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Antibodies to amino acid 200–239 (p200) of ro52 as serological markers for the risk of developing congenital heart block

Clinical and Experimental Immunology Antibodies to amino acid 200–239 (p200) of Ro52 as serological
markers for the risk of developing congenital heart block
L. Strandberg,*,** O. Winqvist,†,** S.-E. Sonesson,‡ S. Mohseni,† Maternal autoantibodies to the p200-epitope of Ro52 have been suggested to
S. Salomonsson,* K. Bremme,‡ correlate with development of congenital heart block. The aim of the present
J. P. Buyon,§ H. Julkunen¶ and study was to evaluate the clinical relevance and predictive value of p200-
M. Wahren-Herlenius* antibodies in high-risk pregnancies. Sera from 515 Finnish, Swedish and
*Rheumatology Unit, †Clinical Immunology Unit, American women were included in the study. Sera originated from 202
Department of Medicine, ‡Department of Woman and Child Health, Karolinska Institutet, mothers with an infant affected by second- or third-degree atrioventricular
Stockholm, Sweden, §New York University School block (AVB), 177 mothers with rheumatic disease having infants with normal
of Medicine, New York, USA, and ¶Division of heart rate and female blood donors (n = 136). A novel serological assay for
Rheumatology, Department of Internal Medicine, Ro52 p200-antibodies with intra- and inter-assay variability of 3% and 3·8%
Helsinki University Central Hospital, Finland respectively was developed. Mothers of children affected by AVB II-III had
significantly higher p200-antibody levels than mothers with rheumatic
disease having children with normal heart rate (P < 0·001). In the Swedish
cohort, a distinction between foetuses with normal conduction, AVB I, AVB II
and III was possible. A significant difference in anti-p200 levels between AVB
I and AVB II-III groups compared with foetuses with normal conduction
(P < 0·05 and P < 0·01) was observed. Using p200-antibodies as a second step
Accepted for publication 19 June 2008 analysis in Ro52-positive pregnancies increased the positive predictive value
Correspondence: M. Wahren-Herlenius, for foetal cardiac involvement (AVB I, II or III) from 0·39 (0·27–0·51) to 0·53
Rheumatology Unit, Department of Medicine, (0·37–0·68). In conclusion, Ro52 p200-antibodies may occur in women with
Karolinska Institutet, SE-171 76 Stockholm, unaffected children, but levels are significantly higher in mothers of children
with congenital heart block and are suggested as a relevant marker in evalu-
ating the risk for foetal AV block.
**These authors contributed equally.
Keywords: autoantibodies, congenital heart block, p200, Ro52
first-degree atrioventricular (AV) block [4], and case reports suggest that introduction of steroid treatment before a com- Congenital heart block is a potentially lethal condition, plete block has developed can inhibit progression or even affecting foetuses of women with Ro/SSA autoantibodies.
revert the block [4,13–18], stressing the importance of iden- During pregnancy, autoantibodies from the mother are tifying the high-risk pregnancies and of close monitoring transported across the placenta and may affect the develop- during susceptibility weeks.
ing child. Congenital heart block is a rare disease with an Maternal antibodies to the amino acid (aa) 200–239 incidence of 1/15 000–20 000 in the general population [1], (p200) of the Ro52 protein have been suggested as a sero- but the prevalence of complete congenital heart block in logical marker for an increased risk of having a child with women with anti-Ro/SSA antibodies is about 2% [2], congenital heart block [19]. To investigate the clinical rel- and higher in subpopulations of Ro/SSA-positive mothers evance and predictive value of Ro52-p200 antibodies, we defined by their autoantibody profiles [3–7]. The mortality developed a novel highly reproducible assay, taking into of affected infants is 15–30% [8,9], and the majority of account the structural features and constraints of the alpha- live born children will need life-long pacemaker implants helical p200 peptide. Congenital heart block is a rare disease, [9–12]. Congenital heart block usually develops during and to investigate a substantial number of cases we used the the 18–24th week of gestation. It may be initiated as a developed assay to analyse biobanks of sera from mothers of 2008 British Society for Immunology, Clinical and Experimental Immunology, 154: 30–37
Serological marker for risk of CHB Table 1. Patients included in the study. Number of Finnish, Swedish and American mothers in the study, their diagnoses, presence of Ro52 autoanti-
bodies and pregnancy outcome.
