Mehdi Alizadeh, Christine Legras, Gilbert Semana, Philippe Le Bouteiller, Bernard Genetet, and Ren6e Fauchet
ABSTRACT: HLA-G gene polymorphism was ana- lyzed by RFLP using seven restriction enzymes and an HLA-G locus-specific probe. Hybridization of 55 DNAs digested with three enzymes (Taq I, Pst I, and Bgl II) revealed two polymorphic bands in each case. RFLP pat- terns obtained with Taq I and Pst I corresponded to the same allelic polymorphism and differed from the Bgl II polymorphism. Combining both polymorphisms enabled determination of four alleles. Allelic frequencies were
calculated: 40% of the subjects tested had allele 1, 36% had allele 2, 22% had allele 3, and 2% had allele 4. Analyzing the complete HLA class I phenotype revealed strong linkage disequilibrium with the HLA-A locus. The polymorphism described is located in the 3' flanking re- gion of the gene. Moreover, extended HLA-A haplo- types were constructed by combining the HLA-G poly- morphism with other class-I-sequence polymorphisms. Human Immunology 38, 206-212 (1993)
The class I HLA multigene family is thought to include at least 18 genomic D N A sequences [1, 2]. Three mem- bers of this family encode the classic transplantation antigens HLA-A, -B, and -C [3]. These highly polymor- phic molecules are involved in processed antigen pre- sentation to cytolytic T lymphocytes [4]. The polymor- phism of these class I proteins is believed to be important for the diversity of immunologic responsive- ness within populations and constitutes a major barrier to tissue transplantation.
In addition to the HLA-A, -B, -C genes, three non- classic class I genes - -HLA-E [5], HLA-F [6], and HLA- G [7 ] - -were characterized. These genes were se- quenced and shown to encode intact class I proteins,
From the Histocompatibility Laboratory (M.A., C.L., G.S., B.G., R.F.), Regional Center for Blood Transfusion, Rennes; INSERM UIO0 (P.I.B.), CHU Purpan; and the Hematology-Immunology Laboratory (R.F.), CHRU, Rennes, France.
Address reprint requests to Dr. G. Semana, Laboratoire d'Histocom- patibilit~, Centre R~gional de Transfusion Sanguine, Rue Pierre Jean Gineste, 35000 Rennes, France.
Received (U) July 7, 1993: accepted September 10, 1993.
distinct from the classic class I antigens. Proteins en- coded by these sequences can be expressed in a human HLA-nu l l B-lymphoblastoid cell line after DNA-medi- ated gene transfer [8]. HLA-G transcripts have been detected on certain trophoblast cell populations [9, 10], in fetal liver [11], and in the anterior eye [12]. This pattern of expression supports the hypothesis that HLA-G is invovled in inducing immune tolerance.
This report describes the analysis of HLA-G gene polymorphism by restriction Fragment-length polymor- phism (RFLP) using seven restriction endonucleases and an HLA-G locus-specific probe. Combining this poly- morphism with those of other new class I sequences (HLA-90, HLA-80, HLA-92, HLA-21, and HLA-F) en- abled us to determine extended HLA-A haplotypes.
M A T E R I A L S A N D M E T H O D S
DNA. For this study, a local panel of 55 unrelated Cau- casian subjects was chosen to represent all serologically defined HLA-A specificities. HLA-A allelic frequencies in this selected panel were A1 (11%), A2 (16%), A3
(10%), A9 (10%), A10 (7%), A l l (11%), A19 (8%), A28 (8%), A29 (9%), A32 (7%), and A33 (3%). DNAs from 25 homozygous reference cell lines from the Tenth International Histocompatibility Workshop were also used. The segregation study was performed on DNAs from three two-generation families.
Restriction enzymes. All of the DNAs were digested with seven restriction enzymes: Hind III, Taq I, Pst I, Bgl II, BamHI, EcoR I, and Msp I.
Probe. The HLA-G locus-specific probe was prepared by polymerase chain reaction (PCR) amplification [13] of an HLA-G clone provided by H. T. Orr, with the following primers: G5'--TGA ACC AGC TGC CCT G and G3'--ccA TCA ATC TCT CTr GGA. The amplification prod- uct of 244 bp is specific for the 3' untranslated region of the HLA-G gene. The specific probe was selected ifi this region because analyzing HLA class I gene sequences showed that probes constructed from the 3' untrans- lated region can be used to detect variations specifically among HLA class I genes, thus avoiding cross-hybrid- izations under high-stringency conditions. The probe specificity was confirmed by hybridization with Hind III-digested DNAs, showing an expected unique frag- ment of 6.0 kb. HLA-G gene polymorphism was stud- ied by using RFLP after hybridization of digested DNAs with the specific probe.
