Chromosoma (Berl.) 59, 137-145 (1976)
CHROMOSOMA 9 by Springer-Verlag 1976
A Possible Active Segment on the Inactive Human X Chromosome Eeva Therman 1, Gloria E. Sarto 2, C. Dist~che 3, and Carter Denniston 1 t Department of Medical Genetics, University of Wisconsin, Madison, Wisconsin 53706, U.S.A., 2 Department of Obstetrics and Gynecology, Northwestern University, Chicago, Illinois, U.S.A., 3 Laboratoire de Cytog~n+tique, Universit6de Li+ge, Belgium
Abstract. An i d i c ( X p - ) in which the two X chromosomes are attached short arm to short arm, and which thus has two b regions (the Q-dark segment next to the centromere on Xp) between the inactivation centers, assumed to be situated on the Q-dark region next to the centromere on Xq, showed 63.8% bipartite Barr bodies as compared with 22.2% formed by i d i c ( X q - ) . In addition, the mean distance of the two parts of the Barr bodies in the fibroblasts of a patient with i d i c ( X p - ) is significantly greater than in the cases with one or no b region. Contrary to the other patients with abnormal X chromosomes, the buccal cells of a woman i d i c ( X p - ) showed a number of bipartite Barr bodies. - To explain these observations we have put forward the hypothesis that the b region on the Xp always remains active and thus, when the rest of the chromosome forms a Barr body, this segment is extended, allowing the two parts of the X chromatin to get farther apart and at the same time increasing the percentage of bipartite bodies.
Introduction
It is a well-established fact that in each mammalian somatic cell only one X chromosome, or its equivalent, is active, regardless of how many X chromosomes the cell may possess. Two hypotheses, which do not necessarily exclude each other, have been presented to explain the observation that persons with the XO sex chromosome constitution or with more than two X chromosomes may show numerous anomalies. According to one hypothesis, the damage has been done by the abnormal X chromosome constitution before inactivation, which in the human embryo is supposed to take place at the 1000~000 cell stage (cf. Lyon, 1974). The other hypothesis assumes that part of the inactive X chromosome(s) always remains active and, hence, a missing X or too many X chromosomes would interfere with the genetic balance of the cell during development.
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T h e r m a n a n d P a t a u (1974; see also L y o n , 1974) p u t f o r w a r d the h y p o t h e s i s t h a t if p a r t o f the inactive X c h r o m o s o m e r e m a i n s active it is m o s t p r o b a b l y the centric region, a n d m o r e p a r t i c u l a r l y the Q - d a r k r e g i o n next to the centrom e r e on the s h o r t arm. T h e p r e s e n t s t u d y p r o v i d e s f u r t h e r evidence in s u p p o r t o f this hypothesis.
Material and Methods Fibroblast cultures (cf. DeMars and Nance, 1964) were prepared for X chromatin studies from five persons with the following sex chromosome constitutions: XX, Xi(Xq), Xidic(Xq-) (Therman et al., 1974a, b), Xidic(Xp-) (Dist+che et al., 1972), and a mosaic XO/XXp+/XXp+Xp+ (the fibroblast cultures did not contain any cells with 47 chromosomes) (Daly et al., 1977). In addition, buccal smears were studied from a patient with idic(Xp-) (de la Chapelle and Stenstrand, 1974). The analysis of the bipartite Barr bodies in the previous cases and in the patient with idic(Xp-) (Dist~che et al., 1972) was performed as follows: fibroblast cultures grown on coverslips and fixed in acetic acid-ethanol (1:3) were stained by using the Feulgen technique (Darlington and La Cour, 1970); the color was subsequently fortified with acid fuchsin. The frequency of bipartite Barr bodies was determined from 500 consecutive, presumably diploid, X chromatin positive cells. Only cells in which at least one part of the X chromatin touched the rim of the nucleus were included in the count. To determine the distance between the two parts of a bipartite Barr body, 30 consecutive nuclei with such bodies were photographed and the distance from the center of one part to the center of the other was measured. The frequency of cells with different Barr body constitutions was determined in the fibroblast culture of this case and in a buccal smear of another patient with idic(Xp-) (de la Chapelle and Stenstrand, 1974).
