Mol. Hum. Reprod. Advance Access originally published online on January 29, 2004
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Molecular Human Reproduction, Vol. 10, No. 4, pp. 237-246, 2004
© European Society of Human Reproduction and Embryology 2004
Association between HLA-G genotype and risk of pre-eclampsia: a case–control study using family triads
1Department of Clinical Biochemistry, Copenhagen University Hospital, H:S Hvidovre Hospital, 30 Kettegaard Allé, DK-2650 Hvidovre, 2Department of Social Medicine, University of Copenhagen, 3 Blegdamsvej, DK-2200 Copenhagen, 3Department of Epidemiology Research, Danish Epidemiology Science Centre, Statens Serum Institut, 5 Artillerivej, DK-2300 Copenhagen and 4Department of Clinical Biochemistry 3011, Copenhagen University Hospital, Rigshospitalet, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark
5 To whom correspondence should be addressed. e-mail: hviid{at}dadlnet.dk
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ABSTRACT |
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Pre-eclampsia affects 2–7% of all pregnancies with varying severity and is a leading cause of maternal and fetal mortality and morbidity. The aetihology involves almost certainly a combination of genetic predisposition with maternal and fetal contributions and environmental factors. Research points towards pathologies in the placenta as the triggering factor which leads to systemic endothelial dysfunction in the mother, probably as the result of interaction with released placental factors circulating in the maternal blood. One prominent hypothesis regarding the aetiology of pre-eclampsia suggests that it is caused by immune- maladaptation. The MHC class Ib gene, HLA-G, is expressed in the placenta and seems to have immunomodulatory functions. Aberrant HLA-G mRNA and protein expression in pre-eclamptic placentas have been reported. Here, we have investigated detailed HLA-G genotypes in a case–control study of 155 family triads of mother, father and newborn. Among primiparas, an overrepresentation of a homozygous HLA-G genotype was detected in the 40 pre-eclamptic offspring compared to the 70 controls [P = 0.002, Fisher’s exact test; odds ratio 5.57 (95% CI 1.79–17.31)]. Further analyses suggested that the differences between pre-eclamptic cases and controls primarily were accomplished by a different transmission from the father of a 14 bp deletion/insertion polymorphism in exon 8 (P = 0.006, Fisher’s exact test), which previously has been linked to differences in the levels of HLA-G expression and in HLA-G mRNA splicing. The results may also indicate that combined mother–child HLA-G genotypes could influence the risk of developing pre-eclampsia. Overall, the study suggests that HLA-G genotypes and expression might have a significant influence on development of pre-eclampsia.
Key words: Key words: HLA-G/genotype/family triad/polymorphism/pre-eclampsia
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Introduction |
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Pre-eclampsia affects 2–7% of all pregnancies and is a leading cause of maternal and fetal mortality and morbidity worldwide (Douglas and Redman, 1994; Walker 2000). The aetiology and pathogenesis most likely involve a combination of maternal–fetal genetic predisposition and environmental factors (Roberts et al., 1989; Roberts and Cooper, 2001).
One of the leading hypotheses of the pathogenesis of pre-eclampsia is the immunological maladaptation theory, which proposes that the maternal immune system does not adapt adequately to the semi-allogenic fetus. Considering the immunological maladaptation hypothesis, candidate genes for the condition should be sought among genes with immunological functions. Some attention has been given to genes in the human MHC on the short arm of chromosome 6, the HLA. Nearly all studies have investigated the classical HLA class Ia and II gene loci and products. The results are inconclusive (Kilpatrick, 1999). However, the relevance of these gene loci as actual candidate genes can be debated because, in contrast to almost all other tissues, the trophoblasts in the placental–decidual contact zone do not express the classical HLA antigens (HLA-A, -B and -DR) but HLA-C and -E antigens, and most prominently, the non-classical HLA class Ib molecule, HLA-G (Le Bouteiller et al., 1996). Therefore, after the characterization of the HLA-G gene and its expression pattern (Geraghty et al., 1987; Kovats et al., 1990), we found HLA-G as one obvious candidate gene in the MHC region. Few studies have addressed HLA-G genetics in pre-eclampsia, with conflicting results (Humphrey et al., 1995; Bermingham et al., 2000; Aldrich et al., 2000; O’Brien et al., 2001; Carreiras et al., 2002). Harrison et al. (1993) were the first to report a 14 bp sequence polymorphism in the 3' untranslated region (UTR) of exon 8 of the HLA-G gene. A study by Humphrey et al. (1995) did not find an association of the 14 bp deletion polymorphism in exon 8 with pre-eclampsia/eclampsia. Another study investigated the possible importance of the HLA-G*0105N allele, with a frameshift mutation in exon 3, in pre-eclamptic placental samples versus controls but found no such association (Aldrich et al., 2000). A recent study by Carreiras et al. (2002) observed a preferential inheritance of maternal HLA-G*0104 alleles among 25 pre-eclamptic mother–child pairs. In a study of seven placental biopsies from pre-eclamptic pregnancies, an association with a codon 93 (CAT)/14 bp insertion (exon 8) allelic variant of HLA-G was observed (O’Brien et al., 2001). Furthermore, lower levels of HLA-G mRNA, especially the HLA-G3 isoform, were observed in the pre-eclamptic placental biopsies compared to controls. In a detailed study, we found significantly lower levels of HLA-G mRNA and differences in HLA-G mRNA alternative splicing associated with the presence of the 14 bp polymorphism in exon 8 (Hviid et al., 2003). So the 14 bp polymorphism may affect HLA-G mRNA stability.