Statistica/ analysis. HLA-G/HLA-A linkage disequi- librium was evaluated using a 2 × 2 contingency
chi-squared test by comparison of expected HLA-G/ HLA-A allele association frequencies with observed fre- quencies. For the test, each locus was considered as di- allelic: the tested allele versus all of the others.
RESULTS AND DISCUSSION
HLA-G gene polymorphism. Hybridization with the HLA-G locus-specific probe revealed polymorphic frag- ments with three restriction enzymes: Taq I, Pst I, and Bgl II. With each enzyme, two polymorphic bands were observed: Taq I, 3.9 and 1.6 kb; Pst I, 1.9 and 1.5 kb; and Bgl II, 12 and 4 kb (Fig. 1). Comparing Taq I and Pst I hybridization with the same DNAs revealed that both RFLP patterns corresponded to the same allelic polymorphism, different from the Bgl II-associated polymorphism. Association of the Bgl II fragments with the Taq I or Pst I bands enabled the determination of four alleles. Allelic frequencies were calculated for each allele (Table 1) and the linkage disequilibrium between HLA-A and HLA-G loci was analyzed (Table 2). A strong linkage disequilibrium was observed: allele 1 was associated with HLA-A2, -A3, -A28, and -A29; allele 2 with HLA-A1, -A9, -A10, and -A33; and allele 3 with HLA-A11. Allele 4 was only associated with HLA-A32 (n -- 2), but most of the HLA-A32 cells were associated with allele 3 (n -- 5). Regarding HLA-A19 subtypes other than A29, A32, and A33, we observed two HLA-G gene associations: allele 1 (n -- 1) and allele 2 (n = 6). Thus, HLA-A19 specificity may be associated with all four alleles. For HLA-A28 specificity, one cell
FIGURE 1 HLA-G gene polymorphism RFLP pat- terns. Hybridizations of DNAs digested with Taq I, Pst I, and Bgl II restriction enzymes revealed in each case two polymorphic bands: Taq I, 3.9-1.6 kb; Pst I, 1.9-1.5 kb; and Bgl II, 12-4 kb.
23.1 KbD.-
9.4 Kb e,.
6.5 Kb m,-
q.2 Kb ~- 3.9 Kb
2.3 Kb w,- 2 Kb o"
1.6 Kb 1.9 Kb
xb
~ l J ~ l 2 Kb
• ,e lm, iS Kb
Taq I Pst I Bgl II
208 M. Alizadeh et al.
TABLE 1 HLA-G gene allele frequencies
HLA-G alleles Gene kb AUelic frequency (%)
Allele 1 Taq I 3.9 40 Pst I 1.9 Bgl II 12
Allele 2 Taq I 1.6 36 Pst I 1.5 Bgl II 12
Allele 3 Taq I 1.6 22 Pst I 1.5 Bgl II 4
Allele 4 Taq I 3.9 2 Pst I 1.9 Bgl II 4
was associated with allele 1 whereas the others were found to be related to allele 3 (n = 6).
HLA-G gene polymorphism location. HLA-G gene se- quence analysis showed that, in intron 5, three restric- tion sites were located in a short region of 80 bp (Taq I, 3154; Pst I, 3207; and Taq I, 3230) and that a Bgl II site was located in exon 3 at position 1631. To determine whether the described polymorphism is correlated with a modification of these restriction sites, two fragments corresponding respectively to regions 2633-3541 and 1167-1732 were specifically amplified by PCR using appropriate primers: PL1, PR1, PL2, and PR2 (Fig. 2). PCR products obtained from different polymorphic al- leles were secondarily digested with the informative re-
striction enzymes. In each case, the polymorphic sites were respected, showing that the polymorphism is not generated by a mutation or a deletion in these regions. Considering the location of the HLA-G probe comple- mentary sequence as well as the restriction fragments obtained, the polymorphic sites were deduced to be located in the 3' flanking region of the gene (Fig. 2).
