WO2008056937A1 - Method and kit for determining chimerism after stem cell transplantation using mitochondrial dna microsatellites as markers - Google Patents

Method and kit for determining chimerism after stem cell transplantation using mitochondrial dna microsatellites as markers Download PDF

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WO2008056937A1
WO2008056937A1 PCT/KR2007/005592 KR2007005592W WO2008056937A1 WO 2008056937 A1 WO2008056937 A1 WO 2008056937A1 KR 2007005592 W KR2007005592 W KR 2007005592W WO 2008056937 A1 WO2008056937 A1 WO 2008056937A1
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poly
sequence
markers
stem cell
mtdna
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Myunggeun Shin
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Industry Foundation Of Chonnam National University
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
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    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
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    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/179—Nucleic acid detection characterized by the use of physical, structural and functional properties the label being a nucleic acid
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Definitions

  • the present invention relates to a method and a kit for determining chimerism after stem cell transplantation using mitochondrial DNA hypervariable region (HV) as a marker, and more specifically, to the method and the kit for qualitatively and quantitatively determining chimerism after stem cell transplantation using mitochondrial DNA hypervariable region, 303 poly C or 16184 poly C, as a marker.
  • HV mitochondrial DNA hypervariable region
  • Allogenic bone marrow transplantation was successfully performed in a human body for the first time at the end of I960' s. Thereafter, allogenic bone marrow transplantation has been considered to be a critical therapy for complete recovery of a wide variety of diseases including intractable hematological disorders. Further, applied diseases and operating methods thereof have been diversified. Success or non-success of stem cell transplantation primarily depends on recurrence of a primary disease, graft rejection, and graft- versus-host disease.
  • chimerism In order to observe engraftment of a transplant and to determine recurrence prior to hematologic relapse, chimerism has been inspected.
  • allogenic stem cell transplantation hematopoietic cells of a patient are completely replaced by the cells derived from a donor, while the residual cells remain ones from the patient, thereby forming complete chimerism (CC) .
  • the hematopoietic cells form mixed chimerism (MC) where the cells from the patient and ones from the donor are present together during a certain period of time after bone marrow transplantation, which is then converted to complete chimerism with the lapse of time.
  • MC mixed chimerism
  • erythrocyte phenotype test As methods for determining chimerism, erythrocyte phenotype test, immunoglobulin isotype analysis, or cytogenetic analysis at the metaphase of cell division has been previously employed, but none of them could give satisfactory results in terms of sensitivity and specificity.
  • the methods have the limitation that the detection cannot be made until a sufficient amount of regenerated cells are obtained after the stem cell transplantation, and thus, have difficulties in the diagnosis of initial graft rejection or acute graft-versus-host disease.
  • Y-chromosome specific fluorescent in situ hybridization (FISH) is currently used in many laboratories due to the advantage that quantitative analysis is available with a relatively high sensitivity when a donor and a patient has different sexualities.
  • VNTR variable number of tandem repeat
  • STR short tandem repeat
  • VNTR or STR VNTR/STR
  • restriction fragment length polymorphism using DNA enabled discrimination between a donor and a patient.
  • RFLP restriction fragment length polymorphism
  • the method is not appropriate to determine chimerism in a patient with bone marrow transplantation, in that it is time- consumptive and labor-intensive, and requires a large amount
  • Mitochondrial DNA is a gene with the size of 16,659 bases, present in mitochondria of cytoplasm. It is an independent intracellular genome to regulate generation of ATP required by cells and tissues and apoptosis. Mitochondrial DNA' s are directly exposed to free oxygen in the cell, which is produced during the energy generation, and has poor ability to repair damaged DNA' s, thereby showing from 10- to 20-fold frequency of genetic variation as compared to nuclear DNA' s. In addition, mtDNA' s are dispersed in a cell, and several thousands of copies thereof are present in a single cell. Thus, they provide a number of target molecules, to give a high sensitivity.
  • mtDNA hypervariable region nucleotide position (np) 16024 ⁇ 16569 and 1-576) for samples from patients who had received stem cell transplantation, and evaluated the utility of total six (6) markers of hypervariable region and of coding region of mtDNA (np 303 poly C, np 16184 poly C, np 514 CA repeat, np 3566 poly C, np 12385 poly C and np 12418 poly A) for determining chimerism after stem cell transplantation. As a result, they confirmed that mtDNA hypervariable region DNA 303 poly C and 16184 poly C could be efficiently used for determination of chimerism after stem cell transplantation, and completed the present invention.
  • an object of the present invention is to provide a method for discriminating cells from individuals by using mtDNA hypervariable region, nucleotide position 303 poly C and/or 16184 poly C, as a marker (s) .
  • Another object of the present invention is to provide a method for determining chimerism after stem cell transplantation by using mtDNA hypervariable region, nucleotide position 303 poly C and/or 16184 poly C, as a marker (s) .
  • Still another object of the present invention is to provide a kit for use in the above-described methods, which comprises primers for mtDNA hypervariable region, 303 poly C and/or 16184 poly C, as a marker (s) .
  • One aspect of the present invention relates to a method for discriminating cells from individuals, which comprises performing length polymorphism analysis for one or more genes selected from the group consisting of mtDNA hypervariable region, nucleotide position 303 poly C and 16184 poly C, as a marker (s) .
