Clinical and cytological aspects of sex chromosome activity

Transcription

1 Hereditas Hereditas 86: (1977) Clinical and cytological aspects of sex chromosome activity ERICA BUHLER Department of Genetics, Children s Hospital, University of Basel, Switzerland BUHLER, E Clinical and cytological aspects of sex chromosome activity. ~ Lund, Sweden. ISSN Received February 10, : The first known example of a human female with more than one euchromatic X chromosome in somatic cells is presented, viz. a child with 2 normal X chromosomes and the long arm of a third X translocated onto one of the No. 22 chromosomes. In cultured lymphocytes there occurred, in addition to the ordinary Lyon pattern of X chromosome inactivation, 2 other patterns in which (I) 2 normal X chromosomes, and (2) one X and the t(x;22) were euchromatic. As the inactivation within the translocation chromosome did not spread onto chromosome 22, the clinical symptoms of the child had to be attributed to the excess of active X material, not to monosomy 22. Contrary to this, absence of heterochromaty of the Y chromosome does not influence the phenotype, as shown by the case of mixed gonadal dysgenesis with non-fluorescing Y presented. Possible reasons for this difference include: (I) The differences between facultative X and constitutive Y heterochromatin; (2) Different cytologic criteria for heterochromatin may not mean the same kind of inactivity; (3) Differences in phenotypic expression may be due to different chromosomal behaviour among tissues; (4) Late replication and decondensation may not equate genetic inactivity in terms of transcriptional inactivity. Erica Buhler, Department of Genetics, Children s Hospitul, University of Basel, CH-4000 Busel, Switzerland Cytological features of heterochromatin are: (I) Late replication as shown either with 3H-thymidine as heavy labelling in late S, with BUdR as decondensation or condensation delay or similar and combined methods. (2) Heterochromatin staining, i.e. positive C-banding and/or positive G-banding and negative R- banding. (3) Intense fluorescence (4) Condensation in interphase It is generally accepted that heterochromatin is genetically inactive. But while constitutive heterochromatin, such as the human Y, remains so, facultative heterochromatin, such as the human X, can change its state of activity. In addition to this basic difference, there also exist differences in staining, replication and condensation behaviour between different types and locations of heterochromatin. In the first part of our discussion we shall deal with functional aspects of X chromosome activity and try to correlate it with its cytological behaviour and compare it to the human Y chromosome. In the second part I shall present relevant examples of change in heterochromatic behaviour of X and Y chromosomes and discuss its implications on development and phenotype. X chromosome According to Lyon s hypothesis all X material in excess of one X chromosome is inactivated early in intrauterine life in somatic cells of the human. This inactivation is thought to be permanent. In germ cells, however, there is evidence for changes in activity of the X chromosome. OHNO and coworkers (1962) were the first to observe that the condensed X typically seen in somatic cells, the sex chromatin body, was absent from female premeiotic germ cells. Direct evidence that both X chromosomes are active has been provided by GARTLER et al. (1973) by demonstration of the hybrid interaction product in oocytes of G6PD (glucose-6-phosphate dehydrogenase) heterozygotes. However, in germ cells of very young female embryos (up to 12 weeks) the two single electrophoretic bands of the G6PD alleles were found, which means that at this stage of development there is only one active X in the germ cells just as in somatic cells. There is, thus, a change from the inactive to the active state by one X in female germ

2 64 E. BUHLER Hrrediias 86 (1977)

