A PROCESS TO DETERMINE HETEROZYGOTE GENOMIC DNA DUPLICATION AND DELETION

Abstract: The present invention relates to a process to determine heterozygote genomic DNA duplication or deletion, and to a test kit for the determination of a disease related to a heterozygote duplication or deletion of the 17p11.2-12 genomic DNA. Further, the invention relates to a process for the determination of Charcot-Marie-Tooth 1A (CMT1A) or Heriditary neuropathy with liability to pressure palsies (HNPP) heterozygosity. Further, the present invention also provides polynucleotide molecules and kits which can be used to determine said diseases.

Description: Field of invention The present invention relates to a process to determine heterozygote genomic DNA duplication or deletion. More specifically, the invention relates to processes for the determination of heterozygote duplication or deletion related to the genomic area 17pll.2-12. More specifically, the present invention relates to a process for the determination of Charcot-Marie-Tooth 1A (CMTIA) or Hereditary neuropathy with liability to pressure palsies (HNPP) heterozygosity. Further, the present invention also provides polynucleotide molecules and kits that can be used to determine said diseases. Background of the invention Recombination between repeated sequences at various loci of the human genome are known to give rise to DNA rearrangement associated with many genetic disorders. Unequal crossing over between flanking repeats has recently been implicated in several deletion and duplication syndromes such as CMT, HNPP, Smith-Magenis syndrome, Williams syndrome, Prader illi / Angelman syndrome, DiGeorge syndrome, INK4 haploinsufficiency and Pelizaeus-Merzbacker disease. As more of these deletion/duplication syndromes are studied in detail, a common mechanism for the development of these syndromes is suggested. The present invention describes a process for the determination of the reciprocal duplication/deletion. A preferred aspect of the present invention relates to processes with reference to the CMT and HNPP. Charcot-Marie-Tooth (CMT) disease is the most common form of hereditary motor and sensory neuropathy (HMSN) . CMT has been classified into a demyelinating (CMTl) and an axonal (CMT2) form. Of CMTl about 70% of the cases are caused by dominantly inheritance of a 1.5 Mb duplication at 17pll.2-12 encompassing the peripheral myelin protein PMP22 gene. Hereditary neuropathy with liability to pressure palsies (HNPP) is caused by the reciprocal deletion of the same 1.5 Mb region (11). Individuals heterozygote for the CMTIA duplication thus have three copies of the PMP22 gene, and individuals heterozygote for the HNPP deletion have only one copy of the PMP22 gene. For testing of heterozygote duplication and deletion several approaches are available. Molecular diagnosis by hybridisation based methods includes Southern blotting, quantitative photostimulated luminescence (PSL) and pulsed-field gel electrophoresis (PFGE) . Less frequently used methods are fluorescence-in situ hybridisation (FISH) , polymorphic markers and endpoint quantitative PCR. The purpose of the present invention is to provide a method which is superior to the above mentioned prior art methods. In the present study we have used real-time quantitative PCR for detecting the genomic DNA duplication and deletion. The kinetics of real-time PCR uses standard PCR in conjunction with a fluorescent TaqMan methodology, and a fluorescence detector, for instance an ABI Prism 7700 Sequence Detector, which is capable of measuring fluorescence in real time. A characteristic parameter of the PCR reaction is the C t (threshold cycle) . This parameter is measured when the PCR amplification is still in the exponential phase, rather than the amount of PCR product accumulated after a fixed number of cycles as by endpoint quantitative PCR. None of the reaction components are limited during the exponential phase, making the results obtained by this method highly reproducible. This greatly improves the precision of DNA quantification. By a comparative Ct method the relative amount of gene in a sample, or as in our case the relative DNA gene number or copy number, can be quantified. By real-time quantitative PCR we have developed a new method with a high sample throughput which can detect both a heterozygote duplication and deletion in a given genomic region. This PCR reaction is very fast and reliable results can be obtained in less then two hours. The results obtained by the method of the present invention show that this is a highly sensitive and specific method and should find broad applications in clinical and research settings . Thus, the present invention relates to a process to determine heterozygote genomic DNA duplication and deletion, characterised in that said heterozygote duplication or deletion