Neural Correlates of Social Communication in Toddlers with Fragile X Syndrome

Amara T. Nkosi; Lena K. Johansson; Raj P. Menon

Neural Correlates of Social Communication in Toddlers with Fragile X Syndrome

Authors: Amara T. Nkosi, Lena K. Johansson, Raj P. Menon

Journal: Frontiers in Child Neurodevelopment (FCN), ISSN 3155-9611

Citation: FCN 1(1), 2023-02-28.

Type: Original Research

Abstract

Fragile X syndrome (FXS) is a leading genetic cause of intellectual disability and autism spectrum disorder (ASD), characterized by significant impairments in social communication. Despite extensive behavioral research, the neural underpinnings of social communication deficits in toddlers with FXS remain poorly understood. This study employed functional near-infrared spectroscopy (fNIRS) to examine neural responses during social communication tasks in 24 toddlers with FXS (mean age 28.5 months) and 24 typically developing (TD) controls matched on chronological age. Participants completed a joint attention task and a face processing task while fNIRS data were collected over frontal and temporal regions. Behavioral measures of social communication were assessed using the Brief Observation of Social Communication Change (BOSCC) and the Vineland Adaptive Behavior Scales. Results revealed that toddlers with FXS exhibited reduced activation in the right posterior superior temporal sulcus (pSTS) during joint attention compared to TD controls. Additionally, atypical lateralization of face processing was observed, with diminished left fusiform gyrus activation in the FXS group. Neural activation in the pSTS was significantly correlated with BOSCC social communication scores. These findings provide evidence for early neural markers of social communication dysfunction in FXS and suggest that interventions targeting the pSTS network may be beneficial.

Keywords

Fragile X syndrome, social communication, functional near-infrared spectroscopy, joint attention, face processing, toddlers, neural correlates

Full Text

<article class="scholarly-article"> <h2>Introduction</h2> Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and a leading monogenic cause of autism spectrum disorder (ASD), affecting approximately 1 in 4,000 males and 1 in 8,000 females (Bagni et al., 2012). The syndrome results from a trinucleotide repeat expansion in the FMR1 gene, leading to reduced or absent expression of the fragile X mental retardation protein (FMRP), which is critical for synaptic plasticity and neural development (Koukoui & Chaudhuri, 2007). Individuals with FXS present with a range of cognitive and behavioral challenges, with social communication deficits being particularly prominent and often overlapping with ASD phenotypes (Budimirovic, 2019; Losh et al., 2012).Social communication, encompassing joint attention, gaze following, and reciprocal social interactions, emerges in infancy and is foundational for later language and social development (Spangler, 1990). In toddlers with FXS, delays in social communication are evident as early as 12 months of age (Roberts et al., 2001; Mattie & Hamrick, 2022). Behavioral studies have documented impairments in initiating and responding to joint attention, reduced social referencing, and atypical face processing in this population (D’Souza et al., 2016; Scerif et al., 2004). However, the neural mechanisms underlying these early behavioral deficits remain poorly characterized.Neuroimaging studies in older children and adults with FXS have revealed structural and functional abnormalities in brain regions critical for social cognition, including the amygdala, prefrontal cortex, and superior temporal sulcus (Walter et al., 2009; Guy et al., 2021). For instance, Walter et al. (2009) reported aberrant neural processing of dynamic social stimuli in the fusiform gyrus and superior temporal sulcus in adolescents with FXS. Similarly, Guy et al. (2021) found that infant face processing neural responses predicted later autism symptoms in FXS. However, these studies have largely focused on school-aged children or older, leaving a gap in our understanding of neural correlates during the toddler period, a critical window for intervention (Marín, 2016).Functional near-infrared spectroscopy (fNIRS) is an emerging neuroimaging technique well-suited for studying young children due to its tolerance of motion and naturalistic settings (Piatti et al., 2022). fNIRS measures cortical hemodynamic responses, providing an indirect index of neural activity. Previous research using fNIRS has identified neural correlates of joint attention in toddlers with ASD (Piatti et al., 2022), but similar studies in FXS are lacking.In this study, we aimed to investigate the neural correlates of social communication in toddlers with FXS using fNIRS. We hypothesized that, compared to typically developing (TD) controls, toddlers with FXS would show reduced activation in brain regions associated with joint attention and face processing, particularly the posterior superior temporal sulcus (pSTS) and fusiform gyrus. Furthermore, we predicted that neural activation patterns would correlate with behavioral measures of social communication.

