Pediatr Cardiol (2015) 36:165–170 DOI 10.1007/s00246-014-0981-8
ORIGINAL ARTICLE
The Value of Acetylcholine Receptor Antibody in Children with Postural Tachycardia Syndrome Jiawei Li • Qingyou Zhang • Ying Liao • Chunyu Zhang • Hongjun Hao • Junbao Du
Received: 15 April 2014 / Accepted: 22 July 2014 / Published online: 3 August 2014 Ó Springer Science+Business Media New York 2014
Abstract Postural tachycardia syndrome (POTS) is characterized by symptoms of orthostatic intolerance. Antibodies of acetylcholine receptor (AChR-ab) affect acetylcholine transmission between the ganglia and result in imbalance of the autonomic nervous system in POTS. This study was designed to analyze the clinical characteristics of POTS patients with AChR-ab positive and explore the value of AChR-ab in children with POTS. In 82 children with POTS, twenty patients (24.39 %) were found as AChR-ab positive. Their clinical characteristics and hemodynamic responses to orthostatic changes were compared with the remaining 60 patients with negative AChRab. Symptoms of POTS children with AChR-ab positive were significantly severe than those of AChR-ab negative patients (p = 0.001). Preceding infection was predominant in patients with AChR-ab positive compared with that of patients with AChR-ab negative (p \ 0.001). Syncope and fatigue were more common in the AChR-ab positive patients (p \ 0.05). The change of upright heart rate was increased significantly in AChR-ab positive patients compared with AChR-ab negative cases (p = 0.013). Multiple logistic regression analysis revealed that preceding infection (OR 22.356, 95 % CI 2.151–34.920), syncope (OR 11.570, 95 % CI 2.098–63.810), and fatigue (OR 11.145, 95 % CI 1.658–74.911) were independent risk factors for
Jiawei Li and Qingyou Zhang have contributed equally to this work. J. Li Q. Zhang Y. Liao C. Zhang J. Du (&) Department of Pediatrics, Peking University First Hospital, Xi-An Men Street No. 1, West District, Beijing 100034, China e-mail: [email protected] H. Hao Department of Neurology, Peking University First Hospital, Beijing 100034, China
POTS with AChR-ab positive. In conclusion, POTS with positive AChR-ab was a heterogeneous disorder. Preceding infection, syncope and fatigue were their main clinical characteristics. Keywords Acetylcholine receptor antibody Children Clinical characteristics Orthostatic intolerance Postural tachycardia syndrome
Introduction Postural tachycardia syndrome (POTS) is characterized by symptoms of orthostatic intolerance in association with excessive tachycardia and relief of these symptoms with recumbence [4, 7]. The clinical features of POTS vary with patients, and symptom severity can be quite disabling in pediatric populations, as these disorders have significant social and health impacts on patients [6]. Although the exact mechanism responsible for POTS has not yet been fully understood, one of the most confident etiologies is the imbalance in the autonomic nervous system of the sympathetic and parasympathetic nervous systems [7, 17]. Acetylcholine, a primary neurotransmitter in the autonomic ganglia in autonomic nervous system including both the sympathetic and parasympathetic nervous systems, can be mediated by the acetylcholine receptor (AChR) of the autonomic ganglion. As preliminary studies reported, acetylcholine receptor antibodies (AChR-ab) were found in some patients with POTS [14]. Antibodies of ganglionic AChR affect acetylcholine transmission between the ganglia, causing imbalance in the autonomic nervous system [17]. Pyridostigmine, an acetylcholinesterase inhibitor, contrary to the role of AChR-ab in the autonomic nervous system, markedly reduced upright heart rate (HR) and
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attenuated the symptoms of POTS patients [9, 12]. Researchers reported autoimmunoreactive IgGs in patients with POTS, supporting the notion that AChR-ab, mainly a form of IgGs, played a role in the pathogenesis of POTS as well [19]. Some authors described a subtype of POTS which was regarded as ‘‘autoimmune’’ or ‘‘neuropathic’’ POTS that was patients with detectable levels of antibodies to the AChR (AChR-ab positive POTS) [7, 10, 14]. As a heterogeneous group of POTS, AChR-ab positive POTS patients may have specific clinical features. Effectively identifying these clinical features would greatly enable us to deepen the understanding of etiology and treatment for POTS. This study was designed to analyze the clinical features of POTS patients with positive AChR-ab and explore the diagnostic value of serum AChR-ab in children with POTS.
