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

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.

SUBJECT OF THE INVENTION
 The invention is concerned with a screening method for diagnosing the
 etiology of spontaneous pregnancy loss in women. More particularly, the
 inventive method involves the use of quantitative X chromosome
 inactivation analysis for the prediction of the presence of gene-linked
 recurrent spontaneous pregnancy loss.
 BACKGROUND OF THE INVENTION
 Recurrent spontaneous abortion (RSA) is a major health concern to women,
 affecting about 17% of couples wishing to have children. The diagnostic
 evaluation of RSA is extensive and complex, with many different
 etiologies, each causing a small proportion of the total cases. The
 etiologies can be grouped into five categories: anatomic, infectious,
 hormonal, immunological, and genetic, thereby requiring the collaborative
 efforts of many medical specialists. Despite such thorough (and expensive)
 diagnostic workups, it has been estimated that the specific cause for RSA
 remains unknown in 37-79% of affected women (Stephenson, Fertil. Steril.
 66:24 (1996)).
 It has been assumed that a large portion of idiopathic RSA is genetic in
 origin. To date, however, the standard genetic evaluation consists solely
 of parental and abortus karyotyping. This identifies parental defects,
 such as balanced translocations that can cause RSA, and it ascertains
 fetal aneuploidy, a common cause of spontaneous abortion that
 intrinsically has little recurrence risk. As a result, the published total
 "genetic" contribution to RSA is essentiually the frequency of
 translocations in the cohort of women with RSA, which is only about 3%.
 This is quite likely an underestimation, as subcytogenetic defects are
 almost certainly a significant cause of RSA.
 It is possible that either autosomal or X-linked recessive lethal traits
 could cause RSA. Identification of recessive lethal traits has been
 difficult, as heterozygous carriers of such traits would appear
 phenotypically normal. A subset of lethal traits may cause an increased
 frquency of spontaneous abortions in carriers, if the X-linked hemizygous
 trait produces a clinically detectable pregnancy. Extended pedigrees in
 which women exhibit spontaneous abortion are available, but are difficult
 to analyze, because of the high population prevalence of spontaneous
 abortion and the assumed extensive genetic and etiologic heterogeneity.
 Thus, a method for clinically ascertaining carriers of autosomal lethal
 defects for recurrent spontaneous pregnancy loss remains problematical,
 although higly desirable.
 There have been many efforts to characterize single genes that may cause
 miscarriage. However, there are approximately 100,000 genes in the human
 genome, which makes the search for such a single gene an almost
 intractable problem.
 The present inventors have investigated the possibility that carriers of
 X-linked recessive lethal traits for recurrent spontaneous abortion
 manifest the molecular phenotype of nonrandom (skewed) X chromosome
 inactivation, and have invented a screening test for identifying and
 diagnosing a subset of women with recurrent spontaneous abortions. The
 test is based on a quantitative determination of the frequency of skewed X
 chromosome inactivation. This novel test, which is described below, can
 identify a lethal defect in a significant percentage of all of the genes
 in a woman. In essence, the test allows many of the lethal gene defects in
 the human genome to be surveyed at one time.
 SUMMARY OF THE INVENTION
 The screening test method of the invention provides for the identification
 and establishment of the risk for women who carry an X-linked lethal gene
 which increases their risk of recurrent spontaneous pregnancy loss.
 The inventive test involves the quantitative determination in female
 subjects of the frequency of highly nonrandom (skewed) X chromosome
 inactivation, compared to that of appropriate control women.
 The inventive test method is performed on DNA extracted from readily
 available cells from subjects, including, but not limited to, peripheral
 blood leukocytes, oral mucosal cells, muscle biopsies, or any other
 appropriate tissue.
 These and other aspects of the invention will become apparent by reference
 to the specification and claims below.

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.
 Additional embodiments, falling within the scope of the invention, may
 become apparent to the reader from the information provided herein.
