Am. J. Hum. Genet. 49:917-931, 1991

Nonuniform Linkage Disequilibrium within a I ,500-kb Region of the Human Immunoglobulin Heavy-Chain Complex Michael A. Walter' and Diane W. Cox Research Institute, The Hospital for Sick Children, Toronto; and Departments of Medical Genetics and Paediatrics, University of Toronto

Summary We have characterized 10 VH polymorphic loci of the VH2, VH3, VH4, and VH5 families. Eight of 10 VH polymorphisms were found to be insertion/deletion polymorphisms, probably the result of nonhomologous recombination over the course of evolution of the current human VH repertoire. The 10 VH polymorphic loci were analyzed in 10 three-generation and 10 two-generation Canadian caucasoid families. Linkage disequilibrium (allelic association) was measured between pairs of VH polymorphic loci, and 12 significant associations were found. The degree of linkage disequilibrium measured between IGH polymorphic loci was then compared with the physical distance separating the loci. The physical distance between IGH polymorphic loci does not entirely determine the degree of linkage disequilibrium between polymorphic loci. Two regions, one in the VH region (between VH3f-2 and VH5-2 and one in the CH region (between C8 and Cy3), were found to have linkage disequilibrium values approximately 1/3,000 of that observed in other portions of the IGH region. The previous identification of recombinants in the C8-toCy3 region indicates that these areas of low linkage disequilibrium are consistent with the presence of recombination hot spots. The observed high amount of recombination in the subtelomeric portion of chromosome 14 therefore appears to be the result of specific hot spots for recombination, rather than a general increase in recombination in this region.

Introduction The IGH gene complex comprises approximately 80 variable (VH) gene segments, at least 20 diversity (DH) elements, six functional joining (JH) segments, and a constant (CH) region comprising nine genes (Cyl-Cy4, Cal and Ca2, CRi, C5, and Cs) and two pseudogenes (Cony and Codi1). In the course of B-cell development, VH, DH, JH, and CH gene segments are juxtaposed to produce an IgH peptide. In combination with the light-chain peptide, the VH-, DH-, and JH-encoded amino acid sequences of the heavychain peptide are responsible for antigen recognition and binding, while the CH-encoded sequences are re-

sponsible for antibody-effector function. Received March 19, 1991; revision received July 16, 1991. Address for correspondence and reprints: Dr. Diane W. Cox, Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. 1. Present address: Imperial Cancer Research Fund, Lincoln's Inn Fields, London, United Kingdom. i 1991 by The American Society of Human Genetics. All rights reserved. 0002-9297/91 /4905-0002$02.00

The IGH gene complex is approximately 2,500 kb in size (Berman et al. 1988; Matsuda et al. 1988; Walter et al. 1990). Complete physical maps of the CH region have been described elsewhere (Bottaro et al. 1989; Hofker et al. 1989). Considerable genetic variation in the CH genes has been observed. This includes protein polymorphisms of the Cyl, Cy2, and Cy3 genes (the GM allotypes) and of the Ca2 gene (the Am allotypes) (for a review, see Schanfield and van Loghem 1986). DNA RFLPs have been described for the switch regions of the CRi, Cal, and Ca2 genes and for the C8, Cy3, Cyl, Cony, C'y2, and Cy4 genes (Bech-Hansen et al. 1983; Ghanem et al. 1988; Benger and Cox 1989). The human VH gene segments have been subdivided into six families (VH1-VH6) based on DNA homology (Kabat et al. 1987; Lee et al. 1987; Schroeder et al. 1987; Shen et al. 1987; Berman et al. 1988; Buluwela and Rabbitts 1988). A detailed restriction map of the VH region has been recently reported (Walter et al. 1990). As is true for the CH region, the degree of genetic polymorphism in the hu917

