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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES

Objective

Recent evidence suggests that anti-Ro/SSA antibodies, strongly associated with the development of congenital heart block, may also be arrhythmogenic for the adult heart. In fact, anti-Ro/SSA–positive patients with connective tissue disease (CTD) frequently display corrected QT (QTc) prolongation associated with an increase in ventricular arrhythmias. However, QTc prolongation prevalence markedly differs throughout the studies (10–60%), but the reason why is not yet clear. The aim of this study was to evaluate whether anti-Ro/SSA–associated QTc prolongation in adult patients with CTD is related to antibody level and specificity.

Methods

Forty-nine adult patients with CTD underwent a resting 12-lead electrocardiogram recording to measure QTc interval, and a venous withdrawal to determine anti-Ro/SSA antibody level and specificity (anti–Ro/SSA 52 kd and anti–Ro/SSA 60 kd) by immunoenzymatic methods and Western blotting.

Results

In our population, a direct correlation was demonstrated between anti–Ro/SSA 52-kd level and QTc duration (r = 0.38, P = 0.007), patients with a prolonged QTc had higher levels of anti–Ro/SSA 52 kd with respect to those with a normal QTc (P = 0.003), and patients with a moderate to high level (≥50 units/ml) of anti–Ro/SSA 52 kd showed a longer QTc interval (P = 0.008) and a higher QTc prolongation prevalence (P = 0.008) than those with a low positive/negative level (<50 units/ml). On the contrary, no association was found between QTc and anti–Ro/SSA 60-kd level.

Conclusion

In anti-Ro/SSA–positive adult patients with CTD, the occurrence of QTc prolongation seems strictly dependent on the anti–Ro/SSA 52-kd level. This finding, possibly explaining the different QTc prolongation prevalence reported, strengthens the hypothesis that an extremely specific autoimmune cross-reaction is responsible for the anti-Ro/SSA–dependent interference on ventricular repolarization.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES

A large body of evidence links the presence of circulating anti-Ro/SSA antibodies in the mother with the risk for the newborn of developing an uncommon but severe syndrome named neonatal lupus, whose main challenge is the congenital heart block (CHB) (1, 2).

On the contrary, although being frequently detected in adult patients with autoimmune disorders, particularly connective tissue diseases (CTDs) (3), anti-Ro/SSA antibodies do not seem to be associated with the development of conduction disturbances. As a consequence, it is generally accepted that the adult heart is resistant to the anti-Ro/SSA–driven immunologic damage.

However, recent evidence from our own and other institutions suggests that anti-Ro/SSA may be arrhythmogenic also for adults (4), more often as the result of a significant interference on ventricular repolarization (5). In particular, corrected QT (QTc) interval prolongation appears to be the most frequent electrocardiographic abnormality in adults with circulating anti-Ro/SSA, although showing a prevalence markedly different among the available studies, ranging from approximately 10% to 60% (6–10). Moreover, some data demonstrated the existence of an increased risk of ventricular arrhythmias, also life threatening, in anti-Ro/SSA–positive patients displaying QTc prolongation (10, 11).

Although the exact mechanisms responsible for the anti-Ro/SSA–associated QTc prolongation are not well known as yet, the possibility has been recently suggested that this antibody may inhibit rapid cardiac delayed rectifier potassium current (IKr) through a direct interaction with the potassium human ether-a-go-go–related gene (ERG) channel on the cardiomyocyte, thereby impairing ventricular repolarization (11).

On the basis of such a specific and direct autoantibody–ion channel interaction, we hypothesize that both the anti-Ro/SSA circulating levels and, as a consequence, the number of potassium channels blocked on the cardiomyocyte membrane, and the specific anti-Ro/SSA subtype involved (i.e., recognizing the 52- or 60-kd subunit), may represent crucial factors to produce (or not) a significant interference on cardiac cell electrophysiology in terms of QTc prolongation. This consideration might explain why only a percentage of anti-Ro/SSA–positive patients presents a QTc prolongation, and why this percentage is so variable throughout the studies (9).

Interestingly, a recent prospective study performed by Jaeggi and colleagues in 186 antibody-exposed fetuses and infants highlighted the importance of the maternal anti-Ro/SSA level in the development of cardiac neonatal lupus, as complete CHB occurred only in children from mothers with moderate to high (≥50 units/ml) circulating antibody concentrations (12).