 EXAMPLES
 Example 1
 Laboratory Methods
 DNA Extraction
 Peripheral blood was collected in EDTA tubes, and DNA isolated from
 leukocytes, as described elsewhere (Pegoraro et al., 1994, above).
 Muscle DNA was isolated from cryosections of muscle biopsies, as described
 elsewhere (Pegoraro et al., 1995, above).
 To isolate oral (cheek) mucosal cellular DNA, mouthwashing was done for 30
 s in about 10 ml of 3% sucrose, and the rinse collected in tubes
 containing 10 mM EDTA as a preservative. Cells were isolated from this
 suspension by centrifugation at 14,000.times.g for 20 min. Cells were
 solubilized in lysis buffer. Proteinase K digestion in PCR buffer was done
 at 60 C for 2 hr. Samples were extracted with phenol-chloroform and
 chloroform, and the DNA was concentrated by use of Amicon MICROCON
 microconcentrators.
 X-chromosome Inactivation Method
 X-inactivation patterns in the aforementioned DNA samples were quantified
 by the use of fluorescent PCR as described elsewhere (Allen et al., 1992,
 above; Pegoraro et al., 1994, above). In brief, the methylation status of
 the androgen receptor promoter adjacent to a highly polymorphic CAG repeat
 in the 5' end of the coding region of the androgen receptor gene was
 assessed by the use of methylation-sensitive restriction enzymes
 HpaII/C/oI). PCR products, both before and after digestion, were
 electrophoresed on an ABI 373A automated sequencer, and peak heights were
 analyzed by the use of GENESCAM software (Applied Biosystems). Corrections
 for preferential PCR of alleles, as well as spontaneous X inactivation,
 were done according to Pegararo et al., 1994, above. Ratios of at least
 90%:10% were used as the cut off points to classify the patient as having
 a highly skewed X inactivation pattern.
 Statistical analyses, cytogenetic analyses and physical mapping of DNA were
 done according to standard methods (Pegoraro et al., 1997, above).
 Example 2
 Association Between Idiopathic RSA and X Lethal Loci
 A limited case-control study was done using the techniques described in
 Example 1, above. In this study the frequency of highly skewed X
 chromosome inactivation in women with two or more unexplained spontaneous
 abortions, was compared to female controls. In this study skewed X
 chromosome was defined as preferential use of one chromosome in greater
 than or equal to 90% of peripheral leukocytes, and assumed that such
 extreme skewing reflects the carrier state for X-linked lethal conditions.
 It was found that 4 of 34 (11.7%) women with unexplained spontaneous
 abortions and one of 62 (1.6%) control women to have skewed X inactivation
 by these criteria. This represents a statistically significant odds ratio
 of 8.13 with 95% confidence limits of 55.9 to 1.18.
 These data demonstrate that the X chromosome inactivation assay is a
 powerful method for ascertaining cell-autonomous X-linked recessive lethal
 defects. Identification of such women is of significant clinical and
 counseling benefit.
 Example 3
 Association Between Idiopathic RSA and X-linked Lethal Loci
 In another study, similar to that described in Example 2, women
 characterized with a number of spontaneous abortions underwent a complete
 evaluation to rule out any of the known causes of RSA described above
 (Stephanson 1996, above). The tests performed were as follows:
 cytogenetic-parental and abortus karyotyping;
 anatomic-hysterosalpingogram; infections-cervical cultures for mycoplasma,
 ureaplasma, gonnococcus, and chlamydia; immunologic-anticardiolipin
 antibodies, antinuclean antibodies, and lupus anticoagulant; and,
 hormonal-serum progesterone, late luteal phase endometrial biopsy, and
 thyroid stimulating hormone.
 The controls were women from the same demographic region with no known
 history of spontaneous abortion.
 Further, the cases and controls were age-distribution matched, and the age
 distribution between the two groups was about the same.