Methods Subjects This prospective study involved children who were admitted to the Department of Pediatrics, Peking University First Hospital and were diagnosed as having POTS by the head-up tilt test (HUTT) between June 2012 and December 2013. Totally, 82 POTS patients aged 11.09 ± 2.56 years were included in this study, of whom 43 (52.44 %) were girls and 39(47.56 %) were boys. The physical examination, 12-lead electrocardiography, and electroencephalogram were normal in all subjects enrolled. The study protocol adhered to the principles of the Declaration of Helsinki and conformed to the criteria of the Ethics Committee of Peking University First Hospital (Beijing, China). All participant’s guardians were fully informed of the purpose and methods of our study, and informed consent was obtained. Diagnostic Criteria The present study referred to the following criteria for POTS [1, 2, 13, 21]. (1) A child has a normal HR when supine and no evidence of cardiovascular disease; (2) after standing up, a child has C2 of the following symptoms: dizziness, chest distress, headache, palpitations, pallor, tremulousness, amaurosis, fatigue, or syncope, which should be relieved or diminished by recumbence and should occur repeatedly for C1 month; (3) in addition to symptoms of orthostatic intolerance (OI), the child displayed a HR increase C40 beats/min or a HR [120 beats/ min within the first 10 min during HUTT. Simultaneously, the decrease in blood pressure should be \20/10 mm Hg; and (4) a child without other diseases that manifested
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Pediatr Cardiol (2015) 36:165–170 Table 1 Standard of symptom scoring for orthostatic intolerance Score
Frequency of orthostatic intolerance symptoms
0
Never
1
Once per month
2
Twice to 4 times per month
3
Twice to 7 times per week
4
More frequent than once per day
symptoms in the autonomic nervous system (e.g., anemia, arrhythmia, hypertension, and endocrine disorders) as well as cardiac or neurologic diseases that would induce syncope. History Collection and Symptom Scoring All children or their parents were asked to report the patient’s symptom burden by using a standardized questionnaire. Its main content included disease frequency, duration, incentives, concomitant symptoms, family history of OI, history of allergy, and preceding infection. To avoid inter-rater variability, all interviews were performed by the same investigator who was blind for HUTT and the results of AChR-ab at the time of interview. To determine the severity of symptoms during the study period, a structured questionnaire of scoring which demonstrated validity and reliability was employed, which was detailed in the previous published literature [20–22]. Scorings were based mainly on the typical symptoms of OI, including syncope, dizziness or lightheadedness, chest tightness, nausea, palpitation, and headache. A numerical value for each symptom was determined by its frequency, and the total score was the sum of all the symptoms scores (Table 1). Protocol for HUTT HUTT is the standard method for assessing POTS. The protocol for HUTT was according to the previously published literatures [1, 21]. As preparation, participants were told to avoid caffeine, high-fat foods, and vasoactive drugs with C4 half-lives. All examinations were performed in a quiet environment with dim light and comfortable temperature. After blood pressure and HR were recorded at the baseline and after the subjects had been in a supine position for at least 10 min, they were positioned upright on the tilt table at an angle of 60 degree, with a footboard being used to bear the subject’s weight. Blood pressure and HR monitoring and electrocardiography were performed simultaneously during the test, by using a Dash 2000 Multi-Lead Physiological Monitor (GE Company, New York, New York). The test would be terminated when a
Pediatr Cardiol (2015) 36:165–170 Table 2 Baseline characteristics and hemodynamic parameters in POTS patients with a different AChR-ab status
POTS postural tachycardia syndrome, AChR-ab acetylcholine receptor antibody, IQR interquartile range, OI orthostatic intolerance, SBP systolic blood pressure, DBP diastolic blood pressure, HR heart rate, Delta SBP changes of SBP from supine to standing, Delta HR heart rate increase to head-up tilt test *
p \ 0.05 versus AChR-ab negative group, **p \ 0.01 versus AChR-ab negative group
167
Items
AChR-ab negative group (n = 62)
AChR-ab positive group (n = 20)
Statistics
p value
Age (years)
10.93 ± 2.65
11.60 ± 2.23
t = -1.018
Sex ratio, no. of female (%)
34 (54.84)
9 (45.00)
v2 = 0.587
0.444
Symptom score
6.53 ± 3.39
9.75 ± 3.77**
t = -3.595
0.001
Symptom duration (mo), median (IQR)
4.00 (2.00–23.00)
11.00 (1.82–24.00)
z = -0.666
0.506
Family history of OI, no. (%)
13 (20.97)
6 (30.00)
v2 = 0.693
0.405
2
Allergic history, no. (%)
11 (17.74)
2 (10.00)
v = 0.223
Preceding infection, no. (%) Supine SBP (mmHg)
14 (22.58) 99.44 ± 7.98
14 (65.00)** 101.15 ± 8.49
v2 = 12.321 t = -0.822
0.312
0.637 \0.001 0.413
Supine DBP (mmHg)
56.97 ± 7.72
57.05 ± 6.37
t = -0.043
0.966
Supine HR (beats/min)
75.71 ± 11.26
74.50 ± 5.99
t = -0.618
0.648
Standing SBP (mmHg)
107.66 ± 9.99
110.25 ± 10.39
t = -0.998
0.321
Standing DBP (mmHg)
65.32 ± 8.28
64.25 ± 9.27
t = -0.489
0.626
Standing HR (beats/min)
122.23 ± 13.12
128.30 ± 12.22
t = -1.830
0.071
Delta SBP (mmHg)
8.23 ± 7.32
9.10 ± 10.34
t = -0.418
0.677
Delta HR (beats/min)
46.52 ± 11.18
53.80 ± 11.20*
t = -2.532
0.013
positive response of increment in HR C40 beats/min or an absolute HR C120 beats/min appeared within 10 min and a diagnosis of POTS would be made. AChR-ab Detecting After being fasting for 8–12 h and rested for at least 10 min, patients were kept in supine position, and a 2 mL aliquot of blood was obtained by venipuncture into a tube with ethylenediaminetetra-acetic acid. The blood was centrifuged at 2000g for 5 min at 4 °C. Serum samples were stored at 20 °C until assay. AChR-ab were measured by enzymelinked immunosorbent assay (ELISA) in a standardized manner. Qualitative ELISA Kit (CUSABIO, human AChR antibody ELISA Kit, Wuhan, China) was used following manufacturer’s guideline. The 96-well flat-bottomed polystyrene plates coated with purified AChR were prepared in the kit. The sample serum (1:10) was added into every reaction hole and incubated overnight at 4 °C. After that, the plates were washed for four times and incubated with HRPconjugated goat-anti-human IgG (1:1000) for another 2 h at 37 °C. Chromogenic reagent A and B solutions were added each 50 lL after four washes, with the seal plate film sealed the reaction hole. The reaction was allowed to develop at 37 °C in the dark for 15 min. Finally, 50-lL termination solution was joined in every reaction well to terminate the chromogenic reaction. Results were read by a microplate reader at an optical density (OD) of 450 nm, and expressed as OD values standard deviation (SD). Positive status of AChR antibodies was defined as a ratio (sample OD/negative OD) C2.1. Otherwise, the status of AChR-ab was defined negative.