Several studies of HLA-G mRNA and protein expression in pre-eclamptic placentas and trophoblast compared to control placentas have shown the same trend of an aberrant or absent expression of HLA-G; however, some discrepancy seems to exist (Colbern et al., 1994; Hara et al., 1996; Lim et al., 1997; Goldman-Wohl et al., 2000; O’Brien et al., 2001). The question remains whether this is genetically determined or a consequence of another more fundamental abnormality, or a combination of both?
To clarify these issues, we conducted a case–control study of HLA-G genotypes in 155 family triads of mother, father and the child including 57 families in which the woman had severe pre-eclampsia during the pregnancy and 98 control family triads. We tested the following three a priori hypotheses: (i) HLA-G alleles are differently distributed between cases and controls; (ii) the specific +14 bp/+14 bp or codon 93 (CAT) +14 bp/codon 93 (CAT) +14 bp HLA-G genotypes are more frequent in the fetuses of pre-eclamptic pregnancies than in controls; (iii) the few variations in the amino acid sequence of HLA-G might form the basis for histo-incompatibility between mother and fetus at the contact zone. Furthermore, as pre-eclampsia occurs 2–3 times more often in nulliparas than multiparas, we analysed both the entire data and the data restricted to primipara triads.
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Samples and extraction of genomic DNA
The participants were recruited to the study from two sources: from the Danish National Birth Cohort (DNBC) and from births at a large obstetric department in Copenhagen. The Danish National Birth Cohort is a population-based cohort including 100 000 pregnancies in Denmark recruited from 1997 to 2002. Information on exposures during pregnancy is collected by means of telephone interviews, and blood samples taken in pregnancy and from the umbilical cord at birth are stored in a biobank (Olsen et al., 2001). From the DNBC we identified a total of 63 potential cases who fulfilled the following inclusion criteria: birth of a single live-born child in 1998 or 1999, the pregnancy or birth was registered in the Danish Hospital Discharge Register with a diagnosis of severe pre-eclampsia or eclampsia (ICD-10 codes O14.1 or O15), and for whom blood samples from the mother and the child were stored in the biobank. In Denmark, the diagnosis of severe pre-eclampsia requires a blood pressure >180/110 mmHg and/or the presence of severe subjective symptoms and/or proteinuria of
5 g/l. The potential case mothers were contacted by letter and asked if she and the father of the child would participate in this particular study. From families who agreed to participate, two different kinds of oral buccal cell samples (oral buccal swab material on filter paper and brush) from the father were obtained for analysis together with stored blood samples from the mother and the child. Altogether, 43 of the potential family triads were recruited to the study; the full HLA-G genotyping of all triad members failed in two families, leaving 41 for analyses. For each of the case families, four potential controls were identified matched on the following criteria: same year and place of birth as the case child, same parity, and no diagnosis of hypertension, pre-eclampsia or eclampsia (ICD-10 codes O10-O16) registered in the Danish Hospital Discharge Register for the actual pregnancy or birth. For each case family included in the study, the goal was to recruit two control families, and oral cheek samples were collected as for the cases. A total of 69 control families was finally included; 76 triads were recruited but in seven families full HLA-G genotyping failed. Samples from the mothers were full blood stains on filter paper collected during pregnancy, and samples from the children were umbilical vein blood stains. These samples were kept at –20°C. Furthermore, women who had attended and delivered at a large obstetric hospital department in Copenhagen were also asked for participation in the study. Sixteen family triads with a history of pre-eclampsia during the pregnancy and 29 control family triads with a pregnancy without pre-eclampsia were included. All the cases had a diagnosis of severe pre-eclampsia in the Danish National Hospital Discharge Register and in their medical records. When data from their medical records were collected, the pre-eclampsia diagnoses corresponded to the clinical and paraclinical data. Samples from these family triads were collected either as regular blood samples, blood stains on filter paper, or oral swab samples on filter paper or brushes. Only single pregnancies were included in the study. From the two sources, a total of 57 family triads with severe pre-eclampsia/eclampsia and 98 control family triads were included in the study; a number of these, 40 and 70 respectively, were first pregnancies.
All included mothers and fathers gave written informed consent and the study was approved by the local Research and Ethics Committee.
Genomic DNA was extracted from the peripheral vein blood samples using a commercial kit, DNA Isolation Kit for Mammalian Blood (Roche, Germany), according to the manufacturer’s directions; the method was based on a salting-out procedure (Miller et al., 1988). Genomic DNA was extracted from oral buccal cell swab samples on filter cards and from brushes using a commercial kit (Qiagen, Germany), according to the manufacturer’s directions. Genomic DNA was extracted from blood stains on filter cards using a method described elsewhere (Rudbeck and Dissing, 1998).
