A comprehensive search for segregation distortion in HLA

A Comprehensive Search for Segregation Distortion in HLA William Klitz, Sing Kai Lo, Meinhard Neugebauer, Max P. Bout, Ekkehard D. Albert, and Glenys Thomson

ABSTRACT: Segregation distortion, the non-Mendelian segregation of gametes, has been wdl docur,w.nted among diverse groups of organisms. These cases are characteriz¢d by extreme segregation ratios found only ia males. Previous reports have suggosted the existence of segregation distortion oporating in the HLA system of humans, a tightly linked complex of genes which regula#os the immune system. In mice, some alleles of the Tit complex, which £~ linked to 14-2 (the HLA hemdogue of mice), cause extreme segregation distortion in wild mice populations. Here we report on the examination of a large body of podigree data on nondiseased families, scord for the alldos of fire HLA region loci. We searched for segregation distortion on the basis of fiv¢ different mulch of inheritance: alldic, haplotypic, genotypic, diffuse occurrence in families, and autosomal effects on the sex ratio. There was no clear evidencefor segregation distortion. In particular, the p~sibility of extreme levels of segregation distortion was firmly rejected in th¢ populations examined, thus reducing the likelihood of common distortion-causing HLA associated haplotypes in our species.

INTRODUCTION Animals have a reduced haploid phase of their life cycle in which haploid individuals remain single cells, metabolically and behaviorly oriented to the/nanguration of the next diploid generation beginning with fertilization. The nourishment, transport, and morphology of the gametes is largely determined by the diploid "parents." The generally short-lived and passive existence of the gametes suggests few opportunities for expression of the haploid genome and differential transmission of gametes. Nonetheless, several cases violating Mendel's law of equal segregation are known (see e.g., [1]). Segregation distortion is observed when differing gametes are transmitted unevenly, and is due to events which differentiate the haploid products of meiosis. Segregation distortion might be the result of events occurring at any t/me during gametic life, from the chromosomal reduction of meiosis to the union of the haploid geaomes following fertilization. Evolutionary models predict equal segregation ratios. Eshel [2] argued that when segregation distortion occurs, it will be reduced by unlinked modifying genes, resulting in Mendelian segregation. Similarly, Lloyd [3] has suggested that From the Department of Genetics, Unirersity of California. Berkeley, California: Department of Statistics, Rutgers Unirersity. New Brunswick Neu, Jersey: Institut fur Medizinische Statistik, Uniz,,rsitaet Bonn. Sigmund Freud Str. 25. 5300 Bonn FRG: and Labor fur lmmungenetiL Kinderpdiklinik der Unirersitaet. Penenkoferstr. 8a. 8000 Munich FRG. Address reprint requeststo Dr. William Klitz, DepartmentofGenetics, UniversityofCalifornia, Berkeley, CA 94720. ReceivedMay 29, 1986; acceptedOcto&r 7, 1986. Human Immunology 18, 163-180 (1987) © Elsevier Science Publishing Co.. Inc., 1987 52 Vanderbilt Ave., New York. NY 10017

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164

W. Klitz etal. genes having different functions will segregate equally (or assort independently) as an optimum solution to cohabitation at one locus (or in one genome). Segregation distortion has a powerful impact on most aspects of conventional diploid selection theory [4]. Selection under haploid conditions results in even slight selective differences among gametes having an immediate effect on ~dlele frequencies. Looking, for example, at a one locus, two allele model, the rate of change in allele frequencies is twice as fast under haploid selection as under additive diploid selection, given identical selection coefficients [5]. On this basis, we might expect that if divergent segregation ratios occur, the gametes having less than equal segregation would not be expected to last long in a population in the absence of compensating selection from other parts of the life history. This might explain the observed rarity of segregation distortion in natural populations. The detection of segregation distortion requires a method of identifying specific gametes or chromosomes from heterozygous individuals, and family data to follow the transmission of identified gametes. Unless the distortion is great, large sample sizes are also necessary. With the exception of sex ratio distortions, most documented cases of segregation distortion are extreme, usually exceeding 80% (e.g., in mice [6], corn, [7], and Drosophila [8]). This may only reflect the low power of smaller sample sizes in detecting less extreme departures from a 50:50 transmission ratio. The extent of genetic variation producing segregation d~stortion in natural populations occurring with small departures from uniform gametic transmission has not received much attention (see [9] for a rece~lt exception). Since sperm are much more mobile and n~merous, and since the chromosomes of sperm require extreme condensation and packaging, compared to eggs, it is understandable that known cases of segregation distortion involve differential sperm transmission. Argaments and data supporting two contrasting ideas have been presented: a strictly diploid genotypic contribution to sperm morphology and chromosome packaging [10], and the expression of the haploid genome during sperm development [11], which then influences gametic survival. The major histocompatibility complex (MHC) in humans, known as the HLA region and located on chromosome 6, presents a logical site in which to search for segregation distortion. The MHC of vertebrates contain~ several loci coding for cell surface proteins which regulate the immune system [12]. These loci are characterized by very high levels of polymorphism, and have been extensively studied in both humans and mice. The mouse MHC (H-2) region is at the distal end of the T/t complex, which codes for male sterility genes and for proteins governing embryonic differentiation (reviewed in [13]). Segregation distorting t haplotypes are common in wild mice populations, and are typically transmitted by sperm in excess of 90% compared to the normal allele [6]. In addition, the sex determining element, Bkm, has been found in the H-2 region [14]. Previous reports of segregation distortion in the HLA complex [15-17], observations of HLA expression on sperm [ 18,19], and availability of extensive pedigree data and population samples recently compiled for HLA loci [20] suggest the value of an intensive examination of the region.

