Correlations Between Linguistic Phenotype and GeneticAlterations in Rett Syndrome Alessandra Falzone (amfalzone@unime.it) Department of Cognitive Science, University of Messina, 6 Via Concezione Messina, Italy Antonio Gangemi (antgangemi@unime.it) Department of Cognitive Science, University of Messina, 6 Via Concezione Messina, Italy AntoninoErrante(aerrante@libero.it) Department of Neurosciences, University of Parma, 39 Via Volturno Parma, Italy Paola Pennisi (paola.pennisi@ifc.cnr.it) Clinical Physiology Institute, National Research Council of Italy (IFC-CNR) Messina, Italy Rosa Angela Fabio (rafabio@unime.it) Department of Cognitive Science, University of Messina, 6 Via Concezione Messina, Italy Abstract Recently, the FOXG1 gene, localised in chromosome 14 Rett syndrome (RTT) is a neurodevelopmental disorder has been identified as the first autosomal gene associated to mainly caused by mutations in the MECP2 gene affecting RTT, in particular to the congenital variant (Ariani et al., around 1 in 10,000 female births. Clinical manifestations 2008).These results demonstrated that RTT presents genetic include severe linguistic and motor impairments that are the and clinical heterogeneity and they provide data for core of phenotype symptoms. Some patients show a moderate molecular bases to understand pathogenic mechanisms of level of conservation of linguistic functions while others lose the disease and to establish targeted therapeutic strategies. the use of functional verbal communication. This paper aims After its nosographical description, RTT was classified at correlating residual linguistic capacity, connected to breathing alterations, to specific RTT genotype. In particular, among Pervasive Developmental Disorders (APA, 2000), the relation between breathing alterations and the pathological but it has been moved to the genetic disorder category severity caused by locus’mutation is investigated. because of its primary ethiology (DSM V, APA, 2013). The clinical features suggest that this disorder is the result Keywords:Rett Syndrome; Language Impairment; of a primary disturbance of neuronal development Genotype/Phenotype correlation. (Armstrong et al., 1995), perhaps resulting in maturational arrest in selective brain regions (Armstrong, 1995). The Introduction basis of this maturational arrest could, for example, be Rett syndrome (RTT) is a neurodevelopmental disorder, defective neurotransmitters systems that fail to provide affecting around 1/10.000 female births (Chahrour & normal trophic factors. Zoghbi2007). Females are primarily affected, even though a From a functional point of view RTT shows a latent few cases of males are reported in the literature (Leonard et period in which symptoms are not almost evident and a first al., 2001; Cohen et al., 2002). In 1999 the mutational clinical stage in which symptoms arise. In late infancy, after screening of candidate genes in the region Xq28 allowed the a period of superficially normal but subtly flawed identification of MECP2 gene as a cause of the standard development, RTT patients undergo striking developmental form. Ten years later, different research studies regarding regression. RTT is characterized by the loss of pre-existing the causes of RTT variations have also been carried hand use – such as object reach, grasp, and manipulation, out(Amir et al., 1999; Guy et al., 2011).Another study and by the appearance of distinctive hand stereotypies – demonstrated that a second gene called CDKL5 localised in such as hand wringing, tapping, and mouthing (Fabio et al., the X chromosome, is involved in the variant with early 2009). Post-regression patients, even though persons with onset convulsions (Weaving et al., 2005). severe intellectual disabilities, often regain social interest 605 and some verbal communication capabilities (Antonietti et presents in 14 girls with RTT and the languagecapabilities al., 2008; Castelli et al., 2013). (comprehension and production) that they still present. In our position, this relation is mediated by breathing alteration linked with genetic mutation: the locus of mutation (before Genotype characterization of RTT or after the nuclear localization signal) is correlated with the Currently, gene MECP2 mutations (Xq28) are found in the severity of breathing phenotype and consequently with majority of cases (90%) of standard Rett and in 30% of speech. cases of atypical RTT. Since there are many clinically documented RTT cases who don’t present the MECP2 Linguistic phenotype in RTT mutation, many scientists in this field believe that the RTT is characterized by severe alteration in motor and pathology is caused by genetic heterogeneity. speech capabilities that are considered as inclusive MECP2 gene is particularly expressed in the neuronal tissue diagnostic criteria (Neul et al., 2010). during specific developmental stages (Jung et al.,2003; In particular, the residual linguistic abilities are very Balmer et al., 2003; Shahbazian et al., 2002).More than 200 different and each individual could manifest various degrees different mutations of the MECP2 gene have been reported of severity in language production: whereas some RTT in the Rett Base (IRSAMECP2 Variation Database) but patients completely lose their activity in verbal sound eight mutations (Arg106Trp, Arg133Cys, Thr158Met, production, which is functional to communication, others Arg168X, Arg255X, Arg270X, Arg294X, Arg306Cys) preserve functional vocal sound and/or words (Budden, affect around 67% of RTT females. A remaining 10% of 1997; De Bona et al., 2000; Fabio et al., 2009; Zappella et RTT cases show a large group of C-terminal frameshift al. 2001). mutations. Linguistic deficits typically arise after the regression Several studies have reported genotype-phenotype phase: indeed, RTT females are characterized by a normal correlations, but with conflicting results. Most authors language development comparable with a healthy one reporting data from different cohorts of RTT patients before the regression phase. They often exhibit babbling and demonstrated that no correlation exists between missense phonological coupling except for the early onset variant. vs. truncating mutations, whereas others reported that the It has been demonstrated that linguistic competence levels truncating defects are more severe than the missense ones. are correlated to the language acquisition stage in which Studies aimed at comparing mutations affecting the different individuals were at the onset of regression (Marschik et al., functional domains share the opinion that defects affecting 2012).Few studies have evaluated a genotype/phenotype the C-terminal domain give a milder clinical score. correlation between linguistic residuals and specific Differences in clustering the mutations, the heterogeneity in genotype. Uchino and colleagues (2001) tried to correlate the size of the analyzed cohorts, the selected clinical the grade of disability in locomotion and that of parameters, and variation in the age of the subjects are likely microcephalus with a language disability in RTT in a to explain the conflicting results. preliminary study and after they correlated language RTT to Given that conflicting findings about genotype–phenotype the loci of MECP2 mutation. This correlation was found on relationships in RTT are still under discussion, it seems the basis of a qualitative evaluation of spoken language. worthwhile to further look into this relationship in order to This study proposes a genotype/linguistic phenotype overcome some methodological flaws. correlation based on an articulation capability test by a In a previous study, Fabio et al. (2014) examined the effect phonetic evaluation test (Fanzago, 1983). Our hypothesis of MECP2 mutations on the phenotypic variability within a moves off the assumption that linguistic alterations in RTT group of 114 RTT patients, focusing on specific derive from alterations in breathing and facial-laryngeal methodological issues. More precisely, the study was muscle’ coordination rather than from general motor performed taking into account what was recommended by disease. As a consequence, genotypes producing severe Ham et al. (2005) concerning the weak points of the breathing and facial-laryngeal muscles alteration are previous studies. The results showed that a specific kind of expected to show a severe linguistic phenotype. genotypes can be associated with the severity of symptoms Indeed, girls with RTT show a complex breathing showed by RTT patients. On the basis of these results, our phenotype that includes hypoventilation, hyperventilation, study aims at providing a phenotype/genotype correlation in apnea and breath hold terminated by Valsalva maneuvers relation to a specific cognitive process, i.e. language (Katz et al., 2009). It seems that modifications of subcortical production and comprehension. There are different nuclei in the brainstem, which regulate breathing rhythm, linguistic phenotype variants in RTT. A restricted sample are connected to speech motor control (Ogier& Katz, 2008, shows the presence of verbal speech (Preserved Speech Ramirez et al., 2013). Many studies show that the brainstem Variant or Zappella variant (Renieri et al. 2009). This form respiratory network (Trevarthen& Daniel, 2005) is affected presents a less severe clinical condition, i.e. regular skull in RTT, but to date this aspect has not yet been analyzed for dimensions and a relevant reduction of epileptic seizures speech. On the basis of previous research on residual and breathing alterations. In the present study we aim at linguistic capacity in RTT patients and on the basis of the correlating the relationships between the genetic mutations evaluation of residual communication ability (both 606 comprehension and articulation) (cf. Fabio et al., 2009) real administered to girls and each table was larger (42 cm x linguistic phenotype to genotype has been correlated. 