DE10102687A1 - Diagnostic kit for prenatal detection of trisomy 13, comprises primer pairs specific for amplification of short tandem repeat regions in chromosome 13 - Google Patents

Abstract

Diagnostic kit for detecting trisomy 13 in a human fetus comprises at least two pairs of oligonucleotide primers suitable for amplification, by polymerase chain reaction (PCR), of both strands of a target DNA sequence, i.e. a short tandem repeat region (X) of human chromosome 13. Independent claims are also included for the following: (1) microarray for detecting trisomy 13, especially a DNA chip, comprising an array of many separate fields, at least one of which carries an oligonucleotide able to hybridize to DNA that is part of X; and (2) method for detecting trisomy 13.

Description

The present invention relates to a diag nostic kit for the detection of a trisomy 13 one human fetus and a microarray therefor and a method for detecting a trisomy 13. Such methods, diagnostic kits and microarrays are required in prenatal diagnostics.

Usually the genetic information of Ge generation passed on unchanged to generation where in the gene inventory via the mechanism of "meiosis" part combined and handed down in each parent becomes. However, it can have certain properties of genes and mutagenic factors genes / mutations in the genetic material occur that according to the type, mode of origin and level of events can be classified differently. With so-called chro Mosome mutations or aberrations enter structure changes in the chromosomes. These aberrations  are mostly caused by errors in "meiosis" and can be numerical or structural in character to have. In the case of numerical aberration, it gives way either the number of a single chromosome (Trisomy, monosomy) or that of an entire chromosome rate (polyploidy) from the norm. One of the most The most common trisomy is trisomy 13 because in the case of trisomy 13 live births affected Fetuses are observed.

Trisomy 13 occurs with an average frequency from 1: 5000 live births. Most be affected children die in the 1st year of life, only 10% are getting older. The average lifespan is only few months. The main features of trisomy 13 are Wed. Cro- or anophthalmia, hypotelorism, one or double-sided cleft lip and palate, Holopros encephaly, scalp defects, deep-seated and dyspla static ears, postaxial polydactyly, heart defects and malformations of the genitourinary system.

The currently used methods of prenatal Trisomy 13 diagnostics are almost entirely based on invasive procedures such as B. amniocentesis or the chorionic villus, and are accordingly with a high health risk for mother and that adolescent child connected. In addition, they are Time available analysis or detection methods for trisomy 13 such as B. cytogenetic analyzes or fluorescence in situ hybridization is very expensive intensive and time-consuming, so that they are not routine ßig in all pregnant women but only in pre- are from corresponding medical indications be performed.

The object of the present invention is therefore to develop methods  a diagnostic kit and a microarray To provide with those with no major risks a trisomy 13 of the fetus for the fetus or mother simple way can be detected.

This task is performed by the diagnostic kit according to An saying 1, the microarray according to claim 19 and that Method according to claim 23 solved. Advantageous Wei further developments of the diagnostic kit according to the invention, the microarrays according to the invention and the inventive his procedures are dependent in the respective Given claims.

The detection of trisomy 13 according to the present Invention is based on a quantitative PCR method and involves the amplification of on chromosome 13 localized so-called "short tandern repeats" (STR's) or "mini satellite DNS's". It acts hypervariable, short DNA sequence motifs (2-4 bp) in the form of so-called tandem repetitions, i.e. H. successive units with a total length of 100-1000 bp. The use of automati sizable fluorescence-based analysis methods leaves an inexpensive and very short time Sample processing, so that the Ver routinely accessible to all pregnant women can be made. In conjunction with one too Methodology according to the invention for non-invasive gain There is also no need for fetal, nucleated cells all health risks for mother and that growing child.

The non-invasive trisomy 13 according to the invention Diagnostic procedures have two important aspects. For one, 13 is used for trisomy detection quantitative multiplex PCR method used  advantageously three different STR-DNA regions of chromosome 13 specifically amplified. benefit the three STR areas are suitable for this D13S631, D13S258 and D13S303. Such STR DNA Regions are extremely heterogeneous, making a diploid Individual usually two different STR alleles contributes to the creation of two PCR fragments of different lengths but the same quantity. In the case of a triploid individual, the three lead Copies of chromosome 13 to either generate three PCR fragments of different lengths (the 3rd Alleles are completely heterogeneous or heterozygous), or there are only two different PCR amplificates generated, but in a ratio of 2: 1 or 1: 2 (2 out of 3 alleles are homogeneous or homozygous). This happens in rare cases called "equivocal" fragment generation, d. H. the Amplification of a specific area of STR DNA leads to the generation of a singular fragment. In In this case it is not possible to make a statement about To meet diploidy / triploidy since both two there could also be three copies of chromosome 13, carry the alleles of the same size. To make this "Diagno to exclude them "is advantageously a Multiplex PCR method proposed in which three STR DNA regions of chromosome 13 simultaneously to be analyzed.

