Human Reproduction, Vol. 14, No. 11, 2735-2738,
November 1999
© 1999 European Society of Human Reproduction and Embryology
Paternal sex chromosome aneuploidy as a possible origin of Turner syndrome in monozygotic twins: Case report
1 Dept de Biologia Cel.lular i Fisiologia, Unitat de Biologia, Facultat de Medicina, Universitat Autònoma de Barcelona, 2 Servei de Pediatria, Hospital Universitari Materno-Infantil Vall d'Hebrón, Barcelona and 3 Dept de Biologia Cellular i Fisiologia, Unitat de Biologia Cellular, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain
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Abstract |
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The meiotic or mitotic origin of most cases of Turner syndrome remains unknown, due to the difficulty in detecting hidden mosaicisms and to the lack of meiotic segregation studies. We have had the opportunity to study one pair of monozygotic twins concordant for Turner syndrome of paternal origin. The paternal origin of the single X chromosome was determined by polymerase chain reaction (PCR) amplification. No mosaicism was detected for the X or Y chromosome. In this case, a meiotic error during gametogenesis would be a likely origin of X monosomy. To determine if meiotic errors are more frequent in the father of these monozygotic twins concordant for Turner syndrome of paternal origin, molecular studies in spermatozoa were conducted to analyse sex chromosome numerical abnormalities. A total of 12520 sperm nuclei from the twins' father and 85338 sperm nuclei from eight normal donors were analysed using three-colour fluorescent in-situ hybridization. There were significant differences between the twins' father and control donors for XY disomy (0.22 versus 0.11%, P < 0.001) and total sex chromosome disomy (0.38 versus 0.21%, P < 0.001). These results could indicate an increased tendency to meiotic sex chromosome non-disjunction in the father of the Turner twins.
Key words: aneuploidy/spermatozoa/Turner syndrome/twins
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Introduction |
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Turner syndrome is one of the most common sex chromosome abnormalities, affecting an estimated 1–2% of all clinically recognized pregnancies (Jacobs, 1992
). Experimental evidence from restriction fragment length polymorphisms (RFLP) has demonstrated that 70–80% of 45,X cases are paternal in origin (Hassold et al., 1992; Lorda-Sánchez et al., 1992; Chu et al., 1994). The loss of a sex chromosome could occur as a result of non-disjunction or anaphase lag at either a meiotic division during gametogenesis, or an early cleavage division of a normal zygote. Some authors have suggested that most 45,X liveborns arise from a mitotic error in an early zygote and are actually cryptic mosaics for a normal cell line in some critical organ or tissue (Hook and Warburton, 1983; Held et al., 1992). However, the presence of X and Y cryptic mosaicism found in 45,X patients seems not to be so frequent (Lorda-Sánchez et al., 1992; Jacobs et al., 1997). So far, the meiotic or mitotic origin of most 45,X cases remains unknown due to the difficulty to detect hidden mosaicisms and to the lack of meiotic segregation studies. Monozygotic twinning in Turner syndrome is not frequent, although it has been reported previously (for review, see Machin, 1996). Usually, the twin pairs are discordant for Turner syndrome; in that case, the origin of the syndrome can be traced to a postzygotic malsegregation of chromosomes, resulting in sex chromosome mosaicism. On the other hand, the origin of monozygotic twins concordant for Turner syndrome is more likely to result from a meiotic error during gametogenesis.
In this paper, we present a case of monozygotic twins concordant for Turner syndrome and paternal in origin (loss of the paternal sex chromosome). In order to study the possible meiotic or mitotic origin of the X monosomy in these monozygotic twins we have carried out a sex chromosome aneuploidy analysis in the spermatozoa from their father, to find out if there is an increase in the rate of sex chromosome disomy/nullisomy. The analysis of paternal gametes may help to elucidate the mechanisms involved in the origin of Turner syndrome.