METHODS We describe five different approaches to revealing segregation distortion, each of which detects a distinctive basis for the genetic mode of action of the phenomenon. Here we consider segregation distortion due on[y to altered sperm transmission, so our search for any departures in transmission includes the contrast of maternal and paternal transmission ratios. Figure 1 shows the five different

Segregation Distortion in HLA

165 Hapiotypic

AIlelic

Diffuse

Genotypi¢

By Sex

Father -~ Family 1

Father

-~

F,lmily 2

Fathee Family3 ~ FIGURE 1 ~ive models for the i~fluence of genetic factors on the differential transmission of sperm. Genetic labels and chromosomes in the sperm heads identify the contrasting types. The absence of a given specificity is indicated with a superscdpted line.

models of segregation distortion, depicted as differential movement of sperm. Below, we define each type of segregation distortion, describe its method of detection, and provide an example from the literature.

Alld¢ effect. In this case, we examine whether a partkLxlar allele produces segregation distortion or serves as a marker for it. The offspring of informative (heterozygous) m a d n ~ ~re examined tbr deviations from the Mendelian r~do. Due to large sample si~es and the ability to use the entire informative sample, the power of this test is high comp~ed to the other approaches. The effect of a single allele or haplo~ype can also be detected by comparing the expected binomial distribution of segregation, conditional on sibship size, to the observed. For example, for an autosomal gene transmitted by one parent, sibships of size two are expected to have an 0Jielic distribution of one fourth with both sibs carrying the al|e|e, one half with one of the sibs carrying the allele, and one fourth with neither sib having the allele, with appropriate extensions to sibsMps of s/ze three, etc. Deviations from these expectations detect segregation distortion. With sufficient sample sizes this "sibship test" can add discrimination to the search for specific aileie or haplotype effects. The segregation distortion observed in the

166

W. Kli~ et al. T/t complex in mice appears to be due to the alleles of one or more loci on a chromosome [13].

Haplotype affect. In this approach, we determine whether a cluster of alleles from the loci ~ , a single chromosome produces the segregation distortion, or several alleles at different loci (i.e., a haplotype) mark the presence of an unidentified "distorter" locus. The offspring produced from an individual carrying a uniquely identifiable multilocus haplorype are examined for deviations from the expected 50:50 transmission ratio. Three locus haplorypes are examined here for the purpose of more adequately identifying unique chromosomes, than would be possible with two locus haplotypes. In a polymorphic system most multilocus haplotypes will be rare, so a limited fraction of the population is useable. It has been suggested that some HLA haplotypes produce segregation distortion [16,17].

Genotype efflct. To determine whether a particular genotype produces the effect, the ratio of offspring from the genotypes of informative rantings is examined. Small sample sizes due to the many genotypes present in polymorphic loci inevitably reduces the power of the test and the proportion of the population examined. We are not aware of cases of simple genotypic distorti,:i, az described here, but the t~g~tly linked responder and distorter loci of the SD system in Drosophila [8] would mimic this effect.

Diffuse family effect. The apparently sporadic occurrence of segregation distortion among families, but consistently within families, can reveal the action of genetic effects from elsewhere in the genome. Environmental effects acting through the tested loci would also be revealed by this method. This test consists of a comparison of the observed to expected haplotype sharing distribution of sib pairs. This procedure was originally developed to identify genetic components of disease (see, e.g., [21]). Unlinked modifier loci as found affecting the segregation d;stomion ~y~tem, SD, in Drosophila [22] produce the diffuse thmily effect. We are aware of no cases of the diffuse family effect due to gene X environment interactions.

Sex ratio efj~ct. Influenced by autosomal genes, the heterogametic sex may transmit uneven numbers of the two sex chromosomes, and thereby influence the sex ratio. Although many instances of uneven primary sex ratios are known, until recently, imzapopulational genetic variation has not been identified [23,24]. The discovery of sex determining factors in the H-2 region of mice [14,25] suggests the possibility of a sex ratio effect in the HLA region. In addition, sex ratio distortion due to an HLA allele has been suggested [26]. DATA The data used in this study is taken from the 9th International Histocompatibility Workshop files consisting of approximately 700 randomly chosen families having some 3000 offspring [20], utilizing the deduced haplotype files produced by the use of the F~anily Analysis Program (FAP) [27]. The FAP program produces, for all individuals in a pedigree, a list of diplotypes, each with its corresponding likelihood. The likelihoods of all of the possible diplotypes of an individual sum to one. We used the first (most likely) diplotype, as determined from the analysis. The likelihood of 85% of the first diplotypes exceeds 0.90. The families were typed for four histocompatibility loci, HLA-A,B,C, and DR. Smaller numbers of these same families were typed for the four HLA coded complement loci C2,

Segregation Distortion in HLA

167

Bf, C4A, and C4B and for the giyoxylase I locus (GLO). The eight HLA region loci are located on a 1.8 cM segment of the short arm of chromosome 6, and the GLO locus is approximately 5 cM proximal to the HLA region [28]. The principal loci and number of alleles (in parenthesis) in this data set are HLA-A (15), HLA-B (28), HLA-C (8), an0 HLA-DR (10). Blank ~leles present in the histocompadbility loci are composite allelic classes coding for proteins for which no andsera are known. Blank alleles are treated as recessives in haplo~pe assignment. Ethnically, the data consists of 72% Caucasi~, 16% Japanese, and 6% Chinese with the remaining 6% comprised of small numbers of other groups. The three most common ethnic groups were tested separately.

