Am. J. Hum. Genet. 49:1034-1040, 1991

A Molecular Study of X Isochromosomes: Parental Origin, Centromeric Structure, and Mechanisms of Formation Isabel Lorda-Sanchez, Franz Binkert, Marco Maechler, and Albert Schinzel Institute of Medical Genetics, University of Zurich, Zurich

Summary Fourteen individuals with an i(Xq) or idic(Xq) were studied using RFLP analysis in order to determine both parental origin and extent of heterozygosity of the isochromosome and to search for the presence of shortarm material. In five cases the isochromosome was paternally derived, while nine patients had a maternal i(Xq). The analysis of heterozygosity of the nine maternally derived isochromosomes by using Xq markers showed heterozygosity in two cases, suggesting an origin from two homologous X chromosomes. Homozygosity was found at all informative loci in seven cases, which therefore are probably the product of either centromere misdivision or sister-chromatid exchange. Presence of Xp markers was seen both in the three i(Xq) chromosomes which appeared dicentric by cytogenetic analysis and in three additional cytogenetically monocentric cases. Mean parental ages were greater for the maternally derived cases as compared with the paternally derived cases.

Introduction

The isochromosome of the long arm of the X chromosome, i(Xq), is the most common structural aberration found in patients with Turner syndrome (Schmid et al. 1974). Although the classical definition of an isochromosome implies a single functional centromere separating two arms which are mirror images of one another, this term has been used to designate a broader group of chromosome rearrangements, including isodicentrics and duplications of genetically nonidentical arms (Van Dyke 1988). The recent application of molecular analysis for determining parental origin and structure of X isochromosomes has confirmed an approximately equal frequency of maternal and paternal origins of the i(Xq), as previously had been estimated by using Xg blood grouping (Otto and Otto 1981; Jacobs et al. 1990), and it has been shown that some i(Xq) chromosomes which appeared monocentric at the cytogenetic level Received March 27, 1991; revision received May 28, 1991. Address for correspondence and reprints: A. Schinzel, Institut fdr Medizinische Genetik, Ramistrasse 74, CH-8001 Zurich, Switzerland. i 1991 by The American Society of Human Genetics. All rights reserved. 0002-9297/91 /4905-0014$02.00

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were in fact dicentric (Sharp et al. 1990). So far, however, no case with heterozygosity of the two arms of the i(Xq) has been found, and therefore it has been suggested that Xq isochromosomes in general originate from one X chromosome through either centromere misdivision or sister-chromatid exchange (Harbison et al. 1988). We present here a molecular study of 11 cases with an apparently monocentric isochromosome Xq and of three cases with a dicentric Xq chromosome, including the first two cases of X isochromosomes in which heterozygosity of the two Xq arms was found. Material and Methods The patients were referred to the Institute of Medical Genetics of the University of Zurich for cytogenetic analysis because of the suspicion of Turner syndrome. Blood lymphocyte chromosome preparations were made, and the chromosomes were GTG- or QFQbanded. A mean of 30 mitoses were scored for each individual, to detect possible mosaicism. CBG-banding was done in four cases (Xs9, Xsl 1, Xs28, and Xs35) to confirm the presence or absence of two centromeres. DNA was extracted from blood of the patients and

Origin of X Isochromosomes

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Table I Summary of Origin of "Isochromosome," Cytogenetic and Molecular Observations, and Parental Ages at Birth of 14 Investigated Patients

Case, Karyotype

Origin of iXqa

Xs9, 46,X,i(Xq) ....................

Maternal (1)

(DXS14)

Maternal Age (years)

Paternal Age (years)

-

25

24

-

19 27

39

-

24

34

+

26 38

33 45

-

44 21

57 29

23

23

+

28

28

-

25 33

23

+

35

32

+

34

34

Presence of Xp

Informative Loci on Xq (homo-/heterozygosity)

DXYS1, DXS178, F9, and DXS374

Xsll, 45,X/46,X,idic(Xq)/ 47,X,idic(Xq),idic(Xq)

(1:28:1) ...................... Xs12, 45,X/46,X,i(Xq)(1:1) ....

Xs14, 45,X/46,X,i(Xq)(6:1) .... Xs16, 45,X/46,X,i(Xq) .......... XS17, 46,X,i(Xq) ..................

Xsl9, 46,X,i(Xq) .................. Xs2O, 46,X,i(Xq) .................. Xs26, 46,X,i(Xq) .................. Xs28, 45,X/46,X,idic(Xq)/ 47,X,idic(Xq),idic(Xq) (13:15:2) ...................... Xs32, 45,X/46,X,i(Xq) (1:4) ............. ......... Xs33, 46,X,i(Xq) ..................

