Title:  Screening test for the lethal genetic trait of recurrent spontaneous pregnancy loss

United States Patent:  6,268,145

Assignee:  Children's National Medical Center (Washington, DC); University of Pittsburgh (Pittsburg, P)

Filed:  September 22, 1999

A screening test to identify women carrying a lethal genetic trait that predisposes to recurrent spontaneous pregnancy loss. The test method involves the quantitative determination of the frequency of highly skewed X chromosome inactivation in DNA derived from tissue cells of female patients, relative to appropriate normal control women. "Highly skewed" is defined as preferential use of one chromosome in at least 90% of the patient's cells being tested. Suitable test tissues include, but are not limited to, peripheral leukocytes, oral mucosal cells, and biopsy material.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Predictive Model

For the purposes of this application, "highly skewed (nonrandom) X-chromosome inactivation" is defined as follows. The X chromosome is unique in that it undergoes transciptional silencing (inactivation) to achieve sex-independent dosage equilibrium of most X-linked genes. X inactivation occurs early in embryogenesis, at the 64 to 100-cell stage. It is thought to be a stochastic event, that is, each cell is equally likely to inactivate either the maternal or paternal allele, and the choice of alleles is not influenced by neighboring cells. Therefore, in normal females, approximately half the cells transcribe genes on the maternally inherited X-chromosome, and the other half transcribe genes on the paternally inherited X chromosome.

Given this feature of X chromosome inactivation, it is unusual to find women with nonrandon (skewed) X inactivation, that is, the preferential use of either the maternally inherited or paternally inherited allele in a preponderance of that female's cells. When this phenomenon is observed, it may be associated with a defect on one of the X chromosomes.

The present inventors suggest that this model of cell selection during embryonic development, which also yields skewed X inactivation in an X:autosome translocation carrier, predicts that functional hemizygosity for even a single vital X-linked gene would also yield skewed X inactivation in a female carrier. Such a carrier may be spared any clinical phenotype, but, as the result of the selection against those cells missing the vital gene, she would manifest the the molecular phenotype of skewed X inactivation. All males are hemizygous for genes on the X chromosome and male pregnancies inheriting the maternal X chromosome with the lethal gene, would not be viable and would spontaneously abort sometime after conception. The inventors predict that, as such, female carriers of X-linked lethal traits will show extremely skewed X chromosome inactivation and may have recurrent pregnancy loss of a male fetus as a result. As these women would abort half of all male embryos their risk of spontaneous abortion increases from a population risk of 15% to a combined risk of up to 40% (15%+25%), assuming that carriers of X-linked lethal traits do not constitute a major proportion of all femsales who experience single pregnancy losses.

Invention Based on Model

Based on their testing of the model, the inventors have invented, and reduced to practise, a screening test for determining if women with recurrent spontaneous abortion (a.k.a., pregnancy loss, miscarriage) are genetically predisposed to pregnancy loss due to an X-linked recessive lethal gene mutation.

The screening test method determines quantitatively the X-chromosome inactivation patterns in women, and is used to identify those women who are preferentially using one chromosome (skewed inactivation), relative to previously tested appropriate normal controls.

As used herein, "highly skewed" is defined as preferential use of one X chromosome in at least 90% of the tissue tested, e.g., peripheral leukocytes. Higher cut off points, e.g., at least 95%, may also be employed to increase even further the reliability of the conclusions from X chromosome inactivation analyses.

The screening test method is carried out on DNA extracted from any convenient cells from a subject. Such convenient cells include blood peripheral leukocytes (Pegoraro et al. Am. J. Hum. Gen. 54:989 1994)), oral mucosal cells (Pegoraro et al. Neurology 45:677 (1995), and cryopreserved muscle biopsies (see, e.g., Lawton et al., J. Oral Pathol. Med. 21:265 (1992); Vogelstein et al, Cancer Res. 47:4806 (1987); Miller et al., Nucleic Ac. Res. 16:1215 (1988); and Pegoraro et al., Am. J. Hum. Gen. 61:160 (1997)), although other tissues may also be used for this purpose. Details of standard methods for DNA extraction from tissues are provided in these references.

X-inactivation patterns in DNA extracted from tissues may be quantified by the use of fluorescent PCR as described elsewhere (Allen et al., Am. J. Hum. Gen. 51:1229 (1992); Pegoraro et al., 1994, Pegoraro et al. 1997). These procedures are summarized in Example 1, below.

Statistical analyses of X-inactivation results may be performed as described in detail in Pegoraro et al., 1997. This same reference also provides details of standard methods for hypervariable-repeat analyses, linkage analyses, cytogenetic analyses, and physical mapping of polymorphisms.

Claim 1 of 7 Claims

We claim:

1. A screening test for identifying female carriers of a X chromosome-linked recessive lethal genetic defect leading to recurrent spontaneous pregnancy loss in said females, comprising determining the frequency of highly skewed X chromosome inactivation in female patients compared to control females, said X chromosome inactivation analyses being conducted on DNA from test cells derived from said women.

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