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The origin of the extra Y chromosome in males with a 47,XYY karyotype
Introduction
Results
Discussion
Materials And Methods
Subjects
Molecular analysis
Acknowledgements
References
The origin of the extra Y chromosome in males with a 47,XYY karyotype
Received June 16, 1999; Revised and Accepted September 1, 1999
The presence of an extra Y chromosome in males is a relatively common occurrence, the 47,XYY karyotype being found in ~1 in 1000 male births. The error of disjunction must occur either during paternal meiosis II or as a post-zygotic mitotic error, both of which are rare events for other chromosomes. It is therefore of interest to determine when errors of Y chromosome disjunction occur. It is possible to distinguish between the different mechanisms of non-disjunction by analysing DNA polymorphisms at the distal tip of the Xp/Yp pseudoautosomal region in 47,XYY males, their parents and in some cases paternal grandparents. A cohort of 28 non-mosaic 47,XYY males was analysed. The results show that there are at least two mechanisms causing non-disjunction of the Y chromosome. In 16 of the 19 cases from which parents were available, the extra Y was generated by non-disjunction at meiosis II after a normal chiasmate meiosis I. Three cases were due to either a post-zygotic mitotic error or non-disjunction at meiosis II after a nullichiasmate meiosis I. Of the nine cases with no parental DNA available, at least four were due to meiosis II non-disjunction following a normal chiasmate meiosis I.
INTRODUCTION
Trisomy occurs in ~4% of all clinically recognized pregnancies and is the most common chromosome abnormality in humans (1). Although most trisomies are the result of maternal meiotic non-disjunction, this is not so for all chromosomes. There is evidence for chromosome-specific mechanisms of non-disjunction; for example, chromosome 2 has a higher proportion of paternal errors than other autosomes, and most cases of trisomy 7 are post-zygotic mitotic (PZM) errors (2). In some cases, perturbations or absence of recombination are associated with non-disjunction both at meiosis I (MI) and meiosis II (MII). For example, Hassold et al. (3) concluded that most paternally derived 47,XXYs result from a meiosis with no X/Y recombination, and Lamb et al. (4) and Savage et al. (5) found that MII chromosome 21 non-disjunction was associated with aberrant recombination at MI.
The occurrence of a 47,XYY karyotype is relatively common, being found in ~1 in 1000 male births (6), and is the only aneuploidy which is not selected against before birth (7). Although the great majority of trisomies are the result of an error during maternal meiosis, most frequently at MI, an additional Y chromosome must be of paternal origin and can only arise by paternal MII non-disjunction or by PZM non-disjunction, as there is evidence that spermatogonia and/or spermatocytes with an additional Y chromosome are selected against during gametogenesis (8,9). Both MII non-disjunction and PZM non-disjunction of paternal origin are relatively rare events for the X chromosome and most autosomes (7), so it is of interest to know the mechanism generating the additional chromosome in 47,XYY males. There are three plausible mechanisms by which an extra Y chromosome can be generated: (i) non-disjunction at male MII following a normal chiasmate MI in which recombination occurs between the X and Y chromosomes within the Xp/Yp pseudoautosomal region (MII-C); (ii) non-disjunction at male MII following an MI in which recombination between the X and Y chromosomes does not occur (MII-NC); or (iii) PZM non-disjunction.
By analysing DNA polymorphisms at the distal tip of the Xp/Yp pseudoautosomal region (PAR) in probands, their parents and their paternal grandparents, it is possible in principle to distinguish between these three mechanisms, although in practice an MII-NC cannot be distinguished from PMZ non-disjunction unless grandparental DNA is available, and even then it is only possible in 50% of PZM cases (Fig. 1). During normal MI in males, a single recombination occurs in the Xp/Yp PAR between one chromatid of the Y chromosome and one chromatid of the X (10,11). If non-disjunction then occurs at MII, both the non-recombined and the recombined Y chromatids segregate into the same gamete. Thus in informative cases for markers distal to the recombination point, heterozygosity in the father is maintained in the 47,XYY offspring. All 47,XYY males with three alleles of a marker distal to where recombination occurs must therefore have arisen via an MII-C non-disjunction, both paternal alleles being present, and an allele from the maternal X chromosome. The occurrence of double cross-overs could lead to false assignment of the causative mechanism; however, although this can occur, it is thought to be extremely rare (12).