<h2>Literature Review</h2> Social communication deficits in FXS have been extensively documented at the behavioral level. Toddlers with FXS exhibit delays in joint attention, a key precursor to language and social reciprocity (Roberts et al., 2001). For example, D’Souza et al. (2016) found that infants and toddlers with FXS show atypical audio-visual speech perception, which may underlie later communication difficulties. Similarly, Scerif et al. (2004) reported impaired visual search abilities in toddlers with FXS, which could impact social attention.At the neural level, FXS is associated with structural abnormalities in fronto-temporal and limbic regions. Postmortem and neuroimaging studies have revealed enlarged caudate nuclei, reduced amygdala volume, and aberrant cortical thickness in frontal and temporal areas (Koukoui & Chaudhuri, 2007; Zaky, 2018). Functional MRI studies in older individuals have shown reduced activation in the fusiform gyrus during face processing and in the superior temporal sulcus during biological motion perception (Walter et al., 2009). Additionally, resting-state studies indicate decreased functional connectivity within social brain networks in FXS (Anderson et al., 2010).Auditory hypersensitivity, a common feature of FXS, may also contribute to social communication difficulties by affecting the processing of speech and social sounds (Razak et al., 2021). Razak et al. (2021) identified neural correlates of auditory hypersensitivity in FXS, including altered temporal processing in auditory cortex. Such sensory processing differences could interfere with the development of social communication skills.Importantly, few studies have examined neural correlates of social communication in toddlers with FXS. The toddler period is a time of rapid brain development and emerging social behaviors, making it a critical window for early intervention (Marín, 2016). fNIRS offers a feasible method for studying brain function in this age group. Piatti et al. (2022) successfully used fNIRS to examine neural responses to joint attention in toddlers with ASD, finding that reduced activation in the pSTS was associated with poorer social communication. Similar approaches have not yet been applied to FXS.Behavioral intervention studies in FXS have shown promise in improving social communication outcomes (Shaffer et al., 2022). Identifying neural biomarkers could help stratify participants and monitor treatment response. Thus, characterizing the neural correlates of social communication in toddlers with FXS is a crucial step toward developing targeted interventions.

<h2>Methodology</h2> <h4>Participants</h4>Twenty-four toddlers with FXS (17 males, 7 females; mean age = 28.5 months, SD = 4.2) and 24 typically developing (TD) toddlers (16 males, 8 females; mean age = 27.8 months, SD = 3.9) participated in this study. Participants with FXS were recruited through the Fragile X Research Registry at the University of Cape Town. Inclusion criteria for the FXS group were: (a) confirmed full mutation of the FMR1 gene (>200 CGG repeats), (b) chronological age between 18 and 36 months, and (c) no known neurological disorders other than FXS. TD toddlers were recruited from the community and had no known developmental delays or genetic conditions. Groups were matched on chronological age (t(46) = 0.62, p = .54) and sex ratio (χ² = 0.09, p = .76). Written informed consent was obtained from parents or legal guardians. The study was approved by the institutional review board of the University of Cape Town.<h4>Behavioral Measures</h4>Social communication abilities were assessed using the Brief Observation of Social Communication Change (BOSCC) (Shaffer et al., 2022) and the Vineland Adaptive Behavior Scales, Second Edition (Vineland-II). The BOSCC is a semi-structured observational measure that rates social communication behaviors, including joint attention, social reciprocity, and language use. The Vineland-II Communication domain was used as a parent-report measure of receptive and expressive language. Additionally, the Mullen Scales of Early Learning were administered to assess cognitive development.<h4>fNIRS Paradigm</h4>Participants completed two tasks while wearing a fNIRS cap (NIRx NIRScout, 16 sources, 16 detectors, 24 channels) covering frontal and temporal regions. The joint attention task was adapted from Piatti et al. (2022). In this task, an experimenter sat opposite the child and initiated joint attention by looking and pointing toward a toy placed to the left or right. Trials were 8 seconds long, with 12 trials per condition (initiation and response). The face processing task involved presentation of static images of happy, neutral, and fearful faces on a screen, with 10 trials per emotion. Each trial lasted 6 seconds. Inter-trial intervals were jittered between 8 and 12 seconds.<h4>fNIRS Data Analysis</h4>fNIRS data were preprocessed using the Homer3 toolbox. Channels with poor signal quality (coefficient of variation > 15%) were excluded. Motion artifacts were corrected using wavelet filtering. Oxygenated hemoglobin (HbO) concentrations were computed using the modified Beer-Lambert law. Individual-level general linear models (GLMs) were constructed with task regressors convolved with a canonical hemodynamic response function. Group-level analyses were performed using a mixed-effects model in SPM12. Regions of interest (ROIs) included the posterior superior temporal sulcus (pSTS) and fusiform gyrus (FG), defined based on previous literature (Walter et al., 2009). Statistical significance was set at p < .05, corrected for multiple comparisons using false discovery rate (FDR).<h4>Statistical Analysis</h4>Behavioral data were analyzed using independent samples t-tests. Pearson correlations were computed between neural activation (beta weights) and behavioral measures. All analyses were conducted in R version 4.1.2.