29,7 cm). Girls have to choose between two items (one In order to do this, speech ability has to be evaluated by target and one distractor) placed separately in front of them. using the Fanzago test, an Italian phonetic evaluation Both items (target and distractor) were shown 3 times and instrument which follows the typical degree of language the spatial position of the target and distractor was acquisition phase in phonological difficulties aspects. Four randomized. When the girl replied with two consecutive and parameters of the Fanzago test were used: the number of correct answers the examiner presented the following table; vowels spontaneously produced, the number of consonants when the girl replied wrongly three times, the test was spontaneously produced, the number of vowels with elicited interrupted. For linguistic measure, Fanzago test was used. denomination and the number of consonants with elicited The Fanzago phonetic articulation test is used to evaluate denomination. The spontaneous production of language the articulation capabilities in children and it is a good sounds and the presence of breathing alteration at baseline measure of their phonetic development. This instrument is condition and during a cognitive task were evaluated. based on spontaneous/repetition elicited denomination of The aims of the present study are two: the first one is to 114 figures grouped in 22 tables. Each table represents one analyze the correlation between breathing dysfunctions and image whose name starts with a vocal sound and other speech. The second one is toanalyze data related to objects in which the same sound is placed in a second or linguistic phenotype and specific RTT genotype in a pilot third position or is coupled with a vowel or consonant study. sounds. This study uses items which represent perceptively salient and commonly used objects for RTT. Methods During the assessment, behavioural breathing parameters Participants were evaluated. All the girls were video taped, two Twenty-one girls with a diagnosis of RTT, ranging from observers independently transcribed the first five minutes of age 4 to 31 (mean= 16,34 years, SD=5,98), took part in the each tape (one during sleep and one in the waking state). experiment. Their families had been contacted by the Italian The final transcription was then coded independently by Rett Association, which asked them to participate in the both observers for the behavioural breathing parameters. study.All of the participants were diagnosed with RTTand The inter-rater agreement concordance was high (Kappa all of them were positive to MECP2 mutation. index = .98). A general assessment was carried out by a psychologist through the Vineland Adaptive Behavior Scale (VABS) Procedure (Sparrow et al., 1984), the standardized test for the Rett All the activities were performed in a setting suitable for Assessment Rating Scale (RARS, Fabio et al., 2005) and language activity with patients: all distracting stimuli were Modified Colored Progressive Matrices. removed so the girls focused only on the task. The Fanzago phonetic articulation test was administered After the initial assessment, each girl was evaluated at to evaluate the status of vocal sound articulation and breathing baseline. After the Fanzago test was administered objective production articulated voice functional to starting with spontaneous vocal production. In the second communication. Behavioural breathing parameters were step girls had to produce phonemes at the request of the evaluated: all the girls showed breathing alteration and none linguistic therapist. In this study, girls are requested to required any Valsalva maneuvers. produce first vowel sounds, that are easier to articulate and that appear early on during normal development. Materials All correct phoneme (spontaneous/elicited) production The assessment consisted in functional, cognitive and was marked on the specific template, in which therapists can linguistic scales administering. In particular the Vineland write if girls utter the entire word painted in the image Adaptive Behavior Scales were used for functional during spontaneous or elicited production. assessment. In order to complete the functional In order to check the effective results of the requested characterization the Rett Assessment Rating Scale (RARS) sound production task, the alteration of breathing rhythms was administered. This is a standardized scale used to during a cognitive, not linguistic