Chandler, P.

Disteche, C.M.

Ferguson-Smith, M.A.

Goulmy, E.A.J.M.

Page, D.C.

Simpson, E.

Leiden University Scholary Publications

Repnnted from Nature, Vol 326, No 6116 pp 876 878, 30 April 1987© Macmülan Journals Ltd , 1987\ >Separation of the genetic lod for theH-Y antigen and for testisdetermination on human Υ chromosomeElizabeth Simpson*, Phillip Chandler*, Eis Goulmyf,Christine M. Disteche*, Malcolm A. Ferguson~Smith§& David C. Page11* Transplantation Biology Section, Chnical Research Centre,Harrow, Middlesex HAI 3UJ, UKt Department of Immunohaematology and Blood Bank AZL,University Hospital Leiden, The Netherlands^Department of Pathology, University of Washington, Seattle,Washington 98195, USA§ Duncan Guthne Institute of Medical Genetics,University of Glasgow, Glasgow, G3 8SJ, UK|| Whitehead Institute, Cambridge Massachusetts 02142, USAThe mammalian Υ chromosome encodes a testis-determining factor(iermed TDF in the human), a master regulator of sex differenti-ation. Embryos with a Υ chromosome develop testes and becomemales whereas embryos iacking a Υ chromosome develop ovariesand become females. Expression of H-Y, a minor histocompatibil-ity antigen, may also be controlled by a gene on the Υ chromosome,and it has been proposed that this antigen is the testis-determiningfactor1. We have tested the postulated identity of H-Y and TDFin the human. H-Y typing with Τ cells was carried out on a seriesof sex-reversed humans (XX males and XY females), each shownby DNA hybridization to carry pari bnt not all of the Υ chromo-some. This deletion analysis maps the gene for H-Y to the longarm or centromeric region of the human Υ chromosome, far fromthe TDF locus, which maps to the distal Short arm.H-Y typing of sex-reversed mice has shed hght on the relaüon-ship of this antigen to gonadal sex determination XX Sxr (sexreversed) mice are both male and H-Y positive owing to thepresence of a small portion of the Υ chromosome2 3, consistentwith a testis-determimng role for H-Y However, a derivativeof Sxr known as Sxr' produces XX Sxr' and XO Sxr' mice thatare phenotypically male but H-Y negative4 5, demonstrating thatthe antigen is not required for testis determinationIn humans, DNA hybridization studies using Y-specificprobes of mdividuals with only parts of the normal Υ chromo-some have yielded an eight-mterval deletion map of the Υchromosome Such studies of XX males and XY females thatcarry part but not all of the Υ chromosome, have demonstratedthat interval 1, representmg a portion of the distal short arm ofthe Y, contains TDF6 7 Similar studies of another series of XXmales, using a different set of Y-DNA probes, hkewise showthem to possess sequences both withm and distal to Ypl 1 2 (refs8 and 9) We reasoned that lf there is a Y-chromosomal generesponsible for H-Y antigen expression then it should be poss-lble to map that gene by H-Y typing mdividuals with well-charactenzed Υ deletionsH-Y-specific T-cell clones and lines have been denved in vitrofrom transfused human female aplastic anaemia patients10These H-Y specific Τ cells, whose response is HLA restncted,were used to H-Y type Epstein-Barr-virus-transformed B-celllines from deleted-Y mdividuals Because only HLA-A2 orB7-restncted H-Y-specific T-cell clones were available at thetime of this study, we were able to H-Y type only those deleted-Ymdividuals who were HLA-A2 and/or -B7 Variante of HLA-A2and -B7 which cannot be recognized by allogeneic or HLA-restncted Τ cells have been descnbed11 Accordingly, in eachcase, the HLA type was determmed serologically and confirmedusing alloreactive cytotoxic Τ cellsTable 1 shows the results of HLA and H-Y typing of eightdeleted-Y mdividuals (six 46.XX males and two 46,XY females)All six XX males were negative for H-Y These include threeXX males, LGL105, WHT950 and WB, who carry intervals 1,2 and 3 of the Υ chromosomal short arm (refs 6 and 12, D C Ρunpubhshed results, see