DNA sequences related to fragile X syndrome

Details DNA sequences related to fragile X syndrome DNA sequences related to fragile X syndrome

1993-09-09

2001-03-06

Zitomer, Stephanie (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C435S320100, C536S023100, C536S023500, C536S024310, C536S024330

Reexamination Certificate

active

06197500

ABSTRACT:

TECHNICAL FIELD
The invention relates generally to genetic diagnosis of humans. In particular, the invention concerns identification of individuals having particular DNA sequences predictive for Fragile X Syndrome.
BACKGROUND ART
Fragile X Syndrome is the most common form of familial mental retardation and affects about one in 2,500 children. The syndrome is characterized by the presence of a cytogenetically detectable fragile site in band q27.3 near the end of the long arm of the X chromosome which, if not the cause of the disorder, is closely associated with it. The diagnostic molecular genetics of the Fragile X Syndrome has been reviewed by Sutherland, G. R. et al. (
Clinical Genet.
(1990) 37:2-11). An additional review is found by Nussbaum, R. L. et al (
Ann. Rev. Genet.
(1986) 20:109-145).
Identification of the DNA spanning and including the fragile site has been reported by Kremer et al (
Am. J. Human Genetics
(1991) 49:656-661) and Heitz et al. (
Science
(1991) 251:1236). Characterization of the fragile site has indicated a particular region of instability within a 5.0 KB EcoRI restriction fragment, with the instability segregating with the Fragile X genotype (Yu et al.,
Science
(1991) 252:1179). The region of instability has further been localized to a 1 KB Pst I fragment containing a P(CCG)
n
repeat. The Fragile X genotype is characterized by an increased amount of unstable DNA that maps to the repeat (Kremer et al.,
Science
(1991) 252:1711). The availability of the cloned DNA makes possible the use of the DNA as a probe to detect length polymorphism of the p(CCG)
n
to characterize the genotype of an individual at that locus (Kremer et al., supra), thereby obviating problems with cytogenetic visualization at the fragile site (Webb et al.,
Prenatal Diagnosis
(1989) 9:771-781).
Additional diagnostic tools are available in the form of polymorphic microsatellite markers linked to the fragile site at Xq27.3 (FRAXA). Richards, et al., (
Am. J. Hum. Genet.
(1991) 48:1051-1057) have described polymorphisms associated with length variation in dinucleotide microsatellite repeats in the vicinity of Xq27.3. These markers have a recombination frequency of 1% and 7%, respectively, in two-point linkage analysis in 31 Fragile X families.
Thus, the availability of cloned DNA spanning the fragile site provides reagents uniquely suited for the detection of the Fragile X allele in appropriate subjects. Furthermore, techniques of gene therapy could be used to replace or compensate for the pathologic Fragile X sequence in affected cell types.
DISCLOSURE OF THE INVENTION
The invention provides a human DNA sequence corresponding to the Fragile X locus and provides a source for suitable probes for diagnosis and sequences useful for modification in therapy. The obtention of this sequence from the fragile site thus permits an improvement in diagnostic techniques as well as the possibility for genetic manipulation to overcome the disorder.
In one aspect, the invention is directed to an isolated and purified DNA molecule of no more than 275 kb which includes the fragile site. In another aspect, the invention is directed to a subsequence contained in this larger DNA of no more than 150 kb, which includes the fragile site. In still another aspect, the invention is directed to a DNA probe which crosses the fragile site, and to the corresponding normal sequence useful in replacement therapy.
In still other aspects, the invention is directed to methods to determine the presence or absence of the Fragile X allele in a subject which method comprises probing DNA isolated from the subject with the probe of the invention. Affected individuals appear to have an amplification of a (CCG)
n
repeat sequence at the fragile site which gives a band of different size than a normal individual when Southern blots are probed with the probe of the invention.
In another aspect, the invention is directed to oligonucleotides useful as primers in the polymerase chain reaction amplification of polymorphic microsatellite AC repeats closely linked to the Fragile X locus. Thus, these primers may be used to identify alleles of the microsatellite regions which vary in AC repeat length, thereby providing a method for screening for a microsatellite repeat sequence allele predictive of inheritance of the Fragile X allele.
In still another aspect, the invention is directed to methods to correct the fragile site by substituting the normal DNA contained in this region or otherwise compensating for this defect, such as by administration of the normal protein product or by antibodies directed against the protein product.


REFERENCES:
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patent: WO90/05194 (1990-05-01), None
patent: WO91/09140 (1991-06-01), None
patent: WO92/14840 (1992-09-01), None
patent: WO92/20825 (1992-11-01), None
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Grant R. Sutherland et al., “Diagnostic Molecular Genetics of the Fragile X”,Clinical Genetics, 1990:37 pp. 2-11.
R.L. Nussbaum et al., “Fragile X Syndrome: A Unique Mutation in Man”,Ann. Rev. Genet. 1986, pp. 109-145.
T.P. Webb, “Missed Prenatal Diagnosis of Fragile-X Syndrome”,Prenatal Diagnosis, 1989, vol. 9, pp. 777-781.
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D. Heitz et al., “Isolation of Sequences That Span the Fragile X and Identification of a Fragile X-Related CpG Island”,Science, Mar. 8, 1991, vol. 251, pp. 1236-1239.
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John A. Sved et al., “Population Genetic Consequences of the Fragile-X Syndrome, Based on the X-Inactivation Imprinting Model”,Am. J. Hum. Genet. 46:443-451, 1990.
Charles D. Laird, “Possible Erasure of the Imprint on a Fragile X Chromosome When Transmitted by a Male”,American Journal of Medical Genetics38:391-395 (1991).
Hermann-Josef Lüdecke et al., “Construction and Characterization of Band-Specific DNA Libraries”,Hum Genet(1990) 84:512-516.
Stephen T. Warren et al., “Isolation of the Human Chromosomal Band Xq28 Within Somatic Cell Hybrids by Fragile X Site Breakage”,Proc. Natl Acad. Sci. USA, vol. 87, pp. 3856-3860, May 1990.
I. Oberlé et al., “Instability of a 550-Base Pair DNA Segment and Abnormal Methylation in Fragile X Syndrome”,Science, vol. 252, pp. 1097-1102, May 24, 1991.
F. Rousseau et al., “Four Chromosomal Breakpoints and Four New Probes Mark Out a 10-cM Region Encompassing the Fragile-X Locus (FRAXA)”,Am. J. Hum. Genet., 48:108-116, 1991.
David T. Burke et al., “Cloning of Large Segments of Exogenous DNA into Yeast by Means of Artificial Chromosome Vectors”,Science, vol. 236, pp. 806-812, May 15, 1987.
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