Maternal and Fetal Human Leukocyte Antigen Class Ia and II Alleles in Severe Preeclampsia and Eclampsia

ORIGINAL ARTICLE Maternal and fetal human leukocyte antigen class Ia and II alleles in severe preeclampsia and eclampsia

J Emmery1, R Hachmon2, CW Pyo2, WC Nelson2, DE Geraghty2, AMN Andersen3, M Melbye4,5 and TVF Hviid1

A line of investigations indicate that genes in the human leukocyte antigen (HLA) complex are involved in a successful acceptance of the semiallogeneic fetus during pregnancy. In this study, associations between specific HLA class Ia (HLA-A and -B) and class II (HLA-DRB1, -DQA1, -DQB1, -DPA1 and -DPB1) alleles and the risk of developing severe preeclampsia/eclampsia were investigated in a detailed and large-scale study. In total, 259 women diagnosed with severe preeclampsia or eclampsia and 260 matched control women with no preeclampsia, together with their neonates, were included in the study. HLA genotyping for mothers and neonates was performed using next-generation sequencing. The HLA-DPB1*04:01:01G allele was significantly more frequent (Pc = 0.044) among women diagnosed with severe preeclampsia/eclampsia compared with controls, and the DQA1*01:02:01G allele frequency was significantly lower (Pc = 0.042) among newborns born by women with severe preeclampsia/ eclampsia compared with controls. In mothers with severe preeclampsia/eclampsia, homozygosity was significantly more common compared with controls at the HLA-DPB1 locus (Pc = 0.0028). Although the current large study shows some positive results, more studies, also with a functional focus, are needed to further clarify a possible role of the classical HLA genes in preeclampsia.

Genes and Immunity (2016) 17, 251–260; doi:10.1038/gene.2016.20; published online 28 April 2016

INTRODUCTION mechanism for the findings has not been clearly elucidated. In Preeclampsia is the leading complication of pregnancy affecting normal conditions, the trophoblast cells do not express HLA class 2–8% of pregnant women in the world.1,2 The immune system is Ia (except HLA-C) and HLA class II that might be part of the believed to play an important role and evidence points toward a mechanisms protecting the fetus from a maternal immune maternal maladaption leading to an immunological reaction response. Studies have reported associations between pregnancy against the semiallogeneic fetus.3,4 Several genes controlling complications and placental trophoblast expression of HLA class II, indicating that the induction of HLA class II molecules might be immune responses are located in the human leukocyte antigen 21 (HLA) region on the short arm of chromosome 6. Genes and involved in fetal rejection responses. However, most studies polymorphisms in the HLA region have been shown to play an have only examined HLA class II allele variation in mothers. The polymorphic HLA class Ia, HLA-A and -B, are widely important role during pregnancy and have been associated with expressed and play an important role in adaptive immune preeclampsia in some previous studies.5 The nonclassical HLA rejection by presenting peptides from intracellular degraded class Ib gene, HLA-G, has drawn much attention because of its proteins on the cell surface for recognition by T cells. A possible expression at the fetomaternal interface together with its association between the HLA-A and -B antigens in women with immunoregulatory functions. Furthermore, associations between preeclampsia and their neonates has been suggested but most polymorphisms in the HLA-G gene and the manifestation of studies have not supported such an association.13–16,20 However, preeclampsia have been demonstrated in some studies, but 6–10 maternal homozygosity at the HLA-A and -B loci has been not all. reported as a possible risk factor for preeclampsia.15,20,22 In contrast to the nonclassical HLA-G gene, the genes coding In addition to studies investigating the HLA allele frequency, the classical HLA class II antigens are very polymorphic and are other studies have focused on the HLA allele sharing between the expressed by antigen-presenting cells. Despite the lack of mother and fetus. Several of these studies have found an expression of HLA class II at the fetomaternal interface, several increased sharing of HLA alleles between the mother and fetus/ studies have examined a possible association between a number father in cases of preeclampsia.15,20,23–25 The results have 11–16 of HLA class II alleles and the occurrence of preeclampsia. implicated several HLA genes, but have so far been inconsistent. Especially, analyses of the HLA-DRB1 gene in relation to However, some findings indicate an increased risk for developing preeclampsia has been investigated but the studies have yielded preeclampsia associated with HLA sharing at the HLA-A, -B or -DR – conflicting results.12,14,17 20 Based upon the results from these loci.23–25 HLA histocompatibility is critical for transplantation; on various studies, it has not yet been possible to link a specific the other hand, HLA histoincompatibility has been associated with HLA-DRB1 allele consistently to the occurrence of preeclampsia. successful pregnancy. It has been speculated that histoincompat- In addition to this, the pathophysiological or immunological ibility between the mother and the fetus might be necessary for

1Centre for Immune Regulation and Reproductive Immunology (CIRRI), Department of Clinical Biochemistry, Zealand University Hospital (Roskilde) and University of Copenhagen, Roskilde, Denmark; 2Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; 3Department of Public Health, University of Copenhagen, Copenhagen, Denmark; 4Department of Epidemiology Research, Statens Serum Institut, Copenhagen, Denmark and 5Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA. Correspondence: Professor TVF Hviid, Centre for Immune Regulation and Reproductive Immunology (CIRRI), Department of Clinical Biochemistry, Zealand University Hospital (Roskilde) and University of Copenhagen, 7-13 Køgevej, Roskilde DK-4000, Denmark. E-mail: [email protected] Received 23 January 2016; revised 25 March 2016; accepted 29 March 2016; published online 28 April 2016 HLA class Ia and II in severe preeclampsia J Emmery et al 252 initiation of maternal immunosuppressive networks at the eclampsia and is considered as a manifestation of severe fetomaternal interface that might prevent fetal rejection and preeclampsia. The results are listed in Tables 1–9. facilitate cytotrophoblastic invasion of the maternal spiral In Tables 1–7, the allele frequencies in the seven examined arteries.24 genes for both mothers and neonates are listed. There was no In an attempt to clarify the discrepancies between the previous significant difference in the distributions of HLA-A and -B alleles and mostly modest-sized studies, the current study, which to our between the severe preeclampsia group and the control group knowledge is the largest and most extensive study, was (Tables 1 and 2). Concerning the distribution of HLA-DQA1 alleles, performed. The objective was to investigate whether specific the frequency in newborns born by women diagnosed with HLA class Ia and II alleles are more common in women who severe preeclampsia was 20.9%, in newborns by women develop severe preeclampsia and their neonates as compared diagnosed with eclampsia 18.5% and in newborns born by control with controls. In addition to this, the effect of sharing allele groups women 28.5%. Thus, the DQA1*01:02:01G allele was significantly between mother and fetus at each of the HLA genes (HLA-A, -B, lower among newborns with severe preeclampsia/eclampsia -DPA1, -DPB1, -DQA1, -DQB1 and -DRB1) was investigated compared with controls (Pc = 0.042) (Table 4). Concerning the as well the total sharing of allele groups across all seven genes HLA class II genes, the frequency of the HLA-DPB1*04:01:01G allele combined. The impact of maternal and fetal HLA homozygosity in eclamptic women was 46.7%, in women with severe on the occurrence of severe preeclampsia/eclampsia was also preeclampsia 52.7% and in the control women 42.3%. Thus, the investigated. HLA-DPB1*04:01:01G allele was significantly more frequent among women diagnosed with severe preeclampsia/eclampsia compared with the control group (Pc = 0.044) (Table 7). The allele distribution RESULTS for the HLA-DPA1, -DQB1 and -DRB1 genes did not show any Distribution of HLA alleles significant differences in neither mothers nor neonates between Severe preeclampsia and eclampsia were combined into one the control group and the group diagnosed with severe category in the statistical analyses, as preeclampsia often precedes preeclampsia/eclampsia (Tables 3, 5 and 7).