  • Another aspect of the invention relates to a method for determining chimerism after stem cell transplantation, which comprises the steps of :
  • Still another aspect of the invention relates to a kit for use in the above-described methods, which comprises one or more forward primers labeled with detectable labels and one or more reverse primers for one or more genes selected from the group consisting of mtDNA hypervariable region, 303 poly C and 16184 poly C, as a marker (s) .
  • length polymorphism analysis can be performed by polymerase chain reaction (PCR) with a forward primer labeled with a detectable label and a reverse primer for 303 poly C or 16184 poly C.
  • the forward primer and the reverse primer for 303 poly C may have the base sequence of Sequence ID Nos . 1 and 2 , respectively, and the forward primer and the reverse primer for 16184 poly C may have the base sequence of Sequence ID Nos. 3 and 4, respectively.
  • Fig. 1 depicts the results of determining chimerism after stem cell transplantation by using mtDNA hypervariable region HV2 , 303 poly C, as a marker;
  • Fig. 2 depicts the results of determining chimerism after stem cell transplantation by using mtDNA hypervariable region HVl, 16184 poly C, as a marker;
  • Fig. 3 depicts the results of identifying mtDNA 16184 poly C marker by nucleotide sequencing
  • Fig. 4 depicts the results of comparatively evaluating sensitivity of mtDNA 303 poly C marker and nuclear DNA D18S51 marker .
  • the present inventors analyzed the base sequence of mtDNA regulatory region, profiled length polymorphism of mtDNA hypervariable region in Koreans, and analyzed genetic diversity of mtDNA hypervariable region. Further, in order to determine chimerism in patients with stem cell transplantation from various hematological disorders by using mtDNA, the inventors selected six markers in the mitochondrial DNA hypervariable region, and compared them with nuclear DNA markers, to ensure their utility through sensitivity and specificity tests. More specifically, length polymorphism analysis was performed for 16184 poly C (16184CCCCCTCCCC16193 , 5CT4C) , 303 poly C (303CCCCCCCTCCCCC315, 7CT5C) and 514 (CA) repeat
  • D1S80, F13A, HUM FABP2 , HUM RENA-4 and amelogenin were subjected to length polymorphism analysis according to the same procedures as mtDNA markers, and evaluated for their utility as compared to the mtDNA markers.
  • three markers 303 poly C, 16184 poly C and 514 (CA) repeat
  • CA 514
  • the mtDNA markers 303 poly C and 16184 poly C which exhibit more than 50% of utility, were selected as novel markers to determine chimerism after stem cell transplantation, with comparing with the nuclear DNA markers.
  • HV2 comprising 303 poly C and 514 (CA) repeat
  • HVl comprising 16184 poly C
  • E. coli cells were transformed with each purified PCR product and cultured at large scale, and then, recombinant DNA was extracted from the cells and sequenced.
  • cell mixing experiment was carried out, comparing with nuclear DNA markers. The peak area of each marker was calculated to obtain the proportion (%) of donor cells. As a result, it was confirmed that the sensitivity of mtDNA markers was remarkably higher than that of nuclear markers. [Best Mode]
  • Example 1 Discovery of novel markers to determine chimerism after stem cell transplantation
  • mtDNA hypervariable region nucleotide position (np) 16024-16569 and 1-576)
  • evaluation of utility for determining chimerism after stem cell transplantation for total six (6) markers in mtDNA hypervariable and coding regions were carried out for samples from donors, patients (recipients) and patients with allogenic stem cell transplantation, respectively, for 27 cases who had received the stem cell transplantation from various hematological disorders (12 cases of acute myelocytic leukemia, 10 cases of acute lymphocytic leukemia, 2 cases of aplastic anemia and 3 cases of chronic myeloproliferative disorder) .
  • PCR product of about 100 bp including these regions was obtained and subjected to capillary electrophoresis by using ABI Prism 3100 Genetic Analyzer and Gene Scan Analysis Software version 3.1 (from Applied Biosysterns) .
  • a fluorophore, HEX (2' , 4 ' , 5' , 7' - tetrachloro-6-carboxy-4, 7-dichlorofluorescein) was labeled at each 5' -terminus of the forward primers.
  • PCR was performed with a reaction mixture with a total volume of 50 ⁇ 1 consisting of 1OX buffer (5 ⁇ 1) , dNTP (400 ⁇ M) , Taq DNA polymerase (2.5 U) (TaKaRa LA TaqTM), each primer (20 pmoles) , sterile distilled water (33.5 ⁇ 1) and genomic DNA (5 ng) .
  • PCR was performed with 35 cycles of heat denaturation at 96 ° C for 1 minute and at 94 ° C for 15 seconds, annealing at 56 ° C for 30 seconds, and extension at 72 ° C for 20 seconds, followed by final extension at 72 ° C for 5 minutes.
  • denaturation was performed at 95 ° C for 5 minutes, and then, reaction was carried out at 4 "C, with a composition consisting of the 50 -fold diluted PCR product (1 ⁇ 1) , deionized formamide (13.5 ⁇ 1) and ROX internal standard for length genotype analysis (from Applied Biosystems) (0.5 ⁇ 1).
  • the denatured PCR product was subjected to length genotype analysis by using ABI Prism 3100 Genetic Analyzer and Gene Scan Analysis Software version 3.1 (from Applied Biosystems) . In order to obtain accurate results, at least two different PCR reactions were carried out for each sample, and repetitive experiments were performed. Primers used for individual mtDNA markers are shown in the following Table 1.