The second aspect concerns the enrichment / isolation fetal cells from peripheral maternal blood or from amniotic fluid. Advantageously, he this is followed by an accumulation of fetal, nuclear or DNA-containing erythrocytes from a maternal blood sample since this is already in early pregnancy stages reproducible and in relatively high amounts in circulate maternal blood.  

The accumulation of fetal cells can advantageously len from a maternal blood sample (whole blood, blood plasma, blood serum) or from amniotic fluid of the cheek ren, such as fetal erythrocytes from pe peripheral, maternal blood, through a Percoll Density gradient centrifugation or by FACS flow cytometry is done.

The following are some examples of the invention inventive kit and the inventive method given.

Fig. 1A shows the results from control blood of healthy volunteers;

FIG. 1B the results of control of blood Triso mie 13 patients;

Fig. 1C, the results of pure water as a control.

Figure 2A shows the results from anion fluid in a healthy fetus;

FIG. 2B the results from Anmionfluid in a fetus with trisomy 13 and

The following are two examples of the richness fetal erythrocytes from peripheral maternal Given blood.

In a first example, the enrichment takes place by Percoll ™ density gradient centrifugation. With a constant centrifugal force and mediumvis cosiness, the sedimentation rate of particles is proportional  to particle size. This regularity uses the density gradient centrifuge method gation, in this example Percoll ™ as Zen centrifugation medium is used. At Percoll ™ han it is a silica derivative that comes standard for the enrichment / separation of subcellular particles is used.

First, a continuous Percoll gradient is created that covers a density range of 1.02-1.13 g / ml. For this, 14 ml of a Percoll ™ NaCl solution (0.15 M NaCl), which has a density of 1.07 g / ml, are centrifuged for 30 min at 20,000 g in a type F 0630 fixed-angle rotor (Beckman Coulter). Subsequently, the continuous gradient thus generated is layered with 5 ml of EDTA whole blood, ie in EDTA buffer for example in standardized, commercially available EDTA tubes, whole blood, and centrifuged for 15 min at 1000 g. After this centrifugation step, the platelets remain in the serum layer above the gradient and are removed with a Pasteur pipette. A second centrifugation step at 1000 g for 15 min finally leads to the separation of the remaining, different blood cell types according to their respective isopycnic densities. The sedimentation layer with mononuclear blood cells is then removed with a Pasteur pipette, washed three times with phosphate buffer saline (PBS, pH = 7.4) and finally resuspended in 1 ml PBS. Phosphate buffer saline (PBS, pH = 7.4) contains KCl 0.2 g / l, KH ₂ PO ₄ 0.2 g / l, NaCl 8.0 g / l and Na ₂ HPO ₄ in aqueous solution 1.15 g / l and is sold, for example, by Life Technologies (Cat.-No .: 14190-094). The DNA of the mononuclear blood cells is then isolated by the standard procedures of the "QIAamp Blood Kit" (Qiagen, Hilden) and can be used for a multiplex PCR.

In a second example, the fetal, core containing erythrocytes by means of FACS flow cyto metry (FACS = fluorescence-associated cell sorting) isolated. First, everyone is enriched mono-nuclear blood cells using Percoll density gradient centrifugation (see above). The sediments tation layer with mononuclear blood cells is there taken with a Pasteur pipette, three times with Phosphate buffer saline (PBS, pH = 7.4) washed and finally resuspended in 1 ml PBS.

Following the density gradient centrifugation, 800 μl of the blood cell suspension obtained are for 60 min at 4 ° C. with a phycoerythrin-conjugated monoclonal antibody, manufacturer: BD PharMingen (Cat.No .: 32595A) clone: GA-R2 (HIR2) isotype: Mouse IgG _2b , K against the glycophorinA surface protein and a fluorescein isothiocyanate (T9-FITC) -conjugated monoclonal antibody, manufacturer: BD PharMingen (Cat.-No .: 30984X) clone: CB38 (NL07) isotype: Mouse IgM , κ incubated or labeled against the transferrin receptor (CD36). A third fluorescence channel is used for the negative discrimination of T, B and NK cells. The final concentration of both antibodies is 0.2 µg per 10 ⁷ cells. The labeled cells are washed twice with PBS and then 10 ⁷ cells are resuspended in 1 ml PBS.

