Satellited Y chromosomes: Structure, origin, and clinical significance

Hum Genet (1984) 67 : 72-85

© Springer-Verlag 1984

Satellited Y chromosomes: Structure, origin, and clinical significance

M. Schmid 1, T. Haaf 1, Eva Solleder 1, W. Schempp 2, M. Leipoldt 3, and H. Heilbronner 4

Institut f'tir Humangenetik, Universit~it Wfirzburg, Koellikerstr. 2, D-8700 Wtirzburg, Federal Republic of Germany 2 Institut ffir Humangenetik, Universitgt Freiburg, Albertstr. 11, D-7800 Freiburg i. Br., Federal Republic of Germany 3 Institut fiat Humangenetik, Universit/it G6ttingen, Nikolausberger Weg 5a, D-3400 G6ttingen, Federal Republic of Germany 4 Institut f'dr Humangenetik, Universit/it Ttibingen, Wilhelmstr. 27, D-7400 TiJbingen, Federal Republic of Germany

Summary. Three cases of inherited satellited Y chromosomes (Yqs) were analysed using several cytogenetic techniques. The cytogenetic data of the 14 cases of Yqs chromosomes described to date were reviewed. All Yqs chromosomes carry an active nucleolus organizer region (NOR) in their long arm and must have developed from translocations involving the short arms of the acrocentric autosomes. The structure of the heterochromatic satellite region in the Yqs chromosomes shows conspicuous inter-familial differences; this permits the reconstruction of the translocations from which the various Yqs were derived. Some causal factors leading to the development of Yqs chromosomes are considered: the specific localization of the four satellite DNAs and highly methylated DNA sequences in the karyotype, and some new experimental data on the spatial arrangement of heterochromatic regions in interphase nuclei. These provide distinct evidence for a preferential involvement of the autosomes 15 and 22 in the translocations with the Y heterochromatin. All clinical reports documenting Yqs males born with malformations were reviewed. It appears that the presence of an extra NOR and NOR-associated heterochromatin in the Yqs chromosomes does not cause any phenotypic abnormalities (as long as the Y euchromatin is intact). The possibility that a Yqs chromosome predisposes to non-disjunc- tion and/or to translocations of other chromosomes is discussed.

Introduction

The so-called satellited Ychromosomes (Yqs) belong to the very rare structural variations in the human karyotype. In this type of structural rearrangement, parts of the short arm of an acrocentric autosome are translocated at the long arm of the Y chromosome, causing its satellited appearance. The first case of such a Yqs chromosome was described by Genest et al. (1967) in a patient with Down syndrome. The chromosome analysis of this patient demonstrated a trisomy 21 and "a Y chromosome the size of a group-F (19-20) autosome, with achromatic filaments terminating in euchromatic small knobs attached to the end of the long arms. These structures could not be differentiated morphologically from satellites of group D or G autosomes" (Genest et al. 1967). This description of the appearance of the rearranged Yqs chromosome can on the

Offprint requests to: M. Schmid

whole be used for all the other Yqs chromosomes found hence. Furthermore, this peculiar satellited Y chromosome was traced back to the great-grandfather of the patient (Family A of Genest et al. 1967, Table 1).

Four teen further cases of Yqs chromosomes have been described in the meantime (Tables 1 and 2). In 13 of these cases the Yqs chromosome occurred within the family line (S chmid 1969; Genest 1972, 1973, 1978; Genest et al. 1983; Hayata et al. 1975, 1977; Howard-Peebles and Stoddard 1976; Howell et al. 1978; Stella et al. 1980; Giraldo et al. 1981; Bordson and Varela 1982; Bayless-Underwood et al. 1983) and in one case the translocation occurred de novo (Turleau et al. 1978). The detailed pedigree analysis of the Family B of Genest (1973, 1978) and Genest et al. (1983) led to the surprising result that this Yqs chromosome has been transmitted in patrilineary fashion for 315 years ! The presence of a distinct achromatic constriction and terminal satellites in the Yqs chromosomes suggested that in the course of the structural rearrangement the nucleolus organizer region (NOR) was translocated from the short arm of an acrocentric chromosome onto the Y chromosome. An indication for this was provided by the many close associations observed between the Yqs chromosomes and the nucleolar constrictions in the short arms of the acrocentric autosomes. The definitive confirmation of the fact that the achromatic constrictions in the Yqs chromosomes are indeed NORs was provided by staining techniques which specifically label the NORs (N-banding, silver (Ag)-staining).

In the present study, three new Yqs chromosomes are introduced. Several special cytogenetic techniques were used in order to characterize these Yqs chromosomes. All cases with Yqs chromosomes previously described in the literature were reviewed and the minor or major malformations found in some probands with a satellited Y chromosome are discussed. The factors contributing to the origin of the human Yqs chromosomes are reviewed, and the NOR-bearing sex chromosomes observed in other mammalian species are taken into consideration.

Case reports

In two of the current cases the satellited Y chromosomes were demonstrated in the metaphases of the amniotic fluid cells in tile course of intrauterine diagnosis. Prenatal diagnosis was performed because of the advanced age of the parents. There were no indications for a chromosomal analysis provided by

73

Fig. la-s. Yqs chromosomes of Case B showing conventional Giemsa staining (a-e), C-bands (d-f), Ag-stained NORs (g-i), distamycin A-induced undercondensation of the Y heterochromatin (k-m), distamycin A/DAPI fluorescence (n-p), and quinacrine fluorescence (q-s). The upper row presents the Yqs from six metaphases each, the two lower rows show partial metaphases where the Yqs chromosomes (large arrows) participate in satellite associations with acrocentric autosomes. Note the distinct satellites and nucleolar constrictions (a), the large amount ofheterochromatin (d) and the conspicuous Ag-stained NORs (g). In the Yqs chromosomes from distamycin A-treated lymphocytes (k) a small segment located at the distal end of the heterochromatin (short arrows) escapes the induced undercondensation. The heterochromatin of this Yqs chromosome fluoresces brightly with distamycin A/DAPI (n) and quinacrine (q)

the family histories. The Yqs chromosomes were also demonstrated in the metaphases from blood lymphocytes from the fathers of these two male fetuses (Cases B and W in this report).

The third case is a 17-year-old male patient with growth retardation (153 cm body length, 61kg body weight). Aside from the retarded growth, the clinical examination presented no abnormalities. A satellited Y chromosome was found in the metaphases of the lymphocytes both from the patient (Case K in this report) as well as from his father.

