EP1238108A2 - Procede pour la detection et l'evaluation d'une region d'adn methylee de maniere potentiellement aberrante sur le chromosome x ou de la clonalite - Google Patents

Procede pour la detection et l'evaluation d'une region d'adn methylee de maniere potentiellement aberrante sur le chromosome x ou de la clonalite

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Publication number
EP1238108A2
EP1238108A2 EP00984601A EP00984601A EP1238108A2 EP 1238108 A2 EP1238108 A2 EP 1238108A2 EP 00984601 A EP00984601 A EP 00984601A EP 00984601 A EP00984601 A EP 00984601A EP 1238108 A2 EP1238108 A2 EP 1238108A2
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Prior art keywords
dna region
methylated
methylation
region
potentially
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German (de)
English (en)
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Oskar A. Haas
Andreas Wienhäusel
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Forschungsinstitut fur Krebskranke Kinder
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Forschungsinstitut fur Krebskranke Kinder
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • a method for the detection and evaluation of a potentially aberrantly methylated DNA region on the X chromosome or the clonality is made available, the use of such a method and a kit for carrying out the method.
  • the DNA, carrier of the genetic information is densely packed in structural units, the chromosomes, in the cell nucleus of the eukaryotes. Changes in the number or structure of the chromosomes as well as changes in the DNA itself, without these microscopic changes in the chromosomes causing, usually result in serious clinical pictures in humans. Diseases can also be caused by epigenetic changes such as aberrant DNA methylation.
  • the methylation of the DNA takes place through covalent binding of methyl groups to the nucleotides and represents an important regulatory mechanism: In the case of the mammalian genome, active genes or their regulatory units are often under-methylated, while inactive DNA sections are often characterized by strong methylation.
  • methyl cytosine is preferably found in methylated DNA as mCpG in the CpG islands of DNA. Most of these are found in regulatory DNA units and are not methylated.
  • imprinted genes - depending on the parental origin of the respective gene or chromosome, usually unmethylated active DNA - and methylated inactive DNA sections. Thus "imprinted genes” are expressed by only one of the two homologous chromosomes.
  • methylation of DNA e.g. by cutting with methylation-sensitive enzymes, by mC-specific antibodies, by genomic sequencing of bisulfite-deactivated DNA, selective hydrazine cleavage of non-methylated DNA, etc.
  • a DNA section shows defective (aberrant) methylation (i.e. hypermethylation of physiologically unmethylated DNA or hypomethylation of pyhsiologically methylated DNA), this usually leads to a deregulation of the physiological function of this DNA section and can result in a disease.
  • determining the phenotype is not enough to diagnose the particular disease.
  • an examination of the patient's genetic material is usually necessary, in particular the methylation of a specific DNA segment.
  • a number of diseases are caused by a change in the DNA on an X chromosome.
  • a DNA region on the X chromosome can be methylated so that this gene is is activated.
  • a change (mutation) of the DNA for example by expansion of a repeat region, can disrupt gene function and can also be methylated (FraX-A, FraX-E, FraX-F (see Carrel et al., American Journal of Medical Genetics 64: 27-30 (1996); Hirst et al., Hum. Molec. Genet. 2: 197-200, 1993; Parrish et al., Nature Genet.
  • diseases can be caused by changes in DNA sections or entire chromosomes (- areas) due to duplication, insertion, translocation. If such a change affects the X chromosome already in the early developmental stages of an individual, one has to speak in many cases of X chromosomal diseases. Even with sporadic diseases, as is the case for many If tumor diseases are true, X-chromosomal changes could be detected (Gale et al., Blood, Vol. 83 , No.
  • ISSN 0003-4800; Leal et al., Hum. Genet., 1994; 94 (4): 423-6; ISSN: 0340-6717; MacDonald et al., Hum. Mol Genet., 1994; 3 (8): 1365-71; ISSN: 0964-6906; and Martinez-Pasarell et al., Horm. Res., October 1999; 51 (5): 248-252; ISSN: 0301- 0163).
  • the affected gene or the affected locus is examined using cytogenetic, Southern blot, PCR, reverse transcription PCR (RT-PCR) or immunological analyzes. In these studies, the sequence, expression, extent of methylation, and the like are examined.
  • Fragile X syndrome (FX or FraX-A syndrome) is an example of an inherited disorder, particularly in men.
  • the clinical phenotype of FX syndrome shows a low to iQ
  • I'MR-1 alleles with a large number of repeat units can be reactivated in vitro by treatment with demethylating agents, such as 5-azadeoxycitidine.
  • demethylating agents such as 5-azadeoxycitidine.
  • FX syndrome Common methods for evaluating FX syndrome include cytogenetic, Southern blot, PCR, reverse transcription PCR (RT-PCR) and immunohistochemical analyzes. These studies usually analyze the size of the expanded repeat region, the Denovo methylation of these gene regions or the gene expression.
