Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-07-27
2001-04-10
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023740
Reexamination Certificate
active
06214558
ABSTRACT:
FIELD OF THE INVENTION
This invention provides methods useful for disease diagnosis by detecting chromosomal aberrations in tissue or body fluid samples. Methods of the invention are especially useful in detecting fetal chromosomal aberrations in blood or in amniotic fluid.
BACKGROUND OF THE INVENTION
Typically, prenatal diagnostic tests include fetal karyotype analysis. Traditional methods rely on invasive techniques, such as amniocentesis and chorionic villus sampling, both of which incur some risk of fetal injury or loss. See, e.g., Sundberg, et. al., Lancet, 350: 697-703 (1997). As an alternative to sampling the fetal environment, current techniques often involve isolating fetal cells from maternal blood. See, e.g., Liou, et al.,
Ann. N. Y. Acad. Sci
., 7: 237-241 (1994). Nucleated red blood cells are reported to be a good cell type for analysis because those cells have sufficient DNA for analysis, they are present in maternal blood, they are easily identified based on their morphology, and they have a known gestational life span. Lamvu, et al.,
Obstet. Gynecol. Surv
., 52: 433-437 (1997).
Whether taken from chorionic villi, amniotic fluid, or maternal blood, samples commonly are karyotyped to determine chromosomal abnormalities, such as aneuploidies. Cytogenic techniques with high-resolution banding frequently have been used for diagnosis of gross fetal chromosomal abnormalities. However, small chromosomal abnormalities (i.e., those involving changes of less than about 5 million base pairs) are difficult, if not impossible, to detect using gross cytogenetic tests. Accordingly, preferred techniques for diagnosis of both gross abnormalities and smaller nucleic acid mutations include molecular cytogenetic techniques, such as fluorescence in situ hybridization (FISH). For example, fetal cells from maternal blood have been isolated using density gradient centrifugation, and magnetic cell sorting, and counted using FISH. Jansen, et al.,
Prenat. Diagn
., 17(10): 953-959 (1997). The FISH technique, either alone, or in conjunction with fluorescent polymerase chain reaction, has become a primary technique for diagnosis of prenatal genetic abnormalities in samples from chorionic villi, amniotic fluid, blood, and whole cells, obtained, for example, from transcervical sampling. While FISH is adequate for screening for common chromosomal aneuploidies, such as trisomy 21, trisomy 13, XXY, and monosomy X, it is not useful for detecting chromosome deletions or rearrangements, which are common in, for example, chromosomes 12, 14, and 17. Lamvu, supra.
Accordingly, further techniques are necessary and desirable for detecting fetal chromosomal abnormalities. Such techniques are presented herein.
SUMMARY OF THE INVENTION
The present invention provides methods for detecting and diagnosing fetal chromosomal aberrations. Methods of the invention comprise detecting in a tissue or body fluid sample, a statistically-significant variation in fetal chromosome number or composition. Practice of the invention permits, for example, detection of aneuploidies, as well as partial chromosomal deletions, nucleic acid substitutions, rearrangements, additions, and the like. A preferred use of the methods is to reliably detect a fetal chromosomal aberration in a chorionic villus sample, amniotic fluid sample, maternal blood sample, or other tissue or body fluid. A fetal chromosomal aberration may be a reduction in chromosome number (e.g., XO), an increase in chromosome number (e.g., XXY, trisomy 21), a chromosomal translocation, a chromosomal deletion (e.g., loss of heterozygosity), or a chromosomal rearrangement. The invention takes advantage of several important insights which permit, for example, reliable detection of a chromosomal deletion or addition. Methods of the invention are useful for the detection and diagnosis of a nucleic acid abnormality, such as a loss of heterozygosity or, more generally, a gross chromosomal abnormality. Detection of such abnormalities may be indicative of a fetal disease or a developmental abnormality (e.g., proper morphological development, proper metabolic development, etc.).
For purposes of the present invention, unless the context requires otherwise, a “mutation” includes modifications, rearrangements, deletions, substitutions, and additions in a portion of genomic DNA or its corresponding mRNA.
In general, the invention comprises methods for counting (i.e. enumerating) the number of a target fetal chromosomal nucleic acid present in a sample. That number is compared with the number of a reference fetal chromosomal nucleic acid. The reference nucleic acid number may be determined from within the sample, or may be an external standard representing the numerical range considered to be indicative of a normal, intact karyotype (i.e., a diploid number of chromosomes). Methods of the invention determine whether any difference between the number of target and reference nucleic acids is statistically significant, a statistically significant difference being indicative of a fetal chromosomal abnormality.
A useful reference number of a nucleic acid is the number of molecules of a reference nucleic acid chosen such that the numbers of molecules of the target and reference nucleic acids are identical in normal fetal cells (i.e., cells not having an aneuploidy or mutation). The enumerative methods of the invention are useful to identify a statistically-significant difference between quantities of target and reference nucleic acids, and to correlate any difference, to a degree of defined statistical confidence, with the presence in the sample of an aneuploidy or mutation.
In a preferred embodiment, an enumerative amount (number of copies) of a target nucleic acid (i.e., chromosomal DNA or portion thereof) in a sample is compared to an enumerative amount of a reference nucleic acid. The reference number is determined by a standard (i.e., expected) amount of the nucleic acid in a normal karyotype or by comparison to a number of a nucleic acid from a non-target chromosome in the same sample, the non-target chromosome being known or suspected to be present in an appropriate number (i.e., diploid for the autosomes) in the sample. A statistically-significant difference between the two enumerative amounts is indicative of an aneuploidy or mutation in the target chromosome.
Also in a preferred general embodiment of the invention, an enumerative amount of a maternal allele on a chromosome is compared to an enumerative amount of the corresponding region of a paternal allele on the same chromosome. A statistically-significant difference between the two amounts is indicative of an aneuploidy or mutation in the chromosome.
In a preferred embodiment, enumerative detection of a chromosomal aberration is accomplished by exposing a nucleic acid sample to first and second radionucleotides. The radionucleotides may be single nucleotides or oligonucleotide probes. The first radionucleotide is capable of hybridizing to a target fetal chromosomal region (i.e., on a chromosome suspected to contain an aberration). The second radionucleotide is capable of hybridizing to a region of a reference fetal chromosome known not to contain an aberration. After washing to remove unhybridized radionucleotides, the number of each of first and second radionucleotides is counted. A statistically-significant difference between the number of first and second radionucleotides is indicative of a the fetal chromosomal abnormality. In a highly-preferred embodiment, fetal chromosomal material is isolated from maternal and other material prior to the exposing steps. Also in a highly-preferred embodiment, the number of the fetal chromosomal region is compared to a predetermined reference standard that provides a range of normal values. A target number within the range is indicative of a “normal” chromosome number for the target (i.e., the chromosome contains aneuploidies).
In preferred methods of the invention, first and second radionucleotides are isolated from other sample components by, for example, gel electrophoresis, chroma
Lapidus Stanley N.
Shuber Anthony P.
Exact Laboratories, Inc.
Houtteman Scott W.
Testa Hurwitz & Thibeault LLP
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