Journal of Immunogenetics (1983) 10,25-29.

H L A A N D R E P R O D U C T I O N ?

M . J. G I P H A R T A N D J . D ’ A M A R O

University Hospital Leiden, Leiden, The Netherlands

(Received 16July 1982)

S U M M A R Y

In mice it has been shown that mating preference is genetically associated with the Major Histocompatibility Complex (MHC). In the present study, an approach is described to study homologous aspects of the MHC in man.

After selection of families with one or more children, a given parental HLA antigen was selected and the frequencies of the spouse’s HLA antigens were determined. Assuming a random distribution, these frequencies should not be statistically different from those of the total population.

Evidence is presented that this distribution is not random for a number of maternal and paternal HLA antigens.

I N T R O D U C T I O N

The prime biological function of the Major Histocompatibility Complex (MHC) is related to immune processes. The MHC plays a keyrole in the recognition of foreign antigens by T lymphocytes and in the subsequent regulatory steps of the immune response. However, observations have been reported in the literature showing associations of the MHC with non-immunological traits in the mouse (Ivanyi, 1978). Also, in man it has been reported that non-immunological traits are associated with the MHC. Dupont et al. (1977) found that the MHC is in tight linkage with a gene coding for the enzyme 21-hydroxylase. Also, association of the MHC with urinary pepsinogen has been described, both in man and mice (Weitkamp, 1978; Szymura & Klein, 1981). Recently we have published evidence that HLA-B I8 is associated with increased male offspring (Giphart & D’Amaro, 198 1).

Experiments in mice have shown the existence of mating preference (Yamazaki et ul., 1976). It has been shown that this mating preference is associated with the presence of olfactory receptors (Yamazaki et a[., 1979). The genes coding for these receptors are associated with the MHC.

It was tempting to find an approach to study homologous aspects of the MHC in mar.. Families with one or more children were selected from our file of over 70,000 HLA-typed individuals. If, in the parents, a given maternal antigen was selected, the frequencies of the paternal HLA antigens should not be statistically different from those of the total paternal

Correspondence: Dr M. J. Giphart, Department of Irnmunohaematology, University Hospital Leiden, bldg. 23, Rijnsburgerweg 10,2333 AA LEIDEN, The Netherlands.

0305-181 1/83/0200-0025%02.00 62 1983 Blackwell Scientific Publications 25

26 M . J. Giphart and J. D’Arnaro population, given a random distribution. We will present evidence that this distribution is not random for a number of maternal and paternal HLA antigens.

M A T E R I A L S A N D M E T H O D S

The HLA phenotypes of more than 3000 Dutch families were used for this analysis. The standard NIH-lymphocytotoxicity test was used to recognize the following HLA determinants: HLA-A1,2,3,9,10,11,28,~19; HLA-B5,7,8,12,13,14,15,w16,17,18,~2 1, w22,27,w35,37,40; H L A - C w l , w 2 , ~ 3 , ~ 4 , ~ 5 , ~ 6 (Bodmer et al., 1977). Those antigens represent 99% of the HLA-A, 97% of the HLA-B and 50% of the H L A - C locus gene frequencies.

Families were selected on the basis of one or more children and on the presence of complete serologically determined parental phenotypes. The phenotype frequencies of the maternal and paternal HLA antigens were determined and compared to each other and to the Dutch population (D’Amaro, 1978). In order to test the null hypothesis that there was no selection in the choice of partners based on traits which are coded for by genes which may be linked to those in the MHC region, the following protocol was deviced. For each of the 30 HLA antigens mentioned above, couples were selected on the basis of the presence of that antigen in the maternal phenotype. The phenotype frequencies of all of the 30 antigens were determined in their partners, i.e. in a subset of the paternal population. Those frequencies were compared to those in the total paternal population, the expected frequency in the absence of any MHC associated selection. The same procedure was followed after selection for the presence of each antigen in the paternal phenotypes. The significance of the differences in the observed and expected frequencies was assessed with a chi squared test. The corresponding P values were corrected for the total of 1800 comparisons (2 x 30 x 30) using the method of Edwards (1974).

The analysis of the material was extended in order to answer the following questions: Is there any influence of the age of the parents at birth of their first child with respect to its sex? Parents with known birthdates (and with children with known birthdates) were divided into 10 age groups and in each group the total number of male and female children were counted. Given the observed immune reactivity of females to the male Y-antigen (Goulmy et af., 1977), is there any influence on the distribution of male and female children after the birth of a male child? The numbers of male and female children were counted after the birth of first male or female child.