class 3 XX males in Fig 1) and threeXX males, RH, JT and JM, with similar portions of the Υ shortarm detected using a different set of Y-DNA probes8 9 WhereasTDF is in interval 1 of the Υ chromosome6 7, these resultsindicate that the gene for H-Y lies outside intervals 1, 2 and 3This conclusion is reinforced by the finding that both XYfemales are positive for H-Y XY female WHT1003 has adeletion of intervals 1, 2 and 3 on the Υ short arm, whereas XYfemale WHT715 has a deletion of intervals 1, 2 and 4A (refs 7and 13, see class 1 and 2 XY females in Fig 1) Taken together,these results with XX males and XY females strongly suggestthat the gene for H-Y maps somewhere in intervals 4B to 7 ofthe Υ chromosome, that is, near the centromere or on the longarm (refs 6 and 7, Fig 1)The finding that XX Sxr' male mice were H-Y negativestrongly suggested that H~Y is not the testis determinantHowever, it might be that the Sxr' mutation had affected theantigenicity but not the testis-determining capability of H-YThere is an earher report of human XX males with negativeH-Y type and an XY female with positive H-Y type withcytotoxic Τ cells14 However, the XX males had not been testedfor the presence of Y-chromosomal DNA and the XY femalehad not been tested for deletions of Υ DNA sequences Theresults reported here are not subject to such ambiguities ofInterpretation In the present study, H-Y typing was carried outon cells from eight sex-reversed mdividuals with well-character-lzed deletions of portions of the Υ chromosome The combma-tion of negative and positive findings reported here demonstratesunequivocally that the gene for H-Y maps far from interval 1,which contains TDF and that H-Y antigen is not the testis-determimng factor H-Y antigen was onginally defined by graftrejection15 The in vitro T-cell assays of the type used for H-Ytyping in this study have been shown to recognize the samehistocompatibility antigen as that defined by graft rejection16In contrast, serological Identification of a male antigen assumedto be H-Y (and now called serologically detected male antigen,SDM) has been problematical In particular, there is doubtconcerning whether the SDM antigen is identical to the H-Yhistocompatibility antigen5 1 7 The implications of the presentstudy, therefore, are hmited to the H-Y antigen and do notextend to the SDM antigenWe are currently examining additional deleted-Y mdividualsin an efiort to map the H-Y locus more precisely The possibilitythat the H-Y gene might be identical to or near a spermatogenesisfactor on the long arm of the human Υ chromosome18 is ofparticular interest, because in mice H-Y (or a closely linkedgene) has been imphcated in spermatogenesis19 Though theresults reported here imply that H-Y has no function in gonadafsex determination, lt may be involved in testis function at a laterstage of developmentWe thank Eis Blokland and Jos Pool for isolating H-Y specificT-cell lines and clones and for assistance with the typing, Albertde la Chapelle, Joseph Hersh and Martin Crawfurd for pro vidingcell lines and blood samples, and Lesley Snadden for help insetting up and maintaining EB transformed lymphoblastoidlines This work was supported in part by grants from the MRCTDFXX Males ~ class 3Tclass 1XYFemales 2IEEIFig 1 Eight interval deletion map of the human Υ chromosome(based on refs 7 and 13) The results of H-Y typing of mdividuals(from Table 1) are summanzed The short arm, centromere andlong arm of the Υ chromosome are denoted by, respectively 'ρ ,cen' and 'q TDF gene for testis determining factor Honzontallines, portions of the chromosome present in the designatedindividualExperiment1234Table 1 HLA andKaryotype/sexXX<JXX<?