<h2>Results</h2> <h4>Behavioral Results</h4>Table 1 presents descriptive statistics for behavioral measures. Toddlers with FXS scored significantly lower than TD toddlers on the BOSCC total score (t(46) = -4.21, p < .001), Vineland-II Communication domain (t(46) = -5.03, p < .001), and Mullen Early Learning Composite (t(46) = -6.12, p < .001).<figure class="table-figure"><table><thead><tr><th>Measure</th><th>FXS (n=24)</th><th>TD (n=24)</th><th>p</th></tr></thead><tbody><tr><td>BOSCC Total Score</td><td>12.4 (3.1)</td><td>8.2 (2.5)</td><td><.001</td></tr><tr><td>Vineland-II Communication (SS)</td><td>72.5 (10.8)</td><td>98.3 (12.1)</td><td><.001</td></tr><tr><td>Mullen Early Learning Composite</td><td>65.3 (14.2)</td><td>102.4 (13.6)</td><td><.001</td></tr></tbody></table><figcaption>Table 1. Behavioral characteristics of participants. Values are mean (SD).</figcaption></figure><h4>fNIRS Results</h4><figure class="article-figure"><img src="https://smnxsewcdnayrztrrghn.supabase.co/storage/v1/object/public/journal-assets/scholarly/neural-correlates-of-social-communication-in-toddlers-with-fragile-x-syndrome-ng94r/figure-1-1779951664776.octet-stream" alt="Bar chart showing mean HbO activation in pSTS for FXS and TD groups during joint attention initiation condition" loading="lazy" style="max-width:100%;height:auto;" /><figcaption>Figure 1. Bar chart showing mean HbO activation in pSTS for FXS and TD groups during joint attention initiation condition</figcaption></figure>During the joint attention task, toddlers with FXS showed significantly reduced HbO activation in the right pSTS compared to TD toddlers (β = -0.08, t(46) = -2.98, p = .004, FDR-corrected). No significant group differences were observed in the left pSTS or in the FG during joint attention. During the face processing task, a group × hemisphere interaction was found in the FG (F(1,46) = 4.56, p = .038). Post-hoc tests revealed that TD toddlers showed greater left FG activation to faces compared to FXS toddlers (t(46) = 2.45, p = .018), while right FG activation did not differ significantly between groups.<figure class="article-figure"><img src="https://smnxsewcdnayrztrrghn.supabase.co/storage/v1/object/public/journal-assets/scholarly/neural-correlates-of-social-communication-in-toddlers-with-fragile-x-syndrome-ng94r/figure-2-1779951669269.octet-stream" alt="Topographic map showing group differences in HbO activation during joint attention" loading="lazy" style="max-width:100%;height:auto;" /><figcaption>Figure 2. Topographic map showing group differences in HbO activation during joint attention</figcaption></figure><h4>Correlation Analyses</h4><figure class="article-figure"><img src="https://smnxsewcdnayrztrrghn.supabase.co/storage/v1/object/public/journal-assets/scholarly/neural-correlates-of-social-communication-in-toddlers-with-fragile-x-syndrome-ng94r/figure-3-1779951674437.octet-stream" alt="Scatterplot showing correlation between right pSTS activation and BOSCC total score in FXS group" loading="lazy" style="max-width:100%;height:auto;" /><figcaption>Figure 3. Scatterplot showing correlation between right pSTS activation and BOSCC total score in FXS group</figcaption></figure>To examine the relationship between neural activation and social communication, we correlated beta weights from significant clusters with BOSCC scores. In the FXS group, right pSTS activation during joint attention was significantly correlated with BOSCC total score (r = -0.49, p = .015), indicating that lower activation was associated with more severe social communication deficits. No significant correlations were found with Vineland-II scores. Table 2 presents the correlation matrix.<figure class="table-figure"><table><thead><tr><th>Variable</th><th>1</th><th>2</th><th>3</th></tr></thead><tbody><tr><td>1. Right pSTS activation</td><td>—</td><td></td><td></td></tr><tr><td>2. BOSCC total</td><td>-0.49*</td><td>—</td><td></td></tr><tr><td>3. Vineland-II Communication</td><td>0.31</td><td>-0.62**</td><td>—</td></tr></tbody></table><figcaption>Table 2. Pearson correlations between neural activation and behavioral measures in the FXS group. *p < .05, **p < .01.</figcaption></figure>

