Abstract: Background and Objective: Phospholipids fatty acids are major structural components of neuronal cell membranes and play a fundamental role in membrane function, modulating fluidity. Changes in the metabolism of fatty acids have been demonstrated in several neuro-psychiatric disorders both in clinical and animal studies. Also in Rett Syndrome very long chain fatty acid and carnitine levels may be decreased. The aim of this work was to evaluate changes in long chain poly-unsaturated fatty acid status and functional neurophysiological, neuropsychological and behavioural functions at three and six months in a trial of long chain poly-unsaturated fatty acid supplementation in girls with Rett Syndrome. Materials and Methods: The study included a randomized, 3-6months, omega 3 placebo-controlled, one-way crossover trial with 22 girls with classical Rett Syndrome (mean age 13.25, range 6-20 years). The patients were randomized to long chain poly-unsaturated fatty acid (250 mg below 15 kg, 500 mg between 15 and 26 kg or 750 mg if over 26 kg) or placebo (maize oil) for 6 months. Patients underwent computerized EEG (electroencephalogram) video-polygraphic recordings during wakefulness. Detailed, specific assessment tools were used to measure attention and discrimination before and after supplementation. Results: Results show that despite the proven assimilation of long chain poly-unsaturated fatty acid, in neurophysiological parameter no statistically significant result emerged. Neuropsychological and behavioural measurements pre-test and post-test showed weak modifications. Conclusion: This study indicates that the effects of long chain poly-unsaturated fatty acid supplementation in girls with RTT in terms of neuropsychological and behavioural parameters are weak.
INTRODUCTION
Neural cell membranes are essentially structured by phospholipids fatty acids, which play a fundamental role in their function, modulating cell membrane fluidity. Long-chain poly-unsaturated fatty acids (LCPUFA), eicosapentaenoic acid (EPA) and docosahexaenoic (DHA) are essential nutrients to maintain human health and lower the risk of cardiovascular diseases and neurodegeneration1. These compounds are important building blocks of neuronal membrane, to which they are anchored by phospholipid molecules. It has been hypothesized that they may have a role in facilitating the transmission of signals between neurons2. The DHA is a key component for the formation and function of the nervous system, particularly the brain and the retina3. The LCPUFA are important inflammatory mediators and have been considered to be beneficial in the prevention or treatment of several diseases4.
Recently, several studies have investigated the role of nutrition in behaviour and learning disorders in children, above all regarding omega-3 and omega-6 fatty acids: A link between neuro developmental disorders and omega 3 fatty acid deficiency has been suggested5 and the supplementation of a combination of EPA and DHA, in children with reading-writing disorder (RWD), dyspraxia (i.e., motor coordination difficulties or developmental coordination disorder), DCD and attention deficit hyperactivity disorder (ADHD) related symptoms has reported to be beneficial6-12.
Among the disorders of neurodevelopment, very long fatty acids (VLCFA) and carnitine levels have been demonstrated to be low in Rett Syndrome (RTT)13. RTT named after the Austrian pediatrician who first described it in 1965, is a childhood neurological developmental disorder. Incidence is estimated from 1/10,000 to 1/15,000 in new- born females14. It mostly affects females, even though a few cases of males are reported in literature15-18. In the past, RTT was generically associated with the autism spectrum disorder, but recently it has been identified as a genetic disorder with a specific biological marker for diagnosis: aetiology is due to a mutation of gene MECP2 on the X-chromosome19,20. Stradomska et al.13 analyzed VLCFA levels in an RTT group and discovered that levels were lower compared to the control group. The supplementation of L-carnitine for 2 months resulted in an increase in VLCFA levels, thus demonstrating a relationship between low VLCFA levels and low carnitine levels in patients with RTT. It may be hypothesized that low carnitine levels interfere with transportation to mitochondria, inhibiting the oxidation of long-chain fatty acids and resulting in a compensation, to a certain extent, by intensified β-oxidation of VLCFA in the peroxisomal system. Low VLCFA was considered secondary to the pathogenesis of RTT, but it may also be suggested that abnormalities in their levels are somehow involved in the development of the disease. More recently De Felice et al.21 analyzing VLCFA in RTT, described the "fatty acid paradox" of the disease. The researchers are of the opinion that fatty acids might represent a molecular target for the disease, even though it is genetically determined. Moreover, they suggested that RTT should be regarded as a neurodevelopmental disease resulting from a disorder of the complex lipid metabolism, in a background of redox imbalance shifted to a pro-oxidant condition.
