Environmental enrichment intervention for Rett syndrome: an individually randomised stepped wedge trial

Rett syndrome is caused by a pathogenic variant in the MECP2 gene [1] resulting in major consequences for the development of motor functioning and cognitive skills. Motor effects include loss of hand function skills during a regression period [2] with a slow decline in gross motor skills over time [3]. Environmental enrichment models are components of most early intervention programs for children with a neurodevelopmental disability evaluated most frequently in cerebral palsy [4]. Although similar programs also exist for Rett syndrome, their evidence base is poor [5]. In a single subject design study with three affected girls, participation in conductive education improved gross motor skills [5].

One of the effects of impaired MECP2 function is reduced production of Brain Derived Neurotrophic Factor (BDNF), a protein required for normal neuronal development and brain function [6]. There is some clinical evidence of a role for BDNF in Rett syndrome pathogenesis [7‐9]. Relationships between the type of BDNF polymorphism and phenotype have been demonstrated, with more clinical severity and earlier seizure onset when limited to the p.Arg168* mutation [7]. Overall, the age of seizure onset has been observed as earlier [9] or slightly later [7] in individuals with the heterozygous (Val/Met) irrespective of mutation type. In another study, no child with a heterozygous (Val/Met) BDNF polymorphism developed epilepsy earlier than 2 years [8]. Environmental enrichment reduces cellular and behavioural deficits in animal models of disorders including Huntington’s disease, Fragile X and Down syndrome [10], and clinical improvements have been found in Parkinson’s and Alzheimer’s diseases [11] and cerebral palsy [4]. When housed in an enriched environment, MECP2 null mice have demonstrated improved motor abilities and increased levels of BDNF in the brain [12]. Environmental enrichment could benefit children in the early stages of Rett syndrome and increased BDNF levels could be associated with improvements. Environmental enrichment could effect wellbeing more generally. For example, difficulties getting to sleep and staying asleep [13] occur frequently in Rett syndrome [14]. Exercise could improve these symptoms and potentially improve behavioural outcomes such as mood [15].

Based on the incidence of Rett syndrome in Australia [16], approximately 1000 girls of the 8.6 million girls born each year in China and approximately 222 of the nearly 2 million girls born each year in the US will have Rett syndrome. Whilst Rett syndrome is rare, many girls are affected globally. This study examined the effects of environmental enrichment on gross motor skills and BDNF protein levels in Rett syndrome. As secondary outcomes, sleep quality and mood were also evaluated.

The study was discussed with 13 families registered with InterRett of whom 12 (92.3%) provided informed consent. The mean (SD) age of the children at the first baseline assessment was 3.0 (1.1) years ranging from 1.5 to 5.2 years. Their mean (SD) age at regression had been 1.5 (0.4) years ranging from 1 to 2.3 years. Each had a pathogenic MECP2 mutation and most of the common mutation categories were represented (C-terminal [n = 1], Early truncating [n = 1], Large deletion [n = 1], p.Arg168* [n = 1], p.Arg255* [n = 2], p.Arg270* [n = 3], p.Arg294* [n = 1] and p.Thr158Met [n = 2]). The BDNF polymorphism was homozygous (Val/Val) in three girls, heterozygous (Val/Met) in seven and negative (Met/Met) in two girls. At the first baseline assessment, five girls walked independently, four walked with assistance and three were unable to walk. One girl aged 18 months (p.Arg255*, Val/Met BDNF polymorphism) had been diagnosed with epilepsy prior to the commencement of the study which was managed with Levitircetam, and two girls developed epilepsy during the intervention period and commenced Valproate.

Mean (SD) values for each of the outcomes at each of the assessments are shown in Table 1. The RSGMS score was similar at baseline assessments 1 and 2, whether the duration of baseline was 1 month (ICC 0.977 [CI 0.751, 0.998], p = 0.003), two (ICC 0.987 [CI 0.813, 0.999], p = 0.001) or 3 months (ICC 0.976 [CI 0.772, 0.998], p = 0.003). The baseline blood BDNF levels were less variable when the duration of baseline was 1 month (ICC 0.615 [CI -0.31, 0.968], p = 0.120) than for 2 months (ICC 0.223 [CI -0.957, 0.969], p = 0.392) and 3 months (ICC 0.309 [CI -1.042, 0.939], p = 0.327), but the average differences were small at 27.7 ng/ml, 40.6 ng/ml and 9.9 ng/ml respectively. In total, video for 116/900 gross motor items were missing over the five testing occasions equating to 2 of 15 items per assessment per participant. Most missing items were the most complex skills and the previously observed level was carried forward as a conservative estimate. Because of illness (one each with lower respiratory tract infection and seizures), two gross motor assessments and post intervention blood tests could not be measured.