Statistical Analysis Statistical analysis was performed using SPSS software, version 13.0 (SPSS, Chicago, Illinois, U.S.A.). Continuous variables are presented as the mean ± SD, and discrete data are presented as the median (interquartile range, IQR) or as percentages. The single Kolmogorov–Smirnov test was used to verify whether the data were normally distributed. Comparisons between two groups were performed using the independent t test for measurement data, Chi square test for categorical variables, and the rank sum test for discrete data. Logistic regression analysis was used to identify independent predictors for POTS of positive AChR-ab. A p value of \ 0.05 was considered statistically significant.
Results Clinical Characteristics of POTS Patients with AChR-ab Positive The clinical features of POTS patients with a different AChR-ab are summarized in Table 2. AChR-ab positive status was presented in 20 (24.39 %) of the children with POTS. Most patients had experienced symptoms for a few months before diagnosis. The median duration of symptoms was 9.5 months, with interquartile range of 2–24 months. As was shown in Table 2, age, sex ratio, symptom duration, family history, and allergic history did not differ between the AChR-ab positive and AChR-ab negative groups (p [ 0.05). Symptom scores of AChR-ab
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Pediatr Cardiol (2015) 36:165–170 Table 3 Association of the factors for POTS patients with AChR-ab positive Indices
B
OR (95 % CI)
p value
Syncope
2.448
11.570 (2.098–63.810)
Fatigue
2.411
11.145 (1.658–74.911)
0.013
Preceding infection
3.107
22.356 (2.151–34.920)
\0.001
0.005
POTS postural tachycardia syndrome, AChR-ab antibodies to acetylcholine receptor, OR odds ratio, CI confidence interval
Fig. 1 The frequency of orthostatic symptoms prescribed in children POTS with a different AChR-ab status. POTS postural tachycardia syndrome, AChR-ab antibodies to acetylcholine receptor, *p \ 0.05 versus AChR-ab negative group
positive POTS were significantly higher than that of AChR-ab negative patients (t = -3.595, p = 0.001). Preceding infection was predominant in patients with AChRab positive (65 %) but it was found in only 22.58 % of patients with AChR-ab negative (v2 = 12.321, p \ 0.001). Commonly reported symptoms of study subjects were detailed (Fig. 1). Dizziness was the most common symptom in both groups. Compared with AChR-ab negative patients, orthostatic syncope and fatigue were more common in the AChR-ab positive patients (p \ 0.05). Other symptoms, such as shortness of breath, gastrointestinal symptoms, fatigue, pallor, blurred vision, palpitation, sweating, and tremulousness were not statistically different between the two groups (p [ 0.05). Hemodynamics of AChR-ab Positive POTS Patients The hemodynamic data of POTS patients in HUTT are shown in Table 2. There were no statistical differences of hemodynamic data between the two groups in supine or standing systolic blood pressure (SBP), diastolic blood pressure (DBP), HR, and postural change in SBP (p [ 0.05). However, change of HR (delta HR) increased significantly in AChR-ab positive group than in AChR-ab negative group (t = -2.532, p = 0.013). Logistic Regression Analysis of Independent Risk Factors for AChR-ab Positive POTS We used forward-stepwise regression analysis and entered gender, age, delta HR, syncope, fatigue, and preceding infection as independent variables. Multiple logistic regression analysis revealed that preceding infection (OR 22.356, 95% CI 2.151–34.920, p = 0.005), orthostatic syncope (OR: 11.570, 95%CI: 2.098-63.810, p = 0.013), and fatigue (OR: 11.145, 95%CI: 1.658-74.911, p \ 0.001) were independent risk factors for POTS with AChR-ab positive (Table 3).