Paternal (1) Paternal (1 and 2) Paternal (1 and 2) Paternal (1) Maternal (1 and 2) Maternal (1) Maternal (2) Maternal (2)

DXS1, DXYS1, DXS17, DXS11, DXS10, and DXS52 DXS1, DXS11, and DXS52 DXS1, DXS178, DXS17, DXS304, and DXS52 DSX52 +

Maternal (1 and 2)

DXYS1, DXS10, F9, DXS296, and DXS52

Xs34, 46,X,i(Xq) ..................

Paternal (1) Maternal (1 and 2) Maternal (1)

Xs35, 45,X/46,X,idic(X)(1:9)

Maternal (1)

a

23

+

DXYS1, DXS13, DXS304, and DSX52 DXS3, DXS10, DXS296, and DXS52 DXS178, DXS296, and DXS52

Numbers in parentheses denote how origin was determined: 1

their parents. In cases Xsl9, Xs32, and Xs33, no blood from the father was available. In case Xsl9, however, the paternal genotype was inferred by examining a sister of the proband. In case Xs2O, DNA was extracted from the maternal grandparents as well. The parental origin of the isochromosome was determined using two highly polymorphic probes: M270, localized in the short arm near the centromere (locus DXS2SS; Fraser et al. 1989), and F814, localized in the distal part of the long arm (loci DXSS2 and F8; Heilig et al. 1988). In addition, in the cases in whom the initial analysis indicated maternal origin of the isochromosome, the following battery of probes detecting RFLPs on the long arm of the X chromosome was used in order to determine the reduction/nonreduction of maternal heterozygosity to homozygosity

=

by M2703; 2

=

by F814.

in the proband: p8 (DXS1), psptPGK (PGK1), pDP34 (DXYS1), pA13R1 (DXS87), p19.2 (DXS3), p212.9 (DXS178), S9 (DSX17), p22.33 (DXS11), p43.15 (DXS42), 6A1 (DXS10), cll (DXS14E), F9 (F9), 4D-8 (DXS98), VK21A (DXS296), U6.2 (DXS304), F8C (F8C), lA1 (DXS374), and F814 (DXSS2) (for references, see Mandel et al. 1989). The possible existence of a second, inactive centromere was investigated by testing for the presence of material from the short arm by using the probe 58.1 (DXS14), which maps to the short X arm 4 cM from the centromere. As Xq and Xp controls, we used, respectively, the probe S9 (DXS17), located at Xq22, and TaklO (DXS89), located at Xp22.2 (for references, see Mandel et al. 1989). Densitometric analysis was used to quantitate band intensities. Paternity was

1036

Lorda-Sanchez et al.

tested in all complete families by using the minisatellite probe 33.15 (Jeffreys et al. 1985). Results

Cytogenetic Analysis

The results of the cytogenetic studies are summarized in table 1. Eleven of the 14 cases had an apparently monocentric isochromosome, and four of these 11 were mosaics with a 45,X cell line. Three cases (Xsl 1, Xs28, and Xs25) had a dicentric isochromosome [idic(Xq)]. All three cases showed mosaicism with a 45,X cell line in addition to the 46,X,idic(Xq). In two cases (Xsl1 and Xs28), a few mitoses with two isochromosomes, [47,X,idic(Xq), idic(Xq)], were also found. Parental Origin

The parental origin of the isochromosome could be determined in all 14 families by using the probe(s) M271 and/or F814 (table 1). In five cases (Xsll, Xs12, Xs14, Xs16, and Xs32), the isochromosome was paternally derived. In the remaining nine cases, including two of the three dicentric isochromosomes, the origin of the isochromosome was maternal. In no case could paternity be excluded by genetic fingerprinting. Molecular Detection of Short-Arm Material

The densitometric analysis of the bands from the probe 58.1 (DXS1 4) and from controls S9 (DXS1 7) and TaklO (DXS89) confirmed the presence of material from proximal Xp in the dicentric isochromosomes (Xsl 1, Xs28, and Xs35) and demonstrated the latter in three additional cases with cytogenetically monocentric, maternally derived isochromosomes (Xs17, Xs26, and Xs34) (fig. 1). In cases Xsl 1, Xsl7, and Xs26, the ratio of the integrated optical densities for p58.1:TAK10 was three times that for the control XY (fig. 1), while it was only twice as much in case Xs34. These patterns of band densities suggest the .presence of (a) two copies of probe p58.1 in the i(Xq) of cases Xsll, Xsl7, and Xs26 and (b) a single dose for probe p58.1 in the i(Xq) of case Xs34. Heterozygosity at the locus DXS14 in the isochromosome of case Xs28 also suggests the presence of two copies of the probe p58.1. Dosage could not be estimated in case Xs35 because of the high degree of mosaicism. All these cases gave only one signal from the normal X when the probe M27I (DXS255; Xpl1.22) was used. From this result it can be inferred that the

