Hum Genet (1982) 61 : 110-112

© Springer-Verlag 1982

H-Y Antigen in Male Patients with X Polysomies

Wolfgang Engel ~, Barbara Klemme ~, Hans-Dieter Probeck ~, and Cbrista Fonatsch 2

~ Institut ffir Humangenetik der Universit/it G6ttingen, Nikolausberger Weg 5 a, D-3400 G6ttingen, Federal Republic of Germany z Abteilung Humangenetik, Zentrum Kinderheilkunde und Humangenetik der Medizinischen Hochschule Hannover, Karl-Wiechert-Allee 9, D-3000 Hannover 61, Federal Republic of Germany

Summary. H-Y antigen was examined in eight male patients with X polysomies, namely four patients with 47,XXY, one patient with 48,XXXY, two patients with 49,XXXXY, and one patient with the mosaic 47,XXY/49,XXXXY. In all patients the H-Y antigen titers were lower than in normal 46,XY males. However, a linear correlation between the number of additional X chromo- somes and the reduction of H-Y antigen titers could not be demonstrated. Such a correlation would be expected if the gene for the repressor of H-Y antigen expression is active also on the additional X chromosomes.

Introduction

The histocompatibility antigen H-Y seems to be the testis- determining factor of the bipotent gonadal anlage in mammals. The structural gene for this antigen was originally assigned to the Y chromosome. Because of the observed H-Y antigen positivity in patients with Turner syndrome (Wolf et al. 1980a; Koo et al. 1981 ; Engel et al. 1982) and in XO mice (Engel et al. 1981), the Y- chromosomal localization of the gene became questionable, and its X or autosomal localization was favored. The intermediary titers of H-Y antigen observed in Turner patients and in XO mice indicate that in a normal H-Y antigen-negative female, gene products coded by both X chromosomes are absolutely neces- sary to suppress the expression of the H-Y antigen gene. Wolf et al. (1980 b) claimed that active genes for a repressor were present on both X chromosomes and assigned them to the short arm of the X chromosome in the segment Xp 223. In the normal XY male the H-Y antigen positivity is sustained by the interaction between a Y-coded inducer and the X-coded repressor, thus leading to the expression of the H-Y antigen gene.

It is well known that in both sexes only one X chromosome is active while additional X chromosomes are subjected to inactivation. However, if in the XX female the genes for the repressor for the H-Y antigen gene are active on both X chromo- somes, the question arises if in X polysomies in males these genes on the additional X chromosomes are active also. In this case one would expect lower H-Y antigen titers in men with X polysomies than in normal 46,XY males, as mentioned by Wolf et al. ( 1980 b). We report here the results of H-Y antigen investigations in eight male patients with X polysomies, namely, four patients with 47,XXY; one patient with 48,XXXY; two patients with 49,XXXXY; and one patient with the mosaic 47,XXY/ 49,XXXXY.

Offprint requests to: W. Engel

Materials and Methods

All patients investigated were known to us as the results of chromosomal studies performed in our laboratories. From 20 to 30 ml of heparinized blood from each patient was sent to us by mail, and chromosomal analyses and H-Y antigen investigations were performed simultaneously.

Cytogenetic Studies. Metaphase chromosomes from peripheral lymphocytes were obtained by standard culture methods. G- banded chromosomes were prepared according to Sperling and Wiesner (1972).

H-Y Antigen Typing. The procedure used for H-Y antigen testing was the same as described earlier (Engel et al. 1982). Antiserum to H-Y antigen was raised in isogenic Lewis/Han rats by means of six weekly intraperitoneal injections of 3 x 107 male spleen cells into female rats. The cells were treated with 0.2% glutaral- dehyde solution in PBS for 20min in ice and subsequently washed three times in PBS. Blood was taken 1 week after the last injection; the serum was heat-inactivated at 56°C for 30 min and absorbed with an equal volume of packed human female A~B erythrocytes. Rabbit complement was selected according to low cytotoxicity on target cells, namely, the cultured human male lymphoid cell line "Raji" and the cultured human female lymphoid cell line "B JAB."

Leukocytes of the patients and of two male and three female controls were prepared from the buffy coat by wash-centrifuga- tion, and portions of 1 x 107 cells (about 5% dead cells) were used for the absorption of 30~tl anti-H-Y antiserum. All cell suspensions were scored as coded samples. In the final cytotoxic test 1 lal of absorbed antiserum was incubated with 103 each of Raji and B JAB cells for 45 min at room temperature, followed by incubation with 5~tl rabbit complement (45rain at room temperature). After the liquid was removed, 1 ~tl of 0.1% trypan blue-Hanks solution was added, and lysed Raji or B JAB cells were counted.

Results and Discussion

H-Y antigen investigations were done in four patients with 47,XXY; one patient with 48,XXXY; two patients with 49,XXXXY; and one patient with the mosaic 47,XXY/ 49,XXXXY. The results are given in Fig. 1 a-g. Compared with the H-Y antigen titers in normal 46,XY males, the titers in all patients with X polysomies are reduced.