of said genomic DNA are determined by the following steps; a) isolating genomic DNA or cDNA from a sample of biological material, and b) performing nucleic acid amplification of said target polynucleotide genomic DNA by using primers capable of hybridising to said genomic DNA, and c) determining the copy number of said genomic DNA relative to an internal polynucleotide standard, by the use real time quantitative PCR, and d) that the amount of PCR product is detected during the exponential phase of the PCR, and that the parameter C t (the threshold cycle) is used to determine the initial target copy number of said genomic DNA. Preferred embodiments of the invention is related to genomic DNA's selected from the group comprising 17pll.2-12, 17pll.2, 7qll.2, 15qll.2, 22qll, Xq22. Further, the present invention can preferably be employed to determine disorders or diseases selected from the group comprising Charcot-Marie-Tooth 1A (CMTIA) , Hereditary neuropathy with liability to pressure palsies (HNPP) , Smith- Magenis syndrome, Williams syndrome, Prader Willi / Angelman syndrome, DiGeorge syndrome, INK4 haploinsufficiency and Pelizaeus-Merzbacker disease. Preferred embodiments of the invention relates to processes to determine CMTIA and HNPP. The present invention also relates to primers capable of hybridising to a gene selected from the group comprising the PMP22 (peripheral myelin protein 22) in CMT1A/HNPP, UBE3A in Angelman syndrome and PLP (proteolipid protein) in Pelizaeus- Merzbacker disease. The present invention also relates to a test kit for the determination of a disease related to a heterozygote duplication or deletion of the 17pll.2-12 genomic DNA, characterised in that the kit comprises; a first set of primers capable of hybridising to said 17pll.2-12 genomic DNA, a second primers capable of hybridising to a reference nucleotide. Preferred primers comprises SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. Experimental section As indicated above, the present invention will be exemplified with reference to CMTIA duplication and HNPP deletion of the 17pll.2-12 genomic DNA. Example 1 Patients . 38 CMTl patients and 15 asymptomatic family members from 15 separate families, 4 HNPP patients and 30 normal controls were studied. Clinical and neurophysiological examinations were performed on all patients and family members, and the diagnosis of CMTl and HNPP were established using accepted clinical and neurophysiological criteria, such as reduced nerve conduction. Informed consent was obtained from each participant and the study has been approved by the Regional ethical committee. Example 2 DNA extraction. Genomic DNA was extracted from whole blood with the QUIAamp DNA Blood kit (Qiagen) as described by the manufacturer. DNA extracted by phenol/chloroform was used in some experiments. DNA or cDNA can also be extracted from other body fluids or tissue . Example 3 Real time quantitative PCR. Primers and probes were selected by the assistance of Primer Express software program ( PE Biosystems) and ordered from Eurogentec (Belgium) . A presently preferred embodiment of the invention uses primers and probes directed towards the PMP22 gene which is localised m the 1.5 MB region 17pll.2-12. In this experimental set-up we used the following primers and probes : PMP22 exon 3 forward primer: 5 ~ -TCTGTCCAGGCCACCATGA- 3 * (SEQ ID NO: 1) PMP22 exon 3 reverse primer: 5'-GAAGAGTTGGCAGAAGAACAGGA-3~ (SEQ ID NO: 2) PMP22 exon 3 TaqMan probe: 5 -CCTGTCGATCATCTTCAGCATTCTGTCTCTG-3 (SEQ ID NO: 3) Human serum albumin exon 12 forward primer: 5 ' -TGTTGCATGAGAAAACGCCA-3' (SEQ ID NO: 4) Human serum albumin exon 12 reverse primer: 5~- GTCGCCTGTTCACCAAGGAT-3 ' (SEQ ID NO: 5) Human serum albumin exon 12 TaqMan probe: 5 ' - AAGTGACAGAGTCACCAAATGCTGCACAG-3 ' . (SEQ ID NO: 6) The TaqMan probes (SEQ ID NO: 4 and SEQ ID NO: 6) contain a fluorophore 5 ' FAM as reporter and 3'TAMRA as quencher. The relative concentrations of appropriate primei , probe and template resulting in the highest intensity and specificity of reporter fluorescent signal were based on optimising preruns. Each assay included samples in triplicates for both the PMP22 gene and the albumin gene. In each assay (96 wells) 13 patients / family members, two normal controls and a no template control (background) were included. A PCR mastermix, representing all PMP22 or albumin samples, were aliquoted to each sample tube to a final volume of 45 μl . The PCR reaction mix: 2x TaqMan Universal PCR Master mix (25 μl), 900 nM (5 μl) forward primer, 900 nM (5 μl) reverse primer and 200 nM (5 μl) probe. DNA (100 ng / 5μl) was diluted to a stock solution before aliquoted to each sample tube containing the PCR reaction mix of PMP22 and albumin triplicate reactions. The 96 well sample tray was centrifuged at 2800 rpm for 1 min. The PCR was performed in the