Observations F o r convenience we have, o n the basis o f the Q - b a n d i n g , d i v i d e d the h u m a n X c h r o m o s o m e into f o u r regions (Fig. 1). The Q - b r i g h t r e g i o n on the distal e n d o f the Xp, is labelled a ; the relatively d a r k segment next to the c e n t r o m e r e is b. T h e c o r r e s p o n d i n g Q - d a r k r e g i o n on the X q is c; this is k n o w n to be s o m e w h a t s h o r t e r t h a n b ( T h e r m a n et al., 1974a; D a l y et al., 1977). The rest o f the Xq, w h i c h is Q-bright, constitutes the d segment. T h e m a i n o b s e r v a t i o n s c o n c e r n the q u a l i t y a n d incidence o f b i p a r t i t e Barr b o d i e s in six i n d i v i d u a l s : in each the X c h r o m a t i n was f o r m e d b y a s t r u c t u r a l l y different X c h r o m o s o m e , one n o r m a l a n d five a b n o r m a l ; in the five latter cases, the a b n o r m a l X c h r o m o s o m e was i n a c t i v a t e d ( G o u w et al., 1970; Dist6che et al., 1972; de la C h a p e l l e a n d S t e n s t r a n d , 1974; T h e r m a n et al., 1974b; D a l y et al., 1977). Some o f the d a t a have been p u b l i s h e d p r e v i o u s l y ( T h e r m a n et al., 1974a, D a l y et al., 1976) a n d are p r e s e n t e d only d i a g r a m m a t i c a l l y (Fig. 2) as a c o m p a r i s o n to the i d i c ( X p - ) which is n o w u n d e r special study. The normal X a n d i(Xq) need no c o m m e n t . C h r o m o s o m e X p + is in essence a n i s o c h r o m o s o m e with a p o r t i o n o f the short a r m (segment b or p a r t o f it) inserted ( D a l y et al., 1977). I n each o f the three isodicentrics, one c e n t r o m e r e is inactivated. I n the i d i c ( X q - ) in w h i c h the two X c h r o m o s o m e s are a t t a c h e d l o n g a r m to long a r m we d e t e r m i n e d t h a t s o m e 5% o f each c h r o m o s o m e was missing ( T h e r m a n et al., 1974b). I n the o t h e r two isodicentrics in which the c h r o m o s o m e s are a t t a c h e d s h o r t a r m to s h o r t a r m , there is also a small percent-
An Active Segment in the Inactive H u m a n X
139
Fig. 1. Segments a, b, c, and d on five Q-banded active h u m a n X chromosomes. The bar represents 5 I-tm idic
x
i(Xq)
Xp+
(Xq-)
idio
(Xp -)
ii
Region b Inactivation _ center
m
Bctrr bodies
0%
4.4%
18.2%
22.2%
63.8%
Fig. 2. The relative positions of the X inactivation center on the Xq and the presumably always-active b region on Xp in five structurally different h u m a n X chromosomes and the incidence and hypothetical structure of bipartite Barr bodies formed by them
age of each chromosome missing (Dist~che et al., 1972; de la Chapelle and Stenstrand, 1974). The normal X chromosome forms practically no bipartite Barr bodies: only one questionable bipartite body was found in 500 sex chromatin positive cells (Therman et al., 1974a). In the fibroblasts that had i(Xq), 4.4% of the sex chromatin positive cells had bipartite bodies; the parts were discrete, although some were lying close to each other. The X p + chromosome formed 18.2% bipartite bodies. In the fibroblast culture with idic(Xq-), 22.2% of the X chromatin bodies were bipartite. In contrast to the i(Xq) and X p + chromosomes, connecting strands were often visible between the two parts of the Barr body even when they were lying fairly far apart.
E. Therman et al.
140 Table 1. Frequency of different types of Barr bodies formed by two idic(Xp - ) chromosomes in buccal cells (de la Chapelle and Stenstrand, 1974) and in fibroblasts (Dist~che et al., 1972) Source
Buccal cells Fibroblasts
Number of nuclei with the following Barr body constitution 0
1
Bipartite
Total
156 114
64 36
23 63
243 213
Fig, 3a-e. Barr bodies from diploid fibroblasts formed by idic(Xp-), a One large X chromatin body. b Barr body in which the division into two parts is faintly discernible, c-e Bipartite Barr bodies. The bar represents 8 gm
An Active Segment in the Inactive H u m a n X
10~
5
0 I0
5 tj
c 0 ~6 I0
141
i(Xc I)
L L
idlc (Xcl-)
Xp+
.El
E Z
5
0 I0
idic ( X p - )
0
i 2 3 4 ,5 6 Distance of centers of pclrts of X body (/zm)
Fig. 4. Distances of the two parts of bipartite Barr bodies formed by four abnormal X chromosomes (no connection was visible between the parts)
In the fibroblast culture of the patient with idic(Xp-), the percentage of X chromatin positive cells was 46.5% (Table 1). Of 500 Barr bodies in presumably diploid cells, 63.8% were bipartite (Fig. 3). The two portions of a bipartite body added up to one full-sized X chromatin body (Fig. 3). Often the Barr body appeared very large and "fuzzy". Usually no connections were visible between the two parts. O f the buccal cells, 9.5% in the other i d i c ( X p - ) case (de la Chapelle and Stenstrand, 1974) revealed bipartite X chromatin bodies (Table 1), whereas no such bodies have been observed in the buccal smears of the patients with i d i c ( X q - ) or with X p + . The results of the measurements of the distances between the two parts of a bipartite Barr body are presented in Figure 4 and Table 2. It is clear from Table 2 that the mean distances are heterogeneous. A Kruskal-Wallis test confirms this (P < 0.0001). (The mean ranks for the location tests for each chromosome type are shown in Table 2.) To test the specific hypotheses,/~i = #j,
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Table 2. Distances (in microns) between parts of bipartite Barr bodies
Chromosome Type
Sample size (ni) Mean (/~/) Variance (a~) Mean rank for location test Mean rank for spread test
(1) i(Xq)
(2) idic(Xq-)
(3) Xp+
(4) idic(Xp-)
20 1.20 0.1125 16.08 28.38
20 1.89 0.1849 44.23 36.30
26 2.03 0.7696 44.69 50.58
30 3.45 1.3489 76.27 68.25
i+-j,
we have used the multiple comparison method of Nemenyi (see Miller, 1966, p. 165). Except for the comparison,/~2 with/~3, all hypotheses are rejected at the 1% level. We conclude, therefore, that gl < g2--~g3 < I-t4. In addition, it appears as if the variance of distance goes up approximately with the square of the mean, and a log transformation does, indeed, stabilize the variances fairly well. To (roughly) test differences in spread among the four distributions we have subtracted from each measurement its own (chromosome specific) median, taken absolute values and repeated the Kruskal-Wallis and Nemenyi analyses on these absolute deviations. This reduces the problem to one of comparing location parameters. ( T h e mean ranks used for these tests of spread are shown in Table 2.) The heterogeneity among spreads (variances) is significant (P < 0.001); and the multiple comparison tests indicate the same pattern of inequalities as for the means, i.e., a 2 < o - ~ a 2 < a ] . It is quite possible that, in fact, az < 0.2, but the sample sizes are simply too small for us to detect the difference. In sum, the distance data are, in general, compatible with the hypothesis proposed in this paper.