Table 1. The human leukocyte antigen (HLA)-A allele distribution in control newborns (n = 243) and their mothers (n = 259), and in newborns (n = 227) and their mothers (n = 257) diagnosed with severe preeclampsia or eclampsia

HLA-A allele Newborn* Mother**

Control Severe Eclampsia Control Severe Eclampsia preeclampsia preeclampsia

n % n % n % n % n % n %

A*01:01:01G 89 18.3 66 16.5 13 24.1 93 18.0 85 18.7 12 20.0 A*02:01:01G 166 34.2 139 34.8 16 29.6 145 28.0 162 35.7 24 40.0 A*02:02 0 0.0 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 A*02:03:01G 1 0.2 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 A*02:05:01G 3 0.6 0 0.0 0 0.0 6 1.2 3 0.7 0 0.0 A*02:121 0 0.0 0 0.0 0 0.0 0 0.0 1 0.2 0 0.0 A*02:152 0 0.0 0 0.0 0 0.0 0 0.0 1 0.2 0 0.0 A*03:01:01G 84 17.3 49 12.3 11 20.4 85 16.4 60 13.2 6 10.0 A*11:01:01G 23 4.7 19 4.8 1 1.9 25 4.8 14 3.1 2 3.3 A*23:01:01G 8 1.6 8 2.0 0 0.0 7 1.4 8 1.8 1 1.7 A*24:02:01G 32 6.6 32 8.0 4 7.4 44 8.5 27 5.9 4 6.7 A*24:03:01G 1 0.2 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 A*25:01:01G 7 1.4 12 30 1 1.9 13 2.5 15 3.3 1 1.7 A*26:01:01G 8 1.6 8 20 1 1.9 8 1.5 7 1.5 1 1.7 A*26:08 1 0.2 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 A*29:01:01G 2 0.4 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 A*29:02:01G 7 1.4 9 2.3 1 1.9 15 2.9 13 2.9 1 1.7 A*30:01:01G 0 0.0 5 1.3 0 0.0 3 0.6 4 0.9 1 1.7 A*30:02:01G 1 0.2 2 0.5 1 1.9 2 0.4 2 0.4 1 1.7 A*30:04:01 3 0.6 0 0.0 0 0.0 2 0.4 2 0.4 0 0.0 A*31:01:02G 10 2.1 14 3.5 1 1.9 13 2.5 16 3.5 1 1.7 A*32:01:01G 12 2.5 18 4.5 2 3.7 19 3.7 11 2.4 3 5.0 A*33:01:01G 2 0.4 1 0.3 0 0.0 5 1.0 1 0.2 0 0.0 A*33:03:01G 1 0.2 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 A*34:02:01 0 0.0 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 A*36:01 0 0.0 1 0.3 0 0.0 0 0.0 0 0.0 0 0.0 A*66:01:01G 2 0.4 0 0.0 0 0.0 2 0.4 1 0.2 0 0.0 A*68:01:01G 7 1.4 2 0.5 0 0.0 8 1.5 3 0.7 0 0.0 A*68:01:02G 15 3.1 11 2.8 1 1.9 16 3.1 13 2.9 0 0.0 A*68:02:01G 1 0.2 3 0.8 1 1.9 2 0.4 4 0.9 2 3.3 A*74:03 0 0.0 1 0.3 0 0.0 0 0.0 1 0.2 0 0.0 Total 486 100.0 400 100.0 54 100.0 518 100.0 454 100.0 60 100.0 *P = 0.4948; χ2 = 12.40; d.f. = 13. **P = 0.1374; χ2 = 18.56; d.f. = 13. For the χ2 test, the alleles were grouped in main allele types A*01, *02, *03, *11, *23, *24, *25, *26, *29, *30 and *31; *32, A*68 and *74 were grouped together; and the remaining alleles were grouped together.

Table 2. The human leukocyte antigen (HLA)-B allele distribution in control newborns (n = 241) and their mothers (n = 259), and in newborns (n = 224) and their mothers (n = 255) diagnosed with severe preeclampsia or eclampsia