HLA-A region extended haplotypes. In the same way, we analyzed other HLA class-I-related gene polymor- phisms: HLA-92, HLA-80, HLA-21, HLA-90, HLA-F, and HLA-E. Except for HLA-E, we described a poly- morphism for all of the other genes. Informative en- zymes and the number of alleles obtained are described in Table 3. These polymorphisms are illustrated in Fig. 3. HLA-A associations were described for each locus (Table 4): in all of the cases, except for HLA-9.2, a strong linkage disequilibrium with HLA-A was assessed by using the same statistical analysis as that for the HLA-G study (data not shown). Combining HLA-G gene polymorphism with other polymorphisms ob- served enabled the determination of putative extended HLA-A-related haplotypes (Table 5) that have to be confirmed by segregation analysis on an extensive panel of two generation families. In most cases, different ex- tended haplotypes could be determined for the same HLA-A serologically defined specificity. It is worth not- ing that very frequently the telomeric region to HLA-A was conserved for the same specificity (A1, A2, A3, A29, and A10) while the centromeric region involving the HLA-92 locus was different, suggesting a hot spot of recombination between HLA-80 and HLA-92 loci.
TABLE 2 HLA-G/HLA-A allele associations
Expected Observed H L A - G / H L A - A frequencies frequencies
Expected and observed frequencies of HLA-A and HLA-G allele associations are signiicantly different in 71% of the cases, suggesting a linkage disequilibrium between HLA-A and HLA-G loci. NS, not significant.
Polymorphism of the HLA-G Gene 209
I I Ex2
f ! ! I . . , Ex3 E ~ rr.J~ Ex7 E.x8
1631 3154 323~ i 3207
J PL1 PR1 PL2 PR2 1167 1732 2633 3541
l Bgl lt-Taq I-Pst I conserved restdction
sites
PL1 : 5' GAG GAG ACA CC-G AAC ACC 3'
PR1 : 5" CTG CAG CGT CTC CTT CCC 3'
PL2 : 5' TAA GGA GGG AG/K TGG AGG 3"
PR2 : 5" AGC CCC CCC GGG GTG 3'
3 '
l 4700 4800 5100 5600 710(3 13600
Polymorphic site location
I Taq I
I Pst l
I Bgl II
FIGURE 2 Location of the de- tected HLA-G gene polymorphism. HLA-G gene organization and se- quence were established previously. Exons (Ex) are indicated by black bars. The two amplified fragments are shown by double lines; the specific primers used for PCR amplification are indicated by arrows and their se- quences are given below. The num- bers refer to restriction site and primer locations. In the 3' flanking region of the gene, restriction sites are deduced from the RFLP fragment sizes.
HLA-92 / Taq I HLA-F / Hind III
4.3 Kb
3.3 Kb
2.4 Kb
5.4 Kb S.2 Kb
FIGURE 3 HLA class-I-related gene polymorphism: RFLP patterns. Four enzyme-probe combinations are illustrated: the HLA-21-Taq I combination generates seven polymorphic bands, the HLA-92-Taq I combination generates three poly- morphic bands, the HLA-F-Hind III combination generates two polymorphic bands, and the HLA-90-Msp I combination generates four polymorphic bands
HLA-21 I Taq ! HLA-Im I MIp I
8.9 Kb $ Kb
7 Kb
6 Kb
4.2 Kb
3.5 Kb
3.4 Kb
12 Kb
3.5 Kb
3.2 Kb
2.1 Kb
C O N C L U S I O N
The present study describes a genomic polymorphism of HLA-G gene. Since this polymorphism was located in the 3' flanking region of the gene, it may reflect a linkage disequilibrium with a polymorphism in the cod- ing region not evidenced by the enzyme-probe combi- nations used in this study. This hypothesis is currently under study by sequence analysis of HLA-G gene homozygous cells. Moreover, we showed that, taking HLA-G gene polymorphism into account in association with the polymorphism of other class I genes or pseudo- genes in the HLA-A region, putative extended haplo- types may be characterized that discriminate different variants in the same HLA-A serologically defined speci- ficity. Knowledge of these HLA extended haplotypes may be useful for understanding the HLA class I genes and disease associations such as for the selection of do- nor-recipient pairs in bone marrow transplantation.