PCR amplification of exons 2, 3, 4 and 8 of the HLA-G gene
The following four sets of primers were used to amplify exons 2, 3, 4 and 8 of the HLA-G gene: exon 2: HLAGEX2A (5'-GGGTCGGGCGGGTCTCAA-3') and BHLAGEX2 (5'-TCCGTGGGGCATGGAGGT-3'); exon 3: 5HLGIN2 (5'-CCCAGACCCTCTACCTGGGAGA-3') and GI3/3 (5'GGCCAGGCT GAGAGGTCTACAGGAGATCA-3'); exon 4: HLAGEX4A (5'-CCATGA GAGATGCAAAGTGCT-3') and BHLAGEX4 (5'-TGCTTTCCCTAA CAGACATGAT-3'); exon 8: GE14HLAG (5'-GTGATGGGCTGT TTAAAGTGTCACC-3') and RHG4 (5'-GGAAGGAATGCAGTT CAGCATGA-3'). The PCR and thermocycling conditions were in general as described previously (Hviid et al., 2002). However, for amplification of exon 3, the PCR conditions were: Final volume of 50 µl containing 75 mmol/l Tris–HCl (pH 8.8 at 25°C), 20 mmol/l (NH4)2SO4, 0.01% (v/v) Tween 20, 2.0 mmol/l MgCl2, 0.2 mmol/l of each dNTP, 20 pmol of each primer and 1.5 U of Taq polymerase; thermocycling conditons: initial denaturation at 94°C for 2 min, 36–40 cycles of 94°C for 30 s, 62°C for 90 s, 72°C for 120 s, and a final extension step at 72°C for 10 min. For blood samples, the number of amplifications was 36–38. For DNA extracted from blood stains and oral buccal swab or brush samples, generally 2 µl of DNA extract (blood stains) and 5 µl (swab samples) were used in each PCR set-up and the number of amplifications was 40.
DNA sequencing of exons 2 and 3 of the HLA-G gene
As HLA class I alleles are nomally defined based on polymorphisms in exons 2 and 3 at the DNA level the PCR products of exons 2 and 3 of the HLA-G gene were directly DNA-sequenced using an ABI Prism Big Dye Terminator cycle sequencing kit (Applied Biosystems, USA) and an ABI Prism 310 Genetic Analyzer (Applied Biosystems). The primers used in the direct DNA sequencing reaction were: exon 2, SEKHLAGEX2 (5'-AGATCA CGACCCCCACCTCCAT-3'); and exon 3, SEKHGEX3 (5'-GGTGGG TCCGGGCGAGGGCGAGGCT-3') and in some instances for control sequencing, primers HLAGEX2A and GI3/3.
Genotyping of exons 4 and 8 of the HLA-G gene
For detecting the HLA-G polymorphism at codon 258 which defines the HLA-G allele G*0106, the exon 4 PCR product was incubated with the restriction endonuclease Eco72I (MBI Fermentas), and for verfication of positive samples also subsequently with NspI; the resulting DNA fragments were analysed by electrophoresis on agarose gels (3% NuSieve GTG agarse; FMC) and stained with ethidium bromide, as described previously (Hviid et al., 2002). For control of the procedure, selected samples were DNA-sequenced using the primers HLAGEX4A and BHLAGEX4.
For genotyping of the 14 bp deletion polymorphism in the 3'-UTR in exon 8 of the HLA-G gene, the PCR products of exon 8 were analysed by electrophoresis on agarose gels (4% NuSieve GTG agarose; FMC) and stained with ethidium bromide. For control, some of the PCR products were sequenced.
Statistical analysis
HLA-G genotype frequencies were compared to Hardy–Weinberg expectations using 
2-tests for each of the six groups (mothers, fathers, children, for cases and controls respectively), both for alleles and the 14 bp deletion polymorphism in exon 8 separately, using the HWE linkage utility program from Dr. Jurg Ott (http://linkage.rockefeller.edu/ott/linkutil.htm). For these analyses, the following HLA-G alleles were grouped together: G*010401 and G*010403 in one group, and the rare alleles were in another group (G*010105, G*010108, G*G3d5, G*0101c, G*0101g, G*0101x, G*0103, G*0103x and G*0105N).
HLA-G allele frequencies and HLA-G genotypes and polymorphism frequencies based upon the 14 bp deletion polymorphism in exon 8 or the codon 93 polymorphism of mothers, fathers and children in the pre-eclampsia group and the control group were compared using the 2-test or Fisher’s exact test whichever was appropriate. Furthermore, we investigated the differences in HLA-G histo-incompatible combinations of the mother and her fetus/child between the pre-eclampsia group and the control group.
Allelic association was compared between cases and controls. Taking advantage of the triad data, another type of test was performed. The transmission/disequilibrium test (TDT) statistic was calculated as follows: TDT 2 = (a – b)2/(a + b) (Spielman et al., 1993). The P-value is calculated from a 2-table with 1 df. Here, a is the number of times that the allele of interest is transmitted, and b is the number of times that it is not transmitted.