HLA-B allele Newborn* Mother**

Control Severe Eclampsia Control Severe Eclampsia preeclampsia preeclampsia

n % n % n % n % n % n%

B*07:02:01G 74 15.4 57 14.5 11 20.4 74 14.3 73 16.2 6 10.0 B*07:05:01G 2 0.4 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 B*08:01:01G 50 10.4 54 13.7 8 14.8 66 12.7 65 14.4 7 11.7 B*13:01:01G 1 0.2 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 B*13:02:01G 3 0.6 9 2.3 1 1.9 10 1.9 9 2.0 2 3.3 B*14:01:01 1 0.2 4 1.0 0 0.0 1 0.2 3 0.7 0 0.0 B*14:02:01 4 0.8 6 1.5 0 0.0 8 1.5 7 1.6 1 1.7 B*15:01:01G 48 10.0 33 8.4 8 14.8 40 7.7 36 8.0 9 15.0 B*15:07:01G 0 0.0 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 B*15:08 0 0.0 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 B*15:17:01G 0 0.0 0 0.0 0 0.0 1 0.2 1 0.2 0 0.0 B*15:18:01G 1 0.2 1 0.3 0 0.0 0 0.0 0 0.0 0 0.0 B*15:24 0 0.0 0 0.0 0 0.0 0 0.0 1 0.2 0 0.0 B*15:78:01 0 0.0 1 0.3 0 0.0 0 0.0 1 0.2 0 0.0 B*18:01:01G 19 3.9 14 3.6 2 3.7 17 3.3 20 4.4 2 3.3 B*18:15 0 0.0 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 B*27:02:01 2 0.4 2 0.5 0 0.0 2 0.4 1 0.2 0 0.0 B*27:03 0 0.0 1 0.3 0 0.0 0 0.0 1 0.2 0 0.0 B*27:04:01G 0 0.0 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 B*27:05:02G 18 3.7 15 3.8 0 0.0 20 3.9 18 4.0 0 0.0 B*27:05:05 1 0.2 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 B*27:10 1 0.2 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 B*27:14 1 0.2 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 B*35:01:01G 25 5.2 27 6.9 2 3.7 34 6.6 26 5.8 2 3.3 B*35:02:01G 3 0.6 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 B*35:03:01G 4 0.8 3 0.8 0 0.0 6 1.2 2 0.4 0 0.0 B*35:08:01 2 0.4 2 0.5 0 0.0 1 0.2 3 0.7 0 0.0 B*37:01:01G 11 2.3 6 1.5 1 1.9 9 1.7 12 2.7 1 1.7 B*38:01:01 3 0.6 3 0.8 0 0.0 4 0.8 6 1.3 0 0.0 B*38:02:01G 1 0.2 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 B*39:01:01G 4 0.8 7 1.8 0 0.0 3 0.6 5 1.1 0 0.0 B*39:06:02 1 0.2 2 0.5 0 0.0 5 1.0 5 1.1 0 0.0 B*39:09 1 0.2 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 B*39:79 1 0.2 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 B*40:01:01G 50 10.4 29 7.4 3 5.6 46 8.9 31 6.9 6 10.0 B*40:02:01G 4 0.8 5 1.3 0 0.0 5 1.0 4 0.9 2 3.3 B*40:03 0 0.0 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 B*41:01 2 0.4 2 0.5 0 0.0 4 0.8 1 0.2 0 0.0 B*41:02:01 4 0.8 0 0.0 0 0.0 2 0.4 0 0.0 1 1.7 B*42:01:01 3 0.6 2 0.5 1 1.9 0 0.0 1 0.2 0 0.0 B*44:02:01G 55 11.4 41 10.4 9 16.7 49 9.5 39 8.7 9 15.0 B*44:03:01G 13 2.7 19 4.8 0 0.0 19 3.7 24 5.3 0 0.0 B*44:04 1 0.2 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 B*44:05:01 0 0.0 2 0.5 0 0.0 3 0.6 1 0.2 0 0.0 B*44:06 0 0.0 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 B*45:01:01G 4 0.8 1 0.3 1 1.9 7 1.4 4 0.9 1 1.7 B*47:01:01G 2 0.4 1 0.3 0 0.0 2 0.4 3 0.7 0 0.0 B*48:01:01G 0 0.0 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 B*49:01:01G 6 1.2 6 1.5 0 0.0 8 1.5 5 1.1 0 0.0 B*50:01:01 3 0.6 1 0.3 0 0.0 2 0.4 2 0.4 0 0.0 B*51:01:01G 20 4.1 12 3.0 2 3.7 25 4.8 15 3.3 2 3.3 B*51:05 0 0.0 1 0.3 0 0.0 0 0.0 0 0.0 1 1.7 B*51:08 1 0.2 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 B*52:01:01G 4 0.8 4 1.0 0 0.0 5 1.0 2 0.4 0 0.0 B*53:01:01 0 0.0 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 B*54:01:01G 2 0.4 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 B*55:01:01G 8 1.7 6 1.5 2 3.7 6 1.2 5 1.1 2 3.3 B*56:01:01G 4 0.8 2 0.5 0 0.0 4 0.8 0 0.0 0 0.0 B*57:01:01G 11 2.3 11 2.8 3 5.6 12 2.3 14 3.1 6 10.0 B*57:02:01 1 0.2 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 B*58:01:01G 1 0.2 1 0.3 0 0.0 1 0.2 3 0.7 0 0.0 B*59:01:01G 0 0.0 1 0.3 0 0.0 0 0.0 0 0.0 0 0.0 B*67:01:01 1 0.2 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 B*78:02:01 0 0.0 0 0.0 0 0.0 0 0.0 1 0.2 0 0.0 Total 482 100.0 394 100.0 54 100.0 518 100.0 450 100.0 60 100.0 *P = 0.8489; χ2 = 9.519; d.f. = 15. **P = 0.7782; χ2 = 10.63; d.f. = 15. For the χ2 test, the alleles were grouped in the main allele types B*07, *08, *14, *15, *18, *27, *35, *37, *40, *44, *49, *51 and *55; *57, B*38 and *39 were grouped together; and the remaining alleles were grouped together.

Table 3. The human leukocyte antigen (HLA)-DRB1 allele distribution in control newborns (n = 244) and their mothers (n = 260), and in newborns (n = 229) and their mothers (n = 256) diagnosed with severe preeclampsia or eclampsia

