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Rescue from the effects of trisomy 13q32→qter owing to skewed X inactivation in a der(X)t(X;13)(p21;q32) carrier
  1. Department of Molecular Medicine, Clinical Genetics Unit, Karolinska Institutet, S-171 76 Stockholm, Sweden

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    Editor—X;autosome translocations are very rare and occur at an estimated frequency of 1:300 000.1According to the hypothesis of Lyon,2 there is a random and irreversible inactivation of one of the two X chromosomes in the female, occurring at an early stage of development. In patients with an X;autosome translocation, X inactivation occurs at random but is followed by cellular selection, favouring the better genetic balance.3 Accordingly, nearly 95% of patients with balanced X;autosome translocations show a skewed inactivation of the normal X chromosome in almost all cells, thereby avoiding somatic monosomy or X chromosome disomy, while patients with unbalanced X;autosome translocations have the der(X) constantly inactivated in 91% of the cases in order to obtain the most optimal balance of the genome.1 We report here a woman who was referred for chromosome analysis because of four consecutive first trimester spontaneous miscarriages following the birth of a healthy daughter.

    The patient is short (152 cm), but otherwise healthy, with no dysmorphic features or malformations. Chromosome analysis of peripheral blood showed an apparently pure Xp deletion using conventional banding techniques. Chromosome microdissection of the aberrant Xp was performed according to Senger et al,4using an inverted phase contrast microscope (Axiovert 135) and a micromanipulator (Narishige MMO-2YD). Six fragments containing the whole aberrant Xp were excised and transferred to a 10 nl collection drop containing 10 mmol/l TRIS-HCl, pH 7.5, 10 mmol/l NaCl, 0.1% SDS, and 0.5 mg/ml proteinase K. After digestion with proteinase K, the collection drop was transferred to a 250 μl reaction tube containing 5 μl of PCR mixture: 5 μmol/l 6-MW-primer - 5′CCG ACT CGA GNN NNN NAT GTG G 3′ - , 200 μmol/l of each dNTP, 0.83 μl of Thermo sequenase buffer, and 4 U Thermo sequenase (Amesham Life Science). Degenerate oligonucleotide primed PCR (DOP-PCR) was performed in a Perkin Elmer Thermal Cycler 2400 according to Teleniuset al 5 with minor modifications. After initial denaturation at 96°C for five minutes, eight low temperature cycles were run including annealing at 30°C for one minute 10 seconds, 37°C for one minute, and 95°C for 30 seconds. Then 45 μl PCR mixture (1.1 μmol/l 6-MW, 220 μmol/l of each dNTP, 2.5 mmol/l MgCl2, 4.5 μl Stoffel buffer, and 5 U AmpliTaq DNA polymerase Stoffel fragment (Perkin Elmer, Cetus)) was added and 32 cycles of 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for one minute were run. The amplified material was labelled with Spectrum Orange-dUTP (Vysis) in 35 additional DOP-PCR cycles. Chromosome specific libraries from the X chromosome and chromosome 13 were PCR labelled with biotin-16-dUTP (Boehringer Mannheim) and Spectrum Orange (Vysis), respectively, and used for forward painting. FISH was performed as previously described.6 The X inactivation pattern was analysed by adding BrdU in late S phase during the last cell cycle before cell harvest, as described by Rooney and Czepulkowski.7

    Chromosome analysis showed a short p arm of one of the X chromosomes, which initially was interpreted as a pure terminal deletion (fig 1A, left). In order to confirm this, FISH analysis using an X chromosome specific library was performed, which failed to label the distal part of the p arm of the aberrant X chromosome (fig 1B). Chromosome microdissection of the aberrant Xp, followed by DOP-PCR amplification and reverse painting, showed an origin from 13q32→qter (fig 1C), and thus an unbalanced translocation 46,X,der(X)t(X;13)(p21;q32). This was also confirmed using a chromosome 13 specific library (fig 1D). X inactivation studies showed a skewed X inactivation, with the der(X) consistently inactivated in 44 analysed cells (fig 1A, right).