Maternal diagnoses (AVB II/III, NHR)† (AVB II/III, NHR) †Figures in parenthesis refer to the diagnoses of the foetus of the mothers. ‡NHR: normal heart rate. Infants without second- or third-degree heart block. The group includes both foetuses with AVB I and normal conduction. §Other refers to other rheumatic diseases (RA, MCTD, UCTD, scleroderma)or the presence of Ro52 antibodies without a defined autoimmune disease. ¶Thirteen cases with signs of first-degree heart block and 41 with normalconduction as mid-gestational foetuses.
foetuses with congenital heart block in Finland, Sweden and confirmed by high performance liquid chromatography the United States, including a total of 515 sera with 202 cases (HPLC) and mass spectrometry.
of AVB II-III.
Enzyme-linked immunosorbent assay for antibodies
binding the p200 peptide
Materials and methods
High-binding 96-well plates (Nunc) were coated with 100 ml Patients and controls
of 3 mg/ml streptavidin diluted in water. Plates were incu-bated at +4°C for 2 days, and then dried at 37°C and stored The study included sera from 515 women (Table 1). These at +4°C until use. Plates were washed four times with wash women were from Helsinki University Hospital in Finland buffer (0·15 M NaCl, ¥0·006 M NaH (n = 194) and Karolinska University Hospital in Sweden 2PO4·H2O, 20% NaN3/ 0·05% Tween-20/2% BSA) and unspecific binding blocked (n = 169) and the US Registry for Neonatal Lupus (n = 152).
with 200 ml 4% BSA in PBS. Plates were washed once with A total of 202 cases of AVB II-III were included. One PBS and coated for at least 6 h at room temperature with hundred and seventy-seven sera originated from mothers 100 ml of 3 mg/ml biotin-p200 peptide in coating buffer with rheumatic disease and/or Ro52 antibodies giving birth to infants without AVB II-III. These were classified as normal 2CO3, 0·07 M NaHCO3, 0·1% NaN3). Plates were washed four times with wash buffer. One hundred ml serum was added per well at a dilution of 1:300 and plates were The details of the Finnish [20] and US [21] patients and incubated by shaking at room temperature for 2 h. Plates collection of corresponding samples have been described were washed four times and affinity-purified alkaline phos- previously. All Swedish patients were systematically fol- phatase (AP)-conjugated, rabbit anti-human IgG antibodies lowed with foetal Doppler echocardiography during mid- (Dakopatts, Glostrup, Denmark) were added at a dilution of trimester pregnancy, and the group with normal heart rate 1:1000. Plates were washed four times with wash buffer. As was further divided into two groups based on the foetal substrate, phosphatase substrate tablets (Sigma, St Louise, findings; AVB I was defined as at least two examinations MO, USA) were dissolved in diethanolamine pH 9·8, and where the Doppler atrioventricular time intervals exceeded 100 ml incubated in the wells for 2 h at room temperature for the 95% reference range based on recordings from 284 detection of IgG. The absorbance was measured at 405 nm women with normal pregnancies [4,22], and those with using a Sunrise absorbance reader (Tecan) and the Magellan normal atrioventricular conduction (NC). Twenty-five of V 3·11 software. A p200 index was calculated based on a ratio the Swedish patients have been previously described [4,23].
with one high-titre patient selected as standard where the Sera were sampled from the mothers during or after p200 index = [(OD sample - OD negative control)/(OD pregnancy. Sera from 136 female Finnish and Swedish blood positive control - OD negative control)] donors between 18 and 54 years of age were used as normal assays in this study were run in the same laboratory, at the control sera (Table 1). Human ethical review boards in Department of Clinical Immunology, Karolinska Institute.
the respective countries approved the investigations, and Ro52 autoantibodies were detected in routine serology at informed consent was given by the mothers.
the respective hospital, or by ELISA as previously described[3].