  • mtDNA markers 303 poly C and 16184 poly C which exhibited more than 50% of utility, were identified as novel markers to determine chimerism after stem cell transplantation, as compared to nuclear DNA markers .
  • the results of evaluating chimerism after stem cell transplantation by using 303 poly C and 16184 poly C markers are shown in Fig. 1 and Fig. 2, respectively.
  • PCR was performed for HV2 comprising 303 poly C and 514 (CA) repeat, and HVl comprising 16184 poly C.
  • F15 5 ' -CACCCTATTAACCACTCACG-3 ' : Sequence ID No. 11
  • R484 5 ' -TGAGATTAGTAGTATGGGAG-3 ' : Sequence ID No. 12
  • F361 5'-ACAAAGAACCCTAACACCAGC-S' : Sequence ID No. 13
  • R921 5'-ACTTGGGTTAATCGTGTGACC-S' : Sequence ID No.
  • Each PCR product was subjected to electrophoresis on a 1% agarose gel stained with ethidiura bromide (0.25 u g/ml) , and excised with a razor.
  • the excised PCR product was purified by using SolGent Agarose Gel Extraction Kit (SolGent, Daej eon, Korea) , and quantitatively analyzed by dissolving it in an extraction buffer (10 inM Tris-Cl, pH 8.5).
  • Individual purified PCR product was inserted to pGEM-T Easy Vector (from Promega, Madison, CA) . Transformation was performed with competent E.
  • Example 3 Tests for sensitivity and specificity of mtDNA markers
  • mtDNA marker 303 poly C
  • D18S51 nuclear DNA marker
  • Proportion (%) of donor (F+G)xl00 / (A+B+C+D+E+F+G) [A, B, C, D, E: markers from recipient (R) ; F, G: markers from donor (D) ]
  • Proportion (%) of donor (C+D)xl00 / (A+B+C+D) [A, B: markers from recipient (R) ; C, D: markers from donor (D) ]
  • a reaction mixture for gene scan analysis of mtDNA markers consisted of 10-fold concentrated buffer solution (5 ⁇ 1) , synthetic nucleotide mixture (400 ⁇ M) , thermophilic polymerase (2.5 U) (from TaKaRa), a mixture of forward primer labeled with a fluorophore and reverse primer for individual mtDNA markers (20 pmoles) , sterile distilled water (33.5 ⁇ 1) and genomic DNA (5 ng) , with a total volume of 50 ⁇ 1.
  • PCR was carried out with 35 cycles of heat denaturation at 96 ° C for 1 minute and at 94 ° C for 15 seconds, annealing at 56 ° C for 30 seconds and extension at 72 ° C for 20 seconds, followed by final extension at 72 ° C for 5 minutes. From 50 ⁇ 1 of the PCR product, 1 ⁇ 1 was taken, and mixed with formaldehyde (13.7 ⁇ 1) and ROX internal standard (0.3 ⁇ 1) , and the mixture was heat-treated at 95 ° C for 5 minutes. Length polymorphism analysis was performed by using ABI Prism 3100 Genetic Analyzer and Gene Scan Analysis Software version 3.1 (from Applied Biosystems) .
  • the present invention employs shorter markers than prior length polymorphism analyses of mtDNA, and thus, facilitates discrimination of mtDNA hypervariable regions from a donor and a recipient before and after transplantation to provide easy and simple qualitative method for determining chimerism of stem cells after allogenic bone marrow transplantation.
  • the present invention can be used even with a small amount of DNA to detect up to 0.1 base of the mtDNA with a high variability, and accurate size, and area and height of peak can be measured, to provide a method for quantitative determination of the proportion (%) of chimerism.
  • the present invention provide a more accurate method with a higher sensitivity than those using nuclear DNA markers which have been currently used for determining chimerism after stem cell transplantation.
  • the invention is useful not only for discriminating cells from a donor and a patient, but also for determining chimerism of stem cells after stem cell transplantation, so as to predict the possibility of graft rejection or recurrence.
  • Sequence ID No. 1 is a base sequence of forward primer F264 for mitochondrial DNA 303 poly C;
  • Sequence ID No. 2 is a base sequence of reverse primer R391 for mitochondrial DNA 303 poly C;
  • Sequence ID No. 3 is a base sequence of forward primer F16142 for mitochondrial DNA 16184 poly C;
  • Sequence ID No. 4 is a base sequence of reverse primer R16251 for mitochondrial DNA 16184 poly C; Sequence ID No. 5 is a base sequence of forward primer F467 for mitochondrial DNA 514 (CA) repeat;
  • Sequence ID No. 6 is a base sequence of reverse primer R556 for mitochondrial DNA 514 (CA) repeat; Sequence ID No. 7 is a base sequence of forward primer F3529 for mitochondrial DNA 3566 poly C;
  • Sequence ID No. 8 is a base sequence of reverse primer R3617 for mitochondrial DNA 3566 poly C;
  • Sequence ID No. 9 is a base sequence of forward primer F12360 for mitochondrial DNA 12385 poly C and 12418 poly A;
  • Sequence ID No. 10 is a base sequence of reverse primer R12465 for mitochondrial DNA 12385 poly C and 12418 poly A;
  • Sequence ID No. 11 is a base sequence of forward primer Fl5 for mitochondrial DNA 303 poly C
  • Sequence ID No. 12 is a base sequence of reverse primer R484 for mitochondrial DNA 303 poly C
  • Sequence ID No. 13 is a base sequence of forward primer F4361 for mitochondrial DNA 514 (CA) repeat;
  • Sequence ID No. 14 is a base sequence of reverse primer R921 for mitochondrial DNA 514 (CA) repeat;
  • Sequence ID No. 15 is a base sequence of forward primer F15971 for mitochondrial DNA 16184 poly C.