ABI Prism 7700 Sequence Detection System (PE Biosystems) which detects the signal from the fluoregenic probe during the exponential phase of the PCR reaction. The thermal cycling conditions were 2 min. at 50°C and 10 min. at 95°C. Thermal cycling conditions of 40 cycles at 95°C for 15 sec. and 65°C for 1 min. TaqMan Universal PCR Master mix, Microamp™ Caps and Microamp™ Reaction tubes were all from PE Biosystems . Example 4 Comparative C t method. The parameter C t (threshold cycle) is defined as the point at which the amplification plot, representing the fractional cycle number of fluorescence generated by cleavage of the probe (ΔRn) , passes a fixed threshold above baseline. Ct is reported as the cycle number at this point. The threshold is automatically set at 10 standard deviations (SD) above the mean of baseline emission representing the background level, and is calculated from cycles 1-15. However, this threshold was manually adjusted above background level (calculated from cycles 1-15) and to the start of the logarithmic curve. A comparative Ct method, as previously described by Livak (Livak KJ. Comparative Ct method. ABI Prism 7700 Sequence Detection System. User Bulletin no .2 : PE Applied Biosystems; 1997) , detects relative gene expression or also the relative gene number. Amplicons are run as triplicates in separate tubes to show relative quantity of PMP22 genes normalised to endogenous control genes of albumin. By the use of a calibrator sample, represented by DNA from a normal healthy control individual, the gene copy number of unknown samples are determined based on a comparison with genes of known copy number. The calibrator sample is analysed on every assay plate together with the unknown sample of interest. The following formula can be used to show the gene copy number by PMP22 duplication or deletion: 2 - (ΔΔCt ) where ΔΔCt = (ΔCt albumin (calibrator sample) - ΔCt PMP22 (calibrator sample) ) - (ΔCt albumin (patient sample) - ΔCt PMP22 (patient sample) ) . ΔCt represents the mean Ct value of each sample triplicate. The ΔΔCt PMP22 range of sample is determined by calculating the expression 2 ~ ( ΔΔCT + _s ) , where s = SD of the difference calculated from the Ct SD of both PMP22 and albumin. This formula is based on the assumption that the rate of Ct change versus the rate of target DNA copy change is identical for both the PMP22 gene of interest and albumin, and also that doubling of target gene copy number results in a one cycle decrease in the measured Ct . This was tested in a DNA dilution assay. Results By the use of ABI Prism 7700 Sequence Detection system the duplication of PMP22, respective deletion was detected by the comparative Ct method which estimates the number of gene copies. Table 1 shows the results of 38 CMTl patients and 15 asymptomatic family members of 15 separate families, 30 normal controls and 4 separate HNPP patients diagnosed by real-time quantitative PCR. Comparative Ct method where ΔΔCt ratio (2 ~ ΔΛCt > defines the PMP22 gene copy number in normal, in CMTIA duplication and in HNPP deletion. Amplicons are run as triplicates in separate tubes to quantify the relative ratio of PMP22 genes normalised to the endogenous control of albumin. The ΔΔCt range given for PMP22 in CMTIA and HNPP patient relative to a normal control is determined by ΔΔCt upper range and ΔΔCt lower range (2 _ΔΔCt+ "s ) which includes the standard deviation of difference calculated from the Ct standard deviation of both PMP22 and albumin. Figure 1A-C shows a amplification plot of PMP22 and albumin from the same run, all in triplicates of a) a normal healthy control (ΔΔCt ratio = 1.00, SD PMP22 = 0.12, SD albumin = 0.13), b) a PMP22 duplication positive CMTIA patient (ΔΔCt ratio = 2.02, SD PMP22 = 0.06, SD albumin = 0.02), c) a PMP22 deletion positive HNPP patient (ΔΔCt ratio = 0.55, SD PMP22 = 0.09, SD albumin = 0.03). The ΔRn value reflects the quantity of fluorescent probe degraded and fit an exponential function generating an amplification plot for each well. The ΔRn is plotted on the y-axis, and time represented by cycle number is plotted on the x-axis. When sufficient hybridisation probe has been cleaved by 5'nuclease activity of the Taq DNA polymerase, the intensity of reporter (FAM) fluorescent emission increases above threshold. The threshold was manually put above the background level calculated from cycles 1-15. The point at which the amplification plot crosses the threshold is defined as Ct, and Ct is reported as the cycle number at this point. The ΔΔCt ratio was calculated by comparative Ct method. (Figure reprint: ABI Prism Sequence Detector 7700 software program, PE Biosystems) Normal control (copy number: 2) The PCR amplification plot of a normal healthy control sample in Figure la illustrates that the Ct value of PMP22 and albumin triplicates was almost