A is used for the isolation of nucleated erythrocytes FACS Vantage SE flow cytometer (Becton-Dickinson) used. The system is water-cooled Double wavelength argon laser (emission wavelengths  488 nm and 365 nm - UV) and an air-cooled Helium neon (HeNe) laser configured and mitfluo resected beads (Becton Dickinson) calibrated. The CellQuest program, manufacturer: Becton Dickinson, Version: 3.3 (Cat.-No .: 342182) is used for the data acquisition, instrument control, as well as the statistical evaluation. Sorting the blood cells len takes place with cell rates of 20000-25000 cells per second, the cell size ("forward scatter"), and the granularity, as well as the surface structure ("side scatter"), the emission of green fluorescence (Transferin receptor, T9-FITC), the emission of the orange fluorescence (GlycophorinA, KC16-Rd) as well as the Red fluorescence emission for the remaining para meters, such as CD45, CD3, CD19 and CD16 / 56 (APC or Cy5 marked) can be used as selection criteria. To further discriminate against nucleated The core dye will ensure cells Hoechst 33342 used. The suggestion is made simultaneously with the UV line of the argon laser. The sorted cells are placed directly in a 1.5 ml reactor transfer container filled with 1 ml PBS and can be stored at -20 ° C.

Again, the DNA of the mononuclear blood cells then through the standard procedures of the "QIAamp Blood Kit "(from Qiagen, Hilden) isolated and for a Multiplex PCR as described below be set.

With the isolated DNA from nucleated fetal A red cell multiplex PCR is carried out. The primers were given in Table I. Sequences used. Kits according to the invention included at least one of the primers given in Table I. pairs. Here a kit was used, which all in  Table I contained primer pairs. The Pri PTR13S631F and PTR13S631R are forward (F) - and Reverse (R) primer to amplify the STR D13S631, the primers PTR13S258F and PTR13S258R for amplification on the STR D13S258 and the primers PTR13S303F and PTR13S303R to amplify the STR D13S303. In the The "Marking" column is on for the respective primer given whether and with which fluorescent dyes he is marked. Furthermore, in the fourth column of Table I the size of the amplified section in Base pairs specified. As can be seen, lie the respective amplified sections are sufficient apart so that they are uniquely identified that can.

Table I

PCR primers

The PCR was then carried out with the in Table II given parameters. A PCR Mastermix used, its composition in Tabel le III is specified. The PCR was carried out on a PCR block cycler PE 9700 from Applied Biosystems carried out.  

Table II

PCR parameters

Table III

Reaction batch (reaction volume 50 µl)

Taq buffer refers to a Taq DNA polymerase Storage Buffer (Taq Buffer B), which is in aqueous solution contains: Tris-HCl 20 mM (pH = 8.0 at 25 ° C), KCl 100 mM, EDTA 0.1 mM, DTT 1 mM, glycerol 50% (v / v), Tween 20 0.5% (v / v), Nonidet-P40 0.5% (v / v) and at for example from Promega Corp. (Cat.-No: M166B) ver is driven.

The results of the PCR were evaluated using ABI fragment analysis. For this purpose, the individual PCR batches were initially used undiluted, 1 µl being used for the ABI fragment analysis. Control blood from healthy volunteers (see FIG. 1A) and from trisomy 13 patients (see FIG. 1B) was initially used as the PCR template. The control blood of trisomy 13 patients was typed here for verification by standard cytogenetic methods.

The documentation of this fragment analysis can be seen in FIG. 1. In Fig. 1A is seen immediately that the fragments', 2 and 2 ', denoted by 1 and 1 or 3 or 3', respectively generate approximately the same high fluorescence signals. With 1 , 1 'the fragments are designated by the primers PTR13S631 (F and R), with 2 , 2 ' the fragments by the primers PTR13S258 (F and R) and with 3 , 3 'the fragments amplified by the primers PTR13S303 (F and R).

From Fig. 1A it can thus be seen that there are only two chromosomes 13 Lich here, ie that the pro band has no trisomy 13 .

