which a control program is recorded (stored). Common forms of non-transitory computer-readable media include, for example, a floppy disk, a flexible disk, a hard disk, a magnetic tape, or any other magnetic storage medium, a CD-ROM, a DVD, or any other optical medium, a RAM, a PROM, an EPROM, a FLASH-EPROM, or other memory chip or a cassette, or any other non-transitory medium from which a computer can read and use.

[0189] Alternatively, one or more steps of the method may be implemented in a transient medium such as a transmissible carrier wave, wherein the control program is embodied as a data signal using a transmission medium such as acoustic or light waves, such as those generated during radio wave and infrared data communications.

[0190] The exemplary methods can be implemented on one or more general purpose computers, special purpose computers, programmed microprocessors or microcontrollers and peripheral integrated circuit components, ASICs or other integrated circuits, digital signal processors, hard-wired electronic or logic circuits such as discrete component circuits, programmable logic devices such as PLDs, PLAs, FPGAs, graphics card CPUs (GPUs) or PALs, etc. In general, a finite state machine can be executed, and thus a finite state machine can be executed. Figure 1 Any device of the flow chart shown can be used to perform one or more steps of the risk stratification method to illustrate the treatment regimen selection in prostate cancer patients. As will be appreciated, although the steps of the method can be all performed by a computer, in some embodiments one or more steps can be at least partially performed manually.

[0191] Computer program instructions may also be loaded onto a computer, other programmable data processing apparatus, or other device, so that a series of operational steps are performed on the computer, other programmable device, or other device to produce a computer-implemented process, such that the instructions executed on the computer or other programmable device provide a process for implementing the functions / actions specified herein.

[0192] Any reference signs in the claims should not be construed as limiting the scope.

[0193] The present invention relates to a method for postoperative risk stratification of a prostate cancer subject, comprising determining a transmembrane protease serine 2-ETS-related gene (TMPRSS2-ERG) fusion status in a biological sample obtained from the subject, determining a gene expression profile of each of one or more phosphodiesterase 4D variants in a biological sample obtained from the subject, determining an expression-based risk score for the subject based on the gene expression profile of the selected phosphodiesterase 4D variants, and determining a postoperative prognostic risk score for the subject based on the expression-based risk score and postoperative clinical variables of the subject, wherein the selected phosphodiesterase 4D variant is selected based on the TMPRSS2-ERG fusion status, wherein if the TMPRSS2-ERG fusion status is positive, the selected phosphodiesterase 4D variant is selected as a first phosphodiesterase 4D variant, and if the TMPRSS2-ERG fusion status is negative, the selected phosphodiesterase 4D variant is selected as a second phosphodiesterase 4D variant. This can allow for improved stratification of subjects in the postoperative setting, which can lead to better postoperative secondary treatment decisions. For example, a postoperative prognostic risk score could allow for better recommendations regarding whether to select specific postoperative secondary treatments for certain subgroups of prostate cancer patients.

[0194] The attached sequence listing entitled 2017PF02537_Sequence Listing_ST25 is incorporated herein by reference in its entirety.