3 Hereditas 86 (1977) SEX CHROMOSOME ACTIVITY 65 Fig. 2. Decondensation of heterochromatic human X with BrdU; upper row: G-staining; lower row: Q-staining. cells during fetal development, probably at the onset of meiosis. The contrary might be true for the X in male germ cells. In spermatogonia the X has been shown to be decondensed and synthesizing RNA. Later it becomes condensed and late replicating and ceases RNA synthesis (MONESI 1965; ODARTCHENKO and PAVILLARD 1970). These findings suggest that the X passes from an active to an inactive state at the onset of meiosis in the germ cells of the male. Despite these facts in germ cells, X inactivation, once established, is believed to be permanent in somatic cells of eutherian mammals. The presumed cytological equivalents to genetic inactivity in the X chromosome are shown in Fig. 1-3 and include late labelling with 3H thymidine (Fig. l), decondensation with BrdU (Fig. 2) and condensation in interphase (Fig. 3). Y chromosome The long arm of the Y chromosome consists mainly of heterochromatin, only the euchromatic short arm and the paracentromeric long arm seem to be genetically active. These parts are strongly male determining. The H-Y antigen and the testis-determining gene product are probably identical (although we do not know whether we are dealing with a structural gene or regulatory genes). The gene(s) coding for it c I I c Fig. 3. Condensation of the human X chromosome; sex chromatin in cell from the amniotic fluid. Fig. 4. The G group chromosomes of 2 cells; late labelling of the Y chromosome with 3H-thymidine.

4 66 E. BUHLER Hereditus 86 (1977) Fig. 5. Decondensation of the human Y chromosome with BrdU; upper row: G-staining, lower row: Q-staining. are either located in the proximal long arm as suggested by SIEBERS et al. (1973) or in the short arm, for which there is new evidence (Koo et al. 1976). Other features in the male, such as testicular maturation, spermatogenesis, body growth, skeletal and dental maturation also seem to be Y dependent, at least partly (BUHLER and STALDER 1976). The distal long arm of the Y, however, is heterochromatic and, as such, probably genetically inactive. It consists of constitutive heterochromatin, a term used to designate repeated nucleotide sequences mostly contained in the satellite fraction of DNA. Just as facultative heterochromatin, the Y chromosome is late replicating, possibly not as late as the X. It shows condensation delay with BrdU and condensation in interphase. Besides, it fluoresces brilliantly with quinacrine, a feature attributed to its AT richness and to its being lighter than the main band fraction of DNA. Satellite DNA, heavier than the main band and either poor in AT sequences or containing AT sequences interrupted by GC sequences, does not exhibit intense Q fluorescence in spite of being constitutively heterochromatic. Thus, intense Q fluorescence is a unique feature of the type of satellite DNA contained in the human Y. Other heterochromatin staining properties, such as C- banding and G-11 staining are shared with other constitutively heterochromatic blocks in some auto- Fig. 6. Intense fluorescence with quinacrine in metaphase of human YY individual (left); condensation in interphase (right)

5 Hereditas 86 (1977) SEX CHROMOSOME ACTIVITY 61 somes. The well-known polymorphisms of these chromosomal regions which lack phenotypic expression suggest that these areas are genetically inactive. The cytological equivalents to genetic inactivity in the constitutive heterochromatin of the human Y are shown in Fig. 4-7 and include late labelling with 3H-thymidine (Fig. 4), decondensation with BrdU (Fig. 5), intense fluorescence with quinacrine (Fig. 6) and positive staining with Giemsa-11, quinacrine and C-banding (Fig.7). Case reports First case The first case to be discussed is apparently the first female with more than one euchromatic X in somatic cells. P.W. is a physically and mentally retarded baby of 8 months (Fig. 8). She is odd-looking with mongoloid slant to the eyes, beaked nose, receding chin, low-set ears, epicanthal folds and congenital heart disease. Hypotonicity changes with hypertonic spells. IQ on the Biihler-Hetzer scale is 54. However, mental development and intellectual achievements seem to be better than physical and motor development. Fig. 9 shows an R-banded karyotype of the little girl. One chromosome 22 was found to be replaced by a C-like chromosome, the long arm of which had the banding pattern of an X long arm, the short arm corresponding to the missing 22 chromosome. Fig. 10 shows her mother s karyotype. She had the same translocation chromosome, but only one normal X and an E-like chromosome with a median band in its long arm. This chromosome consists of the short Fig. 7. Positive heterochromatin staining of the Y chromosome from 4 metaphases; from left to right: Giemsa 11, quinacrine, C-banding. arm, centromere and proximal long arm of the second X. Thus, the mother was a balanced translocation carrier of an X/22 translocation. The child is trisomic for most of an X long arm and monosomic for a 22 short arm. Both anomalies, trisomy X as well as monosomy 22p are known to cause few if any clinical Fig. 8. Appearance of the patient (case I).