The analyses were performed on the total data material and replicated in a subgroup restricted to first pregnancies.
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Detected HLA-G alleles, allele frequencies and genotype distributions
Fifty-seven family triads with an episode of severe pre-eclampsia and 98 control family triads were HLA-G-genotyped. HLA-G alleles were defined, whenever possible, according to World Health Organization acknowledged and published alleles. The organization of the HLA-G gene is shown in Figure 1 together with the localization of some of the polymorphisms. The detected HLA-G alleles, based on polymorphisms in exons 2, 3, 4 and 8 of the HLA-G gene, have all been reported previously (Hviid et al., 2002), except two alleles which to our knowledge have never been reported before: a variant of G*010101 which has CAT at codon 93 instead of CAC, and a variant of G*0103 with CAC at codon 188 instead of CAT and has the 14 bp polymorphism deleted in exon 8. We have designated these two alleles G*0101x and G*0103x respectively, although some uncertainty remains whether the original described G*0103 allele actually has CAT or CAC at codon 188 (Kirszenbaum et al., 1999). The overall distribution of alleles in the six groups (mothers, fathers, offspring, pre-eclamptic and controls respectively) is listed in Table I; primipara triads are listed in Table II, and the 14 bp polymorphism in exon 8 in Tables III and IV. HLA-G genotypes in all six groups were in Hardy–Weinberg equilibrium both for the overall distribution of HLA-G alleles and for the 14 bp deletion polymorphism in exon 8 tested separately. All studied triads were tested, and primipara were also tested as a subgroup.
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When the distributions of the HLA-G alleles in the pre-eclampsia and control triads were compared for the entire data (Table I), no obvious differences were observed. However, the G*0105N allele with a one nucelotide deletion mutation in exon 3 was not observed at all in the pre-eclampsia triads but was observed at a very low frequency in the control triads. When the data from the primipara triads were analysed exclusively, the G*010101, and to some extent the G*010401 alleles, were slightly more frequent in the control triads whereas the opposite trend for the alleles G*010102, G*0103 and G*0106 was observed (Table II). The main differences between these two groups of alleles are for G*010101 and G*010401, CAC at codon 93 and the absence of the 14 bp sequence polymorphism in the 3' UTR of exon 8, whereas G*010102 and G*0106 both have CAT at codon 93 and includes the 14 bp sequence.
Association between the presence of the 14 bp sequence polymorphism in exon 8 of the HLA-G gene and pre-eclampsia in primipara triads
In Table III, the results of the genotyping of the 14 bp deletion polymorphism in exon 8 of the HLA-G gene are shown for the entire data. A total of 22.8% of the children in the pre-eclampsia triads carried the +14 bp/+14 bp genotype compared to 11.2% of the control children, resulting in an odds ratio (OR) of 2.34 [95% confidence interval (CI) 0.97–5.64]. The data were, however, too sparse to gain statistical significance. When the primipara triads were analysed (Table IV) the hypothesized differences between the distributions of the 14 bp polymorphism in pre-eclamptic cases and controls emerged, especially concerning the fathers and the offspring. The allele frequency of the +14 bp HLA-G allele in the pre-eclampsia offspring was 52.5%, whereas 31.4% of the control offspring had this allele (P = 0.003; Fisher’s exact test). Regarding the genotype, a total of 30.0% of the pre-eclampsia offspring were +14 bp/+14 bp positive and only 7.1% of the control offspring had that genotype; and the frequency of the –14 bp/–14 bp genotype was 25.0% in the pre-eclampsia offspring but 44.3% in the controls (P = 0.004; 2 = 11.21, df = 2). This resulted in an OR of 5.57 (95% CI 1.79–17.31; P = 0.002) when +14 bp/+14bp homozygotes versus the other genotypes were tested (Fisher’s exact test). The distributions of the 14 bp polymorphism HLA-G alleles in the pre-eclampsia and control fathers were also significantly different with more +14 bp alleles in the pre-eclamptic cases (P = 0.045, Fisher’s exact test).
A trend towards more +14 bp HLA-G alleles in combined mother–child genotypes in cases with pre-eclampsia compared to controls
Since a specific gene involved in pre-eclampsia may have functional significance in both the mother and the fetus, mother–child combinations of the 14 bp deletion polymorphism of the HLA-G gene in primipara cases and controls were analysed (Table V). The +14 bp allele was absent in only 12.5% of all pre-eclampsia mother–child pairs compared with 28.6% of control pairs. Further, four +14 bp alleles were present in 15.0% of pre-eclampsia mother–child pairs compared with 2.9% of control pairs (P = 0.025; 2-test for trend = 5.03, df = 1).