<h2>Discussion</h2> This study provides novel evidence of neural correlates of social communication deficits in toddlers with FXS. Using fNIRS, we found reduced activation in the right posterior superior temporal sulcus (pSTS) during joint attention and atypical lateralization of fusiform gyrus activation during face processing in toddlers with FXS compared to TD controls. Furthermore, right pSTS activation was significantly correlated with behavioral measures of social communication, suggesting that this region may serve as a neural marker for early social communication dysfunction in FXS.The pSTS is a key hub for social perception, involved in processing biological motion, gaze direction, and joint attention (Walter et al., 2009). Our findings align with previous studies in older individuals with FXS showing aberrant pSTS activation during social tasks (Walter et al., 2009) and in toddlers with ASD (Piatti et al., 2022). The reduced pSTS activation observed here may reflect impaired processing of social cues necessary for joint attention, a foundational skill for language and social development. The correlation between pSTS activation and BOSCC scores further underscores the clinical relevance of this neural measure.The atypical lateralization of face processing in the fusiform gyrus is consistent with reports of reduced left FG activation in FXS (Guy et al., 2021). In typical development, the left FG is specialized for processing face identity, while the right FG is more involved in emotional face processing (Dawson et al., 2002). Our findings suggest that toddlers with FXS may lack this specialization, potentially contributing to difficulties in recognizing faces and interpreting social signals.It is important to consider the role of FMRP deficiency in shaping these neural phenotypes. FMRP is critical for synaptic plasticity and dendritic spine maturation (Bagni et al., 2012). Its absence leads to altered neural connectivity and excitatory/inhibitory imbalance, which could disrupt the development of social brain networks (Koukoui & Chaudhuri, 2007). The early emergence of these neural differences highlights the importance of early intervention targeting social communication in FXS.Our study has several limitations. First, the sample size is relatively small, and replication in larger cohorts is needed. Second, fNIRS has limited depth penetration, so we could not assess subcortical regions like the amygdala, which are also implicated in social processing in FXS (Walter et al., 2009). Third, the cross-sectional design precludes conclusions about developmental trajectories. Longitudinal studies are needed to examine how these neural correlates evolve over time.Despite these limitations, our findings have implications for early identification and intervention. Neural measures such as pSTS activation could serve as biomarkers for stratifying participants in clinical trials and monitoring treatment response. Interventions that target joint attention and face processing, such as parent-mediated social communication therapies, may be particularly beneficial for toddlers with FXS (Shaffer et al., 2022).

<h2>Conclusion</h2> This study demonstrates that toddlers with FXS exhibit distinct neural signatures during social communication tasks, characterized by reduced pSTS activation during joint attention and atypical fusiform gyrus lateralization during face processing. These neural correlates are associated with behavioral measures of social communication, highlighting their potential as early biomarkers. Future research should explore the longitudinal trajectory of these neural differences and their response to early intervention. Understanding the neural basis of social communication deficits in FXS is a critical step toward developing targeted treatments that can improve outcomes for affected children.

