WO1993018184A1 - Detection de la trisomie 21; materiaux et procedes - Google Patents

Detection de la trisomie 21; materiaux et procedes Download PDF

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WO1993018184A1
WO1993018184A1 PCT/GB1993/000437 GB9300437W WO9318184A1 WO 1993018184 A1 WO1993018184 A1 WO 1993018184A1 GB 9300437 W GB9300437 W GB 9300437W WO 9318184 A1 WO9318184 A1 WO 9318184A1
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chromosome
polynucleotides
nucleotide probe
cells
cosmids
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Malcolm Andrew Ferguson-Smith
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Lynxvale Ltd
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Lynxvale Ltd
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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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates to the detection of trisomy 21.
  • Down's Syndrome is one of the most commonly occurring conditions resulting from a chromosomal abnormality. Most usually it is caused by the inheritance of three chromosome 21s instead of the usual two. In some cases, it is caused by the inheritance of two complete chromosome 21s plus the inheritance of a part of a further chromosome 21 which is connected to a part of another chromosome.
  • hCG human chorionic gonadotrophin
  • AFP alphafetoprotein
  • the mother is then offered a fetal screen by amniocentesis.
  • This is a technique in which a sample of the amniotic fluid surrounding the fetus is withdrawn for analysis. Obviously since the technique is invasive, there is some risk (albeit small) that it could endanger the pregnancy.
  • the amniotic fluid contains small numbers of fetal cells.
  • the test for trisomy 21 involves standard cytogenetic analysis on these cells. The cells have to be cultured, both to increase the cell sample size and so that they are actively dividing. This is because the tests depend on the analysis of metaphase chromosomes in hypotonic- reated, acetic-alcohol-fixed, air-dried preparations. The number of chromosome 21s within each cell are then simply counted.
  • the problem with the fetal screen by amniocentesis is that it is time consuming and mothers have to wait for 2-3 weeks for results. The wait is understandably harrowing. Even more importantly, if trisomy 21 is detected, the pregnancy will be already somewhat advanced making elected abortion a more difficult and painful experience.
  • chromosome 21 analysis in interphase appears to be more difficult than for many other chromosomes because no reliable chromosome 21-specific repeat probe is available.
  • a number of nucleotide probes have already been used in an attempt to establish a quick, reliable diagnostic test for trisomy 21.
  • unique probes comprising selected single copy sequences from chromosome 21 may be used.
  • Plasmid based probes can only hold insert base sequences in the order of 5kb. The signals using such plasmid" probes are quite small and difficult to detect in comparison to non-specific background signals.
  • Cosmid based probes which can hold base sequence inserts in the order of 40kb are more useful. Nevertheless, they have been found to be only 50-70% reliable at best and this is still unsatisfactory for diagnostic purposes.
  • Yac (yeast artificial chromosome) based probes have the advantage that they can hold base sequence inserts of large size e.g. several hundred kb. In theory then, such probes should be more specific and therefore reliable.
  • a cosmid contig comprises 2 or more cosmids each holding a base sequence insert.
  • the individual base sequence inserts of each cosmid of the contig overlap with one another. In this way, the cosmid contig provides a probe which spans a stretch of nucleotide sequence greater than each individual cosmid insert.
  • the cosmid contig probe is for chromosome 21. It maps to 21q22 of chromosome 21 and may comprise a nucleotide sequence which hybridises with the chromosome 21 nucleotide sequence provided by probe pGSB3.
  • the present invention provides a nucleotide probe for the diagnosis of trisomy 21 which comprises a cosmid contig having at least first and second cosmids which contain different, but overlapping respective first and second polynucleotides which map to 21q22 and wherein at least one of the first and second polynucleotides comprises a nucleotide sequence which hybridizes with part or all of the 6.4kb EcoRI fragment in D21S19 which is characteristic of 21q22.3.
  • Both of the first and second polynucleotides may comprise a nucleotide sequence which hybridizes with part or all of the 6.4kb EcoRI fragment.
