Maternal and sibling microchimerism in twins and triplets discordant for neonatal lupus syndrome-congenital heart block
A. M. Stevens1,2,
H. M. Hermes1,
N. C. Lambert1,
J. L. Nelson1,3,
P. L. Meroni4 and
R. Cimaz5
1 Fred Hutchinson Cancer Research Center, 2 Department of Pediatrics and 3 Department of Medicine, University of Washington, USA, 4 Istituto Auxologico and 5 Istituti Clinici di Perfezionamento, Università di Milano, Italy.
Correspondence to: A. M. Stevens, Immunogenetics, D2-100, P.O. Box 19024, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. North, Seattle, WA 98109, USA. E-mail: astevens{at}fhcrc.org
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Abstract
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Objective. Neonatal lupus syndrome-congenital heart block (NLS-CHB) is an acquired autoimmune disease in which maternal autoantibodies are necessary but not sufficient for disease. Maternal myocardial cells have been found in the hearts of patients with NLS-CHB, suggesting that maternal microchimerism may also play a role. In this study we asked whether levels of microchimerism in the blood are associated with NLS-CHB in discordant twins and triplets.
Methods. Human leucocyte antigen (HLA)-specific and Y-chromosome-specific real-time quantitative polymerase chain reaction (PCR) was used to quantitatively assay maternal and sibling microchimerism in peripheral blood. Because of HLA allele sharing in families, it was not always possible to distinguish between multiple sources of microchimerism.
Results. In one family, maternal and/or sibling microchimerism was detected in two triplets who had CHB, but not in the triplet with transient hepatitis. Levels ranged from 4 to 948 genome-equivalents of foreign deoxyribonucleic acid per million host genome-equivalents (gEq/million). Over the first year levels of sibling microchimerism decreased in the triplet with complete CHB and increased in the triplet who progressed from first- to second-degree CHB. In a second family, maternal and/or sibling microchimerism was detected in the healthy twin (1223 gEq/million) but not in the twin with CHB.
Conclusions. Maternal and/or sibling microchimerism was detectable in the blood of infant twins and triplets discordant for NLS. Microchimerism in the blood was not specific for NLS-CHB, although in one family levels correlated with disease. Thus, microchimerism in the blood and/or tissues may be involved in the pathogenesis or progression of NLS-CHB, but additional factors must also contribute. Further investigation is warranted.
KEY WORDS: Microchimerism, Maternal, Neonatal lupus, Congenital heart block
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Introduction
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During pregnancy maternal cells pass into the fetus where they may remain indefinitely in the child's blood and tissues, a state referred to as maternal microchimerism [14]. Chimeric cells can also be derived from a twin [5]. Because some autoimmune diseases resemble graft versus host disease after haematopoietic stem cell transplantation, maternal microchimerism has been suggested to play a role in the pathogenesis of idiopathic inflammatory diseases [1, 4, 68].
Neonatal lupus syndrome (NLS) is an important model of acquired autoimmune disease that involves the fetus and mother (reviewed in [9, 10]). NLS, with associated congenital heart block (CHB), rash, hepatitis, thrombocytopenia and neutropenia, occurs in the fetus in association with specific anti-Ro (SSA) and anti-La (SSB) autoantibodies in the mother. It has been proposed, with convincing supportive evidence, that autoantibodies play a role in the pathogenesis of NLS. Maternal autoantibodies, however, are not sufficient to cause NLS. Over 95% of women who are pregnant and have antibodies to SSA and/or SSB give birth to healthy infants, although some of these infants may have occult signs of NLS [11]. Twins, even monozygotic, both of which have been exposed to the same maternal autoantibodies, are often discordant for disease [9]. Therefore, more than humoral autoimmunity is important in the pathogenesis of NLS.
Because of the finding that maternal cells commonly pass into the fetus during pregnancy [2], maternal cells were recently investigated in the organs of NLS patients who died of CHB [8]. In this study, maternal cells were increased in the hearts of infants with disease compared with controls. Moreover, maternal cells were differentiated cardiac myocytes, suggesting that maternal cells act either as targets for the inflammatory response or as regenerative cells after injury. Circulating maternal cells may have migrated to the heart and other tissues in NLS, in which case they may also be found in the blood. The present study addresses the association of circulating microchimerism with NLS in twins and triplets discordant for disease.