From the intermediary titers of H-Y antigen in Turner patients with 45,XO and 45,XO/46,XX, in patients with

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Fig. la-g. Cytotoxicity of anti-H-Y antiserum to Raji and B JAB cells in the presence of rabbit complement after absorption with leukocytes from patients with X polysomies and controls. • • Female controls; zx • male controls; o x patients, a-c 47,XXY; d o, 47,XXY, x, 47,XXY/49,XXXXY; e 48,XXXY; f-g 49,XXXXY. Each titer curve is the result of a single experiment. A = all absorbed antisera were tested for cytotoxic activity on H-Y antigen-negative female lymphoblastoid B JAB cells in the presence of complement (control); mean values and deviations in all experiments performed with the cells of a patient are given. Complement control with Raji and B JAB cells was 10%

4 6 , X X p - or i sochromosome for the long arm of the X[46,Xi (Xq)], and in XO mice, it was concluded tha t in X X females, genes for a repressor on bo th X chromosomes are active and are engaged in the suppress ion of the H-Y structural gene (Wolf et al. 1980a, b; Engel et al. 1981, 1982). According to this hypothesis an increasing n u m b e r of X chromosomes in the male with one Y ch romosome should lead to a reduct ion of the H-Y ant igen titers (Wolf et al. 1981). However , a clear corre la t ion between the n u m b e r of X chromosomes and the H-Y ant igen titer was not observed in our patients. This result can hardly be explained. It can be hypothes ized tha t leukocytes are no t well suited for the H-Y ant igen determinat ion. Possibly due to the ch romosomal abnormal i ty , the n u m b e r of younger leukocytes in the b lood increases in corre la t ion with the n u m b e r of X chromosomes , and younger leukocytes might have more H-Y ant igen molecules on the cell membrane . But also a genetic explanat ion is possible.

The gene for the repressor of H-Y ant igen gene expression was assigned to tha t segment of the shor t a rm of the X chromo- some where the gene for s teroid sulfatase is located (Tiepolo et al. 1980; Mtiller et al. 1980 a). Similar to the gene for the H-Y ant igen repressor, the gene for steroid sulfatase is active on bo th X chromosomes of the normal X X female (Shapiro et al. 1979; Mtiller et al. 1980b). However, it was found that in cases with supernumera ry X chromosomes steroid sulfatase activity is not linearly correlated with the n u m b e r of X chromosomes as would be expected for the steroid sulfatase genes being active also in the addi t ional X chromosomes (Ropers et al. 1981). This result

might indicate tha t in cases with X polysomies the act ivat ion- inact ivat ion pa t te rn of addi t ional X chromosomes is dis turbed. In consequence of this hypothesis , our result of a nonl inear decrease of H-Y ant igen titers in XXY, XXXY, and X X X X Y males can be explained in two different ways: (1) the repressor

genes on the addi t ional X chromosomes are part ial ly or even totally inactivated, and (2) the s tructural gene for H-Y ant igen is located on that par t of the X ch romosome which is normal ly inact ivated in the X X female. In cases with addi t ional X chromo- somes this gene escapes to some extent the inact ivat ion process while the genes for the repressor are active on all addi t ional X chromosomes.

In this case one has to assume tha t in the normal female with XX, two doses of repressor genes are necessary to suppress the H-Y ant igen gene on the active X chromosome. Fur the rmore , it can be speculated tha t in females with addi t ional X chromo- somes, H-Y ant igen positivity can be found. It should be ment ioned here tha t Ohno (1979) pointed out tha t the localiza- t ion of the H-Y st ructural gene on the X ch romosome is the only al ternative to its Y-chromosomal localization. H-Y ant igen typing of females with X polysomies is now being done in our laboratory.

Acknowledgements. We would like to thank Mrs. Bettina Pape for her excellent technical assistance and Mrs. Monika Pelz for preparing the manuscript. This work was supported by the Deutsche Forschungs- gemeinschaft and the Stiftung Volkswagenwerk.

112

References

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Engel W, Klemme B, Probeck H-D, Hansmann I (1982) H-Y antigen in Turner-patients with different sex cbromosome constitutions. Hum Genet 59 : 333-336

Koo GC, Tada N, Chaganti R, H~immerling U (1981) Application of monoclonal anti-H-Y antibody for human H-Y typing. Hum Genet 57: 64-67

Miiller CR, Westerveld A, Migl B, Franke W, Ropers HH (1980a) Regional assignment of the gene locus for steroid sulfatase. Hum Genet 54:201-204

Mialler CR, Migl B, Traupe A, Ropers HH (1980b) X-linked steroid sulfatase: evidence for different gene dosage in males and females. Hum Genet 54:197-199

Ohno S (1979) Major sex-determining genes. Springer Verlag, Berlin Heidelberg New York

Ropers HH, Migl B, Zimmer J, Fraccaro M, Maraschio PP, Westerveld A (1981) Activity of steroid sulfatase in fibroblasts with numerical and structural X-chromosome aberrations. Hum Genet 57 : 354-356

Shapiro L J, Mohandas T, Weiss R (1979) Non-inactivation of an X- chromosome locus in man. Science 204:1224-1226

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Tiepolo L, Zuffardi O, Fraecaro M, diNatale D, Gargantini L, Mtiller CR, Ropers HH (1980) Assignment by deletion mapping of steroid sulfatase X-linked ichthyosis locus to Xp 223. Hum Genet 54: 205-206

Wolf U, Fraccaro M, Mayerova A, Heeht I, Zuffardi O, Hameister H (1980a) Turner syndrome patients are H-Y positive. Hum Genet 54:315-318

Wolf U, Fraccaro M, Mayerova A, Hecht T, Maraschio PP, Hameister H (1980b) A gene controlling H-Y antigen on the X-chromosome. Tentative assignment by deletion mapping to Xp 223. Hum Genet 54:149-154

Wolf U, MiJller U, Ohno S (1981) H-Y antigen and sex determination in mammals. Chromosomes Today 7:258-270

Received April 22 / Revised May 27, 1982