R E S U L T S

The selected families from our files of HLA-typed individuals were analysed for the parental phenotype frequencies of 30 HLA-A, -B and -C-determinants. The results are set out in Table 1. The observed phenotype frequencies are not significantly different from each other, nor are they significantly different from those in a large random sample of the Dutch population (D’Amaro, 1978).

The analyses of the phenotype frequency distributions in subsets of the parents, selected for the presence of a specific HLA antigen in their partners, yielded two matrices of 30 x 30 comparisons for the maternal and paternal subsets.

Most of these comparisons did not reveal any significant differences from the expected frequency distributions. However, the distribution of one maternal and three paternal

HLA and reproduction? 27 TABLE 1. Parental HLA-phenotype frequencies

HLA 0 d HLA 9 CT HLA 0 d

A1 A2 A3 A9 A 10 A l l A28 Awl9 B5 B7 B8

0.3292 0.5018 0.2792 0.1944 0.0845 0.1316 0,0852 0.1890 0.1402 0.2464 0.2428

0.3 148 0.5057 0.29 13 0,2086 0.0821 0.1186 0.0935 0.2156 0.1213 0.2567 0-2637

B12 B13 B 14 B15 Bw16 817 Bi8 Bw21 Bw22 B27 Bw35

0.2014 0.0461 0.048 1 0.1731 0-0586 0.0750 0.0787 0.0246 0.0589 0.b919 0.1644

0.2141 0.0467 0.039 1 0.1782 0.0679 0.0822 0.0743 0.0367 0.0548 0.0650 0.1588

837 840

c w 1 cw2 cw3 cw4 cw5 Cw6

0.0356 0.1756

0.0721 0.1389 0.325 1 0.1597 0.1092 0.1760

0.0382 0.1421

0.0609 0.1006 0.2594 0,1572 0.1521 0.1956

phenotypes were significantly different after correction for the large number of comparisons. Those values are set out in Table 2. In all four cases there were significantly increased frequencies in the spouses. Therefore, those associations appear to be acting in a positive way, although nothing can be concluded as to what the biological function or significance may be.

TABLE 2. Disparate HLA antigen distributions in spouses of individuals with selected antigens

Selected Spouse’s Observed Expected Observed Expected antigen antigen + ve + ve -ve -ve X 2 PC+

d C w 4 ? A w l 9 106 67.67 252 290.33 26.77 0.025 QA2 d B 4 0 317 239.80 1371 1448.20 28.97 0.016 QAw19 d C w 4 106 6’7.91 326 364.09 25.36 0.033 QB7 d B40 180 120.89 671 730.11 33.68 0.007

* Pc = P value after corrections for 1800 comparisons.

The analysis of the sex distribution of children after the birth of a male or a female child shows no significant differences (Table 3). Hence, immunological mechanisms based on the female anti-male immunity as observed after bone marrow transplantation (Goulmy et al., 1977) do not seem to operate in human reproduction.

Parental ages do not have any significant influence on the sex of their children (Table 4).

TABLE 3. Sex distributions after birth of male or female child

1st child is male 1st child is female

Number of male Number of female Number of male Number of female children children children children

760 67 1 758 687 N.S.

M. J. Giphart and J. D ’A mar0 TABLE 4. Parental ages at birth of their first child

Mother’s age Father’s age

Number of children Number of children Age group Male Female Male Female

16-20 21-2s 26-30 31-35 36-40 41-45 45-50 s 1-55 56-60

60

48 215 221

51 1s 0 0 0 0 0

4s 23 1 20 1

58 14 0 0 0 0 0

12 131 249 108 44

8 3 1 0 0

12 127 238 118 39 14

1 0 0 0

D I S C U S S I O N

The observed ‘preferental association’ of some HLA-determinants in males and females may indicate a positive selection bias based on traits which may be coded for by genes which are either in the MHC region or closely linked to their genes within the region.

Some information obtained from studies in the mouse may be helpful in this respect. The H-2 type in the mouse can be obtained from certain measurements from X-rays of the mandible (Festing, 1973). Also, it was shown that the MHC controls body mass and organ-size (Ivanyi, 1972; Gregorova et al., 1977). Thus, it may be concluded that the MHC co-influence ‘appearance’. If this is of importance in partner choice and if the traits in man are also associated with the MHC, the results could be explained in this way.