6 68 E.BUHLER Hereditas 86 (1977) I b X=h xx c I v F1 N a I c N I I 1L N N -2 0 *I..- I a

7 Hereditus 86 (1977) SEX CHROMOSOME ACTIVITY 69 r! -c 1 n I (Y 4 c II) c u 5 L n I h 0 *, N L 5 i u 5 m c * I d " 4-0 4

8 70 E. BUHLER Hereditus 86 (1977) a C Fig. I 1. Sex chromatin patterns; a: 1 small Barr body; b: 1 normal-sized body; c: I normal-sized and 1 small body; d: 2 normal-sized bodies. abnormalities. Triple X females can be slightly mentally retarded, tall or subfertile. Carriers of a Robertsonian translocation involving chromosome 22 with consecutive loss of its short arm are phenotypically normal. Sex-chromatin examination in several tissues revealed 4 different patterns: Normal-sized single Barr bodies, small bodies and double bodies, some of which were of unequal size (Fig. I I). Labelling of lymphocyte cultures with 3H-thymidine and BrdU during the final 7 hours of DNA synthesis revealed three different patterns of X inactivation: (1) Inactivation of the X long arm only, translocated onto chromosome 22, leaving 2 entire Xs euchromatic (Fig. 12: Type 1). (2) Inactivation of one entire X, leaving the other X and the long arm of the X/22 translocation chromosome euchromatic (Fig. 13: Type 2). (3) Inactivation of one X and the long arm of the translocation chromosome, leaving just one X active (Fig. 14: Type 3). This is the pattern to be expected according to Lyon's hypothesis of inactivation of all X material in excess of one. The sex chromatin findings can be interpreted as follows: One small body is thought to correspond to the cells with two active Xs, only the long arm of the translocation X being inactivated. The normal-sized Barr body is derived from one normal-sized heterochromatic X; in these cells one whole X and the long arm of the translocation X are euchromatic. In the normal cells - normal in the sense of Lyon's b d hypothesis - double bodies of different size are to be expected. The equally-sized bodies probably are derived from cells with the same inactivation pattern, the apparent equal size being explained by technical factors and observational bias, because no cells were found in lymphocyte cultures with two whole Xs inactivated and only the long arm of the translocation chromosome euchromatic. Cytologically, there is no doubt that euchromatization has taken place in the X chromosomes of somatic cells in this child. The mechanism by which this re-activation or possibly non-inactivation has been accomplished remains obscure. RAO and IHAN- WAR (1975) presented autoradiographic evidence of euchromatization of the partially facultatively heterochromatic X of an Indian rat in 13'>/:, of bone marrow cells. Sex-chromatin negative XXY triploid cells seem to be another example of the spontaneous occurrence of 2 active Xs in somatic cells. Experimental attempts to induce reactivation of inactivated Xs by hybridization or exposure to selective pressure have mostly been unsuccessful (MIGEON 1972). The only proven instance of derepression of a single gene coding for HGPRT (hypoxanthine-guanine phosphoribosyl-transferase) on the inactive human X has been achieved by hybridization of man and mouse somatic cells by KAHAN and DEMARS (1975). The phenotypic effect of the chromosomal anomaly in this case was first thought to be due to spread of inactivation onto the translocated 22 so that the child would, in fact, be monosomic for an entire chromosome 22. However, Fig. 12 and 14 show that there is no evidence of decondensation with BrdU or late replication with 'H-thymidine of the translocated chromosome 22. Therefore the reason for the failure to thrive and the odd appearance of the child must be sought in the genetic unbalance caused by non-inactivation of all X material in excess of one X. Maybe I should here cite a finding of TAKACI and OSHIMURA (1973) in a study designed primarily to examine the relationship between fluorescence and Giemsa banding of the heterochromatic X in embryonic and adult mouse cells. They observed in 230 metaphases 3 in which they believed that reversal of chronology of replication of the X may have occurred. These 3 instances were encountered in embryos, underdeveloped for their age, a possible equivalent to our patient, who has always been too small and underweight for her age. In this case, lack of late replication, decondensation with BrdU and condensation in interphase suggest genetic activity of more than one X and, hence, genetic unbalance, which is reflected in phenotypical alterations.