STATISTICS The loglinear statistical model [29] is well suited to detect segregation distortion due to alleles, haplo~pes, and genotypes. The three factors determining the value of a cell entry in this model are transmission (plm or minus), parental sex (father or mother), and the genetic factor (the array of alleles, haplotypes, or genotypes). Three-way interaction must exist ~,rnongthese factors to demonstrate segregation distortion, that is, (1) distorted transmission frequencies must be present; (2) only in fathers; and (3) differentially among a particul~ genetic category, e.g., only in DR3 among the DR alleles. One strength of this statistical model is that the problem of experimentwise error (which occurs due to multiple testing) is inherendy solved. The expected frequency of a given cell, f~i~,in the full three-factor model is In(

fijk ) = /~ + ~i + ~j + ~'~ + o~-~j + cqyk + •iYk + ~'~f/k,

where ~ is the grand mean and ~, ~, and y and their two- and three-way combinations describe the possible influence of each of the three factors. Discovery of the best fitting model in log,linear analysis of frequency data is arrived at by first testing the most complicated (full) model against the next most complete model, and continuing in this fashion until f~gnificant terms in the tested model herald the best fit. In this case, however, the first step, testing for the presence of a three-way term, satisfies the hypothesis of segregation distortion. The temptation to search for possible diploid selection or typing effects in the lo#inear model, in the nonindependence of the two-way term--transmission X genetic c o m ~ n e n t - - w a s tempered by the following considemtlon. A confounding influence inherent in foamilydata is created by the spurious nonlndependence of the two-way parental sex X genetic component term, caused by variable numbers of both offspring per family and e f families per genetic category. This rendered dissection of the significance of the twOoWay terms/mpossible. Instead of the loglinear model, two-way goodnes~ of fit tests were employed to search for effects due to typing or diploid selection (see below). The three factor log,linear analysis was carried out for alleles and genotypes from each of the four histocompatlbility loci, and from the haplotypes arising from five different sets of three loci taken from the loci A, C, B, DR, and GLO. Use of the entire population of informative parents by combining rare classes of alleles, haplotypes, and genotypes where necessary to achieve minimum sample size resulted in testing for the desired h l transmission ratio. Although not so effective as the three-factor log-linear model, and requiring interpretation of significance in multiple testing, goodness of fit tests, using the external hypothesis of 1:1 transmission, carried out separately for fathers, mothers, and parents corn-

168

W. Klitz et al. bined, was used to test for allelic, haplotypic, and genotypic effects. The presence of a significant heterogeneity term in the two-way goodness of fit test was used to signal possible segregation distortion, while tests for individual transmission categories (e.g., for haplotype A 1BSDR3) identified the direction and magnitude of each particular case. We searched for segregation distortion occurring sporadically among families by comparing the expected to observed frequencies of sib pair configurations for a given sibship size by X 2 testing. Both the magnitude of the X 2 values as well their consistency among sibships of different sizes are necessary to evaluate the diffuse family effect. The normal human sex ratio is not 1:1, and in fact varies among ethnic groups [24]. To check for the influence of HLA alleles on the sex ratio, we tested the sex ratio of offspring informative for each allele against the sex ratio of the total observed population less the sex ratio of the allele being tested. Although we test each of the five different types of segregation distortion separately, we are in fact slicing the pie of a single data set in various ways. This means that the existence of one type of distortion may be detectable in its own test, as well as in the tests for other types of distortion. For example, an allelic effect detected because of distorted transmission of a particular allele might also appear in the tests on those haplotypes subdividing the responsible allele. On the positive side, this might distinguish haplotype from allele effects. On the other hand, extensive segregation distortion detected among the various types could create serious difficulties in interpretation, because of the fundamental nonindependence of the tests for the various models of genetic determination.

RESULTS The test for three-way independence of parental class X genotype X transmission, our prima,-y test for segregation distortion in the HLA region, was performed for the alleles at each of four loci (A, C, B, and DR), the genotypes at each of four loci, and five different sets of three-locus haplotypes. Only the Caucasian sample was large enough to give meaningful results. In general, there was no evidence for significant three-way interaction. An exception occurred in the case of the alleles of HLA-C, which tested significant for the three-way term at the 0.05 level. Interpretation of the deviation in transmission of a particular allele, haplotype, or genotype showing no evidence for segregation distortion on the basis of goodness of fit tests is possible only in the context of sample size. Setting alpha to 0.05 and beta to 0.20, the detectable deviations in transmission frequency ranged from a minimum of 0.50 plus or minus 0.03 for the HLA-A2 sample of Caucasian mothers with 983 informative transmissions, to 0.50 plus or minus 0.40 for our minimum expected cell number of five (having a total of ten transmissions). Allele Effect The results of three-way interaction and goodness of fit tests on allelic tranmission for each of the histocompatibility loci are summarized in Table 1, with the nominally significant departures for individual alleles shown in Table 2. Variation at the HLA-A locus in the Caucasian sample showed heterogeneity in transmission rates for both parents separately and combined. The low transmission rate of the AO allele in both parents (Table 2) played a major role in this: after retesting without the null allele, significant heterogeneity disappeared