Figure 1. The origin of the extra Y chromosome in 47,XYY males. ABC, alleles of a polymorphism at the distal tip of the Xp/Yp PAR, distal to the site of the single X/Y recombination during MI. MII-C, meiosis II non-disjunction of the Y after a chiasmate MI. In 100% of such cases, the XYY proband carries paternal alleles A and B on the Y chromosomes. Allele B was transfered from the paternal X to one chromatid of the Y during MI recombination. Non-disjunction at MII results in both Y chromatids segregating to the same gamete, thus heterozygosity in the father is maintained in the XYY offspring. MII-NC, meiosis II non-disjunction of the Y following a nullichiasmate MI. In 100% of such cases, the XYY proband will carry paternal allele A on both Y chromosomes. The nullichiasmate paternal MI results in allele A remaining on both chromatids, thus heterozygosity (AB) in the father is reduced to homozygosity (AA) in the XYY offspring. PZM, post-zygotic mitotic non-disjunction. All such cases originate as a 46,XY conceptus, the single Y subsequently replicating. Assuming normal recombination at MI and transfer of the paternal X allele B to one chromatid of the Y, in 50% of cases the Y chromosome originates from the chromatid carrying the A allele and in 50% of cases from that carrying the B allele. Analysis of the paternal grandparents is necessary to ascertain paternal phase; the presence of the grandmother's allele indicates exclusively a PZM mechanism whereas the presence of the grandfather's allele does not distinguish between MII-NC and PZM cases.
47,XYY males with only one of their father's distal Xp/Yp alleles, heterozygosity in the father being reduced to homozygosity in the offspring, must have arisen either via MII-NC non-disjunction in which there is no transfer of the father's distal Xp allele to one Yp chromatid, or via PZM non-disjunction in which two copies of the same Y segregate to one daughter cell.
It is possible to distinguish MII-NC non-disjunction from PZM non-disjunction but not in all cases. For PZM errors, it is assumed that normal X/Y pairing and recombination occur in the PAR during MI. For markers distal to the recombination, on average in 50% of cases the paternal grandfather's allele will be present and in 50% of cases the paternal grandmother's allele. However, MII-NC non-disjunction would result in both Y chromosomes carrying the same grandpaternal allele, as is the case with 50% of those generated via PZM non-disjunction. The presence of the paternal grandmother's allele therefore indicates a PZM origin of the extra Y chromosome, but when the paternal grandfather's allele is present it is not possible to distinguish between MII-NC and PZM.
In the present study, two polymorphisms, DXYS233 (13) and CA-SHOX (14), at the distal tip of the Xp/Yp PAR were analysed in a cohort of 28 47,XYY males and their parents, and paternal grandparents when available.
RESULTS
Results are shown in Tables 1, 2 and 3. Of the 19 cases in which parents were analysed, 18 were informative for at least one of the DXYS233 and CA-SHOX polymorphisms. The remaining case (no. 16) was informative for the DXYS228 polymorphism. Heterozygosity in the father was maintained in the 47,XYY offspring in 16/19 informative cases (Table 1). In three cases, heterozygosity in the father was reduced to homozygosity in the 47,XYY son. Paternal grandparents were available in only three cases (Table 2) of which two were due to MII-C non-disjunction and in the third the grandpaternal allele was present. In nine cases, parents of the 47,XYY proband were not available and, in four of these, three alleles were seen with one or both of the DXYS233 and CA-SHOX polymorphisms (Table 3). Of 15 cases showing three alleles with DXYS233 or CA-SHOX at the distal end of the PAR, 12 were informative for DXYS228 at the proximal end of the PAR. All 12 showed reduction of heterozygosity in the father to homozygosity in the proband, thus confirming that the expected recombination within the PAR had occurred (data not shown). One case (no. 16) was uninformative for DXYS233 and CA-SHOX but had three alleles at the DXYS228 locus, indicating that recombination must have occurred proximal to DXYS228.