Figure I Autoradiographs of Southern blots showing Msp RFLPs on X chromosome that are detected by three different probes: Tak 10 (DXS89; Xp22), p58.1 (DXS14; Xp1l), and S9 (DXS17; Xq22). The density values are presented as integrated optical density (IOD). Comparison of data for case Xs9 (b) with those for the XY control (a) shows that the ratio of IOD p58.1:TAK10 is similar, indicating that case Xs9 has the same number of copies of the proximal and distal probes of the short arm; the ratio of IOD p58.1:S9 is, however, three times lower than that of the XY control, suggesting that in case Xs9 the isochromosome does not present a copy of p58.1. Comparison of the data for case Xs1 1 (c) and those of the control (a) shows that the ratio of IOD p58.1:TAK10 in case Xs11 is three times that in the XY control, while the ratio of IOD p58.1: S9 is similar to that of the XY control. Therefore, case Xsl 1 shows the same number of copies of the probes p58.1 and S9, which is three times the number of copies of probe TAK10. These findings suggest a duplication of the probe p58.1 in the isochromosome of the proband.

breakpoints on the short arms are somewhere between the loci DXS14 and DXS2SS. HomozygositylHeterozygosity of the i(Xq) The application of a battery of Xq markers in the maternally derived cases allowed maternal heterozygosity at different loci to be determined. The informative loci for each case are presented in table 1. In seven cases (Xs9, Xs17, Xsl9, Xs2O, Xs26, Xs33, and Xs3S), all the informative loci showed a reduction of the maternal heterozygosity to homozygosity in the isochromosome of the proband (fig. 2a). The isochromosome of Xs28 showed both homozygosity at some loci (DXS1 0, F9, and DXS2 96) on the middle part of the Xq and heterozygosity at some loci located proximally (DXYS1 ) and distally (DXS52), on the long arm of the X chromosome (fig. 2b), as well as at the locus DXS1 4, localized on the short arm close to the centromere (fig. 3). M271 (DXS2SS), however,

1037

Origin of X Isochromosomes HETEROZYGOSITY

a

b

Autoradiographs of Southern blots showing Bcl Figure 2 RFLPs on X chromosome that are detected by probe F814. Lane 1, Mother. Lane 2, Proband. Lane 3, Father. a, Case Xs26. The proband shows the paternal X allele and a double dosage of one maternal X allele, indicating a reduction of the maternal heterozygosity to homozygosity in the isochromosome of the proband. b, Case Xs28. The patient's DNA shows the paternal X allele and both maternal X alleles, indicating nonreduction of the maternal

heterozygosity to homozygosity in the isochromosome of the proband.

only one signal from the paternal normal X and is therefore not present in the isochromosome. Thus, the isochromosome of case Xs28 seems to be a dicentric X with two genetically different long arms separated by an intercentromeric segment that includes segments from two different short arms attached to each other at breakpoints between the loci DXS1 4 and DXS255 (fig. 3). In case Xs34, no reduction from the maternal heterozygosity could be found in the isochromosome of the proband-at any of the four informative loci (DXS3, DXS1 O, DXS2 96, and DXSS2). Whether recombination took place between both arms prior to or after the formation of the isochromosome could not be determined. gave

Meiotic Recombination In case Xs2O, the DNA from the maternal grandparents was analyzed with the probes for which the mother was heterozygous (DXS1, DXS1 78, DXS1 7,

Proposed structure of dicentric isochromosome in Figure 3 case Xs28, based on results of RFLP analysis. The proband showed reduction of the maternal heterozygosity to homozygosity at some loci (DXS10, F9, and DXS296) on the middle part of the Xq and presented both maternal alleles (heterozygosity) at some loci located proximal (DXYS1) and distal (DXS52) on the long arm of the X chromosome, as well as at the locus DXS14, localized on the short arm close to the centromere.