Discussion
Two hypotheses would explain the fact that although all but the equivalent of one X chromosome is inactivated in a human cell, individuals with one X missing or with too many X chromosomes may exhibit numerous abnormalities. We favor the hypothesis that a segment of the inactive X chromosome always remains active. It seems difficult to imagine how all the abnormal features in, say, a patient with Turner's syndrome could have originated before the time of X inactivation. Possibly relevant to this is a family described by Allderdice et al. (1971). The mother had a balanced reciprocal translocation with most of the Xq attached to 14q. The son had both translocation chromosomes, i.e. X q - and 14q+, and an extra 14q+ chromosome. One of the 14q+ chromosomes was late-labelling and formed the Barr body. The son was reported to display only symptoms characteristic of the Klinefelter syndrome. In this case apparently no damage had been done by the extra chromosome 14 prior to its inactivation, which presumably had spread from the attached Xq. Trisomy for chromosome 14, when it is not inactivated, is clearly lethal since no child
An Active Segment in the Inactive Human X
143
with this trisomy is known to have been born alive. The obvious conclusion is that no genes on chromosome 14 have been active before Barr body formation in the Klinefelter son mentioned above. If this were so, it seems improbable that the X chromosome would have numerous genes active before this stage. Therman and Patau (1974) proposed a hypothesis that if a region remained active on the inactive human X chromosome, it would most probably be the Q-dark region next to the centromere on the short arm (segment b in Fig. 1). This was based on two observations: first, the centric region on the inactive X is relatively early-labelling. (The characteristic of early-labelling applies even more to the Q-dark region on the Xq; however, we have so far no other evidence that this segment would stay active.) And second, it is known that the Xg a gene which is assumed to be situated in one end of the Xp linkage group (cf. McKusick and Chase, 1973) always remains active, at least on a normal X chromosome. If this gene were located in the centromere end of the linkage group, it might well come within the Q-dark region b. The present observations give added support to this hypothesis. Based on the extreme rarity, or, more probably, the non-existence of certain types of abnormal X chromosomes (Therman et al., 1974a; de la Chapelle and Schr6der, 1975) on the one hand, and on the formation of bipartite Barr bodies by some others on the other hand, Therman et al. (1974a) proposed that an X inactivation center, without which the human X chromosome would not be able to form a Barr body, was situated on the proximal region of Xq. Lately we have been able to limit this segment to somewhat less than the Q-dark c region next to the centromere on the Xq (unpublished observations). If a chromosome has two inactivation centers, a proportion of the Barr bodies will be bipartite. The incidence of bipartite bodies and the mean distance between the two parts seemed to be positively correlated with the distance of the assumed inactivation centers on the chromosome when the bipartite bodies formed by i(Xq) and by i d i c ( X q - ) were compared. With the study of a mosaic Turner patient 45,X/46,XXp+/47,XXp + Xp + (Daly et al., 1977), there appeared another factor that seemed to influence the incidence of bipartite Barr bodies. As mentioned above, the X p + chromosome is essentially an i(Xq) with the Q-dark region, or part of it, inserted between the two Xq arms. The distance between the two presumed inactivation centers on the Xp + was not much greater than in i(Xq), but 18.2% of the Barr bodies formed by X p + were bipartite as compared with 4.4% for i(Xq). Furthermore, if the percentage of bipartite bodies and the mean distance between the two parts were influenced only by the distance between the X inactivation centers on the chromosome, one would have expected a significant difference in the incidence of bipartite bodies formed by i d i c ( X q - ) and by X p + since in the former the distance between the two inactivation centers is almost twice the length of the Xq (Fig. 2), whereas in the latter the centers are not much further apart than in i(Xq) (Fig. 2). However, this did not prove to be the case. What could be the reason for this discrepancy? In i(Xq) and in i d i c ( X q - ) there is no b region between the inactivation centers, whereas in X p + there is (Fig. 2). If this region remained active, it would presumably be in an interphasic, non-condensed state. This would allow the parts of a bipartite body to
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get further apart and naturally at the same time increase the frequency of such bodies (Fig. 2). The crucial case to test this hypothesis was obviously an i d i c ( X p - ) in which two presumably active b regions are situated between the inactivation centers. Our expectation was that the incidence of bipartite Barr bodies would be higher and the distance between the two parts greater than in the other cases studied so far (Fig. 2). Both predictions proved to be correct. The incidence of bipartite bodies is 63.8% as compared with 4.4% for i(Xq), 18.2% for Xp + and 22.2% for i d i c ( X q - ) (Fig. 2). The mean distance between the parts is also significantly greater than in the other cases (Fig. 4). A couple of other observations agree also with this hypothesis. Buccal cells of a patient with i d i c ( X p - ) showed 9.5% bipartite X chromatin bodies, not seen in the other cases. And the clean separation of the two parts of a bipartite body formed by i d i c ( X p - ) in the fibroblasts might also reflect the long stretch of an uncoiled chromosome between the parts. The centric region between the inactivation centers in i(Xq) and Xp § may have a somewhat similar effect. The b region has other surprising qualities. We (Daly et al., 1977) measured the Q-regions (a, b, c, and d in Fig. 1) of the active X in six cells each of ten males and in the XO cells of the mosaic mentioned above. As a comparison the same regions were measured on the active X in three females who had an abnormal inactive X chromosome. In all cases in which the cells had only one X chromosome, the