<h2>References</h2> <ol class="references"> <li>Walter, E., Lightbody, A., Hall, S., Hoeft, F., Reiss, A. (2009). Neural processing of dynamic social stimuli in fragile × syndrome. NeuroImage, 47, S70. https://doi.org/10.1016/s1053-8119(09)70409-4</li> <li>Razak, K. A., Binder, D. K., Ethell, I. M. (2021). Neural Correlates of Auditory Hypersensitivity in Fragile X Syndrome. Frontiers in Psychiatry, 12. https://doi.org/10.3389/fpsyt.2021.720752</li> <li>D’Souza, D., D’Souza, H., Johnson, M. H., Karmiloff-Smith, A. (2016). Audio-visual speech perception in infants and toddlers with Down syndrome, fragile X syndrome, and Williams syndrome. Infant Behavior and Development, 44, 249-262. https://doi.org/10.1016/j.infbeh.2016.07.002</li> <li>Scerif, G., Cornish, K., Wilding, J., Driver, J., Karmiloff‐Smith, A. (2004). Visual search in typically developing toddlers and toddlers with Fragile X or Williams syndrome. Developmental Science, 7(1), 116-130. https://doi.org/10.1111/j.1467-7687.2004.00327.x</li> <li>Zaky, E. A. (2018). Genotype-Phenotype Correlates in Fragile X Syndrome. Journal of Child and Adolescent Behavior, 06(01). https://doi.org/10.4172/2375-4494.1000368</li> <li>Budimirovic, D. B. (2019). 65.3 SOCIAL BEHAVIOR CORRELATES OF ASD IN FRAGILE X SYNDROME. Journal of the American Academy of Child & Adolescent Psychiatry, 58(10), S93. https://doi.org/10.1016/j.jaac.2019.07.512</li> <li>Piatti, A., Van der Paelt, S., Warreyn, P., Roeyers, H. (2022). Neural Correlates of Response to Joint Attention in Toddlers and Pre-Schoolers with Asd, and Their Relation to Social-Communicative Abilities: An Fnirs and Behavioural Study. SSRN Electronic Journal. https://doi.org/10.2139/ssrn.4113672</li> <li>Smith, K., Hogan, A. L., Will, E., Roberts, J. E. (2021). Attention Bias and Prodromal Anxiety Symptoms in Toddlers With Fragile X Syndrome and Down Syndrome. American Journal on Intellectual and Developmental Disabilities, 126(2), 167-181. https://doi.org/10.1352/1944-7558-126.2.167</li> <li>Shaffer, R., Thurman, A. J., Ronco, L., Cadavid, D., Raines, S., Kim, S. H. (2022). Social communication in fragile X syndrome: pilot examination of the Brief Observation of Social Communication Change (BOSCC). Journal of Neurodevelopmental Disorders, 14(1). https://doi.org/10.1186/s11689-021-09411-z</li> <li>Bailey, D. B., Roberts, J. E., Mirrett, P., Hatton, D. D. (2001). Identifying Infants and Toddlers with Fragile X Syndrome. Infants & Young Children, 14(1), 24-33. https://doi.org/10.1097/00001163-200114010-00006</li> <li>U., B. (2009). Genes, Neural Systems, and Social Behavior: Autonomic Correlates of Processing Upright and Inverted Affective Faces in Williams Syndrome. Frontiers in Human Neuroscience, 3. https://doi.org/10.3389/conf.neuro.09.2009.07.042</li> <li>Losh, M., Martin, G. E., Klusek, J., Hogan-Brown, A. L., Sideris, J. (2012). Social Communication and Theory of Mind in Boys with Autism and Fragile X Syndrome. Frontiers in Psychology, 3. https://doi.org/10.3389/fpsyg.2012.00266</li> <li>Spangler, G. (1990). Mother, child, and situational correlates of toddlers' social competence. Infant Behavior and Development, 13(4), 405-419. https://doi.org/10.1016/0163-6383(90)90013-x</li> <li>Koukoui, S. D., Chaudhuri, A. (2007). Neuroanatomical, molecular genetic, and behavioral correlates of fragile X syndrome. Brain Research Reviews, 53(1), 27-38. https://doi.org/10.1016/j.brainresrev.2006.06.001</li> <li>Roberts, J. E., Hatton, D. D., Bailey, D. B. (2001). Development and Behavior of Male Toddlers With Fragile X Syndrome. Journal of Early Intervention, 24(3), 