Mark C. Lanasa

Familial Skewed X Inactivation: A Molecular Trait Associated with High Spontaneous-Abortion Rate Maps to Xq28

We report a family ascertained for molecular diagnosis of muscular dystrophy in a young girl, in which preferential activation (> or = 95% of cells) of the paternal X chromosome was seen in both the proband and her mother. To determine the molecular basis for skewed X inactivation, we studied X-inactivation patterns in peripheral blood and/or oral mucosal cells from 50 members of this family and from a cohort of normal females. We found excellent concordance between X-inactivation patterns in blood and oral mucosal cell nuclei in all females. Of the 50 female pedigree members studied, 16 showed preferential use (> or = 95% cells) of the paternal X chromosome; none of 62 randomly selected females showed similarly skewed X inactivation was maternally inherited in this family. A linkage study using the molecular trait of skewed X inactivation as the scored phenotype localized this trait to Xq28 (DXS1108; maximum LOD score [Zmax] = 4.34, recombination fraction [theta] = 0). Both genotyping of additional markers and FISH of a YAC probe in Xq28 showed a deletion spanning from intron 22 of the factor VIII gene to DXS115-3. This deletion completely cosegregated with the trait (Zmax = 6.92, theta = 0). Comparison of clinical findings between affected and unaffected females in the 50-member pedigree showed a statistically significant increase in spontaneous-abortion rate in the females carrying the trait (P < .02). To our knowledge, this is the first gene-mapping study of abnormalities of X-inactivation patterns and is the first association of a specific locus for recurrent spontaneous abortion in a cytogenetically normal family. The involvement of this locus in cell lethality, cell-growth disadvantage, developmental abnormalities, or the X-inactivation process is discussed.

The X Chromosome and Recurrent Spontaneous Abortion: The Significance of Transmanifesting Carriers

If, as we have argued, a subset of female carriers of X-linked recessive lethal traits are genetically predisposed to spontaneous abortion, and if such carriers can reliably be ascertained via their skewed pattern X chromosome inactivation, it becomes possible to test the hypothesis that X-linked recessive lethal traits are a significant cause of RSA in the general population. Intrinsic to this argument are the assumptions that the trait is cell autonomous—that is, that it causes death or growth disadvantage to the cells with the mutant X active and that hemizygous males survive at least until the pregnancy is clinically observable through a positive bHCG test.To test this hypothesis, we have initiated a case-control study wherein we compare the frequency of highly skewed X chromosome inactivation in women with two or more unexplained spontaneous abortions to the frequency in female controls (Lanasa et al. 1998xSee all References1998). The women characterized with idiopathic RSA have undergone a complete evaluation to rule out any of the known causes of RSA described above (Stephenson 1996xFrequency of factors associated with habitual abortion in 197 couples. Stephenson, MD. Fertil Steril. 1996; 66: 24–29Abstract | Full Text PDF | PubMedSee all References1996). The controls are women from the same demographic region, with no known history of spontaneous abortion; furthermore, the cases and controls are age-distribution matched, so that the distribution of ages between the two groups is the same. Defining skewed X inactivation as preferential use of one X chromosome in ≥90% of peripheral leukocytes, we have found 7 (14.6%) of 48 to have skewed X inactivation. In contrast, only 1 (1.5%) of 68 control females exhibit this extent of nonrandom X inactivation. This finding is statistically significant, with P < .01 (Fishers exact test, one-tailed).The frequency of nonrandom X inactivation is somewhat lower (1.5%) in our control group than has been reported previously. Other groups have estimated that the frequency of skewed inactivation (at the level of ≥90% silencing of one copy of the chromosome) as 3.2% (Gale et al. 1997xAcquired skewing of X-chromosome inactivation patterns in myeloid cells of the elderly suggests stochastic clonal loss with age. Gale, RE, Fielding, AK, Harrison, CN, and Linch, DC. Br J Haematol. 1997; 98: 512–519Crossref | PubMedSee all References1997) or 3.5% (Plenge et al. 1997xA promoter mutation in the XIST gene in two unrelated families with skewed X-chromosome inactivation. Plenge, RM, Hendrich, BD, Schwartz, C, Arena, JF, Naumova, A, Sapienza, C, Winter, RM et al. Nat Genet. 1997; 17: 353–356Crossref | PubMed | Scopus (189)See all References1997) in women of the same age in the population at large. Although case-control comparisons across studies may be perilous, it is interesting to note that, even when these higher estimates of skewing frequency are used for the control group, the frequency we observe in our group of RSA-affected women remains significant at the level of P < .05 (Fishers exact test, one-tailed).Although the results presented here are clearly preliminary, it is interesting to speculate about the frequency of X-linked recessive lethal traits in the general population. Given a frequency of idiopathic RSA of 1 in 250 in the general population, and an affection rate of ∼1 in 7 in our case population, the population prevalence of X-linked lethals leading to RSA could be as high as 1 in 1,750. In fact, our ascertainment methodology will miss a large number of carriers, since, on average, a carrier would have to become pregnant five times to show two spontaneous abortions. Furthermore, there is great selective pressure against such traits. As X-linked recessive lethal traits can be passed on only to daughters, the carrier frequency should be halved in each generation. If 1 in 1,750, in fact, approximates the carrier frequency, then the new mutation rate must be 1 in 3,500. Since the highest-known single-gene–mutation rate is that of dystrophin at 1 in 10,000, a mutation rate of 1 in 3,500 indicates extensive genetic heterogeneity. This is consistent with the hypothesis that there are a significant number of vital genes on the X chromosome.RSA is a major womens health concern. As the application of molecular genetics to RSA advances, it will be possible to begin characterizing those genes that cause spontaneous abortion in the recessive state. The X chromosome inactivation assay affords a methodology by which female carriers of X-linked recessive lethal defects can be identified. Over time, then, by assembling familial pedigrees, the individual causative genes can be identified. The X-inactivation assay 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.