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Discussion POTS is not a single condition, but rather a heterogeneous group of disorders resulting in a similar physiological state [6, 7, 10]. Due to multiple mechanisms of potential pathophysiology, patients with POTS have varied manifestations [4, 7]. Successful treatment depends on identifying the etiology and pursuing a comprehensive treatment program. Several authors attempted to classify POTS into subtypes, based on clinical and ancillary test results. In some recent studies of adult cases, researchers described three groups of patients with POTS [7, 10, 14]. The first comprised ‘‘hypovolemic’’ POTS, defined by a decrease in 24 h urinary sodium excretion. The second group comprised ‘‘hyperadrenergic’’ POTS, where patients with increased plasma norepinephrine levels after standing ([600 pg/mL). The third group comprised ‘‘neuropathic’’ or ‘‘autoimmune’’ POTS, where patients were AChR-ab positive, as we studied in this article. We found that AChR-ab positive POTS had frequent syncope and fatigue, with increased changes of HR and symptom scores compared with AChR-ab negative POTS. Preceding infection was also predominant. Multiple logistic regression analysis revealed that preceding infection, syncope, and fatigue were independent risk factors for POTS with AChR-ab positive. AChR-ab, which consist mainly of the IgG, were autoimmune antibodies that can block the membrane of AChR and inhibited AChR current [18]. Nicotinic AChR was divided into two subtypes, N1 and N2 types. N1 type is located in postsynaptic membrane of ganglion, raising excitability of autonomic ganglia postganglionic neurons; while N2 type is located in the membrane of endplate, which can regulate endplate potentials and contract muscles. When AChR-ab is identified and bond with AChR of N1 subtype, they inhibited ligand-gated cation channel of the AChR open and decreased cholinergic transmission in the autonomic ganglia of both the sympathetic and parasympathetic tones. Thus, the autonomic nervous function of POTS patients was impaired [16, 18]. Gibbons et al. [3] reported that serum AChR-ab levels in AAG correlated with the severity of autonomic dysfunction. Considering
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that POTS was a form of AAG with relatively mild symptoms [17], this result was comparable with what we found that AChR-ab positive POTS had more serious symptoms. AChR-ab is attached to N2 subtype of AChR in postsynaptic membrane of neuromuscular junction and caused neuromuscular transmission disorders. Symptom of fatigue therefore appeared, and this was also the pathogenesis of myasthenia gravis [15]. The hemodynamic change from the supine to upright position is that amount of blood flow from the chest to limbs and abdomen. The reduction in cardiac filling exerts continuous inhibitory action on the cardiovascular areas of the medulla (in particular in the nucleus tractus solitary), and the decline arterial pressure causes stimulation of the low pressure receptors that lie in the aortic arch and the carotid sinus. These changes produce an increase in sympathetic output that results in an increase in HR and systemic vasoconstriction, thus maintaining normal perfusion of brain. The rapid HR then reaches to a steady state by the role of vagus nerve. The failure of the system can result in an inability to respond in a normal manner to sudden changes in posture and cause illness of POTS [8]. Acetylcholine is the primary neurotransmitter of the autonomic ganglia in both the sympathetic and parasympathetic nervous systems. AChR-ab can impact sympathetic modulation and cause failure of the peripheral vasculature to vasoconstriction appropriately during upright posture [17, 19]. In the meantime, HR and cardiac contractility may increase sufficiently to maintain blood pressure at relatively normal levels. However, such mechanism usually cannot be fully compensatory or cannot be able to be maintained for a long time, thus resulting in reduction of cerebral blood flow in POTS patients [8]. That is why those POTS patients with AChR-ab positive were more vulnerable to orthostatic syncope. AChR-ab also interferes parasympathetic function of slowing HR. This was keeping with what we found that the AChR-ab positive POTS with HR variation was greater than AChR-ab negative POTS. In addition, researchers reported that about 15 percent of POTS patients were seropositive for AChR antibodies in adults [14], while the incidence in children POTS was unknown. We reported that the percentage of AChR-ab positive was 24.39 % for children POTS, which was higher than that of adults. We speculated that it was because children had relatively low immunity and were more vulnerable to infection. Meanwhile, authors reported the relationship between infection and POTS [7, 14], which was also verified by our result that preceding infection was the risk factor of POTS patients with AChR-ab positive. Mechanisms explained were as follows: when patients had bacterial or viral infection, immune tolerance was broken; or the structure of antigen cluster in viral surface and
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AChR cluster was similar and common antigen-antivirus antibodies were produced, and immune cross reactions appeared; moreover, virus infected and sensitized AChR of muscle-like epithelial cells and other cells in thymus [11]. These reactions can induce AChR-ab which breaks the balance of the autonomic nervous system. It should be taken seriously in clinical diagnosis and treatment of POTS that preceding infection induces serum AChR-ab and POTS symptoms. Greater attention has been paid to the targeted treatments in patients with POTS [9]. In this paper, we analyzed the clinical features of AChR-ab positive POTS so as to help clinicians consider this group of patients based on the clinical features. Of course, timely detection of the AChR-ab was meaningful for the cases. Therefore, our single center experience suggested that targeted therapy should be considered based on specific clinical features of this group of POTS cases, which would be preferably confirmed by laboratory antibody testing. Study Limitations The present study has the following limitations. The number of AChR-ab positive POTS patients is relatively small (n = 20). Further studies with larger numbers of cases are needed. However, the present study on POTS patients with or without positive AChR-ab, using ELISA as a simple, stable and reliable method, would greatly deepen the understanding of this heterogeneous disorder.
Conclusion AChR-ab positive POTS patients are a heterogeneous group with preceding infection, syncope, and fatigue as their main clinical characteristics. Etiological treatments of acetylcholinesterase inhibitors and immunotherapy might be the best choice for them [5, 12]. Effectively identifying POTS patients with AChR-ab positive is important for an individualized treatment of POTS. Acknowledgments This work was supported by grants from the Beijing Committee of Science and Technology, China (Z121107001012051) and the National Twelfth 5-Year Plan for Science and Technology Support (2012BAI03B03). Conflict of interest The authors declare that they have no conflicts of interest.