Materials and methods

The chromosome preparations of Cases B, K, and W as well as five additional, unrelated normal males without Yqs chromosomes were obtained from the peripheral blood according to the method of Pfeiffer (1974). Chromosome medium la (Gibco Bio-Cult) was used for the conventional cultures as well as for the cultures treated with distamycin A and 5-azacytidine (0.5 ml blood per 3.5 ml medium). The staining methods were as follows:

C-banding (Figs. ld-f , 3b, 4n) was done according to the method of Sumner (1972), and silver (Ag)-staining of the NORs (Figs. lg-i , 3c, 5d-f) according to the method of Goodpasture and Bloom (1975).

Q-banding (Figs. lq-s , 3d, 4f). Quinacrine mustard was used according to the technique of Caspersson et al. (1970). The fluorescence photomicroscopy was performed using a Zeiss fluorescence microscope equipped with an HBO 50W/AC

lamp and a filter combination 450-490/FT 510/LP 520. Every fluorescing metaphase of which a karyotype was to be prepared was photographed with three different exposure times; this permits the detection of the very tiny satellites on the Yqs chromosomes in those photographs with long exposure times (Overton et al. 1976).

Distamycin A/DAPI-staining (Figs. in-p , 3e, 4g). A simple modification of the original technique of Schweizer et al. (1978) was performed successfully. The slides were flooded with distamycin A (0.1 mg/ml in McIlvaine buffer, pH 7.0) and covered with a coverslip. After 15-min incubation in the dark at room temperature, the slides were rinsed in McIlvaine buffer and stained with DAPI (0.2 gg/ml in McIlvaine buffer) for 10 min. Finally, the preparations were rinsed in McIvaine buffer for 10 s and mounted in a saturated saccharose solution. For microscopic analysis, a 2 FL filter (Zeiss) was used in the fluorescence microscope.

Bromodeoxyuridine (BrdU)-replication patterns (Fig. 4a-e, h-l , o, p). For the demonstration of the replication patterns on metaphase chromosomes, 10 gg BrdU/ml and 0.5 gg fluoro- deoxyuridine (FdU)/ml were added to the blood cultures 7 h before harvest. The metaphase chromosomes were differen- tially stained with the Hoechst-Giemsa technique according to the method of Schempp and Meer (1983) and with acridine orange (Couturier et al. 1973).

DistamyeinA-in vitro treatment(Figs. 1k-m, 2a-e). The condensation of the Y heterochromatin was prevented by treating the blood cultures with the oligopeptide antibiotic distamycin A according to the technique of Schmid (1979).

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Fig. 2a-e. Giemsa-stained Yqs chromosomes (large arrows) of Case B from distamycin A-treated lymphocytes, a Metaphase with diplo-chromosomes; the inset shows a higher magnification of the Yqs diplo-chromosome in satellite association with a G-group diplo-chromosome. Note that a small segment at the distal end of both Yqs chromosomes escapes the distamycin A-induced undercondensation of the heterochromatin (small arrows), b-e Partial metaphases with multiple satellite associations between the Yqs chromosome and two to six acrocentric autosomes

Fig. 3a-f. Yqs chromosomes of Case K showing Giemsa staining (a), C-bands (b), Ag-stained NORs (e), quinacrine fluorescence (d), distamycin A/DAPI fluorescence (e), and 5-azacytidine-induced undercondensations (13. Note the relatively small amount ofheterochromatin (b) (compare with Fig. la, d). The heterochromatin of this Yqs chromosome shows bright distamycin A/DAPI fluorescence but no bright quinacrine fluorescence (d). In metaphases from 5-azacytidine-treated lymphocytes (13 distinct undercondensations can be observed in the heterochromatin of chromosomes 1, 9, 15, 16, and Y; the chromosomes were previously identified by quinacrine banding. The inset in (I3 shows the Yqs chromosomes from three metaphases; the small arrows indicate the position of the satellites, g, h Giemsa-stained Y chromosomes and autosomes 1, 9,15, and 16 from 5-azacytidine-treated lymphocytes of normal male probands without Yqs chromosomes. Note that the undercondensed heterochromatin of these normally structured Y chromosomes (g) is distinctly longer than that of the Yqs chromosomes (13

5-Azacytidine-in vitro treatment (Figs. 3f-h, 5a-c). The undercondensation of the heterochromatin in the chromosomes Y, 1, 9, 15, and 16 as well as the demonstration of the somatic pairings between these heterochromatic chromosome regions was achieved with the cytidine analogue 5-azacytidine (Viegas- P6quignot and Dutril laux 1976; Schmid et al. 1983). 5-Aza- cytidine (Serva) was dissolved in twice-distilled water and 100 gl each of this solution was added to the blood cultures at 5 x 10 -7 to 2 x 10 -7 (final concentration in the culture) 7h before fixation of the cells.

Results

The satellited Y chromosomes of the three probands B, K, and W could be recognized in the conventionally stained prepara-

tions (Figs. 1a-c, 3a, 4m). In all three cases the satellites were always demonstrated in the long arms of the Y chromosome; the short arms never showed any indication of satellites. In the middle metaphase stage the long arms of the Y chromosomes exhibited the usual degree of close chromatid apposition characteristic for the human Y chromosome (Schmid 1967). Without the satellites and satellite stalks (nucleolar constrictions) these Y chromosomes were slightly shorter than the group-F chromosomes (Fig. 4a, Table 1). Regarding the size of the satellites and the length of the satellite stalks, there were definite inter-individual differences among the three Yqs chromosomes. Both the satellites as well as the satellite stalks were distinctly larger in the Yqs ofproband B than they were in the Yqs of probands K and W (compare Figs. la, 3a, 4m). The satellites and satellite stalks were least pronounced in proband

75

Fig. 4. Yqs chromosomes of the Case W. a Karyotype with R-bands after BrdU treatment and acridine orange staining. All autosomes and the X chromosome exhibit their normal replication patterns. The inset shows a photographic overexposure of the Yqs chromosome; note the satellites at the distal end of the Yqs chromosome (small arrow) which replicate earlier than the terminal Y heterochromatin, b, e Three examples each of the two types of replication patterns in the long arms of the Yqs chromosomes and in (d) and (e) the only type found in the normally structured Y chromosomes of control probands for comparison. The same differences were found after differential staining with the Hoechst-Giemsa te ch- nique (h-l); the small arrows in (e) and (i) point to the satellites, f Q uinacrine-stained Yqs chromosomes without bright heterochromatin fluorescence, g Distamycin A/DAPI staining showing distinctly labeled heterochromatin (large arrow), m Conventional Giemsa staining, n C-bands; note the very small nucleolar constrictions, satellites, and heterochromatic regions (small arrow), o, p Examples of satellite associations between the Yqs chromosome (large arrows) and acrocentric autosomes from BrdU-treated lymphocytes (Hoechst-Giemsa staining)