9 Hiwditua 86 (1977) SEX CHROMOSOME ACTIVITY 7 1 X * X 8 X r 1' d

10 72 E. BUHLER Hereditas 86 (IY77) Fig. IS. Case 2, the G group chromosomes in Q-banding (rows I, 3, 5-7). autoradiography (row 2) and C-banding (rows 4, 8-10), Note non-fluorescing Y! Second case In the second case to be presented, change of heterochromatic behaviour of a Y chromosome does not seem to have changed the phenotype of the individual concerned. C.K. was a phenotypical female with some features of Turner s syndrome and mental subnormality. This case has been published 2 years ago (BUHLER et al. 1974). No sex chromatin and no Y chromatin were found on several occasions. Chromosome examination revealed 75% XO cells and 25% XY cells, the Y lacking heterochromatic behaviour although its size was normal. First an X deletion was suspected, but the consistant non-late replication and negative centromere staining with the C-banding method suggested a Y rather than the short arm of a second X (Fig. 15). Histological findings in the gonads at gonadectomy proved this assumption: remnants of male ducts were found within the streaks. The otherwise female (or Turner) phenotype of the patient can be explained by the XO cell line. It is known that XOjXY individuals can display any phenotype between almost normal male and almost normal female. The Y must have exerted its maledetermining influence early in fetal development in this case, testicular tissue has undergone secondary degeneration, some male duct structures remained. Since the male-determining factors are located on the non-fluorescent pericentromeric regions of the Y chromosome, the change in heterochromatic behaviour of the distal Yq cannot affect sex differentiation in these cases. All other Y-dependent features are thought to be caused by factors located on the euchromatic parts of the Y as well. Thus, we would not expect any phenotypic effects with this chromo-

11 Hurudita.v 86 (IY77) SEX CHROMOSOME ACTIVITY 73 soma1 anomaly, concerning any of the above-mentioned traits. Furthermore, since the phenotype of this and other XOjXY cases with non-fluorescing Y was not different from cases with fluorescing Y, we must assume that cytological euchromatization of the Y apparently is not equivalent to functional euchromatization or genetic activation, as seems to be the case in X-chromosome euchromatization. Discussion What could be the reason for this difference? (1) X-chromosomal heterochromatin is facultative heterochromatin, Y-chromosomal heterochromatin is constitutive heterochromatin. Constitutive heterochromatin probably is genetically inert due to its chemical make-up in the sense of transcriptional inactivity. It is made up by special, mostly repeated satellite DNA sequences, histone and non-histone proteins. Stainability and replication pattern may be the direct consequences of this chemical constitution, mainly of its DNA configuration. This configuration can be altered without affecting transcriptional activity as long as the proteins are unaltered. It has been shown in vitro that sequences which are usually not represented in nuclear RNA can be transcribed after salt extraction of H1 histone and some non-histone proteins ( KLUKAS 1976). Therefore, we can assume that in the constitutive heterochromatin of the human Y a special pattern of these proteins exists which prevents the DNA from being transcribed. Thus, loss of heterochromatic behaviour of the Y chromosome owing to altered DNA configuration would not affect genetic activity or inactivity. In facultative heterochromatin the unspecific reaction to heterochromatin staining suggests a chemical composition of the DNA different from that in the constitutive heterochromatin. Although replication and condensation behaviour are somewhat similar, the sequence of activation ~ inactivation events of the X in human germ cells suggests some alterations in histone and non-histone proteins controlling transcription. Thus, euchromatization of the human X might be due to protein alterations rather than to DNA changes, and would, indeed, affect genetic activity and hence, phenotypic expression. (2) We do not know the exact relationships between late labelling with 3H-thymidine, decondensation with BrdU, condensation in interphase, heterochromatin staining, intense fluorescence. They may indicate, though not exactly, the same kind of inertness. Late labelling certainly depends on DNA replication directly. Condensation delay with BrdU and precococious condensation may be consequences of structural changes secondary to DNA protein interactions or even directly to action on the protein structure of chromatin. Most probably late replication is more equivalent to inactivation than condensation, heteropycnosis or staining reactions. GARTLER et al. (1973) showed that in clones of cells of G6PD heterozygotes with the B-variant inactivated, the A-variant is active in all cells, although half of them show no sex chromatin body. This means that also in cells where the heterochromatic X is not condensed it can still be inactivated. This apparently can apply to constitutive heterochromatin as well: in certain animals late replication of constitutive heterochromatin occurs also in tissues in which no chromocenters are formed. (3) Undetected differences in heterochromatic behaviour of the sex chromosomes between tissues may help explain the differences in phenotypic expressivity. We do not know whether or not X inactivation follows the same pattern in all other tissues of our first case. and we do not know if the Y in cases of XOjXY with non-fluorescing Y is non-fluorescing in all tissues. Besides, it is remarkable that a nonfluorescing Y has never been found in a non-mosaic XY individual so far. Whether or not the cytological euchromatization would have an effect in such a case we do not know. Maybe, such individuals are not viable at all, which could mean that euchromatization does have a phenotypic effect and an euchromatic Y long arm may be functioning in the genetic sense. In conclusion, we must confess that we do not know to what extent we can equate all the mentioned aspects of euchromatin and heterochromatin with genetic activity or inactivity in terms of transcription. Hopefully, evaluation of our first case in more detail - examination of more tissues, quantitative assays of X-chromosomal enzymes, preferably in cloned cells ~ and of our second case by studying H-Y antigen and Y specific DNA, as well as detection of more cases with altered behaviour of sex chromosomes, together with further experimental attempts at derepression of inactive chromosomes, will help to elucidate some of these complex interrelationships. Finally J would like to suggest that all cases of triple X females with associated symptoms other than slight mental retardation, abnormal pattern of growth and fertility should be examined with appropriate methods to clarify replication behaviour of the extra X.