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Significance of the codon 93 (CAT) polymorphism in the pre-eclampsia triads
When the distributions of genotypes of the codon 93 polymorphism in exon 3 (CAC/CAT; His/His) of the HLA-G gene were compared between the pre-eclampsia and control primipara groups, a significant difference between the paternal distributions was observed with 65.0% of the pre-eclampsia fathers being heterozygotes and only 32.9% of the control fathers (P = 0.005;
2 = 10.75, df = 2). The distribution of the primipara pre-eclampsia fathers was actually not in Hardy–Weinberg equilibrium concerning the codon 93 CAC/CAT polymorphism (P = 0.025; 2 = 5.02, df = 1); more heterozygotes were observed than expected. The comparison of the offspring genotype distributions was not significant (P = 0.120; 2 = 4.24, df = 2). However, more homozygotes (CAT/CAT) were observed in the offspring from pre-eclamptic pregnancies compared to controls (17.5% versus 5.7%). These findings are not surprising as a linkage disequilibrium exists between the codon 93 CAT polymorphism and the presence of the 14 bp in exon 8, which in part defines especially the alleles G*010102 and G*0106 that were observed more frequently in the pre-eclampsia triads (Tables I and II). Furthermore, a comparison of the combined CAT,+14 bp/CAT,+14 bp genotype versus other genotypes in the offspring in the cases of primiparity resulted in a P-value of 0.035 (Fisher’s exact test).
Importance of the paternal transmission of the +14 bp HLA-G allele to the offspring in pre-eclampsia triads
In Tables VI and VII, the genotype frequencies of the 14 bp polymorphism in exon 8 of the HLA-G gene within the pre-eclampsia and control triads are listed. TDT analysis was used to compare the frequency of transmitted and non-transmitted +14 bp HLA-G alleles from heterozygous parents using both the pre-eclampsia and control primipara triad data sets (Table VIII). It shall be noted that TDT analysis is not normally performed in a control group; the test is designed so that the parents in the case triads act as controls. However, as control triad data exist in this study because of the case–control design, we thought it reasonable to perform TDT analysis in the control triads as well, as opposite trends of transmission would strengthen results found in the case triads. TDT were performed when parent-of-origin was not considered or when paternal or maternal transmissions were considered separately. In the pre-eclampsia triads no significant results were observed, but a tendency to a more frequent paternal transmission of the +14 bp sequence to the offspring was observed (P = 0.109; 2 = 2.57, df = 1). This is especially interesting because the opposite transmission is observed in the control triads with the transmission of an HLA-G allele with the 14 bp deleted in 20 out of 26 heterozygous fathers (P = 0.006; 2 = 7.54, df = 1). These data can also be analysed in a Fisher’s exact test, which results in P = 0.006. This also holds significance when heterozygous parents from primipara triads were analysed together (P = 0.043; 2 = 4.08, df = 1). Using the entire control triad data set, the TDT test on paternal transmission resulted in a P-value of 0.042 (2 = 4.12, df = 1).
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No association between the transmission of the G*0104 alleles and pre-eclampsia
Because a previous study had reported an association between the G*0104 alleles (with the 14 bp sequence in exon 8 deleted) and parental transmission in pre-eclamptic cases (Carreiras et al., 2002), TDT analysis of this allele was performed. No significant results or trends were observed.
No evidence for HLA-G antigen incompatibility between the pregnant woman and her fetus in pre-eclampsia
To test an HLA-G antigen incompatibility hypothesis in pre-eclampsia, the number of children in the pre-eclampsia triads who carried an HLA-G allele with an amino acid polymorphism inherited from the father and which the pregnant woman did not carry was compared to control triads (Table IX). This was observed in 19.3% of the pre-eclampsia triads and in 13.2% of the control triads for the entire data, which was not significant (P = 0.360; Fisher’s exact test). The results for the primipara were rather similar and non-significant (Table IX). Furthermore, there was no significant difference between the paternal genotypes in the pre-eclampsia triads versus the control triads concerning the numbers of shared HLA-G alleles with amino acid polymorphisms (G*0101, G*0103, G*0104, G*0105N and G*0106) (P = 0.111; 2 = 4.40, df 2; primipara: P = 0.884; 2 = 0.25, df 2). The mean number of shared alleles with identical amino acid sequences of HLA-G between parents was 1.47 in the pre-eclampsia triads and 1.39 in the control triads; for the primipara triads it was 1.53 and 1.49 respectively.
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In this study we found indications for an association between fetal HLA-G genotype and pre-eclampsia in primiparas and significant differences in the paternal transmission of HLA-G alleles. Based on epidemiological and laboratory research, one of the leading hypotheses regarding the aetiology of pre-eclampsia involves a theory of immunological maladaptation to the invading fetal trophoblast and the semi-allogenic fetus. Both HLA-G mRNA and protein expression have in previous studies been shown to be aberrant in pre-eclamptic placentas compared to controls although some discrepancy exists. Therefore, as the trophoblast does not express classical HLA molecules but predominantly an HLA class Ib gene, HLA-G, we investigated the genetics of this candidate gene in cases of pre-eclampsia and in a proper control group. This study of HLA-G genetics and pre-eclampsia with detailed DNA sequence analysis of nearly 500 persons in case and control triad families enabled traditional case–control analysis and case–parent triad statistical analyses.