Fig. 5. a Giemsa-stained metaphase from a 5-azacytidine-treated lymphocyte of a normal male without Yqs chromosome. The chromosomes were previously identified by quinacrine banding. Note the distinct undercondensations of the heterochromatin in the chromosomes 1, 9,15,16, and Y as well as the somatic pairing between the undercondensed heterochromatin of an autosome 15 and the Y chromosome (arrow). b Selected examples of somatic pairing configurations between the undercondensed heterochromatin ofautosomes 15 and normal Y chromosomes found in 5-azacytidine-treated lymphocytes, e Somatic pairings between the undercondensed heterochromatin ofautosomes 1, 9, 16, and Y chromosomes. The position of the chromosome arms is illustrated by the camera lucida drawings beneath the photographic representations. The undercondensed heterochromatic regions are always turned toward each other and in some cases are in direct contact, d-f Three lymphocyte nuclei from a normal male proband possessing a normally structured Y chromosome prestained with quinacrine (left column) and restained with silver (right column). Note that the Y heterochromatin is always closely associated with a cluster of Ag-positive NORs of the acrocentric autosomes (a~,ows)

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W (Fig. 4m); in approximately 30% of the metaphases analysed, these could not be identified.

The specific Ag-labeling of the NORs was always localized within the satellite stalks of the Yqs chromosomes and in no case extended into the adjacent heterochromatic regions (Figs. 1g-i, 3 c). This was especially distinct in the preparations which were prestained with quinacrine and subsequently treated with silver. In the short arms of the normally structured acrocentric chromosomes, the dark silver-label is also always localized within the satellite stalks (Goodpasture et al. 1976).

The C-banding technique enabled the demonstration of a distinct constitutively heterochromatic region in the long arm (band Yq12) in the Yqs chromosomes of all three probands (Figs. 1d-f, 3b, 4n). However, the size of this heterochromatic band showed remarkable inter-individual differences. In the Yqs chromosome of proband B the heterochromatic region constituted approximately two-thirds of the long arm (Fig. ld- f). By comparison, it encompassed one-half of the long arm in the Yqs of proband K (Fig. 3b) and only one-third of the long arm in the Yqs ofproband W (Fig. 4n). Differences among the three Yqs chromosomes could also be observed with quinacrine-banding and distamycin A/DAPI-staining. Thus in the Yqs of proband B the brightly fluorescing heterochromatin in the distal two-thirds of the long arm could be demonstrated both with quinacrine and with distamycin A/DAPI (Fig. 1n-s). The quinacrine-bright heterochromatin in this Yqs consisted of two closely adjacent bands (Fig. 1q-s), which are well known from observations made on normal Y chromosomes (for review see Btihler 1980). In the Yqs chromosomes of the probands K and W, only the distamycin A/DAPI-bright heterochromatin was present (Figs. 3e, 4g). No remnants of the quinacrine-positive heterochromatin could be seen even after the photographs were over-exposed (Figs. 3 d, 4f). This finding can be explained if the position of the distamycin A/DAN- and quinacrine-bright heterochromatin in the normally structured Y chromosomes is taken into account. Whereas quinacrine stains the terminal portion of the Y heterochromatin, the region fluorescing brightly with distamycin A/DAPI is situat- ed further proximally (Fig. 7a; W. Schnedl, personal commu- nication). This indicates that in the translocation leading to the Yqs chromosomes of probands K and W, the break occurred within the distamycin A/DAPI-bright heterochromatin; this resulted in the loss of the terminal, quinacrine-bright heterochromatin (Fig. 7c). In contrast to this, the break leading to the development of the Yqs ofproband B must have been localized within the quinacrine-bright heterochromatin; this led to the loss of only a small portion of the Y chromosome (telomeric region), which is confirmed by the presence of both categories of heterochromatin (Fig. 7d).

The in vitro treatment of the lymphocytes with 100 lag distamycin A/ml for the last 24 h of culture prevents the normal condensation of the entire constitutive heterochromatin in the long arm of the Y chromosome (Prantera et al. 1979; Schmid 1979). This undercondensed heterochromatin appears as a thin chromatin thread suspended at the normally condensed euchromatic region. It was noticed that in the Yqs chromosomes a small segment localized at the distal end of the constitutive heterochromatin escapes the distamycin A-induced undercondensation (Fig. lk-m). These segments appear par- ticularly distinctly in metaphases with diplo-chromosomes, which are quite common in distamycin A-treated cultures (Fig. 2a). It must be assumed that these distamycin A-insensi- tive segments are the centromere-adjacent portions (band p11) of the short arms of the acrocentric chromosomes, which were

translocated onto the Y chromosomes along with the NORs and the satellites. A schematic representation of such a translocation and the Yqs chromosome resulting from it are shown in Fig. 7d.

The cytidine analogue 5-azacytidine induces very distinct undercondensations in the constitutive heterochromatin of the Y chromosome and the autosomes 1, 9,15, and 16 if applied in very low doses during the last hours of culture (Viegas- Pdquignot and Dutrillaux 1976; Schmid et al. 1983). The undercondensed heterochromatin appears as long and only weakly stainable threads; the euchromatin of these chromosomes condenses in a normal fashion (Fig. 3f-h). Just as in the Yqs chromosomes from distamycin A-treated lymphocytes, very small, normally condensed segments could be recognized at the distal end of the undercondensed heterochromatin in the 5-azacytidine-treated Yqs as well (Fig. 3f). In addition, the length of the undercondensed heterochromatin in the Yqs of the probands K and W was distinctly shorter than in the Y chromosomes of the control probands (compare Fig. 3 f and g). The latter can be explained by the decreased amount of constitutive heterochromatin in these Yqs. In the metaphases from the 5-azacytidine-treated lymphocyte cultures, the undercondensed heterochromatid regions of the chromosomes 1, 9, 15, 16, and Y exhibit very conspicuous somatic pairings (Fig. 5b, c; Schmid et al. 1983). It can for several reasons be assumed that the somatic pairings between the heterochromatin of the chromosomes 15 and Y favor the origin of Yqs chromsomes (see Discussion).