In Fig. 1B it can be seen that the fragments designated 1 , 1 'are in a size ratio of 2: 1. Furthermore, in the range between 240 base pairs and 280 base pairs and between 440 bp and 470 bp, a triple of fragments 2 , 2 ', 2 "or 3 , 3 ', 3 " can be seen, which show approximately the same signal strengths. Both the ratios of the fragment pair 1 , 1 'and the fragment triple 2 , 2 ', 2 "and 3 , 3 ', 3 " clearly indicate the presence of a trisomy 13 in the patient examined here.

In Fig. 1C, a control is shown, was measured in which only pure water. It should be noted that there was no significant amplification of DNA here and therefore no significant signals can be seen in the relevant areas.

Fig. 2, in which the same elements are denoted by the same reference numerals as in Fig. 1, shows the results of an examination of samples of Amnion liquid ( Fig. 2A and 2B). As a starting material for the extraction of DNA, amniotic fluid was used in this example, as is obtained in amniotic centering. The DNA isolation was carried out completely as in the example above using the "QIAamp DNA Blood Kit" from Qiagen, Hilden. The further amplification and evaluation of this sample, he followed exactly the same protocol as described for FIG. 1. An ABI fragment analysis was then carried out, the results of which are shown in FIGS . 2A and 2B.

2A is as shown in FIG., There are the time signals jewei 1, 1 'and 3, 3' of the amplifizerten fragments about the same. Furthermore, only a single signal 2 can be seen, which, however, is about twice as high. It is therefore a healthy fetus whose cells in the amniotic fluid have been determined.

In Fig. 2B it can be seen that the fragments, which are denoted by 1 , 1 ', are in a size ratio of 2: 1. Furthermore, in the range between 240 base pairs and 280 base pairs and between 440 bp and 470 bp, a triple of fragments 2 , 2 ', 2 "or 3 , 3 ', 3 " can be seen, which show approximately the same signal strengths. Both the ratios of the fragment pair 1 , 1 'and the fragment triple 2 , 2 ', 2 "and 3 , 3 ', 3 " clearly indicate the presence of a trisomy 13 in the patient examined here.

Claims (32)