HLA-DRB1 allele Newborn* Mother**

Control Severe Eclampsia Control Severe Eclampsia preeclampsia preeclampsia

n % n % n % n % n % n %

DRB1*01:01:01G 42 8.6 31 7.7 8 14.8 43 8.3 40 8.8 7 11.7 DRB1*01:02:01 1 0.2 3 0.7 0 0.0 2 0.4 3 0.7 0 0.0 DRB1*01:03 1 0.2 5 1.2 0 0.0 2 0.4 4 0.9 0 0.0 DRB1*03:01:01G 54 11.1 56 13.9 7 13.0 81 15.6 73 16.2 7 11.7 DRB1*03:04:01 1 0.2 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 DRB1*04:01:01 61 12.5 61 15.1 10 18.5 57 11.0 62 13.7 10 16.7 DRB1*04:02:01 2 0.4 0 0.0 0 0.0 5 1.0 4 0.9 0 0.0 DRB1*04:03:01 3 0.6 1 0.2 0 0.0 3 0.6 0 0.0 0 0.0 DRB1*04:04:01 13 2.7 13 3.2 2 3.7 20 3.8 17 3.8 2 3.3 DRB1*04:05:01 4 0.8 2 0.5 0 0.0 1 0.2 1 0.2 0 0.0 DRB1*04:07:01G 5 1.0 1 0.2 0 0.0 6 1.2 1 0.2 2 3.3 DRB1*04:08:01 3 0.6 2 0.5 0 0.0 6 1.2 4 0.9 0 0.0 DRB1*07:01:01GG 40 8.2 51 12.6 5 9.3 44 8.5 56 12.4 5 8.3 DRB1*07:14 0 0.0 0 0.0 0 0.0 0 0.0 1 0.2 0 0.0 DRB1*08:01:01G 15 3.1 12 3.0 3 5.6 14 2.7 13 2.9 3 5.0 DRB1*08:02:01 0 0.0 0 0.0 0 0.0 2 0.4 0 0.0 0 0.0 DRB1*08:03:02 2 0.4 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 DRB1*08:04:01 2 0.4 1 0.2 0 0.0 1 0.2 2 0.4 0 0.0 DRB1*08:10 0 0.0 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 DRB1*09:01:02 6 1.2 6 1.5 0 0.0 8 1.5 8 1.8 0 0.0 DRB1*10:01:01 3 0.6 1 0.2 0 0.0 5 1.0 2 0.4 1 1.7 DRB1*11:01:01G 17 3.5 16 4.0 2 3.7 15 2.9 11 2.4 2 3.3 DRB1*11:01:02 0 0.0 1 0.2 0 0.0 0 0.0 0 0.0 0 0.0 DRB1*11:02:01 4 0.8 1 0.2 0 0.0 6 1.2 0 0.0 0 0.0 DRB1*11:03 2 0.4 0 0.0 0 0.0 1 0.2 1 0.2 0 0.0 DRB1*11:04:01 8 1.6 4 1.0 0 0.0 9 1.7 3 0.7 1 1.7 DRB1*11:11:01G 0 0.0 0 0.0 0 0.0 0 0.0 1 0.2 0 0.0 DRB1*12:01:01G 11 2.3 10 2.5 1 1.9 12 2.3 10 2.2 0 0.0 DRB1*12:02:01 1 0.2 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 DRB1*13:01:01G 29 5.9 26 6.4 2 3.7 28 5.4 24 5.3 5 8.3 DRB1*13:02:01 39 8.0 23 5.7 2 3.7 30 5.8 19 4.2 2 3.3 DRB1*13:03:01 2 0.4 3 0.7 0 0.0 3 0.6 5 1.1 1 1.7 DRB1*13:67 1 0.2 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 DRB1*14:01:01G 11 2.3 7 1.7 4 7.4 13 2.5 8 1.8 3 5.0 DRB1*14:04 0 0.0 1 0.2 0 0.0 0 0.0 0 0.0 1 1.7 DRB1*14:05:01 0 0.0 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 DRB1*15:01:01G 91 18.6 57 14.1 6 11.1 87 16.7 72 15.9 7 11.7 DRB1*15:01:02 1 0.2 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 DRB1*15:02:01 4 0.8 5 1.2 0 0.0 6 1.2 2 0.4 0 0.0 DRB1*15:03:01G 1 0.2 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 DRB1*16:01:01 4 0.8 3 0.7 2 3.7 3 0.6 2 0.4 1 1.7 DRB1*16:02:01 3 0.6 1 0.2 0 0.0 3 0.6 0 0.0 0 0.0 DRB1*16:09 1 0.2 0 0.0 0 0.0 0 0.0 3 0.7 0 0.0 Total 488 100.0 404 100.0 54 100.0 520 100.0 452 100.0 60 100.0 *P = 0.4101; χ2 = 11.40; d.f. = 11. **P = 0.6888; χ2 = 8.271; d.f. = 11. For the χ2 test, the alleles were grouped in the main allele types DRB1*01,*03, 04, *07, *08, *11, *12, *13, *14, *15 and *16, whereas DRB1*09 and *10 were grouped together.

HLA antigen sharing between mother and fetus two antigens. However, when corrected for multiple testing The distribution of maternal–fetal HLA sharing at each of the (Bonferroni–Holm) the difference did not reach statistical sig- fi fi seven HLA genes examined, along with the complete HLA sharing ni cance (Pc = 0.21). No statistical signi cant association between across all seven genes, is shown in Table 8. The mother donates HLA sharing and the occurrence of severe preeclampsia/eclampsia one allele to her neonate so there must be at least one shared was observed for the other HLA loci. On comparing sharing across allele for each HLA gene. Looking at the maternal–fetal HLA all seven HLA genes, a general decrease in OR for preeclampsia sharing for each HLA locus separately, a significant difference was was observed. The difference between controls and severe observed at the HLA-DQB1 locus, where the odds ratio (OR) for preeclamptic/eclamptic cases showed a slight trend toward preeclampsia/eclampsia sharing both antigens was 0.57 (95% significance when sharing 10 and 11 antigens, with an OR of confidence interval (CI): 0.35–0.94; P=0.03) compared with 0.38 (95% CI: 0.17–0.84; Pc = 0.07) and 0.38 (95% CI: 0.15–0.94; controls, indicating a reduced risk for preeclampsia when sharing Pc = 0.14) respectively.

Table 4. The human leukocyte antigen (HLA)-DQA1 allele distribution in control newborns (n = 244) and their mothers (n = 260), and in newborns (n = 230) and their mothers (n = 256) diagnosed with severe preeclampsia or eclampsia

HLA-DQA1 allele Newborn* Mother**

Control Severe Eclampsia Control Severe Eclampsia preeclampsia preeclampsia

n % n % n % n % n % n %

DQA1*01:01:01G 58 11.9 50 12.3 11 20.4 66 12.7 57 12.6 11 18.3 DQA1*01:02:01Ga 139 28.5 85 20.9 10 18.5 122 23.5 95 21.0 10 16.7 DQA1*01:03:01G 34 7.0 31 7.6 2 3.7 34 6.5 26 5.8 5 8.3 DQA1*02:01 39 8.0 51 12.6 5 9.3 44 8.5 57 12.6 5 8.3 DQA1*03:01:01G 99 20.3 86 21.2 12 22.2 105 20.2 98 21.7 14 23.3 DQA1*04:01:01G 16 3.3 12 3.0 3 5.6 17 3.3 13 2.9 3 5.0 DQA1*05:01:01G 101 20.7 91 22.4 11 20.4 129 24.8 106 23.5 12 20.0 DQA1*06:01:01G 2 0.4 0 0.0 0 0.0 3 0.6 0 0.0 0 0.0 Total 488 100.0 406 100.0 54 100.0 520 100.0 452 100.0 60 100.0 *P = 0.1524; χ2 = 6.704; d.f. = 4. **P = 0.2961; χ2 = 4.916; d.f. = 4. For the χ2 test, the alleles were grouped in main allele types, whereas DQA1*04 and *06 were 2 a grouped together for the χ test. Newborn: controls vs severe preeclampsia/eclampsia; main allele groups as described: Pc = 0.29; all eight alleles: Pc = 0.042.

