Epilepsy & Behavior

Epilepsy & Behavior 66 (2017) 27–33

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Epilepsy & Behavior

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Effectiveness and tolerability of antiepileptic drugs in 104 girls with Rett syndrome

Aglaia Vignoli, Miriam Nella Savini ⁎, Maria Sonia Nowbut, Angela Peron, Katherine Turner, Francesca La Briola, Maria Paola Canevini

Epilepsy Center, San Paolo Hospital, Department of Health Sciences, Università degli Studi di Milano, Italy article info abstract

Article history: Approximately 60–80% of girls with Rett Syndrome (RTT) have epilepsy, which represents one of the most severe Received 29 July 2016 problems clinicians have to deal with, especially when patients are 7–12 years old. Revised 6 October 2016 The aim of this study was to analyze the antiepileptic drugs (AEDs) prescribed in RTT, and to assess their effec- Accepted 8 October 2016 tiveness and tolerability in different age groups from early infancy to adulthood. Available online xxxx We included in this study 104 girls, aged 2–42 years (mean age 13.9 years): 89 had a mutation in MECP2,5in Keywords: CDKL5,2inFOXG1, and the mutational status was unknown in the remaining 8. Rett syndrome Epilepsy was present in 82 patients (79%). Mean age at epilepsy onset was 4.1 years. Epilepsy We divided the girls into 5 groups according to age: b5, 5–9, 10–14, 15–19, 20 years and older. Antiepileptic drugs Valproic acid (VPA) was the most prescribed single therapy in young patients (b15 years), whereas carbamaze- Age-dependency pine (CBZ) was preferred by clinicians in older patients. The most frequently adopted AED combination in the patients younger than 10 years and older than 15 was VPA and lamotrigine (LTG). Seizures in the group aged 10–14 years were the most difficult to treat, requiring a mean of three different AEDs, often used in combination and mostly including VPA. Seizures in fifteen patients (18%) were considered drug resistant. VPA was reported as the most effective AED in younger girls (in 40% of the patients aged b5years,in19% of the girls aged 5–9 years), and CBZ the most effective in the patients 15 years or older. Adverse reactions did not differ from expected: agitation, drowsiness, and weight loss were the most frequently reported. In our sample, LTG was the least tolerated AED. We did not find correlations with MECP2 mutations in terms of effectiveness or adverse reactions. Conclusion: in this study we observed different effectiveness of AEDs based on age, and suggest that clinicians consider age-dependency when prescribing appropriate AEDs in the RTT population. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Epilepsy is one of the main symptoms in RTT, affecting 60–80% of girls, with onset between 2 and 5 years of age in most patients (mean 4 years) Rett syndrome (RTT) is a severe X-linked neurodevelopmental and evidence of age-dependency. Seizure frequency is higher between 7 disorder mostly affecting females, caused by mutations in the MECP2 and 12 years, and decreases in severity in late adolescence [4,5].Thepres- (methyl-CpG binding protein 2) gene (classic form). Prevalence is ence of seizures is associated with worse clinical outcome regarding hand approximately 1:10,000 girls [1,2]. The classic form is characterized by use, ambulation, and communication [6]. normal development in the first months of life, followed by regression All seizure types have been reported except for typical absences and of acquired motor and language skills, appearance of hand stereotypies, clonic seizures. The most frequently reported were focal seizures, and epilepsy. MECP2 mutations are identified in up to 95% of patients followed by generalized tonic-clonic, tonic, and myoclonic [4,7]. with the classic form. Non-classic forms include the preserved-speech Genotype-phenotype correlations are still controversial, even though (Zappella) variant, the early-seizure (Hanefeld) variant (frequently asso- some mutations are associated with a higher prevalence of epilepsy or se- ciated with CDKL5-cyclin-dependent kinase-like 5-mutations), and the verity of seizures [8]. congenital (Rolando) variant (often caused by FOXG1-fork-head box Seizures are drug-resistant in approximately 30% of cases, and protein G1-mutations) [3,4]. polytherapy with three or more anti-epileptic drugs (AEDs) is necessary in 18% of the patients [4,8]. Only a few studies report on effectiveness of AEDs in these patients, ⁎ Corresponding author at: Epilepsy Center, San Paolo Hospital, Università degli Studi di fi Milano, Via di Rudinì, 8, 20142 Milano, Italy. and no de nite recommendations are currently available. Small series E-mail address: [email protected] (M.N. Savini). with limited numbers of patients have been described, reporting that

Table 1 Number of patients using monotherapy and associations of drugs in the 5 age groups.