A synthetic peptide representing aa 200–239 of Ro52 wassynthesized by Thermo Biosciences, Ulm, Germany, with Statistical analysis was performed using Statistica 7·0 (Stat- biotin conjugated at the N-terminal end. Peptide purity was soft, Tulsa, OK, USA). Shapiro–Wilk's W test demonstrated 2008 British Society for Immunology, Clinical and Experimental Immunology, 154: 30–37
L. Strandberg et al. that our data did not fit a normal distribution, and therefore the Mann–Whitney U-test or Kruskal–Wallis anova wasused for statistical analysis. The level of significance was set at P < 0·05.
The University of British Columbia calculator was used for Bayesian calculations.
Receiver operating characteristics (ROC) curve analysiswas performed to calculate sensitivity and specificity of theELISA for p200 antibodies using the Med Calc program.
Detecting Ro52-p200 antibodies with low intra- and
A novel, highly reproducible assay for detection of Ro52- p200 specific antibodies was developed. To allow free folding of the alpha helical p200 sequence and to give a set orienta-tion to the peptide during assay performance, biotin was Fig. 1. p200 levels in mothers of foetuses with or without AVB
conjugated at the N-terminal end during synthesis before II-III. Antibodies to the p200 peptide in maternal sera were measured coating to streptavidin-plates and subsequent ELISA. This by ELISA. Sera from all mothers included in the study (n = 515) was of importance for the Ro52-derived p200 peptide, as the were tested and p200 antibody levels compared between mothers epitope formation and antigenicity of aa 200–239 of Ro52 is of foetuses with AVB II-III and mothers with rheumatic diseaseand foetuses with normal heart rate (NHR), P < 0·0001. The study dependent on correct folding and structure [24]. Patients population includes mothers from Finland, Sweden and the USA.
with high, intermediate or low p200 antibody levels were Female blood donors were from Finland (n = 31) and Sweden selected for determining intra- and inter-assay variability, (n = 105).
which were established at 3% and 3·8%, respectively.
Ro52-p200 antibodies correlate with AVB II-III
foetuses had NHR in the Swedish and American cohorts(P < 0·05 and P < 0·05, respectively), but not in the Finnish Investigating the sera from the Finnish, Swedish and Ameri- cohort (Fig. 2).
can cohorts, we first performed an analysis of all 515 samplesfrom the different populations together (Table 1). Sera weregrouped as originating from: mothers of children with Maternal diagnosis and p200 levels
AVB II-III, mothers with an autoimmune rheumatic disease We further analysed the p200 antibody levels in Ro52- and/or Ro52-positive mothers who had children with positive women with foetuses affected by AVB II-III and normal heart rate (NHR), and healthy blood donors. We foetuses with NHR in relation to the diagnosis of the found a significantly higher level of p200 specific antibodies mothers (Table 1 and Fig. 3). While a graphic plot of the in sera from mothers of children with AVB II-III, both com- p200 values indicated a trend toward differences (Fig. 3a), pared with mothers of children with NHR (P < 0·0001) and anova analysis of p200 levels did not reveal significant dif- healthy blood donors (P < 0·00001) (Fig. 1).
ferences in the AVB II-III versus NHR pregnancies in theFinnish, Swedish or US cohorts when subdivided according p200 antibodies in Ro52-positive mothers and in the
to maternal diagnoses of Sjögren's syndrome, SLE or other Finnish, Swedish and American cohorts
rheumatic disease (denoted ‘Other', and including rheuma- While the difference in maternal p200 antibody levels toid arthritis, myositis, undifferentiated connective tissue between the groups with children affected by, and not disease, scleroderma or asymptomatic women with Ro52 affected by, AVB II-III was statistically significant, a central antibodies). However, by using the Mann–Whitney U-test a question is whether p200 antibodies are a more sensitive significant difference was observed in p200 antibody levels in and specific marker of congenital heart block than Ro52 the diagnostic group ‘Other' in mothers of foetuses with AVB antibodies. We therefore performed an analysis of p200 anti- II-III compared with foetuses with NHR (P < 0·005).
body levels exclusively in Ro52-positive sera. In the analysis When p200 antibody levels were analysed in relation to we noted substantial differences in p200 levels between the maternal diagnosis in the Finnish, Swedish and US cohorts, three populations (Fig. 2), therefore Ro52-positive sera from a difference was noted between mothers with Sjögren's each cohort were analysed separately. We found significantly syndrome and mothers with SLE in the Finnish material higher levels of p200 antibodies in sera from mothers of (P < 0·05) (Fig. 3b). The difference was not significant in foetuses with AVB II-III compared with mothers whose patients from the other countries.
