Fragile X syndrome (FXS) causes inherited mild-to-severe intellectual disability and is the most frequent monogenic cause of autism spectrum disorder (ASD) worldwide (1, 2). FXS arises from a loss of function mutation in the X-linked FMR1 gene due to its transcriptional silencing resulting from hypermethylation of an abnormally expanded CGG trinucleotide repeat in the 5′ untranslated region (3). The transcriptional silencing of FMR1 results in the absence or reduction of Fragile X messenger ribonucleoprotein (FMRP), which is an RNA-binding protein involved in synapse maturation and neural circuit function (4). Accordingly, a disruption in functional integration within brain networks occurs and accounts for neurobehavioral dysfunctions in FXS individuals, which range from developmental delay with general cognitive impairment and severe disability to milder cases with impulsivity, increased response to sensitive stimuli, social anxiety and phobias, hyperactivity, attention deficit, and autism spectrum disorder (5). Language development and literacy are compromised in FXS individuals, with language delays that have been described in the areas of overall communication abilities and the specific domain of expressive, receptive, and pragmatic language and speech intelligibility (6, 7). Common language deficits observed in FXS individuals include delayed language development, articulation deficits, and language processing difficulties, with a consequent impact on social communication skills. The pathophysiological alterations that underlie language deficits in Fragile X syndrome are not fully understood, but a previous study suggests a key role of functional connectivity disruption between the frontal and temporal cortex and exaggerated frontal gamma power before speech onset (8). Accordingly, in the last few years, neurotransmission and synaptic deficits have been shown in the Fmr1 knockout (KO) mouse models, which show common phenotypes with FXS patients, opening new potential targets for therapeutic interventions (4, 9). Indeed, a key function of FMRP is to inhibit protein translation at the synapse, with a consequent upregulation of metabotropic glutamate receptors (mGluRs) 1 and 5 and exaggerated hyperexcitability and long-term depression (LTD) mechanisms (10). Defects in the inhibitory GABAergic system have also been identified in the amygdala, cerebral cortex, and cerebellum in Fmr1 knockout mice models, and they are associated with symptoms such as seizures, anxiety, and attention processing deficit (11), which characterize FXS (12–15). Current pharmacological treatments available for FXS are limited to the use of antiglutamatergic drugs, mood stabilizers, and serotonin reuptake inhibitors to control anxiety and depression. However, no effective treatment has been identified to treat cognitive and autistic behavior, with interventions mainly focused on symptom management. Non-invasive brain stimulation techniques have been widely used to investigate the neurophysiological basis of neurological conditions and to treat several neuropsychiatric symptoms (16–18). Anodal prefrontal transcranial direct current stimulation (tDCS) applied over language areas including Broca’s area has been used to improve articulation and speech output in autism spectrum disorder patients (19) and individuals with chronic aphasia (20–23). Based on these premises, the current study aimed to evaluate the safety, tolerability, and efficacy of prefrontal tDCS combined with standard speech therapy sessions to enhance linguistic function in FXS patients. To detect possible in vivo neurophysiological changes in cortical excitability and cortical oscillations underlying cognitive effects, we collected transcranial magnetic stimulation combined with electroencephalography (TMS-EEG) recordings over the prefrontal cortex.

All procedures are well tolerated with complete adherence to the treatment when each patient completes the 15 sessions. Regarding assessment, one patient was not compliant with the TMS-EEG evaluation. tDCS procedures were well tolerated, and no adverse events (AEs) were reported. The tDCS is a safe technique to perform neuromodulation, in the literature are not reported any reports of serious AEs or irreversible injury (36). Slight side effects were reported in other studies in the form of burn-like lesions and contact dermatitis. Mania or hypomania was also reported in unipolar and bipolar depression patients without a clear causal relationship between tDCS treatments. Finally, an isolated case of seizure was reported in the literature but the involvement of tDCS was not confirmed (37).