Group 1 (b5 years) Group 2 (5–9 years) Group 3 (10–14 years) Group 4 (15–19 years) Group 5 (N20 years) n=60 n=66 n=47 n=35 n=18

Monotherapy 45 (75%) 47% VPA 43 (65%) 65% VPA 25 (53%) 56% VPA 18 (51%) 39% CBZ 6 (33%) 66% CBZ 9% CBZ 32% CBZ 40% LTG 33% LTG 33% VPA 19% LTG 20% CBZ 28% VPA 16% LEV 12% CLB 14% CLB 12% PB Associations of AEDs 14 (23%) 17 (26%) 17 (36%) 9 (26%) 8 (44%) No tp 1 (2%) 1 (2%) 2 (4%) 2 (6%) 1 (5%) Lost at f-up 0 5 (7%) 3 (6%) 6 (17%) 3 (17%)

AEDs, antiepileptic drugs; Tp, therapy; VPA, valproic acid; CBZ, carbamazepine; LTG, lamotrigine; PB, phenobarbital; LEV, levetiracetam; CLB, clobazam. the most frequently used AEDs were valproic acid (VPA), lamotrigine 3. Statistical analysis (LTG), topiramate (TPM), and carbamazepine (CBZ) [9]. Tolerability represents another open issue, with few reports available. Adverse events All collected data were transferred into an electronic database and seem to be similar to the ones observed in the general population [4]. processed using the Statistical Package for the Social Sciences (SPSS, Even though the age-dependency of effectiveness and tolerability IBM, Chicago, IL, U.S.A.) for Macintosh version 22.0. profile of AEDs seems evident in clinical practice, this aspect has not Based on the aforementioned MECP2 classification, we compared been investigated in patients with RTT thus far. patients with and without epilepsy with the non-parametric test χ2. In this study, we analyzed the AEDs prescribed, the effectiveness of Results were considered statistically significant for p-values b 0.05 treatment, and the adverse reactions in five age groups of RTT girls (two-sided). from early infancy to adulthood. 4. Results 2. Methods We included in this study 104 female patients aged 2–42 years This retrospective study was carried out at the Epilepsy Center of the (mean age 13.9 years). San Paolo Hospital, University of Milan (Italy). Data were obtained from Results of genetic tests were available in 100 patients: 89 girls had a a dedicated database of RTT patients, meeting the diagnostic criteria mutation in MECP2,5inCDKL5,2inFOXG1, and in 4 patients no muta- proposed by Neul in 2010 [3]. The institutional review board of San tions were identified. Data about genetic analyses were not available in Paolo Hospital, Milan, approved the study. four patients. We collected data on genetic tests performed, mutations revealed, 83 girls presented classic form of RTT, 6 were classified as Preserved motor skills, and clinical problems. Epilepsy characteristics (presence, Speech Variant, 5 as early seizure variant, and 2 as congenital variant. age of onset, seizure type, frequency, and EEG abnormalities) and phar- Mean age at RTT diagnosis was 5.95 years. Regression was reported macologic treatment (monotherapy, combination of treatment, effec- in 76% (80) of the patients; mean age at regression was 16.1 months. tiveness, adverse events) were reviewed. We classified epileptic seizures according to the 2010 International 4.1. Epilepsy Classification of Seizures, proposed by ILAE in 2010 [10]. As proposed by ILAE, we considered drug resistance as the failure of Epilepsy was present in 79% (82 pts), with mean age at onset of adequate trials of two tolerated and appropriately chosen and used AED 4.1 years (7 days–14 years). schedules (either as monotherapies or in combination) to achieve Seizures were classified as focal in 47.6% (39) of the patients, gener- sustained seizure freedom [11]. alized tonic-clonic in 18.3% (15), tonic in 15.8% (13), spasms in 5% (4), Effectiveness was determined considering clinical data and caregivers' atonic in 4% (3), atypical absences in 2% (2), and myoclonic seizures in reports for a minimum follow-up period of one year [11]. 2% (2). We decided to evaluate antiepileptic treatment in different age EEG recordings showed bilateral fronto-centro-temporal abnor- groups: b5 years (Group 1), 5–9 years (Group 2), 10–14 years (Group malities in 52%, focal abnormalities in 19%, and multifocal spikes in 3), 15–19 years (Group 4), 20 years and older (Group 5). 13%. Slow diffuse rhythms were identified in 5% of the girls, We divided MECP2 mutations into three groups based on type generalized abnormalities in 3% of the patients, and in 7% EEG and localization on the gene: early truncating (within the Nuclear resulted normal for age. Localization Signal-NLS-domain), late truncating (after the NLS do- Data about antiepileptic treatment in each of the five age groups are main), and missense. Based on this classification, we attempted summarized in Tables 1 and 2. correlations between genotype and AED treatment. We did not With respect to MECP2 mutations, 39% of the patients (35) showed consider mutations in CDKL5 and FOXG1, since only seven patients Early Truncating changes, 19% (17) had Late Truncating mutations, had changes in these genes in our cohort. and 36% (32) carried missense mutations. Mutations were not