Recent studies showed that RTT has gradually become a cross-disciplinary topic of interest: Genetic research efforts have combined with those of neuropsychology and behavioural sciences with the attempt of defining the RTT phenotype22-25. For example, De Felice et al.26 investigated a total of 20 patients in stage I and give oral supplementation with ω-3 PUFAs-containing fish oil for 6 months or no treatment. They assessed results only on the base of a global clinical severity score27, based on 13 individual, ordinal categories measuring clinical features common in RTT. De Felice et al.28 also investigated 24 RTT girls at different clinical stages, supplemented with ω-3 PUFAs as fish oil for 12 months and compared to matched healthy controls. Following ω-3 PUFAs supplementation their results indicate that ω-3 PUFAs were able to modulate plasma protein expression in RTT, having a major impact on the modulation of the acute phase response. Again, no neuropsychological data is provided. In the reviews of Benatti et al.29 and De Felice et al.21 no mention of precise neuropsychological scales were made. In the theoretical work of Kirby et al.30 the researchers refer mainly to typical developing subjects and other type of symptomatology, moreover they had not studied RTT syndrome and, given the fluctuating results of the supplementation with omega-3 fatty acids, they conclude that further research is required.
Summarizing, most of the studies in RTT have used only clinical subjective measures or clinical physiological measures, the novelty of the present researcher is that it is used not only clinical measures, but also objective measures related to neuropsychological parameters.
In addition, the application of new technological approaches, developed by researchers in psychology and engineering, have improved the opportunities of defining attention and cognitive processes in girls with RTT18,31-45. In this study, changes in LCPUFA status and functional neurophysiological, neuropsychological and behavioural functions at three and six months in a trial of LCPUFA supplementation in girls with RTT were analyzed. More precisely, a standardized Rett Assessment Rating Scale (RARS)46 was evaluated and used eye tracker technology to objectively measure recognition, matching of pairs (the same) and semantic categorization (similar).
MATERIALS AND METHODS
Participants: Twenty-two girls with RTT (mean age = 13.25 years, range = 6-20) were classified as clinical stage III (characterized by prominent hand apraxia/dyspraxia, apparently preserved ambulation ability and some communicative ability, mainly eye contact) or stage IV (late motor deterioration, with progressive loss of ambulation ability), according to Hagberg47. Demographic, developmental, clinical, behavioural and genetic information, collected from all available sources, such as parent/caregiver reports of past history and current behaviour and features, previous clinical reports and direct observation and examination of the girls, were entered into a database. Neurological examination focused on epilepsy characteristics (age at seizure onset, seizure types, course of epilepsy and antiepileptic drugs). With regard to MeCP2 mutations, four patients had R306C; two patients each had R270X, R294X and R255X; four had C-terminal deletions and the remaining four had T322A, R106W, R133C and R168X.
Mean age at epilepsy onset was 5.1 years (range = 1-14); seizures were focal in eight patients, tonic in five, generalized tonic–clonic in three, myoclonic in one and atonic in one. Seizures were controlled in four cases and occurred sporadically in five patients. In the other cases, epilepsy was drug resistant and seizure frequency was described as monthly in two, weekly in six and daily in one patient. Eleven girls were on antiepileptic drug (AED) monotherapy and six on polytherapy. In one patient, therapy was discontinued because of seizure freedom. Detailed clinical, neurophysiological, behavioural and genetic features of the patients were listed in Table 1. This study was performed between 2016 and 2017.
Measurements: Clinical assessments were made during three exams in the hospital: at baseline, 3 months (before switching to open treatment with LCPUFA) and 6 months. At the beginning of the study, informed consent was signed by the parents, inclusion and exclusion criteria were assessed and the investigators carried out a medical evaluation, including medical and neuropsychiatric history. A physical examination, including height and weight, pulse and blood pressure, as well as a neuromotor examination were performed.