  • WO 92/14840 describes the detection of fragile X syndrome by means of restriction enzymes, these restriction enzymes, for example, only cutting the sites which have non-methylated cytosines. In contrast to unmethylated DNA, the methylated DNA is thus digested, the digestive products being detectable.
  • WO 91/09140 relates to an oligonucleotide with a specific sequence which binds to a specific location, M54, the region of the fragile gene. Patients with fragile X syndrome can be detected by in situ hybridization with the labeled oligonucleotide.
  • WO 92/20825 also relates to a method for diagnosing fragile X syndrome, the amount of mRNA being determined, for example, by RT-PCR or the amount of protein, for example, by means of immunological methods of the FMR-1 gene.
  • Another method of detecting fragile X syndrome is to determine the length of the repeat area. This is done, for example, by digestion of the FMR-1 gene with restriction enzymes or PCR methods with primers that are specific for the repeat region of the FMR-1 gene and subsequent gel electrophoresis.
  • the method according to US Pat. No. 5,658,764 for the detection of the fragile X syndrome also includes a size measurement of the GC-rich sequence with the aid of PCR methods.
  • a disadvantage of PCR is that it is not possible to differentiate between a full mutation and a premutation, especially if there is a borderline case full mutation / premutation, i.e. that the length of the repeat region is around 200 units and the sequence is partially methylated or not methylated. Furthermore, the distinction between a full mutation and a mosaic (e.g. a full mutation on one allele and a premutation or an expansion in the normal range on the other allele) is problematic or impossible. Furthermore, these known methods are very time-consuming, since several process steps are necessary. These methods also show a high "false negative" rate in women.
  • the Das et al. "Methylation analysis of the fragile X syndrome by PCR” (Genetic testing, Volume 1, Number 3, 1997/98) relates to a method for the detection of the fragile X syndrome by means of methylation-specific PCR (MS-PCR).
  • MS-PCR methylation-specific PCR
  • the unmethylated cytosine residues are converted into uracil by adding an agent, while the methylated cytosine residues are not being transformed.
  • the uracil in the modified DNA is replaced by thymidine.
  • primers are selected which result in different product lengths, it can be determined, depending on the PCR product, whether or not the particular site of the FMR-1 gene is methylated.
  • the normal and premium alleles are distinguished from the full mutation and mosaic sequences — amplification of the methylated sequence — by this MS-PCR.
  • Normal and award-winning sequences must then be differentiated using a conventional PCR (page 152, column 2, 2nd paragraph). In conventional PCR, only normal sequences (5-50 repeat units) but not pre-selected sequences (50-200 repeat units) are amplified (see FIG. 2B).
  • a GCC repeat expansion that is present next to a CpG island in the Xq28 includes over 200 GCC units, compared to 6-25 GCC units in normal people.
  • the CpG island is methylated in FraX-E positive individuals. These patients have mental retardations (see Knight et al., Am. J. Hum. Gent. 53: A79, 1993; Knight et al., Cell 74: 127-134, 1993).
  • a gene, FMR2 has been identified that is transcribed distal to the CpG island in FraX-E and is down-regulated by repeat expansion & methylation (Gu et al., Nature Gent. 13: 109-113).
  • the disadvantages of the known determination techniques are that several different procedural steps are often necessary to diagnose the diseases and the disease variant. In many cases, it is not possible to make a clear statement about the clinical picture, since the determination used only allows statements about a specific gene area.
  • the goal of the present! The invention is accordingly to provide a method for diagnosing a potentially aberrantly methylated DNA region on the X chromosome or the clonality, which can be carried out quickly and easily with as few procedural steps as possible, a distinction being made between the different variants, and that Procedures can be used in both male and female patients to differentiate between the different genotypes.
  • the extent of the methylation of the potentially aberrantly methylated DNA region and / or the extent of the methylation and, if appropriate, the sequence variant of a polymorphic DNA region of the X chromosome, which can be either physiologically or aberrantly methylated, is determined in a sample and subsequently the presence and, if appropriate, the variant and the extent of the potential aberration or the clonality is diagnosed from the comparison of the methylation determinations and, if appropriate, the sequence variant, or
  • Chromosome which can be either physiologically or aberrantly methylated, is determined and then the presence and optionally the variant and the extent of the potential aberration is diagnosed from the comparison of the methylation determinations and the sequence variant.
  • the extent of methylation in the context of the present invention is understood to mean the ratio of methylated and unmethylated allele in a chromosome, ie that the methylated and unmethylated DNA are determined simultaneously in one reaction. Through the simultaneous determination, an accurate and quantitative statement can be made regarding the ratio of methylation and non-methylation of a specific sequence. This makes it possible to diagnose the extent and variant of the particular disease.