After 5 weeks of treatment (tDCS coupled with speech therapy), we observed a significant improvement in the word, non-word, and phrase writing test scores in the post-treatment evaluation with respect to the baseline evaluation (T0: 47.94 ± 38.99; T1: 58.13 ± 40.28; Z = −2.937; p = 0.004; r = 0.519). We also found a significant improvement in the word, non-word, and phrase reading test scores (T0: 53.81 ± 39.15; T1: 67.12 ± 38.55; Z = −3.297; p = 0.001; r = 0.58) and in the word, non-word, and phrase repetition test scores (T0: 87.69 ± 24.18; T1: 91.63 ± 19.77; Z = −3.069; p = 0.002; r = 0.54). Moreover, we observed a statistically significant amelioration in the TROG-2 test score after the treatment (T0: 42.25 ± 18.44; T1: 58.5 ± 17.56; Z = −3.53; p < 0.001; r = 0.624). Finally, we found a trend toward increasing the copy test core post-treatment compared to the pre-treatment evaluation (T0: 7.75 ± 3.62; T1: 8.5 ± 3.46; p = 0.057), and the complete results and effectiveness improvement are reported in Table 2.

TMS-EEG analysis revealed a significant positive cluster (p = 0.047) relating to the differences before and after the treatment recorded with the right DLPFC stimulation condition (Figure 2). The positive cluster included the signal from left frontal electrodes (AF7, AF3, AFz, F7, F5, F3, F1, Fz, F2, FT7, FC5, FC3, FC1, FCz, and C5) in the time interval of 26 to 51 ms (Figures 2A,B). Considering that the signal post-treatment of this cluster was negative on average, as shown in the topography in Figures 2C,D, this result indicated that the TEP amplitude in left frontal sites decreased after the treatment concerning the baseline. The statistical comparison performed on all electrodes in the time window from 7 to 60 ms after the TMS pulse did not reveal any spatiotemporal differences for the left DLPFC stimulation condition (all p > 0.05). For the time-frequency analysis, any cluster results were not significant for both the stimulation conditions (all p > 0.05).

Post-treatment neuropsychological measures (reading, writing, repetition, word copy, and TROG-2) have been correlated with the mean values of significant clusters emerging from neurophysiological comparisons. A negative correlation between the total scores of the reading test (EDL-II) and the F2 component was found (R = − 0.573; p = 0.025). A negative correlation between the total scores of the writing test (EDL-II) and the F2 component was found (R = − 0.654; p = 0.008). Finally, a negative correlation between the total scores of the TROG-2 Test and the AFz component was found (R = − 0.549; p = 0.034). Overall, correlational analysis results showed that patients with better linguistic performances after treatment had reduced frontal excitability.