    Figure 1

    The normal X chromosome is indicated by an arrow and the der(X) by an arrowhead. (A) The patient’s X chromosomes, GTG banded (left) and after replication staining (right). (B) FISH on a metaphase from the patient using an X chromosome specific library. The distal part of the p arm of the der(X) is unlabelled. (C) Reverse painting on a metaphase from the patient using the microdissected library of the short arm of the der(X). As expected, the library labels the whole aberrant Xp, but also half the p arm of the normal X and the distal part of 13q. (D) FISH on a metaphase from the patient using a chromosome 13 specific library. Both chromosomes 13 are fully labelled (and there are unspecific signals on the p arms of the acrocentric chromosomes) as well as the distal part of the short arm of the der(X), indicating partial trisomy 13.

    X;autosome translocations are rare events that are most often combined with skewed X inactivation. We describe here a patient with partial trisomy of distal 13q owing to an unbalanced reciprocal translocation 46,X,der(X)t(X;13)(p21;q32), but apparently no clinical symptoms other than short stature and repeated spontaneous miscarriages. Partial trisomy 13q32→qter has previously been described in patients with severe mental retardation, epilepsy, microcephaly, microphthalmia, frontal bossing, haemangiomata, high arched palate, malformed ears, hexadactyly, umbilical and inguinal hernia, cryptorchidism, and congenital heart defects.8None of these symptoms was present in our patient who was referred for chromosome analysis because of repeated early spontaneous miscarriages. The most likely explanation is that the der(X) was found to be constantly inactivated, thereby spreading the inactivation to the small autosomal segment and thus eliminating any adverse effects of the partial trisomy 13q. In addition, the patient is monosomic for the distal half of the short arm of the X chromosome (Xp21→pter). Women with pure Xp deletions show various phenotypes, ranging from normal to Turner syndrome.9 Deletions distal to Xp21 are almost without exception found in patients with a normal female phenotype, who are often, albeit not consistently, short. This is in agreement with no Turner stigmata and short stature observed in our patient.

    The severity of an abnormal phenotype can be mitigated in whole or in part by skewed X inactivation which itself results from selection pressure. In patients with X;autosomal translocations, the skewed X inactivation prevents the development of symptoms by favouring the better genetic balance, which means that the normal X is preferentially inactivated in balanced cases, whereas the der(X) is consistently inactivated in the majority of unbalanced X;autosome translocations. Nevertheless, the percentage of cells in which the der(X) is inactivated may vary, as well as the extent of the spreading of inactivation into the autosomal segment.10 Indeed, variable spreading of inactivation between cells in the same person has been reported, which was shown to be the result of ineffective maintenance or instability of the inactivation.11 All these factors may have an influence on the phenotype, which makes it difficult to evaluate the risk of adverse effects in X;autosome translocation cases, for example, in the prenatal situation. Skewed X inactivation may also be the indirect mechanism of disease in female carriers of X linked disorders such as Duchenne muscular dystrophy and Fabry disease. This has been shown in cases of discordant monozygotic twins12-14 and affected carrier daughters of unaffected carrier mothers.15 The patient’s medical history of repeated spontaneous miscarriages may be explained by male unbalanced conceptuses with partial trisomy 13q as well as nullisomy for the distal part of Xp. The healthy daughter of our patient was found to have inherited the same unbalanced translocation from her mother and so far she has developed normally. Although her X inactivation status has not been studied, a similar pattern to her mother’s should be expected.

    In conclusion, apparently pure deletions of the X chromosome should be investigated further in order to exclude X;autosome translocations, which may have an influence on the phenotype. In the present case, we found an unbalanced translocation with partial trisomy of 13q32→qter using reverse painting, which, in contrast to forward painting, identified not only the chromosomal origin but also which part of chromosome 13 was involved. X inactivation studies showed that the der(X) was constantly inactivated and owing to the skewed X inactivation there were no serious symptoms apart from repeated early miscarriages.


    We thank Dr The-Hung Bui for valuable comments on the manuscript. This work was supported by the Swedish Medical Research Council and Magnus Bergvalls Stiftelse.