  • a potentially aberrantly methylated DNA region is understood to mean a DNA region which is methylated or unmethylated. If this DNA region is affected by methylation, aberrant methylation can cause a disease.
  • a quantitative methylation analysis which is meaningful when determining the clonality (clone size), is suitable for the clarification of all X-chromosomal diseases and also those diseases which are based on a deviation from the normal number of X-chromosomes (Klinefelter, Turner, Multiple X syndrome), or also those diseases whose affected cell population (clone) has X aneuploidy. This is very common in various tumor diseases and haematological neoplasms. The detection and determination of the clone size / clonality of these neoplastic diseases is an important parameter for diagnosis and monitoring. In the case of autosomal diseases that affect imprinted genes, the situation is similar.
  • the extent of methylation in a safely physiologically methylated DNA region of the X chromosome can also be determined.
  • the certainly physiologically methylated DNA region affects only one allele of the X chromosome, while the other allele is not methylated.
  • this additional control serves as an internal standard.
  • a clinical picture is determined by evaluating the potentially aberrantly methylated DNA region and the polymorphic DNA region.
  • a polymorphic DNA region is understood to mean the variability of a DNA sequence, for example a DNA segment which has different alleles in a population, such as the length of a specific DNA segment in the case of microsatellite repeats or Nucleotide and amino acid polymorphisms.
  • the simultaneous determination of the extent of the methylation of the sequence variant together with the determination of the extent of the methylation of the potentially aberrantly methylated DNA region allows a very good statement regarding the variant or the clone size of the corresponding disease.
  • the extent of methylation can be determined by various methods, e.g. Methylation-specific restriction enzymes, oligonucleotides which specifically recognize and hybridize DNA segments which have been modified specifically for methylation, etc.
  • the sequence variant of the polymorphic DNA region can also be determined in various ways, for example by means of PCR or with the aid of restriction enzymes.
  • the extent of the methylation and, if appropriate, the sequence variant are determined by means of a methylation-specific PCR (MS-PCR), using primers with which a methylated or not methylated DNA region are amplified specifically for each methylation.
  • MS-PCR methylation-specific PCR
  • the technique of MS-PCR is a simple method to distinguish between methylated sequences and non-methylated sequences and, because methylation or non-methylation is often an indication of a genetic disease or causes it, for diagnosis used by patients.
  • the MS-PCR is based on the principle that primers are used that are specific for a methylated or non-methylated sequence, so that one or the other primer hybridizes with the sequence, depending on whether it is methylated or not methylated .
  • the PCR products then suggest methylation or non-methylation.
  • the primers can be selected such that the PCR product for the methylated sequence has a certain length and the PCR product for the unmethylated sequence has a different length. Because of their size, the PCR products conclusion about the methylation of the amplified DNA sections.
  • a common method for obtaining MS primers is to specifically change the methylated and / or the non-methylated sequence and to construct corresponding primers which are specific for the changed sequences.
  • One way to specifically change the DNA methylation is to treat the DNA with a deaminating agent, e.g. Sodium bisulfite. Sodium bisulfite converts the unmethylated cytosine to uracil, which is replaced by thymidine in the subsequent DNA amplification.
  • This method thus produces different DNA sequences based on originally homologous but differently methylated alleles.
  • the primers that hybridize with the unmethylated sequence have thymidine instead of cytosine.
  • the primers which are specific for the methylated sequence continue to have cytosine at the sites of the methylated Cs.
  • the primers are chosen such that the PCR product of the methylated sequence always has a different length than the PCR product of the non-repeat region, regardless of the length of the repeat region. methylated sequence.
  • the determination by means of MS-PCR therefore ensures a particularly time-saving and efficient method, since little DNA is used and less work is required.
  • the result of the PCRs is obtained at the same time. No further process steps are necessary, such as Southern blot, hybridization processes, etc.
  • the MS-PCR primer is or are carried out on an antisense strand or on antisense strands.
  • These temperature conditions are gentle on the reaction components and the DNA polymerase.
  • the MS-PCRs are preferably carried out in duplex reactions. This means that the PCRs for the amplification of a specific methylated sequence as well as the same specific non-methylated sequence are carried out in one reaction, for example the PCRs for the amplification of the polymorphic methylated and non-methylated DNA region. This ensures that the reaction products that are obtained can be clearly assigned in the subsequent analysis based on their size, and so the result can be clearly interpreted.
  • the MS-PCR for the determination of the potentially aberrantly methylated DNA region and the MS-PCR for the determination of the safely physiologically methylated DNA region are advantageously carried out in a common multiplex reaction. Since the MS-PCRs generally relate to two different genes and thus two different sequences, the MS-PCRs are not influenced by one another. The determination of the polymorphic DNA region would be carried out in a separate duplex reaction in order to be able to clearly determine the polymorphism, e.g. So that the size of variable PCR products does not overlap with other PCR products in the subsequent evaluation.