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ORIGINAL ARTICLE
The Value of Acetylcholine Receptor Antibody in Children with Postural Tachycardia Syndrome Jiawei Li • Qingyou Zhang • Ying Liao • Chunyu Zhang • Hongjun Hao • Junbao Du
Received: 15 April 2014 / Accepted: 22 July 2014 / Published online: 3 August 2014 Ó Springer Science+Business Media New York 2014
Abstract Postural tachycardia syndrome (POTS) is characterized by symptoms of orthostatic intolerance. Antibodies of acetylcholine receptor (AChR-ab) affect acetylcholine transmission between the ganglia and result in imbalance of the autonomic nervous system in POTS. This study was designed to analyze the clinical characteristics of POTS patients with AChR-ab positive and explore the value of AChR-ab in children with POTS. In 82 children with POTS, twenty patients (24.39 %) were found as AChR-ab positive. Their clinical characteristics and hemodynamic responses to orthostatic changes were compared with the remaining 60 patients with negative AChRab. Symptoms of POTS children with AChR-ab positive were significantly severe than those of AChR-ab negative patients (p = 0.001). Preceding infection was predominant in patients with AChR-ab positive compared with that of patients with AChR-ab negative (p \ 0.001). Syncope and fatigue were more common in the AChR-ab positive patients (p \ 0.05). The change of upright heart rate was increased significantly in AChR-ab positive patients compared with AChR-ab negative cases (p = 0.013). Multiple logistic regression analysis revealed that preceding infection (OR 22.356, 95 % CI 2.151–34.920), syncope (OR 11.570, 95 % CI 2.098–63.810), and fatigue (OR 11.145, 95 % CI 1.658–74.911) were independent risk factors for
Jiawei Li and Qingyou Zhang have contributed equally to this work. J. Li Q. Zhang Y. Liao C. Zhang J. Du (&) Department of Pediatrics, Peking University First Hospital, Xi-An Men Street No. 1, West District, Beijing 100034, China e-mail: [email protected] H. Hao Department of Neurology, Peking University First Hospital, Beijing 100034, China
POTS with AChR-ab positive. In conclusion, POTS with positive AChR-ab was a heterogeneous disorder. Preceding infection, syncope and fatigue were their main clinical characteristics. Keywords Acetylcholine receptor antibody Children Clinical characteristics Orthostatic intolerance Postural tachycardia syndrome
Introduction Postural tachycardia syndrome (POTS) is characterized by symptoms of orthostatic intolerance in association with excessive tachycardia and relief of these symptoms with recumbence [4, 7]. The clinical features of POTS vary with patients, and symptom severity can be quite disabling in pediatric populations, as these disorders have significant social and health impacts on patients [6]. Although the exact mechanism responsible for POTS has not yet been fully understood, one of the most confident etiologies is the imbalance in the autonomic nervous system of the sympathetic and parasympathetic nervous systems [7, 17]. Acetylcholine, a primary neurotransmitter in the autonomic ganglia in autonomic nervous system including both the sympathetic and parasympathetic nervous systems, can be mediated by the acetylcholine receptor (AChR) of the autonomic ganglion. As preliminary studies reported, acetylcholine receptor antibodies (AChR-ab) were found in some patients with POTS [14]. Antibodies of ganglionic AChR affect acetylcholine transmission between the ganglia, causing imbalance in the autonomic nervous system [17]. Pyridostigmine, an acetylcholinesterase inhibitor, contrary to the role of AChR-ab in the autonomic nervous system, markedly reduced upright heart rate (HR) and
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attenuated the symptoms of POTS patients [9, 12]. Researchers reported autoimmunoreactive IgGs in patients with POTS, supporting the notion that AChR-ab, mainly a form of IgGs, played a role in the pathogenesis of POTS as well [19]. Some authors described a subtype of POTS which was regarded as ‘‘autoimmune’’ or ‘‘neuropathic’’ POTS that was patients with detectable levels of antibodies to the AChR (AChR-ab positive POTS) [7, 10, 14]. As a heterogeneous group of POTS, AChR-ab positive POTS patients may have specific clinical features. Effectively identifying these clinical features would greatly enable us to deepen the understanding of etiology and treatment for POTS. This study was designed to analyze the clinical features of POTS patients with positive AChR-ab and explore the diagnostic value of serum AChR-ab in children with POTS.