A detailed study of the replication patterns after 5-BrdU incorporation followed by Hoechst 33258-Giemsa staining or acridine orange fluorescence staining, could be performed on the Yqs ofProband W. The replication patterns ofnon-satellited Y chromosomes from two control probands were used for comparison. The analysis was done on early replication stages (corresponding to those of R-banded chromosomes) in prometaphasic chromosomes (Fig. 4a). Those chromosome regions substituted with 5-BrdU during DNA replication stain dull with acridine orange (pale blue with Giemsa), whereas those incorporating thymidine exhibit bright acridine orange fluorescence (dark red Giemsa staining). The replication patterns of the euchromatic portion of the Yqs chromosome (short arm and proximal region of the long arm) were the same as in the Y chromosomes of the control probands (compare Fig. 4b, c and d, e; Fig. 4h, i and k, 1). Between two and four early replicating bands could be demonstrated. The same bands were also found in an extensive study on the high resolution replication patterns of the human Y chromosome (Schempp and Mfiller 1982). The patterns of replication of the constitutive heterochromatin in the long arm of the Yqs chromosome could be categorized into two different types. One type was the same as in the Y chromosomes of the control probands: the entire distal heterochromatin in the long arm repli- cated late, as shown by the dull acridine orange fluorescence (Fig. 4b, d, e) or pale blue Giemsa staining (Fig. 4h, k, 1). In contrast to this, the second type of the Yqs showed small segments at the distal end of the long arm which obviously belonged to an earlier replication than the heterochromatin and which thus exhibited a bright acridine orange fluorescence (Fig. 4a, c) or dark red Giemsa staining (Fig. 4i). It seems rea- sonable to interpret these segments as the satellites on the distal end of the Yqs, which complete their DNA replication earlier than the Y heterochromatin and later than the Y euchromatin. Furthermore, the analysis of the replication patterns in the prometaphasic stages permitted the exclusion of structural

78

changes in the autosomes and in the X chromosome (Fig. 4a). Since none of the ten autosomes presented an anomaly of the short arm, it can be assumed that the reciprocal translocation between the short arm of an acrocentric chromosome and the long arm of the Y occurred in one of the male ancestors of Proband W.

Like all the other Yqs chromosomes described to date, the Yqs in the present study also showed very conspicuous associations of their satellited long arms with the satellited short arms of the acrocentric autosomes. A series of such satellite associations as found in the differently stained preparations is shown in Figs. 1, 2, and 4. One hundred Giemsa-stained metaphases from conventional lymphocyte cultures from each of the three probands were scored for the presence or absence of satellite associations. The frequencies with which the three Yqs chromosomes participated in satellite associations were very different (Table 1). As has already been observed in earlier studies on the association frequencies of acrocentric autosomes (Schmid et al. 1974; Hayata et al. 1977), the association frequencies of the Yqs chromosomes are largely determined by the length of their nucleolar constrictions: the longer the nucleo- lax constriction, the higher the frequency of association. Thus the Yqs of the Probands B and K which had prominent nucleolar constrictions, were involved in satellite associations with the acrocentric autosomes in 18% and 21% of the metaphases, respectively. By comparison, the Yqs ofproband W whose nucleolar constriction was much less pronounced, associated in only 10% of the metaphases (Table 1). In situ hybridization experiments with radioactively labeled ribosomal RNA to mitotic chromosomes have ascertained that the number of 18S and 28S ribosomal RNA genes in the nucleolar constrictions of the NOR-carrying chromosomes are variable (Henderson et al. 1973; Evans et al. 1974). Furthermore, a positive correlation was found between the content of ribosomal RNA genes of a given NOR-carrying chromosome (as estimated by in situ hybridization) and its association frequency (Warburton et al. 1976). For these reasons, it must be assumed that more ribosomal RNA genes are localized in the longer nucleolar constrictions of the Yqs chromosomes of the Probands B and K than in the Yqs of the Proband W.

It should be noted that the association frequencies of the NOR-carrying chromosomes are very highly influenced by the culture conditions. Among other things, there were distinctly increased association frequencies of all NOR-carrying chromosomes in the distamycin A-treated cell cultures. Thus the analysis of the association frequency of the Yqs in the distamycin A-treated cells ofProband B resulted in values twice that of the conventional cultures (Table 1). The multiple associations, involving three or more NOR-carrying chromosomes (Fig. 2b- e), were especially frequent in the distamycin A-treated cultures (30 of 100 associations). An increase in the frequencies of satellite associations by in vitro treatment with distamycin A was also noted in the cells from amniotic fluid (Jotterand-Bel- lomo 1983).

Discussion

1. Structure of the satellited Y-chromosomes

There is no doubt that the satellited Y chromosomes studied in the present paper as well as all of those described previously carry an active NOR in the long arm. Both the high frequencies of association of the Yqs with the acrocentric autosomes (Table 1) as well as the positive Ag-staining of their satellite

stalks are conclusive of this. The Ag-staining in mitotic metaphase chromosomes only demonstrates those NORs which were active in the preceeding interphase nuclei (Miller et al. 1976a, b; Engel et al. 1977; Schmid et al. 1982). Cytochemical and biochemical analyses showed that the Ag-stained material consists ofnonhistone proteins rich in sulfhydril and disulfide groups (Ag-staining proteins B23 and C23) which rapidly asso- ciate with the newly transcribed ribosomal RNA (Buys and Osinga 1980; Busch et al. 1982). Residues of these proteins remain associated with the NORs up into the metaphase and can therefore be demonstrated with Ag-staining. Therefore it could be concluded with certainty that the satellited Y chromosomes have an active role in nucleolus organization.