1. Diagnostic kit for detecting a trisomy 13 of a human fetus
at least two pairs of oligonucleotides (reverse primer, forward primer),
wherein the two oligonucleotides of each pair are suitable as primers for amplification by means of the polymerase chain reaction in each case one of the two complementary strands of a sought DNA segment, and
wherein the DNA segment sought is a short tandem repeat region (STR DNA region) of human chromosome 13. 2. Diagnostic kit according to the preceding claim, characterized in that it contains a total of three different pairs of oligonucleotides (reverse primer, forward primer),
the two oligonucleotides of each pair being suitable as primers for amplification by means of the polymerase chain reaction in each case one of the two complementary strands of different short tandem repeat DNA regions of the human chromosome 13. 3. Diagnostic kit according to one of the preceding An sayings, characterized in that the short  Tandern repeat area D13S631, D13S258 and / or D13S303. 4. Diagnostic kit according to one of the preceding An sayings, characterized in that it is the for Performing a polymerase chain reaction contains required substances. 5. Diagnostic kit according to one of the preceding An sayings, characterized in that it is used for Performing a polymerase chain reaction required substances a buffer solution, Mag nesium chloride, deoxynucleotide triphosphates, and / or contains a heat-stable polymerase. 6. Diagnostic kit according to the preceding claim, characterized in that it is heat-stable Polymerase a polymerase from Thermus aquaticus (Taq polymerase) contains. 7. Diagnostic kit according to one of the preceding An sayings, characterized in that it as a Po a DNA sample with at least one egg contains the searched DNS sections. 8. Diagnostic kit according to one of the preceding An sayings, characterized in that at least one of each of the two oligonucleotides Pair of oligonucleotides with fluorophores  are marked. 9. Diagnostic kit according to the preceding claim, characterized in that the oligonucleotides of different pairs with different fluo rophores are marked. 10. Diagnostic kit according to claim 9, characterized records that the oligonucleotides of the first and of the second pair with different fluoropho are marked. 11. Diagnostic kit according to one of the preceding claims, characterized in that the oligo nucleotides of the pairs have the following sequences:
GGCAACAAGAGCAAAACTCT and TAGCCCTCACCATGATTGG and / or
ACCTGCCAAATTTTACCAGG and GACAGAGAGAGGGAATAAACC and / or
ACATCGCTCCTTACCCCATC and TGTACCCATTAACCATCCCCA 12. Diagnostic kit according to the preceding claim, characterized in that the oligonucleotide with the following sequence
ACCTGCCAAATTTTACCAGG
is labeled with the fluorophore 5'-NED. 13. Diagnostic kit according to one of the two preceding claims, characterized in that the oligonucleotide with the sequence
GGCAACAAGAGCAAAACTCT and / or
ACATCGCTCCTTACCCCATC
is labeled with the fluorophore 4,7,2 ', 4', 5 ', 7'-hexachloro-6-carboxyfluorescein. 14. Diagnostic kit according to one of the preceding claims, characterized in that it
instructions on how to perform DNS isolation and / or
instructions for performing the polymerase chain reaction and / or
contains instructions for performing a fragment analysis. 15. Diagnostic kit according to one of the preceding An sayings, characterized in that it is a Scheme for evaluating the measurement results obtained contains nisse. 16. Diagnostic kit according to one of the preceding An sayings, characterized in that there is a Nucleic acid microarray (DNA chip) contains where a number of cells (fields) on the array has and in at least one cell an oligonucleotide Kleotid is arranged, that with the searched DNA section hybridizes.   17. Diagnostic kit according to the preceding claim, characterized in that in at least one another cell of the microarray another oli gonucleotide is arranged and the sequence of the Oligonucleotides contained in at least one Cell is arranged to differ from the sequence of the distinguishes further oligonucleotides. 18. Do the diagnostic kit after either one the claims, characterized in that in at least two cells each an oligonucleo tid is arranged, the in different Arranged cells each with oligonucleotides different searched DNA sections hybridi Sieren. 19. Microarray for the detection of a trisomy 13, at for example DNS chip, with an arrangement of several separate cells (Fel dern), characterized in that in at least an oligonucleotide is arranged in a cell, that hybridizes to a stretch of DNA that Part of short tandem repeat areas of the human chromosome 13 is. 20. Microarray according to claim 19, characterized records that in at least one other cell another oligonucleotide is located where in the sequence of the oligonucleotide that in the  at least one cell is arranged from the sequence of the further oligonucleotide under separates. 21. Microarray according to claim 19 or 20, characterized ge indicates that in at least two cells each because an oligonucleotide is arranged, wherein the oligo arranged in different cells nucleotides with different searched DNA Hybridize sections. 22. Microarray according to one of claims 19 to 21, characterized in that the short tandem Repeat area of area D13S631, D13S258 and / or D13S303. 23. Method for detecting a trisomy 13 of a human fetus, characterized in that in a maternal blood sample or amniotic fluid at least two searched DNS sections, that are part of a short tandem repeat area (STR-DNA area) of the human chromosome 13, by a polymerase chain reaction using at least two different pairs amplified by oligonucleotides, wherein the two oligonucleotides of at least one Pair for amplification one of the com complementary strands of the two searched DNA Sections are suitable, and finally the DNA amplified quantitatively is pointed.   24. The method according to the preceding claim, because characterized in that before amplification the DNA-containing components in the maternal Blood sample or the amniotic fluid first be concentrated. 25. The method according to the preceding claim, there characterized by that for concentration sedimentation of the DNA-containing components the maternal blood test (blood set) or the Am ionic liquid is carried out. 26. Procedure according to one of the two previous Claims, characterized in that the on concentration of the DNA-containing components Lysis nucleated fetal contained therein Erythrocytes performed and then the centrifuged cell-free DNA and for the further diagnosis is obtained. 27. The method according to claim 23, characterized in net that the blood from the maternal blood sample plasma / blood serum is obtained and then the DNA sections searched for by a polyme rase chain reaction using the ent in the kit holding oligonucleotides are amplified and finally the amplified DNA is detected.   28. The method according to any one of claims 23 to 27, characterized in that as short tandem Repeat area of area D13S6311, D13S258 and / or D13S303 is used. 29. The method according to any one of claims 23 to 28, characterized in that to prove the plicated DNA from fluorescently labeled Fluorescence radiation emitted by oligonucleotides is detected. 30. The method according to any one of claims 23 to 29, characterized in that a kit according to a of claims 1 to 19 is used. 31. Use of a kit, an oligonucleotide Microarray and / or a method according to one of the preceding claims on prenatal, non-invasive detection of a trisomy 13 ei human fetus. 32. Use according to the preceding claim prenatal, non-invasive detection of a tri somie 13 of a human fetus from a ma ternal blood test or amniotic fluid.