  • the cosmid contig may comprise at least a further cosmid which contains a respective further polynucleotide which overlaps with at least one of said first and second polynucleotides.
  • the further polynucleotide may overlap with both of said first and second polynucleotides.
  • the further polynucleotide may comprise a nucleotide sequence which hybridizes with part or all of said 6.4kb EcoRI fragment.
  • the present invention also provides a cosmid contig which comprises a plurality of cosmids which each contain different polynucleotides, each of the polynucleotides overlapping the polynucleotides of the other of said cosmids in the plurality, and mapping to 21q22 and wherein each of the polynucleotides comprises a nucleotide sequence which hybridizes with part or all of said 6.4kb EcoRI fragment.
  • the polynucleotides may comprise part or all of, or part or all of a substantially homologous variant of a chromosome 21 specific sequence as present in cosmids CCMP21.2, CCMP21.3, CCMP21.4, CCMP21.5 and CCMP21.6 which are each characterised by the restriction map as shown in figure 1.
  • the present invention also provides a nucleotide probe for the diagnosis of trisomy 21 which comprises a cosmid contig having at least first and second cosmids which contain different, but overlapping respective first and second polynucleotides which map to 21q22 wherein at least one of said polynucleotides comprises part or all of, or part or all of a substantially homologous variant of r a chromosome 21 specific sequence as present in cosmids CCMP21.2, CCMP21.3, CCMP21.4, cCMP21.5 and
  • the cosmid contig may comprise cosmids cCMP21.2 and CCMP21.6, the polynucleotides of which span a substantially 55kb portion of 21q22 of chromosome 21.
  • the cosmid contigs may be tagged with a detectable label.
  • kits and reagents having as a component a cosmid contig as provided by the present : " ivention.
  • the preparations may contain other materials for detecting the specific binding of the cosmid contig to chromosome 21.
  • ex vivo methods for the diagnosis of trisomy 21 comprise the steps of (a) isolating a sample of cells of fetal origin; (b) applying a cosmid contig as provided by the present invention to the cell sample; and (c) detecting the number of hybridization signals indicating the specific binding of the probe to chromosome 21 in the nuclei of individual cells in the sample.
  • the method may also comprise the step of washing to cell sample to remove any cosmid contig not hybridized to chromosome 21.
  • the cosmid contig probe as herein provided enables the identification of other sequences specific to chromosome 21 (and more specifically to 21q22 of chromosome 21) . These sequences will locate to either side of the chromosome 21 sequence which hybridises with the cosmid contig herein provided. This can be achieved by chromosome walking using cosmid end clones (Wahl GM et al 1987 Proc. Natl. Acad. Sci 84: 2160-2164). Any such sequences identified by use of the cosmid contig and which are specific to chromosome 21 and more particularly specific to the region 21q22 will also be useful as diagnostic probes.
  • the cosmid contig can be further extended by chromosome walking in which overlapping cosmids at the extremes of the contig are examined by restriction enzyme digestion and gel electrophoresis for the presence of fragments derived from the non- overlapping extremes of the two cosmids. These fragments are then excised from the gel, labelled and suitable fragments used to screen a cosmid library for new cosmids which will overlap with but extend out from the current contig.
  • cosmids from the extremes of the contig can be partially sequenced and oligonucleotides synthesised to allow polymerase chain reaction (PCR) amplification of DNA sequences unique to the original cosmids. Further cosmids from a library can then be screened by PCR with such primers to identify new cosmids which will overlap with but extend out from the current contig.
  • PCR polymerase chain reaction
  • the present invention also provides these further probes for trisomy 21 identified by use of the cosmid contig herein provided.
  • Figure 1 shows a restriction map for a 55kb cosmid contig based on restriction enzyme digestion with EcoRI, BamHI, Clal, Xhol and EcoRV in single and double digest combinations. The figure also shows the relative location of the EcoRI 6.4kb D21S19 insert;
  • Figure 2 shows flow-sorted chromosome 21 library, pBS-21, and DOP-PCR 21 paints hybridized with normal cultured cells.