 Defining X inactivation as preferential use of one X chromosome in at least
 90% of peripheral leukocytes, it was found that 7 (14.6%) of 48 subjects
 had skewed X chromosome inactivation (Table 1, FIG. 1), and only 1 (1.5%)
 of 68 control females demonstrating such inactivation (Table 1). This
 result is statistically significant (P&lt;0.01). The distribution ratios for
 both cases and controls are shown in FIG. 2.
 TABLE 1
 X Chromosome Inactivation Tests in Women with Recurrent
 Spontaneous Abortion
 X Inactivation
 Skewed &gt;90%
 Category n (%) Random Total
 RSA cases 7 (14.6) 41 48
 Controls 1 (1.5) 66 67*
 * P &lt; .01
 The skewing of X chromosome inactivation in RSA is shown in FIG. 1. Genomic
 DNA samples from women with RSA were subjected to PCR amplification of
 highly polymorphic HUMARA locus with fluorescent primers. One
 gravida5para0 (G5PO) woman was heterozygous at this locus (upper trace).
 Digestion of genomic DNA with methylation-sensitive restriction enzymes
 prior to PCR at the HUMANA locus permitted accurate quantitation of
 x-inactivation patterns (lower trace). The G5PO woman showed 100% skewing.
 X-inactivation analysis of HUMANA was performed as in Example 1. Use of
 the highly polymorphic HUMANA locus afforded 91% of individuals
 informative for the X inactivation assay method.
 The data are also presented in graphical form (FIG. 2). X-inactivation
 values are reported as the percentage of activity of the more-active
 alleles; thus, the data range is 50% to 100%, inclusive.
 Example 4
 Inheritance Pattern
 Although the method of the invention does not require knowledge of the
 chromosomal locus of the molecular trait associated with high spontaneous
 pregnancy loss, such information may provide additional specificity to the
 method, particularly in combination with the use of labeled polynucleotide
 probes.
 Pedigree analysis of one large family with a high incidense of RSA showed
 that all affected females were born to affected mothers; there was no
 male-to-female transmission, nor were there "isolated" cases (i.e.,
 affected female child but nonaffected mother).
 Because of this inheritance pattern, we tested an inheritance model of a
 fully penetrant monogenic X-linked dominant trait causing inactivation of
 the X chromosome harboring this trait. This involved an X-chromosome
 linkage search using 27 variable repeats spaced on an average of 10 cM
 apart on the X chromosome. Markers were typed on the ABI autosequencer, by
 use of fluorescent multiplex analysis. Two-point linkage analysis were
 performed, and LOD scores were computed. A statistically significant LOD
 score (&gt;2 for X-linked disorders) was obtained for marker DXS1108 in
 chromosome locus Xq28.
 Further testing of markers in Xq28 showed that an intronic CA repeat in
 intron 13 of F8C suggested a lack of inheritance of alleles bextwen all
 informative affected female family members, suggestive of a deletion. The
 highest two-point maximum LOD score (Zmax) w3as observed for marker F8C
 between the trait and this putative deletion (Zmax=6.92 at theta=0). All
 females who carried the deletion (either by noniheritance of F8C or
 linkage to DXS1108, or both) showed highly skewed X inactivation (at least
 95%). In addition, all the females scored as "unaffected" did not inherit
 the deleted X chromosome.
 No other marker outside the Xq28 region showed a positive LOD score. Other
 studies identified the proximal boundaries of the deletion mutation
 between introns 19 and 22 of F8C, while the distal boundaries of the
 deletion were in a 800-kb interval upstream of the 5' end of F8C.
 General Conclusions
 The present X chromosome inactivation assay affords a screening test by
 which female carriers of X-linked recessive lethal defects producing
 recurrent spontaneous abortion can be identified. By continuing to monitor
 women with RSA and their extended family members, the individual causative
 genes can be identified. The X-chromosome assay method should become an
 important diagnostic tool in the clinical evaluation of women with RSA, as
 secondary skewed X inactivation will be the common denominator by which
 carriers of X-linked recessive lethal traits can be identified.