Since only A2-B40 is a recognized, but low level, linkage disequilibrium in the Dutch Caucasoid population, another possible interpretation of our results would be that we are looking at a possible predictor of future linkage disequilibria.

If the observed associations are stable and specially the ones of the alternative loci, a cross-over event during meiosis would link these HLA-determinants in one haplotype. Given the genetic distance of the HLA-A, -B and -C loci, this would not be an extremely rare event (the cross-over frequency between HLA-A and -B is in the order of 1% (D’Amaro, 1978).

Whether or not the mechanism of ‘mating preference’ as described for mice (Yamazaki, 1979) is applicable based on the MHC to man is not testable since nothing is known about olfactory receptors in man for MHC-products, although it is known that receptors show a polymorphism with respect to their expressions; i.e. they may be absent or non-functional in some but not in other people. Furthermore, it seems likely that olfactory receptors can work autonomously.

The results shown, however, make one thing clear, namely the increasing biological complexity of the Major Histocompatibility Complex.

A C K N O W L E D G M E N T S

This work was supported in part by the Dutch Organization for Health Research (TNO), the Dutch Foundation for Medical Research (FUNGO) which is subsidized by the Dutch

HLA and reproduction? 29

Organization for the Advancement of Pure Research (ZWO), the J. A. Cohen Institute for Radiopathology and Radiation Protection (IRS), and the National Institutes of Health (NIH), no. NOI-AI-82553.

R E F E R E N C E S

BODMER, W.F., BATCHELOR, J.R., BODMER, J.G., FESTENSTEIN, H. & MORRIS, P. (eds.) (1977)

D’AMARO, J. (1978) HLA Polymorphisms in The Netherlands. Thesis, State University, Leiden. DUPONT, B., OBERFIELD, S., SMITHWICK, E.M., LEE, T.S. & LEVINE, L.S. (1977) Close genetic linkage between

EDWARDS. J.H. (1974) HLA and disease. The detection of associations. Journal of Immunogenefics, 1,249. FESTING, M. (1973) A multivariate analysis of subline divergence in the shape of the mandible in C57BLlgr

mice. Genetical Research, 21, 121. GIPHART, M.J. & D’AMARO, J. (1981) The association of HLA-B18 with increased male offspring in paternal

backcross matings. Tissue Antigens, 15, 329. GOULMY, E., TERMUTELEN, A., BRADLEY, B.A. & VAN ROOD, J.J. (1977) Y-antigen killing by T-cells of

women is restricted by HLA. Nature, 266, 544. GREGOROVA, S., I V ~ N Y I , P., SIMONOVA, D., MICKOVA, M. (1977) H-2 associated differences in

androgen-influenced organ weights of A and C57BL/10 mouse strains and their crosses. Immunogenetics, 4, 301.

IVANYI, P. (1978) Some aspects of the H-2 system, the major histocompatibility system in the mouse. Proceedings of The Royal Sociev, London, B, 202, 117.

IvANYi, P. GREGOROVA, S., MICKOVA, M. (1972) Genetic differences in thymus lymph node, testes and vesicular gland weights among inbred mouse strains. Association with the Major Histocompatibility (H-2) system. Folia Biologicu Prahn, 18, 8 1.

SZYMURA, J.M. & KLEIN, J. (1981) Linkage of a gene controlling urinary pesinogen with the major histocompatibility complex of the mouse. Immunogenefics, 13, 267.

WEITKAMP, L.R. (1978) Further data concerning the linkage relationships of loci for urinary pepsinogen and HLA. Cytogenetics and Cell Genetics, 22, 341.

YAMAZAKI, K., BOYSE, E.A., MIKE, V., THALER, H.T., MATHIESON, B.J., ABBOTT, J., BOYSE, J., ZAYAS, Z.A. & THOMAS, L. (1976) Control of mating preference in mice by genes in the major histocompatibility complex. Journal of Experimental Medicine, 144,1324.

YAMAZAKI, K., YAMAGUCHI, M., BARANOSKY, L., BARD, H., BOYSE, E.A. & THOMAS, L. (1979) Recognition among mice. Evidence from the use of a Y-maze differentially scented by congenic mice of different Major Histocompatibility types. Journal of Experimental Medicine, 150, 755.

Hisfocompatibilify Testing, Munksgaard, Copenhagen.

HLA and congenital adrenal hyperplasia (2 1-Hydroxylase deficiency). Lancet, ii, 1309.