identical. CMTIA, duplication of PMP22 (copy number: 3) In samples positive for the PMP22 duplication the Ct value of PMP22 decreased compared to albumin. This is shown in Figure lb. HNPP, deletion of PMP22 (copy number: 1) In samples positive for the PMP22 deletion the Ct value of PMP22 was increased when compared with albumin (Figure lc) . The triplicate runs of PMP22 and albumin show an almost complete overlap of parallel amplification plots. For all samples the Ct SD was minimal (mean 0.07, range 0.01-0.17). The low SD of the triplicates is critical for the reliability of the assay and is a result of 1) precise pipetting of PCR reaction mix and DNA template, and 2) high quality DNA template (see below) . High quality DNA templates are obtained by DNA extraction by the QIAamp DNA Blood. DNA extracted by phenol/chloroform resulted in triplicate amplification plots of lower satisfactory quality, with higher SD (mean 0.3, range 0.1- 0.5) . In order to detect the rather small increase or decrease in copy number (i.e. from 2 to 3 copies, and from 2 to 1 copy for respective duplication and deletion) , it is essentially to use assay conditions which enables highly reproducible results. In PMP22 deletion (Figure lc) the Ct value of PMP22 was higher than for albumin. Interestingly, during the logarithmic exponential growth phase of the PCR reaction, amplification plot of PMP22 deletion crosses the curve of albumin. This indicates that the PCR of PMP22 is more effective than for albumin in reaching a higher total PCR amplicon level after 40 PCR cycles. In a triplicate PMP22 and albumin DNA dilution assay of 25ng, 50ng, 75ng and lOOng template, a doubling of DNA template/gene copy number resulted in a Ct value reduction ranging from 0.90-1.10. After 40 PCR cycles the final number of PCR amplicons was the same in these four parallel reactions for PMP22 respectively albumin. This is in accordance with the requirements for real-time quantitative PCR assay where doubling of DNA template results in a Ct reduction of one cycle, but the same final PCR amplicon level. Table 1 illustrates the PMP22 ratios (2 ΔΔCt >) by duplications, deletions and in normals obtained by the comparative Ct method, showing no overlap in ratios in-between the three separate groups. The ΔΔCt PMP22 range (2 " <ΔΔCT +/_s) ) includes the SD from both PMP22 and albumin resulting in an increase from 2 to 3 gene copies and this was seen as a PMP22 ΔΔCt value decrease > 0.60 when compared to albumin and in relation to the normal control. The reduction in Ct value when estimated by the comparative Ct method, gave a ΔΔCt ratio > 1.50. By PMP22 deletions reduction from 2 to 1 in gene copy and this was seen as a PMP22 ΔΔCt value increase > 0.85 when compared to albumin and in relation to the normal control. The increase in Ct value when estimated by comparative Ct method (ΔΔCT) gave a ΔΔCt ratio < 0.55. These results show that the method is sensitive enough to detect an increase in gene copy number from 2 to 3, and also to detect an decrease in gene copy number from 2 to 1. To our knowledge this is the first time this quantitative PCR method has been used to detect a difference in copy number of only 50%, i.e. either one additional copy or a reduction of one copy. From Bieche I, et . al., "Novel approach to quantitative polymerase chain reaction using real- time detection: application to the detection of gene amplification in breast cancer", Int J Cancer 1998 ; 78 (5) : 661-6, it is known that the PCR method can be used to determine a doubling of copy number, i.e. from 2 to 4 copies. 34 of the 38 CMTl patients (89.5%) were by the real-time quantitative PCR method in accordance with the present invention positive for PMP22 duplication. All four HNPP patients were positive for the PMP22 deletion. None of the asymptomatic family members or normal controls was positive. As mentioned above, real time quantitative PCR has previously been used to detect relative gene amplification by a doubling or more, and also a germ line deletion (35) . However, the present invention describes a novel method, which is sensitive enough to detect a small difference in gene dosage, from two to three gene copies by PMP22 duplication in CMTIA and a reduction from two to one gene copy by PMP22 deletion in HNPP. The results indicate that the method of the present invention is very suitable for diagnosing CMTIA duplication and HNPP deletion. The method in accordance with the present invention has several advantages over other prior art screening methods. Hybridisation-based techniques as Southern blotting, PSL and PFGE are time consuming (several days) , require radioisotopes and relatively large amounts of high-quality genomic DNA. FISH is also time consuming, costly and requires specific equipment and trained personnel. Although polymorphic markers is a PCR based