Table 1. Results of the analysis of 19 47,XYY probands with polymorphisms DXYS233 and CA-SHOX at the distal end of the PAR
| Case | Polymorphism | Alleles | R/NR | Causative mechanism | ||
| Mother | Father | Proband | ||||
| 1 | DXYS233 | 1.3 | 1.2 | 1..2 | ni | MII-C |
| CA-SHOX | 1.2 | 2.3 | 1.2.3 | NR | ||
| 2 | DXYS233 | 2 | 1.3 | 1.2.3 | NR | MII-C |
| CA-SHOX | 1 | 1.2 | 1.2 | ni | ||
| 3 | DXYS233 | 1.2 | 1.3 | 1.3 | ni | MII-C |
| CA-SHOX | 2 | 1.3 | 1.2.3 | NR | ||
| 4 | DXYS233 | - | 1 | 1 | ni | MII-C |
| CA-SHOX | 1.4 | 2.3 | 1.2.3 | NR | ||
| 5 | DXYS233 | 2.4 | 1.3 | 2.3 | R | MII-NC/PZM |
| CA-SHOX | 1.2 | 1.3 | 2.3 | R | ||
| 6 | DXYS233 | 1.3 | 1 | 1 | ni | MII-C |
| CA-SHOX | 2.3 | 1.4 | 1.3.4 | NR | ||
| 7 | DXYS233 | 3 | 1.2 | 1.2.3 | NR | MII-C |
| CA-SHOX | 1.2 | 3 | 2.3 | ni | ||
| 11 | DXYS233 | - | 1.3 | 1.2.3 | NR | MII-C |
| CA-SHOX | - | 1.2 | 1.2 | ni | ||
| 12 | DXYS233 | 1.3 | 1.2 | 1 | R | MII-NC/PZM |
| CA-SHOX | 1.4 | 2.3 | 1.2 | R | ||
| 13 | DXYS233 | 1 | 1.2 | 1.2 | ni | MII-C |
| CA-SHOX | 1.4 | 2.3 | 1.2.3 | NR | ||
| 14 | DXYS233 | 2.3 | 1.3 | 1.3 | ni | MII-C |
| CA-SHOX | 2.3 | 1.2 | 1.2.3 | NR | ||
| 15 | DXYS233 | - | 1 | 1.2 | ni | MII-C |
| CA-SHOX | - | 2.3 | 1.2.3 | NR | ||
| 16 | DXYS233 | 2.3 | 1 | 1.2 | ni | MII-C |
| CA-SHOX | 2.3 | 1 | 1.3 | ni | ||
| DXYS228a | 1.2 | 1.3 | 1.2.3 | NR | ||
| 17 | DXYS233 | 2.3 | 2.4 | 2 | R | MII-NC/PZM |
| CA-SHOX | 1.3 | 1.2 | 1 | R | ||
| 18 | DXYS233 | 1.2 | 2.3 | 1.2.3 | NR | MII-C |
| CA-SHOX | 2.3 | 1.2 | 1.2 | ni | ||
| 19 | DXYS233 | 2.3 | 3.4 | 3.4 | ni | MII-C |
| CA-SHOX | 2.5 | 3.5 | 2.3.5 | NR | ||
| 22 | DXYS233 | 2.5 | 1.4 | 1.2.4 | NR | MII-C |
| CA-SHOX | 1.2 | 2 | 2 | ni | ||
| 23 | DXYS233 | 1.2 | 1.2 | 1.2 | ni | MII-C |
| CA-SHOX | 1.3 | 2.4 | 2.3.4 | NR | ||
| 30 | DXYS233 | - | - | 1.2 | - | MII-C |
| CA-SHOX | 1.3 | 2.3 | 1.2.3 | NR | ||
aCase 16 was non-informative for DXYS233 and CA-SHOX but was found to have three alleles for DXYS228 near the proximal end of the PAR.