Segregation Distortion in HLA TABLE 1

169

Tests o f significance for heterogeneity in transmission among alleles at each o f four H L A loci ~ G o o d ~ s s o f fit

Locus HLA-A

HLA-C

HLA-B

HLA-DR

Population

Alleles tested

Loglinear 3 way interact/on

Caucasian

15

as

Chinese

7

Japanese

7

Caucasian

9

Chinese

5

Japanese

5

Caucasian

30

Ch/nese

14

Japanese

15

Caucasian

13

Chinese

7

Japanese

10

Parent

Total infommtive cases

Allele x transmission heterogeneity

Father Mother Father Mother Father Mother

2071 2064 162 157 376 373

"~ , ,

Father Mother Father Mother Father Mother

1807 1878 165 156 351 336

as ns "* g

as

Father Mother Father Mother Father Mother

2171 2078 194 192 461 474

* ~. as * as ~ ns ns X n$ ns/

ns

Father Mother Father Mother Father Mother

1750 1748 96 92 385 419

nsl "*?

*

as

as} as

ns

a$~ n nsp s ns

as

O

as} ns * --~ .,.

as]

"Tests of samples on parents combined are indicated wizh br0~kezs. S/gnific0ace levels are p < 0.05. "; p < 0.01, **; p < 0.001, o**.

in each of the parents tested separately, and dropped (to <0.05) in the combined sample. In the Chinese sample, fathers transmitted the A2 allele in ercess, sufficient to create heterogeneity in both the father and combined tests. The log-linear test for three-way interaction at HLA-C was significant at p<0.05. The HLA-C mothers showed significant heterogeneity in allelic transmission by goodness of fit (Table 1). The contrast in parental transmission for the three common alleles Cw4, CwS, and CO (Table 2), all contribute to the three-way interaction. Furthermore, the CO contributions of fathers was in excess (as predicted by our conditions for bona fide segregation distort/on). The CO allele was •,dso transmitted in excess in Chinese mothers. The fathers and combined tests for HLA-B alleles in the Caucasian sample (Table 1) were both significant. O f the individual alleles, B42 fathers with no tranmissions to 13 offspr/ng were the most divergent. Deficient and excess transmission for two B alleles (B35 and B62) in the Chinese fathers crew,ted significant differences in the transmission among B alleles in this group.

170

TABLE

W. Klitz e t al.

2

N o m i n a l l y significant d e p a r t u r e s f r o m 1 : 1 t r a n s m i s s i o n in H L A a l l e l e s ~

Caucasian Total offsp

Freq tran

&llele

Parent

A2

Father Mother Father Mother Father Mother Father Mother Father Mother

438 429 128 167 75 71 13 24

.543 ~ ® .543 .531 ~ . .587" .373" .535 .231 ~ , , , o208"~

Father Mother Father Mother Father Mother

442 457 303 305 792 827

.489 .556" .492 ~, .407"* .545* .493

Father Mother Father Mother Father Mother Father Mother Father Mother Father Mother Father Mother

409 409 39 25 13 8 f04 67

.499 .560" .667" .520 .000"#" ~ . .750 .433 { .403

A24 A26 A33 A0

C4 C5 CO

B35 B41 B42 B52 B62 B64 B0

DR2 DR3 DR4 DRIO DRI2 DRO

Father Mother Father Mother Father Mother Father Mother Father Mofl,er Father Mother

42 58

Chinese Total offsp

Freq tran

90 106 54 43

.656 "° .538 .333" .395

60 64

.517 .641"

26 12

.269" .500

47 54

.702" .593

Total offsp

Freq tran

13 53

.231" .472

61 96

.344" .510

17 14

.647 .214"

126 L38

.524 .6~9"

143 201

.406" .507

.333" .414

II 4

40 31 79 98

Japanese

.909 ~ , , .750

.475 ~. .258 °. .430 ~ ,. .347 "" 77 74

.403 ~ . . .432

"Tests on samples of the parents combined are indicated with brackets. Significancelevels are p < 0.05, "; p < 0.01, °°; p < O.OOl, so..