On this basis, the aim of this study was to evaluate whether the anti-Ro/SSA level and specificity rather than their presence per se may represent critical factors to induce the appearance of QTc prolongation in adult patients with CTDs.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES

Study population.

Twenty-five consecutive anti-Ro/SSA– positive and 24 consecutive anti-Ro–negative CTD patients admitted to our institution for ambulatory evaluation were enrolled in the study. Anti-Ro/SSA positivity was determined by fluoroenzyme immunoassay (FEIA), an immunoenzymatic method routinely used in our institution to assess the presence and the level of total anti-Ro/SSA, irrespective of the specific subtype(s) present (see below). Demographic, clinical, and echocardiographic characteristics and ongoing treatment of study patients are detailed in Table 1. None of the patients assumed drugs potentially influencing QTc interval (class I and class III antiarrhythmics, antihistamines, quinolone and macrolide antibacterials, azole antifungals, phenothiazines, tricyclic antidepressants, cisapride), except for hydroxychloroquine. Patients did not present electrocardiogram (EKG) and/or echocardiographic abnormalities and/or a history consistent with coronary artery disease or other organic cardiomyopathies, nor were they affected with diabetes mellitus, renal failure, or electrolyte imbalances (Na+, K+, Ca++, or Mg++). Six of 49 patients were affected by mild arterial hypertension, but none presented echocardiographic findings suggesting left ventricular hypertrophy. Patients with bundle branch block (standard QRS duration >120 msec) were excluded from the study.

Table 1. Demographic, clinical, echocardiographic, and electrocardiographic characteristics and ongoing treatment in anti-Ro/SSA–positive versus –negative patients (FEIA)*
 Positive patients (n = 25)Negative patients (n = 24)P
  • *

    Patients having a total anti-Ro/SSA level >7 units/ml as determined by fluoroenzyme immunoassay (FEIA) were considered as anti-Ro/SSA positive. NS = not significant; SLE = systemic lupus erythematosus; SS = Sjögren's syndrome; UCTD = undifferentiated connective tissue disease; MCTD = mixed connective tissue disease; QTc = corrected QT; IVIG = intravenous immunoglobulin; ACE = angiotensin-converting enzyme.

  • Values of probability <0.05 were considered as significant.

  • Prednisone-equivalent dosage.

Age, mean ± SD years44.6 ± 9.340.9 ± 14.6NS
Sex, F/M21/421/3NS
Diagnosis, no.   
 SLE913 
 SS133 
 UCTD26 
 MCTD12 
Disease duration, mean ± SD years7.7 ± 6.16.3 ± 5.5NS
Patients with concomitant diseases, no.66 
 Autoimmune thyroiditis43 
 Hypertension33 
Echocardiographic findings, mean ± SD   
 Ejection fraction, %61.8 ± 5.862.7 ± 3.8NS
 Left atrial size, mm33.4 ± 4.631.9 ± 5.3NS
 Left ventricular internal dimension, mm45.5 ± 4.744.6 ± 3.9NS
 Aortic root, mm30.2 ± 2.831.1 ± 4.0NS
 Posterior wall thickness, mm8.6 ± 1.58.7 ± 1.3NS
 Ventricular septum, mm9.0 ± 1.49.1 ± 1.4NS
 Estimated pulmonary artery pressure, mm Hg25.6 ± 7.928.1 ± 6.2NS
Heart rate, mean ± SD beats/minute71.0 ± 11.770.1 ± 10.2NS
QTc, mean ± SD msec458.0 ± 29.0442.8 ± 23.10.04
Patients with prolonged QTc (≥460 msec), no. (%)12 (48)4 (17)0.03
Ongoing treatment   
 Steroids, no. (mean dosage)11 (5.3 mg/day)10 (7.7 mg/day)NS
 Hydroxychloroquine, no. (mean dosage)9 (355 mg/day)10 (330 mg/day)NS
 Azathioprine, no. (mean dosage)2 (100 mg/day)2 (125 mg/day)NS
 IVIG, no. (mean dosage)1 (10 gm/month)3 (20 gm/month)NS
 Leflunomide, no. (mean dose)1 (20 mg/day)1 (20 mg/day)NS
 Methotrexate, no. (mean dosage)1 (15 mg/week)0NS
 Pilocarpine, no. (mean dose)3 (12.5 mg/day)1 (15 mg/day)NS
 ACE/angiotensin II receptor inhibitors, no.32NS
 Diuretics, no.30NS
 Antiadrenergic drugs, no.21NS
 Thyroxine, no.11NS
 No therapy, no.24NS

The local ethical committee approved the study, and patients gave their oral and written informed consent in accordance with the principles of the Declaration of Helsinki.

