Increase of angiotensin II type 1 receptor auto-antibodies in Huntington’s disease

In 132 HD participants with genetically definite HD from all stages of the disease serum anti-AT1R antibodies were analysed via ELISA. The main demographic and clinical characteristics of HD subjects at the time point of investigation are reported in Table 1. 46 participants were smokers, 18 had an infection, 16 reported any kind of an allergy, 36 were on serotonin reuptake inhibitors as an antidepressant medication, 59 on an anti-dopaminergic drug and 78 took any kind of other medication.The mean anti-AT1 antibody titer in the HD cohort was determined as 20.5 ± 12.8 U/ml which was significantly higher than in healthy controls (mean titer: 8.6 ± 4.9 U/ml, Figure 1). Upon analysis of ranks close to quartiles 23.8% of values were < 10 U/ml, 23.5% between 10–16 U/ml, 25.8% between 16–35 U/ml and 25% ≥ 35 U/ml. Assuming values > 15 U/ml as relevant titers, 71 of 132 patients (53.8%) display relevant anti-AT1R antibodies. Assuming 20 U/ml as relevant cutoff, 50 of 132 HD individuals (37.9%) displayed relevant anti-AT1R serum antibodies. A total of 29 patients (21.9%) showed titers reaching the ceiling value > 40 U/ml. There were no significant titer differences between males and females.

In a selected cohort of nine patients, follow-up samples were collected one year later. Upon follow-up anti-AT1R antibody titers were surprisingly stable and only differed by a mean of 3.7 ± 4.3 U/ml (p = not significant). Relevant titer changes (assuming the cutoff of 20 U/ml) occurred only in 2/9 (22.2%) cases.

To further assess the significance of our finding in HD individuals, we additionally analyzed the presence of anti-AT1R antibodies in a cohort of a total of 97 MS patients (baseline data given in Table 2). In the total cohort, 20/97 (20.6%) patients displayed titers > 15 U/ml, 13/97 (13.4%) displayed titers > 20 U/ml and 2/97 (2.1%) showed titers reaching the ceiling value of > 40 U/ml. The mean titer was 11.1 ± 8.6 U/ml which was significantly lower than in HD individuals (Figure 1, p < 0.001). In the total MS cohort, antibody titers did not correlate with disease duration, disease course (RR-MS vs. SP-MS), disease severity as measured by EDSS or immunomodulatory treatment. However, MS patients with active disease defined as relapse or MRI activity in the previous 3 months displayed higher antibody titers than patients with stable relapsing-remitting or progressive disease (mean 25.6 ± 11.8 versus 12.0 ± 7.1 U/ml, n = 9 versus 89; p = 0.0005).

In a next step, we correlated absolute antibody values with clinical features of our HD cohort. Parameters included baseline information (age, gender, height, body weight), medication (neuroleptics, SSRI, other), information of disease onset (onset of motor symptoms, onset of psychiatric symptoms), scales for clinical disease stage (UHDRS, independency scale (IS), total functional capacity scale (TFC), disease duration and CAG repeats, measures of neuropsychology (CS) and motor function (MS) including more quantitative motor measures like tapping or peg board results and finally infection, smoking and allergy (Table 3). Antibody titers significantly correlated with motor onset of disease (r = -0.23; p = 0.02), duration of disease (r = 0.18, p = 0.035), UHDRS-MS (r = 0.22, p = 0.012), TFC (r = -0.18, p = 0.03) and IS (r = -0.21, p = 0.016) as well as disease burden score (r = 0.18, p = 0.04). The higher the antibody titer, the earlier was the onset of disease. Yet, antibody titers also significantly correlated with smoking (r = -0.26, p = 0.005) and the presence of infection at sampling (r = 0.27, p = 0.002), but not with allergies. Similar results were obtained when correlating these clinical features with quartiles of anti-AT1R antibodies or when assuming 15 U/ml as cutoff for positive titers (data not shown). In contrast, antibody titers did not correlate with smoking in the healthy control group (r = 0.022, p = 0.76).

After exclusion of HD patients with more severe stages of the disease (Shoulson stage 4 and 5; n = 107 remaining), there were no significant correlations of absolute antibody titers with clinical data. Yet in this very subgroup of patients, the presence of anti-AT1R antibodies (cutoff 20 U/ml) did correlate with motor onset of disease (r = -0.28; p = 0.016) and psychiatric onset of disease (r = -0.36; p = 0.026).

