Mapping the H-Y gene.

Development 101 Supplement 157 161 (1987)

Pnnted m Gicdt Bntdin © T b c Company of Biologists Ljmjtcd 1987 157

Mapping the H-Y gene

ELIZABETH SIMPSON1, PHILLIP CHANDLER1, ANNE McLAREN2, ELS GOULMY3, CHRISTINE Μ DISTECHE4, DAVID C PAGES and MALCOLM Α FERGUSON-SMITH6

'Transplantation Biology Sectton Climcal Research Ccntrc Watford Road Harrow Middlcsex I/Al 3UI UK 2MRC Mammahan Development Umt Wolfton IIou.se 4 Stephenson Wav London NWI 2HC UK ^Depl Immunohaematology & Bioeid Bank AZL Urmemty Hospital I eiden The Nelherlundt, 4Depl Pathology Univasity of Washington Seattle Washington 98195 USA

^Whüehead Institute 9 Cambridge Center Cambridge Massachusetts 02142 USA

'Duncan Guthnc Institute of Medieal Genelies Unnersity oj Glasgow Glasgow G3 8SJ UK

Summary

This paper uses cytotoxic and proliferative Τ cell

clones speeifie for H-Y and restricted by MHC mol-ecules to type mice and humans inheriting incomplete portions of the Υ chromosome. The data have allowed us to map the H-Y antigen gene Hya in mouse to a Position closely linked with, but separable from, Tdy on the Sxr fragment and thus presumably to a position of the normal mouse Υ chromosome near the centro-mere. The human H-Y gene maps between deletion intervals 4B and 7, separate from TDF which is on interval 1. We are currently testing cells from a

number of additional patients who have inherited diff erent portions of the Υ chromosome to pinpoint the mapping more closely. It is of interest that in mouse a Y-linked gene Controlling spermatogenesis (Spy) maps near Hya on the Sxr fragment: they could be the same or closely linked genes. In man, a gene Controlling spermatogenesis maps to Yq and the data so far do not exelude that it could be eoineident with the H-Y gene. Key words Η Υ gene cytotoxic Τ teil, sex leversed mice sex reversed humans

Introduction: Τ cell recognition of H-Y

The male speeifie transplantation antigen, H-Y, is controlled by a gene located on the Υ chromosome in both humans and mice H-Y is a member of a family of mmor histocompatibihty (H) antigens, each charactenzed by their abihty to stimulate certain immune responses of Τ lymphocytes (Loveland & Simpson, 1986) At one time, the examination of Η Υ expression was hmited to grafting expenments but since the advent of methods for generatmg speeifie cytotoxic and proliferative Τ cell responses in vitro and of maintaining these as cloned lmes following the introduction of Τ cell growth factors, II Υ expression can be tested in vitro as well (Simpson McLaren, Chandler & Tomonan, 1984, Simpson et al 1987) This approach has been particularly uscful for exa minmg the H-Y phenotype of individuals from outbred populations who are not so amcnable to the grafting approach One constraint on such in vitro testing wilh H-Y speeifie Τ cells is the nced to identify the major histocompatibihty complex (MHC Η LA

in man, H-2 in mouse) alleles of the individual to be typed, since the recognition of II-Y, hke othei minor Η antigens, is MHC restricted (Simpson & Gordon 1977) Τ cells recognize Η Υ only when it is associated with a particuiar self-MHC ailele, so an appropnate panel of Η Y-specific Τ cells in necessary to Η Υ type individuals of dilferent MHC allotypes