In the same visit, the patients underwent a venous withdrawal to determine anti-Ro/SSA and a resting 12-lead EKG recording to measure the length of QTc interval.

Laboratory tests.

Total anti-Ro/SSA antibodies were assessed with FEIA by the EliA system (Phadia). The test uses a mixture of 60-kd and 52-kd Ro proteins as revealing antigens, and has a cutoff value of 7 units/ml as a positive result. On the basis of the results of this test, patients were classified as positive or negative, as already mentioned, and the titer of the positivity was measured. Anti-Ro/SSA subspecificities were determined by enzyme-linked immunosorbent assay (ELISA), with a cutoff value of 5 units/ml as positivity (Varelisa anti–Ro/SSA 52 kd and Varelisa anti–Ro/SSA 60 kd; Phadia). Both techniques from Phadia utilized human recombinant antigens produced in the baculovirus/insect cell system. Moreover, in all sera, the immune Western blot analysis was performed by a commercial kit (Marblot HEp-2 cells; MarDx). This test, utilizing strip Marblot, uses HEp-2 cells as the source of extractive 60-kd and 52-kd Ro antigens, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Notably, despite being more sensitive than ELISA in detecting anti-Ro/SSA, particularly anti–Ro/SSA 52 kd (13), Western blot analysis is a qualitative test only. On this basis, and in consideration that the first aim of the study was to evaluate the relationship between anti-Ro/SSA level and QTc duration, the ELISA methodology did provide the main data of the study. However, the concomitant Western blot analysis testing allowed recognition of false anti-Ro/SSA–negative patients with QTc prolongation, thereby obtaining a more realistic analysis of the anti-Ro/SSA level–QTc correlation (see below).

EKG recordings.

QTc interval was manually measured on a 12-lead EKG, according to standard criteria. We decided to employ such a method following the results of a pilot phase involving the first 26 patients enrolled, in which we measured the QTc (the longest in the 12 leads) in the same patients and in the same visit either with a manual or a computerized method, the latter based on an ambulatory 12-channel EKG recording system (Prima-Holter, Cardioline, Remco), for a 5-minute period. As the QTc values obtained with the 2 techniques were almost overlapping (only a 0.5 msec difference in the mean QTc; P = 0.93 by 2-tailed Mann-Whitney test), with a high correspondence between the 2 measurements in the single patient (r = 0.79, P < 0.0001 by Pearson's correlation test), we decided to complete the study using the manual method only, as it is faster and more practical.

In more detail, the QT interval was measured from the onset of the Q wave or the onset of the QRS complex to the end of the T wave, defined as the return to the T-P baseline. When U waves were present, the QT interval was measured to the nadir of the curve between the T and U waves. QT interval, determined as the longest hand-measured QT interval in any lead (14), was corrected for heart rate by Bazett's formula to yield the QTc value. The QTc value was calculated by dividing the QT interval by the square root of the R-R interval. QTc was measured from 3 nonconsecutive beats (mean value) by a single investigator (MA) who was blinded to the patient's antibody status. In light of the large prevalence of women in the study population (87%), QTc was considered prolonged if ≥460 msec, in accordance with the 2009 American Heart Association/American College of Cardiology Foundation/Heart Rhythm Society recommendations (14).

Statistical analysis.

Spearman's rank correlation test was employed for detecting any correlation among QTc and laboratory, demographic, clinical, and echocardiographic parameters. Statistical evaluation of the differences between the 2 groups was performed by the 2-tailed Student's t-test for unpaired data for continuous variables normally distributed, and the 2-tailed Mann-Whitney test for continuous variables not normally distributed. The 2-sided Fisher's exact test was performed to evaluate categorical variables. In any case, P values less than 0.05 were considered significant (GraphPad-InStat, version 3.06 for Windows 2000; Microsoft).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES

Anti-Ro/SSA level and specificity: findings of FEIA and ELISA analysis.