  • Sequence ID No. 16 is a base sequence of reverse primer R16451 for mitochondrial DNA 16184 poly C.

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Abstract

Disclosed are a method and a kit for qualitatively and quantitatively determining chimerism after stem cell transplantation using mitochondrial DNA (mtDNA) hypervariable (HV) region, nucleotide position 303 poly C and/or 16184 poly C, as a marker (s). The method is easy and simple, and has extremely high sensitivity and specificity as compared to ones using nuclear DNA markers for determining chimerism after stem cell transplantation.

Description

[DESCRIPTION] [invention Title]
Method and kit for determining chimerism after stem cell transplantation using mitochondrial DNA microsatellites as markers
[Technical Field]
The present invention relates to a method and a kit for determining chimerism after stem cell transplantation using mitochondrial DNA hypervariable region (HV) as a marker, and more specifically, to the method and the kit for qualitatively and quantitatively determining chimerism after stem cell transplantation using mitochondrial DNA hypervariable region, 303 poly C or 16184 poly C, as a marker.
[Background Art] [Disclosure]
[Technical Problem]
Allogenic bone marrow transplantation was successfully performed in a human body for the first time at the end of I960' s. Thereafter, allogenic bone marrow transplantation has been considered to be a critical therapy for complete recovery of a wide variety of diseases including intractable hematological disorders. Further, applied diseases and operating methods thereof have been diversified. Success or non-success of stem cell transplantation primarily depends on recurrence of a primary disease, graft rejection, and graft- versus-host disease.
In order to observe engraftment of a transplant and to determine recurrence prior to hematologic relapse, chimerism has been inspected. In allogenic stem cell transplantation, hematopoietic cells of a patient are completely replaced by the cells derived from a donor, while the residual cells remain ones from the patient, thereby forming complete chimerism (CC) . The hematopoietic cells form mixed chimerism (MC) where the cells from the patient and ones from the donor are present together during a certain period of time after bone marrow transplantation, which is then converted to complete chimerism with the lapse of time.
As methods for determining chimerism, erythrocyte phenotype test, immunoglobulin isotype analysis, or cytogenetic analysis at the metaphase of cell division has been previously employed, but none of them could give satisfactory results in terms of sensitivity and specificity. In particular, the methods have the limitation that the detection cannot be made until a sufficient amount of regenerated cells are obtained after the stem cell transplantation, and thus, have difficulties in the diagnosis of initial graft rejection or acute graft-versus-host disease. Y-chromosome specific fluorescent in situ hybridization (FISH) is currently used in many laboratories due to the advantage that quantitative analysis is available with a relatively high sensitivity when a donor and a patient has different sexualities. In certain regions of human genome, base sequences with a certain size are repeated, and the number of repetition varies among individuals. Among them, the genetic locus of VNTR (variable number of tandem repeat) consists of minisatellite cores with a length of 8-50 base pairs, and the genetic locus of STR (short tandem repeat) consists of microsatellite cores with a length of 2-8 base pairs. The VNTR or STR (VNTR/STR) genetic locus is inherited according to Mendelism, and individuals have different tandem repeats. Since the genetic loci with high heterozygosity provide a high power of discrimination, they have been utilized to determine chimerism after allogenic stem cell transplantation. In later 1980' s, restriction fragment length polymorphism (RFLP) using DNA enabled discrimination between a donor and a patient. However, the method is not appropriate to determine chimerism in a patient with bone marrow transplantation, in that it is time- consumptive and labor-intensive, and requires a large amount
(5-10 μ g) of DNA even though only a very small number of hematopoietic cells are available at the early stage after the transplantation. Since later 1980' s, gene amplification by polymerase chain reaction (PCR) has been generalized to enable the discovery and genotype analysis of VNTR/STR genetic loci. However, these markers showed remarkable differences in their utility depending upon donors and patients, and have not been standardized yet. Most of all, they have problems in terms of sensitivity and reproducibility, particularly, in quantitatively determining chimerism in case of a small number of donor cells or at the early stage after transplantation.