In contrast, anti-AT1R antibodies did not correlate with age, gender, body height or body weight, medication, motor or neuropsychological function, CAG repeat length, Shoulson scale, years to onset or disease duration in HD individuals.

After numerical calculation of antibody titers >40 to obtain exact values (n = 132, mean 32.7 ± 37.4; range: 2.9 to 146.5), titers did not correlate with motor age of onset any more, but we now found stronger correlations with duration of disease (r = 0.244, p = 0.005), UHDRS-MS (r = 0.367, p < 0.001), TFC (r = -0.344, p < 0.001) and IS (r = -0.415, p < 0.001) as well as disease burden score (r = 0.302, p = 0.001). In addition, there was a significant correlation with cognitive sum score (r = -0.293, p = 0.001), and expanded CAG (r = 0.193, p = 0.027). Antibody titers still significantly correlated with smoking (r = -0.264, p = 0.004) and the presence of infection at sampling (r = 0.315, p < 0.001), but not with allergies.

In regression analyses, we modeled the prediction of antibody titers by smoking and infection versus HD related variables in step wise models. Smoking as well as infection were significant predictors for the presence of anti-AT1R antibodies and explained 15% of the variance (r² = 0.151). When including the HD relevant variables independence scale (IS), CAG expanded and Shoulson scale as well as “motor” or “psychiatric onset” of disease to the model, IS alone explains 25.8%, IS and CAG expanded together explain 34.8% and all variables explain 41.5% of the variance (r² = 0.415). A final stepwise model included independence scale (IS) as predictor and as next steps the variables “onset motor” and smoking. IS alone explains 14.5% of the variance, IS and onset motor together explain 19.2% of the variance, IS and “onset motor” and smoking together explain 23.7% of the variance (Table 4).

A covariate analysis of HD patients suffering from psychiatric symptoms (“psychiatric onset”) showed that only the presence of anti-AT1R antibodies (cutoff 20 U/ml), but not smoking or infection was a significant covariate. Further covariate analyses of the variables disease burden score, duration of disease or Shoulson scale after grouping according to the presence of antibody titers and smoking as well as infection as co-variates revealed infection as significant covariate in all analyses.

Upon exclusion of smokers and participants with infection from the HD cohort, a total of 55 individuals remained left for further analyses. The mean anti-AT1R antibody titer in this group was 21.1 ± 12.1 U/ml which was still significantly higher than in the MS cohort and in healthy controls (each p < 0.0001). Assuming an antibody titer of 20 U/ml as cutoff, statistical analyses by Mann–Whitney testing confirmed significant differences between patients with or without significant anti-AT1R antibody titers for “onset motor” (p = 0.046) and “onset psychiatric” (p = 0.044). For the variables “onset” and “years to onset”, there was a trend in this cohort towards earlier time points in the antibody positive subgroup (n = 55, p = 0.096).

HD subjects were recruited at the Department of Neurology, Ruhr University of Bochum. 132 genetically confirmed HD mutation carriers (72 (54.5%) women and 60 (45.4%) men; mean age 46.9 ± 12.8 years) were screened for anti-AT1R antibodies. 30 of the 132 HD mutation carriers were classified as pre-manifest (preHD), based on expert raters’ assessments of motor signs which were not sufficient for the diagnosis of HD (Diagnostic Confidence Level [DCL], item 17 of the UHDRS Motor Assessment) [36]. Sixteen participants of the 102 manifest HD participants were in Shoulson stage I, 35 in stage II, 26 in stage III, seventeen in stage IV and eight in Shoulson stage V, respectively (see also Table 1) [37]. In a subgroup of nine patients, follow-up was after 12 months. At each assessment, the following data and scores were determined: age, gender, weight, height and smoking behavior as well as scores on the motor (MSc), cognitive (verbal fluency test, symbol digit modalities test, Stroop color, Stroop word and Stroop interference, summarized as cognitive score (CS)), and independence (IS), as well as functional sections (TFC) of the validated Unified Huntington’s Disease rating scale (UHDRS) [36]. If possible, fine motor skills were measured by simple (tapping; higher motor impairment leads to lower test results) or complex (pegboard; higher motor impairment leads to higher test results) quantitative movement tests [38, 39]. Years to disease onset (YTO) for the preHD subjects were calculated by subtracting the subject’s age at the time of investigation from his or her estimated onset age using Langbehn’s formula [40]. Moreover we calculated the disease burden score (DBS = [CAG repeat - 35.5] × age) for each subject [41]. Additionally, patients were interviewed about medical problems including allergy, severe other diseases, medication intake and the presence of infections. Infections were excluded clinically for outpatients and excluded or confirmed clinically and by determination of the C-reactive protein for inpatients (all cases with Shoulson stage III to V) and all cases clinically suspicious for an infection. Patients with severe other diseases, such as a carcinoma, were excluded.