FEIA analysis demonstrated a mean ± SD total anti-Ro/SSA concentration of 154.5 ± 95.8 and 0.9 ± 0.9 units/ml in positive (n = 25) and negative (n = 24) patients, respectively (P < 0.0001 by Mann-Whitney test). Sixteen patients displayed reactivity for anti–Ro/SSA 52-kd antibodies (mean ± SD level 79.5 ± 68.9 units/ml), and 19 for anti–Ro/SSA 60-kd antibodies (mean ± SD level 77.9 ± 57.3 U/ml). Five and 8 patients showed an isolated positivity for anti–Ro/SSA 52-kd antibodies and anti–Ro/SSA 60-kd antibodies, respectively, while both antibodies were demonstrated in 11 subjects. Results of FEIA and ELISA analyses were concordant, with the exception of 3 patients (2 patients positive for total anti-Ro/SSA only, without any reactivity for anti–Ro/SSA 52 kd or anti–Ro/SSA 60 kd; 1 patient positive for anti–Ro/SSA 60 kd only, without any reactivity for total anti-Ro/SSA).

Eight patients also revealed positivity for anti-La/SSB antibodies with FEIA analysis. All of these subjects were also anti-Ro/SSA positive, with a very similar prevalence among anti–Ro/SSA 52-kd and anti–Ro/SSA 60-kd patients (6 [37%] of 16 versus 8 [42%] of 19).

Concerning the distribution of anti-Ro/SSA subtypes in specific CTDs, particularly Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE), we could simply report the values observed, as the size of the samples did not allow a reliable statistical evaluation of the differences. Indeed, with respect to SLE patients, SS subjects displayed an apparently higher prevalence of both anti–Ro/SSA 52 kd (10 [62%] of 16 versus 4 [18%] of 22) and anti–Ro/SSA 60 kd (10 [62%] of 16 versus 7 [32%] of 22). However, when we selectively considered the anti-Ro/SSA–positive patients, only the frequency of anti–Ro/SSA 52 kd positivity resulted in clearly different results in SS versus SLE patients (10 [77%] of 13 versus 4 [40%] of 10), while the frequency of anti–Ro/SSA 60 kd did not (10 [77%] of 13 versus 7 [70%] of 10).

Findings of QTc interval in relation to FEIA/ELISA results.

Considering the population as a whole, 16 patients (33%) exhibited QTc prolongation. The mean ± SD QTc (458.0 ± 29.0 versus 442.8 ± 23.1 msec; P = 0.04) and the prevalence of patients with QTc prolongation (12 [48%] of 25 versus 4 [17%] of 24; P = 0.03) were significantly higher in the anti-Ro/SSA–positive versus –negative group (Table 1). Moreover, a significant direct correlation was demonstrated between the total anti-Ro/SSA level and the QTc value in the entire population (r = 0.35, P = 0.013) (Table 2). When we considered the circulating concentration of the 2 specificities separately, this association persisted, even becoming stronger with the anti–Ro/SSA 52-kd subtype only (r = 0.38, P = 0.007) (Figure 1A), while no relationship was demonstrated with the anti–Ro/SSA 60-kd level (r = 0.24, P = not significant [NS]) or with other demographic, clinical, therapeutic, and echocardiographic parameters potentially influencing QTc duration (Table 2).

Table 2. Correlation of laboratory, demographic, clinical, and echocardiographic parameters with corrected QT values (n = 49)*
 rP
  • *

    Statistical analysis was performed with Spearman's rank correlation. NS = not significant.

  • Values of probability <0.05 were considered as significant.

Total anti-Ro/SSA level, units/ml0.350.013
Anti–Ro/SSA 52-kd level, units/ml0.380.007
Anti–Ro/SSA 60-kd level, units/ml0.24NS
Age, years0.08NS
Disease duration, years−0.15NS
Hydroxychloroquine use−0.03NS
Ejection fraction, %−0.08NS
Ventricular septum thickness, mm−0.03NS
thumbnail image

Figure 1. Relationship between corrected QT (QTc) duration and anti–Ro/SSA 52-kd level A, in the entire study population, and B, in the patients with concordant enzyme-linked immunosorbent assay and Western blot analysis results.