Mitochondrial DNA (mtDNA) is a gene with the size of 16,659 bases, present in mitochondria of cytoplasm. It is an independent intracellular genome to regulate generation of ATP required by cells and tissues and apoptosis. Mitochondrial DNA' s are directly exposed to free oxygen in the cell, which is produced during the energy generation, and has poor ability to repair damaged DNA' s, thereby showing from 10- to 20-fold frequency of genetic variation as compared to nuclear DNA' s. In addition, mtDNA' s are dispersed in a cell, and several thousands of copies thereof are present in a single cell. Thus, they provide a number of target molecules, to give a high sensitivity. Further, HV regions and mtDNA microsatellites with large genetic variation have been traditionally utilized in the discrimination of individuals at the level of cells or tissues . [Technical Solution] The present inventors carried out base sequence analysis of mtDNA hypervariable region (nucleotide position (np) 16024~16569 and 1-576) for samples from patients who had received stem cell transplantation, and evaluated the utility of total six (6) markers of hypervariable region and of coding region of mtDNA (np 303 poly C, np 16184 poly C, np 514 CA repeat, np 3566 poly C, np 12385 poly C and np 12418 poly A) for determining chimerism after stem cell transplantation. As a result, they confirmed that mtDNA hypervariable region DNA 303 poly C and 16184 poly C could be efficiently used for determination of chimerism after stem cell transplantation, and completed the present invention.
Thus, an object of the present invention is to provide a method for discriminating cells from individuals by using mtDNA hypervariable region, nucleotide position 303 poly C and/or 16184 poly C, as a marker (s) .
Another object of the present invention is to provide a method for determining chimerism after stem cell transplantation by using mtDNA hypervariable region, nucleotide position 303 poly C and/or 16184 poly C, as a marker (s) .
Still another object of the present invention is to provide a kit for use in the above-described methods, which comprises primers for mtDNA hypervariable region, 303 poly C and/or 16184 poly C, as a marker (s) .
One aspect of the present invention relates to a method for discriminating cells from individuals, which comprises performing length polymorphism analysis for one or more genes selected from the group consisting of mtDNA hypervariable region, nucleotide position 303 poly C and 16184 poly C, as a marker (s) .
Another aspect of the invention relates to a method for determining chimerism after stem cell transplantation, which comprises the steps of :
1) performing length polymorphism analysis for one or more genes selected from the group consisting of mtDNA hypervariable region, nucleotide position 303 poly C and 16184 poly C, in a donor and a recipient before the stem cell transplantation, as a marker (s) ; and
2) performing length polymorphism analysis for said marker (s) in the recipient after the stem cell transplantation, to calculate the proportion of the donor's marker (s) .
Still another aspect of the invention relates to a kit for use in the above-described methods, which comprises one or more forward primers labeled with detectable labels and one or more reverse primers for one or more genes selected from the group consisting of mtDNA hypervariable region, 303 poly C and 16184 poly C, as a marker (s) . According to one embodiment of the present invention, length polymorphism analysis can be performed by polymerase chain reaction (PCR) with a forward primer labeled with a detectable label and a reverse primer for 303 poly C or 16184 poly C. The forward primer and the reverse primer for 303 poly C may have the base sequence of Sequence ID Nos . 1 and 2 , respectively, and the forward primer and the reverse primer for 16184 poly C may have the base sequence of Sequence ID Nos. 3 and 4, respectively. [Brief Description of Drawings]
Fig. 1 depicts the results of determining chimerism after stem cell transplantation by using mtDNA hypervariable region HV2 , 303 poly C, as a marker;
Fig. 2 depicts the results of determining chimerism after stem cell transplantation by using mtDNA hypervariable region HVl, 16184 poly C, as a marker;
Fig. 3 depicts the results of identifying mtDNA 16184 poly C marker by nucleotide sequencing; and
Fig. 4 depicts the results of comparatively evaluating sensitivity of mtDNA 303 poly C marker and nuclear DNA D18S51 marker .
Hereinafter, the present invention will be described in detail.
The present inventors analyzed the base sequence of mtDNA regulatory region, profiled length polymorphism of mtDNA hypervariable region in Koreans, and analyzed genetic diversity of mtDNA hypervariable region. Further, in order to determine chimerism in patients with stem cell transplantation from various hematological disorders by using mtDNA, the inventors selected six markers in the mitochondrial DNA hypervariable region, and compared them with nuclear DNA markers, to ensure their utility through sensitivity and specificity tests. More specifically, length polymorphism analysis was performed for 16184 poly C (16184CCCCCTCCCC16193 , 5CT4C) , 303 poly C (303CCCCCCCTCCCCC315, 7CT5C) and 514 (CA) repeat
(514CACACACACA523, (CA)5 repeat) positioned at HVl and HV2 , non-coding region of mtDNA, and 3566 poly C (3566CCCCCC3571, 6C) , 12385 poly C (12385CCCCCC12390, 5C) and 12418 poly A
(12418AAAAAAAA12425, 8A), and their utility was evaluated for determination of chimerism after stem cell transplantation. In addition, seven (7) markers of nuclear DNA (D12S391, D18S51,
D1S80, F13A, HUM FABP2 , HUM RENA-4 and amelogenin) were subjected to length polymorphism analysis according to the same procedures as mtDNA markers, and evaluated for their utility as compared to the mtDNA markers. As a result, it was confirmed that three markers (303 poly C, 16184 poly C and 514 (CA) repeat) corresponding to HV, non-coding region, among the six ratDNA markers, showed 63%, 67% and 11% of utility for determining chimerism after stem cell transplantation, respectively. Thus, the mtDNA markers 303 poly C and 16184 poly C, which exhibit more than 50% of utility, were selected as novel markers to determine chimerism after stem cell transplantation, with comparing with the nuclear DNA markers.