H-Y expression in sex-reversed mice

CBA)F, (H-2b/k) females lmmumzed with (C57BL/ 10 (H 2b) and CBA (H 2k) male cells, respectively, are given in Table 1 (Simpson, 1982) Table 2 shows the MHC restnction and H-Y specihcity of proliferat-ive Τ cell clones isolatcd Irom similar MLC using spieen cells from C57BL/6 (H-2b) and C3H (H-2k) female mice lmmumzed with syngeneic male cells (Simpson, 1985) H-Y specific cytotoxic Τ cells and clones were used to type cells from a panel of mice carrying the sex-reversing mutation Sxr (Table 3) These include XXSxr males and T16HX&tr females carrying the T16H, X-autosome translocation, which IS invanably active, so that the XSxr of paternal ongin IS inactive This permits the female development of these individudls, smee Sxr is presumably mactive m the majonty of cells, at least dunng gonadogenesis (McLaren & Monk, 1982) The results in Table 3 indicate that each of the XXSxr and XY males were

H-Y positive with the cytotoxic Τ cells and Τ cell clones appropnate for their H-2 haplotype These mice are from a nomnbred colony in which H-2k and H-2b are segregating Each of the XX females is H-Y negative, whilst of the mne T\6HXSxr females, eight are clearly H-Y positive, indicating that in adult hfe, at least, the gene Controlling expression of the H-Y antigen, Hya, on Sxr is expressed in some spieen cells The mnth mouse, number 39, was phenotypically Η Υ negative she was subsequently progeny tested (T16HSxr females, unhke XXSxr males, are tertilc) and since all of the non-XY progeny inhenting her Sxr were H-Y negative, lt was clear that a mutation had altered her Sxr fragment This vanant is now desig-nated Sxr' (McLaren et al 1984) XOSxr' male mice are also H-Y negative when tested by Τ cells in vitro so that XXSxr' and T16HXSxr' mice are not H-Y negative merely because Sxr' in them is inactivated

Table 1. H-Y responset, in H-2b homozygotes and H-2blk heterozygotes

Rcspondcr H 2 female Κ Α Ε BIO b b (b)

(BlOxCBA)Fi b b (b) k k k

Per cent specific lysis of target cells

lable Stimul tting cells (KID) Nonc C 57BL/6cf C57BI / 6 $ BIO A(5R)cf BIO A(4R)cf B10A(2R)cf bml2cf bml4cf CBAcf CBA? C3H OHcf BIO Acf D b b/ k at Α Pnming and boostmg antigen BlOcf CBAcf Target cell BlOcf BIO? C3Hcf C3II SWcf BIO A(2R)cf BIO A(2R)$ BIO A(5R)cf CBAcf CBA$ BIO Acf C3H OHcf BlOcf Κ b b k b k k b k k k d b Η 2 Α Ε b (b) b (b) k k b (b) k k k k b b k k k k k k d d b b D b b k b b b d k k d k b Restncting specificity Correctcd* for Η Υ % lysis rccognition 33 3 2 5 7 3 38 5 30 6 2 2 3 9 31 1 2 4 4 6 35 1 1 2 Η 2Dl Η 2Dk

Ί / 4 J as determincd fiom a four point regrcssion curve

2. Prohferative bbb bbb bbd kkb kkb bb bbb kkk kkk ddk kkd 2 1 responses oj H-Y-specific Γ Clone (ongin 1(B6A') 199 26637 389 3108S 219 177 and r cell clones estnction speciiicity) 10 2(B6 D' 541 241 455 1988 3 558° 270175 172 956 1 5 025 ) C 3 ( C 3 H D ' ) 108970 557 176428 648 D it 1 Irom a sepuate expenment in which the Stimulation by C 57BL/6cf was 65 434 ind mcdium only w is 1575

(Simpson, 1986) XXSxr' and T16HXSxr' mice are also H-Y negative when tested tor lts presence by transplantation, arguing for the identity of H-Y detected by these two methods, one in vitro and one in

vivo, and for the absence of H-Y antigen from all cells

in the body (Simpson et al 1986) Sxr' has lost Hya or the ability to express this gene, but still causes sex reversal in XXSxr' males, therefore the Y-chromo-some-associated testis determinmg gene Tdy on Sxr IS clearly separated from Hya by this mutation, although the two genes are closely hnked on Sxr and therefore presumably on the portion of the normal Υ chromosome, close to the centromere, where Tdy and