Table 2 Number of AEDs used in the ﬁve age groups. AEDs, antiepileptic drugs.

N AED Group 1 (b5 years) Group 2 (5–9 years) Group 3 (10–14 Group 4 (15–19 Group 5 (N20 n=60 n=66 years) years) years) n=47 n=35 n=18

1 AED 35 58% 31 47% 14 30% 19 54% 6 33% 2 AEDs 12 20% 16 24% 8 16% 15 43% 8 45% 3 AEDs 5 8% 10 15% 19 41% 0 0% 2 11% N3 AEDs 8 13% 9 14% 6 13% 1 3% 2 11% A. Vignoli et al. / Epilepsy & Behavior 66 (2017) 27–33 29 classifiable in 6% of the patients (5) due to missing reports. By compar- of 4 taking zonisamide (ZNS) had appetite loss and the drug was ing the individuals with and without epilepsy in each group, we found stopped in both cases. One out of 40 taking VPA developed alopecia. that epilepsy was significantly more frequent only in the group with Two out of 8 taking TPM showed appetite loss and agitation (in one missense mutations (p = 0.002) (Table 3). case TPM was suspended), and in one out of 4 taking LEV agitation was reported (with discontinuation of therapy). 4.2. Effectiveness of AEDs No adverse events were recorded in Groups 4 and 5. Agitation was reported in association with 5 different drugs: LTG We found that AED treatment was effective in 65% of the girls (53 (3 pts), LEV (2), VPA (1), TPM (1), ESM (1). Sedation was associated patients), while seizures in 18% (15 patients) were drug resistant. In with CBZ (1 patient), LTG (1), LEV (1), and CLB (1). Appetite loss oc- the remaining, seizures occurred occasionally (3 patients) or we did curred with ZNS (2), TPM (1), and ESM (1). not have enough data to determine effectiveness (11 patients). No serious adverse events were reported. In Group 1, monotherapy with VPA was effective in 40% of patients Adverse effects are summarized in Fig. 3. (6), CBZ in 20% (3), LEV in 13% (2), vigabatrin (GVG) and LTG in 7% of patients each (1 patient), and combinations of AEDs in 13% (2). 5. Discussion In Group 2 monotherapy with VPA was effective in 19% of patients (3), CBZ in 13% (2), LEV, LTG and adrenocorticotropic hormone In this study, we considered effectiveness and tolerability of AED (ACTH) in 6% each (1 patient) and combinations of AEDs in 42% (8). treatment in a well-studied Italian population of patients diagnosed In Group 3 CBZ, LTG, GVG and TPM resulted effective in 11% each (1 with RTT, with a long follow-up including patients from early infancy patient), and in 55% of patients, combinations of AEDs were needed (5). to adulthood. Few studies have evaluated AED treatment, and none of In Group 4 CBZ showed good effectiveness in 50% of patients (4), LTG them analyzed data according to different age groups [5,6,8,12]. in 25% (2), clobazam (CLB) and VPA in 12% each (1 patient). Antiepileptic drug treatment is currently based on the epileptologist's In Group 5, CBZ was effective in 40% of patients (2), and associations choice and experience, since age-matched prospective studies are difficult of AEDs were effective in 60% (3). to perform given the relatively low prevalence of this rare disease [12,15]. AEDs effectiveness in the five age groups is summarized in Fig. 1. Our study population was fairly representative of the general char- When combinations of AEDs were needed, they included VPA for the acteristics of girls with RTT, as reported in the literature [13,14]. majority of patients. VPA and LTG resulted as the most frequently used According to the documented age dependency of epilepsy in differ- combinations (13 out of 58 patients). Other AEDs used in combination ent epilepsy syndromes, (e.g. Angelman Syndrome) [16] and suggestive with VPA were CBZ, TPM, and CLB (Fig. 2). clinical evolution of epilepsy in RTT, we divided our patients into 5 age AEDs discontinuation was possible in two patients of the last 2 groups to evaluate the infl uence of age on epilepsy treatment outcome. groups (N15 years), in one case with good results, in the other with re- VPA was the most frequently used AED in girls younger than appearance of seizures after 4 years. 