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Patients and methods
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Patients
Informed consent was obtained from the parents, and the hospital review board at the Istituto Auxologico-Università di Milano approved the study protocol. In the first family (triplets reported previously [12]), fetal echocardiography was performed on a 29-yr-old healthy woman with a triplet pregnancy after in vitro fertilization. Triplet A (female) showed a mild pericardial effusion at 21 weeks gestation and developed tricuspid regurgitation and complete AV block by 24 weeks. Triplet B (female) developed enlarged ventricles and tricuspid regurgitation by 25 weeks. The mother had no history of autoimmune disease. Her serum contained autoantibodies specific for SSA/Ro and SSB/La. Dexamethasone treatment was administered between 24 and 31 weeks, and the babies were delivered by Caesarean section at 31 weeks because of premature rupture of membranes. After birth, complete heart block was confirmed in triplet A, who later had a pacemaker placed. First-degree heart block was detected in triplet B, which progressed to periodic second-degree AV block (Wenkebach type). Triplet C (male) had a transient increase in transaminase levels, but normal electrocardiograms.
In the second family (twins), a 29-yr-old woman with Ro/SSA+ subacute cutaneous lupus erythematosus had a previous pregnancy complicated by congenital heart block and intrauterine fetal death the year prior to this pregnancy. She presented with a dichorionic, diamniotic twin pregnancy. Heart block was detected at week 20 in twin A (male), with modest hydropericardium and a heart rate of 52 beats per minute. The patient was treated with dexamethasone from week 21 to week 33. A Caesarean section was performed at week 34. The male twin had a heart rate of 42 beats per minute and pulmonary valve stenosis, and was treated with orciprenaline. A pacemaker was placed at 5 weeks of age. Twin B (female) remained healthy, with no signs of NLS and a normal electrocardiogram.
HLA genotyping
Deoxyribonucleic acid (DNA)-based human leucocyte antigen (HLA) typing was conducted on all five NLS subjects, their two mothers and the grandmother of the triplets to determine specific DRB1, DQA1 and DQB1 alleles. DNA was extracted from whole heparinized blood, peripheral blood mononuclear cells or fingernail clippings. DQA1 and DQB1 alleles were determined by sequence-specific oligonucleotide probe (SSOP) typing with methods similar to those previously described [13] to which other probes were added to detect newly identified alleles of DQA1 and DQB1 [14]. Alternatively, some DRB1 low-resolution assays and DQB1 typing was done using a DYNAL RELITM SSO HLA-DRB or DQB typing kit (DYNAL, Oslo, Norway) following the manufacturer's instructions with subsequent direct sequencing to determine the specific allele. For some subjects DQA1 alleles were inferred based on allele level typing results for both DRB1 and DQB1.
Real-time quantitative HLA-specific polymerase chain reaction (PCR) assays
In female subjects, male sibling DNA was detected by a Q-PCR assay specific for the Y-chromosome sequence DYS14, and for male and female subjects, non-shared HLA sequences were used as targets for real-time quantitative PCR assays to detect and quantify maternal and sibling DNA by previously reported methods [4, 15]. For quantitative measurement of total DNA tested per well a standard curve of ß-globin using a dilution of the equivalent DNA of 5025 000 cells was used. A conversion factor of 6.6 pg of DNA per cell was used to express the results as genome equivalents (gEq) per million maternal gEq. For each subject six to 12 aliquots between 5000 (33 ng) and 20 000 (132 ng) gEq-cells were assayed, including a duplicate for ß-globin. Sensitivity was determined by testing dilutions of the equivalent DNA of 0.5500 cells (11000 copies) homozygous for the HLA-specific allele in a background of 10 000 cell-equivalents negative for that HLA allele, allowing for quantification of microchimerism in the range of 0.01 to 10%.
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Results
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HLA typing was performed for the patients and family members in order to identify non-shared HLA alleles that could be used as targets to detect and quantify microchimeric DNA from mothers and siblings (Table 1).
An example of the real-time PCR assay is shown in Fig. 1. Figures 1A and B demonstrate the standard curve. Figures 1C and D are representative of assays of NLS DNA. In the first case (Fig. 1C) chimeric DRB4 DNA was detected in DNA samples from triplet 1B in nine of 10 wells at the DNA equivalent level of one to three cells per well. By normalizing to 1 million cell-equivalents, this sample was estimated to contain the DNA equivalent of 321 chimeric cells (from her sister) per million host cells. In the second example (Fig. 1D), no DRB4 was amplified in DNA from triplet 1C, suggesting that there was no chimerism from triplet 1A in this sample.
We detected chimeric DNA in eight of 11 blood samples (Table 2). Two of the triplets and one of the twins carried foreign DNA. Because of the high level of HLA allele sharing in families, in no sample was it possible to specifically assay maternal DNA. In triplet 1C and twin 2A, however, no maternal or sibling DNA was detected, demonstrating that maternal microchimerism was present at a level of less than 1 per 100,000 (the number of cell-equivalents assayed). Thus, although the HLA-specific assays could not specifically distinguish whether microchimerism was maternal in positive samples, they could identify samples without detectable maternal DNA.