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Case report |
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Clinical report
The twins, two 13-year-old girls, were born after 38 weeks of a normal pregnancy. They were monochorionic and diamniotic. At birth, the father was 35 years old and the mother 33 years old. The mother had had two previous pregnancies: a miscarriage at 3 months and a healthy girl.
At 4.5 years of age, the twins were referred for clinical and laboratory analyses and follow-up. At that time, physical examination revealed that both twins had short stature, triangular face, low set ears, widely spaced nipples, micrognathia, short neck without pterygium, shield-like thorax, cubitus valgus and IQ (WISCH) of 98 and 72 respectively. No cardiovascular or renal abnormalities were detected. Standard cytogenetic studies of 50 metaphases from peripheral blood showed in each case only a 45,X cell population, and homozygosity tests demonstrated that both twins were identical for the 15 blood markers analysed, making the probability of monozygosity >99.95%.
All blood and semen donors involved in this study gave their informed consent prior to the study, which was approved by our institutional ethics committee.
Molecular studies
DNA extraction from peripheral blood of the Turner twins and their parents was carried out using a standard salt procedure (Miller et al., 1988). It was not possible to obtain permission to study fibroblasts or other somatic tissues. The parental origin of the single sex chromosome was determined by PCR amplification of five X chromosome microsatellites (Figure 1a): DMD49 (Clemens et al., 1991), DYS II (Feener et al., 1991), DXS1283E (Yen and Lin, 1994), AR (Mahtani and Willard, 1993) and DXS52 (Richards et al., 1991). DNA amplification was performed in a final reaction volume of 50 µl containing 1% of standard PCR buffer (Ecogen), 250 µmol/l of dNTPs (Perkin Elmer), 1.5 mmol/l MgCl2, 0.8 µmol/l of each primer (Research Genetics Inc.), 0.5 IU Taq polymerase (Ecogen) and 0.4–1 µg of DNA. Samples were processed in a Perkin Elmer thermal cycler from 24 to 30 cycles. Specific PCR conditions for each primer are described in Table I. Samples were run on a 6% acrylamide:bisacrylamide (19:1) gel at 12 mA for 13–16 h; the gel was then stained with ethidium bromide. DXS52 polymorphism was characterized on a 1% agarose gel owing to its size (700–3000 pb). Electrophoresis was carried out at 100 V for 1 h.
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Three Y chromosome-specific segments (Kocova et al., 1995
) were amplified by PCR to detect possible Y hidden mosaicism (Figure 1b
). Internal controls were simultaneously amplified with SRY (DYS II located in Xp), DYZ1 and DYZ3 (IR5 located in chromosome 19) (Seino et al., 1990).
Three-colour FISH in spermatozoa
Semen samples were obtained from eight healthy control men (from 22 to 55 years of age) and the Turner twins' father (48 years old). Six control donors were of proven fertility. The semen samples were processed for three-colour fluorescent in-situ hybridization (FISH) analysis of spermatozoa as previously described (Vidal et al., 1993).
We applied three-colour FISH with DNA centromeric probes for chromosomes X (Spectrum green, Vysis Inc.), Y (Spectrum orange, Vysis Inc.) and 6 (1:1 mix of Spectrum green and Spectrum orange, Vysis Inc.). FISH incubation and detection were performed according to manufacturer's instructions. Chromosome 6 provided an internal control to characterize diploid and disomic cells, as well as non-hybridized cells.
Slides were analysed under an Olympus AX70 epifluorescence microscope equipped with a FITC/Texas Red/DAPI triple-band pass filter and single-band pass filter for DAPI, Texas Red and FITC.
Scoring criteria
Sperm nuclei were scored only if they were intact and non-overlapped. Two spots of the same colour were scored as two copies of the corresponding chromosome when they were comparable in brightness and size and were separated from each other by a distance longer than the diameter of each signal. All ambiguous signals were examined by at least one additional independent observer.