  • (a,b) Hybridization with the chromosome 21 library showed signals on chromosome 21 and cross- hybridization signals on the other acrocentric chromosomes and diffuse domains in many interphase cells.
  • Figure 3 shows distributions of signals in cultured normal interphase cells (lymphocytes) hybridized with different types of chromosome 21-specific probes
  • Figure 4 shows hybridization of a YAC clone, HY70, with normal cultured cells.
  • (a,b) Hybridization with HY70 using human placental DNA as a competitor showed signals on chromosome 21q22 and cross-hybridization on the short arms of other acrocentric chromosomes; these cross hybridization signals in the interphase cells are similar to the true chromosome 21 signals.
  • Figure 5 shows a comparison of the hybridization efficiency of cosmids containing different sizes of insert.
  • the results suggest that the larger the insert, the more cells that showed complete hybridization;
  • Figure 6 shows the cosmid contig hybridization.
  • (a,b) Normal cultured cells showed signals on chromosome 21q22 and two compact signals in interphase nuclei.
  • (c,d) Uncultured amniotic fluid cells showed three signals in trisomy 21 cells and two signals in normal cells.
  • genomic DNA for library construction was partially digested with Mbol, dephosphorylated and fractionated on a 25-45% sucrose velocity gradient. The appropriate fractions were ligated into the Bam HI site of the cosmid vector pWE15, packaged and then plated out at a density of 125000 colonies per 200mm x 200mm nitrocellulose filter. Replica filters were produced from the primary filters for screening purposes and the master filters were stored on a 25% glycerol-containing medium at -70 C C. Library construction was essentially as described by Warner, J.P. et al 1992 in Molecular Genetic Analysis of Populations: a Practical Approach, Oxford IRL Press 189 223.
  • D21S19 is the Genome Database Accession number; the probe also has the designation pGSB3) specific to chromosome 21. Further information on the probe D21S19 is given in Human Gene Mapping II, London Conference, Cytogenet. + Cell Genet. Vol.58, Nos.3-4, 1991 Ed. Klinger H.P.
  • D21S19 is on public deposit at European Collection of Animal Cell Cultures (ECACC), PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wilts SP4 OJG, United Kingdom under accession number P92022713 (deposit made on 27 February 1991). Details of the probe are given below:
  • Figure .1 shows the five cosmids, their inter ⁇ relationships and restriction enzyme maps.
  • the complete 55kb contig is contained in the two overlapping cosmids CCMP21.2 and cCMP21.6. These two cosmids were used together as a probe as described hereafter.
  • the cosmid DNA was prepared according to a standard alkaline lysis method.
  • each individual cosmid contains a nucleotide sequence insert of approximately 40kb. Together however, they map a chromosome 21 nucleotide- sequence of approximately 55kb.
  • the nucleotide sequence inserts of cosmids CCMP21.3 and CCMP21.4 map within the nucleotide sequence insert of cosmid cCMP21.2.
  • the nucleotide sequence insert of cosmid CCMP21.5 maps within the nucleotide sequence insert of cosmid CCMP21.6. Therefore a contig based on cosmids CCMP21.2 and CCMP21.6 alone, will be sufficient to give the maximum available base coverage of chromosome 21.
  • restriction enzyme maps of the cosmids as shown in figure 1 provides a finger print which characterises the cosmids. Restriction enzymes were supplied by Boehringer Mannheim and used according to the manufacturers instructions. Use of the Cosmid Contig and comparitive studies
  • the chromosome 21 specific library (pBS-21) was constructed by subcloning Hind III digested inserts from a recombinant DNA phage library containing large inserts from chromosome 21, into Bluescribe plasmids.