The reported incidences of pre-eclampsia vary between populations. The variations may be due to genetic differences and other risk factors between populations, but is probably also due to variations in diagnostic criteria. It may be seen that pregnancies in which the mother has had episodes with proteinuria and high blood pressure are erroneously diagnosed as pre-eclamptic. To avoid bias arising from misclassification of cases, we used restrictive inclusion criteria in the case group, and consequently only severe cases of pre-eclampsia were included in the study. Misclassification in the control group is less crucial, but to avoid such misclassification all pregnancies with a diagnosis indicating pre-eclampsia or hypertension in pregnancy were excluded from the potential control group.
Interestingly, homozygosity for the 14 bp polymorphism sequence in the 3'UTR end (exon 8; position 3741; Geraghty et al., 1987) of the HLA-G gene, which is associated with lower HLA-G mRNA and protein levels and unique alternative splicing patterns (Hiby et al., 1999; O’Brien et al., 2001; Rebmann et al., 2001; Hviid et al., 2003), was found in a larger number of pre-eclamptic offspring (30.0%) compared to controls (7.1%), being significant for the primipara subgroup (P = 0.002; OR = 5.57 95% CI 1.79–17.31).
Further analyses using TDT showed that the HLA-G alleles including the 14 bp sequence in exon 8 were preferentially transmitted by heterozygous pre-eclampsia parents (although it did not reach statistical significance) and the opposite was observed in the control parents with the HLA-G alleles with the 14 bp sequence deleted as the allele most often transmitted. When the analysis was divided between heterozygous mothers and fathers, it was shown that this transmission pattern was a consequence of the transmission from the fathers solely (P = 0.006; Fisher’s exact test). The maternal transmissions were random (Table VIIIVIII). In the TDT analyses, the control families were included. This was done to ensure that there was no systematic genotyping error which would give the appearance of an overtransmission. Furthermore, for alleles that confer significant risk, an undertransmission of the risk-associated allele in the control sample should be expected. The fact that there is a significant undertransmission of the +14 bp allele in the controls strengthens the results of this study.
Similar results as described for the 14 bp polymorphism were found for the codon 93 (CAC/CAT) polymorphism, reflecting the linkage disequilibrium between these two polymorphisms. This makes the two alleles, G*010102 and G*0106, together with G*0103 which also includes the 14 bp sequence, the primary pre-eclampsia susceptibility HLA-G alleles. Furthermore, a trend towards a higher number of +14 bp HLA-G alleles combined in mother–child pairs in pre-eclampsia primiparas compared to controls (P = 0.025) was observed. If only the inheritance of a +14 bp allele from the father confers risk as indicated by the TDT, then more mother–child pairs with exclusively +14 bp alleles would not be expected. What seems to confer risk is both the inheritance of a +14 bp allele from the father and a +14 bp/+14 bp genotype of the child. This would imply more mothers with at least one +14 bp allele in the pre-eclampsia group. Table V shows differences between cases and controls regarding two +14 bp alleles or two –14 bp alleles for the mother which indicate that the mother’s HLA-G genotype may also confer risk. However, it cannot be determined whether it just reflects that the chance of having an identical homozygous child (e.g. +14 bp/+14 bp) in a homozygous mother with a heterozygous father is greater than if the mother is heterozygous or whether both the homozygous genotype of the child and the mother add to the mechanisms of pathogenesis (or in reduced risk of pre-eclampsia for the controls)—but apparently only in combination. We found no significant differences between the genotype distributions of the 14 bp HLA-G allele in the pre-eclampsia and control groups regarding the mothers (Tables III and IV).
HLA-G alleles including the 14 bp sequence in exon 8 are associated with lower HLA-G expression than the alleles with the sequence deleted and to differences in patterns of alternative splicing (O’Brien et al., 2001; Rebmann et al., 2001; Hviid et al., 2003). We have found that several alternatively spliced HLA-G mRNA isoforms, including the 14 bp sequence in the 3'UTR end of exon 8 of the HLA-G gene, are expressed at a significantly lower level than the corresponding HLA-G mRNA isoforms with the 14 bp deleted. Characteristic HLA-G mRNA isoform expression patterns were associated with specific HLA-G genotypes and alleles. In alleles with the 14 bp sequence, an additional alternative splicing was observed, with the first 92 bp of exon 8 spliced out. In this regard, the G*010103 allele seems extraordinary, and the total amounts of HLA-G mRNA transcripts may be higher with this allele than with G*010101; however, the consequences of this for protein expression are not clear (Hviid et al., 2003). A low or aberrant HLA-G expression, perhaps reflected in differences in alternative splicing, may have several functional consequences. The protection of the trophoblast cells against natural killer (NK) cell-mediated lysis may be compromised. Low concentrations of HLA-G purified from first trimester placentas, in a mixed lymphocyte reaction, seems to augment the release of Th1-type cytokines and the allocytotoxic T lymphocyte (CTL) response, whereas high concentrations of HLA-G in the cell culture medium results in an increase in Th2-type cytokines and a decrease in Th1 cytokines and of the CTL response (Maejima et al., 1997; Kapasi et al., 2000). A change to a possibly deleterious but predominantly Th1-type cytokine response has been reported in pre-eclampsia (Saito et al., 1999; Rein et al., 2002). These possible effects of low HLA-G expression may affect placentation and also the endothelial cells systemically in the pregnant woman.