Download figure to PowerPoint

These results were strengthened by the fact that comparing the mean antibody level in patients with or without QTc prolongation, we found that patients with a prolonged QTc had higher levels of total anti-Ro/SSA and anti–Ro/SSA 52 kd, but not anti–Ro/SSA 60 kd, with respect to those with a normal QTc (Table 3). Moreover, a longer QTc interval was detected in the patients with the highest anti-Ro/SSA antibody titer. In fact, classifying the patients on the basis of the antibody level in accordance with Jaeggi et al (12), we found that in the group with a moderate to high level (≥50 units/ml), the mean QTc interval and the prevalence of QTc prolongation were both significantly higher in comparison to the group with low positive/negative antibody levels (<50 units/ml) (Tables 4 and 5). Also in this case, a statistically significant difference existed only when considering the level of total anti-Ro/SSA or anti–Ro/SSA 52 kd (not anti–Ro/SSA 60 kd). Moreover, it must be noted that in all of these comparisons, the significant difference for total anti-Ro/SSA always reached a higher level of statistical significance, when in the comparison the anti–Ro/SSA 52-kd subtype only was considered (Tables 3–5).

Table 3. Mean ± SD circulating levels of anti–Ro/SSA antibodies in connective tissue disease patients with or without QTc prolongation (≥460 msec)*
 Patients with QTc ≥460 msec (n = 16)Patients with QTc <460 msec (n = 33)P
  • *

    Statistical analysis was performed with the Mann-Whitney test. QTc = corrected QT; NS = not significant.

  • Values of probability <0.05 were considered as significant.

Total anti-Ro/SSA, units/ml116.0 ± 104.062.1 ± 98.90.027
Anti–Ro/SSA 52 kd, units/ml50.5 ± 62.414.7 ± 45.20.003
Anti–Ro/SSA 60 kd, units/ml50.0 ± 63.823.7 ± 44.7NS
Table 4. Comparison of mean ± SD corrected QT values (msec) in connective tissue disease patients with moderate to high positive (≥50 units/ml) and low positive/negative (<50 units/ml) circulating levels of anti-Ro/SSA*
 Patients with antibody levels ≥50 units/mlPatients with antibody levels <50 units/mlP
  • *

    Statistical analysis was performed with unpaired t-test. NS = not significant.

  • Values of probability <0.05 were considered as significant.

Total anti-Ro/SSA461.6 ± 30.1443.5 ± 22.50.021
Anti–Ro/SSA 52 kd470.5 ± 27.2445.4 ± 24.90.008
Anti–Ro/SSA 60 kd457.5 ± 34.9448.3 ± 24.1NS
Table 5. Prevalence of corrected QT prolongation (≥460 msec) in connective tissue disease patients with moderate to high positive (≥50 units/ml) and low positive/negative (<50 units/ml) circulating levels of anti-Ro/SSA antibodies*
 Patients with antibody levels ≥50 units/mlPatients with antibody levels <50 units/mlP
  • *

    Values are the number/total (percentage). Statistical analysis was performed with Fisher's exact test. NS = not significant.

  • Values of probability <0.05 were considered as significant.

Total anti-Ro/SSA10/19 (53)6/30 (20)0.028
Anti–Ro/SSA 52 kd7/10 (70)9/39 (23)0.008
Anti–Ro/SSA 60 kd6/12 (50)10/37 (27)NS

Concerning the distribution of QTc prolongation among the different diseases, the size of the samples allowed a comparison (although not statistically evaluable) between the 2 subgroups of SS and SLE patients only. With respect to those affected with SLE, SS patients apparently displayed a higher prevalence of QTc prolongation, considering the 2 populations either as a whole (3 [14%] of 22 versus 7 [44%] of 16) or the anti-Ro/SSA–positive patients only (3 [30%] of 10 versus 7 [54%] of 13).