  • the plasmid DNA was prepared according to standard methods, (iii) Chromosome 21 specific YACs HY70, HY94, HY7 and HY8 which map to 21q22 were supplied as a gift.
  • the insert sizes of both HY70 and HY94 are approximately 300kb.
  • the insert sizes of HY7 and HY8 are approximately 90kb.
  • YEPD 1% yeast extract, 2% trytone, 2% glucose
  • the cell pellet was resuspended in 5.0ml of 50 mM Tris pH 7.4, 20 mM EDTA, 1% SDS and incubated at 65°C for 30 min and centrifuged at 10,000 rpm for 10 min. The supernatant was transferred to a clean centrifuge tube and 2 x volumes of ethanol were added and the DNA pellet resuspended in 3.0ml of 10 mM Tris pH 7.4, 1 mM EDTA, 50 ug/ml RNase and incubated at 37°C for 30 min. Phenol/chloroform extraction was carried out, followed by precipitation with isopropanol. The DNA was centrifuged down and the pellet air dried and resuspended in TE (-50 mM Tris pH8.0, 1 mM EDTA).
  • the probe DNA was chemically modified by nick translation with biotin-11-dUTP (Sigma Chemical Co., Poole, Dorset, UK) and precipitated by ethanol. Labelled probe DNA was resuspended in TE to a final concentration of 100 ng/ul.
  • Chromosome 21 DOP-PCR paints were generated by DOP-PCR (Degenerate Oligonucleotide Primed Polymerase Chain Reaction) direct amplification of a small number of flow-sorted chromosome 21s.
  • Biotinylation was achieved by subjecting 300 ng of the primary PCR product in reaction supplemented with dUTP- 11-biotin to further amplification.
  • Uncultured amniotic fluid samples were obtained from East Boothn Regional Cytogenetics Laboratory. Approximately 1.0-2.0 ml of each amniotic fluid sample was used for in situ hybridisation. Samples were centrifuged down at 400g for 8 min. The cell pellet was resuspended in 2 ml of saline. 2 ml of methanol: acetic acid (3:1) was then added. Cells were re-centrifuged down, resuspended in 0.5 ml of methanol: acetic acid (3:1) and stored at -20"C for use. All the samples had been stored at least 6 months. The longest storage was 10 months.
  • Lymphoblastoid cell lines were used for chromosome preparation according to standard methods. Briefly, amethopterin to a final concentration 10"? molar was added to cultures which were ready for harvesting. Cultures were then incubated for a further 16-18 hours, BrdU (5-bromo,2-deoxyuridine) was added to the cultures to a final concentration of 10 ug/ml, followed by further incubation for 5 hours. Finally, Colcemid was added to a final concentration of lO ⁇ g/ml followed by incubation for a further 30 min. before harvesting. Cells were centrifuged at 200g for 8 min. , resuspended in 75 mM potassium chloride solution and incubated at 37°C for 8 min.
  • Amethopterin to a final concentration 10"? molar was added to cultures which were ready for harvesting. Cultures were then incubated for a further 16-18 hours, BrdU (5-bromo,2-deoxyuridine) was added to the
  • the slides were dehydrated in ethanol series (70%, 70%, 90%, 90%).
  • the hybridisation mixture (15 ⁇ l total volume consisting of 50-100 ng biotinylated probe DNA, 5-10 ug of human placental DNA, 50% formamide, 2xSSC, 10% dextran sulphate, 1% SDS and lx Denhardt's) was denatured at 70 C C for 10 min and incubated at 37°C for 30-60 min.
  • the hybridisation reaction was sealed under a coverslip and incubated at 42 C C for 14-16 hours. After hybridisation, the coverslips were removed by rinsing in 2xSSC and the slides were washed twice on 50% formamide/lxSSC at 42°C for 5 min, washed twice in 2xSSC at 42°C for 5 min and blocked in 4XTNFM (4xSSC, 0.05% Tween-20, 5% non-fat milk, spun to remove solid before use) at 37 C C for 20-30 min. The slides were then treated with alternating layers of fluoresceinated avidin and biotinylated goat anti-avidin, both at 5 ug/ml concentration in 4XTNFM buffer, for 30 min at 37°C until two layers of avidin were applied.