15 years (group, 1, 2 and 3), followed by CBZ, LTG and CLB. CBZ became the most adopted monotherapy for patients older than 4.3. Adverse effects 15 years, followed by LTG and VPA. Almost half of the patients younger than 10 years (groups 1 and 4.3.1. Group 1 2) and those aged 15–19 years (group 4) needed only one AED, Two patients out of 47 taking VPA presented adverse events: in one while only 30% of the patients between 10 and 14 years (group case agitation was reported and in the other worsening of ambulation 3) needed only one AED, and 41% of them needed three AEDs. This appeared, requiring discontinuation in the latter case. Two patients finding suggests that seizures in this group are the most difficult to out of 10 taking LTG showed nocturnal hyperactivity and drowsiness treat, as already evidenced in the literature [4]. Physical and hor- during the day. Two out of 4 taking ethosuximide (ESM) experienced monal changes occurring at that age could be the speculative mech- agitation and appetite loss, and treatment was stopped in both. One pa- anism as an alteration in the phenotypic expression of epilepsy tient out of 14 taking CBZ showed drowsiness (CBZ had to be during puberty is well documented in some genetic syndromes [17]. suspended), and one patient out of 7 taking LEV showed agitation. By analyzing the effectiveness of AEDs in different age groups, monotherapy with VPA showed good seizure control in younger girls, 4.3.2. Group 2 especially in the group of patients younger than 5 years of age, decreas- Two patients out of 9 taking CLB had drowsiness and dizziness (CLB ing afterwards, whereas CBZ provided very good effectiveness in girls was stopped), two patients out of 7 taking LEV showed tremors and older than 15 years. drowsiness (therapy was interrupted in one case), one patient out of VPA and CBZ have already been reported as effective AEDs in RTT 12 taking LTG had agitation (LTG was stopped), and 1 out of 5 taking [12], but the age differences of included patients may influence the dis- VPA presented with dizziness. cordant outcomes reported [15]. LTG was frequently used in monotherapy and showed good and 4.3.3. Group 3 increasing effectiveness in the first four age groups, but it is Three patients out of 14 taking LTG showed mild agitation (2 indi- necessary to note that it is usually administered in girls older than viduals) and skin rash (1 patient, with interruption of LTG). Two out 12 years in Italy due to regulatory issues.

Table 3 Prevalence of epilepsy in different MECP2 mutations.

Mutations classiﬁcation MECP2 Epilepsy No epilepsy (N = 68) (N = 16)

Early truncating (within NLS) 26 (38.2%) 9 (56.2%) Ns 0.063 Late truncating (after NLS) 13 (19.1%) 4 (25.0%) Ns 0.366 Missense 29 (42.7%) 3 (18.8%) Sig 0.002

NLS, nuclear localization signal; Ns, Not statistically signiﬁcant; Sig, Signiﬁcant. 30 .Vgoie l plpy&Bhvo 6(07 27 (2017) 66 Behavior & Epilepsy / al. et Vignoli A. – 33

Fig. 1. AED Effectiveness in different age groups. CBZ, carbamazepine; CLB, clobazam; ESM, ethosuximide; GVG, vigabatrin; LEV, levetiracetam; LTG, lamotrigine; NZP, nitrazepam; OXC, oxcarbazepine; PB, phenobarbital; PHT, phenytoin; TPM, topiramate; VPA, valproic acid; ZNS, zonisamide. .Vgoie l plpy&Bhvo 6(07 27 (2017) 66 Behavior & Epilepsy / al. et Vignoli A. – 33