Hya are normally located (Simpson, 1986) Another

mutation which provides evidence foi the lmkage of

Tdy and Hya is Υl desenbed by Eichel & Washburn (1986) Υ' is apparcntly a rearranged Υ chromosome in which the painng region is located close to the centromere amongst the sperm generated by carner males is an XY, bearmg a paternal X to which the greater part of the Υ is attached The XXY mice created by the fertihzation of a normal X bearmg ovum with such a sperm are Η Υ positive and phenotypically male, with aspermatogemc testes (hke XXSxr Simpson et al 1983)

H-Y expressed in sex-reversed humans

The investigation of the position of the human H-Y gene on the Υ chromosome has produced findmgs which are in parallel with those of mice, sjnce they clearly separate the testis-determming factor,

TDF, from the H-Y gene, but in man the lmkage

between these two genes, unhke mouse, is not at all close (Simpson et al 1987)

H-Y typing in man is possible because of the Isolation of Τ cell clones specific tor H-Y from tiansfused spontaneously recovered female aplastic anaemia patients (Goulmy, 1985) Clones currently available are either HLA-A2 or HLA-B7 restneted, so this hmits our ability to type cells from mdividuals carrying one or both of these alleles, fortunately, this includes more than 50 % of the population For the locah/ation of the Η Υ gene in man, potentially informative patients are those who have inherited a partly deleted paternal Υ chromosome or a translo-cated Υ chromosome fragment Such patients are in two phenotypic categones XX males and XY fe-males The six fe-males desenbed here have mhented variable portions of Yp whilst the two females possess Yq and a variable portion of the Yp Table 4 shows the results of HLA and H-Y typing lymphoblastoid Β

Table 3. Η Υ typing by CML and prohferation of Η Υ specific clones of normal mice and of mice of both sex phenotypes carrying Sxr Γ ρίΚ lrlrlpr) from mouse None 30 XX? 32 33 34 4 I 16HXSJC/5 13 35 36 37 38 39 40 41 42 XXSxicf 43 47 31 XYcf 4-) 46 C 3(Dk) 1016 2 174 929 1487 591 26 406 5S269 6620S 42014 46 7^3 64(40 1 X)4 (Π49 47141 S2529 40 797 899 549 317b 12092

Prohfci ation of Η Υ specific elonc (restuction speeihcity)

10 2(Dl) 301 1078 1312 552 649 651 379 828 1 531 586 1255 1648 1613 778 1229 341 72 400 32 610 1451 635 2KA') 765 197 489 245 3 932 3 252 3 086 518 2526 2 053 1 160 1685 4 149 7653 704 225 ND 30472 579 30 CML anti Η Yk 0 9 - 1 2 14 4 3 0 20 0 30 6 26 4 23 0 23 3 29 4 1 3 25 2 25 b ND ND - 0 7 - 3 7 29 3 16 9 typing with anti Η Υ1 1 1 2 0 - 7 7 1 7 8 5 6 4 8 4 - 1 1 5 S 2 0 1 9 3 1 6 2 ND ND 12 4 29 4 2 7 1 1 Η 9 Π Δ type k k k k k k k k k k k k k k k b b k k II Yl type — -+ + + + + + -+ + + + + + +

H>i method of piolifu ilion SCL legend lor 1 ible 2

( MI Ά cytotoxieily it Λ 1/10 1 liom 12 point icgicssion inalysis II 2 type est lbhshed with illospceilic cytotoxic 1 cells

cell hnes from these patients and appropnate A2 and B7-positive normal male and female controls, with cytotoxic Τ cells it IS important to confirm serological HLA typing with Τ cells, since vanants of A2 and B7 exist which are not distinguishable serologically but which cannot be recogmzed by allospecific or MHC-restncted Τ cells (Horai, von der Poel & Goulmy, 1982) Α negative Η Υ typing can thus only be mterpreted as such in face of a positive allotyping for the restnction element with Τ cells (A2 or B7 in the case of individuals shown in Table 4)