  • 4XTNFM 4xSSC, 0.05% Tween-20, 5% non-fat milk, spun to remove solid before use
  • the slides were washed three times at 42°C in 4XTNFM for 5 min. After the last washes in 4XTNFM, the slides were rinsed in 4XSCC, 0.05% Tween- 20 for 5 min, dehydrated by passage through an ethanol series and then mounted in 0.6 ug/ml propidium iodide (PI) and 3 ug/ml DAPI in Citifluor (Citifluor Ltd) for chromosome preparations and 0.6 ug/ml PI in Citifluor for preparations of uncultured cells.
  • PI propidium iodide
  • DAPI DAPI in Citifluor
  • cosmid contig equal amounts of cosmids CCMP21.2 and CCMP21.6 were used to provide the total DNA of 50-100ng per slide.
  • Fluorescence microscopy with appropriate filters was used to analyse the slides. Confocal laser scanning microscopy (MRC-600, Biorad Microscience Ltd) was used to collect and store the images. Approximately 200 cells were scored to give a distribution of signals in cultured interphase cells hybridised with the different types of chromosome 21 specific probes. The study of uncultured amniotic fluid cells by in situ hybridisation with the chromosome 21 cosmid contig was carried out in a blind study. Samples were coded and scored without knowledge of the karyotype. Approximately 50 cells per slide were counted, although approximately 100 cells were counted if the signal distribution suggested an abnormal result.
  • DOP-PCR paints showed more intense signals on chromosome 21, but suffered from the same problem of cross hybridisation signals as the pBS-21 library (Figs. 2c and 2d).
  • the results for signal distribution in the nuclei were similar to those obtained using the pBS-21 library (Fig 3) .
  • Hybridisation with YACs HY70, HY94, HY7 and HY8 on metaphase spreads showed hybridisation signals on chromosome 21q22. Hybridisation signals were also shown on the short arms of other acrocentric chromosomes when human placental DNA was used as a competitor DNA
  • Fig.4a The hybridization signals from YACs HY70 and HY94 were brighter on chromosome 21q22 than those from YACs HY7 and HY8. The signals in the interphase nuclei could not be interpreted, as cross hybridisation signals on other acrocentric chromosomes were easily mistaken for real chromosome 21 signals (Fig.4b). Hybridisation with YAC HY70 using total yeast DNA as competitor, only showed signals on chromosome 21q22 (Fig.4c) and two clear signals in approximately 65% normal interphase cells (Fig.4d and Fig.3).
  • Hybridization with the cosmid CCMP21.5 (insert size 35kb) showed two signals in 56% of normal cultured interphase cells and hybridization with the cosmid CCMP21.2 (insert size 46kb) showed two signals in 69% of normal cultured interphase cells.
  • hybridization with the cosmid contig showed two signals in 85% of normal cultured interphase cells.
  • the hybridization signals produced by the contig were intense and compact on chromosome 21q22 (fig.6a) and in interphase nuclei ( fig.6b) .
  • Fluorescence in situ hybridization with chromosome-specific repeat probes can detect the number of copies of particular chromosomes in interphase nuclei rapidly and without cell culture (e.g. Guyot B. et al., 1988, Prenat. Diagn. 8 ⁇ 435-493). This technique has practical application in prenatal diagnosis.
  • the reliability of the analysis by fluorescence in situ hybridization depends largely on the specificity of the probe and the hybridization efficiency.
  • chromosome-specific repeat probes are almost ideal for interphase analysis, as the hybridization signals produced by these probes are intense and well localized (e.g. Guyot et al., 1988 supra).