210
TABLE 3 HLA class-I-related gene alleles
HLA class-I-related genes ÷ kb
Alleles HLA-92 HLA-80 HLA-21 HLA-90 HLA-F
1 Taq l 4.3 Taq I 11.5 TaqI 7 MspI Taq I 4.2
2 TaqI 3-3 T a q l 12 TaqI 7 MspI Taq I 6
3 Taq I 2.4 Taq I 8.9 Msp I Taq I 4.2
4 Taq I 8 Msp I Taq I 4.2
5 Taq I 3.5
6 Taq I 3.4
7 Taq I 8.9 Taq I 3.5
12 Hind III 5.4 Bgl II 10.5
3.5 Hind III 5.2 Bgl II 13.3
3.2 Hind IIl 5.2 Bgl II 10.5
2.8
Each allele was characterized by one or two fragments. Two alleles were described for HLA-80, three alleles for HLA-92 and HLA-F, four alleles for HLA-90, and seven alleles for HLA-21.
TABLE 4 HLA class-I-related gene frequency and HLA-A association
Each line characterizes an extended haplotype including seven polymorphic loci. The number indicates the corresponding allele of each locus as described in Table 2. ND, not determined.
ACKNOWLEDGMENTS The HLA-G-specific clone was kindly provided by Dr. H.T. Orr. This work was supported by I N S E R M (CRE 89-90-91) and the Fondation pour la Recherche M3dicale.
R E F E R E N C E S
1. Koller BH, Geraghty DE, DeMars R, Duvick L, Rich SS, Orr HT: Chromosomal organization of the human major histocompatibility complex class I gene family. J Exp Med 169:469, 1989.
2. Geraghty DE, Koller BH, H a n s e n J A , Orr HT: The HLA class I gene family includes at least six genes and twelve pseudogenes and gene fragments. J Immunol 149:1934, 1992.
3. Ploegh HL, Orr HT, StromingerJL: Major histocompati-
bility antigens: the human (HLA-A, -B, -C) and murine (H-2K, H-2D) class I molecules. Cell 24:287, 1981.
4. Bjorkman PJ, Saper MA, Samraoui B, Bennet WS, Strominger JL, Wilkey DC: The foreign antigen binding site and T cell recognition regions of class I histocompati- bility antigens. Nature 329:512, 1987.
5. Koller BH, Geraghty DE, Shimuzu Y, DeMars R, Orr HT: HLA-E: a novel HLA class I gene expressed in rest- ing T lymphocytes. J Immunol 141:897, 1988.
6. Geraghty DE, Wei X, Or r HT, Koller BH: Human leu- kocyte antigen F (HLA-F): an expressed HLA gene com- posed of a class I coding sequence linked to a novel tran- scribed repeti t ive element. J Exp Med 171:1, 1990.
7. Geraghty DE, KoUer BH, Orr HT: A human major histo- compatibility complex class I gene that encodes a protein with a shortened cytoplasmic fragment. Proc Natl Acad Sci U S A 84:9145, 1987.
212 M. Alizadeh et al.
8. Shimuzu Y, Geraghty DE, Koller BH, Orr HT, De Mars R: Transfer and expression of three cloned human non- HLA-A, -B, -C class I major histocompatibility complex genes in mutant lymphoblastoid cells. Proc Natl Acad Sci USA 85:227, 1988.
9. Ellis SA, Palmer MS, McMichael AJ: Human trophoblast and the chariocarcinoma cell line Bewo express a trun- cated HLA class I molecule. J Immunol 144:731, 1990.
10. Kovats SE, Main EK, Librach C, Subblebine M, Fisher J, De Mars R: A class I antigen HLA-G, expressed in hu- man trophoblasts. Science 248:220, 1990.
11. Houlihan JM, Biro PA, Fergar-Payne A, Simpson KL, Holmes CH: Evidence for the expression of non-HLA-A,
12.
13.
-B, -C class I genes in the human fetal liver. J Immunol 149:668, 1992.
Ishitani A, Geraghty DE: Alternative splicing of HLA-G transcripts yields proteins with primary structures resem- bling both class I and class II antigens. Proc Natl Acad Sci USA 89:3947, 1992.
Alizadeh M, Semana G, Genetet B, Fauchet R: Fast prep- aration of HLA class I specific probes by polymerase chain reaction: applications to genomic polymorphism and RNA transcription studies. In Tsuji K, Aizawa M, Sasasuki T (edts): HLA 1991, Proceedings of the 11th International Histocompatibility Workshop and Confer- ence, vol 2. Oxford Science, 1992, p 203.