Segregation Distortion in HLA

171

For the DR locus, a deficiency of maternal transmission in both DR10 ~nd DR12 accounted for the heterogeneity in Caucasians. A peculiar and contrasting transmission bias occurred for the small samples of DR3 from the Chinese and Japanese population samples (Table 2). These DR3 effects are the primary cause of the significant heterogeneity values in the combined parental tests found in each of these populations. Notice that the DRO allele was transmitted with a slight deficit in the combined Japanese sample. The recessive blank alleles were frequent contributors to the nonindependence found in the allele X transmission tests, and played a role in the one significant test for three-way interaction (in the HLA-C locus). An overall test for transmission distortion on just the blank alleles combining the data across the three populations to contrast the four loci showed undertranmission for loci A, B, and DR (at p<0.05, 0. I0, and 0.05, respectively) and slight overtranmission at locus C (p<0.10). The heterogeneity term was highly significant (p<0.001), suggesting that the CO blank classes stand apart from AO, BO, and DRO. These blank allele effects are best explained by our use of the haplotype deduction procedures applied to this data. The result of always choosing the most likely haplotype estimated for an individual in a pedigree is that the transmission of rare recessives (AO, BO, and DRO) are underestimated while the transmission of common recessives (CO) are overestimated. We also examined families of sibship sizes from two to seven for deviation from the binomial expectations of transmission. None of the sibships, conditioned on family size, revealed deviations from expected proportions. Further, as a measure of data consistency, we were unable to detect any trends in transmission based on family size. HaplotTpe Effe~s The most common three-locus haplotypes were tested as fvllows (numbers in parenthesis): for the Caucasian sample A-C-B (24), C-B-DR (39), A-B-DR (19), A-DR-GLO ( 17 ), and B-DR-GLO ( 13 ), and for the Japanese sample A-C-B (10), C-B-DR (12), and A-B-DR (6). Tests for three-way interaction were carried out on each of the Caucasian three-locus defined haplotypes with no interacrion detected. Goodness of fit tests for igeterogeneity run separately for the father, mother, and combined samples revealed three instances of statistical significance, all in the Caucasian samples. The transmission of C-B-DR haplotypes (marking one map unit of chromosome 6) was heterogeneous in the mothers and combined samples, and the ADR-GLO haplotypes (marking some seven map units) was heterogeneous in mothers. The transmission frequencies of all nominally significant departures from 1:1 transmission among all the haplotypes tested is shown in Table 3. T,~ere is no pattern of excess or deficit transmission in any of the three-locus haplotype groups. The C-B-DR haplotypes, hemrogeneous in transmission rates for the maternal Caucasian sample, has, for example, two haplotypes tr~rlsmitted in excess and four in deficit. Because of previous reports of segregation distortion due to the B8-DR3GLO2 hapiotype transmitted by males [16], we have tabulated the results of both the former and present studies for this haplotype in Table 4. The 9th Workshop data shows no s/gnificant departures from the expected ratio for either sex, while the data of Awdeh et al. [ 16] show 83% of transm/tted male haplotypes coming from B8-DR3-GLO2. Awdeh et al. [16] defined this haplotype with the complement alleles, C2-1,Bf-S,C4A-QO, and C4B-1. When these additional

172

W. Klitz et al.

TABLE 3

Three locus haplotypes showing nominally significant departures from Mendelian transmission ~ 'Significance Father

Hoplotype

Mother

Population

n

Freq.

n

Freq.

Combined

A2 A30 A2 A24

C7 C6 C5 CI

BI8 BI3 B44 B54

Caucasian Caucasian Caucasian Japanese

46 48 133 32

.326* .563 .429 .500

40 48 133 75

.500 .667" .453 .613"

C7 C6 C0 C2 C6 C7 CO CO C3 C3

B8 BI3 B18 B27 B37 B44 B51 B51 B35 B61

DR3 DR7 DR2 DR4 DRIO DR11 DR4 DR7 DR4 DR4

Caucasian Caucastan Caucastan Caucasian Caucastan Caucastan Caucasian Caucasian Japanese Japanese

188 71 33 22 12 11 31 16 37 11

.41~-* .535 .758" .445 .583 .727 .323" .563 .324* .091"*

232 "62 29 11 16 20 16 36 25 13

.474 .645* .690* .182" .125" .250* .375 .333" .440 .462

* * ***

A30 A2 A25 A2

BI3 BSl BI8 B61

DR7 DRII DR2 DR9

Caucasian

.500 .515 .645 818"

31 24 2i 17

.742** .292* .714 .529

*

Japanese

30 33 31 11

A1 A3 A2 A2

DR2 DR2 DR7 DRI3

GLO2 GLOI GLO1 GLO2

Caucasian Caucasian Caucasian Caucasian

25 33 38 12

.480 .515 .474 .333

14 12 11 20

.143"* .833" .182" .150"*

GLOI

Caucasian

12

.500

16

.250*

B35 DRI

Caucasian Caucasian

* *

* * *

*

***

*Thesignific~ levelsare: *, p < 0.05; ®%p < 0.01; ®®*,p < 0.001.

spe::ificitie~ are taken into account for the 9th Workshop data, the sample size deO:eases by approximately one half (mostly because the complement loci were less frequently typed in the 9th Workshop), but the deviation o f male transmission remains unchanged (45% males, p--0.63). The transmission rates o f the BSDR3-GLO2 haplotype in the two separate samples are idem!cal for the mothers, but very different for the fathers (p<0.001), as revealed by ,the goodness o f fit test. ~. G e n o t y p e Effects Tests for three-way interaction and goodness o f fit tests on the most common HLA genotypes in the Caucasian sample (numbers in parenthesis) were performed on the A (21), C (23), B (23), and D R (30) loci. The three-way interaction term from the log-Iinear model was uniformly nonsignificant. Only the combined parental sample o f the A/A genotypes was heterogeneous under goodness of fit tests. All i~ominally significant dep~xtares from Mendelian transmission for the