2008 British Society for Immunology, Clinical and Experimental Immunology, 154: 30–37
Serological marker for risk of CHB Increased levels of antibodies specific for p200 are
found in AVB I, II and III
Congenital heart block may be initiated or present as a first degree heart block [4]. In the present study, all Swedishpatients (n = 64) were systematically followed by serial Doppler echocardiographic recordings during pregnancy to detect signs of both first, second and third degree heartblock. This made it possible to identify foetuses with signs of AVB I in the group of foetuses with NHR. By using anova itwas demonstrated that the degree of foetal cardiac involve- ment was a highly significant factor of variance for the maternal p200 antibody levels (P < 0·005). Mothers of both foetuses with AVB II-III and those with signs of AVB I hadhigher p200 levels than those with a foetus without cardiac involvement (Fig. 4). No difference was found between mothers where the foetus had signs of AVB II-III or Determination of the p200 assay performance and
predictive value of p200 antibodies
Fig. 3. Maternal diagnosis and p200 levels in Finnish, Swedish and
To evaluate the ability of the assay to discriminate AVB cases American patients. (a) The nationally grouped Ro52-positive mothers (AVB I, II, III) from cases without AVB, including normal were stratified also by their diagnosis of SS, SLE or other rheumatic controls, a receiver operating characteristics (ROC) curve disease (including RA, MCTD, UCTD, scleroderma or asymptomatic analysis was performed in the Swedish cohort. The cut-off women with Ro52 antibodies). (b) Analysing the Ro52-positivemothers, disregarding the infant diagnosis, the Finnish mothers withSS have significantly higher levels of p200 levels than mothers with p < 0·05 p < 0·05 SLE (P < 0·05).
p < 0·001 p < 0·05 p < 0·01 Fig. 4. p200 antibody levels are equally high in AVB I and AVB II-III.
Fig. 2. Levels of p200 antibodies in Ro52-positive Finnish, Swedish
In the Swedish cohort all pregnancies were systematically followed by and American mothers of foetuses with or without AVB II-III. The foetal Doppler echocardiography, allowing further distinction of foetal cohorts were separated to allow comparisons between the three diagnosis into AVB II-III, AVB I and normal conduction (NC). p200 nationalities. Only Ro52-positive women are included. Separating antibody levels in mothers with AVB II-III as well as AVB I foetuses the populations, the p200 levels are significantly different between were significantly higher than p200 antibody levels in mothers of mothers of foetuses with AVB II-III and mothers of foetuses with foetuses with NC. There was not a significant difference in p200 normal heart rate (NHR) in the Swedish and American populations, antibody levels between mothers of foetuses with AVB I and AVB but not in the Finnish population.
2008 British Society for Immunology, Clinical and Experimental Immunology, 154: 30–37
L. Strandberg et al. tions for testing can be established, and low intra- and inter- assay variability was obtained. Using the assay for analysis ofthe combined serological material from Finland, Sweden andthe USA, there was a significant difference in p200 antibody Sensitivity= 90.5 levels between mothers of children affected and not affected by AVB II-III, but also an overlap in p200 levels between the two groups of mothers. The material was therefore stratified to identify potential groups of patients where p200 antibod-ies could be of practical clinical value. Further, we limitedanalysis of p200 levels to Ro52-positive sera, as the main objective of the study was to relate the value of p200 anti- body analysis to Ro52 antibody analysis. In stratification, adifference in p200 levels between the three national cohorts became evident, where Finnish and American sera had gen-erally lower p200 antibody levels than Swedish sera, and alsowhen only Ro52-positive sera were included in analysis. In Swedish and American Ro52-positive sera, the p200 anti- body levels were significantly higher in mothers of infantswith AVB II-III than in mothers of infants with normal heart rate. However, another recent report analysing the levels of Fig. 5. Discrimination performance of the p200 antibody ELISA.