Claims

1. Use of primers and / or probes in the preparation of a kit for a method for postoperative risk stratification of a prostate cancer subject as follows, wherein the primers and / or probes comprise primers and / or probes for determining the fusion state of a transmembrane protease serine 2-ETS-related gene (TMPRSS2-ERG) in a biological sample derived from the prostate of a prostate cancer subject, and primers and / or probes for determining phosphodiesterase 4D variant 5 (PDE4D5), phosphodiesterase 4D variant 7 (PDE4D7) and phosphodiesterase 4D variant 9 (PDE4D9) in a biological sample derived from the prostate of the subject, and wherein the kit comprises a computer program product storing instructions for calculating a postoperative prognostic risk score based on a gene expression profile of a selected phosphodiesterase 4D variant of the subject and a postoperative prostate cancer risk assessment (CAPRA-S) score, The method comprises: - determining the fusion status of the transmembrane protease, serine 2-ETS related gene (TMPRSS2-ERG) in a biological sample derived from the prostate of said subject, - determining the gene expression profile of each of phosphodiesterase 4D variant 5 (PDE4D5), phosphodiesterase 4D variant 7 (PDE4D7) and phosphodiesterase 4D variant 9 (PDE4D9) in a biological sample derived from the prostate of said subject, - determining an expression-based risk score for said subject based on the gene expression profile of the selected phosphodiesterase 4D variant, and - determining a post-operative prognostic risk score for the subject based on the subject's expression-based risk score and the CAPRA-S score, wherein the selected phosphodiesterase 4D variant is selected based on TMPRSS2-ERG fusion status, wherein if the TMPRSS2-ERG fusion status is positive, the selected phosphodiesterase 4D variant is selected as phosphodiesterase 4D variant 7, and if the TMPRSS2-ERG fusion status is negative, the selected phosphodiesterase 4D variant is selected as a second phosphodiesterase 4D variant 5 (PDE4D5) or phosphodiesterase 4D variant 9 (PDE4D9), and wherein the expression-based risk score is determined as follows: EBRS=(((PDE4D_norm+A)*B)+1), Wherein "EBRS" is an expression-based risk score, "PDE4D_norm" is a normalized phosphodiesterase 4D variant gene expression profile value, and A and B are variables, wherein when the phosphodiesterase 4D variant is PDE4D7, A is 6-8 and B is 0.4-0.45, wherein when the phosphodiesterase 4D variant is PDE4D5, A is 4-6 and B is 0.5-0.6, wherein when the phosphodiesterase 4D variant is PDE4D9, A is 3-5 and B is 0.5-0.

6.

2. The use of claim 1, wherein the CAPRA-S score is categorized, wherein a number of points in the range of 1-3 are added to the postoperative prognostic risk score according to the category.

3. The use of claim 1, wherein the expression-based risk score is a value in the range of 1-5, wherein a number of points in the range of 0-3 are added to the postoperative prognosis risk score according to the value, wherein if the expression-based risk score value is in the range of 1 to <2, 3 points are added to the postoperative prognosis risk score, and if the expression-based risk score value is in the range of 2 to <3, 2 points are added to the postoperative prognosis risk score, if the expression-based risk score value is in the range of 3 to <4, 1 point is added to the postoperative prognosis risk score, and if the expression-based risk score value is in the range of 4 to <5, no point is added.

4. The method of claim 1, wherein the method further comprises: - based on the post-operative prognostic risk score, recommending post-operative secondary treatment for the subject, Wherein, the secondary treatment after surgery is selected from: radiotherapy, hormone therapy, chemotherapy, immunotherapy or any combination thereof.

5. The method of claim 1, wherein the method further comprises: - normalizing the gene expression profile of the selected phosphodiesterase 4D variant to one or more reference genes selected from the group consisting of: Homo sapiens hypoxanthine phosphoribosyltransferase 1 (HPRT1), tubulin - α-1b (TUBA1B), Homo sapiens pumilio RNA-binding family member (PUM1) and Homo sapiens TATA box binding protein (TBP), Wherein the expression-based risk score is determined based on a normalized gene expression profile.

6. The use of claim 5, wherein the reference genes include at least two or at least three or all of HPRT1, TUBA1B, PUM1 and TBP.

7. The use of claim 5, wherein determining the gene expression profile of the selected phosphodiesterase 4D variant comprises performing RT-qPCR on RNA extracted from the biological sample, wherein a Cq value is determined for the selected phosphodiesterase 4D variant and for each of one or more reference genes, and wherein determining the expression-based risk score comprises normalizing the Cq value of the selected phosphodiesterase 4D variant using the Cq value of each of the one or more reference genes, and calculating the expression-based risk score as a linear function of the normalized Cq values.