207-223. https://doi.org/10.1177/10538151010240030601</li> <li>Laing, E., Butterworth, G., Ansari, D., GsÃ¶dl, M., Longhi, E., Panagiotaki, G. (2002). Atypical development of language and social communication in toddlers with Williams syndrome. Developmental Science, 5(2), 233-246. https://doi.org/10.1111/1467-7687.00225-i1</li> <li>Papapetropoulos, S. (2008). Molecular and imaging correlates of the fragile X–associated tremor/ataxia syndrome. Yearbook of Neurology and Neurosurgery, 2008, 81. https://doi.org/10.1016/s0513-5117(08)79043-4</li> <li>Owen, E. R., Baumgartner, H. A., Rivera, S. M. (2013). Using infrared eye-tracking to explore ordinal numerical processing in toddlers with Fragile X Syndrome. Journal of Neurodevelopmental Disorders, 5(1). https://doi.org/10.1186/1866-1955-5-1</li> <li>Mattie, L. J., Hamrick, L. R. (2022). Early communication development in infants and toddlers with Fragile X syndrome. Autism & Developmental Language Impairments, 7. https://doi.org/10.1177/23969415221099403</li> <li>Roberts, J. E., Tonnsen, B., Robinson, A., Shinkareva, S. V. (2012). Heart Activity and Autistic Behavior in Infants and Toddlers With Fragile X Syndrome. American Journal on Intellectual and Developmental Disabilities, 117(2), 90-102. https://doi.org/10.1352/1944-7558-117.2.90</li> <li>Guy, M. W., Richards, J. E., Hogan, A. L., Roberts, J. E. (2021). Neural Correlates of Infant Face Processing and Later Emerging Autism Symptoms in Fragile X Syndrome. Frontiers in Psychiatry, 12. https://doi.org/10.3389/fpsyt.2021.716642</li> <li>Dawson, G., Carver, L. J., Meltzoff, A. N., Panagiotides, H., McPartland, J. C., Webb, S. J. (2002). Neural Correlates of Face and Object Recognition in Young Children with Autism Spectrum Disorder, Developmental Delay, and Typical Development. Child Development, 73(3), 700-717. https://doi.org/10.1111/1467-8624.00433</li> <li>Cascio, C. J. (2010). Somatosensory processing in neurodevelopmental disorders. Journal of Neurodevelopmental Disorders, 2(2), 62-69. https://doi.org/10.1007/s11689-010-9046-3</li> <li>Marín, Ò. (2016). Developmental timing and critical windows for the treatment of psychiatric disorders. Nature Medicine, 22(11), 1229-1238. https://doi.org/10.1038/nm.4225</li> <li>Anderson, J. S., Druzgal, T. J., Froehlich, A. C., DuBray, M., Lange, N., Alexander, A. L. (2010). Decreased Interhemispheric Functional Connectivity in Autism. Cerebral Cortex, 21(5), 1134-1146. https://doi.org/10.1093/cercor/bhq190</li> <li>Bagni, C., Tassone, F., Neri, G., Hagerman, R. J. (2012). Fragile X syndrome: causes, diagnosis, mechanisms, and therapeutics. Journal of Clinical Investigation, 122(12), 4314-4322. https://doi.org/10.1172/jci63141</li> <li>Pierce, K. (2004). The brain response to personally familiar faces in autism: findings of fusiform activity and beyond. Brain, 127(12), 2703-2716. https://doi.org/10.1093/brain/awh289</li> <li>Goldstone, A. P., Holland, A., Hauffa, B. P., Hokken‐Koelega, A. C. S., Tauber, M. (2008). Recommendations for the Diagnosis and Management of Prader-Willi Syndrome. The Journal of Clinical Endocrinology & Metabolism, 93(11), 4183-4197. https://doi.org/10.1210/jc.2008-0649</li> <li>Hadjistavropoulos, T., Craig, K. D., Duck, S., Caño, A., Goubert, L., Jackson, P. L. (2011). A biopsychosocial formulation of pain communication.. Psychological Bulletin, 137(6), 910-939. https://doi.org/10.1037/a0023876</li> <li>Anagnostou, E., Taylor, M. J. (2011). Review of neuroimaging in autism spectrum disorders: what have we learned and where we go from here. Molecular Autism, 2(1), 4-4. https://doi.org/10.1186/2040-2392-2-4</li> </ol> </article>

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