  • a method is particularly preferably provided in which the polymorphic DNA region is a DNA region with a repeat polymorphism, which is preferably related to the potentially aberrantly methylated DNA region, the length of the repeat region being determined as a sequence variant and then determining both the presence and the extent of the potential aberration.
  • repeat polymorphism is understood to mean an area which has a repeated repetition of a repeat unit, ie a certain sequence of several nucleotides.
  • the number of repetitions, the so-called “repeat units”, differs depending on the gene or disease, the extent of the disease generally being related to the length of the repeat region.
  • a repeat region that has a particularly unnatural length is usually methylated at the same time, if this DNA region is normally not methylated. In most cases this is also the case For example, the adjacent or potentially aberrantly methylated region is correspondingly strongly methylated, so that the determination of the potentially aberrantly methylated DNA region and the determination of the polymorphic DNA region yield additional results and can therefore be seen as additional controls.
  • the polymorphic DNA region of the CGG trinucleotide repeat region of the FMR-1 gene is particularly preferred.
  • the determination in this area gives a repeatable and unambiguous result. If the determination is carried out, for example, with the aid of MS-PCR, both the length of the repeat region and the extent of the methylation can be determined in a single method step if the primers are selected such that they include the entire repeat region .
  • the CGG trinucleotide repeat region of the FMR-1 gene is a sequence region which provides information about diseases, in particular about the fragile X syndrome, since the length of the repeat region is characteristic for the variant of the disease.
  • polymorphic DNA region is in the first untranslated exon of the FMR-1 gene. This is a region which is advantageous for the determination and leads to an error-free result.
  • the potentially aberrantly methylated DNA region is the FMR-1 gene or a part thereof. This ensures a simplified procedure since only the FMR-1 gene region is necessary for at least part of the analysis. This could be used for the analysis isolated from the rest of the DNA, and the analysis could also be carried out on isolated X chromosomes.
  • the potentially aberrantly methylated DNA region is the 5 'untranslated region of the FMR-1 gene, ie the FMR-1 promoter, or a part thereof.
  • the potentially aberrantly methylated DNA region is close to the CGG repeat region, since this is in the first untranslated exon. In this way, the entire gene, which could possibly disintegrate in the course of the deamination, does not have to be used for the analysis. If the potentially aberrantly methylated DNA region is in the FMR-1 promoter, a shorter DNA section can be used for the determination.
  • the safely physiologically methylated DNA region is a DNA region that is securely methylated on the active X chromosome. Its methylation pattern is therefore opposite to that of the FMR-1 gene, which is methylated on the inactive X chromosome.
  • the ratio of the methylated to the non-methylated DNA region of the one gene is compared with the ratio of the reciprocal methylated DNA region (methylated: non-methylated) and also with the ratio of the methylated repeat Polymorphism compared to unmethylated repeat polymorphism (preferably of the FMR-1 gene).
  • the safely physiologically methylated DNA region is a region that comprises the XIST gene or parts thereof.
  • the XIST gene is methylated on the active chromosome X, so that it has a reciprocal methylation pattern to the FMR-1 gene.
  • the determination using the XIST gene as an internal standard provides a reliable, unambiguous and simple method for the detection and evaluation of a potentially aberrantly methylated DNA region on the X chromosome, especially in women.
  • the DNA region which is certainly physiologically methylated is a region in the XIST gene promoter. It has been found that the methylation of the promoter of the XIST gene is easy to determine and leads to reliable results.
  • the potentially aberrantly methylated DNA region and the polymorphic DNA region are two non-overlapping sequences. This ensures that even if both DNA regions are on the FMR-1 gene, two separate, independent DNA regions are examined, so that the results of the examination - 17 - the two areas complement each other, so that possible confusion errors can be recognized early.
  • two or more potentially aberrantly methylated DNA regions on the X chromosome are detected and evaluated at the same time.
  • the clinical pictures with regard to FraX-A, FraX-E and FraX-F can be determined at the same time, whereby an exact and reliable result is obtained for each individual clinical picture without the results of the different clinical pictures influencing one another.
  • Another aspect of the present invention is the use of a method according to the invention described above for the detection and evaluation of X-chromosomal diseases.
  • These diseases have already been described above, which are diseases which are caused by aberrant methylation in at least one place in the X chromosome.
  • a specific, potentially aberrantly methylated DNA region and possibly a physiologically methylated DNA region of the X chromosome and / or a specific polymorphic DNA region of the X chromosome is analyzed.