Segregation Distortion in HLA TABLE 4

173

C o m p a r i s o n o f transmission frequencies o f the B S - D R 3 - G L O 2 h a p l o t y p e f r o m t w o d a t a sets " Pmpomon transmitted

No. families

No. children

p value

Awdeh e¢ al [16]*: Mammal transmission Paternal transmission

0.35 0.83

12 15

34 41

ns <0.0Ol

9th IHW~: Maternal transmission Paternal transmission

0.475 0.375

14 6

61 24

as us

"In Awdehet al. [16]. the haplotypeis definedto includethe complement~eles C2C-BfS-C4AO-C4BLTaken from a tot~ of 151 normalfmnilie$ and nondiseasedindlvidualsfrom dlse~.,edfmrfilies. Jgth IHW: 9th lnteraadon~ Hiztocompadbilits'Workshopdata ex~unincdin this studs,[20]. g e n o t y p i c tests a r e s h o w n in T a b l e 5. W h e n t h e c o m m o n A 2 allele f o r m s a h e t e r o z y g o t e w i t h t h e rare recessive b l a n k allele, A 2 is c o n s i s t e n d y transmkxed (in 15 o f 16 i n f o r m a t i v e transmissions). T h i s is ¢ x # a i n a b l e o n t h e basis o f t h e h a p l o t y p e d e d u c t i o n p r o c e d u r e d e s c r i b e d above. W h e n this s a m p l e is r e m o v e d , the heterogeneity term mentioned above disappe~s. D i f f u s e F a m i l y Effec~ T o e x a m i n e t h e possibility o f a diffuse family effect o n ~egregation, t h e sib p a i r configurations for s i b s h i p sizes o f t w o t o seven w e r e c o m p u t e d eaong with t h e e x p e c t e d relative f r e q u e n c y o f each configuration. T h e s e e x p e c t e d f r e q u e n c i e s TABLE 5

N o m i m d i y significant d e p a r t u r e s f r o m M e n d e l i a n transmission in H L A g e n o t y p e s f r o m t h e Caucasian p o p u l a t i o n s a m p l e Father

Genov/p¢

n

Mother freq.

n

freq.

A2/A24 A2/A0 AUA28 A|/A11 ALIA32

156 8 47 66 28

.423 .875 ,660 .500 .643

! 59 8 37 32 23

.465 1.000 .622 .656" .652

C0/C7 C0/C4 C6/C5 C6/C1

249 109 27 25

.570 ® .495 .630 .720

277 142 34 16

.513 .408" .618 .375

B44/B7 B44/BS1 B44/B62 B7/Bt8 BS/B18

76 54 60 29 22

.553 .574 .500 .724 ® .227 ®®

78 32 33 26 25

.359" .719 ) ,697" .538 .520

DR3/DR0

29

£90 °

24

.542

Combined

"

"~"

¢,, ®

174

W. Klitz et al.

TABLE 6

Sporadic segregation distortion investigated by h a p l o t y p e sharing a m o n g sib pairs ~

Sibship size

Sample size

Total ~ (d0

2 3 4

28 I00 348

1.79(2) 1.01(3) 6.68(7)

Haplotylms shared

5 6 7

tOt 34 22

2

1

0

Exp. freq.

Obs. freq.

6 3 3 2

0 3 0 4

0 0 3 0

0.0156 0.1250 0.0625 0.0938

0.017 0.152 0.063 0.086

1

4

1

O.1875

O.184

1 2 0

3 0 4

2 4 2

0.3750 0.0469 0.0938

0.345 0.049 O.103

5.35(9) 9.52(14) 11.40(20)

aThe eight possiblesibpalr configurationsare shown for the sibship of size four.

TABLE 7

N o m i n a l l y significant d e p a r t u r e s in t he sex ratio (expressed as p r o p o r t i o n sons) based o n t he H L A allele o f the parents ~ Fathers

Mothers

Allele

Population

n

Freq.

n

Freq.

A2 A2 A2 A23 A24 A30

Caucasian Chinese Japanese Caucasian Caucasian Chinese

981 90 174 73 4 ~8 33

0.484 0.467 0.374 ~ 0.630" 0.445 ~ 0.394

983 106 205 77 429 36

0.486 0.472 0.376 ®** 0.5 ~ 1 0.517 0.639 ®

C1 C3 C4 CO

Chinese Caucasian Caucasian Caucasian

33 418 442 792

0.485 0.450* 0.527 0.490

5! 507 457 827

0.353 ®~ 0.489 0.538* 0.461"*

B7 B18 B35 B39 B39 B46 BSI

Japanese Caucasian Japanese Chinese Japanese Chinese Caucasian

47 271 102 26 41 19 306

0.447 0.494 0.520 0.692* 0.488 0.526 0.500

53 236 88 4 45 33 255

0.604"* 0.555* 0.545" 1.000 0.289* 0.303 ~* 0.424*

DR13

Japanese

38

0.658 ®.