DXS304, and DXSS2). Alleles from both grandparents were present in the isochromosome of the proband (table 2). Thus, at least one recombination had occurred between both maternal X chromosomes prior to or after the formation of the isochromosome. In case Xs28, the alternate reduction/nonreduction of the maternal heterozygosity to homozygosity at some loci on the Xq (fig. 3) in the proband indicates that at least two meiotic recombinations between both maternal X chromosomes are involved in the formation of the dicentric isochromosome. Parental Age

The mean + SD maternal and paternal ages for all cases for whom data were available were 28.7 ± 7.0 and 32.6 ± 9.9 years, respectively. A significantly (P = .036) higher mean maternal age was found for cases with maternally derived isochromosome (31.2 + 7.5) versus paternally derived ones (24.2 ± 3). However, a broad range in maternal ages for the patients with maternally derived isochromosomes was found (table 1). This diversity did not correlate either with the different pericentromeric structures or with homozygosity or heterozygosity of both Xq arms of the isochromosomes. A higher but not significant

Lorda-Sanchez

1038 Table 2

et

al.

twice as many opportunities to generate X isochromosomes formed from one single X chromosome and, second, the possibility of generating X-X translocations or exchanges between homologous X chromosomes which cytogenetically cannot be distinguished from isochromosomes. However, when molecular analysis is used, an i(Xq) originating from an X-X translocation should show heterozygosity of both Xq

Analysis of Recombination in Maternally Derived Isochromosome of Patient Xs2O, Using RFLPs on Xq

Locus, Probe

arms.

DXS1 (Xqll.2), p8 .......... DXS178 (Xq22), p212/9 DXS17 (Xq22), S9 ........... DXS304 (Xq28), U6.2 ...... DXS52 (Xq28), F814 ........ ...

1 1 1 1 3

2 1 1 1 1

2 1 1 1 1

1 2 2 2 3

1 2 2 2 3

11 22 22 11 11

2 1 2 1 2

2 1 2 1 2

(P = .273) increase of mean paternal age was also found for cases in whom the origin was maternal (34.0 ± 11.5), compared with those paternal in origin (30.4 + 7). Discussion

The first studies of parental origin of the isochromoby using the Xg blood group suggested that the i(Xq) originated with equal probability in the fathers and mothers of the patients (Sanger et al. 1971; Otto and Otto 1981). DNA analysis showed a slight excess of maternally derived isochromosomes, which was not significantly different from a 1:1 ratio: in 42 published cases (including the present study) in whom the origin of the X isochromosome was determined by using RFLP(s), the origin was maternal in 57% and paternal in 43% (see table 3). The slight excess of maternal origin i(Xq) might be explained by the fact that the female has two X chromosomes. This offers, first, somes

To the best of our knowledge, no case of an i(Xq) showing heterozygosity has been reported prior to the present study, but only in a few previous cases were more than one informative loci investigated (Callen et al. 1987; Harbison et al. 1988). Thus, these authors believed that Xq isochromosomes result from either centromeric misdivision or sister-chromatid exchange of one X chromosome. Demonstration of heterozygosity at some loci on Xq in two maternally derived isochromosomes studied in the present report, however, suggests that either interchanges between homologous X chromosomes or X-X translocations are indeed possible modes of origin of i(Xq) chromosomes. In studies of the origin of 21 q isochromosomes (dup2lq) by using RFLPs, a few cases with complete or partial heterozygosity of the dup2l q were also found, although the majority of true isochromosomes are derived from a single parental chromosome 21 (Antonarakis et al. 1990; Shaffer et al. 1991). The heterozygosity detected in the isochromosomes of cases Xs28 and Xs34 suggests that they might be the result of an exchange between homologous chromosomes. Nevertheless, the results of molecular analyses show that the possible mechanisms of formation are different in these two cases. The dicentric isochromosome in case Xs28, which also shows recombina-

Table 3 Summary of Studies of Parental Origin of "Isochromosomes"

45,X/46,X,i(Xq) Maternal Paternal

ORIGIN OF i(Xq)a

Paternal

Study (no. of cases) Callen et al. 1987 (5) ................. Harbison et al. 1988 (8) ............. Connor and Loughlin 1989 (6) Deng et al. 1990 (1) Jacobs et al. 1990 (7) ................. Villamar et al. 1990 (1) .............. Present study (14) ...................... Total (42) ......................... .....

a

2 2 4 4 (2) 3 1 5 (1)9 (3) 18 (43%)

Maternal 1 3 (1)

3

1

6 (2) 2 1 4

1 3 5 24 (57%)

46,X,i(Xq) Paternal Maternal

2 2

4 1

2

2 1 1

3

_ 15 (52%)

6 14 (48%)

3 (23%)

Numbers in parentheses are number of cases in whom cytogenetic analysis revealed dicentric isochromosome.