The deletion map shown in Fig 1 is based on Vergnaudeia/ (1986), Distecheeia/ (1986) and Page (1986) and includes the summanzed H-Y results of Table 4 as well as unpubhshed data on class I XX males Since MX class 3 males were H-Y negative it is clear that the gene for H-Y does not map to deletion interval 1-3 on Yp (TDF is in interval 1, see also Affaia et al 1986) Likewise the gene for Η Υ is

excluded irom interval 4A, since the class 2 XY female is H-Y positive and lacks this portion of Yp The H-Y gene thus maps between mtervals 4B and 7, far from TDF in interval 1

Conclusion

In summary, these data, using cytotoxic and prohfer ative Τ cell clones specific for H-Y and restncted by MHC molecules to type mice and humans mhenting incomplete portions of the Υ chromosome, have allowed us to map the H-Y antigen gene Hya m mouse to a position closely hnked with, but separable from, Tdy on the Sxr fragment and thus presumably to a portion of the normal mouse Υ chromosome near the centromere The human Η Υ gene maps between deletion intervals 4B and 7, separate from TDF which is on interval 1 We are currently testing cells Irom a Table 4. HLA and H-Y typing of Β cell hnes from XX males, XY females and normal controls

Exp Karyotype/ sex XX cP XXcf XX cf XYcf XX $ XX cf XYcf XX? XXcf XXcf XYcf

xx 9

XY? XY$ XYcf XX Ρ Individual RH JT LGL 105 Normal male Normal female WB Normal male Normal female WH1 950 JM Normal male Normal female WHT1003 (eise 1) Wffl 715 (casc 2) Fathcr ol easc 2 Mothcr of case 2 HLA' serology Α 2 3 2 2 3 1 2 2 11 2 9 1 2 2 11 1 3 3 28 9 3 24 3 3 28 3 29 Β 21 40 44 45 35 44 8 8 44 17 18 8 8 44 7 7 7 44 7 7 13 7 7 40 7 α-Α2| 18 24 13 20 17 37 25 17 αΒ7 76 62 ND 54 55 57 52 36 O-HY/A2I 9 3 4 38 8 0 17 3 αΉ Υ/Β7 9 1 40 0 70 69 61 6 Η Υ phenotype _ -+ -+ -+ -+ + + — 111 Α serology ptrformed by Lorna Kennedy at ICRF Lincoln s Inn Fields or Donald Palmcr of Depl Immunology RPMS Pci cent specific lysis of target cells at Α Γ/10 1 as dctermineci irom a 6 point regression curvc

Modihcd irom table 1 Simpson α al 1987

XX m lies XY lemalcs Class 1 3 Class 1 2 TDF 4A 4B _{Η Υ}

number of addiüonal patients who have inhented different portions of the Υ chromosome to pinpomt

the mapping more closely It IS of mterest that in mouse a Y-hnked gene, Spy, Controlling spermato-genesis maps near Hya (Burgoyne, Levy & McLaren, 1986, for discussion see Burgoyne, this Symposium) on the Sxr fragment they could be the same or closely hnked genes In man, a gene Controlling spermato-genesis maps to Yq (Tieopolo & Zuffardi, 1976), and the data so far do not exclude the possibihty that it could be coincident with the H-Y gene

References

AITARA, Ν Α , FERGUSON-SMITH, Μ Α , ToLMir, J , KWOK, Κ , MITCHELL, Μ , JAMIESON, D , COOKE, Α &

FLORFNTIN, L (1986) Variable transfer of Y-specrfic sequences m XX males Nudeic Aads Res 14, 5375-5387

BURGOYNE, Ρ , LFVY, Ε & MCLAREN, Α (1986)