~ i f

35

0.457

"Although n o t nominallysignificant,the sex ratios of the offspringof A2 Caucasian and A2 Chinese parents are shown for comparison with the Japanese A2 rados. The sex ratiosof the total s~mpleswere 0.491 (1,249:1,295) for the Caucasian samples, 0.495 (104:106) for the Chinese,and 0.436 (240:310) for the Japanese.

Segregation Distortion in HLA

175

are compared to the observed data in Table 6. (The l~ger family sizes solicited during the collection of the 9th Workshop data is reflected here.) We use sibship size fore as an example of the analysis by showing the eight different possible sibships along with their expected and observed frequencies. A family in the first configuration has all six pairs sharing two haplotypes (all inherit the same haplotype from the mother and the father); this configuration has an expected frequency of 0.0156, compared to the observation of six (0.0172) of the 348 size four families in this category. When the X2 values of these differences were computed for each size sibship to detect any distortion within each family size, no significant deviations were found. There was no significant heterogeneity in segregation ratios among the sibships of different sizes (X2 = 7.58, tdf = 5]). 8¢x Ratio We examined the influence of the HLA region upon sex ratio by searching fo~ deviations in the sex ratio of offspring of fathers and mothers having a given allele. For the expected sex ratio, we used the sex ratio of the entire ~ p l e of each ethnic group, minus the allele being tested. For exan~ple, the 109 sons and 106 daughters produced by A32 Caucasian mothers were compared to the proportion of 1140 sons and 1187 daughters found in the remainder of the Caucasian population sample. Those cases with nominal|y significant (i.e., p<0.05) deviations occurred at a rate of 0.075 (of 253 tests), slightly over that expected by chance (Table 7). In the Japanese, the overall sex ratio, expressed as the proportion sons, was 0.436 (of 550 offspring). Most of this "excess of daughters" was identifiable with parents possessing the A2 allele. The sex ratio of the offspring of A2 bearing Caucasian and Chinese parents is Mso less than the population means, but not significant. DISCUSSION No clear evidence for segregation distortion in the major histocompatibility complex of humans operating through dif~brendal sperm transmission by fathers was uncovered in this study. This result must be viewed against the background of our statistical ability to detect a given deviation. The 9th Imemational Histocompatibility Workshop data is the ~rgest and highest quality HLA population data ever compiled. In particular, this quality (described in [20]) is attributable to the most discriminatory antisera e~ssembted, ped/gree data on all individuals, and computer typing of haplotypes. Despite these strengths, the limits of the available s~arnple sizes permitted us to detect segregation distortion of at best -+ 3% (in A2 Caucasian parents). The discrimination was much worse in most cases, in general, if segregation distortion is operating in the HLA region, it will be very difficult to detect in agiven ~llele, haplotype, or genot~pe, ffthe distortion is < -+ 10% (requiting n>220). In several instances, it was possible to sample only a small portion of the total genetic variation of a category. For example, the Caucasian s ~ p l e of HLA-A-CoB haplotypes allowed us to examine only 20 (or less than 5%) of the known A-C-B haplotypes. Nominally significant deviations in transmission were recorded in several inst~,ee~. Mos~ of these are undoubtedly due simply to sampling effects, and the spurious influence from the blank alleles. Nonetheless, we have listed all of these instances in Tables 2, 3, 5, and 7, with the thought of this serving as a useful backdrop for future studies. O f these several cases, ~:heinstance of the Japanese A2 families which favor daughters in particubx deserves further sampling. Our ability to detect segregation distortion of < 5% (as has been found in

176

W. Klitz et al. Drosophila, see [9]) is poor, and is not likely to improve in the absence of population sampling several times larger than undertaken for the 9th Workshop. We can, however, speak with confidence on the absence of more extreme segregation ratios, as known in the t haplotypes of mice. To what extent could selection in the diploid part of the life cycle confound our interpretation of observed segregation ratios in family data, and has the HLA region been implicated as being under diploid selection? In general, the effects of segregation distortion can be separated from the various modes of diploid selection because of the anticipated involvement of only males in the unequal transmission ofgaraetes. For the HLA region, it has been suggested that selection on the diploid phase might occur through mating preference, differential viability, or maternal-fetal interactions. The influence of a mating preference associated with the MHC has been documented in mice [30]. Rosenberg et al. [31] noted an HLA associated mating preference, but the correlations were based on a sample of mixed ethnic groups, which do not comprise a randomly mating population, so the observed associations were probably not HLA specific. In another study [32], 30 HLA antigens were examined in some 3000 Dutch ~hmilies. Four m~ting types exhibited an excess prevalence. Two of these were the reciprocal rantings of Awl9 and Cw4. The others were A2 and B7 mothers paired with B40 fathers. The antigen Awl9 has subsequently been split into five new specificities, and B40 has been split into two (see [33]). Fu~her studies are needed to establish any HLA mating preference. A viability selection model compatible with the pattern of disequilibrium found amoniz HLA loci is simple dominance, in which the presence of a certain allele or haplotype is protective during a selection episode (i.e., an epidemic), while individuals lacking that specificity are subject to mortality. If this selection acts prenatally, as, for example, has been suggested for type I diabetes [34], then the effects of prenatal mortality and segregation distortion could give similar results. In particular, results mimicking allelic or haplotypic segregation distortion would occur, appearing as an exce~s of a given haplotype or allele. However, this excess would appear in both l~arents, thereby signaling a diploid effect. Viability selection has been suggested to operate on MHC variation due to differential suscep:ibility to microbial pathogens [35]. Although the record of past viability selection may be stamped on the HLA region through patterns of linkage disequilibrium [36], except for the possibility of prenatal mortalky, mortality due to infectious dise~e is not likely to be reflected in the individuals and generations from which the 9th Workshop families were drawn. HLA associated susceptibility to autoimmune disease does distort segregation ratios in a characteristic way. These variant transmission ratios identify particular susceptibility haplotypes, and not segregation distortion as such [37]. Many studies support the observation of increased levels of maternal-paternal HLA matching in couples .,mffering high rates of spontaneous abortions (see, e.g., [38]). This has been interpreted as a requirement for stimulation of the maternal immune system for acceptance of the fetm~. Common alleles will be at a disadvantage in this case because fetal-maternal matching will occur more frequently. In fact, given random mating, the relative exposure to this type of selection for a comcnon vs. rare allele is as the square of the allele (or h~plotype) frequency. The pedigree structure in which HLA data is often collected to assist in typing has made HLA a convenient system to examine segregation distortion. The initial published reports showed consistently even transmission [39--4 I], utilizing early defi~:itions of the HLA-A and HLA-B alleles and up to 535 families. More