The examined Yqs chromosomes of the three probands showed distinct differences regarding the size of the satellites, the nucleolar constrictions, and of the constitutive heterochromatin in the long arms. Furthermore, differences between these Yqs chromosomes were determined following quinacrine and distamycin A/DAPI staining. The cytogenetic data derived from the 12 other cases with Yqs chromosomes (Table 1) also show that the structure of satellited Y chromosomes is not uniform. These differences can be interpreted as follows: 1) The Yqs chromosomes developed from normally structured Y chromosomes by means of reciprocal translocations with the short arms of acrocentric chromosomes. The breaks which occurred in the course of this in the long arm of the Y chromosomes were obviously localized in different sites within the heterochromatic band Yql2 (Fig. 7). Thus in the cases of Genest et al. (1983), Howard-Peebles and Stoddard (1976), Tur- leau et al. (1978), Giraldo et al. (1981), Bordson and Varela (1982), and in the present Case B, the breaks must have occurred in the distal region of the band Yql2, because the heterochromatin in these Yqs still constitutes approximately two-thirds of the long arms (Fig. 7d). In contrast to this, the small remainders of heterochromatin in the Yqs described by Stella et al. (1980), Shabtai et al. (1981), Bayless-Underwood et al. (1983), and the lack of distamycin A/DAPI-positive heterochromatin in the present Cases K and W can be explained by breaks in the proximal portion of the band q12 of the originally normally structured Y chromosome (Fig. 7c). Yqs chromosomes where the entire C-band-positive heterochromatin in the long arm is lacking and where the nucleolar constriction is directly adjacent to the euchromatic band Yqll, are very rare (Fig. 7b). The very small Yqs described by Hayata et al. (1975, 1977) was such a heterochromatin deficient Yqs. 2) The remaining differences in the morphology of the different Yqs chromosomes can be explained by the heterogeneity of the chromosome segments translocated from the short arms of the acrocentric autosomes onto the Y chromosomes. It is well known that the length of the nucleolar constrictions, the size of the satellites, and the quinacrine fluorescence of the satellites in the short arms of the acrocentric autosomes are extremely variable and constitute a constant, characteristic feature of each individual autosome (e.g., McKenzie and Lubs 1975; Schned11978). The short arms of those acrocentric autosomes which were involved in the reciprocal translocation with the Y chromosomes must have been structured in different ways. Prominent satellites with pronounced nucleolar constrictions were translocated on the Yqs chromosomes described by Genest et al. (1967), Genest (1972, 1973, 1978), Howard-Peebles and Stoddard (1976), Hayata et al. (1975,1977), Stella et al. (1980), Giraldo et al. (1981), Bordson and Varela (1982), and Bayless-Underwood et al. (1983). This was also true for the Yqs of the present Probands B and K (Figs. la, 3 a, Table

79

Table 2. The indication for chromosome analysis, the karyotype determined in the index patient and the origin of the satellited Y chromosomes (Yqs) in 18 families

References Index Indication for the chromosome analysis Karyotype Origin of patient of the index the Yqs (see patient Fig. 6)

Genest et al. (1967), ¢ Down syndrome 47,XYqs Familial Family A

Genest et al. (1972, 1973, XI-11 Father of a girl with Down syndrome 46,XYqs Familial 1978, 1983), Family B

Schmid (1969) III-2 Congenital heart defect and hexadactyly; two sisters 46,XX,fra 17p Familial of the patient (III-1, III-3) died with congenital heart defects; the father of the patient (II-4) with the karyotype 46,XYqs, fra 17p

Hayata et al. (1975, 1977) 1I -1 Severe pancytopenia and hepatosplenomegaly; no 46,XYqs Familial cogent evidence of leukemia. The father (I-l) died at the age of 55 years of pulmonary embolism; one brother (II-4) died at the age of 4 years, another (II-5) at 2, both of unknown causes

Howard-Peebles and Stoddard II-1 (1976)

Turleau et al. (1978) II-1

Stella et al. (1980) II-4

Giraldo et al. (1981), Family A VIII-2

Family B VII-2

Family C VIII-6

Family D VIII-8

II-1 Shabtai et al. (1981)

Bordson and Varela (1982)

Bayless-Underwood et al. (1983)

Present Case B

Present Case K

Present Case W

V-1

Family A IV-2

Family B III-2

¢

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Down syndrome 47,XYqs,+21, Familial inv(9)(p13q21)

Son of first cousins. Multiple malformations: slanting lid axes, deep-set eye sockets, amaurosis, deep-set ears with pits, small mouth, micrognathia, cardiopathy and hepatomegaly, underweight, hypotonia

One spontaneous abortion by wife; one son (111-3) died due to multiple malformations: ectopic bladder, bilateral polycystic kidney, anal atresia, undescended testes, patent ductus arteriosus, atelectasis of the lungs, hypertrophy of the left ventricle, bilateral valgus foot

Population screening

Genetic counseling for consanguinity; the first son (VIII-4) with minimal brain dysfunction and autistic behavior

Sterility, gynecomastia, small testes

Cryptorchidism

Minor malformations: asymmetric face, symmetric exophthalmos, mongolian eye slant, small and low set malformed ears, mild hypotonia, bronchial asthma

Two consecutive abortions by wife at 15 and 10 weeks of pregnancy

Prenatal diagnosis due to maternal family history of Down syndrome

Prenatal diagnosis due to increased age of the parents

Prenatal diagnosis due to increased age of the parents (see Case report)

Stunted growth (see Case report)

Prenatal diagnosis due to increased age of the parents (see Case report)

46,XYqs De novo translocation

46,XYqs Familial

46,XYqs Familial

46,XYqs Familial

47,XXYqs Familial

46,XYqs Familial

46,XYqs Familial

46,XYqs Familial

46,XYqs Familial

46,XYqs Familial

46,XYqs Familial

46,XYqs Familial

46,XYqs Familial

80

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VII

2 " I ; "x i S c h m i d (1969)

Gira ldo et al. (1981) F - -(?) . . . . . . . ~ . . . . . .

F m! J

, i I Fam.Cl~

/

Hayata et al. (1975,1977)

Stella et al. (1980) Shabtai et a1.(1982)

- - - - @ I i o ~ ~ I ~ I

#11 III

Bordson and Varela ~'I ~ (1982)I

J:~,, i ,,

~" ~ | IV IV

~" J;]v, li ~" Fam, D m~ vii

I--/ , i v,, /

~m m ~ ~ ~ ii /

Bayless-Underwood et al. (1983)

Turleau et al. (1978)

II /

Key:

[ ] 46,XYqs male V j ~ obligate 46,XYqs carrier

"[~" possible 46 XYqs carrier VI " ~ 46,XX,fra17p

H o w a r d - P e e b l e s and [ ] 46,XYqs, fra17p S toddard (1976) [ ] unbalanced karyotype

r]C)normal karyotype I ~ [ . [ ~ not examined

' • abortion II ~ r [ ~ B i ~ ) { ~ ( ~ ) ~ ) ~ ) ( ~ ) / index patient

7 --.9-- possible consanguinity • de novo Yqs

intrauterine diagnosis

Family A Family B

ill 2 ~ III

IV /

Fig. 6. Pedigrees of the previously reported families with Yqs chromosomes. In the pedigree described by Genest et al. (1973,1983) only the male descendants are registered. For the pedigree of Howard-Peebles and Stoddard (1976) the information supplied by Howell et al. (1978) was used also. The numbers at the bottom-left of some probands are refered to in the text and in Table 2

81

P12"

Eb

r-q 1' .