A cohort of MS patients from Bochum or NeuroCure Center, Berlin, Charité was enrolled in this study. Diagnosis was confirmed by the treating neurologist based on the revised 2010 McDonald clinical and radiologic criteria for MS [42]. 59.8% of MS patients were female and 40.2% male, mean age was 39.3 ± 10.2 years, 80.4% had relapsing remitting (RR-MS) and 19.6% had secondary progressive MS (SP-MS, see Table 2). 69.1% of patients received a disease modifying therapy with injectables (beta interferon or glatiramer acetate), 15.5% were without treatment, 12.3% received a second line therapy (natalizumab, mitoxantrone) and 3.1% were on other treatments. Data from MS patients were correlated with clinical course, disease modifying treatment, relapse and MRI activity and disability (expanded disability score scale, EDSS). Active disease was defined as relapse or MRI activity in the last 3 months prior to serum sampling.

Healthy controls were recruited at the University Hospitals of Goettingen and Erlangen after obtaining informed consent. 70.5% of control individuals were female and 29.5% male. Controls were matched to HD subjects for age (47.5 ± 9.8 years) and smoking status (34.4% smokers in the control group vs 34.8% in the HD group, see also Table 1.

Written informed consent to use serum samples for research purposes was obtained from each individual. Samples were collected in a standardized manner and were analyzed for antibodies against the AT1R. A commercially available sandwich enzyme linked immunosorbent assay (ELISA; CellTrend GmbH, Luckenwalde, Germany) to measure anti-AT1R autoantibodies was employed as described previously [13‐15]. Briefly, microtiter 96-well polystyrene plates were coated with extracts from Chinese hamster ovary cells overexpressing the human AT1R. To maintain the conformational epitopes of both receptors, 1 mM calcium chloride was added to each buffer. Duplicate samples of a 1:100 serum dilution were incubated at 4°C for 2 h. After washing steps, plates were incubated for 60 min with a 1:20000 dilution of horseradish peroxidase-labelled goat anti-human IgG (Jackson, West Grove, Pennsylvania, USA) used for detection. In order to obtain a standard curve, plates were incubated with a ready-to use standard at 2.5, 5, 10, 20, and 40 U/ml. The assay was validated as compared to a cardiomyocyte bioassay (for further assay details see also Dragun) [43]. The ELISA was validated according to the Food and Drug Administration’s ‘Guidance for Industry: Bioanalytical Method Validation’. The inter-assay variability was 7% and the intra-assay variability was 6%. Persons who were unaware of the patients’ characteristics performed the assays. Values reaching the ceiling of > 40 U/l were counted as “40”, values equal to or below the detection limit of 2.5 U/l were counted as “2.5”. In post-hoc analyses, numerical calculations were performed to obtain exact values for titers reaching the ceiling value > 40 U/ml.

The study was approved by the local ethics committees in Erlangen, Goettingen, Bochum and Berlin (Nr 3184–08 Ruhr-University Bochum and ZS EK 13 255/07 Berlin) and was conducted in accordance to the Declaration of Helsinki in its currently applicable form, the guidelines of the International Conference on Harmonization of Good Clinical Practice (ICH-GCP) and the applicable German laws. Participants or their legal guardians gave written informed consent.

Statistical analysis of baseline data and comparison between groups was performed using chi-square-, Mann–Whitney U- and Kruskal Wallis tests calculated via PRISMS (GraphPad Software, Inc., La Jolla, CA, USA). Testing of probability distributions in our samples was performed using Kolmogorow-Smirnov-test. The association between anti-AT1R antibodies of each participant and type of onset, smoking (defined as regular nicotine consumption ever day; yes/no), infection (yes/no), UHDRS-MS, IS, TFS, CS and DBS was tested by correlation and linear regression models controlling for anti-AT1R antibody titers. For correlation analysis, Spearman-Rho analysis was used for non-parametric data and Pearson analysis if data showed normal distribution. Testing for significance was achieved by using Wald- chi-square-test. Correlation analysis and linear regression were computed using PASW-Statistics 18 (IBM SPSS, NY, USA). Significance was accorded to p-values *p < .05; **p < .01; ***p < .001.