b region turned out to be significantly longer than in cells which had two X chromosomes. This applied also to the different types of cells in the mosaic. At the same time no differences were observed in the other Q-regions a, c, and d. It may be remembered in this connection that the whole inactive X chromosome has been found to be shorter than the active X in at least some cell cultures (Takagi and Oshimura, 1973; Sarto et al., 1974). The working hypothesis proposed by Daly et al. (1977) was that the b region always remains active, but may have different states of activity, reflected in its length. The more active, the longer it would be. The amount of activity would depend on the number of X chromosomes in the cell. We are now testing this hypothesis by measuring the Q-regions, particularly the b region, on the active X in a patient with X X X X Y sex chromosome constitution and in other persons with different X chromosome combinations. It is an established fact that the lack of the Xp causes more symptoms than a deletion of corresponding length of the long arm (Therman and Patau, 1974). It will be interesting to see, when different types of deleted X chromosomes are compared, whether or not it is the lack of the b region which is especially important as a cause of the symptoms characteristic of Turner's syndrome. A woman with the chromosome constitution 4 5 , X / 4 6 , X X p - may be relevant in this connection (Buckton et al., 1971). Apart from a shortish stature (148.5 cm), she was phenotypically normal including a normal sex development. We would like to think that this surprising lack of symptoms may depend on the presence of the b region on the X p - chromosome.
Acknowledgements. This is paper No. 1951 from the Genetics Laboratory, University of Wisconsin. It has been aided by the grants GM 15422 and HD 03084-03 from the National Institutes of
An Active Segment in the Inactive Human X
145
Health, and by the F.N.R.S. and the F.R.S.M. (Belgium). We are grateful to Dr. A. de la Chapelle for letting us study buccal smears of his case of idic(Xp-). We wish to thank Dr. Peter Nemenyi for his guidance concerning the problem of multiple comparisons. The photographic work has been done by Mr. Walter Kugler, Jr.
References Allderdice, P.W., Miller, O.J., Klinger, H.P., Pallister, P.D., Opitz, J.M.: Demonstration of a spreading effect in an X-autosome translocation by combined autoradiographic and quinacrinefluorescence studies. (Abstr.) Exc. Med. Int. Congr. Set. 233, 14-15 (1971) Buckton, K.E., Jacobs, P.A., Rae, L.A., Newton, M.S., Sanger, R.: An inherited X-autosome transtocation in man. Ann. hum. Genet. (Lond.) 35, 171-178 (1971) Chapelle, A. de la, Schr6der, J.: Reappraisal of a 46,X,i(Xp) karyotype as 46,Xdel(Xq). Hereditas (Lurid) 80, 137 140 (1975) Chapelle, A. de la, Stenstrand, K. : Dicentric human X chromosomes. Hereditas (Lund) 76, 259 268 (i974) Daly, R.F., Patau, K., Therman, E., Sarto, G.E.: Structure and Barr body formation of an X p + chromosome with two inactivation centers. Amer. J. hum. Genet. 29 (!n press 1977) Darlington, C.D., La Cour, F.L.: The handling of chromosomes. Darien, Conn.: Hafner PuN. Co. 1970 DeMars, R., Nance, W.E.: Electrophoretic variants of glucose-6-phosphate dehydrogenase and the single-active-X in cultivated human cells. Wistar Inst. Syrup. Monogr. 1, 35-48 (1964) Dist6che, C., Hagemeijer, A., Frederic, J., Progneaux, D. : An abnormal large human chromosome identified as an end-to-end fusion of two X's by combined results of the new banding techniques and microdensitometry. Clin. Genet. 3, 388 395 (1972) Gouw, W.L., Coenegracht, J.M., Stalder, G. : A very large metacentric chromosome in a woman with symptoms of Turner's syndrome. Cytogenetics 3, 427 440 (1964) Lyon, M.F. : Mechanisms and evolutionary origins of variable X-chromosome activity in mammals. Proc. roy. Soc. Lond. B 187, 243-268 (1974) McKusick, V.A., Chase, G.A.: Human genetics. Ann. Rev. Genet. 7, 435-473 (1973) Miller, R.: Simultaneous statistical inference. New York: MacGraw Hill i966 Sarto, G.E., Therman, E., Patau, K. : Increased Q-fluorescence of an inactive X q - chromosome in man. Clin. Genet. 6, 1-5 (1974) Takagi, N., Oshimura, M. : Fluorescence and Giemsa banding studies of the allocyclic X chromosome in embryonic and adult mouse cells. Exp. Cell Res. 78, 127 135 (1973) Therman, E., Patau, K. : Abnormal X chromosomes in man : origin, behavior and effects. Humangenetik 25, l 16 (1974) Therman, E., Sarto, G.E., Patau, K.: Center for Barr body condensation on the proximal part of the human Xq: a hypothesis. Chromosoma (Berl.) 44, 361-366 (1974a) Therman, E., Sarto, G.E., Patau, K.: Apparently isodicentric but functionally monocentric X chromosome in man. Amer. J. hum. Genet. 26, 83 92 (1974b)
Received July 2~September 21, 1976 / Accepted September 21, 1976 by H. Bauer Ready for press September 21, 1976
CHROMOSOMA 9 by Springer-Verlag 1976
A Possible Active Segment on the Inactive Human X Chromosome Eeva Therman 1, Gloria E. Sarto 2, C. Dist~che 3, and Carter Denniston 1 t Department of Medical Genetics, University of Wisconsin, Madison, Wisconsin 53706, U.S.A., 2 Department of Obstetrics and Gynecology, Northwestern University, Chicago, Illinois, U.S.A., 3 Laboratoire de Cytog~n+tique, Universit6de Li+ge, Belgium
Abstract. An i d i c ( X p - ) in which the two X chromosomes are attached short arm to short arm, and which thus has two b regions (the Q-dark segment next to the centromere on Xp) between the inactivation centers, assumed to be situated on the Q-dark region next to the centromere on Xq, showed 63.8% bipartite Barr bodies as compared with 22.2% formed by i d i c ( X q - ) . In addition, the mean distance of the two parts of the Barr bodies in the fibroblasts of a patient with i d i c ( X p - ) is significantly greater than in the cases with one or no b region. Contrary to the other patients with abnormal X chromosomes, the buccal cells of a woman i d i c ( X p - ) showed a number of bipartite Barr bodies. - To explain these observations we have put forward the hypothesis that the b region on the Xp always remains active and thus, when the rest of the chromosome forms a Barr body, this segment is extended, allowing the two parts of the X chromatin to get farther apart and at the same time increasing the percentage of bipartite bodies.