Table 2. Results of analysis of the three 47,XYY probands from whom parents and paternal grandparents were available
| Case | Polymorphism | Alleles | Causative mechanism | ||||
| Mother | Father | Proband | GF | GM | |||
| 17 | DXYS233 | 2.3 | 2.4 | 2 | - | 1.4 | MII-NC/PZM |
| CA-SHOX | 1.3 | 1.2 | 1 | - | 2.4 | ||
| 19 | DXYS233 | 2.3 | 3.4 | 3.4 | 3.4 | 1.3 | MII-C |
| CA-SHOX | 2.5 | 3.5 | 2.3.5 | 4.5 | 1.3 | ||
| 22 | DXYS233 | 2.5 | 1.4 | 1.2.4 | 3.4 | - | MII-C |
| CA-SHOX | 1.2 | 2 | 2 | 2.3 | - | ||
Table 3. Results of analysis of the nine 47,XYY probands from whom parents were unavailable
| Case | Polymorphism | Alleles | Causative mechanism |
| 20 | DXYS233 | 1.2.3 | MII-C |
| CA-SHOX | 1.2.3 | ||
| 9 | DXYS233 | 1.2.3 | MII-C |
| CA-SHOX | 1.2.3 | ||
| 10 | DXYS233 | 1.2 | ni |
| CA-SHOX | 1.2 | ||
| 24 | DXYS233 | 1 | ni |
| CA-SHOX | 1 | ||
| 27 | DXYS233 | 1 | ni |
| CA-SHOX | 1 | ||
| 29 | DXYS233 | 1.2 | ni |
| CA-SHOX | 1 | ||
| 26 | DXYS233 | 1.2.3 | MII-C |
| CA-SHOX | 1 | ||
| 21 | DXYS233 | 1.2 | MII-C |
| CA-SHOX | 1.2.3 | ||
| 25 | DXYS233 | 1.2 | ni |
| CA-SHOX | 1.2 |
DISCUSSION
The results show that the extra Y chromosome in 47,XYY males can be generated by at least two mechanisms. Of 19 informative cases where parents were analysed, 16 (84%) were the result of MII-C non-disjunction and three (16%) were the result of either PZM error or MII-NC error. On average, 50% of PZM cases can be distinguished from MII-NC non-disjunction by analysing DNA from the parents and paternal grandparents of the proband; however, in no cases were we able to do this. DNA from the paternal grandparents was only available for three cases (Table 2) of which two were due to MII-C non-disjunction. In the remaining case, where heterozygosity in the father was reduced to homozygosity in the 47,XYY offspring, the grandpaternal allele was present and it was therefore impossible to distinguish PZM non-disjunction from MII-NC non-disjunction.
Of nine cases where parents were unavailable, four had three alleles for one or both of the distal Xp/Yp polymorphisms analysed; thus, of this group, at least 4/9 were caused by MII-C non-disjunction, the remainder being uninformative.
A comparison with paternal non-disjunction in other chromosomes is only possible with chromosome 21 because this is the only chromosome in which appreciable numbers of paternal MII or PZM errors have been identified. For both chromosome 21 and the Y, the extra paternally inherited chromosome is generated predominantly by MII-C non-disjunction. Savage et al. (5) analysed 38 cases of trisomy 21 of paternal MII or PMZ origin of which 28 (74%) were the result of MII-C non-disjunction and 10 (26%) were assumed to be of PZM origin. Although the numbers are not large, the figures for the current 47,XYY trisomy study are similar, with 84% being caused by MII-C non-disjunction and 16% caused by either MII-NC non-disjunction or PZM error.
The 10 trisomy 21 cases assumed to be of PZM origin by Savage et al. (5) could also have been caused by MII-NC error. However, this is unlikely in the majority of cases because studies of trisomy 21 of apparent PZM origin show equal numbers of maternally and paternally derived cases (5), which is consistent with a PZM event occurring randomly involving either chromosome 21 homologue. Furthermore, in those of maternal origin, there is no maternal age effect (15), which argues against a meiotic error. The evidence therefore suggests that for chromosome 21 these cases are much more likely to be of PZM origin.