Methods Subjects This prospective study involved children who were admitted to the Department of Pediatrics, Peking University First Hospital and were diagnosed as having POTS by the head-up tilt test (HUTT) between June 2012 and December 2013. Totally, 82 POTS patients aged 11.09 ± 2.56 years were included in this study, of whom 43 (52.44 %) were girls and 39(47.56 %) were boys. The physical examination, 12-lead electrocardiography, and electroencephalogram were normal in all subjects enrolled. The study protocol adhered to the principles of the Declaration of Helsinki and conformed to the criteria of the Ethics Committee of Peking University First Hospital (Beijing, China). All participant’s guardians were fully informed of the purpose and methods of our study, and informed consent was obtained. Diagnostic Criteria The present study referred to the following criteria for POTS [1, 2, 13, 21]. (1) A child has a normal HR when supine and no evidence of cardiovascular disease; (2) after standing up, a child has C2 of the following symptoms: dizziness, chest distress, headache, palpitations, pallor, tremulousness, amaurosis, fatigue, or syncope, which should be relieved or diminished by recumbence and should occur repeatedly for C1 month; (3) in addition to symptoms of orthostatic intolerance (OI), the child displayed a HR increase C40 beats/min or a HR [120 beats/ min within the first 10 min during HUTT. Simultaneously, the decrease in blood pressure should be \20/10 mm Hg; and (4) a child without other diseases that manifested
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Pediatr Cardiol (2015) 36:165–170 Table 1 Standard of symptom scoring for orthostatic intolerance Score
Frequency of orthostatic intolerance symptoms
0
Never
1
Once per month
2
Twice to 4 times per month
3
Twice to 7 times per week
4
More frequent than once per day
symptoms in the autonomic nervous system (e.g., anemia, arrhythmia, hypertension, and endocrine disorders) as well as cardiac or neurologic diseases that would induce syncope. History Collection and Symptom Scoring All children or their parents were asked to report the patient’s symptom burden by using a standardized questionnaire. Its main content included disease frequency, duration, incentives, concomitant symptoms, family history of OI, history of allergy, and preceding infection. To avoid inter-rater variability, all interviews were performed by the same investigator who was blind for HUTT and the results of AChR-ab at the time of interview. To determine the severity of symptoms during the study period, a structured questionnaire of scoring which demonstrated validity and reliability was employed, which was detailed in the previous published literature [20–22]. Scorings were based mainly on the typical symptoms of OI, including syncope, dizziness or lightheadedness, chest tightness, nausea, palpitation, and headache. A numerical value for each symptom was determined by its frequency, and the total score was the sum of all the symptoms scores (Table 1). Protocol for HUTT HUTT is the standard method for assessing POTS. The protocol for HUTT was according to the previously published literatures [1, 21]. As preparation, participants were told to avoid caffeine, high-fat foods, and vasoactive drugs with C4 half-lives. All examinations were performed in a quiet environment with dim light and comfortable temperature. After blood pressure and HR were recorded at the baseline and after the subjects had been in a supine position for at least 10 min, they were positioned upright on the tilt table at an angle of 60 degree, with a footboard being used to bear the subject’s weight. Blood pressure and HR monitoring and electrocardiography were performed simultaneously during the test, by using a Dash 2000 Multi-Lead Physiological Monitor (GE Company, New York, New York). The test would be terminated when a
Pediatr Cardiol (2015) 36:165–170 Table 2 Baseline characteristics and hemodynamic parameters in POTS patients with a different AChR-ab status
POTS postural tachycardia syndrome, AChR-ab acetylcholine receptor antibody, IQR interquartile range, OI orthostatic intolerance, SBP systolic blood pressure, DBP diastolic blood pressure, HR heart rate, Delta SBP changes of SBP from supine to standing, Delta HR heart rate increase to head-up tilt test *
p \ 0.05 versus AChR-ab negative group, **p \ 0.01 versus AChR-ab negative group
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Items
AChR-ab negative group (n = 62)
AChR-ab positive group (n = 20)
Statistics
p value
Age (years)
10.93 ± 2.65
11.60 ± 2.23
t = -1.018
Sex ratio, no. of female (%)
34 (54.84)
9 (45.00)
v2 = 0.587
0.444
Symptom score
6.53 ± 3.39
9.75 ± 3.77**
t = -3.595
0.001
Symptom duration (mo), median (IQR)
4.00 (2.00–23.00)
11.00 (1.82–24.00)
z = -0.666
0.506
Family history of OI, no. (%)
13 (20.97)
6 (30.00)
v2 = 0.693
0.405
2
Allergic history, no. (%)
11 (17.74)
2 (10.00)
v = 0.223
Preceding infection, no. (%) Supine SBP (mmHg)
14 (22.58) 99.44 ± 7.98
14 (65.00)** 101.15 ± 8.49
v2 = 12.321 t = -0.822
0.312
0.637 \0.001 0.413
Supine DBP (mmHg)
56.97 ± 7.72
57.05 ± 6.37
t = -0.043
0.966
Supine HR (beats/min)
75.71 ± 11.26
74.50 ± 5.99
t = -0.618
0.648
Standing SBP (mmHg)
107.66 ± 9.99
110.25 ± 10.39
t = -0.998
0.321
Standing DBP (mmHg)
65.32 ± 8.28
64.25 ± 9.27
t = -0.489
0.626
Standing HR (beats/min)
122.23 ± 13.12
128.30 ± 12.22
t = -1.830
0.071
Delta SBP (mmHg)
8.23 ± 7.32
9.10 ± 10.34
t = -0.418
0.677
Delta HR (beats/min)
46.52 ± 11.18
53.80 ± 11.20*
t = -2.532
0.013
positive response of increment in HR C40 beats/min or an absolute HR C120 beats/min appeared within 10 min and a diagnosis of POTS would be made. AChR-ab Detecting After being fasting for 8–12 h and rested for at least 10 min, patients were kept in supine position, and a 2 mL aliquot of blood was obtained by venipuncture into a tube with ethylenediaminetetra-acetic acid. The blood was centrifuged at 2000g for 5 min at 4 °C. Serum samples were stored at 20 °C until assay. AChR-ab were measured by enzymelinked immunosorbent assay (ELISA) in a standardized manner. Qualitative ELISA Kit (CUSABIO, human AChR antibody ELISA Kit, Wuhan, China) was used following manufacturer’s guideline. The 96-well flat-bottomed polystyrene plates coated with purified AChR were prepared in the kit. The sample serum (1:10) was added into every reaction hole and incubated overnight at 4 °C. After that, the plates were washed for four times and incubated with HRPconjugated goat-anti-human IgG (1:1000) for another 2 h at 37 °C. Chromogenic reagent A and B solutions were added each 50 lL after four washes, with the seal plate film sealed the reaction hole. The reaction was allowed to develop at 37 °C in the dark for 15 min. Finally, 50-lL termination solution was joined in every reaction well to terminate the chromogenic reaction. Results were read by a microplate reader at an optical density (OD) of 450 nm, and expressed as OD values standard deviation (SD). Positive status of AChR antibodies was defined as a ratio (sample OD/negative OD) C2.1. Otherwise, the status of AChR-ab was defined negative.