technique, a successful diagnosis is dependent on the informativeness of the markers which may be limited. Real-time quantitative PCR is a precise and reproducible method since it is based on Ct values obtained in the exponential phase of the amplification reaction rather than endpoint measurements were only a slight difference in limiting components will have a dramatic effect on the amount of PCR product. The higher the initial amount of genomic DNA, the sooner accumulated product is detected in the PCR process and the lower Ct value. The use of target specific fluorogenic probes implicates only amplification of the intended sequence. It is thus essential to design probes that effectively hybridised with the gene or genomic area of interest. During the early cycles when PCR fidelity is highest and none of the reaction components is limited, the reaction is characterised by an exponential increase in target amplification. As the reaction components become limited, the rate of target amplification decreases until a plateau phase is reached and there is little or no net increase in PCR product. This is the main reason why Ct is a more reliable measure of starting copy number than by endpoint measurement of the accumulated PCR product. As illustrated by PMP22 deletion, the Ct value of PMP22 is higher than for albumin, but interestingly, during the exponential growth phase of the PCR the curve of PMP22 crosses the curve of albumin in reaching a higher total number of PCR amplicons at the plateau phase compared to albumin. Consequently, PCR of PMP22 amplicons is more effective than for albumin, and in determining the copy number of genes, these results would have been misinterpreted if analysed by endpoint PCR. The present invention is thus highly advantageous over the prior art methods. Real-time quantitative PCR needs DNA of very high quality. This was achieved by DNA purified by QIAamp DNA Blood kit were the PMP22 and albumin sample-runs resulted in a much lower Ct SD (mean 0.07) than sample-runs with DNA template purified by standard phenol / chloroform extraction (Ct SD mean 0.3). A low SD of the triplicates was also critical for the high sensitivity and reproducibility of the assay. By real-time quantitative PCR 34 of the 38 CMTl patients were diagnosed positive for the PMP22 duplication and all 4 HNPP patients for the PMP22 deletion. The CMTIA duplication has previously been found in 33 of the patients by quantitative photostimulated luminescence (PSL) imaging in combination with REP-PCR (unpublished results) . The one case positive for the PMP22 duplication that was only found by real-time quantitative PCR had 6 other family members that were also positive. The results show that PSL imaging, both alone and in combination with REP-PCR has a somewhat lower sensitivity than real-time quantitative PCR. Four of 38 CMTl cases, all single patients of separate families, were negative for the duplication by all of the diagnostic tests used. These patients might either have mutations in the PMP22 gene, P0 gene (CMTIB) or connexin 32 gene (CMTIX) or another subtype of CMT. In conclusion, by real-time quantitative PCR we disclose three distinct separate groups of defined ratios representing the PMP22 gene copy number by duplication in CMTIA, deletion in HNPP and in normal. The sample throughput of the method is high since 96 separate reactions (13 separate individuals) can be analysed in two hours. The method is highly sensitive, specific and reproducible. It requires minimal template and involves no radioisotopes . The simplicity of this closed tube system, in eliminating post-PCR handling, removes potential sources of error by post-PCR processing including the chances of carryover contamination. The method of the present invention should find broad application not only for duplication in CMTIA and deletion in HNPP, but also in other diseases. SEQUENCE LISTING <110> Forinnova AS <120> A process to determine heterozygote genomic DNA duplication and deletion <130> Forinnova, PCT-søknad, kvant PCR <140> <141> <160> 6 <170> Patentln Ver. 2.1 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence description of Artificial Sequence : primer <400> 1 tctgtccagg ccaccatga 19 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Description of Artificial Sequence : primer <400> 2 gaagagttgg cagaagaaca gga 23 <210> 3 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence description of Artificial Sequence : primer <400> 3 cctgtcgatc atcttcagca ttctgtctct g 31 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Description of Artificial Sequence :primer <400> 4 tgttgcatga gaaaacgcca 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence : Description of Artificial Sequence :primer <400> 5 gtcgcctgtt caccaaggat 20 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence : Description of Artificial Sequence :primer <400> 6 aagtgacaga gtcaccaaat gctgcacag 29