  • chromosome 21-specific library The successful detection of chromosome 21 abnormalities in metaphase and cultured interphase cells using a chromosome 21-specific library has been reported (Pinkel et al., 1988, Proc. Natl. Acad. Sci., 8_5, 9138-9142). However, the results have been found to be inadequate for uncultured amniotic fluid cells.
  • a comparison is made for different types of chromosome 21-specific probes for aneuploidy analysis of chromosome 21 in interphase cells.
  • the chromosome 21-specific library, pBS-21 showed hybridization signals on chromosome 21 and cross-hybridization signals on other acrocentric chromosomes.
  • Cosmids and YAC clones have large inserts and could be used as alternatives to repeat probes.
  • the YAC clone (HY70) showed intense hybridization signals on chromosome 21q22, but also signals on the short arms of other acrocentric chromosomes when human placental DNA was used as a competitor. These cross hybridization signals in interphase cells can be easily mistaken for the true chromosome 21 signals and make it impossible to determine the copy number of chromosome 21. They were shown to arise from yeast ribosomal repeat sequences, as total yeast DNA used as a competitor was found to suppress them (figs.4c and 4d).
  • amniotic fluid cells obtained by amniocentesis.
  • Cells isolated from uncultured amniotic fluid are of variable quality. Most of the cells (>80%) are degenerate sqaumous epithelial cells which are unsuitable for in situ hybridisation; others are covered by cytoplasm, which causes unacceptable background signals. This means that to be diagnostically useful and to avoid the culture requirement, the nucleotide probe must be both efficient and highly specific for chromosome 21.
  • the signal distributions in uncultured amniotic fluid cells were more variable between individual samples compared with the signal distributions in cultured cells. Trisomy 21 cases can be detected easily, as 34-51% of trisomic cells showed three signals and 31-39% of cells showed two signals compared with ⁇ 10% of normal cells which showed three signals and >60% of normal cells which showed two signals (table 1). Only 50 cells need to be counted to give a diagnostic signal distribution. In two samples, only 20-30 cells were available for analysis but the signal distributions clearly showed them to be normal. The successful diagnosis of the four trisomy 21 cases in the 49 uncultured amniotic fluid samples with this contig suggests that it is a suitable probe for diagnostic use. As analysis of uncultured cells has the potential to provide a result within 48 h or amniocentesis, it should improve the patient acceptability of second-trimester prenatal diagnosis.
  • the contig probe on fetal cells derived from amniotic fluid.
  • the probe may be effectively used on any cells of fetal origin.
  • the contig probe could be used on fetal cells isolated from maternal blood thus providing a diagnostic procedure which does not endanger the pregnancy.
  • the probe can be used on any source of fetal cells.
  • the use of the cosmid contig as herein described provides a reliable and robust probe for the diagnosis of trisomy 21. It offers the opportunity for rapid prenatal diagnosis of trisomy 21 as there is no requirement for cell culture.
  • the cosmid contig probe provided more reliable results than those obtained with the other types of probes. Distribution of Signals in Uncultured, Amniotic Fluid Cells Hybridized with the 21q Cosmid Contig

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Abstract

L'invention concerne des sondes nucléotidiques destinées au diagnostic de la trisomie 21. Ces sondes ont pour base des groupes de cosmides contigus. Ces cosmides contiennent des polynucléotides cartographiant jusqu'à 21q22.
PCT/GB1993/000437 1992-03-03 1993-03-03 Detection de la trisomie 21; materiaux et procedes Ceased WO1993018184A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0430402A2 (fr) * 1989-12-01 1991-06-05 The Regents Of The University Of California Méthodes et compositions pour la coloration de chromosomes particuliers

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0430402A2 (fr) * 1989-12-01 1991-06-05 The Regents Of The University Of California Méthodes et compositions pour la coloration de chromosomes particuliers

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AMERICAN JOURNAL OF HUMAN GENETICS vol. 51, no. 1, July 1992, CHICAGO, USA pages 55 - 65 K. KLINGER ET AL. *
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