Spennatogenic failure in male mice lackmg Η Υ antigen Nature, Lond 320, 170-172

DISTECHE, C Μ , CASANOVA, Μ , SAAL, Η , FRIEDMAN, C , SYBERT, V , GRAHAM, J , THULINE Η , PAGE, D C

& FFLLOUS, Μ (1986) Small deletions of the short arm of the Υ chromosome in 46,Χ Υ females Proc riatn

Acad Su USA 83, 7841-7844

EICHER, Ε Μ & WASHBURN, L L (1986) Genetic control of pnmary determination in mice Α Rev

Genet 20, 327-360

GOULMY, Ε (1985) Class-restnction human cytotoxic Τ lymphocytes directed against minor transplantation antigens and their possible lole in organ

transplantation Progress in Allergy 36 44-72

HORAI, S , VAN DrR POFL, J J & GOULMY, Ε (1982)

Diffeiential lecogmtion of the serologically defined FILA-A2 antigen by allogeneic cytotoxic Τ cells

Immunogenetics 16, 135-142

LOVFIAND, Β & SIMPSON, Ε (1986) The non-MHC transplantation antigens neither wcak nor minor

Immunology Today 7, 223-229

MCLAREN, Α & MONK, Μ (1982) Feitile females pioduced by inactivation fiom X chromosome of 'sex

revened' mice Nature, Lond 300, 446-448

MCLAREN, Α , SIMPSON, Ε , TOMONORI, Κ , CHANDLER, Ρ

& HOGG, Η (1984) Male sexual differentiation in mice lackmg H-Y antigen Nature, Lond 312, 552-555 PAGE, D C (1986) Sex reversal deletion mapping the

male-determimng function of the human Υ

chromosome Cold Spring Harbor Symp quant Biol 51, 229-235

SIMPSON, Ε (1982) The role of H-Y as a minoi transplantation antigen Immunology Today 3, 97-106 SIMPSON, Ε (1985) Recogmtion of minor transplantation

antigens the role of H-2 and other Ir genes In Major

Histocompatibihty System the Gorer Symposium (ed Ρ

Β M e d a w a r & T Lehner), pp 37-55 Oxford Blackwell

SIMPSON, Ε (1986) The H-Y antigen and sex reversal

Cell 44, 813-814

SIMPSON, Ε , CHANDLER, Ρ , GOULMY, Ε DISTFCHE, C Μ , FERGUSON-SMITH, Μ Α & PAGE, D C (1987)

Separation of the genetic loci for the Η Υ antigen and for testis determination on human Υ chromosome

Nature, Lond 326, 876-878

SIMPSON, Ε , CHANDLER, Ρ , HUNT, R , HOGG, Η ,

TOMONARI, Κ & MCLAREN, Α (1986) Η Υ Status of

X/XSxr' male mice in vivo tests Immunology 57,

345-349

SIMPSON, Ε , CHANDLER, Ρ , WASHBURN, L L , BUNKER

Η Ρ & EICHFR, Ε Μ (1983) H-Y typing of

karyotypically abnormal mice Differentiation 23 Suppl. S116-120

SIMPSON, Ε & GORDON, R D (1987) Responsiveness to H-Y antigen Ir gene complementation and target cell specificity Immunol Rev 35, 59-75

SIMPSON, Ε , MCLARFN, Α , CHANDIER, Ρ & TOMONARI,

Κ (1984) Expression of H-Y antigen by female mice carrymg Sxr Translocaüon 37, 17-21

TIFOPOIO, L & ZUFTARDI, Ο (1976) Locahzation of factors Controlling spermatogenesis in the non-fluorescent portion of the human Υ chromosome long arm Human Genet 34, 119-124

TOMARI, Κ (1983) Antigen and MI IC lestnction specificity of two types of cloned male-specific Τ cell hnes / Immunol 131, 1641-1645

VERGNAUD, G , PAGE, D C , SIMMLER, Μ C , BROWN L , ROUYER, F , NOFL, Β , BOTSTFIN D DT Ι Λ