ΧΧ<ίXY<SXX2ΧΧ<ίXYrJXX2XXcJXXSXY6XX$XY9XY5ΧΥ<ίXX9Η-Υ typing of B-ceIIIndividualRHJTLGL 105Normal maleNormal femaleWBNormal maleNormal femaleWHT95OΜNormal maleNormal femaleWHT 1003 (case 1)WHT715 (case 2)Father of case 2Mother of case 2lines from XX males,HLA serology*A2A2A2A2A2A2A2A2B7B7B7B7B7B7B7B7XY females and normal controlsSpecific lysis (%)aA-2182413201237251ZαΒΊ7662ND5455575236aH-Y/A-29343880173«H-Y/B7914007069616H-Y phenotype---+--+---+-+++-The H-Y-specific cytotoxic Τ cells were T-cell lines or clones derived from the peripheral blood of transfused female aplastic anaemia patients10.The alloreactive cytotoxic Τ cells were derived from the peripheral blood of healthy individuals". They were used in the cytotoxic assays 5-7 daysafter aliquots previously stored in liquid nitrogen had been thawed and restimulated in vitro with appropriate antigen bearing irradiated peripheralblood cells in the presence of interleukin-2 (IL-2) in RPMI medium containing penicillin, streptomycin, glutamine, HEPES and 10% fetal calfserum (FCS) or human serum. The target cells were Epstein-Barr virus (EBV)-transformed B-cell lines grown in vitro. Immediately before theassay, aliquots were iabelled with 51Cr-labelled sodium chromate, washed and dispensed at 5 x l 0 3 cells per well in round-bottomed microtitrewells. Triplicate wells were used at each Α: Τ ratio3·4·1 4. Per cent specific lysis given is that at an attacker-to-target ratio of 10:1, the figure beingtaken from a regression analysis of a three or four point titration curve. Underlined figures are positive values on titration curves whose r2 valuelay between 0.70 and 1.00 (refs 3, 4 and 14). ND, Not done.* HLA serology of the B-cell lines from sex-reversed patients and the parents of one was performed either by Lorna Kennedy at the ICRFLincoln's Inn Field, or by Donald Palmer in the Department of Immunology, Hammersmith Hospital. Normal male and female cells in experiments1-3 were EBV-transformed B-cell lines from Professor A. Rickinson of Birmingham: the individuals from which they had been obtained wereserologically identified as A2 or B7 by him.(MAFS), the NIH, the March of Dimes, the American CancerSociety, the Dutch Foundation for Medical Health Research(Medigon), which is subsidized by the Dutch Organisation forthe Advancement of Pure Research (ZWO) and the J. A. CohenInstitute for Radiophathology and Radiation Protection (IRS).Received 3 February, accepted 5 March 19871 Wachtel, S S , Ohne-, S , Koo, G C & Boyse, Ε Α Nature 257, 235-236 (1975)2 Bennett, D et al NaMre 265, 255-257 (1977)3 Simpson, Ε , Edwards, Ρ, Wachtel, S S , McLaren, Α & Chandler, Ρ Immunogenetics 13,355-358 (1981)4 McLaren, Α , Simpson, Ε , Tomonan, Κ, Chandler, Ρ & Hogg, Η Nature 312, 552-555(1984)5 Simpson, Ε Cell 44, 813-814 (1986)6 Vergnaud, G el al Am. J hum Genel 38, 109-124 (1986)7 Page, D C. Cold Spring Harb Symp quant Biol 51, 229-235 (1986)8 Affara, Ν Α el al Nucleic Aads Res 14, 5353-5373 (1986)9 Affara, Ν Α et al Nucleic Aads Res 14, 5375-5387 (1986)10 Goulmy, Ε Progress in Allergy 36, 44-72 (1985)11 Horai, S, van der Poel, J J & Goulmy, Ε Immunogenettcs 16, 135-142 (1982)12 Andersson, Μ , Page, D C & de la Chapelle, Α Science 233, 786-788 (1986)13 Disteche, C Μ et al Proc natn Acad Sa USA 83,7841-7844(1986)14 Goulmy, Ε ,van Leeuwen, Α , Blokland, Ε .Sachs, Ε S &Geraedts,J Ρ Μ Immunogenettcs17, 523-531 (1983)15 Elchwald, Ε J & Silmser, C R Transplant Iluil 2, 148 (1955)16 Simpson, Ε et al Immunology 57, 345-349 (1986)17 Sllvers, W Κ , Gasser, D L & Eicher, Ε Μ Cell 28, 439-440 (1982)18 Tiepolo, L & Zuflardi, Ο Hum Genet 34,119-124(1976)19 Burgoyne, Ρ S, Levy, Ε R & McLaren, Α Nature 320, 170-172 (1986)l'nntcd in Grcat Bnrain by 7urneigrttpJiic Limiled Basirigsfoke Hampshire

Separation of the genetic loci for the H-Y antigen and for testis determination on human Y chromosome.

https://scholarlypublications.universiteitleiden.nl/access/item%3A3138085/download

Separation of the genetic loci for the H-Y antigen and for testis determination on human Y chromosome.

Abstract

Similar works

Full text

Available Versions

Leiden University Scholary Publications