Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1991-08-29
2001-01-30
Zitomer, Stephanie (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S091200, C536S023500, C536S024330, C530S350000
Reexamination Certificate
active
06180337
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of molecular diagnosis of the fragile X syndrome.
BACKGROUND
The fragile X syndrome is the most frequently encountered form of inherited mental retardation in humans and has a prevalence estimated to be 1/1250 males. The fragile X syndrome segregates as an X-linked dominant disorder with reduced penetrance. Either sex when carrying the fragile X mutation may exhibit mental deficiency. It has been shown that approximately 30% of carrier females are penetrant and that 20% of males carrying the fragile X chromosome are normal but may transmit the disorder and have fully penetrant grandsons. In addition to the mental retardation which is variable in severity, penetrant males exhibit additional phenotypic involvement including macroorchidism and distinctive facies. Since fully penetrant males rarely reproduce, it has been suggested that the frequency of new mutations of the fragile X site may be as high as 1/3000 germ cells to maintain the population frequency.
The fragile X syndrome, as implied by its name, is associated with a fragile site expressed as an isochromatid gap in the metaphase chromosome at map position Xq 27.3. The fragile X site is induced by cell culture conditions which perturb deoxypyrimidine pools and is rarely observed in greater than 50% of the metaphase spreads. Neither the molecular nature of the fragile X site, nor its relationship to the gene responsible for the clinical expression of the syndrome is understood. However, based upon genetic linkage studies, as well as in situ hybridizations, the fragile X site and its associated gene are tightly linked if not coincident.
The present application provides a new procedure for detecting the fragile X site at the molecular level. It provides a molecular method for the diagnosis of the fragile X syndrome, describes a unique open reading sequence at the suspected gene locus and provides probes to the fragile X region.
SUMMARY OF THE INVENTION
An object of the present invention is a method for diagnosing fragile X syndrome.
A further object of the present invention is the provision of a sequence of the FMR-1 gene.
An additional object of the present invention is a method of detecting the fragile X syndrome by measuring the mRNA or protein from the FMR-1 gene.
Another object of the present invention is a method of detecting the fragile X syndrome by measuring CGG repeats.
A further object of the present invention is a method of detecting the fragile X syndrome by measuring the methylation associated with a CpG island.
Thus in accomplishing the foregoing objects there is provided in accordance with one aspect of the present invention as a composition of matter, a 3.8 kb cDNA clone containing the FMR-1 gene. A further aspect is a 5222 bp genomic DNA sequence containing at least a fraction of the FMR-1 gene.
A further embodiment of the present invention is a group of cosmid probes for the selection of the FMR-1 gene in the fragile X syndrome.
An additional embodiment of the present invention is a method of detecting fragile X syndrome comprising the steps of digesting DNA from an individual to be tested with a restriction endonuclease and detecting the restriction fragment length polymorphism with hybridization to probes within the fragile X locus and southern blot analysis. In a preferred embodiment of the present invention, the probe is pE5.1 and the restriction endonucleases are selected from the group consisting of EcoR I, Pst I, Xho I and BssH II.
Alternate embodiments of the present invention include detecting the fragile X syndrome by measuring the expression of the FMR-1 gene either as the amount of mRNA expressed or as the amount of FMR-1 protein produced. Another embodiment of the present invention includes a method of detecting X-linked disease comprising the steps of detecting variation in the (CGG)
n
repeat at the 5′ end of the FMR-1 gene by measuring the length of the repeat, wherein n for normal ranges between 16 and 30 and n for X-linked disease is greater than 30. A variety of methods are available to detect the dosage measurements of the repeat. These procedures can be selected from the group consisting of visual examination, densitometry measurement, quantitative radioactivity and quantitative fluorescence as well as pulsed field gel electrophoresis and fluorescence in situ hybridization.
Other and further objects, features and advantages will be apparent and eventually more readily understood from a reading of the following specification and by reference to the accompanying drawings forming a part thereof, wherein examples of the presently preferred embodiments of the invention are given for the purpose of the disclosure.
REFERENCES:
Vincent et al. Nature vol 349 pp 624-626 Issued Feb. 14, 1991.
D. Heitz et al Science vol 251 pp 1236-1239 Issued Mar. 8, 1991.
Ludecke et al. Nature vol 338 pp 348-350 Issued Mar. 23, 1989.
M. Pieretti, et al., “Absence of Expression of the FMR-1 Gene in Fragile X Syndrome”Cell66:817-822 (1991).
J.S. Sutcliffe, et al., “DNA methylation represses FMR-1 transcription in fragile X syndrome”Human Molecular Genetics1:397-400 (1992).
D. Devys, et al., “The FMR-1 protein is cytoplasmic, most abundant in neurons and appears normal in carriers of a fragile X premutation”Nature Genetics4:335-340 (1993).
H. Siomi, et al., “The Protein Product of the Fragile X Gene, FMR1, Has Characteristics of an RNA-Binding Protein”Cell74:291-298 (1993).
C. Verheij, et al., “Characterization and localization of the FMR-1 gene product associated with fragile X syndrome”Nature363:722-724 (1993).
D.P.A. Kuhl, et al., “Fragile X Syndrome Protein FMRP Associates with the Microfilament Fraction of the Cellular Cytoskeleton” Submission to Cell.
Caskey C. Thomas
Fu Ying-Hui
Nelson David L.
Oostra Ben A.
Pieretti Maura
Baylor College of Medicine
Fulbright & Jaworski LLP
Zitomer Stephanie
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