Introduction
It is a well-established fact that in each mammalian somatic cell only one X chromosome, or its equivalent, is active, regardless of how many X chromosomes the cell may possess. Two hypotheses, which do not necessarily exclude each other, have been presented to explain the observation that persons with the XO sex chromosome constitution or with more than two X chromosomes may show numerous anomalies. According to one hypothesis, the damage has been done by the abnormal X chromosome constitution before inactivation, which in the human embryo is supposed to take place at the 1000~000 cell stage (cf. Lyon, 1974). The other hypothesis assumes that part of the inactive X chromosome(s) always remains active and, hence, a missing X or too many X chromosomes would interfere with the genetic balance of the cell during development.
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T h e r m a n a n d P a t a u (1974; see also L y o n , 1974) p u t f o r w a r d the h y p o t h e s i s t h a t if p a r t o f the inactive X c h r o m o s o m e r e m a i n s active it is m o s t p r o b a b l y the centric region, a n d m o r e p a r t i c u l a r l y the Q - d a r k r e g i o n next to the centrom e r e on the s h o r t arm. T h e p r e s e n t s t u d y p r o v i d e s f u r t h e r evidence in s u p p o r t o f this hypothesis.
Material and Methods Fibroblast cultures (cf. DeMars and Nance, 1964) were prepared for X chromatin studies from five persons with the following sex chromosome constitutions: XX, Xi(Xq), Xidic(Xq-) (Therman et al., 1974a, b), Xidic(Xp-) (Dist+che et al., 1972), and a mosaic XO/XXp+/XXp+Xp+ (the fibroblast cultures did not contain any cells with 47 chromosomes) (Daly et al., 1977). In addition, buccal smears were studied from a patient with idic(Xp-) (de la Chapelle and Stenstrand, 1974). The analysis of the bipartite Barr bodies in the previous cases and in the patient with idic(Xp-) (Dist~che et al., 1972) was performed as follows: fibroblast cultures grown on coverslips and fixed in acetic acid-ethanol (1:3) were stained by using the Feulgen technique (Darlington and La Cour, 1970); the color was subsequently fortified with acid fuchsin. The frequency of bipartite Barr bodies was determined from 500 consecutive, presumably diploid, X chromatin positive cells. Only cells in which at least one part of the X chromatin touched the rim of the nucleus were included in the count. To determine the distance between the two parts of a bipartite Barr body, 30 consecutive nuclei with such bodies were photographed and the distance from the center of one part to the center of the other was measured. The frequency of cells with different Barr body constitutions was determined in the fibroblast culture of this case and in a buccal smear of another patient with idic(Xp-) (de la Chapelle and Stenstrand, 1974).