Furthermore, in order to verify genetic variation of length polymorphism for mtDNA markers, HV2 comprising 303 poly C and 514 (CA) repeat, and HVl comprising 16184 poly C were subjected to PCR, and then, purified. E. coli cells were transformed with each purified PCR product and cultured at large scale, and then, recombinant DNA was extracted from the cells and sequenced. In order to test sensitivity and specificity of mtDNA markers, cell mixing experiment was carried out, comparing with nuclear DNA markers. The peak area of each marker was calculated to obtain the proportion (%) of donor cells. As a result, it was confirmed that the sensitivity of mtDNA markers was remarkably higher than that of nuclear markers. [Best Mode]
Hereinafter, the present invention will be specifically described with reference to Examples, which are provided only for better understanding of the present invention, but should not be construed to limit the scope of the invention in any manner .
Example 1: Discovery of novel markers to determine chimerism after stem cell transplantation
Base sequence analysis of mtDNA hypervariable region (nucleotide position (np) 16024-16569 and 1-576) , and evaluation of utility for determining chimerism after stem cell transplantation for total six (6) markers in mtDNA hypervariable and coding regions (np 303 poly C, np 16184 poly C, np 514 CA repeat, np 3566 poly C, np 12385 poly C and np 12418 poly A) were carried out for samples from donors, patients (recipients) and patients with allogenic stem cell transplantation, respectively, for 27 cases who had received the stem cell transplantation from various hematological disorders (12 cases of acute myelocytic leukemia, 10 cases of acute lymphocytic leukemia, 2 cases of aplastic anemia and 3 cases of chronic myeloproliferative disorder) .
First, 10 ml of blood was taken in a heparin tube from individual stem cell donors and recipients prior to transplantation for 27 cases with various hematological disorders. As a sample after transplantation, bone marrow and peripheral blood were taken from a patient on about 20 days and about 90 days after transplantation, and mixed in a culture medium in the ratio of 1:1, and the mixture was subjected to density gradient centrifugal sedimentation to isolate buffy coat. From the buffy coat, genomic DNA was isolated with a genomic DNA extraction kit.
In order to precisely detect length polymorphic variation of 16184 poly C (16184CCCCCTCCCC16193 , 5CT4C) , 303 poly C (303CCCCCCCTCCCCC315, 7CT5C) and 514 (CA) repeat (514CACACACACA523, (CA)5 repeat), which are positioned in HVl and HV2 , non-coding regions of mtDNA, and 3566 poly C (3566CCCCCC3571, 6C), 12385 poly C (12385CCCCCC12390 , 6C) and 12418 poly A (12418AAAAAAAA12425 , 8A), PCR product of about 100 bp including these regions was obtained and subjected to capillary electrophoresis by using ABI Prism 3100 Genetic Analyzer and Gene Scan Analysis Software version 3.1 (from Applied Biosysterns) . A fluorophore, HEX (2' , 4 ' , 5' , 7' - tetrachloro-6-carboxy-4, 7-dichlorofluorescein) , was labeled at each 5' -terminus of the forward primers. PCR was performed with a reaction mixture with a total volume of 50 μ 1 consisting of 1OX buffer (5 μ 1) , dNTP (400 μ M) , Taq DNA polymerase (2.5 U) (TaKaRa LA Taq™), each primer (20 pmoles) , sterile distilled water (33.5 μ 1) and genomic DNA (5 ng) . PCR was performed with 35 cycles of heat denaturation at 96 °C for 1 minute and at 94 °C for 15 seconds, annealing at 56°C for 30 seconds, and extension at 72 °C for 20 seconds, followed by final extension at 72 °C for 5 minutes. Upon completion of PCR, denaturation was performed at 95 °C for 5 minutes, and then, reaction was carried out at 4 "C, with a composition consisting of the 50 -fold diluted PCR product (1 μ 1) , deionized formamide (13.5 μ 1) and ROX internal standard for length genotype analysis (from Applied Biosystems) (0.5 μ 1). The denatured PCR product was subjected to length genotype analysis by using ABI Prism 3100 Genetic Analyzer and Gene Scan Analysis Software version 3.1 (from Applied Biosystems) . In order to obtain accurate results, at least two different PCR reactions were carried out for each sample, and repetitive experiments were performed. Primers used for individual mtDNA markers are shown in the following Table 1.
[Table l] Primers used for individual mtDNA markers mtDNA Start mtDNA Base sequence of primer (5'→ 3') marker Position region
1 303 poly C 303 D- loop Forward : F264 (HEX-5'-CTT TCC ACA CAG ACA
TCA TAA C-3' )
Reverse : R391 (5'-ATC TGG TTA GGC TGG TGT
TAG-3' )
2 16184 poly 16184 D- loop Forward: F16142 (HEX-5'-CTT GAC CAC CTG
C TAG TAC ATA- 3 ' )
Reverse : R16251 (5'-GGA GTT GCA GTT GAT
GTG TGA- 3')
3 514 (CA) 514 D- loop Forward : F467 (HEX-5'-CCC ATA CTA CTA ATC repeat TCA TCA A- 3' )
Reverse : R556 (5'-TTT GGT TGG TTC GGG GTA
TG- 3' )
4 3566 poly C 3566 NDl Forward : F3529 (HEX-5'-CCG
Figure imgf000014_0001
TTA GCT
CTC ACC AT- 3' )
Reverse : R3617 (5'-AAT AGG AGG CCT AGG
TTG AG-3 ')
5 12385 poly 12385 and ND5 Forward : F12360 (HEX-5'-CAC CCT AAC CCT
C and 12418 12418 GAC TTC C-3' ) poly A Reverse : R12465 (5'-GGT GGA TGC GAC AAT
GGA TT- 3 ') (base sequence of F264: Sequence ID No. 1, base sequence of R391: Sequence ID No. 2, base sequence of F16142 : Sequence ID No. 3, base sequence of R16251: Sequence ID No. 4, base sequence of F467: Sequence ID No. 5, base sequence of R556: Sequence ID No. 6, base sequence of F3529: Sequence ID No . 7, base sequence of R3617: Sequence ID No. 8, base sequence of F12360: Sequence ID No. 9, base sequence of R12465: Sequence ID No. 10)
For the seven markers of nuclear DNA (D12S391, D18S51, D1S80, F13A, HUM FABP2 , HUM RENA-4, and amelogenin) , length polymorphism was also analyzed according to the same procedures as the mtDNA markers . The data of mtDNA markers were compared with those of nuclear DNA markers to evaluate their utility.