  • the FMR-1 promoter is determined as a potentially aberrantly methylated DNA region and / or the XIST gene or a part thereof is determined as a safely physiologically methylated DNA region and / or the CGG trinucleotide region of the FMR-1 gene is determined as the polymorphic DNA region.
  • the CpG island in Sq28 is advantageously a potentially aberrantly methylated DNA region and / or the GCC trinucleotide region is a polymorphic DNA region and / or the XIST gene or a part thereof is safely physiologically methylated DNS range determined.
  • the (GCCGTC) n (GCC) m region is advantageously used as the polymorphic DNA region and / or the adjacent CpG island as the potentially aberrantly methylated DNA region and / or the XIST gene or part of it is determined to be a safely physiologically methylated DNA region.
  • the CGG nucleotide repeat region of the FMRI gene is preferably determined as the polymorphic DNA region.
  • the analysis according to the invention enables a clear and rapid distinction to be made between the different genotypes of fragile X syndrome, even in female patients in which the methylation analysis does not provide a clear result, since - in the case of the repeat region - this is competed in the PCR, in particular with a length of more than 200 units, by a repeat of normal length.
  • the sample can be clearly assigned to a specific genotype, since in normal and premutation this DNA region is not methylated, but is methylated in the case of the full mutation.
  • the potentially aberrantly methylated DNA region in addition to the determination of the potentially aberrantly methylated DNA region, also a certainly physiologically methylated one DNS range is analyzed, it can be used as an internal standard. If the potentially aberrantly methylated DNA region cannot be detected due to deletions, the securely methylated DNA region (eg the XIST gene) is detected in any case. This ensures that the method has worked and that the potentially aberrantly methylated DNA region is at least partially missing, ie deleted (eg FMR-1 gene).
  • the number of normal or premutation repeat units can simply be calculated from the length of the MS-PCR products. In this way, a method is made available in which the degree of methylation and the length of the repeat regions are determined simultaneously.
  • the degree of fragile X syndrome is thus determined in a single procedural step (either a single MS-PCR reaction or several MS-PCR reactions side by side).
  • MS-PCR is a fast and reliable procedure that is inexpensive and can be carried out in any laboratory. Furthermore, only a small amount of DNA is necessary for this analysis. Furthermore, no additional, often time-consuming and costly procedures need to be carried out, such as immunological or Southern blot procedures.
  • non-methylated repeat region as a polymorphic DNA region
  • FMR-1 DNA region eg the promoter (as a potentially aberrant methylated DNA region).
  • Mosaic full mutation / premutation the repeat region of the allele with the full mutation is not amplified, the unmethylated repeat region of the premutation allele is amplified, having (48-) 54 to 200 units.
  • the methylated FMR-1 promoter region is amplified.
  • the ratio of the non-methylated to the methylated FMR-1 DNA region and from the non-methylated to the methylated XIST gene is 1: 1. Since both homologs have identical repeat region lengths, only one non-methylated and one methylated FMR-1 repeat region are visible.
  • Women with a premutation have a similar pattern to heterozygous women, with the difference that a methylated and a non-methylated FMR-1 repeat region of an allele are (48-) 54 to 200 units in length.
  • methylation ratio of the FMR-1 DNA region promoter region is 3: 1 (methylation: non-methylation), while the methylation ratio of the XIST gene continues to be 1: 1.
  • Women with a full nutation and either skewed X inactivation or mosaic have the same pattern as affected women with random X inactivation. Skewed X inactivation and mosaics can be identified by quantitative assessment of the methylation ratio of the FMR-1 promoter region with that of the XIST gene and the FMR-1 repeat region.
  • the methylation ratio of the XIST gene remains 1: 1 in all cases, since each cell still contains an active and an inactive chromosome.
  • the methylation ratio of the portion of the further gene (XIST) is a standard.
  • the methylation ratio of the FMR-1 promoter region and that of the repeat region shifts towards non-methylation or methylation, depending on whether the normal chromosome or which has a skewed X inactivation with the expanded allele.
  • a similar pattern can be seen in mosaic women with a normal cell population and one with a full mutation.
  • the present invention relates to a kit of the type mentioned at the outset, wherein it contains primer sets for the specific amplification of the methylated and unmethylated DNA variant in each case of a potentially aberrantly methylated DNA region and / or a safely physiologically methylated DNA region.
  • Region of the X chromosome and / or a polymorphic DNA region of the X chromosome which can be either physiologically or aberrantly methylated.
  • This kit preferably comprises, in addition to the primer sets, further substances necessary for carrying out an MS-PCR: a substance for converting the methylated or non-methylated sequence (for example sodium bisulfite for converting the non-methylated cytosine residues), the necessary enzymes, Buffers, etc.