Walter and Cox

918

man VH region is high. RFLPs of the VH2 (Johnson et al. 1984; Walter and Cox 1988), VH3 (Walter et al. 1987; Walter and Cox 1988; Chen et al. 1989; Souroujon et al. 1989; van Dijk et al. 1989), and VHS (Sam et al. 1988) families have been described. The genetic linkage map of the subtelomeric region of chromosome 14 (14q32 to 14qter) has been reported to span a distance of more than 60 cM (Nakamura et al. 1989) in a region of approximately 10 Mb (Hofker et al. 1990). On the basis of the observation that chiasmata are preferentially found in these regions (Laurie and Hulton 1985), it could be inferred that subtelomeric regions in general undergo a high degree of recombination. The IGH gene cluster has been mapped to 14q32.33 (Cox et al. 1982; Benger et al., in press). On the basis of both the localization of this large gene cluster to a region of increased recombination and the large number of IGH polymorphisms that are available in this gene cluster, we investigated the relation between physical and genetic organization over an approximately 1,500-kb region of the IGH locus. Material and Methods DNA Extraction DNA for polymorphism screening was obtained from peripheral blood of six randomly selected Canadian individuals of caucasoid descent (Madisen et al. 1987). Leukocytes were an appropriate source of DNA for the study of nonrearranged VH genes because leukocyte DNA and fibroblast DNA digested with restriction endonucleases and hybridized with VH gene probes showed similar hybridizing fragments (data not shown). DNA from the 10 three-generation and 10 two-generation Canadian Caucasian families, analyzed here for the distributions of the IGH polymorphisms, was originally obtained during a study of al-antitrypsin deficiency. Results were obtained from unrelated family members and from 10 additional chromosomes of independent origin in incomplete pedigrees. DNA samples were also obtained from the peripheral blood leukocytes of 21 volunteers of Oriental (Chinese) ancestry. Electrophoresis Conditions Three micrograms of DNA was digested with 9 units of restriction endonuclease in manufacturer's recommended buffer overnight and electrophoresed in 0.8% agarose gels (with 0.8 gg ethidium bromide/ ml) for 1,200-1,600 V/h. Gels were then treated with

0.25 N HCl for 15 min and were transferred to Hybond® nylon membrane according to the manufacturer's (Amersham) recommendations. Filters were baked for 2 h, prehybridized for at least 1 h, and hybridized at 420C for 18-24 h with 1-5 x 106 cpm of nick-translated or oligo-labeled probes/ml hybridization solution. Prehybridization and hybridization solutions were as described elsewhere (Linsley et al. 1983). Membranes were washed in 2 x SSC for 30 min and once for 1 h in 0.1 x SSC, 0.1 % SDS at either 52°C (for the VH2, VH3f, and VHS probes) or 650C (for the JH, DH, VH1, VH3, VH4, and VH6 probes). Membranes were exposed to Kodak XAR-5 film at - 700C, using intensifying screens. DNA Probes

DNA probes used in the present study were the following: VH2 is a 1.2-kb EcoRI/BamHI fragment subcloned from VCE-i and containing only VH sequences (Walter and Cox 1988). VH3f is a 2.2-kb EcoRI fragment flanking the VH3 gene segment VH26 and is derived from XVH26 (Matthyssens and Rabbitts 1980). VH4 is the 245-bp ApaI/EagI fragment of 58P2X, a VH4-containing cDNA clone (Schroeder et al. 1987). VHS is the 221-bp PstI fragment of 2-V and is used to detect the VHS family (Berman et al. 1988). VH6 cDNA is the 240-bp PstI fragment of 1SPIC (Schroeder et al. 1988). Cla2O is the truncated 20-kb ClaI fragment derived from cosmid clone C17p3 and containing the VH6 gene segment as well as 3'- and 5'-flanking sequences (Schroeder et al. 1988). Statistical Analysis

Linkage disequilibrium, or allelic association, is the nonrandom association between an allele at one locus and an allele at a second, closely linked locus. As an estimate of linkage disequilibrium, D' values were computed as D/Dmax (Lewontin 1964), where D = h - plql. pl and p2 are the frequencies of the alleles of one locus, and qi and q2 are the frequencies of the second locus; pl and qi are the rare alleles of each polymorphism; h is the frequency of the haplotype containing the rare allele of each polymorphism. If DO, then Dmax is the lesser of plq2 and p2ql. D' has the same range, from - 1 to 1, for all combinations of allelic frequencies at two loci and incorporates both absolute and complete association (Hendrick 1988). X2 Values for pairwise comparisons of different IGH polymorphic loci were calculated as n(D)2/plp2qlq2), with 1 df, where n is the sample size of the pair-

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Walter and Cox

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Figure 2 Autoradiograms of 10 polymorphic human VH region loci. VH2, BglII-digested DNA probed with VCE-1. VH3f, EcoRIdigested DNA probed with VH3f. VH4, BglII-digested DNA samples probed with VH4 family probe. VHS, BglII-digested DNA samples probed with VHS family probe. VH polymorphic fragments are numbered on the right of each gel, and sizes of polymorphic fragments are indicated on the left of each gel.

wise comparison. Standard error (SE) was calculated for polymorphisms as (plp2/n)OS The distributions of 10 VH polymorphisms in two different racial groups, Caucasians and Chinese, were compared by X2 analysis. .