Statistical Analysis Statistical analysis was performed using SPSS software, version 13.0 (SPSS, Chicago, Illinois, U.S.A.). Continuous variables are presented as the mean ± SD, and discrete data are presented as the median (interquartile range, IQR) or as percentages. The single Kolmogorov–Smirnov test was used to verify whether the data were normally distributed. Comparisons between two groups were performed using the independent t test for measurement data, Chi square test for categorical variables, and the rank sum test for discrete data. Logistic regression analysis was used to identify independent predictors for POTS of positive AChR-ab. A p value of \ 0.05 was considered statistically significant.
Results Clinical Characteristics of POTS Patients with AChR-ab Positive The clinical features of POTS patients with a different AChR-ab are summarized in Table 2. AChR-ab positive status was presented in 20 (24.39 %) of the children with POTS. Most patients had experienced symptoms for a few months before diagnosis. The median duration of symptoms was 9.5 months, with interquartile range of 2–24 months. As was shown in Table 2, age, sex ratio, symptom duration, family history, and allergic history did not differ between the AChR-ab positive and AChR-ab negative groups (p [ 0.05). Symptom scores of AChR-ab
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Pediatr Cardiol (2015) 36:165–170 Table 3 Association of the factors for POTS patients with AChR-ab positive Indices
B
OR (95 % CI)
p value
Syncope
2.448
11.570 (2.098–63.810)
Fatigue
2.411
11.145 (1.658–74.911)
0.013
Preceding infection
3.107
22.356 (2.151–34.920)
\0.001
0.005
POTS postural tachycardia syndrome, AChR-ab antibodies to acetylcholine receptor, OR odds ratio, CI confidence interval
Fig. 1 The frequency of orthostatic symptoms prescribed in children POTS with a different AChR-ab status. POTS postural tachycardia syndrome, AChR-ab antibodies to acetylcholine receptor, *p \ 0.05 versus AChR-ab negative group
positive POTS were significantly higher than that of AChR-ab negative patients (t = -3.595, p = 0.001). Preceding infection was predominant in patients with AChRab positive (65 %) but it was found in only 22.58 % of patients with AChR-ab negative (v2 = 12.321, p \ 0.001). Commonly reported symptoms of study subjects were detailed (Fig. 1). Dizziness was the most common symptom in both groups. Compared with AChR-ab negative patients, orthostatic syncope and fatigue were more common in the AChR-ab positive patients (p \ 0.05). Other symptoms, such as shortness of breath, gastrointestinal symptoms, fatigue, pallor, blurred vision, palpitation, sweating, and tremulousness were not statistically different between the two groups (p [ 0.05). Hemodynamics of AChR-ab Positive POTS Patients The hemodynamic data of POTS patients in HUTT are shown in Table 2. There were no statistical differences of hemodynamic data between the two groups in supine or standing systolic blood pressure (SBP), diastolic blood pressure (DBP), HR, and postural change in SBP (p [ 0.05). However, change of HR (delta HR) increased significantly in AChR-ab positive group than in AChR-ab negative group (t = -2.532, p = 0.013). Logistic Regression Analysis of Independent Risk Factors for AChR-ab Positive POTS We used forward-stepwise regression analysis and entered gender, age, delta HR, syncope, fatigue, and preceding infection as independent variables. Multiple logistic regression analysis revealed that preceding infection (OR 22.356, 95% CI 2.151–34.920, p = 0.005), orthostatic syncope (OR: 11.570, 95%CI: 2.098-63.810, p = 0.013), and fatigue (OR: 11.145, 95%CI: 1.658-74.911, p \ 0.001) were independent risk factors for POTS with AChR-ab positive (Table 3).