Neurophysiological assessment: All patients were evaluated with a wakefulness EEG video-polygraphic recording using a computerized EEG System (Micromed System Plus, Micromed s.r.l., Mogliano Veneto, TV, Italy).
| Table 1: | Clinical, EEG and behavioural characteristics of patients |
1Composite rett assessment rating scale (RARS), from the sum of the points in various domains (communication, daily living skills, socialization, motor skills) of the RARS, 2Composite adaptive behavioural scores, from the sum of the points in various domains (communication, daily living skills, socialization, motor skills) of the vineland adaptive behavioural scale |
|
Scalp electrodes were located according to the International 10/20 system. The EMG electrodes were used for deltoid muscles and/or distal muscles if tremor was present, together with ECG and breathing effort assessment. Video/EEG recording typically lasted 20-30 min.
The EEGs were evaluated independently by two of the authors and were classified according to Glaze et al.48 and Vignoli et al.49 as EEG stage III (moderate to marked slowing of background activity with dominant theta and delta activity) or IV (no occipital dominant rhythm and marked slowing of background activity). Moreover, four subgroups of EEG abnormalities were identified: (1) Theta slow activity over the frontal and central regions (eight cases), (2) Fronto centro parietal spikes (eight cases), (3) Generalized spike and waves (one case) and (4) Diffuse subcontinuous spike and waves suggestive of epileptic encephalopathy (one case).
Behavioural measurements
Vineland adaptive behavior scales: The Vineland Adaptive Behavior Scales50 were developed to support the diagnosis of intellectual and developmental disabilities. The scales are displayed into four domains: Communication (receptive, expressive, written); daily living (personal, domestic, community); socialization (interpersonal relationships, play and leisure time, coping skills) and motor skills (gross, fine). The reliability of the scales was established as follows: split-half, 0.73-0.93 for the communication domain, 0.83-0.92 for daily living skills, 0.78-0.94 for Socialization, 0.70-0.95 for motor skills, 0.84-0.98 for adaptive behaviour composite, 0.77-0.88 for Maladaptive Behaviour (Survey Form) (0.80 and 0.90 sec for the Survey Form). Note that interrater reliability coefficients for the Survey and Expanded forms ranged from 0.62-0.75. Standard error of measurement (SEM) ranged from 3.4-8.2 over the four domains and from 2.2-4.9 for the Adaptive Behaviour Composite, on the Survey Form.
Rett assessment rating scale: The Rett Assessment Rating Scale (RARS) is used to evaluate subjects with RTT46. The structure of RARS is similar to that of the Childhood Autism Rating Scale (CARS)51 Gilliam Autism Rating Scale (GARS)52 and Asperger Syndrome Diagnostic Scale (ASDS)53. The items in RARS were constructed following the diagnostic criteria of the Rett Syndrome Diagnostic Criteria Working Group (RSDCWG) and recent research and clinical experience54. A total of 31 items were generated as representative of the profile of RTT. Each item concerns a specific phenotypic characteristic and describes four increasing levels of its severity. Each item is provided with a brief glossary explaining its meaning in a few words. Each item is rated on a 4-point scale, where 1 = within normal limits, 2 = infrequent or low abnormality, 3 = frequent or medium-high abnormality and 4 = strong abnormality. Intermediate ratings are possible; for example, an answer between 2-3 points is rated as 2.5. For each item, the evaluator circles the number corresponding to the best description of the patient. After a patient has been rated on all 31 items, a total score is computed by summing the individual ratings. This total score allows the evaluator to identify the level of severity of RTT considered as a continuum ranging from mild to profound symptoms.
RARS was standardized throughout a process that involved a sample of 220 patients with RTT, demonstrating the instrument to be statistically valid and reliable. More precisely, normal distribution analyses of the scores were computed and the mean scores of the scale were similar to the median and the mode. Skewness and kurtosis values, calculated for the distribution of the total score, were 0.110 and 0.352, respectively. Distribution was found to be normal. Cronbach’s α was used to determine the internal consistency for the whole scale and subscales. Total a was 0.912 and internal consistency of the subscales was high (0.811-0.934).