12 Proc. Cyrogenet. 74 E.BUHLER Hereditas N6 (1977) Literature cited BUHLER, E. M., B~HLER, U. K., TSUCHIMOTO, T. and STALDER. G. R Nan-fluorescent Y chromosome. - Helv. Paediat. Acta 29: BUHLER, E. M. and STALDER, G. R The human Y chromosome. - In Medical Genetics (Ed. G. SZABO and Z. PAPP), Akadimiui Kiado, Budupest, (in press) GARTLER, S. M., CHEN, S. H., FIALKOW. P. J., GIBI-ETT. E. R. and SINGH. S X chromosome inactivation in cells from an individual heterozygous for 2 X-linked genes. Nature New Bid. 236: GARTLER, S. M., LISKAY, R. M. and GANT, N Two functional X chromosomes in foetal oocytes. -- Exp. CeN Res. 82: KAHAN, B. and DEMARS, R Localized derepression on the human inactive X chromosome in mouse-human cell hybrids. ~~~ Nut. Acad. Sci. 72: KLUKAS, C. K Nan-histone proteins and transcription. - Nuture 263: Koo. G. C., WACHTEL, S. S. and BREG, W. R Mapping the locus of the H-Y antigen. - Birth Dy/ects: Original Article Serie.s. I2 (7): MIOEON, B. R Stability of X chromosomal inactivation in human somatic cells. - Narure New Bid. 239: MONESI, V Differential rate of RNA synthesis in the autosomes and sex chromosomes during male meiosis in the mouse. - Chromosomu 17: ODARTCHENKO, N. and PAVILLARD, M Late DNA replication in male mouse meiotic chromosomes. - Science 167: OHNO, S., KLINGER, H. P. and ATKIN, N. B Human oogenesis. Cyrogenetics I: RAO. S. R. V. and IHANWAR, S. C Is late replication of the inactive X chromosome irreversible in all cells of mammals? ~ Cell Genet. 14: SIEBERS, J. W., VOGEL, W., HEPP, H.. BOLZE, H, and DITTRICH, A Structural aberrations of the Y chromosome and the corresponding phenotype. - Humangenetik 19: TAKAGI, N. and OSHIMURA, M Fluorescence and Giemsa banding studies of the allocyclic X chromosome in embryonic and adult mouse cells..- Exp. Cell Res. 78: I35