Observations F o r convenience we have, o n the basis o f the Q - b a n d i n g , d i v i d e d the h u m a n X c h r o m o s o m e into f o u r regions (Fig. 1). The Q - b r i g h t r e g i o n on the distal e n d o f the Xp, is labelled a ; the relatively d a r k segment next to the c e n t r o m e r e is b. T h e c o r r e s p o n d i n g Q - d a r k r e g i o n on the X q is c; this is k n o w n to be s o m e w h a t s h o r t e r t h a n b ( T h e r m a n et al., 1974a; D a l y et al., 1977). The rest o f the Xq, w h i c h is Q-bright, constitutes the d segment. T h e m a i n o b s e r v a t i o n s c o n c e r n the q u a l i t y a n d incidence o f b i p a r t i t e Barr b o d i e s in six i n d i v i d u a l s : in each the X c h r o m a t i n was f o r m e d b y a s t r u c t u r a l l y different X c h r o m o s o m e , one n o r m a l a n d five a b n o r m a l ; in the five latter cases, the a b n o r m a l X c h r o m o s o m e was i n a c t i v a t e d ( G o u w et al., 1970; Dist6che et al., 1972; de la C h a p e l l e a n d S t e n s t r a n d , 1974; T h e r m a n et al., 1974b; D a l y et al., 1977). Some o f the d a t a have been p u b l i s h e d p r e v i o u s l y ( T h e r m a n et al., 1974a, D a l y et al., 1976) a n d are p r e s e n t e d only d i a g r a m m a t i c a l l y (Fig. 2) as a c o m p a r i s o n to the i d i c ( X p - ) which is n o w u n d e r special study. The normal X a n d i(Xq) need no c o m m e n t . C h r o m o s o m e X p + is in essence a n i s o c h r o m o s o m e with a p o r t i o n o f the short a r m (segment b or p a r t o f it) inserted ( D a l y et al., 1977). I n each o f the three isodicentrics, one c e n t r o m e r e is inactivated. I n the i d i c ( X q - ) in w h i c h the two X c h r o m o s o m e s are a t t a c h e d l o n g a r m to long a r m we d e t e r m i n e d t h a t s o m e 5% o f each c h r o m o s o m e was missing ( T h e r m a n et al., 1974b). I n the o t h e r two isodicentrics in which the c h r o m o s o m e s are a t t a c h e d s h o r t a r m to s h o r t a r m , there is also a small percent-
An Active Segment in the Inactive H u m a n X
139
Fig. 1. Segments a, b, c, and d on five Q-banded active h u m a n X chromosomes. The bar represents 5 I-tm idic
x
i(Xq)
Xp+
(Xq-)
idio
(Xp -)
ii
Region b Inactivation _ center
m
Bctrr bodies
0%
4.4%
18.2%
22.2%
63.8%
Fig. 2. The relative positions of the X inactivation center on the Xq and the presumably always-active b region on Xp in five structurally different h u m a n X chromosomes and the incidence and hypothetical structure of bipartite Barr bodies formed by them
age of each chromosome missing (Dist~che et al., 1972; de la Chapelle and Stenstrand, 1974). The normal X chromosome forms practically no bipartite Barr bodies: only one questionable bipartite body was found in 500 sex chromatin positive cells (Therman et al., 1974a). In the fibroblasts that had i(Xq), 4.4% of the sex chromatin positive cells had bipartite bodies; the parts were discrete, although some were lying close to each other. The X p + chromosome formed 18.2% bipartite bodies. In the fibroblast culture with idic(Xq-), 22.2% of the X chromatin bodies were bipartite. In contrast to the i(Xq) and X p + chromosomes, connecting strands were often visible between the two parts of the Barr body even when they were lying fairly far apart.
E. Therman et al.
140 Table 1. Frequency of different types of Barr bodies formed by two idic(Xp - ) chromosomes in buccal cells (de la Chapelle and Stenstrand, 1974) and in fibroblasts (Dist~che et al., 1972) Source
Buccal cells Fibroblasts
Number of nuclei with the following Barr body constitution 0
1
Bipartite
Total
156 114
64 36
23 63
243 213
Fig, 3a-e. Barr bodies from diploid fibroblasts formed by idic(Xp-), a One large X chromatin body. b Barr body in which the division into two parts is faintly discernible, c-e Bipartite Barr bodies. The bar represents 8 gm
An Active Segment in the Inactive H u m a n X
10~
5
0 I0
5 tj
c 0 ~6 I0
141
i(Xc I)
L L
idlc (Xcl-)
Xp+
.El
E Z
5
0 I0
idic ( X p - )
0
i 2 3 4 ,5 6 Distance of centers of pclrts of X body (/zm)
Fig. 4. Distances of the two parts of bipartite Barr bodies formed by four abnormal X chromosomes (no connection was visible between the parts)
In the fibroblast culture of the patient with idic(Xp-), the percentage of X chromatin positive cells was 46.5% (Table 1). Of 500 Barr bodies in presumably diploid cells, 63.8% were bipartite (Fig. 3). The two portions of a bipartite body added up to one full-sized X chromatin body (Fig. 3). Often the Barr body appeared very large and "fuzzy". Usually no connections were visible between the two parts. O f the buccal cells, 9.5% in the other i d i c ( X p - ) case (de la Chapelle and Stenstrand, 1974) revealed bipartite X chromatin bodies (Table 1), whereas no such bodies have been observed in the buccal smears of the patients with i d i c ( X q - ) or with X p + . The results of the measurements of the distances between the two parts of a bipartite Barr body are presented in Figure 4 and Table 2. It is clear from Table 2 that the mean distances are heterogeneous. A Kruskal-Wallis test confirms this (P < 0.0001). (The mean ranks for the location tests for each chromosome type are shown in Table 2.) To test the specific hypotheses,/~i = #j,
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Table 2. Distances (in microns) between parts of bipartite Barr bodies
Chromosome Type
Sample size (ni) Mean (/~/) Variance (a~) Mean rank for location test Mean rank for spread test
(1) i(Xq)
(2) idic(Xq-)
(3) Xp+
(4) idic(Xp-)
20 1.20 0.1125 16.08 28.38
20 1.89 0.1849 44.23 36.30
26 2.03 0.7696 44.69 50.58
30 3.45 1.3489 76.27 68.25
i+-j,
we have used the multiple comparison method of Nemenyi (see Miller, 1966, p. 165). Except for the comparison,/~2 with/~3, all hypotheses are rejected at the 1% level. We conclude, therefore, that gl < g2--~g3 < I-t4. In addition, it appears as if the variance of distance goes up approximately with the square of the mean, and a log transformation does, indeed, stabilize the variances fairly well. To (roughly) test differences in spread among the four distributions we have subtracted from each measurement its own (chromosome specific) median, taken absolute values and repeated the Kruskal-Wallis and Nemenyi analyses on these absolute deviations. This reduces the problem to one of comparing location parameters. ( T h e mean ranks used for these tests of spread are shown in Table 2.) The heterogeneity among spreads (variances) is significant (P < 0.001); and the multiple comparison tests indicate the same pattern of inequalities as for the means, i.e., a 2 < o - ~ a 2 < a ] . It is quite possible that, in fact, az < 0.2, but the sample sizes are simply too small for us to detect the difference. In sum, the distance data are, in general, compatible with the hypothesis proposed in this paper.