We and others have found polymorphisms in the promoter region of the HLA-G gene (Hviid et al., 1999; Ober et al., 2003). However, the relevance of these polymorphisms in the interpretation of the results of this study is not quite clear.
Several attempts have been made to determine risk factors for pre-eclampsia. Epidemiological studies have shown that it occurs more often in nulliparous than multiparous women, and multiparous women changing partner also have a higher risk in a subsequent pregnancy (Walker 2000). Furthermore, a long period of cohabitation preceeding pregnancy protects against developing pre-eclampsia, and there seems to be some evidence that the use of barrier contraceptive methods increases the risk of developing pre-eclampsia (Taylor 1997; Dekker et al., 1998). The risk is elevated if other women in the family (mother, grandmother, sister) have had pre-eclampsia (Cooper et al., 1993). These and other observations have led to speculations regarding an immunological basis or component in pre-eclampsia because previous exposure to foreign or paternal antigens appears to reduce the risk of pre-eclampsia. A study by Lie et al. (1998) concluded that both the mother and the fetus contribute to the risk of pre-eclampsia, the contribution of the fetus being affected by paternal genes. The predominant transmission of the +14 bp sequence HLA-G allele, from the father to the offspring in primipara pre-eclampsia triads, and the opposite predominance of transmission of the 14 bp deleted HLA-G allele, from the father to the offspring in the primipara control triads might be an example of a fetal genetic contribution working through paternal genes.
Histological studies of pre-eclamptic placentas have revealed that the interstitial cytotrophoblast invasion is often weak and that the endovascular invasion is nearly absent (Khong et al., 1986). A report by Blaschitz et al. (1997) showed that endovascular trophoblast cells express HLA-G protein. In general, extravillous cytotrophoblasts express HLA-G and several studies have shown that expression of HLA-G protects against NK-mediated cell lysis (Ponte et al., 1999). It has been reported that clusters of extravillous trophoblasts were insularly devoid of the staining for HLA-G with an anti-HLA-G-specific antibody in pre-eclamptic patients, unlike normal staining in women with an uncomplicated pregnancy (Hara et al., 1996). Furthermore, in primary cell culture experiments of cytotrophoblasts from pre-eclamptic placentas and normal placentas, both HLA-G protein and mRNA were significantly decreased in pre-eclampsia (Lim et al., 1997). Another study, using RNA in-situ hybridization analysis, found that in nine out of 10 pre-eclamptic placentas HLA-G expression was absent or reduced compared to normal placentas (Goldman-Wohl et al., 2000). In contrast to these studies, a recent immunohistochemistry study found no differences between HLA-G expression in the placenta of severe pre-eclampsia and controls (Datema et al., 2003). But in this study only four pre-eclampsia and four control placental biopsies were investigated. However, in summary, all these studies need to be interpreted carefully; differences in HLA-G mRNA and HLA-G protein expression may exist.
We did not find any implication of the HLA-G null allele, HLA-G*0105N, with a one base deletion in exon 3 resulting in a frame-shift, in pre-eclampsia. However, the frequency is low in North-Europeans compared to Africans, for example. This finding is in accordance with a study by Aldrich et al. (2000) of African-Americans with a history of pre-eclampsia and controls. However, that this allele is only found in the control triads is curious, and this allele actually has CAT at codon 93 and the 14 bp sequence in exon 8 as the G*010102 allele. We have no explanation for this finding, and it should be a subject for further investigations. Based on these two studies of G*0105N it can be suggested that the HLA-G2 isoform (and more possibly the soluble G6 isoform/sG2) may be more important in pre-eclampsia, and in pregnancy in general, than the G1 isoforms.
In a recent study of 25 mother–child pairs with pre-eclampsia and 22 control mother–child pairs, six of seven neonates of control mothers inherited HLA-G*0104 from their father whereas five of six infants from pre-eclamptic mothers inherited the G*0104 allele from their mother (P = 0.02) (Carreiras et al., 2002). This observation was not reproduced in this study.
The literature on HLA and pre-eclampsia is also conflicting with respect to the degree of sharing of alleles between couples and thereby the mother and fetus (Taylor, 1997; Kilpatrick, 1999). To test if HLA-G antigen incompatibility might be involved in the aetiology of pre-eclampsia, we tested if the number of fetuses in the pre-eclamptic triads in a greater proportion than the control triads might carry an HLA-G allele with an amino acid polymorphism inherited from the father and which the pregnant woman does not carry. HLA-G, being the major HLA gene expressed by the trophoblasts, must be the obvious HLA candidate to investigate in this regard. No evidence for HLA-G antigen incompatibility, or the opposite, in pre-eclampsia was observed (P = 0.360 for the entire data set and P = 0.158 for primipara; Table IX). For HLA-DR, increased parental compatibility in pre-eclampsia has been reported (Redman et al., 1978; de Brunori et al., 2000).