The results are shown in the following Table 2.
[Table 2] Evaluation of utility of mtDNA markers and nuclear DNA markers
Figure imgf000015_0001
Figure imgf000016_0001
As shown in the above Table 2, three (3) markers corresponding to hypervariable region, non-coding region (303 poly C, 16184 poly C and 514 (CA) repeat) , among the six mtDNA markers showed 63%, 67% and 11% of utility, respectively, for determining chimerism after stem cell transplantation. Thus, mtDNA markers 303 poly C and 16184 poly C, which exhibited more than 50% of utility, were identified as novel markers to determine chimerism after stem cell transplantation, as compared to nuclear DNA markers . The results of evaluating chimerism after stem cell transplantation by using 303 poly C and 16184 poly C markers are shown in Fig. 1 and Fig. 2, respectively.
Example 2: Verification of length polymorphic variation with mtDNA markers
In order to verify length polymorphic variation with mtDNA markers, PCR was performed for HV2 comprising 303 poly C and 514 (CA) repeat, and HVl comprising 16184 poly C. As primers, F15 (5 ' -CACCCTATTAACCACTCACG-3 ' : Sequence ID No. 11) and R484 (5 ' -TGAGATTAGTAGTATGGGAG-3 ' : Sequence ID No. 12) were used for 303 poly C, and F361 (5'-ACAAAGAACCCTAACACCAGC-S' : Sequence ID No. 13) and R921 (5'-ACTTGGGTTAATCGTGTGACC-S' : Sequence ID No. 14) were used for 514 (CA) repeat, to obtain the PCR product with the size of 470 bp and 560 bp, respectively. For 16184 poly C, F15971 (5'- TTAACTCCACCATTAGCACC-3' : Sequence ID No. 15) and R16451 (5'- GCGAGGAGAGTAGCACTCTTG-3' : Sequence ID No. 16) were used as primers, to obtain the PCR product with the size of 480 bp .
Each PCR product was subjected to electrophoresis on a 1% agarose gel stained with ethidiura bromide (0.25 u g/ml) , and excised with a razor. The excised PCR product was purified by using SolGent Agarose Gel Extraction Kit (SolGent, Daej eon, Korea) , and quantitatively analyzed by dissolving it in an extraction buffer (10 inM Tris-Cl, pH 8.5). Individual purified PCR product was inserted to pGEM-T Easy Vector (from Promega, Madison, CA) . Transformation was performed with competent E. coli JM 109 cells, and 8-12 white colonies (containing the recombinant plasmid) were selected and cultured at large scale, and then, the recombinant DNA was extracted. As shown in Fig. 3, length polymorphic variation was confirmed by nucleotide sequencing.
Example 3: Tests for sensitivity and specificity of mtDNA markers
In order to test sensitivity and specificity of the mtDNA markers, cell mixing test was carried out, comparing with nuclear DNA markers. Leukocytes were separated from blood of individual donors and patients, counted and mixed in a ratio of donor cells to patient cells of 1:99, 5:95, 10:90, 20:80, 30:70 and 40:60 to test sensitivity of mtDNA marker (303 poly C) and nuclear DNA marker (D18S51) . As shown in Fig. 4, the peak area of individual marker was calculated, and the sensitivity was compared according to each mixed ratio of cells by using the following formulas:
1) mtDNA 303 poly C marker:
Proportion (%) of donor = (F+G)xl00 / (A+B+C+D+E+F+G) [A, B, C, D, E: markers from recipient (R) ; F, G: markers from donor (D) ]
2) Nuclear DNA D18S51 marker:
Proportion (%) of donor = (C+D)xl00 / (A+B+C+D) [A, B: markers from recipient (R) ; C, D: markers from donor (D) ] A reaction mixture for gene scan analysis of mtDNA markers consisted of 10-fold concentrated buffer solution (5 μ 1) , synthetic nucleotide mixture (400 μ M) , thermophilic polymerase (2.5 U) (from TaKaRa), a mixture of forward primer labeled with a fluorophore and reverse primer for individual mtDNA markers (20 pmoles) , sterile distilled water (33.5 μ 1) and genomic DNA (5 ng) , with a total volume of 50 μ 1. PCR was carried out with 35 cycles of heat denaturation at 96 °C for 1 minute and at 94 °C for 15 seconds, annealing at 56°C for 30 seconds and extension at 72 °C for 20 seconds, followed by final extension at 72 °C for 5 minutes. From 50 μ 1 of the PCR product, 1 μ 1 was taken, and mixed with formaldehyde (13.7 μ 1) and ROX internal standard (0.3 μ 1) , and the mixture was heat-treated at 95 °C for 5 minutes. Length polymorphism analysis was performed by using ABI Prism 3100 Genetic Analyzer and Gene Scan Analysis Software version 3.1 (from Applied Biosystems) .