IGH Physical Map

The physical positions of the IGH polymorphisms investigated here are shown in figure 1. The physical locations of the VH polymorphic loci are from Walter et al. (1990, 1991), and the locations of the CH polymorphic loci are from Hofker et al. (1989). Exact physical distances (d; in kb) between compared polymorphisms were available for CH-region polymorphisms. In the VH region, the physical distance separating paired loci was measured from the midpoint of the interval in which each VH locus was located, since the exact locations of VH polymorphisms are unknown. Results VH2 Polymorphisms

DNA extracted from the leukocytes of six random Caucasian individuals was digested with each of 10 restriction endonucleases to detect VH region RFLPs. The VH2 family probe detected polymorphisms in

BglII-digested DNA, as described elsewhere (Johnson et al. 1984; Walter and Cox 1988). These three polymorphisms of VH2 gene segments, designated here as VH2-2, VH2-4, and VH2-5, are shown in figure 2. VH2-2 has a 12.0-kb fragment with no allelic counterpart. VH2-4 has two alleles-one of 7.3 and one of 7.0 kb. VH2-5 has one detectable allele-a 3.4-kb fragment. For this subfamily, as for all others, scoring of the presence of one or two doses of a specific gene was done by comparison with a constant band detected by each probe. Densitometric analysis was carried out in trial comparisons, as also was done in a previous mapping study (Walter et al. 1991), and confirmed the visual scoring. VH3 Polymorphisms

The complex patterns of hybridization detected with the VH3 family probe precluded the characterization of most of the genetic variation observed (data not shown). To avoid complications due to the large number of VH3 family members, the VH3-flankingregion probe, VH3f, was used to detect VH3 polymorphic loci. The polymorphisms detected with VH3f in EcoRI-digested DNA have been characterized elsewhere (Walter and Cox 1988). These three VH3 polymorphic loci, designated here as VH3f-1, VH3f-2, and VH3f-4, are shown in figure 2. VH3f-1 has a

921

Nonuniform Recombination in the IGH Region

6.7-kb fragment with no allelic counterpart. VH3f-2 has two alleles-a 6.0-kb fragment and a 4.6-kb fragment. VH3f-4 was scored as the presence or absence of a 3.5-kb fragment. VH4 Polymorphisms

The VH4 family probe detects polymorphic loci in DNA digested with Spel, BglII, EcoRI, ApaI, BamHI, and PstI. All enzymes tested detected VH4 polymorphisms. Thirteen of the 15 fragments detected with the VH4 family probe in BgIII-digested DNA were variable. Of these, three VH4 polymorphic lociVH4-8, VH4-1 1, and VH4-1 5-were selected for detailed analysis (fig. 2). VH4-8 is a 4.6-kb fragment to which no allelic counterpart could be assigned. VH411 was scored as the presence or absence of a 3.8-kb fragment. VH4-15 was characterized as the variable presence of a 5.8-kb BglII fragment. The other VH4 variable bands were not studied further, either because of their infrequent nature or because of failure to consistently detect the polymorphic fragments. VHS Polymorphisms

Hybridization of the VH5 family probe to the panel of DNA from six randomly selected individuals revealed that the number of VH5 gene segments in the normal Canadian Caucasian population is polymorphic (Sam et al. 1988). The variable presence of a VH5 hybridizing fragment was detected in DNA digested with RsaI, Dral, EcoRI, HindIII, Spel, and BglII. The polymorphic VH5 locus, VH5-2, was characterized by the appearance or absence of an 8.0-kb fragment in BglII-digested DNA (fig. 2). This enzyme was convenient to use, as the same Southern blots used earlier to characterize the VH2 and VH4 family polymorphisms could be reused. VH6 Region Polymorphisms