Claims: C L A I M S 1. A process to determine heterozygote genomic DNA duplication or deletion, characterised in that said heterozygote duplication or deletion of said genomic DNA are determined by the following steps; a) isolating genomic DNA or cDNA from a sample of biological material, and b) performing nucleic acid amplification of said target polynucleotide genomic DNA by using primers capable of hybridising to said genomic DNA, and c) determining the copy number of said genomic DNA relative to an internal polynucleotide standard, by the use real time quantitative PCR, and d) that the amount of PCR product is detected during the exponential phase of the PCR, and that the parameter C t (the threshold cycle) is used to determine the initial target copy number of said genomic DNA. 2. Process in accordance with claim 1, characterised in that the quantitative PCR uses a TaqMan probe which is a fluorogenic probe with a reporter fluorescent dye and a quencher fluorescent dye, wherein a DNA polymerase cleaves the reporter dye from the TaqMan probe during each extension cycle, and that the released reporter fluorescence emits its characteristic fluorescence, thus making it possible to monitor the number of amplification cycles. 3. Process in accordance with one of the claims 1-2, characterised in that said genomic DNA is selected from the group comprising 17pll.2-12, 17pll.2, 7qll.2, 15qll.2, 22qll, Xq22. 4. Process in accordance with claim 3, characterised in that said genomic DNA is 17pll.2-12. 5. Process in accordance with one of the claims 1-2, characterised in that the determination of said heterozygote duplication or deletion of said genomic DNA can be used to determine disorders or diseases selected from the group comprising Charcot-Marie-Tooth 1A (CMTIA) , Hereditary neuropathy with liability to pressure palsies (HNPP) , Smith- Magenis syndrome, Williams syndrome, Prader Willi / Angelman syndrome, DiGeorge syndrome, INK4 haploinsufficiency and Pelizaeus-Merzbacker disease. 6. Process in accordance with claim 5, characterised in that said disease or disorder is Charcot-Marie-Tooth 1A (CMTIA) . 7. Process in accordance with claim 5, characterised in that said disease or disorder is CMT type HNPP. 8. Process in accordance with one of the claims 1 - 7, characterised in that the primers directed towards said genomic DNA are capable of hybridising to a gene selected from the group comprising the PMP22 (peripheral myelin protein 22) in CMTIA/HNPP, UBE3A in Angelman syndrome and PLP (proteolipid ^ protein) in Pelizaeus-Merzbacker disease. 9. Process in accordance with claim 8, characterised in that said primers are capable of hybridising to the PMP22 gene. 10. Process in accordance with claim 9, characterised in that said primers are capable of hybridising to the exon 3 of PMP22 11. Process in accordance with one of the claims 1-10, characterised in that said primers directed towards the PP22 gene comprises SEQ ID NO: 1 (forward primer), SEQ ID NO: 2 (reverse primer) and SEQ ID NO: 3 (TaqMan probe) . 12. Process in accordance with one of the claims 1-11, characterised in that said standard polynucleotide is serum albumin exon 12. 13. Process in accordance with claim 12, characterised in that the primers capable of hybridise to the albumin standard comprises SEQ ID NO: 4 (forward primer) , SEQ ID NO: 5 (reverse primer) and SEQ ID NO: 6 (TaqMan probe) . 14. Process in accordance with one of the claims 1-13, characterised in that the biological material is selected from the group comprising; whole blood and other body fluids or tissues . 15. A test kit for the determination of a disease related to a heterozygote duplication or deletion of the 17pll.2-12 genomic DNA, characterised in that the kit comprises; - a first set of primers capable of hybridising to said 17pll.2-12 genomic DNA, - a second set of primers capable of hybridising to a reference nucleotide. 16. Test kit in accordance with claim 15, characterised in that the first set of primers comprises SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. 17. Test kit in accordance with claim 15, characterised in that the reference nucleotide is the gene coding for albumin exon 3. 18. Test kit in accordance with claim 15, characterised in that the second set of primers comprises SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. 19. Test kit in accordance with one of the claims 15-18, characterised in that the test kit further comprises buffers and enzymes required to conduct the quantitative PCR reaction.

Inventors: AARSKOG NINA K, VEDELER CHRISTIAN A, AARSKOG, NINA, K., VEDELER, CHRISTIAN, A.
Applicants: AARSKOG NINA K, FORINNOVA AS, VEDELER CHRISTIAN A, AARSKOG, NINA, K., VEDELER, CHRISTIAN, A.
Application year: 2001
Main classification: C12Q1
IPC codes: C12Q1