Does this also suggest that those 47,XYYs shown to be the result of either MII-NC non-disjunction or PZM error are more likely to be caused by PZM error? Chromosome 21 and the Y are similar in size and, on average, only one recombination occurs on chromosome 21 during male meiosis, as is the case with the Y. However, there are distinct differences in the way in which pairing occurs during meiosis. Chromosome 21 pairs along its whole length, whereas the X and Y chromosomes are only homologous in the PARs. MII-NC non-disjunction seems inherently less likely because it requires two uncommon events. However, nullichiasmate MI is known to occur in male meiosis, Hassold et al. (3) concluding that most paternally derived 47,XXYs result from a meiosis in which the X and the Y did not recombine. If this was due to the X and Y not pairing at all during MI, then the Y could be misaligned on the metaphase plate. This misalignment could continue into MII, resulting in the two Y chromatids migrating to the same pole and generating a 24,YY gamete. There is also some evidence that perturbations of X chromosome recombination in MI are associated with non-disjunction at MII (16) although it is not known whether MI with no recombination predisposes to MII non-disjunction.
There seems to be no compelling weight of evidence either way to suggest whether either MII-NC non-disjunction or PZM error is more likely to occur in Y chromosome non-disjunction. The question can only be answered by analysing significant numbers of cases with paternal grandparents. If grandmaternal and grandpaternal alleles at distal Xp/Yp are represented in equal numbers, then we must assume that PZM non-disjunction is the predominant mechanism. If the grand-paternal allele is present exclusively, then MII-NC non-disjunction must predominate. If there are more cases with a grandpaternal than with a grandmaternal allele, the difference is likely to be the incidence of the latter mechanism.
Whatever the true incidence of MII-NC and PZM non-disjunction, it is clear that the great majority of 47,XYY males result from an MII-C non-disjunction.
MATERIALS AND METHODS
Subjects
The subjects of the analysis were 28 males with a non-mosaic 47,XYY karyotype as ascertained by cytogenetic analysis of peripheral blood cells. Nineteen patients were analysed together with parental DNA, and in three of these DNA from paternal grandparents was also available. In nine cases, a sample was obtained from the proband only.
Molecular analysis
DNA was extracted from whole blood using a standard salt precipitation technique (17). Four (CA)n repeat polymorphisms were analysed, DXYS233 (13) and CA-SHOX (14) at the distal end of the PAR and DXYS228 (13) as a control at the proximal end of the PAR. Primer sequences were as follows: DXYS233, F 5[prime]-TGATTTCCATCCTGGGGT-3[prime], R 5[prime]-GTGGGAATTCGAGGCTG-3[prime]; CA-SHOX, F 5[prime]-CATGTCATATATATATGTGATCC-3[prime], R 5[prime]-GACACAGAAATCCTTCATAAAAT-3[prime]; DXYS228, F 5[prime]-ATTAGCAGTTCACAGAGCCC-3[prime], R 5[prime]-ACGTGGGAGCAATAGTTCA-3[prime].
One primer of each pair was fluorescently end-labelled. PCRs were carried out with 2.5 ng/µl of each primer, 200 nM each dNTP, 5 ng/µl DNA template and 0.05 U/µl `Hotstar' Taq polymerase (Qiagen, Crawley, UK) using the manufacturer's buffer. Prior to thermal cycling, the Taq polymerase was activated by heating the reaction mix for 15 min at 94°C and amplification achieved by 30 cycles of 30 s at 94°C, 30 s at 55°C and 30 s at 72°C. After PCR amplification, the products were diluted 1 in 10 and 1 µl was loaded onto an ABI 377 automated sequencer.
ACKNOWLEDGEMENTS
We are very grateful to the patients and their families who made this study possible, to the clinicians who referred the cases to us, and to our colleagues within the Wessex Regional Genetics Laboratory who undertook all the cytogenetic analysis.
REFERENCES
+To whom correspondence should be addressed. Tel: +44 1722 336262 ext. 4080; Fax: +44 1722 338095; Email: drobinso{at}hgmp.mrc.ac.uk
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