Ro52-p200 antibodies in sera from the Research Registry for ROC analysis of the Swedish cohort including healthy controls Neonatal Lupus did not identify p200-binding antibodies as optimizes the cut-off at the p200 index = 30, which results in a specific for sera of mothers of children with complete con- sensitivity of 90·5% and a specificity 96% and a positive likelihood genital heart block [25]. A potential reason for the discrep- ratio (+LR) of 23 of cardiac involvement (AVB I, II or III).
ancy between the studies is the difference in assay format.
Different conditions were used for peptide coating; either point of a p200 index = 30 gave the optimal assay perfor- attaching p200 peptide directly to the plastic ELISA well mance with a sensitivity of 90·6% and a specificity of 96% surface [25], or by a biotin–streptavidin interaction via a (Fig. 5). The positive likelihood ratio defined as sensitivity/ biotin molecule conjugated to the p200 peptide during syn- (1-specificity) was 23, indicating that a positive assay result thesis (the present study). The latter allows free folding of the is likely to identify presence of disease in the form of peptide and creates a set orientation during assay perfor- mance, which is of importance as the epitope formation and A potential clinical use of analysing p200 antibody levels antigenicity of aa 200–239 of Ro52 is strongly dependent on would be to guide the clinician as to the risk of congenital correct folding and structure [24]. A further difference, heart block in an individual pregnancy. By using the p200 besides serum dilution and incubation times, is that the pre- assay as a second step analysis in Ro52-positive women, the vious study [25] used reagents to detect p200 specific anti- positive predictive value for detecting foetuses with AVB bodies that may bind to several Ig-isotypes, and recorded the II-III increased from 0·17 (0·07–0·27, 95% confidence range) collective signal generated [26]. The present study focused to 0·23 (0·10–0·35). For detecting foetuses with AVB I-III the detection at p200 antibodies of the IgG isotype only, as these corresponding increase in probabilities was from 0·39 (0·27– are the immunoglobulins that are transferred across the pla- 0·51) to 0·53 (0·37–0·68). The odds of obtaining a positive centa during pregnancy.
p200 antibody test result in a Ro52-positive AVB II-III and In Finnish sera the difference in p200 antibody levels AVB I-III pregnancy compared with a woman with a NC between mothers of children with AVB II-III and with NHR foetus (+ LR) were 1·42 (1·06–1·91) and 1·73 (1·24–2·42), was not significant, although p200 antibodies were detected respectively. The negative likelihood ratios, the odds of in the sera from mothers of children with AVB II-III. The obtaining a negative p200 antibody test result in Ro52- explanation for this could be that in the Finnish series positive women with a AVB II-III or AVB I-III foetus com- mothers with children having non-diagnosed AVB I may be pared with a NC pregnancy, were 0·27 (0·04–1·81) and 0·18 present in the NHR group, as these pregnancies were not monitored by Doppler echocardiography. Another factorthat might explain why a difference between the groups was not observed in the Finnish cohort is that most of the sera In the present collaborative study we examined the relevance from control pregnancies originated from women with of specific Ro52 antibodies as maternal serological indicators primary Sjögren's syndrome. Analysis revealed that in the of high risk for congenital heart block in the foetus during total Finnish cohort mothers with Sjögren's syndrome as a pregnancy. Assay development confirmed that stable condi- group had higher p200 antibody levels, which is in line with 2008 British Society for Immunology, Clinical and Experimental Immunology, 154: 30–37
Serological marker for risk of CHB previous reports where higher Ro and La antibody levels of autoantibody positive women has been described, have been observed [27].
although it is rare [20,32]. It is possible that foetal AVB I is The general difference in antibody levels between the an indication of later conduction pathologies in life, but no whole national cohorts could be due to factors such as the data presently support this possibility. Prospective longitu- time of sampling of sera in relation to the affected pregnancy dinal follow-up studies will be required in order to under- or storage conditions, and ethnic or genetic differences.
stand whether foetuses with signs of AVB I in utero are at Studies of congenital heart block and collection of sera have risk for late cardiac complications, and these are underway been running for close to two decades in the Finnish and in our clinic.