  • a substance for converting the methylated or non-methylated sequence for example sodium bisulfite for converting the non-methylated cytosine residues
  • the necessary enzymes for example sodium bisulfite for converting the non-methylated cytosine residues
  • Buffers etc.
  • This Ki can be particularly advantageous; for the detection and evaluation of X-chromosomal diseases using primer sets for the methylation-specific amplification of a DNA re- gion with a repeat polymorphism for the determination of the polymorphic DNA region. The extent of methylation and the length of the repeat polymorphism are determined with these primer sets in order to test the degree of the disease and the clonality thereof.
  • chromosomal diseases are, for example: FraX-A, FraX-E, FraX-F, X-chromosomal retardation, clonality, etc.
  • the kit preferably comprises primer sets for amplifying a polymorphic DNA region of the CGG nucleotide repeat region in the first exon of the FMR-1 gene. Since the repeat range of the FMR-1 gene, as already described above, ensures a reliable and clear statement about the degree of disease of the fragile X syndrome or the clone size, a quick and safe analysis procedure can be provided with the help of this kit.
  • the kit comprises primer sets for the amplification of the XIST gene or parts thereof for the determination of the safely physiologically methylated DNA region.
  • the XIST gene has a methylation pattern that is reciprocal to that of the FMR-1 gene. The methylation pattern in the result allows an exact interpretation regarding the variant of the fragile X syndrome.
  • the kit comprises primer sets for amplifying the FMR-1 gene or a part thereof for determining the potentially aberrantly methylated DNA region.
  • the kit advantageously comprises primer sets for amplifying the FMR-1 gene promoter or a part thereof. The preferred analysis method described above can thus be carried out.
  • primer sets are each provided as duplex sets, spatially separated from one another. This ensures, as described above, that clear and easily interpretable results are obtained.
  • the primer sets for the amplification of the safely physiologically methylated DNA region and the potentially aberrantly methylated DNA region are provided together in a multiplex set or 4-plex set.
  • the methylation-specific PCR reactions become two DNA regions are carried out simultaneously in a reaction mixture, which makes the process time-saving. This is possible because the two genes (FMR-1 and XIST) have different sequences, so that the two PCR reactions do not influence each other.
  • EDTA anticoagulated, peripheral blood samples from healthy and fragile X syndrome patients were stored in 500 ⁇ l aliquots at -20 ° C until DNA extraction.
  • DNA was extracted from 80 ⁇ l blood with DNAzol TM (Vienna Lab, Vienna, Austria) and resuspended in 30 ⁇ l sterile water.
  • 0.5 ⁇ g DNA was according to the protocols of Zeschnigk et al. "A single-tube PCR test for the diagnosis of Angelman and Prader-Willi syndrome based on allelic methylation differences at the SNRPN locus.” Eur. J. Hum. Genet. 5, 94-8 (1997); "Imprinted segments in the human geno e: different DNA methylation patterns in the Prader-Willi / Angelman syndrome region as determined by the genomic sequencing method.” Hum. Mol. Genet. 6, 387-95 (1997), the deamination at 55 ° C. for two hours Add 8 ⁇ l of polyacrylic carrier, which shortens the DNA precipitation to 10 min at -20 ° C. The deaminated DNA was dissolved in 20 ul sterile water.
  • a multiplex PCR reaction mixture two forward primers (PUF, PMF), which are specific for unmethylated and methylated DNA, and a common reverse primer (PR), which are specific for the deaminated, unmethylated and methylated FMR- 1 promoter, and two forward primers (XUF, XMF) and two backward primers (XUR, XMR), which are specific for the deaminated, unmethylated and methylated XIST promoter, were added.
  • the duplex PCR mix comprises two forward primers (RUF, RMF) and two reverse primers (RUR, RMR), which are specific for the deaminated, unmethylated and methylated triplet repeat region.
  • An additional primer pair detects a further methylated sequence in the FMR-1 promoter (FMF, FMR), but cannot be combined with the other primers.
  • the PCR was carried out in a reaction volume of 25 ⁇ l under oil, 1 ⁇ l of the 20 ⁇ l deaminated DNA from patients and normal controls being used in each case.
  • the amplification buffer F-511 (10 mM Tris, pH 8, 8, 50 mM KC1, 1.5 mM MgCl 2 , 0.1% Triton-X-100; Finnzymes Oy, Espoo, Finland) ( DYN) used; Optimized buffer EXT (50 mM Tris, 15 mM NH4CI, 1.5 mM MgCl 2 , 0.1% Triton-X-100, pH 9.0) was used for the duplex reaction with 4% DMSO and 60 mM TMAC and used for FMP amplification without amplifier (DMSO, TMAC).
  • the dNTPs concentrations were 200 ⁇ M of each nucleotide. Table 1 lists the optimal primer concentrations.