The results are shown in the following Table 3. [Table 3]
Figure imgf000019_0001
As shown in Table 3, since a cell contain about thousands copies of mitochondria, the sensitivity of mtDNA markers was confirmed remarkably higher than that of nuclear DNA markers, [industrial Applicability]
The present invention employs shorter markers than prior length polymorphism analyses of mtDNA, and thus, facilitates discrimination of mtDNA hypervariable regions from a donor and a recipient before and after transplantation to provide easy and simple qualitative method for determining chimerism of stem cells after allogenic bone marrow transplantation. In addition, the present invention can be used even with a small amount of DNA to detect up to 0.1 base of the mtDNA with a high variability, and accurate size, and area and height of peak can be measured, to provide a method for quantitative determination of the proportion (%) of chimerism. Since several thousands copies of mitochondrial DNA exist in a cell, the present invention provide a more accurate method with a higher sensitivity than those using nuclear DNA markers which have been currently used for determining chimerism after stem cell transplantation. Thus, the invention is useful not only for discriminating cells from a donor and a patient, but also for determining chimerism of stem cells after stem cell transplantation, so as to predict the possibility of graft rejection or recurrence.
[Free Text of Sequence List]
Sequence ID No. 1 is a base sequence of forward primer F264 for mitochondrial DNA 303 poly C;
Sequence ID No. 2 is a base sequence of reverse primer R391 for mitochondrial DNA 303 poly C;
Sequence ID No. 3 is a base sequence of forward primer F16142 for mitochondrial DNA 16184 poly C;
Sequence ID No. 4 is a base sequence of reverse primer R16251 for mitochondrial DNA 16184 poly C; Sequence ID No. 5 is a base sequence of forward primer F467 for mitochondrial DNA 514 (CA) repeat;
Sequence ID No. 6 is a base sequence of reverse primer R556 for mitochondrial DNA 514 (CA) repeat; Sequence ID No. 7 is a base sequence of forward primer F3529 for mitochondrial DNA 3566 poly C;
Sequence ID No. 8 is a base sequence of reverse primer R3617 for mitochondrial DNA 3566 poly C;
Sequence ID No. 9 is a base sequence of forward primer F12360 for mitochondrial DNA 12385 poly C and 12418 poly A;
Sequence ID No. 10 is a base sequence of reverse primer R12465 for mitochondrial DNA 12385 poly C and 12418 poly A;
Sequence ID No. 11 is a base sequence of forward primer Fl5 for mitochondrial DNA 303 poly C; Sequence ID No. 12 is a base sequence of reverse primer R484 for mitochondrial DNA 303 poly C;
Sequence ID No. 13 is a base sequence of forward primer F4361 for mitochondrial DNA 514 (CA) repeat;
Sequence ID No. 14 is a base sequence of reverse primer R921 for mitochondrial DNA 514 (CA) repeat;
Sequence ID No. 15 is a base sequence of forward primer F15971 for mitochondrial DNA 16184 poly C; and
Sequence ID No. 16 is a base sequence of reverse primer R16451 for mitochondrial DNA 16184 poly C.

Claims

[CLAIMS] [Claim l]
A method for determining chimerism after stem cell transplantation, which comprises the steps of: 1) performing length polymorphism analysis for one or more genes selected from the group consisting of mtDNA hypervariable region, nucleotide position 303 poly C and 16184 poly C, as a marker (s), in a donor and a recipient before the stem cell transplantation; and 2) performing length polymorphism analysis for said marker (s) of the recipient after the stem cell transplantation, to calculate the proportion of the donor's marker (s) .
[Claim 2]
The method according to claim 1, wherein the length polymorphism analysis for 303 poly C is performed by polymerase chain reaction (PCR) with a forward primer of
Sequence ID No. 1 labeled with a detectable label and a reverse primer of Sequence ID No. 2.
[Claim 3] The method according to claim 1, wherein the length polymorphism analysis for 16184 poly C is performed by PCR using a forward primer of Sequence ID No. 3 labeled with a detectable label and a reverse primer of Sequence ID No. 4.
[Claim 4] A kit for use in the method according to claim 1, which comprises one or more forward primer labeled with detectable labels and one or more reverse primer for one or more genes selected from the group consisting of mtDNA hypervariable region DNA, nucleotide position 303 poly C and 16184 poly C, as a marker (s) .
[Claim 5]
The kit according to claim 4, wherein the forward primer and the reverse primer for 303 poly C have the base sequence of Sequence ID Nos . 1 and 2, respectively, and the forward primer and the reverse primer for 16184 poly C have the base sequence of Sequence ID Nos. 3 and 4, respectively.
PCT/KR2007/005592 2006-11-07 2007-11-07 Method and kit for determining chimerism after stem cell transplantation using mitochondrial dna microsatellites as markers WO2008056937A1 (en)

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