Segregation Dismrtian in HLA

177

recently, Cudworth et ~. [ 15] reported randomly selected healthy families typed for HLA-A and HLA-B to have significant deviations from Mende~an s e l e c t i o n in the fathers bearing a chromosome carrying A1-BS. Awdeh et al. [16] found nonrandom transmission in fathers carrying a haplotype consisting of alleles from seven loci from HLA-B to GLO. This study udlized 151 Caucasian families, some of which had been selected because of particular diseases. All of these studies looked for allelic and haplotypic mmsmission as defined here, except for [39], who developed a method similar to the diffuse effect, also described here. Gene expression of the haploid genome is perhaps the s/ropiest model for differential sperm transmission. Post-meio& gene transcription does apparemly occur [42,43], but the contribution of any resulting"meiotic" proteins to creating variation in sperm success is unknown. The expression of MHC genes on sperm has been discussed for some years (see [44]), and evidence remains commdic¢ory for the expression of post epidydimal MHC proteins on sperm (for differing views, see [1%45-48]. The T/t region of mice is a well-documented case of mammalimi seg~'ega¢ioa distortion [6,13]. The distortion is due to unequal tamnsmission of t bearing haplotypes in he:erozygous males. These haplotypes are lethal or semilethal when homozygous and so depend upon segregation distortion to maintain their h/gh frequencies in natural populations. Another characteristic feature of t-haplo~es is suppressed recombination. The MHC of mice is embedded in the Tit region in which particular H-2 hapio~pes show some association with various t bearing haplotypes [49]. It has been suggested that the HLA complex, like H-2, is also closely associated with a human Tit re#on [16,17]. A similar suite of traits for a human T/t region found in the mouse Tit re#on would include t-chromosomes causing suppressed crossing-over, homozygous lethality balanced by segregation distortion in fathers heterozygous for t-heplo~pes, and very high linkage disequilibrium of t-bearing HLA heplotypes. Using the mouse model, then, a sample of HLA haplotypes should reveal high linkage disequilibrium in one or more haplo~pes, which also have distorted transmission ratios in males. Pedigree data should also show an absence of crossovers in the suspect haplot~ms. Linkage disequilibrium is a well-known feature of the HLA system (see, e.g., [33]), and this has been attributed in part to selection [36]. Pmicular sets of haplot~pes often characterize the different ethnic groups [33]. In any case, there are then nonrandom dusters of alleles at the loci of a single chromosome. One of the most common HLA-B alleles in Caucasian populations (at p = O.10), B8 is Mso a member of A locus and DR locus haplo~pes showing the highest disequilibrium of any common three-locus haplotype in the popuhtion (A1 B8 DR3). Cudworth et M. [15] reported enhanced transmission ofA1-138 haplo~/pes in males, and Awdeh et al. [16] reported a muldtocus haplo~/pe including BSC2(1)-B~$~C4A0-C4B1-DR3-GLO2 (but not A1) to also show enhanced transmission. The data presented hero has not corroborated either of these reports (Table 4). Two additional lines of evidence are available for establishing evidence on the existence of a Tit re#on tightly linked to HLA in humans. HLA hapio~pespecific crossover suppression has not been observed [50], but neural tube defects, a possible indicaebn of t-heplotype gene expression [13], have been associaeed with HLA-A, but not HLA-B or HLA-DR alleles [51]. TMs evidence would put the human T/z complex on the HLA-A side of the HLA region, and tend to diminate a major impac~ of crossover suppression on i:~L~L This is in contrast to the data of Awdeh et ai. [16] whose suspect haplo~/pe excluded the HLA-A locus. If the T/t region of humans is linked to the HLA complex on

178

W. Klitz et al. chromosome 6, it does not carry t-haplotypes in high frequencies causing the range o f effects as found in the mouse. ACKNOWLEDGMENTS Research supported by NIH grants HD12731 and GM353226, by Deutsche Forschungsgemeinschaft SFB 113 B3 + B8, and by University of California at Berkeley IBM ACIS.

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