task was evaluated in this evaluate subjects with RTT (Fabio et al., 2005). It is study. constructed by following the diagnostic criteria for RTT proposed by DSM-IV-TR (APA, 2010) and recent research Results and clinical experience. It follows a structure similar to that Before proceeding with the analysis of the genotype- used for the diagnosis of the pervasive developmental phenotype correlation, Pearson’s correlation coefficient disorders included in the same nosographical category as between the sum of each type of respiratory dysfunctions RTT (i.e. Childhood Autism Rating Scale, CARS). and the four parameters of Fanzago test were calculated For cognitive measure, Modified Raven’s Coloured Results show that breathing problems display an inverse Progressive Matrices were used (Antonietti et al., 2003). correlation with each of the Fanzago parameters, namely Differently from the standard Raven’s Colored Progressive with the number of vowels spontaneously produced (r(21)= Matrices, in this adapted scale the A series was -.378, p<.137), the number of consonants spontaneously 607 produced (r (21)= -.61, p<.001), the number of vowels with elicited denomination (r(21)= -.498, p<.03), and the number of consonants with elicited denomination (r(21)= -.29, p<.23). To proceed with the genotype-phenotype correlation, since the number of participants was low, dichotomized scores to classify participants into a particular breathing type were used. Based on high (> median) or low (≤ median) scores on respiratory rhythm (Mdn = 1.2), mild breathing problems (Mdn = 0.6), and severe breathing problems (Mdn = 2,8), participants were placed within one of the two type categories. Since some of the girls with RTT shows only clinical features and not mutation in MECP2, only 14 patients were included in the analysis. As shown in figure 1, patients with a truncating mutation after NLS manifested a lower degree of impairment than patients with a truncating mutation within NLS in the Fig. 2 Genomic structure of the MECP2 gene and breathing dysfunctions (χ2 (2, N = 14) = 1.74, p<.05). localization of vowels with elicited denomination Conclusion In this work, a pilot study related to correlations between severity of breathing disorders and the residuals of speech capacity have been conducted. The results showed that the intensity of the parameters of the breathing dysfunction conditions speech production, in relation to the specific type of mutation in MECP2 in RTT patients. In particular comparisons between the truncating mutations differently affecting functional domains induce support for the idea that the crucial factor that leads to different phenotypes is the integrity of NLS (nuclear localization signal). Indeed, with reference to general phenotype, a milder form is linked to the possibility of protein to penetrate into the nucleus and link to Methylated CpG, maintaining its residual expression. Fig. 1 Genomic structure of the MECP2 gene and In this study the specific linguistic phenotype correlated to localization of breathing problems in the coding regions the the two kinds of mutations were clearly shown by the difference in the breathing scores and in the level of the With reference to the number of vowels with elicited vowels denomination with elicitation. denomination, based on high (> median) or low (≤ median) This pilot offers new possibility of genotype/phenotype scores on respiratory rhythm (Mdn = 1), low level of vowels correlation in a genetic syndrome because differently from with elicited denomination (Mdn = 0.4), and high level of other correlational study, the present one is focused on vowels with elicited denomination (Mdn = 2,7), participants specific cognitive process (i.e. language) rather than the were placed within one of the two type categories. As generic phenotype (i.e. both cognitive impairments and all shown in figure 2, patients with a truncating mutation after the clinical feature damage). NLS manifested a higher level of vowels denominations The present work analyzed samples with a limited number than patients with a truncating mutation within NLS (χ2 (2, of patients, for this reason it is just a pilot study and more N = 14) = 2.15, p<.05). With reference to the number of data has to be collected. The most important innovation consonants with elicited denomination (namely the introduced in the study was the use of the correlation syllables), only two girls (P376S and T442T) were able to between breathing and language in relation to the specific repeat a high number of syllables (respectively 12 and 10). genotype. For this reason the relative figure was not produced. References Adler, D. A., Quaderi, N. A., Brown, S. D., Chapman, V. M., Moore, J., Tate, P., Disteche, C. M. (1995). 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