Humphrey et al. (1995) investigated the presence or absence of the 14 bp sequence in the 3' untranslated region of the HLA-G gene in pre-eclamptic/eclamptic patients and control groups. No relationship between susceptibility to pre-eclampsia or being born of a pre-eclamptic pregnancy and the 14 bp insertion was detected. However, parts of this study were based on linkage analysis of maternally expressed genes, and to examine the effect of fetally expressed HLA-G on pre-eclampsia susceptibility only 13 individuals were examined with no well-defined control group of individuals born of non-pre-eclamptic pregnancies. A few recent genome-wide scans for pre-eclampsia susceptibility loci have not revealed a candidate locus in the MHC region on chromosome 6 (Arngrimsson et al., 1999; Moses et al., 2000; Lachmeijer et al., 2001). But these were only genome scans for maternal susceptibility loci; offspring or fathers were not studied. The present study suggests that HLA-G is a susceptibility locus in the fetus/offspring of pre-eclamptic pregnancies.
Recently, O’Brien et al. (2001) have studied the distribution of the 14 bp polymorphism in exon 8 together with the polymorphism at codon 93 (CAC/CAT) in the beginning of exon 3 in a small number of placentas (n = 7) from cases of mild pre-eclampsia and 11 control placental biopsies. An association of the +14 bp/+14 bp genotype with (mild) pre-eclampsia was found. Furthermore, the authors found a significant association of the CAT/+14 bp allele (or the +14 bp polymorphism alone) and (mild) pre-eclampsia. An overall reduced expression of HLA-G mRNA isoforms was also detected in the +14 bp/+14 bp samples together with an absence of the HLA-G3 mRNA isoform. We have confirmed these observations and furthermore shown rather marked differences in HLA-G mRNA alternative splicing and expression levels associated with different HLA-G alleles, partly based on the 14 bp sequence polymorphism, in first trimester trophoblast samples and sorted cells (Hviid et al., 2003). However, a previous study of 65 pre-eclampsia offspring and 74 control offspring from primigravidae did not observe this association to +14 bp/+14 bp homozygosity in the offspring’s HLA-G genotype from pre-eclamptic pregnancies (Bermingham et al., 2000). Instead, a high frequency of heterozygous –14 bp/+14 bp was observed in the offspring, which actually deviated from Hardy–Weinberg expectations. We have no explanation for the discrepancies in these findings.
Two other possibilities shall briefly be discussed concerning HLA-G and the pathogenesis of pre-eclampsia. First, it has been suggested that the pathology often observed in the placenta in pre-eclampsia could involve viral infection, although this is highly controversial and no proof exists (Arechavaleta-Velasco et al., 2002). Both human cytomegalovirus (HCMV) and herpes simplex virus 1 have been shown to interfere with and reduce HLA-G expression in the placenta (Schust et al., 1996; Fisher et al., 2000). A recent study found that the presence of HCMV sequences in women carrying certain HLA alleles increased the relative risk of developing pre-eclampsia up to 40 times (Carreiras et al., 2002). Second, the fact that the incidence of pre-eclampsia in a subsequent pregnancy seems to be higher in multiparous women changing partner compared to no change in partner, might be explained by the involvement of some kind of specific immunity. It can be hypothesized that HLA-G expression has an immunomodulatory role in controlling a possible maternal alloresponse against the potential semi-allogenic fetal tissue (Carosella et al., 1999). An alloresponse not primarily directed against the trophoblast because of its lack of HLA class Ia and II antigens but, for example, against fetal leukocytes passing from the fetus to the mother. Finally, the results in this study concerning HLA-G polymorphism and pre-eclampsia are significant in the subgroup of primipara triads, but only borderline significant in the entire data set including multiparous women. It can be speculated that the expression of HLA-G in the placenta during the first pregnancy could be involved in a form of priming of the maternal immune response during the confrontation with the fetus. In some pregnancies with low and/or aberrant HLA-G expression, perhaps in regard to patterns of alternative splicing, this interaction might be insufficient during the first pregnancy and be one factor in the development of pre-eclampsia, but in subsequent pregnancies the priming in the first pregnancy may lead to an increased and appropriate response in the mother.
In conclusion, our results are in favour of a role for an immunological contribution, and a genetic component being involved, in pre-eclampsia.
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We wish to thank the participating families who contributed with samples for analysis. We also thank Kenn Schultz-Nielsen and Vanja Orozova-Bekkevold for assistance with extracting the data from the Danish National Birth Cohort. The Danish National Research Foundation has established the Danish Epidemiology Science Centre that initiated and created the Danish National Birth Cohort. The cohort is furthermore a result of a major grant from this foundation. Additional support for the Danish National Birth Cohort is obtained from the Pharmacy Foundation of 1991, the Egmont Foundation, the March of Dimes Birth Defects Foundation, and the Augustinus Foundation. This paticular study was supported by The Pharmacy Foundation of 1991, The Copenhagen Hospital Corporation, The A.P.Møller Foundation for the Advancement of Medical Science, The Plasmid Foundation and The Danish Medical Association Research Fund.
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Submitted on September 7, 2003; resubmitted on November 26, 2003; accepted on December 1, 2003.
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