Reply to Migeon and Haisley-Royster

To the Editor:We thank Drs. Migeon and Haisley-Royster (1998 [in this issue])xFamilial skewed X inactivation and X-linked mutations: unbalanced X inactivation is a powerful means to ascertain X-linked genes that affect cell proliferation. Migeon, BR and Haisley-Royster, C. Am J Hum Genet. 1998; 62: 1555–1557Abstract | Full Text | Full Text PDF | PubMed | Scopus (21)See all References)Migeon and Haisley-Royster (1998 [in this issue]) for their interest in our research. We are, however, a bit puzzled by their letter to the editor, since they write that they disagree with the interpretation of our results yet then restate what was already written in our previously published article (Pegoraro et al. 1997xFamilial skewed X inactivation: a molecular trait associated with high spontaneous-abortion rate maps to Xq28. Pegoraro, E, Whitaker, J, Mowery-Rushton, P, Surti, U, Lanasa, M, and Hoffman, EP. Am J Hum Genet. 1997; 61: 160–170Abstract | Full Text PDF | PubMedSee all ReferencesPegoraro et al. 1997).The 50-member pedigree that we reported showed an X-linked dominant disorder with male lethality. There is no question of this fact, because we found a deletion mutation of Xq28 associated with skewed X inactivation and recurrent pregnancy loss (LOD = 6.92). The deletion included the factor VIII gene, yet there were no males from 50 females with factor VIII deficiency, again clearly proving that this family had an X-linked dominant disorder with male lethality.Drs. Migeon and Haisley-RoysterxFamilial skewed X inactivation and X-linked mutations: unbalanced X inactivation is a powerful means to ascertain X-linked genes that affect cell proliferation. Migeon, BR and Haisley-Royster, C. Am J Hum Genet. 1998; 62: 1555–1557Abstract | Full Text | Full Text PDF | PubMed | Scopus (21)See all ReferencesMigeon and Haisley-Royster appear to wish to address two issues: (1) interpretation of the likely mechanisms that would cause X-inactivation skewing in the females in this family; and (2) transcriptional timing of the deleted gene or gene products in Xq28 and the observed effect on miscarriage detection. There is very little to disagree with in Drs. Migeon and Haisley-RoystersxFamilial skewed X inactivation and X-linked mutations: unbalanced X inactivation is a powerful means to ascertain X-linked genes that affect cell proliferation. Migeon, BR and Haisley-Royster, C. Am J Hum Genet. 1998; 62: 1555–1557Abstract | Full Text | Full Text PDF | PubMed | Scopus (21)See all ReferencesMigeon and Haisley-Roysters interpretation of our results; they suggest that a growth disadvantage is probably playing a role, which is precisely what we stated in our discussion. We, too, feel that growth disadvantage is the most likely mechanism causing skewing of X-chromosome inactivation. However, in the absence of characterization of the causative genes in Xq28, it seems unreasonable to dismiss the possibility that the gene(s) may actually be involved in the process of X inactivation. This is the least likely mechanism, but it does not seem to warrant exclusion from discussion.The timing of transcription of the gene products in Xq28 undoubtedly affects when the miscarriage occurs. In fact, the issue of timing is central to the inferred genetic mechanism. A cell-lethal trait expressed very early in embryonic development would be undetectable or perhaps would cause a “biochemical pregnancy.” Activation later in embryonic life would still cause male lethality but would be less likely to cause complete skewing of X inactivation in multiple tissues in the heterozygous female. In view of this delicate balance in timing, we feel that the genes in question are most likely to be transcribed early in fetal development and to impart a growth disadvantage rather than being cell lethal. The size of the deletion mutation, however, is less important to when the miscarriage occurs: size is simply being used as a surrogate to the assumed importance of the deletion region and gene(s) contained in that region. In the end, this is all an exercise in mental gymnastics, since the characterization of the causative gene(s) will enlighten us all as to the true mechanism.