Table 5. The human leukocyte antigen (HLA)-DQB1 allele distribution in control newborns (n = 244) and their mothers (n = 259), and in newborns (n = 230) and their mothers (n = 257) diagnosed with severe preeclampsia or eclampsia

HLA-DQB1 allele Newborn* Mother**

Control Severe Eclampsia Control Severe Eclampsia preeclampsia preeclampsia

n % n % n % n % n % n %

DQB1*02:01:01G 84 17.2 91 22.4 10 18.5 114 22.0 114 25.1 8 13.3 DQB1*03:01:01G 77 15.8 64 15.8 8 14.8 81 15.6 58 12.8 13 21.7 DQB1*03:02:01G 61 12.5 49 12.1 8 14.8 64 12.4 64 14.1 6 10.0 DQB1*03:03:02G 16 3.3 22 5.4 2 3.7 20 3.9 25 5.5 4 6.7 DQB1*03:04 2 0.4 0 0.0 0 0.0 1 0.2 1 0.2 0 0.0 DQB1*03:05:01 0 0.0 1 0.2 0 0.0 0 0.0 0 0.0 0 0.0 DQB1*04:01:01G 0 0.0 1 0.2 0 0.0 0 0.0 0 0.0 0 0.0 DQB1*04:02:01 16 3.3 12 3.0 3 5.6 16 3.1 13 2.9 3 5.0 DQB1*05:01:01G 48 9.8 41 10.1 8 14.8 51 9.8 50 11.0 8 13.3 DQB1*05:02:01G 6 1.2 3 0.7 2 3.7 5 1.0 4 0.9 1 1.7 DQB1*05:03:01G 11 2.3 8 2.0 3 5.6 15 2.9 7 1.5 3 5.0 DQB1*06:01:01G 5 1.0 5 1.2 0 0.0 5 1.0 2 0.4 0 0.0 DQB1*06:02:01G 93 19.1 58 14.3 6 11.1 88 17.0 72 15.9 7 11.7 DQB1*06:03:01G 30 6.1 28 6.9 2 3.7 28 5.4 26 5.7 5 8.3 DQB1*06:04:01G 38 7.8 22 5.4 2 3.7 29 5.6 18 4.0 2 3.3 DQB1*06:09 1 0.2 1 0.2 0 0.0 1 0.2 0 0.0 0 0.0 Total 488 100.0 406 100.0 54 100.0 518 100.0 454 100.0 60 100.0 *P = 0.3146; χ2 = 9.337; d.f. = 8. **P = 0.8028; χ2 = 1.633; d.f. = 4. For the χ2 test, the alleles were grouped in the main allele types DQB1*02, *03, *04, *05 and *06.

Increased HLA homozygosity associated with severe with eclampsia and 66.5% of the control women were homo- preeclampsia/eclampsia zygous; however, this was not statistically significant (Pc = 0.086). The association between maternal and fetal HLA homozygosity/ In addition, at the HLA-DPB1 locus a clearly significant difference heterozygosity and preeclampsia/eclampsia was investigated for between controls and severe preeclampsia/eclampsia was each of the seven HLA genes (Table 9). No significant association observed, where 32.7% of women with preeclampsia, 26.7% of was found between fetal HLA homozygosity/heterozygosity women with eclampsia and 18.1% of control women were and severe preeclampsia/eclampsia compared with controls. homozygotes (Pc = 0.0028). The distribution at the HLA-B, -DQA1, fi Homozygosity was more common in cases compared with -DQB1 and -DRB1 alleles did not differ signi cantly among controls at the HLA-A locus, where 20.7% of women with severe preeclamptic women and controls. preeclampsia, 23.3% of women with eclampsia and 13.9% of control women were homozygotes. This difference was, however, Logistic regression analyses not statistically significant (Pc = 0.086). Concerning the HLA-DPA1 In logistic regression analyses both maternal HLA-DPB1 homo- locus, 80% of women with severe preeclampsia, 74.4% of women zygosity and fetal HLA-DQA1 homozygosity were significantly

Table 6. The human leukocyte antigen (HLA)-DPA1 allele distribution in control newborns (n = 244) and their mothers (n = 260), and in newborns (n = 230) and their mothers (n = 257) diagnosed with severe preeclampsia or eclampsia

HLA-DPA1 allele Newborn* Mother**

Control Severe Eclampsia Control Severe Eclampsia preeclampsia preeclampsia

n % n % n % n % n % n %

DPA1*01:03:01G 416 85.2 350 86.2 47 87.0 433 83.3 396 87.2 54 90.0 DPA1*01:04 2 0.4 0 0.0 0 0.0 3 0.6 1 0.2 0 0.0 DPA1*01:05 1 0.2 1 0.2 0 0.0 1 0.2 0 0.0 0 0.0 DPA1*01:06:01 0 0.0 1 0.2 0 0.0 0 0.0 0 0.0 0 0.0 DPA1*02:01:01 35 7.2 22 5.4 3 5.6 36 6.9 23 5.1 2 3.3 DPA1*02:01:02 19 3.9 18 4.4 2 3.7 27 5.2 24 5.3 2 3.3 DPA1*02:01:04 0 0.0 0 0.0 0 0.0 2 0.4 0 0.0 0 0.0 DPA1*02:02:01 1 0.2 1 0.2 1 1.9 2 0.4 0 0.0 0 0.0 DPA1*02:02:02 14 2.9 11 2.7 1 1.9 16 3.1 9 2.0 2 3.3 DPA1*03:01 0 0.0 2 0.5 0 0.0 0 0.0 1 0.2 0 0.0 Total 488 100.0 406 100 54 100.0 520 100.0 454 100.0 60 100.0 *P = 0.6943; χ2 = 0.1544; d.f. = 1. **P = 0.0728; χ2 = 3.219; d.f. = 1. For the χ2 test, all the DPA1*01 alleles were grouped together, and all DPA1*02 and *03 sub- alleles were grouped together.

Table 7. The human leukocyte antigen (HLA)-DPB1 allele distribution in control newborns (n = 244) and their mothers (n = 260), and in newborns (n = 230) and their mothers (n = 256) diagnosed with severe preeclampsia or eclampsia