On the contrary, when (on the basis of the above findings) our evaluation specifically regarded the patients with a moderate to high level (≥50 units/ml) of anti–Ro/SSA 52 kd, the frequency of QTc prolongation in SS and SLE practically coincided (5 [71%] of 7 versus 2 [67%] of 3).

Finally, anti-La/SSB status did not seem to influence the occurrence of QTc prolongation. In fact, no association existed between the circulating antibody level and the QTc length (r = 0.22, P = NS), and no difference in the prevalence of QTc prolongation was found in anti–Ro/SSA 52-kd–positive patients with or without concomitant anti-La/SSB positivity (3 [50%] of 6 versus 5 [50%] of 10).

Findings of Western blot analysis.

Although the above results are rather clear, during the analysis of the data we noted that a patient with marked QTc prolongation displayed partially conflicting results from the FEIA and ELISA analyses. In fact, she had a moderate to high positivity for total anti-Ro/SSA (83 units/ml) in FEIA, but just a low reactivity only (8.6 units/ml) for anti–Ro/SSA 60 kd in ELISA (anti–Ro/SSA 52-kd antibodies were negative at 0.9 units/ml). These data also conflicted with the result of a previous Western blot analysis (performed in 2000) clearly demonstrating the presence of anti–Ro/SSA 52 kd in the serum. Because of this, we repeated Western blot analysis, which confirmed the 2000 datum. On this basis, suggesting that the ELISA test was probably unable to detect the specific anti–Ro/SSA 52-kd subtype present in the patient (possibly explaining the antibody “concentration gap” existing between the results obtained in FEIA and ELISA), we decided to perform the Western blot analysis in all of the 49 patients included in the study.

With respect to the immunoenzymatic methods, Western blot analysis demonstrated a higher sensibility in selectively detecting anti–Ro/SSA 52 kd. In fact, anti–Ro/SSA 52-kd antibodies were found in 25 of 49 patients (versus 16 demonstrated by ELISA), whereas anti–Ro/SSA 60-kd antibodies were revealed by Western blot analysis in only 14 patients (versus 20 with ELISA). Interestingly, 5 of 11 ELISA-negative subjects displaying anti–Ro/SSA 52-kd positivity with Western blot analysis had a prolonged QTc; moreover, 6 subjects with anti–Ro/SSA 52 kd in Western blot analysis were totally negative for anti-Ro/SSA (not only in ELISA but also in FEIA), and 2 of them showed a QTc prolongation. As a consequence, when detecting the presence of anti-Ro/SSA by combining the immunoenzymatic methods (ELISA and FEIA) and Western blot analysis results, the number of patients with QTc prolongation displaying anti-Ro/SSA positivity further increased from 75% (12 of 16) to 87.5% (14 of 16).

Moreover, the fact that several patients showing anti–Ro/SSA 52 kd in Western blot analysis were negative when evaluated in ELISA (<5 units/ml) suggests that in these subjects, the anti–Ro/SSA 52-kd concentration detected by the immunoenzymatic method was not indicative of the antibody titer really present in the blood, thus possibly underestimating the QTc–anti-Ro/SSA 52-kd level correlation. Accordingly, when we considered only those patients showing a concordance between ELISA and Western blot analysis results (n = 38), the strength of the correlation existing between the 2 parameters markedly increased (r = 0.47, P = 0.003) (Figure 1B).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES

The main findings of the present study are the following: in adult patients with CTD, the occurrence of QTc prolongation is specifically associated with the presence of circulating anti–Ro/SSA 52 kd, with the length of QTc interval directly correlating with the level of this anti-Ro/SSA subtype in the serum. Moreover, the combined use of immunoenzymatic methods and Western blot analysis seems to be the most sensible approach for the recognition of anti-Ro/SSA–positive CTD patients with QTc prolongation.

Increasing evidence suggests that QTc prolongation is a relatively common finding in adult patients with CTD and circulating anti-Ro/SSA antibodies, with a possible higher risk of developing life-threatening ventricular arrhythmias with respect to anti-Ro/SSA–negative CTD patients (9). However, the available studies on this topic reported nonhomogeneous results, particularly regarding the actual prevalence of QTc prolongation.