Compared to baseline, the within-person RSGMS score increased by 3.4 (95%CI 1.5, 5.3) points after 2 months of treatment, 5.7 (95%CI 3.3, 8.2) after 4 months, and 8.2 (95%CI 5.1, 11.2) after 6 months of treatment, adjusting for age of regression and current age. RSGMS scores increased 1.1 (95%CI 0.1, 2.2) point per additional month in age at the time of regression. For the 10 girls with post intervention BDNF data, there was a 10.3 (95%CI 3.2, 17.4) point increase in RSGMS score after 6 months of treatment adjusting for the effects of age at regression, current age and BDNF levels. Compared to baseline, BDNF levels increased by 321.4 ng/ml (95%CI 272.0, 370.8) after 6 months of treatment, adjusting for age at regression, current age and RSGMS score. BDNF levels increased an additional 39.0 ng/ml (95%CI 15.2, 62.4) for each additional year in age, adjusting for age at regression and the RSGMS score (Table 2).

Adjusting for covariates, BMI decreased slightly after 2 months of treatment (−0.6 kg/m² [−1.2. -0.1]) but thereafter increased and after 6 months of treatment was similar to baseline values (−0.3 kg/m² [−1.2, 0.6]); the DIMS subscale scores remained similar to baseline values throughout the treatment period as did the mood subscale of the RSBQ (Table 3).

The median (range) RSGMS and blood BDNF levels at each assessment are presented in Table 4 for each of the BDNF polymorphisms. Girls with the heterozygous polymorphism (Val/Met) had the lowest median RSGMS scores and blood BDNF levels at baseline but the magnitude of increase after 6 months of treatment was similar to girls with the Val/Val or Met/Met polymorphisms. The increase in score as a proportion of the baseline score was highest in girls with the Val/Met polymorphism (62%) compared with those with the Val/Val (29%) or Met/Met (32%) polymorphisms (Table 4).

Motor learning and exercise supplemented with rich social, cognitive and other sensory experiences had positive effects on motor functioning in Rett syndrome. Adjusting for covariates, participants gained on average eight points on the 45-point motor scale after 6 months treatment, much greater in magnitude than the response observed in the conductive education study after 6 months [5]. Importantly, gross motor improvement was observed for children with different abilities at baseline and included achievement of independent sitting, walking or transition skills such as sitting to standing. We did find that children with a later age at regression, usually observed with MECP2 mutations associated with milder clinical severity [30], gained slightly more gross motor skills over the treatment period. It is not known whether a longer treatment period was necessary to achieve greater gross motor gains for those who had experienced earlier regression.

General health issues were not compromised. Growth and sleep patterns were maintained over the study period and the intervention did not appear to affect mood, although the mood subscale of the RSBQ developed for Rett syndrome may not have been sensitive to changes in mood in preschool children. Two girls in the current study developed epilepsy during the intervention period which was not inconsistent with the natural history of Rett syndrome where 50% develop epilepsy by 5 years of age [31]. We observed that an environmental enrichment intervention could be safely delivered to girls with Rett syndrome.

To our knowledge, this is the first intervention study investigating serum BDNF levels as an outcome in a genetically characterised sample of children with Rett syndrome. Baseline BDNF levels in our sample were generally lower than in children in the general population measured using the same assay [32], similar to animal studies where BDNF protein levels in the brain of MecP2 mutant mice were reduced compared with wild-type levels [33]. In contrast, BDNF levels in children with autism spectrum disorder [34] and Down syndrome are high [35] compared with the general population. BDNF levels vary with factors such as the time of the day [24, 36] and age [36], and further standardized assessment in children with Rett syndrome is necessary to understand BDNF production in Rett syndrome and explain any variance between different neurodevelopmental disorders.