10 (77%)

Origin of X Isochromosomes

tion and two genetically different copies of the Xp proximal probe 58.1, might be the result either of a premeiotic X-X translocation with a later meiotic recombination or of a homologous X-chromosome short-arm interchange. This failure might be caused by a paracentric inversion or by an "inversion loop," although the small intercentromeric distance seen in this case makes the latter mechanism less likely. In case Xs34, complete heterozygosity suggests a premeiotic formation of the i(Xq), without subsequent meiotic recombination, and the presence of only one single dose of probe 58.1 indicates an asymmetrical X-X rearrangement.

Complete homozygosity at all informative loci of the i(Xq) was found both in seven of nine cases with maternal origin that were studied in the present investigation and in all six maternally derived cases investigated by Callen et al. (1987) and Harbison et al. (1988). This suggests that the isochromosome was formed from two genetically identical sister chromatids, which, of course, should be the case in all the paternally derived isochromosomes as well. The observation, in case Xs2O, of meiotic recombination between both maternal X chromosomes, besides a complete homozygosity of the isochromosome, makes its formation at the premeiotic or meiotic stage unlikely and therefore suggests an origin either in the zygote or at an early postzygotic stage. As no mosaicism with a normal (46,XX) cell line was found in any of the present cases, the timing of formation of the i(Xq) in Xs2O (and possibly in the case of further i(Xq) chromosomes in this and other series) can be narrowed to the short period of the first mitotic divisions following zygote formation. Presence of Xp material was demonstrated in three additional i(Xq) cases in whom cytogenetic analysis failed to detect a dicentric isochromosome. Thus, as suggested by Sharp et al. (1990), it seems that there are a greater number of dicentric isochromosomes than have been identified by cytogenetic analysis, and, therefore, short-arm interchanges seem to be, along with centromere misdivision, a common mechanism of i(Xq) formation. In those cases in whom the origin of the isochromosome was studied, mosaicism with a 45,X cell line was found in eight (57%) of the 14 cases of the present study and in 29 (70%) of the 42 cases described in the literature (table 3). It is interesting to point out that in the present study no paternally derived case without mosaicism was found. Of the 13 cases from the literature who did not have apparent mosaicism, only three

1039

cases were paternally derived. This difference, however, was not significant (P = .082). Mosaicism with a 45,X cell line might be the result of an isochromosome formation during the first mitotic division of the zygote with loss of the short arms, as occurred in at least a part of our homozygotic isochromosomes (see above). Alternatively, as originally suggested by de la Chapelle et al. (1966), it might also be the result of mitotic instability of some isochromosomes, instability which seems to be higher in dicentric i(Xq)s than in monocentric i(Xq)s (Schmid et al. 1974; Hsu et al. 1978), and might depend on the length of the distance between the two centromeres. The three cases (Xsl7, Xs26, and Xs34) in whom a dicentric isochromosome was only inferred from the molecular analysis and who hence had the smallest amount of Xp material showed no mosaicism with a 45,X cell line. A significantly higher (P = .036) mean maternal age for the maternally derived cases compared with the paternally derived cases has been found in the present study, confirming the tendency shown in other recent studies of isochromosomes X and 21 (Antonarakis et al. 1990; Jacobs et al. 1990). The large variance in maternal ages for the patients with maternally derived isochromosomes did not seem to correlate either with the different pericentromeric structures or with genetic identity/nonidentity of both Xq arms. Maternal age might depend on the time of formation: if maternal age was increased only for the cases originating prior to or during meiosis, no age effect would be expected in cases formed postzygotically; indeed, the mother of case Xs2O was only 21 years old at delivery of the patient. A potential paternal age effect in 46,X,i(Xq) Turner syndrome patients has long been controversial. Carothers et al. (1989) could not find a universal paternal age effect for this condition, but they suggested an increased paternal age for the paternally derived cases. The lower mean parental age for our five paternally derived X isochromosomes does not, however, support this assumption (table 1). Thus, the overall increase of parental ages for i(Xq) seems to be only a consequence of the increased maternal age for the cases with a maternally derived X isochromosome.

Acknowledgments I.L.-S. received a Swiss "Bundesstipendium" for 2 years. The work was supported by a grant from the EMDO Foundation, Zurich, to I.L.-S. and A. S. and by a grant from the

1040 Stiftung fur wissenschaftliche Forschung der Universitat Zurich to A.S. We are grateful to Drs. I. Craig, A. Jeffreys, J. L. Mandel, and J. Weissenbach for providing the probes. We would also like to thank the patients, their parents, their clinicians, and, especially, Drs. 0. Tonz, E. A. Werder, and M. Zachmann for their collaboration.

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