Fig. 2. Combinations of AEDs used in different age groups. CBZ, carbamazepine; CLB, clobazam; ESM, ethosuximide; GVG, vigabatrin; LEV, levetiracetam; LTG, lamotrigine; NZP, nitrazepam; OXC, oxcarbazepine; PB, phenobarbital; PHT, phenytoin; TPM, topiramate; VPA, valproic acid; ZNS, zonisamide. 31 32 .Vgoie l plpy&Bhvo 6(07 27 (2017) 66 Behavior & Epilepsy / al. et Vignoli A. – 33

Fig. 3. Adverse events reported. CBZ, carbamazepine; CLB, clobazam; ESM, ethosuximide; LEV, levetiracetam; LTG, lamotrigine; TPM, topiramate; VPA, valproic acid; ZNS, zonisamide. A. Vignoli et al. / Epilepsy & Behavior 66 (2017) 27–33 33

Other potentially useful drugs (such as LEV, TPM) were used in a re- References stricted number of patients because of potential effects on nutritional [1] SJ M, Weber P, Lütschg J. Rett syndrome: clinical and electrophysiologic aspects. and behavioral aspects. Therefore, we could not obtain data on their Pediatr Neurol 2007 Feb;36(2):95–100. effectiveness. [2] Hagberg B, Hagberg G. Rett syndrome: epidemiology and geographical variability. The combinations of drugs most often used and with the best Eur Child Adolesc Psychiatry 1997;6(Suppl 1):5–7. [3] Neul JL, Kaufmann WE, Glaze DG, Christodoulou J, Clarke AJ, Bahi-Buisson N, et al. results for seizure control included VPA (see Fig. 1). We noticed a Rett syndrome: revised diagnostic criteria and nomenclature. Ann Neurol 2010 relatively poor positive effect of benzodiazepines in monotherapy Dec;68(6):944–50. and in combination with other AEDs. This finding can be explained [4] Krajnc N. Management of epilepsy in patients with Rett syndrome: perspectives and – both by previous human [18] and cellular studies [19], indicating considerations. Ther Clin Risk Manag 2015 Jun 10;11:925 32. [5] Vignoli A, La Briola F, Peron A, Turner K, Savini M, Cogliati F, et al. Medical care of ad- that GABAergic transmission is modified in RTT. olescents and women with Rett syndrome: an Italian study. Am J Med Genet A 2012 The most frequent adverse events were not age-specific and were Jan;158A(1):13–8. not different from those expected in the general epilepsy population [6] Glaze DG, Percy AK, Skinner S, Motil KJ, Neul JL, Barrish JO, et al. Epilepsy and the natural history of Rett syndrome. Neurology 2010 Mar 16;74(11):909–12. [15,20,21]. In general, LTG was the least tolerated AED. [7] Buoni S, Zannolli R, Felice CD, Saponari S, Strambi M, Dotti MT, et al. Drug-resistant The results from various studies strongly indicate that AED use may epilepsy and epileptic phenotype-EEG association in MECP2 mutated Rett syn- have negative impact on bone health [22]. Although in RTT populations drome. Clin Neurophysiol 2008 Nov;119(11):2455–8. [8] Nissenkorn A, Levy-Drummer RS, Bondi O, Renieri A, Villard L, Mari F, et al. Epilepsy VPA has particularly been linked with increased risk of bone fractures in Rett syndrome—lessons from the Rett networked database. Epilepsia 2015 Apr; [23], the use of VPA was not linked to a higher risk of bone fractures in 56(4):569–76. our population. [9] Dolce A, Ben-Zeev B, Naidu S, Kossoff EH. Rett syndrome and epilepsy: an update for child neurologists. Pediatr Neurol 2013 May;48(5):337–45. Comparing the patients with and without epilepsy according to [10] Berg AT, Berkovic SF, Brodie MJ, Buchhalter J, Cross JH, van Emde BW, et al. Revised mutational status, we found that epilepsy was significantly more terminology and concepts for organization of seizures and epilepsies: report of the frequent only in the group with missense mutations (p = 0.002), ILAE commission on classification and terminology, 2005—2009. Epilepsia 2010 fi Apr;51(4):676–85. while the difference was not statistically signi cant in the groups [11] Kwan P, Arzimanoglou A, Berg