Fig.Ta-f. Schematic representation of different types of reciprocal translocations which can produce Yqs chromosomes, a Normally structured acrocentric autosome and Y chromosome. Dark sector: quinacrine bright heterochromatin. Hatched sector: distamycin A/ DAPI bright heterochromatin. Dotted line (band p12): NOR. Note that the bands pll and p13 in the acrocentric autosome are also heterochromatic. The wavey lines at the two chromosomes indicate the pre- sumed breakage points. For the sake of simplicity, only one of the possible breakage points (band pll) in the acrocentric autosome was taken into account, b Break at the boundary of euchromatic band Yqll and heterochromatic band Yql2. e Break within the distamycin A/DAPI bright region of the heterochromatic band Yql2. d Break within the quinacrine bright region of the heterochromatic band Yql2. Note the structural differences between the resulting Yqs chromosomes, e, fReciprocal translocations between an acrocentric autosome lacking its satellite (band p13) and a normally structured Y chromosome: this produces a Yqs chromosome with nucleolus organizer region (satellite stalk) but without satellites in its long arm

1). By comparison, only small satellites were translocated from the acrocentric autosomes onto the Y chromosomes in the cases of Turleau et al. (1978) and in the present Case W (Fig. 4m). Finally, in the Yqs described by Shabtai et al. (1981), the satellites could only be surmised but were never clearly seen (Table 1). The only Yqs chromosome whose satellite showed a similarly bright fluorescence as the terminal heterochromatin of the band Yql2 was that of the present Case B (Fig. 1q-s); all other Yqs chromosomes had a satellite whose quinacrine fluorescence was approximately the same intensity as the proximal euchromatic band Yqll (Figs. 3d and 4f, Table 1).

It needs to be considered that acrocentric chromosomes lacking the satellites in their short arms are not rare. In these

I I~ III IV 5 "M d ' D 5 "A II 111 IV 5"M d'D 5-A

ttttt; 1i11!t!

Fig. 8. The localization of the four satellite DNAs (I-IV), 5-methylcytosine enriched DNA (5-M), distamycin A/DAPI bright heterochromatin (d-D), and 5-azacytidine-sensitive heterochromatin (5-A) in the Y chromosome, the five acrocentric autosomes and autosomes 1, 9, and 16. The satellite DNAs I-IV are distributed according to the satellite maps of Miklos and John (1979). Every chromosome was divided into equal sized segments, each corresponding to one-half the length of chromosome 21. The major sites of hybridization to such segments are shown as black sectors. The threshold values used in the quantitative evaluation of the autoradiographs were 1.5 grains/ segment for satellites I, II, and IlI, and 2.0 grains/segment for satellite IV. Chromosome 16 carries satellite II (hatched sector); however, its amount falls below the threshold values used by Miklos and John (1979). The hatched area in the acrocentric autosome 22 indicates a very low amount of highly methylated DNA as detected by immuno- fluorescence technique and antisera specific for 5-methylcytosine (Schnedl et al. 1975). Note that only the distamycin A/DAPI positive heterochromatin is 5-azacyticline-sensitive (compare with Figs. 3f and 5a)

acrocentrics, residual parts of the NOR can sometimes be seen in the form of stalks (Schmid et al. 1974). If one of these par- tially deleted short arms is translocated on a Y chromosome, it forms a Yqs with a NOR localized on the end of the long arm, but without a satellite (Fig. 7e, f). This kind of Yqs chromosome, for which the Yqs observed by Shabtai et al. (1981) serves as a good example, can be easily overlooked in the metaphases if the preparations are not Ag-stained. There is therefore the possibility that Yqs chromosomes do occur in a greater frequency than can be concluded from the few cases observed to date. More extensive studies with the simple method of Ag- staining for NORs are necessary to determine the frequency of this structural variation of the Y chromosome in the population.

It must be concluded from the structural differences between the different Yqs chromosomes occurring in family lines (Tables 1 and 2) that these Yqs are not of monophyletic origin, but instead developed several times independent of each other. The differences in ethnic origin of the 14 families also suggest this. Finally, de novo origin ofa Yqs could be un- equivocally demonstrated in the case reported by Turleau et al. (1978). A normally structured Y chromosome was found in the father of a child with Yqs born with multiple malformations (I-l, II-1 in Fig. 6). The paternity was confirmed by extensive analysis of red blood cell enzymes and serum types.

82

2. Y/Autosomal translocations and the origin of Yqs chromosomes

As was repeatedly presented, the exchanges between the long arms of the Y chromosomes and the short arms of the acrocentric autosomes are supposed to be reciprocal. It can therefore be expected that portions of the Y heterochromatin will be translocated to the short arms of the acrocentric autosomes participating in these translocations (Fig. 7a-d). A consider- able number of acrocentric autosomes with large, quinacrine- bright short arms were found (see review by Smith et al. 1979). The proof that these brightly fluorescing short arms actually consisted ofY heterochromatin and not of enlarged autosomal heterochromatin was furthered by the identification of two Y- specific, repetitive DNA sequences (2.1 and 3.4 kilobases in length) using Hae III restriction endonuclease analysis of the DNA from female carriers of such acrocentrics (Kinross et al. 1978; Cooke and Noel 1979; Schmid et al. 1983) and/or by the specific undercondensation of the translocated Y heterochromatin in distamycin A-treated lymphocytes (Schmid 1979; Cohen et al. 1981; Funderburk et al. 1982; Schmid et al. 1983). The reviews of the Y/autosomal translocations described to date show that in 70% of the cases the Y heterochromatin was translocated on the short arm of an acrocentric autos6me. Of these in turn, chromosome 15 is the most frequently involved with a rate of 52%, followed by chromosome 22 (33%), chromosome 21 (7%), and chromosomes 13 and 14 (4% each) (Smith et al. 1979; Davis 1981). These numbers clearly show that the short arms of chromosomes 15 and 22 participate in translocations with the Y chromosome much more frequently than expected, whereas chromosomes 13, 14, and 21 are underrepre- sented.