  • the amplifications were carried out on a Biometra TrioBlock (Biometra, Goettingen, Germany) and started with a Dynazyme 501L unit (Finnzymes Oy, Espoo, Finland), with a first denaturation step at 95 ° C. for 5 min.
  • the multiplex PCR profiles were 33 cycles 95 ° C / 30 s [program 1], 60 ° C / 20 s, 72 ° C / 40 s [program 2].
  • Duplex and FMP profiles were 35 cycles at 95 ° C / 45 s, 63 ° C / 1 min and 72 ° C / 1 min.
  • PCR products (5 ul) were separated in 0.5x TBE buffer (90mM Tris, 90mM borate, 2mM EDTA, pH 8.0) on NOVEX-TBE gels (Novex, San Diego, California, USA ). The bands were detected by staining with ethidium bromide (EtBr). Densitometric analyzes were performed with KODAK-1D TM 2.0.2 software package (Kodak, New Haven, CT, USA).
  • the number of repeat units for normal individuals and premutation carriers could be calculated from the length of the PCR products.
  • Table 2 lists the results to be expected, where "-" means no PCR product, "+” means a PCR product and "2+” means two products with different lengths.
  • the results to be expected are shown schematically in FIG. 2, the same numbering as in Table 2 being used.
  • the ratio of unmethylated to methylated FMR-1 promoter and that of the XIST gene promoter is about 1: 1. Since both homologs have identical repeat region lengths, only one unmethylated and one methylated FMR-1 repeat region are visible in each case.
  • Women with a premutation have a similar pattern to heterozygous women, with the difference that a methylated and a non-methylated FMR-1 repeat region have a length between (48-) 54 to 200 units.
  • Skewed X inactivation and mosaic can be identified by semi-quantitative comparison of the methylation ratio of the FMR-1 promoter and the FMR-1 repeat region with that of the gene portion of the XIST gene.
  • the methylation ratio of the XIST gene remains 1: 1 in all cases, since each cell continues to carry 1 active and 1 inactive chromosome.
  • the methylation ratio of the FMR-1 promoter and that of the repeat region shifts towards non-methylation or methylation, depending on whether the normal chromosome or the one with the expanded allele has skewed X inactivation.
  • a similar pattern can be seen in mosaic women with a normal cell population and one with a full mutation.
  • FIG. 3a and 3b show gel electrophoresis of the MS-PCR products, with FIG. 3a the products of the multiplex reaction (FMR-1 promoter, XIST promoter) and FIG. 3b the products of the duplex reaction ( FMRI repeat expansion) shows, using the same numbering as in Table 2, and n means the number of repeat units.
  • negative control nodenta DNA that was not deaminated before amplification
  • Table 3 shows the results of a densitometric analysis of the multiplex PCR products from female patients.
  • the ratio of unmethylated and methylated XIST products, specific for the inactive or active X chromosome, remained stable within all female samples (mean (XM / XU) 1.033 ⁇ 0.21).
  • hum-A (um): SEQ. ID.Nr. 18
  • hum-B2 (m): SEQ. ID. r. 19
  • hum-C (com) SEQ. ID.Nr. 20
  • hum-A hybridizes with the unmethylated sequence
  • hum-B2 hybridizes with the methylated sequence
  • hum-C hybridizes in both cases.
  • 25 ul hum-A [20 pmol / ul] 37.5 ul hum-B2 [20 pmol / ul]
  • 50 ul hum-C [20 pmol / ul] 672 ul AD
  • 100 ul DYN and 100 are used per batch ul dNTP's [2 M] used.
  • the PCR profiles were 33 cycles 95 ° C / 20 ⁇ , 54 ° C / 40 s, 72 ° C / 40 s [program 3].
  • Table 4 shows the results of a densitometric analysis of the multiplex PCR products, the results being shown in the gel shown in FIG. 4.
  • the numbering of the Bands in the gel of Fig. 4 correspond to the numbering of the columns in Table 4.
  • the net intensities are calculated from the ratios of unmethylated and methylated products, the mean (A + A ') / 2 being all the more 0.5 deviates (ie 50% of the total cell population), the more skewed. Patients 15, 16 and 19 are heavily skewed.
  • Multiplex-MS-PCRs are performed on patients in order to detect and evaluate the occurrence and extent of any aberrant methylation of the promoter region of FraX-A, FraX-E and FraX-F in a single reaction.
  • Table 5 shows the primers used [20 pmol / ⁇ l], the amounts of the substances used for the MS-PCRs and the expected product sizes, the concentration of the d TPs being 2 mM. 5 shows the result (in the form of an image of the gel) of these multiplex MS-PCRs [program 1], where A is the size standard, B normal women, C normal men, D male patients with FraX -E disease and E native non-deaminated DNA. It can be seen that compared to normal men, the diseased male patients show a band of the order of 248 bp, but no FraX-F bands (191 bp) occur. All patients show control (unmethylated promoter, PU).