Neuropsychological measurements: Eye tracker technology was applied to measure recognition, matching of pairs (the same) and semantic categorization (the similar). Three tasks were carried out: (1) Response to verbal instruction (look at the dog, etc.), (2) Recognition and matching of pairs (look at the one that is the same) and (3) Semantic categorization (look at the one that is similar). The pictures used were of items familiar to the children, according to their parents (Fig. 1).
Nine different images were split into three categories: fruit (apple, grapes and banana), animals (dog, cat and horse) and emotions (happy, sad and angry). Each object was presented for 10 sec. Fixation length (seconds) was measured using an Eye gaze device in order to record the subject’s visual scanning response to visual computer screen stimulation. This device records ocular movements such as the location and duration of ocular fixations (i.e., pause of eye movement on an object of interest) and saccadic movements (i.e., rapid movements between fixations)55.
Eye gaze was employed in a microcomputer with a 15-in. LCD monitor and a Matrox-like video plaque, which captures the signals sent from a video camera equipped with lenses sensitive to high-speed infrared light. The camera also has an LED that emits low-intensity infrared light directly onto the retina of the person sitting in front of the monitor.
| Fig. 1(a-c): | Examples used for the eye tracker technology. Three tasks were designed, (a) Response to verbal instruction (look at the dog, etc.), (b) Recognition and matching of pairs (look at the one that is the same) and (c) Semantic categorization (look at the one that is similar). The images used were of objects familiar to the children, according to their parents |
The direction of the gaze is determined according to the Pupil Center/Corneal Reflection Method. The Passive Gaze Tracking software (LC Technologies, Inc., Sao Paulo, Brazil) was used to generate gaze point, pupil diameter during visual scanning and eyeball position. The girls sat on their parent’s lap, about 20 cm away from the monitor.
Procedure: Each girl was assessed with eye gaze for the neuropsychological aspect, on the same day and in direct succession. Instead, parents were requested to fulfil behavioural measurements in a separate session.
Design: The design of the study was randomized placebo-controlled consisting of two 3 month periods (Study Periods 1 and 2). The first 3-month period was double-blind. The manufacturer of LCPUFA supplied consecutively numbered identical bottles of which 50% contained active treatment and 50% placebo in random order according to a code list that was not accessible to the investigators. The code was opened by a third party when all patients had completed the study, after 6 months. At the start of study period 1, the patients were randomized to LCPUFA (250 mg below 15 kg, 500 mg between 15 and 26 kg or 750 mg if >26 kg) or placebo (maize oil) for 3 months. At the start of study period 2 the groups of patients were switched in order to allow the treatment to all girls.
Statistical analyses: Data were analyzed using SPSS 20.0 for Mac. The descriptive statistics of the dependent variables were tabulated and examined. Alpha-level was set at 0.05 for all statistical tests. In the case of significant effects, the effect size of the test was reported. The effect sizes were computed and categorized according to Cohen56. The paired-samples t-test was used to compare the groups at the base-line and three month periods and at baseline and six-month periods. With reference to each of the categories, t-test for independent group on the means of the three tasks was applied.
RESULTS
With reference to the assimilation of LCPUFA supplementation, plasmatic DHA levels showed no statistical differences using t-test in pre-test phase, but in post-test t-test shows statistical significance (t (20) = 3.11, p<0.01). In addition, DHA in phospholipids circulating levels showed no statistical differences using t-test in pre-test phase, but in post-test 2 t-tests show statistical significance (t (20) = 2.81, p<0.001).
These results suggested that there was some assimilation of LCPUFA supplementation (Table 2).
Despite these results, neurophysiological parameter results showed no statistically significant results. No statistical significance emerges from t-test results.
Weak results also emerged from neuropsychological and behavioural measurements comparing pre-test and post-test measurements. With reference to the Vineland Adaptive Behavior and RARS scale no statistical significance with t-test emerges between baseline measurements and three-month measurements and between baseline and six months measurements.
With reference to neuropsychological measurements, Table 3 showed the means and standard deviation of the length of fixations of the correct target.
With reference to each of the categories, t-test for independent group on the means of the three tasks was applied (Table 3). With reference to baseline measurements, t-test for independent group shows no statistical significance; with reference to the three-month measurements, the t-test for independent group shows statistical significance only with fruit and animal categories, with reference to the six-month measurements, the t-test for independent group shows statistical significance in all categories (Table 3).