There are two mutually complementary explanations for the overproportionally frequent involvement of chromosomes 15 and 22 in translocations with the Y heterochromatin: (1) Very similar or identical DNA sequences are localized in the constitutive heterochromatin of the short arms of chromosomes 15 and 22 and in the long arm of the Y chromosome, which promotes pairing and crossing over between these non- homologous chromosome regions. (2) In the interphase nuclei, the heterochromatic regions of chromosomes 15, 22, and Y are arranged in very close spatial contact ("somatic pairing"), which makes a translocation between them more likely. As far as the DNA sequences are concerned, the studies with the in situ hybridization technique (for review see Gosden et al. 1975; Miklos and John 1979) have shown that high concentrations of all four satellite DNAs I - IV are localized in the heterochromatin of chromosomes 15 and Y; the heterochromatin of chromosome 22 is enriched with the satellite DNAs I, III, and IV, whereas the amount of satellite DNA II found here falls below the threshold values used in the quantitative evaluation of the autoradiographs (Fig. 8). In the heterochromatin of the remaining acrocentric autosomes (Nos. 13, 14, and 21), only satellite IV is present in high concentrations. Using an immu- nofluorescence technique and antisera specific for 5-methylcytosine, it was furthermore possible to demonstrate that the heterochromatic regions of chromosomes 15 and Y and, to a lesser degree, chromosome 22 contain highly methylated DNA (Fig. 8) (Miller et al. 1974; Schnedl et al. 1975). These observations show that the agreement of the highly repetitive DNA is in fact greater between the heterochromatin of chromosomes 15, 22, and Y than it is between the heterochromatin of chromosomes 13, 14, 21, and the Y chromosome.

Somatic pairings between the heterochromatin of chromosomes 15 and Y have also been recently demonstrated success-

fully (Schmid et al. 1983a). The condensation of the heterochromatin in chromosomes 1, 9, 15, 16, and Y during the mitotic prophase is inhibited by the in vitro treatment of cell cultures with 5-azacytidine (Figs. 3g, h and 5a). As a consequence, the somatic pairings between these heterochromatic regions remain intact from the interphase nuclei up into metaphase and are thus susceptible to analysis (Fig. 5b, c). The examples shown in Fig. 5b illustrate the strength of the somatic pairings between the heterochromatin of chromosomes 15 and Y, and why these promote the development of Yqs chromosomes. The translocation only exchanges terminally located heterochromatin and a NOR. Since the heterochromatin is genetically inert and as the number of active NORs is inter-individ- ually variable anyway, the gametes of the carrier of such a translocation are at no selective disadvantage. Both the Yqs chromosome as well as the chromosome 15 with the translocated Y heterochromatin can be transmitted without effect on the phenotype of the progeny, even when they segregate indepen- dently of each other after the meiotic divisions; no imbalance of genetically relevant material can occur. The situation is completely different, however, when reciprocal translocations occur due to somatic pairings between the Y heterochromatin and the pericentromeric heterochromatin ofautosomes 1, 9, or 16 (Fig. 5c). In these cases, the entire arm of one of these autosomes is translocated on the Y chromosome. The chromosomal balance of the progeny would only be secured if the rearranged Y chromosome segregates in the same gamete to- gether with the rearranged autosome. For this reason, the translocations between the heterochromatin of the Y chromosome and the non-acrocentric autosomes are subject to high postmeiotic and postzygotic selective pressure.

The mitotic condensation of the heterochromatin in the short arms of the acrocentric autosomes Nos. 13, 14, 21, and 22 is not inhibited by 5-azacytidine (Figs. 5a and 8). The somatic pairings between these acrocentrics and the Y heterochromatin can therefore not be analysed by in vitro treatment of the cultures with 5-azacytidine. However, there are definite indications for the existence of somatic pairings between the short arms of all acrocentric autosomes and the Y heterochromatin in the interphase nuclei. It can thus be shown on quinacrine- and subsequently Ag-stained nuclei that the brightly fluorescing Y heterochromatin (Y body) is always localized in the middle of all active, Ag-positive NORs (Fig. 5d-f). It can be stated in summary that both the common satellite DNAs in the heterochromatin of chromosomes 15, 22, and Y as well as the somatic pairings between the Y heterochromatin and the heterochromatin of all acrocentric autosomes, constitute decisive factors for the development of Yqs chromosomes.

3. Clinical significance of the satellited Y chromosomes

As discussed in the previous section, the presence of a Yqs chromosome should cause no phenotypic abnormalities in the carriers. Most of the Yqs chromosomes described are indeed family marker chromosomes, transmitted in patrilineary fashion for several generations, which did not lead to progeny with specific phenotypic malformations in these families (Fig. 6). The most impressive example of this is the French- Canadian family described by Genest et al. (1973, 1983). In this family, the Yqs arose more than 300 years ago and is the oldest translocation chromosome yet reported in the human species. This Yqs was only discovered because one of the 16 male descendants in the eleventh generation (XI-11 in Fig. 6) had a child with trisomy 21.

Table 3. Summary of NOR-carrying sex chromosomes in mammals (according to Merry et al. 1983) a

83

Species NOR-carrying chromosomes b

Autosomes X Y

Hylobates concolor (whi te-cheeked gibbon)

Hylobates syndactylus (siamang)

Ammospermophilus harisii (antelope squirrel)

Nyctereutes procyonoides (raccoon dog)

Canisfamiliaris (domestic clog)

Callithrix ssp. (Brazilian marmosets)

Lagorchestes conspicillatus (hare wallaby)

Carollia castanea (Allen's fruit bat)

Carollia perspicillata (Seba's fruit bat)

Monodelphis domestica (gray short-tailed opossum)

q-

+

+

+

+

+

+

a The position of the NORs in the sex chromosomes is a constitutive property of the karyotypes of these species and not an occasionally occurring rearrangement in individual animals

b Demonstrated by in situ hybridization of radioactively labeled 18S and 28S ribosomal RNA to mitotic chromosomes and/or Ag-staining

Table 2 shows that the abnormal phenotypes diagnosed in some of the carriers of an inherited Yqs chromosome are of very heterogeneous nature. No specific anomaly can be corre- lated with the 46,XYqs karyotype. Neither was there an incrased incidence of infertility or a remarkable number of spontaneous abortions in the families of the index patients. This leaves the conclusion that the malformations found in some of the families with a satellited Y chromosome are incidental findings bearing no relation to this structural variation of the Y chromosome. This is confirmed by the fact that three of the inherited Yqs chromosomes were found by chance in prenatal diagnoses performed due to the advanced age of the parents (Family A of Bayless-Underwood et al. 1983; current Cases B and W), and another one in the course ofa cytogenetic population study on chromosomal polymorphisms (Family A of Giraldo et al. 1981).