Discussion
Two hypotheses would explain the fact that although all but the equivalent of one X chromosome is inactivated in a human cell, individuals with one X missing or with too many X chromosomes may exhibit numerous abnormalities. We favor the hypothesis that a segment of the inactive X chromosome always remains active. It seems difficult to imagine how all the abnormal features in, say, a patient with Turner's syndrome could have originated before the time of X inactivation. Possibly relevant to this is a family described by Allderdice et al. (1971). The mother had a balanced reciprocal translocation with most of the Xq attached to 14q. The son had both translocation chromosomes, i.e. X q - and 14q+, and an extra 14q+ chromosome. One of the 14q+ chromosomes was late-labelling and formed the Barr body. The son was reported to display only symptoms characteristic of the Klinefelter syndrome. In this case apparently no damage had been done by the extra chromosome 14 prior to its inactivation, which presumably had spread from the attached Xq. Trisomy for chromosome 14, when it is not inactivated, is clearly lethal since no child
An Active Segment in the Inactive Human X
143
with this trisomy is known to have been born alive. The obvious conclusion is that no genes on chromosome 14 have been active before Barr body formation in the Klinefelter son mentioned above. If this were so, it seems improbable that the X chromosome would have numerous genes active before this stage. Therman and Patau (1974) proposed a hypothesis that if a region remained active on the inactive human X chromosome, it would most probably be the Q-dark region next to the centromere on the short arm (segment b in Fig. 1). This was based on two observations: first, the centric region on the inactive X is relatively early-labelling. (The characteristic of early-labelling applies even more to the Q-dark region on the Xq; however, we have so far no other evidence that this segment would stay active.) And second, it is known that the Xg a gene which is assumed to be situated in one end of the Xp linkage group (cf. McKusick and Chase, 1973) always remains active, at least on a normal X chromosome. If this gene were located in the centromere end of the linkage group, it might well come within the Q-dark region b. The present observations give added support to this hypothesis. Based on the extreme rarity, or, more probably, the non-existence of certain types of abnormal X chromosomes (Therman et al., 1974a; de la Chapelle and Schr6der, 1975) on the one hand, and on the formation of bipartite Barr bodies by some others on the other hand, Therman et al. (1974a) proposed that an X inactivation center, without which the human X chromosome would not be able to form a Barr body, was situated on the proximal region of Xq. Lately we have been able to limit this segment to somewhat less than the Q-dark c region next to the centromere on the Xq (unpublished observations). If a chromosome has two inactivation centers, a proportion of the Barr bodies will be bipartite. The incidence of bipartite bodies and the mean distance between the two parts seemed to be positively correlated with the distance of the assumed inactivation centers on the chromosome when the bipartite bodies formed by i(Xq) and by i d i c ( X q - ) were compared. With the study of a mosaic Turner patient 45,X/46,XXp+/47,XXp + Xp + (Daly et al., 1977), there appeared another factor that seemed to influence the incidence of bipartite Barr bodies. As mentioned above, the X p + chromosome is essentially an i(Xq) with the Q-dark region, or part of it, inserted between the two Xq arms. The distance between the two presumed inactivation centers on the Xp + was not much greater than in i(Xq), but 18.2% of the Barr bodies formed by X p + were bipartite as compared with 4.4% for i(Xq). Furthermore, if the percentage of bipartite bodies and the mean distance between the two parts were influenced only by the distance between the X inactivation centers on the chromosome, one would have expected a significant difference in the incidence of bipartite bodies formed by i d i c ( X q - ) and by X p + since in the former the distance between the two inactivation centers is almost twice the length of the Xq (Fig. 2), whereas in the latter the centers are not much further apart than in i(Xq) (Fig. 2). However, this did not prove to be the case. What could be the reason for this discrepancy? In i(Xq) and in i d i c ( X q - ) there is no b region between the inactivation centers, whereas in X p + there is (Fig. 2). If this region remained active, it would presumably be in an interphasic, non-condensed state. This would allow the parts of a bipartite body to