The VH6 gene family is unusual, as it contains only single gene segment member, called VH6. VH6 is the most 3' VH gene segment and marks the beginning of the VH region, 77 kb from the constant region Cg gene (Schroeder et al. 1988). A VH6 cDNA probe, isolated to contain only VH sequences, was used to detect polymorphisms. No VH6 polymorphisms were detected in TaqI, PstI, RsaI, BstEII, BglI, BglII, BamHI, or EcoRI digests of DNA samples. No polymorphic loci were detected with a second VH6containing probe, Cla2O, in PstI, BglII, EcoRI, TaqI, AvaIl, DraI, or RsaI digests of DNA, despite the fact that Cla2O is 20 kb in length. a

Table I Allelic Designations and Frequencies of 10 VH Polymorphic Loci

ALLELE

VH FAMILY (enzyme) AND Locusa

VH2 (BglII): 2 ........... 4

...........

5

...........

VH3f (EcoRI): 1 ........... 2

...........

4

...........

VH4 (BgIlI): 8 ...........

11 15

...........

...........

VH5 (BglII): 2 ...........

No. OF CHROMOSOMES TYPED

Sizeb (kb)

Mean ± SE

12.0 0 7.5 7.0 3.4 0

.28 .72 .06 .94 .45 .55

.04 .04 .02 .02 .04 .04

134

6.7 0 6.0 4.6 0 0

.28 .72 .83 .17 .13 .87

.04 .04 .03 .03 .03 .03

134

4.6 0 3.8 0 5.8 0

.38 .62 .60 .40 .11 .89

.05 .05 .05 .05 .03 .03

8.0 0

.50 .50

.04 .04

Frequency

134 134

134 134

88 87 89 110

a VH2 polymorphic loci were detected with the VH2 family probe, VH3f loci with probe VH3f, VH4 loci with the VH4 family probe, and VH5 polymorphic locus with the VH5 probe. b A zero (0) indicates that an allelic counterpart to the polymorphic was not found.

Allele Frequencies and Designations of the VH Polymorphisms

The distribution of the 10 VH polymorphic loci were analyzed in 10 two-generation and 10 threegeneration families. The distributions of the VH2 and VH3f polymorphisms in these families have been reported elsewhere (Walter and Cox 1988). The allelic frequencies of the 10 VH polymorphisms are shown in table 1. The alleles of all VH polymorphic loci were in Hardy-Weinberg equilibrium. Linkage Disequilibrium in the VH Region On the chromosomes obtained from the study of the Caucasian families the distributions of the alleles of the 10 polymorphic loci of the VH2, VH3, VH4, and VH5 families were analyzed to determine the extent of linkage disequilibrium. X2 Analysis in 2 x 2 contingency tables was carried out for all pairwise

Walter and Cox

922 Table 2

x2 and Associated Sample Sizes of Pairwise Comparisons of VH4-15

VH4-15... VH3f-1 VH2-2 VH4-11 .. VH2-4 VH3f-4 VH4-8 VH3f-2 VH2-5

....

.....

.....

....

.....

....

.....

VH5-2

.....

10 VH Polymorphic Loci

VH3f-1

VH2-2

VH4-11

VH2-4

VH3f-4

VH4-8

VH3f-2

VH2-5

VH5-2

4.82*

4.82* 134**

.68 15.2** 15.2**

.67 3.25 3.25 .01

.18 3.68* 3.68* .57 .05

.34 .13 .13 .43 .69 6.55*

.04 5.68* 5.68* .05 6.45* 1.74 .32

.54 .51 .51 .14 .22 .15 .90 .46

1.40 .70 .70 1.36 .01 .01 .84 .01

87 87 88 89 88 88 89 86 82

134 87 134 134 88 134 134 110

87 134 134 88 134 134 110

87 87 86 87 86 80

134 88 134 134 110

88 134 134 110

88 87 81

134 110

53.9* 110

NOTE. -Numbers above the diagonal are X2 values; numbers below the diagonal are sample sizes. Underlined values were calculated by Fisher's exact method. * P *

.05. P < .01.