In fact, in a cohort of adult patients affected with several types of CTDs, we found that more than one-half (58%) of anti-Ro/SSA–positive subjects displayed a prolonged QTc, with mean QTc values significantly longer in positive versus negative patients (6). Accordingly, a similar prevalence of anti-Ro/SSA–associated QTc prolongation (46%) was demonstrated in a further 24-hour EKG monitoring study also showing that this EKG abnormality lasted throughout the day and was associated with the presence of complex ventricular arrhythmias (10). However, partially conflicting results were obtained by other studies. Costedoat-Chalumeau and colleagues (7) did not observe any significant differences in mean QTc interval when comparing anti-Ro/SSA–positive with –negative patients affected with CTD, while Gordon et al (15), despite not observing significant differences between the 2 groups, however, found a slightly longer QTc in the Ro/SSA–positive group, and this difference was very close to statistical significance (P = 0.063). It should be stressed that in both of these studies, the prevalence of SLE patients was particularly high (approximately 90% and 70%, respectively), while in the 2 studies performed in our institution, it ranged between 20% and 30%. As a result, it is possible that in SLE patients the QTc prolongation is less frequent and therefore less easily detectable than in the other CTDs.

Data from a Canadian group seem to confirm this view. Bourre-Tessier and colleagues (8) performed 2 consecutive large studies on 150 and 278 patients affected with SLE (310 patients in total, 118 participating in both studies). Using a multivariate logistic regression analysis, the authors found a 5.1–12.6 times higher risk of QTc prolongation in the anti-Ro/SSA–positive group than in negative patients. Interestingly, the percentage of anti-Ro/SSA–positive patients with QTc prolongation was far lower in these studies (11–16%) with respect to our studies (∼50%) (9). However, despite having a comparable percentage of QTc prolongation (16%) and patients with very similar characteristics, Costedoat-Chalumeau et al (7) did not find any difference between the 2 groups, unlike Bourre-Tessier et al (8). The reason may consist of the fact that the phenomenon is probably less frequent in SLE patients, thus requiring a larger sample size to be observed in a significant way. Taking this into consideration, it also seems conceivable how in the study by Gordon et al (15), in which the preponderance of SLE patients was less marked, differences in QTc approached statistical significance.

The results of the present study, strongly suggesting the selective involvement of the anti–Ro/SSA 52-kd subtype in the development of anti-Ro/SSA–associated QTc prolongation, may help clarify the pathogenetic basis of this phenomenon. In fact, it has been demonstrated that in SLE patients, the prevalence (16) and the mean signal intensity (17) of anti–Ro/SSA 52-kd antibodies are both significantly lower than in other CTDs, particularly SS. On this basis, and in consideration of the critical role here emerging for anti–Ro/SSA 52-kd circulating levels in the development of QTc prolongation, it seems well plausible that this EKG abnormality is less common in SLE patients, thus putatively accounting for the differences among the available studies. Despite the small size of the samples, the findings of the present study of an anti–Ro/SSA 52 kd and QTc prolongation prevalence in SS versus SLE seem to be consistent with such a view.

At the moment, the specific mechanisms possibly linking the presence of anti-Ro/SSA with a prolonged QTc have not been clearly elucidated. However, in an emblematic case report, Nakamura and colleagues (11) showed that both the serum and purified IgG from an anti-Ro/SSA–positive patient with marked QTc prolongation (700 msec) and life-threatening ventricular arrhythmias (torsades de pointes) were able to directly bind the human ERG potassium channel and selectively block the related IKr, one of the main currents involved in ventricular repolarization. Moreover, many data from Karnabi and Boutjdir (18) clearly demonstrated the ability of anti-Ro/SSA antibodies, particularly anti–Ro/SSA 52 kd, to cross-react with and inhibit calcium channels (L type and T type), thus providing a possible explanation for the development of conduction abnormalities in the fetus. This evidence, together with the fact that potassium and calcium channels share many structural similarities since they belong to the same superfamily of the voltage-gated ion channels (19), suggests the intriguing hypothesis that rhythm disturbances observed in the fetus and the adult are the consequence of an autoimmune reaction involving anti–Ro/SSA 52 kd and specific cardiac ionic channels (9). This view, implying the existence of a molecular mimicry between these channels and the Ro 52-kd protein, is further strengthened by the significant and selective relationship between the QTc interval and anti–Ro/SSA 52-kd level shown in the present study. Moreover, Ro 60-kd and Ro 52-kd proteins, although physically and functionally strictly associated, display relevant differences either in the molecular structure or in epitope recognition. In fact, while in Ro 60-kd protein, which possesses an RNA binding domain, the autoepitopes recognized are highly conformational, in the context of the Ro 52-kd protein, showing zinc finger and leucine zipper domains without a specific RNA binding site, the autoantibodies recognize only linear epitopes (usually placed in the leucine zipper site) (3). These differences appear to be consistent with the finding that only 1 of the 2 subtypes of anti-Ro/SSA is associated with the QTc interval and then putatively implicated in this autoimmune cross-reaction.