144
E. Therman et al.
get further apart and naturally at the same time increase the frequency of such bodies (Fig. 2). The crucial case to test this hypothesis was obviously an i d i c ( X p - ) in which two presumably active b regions are situated between the inactivation centers. Our expectation was that the incidence of bipartite Barr bodies would be higher and the distance between the two parts greater than in the other cases studied so far (Fig. 2). Both predictions proved to be correct. The incidence of bipartite bodies is 63.8% as compared with 4.4% for i(Xq), 18.2% for Xp + and 22.2% for i d i c ( X q - ) (Fig. 2). The mean distance between the parts is also significantly greater than in the other cases (Fig. 4). A couple of other observations agree also with this hypothesis. Buccal cells of a patient with i d i c ( X p - ) showed 9.5% bipartite X chromatin bodies, not seen in the other cases. And the clean separation of the two parts of a bipartite body formed by i d i c ( X p - ) in the fibroblasts might also reflect the long stretch of an uncoiled chromosome between the parts. The centric region between the inactivation centers in i(Xq) and Xp § may have a somewhat similar effect. The b region has other surprising qualities. We (Daly et al., 1977) measured the Q-regions (a, b, c, and d in Fig. 1) of the active X in six cells each of ten males and in the XO cells of the mosaic mentioned above. As a comparison the same regions were measured on the active X in three females who had an abnormal inactive X chromosome. In all cases in which the cells had only one X chromosome, the b region turned out to be significantly longer than in cells which had two X chromosomes. This applied also to the different types of cells in the mosaic. At the same time no differences were observed in the other Q-regions a, c, and d. It may be remembered in this connection that the whole inactive X chromosome has been found to be shorter than the active X in at least some cell cultures (Takagi and Oshimura, 1973; Sarto et al., 1974). The working hypothesis proposed by Daly et al. (1977) was that the b region always remains active, but may have different states of activity, reflected in its length. The more active, the longer it would be. The amount of activity would depend on the number of X chromosomes in the cell. We are now testing this hypothesis by measuring the Q-regions, particularly the b region, on the active X in a patient with X X X X Y sex chromosome constitution and in other persons with different X chromosome combinations. It is an established fact that the lack of the Xp causes more symptoms than a deletion of corresponding length of the long arm (Therman and Patau, 1974). It will be interesting to see, when different types of deleted X chromosomes are compared, whether or not it is the lack of the b region which is especially important as a cause of the symptoms characteristic of Turner's syndrome. A woman with the chromosome constitution 4 5 , X / 4 6 , X X p - may be relevant in this connection (Buckton et al., 1971). Apart from a shortish stature (148.5 cm), she was phenotypically normal including a normal sex development. We would like to think that this surprising lack of symptoms may depend on the presence of the b region on the X p - chromosome.
Acknowledgements. This is paper No. 1951 from the Genetics Laboratory, University of Wisconsin. It has been aided by the grants GM 15422 and HD 03084-03 from the National Institutes of
An Active Segment in the Inactive Human X
145
Health, and by the F.N.R.S. and the F.R.S.M. (Belgium). We are grateful to Dr. A. de la Chapelle for letting us study buccal smears of his case of idic(Xp-). We wish to thank Dr. Peter Nemenyi for his guidance concerning the problem of multiple comparisons. The photographic work has been done by Mr. Walter Kugler, Jr.
References Allderdice, P.W., Miller, O.J., Klinger, H.P., Pallister, P.D., Opitz, J.M.: Demonstration of a spreading effect in an X-autosome translocation by combined autoradiographic and quinacrinefluorescence studies. (Abstr.) Exc. Med. Int. Congr. Set. 233, 14-15 (1971) Buckton, K.E., Jacobs, P.A., Rae, L.A., Newton, M.S., Sanger, R.: An inherited X-autosome transtocation in man. Ann. hum. Genet. (Lond.) 35, 171-178 (1971) Chapelle, A. de la, Schr6der, J.: Reappraisal of a 46,X,i(Xp) karyotype as 46,Xdel(Xq). Hereditas (Lurid) 80, 137 140 (1975) Chapelle, A. de la, Stenstrand, K. : Dicentric human X chromosomes. Hereditas (Lund) 76, 259 268 (i974) Daly, R.F., Patau, K., Therman, E., Sarto, G.E.: Structure and Barr body formation of an X p + chromosome with two inactivation centers. Amer. J. hum. Genet. 29 (!n press 1977) Darlington, C.D., La Cour, F.L.: The handling of chromosomes. Darien, Conn.: Hafner PuN. Co. 1970 DeMars, R., Nance, W.E.: Electrophoretic variants of glucose-6-phosphate dehydrogenase and the single-active-X in cultivated human cells. Wistar Inst. Syrup. Monogr. 1, 35-48 (1964) Dist6che, C., Hagemeijer, A., Frederic, J., Progneaux, D. : An abnormal large human chromosome identified as an end-to-end fusion of two X's by combined results of the new banding techniques and microdensitometry. Clin. Genet. 3, 388 395 (1972) Gouw, W.L., Coenegracht, J.M., Stalder, G. : A very large metacentric chromosome in a woman with symptoms of Turner's syndrome. Cytogenetics 3, 427 440 (1964) Lyon, M.F. : Mechanisms and evolutionary origins of variable X-chromosome activity in mammals. Proc. roy. Soc. Lond. B 187, 243-268 (1974) McKusick, V.A., Chase, G.A.: Human genetics. Ann. Rev. Genet. 7, 435-473 (1973) Miller, R.: Simultaneous statistical inference. New York: MacGraw Hill i966 Sarto, G.E., Therman, E., Patau, K. : Increased Q-fluorescence of an inactive X q - chromosome in man. Clin. Genet. 6, 1-5 (1974) Takagi, N., Oshimura, M. : Fluorescence and Giemsa banding studies of the allocyclic X chromosome in embryonic and adult mouse cells. Exp. Cell Res. 78, 127 135 (1973) Therman, E., Patau, K. : Abnormal X chromosomes in man : origin, behavior and effects. Humangenetik 25, l 16 (1974) Therman, E., Sarto, G.E., Patau, K.: Center for Barr body condensation on the proximal part of the human Xq: a hypothesis. Chromosoma (Berl.) 44, 361-366 (1974a) Therman, E., Sarto, G.E., Patau, K.: Apparently isodicentric but functionally monocentric X chromosome in man. Amer. J. hum. Genet. 26, 83 92 (1974b)
Received July 2~September 21, 1976 / Accepted September 21, 1976 by H. Bauer Ready for press September 21, 1976