DISCUSSION
The aim of the present study was to evaluate changes in LCPUFA status and functional neurophysiological, neuropsychological and behavioural functions, at three and six months, in a trial of LCPUFA supplementation in girls with RTT. The novelty of the present paper was that it used not only clinical measures, but also objective measures related to neuropsychological parameters. The main results indicated that there was some assimilation of LCPUFA supplementation, but neuropsychological and behavioural measurements pre-test and post-test showed weak modifications.
With reference to LCPUFA, despite plasmatic DHA levels and phospholipids circulating levels showing statistical significance at 6 months, meaning that LCPUFA assimilation of supplementation took place, neither neurophysiological parameters nor behavioural measurements (Vineland Adaptive Behavior Scales and RARS) showed statistical significance emerging between baseline measurements and three-month measurements and between baseline and six-month measurements.
Despite neuropsychological measurements showing no significant differences, it is important to note that the length of fixations of the correct target showed an increase. This result indicated that attentional and discrimination measurements appeared to be influenced by LCPUFA supplementation. Thus, it can be speculated that LCPUFA supplementation appears to be useful only in attentional measurements but may be beneficial in improving cognitive functioning in patients with very mild symptoms. Patients with RTT showed increased lipid peroxidation and a dysregulated pattern of metallothionein expression57, indicating an alteration of the oxidative stress pattern58-62.
| Table 2: | Docosahexaenoic acid levels with and without LCPUFA supplementation |
| Table 3: | Means and standard deviation of the length of fixation of the correct target |
| M: Mean, DS: Standard deviation, df: Degree of freedom | |
It has been proven that omega-3 phospholipids have an anti-inflammatory activity, lowering C-reactive protein63-64 and may partially reduce RTT severity in early phases of the disease26,65-67.
Only few studies have examined the possible effects on brain functions in healthy young adults of LCPUFA supplementation. Some studies indicate that DHA supplementation improves memory in healthy, young adults whose habitual diets were low in DHA68-73; a systematic review and meta-analysis provided evidence for the effects of LCPUFA on cognitive protection from observational, preclinical and clinical studies74. Jiao et al.75 in a systematic review and meta-analysis of randomized controlled trials, investigated the effect of n-3 PUFA supplementation on cognitive function throughout the life span from infancy to old age. All these works claimed that n-3 PUFA supplements may significantly improve cognitive development in infants, but do not improve cognitive performance in children, adults, or the elderly. n-3 PUFA intake, especially DHA supplements, may benefit cognitive development during infancy.
Considering the current state of knowledge, the present study is partially in agreement with previous findings, as a positive effect of LCPUFA was found only in attention measurements, but not in neurophysiological and behavioural functions.
Previous studies regarding the effects of omega-3 fatty acids mainly analyzed general factors such as global scale of severity or parent’s judgement21. To the best of our knowledge, this is the first study that has measured behavioural and cognitive factors directly evaluating the performance of girls with RTT after an interventional study. Overall, this is a study with essentially negative results. The double-blind randomized placebo-controlled trial demonstrated that LCPUFA supplementation for 3 and 6 months was not statistically superior to placebo in the entire group of girls with RTT. Because of the limited number of patients included, due to the rarity of RTT, no firm conclusions can be drawn. However, further research in subjects with RTT should involve wider sample size and should provide randomized placebo-controlled trial.
CONCLUSION
The effects of LCPUFA on functional neurophysiological, neuropsychological and behavioural functions of girls with RTT have been investigated in the current study. While the major results are broadly negative, the major findings of this work reveal that LCPUFA produces a positive effect only on attention.
ACKNOWLEDGMENTS
We would like to thank all patients and their family and AIRETT for their cooperation, effort and kindness throughout the study.
SIGNIFICANCE STATEMENT
This study investigated the effect of long chain poly-unsaturated fatty acid supplementation in girls with a genetic syndrome, Rett Syndrome. The results indicated that long-chain poly-unsaturated fatty acids may be beneficial in improving cognitive functioning but only in attention measurements. This study will help researchers use long-chain poly-unsaturated fatty acids in Rett Syndrome. However, many further randomized placebo-controlled trials in RTT should be conducted, involving a wider sample.