American groups, while investigations were more recently Earlier attempts to define a specific antibody profile in initiated by the Swedish researchers. Serum storage time will mothers of children with CHB have demonstrated a preva- therefore naturally vary between the biobanks, and poten- lent, but not unique, anti-Ro 52-kDa antibody response tially also the number of times each individual sample has [33–38]. Fritsch and colleagues recently presented data from been thawed and re-frozen, factors both known to affect IgG longitudinally collected samples before, during and after stability [28]. The time of serum sampling in relation to the pregnancies where congenital heart block occurred [38].
affected pregnancy also differs between the three national Their results indicate that antibodies to aa 1–13, 277–292 materials. The majority of Finnish mothers of children with and 365–382 are elevated during week 18–30 when the congenital heart block were identified by register-based AV block develops, and antibodies to aa 365–382 are of investigations, and blood samples taken after pregnancies, in special interest as they may have a functional impact by some cases several decades after the affected pregnancy. Also cross-reacting with the 5-HT4 receptor. These investigators, the American material includes sera sampled years after however, did not include peptides corresponding to the p200 birth of an affected child. While levels of Ro and La auto- peptide used in the current study, therefore a direct compari- antibodies rarely vary to the extent of making a positive son of results is not feasible. Previous studies have also individual test negative, substantial variations over time suggested that antibodies to Ro 60-kDa have a minor role in have been demonstrated [29–31], and the timing of serum predicting the clinical outcome in Ro- and La-positive sampling may accordingly influence the result. Thus sera mothers [9,34,39], while La antibodies were recently shown may not be directly comparable between the cohorts. Con- to add to the risk of developing congenital heart block [40].
genital heart block is, however, a rare disease and to analyse The present study shows that analysis of p200 antibodies as a any substantial number of patients, collaborative efforts and second step analysis in Ro52-positive women yields a posi- pooling of existing clinical materials is needed. While differ- tive predictive value for detecting foetuses with AVB I-III of ences may exist between the included biobanks and need to 0·53 in the Swedish cohort where this analysis was possible, be kept in mind when interpreting the results, it is important and may thus be clinically helpful in identifying high-risk to evaluate novel potential biomarkers in different materials pregnancies. However, the recurrence of congenital heart to understand their applicability. The analysed serum banks block in subsequent pregnancies is around 20% [8,9], stress- constitute the, or some of the, largest collections worldwide ing that other factors also influence foetal susceptibility or and have taken decades to collect, and until prospective mul- resistance to disease [41,42].
ticentre studies are initiated remain the best alternative for In conclusion, our data from an international collabora- tive study of the relevance of p200 specific Ro52 antibodies Levels of p200 antibodies have been claimed to correlate in congenital heart block demonstrate that p200 antibody with prolongation of the PR-interval [4], while a subse- levels are significantly higher in mothers who have pregnan- quent study did not confirm this observation [25]. The cies complicated by AVB II-III in the foetus. Also, when present study allowed an analysis of p200 antibody levels in analysis was limited to Ro52-positive pregnancies p200 an extended number of mothers of foetuses with AVB I, II antibody levels of AVB II-III were higher than in pregnan- and III, as well as in foetuses without cardiac involvement.
cies with normal heart rate in the foetus in the Swedish and Levels of p200 antibodies were equally high in mothers of American cohorts, but not the Finnish. Our data further children affected by AVB II-III and AVB I, and in Ro52- show that p200 antibody levels are equally high in mothers positive mothers an additional positive predictive value of of foetuses with signs of AVB I as in those of foetuses with p200 antibody analysis in identifying pregnancies compli- signs of AVB II-III, and that a high likelihood ratio and cated by foetal AV block was observed. The clinical long- predictive value for cardiac involvement using p200 anti- term value of detecting AVB I in Ro52-positive pregnancies body levels may be obtained by standardizing the method other than for excluding AVB II-III in the foetus remains for analysis. In combination with foetal Doppler echocar- an open question. This prolongation of the PR interval in diography, determination of Ro52-p200 antibody levels utero appears spontaneously reversible at birth or shortly may prove a valuable clinical tool to identify pregnancies after, and in our opinion in the face of present data and where the risk for congenital heart block is high, and allow collective knowledge does not call for any treatment.
treatment before the condition has progressed into a com- However, progression of AV-block postnatally in children plete AV block.
2008 British Society for Immunology, Clinical and Experimental Immunology, 154: 30–37
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2008 British Society for Immunology, Clinical and Experimental Immunology, 154: 30–37

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