HLA-DPB1 allele Newborn* Mother**

Control Severe Eclampsia Control Severe Eclampsia preeclampsia preeclampsia

n % n % n % n % n % n %

DPB1*01:01:01 23 4.7 20 4.9 2 3.7 32 6.2 25 5.5 2 3.3 DPB1*02:01:02G 51 10.5 45 11.1 7 13.0 61 11.7 49 10.8 6 10.0 DPB1*02:01:04 0 0.0 1 0.2 0 0.0 0 0.0 1 0.2 0 0.0 DPB1*02:02 3 0.6 1 0.2 1 1.9 5 1.0 2 0.4 1 1.7 DPB1*03:01:01G 68 13.9 48 11.8 7 13.0 69 13.3 44 9.7 8 13.3 DPB1*04:01:01Ga 228 46.7 206 50.7 21 38.9 220 42.3 238 52.7 28 46.7 DPB1*04:02:01G 57 11.7 53 13.1 8 14.8 58 11.2 52 11.5 9 15.0 DPB1*05:01:01G 13 2.7 9 2.2 1 1.9 16 3.1 10 2.2 2 3.3 DPB1*06:01 7 1.4 5 1.2 3 5.6 9 1.7 4 0.9 2 3.3 DPB1*08:01 7 1.4 3 0.7 1 1.9 6 1.2 2 0.4 1 1.7 DPB1*09:01:01 5 1.0 2 0.5 0 0.0 3 0.6 4 0.9 0 0.0 DPB1*11:01:01 5 1.0 5 1.2 1 1.9 11 2.1 6 1.3 0 0.0 DPB1*13:01:01G 8 1.6 4 1.0 0 0.0 8 1.5 5 1.1 1 1.7 DPB1*14:01 0 0.0 0 0.0 0 0.0 1 0.2 0 0.0 0 0.0 DPB1*15:01 4 0.8 0 0.0 0 0.0 6 1.2 3 0.7 0 0.0 DPB1*16:01 2 0.4 0 0.0 0 0.0 4 0.8 2 0.4 0 0.0 DPB1*17:01:01G 5 1.0 3 0.7 1 1.9 6 1.2 3 0.7 0 0.0 DPB1*19:01:01G 1 0.2 1 0.2 1 1.9 1 0.2 0 0.0 0 0.0 DPB1*20:01:01 0 0.0 0 0.0 0 0.0 3 0.6 1 0.2 0 0.0 DPB1*23:01:01G 1 0.2 0 0.0 0 0.0 1 0.2 1 0.2 0 0.0 Total 488 100.0 406 100.0 54 100.0 520 100.0 452 100.0 60 100.0 *P = 0.5310; χ2 = 7.052; d.f. = 8. **P = 0.0751; χ2 = 14.26; d.f. = 8. For the χ2 test, the alleles were grouped in the main allele types DPB1*01, *02, *03, *04, *05, *06 and *11; DPB1*08 and *09 were grouped together; and the remaining alleles were grouped together. aMothers: controls vs severe preeclampsia/eclampsia; main allele groups as described: Pc = 0.0072; all 20 alleles: Pc = 0.044.

correlated to occurrence of preeclampsia after adjustment for habits (P = 0.005; OR = 0.57; CI: 0.384–0.84), whereas maternal body mass index and smoking habits (P = 0.001; OR = 2.38; CI: HLA-DPB1 heterozygosity did not show any significant correlation 1.41–4.02 and P = 0.013; OR = 0.38; CI: 0.18–0.81). Fetal HLA-DQA1 (P = 0.767; OR = 1.07; CI: 0.70–1.62). As expected, body mass index heterozygosity was also significantly correlated to occurrence of and smoking habits were statistically significant predictors for preeclampsia, when adjusted for body mass index and smoking occurrence of preeclampsia.

Table 8. Human leukocyte antigen (HLA) sharing across HLA class Ia and HLA class II between mothers and neonates in women who were diagnosed with preeclampsia or eclampsia compared with healthy women

HLA antigen Concordant HLA antigens Control Severe Eclampsia Odds ratio (conﬁdence interval) P-value (Pc) preeclampsia

n % n % n %

HLA-A 1 211 87.2 169 84.5 20 74.1 1.37 (0.89–2.29) 0.24 2 31 12.8 31 15.5 7 25.9 HLA-B 1 219 91.3 177 89.8 25 92.6 1.14 (0.61–2.13) 0.75 2 21 8.8 20 10.2 2 7.4 HLA-DPA1 1 61 25.0 52 25.6 6 22.2 0.99 (0.65–1.50) 1.00 2 183 75.0 151 74.4 21 77.8 HLA-DPB1 1 179 73.4 142 70.0 18 66.7 1.21 (0.81–1.80) 0.42 2 65 26.6 61 30.0 9 33.3 HLA-DQA1 1 193 79.1 160 78.8 24 88.9 0.95 (0.61–1.48) 0.82 2 51 20.9 43 21.2 3 11.1 HLA-DQB1 1 194 79.8 176 86.7 25 92.6 0.57 (0.35–0.94) 0.03 (0.21) 2 49 20.2 27 13.3 2 7.4 HLA-DRB1 1 216 88.5 183 90.6 26 96.3 0.34 (0.40–1.35) 0.36 2 28 11.5 19 9.4 1 3.7 Total HLA sharing 7 28 11.9 37 19.0 4 14.3 1 1.00 8 78 33.1 74 37.9 13 46.4 0.76 (0.43–1.35) 0.39 9 68 28.8 54 27.7 6 21.4 0.60 (0.33–1.09) 0.10 10 27 11.4 15 7.7 0 0.0 0.38 (0.17–0.84) 0.01 (0.07) 11 18 7.6 8 4.1 2 7.1 0.38 (0.15–0.94) 0.04 (0.14) 12 11 4.7 6 3.1 2 7.1 0.50 (0.18–1.40) 0.20 13 5 2.1 0 0.0 1 3.6 0.14 (0.02–1.23) 0.08 14 1 0.4 1 0.5 0 0.0 0.68 (0.04–11.39) 1.00

DISCUSSION eclampsia compared with controls (Pc = 0.042), indicating a 13 To our knowledge, this is the largest and most extensive study protective function of this allele. In a study by Carreiras et al., investigating the association between HLA genes and the an increase of HLA-DQA1*0201 alleles among preeclamptic occurrence of severe preeclampsia and eclampsia. The current mothers and neonates was found, although this result has not study showed that the HLA-DPB1*04:01:01G allele was signifi- been verified in other studies. cantly more frequent (Pc = 0.044) among women diagnosed with Most studies investigating preeclampsia and HLA genotypes severe preeclampsia/eclampsia compared with controls, and the have been focusing on the HLA-DRB1 genotypes. However, the DQA1*01:02:01G allele frequency was significantly lower findings from previous studies are weak and inconsistent 12–14,20,18 (Pc = 0.042) among newborns born by women with severe regarding the HLA-DRB1 genotypes. This study did not preeclampsia/eclampsia compared with controls. Investigation of confirm any of the earlier and conflicting results, finding no fetomaternal HLA sharing demonstrated no significant association significant difference in the distribution of HLA-DRB1 alleles. with the occurrence of severe preeclampsia. Furthermore, in Considering the HLA class Ia genes, some studies have revealed mothers with severe preeclampsia/eclampsia homozygosity was no significant differences in the distribution of HLA-A and -B significantly more common compared with controls at the HLA- alleles between preeclamptic women and control women 13–15 fi DPB1 locus (Pc = 0.0028). together with their neonates. In line with this, no signi cant In this study, it was speculated whether the HLA class Ia and association between preeclampsia and HLA class Ia alleles was class II genes might influence the risk of severe preeclampsia and observed in the current study. eclampsia. The allele frequencies were investigated for all seven Regarding other HLA genes, an association between pree- genes (HLA-A, -B, - DRB1, -DQA1, -DQB1, -DPA1 and -DPB1). Taken clampsia and the distribution of the nonclassical HLA-G alleles has as a whole, frequencies of HLA class Ia and class II alleles in the been reported. Especially polymorphisms in the noncoding region women with severe preeclampsia and their neonates did not of HLA-G have been investigated and have shown to affect the show any significant differences compared with the matched protein expression and reduce the possible immunoregulatory controls. However, when looking at single alleles, the HLA- function of HLA-G.9,26,27 However, the immunogenetic studies of DPB1*04:01:01G allele was significantly more frequent among HLA-G in relation to risk of developing preeclampsia have been women diagnosed with preeclampsia/eclampsia compared with conflicting. the controls (Pc = 0.044). Other studies investigating the associa- When studies investigating the allele distribution of the same tion between HLA-DPB1 alleles are scarce. Honda et al.11 HLA genes and its association to preeclampsia are compared, the investigated HLA-DQB1 and HLA-DPB1 alleles in women with results are often different and inconsistent, making it unlikely that preeclampsia and did not find any significant difference in specific polymorphisms should be a main risk factor for comparison with normal fertile women concerning HLA-DPB1 preeclampsia. However, it is important to emphasize that these alleles. The same study observed that the frequency of the HLA- studies are different regarding several parameters, like HLA DQB1*04 allele was significantly higher in preeclamptic women, genotyping techniques, grading of preeclampsia, inclusion criteria, and this was not verified in the present study. There was no parity and the ethnicity of the subjects. However, evidence that a association between HLA-DQB1 allele frequencies and preeclamp- specific HLA class Ia or class II antigen is associated with sia. Concerning HLA-DQA1, the frequency of the DQA1*01:02:01G preeclampsia risk is not convincing. Yet, HLA molecules are very allele was significantly lower among newborns with preeclampsia/ important in immune recognition, acceptance/rejection of