AT, Brodie MJ, Hauser WA, Mathern G, et al. Defini- with either early or late truncating changes. These results suggest tion of drug resistant epilepsy: consensus proposal by the ad hoc task force of the that if a patient has a truncating mutation, it is hard to predict ILAE commission on therapeutic strategies. Epilepsia 2010;51:1069–77. whether or not she will develop seizures based only on mutation [12] Pintaudi M, Calevo MG, Vignoli A, Baglietto MG, Hayek Y, Traverso M, et al. Antiepilptic drugs in Rett syndrome. Eur J Paediatr Neurol 2015 Jul;19(4):446–52. data. On the other hand, patients with missense mutations seem to [13] Smeets EE, Chenault M, Curfs LM, Schrander-Stumpel CT, Frijns JP. Rett syndrome be more likely to develop seizures. However, the limitation of our and long-term disorder profile. Am J Med Genet A 2009 Feb;149A(2):199–205. analysis is inadequate sample size to perform genotype-fenotype [14] Guerrini R, Parrini E. Epilepsy in Rett syndrome, and CDKL5- and FOXG1-gene- related encephalopathies. Epilepsia 2012 Dec;53(12):2067–78. correlations of each mutation. Further investigations, with larger [15] Huppke P, Köhler K, Brockmann K, Stettner GM, Gärtner J. Treatment of epilepsy in number of patients, are needed to delineate specificprofiles and Rett syndrome. Eur J Paediatr Neurol 2007 Jan;11(1):10–6. treatment options based on mutations. [16] Larson AM, Shinnick JE, Shaaya EA, Thiele EA, Thibert RL. Angelman syndrome in adulthood. Am J Med Genet A 2015 Feb;167A(2):331–44. In conclusion, our study suggests that VPA is the most prescribed [17] Morrell MJ. Epilepsy in women: the science of why it is special. Neurology 1999; AED for girls with RTT younger than 10 years of age, whereas CBZ should 53(4 Suppl 1):S42–8. be considered in girls older than 15 years. Seizures in the group of pa- [18] Yamashita Y, Matsuishi T, Ishibashi M, Kimura A, Onishi Y, Yonekura Y, et al. De- – fi crease in benzodiazepine receptor binding in the brains of adult patients with Rett tients aged 10 14 years were the most dif cult to treat and a combina- syndrome. J Neurol Sci 1998 Feb 5;154(2):146–50. tion of AEDs, frequently including VPA, was necessary. [19] Livide G, Patriarchi T, Amenduni M, Amabile S, Yasui D, Calcagno E, et al. GluD1 is a Based on our findings, we suggest that clinicians consider age- common altered player in neuronal differentiation from both MECP2-mutated and CDKL5-mutated iPS cells. Eur J Hum Genet 2015 Feb;23(2):195–201. dependency of epilepsy treatment when administering AEDs in RTT. [20] Krajnc N, Župančič N, Oražem J. Epilepsy treatment in Rett syndrome. J Child Neurol Newer drugs could be used, but age-related restrictions should be 2011 Nov;26(11):1429–33. considered, due to current regulatory constraints. Other options are [21] Bao X, Downs J, Wong K, Williams S, Leonard H. Using a large international sample represented by non-pharmacological treatments, such as ketogenic to investigate epilepsy in Rett syndrome. Dev Med Child Neurol 2013 Jun;55(6): 553–8. diet and vagus nerve stimulation, but little evidence is available in [22] Svalheim S, Sveberg L, Mochol M, Taubøll E. Interactions between antiepileptic drugs the RTT population. and hormones. Seizure 2015 May;28:12–7. Future studies should focus on new-generation AEDs and the trans- [23] Leonard H, Downs J, Jian L, Bebbington A, Jacoby P, Nagarajan L, et al. Valproate and risk of fracture in Rett syndrome. Arch Dis Child 2010 Jun;95(6):444–8. lation of drugs with proven efficacy in mouse models or induced plurip- otent stem cells (iPS) into human models.

Acknowledgments

The authors would like to acknowledge all the patients and families from AIR (Italian Association for Rett Syndrome) who helped us carry out the study.