Statistical analysis
To determine if there were any significant differences in disomy and diploidy frequencies among the donors, we used a two-tailed Fisher's exact test. The Bonferroni procedure was applied to adjust for multiple comparisons. A Spearman correlation test was employed to study the relationship between the control donor's age and the aneuploidy frequencies.
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Results |
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All five markers for the X chromosome were informative and showed that the twins only inherited one allele from their mother. No Y amplification was detected (Figure 1
). X chromosome and chromosome 19 fragments were simultaneously amplified with Y chromosome specific fragments as internal controls to avoid false-negative results.
We analysed 85338 sperm nuclei from control donors (~10000 per donor), and 12520 from the father of the Turner monozygotic twins by three-colour FISH. Hybridization and decondensation efficiency were in each case higher than 98%. Disomy, diploidy and nullisomy rates obtained in sperm nuclei for control donors and the twins' father are shown in Table II. Interdonor heterogeneity was found for control donors in diploidy and nullisomy frequencies. However, there was no correlation between donor ages and disomy rates.
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There were significant differences between the twins' father and control donors for XY disomy (0.22 versus 0.11%; P < 0.001), and total sex chromosome disomy (0.38 versus 0.21%; P < 0.001) and sex chromosome nullisomy (0.51 versus 0.32%; P < 0.01).
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Discussion |
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The twins reported in this study were monochorionic, diamniotic and monozygotic (with a probability >99.95%) and both displayed the full Turner phenotype. We have established, by PCR amplification, five X chromosome polymorphisms, indicating that the origin of the syndrome for these twins was paternal (loss of the paternal sex chromosome).
These observations suggest that the error leading to X-monosomy occurred before the twinning event, either during early embryo cleavage or paternal spermatogenesis. A mitotic error in an early embryo would produce two mosaic embryos (probably 45,X/46,XX), although the production of two non-mosaic 45,X embryos, in which the normal cell line would only contribute to extra-embryonic tissues, could not be rejected. On the other hand, a meiotic error during paternal spermatogenesis would lead to two non-mosaic 45,X embryos both concordant for Turner syndrome. The possibility of sex chromosome mosaicism has been ruled out in peripheral blood for these twins. Unfortunately, it has not been possible to analyse the placenta or the fibroblasts and we cannot absolutely exclude mosaicism.
The study carried out in the spermatozoa of their father and eight control donors has shown several differences in the sex chromosome aneuploidy rates. The frequencies of XY (0.22%) and total sex chromosome disomy (0.37%) from the twins' father were significantly increased compared with those obtained in our control donors. These data suggest an increase of meiotic errors in the segregation of sex chromosomes. The observed excess of XY spermatozoa has to be the result of non-disjunction during meiosis I, that would produce an excess of XY and nullisomic versus normal spermatozoa for sex chromosomes.
Thus, it seems that in these monozygotic twins, Turner syndrome would have originated through a meiotic error during paternal spermatogenesis. At any rate, this sex chromosome aneuploidy increase in the twins' father compared to control donors is moderate and we cannot exclude that it also could be attributed to individual variations in sperm aneuploidy described by several authors (reviewed by Downie et al., 1997; Egozcue et al., 1997). However, recently, a moderate increase for chromosome 21 disomy has also been found in the spermatozoa of fathers of Down's syndrome children (Blanco et al., 1998). Further studies of larger series of Turner syndrome patients will be needed to elucidate the origin of non-mosaic Turner syndrome.
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Acknowledgments |
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We gratefully acknowledge Dr D. Gallardo (Banc de Sang, Hospital de la Vall d'Hebrón de Barcelona) for performing the homozygosity test. This work received financial support from Dirección General de Enseñanza Superior, Ministerio de Educación y Cultura (DGICYT, project no. PB95-0655) and the Generalitat de Catalunya (CIRIT, 1997 SGR 00030).
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Notes |
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4 To whom correspondence should be addressed
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References |
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Submitted on April 12, 1999; accepted on August 17, 1999.
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