Table 9. Maternal and fetal human leukocyte antigen (HLA) homozygosity/heterozygosity distributions

HLA antigen Neonates* Mother** P-value (Pc)

Control Severe Eclampsia Control Severe Eclampsia preeclampsia preeclampsia

n % n % n % n % n % n %

HLA-A Homozygous 49 20.2 28 14.0 6 22.2 36 13.9 47 20.7 7 23.3 *0.1481 **0.0369 (0.0861) Heterozygous 194 79.8 172 86.0 21 77.8 223 86.1 180 79.3 23 76.7 Total 243 200 27 259 227 30

HLA-B Homozygous 23 9.5 12 6.1 5 18.5 18 6.9 17 7.6 2 6.9 *0.5098 **0.8657 Heterozygous 218 90.5 185 93.9 22 81.5 241 93.1 208 92.4 28 93.1 Total 241 197 27 259 225 30

HLA-DPA1 Homozygous 178 73.0 147 72.4 20 74.1 173 66.5 169 74.4 24 80.0 *1.00 **0.0338 (0.0861) Heterozygous 66 27.0 56 27.6 7 25.9 87 33.5 58 25.6 6 20.0 Total 244 203 27 260 227 30

HLA-DPB1 Homozygous 65 26.6 57 28.1 6 22.2 47 18.1 74 32.7 8 26.7 *0.9176 **0.0004 (0.0028) Heterozygous 179 73.4 146 71.9 21 77.8 213 81.9 152 67.3 22 73.3 Total 244 203 27 260 226 30

HLA-DQA1 Homozygous 59 24.2 36 17.7 7 25.9 53 20.4 49 21.7 4 13.3 *0.1795 **1.00 Heterozygous 185 75.8 167 82.3 20 74.1 207 79.6 177 78.3 26 86.7 Total 244 203 27 260 226 30

HLA-DQB1 Homozygous 36 14.8 27 13.3 3 11.1 33 12.7 36 15.9 2 12.7 *0.5987 **0.5248 Heterozygous 208 85.2 176 86.7 24 88.9 226 87.3 191 84.1 28 87.3 Total 244 203 27 259 227 30

HLA-DRB1 Homozygous 25 10.2 16 7.9 3 11.1 22 8.5 23 10.2 1 3.3 *0.5277 **0.7588 Heterozygous 219 89.8 186 92.1 24 88.9 238 91.5 203 89.8 29 96.7 Total 244 202 27 260 226 30 *P-values for comparisons between cases and controls for the neonates. **P-values for comparisons between cases and controls for the mothers.

transplanted organs and tissue graft, as well as in fetomaternal exposure to paternal/fetal antigens may be protective.28 However, immune tolerance. In line with this, the fetus is semiallogeneic to change in paternity could result in a longer interval between the mother and the relations between the different HLA pregnancies, and this might be an explanation for the increased genotypes might be relevant in the etiology of preeclampsia. risk in first pregnancy with a different father.29 The maternal Instead of looking at specific HLA alleles, earlier studies have immunological protective response to the allogeneic fetus might focused on the relation between preeclampsia and HLA histo- be stimulated before pregnancy by sexual intercourse. This is compatibility between mother and fetus.15,17,20,23,25 Despite the supported by studies showing a significant protection against fact that the fetus is a semiallograft, histoincompatibility between preeclampsia associated with sperm exposure over a prolonged mother and fetus might have a positive effect on the pregnancy. period.23,30,31 A new study examined how fetomaternal histocom- The mechanism by which histoincompatibility should provide a patibility is associated with preeclampsia together with effects of protective response toward the pregnancy has not been fully the women’s past exposure to semen. HLA class I sharing was explained. It could be speculated that the foreign fetal antigens found to strongly increase the risk of preeclampsia, but moderate are recognized by the mother’s immune system that might elicit to high seminal fluid exposure attenuates this risk. Regarding HLA protective immune responses, thereby preventing maternal class II, sharing between fetus and mother only increased the risk immune rejection of the fetus. If the mother and fetus share the of preeclampsia in the context of moderate to high seminal fluid same antigens, this protective immune response might not be exposure.23 stimulated. In line with this, the fetus presents paternal antigens In this study the effect of HLA sharing between mother and and therefore a maternal–paternal immune interaction could also fetus at each of the five HLA class II genes (DRB1, DQA1, DQB1, be important for the pregnancy. The role of maternal–paternal DPA1 and DPB1) and the two HLA class Ia genes (A and B) was immune interaction on the occurrence of preeclampsia can be investigated as well as the total sharing of alleles across all seven seen from the fact that preeclampsia occurs more often in the first genes combined. A couple of studies have examined maternal– pregnancy, and if multiparous women change partner the risk fetal HLA sharing and risk of preeclampsia and have yielded varied increases to the level of the first pregnancy, indicating that prior findings.15,20,23–25,32 In a recent and large study by Biggar et al.,15