The decision concerning a possible karyotype-phenotype correlation in the only case of de novo Yqs chromosome described to date (Turleau et al. 1978) poses more of a problem. In this case, a Yqs chromosome was demonstrated in a newborn male (II-1 in Fig. 6) with severe multiple malformations (Table 2), whereas the father (I-l, Fig. 6) possessed an inconspicuous phenotype and a normally structured Y chromosome. Illegiti- macy could be excluded with a high degree of certainty by the analysis of serologic markers. It was suggested that the translocation between the Y heterochromatin and the short arm of an acrocentric autosome took place during paternal meiosis. A postzygotic translocation seemed improbable as no mosaicism of 46,XY/46,XYqs was found and because none of the acrocentric autosomes in the 46,XYqs cells exhibited a morpholog- ic alteration in the short arms. The breakpoint in the paternal Y chromosome must have occurred in the distal portion of the heterochromatic band Yq12, because this heterochromatin was not visibly reduced (see Fig. 2c of Turleau et al. 1978). As the schematic diagram in Fig. 7d shows, no change in genetically relevant euchromatin occurs in the Yqs chromosome in the course of such a translocation. Besides this, the symptoms shown by the patient we1"e much more severe than those occurring in an aberration involving the euchromatin in the long

arm of the Y chromosome (band Yqll) (see review by Davis 1981). For these reasons, it appears improbable that the de novo Yqs was the causal factor for the malformations found in the patient. It needs to be taken into account that the parents of the patient were first cousins; therefore, the possibility of an auto- somally recessively inherited d isease-as discussed by Turleau et al. (1978)-must also be considered.

The Ag-staining studies on karyotypes of normal male probands have shown that the modal number of Ag-positive NORs per individual lies between 6 and 10 (Mikelsaar et al. 1977). It can already be recognized by this great inter-individual variability in the number of active NORs in diploid cells that no disadvantages can be expected for the carrier of an additional NOR in the Yqs chromosome. At this point, one should also consider the exceptional chromosomal position of the NORs in some other mammals. It has been shown for six species to date that aNOR is localized in the Y chromosomes ofal lmale individuals studied in addition to the NORs in the autosomes (Table 3). Apparently, NORs from autosomes were translocated on the Y chromosomes in the ancestors of these species and these NOR-carrying Y chromosomes subsequently became established in all males by drift in small, inbreeding popula- tions. This produced a sex-specific difference in the NOR- number, which did not constitute a selective disadvantage for the further differentiation of these species. The NOR-carrying X chromosomes in some of these mammals can be assessed in a similar fashion (Table 3).

Some of the earlier studies on Yqs chromosomes already discussed the question of whether these structural variations in the Y chromosome could predispose to aberrations involving other chromosomes. Thus the patient (Family A) of Genest et al. (1967) had both trisomy 21 and the Yqs chromosome. The index patient of Howard-Peebles and Stoddard (1976) was a male with Yqs, trisomy 21, and inversion in the chromosome 9 (Table 2). The index patient of the Family B examined by Genest et al. (1973, 1983) (XI-11 in Fig. 6) was the father of a girl with trisomy 21. Klinefelter syndrome and a satellited Y chromosome (47,XXYqs) were determined in the index patient in the Family C of Giraldo et al. (1981) (VIII-6 in

84

Fig. 6) and in a case recorded by Genes t (1978); the latter is a pat ient whose ancestors came f rom Qu6bec and who were probably related to the French-Canadian Family B of Genes t et al. (1973, 1983). A very unusua l - -and with regard to the etio- logic invo lvement o f the Yqs the most i n t e r e s t i n g - c h r o m o - some aberrat ion was again found in the Family B o f G e n e s t et al. (1983) in a newborn male (XII-2 in Fig. 6) wi th craniosteno- sis and minor malformat ions . The banding analysis of the karyotype revealed that the terminal part of the long arm of a p resumed acrocentr ic au tosome No. 14 was translocated onto the satellite of the Yqs chromosome: t ( Y q s t e r : : 1 4 q 2 4 ~ 14q ter). The clinical picture of the patient was very similar to the phenotypes reported in four o ther cases of partial t r isomies for the terminal part of 14q. The t ranslocat ion mus t have ocurred de novo during paternal meiosis since the father of the pat ient (XI-8 in Fig. 6) had a typical Yqs chromosome. There was an interval of three centuries (nine generat ions) be tween the origin of the Yqs ch romosome in this family (probably in III-3 in Fig. 6) and the t ranslocat ion 14q/Yqs. Taking all unbalanced karyotypes found in the families with Yqs chromosomes into account, it is not easy to decide whe ther the Yqs ch romosome played a role in the deve lopment of addit ional ch romosome aberrations or not. On the o ther hand, the observed tr isomies 21 and Klinefel ter syndromes are among the most f requent of all ch romosomal disorders and could therefore be just if iably considered incidental findings. On the o ther hand, the t ranslocat ion demonst ra ted be tween the satellites of a Yqs and a terminal part o f l 4 q (Genes t et al. 1983) war- rants caut ion with regard to trivializing the risk for unbalanced gametes in the carriers of Yqs chromosomes . W h e t h e r a causal relat ionship exists be tween the presence o f a Y q s ch romosome and the origin of chromosomal abnormali t ies during meiosis or not could be de te rmined by the analysis of ch romosome complements f rom the sperm of Yqs carriers by the fertiliza- t ion of hamster eggs (Rudak et al. 1978; Mart in 1983).

Acknowledgements. We thank Miss Beatrix Geile for technical assist- ance and Mrs. Jutta Jackowski for photographic documentation.

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Received January 9, 1984

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Satellited Y chromosomes: Structure, origin, and clinical significance