An important mechanism underpinning the beneficial effects of environmental enrichment is believed to be increased levels of BDNF, which promotes the survival and growth of neurones, synaptic efficiency and neuroplasticity [6]. In studies of MeCP2 knockout mice, strategies to improve BDNF signalling have included administration of IGF-1 [37] and the provision of an enriched environment [12, 38]. In 12 girls with a MECP2 mutation, peripherally administered IGF-1 was associated with modest improvement in autonomic and behavioural features [39]. General population studies suggest that aerobic exercise is associated with increases in BDNF levels [40]. In our present study, the provision of an enriched and supported environment intervention that included exercise was associated with a threefold increase in serum BDNF levels alongside parallel gains in gross motor skills. Although we cannot discern the ingredients of the intervention that were associated with changes in BDNF levels, we recognize that administration of any exercise regimen for young children with movement deficits requires training and learning in an appealing environment that promotes, rewards and challenges activity.

We also included the polymorphism status of the BDNF gene in our analyses. The substitution of valine for methionine at codon 66 (Val66Met) is a common BDNF polymorphism which can impair intracellular trafficking and secretion of the resulting mature BDNF protein [33] and in Mecp2 knockout mice, reduced dendritic growth and complexity and reduced frequency of postsynaptic currents were found [41]. In Rett syndrome, the presence of the Val/Met BDNF polymorphism has been associated with slightly greater clinical severity [7] consistent with the current study where girls with the Val/Met polymorphism had the poorest gross motor skills at baseline. Two of the three individuals with seizures had the Val/Met polymorphism and the other the Met/Met polymorphism. One of these Val/Met individuals had seizure onset prior to 2 years of age in contrast with another study there were no individual with this genotype experienced seizures prior to 2 years of age [8]. It would be reasonable to expect that not all individuals with Rett syndrome would benefit equally from increased BDNF because of the type of MECP2 mutation or BDNF polymorphism. A six-week high-intensity exercise program resulted in cognitive benefits for children in the general population compared to an active control group, and the greatest improvements were observed in children who were Met carriers compared to those with homozygous BDNF status [42]. Similar to this recent literature, we also observed different responses to training with the change in gross motor scores as a proportion of baseline scores being greatest for girls with the Val/Met polymorphism. In contrast to previous studies of mainly Caucasian girls with Rett syndrome [7‐9], more girls in the current sample had the Val/Met rather than the Val/Val polymorphism similar to other studies of Asian populations [43]. Investigations of larger sample sizes are needed to investigate the relationships between the BDNF polymorphism and responses to an enriched environment.

We did not conduct a conventional randomized controlled trial because of sample size availability and the striking variability in motor abilities between different genotypes, which limited our capacity to randomize into two similar groups. A small proportion of video data was missing and our sample size precluded longitudinal modelling to estimate the missing data. We therefore carried forward the last known value for each item for each child, consistent with our observations that no child lost skill over the course of the intervention and resulting in a conservative estimate of functioning. We also do not know whether increased blood levels of BDNF over the study period were reflected in brain tissues, but we are encouraged by the parallel increases in gross motor skills and evidence that serum BDNF can cross the blood-brain barrier in young animals [44]. We nevertheless implemented strategies to minimise bias. We recruited children with minimal exposure to interventions enabling us to more clearly identify an effect; we randomised individuals to the duration of the baseline period and then assessed under control and intervention conditions; we standardised the timing of blood draws to reduce diurnal variation in serum BDNF level [24]; and we used a blinded assessor to code the video data. We still captured variability in MECP2 mutation and BDNF polymorphism types and different levels of baseline skills but recommend replication of our methods to accumulate more evidence.

Environmental enrichment has long been applied as an intervention to ameliorate the effects of brain disorders on functioning. We suggest that the enriched environment has benefits for children with Rett syndrome and clinicians and therapists should plan therapy and activity opportunities accordingly. New drug, cell and gene therapies for neurological disorders including Rett syndrome are being developed that use BDNF pathways [45]. We propose that behavioural interventions such as environmental enrichment will be an important adjunct in the evaluation of these new therapies because environmental enrichment also has implications for functioning.