combinations of the 10 VH polymorphic loci characterized in the Caucasian families. Statistical significance was considered as P < .05, as determined either by X2 analysis or by Fisher's exact method. The latter is recommended when marginal totals are less than 15 (Steel and Torrie 1980), which was never the case in the present study. Nevertheless, this calculation was used when category numbers were small, as indicated in table 2. X2 Values and sample sizes are shown in table 2. Twelve of the 45 pairwise interactions examined were significant. The large number of comparisons calculated in table 2 make it difficult to obtain an overall impression of the extent of linkage disequilibrium in the VH region. The probability of observing k or more significant values among m tests (which are assumed to be independent) is given by m

P = Z(k)a(1 a) r=k

(Chakravarti et al. 1984), where a = .05 is the significance level. A number of significant interactions, calculated as ma[ma(l - a)]05, would be expected to be observed by chance alone. For the data present in table 2, the observed 12 significant interactions of 45 pairwise comparisons would occur randomly at a probability of 2.4 x 10-6, when 3.29 significant interactions would be expected to be observed by chance alone. The D' values in the VH region fall into two main clusters, each characterized by a high degree of association (defined here as D' > .75, significant at P < .01). The polymorphic loci VH2-2, VH3f-1, and VH4-11

form one cluster of strong association, and VH2-5 and VH5-2 form a second cluster of strong association. Other VH polymorphic loci have significant (P < .05) but weaker associations with the VH2-2, VH3f-1, and VH4-11 cluster (table 2). The members of the second cluster of association, VH2-5 and VH5-2, do not have significant associations with any other VH polymorphic loci. VH2-5 and VH5-2 thus form a distinct set of polymorphisms within the VH region. VH Polymorphisms in Oriental Individuals The allele frequencies of 10 VH polymorphic loci -

VH2-2, VH2-4, VH2-5, VH3f-1, VH3f-2, VH3f-4, VH4-8, VH4-11, VH4-15 and VH5-2-as shown in figure 2, were also investigated in DNA samples from volunteers of Oriental (Chinese) ancestry. The results are summarized in table 3. One VH polymorphism, VH3f-2, has different allele frequencies in Chinese and Caucasian populations (X2 = 7.50, P < .05). All insertion/deletion VH polymorphisms found in Caucasians were also found in the Chinese individuals. Nonvariant fragments in Caucasian DNA samples, detected in BglII-digested DNA by the VH2, VH4, or VH5 family probes and by the VH3f probe in EcoRIdigested DNA, were also nonvariant in the Chinese DNA samples. Therefore the same VH polymorphisms were observed in both Caucasian and Chinese DNA samples and, in nine of 10 situations, at similar allele frequencies. Selection of IGH Polymorphisms for Comparison of Linkage Disequilibrium and Physical Distance Nine of the 10 VH polymorphisms have been physically localized within the VH region, as shown in fig-

Nonuniform Recombination in the IGH Region

923

Table 3 VH Region Polymorphisms in Orientals Compared with Caucasians

Gene and Genotypea VH2-2: 12.0/12.0. 12.0/0.......... 0/0.......... VH2-4: 7.0/7.0....... 7.0/7.3....... 7.3/7.3....... VH2-5: 3.4/3.4....... 3.4/0.......... 0/0.......... VH3f-1: 6.7/6.7....... 6.7/0.......... 0/0.......... VH3f-2 6.0/6.0....... 6.0/4.6....... 4.6/4.6....... VH3f-4: 3.5/3.5....... 3.5/0.......... 0/0.......... VH4-8: 4.6/4.6....... 4.6/0.......... 0/0.......... VH4-1 1: 3.8/3.8....... 3.8/0.......... 0/0.......... VH4-15: 5.8/5.8 .......

5.8/0.......... 0/0.......... VH5-2:

No. of Orientals 1 3 16

16 4

No. of Caucasians 5

26 } 31

0

54 8 O.

7

11

2

37 14J

1 3 16

26 } 31

10 9 2

42 20 0

11

0

2

2 18

14 46

1

X2

.62

3

8 4

22 } 12

2 8 3

10 221 5

0

0

4 9

2.8

9 S

8.0/8.0

.......

2

16

8.0/0

.......

4

27

0/0

.......

8

14

J

5.65

NOTE. -The total number of individuals typed differs between polymorphisms. Solitary chromosomes typed for VH region polymorphisms in incomplete Caucasian pedigrees were not included in this analysis. a Genotype allele designations are in kb; 0 indicates the deletion allele of a VH region insertion/deletion polymorphism. *P>

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