Genes and Immunity (2016) 251 – 260 © 2016 Macmillan Publishers Limited HLA class Ia and II in severe preeclampsia J Emmery et al 259 no association regarding HLA-A, -B, or -DR sharing in 201 cases MATERIALS AND METHODS and 195 controls was found. It should be noted that some of the Subjects cases of preeclampsia/eclampsia investigated in the current study The study is a nested case–control study within the framework of the were also included in the study by Biggar et al.15 In contrast, Danish National Birth Cohort (DNBC) at the Danish National Biobank (DNB) Schneider et al.25 found a significant association between overall at Statens Serum Institut, Copenhagen, Denmark. Genomic DNA from 518 maternal–fetal sharing at the HLA-A, -B, and -DR loci with risk of mothers and their newborn was obtained from DNB/DNBC that includes preeclampsia. When investigating maternal–fetal sharing at each data and biological specimens on 100 000 women who were pregnant in fi Denmark between 1996 and 2002. The present study used either full blood loci, the current study found a trend toward a signi cant fi – stains from the mothers collected on lter paper during pregnancy, difference at the HLA-DQB1 locus, where an increased fetal umbilical cord blood on filter paper from the infant at the time of birth or maternal sharing of two antigens was found among controls blood on filter paper from the infant collected at days 2–5 after birth. The (OR = 0.57; CI: 0.35–0.94). This result is in contrast to an earlier latter originated from the Danish Neonatal Screening Biobank in DNB. study by Triche et al.23 who found that preeclampsia subjects had Cases were included in this project on the basis of the following criteria: 65% higher odds of HLA-DQB1 sharing. In general, the risk of from the DNBC, Caucasian mothers and offspring were selected, preferen- – tially primigravida or alternatively primipara with a diagnosis of severe preeclampsia having maternal fetal sharing across all seven HLA fi fi preeclampsia or eclampsia (ICD-10 (International Statistical Classi cation of genes decreased; however, it was only signi cant before Diseases and Related Health Problems-10th revision) codes O14.1 or O15) – correction when sharing 10 antigens (OR = 0.38; CI: 0.17 0.84) or registered in the Danish Hospital Discharge Register (n = 259). For each case a 11 antigens (OR = 0.38; CI: 0.15–0.94). control was selected matched on the following criteria: same year and place The degree of homozygosity and the occurrence of preeclampsia of birth and restricted to women with no prior history of hypertension, has been studied before, where especially homozygosity at the preeclampsia, eclampsia and/or gestational diabetes. Some samples were HLA-A, -B and -DR locus have been in focus. No association of missing in the biobank and a few analyses of specific genes failed. In total, maternal homozygosity at the HLA-A, -B and -DR locus was found there were 221 complete matched cases and controls in the data set. In the 16,20 fl tables showing the DNA genotyping results for each HLA gene, the total in two studies by the same author. Con icting results have numbers of successful analyzed samples are reported for cases and controls, been obtained in other and more recent studies. An increase in respectively. Some of the cases of preeclampsia/eclampsia investigated in the maternal homozygosity in preeclamptic women has been current study were also included in a study by Biggar et al.15 that was based reported at the HLA-A locus,15,22 the HLA-B locus22,25 and the upon the DNBC biobank as well. The Ethical Committee of Region Zealand, HLA-DR locus.18,19 The current study found a trend for HLA Denmark, approved the project under the number SJ-234. homozygosity more common in mothers with preeclampsia/ eclampsia at the HLA-A locus (Pc = 0.086) compared with controls, DNA extraction and amplification 15 consistent with the result obtained by Biggar et al. In addition, Genomic DNA extraction from filter paper and PKU (phenylketonuria) cards homozygosity among women with preeclampsia was increased at was performed using Extract-N-Amp (Sigma-Aldrich, St Louis, MO, USA). fi the HLA-DPA1 locus (Pc = 0.086) and HLA-DPB1 locus (Pc = 0.0028). Whole genome ampli cation was carried out using REPLI-g Midi Kit Another smaller study found contradictory results, where the (Qiagen, Venlo, Netherlands). These procedures were carried out at Statens Serum Institut (Copenhagen, Denmark), and an aliquot of whole genome incidence of homozygosity of HLA-DPB1 alleles, together with the fi fi ampli ed DNA was sent to the Fred Hutchinson Cancer Research Center HLA-DQB1 alleles, in preeclamptic women was not signi cantly (Seattle, WA, USA) for further analysis. different compared with that of normal fertile women.11 No association between increase in homozygosity and the occurrence HLA genotyping of preeclampsia among neonates was found in this study. The mechanisms by which maternal HLA homozygosity could HLA typing was carried on the Illumina MiSeq (San Diego, CA, USA) using the v2.0 sequencing kit from Scisco Genetics Inc. (Seattle, WA, USA). The increase the risk of preeclampsia are not quite obvious. complete protocol using an amplicon-based PCR followed by sample The findings of this study might be in line with studies suggesting barcoding and data analysis was carried out as previously described.35 that a maternal immunological protective response to the For this study, the approach established complete phased sequence semiallogeneic fetus is stimulated before pregnancy by prior data for exons 2 through 4 for HLA class I and exons 2 and 3 for HLA and prolonged exposure to paternal antigen in seminal class II, all loci. Amplification bias due to the use of whole genome fi fluid.23,30,31,33,34 It can be speculated that if the maternal ampli ed DNA was accounted for by comparing shared haplotypes protective reaction is stimulated by an immune alloreaction to between the mother and child samples. Samples that did not share all loci within a haplotype were retested and rerun at higher sequence foreign paternal HLA antigens, the consequence of maternal density to recover missing genotypes. In total, 8 sample pairs of the 518 homozygosity would be a lower antigenetic variety between examined required retesting and analysis and all initially missing shared mother and father that might reduce the induction of an loci were recovered. immunological response in a future pregnancy. Therefore, couples sharing HLA antigens might have an increased risk of developing Statistical analyses preeclampsia, in line with the generally reduced frequency of HLA Allele frequencies of the seven genes were obtained using IBM SPSS homozygotes in the human population, reflecting a reduced statistic version 22 (IBM, Armonk, NY, USA). Comparisons between fertility among couples with matching HLA antigens. preeclamptic and control groups were performed using the χ2 test and This study provided a detailed and large-scale study of HLA Fisher’s exact test, considering differences as statistically significant when class Ia and II gene alleles in severe preeclampsia and eclampsia. Po0.05. Correction of P-values for multiple comparisons was performed – 36 χ2 A few specific HLA alleles were demonstrated to differ significantly using Bonferroni Holm method, indicated by Pc. To perform the test, at least five observations were required, making it necessary to group the between preeclamptic cases and controls. Nevertheless, the – fl alleles in main allele groups. Maternal fetal HLA sharing was analyzed by results are various and con icting with other studies. Maternal Fisher’s exact test and by calculating OR and 95% CI. When the antigens homozygosity was increased among women with preeclampsia were considered in combination, the range of antigens in common varied compared with controls, reducing the HLA antigenetic variation from 7 to 14. Fisher’s exact test was also used to analyze any significant between partners. Consequently, this might reduce the induction differences in the incidence of homozygosity/heterozygosity between of a maternal protective immune response derived by prior and preeclamptic cases and controls. prolonged exposure to paternal antigens, probably from seminal Finally, logistic regression analyses were performed to assess the impact fl of a number of factors on the likelihood of developing preeclampsia. The uid before pregnancy. model contained body mass index of the mothers, smoking behavior

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