In this study, we evaluated the efficacy of left prefrontal tDCS combined with standard speech therapy to enhance language function in FXS patients. We found that anodal left prefrontal tDCS combined with speech therapy was able to induce a significant amelioration in writing, reading, repetition, and TROG-2 comprehension test scores in our population. Our neurophysiological data suggest that this improvement is accompanied by a parallel decrease after treatment in left prefrontal excitability. Moreover, correlational analysis showed that patients with lower left prefrontal cortical excitability displayed better language function enhancement after treatment. These results indicate that the modulation of the prefrontal cortex induced by the anodal tDCS intervention could be effective in individuals with FXS, and further highlight the alteration between excitatory and inhibitory circuits balance as a target for intervention in individuals with FXS. Our results are in line with previous studies suggesting that the disruption of excitatory glutamatergic and GABAergic inhibitory neurotransmission balance might be responsible for impairment in cognitive function, selective attention deficits, and many of the other behavioral and developmental aspects of FXS, as well as for language deficits (8, 14, 15). Accordingly, a recent study has shown that individuals with FXS often exhibit atypical neurophysiological patterns, including increased activation in the frontal region of the brain during language processing and a negative correlation between the degree of frontal activation and language performance in individuals with FXS (8). This suggests that as frontal activation increases, language abilities tend to decline or remain impaired. Conversely, when frontal activation decreases, there may be an improvement in language skills, as already shown in autism spectrum disorder (39). With this regard, tDCS has been previously shown to modulate functional connectivity between DLPFC and other regions of the brain (40), and this additional mechanism could suggest another possible neurophysiological explanation for our results. In line with this observation, our findings might indicate that excessive left prefrontal activation or over-engagement in the prefrontal region during language processing could potentially hinder language performance. Reduced frontal activity may allow for more efficient neural processing or allocation of cognitive resources to other language-related regions, leading to enhanced language skills. Beneficial cognitive effects induced by tDCS in language function might depend also on improved learning or working-memory modulation, as previously described (41). Recent studies have demonstrated that anodal tDCS over DLPFC was able to enhance naming and speed verbal reaction time performances, thus leading to hypothesize its role in a specific network dedicated to lexical retrieval/selection processing in naming (42). Moreover, the combination of anodal tDCS in Broca’s area with standard speech therapy has been tested in the aphasic patient population with beneficial effects in terms of articulatory defects (21), naming accuracy (43), and verbal fluency (44). In addition, tDCS may also exert non-neuronal effects in FXS, possibly by enhancing the expression of brain-derived neurotrophic factor (BDNF). The BDNF is a neurotrophic factor that supports neurogenesis and survival of neurons by modulating the inflammatory response acting on microglia and reducing the interference with blood–brain barrier disruption (45). Accordingly, microglia play a critical role in the development and maintenance of synapses through bidirectional communication, and its alteration was demonstrated to be a potential contributor to the pathophysiology of FXS (46). Overall, to the best of our knowledge, this was the first study showing high feasibility, tolerability, and safety of tDCS in a cohort of the FXS population. All the participants completed the treatment session successfully, and no significant discomfort was reported at the electrode sites and no side effects were observed, such as seizures, acute mood changes, and irritability. Eleven patients (out of sixteen) reported a persistent tingling sensation under electrodes which did not affect the tDCS and speech therapy session. Although most children with FXS start speech-language intervention early in their lives, there are no current studies that have specifically evaluated language interventions for individuals with FXS. Recent evidence suggests that speech therapy programs that have been designed broadly for individuals with language learning difficulties, including individuals with intellectual and developmental disabilities, may be appropriate for individuals with FXS, even for older children (47, 48). In this context, our study provides novel evidence that combining speech therapy intervention with neuromodulation could be effective in FXS individuals; however, more clinical studies evaluating standard therapy approaches alone for this population are needed. Surprisingly, in our study, we found an enhancement of language function after the combined treatment in an adult sample of FXS patients with a great heterogeneity in language skills. The two non-verbal adults showed progress in auditory discrimination in phonemic and syllabic production, and this is an important result since this trial permits them to approach verbal communication. Twelve participants showed improvement in reading and writing ability as well as in receptive language, and caregivers believe that after treatment, individuals with FXS are more interested in communicating through reading and writing, e.g., they write spontaneously the means of public transport or the name of product and price in the supermarket. Finally, the two high-functioning participants showed improvements in oral reading of stories for oral comprehension, dictation of syllables, real words and non-words, dictation of sentences, dictation of texts, and recounting writing stories, and they were more skillful in studying university books. Our study is limited by the absence of a sham-controlled group and by the small sample of patients. However, we used neurophysiological measures to detect possible cortical changes underpinning cognitive functioning, and our findings are in line with recent literature, which shows that lower left prefrontal cortical excitability displayed better language function enhancement after treatment. Moreover, our conclusion is limited by the lack of assessment of the effects of either prefrontal tDCS or speech therapy separately and the lack of long-term follow-up. Further studies with a randomized, double-blind sham-controlled trial design and a longer follow-up are needed to optimize non-invasive brain stimulation combined with speech therapy in FXS patients.

In conclusion, our study provides evidence that left prefrontal tDCS combined with standard speech therapy might be effective in enhancing language function in FXS patients, mainly through the reduction of frontal cortical hyperexcitability. Considering the impact of language disturbances in FXS individuals and their families and the lack of pharmacological treatment or specific speech intervention, our results suggest that neuromodulation strategies targeting excitation/inhibition neurotransmission imbalance and connectivity disruption could represent a potential new therapeutic tool in FXS.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

The studies involving humans were approved by Santa Lucia Foundation Ethics Committee. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

CP: Data curation, Investigation, Project administration, Writing – original draft. MA: Writing – original draft, Formal analysis, Visualization. RE: Formal analysis, Writing – review & editing. AD: Writing – review & editing, Data curation. MF: Data curation, Writing – review & editing. SP: Writing – review & editing, Conceptualization, Formal analysis. IB: Writing – review & editing, Visualization. AMC: Visualization, Writing – review & editing. SB: Visualization, Writing – review & editing. PC: Visualization, Writing – review & editing. GK: Writing – review & editing, Conceptualization, Methodology, Supervision.