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Discussion POTS is not a single condition, but rather a heterogeneous group of disorders resulting in a similar physiological state [6, 7, 10]. Due to multiple mechanisms of potential pathophysiology, patients with POTS have varied manifestations [4, 7]. Successful treatment depends on identifying the etiology and pursuing a comprehensive treatment program. Several authors attempted to classify POTS into subtypes, based on clinical and ancillary test results. In some recent studies of adult cases, researchers described three groups of patients with POTS [7, 10, 14]. The first comprised ‘‘hypovolemic’’ POTS, defined by a decrease in 24 h urinary sodium excretion. The second group comprised ‘‘hyperadrenergic’’ POTS, where patients with increased plasma norepinephrine levels after standing ([600 pg/mL). The third group comprised ‘‘neuropathic’’ or ‘‘autoimmune’’ POTS, where patients were AChR-ab positive, as we studied in this article. We found that AChR-ab positive POTS had frequent syncope and fatigue, with increased changes of HR and symptom scores compared with AChR-ab negative POTS. Preceding infection was also predominant. Multiple logistic regression analysis revealed that preceding infection, syncope, and fatigue were independent risk factors for POTS with AChR-ab positive. AChR-ab, which consist mainly of the IgG, were autoimmune antibodies that can block the membrane of AChR and inhibited AChR current [18]. Nicotinic AChR was divided into two subtypes, N1 and N2 types. N1 type is located in postsynaptic membrane of ganglion, raising excitability of autonomic ganglia postganglionic neurons; while N2 type is located in the membrane of endplate, which can regulate endplate potentials and contract muscles. When AChR-ab is identified and bond with AChR of N1 subtype, they inhibited ligand-gated cation channel of the AChR open and decreased cholinergic transmission in the autonomic ganglia of both the sympathetic and parasympathetic tones. Thus, the autonomic nervous function of POTS patients was impaired [16, 18]. Gibbons et al. [3] reported that serum AChR-ab levels in AAG correlated with the severity of autonomic dysfunction. Considering
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that POTS was a form of AAG with relatively mild symptoms [17], this result was comparable with what we found that AChR-ab positive POTS had more serious symptoms. AChR-ab is attached to N2 subtype of AChR in postsynaptic membrane of neuromuscular junction and caused neuromuscular transmission disorders. Symptom of fatigue therefore appeared, and this was also the pathogenesis of myasthenia gravis [15]. The hemodynamic change from the supine to upright position is that amount of blood flow from the chest to limbs and abdomen. The reduction in cardiac filling exerts continuous inhibitory action on the cardiovascular areas of the medulla (in particular in the nucleus tractus solitary), and the decline arterial pressure causes stimulation of the low pressure receptors that lie in the aortic arch and the carotid sinus. These changes produce an increase in sympathetic output that results in an increase in HR and systemic vasoconstriction, thus maintaining normal perfusion of brain. The rapid HR then reaches to a steady state by the role of vagus nerve. The failure of the system can result in an inability to respond in a normal manner to sudden changes in posture and cause illness of POTS [8]. Acetylcholine is the primary neurotransmitter of the autonomic ganglia in both the sympathetic and parasympathetic nervous systems. AChR-ab can impact sympathetic modulation and cause failure of the peripheral vasculature to vasoconstriction appropriately during upright posture [17, 19]. In the meantime, HR and cardiac contractility may increase sufficiently to maintain blood pressure at relatively normal levels. However, such mechanism usually cannot be fully compensatory or cannot be able to be maintained for a long time, thus resulting in reduction of cerebral blood flow in POTS patients [8]. That is why those POTS patients with AChR-ab positive were more vulnerable to orthostatic syncope. AChR-ab also interferes parasympathetic function of slowing HR. This was keeping with what we found that the AChR-ab positive POTS with HR variation was greater than AChR-ab negative POTS. In addition, researchers reported that about 15 percent of POTS patients were seropositive for AChR antibodies in adults [14], while the incidence in children POTS was unknown. We reported that the percentage of AChR-ab positive was 24.39 % for children POTS, which was higher than that of adults. We speculated that it was because children had relatively low immunity and were more vulnerable to infection. Meanwhile, authors reported the relationship between infection and POTS [7, 14], which was also verified by our result that preceding infection was the risk factor of POTS patients with AChR-ab positive. Mechanisms explained were as follows: when patients had bacterial or viral infection, immune tolerance was broken; or the structure of antigen cluster in viral surface and
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AChR cluster was similar and common antigen-antivirus antibodies were produced, and immune cross reactions appeared; moreover, virus infected and sensitized AChR of muscle-like epithelial cells and other cells in thymus [11]. These reactions can induce AChR-ab which breaks the balance of the autonomic nervous system. It should be taken seriously in clinical diagnosis and treatment of POTS that preceding infection induces serum AChR-ab and POTS symptoms. Greater attention has been paid to the targeted treatments in patients with POTS [9]. In this paper, we analyzed the clinical features of AChR-ab positive POTS so as to help clinicians consider this group of patients based on the clinical features. Of course, timely detection of the AChR-ab was meaningful for the cases. Therefore, our single center experience suggested that targeted therapy should be considered based on specific clinical features of this group of POTS cases, which would be preferably confirmed by laboratory antibody testing. Study Limitations The present study has the following limitations. The number of AChR-ab positive POTS patients is relatively small (n = 20). Further studies with larger numbers of cases are needed. However, the present study on POTS patients with or without positive AChR-ab, using ELISA as a simple, stable and reliable method, would greatly deepen the understanding of this heterogeneous disorder.
Conclusion AChR-ab positive POTS patients are a heterogeneous group with preceding infection, syncope, and fatigue as their main clinical characteristics. Etiological treatments of acetylcholinesterase inhibitors and immunotherapy might be the best choice for them [5, 12]. Effectively identifying POTS patients with AChR-ab positive is important for an individualized treatment of POTS. Acknowledgments This work was supported by grants from the Beijing Committee of Science and Technology, China (Z121107001012051) and the National Twelfth 5-Year Plan for Science and Technology Support (2012BAI03B03). Conflict of interest The authors declare that they have no conflicts of interest.
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