  • the net intensity of the bands in the gel is measured, the strength of the individual bands from one another indicating the extent of the disease. If, for example, one of the bands specific to the methylated promoter (PM, FraX-E AB, FraX-F AB) is amplified in a ratio of 3: 2 compared to the other band, this is an indication of a disease. In the present gel in FIG. 5, all women have a ratio of 2: 2, from which it can be seen that these women are healthy (neither FraX-A, FraX-E nor FraX-F ill).
  • Example 5 Combination of a duplex PCR with a potentially methylated DNA region
  • a PCR [program 2] is used to amplify the polymorphic gene region (in the present case the FX repeat (RU, RM)) together with the potentially aberrantly methylated gene region (in the example the methylated FMR-1 promoter (FX-JK) ) carried out.
  • the band pattern and the band intensities as shown in Table 6, a disease can be concluded and, if necessary, the extent of the disease can be determined.
  • Table 7 shows the primers used and the PCR products to be expected.
  • FX-K 5 '-GGAAGTGAAATCGAAACGGAGTTGAGC-3' (SEQ ID NO 21)
  • FX-J 5 '-AACGTTCTAACCCTCGCGAAACAATACG-3' (SEQ ID NO 22)
  • Figure 6 shows the separation of the PCR products, where S is the standard, 21 a normal man, 22 a male patient with premutation, 23 a male patient with full mutation, 24 a normal woman (homozygous repeat), 25 a normal woman (heterozygous repeat), 26 a female patient with premutation, 27 a female patient with full mutation, 28 as negative control native undeamined DNA.
  • the unmethylated repeat In a normal man, only the unmethylated repeat is amplified. In a male patient with a premutation, the unmethylated expanded repeat is also amplified. In a male patient with a full mutation, the methylated promoter is amplified and, if appropriate, the methylated repeat (as already mentioned, the repeat is not amplified if it exceeds a certain size).
  • Normal women have an intensity ratio of 2: 2: 2 (methylated promoter: methylated repeat: unmethylated repeat), whereby homozygous women have two identical alleles (same repeat number), heterozygous women have different repeat lengths, so that two different alleles with different repeat lengths (2 x 1) can be seen.
  • Women with a premutation have a ratio of 2: 1: 1 (the expanded repeat cannot be amplified from a certain size or if it is amplified, the normal and the expanded repeat can be seen).
  • this embodiment of the method according to the invention ensures an accurate evaluation of the clinical picture, both in male and in female patients.

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Abstract

L'invention concerne un procédé pour la détection et l'évaluation d'une région D'ADN méthylée de manière potentiellement aberrante sur le chromosome X ou de la clonalité. Selon ce procédé, on détermine dans un échantillon l'étendue de la méthylation de la région d'ADN méthylée de manière potentiellement aberrante et éventuellement l'étendue de la méthylation dans une région D'ADN du chromosome X méthylée physiologiquement sûrement ou l'étendue de la méthylation ainsi qu'éventuellement la variante de séquence d'une région D'ADN polymorphe du chromosome X qui peut être méthylée soit physiologiquement soit de manière aberrante. Puis, on diagnostique, à partir de la comparaison des méthylations déterminées et éventuellement de la variante de séquence, la présence et éventuellement la variante et l'étendue de l'aberration potentielle ou de la clonalité.
EP00984601A 1999-12-03 2000-12-04 Procede pour la detection et l'evaluation d'une region d'adn methylee de maniere potentiellement aberrante sur le chromosome x ou de la clonalite Withdrawn EP1238108A2 (fr)

Applications Claiming Priority (3)

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AT0204199A AT408989B (de) 1999-12-03 1999-12-03 Verfahren zur detektion und evaluierung einer potentiell aberrant methylierten dns-region am x-chromosom oder der klonalität
AT204199 1999-12-03
PCT/AT2000/000324 WO2001040507A2 (fr) 1999-12-03 2000-12-04 Procede pour la detection et l'evaluation d'une region d'adn methylee de maniere potentiellement aberrante sur le chromosome x ou de la clonalite

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CN108715869A (zh) * 2018-06-01 2018-10-30 华南农业大学 一种基于获取特定供体细胞提高哺乳动物克隆效率的方法

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CN107245519A (zh) * 2017-06-06 2017-10-13 俞晓敏 一种pws及as的快速检测方法

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108715869A (zh) * 2018-06-01 2018-10-30 华南农业大学 一种基于获取特定供体细胞提高哺乳动物克隆效率的方法
CN108715869B (zh) * 2018-06-01 2020-12-08 华南农业大学 一种基于获取特定供体细胞提高哺乳动物克隆效率的方法

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