For triplets 1A and 1B samples were available both from 16 weeks and from 1 yr of life. Levels of chimerism changed during this period. In 1A (the infant with third-degree CHB) the level of male DNA (presumably derived from sibling 1C) decreased from 948 to 67 gEq/million. In triplet 1B, who progressed from first- to second-degree CHB, the level of male DNA (presumably from sibling 1C) increased from 4 to 376 gEq/million and the level of DNA from sibling 1A (as quantified by the DRB4 assay) increased from 9 to 321. Thus, in subjects 1A and 1B, levels of sibling microchimerism correlated with progression of disease.
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Discussion
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In this study maternal and/or sibling microchimerism was detected in the blood of infants discordant for NLS-CHB. Discordance for NLS has been reported previously [9], but an explanation for the discordance has not been clear. In the present study, in one family maternal and/or sibling microchimerism correlated with NLS disease activity. That triplet 1B demonstrated increased chimerism during the first year of life is especially interesting, because this infant had CHB which increased in severity during the same period while triplet 1A had stable disease and microchimerism decreased.
There are several shortcomings to this limited study. First, the blood samples cannot be directly compared between subjects because they were drawn at different ages. It is not known how levels of maternal or sibling microchimerism change with age in healthy infants or infants with NLS-CHB. Second, the Q-PCR assays were not always able to distinguish maternal from sibling microchimerism. Because twintwin transfusion is common [5], any or all of the chimerism detected in this study could represent sibling DNA. Finally, the effects of in vitro fertilization on the extent of chimerism are unknown, but could theoretically have had an effect on the studies in Family 1.
A previous study of infants with NLS who died of CHB showed that the frequency of maternal cells in the tissues, especially the myocardium, was more than 20-fold higher than the frequency of maternal cells previously reported either in the blood in the current study or in cord blood in previous studies [2, 8]. The maternal cells appeared to have differentiated into myocardial cells, and only rarely were maternal haematopoietic cells detected. Thus, if maternal cells are involved in the pathogenesis of NLS, they may act within the tissues specifically as immune targets. Allogeneic somatic cells could provide a source of foreign antigens: HLA proteins as well as other allelic proteins. These foreign proteins could provide a chronic inflammatory stimulus. Alternatively, maternal blood cells could act as effectors, attacking host tissues. Finally, maternal cells could act secondarily in tissue repair. Maternal microchimerism could therefore be one of the factors, along with others (such as Fc-gamma polymorphisms, HLA alleles or genes involved in inflammation and fibrosis [9, 10]), that could act together with autoantibodies in the pathogenesis of NLS.
It is known that maternal antibodies bind to the myocardial tissues in fetuses with CHB [10]. Maternal cells in the fetal tissues may be one more additional factor required for a fetus, predisposed by maternal anti-SSA antibodies and maternal HLA molecules, to develop NLS. There is precedence for the requirement of both antigen-specific lymphocytes and autoantibodies for organ-specific autoimmunity in an animal model [16]. The earliest passage of maternal cells into the human fetus has been detected at 13 weeks [17], during the time when a low level of immunoglobulin is also starting to pass into the fetus. Since the CHB occurs at 17 weeks or thereafter, either maternal antibody or maternal cells or a combination of the two could trigger the pathogenic inflammatory response. There is also precedence for disease transmission via maternalfetal cell trafficking. Maternal microchimerism has previously been reported in two other autoimmune diseases: systemic sclerosis and juvenile myositis [1, 4, 6, 7]. However, in these studies maternal microchimerism was also found in the blood of healthy adult and older child controls.
This study demonstrates that microchimerism from a mother or a sibling can be found in immunocompetent infants aged 6 weeks to 1 yr. Maternal DNA in infants has not been reported previously, although it might be expected because maternal microchimerism has been reported in cord blood at comparable levels (1050 000 gEq/million host cells [1822]), and also in the blood of older children and adults, ranging from 2 to 250 gEq/million [1, 4, 7] and in the thymus in infants [23]. In two studies, levels of maternal microchimerism were even higher in tissues [7, 8]. Thus, persistent maternal microchimerism may be a normal part of immune system and/or organ development.
In conclusion, maternal and/or sibling microchimerism has been detected in the blood of infant twins and triplets discordant for NLS. Levels of sibling microchimerism correlated with disease in one family but not in a second family. Thus, microchimerism in the blood and/or tissues may be important in the pathogenesis of NLS-CHB, but other factors are necessary and further investigation is warranted.
The authors have declared no conflicts of interest.
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Submitted 27 June 2004;
revised version accepted 23 September 2004.