Another important result of this work is that Western blot analysis is very effective in detecting anti–Ro/SSA 52 kd, also revealing that a number of ELISA-negative patients with QTc prolongation were actually anti–Ro/SSA 52-kd positive. As a result, the combined use of immunoenzymatic methods and Western blot analysis in our population demonstrated the presence of circulating anti-Ro/SSA in approximately 90% of the patients displaying a prolonged QTc. These data, supporting the need of using both diagnostic methods to improve the recognition of anti–Ro/SSA 52-kd–positive patients (also in the view to assessing the actual risk of delivering a child with CHB), showed that this approach is the most sensible for detecting anti-Ro/SSA–positive CTD patients with QTc prolongation.

Similar to Western blot analysis, previous data also demonstrated that line blot immunoassay (immunoblot) had a higher sensibility in detecting anti–Ro/SSA 52 kd with respect to ELISA and FEIA (13). However, although Western blot analysis utilizes the native Ro 52-kd protein extracted from HEp-2 cells, the antigen employed in immunoblot is a Ro 52-kd protein of recombinant origin (similar to ELISA and FEIA). As a result, although the basis of the different sensibility among the methods is not clear, nevertheless it does not seem related to the characteristics of the Ro 52-kd antigen in use, but rather to technical factors specifically involved in the blotting procedures. Anyway, since only a part of the studies evaluating the possible association between anti-Ro/SSA and QTc prolongation in adults also used Western blot analysis to determine the antibody presence, this datum may contribute to explaining the different prevalence of the phenomenon throughout these studies.

Last but not least, the demonstration that anti–Ro/SSA 52-kd antibodies are a key factor in producing QTc prolongation in our patients suggests the intriguing hypothesis that such autoantibodies may also play a role in the development of life-threatening ventricular arrhythmias in the general population. In fact, in accordance with the recent evidence that Ro 52 kd represents one of the most immunogenic human proteins (20), anti-Ro/SSA antibodies are highly prevalent in the general population (up to approximately 3%), but in most cases (60–70%) totally asymptomatic (21), particularly when anti–Ro/SSA 52-kd positivity occurs alone (22). On this basis, anti–Ro/SSA (specifically anti–Ro/SSA 52 kd), by reducing the repolarization reserve, may be possibly silently involved as a predisposing factor in a number of “idiopathic” life-threatening arrhythmias, including drug-induced torsades de pointes, and sudden unexpected deaths occurring in the general population. Accordingly, in the above-reported case by Nakamura et al (11), the patient was totally asymptomatic for autoimmune disease, and the presence of high circulating levels of anti-Ro/SSA arose only because it was specifically tested (on the basis of the results of previous studies [6, 10]) after the exclusion of all of the known causes of QT prolongation, including drugs, electrolyte and hormone imbalance, structural heart disease, and mutations in the genes responsible for the congenital long QT syndrome. In this sense, anti-Ro–associated QTc prolongation may represent a new form of acquired long QT syndrome of autoimmune origin.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Lazzerini had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Lazzerini.

Acquisition of data. Lazzerini, Acampa, Bellisai, Bacarelli, Dragoni, Fineschi, Simpatico.

Analysis and interpretation of data. Lazzerini, Capecchi, Acampa, Morozzi, Galeazzi, Laghi-Pasini.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES

We thank Dr. Cinzia Montilli for her assistance in the collection of the data.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. AUTHOR CONTRIBUTIONS
  8. Acknowledgements
  9. REFERENCES
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