Genetically confirmed FRDA patients were recruited through the ataxia outpatient clinic of the Innsbruck Medical University between January 2016 and June 2017. Age- and sex-matched healthy controls were enrolled within a previous study . Vitamin deficiency, thyroid dysfunction, diabetes mellitus and/or impaired glucose tolerance were set as general exclusion criteria. Further exclusion criteria for cardiovascular tests were (1) symptomatic cardiomyopathy (New York Heart Association, “NYHA”, stage ≥ 2) and (2) severe ataxia (defined as a SARA score ≥ 28, see below).
Investigations and assessments were conducted in accordance with the Declaration of Helsinki on ethical principles for medical research involving human subjects. The study protocol was approved by the local ethic committee and written informed consent was collected from both patients and controls.
Basic demographic and clinical data as well as results of genetic testing were collected for all patients.
Depending upon age at disease onset, an early onset (≤ 24 years of age) and late-onset (≥ 25 years old) group were defined .
Disease severity was evaluated with the Scale for the Assessment and Rating of Ataxia (SARA) and the Activity of daily living scale (ADL) . The SARA is a semi-quantitative scale which rates the severity of ataxia (from 0 = no ataxia to 40 = most severe ataxia) by evaluating eight items (gait, stance, sitting, speech disturbance, dysmetria at finger chase, tremor at nose–finger test, dysdiadochokinesis and dysmetria at heel-shine test) . A number of studies have demonstrated the sensitivity of SARA in evaluating disease progression in FRDA [4, 14]. The ADL is a subscale of the FARS (Friedreich’s Ataxia Rating Scale) which describes the functional deterioration in performing daily activities (from 0 = no disability to 36 = most severe impairment) . It consists of nine items: speech, swallowing, cutting food/handling utensils, dressing, personal hygiene, falling, walking, quality of sitting position and bladder function. The ADL scale was proved to be an excellent outcome measure in FRDA, which is able to capture disease progression as well as the SARA .
A standard 12-leads ECG and transthoracic echocardiography was performed in all patients. Autonomic symptoms were investigated by means of the SCOPA-Aut questionnaire (Scales for outcomes in Parkinson’s disease—Autonomic) . The SCOPA-Aut includes 25 items belonging to the following domain: deglutition, cardiovascular, gastrointestinal, urinary, pupillary function, thermoregulation and sweating, sexual. Patients are asked to recall the frequency of various symptoms in the 4 weeks preceding the examination and to provide ratings between “never” (= 0) and “often” (= 3). The EQ-5D-3L questionnaire was applied as quality of life measure . Eligible patients for cardiovascular test underwent a screening standing test and the Orthostatic Hypotension Questionnaire (OHQ) was administered .
Cardiovascular autonomic function assessment
Cardiovascular autonomic function testing (CAFT) was performed in a quiet setting, with constant room temperature (22 °C). Patients and controls were instructed not to drink any coffee, tea or taurine-containing beverages on the day of examination and to fast in the 2 h before testing. Sympathomimetic and anticholinergic drugs were stopped 48 h before testing.
Heart rate (HR) and BP were continuously recorded by means of non-invasive monitoring (Task Force® Monitor, CNSystems 2007) using the following protocol: 10 min supine, 10 min 60° head-up tilt, 5 min supine and 5 min active standing. Thereafter, metronomic breathing (6 cycles/minute for 1 min), and the Valsalva maneuver (blowing into a mouthpiece for 15 s at an expiratory pressure of 40 mmHg—3 trials) were performed. The head-up tilt was performed just in patients who have been able to tolerate the screening standing test.
Data from CAFT recording were exported in an excel file for further processing. Mean values of HR, systolic BP and diastolic BP (i.e., average of 15 beat-to-beat values) were collected at the following time points: after 10 min of supine rest, after 3 and 10 min of head-up tilt, after 5 min of supine rest, after 3 and 5 min of active standing. Then, HR and BP changes (Δ) were calculated (i.e., differences between head-up tilt/active standing values and values in the preceding supine position). Deep breathing ratio was calculated as the mean of six ratios which are obtained dividing the longest R–R interval of ECG recording during expiration by the shortest interval during inspiration of the metronomic breathing. To calculate the Valsalva ratio, the best performed trial of Valsalva maneuver was chosen. Valsalva ratio was obtained dividing the highest HR in phase II by the lowest HR in phase IV. During the Valsalva maneuver, a BP rise is observed (1) during the late phase II (II_L), as consequence of a reflex vasoconstriction and (2) during the phase IV, when venous reflow and cardiac output return normal (see also Fig. 1). The magnitude of these BP rises (quantified as BP phase II_L—BP phase II_E and BP phase IV—BP phase I) is an index of vascular adrenergic function which was applied as further autonomic assessment in this protocol .
The baroreflex sensitivity was calculated both in the supine position and during head-up tilt following the sequence method , by means of a customized software (by JP Ndayisaba). For further methodological details, see also .
Sudomotor function assessment
The sudomotor function was evaluated by testing of the skin sympathetic reflex. The Sympathetic Skin Response was recorded with surface electrodes from the sole and dorsum of both feet after ipsi- and contralateral electrical stimulation of the tibial nerve at the medial ankle using a Dantec Keypoint G4 Workstation (Alpine Biomed ApS, Skovlunde, Denmark). Stimuli were applied at an intensity of 30 mA and duration of 0.2 ms.
Statistical analysis was performed with SPSS version 24. Categorical variables are reported as percentages, while continuous variables are reported as mean and standard deviation or median and interquartile range depending on their distribution, tested by means of Shapiro–Wilk test. Levene test was applied to test homogeneity of variance. Comparisons of autonomic function indexes between patients and healthy controls were performed by means of ANOVA for normally distributed variables, with homogeneous variance or unpaired t test if homogeneity of variance was not assumed. Mann–Whitney U test was applied for not normally distributed values. A multiple regression model was applied to estimate the influence of age at examination, GAA1 repeats, disease duration and severity, expressed by SARA or ADL, on general autonomic outcome, expressed by SCOPA-Aut scores. A logarithmic transformation was applied to not normally distributed variables. Statistical significance was set at p < 0.05.