8. A diagnostic kit comprising: at least one primer and / or probe for determining the fusion status of a transmembrane protease, serine 2-ETS related gene (TMPRSS2-ERG) in a biological sample derived from a prostate gland of a subject with prostate cancer, primers and / or probes for determining the gene expression profile of each of phosphodiesterase 4D variant 5 (PDE4D5), phosphodiesterase 4D variant 7 (PDE4D7), and phosphodiesterase 4D variant 9 (PDE4D9) in a biological sample derived from a prostate gland of the subject; and Instructions for calculating a post-surgery prognostic risk score based on the gene expression profile of the subject's selected phosphodiesterase 4D variant and a Cancer of the Prostate Risk Assessment After Surgery (CAPRA-S) score, the instructions being stored on a computer program product that, when executed by a computer, performs a method comprising: - Determine TMPRSS2-ERG fusion status, - determining an expression-based risk score for said subject based on the gene expression profile of said selected phosphodiesterase 4D variant, and - determining a post-operative prognostic risk score for the subject based on the subject's expression-based risk score and the CAPRA-S score, wherein the selected phosphodiesterase 4D variant is selected based on TMPRSS2-ERG fusion status, wherein if the TMPRSS2-ERG fusion status is positive, the selected phosphodiesterase 4D variant is selected as phosphodiesterase 4D variant 7 (PDE4D7), and if the TMPRSS2-ERG fusion status is negative, the selected phosphodiesterase 4D variant is selected as phosphodiesterase 4D variant 5 (PDE4D5) or phosphodiesterase 4D variant 9 (PDE4D9), and wherein the expression-based risk score is determined as follows: EBRS=(((PDE4D_norm+A)*B)+1), Wherein "EBRS" is an expression-based risk score, "PDE4D_norm" is a normalized phosphodiesterase 4D variant gene expression profile value, and A and B are variables, wherein when the phosphodiesterase 4D variant is PDE4D7, A is 6-8 and B is 0.4-0.45, wherein when the phosphodiesterase 4D variant is PDE4D5, A is 4-6 and B is 0.5-0.6, wherein when the phosphodiesterase 4D variant is PDE4D9, A is 3-5 and B is 0.5-0.

6.

9. A computer program product comprising instructions which, when executed by a computer, cause the computer to perform a method comprising: - determining the transmembrane protease, serine 2-ETS related gene (TMPRSS2-ERG) fusion status in a biological sample derived from the prostate of a subject with prostate cancer, - determining the gene expression profile of each of phosphodiesterase 4D variant 5 (PDE4D5), phosphodiesterase 4D variant 7 (PDE4D7) and phosphodiesterase 4D variant 9 (PDE4D9) in a biological sample derived from the prostate of said subject, - determining an expression-based risk score for said subject based on the gene expression profile of the selected phosphodiesterase 4D variant, and - determining a post-operative prognostic risk score for the subject based on the subject's expression-based risk score and a Cancer of the Prostate Risk Assessment After Surgery (CAPRA-S) score, wherein the selected phosphodiesterase 4D variant is selected based on TMPRSS2-ERG fusion status, wherein if the TMPRSS2-ERG fusion status is positive, the selected phosphodiesterase 4D variant is selected as phosphodiesterase 4D variant 7 (PDE4D7), and if the TMPRSS2-ERG fusion status is negative, the selected phosphodiesterase 4D variant is selected as phosphodiesterase 4D variant 5 (PDE4D5) or phosphodiesterase 4D variant 9 (PDE4D9), and wherein the expression-based risk score is determined as follows: EBRS=(((PDE4D_norm+A)*B)+1), Wherein "EBRS" is an expression-based risk score, "PDE4D_norm" is a normalized phosphodiesterase 4D variant gene expression profile value, and A and B are variables, wherein when the phosphodiesterase 4D variant is PDE4D7, A is 6-8 and B is 0.4-0.45, wherein when the phosphodiesterase 4D variant is PDE4D5, A is 4-6 and B is 0.5-0.6, wherein when the phosphodiesterase 4D variant is PDE4D9, A is 3-5 and B is 0.5-0.6.

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